Synthesis, anti-inflammatory, antiplatelet and in silico ealuations of (E)-3-(3-(2,3-dihydro-3-methyl-2-oxo-3H-benzoxazole-6-yl)-1-phenyl-1H-pyrazole- 4-yl)acrylamides

Article abstract of DOI:10.3906/kim-1110-8

A series of (E) -3-(3-(2,3-dihydro-3-methyl-2-oxo-3H -benzoxazole-6-yl)-1- phenyl-1H -pyrazole-4-yl)acrylamides (7a-k) were synthesized and ealuated for their in itro inhibitory actiities on COX-1 and COX-2 isoforms using a human whole blood assay as well as their antiplatelet profile against human platelet aggregation using arachidonic acid as agonists. Among the synthesized deriaties 7a-k, especially compound 7g exhibited dual anti-inflammatory and antiplatelet actiity with selectie COX-2 inhibition. TUeBITAK.

Full text of DOI:10.3906/kim-1110-8

Turk J Chem
36 (2012) , 367 – 382.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1110-8
Synthesis, anti-inflammatory, antiplatelet and in silico
evaluations of (E)-3-(3-(2,3-dihydro-3-methyl-2-oxo-
3H -benzoxazole-6-yl)-1-phenyl-1H -pyrazole-
4-yl)acrylamides
2
3
Sultan BAYTAS¸1,∗, Nilu¨fer Nermin TURAN DURAL , Ye¸sim OZKAN ,
¨
3
4
1,†
˙
¨
¨
¨
Hasan Bolkan S¸IMS¸EK , Tu¨rkiz GURSEL , Serdar UNLU
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Gazi University, Ankara-TURKEY
2Department of Pharmacology, Faculty of Pharmacy, Gazi University, Ankara-TURKEY
3Department of Biochemistry, Faculty of Pharmacy, Gazi University, Ankara-TURKEY
4Pediatric Hematology Unit, Department of Pediatrics, Gazi University School of Medicine,
Ankara-TURKEY
e-mail: baytas@gazi.edu.tr
Received: 07.10.2011
A series of (E)-3-(3-(2,3-dihydro-3-methyl-2-oxo-3H -benzoxazole-6-yl)-1-phenyl-1H -pyrazole-4-yl)acry-
lamides (7a–k) were synthesized and evaluated for their in vitro inhibitory activities on COX-1 and COX-2
isoforms using a human whole blood assay as well as their antiplatelet profile against human platelet aggre-
gation using arachidonic acid as agonists. Among the synthesized derivatives 7a–k, especially compound
7g exhibited dual anti-inflammatory and antiplatelet activity with selective COX-2 inhibition.
Key Words: Anti-inflammatory activity, antiplatelet activity, pyrazoles, COX, TXA2 synthetase
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most frequently prescribed drugs for treatment
of pain, fever, and inflammatory and rheumatic diseases. Studies in this field, aimed at discovering better
∗Corresponding author
†Present address: FARGEM (Pharmaceutical Research and Development Center) Inc., Sancaklar Mevkii, 81100 Du¨zce-TURKEY
367

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
tolerated and potent NSAIDs with fewer side effects characteristic of current NSAIDs have been of interest for
many years. The non-selective inhibition of the 2-isoforms of COX is considered to have been responsible for
the unfavorable side effects associated with the chronic use of NSAIDs. It was assumed that more selective
COX-2 inhibitors would have reduced side effects due to the COX-1 inhibition.1,2 Therefore, it would be
useful to develop COX-2 selective inhibitors, which are demonstrated to possess a significantly enhanced gastric
safety compared to non-selective NSAIDs. Celecoxib and rofecoxib are 2 well known selective COX-2 inhibitors
belonging to the COXIB class. However, rofecoxib and other COX-2 inhibitors have been withdrawn from
the market due to their adverse cardiovascular side effects. Recent studies have shown that COX-2 inhibitors
are associated with increased thromboembolic risk in cardiovascular disease patients. 3−5 A selective COX-2
inhibitor causes reduction of prostacyclin (PGI2) amount. Meanwhile, thromboxane A2 (TxA2) production
is still continued in platelets by COX-1 isoform. It is considered that cardiovascular toxicity of coxibs was
generated because of imbalance between PGI2 and TxA2 levels.6 In a patient who requires effective anti-
inflammatory treatment and has a high risk for both gastrointestinal bleeding and cardiovascular thrombosis,
the use of selective COX-2 inhibitors with antiplatelet agent is recommended.7 Moreover, there is currently no
clear evidence that COX-2 inhibitors represent an independent risk factor in patients at low risk of cardiovascular
diseases. Therefore, the clinical rationale for developing compounds with selective COX-2 inhibition still remains
to be established.5,8
There are several reviews that mainly focus on the molecular and functional bases of the inhibition
of COX enzymes by non-selective and COX-2 selective inhibitors.9,10 The pharmacophores for most of the
selective COX-2 inhibitors consist of a central 1,2-diarylsubstituted 5-membered heterocyclic ring. 2,11−13 Some
1,3-diarylpyrazoles derivatives have also been reported as selective COX-2 inhibitors.14,15 Some studies showed
that preparation of the amide derivatives of currently used NSAIDs might be a useful approach to achieve novel
COX-2 inhibitors. Kalgutgar et al. showed that amide derivatives of indomethacin16 and meclofenamic acid17
selectively inhibited COX-2. Recently, this approach has been used to prepare ester and amide derivatives
of ibuprofen, ketoprofen, and mefenamic acid, and the researchers found that especially ester derivative of
ketoprofen showed more potent analgesic and anti-inflammatory activity than the parent drug.18
The 2-oxo-3H-benzoxazole ring has become an important building block in medicinal chemistry and
has led to the discovery of a number of derivatives endowed with antispasmodic, antitubercular, antibacterial,
antimicrobial, antifungal, and normolipemic effects.19,20 Several 2-oxo-3H -benzoxazole derivatives have been
previously reported as analgesic and anti-inflammatory agents.21,22 Our research group has been interested
for some time in studying the effect of substituting selected aromatic rings in current NSAIDs with alternative
heteroaromatic moieties such as 2-oxo-3H -benzoxazole, 2-oxo-3H -benzothiazole and 3(2H)-pyridazinone.23−25
The (3-methyl-2-oxo-3H-benzoxazole-6-yl)-1H -pyrazole derivatives were found to be potent COX-2 inhibitors;
especially compound A (Figure) demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC50
1 μM, COX-2 IC50 = 0.011 μM).26
=
N-Phenylpyrazole arylhydrazone derivative (B in Figure) was found to be active as an analgesic, anti-
inflammatory and antiplatelet agent. These types of compounds were very effective in antiplatelet activity
on arachidonic acid induced platelet aggregation in rabbit citrated platelet rich plasma possibly acting at the
arachidonic acid cascade level.27,28
Ozagrel is a cinnamic acid derivative having an imidazol-1-yl substituent (Figure) that acts as a selective
368

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
inhibitor of TxA2 synthetase with an IC50 of 11 nM29 Essential structural features of TxA2 synthetase
inhibitors are as follows: a basic nitrogen atom of a substituted pyridine or imidazole ring, and a carboxylic
acid group separated by a unsaturated trans-alkyl chain.6 6-Phenyl-3(2H)-pyridazinones were reported as
antiplatelet agents and the lead compound (IC50 = 25 μM) contained a 3-oxoprop-1-en-1-yl fragment as a
key structural element in the heterocyclic core.30 Optimization studies have shown that removal of the phenyl
group at C-6 and addition of a benzyl group at N-2 increases the antiplatelet activity (Figure, compound C
IC50 = 4.2 μM).
H3C
H3C
NO2
O
N
H
N
O
N
N
H
N
N
N
O
CF3
N
N
O
N(CH3)2
3(2H)-pyridazinone derivative
Cl
IC50=4.2 µM
N-Phenylpyrazole arylhydrazone
(3-methyl-2-oxo-3H-benzoxazole-6-yl)-
1H-pyrazole derivative
derivative
C
B
A
O
N
OH
N
CH3
N
O
ozagrel
O
O
R
N
N
Figure Design of the title compounds.
In the search for new anti-inflammatory and antiplatelet agents, we describe herein the design, syn-
thesis and anti-inflammatory and antiplatelet evaluation of new (E)-3-(3-(2,3-dihydro-3-methyl-2-oxo-3H-
benzoxazole-6-yl)1-phenyl-1H -pyrazole-4-yl) acrylamides, which bear a central pyrazole ring whose 1,3,4-
positions are substituted with phenyl, 3-methyl-2(3H)-benzoxazole moieties and 3-oxoprop-1-en-1-yl fragment,
respectively (Figure). Finally, these derivatives were submitted to in silico oral biodisponibility screening to
analyze their overall potential to qualify for use as a drug.
369

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
Experimental
Chemistry
The chemicals were purchased from commercial vendors and used without purification. Thin-layer chromatog-
raphy (TLC) was performed on Merck 60F254 plates. The reactions were monitored by TLC on silica gel, with
detection by UV light (254 nm) or charring Dragendorff reagent.31 Melting points were determined with an
SMP-II Digital Melting Point Apparatus and are uncorrected (Schorpp Geaetetechnik, Germany). IR spectra
were recorded on a Bruker Vector 22 spectrometer as KBr disks. 1 H-NMR spectra were recorded in CDCl3 or
DMSO-d6 on a Varian Mercury 400 MHz High Performance Digital FT-NMR spectrometer using tetramethyl-
silane as the internal standard at the NMR facility of the Faculty of Pharmacy, Ankara University. All chemical
shifts were recorded as δ (ppm). High resolution mass spectra data (HRMS) were collected in-house using
a Waters LCT Premier XE Mass Spectrometer (high sensitivity orthogonal acceleration time-of-flight instru-
ment) operating in either ESI (+) or ESI (-) methods, also coupled to an AQUITY Ultra Performance Liquid
Chromatography system (Waters Corporation, Milford, MA, USA).Flash chromatography was performed with
r
ꢀ
a Combiflash Rf automated flash chromatography system with RediSep columns (Teledyne-Isco, Lincoln, NE,
USA) using dichloromethane-methanol solvent gradients. Elemental analyses were performed with a LECO-932
(C, H, N, S-Elemental Analyzer) at the Faculty of Pharmacy, Ankara University. Microwave-assisted reactions
were carried out with a Milestone MicroSYNTH Microwave Synthesis System.
2-Oxo-3H -benzoxazole (1)
A dry flask charged with o-aminophenol (10.91 g, 0.1 mol) and urea (12.01 g, 0.2 mol) was placed in the
MicroSYNTH Microwave Synthesis System and irradiated at 400 W for 15 min while the temperature was set
to 140 ◦ C. After the reaction was completed, the flask was cooled to room temperature and the solid was
solved in 5% solution of sodium hydroxide. After acidification with concentrated HCl, the desired product was
obtained. Yield 11.61 g (86%). mp 136 ◦ C (Ref.32 ; 137-138 ◦ C).
3-Methyl-2-oxo-3H-benzoxazole (2)
The described method was used,33 and the reaction product was obtained in a yield of about 90%; mp 83 ◦ C
(Ref.34 ; 83-84 ◦ C).
6-Acetyl-3-methyl-2-oxo-3H-benzoxazole (3)
A dry flask charged with 3-methyl-2-oxo-3H-benzoxazole (7.45 g, 0.05 mol), acetic acid (3.15 mL, 0.055 mol),
and PPA (100 g) was placed in the MicroSYNTH Microwave Synthesis System and irradiated at 300 W for
22 min while the temperature was set to 90 ◦ C. The reaction mixture was poured into ice-cold water. The
precipitated mixture was filtered off, dried, and purified by recrystallization from ethanol–water mixture. Yield
8.5 g (89%). mp 167-168 ◦ C (Ref.35 ; 166-168 ◦ C).
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Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
6-Acetyl-3-methyl-2-oxo-3H-benzoxazolephenyl hydrazone (4)
A solution of 6-acetyl-3-methyl-2-oxo-3H-benzoxazole (10 g, 0.052 mol), phenyl hydrazine (6.27 g, 0.058 mol)
and acetic acid (2 mL, 0.035 mol) in ethanol was stirred for 2 h at reflux and then evaporated. The precipitate
was filtered off and dried. Yield 11.64 g (70%). mp 229-230 ◦ C. FT-IR (KBr) cm−1 1754 (C=O).
3-(2,3-Dihydro-3-methyl-2-oxo-3H-benzoxazole-6-yl)-1-phenyl-1H-pyrazole-4-carboxy aldehyde 5
Method A
In a dry flask, POCl3 (2.8 mL, 0.03 mol) was added dropwise to an ice-cold stirred solution of phenyl
hydrazone of 3-methyl-6-acetyl-2(3H)-benzoxazolone (2.8 g, 0.01 mol) in 10 mL of DMF. The reaction mixture
was allowed to attain room temperature, and was then heated at 50 ◦ C for about 4 h. The resulting mixture
was poured onto crushed ice, neutralized with dilute NaOH and left overnight. The yellow precipitate obtained
was purified by crystallization in toluene. Yield 2.6 g (85%).
Method B
In a dry flask, POCl3 (2.8 mL, 0.03 mol) was added dropwise to an ice-cold stirred solution of phenyl
hydrazone of 3-methyl-6-acetyl-2(3H)-benzoxazolone (2.8 g, 0.01 mol) in 10 mL of DMF. The reaction mixture
was allowed to attain room temperature, and then flask was placed in the MicroSYNTH Microwave Synthesis
System and irradiated at 210 W for 5 min while the temperature was set to 50 ◦ C. The resulting mixture
was poured onto crushed ice, neutralized with dilute sodium hydroxide and left standing overnight. The yellow
precipitate obtained was purified by crystallization in toluene. Yield 3 g (94.3%). mp 255-257 ◦ C. FT-IR (KBr)
cm−1 1759 (C=O), 1688 (C=O). 1 H-NMR (400 MHz, CDCl3) δ 10.05 (s, 1H, CHO), 8.54 (s, 1H, pyrazole-H5),
7.78 (m, 4H, ArH), 7.53 (t, 2H, J = 8.0, ArH), 7.41 (d, 1H, J = 7.6, ArH), 7.08 (d, 1H, J = 8.0, ArH), 3.47
(s, 3H, CH3). Anal. (C18 H13 N3 O3): C, H, N calc. 67.71, 4.10, 13.16 found 67.67, 4.11, 13.21.
(E)-3-(3-(2,3-Dihydro-3-methyl-2-oxo-3H-benzoxazole-6-yl)-1-phenyl-1H-pyrazole-4-yl)acrylic
acid 6
Method A
To a solution of 3-(2,3-dihydro-3-methyl-2-oxo-3H-benzoxazole-6-yl)-1-phenyl-1H -pyrazole-4-carboxy al-
dehyde 5 (3.19 g, 0.01 mol) in pyridine (30 mL) were added malonic acid (4.16 g, 0.04 mol) and piperidine
(1.5 mL, 0.015 mol), and the reaction mixture was refluxed for 5 h. On cooling, the reaction mixture was
poured onto a solution (100 mL) of crushed ice and concentrated HCl (50% by volume) mixture. The resulting
precipitated was filtered off, washed with acidified water and dried. Yield 2.3 g (63.7%).
Method B
A dry flask charged with 3-(2,3-dihydro-3-methyl-2-oxo-3H-benzoxazole-6-yl)-1-phenyl-1H -pyrazole-4-
carboxy aldehyde 5 (3.19 g, 0.01 mol), malonic acid (4.16 g, 0.04 mol), piperidine (1.5 mL, 0.015 mol) and 30
mL of pyridine was placed in the MicroSYNTH Microwave Synthesis System and irradiated at 600 W for 15
min while the temperature was set to 115 ◦ C. The resulting mixture was poured onto a solution (100 mL) of
crushed ice and concentrated HCl (50% by volume) mixture. The resulting precipitated was filtered off, washed
with acidified water and dried. Yield 2.6 g (72%). mp 288-290 ◦ C; FT-IR (KBr) cm−1 1779 (C=O), 1689
371

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
(C=O); 1 H-NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H, COOH), 9.22 (s, 1H, pyrazole-H5), 7.93 (d, 2H, J =
7.6, ArH), 7.59-7.37 (m, 7H, ArH), 6.44 (d, 1H, J = 16, O=C-CH=CH), 3.40 (s, 3H, CH3). ESI-MS 362.11
[M+H]+
General procedure for the preparation of (E)-3-(3-(2,3-dihydro-3-methyl-2-oxo-3H-benzoxazole-
6-yl)1-phenyl-1H-pyrazole-4-yl)acrylamides 7
To a solution of appropriate carboxylic acid derivative (1 mmol) and amine derivative (1 mmol) in 10 mL
of DCM-THF mixture (5:1) were added DMAP (0.2 mmol) and EDCI (1.1 mmol) and the resulting solution
was stirred overnight at room temperature. The reaction mixture was quenched with 1 N HCl and extracted
with DCM. The organic phase was washed with a 1% NaHCO3 solution and brine, dried over Na2 SO4 , and
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ꢀ
evaporated under vacuum. The residue was purified by flash column chromatography (Combiflash Rf) using
DCM-MeOH as eluents.
6-(4-((E)-3-Morpholino-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazole-3-yl)-3-methyl-2-oxo-3H-benzo-
xazolone (7a)
Elution with DCM-MeOH (0%-4%) afforded 7a as a white solid (yield 82%); mp 113-115 ◦ C; FT-IR (KBr)
cm−1 1757 (C=O), 1643 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.19 (s, 1H, pyrazole-H5), 7.90 (d, 2H, J =
8.4, ArH), 7.57 (t, 3H, J=8, ArH), 7.58-7.39 (m, 4H, ArH), 7.19 (d, 1H, J = 15.2, O=C-CH=CH), 3.68-3.56
(m, 8H, morpholine), 3.40 (s, 3H, CH3); HRMS C24 H22 N4 O4 [M+H]+ calc. 431.1719, found m/z 431.1732.
(E)-N-Benzyl-3-(3-(2,3-dihydro-3-methyl-2-oxo-3H-benzoxazol-6-yl)-1-phenyl-1H-pyrazole-4-yl)
acrylamide (7b)
Elution with DCM-MeOH (0%-5%) afforded 7b as a white solid (yield 83%); mp 245-246 ◦ C; FT-IR (KBr)
cm−1 1759 (C=O), 1648 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 8.99 (s, 1H, pyrazole-H5), 8.63 (t, 1H, J =
6, ArH), 7.95 (d, 2H, J = 9.2, ArH), 7.58-7.24 (m, 11H, ArH), 6.54 (d, 1H, J = 15.6, O=C-CH=CH), 4.38
(d, 2H, J = 6.4, OCNH-CH2), 3.40 (s, 3H, CH3); HRMS C24 H22 N4 O3 [M+H]+ calc. 451.1719, found m/z
451.1725.
6-(4-((E)-3-(4-(4-Chlorophenyl)piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazole-3-yl)-3-
methyl-2-oxo-3H-benzoxazolone (7c)
Elution with DCM-MeOH (0%-5%) afforded 7c as a white solid (yield 71%); mp 243-245 ◦ C; FT-IR (KBr)
cm−1 1773 (C=O), 1632 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.20 (s, 1H, pyrazole-H5), 7.91 (d, 2H, J =
7.6, ArH), 7.57-7.37 (m, 9H, ArH), 7.24 (d, 1H, J = 9.2, ArH), 7.22 (d, 1H, J = 15.2, O=C-CH=CH), 6.98
(d, 1H, J = 8.8, ArH), 3.74 (m, 4H, piperazin-H2,6), 3.37 (s, 3H, CH3), 3.16 (m, 4H, piperazin-H3,5); HRMS
C30 H26 ClN5 O3 [M+H]+ calc. 540.0121, found m/z 540.1802.
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Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
6-(4-((E)-3-(4-(4-Fluorophenyl)piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazole-3-yl)-3-
methyl-2-oxo-3H-benzoxazolone (7d)
Elution with DCM-MeOH (0%-4%) afforded 7d as a white solid (yield 68%); mp 250-252 ◦ C; FT-IR (KBr)
cm−1 1776 (C=O), 1632 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.20 (s, 1H, pyrazole-H5), 7.89 (d, 2H, J =
7.6, ArH), 7.57-7.36 (m, 8H, ArH), 7.22 (d, 1H, J = 15.2, O=C-CH=CH), 7.08-6.97 (m, 3H, ArH), 3.74 (m,
4H, piperazin-H2,6), 3.37 (s, 3H, CH3), 3.09 (m, 4H, piperazin-H3,5); HRMS C30 H26 FN5 O3 [M+H]+ calc.
524.2098, found m/z 524.2098.
6-(4-((E)-3-(4-(4-Fluorobenzyl)piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazole-3-yl)-3-
methyl-2-oxo-3H-benzoxazolone (7e)
Elution with DCM-MeOH (0%-5%) afforded 7e as a white solid (yield 65%); mp 126-128 ◦ C; FT-IR (KBr)
cm−1 1758 (C=O), 1643 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.20 (s, 1H, pyrazole-H5), 7.90 (d, 2H, J =
8, ArH), 7.56 (m, 3H, ArH), 7.46-7.34 (m, 6H, ArH), 7.19-7.14 (m, 3H, ArH), 3.55 (m, 4H, piperazin-H2,6),
3.50 (s, 2H, CH2), 3.39 (s, 3H, CH3), 2.42-2.34 (m, 4H, piperazin-H3,5); HRMS C31 H28 FN5 O3 [M+H]+ calc.
538.2254, found m/z 538.2277.
6-(4-((E)-3-(4-(4-Chlorobenzyl)piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazole-3-yl)-3-
methyl-2-oxo-3H-benzoxazolone (7f)
Elution with DCM-MeOH (0%-5%) afforded 7f as a white solid (yield 72%); mp 197-198 ◦ C; FT-IR (KBr) cm−1
1780 (C=O), 1640 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.19 (s, 1H, pyrazole-H5), 7.90 (d, 2H, J = 7.6),
7.56 (m, 3H, ArH), 7.46-7.34 (m, 8H, ArH), 7.17 (d, 1H, J = 15.2, O=C-CH=CH), 3.68-3.56 (m, 4H, piperazin-
H2,6), 3.51 (s, 2H, CH2), 3.39 (s, 3H, CH3), 2.43-2.35 (m, 4H, piperazin-H3,5); HRMS C31 H28 ClN5 O3
[M+H]+ calc. 554.1959, found m/z 554.1985.
6-(4-((E)-3-(4-(Pyridin-4-yl)piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazole-3-yl)-3-
methyl-2-oxo-3H-benzoxazolone (7g)
Elution with DCM-MeOH (0%-4%) afforded 7g as a white solid (yield 57%); mp 263-265 ◦ C; FT-IR (KBr)
cm−1 1776 (C=O), 1648 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.22 (s, 1H, pyrazole-H5), 8.19 (d, 2H, J =
6.4, pyridine-H2,6), 7.91 (d, 2H, J = 8.8, ArH), 7.60-7.39 (m, 7H, ArH), 7.23 (d, 1H, J = 15.2, O=C-CH=CH),
6.87 (d, 2H, J = 6.4, pyridine-H3,5), 3.82-3.69 (m, 4H, piperazin-H2,6), 3.45-3.31 (m, 7H, CH3 , piperazin-H3,5);
HRMS C29 H26 N6 O3 [M+H]+ calc. 507.2145, found m/z 507.2141.
6-(4-((E)-3-(4-(4-Tolyl)piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazole-3-yl)-3-methyl-2-
oxo-3H -benzoxazolone (7h)
Elution with DCM-MeOH (0%-4%) afforded 7h as a white solid (yield 78%); mp 217-219 ◦ C; FT-IR (KBr)
cm−1 1776 (C=O), 1648 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.23 (s, 1H, pyrazole-H5), 7.91 (d, 2H, J =
8, ArH), 7.59-7.39 (m, 7H, ArH), 7.26 (d, 1H, J = 15.2, O=C-CH=CH), 7.05 (d, 2H, J = 8.4, ArH), 6.89 (d,
373

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
2H, J = 8.4, ArH), 3.82-3.71 (m, 4H, piperazin-H2,6), 3.40 (s, 3H, N-CH3), 3.13-3.09 (m, 4H, piperazin-H3,5),
2.21 (s, 3H, Ar-CH3); HRMS C31 H29 N5 O3 [M+H]+ calc. 520.2349, found m/z 520.2359.
6-(4-((E)-3-(4-(4-(Trifluoromethyl)phenyl)piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazo-
le-3-yl)-3-methyl-2-oxo-3H-benzoxazolone (7i)
Elution with DCM-MeOH (0%-4%) afforded 7i as a white solid (yield 63%); mp 235-237 ◦ C; FT-IR (KBr)
cm−1 1776 (C=O), 1632 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.23 (s, 1H, pyrazole-H5), 7.93 (d, 2H,
J = 8.8, ArH), 7.60-7.39 (m, 9H, ArH), 7.23 (d, 1H, J = 15.2, O=C-CH=CH), 7.13 (d, 2H, J = 8.8, ArH),
3.85-3.73 (m, 4H, piperazin-H2,6), 3.40-3.31 (m, 7H, CH3 , piperazin-H3,5); HRMS C31 H26 F3 N5 O3 [M+H]+
calc. 574.2066, found m/z 574.2075.
6-(4-((E)-3-(4-(4-(Methylbenzyl)piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazole-3-yl)-3-
methyl-2-oxo-3H-benzoxazolone (7j)
Elution with DCM-MeOH (0%-4%) afforded 7j as a white solid (yield 77%); mp 207-208 ◦ C; FT-IR (KBr)
cm−1 1778 (C=O), 1640 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.18 (s, 1H, pyrazole-H5), 7.90 (d, 2H,
J = 8, ArH), 7.56 (m, 3H, ArH), 7.46-7.34 (m, 4H, ArH), 7.20-7.13 (m, 5H, ArH), 3.67-3.55 (m, 4H, piperazin-
H2,6), 3.46 (s, 2H, CH2), 3.39 (s, 3H, CH3), 2.42-2.33 (m, 4H, piperazin-H3,5), 2.28 (s, 3H, CH3); HRMS
C32 H31 N5 O3 [M+H]+ calc. 534.2505, found m/z 534.2526.
6-(4-((E)-3-(4-(4-(Trifluoromethyl)benzyl)piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazo-
le-3-yl)-3-methyl-2-oxo-3H-benzoxazolone (7k)
Elution with DCM-MeOH (0%-4%) afforded 7k as a white solid (yield 68%); mp 118-120 ◦ C; FT-IR (KBr)
cm−1 1757 (C=O), 1643 (C=O); 1 H-NMR (400 MHz, DMSO-d6)δ 9.19 (s, 1H, pyrazole-H5), 7.90 (d, 2H, J =
7.6, ArH), 7.71 (d, 2H, J = 8.4, ArH), 7.58-7.55 (m, 5H, ArH), 7.47-7.38 (m, 4H, ArH), 7.17 (d, 1H, J = 15.2,
O=C-CH=CH), 3.70-3.56 (m, 6H, CH2 , piperazin-H2,6), 3.39 (s, 3H, CH3), 2.43-2.35 (m, 4H, piperazin-H3,5);
HRMS C32 H28 F3 N5 O3 [M+H]+ calc. 588.2222, found m/z 588.2230.
Biological assays
Cyclooxygenase inhibition
Human whole blood COX-1 and COX-2 assay
The human whole blood assay, originally developed by Patrignani et al.36 is considered the more biologically
relevant way to assess the inhibition of the cyclooxygenase isoenzymes, COX-1 and COX-2, by a test compound37
In this assay, platelets stimulated upon coagulation are thought to be the main source of COX-1, whereas
monocytes stimulated with LPS are thought to be the source of COX-2. COX-1 activity is determined by the
production of thromboxane B2 (TxB2), while COX-2 activity is determined by the production of prostaglandin
E2 (PGE2).
374

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
For testing the COX-1 activity, fresh blood samples from healthy volunteers who had not taken any
NSAIDs for at least 7 days prior to blood extraction were collected in vacutainer tubes without any anticoagulant.
Aliquots of 500 μL of blood were incubated either with 1 μL of vehicle (DMSO) or 1 μL of test compound
(synthesized compound or indomethacin as reference) solution (10 μM) for 1 h at 37 ◦ C. Plasma was separated
by centrifugation (5 min at 13,000 rpm, 4 ◦ C) and TXB2 levels were measured using the Correlate-EIATM
TXB2 Enzyme Immunoassay Kit from Assay Design Inc. (Ann Arbor, MI, USA). Indomethacin was used as
reference.
For COX-2, fresh blood samples from healthy volunteers who had not taken any NSAIDs for at least
7 days prior to blood extraction were collected in EDTA-containing tubes. Aliquots of 500 μL of blood
were incubated either with 1 μL of vehicle (DMSO) or 1 μL of test compound (synthesized compound or
indomethacin as reference) solution (10 μM) in the presence of LPS (10 μg/mL) for 24 h at 37 ◦ C to induce
COX-2 expression. Plasma was separated by centrifugation (5 min at 13000 rpm, 4 ◦ C) and PGE2 levels were
measured using the Correlate-EIATM PGE2 Enzyme Immunoassay Kit from Assay Design Inc. (Ann Arbor,
MI, USA). Indomethacin was used as reference.
Effect of compounds on platelet aggregation
The platelet aggregation study was carried out using the turbidometric method described by Born et al.38
Freshly drawn venous human citrated blood (sodium citrate 3.8%, 1:9 v/v) from healthy subjects who had not
taken drugs with antiplatelet activity for 10 days were centrifuged with 800 rpm for platelet-rich plasma (PRP)
and 1500 rpm for platelet-poor plasma (PPP). Platelet count in PRP was adjusted to 3.8 × 108 platelets/mL
using PPP.
Aggregation was measured at 37 ◦ C with constant stirring at 1100 rpm by utilizing a Lumi-Aggregometer
(Chrono-Log). The test compound (or the standard inhibitor, aspirin) was dissolved in DMSO. The final
concentration of DMSO in the test cuvette was fixed at 1% (v/v). Final concentrations of synthesized compounds
for antiplatelet activity assay were selected according to their solubility in DMSO. A 400 μL sample of PRP
was placed in the cuvette of the aggregometer and incubated for 5 min with 5 μL of test compound. Then
5 μL of inducer (arachidonic acid [AA], 600 μM, final concentration) was added and the change in the light
transmission was recorded for 5 min. Aspirin was used as reference drug for antiplatelet activity
In silico oral biodisponibility
The theoretical study of oral bioavailability was performed using the molinspiration on-line program
(http://www.molinspiration.com). Lipinski’s rule-of-five describes molecular properties important for a drug
pharmacokinetics in the human body. Poor absorption and permeation are more likely to occur when there
are more than 5 hydrogen-bond donors (HBD), more than 10 hydrogen-bond acceptors (HDA), the molecular
mass (MM) is greater than 500, or the log P value (clogP) is greater than 5. The active compound must be
consistent with at least 3 of the 4 rules.39
375

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
Results and discussion
Chemistry
We focused our synthetic efforts on diaryl heterocyclic ring systems as illustrated in the Scheme. The starting
compound, 3-methyl-2-oxo-3H-benzoxazole (2), was prepared by methylation with dimethyl sulfate of 2-oxo-
3H -benzoxazole (1) which was readily synthesized via the reaction of o-aminophenol and urea. Compound 2 was
then converted to 6-acetyl-3-methyl-2-oxo-3H-benzoxazole (3) via Friedel–Crafts acylation under microwave
conditions (Scheme). Subsequently, the hydrazone derivative 4 was generated by carrying out condensation
in the presence of phenylhydrazine and acetic acid in refluxing ethanol. This hydrazone derivative was then
reacted with POCl3 and DMF using 2 different methods (conventional synthesis and reaction under microwave
conditions) resulting in 1,3-diaryl pyrazole 5 with an aldehyde group at the 4 position. 3-(2,3-Dihydro-3-methyl-
2-oxo-3H -benzoxazole-6-yl)-1-phenyl-1H -pyrazole-4-carboxy aldehyde 5 was then treated with malonic acid in
pyridine to prepare the corresponding α,β-unsaturated carboxylic acid via Knoevenagel condensation reaction
using both conventional and microwave irradiation methods. By treatment of 6 with appropriate amines in
the presence of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI), dimethylaminopyridine
(DMAP), which was used as the carboxylate activator, the resulting amide derivatives 7 were prepared in good
yield (57%-83%).
The chemical structures of these compounds were elucidated by their elemental analysis, and IR, HRMS
and 1 H-NMR spectral data.
The IR spectra of hydrazone 4 showed disappearance of the carbonyl peak at 1675 cm−1 belonging to
the acetyl group of 6-acetyl-3-methyl-2-oxo-3H-benzoxazole (3), and a secondary N-H stretching band was
observed at 3344 cm−1 as a singlet. The IR spectra of compound 5 exhibited a characteristic strong absorption
for the aldehyde carbonyl group at 1687 cm−1 . In the IR spectra of compound 6, a strong absorption band
at 1689 cm−1 and intense O-H stretching absorption in the region of 3300-2500 cm−1 for α,β-unsaturated
carboxylic acid were observed. Final amide derivatives exhibited a characteristic strong absorption in the area
of 1648-1632 cm−1 attributable to the C=O of the amide group.
In the 1 H-NMR spectra of compound 5, 2 singlets were displayed due to the aldehyde group at 10.05 ppm
and pyrazole at 8.54 ppm, each showing the integration for 1 proton. In the 1 H-NMR spectra of compounds
6, the signal of the carboxylic acid was observed at 12.35 ppm. One of the olefinic protons of the alkyl chain
was observed as a doublet at 6.45 ppm with a coupling constant of 16 Hz indicating the (E) isomer. In the
1 H-NMR spectra of the final amide derivatives 7a-k that olefinic proton was observed at about 7.26-7.17 ppm
as a doublet with a coupling constant of 15.2 Hz. Pyrazole gave a singlet at about 9.23-8.99 ppm.
Biological evaluation
The compounds reported herein were tested for their ability to inhibit COX-2 and/or COX-1 using in the vitro
human whole blood assay described by Patrignani et al.36 which is considered the more biologically relevant way
to assess the inhibition of the cyclooxygenase isoenzymes, COX-1 and COX-2, by a test compound37 Preliminary
screenings of both title compounds and indomethacin as reference were performed at a final concentration of
10 μM to determine the percent inhibition of the COX-1 and COX-2 isoforms.
376

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
CH3
CH3
H
N
N
O
NH2
OH
N
a
b
c
O
+
2HN
O
O
O
O
O
NH2
O
CH3
1
2
3
CH3
N
d
CH3
O
e
N
O
N
N
HN
O
N
O
O
H
5
4
CH3
CH3
N
N
g
f
O
O
N
N
O
O
N
N
H
H
C
C
H
H
COOH
6
COR
7
Scheme Reagents and conditions: a) MW irradiation, 400 W, 140 ◦ C, 15 min; b) dimethyl sulfate, NaOH, 30 min; c)
acetic acid, PPA, MW irradiation, 90 ◦ C, 20 min; d) phenyl hydrazine, acetic acid, ethanol, 2 h reflux; e) Method A;
dimethyl formamide, phosphoroxy chloride (POCl3), 50 ◦ C, 4 h; Method B; dimethyl formamide, phosphoroxy chloride
(POCl3), MW irradiation, 210 W, 50 ◦ C, 5 min; f) Method A; malonic acid, pyridine, piperidine, 115 ◦ C, 5 h; Method
B; malonic acid, pyridine, piperidine, MW irradiation, 600 W, 115 ◦ C, 15 min; g) appropriate amine derivative, N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI), dimethylaminopyridine (DMAP), dichloromethane,
rt, overnight.
With regard to COX-1 inhibitory activities (Table 1) only compound 7a (22%), carrying a morpholine
ring at the side chain showed moderate inhibition at 10 μM. Although the inhibitory activities were not very
pronounced, all derivatives demonstrated COX-2 inhibition (1%-39%), except 7a. Compound 7e, having 4-
floro benzyl piperazine at the amide portion, inhibited COX-2 but not COX-1, although this compound was
a poor inhibitor of COX-2 (20%). Interestingly, 4-chlorobenzyl substitution on the piperazine ring resulted
in a derivative (7f) that was inactive against COX-1 at the concentration range tested (10 mM) and COX-2
inhibitory activity of this compound was less pronounced (3%).
With respect to COX selectivities, compounds 7g, 7h, 7i, and 7j showed COX-2 selective inhibitory
properties. Insertion of the 4-pyridyl, 4-methyl and trifluoromethylphenyl group in compounds 7g, 7h, 7i
improved inhibitory action (∼28%). The 4-methyl benzyl piperazine derivative 7j showed the best inhibitory
activity (38%).
377

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
Indomethacin, meclofenamic acid and zomepirac are known to show their activity primarily by inhibition
of COX-1 isoform. By preparing amides and esters through the free carboxylate group in these drugs, COX-2
selective compounds were achieved11,16,17 As expected like these well-established COX-2 inhibitor compounds,
the derivatization of the carboxyl function to amide derivatives in our final compounds did produce molecules
with potent anti-inflammatory activities and with COX-2 selectivity to some extent.
Table 1. Effects of synthesized compounds on in vitro COX-2 and COX-1 enzyme inhibition in human whole blood
assay and in vitro platelet aggregation induced by arachidonic acid.
CH3
CH3
Structure I
Structure II
N
N
O
O
N
N
O
O
N
N
R
N
R
N
O
O
Antiplatelet activity
(inducer-600 μM AA)
Final
Anti-inflammatory activity
Comp.
R
COX-2 inhibition
(%)
COX-1 inhibition
(%)
Inhibition
(%)
Concentration
(μM)
Structure I
OH
morpholin-1-yl
NCH2C6H4
Structure II
4-ClC6H4
6
7a
7b
NI
1.17
16.6
NI
21.88
NI
122
122
122
4.9
1.2
11.2
7c
7d
7e
7f
7g
7h
7i
9.11
8.65
20.32
3.04
28.50
27.57
27.33
38.78
13.32
89.14
NT
NI
NI
1.55
NI
NI
NI
NI
NI
NI
68.57
NT
47.7
61
122
122
61
122
81.3
122
122
NT
122
4.9
2.9
3.7
NI
39.6
4.9
NI
4-FC6H4
CH2(4-FC6H4)
CH2(4-ClC6H4)
pyridine-4-yl
4-CH3C6H4
4-CF3C6H4
7j
7k
CH2(4-CH3C6H4)
CH2(4-CF3C6H4)
NI
11.8
NT
100
Indomethacina)
Aspirinb)
a)Indometacin was used as nonselective COX reference. Indometacin (10 μM) was also tested for determining % inhibition
as reference drug; b)Aspirin was used as reference drug for antiplatelet activity; NI: No inhibition; NT: Not tested.
378

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
The antiplatelet effects obtained in the Born test38 with compounds 6 and 7a-7k are summarized
in Table 1. We used arachidonic acid as inducer of the platelet aggregation and aspirin as reference drug.
Most compounds exhibited no antiplatelet action. Derivatives 7b and 7k which bear benzyl amine and 4-
trifloromethylbenzylpiperazine at the amide portion respectively, were found to be poor inhibitors of platelet
aggregation. However, 4-pyridylpiperazine derivative 7g exhibited moderate inhibitory activity on platelet
aggregation at 61 μM concentration. The structure-activity relationship of TXA2 synthetase inhibitors has
already been well established. It is found that there should be appropriate length between nitrogen atom of
pyridine residue, which is known to selectively inhibit TXA2 synthetase via making chelate with iron, and
carboxylic acid moiety for selective TXA2 synthetase inhibitors40,41 Introducing pyridine moiety to the amide
portion of title compounds resulted in a potent antiplatelet compound and this result was consistent with the
literature.
In silico oral biodisponibility—molecular modeling approach
The synthesized compounds (6, 7a-k) were submitted to an in silico evaluation using a molecular modeling
approach. Good absorption after oral administration is obligatory for anti-inflammatory and antiplatelet medical
Table 2. Parameters for Lipinski rule-of-five for synthesized compounds predicted using a molecular modeling approach.
Predicted oral bioavailibility (Lipinski rule-of-five)a)
Compound
HBA
HBD
M.M.
clogP
2.337
2.288
3.596
4.709
4.127
3.896
4.410
2.742
4.480
4.926
4.181
4.628
6
7
8
7
8
8
9
8
8
8
8
8
8
1
0
1
0
0
0
0
0
0
0
0
0
361.357
430.464
450.498
540.023
509.541
537.595
554.050
506.566
519.605
573.575
533.632
587.602
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
a
To present good theoretical oral bioavailability – number of hydrogen bond acceptors (HBA) ≤ 10 and donors
(HBD) ≤ 5, clogP ≤ 5 and molecular mass ≤ 500.
379

Synthesis, anti-inflammatory, antiplatelet and in silico evaluations ..., S. BAYTAS¸, et al.
use purposes. To predict the drug-like properties of the synthesized compounds, we analyzed these derivatives
according to the rule-of-five developed by Lipinski et al. (Table 2).39 This rule theoretically determines if a
chemical compound has suitable properties to be an orally active drug in humans. According to the rule-of-five,
an orally active drug has fewer than 10 hydrogen bond acceptors and fewer than 5 donors, clogP less than 5
and molecular mass (MM) below 500. Molecules violating more than one of these rules may have problems with
bioavailability. The results showed that compounds 6 and 7a,b fulfilled the Lipinski rule-of-five (Table 2). The
rest of the compounds violated only the criterion for molecular mass.
Conclusions
The synthesis of a series of (E)-3-(3-(2,3-dihydro-3-methyl-2-oxo-3H-benzoxazole-6-yl)1-phenyl-1H -pyrazole-
4-yl)acrylamide derivatives is described along with their preliminary evaluation as potential anti-inflammatory
and antiplatelet agents. The results of the biological evaluation revealed that especially compound 7g exhibited
dual anti-inflammatory and antiplatelet activity with selective COX-2 inhibition. We think that the preliminary
in vitro activity results of this class of compounds might lead to further studies to develop better candidates
for COX-2 selectivity and with potent anti-inflammatory and antiplatelet activities.
Acknowledgment
This investigation was supported by Scientific Research Grant 02/2006-19, awarded by Gazi University BAP.
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382

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