European Journal of Medicinal Chemistry 78 (2014) 35e42

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Short communication

Discovery of 2-iminobenzimidazoles as potent hepatitis C virus inhibitors with a novel mechanism of action Marc Peter Windisch a, *, 3, Suyeon Jo b, 3, Hee-Young Kim a, Soo-Hyun Kim a, Keumhyun Kim a, Sunju Kong b, Hyangsuk Jeong c, Sujin Ahn c, Zaesung No d,1, Jong Yeon Hwang b, *, 2 a

Applied Molecular Virology, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea Medicinal Chemistry Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea DMPK Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea d Late Discovery Program, Institut Pasteur Korea (IP-K), Seongnam-si, Gyeonggi-do, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 January 2014 Received in revised form 11 March 2014 Accepted 11 March 2014 Available online 13 March 2014

In this report we describe 2-iminobenzimidazole (IBI) analogs, identified during the course of a phenotypic high-throughput screening campaign, as novel hepatitis C virus (HCV) inhibitors. A series of IBI derivatives was synthesized and evaluated for their inhibitory activity against infectious HCV. Among the IBIs derivatives studied in this work, we identified promising compounds with high antiviral efficacy, high selectivity index and good microsomal stability. Noteworthy, the IBI series exhibited inhibitory activity on early and late steps of the viral cycle, but not in the HCV replicon system demonstrating a mechanism of action distinct from clinical-stage and approved anti-HCV drugs. Overall, our results suggest that IBIs are predestinated for further exploration as lead compounds for novel HCV interventions. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Hepatitis C virus Phenotypic screening Iminobenzimidazole Entry Release

1. Introduction More than 170 million people worldwide are chronically infected with the hepatitis C virus (HCV) and are at risk of developing lifethreatening liver diseases [1]. Despite the recent approval of two direct-acting antiviral agents (DAAs), the standard of care (SOC) still relies on a combination therapy of pegylated interferon-alpha (PEG-IFNa) and ribavirin (RBV), associated with an unsatisfactory sustained virologic response rate (SVR) of only 70e80% [2e5]. As for the DAAs, PEG-IFNa therapy is accompanied by serious side effects responsible for low adherence to the therapy. This obvious unmet medical need encouraged us to screen small molecule compound libraries for new antiviral agents with a novel mechanism of action (MoA). We screened more than 200,000 small-molecules in a high-

content assay designed to identify compounds that can inhibit all stages of the viral life cycle [6]. In order to enrich for inhibitors with a novel mechanism of action, we counter-screened the active hits in the HCV replicon system, thereby reducing competition with replication inhibitors in clinical development. The inhibitors were finally tested in a series of secondary assays to elucidate their putative MoA. Thereby, we observed that IBI has a novel MoA by interfering with early and late steps in the HCV life cycle. These results prompted us to explore further the potential of the IBIs series as a new anti-HCV lead series. Initial structureeactivity relationship (SAR) studies revealed the functional groups responsible for antiHCV activity and identified a lead candidate suitable for further lead optimization studies. Here we report the results of our SAR and virological studies. 2. Results and discussion

* Corresponding authors. E-mail addresses: [email protected] (M.P. Windisch), [email protected]. kr, [email protected] (J.Y. Hwang). 1 Current address: Gyeonggi Bio-Center, Suwon 443-270, Republic of Korea. 2 Current address: Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea. 3 These authors equally contributed to this work. http://dx.doi.org/10.1016/j.ejmech.2014.03.030 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

2.1. High throughput screening and hit selection 200,000 compounds were screened against the entire HCV life cycle [7]. Briefly, naïve Huh-7 target cells were plated in 384-well microplates, incubated with 6 mM of the test compounds in

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M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42

Fig. 1. Structures of IBI (1a-c) and a known HCV IRES inhibitor (2).

duplicate, and inoculated with cell culture derived HCV (HCVcc). At 72 h post-infection experiments were analyzed and the half maximal effective concentration (EC50) to inhibit viral replication was calculated. In addition, the cytotoxic concentration (CC50) was determined to rule out whether the observed antiviral effects were due to toxicity induced by compounds. Active hits were cheerypicked and confirmed for activity by 10-point dose response curve (DRC) analysis in the primary assay as described previously [7]. Confirmed active hits were triaged in HCV pseudoparticle (HCVpp) and replicon systems to determine inhibitory effects on viral entry and viral RNA replication, respectively. Following this rational, we identified a series of 2iminobenzimidazoles (IBIs, 1aec) that exhibited submicromolar inhibitory activity in the infectious HCVcc assay (Fig. 1). Interestingly, 2-aminobenzimidazole (2), which has a chemical structure resembling IBI 1, has been reported as an in vitro inhibitor of HCV translation by interaction with the viral internal ribosome entry side (IRES) [8,9]. However, the IBIs series was inactive in an HCV genotype 1 and 2 replicon systems, suggesting that IBIs 1 have a MoA distinct from the 2-aminobenzimidazole series (2) which encouraged us to initiate a SAR study. 2.2. Chemistry A general scheme for synthesis of IBIs is depicted in Scheme 1. Commercially available chloronitrobenzenes 3 were

treated with methylamine to give intermediates 4, which were then reduced to methylaminoanilines 5. Cyclization of 5 with 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea 6 gave Boc-protected 2-aminobenzimidazoles 7 in good yields [10]. After deprotection of Boc on compound 7 by treatment of 4 N hydrogen chloride, N-alkylations with three types of reagents including benzyl bromides, phenoxyethyl bromides, and 2bromoacetophenones were conducted to afford the corresponding acyclic IBI analogs 9, 10, and 11, respectively. Several IBI analogs such as compound 16, 20, 23, and 25 were synthesized as shown in Scheme 2. 2-Chlorobenzimidazole 12 was subjected to alkylation with phenoxyethyl bromide to afford intermediate 13, which was condensed with ethanolamine to give intermediate 14. Chlorination by treatment with POCl3 and subsequent cyclization under thermal heating provided the desired cyclic IBI 16 [11]. Compound 20, imidazole-IBI, was synthesized from 2-aminobenzimidazole 17. Alkylation of compound 17 with 4-chlorophenoxyethylbromide gave compound 18, which was then treated with 1-bromobutan-2-one to give intermediate 19. Cyclization of compound 19 under heating led to imidazolo-IBI 20 [12]. Compound 23 bearing cyano in imino group of IBI was obtained from compound 21 by treatment of diphenyl cyanocarbonimidate [10], followed by alkylation with 4-chlorophenoxyethylbromide. Benzimidazolone 25 was synthesized through cyclization of compound 21 with carbonyl diimidazole (CDI), followed by alkylation.

Scheme 1. Reagents and conditions: (a) Methylamine, 5 mol% Cu, H2O, air, 100
C, 8 h; (b) Raney Ni, H2, EtOH, rt, 3 h; (c) p-TsOH, iPrOH, 65
C, 16 h; (d) 4 N HCl, 1,4-dioxane, 100
C, 30 min; (e) BnBr, DMF, rt-150
C; (f) Bromoacetophenone, DMF, rt-150
C; (g) Phenoxyethyl bromide, DMF, rt-150
C.

M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42

37

Scheme 2. Reagents and conditions: (a) 4-ClPhOEtBr, K2CO3, MeCN, 80
C; (b) ethanolamine, microwave, 200
C, 30 min; (c) POCl3, 110
C; (d) TEA, toluene, reflux; (e) 1-bromobutan-2-one, TEA, EtOH, reflux, 2 h; (f) 2-aminoethan-1-ol, microwave, 200
C, 30 min; (g) diphenyl cyanocarbonimidate, MeCN, 100
C, 8 h; (h) CDI, pyridine, THF, 65
C, 1 h.

2.3. Structureeactivity relationship (SAR) study All IBIs were evaluated for their anti-HCV activity and cytotoxicity (Tables 1e3) as described in Section 2.1. First we examined R2 substitution effect (Table 1). IBIs could be divided in three groups (benzyl 9, acetophenonyl 10, and phenoxyethyl 11), depending on the functional group (Fig. 1). IBIs 9 bearing a benzyl moiety exhibited good anti-HCV activities (EC50 ¼ 0.17e0.46 mM), but had a narrow selectivity index (SI ¼ 11.8e34.7). In case of acetophenone substituted IBIs (10), compound 10a and 10c displayed good antiHCV activity (EC50 ¼ 0.073 and 0.079 mM, respectively) with low cytotoxicity value (CC50 ¼ 20 and 8.9 mM, respectively), thereby providing good selectivity index value (SI ¼ 274 and 113, respectively). Compound 10d also showed good antiviral activity (EC50 ¼ 0.046 mM), but provided low selectivity index value (SI ¼ 59) due to increased cytotoxicity (CC50 ¼ 2.7 mM) in comparison with compound 10a. 4-Et substituted IBI 10b and naphthalene IBI 10e had a weaker antiviral activities compared to other substitutions. Phenoxyethyl substituted IBIs (11), had excellent anti-HCV potency (EC50 ¼ 0.016e0.050 mM). Among them the most promising compound was 4-chloro substituted IBI 11a with EC50 value of 0.016 mM and CC50 value of 7.6 mM, thereby providing a high SI of 475. Next, we synthesized several IBI analogs bearing various functional groups to determine essential chemical features of IBIs associated with potency. The scheme for synthesis of these analogs

is depicted in Scheme 2, and their anti-HCV activities are summarized in Table 2. Cyclic-IBI 16 showed similar anti-HCV activity (EC50 ¼ 0.050 mM) compared to acyclic IBI 11a, whereas imidazoleIBI 20, bearing an aromatic characteristic, had strongly reduced anti-HCV activity (EC50 ¼ 6.0 mM). These results indicate that cyclic structure is tolerated but aromatic structure is not favorable for anti-HCV activity. 2-Aminobenzimidazole 18 and IBI 19 with ketone moiety showed at least 100-fold reduced potency compared to IBI 11a. Compound 23 bearing cyano moiety in imine group of IBI was inactive. In addition, replacement of imine moiety in IBI to ketone (25) led to a loss of activity (>20 mM). This result strongly suggested that the imine group in IBI is crucial for anti-HCV activity. We further explored the effect of R1 substituent on anti-HCV activity (Table 3). Four compounds (26ae26d) with chloro substituent in position 4, 5, 6, and 7 were examined. Among chlorosubstituted compounds, 4-Cl substituted IBI (26a) exhibited fourfold improved anti-HCV potency (EC50 ¼ 0.0033) compared to compound 11a, while 5-Cl (26b) and 7-Cl (26d) substituted IBI retained antiviral activities. 6-Chloro substituted IBI (26c) displayed slightly reduced anti-HCV activity. In case of methyl substitution, 5-Me substituted IBI (26f) exhibited highly potent anti-HCV activity with EC50 value of 0.001 mM, while 4-Me and 6-Me substituted IBI (26e and 26g) were less active (EC50 ¼ 0.13 and 0.090 mM, respectively). Among all IBIs tested in this study, compound 26f exhibited the highest selectivity index value (SI ¼ 2900) in the HCVcc system.

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M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42

Table 1 Anti-HCV activity of IBIs 9e11.

No.

R2

EC50 (mM)

CC50 (mM)

SI (CC50/EC50)

9a 9b 9c 10a 10b 10c 10d

4-Et 4-tertBu 4-Ph 4-Cl 4-Et 4-tertBu 4-Ph

0.46 0.17 0.22 0.073 0.38 0.079 0.046

5.8 5.9 2.6 20 9.5 8.9 2.7

12.6 34.7 11.8 274 25 113 59

0.34

4.5

13

0.016 0.050 0.045 0.20

7.6 8.9 2.8 3.4

475 178 62 17

10e

11a 11b 11c 11d

4-Cl 4-Et 4-tertBu 4-Ph

EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate measurements. Table 2 Anti-HCV activity of IBI 16, 18, 19, 20, 23, and 25.

In order to determine metabolic stability, all IBI derivatives listed in Table 3 were tested in human and rat liver microsomes. Chloro-substituted IBIs (26ae26d) displayed an excellent microsomal stability in rat microsomes (t1/2 ¼ 37 or >60 min), whereas compound 11a (t1/2 ¼ 13.5 min) and 26ee26g (t1/2 ¼ 8.7e24.6 min) had a less favorable stability. 2.4. Mechanism of action studies EC50 (mM)

CC50 (mM)

16

0.050

20

18

4.0

8.9

19

2.3

8.5

No.

20

Q

6.0

20

23

>20

>20

25

>20

>20

EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate measurements.

Our primary screening campaign was conducted using the infectious HCV cell culture system, which in principle allows to target any step of the entire viral life cycle. Consequently, the MoA and ultimately the molecular target of active hit compounds remain to be elucidated. In order to determine which step is inhibited by the IBI series, we analyzed the activity of selected derivatives in HCVpp, HCV replicon and HCVcc late assays enabling to monitor viral entry, RNA replication and virus secretion, respectively. Using a viral supernatant transfer assay employing transiently transfected cells (producer cells) secreting infectious viral particle, we monitored inhibition of late stages in the viral life cycle by IBI, as previously

Table 3 Anti-HCV activity and microsomal stability of IBIs 26. No.

R1

EC50 (mM)

CC50(mM)

SI (CC50/EC50)

Metabolic stability Human/rat (t1/2, min)

11a 26a 26b 26c 26d 26e 26f 26g

H 4-Cl 5-Cl 6-Cl 7-Cl 4-Me 5-Me 6-Me

0.016 0.0033 0.011 0.04 0.0097 0.13 0.0010 0.09

7.6 1.5 4.1 9.84 4.3 15 2.9 10.62

475 455 373 246 443 115 2900 118

>60/13.5 >60/>60 >60/>60 >60/>60 >60/37.4 >60/24.6 >60/8.7 >60/18.0

EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate measurements.

M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42

described by Wichroski et al. [13]. No HCV RNA replication inhibition of producer cells was observed (black diamonds), corroborating the finding that the IBI series is inactive in HCV replicon assays (Fig. 2A). Transfer cell culture supernatant of producer cells to naïve target cells revealed a strong dose dependent reduction of secreted infectious HCV particles (blue squares), suggesting that IBIs interfere with a late step of the viral life cycle that affects the production of infectious virions. In addition, we monitored inhibition of viral particle attachment and/or internalization by using HCVpps. In order to demonstrate HCV-specificity of IBIs, we used vesicular stomatitis virus glycoprotein expressing pseudoparticles (VSVGpps) which were capable of transducing Huh-7 target cells in the presence of IBIs. Nonetheless, transduction of HCVpps was inhibited indicating that IBIs interfere with HCV attachment and/or internalization (Fig. 2B). Taken together, our data demonstrate that IBIs exhibit anti-HCV activity by inhibiting early and late steps of the viral cycle, whereas viral RNA replication was not affected. 3. Conclusion In conclusion, a series of iminobenzimidazoles was identified by a target-free phenotypic HTS assay using the infectious HCV cell culture system. Through our preliminary SAR study, we identified a series of promising IBI lead compounds that show excellent potency and safety margin, and an acceptable stability in human and rat liver microsomes. In addition, we demonstrated that the IBIs series inhibits early and late steps of the HCV life cycle. Although further intensive lead optimization and MoA studies are required, the IBI scaffold is not only a new chemical class for anti-HCV drug development, but also an interesting tool compound to study some basic aspects of the HCV life cycle. 4. Experimental section 4.1. Chemistry All materials were obtained from commercial suppliers and used without further purification. Solvents in this study were dried using an aluminum oxide column. Thin-layer chromatography was performed on pre-coated silica gel 60 F254 plates. Purification of intermediates was carried out by normal phase column chromatography (MPLC, Silica gel 230e400 mesh). NMR spectra were recorded on a Varian 400 MHz. LC/MS data were obtained using a Waters 2695 LC and Micromass ZQ spectrometer. Identity of final compounds was confirmed by proton NMR, carbon NMR, and mass spectrometry.

39

4.1.1. The representative procedure for the preparation of N-methyl2-nitroaniline (4, R1 ¼ H) A solution of 1-chloro-2-nitrobenzene (1.57 g, 10 mmol), 40% aqueous methylamine (4.3 mL, 50 mmol), and copper powder (31 mg, 0.5 mmol) was sealed in a 30 mL screw-cap tube and stirred at 100
C for 8 h. The reaction mixture was then cooled-down to room temperature and extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo to give crude product 4 (1.4 g, yield 95%) as oil. The crude product was used directly in next reaction step. 1H NMR (400 MHz, CDCl3) d 8.18 (d, J ¼ 8.0 Hz, 1H), 8.16 (NH, 1H), 7.45 (t, J ¼ 7.8 Hz, 1H), 6.84 (d, J ¼ 8.0 Hz, 1H), 6.65 (t, J ¼ 7.8 Hz, 1H), 3.02 (d, J ¼ 5.2 Hz, 3H); LC/MS (electrospray) m/z (M þ H)þ 153.05. 4.1.2. Representative procedure for the preparation of N1-methylbenzene-1,2-diamine (5, R1 ¼ H) A solution of compound 4 (R1 ¼ H, 760 mg, 5 mmol) and Raney Nickel (3 g) in ethanol was hydrogenated under 1 atm at room temperature for 3 h. The mixture was filtered over a pad of Celite. The filtrate was concentrated in vacuo and purified by silica gel column chromatography (eluent: n-Hex/EA ¼ 5:1) to give the product 5 (366 mg, yield 60%) as oil. 1H NMR (400 MHz, CDCl3) d 6.86 (t, J ¼ 8.4 Hz, 1H), 6.69 (m, 3H), 3.32 (NH, 2H), 2.87 (s, 3H); LC/ MS (electrospray) m/z (M þ H)þ 123.98. 4.1.3. Representative procedure for the preparation of tert-butyl (1-methyl-1H-benzo[d]imidazol-2-yl)carbamate (7, R1 ¼ H) A solution of compound 5 (R1 ¼ H, 100 mg, 0.657 mmol), 1,3bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea 6 (209 mg, 0.722 mmol), and p-toluene sulfonic acid (12 mg, 0.065 mmol) in isopropanol was heated at 65
C for 16 h. The reaction mixture was concentrated in vacuo and diluted with ethyl acetate. The organic layer was successively washed with 1N sodium hydroxide and brine, and dried over sodium sulfate. After concentration in vacuo, the crude product was purified by silica gel column chromatography (eluent : n-Hex/EA ¼ 5 : 1) to give the compound 7 (R1 ¼ H, 121 mg, yield 75%) as solid. 1H NMR (400 MHz, DMSO-d6) d 11.9 (NH, 1H), 7.38 (m, 2H), 7.14 (m, 2H), 3.65 (s, 3H), 1.45 (s, 9H); LC/MS (electrospray) m/z (M þ H)þ 248.03. 4.1.4. Representative procedure for the preparation of 1-methyl-1Hbenzo[d]imidazol-2-amine (8, R1 ¼ H) A solution of compound 7 (R1 ¼ H, 75 mg, 0.303 mmol) and 4 N HCl in 1,4-dioxane solution (1.5 mL, 6 mmol) was heated at 100
C for 30 min. The reaction mixture was then cooled-down to room temperature and the precipitant was collected and dissolved in

Fig. 2. Evaluation of HCV entry and secretion in the presence of IBIs. (A) Transiently transfected Huh-7.5 producer cells secreting HCV particle were treated with increasing IBI 11a concentration [mM] (X-axis). 48 h after treatment, viral cell culture supernatants were transferred to naïve target cells. Viral RNA replication (black diamonds) and cytotoxicity (red diamonds) of transiently transfected producer cells was determined at 72 h post compound treatment. Viral RNA replication of inoculated target cells was determined at 72 h post infection (blue squares). Data were normalized to the 1% DMSO control that was set to 0% inhibition. (B) HCVpp expressing E1/E2 viral glycoproteins of HCV and VSVGpp were incubated with 3.3 mM of IBI 11a. Inhibition of pseudoparticle transduction (%) and cell viability (%) is shown by black bars (left Y-axis) and red bars (right Y-axis), respectively. Data were normalized to the 1% DMSO control that was set to 0% inhibition. Experiments were conducted in triplicates with quadruple measurements (n ¼ 12). Representative experiments are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42

dichloromethane. The organic solution was neutralized with 1 N NaOH solution and washed with brine and dried over sodium sulfate. After concentration in vacuo, the crude solid product was washed with ether to give the pure product 8 (R1 ¼ H, 42 mg, yield 95%) as solid. 1H NMR (400 MHz, CDCl3) d 7.13 (m, 2H), 6.89 (m, 2H), 6.37 (NH, 2H), 3.47 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 148.07. 4.1.5. General procedure for the preparation of compounds 9, 10, 11 A solution of compound 8 (0.203 mmol, 1 eq) and the corresponding halides (0.244 mmol, 1.2 eq) in DMF (2 mL) was stirred at room temperature for 12 h. After the reaction was completed, ethyl acetate was added to the reaction mixture. The precipitate was filtered and washed with ether to give the corresponding product 9, 10, 11 as a salt form. 4.1.5.1. 1-(4-ethylbenzyl)-3-methyl-1H-benzo[d]imidazol-2(3H)imine (9a). Yield 43%; mp. 281.4
C; 1H NMR (400 MHz, DMSO-d6) d 8.99 (NH2, 2H), 7.53 (d, J ¼ 7.6 Hz, 1H), 7.42 (d, J ¼ 7.6 Hz, 1H), 7.31 (t, J ¼ 3.8 Hz, 1H), 7.25 (m, 5H), 5.42 (s, 2H), 3.68 (s, 3H), 2.55 (q, J ¼ 4.9 Hz, 2H), 1.12 (t, J ¼ 7.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) d 150.9, 144.2, 132.8, 131.2, 130.1, 128.8, 128.0, 124.0, 111.1, 110.8, 45.9, 30.3, 28.4, 16.2; LC/MS (electrospray) m/z (M þ H)þ 266.13. 4.1.5.2. 1-(4-(tert-butyl)benzyl)-3-methyl-1H-benzo[d]imidazol2(3H)-imine hydrochloride (9b). Yield 40%; mp. 286.2
C; 1H NMR (400 MHz, DMSO-d6) d 7.55 (d, J ¼ 7.6 Hz, 1H), 7.47 (d, J ¼ 7.6 Hz, 1H), 7.37 (d, J ¼ 8.0 Hz, 2H), 7.28 (m, 2H), 7.24 (t, J ¼ 8.0 Hz, 2H), 5.44 (s, 2H), 3.72 (s, 3H), 1.25 (s, 9H); LC/MS (electrospray) m/z (M þ H)þ 294.16. 4.1.5.3. 1-([1,1’-biphenyl]-4-ylmethyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-imine (9c). Yield 35%; mp. 315.4
C; 1H NMR (400 MHz, DMSO-d6) d 9.04 (NH, 1H), 7.64 (m, 4H), 7.57 (d, J ¼ 7.6 Hz, 1H), 7.31 (m, 8H), 5.52 (s, 2H), 3.70 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 314.16. 4.1.5.4. 1-(4-chlorophenyl)-2-(2-imino-3-methyl-2,3-dihydro-1Hbenzo[d]imidazol-1-yl)ethanone hydrobromide (10a). Yield 45%; mp. 257.2
C; 1H NMR (400 MHz, DMSO-d6) d 8.91 (NH2, 2H), 8.11 (d, J ¼ 6.8 Hz, 2H), 7.73 (d, J ¼ 6.8 Hz, 2H), 7.62 (d, J ¼ 8.0 Hz, 2H), 7.36 (t, J ¼ 8.2 Hz, 1H), 7.29 (t, J ¼ 8.2 Hz, 1H), 6.01 (s, 2H), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 191.1, 151.3, 139.8, 133.4, 131.1, 130.8, 129.6, 124.3, 111.2, 111.0, 50.7, 30.4; LC/MS (electrospray) m/z (M þ H)þ 300.01. 4.1.5.5. 1-(4-ethylphenyl)-2-(2-imino-3-methyl-2,3-dihydro-1Hbenzo[d]imidazol-1-yl)ethanone hydrobromide (10b). Yield 55%; mp. 254.7
C; 1H NMR (400 MHz, DMSO-d6) d 8.94 (NH2, 2H), 8.03 (d, J ¼ 8.0 Hz, 2H), 7.60 (t, J ¼ 7.6 Hz, 2H), 7.48 (d, J ¼ 8.4 Hz, 2H), 7.35 (t, J ¼ 7.8 Hz, 1H), 7.28 (t, J ¼ 7.8 Hz, 1H), 6.02 (s, 2H), 3.75 (s, 3H), 2.73 (q, J ¼ 4.9 Hz, 2H), 1.22 (t, J ¼ 7.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) d 191.4, 151.7, 151.3, 132.5, 130.8, 129.4, 128.9, 124.3, 111.1, 111.0, 50.6, 30.4, 29.0, 16.0 LC/MS (electrospray) m/z (M þ H)þ 294.16. 4.1.5.6. 1-(4-(tert-butyl)phenyl)-2-(2-imino-3-methyl-2,3-dihydro1H-benzo[d]imidazol-1-yl)ethanone hydrobromide (10c). Yield 47%; mp. 265.3
C; 1H NMR (400 MHz, DMSO-d6) d 8.83 (NH2, 2H), 8.03 (d, J ¼ 7.2 Hz, 2H), 7.67 (d, J ¼ 7.2 Hz, 2H), 7.59 (t, J ¼ 9.2 Hz, 2H), 7.36 (t, J ¼ 7.8 Hz, 1H), 7.29 (t, J ¼ 7.8 Hz, 1H), 5.95 (s, 2H), 3.73 (s, 3H), 1.34 (s, 9H); LC/MS (electrospray) m/z (M þ H)þ 322.13. 4.1.5.7. 1-([1,10 -biphenyl]-4-yl)-2-(2-imino-3-methyl-2,3-dihydro1H-benzo[d]imidazol-1-yl)ethanone hydrobromide (10d). Yield 46%;

mp. 271.5
C; 1H NMR (400 MHz, DMSO-d6) d 8.18 (d, J ¼ 8.4 Hz, 2H), 7.96 (d, J ¼ 8.4 Hz, 2H), 7.81 (d, J ¼ 7.6 Hz, 2H), 7.61 (d, J ¼ 8.0 Hz, 2H), 7.54 (t, J ¼ 7.6 Hz, 2H), 7.46 (t, J ¼ 7.2 Hz, 1H), 7.36 (t, J ¼ 7.6 Hz, 1H), 7.29 (t, J ¼ 7.6 Hz, 1H), 6.09 (s, 2H), 3.76 (s, 3H); LC/ MS (electrospray) m/z (M þ H)þ 342.06. 4.1.5.8. 2-(2-imino-3-methyl-2,3-dihydro-1H-benzo[d]imidazol-1yl)-1-(naphthalen-2-yl)ethanone hydrobromide (10e). Yield 48%; mp. 258.0
C; 1H NMR (400 MHz, DMSO-d6) d 8.88 (NH2, 2H), 8.81 (s, 1H), 8.21 (d, J ¼ 8.0 Hz, 1H), 8.13 (d, J ¼ 8.8 Hz, 1H), 8.06 (t, J ¼ 9.2 Hz, 2H), 7.75 (m, 2H), 7.68 (t, J ¼ 9.2 Hz, 2H), 7.38 (t, J ¼ 8.2 Hz, 1H), 7.31 (t, J ¼ 8.2 Hz, 1H), 6.13 (s, 2H), 3.76 (s, 3H); LC/ MS (electrospray) m/z (M þ H)þ 316.14. 4.1.5.9. 1-(2-(4-chlorophenoxy)ethyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-imine hydrobromide (11a). Yield 41%; mp. 300.8
C; 1H NMR (400 MHz, DMSO-d6) d 8.84 (NH2, 2H), 7.66 (d, J ¼ 2.1 Hz, 1H), 7.55 (d, J ¼ 2.1 Hz, 1H), 7.35 (m, 2H), 7.30 (d, J ¼ 8.8 Hz, 2H), 6.85 (d, J ¼ 8.8 Hz, 2H), 4.60 (t, J ¼ 5.0 Hz, 2H), 4.29 (t, J ¼ 5.0 Hz, 2H), 3.65 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 157.2, 150.8, 132.5, 131.8, 130.5, 129.9, 125.4, 124.1, 116.8, 111.5, 110.8, 66.2, 43.0, 30.2; LC/MS (electrospray) m/z (M þ H)þ 302.05. 4.1.5.10. 1-(2-(4-ethylphenoxy)ethyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-imine hydrobromide (11b). Yield 42%; mp. 276.0
C; 1H NMR (400 MHz, DMSO-d6) d 8.84 (NH2, 2H), 7.65 (d, J ¼ 2.1 Hz, 1H), 7.54 (d, J ¼ 2.1 Hz, 1H), 7.34 (m, 2H), 7.06 (d, J ¼ 8.8 Hz, 2H), 6.72 (d, J ¼ 8.8 Hz, 2H), 4.59 (t, J ¼ 5.0 Hz, 2H), 4.26 (t, J ¼ 5.0 Hz, 2H), 3.64 (s, 3H), 2.50 (q, J ¼ 4.9 Hz, 2H), 1.09 (t, J ¼ 7.8 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) d 156.5, 150.7, 136.9, 130.8, 130.5, 129.3, 124.1, 114.8, 111.6, 110.8, 65.9, 43.2, 30.3, 27.9, 16.5; LC/MS (electrospray) m/z (M þ H)þ 296.13. 4.1.5.11. 1-(2-(4-(tert-butyl)phenoxy)ethyl)-3-methyl-1H-benzo[d] imidazol-2(3H)-imine hydrobromide (11c). Yield 48%; mp. 270.4
C; 1 H NMR (400 MHz, DMSO-d6) d 8.88 (NH2, 2H), 7.66 (d, J ¼ 2.1 Hz, 1H), 7.55 (d, J ¼ 2.1 Hz, 1H), 7.33 (m, 2H), 7.23 (d, J ¼ 8.8 Hz, 2H), 6.73 (d, J ¼ 8.8 Hz, 2H), 4.61 (t, J ¼ 5.0 Hz, 2H), 4.27 (t, J ¼ 5.0 Hz, 2H), 3.65 (s, 3H), 1.20 (s, 9H); 13C NMR (100 MHz, DMSO-d6) d 156.2, 150.7, 143.8, 130.8, 130.5, 126.7, 124.0, 114.4, 111.6, 110.8, 65.9, 43.2, 34.3, 31.9, 30.4; LC/MS (electrospray) m/z (M þ H)þ 324.17. 4.1.5.12. 1-(2-([1,1’-biphenyl]-4-yloxy)ethyl)-3-methyl-1H-benzo[d] imidazol-2(3H)-imine hydrobromide (11d). Yield 46%; mp. 291.9
C; 1 H NMR (400 MHz, DMSO-d6) d 8.86 (NH2, 2H), 7.69 (d, J ¼ 2.0 Hz, 1H), 7.56 (m, 5H), 7.41 (t, J ¼ 7.8 Hz, 2H), 7.36 (m, 2H), 7.29 (t, J ¼ 7.4 Hz, 1H), 6.91 (d, J ¼ 8.8 Hz, 2H), 4.63 (t, J ¼ 5.0 Hz, 2H), 4.35 (t, J ¼ 5.0 Hz, 2H), 3.66 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 344.10. 4.1.6. Synthesis of 2-chloro-1-(2-(4-chlorophenoxy)ethyl)-1Hbenzo[d]imidazole (13) 1 H NMR (400 MHz, Acetone-d6) d 7.65 (d, J ¼ 8.4 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.29 (m, 2H), 7.23 (d, J ¼ 6.8 Hz, 2H), 6.89 (d, J ¼ 6.8 Hz, 2H), 4.43 (t, J ¼ 5.2 Hz, 2H), 4.74 (t, J ¼ 5.2 Hz, 2H); LC/MS (electrospray) m/z (M þ H)þ 309. 4.1.7. 2-((1-(2-(4-chlorophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl) amino)ethan-1-ol (14) 1 H NMR (400 MHz, CD3OD) d 7.29 (d, J ¼ 8.4 Hz, 1H), 7.23 (d, J ¼ 8.4 Hz, 1H), 7.18 (m, 2H), 7.03 (d, J ¼ 8.8 Hz, 2H), 6.83 (d, J ¼ 8.8 Hz, 2H), 4.38 (t, J ¼ 5.2 Hz, 2H), 4.23 (t, J ¼ 5.2 Hz, 2H), 3.57 (t, J ¼ 5.4 Hz, 2H), 3.06 (t, J ¼ 5.4 Hz, 2H); LC/MS (electrospray) m/z (M þ H)þ 332.

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4.1.8. Synthesis of N-(2-chloroethyl)-1-(2-(4-chlorophenoxy)ethyl)1H-benzo[d]imidazol-2-amine (15) 1 H NMR (400 MHz, CD3OD) d 7.60 (d, J ¼ 8.4 Hz, 1H), 7.42 (m, 3H), 7.21 (d, J ¼ 8.8 Hz, 2H), 6.84 (d, J ¼ 8.8 Hz, 2H), 4.62 (t, J ¼ 4.8 Hz, 2H), 4.37 (t, J ¼ 4.8 Hz, 2H), 3.89 (m, 4H); LC/MS (electrospray) m/z (M þ H)þ 352. 4.1.9. Synthesis of 9-(2-(4-chlorophenoxy)ethyl)-2,9-dihydro-3Hbenzo[d]imidazo[1,2-a]imidazole (16) Yield 22%; Yellow oil; 1H NMR (400 MHz, CD3OD) d 7.60 (m, 1H), 7.36 (m, 3H), 7.21 (d, J ¼ 8.8 Hz, 2H), 6.86 (d, J ¼ 8.8 Hz, 2H), 4.54 (t, J ¼ 4.8 Hz, 2H), 4.42 (m, 4H), 4.36 (t, J ¼ 4.8 Hz, 2H); 13C NMR (100 MHz, CD3OD) d 156.9, 156.5, 134.9, 129.2, 128.3, 126.2, 124.5, 123.5, 115.8, 111.5, 110.2, 65.6, 49.6, 43.9, 43.4; LC/MS (electrospray) m/z (M þ H)þ 314. 4.1.10. Synthesis of 1-(2-(4-chlorophenoxy)ethyl)-1H-benzo[d] imidazol-2-amine (18) Yield 48%; mp. 180.6
C; 1H NMR (400 MHz, DMSO-d6) d 7.29 (d, J ¼ 6.8 Hz, 2H), 7.20 (d, J ¼ 7.2 Hz, 1H), 7.10 (d, J ¼ 7.2 Hz, 1H), 6.93 (t, J ¼ 3.6 Hz, 1H), 6.90 (m, 3H), 6.39 (NH2, 2H), 4.37 (t, J ¼ 5.0 Hz, 2H), 4.20 (t, J ¼ 5.0 Hz, 2H); LC/MS (electrospray) m/z (M þ H)þ 288.97. 4.1.11. Synthesis of 1-(3-(2-(4-chlorophenoxy)ethyl)-2-imino-2,3dihydro-1H-benzo[d]imidazol-1-yl)butan-2-one (19) Yield 79%; mp. 218.8
C; 1H NMR (400 MHz, DMSO-d6) d 8.83 (NH2, 2H), 7.67 (d, J ¼ 8.4 Hz, 1H), 7.50 (d, J ¼ 8.4 Hz, 1H), 7.33 (m, 4H), 6.87 (d, J ¼ 6.8 Hz, 2H), 5.23 (s, 2H), 4.63 (t, J ¼ 5.0 Hz, 2H), 4.31 (t, J ¼ 5.0 Hz, 2H), 2.61 (q, J ¼ 20.2 Hz, 2H), 1.01 (t, J ¼ 7.4 Hz, 3H); LC/ MS (electrospray) m/z (M þ H)þ 358.98. 4.1.12. Synthesis of 9-(2-(4-chlorophenoxy)ethyl)-2-ethyl-9Hbenzo[d]imidazo[1,2-a]imidazole (20) Yield 70%; 1H NMR (400 MHz, CDCl3) d 7.44 (d, J ¼ 8.0 Hz, 2H), 7.27 (t, J ¼ 3.6 Hz, 1H), 7.17 (m, 3H), 7.08 (s, 1H), 6.72 (d, J ¼ 8.0 Hz, 2H), 4.56 (t, J ¼ 5.0 Hz, 2H), 4.37 (t, J ¼ 5.0 Hz, 2H), 2.74 (q, J ¼ 20.2 Hz, 2H), 1.32 (t, J ¼ 7.4 Hz, 3H); LC/MS (electrospray) m/z (M þ H)þ 340.02. 4.1.13. Synthesis of N-(6,7-dichloro-1-methyl-1,3-dihydro-2Hbenzo[d]imidazol-2-ylidene)cyanamide (22) A solution of compound 21 (600 mg, 3.140 mmol) and diphenyl cyanocarboimidate (748 mg, 3.140 mmol) in MeCN was heated at 100
C for 8 h. The reaction mixture was then cooled-down to room temperature to precipitate the product. The precipitate was collected and dried to afford the pure compound 22 (227 mg, yield 30%). 1H NMR (400 MHz, DMSO-d6) d 13.06 (NH, 1H), 7.38 (d, J ¼ 8.4 Hz, 2H), 7.14 (d, J ¼ 8.4 Hz, 2H), 3.68 (s, 3H); LC/MS (electrospray) m/ z (M þ H)þ 242.02. 4.1.14. Synthesis of N-(4,5-dichloro-1-(2-(4-chlorophenoxy)ethyl)3-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-ylidene)cyanamide (23) A solution of compound 22 (50 mg, 0.207 mmol), 1-(2bromoethoxy)-4-chlorobenzene (73 mg, 0.311 mmol), K2CO3 (86 mg, 0.622 mmol) in DMF (2 mL) was heated at 80
C for 4 h. The reaction mixture was then cooled-down to room temperature and poured into water. The precipitate was filtered and washed with EA to give the compound 23 (32 mg, Yield 40%). mp. 197.5
C; 1H NMR (400 MHz, DMSO-d6) d 7.54 (m, 2H), 7.26 (d, J ¼ 8.8 Hz, 2H), 6.84 (d, J ¼ 8.8 Hz, 2H), 4.66 (t, J ¼ 5.0 Hz, 2H), 4.31 (t, J ¼ 5.0 Hz, 2H), 3.87 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 157.3, 151.0, 131.8, 129.9, 128.8, 127.1, 125.3, 124.7, 116.8, 115.7, 114.2, 110.7, 66.7, 43.3, 32.5; LC/MS (electrospray) m/z (M þ H)þ 396.97.

41

4.1.15. Synthesis of 6,7-dichloro-1-methyl-1,3-dihydro-2H-benzo[d] imidazol-2-one (24) A solution of compound 21 (1.57 mmol, 1.0 equiv), pyridine (3.14 mmol, 2.0 equiv) and CDI (3.14 mmol, 2.0 equiv) in THF (5 mL) was heated at 65
C for 1 h. After reaction was completed, the reaction mixture was poured into EtOAc. The precipitant was collected and washed with EtOAc/Hexane, and dried to give compound 24 (Yield 55%). 4.1.16. Synthesis of 4,5-dichloro-1-(2-(4-chlorophenoxy)ethyl)-3methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (25) White solid; Yield 78%; mp. 167.3
C; 1H NMR (400 MHz, CDCl3) d 7.31 (m, 2H), 7.26 (d, J ¼ 8.4 Hz, 2H), 6.87 (d, J ¼ 8.4 Hz, 2H), 4.22 (m, 4H), 3.61 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 157.4, 154.3, 130.5, 129.8, 127.7, 125.2, 124.9, 122.8, 116.8, 112.8, 109.0, 66.5, 41.2, 30.2; LC/MS (electrospray) m/z (M þ H)þ 372. 4.1.16.1. Synthesis of 4-chloro-1-(2-(4-chlorophenoxy)ethyl)-3methyl-1H-benzo[d]imidazol-2(3H)-imine hydrobromide (26a). Yield 43%; mp. 246.0
C; 1H NMR (400 MHz, DMSO-d6) d 8.98 (NH2, 2H), 7.66 (d, J ¼ 8.8 Hz, 1H), 7.32 (m, 2H), 7.30 (d, J ¼ 8.8 Hz, 2H), 6.84 (d, J ¼ 8.8 Hz, 2H), 4.60 (t, J ¼ 5.0 Hz, 2H), 4.27 (t, J ¼ 5.0 Hz, 2H), 3.88 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 337.01. 4.1.16.2. Synthesis of 5-chloro-1-(2-(4-chlorophenoxy)ethyl)-3methyl-1H-benzo[d]imidazol-2(3H)-imine hydrobromide (26b). Yield 46%; mp. 314.2
C; 1H NMR (400 MHz, DMSO-d6) d 8.98 (NH2, 2H), 7.77 (s, 1H), 7.67 (d, J ¼ 8.8 Hz, 1H), 7.39 (d, J ¼ 8.8 Hz, 1H), 7.29 (d, J ¼ 8.8 Hz, 2H), 6.85 (d, J ¼ 8.8 Hz, 2H), 4.59 (t, J ¼ 5.0 Hz, 2H), 4.27 (t, J ¼ 5.0 Hz, 2H), 3.60 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 337.01. 4.1.16.3. Synthesis of 5-chloro-3-(2-(4-chlorophenoxy)ethyl)-1methyl-1H-benzo[d]imidazol-2(3H)-imine hydrobromide (26c). Yield 43%; mp. 281.3
C; 1H NMR (400 MHz, DMSO-d6) d 9.05 (NH2, 2H), 7.80 (s, 1H), 7.57 (d, J ¼ 8.4 Hz, 1H), 7.38 (d, J ¼ 8.4 Hz, 1H), 7.30 (d, J ¼ 8.8 Hz, 2H), 6.83 (d, J ¼ 8.8 Hz, 2H), 4.63 (t, J ¼ 5.0 Hz, 2H), 4.29 (t, J ¼ 5.0 Hz, 2H), 3.65 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 157.2, 151.4, 131.6, 130.0, 128.3, 125.4, 124.0, 116.7, 112.2, 111.9, 66.1, 43.1, 30.5; LC/MS (electrospray) m/z (M þ H)þ 337.01. 4.1.16.4. Synthesis of 4-chloro-3-(2-(4-chlorophenoxy)ethyl)-1methyl-1H-benzo[d]imidazol-2(3H)-imine hydrobromide (26d). Yield 30%; mp. 287.8
C; 1H NMR (400 MHz, DMSO-d6) d 9.02 (NH2, 2H), 7.57 (d, J ¼ 6.0 Hz, 1H), 7.37 (m, 2H), 7.31 (d, J ¼ 4.8 Hz, 2H), 6.87 (d, J ¼ 4.8 Hz, 2H), 4.85 (t, J ¼ 5.0 Hz, 2H), 4.33 (t, J ¼ 5.0 Hz, 2H), 3.66 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 337.01. 4.1.16.5. Synthesis of 1-(2-(4-chlorophenoxy)ethyl)-3,4-dimethyl-1Hbenzo[d]imidazol-2(3H)-imine hydrobromide (26e). Yield 40%; mp. 242.2
C; 1H NMR (400 MHz, DMSO-d6) d 8.71 (NH2, 2H), 7.48 (d, J ¼ 8.4 Hz, 1H), 7.30 (d, J ¼ 4.8 Hz, 2H), 7.22 (t, J ¼ 7.8 Hz, 1H), 7.09 (t, J ¼ 7.8 Hz, 1H), 6.84 (d, J ¼ 4.8 Hz, 2H), 4.58 (t, J ¼ 5.0 Hz, 2H), 4.27 (t, J ¼ 5.0 Hz, 2H), 3.84 (s, 3H), 2.66 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 316.07. 4.1.16.6. Synthesis of 1-(2-(4-chlorophenoxy)ethyl)-3,5-dimethyl-1Hbenzo[d]imidazol-2(3H)-imine hydrobromide (26f). Yield 55%; mp. 319.8
C; 1H NMR (400 MHz, DMSO-d6) d 8.75 (NH2, 2H), 7.52 (d, J ¼ 8.4 Hz, 1H), 7.37 (s, 1H), 7.29 (d, J ¼ 4.4 Hz, 2H), 7.16 (d, J ¼ 8.0 Hz, 1H), 6.85 (d, J ¼ 4.4 Hz, 2H), 4.56 (t, J ¼ 5.0 Hz, 2H), 4.27 (t, J ¼ 5.0 Hz, 2H), 3.61 (s, 3H), 2.41 (s, 3H); 13C NMR (100 MHz, DMSOd6) d 157.3, 150.6, 133.8, 130.9, 129.9, 128.5, 125.4, 124.8, 116.8, 111.2, 111.0, 66.2, 43.0, 30.2, 21.6; LC/MS (electrospray) m/z (M þ H)þ 316.07.

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M.P. Windisch et al. / European Journal of Medicinal Chemistry 78 (2014) 35e42

4.1.16.7. Synthesis of 3-(2-(4-chlorophenoxy)ethyl)-1,5-dimethyl-1Hbenzo[d]imidazol-2(3H)-imine hydrobromide (26g). White solid; Yield 27%; mp. 303.9
C; 1H NMR (400 MHz, DMSO-d6) d 8.70 (NH2, 2H), 7.41 (m, 2H), 7.30 (d, J ¼ 2.0 Hz, 2H), 7.14 (d, J ¼ 8.0 Hz, 1H), 6.85 (d, J ¼ 2.0 Hz, 2H), 4.54 (t, J ¼ 5.0 Hz, 2H), 4.29 (t, J ¼ 5.0 Hz, 2H), 3.60 (s, 3H), 2.41 (s, 3H); LC/MS (electrospray) m/z (M þ H)þ 316.14. 4.2. Biological evaluation 4.2.1. HCVcc assay Naïve Huh-7 cells were plated in 384-well plates, incubated with serially diluted compounds and inoculated with cell culture adapted HCVcc 2 h after compound treatment. At 72 h postinfection, HCV infection rates and cytotoxicity were determined [7]. 4.2.2. HCVpp assay HCV E1/E2-pseudotyped (or VSV-G pseudotyped as a control) lentiviral particles expressing a GFP reporter gene were produced as described previously [14]. Comparable amounts of HCVpp and VSVGpp were used in every experiment. Briefly, Huh-7 target cells were plated in 384-well plates, treated with compounds for 1 h followed by transduction with HCVpp or VSVGpp. Cell culture supernatants were replaced 12 h later and at 72 h post inoculation cytotoxicity and transduction efficiency were evaluated by determining number of cell nuclei and GFP reporter gene expression, respectively, by confocal microscopy at 20 magnification (ImageXpress ULTRA, Molecular Device). Relative transduction values were obtained using DMSO-treated cells as control. 4.2.3. HCVcc late assay The production of HCV was determined by a cell culture supernatant transfer assay as described previously [13]. Briefly, Huh7 cells transiently transfected with full length HCV RNA genomes (producer cells) secreting infectious virions, were plated in 384well plates and incubated overnight at 37
C. The next day cells were treated with serially diluted compounds at 37
C for 48 h followed by a wash step. The viral cell culture supernatant was clarified by centrifugation 24 h after the washing step and transferred to naïve Huh-7 target cells in 384-well plates. HCV RNA replication in producer cells and in target cells was measured at 72 h post compound treatment and 72 h post inoculation respectively. 4.2.4. HCV replication assay Huh-7 replicon cells stably replicating subgenomic HCV genomes were plated in 384-well plates and incubated at 37
C with serially diluted compounds. 72 h after compound treatment, the EC50 and CC50 values were calculated by non-linear regression using GraphPad Prism (GraphPad software) [7]. 4.3. Microsomal metabolic stability assay Compounds (2 mM final concentration in 0.02% DMSO) were incubated with 0.5 mg/mL human liver (pool of 200, mixed gender, Xenotech) and rat male (BD Gentest) microsomes in potassium phosphate buffer. The reaction was initiated by the addition of NADPH and stopped either immediately or at 10, 20, 30 and 60 min for a precise estimate of clearance. A triple quadrupole Quattro PremierÔ mass spectrometer (Waters, Milford, MA) with electrospray ionization (ESI) was employed for sample analysis. Samples were passed through trapping cartridges (Acquity BEH RP18

50 mm  2.1 mm, 1.7 mm, Waters, Milford, MA) followed by an analytical column. The mobile phases were (A) water with 0.1% of formic acid and (B) acetonitrile with 0.1% of formic acid at a flow rate of 0.4 mL/min. The LC conditions were 5% B at 0 min, a linear gradient from 5 to 50% B over 0.5 min, held at 50% for 0.25 min, then ramped from 50 to 95% over 0.25 min, followed by 95% B for 0.75 min and back to 5% B over 0.25 min, then held at 5% B for the remaining 0.75 min. The percentage of remaining compound was calculated by comparing with the initial quantity at 0 min. Half-life was then calculated based on first order reaction kinetics. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MEST) (No. 2012-00011), Gyeonggi-do and KISTI. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.03.030. References [1] Hepatitis C, WHO, (2012). [2] S. Chevaliez, R. Brillet, E. Lazaro, C. Hezode, J.M. Pawlotsky, Analysis of ribavirin mutagenicity in human hepatitis C virus infection, Journal of Virology 81 (2007) 7732e7741. [3] N. Hayashi, T. Takehara, Antiviral therapy for chronic hepatitis C: past, present, and future, Journal of Gastroenterology 41 (2006) 17e27. [4] M.P. Manns, H. Wedemeyer, M. Cornberg, Treating viral hepatitis C: efficacy, side effects, and complications, Gut 55 (2006) 1350e1359. [5] J. Shepherd, J. Jones, D. Hartwell, P. Davidson, A. Price, N. Waugh, Interferon alpha (pegylated and non-pegylated) and ribavirin for the treatment of mild chronic hepatitis C: a systematic review and economic evaluation, Health Technology Assessment 11 (2007) 1e205 (iii). [6] J.Y. Hwang, M.P. Windisch, S. Jo, K. Kim, S. Kong, H.C. Kim, S. Kim, H. Kim, M.E. Lee, Y. Kim, J. Choi, D.S. Park, E. Park, J. Kwon, J. Nam, S. Ahn, J. Cechetto, J. Kim, M. Liuzzi, Z. No, J. Lee, Discovery and characterization of a novel 7aminopyrazolo[1,5-a]pyrimidine analog as a potent hepatitis C virus inhibitor, Bioorganic & Medicinal Chemistry Letters 22 (2012) 7297e7301. [7] J.Y. Hwang, H.Y. Kim, S. Jo, E. Park, J. Choi, S. Kong, D.S. Park, J.M. Heo, J.S. Lee, Y. Ko, I. Choi, J. Cechetto, J. Kim, J. Lee, Z. No, M.P. Windisch, Synthesis and evaluation of hexahydropyrimidines and diamines as novel hepatitis C virus inhibitors, European Journal of Medicinal Chemistry 70C (2013) 315e325. [8] S. Zhou, K.D. Rynearson, K. Ding, N.D. Brunn, T. Hermann, Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA, Bioorganic & Medicinal Chemistry 21 (2013) 6139e6144. [9] J. Parsons, M.P. Castaldi, S. Dutta, S.M. Dibrov, D.L. Wyles, T. Hermann, Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA, Nature Chemical Biology 5 (2009) 823e825. [10] J.Y. Hwang, H.Y. Kim, D.S. Park, J. Choi, S.M. Baek, K. Kim, S. Kim, S. Seong, I. Choi, H.G. Lee, M.P. Windisch, J. Lee, Identification of a series of 1,3,4trisubstituted pyrazoles as novel hepatitis C virus entry inhibitors, Bioorganic & Medicinal Chemistry Letters 23 (2013) 6467e6473. [11] V.A. Anisimova, M.V. Levchendo, A.F. Pozharskii, Research on imidazo[1,2-a] benzimidazole derivatives. 22. Synthesis of 2,3-dihydroimidazo[1,2-a]benzimidazoles starting from 2-imino-3-(2-hydroxyethyl)benzimidazolines, Chemistry of Heterocyclic Compounds 22 (1986) 732e739. [12] N. Abe, T. Nishiwaki, H. Yamamoto, Syntheses and regioselective cycloaddition reactions of cyclohept[d]imidazo[1,2-a]imidazoles (1,3a,9triazacyclopent[a]azulenes): formation of 2,3-diazacyclohepta[ef]cycl[3.2.2] azine system, Chemistry Letters 11 (1982) 805e806. [13] M.J. Wichroski, J. Fang, B.J. Eggers, R.E. Rose, C.E. Mazzucco, K.A. Pokornowski, C.J. Baldick, M.N. Anthony, C.J. Dowling, L.E. Barber, J.E. Leet, B.R. Beno, S.W. Gerritz, M.L. Agler, M.I. Cockett, D.J. Tenney, High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus, PLoS One 7 (2012) e42609. [14] B. Bartosch, J. Dubuisson, F.L. Cosset, Infectious hepatitis C virus pseudoparticles containing functional E1-E2 envelope protein complexes, Journal of Experimental Medicine 197 (2003) 633e642.