Synthesis of novel pyrazole-thiadiazole hybrid as potential potent and selective cyclooxygenase-2 (COX-2) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.08.062

A series of 1,3,4-trisubstituted pyrazole derivatives (3a-f), (4a-f), and (5a-f) have been synthesized and evaluated for their cyclooxygenase (COX-1 and COX-2) inhibitory activity. The structures of newly synthesized compounds were characterized by IR, ¹H NMR, and mass spectral analysis. All of the compounds showed good inhibition of COX-2 with IC₅₀ of 1.33-17.5 μM. Among these derivatives, compound (5c) was the most potent and selective COX-2 inhibitor (IC₅₀ = 1.33 μM), with a significant selectivity index (SI >60). Molecular docking studies were carried out in order to predict the hypothetical binding mode of these compounds to the COX-2 isoenzyme. The result of present study suggests that pyrazole-thiadiazole hybrid could be an interesting approach for the design of new selective COX-2 inhibitory agents.

Full text of DOI:10.1016/j.bmcl.2014.08.062

Accepted Manuscript
Synthesis of novel pyrazole-thiadiazole hybrid as potential potent and selective
cyclooxygenase-2 (COX-2) inhibitors
S.G. Alegaon, M.B. Hirpara, K.R. Alagawadi, K.K. Hullatti, K. Kashniyal
PII:
S0960-894X(14)00919-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.08.062
BMCL 21960
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
31 March 2014
4 August 2014
28 August 2014
Please cite this article as: Alegaon, S.G., Hirpara, M.B., Alagawadi, K.R., Hullatti, K.K., Kashniyal, K., Synthesis
of novel pyrazole-thiadiazole hybrid as potential potent and selective cyclooxygenase-2 (COX-2) inhibitors,
Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.08.062
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Synthesis of novel pyrazole-thiadiazole hybrid as potential potent and selective
cyclooxygenase-2 (COX-2) inhibitors
Authors:
a
a
a
b
a
S.G. Alegaon *, M.B. Hirpara , K.R. Alagawadi , K. K. Hullatti , K.Kashniyal
a
Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy,
Nehrunagar, Belgaum-590 010, Karnataka, India
b
Department of Pharmacognosy, KLE University’s College of Pharmacy, Nehrunagar,
Belgaum-590 010, Karnataka, India
Address:
*Corresponding author: Shankar G. Alegaon, M.Pharm, Ph.D. Associate Professor,
Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy,
Nehrunagar, Belgaum-590 010, Karnataka, India. Tel.: +91 8312471399; Fax. +91
8
312472387.
E-mail: sgalegaon@klepharm.edu, sgalegaon@gmail.com (S.G.Alegaon)
Abstract
A series of 1,3,4-trisubstituted pyrazole derivatives (3a-f), (4a-f), and (5a-f) have
been synthesized and evaluated for their cyclooxygenase (COX-1 and COX-2) inhibitory
1
activity. The structures of newly synthesized compounds were characterized by IR, H
NMR, and mass spectral analysis. All of the compounds showed good inhibition of
COX-2 with IC of 1.33–17.5 µM. Among these derivatives, compound (5c) was the
50
most potent and selective COX-2 inhibitor (IC = 1.33 µM), with a significant selectivity
50
index (SI >60). Molecular docking studies were carried out in order to predict the
hypothetical binding mode of these compounds to the COX-2 isoenzyme. The result of
present study suggests that pyrazole-thiadiazole hybrid could be an interesting
approach for the design of new selective COX-2 inhibitory agents.
Keywords: Pyrazoles, thiadiazole, COX-1 and COX-2, Cytotoxicity, Docking, Drug
likeness.
1

Pyrazole or 1, 2-diazole is nitrogen containing heterocyclic compounds have drawn
a great attention of the researchers in the recent years due to their wide range of
1-4
5
biological activities such as anti-inflammatory, antipyretic, analgesic, antitubercular,
6
7
8
anticancer, antimicrobial, sodium channel blocker, antidepressant, anxiolytic,
9
10
11
12
13
neuroprotective, antiviral,
antioxidant, anti-influenza and antidiabetic, activity.
besides this, pyrazole bearing drug molecules are extensively used in the management
14
15
of atherosclerosis, inflammatory bowel syndrome, and Alzheimer’s disease. In
addition, several active pharmaceutical ingredients, possessing pyrazole as basic
16
17
18
nucleus, like deracoxib celecoxib atorivodine and atorivodine are already in the
market. On the other hand thiadiazole ring system is one of the privileged structure
fragments in modern medicinal chemistry considering its broad biological spectrum and
affinity for various biotargets of this class heterocyclic compound. They are
antimicrobial, antihypertensive, antidepressant, anticonvulsant, antileishmanial, anti-
19,20
inflammatory, and anticancer agent.
reported as potential anti-inflammatory,
cyclooxygenase-2 (COX-2) and lipooxygenase (LOX) inhibitors.
In addition, thiazolidinones were recently
2
1-23
24,25
anticancer,
as well as selective
26,27
In view of the above
facts and modification in the structure of the targets will alter their pharmacological
properties as well as their physiochemical properties. Keeping in view of this and in
28-30
continuation of our search on pharmacologically active heterocyclic compounds,
in
this paper, we described the synthesis and the structure relationship of series of novel
pyrazole derivatives possessing thiazolidinone and thiadiazole moieties as potential
potent and selective cyclooxygenase-2 (COX-2) inhibitors.
The reaction sequence employed for synthesis of the title compounds (3a-f), (4a-f),
and (5a-f) are illustrated in Scheme 1. The two step procedure was adopted for the
synthesis of 4-formylpyrazole derivatives (2a-c). The reaction of substituted-
acetophenones with phenyl hydrazine in acetic acid at room temperature provided the
corresponding hydrazones (1a-c). Treatment of the hydrazone derivatives (1a-c) with
Vilsmeir-Haack
reagent
(DMF-POCl3)
afforded
the
corresponding
3-(4-
31
substitutedphenly)-1-phenyl-1H-pyrazole-4-carbaldehydes (2a-c). Thiosemicarbazone
derivatives (3a-f) were obtained via condensation of 1,3-diarylpyrazole-4-carbaldehyde
2

derivatives (2a-c) with thiosemicarbazide or 4-phenylthiosemicarbazide in presence of a
catalytic amount of conc. HCl in ethanol: chloroform (7: 3) as the solvent affording in
32
satisfactory yield.
The synthesized thiosemicarbazone derivatives (3a-f) were
characterized by IR and NMR, The IR spectra showed the appearance of characteristic
-1
bands at 3397 and 3295 cm assignable for amino (NH) group, while its proton NMR
spectrum indicated singlet at around δ 8.3 ppm for -CH=N- proton, and singlet signals at
δ 11.76 - 11.16 ppm for the =N-NH- proton. The two amino (-NH ) protons appeared as
2
two singlets at 7.7 and 8.2 ppm due to en-thiol tautomerism, while these protons
disappeared in compounds (4a-f), and (5a-f).
The reaction between ethyl bromoacetate and thiosemicarbazone derivatives
(3a-f) in glacial acetic acid containing anhydrous sodium acetate afforded the
33
corresponding 4-thiazolidinone compounds (4a-f). The proton NMR spectra showed
characteristic singlet at δ 4 ppm attributed to CH of 4-thiazolidinone, their IR spectra
2
-1
showed new band at 1700 cm attributed to a carbonyl group of 4-thiazolidinone. On
the other hand reaction of thiosemicarbazone derivatives (3a-f) with acetic anhydride
34
afforded the corresponding 1,3,4-thiadiazole derivatives (5a-f). Their IR spectra
-1
showed new band at 1660-1700 cm attributed to an acetyl group of 1,3,4-thiadiazol-2-
yl, and their proton NMR spectra showed characteristic new singlet at δ 7 ppm
attributed to CH of 1,3,4-thiadiazolyl in addition to signals due to methyl groups of the
CH -C=O at δ 1.84 - 2.2 ppm. The synthesized compounds were evaluated for their
3
ability to inhibit the COX-1 and COX-2 enzymes using COX inhibitor screening kit
26
(Cayman Chemical, Ann Arbor, MI, USA) according to previously reported method.
Indomethacin and celecoxib were used as reference compounds. The results are given
in Table 1. In vitro COX-1/COX-2 inhibition studies revealed that all tested compounds
(
3a-f, 4a-f, and 5a-f) are more potent inhibitor of COX-2 (IC = 1.33-17.5 µM range)
50
than inhibitors of COX-1 (IC = 42.33 to 80.33 µM range). In the thiosemicarbazone
50
series of compounds (3a-f) containing substituted pyrazole, compound 3c displayed
good COX-2 inhibitory activity (COX-1 IC = 62.33 µM, COX-2 IC = 2.5 µM, SI >24),
50
50
compound 3a also exhibited comparable COX-2 inhibitory potency (COX-1 IC = 56.33
50
µM, COX-2 IC = 3.33 µM, SI >16). In the second series of pyrazole compounds (4a-f)
50
3

containing 2-(hydrazinyl) thiazole-4-one, compound 4c displayed moderate COX-2
inhibitory activity (COX-1 IC = 71.33 µM, COX-2 IC = 8.5 µM, SI 8.39). Among the
50
50
third series of compounds (5a-f) possessing novel pyrazole-thiadiazole hybrid,
compound 5c was identified as the most potent and selective COX-2 inhibitor (COX-1
IC = 80.33 µM, COX-2 IC = 1.33 µM) with an significant COX-2 selectivity index (SI
50
50
>60). Compound 5a and 5b also displayed comparable COX-2 inhibitory potency.
From the structure-activity relationship (SAR) perspective, straight comparison of
inhibitory potency and selectivity index profiles of all three series of compounds (3a-f),
4a-f), and (5a-f) revealed that compounds of 4-thiazolidinones possess comparably low
(
COX-2 inhibitory potency and selectivity, except for compound 4c. However, significant
enhancement of COX-2 inhibitory potency and selectivity was achieved with pyrazole-
thiadiazole hybrid compounds. Subsequently SAR studies were performed to determine
how the substituent of subunits affected the cyclooxygenase inhibitory activities. The
Table 1 revealed that none of the tested compounds displayed inhibitory activity on
COX-1 down to 55 µM. Compounds (3a-f), (4a-f), and (5a-f) with para substituted on
phenyl ring B exhibited significant COX-2 inhibitory activities in order of OCH > Cl > Br.
3
The results demonstrated that an electron donating 4-methoxy functional group may be
helpful to improve COX-2 inhibitory activities while electron withdrawing halogen group
may show some relatively negative effects (Fig 1). The results also indicated that in the
case of constant pyrazole ring C substituents. Amongst eighteen pyrazole compounds
tested, compounds with thiosemicarbazide skeleton (3a-c), (4a-c), and (5a-c) exhibited
better activities than those 4-phenylthiosemicarbazide skeleton (3d-f), (4d-f), and (5d-f).
It can be concluded, that almost all synthesized compounds showed selective COX-2
inhibitory activity. In order to validate the cyclooxygenase-2 (COX-2) inhibitory activity
results, most promising compounds 3c, 5a, 5b, and 5c were selected for docking
studies. Molecular docking study was performed by using AutoDock tools 1.5.6 (ADT
35
4
.2). The 3D structures of compounds in pdb form were prepared by using CS
ChemDraw Ultra 12.0. The 3D crystal structure of COX-1 and COX-2 were downloaded
from Protein Data Bank (PDB ID: 1Q4G and 1CX2, respectively). Hetero atoms, water
molecules were removed and hydrogens were added to the protein for correct
4

calculation of partial atomic charges. The grid box was set at 64, 64, and 64 Å (x, y, and
z) with center x = 22.0, y = 35.0 and z = 210.0 for 1Q4G.pdb and 64, 64, and 64 Å (x, y,
and z) with center x = 25.0, y = 23.0 and z = 19.0 for 1CX2.pdb. The Lamarckin genetic
algorithm (LCA) was applied to search conformers that possessed lowest binding
energy. Results of molecular docking analysis were obtained as estimated free energy
of binding in kal/mol. The molecular docking studies revealed that compounds 3c, 5a,
5
b, and 5c were more selective towards COX-2 than COX-1. The calculated binding
energy of docked compounds towards COX-2 was found to be between -9.85 and -
1.11 kcal/mol, whereas compounds docked to COX-1 exhibited binding energy
1
between -4.12 and -5.92 kcal/mol Table 2. The binding mode of the most active
inhibitors 5a and 5c to the COX-2 protein and interactions are shown in Fig. 2. It was
observed that the compound 5a exhibited binding energy -11.11 kcal/mol, which is
lower than that of the well-known coxib inhibitor, celecoxib (-10.37 kcal/mol) Table 2.
The compound 5a is nicely bond to COX-2 via two hydrogen bonds and a π – cation
interaction. The oxygen atom of 4-acetyl group of compound 5a forms hydrogen bonds
(N----H-N: 2.37 Å) with the amino hydrogen atom of ARG 513 residue and acetamide
group of compound 5a forms hydrogen bonds (N----H-N: 2.37 Å) with the amino
hydrogen atom of ARG 120 and phenyl ring forms a π – cation interaction with the
amino cation (distance: 4.98 Å) of ARG 513. Compound 5c is satisfactorily bond to
COX-2 via three hydrogen bonds and a π – cation interaction. The 4-position nitrogen
atom of 1,3,4-thiadiazole ring forms a hydrogen bond (N----H-N: 1.99 Å) with the amino
hydrogen atom of ARG 513, and sulphur atom of 1,3,4-thiadiazole ring forms a
hydrogen bond (S----H-O: 5.10 Å) with the hydroxyl hydrogen atom of TYR 355, and
amino hydrogen of acetamide group forms a hydrogen bond (N-H----O: 1.85 Å) with the
oxygen atom of GLU 524, and phenyl ring forms a π – cation interaction with the amino
cation (distance: 4.69 Å) of ARG 120. In general, the molecular docking results were
essentially in agreement with the pharmacological data in terms of evaluation of binding
energy and binding mode.
The 3-(4, 5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) cell
36
proliferation assay was used to evaluate cytotoxicity of the synthesized compounds
5

against normal HDF (Human dermal fibroblast) cell line. The cytotoxicity results were
summarized in Table 3. The data showed that most of 1,3,4-trisubstituted pyrazole
derivatives having a quite low toxicity. The compounds 3c, 3d, 4d, 4f, 5d, and 5f inhibited
approximately 38% cell viability at 100 µM, with compound 3a being the least toxic.
37, 38
Analysis of molecular parameters that connected with Lipinski’s rule of five.
Briefly, this simple rule is based upon the observation that most of pharmacological
active agents have a molecular weight (MW) of 500 or less, a log p not more than 5,
number of hydrogen bond donor atoms of 5 or less, and number of hydrogen bond
accepter atoms of 10 or less (N or O) (Table 4). Results showed that most of the
synthesized compounds 3a, 3b, 3c, 3f, 4a, 4b, 4c and 4f obey the Lipinski’s rule of five.
As summarized in Table 3, pyrazole derivatives containing thiadiazole 5a, 5b, and 5c
moiety showed favorable values for involved molecular parameters that fit well with the
Lipinski’s rule of five. Whereas, Compounds 3d, 3e, and 4d showed a LogP values
larger than five, and compounds 5d, 5e and 5f exhibited a molecular weight higher than
5
00, thus violating the Lipinski’s rule of five, but since the number of violations is no
more than one, thus acceptable. These data may suggest the potentiality of these
derivatives as new cyclooxygenase-2 (COX-2) inhibitory agents.
In summary, a series of 1,3,4-trisubstituted pyrazole derivatives have been
synthesized and evaluated for their cyclooxygenase (COX-1 and COX-2) inhibitory
activity. In synthesized compounds, 5c displayed the most potent COX-2 inhibitory
activity with IC of 1.33 µM but weak to COX-1. Molecular docking studies revealed that
50
pyrazole analogues exhibits good interact with active site of the docked receptor
cyclooxygenase-2. The result of present study suggests that pyrazole-thiadiazole hybrid
could be an interesting approach for the design of new selective COX-2 inhibitory
agents.
Acknowledgements
Authors are grateful to Dr. A. D. Taranalli, Principal and Dean Faculty of
Pharmacy, for providing necessary facilities for the research. Authors are also grateful
to NMR Research center, IISC, Bangalore, India, for providing the spectral data.
6

References and notes
1
.
Chandra, N.; Kumar, S.; Kaushik, P.; Kaushik, D.; Roy, S. K.; Gupta, G. K.;
Jachak, S. M.; Kapoor, J. K.; Sharma, P. K. Bioorg. Med. Chem. 2013, 21, 4581.
El-Sayed, M. A. A.; Abdel-Aziz, N. I.; Abdel-Aziz, A. A. M.; El-Azab, A. S. Bioorg.
Med. Chem. 2012, 20, 3306.
2
.
3
4
.
.
Dannhardt, G.; Kiefer, W. Eur. J. Med. Chem. 2001, 36, 109.
Sauzem, P. D.; Sant’Anna, G. D. S.; Machado, P.; Duarte, M. M. M. F.; Ferreira,
J.; Mello, C. F.; Beck, P.; Bonacorso, H. G.; Zanatta, N.; Martins, M. A. P.; Rubin,
M. A. Eur. J. Pharmacol. 2009, 61, 91.
5
.
Alegaon, S. G.; Alagawadi, K. R.; Dadwe, D. H. DOI: 10.1055/s-0033-1363976.
Drug Res (Arzneimittelforschung).
6
7
.
.
Koca, I.; Ozgur, A.; Coskun, K. A.; Tutar, Y. Bioorg. Med. Chem. 2013, 21, 3859.
Tanitame, A.; Oyamada, Y.; Ofuji, K.; Terauchi, H.; Kawasaki, M.; Wachi, M.;
Yamagishi, J. I. Bioorg. Med. Chem. Lett. 2005, 15, 4299.
8
.
Tyagarajan, S.; Chakravarty, P. K.; Zhou, B.; Taylor, B.; Eid, R.; Fisher, M. H.;
Parsons, W. H.; Wyvratt, M. J.; Lyons, K. A.; Klatt, T.; Li, X.; Kumar, S.; Williams,
B.; Felix, J.; Priest, B. T.; Brochu, R. M.; Warren, V.; Smith, M.; Garcia, M.;
Kaczorowski, G. J. Bioorg. Med. Chem. Lett. 2010, 20, 7479.
9
1
.
Gokhan-Kelekci, N.; Koyunoglu, S.; Yabanoglu, S.; Yelekci, K.; Ozgen, O.; Ucar,
G.; Erol, K.; Kendi, E.; Yesilada, A. Bioorg. Med. Chem. 2009, 17, 675.
Shih, S. R.; Chu, T. Y.; Reddy, G.; Tseng, S. N.; Chen, H. L.; Tang, W. F.; Wu,
M. S.; Yeh, J. Y.; Chao, Y. S.; Hsu, J.; Hsieh, H. P.; Horng, J. T. J. Biomed. Sci.
0.
2
010, 17, 13.
1
1
1.
2.
Bandgar, B. P.; Gawande, S. S.; Bodade, R. G.; Gawande, N. M.; Khobragade,
C. N. Bioorg. Med. Chem. 2009, 17, 8168.
Shih, S. R.; Chu, T. Y.; Reddy, G. R.; Tseng, S. N.; Chen, H. L.; Tang, W. F.;
Wu, M. S.; Yeh, J. Y.; Chao, Y. S.; Hsu, J. T.; Hsieh, H. P.; Horng, J. T. J.
Biomed. Sci. 2010. 17, 13. doi: 10.1186/1423-0127-17-13.
1
3.
Shen, D. M.; Brady, E. J.; Candelore, M. R.; DallasYang, Q.; Ding, V. D. H.;
Feeney, W. P.; Jiang, G.; McCann, M. E.; Mock, S.; Qureshi, S. A.; Saperstein,
7

R.; Shen, X.; Tong, X.; Tota, L. M.; Wright, M. J.; Yang, X.; Zheng, S.; Chapman,
K. T.; Zhang, B. B.; Tata, J. R.; Parmee, E. R. Bioorg. Med. Chem. Lett. 2011,
2
1, 76.
1
4.
Liu, X.; Huang, X.; Lin, W.; Wang, D.; Diao, Y.; Li, H.; Hui, X.; Wang, Y.; Xu, A.;
Wu, D.; Ke, D. Bioorg. Med. Chem. Lett. 2011, 21, 2949.
1
1
5.
6.
Lan, P.; Huang, Z. J.; Sun, J. R.; Chen, W. M. Int. J. Mol. Sci. 2010, 11, 3357.
Sakya, S. M.; Cheng, H.; Lundy DeMello, K. M.; Shavnya, A.; Minich, M. L.; Rast,
B.; Dutra, J.; Li, C.; Rafka, R. J.; Koss, D. A.; Li, J.; Jaynes, B. H.; Ziegler, C. B.;
Mann, D. W.; Petras, C. F.; Seibel, S. B.; Silvia, A. M.; George, D. M.; Hickman,
A.; Haven, M. L.; Lynch, M. P. Bioorg. Med. Chem. Lett. 2006, 16, 1202.
Ranatunge, R. R.; Garvey, D. S.; Janero, D. R.; Letts, L. G.; Martino, A. M.;
Murty, M. G.; Richardson, S. K.; Young, D. V.; Zemetseva, I. S. Bioorg. Med.
Chem. 2004, 12, 1357.
1
7.
1
1
2
2
2
2
2
2
8.
9.
0.
1.
2.
3.
4.
5.
Reddy, G. J.; Pallavi, K.; Reddy, R. S.; Reddy, K. S. Indian. J. Chem. 2005, 44,
8
12.
Kumar, D.; Kumar, N. M.; Chang, K. H.; Shah, K. Eur. J. Med. Chem. 2010, 45,
664.
Alegaon, S. G.; Alagawadi, K. R. Letters in Drug Design & Discovery. 2011, 8,
42.
4
6
Hu, J.; Wang, Y.; Wei, X.; Wu, X.; Chen, G.; Cao, G.; Shen, X.; Zhang, X.; Tang,
Q.; Liang, G.; Li, X. Eur. J. Med. Chem. 2013, 64, 292.
Apostolidis, J.; Liaras, K.; Geronikaki, A.; Hadjipavlou-Litina, D.; Gavalas, A.;
Sokovic, M.; Glamoclija, J.; Ciric, A. Bioorg. Med. Chem. 2013, 21, 532.
Helal, M. H. M.; Salem, M. A.; El-gaby, M. S. A.; Aljahdali, M. Eur. J. Med. Chem.
2
013, 65, 517.
Wang, S.; Zhao, Y.; Zhang, G.; Lv, Y.; Zhang, N.; Gong, P. Eur. J. Med. Chem.
011, 46, 3509.
2
Hayrylyuk, D.; Mosula, L.; Zimenkovsky, B.; Vasylenko, O.; Gzella, A.; Lesyk, R.
Eur. J. Med. Chem. 2010, 45, 5012.
8

2
2
6.
7.
Unsal-Tan, O.; Ozadali, K.; Paskin, K.; Balkan, A. Eur. J. Med. Chem. 2012, 57,
9.
5
Eleftheriou, P.; Geronikaki, A.; Litina, D. H.; Vicini, P.; Filz, O.; Filimonov, D.;
Poroikov, V.; Chaudhaery, S. S.; Roy, K. P.; Sexena, A. K. Eur. J. Med. Chem.
2
012, 47, 111.
2
8.
Alegaon, S. G.; Alagawadi, K. R.; Sonkusare, P. V.; Chaudhary, S. M.; Dadwe,
D. H.; Shah, A. S. Bioorg. Med. Chem. Lett. 2012, 22, 1917.
2
3
9.
0.
Alegaon, S. G.; Alagawadi, K. R.; Med. Chem. Res. 2012, 21, 3214.
Alegaon, S. G.; Alagawadi, K. R.; Pawar, S. M.; Vinod, D.; Rajput, U. Med.
Chem. Res. 2014, 23, 987.
3
3
1.
2.
Huang, X. F.; Lu, X.; Zhang, Y.; Song, G. Q.; He, Q. L.; Li, Q. S.; Yang, X. H.;
Wei, Y.; Zhu, H. L. Bioorg. Med. Chem. 2012, 20, 4895.
General procedure for the synthesis of compounds (3a-f). To a solution of 3-(4-
substitutedphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (2a-c) (10 mmol) in 30
ml of ethanol: chloroform (7:3). thiosemicarbazide or 4-phenylthiosemicarbazide
(10 mmol) was added with stirring and resulting reaction mixture was stirred at 80
ºC for 10 hours. The reaction was monitored by thin-layer chromatography (TLC)
for completion. Reaction mixture was cooled to room temperature solid obtained
was filtered, washed with cold ethanol, and recrystallized from ethanol.
3
3.
General procedure for the synthesis of compounds (4a-f). To a solution of1-((3-
(
substituted)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl) methylene) thiosemicarbazide
3a-c) or 1-((3-(substituted)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)methylene)4-
(
phenylthiosemicarbazide (3d-f) (10 mmol) in 15 ml of glacial acetic acid,
ethylbromoacetate, (0.01mol) and anhydrous sodium acetate (10 mmol) were
added with stirring at room temperature. The resulting reaction mixture was
heated under reflux for 8 h. The reaction was monitored by thin-layer
chromatography (TLC) for completion. Reaction mixture was cooled to room
temperature and poured into ice-cold water; the solid precipitated was filtered,
washed with water, dried and recrystallized from ethanol.
9

3
4.
General procedure for the synthesis of compounds (5a-f). To a solution of1-((3-
(
substituted)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)methylene) thiosemicarbazide
3a-c) or 1-((3-(substituted)-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl) methylene)4-
(
phenyl thiosemicarbazide (3d-f) (0.01mol) in acetic anhydride (10ml) was heated
under reflux for 5 hours. The reaction was monitored by thin-layer
chromatography (TLC) for completion. After the reaction mixture was attained at
room temperature, excess acetic anhydride was decomposed by water and the
reaction mixture was stirred for 30 minutes. The separated product was collected
by filtration, washed with water, dried and recrystallized from ethanol.
Autodock tools (ADT) program, Molecular Graphics Laboratory, the Scripps
Research Institute. http://autodock.scripps.edu/.
3
5.
3
3
6.
7.
Mosmann, T. J. Immunol. Methods. 1983, 65, 55.
Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug. Deliv.
Rev. 2001, 46, 3.
3
8.
Molinspiration Cheminformatics, Bratislava, Slovak Republic, Available from:
http://www.molinspiration.com/services/properties.html.
1
0

Caption illustrations for figure
Figure 1. 1,3,4-trisubstituted pyrazole prototype
Figure 2. Docking figures of 5a and 5c in COX-2 protein cavity.
Scheme 1. Reagents and conditions: (a) Dry ethanol, glacial acetic acid (1mL) reflux 5
h; (b) DMF/POCl , 80 ºC; (c) Thiosemicarbazide or 4-phenylthiosemicarbazide, ethanol:
3
chloroform (7:3), reflux 10 h; (d) Ethylbromoacetate, anhydrous sodium acetate, glacial
acetic acid, reflux 8 h; (e) Acetic anhydride, reflux 5 h.
Caption illustrations for table
Table 1.
Data of the in vitro COX-1/COX-2 enzyme inhibition assay of synthesized compounds
a
IC values is the test compound concentration required to produce 50% inhibition of
50
COX-1/COX-2 for means of two determinations and deviation from the mean is <10% of
mean values.
b
selectivity index (COX-1 IC /COX-2 IC ).
50
50
Table 2. Binding energy of compounds (3a, 5a, 5b, and 5c) after docking in to COX-1
and COX-2 active site
Table 3. Cytotoxicity of compounds against HDF (Human dermal fibroblast) cell line
Each experiment was independently performed three times, and data are shown as
means SD.
Table 4. Molecular parameters of 1,3,4-trisubstituted pyrazole derivatives (3a-f), (4a-f),
and (5a-f)
a
b
c
TPSA, topological polar surface area; n-rotb, number of rotatable bonds ; nON,
d
number of hydrogen bond acceptors; nOHNH, number of hydrogen bond donors;
e
miLogP, logarithm of compound partition coefficient between n-octanol and water;
f
MW, molecular weight.
1
1

Table 1.
Data of the in vitro COX-1/COX-2 enzyme inhibition assay of synthesized compounds
b
Compound
R1
R2
COX-1
COX-2
(SI)
a
a
IC (µM)
IC (µM)
50
50
3
3
3
3
3
a
b
c
d
e
Cl
Br
OCH3
Cl
Br
OCH3 C H
Cl
Br
OCH3
Cl
H
H
H
56.33
60.33
62.33
64.5
65.66
42.33
60.66
66.33
71.33
62.33
60.5
62.33
70.33
64.66
80.33
70.33
72.66
64.66
9
3.33
6.33
2.5
8.33
11.66
7.33
10.66
12.66
8.5
15.33
17.5
12.66
2.33
2.5
1.33
3.33
6.5
16.91
9.53
24.93
7.74
5.63
5.77
5.69
5.23
8.39
4.06
C H
6
5
5
5
C H
6
3
f
6
4a
4b
4c
4d
4e
H
H
H
C H
6
5
5
5
Br
C H
3.45
4.92
6
4
f
OCH3 C H
Cl
Br
OCH3
Cl
6
5a
5b
5c
5d
5e
H
H
H
30.18
25.86
60.39
21.12
11.17
19.41
2.9
C H
6
5
5
5
Br
C H
6
5
f
OCH3 C H
-
-
3.33
3.1
0.3
6
Indomethacin
Celecoxib
-
-
32
106.66
a
IC50 values is the test compound concentration required to produce 50% inhibition of
COX-1/COX-2 for means of two determinations and deviation from the mean is <10% of
mean values.
b
selectivity index (COX-1 IC /COX-2 IC ).
50
50

Table 2. Binding energy of compounds (3a, 5a, 5b, and 5c) after docking in to COX-1
and COX-2 active site
COX-1
COX-2
Compound
Binding
Energy
-5.92
Hydrogen bonding
residues
ARG 120, GLN 192,
GLN 351
TYR 355
GLN 192
ARG 197, GLN 350,
GLY 354
GLN 350, TYR 355
Binding
Energy
-9.85
Hydrogen bonding
residues
ARG 120, SER 353,
GLN 192, LEU 352
ARG 120, ARG 513
ARG 513
ARG 120, ARG 513,
TYR 355, GLU 524
ARG 120, ARG 513,
HIS 90
3
c
5a
5b
5c
-5.47
-4.12
-4.93
-11.11
-11.03
-10.08
-10.37
Celecoxib
-6.93

Table 3. Cytotoxicity of compounds against HDF (Human dermal fibroblast) cell line
Compound R1
R2
%Cell viability ±
SD at 100 µM
71.74 ± 1.2
57.63 ± 1.0
63.37 ± 1.2
61.67 ± 2.6
58.74 ± 2.6
52.63 ± 2.1
58.74 ± 3.5
55.63 ± 1.0
59.67 ± 2.6
60.67 ± 1.0
57.37 ± 1.2
63.37 ± 1.2
57.74 ± 3.5
51.63 ± 2.1
56.67 ± 2.6
61.67 ± 2.6
51.37 ± 1.2
61.37 ± 1.2
3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
a
b
c
d
e
f
a
b
c
d
e
f
a
b
c
d
e
f
Cl
Br
OCH3
Cl
Br
OCH3
Cl
Br
OCH3
Cl
Br
OCH3
Cl
Br
OCH3
Cl
Br
H
H
H
C H
6
5
5
5
C H
6
C H
6
H
H
H
C H
6
5
5
5
C H
6
C H
6
H
H
H
C H
6
5
5
5
C H
6
OCH3
C H
6
Each experiment was independently performed three times, and data are shown as
means SD.

Table 4. Molecular parameters of 1,3,4-trisubstituted pyrazole derivatives (3a-f), (4a-f), and (5a-f)
a
b
c
d
e
f
Compound
Rule of 5
TPSA n-rotb
nON nOHNH miLogp
MW
Rule of 5 violation
-
-
≤10
5
5
6
5
5
6
6
6
7
6
6
7
7
7
8
7
7
8
5
5
≤5
3
3
3
2
2
2
1
1
1
0
0
0
1
1
1
0
0
0
1
2
≤5
≤500
≤1
0
0
0
1
1
0
0
0
0
1
2
0
0
0
0
1
1
1
0
0
3
3
3
3
3
a
b
c
d
e
68.24
68.24
77.47
54.24
54.24
63.47
71.65
72.65
80.88
62.86
62.86
72.09
79.59
79.59
88.31
70.80
70.80
80.04
68.53
77.99
5
5
6
7
7
8
5
5
6
5
5
6
4
4
5
5
5
6
4
4
3.88
4.01
3.26
5.31
5.45
4.69
3.68
3.81
3.06
5.09
5.22
4.47
2.69
2.82
2.07
4.63
4.76
4.01
3.98
3.61
355.85
400.30
351.43
431.95
476.40
427.53
395.87
440.32
391.45
471.97
516.42
467.55
439.92
484.37
435.50
516.02
560.47
511.60
357.79
381.37
3
f
4a
4b
4c
4d
4e
4
f
5a
5b
5c
5d
5e
5
f
Indomethacin
Celecoxib
a
b
c
d
TPSA, topological polar surface area; n-rotb, number of rotatable bonds ; nON, number of hydrogen bond acceptors; nOHNH,
e
f
number of hydrogen bond donors; miLogP, logarithm of compound partition coefficient between n-octanol and water; MW,
molecular weight.

Scheme 1. Reagents and conditions: (a) Dry ethanol, glacial acetic acid (1mL) reflux 5
h; (b) DMF/POCl , 80 ºC; (c) Thiosemicarbazide or 4-phenylthiosemicarbazide, ethanol:
3
chloroform (7:3), reflux 10 h; (d) Ethylbromoacetate, anhydrous sodium acetate, glacial
acetic acid, reflux 8 h; (e) Acetic anhydride, reflux 5 h.

Figure 1. 1,3,4-trisubstituted pyrazole prototype

Figure 2. Docking figures of 5a and 5c in COX-2 protein cavity.

GRAPHICAL ABSTRACT
Synthesis of novel pyrazole-thiadiazole hybrid as potential potent and
selective cyclooxygenase-2 (COX-2) inhibitors
a
a
a
b
a
S.G. Alegaon *, M.B. Hirpara , K.R. Alagawadi , K. K. Hullatti , K.Kashniyal
a
Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy,
Nehrunagar, Belgaum-590 010, Karnataka, India
b
Department of Pharmacognosy, KLE University’s College of Pharmacy,
Nehrunagar, Belgaum-590 010, Karnataka, India
A novel class of pyrazole derivatives have been synthesized and investigated for
their in vitro cyclooxygenase inhibitory and cytotoxicity activities. Compound 5c
showed potential selective COX-2 inhibitory activity.