Synthesis and antihyperglycemic evaluation of new 2-hydrazolyl-4- thiazolidinone-5-carboxylic acids having pyrazolyl pharmacophores

Article abstract of DOI:10.1016/j.bmcl.2014.04.064

In the search of new antihyperglycemic agents and following rational approach of drug designing here new 2-hydrazolyl-4-thiazolidinone-5-carboxylic acids (4a-g) with pyrazolyl pharmacophore have been synthesized via thia Michael addition reaction of 1-((3

Full text of DOI:10.1016/j.bmcl.2014.04.064

Accepted Manuscript
Synthesis and antihyperglycemic evaluation of new 2-hydrazolyl-4-thiazolidi-
none-5-carboxylic acids having pyrazolyl pharmacophores
Manisha R. Bhosle, Jyotirling R. Mali, Savita Pal, Arvind K. Srivastava, Ramrao
A. Mane
PII:
S0960-894X(14)00421-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.064
BMCL 21557
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 November 2013
15 March 2014
17 April 2014
Please cite this article as: Bhosle, M.R., Mali, J.R., Pal, S., Srivastava, A.K., Mane, R.A., Synthesis and
antihyperglycemic evaluation of new 2-hydrazolyl-4-thiazolidinone-5-carboxylic acids having pyrazolyl
pharmacophores, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.
2014.04.064
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Graphical Abstract
Synthesis and antihyperglycemic evaluation of new 2-hydrazolyl-4-
thiazolidinone-5-carboxylic acids having pyrazolyl pharmacophores
Manisha R. Bhoslea, Jyotirling R. Malia, Savita Palb, Arvind K.Srivastavab and Ramrao A. Manea*
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad 431004, India
bDivision of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226031, India
E-mail: manera2011@gmail.com

Synthesis and antihyperglycemic evaluation of new 2-hydrazolyl-4-thiazolidinone-5-
carboxylic acids having pyrazolyl pharmacophores
Manisha R. Bhoslea, Jyotirling R. Malia, Savita Palb, Arvind K.Srivastavab and Ramrao A.
Manea*
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad 431004, India
bDivision of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226031, India
E-mail: manera2011@gmail.com
Abstract
In the search of new antihyperglycemic agents and following rational approach of drug designing
here new 2-hydrazolyl-4-thiazolidinone-5-carboxylic acids (4a–g) with pyrazolyl
pharmacophore have been synthesized via thia Michael addition reaction of 1-((3-(4-substituted
phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiosemicarbazides (3a-g) with malic anhydride.
The required precursors, (3a-g) were obtained by condensing known 3-(4-substitutedphenyl)-1-
phenyl-1H-pyrazole-4-carbaldehydes (1a-g) with thiosemicarbazide in ethanol. The newly
synthesized compounds (4a–g) have been evaluated for the antihyperglycemic activity in sucrose
loaded rat model and among these compounds 4d, 4f and 4g have displayed significant
antihyperglycemic activity.
Keywords: Type 2 Diabetes mellitus, Oral Sucrose Tolerance Test, antihyperglycemic activity,
2-hydrazolyl-4-thiazolidinone-5-carboxylic acids, pyrazole
Type 2 diabetes mellitus (T2DM) is most prevalent and serious disease in the world.1
T2DM is also responsible for morbidity, mortality and overall health problems in the developed
and fast developing countries.2 Continuous quest is found to be directed in search of effective
and safer antihyperglycemic/antidiabetic agents for treating T2DM. It has been observed that
several thiazolidinediones (TZDs) are established as clinical agents for treating and controlling
type 2 DM. The thiazolidinediones containing medicaments presently used are pioglitazone,3 and
rosiglitazone.4 Although these synthetic drugs have shown significant therapeutic potential but
each one of them is accompanying with one or other kind of side effects.5

Literature survey reveals that more attention has been found to synthesize 2,4-TZDs
bearing five/six membered biodynamic heterocyclic moieties for obtaining synergetic
antihyperglycemic activity. The acidic functionality of the TZD ring is considered essential for
its binding to PPARγ.6 Thiazolidine-2,4-dione having carboxylic ester appendage at N-3 has also
been reported to have antihyperglycemic activity.7 Recently the saroglitazar has been launched
as antihyperglycemic agent for type 2 DM having noncyclic ethoxy phenyl propanoic acid as one
of the functionality in its molecular frame work.8
The study of pyrazoles has become of much interest. A systematic investigation of this
class of compounds revealed that pyrazole containing pharmacoactive agents play important role
in medicinal chemistry and displayed anti-inflammatory, antibacterial, antifungal, antiviral,
antitubercular, antioxidant and antiandrogenic activities.9-15 It has been also reported that some of
the pyrazole derivatives are emerging as potential antihyperglycemic agents.16 According to
literature survey, there are no reports on the designing of the molecules having 2,4-
thiazolidinediones-5-carboxylic acid and pyrazole ring system in one molecular framework.
Keeping in view the multifarious applications of pyrazoles, carboxy thiazolidinediones
and in continuation of our earlier interest in designing and synthesizing new analogues of
antidiabetic agents, 2,4-thiazolidinediones17 here new 2-hydrazolyl-4-thiazolidinone-5-
carboxylic acids has been synthesized from readily available reactants with hope to obtain the
titled, 2-hydrazolyl-4-thiazolidinone-5-carboxylic acids with enhanced antihyperglycemic
activity with minimal side effects.
The multistep synthetic route (Scheme 1) for the desired/titled compounds has been
developed starting from 4-formyl pyrazoles. Formyl pyrazoles, 3-(4-substitutedphenyl)-1-
phenyl-1H-pyrazole-4-carbaldehydes (1a-g) were synthesized using reported procedure18 i.e.
carrying Vilsmeier Haack reaction of phenyl/aryl hydrazones with POCl3 and DMF. Phenyl
hydrazones were obtained by condensing substituted acetophenones and phenyl hydrazine in
ethanol. Thus obtained pyrazole aldehydes (1a-g) were then condensed with thiosemicarbazide
(2) in ethanol in presence of desiccant, molecular sieves under reflux for 1h and obtained 1-((3-
(4-substituted phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiosemicarbazides (3a-g) with
good to excellent yields (Table 1). The pyrazolyl thiosemicarbazones (3a-g) when allowed to
react with maleic anhydride in refluxed toluene gave new 2-(4-(1-phenyl-3-p-substituted-

pyrazol-4-yl) methylene-2-hydrazolyl)-1,3-thiazolidin-5-yl-acetic acids (4a-g), by thia Michael
addition reaction (Scheme 1, Table 1).
1
The structures of the synthesized compounds have been confirmed from IR, H NMR,
1
13C NMR and MS spectral data. H NMR spectrum of compound 4a indicates the formation of
the product as it shows a characteristic triplet at δ 4.42 ppm which corresponds to the methine
proton of the 4-thiazolidinone ring. Methylene protons of the propionic acid group give signal as
pair of doublet at δ 2.97-3.34 ppm. A broad singlet was appeared at 12.35 ppm for carboxylic
proton. The presence of four characteristics carbon signals are observed at δ 36.80, 43.67, 171.73
13
and 175.40 ppm in C NMR spectrum of 4a due to the carbon of methylene (CH2-COOH),
methine (-CH-), carbonyl (-NH-C=O) and carbonyl of carboxylic acid (-COOH) groups,
respectively which confirms the presence of 4-thiazolidinone ring with carboxylic acid in the
product. The mass spectrum further provided support to the structure, assigned 2-(4-(1-phenyl-3-
-pyrazol-4-yl) methylene-2-hydrazolyl) -1,3-thiazolidin-5-yl-acetic acid (4a) as displays (M+H)+
ion peak at m/z 420 for the molecular formula C21H17N5O3.
Antihyperglycemic activity evaluation: The compounds (4a-g) were evaluated for their
antihyperglycemic activity in male albino rats of Sprague Dawley strain.19
Procurement and Selection of Animals: Male albino rats of Sprague Dawley strain (8 to
10 weeks of age: body weight range 160 20 g) were procured from National Laboratory
Animal Centre (NLAC) of the Institute. All the experiments on animals were conducted in
accordance to instructions of Institute Animal Ethics Committee.
Improvement on Oral Sucrose Tolerance Test (OSTT) in normal rats: The overnight
fasted rats showing blood glucose level between 60 to 80 mg/dl measured by glucometer using
glucostrips were finally selected, divided into groups. Each consisted of five animals each. Rats
of experimental groups were respectively administered the suspensions of the test samples orally
prepared in 1.0 % gum acacia (vehicle) at pre-selected dose levels i.e. 100 mg/kg. The standard
antidiabetic drug i.e. Metformin was administered at 100 mg/kg dose levels. Animals of control
group were given an equal amount of 1.0 % gum acacia and termed as sham treated control. An
oral sucrose load of 10g/kg body weight was given to each animal exactly after 30 min post
administration of the test sample/vehicle. Blood glucose of each rat was again determined from
the tail vein at 30, 60, 90 and 120 min post administration of sucrose by glucostrips. Quantitative
glucose tolerance of each animal was calculated by Area under curve (AUC) method (Prism

Software). The average fall in AUC of experimental group compared to control group was
always termed as % improvement on oral sucrose tolerance test (OSTT) post sucrose load and
also termed as % antihyperglycemic activity in normal rats. As compounds 4a-g, are
thiazolidinone derivatives. The mode of action thiazolidinediones is to increase the insulin
sensitivity by acting on adipose, muscle, and liver to increase glucose utilization and decrease
21-23
glucose production
as well as metformin also decreases hyperglycemia by suppressing
glucose production by the liver (hepatic gluconeogenesis).24 Therefore, for the study of
antihyperglycemic activity of selected compounds, we used metformin as a positive control.
Statistical Analysis: Analysis of statistical significance of differences in measurements
between samples was done by using Dunnet’s test following ANOVA software. It is denoted by
p values. Statistically Significance difference was set at following levels * represents p< 0.05, **
represents p< 0.01 and *** represents p< 0.001.
Effect of test samples in normal rats: Table 2 and Fig 1 Represents the average blood
glucose profile of compound code 4a-4g on the improvement of oral glucose tolerance post
sucrose load in normal rats. Out of 7 compounds tested, compound 4d, 4f and 4g showed
significant reduction on OSTT to the tune of 7.64% (p<0.05), 7.57(p<0.05) and 7.97% (p<0.05),
While the standard antidiabetic drug, metformin showed improvement on OSTT by 14.1%
(p<0.01) at 100 mg/kg in normal rats. Among these 4a-g compounds 4d, 4f and 4g have
considerably reduce the blood glucose level compare to 4a in normal rats. These compounds are
having additional active pharmacophores like fluro, nitro and hydroxyl on one of the benzonoid
ring that probably be enhancing acidic behavior through conjugation/mesomeric effect of the
2,4-TZD scaffold, which may be responsible for their significant activity.
In conclusion we have synthesized new 2-hydrazolyl-4-thiazolidinone-5-carboxylic
acids with pyrazolyl moiety. The newly synthesized compounds 4a-g has been evaluated for the
antihyperglycemic activity by Oral Sucrose Tolerance Test (OSTT) in normal rat model. Among
these compounds 4d, 4f and 4g have displayed significant reduction on OSTT.
All the chemicals used were of laboratory grade. Melting points of all the synthesized
compounds were determined in open capillary tubes and are uncorrected. 1H NMR and 13C NMR
spectra were recorded with a Bruker Avance 400 spectrometer operating at 300 MHz, 200 MHz
and 75 MHz using DMSO-d6 or CDCl3 solvent and tetramethylsilane (TMS) as the internal

standard and chemical shift in δ ppm. Mass spectra were recorded on a Sciex, Model; API 3000
LCMS/MS Instrument. Elemental analyses were done on EA1108 (Carlo-Erba). The purity of
each compound was checked by TLC using silica-gel, 60F254 aluminum sheets as adsorbent and
visualization was accomplished by iodine/ultraviolet light. Metformin and STZ were purchased from
Sigma Aldrich Co., USA. One touch glucometer and glucostrips was purchased from Roche Diagnostics
India Ltd.
N
N
N
EtOH, mol. sieves
1h, reflux
S
N
NH2
NH2
S
N
HN
N
H
H2N
O
R
(3a-g)
(2)
R
(1a-g)
O
O
Toluene
reflux,
3h
O
O
N
N
HN
S
N
COOH
N
R
(4a-g)
Sche
me 1 Synthesis of 2-(4-(1-phenyl-3-p-substituted-pyrazol-4-yl) methylene-2-hydrazolyl) -1,3-
thiazolidin-5-yl-acetic acids

Table 1: Physical data of 1-((3-(4-substituted phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)
thiosemicarbazides (3a-g) and 2-(4-(1-phenyl-3-p-substituted-pyrazol-4-yl) methylene-2-
hydrazolyl) -1,3-thiazolidin-5-yl-acetic acids (4a-g).
Melting Point
(°C)
Entry
Compound
R
Yield (%)b
1
2
-H
92
86
91
94
87
91
82
85
96
80
82
71
78
80
210-212
3a
3b
3c
3d
3e
3f
-CH3
-OCH3
-F
201-202
162-163
218-220
214-216
310-312
215-217
236-238
230-232
242-243
249-250
200-202
260-262
224-226
3
4
5
-Br
6
-NO2
-OH
-H
7
3g
4a
4b
4c
4d
4e
4f
8
9
-CH3
-OCH3
-F
10
11
12
13
14
-Br
-NO2
-OH
4g
bIsolated Yield

Table 2: Effect of compound code 4a-4g and standard antidiabetic drug metformin on the
improvement of Oral Sucrose Tolerance Test (OSTT) in normal rats.
Compound
Dose (mg/dL)
Significance
% improvement
on OSTT
100
100
100
100
100
100
100
100
Nil
4a
-
p<0.05
p<0.05
p<0.05
p<0.05
p<0.05
p<0.05
p<0.01
6.09
6.97
7.64
5.69
7.57
7.97
14.1
4b
4c
4d
4e
4f
4g
Metformin
AUC0-2h
AUC 0-2 h
20000
10000
0
20000
10000
0
7.57%*
7.97%*
7.64%*
14.1%**
14.1%**

Glucose value 0-2 h
Glucose value 0-2 h
200
150
100
50
200
150
100
50
Control
Control
4d (100 mg/kg)
4 f (100 mg/kg)
Metformin(100 mg/kg)
4 g (100 mg/kg)
0
Metformin(100 mg/kg)
0
0
20
40
60
80
100
120
0
30
60
90
120
Time (min)
Time (min)
Fig:1 Effect of compounds code 4d, 4f, 4g and standard antidiabetic drug metformin on the
improvement of Oral Sucrose Tolerance Test (OSTT) in normal rats.
Acknowledgments
The authors are thankful to Professor D. B. Ingle for his invaluable discussions and
guidance. One of the authors, Manisha R. Bhosle, is thankful to Dr. Babasaheb Ambedkar
Marathwada University authorities for awarding Jahagirdar Research Fellowship.
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G.; Jiaping, D. J. Med. Chem. 2001, 44, 2601. (b) Hernández-Vázquez, E; Aguayo-Ortiz,
R. Ramírez-Espinosa, J.J.; Estrada-Soto, S.; Hernández-Luis, F. Eur. J. Med. Chem.
2013, 69, 10.(c) Guzman-Perez, A.; Pfefferkorn, J. A.; Lee, E. C.Y.; Stevens, B. D.;
Aspnes, G. E.; Bian, J.; Didiuk, M. T.; Filipski, K. J.; Moore, D.; Perreault, C.;
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20. Synthesis of 1-((3-(4-phenyl)-1-phenyl-1H-pyrazol-4-yl) methylene) thiosemicarbazide
(3a) A mixture of 3-(4-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (1a) (10 mmol)
and thiosemicarbazide (2) (10 mmol) was refluxed in ethanol in the presence of activated
molecular sieves of size 4A0 (10 g). After 1h of reflux the reaction mixture was filtered to
remove molecular sieves. Ethanol was removed under vacuum. The residual mass was
poured on crushed ice. The solid obtained was filtered and washed with water. The crude
solid was purified by crystallization. Similarly the other compounds, (3b-g) of the series
were prepared. The melting points and the yields of the derivatives are recorded in Table
1 1-((3-(4-phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiosemicarbazide (3a). IR
(KBr, ν cm-1): 3410, 3100, 1597, 1590, 1334 and 1250. 1H NMR (200 MHz, DMSO-d6, δ
ppm): 7.55 (s, 1H, -CH=N-), 8.01 (s, 1H, pyrazoline-H), 8.29-8.77 (m, 10H, Ar-H), 9.10
(s, 1H, NH) 9.36 (s, 1H, NH, tautomeric) and 11.73 (s, 1H, SH, enolic). MS (ESI+, m/z):
322 (M+).
Synthesis of 2-(4-(1-phenyl-3--pyrazol-4-yl) methylene-2-hydrazolyl) -1,3-thiazolidin-5-
yl-acetic acid (4a): 1-((3-(4-substituted phenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)
thiosemicarbazide (3a) (3 mmol) and maleic anhydride (4 mmol) were refluxed in
toluene. Thia Michael addition was monitored by thin layer chromatography. After 3h of
reflux the reaction mixture was cooled. Excess toluene was removed on rota evaporator.
The residual mass was poured on crushed ice and extracted with ethyl acetate. From
extract organic medium was removed under reduced pressure. The residual solid obtained
was dried and purified by crystallization. Similarly the other compounds, (4b-g) of the
series were prepared. The melting points and the yields of the derivatives are recorded in
Table 1
Spectral analysis: 2-(4-(1,3-dipheny--pyrazol-4-yl) methylene-2-hydrazolyl) -1,3-
thiazolidin-5-yl-acetic acid (4a): IR (KBr, ν cm-1): 3433, 3271, 2924, 1643, 1720, 1597
1
and 1215. H NMR (300 MHz, DMSO-d6, δ ppm): 2.97-3.34 (dd, overlapped, 2H,
methylene), 4.42 (t, 1H, methine), 7.23-8.02 (m, 10H, Ar-H), 8.42 (s, 1H), 8.94 (s, 1H),
13
11.31 (s, 1H, NH) and 12.35 (bs, 1H, -COOH). C NMR (75 MHz, DMSO-d6, δ ppm):
36.80, 43.67, 116.68, 118.85, 127.06, 128.49 (2C), 128.59 (2C), 128.84 (2C), 129.48

(2C), 129.59, 132.00, 138.91, 149.09, 151.64, 171.73 and 175.40. MS (ESI+ , m/z): 420
(M+). Elemental Analysis: Anal. Calcd for C21H17N5O3S: C, 60.13; H, 4.09; N, 16.70; S,
7.64 found C, 60.33; H, 4.64; N, 16.57 and S, 7.61.
2-(4-(1-phenyl-3-4-methylphenyl-pyrazol-4-yl) methylene-2-hydrazolyl) -1,3-thiazolidin-
5-yl-acetic acid (4b): IR (KBr, ν cm-1): 3446, 3266, 2937, 1635, 1726, 1599 and 1214. 1H
NMR (300 MHz, DMSO-d6, δ ppm): 2.38 (s, 3H, CH3), 2.85-3.01 (dd, overlapped, 2H,
methylene), 4.37 (t, 1H, methine), 7.30-8.00 (m, 9H, Ar-H), 8.41 (s, 1H), 8.93 (s, 1H),
12.05 (bs, 1H, NH) and 12.40 (bs, 1H, -COOH). 13C NMR (75 MHz, DMSO-d6, δ ppm):
20.93, 36.85, 43.70, 116.60, 118.84, 127.01, 128.47 (2C), 129.13 (2C), 129.60 (2C),
130.83, 138.01, 138.95, 149.19, 151.71, 162.73, 166.75, 171.77 and 175.44. MS (ESI+,
m/z):434 (M+). Elemental Analysis: Anal. Calcd for C22H19N5O3S: C, 60.96; H, 4.42; N,
16.16; S, 7.40 found C, 61.00; H, 4.48; N, 16.20 and S, 7.35.
2-(4-(1-phenyl-3-4-methoxyphenyl-pyrazol-4-yl)
methylene-2-hydrazolyl)
-1,3-
thiazolidin-5-yl-acetic acid (4c): IR (KBr, ν cm-1): 3440, 3260, 2930, 1640, 1725, 1597
1
and 1218. H NMR (300 MHz, DMSO-d6, δ ppm): 3.83 (s, 3H, OCH3), 2.73-3.09 (dd,
overlapped, 2H, methylene), 4.39 (t, 1H, methine), 7.05-7.07 (d, 2H), 7.14-7.99 (m, 7H,
13
Ar-H), 8.42 (s, 1H), 8.92 (s, 1H), 12.30 (bs, 1H, NH) and 12.35 (bs, 1H, -COOH). C
NMR (75 MHz, DMSO-d6, δ ppm): 36.88, 43.74, 55.20, 113.92, 114.29, 116.44, 118.77
(2C), 124.41 (2C), 126.95 (2C), 139.61, 130.00, 138.96, 149.34, 151.48, 159.58, 162.67,
171.83 and 175.46. MS (ESI+, m/z ): 450 (M+). Elemental Analysis: Anal. Calcd for
C22H19N5O4S: C, 58.79; H, 4.26; N, 15.58; S, 7.13 found C, 58.60; H, 4.30; N, 15.52 and
S, 7.20.
2-(4-(1-phenyl-3-4-flurophenyl-pyrazol-4-yl) methylene-2-hydrazolyl) -1,3-thiazolidin-5-
1
yl-acetic acid (4d): IR (KBr, ν cm-1): 3438, 3258, 2935, 1649, 1729, 1592 and 1214. H
NMR (300 MHz, DMSO-d6, δ ppm): 2.86-3.11 (dd, overlapped, 2H, methylene), 4.38 (t,
1H, methine), 7.23-8.20 (m, 10H, Ar-H), 8.94 (s, 1H), 11.31 (s, 1H, NH) and 12.31 (bs,
1H, -COOH). 13C NMR (75 MHz, DMSO-d6, δ ppm): 36.75, 43.70, 114.23, 115.19,
115.40, 117.17 (2C), 127.09 (2C), 128.58 (2C), 129.61, 130.80, 130.88, 138.85, 150.44,
162.84, 163.49, 171.80 and 175.41. MS (ESI+, m/z):438 (M+). Elemental Analysis: Anal.
Calcd for C21H16FN5O3S: C, 57.66; H, 3.69; 4.34; N, 16.01; S, 7.33 found C, 57.60; H,
4.00; N, 16.08 and S, 7.29.

2-(4-(1-phenyl-3-4-nitrophenyl-pyrazol-4-yl) methylene-2-hydrazolyl) -1,3-thiazolidin-5-
1
yl-acetic acid (4f): IR (KBr, ν cm-1): 3432, 3260, 2939, 1641, 1730, 1593 and 1220. H
NMR (300 MHz, DMSO-d6, δ ppm): 2.91-3.13 (dd, overlapped, 2H, methylene), 4.39 (t,
1H, methine), 7.36-8.32 (m, 10H, Ar-H), 8.46 (s, 1H), 11.35 (s, 1H, NH) and 12.33 (bs,
1H, -COOH). 13C NMR (75 MHz, DMSO-d6, δ ppm): 36.73, 43.77, 117.46, 117.98,
118.67, 118.92 (2C), 123.86 (2C), 127.39 (2C), 128.98, 129.67, 131.37, 134.33, 138.79,
148.86, 163.22, 171.75 and 175.40. MS (ESI+, m/z):465 (M+). Elemental Analysis: Anal.
Calcd for C21H16N6O5S: C, 54.31; H, 3.47; N, 18.09; S, 6.90 found C, 54.60; H, 3.40; N,
18.04 and S, 6.89.
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