Novel benzenesulfonylureas containing thiophenylpyrazoline moiety as potential antidiabetic and anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2014.09.044

In the present study a library of twenty six benzenesulfonylureas containing thiophenylpyrazoline moiety has been synthesized. All the compounds were docked against PPAR-γ target. Most of the compounds displayed higher dock score than standard drugs, glibenclamide and rosiglitazone. All the synthesized compounds were primarily evaluated for their antidiabetic effect by oral glucose tolerance test. Further assessment of antidiabetic potential of sixteen active compounds was then done on STZ induced diabetic model. The results of in vivo activity by both the methods were found to be consistent with each other as well as with docking studies. Change in body weight of STZ induced animals post treatment was also assessed at the end of study. In vitro PPAR-γ transactivation assay was performed on active compounds in order to validate docking results and the most active compound 3k was also shown to elevate gene expression of PPAR-γ. Furthermore, the compounds were screened by National Cancer Institute, Bethesda for anticancer effect and two compounds 3h and 3i were selected at one dose level since they exhibited sensitivity towards tumor cell lines (mainly melanoma).

Full text of DOI:10.1016/j.bmcl.2014.09.044

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5298–5303
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel benzenesulfonylureas containing thiophenylpyrazoline moiety
as potential antidiabetic and anticancer agents
a
a
a
a
Chetna Kharbanda a, Mohammad Sarwar Alam a, , Hinna Hamid , Kalim Javed , Syed Shafi , Yakub Ali ,
⇑
Perwez Alam b, M. A. Q. Pasha b, Abhijeet Dhulap c, Sameena Bano a, Syed Nazreen a, Saqlain Haider a
a Department of Chemistry, Faculty of Science, Hamdard University, New Delhi 110 062, India
b Functional Genomics Unit, CSIR-Institute of Genomics & Integrative Biology, Delhi, India
c CSIR Unit for Research and Development of Information Products, Pune 411038, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 June 2014
Revised 28 August 2014
Accepted 16 September 2014
Available online 23 September 2014
In the present study a library of twenty six benzenesulfonylureas containing thiophenylpyrazoline moi-
ety has been synthesized. All the compounds were docked against PPAR-c target. Most of the compounds
displayed higher dock score than standard drugs, glibenclamide and rosiglitazone. All the synthesized
compounds were primarily evaluated for their antidiabetic effect by oral glucose tolerance test. Further
assessment of antidiabetic potential of sixteen active compounds was then done on STZ induced diabetic
model. The results of in vivo activity by both the methods were found to be consistent with each other as
well as with docking studies. Change in body weight of STZ induced animals post treatment was also
Keywords:
2-Acetylthiophene
Sulfonylureas
assessed at the end of study. In vitro PPAR-
in order to validate docking results and the most active compound 3k was also shown to elevate gene
expression of PPAR- . Furthermore, the compounds were screened by National Cancer Institute, Bethesda
c transactivation assay was performed on active compounds
PPAR-
c
c
Antidiabetic
for anticancer effect and two compounds 3h and 3i were selected at one dose level since they exhibited
sensitivity towards tumor cell lines (mainly melanoma).
Anticancer
Gene expression
Ó 2014 Elsevier Ltd. All rights reserved.
The peroxisome proliferator-activated receptors (PPARs) are
ligand-dependent transcription factors which are involved in the
regulation of lipid and glucose metabolism.1 PPARs are classified
The utility of sulfonylureas have been proved as effective oral
antidiabetic agents till date.5 Consequently, the research is still
being pursued by scientists to synthesize new sulfonylurea deriva-
tives with lesser side effects. The mode of action of sulfonylureas is
mainly to stimulate insulin secretion by binding to sulfonylurea
receptors (SURs) of ATP assisted potassium ion (KATP) channels
present in pancreatic b-cells.6 Their mechanistic action on molecu-
lar level has not yet been too intensively studied. The activation of
into three subclasses-
and associated with activation of different target genes by binding
to their regulatory region. One of these, PPAR- regulates gene
expression in response to lipophilic ligands since its ligand binding
a, b and c which are coded by different genes
c
pocket is quite spacious.2 PPAR-
c increases fatty acid flux in adipo-
cytes which on contrary alleviates insulin resistance.3 In addition,
many reports contemplated that during adipocyte differentiation,
PPAR-c is the general known mechanism of thiazolidinediones for
improving a glycemic condition.7,8 But in some recent studies it has
been reported that several marketed sulfonylureas also induce
PPAR-
c
exerts antimitotic action, thereby reduces the risk of cell
in fibroblasts leads to
proliferation. The activation of PPAR-
c
PPAR-
glimepiride, gliquidone and glipizide has been reported to stimu-
late pancreatic insulin secretion by acting as PPAR- agonist in
c transcriptional activity. Sulfonylureas like glibenclamide,
retraction of cell cycle in tumor cells which have exponential
growth rate. It has also been reported that the mutations in
c
PPAR-
c
resulting in loss of function were found to be associated
addition to binding with sulfonylurea receptor (SUR) on the plasma
with risk of cancer.4 Therefore, it is evident that there is close rela-
membrane of pancreatic b-cells.9 It has also been studied that gli-
tion between PPAR-
c
and cancer.
benclamide binds to PPAR-
with respect to marketed PPAR-
c
receptor site in a competitive manner
agonists like pioglitazone and
c
rosiglitazone. Therefore, in the present study we are presenting a
library of twenty six sulfonylurea derivatives which have been
⇑
Corresponding author. Tel.: +91 11 26059688 (5555); fax: +91 11 26059663.
E-mail addresses: msalam@jamiahamdard.ac.in, msalam5555@gmail.com
showing significant antidiabetic activity by acting as PPAR-
c ago-
nists. Increase in body weight which is one of the major side effects
(M.S. Alam).
http://dx.doi.org/10.1016/j.bmcl.2014.09.044
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

C. Kharbanda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5298–5303
5299
O
H
O
Ethanol, 30% NaOH
S
r.t., 12hrs
O
Ar
S
Ar
1a-f
H2
N
NH
SO2NH2
Ar
Ar
Acetone, K2CO3
RCNZ, Reflux
N
N
N
S
N
S
H
N
H
N
O
S
O
SO2NH2
R
2a-f
Z
3a-z
3a, f, j, r, w: R = -CH2C6H5, Z = O
1a, 2a, 3a-e: Ar = thiophenyl
1b, 2b, 3f-i: Ar = 5-methylthiophenyl
1c, 2c, 3j-n: Ar = phenyl
3b, g, k, o, s: R = o-CH3C6H5, Z =O
3c, h, l, p, t, x: R = p-CH3C6H5, Z = O
1d, 2d, 3o-q: Ar = 4-methoxyphenyl
1e, 2e, 3r-v: Ar = N, N-dimethylphenyl
1f, 2f, 3w-z: Ar = anthracen-9-yl
3d, i, m, q, u, y: R = p-OCH3C6H5, Z = O
3e, n: R = n-C4H9, Z = O
3v, z: R = -CH2C6H5, Z = S
Scheme 1.
of sulfonylurea agent has also been evaluated for their effect on
body weight.
drugs glibenclamide and rosiglitazone. It has been found that
glibenclamide (À7.42) exhibited higher docking score than
rosiglitazone (À5.72). Captivatingly, docking score of nineteen
compounds (3a–n, 3p, 3r, 3t, 3v, 3w) was found to be higher than
rosiglitazone and sixteen compounds (3a, 3c–f, 3h, 3i–n, 3p, 3r, 3t,
3w) showed higher docking score than glibenclamide. On careful
examination of different substitutions, it could be inferred that
most of the compounds containing unsubstituted benzyl ring
exhibited higher docking score than both the standards. The
The chalcones (1a–f) obtained by reacting 2-acetyl thiophene
with appropriate aldehyde resulted in the synthesis of key inter-
mediates, that is, pyrazolines (2a–f). The pyrazolines so obtained
were made to react with appropriate aryl isocyanate and aryl
isothiocyanate in the presence of anhydrous potassium carbonate
and acetone by conventional reactions.10,11 The resulting solid
was filtered, washed with water, dried, and crystallized from
acetone (Scheme 1). All reagents were procured from commercial
source and used without further purification unless indicated.
The progress of reaction was monitored by using silica gel GF245
plates (Merck Pvt. Ltd, Germany). The purification of compounds
was carried out by recrystallization from ethanol. 1H NMR, IR, Mass
spectra and CHNS data was also presented to confirm the synthesis
of desired product. 1H NMR spectra of all the synthesized com-
pounds was recorded in parts per million (ppm) using TMS as
the standard and at 300–400 MHz frequency. The appearance of
three double doublets in the range of d 3.00–6.00 ppm confirmed
the formation of pyrazoline ring. The coupling with isocyanate/iso-
thiocyanate was verified by increase in aromatic protons depend-
ing on the substituent. 1H NMR of the compound 3k at 400 MHz
showed singlet at chemical shift 2.09 ppm corresponding to three
methyl protons present on tolyl group attached to sulfonylurea
linkage. The formation of pyrazoline ring was confirmed by the
presence of three double doublets at 3.13 (J = 5.6 Hz, 17.2 Hz),
3.94 (J = 12.0 Hz, 17.2 Hz) and 5.54 ppm (J = 5.6 Hz, 12.0 Hz).
Remaining aromatic protons appeared as multiplet in the region
6.69–7.84 ppm. Mass spectra showing peaks at [M+H]+ or
[MÀH]+ further confirmed the syntheses of desired products.
As discussed earlier, the mode of action of action of sulfonylu-
alignment of compounds in active PPAR-c site was such that
the most of the compounds formed H-bond with different amino
acid residues. In some cases, more than one H-bond formation or
p–p stacking was also found. The images of the compounds
displaying high docking score have been shown in Figure 1 and
dock scores were given in Table S1 (Supplementary material).
The efficacy of administration of all the synthesized compounds
as oral antidiabetic agents has been assessed primarily by loading
glucose on normal rats. It has been observed that in comparison to
standard drug glibenclamide, seven compounds (3d, 3i–n) signifi-
cantly controlled increase in plasma glucose level. However, six-
teen compounds (3a, 3c–f, 3h, 3i–n, 3p, 3r, 3t, 3v) exhibited
significant lowering in plasma glucose level than PPAR-c agonist,
rosiglitazone. The change in plasma glucose level was illustrated
graphically in Figure 2. The result of Glucose Tolerance Test on
in vivo model was consistent with docking studies. Few exceptions
from docking results were related to the compounds with bulky
aryl substitution. For instance for compound 3w docking score
was as high as À11.54 but in vivo antidiabetic activity was less sig-
nificant to standard drug glibenclamide which showed docking
score of À7.42.
The compounds which showed significant glucose tolerance
were further evaluated for their effect in STZ induced diabetic
model.12 It was found that the results of both the studies are in
concurrence. The change in plasma glucose level on 7th and 15th
day of study was assessed and given in Figure 3. The supplementa-
tion of diabetic rats with glibenclamide as well as rosiglitazone
reas can also be through the activation of PPAR-c. Such reports
on sulfonylureas prompted us to carry out molecular docking stud-
ies of synthesized compounds. In order to get better insight of
ligand–protein interaction, all the synthesized compounds were
docked and their dock scores were compared to that of standard

5300
C. Kharbanda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5298–5303
Figure 1. Images of the compounds (3w, k, l) in PPAR-c active site.
Figure 2. Antidiabetic effect of treating wistar rats with synthesized compounds by Oral Glucose Tolerance Test. Data is analyzed by one way ANOVA followed by Dunnett
⁄
⁄⁄
⁄
test and expressed as mean SEM from six observations; represents change as compared to control; indicates p < 0.01 & indicates p < 0.05.

C. Kharbanda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5298–5303
5301
Figure 3. Antidiabetic effect of treating STZ-induced wistar rats with synthesized compounds. Data is analyzed by⁄o⁄⁄ne way ANOVA followed by Dunnett test and expressed as
⁄
⁄⁄
mean SEM from six observations; represents change as compared to diabetic control; indicates p < 0.01 &
indicates p < 0.001.
restored plasma glucose level near to normal but the alleviation
effect of seven synthesized compounds (3d, 3i–n) was compara-
tively more pronounced.
Structure activity relationship has been generated to under-
stand the effect of different substitutions placed around pyrazoline
ring and correlations that have been drawn from it are as follows
(Fig. 4):
ꢀ Most of the compounds containing unsubstituted phenyl group
showed maximum docking score as well as significant in vivo
antidiabetic activity.
ꢀ However substitution on aryl ring linked to urea/thio urea link-
age did not show any particular trend in regards to activity or
docking score.
Figure 5. In vitro PPAR-c transactivation assay on 3T3-L1 cell line.
ꢀ Substituting bulky aryl groups like anthracene significantly
lowered the in vivo antidiabetic activity even if docking score
was high.
compounds was presented in Figure 5. In contrast to in vivo results,
the transactivation activity of glibenclamide as well as active
compounds was found to be quite less than the reference drug,
rosiglitazone. The elevated in vivo activity might be due to the fact
ꢀ Replacing aryl ring with thiophenyl ring exhibited in vivo activ-
ity almost as significant as benzyl ring substitution. However,
thiophenyl ring substituted with methyl group did not bring
significant variation in comparison to unsubstituted thiophenyl
ring.
that sulfonylureas not only activate PPAR-
c but also bind to SU
receptors present in pancreatic cells when acts in vivo.
b
Additionally, the transactivational assay involves different extent
of co-activator recruitment by different ligands which are also
responsible for changes in conformation of target protein.
The treatment with sulfonylurea agents is potentially associ-
ated with increase in body weight. Therefore, in the present study
the change in body weight was carefully observed over the period
of 15 days of study. The induction of STZ resulted in significant
weight loss after 15th day of study (Fig. 6). The standard groups
and groups treated with compounds 3e, 3h, 3n, 3r showed signif-
icant increase in body weight. However, this increase in body
weight was appreciably controlled in groups treated with com-
pounds 3a, 3c–k, 3m, 3p, 3t, 3v in comparison to glibenclamide
and rosiglitazone treated groups.
ꢀ Replacing urea linkage with thiourea linkage significantly
lowered the in vivo antidiabetic activity as well as dock score.
The active compounds (3a, 3c–f, 3h, 3i–n, 3p, 3r, 3t, 3v) were
scrutinized by in vitro PPAR transactivation assay to validate their
mechanism of action. The result of transactivation activity of these
Aryl Ring
4
S
5
3
N
1
N
2
Finally, the effect of the most potent compound 3k on the
expression of PPAR-c target genes were measured in 3T3-L1
O
fibroblasts in comparison to standard drugs, rosiglitazone and
glibenclamide (Fig. 7). The compound 3k significantly enhanced
urea/thiourea linkage aryl group
S
the expression of PPAR-c genes but the result was not as signifi-
cant as rosiglitazone. It was found that rosiglitazone augmented
O
gene expression relatively by 1.5 folds whereas the compound 3k
Figure 4. General structure of synthesized sulfonylurea.

5302
C. Kharbanda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5298–5303
Figure 6. Change in body weight after 15 day study on STZ-induced diabetic rats. Data is analyzed by one way ANOVA followed by Bonferroni ‘t’ test and expressed as
mean SEM from six observations; ⁄ represents significant change as compared to diabetic control; # represents significant change as compared to normal control. ⁄⁄ indicates
⁄
##
#
p < 0.01 & indicates p < 0.05;
indicates p < 0.05 & indicates p < 0.01.
Table 1
Anticancer data of compounds 3h and 3i provided by NCI
Panel
Cell line
Growth percent
NSC: 777382/1 NSC: 777381/1
(3i)
(3h)
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
88.12
87.89
102.86
111.39
94.70
95.29
75.83
126.37
113.81
94.41
83.37
84.69
Non-small cell lung
cancer
A549/ATCC
HOP-62
5.45
81.02
101.55
76.92
HOP-92
87.06
84.91
NCI-H226
NCI-H23
110.15
97.55
110.48
98.53
Figure 7. PPAR-c gene expression evaluation of compound 3k in comparison to
standards, glibenclamide and rosiglitazone. Data is analyzed by one way ANOVA
NCI-H322M
NCI-H460
NCI-H522
102.37
108.95
87.33
103.57
103.16
91.51
⁄
followed by Dunnett test and expressed as mean SEM from three observations;
⁄⁄
represents change as compared to control; indicates p < 0.01.
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
À63.47
111.31
98.77
102.46
119.52
98.51
À54.62
108.61
85.12
99.31
127.85
97.36
caused 0.95 fold elevation. On the other hand, the gene expression
was ameliorated by one fold on treatment with glibenclamide.
Several reports have been extended to prove the anticancer
KM12
potential of PPAR-c and sulfonylureas. Therefore, the structures
SW-620
107.16
106.34
of twenty six compounds have been submitted to NCI for evaluat-
ing their anticancer activity. Among all the synthesized com-
pounds, two compounds (3h, 3i) have been selected by NCI,
Bethesda at one dose level study. Both these compounds 3h and
3i showed mean growth percent of 89.29% and 87.76%, respec-
tively (Table 1). Both these compounds engaged in mild sensitivity
towards melanoma (MALME-3M, M14, MDA-MB-435, SK-MEL-28,
SK-MEL-5, UACC-257, UACC-62) with growth percent less than
62%. The compound 3i also exhibited pronounced sensitivity
(5.45%) towards A549/ATCC cell line cultured from non-small cell
lung cancer.
CNS cancer
Melanoma
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
97.85
104.70
104.31
103.16
99.87
95.62
100.07
98.31
108.18
93.74
98.07
94.12
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
96.81
31.15
52.12
52.30
57.56
48.27
49.98
30.03
31.00
99.74
40.64
58.39
61.95
60.91
47.78
61.42
40.91
38.15
It can be concluded that ten compounds out of twenty six
compounds have been found to possess significant antidiabetic
potential without inducing increase in body weight. Mechanisti-
Ovarian cancer
IGROV1
108.05
106.38
102.84
113.48
106.90
103.44
107.76
98.17
106.40
114.78
99.82
cally, these compounds are engaged in the activation of PPAR-
c
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
as observed from in vitro transactivation assay. One of the com-
pound 3k, which exhibited remarkably high antidiabetic activity
has also shown significant increase in PPAR-
c gene expression.
102.87
Two compounds 3h and 3i also exhibited potency against cancer.
Therefore, these sulfonylurea derivatives can be explored further

C. Kharbanda et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5298–5303
5303
USA, for performing in vitro anticancer screening. The authors
are thankful to Dr. Javed Iqbal, Director, Institute of Life Sciences
(ILS), Hyderabad, for providing necessary in vitro facilities and
Dr. Parimal Misra for his help in performing in vitro studies. One
of the authors, CK is also thankful to University Grant Commission
for providing financial assistance.
Table 1 (continued)
Panel
Cell line
SK-OV-3
Growth percent
NSC: 777382/1 NSC: 777381/1
(3i)
(3h)
82.55
78.27
Renal cancer
786-0
A498
100.87
101.86
93.16
94.74
—
111.79
96.03
87.18
103.79
109.00
96.60
95.78
—
111.21
96.78
85.69
Supplementary data
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
09.044.
Prostate cancer
Breast cancer
PC-3
DU-145
96.33
105.62
86.34
102.33
References and notes
MCF7
MDA-MB-231/
ATCC
HS 578T
BT-549
T-47D
99.04
113.39
97.09
116.79
1. Willson, T. M.; Lambert, M. H.; Kliewer, S. A. Annu. Rev. Biochem. 2001, 70, 341.
2. Nolte, R. T.; Wisely, G. B.; Westin, S.; Cobb, J. E.; Lambert, M. H.; Kurokawa, R.;
Rosenfeld, M. G.; Willson, T. M.; Glass, C. K.; Milburn, M. V. Nature 1998, 395,
137.
3. Tontonoz, P.; Hu, E.; Spiegelman, B. Cell 1994, 79, 1147.
4. Rosen, E. D.; Spiegelman, B. M. J. Biol. Chem. 2001, 276, 37731.
5. Spruce, A. E.; Standen, N. B.; Stanfield, P. R. Nature 1985, 316, 736.
6. Kharbanda, C.; Alam, M. S. Chem. Biol. Inter. 2013, 3, 230.
7. Fukuen, S.; Iwaki, M.; Yasui, A.; Makishima, M.; Matsuda, M.; Shi momura, I. J.
Biol. Chem. 2005, 280, 23653.
97.52
116.23
85.68
—
96.05
74.25
78.79
—
MDA-MB-468
87.76
Mean
89.29
8. Scarsi, M.; Podvinec, M.; Roth, A.; Hug, H.; Kersten, S.; Albrecht, H.; Schwede, T.;
Meyer, U. A.; Rucker, C. Mol. Pharmacol. 2007, 71, 398.
9. Inukai, K.; Watanabe, M.; Nakashima, Y.; Takata, N.; Isoyama, A.; Sawa, T.;
Kurihara, S.; Awata, T.; Katayama, S. Biochem. Biophys. Res. Commun. 2005, 328,
484.
10. Arrault, A.; Rocchi, S.; Picard, F.; Maurois, P.; Pirotte, B.; Vamecq, J. Biomed.
Pharmacother. 2009, 63, 56.
to design more potent antidiabetic and anticancer agents with les-
ser side effects.
11. Martin, J. A.; Brooks, D. A.; Prieto, L.; Gonzalez, R.; Torrado, A.; Rojo, I.; Lopez de
Uralde, B.; Lamas, C.; Ferritto, R.; Dolores Martin-Ortega, M. Bioorg. Med. Chem.
Lett. 2005, 15, 51.
Acknowledgements
12. Kharbanda, C.; Alam, M. S.; Hamid, H.; Bano, S.; Haider, S.; Syed, N.; Ali, Y.;
Javed, K. J. Ethnopharmacol. 2014, 151, 931.
The authors wish to express their thanks to Dr. G. N. Qazi, Vice
Chancellor, Jamia Hamdard for providing necessary research facil-
ities and to the staff members of National Cancer Institute (NCI),