Synthesis and anti-inflammatory evaluation of some new 3,6-disubstituted-1, 2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles bearing pyrazole moiety

Article abstract of DOI:10.1007/s00044-011-9865-0

In the present study, a new series of 3,6-disubstituted- 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles (4aj) have been synthesized by condensing 3-substituted-4-amino-5- mercapto-1,2,4-triazoles (1a-b) with various 3-substitutedpyrazole- 4-carboxylic acids (3

Full text of DOI:10.1007/s00044-011-9865-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3272–3280
DOI 10.1007/s00044-011-9865-0
ORIGINAL RESEARCH
Synthesis and anti-inflammatory evaluation of some new
3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles bearing
pyrazole moiety
•
Shridhar Malladi Arun M. Isloor Prashanth Shetty
•
•
•
•
•
Hoong Kun Fun Sandeep Telkar Riaz Mahmood
Nishitha Isloor
Received: 16 February 2011 / Accepted: 31 October 2011 / Published online: 16 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In the present study, a new series of 3,6-disub-
stituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles (4aj) have
been synthesized by condensing 3-substituted-4-amino-5-
mercapto-1,2,4-triazoles (1a–b) with various 3-substituted-
pyrazole-4-carboxylic acids (3a–e) in the presence of POCl3.
The structures of newly synthesized compounds were char-
acterized by elemental analysis, IR, 1H NMR, 13C NMR, and
mass spectroscopic studies. Structure of the compound 4b
was also confirmed by recording the single crystal X-ray
structure. All the synthesized compounds were screened for
their anti-inflammatory activities by carrageenan induced
paw edema method. Anti-inflammatory screening indicated
that, compounds 4d, 4e, and 4h were found to be biologically
active whereas remaining compounds showed poor anti-
inflammatory activity. Also molecular docking studies were
also performed for compounds which showed good anti-
inflammatory activity.
Keywords Triazolothiadiazoles ꢀ Pyrazole ꢀ
Anti-inflammatory activity
Introduction
In the last few decades, triazoles and their derivatives have
gained significant importance in the field of medicinal
chemistry due to their diversified biological properties like
antibacterial (Holla et al., 2001; Suresh et al., 2007;
Prakash et al., 2004), antifungal (Hirpara et al., 2003),
antiviral (Tarmas et al., 2006), anti-inflammatory
(Tozkoparan et al., 2000), analgesic (Turan-Zitouni et al.,
1999), antimicrobial (Isloor et al., 2009), antihyperten-
sive (Czarnocka-Janowicz et al., 1991), antitubercular
(Kucukguzel et al., 2001), and anticancer (He et al., 2010)
properties. The amino and mercapto groups of 1,2,4-tria-
zoles serve as readily accessible nucleophilic centers for
the preparation of N-bridged heterocycles. Many com-
mercial drugs such as Ribavirin and Taribavirin (antiviral),
Fluconazole and Itraconazole (antifungal) are of triazole
derivatives only.
S. Malladi ꢀ A. M. Isloor (&)
Medicinal Chemistry Division, Department of Chemistry,
National Institute of Technology-Karnataka, Surathkal,
Mangalore 575025, India
e-mail: isloor@yahoo.com
P. Shetty
Department of Pharmacology, Nitte Gulabi Shetty Memorial
College of Pharmacology, Deralakatte, Mangalore 574160, India
H. K. Fun
X-Ray Crystallography Unit, School of Physics, Universiti Sains
Malaysia, 11800 Penang, Malaysia
Inflammation and analgesia underlies a wide variety of
physiological and pathological processes involving a well
established and co-ordinated program between many dif-
ferent immune cells, enzymes, and mediators. Cyclooxy-
genase (COX; prostaglandin G/H synthase, EC 1.14.99.1)
catalyzes the first two steps in the biosynthesis of biolog-
ical mediator prostaglandin, and there lies a great interest
in COX-2 as a key therapeutic target for inflammation
(Rouzer and Marnett, 2009; McKellar et al., 2008;
Jankowski and Hunt, 2008).
S. Telkar ꢀ R. Mahmood
Department of P.G. Studies and Research in Biotechnology
and Bioinformatics, Jnanasahyadri, Kuvempu University,
Shankaraghatta 577451, Karnataka, India
N. Isloor
Biotech Division, Department of Chemical Engineering,
National Institute of Technology-Karnataka, Surathkal,
Mangalore 575025, India
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Many pyrazole derivatives are reported to possess a
wide range of bioactivities. The pyrazole motif makes up
the core structure of numerous biologically active com-
pounds. Thus, some representatives of this heterocycle
exhibit analgesic (Isloor et al., 2000), anti-inflammatory
(Kalluraya et al., 2004), and antimicrobial activity (Isloor
et al., 2009). Much attention was paid to pyrazole as a
potential antimicrobial agent after the discovery of the
natural pyrazole C-glycoside, pyrazofurin which demon-
strated a broad spectrum of antimicrobial activity (Comber
et al., 1992). Our previous studies also indicate that few of
the pyrazole derivatives (Kalluraya et al., 2001; Isloor
et al., 2010) are pharmacologically active.
& Elemental). The reaction between 3a–e and 1a–b
resulted in the formation of cyclized products 4a–j. The
1
absence of NH2 (*d 5.6) and SH (*d 13.4) peaks in H
NMR spectra of 4a–j which were present in 1a–b can be
attributed for the involvement of these functional groups in
the formation of cyclized products and also cyclization was
further confirmed by the absence of NH2 (*3,260 cm-1
)
1
absorption band in the IR spectrum of 4a–j. The H NMR
of 4f showed a singlet at d 13.8 and 8.46 are due to NH and
5H proton of pyrazole ring. A multiplet and a triplet were
observed at d 7.79 and 7.29 (Jortho = 8.6 Hz) which cor-
responds to Ar–H. Aliphatic protons which resonated as
triplet, multiplet, and triplet at d 2.91, 1.75, and 0.91 can be
assigned to –CH2– CH2–CH3, respectively. Similarly, IR
spectrum of 4f exhibited bands at 3403, 3102, 2964, 1603,
and 1483 cm-1 which corresponds to N–H, aromatic C–H,
aliphatic C–H, C=N and C=C, respectively. The mass
spectrum of 4f showed molecular ion peak at m/z = 329
(M ? 1), which is in agreement with the molecular for-
mula C15H13FN6S. Among the synthesized compounds the
structure of 4b was confirmed by single crystal X-ray
analysis (Fig. 1). The spectral values for all the compounds
and C, H, N analyses are given in the experimental part and
the characterization data is provided in Table 1. Similarly,
crystallographic data (Fun et al., 2010) has been provided
in Table 2.
1,3,4-Thiadiazoles exhibit wide spectrum of biological
activities, possibly due to presence of toxophoric [N–C–
S– moiety (Kamotra et al., 2007). They find applications as
antibacterial and anti-inflammatory agents (Mathew et al.,
2007). A triazolo thiadiazole system may be viewed as a
cyclic analog of two very important components, thio-
semicarbazide and biguanide which often display diverse
biological activities. The 1,2,4-triazolo[3,4-b]-1,3,4-thia-
diazole derivatives obtained by reacting the bio-labile
1,2,4-triazole and pyrazole rings together are reported to be
pharmacologically active.
Recently some bis triazolo-thiadiazoles endowed with
excellent anticancer activities have been reported (Holla
et al., 2002). Prompted by these literatures and from our
previous research on heterocycles (Vijesh et al., 2010), we
planned to synthesize new triazolothiadiazole derivatives
containing pyrazole, halogen as substituents and to study
their anti-inflammatory activity.
OH
H
R2
R2
O
O
KMnO4
NaOH
N
N
N
H
N
H
Results and discussion
(3a-e)
(2a-e)
Chemistry
N
N
POCl3
Reflux, 7h
R1
3-Substituted-4-amino-5-mercapto-1,2,4-triazoles (1) were
synthesized as reported in the literature (Dhaka et al., 1974;
Reid and Heindel, 1976). Various 3-substituted-pyrazole-4-
carbaldehydes (2) were also prepared according to
literature method [8]. Oxidation of 3-substituted-pyrazole-
4-carbaldehydes using potassium permanganate in the
presence of base (Lebedev et al., 2005) yielded corre-
sponding acid. Subsequently condensation of 3-substituted-
4-amino-5-mercapto-1,2,4-triazoles (1) with various
3-substituted-pyrazole-4-carboxylic acids (3) in the pres-
ence of phosphorous oxychloride afforded a series of
3,6-disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles
(4a–j). The synthesis of compounds (4a–j) followed the
general pathway outlined in Scheme 1. The structures of
the synthesized compounds (4a–j) were confirmed by
analytical and spectral data (IR, 1H NMR, 13C NMR, Mass
N
SH
NH2
(1a-b)
N
N
R1
N
S
N
R2
N
N
H
(4a-j)
Where R1 = C2H5, C3H7
R2 = C6H5, 4-F-C6H4, 4-OCH3-C6H4, 4-Cl-C6H4, 2,4-Cl2-C6H3
Scheme 1 Synthetic route for 3,6-disubstituted-1,2,4-triazolo-
[3,4-b]-1,3,4-thiadiazoles
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Med Chem Res (2012) 21:3272–3280
In silico studies
The docking of receptor COX-2 with newly synthesized
candidate ligands exhibited well established bonds with one
or more amino acids in the receptor active pocket. Docking
studies were performed for compounds 4d, 4e, 4h, & Dic-
lofenac. The active pocket was considered to be the site
where Diclofenac was complexed in COX-2 of 1PXX. The
active pocket consisted of ten amino acid residues as Val349,
Ser530, Leu352, Tyr385, Tyr348, Trp387, Gly526, Ala527,
Met522, and Leu384 as shown in Fig. 3. The synthesized
ligand molecules having 2D structure were converted to
energy minimized 3D structures and were further used for in
silico protein–ligand docking. Figure 4. Shows the images of
ligands docked separately to COX2 including the considered
standard Diclofenac. Table 3 shows the Binding Energy and
Inhibition Constant of all the four compounds including
standard Diclofenac. In-silico studies revealed that all three
synthesized molecules showed good binding energy toward
Considering the well obtained in vitro results, it was
thought worthy to perform molecular docking studies,
screening for supportive coordination between in silico
studies with in vitro results. Considering COX-2 as the
target receptor, comparative and automated docking studies
with newly synthesized candidate lead compounds was
performed to determine the best in silico conformation.
The Lamarckian genetic algorithm, inculcated in the
docking program AutoDock 4.2, was employed for the
purpose. Figure 2 shows the native crystal structure of
Diclofenac bound to the Cyclooxygenase active site of
COX-2 obtained from Protein Data Bank (http://www.
pdb.org/pdb/home/home.do) with the PDB ID 1PXX
(Rowlinson et al., 2003).
the target protein ranging from -9.1 to -8.76 kJ mol-1
.
Table 2 Crystallographic data for compound 4b
Empirical formula
Formula weight
Temperature (K)
C14H11FN6S
314.35
100
˚
Wavelength (A)
0.71073
Crystal system
Space group
Orthorhombic
Pca21
Cell dimensions
˚
a (A)
35.053 (2)
3.8463 (2)
9.9482 (6)
1341.26 (13)
4
˚
b (A)
˚
c (A)
3
˚
Volume (A )
Z
Density (Mg m-3
)
1.557
F (000)
648
H range for data collection (P)
2.3–30.0
Fig. 1 X-ray crystal structure of compound 4b
Table 1 Characterization data
of the compounds 4a–j
Compounds
R1
R2
Molecular formula (mol. wt)
Yield (%)
M.p. (oC)
4a
4b
4c
4d
4e
4f
C2H5
C2H5
C2H5
C2H5
C3H7
C3H7
C3H7
C3H7
C2H5
C3H7
C6H5
C14H12N6S (296)
68
74
65
72
56
61
67
69
71
77
217–219
206–207
222–224
290–293
219–221
187–189
213–215
220–222
200–205
203–205
4-F–C6H4
4-OCH3–C6H4
4-Cl–C6H4
C6H5
C14H11FN6S (314)
C15H14N6OS (326)
C14H11ClN6S (330)
C15H14N6S (310)
4-F–C6H4
4-OCH3–C6H4
4-Cl–C6H4
2,4-Cl2–C6H3
2,4-Cl2–C6H3
C15H13FN6S (328)
C16H16N6OS (340)
C15H13ClN6S (344)
C14H10Cl2N6S (365)
C15H12Cl2N6S (379)
4g
4h
4i
4j
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Conclusion
A series of new 3,6-disubstituted-1,2,4-triazolo-[3,4-b]-
1,3,4-thiadiazoles bearing pyrazole derivatives were syn-
thesized in reasonable yields. They were characterized by
1H NMR, 13C NMR, mass spectrometry, IR studies, and
elemental analyses. Compound 4b was analyzed for its
molecular structure by single crystal X-ray crystallography.
All the newly synthesized compounds were screened for
anti-inflammatory activity by Carrageenan induced paw
edema method. Among the screened compounds, 4h
showed significant anti-inflammatory activity at percentage
inhibition of 64.7, compared to the standard drug Dic-
lofenac sodium which showed the percentage inhibition at
80.4. Similarly compounds 4d and 4e have showed per-
centage inhibition of 56.9. However remaining compounds
showed poor anti-inflammatory activity. Docking studies
were performed for compounds 4d, 4e, 4h, & Diclofenac
among which 4h showed the best result and can be con-
sidered as the best inhibitor of COX-2.
Compound 4h has propyl and p-chlorophenyl
substituents, which is accounted for their significant
anti-inflammatory activity. Compounds 4d has ethyl and
p-chlorophenyl moiety and 4e has propyl and phenyl
moieties, which has accounted for the moderate activity.
Fig. 2 a Secondary structure of COX-2 (PDB ID 1PXX) complexed
with Diclofenac (complete protein), showing its dimeric nature with
two identical subunits in each monomer. b Chain A (part of complete
protein) complexed with Diclofenac
Experimental
Chemistry
Melting points were determined by open capillary method
and were uncorrected. The IR spectra (in KBr pellets) were
recorded on a Thermo Nicolet avatar 330-FT-IR spectro-
photometer. 1H-NMR and 13C-NMR spectra were recorded
(DMSO-d6) on a Bruker (400 MHz) and Varian (400 MHz)
spectrometer using TMS as internal standard. Chemical shift
values are given in d scales. The mass spectra were recorded
on LC–MS-Agilent 1100 series and API 2000 LC/MS/MS
system. Single crystal X-ray structure was recorded using
Bruker instrument. Elemental analyses were performed on a
Flash EA 1112 series CHNS-O Analyzer. The completion of
the reaction was checked by thin layer chromatography
(TLC) on silica gel coated aluminum sheets (silica gel 60
F254). Commercial grade solvents and reagents were used
without further purification.
Fig. 3 PDB sum’s ligplot results for 1PXX, showing all ten amino
acid residues of active pocket
General procedure for the synthesis of 3-(4-substituted
phenyl)-1H-pyrazole-4-carboxylic acid (3a–e)
Finally considering both in vitro and in silico molecular
docking results, among the synthesized molecules 4h
showed the best result and can be considered as the best
inhibitor of COX-2.
Suitable 3-substituted-pyrazol-4-carbaldehyde (2a–e)
(0.01 mol) was dissolved with stirring in a solution of 2 g
of NaOH in 40 ml of water. The mixture was cooled to
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Med Chem Res (2012) 21:3272–3280
Fig. 4 Showing all ligands
docked in best of its
conformation. a 4d forming 1H
bond with Met522. b 4e forming
no H bonds. c 4h forming 1H
bond with Met522. d Diclofenac
forming 1H bond with Arg120
Table 3 Binding energy (kJ mol-1) and inhibition constant of all the
four compounds including standard Diclofenac
3-Ethyl-6-(3-phenyl-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4a)
Compound
Binding energy
(kJ mol-1
Inhibition constant
IR (KBr, tmax cm-1): 3,390 (N–H-str), 3,066 (aromatic
C–H-str), 2,909 (aliphatic C–H-str), 1,604 (C=N), 1,481
)
4d
-8.76
-8.92
-9.1
406.41 nM
287.8 nM
212.03 nM
3.22 nM
1
(C=C); H NMR (DMSO-d6): d 1.31(t, 3H, CH3), 2.99 (q,
4e
2H, CH2), 7.49–7.72 (m, 5H, Ar–H), 8.43 (s, 1H, pyrazole-
5H), 13.70 (s, 1H, Pyrazole N–H). 13C NMR: 159.9, 152.8,
148.7, 130.2, 129.8, 129.5, 128.9, 109.3, 18.5, 11.1. MS: m/
z = 297 (M ? 1). Anal. calcd. for C14H12N6S: C, 56.74;
H, 4.08; N, 28.36. Found: C, 56.69; H, 4.04; N, 28.39%.
4h
Diclofenac
-7.49
15°C, and a solution of KMnO4 (0.0088 mol) in 40 ml of
water was quickly added. The mixture was stirred for 30 min
at 20°C and then heated to 95–100°C until the solution
becomes completely decolorized. The solution was cooled
and filtered to remove MnO2 precipitate. Then, the filtrate
was acidified with Conc. HCl to pH 3. The resulting solid was
filtered off, washed with water and dried.
3-Ethyl-6-(3-(4-fluorophenyl)-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4b)
IR (KBr, tmax cm-1): 3,397(N–H-str), 3,091 (aromatic C–
H-str), 2,929 (aliphatic C–H-str), 1,605 (C=N), 1,479
(C=C); 1H NMR (DMSO-d6): d 1.30 (t, 3H, CH3), 2.98 (q,
2H, CH2), 7.75–7.79 (m, 2H, Ar–H), 7.30–7.35 (t, 2H,
Jortho = 9 Hz, Ar–H), 8.46 (s, 1H, pyrazole-5H), 13.70 (s,
1H, Pyrazole N–H). 13C NMR: 159.6, 148.7, 131.8, 131.8,
115.9, 115.7, 109.4, 18.5, 11.2. MS: m/z = 315 (M ? 1).
Anal. calcd. for C14H11FN6S: C, 53.49; H, 3.53; N, 26.74.
Found: C, 53.45; H, 3.58; N, 26.77%.
General procedure for the synthesis of 3,6-
disubstituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles
(4a–j)
An equimolar mixture of respective triazole (1) (0.001 mol)
and 3-(4-substitutedphenyl)-1H-pyrazole-4-carboxylic acid
(3) (0.001 mol) was dissolved in 5 ml of dry phosphorous
oxychloride. The resulted solution was further refluxed for
8–9 h. Excess phosphorous oxychloride was then distilled
off and the mixture was gradually poured onto crushed ice
with stirring. The mixture was allowed to stand overnight
and the solid was separated. The separated solid was filtered,
washed thoroughly with cold water, 20% NaHCO3 solution
and recrystallised from a mixture of dioxane and ethanol.
3-Ethyl-6-(3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4c)
IR (KBr, tmax cm-1): 3,350 (N–H-str), 3,111 (aromatic C–
H-str), 2,931 (aliphatic C–H-str), 1,613 (C=N), 1,472
(C=C); 1H NMR (DMSO-d6): d 1.33 (t, 3H, CH3), 2.99 (q,
2H, CH2), 3.83 (s, 3H, –OCH3), 7.05–7.07 (d, 2H,
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J = 8.0 Hz, Ar–H), 7.62–7.65 (d, 2H, J = 8.8 Hz, Ar–H),
8.34 (s, 1H, pyrazole-5H), 13.70 (s, 1H, Pyrazole N–H).
13C NMR: 159.7, 131.0, 114.4, 109.1, 55.7, 18.5, 11.3. MS:
m/z = 327 (M ? 1). Anal. calcd. for C15H14N6OS: C,
55.20; H, 4.32; N, 25.75. Found: C, 55.17; H, 4.36; N,
25.78%.
for C16H16N6OS: C, 56.45; H, 4.74; N, 24.69. Found: C,
56.41; H, 4.78; N, 24.72%.
3-Propyl-6-(3-(4-chlorophenyl)-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4h)
IR (KBr, tmax cm-1): 3,391 (N–H-str), 3,088 (aromatic C–
H-str), 2,928 (aliphatic C–H-str), 1,566 (C=N), 1,476
(C=C); 1H NMR (DMSO-d6): d 0.92 (t, 3H, CH3), 1.74 (m,
2H, CH2), 2.90 (t, 2H, CH2), 7.52–7.53 (d, 2H, J = 7.2 Hz,
Ar–H), 7.73–7.75 (d, 2H, J = 8.4 Hz, Ar–H), 8.57 (s, 1H,
pyrazole-5H), 13.80 (s, 1H, Pyrazole N–H). MS: m/z =
345 (M ? 1), 347 (M ? 2). Anal. calcd. for C15H13ClN6S:
C, 52.25; H, 3.80; N, 24.37. Found: C, 52.29; H, 3.77; N,
24.33%.
3-Ethyl-6-(3-(4-chlorophenyl)-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4d)
IR (KBr, tmax cm-1): 3,360 (N–H-str), 3,173 (aromatic C–
H-str), 2,940 (aliphatic C–H-str), 1,579 (C=N), 1,498
(C=C); 1H NMR (DMSO-d6): d 1.16 (t, 3H, CH3), 2.50 (q,
2H, CH2), 7.46–7.48 (d, 2H, J = 8.8 Hz, Ar–H), 7.78–7.80
(d, 2H, J = 8.4 Hz, Ar–H), 8.37 (s, 1H, pyrazole-5H),
13.70 (s, 1H, Pyrazole N–H). MS: m/z = 331 (M ? 1).
Anal. calcd. for C14H11ClN6S: C, 50.83; H, 3.35; N, 25.41.
Found: C, 50.86; H, 3.31; N, 25.45%.
3-Ethyl-6-(3-(2,4-dichlorophenyl)-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b] [1,3,4]thiadiazole (4i)
3-Propyl-6-(3-phenyl-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4e)
IR (KBr, tmax cm-1): 3,320 (N–H-str), 3,097 (aromatic C–
H-str), 2,926 (aliphatic C–H-str), 1,582 (C=N), 1,479
(C=C); 1H NMR (DMSO-d6): d 1.20 (t, 3H, CH3), 2.89 (q,
2H, CH2), 7.59–7.81 (m, 3H, Ar–H), 8.62 (s, 1H, pyrazole-
5H). 13C NMR: 158.8, 152.5, 148.6, 135.5, 134.9, 134.1,
129.6, 128.1, 111.2, 18.5, 11.01. MS: m/z = 365 (M ?),
367 (M ? 2), 369 (M ? 4). Anal. calcd. for
C14H10Cl2N6S: C, 46.04; H, 2.76; N, 23.01. Found: C,
46.07; H, 2.71; N, 23.08%.
IR (KBr, tmax cm-1): 3,389 (N–H-str), 3,091 (aromatic C–
H-str), 2,918 (aliphatic C–H-str), 1,613 (C=N), 1,479
(C=C); 1H NMR (DMSO-d6): d 0.93 (t, 3H, CH3), 1.77 (m,
2H, CH2), 2.93 (t, 2H, CH2), 7.47–7.71 (m, 5H, Ar–H),
8.43 (s, 1H, pyrazole-5H), 13.70 (s, 1H, Pyrazole N–H).
MS: m/z = 311 (M ? 1). Anal. calcd. for C15H14N6S: C,
58.05; H, 4.55; N, 25.41. Found: C, 50.86; H, 3.31; N,
27.08%.
3-Propyl-6-(3-(2,4-dichlorophenyl)-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b] [1,3,4]thiadiazole (4j)
3-Propyl-6-(3-(4-fluorophenyl)-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4f)
IR (KBr, tmax cm-1): 3,325 (N–H-str), 3,099 (aromatic C–H-
str), 2,875 (aliphatic C–H-str), 1,583 (C=N), 1,472 (C=C);
1H NMR (DMSO-d6): d 0.86 (t, 3H, CH3), 1.62 (m, 2H,
CH2), 2.82 (t, 2H, CH2), 7.59–7.81 (m, 3H, Ar–H), 8.64 (s,
1H, pyrazole-5H), 13.79 (s, 1H, Pyrazole N–H). 13C NMR:
158.8, 152.4, 147.6, 134.9, 134.0, 129.5, 128.0, 111.1, 26.7,
19.9, 13.8. MS: m/z = 379 (M ?), 381 (M ? 2), 383
(M ? 4). Anal. calcd. for C15H12Cl2N6S: C, 47.50; H, 3.19;
N, 22.16. Found: C, 47.47; H, 3.14; N, 22.19%.
IR (KBr, tmax cm-1): 3,403 (N–H-str), 3,102 (aromatic C–
H-str), 2,964 (aliphatic C–H-str), 1,603 (C=N), 1,483
(C=C);1H NMR (DMSO-d6): d 0.92 (t, 3H, CH3), 1.75 (m,
2H, CH2), 2.91 (t, 2H, CH2), 7.74–7.77 (m, 2H, Ar–H),
7.29–7.33 (t, 2H, Jortho = 8.6 Hz, Ar–H), 8.46 (s, 1H,
pyrazole-5H), 13.71 (s, 1H, Pyrazole N–H). MS:
m/z = 329 (M ? 1). Anal. calcd. for C15H13FN6S: C, 54.87;
H, 3.99; N, 25.59. Found: C, 54.82; H, 3.94; N, 25.56%.
3-Propyl-6-(3-(4-methoxyphenyl)-1H-pyrazol-4-yl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4g)
Anti-inflammatory activity
Screening of anti-inflammatory drugs by Carrageenan
induced paw edema method (Hu et al., 2008; Maria
et al., 2008)
IR (KBr, tmax cm-1): 3,360 (N–H-str), 3,081 (aromatic C–
H-str), 2,951 (aliphatic C–H-str), 1,611 (C=N), 1,463
(C=C); 1H NMR (DMSO-d6): d 0.94 (t, 3H, CH3), 1.78 (m,
2H, CH2), 2.93 (t, 2H, CH2), 3.82 (s, 3H, –OCH3), 7.03–
7.06 (d, 2H, J = 8.8 Hz, Ar–H), 7.61–7.63 (d, 2H,
J = 8.8 Hz, Ar–H), 8.32 (s, 1H, pyrazole-5H), 13.68 (s,
1H, Pyrazole N–H). MS: m/z = 341 (M ? 1). Anal. calcd.
The anti-inflammatory activity of the test compounds was
carried out using carrageenan-induced rat paw edema
model according to Winter et al. (1962) by employing 1%
carrageenan solution as the phlogistic agent. Edema was
123

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Med Chem Res (2012) 21:3272–3280
induced in the left hind paw of Wistar rats (200–250 g) by
the sub-plantar injection of 0.1 ml of 1% Carrageenan in
distilled water. Both sexes were used. Each group com-
posed of six animals. The animals which were bred in our
laboratory were housed under standard conditions and
received a diet of commercial food pellets and water
ad libitum during the maintenance but they were entirely
fasted during the experiment period. Our studies were
conducted in accordance with recognized guidelines on
animal experimentation.
drug score via in silico (Sander et al., 2009). Energy of the
molecules was minimized using Dundee PRODRG2 server
(Schuttelkopf and van Aalten, 2004).The energy mini-
mized compounds were then read as input for AutoDock
4.2, in order to carry out the docking simulation (Morris
et al., 2009). All the heteroatoms were removed from the
1PXX.pdb, to make complex less receptor free of any
ligand before docking. The Graphical User Interface pro-
gram ‘‘AutoDock Tools’’ was used to prepare, run, and
analyze the docking simulations. Kollman united atom
charges, solvation parameters and polar hydrogen’s were
added to the receptor for the preparation of protein in
docking simulation. Since ligands are not peptides,
Gasteiger charge was assigned and then non-polar hydro-
gens were merged. AutoDock requires pre-calculated grid
maps, one for each atom type, present in the ligand being
docked and its stores the potential energy arising from the
interaction with macromolecule. This grid must surround
the region of interest (active site) in the macromolecule.
In the present study, the binding site was selected based on
the amino acid residues, which are involved in binding
with Diclofenac of COX-2 as obtained from PDB with ID
1PXX which would be considered as the probable best
accurate region as it is solved by experimental crystallo-
graphic data (Rowlinson et al., 2003). Therefore, the grid
was centered at the region including all the ten amino acid
residues (Val349, Ser530, Leu352, Tyr385, Tyr348,
Trp387, Gly526, Ala527, Met522 and Leu384) that sur-
round active site as in Fig. 2. The grid box size was set at
56, 44, and 54 A° for x, y and z, respectively, and the grid
center was set to 26.472, 25.806, and 12.52 for x, y and z,
respectively, which covered all the ten amino acid residues
in the considered active pocket. AutoGrid 4.0 Program,
supplied with AutoDock 4.0 was used to produce grid
maps. The spacing between grid points was 0.375 ang-
stroms. The Lamarckian Genetic Algorithm (LGA) was
chosen to search for the best conformers. During the
The test compounds were given intraperitoneally 30 min
after Carrageenan injection. The paw volume was measured
plethysmometrically at 0 and 3 h after the carrageenan
injection.Thedifferenceinthe pawvolumeoftheinjectedand
the control were compared for each animal. The percentage
inhibition of edema was calculated using the formula,
% Oedema inhibition ¼ 100 ꢁ ðVtest=VcontrolÞ ꢂ 100
Statistical analysis
All experimental groups were composed of six animals.
Data obtained from animal experiments were expressed as
mean standard error (S.E.M.). The statistical signifi-
cance of difference between groups were assessed by
means of analysis of variance (ANOVA) followed by
Dunnet’s test. The results of the anti-inflammatory studies
have been presented in Table 4.
In silico studies
The ligands were drawn in ChemDraw Ultra 6.0 (Chem
Office package) assigned with proper 2D orientation and
the structure of each compound was analyzed for connec-
tion error in bond order. OSIRIS, an ADMET based Java
library layer that provides reusable cheminformatics func-
tionality and is an entirely in-house developed drug dis-
covery informatics system was used to predict the total
Table 4 Anti-inflammatory
activity of compounds (4a–j)
Treatment
Dose
(mg/kg)
Initial paw
volume (ml) (b)
Paw volume after
3 h (ml) (a)
Edema
volume (a–b)
Percentage
inhibition
Control
Vehicle
10
0.93 0.01
0.80 0.04
0.8 0.08
2.20 0.04
1.05 0.02
1.78 0.01
1.83 0.06
1.90 0.08
1.38 0.02
1.40 0.04
2.00 0.09
2.38 0.01
1.28 0.04
1.93 0.02
1.80 0.02
1.27 0.02
0.25 0.04
0.90 0.09
0.98 0.02
1.05 0.01
0.55 0.04
0.55 0.08
1.05 0.04
0.93 0.02
0.45 0.08
0.95 0.01
0.98 0.09
Diclofenac
80.4**
29.4*
23.5*
17.6
4a
4b
4c
4d
4e
4f
20
20
0.85 0.04
0.85 0.01
0.83 0.09
0.85 0.02
0.93 0.09
0.95 0.04
0.85 0.01
0.98 0.09
0.83 0.08
20
20
56.9**
56.9**
12.0
20
20
4g
4h
4i
20
13.5
20
64.7**
25.5*
23.5*
* P \ 0.05 compared to control
20
** P \ 0.01 compared to
control
4j
20
123

Med Chem Res (2012) 21:3272–3280
3279
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Kalluraya B, Isloor AM, Priya FV, Jagadeesha RL (2004) Synthesis
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sidered for each compound. All the AutoDock docking
runs were performed in Intel CentrinoCore2Duo CPU @
2.20 GHz of IBM system origin, with 2 GB DDR RAM.
AutoDock 4.0 was compiled and run under Windows XP
Service Pack 3 operating system.
2-[4-arylhydrazono-3-methyl-5-oxo-2-pyrazolin-1-yl]thiazoles.
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Acknowledgments The authors are thankful to HOD of Chemistry
Department and Director of National Institute of Technology-
Karnataka, Surathkal, India for providing research facilities and
encouragement. AMI is also thankful to ‘‘Department of Atomic
Energy, Board for Research in Nuclear Sciences, Government of
India’’ for ‘Young Scientist’ award.
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