Design, synthesis, and biological evaluation of new 4-thiazolidinone derivatives substituted with benzimidazole ring as potential chemotherapeutic agents

Article abstract of DOI:10.1007/s00044-012-0057-3

In search of novel antiviral and anticancer agents with promising pharmacotoxicological profile, a study was initiated to synthesize new 2-thioxo-4-thiazolidinones as well as 2-phenylimino-4-thiazolidinones substituted with benzimidazole ring system. The compounds were screened primarily for their antiviral as well as anticancer activities. The synthesis of some novel 5-substituted thiazolidinones was also described. None of the tested compounds showed inhibitory activity against Hepatitis C virus replication. Two 2-phenylimino-4-thiazolidinone derivatives (9a and 10) exhibited significant antiproliferative activity against human colon carcinoma cell line HCT 116 and human hepatocellular carcinoma HEPG2 cell line, respectively. Results also indicated that six thiazolidinone derivatives (5a, 5d, 5e, 5f, 5h, and 9d) showed moderate antiproliferative activity against human breast adenocarcinoma cell line MCF7 in comparison to the standard drug Doxorubicin. Moreover, a docked pose of the most potent three cytotoxic compounds 5a, 5h, and 9d against MCF7 was obtained bound to Human N-acetyl transferase1 NAT1 binding pocket by molecular operating environment module.

Full text of DOI:10.1007/s00044-012-0057-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:707–725
DOI 10.1007/s00044-012-0057-3
ORIGINAL RESEARCH
Design, synthesis, and biological evaluation of new
4-thiazolidinone derivatives substituted with benzimidazole
ring as potential chemotherapeutic agents
•
Georgina N. Masoud Amal M. Youssef
•
•
Magdy M. Abdel Khalek Abeer E. Abdel Wahab
•
•
Ibrahim M. Labouta Aly A. B. Hazzaa
Received: 11 December 2011 / Accepted: 4 April 2012 / Published online: 1 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract In search of novel antiviral and anticancer
agents with promising pharmacotoxicological profile, a
study was initiated to synthesize new 2-thioxo-4-thiazoli-
dinones as well as 2-phenylimino-4-thiazolidinones
substituted with benzimidazole ring system. The com-
pounds were screened primarily for their antiviral as well
as anticancer activities. The synthesis of some novel
5-substituted thiazolidinones was also described. None of
the tested compounds showed inhibitory activity against
Hepatitis C virus replication. Two 2-phenylimino-4-thia-
zolidinone derivatives (9a and 10) exhibited significant
antiproliferative activity against human colon carcinoma
cell line HCT 116 and human hepatocellular carcinoma
HEPG2 cell line, respectively. Results also indicated that
six thiazolidinone derivatives (5a, 5d, 5e, 5f, 5h, and 9d)
showed moderate antiproliferative activity against human
breast adenocarcinoma cell line MCF7 in comparison to
the standard drug Doxorubicin. Moreover, a docked pose of
the most potent three cytotoxic compounds 5a, 5h, and 9d
against MCF7 was obtained bound to Human N-acetyl
transferase1 NAT1 binding pocket by molecular operating
environment module.
Keywords Thiazolidinones and benzimidazoles ꢀ
Antiviral activity ꢀ Breast cancer ꢀ NAT1
Introduction
The life threatening infections caused by viruses have
increased vigorously, especially in immunocompromised
patients. The high mortality associated with Hepatitis C
infection and cancer urgently calls for new, refined thera-
pies, and molecular targets to be directed for the aid of
treating hepatitis infection and cancer. Chronic infection
with Hepatitis C virus represents a major public health
threat. The current treatment Interferon alpha (INF-a),
apart from being costly and having complicated polypep-
tide structure with poor pharmacokinetic profile, shows
successful relapse rate in\50 % of patients (De Francesco
and Carfi, 2007).
Cancer has surpassed heart diseases as the leading cause
of death in the U.S (Edward and Elizabeth, 2005). For most
types of cancer, chemotherapeutic treatments have
achieved limited success to date, which highlight the need
for more efficacious and better-tolerated therapy. Improved
understanding of cancer biology at the molecular level has
led to a shift in treatment paradigm from the traditional
cytotoxics toward more rationally designed targeted
therapies.
G. N. Masoud ꢀ A. M. Youssef (&) ꢀ M. M. Abdel Khalek ꢀ
I. M. Labouta ꢀ A. A. B. Hazzaa
Breast cancer is the most common cancer in the United
Kingdom and worldwide; more than a million women are
diagnosed with breast cancer every year, accounting for a
tenth of all new cancers and 23 % of all female cancer
cases (http://info.cancerresearchuk.org). Breast cancer is a
heterogeneous disease and different breast cancer subtypes
can be identified based on gene expression profiling. Cur-
rent breast cancer therapies, based on Selective Estrogen
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
University of Alexandria, Alexandria, Egypt
e-mail: ayoussef65@yahoo.com
A. E. Abdel Wahab
Department of Medical Biotechnology, Genetic Engineering
and Biotechnology Research Institute (GEBRI),
City for Scientific Research and Technology Application,
Borg El-Arab, Alexandria, Egypt
123

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Med Chem Res (2013) 22:707–725
Cl
Receptor Modulators (SERMs), such as tamoxifen or aro-
matase inhibitors, can be unsuccessful due to intrinsic or
acquired resistance to these therapies (Weinshilboum,
2008).
Cl
S
S
O
After profiling gene expression, identification of some
unexpected overexpressed genes in breast cancer cells has
emerged new targets for drug discovery. Human N-acetyl
transferase 1 (NAT1) is one such protein which has
emerged as a new diagnostic marker or drug target for
breast cancer. Furthermore, specific activity of human
NAT1 in a range of breast cancer cell lines (MCF7, T47D,
MDA-MB-231, and ZR-75-1), which have been proven to
be good models for studying molecular pathways in
estrogen receptor (ER) breast tumors, were analyzed (Zhu
et al., 2006). Among ER-positive lines, the human breast
adenocarcinoma MCF7 is explored, in which NAT1
activity ranged from 1.8 0.4 nmol/min/mg (Wakefield
et al., 2006).
N
O
HN
S
O
Lead compound (A)
IC50 = 0.2 µM
a
a
R
R
b
b
S
S
N
O
N
S
O
N
N
d
N
d
c
c
HN
HN
e
e
a, e: Hydrophobic, b: olefinic, c: free rotation, d: Covalent bonding.
In the course of an initial high throughput screening of a
small molecule compound library, the lead compound (A)
was discovered as a selective Hepatitis C virus (HCV)
NS5B inhibitor (IC50 = 0.2 lM) (Fig. 1).
Fig. 2 Features similarities between the lead and the target com-
pounds
The aim of the present work was to design and syn-
thesize novel heterocycles structurally related to 4-thiazo-
lidinones derivatives with potential anti-HCV activity. The
optimization strategy of the lead compound (A) depends on
the following approaches: Combination of two heterocyclic
systems (4-thiazolidinone and benzimidazole) having
diverse biological activities in one hybrid molecule which
might provide distinguished efficacy and accessible syn-
ergistic activity. Modifications of the aryl functionality in
position 5 of the 4-thiazolidinone moiety. The type of
substituent on the phenyl ring appears to play an important
role in activity. This behavior depends on the nature and
position of substituent as well as the biological target. In
this work, we try to study the influence of versatile electron
donating, electron withdrawing, lipophilic, and hydrophilic
substituent groups. The isosteric replacement of 2-thioxo
group by 2-phenylimino in the 4-thiazolidinone core.
During the anti-HCV testing, some of the newly syn-
thesized compounds showed cytotoxic activity on the tes-
ted cell line. This directed us to explore the cytotoxic
activity of these compounds on three different cell lines.
Most of the tested compounds exhibited antiproliferative
activity against human breast adenocarcinoma cell line
MCF7. In this work, a theoretical docking study was per-
formed on the potent synthesized compounds to illustrate
the preferred binding modes of the thiazolidine ring with
the target protein NAT1. The docking study was performed
by MOE software (MOE Chemical Computing Group Inc;
Montreal). The rationale behind this concept was based on
the finding of several specific inhibitors for NAT1 that
were based on the structure of rhodanine. These were
Comprehensive SAR study was reported on this lead
compound, we have combined the information previously
reported (Powers et al., 2006) into a cohesive picture of
possible structural features required for the inhibition of
NS5B, which could be the presence of lipophilic sub-
stituent on the 5-arylidene moiety, the presence of olefin
moiety for the covalent binding to Cys366 of the target
NS5B protein and free rotation for the group attached to the
N-3 of 4-thiazolidinone ring (Fig. 2).
The incorporation of benzimidazole nucleus, a biologi-
cally accepted pharmacophore, in the 4-thiazolidinone
molecule has made it a versatile heterocycle possessing
wide spectrum of biological activity. Benzimidazole ring is
one of the important fused heterocyclic system that con-
stitutes a part of countless derivatives of biological rele-
vance such as antiviral (Evers et al., 2004; Middleton et al.,
2004; Hirashima et al., 2006; Li et al., 2006), anticancer
(Kumar et al., 2002; Hranjec et al., 2010; Refaat, 2010),
antimicrobial (Ayhan-Kilcigil et al., 1999; Gulgun and
Nurten, 2003), and antifungal activity (Pawar et al., 2004).
Cl
Cl
OH
O
S
NH
S
S
O
N
O
S
HN
S
O
B
A
Fig. 1 Structures of the lead compounds a and b
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Med Chem Res (2013) 22:707–725
709
identified through a screen of a library of 5000 compounds
against five different recombinant NATs from prokaryote
and eukaryote sources (Russell et al., 2009). One of these,
(Z)-5-(20-hydroxybenzylidene)-2-thioxothiazolidin-4-one
(B), demonstrated potent inhibition of human NAT1
(IC50 = 1.1 lM). Docking studies suggested that this
compound interacted with the hydrophobic pocket of the
active site of the enzyme. The crystal structure of this
compound has suggested that it may interact with the
adjacent molecules forming several weak bonds such as phi
and hydrogen bonds (Barreiro et al., 2007). Also, based on
literature, it was found that 2-substituted benzimidazoles
are active against the human breast adenocarcinoma cell
line MCF7 (where NAT1 enzyme is expressed) (Hranjec
et al., 2010).
in the literature (Ohishi et al., 1990). The methylene proton
of the Z-isomer was more downfield (7.9 ppm) than that of
the E-isomer (7.4 ppm) due to the interaction with the
carbonyl group at the 4-position (Sing et al., 2001).
On the other hand, Knoevenagel condensation of rho-
danine with aromatic ketones proceeded in the presence of
ammonium chloride or ammonium acetate with heating
(Sing et al., 2001). Refluxing 3-[(1H-benzo[d]imidazol-2-
yl) methyl]-2-thioxothiazolidin-4-one (3), isatin, ammo-
nium chloride, and ammonium hydroxide in ethanol for
12 h yielded 6, where isatin condenses only with one of its
carbonyl groups at the nucleophilic C-5 active methylene
of rhodanine nucleus giving the required product in a good
yield.
Compounds 7a–c were obtained in good yields through
an addition of 2-amino methyl benzimidazole dihydro-
chloride salt (2) to the selected aryl isothiocyanate in eth-
anol at room temperature (Husain et al., 1982; Rida et al.,
1986). Potassium carbonate was added to liberate the free
base of 2 during reaction.
Results and discussion
Chemistry
3-[(1H-Benzo[d]imidazol-2-yl) methyl]-(4-substituted
phenylimino) thiazolidin-4-ones (8a–c) were prepared by
refluxing 1-[(1H-benzo[d]imidazol-2-yl) methyl]-3-(4-
substituted phenyl) thiourea (7a–c) with equimolar amount
of ethyl bromoacetate in ethanol (Husain et al., 1982; Rida
et al., 1986; Soliman, 1981). The reaction mixture was
cooled and the product was separated without addition of
sodium acetate. The ring closure of compounds 7a–c with
ethyl bromoacetate was confirmed as being oriented toward
the formation of structures 8a–c in form I rather than form
II based on what previously mentioned in literature
(St. Laurent et al., 2004) (Fig. 3).
The synthetic strategies to obtain the target compounds are
depicted in Schemes 1 and 2. The key starting material
2-aminomethyl benzimidazole dihydrochloride salt (2) was
prepared, according to Cescon and Day’s (Cescon and Day,
1962) method. Treating 2 with bis (carboxymethyl) tri-
thiocarbonate in the presence of sodium carbonate afforded
the desired rhodanine 3. This strategy is based on the
method of Holmberg (1910) for preparing N-substituted
rhodanines. It involves the direct cyclization of a wide
variety of amines with bis (carboxymethyl) trithiocarbon-
ate in the presence of sodium carbonate to afford the
desired rhodanine heterocycle in one step (Strube, 1959;
Bulic et al., 2007). Cyclization proceeded successfully
when an aqueous suspension of the previous mixture was
heated in water on a boiling water bath for 5 h. The same
product was obtained in the same yield when the reaction
mixture was refluxed in isopropanol for 18 h. It was found
that coupling of diazonium salts of the appropriate amine
in acetic acid with the 3-[(1H-benzo[d]imidazol-2-yl)
methyl]-2-thioxothiazolidin-4-one (3) in dioxane, at the
nucleophilic C-5 active methylene yielded the corre-
sponding 5-arylazo derivatives (4a–c).
Several aldehydes have been condensed with pseud-
othiohydantoins (8a–c) by heating under reflux with sodium
acetate in acetic acid at 80 °C for several hours to yield the
corresponding 3-[(1H-benzo[d]imidazol-2-yl) methyl]-2-
(4-substituted phenylimino)-5-arylidenethiazolidin-4-ones
(9a–n). Knoevenagel condensation was accomplished by
reaction of 3-[(1H-benzo[d]imidazol-2-yl) methyl]-2-(phe-
nylimino) thiazolidin-4-one (8a) with isatin, ammonium
chloride, and ammonium hydroxide in ethanol for several
hours to afford 10. This was achieved in accordance with
the reported general procedure for aldol condensation of
ketones with 4-thiazolidinones (Brown, 1961).
Knoevenagel condensation of rhodanine 3 with aromatic
aldehydes at the nucleophilic C-5 active methylene was
accomplished using sodium acetate in refluxing acetic acid
to afford 5a–j. Based on literature precedence the isomeric
ratio of products was presumed to be mainly Z in all cases
(Sing et al., 2001). It was reported that the thermody-
namically stable Z-isomer predominated with a ratio of Z:E
isomers 10:1 after recrystallization for all arylidene rho-
danines. The ratio of the two geometrical stereoisomers
Scheme 2 starts with the preparation of 2-b-aminoethyl
benzimidazole hydrochloride (11) as described by Cescon
and Day (1962). Trials to cyclize 2-b-aminoethyl benz-
imidazole hydrochloride (11) to the corresponding rhoda-
nine (12) by heating under reflux with bis (carboxymethyl)
trithiocarbonate in water for 5 h, according to Holmberg
(1910) method, were unsuccessful and no product could be
separated (method B). In another trial for the synthesis of
the target rhodanine (12), refluxing of the above mentioned
1
was readily quantified by H NMR as previously reported
123

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Med Chem Res (2013) 22:707–725
NH2
NH2
1
a
N
N
N
N
H
b
g
N
R1
f
NH
O
O
N
H
N
N
H
N
H
N
N
H
2
NH2.2HCl
S
N
S
S
7 a-c
S
3
a: R1
=
H
b: R1 = CH3
c: R1
= Cl
R1
8 a-c
e
h
c
d
i
N
N
N
N
N
N
O
O
O
O
N
O
N
N
H
N
H
N
N
H
N
N
N
H
H
S
N
H
N
S
N
NH
S
S
CHR'
S
CHR2
S
S
S
N
O
5 a-j
6
N
O
H
R
a = R' = C6H5
b = R' = p-C6
c = R'= p-C6 H4N(Me)2
d = R' = p-C6H4 NO2
e = R' = o-C6 H4 Cl
f = R' = C6 H5 CH=CH
g = R' = p-C6 H4 OCH3
h = R'= 2,5C6H3(OCH3
N
H
4 Cl
R
1 = H
R1
R1
O
N
9 a-n
N
10
H
H
R1
H
H
H
H
H
H
H
H
R2
C6 H5
S
N
9a:
9b:
9c:
9d:
9e:
9f:
S
N
)
)
(p)ClC6 H4
(p)NO2C6 H4
(o)ClC6H4
C6 H5 -CH=CH-
2,5-(OCH3 )C6H3
2,4-(OCH3)C6H3
i = R'= 2,4C6H3(OCH3
j = R'= C5
4 a-c
R
H
4N
a = R= -SO2NHCOCH3
N
b = R=
-SO2NH
9g:
9h:
C5 H4
N
N
CH3
CH3
CH3
CH3
Cl
C6 H5
9i:
c = R = Cl
(p)ClC6H4
9j:
(p)NO2C6 H4
9k:
9l:
9m:
9n:
(o)ClC6 H4
C6H5
(p)ClC6 H4
Cl
Scheme 1 (a) glycine (1.5 equiv), 5.5 N HCl, reflux 30 h; (b) bis
(carboxymethyl) trithiocarbonate (1.0 equiv), Na2CO3, H2O, reflux
5 h; (c) 1ry amine in acetic acid (1.0 equiv), NaNO2 in water,
dioxane, stir at 5 C; (d) R’CHO (1.2 equiv), NaOAc, acetic acid,
reflux 3-4 h; (e, i) Isatin (1.2 equiv.), NH4Cl, NH4OH, ethanol, reflux
12 h; (f), K2CO3, ethanol, rt (overnight); (g) BrCH2COOEt, ethanol,
reflux 20–30 h; (h) R2CHO (1.2 equiv), NaOAc, acetic acid, reflux
12–15 h
NH2
done to synthesize the rhodanine (12) by stirring a mixture
of 2-b-aminoethyl benzimidazole hydrochloride (11), car-
bon disulfide, ethyl bromoacetate, and triethylamine in
methanol at room temperature (Lee and Sim, 2000).
Unfortunately, the infrared (IR) spectrum of the product
lacked the strong absorption band of the C=O function,
indicating that the reaction proceeded via another unex-
pected pathway (method D). It gave the same product as
that obtained from the previously mentioned method.
However, during our attempts to synthesize 3-[2-(1H-
N
a
N
H
NH2.XHCl
X= 1 & 2
NH2
1
11
c/d/e
b/c/d
O
N
N
N
N
H
N
NH
S
S
12
C12H11N3OS2
Mol. Wt.: 277.37
S
13
10H9N3S
Mol. Wt.: 203.26
C
benzo[d]imidazol-2-yl)
ethyl]-2-thioxothiazolidin-4-one
(12) either by condensing 2-b-Aminoethyl benzimidazole
hydrochloride (11) with bis (carboxymethyl) trithiocar-
bonate in the presence of sodium carbonate (method C), or
with carbon disulfide and ethyl bromoacetate in the pres-
ence of triethylamine (method D), a nitrogenous product
was isolated. Elemental analysis and the molecular ion
peak showed this product to have the molecular formula
C10H9N3S. Its IR (cm-1) spectrum revealed a broad band
of medium intensity centered at 3412 (N–H), while
the expected sharp band of (C=O) did not appear.
Scheme 2 (a) b-alanine (1.5 equiv), 5.5 N HCl, reflux 24 h; (b)
S
(1.0 equiv), Na2CO3, H2O, reflux 5 h; (c)
HOOCCH2SCSCH2COOH
S
(1.0 equiv), i-PrOH, reflux 18 h; (d) CS2,
HOOCCH2SCSCH2COOH
BrCH2COOEt, (Et)3N, MeOH, rt; (e) CS2, EtOH, rt
mixture in isopropanol for 18 h was proposed (Bulic et al.,
2007). The spectral data showed that such conditions did
not impact cyclization as expected, and unexpected product
was obtained (method C). Also, many attempts have been
123

Med Chem Res (2013) 22:707–725
711
N
using the hepatocellular carcinoma HEPG2 cell line
infected with the hepatitis-C virus. Monitoring of the HCV
viremia pre-and post-antiviral therapy through the detec-
tion of viral (?)- and/or (-)-RNA strands by the use of
qualitative RT-PCR has become the most frequently used,
reliable, and sensitive technique. Recently, it has been
reported that the detection of the (-)-strand HCV-RNA by
the RT-PCR is a very important tool for understanding the
life cycle of the HCV. It provides a reliable marker for the
diagnosis of HCV and monitors the viral response to
antiviral therapy (El-Awady et al., 1999). Based on these
facts, the adopted method in the present study contributes
to the simultaneous detection of the (?)- and/or (–)-HCV-
RNA strands in HEPG2 hepatoma cells infected with HCV.
Inhibition of viral replication was detected by amplification
of viral RNA segments by the RT-PCR technique both in
the cultivated infected cells alone (as a positive control)
and at the specified dose for each test compound as shown
in Table 1 at optimal temperature. The test compound is
considered to be active when it is capable of inhibiting the
viral replication inside the HCV-infected HEPG2 cells,
as evidenced by the disappearance of the (?)- and/or
(–)-strands viral RNA-amplified products detected by the
RT-PCR (compared to positive control).
HN
R
N
HN
H
S
7 a-c
BrCH2COOEt
no NaOAc
N
N
O
N
H
N
H
N
N
N
S
N
S
R
O
R
Form I
Form II
(more favoured)
(less favoured)
8 a-c
Fig. 3 Possible cyclization modes of 1-[(1H-benzo[d]imidazol-2-yl)
methyl]-3-(4-substituted phenyl) thiourea (7a–c) with ethyl bro-
moacetate
1
The H NMR (ppm) showed two triplets allocated for two
methylene groups (2 9 CH2) two doublets for the benz-
imidazole C4,7-Hs, two doublet of doublet for the benz-
imidazole C5,6-Hs at the expected range of d–ppm scale
and a D2O exchangeable singlet for the NH function at
10.32. The properties of this compound are therefore
in accordance with the proposed structure (13) namely; 3,
4-dihydropyrimido [1, 6-a] benzimidazole-1(2H)–thione.
Unequivocally, the same compound was obtained via
condensing 2-b-Aminoethyl benzimidazole hydrochloride
(11) and carbon disulfide in ethanol the molecular formula
of which is C10H9N3S (method E). In addition, the physical
constant (mp) and IR spectral data of compound 13 coni-
cided with those of that compound, which has been pre-
pared according to a reported procedure (Singh et al.,
1986).
Unfortunately, the ten tested compounds, 4a, 4b, 5c, 5d,
5g, 5h, 6, 9b, 9j, and 10, did not show any inhibition of
HCV replication at the specified doses as shown in Fig. 4,
as all of them showed the appearance of both (?)- and
(-)-strands of HCV RNA at the doses used.
Anticancer activity
Single dose assay
According to the National Cancer Institute- Cairo, struc-
tures are generally selected for screening based on their
ability to add diversity to the NCI small compound
Biological studies
Table 1 Results of the neutral red uptake assay of the thiazolidinone
derivatives
Antiviral activity
Compound ID
IC50 (lM)
Used dose (lM)
Qualitative in vitro anti-HCV screening
4a
4b
5c
5d
5g
5h
6
15.26
30.55
14.76
39.52
54.28
38.95
55.75
32.38
19.71
15.61
0.30
0.611
0.295
0.078
0.108
0.077
1.116
0.647
0.394
0.312
All the newly synthesized compounds were tested for their
cytotoxicity prior to anti-HCV evaluation. Compounds 4a,
4b, 5c, 5d, 5g, 5h, 6, 9b, 9j, and 10 proved to be safe to
human blood (didn’t show hemolysis at reasonable con-
centrations). Thus, they were selected for the anti-HCV
assay. Others were found not suitable for further
processing.
9b
9j
Compounds 4a, 4b, 5c, 5d, 5g, 5h, 6, 9b, 9j, and 10
were investigated for their in vitro action as anti-HCV
10
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Med Chem Res (2013) 22:707–725
collection. The NCI standards aim to study the influence of
versatile electron donating, electron withdrawing, lipo-
philic, and hydrophilic substituent groups on the biological
activity.
The cytotoxicity of 12 representative compounds 5e, 5f,
5g, 5h, 5i, 5j, 9a, 9c, 9d, 9g, 9h, and 10 was evaluated
against three cell lines representing three common forms of
human cancer, i.e., human breast adenocarcinoma cell line
MCF7, colon carcinoma cell line HCT 116, and human
hepatocellular carcinoma cell line HEPG2 by the method
of Skehan (Skehan et al., 1990) at 100-lM concentration.
Regarding the structures of the tested compounds we
can notice that rhodanine derivatives (5e, 5f, 5g, 5h, and
5j) showed higher activity compared to pseudothiohydan-
toin congeners (9a, 9c, 9d, 9g, 9h, and 10) (Table 2). All
the tested compounds were found to possess potential
antitumor activities against all the tested tumor cell lines
with growth percentages less than 25 % at 100 lM con-
centration. In general, all the compounds tended to be more
active against MCF7, than against other tumor cell lines.
Also, study of the results presented in Table 2 indicated
that compounds 5e, 5f, 5g, 5h, 5j, and 9d showed the least
growth percentages against MCF7, while compounds 9a
and 10 were the most active against HCT 116 and HEPG2
cell lines, respectively. Thus, they are likely to be inter-
esting candidates for advanced five dose assay on these cell
lines.
Fig. 4 RT-PCR amplification products on gel electrophoresis: a
represents the PCR product of HCV RNA (?) strands isolated from
HEPG2 cells for the different samples. b represents the PCR product
of HCV RNA (?) strands isolated from peripheral blood mononuclear
cells (PBMC) of infected patients for the different samples. In both
cases identical results were obtained for the (-) strand RNA
Table 2 Growth percentages of 100 lM of thiazolidinones derivatives against three cell lines in primary anticancer study
N
O
N
N
H
R1
CHR2
S
Compound
R1
R2
Growth percentage
Breast MCF7
Most sensitive
cell line
Colon HCT 116
Liver HEPG2
5e
S
(o)ClC6H4
C6H5–CH=CH–
(p)OCH3C6H4
2,5-(OCH3)C6H3
2,4-(OCH3)C6H3
C5H4N
-4.32
-3.93
-5.75
-2.27
0.34
27.6
21.22
16.19
20.8
9.07
14.18
11.83
18.56
17.8
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Liver
–
5f
S
5g
S
5h
S
5i
S
19.2
5j
S
-3.83
-0.69
-0.79
-1.74
-1.19
-1.39
7.07
16.7
22.8
9a
N–Ar
N–Ar
N–Ar
N–Ar
N–Ar
N–Ar
–
C6H5
7
24.4
9c
(p)NO2C6H4
(o)ClC6H4
2,4-(OCH3)C6H3
C5H4N
17.98
13.8
24
9d
18.9
9 g
15.52
22.86
16.2
18.39
19.73
6.8
9 h
10
Isatin nucleus
–
Positive control
379.92
336.08
341.27
123

Med Chem Res (2013) 22:707–725
713
Five dose assay
Table 3 IC50 values of thiazolidinones 5a, 5d, 5e, 5f, 5g, 5h, 5j, 6,
9a, 9d, 10, and doxorubicin as determined by SRB assay
Potential cytotoxicity of compounds 5e, 5f, 5g, 5h, 5j, and
9d as well as compounds 5a, 5d, and 6 against MCF7-
breast cancer cell line, compound 9a against HCT
116-colon cancer cell line and compound 10 against HEPG2-
liver cancer cell line, was tested by the method of Skehan
et al., (1990). The relation between surviving fraction and
drug concentration was plotted to get the survival curve for
each compound (Fig. 5). Also, the IC50 (dose of the com-
pound which caused a 50 % reduction of survival values) for
each compound was determined and shown in Table 3
against the standard antitumor Doxorubicin (Dox.). The
results are also represented graphically in Figs. 6 and 7.
ID
IC50 (lM)*
Cell line
5a
5d
5e
5f
35.05 0.005
39.35 0.010
39.64 0.009
37.35 0.010
46.66 0.018
27.7 0.010
40.85 0.010
35.41 0.25
7.3 0.013
MCF7
MCF7
MCF7
MCF7
MCF7
MCF7
MCF7
MCF7
HCT 116
MCF7
HEPG2
MCF7
HCT 116
HEPG2
5g
5h
5j
6
9a
9d
10
Dox.
34.83 0.08
5.91 0.011
1.28 0.08
1.19 0.09
Molecular modeling
1.08 0.03
As a reference to our modeling and docking study, the
complex of human NAT1 enzyme coupled with (Z)-5-
(20-hydroxybenzylidene)-2-thioxothiazolidin-4-one (B) as
inhibitor was used. The aryl substituent of which was pre-
dicted to extend into a hydrophobic pocket, with contact
residues around the groove being Val93, Phe125, Val216, and
Phe287. This provides a possible explanation for the prefer-
ence for lipophilic substituents. Stabilization also appeared
to be provided by two hydrogen bonding interactions
between Arg127 and both the carbonyl and 20-hydroxy sub-
stituent of rhodanine and the hydroxyl group of the Thr289
(Russell et al., 2009).
* Values were calculated from dose response curves done in triplicate
for each compound. Values were given standard deviation, MCF7
human breast adenocarcinoma, HCT 116 human colon carcinoma,
HEPG2 human hepatocellular carcinoma, Dox. Doxorubicin
The lowest energy conformations for the most potent
inhibitors 5a, 5h, and 9d showed relatively similar binding
modes using the deposited crystal structure of human
NAT1 (2PQT). They performed proper hydrophobic
interactions with amino acids of the hydrophobic pocket at
the binding site, namely, Phe125, Val216, and Phe287, which
confirms the preference of the lipophilic substituents on
Fig. 6 IC50 values of compounds 5a, 5d, 5e, 5f, 5g, 5h, 5j, 6, 9d, and
Doxorubicin against MCF7 cell line
the aryl moiety as it was predicted by our reference com-
pound (B).
Molecular modeling studies of our target compound 5a
indicated that imidazole-N3 and the carbonyl oxygen con-
tributed three hydrogen bonding interactions with NH of
Arg127, further stabilization occurs through an additional
hydrogen bonding interaction between the imidazole-NH
and the hydroxyl group of Phe287 residue of the target
protein (Fig. 8).
Concentration µg/ml
Fig. 5 Surviving fraction and dose effect of 5a, 5d, 5e, 5f, 5g, 5h, 5j,
6, 9d, and Doxorubicin against human breast adenocarcinoma cell
line MCF7
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Med Chem Res (2013) 22:707–725
Compound ID
Compound ID
Fig. 7 IC50 values of compounds 9a, 10, and Doxorubicin against
HCT 116 and HEPG2 cell lines, respectively
In compound 5h, carrying two methoxy groups at the
2nd and 5th positions affects the conformational configu-
ration leading to extending the ligand away from the con-
served residues at the binding site. As a result, 5h changed
its overall conformation to avoid clashes with the protein
binding backbone. However, it showed hydrogen bond
interaction at the 5-OCH3 with Tyr208 residue and better
lipophilic recognition within the binding pocket due to the
hydrophobic aromatic interactions with the surrounding
residues Arg127, Tyr129, Lys188, and Ile189 (Fig. 9).
Fig. 9 Binding mode for compound 5h docked and minimized in the
NAT1 binding pocket by MOE software
interactions with Arg127, Tyr129, Lys188, and Ile189, simi-
larly as in case of compound 5h (Fig. 10).
Although both compounds 5a and 5j are 2-thioxo-4-
thiazolidinone derivatives, yet they displayed contradicting
activity. This could be explained by studying the molecular
electrostatic potential (MEP) of both compounds to explain
this difference in activity. The molecular electrostatic
potential surface of compound 5a (relatively NAT1
inhibitor) showed three intense red (negative) regions in
comparison to compound (B), while compound 5j (rela-
tively inactive toward NAT1) showed 4 intense red
regions. This extra region may lead to non-favorable
interactions with the receptor (Fig. 11).
The pseudothiohydantoin derivative 9d showed restric-
ted flexibility due to the ortho-chloro group in addition to
the bulkiness of the phenylimino group at the C-2 of the
thiazolidinone ring, which keeps the carbonyl group
unexposed for any hydrogen bond interactions. However,
the presence of the lipophilic ortho-chloro group encour-
aged more hydrophobic interactions with the surrounding
residues Val93, Ser102, and Ile106. This in addition to the
Fig. 8 Binding mode for compound 5a docked and minimized in the
NAT1 binding pocket by MOE software
Fig. 10 Binding mode for compound 9d docked and minimized in
the NAT1 binding pocket by MOE software
123

Med Chem Res (2013) 22:707–725
715
Fig. 11 Molecular electrostatic
potential on a Gaussian surface
of compounds a 5a, b B, c 5j.
Red is negative and blue is
positive
Conclusion
position, e.g., compound 5 g has an IC50 = 46.66 lM;
while, compounds 9a and 10 exhibited significant anti-
proliferative activity against colon carcinoma cell line HCT
116 and human hepatocellular carcinoma cell line HEPG2,
respectively.
In the present study, a series of new 2-thioxo-4-thiazoli-
dinones as well as 2-phenylimino-4-thiazolidinones
substituted with benzimidazole ring was synthesized and
screened for their antiviral and anticancer activities. The
ten tested compounds did not show any inhibition of HCV
replication at the specified doses when investigated for
their in vitro action as anti-HCV using the hepatocellular
carcinoma HEPG2 cell line infected with the hepatitis-C
virus. On the other hand, thiazolidinone derivatives (5a,
5d, 5e, 5f, 5h, and 9d) showed moderate antiproliferative
activity (IC50 = 40 lM or less) against human breast
adenocarcinoma cell line MCF7 in comparison to the
standard Doxorubicin (IC50 = 1.28 lM). Regarding the
structures of the tested compounds, we can notice that
rhodanine derivative (5h) with electron donating groups
(methoxy group at 2nd and 5th positions) showed higher
activity (IC50 = 27.7 lM) compared to the unsubstituted
congener 5a. Activity was reduced upon substitution with
electron withdrawing groups, e.g., compounds 5d and 5e,
substituted with nitro and chloro groups, respectively. A
decrease in activity is noticed as the position of methoxy
group changes as going from the 2nd and 5th to the 4th
The overall outcome of the molecular docking study
revealed that the three active compounds 5a, 5h, and 9d
showed relatively similar binding modes in compare to the
lead compound (B). Benzimidazole ring also contributed in
H-bonding interactions, for example 5a. Also, Compound
5h showed hydrogen bonding through its 5-OCH3 group
with Tyr208, which may provide an explanation for the
relatively high potency of 5h over compound 9d.
Experimental
Chemistry
Melting points were determined in open-glass capillaries
on Griffin melting point apparatus and were uncorrected.
The (IR) spectra were recorded on Perkin-Elmer 1430 IR
1
spectrophotometer using the KBr palate technique. H and
13C-NMR spectra were run on a Varian Gemini 200,
123

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Med Chem Res (2013) 22:707–725
300 MHz and Joel 500 MHz spectrophotometers using
tetramethylsilane (TMS) as the internal standard and
DMSO-d6 as the solvent (Chemical shifts in d, ppm).
Splitting patterns were designated as follows: s: singlet; d:
doublet; m: multiplet. The mass spectra were scanned on a
Finnigan mass spectrometer model SSQ/7000 (70 e.V) and
on a gas chromatograph/mass spectrometer Shimadzu
GCMS/Qp2010 Plus operating at 70 eV. Microanalyses
were performed at the Microanalytical Unit, Faculty of
Science, Cairo University, Egypt and the found values
were within 0.4 % of the theoretical values.
and the reaction was monitored by TLC technique. Upon
dilution, a heavy precipitated product was obtained, fil-
tered, washed with water, and purified by crystallization
from proper solvents to give 4a–c; yields (80–90 %).
3-[(1H-Benzo[d]imidazol-2-yl) methyl]-5-
(sulfacetamido phenylazo)-2 thioxothiazolidin-4-one
(4a)
This compound was obtained according to the general
procedure and crystallized from acetic acid as a yellow
crystal, mp: 284–286°C. IR m (cm-1) 3311 (N–H, benz-
imidazole), 3219 (N–H, sulfonamide), 1721 (C=O, rhoda-
nine), 1693 (C=O, acetyl group), 1620 (C=N), and 1217
3-[(1H-Benzo[d]imidazol-2-yl) methyl]-2-
thioxothiazolidin-4-one (3)
1
(C=S). H NMR (300 MHz), d: 1.92 (s, 3H, CO–CH3),
5.45 (s, 2H, CH2–N), 7.14–7.18 (m, 2H, benzimidazole-
An equimolar mixture of 2-aminomethyl benzimidazole
dihydrochloride (2) (1.09 g, 5 mmol) and sodium carbon-
ate (0.53 g, 5 mmol) in 10 ml of water or isopropanol was
heated on a water bath with stirring for 15 min. To the
clear solution, a hot solution of bis (carboxymethyl) tri-
thiocarbonate (1.13 g, 5 mmol) in 15 ml water or isopro-
panol is added at once. The mixture was heated on a water
bath with stirring for 15 min; within 10 min a yellow
precipitate was formed. Heating and stirring were contin-
ued for 5 h for aqueous solution or 18 h for isopropanol
solution. The precipitate was removed by suction filtration.
The solid was transferred to a flask containing 10 ml water,
was heated on a water bath to 70–75 °C, while the lumps
were crushed by a glass rod to obtain a homogenous
mixture. The mixture was then filtered with suction when
hot and the product was washed by rinsing it with small
amount of hot water, dried, and crystallized from ethanol,
yield: 58.1 %, mp: 220–222 °C. IR m (cm-1): 3452 (N–H),
1738 (C=O), 1614 (C=N), 1440, 1332, 1271, 1194 (NCS
amide I, II, III, IV bands respectively). 1H NMR
(500 MHz), d: 4.37 (s, 2H, CH2–C=O), 5.23 (s, 2H, CH2–
N), 7.09–7.13 (m, 2H, benzimidazole-C5,6-Hs), 7.4–7.42
(d, 1H, benzimidazole-C4-H, J = 10 Hz), 7.48–7.5 (d, 1H,
benzimidazole-C7-H, J = 10 Hz), 12.4 (s, 1H, NH, D2O
exchange). Anal. calcd for C11H9N3OS2 (263.33): C,
50.17; H, 3.44; N, 15.96. Found C, 49.61; H, 4.04; N,
15.37.
C
5,6-Hs), 7.42–7.51 (m, 4H, sulfonamide Ar–Hs), 7.84–
7.92 (m, 2H, benzimidazole-C4,7-Hs), 11.32 and 11.94 (s,
1H, diazo-hydrazo-NH, D2O exchange), 11.59 (s, 1H,
sulfacetamide-NH, D2O exchange), 12.52 (s, 1H, benz-
imidazole-NH, D2O exchange). Anal. calcd for
C19H16N6O4S3.‘H2O (497.56): N, 16.88; S, 19.29. Found
N, 16.16; S, 18.41.
3-[(1H-Benzo[d]imidazol-2-yl) methyl]-5-[(2-
sulfadiazinyl) phenylazo]-2-thioxothiazolidin-4-one
(4b)
This compound was obtained according to the general
procedure and crystallized from Ethanol/H2O as a brown-
ish yellow crystal, mp: 177–178°C. IR m (cm-1) 3038 (N–
1
H), 1697 (C=O), 1580 (C=N), and 1160 (C=S). H NMR
(300 MHz), d: 5.45 (s, 2H, CH2–N), 7.06–7.09 (m, 2H,
benzimidazole-C5,6-Hs), 7.60–7.64 (m, 4H, sulfonamide
Ar–Hs), 7.87–8.18 (m, 2H, benzimidazole-C4,7-Hs), 8.46–
8.52 (d, 1H, pyrimidine-C5-H), 8.53 and 8.55 (2s, 2H,
pyrimidine-C4,6-Hs), 11.52 and 11.98 (s, 1H, diazo-
hydrazo-NH, D2O exchange), 12 (s, 1H, sulfacetamide-
NH, D2O exchange), 12.52 (s, 1H, benzimidazole-NH,
D2O exchange). MS, m/z (rel. Abund. %): 423 (6.3)
(M?ꢀ?1), 524 (4.0) (M?), 331 (5.5), 297 (5.3), 255 (13.5),
192 (17.7), 170 (30.4), 162 (15.9), 149 (8.4), 144 (7.0), 128
(13.4), 118 (18.1), 98 (8.6), 96 (38.8), 77 (18.1), 64 (100).
Anal. calcd for C21H16ClN8O3S3 (524.6): C, 48.08; H,
3.07. Found C, 48.03; H, 3.74.
General procedure for the synthesis of compounds 4a–c
To an ice-cooled solution of the appropriate aromatic
amine (3 mmol) in glacial acetic acid (6 ml) a solution of
sodium nitrite (0.21 g, 3 mmol) in water (2 ml) was added.
The mixture was stirred for 10 min at 0–5 °C. The resulted
clear diazonium salt, over a period of 10 min to a stirred,
ice cooled solution of 3-[(1H-benzo[d]imidazol-2-yl)
methyl]-2-thioxothiazolidin-4-one (3) (0.78 g, 3 mmol) in
dioxane (13 ml) was added. Stirring was continued for 4 h
3-[(1H-Benzo[d]imidazol-2-yl) methyl]-5-[4-
(chlorophenylazo)]-2-thioxothiazolidin-4-one (4c)
This compound was obtained according to the general
procedure and crystallized from dioxane/petroleum ether as
an orange crystal, mp: 168–171°C. IR m (cm-1) 3219 (NH),
1699 (C=O), 1603 (C=N), and 1126 (C=S). 1H NMR
123

Med Chem Res (2013) 22:707–725
717
(300 MHz), d: 5.45 (s, 2H, CH2–N), 7.13–7.19 (m, 2H,
benzimidazole-C5,6–Hs), 7.27–7.44 (m, 4H, sulfonamide
Ar–Hs), 7.47–7.53 (m, 2H, benzimidazole-C4,7-Hs), 11.07
and 11.40 (s, 1H, diazo-hydrazo-NH, D2O exchange). MS,
m/z (rel. Abund. %): 402 (2.1) (M?ꢀ?1), 401 (4.0) (M?ꢀ),
199 (7.1), 189 (30.3), 173 (9.2), 163 (6.7), 146 (7.1), 131
(100.0), 126 (32.6), 99 (14.0), 90 (9.3), 77 (11.8), 63 (5.5).
Anal. calcd for C17H12ClN5OS2 (401.89): C, 50.81; H,
3.01; N, 17.43. Found C, 50.08; H, 2.57; N, 16.59.
J = 9), 7.72–7.75 (d, 2H, phenyl-C3,5-Hs, J = 9), 7.92 (s,
1H, C=CH vinylic H), 12.5 (s, 1H, NH, D2O exchange).
Anal. calcd for C18H12ClN3OS2 (385.89): C, 56.02; H,
3.13; N, 10.89. Found C, 55.19; H, 3.33; N, 1057.
(Z) 3-[(1H-Benzo[d]imidazol-2-yl) methyl]-5-(4-
dimethyl aminobenzylidene)-2-thioxothiazolidin-4-one
(5c)
This compound was obtained according to the general pro-
cedure as a reddish orange crystal, yield: 81.2 %, mp: 258–
60 °C. IR m (cm-1): 3433 (NH), 1689 (C=O), 1615-1614
(C=N). 1H NMR (300 MHz), d: 3.04–3.06 (s, 6H,
p-N(CH3)2), 5.42 (s, 2H, CH2–N), 7.85–7.88 (m, 2H, benz-
General procedure for the synthesis of compounds 5a–j
To a mixture of 3-[(1H-benzo[d]imidazol-2-yl) methyl]-2-
thioxothiazolidin-4-one (3) (1 mmol) and the appropriate
aldehyde (1.2 mmol) in glacial acetic acid (10 ml), a slight
excess of anhydrous sodium acetate (1.5 mmol) was added.
The reaction mixture was refluxed for 3–4 h; the reactions
were monitored by thin-layer chromatography technique.
After completion of the reaction, the reaction mixture was
cooled and diluted with water (10 ml). The products pre-
cipitated 5a–5j were filtered, washed with water; dried, and
crystallized from ethanol.
imidazole-C5,6-Hs), 7.12–7.15 (m, 2H, benzimidazole-C4,7
-
Hs), 7.48–7.55 (m, 2H, phenyl-Ar–Hs), 7.72 (s, 1H, C=CH
vinylic H), 12.49 (s, 1H, NH, D2O exchange). MS, m/z (rel.
Abund. %): 396 (11.7) (M?ꢀ?2), 395 (21) (M?ꢀ?1), 394
(54.5) (M?ꢀ), 264 (7.3), 206 (28), 177 (100.0), 131(14.9),
104 (14). Anal. calcd for C20H18N4OS2.1‘H2O (421.51): C,
56.93; H, 4.98; N, 13.28. Found C, 56.53; H, 4.68; N, 13.12.
(Z)-3-[(1H-Benzo[d]imidazol-2-yl) methyl]-5-(4-
nitrobenzylidene)-2-thioxothiazolidin-4-one (5d)
(Z)-3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-
benzylidene-2-thioxothiazolidin-4-one (5a)
This compound was obtained according to the general
procedure as a golden yellow crystal, yield: 88.38 %, mp:
242–244 °C. IR m (cm-1): 3394 (NH), 1732 (C=O), 1605
(C=N). 1H NMR (300 MHz), d: 5.45 (s, 2H, CH2–N),
7.13–7.16 (m, 2H, benzimidazole-C5,6-Hs), 7.5 (m, 2H,
benzimidazole-C4,7-Hs), 7.65–7.67 (d, 2H, phenyl-C2,6-Hs,
J = 6), 7.73–7.75 (d, 2H, phenyl-C3,5-Hs, J = 6), 7.92 (s,
1H, C=CH vinylic H), 12.49 (s, 1H, NH, D2O exchange).
MS, m/z (rel. Abund. %): 398 (3.9) (M?ꢀ?2), 397 (4.2)
(M?ꢀ?1), 396 (15.2) (M?ꢀ), 189 (69.4), 131 (100), 118
(27.1), 89 (41.2), 63 (19.0). Anal. calcd for
C18H12N4O3S2(396.44): S, 16.18. Found S, 15.50.
This compound was obtained according to the general
procedure as a golden yellow crystal, yield: 71 %, mp:
228–230 °C. IR m (cm-1): 3440 (NH), 1703 (C=O), 1559
(C=N). 1H NMR (300 MHz), d: 5.45 (s, 2H, CH2–N),
7.12–7.17 (m, 2H, benzimidazole-C5,6-Hs), 7.44–7.46 (d,
1H, phenyl-C4-H, J = 6 Hz), 7.52–7.62 (m, 4H, phenyl-
C2,3,5,6-Hs), 7.69–7.7 (d, 1H, bezimidazole-C4-H,
J = 3 Hz), 7.72 (d, 1H, benzimidazole-C7-H, J = 1.8 Hz),
7.92 (s, 1H, C=CH vinylic H), 12.49 (s, 1H, NH, D2O
exchange). 13C-NMR, d: 51.53 (CH2–N), 131.21–142.9
(Ar–Cs of benzimidazole and phenyl rings), 157.51
(C=CH), 176.16 (C=O) and 202.92 (C=S). MS, m/z (rel.
Abund. %): 353 (2.0) (M?ꢀ?2), 352 (4.3) (M?ꢀ?1), 351
(15.7) (M?ꢀ), 189 (45), 134 (100), 131 (52.8), 118 (10.4),
90 (14), 77 (13). Anal. calcd for C18H13N3OS2 (351.44): C,
61.52; H, 3.73; N, 11.96. Found C, 60.69; H, 3.71; N,
11.75.
(Z)-3-[(1H-Benzo[d]imidazol-2-yl) methyl]-5-(2-
chlorobenzylidene)-2-thioxothiazolidin-4-one (5e)
This compound was obtained according to the general
procedure as a golden yellow crystal, yield: 93.5 %, mp:
250–252 °C. IR m (cm-1): 3437 (NH), 1712 (C=O), 1596
(C=N). 1H NMR (300 MHz), d: 5.45 (s, 2H, CH2–N),
7.13–7.16 (m, 2H, benzimidazole-C5,6-Hs), 7.45-7.47 (m,
2H, benzimidazole-C4,7-Hs), 7.67–7.8 (m, 4H, phenyl-
(Z)-3-[(1H-Benzo[d]imidazol-2-yl) methyl]-5-(4-
chlorobenzylidene)-2-thioxothiazolidin-4-one (5b)
This compound was obtained according to the general
procedure as a yellow crystal, yield: 78 %, mp: 255–
256 °C. IR m (cm-1): 3433 (NH), 1724 (C=O), 1604
(C=N). 1H NMR (300 MHz), d: 5.45 (s, 2H, CH2–N),
7.13–7.16 (m, 2H, benzimidazole-C5,6-Hs), 7.49 (m, 2H,
benzimidazole-C4,7-Hs), 7.64–7.67 (d, 2H, phenyl-C2,6-Hs,
C3,4,5,6-Hs), 7.92 (s, 1H, C=CH vinylic H), 12.49 (s, 1H,
NH, D2O exchange). MS, m/z (rel. Abund. %): 387 (44.4)
(M?ꢀ?2), 386 (31.6) (M?ꢀ?1), 385 (100) (M?ꢀ), 352
(35.7), 189 (86.3), 131 (83.4), 118 (31.1), 77 (48). Anal.
calcd for C18H12ClN3OS2.‘H2O (394.89): C, 54.69; H,
3.29; N, 10.63. Found C, 54.65; H, 3.712; N, 10.93.
123

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Med Chem Res (2013) 22:707–725
(C=N). 1H NMR (500 MHz), d: 3.8–3.82 (2 s, 6H, phenyl-
(Z)-3-[(1H-Benzo[d]imidazol-2-yl) methyl]-5-(3-
phenyl allylidene)-2-thioxothiazolidin-4-one (5f)
C2,4-OCH3), 5.71 (s, 2H, CH2–N), 6.32 (s, 1H, phenyl-C3-
H), 6.43-6.45 (d, 1H, phenyl-C5-H, J = 10), 7.06–7.08 (d,
1H, phenyl-C6-H, J = 10), 7.18-7.2 (dd, 2H, benzimid-
azole-C5,6-Hs), 7.55-7.57 (2d, 2H, benzimidazole-C4,7-Hs),
7.8 (s, 1H, C=CH vinylic H). MS, m/z (rel. Abund. %): 412
(5.7) (M?ꢀ?1), 411 (10.7) (M?ꢀ), 189 (100), 179 (57.0),
151 (39.5), 131 (52.9), 77 (19.9). Anal. calcd for
C20H17N3O3S2 (411.49): N, 10.21. Found N, 9.99.
This compound was obtained according to the general
procedure as a yellow crystal, yield 87.5 %, mp 248–
50 °C. IR m (cm-1): 3437 (NH), 1704 (C=O), 1578 (C=N).
1H NMR (200 MHz), d: 5.45 (s, 2H, CH2–N), 7.21–7.77
(m, 11 H, benzimidazole, phenyl Hs and 2Hs of conjugated
system), 12.5 (broad signal, 2H, NH and 1H of conjugated
system). MS, m/z (rel. Abund. %): 379 (2.9) (M?ꢀ?2), 378
(5.4) (M?ꢀ?1), 377 (16.5) (M?ꢀ), 189 (76.9), 160 (75.5),
131 (100), 115 (75.7), 77 (37.0). Anal. calcd for
C20H15N3OS2 (377.48): N, 11.13. Found N, 10.8.
(Z)-3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-[(pyridin-
2-yl)methylene]-2-thioxothiazolidin-4-one (5j)
This compound was obtained according to the general
procedure as a yellow crystal, yield: 90.9 %, mp: 254–
256°C. IR m (cm-1): 3377 (NH), 1709 (C=O), 1615 (C=N).
1H NMR (300 MHz), d: 5.67 (s, 2H, CH2–N), 7.23–7.57
(m, 8H, benzimidazole and pyridine-Hs), 7.9 (s, 1H, C=CH
vinylic H), 12.49 (s, 1H, NH, D2O exchange). MS, m/z (rel.
Abund. %): 354 (1.7) (M?ꢀ?2), 353 (5.1) (M?ꢀ?1), 352
(14.5) (M?ꢀ), 319 (7.6), 221 (31.4), 189 (22.5), 135 (100.0),
91 (12.6), 77 (23.9). Anal. calcd for C17H12N4OS2
(352.43): N, 15.90. Found N, 15.29.
(Z)-3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(4-
methoxybenzylidene)-2-thioxothiazolidin-4-one (5g)
This compound was obtained according to the general
procedure as a golden yellow crystal, yield: 97.11%, mp:
250–252 °C. IR m (cm-1): 3438 (NH), 1708 (C=O), 1590
1
(C=N). H NMR (300 MHz), d: 3.82 (s, 3H, C4-OCH3),
5.45 (s, 2H, CH2–N), 7.12–7.16 (m, 2H, benzimidazole-
C5,6-Hs), 7.02-7.04 (d, 2H, phenyl-C3,5-Hs, J = 6), 7.5-
7.52 (m, 2H, benzimidazole-C4,7-Hs), 7.60-7.62 (d, 2H,
phenyl-C2,6-Hs, J = 6), 7.9 (s, 1H, C=CH vinylic H),
12.5 (s, 1H, NH, D2O exchange). Anal. calcd for
C19H15N3O2S2.1‘H2O (408.47): C, 55.81; H, 4.40; N,
10.28; S, 15.66. Found C, 55.70; H, 3.75; N, 10.21; S, 16.00.
3-[3-((1H-Benzo[d]imidazol-2-yl) methyl)-4-oxo-2-
thioxothiazolidin-5-ylidene] indolin-2-one (6)
To a solution of 3-[(1H-benzo[d]imidazol-2-yl) methyl]-2-
thioxothiazolidin-4-one (3) (0.26 g, 1 mmol) and isatin
(0.17 g, 1.2 mmol) in ethanol (12 ml), a solution of
ammonium chloride (0.07 g, 1.5 mmol) in concentrated
ammonium hydroxide (0.05 ml, 1.5 mmol) was added. The
reaction mixture was refluxed for 12 h, cooled, and the
product separated out was filtered, washed with ethanol,
dried, and crystallized from aqueous DMF, yield: 75.94 %,
mp:[300 °C. IR m (cm-1): 3228 (N–H), 1713 (C=O amide
carbonyl of rhodanine), 1692 (C=O amide carbonyl of
(Z)-3-[(1H-Benzo[d]imidazol-2-yl) methyl]-5-(2,5-
dimethoxybenzylidene)-2-thioxothiazolidin-4-one (5h)
This compound was obtained according to the general
procedure as a golden yellow crystal, yield: 97.32 %, mp:
244–246°C. IR m (cm-1): 3441 (NH), 1714 (C=O), 1584
(C=N). 1H NMR (300 MHz), d: 3.79–3.88 (2s, 6H, phenyl-
C2,5-OCH3), 5.45 (s, 2H, CH2–N), 6.99–7.9 (m, 7H, Ar–
1
Hs), 7.96 (s, 1H, C=CH vinylic H), 12.5 (s, 1H, NH, D2O
exchange). 13C-NMR, d: 41.9 (CH2–N), 55.57 (C5-OCH3),
56.03 (C2-OCH3), 113.25–128.76 (Ar–Cs of benzimidazole
and phenyl rings), 147.95 (C=CH), 152.47 (2-OCH3),
153.21 (5-OCH3), 166.61 (C=O) and 193.65 (C=S). MS,
m/z (rel. Abund. %): 413 (27.5) (M?ꢀ?2), 412 (26.3)
(M?ꢀ?1), 411 (100) (M?ꢀ), 378 (44.7), 189 (78.1), 131
(25.9), 77 (16.4). Anal. calcd for C20H17N3O3S2 (411.49):
N, 10.21. Found N, 9.96.
isatin), 1617 (C=N). H NMR (200 MHz), d: 5.52 (s, 2H,
CH2–N), 6.99–8.83 (m, 8H, Ar–Hs), 11.38 (s, 1H, isatin-
NH), 12.55 (s, 1H, benzimidazole-NH). Anal. calcd for
C19H12N4O2S2.‘ H2O (401.45): C, 56.79; H, 3.23; N,
13.94. Found C, 56.15; H, 3.19; N, 13.46.
General procedure for the synthesis of compounds 7a–c
A mixture of 2-aminomethyl benzimidazole dihydrochlo-
ride (2) (0.65 g, 3 mmol), the selected aryl isothiocyanate
derivative (3 mmol) and potassium carbonate (0.41 g,
3 mmol) in ethanol (7 ml) was stirred at room temperature
overnight. The reaction mixture was poured into (15 ml)
ice water and the precipitated product was filtered, washed
with cold water, dried, and crystallized from the appro-
priate solvent.
(Z)-3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(2,4-
dimethoxybenzylidene)-2-thioxothiazolidin-4-one (5i)
This compound was obtained according to the general
procedure as a yellow crystal, yield 85.15 %, mp 248–
250 °C. IR m (cm-1): 3434 (NH), 1700 (C=O), 1576
123

Med Chem Res (2013) 22:707–725
719
1-[(1H-Benzo[d]imidazol-2-yl) methyl]-3-phenyl
thiourea (7a)
(16.1), 89 (10.6), 82 (11.4), 80 (7.1), 77 (35.0), 75 (21.3),
74 (13.4), 73 (16.9), 52 (15.7), 51 (25.2).
This compound was obtained according to the general
procedure and crystallized from ethanol as a white crystal,
yield: 66.1 %, mp: 184–186 °C [reported 181–183°C] [30].
IR m (cm-1): 3367 (NH), 3283 (NH), 1598 (C=N), 1365
(C=S of thiourea).
3-[(1H-benzo[d]imidazol-2-yl) methyl]-2-(4-methyl
phenylimino) thiazolidin-4-one (8b)
The reaction mixture was poured into (15 ml) ice water.
The precipitated product was filtered, washed by water,
dried, and crystallized from toluene, yield: 74.4 %, mp:
224–226°C. IR m (cm-1): 2937 (NH), 1726 (C=O), 1620
(C=N).1H NMR (300 MHz), d: 2.25 (s, 3H, phenyl-CH3),
4.14 (s, 2H, CH2-CO), 5.14 (s, 2H, CH2–N), 6.73–6.89 (2d,
2H, phenyl-C3,5-Hs), 7.11–7.27 (m, 4H, phenyl-C2,6-Hs
and benzimidazole-C5,6-Hs), 7.36–7.54 (m, 2H, benzimid-
azole-C4,7-Hs), 12.36 (s, 1H, NH, D2O-exchange).
1-[(1H-Benzo[d]imidazol-2-yl) methyl]-3-(4-methyl
phenyl) thiourea (7b)
This compound was obtained according to the general
procedure and crystallized from ethanol as a white crystal,
yield: 87.83 %, mp: 204–206 °C (reported 187–188 °C)
[31]. IR m (cm-1): 3289 (NH), 3227 (NH), 1616 (C=N),
1238 (C=S of thiourea).
3-[(1H-Benzo[d]imidazol-2-yl) methyl]-2-(4-
chlorophenylimino) thiazolidin-4-one (8c)
1-[(1H-Benzo[d]imidazol-2-yl) methyl]-3-(4-
chlorophenyl) thiourea (7c)
The precipitated product was triturated with petroleum ether,
filtered, and dried. The product was used without further
purification for the next step, yield: 89.8 %, mp: 260–261°C.
IR m (cm-1): 2930 (NH), 1722 (C=O), 1634 (C=N).
This compound was obtained according to the general
procedure and crystallized from acetonitrile as a white
crystal, yield: 86.49 %, mp: 200–202 °C (reported 195–
197 °C–[30]. IR m (cm-1): 3271 (NH), 3087 (NH), 1617
(C=N), 1333 (C=S of thiourea).
General procedure for the synthesis of compounds 9a–n
To a solution of 1-[(1H-benzo[d]imidazol-2-yl) methyl]-2-
(4-substituted phenylimino) thiazolidin-4-ones (8) (0.32 g,
1 mmol) and the appropriate aldehyde (1.2 mmol) in gla-
cial acetic acid (10 ml) a slight excess of anhydrous
sodium acetate (0.12 g, 1.5 mmol) was added. The reaction
mixture was refluxed for 10 to 12 h; the reaction was
monitored by TLC technique. After completion of the
reaction, the reaction mixture was cooled and diluted with
water (10 ml). The product formed was filtered, washed
with water, dried, and crystallized from the appropriate
solvent to give 9a–n.
General procedure for the synthesis of compounds 8a–c
A mixture of disubstituted thiourea (7a–c) (2 mmol) and
ethyl bromoacetate (0.22 ml, 2 mmol) in ethanol (10 ml)
was heated under reflux for 20–30 h. The reaction mixture
was concentrated under reduced pressure and then cooled.
3-[(1H-Benzo[d]imidazol-2-yl) methyl]-2-
(phenylimino)thiazolidin-4-one (8a)
The precipitated product was filtered, washed with ethanol,
dried, and recrystallized from ethanol, yield; 79 %, mp:
270–272 °C. IR m (cm-1): 3439 (N–H), 1728 (C=O), 1624
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-benzylidene-
2-(phenylimino) thiazolidin-4-one (9a)
1
(C=N), 1586 (C=C). H NMR (300 MHz), d: 4.21 (s, 2H,
This compound was obtained according to the general
procedure and crystallized from ethanol as a golden yellow
crystal, yield: 97.5 %, mp: 280–282°C. IR m (cm-1): 3416
CH2-C=O), 5.37 (s, 2H, CH2–N), 6.80-6.81 (d, 1H, phenyl-
C2-H, J = 1.8 Hz), 6.83 (d, 1H, phenyl-C6-H, J =
1.8 Hz),7.07–7.12 (m, 1H, phenyl-C4-H), 7.29–7.35 (m,
2H, benzimidazole-C5,6-Hs), 7.44-7.46 (d, 1H, benzimid-
azole-C7-H, J = 3.3 Hz), 7.46–7.48 (d, 1H, benzimid-
azole-C4-H, J = 3.3 Hz), 7.68–7.78 (m, 2H, phenyl-C3,5-
Hs). MS, m/z (rel. Abund. %): 322 (11.0) (M?ꢀ), 188 (7.5),
187 (7.1), 159 (14.2), 157 (35.0), 156 (19.7), 155 (100),
154 (41.3), 129 (5.5), 128 (14.6), 127 (24.8), 126 (11.4),
118 (13.8), 104 (10.2), 103 (5.5), 102 (6.7), 101 (15.7), 99
(28.7), 98 (10.6), 92 (9.8), 91 (10.6), 92 (9.8), 91 (10.6), 90
1
(NH), 1710 (C=O), 1633 (C=C), 1589 (C=N). H NMR
(200 MHz), d: 5.35 (s, 2H, CH2–N), 6.82–7.52 (m, 14H,
Ar–Hs), 7.8 (s, 1H, C=CH vinylic H), 12.5 (s, 1H, NH,
D2O exchange). MS, m/z (rel. Abund. %): 412 (1.2)
(M?ꢀ?2), 411 (2.0) (M?ꢀ?1), 410 (7.1) (M?ꢀ), 305 (6.2),
275 (8.1), 263 (5.2), 247 (29.9), 236 (5.9), 220 (5.3), 161
(5.7), 145 (8.7), 134 (100), 118 (17.8), 104 (14.5), 90
(17.6), 89 (16.1), 77 (48.3) and 65 (7.0). Anal. calcd for
C24H18N4OS (410.49): N, 13.65. Found N, 13.91.
123

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Med Chem Res (2013) 22:707–725
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(4-
chlorobenzylidene)-2-(phenylimino) thiazolidin-4-one
(9b)
yellow crystal, yield: 71.1 %, mp: 276–278°C. IR m
(cm-1): 3375 (NH), 1707 (C=O), 1635 (C=C), 1588
(C=N). 1H NMR (500 MHz), d: 5.68 (s, 2H, CH2–N),
6.60–7.57 (m, 12H, Ar–Hs), 7.75 (s, 1H, C=CH vinylic H),
12.52 (s, 1H, NH, D2O exchange). MS, m/z (rel. Abund.
%): 438 (9.0) (M?ꢀ?2), 437 (30.6) (M?ꢀ?1), 436 (100.0)
(M?ꢀ), 248 (89.3), 207 (35.0), 160 (99.4) and 77 (96.7).
Anal. calcd for C26H20N4OS (436.53): C, 71.54; H, 4.62;
N, 12.83; S, 7.35. Found C, 70.97; H, 4.212; N, 12.46; S,
7.078.
This compound was obtained according to the general
procedure and crystallized from aqueous DMF as a yellow
crystal, yield: 78.8 %, mp: 254–256°C. IR m (cm-1): 3375
1
(NH), 1711 (C=O), 1635 (C=C), 1590 (C=N). H NMR
(200 MHz), d: 5.32 (s, 2H, CH2–N), 6.98-8.0 (m, 13H, Ar–
Hs), 7.9 (s, 1H, C=CH vinylic H), 12.46 (s, 1H, NH, D2O
exchange). Anal. calcd for C24H17ClN4OS (444.94): N,
12.59; S, 7.21. Found N, 11.85; S, 6.60.
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(2,5-
dimethoxybenzylidene)-2-(phenylimino) thiazolidin-4-
one (9f)
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(4-
nitrobenzylidene)-2-(phenylimino) thiazolidin-4-one
(9c)
This compound was obtained according to the general
procedure and crystallized from ethanol as a yellow crystal,
yield: 70.5 %, mp: 238-240°C. IR m (cm-1): 3416 (NH),
1710 (C=O), 1636 (C=C), 1591 (C=N). 1H NMR
(200 MHz), d: 3.71 (s, 3H, OCH3 at C4), 3.84 (s, 3H, OCH3
at C2), 5.32 (s, 2H, CH2–N), 6.97–7.57 (m, 12H, Ar–Hs),
7.96 (s, 1H, C=CH vinylic H), 12.51 (s, 1H, NH, D2O
exchange). MS, m/z (rel. Abund. %): 472 (6.0) (M?ꢀ?2),
471 (18.5) (M?ꢀ?1), 470 (59.9) (M?ꢀ), 248 (61.1), 194
(88.7), 131 (53.0) and 77 (100.0). Anal. calcd for
C26H22N4O3S.1‘H2O (497.54) : C, 62.70; H, 5.02; N,
11.25; S, 6.43. Found C, 62.29; H, 4.391; N, 11.65; S,
6.197.
This compound was obtained according to the general
procedure and crystallized from acetic acid as a yellow
crystal, yield: 72.5 %, mp: 300–332°C. IR m (cm-1): 3397
1
(NH), 1713 (C=O), 1631 (C=C), 1588 (C=N). H NMR
(200 MHz), d: 5.36 (s, 2H, CH2–N), 6.97–8.32 (m, 13H,
Ar–Hs), 7.97 (s, 1H, C=CH vinylic H), 12.63 (s, 1H, NH,
D2O exchange). MS, m/z (rel. Abund. %): 457 (5.2)
(M?ꢀ?2), 456 (9.6) (M?ꢀ?1), 455 (28.7) (M?ꢀ), 248 (33.5),
173 (12.5), 131 (90.4) and 77 (100.0). Anal. calcd for
C24H17N5O3S(455.49): C, 63.29; H, 3.76; N, 15.38; S,
7.04. Found C, 63.06; H, 3.744; N, 14.9; S, 6.08.
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(2-
chlorobenzylidene)-2-(phenylimino) thiazolidin-4-one
(9d)
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(2,4-
dimethoxybenzylidene)-2-(phenylimino) thiazolidin-4-
one (9g)
This compound was obtained according to the general
procedure and crystallized from ethanol as a yellow crystal,
yield: 79.5 %, mp: 258–260 °C. IR m (cm-1): 3416 (NH),
1721 (C=O), 1635 (C=C), 1589 (C=N). 1H NMR
(200 MHz), d: 5.34 (s, 2H, CH2–N), 6.94-7.51 (m, 13H,
Ar–Hs), 7.98 (s, 1H, C=CH vinylic H), 12.52 (s, 1H, NH,
D2O exchange). 13C-NMR, d: 40.76 (CH2–N), 131.42-
120.48 (Ar–Cs of benzimidazole and phenyl rings), 134
(C=CH), 147 (C=N-benzimidazole), 148.85 (C–N-phe-
nylimino), 148.93 (C=N-phenylimino) and 165.11 (C=O).
Anal. calcd for C24H17ClN4OS (444.94): C, 64.79; H, 3.85;
N, 12.59. Found C, 65.61; H, 4.099; N, 12.40.
This compound was obtained according to the general
procedure and crystallized from ethanol as a yellow crystal,
yield: 74.9 %, mp: 236–238 °C. IR m (cm-1): 3410 (NH),
1707 (C=O), 1633 (C=C), 1590 (C=N). 1H NMR
(200 MHz), d: 3.72–3.8 (2s, 6H, OCH3 at C2,5), 5.34 (s,
2H, CH2–N), 6.94–7.54 (m, 12H, Ar–Hs), 7.95 (s, 1H,
C=CH vinylic H), 12.51 (s, 1H, NH, D2O exchange). MS,
m/z (rel. Abund. %): 472 (5.4) (M?ꢀ?2), 471 (15.4)
(M?ꢀ?1), 470 (57.8) (M?ꢀ), 248 (58.6), 194 (100.0), 131
(41.1) and 77 (87.5). Anal. calcd for C26H22N4O3S.‘H2O
(479.54): C, 65.06; H, 4.79; N, 11.67. Found C, 64.53; H,
5.36; N, 11.61.
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(-3-
phenylallylidene)-2-(phenylimino)thiazolidin-4-one
(9e)
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-2-
(phenylimino)-5-[(pyridin-2-yl) methylene] thiazolidin-
4-one (9h)
This compound was obtained according to the general
procedure and crystallized from dioxane/ethanol as a
This compound was obtained according to the general
procedure and crystallized from ethanol as a green crystal,
123

Med Chem Res (2013) 22:707–725
721
yield: 96.2%, mp: 268–270 °C. IR m (cm-1): 3385 (NH),
1707 (C=O), 1625 (C=C), 1586 (C=N). 1H NMR
(300 MHz), d: 5.30 (s, 2H, CH2–N), 6.90–7.50 (m, 13H,
Ar–Hs), 7.94 (s, 1H, C=CH vinylic H), 12.59 (s, 1H, NH,
D2O exchange). MS, m/z (rel. Abund. %): 413 (3.6)
(M?ꢀ?2), 412 (9.1) (M?ꢀ?1), 411 (24.0) (M?ꢀ), 280 (7.80),
248 (24.6), 163 (21.3), 145 (23.11), 135 (100.0), 118
(26.9), 104 (22.2), 91 (18.7) and 77 (41.2). Anal. calcd for
C23H17N5OS (411.48): N, 17.02. Found N, 16.90.
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(2-
chlorobenzylidene)-2-(4-methyl phenylimino)
thiazolidin-4-one (9l)
This compound was obtained according to the general
procedure and crystallized from ethanol as a yellow crystal,
yield: 70.2 %, mp: 223–226°C. IR m (cm-1): 3406 (NH),
1708 (C=O), 1637 (C=C), 1605 (C=N). 1H NMR
(200 MHz), d: 2.29 (s, 3H, phenyl-CH3), 5.3 (s, 2H, CH2–
N), 6.8-7.8 (m, 13H, Ar–Hs), 7.85 (s, 1H, C=CH vinylic
H), 12.51 (s, 1H, NH, D2O exchange). Anal. calcd for
C25H19ClN4OS.‘H2O (467.96): C, 64.10; H, 4.27; N,
11.96. Found C, 63.42; H, 4.61; N, 11.68.
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-benzylidene-
2-(4-methyl phenylimino) thiazolidin-4-one (9i)
This compound was obtained according to the general
procedure and crystallized from ethanol as a yellow crystal,
yield: 66.1 %, mp: 270–272 °C. IR m (cm-1): 3390 (NH),
1709 (C=O), 1628 (C=C), 1581 (C=N). 1H NMR
(300 MHz), d: 2.29 (s, 3H, phenyl-CH3), 5.31 (s, 2H, CH2–
N), 6.82–7.59 (m, 13H, Ar–Hs), 7.82 (s, 1H, =CH vinylic-
H), 12.49 (s, 1H, NH, D2O exchange). Anal. calcd for
C25H20N4OS.‘H2O (433.52): C, 69.20; H, 4.84; N, 12.91;
S, 7.38. Found C, 68.47; H, 4.245; N, 12.99; S, 7.02.
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-benzylidene-
2-(4-chlorophenylimino)- thiazolidin-4-one (9m)
This compound was obtained according to the general
procedure and crystallized from ethanol as a yellow crystal,
yield: 63.6 %, mp: 218–221 °C. IR m (cm-1): 3410 (NH),
1718 (C=O), 1630 (C=C), 1590 (C=N). 1H NMR
(500 MHz), d: 5.68 (s, 2H, CH2–N), 6.60-7.57 (m, 13H,
Ar–Hs), 7.75 (s, 1H, C=CH vinylic H), 12.52 (s, 1H, NH,
D2O exchange). MS, m/z (rel. Abund. %): 446 (1.7)
(M?ꢀ?2), 445 (2.9) (M?ꢀ?1), 444 (3.3) (M?ꢀ), 133 (100.0),
104 (10.0), 90 (15.4) and 77 (17.1). Anal. calcd for
C24H17ClN4OS.‘H2O (453.94): C, 63.44; H, 3.96; N,
12.33. Found C, 63.10; H, 4.19; N, 12.41.
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(4-
chlorobenzylidene)-2-(4-methyl phenylimino)
thiazolidin-4-one (9j)
This compound was obtained according to the general
procedure and crystallized from ethanol as a yellow crystal,
yield: 54.7 %, mp: 288–290 °C. IR m (cm-1): 3354 (NH),
1711 (C=O), 1639 (C=C), 1605 (C=N).1H NMR
(300 MHz), d: 2.29 (s, 3H, phenyl-CH3), 5.31 (s, 2H, CH2–
N), 6.82–7.59 (m, 13H, Ar–Hs), 7.82 (s, 1H, =CH vinylic-
H), 12.49 (s, 1H, NH, D2O exchange). MS, m/z (rel.
Abund. %): 460 (6.2) (M?ꢀ?2), 459 (4.6) (M?ꢀ?1), 458
(9.6) (M?ꢀ), 261 (19.3), 170 (24.9), 168 (56.0), 131 (100.0),
118 (24.1), 104 (17.5), 91 (17.7) and 77 (16.9). Anal. calcd
for C25H19ClN4OS.‘H2O (467.96): C, 64.10; H, 4.27; N,
11.96; S, 6.83. Found C, 63.33; H, 3.84; N, 12.21; S, 6.29.
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(4-
chlorobenzylidene)-2-(4-chlorophenylimino)-
thiazolidin-4-one (9n)
This compound was obtained according to the general
procedure and crystallized from ethanol as a yellow crystal,
yield: 87.6 %, mp: 276–277 °C. IR m (cm-1): 3383 (NH),
1710 (C=O), 1628 (C=C), 1583 (C=N). 1H NMR
(500 MHz), d: 5.58 (s, 2H, CH2–N), 6.6-7.57 (m, 12H, Ar–
Hs), 7.75 (s, 1H, C=CH vinylic H), 12.5 (s, 1H, NH, D2O
exchange). Anal. calcd for C24H16Cl2N4OS (479.38): C,
60.13; H, 3.36; N, 11.69. Found C, 59.91; H, 3.74; N, 11.69.
3-[(1H-Benzo[d]imidazol-2-yl)methyl]-5-(4-
nitrobenzylidene)-2-(4-methyl phenylimino)
thiazolidin-4-one (9k)
3-[3-((1H-Benzo[d]imidazol-2-yl) methyl)-4-oxo-2-
(phenylimino) thiazolidin-5-ylidene] indolin-2-one (10)
This compound was obtained according to the general
procedure and crystallized from acetic acid as a yellow
crystal, yield: 69.7 %, mp: [300 °C. IR m (cm-1): 3396
To a solution of 3-[(1H-benzo[d]imidazol-2-yl) methyl]-2-
(phenylimino) thiazolidin-4-one (8a) (0.32 g, 1 mmol) and
isatin (0.17 g, 1.2 mmol) in ethanol (12 ml), a solution of
ammonium chloride (0.07 g, 1.5 mmol) in concentrated
ammonium hydroxide (0.05 ml, 1.5 mmol) was added. The
reaction mixture was refluxed for 12 h, cooled and the
product precipitated was filtered, washed with ethanol,
dried, and crystallized from aqueous DMF, yield: 53.21 %
1
(NH), 1710 (C=O), 1637 (C=C). H NMR (200 MHz), d:
2.3 (s, 3H, phenyl-CH3), 5.3 (s, 2H, CH2–N), 6.95-8 (m,
13H, Ar–Hs), 7.9 (s, 1H, C=CH vinylic H), 12.5 (s, 1H,
NH, D2O exchange). Anal. calcd for C25H19N5O3S
(469.52): C, 63.95; H, 4.08; N, 14.92. Found C, 62.83; H,
3.55; N, 14.74.
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Med Chem Res (2013) 22:707–725
mp:[300 °C. IR m (cm-1): 3228 (N–H); 1713 (C=O amide
carbonyl of rhodanine), 1692 (C=O amide carbonyl of
(100), 131 (83.1), 118 (15.7), 104 (24.0), 90 (36.7), 77
(37.3), 63 (55.9) and 51 (41.4). Anal. calcd for C10H9N3S
(203.26): C, 59.09; H, 4.46; N, 20.9.
1
isatin), 1617 (C=N). H NMR (300 MHz), d: 5.12 (s, 2H,
CH2–N), 6.77–7.61 (m, 13H, Ar–Hs), 10.54 (s, 1H, isatin-
NH, D2O exchange), 12.49 (s, 1H, benzimidazole-NH,
D2O exchange). MS, m/z (rel. Abund. %): 453 (4.9)
(M?ꢀ?2), 452 (8.1) (M?ꢀ?1), 451 (28.1) (M?ꢀ), 333 (14.9),
248 (52.6), 175 131 (100.0) and 77 (73.9). Anal. calcd for
C25H17N5O2S2.2H2O (487.50): C, 61.53; H, 4.30; N, 14.35.
Found C, 61.65; H, 4.54; N, 14.42.
Method E
A mixture of 2-aminoethyl benzimidazole hydrochloride
(11) (0.43 g, 2 mmol) and carbon disulfide (0.12 ml,
2 mmol) in 8 ml methanol was stirred at room temperature
for an overnight. The precipitated product was filtered,
washed with methanol dried (yield 0.38 g, 93.59 %), and
crystallized from ethanol; m.p. 216–218 °C. IR m (cm-1):
3444 (NH), 1615 (C=N), 1110 (C=S).
3,4-Dihydropyrimido [1, 6-a] benzimidazole-1(2H)–
thione (13)
Method C
Biological studies
A mixture of 2-aminoethyl benzimidazole hydrochloride
(11) (0.53 g, 2.5 mmol) and sodium carbonate (0.53 g,
5 mmol) in 10 ml isopropanol was stirred under reflux. The
mixture was heated on a water bath for 15 min, and to the
clear solution, a hot solution of bis (carboxymethyl) tri-
thiocarbonate (0.56 g, 2.5 mmol) in 15 ml isopropanol was
added at once. A yellowish orange precipitate was formed.
Heating and stirring were continued for 18 h. The precip-
itate was removed by suction filtration. The solid was
transferred to a flask containing 5 ml water and heated on a
water bath to 70–75 °C, while the lumps were crushed by a
glass rod to obtain a homogenous mixture. The mixture
was filtered with suction when hot, cleaned by rinsing it
with small amount of hot water, dried as possible, and
crystallized from ethanol, yield: 23.64 %, mp: 216–218 °C.
Antiviral activity
Neutral red uptake assay to measure cytotoxicity
Isolation of lymphocytes from whole human blood by
means of gradient separation by Ficoll-PagueTM Plus (MP
Biomedicals, France), 10 9 104 lymphocyte cells were
seeded per well in 96-well plates and the plates were
incubated in RPMI media containing different concentra-
tions of the test compounds 4a, 4b, 5c, 5d, 5g, 5h, 6, 9b, 9j,
and 10 for 24, 48, 72, and 96 h.
The fraction of viable lymphocyte cells was measured by
the neutral red assay
Method D
The neutral red assay is based on the initial protocol
described by Borenfreund (Borenfreund and Puerner, 1984)
and determines the accumulation of the neutral red dye in
the lysosomes of viable cells (Fotakis and Timbrell, 2006).
Following exposure of different concentrations of each
test chemicals to cells were incubated for 3 h with neutral red
dye (40 lg/ml) dissolved in culture media RPMI. Cells were
then washed using Phosphate Buffered Saline (PBS) and the
addition of 1 ml of elution medium (ethanol/glacial acetic
acid/water 50 %/1 %/49 %) followed by gentle shaking for
10 min so that complete dissolution was achieved. Aliquots
of the resulting solutions were transferred to 96-well plates
and absorbance at 490 nm was recorded using microliter
plate reader spectrophotometer (Biotek, U.S.A).
A mixture of 2-aminoethyl benzimidazole hydrochloride
(11) (0.43 g, 2 mmol), carbon disulfide (0.12 ml, 2 mmol),
ethyl bromoacetate (0.22 ml, 2 mmol) and triethylamine
(0.83 ml, 6 mmol) in 8 ml methanol, was stirred at room
temperature overnight. The precipitated product was fil-
tered, washed with methanol, dried (yield 0.38 g, 93.59%),
and crystallized from ethanol; m.p. 216–218 °C. The
products obtained by method (C) and method (D) are one
and the same compound. The identity of the product was
confirmed by TLC technique, mp, and IR spectra. IR m
(cm-1) : 3441 (N–H), 1614 (C=N), 1111 (C=S of thiourea).
1H NMR (500 MHz), d: 3.19–3.2 (t, 2H, CH2-CH2–N,
J = 6.9 Hz), 3.50-3.53 (t, 2H, CH2–N, J = 6.85 Hz),
7.28–7.29 (d, 1H, benzimidazole-C4-H, J = 3.8 Hz), 7.29–
7.30 (d, 1H, benzimidazole-C7-H, J = 3.8 Hz), 7.59–7.60
(dd, 1H, benzimidazole-C5-H, J = 3.85, 5.75 Hz), 8.76–
8.78 (dd, 1H, benzimidazole-C6-H, J = 3, 6 Hz), 10.32 (s,
1H, NH, D2O exchange). MS, m/z (rel. Abund. %): 205
(3.8) (M?ꢀ?2), 204 (8) (M?ꢀ?1), 203 (53) (M?ꢀ), 145
The simplest estimate of IC50 is to plot x–y and fit the
data with a straight line (linear regression).
Cell culture
HEPG2 cells were washed twice in RPMI1640 media
supplemented with 200 lM L-glutamine and 25 lM
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Med Chem Res (2013) 22:707–725
723
HEPES buffer; 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (all chemicals and media, Cambrex). The
cells were suspended at 2 9 105 cells/ml in RPMI culture
media (RPMI supplemented media, 10% fetal bovine serum
(FBS); Gibco-BRL). The cells were left to adhere on the
polystyrene 6-well plates for 24 h in an incubator (37 °C,
5 % CO2, 95 % humidity). The cells were washed twice
from debris and dead cells using RPMI supplemented media.
Cytotoxicity assay was performed on HEPG2 cell line
by the previously described method.
each of the reverse nested primer and the forward nested
primer, two units of taq DNA polymerase (Promega,
Madison, WI, USA) and 10 ll from the RT-PCR reaction
product in a 19 buffer supplied by the Vendor. A fragment
of 174 bp length was identified in positive samples.
Anticancer activity
Cell lines and culture
The human MCF7, HCT 116, and HEPG2 cell lines were
obtained from the American Type Culture Collection
(ATCC, USA). The MCF7 cells were maintained in
L-glutamine containing Eagle’s MEM (Sigma, Germany),
supplemented with NaHCO3 (2.2 g/l), sodium pyruvate
(110 mg/l), gentamycin (50 mg/l), and 10 % fetal calf
serum (FCS; Gibco, Germany) using 75 cm2-culture flasks
in a humidified atmosphere (95 % air/5 % CO2) at 37 °C.
Measurement of potential cytotoxicity by sulforhoda-
mine-B (SRB) assay
Qualitative in vitro anti-HCV screening
PBMC and HEPG2 cell cultures were prepared as dis-
cussed in EL-Hawash (EL-Hawash et al., 2006), then
infected with 2% HCV-infected serum in RPMI culture
medium containing 8 % FBS. Each of the tested com-
pounds was added at the specified doses. Positive and
negative control cultures were included. After 96 h of
incubation at 37 °C, 5 % CO2, and 95 % humidity, a sec-
ond dose of the test compound was added. The cells were
incubated for a further 96 h followed by total RNA
extraction. The positive strand and its replicating form
(negative strand) were detected by RT-PCR using HCV
specific primers to the 59-untranslated region of the virus.
Single dose assay
The tested compounds 5e, 5f, 5g, 5h, 5i, 5j, 9a, 9c, 9d, 9g,
9h, and 10 were submitted and evaluated at a single con-
centration (100 lM). Primary anticancer assays were per-
formed according to the procedure of Skehan (Skehan
et al., 1990), which is described below. The compounds
were added at a single concentration and the cell culture
was incubated for 48 h. End point determinations were
made with a protein binding dye, sulforhodamine B (SRB).
Results for each test compound were reported as the per-
cent of growth of the treated cells when compared to the
untreated control cells. Since the compounds were dis-
solved in DMSO, it was used as vehicle control. The per-
centage growth was evaluated spectrophotometrically at
570 nm versus controls not treated with test agents.
Using the seven absorbance measurements [time zero,
(Tz), control growth in the absence of drug (C), and test
growth in the presence of drug at 100 lM concentration
(Ti)], the growth percentage for each compound was cal-
culated as
RNA extraction and RT-PCR of HCV RNA
Total RNA was extracted from HEPG2 HCV-infected cells
by the method described by El-Awady (El-Awady et al.,
1999). Briefly, culture cells were mixed with 200 ll of 4 M
guanidinium isothiocyanate containing 25 mM sodium
citrate, 0.5 % sarcosyl, 0.1 M b-mercaptoethanol, and
100 ll sodium acetate. The lysed cells were mixed with an
equal volume of phenol, chloroform, and isoamyl alcohol
(25:24:1, pH 4). After vortexing of the sample, the mixture
was centrifuged at 14 K rpm for 10 min at 4 °C. The
aqueous layer was collected and mixed with an equal vol-
ume of isopropanol. After incubation at -20 °C overnight,
RNA was precipitated by centrifugation at 14 K rpm for
30 min at 4 °C and the precipitated RNA was washed twice
using 70 % ethanol. The complimentary DNA (cDNA) and
the first PCR reaction of the nested PCR detection system
for the HCV RNA was performed in a 50-ll volume single-
step reaction using the Ready-To-Go RT-PCR beads
(Amersham Pharmacia Biotech, Piscataway, NJ, USA),
10 lM from each of the RT downstream primer, PCR for-
ward primer, and reverse primer P2. The thermal cycling
protocol was manipulated as follows: 30 min at 42 °C for
cDNA synthesis followed by 5 min at 95 °C and 30 cycles
of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C. The
nested PCR amplification was performed in 50-ll reaction
mixture containing 0.2 mM from each dNTP, 10 lM from
Growth percentage ¼ ½ðTi ꢁ TzÞ=ðC ꢁ TzÞꢂ ꢃ 100
Five dose assay
Potential cytotoxicity of compounds was tested by the
method of Skehan et al., (1990) as follows Cells were
plated in 96-multiwell plate (5 9 103 cells) for 24 h before
treatment with the compounds 5e, 5f, 5g, 5h, 5j, and 9d as
well as compounds 5a, 5d, and 6 to allow attachment of
cells to the wall of the plate. Different concentrations of the
compounds (0, 5, 12.5, 25, and 50 lg/ml) were added to
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Med Chem Res (2013) 22:707–725
the cell monolayer. Triplicate wells were prepared for each
individual dose. Monolayer cells were incubated with the
compounds under test for 48 h at 37 °C and in atmosphere
of 5 % CO2. After 48 h, cells were fixed with trichloro-
acetic acid, stained for 30 min with 0.4 % (wt/vol) SRB
stain, and dissolved in 1 % acetic acid. Unbound dye was
removed by four washes with 1 % acetic acid, and protein-
bound dye was extracted with 10 mM buffered Tris EDTA
[tris (ethylene diamine tetra acetic acid)] for determination
of color intensity in an ELISA reader. For comparison
purposes, the cytotoxicity of Doxorubicin, a standard
antitumor drug, was evaluated under the same conditions.
The relation between surviving fraction and drug concen-
tration is plotted to get the survival curve of the specified
compound against each tumor cell line.
removed from Cys68 of the deposited structure of human
NAT1 (2PQT) before docking being performed. The NAT1
structure was used after the restoration of the terminal thiol
motif of Cys68. Hydrogen atoms were added to NAT1 and
subsequently minimized by the force MMFF94x field. The
synthesized target compounds were opened by the MOE
software. The docking was done by the default MOE-
DOCK, where the option Rotate Bonds was selected to
give flexible ligand-rigid receptor docking. Thirty con-
formers of the ligand were retained with highest and best
score by default. The top score ligand–receptor docking
was then demonstrated by 2D and 3D ligand–receptor
interactions.
Acknowledgments Authors thank all the members of pharmacol-
ogy unit at the National Cancer Institute, Cairo University, for pre-
paring the cytotoxicity testing.
Conflict of interest None.
Molecular modeling
Energy minimization and conformational search
procedure
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