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European Journal of Medicinal Chemistry 90 (2015) 497e506

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

New 1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamidesinhibit hepatitis C virus replication via suppression of cyclooxygenase-2

Dinesh Manvar a, 1, Sveva Pelliccia b, 1, Giuseppe La Regina b, Valeria Famiglini b,Antonio Coluccia b, Anna Ruggieri c, Simona Anticoli d, Jin-Ching Lee e, Amartya Basu a,Ozge Cevik a, Lucia Nencioni d, Anna Teresa Palamara f, g, Claudio Zamperini h,Maurizio Botta h, Johan Neyts i, Pieter Leyssen i, Neerja Kaushik-Basu a, *,Romano Silvestri b, *

a Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers, The State University of New Jersey, New Jersey Medical School, 185 SouthOrange Avenue, New Jersey 07103, United Statesb Istituto Pasteur e Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universit�a di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italyc Istituto Superiore di Sanit�a, Department of Infectious Parasitic and Immune Mediated Diseases, Viale Regina Elena 299, I-00161 Roma, Italyd Department of Public Health and Infectious Diseases, Sapienza Universit�a di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italye Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan, People's Republic of Chinaf Department of Public Health and Infectious Diseases, Istituto Pasteur e Fondazione Cenci Bolognetti, Sapienza Universit�a di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italyg San Raffaele Pisana Scientific Institute for Research, Hospitalization and Health Care, 00166 Rome, Italyh Dipartimento di Biotecnologia Chimica e Farmacia, Universit�a di Siena, Via Aldo Moro 2, I-53100 Siena, Italyi Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

a r t i c l e i n f o

Article history:Received 9 October 2014Received in revised form15 November 2014Accepted 21 November 2014Available online 27 November 2014

Keywords:HCVSubgenomic replicon 1b genotypeCyclooxigenase-2Pyrazolecarboxamide

* Corresponding authors.E-mail address: romano.silvestri@uniroma1.it (R. S

1 D.M. and S.P. contributed equally to this work.

http://dx.doi.org/10.1016/j.ejmech.2014.11.0420223-5234/© 2014 Published by Elsevier Masson SAS

a b s t r a c t

We report here the synthesis and mechanism of inhibition of pyrazolecarboxamide derivatives as a newclass of HCV inhibitors. Compounds 6, 7, 8 and 16 inhibited the subgenomic HCV replicon 1b genotype atEC50 values between 5 and 8 mM and displayed an even higher potency against the infectious Jc1 HCV 2agenotype. Compound 6 exhibited an EC50 of 6.7 mM and selectivity index of 23 against HCV 1b, andreduced the RNA copies of the infectious Jc1 chimeric 2a clone by 82% at 7 mM. Evaluation of the mode ofanti-HCV activity of 6 revealed that it suppressed HCV-induced COX-2 mRNA and protein expression,displaying an IC50 of 3.2 mM in COX-2 promoter-linked luciferase reporter assay. Conversely, the anti-HCVactivity of 6 was abrogated upon over-expression of COX-2. These findings suggest that 6 as a repre-sentative of these pyrazolecarboxamides function as anti-HCV agents via targeting COX-2 at both thetranscription and translation levels.

© 2014 Published by Elsevier Masson SAS.

1. Introduction

Hepatitis C virus (HCV), a single stranded positive sense RNAvirus, is a causative agent of hepatitis C, a disease that affects 3% ofthe world population, an estimated 200 million people worldwide.The global incidence of HCV infection is underestimated as the

ilvestri).

.

acute infection is generally asymptomatic. Eighty percent of HCVinfections lead to chronic liver disease that has the potential toevolve into fibrosis, cirrhosis and hepatocellular carcinoma [1e3].Both prophylaxis of HCV infection and vaccine development havebeen quite unsuccessful so far [4].

Treatment of HCV infections with peg-IFN, wherein INF-a iscovalently bound to polyethylene glycol, in combination with thesynthetic guanosine analogue Ribavirin, has demonstrated severeadverse effects in a large number of patients. The sustained viralresponse rate to this treatment was also very low in patients

Table 1Anti-HCV activities of compound 4e21.

Compd R1 R2 R3 Replicon 1b, Con 1 strain, Huh 5-2cells (mM)a

SIe

CC50b EC50c EC90d

4 H H H >228 31.7 ± 7.3 ndf >7.25 H H CN >247.6 39.6 ± 7.6 >241 >6.256 H H Me >153.8 6.7 ± 1.7 nd 237 H 4-Cl H 120.2 5.9 ± 1.3 nd 20.48 H 4-Cl CN 137.7 7.8 ± 0.3 nd 17.69 H 4-Cl Me 193.7 16.5 ± 6.0 61.8 11.710 H 3,4-Cl2 H >220.3 39.8 ± 3.2 nd >5.511 H 3,4-Cl2 CN >207.2 108.1 ± 13.4 nd >1.912 4-Cl H H >241.1 26.9 ± 13.1 >206.7 >8.913 4-Cl H CN 202.5 26.2 ± 6.5 nd 7.714 4-Cl H Me >232.2 16.7 ± 2.2 57.1 13.815 4-Cl 4-Cl H >220.3 35.4 ± 6.7 170.9 >6.216 4-Cl 4-Cl CN 151.7 5.5 ± 1.1 129.5 27.317 4-Cl 4-Cl Me >160.1 13.6 ± 2.4 nd >11.818 4-Cl 3,4-Cl2 H >202.7 >159.0 nd >1.319 4-Cl 3,4-Cl2 CN >257.3 50.5 ± 1.5 nd 5.120 4-Cl H H >241.2 >241.2 nd nd21 4-Cl 4-Cl H 62.6 13.7 ± 0.9 nd 4.7

a The data represent an average of at least two independent measurements induplicate ± SD.

b CC50: concentration of compound that reduces the overall cellular metabolicactivity by 50% (MTS-based assay).

c EC50.d EC90: effective concentration of compound that inhibits the virus replication by

50% or 90%, respectively (luciferase-based assay).

D. Manvar et al. / European Journal of Medicinal Chemistry 90 (2015) 497e506498

infected with HCV genotype 1, the predominant genotype in thewestern countries [5,6]. In 2011, the FDA approved the first direct-acting antiviral agents (DAAs) Boceprevir and Telaprevir for thetreatment of chronic hepatitis C genotype 1 infection in combina-tion with pegIFN-2a or -2b, and Ribavirin [7,8]. However, despiteimproved response rates, combination treatments including eitherBoceprevir or Telaprevir show the rapid selection of drug-resistantviral mutants [9], limited antiviral efficacy in patients with HCVgenotypes 2e6 infections and long-term toxic effects [8,10].Therefore, new HCV agents are under investigation towards eithernon-structural proteins or host factors.

In continuation of our studies on pyrazole agents [11] weobserved that N,1-diphenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (4), a pyrazolecarboxamide previously synthesizedby us, well matched structural features of some known HCV in-hibitors (i.e. 1 [12], 2 [13] and 3 [14]) (Chart 1).

In a preliminary screening, compound 4 showed significant in-hibition of the HCV replicon 1b (Con 1 strain) in Huh 5e2 cells withan EC50 of 31.7 mM (Table 1), and it was predicted to have drug-likeproperties by QikProp software (Table 2). These results promptedthe synthesis of the new 1-phenyl-5-(1H-pyrrol-1-yl)pyr-azolecarboxamides 5e21, in order to analyse the potential forproductive modifications of 4, in a quest to discover new potentanti-HCV agents.

2. Chemistry

Carboxamides 5e19 were obtained by reaction of carboxylicacids 22e27 with 1,10-carbonyldiimidazole in anhydrous THF at25 �C, and subsequent treatment of the imidazolide intermediateswith appropriate anilines (Scheme 1). Carboxamides 4, 20 and 21were obtained by reaction of acids 22 or 28 with the appropriateanilines in the presence of BOP reagent and triethylamine inanhydrous DMF at 25 �C. The corresponding carboxylic acids,22e27 [11c] and 28 [15] were synthesized by following our earlierreported procedures.

Chart 1. Structures of compounds 1e21. 4e21: R1 ¼ H, 4-Cl; R2 ¼ H, 4-Cl, 3,4-Cl2;R3 ¼ H, CN, Me.

e SI: selectivity index, CC50/EC50 ratio.f nd: not determined.

3. Results and discussion

3.1. Inhibition of HCV replicon 1b in Huh 5-2 cells

Compared to 4, compound 7 (EC50 ¼ 5.9 mM) bearing a chlorineatom at position 4 of the 3-(phenylaminocarbonyl)pyrazole moiety(R1 ¼ H; R2 ¼ 4-Cl; R3 ¼ H), yielded a 5.4-fold improvement of theantiviral activity. Similarly, the 3-[(4-chlorophenyl)amino-carbonyl]-4-cyanopyrazole derivative 8 (R1¼H; R2¼ 4-Cl; R3¼ CN)(EC50 ¼ 7.8 mM) was 5.1-fold more potent than the parent com-pound 5 (R1 ¼ H; R2 ¼ H; R3 ¼ CN) (EC50 ¼ 39.6 mM). In contrast,when a 3,4-dichlorophenyl moiety was introduced (compounds 10,11,18,19) (R1 ¼ H, 4-Cl; R2 ¼ 3,4-Cl2; R3 ¼ H, CN), a reduction in theantiviral activity was observed. Introduction of a chlorine atom atthe pendant 1-phenyl group of 8, to give the corresponding 1-(4-chlorophenyl)pyrazole derivative 16 (R1 ¼ 4-Cl; R2 ¼ 4-Cl;R3¼ CN) resulted in a strong replicon inhibitionwith EC50¼ 5.5 mM.

Replacement of the cyano substituent at position 4 of the pyr-azole with the methyl group (6, EC50 ¼ 6.7 mM) (R1 ¼ H; R2 ¼ H;R3 ¼ CH3), yielded 5.9- and 4.7-fold improvements in the antiviralactivity relative to 5 and 4, respectively. Furthermore, when thephenylaminocarbonyl moiety was shifted from position 3 to 4 ofthe pyrazole nucleus, a dramatic reduction (>9-fold) of the HCVreplicon activity was observed (compare 20with 12). However, theinhibitory activity was restored when the (4-chlorophenyl)

Table 2Prediction of drug-like properties of 4, 6, 7, 8 and 16 and reference compounds 1e3.

Compd MWa SASAb Volc Donor HBd Accpt HBe QP polrzf Qplog C16g Qplo Pocth Qplo Pwi Qplo Po/wj QplogSk PHOAl

4 328.37 634.61 1084.9 1 3.5 41.45 12.96 17.79 10.03 4.85 �6.235 1006 342.40 651.35 1133.8 1 3.5 43.06 13.18 18.07 9.82 5.13 �6.538 1007 362.37 660.62 1133.4 1 3.5 42.95 13.62 18.78 9.81 5.38 �7.018 1008 387.82 685.95 1187.6 1 5 44.27 14.38 21.36 11.30 4.74 �7.749 10016 422.27 692.02 1215.7 1 5 44.78 14.65 20.41 10.90 5.12 �8.173 1001 446.49 718.22 1302.6 2 9 44.06 13.02 22.91 14.55 2.85 �5.295 88.692 541.57 805.56 1466.9 2 12.5 53.78 16.6 29.5 20.29 2.23 �7.067 603 484.59 855.65 1567.4 1 6.5 56.06 16.3 24.7 11.25 6.24 �7.042 100

a MW, molecular weight.b SASA, total solvent accessible surface area (range, 300e1000 Å2).c Volume, total solvent accessible area (range: 500e2000 Å3).d H-bond donors.e H-bond acceptors.f QPpolrz, Predicted polarizability (range, 13e70 Å3).g QPlogPC16, predicted log of hexadecane/gas partition coefficient (range, 4e18).h QPlogPoct, predicted log of octanol/gas partition coefficient (range, 8e43).i QPlogPw, predicted log of water/gas partition coefficient (range, 5e48).j QPlogPo/w predicted log of octanol/water partition coefficient (range �2 to 6).k QplogS, predicted log of aqueous solubility (range, �6 to 0.5 M).l PHOS per cent human oral absorption.

D. Manvar et al. / European Journal of Medicinal Chemistry 90 (2015) 497e506 499

aminocarbonyl moiety was placed at position 4 of the pyrazole (21,EC50 ¼ 13.7 mM) (R1 ¼ 4-Cl; R2 ¼ 4-Cl, R3 ¼ H).

In summary, the most pronounced antiviral activity was ob-tained when a chlorine atom was introduced at position 4 of thephenylaminocarbonyl moiety (compounds 7, 8, 16). In comparingposition 3 with 4 of the pyrazole, the phenylaminocarbonyl moietyat position 3 resulted in higher selectivity (compare 12 and 15with20 and 21). Compounds 6, 7, 8 and 16 were the most potent in-hibitors of the HCV replicon within this series with inhibitoryconcentrations (EC50 values) in the lowest micromolar range ofconcentration. The most active derivative 16 also elicited thehighest selectivity index (16: EC50 ¼ 5.5 mM; SI ¼ 27.3).

3.2. Inhibition of HCV-Jc1 virus infected Huh 7.5 cells

We investigated the antiviral activity of three representativecompounds 4, 6 and 7 in HCV infected Huh 7.5 cells, using the mostadvanced HCV infectious system based on RNA transfection and

Scheme 1. Synthesis of pyrazole-3-carboxamides 4e19 and pyrazole-4-carboxamides20,21. 4e21, see Table 1 for R1, R2 and R3. Carboxylic acids: 22, R1 ¼ R3 ¼ H; 23, R1 ¼ H,R3 ¼ CN; 24, R1 ¼ 4-Cl, R3 ¼ H; 25, R1 ¼ 4-Cl, R3 ¼ CN; 26, R1 ¼ H, R3 ¼Me; 27, R1 ¼ 4-Cl,R3 ¼ Me; 28, R1 ¼ 4-Cl, R3 ¼ H. Reagents and conditions. (a): (5e19) (i) 1,10-carbon-yldiimidazole, anhydrous THF, 25 �C, 3 h; (ii) aniline (5, 6, 13, 14), 4-chloroaniline (7, 8,9, 15, 16, 17) or 3,4-dichloroaniline (10, 11, 18, 19), 25 �C, 24 h, 10e83%; (b): (4, 20, 21)aniline (4, 20), 4-chloroaniline (21) BOP reagent, triethylamine, anhydrous DMF, 25 �C,12 h, 13e70%.

subsequent infection of Huh7.5 cell line with HCV genotype 2ainfectious chimeric clone Jc1 [16].

In the Jc1 system, the compounds were initially screened at theEC50 values obtained in Huh 5e2 replicon 1b cells. Compounds 4, 6and 7 at concentrations of 10 mM, 7 mM and 6 mM, respectively,reduced the intracellular RNA copies of HCV genome by 70%, 82%and 68% (Chart 2), thus exhibiting higher potency in the Jc1 systemthan in the replicon 1b cells. These results suggest that treatmentwith compound 4 as well as with its analogues 6 and 7 effectivelyinhibited HCV infection in vitro.

3.3. Inhibition of HCV NS5B RdRp

To investigate whether the anti-HCV activity of compounds4e21 is mediated through targeting HCV NS5B, we evaluated theeffect of the compounds on NS5B RdRp activity by the standardprimer-dependent elongation reaction employing poly rA/rU12template-primer and recombinant HCV NS5BCD21 [17]. However,either no inhibition or considerably poor inhibition (data notshown) of RdRp activity of NS5B was observed, suggesting that theobserved anti-HCV effect was not due to the direct inhibition ofHCV NS5B.

Chart 2. Antiviral activity of compounds 4, 6 and 7 on HCV Jc1 infected cells. Huh 7.5cells were infected at 1 m.o.i. in 12 wells plates. Six h post-infection, cells were treatedfor 72 h with either DMSO (control) or compounds 4 (10 mM), 6 (7 mM) or 7 (6 mM).Cells were harvested and processed for total RNA isolation and one step qRT-PCR.*p < 0.05; **p < 0.01.

D. Manvar et al. / European Journal of Medicinal Chemistry 90 (2015) 497e506500

3.4. HCV IRES activity

We next investigated whether the anti-HCV effects of thesecompounds could be correlated to down regulation of HCV IRESactivity. Thus, Huh 7.5 cells were transfected with the pClneo-Rluc-IRES-Fluc reporter plasmid [18] in which Rluc was translated in acap-dependent manner and Fluc was translated via HCV IRES-mediated initiation, and treated with increasing concentrations ofcompound 6, as a representative of this series. The results (data notshown) clearly indicated that 6 had no effect on the HCV IRESmediated translation, thus ruling out interference with HCV IRESmediated translation as the potential mechanism of action.

Chart 3. Effect of 6 on HCV-induced COX-2 expression (A to C) Concentration-dependent inhibition of COX-2 promoter activity (A), protein synthesis (B) and RNAtranscription (C) by 6. For the promoter activity assay (A), MH-14 cells were transientlytransfected with the COX-2 promoter reporter plasmid pCOX-2-Luc and treated for48 h with DMSO (ctr) or the indicated concentrations of 6. The percent decrease ofrelative luciferase activity (RLU) in 6-treated MH-14 cells relative to DMSO-treatedcells, arbitrarily set at 100% for reference is shown. The RLU of pCOX-2-Luc-trans-fected-Huh-7 cells is shown for comparison. For protein and RNA analysis (B, and C),MH-14 cells were incubated with increasing concentrations of 6 for 48 h. Total celllysates were extracted and analyzed by Western blotting with anti-COX-2 and anti-b-Actin (loading control) antibodies. Total RNA in 6-treated MH-14 cells was extractedand quantified by qRT-PCR for relative levels of COX-2 mRNA and HCV RNA, normal-ized against b-Actin mRNA. (C) Densitometric analysis of HCV RNA and COX-2 proteinlevels. Values reported are percent fold change with respect to control cells. Error barsrepresent SD from three independent experiments performed in duplicate. *P < 0.05;**P < 0.01.

3.5. Compound 6 suppresses COX-2 expression

Multiple studies have demonstrated the stimulation of COX-2expression during HCV infection [19]. To investigate whethercompound 6 could attenuate HCV-induced COX-2 expression, weanalyzed the transcription and translation levels of COX-2 incompound 6-treated MH-14 cells. The COX-2 promoter-linkedluciferase reporter assay revealed a dramatic suppression of theCOX-2 promoter reporter in a concentration-dependent manner(Chart 3A). This corresponded to 35% and 68% suppression at2.8 mM and 4.4 mM, respectively of compound 6 concentration, andnear complete abolition above 14 mM, thus yielding an inhibitoryconcentration (IC50) ¼ 3.2 mM. These results indicate that 6 maymediate its antiviral effects through inhibition of COX-2 mRNAtranscripts.

We also investigated the effect of compounds 7, 8 and 16, whichdisplayed EC50 values near similar to 6 and SI > 17, on COX-2transcription. The COX-2 promoter-linked luciferase reporterassay revealed that all 3 compounds decreased luciferase activity,albeit to markedly different extents (Chart 1 of SupplementaryMaterial). Of these, 7 was the most effective, exhibiting a some-what similar trend as observed with 6, of dose-dependent sup-pression of the COX-2 promoter reporter. This was reflected in a60% decrease at 10 mM and ~95% inhibition above 25 mM compound7 concentrations. Compounds 8 displayed ~50% suppression at10 mM which only marginally increased to 65% at 50 mM. Surpris-ingly, compound 16 displayed no such distinct trend of dose-dependent decrease, exhibiting only 25e30% inhibition of COX-2promoter reporter at concentrations of 10e50 mM. This data sug-gests that the efficacy of compound 7 as anti-HCV agent may berelated to its ability to suppress COX-2 at the transcription level,while mechanisms in addition to COX-2 transcript suppressionmaycontribute towards the anti-HCV effects mediated by compounds 8and 16.

To validate the specificity of compound 6 in attenuating COX-2transcription, we analyzed its effect on the transcription of otherHCV-associated host factors such as heme oxygenase-1 (HO-1),Nrf2 and Interferon stimulated genes (ISGs). It was evident fromour findings that even at 50 mM concentration, 6 displayed no effecton the promoter-linked luciferase reporter assay for HO-1, Nrf2 orISGs in MH-14 cells (data not shown), thus ruling out the tran-scription regulation of these host factors as the potential mecha-nism of 6.

We next investigated the effect of 6 on COX-2mRNA and proteinsynthesis in relation to HCV replication inhibition. Towards thisaim, MH-14 cells were incubated with 6 at increasing doses for 48 hand subjected to analysis of endogenous levels of COX-2 proteinand mRNA expression and concomitant expression of HCV RNAlevels (Chart 3B and C). We observed that synthesis of COX-2 pro-tein was suppressed in a concentration dependent manner in 6-treated MH-14 cells (Chart 3B). This was reflected by ~50% decreaseof endogeneous COX-2 expression at 5 mM and near completeabolition above 15 mM, consistent with the trend seen with theCOX-2 promoter-linked luciferase reporter assay (Chart 3A).

The 6-mediated suppression of COX-2 was specific as deducedfrom the levels of b-actin under similar treatment conditions.Notably, suppression of COX-2 protein by 6 (Chart 3B) exhibited acorrelative trend with levels of COX-2 mRNA and a concomitantdecrease in HCV RNA (Chart 3C). This data indicates that compound6 inhibits HCV RNA replication via attenuating COX-2 mRNA tran-scription and consequent suppression of COX-2 protein synthesis.

Table 3ADME Properties of compound 6.

Compd PAMPAa

Papp � 10�6

(cm/s)

Membraneretention (%)

Watersolubilityb

(LogS)

Metabolicstabilityc (%)

Majormetabolites(%)

6 1.45 72.1 �7.69 98.87 1.12

a PAMPA: parallel artificial membrane permeability assay.b LogS: log mol L�1.c % of unmodified parent drug.

D. Manvar et al. / European Journal of Medicinal Chemistry 90 (2015) 497e506 501

3.6. Exogenous expression of COX-2 abrogates 6-mediatedinhibition of HCV RNA replication

To further investigate at the molecular level the role of COX-2down-regulation in 6-mediated inhibition of HCV replication, weresorted to transient overexpression of COX-2 and analyzed theanti-HCV effects of compound 6 under these conditions. MH-14cells were transfected with either a control vector (pcDNA4/myc-His-A) or increasing concentrations of pCMV-COX-2-Myc plasmidand subsequently treated with 6 at 25 mM, a concentration thatinhibited HCV-induced COX-2 expression and HCV replication by>90% (Chart 4A). RT-PCR and Western blotting analysis revealed acomplete knock-down of HCV RNA (top) and COX-2 expression(bottom), in 6-treated cells relative to DMSO treated control cells(Chart 4A, compare lane 2 to 1). A dose-dependent increase ofexogenous COX-2-Myc resulted in a gradual restoration of HCV RNAlevels in 6-treated cells (Chart 4A, lanes 3e6). This corresponded toquantitative recovery of HCV RNA levels by 50% and 100% in 6-treated cells at 0.4 mg and above 0.8 mg of pCMV-COX-2-Mycplasmid, respectively (Chart 4B).

This data indicates that exogenous COX-2 expression abrogates6-mediated anti-HCV activity. Taken together, our findings suggestthat compound 6mediates its anti-HCV effects via targeting COX-2.This supports targeting COX-2 as a viable strategy for anti-HCVinhibitor development and is in agreement with some previousstudies [20e22].

3.7. Prediction of drug-like properties

Drug likeness involves molecular features that mainly describewhether the molecule resembles the properties of known drugs.The drug-like properties of 4, 6, 7, 8 and 16 and reference com-pounds 1e3 (Table 2) as predicted by employing QikProp software[23] fall within the reference ranges (Table 2 caption) of the 95% ofused drugs, with the only violation of the QplogS values. These datahighlight an interesting drug-like profile of these anti-HCV agentsrepresenting a promising basis for further development.

3.8. ADME studies

Compound 6 exhibited excellent plasma andmetabolic stability,and did not behave as a prodrug. Derivative 6 was not hydrolysedby the human plasmatic enzymes for 24 h (Chart 2 ofSupplementary Material). Compound 6 underwent metabolicoxidative CYP-dependent metabolism. Themetabolites hadM.W. ofthe parent compound þ16; such results were in good agreementwith the metabolite structures hypothesized by Metasite software

Chart 4. Dose dependent restoration of 6-mediated suppression of HCV replication upon expCMV-COX-2-Myc plasmid encoding cox-2 and 6 h post-transfection, treated with either Dblotting (WB) for expression of COX-2 and b-Actin as loading control (A, bottom two panels).two panels). B depicts the quantification of HCV RNA and COX-2 expression level withexperiments.

(Chart 3 of Supplementary Material). Compound 6 showed lowmembrane permeability and water solubility (Table 3).

4. Conclusions

The present study describes the synthesis and biological eval-uation of a series of 1-phenyl-5-(1H-pyrrol-1-yl)pyrazolecarbox-amides as a new class of anti-HCV agents. Our results show that thepyrazolecarboxamides inhibited HCV replication without causinghost cellular toxicity. Significantly, four compounds bearing thisscaffold exerted strong antiviral effects on both HCV genotype 1band 2a. Of these, compound 6 proved to be the most promising,exhibiting an EC50 value of 6.7 mM and selectivity index of 23against HCV 1b, and reducing the RNA copies of the infectious Jc1chimeric 2a clone by 82% at 7 mM. The anti-HCV activity of com-pound 6was identified to be mediated by COX-2 inhibition, at boththe transcriptional and translational levels. The broad range andstrong antiviral activities against HCV genotype 1b and 2a of thepyrazolcarboxamide compounds, the interesting molecular mech-anism of anti-HCV activity, that targets host factor dysregulatedduring viral infection, together with their drug likeness properties,serve as a strong basis for further optimization of this new chemicalclass of HCV inhibitors.

5. Experimental protocols

5.1. Chemistry

Melting points (mp) were determined on a Stuart ScientificSMP1 apparatus and are uncorrected. Infrared spectra (IR) were runon a PerkineElmer SpectrumOne FT-ATR spectrophotometer. Bandposition and absorption ranges are given in cm�1. Proton nuclearmagnetic resonance (1H NMR) spectra were recorded on a Bruker400 MHz FT spectrometer in the indicated solvent and corre-sponding fid files processed by MestreLab Research S.L. MestreR-eNova 6.2.1-769 software. Chemical shifts are expressed in d units

ogenous COX-2 expression. MH14 cells were transfected with the indicated amounts ofMSO (control) or 25 mM compound 6 for 72 h. Cell lysates were analysed by WesternIn another set, total RNAs were subjected to qRT-PCR to evaluate HCV RNA levels (A, toprespect to control. The values represent the average ± SD from three independent

D. Manvar et al. / European Journal of Medicinal Chemistry 90 (2015) 497e506502

(ppm) from tetramethylsilane. Column chromatography was per-formed on columns packed with alumina from Merck (70e230mesh) or silica gel from MachereyeNagel (70e230 mesh).Aluminum oxide thin layer chromatography (TLC) cards from Fluka(aluminum oxide precoated aluminum cards with fluorescent in-dicator detectable at 254 nm) and silica gel TLC cards fromMachereyeNagel (silica gel precoated aluminum cards with fluo-rescent indicator detectable at 254 nm) were used for TLC. Devel-oped plates were visualized by a Spectroline ENF 260C/FE UVapparatus. Reagents and solvents from commercial sources wereused without further purification. Organic solutions were driedover anhydrous Na2SO4. Evaporation of the solvents was carried outon a Büchi rotavapor R-210 equipped with a Büchi V-855 vacuumcontroller and a Büchi V-700 or V-710 vacuum pump. Elementalanalyses of the compounds were found within ±0.4% of the theo-retical values. For RdRp assays, compoundswere dissolved in DMSOas 10 mM stocks and serially diluted in DMSO immediately prior tothe use.

5.1.1. General procedure for the synthesis of carboxamides 5e19.Example. 4-Cyano-N,1-diphenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (5)

To a mixture of 4-cyano-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyr-azole-3-carboxylic acid (0.25 g, 1.0 mmol) (22) [11c] in anhydrousTHF (14 mL) was added 1,10-carbonyldiimidazole (0.220 g,1.3 mmol) under Ar stream. The reaction mixture was stirred for2 h at room temperature. Aniline (0.170 g, 0.16 mL, 1.8 mmol) wasadded and the reaction was stirred at room temperature for 24 h.The mixture was diluted with water and extracted with ethyl ace-tate. The organic layer was washed with brine, dried and filtered.Removal of the solvent gave a residue that was purified by a firstcolumn chromatography (silica gel, ethyl acetate/n-hexane 1:1 aseluent) and a second column chromatography (silica gel, chloro-form/ethanol 9:1 as eluent) to provide 5 (0.14 g, 44%), mp180e185 �C (from ethanol). 1H NMR (DMSO-d6): d 6.29e6.31 (m,2H), 6.99e7.01 (m, 2H), 7.14 (t, J ¼ 6.9 Hz, 1H), 7.35e7.40 (m, 4H),7.49e7.51 (m, 3H), 7.81e7.83 (d, J ¼ 7.8 Hz, 2H), 10.60 ppm (br s,disappeared on treatment with D2O, 1H). IR: n 1684, 3299 cm�1.Anal. Calcd. for C21H15N5O: C, 71.38%; H, 4.28%; N, 19.82%. Found: C,71.11%; H, 4.20%, N, 19.60%.

5.1.2. N,1-diphenyl-5-(1H-pyrrol-1-yl)-4-methyl-1H-pyrazole-3-carboxamide (6)

Was synthesized as 5 from 4-methyl-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxylic acid (26) and aniline. Yield 62% mp147e150 �C (from ethanol). 1H NMR (DMSO-d6): d 2.16 (s, 3H),6.27e6.29 (m, 2H), 6.90e6.92 (m, 2H), 7.10 (t, J ¼ 7.2 Hz, 1H),7.24e7.26 (d, J¼ 6.7, 2H), 7.32e7.43 (m, 5H), 7.81e7.83 (d, J¼ 8.4 Hz,2H), 10.16 ppm (br s, disappeared on treatment with D2O, 1H). 13CNMR (DMSO-d6) d 111.0, 115.0, 120.8, 123.3, 123.4, 124.1, 128.6,129.0, 129.5, 138.1, 138.4, 139.0, 143.8, 161.0 ppm. IR: n 1666,3362 cm�1. Anal. Calcd. for C21H18N4O: C, 73.67%; H, 5.30%; N,16.36%. Found: C, 73.49%; H, 5.22%, N, 16.18%.

5.1.3. N-(4-Chlorophenyl)-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (7)

Was synthesized as 5 from 22 and 4-choroaniline. Yield 75%, mp140e145 �C (from ethanol). 1H NMR (DMSO-d6): d 6.21e6.22 (m,2H), 6.87e6.89 (m, 2H), 7.09e7.10 (m, 1H), 7.29e7.31 (m, 2H),7.39e7.46 (m, 5H), 7.86e7.88 (dd, J¼ 9.2 and 1.8 Hz, 2H), 10.40 ppm(br s, disappeared on treatment with D2O, 1H). 13C NMR (DMSO-d6)d 99.9, 103.6, 111.0, 122.4, 123.0, 124.6, 127.9, 128.9, 129.2, 129.7,138.0,140.8,146.7,159.9 ppm. IR: n 1678, 3382 cm�1. Anal. Calcd. forC20H15ClN4O: C, 66.21%; H, 4.17%; N, 15.44%; Cl, 9.77%. Found: C,66.02%; H, 4.15%, N, 15.24%; Cl, 9.51%.

5.1.4. N-(4-Chlorophenyl)-4-cyano-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (8)

Was synthesized as 5 from 23 and 4-choroaniline. Yield 42%, mp197e201 �C (from ethanol). 1H NMR (DMSO-d6): d 6.29e6.31 (m,2H), 6.98e7.01 (m, 2H), 7.38e7.44 (m, 4H), 7.48e7.52 (m, 3H),7.86e7.88 (d, J ¼ 8.6 Hz, 2H), 10.76 ppm (br s, disappeared ontreatment with D2O, 1H). 13C NMR (DMSO-d6) d 89.9, 111.9, 112.0,122.8, 122.9, 125.2, 128.5, 129.0, 129.8, 130.3, 136.8, 137.4, 145.5,146.9, 157.9 ppm. IR: n 1678, 3318 cm�1. Anal. Calcd. forC21H14ClN5O: C, 65.04%; H, 3.64%; N, 18.06%; Cl, 9.14%. Found: C,64.87%; H, 3.58%, N, 17.88%; Cl, 8.90%.

5.1.5. N-(4-chlorophenyl)-4-methyl-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (9)

Was synthesized as 5 from 26 and 4-choroaniline. Yield 12%, mp147e150 �C (from ethanol). 1H NMR (DMSO-d6): d 2.15 (s, 3H),6.25e6.27 (m, 2H), 6.89e6.91 (m, 2H), 7.23e7.25 (m, 2H), 7.37e7.41(m, 5H), 7.86e7.88 (d, J ¼ 8.8 Hz, 2H), 10.34 ppm (br s, disappearedon treatment with D2O, 1H). IR: n 1667, 3357 cm�1. Anal. Calcd. forC21H17ClN4O: C, 66.93%; H, 4.55%; N, 14.87%; Cl, 9.41%. Found: C,66.77%; H, 4.48%, N, 14.70%; Cl, 9.28%.

5.1.6. N-(3,4-Dichlorophenyl)-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (10)

Was synthesized as 5 from 22 and 3,4-dichloroaniline. Yield 51%,mp 142e146 �C (from ethanol). 1H NMR (DMSO-d6): d 6.20e6.22(m, 2H), 6.87e6.89 (m, 2H), 7.11 (s, 1H), 7.30e7.32 (m, 2H),7.44e7.46 (m, 3H), 7.60e7.62 (d, J ¼ 8.8 Hz, 1H), 7.84e7.86 (dd,J¼ 2.2 and 8.6 Hz, 1H), 8.20 (s, 1H), 10.57 ppm (br s, disappeared ontreatment with D2O, 1H). IR: n 1693, 3367 cm�1. Anal. Calcd. forC20H14Cl2N4O: C, 60.47%; H, 3.55%; N, 14.10%; Cl, 17.85%. Found: C,60.35%; H, 3.50%, N, 13.95%; Cl, 17.68%.

5.1.7. 4-Cyano-N-(3,4-dichlorophenyl)-1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (11)

Was synthesized as 5 from 23 and 3,4-dichloroaniline. Yield 10%,mp 213e219 �C (from ethanol). 1H NMR (DMSO-d6): d 6.29e6.31(m, 2H), 6.99e7.01 (m, 2H), 7.38e7.40 (m, 2H), 7.49e7.51 (m, 3H),7.63e7.65 (d, J ¼ 8.4 Hz, 1H), 7.84e7.86 (d, J ¼ 8.8 Hz, 1H), 8.21 (s,1H), 10.92 ppm (br s, disappeared on treatment with D2O, 1H). IR: n1692, 3351 cm�1. Anal. Calcd. for C21H13Cl2N5O: C, 59.73%; H, 3.10%;N, 16.59%; Cl, 16.79%. Found: C, 59.56%; H, 3.04%, N, 16.48%; Cl,16.51%.

5.1.8. 1-(4-Chlorophenyl)-N-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (12)

Was synthesized as 5 from 1-(4-chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxylic acid (24) and aniline. Yield 44%, mp190e195 �C (from ethanol). 1H NMR (DMSO-d6): d 6.23e6.26 (m,2H), 6.89e6.91 (m, 2H), 7.08e7.12 (m, 2H), 7.28e7.37 (m, 4H),7.52e7.54 (d, J ¼ 8.2 Hz, 2H), 7.80e7.82 (d, J ¼ 8.0 Hz, 2H),10.24 ppm (br s, disappeared on treatment with D2O, 1H). IR: n1675, 3391 cm�1. Anal. Calcd. for C20H15ClN4O: C, 66.21%; H, 4.17%;N, 15.44%; Cl, 9.77%. Found: C, 65.96%; H, 4.12%, N, 15.19%; Cl, 9.61%.

5.1.9. 1-(4-Chlorophenyl)-4-cyano-N-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (13)

Was synthesized as 5 from 1-(4-chlorophenyl)-4-cyano-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxylic acid (25) and aniline. Yield40% mp 234e238 �C (from ethanol). 1H NMR (DMSO-d6):d 6.31e6.34 (m, 2H), 7.00e7.02 (m, 2H), 7.15 (t, J ¼ 7.08 Hz, 1H),7.35e7.40 (m, 4H), 7.59e7.61 (d, J ¼ 8.08 Hz, 2H), 7.81e7.83 (d,J ¼ 7.68 Hz, 2H), 10.61 ppm (br s, disappeared on treatment withD2O, 1H). IR: n 1685, 3337 cm�1. Anal. Calcd. for C21H14ClN5O: C,

D. Manvar et al. / European Journal of Medicinal Chemistry 90 (2015) 497e506 503

65.04%; H, 3.64%; N, 18.06%; Cl, 9.14%. Found: C, 64.82%; H, 3.58%, N,17.83%; Cl, 8.95%.

5.1.10. 1-(4-Chlorophenyl)-4-methyl-N-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (14)

Was synthesized as 5 from 1-(4-chlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxylic acid (27) and aniline. Yield83% mp 155e158 �C (from ethanol). 1H NMR (DMSO-d6): d 2.14 (s,3H), 6.28e6.30 (m, 2H), 6.91e6.93 (m, 2H), 7.10 (t, J ¼ 6.7 Hz, 1H),7.22e7.25 (m, 2H), 7.32e7.36 (m, 2H), 7.47e7.50 (m, 2H), 7.80e7.82(d, J ¼ 7.9 Hz, 2H), 10.19 ppm (br s, disappeared on treatment withD2O, 1H). IR: n 1680, 3355 cm�1. Anal. Calcd. for C21H17ClN4O: C,66.93%; H, 4.55%; N,14.87%; Cl, 9.41%. Found: C, 66.80%; H, 4.49%, N,14.70%; Cl, 9.28%.

5.1.11. N,1-bis(4-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (15)

Was synthesized as 5 from 24 and 4-choroaniline. Yield 44%, mp198e200 �C (from ethanol). 1H NMR (DMSO-d6): d 6.24e6.26 (m,2H), 6.90e6.92 (m, 2H), 7.11 (s, 1H), 7.28e7.30 (d, J ¼ 8.7 Hz, 2H),7.39e7.42 (d, J¼ 8.5 Hz, 2H), 7.52e7.54 (d, J¼ 8.0 Hz, 2H), 7.85e7.87(d, J ¼ 8.8 Hz, 2H), 10.42 ppm (br s, disappeared on treatment withD2O, 1H). IR: n 1685, 3395 cm�1. Anal. Calcd. for C20H14Cl2N4O: C,60.47%; H, 3.55%; N,14.10%; Cl,17.85%. Found: C, 60.32%; H, 3.49%, N,13.88%; Cl, 17.59%.

5.1.12. N,1-bis(4-chlorophenyl)-4-cyano-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (16)

Was synthesized as 5 from 25 and 4-choroaniline. Yield 47%, mp255e258 �C (from ethanol). 1H NMR (DMSO-d6): d 6.31e6.34 (m,2H), 6.99e7.02 (m, 2H), 7.38e7.44 (m, 4H), 7.58e7.61 (d, J ¼ 8.7 Hz,2H), 7.85e7.88 (d, J ¼ 8.7 Hz, 2H), 10.78 ppm (br s, disappeared ontreatment with D2O, 1H). IR: n 1685, 3323 cm�1. 13C NMR (DMSO-d6) d 90.2, 99.9, 111.8, 112.2, 122.8, 122.9, 126.9, 128.5, 129.0, 129.9,134.9, 135.6, 137.3, 145.6, 147.0, 157.8 ppm. Anal. Calcd. forC21H13Cl2N5O: C, 59.73%; H, 3.10%; N, 16.59%; Cl, 16.79%. Found: C,59.50%; H, 3.06%, N, 16.41%; Cl, 16.51%.

5.1.13. N,1-bis (4-Chlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (17)

Was synthesized as 5 from 1-(4-chlorophenyl)-4-methyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxylic acid (29) and 4-chloroaniline. Yield 11%, mp 179e182 �C (from ethanol). 1H NMR(DMSO-d6): d 2.14 (s, 3H), 6.28e6.30 (m, 2H), 6.91e6.93 (m, 2H),7.22e7.24 (d, J¼ 8.8 Hz, 2H), 7.39e7.41 (d, J¼ 8.8 Hz, 2H), 7.48e7.51(d, J ¼ 8.8 Hz, 2H), 7.85e7.87 (d, J ¼ 8.8 Hz, 2H), 10.36 ppm (br s,disappeared on treatment with D2O, 1H). IR: n 1669, 3367 cm�1.Anal. Calcd. for C21H16Cl2N4O: C, 61.33%; H, 3.92%; N, 13.62%; Cl,17.24%. Found: C, 61.15%; H, 3.88%, N, 13.46%; Cl, 17.01%.

5.1.14. 1-(4-Chlorophenyl)-N-(3,4-dichlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (18)

Was synthesized as 5 from 24 and 3,4-dichloroaniline. Yield53%, mp 165e171 �C (from ethanol). 1H NMR (DMSO-d6):d 6.23e6.25 (m, 2H), 6.89e6.91 (m, 2H), 7.12 (s, 1H), 7.29e7.31 (d,J¼ 8.8 Hz, 2H), 7.53e7.55 (d, J¼ 8.8 Hz, 2H), 7.60e7.63 (d, J¼ 8.8 Hz,1H), 7.83e7.86 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 8.20 (s, 1H), 10.59 ppm(br s, disappeared on treatment with D2O, 1H). IR: n 1687,3380 cm�1. Anal. Calcd. for C20H13Cl3N4O: C, 55.64%; H, 3.04%; N,12.98%; Cl, 24.64%. Found: C, 55.46%; H, 2.98%, N,12.70%; Cl, 24.50%.

5.1.15. 1-(4-Chlorophenyl)-4-cyano-N-(3,4-dichlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carbo-xamide (19)

Was synthesized as 5 from 25 and 3,4-dichloroaniline. Yield 11%,mp 237e244 �C (from ethanol). 1H NMR (DMSO-d6): d 6.31e6.33

(m, 2H), 6.99e7.01 (m, 2H), 7.38e7.40 (d, J ¼ 8.4 Hz, 2H), 7.59e7.65(m, 3H), 7.82e7.85 (dd, J ¼ 2.4 and 8.8 Hz, 1H), 8.19 (s, 1H),10.93 ppm (br s, disappeared on treatment with D2O, 1H). IR: n1693, 3346 cm�1. Anal. Calcd. for C21H12Cl3N5O: C, 55.23%; H, 2.65%;N, 15.33%; Cl, 23.29%. Found: C, 55.03%; H, 2.61%, N, 15.12%; Cl,22.95%.

5.1.15.1. General procedure for the synthesis of carboxamides 4, 20and 21. Example. 1-(4-Chlorophenyl)-N-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-4-carboxamide (20). A mixture of 1-(4-chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-4-carboxylic acid (28) [15] (0.25 g,0.87 mmol), aniline (0.23 g, 0.24 mL, 2.61 mmol), BOP reagent(0.35 g, 0.87 mmol) and triethylamine (0.26 g, 0.36 mL, 2.61 mol) inanhydrous DMF (8 mL) was stirred at room temperature overnight.The reaction mixture was diluted with brine and extracted withethyl acetate. The organic layer was washed with brine, dried andfiltered. Removal of the solvent gave a residue that was purified bycolumn chromatography (silica gel, n-hexane/ethyl acetate 2:1 aseluent) to provide 20 (40 mg, 13%), mp 181e185 �C (from ethanol).1H NMR (DMSO-d6): d 6.21e6.23 (m, 2H), 6.90e6.92 (m, 2H), 7.06 (t,J ¼ 7.4 Hz, 1H), 7.10e7.13 (dd, J ¼ 1.8 and 8.7 Hz, 2H), 7.29e7.32 (m,2H), 7.46e7.48 (dd, J ¼ 2.1 and 8.9 Hz, 2H), 7.60e7.61 (d, J ¼ 7.08,2H), 8.43 (s, 1H), 9.77 ppm (br s, disappeared on treatment withD2O, 1H). IR: n 1665, 3386 cm�1. Anal. Calcd. for C20H15ClN4O: C,66.21%; H, 4.17%; N, 15.44%; Cl, 9.77%. Found: C, 65.96%; H, 4.12%, N,15.30%; Cl, 9.60%.

5.1.16. N,1-bis(4-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1H-pyrazole-4-carboxamide (21)

Was synthesized as 20 from 28 and 4-chloroaniline. Yield 59%,mp 177e181 �C (from ethanol). 1H NMR (DMSO-d6): d 6.20e6.22(m, 2H), 6.88e6.90 (m, 2H), 7.11e7.13 (d, J ¼ 8.6 Hz, 2H), 7.36e7.38(d, J ¼ 8.9 Hz, 2H), 7.46e7.48 (d, J ¼ 8.6 Hz, 2H), 7.65e7.67 (d,J ¼ 8.9 Hz, 2H), 8.42 (s, 1H), 10.00 ppm (br s, disappeared ontreatment with D2O, 1H). IR: n 1670, 3388 cm�1. Anal. Calcd. forC20H14Cl2N4O: C, 60.47%; H, 3.55%; N, 14.10%; Cl, 17.85%. Found: C,60.38%; H, 3.53%, N, 13.88%; Cl, 17.62%.

5.1.17. N,1-Diphenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide (4)

Was synthesized as 20 from 22 and aniline. Yield 70%, mp114e115 �C (from ethanol). 1H NMR (CDCl3): d 6.29e6.31 (m, 2H),6.67e6.69 (m, 2H), 7.03e7.04 (m, 1H), 7.15e7.20 (m, 1H), 7.23e7.29(m, 2H), 7.38e7.43 (m, 5H), 7.77e7.75 (m, 2H), 8.80 ppm (br s,disappeared on treatment with D2O, 1H). IR: n 1672, 3377 cm�1.Anal. Calcd. for C20H16N4O: C, 73.15%; H, 4.91%; N, 17.06%. Found: C,72.95%; H, 4.77%, N, 16.88%.

5.2. Purity determination

Compound 6 was dissolved in DMSO (1 mM) and analyzed byLC-UV-MS chromatography. LC analysis were performed with anAgilent 1100 LC/MSD VL system (G1946C) (Agilent Technologies,Palo Alto, CA) having vacuum solvent degassing unit, binary high-pressure gradient pump, 1100 series UV detector, and 1100 MSDmodel VL benchtop mass spectrometer. Chromatographic profileswere obtained using a Varian Polaris C18-A column (150e4.6 mm,5 mm particle size) and gradient elution: eluent A ¼ ACN, eluentB ¼ water. The analysis started with 2% of A, which rapidlyincreased up to 70% in 12 min and then slowly up to 98% in 20 min.The flow rate was 0.8 mL min�1 and injection volume was 20 mL.The Agilent 1100 series mass spectra detection (MSD) single-quadrupole instrument carried an orthogonal spray API-ES (Agi-lent Technologies, Palo Alto, CA). Nitrogen was used as nebulizingand drying gas. The pressure of the nebulizing gas, flow of the

D. Manvar et al. / European Journal of Medicinal Chemistry 90 (2015) 497e506504

drying gas, capillary voltage, fragmentor voltage, and vaporizationtemperature were set at 40 psi, 9 L/min, 3000 V, 70 V, and 350 �C,respectively. UV detection was monitored at 254 nm. The LC-ESI-MS determination was performed by operating the MSD in thepositive ion mode. Spectra were acquired over the scan range m/z100e1500 using a step size of 0.1 mm. The compound purity wasdetermined by measuring the peak areas detected at 254 nm.

5.3. Pharmacokinetic studies

5.3.1. Water solubility assayCompound 6 (1 mg) was added as a powder to 1 mL of water.

The sample was shaken at 20 �C for 24 h. The suspensionwas takenoff by a 0.45 mm nylon filter (Acrodisc), and the solubilized com-pound was determined by LC-UV-MS assay. The experiments werecarried out in triplicate. Chromatographic analysis was performedas above reported for purity determination. Quantification ofcompound 6 was assessed by means of calibration curves of stan-dard solutions in methanol.

5.3.2. Parallel artificial membrane permeability assay (PAMPA)Donor solution of compound 6 (0.5 mM) was prepared by

diluting 1 mM DMSO stock solution with phosphate buffer (pH 7.4,25 mM). Filters were coated with 5 mL of a 1% (w/v) dodecane so-lution of phosphatidylcholine for intestinal permeability. The donorsolution (150 mL) was added to each well of the filter plate. To eachwell of the acceptor plate were added 300 mL of 1:1 DMSO -phosphate buffer solution. The experiments were carried out inthree plates on different days. The sandwich was incubated for5 h at room temperature under gentle shaking. After incubation,the plates were separated, and samples were taken from bothreceiver and donor sides and analyzed using LC with UV detectionat 254 nm. LC analysis were performed with a PerkineElmer series200 provided with an UV detector (PerkineElmer 785A, UV/visDetector). Chromatographic separations were achieved using aPolaris C18-A column (150e4.6 mm, 5 mm particle size) at a flowrate of 0.8 mL min�1 and a mobile phase of 6:4 ACN-water.

Permeability (Papp) for PAMPA was calculated according to thefollowing equation, obtained from Wohnsland and Faller [24] andSugano et al. [25] and modified in order to obtain permeabilityvalues in cm/s.

Papp ¼ VDVA

ðVD þ VAÞAt� lnð1� rÞ

where VA is the volume in the acceptor well, VD is the volume in thedonor well (cm3), A is the effective area of the membrane (cm2), t isthe incubation time (s) and r the ratio between drug concentrationin the acceptor and equilibrium concentration of the drug in thetotal volume (VD þ VA). Drug concentration is estimated by usingthe peak area integration.

Membrane retention (%) was calculated according to thefollowing equation:

%MR ¼ ½r � ðDþ AÞ�100Eq

where r is the ratio between drug concentration in the acceptor andequilibrium concentration, D, A, and Eq represent drug concentra-tion in the donor, acceptor and equilibrium solution, respectively.

5.3.3. Microsomal stability assayCompound 6 in DMSO solution was incubated at 37 �C for

60 min in 25 mM phosphate buffer (pH 7.4), 5 mL of human livermicrosomal protein (0.2 mg/mL), in the presence of an NADPH-

generating system at a final volume of 0.5 mL and concentrationof 50 mM; DMSO did not exceed 2%. The reaction was cooled on iceand quenched by adding 1.0 mL of ACN. The reaction mixtures werecentrifuged for 15 min at 10,000 rpm, and the parent drug and itsmetabolites were determined by LC-UV-MS. The chromatographicanalysis was performed as above reported. The percentage of un-metabolized compound was calculated using reference solutions.The experiments were performed in triplicate and the metabolicbehavior was predicted by means of Metasite software.

5.3.4. Plasma stability assayTo determine the enzymatic stability, pooled human plasma

(750 mL), phosphate buffer (pH 7.4, 700 mL), and 50 mL of 3.0 mMsolution of 6 in DMSO (final concentration 100 mM)weremixed in atest tube. The mixture was incubated at 37 �C. At the time points of0 h and 24 h aliquots of a 150 mL were removed, mixed with 600 mLof cold ACN and centrifuged at 5000 rpm for 15 min. The super-natant was removed and analyzed by HPLC. The stability waschecked by HPLC with UV-MS detector as above reported.

5.4. Biological assays

5.4.1. Cell cultureMH-14 cells expressing the HCV 1b subgenomic replicon were

cultured in Dulbecco's modified Eagle's medium (DMEM; Invi-trogen Inc., Gaithersburg, MD) supplemented with 10% fetal bovineserum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 0.5 mg/mL G418. Huh 5.2 cells bearing the HCV 1bI389luc-ubineo/NS3-3’/5.1 replicon were cultured in DMEM sup-plemented with 10% FCS, nonessential amino acids, 100 U/mlpenicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 0.5 mg/mL G418. Huh7.5 were grown in DMEM supplemented with 10%FBS, 0.3 mg/ml L-glutamine, 100 U/ml penicillin, 100 mg/ml strep-tomycin and non-essential amino acids. All cells were cultured at37 �C in 5% CO2 atmosphere at 95e99% relative humidity.

5.4.2. HCV Jc1 infection assayThe inhibitory effect of compounds 4, 5 and 6 on HCV infection

was assayed employing the HCV genotype 2a infectious chimericclone Jc1 (generous gift of C. Rice). Briefly, HCV-Jc1 virus stock of105 TCID50/mL was used to infect Huh7.5 cell lines at 1 m.o.i., in 12multi wells plates. Six hours post viral adsorption, cells werewashed with PBS, supplemented with fresh media and incubatedwith the compounds at concentration specified in Chart 1 legend.Seventy-two hours post-treatment, cells were harvested and pro-cessed for total RNA extraction employing RNeasyMini Kit (Qiagen)in accordance with the manufacturer's protocol.

5.4.3. HCV RNA quantitation by qRT-PCRQuantitative One Step RT-PCRwas carried out using an ABI 7000

real Time PCR System (Applied Biosystem). 5 mL of total RNA wasmixed with 2� TaqMan One Step RT-PCR Master Mix (AppliedBiosystem and 1 mM forward (TCCCGGGAGAGCCATAGT), 1 mMreverse primers (CCCAGTCTTCCCGGCAATT) and 200 nM probe(FAM- CACCGGTTCCGCAGACC-TAMRA). In vitro transcribed geno-type 2a RNAwas used as a standard to quantify the copy numbers ofviral RNA.

5.4.4. Cell based inhibition and cytotoxicity assaysInhibitory potency (EC50, EC90 values) and cytotoxicity (CC50

values) of the compounds were evaluated in Huh 5.2 cells asdescribed previously [26].

D. Manvar et al. / European Journal of Medicinal Chemistry 90 (2015) 497e506 505

5.4.5. NS5B RdRp inhibition assayThe effect of the compounds on in vitro NS5B RdRp activity was

investigated employing recombinant NS5BD211b (HC-J4) and polyrA-U12 template-primer as described previously [27]. NS5B activitywas determined relative to DMSO treated control that was set at100%.

5.4.6. Transfection and luciferase reporter assayMH-14 cells were seeded in 48well plate (1�105 cells/well) and

12 h post seeding, transfected with 300 ng of gene specific reporterplasmid pCOX-2-FLuc [20,26,28], pHO-1-Luc [29], pISRE-Luc [27],or p3xARE-Luc [30] using LipoD293 transfection reagent in accor-dance with the manufacturer's protocol (SignaGen Lab, USA). Eachtransfection complex contained 30 ng of Renilla luciferase reporterplasmid (pRL-SV40) as internal control for normalization of thetransfection efficiency. Six hours post-transfection, cells wereincubated with increasing concentrations of 6 (quarter log di-lutions) or DMSO (control) for 48 h. Luciferase activity assay wasperformed using the Dual-Glo Luciferase Assay Kit (Promega, USA)in accordance with the manufacturer's instructions.

To study the effect of 6 on HCV IRES mediated translation,Huh7.5 cells were seeded in 48 well plate and transfected withpClneo-Rluc-IRES-Fluc (300 ng/well) reporter plasmid, in whichRluc was translated in a cap-dependent manner and Fluc wastranslated via HCV IRES-mediated initiation [18]. Six hours post-transfection, the cells were incubated with increasing concentra-tions of compound 6 or DMSO (control) for 48 h. Luciferase activityassay was performed employing the Dual-Glo Luciferase Assay Kit.

5.4.7. Western blottingEqual amounts of whole cell lysates (Dc protein assay, Bio-Rad)

were resolved over 8% SDS-PAGE and gels were transferred to PVDFmembrane by Trans Blot semi-dry transfer apparatus (Bio-Rad) andblots were blocked in 3% BSA for 1 h and probed with anti-NS5A,anti-NS5B, anti-COX-2 and b-Actin antibodies.

5.4.8. RT-PCRTotal RNA was isolated either using Triazol reagent (Life Tech-

nologies) or RNeasy mini kit (Promega Corp.). Isolated RNA's werequantified using NanoDrop (ND1000, NanoDrop Technologies), andequal amount of RNA's were reverse transcribed using SuperscriptII reverse transcriptase (Life Technologies) using HCV specific oroligo dT18 as a primer by following manufacturer's protocol in afinal volume of 20 mL. Two micro liters of synthesized cDNA's wereused for down-stream PCR applications using gene specific primersand Taq DNA polymerase (SigmaeAldrich) in a final volume of50 mL by following manufacturer's protocol. For Cox-2 forwardprimer was 50-TGCAAGTTCTCCCGCTCC-30 and reverse primer was50-CAGCATTTTGCCATCTTGTGAC-30. The forward and reverseprimer sequences for b-Actinwas 50- AGCGAGCATCCCCCAAAGTT-30

and 50- GGGCACGAAGGCTCATCATT-30, respectively. The HCVprimers sequences were 50-CGGGAGAGCCATAGTGG-30 for forwardand 50-AGTACCACAAGGCCTTTCG-30 for reverse primer.

Acknowledgments

The authors are grateful to Dr. Charles Rice for the Jc1 viruschimera construct and Huh 7.5 cells. We thank Drs. Ralf Bar-tenschlager and Kunitada Shimotohno for the kind gift of Huh 5-2andMH-14 cells, respectively. We acknowledge the generous gift ofthe reporter plasmids pCIneo-Rluc-IRES-Fluc, pHO-1-Luc andp3xARE-Luc from Drs. Naoya Sakamoto, Anupam Agarwal, and Dr.Being-Sun Wung, respectively. This research project was partlysupported by Istituto Pasteur-Fondazione Cenci Bolognetti (Grant2012) to R.S., Istituto Superiore di Sanit�a intramural fundings to

A.R., Italian Ministry of Instruction, Universities and Research(Projects PON01-01802 and PRIN 2010e2011 Grant2010PHT9NF_005) to A.T.P., and the National Institute of HealthResearch Grant CA153147 to N.K.-B.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2014.11.042.

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