Novel 1,3-diarylpyrazole acrylamides: Synthesis, antiplatelet activity screening, and in silico evaluation studies

Article abstract of DOI:10.1007/s00044-013-0580-x

(E)-3-[3-(pyridin-4-yl)-1-phenyl-1H-pyrazole-4-yl]acryl amides were evaluated for their antiplatelet activities. Compounds 4o and 4r were found as active derivatives showing a potent inhibitory activity on the arachidonic acid-induced aggregation, with IC₅₀ of 20.2 and 30.3 μM, respectively. Compounds 4j, 4k, and 4u presented significant inhibitor effect on collagen-induced platelet aggregation (73.1, 82.5, and 86.7 %, respectively). All synthesized compounds demonstrated good drug-likeness and drug-score values in silico evaluations.

Full text of DOI:10.1007/s00044-013-0580-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0580-x
ORIGINAL RESEARCH
Novel 1,3-diarylpyrazole acrylamides: synthesis, antiplatelet
activity screening, and in silico evaluation studies
Sultan Nacak Baytas
Yesim Ozkan Serdar Unlu
•
•
Nazan Inceler
•
•
M. Fethi Sahin
Received: 8 November 2012 / Accepted: 11 March 2013
Ó Springer Science+Business Media New York 2013
Abstract (E)-3-[3-(pyridin-4-yl)-1-phenyl-1H-pyrazole-
4
aggregation are key events in homeostasis and thrombosis
(clot formation). Hyperactivity of platelets increases the risk
of various vascular diseases, such as unstable angina, acute
myocardial infarction, transient ischemic attacks, stroke, and
complications following percutaneous coronary intervention
(Fuster et al., 1992; Davis and Thomas, 1985; Trip et al.,
1990; Jackson and Schoenwaelder, 2003). Thus, antiplatelet
therapy is a basic tool in the prevention and treatment of
atherothrombotic diseases. In injured vessels, blood aggre-
gation is generated as a physiological defense reaction by
releasing biologically active compounds such as adenosine
diphosphate (ADP), thrombin, and prostaglandin endoper-
oxide. Agonists such as thrombin, arachidonic acid (AA),
thromboxane A (TxA ), platelet-activating factor (PAF),
-yl]acryl amides were evaluated for their antiplatelet
activities. Compounds 4o and 4r were found as active
derivatives showing a potent inhibitory activity on the
arachidonic acid-induced aggregation, with IC50 of 20.2
and 30.3 lM, respectively. Compounds 4j, 4k, and 4u
presented significant inhibitor effect on collagen-induced
platelet aggregation (73.1, 82.5, and 86.7 %, respectively).
All synthesized compounds demonstrated good drug-like-
ness and drug-score values in silico evaluations.
Keywords Antiplatelet activity ꢀ 1,3-Diarylpyrazoles ꢀ
Acryl amides ꢀ Arachidonic acid ꢀ Collagen
2
2
and collagen are able to induce platelet aggregation. Among
these agonists, AA is one of the most powerful agonists for
platelet activation (Fuster et al., 1992; Arita and Hansaki,
1989; Needleman et al., 1986). Currently, different anti-
platelet agents such as ticlopidine, clopidogrel, glycoprotein
IIb/IIIa inhibitors, and acetylsalicylic acid (ASA) are avail-
able for clinical use. However, they have certain disadvan-
tages such as notable side effects (i.e., gastric erosion,
agranulocytosis) and inefficient therapy (Storey, 2001;
Schros, 1995; Bennett, 2001; Berger, 1999; McKee et al.,
Introduction
Cardiovascular diseases are responsible for the largest
number of natural deaths worldwide. Cardiac conditions
involving thrombotic disorders include acute myocardial
infarction, valvular heart disease, unstable angina, and atrial
fibrillation (Yusuf et al., 2001). Platelet adhesion and
S. N. Baytas (&) ꢀ N. Inceler ꢀ S. Unlu ꢀ M. Fethi Sahin
Division of Pharmaceutical Sciences, Department
of Pharmaceutical Chemistry, Faculty of Pharmacy,
Gazi University, Etiler, 06330 Ankara, Turkey
e-mail: sbaytas@gmail.com
2002). Therefore, studies for developing safer antiplatelet
agents with fewer side effects have recently been the goal of
many researchers.
Essential structural features of TxA synthetase inhibi-
2
tors are a basic nitrogen atom of a substituted pyridine or
imidazole ring, and carboxylic acid group separated by an
unsaturated trans-alkyl chain (De Leval et al., 2004). It is
found that there should be appropriate length between
nitrogen atom of pyridine residue, which is known to
Y. Ozkan
Department of Biochemistry, Faculty of Pharmacy,
Gazi University, Ankara, Turkey
Present Address:
S. Unlu
FARGEM (Pharmaceutical Research and Development Center)
Inc., Sancaklar Mevkii, 81100 D u¨ zce, Turkey
selectively inhibit TxA synthetase via making chelate with
2
iron, and carboxylic acid moiety for selective TxA2
123

Med Chem Res
synthetase inhibitor (Lizuka et al., 1981; Tanouchi et al.,
981). Ozagrel is a cinnamic acid derivative having imi-
isoforms using human whole blood assay as well as their
antiplatelet profile against human platelet aggregation
using arachidonic acid, as agonists. Especially, a 4-pyr-
idylpiperazin amide derivative exhibited the best inhibi-
tory activity on platelet aggregation. Based on these
results, we aimed to introduce pyridine moiety to central
pyrazole ring instead of benzoxazole ring, and searched
its effect on platelet aggregation. In the search for new
antiplatelet agents, we describe herein the design, syn-
thesis, and antiplatelet activities of new (E)-3-[3-(pyridin-
4-yl)-1-phenyl-1H-pyrazole-4-yl]acryl amides, bearing a
central pyrazole ring substituted with phenyl, pyridine
moieties, and 3-oxoprop-1-en-1-yl fragment at 1,3, and 4
positions, respectively (Fig. 1). Finally, these derivatives
were submitted to an in silico oral biodisponibility
screening to analyze their overall potential to qualify for a
drug.
1
dazol-1-yl substituent (Fig. 1) that acts as a selective
inhibitor of TxA2 synthetase with an IC50 of 11 nM.
(
Silveira et al., 1993). In particular, an N-Phenylpyrazole
arylhydrazone derivative compound (Fig. 1) possesses
analgesic, anti-inflammatory, and antiplatelet activities.
These types of compounds were found to be 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
(
Loo et al., 1987).
Microwave irradiation dominates over usual conven-
tional methods due to its non-hazardous and eco-friendly
nature. The microwave induced organic reactions have
received considerable attention due to their simplicity and
operational convenience. The use of microwave irradiation
as a source of heat in synthetic chemistry offers a prom-
ising alternative (Jyothi et al., 2007; Kalluraya et al.,
2
008). Microwave-assisted heating under controlled con-
Results
ditions is a priceless technology because it often dramati-
cally reduces reaction times, with increased yield
Chemistry
(
Escalante and Diaz-Coutino, 2009). Nowadays, micro-
wave-assisted organic synthesis has become a new era in
the field of synthetic chemistry (Algul et al., 2009).
In our previous study (Baytas et al., 2012), a series of
The synthesis of (E)-3-[1-phenyl-3-(pyridin-4yl)-1H-
pyrazole-4-yl]acrylamides, starting from 3-[1-phenyl-3-
(pyridin-4-yl)-1H-pyrazol-4-yl]acrylic acid 3 and amine
derivatives, has been accomplished as outlined in
Scheme 1. At first, the hydrazone derivative 1 was gener-
ated by carrying out a condensation reaction in the pres-
ence of 4-acetylpyridine, phenylhydrazine, and acetic acid
in refluxing ethanol. This hydrazone derivative was then
(
E)-3-[3-(2,3-dihydro-3-methyl-2-oxo-3H-benzoxazole-6-
yl)-1-phenyl-1H-pyrazole-4-yl]acrylamides (A in Fig. 1)
have been synthesized using both conventional and
microwave-assisted methods and evaluated for their
in vitro inhibitory activities on COX-1 and COX-2
Fig. 1 Structure of Ozagrel, the
lead compound (A) and general
structure of the synthesized
compounds
H C
NO2
3
N
N
N
H
O
N
N
OH
N
N(CH3)2
ozagrel
N-Phenylpyrazole arylhydrazone
derivative
CH3
O
N
N
O
O
O
R
N
N
N
N
N
N
N
A
1
23

Med Chem Res
Scheme 1 a Phenyl hydrazine,
acetic acid, ethanol, 2 h reflux;
b Method A; dimethyl
N
N
a
b
N
CH3
N
N
formamide, POCl
Method B; dimethyl formamide,
POCl , MW irradiation, 210 W,
3
, 50 °C, 4 h;
HN
O
H
N
1
3
O
50 °C, 20 min; c Method A;
malonic acid, pyridine,
2
piperidine, 115 °C, 5 h; Method
B; malonic acid, pyridine,
piperidine, MW irradiation,
6''
N
5''
6
00 W, 115 °C, 15 min; d
2
''
N
appropriate amine derivative,
ethyl chloroformate,
6
N
N
5
3''
d
c
N
4
triethylamine, dichloromethane,
rt, overnight
N
5'
3
2
R
O
3
COOH
4
-1
reacted with POCl and DMF using two different methods
3
secondary N–H stretching band was observed at 3274 cm
(
i.e., conventional synthesis and reaction under microwave
as singlet. The IR spectra of compound 2 exhibited a
characteristic strong absorption for aldehyde carbonyl
conditions) resulting in 1,3-diaryl pyrazole 2 with aldehyde
group at the 4 position. 1-Phenyl-3-(pyridin-4-yl)-1H-pyr-
azole-4-carbaldehyde 2 was then treated with malonic acid
in pyridine to prepare the corresponding a,b-unsaturated
carboxylic acid via Knoevenagel condensation reaction.
We focused our attention on the microwave-assisted syn-
thesis technique after obtaining compounds 2 and 3 by
classical heating method. Thus, synthesis of compounds 2
and 3 is performed in a microwave oven due to its low cost
and ready availability. In order to synthesize compound 2,
-1
group at 1669 cm . In the IR spectra of compound 3,
and intense O–H
-1
strong absorption band at 1681 cm
-1
stretching absorption in the region of 3300–2500 cm for
a,b-unsaturated carboxylic acid were observed. Final amide
derivatives exhibited a characteristic strong absorption in the
-1
area of 1664–1641 cm attributable to the C=O of amide
group.
1
The H-NMR spectrum of the compound 2 showed the
disappearance of the methyl proton signal and N–N–H
1
signal. In the H-NMR spectra of compound 2, two singlets
were displayed due to aldehyde group at 10.04 ppm and
phosphoroxy chloride (POCl ) was added dropwise to an
3
ice-cold stirred solution of 4-(1-(2-phenylhydrazon-
o)ethyl)pyridine 1 in DMF. The reaction mixture was
allowed to attain room temperature, and then heated at
pyrazole-H at 9.43 ppm, each showing the integration for
5
1
one proton. In the H-NMR spectra of compound 3, signal
5
0 °C using conventional method or in microwave oven.
of the carboxylic acid was not observed due to proton
exchange. One of the olefinic protons of alkyl chain was
observed as doublet at 6.51 ppm with coupling constant
Following the work-up, the desired compounds were
obtained. Comparing the two methods utilized, the micro-
wave-assisted synthesis technique dramatically cut down
reaction time (i.e., for compound 2; conventional method
1
16 Hz indicating the (E) isomer. In the H-NMR spectra of
final amide derivatives 4a–v, that olefinic proton was
observed at about 7.26–7.17 ppm as doublet with coupling
constant 15.2 Hz. Pyrazole-H5 gave a singlet at about
9.23–8.99 ppm.
5
h, microwave-assisted synthesis 20 min), and increased
the product yields as shown in experimental part (con-
ventional method yield 80 %, microwave-assisted synthe-
sis yield 92 %). By treatment of 3 with appropriate amines
in the presence of triethylamine and ethyl chloroformate
which was used as the carboxylate activator, the resulting
amide derivatives 4 were prepared in good yield
Biological evaluation
The antiplatelet effects obtained in the Born test (Born and
Cross, 1963), with compounds 3, 4a–v are summarized in
Fig. 2 and Table 2. We used arachidonic acid or collagen
as inducer of the platelet aggregation. The antiplatelet
activity was measured in vitro on the responses induced by
AA (700 lM) or collagen (5 lg/ml) on citrated platelet-
rich human plasma. The initial screening at 100 lM
concentrations revealed the compound 4o and 4r as
the most important derivatives in antiplatelet activity
(
40–88 %). Compounds were purified by automated flash
chromatography and checked for purity with UPLC before
being tested in biological assays (purity was [97 %).
The structures of these compounds were confirmed by
1
high-resolution mass spectrometry (HRMS), IR, H-, and
1
C-NMR spectral data (Table 1).
3
The IR spectra of hydrazone 1 showed disappearance of
the carbonyl peak of acetyl group of 4-acetylpyridine;
123

Med Chem Res
Table 1 Synthesized 1,
-pyrazole acryl amide
derivatives 4a–v
3
N
N
N
R
4
O
Comp.
R
Comp.
4h
R
Comp.
4o
R
4
a
N
N
N
N
NH
O
CH3
4
b
4i
4j
4p
4r
N
N
Cl
N
N
OCH3
OCH
3
NH
H CO
OCH3
3
4
c
N
N
H
N
N
N
N
N
N
N
Cl
4
d
4k
4l
4s
4t
H
N
N
N
CH3
N
O
4
e
N
N
N
O
H C
CH3
N
N
3
O
4
f
4m
4n
4u
4v
N
N
CF3
N
O
N
N
O
4
g
O
N
N
N
N
N
OH
H CO
3
screening studies. Compound 4o, bearing benzylamine
moiety at amide portion, was the most active derivative
showing a potent inhibitory activity on the AA-induced
aggregation, with an IC50 of 20.2 lM (Table 2). The
inhibitor effect of this compound on the collagen-induced
aggregation was less pronounced (inhibition 54.9 %)
(4f, 4h, 4j, 4k, 4l, and 4m) out of 14 prove to be effective
as inhibitor on the AA-induced aggregation, although with
lower potency than the 4o and 4r (inhibition 5–11 %).
Interestingly, these piperazine amides showed moderate to
high inhibitor activity on the collagen-induced aggregation
(25.0, 42.0, 73.1, 82.5, 50.0, and 68.9 %, respectively).
Compounds 4t and 4u were weakly active at 100 lM
concentration on AA-induced aggregation. Moreover, 4t
and 4u were very active at the screening concentration in
the collagen-induced test (56.5 and 86.7 %, respectively).
The rest of the compounds were quite inactive at test
conditions.
(
Fig. 2). Compound 4r, a non aromatic amide having
cyclohexyl substitution at the amide portion, also dis-
played potency on AA-induced aggregation test and its
IC50 value was 30.3 lM. Compound 4r presented a high
activity in the collagen-induced assay (71.8 %). As con-
cerns substituted piperazine amides 4a–n, six compounds
1
23

Med Chem Res
Fig. 2 Effect of (E)-3-[1-
Phenyl-3-(pyridin-4-yl)-1H-
pyrazol-4-yl]acryl amide
120
Collogen
Arachidonic acid
1
00
derivatives 3, 4a–v (100 lM)
and aspirin on in vitro platelet
aggregation of human citrated
platelet-rich plasma induced by
arachidonic acid and collagen
8
6
4
2
0
0
0
0
0
3
4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4r 4s 4t 4u 4v ASA
Compound
Table 2 IC50 (lM) of compound 4r and 4o on AA-induced platelet
aggregation
In this study, we also examined the drug-score. The drug
score combines drug-likeness, clog P, log S, molecular
weight, and toxicity risks in one handy value, and may be
used to judge the compound’s overall potential to qualify
for a drug. The results showed that the all synthesized
compounds including the two potent ones (i.e., 4r and 4o)
demonstrated good drug-score values (Table 3). Moreover,
we used the Osiris program for prediction of the overall
toxicity of the derivatives as it may point to the presence of
some fragments generally responsible for the mutagenic,
tumorigenic, irritant, or reproductive effects in these mol-
ecules. All compounds presented a low in silico toxicity
risk profile.
Compound
4o
4r
ASA
7.7
IC50 (lM)
20.2
30.3
Lipinski’s Rule of five and drug-likeness profile
The synthesized compounds (4a–v) were submitted for an
in silico evaluation. To predict drug-like properties of
synthesized compounds, we analyzed these derivatives
according to the rule-of-five developed by Lipinski et al.,
(
2001). Predictions of ADME properties for the compounds
are given in Table 3. Calculated physicochemical proper-
ties (http://www.molinspiration.com) showed that most of
the compounds fulfilled the Lipinski ‘rule-of-five’. The
compounds 4f and 4i violated only the criteria for molec-
ular weight. Theoretically, these compounds should present
good passive oral absorption, and differences in their bio-
activities cannot be attributed to this property. All com-
pounds exhibited favorable clog P values. The polar
surface areas of synthesized compounds were relatively
small in comparison with the average value for acceptable
Discussion
Antiplatelet drugs are intended to prevent and/or reverse
platelet aggregation in arterial thrombosis, most promi-
nently in myocardial infarction and ischemic stroke.
Aspirin is widely used to reduce the risk of myocardial
infarction and thromboembolic disease. The mechanism of
action of ASA is related to its capacity to permanently
inactivate the platelet PGHS by acetylation of the serine
amino acid residue (Mason et al., 2005; Patrignani, 2003).
Different antiplatelet agents such as ticlopidine, clopido-
grel, glycoprotein IIb/IIIa inhibitors are also available for
clinical use. But, they have certain disadvantages such as
notable side effects (i.e., gastric erosion, agranulocytosis)
and inefficient therapy.
´
2
˚
drug molecules (\90A ).
Currently, there are many approaches to assess a com-
pound drug-likeness based on topological descriptors, fin-
gerprints of molecular drug-likeness structure keys (Tetko,
2
005). In this study, we used the Osiris program (http://
www.organic-chemistry.org/prog/peo) for calculating the
fragment-based drug-likeness of the compounds (Table 3).
Interestingly, all compounds demonstrated good drug-like-
ness values (from 6.92 to 0.15) except compounds 4f (having
trifluoromethylphenylpiperazine ring) and 4r (bearing
cyclohexyl moiety) (-3.44 and -4.17, respectively).
In our previous study, we verified the antiplatelet activity
of (E)-3-[3-(2,3-dihydro-3-methyl-2-oxo-3H-benzoxazole-
6-yl)-1-phenyl-1H-pyrazole-4-yl]acrylamides (Baytas et al.,
2012) and determined the 6-(4-((E)-3-(4-(Pyridin-4-yl)
123

Med Chem Res
Drug-score
Table 3 Calculated ADME properties of synthesized compounds
Compd. Predicted oral bioavailability
Drug-likeness
a
b
c
d
e
f
HBA
HBD
M.W
clog P
LogS
TPSA
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
a
b
c
d
e
f
6
6
6
6
6
6
7
7
9
8
7
8
8
8
5
6
5
5
6
5
5
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
0
0
0
435.5
469.9
469.9
449.5
463.5
503.5
465.5
479.5
525.6
437.4
436.5
453.5
457.5
447.5
380.4
410.4
372.4
330.3
360.4
358.4
448.5
3.45
4.13
4.11
3.90
4.36
4.35
3.46
3.84
3.28
2.17
2.17
2.05
1.45
1.02
3.02
3.08
3.53
1.99
1.71
2.77
4.44
-3.76
-4.50
-4.50
-4.11
-4.45
-4.54
-3.78
-4.08
-3.82
-3.03
-2.97
-3.25
-2.58
-1.63
-4.03
-4.05
-4.28
-3.47
-2.41
-3.30
-4.60
54.26
54.26
54.26
54.26
54.26
54.26
63.49
63.49
81.96
80.04
67.15
84.47
80.57
83.72
59.81
69.05
59.81
59.81
60.26
51.02
51.02
6.92
5.8
0.71
0.58
0.58
0.65
0.59
0.28
0.67
0.62
0.60
0.77
0.77
0.74
0.76
0.78
0.57
0.64
0.36
0.71
0.79
0.63
0.55
6.11
3.95
5.32
-3.44
6.46
4.69
5.65
6.52
5.23
5.23
3.99
2.75
0.15
1.33
-4.17
0.81
1.48
0.24
3.39
g
h
i
j
k
l
m
n
o
p
r
s
t
u
v
Number of hydrogen bond acceptor
Number of hydrogen bond donor
Molecular weight
b
c
d
Calculated lipophilicity
e
Solubility parameter
f
´
2
˚
Topological polar surface area (A )
piperazin-1-yl)-3-oxoprop-1-enyl)-1-phenyl-1H-pyrazole-
-yl)-3-methyl-2-oxo-3H-benzoxazolone as lead com-
pound (A in Fig. 1). With respect to the knowledge that the
in one handy value that may be used to judge the drug
potential of a compound. Although the Osiris risk alerts are
not a fully reliable toxicity prediction, the theoretical low-
toxicity profile of these compounds reinforces further
synthetic and biological exploration for the development of
new antiplatelet drugs.
3
TxA synthetase inhibitors have substituted pyridine or
2
imidazole ring, we aimed at modifying structural features
of the lead compound. Our strategy to optimize the activity
was the replacement of the benzoxazolone ring of the
central pyrazole with the pyridine ring. In particular, the
structural changes in the 1,3-diaryl part of compounds did
yield substantial improvement of inhibition of platelet
aggregation comparison with the previously prepared
derivative A (Fig. 1).
Conclusion
A series of new 1,3-diarylpyrazole-4-acrylic acid deriva-
tives as inhibitors of AA-induced and collagen-induced
platelet aggregation were investigated with a focus on the
role of the pyridine replacement of one of the aryl groups in
the diarylpyrazole template. Furthermore, the microwave-
assisted technique performed, presented the minimum use
of the hazardous phosphorus oxychloride, short reaction
time and increased yield compared to conventional
method. Based on our biological and theoretical results, we
identified some of the synthesized 1,3-diarylpyrazole
It has been also determined the suitability antiplatelet
purposes where a good absorption after oral administration
is obligatory. Physicochemical properties of synthesized
compounds were evaluated in silico, and found that all
compounds should present good passive oral absorption.
All synthesized compounds demonstrated good drug-like-
ness and drug-score values. The drug-score combines drug
likeness, clogP, logS, molecular weight, and toxicity risks
1
23

Med Chem Res
derivatives as potential lead compounds and significant
inhibitors (4o, IC50 of 20.2 lM; 4r, IC50 of 30.3 lM on
AA-induced platelet aggregation; 4j 73.1 %, 4k 82.5 %,
and 4u 86.7 % inhibition on collagen-induced platelet
aggregation), to be further in vitro and in vivo investigated.
4-(1-(2-Phenylhydrazono)ethyl)pyridine 1
A solution of 4-acetylpyridine (0.052 mol), phenyl hydra-
zine (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 precipitated was filtered off and dried. Yield 85 %,
m.p. 147 °C [Lit. m.p. (Chu and Teague, 1958):
-
1
Experimental
148–149 °C]; IR (FTIR/FTNIR-ATR): 3227 cm (N–H),
-1 1
1
941 cm
J = 6.4 Hz), 7.65 (2H, d, J = 6 Hz), 7.59 (1H, s), 7.31
2H, t, J = 7.6 Hz), 7.21 (2H, d, J = 7.6 Hz), 6.93 (1H,
(C=N); H-NMR (CDCl ) d: 8.59 (2H, d,
3
Chemistry
(
1
3
The chemicals were purchased from the commercial ven-
dors and were used without purification. Thin-layer chro-
matography (TLC) was performed on Merck 60F254 plates.
Reactions were monitored by TLC on silica gel, with
detection by UV light (254 nm) or charring Dragendorff
reagent (Stahl, 1969). Melting points were determined with
an SMP-II Digital Melting Point Apparatus and are uncor-
rected (Schorpp Geaetetechnik, Germany). Elementary
analyses were performed on a Leco CHNS 932 analyzer and
satisfactory results ± 0.4 % of calculated values (C, H, N)
were obtained. IR spectra were obtained in-house using a
Perkin Elmer Spectrum 400 FTIR/FTNIR spectrometer
equipped with a Universal ATR Sampling Accessory.
dd, J = 6.8 Hz, J = 7.6 Hz), 2.22 (3H, s); C-NMR
(CDCl ) d: 170.2, 150.6, 142.3, 138.7, 130.4, 125.7, 125.2,
120.0, 114.3, 114.1, 17.1; HRMS C13
212.1188, Found m/z 212.1180; Anal. Calc.(%) for
. C: 73.91 H: 6.20 N: 19.89, Found C: 74.21 H:
3
?
H N [M?H] Calc.
14 3
C H N
13 13 3
6.22 N: 19.38.
1-Phenyl-3-(pyridin-4-yl)-1H-pyrazole-4-carbaldehyde 2
Method A In a dry flask, phosphoroxy chloride (POCl
)
3
(16.77 ml, 0.18 mol) was added dropwise to an ice-cold
stirred solution of 4-(1-(2-phenylhydrazono)ethyl)pyridine
(12.71 g, 0.06 mol) in 100 ml DMF. The reaction mixture
was allowed to attain room temperature, and then heated at
50 °C for about 5 h. The resulting mixture was poured onto
crushed ice, neutralized with dilute NaOH and left over-
night. The yellow precipitate obtained was purified by
crystallization in acetone–water mixture. Yield 12 g
(80 %).
1
H-NMR spectra were recorded in CDCl or DMSO-d on a
6
3
Varian Mercury 400 MHz FT-NMR spectrometer using
tetramethylsilane as the internal standard at the NMR
facility of Faculty of Pharmacy, Ankara University; values
1
3
are given in d (ppm) and J values are in Hz. C-NMR
spectra were recorded in CDCl3 on a Varian Mercury
3
00 MHz FT-NMR spectrometer using tetramethylsilane as
the internal standard at the NMR facility of FARGEM
Pharmaceutical Research and Development Center) Inc.
Method B In a dry flask, POCl (16.77 ml, 0.18 mol) was
3
(
added dropwise to an ice-cold stirred solution of 4-(1
-(2-phenylhydrazono)ethyl)pyridine (12.71 g, 0.06 mol) in
100 ml DMF. The reaction mixture was allowed to attain
room temperature, and then flask was placed in MicroS-
YNTH microwave synthesis system and irradiated at
210 W for 20 min while the temperature was set to 50 °C.
The resulting mixture was poured onto crushed ice, neu-
tralized with dilute sodium hydroxide and left standing
overnight. The yellow precipitate obtained was prufied by
crystallization in acetone–water mixture. Yield 13.8
(92 %), m.p. 147–149 °C [Lit. mp (Badadhe et al., 2011):
High-resolution mass spectra data (HRMS) were collected
in-house using a Waters LCT Premier XE Mass Spec-
trometer (high sensitivity orthogonal acceleration time-
of-flight instrument) operating in ESI (?) method, also
coupled to an AQUITY ultra performance liquid chroma-
tography system (Waters Corporation, Milford, MA, USA).
All compounds were of[97 % purity. Flash chromatogra-
Ò
phy was performed with a Combiflash Rf automated flash
chromatography system with RediSep columns (Teledyne-
Isco, Lincoln, NE, USA) using dichloromethane-methanol
solvent gradients. Microwave-assisted reactions were car-
ried out with a Milestone MicroSYNTH microwave syn-
thesis system. Calculation of important physicochemical
properties (logP, number of hydrogen bond donors and
acceptors and total polar surface area) was performed using
mol inspiration cheminformatics software at URL http://
www.molinspiration.com. Determination of drug-likeness
and drug score was done using Osiris program at URL
http://www.organic-chemistry.org/prog/peo.
-1
1
148 °C]; IR (FTIR/FTNIR-ATR): 1669 cm (C=O); H-NMR
(DMSO- d ) d: 10.04 (1H, s), 9.43 (1H, s), 8.73 (2H, d,
6
J = 1.6 Hz), 8.02-7.97 (4H, m), 7.60 (2H, dd, J = 7.6 Hz,
J = 8.4 Hz), 7.46 (1H, dd, J = 7.2 Hz, J = 7.6 Hz);
1
3
C-NMR (DMSO- d
139.1, 136.9, 130.4, 128.7, 123.4, 123.3, 120.0; HRMS
) d: 185.0, 150.7, 150.3, 139.2,
6
?
C
H
N
O [M?H] Calc. 250.0980, Found m/z 250.
0980. Anal. Calc. (%) for C15 O C: 72.28 H:
4.45 N: 16.86; Found C: 72.20 H: 4.42 N: 17.07.
15
12
3
H N
11 3
123

Med Chem Res
(E)-3-[1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl]acrylic
acid 3
1-Phenyl-4-[(2E)-3-[1-phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-
yl]prop-2-enoyl] piperazine 4a Elution with DCM-MeOH
(
0–5 %) yielded 4a as a white solid. (Yield 48 %, m.p.
-
1
Method A To a solution of 1-phenyl-3-(pyridin-4-yl)-1H-
pyrazole-4-carbaldehyde 2 (2.49 g, 0.01 mol) in pyridine
208–209 °C); IR (FTIR/FTNIR-ATR): 1645 cm (C=O);
1
H-NMR (CDCl ) d: 8.71 (2H, d, J = 6.4 Hz), 8.25 (1H,
3
(
(
30 ml), malonic acid (4.16 g, 0.04 mol) and piperidine
1.5 ml, 0.015 mol) were added, and the reaction mixture
s), 7.80 (1H, d, J = 15.2 Hz), 7.79 (2H, d, J = 7.6 Hz),
7.67 (2H, d, J = 6.0 Hz), 7.56–7.53 (2H, m), 7.39 (1H, t,
J = 7.6 Hz), 7.32 (2H, t, J = 7.2 Hz), 6.96–6.90 (3H, m),
6.79 (1H, d, J = 15.2 Hz), 3.90 (2H, s), 3.75 (2H, s),
was refluxed for 5 h. On cooling, the reaction mixture was
poured onto a solution (100 ml) of crushed ice and con-
centrated HCl (50 % by volume) mixture, then, pH was
adjusted to 7. The resulting precipitate was filtered off,
washed with acidified water, and dried. Yield 2.3 g
1
3
3.24–3.21 (4H, m); C-NMR (CDCl ) d: 165.1, 160.5,
3
151.0, 149.7, 146.1, 141.1, 139.9, 139.4, 139.3, 132.7,
129.9, 129.5, 127.8, 126.9, 123.1, 120.9, 119.6, 118.0,
?
116.9, 50.7, 49.5, 45.9, 42.3; HRMS C H N O [M?H]
27 26 5
(
63.7 %).
Calc. 436.2137, Found m/z 436.2122.
Method B The dry flask charged with 1-phenyl-3-
pyridin-4-yl)-1H-pyrazole-4-carbaldehyde (2.49 g,
.01 mol), malonic acid (4.16 g, 0.04 mol), piperidine
1.5 ml, 0.015 mol), and 30 ml pyridine was placed in
(
2
1-(4-Chlorophenyl)-4-[(2E)-3-[1-phenyl-3-(pyridin-4-yl)-1H-
pyrazol-4-yl]prop-2-enoyl]piperazine 4b Elution with
DCM-MeOH (0–2 %) gave 4b as a white solid (Yield
76 %, m.p. 219–221 °C); IR (FTIR/FTNIR-ATR):
0
(
MicroSYNTH microwave synthesis system and irradiated
at 600 W for 15 min while the temperature was set to
-
1
1
1642 cm
(C=O); H-NMR (CDCl ) d: 8,72 (2H, d,
3
1
15 °C. The resulting mixture was poured onto a solution
100 ml) of crushed ice and concentrated HCl (50 % by
J = 5.6 Hz), 8.25 (1H, s), 7.82 (1H, d, J = 15.2 Hz),
7.78–7.76 (2H, m), 7.66 (2H, d, J = 6 Hz), 7.53–7.50 (2H,
m), 7.39–7.36 (1H, m), 7.24 (2H, d, J = 8.4 Hz), 6.86 (2H,
d, J = 8.4 Hz), 6.74 (1H, d, J = 15.2 Hz), 3.88 (2H, s),
(
volume) mixture, then, pH was adjusted to 7. The
resulting precipitate was filtered off, washed with acidi-
fied water, and dried. Yield 78 %, m.p. 287–288 °C;
-1
1
3
3.74 (2H, s), 3.18 (4H, t, J = 5.2 Hz); C-NMR (CDCl3)
d: 165.1, 160.5, 149.7, 149.6, 141.1, 139.9, 132.8, 129.8,
129.3, 127.8, 126.9, 125.8, 123.1, 119.6, 119.1, 118.0,
-
1
IR (FTIR/FTNIR-ATR): 1681 cm
(C=O), 2440 cm
1
(
C=C); H-NMR (DMSO- d ) d: 9.27 (1H, s), 8.73 (2H, d,
6
?
J = 6 Hz), 7.95 (2H, d, J = 8.4 Hz), 7.66 (2H, d,
J = 6.4 Hz), 7.60–7.53 (3H, m), 7.41 (1H, t, J = 7.6 Hz),
50.7, 49.5, 45.9, 42.3; HRMS C H ClN O [M?H]
2
7
25
5
Calc. 470.1748, Found m/z 470.1730.
1
3
6
1
1
.49 (1H, d, J = 16 Hz); C-NMR (DMSO- d ) d: 168.2,
6
50.9, 149.9, 140.1, 139.4, 133.9, 130.3, 129.6, 128.0,
?
1-(3-Chlorophenyl)-4-[(2E)-3-[1-phenyl-3-(pyridin-4-yl)-1H-
pyrazol-4-yl]prop-2-enoyl]piperazine 4c Elution with
DCM-MeOH (0–2 %) yielded 4c as a white solid (Yield
60 %, m.p. 231–232 °C); IR (FTIR/FTNIR-ATR):
23.3, 120.3, 119.5, 118.3; HRMS C H N O [M?H]
7 14 3
1
Calc. 292.1086, Found m/z 292.1089; Anal Calc. (%) for
C H N O C: 70.09 H: 4.50 N: 14.42; Found C: 69.92
1
7 13 3
-
1
1
H: 4.65 N: 14.34.
1644 cm
J = 5.6 Hz), 8.25 (1H, s), 7.82 (1H, d, J = 15.2 Hz),
.79–7.77 (2H, m), 7.66 (2H, d, J = 6 Hz), 7.51 (2H, m),
(C=O); H-NMR (CDCl ) d: 8.72 (2H, d,
3
7
General procedure for the preparation of (E)-3-[1-phenyl-
-(pyridin-4-yl)-1H-pyrazol-4-yl]acrylamides 4
7.38 (1H, m), 7.2 (1H, t, J = 8 Hz), 6.89–6.78 (3H, m),
6.74 (1H, d, J = 15.2 Hz), 3.88 (2H, s), 3.74 (2H, s), 3.23
3
1
3
(
4H, m); C-NMR (CDCl ) d: 165.1, 152.0, 149.6, 149.5,
3
To the solution of acid derivatives (1 mmol), triethylamine
2 mmol) and ethyl chloroformate (1 mmol), were added,
141.2, 139.4, 135.2, 132.8, 130.4, 129.9, 127.9, 127.0,
(
123.2, 120.4, 119.6, 119.1, 117.9, 116.6, 116.5, 114.6,
?
followed by stirring at 0 °C for 30 min. After addition of
the appropriate amine derivative (1.2 mmol), the mixture
was stirred for an additional 1 h at 0 °C. Then, the reaction
mixture was warmed to room temperature, and kept stirring
overnight. After the solvent was evaporated under reduced
pressure, acetone was added, filtered, and evaporated.
Residue was dissolved in DCM and the organic phase was
49.5, 49.0, 45.6, 42.3; HRMS C H N OCl [M?H]
7 25 5
2
Calc. 470.1748, Found m/z 470.1736.
1-(4-Methylphenyl)-4-[(2E)-3-[1-phenyl-3-(pyridin-4-yl)-1H-
pyrazol-4-yl]prop-2-enoyl]piperazine 4d Elution with
DCM-MeOH (0–2 %) gave 4d as a white solid (Yield
60 %, m.p. 241–242 °C); IR (FTIR/FTNIR-ATR):
-
1
1
washed with a 1 % NaHCO solution and brine, dried over
3
1643 cm (C=O); H-NMR (DMSO-d ) d: 9.28 (1H, s),
6
Na SO , and evaporated under vacuum. The residue was
2
8.73 (2H, d, J = 6.4 Hz), 7.94 (2H, d, J = 9.6 Hz), 7.65
(2H, d, J = 6.4 Hz), 7.59 (2H, m), 7.55 (1H, d,
J = 15.2 Hz), 7.42 (1H, m), 7.28 (1H, d, J = 15.2 Hz),
4
Ò
purified by flash column chromatography (Combiflash Rf)
using DCM-MeOH as eluents.
1
23

Med Chem Res
7
.05 (2H, d, J = 8.4 Hz), 6.90 (2H, d, J = 8.4 Hz), 3.83
1-(2-Ethoxyphenyl)-4-[(2E)-3-(1-phenyl-3-(pyridin-4-yl)-1H-
pyrazol-4-yl)prop-2-enoyl]piperazine 4h Elution with
DCM-MeOH (0–5 %) yielded 4h as a white solid (Yield
62 %, m.p. 133–135 °C); IR (FTIR/FTNIR-ATR):
1
2H, s), 3.72 (2H, s), 3.11 (4H, m), 2.21 (3H, s); C-NMR
3
(
(
CDCl ) d: 165.2, 149.6, 149.5, 139.4, 135.2, 132.8, 130.4,
3
1
29.9, 127.9, 127.0, 125.5, 123.2, 119.6, 118.1, 117.3,
?
0.7, 49.5, 45.9, 42.3, 20.9; HRMS C H N O [M?H]
28 28 5
-1
1
5
1643 cm
J = 6.0 Hz), 8.24 (1H, s), 7.81 (1H, d, J = 15.2 Hz), 7.77
2H, d, J = 7.2 Hz), 7.66 (2H, d, J = 6.0 Hz), 7.53 (2H,
(C=O); H-NMR (CDCl ) d: 8.72 (2H, d,
3
Calc. 450.2294, Found m/z 450.2272.
(
1
-(2,3-Dimethylphenyl)-4-[(2E)-3-[1-phenyl-3-(pyridin-4-yl)-
H-pyrazol-4-yl]prop-2-enoyl]piperazine 4e Elution with
m), 7.38 (1H, t, J = 7.6 Hz), 7.01–6.87 (4H, m), 6.82 (1H,
d, J = 15.2 Hz), 4.12–4.07 (2H, m), 3.92 (2H, s), 3.78 (2H,
1
DCM-MeOH (0–5 %) gave 4e as a white solid (Yield
1
3
s), 3.12 (4H, t, J = 4.8 Hz), 1.48 (3H, m); C-NMR
(CDCl ) d: 165.1, 150.2, 149.5, 149.1, 141.6, 140.4, 139.3,
132.3, 129.9, 129.0, 127.8, 126.9, 126.2, 123.8, 123.2,
4
1
1 %, m.p. 207–209 °C); IR (FTIR/FTNIR-ATR):
3
-
1
1
645 cm
(C=O); H-NMR (CDCl ) d: 8.71 (2H, d,
3
J = 6.0 Hz), 8.24 (1H, s), 7.82 (1H, d, J = 15.2 Hz), 7.79
2H, d, J = 7.6 Hz), 7.68 (2H, d, J = 6.4 Hz), 7.52 (2H,
121.1, 119.6, 119.5, 118.6, 118.4, 112.5, 63.8, 51.3, 50.5,
?
[M?H] Calc.
(
46.2, 42.6, 15.1; HRMS C29
H
30
N
5
O
2
m), 7.39 (1H, t, J = 7.6 Hz), 7.11 (1H, m), 6.95 (1H, d,
J = 7.2 Hz), 6.89 (1H, d, J = 8.4 Hz), 6.81 (1H, d,
J = 15.2 Hz), 3.74 (4H, s), 2.91 (4H, s), 2.29 (3H, s), 2.26
480.2400, Found m/z 480.2381.
1-[(2E)-3-(1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)prop-2-
enoyl]-4-(2,3,4-trimethoxybenzyl)piperazine 4i Elution with
DCM-MeOH (0–5 %) gave 4i as a white solid (Yield
60 %, m.p. 148–150 °C); IR (FTIR/FTNIR-ATR):
1
3
(
3H, s); C-NMR (CDCl ) d: 165.2, 150.9, 149.6, 141.1,
3
1
39.9, 138.4, 132.4, 127.8, 127.2, 126.1, 125.8, 123.2,
1
19.6, 119.5, 118.3, 117.0, 52.6, 52.2, 46.3, 42.5, 20.4,
?
4.1; HRMS C H N O [M?H] Calc. 464.2450, Found
m/z 464.2443.
-1
1
1
1649 cm
J = 6.0 Hz), 8.21 (1H, s), 7.78 (2H, d, J = 7.6 Hz), 7.76
1H, d, J = 15.2 Hz), 7.66 (2H, d, J = 6.0 Hz), 7.52 (2H,
(C=O); H-NMR (CDCl ) d: 8.71 (2H, d,
3
2
9 30 5
(
1
-[(2E)-3-[1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl]prop-2-
m), 7.38 (1H, m), 6.99 (1H, d, J = 8 Hz), 6.74 (1H, d,
J = 15.2 Hz), 6.66 (1H, d, J = 8.4 Hz), 3.89 (3H, s), 3.88
(3H, s), 3.86 (3H, s), 3.73 (2H, s), 3.57 (2H, s), 3.51 (2H,
enoyl]-4-[3-(trifluoromethyl)phenyl] piperazine 4f Elution
with DCM-MeOH (0–5 %) yielded 4f as a white solid
1
3
(
Yield 40 %, m.p. 182–185 °C); IR (FTIR/FTNIR-ATR):
s), 2.50 (4H, m); C-NMR (CDCl ) d: 165.1, 152.8, 150.5,
3
142.4, 140.3, 139.9, 139.4, 133.8, 129.9, 127.7, 126.8,
-
1
1
1
647 cm
(C=O); H-NMR (CDCl ) d: 8.71 (2H, d,
3
J = 6.0 Hz), 8.25 (1H, s), 7.83 (1H, d, J = 15.2 Hz), 7.78
2H, d, J = 7.2 Hz), 7.66 (2H, d, J = 6.0 Hz), 7.52 (2H,
123.0, 119.6, 118.8, 117.9, 107.1, 61.4, 61.0, 56.6, 56.2,
?
[M?H] Calc.
(
53.1, 52.5, 45.6; HRMS C31
H
34
N
5
O
4
m), 7.40–7.36 (2H, m), 7.15 (2H, d, J = 7.6 Hz), 7.09 (1H,
d, J = 8.4 Hz), 6.78 (1H, d, J = 15.2 Hz), 3.91 (2H, s),
540.2611, Found m/z 540.2608.
1
.76 (2H, s), 3.29–3.26 (4H, t, J = 5.2 Hz); C-NMR
3
3
2-{4-[(2E)-3-(1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)prop-
2-enoyl]piperazine-1-yl}pyrimidine 4j Elution with DCM-
MeOH (0–2 %) yielded 4j as a white solid (Yield 78 %,
(
CDCl ) d: 165.1, 151.1, 149.2, 144.6, 141.5, 139.3, 132.9,
3
1
1
31.6, 131.5, 130.5, 129.9, 127.9, 127.0, 123.2, 119.6,
-
1
19.5, 119.2, 117.9, 117.0, 112.9, 49.9, 49.5, 45.9, 42.3;
?
m.p. 246–248 °C); IR (FTIR/FTNIR-ATR): 1648 cm
1
HRMS C H F N O [M?H] Calc. 504.1986, Found m/z
2
(C=O); H-NMR (DMSO-d ) d: 9.25 (1H, s), 8.71 (2H, d,
6
8 25 3 5
5
04.1971.
J = 6.0 Hz), 8.37 (2H, d, J = 4.4 Hz), 7.89 (2H, d,
J = 8.0 Hz), 7.63 (2H, d, J = 6.0 Hz), 7.56 (2H, t,
J = 8.0 Hz), 7.51 (1H, d, J = 15.2 Hz), 7.39 (1H, m), 7.27
(1H, d, J = 15.2 Hz), 6.65 (1H, m), 3.78–3.64 (8H, m);
1
1
-(2-Methoxyphenyl)-4-[(2E)-3-[1-phenyl-3-(pyridin-4-yl)-
H-pyrazol-4-yl]prop-2-enoyl]piperazine 4g Elution with
1
3
DCM-MeOH (0–5 %) gave 4g as a white solid (Yield
C-NMR (CDCl
3
) d: 165.3, 161.8, 158.0, 149.9, 140.9,
139.4, 132.8, 129.9, 129.8, 127.9, 126.9, 123.1, 119.6,
7
1
2 %, m.p. 152–153 °C); IR (FTIR/FTNIR-ATR):
-1 1
645 cm
(C=O); H-NMR (CDCl ) d: 8.71 (2H, d,
117.9, 110.8, 45.8, 44.0, 43.8, 42.6; HRMS C25
?
[M?H] Calc. 438.2029, Found m/z 438.2019.
H
24
N
7
O
3
J = 6.4 Hz), 8.24 (1H, s), 7.79 (1H, d, J = 15.2 Hz), 7.78
2H, d, J = 7.6 Hz), 7.66 (2H, d, J = 6.0 Hz), 7.52 (2H, m),
(
7
.38 (1H, t, J = 7.6 Hz), 7.05 (1H, m), 6.94–6.89 (3H, m),
.78 (1H, d, J = 15.2 Hz), 3.93 (2H, s), 3.90 (3H, s), 3.77
1-[(2E)-3-(1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)prop-
2-enoyl]-4-pyridin-4-ylpiperazine 4k Elution with DCM-
MeOH (0–5 %) gave 4k as a white solid (Yield 88 %, m.p.
6
13
(2H, s), 3.10 (4H, m); C-NMR (CDCl ) d: 165.1, 152.4,
3
-
1
1
1
5
4
49.6, 145.6, 141.2, 140.5, 139.4, 132.5, 129.9, 127.8, 126.9,
228–230 °C); IR (FTIR/FTNIR-ATR): 1645 cm (C=O);
1
23.9, 123.1, 121.2, 119.6, 119.3, 118.7, 118.2, 111.4, 55.6,
H-NMR (DMSO-d ) d: 9.28 (1H, s), 8.74 (2H, d,
6
?
2.2, 51.8, 46.2, 42.6; HRMS C H N O [M?H] Calc.
66.2243, Found m/z 466.2241.
J = 6.0 Hz), 8.20 (2H, d, J = 6.4 Hz), 7.94 (2H, d,
J = 9.6 Hz), 7.66 (2H, d, J = 6.4 Hz), 7.60 (2H, m), 7.55
28 28 5 2
123

Med Chem Res
(
1H, d, J = 15.2 Hz), 7.43 (1H, t, J = 6.8 Hz), 7.27 (1H,
d, J = 15.2 Hz), 6.88 (2H, d, J = 6.8 Hz), 3.83–3.71 (4H,
(2E)-N-benzyl-3-(1-phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-
yl)acrylamide 4o Elution with DCM-MeOH (0–5 %)
gave 4o as a white solid (Yield 48 %, m.p. 205–207 °C);
-1
1
3
m), 3.43 (4H, m); C-NMR (CDCl ) d: 165.3, 154.8,
1
3
-
1
50.2, 149.7, 140.2, 139.9, 133.4, 129.9, 127.8, 127.0,
IR (FTIR/FTNIR-ATR): 1651 cm
(C=O), 3247 cm
(N–H); H-NMR (DMSO-d ) d: 9.06 (1H, s), 8.73 (2H, d,
6
1
1
23.0, 119.6, 118.8, 117.1, 108.5, 46.0, 45.4, 42.1, 41.8;
?
HRMS C H N O [M?H] Calc. 437.2090, Found m/z
J = 5.6 Hz), 8.68 (1H, m), 7.97 (2H, d, J = 10.0 Hz), 7.67
(2H, d, J = 6.0 Hz), 7.57 (2H, m), 7.51 (1H, d,
J = 15.6 Hz), 7.42 (1H, m), 7.35–7.24 (5H, m), 6.58 (1H,
2
6 25 6
4
37.2082.
1
3
1
-(2-Furoyl)-4-[(2E)-3-(1-phenyl-3-(pyridin-4-yl)-1H-pyr-
d, J = 15.6 Hz), 4.39 (2H, d, J = 6.4 Hz); C-NMR
(CDCl ) d: 165.7, 149.9, 139.3, 139.0, 137.9, 130.7, 129.8,
129.2, 128.9, 128.1, 127.8, 126.7, 123.1, 121.7, 119.5,
azol-4-yl)prop-2-enoyl]piperazine 4l Elution with DCM-
MeOH (0–5 %) yielded 4 l as a white solid (Yield 86 %,
3
-
1
?
m.p. 183–185 °C); IR (FTIR/FTNIR-ATR): 1647 cm
(
118.2, 114.1, 44.0; HRMS C24H N O [M?H] Calc.
21 4
1
C=O); H-NMR (DMSO-d ) d: 9.26 (1H, s), 8.74 (2H, d,
381.1715, Found m/z 381.1705.
6
J = 6.4 Hz), 7.92 (2H, d, J = 7.6 Hz), 7.87 (1H, d,
J = 2.4 Hz), 7.65 (2H, d, J = 6.4 Hz), 7.59 (2H, m), 7.54
(2E)-N-(4-Methoxybenzyl)-3-(1-phenyl-3-(pyridin-4-yl)-
1H-pyrazol-4-yl)acrylamide 4p Elution with DCM-
MeOH (0–5 %) yielded 4p as a white solid (Yield 45 %,
(
1H, d, J = 15.2 Hz), 7.42 (1H, t, J = 7.2 Hz), 7.25 (1H,
d, J = 15.2 Hz), 7.06 (1H, d, J = 3.6 Hz), 6.66 (1H, t,
1
3
-1
J = 5.2 Hz), 3.78–3.67 (8H, m); C-NMR (CDCl ) d:
m.p. 221–223 °C); IR (FTIR/FTNIR-ATR): 1650 cm
3
-
1
1
1
77.2, 165.1, 159.8, 149.9, 147.6, 144.3, 140.7, 139.3,
(C=O), 3282 cm (N–H); H-NMR (DMSO-d ) d: 9.04
6
1
33.3, 129.9, 127.9, 127.0, 123.1, 119.1, 119.0, 117.4,
?
(1H, s), 8.73 (2H, d, J = 6.0 Hz), 8.60 (1H, m), 7.95 (2H,
d, J = 8.8 Hz), 7.66 (2H, d, J = 6.0 Hz), 7.56 (2H, m),
7.49 (1H, d, J = 15.6 Hz), 7.40 (1H, t, J = 7.6 Hz), 7.22
1
11.8, 45.8, 44.0, 43.8, 42.6; HRMS C H N O [M?H]
26 24 5 3
Calc. 454.1879, Found m/z 454.1871.
(
2H, d, J = 9.2 Hz), 6.90 (2H, d, J = 8.4 Hz), 6.55 (1H, d,
1
3
1-[(2E)-3-(1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)prop-2-
J = 15.6 Hz), 4.32 (2H, d, J = 6.0 Hz), 3.73 (3H, s); C-
NMR (CDCl ) d: 165.4, 159.2, 149.9, 149.6, 141.8, 139.3,
130.6, 130.3, 129.9, 129.6, 127.9, 127.1, 123.2, 122.0,
enoyl]-4-(tetrahydrofuran-2-yl-carbonyl) piperazine 4m Elu-
tion with DCM-MeOH (0–5 %) gave 4 m as a white solid
3
(
Yield 43 %, m.p. 177–178 °C); IR (FTIR/FTNIR-ATR):
119.6, 118.6, 114.3, 55.6, 43.7; HRMS C25
?
[M?H] Calc. 411.1821, Found m/z 411.1806.
H
23
N
O
4
2
-
1
1
1
646 cm
(C=O); H-NMR (CDCl ) d: 8.71 (2H, d,
3
J = 6.0 Hz), 8.25 (1H, s), 7.81 (1H, d, J = 15.2 Hz), 7.78
2H, d, J = 7.6 Hz), 7.65 (2H, d, J = 5.6 Hz), 7.52 (2H,
m), 7.39 (1H, m), 6.75 (1H, d, J = 15.2 Hz), 4.61 (1H, s),
(
(2E)-N-Cyclohexyl-3-(1-phenyl-3-(pyridin-4-yl)-1H-pyrazol-
4-yl)acrylamide 4r Elution with DCM-MeOH (0–5 %)
gave 4r as a white solid (Yield 60 %, m.p. 234–235 °C);
1
3
3
.92–3.53 (10H, m), 2.34–1.90 (4H, m);
C-NMR
-
1
-1
(
CDCl ) d: 177.2, 165.1, 149.9, 147.6, 140.7, 139.3, 133.3,
IR (FTIR/FTNIR-ATR): 1649 cm
3277 cm
H-NMR (CDCl ) d: 8.70 (2H, d, J = 6.0 Hz), 8.17 (1H,
3
(N–H);
3
1
1
4
4
29.9, 127.9, 123.6, 119.6, 118.7, 69.8, 55.6, 45.8, 44.0,
?
3.8, 42.6, 26.0; HRMS C H N O [M?H] Calc.
58.2192, Found m/z 458.2177.
s), 7.75 (2H, d, J = 8.0 Hz), 7.70 (1H, d, J = 15.6 Hz),
7.65 (2H, d, J = 6.0 Hz), 7.52 (2H, m), 7.38 (1H, m), 6.23
2
6 28 5 3
(
1H, d, J = 15.2 Hz), 4.45 (1H, d, J = 8.0 Hz), 3.92 (1H,
1
3
2
-(2-{4-[(2E)-3-(1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-
m), 2.00–1.62 (6H, m), 1.45–1.12 (4H, m); C-NMR
(CDCl ) d: 166.5, 149.9, 143.9, 140.4, 139.9, 133.4, 129.9,
129.3,127.9, 126.3, 121.1, 52.1, 32.3, 26.2, 25.0; HRMS
yl)prop-2-enoyl]piperazine-1-yl}ethoxy)ethanol 4n Elu-
tion with DCM-MeOH (0–5 %) yielded 4n as a white solid
3
?
(
Yield 41 %, m.p. 113–115 °C); IR (FTIR/FTNIR-ATR):
C H N O [M?H] Calc. 373.2028, Found m/z 373.2010.
23 25 4
-
1
1
1
641 cm
(C=O); H-NMR (CDCl ) d: 8.70 (2H, d,
3
J = 6.4 Hz), 8.23 (1H, s), 7.78 (2H, d, J = 7.2 Hz), 7.77
1H, d, J = 15.6 Hz), 7.66 (2H, d, J = 5.6 Hz), 7.52 (2H,
m), 7.39 (1H, t, J = 7.6 Hz), 6.73 (1H, d, J = 15.6 Hz),
.78 (1H, s), 3.73 (2H, m), 3.69 (4H, m), 3.64 (4H, m), 2.64
(2E)-N-Cyclopropyl-3-(1-phenyl-3-(pyridin-4-yl)-1H-pyra-
zol-4-yl)acrylamide 4s Elution with DCM-MeOH
(0–5 %) yielded 4s as a white solid (Yield 61 %, m.p.
(
-
1
3
224–225 °C); IR (FTIR/FTNIR-ATR): 1664 cm (C=O),
1
3
-1 1
(
2H, m), 2.58 (4H, t, J = 5.2 Hz); C-NMR (CDCl ) d:
3238 cm
(N–H); H-NMR (CDCl ) d: 8.69 (2H, d,
3
3
1
65.1, 151.2, 150.3, 149.9, 141.1, 139.4, 132.7, 129.9,
J = 6.4 Hz), 8.16 (1H, s), 7.75 (2H, m), 7.68 (1H, d,
J = 13.6 Hz), 7.63 (2H, d, J = 6.0 Hz), 7.52 (2H, t,
J = 8.0 Hz), 7.38 (1H, m), 6.19 (1H, d, J = 16.0 Hz), 5.77
1
29.8, 126.9, 123.1, 123.0, 119.6, 118.9, 117.5, 72.69,
?
4.5, 61.9, 57.8, 56.8, 49.9; HRMS C H N O [M?H]
25 30 5 3
6
1
3
Calc. 448.2349, Found m/z 448.2332.
(1H, s), 2.87 (1H, m), 0.86–0.54 (4H, m); C-NMR
1
23

Med Chem Res
(
CDCl ) d: 167.2, 149.9, 149.3, 139.3, 130.1, 129.9, 127.8,
27.1, 123.1, 121.2, 119.5, 118.6, 114.0, 23.1, 6.9; HRMS
blood (sodium citrate 3.2 %, 1:9 v/v) from healthy sub-
jects, who had not taken drugs with antiplatelet activity for
10 days, was centrifuged at 800 rpm for 10 min to obtain
PRP. After separation, samples were centrifuged at
3,500 rpm for 10 min to obtain PPP. Platelet count in PRP
3
1
?
C H N O [M?H] Calc 331.1559, Found m/z 331.1545.
2
0 19 4
4-[(2E)-3-(1-Phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)
prop-2-enoyl]morpholine 4t Elution with DCM-MeOH
8
was adjusted to 3.8 9 10 platelets/ml using PPP.
(
0–5 %) gave 4t as a white solid (Yield 40 %, m.p.
-
Throughout the experiments, the volunteers were treated
under the audit of Gazi University’s Commission of Ethics
according to the suggested international ethical guidelines
(Permission No: 303).
1
2
1
36–238 °C); IR (FTIR/FTNIR-ATR): 1647 cm (C=O);
H-NMR (CDCl ) d: 8.72 (2H, d, J = 6.0 Hz), 8.23 (1H, s),
3
7
.81 (1H, d, J = 15.2 Hz), 7.78 (2H, d, J = 7.6 Hz), 7.66
2H, d, J = 6.0 Hz), 7.52 (2H, m), 7.39 (1H, m), 6.72 (1H,
(
1
3
d, J = 15.2 Hz), 3.73 (6H, s), 3.59 (2H, s); C-NMR
CDCl ) d: 162.8, 149.9, 141.8, 139.9, 132.7, 129.9, 128.9,
Platelet aggregation studies Platelet aggregation was
measured by using an aggregometer (APACT 4004, LABi-
Tec, Ahrensburg, Germany) according to the turbidimetric
method described by Born et al., (1963). The test compound
(or the standard inhibitor, aspirin) was dissolved in DMSO.
PRP was incubated at 37 °C with constant stirring at
1,100 rpm and stimulated with arachidonic acid (AA, final
concentration 700 lM) or collagen (final concentration
5 lM). 199 ll sample of PRP was placed in the cuvette of
aggregometer and incubated for 5 min with 0.5 ll of test
compound (or standard inhibitor, or DMSO) before addition
of 10 ll inducer. The changes in optical density were
monitored for 3 min. All experiments were performed in
triplicate. Inhibition of platelet aggregation was expressed as
percentage of inhibition using the following equation:
(
3
1
27.9, 126.7, 123.2, 119.6, 118.9, 117.2, 114.0, 66.6, 46.2;
?
HRMS C H N O [M?H] Calc. 361.1655, Found m/z
2
1 21 4 2
3
61.1644.
-{4-[(1E)-3-Oxo-3-(piperidin-1-yl)prop-1-en-1-yl]-1-phe-
nyl-1H-pyrazol-3-yl}pyridine 4u Elution with DCM-MeOH
4
(
0–5 %) yielded 4u as a white solid (Yield 44 %, m.p.
-
1
1
1
92–194 °C); IR (FTIR/FTNIR-ATR):1646 cm (C=O);
H-NMR (CDCl ) d: 8.71 (2H, d, J = 6.4 Hz), 8.22 (1H,
3
s), 7.78 (2H, d, J = 7.2 Hz), 7.66 (1H, d, J = 16.0 Hz),
7
.65 (2H, d, J = 6.4 Hz), 7.52 (2H, m), 7.38–7.34 (1H,
m), 6.78 (1H, d, J = 15.2 Hz), 3.66 (2H, s), 3.52 (2H, s),
1
3
1
1
1
.69–1.61 (6H, m); C-NMR (CDCl ) d: 165.0, 149.9,
3
49.7, 140.8, 139.4, 131.7, 129.8, 127.6, 126.8, 123.0,
%
Inhibition
ꢀ
19.5, 119.3, 118.8, 114.1, 47.2, 43.6, 27.0, 25.8, 24.8;
?
ꢁ
Maximum aggregation of compound treated PRP
HRMS C H N O [M?H] Calc. 359.1872, Found m/z
2
¼
1 ꢁ
2 23 4
Maximum aggregation of DMSO treated PRP
3
59.1871.
ꢂ 100
4
1
-{4-[(1E)-3-(4-Benzylpiperidin-1-yl)-3-oxoprop-1-en-1-yl]-
-phenyl-1H-pyrazol-3-yl}pyridine 4v Elution with
Lipinski’s rule of five and drug-likeness profile In order to
explore the bioavailability of synthesized derivatives, the-
oretical calculations were carried out to predict some
physicochemical properties of synthesized compounds.
The bioavailability of the compounds was assessed using
ADME (absorption, distribution, metabolism, and elimi-
nation) prediction methods. In particular, we calculated the
compliance of compounds to the Lipinski’s rule of five
(Lipinski et al., 2001). Lipinski’s ‘rule-of-five’ and the
later addition of other parameters such as polar surface area
(PSA) (Ertl et al., 2000) describes molecular properties
important for drug pharmacokinetics in the human body.
This approach has been widely used as a filter for sub-
stances that would likely be further developed in drug
design programs. Poor absorption and permeation are more
likely to occur when there are more than five hydrogen-
bond donors (HBD), more than ten hydrogen-bond accep-
tors (HDA), the molecular mass (MM) is greater than 500,
or the logP value (clogP) is greater than five. The topo-
logical polar surface area (TPSA) should be smaller than
DCM-MeOH (0–5 %) gave 4v as a white solid (Yield
-1
5
6 %, m.p. 162–163 °C); IR (FTIR/FTNIR-ATR): 1645 cm
1
(C=O); H-NMR (CDCl ) d: 8.71 (2H, d, J = 6.4 Hz), 8.21
3
(1H, s), 7.84 (2H, d, J = 7.2 Hz), 7.75 (1H, d, J = 15.2 Hz),
7.66 (2H, d, J = 6.4 Hz), 7.52 (2H, t, J = 8.0 Hz), 7.38 (1H,
m), 7.31 (1H, m), 7.21 (2H, t, J = 7.6 Hz), 7.15 (2H, d,
J = 8.0 Hz), 6.76 (1H, d, J = 15.2 Hz), 4.72–3.96 (2H, d,
J = 12.8 Hz), 3.06–2.99 (2H, m), 2.62–1.73 (6H, m), 1.26-
1
3
1
1
1
.22 (2H, m); C-NMR (CDCl ) d: 165.0, 149.8, 141.0, 139.7,
3
37.8, 131.3, 129.8, 129.3, 128.5, 126.8, 126.3, 123.1, 119.6,
19.3, 118.8, 114.1, 46.3, 43.1, 42.9, 38.5; HRMS C H N O
?
M?H] Calc. 449.2341, Found m/z 449.2329.
29 29 4
[
Biological assays
Antiplatelet activity
Preparation of platelet rich plasma (PRP) and platelet
poor plasma (PPP) Freshly drawn venous human citrated
123

Med Chem Res
˚
0 A (Brueggemeier et al., 2005). Molecules violating
http://www.molinspiration.com/cgi-bin/properties
http://www.organic-chemistry.org/prog/peo
9
more than one of these rules may have problems with
bioavailability.
Jackson SP, Schoenwaelder SM (2003) Antiplatelet therapy: in search
of the ‘magic bullet’. Nat Rev Drug Discov 2:775–789
Jyothi C, Rai SN, Kalluraya B (2007) Environmentally benign
synthesis of sydnone containing 1,3,4-thiadiazines under micro-
wave and solvent free conditions. J Chem Sci 119:299–302
Kalluraya B, Rao JN, Sujith KV (2008) Microwave assisted one-pot
synthesis of some 2,5-disubstituted 1,3,4-oxadiazoles. Indian J
Heterocycl Chem 17:359–362
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001) Exper-
imental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv
Drug Deliv Rev 46:3–26
Acknowledgments This investigation was supported by Scientific
Research Grant 02/2009-21, awarded by Gazi University BAP.
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