Synthesis of novel diflunisal hydrazide-hydrazones as anti-hepatitis C virus agents and hepatocellular carcinoma inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.10.041

Hepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver cirrhosis and hepatocellular carcinoma (HCC). The objective of our research was to develop effective agents against viral replication. We have previously identifi

Full text of DOI:10.1016/j.ejmech.2015.10.041

Accepted Manuscript
Synthesis of Novel Diflunisal Hydrazide-Hydrazones as Anti-Hepatitis C Virus Agents
and Hepatocellular Carcinoma Inhibitors
Sevil Şenkardeş, Neerja Kaushik-Basu, İrem Durmaz, Dinesh Manvar, Amartya Basu,
Rengül Atalay, Ş.Güniz Küçükgüzel
PII:
S0223-5234(15)30323-8
10.1016/j.ejmech.2015.10.041
EJMECH 8176
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 March 2015
Revised Date: 23 October 2015
Accepted Date: 25 October 2015
Please cite this article as: S. Şenkardeş, N. Kaushik-Basu, İ. Durmaz, D. Manvar, A. Basu, R. Atalay,
Ş.G. Küçükgüzel, Synthesis of Novel Diflunisal Hydrazide-Hydrazones as Anti-Hepatitis C Virus Agents
and Hepatocellular Carcinoma Inhibitors, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.10.041.
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ACCEPTED MANUSCRIPT
Synthesis of Novel Diflunisal Hydrazide-Hydrazones as Anti-Hepatitis C Virus Agents
and Hepatocellular Carcinoma Inhibitors
Sevil Şenkardeş1, Neerja Kaushik-Basu2, İrem Durmaz3, Dinesh Manvar2, Amartya Basu2,
Rengül Atalay4, Ş.Güniz Küçükgüzel1*
1Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpaşa 34668
İstanbul, Turkey
2Rutgers-New Jersey Medical School, Department of Microbiology, Biochemistry and Molecular Genetics,
Newark, NJ 07103, New Jersey, USA
3Bilkent University, Department of Molecular Biology and Genetics, 06800 Bilkent Ankara, TURKEY.
4Cancer Systems Biology Laboratory, Graduate School of Informatics Middle East Technical University
ODTU 06800 Ankara TURKEY
ABSTRACT
Hepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver
cirrhosis and hepatocellular carcinoma (HCC). The objective of our research was to develop
effective agents against viral replication. We have previously identified the hydrazide-
hydrazone scaffold as a promising hepatitis C virus (HCV) and hepatocelluler inhibitor.
Herein we describe the design a number of 2',4'-difluoro-4-hydroxy-N'-(arylmethylidene)
biphenyl-3-carbohydrazide (3a-t) as anti-HCV and anticancer agents. Results from evaluation
of anti-HCV activity indicated that most of the synthesized hydrazone derivatives inhibited
viral replication in the Huh7/Rep-Feo1b and Huh 7.5-FGR-JCI-Rluc2A reporter systems.
Antiproliferative activities of increasing concentrations of 2',4'-difluoro-4-hydroxy-N'-(2-
pyridyl methylidene)biphenyl-3-carbohydrazide 3b and diflunisal (2.5-40 ꢀM) were assessed
in liver cancer cell lines (Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475) with
sulforhodamine B assay for 72h. Compound 3b with 2-pyridinyl group in the hydrazone part
exhibited promising cytotoxic activity against all cell lines with IC50 values of 10, 10.34 16.21
4.74, 9.29 and 8.33 µM for Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475cells,
respectively, and produced dramatic cell cycle arrest at SubG1/G0 phase as an indicator of
apoptotic cell death induction.
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Running Title
Synthesis of Diflunisal hydrazones as anti-HCV and anticancer agents
Keywords: Diflunisal, Hydrazide-hydrazone, Hepatitis C, Hepatocellular carcinom, antiviral.
Correspondence: Prof.Ş.Güniz Küçükgüzel
Adress : Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpaşa
34668 İstanbul, Turkey
Tel : +9 0216 414 29 62
Fax : +90216 345 29 52
e mail: gkucukguzel@marmara.edu.tr
* This work was partly presented at the 4th International Meeting on Pharmacy and Pharmaceutical and
Sciences, İstanbul-TURKEY, 18-23 September, 2014.
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1. Introduction
Hepatitis C virus (HCV) is an enveloped virus that is classified in the hepacivirus genus of the
Flaviviridae family [1]. The virus RNA genome encodes a polyprotein, which is
posttranslationally processed by host and virus proteases into 10 mature proteins, of which 4
are structural proteins (C, E1, E2, and p7) and 6 nonstructural proteins (NS2, NS3, NS4A,
NS4B, NS5A, and NS5B) [2]. HCV is a worldwide infectious pathogen that causes chronic
liver diseases, including hepatic fibrosis, hepatic cirrhosis and hepatocellular carcinoma
(HCC) [3]. Until recently, HCV-infected patients were treated with a combination of
pegylated interferon- α (IFN- α) and the nucleoside analog ribavirin. However, this therapy
had limited effectiveness especially in context of patients infected with HCV genotype 1.
Furthermore, treatment with interferon is associated with numerous side effects. Recently,
new anti-HCV therapies utilizing the direct acting antivirals (DAAs) against the viral
proteins HCV NS3-4A protease and NS5B polymerase have been approved. These therapy
although more promising have complicated dosing regimens limiting patient compliance [4-7]
urther, the selection of HCV drug resistant variants continues to remain a concern [8-9]. On
the other hand, acute and chronic liver diseases that are caused by an infection with hepatitis-
C virus (HCV), such as hepatocellular carcinoma and liver cirrhosis have received much
attention over the past decade. Recently, HCV is believed to act as carcinogen by virtue of the
increased risk of hepatocellular carcinoma among persistently infected patients with chronic
active hepatitis. Therefore, it is important to develop new, safer and even more effective
agents against HCV infection and resistance emergence.
Diflunisal derivatives [10-14] (Fig. 1) have been reported to possess diverse biological
activities such as anticancer, anti-HCV, anticonvulsant, antimicrobial and anti-inflammatory
properties [15]. In medicinal chemistry, the presence of a hydrazide-hydrazone group in
compounds, has usually led to the development of clinically relevant biological molecules
with antimicrobial, anticancer [16-17] and antiviral properties [18].
Fig. 1. Diflunisal derivatives
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Recently, our group reported the synthesis of novel hydrazide-hydrazone derivatives and their
HCV NS5B inhibition effects [19] (Fig. 2).
Fig. 2. Hydrazones of anti-HCV activity.
Diflunisal is a difluorophenyl derivative of salicylic acid and a non-steroidal drug with
analgesic, anti-inflammatory and antipyretic properties. It is a peripherally-acting non-
narcotic analgesic drug which functions as a prostaglandin synthase inhibitor. In animals,
prostaglandins sensitize afferent nerves and potentiate the action of bradykinin in inducing
pain. Since prostaglandins are known to be among the mediators of pain and inflammation,
the mode of action of diflunisal may be due to a decrease of prostaglandins in peripheral
tissues.
Herein, we report our ongoing efforts towards development of more effective anti-HCV
agents. We focused our attention on the hydrazide-hydrazone moiety. Thus, a new series of
hydrazide-hydrazone derivatives were synthesized from diflunisal and evaluated for their anti-
HCV activity in vitro and anticancer activity against hepatocellular cancer cell lines.
2. Results and discussion
2.1. Chemistry
Methyl 2′,4′-difluoro-4-hydroxybiphenyl-3-carboxylate [1] was prepared by the
reaction of diflunisal and methanol in the presence of a few drops of concentrated sulfuric
acid. 2′,4′-Difluoro-4-hydroxybiphenyl-3-carboxylic acid hydrazide [2] was prepared by
heating hydrazine-hydrate and [1] in methanol [10]. After condensing hydrazide with
substituted aldehydes in ethanol, novel 2',4'-difluoro-4-hydroxy-N'-(arylmethylidene)
biphenyl-3-carbohydrazide [3a-t] were obtained. The synthesis of novel series of hydrazide-
hydrazones 3a-t was performed as outlined in Scheme 1. All synthesized compounds were
checked for purity using TLC and HPLC-UV/DAD and were characterized by their melting
points, 1H-NMR, 13C-NMR and HR-MS spectral data.
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Scheme 1. The synthetic route for the preparation of the target compounds (3a-t).
The FT-IR spectra of hydrazones showed absorption bands at 1583-1614 cm-1 due to
C=N groups. Moreover, C=O absorption bands were observed between 1633-1647 cm-1. The
absorption bands associated with other functional groups appeared in the expected regions. In
1
the H-NMR spectra of all hydrazones, the signal representing the azomethine CH protons
appeared at 8.41-9.11 ppm, whereas a D2O-exchangeable signal due to NH amidic proton (-
CONHN=CH-) and -OH resonated at 11.78-12.28 ppm. The other protons appeared at the
13
expected chemical shifts and integral values. C-NMR spectrum of 3a-t showed the absence
of the C=O signal at δ 164.59-165.38 ppm and -N=CH 144.70-149.32 ppm. Besides, the
heteronuclear multiple bond correlation (HMBC) spectrum of compound 3o also confirmed
1
the detection of long-range H-13C couplings. HR-EI (for 3j and 3o) and DART-MS (for 3d
and 3s) confirmed the molecular weights and empirical formulae of compounds with less than
1 mmu bias between calculated and experimental m/z values of either molecular or fragment
ions. The major fragmentation pattern involved the cleavage of the CONH-NH= amide bond
m/z 249. The species m/z 232 and m/z177 in compounds (3j, 3o) may results from loss of -
NH2 and subsequent loss of CO in the phenyl ring.
2.2. Biological evaluation
2.2.1. Anti-HCV effect of diflunisal hydrazide-hydrazones
In order to identify potential anti-HCV agents, we employed two reporter cell lines,
Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A (Table 1). These cells carry the
autonomously replicating HCV RNA of genotype 1b and 2a in the firefly and Renilla
luciferase reporters, respectively [20-24]. Therefore levels of their respective luciferase serves
as an indicator of HCV RNA replication. The effect of the compounds on cell viability was
investigated in the Huh7.5 parental cells by the MTS assay and employed to compute 50%
cytotoxicity values (CC50) (Table 1) .
Table 1. Anti-HCV activity of diflunisal hydrazide-hydrazones (3a-t).
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The nineteen compounds displayed a wide range of cytotoxicity in the parental Huh7.5 cells.
Among these 3f, 3g, 3i, 3k, 3n, 3o, 3p and 3t exhibited CC50 < 50 µM, suggesting that these
compounds may be detrimental to cell viability. In contrast, 3c, 3m and 3r with CC50 > 200
µM, proved to be the least cytotoxic. The remaining compounds displayed cytotoxicity in the
interim range with compounds 3a, 3d, 3e, 3h, and 3l exhibiting CC50 > 50 µM, while 3b, 3j
and 3s displaying CC50 > 100 µM.
We next screened the compounds at 50 µM concentration for their anti-HCV activity. With
the exception of 3d which exhibited 33% and 23% inhibition against 1b and 2a replicon
reporters respectively, all others displayed ≥58% inhibition. Notably, all compounds with the
exception of 3g, 3h, 3r and 3a exhibited higher inhibition of 2a replicons relative to 1b,
suggesting higher antiviral potency of the compounds against HCV genotype 2a. Compounds
exhibiting ≥70% inhibition against 1b replicon and CC50 ≥ 50 µM were further characterized
in terms of their EC50 values. This resulted in the identification of 6 compounds 3a, 3b, 3c,
3h, 3m and 3r which met this cut-off criteria and displayed EC50 values ranging between 3.9-
16.5 µM and selectivity index (SI) between 3-25. Among these, compound 3b, 2',4'-difluoro-
4-hydroxy-N'-[(pyridin-2-yl)methylidene]biphenyl-3-carbohydrazide appeared the most
promising with an EC50 of 3.9 and SI >25.6.
To identify the mechanism of action of these compounds against HCV, we investigated if the
compounds target HCV NS5B. Towards this end, we screened the compounds in vitro for
their ability to inhibit NS5B RdRp activity. As seen in Table 1, the compounds displayed no
inhibition or ≤18% inhibition of HCV NS5B, thus suggesting that the compounds function
through a mechanism other than targeting HCV NS5B.
Further studies are in progress to optimize the structures of hydrazide-hydrazone derivatives
with the aim of increasing their anti-HCV potency.
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2.2.2. Anticancer activity of compound 3b against liver cancer cell lines
In order to determine the potential anticancer activity of the obtained diflunisal hydrazide
hydrazone 3b and diflunisal, we evaluated their cytotoxic activity on liver cancer cell lines
(Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU-475) with the sulforhodamine B assay
(2.5–40 ꢀM) for 72h as described before [25] (Fig. 3). Compound 3b was bioactive in all six
of the cancer cell lines with IC50 values in micromolar ranges (Table 2). In this study,
diflunisal was included as an experimental control.
Fig. 3. Cytotoxicity induced by the compounds on liver cancer cell lines (Huh7, HepG2,
Hep3B, Mahlavu, FOCUS and SNU-475). The cells were treated with increased concentration
of the compound (2.5–40 ꢀM) for 72h. NCI-60 SRB assay was then performed. Absorbance
values were obtained and normalized to DMSO control. The experiment was conducted in
triplicate.
Table 2. IC50 values of 3b on liver cancer cell lines.
Compound 3b, bearing 2-pyridinyl group in the hydrazone core inhibited cell proliferation
with IC50 values between 4.74-16.21 µM as shown in Table 2. While Diflunisal showed no
inhibition against Huh7, HepG2, Hep3B, and SNU-475 liver cells, compound 3b by contrast
demonstrated anticancer activity against the same cells with IC50 values 10.0, 10.34, 16.21
and 8.33 ꢀM, respectively. Compound 3b exhibited the highest growth inhibitory activity
against Mahlavu lines (IC50=4.74 µM).
2.2.2.1. Effect of diflunisal and compound 3b on the morphology of Huh 7 and Mahlavu cells
It is known that a cell that is undergoing apoptosis exhibits nuclear condensation and DNA
fragmentation, which can be detected by staining with Hoechst 33258 and fluorescence
microscopy. To examine the nature of cell death induced upon compound treatment, we next
analyzed the changes in cell morphology with light microscopy. Towards this end, human
liver cancer cells were treated with diflunisal and compound 3b at their IC50 values for 72 h
(Table 2) and compared against DMSO treated controls. As shown in Fig. 4, compound 3b
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induced cell death with diverse morphologies. In Huh7 cells, treatment with 3b at IC50
concentration, resulted in nuclear condensation and DNA fragmentation in the cells, which
was in parallel with their cell cycle analysis but no such morphological change were observed
upon diflunisal (Fig. 4).
Fig. 4. Nuclear Hoechst (33258) staining (40x) of liver cancer cells treated with IC50
concentrations of the compounds or DMSO control for 72h.
2.2.2.2. Effect of diflunisal and compound 3b on cell cycle arrest in Huh 7 and Mahlavu cells
In order to investigate the effects of the compounds on cell cycle, we treated the liver cancer
cells with DMSO or compounds at their IC50 concentrations for 72h and stained the cells with
propidium iodide. This assay revealed that compound 3b induced SubG1/Go arrest in
Mahlavu cells lines, with the effect being more prounced in Mahlavu cells (Fig. 5).
Fig. 5. Cell cycle distribution of liver cancer cells (Huh7 and Mahlavu) treated with IC50
concentrations of the compounds or DMSO control for 72h.
2.2.2.3. Apoptosis induction by compound 3b
The changes observed in fluorescent microscopy together with SubG1 cell cycle arrest
suggested that the compound induced apoptotic cell cycle arrest. In order to confirm this, the
presence of cleaved PARP (an indicator of apoptitic induction) was investigated in cells
treated with DMSO control, diflunisal or 3b according to IC50 concentrations for 72h. In
Huh7 cells, no cleaveage could be observed. On the other hand, compound 3b caused
cleavage of PARP protein indicating that 3b induced apoptosis in Mahlavu liver cancer cell
line (Fig. 6).
Fig. 6. Western blot results of liver cancer cells treated with IC50 concentrations of the
compounds or DMSO control for 72h.
3. Conclusion
In this study, a series of of 2',4'-difluoro-4-hydroxy-N'-(arylmethylidene)biphenyl-3-
carbohydrazide [3a-t] have been synthesized and evaluated for their anti-HCV and anticancer
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activity. The results revealed increase in cleaved PARP (a marker for apoptosis) in Mahlavu
cells treated with 3b indicating induction of apoptosis. In Huh7 cells no cleaved fragment was
observed. According to FACS analysis, in both cell lines, treatment with compound 3b
resulted in SubG1 cell cycle arrest. Together, this data indicates that compound 3b may be a
promising lead candidate for further optimization and development as a prospective anti-HCV
and hepatocellular carcinoma inhibitory agent.
4. Experimental section
4.1. Chemistry
All reagents and solvents were obtained from commercial suppliers and were used
without further purification. Merck silica gel 60 F254 plates were used for analytical TLC.
Melting points wee determined using Schmelzpunktbestimmer SMP II apparatus and were
1
uncorrect. H and 13C- NMR spectra were recorded on 300 MHz or 500 MHz Varian UNITY
INOVA or HD BRUKER 300 MHz Ultrashield TM spectrometer. Elemental analyses were
determined by CHNS-932 (LECO). FT-IR spectra were recorded on a Schimadzu FTIR-
8400S spectrometer. HR-MS mass spectra were acquired using a Jeol JMS-700 spectrometer.
Purity of the synthesized compounds have been demonstrated by HPLC analysis using
reversed-phase chromatography. The liquid chromatographic system consists of an Agilent
Technologies 1100 series instrument equipped with a quaternary solvent delivery system and a
model Agilent series G1315 A photodiode array detector. A Rheodyne syringe loading sample
injector with a 50-µL sample loop was used for the injection of the analytes. Chromatographic
data were collected and processed using Agilent Chemstation Plus software. The separation
was performed at ambient temperature using a reverse phase ACE C18 (4.0x100 mm) column.
All experiments were performed in gradient mode. ACN/H2O system was used as gradient
system: 50:50 from 0 to 3 min; 75:25 to 50:50 from 3 to 6 min; 100:0 to 75:25 from 6 to 12
min; the flow rate was 1.0 mL/min with monitoring at 254 nm.
4.1.1. Methyl 2′,4′-difluoro-4-hydroxybiphenyl-3-carboxylate (1) and 4.1.2. 2′,4′-Difluoro-4-
hydroxybiphenyl-3-carboxylic acid hydrazide (2) were prepared described in the literature
[10].
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4.1.3. General procedure for the synthesized of the compounds
A mixture of appropriate aldehydes and an equimolar amount of diflunisal hydrazide in
ethanol was refluxed for 2h and novel 2',4'-difluoro-4-hydroxy-N'(arylmethylidene)biphenyl-
3-carbohydrazide [3a-t] were obtained.
4.1.3.1. 2',4'-Difluoro-4-hydroxy-N'-[(thiophen-2-yl)methylidene]biphenyl-3-carbohydrazide
(3a)
o
Dark brown solid; Yield: 90%; HPLC: tR (min.): 8.94; Mp 251 C; FTIR (cm-1): 3242 (O-
1
H&N-H), 3072, 3032 (=C-H arom.), 1637 (C=O), 1608 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7.08 (d, 1H, J=8.4 Hz, H5), 7.22 (t, 1H, H11), 7.37 (t, 1H, H9), 7.49-
7.65 (m, 4H, Ar-H), 7.72 (d, 1H, J=5.1 Hz, H14),7.99 (s, 1H, H2), 8.67 (s, 1H, CH=N), 11.90
(s, 2H, Ar-OH &CO-NH-N=CH); Anal. Calcd for C18H12F2N2O2S: C, 60.33; H, 3.38; N, 7.82;
S, 8.95; found C, 59.82; H, 3.36; N, 7.79; S, 9.42.
4.1.3.2. 2',4'-Difluoro-4-hydroxy-N'-[(pyridin-2-yl)methylidene]biphenyl-3-carbohydrazide
(3b)
o
Light yellow solid; Yield: 80%; HPLC: tR (min.): 2.76; Mp 241 C; FTIR (cm-1): 3254 (O-
1
H&N-H), 3076, 3045 (=C-H arom.), 1639 (C=O), 1614 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7.10 (d, 1H, J=8.5 Hz, H5); 7.22 (t, 1H, H11); 7.35-8.65 (m, 8H, Ar-
H); 8.48 (s, 1H, CH=N); 11.85 (s, 1H, Ar-OH); 12,07 (s, 1H, CO-NH-N=CH); Anal. Calcd
for C19H13F2N3O2: C, 64.59; H, 3.71; N, 11.89; found C, 64.32; H, 3.78; N, 11.80.
.
4.1.3.3. 2',4'-Difluoro-4-hydroxy-N'-[(pyridin-3-yl)methylidene]biphenyl-3-carbohydrazide
(3c)
o
Off-white solid; Yield: 88%; HPLC: tR (min.): 2.39; Mp 277-278 C; FTIR (cm-1): 3242 (O-
1
H&N-H), 3041, 3032 (=C-H arom.), 1629 (C=O), 1608 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7,06 (d, 1H, J=8.7 Hz, H5); 7,22 (t, 1H, H11); 7,39 (t, 1H, H9); 7.43-
8.65 (m, 6H, Ar-H); 8.02 (s, 1H, H2); 8.88 (s, 1H, CH=N); 11.92 (s, 1H, Ar-OH); 12.05 (s,
1H, CO-NH-N=CH); Anal. Calcd for C19H13F2N3O2: C, 64.59; H, 3.71; N, 11.89; found C,
63.75; H, 3.76; N, 11.74.
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4.1.3.4. 2',4'-Difluoro-4-hydroxy-N'-[(pyridin-4-yl)methylidene]biphenyl-3-carbohydrazide
(3d)
o
Off-white solid; Yield: 86%; HPLC: tR (min.): 2.51; Mp 261-263 C; FTIR (cm-1): 3251 (O-
1
H&N-H), 3036 (=C-H arom.), 1646 (C=O), 1608 (C=N); H-NMR (300 MHz), (DMSO-
d6/TMS) δ ppm: 7,11 (d, 1H, J=8.7 Hz, H5); 7.22 (t, 1H, H11); 7.37 (t, 1H, H9); 7.59-7.71
&8,66-8,68 (m, 6H, Ar-H); 8.02 (s, 1H, H2); 8.44 (s, 1H, CH=N); 12.12 (s, 2H, Ar-OH &CO-
NH-N=CH); DART-MS; (m/z, Calcd./Found): 354,1048/354,1047 [C19H13F2N3O2+H]+;
707.2024/707.2009 [2(C19H13F2N3O2)+H]+; Anal. Calcd for C19H13F2N3O2: C, 64.59; H, 3.71;
N, 11.89; found C, 64.40; H, 4.06; N, 11.67.
4.1.3.5.
2',4'-Difluoro-4-hydroxy-N'-[(5-methylthiophen-2-yl)methylidene]biphenyl-3-
carbohydrazide (3e)
o
Light brown solid; Yield: 95%; HPLC: tR (min.): 9.03; Mp 242 C; FTIR (cm-1): 3246 (O-
1
H&N-H), 3076, 3047 (=C-H arom.), 1639 (C=O), 1610 (C=N); H-NMR (300 MHz),
(CD3COCD3-d6/TMS) δ ppm: 2.52 (s, 3H, methyl); 6.87 (d, 1H, J=3.6 Hz, H4, thiophene);
7.08 (d, 1H, J=8.7 Hz, H5); 7.22 (t, 1H, H11); 7.30 (d, 1H, J=3.6 Hz, H3, thiophene); 7.38 (t,
1H, H9); 7.56-7.64 (m, 2H, Ar-H); 7.99 (s, 1H, H2); 8.59 (s, 1H, CH=N); 11.78 (s, 1H, Ar-
OH); 11.99 (s, 1H, CO-NH-N=CH); 13C-NMR (150 MHz) (DMSO-d6/TMS) δ ppm: 15.86,
104.93,112.47, 116.79,118.02,124.54, 125.50, 126.90, 129.17, 132.16, 132.39, 134.45,
136.93, 143.95, 144.70 (-N=CH), 158.66&160.31 (C-F), 159.11, 161.14&162.77 (C-F),
164.59 (-C=O); Anal. Calcd for C19H14F2N2O2S: C, 61.28; H, 3.79; N, 7.52; S, 8.61; found C,
60.78; H, 3.66; N, 7.33; S, 9.45.
.
4.1.3.6.
2',4'-Difluoro-4-hydroxy-N'-[(3-bromophenyl)methylidene]biphenyl-3-
carbohydrazide (3f)
White solid; Yield: 81%; HPLC: tR (min.): 4.75; Mp 262 oC; FTIR (cm-1): 3232 (O-H&N-H),
1
3047 (=C-H arom.), 1606 (C=O), 1606 (C=N); H-NMR (300 MHz), (DMSO-d6/TMS) δ
ppm: 7.09 (d, 1H, J=8.4Hz, H5); 7.21 (t, 1H, H11); 7.37 (t, 1H, H9); 7.42-7.95 (m, 6H, Ar-H);
8.01 (s, 1H, H2); 8.41 (s, 1H, CH=N); 11.94 (2s, 2H, CO-NH-N=CH&Ar-OH). 13C-NMR
(150 MHz) (DMSO-d6/TMS) δ ppm: 104.92, 112.49, 117.04, 118.08, 122.64, 124.50, 125.61,
126.85, 129.54, 129.77, 131.53, 132.15, 133.30, 134.57, 136.98, 147.49 (-N=CH),
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158.65&160.29 (C-F); 158.80, 161.13&162.77 (C-F); 164.79 (-C=O); Anal. Calcd for
C20H13BrF2N2O2: C, 55.70; H, 3.04; N, 6.50; found C, 55.34; H, 2.93; N, 6.47.
4.1.3.7.
2',4'-Difluoro-4-hydroxy-N'-[(2-chlorophenyl)methylidene]biphenyl-3-
carbohydrazide (3g)
o
Light yellow solid; Yield: 86%; HPLC: tR (min.): 9.65; Mp 269-270 C; FTIR (cm-1): 3261
1
(O-H&N-H), 3078 (=C-H arom.), 1639 (C=O), 1614 (C=N); H-NMR (300 MHz), (DMSO-
d6/TMS) δ ppm: 7.06 (d, 1H, J=8.4Hz, H5); 7.22 (t, 1H, H11); 7.39 (t, 1H, H9); 7.33-7.70 (m,
5H, Ar-H); 8.02 (s, 1H, H2); 8.84 (s, 1H, CH=N); 12.28 (s, 2H, CO-NH-N=CH&Ar-OH). 13C-
NMR (75 MHz) (DMSO-d6/TMS) δ ppm: 104.90, 112.57, 116.73, 118.28, 124.60, 124.77,
125.05, 127.49, 128.13, 129.27, 130.43, 131.88, 132.17, 133.80, 134.60, 144.91 (-N=CH),
157.91&160,33 (C-F), 159.98, 161.20&163.44 (C-F), 165.38 (-C=O); Anal. Calcd for
C20H13ClF2N2O2: C, 62.11; H, 3.39; N, 7.24; found C, 61.39; H, 3.23; N, 7.08.
4.1.3.8.
2',4'-Difluoro-4-hydroxy-N'-[(2-chloro-6-fluorophenyl)methylidene]biphenyl-3-
carbohydrazide (3h)
o
Light yellow solid; Yield: 78%; HPLC: tR (min.): 9.38; Mp 260 C; FTIR (cm-1): 3227 (O-
1
H&N-H), 3080, 3051 (=C-H arom.), 1647 (C=O), 1602 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7.09 (d, 1H, J=8.4 Hz, H5); 7.22 (t, 1H, H11); 7.33-7.70 (m, 6H, Ar-
H); 8.01 (s, 1H, H2); 8.69 (s, 1H, CH=N); 11.92 (s, 1H, Ar-OH); 12.11 (s, 1H, CO-NH-
N=CH); Anal. Calcd for C20H12ClF3N2O2: C, 59.35; H, 2.99; N, 6.92; found C, 58.74; H,
2.92; N, 6.75.
4.1.3.9.
2',4'-Difluoro-4-hydroxy-N'-[(2,6-dichlorophenyl)methylidene]biphenyl-3-
carbohydrazide (3i)
o
Dark yellow solid; Yield: 95%; HPLC: tR (min.): 9.76; Mp 208 C; FTIR (cm-1): 3244 (O-
1
H&N-H), 3074, 3032 (=C-H arom.), 1637 (C=O), 1608 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7.10 (d, 1H, J=8.7 Hz, H5); 7.22 (t,1H, H11); 7.36 (t, 1H, H9); 7.42-
7.67 (m, 5H, Ar-H); 8.02 (s, 1H, H2); 8.66 (s, 1H, CH=N); 11.87 (s, 1H, Ar-OH); 12.16 (s,
1H, CO-NH-N=CH); Anal. Calcd for C20H12Cl2F2N2O2: C, 57.03; H, 2.87; N, 6.65; found C,
56.92; H, 2.79; N, 6.62.
12

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4.1.3.10.
2',4'-Difluoro-4-hydroxy-N'-[(2,6-difluorophenyl)methylidene]biphenyl-3-
carbohydrazide (3j)
Off-white solid; Yield: 90%; HPLC: tR (min.): 8.89; Mp 280 oC; FTIR (cm-1): 3232 (O-H&N-
1
H), 3046, 3022 (=C-H arom.), 1647 (C=O), 1608 (C=N); H-NMR (300 MHz), (DMSO-
d6/TMS) δ ppm: 7.09 (d, 1H, J=8.7 Hz, H5); 7.23-7.66 (m, 7H, Ar-H); 8.01 (s, 1H, H2); 8.61
(s, 1H, CH=N); 12.16 (s, 2H, Ar-OH&CO-NH-N=CH). HRMS (EI, m/z): monoisotopic mass
for C20H12F4N2O2 (Calcd. / Found): 388.0834/ 388.0842; Anal. Calcd for C20H12F4N2O2: C,
61.86; H, 3.11; N, 7.07; found C, 61.16; H, 2.98; N, 7.07.
4.1.3.11.
2',4'-Difluoro-4-hydroxy-N'-[(3,4-dichlorophenyl)methylidene]biphenyl-3-
carbohydrazide (3k)
o
Off-white solid; Yield: 82%; HPLC: tR (min.): 9.53; Mp 258-260 C; FTIR (cm-1): 3255 (O-
1
H&N-H), 3076, 3037 (=C-H arom.), 1639 (C=O), 1614 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7.10 (d, 1H, J=8.7 Hz, H5); 7.21 (t, 1H, H11); 7.38 (t, 1H, H9); 7.57-
8.01 (m, 4H, Ar-H); 7.75 (s, 1H, H14); 7.78 (s, 1H, H2); 8.43 (s, 1H, CH=N); 11.96 (s, 1H,
Ar-OH); 12.03 (s, 1H, CO-NH-N=CH); Anal. Calcd for C20H12Cl2F2N2O2: C, 57.03; H, 2.87;
N, 6.65; found C, 56.66; H, 3.00; N, 6.63.
4.1.3.12.
2',4'-Difluoro-4-hydroxy-N'-[(5-ethylthiophen-2-yl)methylidene]biphenyl-3-
carbohydrazide (3l)
o
Dark brown solid; Yield: 93%; HPLC: tR (min.): 9.28; Mp 196 C; FTIR (cm-1): 3228, 3213
1
(O-H&N-H), 3074 (=C-H arom.), 1635 (C=O), 1606 (C=N); H-NMR (300 MHz), (DMSO-
d6/TMS) δ ppm: 1.25 (t, 3H, methyl); 2.83 (q, 2H, ethyl); 6.89-7.65 (m, 7H, Ar-H); 8.00 (s,
1H, H2); 8.58 (s, 1H, CH=N); 11.79 (s, 1H, Ar-OH); 12.11 (s, 1H, CO-NH-N=CH); Anal.
Calcd for C20H16F2N2O2S: C, 62.16; H, 4.17; N, 7.25; S: 8.30; found C, 61.95; H, 4.27; N,
6.90; S, 9.18.
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4.1.3.13.
2',4'-Difluoro-4-hydroxy-N'-[(4-cyanophenyl)methylidene]biphenyl-3-
carbohydrazide (3m)
o
Light yellow solid; Yield: 82%; HPLC: tR (min.): 8.23; Mp 284-285 C; FTIR (cm-1): 3227
1
(O-H&N-H), 3066, 3022 (=C-H arom.), 1633 (C=O), 1612 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7.11 (d, 1H, J=8.4Hz, H5); 7.22 (t, 1H, H11); 7.39 (t, 1H, H9); 7.57-
7.94 (m, 6H, Ar-H); 8.01 (s, 1H, H2); 8.52 (s, 1H, CH=N); 12.06 (s, 2H, CO-NH-N=CH&Ar-
13
OH). C-NMR (75 MHz) (DMSO-d6/TMS) δ ppm: 104.95, 112.62, 117.16, 118.01, 119.10,
124.44 (CN), 124.62, 125.64, 128.25, 129.65, 132.10, 133.63, 134.63, 139.02, 147.20 (-
N=CH); 157.93&160.26 (C-F); 159.74, 161.21&163.94 (C-10); 164.80 (-C=O); Anal. Calcd
for C21H13F2N3O2: C, 66.84; H, 3.47; N, 11.14; found C, 66.30; H, 3.44; N, 11.05.
4.1.3.14.
2',4'-Difluoro-4-hydroxy-N'-[(2,2-difluoro-1,3-benzodioxol-5-yl)methylidene]
biphenyl-3-carbohydrazide (3n)
o
White solid; Yield: 82%; HPLC: tR (min.): 9.64; Mp 244-245 C; FTIR (cm-1): 3244 (O-
1
H&N-H), 3080, 3024 (=C-H arom.), 1641 (C=O), 1583 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7.11 (d, 1H, J=8.5Hz, H5); 7.22 (t, 1H, H11); 7.38 (t, 1H, H9); 7.51-
7.65 (m, 5H, Ar-H); 8.02 (s, 1H, H2); 8.47 (s, 1H, CH=N); 11.94 (s, 2H, CO-NH-N=CH&Ar-
OH). 13C-NMR (150 MHz) (DMSO-d6/TMS) δ ppm: 104.93, 108.04, 110.95, 112.41, 116.95,
118.07, 124.51, 125.31, 125.45, 129.97& 131.95&133.31 (C-F); 124.55, 131.71, 132.14,
134.52, 143.83, 144.42, 147.33 (-N=CH); 158.66&160.30 (C-F); 159.10, 161.12. 162.80 (C-
F), 164.79 (-C=O); Anal. Calcd for C21H12F4N2O4: C, 58.34; H, 2.80; N, 6.48; found C, 57.86;
H, 2.73; N, 6.37.
4.1.3.15.
2',4'-Difluoro-4-hydroxy-N'-{[3-(trifluoromethyl)methylidene}biphenyl-3-
carbohydrazide (3o)
White solid; Yield: 92%; HPLC: tR (min.): 9.72; Mp 221 oC; FTIR (cm-1): 3257 (O-H&N-H),
1
3047 (=C-H arom.), 1641 (C=O), 1608 (C=N); H-NMR (500 MHz), (DMSO-d6/TMS) δ
ppm: 7.09 (d, 1H, J=8.7 Hz, H5); 7.20 (t, 1H, H11); 7.35 (t, 1H, H9); 7.57-8.04 (m, 6H, Ar-H);
8.08 (s, 1H, H2); 8.54 (s, 1H, CH=N); 12.02 (s, 2H, CO-NH-N=CH&Ar-OH) (exchangeable
13
with D2O). C-NMR (125 MHz) (DMSO-d6/TMS) δ ppm: 105.16, 112.78, 117.34, 118.25,
121.45&123.62&125.78&127.95 (-CF3); 123.87, 124.76, 125.86, 127.23, 129.83, 130.28,
14

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130.79, 131.89, 132.38, 134.79, 135.97, 147.71 (-N=CH); 158.74&160.71 (C-F), 159.02,
161.22. 163.18 (C-F), 165.05 (-C=O). HRMS (EI,m/z): monoisotopic mass for C21H13F5N2O2
(Calcd./Found): 420.0897/ 420.0894; Anal. Calcd for C21H13F5N2O2: C, 60.01; H, 3.12; N,
6.66; found C, 59.69; H, 3.45; N, 6.57.
4.1.3.16.
2',4'-Difluoro-4-hydroxy-N'-{[4-(trifluoromethyl)phenyl]methylidene}biphenyl-3-
carbohydrazide (3p)
o
Yellow solid; Yield: 81%; HPLC: tR (min.): 9.69; Mp 260 C; FTIR (cm-1): 3261 (O-H&N-
1
H), 3074, 3049 (=C-H arom.), 1633 (C=O), 1616 (C=N); H-NMR (300 MHz), (DMSO-
d6/TMS) δ ppm: 7.10 (d, 1H, J=8.4Hz, H5); 7.22 (t, 1H, H11); 7.39 (t, 1H, H9); 7.57-7.97 (m,
6H, Ar-H); 8.02 (s, 1H, H2); 8.54 (s, 1H, CH=N); 11.91 (s, 1H, Ar-OH) 12.04 (s, 1H, CO-
NH-N=CH); Anal. Calcd for C21H13F5N2O2: C, 60.01; H, 3.12; N, 6.66; found C, 59.58; H,
3.01; N, 6.64.
4.1.3.17.
2',4'-Difluoro-4-hydroxy-N'-[(napthalen-1-yl)methylidene]biphenyl-3-
carbohydrazide (3r)
o
Yellow solid; Yield: 80%; HPLC: tR (min.): 9.41; Mp 266-268 C; FTIR (cm-1): 3265 (O-
1
H&N-H), 3078, 3047 (=C-H arom.), 1637 (C=O), 1612 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7.13 (d, 1H, J=8.4Hz, H5); 7.24 (t, 1H, H11); 7.41 (t, 1H, H9); 7.60-
8.95 (m, 10H, Ar-H); 9.11 (s, 1H, CH=N); 12.01 (s, 2H, Ar-OH&CO-NH-N=CH);
Anal. Calcd for C24H16F2N2O2: C, 71.64; H, 4.01; N, 6.96; found C, 71.34; H, 4.07; N, 6.98.
4.1.3.18.
2',4'-Difluoro-4-hydroxy-N'-[(napthalen-2-yl)methylidene]biphenyl-3-
carbohydrazide (3s)
o
Light yellow solid; Yield: 89%; HPLC: tR (min.): 9.88; Mp 279 C; FTIR (cm-1): 3244 (O-
1
H&N-H), 3047, 3039 (=C-H arom.), 1651 (C=O), 1606 (C=N); H-NMR (300 MHz),
(DMSO-d6/TMS) δ ppm: 7.10 (d, 1H, J=8.4Hz, H5); 7.22 (t, 1H, H11); 7.38 (t, 1H, H9); 7.57-
8.05 (m, 9H, Ar-H); 8.25(s, 1H, H2); 8.61 (s, 1H, CH=N); 11.95 (s, 1H, Ar-OH&CO-NH-
N=CH). 13C-NMR (150 MHz) (DMSO-d6/TMS) δ ppm: 104.93, 112.49, 116.99, 118.06,
15

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123.19, 124.52, 127.29, 127.76, 128.27, 128.86, 129.04, 129.41, 129.54, 132.15, 132.21,
132.27, 133.27, 134.32, 134.54, 149.32 (-N=CH), 158.67&160.14, 159.04, 160.31&162.77,
164.87 (-C=O). DART-MS; (m/z, Calcd /Found): 403.1253/403.1258 [C24H16F2N2O2+H]+ ;
805.2432/805.2445 [2(C24H16F2N2O2)+H]+; Anal. Calcd for C24H16F2N2O2: C, 71.64; H, 4.01;
N, 6.96; found C, 71.64; H, 3.82; N, 6.86.
4.1.3.19.
2',4'-Difluoro-4-hydroxy-N'-[(3-phenoxyphenyl)methylidene]biphenyl-3-
carbohydrazide (3t)
o
Yellow solid; Yield: 85%; HPLC: tR (min.): 9.21; Mp 198 C; FTIR (cm-1): 3257 (O-H&N-
1
H), 3064, 3020 (=C-H arom.), 1635 (C=O), 1614 (C=N); H-NMR (300 MHz), (DMSO-
d6/TMS) δ ppm: 6.98-7.64 (m, 14H, Ar-H); 8.01 (s, 1H, H2); 8.44 (s, 1H, CH=N); 11.82 (s,
1H, Ar-OH); 11.94 (s, 1H, CO-NH-N=CH); Anal. Calcd for C26H18F2N2O3: C, 70.26; H, 4.13;
N, 6.32; found C, 69.90; H, 4.13; N, 6.32.
4.2. Biological studies
4.2.1.1. Cell Culture
Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A replicon reporter cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum, 5%
antibiotic and 0.5 mg/ mL G418. All cells were cultured at 37oC and 5% CO2.
4.2.1.2. HCV Replicon Based Luciferase Reporter Assays
The Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A replicon reporter cells have been
described previously [20, 21]. To evaluate the anti-HCV activity of the compounds, HCV
replicon reporter cells were seeded in 96 well plate at a confluence of 1x104cells/well. Eight
hours post seeding, the cells were treated with the individual compounds (100 µM/well) or
equivalent amounts of DMSO for 42 h. Cell viability was measured in the parental Huh7.5
cells by the colorimetric MTS assay employing the CellTiter 96AQueous One Solution assay
reagent (Promega, USA). The anti-HCV activity of the compounds was evaluated as the
relative levels of the luciferase signals in compound treated cells versus DMSO controls.
16

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4.2.1. 3. NS5B inhibition assay
The biological activity of the compounds against NS5B polymerase were evaluated in a
reaction buffer containing 20 mM Tris-HCl (pH 7.0),100 mM NaCl, 100 mM sodium
glutamate, 0.1 mM DTT, 0.01% BSA,0.01% Tween-20, 5% glycerol, 20 U/mL of RNase Out,
0.25 µM of polyrA/U12, 25 µM UTP, 2 µCi [alpha-32P]UTP, 300 ng of NS5BC∆21and 1.0
mM MnCl2 with or without inhibitors (100 µM) in a total volume of 25 µL for 1 h at 30oC as
previously described [26, 27]. Reactions were terminated by the addition of ice-cold 5% (v/v)
trichloroacetic acid(TCA) containing 0.5 mM pyrophosphate. Reaction products were
precipitated on GF-B filters and quantified on a liquid scintillation counter. NS5B activity in
the presence of DMSO control was set at 100% and that in the presence of the compounds
was determined relative to this control.
4.2.2. Anticancer activity against hepatocelluler cell lines
4.2.2.1. Cell culture
Human liver cancer cell lines were grown in the standard growth medium medium (2 mM L-
glutamine, 0.1 mM nonessential amino acids, 100 units/mL penicillin, 100 lg/mL
streptomycin, 10% FCS in DMEM (Gibco, Invitrogen) or RPMI (Gibco, Invitrogen) and
incubated at 37 oC under 5% CO2 .
4.2.2. 2. Sulforhodamine B (SRB) assay for cytotoxicity screening
Human liver cancer cells were cultured in 96-well plates (1000-3000 cell/well) and for 24h.
They were treated with increasing concentrations of the compounds (2.5–40 ꢀM). Cell were
washed with 1xPBS (CaCl2-, MgCl2-free) (Gibco, Invitrogen) at the end of 72h incubation
period. Fixation was performed using 10% (v/v) trichloroacetic acid (MERCK). Finally, 0.4%
(m/v) of sulforhodamine (Sigma–Aldrich) in 1% acetic acid solution was added to each well
for staining process. The bound sulforhodamine B was then solubilized usin 10 mM Tris-base.
The absorbance values were obtained at 515 nm.
17

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4.2.2. 3. Hoechst stain
Human liver cancer cells (Huh7 and Mahlavu) were inoculated in 6-well plates for 24h. The
cells were treated with IC50 concentrations of the compounds for 72 h. Hoescht 33258
(Sigma–Aldrich) staining was used to determine nuclear condensation. Cells were fixed with
1 mL of cold methanol and the samples were incubated with 3 lg/mL of Hoescht, and
examined under fluorescent microscopy (40x).
4.2.2.4.Western blotting
Human liver cancer cells were cultured in 100mm culture dish. 24h later, growth medium was
replaced and cells were treated with IC50 concentrations of the compounds or DMSO controls.
At the end of 72h incubation, samples were scraped and collected for western blot analysis.
Anti-PARP antibody (Cell Signaling, 9532) and anti-β-actin antibody (Sigma, 5441) were
used as primary antibodies. Anti-rabbit (6154) and anti-mouse (0168) secondary antibodies
were used.
4.2.2.5. Cell cycle analysis
Cells were treated with IC50 concentrations of the compounds or 72h. Then samples were
stained with propidium iodide which binds to DNA and analyzed with MUSE Cell Cycle
analyzer kit.
Acknowledgements
This work was supported by The Scientific and Technical Research Council of Turkey
(TÜBİTAK), Research Fund Project Number: 112S013. Anti-HCV studies were supported by
funds from the UMDNJ Health Foundation to N.K. -B. Diflunisal was supplied by Sanovel
Pharmaceutical Industry Inc. The authors are grateful to Jürgen Gross from the Institute of
Organic Chemistry, University of Heidelberg, for his generous help in obtaining HR-EI and
DART-MS mass spectra of the synthesized compounds. We acknowledge Dr. Hengli Tang
for generously sharing the Huh7.5-FGR-JC1-Rluc2A replicon reporter cells.
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Captions of the Figures
Fig. 1. Diflunisal derivatives .
Fig. 2. Hydrazones of anti-HCV activity.
Fig. 3. Cytotoxicity induced by the compounds on liver cancer cell lines (Huh7, HepG2,
Hep3B, Mahlavu, FOCUS and SNU-475). The cells were treated with increased concentration
of the compound (2.5–40 ꢀM) for 72h. NCI-60 SRB assay was then performed. Absorbance
values were obtained and normalized to DMSO control. The experiment was conducted in
triplicate.
Fig. 4. Nuclear Hoechst (33258) staining (40x) of liver cancer cells treated with IC50
concentrations of the compounds or DMSO control for 72h.
Fig. 5. Cell cycle distribution of liver cancer cells (Huh7 and Mahlavu) treated with IC50
concentrations of the compounds or DMSO control for 72h.
Fig. 6. Western blot results of liver cancer cells treated with IC50 concentrations of the
compounds or DMSO control for 72h.
Captions of the Tables
Table 1. Anti-HCV activity of diflunisal hydrazide-hydrazones (3a-t)
Table 2. IC50 values of 3b on liver cancer cell lines.
Captions of the Schemes
Scheme 1. The synthetic route for the preparation of the target compounds (3a-t)
Graphical abstract
21

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22

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Table 1. Anti-HCV activity of diflunisal hydrazide-hydrazones (3a-t)
Huh7/Rep-Feo1b
Huh7.5-FGR-
Anti-NS5B
Activity
CC50
Comp.
JC1-Rluc2A
(µM)a
(% inhibition)b
EC50 (µM)
(Inhibition%)c
SIe
(% Inh., 50
d
µM)f
>50
>100
>200
>50
91 ± 2
79 ± 3
83 ± 6
33 ± 2
64 ± 2
81 ± 4
90 ± 2
15.6 ± 1.1
3.,9 ± 1.1
8.,1 ± 3.0
ND
>3.2
>25.6
>24.7
ND
17 ± 8
NI
3a
3b
3c
3d
3e
3f
98 ± 1
90 ± 3
8 ± 5
7 ± 4
8 ± 3
9 ± 2
7 ± 4
88 ± 10
23 ± 13
>50
ND
ND
95 ± 2
92 ± 5
<50
ND
ND
<50
ND
ND
3g
78 ± 11
>50
83 ± 2
3.9 ± 1.,7
>12.8
11 ± 9
3h
74 ± 14
96 ± 3
72 ± 15
95 ± 2
96 ± 2
82 ± 4
95 ± 2
99 ± 1
99 ± 1
<50
>100
<50
72 ± 5
69 ± 2
66 ± 3
65 ± 2
77 ± 5
65 ± 1
87 ± 3
86 ± 3
79 ± 3
65 ± 13
92 ± 2
ND
ND
ND
ND
NI
NI
3i
3j
ND
ND
NI
3k
3l
>50
ND
ND
NI
>200
<50
11.8 ± 0.5
ND
>16.9
ND
NI
3m
3n
3o
3p
3r
3s
NI
<50
ND
ND
18 ± 4
12 ± 6
NI
<50
ND
ND
>200
>100
<50
16.5 ± 4.2
ND
>6.1
ND
70 ± 10
58 ± 11
NI
ND
ND
14 ± 2
3t
99 ± 1
aCC50 values were evaluated in Huh 7.5 parental cells by the MTS assay. Anti-HCV activity of the compounds was
determined at 50 µM against Huh7.5-FGR-JC1-Rluc2Ab and Huh7/Rep-Feo1bc replicon reporter cells. EC50 values
d
were computed from dose-response curves at 8-10 concentrations of the compounds employing CalcuSyn V2 software.
Cells treated with equal amounts of DMSO served as control in all experiments. eSI: selectivity index represents the
ratio of CC50 to EC50. The values shown are an average of three independent experiments with standard deviation.
fPercent inhibition of HCV NS5B RdRp activity was determined at 50 µM concentration of the indicated compound.
The data represents an average of three independent measurements. NS5B RdRp activity in the absence of the inhibitor
was taken as 100 percent after subtraction of residual background activity. NI, no inhibition; ND, not determined.

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Table 2. IC50 values of 3b on liver cancer cell lines.
Cell line Diflunisal
3b
IC50 (µM)
10
10.34
16.21
4.74
IC50 (µM)
NO INHIBITION
NO INHIBITION
NO INHIBITION
51.7
Huh7
HepG2
Hep3B
Mahlavu
Focus
29.8
9.29
NO INHIBITION
8.33
SNU-475

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O
O
O
OH
OH
CH3OH/H2SO4
F
OH
F
F
F
1
Diflunisal
NH2NH2.H2O/CH3OH
O
O
NH
NH2
OH
NH
N CH-Ar
OH
Ar-CHO/EtOH
F
F
F
F
2
3a-t
Scheme 1. The synthetic route for the preparation of the target compounds (3a-t)

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F
F
F
A
S
F
OH
F
NH
N
A
F
NHN
O
O
O
OH
HO
X
N
NH
A
N
X=O, N, S
F
F
F
CH3
H3C
F
S
N
N
N
O
NH
F
O
O
OH
OH
Fig. 1. Diflunisal derivatives

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CH3
NH
N
O
F
F
CH3
F
NH
O
N
O
CH3
Fig. 2. Hydrazones of anti-HCV activity.

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3b
100
80
60
40
20
0
40 μM
20 μM
10 μM
5 μM
2.5 μM
Huh7
HepG2
Hep3B
Mahlavu
Focus
SNU-475
DIFLUNISAL
100
80
60
40
20
0
40 μM
20 μM
10 μM
5 μM
2.5 μM
Huh7
HepG2
Hep3B
Mahlavu
Focus
SNU-475
Fig. 3. Cytotoxicity induced by the compounds on liver cancer cell lines (Huh7, HepG2,
Hep3B, Mahlavu, FOCUS and SNU-475). The cells were treated with increased concentration
of the compound (2.5–40 µM) for 72h. NCI-60 SRB assay was then performed. Absorbance
values were obtained and normalized to DMSO control. The experiment was conducted in
triplicate.

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Fig. 4. Nuclear Hoechst (33258) staining (40x) of liver cancer cells treated with IC50 concentrations of
the compounds or DMSO control for 72h.