Novel 4-thiazolidinones as non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase

Article abstract of DOI:10.1002/ardp.201400247

In continuation of our efforts to develop new derivatives as hepatitis C virus (HCV) NS5B inhibitors, we synthesized novel 5-arylidene-4-thiazolidinones. The novel compounds 29-42, together with their synthetic precursors 22-28, were tested for HCV NS5B inhibitory activity; 12 of these compounds displayed IC₅₀ values between 25.3 and 54.1 μM. Compound 33, an arylidene derivative, was found to be the most active compound in this series with an IC₅₀ value of 25.3 μM. Molecular docking studies were performed on the thumb pocket-II of NS5B to postulate the binding mode for these compounds.

Full text of DOI:10.1002/ardp.201400247

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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–13
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Full Paper
Novel 4-Thiazolidinones as Non-Nucleoside Inhibitors of Hepatitis
C Virus NS5B RNA-Dependent RNA Polymerase
1
1
2
1
2
2
˙
Gizem C¸ akır , Ilkay Ku¨ c¸u¨ kgu¨ zel , Rupa Guhamazumder , Esra Tatar , Dinesh Manvar , Amartya Basu ,
Bhargav A. Patel3, Javairia Zia2, Tanaji T. Talele3, and Neerja Kaushik-Basu2*
1
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Marmara University, HaydarpasSa,
Istanbul, Turkey
Department of Biochemistry & Molecular Biology, New Jersey Medical School, The State University of
New Jersey, Newark, NJ, USA
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University,
Queens, NY, USA
˙
2
3
In continuation of our efforts to develop new derivatives as hepatitis C virus (HCV) NS5B inhibitors, we
synthesized novel 5-arylidene-4-thiazolidinones. The novel compounds 29–42, together with their
synthetic precursors 22–28, were tested for HCV NS5B inhibitory activity; 12 of these compounds displayed
IC50 values between 25.3 and 54.1 mM. Compound 33, an arylidene derivative, was found to be the most
active compound in this series with an IC50 value of 25.3 mM. Molecular docking studies were performed
on the thumb pocket-II of NS5B to postulate the binding mode for these compounds.
Keywords: Antiviral agents / Hepatitis C / HCV NS5B polymerase / Molecular modeling /
4-Thiazolidinones
Received: July 2, 2014; Revised: September 7, 2014; Accepted: October 1, 2014
DOI 10.1002/ardp.201400247
challenging due to HCV diversity and antiviral host immunity,
Introduction
resulting in only a few vaccine candidates being tested in
phase I/II trials [4]. Treatment of HCV has been an unmet
clinical need as efficacy of the most widely used pegylated
interferon-alpha (PEG-IFN-alpha) plus ribavirin (RBV) treat-
ment regimen is limited against various HCV genotypes
and in addition associated with drawbacks of specificity and
toxicity.
Hepatitis C virus (HCV) was identified in 1989 as the causative
agent for non-A, non-B hepatitis infection [1] and currently
has an estimated prevalence of 180–200 million people
worldwide [2]. HCV infection is the principle cause of
mortality related to hepatocellular carcinoma and is a leading
indication for liver transplantation. HCV is also marked by its
lymphotropic characteristics with patients developing cryo-
globulinemia, B-cell non-Hodgkin’s lymphoma, and mono-
clonal gammopathies [3].
Following identification of HCV as an enveloped, positive-
stranded RNA virus with 9.6 kb genome encoding three
structural (Core, E1, and E2) and seven nonstructural (p7,
NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins [5–7],
development of small molecule directly acting antivirals
(DAA) against HCV yielded two NS3-4A protease inhibitors:
To date, there is no prophylactic vaccine available against
HCV. HCV vaccine development has proved particularly
boceprevir (Victrelis1
)
and telaprevir (Incivek1). Upon
clinical use of approved boceprevir and telaprevir in
combination with Peg-IFN-a and RBV for the treatment
of genotype 1 chronic hepatitis C in adult patients, the
effectiveness of anti-HCV therapy has increased to 70% from
40 to 50%. However, since HCV exhibits genetic heteroge-
neity and a high mutation rate, four new DAA treatment
options have been suggested; HCV NS3 protease inhibitors
simeprevir (Olysio1) and faldaprevir together with Peg-IFN-a
˙
Correspondence: Prof. Ilkay Ku¨ c¸u¨ kgu¨ zel, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Marmara University,
HaydarpasSa, 34668 Istanbul, Turkey.
E-mail: ikucukguzel@marmara.edu.tr
Fax: þ90 216 3452952
ꢀAdditional correspondence: Prof. Neerja Kaushik-Basu
E-mail: kaushik@njms.rutgers.edu
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.
and RBV for the treatment of genotype
nucleotide NS5B polymerase inhibitor sofosbuvir (Sovaldi1
plus RBV with and without Peg-IFN-a for the treatment of
1 patients;
)
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genotypes 1–6 patients and genotypes 1–4 patients, respec-
tively, sofosbuvir together with NS5A replication inhibitor
daclatasvir for genotypes 1–3 patients [8–10].
transcriptase (HIV-RT) [25–27]. The therapeutic potential
of the thiazolidinone scaffold against HCV NS5B was
first reported employing 20,40-difluoro-4-hydroxybiphenyl-3-
carboxylic acid[2-(5-nitro-2-furyl/substituted phenyl)-4-thia-
zolidinone-3-yl]amides [21, 28]. Subsequently, 2,3-diarylthia-
zolidinones were reported to exhibit significant differences
in activity against HCV RdRp and HIV-RT [27, 29–31].
Existing literature reports also presents similar chemotypes
carrying thiazolone [1, 16, 32], 2-thioxo-1,3-thiazolidinone
(rhodanine) [33, 34], and iminothiazolidinone (prominently
BMS 824) [35] scaffolds as inhibitors of HCV NS5B. Recently,
we reported 2-[[5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl]-
imino]-5-(2,6-dichlorobenzylidene)-1,3-thiazolidin-4-one and
2-[[5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl]imino]-5-(2,6-di-
chlorobenzylidene)-1,3-thiazolidin-4-one as HCV NS5B inhib-
itor leads with IC50 value of 5.6 and 19.8 mM, respectively [36].
In continuation of our efforts to develop anti-HCV NS5B
inhibitors, we synthesized new derivatives of thiazolidinones
bearing modifications at the aryl moiety of 5-aryl-1,3,4-
thiadiazole and replacement of the arylidene core with 2,6-
dichloro or 3-fluoro substituted benzylidene groups (Fig. 2).
The synthesized compounds were evaluated for their in vitro
NS5B RdRp inhibitory potential followed by cellular antiviral
toxicity profiling. Further binding interactions of the most
active compound with NS5B were elucidated through docking
study.
Another DAA approach against HCV has focused on
targeting its NS5B RNA-dependent RNA polymerase (RdRp),
which catalyzes HCV RNA replication and has no functional
equivalent in the host [11, 12]. NS5B, comprising 531 amino
acid residues, is folded into characteristic fingers, palm,
and thumb subdomains of the polymerase protein family.
Extensive crystallographic studies has facilitated the identifi-
cation of the binding sites for nucleoside triphosphate
substrate and validation of the five allosteric pockets on
NS5B. Thumb pocket-I (TP-I) and TP-II are located in the thumb
subdomain, palm pocket-III (PP-III) and PP-IV are located
in the palm subdomain and PP-V is located in the finger
subdomain [13–15].
Small molecule inhibitors binding to allosteric pockets of
NS5B are considered to have the advantage of being more
selective in addition to demonstrating fewer off-target
side effects compared to nucleoside or nucleotide analogs
as there are no homologous cellular enzymes, which bind
to these allosteric inhibitors [16, 17]. Non-nucleoside
allosteric inhibitors of HCV RdRp have been reported to
include diverse chemical scaffolds as benzo-1,2,4-thiadiazines,
1,5-benzodiazepines, benzo[de]-isoquinolines, pyrazolo[1,5-
a]pyrimidines, acrylic acid, anthranilic acid, indole-2-carbox-
ylic acid, rhodanine, amino-substituted isothiazole, benzo-
furan, 2-oxy-6-fluorobenzamide, piperazine-2-carboxamide,
indole, and phenylalanine [18]. Clinically validated drugs
(Fig. 1) such as telaprevir (VX-222), ABT-072, tegobuvir (GS-
9190), filibuvir (PF 868554), setrobuvir (ANA598), and VCH-916
hold promise as non-nucleoside NS5B polymerase inhibitors
of HCV [19].
Results and discussion
Chemistry
The target compounds were synthesized using a stepwise
reaction protocol (Scheme 1). Thiosemicarbazones 1–7
were obtained by reaction of thiosemicarbazide and appro-
priate aldehyde. 2-Amino-1,3,4-thiadiazole derivatives 8–14
were obtained by oxidative cyclization of thiosemicarbazones
1–7 in the presence of ferric chloride. A suggested mechanism
for this reaction is depicted in Scheme 2. According to the
proposed cyclization mechanism, reaction is initiated by
deprotonation, forming a radical at thiosemicarbazone N2.
After resonance of this radical, the thiol radical attacks toward
4-Thiazolidinones have been reported to possess a wide
range of biological activities including antiproliferative,
antibacterial, antifungal, and antimycobacterial functions
[20–24]. These compounds are also known to target cyclo-
oxygenase (COX) enzymes thus making thiazolidinones
excellent candidates to develop as anti-tumor and anti-
inflammatory agents [23, 24]. Further, thiazolidinone com-
pounds have been shown to inhibit the HIV-1 reverse
Figure 1. Small molecule inhibitors of HCV NS5B on clinical trials.
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Figure 2. Structures of existing 4-thiazolidinones, 2-thioxo-4-thiazolidinones, 4-thiazolones, and the newly designed compounds
22–42.
Scheme 1. Synthetic route to compounds 1–42. Reagents and conditions: (a) Ethanol, acetic acid, reflux; (b) FeCl3, ethanol, reflux;
(c) ClCH2COCl, TEA, DCM, rt; (d) NH4SCN, ethanol/dioxane, reflux; (e) R2-CHO, CH3ONa, methanol, reflux.
azomethine carbon to form the ring and expulsion of another
hydrogen radical finally yields 5-aryl-1,3,4-thiadiazole-2-
amines 8–14 [37]. Syntheses of 2-chloro-N-[5-(aryl)-1,3,4-
thiadiazol-2-yl]acetamides 15–21 were carried out by the
reaction of the appropriate amines with chloroacetylchloride
in the presence of TEA. Cyclization of chloroacetamides 15–21
in the presence of ethanolic solution of ammonium thio-
cyanate in dioxane afforded 2-{[5-(substituted phenyl)-
1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-ones 22–28. In
earlier studies, 1,3-thiazolidin-4-ones were obtained by the
reaction of chloroacetamides with ammonium thiocyanate
in ethanol [24, 38–41] or acetone [42], or DMF (under MW
irradiation) [43].
The target compounds 29–42 were obtained by the reaction
of 4-thiazolidinones 22–28 with commercially available
aldehydes in the presence of sodium methoxide. This method
was different from earlier studies in which 4-thiazolidinones
were reacted with aldehydes in acetic acid medium buffered
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Scheme 2. The mechanism of 1,3,4-thiadiazole ring closure via oxidative cyclization of thiosemicarbazones 1–7.
with sodium acetate or in ethanol and piperidine. However,
use of sodium methoxide in methanolic medium instead of
sodium acetate in acetic acid provided shorter reaction
times [44].
32, 34–36, and 42 exchanged with deuterium in DMSO-d6.
–
Compounds 29–42 exhibited Ar–CH C resonances at 7.75–
–
7.78 ppm separately within the aromatic peaks range of 7.33–
7.81 ppm. It is known that the 5-arylidene-1,3-thiazolidinones
may exist as E/Z geometrical isomers due to the rotational
All synthesized compounds were checked for purity using
TLC, HPLC-UV/DAD, and elemental analyses. The new com-
pounds 12, 16, 17, 19, 21–24, 26, 28, and 29–42 were
characterized by their melting points and spectral data (IR,
1H NMR, and MS) and 13C NMR spectra were recorded for
compounds 22–24, 26–28, 31, 37, and 41. Absorption bands at
–
restriction about C C double bond. In Z isomer, the methine
–
proton resonates at higher chemical shift values due to the
–
deshielding effect of the adjacent C O group while it
–
resonates at lower chemical shifts due to comparatively less
deshielding effect of the sulfur in E isomer [22, 38, 47, 49]. To
this respect, we decided that 5-arylidene-1,3-thiazolidin-4-
ones 29–42 were the Z isomers.
1715 cmꢁ1 were attributed to the C O stretching band of
–
–
2-chloro-N-[5-(2-chloro-6-fluorophenyl)-1,3,4-thiadiazol-2-yl]-
acetamide 19, which was not seen in 5-(2-chloro-6-fluoro-
phenyl)-1,3,4-thiadiazole-2-amine 12 IR spectrum. In the NMR
spectrum of 12 N–H protons were determined at 7.38–
7.64 ppm with aromatic protons. However, the N–H proton
of 19 was described at 13.22 ppm. After the cyclization of this
compound 19, we obtained 2-{[5-(2-chloro-6-fluorophenyl)-
1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one 26 for which
N–H proton was determined at 12.69 ppm and CH2 protons
were determined at 4.40 ppm.
While the carbonyl carbon of the thiazolidinone ring
appeared at 174 ppm, C2 of thiazolidinone ring for com-
pounds 29–42 appeared with aromatic carbons. Methylidene
carbon of arylidene moiety showed resonance around 120–
130 ppm in accordance with the literature [36, 44, 50].
High-resolution electrospray ionization (ESI) mass spectra
(HRMS) of compounds 32 and 39 confirmed their molecular
weights and empirical formulae, with less than 5 mmu bias
between calculated and experimental m/z values of molecular
ions. Low-resolution ESI mass spectra of compounds 22–39
and 41 were recorded in either positive or negative ionization
mode. The LC–MS/MS (ESI) analysis of the synthesized
compounds gave correct molecular ion peaks corresponding
to (MþH)þ in positive ionization and (MꢁH)ꢁ in negative
ionization mode in each case.
Absorption bands at 1738–1709 cmꢁ1 were attributed to the
–
C O stretching bands of 1,3-thiazolidine-4-one compounds
–
22–28 and provided confirmatory evidence for ring clo-
sure [21, 44]. Another support for thiazolidinone ring closure
was the detection of signals at 4.15–4.40 ppm, due to the
presence of endocyclic –S–CH2 protons [45, 46]. NH protons of
compounds 22–28 were determined at 12.43–12.69 ppm
accounting for a lactam proton but not for an imine proton,
which is expected to exhibit lower chemical shift values [40,
47]. 13C NMR data of the thiazolidinones 22–28 have also
supported the carbon framework [21, 36, 48]. The carbonyl
carbon of the thiazolidinone ring appeared at about 174 ppm,
the signal of the thiazolidinone C5 appeared at 36 ppm and
the signal at about 167–171 ppm was attributed to C2 of
thiazolidinone ring. Disappearance of –S–CH2 signals in the
1H NMR spectra of 5-arylidene-1,3-thiazolidinone-4-ones 29–
42 indicates that active methylene group of 4-thiazolidinones
reacted with selected aldehydes. N–H protons of compounds
were determined at 13.16–13.23 ppm, but for compounds 31,
Biological activity
Inhibition of NS5B RdRp activity by compounds 22–42
The anti-NS5B RdRp activity of the target compounds 22–42
(Table 1) was determined against recombinant NS5BCD21 1b
in a primer-dependent elongation assay as described previ-
ously [28, 51]. Preliminary screening to identify candidate
NS5B inhibitors was first conducted at 50 mM compound
concentration and those exhibiting ꢂ50% inhibition of NS5B
RdRp activity at this concentration were further investigated
for IC50 value evaluation. This investigation led to the
identification of 12 compounds satisfying this criterion, while
nine compounds exhibited no inhibition or ꢃ50% inhibition
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Table 1. Anti-HCV effects in cell-based assays, anti-NS5B RdRp activity, and calculated lipophilicity values of compounds
22–42.
Cell
Huh7/
NS5B RdRp
Inhibition
(%)c)
viability
(%)a)
Rep-Feolb
inhibition (%)b)
IC50
Compound
Lab. ID code
R1
R2
(mM)
log Pd)
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
KUC110142
KUC110132
KUC110136
KUC110124
KUC110128
KUC110116
KUC110120
KUC110144
KUC110134
KUC110138
KUC110126
KUC110130
KUC110118
KUC110122
KUC110143
KUC110133
KUC110137
KUC110125
KUC110129
KUC110117
KUC110121
3-Pyridyl
–
99 ꢄ 8
90 ꢄ 5
71 ꢄ 1
71 ꢄ 8
68 ꢄ 2
85 ꢄ 6
68 ꢄ 2
45 ꢄ 4
32 ꢄ 2
28 ꢄ 1
36 ꢄ 3
28 ꢄ 2
ND
NI
6 ꢄ 5
NI
ND
0.81
1.66
1.70
2.29
2.45
2.78
2.78
3.09
3.81
3.85
4.24
4.33
4.66
4.76
4.01
4.82
4.74
5.07
5.35
5.59
5.70
2-Fluorophenyl
3-Fluorophenyl
2-Chlorophenyl
2-Chloro-6-fluorophenyl
2,4-Dichlorophenyl
2,6-Dichlorophenyl
3-Pyridyl
–
NI
ND
–
25 ꢄ 2
NI
NI
ND
–
9 ꢄ 1
NI
ND
–
34 ꢄ 3
NI
ND
–
NI
ND
–
40 ꢄ 4
69 ꢄ 4
74 ꢄ 2
79 ꢄ 3
89 ꢄ 3
97 ꢄ 2
ND
31 ꢄ 2
71 ꢄ 2
79 ꢄ 5
86 ꢄ 1
55 ꢄ 5
83 ꢄ 1
ND
ND
3-Fluorophenyl
3-Fluorophenyl
3-Fluorophenyl
3-Fluorophenyl
3-Fluorophenyl
3-Fluorophenyl
3-Fluorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
47.3 ꢄ 2.4
44.6 ꢄ 3.9
34.8 ꢄ 2.8
ND
2-Fluorophenyl
3-Fluorophenyl
2-Chlorophenyl
2-Chloro-6-fluorophenyl
2,4-Dichlorophenyl
2,6-Dichlorophenyl
3-Pyridyl
25.3 ꢄ 1.7
ND
30 ꢄ 3
29 ꢄ 3
26 ꢄ 1
30 ꢄ 10
41 ꢄ 2
33 ꢄ 1
23 ꢄ 2
26 ꢄ 1
97 ꢄ 2
92 ꢄ 1
97 ꢄ 1
77 ꢄ 6
88 ꢄ 4
84 ꢄ 5
58 ꢄ 4
88 ꢄ 4
84 ꢄ 3
74 ꢄ 9
80 ꢄ 1
80 ꢄ 2
88 ꢄ 2
90 ꢄ 1
91 ꢄ 1
90 ꢄ 1
37.2 ꢄ 10.2
49.5 ꢄ 2.9
54.1 ꢄ 10.6
50.5 ꢄ 2.3
40.2 ꢄ 4.0
38.5 ꢄ 4.2
42.5 ꢄ 1.9
41.5 ꢄ 1.7
2-Fluorophenyl
3-Fluorophenyl
2-Chlorophenyl
2-Chloro-6-fluorophenyl
2,4-Dichlorophenyl
2,6-Dichlorophenyl
a)Huh7 and b)Huh7/Rep-Feo1b cells were treated with the indicated compounds for 48 h for cell viability (100 mM) and anti-HCV
activity (50 mM) evaluations. Cells treated with equal amounts of DMSO served as control. The data represents the average ꢄ SD
of three independent experiments in duplicate.
c)NS5B RdRp inhibition was determined in vitro at 50 mM concentration of the indicated compound. The IC50 values were
determined from dose–response curves employing 8–12 concentrations of the compound in duplicate in two independent
experiments. Curves were fitted to data points using nonlinear regression analysis and IC50 values interpolated employing
Calcusyn software. NI, no inhibition; ND, not determined. Compound 34 was not soluble at 5 mM concentration and not
included in the screening.
d)log P values were calculated using ALOGPS 2.102 log P/log S calculation software (http://www.vcclab.org).
of NS5B RdRp activity at this concentration. Of these,
compounds 22–28 bearing 1,3-thiazolidine-4-one moiety
displayed 6–31% inhibition whereas their corresponding
arylidene derivatives 29–42 exhibited 71–91% NS5B RdRp
inhibitory activity. Compounds 29–42 were further screened
for their NS5B inhibition potency and yielded IC50 values
between 25.3 and 54.1 mM. Among these, compound 33 with
IC50 value of 25.3 mM was the most active of the arylidene
derivatives. Together this data suggests that arylidene moiety
attached to the 1,3-thiazolidine-4-one ring may be essential
for anti-HCV NS5B activity.
thiazolidine-4-one moiety bearing compounds 22–28 exhibit-
ing ꢂ68% cellular viability at 100 mM concentration in
contrast to ꢃ45% cellular viability observed with the
corresponding arylidene derivatives 29–42 (Table 1). This
data suggests that toxicity of 1,3-thiazolidine-4-one com-
pounds 29–42 increased by integration of arylidene core.
Furthermore, there appeared to be an inverse correlation
between the effect of the compounds on cellular viability and
their antiviral efficacy. Thus, compounds 22, 23, and 25 with
ꢂ85% cellular viability exhibited no effect on HCV RNA
replication at 50 mM concentration. The remaining four 1,3-
thiazolidine-4-one bearing compounds displayed ꢂ68%
cellular viability but ꢃ40% inhibition of HCV RNA replication
at the aforementioned concentrations. By contrast, com-
pounds 29–42 carrying the arylidene moiety on the 1,3-
thiazolidine-4-one scaffold exhibited ꢂ70% inhibition of HCV
RNA replication but displayed ꢃ45% cellular viability. It may
be noted though that this inhibition may partly be attributed
to the toxicity of the compounds in cell-based assay.
High throughput cell-based anti-HCV screening of
compounds
The parental Huh7 cells were employed for screening the
effect of the compounds on cell viability while the Huh7/Rep-
Feo1b replicon cells served as reporters for the anti-HCV
activity of the compounds in cell-based assay [52, 53]. This
investigation yielded
a clear cut trend, with the 1,3-
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With the objective of gaining some insight into the chemical
properties of this group of compounds, we derived their log P
values (Table 1) using ALOGPS 2.102 log P/log S calculation
software (http://www.vcclab.org). The derived log P values
were least for compound 22 and highest for compound 42.
It was apparent from this data that compounds with log P
values between 3.5 and 4.5 exhibited better inhibition of
NS5B RdRp activity, whereas a decrease in hydrophobicity
of the compounds to log P values below 2.8 negatively
impacted both their ability to inhibit in vitro NS5B polymerase
activity as well as the anti-HCV activity in cell-based assay.
This observation may assist in efforts to design better 1,3-
thiazolidine-4-one based inhibitors.
Ile482, and Leu497. The backbone “NH” of the dipeptide
fragment formed by Ser476 and Tyr477 may interact
electrostatically with the thiazolidinone ring and the
bridging imine nitrogen. The protonated amine nitrogen
in the side-chain of Lys533 forms a cation–pi interaction
with the thiadiazole ring. Moreover, the N-3 atom of the
thiadiazole ring interacts with the guanidine side-chain of
Arg422 through electrostatic bonding. The 2-chloro group in
the 2-chloro-6-fluorophenyl ring forms electrostatic inter-
actions with the polar side-chains of Asn527 and Arg422.
These docking studies will facilitate further lead optimiza-
tion efforts.
Molecular modeling studies
Docking model of the most potent compound 33 at thumb
Conclusions
pocket-II of NS5B
As a part of our continuing efforts to identify potent
inhibitors of HCV NS5B polymerase, a novel series of 5-
arylidene-4-thiazolidinones were synthesized and evaluated
for their NS5B RdRp inhibitory activity. Compounds 22–28
displayed 6–31% inhibition whereas their corresponding
arylidene derivatives 29–42 exhibited 71–91% NS5B RdRp
inhibitory activity. Compounds 29–42 were further screened
for their NS5B inhibition potency and yielded IC50 values
between 25.3 and 54.1 mM. By performing a high throughput
cell-based anti-HCV screening for compounds 22–42; com-
pounds 22–28 were observed to exhibit ꢂ68% cellular
viability at 100 mM concentration in contrast to compounds
29–42 with ꢃ45% cellular viability. Docking model of
compound 33 with TP-II of NS5B will assist future lead
optimization efforts. Outcomes of all these efforts will be
The docking studies for the compounds reported here were
performed on thumb pocket-II (TP-II) of NS5B using X-ray co-
crystal structure of HCV NS5B-PF868554 (PDB ID: 3FRZ) [54]
following a similar protocol as mentioned in our previous
report [36]. Extra precision (XP) docking results show a
correlation between the glide gscore representing the
binding energy to the observed IC50 values viz. 33 (ꢁ5.901
kcal/mol) < 31 (ꢁ5.626 kcal/mol) < 35 (ꢁ4.537 kcal/mol).
A
ribbon representation of the docked complex of compound
33 with that of allosteric site on the thumb domain is
displayed in Fig. 3. The guanidine-side chain of Arg501 forms
a cation–pi interaction with the 3-fluorophenyl ring. In
addition, the 3-flurophenyl ring is in vicinity to the side-
chains of non-polar residues Leu419, Met423, Tyr477,
Figure 3. XP docking model of compound 33 at thumb pocket-II of NS5B. (A) NS5B protein is shown in ribbon (pale green)
presentation. Selected amino acids are depicted as sticks with the atoms colored as carbon – wheat, hydrogen – white, nitrogen –
blue, oxygen – red, sulfur – yellow) whereas the inhibitor is shown as ball and stick model with the same color scheme as above except
carbon atoms are represented in orange. The image was generated using pymol v1.6. (B) A 2D representation of the docked model in
˚
panel A with the residues within 5 A of compound 33 of NS5B is shown. The amino acids are shown as colored bubbles; purple
indicates charged, cyan indicates polar, and green indicates hydrophobic residues. Red line shows cation–pi interaction.
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4-Thiazolidinones as Inhibitors of HCV NS5B Polymerase
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taken into consideration for designing new analogs with
lower cellular toxicity.
General procedure for the synthesis of 2-
(arylmethylidene)hydrazinecarbothioamides (1–7)
The ethanolic solution of thiosemicarbazide (30 mmol) was
heated under reflux with various aromatic aldehydes (30 mmol)
in the presence of a few drops of acetic acid. The crude products
1–7 were filtered and cyrstallized from appropriate solvent.
Experimental
Chemistry
All solvents and reagents were obtained from commercial
sources and used without further purification. The purity of
the compounds was confirmed by the thin-layer chromatog-
raphy (TLC) performed on Merck silica gel 60 F254 aluminum
sheets (Merck, Darmstadt, Germany), using developing
systems: S1: chloroform/methanol/acetic acid (93:5:2 v/v/v)
and S2: acetone/DMF/ethanol (10:5:1 v/v/v). Spots were
detected under UV light at l ¼ 254 and 366 nm.
2-(Pyridin-3-ylmethylidene)hydrazinecarbothioamide (1)
Yield 46%. m.p. 218–219˚C (MeOH–CH3COOH) (lit. 216˚C [57]).
TLC Rf: 0.70 (S1). HPLC tR (min): 1.28. IR, n (cmꢁ1): 3391, 3263
–
–
–
(N–H str), 1627–1591 (C N str), 1360–1264 (C S str).
–
2-(2-Fluorobenzylidene)hydrazinecarbothioamide (2)
Yield 97.4%. m.p. 184–185˚C (EtOH) (lit. 183–184˚C [58]). TLC
Rf: 0.70 (S1). HPLC tR (min): 7.6. IR, n (cmꢁ1): 3432, 3370 (N–H
–
str), 1593 (C N str), 1244 (C S str).
–
–
All melting points were determined using Kleinfeld SMP-II
basic model point apparatus and are uncorrected. Elemental
analyses were obtained using Leco CHNS-932 and are
consistent with the assigned structures. High-resolution
electron impact mass spectra of compounds 32 and 39 were
recorded on Jeol JMS-700 High Resolution instrument. ESI
positive and negative ionization (low resolution) mass spectra
of the synthesized compounds were obtained using AB SCIEX
API 2000 LC–MS/MS instrument.
–
2-(3-Fluorobenzylidene)hydrazinecarbothioamide (3)
Yield 84.6%. m.p. 190–192˚C (EtOH) (lit. 190˚C [59]). TLC Rf:
0.80 (S1). HPLC tR (min): 5.22. IR, n (cmꢁ1): 3391, 3235 (N–H str),
–
–
1576 (C N str), 1361–1294 (C S str).
–
–
2-(2-Chlorobenzylidene)hydrazinecarbothioamide (4)
Yield 78.2%. m.p. 220˚C (EtOH) (lit. 220–221˚C [60]). TLC Rf:
0.68 (S1). HPLC tR (min): 8.22. IR, n (cmꢁ1): 3414, 3245 (N–H str),
Infrared spectra were recorded on a Shimadzu FT-IR
Affinity-1 and data are expressed in wavenumber (cmꢁ1).
NMR spectra were recorded on Bruker AVANCE DPX 300–
600 MHz for 1H NMR and 13C NMR. The chemical shifts were
expressed in ppm downfield from tetramethylsilane (TMS)
using DMSO-d6 as solvent. The high-pressure liquid chro-
matographic system consists of an Agilent 1100 series
instrument equipped with quaternary solvent delivery
system and a model Agilent series G1315 B photodiode
array detector. A Rheodyne syringe loading sample injector
with 50 mL sample loop was used for the injection of the
analytes. Chromatographic data were collected and proc-
essed using Agilent ChemStation plus software. The
separation was performed at ambient temperature by
using reversed phase ACE C18 (4 mm ꢅ 100 mm ꢅ 5 mm)
column. All experiments were performed in gradient mode.
The mobile phase was prepared by mixing acetonitrile and
TEA-pH 4.50 phosphate buffer (80:20 v/v during 0–2 min,
70:30 v/v during 2–4 min, 60:40 v/v during 4–6 min, 50:50 v/v
during 6–8 min, 25:75 v/v during 8–10 min, 0:100 v/v during
10–18 min, 40:60 v/v during 18–20 min, 80:20 v/v during 20–
22 min) and filtered through a 0.45 mm pore filter and
subsequently degassed by ultrasonication, prior to use.
Solvent delivery was employed at a flow rate of 0.8 mL/min.
Detection of the analytes was carried out at 210, 230, 254,
and 280 nm.
–
–
–
1591 (C N str), 1374–1278 (C S str).
–
2-(2-Chloro-6-fluorobenzylidene)hydrazine-
carbothioamide (5)
Yield 57.7%. m.p. 231˚C (EtOH) (lit. 228˚C [61]). TLC Rf: 0.66
(S1). HPLC tR (min): 8.53. IR, n (cmꢁ1): 3406, 3229 (N–H str), 1595
–
–
–
–
(C N str), 1285 (C S str).
2-(2,4-Dichlorobenzylidene)hydrazinecarbothioamide (6)
Yield 59.3%. m.p. 240˚C (EtOH) (lit. 238˚C [59]). TLC Rf: 0.62
(S1). HPLC tR (min): 10.22. IR, n (cmꢁ1): 3444, 3323 (N–H str),
–
–
–
1587 (C N str), 1386–1275 (C S str).
–
2-(2,6-Dichlorobenzylidene)hydrazinecarbothioamide (7)
Yield 27%. m.p. 238˚C (EtOH) (lit. 236–237˚C [62]). TLC Rf: 0.68
(S1). HPLC tR (min): 8.00. IR, n (cmꢁ1): 3406, 3235 (N–H str), 1590
–
–
–
–
(C N str), 1290 (C S str).
General procedure for the synthesis of 5-(aryl)-1,3,4-
thiadiazol-2-amines (8–14)
Compounds 1–7 (1 mmol) were dissolved in ethanol and
ethanolic ferric chloride solution (4 mmol) was added. The
reaction mixtures were heated under reflux for 16–20 h.
The mixtures were neutralized using ammonia solution,
filtered, and washed with water, dried, and cyrstallized from
appropriate solvent to obtain products 8–14.
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 ALOGPS 2.102 log P/log S calculation software [55, 56].
The calculated log P values for the compounds are given in
Table 1.
5-(Pyridin-3-yl)-1,3,4-thiadiazol-2-amine (8)
Yield 88%. m.p. 236–239˚C (EtOH) (lit. 236–238˚C [63]). TLC Rf:
0.71 (S1). HPLC tR (min): 1.18. IR, n (cmꢁ1): 3259, 3222 (N–H str),
–
1643 (N–H bending), 1575 (C N str).
–
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Archiv der Pharmazie
5-(2-Fluorophenyl)-1,3,4-thiadiazol-2-amine (9)
2-Chloro-N-[5-(3-fluorophenyl)-1,3,4-thiadiazol-2-yl]-
Yield 51%. m.p. 226–227˚C (EtOH) (lit. 223–225˚C [64]). TLC Rf:
0.59 (S1). HPLC tR (min): 6.29. IR, n (cmꢁ1): 3231 (N–H str), 1634–
acetamide (17)
Yield 64%. m.p. 239˚C (MeOH). TLC Rf: 0.69 (S1). HPLC tR (min):
7.78. IR, n (cmꢁ1): 3180 (N–H str), 1705 (amide C O), 1565 (C N
–
–
–
–
–
1613 (N–H bending), 1581 (C N str).
–
str). 1H NMR: d (ppm) 4.50 (s, 2H, CH2), 7.31–7.43 (m, 1H, ArH),
7.56–7.63 (m, 1H, ArH), 7.75–7.83 (d, 2H, J ¼ 7.5 Hz, ArH), 13.13
(s, 1H, NH). Anal. calcd. for C10H7ClFN3OS (271.70): C, 44.21; H,
2.60; N, 15.47; S, 11.80%. Found C, 43.96; H, 2.59; N, 15.31; S,
11.75%.
5-(3-Fluorophenyl)-1,3,4-thiadiazol-2-amine (10)
Yield 86%. m.p. 229–230˚C (EtOH) (lit. 234–236˚C [64]). TLC Rf:
0.55 (S1). HPLC tR (min): 4.43. IR, n (cmꢁ1): 3256 (N–H str), 1634–
–
1612 (N–H bending), 1587 (C N str).
–
5-(2-Chlorophenyl)-1,3,4-thiadiazol-2-amine (11)
Yield 54%. m.p. 190–192˚C (EtOH/DMF) (lit. 192–195˚C [65]).
TLC Rf: 0.56 (S1). HPLC tR (min): 6.98. IR, n (cmꢁ1): 3273 (N–H
2-Chloro-N-[5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl]-
acetamide (18)
Yield 97.2%. m.p. 215–217˚C (MeOH) (lit. 210–211˚C [39]). TLC
Rf: 0.73 (S1). HPLC tR (min): 9.71. IR, n (cmꢁ1): 3192 (N–H str),
–
str), 1638 (N–H bending), 1566 (C N str).
–
–
–
1709 (amide C O), 1575 (C N str).
–
–
5-(2-Chloro-6-fluorophenyl)-1,3,4-thiadiazol-2-amine (12)
Yield 72%. m.p. 182–184˚C (ACN). TLC Rf: 0.53 (S1). HPLC tR
2-Chloro-N-[5-(2-chloro-6-fluorophenyl)-1,3,4-thiadiazol-
2-yl]acetamide (19)
(min): 6.68. IR, n (cmꢁ1): 3378, 3262 (N–H str), 1600 (N–H
1
–
bending), 1568 (C N str). H NMR: d (ppm) 7.38–7.64 (m, 5H,
Yield 82%. m.p. 231–233˚C (MeOH). TLC Rf: 0.66 (S1). HPLC tR
–
(min): 9.77. IR, n (cmꢁ1): 3180 (N–H str), 1715 (amide C O),
–
ArH, NH2). Anal. calcd. for C8H5ClFN3S (229.66): C, 41.84; H,
2.19; N, 18.30; S, 13.96%. Found C, 41.59; H, 2.48; N, 17.93; S,
13.94%.
–
1
–
1570 (C N str). H NMR: d (ppm) 4.51 (s, 2H, CH ), 7.44–7.71 (m,
–
2
3H, ArH), 13.22 (s, 1H, NH). Anal. calcd. for C10H6Cl2FN3OS
(306.14): C, 39.23; H, 1.98; N, 13.73; S, 10.47%. Found C, 39.48;
H, 2.28; N, 13.66; S, 10.38%.
5-(2,4-Dichlorophenyl)-1,3,4-thiadiazol-2-amine (13)
Yield 70.7%. m.p. 229˚C (EtOH/DMF) (lit. 240–242˚C [39]). TLC
Rf: 0.48 (S1). HPLC tR (min): 9.28. IR, n (cmꢁ1): 3288, 3279 (N–H
2-Chloro-N-[5-(2,4-dichlorophenyl)-1,3,4-thiadiazol-2-yl]-
acetamide (20)
–
str), 1613 (N–H bending), 1587 (C N str).
–
Yield 63.6%. m.p. 248–250˚C (MeOH) (lit. 246–247˚C [39]). TLC
Rf: 0.70 (S1). HPLC tR (min): 10.41. IR, n (cmꢁ1): 3174 (N–H str),
5-(2,6-Dichlorophenyl)-1,3,4-thiadiazol-2-amine (14)
Yield 72.2%. m.p. 254–255˚C (EtOH/DMF) (lit. 257–259˚C [66]).
TLC Rf: 0.48 (S1). HPLC tR (min): 8.42. IR, n (cmꢁ1): 3288, 3279
–
–
–
1708 (amide C O), 1581 (C N str).
–
–
(N–H str), 1613 (N–H bending), 1558 (C N str).
–
2-Chloro-N-[5-(2,6-dichlorophenyl)-1,3,4-thiadiazol-2-yl]-
acetamide (21)
General procedure for the synthesis of 2-chloro-N-[5-(aryl)-
1,3,4-thiadiazol-2-yl]acetamides (15–21)
Yield 40.3%. m.p. 218–220˚C (EtOH). TLC Rf: 0.74 (S1). HPLC tR
(min): 8.09. IR, n (cmꢁ1): 3175 (N–H str), 1722 (amide C O),
–
–
1
–
–
Compounds 8–14 (5 mmol) were dissolved in DCM and TEA
(6 mmol) was added to the reaction mixtures. Chloroacetyl
chloride (10 mmol) was slowly added to the reaction mixtures.
The reaction mixtures were heated for 2 h under reflux. The
crude products were filtered, dried, and cyrstallized from
appropriate solvent to obtain products 15–21.
1578 (C N str). H NMR: d (ppm) 4.52 (s, 2H, CH ), 7.60–7.73 (m,
2
3H, ArH), 13.20 (s, 1H, NH). Anal. calcd. for C10H6Cl3N3OS
(322.60): C, 37.23; H, 1.87; N, 13.03; S, 9.94%. Found C, 37.56;
H, 2.02; N, 12.95; S, 9.83%.
General procedure for the synthesis of 2-{[5-(aryl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-ones (22–28)
Compounds 15–21 (5 mmol) were dissolved in 1,4-dioxane.
Ten millimoles of ammonium thiocyanate was dissolved in
ethanol and added to the reaction mixtures and heated for
6 h under reflux. The solvent was evaporated under vacuum.
The crude products were filtered, dried, and crystallized from
appropriate solvent.
2-Chloro-N-[5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl]-
acetamide (15)
Yield 49%. m.p. 270–272˚C (MeOH) (lit. 282–283˚C [67]). TLC
Rf: 0.83 (S1). HPLC tR (min): 8.50. IR, n (cmꢁ1): 3102 (N–H str),
–
–
1704 (amide C O), 1591 (C N str).
–
–
2-Chloro-N-[5-(2-fluorophenyl)-1,3,4-thiadiazol-2-yl]-
acetamide (16)
2-{[5-(Pyridin-3-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-
thiazolidin-4-one (22)
Yield 37%. m.p. 250–254˚C (EtOH). TLC Rf: 0.76 (S1). HPLC tR
Yield 83%. m.p. 271˚C (MeOH). TLC Rf: 0.72. HPLC tR (min): 9.80
(S1). IR, n (cmꢁ1): 3190 (N–H str), 1705 (amide C O), 1583 (C N
–
–
–
–
str). 1H NMR: d (ppm) 4.50 (s, 2H, CH2), 7.36–7.82 (m, 3H, ArH),
(min): 9.90. IR, n (cmꢁ1): 3117 (N–H str), 1730 (C O str), 1553
–
–
1
–
8.23–8.28 (m, 1H, ArH), 13.13 (s, 1H, NH). Anal. calcd. for
(C N str). H NMR: d (ppm) 4.35 (s, 2H, SCH ), 7.43–9.32 (m, 4H,
ArH), 12.66 (s, 1H, lactam NH). 13C NMR: d (ppm) 36.51
–
2
C10H7ClFN3OS (271.70): C, 44.21; H, 2.60; N, 15.47; S, 11.80.
Found C, 43.78; H, 2.59; N, 15.17; S, 11.65.
(thiazolidinone C5), 125.07, 127.06, 135.26, 148.27, 152.21
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(Ar–C), 167.50 (thiazolidinone C2), 174.85 (thiazolidinone C4).
LC–MS-(ESI): Calculated: Mmi: 277.0092, (MþH)þ: 278.0165,
(MꢁH)ꢁ: 276.0019. Found: (MþH)þ: 277.9468, (MꢁH)ꢁ:
275.9249.
calcd. for C11H6ClFN4OS2 (328.77): C, 40.19; H, 1.84; N, 17.04; S,
19.51%. Found C, 40.62; H, 2.15; N, 16.89; S, 19.56%. LC–MS-
(ESI): Calculated: Mmi: 327.9655, (MþH)þ: 328.9728, (MꢁH)ꢁ:
326.9582. Found: (MþH)þ: 328.8571, (MꢁH)ꢁ: 326.8817.
2-{[5-(2-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-
thiazolidin-4-one (23)
2-{[5-(2,4-Dichlorophenyl)-1,3,4-thiadiazol-2-yl]imino}-
1,3-thiazolidin-4-one (27)
Yield 69%. m.p. 269–271˚C (EtOH). TLC Rf: 0.81 (S1). HPLC tR
Yield 11%. m.p. 190˚C (EtOH) (lit. 186–188˚C [39]). TLC Rf: 0.86
(min): 8.55. IR, n (cmꢁ1): 3109 (N–H str), 1738 (C O str), 1558
(S1). HPLC tR (min): 9.29. IR, n (cmꢁ1): 3120 (N–H str), 1736–1722
–
–
1
1
–
–
–
–
–
–
(C N str). H NMR: d (ppm) 4.35 (s, 2H, SCH ), 7.61–7.99 (m, 3H,
(C O str), 1591 (C N str). H NMR: d (ppm) 4.40 (s, 2H, SCH ),
2
2
ArH), 8.42–8.47 (m, 1H, ArH), 12.63 (s, 1H, lactam NH). 13C
NMR: d (ppm) 36.46 (thiazolidinone C5), 117.07 and 117.36,
118.54 and 118.69, 126.11 and 126.15, 128.71, 133.56 and
133.68, 141.77 and 141.97, 156.92, 157.02 and 157.60, 160.925
(Ar–C), 167.16 (thiadiazole C2), 171.94 (thiazolidinone C2),
172.01 (thiadiazole C5), 174.79 (thiazolidinone C4). Anal.
calcd. for C11H7FN4OS2 (294.33): C, 44.89; H, 2.40; N, 19.04;
S, 21.79%. Found C, 45.44; H, 2.55; N, 18.73; S, 21.60%. LC–MS-
(ESI): Calculated: Mmi: 294.0045, (MþH)þ: 295.0118, (MꢁH)ꢁ:
292.9972. Found: (MþH)þ: 294.9925, (MꢁH)ꢁ: 292.9662.
7.20, 7.37, 7.54 (3s, 1H, sec amine NH), 7.88 (d, 1H, J ¼ 8.7 Hz,
ArH), 8.14 (s, 1H, ArH), 8.41 (d, 1H, J ¼ 8.4 Hz, ArH), 12.68 (s, 1H,
lactam NH). 13C NMR: d (ppm) 36.55 (thiazolidinone C5),
128.53, 129.97, 130.86, 132.41, 132.81, 136.66 (Ar–C), 159.30
(thiadiazole C2), 167.49 (thiazolidinone C2), 172.38 (thiadia-
zole C5), 174.83 (thiazolidinone C4). LC–MS-(ESI): Calculated:
M
mi: 343.9360, (MþH)þ: 344.9432, (MꢁH)ꢁ: 342.9287. Found:
(MþH)þ: 344.7782, (MꢁH)ꢁ: 342.8303.
2-{[5-(2,6-Dichlorophenyl)-1,3,4-thiadiazol-2-yl]imino}-
1,3-thiazolidin-4-one (28)
2-{[5-(3-Fluorophenyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-
thiazolidin-4-one (24)
Yield 80%. m.p. 229–230˚C (EtOH). TLC Rf: 0.91 (S1). HPLC tR
(min): 8.05. IR, n (cmꢁ1): 3174 (N–H str), 1724 (C O str), 1556
–
–
1
–
–
Yield 37%. m.p. 258–259˚C (EtOH). TLC Rf: 0.85 (S1). HPLC tR
(C N str). H NMR: d (ppm) 4.29 (s, 2H, SCH ), 7.28–7.85 (m, 3H,
2
(min): 7.53. IR, n (cmꢁ1): 3109 (N–H str), 1736 (C O str), 1557
ArH), 12.60 (s, 1H, lactam NH). 13C NMR: d (ppm) 36.47
(thiazolidinone C5), 128.68, 129.38, 133.79, 135.55 (Ar–C),
158.12 (thiadiazole C2), 167.67 (thiazolidinone C2), 173.06
–
–
1
–
(C N str). H NMR: d (ppm) 4.31 (s, 2H, SCH ), 7.55–7.95 (m, 4H,
–
2
ArH), 12.58 (s, 1H, lactam NH). 13C NMR: d (ppm) 36.44
(thiazolidinone C5), 124.11, 124.15, 132.19, 132.31, 132.77,
136.89 (Ar–C), 164.61 (thiadiazole C2), 167.23 (thiazolidinone
C2), 171.26 (thiadiazole C5), 174.78 (thiazolidinone C4). Anal.
calcd. for C11H7FN4OS2 (294.33): C, 44.89; H, 2.40; N, 19.04; S,
21.79%. Found C, 44.92; H, 2.68; N, 18.78; S, 21.61%. LC–MS-
(ESI): Calculated: Mmi: 294.0045, (MþH)þ: 295.0118, (MꢁH)ꢁ:
292.9972. Found: (MþH)þ: 295.0114, (MꢁH)ꢁ: 292.9281.
(thiadiazole C5), 174.77 (thiazolidinone C4). Anal. calcd. for
1
C
11H6Cl2N4OS2 ꢆ 2H2O (354.23): C, 37.30; H, 1.99; N, 15.82; S,
18.10%. Found C, 36.99; H, 2.41; N, 16.11; S, 17.62%. LC–MS-
(ESI): Calculated: Mmi: 343.9360, (MþH)þ: 344.9433, (MꢁH)ꢁ:
342.9287. Found: (MþH)þ: 344.7846, (MꢁH)ꢁ: 342.8371.
General procedure for the synthesis of 5-(substituted
benzylidene)-2-{[5-(aryl)-1,3,4-thiadiazol-2-yl]imino}-1,3-
thiazolidin-4-ones (29–42)
2-{[5-(2-Chlorophenyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-
thiazolidin-4-one (25)
The solution of compounds 22–28 (1.5 mmol) was dissolved in
sodium methoxyde (3 mmol) by heating. An excess amount of
appropriate aldehyde (1.8 mmol) was dissolved in methanol
and added to this mixture and refluxed for 8–10 h. The
reaction mixture was cooled at room temperature and 50 mL
ice-cold water was poured into it and this mixture was
neutralized with glacial acetic acid. The precipitated solid was
filtered, washed with water, and then recrystallized from
ethanol.
Yield 32%. m.p. 228–229˚C (EtOH–water) (lit. 222–224˚C [43]).
TLC Rf: 0.82 (S1). HPLC tR (min): 9.16. IR, n (cmꢁ1): 3179 (N–H
1
–
–
–
str), 1709 (C O str), 1553 (C N str). H NMR: d (ppm) 4.15 (s,
–
2H, SCH2), 7.52–8.16 (m, 4H, ArH), 12.43 (s, 1H, lactam NH).
Anal. calcd. for C11H7ClN4OS2 (310.78): C, 42.51; H, 2.27; N,
18.03; S, 20.64%. Found C, 42.73; H, 2.45; N, 17.96; S, 20.20%.
LC–MS-(ESI): Calculated: Mmi: 309.9750, (MþH)þ: 310.9826,
(MꢁH)ꢁ: 308.9677. Found: (MþH)þ: 310.9221, (MꢁH)ꢁ:
308.9561.
5-(3-Fluorobenzylidene)-2-{[5-(pyridin-3-yl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (29)
Yield 83%. m.p. 305–306˚C (EtOH). TLC Rf: 0.90 (S2). HPLC tR
2-{[5-(2-Chloro-6-fluorophenyl)-1,3,4-thiadiazol-2-yl]-
imino}-1,3-thiazolidin-4-one (26)
Yield 86%. m.p. 183–185˚C (EtOH). TLC Rf: 0.70 (S1). HPLC tR
(min): 9.33. IR, n (cmꢁ1): 3077 (N–H str), 1721 (C O str), 1599
–
–
1
(min): 8.45. IR, n (cmꢁ1): 3164 (N–H str), 1715 (C O str), 1580
(C N str). H NMR: d (ppm) 7.36–7.72 (m, 9H, ArH, CH), 13.16
–
–
–
–
–
–
1
–
(C N str). H NMR: d (ppm) 4.40 (s, 2H, SCH ), 7.70–7.96 (m, 3H,
(s, 1H, lactam NH). Anal. calcd. for
C
17H10FN5OS2 ꢆ H2O
–
2
ArH), 12.69 (s, 1H, lactam NH). 13C NMR: d (ppm) 36.57
(thiazolidinone C5), 126.92, 126.97, 134.13, 134.26, 134.58,
134.61 (Ar–C), 162.57 (thiadiazole C2), 167.77 (thiazolidinone
C2), 173.22 (thiadiazole C5), 174.88 (thiazolidinone C4). Anal.
(401.44): C, 50.86; H, 3.01; N, 17.45; S, 15.98%. Found C,
51.46; H, 3.00; N, 17.72; S, 15.93%. LC–MS-(ESI): Calculated:
M
mi: 383.0311, (MþH)þ: 384.0383, (MꢁH)ꢁ: 382.0238. Found:
(MþH)þ: 383.8690, (MꢁH)ꢁ: 381.9213.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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–
5-(3-Fluorobenzylidene)-2-{[5-(2-fluorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (30)
C–H), 7.88 (s, 1H, ArH), 8.18–8.21 (d, 1H, ArH, J ¼ 8.4 Hz), NH
–
proton exchanged with DMSO. Anal. calcd. for
1
Yield 70%. m.p. 277–279˚C (EtOH). TLC Rf: 0.94 (S2). HPLC tR
C
18H9Cl2FN4OS2 ꢆ 2H2O (460.33): C, 46.96; H, 2.19; N, 12.17;
(min): 11.48–11.73. IR, n (cmꢁ1): 3149 (N–H str), 1702 (C O str),
S, 13.93%. Found C, 47.44; H, 2.41; N, 11.99; S, 13.68%. LC–MS-
(ESI): Calculated: Mmi: 449.9578, (MþH)þ: 450.9652, (MꢁH)ꢁ:
448.9506. Found: (MþH)þ: 450.9312, (MꢁH)ꢁ: 448.7850.
–
–
1
–
–
1568 (C N str). H NMR: d (ppm) 7.33–7.75 (m, 7H, ArH), 7.78 (s,
–
1H, CH), 8.22–8.26 (s, 1H, ArH), 13.18 (s, 1H, lactam NH). Anal.
–
calcd. for C18H10F2N4OS2 (400.42): C, 53.99; H, 2.52; N, 13.99; S,
16.02%. Found C, 53.32; H, 2.56; N, 13.72; S, 15.73%. LC–MS-
(ESI): Calculated: Mmi: 400.0264, (MþH)þ: 401.0337, (MꢁH)ꢁ:
399.0191. Found: (MþH)þ: 400.8339, (MꢁH)ꢁ: 398.8876.
5-(3-Fluorobenzylidene)-2-{[5-(2,6-dichlorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (35)
Yield 81%. m.p. 266–267˚C (EtOH). TLC Rf: 0.96 (S2). HPLC tR
(min): 10.94. IR, n (cmꢁ1): 3142 (N–H str), 1729 (C O str), 1597
–
–
1
–
–
–
–
5-(3-Fluorobenzylidene)-2-{[5-(3-fluorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (31)
Yield 83%. m.p. 293˚C (EtOH). TLC Rf: 0.96 (S2). HPLC tR
(C N str). H NMR: d (ppm) 7.33–7.82 (m, 8H, ArH, C–H), NH
proton exchanged with DMSO. Anal. calcd. for C18H9Cl2FN4OS2
(451.32): C, 47.90; H, 2.01; N, 12.41; S, 14.21%. Found C, 47.94;
(min): 10.97. IR, n (cmꢁ1): 3116 (N–H str), 1717 (C O str), 1596
H, 2.35; N, 11.89; S, 13.26%. LC–MS-(ESI): Calculated: Mmi:
–
–
1
–
–
–
(C N str). H NMR: d (ppm) 7.36–7.76 (m, 9H, ArH, CH), NH
449.9578, (MþH)þ: 450.9652, (MꢁH)ꢁ: 448.9506. Found:
–
proton exchanged with DMSO.13C NMR: d (ppm) 114.10,
114.42, 117.60, 117.91, 118.58, 118.85, 124.23, 126.33,
132.18, 132.75, 136.09, 136.21, 159.50 (ArC, thiazolidinone
(MþH)þ: 450.6951, (MꢁH)ꢁ: 448.7866.
5-(2,6-Dichlorobenzylidene)-2-{[5-(pyridin-3-yl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (36)
–
C5, CH–Ar), 164.67 (thiazolidinone C , thiadiazole C ),
–
2
5
167.53 (thiazolidinone C4), 170.84 (thiadiazole C2). Anal.
calcd. for C18H10F2N4OS2 (400.42): C, 53.99; H, 2.52; N, 13.99;
S, 16.02%. Found C, 53.72; H, 2.84; N, 13.94; S, 15.99%. LC–
MS-(ESI): Calculated: Mmi: 400.0264, (MþH)þ: 401.0337,
(MꢁH)ꢁ: 399.0191. Found: (MþH)þ: 400.8158, (MꢁH)ꢁ:
398.8927.
Yield 79%. m.p. 292–294˚C (EtOH). TLC Rf: 0.90 (S2). HPLC tR
(min): 9.88. IR, n (cmꢁ1): 3066 (N–H str), 1717 (C O str), 1583
–
–
1
–
–
(C N str). H NMR: d (ppm) 7.52–7.64 (m, 7H, ArH), 7.75 (s, 1H,
–
CH), NH proton exchanged with DMSO. Anal. calcd. for
–
C17H9Cl2N5OS2 (434.32): C, 47.01; H, 2.09; N, 16.12; S, 14.77%.
Found C, 47.14; H, 2.35; N, 16.06; S, 14.73%. LC–MS-(ESI):
Calculated: Mmi
:
432.9625, (MþH)þ: 433.9698, (MꢁH)ꢁ:
5-(3-Fluorobenzylidene)-2-{[5-(2-chlorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (32)
431.9558. Found: (MþH)þ: 433.7563, (MꢁH)ꢁ: 431.8460.
Yield 22%. m.p. 310–312˚C (EtOH). TLC Rf: 0.58 (S2). HPLC tR
5-(2,6-Dichlorobenzylidene)-2-{[5-(2-fluorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (37)
(min): 11.25. IR, n (cmꢁ1): 3130 (N–H str), 1726 (C O str),
–
–
1
–
–
–
–
1597 (C N str). H NMR: d (ppm) 7.19–8.11 (m, 9H, ArH, CH),
Yield 40%. m.p. 260–262˚C (EtOH). TLC Rf: 0.90 (S2). HPLC tR
(min): 11.77, 12.11. IR, n (cmꢁ1): 3136 (N–H str), 1723 (C O str),
–
NH proton exchanged with DMSO. Anal. calcd. for
18H10ClFN4OS2 (416.88): C, 51.86; H, 2.42; N, 13.44; S,
–
1
–
–
C
1578 (C N str). H NMR: d (ppm) 7.39–7.70 (m, 6H, ArH), 7.75
–
15.38%. Found C, 51.82; H, 2.54; N, 13.34; S, 14.65%. HR-
MS (ESI), m/z 417.0038 (MHþ, C18H10ClFN4OS2), required:
417.0041. LC–MS-(ESI): Calculated: Mmi: 415.9968, (MþH)þ:
417.0041, (MꢁH)ꢁ: 414.9896. Found: (MþH)þ: 416.7717,
(MꢁH)ꢁ: 414.8577.
(s, 1H, CH), 8.16–8.22 (m, 1H, ArH), 13.18 (s, 1H, lactam NH).
–
13C NMR: d (ppm) 117.54, 117.81, 118.10, 126.30, 128.62,
129.16, 131.36, 131.86, 132.20, 132,91, 136.15, 159.41, 160.78
–
(ArC, thiazolidinone C5, CH–Ar), 164.54 (thiazolidinone C ),
–
2
161.30 (thiadizole C5), 167.48 (thiazolidinone C4), 171.61
(thiadiazole C2). Anal. calcd. for C18H9Cl2FN4OS2 (451.32):
C, 47.90; H, 2.01; N, 12.41; S, 14.21%. Found C, 47.83; H, 2.07;
N, 12.29; S, 14.11%. LC–MS-(ESI): Calculated: Mmi: 449.9578,
(MþH)þ: 450.9652, (MꢁH)ꢁ: 448.9506. Found: (MþH)þ:
450.6488, (MꢁH)ꢁ: 448.7859.
5-(3-Fluorobenzylidene)-2-{[5-(2-chloro-6-fluorophenyl)-
1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (33)
Yield 38%. m.p. 264–266˚C (EtOH). TLC Rf: 0.72 (S2). HPLC tR
(min): 9.72. IR, n (cmꢁ1): 3081 (N–H str), 1725 (C O str), 1597
–
–
1
–
–
–
(C N str). H NMR: d (ppm) 7.34–7.84 (m, 8H, ArH, CH), 13.16
–
(s, 1H, lactam NH). Anal. calcd. for C18H9ClF2N4OS2 (434.87): C,
49.71; H, 2.09; N, 12.88; S, 14.75%. Found C, 49.56; H, 2.47; N,
12.29; S, 14.31%. LC–MS-(ESI): Calculated: Mmi: 433.9874,
(MþH)þ: 434.9947, (MꢁH)ꢁ: 432.9802. Found: (MþH)þ:
434.7592, (MꢁH)ꢁ: 432.8572.
5-(2,6-Dichlorobenzylidene)-2-{[5-(3-fluorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (38)
Yield 79%. m.p. 260–262˚C (EtOH). TLC Rf: 0.93 (S2). HPLC tR
(min): 11.12, 11.42. IR, n (cmꢁ1): 3123 (N–H str), 1717 (C O str),
–
–
1
–
–
1575 (C N str). H NMR: d (ppm) 7.38–7.44 (m, 1H, ArH), 7.51–
–
7.66 (m, 5H, ArH, CH), 7.75 (d, 2H, ArH), 13.18 (s, 1H, lactam
–
5-(3-Fluorobenzylidene)-2-{[5-(2,4-dichlorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (34)
NH). Anal. calcd. for C18H9Cl2FN4OS2 (451.32): C, 47.90; H, 2.01;
N, 12.41; S, 14.21%. Found C, 47.61; H, 2.38; N, 12.11; S,
13.97%. LC–MS-(ESI): Calculated: Mmi: 449.9578, (MþH)þ:
450.9652, (MꢁH)ꢁ: 448.9506. Found: (MþH)þ: 450.6702,
(MꢁH)ꢁ: 448.7804.
Yield 48%. m.p. 297–298˚C (EtOH). TLC Rf: 0.70 (S2). HPLC tR
(min): 10.26. IR, n (cmꢁ1): 3156 (N–H str), 1717 (C O str), 1594
–
–
–
–
1
(C N str). H NMR: d (ppm) 7.33–7.66 (m, 5H, ArH), 7.78 (s, 1H,
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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4-Thiazolidinones as Inhibitors of HCV NS5B Polymerase
Archiv der Pharmazie
5-(2,6-Dichlorobenzylidene)-2-{[5-(2-chlorophenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (39)
concentration were investigated further for their IC50 values
employing 8–10 concentrations of the serially diluted com-
pounds. The IC50 values were analyzed from dose–response
curves utilizing Graphpad prism 3.03 software.
Yield 97%. m.p. 251–252˚C (EtOH). TLC Rf: 0.92 (S2). HPLC tR
(min): 12.06. IR, n (cmꢁ1): 3056 (N–H str), 1709 (C O str), 1592
–
–
–
–
1
(C N str). H NMR: d (ppm) 7.15–7.71 (m, 7H, ArH), 7.76 (s, 1H,
–
CH), 13.19 (s, 1H, lactam NH). Anal. calcd. for C18H9Cl3N4OS2
2H2O (476.79): C, 45.34; H, 2.11; N, 11.75; S, 13.45%. Found C,
Cell-based anti-HCV screening
–
1
ꢆ
The effect of the compounds on cell viability and anti-HCV
activity was investigated employing Huh7 parental and Huh7/
Rep-Feo1b replicon reporter cells, respectively, as previously
described [52, 53]. Briefly, 1 ꢅ 104 cells were screened at
compound concentrations of 100 and 50 mM for 48 h for
evaluation of cell viability and antiviral effect, respectively.
The concentration of DMSO in cell culture was kept constant
at 0.5%. Cell viability was measured by the colorimetric MTS
assay employing the CellTiter 96AQueous One Solution assay
reagent (Promega, USA). Inhibitory effect of the compounds
on HCV RNA replication was measured as the relative levels of
firefly luciferase signals in compound-treated cells versus
DMSO controls.
45.28; H, 2.24; N, 11.71; S, 13.40%. HR-MS (ESI), m/z 466.9357
(MHþ, C18H9Cl3FN4OS2), required: 466.9356. LC–MS-(ESI):
Calculated: Mmi
464.9210. Found: (MþH)þ: 466.8894, (MꢁH)ꢁ: 464.9371.
:
465.9283, (MþH)þ: 466.9356, (MꢁH)ꢁ:
5-(2,6-Dichlorobenzylidene)-2-{[5-(2-chloro-6-
fluorophenyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-
4-one (40)
Yield 20%. m.p. 226–227˚C (EtOH). TLC Rf: 0.73 (S2). HPLC tR
(min): 9.28. IR, n (cmꢁ1): 3089 (N–H str), 1710 (C O str), 1596
–
–
1
–
–
–
–
(C N str). H NMR: d (ppm) 7.44–7.80 (m, 7H, ArH, CH), 13.23
(s, 1H, lactam NH). Anal. calcd. for C18H8Cl3FN4OS2 (485.77): C,
44.51; H, 1.66; N, 11.53; S, 13.20%. Found C, 44.32; H, 1.97; N,
11.29; S, 13.17%.
Molecular modeling
The compounds were built and prepared for docking using
5-(2,6-Dichlorobenzylidene)-2-{[5-(2,4-dichlorophenyl)-
1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (41)
Yield 39%. m.p. 282–283˚C (EtOH). TLC Rf: 0.80 (S2). HPLC tR
Ligprep v2.7 and Macromodel v10.1 implemented in
¨
Maestro v9.5 (Schrodinger, LLC, New York, NY, 2013)
following the steps mentioned previously [69]. The X-ray
crystal structure of PF868554 bound HCV NS5B (PDB
ID:3FRZ) [54] was used for docking into TP-II. The protocols
for protein preparation, grid generation, and docking
simulations were essentially the same as those mentioned
in our earlier report except the modeling tools used were
(min): 10.28. IR, n (cmꢁ1): 3089 (N–H str), 1706 (C O str), 1583
–
–
1
–
–
(C N str). H NMR: d (ppm) 7.51–7.65 (m, 3H, ArH), 7.75 (s, 1H,
CH), 7.88–8.15 (m, 3H, ArH), 13.20 (S, 1H, lactam NH). 13C
–
–
NMR: d (ppm) 128.52, 129.96, 130.85, 132.40, 132.81, 136.66
–
(ArC, thiazolidinone C5, CH–Ar), 167.48 (thiazolidinone C ),
–
2
¨
159.28 (thiadizole C5), 172.37 (thiazolidinone C4), 174.83
(thiadiazole C2). Anal. calcd. for C18H8Cl4N4OS2 (502.22): C,
43.05; H, 1.61; N, 11.16; S, 12.77%. Found C, 43.03; H, 1.87; N,
10.83; S, 12.35%.
those implemented in Maestro v9.5 (Schrodinger, LLC) [36].
The highest scoring pose of compound 33 within TP-II was
used for graphical analysis. All computations were carried
out on a Dell Precision 490 dual processor with Linux OS
(Ubuntu 12.04 LTS).
5-(2,6-Dichlorobenzylidene)-2-{[5-(2,6-dichlorophenyl)-
1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (42)
Yield 17%. m.p. 238–240˚C (EtOH). TLC Rf: 0.94 (S2). HPLC tR
This work was supported by the Research Fund of Marmara
University, project number: SAG-C-YLP-071211-0303 and New
Jersey Health Foundation grant. The authors are grateful to
Dr. Ju¨ rgen Gross from the Institute of Organic Chemistry,
University of Heidelberg, for his generous help on obtaining
HR-ESI mass spectra of the compounds 32 and 39.
(min): 10.59. IR, n (cmꢁ1): 3128 (N–H str), 1733 (C O str), 1586
–
–
(C N str). 1H NMR: d (ppm) 7.49–7.75 (m, 7H, ArH, CH), NH
–
–
–
–
proton exchanged with DMSO. Anal. calcd. for C18H8Cl4N4OS2
(502.22): C, 43.05; H, 1.61; N, 11.16; S, 12.77%. Found C, 43.14;
H, 1.80; N, 10.86; S, 12.17%.
The authors have declared no conflict of interest.
Biological studies
NS5B inhibition assay
The effect of the compounds on HCV NS5B RdRp activity was
evaluated on poly rA-U12 template primer in the presence of
[a-32P]UTP and MnCl2 as described previously [51, 68].
Reactions in the presence of the compound or DMSO were
incubated at 30˚C for 1 h and terminated by the addition of
5% TCA. The nascent radiolabeled RNA was precipitated on
GF-B filters and counted in a liquid scintillation counter. NS5B
activity in the presence of DMSO was set at 100% and that in
the presence of the compounds was determined relative to
this control. Compounds exhibiting ꢂ50% inhibition at 50 mM
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Archiv der Pharmazie
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