Synthesis, antiamoebic and molecular docking studies of furan-thiazolidinone hybrids

Article abstract of DOI:10.1016/j.ejmech.2016.08.053

In continuation of our previous work, a series of furan-thiazolidinone hybrids was prepared by Knoevenagel condensation of 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one with different aryl aldehydes in presence of strong base. Some members o

Full text of DOI:10.1016/j.ejmech.2016.08.053

Accepted Manuscript
Synthesis, antiamoebic and molecular docking studies of furan-thiazolidinone hybrids
Mohammad Fawad Ansari, Shadab Miyan Siddiqui, Kamal Ahmad, Fernando
Avecilla, Sudhaker Dharavath, Samudrala Gourinath, Amir Azam
PII:
S0223-5234(16)30706-1
10.1016/j.ejmech.2016.08.053
EJMECH 8849
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 July 2016
Revised Date: 21 August 2016
Accepted Date: 22 August 2016
Please cite this article as: M.F. Ansari, S.M. Siddiqui, K. Ahmad, F. Avecilla, S. Dharavath, S. Gourinath,
A. Azam, Synthesis, antiamoebic and molecular docking studies of furan-thiazolidinone hybrids,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.053.
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ACCEPTED MANUSCRIPT
Synthesis, Antiamoebic and Molecular Docking Studies of Furan-thiazolidinone Hybrids
1
1
2
3
Mohammad Fawad Ansari , Shadab Miyan Siddiqui , Kamal Ahmad , Fernando Avecilla , Sudhaker
4
4
1
Dharavath , Samudrala Gourinath , Amir Azam *
1
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, 110 025, New Delhi, India
2
Centre for Interdisciplinary Research in Basic Science, Jamia Nagar, 110 025, New Delhi, India
3
Departamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira, 15071 A Coruña,
Spain.
4
School of Life Sciences, Jawaharlal Nehru University, New Delhi - 110067
*
Corresponding author. Tel.: +91 11 2698 1717/3253;
fax: +91 11 2698 0229/1232. E-mail address: amir_sumbul@yahoo.co.in (A. Azam)
Compound 6
Antiamoebic activity IC (µM) ± S.D = 0.67±0.042
5
0
Cytotoxicity IC (µM) ± S.D = >400 (48 h) >250 (72h)
5
0
Inhibition Assay IC (µM) = 144.3
5
0

ACCEPTED MANUSCRIPT
Synthesis, Antiamoebic and Molecular Docking Studies of Furan-thiazolidinone
Hybrids
1
1
2
3
Mohammad Fawad Ansari , Shadab Miyan Siddiqui , Kamal Ahmad , Fernando Avecilla ,
4
4
1
Sudhaker Dharavath , Samudrala Gourinath , Amir Azam *
1
Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, 110 025, New Delhi, India
2
Centre for Interdisciplinary Research in Basic Science, Jamia Nagar, 110 025, New Delhi,
India
3
Departamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira,
1
5071 A Coruña, Spain.
4
School of Life Sciences, Jawaharlal Nehru University, New Delhi - 110067
Abstract: In continuation of our previous work, a series of furan-thiazolidinone hybrids was
prepared by Knoevenagel condensation of 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-
thiazolidin-4-one with different aryl aldehydes in presence of strong base. Some members of
the series exhibited remarkable antiamoebic activity and cell viability. Three compounds (3, 6
and 11) showed excellent binding energy for Entamoeba histolytica O-acetyle-L-serine
sulfohydrolase and Entamoeba histolytica thioredoxin reductase. These compounds
demonstrated significant inhibition of O-acetyle-L-serine sulfohydrolase. The promising
antiamoebic activity and enzymatic assay of 3, 6 and 11 make them promising molecules for
further lead optimization in the development of novel antiamoebic agents.
Keywords: Furan, Thiazolidinone, Antiamoebic activity, MTT assay, Thioredoxin
Reductase.
*
Corresponding author. Tel.: +91 11 2698 1717/3254; fax: +91 11 2698 0229. E-mail
address: amir_sumbul@yahoo.co.in (A. Azam).
1

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1
. Introduction
Entamoeba histolytica (E. histolytica), the causative agent of amoebiasis has been listed by
the National Institute of Health as a category B priorty biodefense pathogen in the United
States [1]. This water borne pathogen possesses contact-dependent cell killing activity and
causes up to 100,000 fatalities per annum worldwide [2-3]. There is currently no vaccine
against E. histolytica [4]. Metronidazole (MNZ) is the first line medicament against
amoebiasis but long term use of the drug produces plenty of perilous side effects [5-6].
Furthermore, resistance to MNZ has been reported and the treatment failure may emerge as a
major public health issue [7]. E. histolytica O-acetyl serine sulfhydrylase (EhOASS) and E.
histolytica thioredoxin reductase (EhTrR) are two important enzymes that play crucial role in
the life cycle of the parasite. The growth and survival of E. histolytica depend upon the
cysteine biosynthetic pathway [8]. O-acetyl serine sulfhydrylase catalyses the last step of the
cysteine biosynthetic pathway. Cysteine, which is the product of this pathway, is the only
anti-oxidative thiol in E. histolytica and plays significant role in maintaining the redox
balance in the organism [9]. E. histolytica thioredoxin reductase is also an important enzyme
that maintains intracellular redox balance [10]. Thioredoxin reductase (TrR) catalyzes the
reversible transfer of reducing equivalents between NADPH and thioredoxin [11-12]. In view
of the importance of E. histolytica O-acetyl serine sulfhydrylase and E. histolytica
thioredoxin reductase, these enzymes could be good drug target as both of these enzyme
homologues are absent in mammalian hosts.
It has been demonstrated that the additive or even significant synergistic effects can be
achieved by molecular hybridization of two or more different medicinally active scaffolds
into a single chemical entity [13]. Furan, a well-studied five-membered heterocyclic, has
2

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been accounted to display various biological activities such as antibacterial, antiviral,
antifungal, antitumor, antiinflamatory and antiglycemic [14]. Furan constitutes the basic core
of antiamoebic drug diloxanide furoate (Figure 1) [15]. In our previous studies, some
compounds bearing furan ring in conjugation with other scaffolds have been found to exhibit
remarkable antiamoebic activities (Figure 2) [16]. Recently we have synthesized some novel
thiazolidinone derivatives starting from 2-methylpropane-1-amine that demonstrated better
growth inhibitory potential against E. histolytica (Figure 3) [17]. Encouraged by the results of
the previous studies [16-17] and the fact that the combination of thiazolidinones with other
heterocyclic rings produce various biological activities [18], we hypothesized that the
thiazolidinone derivatives incorporating a heterocyclic ring might exhibit synergistic effects.
Therefore, in this paper, we herein report the synthesis, antiamoebic activity, molecular
docking and enzymatic assay of furan-thiazolidinone hybrids (Figure 4).
2
. Results and Discussion
2
.1. Chemistry
The synthetic pathway leading to the title compounds (3-17) is depicted in Scheme
(1). 1-(Furan-2-ylmethyl)-3-phenylthiourea (1) was synthesized by reacting phenyl
isothiocyanate with furfuryl amine in presence of toluene. Sodium acetate assisted cyclization
of compound 1 with chloroacetic acid afforded 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-
thiazolidin-4-one (2). Finally, Knoevenagel condensation of compound 2 with different aryl
aldehydes in presence of strong base furnished the target furan-thiazolidinone hybrids (3-17).
1
The structures of all the target compounds (3-17) were elucidated on the basis of FT-IR, H
13
NMR, C NMR and ESI-MS. Additional supports for the proposed structure confirmed by
X-ray crystallographic studies. The purity of the compounds was confirmed by the elemental
analyses.
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2
.2. Single crystal structure
Single crystal structures of 2, 6, 8, 13 and 15
The asymmetric units of 2, 6, 13 and 15 contain one molecule of furan-thiazolidinone
hybrids. The asymmetric unit of 8 contains two molecules of furan-thiazolidinone hybrids
and one molecule of cyclohexane. The cyclohexane ring is in a chair form. The furan and
phenylimino rings are rotated respect to the carbon atom, C5, and nitrogen atom, N2,
respectively, in different forms in the five compounds (Figures 5 to 9). Crystal data and
details of the data collection and refinement for the compounds 2, 6, 8, 13 and 15 are
mentioned in Table 1. Bond lengths and angles are summarized in supplementary data (Table
SI1). In 8, the presence of intermolecular hydrogen bonds between the two molecules of
furan-thiazolidinone hybrids determinate the crystal packing (Table SI2 in supplementary
data). In the solid state, the compounds 2 and 8 form π-π stacking interactions between
thiazolidinone rings, which are in anti cofacial configuration respect to thiazolidinone units
(Figures 10 and 11) [19]. The distances between centroids are: dc1-c2 = 3.777 Å [c1 (N1J-C6J-
S1J-C13J-C14J), c2 (N1B-C6B-S1B-C13B-C14B) and repeated for other centroids] in 2, and
dc3-c4 = 3.562 Å [c3 (N4C-C31C-S2G-C29C-C30C), c4 (N4G-C31G-S2G-C29G-C30G)] and
repeated for other centroids] in 8. In 6, several π-π stacking interactions between
dimethoxybenzylidene and thiazolidinone groups of different frameworks determinate the
crystal packing, and form antiparallel dimers, [20] (Figure 12): dC13D-C18H = 3.585 Å, d -c
c5 18D
=
=
3.330 Å [c5 (C16H, C17H, C18H, C19H, C20H, C21H)], dC16D-C16H = 3.396 Å and dc6-C15H
3.330 Å [c6 (C16D, C17D, C18D, C19D, C20D, C21D)]. In 13, π-π-stacking interactions
between π clouds of chlorobenzylidene groups of different frameworks are the principal
interactions present in the crystal packing, and form antiparallel dimers, dC15J-c7 = 3.502 Å [c7
(C16C-C17C-C18C-C19C-C20C-C21C) and repeated for other centroids] (Figure 13). In 15,
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π-π-stacking interactions between π clouds, of the isopropoxybenzylidene and thiazolidinone
groups of different frameworks form antiparallel dimers, dc8-c9 = 3.696 Å [c8 (N1J-C6J-S1J-
C13J-C14J), c9 (C16A-C17A-C18A-C19A-C20A-C21A) and repeated for other centroids]
(Figure 14).
2
2
.3. Biology
.3.1. Antiamoebic activity
All the hybrid molecules (3-17) were evaluated in vitro for antiamoebic activity
against HM1: IMSS strain of E. histolytica by microdilution method [21]. The antiamoebic
activity was compared with the standard amoebicidal drug MNZ that had 50% inhibitory
concentration (IC ) 1.64 ꢀM. The results manifested that 1-(furan-2-ylmethyl)-3-
5
0
phenylthiourea (1; IC = 8.22 ꢀM) had higher IC value than the standard drug. However,
5
0
50
compound 2 (IC = 3.57 ꢀM), which integrates furan and thiazolidinones moieties together
5
0
in single molecular skeleton had lower IC50 value than compound 1. The compound 1 and
compound 2 having different structure were found to exhibit different biological behaviour
that suggest that the molecular hybridization of furan and thiazolidinone had an excellent
impact on antiamoebic activity. Moreover, Knoevenagel condensation of compound 2
furnished the compounds (3-17; IC50 = 0.27-2.97 ꢀM) having better antiamoebic potential
than compound 2. Among all the furan-thiazolidinone hybrids, seven compounds (3, 6, 8, 11,
1
2, 14 and 16) exhibited less IC value than MNZ. Compound 11 (IC = 0.27 ꢀM) having
50 50
heterocyclic substituent was found to be endowed with maximum antiamoebic activity. The
biological behavior of the compounds revealed that the compound with hydroxyl (8, IC =
5
0
1
.04 ꢀM), methoxy group (16, IC50 = 1.35 ꢀM) and methyl (14, IC50 = 1.48 ꢀM) at para
position displayed remarkable activity. However, the introduction of nitro (5, IC = 2.87
5
0
ꢀ
M), isopropoxy (15, IC = 2.20 ꢀM), ethoxy (9, IC = 2.03 ꢀM) and chloro (17, IC = 2.33
50 50 50
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ꢀ
M) group at the same position had negative effect on antiamoebic activity. The results
suggest that the substituents present on position 5 of thiazolidinone ring play significant role
in the activity against HM1: IMSS strain of E.histolytica. However, it can be concluded that
the entire molecular-skeleton of the particular compound is concerned with its IC value and
5
0
therefore, responsible for its antiamoebic activity.
2
.3.2. Cytotoxic activity
Cytotoxicity of active compounds has been studied using the MTT cell viability assay
on Chinese hamster ovary (CHO) normal cell lines. MTT is a tetrazolium salt, which can be
easily reduced to a colored formazan product with the help of reducing enzymes present only
in metabolically active or dividing cells [22]. The enzyme succinate dehydrogenase and
cytochrome oxidase present in mitochondria of active cells helps in the reduction of MTT
[
23]. A confluent population of CHO cells was treated with increasing concentrations of
compounds and the number of viable cells was measured after 48 h and 72 h by MTT cell
viability assay based on mitochondrial reduction of the yellow MTT tetrazolium dye to a
highly colored blue formazan product. This assay usually shows high correlation with
number of live cells and cell proliferation. The concentration range for all the active
compounds (3, 6, 8, 11, 12, 14, 16 and MNZ) is mentioned in (Figure 15), which illustrates
that all the active compounds and metronidazole were least-cytotoxic in the concentration
range of 2.5-50 µM for 48 h and 2.5-25 µM for 72 h. At 100 µM for 48 h and 50 µM for 72
h, only three compounds 3, 6 and 11 showed maximum viability and least cytotoxicity
respectively (Table 2). Therefore, it can be concluded that the cytotoxicity of all the
compounds (3, 6, 8, 11, 12, 14 and 16) was found to be concentration-dependent and all the
screened compounds were least-cytotoxic against the Chinese hamster ovary (CHO) normal
cell lines in the concentration range of 2.5-50 µM.
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2
.4 Drug –likeness properties
The molecular properties of synthesized compounds with known inhibitors (Table
SI3) were analyzed by Lipinski’s rule of five. Interestingly all the synthesized compounds
and known inhibitors have molecular weight in the range 171– 450 (< 500). Low molecular
weight drug molecules (<500) are transported, diffuse and absorbed without difficulty as
compared to heavy molecules. Molecular weight is an important aspect in curative drug
action; if molecular weight increases from certain limit, the large size of the compounds also
increases correspondingly, which in turn affects the drug action [24]. Number of hydrogen
bond acceptors (O and N atoms) and number of hydrogen bond donors (NH and OH) in the
synthesize compounds and known inhibitors were establish to be within Lipinski’s limit
range from 4-9 & 0-2 i.e. less than 10 and 5 respectively [25].
2
.4.1 Pharmacokinetics and toxicity
Most of drugs fail in clinical trials due to weak pharmacokinetic properties and
cellular toxicity. Therefore, in silico pharmacokinetic profile of selected compounds was
evaluated to fetch the putative bioavailability for EhTrR and EhOASS inhibitors provided in
supplementary data (Table SI4). Physicochemical properties, especially aqueous solubility
(logS), lipophilicity (clogP), polar surface area (PSA), and molecular weight (MW) are
directly related to the absorption and bioavailability of a drug molecule [26]. These properties
directly affect the movement of a drug from the site of administration into the blood. The
CYPs (cytochrome P450) play a significant role in drug metabolism and are equally
important for disposition of drugs in body, their pharmacological and toxicological effects
[
27]. Here, ADMET (DS3.5) was used to get the probable pharmacokinetic profile of
molecules. It utilizes QSAR models to compute the ADMET related properties for small
molecules. Alog P value (Lipophilicity) is significant property for the prediction of per oral
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bioavailability of drug molecules [24]. Results showed that all virtual synthesized compounds
have ideal AlogP value ≤ 5, except synthesized compounds 9, 13, 14, 15 and 17 (logP =
5
.226, 5.558, 5.379, 5.603and 5.558 respectively). However, logP value up to 6 is
considerable for a drug molecule [28]. Similarly, all hits showed moderate to good range of
solubility level (solubility level = 0 to 4). The compound 11 showed better solubility with
level 2, AlogP = 3.885, having good absorption (level 0) and moderate hepatotoxicity (level
1
). Only one compounds 15 showed hepatotoxicity probability score ≤ 0.5. The observed
human intestinal absorption (HIA) value is excellent for all molecules. The blood-brain-
barrier (BBB) penetration ability of compounds is high when prediction value is zero and is
least for the prediction value of 4. All hybrid molecules showed better ability, except
compound 4 and 5 (BBB = 4). The known EhTrR and EhOASS inhibitors, MNZ, 2-{1-[2-(2-
methyl-5-nitro-imidazol-1-yl)-ethyl]-1H-[1,2,3]triazol-4-yl}-pyridine (A) and 4-hydroxy-2-
[
2-(1H-indol-3-yl)-2-oxoethyl]sulfanyl- 1H-pyrimidin-6-one (W), are showed good logP
value ≤ 5. All have good human intestinal absorption value; however, MNZ and A showed
lower probability score of hepatotoxicity, except W higher probability score of
hepatotoxicity. The antiamoebic drug MNZ having a good ADMET profile (logP = -0.337,
BBB = 3, solubility = 4 and HIA = 0), showed less hepatotoxicity effect and is also not
reactive to CYP2D6. All the compounds except 2, 5, 8, 9, 11 and 15 exhibited <0.5
probability value for CYP2D6 enzyme. For good drug ability, the ideal plasma protein
binding (PPB) level is 0. All synthesized compounds had > 0 probability PPB activity,
whereas MNZ and A showed level-0. PSA is dependent on the conformation and hydrogen-
bonding. It implies the single low-energy conformer of the molecule. For activity of a drug,
the optimum value of PSA is ≤ 90 Å [29]. The hydrogen bonding and logP value are the
important two descriptors to define the PSA of drug molecule. All predicted compounds
showed significant PSA, whereas A and W showed higher PSA value ≥ 90 Å. The computer-
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aided toxicity predictor, TOPKAT was used to examine the cellular toxicity of synthesize
compounds. The carcinogenic and mutagenic effect of compounds with WOE Prediction
(weight of evidence) and Ames Prediction was our primary goal. It comprises of various
models and toxicity endpoints (irritation, teratogenicity, sensitization, neuro-toxicity and
immunotoxicology) that are often employed in drug development. All the selected
compounds showed Ames probability score ≤ 7 and are not mutagens except compounds 4
and 5 which has score ≥ 7 and is anticipated as mutagen. All EhTrR and EhOASS inhibitors
under study are predicted as non-mutagen, except MNZ and A. Other toxicity predictor WOE
was employed to examine the relative level of certainty of compounds that may cause cancer
in humans. All compounds were found to be non-carcinogenic, except five virtual compounds
(2, 4, 5, 10 and 11). MNZ and W are predicted as carcinogenic. The ADMET score and
TOPKAT property data of virtual synthesized compounds with standard drug, suggested that
selected molecules may be exploited as bioactive compounds.
2
.5 Molecular docking
All the title compounds (3-17) with reference drug MNZ were targeted for molecular
docking studies using rigid docking method. Auto docking 4.2 was used to determine the
orientation of inhibitors bound in the active site of the EhTrR and EhOASS protein (4A5L
and 3BM5) [30]. The docking of inhibiting ligand molecules 6, 8, 12, 14 and 16 with the
protein 4A5L reveals that all the inhibitors are in bonding with more than one amino acid
residues except 3, 11, and 16 and thereby they occupy the active pockets of the protein 4A5L
shown in Figure 16 [C, D, E, F, G, H and I]. Theoretically all the seven inhibitor molecules
(3, 6, 8, 11, 12, 14 and 16) exhibit very good binding energy compared with reference drug
MNZ and A as shown in Table 3.
9

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The docking of inhibiting ligand molecules 3, 6, 8, 11, 12, 14 and 16 with the protein
4
A5L reveals that all the inhibitors are in bonding with amino acid residues and thereby they
occupy the active pockets of the protein 3BM5 shown in Figure 17 [C, D, E, F, G, H and I].
Theoretically all the seven hybrids displayed significant binding energy than the reference
drug MNZ and W as shown in Table 4. The inhibitor 3, 6, 8, 12, 14 and 16 formed hydrogen
bonds with bond distances range from 2.3 to 3.6 Å with amino acid residues of protein 4A5L
and with minimum inhibitory constant as shown in Table 3. Table 4 showed the hydrogen
bond distance of inhibitors in the range 3.0 Å - 3.5 Å with amino acid residues of protein
3
BM5 and with minimum inhibitory constant. Compounds 3, 6, 8, 11, 12, 14 and 16 were
found to have very good binding energy for EhTrR and EhOASS. Therefore, these
compounds were good inhibitors of the target protein EhTrR and EhOASS. Among all the
docked compounds (2-17), only three compounds 3, 6 and 11 showed excellent binding
energy for EhTrR and EhOASS, therefore, these may be considered as good inhibitors of
proteins (4A5L and 3BM5).
2
.6 Enzymatic Inhibition assay
Among all the hybrids, three hybrids (3, 6 and 11) with most promising antiamoebic
activity were selected for enzymatic inhibition assay against E. histolytica O-acetyle-L-serine
sulfohydrolase. The enzyme EhOASS was expressed and purified as described previously
[
31]. Inhibitory kinetics analysis was carried out for EhOASS with varied concentration of
furan-thiazolidinone hybrids to measure the decrease in enzymatic activity. The rate of a
reaction was found to significantly decrease with increasing concentration of inhibitor. To
explore the inhibition of furan-thiazolidinone hybrids, IC50 values were calculated by
measuring normalized inhibition with increasing log inhibition concentration (Figure 18).
Our biochemical experimental results showed that compound 3, 6 and 11 inhibited OASS
1
0

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with IC50 in the range of 137.9-157.5 ꢀM concentration Table 2. Further these molecules
could be expected to inhibit thioredoxin reductase and other enzyme in E. histolytica.
3
. Conclusion
All the synthesized hybrids were capable of inhibiting the growth of E. histolytica out of
which seven compounds (IC = 0.27–1.48 ꢀM) were found to have better efficacy than the
5
0
standard drug MNZ (IC50 = 1.64 ꢀM) and the activity was substituent dependent. All the
seven hybrids offered remarkable cell viability. Among all hybrids, three compounds (3, 6
and 11) showed excellent binding energy for E. histolytica O-acetyle-L-serine sulfohydrolase
and E. histolytica thioredoxin reductase. In summary, three compounds (3, 6 and 11) showed
most promising antiamoebic activity and significant inhibition of O-acetyle-L-serine
sulfohydrolase, therefore these molecules may be subjected to further investigations for the
development of novel effective antiamoebic agents.
4
. Experimental
All the required chemicals were purchased from Merck and Aldrich Chemical Company
(USA). Precoated aluminium sheets (Silica gel 60 F254, Merck Germany) were used for thin-
layer chromatography (TLC) and spots were visualized under UV light. The melting points
were recorded on Veego instrument with model specifications REC-22038 A2 and are
uncorrected. Elemental analyses were performed on Elementar Vario analyzer and the results
are within ±0.4% of the theoretical values. IR spectra were acquired at Bruker FT-IR
1
13
spectrophotometer. H and C NMR were recorded on a Bruker Spectrospin DPX 400 MHz
and Bruker Spectrospin DPX 100 MHz spectrometer respectively, using CDCl as a solvent
3
and trimethylsilane (TMS) as the internal standard. Splitting patterns are designated as
follows; s, singlet; d, doublet; t, triplet; m, multiplet. Chemical shift values are given in ppm.
Mass spectra were recorded by ESI-MS (AB-Sciex 2000, Applied Biosystem).
1
1

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4
.1. General procedure for the synthesis of 1-(furan-2-ylmethyl)-3-phenylthiourea (1)
On treating phenylisothiocyanate (70 mmol) with furfuryl amine (70 mmol) in toluene white
precipitates were obtained which were collected by filtration, washed with toluene, and dried
to afford 1-(furan-2-ylmethyl)-3-phenylthiourea (1).
4
.1.1. 1-(Furan-2-ylmethyl)-3-phenylthiourea (1) Yield: 93%; white solid; m.p: 103°C;
Anal. calc. (%) for C H N OS: C 62.04, H 5.21, N 12.06, S 13.80. found: C 62.24, H 5.29,
1
2
12
2
-1
1
N 12.23, S 13.97; FT-IR νmax (cm ): 3279 (NH), 3059 (C-H, Ar-H), 1238 (C=S); HNMR
CDCl3) δ (ppm): 8.36 (s, 1H, NH), 7.43 (t, 2H, J=7.60 Hz, Ar-H), 7.33 (s, 1H, S=C-NH),
.30 (t, 1H, J=7.20 Hz, Ar-H), 7.22 (d, 2H, J=7.60 Hz, Ar-H), 6.33-6.27 (m, 3H, Ar-H), 4.85
(
7
13
(
d, 2H, J=5.2 Hz, CH ); C NMR (CDCl3) δ (ppm): 180.46, 150.25, 142.42, 136.01, 130.25,
2
1
27.35, 125.12, 110.51, 108.18, 42.34; ESI-MS: m/z = 232.30
4
.2. General procedure of synthesis of 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-
thiazolidin-4-one (2)
-(Furan-2-ylmethyl)-3-phenylthiourea (30 mmol) was dissolved in ethanol and then
1
anhydrous sodium acetate (60 mmol) and chloroacetic acid (37.5 mmol) were added in the
reaction mixture. The suspension was heated under reflux for 12 h and then the solvent was
evaporated. Water was added and the aqueous layer was extracted with ethyl acetate. The
organic layer was washed with saturated sodium bicarbonate and brine. The combined
organic extracts were dried over Na SO and concentrated in vacuo, consequently yellow
2
4
solid product 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one (2) was obtained.
4
.2.1. 3-(Furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one (2) Yield: 90%; light
brown solid; m.p: 95°C; Anal. calc. (%) for C H N O S: C 61.75, H 4.44, N 10.29, S 11.77.
1
4
12
2
2
-1
found: C 61.54, H 4.62, N 10.11, S 11.52; FT-IR νmax (cm ): 1723 (-N-C=O), 1620 (C=N),
1
2

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1
1
376 (tert. N), 754 (C-S-C); H NMR (CDCl3) δ (ppm): 7.38-7.33 (m, 3H, Ar-H), 7.17 (t,
1
H, J=7.6 Hz, Ar-H), 6.98 (d, 2H, J=7.2, Ar-H), 6.43 (d, 1H, Ar-H), 6.35-6.33 (m, 1H, Ar-
1
3
H), 5.04 (s, 2H, CH ), 3.81 (s, 2H, CH ); C NMR (CDCl ) δ (ppm): 171.31, 153.40, 149.09,
2
2
3
1
2
47.86, 142.37, 129.28, 124.70, 121.01, 110.50, 109.60, 39.06, 32.68; ESI-MS: m/z =
72.32.
4
.3. General procedure of synthesis of Furan-thiazolidinone hybrids (3-17)
A mixture of 3-(Furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one (1 mmol),
different aldehydes (1 mmol), hexahydropyridine (1.15 mmol), and ethanol (35 mL) were
heated under reflux for 11-12 h. The reaction mixture was cooled to room temperature and
the precipitates were obtained which were filtered, washed and recrystallized from ethanol to
furnished final products (3-17).
4
.3.1.
5-(3,
4-Dimethoxybenzylidene)-3-(furan-2-ylmethyl)-2-(phenylimino)-1,
3-
thiazolidin-4-one (3) Yield: 83%; yellow solid; m.p. 126°C; Anal. Calc. C H N O S: C
23
20
2
4
-1
6
5.70, H 4.79, N 6.66, S 7.63%. found: C 65.52, H 4.63, N 6.42, S 7.55%; FT-IR νmax (cm ):
1
1
710 (-N-C=O), 1643 (C=N), 1594 (C=C), 1374 (tert. N), 760 (C-S-C); H NMR (CDCl ) δ
3
(
ppm): 7.70 (s, 1H, =CH), 7.38 (t, 3H, J=7.6 Hz, Ar-H), 7.18 (t, 1H, J=7.6, Ar-H), 7.05-7.02
m, 1H, Ar-H), 7.01 (d, 2H, J=7.2 Hz, Ar-H), 6.91 (s, 1H, Ar-H), 6.87 (d, 1H, J=8.4 Hz, Ar-
(
H), 6.43 (s, 1H, Ar-H), 6.32-6.31 (m, 1H, Ar-H), 5.15 (s, 2H, CH ), 3.87 (s, 3H, OCH ), 3.83
2
3
1
3
(
s, 3H, OCH ); C NMR (CDCl ) δ (ppm): 166.49, 149.69, 149.21, 149.11, 148.05, 142.39,
3 3
1
5
31.33, 129.33, 126.67, 124.88, 123.42, 121.24, 118.95, 113.06, 111.26, 110.48, 109.49,
5.99, 39.06; ESI-MS: m/z = 420.48.
4
.3.2. 5-(3-Nitrobenzylidene) - 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one
(
4) Yield: 85%; off white solid; m.p. 153°C; Anal. Calc. C H N O S: C 62.21, H 3.73, N
21 15 3 4
-1
1
0.36, S 7.91%. found: C 62.49, H 3.85, N 10.57, S 7.64 %; FT-IR νmax (cm ): 1710 (-N-
1
3

ACCEPTED MANUSCRIPT
1
C=O), 1639 (C=N), 1595 (C=C), 1340 (tert. N), 767 (C-S-C); H NMR (CDCl ) δ (ppm):
3
8
.27 (s, 1H, Ar-H), 8.20 (d, 1H, J=7.6 Hz, Ar-H), 7.80 (s, 1H, =CH), 7.74 (d, 1H, J=7.6 Hz,
Ar-H), 7.61 (t, 1H, J=8.0 Hz, Ar-H), 7.43-7.40 (m, 3H, Ar-H), 7.24 (t, 1H, J=7.6 Hz, Ar-H),
1
3
7
.02 (d, 2H, J=7.6 Hz, Ar-H), 6.48 (s, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 5.20 (s, 2H, CH ); C
2
NMR (CDCl ) δ (ppm): 165.69, 148.81, 148.67, 148.14, 147.55, 142.54, 135.47, 134.63,
3
1
30.07, 129.48, 128.01, 125.30, 125.18, 124.48, 123.90, 121.02, 110.52, 109.75, 39.39; ESI-
MS: m/z = 405.42.
4
.3.3. 5-(4-Nitrobenzylidene) - 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one
(
5) Yield: 83%; yellow solid; m.p. 228°C; Anal. Calc. C H N O S: C 62.21, H 3.73, N
21
15
3
4
-1
1
0.36, S 7.91%. found: C 62.32, H 3.77, N 10.21, S 7.96 %; FT-IR νmax (cm ): 1704 (-N-
1
C=O), 1637 (C=N), 1590 (C=C), 1372 (tert. N), 756 (C-S-C); H NMR (CDCl ) δ (ppm):
3
8
.25 (d, 2H, J=8.4 Hz, Ar-H), 7.79 (s, 1H, =CH), 7.59 (d, 2H, J=8.4 Hz, Ar-H), 7.43-7.40 (m,
H, Ar-H), 7.25-7.22 (m, 1H, Ar-H), 7.02 (d, 2H, J=7.2 Hz, Ar-H), 6.48 (s, 1H, Ar-H), 6.36
3
1
3
(
s, 1H, Ar-H), 5.23 (s, 2H, CH ); C NMR (CDCl ) δ (ppm): 165.67, 148.73, 148.20, 147.60,
2 3
1
3
42.58, 139.86, 130.33, 129.49, 127.93, 126.28, 125.34, 124.21, 121.03, 110.54, 109.82,
9.40; ESI-MS: m/z = 405.42
4
.3.4.
5-(2,
5-Dimethoxybenzylidene)-3-(furan-2-ylmethyl)-2-(phenylimino)-1,
3-
thiazolidin-4-one (6) Yield: 83%; yellow solid; m.p. 99°C; Anal. Calc. C H N O S: C
2
3
20
2
4
-
1
6
1
5.70, H 4.79, N 6.66, S 7.63%. found: C 65.61, H 4.57, N 6.33, S 7.74 %; FT-IR νmax (cm ):
1
710 (-N-C=O), 1633 (C=N), 1588 (C=C), 1360 (tert. N), 750 (C-S-C); H NMR (CDCl ) δ
3
(ppm): 8.13 (s, 1H, =CH), 7.38-7.35 (m, 3H, Ar-H), 7.19 (t, 1H, J=7.2 Hz, Ar-H), 7.01 (d,
2
H, J=7.6 Hz, Ar-H), 6.90 (s, 1H, Ar-H), 6.86-6.81 (m, 2H, Ar-H), 6.45 (s, 1H, Ar-H), 6.34
13
(
s, 1H, Ar-H), 5.18 (s, 2H, -CH ), 3.81 (s, 3H, OCH ), 3.72 (s, 3H, OCH ); C NMR
2 3 3
(
CDCl ) δ (ppm): 166.26, 153.38, 152.81, 149.72, 149.29, 147.96, 142.32, 129.29, 126.58,
3
1
4

ACCEPTED MANUSCRIPT
1
24.82, 123.67, 121.88, 121.19, 116.02, 114.61, 112.02, 110.45, 109.42, 56.08, 55.81, 39.03;
ESI-MS: m/z = 420.48
4
.3.5. 5-(1, 3-Benzodioxol-5-ylmethylidene)-3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-
thiazolidin-4-one (7) Yield: 85%; yellow solid; m.p. 130°C; Anal. Calc. C H N O S: C
22
16
2
4
-1
6
5.33, H 3.99, N 6.93, S 7.93%. found: C 65.61, H 4.15, N 7.21, S 8.19 %; FT-IR νmax (cm ):
1
1
701 (-N-C=O), 1641 (C=N), 1583 (C=C), 1365 (tert. N), 744 (C-S-C); H NMR (CDCl ) δ
3
(ppm): 7.68 (s, 1H, =CH), 7.41-7.38 (m, 3H, Ar-H), 7.20 (t, 1H, J=7.2 Hz, Ar-H), 7.02-6.97
(m, 3H, Ar-H), 6.92 (s, 1H, Ar-H), 6.84 (d, 1H, J=8.0 Hz, Ar-H), 6.46 (s, 1H, Ar-H), 6.35 (s,
1
3
1
1
1
H, Ar-H), 6.00 (s, 2H, OCH ), 5.17 (s, 2H, CH ); C NMR (CDCl ) δ (ppm): 166.44,
2 2 3
49.54, 149.22, 149.11, 148.33, 148.07, 142.36, 131.14, 129.38, 128.01, 125.99, 124.90,
21.19, 119.18, 110.46, 109.46, 109.08, 108.93, 101.72, 39.09; ESI-MS: m/z = 404.43
4
.3.6. 5-(4-Hydroxybenzylidene) - 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-
one (8) Yield: 83%; brown solid; m.p. 153°C; Anal. Calc. C H N O S: C 67.00, H 4.28, N
2
1
16
2
3
-
1
7
1
1
.44, S 8.52%. found: C 65.26, H 4.41, N 7.66, S 8.59 %; FT-IR νmax (cm ): 1700 (-N-C=O),
1
626 (C=N), 1577 (C=C), 1369 (tert. N), 761 (C-S-C); H NMR (CDCl ) δ (ppm): 7.71 (s,
3
H, =CH), 7.41-7.37 (m, 3H, Ar-H), 7.31 (d, 2H, J=8.4 Hz, Ar-H), 7.21 (t, 1H, J=7.6 Hz, Ar-
H), 7.03 (d, 2H, J=7.6 Hz, Ar-H), 6.81 (d, 2H, J=8.8 Hz, Ar-H), 6.44 (s, 1H, Ar-H), 6.37 (bs,
1
3
1
1
1
H, OH), 6.33 (s, 1H, Ar-H), 5.17 (s, 2H, CH ); C NMR (CDCl ) δ (ppm): 166.88, 159.73,
2 3
50.12, 149.28, 148.22, 142.32, 132.32, 131.82, 129.34, 124.98, 124.78, 121.26, 117.15,
16.66, 110.46, 109.37, 39.40; ESI-MS: m/z = 376.42.
4
.3.7. 5-(4-Ethoxylbenzylidene) - 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-
one (9) Yield: 84%; off white solid; m.p. 107°C; Anal. Calc. C H N O S: C 68.30, H 4.98,
2
3
20
2
3
-1
N 6.93, S 7.93%. found: C 68.53, H 4.87, N 6.78, S 7.74%; FT-IR νmax (cm ): 1706 (-N-
1
C=O), 1635 (C=N), 1588 (C=C), 1373 (tert. N), 741 (C-S-C); H NMR (CDCl ) δ (ppm):
3
1
5

ACCEPTED MANUSCRIPT
7
.74 (s, 1H, =CH), 7.42-7.37 (m, 5H, Ar-H), 7.22 (t, 1H, J=7.6 Hz, Ar-H), 7.03 (d, 2H, J=7.2
Hz, Ar-H), 6.91 (d, 2H, J=8.8 Hz, Ar-H), 6.46 (s, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 5.18 (s, 2H,
13
CH ), 4.07 (m, 2H, CH ), 1.44 (t, 3H, J=7.2, CH ); C NMR (CDCl ) δ (ppm): 166.60,
2
2
3
3
1
1
60.32, 149.88, 149.29, 148.21, 142.34, 131.91, 131.23, 129.35, 126.17, 124.81, 121.25,
18.28, 115.02, 110.46, 109.42, 63.68, 39.04, 14.69; ESI-MS: m/z = 404.48
4
.3.8. 5-Benzylidene-3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-one (10)
Yield: 83%; dark yellow solid; m.p. 120°C; Anal. Calc. C H N O S: C 69.98, H 4.47, N
2
1
15
3
3
-
1
7
1
1
.77, S 8.90%. found: C 69.71, H 4.64, N 7.71, S 8.54 %; FT-IR νmax (cm ): 1711 (-N-C=O),
1
631 (C=N), 1586 (C=C), 1371 (tert. N), 727 (C-S-C); H NMR (CDCl ) δ (ppm): 7.79 (s,
3
H, =CH), 7.45-7.33 (m, 8H, Ar-H), 7.29 (t, 1H, J=7.6 Hz, Ar-H), 7.03 (d, 2H, J=7.6 Hz, Ar-
13
H), 6.47 (s, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 5.19 (s, 2H, -CH ); C NMR (CDCl ) δ (ppm):
2
3
1
1
66.31, 149.52, 149.18, 148.03, 142.38, 133.74, 131.24, 129.95, 129.79, 129.36, 129.01,
24.91, 121.51, 121.19, 110.47, 109.47, 39.14; ESI-MS: m/z = 360.42
4
4
.3.9. 5-(Pyridin-2-ylmethylidene)-3-(furan-2-ylmethyl)-2-(phenylimino) - 1, 3-thiazolidin-
-one (11) Yield: 83%; yellow solid; m.p. 196°C; Anal. Calc. C H N O S: C 66.46, H 4.18,
20
15
3
2
-1
N 11.63, S 8.87%. found: C 66.33, H 4.37, N 11.54, S 8.56 %; FT-IR νmax (cm ): 1705 (-N-
1
C=O), 1635 (C=N), 1581 (C=C), 1373 (tert. N), 750 (C-S-C); H NMR (CDCl ) δ (ppm):
3
8
.61 (s, 1H, Ar-H), 7.67 (s, 1H, =CH), 7.65 (m, 1H, Ar-H), 7.42 (d, 1H, J=8.0 Hz, Ar-H),
.39-7.35 (m, 3H, Ar-H), 7.18-7.13 (m, 2H, Ar-H), 7.03 (d, 2H, J=7.2 Hz, Ar-H), 6.43-6.42
7
1
3
(
m, 1H, Ar-H), 6.32-6.31 (m, 1H, Ar-H), 5.16 (s, 2H, CH2); C NMR (CDCl ) δ (ppm):
3
1
66.48, 153.12, 152.28, 149.31, 149.29, 147.93, 142.34, 136.72, 129.31, 126.98, 126.78,
26.63, 124.63, 122.85, 121.32, 110.49, 109.37, 38.52; ESI-MS: m/z = 361.41.
1
4
.3.10. 5-(3, 4, 5-Trimethoxybenzylidene)-3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-
thiazolidin-4-one (12) Yield: 83%; off white solid; m.p. 102°C; Anal. Calc. C H N O S: C
24
22
2
5
1
6

ACCEPTED MANUSCRIPT
-1
6
3.98, H 4.92, N 6.22, S 7.12%. found: C 63.91, H 4.85, N 6.44, S 7.31 %; FT-IR νmax (cm ):
1
1
710 (-N-C=O), 1641 (C=N), 1593 (C=C), 1372 (tert. N), 759 (C-S-C); H NMR (CDCl ) δ
3
(ppm): 7.67 (s, 1H, =CH), 7.36-7.32 (m, 3H, Ar-H), 7.16 (d, 1H, J=7.2 Hz, Ar-H), 7.00 (d,
2
H, J=7.6 Hz, Ar-H), 6.63 (s, 2H, Ar-H), 6.43 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 5.15 (s, 2H,
1
3
-
CH ), 3.84 (s, 3H, OCH ), 3.79 (s, 6H, OCH ); C NMR (CDCl ) δ (ppm): 166.19,
2 3 3 3
1
1
53.49,149.18, 147.83, 142.36, 139.95, 139.92, 131.34, 129.26, 124.94, 121.17, 120.69,
10.46, 109.44, 107.49, 107.47, 60.93, 56.30, 39.14; ESI-MS: m/z = 450.50.
4
.3.11. 5-(3-Chlorobenzylidene) - 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-
one (13) Yield: 83%; yellow solid; m.p. 103°C; Anal. Calc. C H N O SCl: C 63.87, H
2
1
15
2
2
-1
3
.83, N 7.09, S 8.12%. found: C 63.61, H 3.57, N 7.25, S 8.47 %; FT-IR νmax (cm ): 1712 (-
1
N-C=O), 1636 (C=N), 1589 (C=C), 1371 (tert. N), 754 (C-S-C); H NMR (CDCl ) δ (ppm):
3
7
.70 (s, 1H, =CH), 7.43-7.39 (m, 4H, Ar-H), 7.32 (s, 3H, Ar-H), 7.23 (t, 1H, J=7.2 Hz, Ar-
H), 7.03 (d, 2H, J=7.2 Hz, Ar-H), 6.47-6.46 (m, 1H, Ar-H), 6.36-6.35 (m, 1H, Ar-H), 5.19 (s,
1
3
2
1
1
H, CH2); C NMR (CDCl ) δ (ppm): 166.00, 149.00, 148.83, 147.77, 142.46, 135.50,
3
35.07, 130.22, 129.77, 129.70, 129.48, 129.43, 127.60, 125.09, 123.20, 121.11, 110.50,
09.62, 39.25; ESI-MS: m/z = 394.87
4
.3.12. 5-(4-Methylbenzylidene) - 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-
one (14) Yield: 87%; pale yellow solid; m.p.121°C; Anal. Calc. C H N O S: C 70.57, H
2
2
18
2
2
-1
4
.85, N 7.48, S 8.56%. found: C 70.32, H 4.81, N 7.33, S 8.27 %; FT-IR νmax (cm ): 1708 (-
1
N-C=O), 1639 (C=N), 1593 (C=C), 1361 (tert. N), 755 (C-S-C); H NMR (CDCl ) δ (ppm):
3
7
.77 (s, 1H, =CH), 7.42-7.38 (m, 3H, Ar-H), 7.35 (d, 2H, J=8.0 Hz, Ar-H), 7.21 (d, 3H, 8.0
Hz, Ar-H), 7.04 (d, 2H, J=7.6 Hz, Ar-H), 6.46 (m, 1H, Ar-H), 6.36-6.34 (m, 1H, Ar-H), 5.19
1
3
(
s, 2H, -CH ), 2.36 (s, 3H, CH ); C NMR (CDCl ) δ (ppm): 166.46, 149.72, 149.23,148.10
2 3 3
,
142.36 ,140.41, 131.38, 130.96, 130.03, 129.77, 129.35, 124.86, 121.22, 120.24, 110.46,
09.45, 39.08, 21.51; ESI-MS: m/z = 374.45
1
1
7

ACCEPTED MANUSCRIPT
4
.3.13. 5-(4-Isopropoxybenzylidene)
-
3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-
thiazolidin-4-one (15) Yield: 83%; yellow solid; m.p. 103°C; Anal. Calc. C H N O S: C
2
4
22
2
3
-
1
6
1
8.88, H 5.30, N 6.69, S 7.66%. found: C 68.61, H 5.37, N 6.61, S 7.47 %; FT-IR νmax (cm ):
1
708 (-N-C=O), 1636 (C=N), 1594 (C=C), 1375 (tert. N), 770 (C-S-C); H NMR (CDCl ) δ
3
(ppm): 7.71 (s, 1H, =CH), 7.39-7.34 (m, 5H, Ar-H), 7.19 (t, 1H, J=7.6 Hz, Ar-H), 7.01 (d,
2
5
H, J=8.0 Hz, Ar-H), 6.87 (d, 2H, J=8.8 Hz, Ar-H), 6.44 (s, 1H, Ar-H), 6.33 (s, 1H, Ar-H),
13
.16 (s, 2H, CH ), 4.58-4.52 (m, 1H, CH), 1.32 (d, 6H, J=6.0 Hz, CH ); C NMR (CDCl ) δ
2
3
3
(ppm): 166.66, 159.39, 149.96, 149.28, 148.20, 142.37, 131.97, 131.30, 129.36, 125.91,
1
24.82, 121.26, 118.11, 116.01, 110.48, 109.45, 70.08, 39.04, 21.94; ESI-MS: m/z = 418.50
4
4
.3.14. 5-(4-Methoxybenzylidene) - 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-
-one (16) Yield: 83%; yellow solid; m.p. 135°C; Anal. Calc. C H N O S: C 67.67, H 4.65,
22
18
2
3
-1
N 7.17, S 8.21%. found: C 67.39, H 4.32, N 6.94, S 8.31 %; FT-IR νmax (cm ): 1706 (-N-
1
C=O), 1633 (C=N), 1586 (C=C), 1368 (tert. N), 743 (C-S-C); H NMR (CDCl ) δ (ppm):
3
7
.74 (s, 1H, =CH), 7.40-7.39 (m, 5H, Ar-H), 7.21 (t, 1H, J=7.6 Hz, Ar-H), 7.04 (d, 2H, J=7.2
Hz, Ar-H), 6.92 (d, 2H, J=8.4 Hz, Ar-H), 6.46 (s, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 5.18 (s, 2H,
1
3
CH ), 3.82 (s, 3H, OCH ); C NMR (CDCl ) δ (ppm): 166.54, 160.90, 149.81, 149.31,
2
3
3
1
1
48.20, 142.33, 131.87, 131.11, 129.34, 126.40, 124.82, 121.24, 118.54, 114.57, 110.46,
09.40, 55.39, 39.06; ESI-MS: m/z = 390.45
4
.3.15. 5-(4-Chlorobenzylidene) - 3-(furan-2-ylmethyl)-2-(phenylimino)-1, 3-thiazolidin-4-
one (17) Yield: 85%; off white solid; m.p: 113°C; Anal. Calc. C H N O SCl: C 63.87, H
2
1
15
2
2
-1
3
.83, N 7.09, S 8.12%. found: C 63.96, H 4.01, N 7.26, S 8.17 %; FT-IR νmax (cm ): 1712 (-
1
N-C=O), 1646 (C=N), 1593 (C=C), 1377 (tert. N), 733 (C-S-C); H NMR (CDCl ) δ (ppm):
3
7
.72 (s, 1H, =CH), 7.43-7.39 (m, 3H, Ar-H), 7.36 (s, 4H, Ar-H), 7.23 (t, 1H, J=7.2 Hz, Ar-
H), 7.03 (d, 2H, J=7.6 Hz, Ar-H), 6.47 (m, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 5.19 (s, 2H, -CH2);
1
3
C NMR (CDCl ) δ (ppm): 166.17, 149.10, 149.02, 147.91, 142.48, 135.81, 132.16, 131.08,
3
1
8

ACCEPTED MANUSCRIPT
1
3
29.75, 129.43, 129.33, 125.06, 122.10, 121.15, 110.52, 109.65, 39.19; ESI-MS: m/z =
94.87
5
. X-Ray crystal structure determination
Three-dimensional X-ray data were collected on a Bruker Kappa Apex CCD diffractometer at
low temperature for 2, 6, 8, 13 and 15 by the scan method. Reflections were measured
from a hemisphere of data collected from frames, each of them covering 0.3º in . A total of
5012 for 2, 56226 for 6, 98155 for 8, 60473 for 13 and 28530 for 15 reflections measured
φ-ω
ω
4
were corrected for Lorentz and polarization effects and for absorption by multi-scan methods
based on symmetry-equivalent and repeated reflections. Of the total, 3592 for 2, 4254 for 6,
5
450 for 8, 5121 for 13 and 3681 for 15, independent reflections exceeded the significance
level ( F /  F ) > 4.0, respectively. After data collection, in each case an empirical
σ
absorption correction (SADABS) [32] was applied, and the structures were solved by direct
2
methods and refined by full matrix least-squares on F data using SHELX suite of programs
[
33]. In 2, 13 and 15 hydrogen atoms were located in difference Fourier map and left to refine
freely. In 6, hydrogen atoms were located in difference Fourier map and left to refine freely,
except for C(22) and C(23), which were included in calculation position and refined in the
riding mode. In 8, hydrogen atoms were located in difference Fourier map and left to refine
freely, except for O(3), C(4S) and O(6), which were included in calculation position and
refined in the riding mode. Refinements were done with allowance for thermal anisotropy of
all non-hydrogen atoms. A final difference Fourier map showed no residual density outside:
–
3
–3
–3
0
.415 and -0.215 e.Å for 2, 0.343 and -0.287 e.Å for 6, 0.657 and -0.584 e.Å for 8, 0.689
–
3
–3
and -0.577 e.Å for 13 and 0.533 and -0.344 e.Å for 15. A weighting scheme w =
2
2
2
2
2
2
1
/[σ (F ) + (0.048200 P) + 0.388800 P] for 2, w = 1/[σ (F ) + (0.043600 P) + 0.630100
o o
2
2
o
2
2
2
o
2
P] for 6, w = 1/[σ (F ) + (0.057000 P) + 3.064500 P] for 8, w = 1/[σ (F ) + (0.054700 P)
1
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2
2
2
+
0.500400 P] for 13 and w = 1/[σ (F ) + (0.067300 P) + 0.191600 P] for 15, where P =
o
2
2
(
|F | + 2|F | )/3, were used in the latter stages of refinement. Further details of the crystal
o c
structure determination are given in Table 1. CCDC 1472093-1472097 contain the
supplementary crystallographic data for the structures reported in this paper. These data can
be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
(+44) 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk. Supplementary data associated with
this article can be found, in the online version, at doi: $$$$$.
6
. Pharmacological Evaluation
6
.1. Antiamoebic activity
All the desired compounds (1-17) were screened for in vitro antiamoebic activity against
HM1: IMSS strain of E. histolytica by microdilution method [21]. E. histolytica trophozoites
were cultured in culture tubes by using Diamond TYIS-33 growth medium [34]. The test
compounds (1mg) were dissolved in DMSO (40 µL, level at which no inhibition of amoeba
occurs) [35]. The stock solutions of the compounds were prepared freshly before use at a
concentration of 1 mg/mL. Two-fold serial dilutions were made in the wells of 96-well
microtiter plate (costar). Each test includes metronidazole as a standard amoebicidal drug,
control wells (culture medium plus amoebae) and a blank (culture medium only). All the
experiments were carried out in triplicate at each concentration level and repeated thrice. The
amoeba suspension was prepared from a confluent culture by pouring off the medium at 37
°
C and adding 5 mL of fresh medium, chilling the culture tube on ice to detach the organisms
from the side of flask. The number of amoeba/mL was estimated with the help of a
haemocytometer, using trypan blue exclusion to confirm the viability. The suspension was
5
diluted to10 organism/mL by adding fresh medium and 170 ꢀL of this suspension was added
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to the test and control wells in the plate so that the wells were completely filled (total volume,
4
3
40 mL). An inoculum of 1.7 x 10 organisms/well was chosen so that confluent, but not
excessive growth, took place in control wells. Plate was sealed and gassed for 10 min with
nitrogen before incubation at 37 °C for 72 h. After incubation, the growth of amoeba in the
plate was checked with a low power microscope. The culture medium was removed by
inverting the plate and shaking gently. Plate was then immediately washed with sodium
chloride solution (0.9%) at 37 °C. This procedure was completed quickly and the plate was
not allowed to cool in order to prevent the detachment of amoeba. The plate was allowed to
dry at room temperature and the amoebae were fixed with methanol and when dried, stained
with (0.5%) aqueous eosin for 15 min. The stained plate was washed once with tap water,
then twice with distilled water and then allowed to dry. A 200 µL portion of 0.1 N sodium
hydroxide solutions was added to each well to dissolve the protein and release the dye. The
optical density of the resulting solution in each well was determined at 490 nm with a
microplate reader. The % inhibition of amoebal growth was calculated from the optical
densities of the control and test wells and plotted against the logarithm of the dose of the drug
tested. Linear regression analysis was used to determine the best fitting line from which the
IC value was found. The IC values are reported in Table 2.
5
0
50
6
6
.2 Toxicity assays
.2.1. Cell viability (MTT) assay:
The Chinese hamster ovary (CHO) cells were obtained from NCCS (Pune, India). The cells
were cultured in Roswell Park Memorial Institute (RPMI) (Sigma Aldrich) with 10% foetal
bovine serum (Gibco-life technologies) and 1% penicillin-streptomycin-neomycin. The effect
of compounds 3, 6, 8, 11, 12, 14, 16 and the standard drug MNZ on cell proliferation was
measured by using an MTT-based assay [23]. Briefly, the cells (5,000/well) were incubated
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in triplicate in a 96-well plate in the presence of various concentrations of compounds 3, 6, 8,
1
1, 12, 14, 16 as well as MNZ or vehicle DMSO (dimethyl sulphoxide) alone in a final
0
volume of 100 µL at 37 C and 5% CO in and humidified atmosphere chamber for 48 h and
2
7
2 h. At the end of this time period, 20 µL of MTT solution (5 mg/mL in PBS) was added to
0
each well, and the cells were incubated at 37 C in a humidified atmosphere chamber for 4 h.
After 4 h, the supernatant was removed from each well. The colored formazan crystal
produced from MTT was dissolved in 100 µL of DMSO, and then the absorbance value was
measured at 570 nm by a multi-scanner auto reader (Bio-Rad). The following formula was
used for the calculation of the percentage of cell viability [36]:
CV= (absorbance of the experimental samples/ absorbance of the control sample) x 100
6
.3 In silico pharmacokinetics analysis
.3.1. Measuring drug -likeness
6
Drug like properties of compounds were analyzed by the online tool Molinspiration server
http://www.molinspiration.com). A Molinspiration support for calculation of important
(
molecular properties is based on Lipinski Rules of five such as (molecular weight, number of
hydrogen bond donors and acceptors) [25].
6
.3.2. ADMET Predictions
Discovery studio 3.5 (Accelrys San Diego, USA) was used to generate ADMET values. The
Absorption, Distribution, Metabolism, Excretion and Toxicity commonly abbreviated as
ADMET properties are considered before designing a drug as these properties plays an
important role in clinical phases. Administration of these properties before drug designing
leads to cost savings in drug design [37]. These studies resulted in identification of
antiamoebic compounds. The after effects of drug intake are administered by TOPKAT. It
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assesses the toxicological endpoints by Quantitative Structure Toxicity Relationship (QSTR).
Ames mutagen predication, Ames probability, Ames enrichment and weight of evidence are
tested by toxicity profile of the compounds.
6
.3.3. Preparation of ligands
The structures of ligands were drawn by using Chemdraw ultra 8.0. The structure of ligand
was copied in a smile form and then translated using online smile translator
(http://cactus.nci.nih.gov/translate/). The PDB file of ligand was then generated and this PDB
file was converted into PDBQT file by such process like (detect root, chose the torsion and
set the number of torsion) with using Auto Dock Tools (ADT) version 1.5.6 from the Scripps
Research Institute.
6
.3.4. Selection and preparation of target protein
The crystal structure of E. histolytica thioredoxin reductase (EhTrR) and E. histolytica O-
acetyl serine sulfhydrylase (EhOASS) were selected as a drug target. Its structure (PDB ID:
4
A5L and 3BM5) has been retrieved from RCSB protein Data Bank. All water molecules
were removed and on the final stage hydrogen atoms were added to the target protein
molecule. Then PDB file was converted into PDBQT file by using Auto Dock Tools (ADT)
version 1.5.6 from the Scripps Research Institute.
6
.3.5. Molecular docking
Molecular docking stimulation using the ligand molecules with EhTrR (PDB ID: 4A5L) and
EhOASS (PDB ID: 3BM5) were conducted using Autodock 4.2 docking suite by employing
Lamarckian genetic algorithm [30]. The ligands were set to explore and flexible to rotate
most probable binding poses, while receptor was kept rigid. The grid maps representing the
center of active site pocket for the ligand were calculated with Autogrid. The dimensions of
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the grid for EhTrR and EhOASS were 64 × 64 × 64 grid points with aspacing of 0.575 Å
between the grid points but centered on the ligand for both receptor (-6.670, 3.419 and -
1
4.903 coordinates and -5.500, 26.609 and 0.418 coordinates). In the present docking study
was performed by each run with population of 150 individuals, rate of gene mutation 0.02,
cross-over rate 0.8 and the remaining parameters were set as default. Ten poses docking
conformations were generated and the best docked conformation was selected based on the
Autodock binding energy (Kcal/mol), for further analysis. Finally, the results generated were
visualized by PyMOL viewer for analysis minimum binding energy (Kcal/mol), Ki
(
Inhibition constant) value (ꢀM), and hydrogen and hydrophobic interaction of the docked
inhibitor to the modeled structure.
6
.5 Enzymatic Inhibition Assay
The sulfhydrylase activity was monitored using TNB (5-thio-2-nitrobenzoate) as an
alternative substrate as described previously [38]. O-acetyl L-serine (OAS), 5, 5’-Dithiobis-
(2-Nitrobenzoic Acid) (DTNB) and HEPES buffer were purchased from sigma. The
disappearance (decrease in color of TNB as it was utilized in reaction) of TNB was measured
continuously with increasing concentration of Inhibitor at 412 nm using Uv-visible
spectrophotometer (GE Healthcare). A typical assay contained 100mM HEPES pH 7.0, 50
mM TNB, 90 ꢀg EhOASS and inhibitor concentration was varied from 0.25 to 500 ꢀM. This
reaction mixture was incubated at room temperature for 30 min. The reaction was started by
the addition of substrate OAS and a decrease in enzyme activity is monitored over a fixed
interval of time in the presence of varied concentrations of inhibitor. The pattern of the
standard reaction is compared with the ones with the inhibitor and percentage decrease in
activity calculated using the equation:
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1
00 − ((decrease in absorbance for reactions with inhibitor/decrease in absorbance for
standard reaction) × 100) [39].
To determine the IC for inhibitors, its concentration was varied from 0.25 to 500 ꢀM, while
5
0
keeping the OASS concentration constant at 20 ꢀM.
Acknowledgement
One of the authors MFA is thankful to University Grant Commission, India for providing
BSR meritorious fellowship.
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