Novel benzothiazole based sulfonylureas/sulfonylthioureas: Design, synthesis and evaluation of their antidiabetic potential

Article abstract of DOI:10.1039/c5nj03589a

In the present study, twenty-eight benzothiazole based sulfonylureas/sulfonylthioureas were synthesized and were assessed for their antidiabetic effect in a normoglycemic rat model by the in vivo oral glucose tolerance test (OGTT). All the synthesized com

Full text of DOI:10.1039/c5nj03589a

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Novel benzothiazole based sulfonylureas/
sulfonylthioureas: design, synthesis and evaluation
Cite this:DOI: 10.1039/c5nj03589a
of their antidiabetic potential†
Chetna Kharbanda,a Mohammad Sarwar Alam,*a Hinna Hamid,*a Kalim Javed,a
Sameena Bano,a Yakub Ali,a Abhijeet Dhulap,b Parwez Alamc and M. A. Q. Pashac
In the present study, twenty-eight benzothiazole based sulfonylureas/sulfonylthioureas were synthesized
and were assessed for their antidiabetic effect in a normoglycemic rat model by the in vivo oral glucose
tolerance test (OGTT). All the synthesized compounds were studied for their interactions inside the
PPAR-g receptor site through a docking study. Subsequently, in vitro PPAR-g transactivation assay was
performed on ten active compounds 7c, 7d, 7i–l, 8c, 8d, 8g, and 8h which showed potent antidiabetic
activity in the OGTT (better than standard drugs) and also showed a good dock score with the PPAR-g
receptor site. These active ten compounds were also found to transactivate PPAR and therefore were
assessed for their antidiabetic potential on the STZ induced diabetic model. Effects of these compounds
on body weight were also monitored during the course of study. Furthermore, the most effective com-
pound 7j was evaluated for its effect on PPAR-g gene expression.
Received (in Montpellier, France)
17th December 2015,
Accepted 17th May 2016
DOI: 10.1039/c5nj03589a
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sulfonylurea derivatives with lesser side effects. Sulfonylureas
mainly act by stimulating insulin secretion by binding to sulfonyl-
Introduction
The peroxisome proliferator-activated receptor-g (PPAR-g) is abun- urea receptors (SURs) of ATP sensitive potassium ion channels
dantly found in adipose tissue, macrophages, skeletal muscles and present in pancreatic b-cells.3 Though thiazolidinediones repre-
intestinal cells. Important functions of PPAR-g include regulation sent a class of antidiabetic compounds generally known as PPAR-g
of glucose homeostasis, adipogenesis and insulin sensitivity.1 agonists, several marketed sulfonylureas like glibenclamide,
Activation of PPAR-g stimulates the insulin dependent glucose glimepiride, gliquidone and glipizide have recently been proven
transporter GLUT4 protein which accelerates the transportation of to improve insulin sensitization by acting as PPAR-g agonists in
fatty acids synthesized from glucose. This process increases both addition to binding with SU-receptors. It has been studied that
the storage capacity and fatty acid flux in the adipocyte which in glibenclamide competitively binds to the PPAR-g receptor site
turn helps in glucose homeostasis. In addition, the ligand-binding with respect to marketed PPAR-g agonists like pioglitazone and
domain of PPAR-g contains a large binding pocket that allows the rosiglitazone.4,5 Benzothiazole derivatives encompass multiple
interaction of diverse types of ligands to form ligand–receptor applications in bioorganic and medicinal chemistry due to a
complexes.2
unique bicyclic ring system of benzothiazole. The pharma-
Despite being primitive, sulfonylureas are still being used as cological profile of compounds containing the benzothiazole
oral antidiabetic agents either alone or in combination with moiety comprises antimicrobial,6–10 anticancer,11 anthelmintic12
other classes of oral antidiabetic drugs. Their intriguing anti- and anti-diabetic13 activities.
diabetic property is still provoking scientists to synthesize new
Therefore, the present work emphasizes the synthesis of
benzothiazole based sulfonylureas as an approach to design new
antidiabetic agents along with subsequent control over weight gain.
The effect of these synthesized compounds as PPAR-g agonists was
also studied.
a Department of Chemistry, Faculty of Science, Hamdard University,
New Delhi-110 062, India. E-mail: msalam@jamiahamdard.ac.in,
msalam5555@gmail.com, hhmaid@jamiahamdard.ac.in; Fax: +91 11 26059663;
Tel: +91 11 26059688 (5555), (5559)
b CSIR Unit for Research and Development of Information Products,
Results and discussion
Chemistry
Pune-411038, India
c Functional Genomics Unit, CSIR-Institute of Genomics & Integrative Biology,
Delhi, India
The synthetic route used to synthesize benzothiazole based
sulfonylurea derivatives is outlined in Scheme 1. The structural
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5nj03589a
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Scheme 1 Synthesis of benzothiazole based sulfonylurea derivatives.
1
elucidation was based on H NMR, 13C NMR, IR, mass spectra came near to normal after 90 min for ten compounds 7c, 7d,
and CHNS data of synthesized compounds. 1H NMR spectra of 7i–l, 8c, 8d, 8g and 8h.
all the synthesized compounds were recorded at 300–500 MHz
frequency in parts per million (ppm) using TMS as the standard. The
appearance of three double doublets in the range of d 3.00–6.00 ppm
confirmed the formation of the pyrazoline ring. Coupling with
benzoisocyanate/benzoisothiocyanate was verified by increase
in signals due to aromatic protons depending on the substituent
and from the presence of the singlet of CH2 at around d 4.65 ppm.
Mass spectra showing specific peaks at [M + 1]+ further con-
firmed the syntheses of desired products. The structure of each
compound indicating different substitutions has been provided
in Fig. S1 (ESI†).
Docking studies
As mentioned earlier, sulfonylureas can act through the activa-
tion of PPAR-g in addition to their action on K+
channels.
ATP
Therefore, before proceeding further we first carried out mole-
cular docking studies of synthesized compounds against the
PPAR-g target. Dock scores of all the synthesized compounds
were compared with the standard drug, rosiglitazone, which is
a PPAR-g agonist. Interestingly, eighteen (7a–d, 7f, 7i–o, 8c,
8d, 8f–h, and 8j) out of twenty eight synthesized compounds
showed a higher dock score than rosiglitazone (ꢀ5.72). It
should be noted that a negative value indicates low energy
which in turn indicates likely binding interactions to make a
Oral glucose tolerance test (OGTT)
Primarily, all the synthesized compounds were scrutinized by stable system i.e. the more negative the value, the more stable
loading glucose in normal rats to assess their antidiabetic the system will be. Careful examination of docking images
potential. From the results of the oral glucose tolerance test, showed that most of the compounds were either deeply buried
it was observed that ten compounds namely 7c, 7d, 7i–l, 8c, 8d, inside the receptor site or formed H-bonds with amino acid
8g and 8h significantly lowered the plasma glucose level as residues of the receptor site. Some of the compounds displayed
compared to the standard drug glibenclamide. Moreover, the more than one H-bonding interactions. The images of the com-
alleviation in the plasma glucose level that was observed for a pounds displaying a high docking score are shown in Fig. 2 and
PPAR-g agonist, rosiglitazone, after 90 min was less than twelve dock scores are given in Table S1 (ESI†). The ligand interaction
compounds. The results of the OGT test are illustrated in Fig. 1. diagram of compound 7k with the highest dock score of ꢀ10.06
It was seen that the plasma glucose concentration of animals in showed the presence of p–p stacking as well as H-bonding with
the control group suddenly increased after glucose load while LYS-261 and ARG-280 and compound 8g (ꢀ10.03) formed
the groups administered with synthesized compounds showed multiple H-bonds with LYS-261 present in the receptor site
a gradual increase in the plasma glucose level. The increase in while the dock score of rosiglitazone was only ꢀ5.72 though it
the plasma glucose level of groups administered with synthe- showed p–p stacking with the GLU-272 amino acid residue of
sized compounds started reducing with the passage of time and the receptor site.
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Fig. 1 Antidiabetic effect of treating wistar rats with synthesized compounds by the Oral Glucose Tolerance Test. Data are analyzed by one way
ANOVA followed by the Dunnett test and expressed as mean ꢂ SEM from six observations; * represents a change as compared to control; ** indicates
p o 0.01 & * indicates p o 0.05.
In vitro PPAR transactivation assay. To confirm that the to normal. Remaining four compounds (7c, 7d, 8c and 8d)
synthesized compounds are acting through transactivation of assuaged the plasma glucose level almost as appreciably as
PPAR-g, ten compounds 7c, 7d, 7i–l, 8c, 8d, 8g and 8h (that standard drugs, glibenclamide and rosiglitazone.
showed good oral glucose tolerance and have a high dock score)
Structure–activity relationship
were scrutinized by in vitro PPAR transactivation assay (Table 1).
All the active compounds showed moderate alleviation of PPAR
in transactivation assay. Captivatingly, compound 7k which
exhibited highest dock score transactivated PPAR by only 54.93%
as compared to rosiglitazone which showed 81.68% elevation in
PPAR transactivation. Another two compounds 7j and 8h showed
PPAR transactivation as much as compound 7k i.e. 54.01 and
54.29%, respectively. The synthesized compounds showed high
in vivo antidiabetic activity but less PPAR transactivation than
rosiglitazone which inferred that PPAR transactivation is one of the
targets aimed by these synthesized compounds in order to exhibit
antidiabetic activity in a more complex in vivo system.
To understand the effect of different substitutions on in vivo anti-
diabetic activity, correlations have been drawn as follows (Fig. 4):
ꢁ Sulfonylureas derived from p-chloroacetophenone are
in general more biologically active than p-bromoacetophenone
derivatives.
ꢁ Fluoro substitution on the aryl ring exhibited higher anti-
diabetic activity than other substitutions while chloro substitu-
tion on the aryl group significantly lowered in vivo activity.
ꢁ Replacing the aryl ring with heterocycle like thiophene also
displayed significant biological activity.
ꢁ Substitution of methyl and methoxy on the aryl ring lowered
biological activity.
Antidiabetic activity on STZ-induced diabetic rats
ꢁ In the case of p-chloro derivatives, sulfonylureas are more
active than sulfonylthioureas while the trend is reverse in p-bromo
derivatives with few exceptions.
Ten compounds (7c, 7d, 7i–l, 8c, 8d, 8g, and 8h) were further
evaluated for their effect in the STZ induced diabetic model.
It was found that the supplementation of diabetic rats with
compounds 7i–l, 8g and 8h caused significant lowering of the
Body weight
plasma glucose level on 15th day of the study (Fig. 3). These six Increase in body weight is one of the closely associated con-
compounds restored the plasma glucose level almost close cerns of treatment with sulfonylureas. Therefore, the change in
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Fig. 2 Docking images of compounds 7k, 8g and rosiglitazone.
Table 1 In vitro PPAR-g transactivation assay on the 3T3-L1 cell line
body weight post treatment with active compounds (7i–l, 8g
and 8h) was carefully observed over the period of 15 days of
study. In contrast to STZ induced control where significant
weight loss was observed, the animals treated with glibenclamide
and rosiglitazone exhibited increase in body weight. However, the
treatment with active compounds 7i, 7j, 7l and 8g maintained the
body weight and did not allow much increase up to 15 days of
study (Fig. 5).
Compounds
% PPAR transactivation (mean ꢂ SEM)
Control
Rosiglitazone
7c
7d
7i
7j
7k
7l
8c
8d
8g
8h
0.15 ꢂ 0.001
81.68 ꢂ 0.001
43.48 ꢂ 0.001
43.69 ꢂ 0.002
45.85 ꢂ 0.001
54.01 ꢂ 0.001
54.93 ꢂ 0.001
49.58 ꢂ 0.001
43.11 ꢂ 0.001
41.49 ꢂ 0.001
53.21 ꢂ 0.001
54.29 ꢂ 0.002
PPAR-c gene expression
The most potent compound 7j was evaluated for its effect on
the expression of PPAR-g target genes. The expression of PPAR-g
genes was measured in 3T3-L1 fibroblasts in comparison with
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Fig. 3 Antidiabetic effect of synthesized compounds on STZ induced diabetic animals. Data are analyzed by one way ANOVA followed by the Dunnett
test and expressed as mean ꢂ SEM from six observations; * represents a change as compared to diabetic control; ** indicates p o 0.01 & *** indicates
p o 0.001.
Fig. 4 General structure of synthesized benzothiazole based sulfonylurea.
Fig. 6 PPAR-g gene expression evaluation of compound 7j in comparison
with standards, glibenclamide and rosiglitazone. Data are analyzed by one
way ANOVA followed by the Dunnett test and expressed as mean ꢂ SEM
from three observations; * represents change as compared to control;
** indicates p o 0.01. PCR was performed in triplicate and was repeated
two times for each gene and each sample. Relative transcript quantities
were calculated using the Ct method with b-actin as the endogenous
reference gene.
Glibenclamide, on the other hand, showed less significant
Fig. 5 Change in body weight after a 15 day study on STZ-induced
elevation in gene expression as compared to compound 7j
diabetic rats. Data are analyzed by one way ANOVA followed by the
and rosiglitazone.
Bonferroni ‘t’ test and expressed as mean ꢂ SEM from six observations;
* represents a significant change as compared to diabetic control; # represents
a significant change as compared to normal control. ** Indicates p o 0.01 &
* indicates p o 0.05; ## indicates p o 0.05 & # indicates p o 0.01.
Conclusion
It can be concluded that four compounds 7i, 7j, 7l and
standard drugs, rosiglitazone and glibenclamide (Fig. 6). The 8g exhibited significant antidiabetic activity as compared to
standard drug, glibenclamide, amplified the expression of PPAR-g standard drugs without showing a much increase in body
genes to lesser extent than rosiglitazone despite having strong weight. The most active compound 7j also showed an increase
in vivo antidiabetic activity. Similarly, compound 7j signifi- in PPAR-g gene expression. Therefore, the present study
cantly enhanced the expression of PPAR-g genes more than demonstrated that the antidiabetic effect of these compounds
glibenclamide but less than rosiglitazone. It was found that might partially be due to the activation of PPAR-g. Furthermore,
rosiglitazone increased gene expression by 1.5 fold while it can be concluded that the combination of benzothiazole
the treatment with compound 7j caused 1.22 fold elevation. with sulfonylurea allows us to design more of the biologically
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active molecules that can lead to discovery of potential (C aromatic), 155.50 (CQN), 159.85 (N1–C benzothiazolyl),
ꢀ
drug candidates.
170.30 (CQO). FAB-MS m/z: 608 [M + 1]+. Elemental analysis:
calculated for C28H22ClN5O3S3; C = 55.30, H = 3.65, N = 11.52,
S = 15.82. Found: C = 55.35, H = 3.71, N = 11.56, S = 15.87.
{2-[3-(4-Chloro-phenyl)-5-thiophen-2-yl-4,5-dihydro-pyrazol-1-
yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (7d). Yield = 59.2%,
m.p. 181–182 1C, Rf = 0.56, toluene : ethyl acetate : formic acid,
Experimental data
General synthesis of final products (7a–p and 8a–l)
p-Chloroacetophenone (1) and p-bromoacetophenone (2) were 5 : 4 : 1. IR nmax (KBr): 3361, 3239 & 1551 (N–H), 1594 (CQN), 1321
1
made to react with appropriate aldehyde to give corresponding & 1148 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm): 3.57
chalcones, 3a–h and 4a–f. The synthesis of intermediate (1H, dd, J = 5.6 Hz, 18.0 Hz), 4.12 (1H, dd, J = 11.5 Hz, 17.2 Hz),
pyrazolines (5a–h and 6a–f) was carried out by reacting synthe- 4.63 (2H, s, CH2), 6.20 (1H, dd, J = 6.0 Hz, 12.0 Hz), 6.48 (2H, s,
sized chalcones (1.0 mmol) with 1.2 mmol of 2-hydrazino- NH), 6.97–8.43 (15H, m, Ar). 13C NMR (125 MHz, DMSO, dppm):
benthothiazole-6-sulfonic acid amide.14 The pyrazolines were 42.79 (C4), 57.41 (C5), 63.18 (CH2), 112.01–129.56 (C aromatic),
reacted with appropriate benzylisocyanate or benzylisothio- 155.58 (CQN), 156.49 (N1–C benzothiazolyl), 158.44 (CQS).
ꢀ
cyanate in the presence of anhydrous potassium carbonate FAB-MS m/z: 624 [M + 1]+. Elemental analysis: calculated for
and acetone by conventional reactions.15 After reaction com- C28H22ClN5O2S4; C = 53.88, H = 3.55, N = 11.22, S = 20.55. Found:
pletion, the reaction mixture was carefully concentrated and C = 53.95, H = 3.61, N = 11.27, S = 20.62.
filtered. The solid so obtained was washed with water, dried,
{2-[3-(4-Chloro-phenyl)-5-(2-chloro-phenyl)-4,5-dihydro-pyrazol-
and crystallized from acetone (Scheme 1). The compounds were 1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (7e). Yield = 64.2%,
finally recrystallized from ethanol to give pure desired products m.p. 198–199 1C, Rf = 0.55, toluene : ethyl acetate : formic acid,
(7a–p and 8a–l).
5 : 4 : 1. IR nmax (KBr): 3362, 3237 & 1541 (N–H), 1595 (CQN), 1318
{2-[3-(4-Chloro-phenyl)-5-(4-methoxy-phenyl)-4,5-dihydro-pyrazol- & 1147 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm): 3.61
1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (7a). Yield = 64.2%, (1H, dd, J = 5.6 Hz, 17.2 Hz), 4.26 (1H, dd, J = 11.6 Hz, 17.2 Hz),
m.p. 148–149 1C, Rf = 0.57, toluene : ethyl acetate : formic acid, 4.66 (2H, s, CH2), 6.18 (1H, dd, J = 6.0 Hz, 12.0 Hz), 6.49 (2H, s,
5:4:1. IR nmax (KBr): 3363, 3242 & 1541 (N–H), 1595 (CQN), 1321 NH), 7.11–7.92 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm):
1
& 1151 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO, dppm): 3.55 (1H, 46.25 (C4), 53.87 (CH2), 63.96 (C5), 100.18–129.47 (C aromatic),
ꢀ
dd, J = 5.2 Hz, 18.0 Hz), 3.71 (3H, s, OCH3), 4.25 (2H, s, CH2), 4.63 153.58 (CQN), 158.41 (N1–C benzothiazolyl), 171.90 (CQO).
ꢀ
(1H, dd, J = 12.0 Hz, 17.6 Hz), 5.79 (1H, dd, J = 6.0 Hz, 12.0 Hz), 6.25 FAB-MS m/z: 636 [M + 1]+. Elemental analysis: calculated for
(2H, s, NH), 7.20–7.38 (16H, m, Ar). 13C NMR (125 MHz, DMSO, C30H23Cl2N5O2S3; C = 56.60, H = 3.64, N = 11.00, S = 10.07. Found:
d
ppm): 43.41 (C4), 53.57 (CH2), 57.16 (C5), 63.47 (OCH3), 113.95– C = 56.66, H = 3.68, N = 11.07, S = 10.09.
ꢀ
143.76 (C aromatic), 154.80 (CQN), 155.23 (N1–C benzothiazolyl),
{2-[3-(4-Chloro-phenyl)-5-(2-chloro-phenyl)-4,5-dihydro-pyrazol-
ꢀ
159.56 (CQO). FAB-MS m/z: 632 [M + 1]+. Elemental analysis: 1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (7f). Yield = 59.7%,
calculated for C31H26ClN5O4S2; C = 58.90, H = 4.15, N = 11.08, m.p. 183–184 1C, Rf = 0.55, toluene : ethyl acetate : formic acid,
S = 10.14. Found: C = 58.92, H = 4.18, N = 11.11, S = 10.17.
5:4:1. IR nmax (KBr): 3361, 3233 & 1539 (N–H), 1590 (CQN),
{2-[3-(4-Chloro-phenyl)-5-(4-methoxy-phenyl)-4,5-dihydro-pyrazol- 1314 & 1141 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm):
1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (7b). Yield = 61.7%, 3.59 (1H, dd, J = 5.6 Hz, 17.4 Hz), 4.23 (1H, dd, J = 11.5 Hz, 17.6 Hz),
m.p. 149–150 1C, Rf = 0.57, toluene : ethyl acetate : formic acid, 4.62 (2H, s, CH2), 6.08 (1H, dd, J = 6.0 Hz, 12.0 Hz), 6.36 (2H, s, NH),
5:4:1. IR nmax (KBr): 3363, 3241 & 1539 (N–H), 1592 (CQN), 1318 7.16–8.26 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm):
& 1148 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO, dppm): 3.58 (1H, 43.47(C4), 57.61 (C5), 63.69 (CH2), 112.00–132.14 (C aromatic),
1
dd, J = 5.6 Hz, 17.4 Hz), 3.72 (3H, s, OCH3), 4.26 (2H, s, CH2), 4.53 154.61 (CQN), 155.48 (N1–C benzothiazolyl), 158.98 (CQS).
ꢀ
(1H, dd, J = 11.2 Hz, 17.6 Hz), 5.68 (1H, dd, J = 6.0 Hz, 12.2 Hz), 5.89 FAB-MS m/z: 652 [M + 1]+. Elemental analysis: calculated for
(2H, s, NH), 7.12–8.04 (16H, m, Ar). 13C NMR (125 MHz, DMSO, C30H23Cl2N5O2S3; C = 55.21, H = 3.55, N = 10.73, S = 14.74. Found:
dppm): 43.56 (C4), 57.68 (C5), 63.28 (CH2), 54.31 (OCH3), 110.41– C = 55.24, H = 3.58, N = 10.78, S = 14.77.
129.32 (C aromatic), 154.81 (CQN), 155.64 (N1–C benzothiazolyl),
{2-[3-(4-Chloro-phenyl)-5-(4-chloro-phenyl)-4,5-dihydro-pyrazol-
ꢀ
158.60 (CQS). FAB-MS m/z: 649 [M + 1]+. Elemental analysis: 1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (7g). Yield = 65.3%,
calculated for C31H26ClN5O3S3; C = 57.44, H = 4.04, N = 10.80, m.p. 234–235 1C, Rf = 0.55, toluene : ethyl acetate : formic acid,
S = 14.84. Found: C = 57.45, H = 4.06, N = 10.85, S = 14.91.
5 : 4 : 1. IR nmax (KBr): 3362, 3240 & 1545 (N–H), 1601 (CQN), 1320
1
{2-[3-(4-Chloro-phenyl)-5-thiophen-2-yl-4,5-dihydro-pyrazol-1- & 1149 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm): 3.68
yl]-benzothiazole-6-sulfonyl}N0-benzylurea (7c). Yield = 64.4%, (1H, dd, J = 6.0 Hz, 18.0 Hz), 4.15 (1H, dd, J = 12.2 Hz, 18.0 Hz),
m.p. 236–237 1C, Rf = 0.55, toluene : ethyl acetate : formic acid, 4.67 (2H, s, CH2), 5.87 (1H, dd, J = 5.6 Hz, 12.0 Hz), 6.09 (2H, s,
5 : 4 : 1. IR nmax (KBr): 3361, 3244 & 1541 (N–H), 1591 (CQN), NH), 7.02–8.01 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm):
1
1318 & 1148 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm): 44.12 (C4), 54.18 (CH2), 63.25 (C5), 110.50–125.98 (C aromatic),
ꢀ
3.58 (1H, dd, J = 5.5 Hz, 17.8 Hz), 4.15 (1H, dd, J = 11.2 Hz, 17.6 153.48 (CQN), 155.74 (N1–C benzothiazolyl), 158.16 (CQO).
ꢀ
Hz), 4.71 (2H, s, CH2), 5.88 (1H, dd, J = 5.6 Hz, 11.5 Hz), 6.09 FAB-MS m/z: 636 [M + 1]+. Elemental analysis: calculated for
(2H, s, NH), 6.98–8.01 (15H, m, Ar). 13C NMR (125 MHz, DMSO, C30H23Cl2N5O3S2; C = 56.60, H = 3.64, N = 11.00, S = 10.07. Found:
d
ppm): 43.33 (C4), 53.86 (CH2), 63.73 (C5), 104.22–131.37 C = 56.62, H = 3.69, N = 11.11, S = 10.08.
ꢀ
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{2-[3-(4-Chloro-phenyl)-5-(4-chloro-phenyl)-4,5-dihydro-pyrazol- 1319 & 1151 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm):
1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (7h). Yield = 61.3%, 3.82 (1H, dd, J = 6.0 Hz, 17.8 Hz), 4.22 (1H, dd, J = 11.6 Hz, 17.2 Hz),
m.p. 168–169 1C, Rf = 0.57, toluene : ethyl acetate : formic acid, 4.67 (2H, s, CH2), 5.86 (1H, dd, J = 6.0 Hz, 12.0 Hz), 6.17 (2H, s, NH),
5:4:1. IR nmax (KBr): 3362, 3241 & 1545 (N–H), 1595 (CQN), 1318 7.12–8.94 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm): 43.95
& 1151 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO, dppm): 3.69 (1H, (C4), 57.68 (C5), 63.49 (CH2), 110.46–132.53 (C aromatic), 154.31
dd, J = 5.6 Hz, 18.0 Hz), 4.14 (1H, dd, J = 12.0 Hz, 17.4 Hz), 4.61 (2H, (CQN), 156.40 (N1–C benzothiazolyl), 158.24 (CQS). FAB-MS m/z:
ꢀ
s, CH2), 5.89 (1H, dd, J = 6.0 Hz, 12.0 Hz), 6.93 (2H, s, NH), 6.94–8.16 637 [M + 1]+. Elemental analysis: calculated for C30H23ClFN5O2S3;
(16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm): 43.59 (C4), 57.36 C = 56.64, H = 3.64, N = 11.01, S = 15.12. Found: C = 56.67, H = 3.68,
(C5), 63.42 (CH2), 112.14–132.56 (C aromatic), 155.72 (CQN), 156.39 N = 11.04, S = 15.17.
(N1–C benzothiazolyl), 158.64 (CQS). FAB-MS m/z: 652 [M + 1]+.
{2-[3-(4-Chloro-phenyl)-5-(4-dimethylamino-phenyl)-4,5-dihydro-
ꢀ
Elemental analysis: calculated for C30H23Cl2N5O2S3; C = 55.21, H = pyrazol-1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (7m). Yield =
3.55, N = 10.73, S = 14.74. Found: C = 55.27, H = 3.61, N = 10.75, 67.2%, m.p. 268–269 1C, Rf = 0.54, toluene : ethyl acetate : formic
S = 14.79.
acid, 5:4:1. IR nmax (KBr): 3361, 3238 & 1539 (N–H), 1588 (CQN),
{2-[3-(4-Chloro-phenyl)-5-phenyl-4,5-dihydro-pyrazol-1-yl]- 1318 & 1147 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO, dppm): 2.87
benzothiazole-6-sulfonyl}N0-benzylurea (7i). Yield = 67.0%, m.p. (6H, s, N(CH3)2), 3.59 (1H, dd, J = 5.6 Hz, 18.0 Hz), 4.03 (1H, dd,
161–162 1C, Rf = 0.54, toluene: ethyl acetate: formic acid, 5 : 4 : 1. J = 12.2 Hz, 17.6 Hz), 4.68 (2H, s, CH2), 5.76 (1H, dd, J = 6.0 Hz,
IR nmax (KBr): 3367, 3245 & 1541 (N–H), 1596 (CQN), 1323 & 1151 12.0 Hz), 6.23 (2H, s, NH), 7.03–8.52 (16H, m, Ar). 13C NMR
cmꢀ1 (SO2No). 1H NMR (500 MHz, DMSO, dppm): 3.48 (1H, dd, (125 MHz, DMSO, dppm): 43.26 (C4), 54.10 (N(CH3)2), 55.36 (CH2),
ꢀ
ꢀ
J = 6.5 Hz, 17.0 Hz), 3.95 (1H, dd, J = 11.5 Hz, 17.5 Hz), 4.62 (2H, 63.40 (C5), 112.96–130.19 (C aromatic), 150.34 (CQN), 155.24
s, CH2), 5.87 (2H, s, NH), 6.40 (1H, dd, J = 5.5 Hz, 12.5 Hz), (N1–C benzothiazolyl), 159.27 (CQO). FAB-MS m/z: 646 [M + 1]+.
ꢀ
6.94–8.16 (17H, m, Ar). 13C NMR (125 MHz, DMSO, dppm): 43.42 Elemental analysis: calculated for C32H29ClN6O3S2; C = 59.57,
(C4), 43.98 (CH2), 63.94 (C5), 100.00–135.37 (C aromatic), H = 4.53, N = 13.03, S = 9.94. Found: C = 59.61, H = 4.56,
ꢀ
153.50 (CQN), 155.85 (N1–C benzothiazolyl), 158.55 (CQO). N = 13.08, S = 10.02.
ꢀ
FAB-MS m/z: 602 [M + 1]+. Elemental analysis: calculated for
{2-[3-(4-Chloro-phenyl)-5-(dimethylamino-phenyl)-4,5-dihydro-
C30H24ClN5O3S2; C = 59.84, H = 4.02, N = 11.63, S = 10.65. Found: pyrazol-1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (7n).
C = 59.88, H = 4.10, N = 11.68, S = 10.67. Yield = 62.3%, m.p. 190–191 1C, Rf = 0.55, toluene : ethyl
{2-[3-(4-Chloro-phenyl)-5-phenyl-4,5-dihydro-pyrazol-1-yl]- acetate : formic acid, 5 : 4 : 1. IR nmax (KBr): 3365, 3239 & 1541
benzothiazole-6-sulfonyl}N0-benzylthiourea (7j). Yield = 63.7%, (N–H), 1597 (CQN), 1321 & 1150 cmꢀ1 (SO2No). 1H NMR
m.p. 204–205 1C, Rf = 0.55, toluene : ethyl acetate : formic acid, (400 MHz, DMSO, dppm): 2.89 (6H, s, N(CH3)2), 3.61 (1H, dd,
5 : 4 : 1. IR nmax (KBr): 3368, 3244 & 1547 (N–H), 1598 (CQN), J = 5.2 Hz, 17.0 Hz), 4.12 (1H, dd, J = 12.0 Hz, 17.6 Hz), 5.87 (1H,
1
1322 & 1152 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm): dd, J = 6.0 Hz, 12.0 Hz), 6.11 (2H, s, NH), 7.08–7.94 (16H, m, Ar).
3.84 (1H, dd, J = 6.0 Hz, 17.8 Hz), 4.18 (1H, dd, J = 11.2 Hz, 17.4 13C NMR (125 MHz, DMSO, dppm): 43.53 (C4), 54.68 (N(CH3)2),
ꢀ
Hz), 4.66 (2H, s, CH2), 5.89 (1H, dd, J = 6.0 Hz, 11.6 Hz), 6.18 57.43 (C5), 63.47 (CH2), 112.81–134.32 (C aromatic), 154.32
(2H, s, NH), 7.04–8.01 (17H, m, Ar). 13C NMR (125 MHz, DMSO, (CQN), 155.76 (N1–C benzothiazolyl), 158.55 (CQS). FAB-MS
ꢀ
d
ppm): 43.59 (C4), 57.29 (C5), 63.52 (CH2), 100.20–132.41 m/z: 661 [M + 1]+. Elemental analysis: calculated for C32H29ClN6O2S3;
(C aromatic), 154.58 (CQN), 155.45 (N1–C benzothiazolyl), C = 58.12, H = 4.42, N = 12.71, S = 14.55. Found: C = 58.16, H = 4.44,
ꢀ
158.35 (CQS). FAB-MS m/z: 618 [M + 1]+. Elemental analysis: N = 12.75, S = 14.60.
calculated for C30H24ClN5O2S3; C = 58.29, H = 3.91, N = 11.33,
S = 15.56. Found: C = 58.35, H = 3.96, N = 11.41, S = 15.62.
{2-[3-(4-Chloro-phenyl)-5-(4-methyl-phenyl)-4,5-dihydro-pyrazol-
1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (7o). Yield = 54.2%,
{2-[3-(4-Chloro-phenyl)-5-(4-fluoro-phenyl)-4,5-dihydro-pyrazol- m.p. 144–145 1C, Rf = 0.55, toluene : ethyl acetate : formic acid,
1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (7k). Yield = 55.7%, 5:4:1. IR nmax (KBr): 3363, 3242 & 1541 (N–H), 1592 (CQN), 1319
m.p. 158–159 1C, Rf = 0.56, toluene : ethyl acetate : formic acid, & 1154 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO, dppm): 3.10 (3H,
5 : 4 : 1. IR nmax (KBr): 3365, 3242 & 1542 (N–H), 1592 (CQN), 1321 s, CH3), 3.41 (1H, dd, J = 6.0 Hz, 17.4 Hz), 3.96 (1H, dd, J = 11.6 Hz,
1
& 1151 cmꢀ1 (SO2No). H NMR (500 MHz, DMSO, dppm): 3.60 17.4 Hz), 4.69 (2H, s, CH2), 5.81 (1H, dd, J = 6.0 Hz, 12.0 Hz), 6.09
(1H, dd, J = 5.6 Hz, 17.8 Hz), 4.27 (1H, dd, J = 11.5 Hz, 17.6 Hz), (2H, s, NH), 7.04–8.03 (16H, m, Ar). 13C NMR (125 MHz, DMSO,
4.41 (2H, s, CH2), 5.86 (1H, dd, J = 5.6 Hz, 11.5 Hz), 6.21 (2H, s,
dppm): 20.12 (CH3), 43.61 (C4), 57.56 (C5), 64.02 (CH2), 112.01–
NH), 6.94–7.85 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm): 132.17 (C aromatic), 154.90 (CQN), 155.95 (N1–C benzothiazolyl),
ꢀ
43.67 (C4), 59.79 (CH2), 63.72 (C5), 100.00–137.87 (C aromatic), 159.61 (CQS). FAB-MS m/z: 616 [M + 1]+. Elemental analysis:
ꢀ
143.76 (CQN), 155.23 (N1–C benzothiazolyl), 158.47 (CQO). calculated for C31H26ClN5O3S2; C = 60.43, H = 4.25, N = 11.37,
ꢀ
FAB-MS m/z: 620 [M + 1]+. Elemental analysis: calculated for S = 10.41. Found: C = 60.49, H = 4.28, N = 11.42, S = 10.45.
C30H23ClFN5O3S2; C = 58.11, H = 3.74, N = 11.29, S = 10.34. Found:
C = 58.20, H = 3.81, N = 11.35, S = 10.38.
{2-[3-(4-Chloro-phenyl)-5-(4-methyl-phenyl)-4,5-dihydro-pyrazol-
1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (7p). Yield =
{2-[3-(4-Chloro-phenyl)-5-(4-fluoro-phenyl)-4,5-dihydro-pyrazol- 57.5%, m.p. 198–199 1C, Rf = 0.55, toluene : ethyl acetate: formic
1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (7l). Yield = 59.4%, acid, 5 : 4 : 1. IR nmax (KBr): 3364, 3241 & 1542 (N–H), 1592
m.p. 220–221 1C, Rf = 0.57, toluene : ethyl acetate : formic acid, (CQN), 1318 & 1154 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO,
5:4:1. IR nmax (KBr): 3364, 3244 & 1541 (N–H), 1600 (CQN),
dppm): 3.19 (3H, s, CH3), 3.39 (1H, dd, J = 6.0 Hz, 18.0 Hz),
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3.95 (1H, dd, J = 11.6 Hz, 17.6 Hz), 4.75 (2H, s, CH2), 5.85 (1H, 154.80 (CQN), 155.23 (N1–C benzothiazolyl), 178.94 (CQS).
ꢀ
dd, J = 5.6 Hz, 12.0 Hz), 6.25 (2H, s, NH), 7.04–8.05 (16H, m, Ar). FAB-MS m/z: 667 [M + 1]+. Elemental analysis: calculated for
13C NMR (125 MHz, DMSO, dppm): 20.32 (CH3), 43.69 (C4), 57.41 C28H22BrN5O2S4; C = 50.29, H = 3.32, N = 10.47, S = 19.18. Found:
(C5), 63.52 (CH2), 112.21–132.41 (C aromatic), 154.92 (CQN), C = 50.25, H = 3.31, N = 10.41, S = 19.22.
155.63 (N1–C benzothiazolyl), 161.21 (CQS). FAB-MS m/z:
{2-[3-(4-Bromo-phenyl)-5-(4-chloro-phenyl)-4,5-dihydro-pyrazol-
ꢀ
632 [M + 1]+. Elemental analysis: calculated for C31H26ClN5O2S3; 1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (8e). Yield = 65.3%,
C = 58.89, H = 4.15, N = 11.08, S = 15.22. Found: C = 58.92, H = m.p. 217–218 1C, Rf = 0.55, toluene : ethyl acetate : formic acid,
4.22, N = 11.12, S = 15.25.
5 : 4 : 1. IR nmax (KBr): 3365, 3241 & 1542 (N–H), 1594 (CQN), 1318
{2-[3-(4-Bromo-phenyl)-5-(4-methoxy-phenyl)-4,5-dihydro-pyrazol- & 1149 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm): 3.40
1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (8a). Yield = 59.8%, (1H, dd, J = 4.8 Hz, 18.0 Hz), 4.13 (1H, dd, J = 10.4 Hz, 17.6 Hz),
m.p. 139–140 1C, Rf = 0.57, toluene : ethyl acetate : formic acid, 4.31 (2H, s, CH2), 5.91 (1H, dd, J = 6.8 Hz, 11.6 Hz), 6.23 (2H, s,
5:4:1. IR nmax (KBr): 3365, 3241 & 1542 (N–H), 1594 (CQN), 1318 NH), 7.00–7.82 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm):
1
& 1149 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO, dppm): 3.65 (1H, 43.67 (C4), 53.99 (CH2), 63.58 (C5), 110.42–128.37 (C aromatic),
ꢀ
dd, J = 6.0 Hz, 17.4 Hz), 3.83 (3H, s, OCH3), 4.43 (1H, dd, J = 11.6 Hz, 153.86 (CQN), 163.17 (N1–C benzothiazolyl), 168.52 (CQO).
ꢀ
17.6 Hz), 4.67 (2H, s, CH2), 5.88 (1H, dd, J = 6.0 Hz, 12.0 Hz), 6.12 FAB-MS m/z: 680 [M + 1]+. Elemental analysis: calculated for
(2H, s, NH), 6.99–8.04 (16H, m, Ar). 13C NMR (125 MHz, DMSO, C30H23BrClN5O3S2; C = 52.91, H = 3.40, N = 10.28, S = 9.42. Found:
d
ppm): 43.57 (C4), 57.38 (CH2), 63.88 (C5), 64.53 (OCH3), 109.36– C = 52.94, H = 3.41, N = 10.31, S = 9.47.
ꢀ
128.97 (C aromatic), 155.90 (CQN), 158.05 (N1–C benzothiazolyl),
{2-[3-(4-Bromo-phenyl)-5-(4-chloro-phenyl)-4,5-dihydro-pyrazol-
ꢀ
171.19 (CQO). FAB-MS m/z: 676 [M + 1]+. Elemental analysis: 1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (8f). Yield = 59.8%,
calculated for C31H26BrN5O4S2; C = 55.03, H = 3.87, N = 10.35, m.p. 212–213 1C, Rf = 0.57, toluene : ethyl acetate : formic acid,
S = 9.48. Found: C = 55.07, H = 3.90, N = 10.38, S = 9.52.
5:4:1. IR nmax (KBr): 3365, 3242 & 1539 (N–H), 1589 (CQN),
{2-[3-(4-Bromo-phenyl)-5-(4-methoxy-phenyl)-4,5-dihydro-pyrazol- 1321 & 1152 cmꢀ1 (SO2No). H NMR (300 MHz, DMSO, dppm):
1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (8b). Yield = 67.0%, 3.68 (1H, dd, J = 5.1 Hz, 17.1 Hz), 4.16 (1H, dd, J = 11.7 Hz, 17.1 Hz),
m.p. 269–270 1C, Rf = 0.57, toluene : ethyl acetate : formic acid, 4.66 (2H, s, CH2), 5.85 (1H, dd, J = 4.8 Hz, 12.0 Hz), 6.27 (2H, s, NH),
5:4:1. IR nmax (KBr): 3363, 3241 & 1542 (N–H), 1594 (CQN), 7.06–8.85 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm): 43.49
1
1
1320 & 1151 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm): (C4), 54.72 (C5), 63.44 (CH2), 100.01–134.08 (C aromatic), 154.84
3.67 (1H, dd, J = 5.4 Hz, 17.2 Hz), 4.00 (3H, s, OCH3), 4.79 (1H, dd, (CQN), 155.54 (N1–C benzothiazolyl), 168.14 (CQS). FAB-MS m/z:
ꢀ
J = 12.0 Hz, 17.6 Hz), 5.98 (1H, dd, J = 6.0 Hz, 12.0 Hz), 6.23 (2H, s, 695 [M + 1]+. Elemental analysis: calculated for C30H23BrClN5O2S3;
NH), 7.01–8.07 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm): C = 51.69, H = 3.33, N = 10.05, S = 13.80. Found: C = 51.72, H = 3.36,
43.26 (C4), 54.64 (CH2), 58.15 (C5), 64.36 (OCH3), 110.89–132.02 N = 10.06, S = 13.83.
ꢀ
(C aromatic), 155.49 (CQN), 158.37 (N1–C benzothiazolyl), 159.14
{2-[3-(4-Bromo-phenyl)-5-(4-fluoro-phenyl)-4,5-dihydro-pyrazol-
ꢀ
(CQS). FAB-MS m/z: 692 [M + 1]+. Elemental analysis: calculated for 1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (8g). Yield = 65.9%,
C31H26BrN5O3S3; C = 53.75, H = 3.78, N = 10.11, S = 13.89. Found: m.p. 163–164 1C, Rf = 0.55, toluene: ethyl acetate : formic acid,
C = 53.77, H = 3.81, N = 10.16, S = 13.92.
5 : 4 : 1. IR nmax (KBr): 3362, 3239 & 1541 (N–H), 1595 (CQN), 1321
1
{2-[3-(4-Bromo-phenyl)-5-thiophen-2-yl-4,5-dihydro-pyrazol-1- & 1149 cmꢀ1 (SO2No). H NMR (300 MHz, DMSO, dppm): 3.42
yl]-benzothiazole-6-sulfonyl}N0-benzylurea (8c). Yield = 62.3%, (1H, dd, J = 4.8 Hz, 17.1 Hz), 4.12 (1H, dd, J = 11.7 Hz, 17.6 Hz),
m.p. 207–208 1C, Rf = 0.57, toluene : ethyl acetate : formic acid, 4.66 (2H, s, CH2), 5.89 (1H, dd, J = 4.8 Hz, 11.7 Hz), 6.04 (2H, s,
5 : 4 : 1. IR nmax (KBr): 3367, 3245 & 1541 (N–H), 1591 (CQN), NH), 7.06–8.88 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm):
1
1321 & 1151 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm): 43.56 (C4), 53.72 (CH2), 63.76 (C5), 112.50–128.42 (C aromatic),
ꢀ
3.68 (1H, dd, J = 5.6 Hz, 17.4 Hz), 4.22 (1H, dd, J = 11.8 Hz, 17.6 153.65 (CQN), 157.03 (N1–C benzothiazolyl), 158.95 (CQO).
ꢀ
Hz), 4.67 (2H, s, CH2), 5.87 (1H, dd, J = 5.6 Hz, 12.0 Hz), FAB-MS m/z: 664 [M + 1]+. Elemental analysis: calculated for
6.22 (2H, s, NH), 7.06–8.19 (15H, m, Ar). 13C NMR (125 MHz, C30H23BrFN5O3S2; C = 54.22, H = 3.49, N = 10.54, S = 9.65. Found:
DMSO, dppm): 43.22 (C4), 48.58 (CH2), 63.87 (C5), 100.09–134.27 C = 54.25, H = 3.47, N = 10.61, S = 9.67.
ꢀ
(C aromatic), 153.61 (CQN), 155.85 (N1–C benzothiazolyl),
{2-[3-(4-Bromo-phenyl)-5-(4-fluoro-phenyl)-4,5-dihydro-pyrazol-
ꢀ
158.29 (CQO). FAB-MS m/z: 652 [M + 1]+. Elemental analysis: 1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (8h). Yield = 65.5%,
calculated for C28H22BrN5O3S3; C = 51.53, H = 3.40, N = 10.73, m.p. 157–158 1C, Rf = 0.57, toluene : ethyl acetate : formic acid,
S = 14.74. Found: C = 51.57, H = 3.46, N = 10.75, S = 14.79.
5:4:1. IR nmax (KBr): 3365, 3241 & 1542 (N–H), 1594 (CQN),
{2-[3-(4-Bromo-phenyl)-5-thiophen-2-yl-4,5-dihydro-pyrazol-1- 1318 & 1149 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO, dppm):
yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (8d). Yield = 63.2%, 3.58 (1H, dd, J = 5.6 Hz, 18.0 Hz), 4.22 (1H, dd, J = 5.6 Hz, 17.4 Hz),
m.p. 190–191 1C, Rf = 0.56, toluene : ethyl acetate : formic acid, 4.69 (2H, s, CH2), 5.87 (2H, s, NH), 6.18 (1H, dd, J = 6.4 Hz, 12.0 Hz),
5 : 4 : 1. IR nmax (KBr): 3361, 3239 & 1539 (N–H), 1589 (CQN), 1320 6.98–8.94 (16H, m, Ar). 13C NMR (125 MHz, DMSO, dppm): 43.62 (C4),
1
& 1148 cmꢀ1 (SO2No). H NMR (500 MHz, DMSO, dppm): 3.62 57.79 (C5), 63.49 (CH2), 112.15–132.43 (C aromatic), 154.81 (CQN),
(1H, dd, J = 5.5 Hz, 17.0 Hz), 4.29 (1H, dd, J = 11.5 Hz, 17.0 Hz), 155.40 (N1–C benzothiazolyl), 158.72 (CQS). FAB-MS m/z: 680
ꢀ
4.67 (2H, s, CH2), 6.19 (1H, dd, J = 6.0 Hz, 11.5 Hz), 6.47 (2H, s, [M + 1]+. Elemental analysis: calculated for C30H23BrFN5O2S3;
NH), 6.94–8.37 (15H, m, Ar). 13C NMR (125 MHz, DMSO, dppm): C = 52.94, H = 3.41, N = 10.29, S = 14.13. Found: C = 52.95,
43.67 (C4), 59.79 (C5), 63.47 (CH2), 100.00–137.87 (C aromatic), H = 3.46, N = 10.31, S = 14.15.
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{2-[3-(4-Bromo-phenyl)-5-(4-dimethylamino-phenyl)-4,5-dihydro- Biological experiment
pyrazol-1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (8i). Yield =
62.1%, m.p. 164–165 1C, Rf = 0.56, toluene : ethyl acetate: formic
acid, 5 : 4 : 1. IR nmax (KBr): 3362, 3244 & 1541 (N–H), 1592
(CQN), 1319 & 1151 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO,
Animals
Male rats of Wistar strain16 were obtained from the Central Animal
house at Hamdard University, New Delhi. Animals were housed at
an ambient temperature (25 ꢂ 2 1C) with ad libitum access to water
and food. The experiments were performed in compliance with
Institutional Animals Ethics Committee guidelines, approved by
the Institutional Animal Ethics Committee of the University, Jamia
Hamdard, New Delhi, India (Registration No. 757-CPSCEA).
d
ppm): 2.86 (6H, s, N(CH3)2), 3.61 (1H, dd, J = 5.6 Hz, 17.2 Hz),
4.21 (1H, dd, J = 11.6 Hz, 17.8 Hz), 4.67 (2H, s, CH2), 5.76 (1H, dd,
J = 6.0 Hz, 11.5 Hz), 6.23 (2H, s, NH), 7.03–8.51 (16H, m, Ar).
13C NMR (125 MHz, DMSO, dppm): 43.38 (C4), 54.09 (CH2), 56.24
ꢀ
(N(CH3)2), 63.57 (C5), 112.30–131.62 (C aromatic), 153.42 (CQN),
155.61 (N1–C benzothiazolyl), 164.11 (CQO). FAB-MS m/z: 689
ꢀ
In vivo antidiabetic activity by the OGTT
[M + 1]+. Elemental analysis: calculated for C32H29BrN6O3S2;
C = 55.73, H = 4.24, N = 12.19, S = 9.30. Found: C = 55.76,
H = 4.29, N = 12.21, S = 9.32.
All the synthesized compounds (7a–p and 8a–l) were evaluated for
oral glucose tolerance by the earlier reported method.17 Overnight
fasted animals (130–180 g) were randomly divided into thirty one
groups of five rats. The control group comprising healthy rats was
administered with the vehicle (10% carboxymethylcellulose)
only. Two groups of animals were administered with standard
drugs glibenclamide (30 mg kgꢀ1 b.w.) and rosiglitazone
(36 mg kgꢀ1 b.w.). An oral dose of 30 mg kgꢀ1 b.w. synthesized com-
pounds suspended in 10% CMC was given to the remaining animal
groups. Post 30 minutes of dosing, 3 g kgꢀ1 b.w. glucose solution
was given to all animals. Blood samples were withdrawn from retro-
orbital plexus just prior to glucose load and after 30 and 90 min. The
working solution of each sample was prepared and quantified
according to the manufacturer’s instructions in the GOD-POD kit.18
{2-[3-(4-Bromo-phenyl)-5-(4-dimethylamino-phenyl)-4,5-dihydro-
pyrazol-1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (8j). Yield =
62.4%, m.p. 211–212 1C, Rf = 0.56, toluene : ethyl acetate : formic
acid, 5 : 4 : 1. IR nmax (KBr): 3365, 3241 & 1542 (N–H), 1594
(CQN), 1318 & 1149 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO,
dppm): 2.87 (6H, s, N(CH3)2), 3.61 (1H, dd, J = 5.5 Hz, 18.0 Hz),
4.23 (1H, dd, J = 12.0 Hz, 17.2 Hz), 4.66 (2H, s, CH2), 5.76 (1H,
dd, J = 5.6 Hz, 11.8 Hz), 6.29 (2H, s, NH), 7.01–8.05 (16H, m, Ar).
13C NMR (125 MHz, DMSO, dppm): 43.62 (C4), 54.31 (N(CH3)2),
ꢀ
57.79 (C5), 63.49 (CH2), 112.21–132.43 (C aromatic), 154.81
(CQN), 155.40 (N1–C benzothiazolyl), 158.99 (CQS). FAB-MS
ꢀ
m/z: 705 [M + 1]+. Elemental analysis: calculated for C32H29BrN6O2S3;
C = 54.46, H = 4.14, N = 11.91, S = 13.63. Found: C = 54.50, H = 4.16,
N = 11.94, S = 13.68.
Docking studies
The crystal structure of PPAR-g protein (accession number 3CS8)
was downloaded from the protein data bank.19 The structure of
the PPAR-g receptor contains two identical monomeric chains
(A & B). For the ease of understanding, one of the chains (chain B)
was eliminated while the water molecules in close proximity of
ligands were retained. 3D-structures of proteins and ligands were
prepared using the LigPrep module and energy minimization was
carried out using the OPLS 2005 force field in Schrondinger
software. The chiralities of ligands were maintained during this
step. The minimized reference protein was used to generate a
grid which was used to dock new ligands. Docking studies on the
low energy state of ligands were performed using Glide software
with extra precision. The glide score and ligand properties of all
the ligands were inscribed by XP descriptor information.
{2-[3-(4-Bromo-phenyl)-5-(4-methyl-phenyl)-4,5-dihydro-pyrazol-
1-yl]-benzothiazole-6-sulfonyl}N0-benzylurea (8k). Yield = 61.0%,
m.p. 225–226 1C, Rf = 0.55, toluene : ethyl acetate : formic acid,
5 : 4 : 1. IR nmax (KBr): 3362, 3242 & 1540 (N–H), 1591 (CQN),
1
1319 & 1147 cmꢀ1 (SO2No). H NMR (400 MHz, DMSO, dppm):
2.81 (3H, s, CH3), 3.40 (1H, dd, J = 6.0 Hz, 17.2 Hz), 4.11 (1H, dd,
J = 11.6 Hz, 18.0 Hz), 4.67 (2H, s, CH2), 5.90 (1H, dd, J = 6.0 Hz,
12.4 Hz), 6.19 (2H, s, NH), 7.07–8.43 (16H, m, Ar). 13C NMR
(125 MHz, DMSO, dppm): 21.11 (CH3), 43.56 (C4), 58.16 (CH2),
ꢀ
63.49 (C5), 121.05–138.40 (C aromatic), 155.71 (CQN), 158.45
(N1–C benzothiazolyl), 164.41 (CQO). FAB-MS m/z: 660 [M + 1]+.
ꢀ
Elemental analysis: calculated for C31H26BrN5O3S2; C = 56.36,
H = 3.97, N = 10.60, S = 9.71. Found: C = 56.38, H = 3.99,
N = 10.61, S = 9.76.
{2-[3-(4-Bromo-phenyl)-5-(4-methyl-phenyl)-4,5-dihydro-pyrazol-
1-yl]-benzothiazole-6-sulfonyl}N0-benzylthiourea (8l). Yield =
In vitro PPAR transactivation assay
59.8%, m.p. 221–222 1C, Rf = 0.56, toluene : ethyl acetate : formic Human embryonic kidney (HEK 293) cells were cultured in
acid, 5 : 4 : 1. IR nmax (KBr): 3365, 3240 & 1542 (N–H), 1591 DMEM containing 10% FBS at 37 1C in a 5% CO2 humidified
(CQN), 1321 & 1149 cmꢀ1 (SO2No). 1H NMR (400 MHz, DMSO, atmosphere.20 When 70–80% cell confluency was obtained, cells
dppm): 3.11 (3H, s, CH3), 3.86 (1H, dd, J = 6.4 Hz, 17.6 Hz), 4.62 were inoculated in a 96 well plate containing approximately
(1H, dd, J = 11.5 Hz, 17.2 Hz), 4.71 (2H, s, CH2), 5.86 (1H, dd, 70000 cells per well. Transfection of cells was done with 2.5 mL
J = 5.5 Hz, 12.0 Hz), 6.28 (2H, s, NH), 7.02–8.01 (16H, m, Ar). of PPRE-Luc, 6.67 mL of PPAR-g, 1.0 mL of renilla and 20 mL of
13C NMR (75 MHz, DMSO, dppm): 19.08 (CH3), 43.65 (C4), 47.95 lipofectamine. Test compounds (10 mM) were added to cells for
(C5), 63.21 (CH2), 119.50–137.75 (C aromatic), 156.12 (CQN), 24 h after 5 h of transfection. Cells were collected with the help of
165.81 (N1–C benzothiazolyl), 178.94 (CQS). FAB-MS m/z: 676 lysis buffer and luciferase activity was monitored on the lumino-
ꢀ
[M + 1]+. Elemental analysis: calculated for C31H26BrN5O2S3; meter by using a luciferase assay kit. The manufacturer’s instruc-
C = 55.02, H = 3.87, N = 10.35, S = 14.22. Found: C = 55.05, tions were followed while using the luciferase kit. Glibenclamide
H = 3.91, N = 10.41, S = 14.27.
and rosiglitazone were taken as reference drugs.
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Paper
NJC
In vivo antidiabetic activity on the diabetic model
facilities The authors are thankful to Dr Javed Iqbal, former
Director, Institute of Life Sciences (ILS), Hyderabad, for provid-
ing necessary in vitro facilities and Dr Parimal Misra for his help
in performing in vitro studies. CK is also thankful to University
Grant Commission for providing financial assistance.
A fresh solution of streptozotocin (45 mg kgꢀ1 b.w.) in citrate buffer
(pH 4.5, 0.1 M) was administered intraperitoneally to prepare a
diabetic model. The blood glucose was monitored daily to ensure the
progression of diabetes. Animals having blood glucose levels more
than 250 mg dLꢀ1 were divided into thirty two groups. Apart from
these animals, a group of healthy rats comprised a control group References
and was administered with the vehicle only. The diabetic control
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administered with 30 mg kgꢀ1 b.w. of synthesized compounds. The
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Gene expression
3T3-L1 cells (American Type Culture Collection) were seeded in a 24
well plate using DMEM containing 10% calf serum (Invitrogen). The
mixture was incubated for 24 h to attain 70% confluency. Cells were
treated with 10 mM of compound 7j, 10 mM standards (glibenclamide
and pioglitazone) as positive control and DMSO as negative control
and were again incubated for 24 h at 37 1C and 5% CO2. Cells were
transferred to 1.5 ml micro centrifuge tubes after 24 h and the total
RNA was isolated by TRI Reagents (Molecular Research Centre). The
quantity and quality of RNA were determined on a NanoDrop
ND-2000c spectrophotometer and integrity was verified on a 1.5%
agarose gel. The total RNA (1 mg) so obtained was used to generate
cDNA using an EZ-first strand cDNA synthesis kit for RT (reverse
transcription)-PCR (Biological Industries). Pearl Primer software was
used to design primers of PPAR-g and b-actin for real-time PCR.
Reactions were run at 95 1C for 10 min followed by 40 cycles of 95 1C
for 15 s and 60 1C for 1 min.21 Real-time PCR was performed in
triplicate on an ABI Prism 7300 Sequence Detection System (Applied
Biosystems) using the SYBR Green PCR Master Mix (Applied Bio-
systems). PCR was repeated two times for each gene and each
sample. Relative transcript quantities were calculated using the Ct
method with b-actin as the endogenous reference gene.
Conflict of interest
The authors have no conflict of interest.
21 N. Syed, M. S. Alam, H. Hamid, M. Shahar Yar, S. Shafi,
A. Dhulap, P. Alam, M. A. Q. Pasha, S. Bano, M. M. Alam,
S. Haider, Y. Ali, C. Kharbanda and K. K. Pillai, Eur. J. Med.
Chem., 2014, 87, 175–185.
Acknowledgements
The authors wish to express their thanks to Dr G. N. Qazi, Vice
Chancellor, Jamia Hamdard for providing necessary research
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2016