Synthesis and structure-activity-relationship studies of thiazolidinediones as antiplasmodial inhibitors of the Plasmodium falciparum cysteine protease falcipain-2

Article abstract of DOI:10.1016/j.ejmech.2014.11.061

Following a structure-based virtual screening, a series of 2,4 thiazolidinediones was synthesized in order to explore structure activity relationships for inhibition of the Plasmodium falciparum cysteine protease falcipain-2 (FP-2) and of whole cell antiparasitic activity. Most compounds exhibited low micromolar antiplasmodial activities against the P. falciparum drug resistant W2 strain. The most active compounds of the series were tested for in vitro microsomal metabolic stability and found to be susceptible to hepatic metabolism. Subsequent metabolite identification studies highlighted the metabolic hot spots. Molecular docking studies of a frontrunner inhibitor were carried out to determine the probable binding mode of this class of inhibitors in the active site of FP-2.

Full text of DOI:10.1016/j.ejmech.2014.11.061

European Journal of Medicinal Chemistry 90 (2015) 507e518
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and structureeactivity-relationship studies of
thiazolidinediones as antiplasmodial inhibitors of the Plasmodium
falciparum cysteine protease falcipain-2
Rajni Kant Sharma a, Yassir Younis a, Grace Mugumbate a, Mathew Njoroge a, Jiri Gut b,
, c, d, *
Philip J. Rosenthal b, Kelly Chibale a
a Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
b Department of Medicine, University of California, San Francisco, CA 94143, USA
c Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
d South African Medical Research Council, Drug Discovery and Development Unit, University of Cape Town, Rondebosch 7701, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Following a structure-based virtual screening, a series of 2,4 thiazolidinediones was synthesized in order
to explore structure activity relationships for inhibition of the Plasmodium falciparum cysteine protease
falcipain-2 (FP-2) and of whole cell antiparasitic activity. Most compounds exhibited low micromolar
antiplasmodial activities against the P. falciparum drug resistant W2 strain. The most active compounds
of the series were tested for in vitro microsomal metabolic stability and found to be susceptible to hepatic
metabolism. Subsequent metabolite identification studies highlighted the metabolic hot spots. Molecular
docking studies of a frontrunner inhibitor were carried out to determine the probable binding mode of
this class of inhibitors in the active site of FP-2.
Received 4 August 2014
Received in revised form
25 November 2014
Accepted 29 November 2014
Available online 2 December 2014
Keywords:
Antiplasmodial agents
Thiazolidinone
Falcipain-2
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
FP-2 inhibitors [6e20]. In an effort to identify potential antimalarial
inhibitors of FP-2, structure-based virtual screening of the ZINC
Malaria is a major cause of morbidity and mortality. According
to the 2012 World Health Organization (WHO) report, malaria is
responsible for an estimated 500,000e900,000 deaths each year,
especially among children and pregnant women [1]. Plasmodium
falciparum, the most virulent human malaria parasite and Plasmo-
dium vivax are responsible for more than 95% of malaria cases in the
world. In light of established and continuing development of
resistance of P. falciparum to most antimalarial drugs, the identifi-
cation and characterization of novel antimalarial chemotypes is an
important priority [2e4].
The P. falciparum cysteine protease falcipain-2 (FP-2), is a key
enzyme in the life cycle of P. falciparum since it is involved in the
digestion of host haemoglobin in the digestive vacuole of the
parasite, providing amino acids for its survival and development
[5]. This makes FP-2 an attractive target for antimalarial drug
design. Hence, to date, many chemotypes have been identified as
database, which contains more than 13 million drug like com-
pounds, was previously carried out. A total of 32 high ranked
compounds were purchased from the SPECs chemical database
(www.specs.net), and 14 compounds with activity against the drug
resistant P. falciparum W2 strain and FP-2 were identified [21].
Amongst these structures is a thiazolidine-2,4-dione (TZD) deriv-
ative, TZD 4 (Fig. 1), which exhibited modest activity against FP-2
(IC50 ¼ 25.5
m
M) and P. falciparum (EC50 ¼ 0.45
mM). Compound
TZD 4 was selected for subsequent hit validation through resyn-
thesis and biological retesting. Although thiazolidinedione de-
rivatives have been reported to have antimicrobial, anti-
proliferative, anti-viral, anti-convulsant, anti-inflammatory and
anti-cancer activities [22e28], to our knowledge their anti-
plasmodial activity has not previously been studied.
In this paper we report the synthesis and structureeactivity-
relationship studies around the thiazolidinedione core. In addition,
we report microsomal metabolic stability profiling and molecular
docking studies of frontrunner compounds.
* Corresponding author. Department of Chemistry, University of Cape Town,
Rondebosch 7701, South Africa.
E-mail address: Kelly.Chibale@uct.ac.za (K. Chibale).
http://dx.doi.org/10.1016/j.ejmech.2014.11.061
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

508
R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
Table 1
In vitro antiplasmodial activities of TZD 4, 4aev compounds against FP-2 and
P. falciparum (IC50 mM).
Cpd
R
P. falciparum
EC50 M)
FP2 IC50 Cpd R
M)
W2 EC50 FP2 IC50
M) M)
(
m
(
m
(
m
(m
Fig. 1. Chemical structure of the thiazolidine-2,4-dione derivative TZD 4 and two sites
of modifications as depicted by blue and pink rings. (For interpretation of the refer-
ences to colour in this figure legend, the reader is referred to the web version of this
article.)
TZD
4
0.88
>50
4k
>10
35.28
4a
4b
4c
4.65
>50
>50
4l
>10
>10
>10
44.97
33.25
2. Results and discussion
2.1. Chemistry
>10
4m
4n
The target compounds TZD 4, 4aet were synthesized using an
efficient 3-step synthetic route (Scheme 1; a). The synthesis
commenced with commercially available 1-phenyl piperazine 1,
which was reacted with chloroacetyl chloride in the presence of
triethylamine at 0 ꢀC for 1 h to give 2-chloro-1-(4-phenylpiperazin-
1-yl)ethanone 2. This intermediate and 2,4-thiazolidinedione 5, in
acetone under basic conditions, were heated for 24 h to yield the
key intermediate 3, which was reacted with appropriate
substituted benzaldehydes using the Knoevenagel condensation
reaction under microwave irradiation to obtain a library of C-5
substituted analogues [29]. By fixing the trimethoxyphenyl sub-
stituent at the C-5 position and varying substituents at the N-3
position, two sets of the target compounds 9aej and 11aei were
synthesized as shown in (Scheme 1; b, c). For the synthesis of an-
alogues 9aej, the free acid intermediate 7 was prepared and sub-
>10
>50
>50
>50
4d
4e
4f
>10
>10
>10
4o
4p
4q
>10
>10
>10
>50
>50
>50
13.44
18.04
4g
4h
>10
>10
15.80
23.88
4r
4s
>10
>10
45.71
45.33
38.80
4i
4j
>10
>10
23.89
35.28
4t
3
>10
>10
jected to
a Knoevenagel condensation reaction with 2,4,6-
trimethoxybenzaldehyde to obtain the key intermediate 8 as
shown in Scheme 1; b. The intermediate 8 was reacted with
appropriate substituted piperazines to deliver the target com-
pounds 9aej in moderate yield. The second set of N-3 linked ana-
logues 11aei was synthesized by a similar synthetic protocol as
described in Scheme 1; c.
H
>50
ART
CQ
E64
0.019
0.033
ART
CQ
0.019
0.033
0.047 E64
0.047
2.2. Biological evaluation
were included as controls in all experiments. A first set of analogues
was designed in order to explore the SAR around the trimethox-
yphenyl group of TZD 4 at position 5, as shown in Table 1. In this
work, the resynthesised TZD 4 exhibited relatively weak activity
The activities of all synthesized compounds were determined
in vitro against FP-2 and the chloroquine resistant W2 strain of
P. falciparum (Tables 1 and 2). E-64, chloroquine and artesunate
Scheme 1. Reagents and conditions: (a) (i) chloroacetyl chloride, Et3N, DCM, 0 ꢀC, 1 h; (55%) (ii) 2,4 thiazolidinedione, K2CO3, acetone, 60e70 ꢀC, 24 h; (52%) (iii) substituted
aldehydes, NH4OH, toluene, MW, 170 ꢀC, 20e30 min; (b) (i) bromoacetylmethylester, NaH, DMF,
0 4 h; (52%) (iii) 2,4,6-
ꢀCe25 ꢀC, 24 h; (ii) HBr (48%), 100 ꢀC,
trimethoxybenzaldehyde, piperidine, AcOH, 50 ꢀC, 5 h; (69%) (iv) substituted piperazines, EDCI, HOBt$H2O, DIPEA, DMF, 0 ꢀCe25 ꢀC, 24 h; (c) (i) 2,4,6-trimethoxybenzaldehyde,
piperidine, AcOH, 50 ꢀC, 5 h; (76%) (ii) alkyl/aryl halides, NaH, DMF, 0 ꢀCe25 ꢀC, 14 h. For complete structure see Tables 1e2.

R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
509
Table 2
In vitro antiplasmodial activities of 8, 9aej, 10 and 11aei compounds against the W2 (EC50 mM) strain and FP-2 enzyme of P. falciparum (IC50 mM).
.
Cpd
R
W2 EC50
(
mM)
FP2 IC50
(
m
M)
Cpd
R
W2 EC50
(m
M)
FP2 IC50 (mM)
8
9a
CH2CO2H
>10
5.28
>50
>50
10
11a
H
CH3
>10
>10
47.84
>50
9b
9c
9d
9e
>10
3.56
1.6
>50
>50
>50
>50
11b
11c
11d
11e
CH2CH3
>10
>10
8.88
>10
>50
36.24
43.35
29.08
1.21
9f
4.70
1.11
0.55
33.75
12.64
16.25
11f
11g
11h
11i
8.75
>10
>50
>50
>50
29.08
9g
9h
>10
9i
9j
1.43
3.97
11.23
12.24
>10
ART
CQ
0.019
0.033
ART
CQ
0.019
0.033
E64
0.047
E64
0.047
Cpd: compound.
against P. falciparum and FP-2, with EC50
¼
0.88
m
M
and
resulted in improved enzyme inhibitory activity for 4e
M). Significantly reduced
enzyme activity was observed on introduction of para halogen or
di-halogen substitutions on the phenyl ring as in 4d, 4o, 4n and 4p
IC50 > 50 M respectively, compared to previously reported activ-
m
(IC50 ¼ 13.44
m
M) and 4f (IC50 ¼ 18.04
m
ities [21]. Removal of one or two methoxy substituents in TZD 4, did
not enhance inhibition of FP-2 as shown by activities of 4aec
(IC50 > 50
mM). However, introduction of strong electron with-
(IC50 > 50
mM). Interestingly, ortho halogen substitutions resulted in
drawing groups (OCF3, CF3) at the para position of the phenyl ring
increased enzyme inhibitory activity, as exemplified by 4g
(IC50 ¼ 15.80
substituted
m
M) and 4h (IC50 ¼ 23.88
compound 4s displayed
M), but replacement of the methyl group with iso-
M).
m
M). The meta methyl
modest potency
(IC50 ¼ 45.33
m
propyl 4i led to marked improvement in potency (IC50 ¼ 23.89
m
However, no improvement in activity against P. falciparum was
observed.
The next phase of SAR exploration was directed at optimising
position 3 of the thiazolidinedione core while fixing the trime-
thoxyphenyl group, as shown in Table 2. Removal or replacement of
the right hand side of compound TZD 4 with aliphatic alkyl moi-
eties or substituted benzyl groups revealed no significant
improvement in FP-2 inhibitory activity as in 8, 11a, 11b and 11feh
(IC50 > 50 mM). Interestingly, introducing a chloro substituent at the
ortho, meta and para positions of the phenyl ring led to significant
improvement in enzyme inhibitory and W2 activities, as demon-
strated by 9g (FP-2 IC50 ¼ 12.64
IC50 ¼ 16.25 M; W2 EC50 ¼ 0.55
M). Similarly, introducing the electron with-
m
m
M; W2 EC50 ¼ 1.11
m
M), 9h (FP-2
m
M), and 9i (FP-2 IC50 ¼ 11.23
m
M;
W2 EC50 ¼ 1.43
m
drawing cyano group at the para position of the phenyl ring
resulted in moderate enzyme inhibitory and whole cell potency, as
Fig. 2. Comparison of the docked (pink) and crystal structure (green) conformations of
E64. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
demonstrated by compound 9j (FP-2 12.24
mM; W2 3.97
mM), while
compound 9e (FP-2 IC50 > 50
mM; W2 EC50 ¼ 1.21
mM) with the para

510
R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
Fig. 3. Binding mode of compound 9i (2D structure) in binding pocket of FP2. A) Electrostatic potential (red e negative and blue e positive) surface structure of FP-2 showing
subsites S1, S2, and S3. The yellow sticks and balls (coloured by atom type) depict the docked conformation of compound 9i in the binding site of FP-2. C) 2D representation of
interactions between compound 9i and some amino acid residues in S1 and S2 subsites of FP-2 (green e hydrophobic, cyan-polar, pink ionic residues and yellow shows solvent
exposed atoms in the ligand). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
methoxy substituent showed no enzyme inhibition at the highest
concentration tested, but retained whole cell activity (W2
antiplasmodial activity, as exemplified by compounds 9c (FP-2
IC50 > 50
EC50 ¼ 1.6
m
M; W2 EC50 ¼ 3.56
mM), 9d (FP-2 IC50 > 50 mM; W2
EC50 ¼ 1.21
mM). Replacement of the phenyl ring with cyclohexyl
mM). Thus, a lack of correlation between FP2 inhibitory
and 4-pyridyl substituents also resulted in compounds with no
enzyme inhibitory activity at the highest concentration tested, but
activity and whole cell activity was evident, suggesting that com-
pounds were acting against other yet unknown plasmodial targets.
2.3. Molecular modelling
Table 3
Results from the in vitro microsomal stability of TZD 4, 9g, 9h, 9i, 9j.
Amongst the tested analogues, compound 9i displayed the
highest inhibitory activity against FP-2. In order to further under-
stand interactions between FP-2 and compound 9i, docking cal-
culations were carried out using Glide 5.7 [30,31]. Out of the four
crystal structures of FP-2 reported in the Protein Databank, the
structure co-crystallized with a low molecular weight, epox-
ysuccinate (E64) (PDB ID: 3BPF) was used. The co-crystallized in-
hibitor was removed from the binding pocket and to validate our
docking calculations the inhibitor, E64 was redocked to FP-2. When
compared with the crystal structure, the docked conformation of
E64 gave rmsd of ~1.94 Å and had a Glide score of ꢁ5.48 kcal/mol
(Fig. 2). Thus the crystal structure conformation was regenerated.
Docked conformations of compound 9i were ranked based on their
Glide scores and the best conformation, shown in Fig. 3, gave a
Glide score of ꢁ5.50 kcal/mol. In this conformation the chlor-
ophenyl group occupied the S2 subsite whilst the trimethox-
yphenyl group was in the S1 subsite. The thiazolidine-2,4-dione
Compound LM species % Remaining Predicted half-life (min) Solubility
(
m
M)
pH 2 pH 6.5
TZD 4
9g
Human
Rat
Mouse
Human
Rat
Mouse
Human
Rat
Mouse
Human
Rat
5.4
4.3
6.1
34.0
20.2
1.7
12.9
31.1
3.0
47.2
37.7
7.7
15.7
19.8
6.3
7.1
6.6
7.4
19.3
13.0
5.1
10.2
17.8
5.9
27.7
21.4
8.1
11.2
12.8
7.5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
9h
9i
Mouse
Human
Rat
9j
Mouse

R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
511
OMe
O
OMe
O
O-demethylation
N
S
MeO
N
N
N
OMe
S
MeO
N
N
O
OMe
O
OH
O
O
M4
O-demethylation
M3
Oxidation
OMe
O
N
S
MeO
N
N
OMe
O
O
TZD 4
OMe
O
OMe
O
N
S
MeO
O-demethylation
N
NH
OMe
N
S
MeO
O
O
N
N
OMe
OH
O
O
M1
N-dearylation
M2
O-demethylation + Oxidation
Fig. 4. Proposed microsomal metabolites of TZD 4.
(TZD) scaffold was placed in a position close to the active site of the
enzyme but at a distance that prevents covalent bond formation
with Cys42 (Fig. 3A). The docked conformation of 9i was stabilised
by hydrophobic interactions between the chlorophenyl group and
hydrophobic residues Ile85, Gly83, Leu84, and Ala175 in S2 (green
circles in Fig. 3B) and additionally, there are dipoleedipole in-
teractions between His174 ND1 and atom O1 in compound 9i,
which are ~4.23 Å apart (Fig. 3B). Occupation of the S2 subsite, a site
also occupied by E64, is important for specificity of cysteine pro-
teases [32]. Therefore, the inhibitory activity of 9i against FP-2 may
be attributed to its conformation and interaction with residues like
His174 that are important for enzyme inhibition activity.
3. Conclusions
Analogues of substituted thiazolidine-2,4-diones based on a hit
identified from virtual screening of the commercial ZINC database
for falcipain-2 inhibition were synthesized using relatively facile
synthetic routes. The in vitro biological evaluation of these com-
pounds against FP-2 and P. falciparum revealed several that had IC50
values in the low micromolar range. In particular, the presence of a
trimethoxy-substituted phenyl ring attached to C-5 of the core 2,4-
thiazolidinedione was found to be necessary for antiplasmodial
activity while at position 3, a phenyl substituted piperazine ring
was necessary for optimal activity. Modifications to the hit scaffold
and in silico docking studies on falcipain-2 helped to delineate the
tentative SAR of these compounds and provide insight into the
requirements for activity.
2.4. Microsomal stability and metabolite identification studies
Despite their notable plasmodial growth inhibition, selected
compounds from this series were found to be metabolically un-
stable and prone to rapid degradation in human, rat and mouse
hepatic microsomes in vitro. Metabolite identification studies
revealed that metabolism occurred at the terminal phenyl sub-
stituents on both ends of the molecules, mostly through ring hy-
droxylation or O-dealkylation.
Based on these data, the thiazolidinedione class of compounds
appears to be a promising novel chemotype for anti-malarial drug
discovery. Since demonstrating in vivo efficacy in an appropriate
mouse model is a prerequisite before a lead optimization campaign
may be undertaken, addressing poor microsomal metabolic sta-
bility remains the priority before additional SAR explorations to
improve antiplasmodial potency and other desirable properties can
be undertaken.
The selected analogues TZD 4, 9g, 9h, 9i and 9j were evaluated
for their in vitro microsomal metabolic stability using human, rat
and mouse liver microsomes as a preliminary indication of the
likely in vivo metabolic clearance. All the compounds showed poor
metabolic stability across the three species as indicated in Table 3.
Metabolite identification studies were therefore carried out in or-
der to understand the sites of metabolism contributing to this rapid
clearance. The metabolism of TZD 4 proceeds mainly by hydrox-
ylation on the unsubstituted phenyl ring. This metabolite accounts
for about 50% of the metabolism. O-Dealkylation, N-dearylation and
a combination of hydroxylation and O-dealkylation were also
detected as metabolites. Substitution on the phenyl ring, as in 9j,
shifts the metabolic hotspot to the other side of the molecule with
O-dealkylation being the major metabolite (>50%) (Fig. 4).
2.5. Cytotoxicity studies
4. Experimental section
The in vitro cytotoxicity of the most active compounds TZD 4
and 9i was evaluated against a mammalian cell-line, Chinese
Hamster Ovarian (CHO) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) assay. No significantly cyto-
4.1. Chemistry
4.1.1. General
toxicity was observed for TZD 4 (IC50 53.7
(IC50 > 188.3
M; SI ¼ 131.6).
mM; SI ¼ 30.5) or 9i
1H NMR spectra were recorded on a Varian Mercury Spec-
trometer at 300 MHz or a Varian Unity Spectrometer at 400 MHz
m

512
R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
with Me4Si as internal standard. 13C NMR spectra were recorded at
75 and 100 MHz on a Varian Mercury Spectrometer or at 100 MHz
on Varian Unity Spectrometer with Me4Si as internal standard.
High resolution mass spectra were recorded on a VG70 SEQ
micromass spectrometer. Melting points were determined using a
Reichert-Jung Thermovar hot-stage microscope and are uncorrec-
ted. Analytical thin-layer chromatography (TLC) was performed on
aluminium-backed silica-gel 60 F254 (70e230 mesh) plates. Column
chromatography was performed with Merck silica-gel 60
(70e230 mesh).
m), 3.21e3.12 (4H, m); 13C NMR (100 MHz, CDCl3)
d: 168.6, 166.3,
163.4, 163.2, 160.2, 150.8, 131, 130.1, 129.2 (2C), 120.7, 118.1, 116.8,
115.6 (2C),105.5, 98.6, 55.5 (2C), 49.5, 49.3, 44.8, 42.2, 42.1; EIMS m/
0
z: 467.09; HPLC (% purity): 99%, tr ¼ 8.32.
4.1.3.3. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(3-
methoxybenzylidene)1,3-thiazolidine-2,4-dione (4b). (42 mg, 61%)
yellow solid; Mpt. 189 ꢀC; Rf 0.38 (35% EtOAcehexane); 1H NMR
(400 MHz, CDCl3)
d
: 7.9 (1H, s), 7.38 (1H, t, J ¼ 7.3 Hz), 7.32e7.25
(2H, m), 7.11 (1H, d, J ¼ 8.3 Hz), 6.95e6.92 (5H, m), 4.59 (2H, s), 3.96
(3H, s), 3.80e3.67 (4H, m), 3.28e3.19 (4H, m); 13C NMR (100 MHz,
4.1.2. Synthesis of 2,4 thiazolidinedione derivatives
CDCl3) d: 167.8, 165.9, 162.9, 160, 150.7, 134.5, 134.4, 130.2, 129.3
4.1.2.1. 1-(Chloroacetyl)-phenyl piperazine (2). To
a
solution of
(2C), 122.7, 121.8, 120.9, 116.9 (2C), 116.6, 115, 55.4, 49.6, 49.4, 44.8,
0
phenyl piperazine (2.0 g, 0.0123 mol) in DCM (60 ml), TEA (2.2 ml,
1 equiv) was added at RT and cooled the reaction mass to 0 ꢀC.
Chloroacetyl chloride (1.2 g, 1 equiv) was added drop wise at 0 ꢀC
and stirred for 1 h at RT. The saturated NaHCO3 solution (60 ml) was
added and stirred for 15 min at RT. The organic layer was separated
and washed with brine, dried over MgSO4, filtered and concen-
trated. The concentrated mass was purified by silica gel CC using
25e30% EtOAcehexane to give 1-(chloroacetyl)-phenyl piperazine
(1.6 g, 55%) 2 as off white solid. Rf 0.63 (30% EtOAcehexane); Mpt.
42.3 (2C); EIMS m/z: 437.14; HPLC (% purity): 98.2%, tr ¼ 7.65.
4.1.3.4. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(4-
methoxybenzylidene)1,3-thiazolidine-2,4-dione (4c). (46 mg, 42%)
yellow solid; Mpt. 238 ꢀC; Rf 0.60 (35% EtOAcehexane); 1H NMR
(400 MHz, CDCl3)
d
: 7.81 (1H, s), 7.41 (2H, d, J ¼ 8.6 Hz), 7.24e7.18
(2H, m), 6.92 (2H, d, J ¼ 8.8 Hz), 6.89e6.84 (3H, m), 4.51 (2H, s), 3.79
(3H, s), 3.73e3.60 (4H, m), 3.22e3.13 (4H, m); 13C NMR (100 MHz,
CDCl3) d: 168, 166.2, 163, 161.5, 150.6, 134.3, 132.2, 129.3 (2C), 125.9
(2C), 121.1, 120.9, 118.4, 116.9 (2C), 114.8, 55.4, 49.6, 49.4, 44.8, 42.2
77 ꢀC; 1H NMR (400 MHz, CDCl3)
(3H, m), 4.11 (2H, s), 3.80e3.77 (4H, m), 3.70e3.67 (4H, m); 13C
NMR (100 MHz, CDCl3) : 165.1, 150.8, 130.8 (2C), 120.7, 116.8 (2C),
49.7, 49.3, 46.2, 40.7, 38.7.
d
: 7.30e7.25 (2H, m), 6.94e6.92
0
(2C); EIMS m/z: 437.14; HPLC (% purity): 98.2%, tr ¼ 8.70.
d
4.1.3.5. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(4-
fluorobenzylidene)1,3-thiazolidine-2,4-dione (4d). (37 mg, 56%)
white solid; Mpt. 215 ꢀC; Rf 0.62 (35% EtOAcehexane); 1H NMR
4.1.2.2. 3-(2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl)thiazolidine-2,4-
dione (3). The mixture of 2 (1.0 g, 0.418 mmol), TZDs (0.981 g,
2 equiv) and K2CO3 (1.15 g, 2 equiv) in acetone (130 ml) was stirred
for 24 h at 60e70 ꢀC under N2. The reaction mass was filtered and
concentrated to yellowish mass. The crude mass was purified on
silica gel CC at 30e40% EtOAc:hexane afforded 3-(2-oxo-2-(4-
phenylpiperazin-1-yl)ethyl)thiazolidine-2,4-dione (700 mg, 52%)
3 as white solid. Rf 0.50 (30e40% EtOAcehexane); Mpt. 175 ꢀC; 1H
(400 MHz, CDCl3)
d
: 7.83 (1H, s), 7.47 (2H, dd, J ¼ 2.1, 5.2 Hz),
7.26e7.21 (2H, m), 7.14e7.08 (2H, m), 6.91e6.85 (3H, m), 4.53 (2H,
s), 3.75e3.61 (4H, m), 3.23e3.12 (4H, m); 13C NMR (100 MHz,
CDCl3) d: 167.6, 165.9, 165, 162.9, 162.4, 150.5, 133.1, 132.3, 132.2,
129.5 (2C), 129.3, 121.1, 117, 116.8, 116.6, 116.4, 49.7, 49.4, 44.7, 42.3,
0
42.2; EIMS m/z: 425.1; HPLC (% purity): 95.1%, tr ¼ 8.35.
NMR (400 MHz, CDCl3)
(2H, s), 3.8 (2H, s), 3.35e3.36 (4H, m), 3.0e2.92 (4H, m); 13C NMR
(100 MHz, CDCl3) : 171.5, 171.1, 162.8, 150.7, 129.3 (2C), 126.9 (2C),
d
: 7.07e7.02 (2H, m), 6.7e6.67 (3H, m), 4.20
4.1.3.6. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(4-
trifluoromethoxybenzylidene)
(34 mg, 44%) yellow solid; Mpt. 190 ꢀC; Rf 0.64 (35%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 7.80 (1H, s), 7.55 (2H,
1,3-thiazolidine-2,4-dione
(4e).
d
116.9, 49.5, 49.3, 44.7, 42.3 (2C), 33.9; EIMS m/z: 319.1; HPLC (%
d
0
purity): 99%, tr ¼ 5.76.
dd, J ¼ 2.1, 6.8 Hz), 7.32e7.28 (4H, m), 6.99e6.93 (3H, m), 4.60 (2H,
s), 3.82e3.68 (4H, m), 3.30e3.20 (4H, m); 13C NMR (100 MHz,
4.1.3. General procedure for the synthesis of TZD 4, 4aet
CDCl3) d: 167.3, 165.8, 162.8, 154.6, 150.3, 132.5, 131.7 (2C), 129.3
The intermediate
3
(1 equiv), substituted benzaldehydes
(3C),122.4 (2C),121.3,117.1 (4C), 49.8, 49.6, 44.7, 42.4, 42.2; EIMS m/
0
(1 equiv) and ammonium acetate (2 equiv) in toluene (4 ml) were
irradiated on microwave at 125 ꢀC, 220 W for 20 min. The solvent
was evaporated on vacuo and crude mixture was purified on silica
gel CC yielded compounds 4, 4aet.
z: 491.05; HPLC (% purity): 98.3%, tr ¼ 9.20.
4.1.3.7. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(4-
trifluoromethylbenzylidene)
(40 mg, 33%) yellow solid; Mpt. 202 ꢀC; Rf 0.58 (40%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 7.86 (1H, s), 7.66 (2H,
1,3-thiazolidine-2,4-dione
(4f).
4.1.3.1. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(2,4,6-
d
trimethoxybenzylidene)
(50 mg, 64%) yellow solid; Mpt. 220 ꢀC; Rf 0.56 (60%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 8.23 (1H, s),
1,3-thiazolidine-2,4-dione
(TZD
4).
d, J ¼ 8.2 Hz), 7.55 (2H, d, J ¼ 8.1 Hz), 7.26e7.21 (2H, m), 6.88e6.84
(3H, m), 4.53 (2H, s), 3.73e3.60 (4H, m), 3.21e3.12 (4H, m); 13C
d
NMR (100 MHz, CDCl3) d: 167.1, 165.6, 162.7, 150.7, 136.6, 132.2,
7.29e7.25 (2H, m), 6.95e6.92 (3H, m), 6.10 (2H, s), 4.55 (2H, s), 3.87
(3H, s), 3.86 (3H, s), 3.85 (3H, s), 3.78e3.67 (4H, m), 3.27e3.18 (4H,
130.1 (3C), 129.3 (3C), 126.1 (2C), 124.3, 120.9, 116.9 (2C), 49.5, 49.3,
0
44.8, 42.5, 42.3; EIMS m/z: 475.05; HPLC (% purity): 93.5%, tr ¼ 9.05.
m); 13C NMR (100 MHz, CDCl3)
d: 167, 164.3 (2C), 163.5, 160.1 (2C),
150.8, 129.2, 128.6 (2C), 120.7, 120.2, 116.8 (2C), 104, 90.4 (2C), 55.5
(2C), 55.4, 49.5, 49.3, 44.8, 42.2, 41.8; EIMS m/z: 497.16; HPLC (%
4.1.3.8. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(2-
chlorobenzylidene)1,3-thiazolidine-2,4-dione (4g). (30 mg, 44%)
white solid; Mpt. 166 ꢀC; Rf 0.72 (40% EtOAcehexane); 1H NMR
0
purity): 97.2%, tr ¼ 8.41.
(400 MHz, CDCl3)
d
: 8.26 (1H, s), 7.54 (1H, dd, J ¼ 2.3, 5.7 Hz), 7.47
4.1.3.2. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(2,4-
dimethoxybenzylidene)1,3-thiazolidine-2,4-dione (4a). (38 mg, 52%)
yellow solid; Mpt. 198 ꢀC; Rf 0.60 (40% EtOAcehexane); 1H NMR
(1H, dd, J ¼ 2.3, 5.4 Hz), 7.38e7.35 (2H, m), 7.32e7.25 (2H, m),
6.98e6.92 (3H, m), 4.6 (2H), 3.82e3.68 (4H, m), 3.30e3.20 (4H, m);
13C NMR (100 MHz, CDCl3)
d: 167.6, 165.4, 162.9, 150.3, 136, 131.8,
(400 MHz, CDCl3)
d: 8.18 (1H, s), 7.33 (1H, d), 7.25e7.19 (2H, m),
131.3, 130.6, 130.4, 129.3 (2C), 128.9, 127.2, 124.5, 121.1, 116.9 (2C),
6.88e6.83 (3H, m), 6.52 (1H, dd, J ¼ 2.3, 8.7 Hz), 6.40 (1H, d,
49.7, 49.5, 44.7, 42.3, 42.2; EIMS m/z: 440.99; HPLC (% purity): 99%,
0
J ¼ 2.2 Hz), 4.51 (2H, s, H), 3.80 (3H, s), 3.79 (3H, s), 3.73e3.61 (4H,
tr ¼ 7.83.

R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
513
4.1.3.9. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(2-
bromobenzylidene)1,3-thiazolidine-2,4-dione (4h). (28 mg, 37%)
white solid; Mpt. 138 ꢀC; Rf 0.68 (40% EtOAcehexane); 1H NMR
4.1.3.15. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(3,4-
difluorobenzylidene)1,3-thiazolidine-2,4-dione (4n). (50 mg, 45%)
yellow solid; Mpt. 188 ꢀC; Rf 0.74 (30% EtOAcehexane); 1H NMR
(400 MHz, CDCl3)
d
: 8.14 (1H, s), 7.62 (1H, d, J ¼ 7.9 Hz), 7.47 (1H, d,
(400 MHz, CDCl3):
d
7.74 (1H, s), 7.28 (1H, dd, J ¼ 1.8, 8.2 Hz), 7.24
J ¼ 7.8 Hz), 7.35 (1H, t, J ¼ 7.3 Hz), 7.26e7.19 (3H, m), 6.89e6.84 (3H,
(1H, d, J ¼ 2.1 Hz), 7.23e7.18 (3H, m), 6.89e6.85 (3H, m), 4.52 (2H,
m), 4.53 (2H, s), 3.72e3.60 (4H, m), 3.21e3.13 (4H, m); 13C NMR
s), 3.73e3.59 (4H, m), 3.22e3.12 (4H, m); 13C NMR (100 MHz,
(100 MHz, CDCl3)
d
: 167.6, 165.3, 162.8, 150.7, 133.7, 133.6, 133.2,
CDCl3) d: 167.1, 165.6, 162.7, 150.6, 131.8, 130.4, 129.3 (2C), 126.8,
131.5, 129.3 (2C), 129, 127.9, 126.2, 124.6, 120.8, 116.9 (2C), 49.5,
49.3, 44.8, 42.3 (2C); EIMS m/z: 485.06; HPLC (% purity): 98%,
122.7, 121, 118.8, 118.6, 118.4, 118.2, 116.9 (2C), 49.6, 49.4, 44.8, 42.4,
0
42.3; EIMS m/z: 443.04; HPLC (% purity): 95.6%, tr ¼ 8.57.
0
tr ¼ 9.03.
4.1.3.16. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(4-
chlorobenzylidene)1,3-thiazolidine-2,4-dione (4o). (43 mg, 39%)
light yellow solid; Mpt. 240 ꢀC; Rf 0.64 (35% EtOAcehexane); 1H
4.1.3.10. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(3-
isopropylbenzylidene)1,3-thiazolidine-2,4-dione (4i). (36 mg, 32%)
white solid; Mpt. 170 ꢀC; Rf 0.54 (30% EtOAcehexane); 1H NMR
NMR (400 MHz, DMSO)
d
: 7.96 (1H, s), 7.67 (2H, d, J ¼ 8.6 Hz), 7.60
(400 MHz, CDCl3)
d
: 7.86 (1H, s), 7.34 (1H, d, J ¼ 7.6 Hz), 7.30 (1H, d,
(2H, d, J ¼ 8.7 Hz), 7.25e7.20 (2H, m), 6.96 (2H, d, J ¼ 7.8 Hz), 6.79
(1H, t, J ¼ 7.2 Hz), 4.66 (2H, s), 3.69e3.57 (4H, m), 3.22e3.10 (4H,
J ¼ 1.8 Hz), 7.29e7.19 (4H, m), 6.89e6.84 (3H, m), 4.52 (2H, s),
3.73e3.60 (4H, m), 3.21e3.11 (4H, m), 2.89 (1H, sep, J ¼ 6.9 Hz), 1.21
m); 13C NMR (100 MHz, CDCl3)
d: 167.4, 165.8, 162.8, 150.2, 136.7,
(6H, m); 13C NMR (100 MHz, CDCl3)
d: 168, 166, 163, 150, 149.9,
132.9, 131.7, 131.3, 129.5 (2C), 129.3 (2C), 122.1, 121.1, 120, 117 (2C),
49.7, 49.4, 44.7, 42.4, 42.2; EIMS m/z: 441.02; HPLC (% purity): 99%,
134.8,133, 129.3 (2C), 129.2, 129, 128.3,127.7, 121.1, 120.9,116.9 (2C),
49.6, 49.4, 44.8, 42 (2C), 34, 23.8 (2C); EIMS m/z: 449.14; HPLC (%
0
tr ¼ 9.31.
0
purity): 96.4%, tr ¼ 9.96.
4.1.3.17. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(2,4-
dichlorobenzylidene)1,3-thiazolidine-2,4-dione (4p). (50 mg, 42%)
yellow solid; Mpt. 223 ꢀC; Rf 0.60 (35% EtOAcehexane); 1H NMR
4.1.3.11. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(2,5-
ditrifluoromethylbenzylidene)
(42 mg, 30%) yellow solid; Mpt. 156 ꢀC; Rf 0.72 (25%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 8.07 (1H, s),
1,3-thiazolidine-2,4-dione
(4j).
(400 MHz, CDCl3)
d
: 8.11 (1H, s), 7.44 (1H, d, J ¼ 2.0 Hz), 7.41 (1H, d,
d
J ¼ 8.2 Hz), 7.28 (1H, dd, J ¼ 2.0, 8.4 Hz), 7.25e7.19 (2H, m),
7.85e7.83 (2H, m), 7.71 (1H, d, J ¼ 8.7 Hz), 7.25e7.21 (2H, m),
6.88e6.83 (3H, m), 4.53 (2H, s), 3.74e3.60 (4H, m), 3.22e3.12 (4H,
6.89e6.85 (3H, m), 4.53 (2H, s), 3.74e3.60 (4H, m), 3.22e3.13 (4H,
m); 13C NMR (100 MHz, CDCl3)
d: 167.1, 165.3, 162.7, 150.6, 136.8,
m); 13C NMR (100 MHz, CDCl3)
d: 166.4, 164.4, 162.6, 150.6, 134.5,
136.6, 130.4 (2C), 130.3, 129.6, 129.3 (2C), 127.7, 124.9, 121, 116.9
(2C), 49.6, 49.4, 44.8, 42.4, 42.3; EIMS m/z: 475.04; HPLC (% purity):
133.4, 129.3 (2C), 128.4, 128.1, 127.6 (2C), 127.5, 126.5 (2C), 125.9,
121, 116.9 (2C), 49.6, 49.4, 44.8, 42.5, 42.3; EIMS m/z: 543.06; HPLC
0
99%, tr ¼ 9.55.
0
(% purity): 98%, tr ¼ 9.4.
4.1.3.12. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(2-
4.1.3.18. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(3-
carboxybenzylidene)1,3-thiazolidine-2,4-dione (4q). (46 mg, 41%)
white solid; Mpt. 214 ꢀC; Rf 0.48 (15% MeOHeDCM); 1H NMR
trifluoromethoxybenzylidene)
(32 mg, 27%) yellow solid; Mpt. 167 ꢀC; Rf 0.52 (35%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 8.07 (1H, s), 7.54 (1H,
1,3-thiazolidine-2,4-dione
(4k).
d
(400 MHz, DMSO)
d
: 8.21 (1H, s), 8.04 (1H, d, J ¼ 7.5 Hz), 7.98 (1H, s),
dd, J ¼ 1.8, 7.5 Hz), 7.41 (1H, dd, J ¼ 1.8, 7.8 Hz), 7.36 (1H, dd, J ¼ 1.5,
7.71 (1H, d, J ¼ 7.0 Hz), 7.54 (1H, t, J ¼ 7.7 Hz), 7.24e7.20 (2H, m),
6.98e6.79 (3H, m), 4.66 (2H, s), 3.68e3.58 (4H, m), 3.22e3.10 (4H,
7.5 Hz), 7.26e7.19 (2H, m), 6.89e6.84 (4H, m), 4.53 (2H, s),
3.73e3.60 (4H, m), 3.23e3.12 (4H, m); 13C NMR (100 MHz, CDCl3)
d:
m); 13C NMR (100 MHz, DMSO)
d: 169.6, 167.5, 165.7, 163.7, 151.1,
167.4, 165.4, 162.8, 150.7, 148.1, 131.7, 131.4, 129.3 (2C), 129, 127.2,
127.1, 126.8, 124.7, 121.3, 120.9, 116.9 (2C), 49.6, 49.4, 44.8, 42.4,
138, 133.9, 132.3, 131.9, 130.8 (2C), 129.4 (2C), 121.5, 119.9 (2C), 116.4
(2C), 49, 48.6, 44.4, 43.1, 42; EIMS m/z: 451.05; HPLC (% purity): 99%,
0
42.3; EIMS m/z: 491.11; HPLC (% purity): 98.9%, tr ¼ 9.15.
0
tr ¼ 6.89.
4.1.3.13. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(3-
4.1.3.19. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(4-
methylbenzylidene)1,3-thiazolidine-2,4-dione (4r). (48 mg, 45%)
yellow solid; Mpt. 239 ꢀC; Rf 0.64 (35% EtOAcehexane); 1H NMR
trifluoromethoxybenzylidene)1,3-thiazolidine-2,4-dione
(40 mg, 32%) yellow solid; Mpt. 160 ꢀC; Rf 0.64 (35%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 7.82 (1H, s), 7.45 (1H,
(4l).
d
(400 MHz, CDCl3)
(4H, m), 6.90e6.84 (3H, m), 4.53 (2H, s), 3.72e3.60 (4H, m),
3.21e3.12 (4H, m), 2.34 (3H, s); 13C NMR (100 MHz, CDCl3)
: 168,
d
: 7.84 (1H, s), 7.35 (2H, d, J ¼ 8.1 Hz), 7.26e7.19
t, J ¼ 7.8 Hz), 7.38 (1H, dd, J ¼ 1.2, 6.3 Hz), 7.29e7.19 (4H, m),
6.90e6.84 (3H, m), 4.53 (2H, s), 3.74e3.60 (4H, m), 3.23e3.12 (4H,
d
m); 13C NMR (100 MHz, CDCl3)
d: 167.2, 165.6, 162.7, 150.6, 149.7,
166.1, 163, 150.7, 141.3, 134.5, 130.5, 130.3, 130.1, 130, 129.4, 129.3
(2C), 120.9, 120.2, 116.9, 116.8, 49.6, 49.4, 44.8, 42.2 (2C), 21.5; EIMS
135.2, 132.3, 130.7, 129.3 (2C), 128 (2C), 123.6, 122.5, 122.2, 120.9,
116.9 (2C), 49.6, 49.4, 44.8, 42.4, 42.3; EIMS m/z: 491.10; HPLC (%
0
m/z: 421.13; HPLC (% purity): 98.9%, tr ¼ 9.05.
0
purity): 99%, tr ¼ 9.19.
4.1.3.14. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(3-
fluorobenzylidene)1,3-thiazolidine-2,4-dione (4m). (42 mg, 40%)
yellow solid; Mpt. 199 ꢀC; Rf 0.58 (30% EtOAcehexane); 1H NMR
4.1.3.20. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-(3-
methylbenzylidene)1,3-thiazolidine-2,4-dione (4s). (38 mg, 36%)
yellow solid; Mpt. 194 ꢀC; Rf 0.66 (35% EtOAcehexane); 1H NMR
(400 MHz, CDCl3)
d
: 7.81 (1H, s), 7.24e7.35 (1H, m), 7.26e7.19 (3H,
(400 MHz, CDCl3)
d
: 7.84 (1H, s), 7.32 (1H, d, J ¼ 8.2 Hz), 7.28e7.25
m), 7.17e7.12 (1H, m), 7.06 (1H, dd, J ¼ 2.5, 8.3 Hz), 6.90e6.85 (3H,
(3H, m), 7.23 (1H, d, J ¼ 1.2 Hz), 7.21e7.17 (1H, m), 6.95e6.92 (3H,
m), 4.53 (2H, s), 3.74e3.60 (4H, m), 3.22e3.12 (4H, m); 13C NMR
m), 4.53 (2H, s), 3.74e3.62 (4H, m), 3.21e3.12 (4H, m), 2.34 (3H, s);
(100 MHz, CDCl3)
d
: 167.4, 165.7, 164.2, 162.8, 150.6, 135.3, 132.8,
13C NMR (100 MHz, CDCl3)
d: 168, 166, 163, 150.7, 139, 134.6, 133.2,
130.8, 130.7, 129.3, 125.8, 123.1, 121, 117.6, 117.4, 116.9, 116.7, 49.6,
131.4, 130.9, 129.3 (2C), 129.1, 127.3, 120.9, 120.2, 116.9 (2C), 49.6,
49.4, 44.8, 42.4, 42.3; EIMS m/z: 425.08; HPLC (% purity): 98.9%,
49.4, 44.8, 42.3 (2C), 21.3; EIMS m/z: 421.14; HPLC (% purity): 99.2%,
0
0
tr ¼ 8.19.
tr ¼ 7.50.

514
R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
4.1.3.21. 3-[2-Oxo-2-(4-phenylpiperazin-1-yl)ethyl]-5-benzylidene-
1H NMR (400 MHz, CD3OD)
3.89 (6H, s), 3.87 (3H, s), 3.67e3.62 (4H, m), 3.05e2.93 (4H, m); 13
NMR (100 MHz, CDCl3) : 169.7,167.5, 166.9,164.3,160.2 (2C), 128.8,
d: 8.19 (1H, s), 6.26 (2H, s), 4.60 (2H, s),
1,3-thiazolidine-2,4-dione (4t). (38 mg, 60%) white solid; Mpt.
C
211 ꢀC; Rf 0.70 (25% EtOAcehexane); 1H NMR (400 MHz, DMSO)
d:
d
7.86 (1H, s), 7.46e7.36 (5H, m), 7.24e7.18 (2H, m), 6.89e6.84 (3H,
119.9, 102.7, 90.4 (2C), 55.5 (2C), 55.4, 44.5 (2C), 43, 41.9, 41.8; EIMS
m), 4.52 (2H, s), 3.73e3.60 (4H, m), 3.22e3.12 (4H, m); 13C NMR
m/z: 421.26; HPLC (% purity): 99%, tr ¼ 14,41.
0
(100 MHz, DMSO) d: 167.9, 166, 162.9, 150.6, 134.4, 133.2, 130.5 (2C),
130.2 (2C), 129.3, 129.2 (2C), 121.4, 120.9, 117, 116.9, 49.6, 49.2, 44.8,
4.1.4.2. 3-[2-Oxo-2-(4-methylpiperazin-1-yl)ethyl]-5-(2,4,6-
0
42.3 (2C); EIMS m/z: 407.10; HPLC (% purity): 99%, tr ¼ 8.58.
trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione
(60 mg, 61%) white solid; Mpt. 183 ꢀC; Rf 0.76 (5% MeOHeEtOAc);
1H NMR (400 MHz, CDCl3)
: 8.14 (1H, s), 6.02 (2H, s), 4.42 (2H, s),
3.78 (6H, s), 3.77 (3H, s), 3.59e3.48 (4H, m), 2.43e2.36 (4H, m), 2.27
(3H, s); 13C NMR (100 MHz, CDCl3)
: 169.8, 167, 164.3, 163.3, 160.1
(9b).
4.1.3.22. 2-(2,4-Dioxo-1,3-thiazolidin-3-yl)acetic acid (7). To a so-
lution of TZD (1 g, 8.53 mmol) in dry DMF (5 ml), NaH (340 mg,
1 equiv) was added and stirred for 10 min at room temperature. The
bromoacetic ester (786 mg, 1 equiv) was added drop wise in the
reaction mass and stirred for 24 h at room temperature. The reac-
tion mass was extracted with H2O (20 ml) and DCM (20 ml). The
organic layer was washed with 5% HCl solution, dried over MgSO4,
concentrated and purified on silica gel CC using 50% ethyl-
acetateehexane afforded methyl 2-(2,4-dioxo-1,3-thiazolidin-3-yl)
acetate (1.4 g, 87%) (8) as yellow liquid. Rf 0.46 (50%
d
d
(2C), 128.5, 120.2, 104.6, 90.4 (2C), 55.5 (2C), 55.4, 54.6, 54.3, 45.8,
0
44.5, 42, 41.8; EIMS m/z: 435.13; HPLC (% purity): 99%, tr ¼ 11.17.
4.1.4.3. 3-[2-Oxo-2-(4-cyclohexylpiperazin-1-yl)ethyl]-5-(2,4,6-
trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione (9c). (65 mg,
44%) light yellow solid; Mpt. 182 ꢀC; Rf 0.72 (5% MeOHeEtOAc); 1H
NMR (400 MHz, CDCl3)
(6H, s), 3.85 (3H, s), 3.61e3.48 (4H, m), 2.63e2.54 (4H, m), 2.30 (1H,
m), 1.82e1.18 (10H, m); 13C NMR (100 MHz, CDCl3)
: 169.8, 167,
d: 8.21 (1H, s), 6.10 (2H, s), 4.49 (2H, s), 3.86
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
eNCH2COOCH3), 3.97 (2H, s, H5), 3.69 (3H, s, eNCH2COOCH3); 13
NMR (100 MHz, DMSO) : 170.9, 170.5, 166.6, 52.8, 41.9, 33.8; EIMS
d: 4.27 (2H, s,
C
d
d
164.2, 163.1, 160.1 (2C), 128.6, 120.3, 104.6, 90.4 (2C), 63.4, 55.4 (2C),
m/z: 189.03. The yellow liquid was refluxed with 40% HBr(10 ml) for
4 h at 110 ꢀC. Water (10 ml) was added and reaction mass was
extracted with ethylacetate (50 ml ꢂ 3). The combined organic
layer was dried over MgSO4 filtered and concentrated on vacuo
gave 2-(2,4-dioxo-1,3-thiazolidin-3-yl)acetic acid (2.1 g, 52%) (7) as
pale white solid. Rf 0.22 (EtOAc); Mpt. 143 ꢀC; 1H NMR (400 MHz,
55.3, 48.9, 48.4, 45.3, 42.7, 41.9, 28.8 (2C), 26.2, 25.8 (2C); EIMS m/z:
0
503.12; HPLC (% purity): 99%, tr ¼ 11.84.
4.1.4.4. 3-[2-Oxo-2-(4-pyridylpiperazin-1-yl)ethyl]-5-(2,4,6-
trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione
(65 mg, 58%) reddish solid; Mpt. 217 ꢀC; Rf 0.38 (15% MeOHeDCM);
1H NMR (400 MHz, DMSO)
(9d).
DMSO)
d
: 4.34 (2H, s), 3.84 (2H, s); 13C NMR (100 MHz, DMSO)
d:
d
: 8.17 (2H, d, J ¼ 6.5 Hz), 8.01 (1H, s),
171.5, 168.5, 168.1, 41.4, 33.2; EIMS m/z: 175.
6.85 (2H, d, J ¼ 6.5 Hz), 6.31 (2H, s), 4.58 (2H, s), 3.86 (6H, s), 3.85
(3H, s), 3.70e3.54 (4H, m), 3.45e3.27 (4H, m); 13C NMR (100 MHz,
4.1.3.23. 2-(5-(2,4,6-Trimethoxybenzylidene)-2,4-dioxo-thiazolidin-
3-yl) acetic acid (8). To a solution of 7 (1 equiv) in absolute ethanol
(10 ml), 2,4,6-trimethoxybenzaldehyde (1.1 equiv), piperidine
(0.6 ml) and AcOH (2e4 drops) was added and reaction mass was
stirred for 5 h at 40e50 ꢀC. The reaction solvent was evaporated and
water(40 ml) was added. The aqueous layer was extracted with
ethylacetate (50 ml ꢂ 2) and then pH of aqueous layer was acidified
to 3 pH using 5% HCl solution. The acidified aqueous layer was
extracted with ethylacetate (50 ml ꢂ 3). The combined organic
layer was dried over MgSO4 filtered, concentrated followed by silica
gel CC using 5e15% MeOHeDCM afforded 2-(5-(2,4,6-
trimethoxybenzylidene)-2,4-dioxo-thiazolidin-3-yl) acetic acid
(1.4 g, 69%) 8 as yellow solid. Mpt. 184 ꢀC; Rf 0.48 (15% MeOH-DCM);
CDCl3) d: 169.7, 167, 164.4, 163.8, 160.2 (2C), 154.8, 148.7 (2C), 128.8,
119.8, 108.5, 108.4, 108.3, 90.4 (2C), 55.5 (2C), 55.4, 45.6 (2C), 44,
0
41.7, 41.3; EIMS m/z: 498.08; HPLC (% purity): 99%, tr ¼ 10.96.
4.1.4.5. 3-[2-Oxo-2-(4-(4-methoxyphenyl)piperazin-1-yl)ethyl]-5-
(2,4,6-trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione
(50 mg, 42%) yellow solid; Mpt. 182 ꢀC; Rf 0.56 (35%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 8.21 (1H, s), 6.92 (2H,
(9e).
d
d, J ¼ 8.8 Hz), 6.85 (2H, d, J ¼ 9.0 Hz), 6.10 (2H, m), 4.55 (2H, m), 3.86
(6H, s), 3.85 (3H, s), 3.77 (3H, s), 3.78e3.66 (4H, m), 3.13e3.06 (4H,
m); 13C NMR (100 MHz, CDCl3)
d: 169.8, 167, 164.3 (2C), 163.4, 160.1
(2C), 145.2, 128.6, 120.2, 119.1 (2C), 114.6 (2C), 104, 90.4 (2C), 55.5
(2C), 55.4, 51.1, 44.9 (2C), 42.3, 41.9 (2C); EIMS m/z: 527.09; HPLC (%
1H NMR (400 MHz, DMSO)
3.86 (6H, s), 3.85 (3H, s); 13C NMR (100 MHz, DMSO)
d: 8.02 (1H, s), 6.30 (2H, s), 4.24 (2H, s),
purity): 99%, tr ¼ 13.47.
0
d
: 168.6, 166.4
(2C), 165, 160.2 (2C), 127.9, 119.3, 103.5, 91.5 (2C), 56.2 (3C), 42.5;
4.1.4.6. 3-[2-Oxo-2-(4-phenoxypiperazin-1-yl)ethyl]-5-(2,4,6-
trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione (9f). (62 mg,
52%) white solid; Mpt. 126 ꢀC; Rf 0.30 (EtOAc); 1H NMR (300 MHz,
0
EIMS m/z: 353.06; HPLC (% purity): 98%, tr ¼ 12.44.
4.1.4. General procedure for the synthesis of 9aej
DMSO)
3.84 (6H, s), 3.83 (3H, s), 3.60e3.49 (8H, m); 13C NMR (100 MHz,
CDCl3) : 170.6, 169.7, 167, 164.4, 163.8, 160.2 (2C), 135.1, 130.1, 128.7
d: 8.04 (1H, s), 7.46e7.41 (5H, m), 6.31 (2H, m), 4.55 (2H, s),
The solution of 8 (1 equiv) and substituted phenyl piperazine
(1 equiv) in dry DMF (5 ml) was cooled to 0 ꢀC. The N-ethyl-N0-
d
(dimethyaminopropyl)carbodimide
hydrochloride
(EDC$HCl)
(2C), 128.6, 127.1 (2C), 119.9, 104.5, 90.4 (2C), 55.8 (2C), 55.4, 44.8,
0
(1 equiv), 1-hyroxybenzotriazole hydrate (HOBt$H2O) (1 equiv) and
DIPEA (2 equiv) were added and the resulting mixture stirred for
24 h at room temperature. H2O (40 ml) was added to reaction mass
and extracted with ethylacetate (40 ml ꢂ 3). The combined organic
layer was washed with saturated aqueous NaHCO3 solution (30 ml),
brine (30 ml), dried over MgSO4 and concentrated to give crude
mass which were purified on silica gel CC to afforded desired
products 9aej.
41.8 (4C); EIMS m/z: 525.04; HPLC (% purity): 99%, tr ¼ 12.51.
4.1.4.7. 3-[2-Oxo-2-(4-(2-chlorophenyl)piperazin-1-yl)ethyl]-5-
(2,4,6-trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione
(60 mg, 50%) yellow solid; Mpt. 179 ꢀC; Rf 0.68 (50%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 8.23 (1H, s), 7.38 (1H,
(9g).
d
d, J ¼ 8.2 Hz), 7.22 (1H, t, J ¼ 8.2 Hz), 7.03e6.99 (2H, m), 6.1 (2H, s),
4.55 (2H, s), 3.86 (6H, s), 3.85 (3H, s), 3.81e3.67 (4H, m), 3.12e3.03
(4H, m); 13C NMR (100 MHz, CDCl3)
d: 169.8, 167, 164.3, 163.6, 160.1
4 .1. 4 .1. 3 - [ 2 - O x o - 2 - ( p i p e ra z i n - 1 - yl ) e t hyl ] - 5 - ( 2 , 4 , 6 -
(2C), 151.8, 135.1, 130.2, 129.7, 128.6, 120.3, 116.5, 114.5, 104.6, 90.4
trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione
(9a).
(2C), 55.6, 55.5, 55.4, 48.9, 48.8, 44.6, 42, 41.8; EIMS m/z: 531.05;
(30 mg, 42%) yellow solid; Mpt. 109 ꢀC; Rf 0.42 (20% MeOHeDCM);
HPLC (% purity): 99%, tr ¼ 14.41.
0

R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
515
4.1.4.8. 3-[2-Oxo-2-(4-(3-chlorophenyl)piperazin-1-yl)ethyl]-5-
(2,4,6-trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione (9h).
(55 mg, 46%) yellow solid; Mpt. 175 ꢀC; Rf 0.70 (50%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 8.23 (1H, s), 7.17 (1H,
t, J ¼ 8.0 Hz), 6.88 (1H, d, J ¼ 1.4 Hz), 6.86 (1H, d, J ¼ 7.6 Hz), 6.78
(1H, d, J ¼ 7.6 Hz), 6.1 (2H, s), 4.54 (2H, s), 3.86 (6H, s), 3.85 (3H, s),
3.77e3.66 (4H, m), 3.26e3.20 (4H, m); 13C NMR (100 MHz, CDCl3)
4.1.5.2. 3-Ethyl-5-(E/Z)(2,4,6-trimethoxybenzylidene)-1,3-
thiazolidine-2,4-dione (11b). (58 mg, 66%) yellow solid; Mpt.
123 ꢀC; Rf 0.60 (50% EtOAcehexane); 1H NMR (400 MHz, CDCl3)
d:
d
8.12 (1H, s), 6.02 (2H, s), 3.79 (6H, s), 3.78 (3H, s), 3.68 (2H, q,
J ¼ 7.0 Hz), 1.18 (3H, t, J ¼ 7.0 Hz); 13C NMR (100 MHz, CDCl3)
d:
169.6, 167.7, 164.1, 160 (2C), 127.7, 120.4, 104.6, 90.1 (2C), 55.4 (3C),
0
36.5, 13.1; EIMS m/z: 323.08; HPLC (% purity): 99%, tr ¼ 13.88.
d: 169.8, 167, 164.3, 163.5, 160.1 (2C), 148.5, 130.7, 129, 128.6, 127.7,
124.4, 120.6, 120.2, 104.6, 90.4 (2C), 55.5 (2C), 55.4, 51.3, 50.9, 45.2,
4.1.5.3. 3-Benzyl-5-(E/Z)(2,4,6-trimethoxybenzylidene)-1,3-
0
42.6, 41.9; EIMS m/z: 531.03; HPLC (% purity): 99%, tr ¼ 14.54.
thiazolidine-2,4-dione (11c). (56 mg, 54%) yellow solid; Mpt.164 ꢀC;
Rf 0.62 (50% EtOAcehexane); 1H NMR (400 MHz, CDCl3)
d
: 8.14 (1H,
s), 7.36 (2H, d, J ¼ 7.6 Hz), 7.26e7.19 (3H, m), 6.02 (2H, m), 4.79 (2H,
m), 3.78 (6H, s), 3.77 (3H, s); 13C NMR (100 MHz, CDCl3)
: 170.6,
4.1.4.9. 3-[2-Oxo-2-(4-(4-chlorophenyl)piperazin-1-yl)ethyl]-5-
d
(2,4,6-trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione
(56 mg, 47%) yellow solid; Mpt. 187 ꢀC; Rf 0.70 (50%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 8.2 (1H, s), 7.20 (2H,
(9i).
167.1, 164.1, 160.2 (2C), 135.7, 128.8, 128.5, 128.1, 128, 127.9 (2C),
120.2, 104.5, 90.1 (2C), 55.5 (2C), 55.4, 44.8; EIMS m/z: 385.1; HPLC
d
0
(% purity): 99%, tr ¼ 15.92.
d, J ¼ 8.9 Hz), 6.83 (2H, d, J ¼ 8.9 Hz), 6.07 (2H, s), 4.52 (2H, s), 3.83
(6H, s), 3.79 (3H, s), 3.76e3.64 (4H, m), 3.20e3.12 (4H, m); 13C NMR
(100 MHz, CDCl3) d: 169.8, 167, 164.3, 163.4, 160.1 (2C), 145.2, 128.6
(2C), 120.2, 119.1 (2C), 114.6 (2C), 104, 90.4 (2C), 55.5 (2C), 55.4, 51.1
(2C), 44.9, 42.3, 41.9; EIMS m/z: 531.01; HPLC (% purity): 99%,
4.1.5.4. 3-(4-Methylbenzyl)-5-(E/Z)(2,4,6-trimethoxybenzylidene)-
1,3-thiazolidine-2,4-dione (11d). (70 mg, 64.8%) yellow solid; Mpt.
167 ꢀC, Rf 0.68 (35% EtOAcehexane); 1H NMR (400 MHz, CDCl3)
d:
0
tr ¼ 14.38.
8.14 (1H, s), 7.27 (2H, d, J ¼ 7.9 Hz), 6.92 (2H, d, J ¼ 7.7 Hz), 6.03 (2H,
m), 4.76 (2H, m), 3.79 (9H, s), 2.25 (3H, s); 13C NMR (100 MHz,
CDCl3) d: 167.1,164.2,160 (3C),137.6,132.8,129.2,129.1,128.9,128.8,
4.1.4.10. 3-[2-Oxo-2-(4-(4-cyanophenyl)piperazin-1-yl)ethyl]-5-
128, 120.4, 104.6, 90.4 (2C), 55.5, 55.4 (2C), 44.5, 21.1; EIMS m/z:
(2,4,6-trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione
(50 mg, 48%) yellow solid; Mpt. 205 ꢀC; Rf 0.58 (70%
EtOAcehexane); 1H NMR (300 MHz, CDCl3)
: 8.14 (1H, s), 7.42 (2H,
(9j).
0
399.09; HPLC (% purity): 99%, tr ¼ 15.65.
d
d, J ¼ 8.9 Hz), 6.78 (2H, d, J ¼ 8.9 Hz), 6.02 (2H, s), 4.46 (2H, s), 3.78
4.1.5.5. 3-(2,4-Difluorobenzyl)-5-(2,4,6-trimethoxybenzylidene)-1,3-
thiazolidine-2,4-dione (11e). (50 mg, 43%) yellow solid; Mpt.147 ꢀC;
(9H, s), 3.74e3.62 (4H, m), 3.39e3.28 (4H, m); 13C NMR (100 MHz,
Rf 0.62 (30% EtOAcehexane); 1H NMR (400 MHz, CDCl3)
d: 8.17 (1H,
CDCl3) d: 169.7, 167, 164.4, 163.8, 160.2 (2C), 150.2, 133.6, 128.8,
128.7, 119.8, 119.6, 114.6, 104.5, 104.4, 101.4, 90.4, 90.3, 55.6, 55.5,
55.4, 47 (2C), 44.2, 41.8, 41.5; EIMS m/z: 522.01; HPLC (% purity):
s), 7.30 (1H, d, J ¼ 8.1 Hz), 6.78 (1H, dd, J ¼ 2.6, 8.6 Hz), 6.73 (1H, d,
J ¼ 2.5 Hz), 6.04 (2H, m), 4.8 (2H, s), 3.80 (6H, s), 3.79 (3H, s); 13C
0
99%, tr ¼ 13.
NMR (100 MHz, CDCl3) d: 169.4, 167, 164.4 (2C),160.1 (2C), 131.4,
131.3, 128.5, 119.7, 111.4, 104.5, 104.2, 103.6, 90.4 (2C), 55.5 (2C),
0
55.4, 37.8; EIMS m/z: 421.11; HPLC (% purity): 99%, tr ¼ 15.11.
4.1.4.11. 5-(2,4,6-Trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione
(10). To a solution of TZD (1 equiv) in AcOH (4 ml), 2,4,6-
trimethoxybenzaldehyde(1 equiv) and NH4OAc (2 equiv) was
added and reaction mass was stirred for 16e24 h at 110e115 ꢀC.
Filter the reaction mass and washed with EtOH and hexane. The
crude mass was dried in oven at 80e100 ꢀC afforded 10 as yellow
solid(580 mg, 76%) yellow solid; Mpt. 271 ꢀC; Rf 0.48 (50%
4.1.5.6. 3-(4-Fluorobenzyl)-5-(E/Z)(2,4,6-trimethoxybenzylidene)-
1,3-thiazolidine-2,4-dione (11f). (60 mg, 55%) yellow solid; Mpt.
166 ꢀC; Rf 0.80 (50% EtOAcehexane); 1H NMR (400 MHz, CDCl3)
d:
8.14 (1H, s), 7.38 (1H, d, J ¼ 5.3 Hz), 7.34 (1H, d, J ¼ 5.3 Hz), 6.92 (2H,
d, J ¼ 8.9 Hz), 6.04 (2H, m), 4.76 (2H, s), 3.8 (6H, s), 3.79 (3H, s); 13
C
EtOAcehexane); 1H NMR (400 MHz, DMSO)
s), 3.83 (9H, s); 13C NMR (100 MHz, DMSO)
d
: 7.85 (1H, s), 6.28 (2H,
NMR (100 MHz, CDCl3) d: 167.1, 164.3, 163.7, 160.1 (2C), 131.6 (2C),
d: 169.7, 168.7, 164.6, 160
130.8, 130.7, 128.3, 120, 115.5, 115.3, 104.5, 90.4 (2C), 55.5 (2C), 55.4,
44; EIMS m/z: 403.07; HPLC (% purity): 99%, tr ¼ 15.17.
(2C), 126.2, 122.2, 103.6, 91.5 (2C), 56.1 (3C); EIMS m/z: 295.01;
0
0
HPLC (% purity): 99% tr ¼ 12.32.
4 .1. 5 . 7 . 3 - ( 4 -Tr i fl u o r o m e t h y l b e n z y l ) - 5 - ( E / Z ) ( 2 , 4 , 6 -
4.1.5. General procedure for the synthesis of 11aei
trimethoxybenzylidene)-1,3-thiazolidine-2,4-dione
(54 mg, 44%) yellow solid; Mpt. 191 ꢀC; Rf 0.64 (50%
EtOAcehexane); 1H NMR (400 MHz, CDCl3)
: 8.17 (1H, s), 7.52 (2H,
d, J ¼ 8.4 Hz), 7.48 (2H, d, J ¼ 7.6 Hz), 6.04 (2H, m), 4.85 (2H, m), 3.8
(6H, s), 3.79 (3H, s); 13C NMR (100 MHz, CDCl3)
: 169.4, 166.9,
(11g).
To a solution of 10 (1 equiv) in dry DMF (5 ml), NaH (1 equiv)
was added and stirred for 10 min at room temperature. The alkyl/
substituted aryl halide (1 equiv) was added in the reaction mass
and stirred for 14 h at room temperature. The reaction mass was
extracted with H2O (20 ml) and ethylacetate (20 ml). The organic
layer was washed with 5% HCl solution, dried over MgSO4,
concentrated and purified on Si gel CC using binary solution of ethyl
acetate: hexane at different polarities afforded compounds 11aei
as yellow solids.
d
d
164.5, 160.2 (2C), 139.6, 129.6, 128.5 (2C), 125.5, 125.4 (2C), 119.8,
104.6, 90.6 (2C), 55.4 (3C), 44.2; EIMS m/z: 453.07; HPLC (% purity):
0
99%, tr ¼ 16.01.
4.1.5.8. 3-(3-Methoxybenzyl)-5-(E/Z)(2,4,6-trimethoxybenzylidene)-
1,3-thiazolidine-2,4-dione (11h). (48 mg, 42%) yellow solid; Mpt.
4.1.5.1. 3-Methyl-5-(E/Z)(2,4,6-trimethoxybenzylidene)-1,3-
158 ꢀC; Rf 0.56 (50% EtOAcehexane); 1H NMR (400 MHz, CDCl3)
d:
thiazolidine-2,4-dione (11a). (60 mg, 72%) yellow solid; Mpt.180 ꢀC;
8.14 (1H, s), 7.14 (1H, t, J ¼ 7.9 Hz), 6.95e6.91 (2H, m), 6.75 (1H, dd,
Rf 0.58 (50% EtOAcehexane); 1H NMR (400 MHz, CDCl3)
d
: 8.13 (1H,
s), 6.03 (2H, s), 3.79 (6H, s), 3.78 (3H, s), 3.12 (3H, s); 13C NMR
(100 MHz, CDCl3) : 169.9, 167.5, 164.2, 160.9 (2C), 127.9, 122.4,
J ¼ 2.5, 8.2 Hz), 6.02 (2H, m), 4.76 (2H, m), 3.79 (6H, s), 3.78 (3H, s),
3.72 (3H, s); 13C NMR (100 MHz, CDCl3)
d: 169.6, 167.1, 164.2, 160.1
d
(2C), 159.7, 151.5, 137.2, 129.6, 128.1, 121, 114.2, 113.6, 104.6, 90.4
104.5, 90.4 (2C), 55.5 (2C), 55.4, 29.6; EIMS m/z: 309.06; HPLC (%
(2C), 55.5 (2C), 55.4, 55.2, 44.7; EIMS m/z: 415.08; HPLC (% purity):
0
0
purity): 99%, tr ¼ 13.24.
99%, tr ¼ 15.12.

516
R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
4.1.5.9. 3-Adamentyl-5-(2,4,6-trimethoxybenzylidene)-1,3-
active site and the grid centres were defined by the co-crystallised
ligand E64.
thiazolidine-2,4-dione (11i). (60 mg, 51%) yellow solid; Mpt. 272 ꢀC;
Rf 0.68 (5% IPAeDCM); 1H NMR (400 MHz, DMSO)
d
: 7.87 (1H, s),
6.28 (2H, m), 3.83 (9H, s), 2.30e1.67 (15H, m); 13C NMR (100 MHz,
DMSO) : 169.7, 168.7, 164.6, 160 (2C), 126.2, 122.2, 103.6, 90.5 (2C),
4.3.2. Ligand preparation and docking
d
To validate our docking calculations, non-covalent docking ex-
periments were performed using E64 with the epoxide ring
opened. ConfGen version 2.3, on Maestro, generated different
conformations of the E64 and compound 9i, which were later
docked into the target, FP-2. In the docking process, standard-
precision (SP) and extra-precision (XP) docking calculations were
respectively adopted with default Glide docking parameters to
generate the minimized poses using 5000 energy minimisation
steps. The Glide scoring function (G-Score) was used to select the
final 5 poses for each ligand and the conformation with lowest
binding score was selected. Maestro was used to view and analyze
the best docked conformations.
56.1 (3C), 49.3, 39.6 (3C), 35.3 (3C), 32.6 (3C); EIMS m/z: 429.07;
HPLC (% purity): 99%.
4.2. Evaluation of in vitro antiplasmodial activity
4.2.1. Enzyme inhibition
Falcipain-2 was recombinantly expressed in Escherichia coli and
refolded to the active enzyme as previously described [33]. IC50
values against recombinant falcipain-2 was determined as follows.
The substrate (H-D-Val-Leu-Arg-AFC), added to a final concentra-
tion of 30
different concentrations of tested inhibitors in the reaction buffer
(100 mM sodium acetate, pH 5.5, 500 M CHAPS, and 10 mM DTT).
mM for FP-2, was mixed at room temperature with
m
Inhibitor solutions were prepared from stocks in DMSO (maximum
concentration of DMSO in the assay was 1%).
4.4. Metabolite ID incubations
The reaction was started with the addition of the 0.25 nM FP-2
recombinant enzyme. Fluorescence was monitored for 1 h at room
temperature in a SpectraMax Plus 384 spectrofluorometer. IC50
values were determined from plots of percent activity over con-
centration using GraFit software.
4.4.1. In vitro metabolism incubations
The test compound (10 m
M) was incubated at 37 ꢀC with a so-
lution of human (pooled human mixed gender) or rat (male rat IGS)
liver microsomes (Xenotech) and NADPH (1 mM) in phosphate
buffer (100 mM, pH 7.4) which contained magnesium chloride
(5 mM) for 1 h while shaking. An equal volume of ice cold aceto-
nitrile was added to stop the reaction and to precipitate the pro-
teins. After centrifuging the mixture at 14,000 rpm for 30 min the
supernatant was transferred to an HPLC vial. Control samples with
no NADPH, no microsomes and a T0 sample were also included and
processed in the same manner.
4.2.2. Cultured malaria parasites inhibition
The effects of inhibitors were studied against W2-strain
P. falciparum, obtained from the Malaria Research and Reference
Reagent Resource Center and cultured with human erythrocytes at
2% haematocrit in RPMI medium with either 10% human serum or
0.5% AlbuMAX II serum substitute. Synchrony was maintained with
sorbitol. Cultures were incubated with serial dilutions of test
compounds from 1000ꢂ stocks in DMSO for 48 h beginning at the
ring stage. The medium was changed after 24 h, with maintenance
of the appropriate inhibitor concentration. After 48 h, when control
cultures contained nearly all new ring-stage parasites, parasites
were fixed with 1% formaldehyde in PBS, pH 7.4, for 48 h at room
temperature and then labelled with YOYO-1 (1 nM; Molecular
Probes) in 0.1% Triton X-100 in PBS. Parasitaemias were determined
from dot plots (forward scatter vs fluorescence) acquired on a
FACSort flow cytometer using CELLQUEST software (Becton Dick-
inson). IC50 values for growth inhibition were determined with
GraphPad Prism software from plots of percentage parasitaemia
compared to controls (untreated parasites) over inhibitor concen-
tration. In each case, the goodness of curve fit was documented by
R2 values of ꢃ0.95.
4.4.2. Analysis
The samples were analysed using an Agilent 1200 Rapid Reso-
lution (600 bar) HPLC, with a diode array detector, coupled to an AB
SCIEX 4000 QTRAP MS (ABSciex, Johannesburg, South Africa). For
elution, 0.1% formic acid in water (with 5% acetonitrile) and 0.1%
formic acid in 95% acetonitrile were used as mobile phases A and B
respectively. A Kinetex C18 (150 mm ꢂ 2.1 mm), packed with 2.6
mm
fused-core particles (Separations, Johannesburg, South Africa) was
used for the analysis. The gradient run from 0% to 90% B in 9 min,
where it was held for 3 min, before re-equilibration to the starting
conditions. The total run time was 15 min. The LC-MS method was
created using Lightsight v2.1 (AB Sciex) which relied on Analyst v.
1.5 method files created during direct infusion of the compounds.
The methods were in electrospray positive mode and consisted of
full scans coupled through information dependent acquisition to
two enhanced product ion scans. The scan range was 100e600
using a scan rate of 4000 Da/s giving a total cycle time of 1.02 s.
Collision energy was set to 30 15 for 1 and 40 15 for 9. The
source temperature was 500 ꢀC, ion spray voltage 5000 V and
curtain gas 20 psi. The sheath gas (gas 1) and nebulizer gas (gas 2)
were set to 50 psi.
4.3. Molecular modelling studies
4.3.1. Protein structure preparation
Four crystal structures are available for FP-2: 3BPF (FP-2 cova-
lently linked to E64 at 2.9 Å resolution) [34], 1YVB (FP-2 covalently
linked to cystatin at 2.7 Å resolution) [35], 2GHU (not complexed at
3.1 Å resolution) [36] and 2OUL (FP-2 covalently linked to chagasin
at 2.2 Å resolution) [37]. Although both 1YVB and 2OUL had better
resolution than 3BPF, they are complexed to large inhibitors (over
100 amino acids). In contrast, 3BPF contains E64, a low molecular-
weight, non-selective inhibitor that is comparable to our class of
compounds. The protein was prepared using the Protein Prepara-
tion Wizard incorporated in Maestro [38] by adding missing
hydrogen and side chains. All co-crystallised water molecules and
bad contacts were removed. The receptor grid generation tool on
Glide 5.7 was used to generate grid maps that covered the target's
An extra set of runs was done with the same set of conditions as
above but passing the eluent through the diode array detector
(DAD). An Ascentis C18 (150 mm ꢂ 4.6 mm) was used instead of the
column above to reduce the system back pressure and allow DAD
detection.
Metabolites were identified by comparison of sample and con-
trol runs using Lightsight v2.1. A neutral loss and precursor frag-
ment filter was also used post-acquisition using the IDATrace
Extractor script in analyst 1.5 to ensure that all parent derived
peaks were detected. Comparing their fragmentation patterns to
that of the parent compound tentatively identified the metabolites.

R.K. Sharma et al. / European Journal of Medicinal Chemistry 90 (2015) 507e518
517
Falcipain inhibitors: optimization studies of the 2-pyrimidinecarbonitrile lead
series, J. Med. Chem. 53 (2010) 6129e6152.
4.5. Cytotoxic assay
[13] A. Breuning, B. Degel, F. Schulz, C. Büchold, M. Stempka, U. Machon, et al.,
Michael acceptor based antiplasmodial and antitrypanosomal cysteine pro-
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1951e1963.
Test samples were screened for in vitro cytotoxicity against a
mammalian cell-line, Chinese Hamster Ovarian (CHO) using the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
ꢀ
[14] J.N. Domínguez, C. Leon, J. Rodrigues, N. Gamboa de Domínguez, J. Gut,
(MTT)-assay. The MTT-assay is used as a colorimetric assay for
cellular growth and survival, and compares well with other avail-
able assays [39] and [40]. The tetrazolium salt MTT was used to
measure all growth and chemosensitivity. The test samples were
tested in triplicate. The test samples were prepared to a 20 mg/ml
stock solution in 100% DMSO. Test compounds were stored
at ꢁ20 ꢀC. Dilutions were prepared on the day of the experiment.
Emetine was used as the reference drug in all experiments. The
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P.J. Rosenthal, J.H. McKerrow, K. Chibale, Synthesis and structureeactivity
relationships of parasiticidal thiosemicarbazone cysteine protease inhibitors
against Plasmodium falciparum, Trypanosoma brucei, and Trypanosoma cruzi,
J. Med. Chem. 47 (2004) 3212e3219.
initial concentration of emetine was 100
diluted in complete medium with 10-fold dilutions to give 6 con-
centrations, the lowest being 0.001 g/ml. The same dilution
mg/ml, which was serially
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cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium fal-
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[18] E. Guantai, K. Ncokazi, T.J. Egan, J. Gut, P.J. Rosenthal, R. Bhampidipati,
A. Kopinathan, P.J. Smith, K. Chibale, Enone- and chalcone-chloroquinoline
hybrid analogues: in silico guided design, synthesis, antiplasmodial activity,
in vitro metabolism, and mechanistic studies, J. Med. Chem. 54 (2011)
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m
technique was applied to the test samples. The highest concen-
tration of solvent to which the cells were exposed had no
measurable effect on cell viability (data not shown). The 50%
inhibitory concentration (IC50) values were obtained from full
doseeresponse curves, using a non-linear doseeresponse curve
fitting analysis via GraphPad Prism v. 4 software.
^
[19] J. Lavrado, G.G. Cabal, M. Prudencio, M.M. Mota, J. Gut, P.J. Rosenthal, C. Diaz,
R.C. Guedes, D.V.J.A. Santos, E. Bichenkova, K.T. Douglas, R. Moreira, A. Paulo,
Incorporation of basic side chains into cryptolepine scaffold: structur-
eeantimalarial activity relationships and mechanistic studies, J. Med. Chem.
54 (2011) 734e750.
Acknowledgements
[20] P. Gibbons, E. Verissimo, N.C. Araujo, V. Barton, G.L. Nixon, R.K. Amewu,
J. Chadwick, P.A. Stocks, G.A. Biagini, A. Srivastava, P.J. Rosenthal, J. Gut,
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R.J. Guedes, Novel anti-plasmodial hits identified by virtual screening of the
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The University of Cape Town, South African Medical Research
Council, and South African Research Chairs Initiative of the
Department of Science and Technology, administered through the
South African National Research Foundation are gratefully
acknowledged for support (K.C.).
[22] P.S. Kenderekar, R. Siddiqui, P. Patil, S. Bhusare, R. Pawar, Synthesis and
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