2-Heteroarylimino-5-arylidene-4-thiazolidinones as a new class of non-nucleoside inhibitors of HCV NS5B polymerase

Article abstract of DOI:10.1016/j.ejmech.2013.08.043

Hepatitis C virus (HCV) NS5B polymerase is an important and attractive target for the development of anti-HCV drugs. Here we report on the design, synthesis and evaluation of twenty-four novel allosteric inhibitors bearing the 4-thiazolidinone scaffold as

Full text of DOI:10.1016/j.ejmech.2013.08.043

Accepted Manuscript
2-Heteroarylimino-5-arylidene-4-thiazolidinones as a new class of non-nucleoside
inhibitors of HCV NS5B polymerase
İlkay Küçükgüzel, Gökhan Satılmış, K.R. Gurukumar, Amartya Basu, Esra Tatar,
Daniel B. Nichols, Tanaji T. Talele, Neerja Kaushik-Basu
PII:
S0223-5234(13)00560-6
10.1016/j.ejmech.2013.08.043
EJMECH 6390
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 June 2013
Revised Date: 27 August 2013
Accepted Date: 31 August 2013
Please cite this article as: İ. Küçükgüzel, G. Satılmış, K.R. Gurukumar, A. Basu, E. Tatar, D.B. Nichols,
T.T. Talele, N. Kaushik-Basu, 2-Heteroarylimino-5-arylidene-4-thiazolidinones as a new class of non-
nucleoside inhibitors of HCV NS5B polymerase, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2013.08.043.
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2
-Heteroarylimino-5-arylidene-4-thiazolidinones as a new class of
non-nucleoside inhibitors of HCV NS5B polymerase
1
,*
1
2
2
1
Đlkay Küçükgüzel , Gökhan Satılmıꢀ , K. R. Gurukumar , Amartya Basu , Esra Tatar ,
2
3
2,*
Daniel B. Nichols , Tanaji T. Talele , and Neerja Kaushik-Basu
1
Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
Haydarpaꢀa 34668 Đstanbul, Turkey
2
New Jersey Medical School, Department of Biochemistry & Molecular Biology, NJ 07103, USA
3
St. John’s University, Department of Pharmaceutical Sciences, College of Pharmacy and
Health Sciences, Queens, NY 11439, USA
Corresponding authors
Tel: +90 216 349 12 16; fax: +90 216 345 29 52 (Đ. K.); Tel: +1 973 972 8653; fax: +1 973 972
5
594 (N.K.-B.)
E-mail Addresses: ikucukguzel@marmara.edu.tr (Đlkay Küçükgüzel), kaushik@umdnj.edu
Neerja Kaushik-Basu)
(
Abbreviations: AP, allosteric pocket; HCV, hepatitis C virus; NI, nucleoside inhibitor; NNI,
non-nucleoside inhibitor; PP-I, palm pocket-I; RdRp, RNA-dependent RNA polymerase; SP,
subpocket; TP, thumb pocket
1

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Abstract
Hepatitis C virus (HCV) NS5B polymerase is an important and attractive target for the
development of anti-HCV drugs. Here we report on the design, synthesis and evaluation of
twenty-four novel allosteric inhibitors bearing the 4-thiazolidinone scaffold as inhibitors of HCV
NS5B polymerase. Eleven compounds tested were found to inhibit HCV NS5B with IC values
5
0
ranging between 19.8-64.9 µM. Compound 24 was the most active of this series with an IC of
5
0
5
2
.6 µM. A number of these derivatives further exhibited strong inhibition against HCV 1b and
a genotypes in cell based antiviral assays. Molecular docking analysis predicted that the
thiazolidinone derivatives bind to the NS5B thumb pocket-II (TP-II). Our results suggest that
further optimization of the thiazolidinone scaffold may be possible to yield new derivatives with
improved enzyme- and cell-based activity.
Keywords: Antiviral agents; HepatitisC; HCVNS5B polymerase; Thiazolidinones
2

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1
. Introduction
Hepatitis C virus (HCV) is an important human pathogen of global public health
significance with an estimated 3% of the world population infected [1, 2]. The enveloped, ~9.6
kb positive-stranded RNA virus encodes for a large polyprotein of ~ 3010 amino acids in length
[
3, 4]. Persistent HCV infection is the leading cause of severe hepatitis that often progresses to
cirrhosis, steatosis, and hepatocellular carcinoma [5-7]. HCV is also a lymphotropic virus
causing several lymphoproliferative disorders such as cryoglobulinemia, B-cell non-Hodgkin’s
lymphoma and monoclonal gammopathies [8].
To date, there is no prophylactic vaccine against HCV. The emergence of FDA approved
®
®
HCV protease inhibitors Boceprevir (Victrelis ) and Telaprevir (Incivek ) has significantly
improved sustained virological response in treating HCV genotype 1 infections [9-11]. These
HCV protease inhibitors block the enzymatic activity of the NS3-4A viral protease, thereby
inhibiting the protease from cleaving the HCV polyprotein into individual replicase components
[
9-11]. However, these inhibitors must be used in combination with pegylated-interferon (IFN)
and ribavirin to avoid selection for protease resistant HCV variants. Both IFN-related side
effects and a complicated dosing regimen that may limit patient compliance remain major
concerns in current HCV therapeutics [9-11]. Therefore, the search for novel directly acting
antivirals with reduced side-effects and high therapeutic index that target multiple HCV
genotypes continues to be an area of high priority.
Most small molecule inhibitor approaches to HCV have focused upon inhibition of
essential viral targets, such as the NS3-4A protease (analogous to HIV protease) [12].
Additionally, the HCV non-structural protein 5B (NS5B) represents an attractive target for anti-
HCV agents for several reasons. NS5B encodes the viral RNA dependent RNA polymerase
(RdRp) and is a critical component of the viral replication machinery [13-15]. Pharmacologic
agents that inhibit NS5B function block replication of viral RNA in tissue culture [16-18].
Additionally, mammalian cells lack proteins with RdRp activity. Therefore, molecules that target
NS5B are not likely to exhibit toxicity in host cells. The structure of the 66 kDa NS5B has been
well characterized. NS5B has the characteristic “right hand” structure of the polymerase family
and contains a catalytic site in the palm domain and RNA interacting regions of the RdRp in the
finger and thumb domains [19-22]. Current NS5B inhibitors have been grouped into two broad
3

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categories. Nucleoside inhibitors (NIs) mimic rNTPs substrates resulting in chain termination
when incorporated into elongating HCV RNA. Non-nucleoside inhibitors (NNIs) are a diverse
set of small molecule inhibitors that function by binding NS5B in one of five allosteric pockets
located on the thumb and palm domains [12, 17, 18]. Presumably, the binding of NNIs to
allosteric pockets results in a conformational change that inhibits NS5B enzymatic activity.
However, current research cannot rule out the possibility that these inhibitors have additional
mechanisms that block NS5B function.
Thiazolidinone compounds possess versatile biological activity including
antiproliferative, antibacterial, antifungal, and antimycobacterial functions [23-27]. These
compounds are also known to target cyclooxygenase (COX) enzymes making thiazolidinone
compounds excellent candidates to develop as anti-tumor and anti-inflammatory agents [25, 26].
Further, thiazolidone compounds have been shown to inhibit the HIV-1 reverse transcriptase [28-
3
0].
Because these compounds showed antiviral activity, we originally investigated
thiazolidinone compounds for the ability to inhibit the HCV NS5B polymerase. We reported
compounds bearing the thiazolidinone motif that non-competitively inhibited HCV NS5B
through binding to an allosteric site within the viral RdRp [31]. We concluded that these
compounds have the potential to be developed into more potent HCV NS5B NNIs.
A literature survey on HCV NS5B polymerase inhibitors clearly indicates that 4-
thiazolidinones could inhibit this enzyme and might be promising candidates for the
development of novel antiviral agents against HCV. The 4-thiazolidinone scaffold appears in the
form of 2,3-diaryl/2-aryl-3-acylamino-4-thiazolidinones (A , A ) [31, 32]; 2-amino-5-arylidene-
1
2
4
1
-thiazolinones (B , B ) [33, 34]; or 3-substituted-2-thioxo-4-thiazolidinones (C , C ) (Scheme
1 2 1 2
) [35, 36]. As can be seen from Figure 1, 5-arylidene derivatives (B and C type compounds)
inhibits NS5B at lower IC values. These findings encouraged us to go further with our ongoing
50
studies on 4-thiazolidinones derivatives. In this study, we designed a new class of potential
inhibitors by the modification of the compounds presented in Figure 1. Design strategy included:
i. protection of the 4-thiazolidinone core; ii. introduction of an arylidene moiety at C5 position of
the core; iii. introduction of arylimino moiety at C2 position of the core instead of aryl, amino or
thioxo functionality; and iv. keeping the N3 position of the core unsubstituted. Here, we report
the synthesis, anti-NS5B activity and anti-HCV activity of these new derivatives. We employed
molecular modeling to predict the binding site of the thiazolidinone compounds on NS5B
4

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polymerase. This study represents an important step in developing these compounds as NS5B
inhibitors.
2
. Results and Discussion
2
.1. Chemistry
The target compounds 9-26 were synthesized using a stepwise reaction protocol starting
from thiosemicarbazide and 4-chloro/4-fluorobenzoyl chloride at first step. Resulting 1-
aroylthiosemicarbazides 1 and 2 were then subjected to cyclization using concentrated sulfuric
acid to give 2-amino-5-aryl-1,3,4-thiadiazoles 3 and 4. Synthesis of 2-chloro-N-[5-(4-
chloro/fluorophenyl)-1,3,4-thiadiazol-2-yl]acetamides 5 and 6 were carried out by the reaction of
the appropriate amines 3 and 4 with chloroacetylchloride in the presence of TEA and DCM at
room temperature. Cyclization of chloroacetamides 5 and 6 in the presence of ammonium
thiocyanate in refluxing ethanol afforded 2-[[5-aryl-1,3,4-thiadiazol-2-yl]imino]-1,3-
thiazolidine-4-ones 7 and 8 in good yield. The target compounds 9-26 were obtained refluxing a
methanolic solution of 4-thiazolidinones 7 or 8 with commercially available aldehydes in the
presence of sodium methoxide. This method was different from the one described by Vicini et.al.
who reacted 4-thiazolidinones with aldehydes in acetic acid buffered medium with sodium
acetate [37]. However, use of sodium methoxide in methanolic medium instead of sodium
acetate in acetic acid, provided shorter reaction times [38].
All synthesized compounds were checked for purity using HPLC–UV/DAD and
elemental analysis. The new compounds 7-26 were characterized by their melting points and
1
13
-1
spectral data ( H NMR, C NMR and HR-MS). Absorption bands at 1720 cm were attributed
to the C=O stretching bands of both 1,3-thiazolidine-4-one compounds 7 and 8 provided
1
confirmatory evidence for ring closure [24]. H NMR chemical shifts were recorded within
expected regions for all synthesized compounds. Support for 4-thiazolidinone ring closure was
the detection of signals at 4.12 and 4.04 ppm, due to the presence of endocyclic -S-CH - protons
2
[
39]. Determination of endocyclic -NH- protons of compounds 7 and 8 at 12.22 and 12.38 ppm
influenced in favor of lactam proton owing to the fact that an imine proton resonates around 9.70
ppm [37, 40]. Disappearance of signals of endocyclic -S-CH - protons in the spectra of
2
compounds 9-26 indicates that active methylene group of 4-thiazolidinones 7-8 reacted with the
5

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selected aldehydes to yield 5-arylidene-1,3-thiazolidine-4-one derivatives 9-26. Assignment of
NH peaks of compounds 7-26 at 12.07-13.66 ppm as one signal may impute to predominant
existence of only one isomer on C=N imino exocyclic double bond [27]. Compounds 9-26
exhibited arylidene Ar-CH=C< resonances at 7.70-7.98 ppm. A survey of literature revealed that;
Ar-CH=C< proton, deshielded by the adjacent C=O, was detected at 7.63–7.97 ppm in 1H NMR
spectra in respect of thermodynamically stable Z configuration of exocyclic C=C bond [25, 38,
1
3
4
1-43]. C-NMR data of the thiazolidinone compounds 7 and 8 have also supported the carbon
framework [24, 44]. The carbonyl carbon of the thiazolidinone ring appeared at 174.57 ppm, the
signal of the thiazolidinone C appeared at about 36 ppm and the signal at about 166 ppm is
5
attributed to C of thiazolidinone ring. Earlier reports reveal that, dinstinctive methylidene carbon
2
of arylidene moiety showed resonance around 120-130 ppm and the mentioned carbon was
evaluated between 133-145 ppm for our representative compounds 9-11, 14, 17-20, 24-25 [45-
4
7].
2
.2. Structure–activity relationship
The HCV NS5B RdRp inhibitory activity of the synthesized compounds 3-26 is shown in
Table 1. Synthetic precursors 1,3,4-thiadiazole-2-amines 3-4 and chloroacetamides 5-6 showed
very low inhibition (15.4-20.0%) and their IC50 values were not determined. This clearly
indicates the necessity of 4-thiazolidinone ring attached to 1,3,4-thiadiazole ring with imino
linkage. Compounds 7 and 8, which bear C5 unsubstituted thiazolidinone ring, showed modest
activity profiles. However, the most active compounds were observed to have an arylidene
moiety at C5 of thiazolidinone ring. This clearly indicates the contribution of these arylidene
groups for stabilizing the binding mode to NS5B active site.
Thiadiazolylimino-4-thiazolidinone backbone was chosen as template for the
development of new HCV NS5B inhibitors. We preferred 4-fluorophenyl and 4-chlorophenyl
moieties at C5 of 1,3,4-thiadiazoles 7-8 as a starting point. The first 4-thiazolidinone compounds
7
-8 were identified as NS5B inhibitors with IC values of 38.6 and 42.5 µM, respectively.
50
Therefore, we decided to perform a SAR survey around 9-26 to explore the contribution of
benzylidene moiety introduced at C5 of 4-thiazolidione ring. Compounds 9-17 had a 4-
fluorophenyl moiety whereas this fluorine atom was replaced with chlorine in 18-26. Most of the
2
-thiadiazolylimino-5-arylidene-4-thiazolidinones 9-26 exhibited appreciable inhibition of HCV
6

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NS5B polymerase at 100 µM ranging between 11.5 and 82.0 percent. Among the compounds
tested, eleven derivatives were shown to exhibit greater than 50% NS5B RdRp inhibition at 100
µM concetration and IC values of these derivatives were determined.
5
0
Among 9-17 series, four representatives exhibited higher than 50% inhibition; IC values
5
0
of these derivatives ranged between 19.8 and 64.9 µM. Of the compounds 18-26, five
compounds exceeded 50% inhibition and their IC values ranged between 5.6 and 56.2 µM.
50
Fluorine substitution of the benzylidene moiety gave the best result in compound 10 which had a
4
-fluorophenyl substitution at C5 of 1,3,4-thiadiazole ring with an IC value of 34.6 µM. In both
50
9
-17 and 18-26 series, a shift of fluorine atom to 2 or 4-position resulted in decrease in activity.
Simultaneous fluorine and chlorine substitution of the benzylidene moiety resulted in zero or
marginal inhibition of NS5B. Three types of chlorine substitution were attempted in compounds
1
3-15 and 22-24 and best positioning was observed to be 2,6-dichloro substitution as evidenced
by compound 24, which had an IC50 value of 5.6 µM. This compound was the most potent
representative of all the synthesized compounds 7-26. Replacing the 4-chlorophenyl of
compound 24 with 4-fluorophenyl whilst keeping 2,6-dichlorobenzylidene constant resulted in
the second most active compound of this study which had an IC value of 19.8 µM.
5
0
Introduction of benzylidene groups with 2,6-dimethoxy or 4-nitro substitution resulted in
marginal or complete loss of activity. It was also noteworthy that the most active derivatives 15
and 24 were the ones with highest Log P values of their series 9-17 (R =F) and 18-26 (R =F),
1
2
respectively. These studies revealed the influence of two different 4-halogenophenyl groups at
C5 position of thiadiazole and several benzylidene moieties at C5 position of 4-thiazolidinone
ring. Compounds 15 and 24 possessing 2,6-dichlorobenzylidene moiety were proven as
promising lead compounds for further development.
2
.3. Correlation between Log P and IC values
50
The lipophilicity of a molecule represented by its Log P value is a valuable index utilized in
rational drug design to predict the physicochemical properties of the molecule in terms of drug
absorption, bioavailability, and hydrophobic drug-receptor interactions. In order to gain insight
into the lipophilicity of the 4-thiazolidinone derivatives, we calculated their Log P values using
ALOGPS 2.102 log P/log S calculation software (http://www.vcclab.org) as previously described
[
48, 49] and then examined the relationship between the 4-thiazolidinone-mediated inhibition of
7

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HCV NS5B (IC values) and their Log P values (Table 1). Compounds 3 to 8 exhibited low
5
0
lipophilicity with Log P values between 1.7 to 2.4, compounds 9, 10, 11, 16 and 17 with Log P
between 3.7 to 4.0 exhibited moderate lipophilicity, while the remaining 4-thiazolidinone
derivatives exhibited relatively higher lipophilicity with Log P values ranging from 4.3 to 4.9.
Compound 24, the most active compound of this series (IC =5.6 µM) exhibited the highest
5
0
lipophilicity, while paradoxically, compound 23 with similar lipophilicity was a poor inhibitor of
HCV NS5B exhibiting only 26% inhibition at 100 µM. Similarly, while compound 7, the least
lipophilic compound of this series (Log P=1.7) displayed modest inhibition of HCV NS5B
(
IC =38.6 µM), compound 3 with similar Log P of 1.7 was the least active NS5B inhibitor
50
among these derivatives. Thus, no clear cut correlation emerged between the lipophilicty of these
compounds and their properties as NS5B inhibitors.
2
.4. High throughput cell-based screening of compounds
Three reporter cell lines BHK-NS5B-FRLuc, Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-
Rluc2A were employed in a preliminary screen to investigate the cytotoxicity and anti-HCV
activities of the compounds at 100 µM (Table 2) [50-53]. The BHK-NS5B-FRLuc carrying a
bicistronic reporter gene (+)FLuc-(-)UTR-RLuc and stably transfected NS5B, permitted the
measure of cellular viability by the firefly luciferase luminescence, and intracellular HCV NS5B
RdRp activity as a ratio of Renilla to firefly luciferase luminescence, thus facilitating the
simultaneous identification of potent non-toxic HCV NS5B RdRp inhibitors [50]. Further
screening of these compounds as anti-HCV agents was carried out in the Huh7/Rep-Feo1b and
Huh7.5-FGR-JC1-Rluc2A cells, which carry the autonomously replicating HCV RNA of
genotype 1b and 2a in the firefly and Renilla luciferase reporters, respectively [51].
In the BHK-NS5B-FRLuc reporter cells, the twenty-three compounds displayed a wide
range of cytotoxicty (0-100%) and inhibition of intracellular NS5B RdRp activity (0-85%) at 100
µM concentration (Table 2). Of these, four compounds (14, 21-23) displayed no cytotoxicty or
inhibition, suggesting that these compounds may not be cell permeable, while five others
(compounds 5, 6, 12, 24 and 25) displayed >85% cytotoxicity and were not investigated for their
inhibition parameters. Compounds 16 and 20 exhibited a similar pattern with ~70% cytotoxicty
and no inhibition, while 9 and 15 displayed a somewhat similar trend in terms of inhibition of
1
1-14%, though the cytotoxicty varied between 56-72%. Compound 4 displayed ~29%
8

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cytotoxicity but did not inhibit, while 3 and 17 exhibited ~10% inhibition and low toxicity of 0-
1
5%. Compounds 10 and 19 displayed 60-86% inhibition though the ~55% cytotoxicty remained
a concern. The remaining four compounds 7, 8, 13, and 18 emerged the most promising,
displaying negligible cytotoxicity (0-15%) and 58-66% inhibition of intracellular NS5B RdRp
activity.
In the parental Huh7.5 cells, all compounds exhibited a remarkably similar trend of
cytotoxicity (Table 2) to that observed in the BHK cells, thus suggesting that the permeability of
the compounds remained similar in these two cell types. Notably, compounds 21-23 which
displayed no cytotoxicity or inhibition in the BHK-NS5B reporter, displayed an identical pattern
in the two replicon reporters, thus reiterating the lack of cell permeability of these compounds.
The other compounds exhibited an overall trend of higher inhibition in the two replicon reporters
compared to the BHK-NS5B reporter, thus suggesting that the compounds in addition to
targeting NS5B polymerase may potentially target other host factors required by HCV for its
replication. This was true for all compounds except 8, which showed a somewhat reduced
inhibition in the 1b replicon reporter compared to the NS5B reporter, thus suggesting that the
inhibition observed in the 1b replicon reporter may be attributed to inhibition of NS5B
polymerase activity alone in these cells. Furthermore, the inhibition of HCV RNA replication
was similar in the two replicon reporters for all compounds, except 4, 8 and 14, which exhibited
1
.6 to 2-fold higher inhibition of HCV RNA replication in the 2a versus 1b replicon reporter.
Interestingly, both 4 and 14 do not inhibit NS5B1b as is evident from the BHK-NS5B reporter
data, while 8 inhibits NS5B1b to a comparable extent in the 1b replicon. Therefore, one may
speculate that 4 and 14 potentially inhibit the 1b replicon by predominantly targeting some
essential host factors required by HCV1b. In case of 2a replicon, 4 and 14 may in addition
display different sensitivities to the NS5B2a polymerase. For compounds 5, 6, 12, 15, 24 and 25
which exhibit >70% cytotoxicity and near complete inhibition of HCV RNA replication in both
reporters, the inhibition observed may be partly attributed to the cellular toxicity effects of these
compounds.
These results suggest that the 4-thiazolidinone derivatives inhibit HCV RNA replication
by either targeting the NS5B polymerase and/or other host factors required by HCV for its
replication. While the exact mechanism needs to be elucidated, the present study provides a
platform to further optimize this scaffold and develop them into potent anti-HCV agents.
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2
.5. Molecular modeling
Since some thiazolone-based non-nucleoside inhibitors of HCV NS5B were reported to
bind to allosteric pocket located on the thumb domain of NS5B [33], we preferably performed
docking studies using thumb pocket-II of NS5B. Glide predicted binding model of the most
potent compound 24 within the thumb pocket-II of NS5B polymerase using the co-crystal
structure of PF-868554-NS5B (PDB ID: 3FRZ) is shown in Figure 2 [54]. The 4-chlorophenyl
ring is stabilized by hydrophobic interactions with the side chains of Leu419, Met423, Tyr477
and Leu497 and through offset type of aromatic-aromatic interaction with the indole ring of
Trp528. The 4-chloro group was found to be involved in electrostatic interactions with the
guanidine group of Arg422. The 1,3,4-thiadiazole ring forms aromatic-aromatic interaction with
Tyr477. The terminal 2,6-dichlorobenzylidene moiety extends to a hydrophobic channel defined
by residues Ile482, Ala486, Leu489, Pro496 and Leu497. One of the chloro atoms on the
benzylidene ring seem to be stabilized through the electrostatic interaction with the guanidine
group of Arg490. The thiazolidinone ring is anchored by Ile482. In addition to hydrophobic
interactions, the N3 and N4 atoms of the thiadiazole ring were found to be located within
hydrogen bonding distances from the backbone –NHs of Tyr477 and Ser476, respectively.
These binding interactions suggested scope for further optimization of
thiazolidinones at the terminal phenyl rings. For example, these phenyl rings may be substituted
with di and/or tri-fluoro groups to enhance electrostatic contacts with the guanidine group of
Arg422 and Arg490.
3
. Conclusions
Our efforts to develop potent inhibitors of HCV NS5B polymerase is a preeminent
activity in the search to find novel therapies for treating HCV infection. A novel series of 5-
arylidene-4-thiazolidinones were synthesized and evaluated as HCV NS5B polymerase
inhibitors. Of the twenty-four compounds, eleven derivatives exhibited modest inhibition of
NS5B RdRp activity resulting in the identification of 2-{[5-(4-chlorophenyl)-1,3,4-thiadiazol-2-
yl]imino}-5-(2,6-dichlorobenzylidene)-1,3-thiazolidin-4-one (24) with an IC value of 5.6 µM
5
0
as the most active of this series. A number of these derivatives further exhibited strong inhibition
against HCV 1b and 2a genotypes in cell based antiviral assays. Binding model of compound 24
1
0

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with thumb pocket-II of NS5B was also studied. Biological data and molecular modeling
outcomes revealed the substantial influence of 5-arylidene moiety upon high inhibition of HCV
NS5B enzyme activity. Currently we are working on optimization strategy for anti-NS5B
activity of lead compounds which involves (i) modification of 1,3,4-thiadiazole ring of
compounds 9-26 or replacement of the same ringwith other heteroaryl rings and (ii) attachment
of various arylmethylidene groups on thiazolidinone ring. These efforts should lead to new
analogs with potentially high affinity towards NS5B and low cellular toxicity.
4
. Experimental
4
.1. Chemistry
All solvents and reagents were obtained from commercial sources and used without
purification. All melting points (ºC, uncorrected) were determined using Kleinfeld SMP-II basic
model melting point apparatus. Elemental analyses were obtained using Leco CHNS-932 and are
consistent with the assigned structures. Infrared spectra were recorded on a Shimadzu FTIR
-1
8
400S and data are expressed in wavenumber ν (cm ). NMR spectra were recorded on Bruker
1
13
AVANCE-DPX 400 at 400 MHz for H-NMR and 100 MHz for C-NMR (DEPT and
Decoupled), the chemical shifts were expressed in δ (ppm) downfield from tetramethylsilane
(
TMS) using DMSO-d as solvent. The liquid chromatographic system consists of an Agilent
6
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 ꢁl sample loop was used for the injection of the analytes. Chromatographic
data were collected and processed using Agilent Chemstataion Plus software. The separation was
performed at ambient temperature by using a reversed phase Nova-Pak C18 (3.9x150 mm, 5 ꢁm
particle size) column. All experiments were performed in gradient mode. The mobile phase was
prepared by mixing acetonitrile and TEA-pH: 4.51 phosphate buffer (50 : 50 v/v during 0-3 min,
7
5 : 25 v/v during 3-6 min, 100 : 0 v/v during 6-9 min, 100 : 0 v/v during 9-15 min, 75 : 25 v/v
during 15-18 min, 50 : 50 v/v during 18-20 min) and filtered through a 0.45 ꢁm pore filter and
subsequently degassed by ultrasonication, prior to use. Solvent delivery was employed at a flow
-1
rate of 1ml.min . Detection of the analytes was carried out at 230, 254, 270, 310 nm.
SMILES were generated from the structures using the ACD/ChemSketch version 12.0
molecular editor (http://www.acdlabs.com) and then log P values were calculated using
1
1

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ALOGPS 2.102 log P/log S calculation software [48, 49]. The calculated log P values for all the
compounds are given in Table 1.
4
.1.1. Synthesis of 1-aroylthiosemicarbazides 1 and 2
The solution of thiosemicarbazide (10 mmol) in 20 ml acetone was reacted with 4-fluorobenzoyl
chloride or 4-clorobenzoyl chloride (10 mmol) in the presence of sodium bicarbonate (10 mmol)
with continous stirring in an ice bath. Acetone was evaporated under vacuo and the solid
precipitated was filtered and washed with water and then recrystallized.
4
.1.1.1. 4-Fluorobenzoylthiosemicarbazide 1
It was obtained as a white solid and was recrystallized from ethanol. HPLC t (min.): 2.77, Mp:
R
1
80°C (lit. 172°C) [55].
4
.1.1.2. 4-Chlorobenzoylthiosemicarbazide 2
It was obtained as a white solid and was recrystallized from ethanol. HPLC t (min.): 2.91, Mp:
R
2
14°C (lit.218-220°C;)[55].
4
.1.2. Synthesis of 2-amino-5-aryl-1,3,4-thiadiazoles 3 and 4
1
-Aroylthiosemicarbazide (compounds 1 or 2) was stirred in concentrated H SO at room
2
4
temperature for 4 h and the crude product was precipitated by using NaOH solution (10%) and
the solid precipitated was filtered and washed with water and then recrystallized.
4
.1.2.1. 2-Amino-5-(4-fluorophenyl)-1,3,4-thiadiazole 3
It was obtained as a white solid and was recrystallized from ethanol. HPLC t (min.): 4.09, Mp:
R
2
40°C (lit. 232-234°C [56]; 230°C; [57]).
4
.1.2.2. 2-Amino-5-(4-chlorophenyl)-1,3,4-thiadiazole 4
It was obtained as a white solid and was recrystallized from ethanol. HPLC t (min.): 5.57, Mp:
R
2
30°C (lit. 214-216°C; [56]/ 226-227°C; [58]).
1
2

ACCEPTED MANUSCRIPT
4
.1.3. Synthesis of 2-Chloro-N-[5-(4-chloro/fluorophenyl)-1,3,4-thiadiazol-2-yl]acetamides 5
and 6
The solution of 5-(4-chloro/fluorophenyl)-1,3,4-thiadiazol-2-amine (5 mmol) in 20 ml
dichloromethane and 1 ml triethylamine was reacted with chloroacetyl chloride (10 mmol) at
5
0ºC for 2 hours. Dichloromethane was evaporated under vacuo and the solid precipitated was
filtered and washed with water and then recrystallized.
4
.1.3.1. 2-Chloro-N-[5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl]acetamide 5
It was obtained as a white solid and was recrystallized from ethanol. HPLC t (min.): 6.21, Mp:
R
1
1
2
52°C, yield 62%. FTIR (cm ): 3185 (N-H), 3050 (=C-H arom), 1705 (C=O), 1576 (C=N). H-
NMR (300 MHz, DMSO-d ): δ 4.49 (s, 2H, -CH -), 7.34-7.41 (m, 2H, Ar-H), 7.98-8.04 (m, 2H,
6
2
Ar-H), 13.08 (s, 1H, NH) ppm. Anal. Calcd. for C H ClFN OS: C, 44.21 ; H, 2.60; N, 15.47; S,
10
7
3
1
1,80 Found: C, 44.63; H, 2.75; N, 14.92; S, 11,73 .
4
.1.3.2. 2-Chloro-N-[5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl]acetamide 6
It was obtained as a white solid and was recrystallized from ethanol HPLC t (min.): 7.59, Mp:
R
2
51°C (lit.243-244°C; [59]).
4
4
.1.4. Synthesis of 2-[[5-(4-Chloro/fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-1,3-thiazolidine-
-ones 7 and 8
A solution of 2-chloro-N-[5-(4-chloro/fluorophenyl)-1,3,4-thiadiazol-2-yl]acetamide (5 mmol)
and ammonium thiocyanate (10 mmol) in 20 ml absolute ethanol were refluxed for 6 hours and
allowed to stand overnight. The precipitate was filtered and washed with water and then
recrystallized.
4
.1.4.1. 2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-1,3-thiazolidine-4-one 7
It was obtained as a brown solid from compound 5 and was recrystallized from ethanol. HPLC tR
1
(
min.): 4.37, Mp: 232°C, yield 87%. FTIR (cm ): 3128 (N-H lactam), 3057 (=C-H arom), 1720
1
3

ACCEPTED MANUSCRIPT
1
(
C=O lactam), 1591 (C=N). H-NMR (300 MHz, CDCl - DMSO-d ): δ 4.04 (s, 2H,
3
6
thiazolidinone-SCH -), 7.56-7.63 (m, 2H, Ar-H), 8.16-8.21 (m, 2H, Ar-H), 12.22 (s, 1H,
2
13
thiazolidinone NH) ppm. C-NMR (300 MHz, CDCl -DMSO-d ): δ ppm 36.44 (thiazolidinone
3
6
C ), 117.03 and 117.33, 127.38, 130.08 and 130.19, 162.55 (Ar-C), 163.87 (thiadiazole C ),
5
2
1
66.93 (thiazolidinone C ), 170.83 (thiadiazole C ), 174.57 (thiazolidinone C ). Anal. Calcd. for
2 5 4
C H FN OS : C, 44.89 ; H, 2.40; N, 19.04. Found: C, 45.171; H, 2.52; N, 18.71.
11
7
4
2
4
.1.4.2. 2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-1,3-thiazolidine-4-one 8
It was obtained as a brown solid from compound 6 and was recrystallized from ethanol. HPLC tR
1
(
min.): 5.62, Mp: 297°C, yield 89%. FTIR (cm ): 3105 (N-H lactam), 3055 (=C-H arom), 1720
1
(
C=O lactam), 1591 (C=N). H-NMR (300 MHz, CDCl - DMSO-d ): δ 4.12 (s, 2H,
3
6
thiazolidinone-SCH -), 7.58 (d, J= 8.22 Hz, 2H, Ar-H), 7.91 (d, J= 8.51 Hz, 2H, Ar-H), 12.38
2
1
3
(
(
(
s, 1H, thiazolidinone NH) ppm. C-NMR (300 MHz, CDCl -DMSO-d ): δ ppm 36.23
3 6
thiazolidinone C ), 129.20, 129.39, 129.91, 136.01 (Ar-C), 163.58 (thiadiazole C ), 166.89
5
2
thiazolidinone C ), 170.83 (thiadiazole C ), 174.57 (thiazolidinone C ). LCMS (ESI-negative
2
5
4
–
ion): 308, 310 [M-2H] . Anal. Calcd. for C H ClN OS : C, 42.51 ; H, 2.27; N, 18.03. Found: C,
1
1
7
4
2
4
3.11; H, 2.37; N, 18.09.
4
.1.5. Synthesis of 2-[[5-(4-chloro/fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(substituted
benzylidene)-1,3-thiazolidin-4-ones 9-26
Compound 7 or 8 (2 mmol) was dissolved in methanolic solution of sodium methoxide (2
mmol). After dissolution by heating, 2-3 mmol corresponding aldehyde was added and reaction
medium was refluxed for 4 h. At the end of reaction, the product was precipitated by pouring the
medium into ice-cold water and neutralizing by acetic acid. Crude product was filtered and
washed with water and then recrystallized from ethanol.
4
.1.5.1.
2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2-fluorobenzylidene)-1,3-
thiazolidin-4-one 9
It was obtained as a brown solid from compound 7 and was recrystallized from ethanol. HPLC tR
1
(
min.): 8.32, Mp: 279°C (d), yield 68%. FTIR (cm ): 3128 (N-H lactam), 3057 and 3010 (=C-H
1
arom), 1720 (C=O lactam), 1591 (C=N). H-NMR (300 MHz, CDCl - DMSO-d ): δ 7.34-7.57
3
6
1
4

ACCEPTED MANUSCRIPT
(m, 4H, Ar-H), 7.61-7.66 (m, 2H, Ar-H), 7.75 (s, 1H, =CH-Ar), 7.94-7.99 (m, 2H, Ar-H), 12.07
1
3
(
s, 1H, thiazolidinone NH) ppm. C-NMR (300 MHz, CDCl -DMSO-d ): δ ppm 116.60 and
3 6
1
1
16.84, 117.13, 121.38 and 121.59, 124.14, 125.95, 126.99 and 127.10, 129.89 and 130.01,
38.21, 139.44, 152.22, 154.71, 158.86, 159.34, 162.43, 164.38 (Ar-C), 133.28 (thiazolidinone
C ), 142.09 (=CH-Ar), 165.74 (thiadiazole C ), 167.16 (thiazolidinone C ), 170.11 (thiadiazole
5
2
2
–
C ), 172.52 (thiazolidinone C ). LCMS (ESI-negative ion): 399, 400 [M-H] . Anal. Calcd. for
5
4
C H F N OS : C, 53.99 ; H, 2.52; N, 13.99. Found: C, 54.06; H, 2.57; N, 13.73.
18
10
2
4
2
4
.1.5.2.
2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(3-fluorobenzylidene)-1,3-
thiazolidin-4-one 10
It was obtained as a brown solid from compound 7 and was recrystallized from ethanol. HPLC tR
-1
(
min.): 8.39, Mp: 276-277°C (d), yield 77%. FTIR (cm ): 3112 (N-H lactam), 3066 and 3022
1
(
=C-H arom), 1720 (C=O lactam), 1602 and 1593 (C=N). H-NMR (300 MHz, CDCl -DMSO-
3
d6): δ 7.32-7.66 (m, 6H, Ar-H), 7.76 (s, 1H, =CH-Ar), 7.95-7,99 (m, 2H, Ar-H), 13.05 (s, 1H,
13
thiazolidinone NH) ppm. C-NMR (300 MHz, CDCl -DMSO-d ): δ ppm 116.84, 117.13,
3
6
1
17.31 and 117.60, 126.10 and 126.97, 129.91 and 130.02, 135.86, 158.90, 161.11, 162.43 (Ar-
C), 131.89 (thiazolidinone C ), 138.58 (=CH-Ar), 164.35 (thiadiazole C ), 165.73
5
2
(
thiazolidinone C ), 167.26 (thiadiazole C ), 170.12 (thiazolidinone C ). LCMS (ESI-negative
2 5 4
–
ion): 399, 400 [M-H] . Anal. Calcd. for C H F N OS : C, 53.99 ; H, 2.52; N, 13.99. Found: C,
1
8
10
2
4
2
5
3.27; H, 2.83; N, 14.23.
4
.1.5.3.
2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(4-fluorobenzylidene)-1,3-
thiazolidin-4-one 11
It was obtained as a dark green solid from compound 7 and was recrystallized from ethanol.
1
HPLC t (min.): 8.58, Mp: 305°C (d), yield 69%. FTIR (cm ): 3113 (N-H lactam), 3057 and
R
1
3
7
012 (=C-H arom), 1716 (C=O lactam), 1585 (C=N). H-NMR (300 MHz, CDCl -DMSO-d ): δ
3
6
.35-7.45 (m, 4H, Ar-H), 7.71-7.75 (m, 2H, Ar-H), 7.78 (s, 1H, =CH-Ar), 7.96-8.00 (m, 2H, Ar-
13
H), 12.07 (s, 1H, thiazolidinone NH) ppm. C-NMR (300 MHz, CDCl -DMSO-d ): δ ppm
3
6
1
1
16.84 and 116.92, 117.14 and 117.21, 124.07, 127.01, 129.91, 130.02 and 130.22, 132.32,
38.47 and 138.52, 139.18, 159.13 (Ar-C), 133.23 (thiazolidinone C ), 139.18 (=CH-Ar),
5
1
5

ACCEPTED MANUSCRIPT
1
64.25 (thiadiazole C ), 165.73 (thiazolidinone C ), 167.40 (thiadiazole C ), 170.19
2
2
5
–
(
thiazolidinone C ). LCMS (ESI-negative ion): 399, 400 [M-H] . Anal. Calcd. for
4
C H F N OS : C, 53.99 ; H, 2.52; N, 13.99. Found: C, 54.62; H,2.49; N,13.89.
18
10
2
4
2
4
.1.5.4.
2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2-chloro-6-fluoro-
benzylidene)-1,3-thiazolidin-4-one 12
It was obtained as a brown solid from compound 7 and was recrystallized from ethanol. HPLC tR
1
(
min.): 8.52, Mp: 287°C (d), yield 76%. FTIR (cm ): 3120 (N-H lactam), 3068 and 3018 (=C-H
1
arom), 1722 (C=O lactam), 1591 (C=N). H-NMR (300 MHz, CDCl -DMSO-d ): δ 7.37-7.62
3
6
(m, 5H, Ar-H), 7.71 (s, 1H, =CH-Ar), 7.97-8.01 (m, 2H, Ar-H), 13.11 (s, 1H, thiazolidinone NH)
–
ppm. LCMS (ESI-negative ion): 433, 434 [M-H] . Anal. Calcd. for C H ClF N OS C, 49.71;
18
9
2
4
2
H, 2.09; N, 12.88. Found: C, 50.08; H, 2.26; N, 12.58.
4
.1.5.5.
2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2-chlorobenzylidene)-1,3-
thiazolidin-4-one 13
It was obtained as a black solid from compound 7 and was recrystallized from ethanol. HPLC tR
1
(
min.): 9.14, Mp: 299°C (d), yield 61%. FTIR (cm ): 3147 (N-H lactam), 3076 and 3036 (=C-H
1
arom), 1720 (C=O lactam), 1599 and 1589 (C=N). H-NMR (300 MHz, CDCl -DMSO-d ): δ
3
6
7
.36-7.53 (m, 2H, Ar-H), 7.55-7.69 (m, 4H, Ar-H), 7.71 (s, 1H, =CH-Ar), 7.92-8.02 (m, 2H, Ar-
–
H), 13.13 (s, 1H, thiazolidinone NH) ppm. LCMS (ESI-negative ion): 415, 416 [M-H] . Anal.
Calcd. for C H ClFN OS C, 51.86; H, 2.42; N, 13.44. Found: C, 52.28; H, 2.44; N, 13.09.
1
8
10
4
2
4
1
.1.5.6.
2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2,4-dichlorobenzylidene)-
,3-thiazolidin-4-one 14
It was obtained as a dark brown solid from compound 7 and was recrystallized from ethanol.
1
HPLC t (min.): 9.72, Mp: 318°C (d), yield 70%. FTIR (cm ): 3129 (N-H lactam), 3064 and
R
1
3
028 (=C-H arom), 1720 (C=O lactam), 1602 and 1577 (C=N). H-NMR (300 MHz, CDCl -
3
DMSO-d ): δ 7.41 (t, J=8.4 Hz, J=8.4 Hz, 2H, Ar-H), 7.71 (s, 2H, =CH-Ar, Ar-H), 7.85 (d,
6
J=5.7 Hz, 2H, Ar-H), 7.98-8.03 (q, 2H, Ar-H), 13.22 (s, 1H, thiazolidinone NH) ppm. LCMS
–
(
ESI-negative ion): 449, 451 [M-2H] . Anal. Calcd. for C H Cl FN OS C, 47.90; H, 2.01; N,
1
8
9
2
4
2
1
6

ACCEPTED MANUSCRIPT
1
2.41. Found: C, 48.58; H, 2.10; N, 12.36.
4
1
.1.5.7.
2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2,6-dichlorobenzylidene)-
,3-thiazolidin-4-one 15
It was obtained as a dark brown solid from compound 7 and was recrystallized from
1
ethanol. HPLC t (min.): 8.75, Mp: 289°C (d), yield 64%. FTIR (cm ): 3130 (N-H lactam), 3084
R
1
and 3020 (=C-H arom), 1718 (C=O lactam), 1593 (C=N). H-NMR (300 MHz, CDCl -DMSO-
3
d6): δ 7.35-7,65 (m, 5H, Ar-H), 7.73 (s, 1H, =CH-Ar), 7.94-7.98 (m, 2H, Ar-H), 13.14 (s, 1H,
13
thiazolidinone NH) ppm. C-NMR (300 MHz, CDCl -DMSO-d ): δ ppm 116.88, 117.77,
3
6
1
1
26.94, 128.99, 129.30 and 129.96, 130.08, 131.95, 158.41 (Ar-C), 132.37 (thiazolidinone C5),
33.56 (=CH-Ar), 164.56 (thiadiazole C ), 165.98 (thiazolidinone C ), 170.14 (thiadiazole C ,
2
2
5
–
thiazolidinone C ). LCMS (ESI-negative ion): 449, 451 [M-2H] . Anal. Calcd. for
4
C H Cl FN OS C, 47.90; H, 2.01; N, 12.41. Found: C, 48.35; H, 2.09; N, 12.27.
18
9
2
4
2
4
.1.5.8.
2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(4-nitrobenzylidene)-1,3-
thiazolidin-4-one 16
It was obtained as a black solid from compound 7 and was recrystallized from ethanol. HPLC tR
1
(
min.): 7.64, Mp: 338°C (d), yield 71%. FTIR (cm ): 3111 (N-H lactam), 3068 and 3024 (=C-H
1
arom), 1718 (C=O lactam), 1589 (C=N). H-NMR (300 MHz, CDCl -DMSO-d ): 7.39 (t, J=8.4
3
6
Hz, J=8.4 Hz, 2H, Ar-H), 7.89-8.02 (m, 5H, =CH-Ar, Ar-H), 8.37-8.56 (m, 2H, Ar-H), 13.66 (s,
–
1
H, thiazolidinone NH) ppm. LCMS (ESI-negative ion): 425, 427 [M-2H] . Anal. Calcd. for
C H FN O S C, 50,58; H, 2,36; N, 16,38. Found: C, 50,67; H, 2,38; N, 15,90.
18
10
5
3 2
4
.1.5.9. 2-[[5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2,6-dimethoxybenzylidene)-1,3-
thiazolidin-4-one 17
It was obtained as a brown solid from compound 7 and was recrystallized from ethanol.
1
Mp: 273°C (d), yield 65%. FTIR (cm ): 3120 (N-H lactam), 3057 (=C-H arom), 1716 (C=O
1
lactam), 1595 (C=N). H-NMR (300 MHz, CDCl -DMSO-d ): δ 4.13 (s, 6H, -CH ), 7.02 (d, J
3
6
3
=8.4 Hz, 2H, Ar-H), 7.59-7.71 (m, 2H, Ar-H), 7.73 (s, 1H, =CH-Ar-H), 8.15-8.25 (m, 3H, Ar-H),
1
3
1
2.95 (s, 1H, tiyazolidinon-NH-) ppm. C-NMR (300 MHz, CDCl -DMSO-d ): δ ppm 56.50(-
3 6
1
7

ACCEPTED MANUSCRIPT
O-CH ), 110.61, 117.04 and 117.33, 125.85, 127.18, 127.37 and 127.41, 158.91 (Ar-C), 130.26
3
(
thiazolidinone C ), 134.04 (=CH-Ar), 164.13 (thiadiazole C ), 165.89 (thiazolidinone C ),
5
2
2
–
1
68.08 (thiadiazole C ), 170.56 (thiazolidinone C ). LCMS (ESI-negative ion): 440, 441 [M-H] .
5 4
Anal. Calcd. for C H FN O S C, 54.29; H, 3.42; N, 12.66. Found: C, 54.22; H, 3.45; N, 12.20.
2
0
15
4
3 2
4
.1.5.10. 2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2-fluorobenzylidene)-1,3-
thiazolidin-4-one 18
It was obtained as a brown solid from compound 8 and was recrystallized from ethanol. HPLC tR
1
(
min.): 9.46, Mp: 284°C (d), yield 78%. FTIR (cm ): 3117 (N-H lactam), 3068 and 3016 (=C-H
1
arom), 1722 (C=O lactam), 1599 (C=N). H-NMR (300 MHz, CDCl – DMSO-d ): δ 7.36 (t,
3
6
J=7.6 Hz, J=7.6 Hz, 1H, Ar-H), 7.49 (d, J=7.1 Hz, 2H, Ar-H), 7.52-7.68 (m, 3H, Ar-H), 7.79 (s,
1
3
1
H, =CH-Ar), 7.93-7.98 (m, 2H, Ar-H), 13.09 (s, 1H, thiazolidinone NH) ppm. C-NMR (300
MHz, CDCl -DMSO-d ): δ 117.36, 117.65 and 117.94, 126.11, 129.28 and 129.97, 136.05 and
3
6
1
36.24, 159.14 and 161.13 (Ar-C), 132.05 (thiazolidinone C ), 145.55 (=CH-Ar), 164.37
5
(
thiadiazole C ), 167.31 (thiazolidinone C ), 170.41 (thiadiazole C ), 171.41 (thiazolidinone C )
2 2 5 4
–
ppm. LCMS (ESI-negative ion): 414, 416 [M-2H] . Anal. Calcd. for C H ClFN OS : C, 51.86;
1
8
10
4
2
H, 2.42; N, 13.44. Found: C, 52.43; H, 2.41; N, 13.00.
4
.1.5.11. 2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(3-fluorobenzylidene)-1,3-
thiazolidin-4-one 19
It was obtained as a brown solid from compound 8 and was recrystallized from ethanol. HPLC tR
1
(
min.): 8.91, Mp: 284°C (d), yield 78%. FTIR (cm ): 3117 (N-H lactam), 3068 and 3016 (=C-H
1
arom), 1722 (C=O lactam), 1599 (C=N). H-NMR (300 MHz, CDCl – DMSO-d ): δ 7.36 (t,
3
6
J=7.8 Hz, J=7.8 Hz, 1H, Ar-H), 7.49 (d, J=7.2 Hz, 2H, Ar-H), 7.59-7.68 (m, 3H, Ar-H), 7.79 (s,
1
3
1
H, =CH-Ar), 7.95-7.98 (m, 2H, Ar-H), 13.09 (s, 1H, thiazolidinone NH) ppm. C-NMR (300
MHz, CDCl -DMSO-d ): δ 117.36, 117.65 and 117.94, 126.11, 129.28 and 129.97, 136.05 and
3
6
1
36.24, 159.14 and 161.13 (Ar-C), 132.05 (thiazolidinone C ), 145.55 (=CH-Ar), 164.37
5
(
thiadiazole C ), 167.31 (thiazolidinone C ), 170.41 (thiadiazole C ), 171.41 (thiazolidinone C )
2 2 5 4
–
ppm. LCMS (ESI-negative ion): 414, 416 [M-2H] . Anal. Calcd. for C H ClFN OS ½ mol
18
10
4
2.
H O : C, 50.76; H, 2.60; N, 13.16. Found: C, 50.60; H, 2.44; N, 13.11.
2
1
8

ACCEPTED MANUSCRIPT
4
.1.5.12. 2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(4-fluorobenzylidene)-1,3-
thiazolidin-4-one 20
It was obtained as a brown solid from compound 8 and was recrystallized from ethanol. HPLC tR
1
(
min.): 9.49, Mp: 318°C (d), yield 78%. FTIR (cm ): 3144 (N-H lactam), 3076 and 3026 (=C-H
1
arom), 1716 (C=O lactam). H-NMR (300 MHz, CDCl -DMSO-d ): δ 7.43 (t, J=6.4 Hz, J=6.4
3
6
Hz, 2H, Ar-H), 7.59 (d, J=6.38 Hz, 2H, Ar-H), 7.75 (t, J=5.1 Hz, J=5.1Hz, 2H, Ar-H), 7.81 (s,
1
3
1
H, =CH-Ar), 7.92-7.96 (m, 2H, Ar-H), 13.03 (s, 1H, thiazolidinone NH) ppm. C-NMR (300
MHz, CDCl -DMSO-d ): δ 116.96 and 117.25, 124.09, 129.26 and 129.97, 130.24, 136.21,
3
6
1
59.35 (Ar-C), 133.26 (thiazolidinone C ), 137.73 (=CH-Ar), 164.82 (thiadiazole C ), 167.43
5 2
(
thiazolidinone C ), 169.65 (thiadiazole C , thiazolidinone C ) ppm. LCMS (ESI-negative ion):
2
5
4
–
4
5
14, 416 [M-2H] . Anal. Calcd. for C H ClFN OS : C, 51.86; H, 2.42; N, 13.44. Found: C,
18
10
4
2
1.32; H, 2.49; N, 13.36.
4
.1.5.13.
2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2-chloro-6-fluoro-
benzylidene)-1,3-thiazolidin-4-one 21
It was obtained as a yellow solid from compound 8 and was recrystallized from ethanol. HPLC
1
t (min.): 9.31, Mp: 300°C (d), yield 70%. FTIR (cm ): 3120 (N-H lactam), 3068 and 3020 (=C-
R
1
H arom), 1726 (C=O lactam), 1597 (C=N). H-NMR (300 MHz, CDCl -DMSO-d ): δ 7.41-7.62
3
6
(m, 5H, Ar-H), 7.69 (s, 1H, =CH-Ar), 7.93 (d, J=8.4 Hz, 2H, Ar-H), 13.32 (s, 1H, thiazolidinone
NH) ppm. LCMS (ESI-negative ion): 448, 451 [M-3H] and 450, 451 [M-H]. Anal. Calcd. for
C H Cl FN OS : C, 47.90; H, 2.01; N, 12.41. Found: C, 47.72; H, 2.28; N, 12.00.
18
19
2
4
2
4
.1.5.14.
2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2-chlorobenzylidene)-1,3-
thiazolidin-4-one 22
It was obtained as a yellow solid from compound 8 and was recrystallized from ethanol.
1
HPLC t (min.): 9.79, Mp: 337°C (d), yield 67%. FTIR (cm ): 3147 (N-H lactam), 3086 and
R
1
3
7
034 (=C-H arom), 1720 (C=O lactam), 1597 (C=N). H-NMR (300 MHz, CDCl -DMSO-d ): δ
3
6
.61-7.70 (m, 6H, Ar-H, =CH-Ar), 7.96 (d, J=8.1 Hz, 3H, Ar-H) 13.15 (s, 1H, thiazolidinone
-
-
NH) ppm. LCMS (ESI-negative ion): 430, 433 [M-3H] and 432, 433 [M-H] . Anal. Calcd. for
1
9

ACCEPTED MANUSCRIPT
C H Cl N OS : C, 49.89; H, 2.33; N, 12.93. Found: C, 49.89; H, 2.36; N, 12.70.
18
10
2
4
2
4
1
.1.5.15.
2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2,4-dichlorobenzylidene)-
,3-thiazolidin-4-one 23
It was obtained as an orange solid from compound 8 and was recrystallized from ethanol. HPLC
1
t (min.): 11.32, Mp: 324°C (d), yield 69%. FTIR (cm ): 3122 (N-H lactam), 3064 and 3024
R
1
(
=C-H arom), 1716 (C=O lactam), 1595 (C=N). H-NMR (300 MHz, CDCl -DMSO-d ): δ 7.43-
3
6
7
.70 (m, 5H, Ar-H), 7.82 and 7.84 (s, 1H, =CH-Ar), 7.95 (d, J=8.4 Hz, 2H, Ar-H), 12.85 (s, 1H,
-
thiazolidinone NH) ppm. LCMS (ESI-negative ion): 467, 468 [M-H] . Anal. Calcd. for
C H Cl N OS : C, 46.22; H, 1.94; N, 11.98. Found: C, 46.08; H, 2.11; N, 11.81.
18
9
3
4
2
4
1
.1.5.16.
2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2,6-dichlorobenzylidene)-
,3-thiazolidin-4-one 24
It was obtained as an orange solid from compound 8 and was recrystallized from ethanol. HPLC
1
t (min.): 9.46, Mp: 324°C (d), yield 69%. FTIR (cm ): 3126 (N-H lactam), 3070 and 3016 (=C-
R
1
H arom), 1718 (C=O lactam), 1585 (C=N). H-NMR (400 MHz, CDCl -DMSO-d ): δ 7.48-7,63
3
6
(m, 5H, Ar-H), 7.72 (s, 1H, =CH-Ar-H), 7.88 (d, J=8.7 Hz, 2H, Ar-H), 13.13 (s, 1H,
13
thiazolidinone NH) ppm. C-NMR (400 MHz, CDCl -DMSO-d ): δ 129.00, 129.10, 129.23,
3
6
1
1
1
29.27 and 129.92, 130.44, 131.91, 132.34, 135.08, 136.26 (Ar-C), 133.54 (thiazolidinone C5),
38.55 (=CH-Ar), 164.44 (thiadiazole C ), 165.96 (thiazolidinone C ), 170.29 (thiadiazole C ),
2
2
5
-
72.50 (thiazolidinone C ) ppm. LCMS (ESI-negative ion): 464, 468 [M-4H] and 467, 468 [M-
4
-
H] . Anal. Calcd. for C H Cl N OS : C, 46.22; H, 1.94; N, 11.98. Found: C, 46.09; H, 2.15; N,
1
8
9
3
4
2
1
1.47.
4
.1.5.17.
2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2,6-dimethoxy-
benzylidene)-1,3-thiazolidin-4-one 25
It was obtained as a yellow solid from compound 8 and was recrystallized from ethanol. HPLC
1
t (min.): 11.22, Mp: 276°C (d), yield 68%. FTIR (cm ): 3115 (N-H lactam), 3049 and 3010
R
1
(
=C-H arom), 1716 (C=O lactam). H-NMR (300 MHz, CDCl -DMSO-d ): δ 3.88 (s, 6H, Ar-
3
6
CH ), 6.77 (d, J=8.4 Hz, 2H, Ar-H), 7.46 (t, 1H, J=11.2 Hz, J=11.2 Hz, Ar-H), 7.58 (d, 2H,
3
2
0

ACCEPTED MANUSCRIPT
J=9.2 Hz, Ar-H), 7.91 (s, 1H, =CH-Ar-H), 7,93 (d, J=11.6 Hz 2H, Ar-H) 12.73 (s, 1H,
13
thiazolidinone NH). C-NMR (400 MHz, CDCl -DMSO-d ): δ 56.27 (-OCH ) 110.35, 125.58,
3
6
3
1
27.01, 129.26, 129.37, 129.92 and 158.68 (Ar-C), 133.82 (thiazolidinone C ), 136.05 (=CH-
5
Ar), 163.81 (thiadiazole C ), 167.85 (thiazolidinone C ), 170.55 (thiadiazole C , thiazolidinone
2
2
5
C ). LCMS (ESI-negative ion): 456, 457 [M-H]. Anal. Calcd. For C H ClN O S C, 52.34; H,
4
20 15
4
3 2
3
.29; N, 12.21. Found C, 51.95; H, 3.33; N, 11.97.
4
.1.5.18.
2-[[5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(4-nitrobenzylidene)-1,3-
thiazolidin-4-one 26
It was obtained as a yellow solid from compound 8 and was recrystallized from ethanol. HPLC
1
t (min.): 8.22, Mp: 319°C (d), yield 76%. FTIR (cm ): 3171 (N-H lactam), 3100 and 3020 (=C-
R
1
H arom), 1710 (C=O lactam), 1583 (C=N), 1550 and 1350 (N-O NO ). H-NMR (300 MHz,
2
CDCl -DMSO-d ): δ 7.61 (d, J=8.7 Hz, 2H, Ar-H), 7.94 (t, J=8.7 Hz, J=8.7 Hz, 4H, Ar-H), 7,98
3
6
(s, 1H, =CH-Ar-H), 8.38 (d, J=8.4 Hz, 2H, Ar-H), 13.17 (s, 1H, thiazolidinone NH) ppm. LCMS
-
(
ESI-negative ion): 441, 443 [M-2H] . Anal. Calcd. for C H ClN O S : C, 48.70; H, 2.27; N,
1
8
10
5
3 2
1
5.78. Found: C, 48.84; H, 2.36; N, 15.
4
4
.2. Biological Studies
.2.1 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,
.1 mM DTT, 0.01% BSA,0.01% Tween-20, 5% glycerol, 20 U/mL of RNase Out, 0.25 µM of
0
32
polyrA/U , 25 µM UTP, 2 µCi [alpha- P]UTP, 300 ng of NS5BCꢂ21and 1.0 mM MnCl with
1
2
2
o
or without inhibitors (100 µM) in a total volume of 25 µL for 1 h at 30 C as previously described
31, 60]. 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.
2
1

ACCEPTED MANUSCRIPT
4
.2.2 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. BHK-NS5B-FRLuc reporter cells were grown in DMEM with 10% fetal
bovine serum, 5% antibiotic-antimycotic, 5% nonessential amino acid, 1 mg/mL G418 and 10
o
µg/mL blasticidin. All cells were cultured at 37 C and 5% CO .
2
4
.2.3 BHK-NS5B-FRLuc Reporter Assay
The effect of the extracts on the intracellular NS5B RdRp activity was evaluated employing the
BHK-NS5B-FRLuc reporter as described previously [50,52, 53]. Briefly, the cells were seeded at
4
a density of 1x10 cells/well in a 96 well plate and 8 h later treated with the individual compound
at 100 µM concentration per well. Cells were lysed 42 h post treatment using passive lysis buffer
and luciferase expression was measured using the Dual-Glo Luciferase Assay Kit (Promega,
USA). The effect of the compounds on cell viability was estimated as the relative levels of firefly
luciferase in compound treated cells versus DMSO controls. The inhibitory effect of the
compounds on the intracellular NS5B RdRp activity was evaluated from the percent reduction in
RLuc to FLuc luminescence signal in extract treated cells versus DMSO controls.
4
.2.4 HCV Replicon Based Luciferase Reporter Assays
The Huh7/Rep-Feo1b replicon reporter cells have been described previously [51-53]. The
Huh7.5-FGR-JC1-Rluc2A replicon cells were generated in Dr. Hengli Tang’s lab as described
herein. Briefly, the 2623 base pair (b.p.) AgeI-BsiWI fragment from the full length HCV 2a
plasmid pFGR-JFH-1 was used to replace the similarly digested 1190 b.p. fragment from the p7-
Rluc2A plasmid, such that the JFH-1 UTR, a neomycin resistance gene, and most of the JFH-1
E1 was incorporated into the p7Rluc2a plasmid. The resulting 14,806 b.p. construct was
linerized with XbaI for in vitro transcription of the viral RNA, and electroporated (10 µg RNA)
into Huh7.5 cells. Cells expressing the HCV proteins and the Renilla luciferase reporter were
selected over a period of 3-4 weeks with 0.5 mg/ mL G418.
To evaluate the anti-HCV activity of the compounds, HCV replicon reporter cells were
4
seeded in a 96 well plate at a confluence of 1x10 cells/well. Eight hours post seeding, the cells
2
2

ACCEPTED MANUSCRIPT
were treated with the individual compounds (100 µM/well) or equivalent amounts of DMSO for
4
2 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.
4
.3. Molecular Modeling
Compounds were built and subjected to LigPrep as per previous reports using Maestro
.0 and Macromodel program v9.7 (Schrödinger, Inc., New York, NY, 2009) [61]. The output
9
obtained from the LigPrep run was used as input for docking simulations. The X-ray co-crystal
structure of HCV NS5B-PF868554 (PDB ID: 3FRZ) was used for docking into thumb pocket-II
[
54]. Protein refinement without crystallographic water molecules, energy grid generation using
bound ligand as a reference, and “Extra Precision” (XP) Glide docking v5.0 (Schrödinger, Inc.,
New York, NY, 2009) was performed with the default parameters. The top scoring pose of
compound 24 within the TP-II was used for graphical analysis. All computations were carried
out on a Dell Precision 470n dual processor with the Linux OS (Red Hat Enterprise WS 4.0).
Acknowledgements
We thank Drs. Jin-Ching Lee, Naoya Sakamoto, and Hengli Tang for the BHK-NS5B-FRLuc,
Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A replicon reporter cells. This work was
supported by the National Institute of Health Research Grant CA153147 and the UMDNJ
foundation grant to N.K.-B. The authors are grateful to Schimadzu Turkey Branch Office, for
their generous help in obtaining LC/MS-ESI mass spectra of the synthesized compounds.
2
3

ACCEPTED MANUSCRIPT
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Figure Legends and Table Headings
Fig. 1.
Structures of existing thiazolidinones (A), thiazolones (B) and rhodanines (C) as
inhibitors of HCV NS5B and the newly designed 5-arylidene-2-heteroarylimino-4-
thiazolidinones.
Fig. 2.
XP-Glide predicted binding model of compound 24 with residues of NS5B TP-II
site. Compound 24 is shown as ball and stick model whereas amino acids are shown
as stick model. Dotted red lines show electrostatic contacts with distances in Å.
Scheme 1. General procedure for the synthesis of compounds 1-26. Reagents and conditions: (a)
o
NaHCO , acetone, 0 C to rt; (b) conc. H SO , rt, 4 h; (c) ClCOCH Cl, TEA, DCM,
3
2
4
2
o
5
4
0 C, 2 h; (d) NH SCN, ethanol, reflux, 6 h; (e) R -CHO, CH ONa, MeOH, reflux,
4 2 3
h.
Table 1. Anti-NS5B RdRp activity and calculated lipophilicity values of compounds 3-26.
Table 2. Anti-HCV Effects in cell based reporter assays.
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