Synthesis of pyrazoline-thiazolidinone hybrids with trypanocidal activity

Article abstract of DOI:10.1016/j.ejmech.2014.07.103

A series of novel 4-thiazolidinone-pyrazoline conjugates have been synthesized and tested for anti-Trypanosoma brucei activity. Screening data allowed us to identify five thiazolidinone-pyrazoline hybrids, which possess promising trypanocidal activity, with IC₅₀ ≤ 1.2 μM. The highest active thiazolidinone-pyrazoline conjugates 3c and 6b (IC values of 0.6 μM and 0.7 μM, respectively) were 6-times more potent antitrypanosomal agents than nifurtimox. In addition, these compounds, as well as 6d and 6e had selectivity index higher than 50, and were more selective than nifurtimox. SAR study included substituent variations at the pyrazoline moiety, modifications of N3 position of the thiazolidinone portion, elongation of the linker between the heterocycles, as well as rhodanine-isorhodanine isomerism. It was also shown that methyl or aryl substitution at the thiazolidinone N3-position is crucial for trypanocidal activity.

Full text of DOI:10.1016/j.ejmech.2014.07.103

European Journal of Medicinal Chemistry 85 (2014) 245e254
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of pyrazolineethiazolidinone hybrids with trypanocidal
activity
Dmytro Havrylyuk a, Borys Zimenkovsky a, Olexandr Karpenko b, Philippe Grellier c,
,
*
Roman Lesyk a
a Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, Lviv 79010, Ukraine
b Enamine Ltd., Alexandra Matrosova 23, Kyiv 01103, Ukraine
c National Museum of Natural History, UMR 7245 CNRS-MNHN, Team BAMEE, CP 52, 57 Rue Cuvier, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 4-thiazolidinoneepyrazoline conjugates have been synthesized and tested for anti-
Trypanosoma brucei activity. Screening data allowed us to identify five thiazolidinoneepyrazoline hy-
Received 20 February 2014
Received in revised form
26 July 2014
Accepted 28 July 2014
Available online 30 July 2014
brids, which possess promising trypanocidal activity, with IC50 ꢀ 1.2
m
M. The highest active thiazolidi-
M, respectively) were 6-times
noneepyrazoline conjugates 3c and 6b (IC50 values of 0.6 M and 0.7
m
m
more potent antitrypanosomal agents than nifurtimox. In addition, these compounds, as well as 6d
and 6e had selectivity index higher than 50, and were more selective than nifurtimox. SAR study
included substituent variations at the pyrazoline moiety, modifications of N3 position of the thiazoli-
dinone portion, elongation of the linker between the heterocycles, as well as rhodanineeisorhodanine
isomerism. It was also shown that methyl or aryl substitution at the thiazolidinone N3-position is crucial
for trypanocidal activity.
Keywords:
Synthesis
Pyrazolines
4-Thiazolidinones
Antitrypanosomal activity
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
oxothiazolylhydrazones against Trypanosoma cruzi-infected cells.
Docking studies suggested that these compounds were potential
Human African trypanosomiasis (HAT) is among the most
serious neglected tropical diseases and is caused by Trypanosoma
brucei gambiense and rhodesiense (West and East African forms,
respectively). It is estimated that about 20 000 people are currently
suffering from HAT [1]. Available treatment still mainly relies on the
drugs developed many decades ago, which in their majority are
toxic, show decreasing efficacy, and involve administration/patient
compliance issues. Therefore, the discovery and development of
novel effective, safe, and affordable antitrypanosomal agents is a
task of high priority.
ligands for the cysteine protease cruzain. The most promising
M)
compound I, has shown to be very active (IC50 (Y strain) ¼ 0.3
m
at non-cytotoxic concentrations towards mammalian cells, and its
potency was comparable to the reference drugs nifurtimox and
benznidazole [18]. The 2-hydrazolyl-4-thiazolidinone-5-carboxylic
acid derivatives II have shown promising activity on the cruzipain
protease [19].
The 4-thiazolidinone derivatives with 4-dialkylaminobicyclo
[2.2.2]octane fragment III were tested against T. brucei rhode-
siense and showed moderate to weak activity (IC50 > 6.12 mM) [20].
The pharmacological activities of thiazolidinone [2e4] and
pyrazoline [5,6] derivatives are of current interest. Thiazolidino-
neediazole hybrids have been reported to possess promising
chemotherapeutic properties including anticancer [7e14] and
antiviral [15e17] activities. On the other hand, thiazolidinones and
pyrazolines (Fig. 1) are of great importance in the design and syn-
thesis of novel biologically active agents that exert trypanocidal
activity [18e26]. Leite et al. [18] tested a small library of aryl-4-
The 2-thioxo-4-thiazolidinone-3-acetic acid derivatives IV were
identified as inhibitors of T. brucei dolicholphosphate mannose
synthase and the most active compounds demonstrated trypano-
cidal activity against cultured bloodstream-form T. brucei (ED50
from 96 to 492 mM) [21]. It should be noted that fused and non-
fused thiazolidinone derivatives were also tested for their trypa-
nocidal activity. Thiopyrano[2,3-d][1,3]thiazoles, which can be
considered as cyclic isosteric mimetics of their synthetic precursors
5-arylidene-4-thiazolidinones without Michael accepting func-
tionalities, have been evaluated as potential antitrypanosomal
agents [22]. The most active analogue V inhibited T. brucei brucei
* Corresponding author.
E-mail addresses: dr_r_lesyk@org.lviv.net, roman.lesyk@gmail.com (R. Lesyk).
and T. brucei gambiense with an IC50 of 0.26 and 0.42
mM,
http://dx.doi.org/10.1016/j.ejmech.2014.07.103
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

246
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 85 (2014) 245e254
Fig. 1. Thiazolidinones and pyrazolines with trypanocidal activity.
respectively. It has been also reported that pyrimidine nucleoside-
thiazolinin-4-one hybrids VI possess moderate activity against
bloodstream-form T. brucei brucei with IC50 of 25e>100 mM [23].
triethyl orthoformate [27]. Considering the critical influence of the
thiazolidinone N3-substituent for the trypanocidal activity [28], the
corresponding 5-ethoxymethylene N3-substituted rhodanines
with methyl (1b), furan-2-ylmethyl (1c) and aryl (1d and 1e)
groups were synthesized in the same conditions. The target thia-
zolidinoneepyrazoline hybrids 2aee, 3aec, 4aed, 5aee, and 6aeg
with a methylidene linking group were obtained by reacting 5-
ethoxymethylene rhodanines 1aee with the appropriate 3,5-
diaryl-2-pyrazolines in refluxing ethanol (Scheme 1). In order to
investigate whether the migration of the thiocarbonyl function
from C2 to C4 position of the thiazolidine ring is crucial for activity,
the 4-thioxothiazolidin-2-one-pyrazoline conjugates 7aeb were
synthesized by using 4-thioxo-2-thiazolidinone (isorhodanine) as
starting material (Scheme 1).
To study the influence of the linking group on the trypanocidal
activity of the thiazolidinoneepyrazoline conjugates, the carboxylic
acid derivative 8 was synthesized by reaction of 1b with glycine in
acetic acid. The coupling of 8 with 3,5-diaryl-2-pyrazolines in the
presence of DCC led to the 2-oxoethylaminomethylene-linked
thiazolidinoneepyrazoline hybrids 9aec (Scheme 2).
In the case of the pyrazoline derivatives, some novel compounds
(e.g. VII) have been identified as inhibitors of the trypanosomal
cysteine protease cruzain with IC50 of 40e230 nM [24]. Therefore
the combination of thiazolidinone and pyrazoline fragments in one
molecule is a perspective approach to design promising anti-
trypanosomal agents (Fig. 1). Thus, among the thiazolidino-
neepyrazoline conjugates, compound VIII was investigated for its
activity against some causative organisms of tropical diseases and
showed the highest activity against T. brucei rhodesiense
(IC50 ¼ 12
mg/ml) [25].
From our previous studies on the in vitro antitrypanosomal ac-
tivity of non-condensed thiazolidineepyrazoline derivatives,
compound IX has identified as a hit agent against T. brucei brucei
(IC50 ¼ 3.01
mg/ml) [26]. To continue this study the 5-pyrazoline
substituted 4-thiazolidinones X were further selected in in vitro
assays against T. brucei brucei and T. brucei gambiense and showed
IC50 from 5.4 to 13.9 mM and 2.5 to 6.7 mM, respectively [16].
In the present study, we designed (Fig. 2) and synthesized new
thiazolidinoneepyrazoline hybrid compounds bearing various
substituents at the thiazolidinone N3 position and pyrazoline ring 3
and 5 positions, and having different linkages between the pyr-
azoline ring and the thiazolidinone scaffold. The resulting de-
rivatives were tested for their ability to inhibit the in vitro growth of
T. brucei gambiense. The chemical modifications of 5-pyrazoline
substituted 4-thiazolidinones resulted in a 4e10-fold increase in
potency (for the most active candidates) as compared to the pyr-
azolineethiazolidinone analogues X [16].
The structure of the newly synthesized heterocyclic-
substituted thiazolidinones was confirmed by elemental analysis
and spectroscopic data (1H NMR, 13C NMR and LCMS). 1H NMR
spectra of all synthesized compounds showed characteristic pat-
terns of an AMX system for the protons at positions 4 and 5 of the
pyrazoline ring. The olefinic proton (]CH) of compounds 2aee,
3aec, 4aed, 5aee and 6aeg showed
~ 7.19e7.95 ppm. In the 1H NMR spectra of 2aec, 7a and 7b, the
broad singlet of the NH proton of the thiazolidinone ring appeared
at ~12.32e12.97 ppm.
Due to the enamine fragment at 5-position of the thiazolidinone
a
singlet at
d
d
2. Results and discussion
ring, compounds 8 and 9aec exist as a mixture of Z and E isomers
(Fig. 3). In the 1H NMR spectra of compound 8, the olefinic proton
2.1. Chemistry
(]CH) resonates as two doublets at
d
¼ 7.63 ppm (Z-isomer) and
d
¼ 7.43 ppm (E-isomer). In addition, the enamine NH proton
The general methods for the synthesis of the new thiazolidi-
noneepyrazoline hybrids are depicted in Schemes 1 and 2.
The synthesis of 5-ethoxymethylidene rhodanine 1a was effec-
ted by reaction of 2-thioxo-4-thiazolidinone (rhodanine) with
appeared as two multiplets at
d
enamine NH proton were also observed in the 1H NMR spectra of
d
¼ 8.93e8.97 ppm (E-isomer) and
¼ 8.28e8.31 ppm (Z-isomer). Two similar multiplets for the
compounds 9aec.

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 85 (2014) 245e254
247
Fig. 2. Design of novel thiazolidinoneepyrazoline hybrids.
2.2. In vitro evaluation of antitrypanosomal activity and
cytotoxicity
combination with eflornithine for the gambiense human African
trypanosomiasis therapy [29], was selected as reference antipara-
sitic drug and exhibited a CC50 value of 78.2
derived cell line (L-6).
Of all the tested compounds, thiazolidinoneepyrazoline hybrids
3c and 6b possessed the highest trypanocidal properties, with IC50
mM against myoblast-
All compounds were screened for inhibitory activity against T.
brucei gambiense (Feo strain). Compounds were first tested at fixed
concentrations of 50, 10 and 1
determined for those showing significant trypanocidal activity at
10 g/ml (>40e50% of parasite growth inhibition) (Table 1). Cyto-
mg/ml. IC50 values were further
values of 0.6 mM and 0.7 mM, respectively (Table 1) and were 6-
m
times more potent antitrypanosomal agents than nifurtimox. In
addition, these compounds, as well as 6d and 6e had selectivity
index higher than 50, and were more selective than nifurtimox. The
doseeresponse curves of hybrids 3c, 6a, 6b, 6d and 6e are depicted
in Fig. 4.
toxicities against myoblast-derived cell line (L-6) were determined
to calculate the selectivity indices (the ratios of cytotoxic CC50
values to antitrypanosomal IC50 values) of the highly active com-
pounds 3b, 3c, 6a, 6b, 6d and 6e. Nifurtimox, which is used in
Scheme 1. Synthesis of 5-(3,5-diaryl-4,5-dihydropyrazol-1-ylmethylene)-2-thioxothiazolidin-4-ones (2aee, 3aec, 4aed, 5aee, 6aeg) and 5-(3,5-diaryl-4,5-dihydropyrazol-1-
ylmethylene)-4-thioxothiazolidin-2-ones (7a, 7b). Reagents, conditions and yields: (a) CH(OC2H5)3, Ac2O, reflux 1 h, [27]; (b) EtOH, reflux 1 h, 78e92%.
Scheme 2. Synthesis of 5-{[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-2-oxo-ethylamino]-methylene}-3-methyl-2-thioxothiazolidin-4-ones. Reagents, conditions and yields: (c)
glycine, AcOH, reflux 3 h, 75%; (d) EtOH, reflux 1 h, 61e75%.

248
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 85 (2014) 245e254
were observed for the inactive N3 furan-2-ylmethyl substituted
compounds 4aed. We observed that compounds bearing 2-
hydroxyphenyl (6a), 4-dimethylaminophenyl (6e), and 4-
chlorophenyl (6f) substituents at position 5 of the 5-aryl-3-
phenylpyrazoline fragment were 5e10 fold more potent than the
corresponding 4-methoxyphenyl analogue 6c. Attachment a hy-
droxyl group to 5-aryl substituent of the 3,5-diarylpyrazoline
fragment yielded the most active N3-aryl substituted hybrids, as
exemplified by 6a and 6b. Surprisingly, the replacement of the 4-
methoxyphenyl substituent (5d) or phenyl substituent (6f) at po-
sition 3 of the respective 5-(4-chlorophenyl)-3-arylpyrazoline
fragments with a 2-naphthalenyl residue (5e and 6g) led to a
substantial loss of activity.
Additionally, we observed that the modification of thiazolidi-
none N3 position is more critical for potent trypanocidal activity in
comparison to the substitution at positions 3 and 5 derivation of
the pyrazoline fragment. Thus, the insertion of a 2-furanylmethyl
substituent at the thiazolidinone N3 position had an unfavorable
effect and led to inactive derivatives (4aed). However, the substi-
tution of the thiazolidinone NH hydrogen by methyl or other aryl
groups significantly increased trypanocidal potency. p-Tolyl
substituted hybrid 5a was 10-fold more potent trypanocidal agent
than the parent compound 2a. A remarkable improvement in ac-
tivity was observed in the case of the N3-(3-acetoxyphenyl) ana-
logues (6), indicating that the 3-acetoxyphenyl group was the most
beneficial to trypanocidal activity in this series of compounds. Thus,
many of the thiazolidinone N3 substituted derivatives synthesized
Fig. 3. Z/E-tautomerism of compounds 8 and 9aec.
The initial SAR study included two distinct modifications of the
thiazolidinoneepyrazoline hybrid structure: variation of sub-
stituents at the pyrazoline moiety and substitution of the thiazo-
lidinone NH hydrogen by methyl, 2-furanylmethyl, and aryl groups.
SAR study revealed that aryl variation in the pyrazoline ring had
no influence on the antitrypanosomal activity or resulted in
ambiguous levels of activity (Table 1). For example, the N3 unsub-
stituted thiazolidinones 2aec with different aryls at 3 and 5 loca-
tions of the pyrazoline ring demonstrated the same range of
trypanocidal activity (IC50 from 10.5 to 14.7 mM). The same trends
Table 1
Anti-trypanosomal activity and cytotoxicity of thiazolidinoneepyrazoline hybrids.
Comp
R1
R2
Ar
% inhibitiona
IC50
m
Mb
SD IC50
Cytotoxicity on myoblast-
derived cell line (L-6)
50
mg/ml
10
m
g/ml
1
m
g/ml
CC50
m
Mb
SD CC50
SId
2a
2b
2c
H
4-OMe
4-NMe2
4-F
4-Cl
4-Cl
Ph
Ph
91.5
74.0
85.4
56.0
97.0
97.1
92.3
76.2
85.0
42.0
97.2
97.2
21.9
30.2
19.9
8.2
97.1
97.2
14.7
10.5
13.2
nd
>1
0.6
3.79
2.2
2.77
nd
Naphthalen-2-yl
Ph
Naphthalen-2-yl
Naphthalen-2-yl
3ac
3b
3c
Me
e
153.7
175.2
4.1
17.0
4-OMe
0.03
292
4a
4-OMe
Ph
46.6
67.3
37.9
nd
nd
4b
4c
4d
4-OMe
4-OMe
4-Cl
4-OMe-C6H4
Naphthalen-2-yl
Naphthalen-2-yl
12.8
54.6
59.6
48.2
32.1
45.3
38.7
19.4
29.0
>98.9
nd
nd
e
nd
nd
O
Me
5a
4-OMe
Ph
17.8
69.2
52.5
1.5
0.31
5b
5c
5d
5e
4-OMe
4-NMe2
4-Cl
4-OMe-C6H4
Ph
4-OMe-C6H4
Naphthalen-2-yl
23.1
5.8
40.2
41.7
55.5
51.9
70.4
59.4
44.5
39.8
62.8
27.6
nd
>20.1
2.5
nd
e
0.81
5.74
4-Cl
21.7
6a
2-OH
Ph
90.9
93.8
83.1
1.1
0.18
13.6
40.0
0.2
12.4
57.1
6b
6c
6d
6e
6f
2-OH
4-OMe
4-OMe
4-NMe2
4-Cl
4-OMe-C6H4
Ph
Naphthalen-2-yl
Ph
89.5
97.2
94.7
81.6
90.3
90.6
91.7
97.5
95.8
84.9
91.4
89.9
90.1
79.3
87.6
53.1
42.1
66.7
0.7
10.8
1.2
1.2
1.9
0.03
0.26
0.23
0.28
0.47
1.92
10.0
>172.5
>184.3
e
e
>143.8
>153.6
O
Ph
6g
4-Cl
Naphthalen-2-yl
16.5
Me
O
7a
7b
9a
9b
H
2-OH
4-OMe
4-OMe
4-Cl
Ph
85.8
87.2
96.1
93.5
95.6
93.3
74.7
96.5
86.9
94.8
61.0
19.7
62.3
52.0
46.6
3.5
18.6
4.6
2.1
1.5
4.4
0.46
4.71
0.66
0.2
0.14
0.7
Naphthalen-2-yl
Ph
Naphthalen-2-yl
Naphthalen-2-yl
Me
9c
4-F
Nifurtimox
78.2
18
17.8
a
Percentage of inhibition of parasite growth at 50, 10 and 1
m
g/ml.
IC50 value is the mean the standard deviation (SD) of three independent experiments (nd e not determined).
Percentage of inhibition of parasite growth were determined at 100, 20 and 2 g/ml.
b
c
m
d
Determined as CC50/IC50
.

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 85 (2014) 245e254
249
3. Conclusions
In the present paper new 4-thiazolidinone based conjugates
with pyrazoline moiety at 5 position are described. Trypanocidal
activity assay of the synthesized compounds has allowed us to
identify thiazolidinoneepyrazoline hybrids 3c and 6b, which were
found to be the most active derivatives, with IC50 values of 0.6
mM
and 0.7 M, respectively. A SAR study for the anti-T. brucei agents
m
was conducted and included substituent variations at the pyrazo-
line moiety, modifications of the thiazolidinone N3 position, elon-
gation of the linker between the two heterocycles, as well as
rhodanineeisorhodanine isomerism. We observed a remarkable
improvement in activity when the thiazolidinone N3 position was
substituted with methyl and aryl groups. Further investigations on
the thiazolidinoneepyrazoline derivatives could lead to more
potent compounds as promising candidates for the development of
new antitrypanosomal chemotherapy.
4. Experimental
4.1. Materials and methods
2-Thioxo-4-thiazolidinone [30], 4-thioxo-2-thiazolidinone [31],
3,5-diaryl-4,5-dihydro-1H-pyrazole [32] and 5-ethoxymethylene
rhodanines 1aee [27] were employed as starting materials and
prepared according to the method described previously.
Fig. 4. The doseeresponse curves of compounds 3c, 6a, 6b, 6d and 6e on Trypanosoma
brucei gambiense growth.
were significantly more active than the N3 unsubstituted conju-
Melting points were measured in open capillary tubes on a
BUCHI B-545 melting point apparatus and are uncorrected. The
}
gates, and had a range of IC50 values of 0.6e1.9
mM (Table 1, Fig. 5).
The further SAR study was directed towards the rhodanine-
eisorhodanine isomerism. To this end, the isorhodanineepyrazo-
line conjugates 7a and 7b were synthesized and tested for their
trypanocidal activity. The biological activity data revealed that the
relative positioning of the thiocarbonyl and carbonyl functions on
the thiazolidinone ring (positions 2 and 4) essentially does not
affect the activity, considering the same range of the trypanocidal
potency of 7b and isosters 2aec. However, the 4-thiocarbonyl 4-
functionalized derivative 7a showed a 3e4 fold and 5-fold in-
crease in potency as compared to 2aec and 7b, respectively. This
finding could be explained by the presence of a hydroxyl group in
the diarylpyrazoline moiety, which also provided the benefit of the
improved activity in compounds 6a and 6b (Table 1, Fig. 5).
elemental analysis (C, H, N) were performed using a PerkineElmer
2400 CHN analyzer. Analyses indicated by the symbols of the ele-
ments or functions were within 0.4% of the theoretical values. The
1H NMR spectra were recorded on Varian Gemini 400 MHz and 13
C
NMR spectra on Varian Mercury-400 100 MHz in DMSO-d6 or
DMSO-d6 þ CCl4 mixture using tetramethylsilane (TMS) as an in-
ternal standard. Chemical shifts are reported in ppm units with use
of
d scale. Mass spectra were obtained using electrospray (ES)
ionization techniques on an Agilent 1100 Series LCMS. Analytical
HPLC were performed on an Agilent 1100 HPLC with DAD (Diode
Array Detection). ESI refers to electrospray ionization, HPLC refers
to high pressure liquid chromatography, LCMS refers to liquid
chromatography coupled with a mass spectrometer, M in the
context of mass spectrometry refers to the molecular peak, MS
refers to mass spectrometer. Purity of all compounds was deter-
mined to be ꢁ95% by HPLC (Agilent 1100 HPLC) with Diode Array
Detection. The peak purity was checked with UV spectra.
Finally, we prepared and tested the 2-oxoethylaminomethylene-
linked thiazolidinoneepyrazoline hybrids 9ae9c and observed that
elongation of the linker between the two heterocycles decreases the
trypanocidal potency as compared to the highly active analogue 3c.
Fig. 5. SAR of trypanocidal thiazolidinoneepyrazoline conjugates.

250
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 85 (2014) 245e254
4.2. Chemistry
4.2.1.5. 5-[5-(4-Chlorophenyl)-3-naphthalen-2-yl-4,5-
dihydropyrazol-1-ylmethylene]-3-methyl-2-thioxothiazolidin-4-one
4.2.1. General procedure for synthesis of 5-(3,5-diaryl-4,5-
dihydropyrazol-1-ylmethylene)-2-thioxothiazolidin-4-ones (2aee,
3aec, 4aed, 5aee, 6aeg)
(3b). Yield 80%, mp 279e280 ꢂC. 1H NMR (400 MHz, DMSO-
d6 þ CCl4):
d 8.26 (s, 1H, arom), 8.08 (brs, 2H, arom), 7.97e8.02 (m,
2H, arom), 7.61e7.63 (m, 2H, arom), 7.57 (s, 1H, ]CH), 7.52 (d, 2H,
J ¼ 8.4 Hz, arom), 7.44 (d, 2H, J ¼ 8.4 Hz, arom), 5.76 (dd, 1H, CH2CH,
J ¼ 11.4, 6.1 Hz), 4.14 (dd, 1H, CH2CH, J ¼ 18.4, 11.4 Hz), 3.61 (dd, 1H,
CH2CH, J ¼ 18.4, 6.1 Hz), 3.29 (s, 3H, CH3). 13C NMR (100 MHz,
A
mixture of 5-ethoxymethylene-2-thioxothiazolidin-4-one
1aee (5 mmol) and appropriate 3,5-diaryl-4,5-dihydro-1H-pyr-
azole (5 mmol) was refluxed in 10 ml of ethanol during 1 h. The
crystalline product was separated by filtration, washed with
ethanol, and dried. Recrystallization from DMFeEtOH (1:1)
rendered the desired product in pure form.
DMSO-d6):
d 186.5 (C]S), 166.3 (C]O), 159.4 (C]N), 144.3, 139.3,
134.1, 133.6, 133.5, 132.7, 129.4, 129.0, 128.9, 128.8, 128.7, 128.0,
127.9, 127.5, 127.2, 123.1, 94.2 (]CH), 64.3 (CHCH2), 41.7 (CHCH2),
30.8 (CH3). LCMS (ESIþ) m/z 464/465 (MþH)þ. Calcd. for
C24H18ClN3OS2: C, 62.13; H, 3.91; N, 9.06; Found: C, 62.37; H, 4.18;
4.2.1.1. 5-[5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-
N, 9.32%.
ylmethylene]-2-thioxothiazolidin-4-one
(2a). Yield
85%,
d 12.92 (s, 1H,
mp
259e260 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
4.2.1.6. 5-[5-(4-Methoxyphenyl)-3-naphthalen-2-yl-4,5-
dihydropyrazol-1-ylmethylene]-3-methyl-2-thioxothiazolidin-4-one
(3c). Yield 86%, mp 250e252 ꢂC. 1H NMR (400 MHz, DMSO-
NH), 7.84e7.89 (m, 2H, arom), 7.56e7.58 (m, 3H, arom), 7.33 (d, 2H,
J ¼ 8.5 Hz, arom), 7.24 (s, 1H, ]CH), 7.00 (d, 2H, J ¼ 8.5 Hz, arom),
5.58 (dd, 1H, CH2CH, J ¼ 11.2, 7.0 Hz), 3.98 (dd, 1H, CH2CH, J ¼ 18.2,
11.2 Hz), 3.77 (s, 3H, OCH3), 3.47 (dd,1H, CH2CH, J ¼ 18.2, 7.0 Hz). 13
C
d6 þ CCl4):
d 8.28 (s, 1H, arom), 8.08 (brs, 2H, arom), 7.97e8.03 (m,
2H, arom), 7.61e7.63 (m, 2H, arom), 7.45 (s, 1H, ]CH), 7.37 (d, 2H,
J ¼ 8.4 Hz, arom), 7.00 (d, 2H, J ¼ 8.4 Hz, arom), 5.66 (dd, 1H, CH2CH,
J ¼ 11.2, 6.7 Hz), 4.10 (dd, 1H, CH2CH, J ¼ 18.1, 11.2 Hz), 3.77 (s, 3H,
NMR (100 MHz, DMSO-d6):
d 198.2 (C]S), 168.4 (C]O), 159.7 (C]
N), 154.7, 144.3, 133.8, 133.2, 130.1, 129.2, 128.6, 127.3, 114.7, 96.5 (]
CH), 64.5 (CHCH2), 55.3 (OCH3), 41.4 (CHCH2). LCMS (ESIþ) m/z 396
(MþH)þ. Calcd. for C20H17N3O2S2: C, 60.74; H, 4.33; N,10.62; Found:
C, 60.40; H, 4.61; N, 10.38%.
OCH3), 3.60 (dd, 1H, CH2CH, J ¼ 18.2, 6.7 Hz), 3.28 (s, 3H, CH3). 13
C
NMR (100 MHz, DMSO-d6): d 193.6 (C]S),186.4 (C]O),166.2,159.6
(C]N), 134.1, 133.4, 132.8, 131.9, 128.9, 128.8, 128.7, 128.6, 128.0,
127.6, 127.2, 123.1, 114.8, 93.8 (]CH), 64.7 (CHCH2), 55.3 (OCH3),
41.5 (CHCH2), 30.8 (CH3). LCMS (ESIþ) m/z 460 (MþH)þ. Calcd. for
4. 2.1. 2. 5-[5-(4-Dimethylaminophenyl)-3-phenyl-4, 5-
dihydropyrazol-1-ylmethylene]-2-thioxothiazolidin-4-one
(2b).
C25H21N3O2S2: C, 65.34; H, 4.61; N, 9.14; Found: C, 65.67; H, 4.39; N,
Yield 78%, mp 289e290 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
9.37%.
d
12.90 (s, 1H, NH), 7.84e7.87 (m, 2H, arom), 7.56e7.58 (m, 3H,
arom), 7.21 (d, 2H, J ¼ 8.8 Hz, arom), 7.19 (s, 1H, ]CH), 6.75 (d, 2H,
J ¼ 8.8 Hz, arom), 5.48 (dd, 1H, CH2CH, J ¼ 11.2, 7.4 Hz), 3.93 (dd, 1H,
CH2CH, J ¼ 18.3, 11.2 Hz), 3.44 (dd, 1H, CH2CH, J ¼ 18.3, 7.4 Hz), 2.91
4.2.1.7. 3-Furan-2-ylmethyl-5-[5-(4-methoxyphenyl)-3-phenyl-4,5-
dihydropyrazol-1-ylmethylene]-2-thioxothiazolidin-4-one
(4a).
Yield 75%, mp 198e200 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
(s, 6H, N(CH3)2). 13C NMR (100 MHz, DMSO-d6):
d 195.9 (C]S),
d
7.87e7.89 (m, 2H, arom), 7.54e7.59 (m, 4H, arom), 7.43 (s, 1H, ]
168.3 (C]O), 159.1 (C]N), 150.7, 132.4, 131.2, 130.3, 129.2, 128.3,
127.2, 126.4, 112.7, 96.2 (]CH), 64.9 (CHCH2), 41.2 (CHCH2), 39.3
(N(CH3)2). LCMS (ESIþ) m/z 409 (MþH)þ. Calcd. for C21H20N4OS2: C,
61.74; H, 4.93; N, 13.71; Found: C, 61.98; H, 5.15; N, 13.58%.
CH), 7.35 (d, 2H, J ¼ 8.1 Hz, arom), 7.01 (d, 2H, J ¼ 8.1 Hz, arom), 6.38
(brs, 1H, arom), 6.31 (brs, 1H, arom), 5.61 (dd, 1H, CH2CH, J ¼ 11.3,
6.5 Hz), 5.13 (s, 2H, CH2), 4.01 (dd, 1H, CH2CH, J ¼ 18.2, 11.3 Hz), 3.78
(s, 3H, OCH3), 3.50 (dd, 1H, CH2CH, J ¼ 18.2, 6.5 Hz). 13C NMR
(100 MHz, DMSO-d6): d 193.2 (C]S), 165.6 (C]O), 159.9, 159.7 (C]
4.2.1.3. 5-[5-(4-Fluorophenyl)-3-naphthalen-2-yl-4,5-
N), 148.6, 142.5, 133.7, 131.7, 131.4, 130.0, 129.2, 128.7, 127.4, 114.8,
110.6, 108.9, 92.9 (]CH), 64.6 (CHCH2), 55.3 (OCH3), 41.5 (CHCH2),
40.5 (CH2). LCMS (ESIþ) m/z 476 (MþH)þ. Calcd. for C25H21N3O3S2:
C, 63.14; H, 4.45; N, 8.84; Found: C, 62.82; H, 4.12; N, 8.55%.
dihydropyrazol-1-ylmethylene]-2-thioxothiazolidin-4-one
(2c).
Yield 82%, mp 296e298 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
d
12.97 (s, 1H, NH), 8.27 (s, 1H, arom), 7.98e8.09 (m, 4H, arom),
7.61e7.63 (m, 2H, arom), 7.46e7.51 (m, 2H, arom), 7.32 (s,1H, ]CH),
7.29 (d, 2H, J ¼ 7.3 Hz, arom), 5.72 (dd, 1H, CH2CH, J ¼ 11.5, 6.5 Hz),
4.13 (dd, 1H, CH2CH, J ¼ 17.9, 11.5 Hz), 3.60 (dd, 1H, CH2CH, J ¼ 17.9,
4.2.1.8. 5-[3,5-Bis-(4-methoxyphenyl)-4,5-dihydropyrazol-1-
ylmethylene]-3-furan-2-ylmethyl-2-thioxothiazolidin-4-one
(4b).
6.5 Hz). 13C NMR (100 MHz, DMSO-d6):
d 196.1 (C]S), 168.5 (C]O),
Yield 88%, mp 262e264 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
165.8 (d, JCF ¼ 200.0 Hz, 1C), 161.3, 158.8 (C]N), 144.3, 136.5, 134.0,
132.8, 132.5, 131.9 (d, JCF ¼ 30.0 Hz, 2C), 129.5, 129.4, 128.8, 128.7,
128.0, 127.6, 127.2, 123.1, 116.3 (d, JCF ¼ 21.0 Hz, 2C), 97.0 (]CH),
64.3 (CHCH2), 41.7 (CHCH2). LCMS (ESIþ) m/z 434 (MþH)þ. Calcd.
for C23H16FN3OS2: C, 63.72; H, 3.72; N, 9.69; Found: C, 63.50; H,
3.45; N, 9.98%.
d
7.82 (d, 2H, J ¼ 8.6 Hz, arom), 7.53 (s, 1H, arom), 7.40 (s, 1H, ]CH),
7.33 (d, 2H, J ¼ 8.5 Hz, arom), 7.14 (d, 2H, J ¼ 8.6 Hz, arom), 6.99 (d,
2H, J ¼ 8.5 Hz, arom), 6.36 (brs, 1H, arom), 6.28 (d, 1H, J ¼ 2.8 Hz,
arom), 5.59 (dd, 1H, CH2CH, J ¼ 11.1, 7.1 Hz), 5.12 (s, 2H, CH2), 3.96
(dd, 1H, CH2CH, J ¼ 18.3, 11.1 Hz), 3.84 (s, 3H, OCH3), 3.77 (s, 3H,
OCH3), 3.46 (dd, 1H, CH2CH, J ¼ 18.3, 7.1 Hz). 13C NMR (100 MHz,
DMSO-d6):
d 192.9 (C]S), 165.5 (C]O), 161.9, 159.9, 159.7 (C]N),
4.2.1.4. 5-[5-(4-Chlorophenyl)-3-phenyl-4,5-dihydropyrazol-1-
148.7, 142.5, 133.6, 131.7, 129.3, 128.6, 122.4, 114.8, 110.6, 108.9, 92.2
(]CH), 64.5 (CHCH2), 55.6 (OCH3), 55.3 (OCH3), 41.6 (CHCH2), 40.5
(CH2). LCMS (ESIþ) m/z 506 (MþH)þ. Calcd. for C26H23N3O4S2: C,
61.76; H, 4.59; N, 8.31; Found: C, 61.48; H, 4.75; N, 8.09%.
ylmethylene]-3-methyl-2-thioxothiazolidin-4-one (3a). Yield 87%,
mp 288e290 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
d 7.84e7.86
(m, 2H, arom), 7.50e7.58 (m, 6H, arom, ]CH), 7.40e7.42 (m, 2H,
arom), 5.72 (dd, 1H, CH2CH, J ¼ 11.3, 6.0 Hz), 4.04 (dd, 1H, CH2CH,
J ¼ 18.1, 11.3 Hz), 3.48 (dd, 1H, CH2CH, J ¼ 18.1, 6.0 Hz), 3.29 (s, 3H,
4.2.1.9. 3-Furan-2-ylmethyl-5-[5-(4-methoxyphenyl)-3-naphthalen-
2-yl-4,5-dihydropyrazol-1-ylmethylene]-2-thioxothiazolidin-4-one
(4c). Yield 91%, mp 224e226 ꢂC. 1H NMR (400 MHz, DMSO-
CH3). 13C NMR (100 MHz, DMSO-d6):
d 193.8 (C]S), 166.3 (C]O),
159.4 (C]N), 144.3, 139.3, 133.7, 133.5, 131.4, 129.9, 129.4, 129.2,
129.0, 127.4, 94.0 (]CH), 64.2 (CHCH2), 41.7 (CHCH2), 30.8 (CH3).
LCMS (ESIþ) m/z 414/416 (MþH)þ. Calcd. for C20H16ClN3OS2: C,
58.03; H, 3.90; N, 8.56; Found: C, 58.37; H, 3.67; N, 8.82%.
d6 þ CCl4):
d 8.28 (s, 1H, arom), 8.08 (brs, 2H, arom), 7.97e8.04 (m,
2H, arom), 7.60e7.63 (m, 2H, arom), 7.55 (brs, 1H, arom), 7.45 (s, 1H,
]CH), 7.37 (d, 2H, J ¼ 8.6 Hz, arom), 7.01 (d, 2H, J ¼ 8.6 Hz, arom),

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 85 (2014) 245e254
251
6.36 (t, 1H, J ¼ 3.0 Hz, arom), 6.31 (d, 1H, J ¼ 3.0 Hz, arom), 5.65 (dd,
1H, CH2CH, J ¼ 11.1, 6.7 Hz), 5.13 (s, 2H, CH2), 4.10 (dd, 1H, CH2CH,
J ¼ 18.2, 11.1 Hz), 3.77 (s, 3H, OCH3), 3.61 (dd, 1H, CH2CH, J ¼ 18.2,
130.2, 129.6, 129.2, 128.5, 128.1, 127.3, 126.5, 112.7, 93.6 (]CH), 65.0
(CHCH2), 41.3 (CHCH2), 39.3 (N(CH3)2), 20.9 (CH3). LCMS (ESIþ) m/z
499 (MþH)þ. Calcd. for C28H26N4OS2: C, 67.44; H, 5.26; N, 11.24;
Found: C, 67.70; H, 5.48; N, 11.52%.
6.7 Hz). 13C NMR (100 MHz, DMSO-d6):
d 193.2 (C]S), 165.7 (C]O),
159.9, 159.7 (C]N), 148.7, 142.5, 134.1, 133.6, 132.8, 131.7, 129.0,
128.8,128.7,128.6,128.0,127.9,127.6,127.2,123.1,114.8,110.6,108.9,
93.1 (]CH), 64.7 (CHCH2), 55.3 (OCH3), 41.5 (CHCH2), 40.6 (CH2).
LCMS (ESIþ) m/z 526 (MþH)þ. Calcd. for C29H23N3O3S2: C, 66.26; H,
4.41; N, 7.99; Found: C, 66.01; H, 4.68; N, 8.27%.
4.2.1.14. 5-[5-(4-Chlorophenyl)-3-(4-methoxyphenyl)-4,5-
dihydropyrazol-1-ylmethylene]-2-thioxo-3-p-tolylthiazolidin-4-one
(5d). Yield 85%, mp 210e211 ꢂC. 1H NMR (400 MHz, DMSO-
d6 þ CCl4):
d
7.88 (d, 2H, J ¼ 8.5 Hz, arom), 7.66 (d, 2H, J ¼ 8.5 Hz,
arom), 7.41 (s, 1H, ]CH), 7.35 (d, 2H, J ¼ 8.6 Hz, arom), 7.28 (d, 2H,
J ¼ 8.1 Hz, arom), 7.11 (d, 2H, J ¼ 8.1 Hz, arom), 7.00 (d, 2H,
J ¼ 8.6 Hz, arom), 5.63 (dd, 1H, CH2CH, J ¼ 11.3, 7.0 Hz), 3.99 (dd, 1H,
CH2CH, J ¼ 18.4, 11.3 Hz), 3.76 (s, 3H, OCH3), 3.49 (dd, 1H, CH2CH,
J ¼ 18.4, 7.0 Hz), 2.35 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
4.2.1.10. 3-Furan-2-ylmethyl-5-[5-(4-chlorophenyl)-3-naphthalen-2-
yl-4,5-dihydropyrazol-1-ylmethylene]-2-thioxothiazolidin-4-one
(4d). Yield 82%, mp 232e234 ꢂC. 1H NMR (400 MHz, DMSO-
d6 þ CCl4):
d 8.28 (s, 1H, arom), 8.09 (brs, 2H, arom), 7.97e8.03 (m,
2H, arom), 7.60e7.65 (m, 2H, arom), 7.51e7.55 (m, 4H, arom, ]CH),
7.46 (d, 2H, J ¼ 7.1 Hz, arom), 6.38 (brs, 1H, arom), 6.31 (brs, 1H,
arom), 5.78 (dd, 1H, CH2CH, J ¼ 10.9, 6.3 Hz), 5.20 (s, 2H, CH2), 4.17
(dd, 1H, CH2CH, J ¼ 18.0, 10.9 Hz), 3.62 (dd, 1H, CH2CH, J ¼ 18.0,
d 194.4 (C]S), 166.3 (C]O), 159.7 (C]N), 158.5, 138.5, 135.9, 133.6,
133.3, 131.8, 129.7, 129.4, 129.0, 128.7, 128.5, 114.7, 94.3 (]CH), 64.8
(CHCH2), 55.3 (OCH3), 41.4 (CHCH2), 20.9 (CH3). LCMS (ESIþ) m/z
520/522 (MþH)þ. Calcd. for C27H22ClN3O2S2: C, 62.36; H, 4.26; N,
8.08; Found: C, 62.61; H, 4.01; N, 8.27%.
6.3 Hz). 13C NMR (100 MHz, DMSO-d6):
d 193.3 (C]S), 165.7 (C]O),
159.7 (C]N), 148.7, 144.3, 142.6, 139.2, 134.1, 133.9, 133.5, 132.7,
129.4, 129.0, 128.9, 128.8, 128.0, 127.9, 127.4, 127.2, 123.1, 110.7,
109.0, 93.6 (]CH), 64.4 (CHCH2), 41.7 (CHCH2), 40.5 (CH2). LCMS
(ESIþ) m/z 531 (MþH)þ. Calcd. for C28H20ClN3O2S2: C, 63.45; H,
3.80; N, 7.93; Found: C, 63.72; H, 4.12; N, 7.68%.
4.2.1.15. 5-[5-(4-Chlorophenyl)-3-naphthalen-2-yl-4,5-
dihydropyrazol-1-ylmethylene]-2-thioxo-3-p-tolylthiazolidin-4-one
(5e). Yield 78%, mp 262e264 ꢂC. 1H NMR (400 MHz, DMSO-
d6 þ CCl4):
d 8.29 (s, 1H, arom), 8.12 (brs, 2H, arom), 7.98e8.04 (m,
2H, arom), 7.62e7.65 (m, 2H, arom), 7.57 (s, 1H, ]CH), 7.52 (d, 2H,
J ¼ 8.4 Hz, arom), 7.45 (d, 2H, J ¼ 8.4 Hz, arom), 7.30 (d, 2H,
J ¼ 8.1 Hz, arom), 7.13 (d, 2H, J ¼ 8.1 Hz, arom), 5.78 (dd, 1H, CH2CH,
J ¼ 11.4, 6.0 Hz), 4.17 (dd, 1H, CH2CH, J ¼ 18.4, 11.4 Hz), 3.62 (dd, 1H,
CH2CH, J ¼ 18.4, 6.0 Hz), 2.36 (s, 3H, CH3). 13C NMR (100 MHz,
4.2.1.11. 5-[5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-
ylmethylene]-2-thioxo-3-p-tolylthiazolidin-4-one (5a). Yield 88%,
mp 214e216 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
d 7.89e7.91
(m, 2H, arom), 7.58e7.61 (m, 3H, arom), 7.43 (s, 1H, ]CH), 7.35 (d,
2H, J ¼ 8.6 Hz, arom), 7.29 (d, 2H, J ¼ 8.1 Hz, arom), 7.11 (d, 2H,
J ¼ 8.1 Hz, arom), 7.00 (d, 2H, J ¼ 8.6 Hz, arom), 5.63 (dd, 1H, CH2CH,
J ¼ 11.5, 6.7 Hz), 4.03 (dd, 1H, CH2CH, J ¼ 18.4, 11.5 Hz), 3.76 (s, 3H,
DMSO-d6):
d 194.5 (C]S), 166.4 (C]O), 159.4 (C]N), 139.4, 138.5,
134.1, 133.6, 133.5, 133.4, 132.8, 129.7, 129.4, 129.0, 128.9, 128.7,
128.5, 128.0, 127.9, 127.5, 127.3, 123.1, 94.5 (]CH), 64.3 (CHCH2),
40.5 (CHCH2), 20.9 (CH3). LCMS (ESIþ) m/z 541/543 (MþH)þ. Calcd.
for C30H22ClN3OS2: C, 66.71; H, 4.11; N, 7.78; Found: C, 66.48; H,
3.87; N, 7.52%.
OCH3), 3.50 (dd, 1H, CH2CH, J ¼ 18.4, 6.7 Hz), 2.35 (s, 3H, CH3). 13
C
NMR (100 MHz, DMSO-d6): 13C NMR (100 MHz, DMSO-d6):
d 194.4
(C]S), 166.3 (C]O), 159.7 (C]N), 159.6, 144.3, 138.5, 133.6, 133.4,
131.9,131.4, 130.1,129.7,129.2,128.6,128.5,127.4,114.8, 93.9 (]CH),
64.6 (CHCH2), 55.3 (OCH3), 41.5 (CHCH2), 20.9 (CH3). LCMS (ESIþ)
m/z 486 (MþH)þ. Calcd. for C27H23N3O2S2: C, 66.78; H, 4.77; N,
8.65; Found: C, 66.43; H, 4.99; N, 8.42%.
4.2.1.16. 5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-
ylmethylene]-3-(3-acetoxyphenyl)-2-thioxothiazolidin-4-one
(6a).
Yield 78%, mp 238e240 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
d
9.94 (s, 1H, OH), 7.89 (d, 2H, J ¼ 5.8 Hz, arom), 7.51e7.55 (m, 3H,
4.2.1.12. 5-[3,5-Bis-(4-methoxyphenyl)-4,5-dihydropyrazol-1-
ylmethylene]-2-thioxo-3-p-tolylthiazolidin-4-one (5b). Yield 92%,
arom), 7.46 (s, 1H, ]CH), 7.19 (d, 2H, J ¼ 7.4 Hz, arom), 7.12 (d, 1H,
J ¼ 7.6 Hz, arom), 7.02 (s, 1H, arom), 6.90 (d, 1H, J ¼ 7.9 Hz, arom),
6.82 (t, 1H, J ¼ 7.1 Hz, arom), 5.75 (dd, 1H, CH2CH, J ¼ 11.5, 5.9 Hz),
3.88 (dd, 1H, CH2CH, J ¼ 18.1, 11.5 Hz), 3.51 (dd, 1H, CH2CH, J ¼ 18.1,
mp 270e272 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
d 7.84 (d, 2H,
J ¼ 8.7 Hz, arom), 7.41 (s, 1H, ]CH), 7.33 (d, 2H, J ¼ 8.6 Hz, arom),
7.28 (d, 2H, J ¼ 8.2 Hz, arom), 7.15 (d, 2H, J ¼ 8.7 Hz, arom), 7.10 (d,
2H, J ¼ 8.2 Hz, arom), 6.99 (d, 2H, J ¼ 8.6 Hz, arom), 5.59 (dd, 1H,
CH2CH, J ¼ 11.2, 6.7 Hz), 3.97 (dd, 1H, CH2CH, J ¼ 18.2, 11.2 Hz), 3.84
(s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.46 (dd, 1H, CH2CH, J ¼ 18.2,
5.9 Hz), 2.27 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
d 194.0
(C]S), 169.0 (C]O), 166.0 (C]O), 160.1, 155.4 (C]N), 150.8, 144.3,
137.0, 133.4, 131.3, 130.2, 129.8, 129.2, 127.3, 126.4, 125.1, 122.6,
122.5, 119.5, 116.1, 93.3 (]CH), 62.1 (CHCH2), 40.5 (CHCH2), 20.9
(CH3). LCMS (ESIþ) m/z 516 (MþH)þ. Calcd. for C27H21N3O4S2: C,
62.90; H, 4.11; N, 8.15; Found: C, 62.63; H, 4.32; N, 8.40%.
6.7 Hz), 2.35 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
d 194.2
(C]S), 166.2 (C]O), 161.9, 159.7 (C]N), 159.6, 138.4, 133.7, 133.4,
131.9, 129.6, 129.3, 128.6, 128.5, 122.5, 114.8, 93.2 (]CH), 64.5
(CHCH2), 55.6 (OCH3), 55.3 (OCH3), 41.5 (CHCH2), 20.9 (CH3). LCMS
(ESIþ) m/z 516 (MþH)þ. Calcd. for C28H25N3O3S2: C, 65.22; H, 4.89;
N, 8.15; Found: C, 65.03; H, 4.63; N, 8.39%.
4.2.1.17. 5-[5-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)-4,5-
dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-2-
thioxothiazolidin-4-one (6b). Yield 82%, mp 266e268 ꢂC. 1H NMR
(400 MHz, DMSO-d6 þ CCl4):
d 10.28 (s, 1H, OH), 7.81 (d, 2H,
4.2.1.13. 5-[5-(4-Dimethylaminophenyl)-3-phenyl-4,5-
dihydropyrazol-1-ylmethylene]-2-thioxo-3-p-tolylthiazolidin-4-one
(5c). Yield 81%, mp 164e166 ꢂC. 1H NMR (400 MHz, DMSO-
J ¼ 8.4 Hz, arom), 7.48e7.53 (m, 2H, arom), 7.36 (s, 1H, ]CH), 7.29
(d, 1H, J ¼ 8.4 Hz, arom), 7.19 (d, 1H, J ¼ 7.8 Hz, arom), 7.11 (d, 1H,
J ¼ 7.8 Hz, arom), 7.06 (d, 2H, J ¼ 8.4 Hz, arom), 7.03 (s, 1H, arom),
6.84 (d, 1H, J ¼ 8.5 Hz, arom), 5.68 (dd, 1H, CH2CH, J ¼ 11.4, 6.4 Hz),
3.86 (s, 3H, OCH3), 3.83 (dd, 1H, CH2CH, J ¼ 17.9, 11.4 Hz), 3.47 (dd,
1H, CH2CH, J ¼ 17.9, 6.4 Hz), 2.28 (s, 3H, CH3). 13C NMR (100 MHz,
d6 þ CCl4): 7.90 (d, 2H, J ¼ 6.5 Hz, arom), 7.57e7.62 (m, 3H, arom),
d
7.38 (s, 1H, ]CH), 7.28 (d, 2H, J ¼ 7.6 Hz, arom), 7.22 (d, 2H,
J ¼ 7.8 Hz, arom), 7.11 (d, 2H, J ¼ 7.4 Hz, arom), 6.75 (d, 2H, J ¼ 7.8 Hz,
arom), 5.54 (dd, 1H, CH2CH, J ¼ 10.6, 6.8 Hz), 3.98 (dd, 1H, CH2CH,
J ¼ 18.6, 10.6 Hz), 3.49 (dd, 1H, CH2CH, J ¼ 18.6, 6.8 Hz), 2.90 (s, 6H,
DMSO-d6):
d 193.8 (C]S), 169.0 (C]O), 166.0 (C]O), 161.8, 160.0,
154.9 (C]N), 150.7, 137.0, 133.3, 132.7, 131.7, 129.7, 129.2, 127.6,
126.4, 122.6, 122.5, 118.3, 114.7, 110.2, 92.8 (]CH), 61.5 (CHCH2),
55.6 (OCH3), 40.5 (CHCH2), 20.9 (CH3). LCMS (ESIþ) m/z 546
N(CH3)2), 2.35 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
d 194.4
(C]S), 166.2 (C]O), 159.7 (C]N), 150.7, 138.5, 133.6, 133.3, 131.3,

252
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 85 (2014) 245e254
(MþH)þ. Calcd. for C28H23N3O5S2: C, 61.64; H, 4.25; N, 7.70; Found:
C27H20ClN3O3S2: C, 60.72; H, 3.77; N, 7.87; Found: C, 60.43; H, 3.95;
N, 7.55%.
C, 61.38; H, 4.05; N, 7.93%.
4.2.1.18. 5-[5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-
4.2.1.22. 5-[5-(4-Chlorophenyl)-3-naphthalen-2-yl-4,5-
dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-2-
thioxothiazolidin-4-one (6g). Yield 91%, mp 260e262 ꢂC. 1H NMR
ylmethylene]-3-(3-acetoxyphenyl)-2-thioxothiazolidin-4-one
(6c).
Yield 77%, mp 218e220 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
d
7.89e7.92 (m, 2H, arom), 7.56e7.60 (m, 3H, arom), 7.52 (d, 1H,
(400 MHz, DMSO-d6 þ CCl4):
d 8.30 (s, 1H, arom), 8.12 (brs, 2H,
J ¼ 8.0 Hz, arom), 7.45 (s, 1H, ]CH), 7.35 (d, 2H, J ¼ 8.6 Hz, arom),
7.24 (d, 1H, J ¼ 8.2 Hz, arom), 7.18 (d, 1H, J ¼ 8.0 Hz, arom), 7.12 (brs,
1H, arom), 7.00 (d, 2H, J ¼ 8.6 Hz, arom), 5.64 (dd, 1H, CH2CH,
J ¼ 11.4, 6.7 Hz), 4.03 (dd, 1H, CH2CH, J ¼ 18.5, 11.4 Hz), 3.76 (s, 3H,
arom), 7.98e8.04 (m, 2H, arom), 7.57e7.64 (m, 4H, arom, ]CH),
7.53 (d, 2H, J ¼ 8.9 Hz, arom), 7.45 (d, 2H, J ¼ 8.4 Hz, arom), 7.26 (d,
1H, J ¼ 7.1 Hz, arom), 7.20 (d, 1H, J ¼ 8.2 Hz, arom), 7.14 (brs, 1H,
arom), 5.79 (dd, 1H, CH2CH, J ¼ 11.3, 6.8 Hz), 4.17 (dd, 1H, CH2CH,
J ¼ 18.1, 11.3 Hz), 3.63 (dd, 1H, CH2CH, J ¼ 18.1, 5.6 Hz), 2.27 (s, 3H,
OCH3), 3.51 (dd, 1H, CH2CH, J ¼ 18.5, 6.7 Hz), 2.27 (s, 3H, CH3). 13
C
NMR (100 MHz, DMSO-d6):
d
194.1 (C]S), 169.0 (C]O), 166.0 (C]
CH3). 13C NMR (100 MHz, DMSO-d6):
d 194.2 (C]S), 169.0 (C]O),
O), 159.8, 159.7, 150.8 (C]N), 144.3, 136.9, 133.5, 131.9, 131.4, 130.1,
129.8, 129.2, 128.6, 127.4, 122.5, 114.8, 93.8 (]CH), 64.6 (CHCH2),
55.3 (OCH3), 41.5 (CHCH2), 20.9 (CH3). LCMS (ESIþ) m/z 530
(MþH)þ. Calcd. for C28H23N3O4S2: C, 63.50; H, 4.38; N, 7.93; Found:
C, 63.22; H, 4.65; N, 7.71%.
166.0 (C]O), 159.5, 150.9 (C]N), 139.4, 136.9, 134.1, 133.7, 133.5,
132.8, 129.8, 129.4, 129.0, 128.9, 128.8, 128.7, 128.0, 127.9, 127.5,
127.2, 126.4, 123.1, 122.7, 122.6, 94.5 (]CH), 64.4 (CHCH2), 41.7
(CHCH2), 20.9 (CH3). LCMS (ESIþ) m/z 584/586 (MþH)þ. Calcd. for
C
31H22ClN3O3S2: C, 63.74; H, 3.80; N, 7.19; Found: C, 63.92; H, 3.61;
N, 7.45%.
4.2.1.19. 5-[5-(4-Methoxyphenyl)-3-naphthalen-2-yl-4,5-
dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-2-
thioxothiazolidin-4-one (6d). Yield 89%, mp 232e234 ꢂC. 1H NMR
4.2.2. General procedure for synthesis of 5-(3,5-diaryl-4,5-
dihydropyrazol-1-ylmethylene)-4-thioxothiazolidin-2-ones (7a, 7b)
A mixture of 5-ethoxymethylene-4-thioxothiazolidin-2-one 1f
(5 mmol) and appropriate 3,5-diaryl-4,5-dihydropyrazole (5 mmol)
was refluxed in 10 ml of ethanol during 1 h. After cooling, the solid
precipitated was filtered off, washed with ethanol and recrystal-
lized from a DMF/ethanol 1:2 mixture.
(400 MHz, DMSO-d6 þ CCl4):
d 8.31 (s, 1H, arom), 8.12 (brs, 2H,
arom), 8.00 (t, 2H, J ¼ 7.9 Hz, arom), 7.62e7.65 (m, 2H, arom),
7.49e7.55 (m, 2H, arom, ]CH), 7.38 (d, 2H, J ¼ 8.4 Hz, arom), 7.25
(d, 1H, J ¼ 7.8 Hz, arom), 7.19 (d, 1H, J ¼ 7.8 Hz, arom), 7.14 (brs, 1H,
arom), 7.01 (d, 2H, J ¼ 8.4 Hz, arom), 5.68 (dd, 1H, CH2CH, J ¼ 11.1,
6.8 Hz), 4.13 (dd, 1H, CH2CH, J ¼ 18.1, 11.1 Hz), 3.77 (s, 3H, OCH3),
3.63 (dd, 1H, CH2CH, J ¼ 18.1, 6.8 Hz), 2.27 (s, 3H, CH3). 13C NMR
4.2.2.1. 5-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-
(100 MHz, DMSO-d6):
d
194.1 (C]S), 169.0 (C]O), 166.0 (C]O),
ylmethylene]-4-thioxothiazolidin-2-one
(7a). Yield
91%,
d 12.32 (s, 1H,
mp
159.8, 159.7, 150.8 (C]N), 136.9, 134.1, 133.5, 132.8, 131.9, 129.8,
129.0, 128.9, 128.8, 128.6, 128.0, 127.9, 127.6, 127.2, 126.4, 123.1,
122.6, 122.5, 114.8, 94.0 (]CH), 64.8 (CHCH2), 55.3 (OCH3), 41.5
(CHCH2), 20.9 (CH3). LCMS (ESIþ) m/z 580 (MþH)þ. Calcd. for
260e261 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
NH), 9.93 (s, 1H, OH), 7.98 (d, 1H, J ¼ 9.8 Hz, arom), 7.88 (brs, 2H,
arom), 7.48e7.57 (m, 3H, arom, ]CH), 7.24e7.28 (m, 2H, arom),
6.88e6.94 (m, 2H, arom), 5.84 (dd, 1H, CH2CH, J ¼ 11.8, 6.1 Hz), 3.90
(dd, 1H, CH2CH, J ¼ 18.0, 11.8 Hz), 3.54 (dd, 1H, CH2CH, J ¼ 18.0,
C
32H25N3O4S2: C, 66.30; H, 4.35; N, 7.25; Found: C, 66.57; H, 4.59; N,
7.03%.
6.1 Hz). 13C NMR (100 MHz, DMSO-d6):
d 186.3 (C]S), 172.5 (C]O),
160.8 (C]N), 155.5, 137.0, 131.5, 130.4, 130.1, 129.4, 129.2, 127.5,
124.7, 119.5, 116.2, 104.4 (]CH), 62.8 (CHCH2), 41.3 (CHCH2). LCMS
(ESIþ) m/z 382 (MþH)þ. Calcd. for C19H15N3O2S2: C, 59.82; H, 3.96;
N, 11.02; Found: C, 60.07; H, 3.71; N, 10.79%.
4.2.1.20. 5-[5-(4-Dimethylaminophenyl)-3-phenyl-4,5-
dihydropyrazol-1-ylmethylene]-3-(3-acetoxyphenyl)-2-
thioxothiazolidin-4-one (6e). Yield 84%, mp 252e254 ꢂC. 1H NMR
(400 MHz, DMSO-d6 þ CCl4):
d 7.86e7.89 (m, 2H, arom), 7.48e7.55
(m, 4H, arom), 7.39 (s, 1H, ]CH), 7.19 (d, 2H, J ¼ 8.2 Hz, arom), 7.10
(d, 1H, J ¼ 7.4 Hz, arom), 7.02 (brs, 1H, arom), 6.73 (d, 2H, J ¼ 8.2 Hz,
arom), 5.52 (dd, 1H, CH2CH, J ¼ 11.5, 6.4 Hz), 3.95 (dd, 1H, CH2CH,
J ¼ 18.0, 11.5 Hz), 3.41 (dd, 1H, CH2CH, J ¼ 18.0, 6.4 Hz), 2.95 (s, 6H,
4.2.2.2. 5-[5-(4-Methoxyphenyl)-3-naphthalen-2-yl-4,5-
dihydropyrazol-1-ylmethylene]-4-thioxothiazolidin-2-one
(7b).
Yield 88%, mp 256e258 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
d
12.58 (s, 1H, NH), 8.34 (s, 1H, arom), 8.00e8.08 (m, 4H, arom), 7.95
N(CH3)2), 2.27 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
d
194.0
(s, 1H, ]CH), 7.61e7.67 (m, 2H, arom), 7.41 (d, 2H, J ¼ 8.3 Hz, arom),
7.04 (d, 2H, J ¼ 8.3 Hz, arom), 5.78 (dd, 1H, CH2CH, J ¼ 11.1, 6.5 Hz),
4.12 (dd, 1H, CH2CH, J ¼ 18.1, 11.1 Hz), 3.79 (s, 3H, OCH3), 3.63 (dd,
(C]S), 169.0 (C]O), 166.0 (C]O), 159.9, 150.8 (C]N), 150.7, 136.9,
133.5, 131.4, 130.2, 129.8, 129.2, 128.1, 127.4, 126.5, 126.4, 122.6,
122.5, 112.7, 93.6 (]CH), 65.0 (CHCH2), 41.3 (CHCH2), 39.4
(N(CH3)2), 20.9 (CH3). LCMS (ESIþ) m/z 543 (MþH)þ. Calcd. for
1H, CH2CH, J ¼ 18.1, 6.5 Hz). 13C NMR (100 MHz, DMSO-d6):
d 186.9
(C]S), 172.5 (C]O), 160.4, 159.8 (C]N), 144.3, 137.0, 134.2, 132.7,
131.5, 129.2, 128.9, 128.8, 128.7, 128.1, 128.0, 127.5, 127.2, 123.4,
114.9, 105.0 (]CH), 65.4 (CHCH2), 55.3 (OCH3), 41.3 (CHCH2). LCMS
(ESIþ) m/z 446 (MþH)þ. Calcd. for C24H19N3O2S2: C, 64.70; H, 4.30;
N, 9.43; Found: C, 64.47; H, 4.58; N, 9.21%.
C
29H26N4O3S2: C, 64.19; H, 4.83; N, 10.32; Found: C, 64.35; H, 4.61;
N, 10.62%.
4.2.1.21. 5-[5-(4-Chlorophenyl)-3-phenyl-4,5-dihydropyrazol-1-
ylmethylene]-3-(3-acetoxyphenyl)-2-thioxothiazolidin-4-one
(6f).
Yield 80%, mp 264e266 ꢂC. 1H NMR (400 MHz, DMSO-d6 þ CCl4):
4.2.3. Synthesis of [(3-methyl-4-oxo-2-thioxothiazolidin-5-
ylidenemethyl)-amino]-acetic acid (8)
d
7.87e7.90 (m, 2H, arom), 7.51e7.60 (m, 7H, arom, ]CH), 7.42 (d,
2H, J ¼ 8.1 Hz, arom), 7.25 (d, 1H, J ¼ 7.2 Hz, arom), 7.18 (d, 1H,
J ¼ 7.2 Hz, arom), 7.13 (brs, 1H, arom), 5.74 (dd, 1H, CH2CH, J ¼ 11.4,
5.8 Hz), 4.07 (dd, 1H, CH2CH, J ¼ 18.6, 11.4 Hz), 3.51 (dd, 1H, CH2CH,
J ¼ 18.6, 5.8 Hz), 2.27 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
A mixture of 5-ethoxymethylene-3-methyl-2-thioxothiazolidin-
4-one 1b (10 mmol) and glycine (12 mmol) was refluxed in 20 ml of
glacial acetic acid during 3 h. The crystalline product was separated
by filtration, washed with ethanol, and dried. Recrystallization from
acetic acid rendered the desired product in pure form.
Yield 75%, mp 222e224 ꢂC. 1H NMR (400 MHz, DMSO-
d
194.3 (C]S), 169.0 (C]O), 166.0 (C]O),159.5,150.8 (C]N), 144.3,
139.3, 136.9, 133.8, 133.5, 131.5, 130.0, 129.8, 129.4, 129.3, 129.0,
127.4, 126.4, 122.7, 122.6, 94.3 (]CH), 64.2 (CHCH2), 41.7 (CHCH2),
20.9 (CH3). LCMS (ESIþ) m/z 534/536 (MþH)þ. Calcd. for
d6 þ CCl4):
d 13.01 (brs, 1H, COOH), 8.93e8.97 (m, 0.10*1H, NH),
8.28e8.31 (m, 0.90*1H, NH), 7.63 (d, 0.90*1H, J ¼ 13.3 Hz, ]CH),

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 85 (2014) 245e254
253
7.43 (d, 0.10*1H, J ¼ 13.3 Hz, ]CH), 4.01e4.06 (m, 2H, CH2), 3.30 (s,
3H, CH3). 13C NMR (100 MHz, DMSO-d6):
190.6 (C]S), 171.2 (C]
O), 166.5 (C]O), 146.2, 89.6 (]CH), 49.0 (CH2), 30.9 (CH3). LCMS
(ESIþ) m/z 233 (MþH)þ. (Z)/(E) ratio (%) 71:29. Calcd. for
C7H8N2O3S2: C, 36.20; H, 3.47; N, 12.06; Found: C, 36.41; H, 3.27; N,
11.83%.
CH), 59.6 (CHCH2), 49.9 (CH2), 41.9 (CHCH2), 30.9 (CH3). LCMS
d
(ESIþ) m/z 505 (MþH)þ. (Z)/(E) ratio (%) 81:19. Calcd. for
C26H21FN4O2S2: C, 61.89; H, 4.19; N, 11.10; Found: C, 61.61; H, 4.43;
N, 11.33%.
4.3. Anti-trypanosomal activity assay
4.2.4. General procedure for synthesis of 5-{[2-(3,5-diaryl-4,5-
dihydropyrazol-1-yl)-2-oxo-ethylamino]-methylene}-3-methyl-2-
thioxothiazolidin-4-ones (9aec)
Bloodstream form of T. brucei gambiense Feo strain were
cultured in HMI9 medium supplemented with 10% FCS at 37 ꢂC
under an atmosphere of 5% CO2 [33]. In all experiments, log-
phase parasite cultures were harvested by centrifugation at
3000 ꢃ g and immediately used. Drug assays were based on the
conversion of a redox-sensitive dye (resazurin) to a fluorescent
product by viable cells as previously described [34]. Drug stock
solutions were prepared in pure DMSO. T. brucei bloodstream
forms (104 cells) were cultured in 96-well plates either in the
absence or in the presence of different concentrations of in-
A
mixture of appropriate 3,5-diaryl-4,5-dihydropyrazole
(5 mmol) and DCC (5 mmol) was stirred at r.t. during 10 min in
10 ml of THF. Carboxylic acid 8 (5 mmol) was added and the re-
action mixture was stirred at rt for 1 h. The mixture was then
filtered and the filtrate was precipitated by the addition of water.
After cooling, the solid precipitated was filtered off, washed with
water and methanol and recrystallized from a DMF/ethanol 1:2
mixture.
hibitors in a final volume of 200
resazurin solution was added in each well at the final concen-
tration of 45 M and fluorescence was measured at 530 nm and
ml. After a 72-h incubation,
4.2.4.1. 5-({2-[5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydropyrazol-
1-yl]-2-oxoethylamino}-methylene)-3-methyl-2-thioxothiazolidin-4-
one (9a). Yield 67%, mp 139e140 ꢂC. 1H NMR (400 MHz, DMSO-
m
590 nm wavelengths after a further 4-h incubation. The per-
centage of inhibition of parasite growth rate was calculated by
comparing the fluorescence of parasites maintained in the
presence of drug to that of in the absence of drug. DMSO was
used as control. Concentration inhibiting 50% of parasite growth
(IC50) was determined from the doseeresponse curve with a drug
d6 þ CCl4):
d 9.07e9.10 (m, 0.20*1H, NH), 8.40e8.42 (m, 0.80*1H,
NH), 7.80 (brs, 2H, arom), 7.70 (d, 1H, J ¼ 12.3 Hz, ]CH), 7.46 (brs,
2H, arom), 7.46e7.53 (m, 3H, arom), 7.16 (d, 2H, J ¼ 6.3 Hz, arom),
6.84 (d, 2H, J ¼ 6.3 Hz, arom), 5.52 (dd, 1H, CH2CH, J ¼ 11.8, 6.1 Hz),
4.52e4.62 (m, 2H, CH2), 3.85 (dd, 1H, CH2CH, J ¼ 16.1, 11.8 Hz), 3.75
(s, 3H, OCH3), 3.29 (s, 3H, CH3), 3.15 (dd, 1H, CH2CH, J ¼ 16.1, 6.1 Hz).
concentrations ranging from 10
mg/ml to 0.625 mg/ml and pre-
sented in M. IC50 value is the mean the standard deviation of
m
13C NMR (100 MHz, DMSO-d6):
d 190.5 (C]S), 166.4 (C]O), 165.8
three independent experiments.
(C]O), 158.7, 155.7 (C]N), 146.7, 133.9, 130.9, 130.7, 128.9, 127.2,
127.0, 114.1, 89.4 (]CH), 59.6 (CHCH2), 55.2 (OCH3), 49.8 (CH2), 41.8
(CHCH2), 30.9 (CH3). LCMS (ESIþ) m/z 467 (MþH)þ. (Z)/(E) ratio (%)
75:25. Calcd. for C23H22N4O3S2: C, 59.21; H, 4.75; N,12.01; Found: C,
59.03; H, 4.95; N, 12.24%.
4.4. In vitro cytotoxicity assay on mammalian cell
Cytotoxicity was evaluated by using a rat myoblast-derived cell
line (L-6). Assays were performed in 96-well plates in RPMI me-
dium containing 25 mM HEPES, pH 7.3, 10% fetal calf serum under
5% CO2 atmosphere, at 37 ꢂC. After trypsin treatment, L-6 cells were
4.2.4.2. 5-({2-[5-(4-Chlorophenyl)-3-naphthalen-2-yl-4,5-
dihydropyrazol-1-yl]-2-oxoethylamino}-methylene)-3-methyl-2-
thioxothiazolidin-4-one (9b). Yield 71%, mp 158e160 ꢂC. 1H NMR
seeded at 5000 cells per well in 100
ml. After 24 h incubation, cells
(400 MHz, DMSO-d6 þ CCl4):
d 9.09e9.12 (m, 0.25*1H, NH),
were washed and two-fold dilutions of drug were added (200
ml per
8.42e8.46 (m, 0.75*1H, NH), 8.06e8.12 (m, 2H, arom), 7.89e7.93
(m, 3H, arom), 7.74 (d, 1H, J ¼ 13.4 Hz, ]CH), 7.54e7.56 (m, 2H,
arom), 7.30e7.35 (m, 4H, arom), 5.64 (dd, 1H, CH2CH, J ¼ 11.4,
4.1 Hz), 4.62e4.70 (m, 2H, CH2), 4.01 (dd, 1H, CH2CH, J ¼ 16.8,
well). Drug stock solutions were prepared in pure DMSO. The final
DMSO concentration in the cultures remained below 1%. Control
cultures were constituted of cultures treated with pure DMSO
instead of drug. The cytotoxicity assay was based on the conversion
of a redoxsensitive dye (resazurin) to a fluorescent product by
viable cells [35]. After 5 days of incubation, resazurin solution was
11.4 Hz), 3.31 (s, 3H, CH3), 3.26 (dd, 1H, CH2CH, J ¼ 16.8, 4.1 Hz). 13
C
NMR (100 MHz, DMSO-d6): d 190.5 (C]S), 166.4 (C]O), 166.1 (C]
O), 155.8 (C]N), 146.7, 144.3, 133.9, 132.7, 132.1, 128.7, 128.6, 128.4,
128.3, 128.1, 127.9, 127.8, 127.6, 127.0, 123.3, 89.5 (]CH), 59.6
(CHCH2), 49.9 (CH2), 41.7 (CHCH2), 30.9 (CH3). LCMS (ESIþ) m/z
521/523 (MþH)þ. (Z)/(E) ratio (%) 70:30. Calcd. for
added in each well at the final concentration of 45 mM. Fluorescence
was measured at 530 nm excitation and 590 nm emission wave-
lengths after a further 4-h incubation. The percentage of inhibition
of cell growth was calculated by comparing the fluorescence of cells
maintained in the presence of drug to that of in the absence of drug.
IC50s were determined from the doseeresponse curves with drug
C
26H21ClN4O2S2: C, 59.93; H, 4.06; N, 10.75; Found: C, 59.68; H,
3.88; N, 10.92%.
concentrations ranging from 10 mg/ml to 10 ng/ml. IC50 value is the
4.2.4.3. 5-({2-[5-(4-Fluorophenyl)-3-naphthalen-2-yl-4,5-
dihydropyrazol-1-yl]-2-oxoethylamino}-methylene)-3-methyl-2-
thioxothiazolidin-4-one (9c). Yield 75%, mp 192e194 ꢂC. 1H NMR
mean the standard deviation of three independent experiments.
The doseeresponse curves have been fitted with two-parameter
log-logistic function (eq. (1)), fixing upper limit at 100% and
lower limit at 0%:
(400 MHz, DMSO-d6 þ CCl4):
d 9.09e9.12 (m, 0.20*1H, NH),
8.42e8.46 (m, 0.80*1H, NH), 8.06e8.12 (m, 2H, arom), 7.90e7.95
(m, 3H, arom), 7.74 (d, 1H, J ¼ 13.4 Hz, ]CH), 7.54e7.57 (m, 2H,
arom), 7.30e7.35 (m, 2H, arom), 7.06e7.10 (m, 2H, arom), 5.65 (dd,
1H, CH2CH, J ¼ 12.0, 3.5 Hz), 4.62e4.71 (m, 2H, CH2), 4.01 (dd, 1H,
À Á
1
f x ¼
;
(1)
1 þ eðbꢃlnðxÞꢄlnðeÞÞ
CH2CH, J ¼ 17.0, 12.0 Hz), 3.31 (s, 3H, CH3), 3.09 (brs, 1H, CH2CH). 13
C
where b is a Hill coefficient that defines the steepness of logistic
curve and e is IC50 and corresponds to the position of curve on x-
axis. The coefficients of determination are presented in Fig. 4 and
have high values, confirming that chosen parsimonious model is
accurate.
NMR (100 MHz, DMSO-d6):
d 190.6 (C]S), 166.4 (C]O), 166.0 (C]
O), 165.8 (d, JCF ¼ 200.0 Hz, 1C), 155.8 (C]N), 146.8, 144.3, 138.1,
133.8, 132.7, 131.9 (d, JCF ¼ 30.0 Hz, 2C), 128.6, 128.5, 128.4, 128.3,
128.1, 128.0, 127.6, 127.0, 123.3, 115.5 (d, JCF ¼ 21.0 Hz, 2C), 89.5 (]

254
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 85 (2014) 245e254
[16] D. Havrylyuk, B. Zimenkovsky, O. Vasylenko, C.W. Day, D.F. Smee, P. Grellier,
Acknowledgment
R. Lesyk, Synthesis and biological activity evaluation of 5-pyrazoline
substituted 4-thiazolidinones, Eur. J. Med. Chem. 66 (2013) 228e237.
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The authors support all people of good will currently struggling
for liberty and justice in Ukraine.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.103.
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