Design and discovery of 2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-phenyl)thiazole derivatives as cardiotonic agents via inhibition of PDE3

Article abstract of DOI:10.1016/j.bmc.2015.08.002

A series of novel 2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-phenyl)thiazole derivatives, 6(a-o) were designed, synthesized and evaluated for inhibitory activity against human PDE3A and PDE3B. In PDE3 assay, entire set of targeted analogs showed consid

Full text of DOI:10.1016/j.bmc.2015.08.002

Accepted Manuscript
Design and discovery of 2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-phe-
nyl)thiazole derivatives as cardiotonic agents via inhibition of PDE3
Li-Min Duan, Hong-Ying Yu, Yan-Long Li, Chun-Juan Jia
PII:
S0968-0896(15)00669-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.08.002
BMC 12506
Reference:
Bioorganic & Medicinal Chemistry
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1 August 2015
4 August 2015
Please cite this article as: Duan, L-M., Yu, H-Y., Li, Y-L., Jia, C-J., Design and discovery of 2-(4-(1H-tetrazol-5-
yl)-1H-pyrazol-1-yl)-4-(4-phenyl)thiazole derivatives as cardiotonic agents via inhibition of PDE3, Bioorganic &
Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.08.002
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Bioorganic & Medicinal Chemistry
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Design and discovery of 2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-
phenyl)thiazole derivatives as cardiotonic agents via inhibition of PDE3
Li-Min Duana#, Hong-Ying Yub#*, Yan-Long Lic, Chun-Juan Jiac
a Department of Geriatric, DaQing Oil Field General Hospital, Da Qing, Heilongjiang 16300, China
b Department of Cardiology, DaQing Oil Field General Hospital, Da Qing, Heilongjiang, 163001, China
c Department of Internal Medicine, The People’s Hospital of Shouguang, Shouguan, 262700, China
#Contributed equally to this work
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of novel 2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-phenyl)thiazole derivatives, 6(a-
o) were designed, synthesized and evaluated for inhibitory activity against human PDE3A and
PDE3B. In PDE3 assay, entire set of targeted analogues showed considerable inhibition of
PDE3A (IC50 = 0.24 0.06 - 16.42 0.14 µM) over PDE3B (IC50 = 2.34 0.13 - 28.02 0.03
µM). Among the synthesized derivatives, compound 6d exhibited most potent inhibition of
PDE3A with IC50 = 0.24 0.06 µM than PDE3B (IC50 = 2.34 0.13 µM). This compound was
further subjected for evaluation of cardiotonic activity (contractile and chronotropic effects) in
comparison with Vesnarinone. Results showed that, it selectively modulates the force of
contraction (63% 5) rather than frequency rate (23% 2) at 100 µM. Docking study of above
compound was also carried out in the active site of PDE3 protein model to give proof to the
mechanism of action of designed inhibitor. Further, in sub-acute toxicity experiment in Swiss-
albino mice, it was found to be non-toxic up to 100 mg/kg dose for 28 days.
Available online
Keywords:
Synthesis,
cardiotonic,
PDE3,
docking,
toxicity study
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Cardiovascular diseases (CVDs) are considered as the
foremost reason of death globally more than any other causes.1
According to the recent estimates of WHO, 17.5 million people
died from CVDs in 2012, representing 31% of all global deaths.
Of these deaths, an estimated 7.4 million were due to coronary
heart disease and 6.7 million were due to stroke.2,3,4 Alone in
United States, about 5.1 million have congestive heart failure
(CHF) which responsible for one in nine deaths in 2009. As a
matter of fact, despite of advances in treatment, roughly half of
the people who develop heart failure die within 5 years of
diagnosis. It was also associated with economic burden which
costs approximately $32 billion each year in US, including cost
of health care services, medications to treat heart failure, and
missed days of work.5,6 Concerning to the sternness of the heart
failures, novel and effective medication are need urgently.
For past few decades, glycosides obtained from digitalis have
been used for the treatment of CHF. However, the clinical utility
of these agents are seriously jeopardized due to their propensity
to cause high life-threatening arrhythmias along with a low
therapeutic index.7,8 These side effects could be overcome by
using a novel class of ‘non-glycoside’ cardiotonic agents which
Figure 1 Pharmacological significance of PDE3 Inhibitors.
safety profile and improved efficacy. The PDE enzymes are
responsible for regulating the cellular levels of cyclic adenosine
monophosphate (cAMP) and cyclic guanosine monophosphate
(cGMP) by catalyzing the hydrolysis of these second messengers,
fig 1.9 Based on the substrate specificity, kinetic properties, and
acts
by
selective inhibition
of
cyclic
nucleotide
phosphodiesterase (PDE) enzymes. These inhibitors offer better

sensitivity to specific inhibitors, tissue distribution and sequence-
derived information, 11 PDE families have been recognized in
mammalian tissues. It includes PDE1, PDE2 to PDE11. Various
PDEs, including PDE-3, are responsible for inducing vascular
relaxation in different vascular beds when they are inhibited.
Thus, inhibition of PDEs by specific inhibitors in cardiovascular
tissues enhances cytosolic levels of cAMP which in turn causes a
reduction in platelet aggregation, smooth muscle cell
proliferation in vitro, and induction of a cardiotonic effect. PDE3
was further sub-divided into two sub-types, viz., PDE3A and
PDE3B, where, PDE3A is found in cardiac tissue, platelets and
vascular smooth muscles, while PDE3B is prominently expressed
in hepatocytes and adipose tissue. Therefore, selective inhibition
of PDE3A offers acute inotropic and vasodilatory effects towards
the treatment of CHFs which warrant the discovery of new
medication to this armamentarium of drugs.10,11
ppm, whereas resonance of pyrazole protons found as a singlet in
the region 8.93-8.89 ppm along with another singlet at 8.40-8.42
ppm for proton of thiazole ring. Finally all the structure of title
compounds was established by mass and elemental analysis.
HCl
S
O
O
S
S
HMTA
N
N
N
N
R
N
N
R
O
O
N
EtOH
79%
R
N
H
NH2
CF3COOH
90%
4
CHO
3
1h, Δ
2
12h, Δ
1
DMF
Where R
a: 4-Cl
b: 3-Cl
c: 2-Cl
d: 4-F
=
NaI
NH2OH
6h, Δ
h: 3-NO2
i: 2-NO2
j: 4-CH3
k: 3-CH3
l: 2-CH3
S
N
N
NaN3
S
N
R
N
N
N
R
DMF, 99%
Δ
N
e: 3-F
N
6 (a-o)
N
H
CN
N
5
f: 2-F
m: 4-OCH3
n: 3-OCH3
g: 4-NO2
o: 2-OCH3
Scheme 1 Synthetic Route for the Preparation of 2-(4-(1H-
tetrazol-5-yl)-1H-pyrazol-1-yl)-4-substituted phenylthiazole 6(a-
o).
Encouraged by the excellent phosphodiesterase activity of
LQFM 021 (fig. 2), a novel pyrazole derivative,11 present study
deals with the synthesis of novel analogues of pyrazole tethered
to other heterocyclic scaffolds i.e.tetrazole-pyrazole-thiazole
conjugates. These constitutive compounds were evaluated for
PDE3 inhibitory and cardiotonic activity. Docking was also
carried out to clarify the interaction of compounds with PDE3.
2.1.2. Pharmacological Activity
The synthesized target compounds were assessed for their
PDE3 (types A and B) inhibitory activities in comparison with
Vesnarinone and the results were shown in Table 1.
Table 1: Enzyme inhibitory assessment data of compounds 5
(a-o) and Vesnarinone.
S
N
N
IC50 (in µM)
Compound
N
N
N
N
N
PDE3A
PDE3B
N
N
N
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
2.45 0.13
2.99 0.05
3.62 0.08
0.24 0.06
1.45 0.12
1.89 0.06
6.23 0.04
6.89 0.05
7.43 0.11
8.15 0.07
9.12 0.08
9.89 0.06
13.44 0.02
14.63 0.03
16.42 0.14
11.21 0.05
3.83 0.06
5.01 0.02
5.92 0.05
2.34 0.13
4.65 0.04
4.72 0.11
7.27 0.07
8.54 0.02
10.21 0.05
16.72 0.03
14.49 0.15
16.62 0.01
25.62 0.07
25.11 0.05
28.02 0.03
14.54 0.03
N
F
N
H
N
H
F
B
A
Figure 2 Figure 2 Structure of LQFM-021 (A) and designed
inhibitor (B).
2.1. Results and discussion
2.1.1. Chemistry
The synthesis of title analogues was depicted in Scheme 1.
Initially, the generation of target analogues begins with the
synthesis of compound 3 in accordance with the method
Vesnarinone
12
described by Finar and Godfrey.
Compound 3a was then
further subjected to Duff’s Condition, where it selectively and
regiospecificaly formylated to afford compound 4.13,14,15 The
synthesis of compound 5 was accomplished via the formation of
the oxime of compound 4 in the presence of hydroxylamine. This
was followed by in situ dehydration in the presence of sodium
iodide and DMF at reflux temperature for 8 h to furnish 5. 16 The
last step corresponds to the synthesis of compound (6) via 1,3-
bipolar cycloaddition between compound 5 and sodium azide
using ammonium chloride as the catalyst in DMF at reflux
temperature for the period determined through TLC.
Among the synthesized compounds, 6d was disclosed as the most
potent inhibitor for both PDE3A (0.24 0.06 µM) and PDE3B
(2.34 0.13µM). While changing the pattern of the substitution
of fluoro group leads to prominently decline in activity against
PDE3B and less against PDE3A. Against PDE3B, the
introduction of chloro group at the para position (6a), showed
considerable decline in potency and this was followed by other
analogues having isomeric replacements. While against the
PDE3A, these compounds showed marginally improved IC50
values. Furthermore, marked decline in inhibitory activity was
reported by compounds 6g, 6h and 6i having isomeric
substitution of nitro group, where they significantly inhibit
PDE3A than PDE3B. It is noteworthy to mention that,
introduction of electron donating substituent, renders compounds
least active and even some cases found less potent that
Vesnarinone as standard. For instance, compound 6j, 6k and 6l,
against PDE3A, showed IC50 values ranging from (8.15 0.07 -
The FT-IR absorption bands of title compounds as presented
in the experimental section showed a strong band at 3452-3498
cm-1 which is attributed to primary N-H stretching frequency.
The presence of C=N group was confirmed at 1628-1678 cm-1.
Moreover, the occurrence of strong band at 2956-2982 cm-1
showed the stretching frequencies of Ar C-H group, whereas the
presence of Cl on phenyl ring was observed at 1092-887 cm-1.
The C-N group was observed at 1208-1268 cm-1. Title
compounds showed another band at 681-693 cm-1 corresponding
9.89
0.06 µM). The same inhibition pattern for these
compounds was observed against PDE3B, but with less potency,
except compound 6k, which found almost equipotent to
Vesnarinone. Rest of the compounds, viz., 6m (13.44 0.02µM),
1
to C-S-C stretching vibration. The H-NMR spectra showed the
resonance of Ar-H in the range of 8.01 to 7.45 ppm. The presence
of protons of N-H groups is confirmed via singlet at 3.59-3.58

6n (14.63 0.03 µM) and 6o (16.42 0.14 µM) showed reduced
activity in comparison to standard (11.21 0.05 µM). In PDE3B
inhibition assay, these compounds found almost ten-to-fourteen
folds less potent then standard. From the results, it was indicated
that, the inhibition potency of the compounds will follow a
comprehensible structural pattern. Results showed that,
compounds containing electron withdrawing substituent found
more potent than electron donating and para is the most
favourable position of substituent than ortho and meta. Thus, the
structural-activity relationships of these inhibitors have been
shown in Fig 3.
was conducted to define the orientation and interaction of 6d with
PDE3. For this study, catalytic domain of human
phosphodiesterase 3b in complex with a dihydropyridazine
inhibitor (1SO2) was downloaded from protein data bank and
heteroatoms including the water molecules were cleaned (clean
geometry) using Discovery Studio. It was selected due to
reasonable homology between PDE3A and PDE3B, where their
identities and positives were obtained as 48% and 67%,
17, 18, 19
respectively, extracted from NCBI-BLAST.
This
homology was further increased to approximately 95% within
20, 21
15Å region in and around active site.
The ligand 6d was
docked in the active site defined over the co-crystallised
dihydropyridazine inhibitor with the help of Binding-Site module
within the Discovery studio 2.5.
On the whole, CDOCKER is a grid-based molecular docking
method that employs CHARMM force fields. This protein was
firstly held rigid while the ligand was allowed to freely rotate
during the refinement. Two hundred random ligand
conformations were then generated from the initial ligand
structure through high temperature molecular dynamics, followed
by random rotations; refinement by grid-based (GRID 1)
simulated annealing, and a final grid-based or full force field
minimisation. In this experiment, the ligand was heated to a
temperature of 700 K in 2000 steps. The cooling steps were set to
5000 steps with 300 K cooling temperature. The grid extension
was set to 10 Å. Hydrogen atoms were added to the structure and
all ionisable residues were set at their default protonation state at
a neutral pH. For ligand 6d, ten ligand binding poses were ranked
according to their CDOCKER energies, and the predicted
binding interactions were analysed. Furthermore, best among the
ten ligand binding pose was chosen and carried out in–situ ligand
minimization using standard protocol.
Figure 3 SAR of the compounds 6 (a-o)
Due to over expression of PDE3A in cardiac tissues than
PDE3B, compound 6d which had showed the best PDE3A
inhibitory activity was tested for its effect on the force of
contraction and frequency rate. For this experiment,
spontaneously beating atria of rat was taken in comparison with
vesnarinone. To avoid the interference of catecholamine release,
reserpine-pretreated animals were used in the study. Results
showed that, it increases the cardiac contractility more efficiently
(63%
5) than Vesnarionone (50%
4) at 100 µM. After
washing of the myocardial preparations, the atria of the subject
have regained the contractility to the pre-drug state. This
behaviour suggests that, the action of compound 6d is reversible
and original state could be achieved after removing the drug. On
the other hand, it does not considerably enhance the force of the
contraction (23% 2) in comparison to Standard (21% 7). This
enables us to understand that, it act as ionotropic agent without
causing significant arrhythmias, Table 2.
Table 2: Effects of the compound 6d on force of contractility
and frequency rate.#
Parameter
Compound
Base
100
1 ×10- 1 × 10-
1
×
6
5
10-4
Contractile 6d
force
4
2
3
5
110
6
129 3 163
5
Vesnarinone 100
6d 100
Vesnarinone 100
105
4
125
3
150
4
123
2
121
7
Frequency
Rate
108
4
114
4
105
3
112
2
#
The effect of each concentration (mol L-1) of a compound was defined
by the difference between the contraction and frequency before and after its
addition to the bathing fluid, and was expressed as a percent variation in
contraction and frequency in respect to the controls. Results are means
SEM from six artria.
With in vitro results in hand, docking study of most active
compound 6d was carried out in the active site of PDE3 protein
model to give proof to the mechanism of action of designed
inhibitor. Docking was conducted with the CDOCKER program
in Discovery Studio 2.5 (Accelrys, San Diego, USA). This study
Figure 4 Three dimensional schematic representation of the
ligand 6d (most active) and 6o (least active) with that of human
phosphodiesterase 3b.

Results revealed that, 6d was efficiently buried and
engulfed in the active site of PDE3 by making close interatomic
contacts. It was found that tetrazole-pyrazole portion of
compound 6d was surrounded by His731, His737, His825 and
Asp822, fig. 4. The formation of one hydrogen bond with
Asp822 with tetrazole ring of the ligand was also revealed which
depicts its better affinity against the target protein. It was also
revealed that, channelling of thiazole and its neighbouring phenyl
fragment deep inside the pocket lined with residues like Tyr737,
Phe559 and Phe991 would contribute to enhanced affinity and
better stabilisation of compound 6d within the active site. Inside
the deep cleft of the active site, compound 6d showed the
formation of two non-covalent pi-pi stacking interaction between
engulfed aromatic residues of the ligand with Phe991, a key
catalytic amino acid residue of PDE3. On the contrary, the
orientation of the ligand was found different in the case of least
active compound 6o. As shown in figure 4, it showed the
formation of two pi-pi interaction with Phe991 along with one H-
bond with Thr952. However the tetrazole ring was found oriented
in deep cleft of the active site in opposite manner to that of
compound 6d. On comparing both ligand (6d and 6o), the
engulfment of phenyl thiazole in deeper cleft lined by Gly940,
Pro941, His948, Gln988 and Thr952 serve as vital factor for the
generation of activity, while distortion from this orientation
render compound non active, i.e., compound 6o.
parameters in Swiss albino mice (Mean SD; n = 6; and P < 0.05
compared to control, 0.1, 1, 10 and 100 mg/kg).
Dose in mg/kg body weight once orally for 28 days
Parameter
Control
0.1
1
10
100
31.2
1.6
29.28
28.04
1.7
32.62
3.2
33.0
Body Weight
2.1
4.5
RBC
(x
(x
7.13
0.8
7.47
1.3
7.83
0.8
7.74
0.5
7.2 1.1
6.1 1.3
10/mm2)
WBC
10/mm2)
6.91
2.6
5.41
0.7
5.68
3.1
6.9 0.2
Haemoglobin
(g/dL)
13.87
0.46
14.5
0.2
15.34
0.45
15.72
0.01
15.24
1.34
Cholesterol
(mg/dL)
138.43
1.52
125.66
1.30
129.03
2.56
135.4
2.04
134.3
0.34
Triglycerides
(mg/dL)
263.69
3.38
254.1
4.61
247.26
2.89
251.86
2.15
255.4
0.22
Total Protein 3.45
3.96
1.12
3.64
0.11
4.12
0.45
4.54
0.36
(mg/dL)
0.45
Creatinine
(mg/dL)
0.04
0.37
0.04
0.07
0.04
0.02
0.04
0.45
0.05
3.5
The docked interaction of LQFM-021 was found
similar to the docked complex of most promising inhibitor 6d, we
found that both ligand shares a common interaction with Phe991,
a key catalytic residue. Moreover, the aromatic portion of the
molecules was found protruded towards the inner cleft lined by
His737, His741, His948, Thr952, Gln988, Gly940, Pro941 and
Thr952.
132.4
6.26
135.5
3.4
136.6
0.5
133.3
8.9
134.02
4.6
ALP (U/L)
2.78
1.82
2.89
0.01
2.61
0.03
2.57
0.05
2.59
2.3
Albumin (g/dL)
SGPT (U/L)
SGOT (U/L)
41.46
17.25
40.12
13.34
41.34
0.03
41.8
2.7
42.1
4.3
Earlier studies suggests that, similar interaction was
observed in the case of other various PDE3A inhibitors,
including Vesnarinone, which suggests that, our designed
molecules could be act in similar fashion.
112.7
10.6
107.8
6.78
109.8
8.45
110.2
12.67
111.6
12.31
To confirm the potential application of compound 6d as
viable lead for drug development, it should posses no toxicity
along with potent bioactivity. After establishing pharmacological
activity of compound 6d, it’s worthwhile to perform its toxicity
evaluation. Thus, in acute toxicity study, it has been found to be
safe upto 1000 mg/kg as a single acute oral dose. Hence, sub
acute experiment was conducted in accordance with the
Organization for Economic Co-operation and Development
(OECD) test guideline No 423, and the same has been illustrated
in experimental section.
3. Conclusions
In conclusion, we have efficiently synthesized 2-(4-(1H-
tetrazol-5-yl)-1H-pyrazol-1-yl)-4-sustituted phenylthiazole 6(a-o)
as cardiotonic agents in good yields. It has been found that,
minor structural variation render compound active. SAR studies
suggest that, compounds containing electron withdrawing
substituent found more potent than electron donating and among
them para is the most favourable position of substituent than
ortho and meta. Together with selective and potent inhibition of
PDE3A along with efficient inotropic action, compound 6d could
be act as a lead for the future drug discovery against CHF.
There were no observational changes; morbidity and mortality
found in experimental animals throughout the period up to 28
days at the dose level of 100 mg/kg body weight, once orally.
The blood and serum samples upon analysis showed no major
changes in total haemoglobin level, RBC count, WBC count,
SGOT, creatinine, triglycerides, cholesterol, albumin (Table 3).
On the basis of gross pathological study of animals, no changes
in any of the organs studied including their absolute and relative
weight were found. Hence, the experiment showed that
compound 6d is well tolerated by the Swiss albino mice up to the
dose level of 100 mg/kg body weight once orally for 28 days in
sub-acute toxicity study.
4. Experimental
4.1. Animals, drugs, chemicals and instruments
Adult male Wistar rats (250–350 g), were kept in controlled
environmental conditions (temperature: 23
2 °C; light–dark
cycle: 7 am to 7 pm) and had a free access to a standard
laboratory diet and water. In order to obtain atrial preparation,
depleted in endogenous catecholamine, the animals were treated
intraperitoneally with reserpine (5 mg/kg b.wt) 24 h before
euthanasia.10 In each experiment, the animals were anaesthetized
with intraperitoneal injection of thiopental 80 mg/kg, and after
midline thoracotomy, the heart was rapidly excised and placed in
Table 3: Effect of compound 6d at 0.1, 1, 10 and 100 mg/kg
body weight once orally for 28 days in sub acute toxicity study
on body weight, heamtological and serum biochemical
a
dissection dish filled with oxygenated Krebs–Henseleit

solution. The enzymes were obtained from BPS BioScience Inc.
All chemicals purchased from Merck and Fluka Chemical Co.
The experiments were performed in compliance with Institutional
Animal Ethical Committee of DaQing Oil Field General
Hospital, China.
CDCl3, TMS) δ ppm: 8.95 (d, 1H J =1.84 Hz, pyrazol), 8.45 (s,
1H thiazole), 8.19 (d, 1H J =1.71 Hz, pyrazol), 8.01–7.98 (m,
2H,Ar-H), 7.45-747 (m, 2H,Ar-H), 3.51 (br–s, 1H, tetrazol, NH);
13C-NMR (100MHz, CDCl3) δ,ppm: 163.4, 157.5, 149.8, 137.6,
134.9, 134.4, 129.6, 129.4, 129.2, 128.8, 125.7, 108.5, 102.3;
Mass : 330.79 (M+1); Elemental analysis for C13H8ClN7S:
Calculated: C, 47.35; H, 2.45; N, 29.73; Found: C, 47.34; H,
2.48; N, 29.75.
All chemicals were of analytical grade and used directly. Melting
points were determined in open capillary tubes with
electrothermal melting point apparatus (MP-1) and are
uncorrected. The completion of reaction was checked by thin
layer chromatography (TLC) using silica gel-G coated Al-plates
(0.5 mm thickness, Merck; Solvent system: benzene:ethyl
acetate) and the plates were illuminated under UV (254 nm) and
evaluated in iodine vapor. The solubility of all the compounds
were tested by using water, chloroform, ethanol, DMF, DMSO,
benzene, acetic acid, ethyl acetate, and dilute acids. FT-IR (in 2.0
cm-1, flat, smooth, abex, KBr) spectra were recorded on Perkin
Elmer-Spectrum RX-I spectrometer. Elemental analysis was
4.2.1.3.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(2-
chlorophenyl)thiazole. (6c)
White yellow-crystals; Yield: 78%; M.p: 216-218 °C; MW:
329.77; Rf: 0.62; FT-IR (νmax; cm-1 KBr): 3462 (N-H stretch), 3047 ,
2961 (C-H stretch), 1649 (C=N stretch), 1215 (C-N stretch), 1185 (C-S),
1
872 (aromatic-Cl), 756, 685 (C-S-C) cm–1; H-NMR (400MHz,
CDCl3, TMS) δ ppm: 8.89 (d, 1H J =1.79 Hz, pyrazol), 8.42 (s,
1H thiazole), 8.23 (d, 1H J =1.75 Hz, pyrazol), 7.73–7.62 (m,
2H,Ar-H), 7.39-736 (m, 2H,Ar-H), 3.58 (br–s, 1H, tetrazol, NH);
13C-NMR (100MHz, CDCl3) δ,ppm: 163.6, 158.4, 152.3, 137.6,
132.5, 132.2, 130.9, 130.2, 129.6, 129.2, 128.9, 108.6, 102.3;
Mass : 330.79 (M+1); Elemental analysis for C13H8ClN7S:
Calculated: C, 47.35; H, 2.45; N, 29.73; Found: C, 47.38; H,
2.43; N, 29.78.
1
carried out on a Vario EL III CHNOS elementor analyzer. H
NMR spectra were recorded on a Bruker Avance II 400 NMR
Spectrometer and the 13C NMR spectra on a Bruker Avance II
100 NMR spectrometer in DMSO-d6 using TMS as the internal
standard. The chemical shifts are reported in parts per million
(ppm, d), and signals are described as s (singlet), d (doublet), t
(triplet), q (quartet), and m (multiplet). Mass spectra were
obtained on VG-AUTOSPEC spectrometer. The developed
tension was recorded isometrically by means of a high-sensitivity
force transducer (type E. Zimmermann, Eipzig-Berlin) connected
to powerlab 8/30 (model 870). Solvents were purified prior to use
according to standard procedure.
4.2.1.4.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-
fluorophenyl)thiazole. (6d)
Brown-crystals; Yield: 62%; M.p: 198-201 °C; MW: 313.31;
Rf: 0.58; FT-IR (νmax; cm-1 KBr): 3472 (N-H stretch), 3058 , 2968
(C-H stretch), 1642 (C=N stretch), 1225 (C-N stretch), 1187 (C-S), 863
(aromatic-F), 758, 682 (C-S-C) cm–1; 1H-NMR (400MHz, CDCl3,
TMS) δ ppm: 8.87 (d, 1H J =1.68 Hz, pyrazol), 8.56 (s, 1H
thiazole), 8.25 (d, 1H J =1.78 Hz, pyrazol), 7.64–7.61 (m, 2H,Ar-
H), 7.25-721 (m, 2H,Ar-H), 3.64 (br–s, 1H, tetrazol, NH); 13C-
NMR (100MHz, CDCl3) δ,ppm: 163.4, 162.8, 158.5, 149.6,
137.3, 130.7, 129.4, 128.6, 116.2, 108.7, 102.4; Mass : 314.38
(M+1); Elemental analysis for C13H8FN7S: Calculated: C, 49.83;
H, 2.57; N, 31.29; Found: C, 49.85; H, 2.57; N, 31.28.
4.2.1. Synthesis of 2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-
sustituted phenylthiazole 6(a-o)
A mixture of 1-(substituted phenylthiazol-2-yl)-1H-pyrazole-
4-carbonitrile (5) (10 mmol), sodium azide (60 mmol), and
ammonium chloride (60 mmol) in 40 mL of DMF was heated at
reflux temperature for 72 h. The completion of the reaction has
been assessed using the TLC using the different polarity of the
solvents. The resultant reaction mixture was then poured into
water and acidified to pH 5. The product was vacuum filtered and
dried to provide compound 6.
4.2.1.5.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(3-
fluorophenyl)thiazole. (6e)
4.2.1.1.
chlorophenyl)thiazole (6a)
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-
Light brown-crystals; Yield: 73%; M.p: 208-211 °C; MW:
313.31; Rf: 0.69; FT-IR (νmax; cm-1 KBr): 3478 (N-H stretch), 3064 ,
2976 (C-H stretch), 1648 (C=N stretch), 1228 (C-N stretch), 1192 (C-S),
White-crystals; Yield: 72%; M.p: 189-191 °C; MW: 329.77;
Rf: 0.64; FT-IR (νmax; cm-1 KBr): 3452 (N-H stretch), 3028 , 2956
(C-H stretch), 1628 (C=N stretch), 1208 (C-N stretch), 1193 (C-S), 1092
(aromatic-Cl), 762, 693 (C-S-C) cm–1; 1H-NMR (400MHz,
CDCl3, TMS) δ ppm: 8.93 (d, 1H J =1.88 Hz, pyrazol), 8.40 (s,
1H thiazole), 8.26 (d, 1H J =1.81 Hz, pyrazol), 7.95–7.93 (m,
2H,Ar-H), 7.53-751 (m, 2H,Ar-H), 3.59 (br–s, 1H, tetrazol, NH);
13C-NMR (100MHz, CDCl3) δ,ppm: 163.5, 158.5, 149.9, 137.6,
134.2, 131.9, 129.6, 129.1, 128.9, 108.5, 102.3; Mass : 330.79
(M+1); Elemental analysis for C13H8ClN7S: Calculated: C, 47.35;
H, 2.45; N, 29.73; Found: C, 47.38; H, 2.45; N, 29.76.
868 (aromatic-F), 762, 676 (C-S-C) cm–1; H-NMR (400MHz,
1
CDCl3, TMS) δ ppm: 8.83 (d, 1H J =1.82 Hz, pyrazol), 8.48 (s,
1H thiazole), 8.23 (d, 1H J =1.74 Hz, pyrazol), 7.58–7.54 (m,
2H,Ar-H), 7.21-718 (m, 2H,Ar-H), 3.56 (br–s, 1H, tetrazol, NH);
13C-NMR (100MHz, CDCl3) δ,ppm: 163.7, 162.1, 158.2, 149.9,
137.6, 134.7, 129.2, 127.4, 123.3, 115.9, 115.2, 108.3, 102.7;
Mass : 314.38 (M+1); Elemental analysis for C13H8FN7S:
Calculated: C, 49.83; H, 2.57; N, 31.29; Found: C, 49.87; H,
2.59; N, 31.32.
4.2.1.6.
fluorophenyl)thiazole. (6f)
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(2-
4.2.1.2.
chlorophenyl)thiazole. (6b)
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(3-
Brown-crystals; Yield: 58%; M.p: 203-205 °C; MW: 313.31;
Rf: 0.62; FT-IR (νmax; cm-1 KBr): 3484 (N-H stretch), 3052 , 2979
(C-H stretch), 1651 (C=N stretch), 1239 (C-N stretch), 1198 (C-S), 862
(aromatic-F), 768, 669 (C-S-C) cm–1; 1H-NMR (400MHz, CDCl3,
White-crystals; Yield: 68%; M.p: 206-208 °C; MW: 329.77;
Rf: 0.71; FT-IR (νmax; cm-1 KBr): 3458 (N-H stretch), 3035 , 2968
(C-H stretch), 1642 (C=N stretch), 1210 (C-N stretch), 1196 (C-S), 887
(aromatic-Cl), 768, 681 (C-S-C) cm–1; 1H-NMR (400MHz,

TMS) δ ppm: 8.87 (d, 1H J =1.85 Hz, pyrazol), 8.45 (s, 1H
thiazole), 8.21 (d, 1H J =1.72 Hz, pyrazol), 7.53–7.51 (m, 2H,Ar-
H), 7.18-716 (m, 2H,Ar-H), 3.52 (br–s, 1H, tetrazol, NH); 13C-
NMR (100MHz, CDCl3) δ,ppm: 163.4, 158.5, 157.9, 152.6,
137.8, 130.9, 129.5, 128.8, 124.8, 123.5, 114.7, 108.5, 102.4;
Mass : 314.38 (M+1); Elemental analysis for C13H8FN7S:
Calculated: C, 49.83; H, 2.57; N, 31.29; Found: C, 49.84; H,
2.57; N, 31.27.
δ ppm: 8.95 (d, 1H J =1.72 Hz, pyrazol), 8.21 (s, 1H thiazole),
8.38 (d, 1H J =1.68 Hz, pyrazol), 7.53–7.49 (m, 2H,Ar-H), 7.22-
721 (m, 2H,Ar-H), 3.52 (br–s, 1H, tetrazol, NH), 2.27 (s, 3H,
CH3); 13C-NMR (100MHz, CDCl3) δ,ppm: 163.2, 158.7, 149.8,
137.6, 131.7, 130.2, 129.5, 128.8, 125.7, 108.5, 102.4, 21.3;
Mass: 310.32 (M+1); Elemental analysis for C14H11N7S:
Calculated: C, 54.36; H, 3.58; N, 31.69; Found: C, 54.37; H,
3.59; N, 31.71.
4.2.1.7.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-
4.2.1.11.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(m-
nitrophenyl)thiazole. (6g)
tolyl)thiazole. (6k)
Yellow-crystals; Yield: 69%; M.p: 223-225 °C; MW: 340.32;
Rf: 0.75; FT-IR (νmax; cm-1 KBr): 3476 (N-H stretch), 3054 , 2972
(C-H stretch), 1659 (C=N stretch),1358 (NO stretch), 1242 (C-N stretch),
Light brown-crystals; Yield: 71%; M.p: 213-214 °C; MW:
309.35; Rf: 0.59; FT-IR (νmax; cm-1 KBr): 3492 (N-H stretch), 3068 ,
2982 (C-H stretch), 2953 (CH3), 1672 (C=N stretch), 1258 (C-N stretch),
1
1
1189 (C-S), 784, 679 (C-S-C) cm–1; H-NMR (400MHz, CDCl3,
1182 (C-S), 785, 682 (C-S-C) cm–1; H-NMR (400MHz, CDCl3,
TMS) δ ppm: 8.78 (d, 1H J =1.73 Hz, pyrazol), 8.42 (s, 1H
thiazole), 8.14 (d, 1H J =1.63 Hz, pyrazol), 7.78–7.73 (m, 2H,Ar-
H), 7.24-722 (m, 2H,Ar-H), 3.62 (br–s, 1H, tetrazol, NH); 13C-
NMR (100MHz, CDCl3) δ,ppm: 163.7, 158.2, 149.7, 147.2,
139.1, 137.5, 129.2, 126.3, 124.4, 108.1, 102.5; Mass : 341.34
(M+1); Elemental analysis for C13H8N8O2S: Calculated: C, 45.88;
H, 2.37; N, 32.93; Found: C, 45.89; H, 2.36; N, 32.93.
TMS) δ ppm: 8.92 (d, 1H J =1.71 Hz, pyrazol), 8.26 (s, 1H
thiazole), 8.32 (d, 1H J =1.58 Hz, pyrazol), 7.75–7.73 (m, 2H,Ar-
H), 7.28-724 (m, 2H,Ar-H), 3.57 (br–s, 1H, tetrazol, NH), 2.34
(s, 3H, CH3); 13C-NMR (100MHz, CDCl3) δ,ppm: 163.3, 158.3,
149.8, 138.9, 137.6, 132.9, 130.4, 129.1, 129.0, 128.9, 124.5,
108.5, 102.3, 21.6; Mass: 310.39 (M+1); Elemental analysis for
C14H11N7S: Calculated: C, 54.36; H, 3.58; N, 31.69; Found: C,
54.39; H, 3.61; N, 31.68.
4.2.1.8.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(3-
nitrophenyl)thiazole. (6h)
4.2.1.12
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(o-
tolyl)thiazole. (6l)
Light yellow-crystals; Yield: 73%; M.p: 216-218 °C; MW:
340.32; Rf: 0.53; FT-IR (νmax; cm-1 KBr): 3484 (N-H stretch), 3057 ,
2971 (C-H stretch), 1654 (C=N stretch),1359 (NO stretch), 1248 (C-N
Dark brown-crystals; Yield: 78%; M.p: 221-224 °C; MW:
309.35; Rf: 0.63; FT-IR (νmax; cm-1 KBr): 3498 (N-H stretch), 3074 ,
2978 (C-H stretch), 2959 (CH3), 1676 (C=N stretch), 1262 (C-N stretch),
1
stretch), 1193 (C-S), 789, 682 (C-S-C) cm–1; H-NMR (400MHz,
1
CDCl3, TMS) δ ppm: 8.94 (d, 1H J =1.68 Hz, pyrazol), 8.38 (s,
1H thiazole), 8.32 (d, 1H J =1.59 Hz, pyrazol), 7.96–7.93 (m,
2H,Ar-H), 7.14-712 (m, 2H,Ar-H), 3.57 (br–s, 1H, tetrazol, NH);
13C-NMR (100MHz, CDCl3) δ,ppm: 163.3, 158.5, 149.9, 148.4,
137.6, 133.9, 132.9, 130.6,129.2, 123.9, 122.7, 108.3, 102.4;
Mass : 341.38 (M+1); Elemental analysis for C13H8N8O2S:
Calculated: C, 45.88; H, 2.37; N, 32.93; Found: C, 45.87; H,
2.37; N, 32.95.
1188 (C-S), 789, 687 (C-S-C) cm–1; H-NMR (400MHz, CDCl3,
TMS) δ ppm: 8.95 (d, 1H J =1.92 Hz, pyrazol), 8.22 (s, 1H
thiazole), 8.38 (d, 1H J =1.57 Hz, pyrazol), 7.54–7.52 (m, 2H,Ar-
H), 7.32-728 (m, 2H,Ar-H), 3.51 (br–s, 1H, tetrazol, NH), 2.18
(s, 3H, CH3); 13C-NMR (100MHz, CDCl3) δ,ppm: 163.2, 158.3,
152.3, 137.4, 136.9, 129.7, 129.5, 129.1, 128.6, 126.2, 122.7
108.1, 102.3, 18.7; Mass: 310.31 (M+1); Elemental analysis for
C14H11N7S: Calculated: C, 54.36; H, 3.58; N, 31.69; Found: C,
54.37; H, 3.56; N, 31.72.
4.2.1.9.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(2-
nitrophenyl)thiazole. (6i)
4.2.1.13.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(4-
methoxyphenyl)thiazole. (6m)
Yellow-crystals; Yield: 62%; M.p: 232-235 °C; MW: 340.32;
Rf: 0.48; FT-IR (νmax; cm-1 KBr): 3489 (N-H stretch), 3052 , 2974
(C-H stretch), 1658 (C=N stretch),1362 (NO stretch), 1256 (C-N stretch),
Dark green-crystals; Yield: 58%; M.p: 235-237 °C; MW:
325.35; Rf: 0.73; FT-IR (νmax; cm-1 KBr): 3482 (N-H stretch), 3079 ,
2975 (C-H stretch), 1672 (C=N stretch), 1668 (C-O stretch), 1268 (C-N
1
1198 (C-S), 795, 686 (C-S-C) cm–1; H-NMR (400MHz, CDCl3,
1
TMS) δ ppm: 8.93 (d, 1H J =1.69 Hz, pyrazol), 8.18 (s, 1H
thiazole), 8.33 (d, 1H J =1.62 Hz, pyrazol), 7.94–7.93 (m, 2H,Ar-
H), 7.21-723 (m, 2H,Ar-H), 3.54 (br–s, 1H, tetrazol, NH); 13C-
NMR (100MHz, CDCl3) δ,ppm: 163.7, 158.2, 152.5, 148.8,
137.5, 135.3, 132.6, 129.6, 128.9, 125.2, 124.4, 108.7, 102.3;
Mass: 341.32 (M+1); Elemental analysis for C13H8N8O2S:
Calculated: C, 45.88; H, 2.37; N, 32.93; Found: C, 45.88; H,
2.39; N, 32.92.
stretch), 1197 (C-S), 782, 681 (C-S-C) cm–1; H-NMR (400MHz,
CDCl3, TMS) δ ppm: 8.92 (d, 1H J =1.86 Hz, pyrazol), 8.12 (s,
1H thiazole), 8.24 (d, 1H J =1.52 Hz, pyrazol), 7.51–7.50 (m,
2H,Ar-H), 7.02-703 (m, 2H,Ar-H), 3.73 (s, 3H, OCH3), 3.48 (br–
s, 1H, tetrazol, NH); 13C-NMR (100MHz, CDCl3) δ,ppm: 163.4,
160.6, 158.5, 149.9, 137.2, 129.3, 128.3, 125.7, 114.8 108.5,
102.3, 55.2; Mass: 326.37 (M+1); Elemental analysis for
C14H11N7OS: Calculated: C, 51.68; H, 3.41; N, 30.14; Found: C,
51.66; H, 3.40; N, 30.18.
4.2.1.10.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(p-
tolyl)thiazole. (6j)
4.1.2.14.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(3-
methoxyphenyl)thiazole. (6n)
Brown-crystals; Yield: 75%; M.p: 195-197 °C; MW: 309.35;
Rf: 0.52; FT-IR (νmax; cm-1 KBr): 3482 (N-H stretch), 3058 , 2976
(C-H stretch), 2946 (CH3), 1664 (C=N stretch), 1252 (C-N stretch), 1189
(C-S), 792, 684 (C-S-C) cm–1; 1H-NMR (400MHz, CDCl3, TMS)
Green-crystals; Yield: 69%; M.p: 241-243 °C; MW: 325.35;
Rf: 0.63; FT-IR (νmax; cm-1 KBr): 3487 (N-H stretch), 3072 , 2974
(C-H stretch), 1678 (C=N stretch), 1670 (C-O stretch), 1261 (C-N stretch),

1
1182 (C-S), 787, 685 (C-S-C) cm–1; H-NMR (400MHz, CDCl3,
Resting tension was adjusted to about 0.5 g in the whole atria and
the initial equilibration period was 40–50 min for each
preparation. Since the atria were isolated from reserpine-
pretreated animals, depletion of catecholamines was verified by
lack of any positive inotropic effect induced by tyramine (1.5
µM). Experiments were performed only in preparations that did
not response to tyramine. The test compounds were added
cumulatively (1–100 µM) and the responses of each
concentration were recorded up to the maximum.
TMS) δ ppm: 8.96 (d, 1H J =1.89 Hz, pyrazol), 8.18 (s, 1H
thiazole), 8.18 (d, 1H J =1.47 Hz, pyrazol), 7.47–7.44 (m, 2H,Ar-
H), 7.01-6.98 (m, 2H,Ar-H), 3.67 (s, 3H, OCH3), 3.53 (br–s, 1H,
tetrazol, NH); 13C-NMR (100MHz, CDCl3) δ,ppm: 163.7, 161.1,
158.4, 149.2, 137.6, 134.2, 130.4, 129.3, 119.8, 114.3, 113.6,
108.5, 102.3, 55.2; Mass: 326.34 (M+1); Elemental analysis for
C14H11N7OS: Calculated: C, 51.68; H, 3.41; N, 30.14; Found: C,
51.64; H, 3.40; N, 30.16.
4.1.2.15.
2-(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-4-(2-
4.4. Sub-acute oral toxicity of compound 6d
methoxyphenyl)thiazole. (6o)
In view of potent cardiotonic activity of 6d in in-vitro model,
sub-acute oral toxicity of the same was carried out in Swiss
albino mice for its further development as viable drug candidate.
Acute toxicity test has been completed and found that compound
has been found to be safe upto 1000 mg/kg as a single acute oral
dose. Hence, sub acute experiment was conducted in the present
study in accordance with the Organization for Economic Co-
operation and Development (OECD) test guideline No 423
(1987).
Green yellow-crystals; Yield: 81%; M.p: 233-235 °C; MW:
325.35; Rf: 0.61; FT-IR (νmax; cm-1 KBr): 3492 (N-H stretch), 3079 ,
2971 (C-H stretch), 1682 (C=N stretch), 1679 (C-O stretch), 1268 (C-N
stretch), 1187 (C-S), 781, 683 (C-S-C) cm–1; H-NMR (400MHz,
1
CDCl3, TMS) δ ppm: 8.93 (d, 1H J =1.85 Hz, pyrazol), 8.23 (s,
1H thiazole), 8.17 (d, 1H J =1.57 Hz, pyrazol), 7.55–7.52 (m,
2H,Ar-H), 7.01-6.97 (m, 2H,Ar-H), 3.78 (s, 3H, OCH3), 3.52
(br–s, 1H, tetrazol, NH); 13C-NMR (100MHz, CDCl3) δ,ppm:
163.6, 158.5, 157.3, 152.7, 137.6, 131.1, 129.7, 129.2, 121.5,
118.9, 111.6, 108.5, 102.3, 56.1; Mass: 326.37 (M+1); Elemental
analysis for C14H11N7OS: Calculated: C, 51.68; H, 3.41; N,
30.14; Found: C, 51.67; H, 3.39; N, 30.15.
For the sub-acute oral toxicity study, 30 mice (15 male and 15
female) were taken and divided into five groups comprising 3
male and 3 female mice in each group weighing between 20 and
25 g. The animals were maintained at 22 5 °C with humidity
control and also on an automatic dark and light cycle of 12 h. The
animals were fed with the standard mice feed and provided ad
libitum drinking water. Mice of group 1 were kept as control and
animals of groups 2, 3, 4 and 5 were kept as experimental. The
animals were acclimatized for 7 days in the experimental
environment prior to the actual experimentation. The test
compound was solubilized in dimethyl sulphoxide and then
suspended in caboxymethyl cellulose (CMC, 0.7%) and was
given at 0.1, 1, 10 and 100 mg/kg body weight to animals of
groups 2, 3, 4 and 5 respectively once orally for 28 days. Control
animals received only vehicle.
4.2. Assessment of PDE3A and PDE3B inhibitory activity
Assays were conducted following the instruction from the
manufacture of the assay kit. The enzymatic reactions were
conducted at room temperature for 60 min in a 50 µl mixture
containing IMAP Reaction Buffer, 100 nM FAM-cAMP, 1 ng
PDE3A or PDE3B and the test compound. The binding reactions
were performed in the present of Binding Reagent (1:600 dilution
in a reagent binding buffer containing 85% Binding Buffer A and
15% Binding Buffer B) at room temperature for 60 min.
Fluorescence intensity was measured at an excitation of 485 nm
and an emission of 528 nm using a BioTek Synergy™ 2
microplate reader. PDE3 activity assays were performed in
duplicates at each concentration. Fluorescence intensity is
converted to fluorescence polarization u\sing the Gen5 software.
The fluorescence polarization data were analyzed using the
computer software, Graphpad Prism. The highest value of
fluorescence polarization (FPt) in each data set was defined as
100% activity. In the absence of PDE3, the value of fluorescent
polarization (FPb) in each data set was defined as 0% activity.
The percent activity in the presence of the compound was
calculated according to the following equation: %binding = (FP–
FPb)/(FPt–FPb) × 100, where FP = the fluorescence polarization
in the presence of the compound, FPb = the fluorescent
polarization in the absence of PDE3, and FPt = the highest
fluorescent polarization in each data set. The IC50 values were
determined from the % activity of the enzymes at 0.01, 0.1, 1, 10
and 100 µM concentrations of synthetic inhibitors using
sigmoidal dose–response curve in Graphpad Prism.
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bathing solution was made up (in mM):NaCl 118, KCl 4.5, CaCl2
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Research Highlight
•
•
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Compound 6d exhibited most potent inhibition of
PDE3A than PDE3B.
It buried deep inside the PDE pocket lined with residues
like Tyr737, Phe559 and Phe991.
It shows no observational changes, morbidity and
mortality in experimental animals in toxicity assay

Graphical