Pyrazole derivatives as inhibitors of arachidonic acid-induced platelet aggregation

Article abstract of DOI:10.1016/j.ejmech.2013.03.048

Antiplatelet drugs are promising therapeutics to intervene with platelet aggregation in arterial thrombosis, most prominently in myocardial infarction and ischemic stroke. Here, we describe the synthesis and structure-activity relationships of potent inhibitors of platelet aggregation based on the 1,5-diarylpyrazol-3-carboxamide scaffold. Analogs from this series demonstrated potent anti-aggregatory activities against arachidonic acid-induced platelet aggregation, as measured by turbidimetric method of Born. 1,5-Diarylpyrazole-3- carboxamides obtained with small-basic amines (7, 8, 50, 51, 61, 62) displayed the strongest activity with IC₅₀ values in low nanomolar range (5.7-83 nM). On the basis of their high potency in cellular environment, these straightforward pyrazole derivatives may possess potential in the design of more potent compounds for intervention with cardiovascular diseases.

Full text of DOI:10.1016/j.ejmech.2013.03.048

European Journal of Medicinal Chemistry 64 (2013) 42e53
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Pyrazole derivatives as inhibitors of arachidonic acid-induced platelet
aggregation
a
a
a
b
c
_
Serkan Levent , Burcu Çalıs¸ kan , Murat Çiftçi , Yes¸ im Özkan , Idil Yenicesu ,
Hüseyin Ünverd, Erden Banoglu a
,
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Taç Sok. No:3, Etiler, Yenimahalle, Ankara 06330, Turkey
b Department of Biochemistry, Faculty of Pharmacy, Gazi University, Yenimahalle, Ankara 06330, Turkey
c Pediatric Hematology Unit and Blood Bank, Faculty of Medicine, Gazi University, Ankara, Turkey
d
ꢀ
Department of Physics, Faculty of Science, Ankara University, Tandogan, Ankara 06100, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Antiplatelet drugs are promising therapeutics to intervene with platelet aggregation in arterial throm-
bosis, most prominently in myocardial infarction and ischemic stroke. Here, we describe the synthesis
and structureeactivity relationships of potent inhibitors of platelet aggregation based on the 1,5-
diarylpyrazol-3-carboxamide scaffold. Analogs from this series demonstrated potent anti-aggregatory
activities against arachidonic acid-induced platelet aggregation, as measured by turbidimetric method
of Born. 1,5-Diarylpyrazole-3-carboxamides obtained with small-basic amines (7, 8, 50, 51, 61, 62) dis-
played the strongest activity with IC50 values in low nanomolar range (5.7e83 nM). On the basis of their
high potency in cellular environment, these straightforward pyrazole derivatives may possess potential
in the design of more potent compounds for intervention with cardiovascular diseases.
Received 25 December 2012
Received in revised form
20 March 2013
Accepted 24 March 2013
Available online 6 April 2013
Keywords:
Pyrazole
Carboxamide
Antiplatelet
Arachidonic acid
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
potent antiplatelet agents to decrease the residual risk for ischemic
events [3,5].
Platelets are key mediators of haemostasis at sites of vascular
injury, however they also mediate pathologic thrombosis.
Thrombus formation through activation of platelets in response to
rupture of an atherosclerotic plaque or endothelial cell erosion is
the major cause of atherothrombotic disease [1e4]. Therefore,
treating patients under thrombotic risk with rapidly acting anti-
platelet drugs is of clinical importance for prevention of morbidity
and mortality [2,5]. However, most of the currently available anti-
platelet drugs, i.e. aspirin and clopidogrel, have limited efficacy and
deleterious side effects [6,7]. Almost half of the world population is
resistant to these drugs correlating to an increased risk of cardio-
vascular events such as recurrent myocardial infarction and stroke
[8,9]. Although the use of dual antiplatelet therapy with aspirin and
clopidogrel has demonstrated significant clinical benefit to reduce
cardiac syndromes after coronary procedures and interventions,
atherothrombotic disease still remains the leading cause of deaths
in Western populations [10]. Therefore, current limitations of an-
tiplatelet therapy continuously trigger the search for novel and
The pyridazine and pyrazole cores were often used as versatile
scaffolds to develop new compounds with wide range of biological
activities and shown that certain compounds having these scaffolds
endowed with inhibitory activity on platelet aggregation [11e16].
In addition, synthesis of diverse pools of small drug-like molecules
possessing vicinal diaryl template was the focus of many medicinal
chemists and our research on vicinal diaryl systems produced a
large number of compounds endowed with interesting pharma-
cological properties against cyclooxygenase and lipoxygenase
pathways [17e21]. Especially, 1-(pyridazin-3-yl)pyrazole-3-
carboxylic acid derivatives having a vicinal diaryl motif endowed
with good inhibitory activity against cyclooxygenase enzymes and
also for the inhibition of 5-lipoxygenase-mediated leukotriene
formation [17,20]. Encouraged with the well-documented anti-
platelet properties associated with these heterocyclic cores, we
screened our own chemical library for their ability to inhibit
arachidonic acid (AA)-induced platelet aggregation leading to the
identification of ethyl 5-(4-chlorophenyl)-1-(6-chloropyridazin-3-
yl)-1H-pyrazole-3-carboxylate (I, Fig. 1) as a developable scaffold
(67% inhibition at 100 mM). In this communication, we present the
synthesis and preliminary results of the structureeactivity re-
lationships (SAR) on newly synthesized pyrazole-3-carboxamide
derivatives having the general structure of I.
* Corresponding author. Tel.: þ90 3122023236; fax: þ90 3122235018.
E-mail addresses: banoglu@gazi.edu.tr, ebanoglu@gmail.com (E. Banoglu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.048

S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
43
acid [20]. Compound 5 was also converted to the corresponding
pyridazinone (28) (a predominant form of hydroxypyridazines)
derivative, by refluxing in glacial acetic acid in the presence of so-
dium acetate [20] (Scheme 2).
Carboxamide derivatives lacking the pyridazine ring at
1-position of pyrazole were prepared as outlined in Scheme 3.
Synthesis of 3-phenylpyrazole-5-carboxylate (31) was accom-
plished by reaction of 3 with hydrazine hydrate in acetic acid [23].
Compound 31 was then methylated by methyl iodide to obtain
N-methylpyrazole derivative (32) in analogy to the published pro-
cedures [24e26]. Since the reaction of unsymmetrical 1,3-
diketones with hydrazine may lead to a tautomeric pyrazole (1H
or 2H) derivative, the methylation of 31 may produce 1- or 2-
methylpyrazole regioisomers. However, the structural verification
of 1-methylpyrazole regioisomer was fully elucidated using HMBC
spectra of 32 in analogy to procedures described before. The HMBC
Fig. 1. Structure of the starting compound and features modified during SAR.
2. Results and discussion
2.1. Chemistry
The pyrazole derivatives studied in this communication were
synthesized as outlined in Schemes 1e6 and the isoxazole de-
rivatives were prepared as shown in Scheme 7. All compounds were
purified by automated flash chromatography and checked for pu-
rity with UPLC before being tested in biological assays (purity
was >97%). The structures of these compounds were confirmed by
high resolution mass spectrometry (HRMS), elemental analysis, IR,
13C NMR and 1H NMR spectral data.
As shown in Scheme 1, the synthesis of methyl 4-(4-
chlorophenyl)-2,4-dioxobutanoate (3) was carried out by Claisen
condensation of the commercially available acetophenone with
dimethyl oxalate in the presence of a strong base [22]. The 1,5-
diarylpyrazole derivative (5) was then conveniently synthesized by
condensation of diketone (3) with 3-chloro-6-hydrazinylpyridazine
(4) in methanol in the presence of 0.5 eq HCl. Nucleophilic substi-
tution of 6-chloropyridazine(5) with sodium methoxidefollowed by
successive ester hydrolysis yielded 6-methoxypyridazine analog in
acid form (6). The structure of 1,5-diarylpyrazole scaffold of 6 was
confirmed using X-ray diffraction as shown in Fig. 2.
The first series of amide derivatives (7e15) were obtained by
coupling of 6 with various amines using ethyl chloroformate as the
carboxyl group activator in the presence of triethylamine. The same
acid derivative was also reacted with various phenols and i-pen-
tanol using N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) as the carboxyl group activator to obtain
the corresponding ester derivatives (16e24). Preparation of 6-
methylsulfonylpyridazine derivative (25) was achieved by reac-
tion of 5 with sodium methane thiolate to obtain the methylthio
intermediate, which was subsequently used to produce methyl-
sulfonyl (25) under controlled oxidation with m-chloroperbenzoic
3
spectra of 32 indicated a remarkable JCH correlation of three pro-
tons at
d
3.8 ppm (N1-CH3) and
d 7.78 ppm (o-ph-H) with quater-
nary sp2 carbon signals at
d
138.3 ppm (C5) and 148.2 ppm (C3),
respectively (Figure S1, Supplementary data). Both compounds (31
and 32) were further hydrolyzed in THFewater mixture in the
presence of LiOH to carboxylic acid derivatives, which were reacted
with selected amines to obtain target carboxamides (33e36). The
synthesis of analogs which lack the 5-phenyl ring of pyrazole was
outlined in Scheme 4. As shown, methyl 2,4-dioxopentanoate (38),
which was prepared from acetone and dimethyl oxalate, was
reacted with 6-chloro-3-hydrazinopyridazine (4) in the presence of
catalytic amount of HCl to obtain the carboxylate 39. As one may
expect, the reaction of unsymmetrical 1,3-diketones with N-aryl-
substituted hydrazines can give two regioisomeric products which
cannot tautomerize. In the case of the reaction of 38 with 6-chloro-
3-hydrazinopyridazine (4), the structure of the resulting derivative
(39) was verified on the basis of observed 1He13C correlations in
HSQS and HMBC spectra (Figure S2, Supplementary data). As
shown, methyl protons at
d
2.8 ppm demonstrated 2JCH correlation
143.9 ppm indicating that the ring
with the sp2 carbon (C5) at
d
closure occurred to put the methyl at 5-position of pyrazole. Sub-
sequent nucleophilic substitution of 39 with sodium methoxide
and ester hydrolysis produced the intermediate carboxylic acid
derivative 40, which was used to prepare the target amide de-
rivatives (41e42).
Chain elongated amide derivatives (45e46) were synthesized as
demonstrated in Scheme 5. The starting intermediate 1,5-
diarylpyrazole-3-propanoic acid (44) was prepared by condensa-
tion of 4,6-dioxo-6-phenylhexanoic acid (43) with HCl salt of
Scheme 1. Synthesis of amide and ester derivatives of 5-(4-chlorophenyl)-1-(6-methoxypyridazin-3-yl)-1H-pyrazole-3-carboxylic acid. Reagents and conditions: i. NaOCH3, MeOH,
rt; ii. MeOH, HCl, rt; iii. a) NaOCH3, MeOH, , b) H2O, ; iv. Amine derivatives, ethyl chloroformate, Et3N, CH2Cl2, rt, or Phenol derivatives, EDC, DMAP, CH2Cl2, rt.
D
D

44
S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
Scheme 2. Synthesis of methylsulfonylpyridazine and pyridazinone derivatives. Re-
agents and conditions: i. a) NaSCH3, THF, , b) NaOH, H2O, , c) MCPA; CH2Cl2, MeOH,
ꢀC / rt, ii. a) CH3COOH, CH3COONa, ; b) LiOH, THF, H2O, 70 ꢀC; iii. Piperidine or
morpholine, EDC, DMAP, CH2Cl2, rt.
D
D
0
D
ceric ammonium nitrate. Compound 56 underwent a palladium-
catalyzed Suzuki cross-coupling reaction with 4-chlorophenyl-
boronic acid to generate 57, which was hydrolyzed (58) and reacted
with selected amines to furnish the desired amide derivatives 59
and 60. Lastly, the pyrrolidine amides (61e62) of methoxypyr-
idazine (6) and methoxyphenyl (49) carboxylic acid intermediates
were also prepared. All starting carboxylic acid intermediates 25,
28, 31, 32, 40, 44, 49 and 58 were reacted with piperidine, mor-
pholine and pyrrolidine to produce the corresponding amides (26e
27, 29e30, 33e36, 41e42, 45e46, 50e51, 59e60, 61e62 were
prepared from pyrrolidine) under conditions using EDC as the
carboxylate activator.
2.2. Structureeactivity relationships
In order to determine the most eminent features of the SAR in
this series, we initially modified the carboxyl side chain of the
starting compound (6) by amidation/esterification with various
amines/phenols of different size, basicity and hydrophobicity with
the aim to improve potency and to trace a tentative SAR profile for
potential anti-platelet agents. Next, we introduced polar sub-
stituents at 6-position of pyridazine, replaced pyridazine with
phenyl and extended the carbon chain in the carboxyl arm of
central pyrazole. Furthermore, the pyrazole derivatives lacking
either the 1-pyridazine or 5-phenyl groups were synthesized. The
final area of our interest in SAR studies was the pyrazole to iso-
xazole scaffold change. By these derivatives, our aim was to explore
how antiplatelet activity would be affected by the presence of a side
chain of varying magnitude and basicity at 3-position of pyrazole
and to understand the potential influence of vicinal diaryl pattern
about the central heterocycle as well as the necessity of central
pyrazole on their biological profiles.
Fig. 2. X-ray crystal structures of 6. Displacement ellipsoids are drawn at 50% proba-
bility level.
3-chloro-6-hydrazinopyridazine (4) in alcohol/triethylamine as
previously reported by our group [17,20]. Amidation of compound
44 with selected amines generated the corresponding amide de-
rivatives (45e46). Isosteric replacement of the pyridazine ring with
phenyl ring was realized by condensation of the HCl salt of 4-
methoxyphenylhydrazine with 3, ester hydrolysis and subsequent
amidation with selected amine derivatives (50e51, Scheme 6).
Isoxazole derivatives were prepared as shown in Scheme 7.
The keto-enol ester 54, which was obtained by reaction of
4-methoxyacetophenone with diethyloxalate, was reacted with
hydroxylamine hydrochloride to produce the isoxazole 55. Bromi-
nation at the 4-position of the isoxazole (56) was achieved with
N-bromosuccinimide with the addition of a catalytic amount of
In an initial screening round, compounds 7e24 were tested at 1
and 10 mM for their ability to inhibit platelet aggregation by the
turbidimetric method of Born [27] employing AA, ADP and collagen
as platelet aggregation inducers (Table 1). Since we intended to
identify potent platelet aggregation inhibitors with improved effi-
ciency versus the starting compound (I), higher concentrations
than 10 mM were not screened as they appeared not pharmaco-
logically relevant and encouraging. Although limited number of
compounds precludes a detailed SAR, a few general features can be
deduced to be taken into account for further work. Analysis of the
aggregation data obtained indicated that amide derivatives as

S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
45
Scheme 3. Synthesis of derivatives lacking pyridazine nucleus. Reagents and conditions: i. NH2NH2$H2O, CH3COOH,
b) SOCl2, CH2Cl2, , c) Piperidine or morpholine, DIEA, CH2Cl2, rt.
D
; ii. CH3I, K2CO3, DMF, 50 ꢀC; iii. a) LiOH, H2O, THF, 70 ꢀC,
D
compared to ester derivatives caused greater inhibition of AA-
induced platelet aggregation at 1 M (Table 1). Furthermore, in
compared to the inhibitory activities of same compounds against
AA-induced platelet aggregation (Table 1).
m
this series, introduction of smaller carboxamide side chains led to
an increased inhibitory activity against AA-induced aggregation of
platelets. Especially, a substantial increase in potency was associ-
ated with amidation with small basic amines, i.e. piperidine and
morpholine (compounds 7 and 8). In addition, secondary amide
derivatives produced by pyridin-4-amine (9) and anilines (10e11)
were also potent inhibitors of AA-induced platelet aggregation.
However, larger phenylpiperazine amides (12e14) led to a dimin-
To determine the exact potency of those compounds that showed
significant inhibition of AA- and collagen-induced platelet aggrega-
tion at 1 mM, selected (potent) compounds (7e11, 13e14, 24 for AA-
induced aggregation; 9e11 for collagen-induced aggregation) were
further investigated in concentration-response studies. As shown in
Table 2, the IC50 values of selected compounds against AA-induced
platelet aggregation were in the range of 0.041e5.49
pounds 7e9 also significantly inhibited the collagen-induced platelet
aggregation with IC50 values of 1.98, 2.5 and 2.8 M, respectively. In
agreement with previous reports [28,29], the IC50 values for the
reference drugs aspirin and indomethacine were 7.76 and 1.62
mM. Com-
ished activity at 1
properties at 10
m
M while still maintaining the antiplatelet
m
m
M. Surprisingly, in the case of 2-pyrimi-
dinylpiperazine derivative (15), a loss of potency was observed at
both concentrations. Lipophilic ester derivatives (16e24) were
much less active at 1 mM as compared to amide derivatives. When
the antiplatelet activity of compounds was tested against collagen-
and ADP-induced platelet aggregation, none of the compounds
showed inhibitory activity against ADP-induced aggregation, while
some (7e11, 16, 24) were able to maintain their antiplatelet prop-
mM
for AA-induced platelet aggregation, respectively, under our assay
conditions. Among all compounds studied for inhibition of AA-
induced platelet aggregation, piperidine (7), morpholine (8), N-pyr-
idin-4-yl (9), N-(4-methoxyphenyl)- (10), and N-(4-chlorophenyl)-
(11) carboxamides were identified as potent antiplatelet agents, with
7 and 8 being the most potent ones with an inhibitory activity about
forty and twenty times than that of indomethacin, respectively
(Table 2). The above results indicated that amide bond formation
with small-sized and more basic amines played a key role in
enhancing antiplatelet activity (comparing 7e9 with 10e11). How-
ever, amidation with less basic and larger phenylpiperazine de-
rivatives (13e14) greatly reduced the potency. It was interesting to
erties against collagen-induced aggregation at 10
active derivatives showing more than 50% inhibition at 10
against collagen-induced aggregation (7e11, 16 and 24) were
rescreened at 1 M to conclude that compounds 7e9 with smaller
mM. The most
mM
m
basic amide side chains were also strong inhibitors of collagen-
induced platelet aggregation although with a reduced potency as
Scheme 4. Synthesis of derivatives lacking phenyl nucleus. Reagents and conditions: i. NaOCH3, MeOH, rt; ii. compound 4, HCl, MeOH, rt; iii. a) NaOCH3, MeOH,
iv. Piperidine or morpholine, EDC, DMAP, CH2Cl2, rt.
D, b) H2O, D;

46
S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
Scheme 5. Synthesis of carboxyl side-chain elongated derivatives. Reagents and conditions: i. a) HCl salt of compound 4, Et3N, MeOH,
D, b) CH3ONa, MeOH, D; ii. Piperidine or
morpholine, EDC, DMAP, CH2Cl2, rt.
see that esterification with i-pentanol (24) also resulted in potent
antiplatelet activity as compared to positive control.
directly to the pyrazole clearly govern the potent inhibition of AA-
induced platelet aggregation. Since amidation of the carboxylic
acid side chain with piperidine (7) and morpholine (8) in methox-
ypyridazine series contributed greatly to the efficiency, we next
evaluated the methoxyphenyl analogs (50 and 51) of respective
amide derivatives, as more lipophilic counterparts. Apparently,
introducing more lipophilicity to molecules even increased the
On the basis of the results from the initial screening, these first
series of compounds were determined to be more potent inhibitors
of AA-induced platelet aggregation, and the carboxamide formation
with smaller basic amines remarkably influenced the activity and
selectivity of the pyrazole molecules toward this pathway. It is well
known that the carbonyl oxygen of carboxamides is the center of
basicity in the processes of protonation and H-bond formation [30].
Therefore, it might be speculated that formation of carboxamides
with basic amines such as piperidine and morpholine results in a
substantial increase in the basicity of this center, most probably
correlating with the potency increase in this type of pyrazole
compounds. For this reason, these carboxamide moieties in mole-
cules were not altered for further SAR approaches (Schemes 2e7)
while concentrating on modifications of the 1,5-diarylpyrazole
skeleton. Our first attempt was to use the most active derivatives
(7 and 8) to pursue SAR on the modification of 6-methoxy of pyr-
idazine ring (Scheme 2). On this basis, moving from methoxy to
methylsulfonyl (26e27) or carbonyl functionality (29e30) led to a
considerable loss of activity indicating that polar substituents at this
position are not well tolerated (Table 2). We next examined the
necessity of the vicinal diaryl pattern of the most active derivatives
(7e8) by successive removal of either aryl groups. Removal of the
pyridazine nucleus strongly disfavored the antiplatelet activity of
potency leading to 50 (IC50 ¼ 0.0079
with respect to corresponding methoxypyridazine analogs (7,
M) (Table 2). In this regard, we
mM) and 51 (IC50 ¼ 0.0073
mM)
IC50 ¼ 0.041
m
M and 8, IC50 ¼ 0.083
m
also prepared additional pyrrolidine carboxamides (61 and 62) of
methoxypyridazine (6) and methoxyphenyl (49) intermediates,
obtaining a great contribution to antiplatelet activity with IC50
values of 0.015 mM and 0.0057 mM, respectively (Fig. 3, Table 2).
Meantime, we also investigated the SAR for the five-membered
heteroaryl ring segment in the current methoxyphenyl de-
rivatives. As a result, the pyrazole to isoxazole switch caused about
600 (59, IC50 ¼ 5.03
m
M) and 2500 (60, IC50 ¼ 18.4
mM) fold loss of
potency irrespective of the carboxamide side chain (Table 2), indi-
cating that pyrazole core was the structural basis for peak activities
in low nanomolar ranges against AA-induced platelet aggregation.
3. Conclusions
We have designed, synthesized and performed biological eval-
uation of a small set of potential antiplatelet pyrazole derivatives by
modifying the scaffold of starting compound (I) from our own
chemical library. Our efforts for increasing the inhibitory efficiency
of (I) against platelet aggregation helped for comprehension of the
key features of these pyrazole-based compounds to direct the
synthesis toward the most potent inhibitors against AA-induced
platelet aggregation. In particular, the different carboxamide side-
chains at 3-position of the pyrazole ring caused a great disparity
in activity among this class of compounds, where only 7e9 with
small-sized carboxamide side arm exhibited significant inhibitory
activity on platelet aggregation induced by both AA and collagen,
specifically favoring the potent inhibition of AA-induced platelet
aggregation. By means of our SAR efforts, a satisfactory explanation
of the necessary scaffold for these pyrazole-based antiplatelet
compounds was provided. As a final outcome, we present com-
pounds 61 and 62 (Fig. 3) as novel potent anti-platelet agents with
IC50 values in the range of two or one-digit nanomolar range (IC50
values of 15 and 5.7 nM, respectively). Further studies will be
dedicated to evaluate the effect of pyridazine and phenyl sub-
stitutions and replacement of pyridazine nucleus with different
heterocycles with a detailed analysis of their molecular pharma-
cology and selectivity toward other platelet-aggregation pathways.
the resulting carboxamide derivatives (33e36; IC50 > 20
Likewise, replacement of 5-phenyl with methyl (41e42) also caused
a dramatic loss of antiplatelet activity (IC50 > 20 M). We also
investigated the effects of introducing a longer amide chain at 3-
position of pyrazole. When the direct connection of amide bond
to the central pyrazole was separated by introduction of a carbon
spacer (45e46), compounds were still active, albeit the potency was
mM).
m
decreased (IC50 ¼ 0.92 and 1.08
mM). Taken together, vicinally diaryl
substituted pyrazole skeleton with a carboxamide connected
4. Experimental section
4.1. Chemistry
Scheme 6. Replacement of pyridazine residue by a phenyl group. Reagents and con-
ditions: i. a) Et3N, MeOH, D; ii. a) LiOH, THF, H2O, D; iii. Piperidine or morpholine, EDC,
1H and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 on
DMAP, CH2Cl2, rt.
a Varian Mercury 400 MHz High Performance Digital FT-NMR

S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
47
Scheme 7. Synthesis of isoxazole derivatives. Reagents and conditions: i. NaOC2H5 EtOH, rt; ii. NH2OH$HCl, EtOH,
acid, 80 ꢀC; v. LiOH, H2O, THF,
; vi. EDC, DMAP, CH2Cl2, rt.
D
; iii. NBS, CAN, AcCN,
D, iv. Pd(PPh)3Cl2, p-chlorophenyboronic
D
spectrometer using tetramethylsilane as the internal standard at
the NMR facility of Faculty of Pharmacy, Ankara University. All
IPDS II single crystal diffractometer (STOE & Cie GmbH, Darmstadt,
Germany). Melting points were determined with an SMP-II Digital
Melting Point Apparatus and are uncorrected (Schorpp Geaete-
technik, Germany). IR spectra were obtained using a Perkin Elmer
Spectrum 400 FTIR/FTNIR spectrometer equipped with a Universal
ATR Sampling Accessory and only carbonyl stretching frequencies
were given. Flash chromatography was performed with a Combi-
flashÒRf automated flash chromatography system with RediSep
columns (Teledyne-Isco, Lincoln, NE, USA) using hexaneeethyl ac-
etate or dichloromethane-methanol solvent gradients. The purity
of the final compounds was determined to be >97% by UPLC with
UV detector. Compounds 3, 4, 31, 32, 38, 43, 44, 54, 55 and 56 were
previously reported and synthesized in analogy to the described
procedures by using conventional methods [17,20,22e26,31]. All
biochemicals used in this study were purchased from Sigmae
Aldrich Chemical Company (St Louis, MO, USA).
chemical shifts were recorded as
d (ppm). Because of the tauto-
meric effect of the pyrazole ring in compounds 33e34, before
obtaining the 13C NMR spectra, this compounds were dissolved in
DMSO-d6, followed by a tiny amount of dry NaH, and 2e3 drops of
D2O were added to the NMR tube and stirred well in order to
prevent the tautomeric effects to obtain a clear 13C NMR spectra.
The structure verification of the compounds 35 and 39 was done by
means of HSQC and HMBC spectra (Varian Mercury 400 FT-NMR).
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 instrument)
operating in ESI (þ) method, also coupled to an AQUITY Ultra
Performance Liquid Chromatography system (Waters Corporation,
Milford, MA, USA). X-ray diffraction data were recorded on an STOE
4.1.1. Methyl 5-(4-chlorophenyl)-1-(6-chloropyridazine-3-yl)-1H-
pyrazole-3-carboxylate (5)
To a mixture of methyl 4-(4-chlorophenyl)-2,4-dioxobutanoate
(3) (0.005 mol) and 6-chloro-3-hydrazino-pyridazine (4) (0.005
mol) in 25 ml methanol, conc. HCl (0.0025 mol) was added. After
Table 1
Structure and activity of compounds 7e24: Percent reduction of platelet aggregation
induced by agonists AA (700
m
M), collagen (2
m
g/ml) and ADP (10
mM).
Cmpd
no
R
AA
Collagen
ADP
10
1
m
M
10
m
M
10
m
M
m
M
7
8
9
Piperidine
Morpholine
4-Aminopiperidine
4-Methoxyaniline
4-Chloroaniline
4-F-phenylpiperazine
4-CH3-phenylpiperazine
4-CF3-piridin-2-ylpiperazine 10.86 65.46
2-Pyrimidinylpiperazine
4-Heptyloxyphenol
4-Methoxyphenol
4-Methylphenol
4-Cyclopentylphenol
4-(Imidazol-1-yl)phenol
3-i-Propylphenol
2-Cl-3-pyridinol
2-Naphthol
92.89 94.77
92.74 94.81
84.87 80.53
94.23 79.93
80.93 74.31
71.13 (76.78)a NIb
70.87 (74.21)a 11.34
73.56 (77.59)a 8.14
72.14 (43.56)a 3.92
70.48 (25.71)a 7.93
Table 2
IC50 values for antiplatelet activity of selected compounds against AA-induced
platelet aggregation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Cmpd no
IC50
(
m
M)
Cmpd no
IC50 (mM)
NI
3.96
79.74
97.59
32.25
20.12
16.02
22.86
NI
NI
6.8
2.7
7
8
9
0.041
0.083
0.11
0.40
0.61
4.67
5.49
1.33
19.6
>20
2.09
12.6
>10
>10
35
36
41
42
45
46
50
51
59
60
61
62
>10
>10
>10
>10
0.92
1.08
0.0079
0.0073
5.03
NI
NI
61.89 (32.24)a NI
10
11
13
14
24
26
27
29
30
33
34
3.87
6.05
93.03
84.25
24.16
NI
23.55 95.00
48.51
NI
NI
86.70
90.05
84.00
86.52
86.31
19.87
NI
5.25
8.97
NI
2.11
NI
42.27
NI
18.4
8.49
NI
0.015
0.0057
7.76
2.36
22.44
3.34
6.77
i-Pentanol
19.00 91.16
66.7 (34.16)a
Aspirin
Indomethacin
a
Percent inhibition at 1
No detectable inhibition.
m
M.
1.62
b

48
S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
(ATR): 2976, 1614, 1092 cmꢁ1. 1H NMR (DMSO-d6):
d 1.51e1.61 (6H,
m), 3.59 (2H, s), 3.75 (2H, s), 3.99 (3H, s), 6.95 (1H, s), 7.31 (2H, m),
7.42 (2H, m), 7.47 (1H, d, J ¼ 8.8 Hz), 7.98 (1H, d, J ¼ 9.6 Hz). 13C NMR
(DMSO-d6): d 24.75, 26.09, 27.05, 43.37, 48.17, 55.69, 110.80, 120.91,
128.27, 129.12, 129.21, 131.00, 134.08, 143.76, 149.21, 152.77, 161.87,
165.19; HRMS (m/z): [M þ H]þ calcd for C20H21ClN5O2: 398.1384;
found 398.1371. Anal. Calcd for C20H20ClN5O2: C, 60.38; H, 5.07; N,
17.60; found C, 59.51; H, 4.86; N, 17.11.
Fig. 3. Structures of the most active compounds 61 and 62 against AA-induced platelet
aggregation.
4.1.6. 4-{[1-(6-Methoxypyridazine-3-yl)-5-(4-chlorophenyl)-1H-
pyrazole-3-yl]carbonyl}morpholin (8)
stirring at rt for 5 h, the obtained precipitate was filtrated, washed
with methanol, and dried. Yield 75%; Mp 162e163 ꢀC; IR (ATR):
Prepared from 2 and morpholin using the procedure 1. Flash
3129, 3084, 2977, 1726, 1094 cmꢁ1; 1H NMR (DMSO-d6):
d 3.87 (3H,
chromatography hexane:EtOAc; yield 67%; Mp 208e209 ꢀC; IR
s), 7.25 (1H, s), 7.37 (2H, d, J ¼ 8.4 Hz), 7.43 (2H, d, J ¼ 8.4), 8.18 (1H,
(ATR): 2976, 1622, 1088 cmꢁ1. 1H NMR (DMSO-d6):
d 3.60e3.64 (6H,
d, J ¼ 8.8 Hz), 8.23 (1H, d, J ¼ 9.6 Hz); 13C NMR (DMSO-d6):
d
52.86,
m), 3.91 (2H, m), 3.99 (3H, s), 7.01 (1H, s), 7.31 (2H, d, J ¼ 8.4 Hz),
112.11, 127.68, 128.67, 129.22, 131.19, 132.58, 134.50, 145.21, 145.88,
7.42 (2H, d, J ¼ 8.4), 7.48 (1H, d, J ¼ 9.2 Hz), 8.00 (1H, d, J ¼ 9.2 Hz).
155.72, 156.56, 162.12; HRMS (m/z): [M
15H11Cl2N4O2: 349.0259; found 349.0257.
þ
H]þ calcd for
13C NMR (DMSO-d6):
d 43.01, 47.86, 55.71, 66.80, 67.16, 111.30,
C
120.93, 128.36, 129.01, 129.25, 131.01, 134.14, 143.88, 148.62, 152.72,
161.86, 165.25. HRMS (m/z): [M þ H]þ calcd for C19H19ClN5O3:
400.1176; found 400.1129. Anal. Calcd for C19H18ClN5O3: C, 57.07; H,
4.54; N, 17.52; found C, 56.67; H, 4.37; N, 17.33.
4.1.2. 5-(4-Chlorophenyl)-1-(6-methoxypyridazin-3-yl)-1H-
pyrazole-3-carboxylic acid (6)
The solution of compound 5 (0.015 mol) and sodium methoxide
(0.045 mol) in 20 ml methanol was heated at reflux for 2.5 h. After
addition of 40 ml water, the reaction mixture was heated at reflux
for another hour, diluted with water, and acidified. The precipitate
formed was washed with water and recrystallized from ethanole
water mixture. Yield 82%; Mp 225e226 ꢀC; IR (ATR): 3381, 2976,
4.1.7. 5-(4-Chlorophenyl)-1-(6-methoxypyridazin-3-yl)-N-(pyridin-
4-yl)-1H-pyrazole-3-carboxamide (9)
Prepared from 2 and pyridine-4-amine using the procedure 1.
Flash chromatography CH2Cl2:MeOH; yield 37%; Mp 247e248 ꢀC. IR
(ATR): 3382, 3130, 2976, 1694, 1091 cmꢁ1 1H NMR (DMSO-d6):
.
2600, 1694, 1089 cmꢁ1; 1H NMR (DMSO-d6):
d
4.04 (3H, s), 7.16 (1H,
d
4.02 (3H, s), 7.27 (1H, s), 7.35 (2H, d, J ¼ 8.4 Hz), 7.45 (2H, d,
s), 7.34 (2H, d, J ¼ 8.8 Hz), 7.45 (2H, d, J ¼ 8.8), 7.52 (1H, d,
J ¼ 8.8 Hz), 7.55 (1H, d, J ¼ 9.2 Hz), 7.84 (2H, d, J ¼ 6.4 Hz), 8.12 (1H,
J ¼ 9.2 Hz), 8.35 (1H, d, J ¼ 9.6 Hz),13.23 (1H, br s); 13C NMR (DMSO-
d, J ¼ 9.2 Hz), 8.46 (2H, d), 10.68 (1H, s, NH). 13C NMR (DMSO-d6):
d6):
d
55.74, 111.31, 120.92, 128.44, 128.94, 129.25, 131.03, 134.19,
d 55.79, 110.08, 114.83, 120.85, 128.75, 129.31, 131.10, 134.37, 145.18,
144.65, 146.30, 152.79, 163.33, 165.36; HRMS (m/z): [M þ H]þ calcd
146.04, 148.23, 151.01, 152.72, 160.91, 165.45. HRMS (m/z): [M þ H]þ
for C15H12ClN4O3: 331.0598; found 331.0602; Anal. Calcd for
calcd for C20H16ClN6O2: 407.1023; found: 407.1035. Anal. Calcd for
C
15H11ClN4O3: C, 54.47; H; 3.35; N, 16.94; found C, 54.04; H, 3.31; N,
C20H15ClN6O2: C, 59.05; H, 3.72; N, 20.66; found C, 58.89; H, 3.66; N,
16.85.
20.57.
4.1.3. Synthesis of amide derivatives 7e15 (procedure 1)
4.1.8. 5-(4-Chlorophenyl)-N-(4-methoxyphenyl)-1-(6-methoxy-
pyridazin-3-yl)-1H-pyrazole-3-carboxamide (10)
Prepared from 2 and p-anisidine using the procedure 1.
Recrystallized from methanol; yield 42%; Mp 209e210 ꢀC. IR
(ATR): 3360, 3136, 3061, 2975, 1675, 1091 cmꢁ1. 1H NMR (CDCl3):
Compound 2 (1.0 mmol) in 10 ml dichloromethane at 0 ꢀC (ice-
bath) was treated with triethylamine (2.2 mmol) and ethyl chlor-
oformate (1.1 mmol). After stirring the reaction mixture at 0 ꢀC
under an argon atmosphere for 30 min, the appropriate amine
derivative (1.1 mmol) was added, and the final mixture was stirred
at room temperature for overnight. After diluting with 15 ml
d
3.82 (3H, s), 4.16 (3H, s), 6.91 (2H, d, J ¼ 9.2 Hz), 7.13 (1H, s), 7.14
(1H, d, J ¼ 9.2 Hz), 7.25 (2H, d, J ¼ 8.8 Hz), 7.34 (2H, d, J ¼ 8.8 Hz),
dichloromethane, the mixture was extracted with
1
N
HCl
7.61 (2H, d, J ¼ 8.8 Hz), 7.68 (1H, d, J ¼ 9.2 Hz), 8.65 (1H, s, NH). 13
C
(3 ꢂ 30 ml), 5% NaHCO3 (3 ꢂ 30 ml) and water (2 ꢂ 30 ml). The
organic phase was dried over sodium sulfate, and evaporated to
dryness. The crude product was purified by flash chromatography
or recrystallized from the appropriate solvent.
NMR (DMSO-d6): d 54.96, 55.09, 109.04, 113.69, 120.03, 121.92,
127.78, 128.29, 128.48, 130.30, 131.48, 133.45, 144.07, 148.27,
152.04, 155.57, 158.88, 164.56. HRMS (m/z): [M þ H]þ calcd for
C
C
22H19ClN5O3: 436.1176; found 436.1183; Anal. Calcd for
22H18ClN5O3: C, 60.62; H, 4.16; N, 16.07; found: C, 60.36; H, 4.17;
4.1.4. Synthesis of ester derivatives 16e24 (procedure 2)
N, 15.99.
To a mixture of compound 2 (0.001 mol), a phenol/alcohol deriv-
ative (0.0011 mol) and DMAP (0.0002 mol) in 30 ml dichloromethane,
4.1.9. N,5-Bis(4-chlorophenyl)-1-(6-methoxypyridazin-3-yl)-1H-
pyrazole-3-carboxamide (11)
N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride
(EDC) (1.1 mmol) was added, and stirred at room temperature over-
night (dicyclohexylcarbodiimide (DCC) was used instead of EDC for
compounds 13 and 15). The reaction mixture was diluted with
dichloromethane and washed with 0.5 N HCl (2 ꢂ 30 ml), 1% NaOH
solution (2 ꢂ 30 ml) and water (2 ꢂ 30 ml). After drying over sodium
sulfate, solvent was evaporated off under reduced pressure and the
crude product was purified by flash chromatography.
Prepared from 2 and 4-chloroaniline using the procedure 1.
Recrystallized with ether-petroleum ether; yield 61%; Mp 254e
255 ꢀC. IR (ATR): 3258, 3120, 3070, 2977, 1671, 1090 cmꢁ1. 1H NMR
(DMSO-d6):
d
4.02 (3H, s), 7.23 (1H, s), 7.35 (2H, d, J ¼ 8.8 Hz), 7.39
(2H, d, J ¼ 8.8 Hz), 7.44 (2H, d, J ¼ 8.8 Hz), 7.54 (1H, d, J ¼ 9.6 Hz),
7.85 (2H, d, J ¼ 8.8 Hz), 8.12 (1H, d, J ¼ 9.2 Hz), 10.45 (1H, s, NH).
13C NMR (DMSO-d6):
d 55.77, 109.94, 120.84, 122.65, 128.16,
128.66, 128.92, 129.25, 129.28, 131.08, 134.29, 138.19, 144.99,
148.66, 152.77, 160.14, 165.39. HRMS (m/z): [M þ H]þ calcd
for C21H16Cl2N5O2: 440.0681; found 440.0663; Anal. Calcd for
4.1.5. 1-{[1-(6-Methoxypyridazine-3-yl)-5-(4-chlorophenyl)-1H-
pyrazole-3-yl]carbonyl}piperidine (7)
Prepared from 2 and piperidine using the procedure 1. Flash
C
21H15Cl2N5O2: C, 57.29; H, 3.43; N, 15.91; found C, 56.77; H, 3.48;
chromatography hexane:EtOAc; yield 77%; Mp 154e155 ꢀC. IR
N, 15.78.

S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
49
4.1.10. 1-{[1-(6-Methoxypyridazine-3-yl)-5-(4-chlorophenyl)-1H-
pyrazole-3-yl]carbonyl}-4-(4-fluorophenyl)piperazine (12)
1091 cmꢁ1. 1H NMR (CDCl3):
d
1.04 (3H, t, J ¼ 7.4 Hz), 1.82 (2H, m),
3.93 (2H, t, J ¼ 6.6 Hz), 4.13 (3H, s), 6.93 (2H, d), 7.15 (4H, m), 7.28
Prepared from 2 and 1-(4-fluorophenyl)piperazine using the
procedure 1. Flash chromatography hexane:EtOAc; yield 47%; Mp
(2H, d, J ¼ 8.4 Hz), 7.35 (2H, d, J ¼ 8.4 Hz), 7.93 (1H, d, J ¼ 9.2 Hz). 13
C
NMR (DMSO-d6): d 11.09, 22.73, 55.81, 70.03, 111.88, 115.79, 120.99,
193e194 ꢀC; IR (ATR): 2977, 1618, 1090 cmꢁ1
;
1H NMR (CDCl3):
123.31, 128.57, 128.62, 129.34, 131.11, 134.45, 144.06, 144.53,
145.14, 152.73, 157.28, 160.82, 165.55. HRMS (m/z): [M þ H]þ calcd
for C24H22ClN4O4: 465.1330; found 465.1318. Anal. Calcd for
C24H21ClN4O4: C, 62.00; H, 4.55; N, 12.05; found C, 61.58; H, 4.58; N,
11.99.
d
3.15 (2H, t), 3.20 (2H, t), 3.99 (2H, t), 4.13 (3H, s), 4.22 (2H, t), 6.89e
7.01 (5H, m), 7.12 (1H, d, J ¼ 9.6 Hz), 7.26 (2H, m), 7.33 (2H, m) 7.70
(1H, d, J ¼ 9.6 Hz). 13C NMR (DMSO-d6):
d
42.50, 47.05, 49.95, 50.51,
55.71, 111.29, 115.94, 116.15, 118.42, 118.50, 120.91, 128.33, 129.03,
129.24, 131.03, 134.16, 143.92, 148.37, 148.79, 152.76, 155.84, 158.18,
161.79, 165.27. HRMS (m/z): [M þ H]þ calcd for C25H23ClFN6O2:
493.1555; found 493.1560; Anal. Calcd for C25H22ClFN6O2: C, 60.91;
H, 4.50; N, 17.05; found: C, 60.63; H, 4.62; N, 16.65.
4.1.15. 4-Methoxyphenyl 5-(4-chlorophenyl)-1-(6-methoxypyridazin-
3-yl)-1H-pyrazole-3-carboxylate (17)
Prepared from 2 and 4-methoxyphenol using the procedure 2.
Recrystallized with methanol; yield 82%; Mp 221e222 ꢀC; IR (ATR):
4.1.11. 1-{[1-(6-Methoxypyridazine-3-yl)-5-(4-chlorophenyl)-1H-
pyrazole-3-yl]carbonyl}-4-(4-methylphenyl)piperazine (13)
Prepared from 2 and 1-(4-methylphenyl)piperazine using the
procedure 1. Flash chromatography hexane:EtOAc; yield 59%; Mp:
2976, 1730, 1092 cmꢁ1. 1H NMR (CDCl3):
d 3.83 (3H, s), 4.13 (3H, s),
6.94 (2H, d, J ¼ 8.8 Hz), 7.15e7.19 (4H, m), 7.28 (2H, d, J ¼ 8.4 Hz),
7.35 (2H, d, J ¼ 8.4 Hz), 7.93 (1H, d, J ¼ 9.2 Hz). 13C NMR (DMSO-d6):
d
55.79, 56.17, 111.83, 115.31, 120.93, 123.27, 128.53, 128.61, 129.31,
193e194 ꢀC. IR (ATR): 3069, 2977, 1639, 1090 cmꢁ1
.
1H NMR
131.11, 134.45, 144.22, 144.53, 145.16, 152.73, 157.85, 160.78, 165.57.
HRMS (m/z): [M þ H]þ calcd for C22H18ClN4O4: 437.1017; found
437.0997; Anal. Calcd for C22H17ClN4O4: C, 60.49; H, 3.92; N, 12.83;
found C, 60.16; H, 4.03; N, 12.67.
(CDCl3): d 2.28 (3H, s), 3.17 (2H, t), 3.23 (2H, t), 3.98 (2H, t), 4.13 (3H,
s), 4.21 (2H, t), 6.87 (2H, d, J ¼ 8.0 Hz), 6.92 (1H, s), 7.09e7.13 (3H,
m), 7.27 (2H, d, J ¼ 8.8 Hz), 7.33 (2H, d, J ¼ 8.8 Hz), 7.72 (1H, d,
J ¼ 9.2 Hz). 13C NMR (DMSO-d6):
d 20.73, 42.52, 47.08, 49.68, 50.27,
55.70, 111.28, 116.91, 120.90, 128.32, 128.98, 129.05, 129.23, 130.12,
131.02, 134.16, 143.91, 148.83, 149.39, 152.76, 161.79, 165.26. HRMS
(m/z): [M þ H]þ calcd for C26H26ClN6O2: 489.1806; found 489.1788;
Anal. Calcd for C26H25ClN6O2: C, 63.86; H, 5.15; N, 17.19; found C,
63.61; H, 5.17; N, 17.02.
4.1.16. 4-Methylphenyl 5-(4-chlorophenyl)-1-(6-methoxypyridazin-
3-yl)-1H-pyrazole-3-carboxylate (18)
Prepared from 2 and 4-methylphenol using the procedure 2.
Flash chromatography CH2Cl2:MeOH; yield 38%; Mp 245e246 ꢀC;
IR (ATR): 3141, 3054, 2977, 1731, 1091 cmꢁ1. 1H NMR (CDCl3):
d 2.37
(3H, s), 4.13 (3H, s), 7.12e7.17 (4H, m), 7.23 (2H, d, J ¼ 8.4 Hz), 7.28
4.1.12. 1-{[1-(6-Methoxypyridazine-3-yl)-5-(4-chlorophenyl)-1H-
pyrazole-3-yl]carbonyl} 4-(5-trifluoromethylpyridine-2-yl)
piperazine (14)
Prepared from 2 and 1-(5-trifluoromethylpyridine-2-yl)pipera-
zine using the procedure 1. Flash chromatography hexane:EtOAc;
yield 71%; Mp 198e199 ꢀC; IR (ATR): 3129, 2978,1612,1079 cmꢁ1. 1H
(2H, d, J ¼ 8.4 Hz), 7.35 (2H, d, J ¼ 8.4 Hz), 7.93 (1H, d, J ¼ 9.2 Hz). 13
C
NMR (DMSO-d6):
d 21.11, 55.81, 111.85, 120.94, 122.15, 128.57,
128.62, 129.33, 130.70, 131.14, 134.48, 136.10, 144.51, 145.20,
148.67, 152.75, 160.61, 165.60. HRMS (m/z): [M þ H]þ calcd for
C
C
22H18ClN4O3: 421.1067; found 421.1074; Anal. Calcd for
22H17ClN4O3: C, 62.79; H, 4.07; N, 13.31; found C, 62.65; H, 4.17; N,
NMR (DMSO-d6):
d
3.71e3.76 (6H, m), 4.00 (3H, s), 4.05 (2H, m), 6.95
13.26.
(1H, d, J ¼ 8.8 Hz), 7.04 (1H, s), 7.33 (2H, d, J ¼ 8.4 Hz), 7.44 (2H, d,
J ¼ 8.4Hz), 7.49 (1H, d, J ¼ 9.6 Hz), 7.82 (1H, dd, J1 ¼8.8Hz, J2 ¼ 2.4Hz),
4.1.17. 4-Cyclopentylphenyl 5-(4-chlorophenyl)-1-(6-methoxy-
pyridazin-3-yl)-1H-pyrazole-3-carboxylate (19)
8.04 (1H, d, J ¼ 8.8 Hz), 8.42 (1H, s). 13C NMR (DMSO-d6):
d 42.33,
44.45, 45.31, 46.64, 55.73, 67.16,107.09,111.38,120.95,128.34,129.02,
129.25, 131.03, 134.16, 135.33, 143.91, 145.91, 148.74, 152.75, 160.68,
161.97, 165.27. HRMS (m/z): [M þ H]þ calcd for C25H22ClF3N7O2:
544.1476; found 544.1464; Anal. Calcd for C25H21ClF3N7O2: C, 55.20;
H, 3.89; N, 18.03; found C, 55.04; H, 3.97; N, 17.76.
Prepared from 2 and 4-cyclopentylphenyl using the procedure
2. Flash chromatography CH2Cl2:MeOH; yield 81%; Mp 212e213 ꢀC;
IR (ATR): 3141, 3063, 2974, 1733, 1090 cmꢁ1 1H NMR (CDCl3):
.
d
1.55e1.85 (6H, m), 2.09 (2H, m), 3.01 (1H, m), 4.13 (3H, s), 7.15e
7.19 (4H, m), 7.26e7.30 (4H, m), 7.35 (2H, d, J ¼ 8.4 Hz), 7.93 (1H, d,
J ¼ 9.2 Hz). 13C NMR (DMSO-d6):
d 25.70, 34.96, 45.45, 55.82, 110.91,
4.1.13. 1-{[1-(6-Methoxypyridazine-3-yl)-5-(4-chlorophenyl)-1H-
pyrazole-3-yl]carbonyl}-4-(pyrimidine-2-yl)piperazine (15)
111.87, 120.97, 122.14, 128.60, 128.73, 129.34, 131.15, 134.48, 144.50,
144.57, 145.20, 148.80, 152.75, 160.65, 165.60. HRMS (m/z):
[M þ H]þ calcd for C26H24ClN4O3: 475.1537; found 475.1520; Anal.
Calcd for C26H24ClN4O3: C, 65.75; H, 4.88; N, 11.80; found C, 65.60;
H, 4.84; N, 11.64.
Prepared from 2 and 1-(2-pyrimidyl)piperazine using the pro-
cedure 1. Flash chromatography CH2Cl2:MeOH; Recrystallized with
methanol; yield 63%; Mp 241e242 ꢀC; IR (ATR): 3056, 2976, 1628,
1092 cmꢁ1. 1H NMR (CDCl3):
d 3.91e3.97 (8H, m), 4.14 (3H, s), 6.55
(1H, t, J ¼ 4.8 Hz), 6.94 (1H, s), 7.13 (1H, d, J ¼ 9.6 Hz), 7.27 (2H, m),
4.1.18. 4-(1H-Imidazol-1-yl)phenyl 5-(4-chlorophenyl)-1-(6-
methoxypyridazin-3-yl)-1H-pyrazole-3-carboxylate (20)
Prepared from 2 and 4-(1H-imidazol-1-yl)phenol using the
procedure 2. Flash chromatography CH2Cl2:MeOH; recrystallized
with ethanol; yield 31%; Mp 207e208 ꢀC. IR (ATR): 2976, 1728,
7.34 (2H, m), 7.75 (1H, d, J ¼ 9.6 Hz), 8.34 (2H, d, J ¼ 4.8 Hz). 13C NMR
(DMSO-d6):
d
42.50, 43.76, 44.56, 46.99, 55.72, 111.18, 111.35,
120.95, 128.33, 129.11, 129.24, 131.06, 134.16, 143.91, 148.80,
152.78, 158.69, 161.81, 162.07, 165.27. HRMS (m/z): [M þ H]þ calcd
for
C
23H22ClN8O2: 477.1554; found 477.1534; Anal. Calcd for
1093 cmꢁ1. 1H NMR (CDCl3):
d
4.15 (3H, s), 7.17 (1H, s, J ¼ 9.6 Hz),
C
23H21ClN8O2: C, 57.92; H, 4.44; N, 23.50; found C, 58.16; H, 4.57; N,
7.20 (1H, s), 7.23 (1H, m), 7.28e7.30 (3H, m), 7.36 (2H, d, J ¼ 8.0 Hz),
22.96.
7.40 (2H, d, J ¼ 8.4 Hz), 7.47 (2H, d, J ¼ 8.4 Hz), 7.86 (1H, s), 7.91 (1H,
d, J ¼ 9.2 Hz). 13C NMR (DMSO-d6):
d 55.04, 111.23, 118.14, 120.24,
4.1.14. 4-Propoxyphenyl 5-(4-chlorophenyl)-1-(6-methoxypyridazin-
3-yl)-1H-pyrazole-3-carboxylate (16)
Prepared from 2 and 4-propoxyphenol using the procedure 2.
Flash chromatography CH2Cl2:MeOH; Recrystallized with meth-
anol; yield 26%; Mp 197e198 ꢀC. IR (ATR): 3133, 3072, 2974, 1727,
121.59, 123.19, 127.78, 127.80, 128.58, 129.87, 130.34, 133.70, 134.82,
135.62, 143.47, 144.45, 148.43, 151.93, 159.68, 164.79. HRMS (m/z):
[M þ H]þ calcd for C24H18ClN6O3: 473.1129; found 473.1107; Anal.
Calcd for C24H17ClN6O3: C, 60.96; H, 3.62; N, 17.77; found C, 60.42;
H, 3.73; N, 17.50.

50
S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
4.1.19. 3-Isopropylphenyl 5-(4-chlorophenyl)-1-(6-methoxypyrida-
zin-3-yl)-1H-pyrazole-3-carboxylate (21)
which was submitted to next step without further purification to
obtain methylsulfonyl derivative (25) as follows: To the solution of
methylthio intermediate in dichloromethaneemethanol mixture
(5:1), m-chloroperbenzoic acid (70%, 0.0016 mol) was added
portion wise at 0 ꢀC. The reaction mixture was stirred for 20 min at
Prepared from 2 and 3-isopropylphenol using the procedure 2.
Flash chromatography CH2Cl2:MeOH; yield 24%; Mp 167e168 ꢀC; IR
(ATR): 3146, 3076, 2974, 1732, 1092 cmꢁ1. 1H NMR (CDCl3):
d 1.27
(6H, d, J ¼ 6.4 Hz), 2.95 (1H, m), 4.13 (3H, s), 7.07e7.37 (10H, m), 7.94
0
ꢀC and kept stirred overnight at room temperature. After evap-
(1H, d, J ¼ 8.8 Hz). 13C NMR (DMSO-d6):
d
24.39, 33.91, 55.80, 111.88,
oration of reaction media, the residue was suspended in ether and
119.86, 120.20, 120.99, 124.91, 128.57, 128.59, 129.34, 130.14, 131.11,
134.44, 144.49, 145.16, 150.90, 151.18, 152.72, 160.52, 165.55. HRMS
(m/z): [M þ H]þ calcd for C24H22ClN4O3: 449.1380; found 449.1384;
Anal. Calcd for C24H21ClN4O3: C, 64.21; H, 4.72; N, 12.48; found C,
63.94; H, 4.78; N, 12.38.
the precipitate was filtered and recrystallized from ethanol. Yield
74%; Mp 232e233 ꢀC. 1H NMR (DMSO-d6):
d 3.46 (3H, s), 7.20 (1H,
s), 7.46 (4H, m), 8.51 (1H, d, J ¼ 8.8 Hz), 8.53 (1H, d, J ¼ 8.8 Hz).
HRMS (m/z): [M þ H]þ calcd for C15H12ClN4O4S: 379.0268; found
379.0257.
4.1.20. 2-Chloropyridin-3-yl 5-(4-chlorophenyl)-1-(6-methoxypyrida-
zin-3-yl)-1H-pyrazole-3-carboxylate (22)
4.1.24. (5-(4-Chlorophenyl)-1-(6-(methylsulfonyl)pyridazin-3-yl)-
1H-pyrazol-3-yl)(piperidin-1-yl)methanone (26)
Prepared from 2 and 2-chloropyridin-3-ol using the procedure
2. Flash chromatography CH2Cl2:MeOH; recrystallized with
ethanol; yield 16%; Mp 187e188 ꢀC. IR (ATR): 1746, 1091 cmꢁ1. 1H
Prepared from 25 and piperidine using procedure 2. Flash
chromatography hexane:EtOAc; yield 65%; Mp 216e217 ꢀC. 1H NMR
(DMSO-d6):
d 1.57e1.73 (6H, m), 3.38 (3H, s), 3.77 (4H, dt), 6.84
NMR (CDCl3):
(2H, d, J ¼ 8.8 Hz), 7.36 (3H, m), 7.70 (1H, m), 7.93 (1H, d, J ¼ 9.2 Hz),
8.37 (1H, m). 13C NMR (DMSO-d6):
55.84, 112.19, 121.01, 125.42,
d
4.14 (3H, s), 7.17, (1H, d, J ¼ 9.6 Hz), 7.22 (1H, s), 7.28
(1H, s), 7.29 (2H, d, J ¼ 8.4 Hz), 7.38 (2H, d, J ¼ 8.8 Hz), 8.27 (1H,
d, J ¼ 9.2 Hz), 8.32 (1H, d, J ¼ 9.2 Hz). 13C NMR (DMSO-d6):
d
d 23.92, 25.29, 26.26, 40.59, 42.59, 47.42, 111.81, 124.81, 127.60,
128.40, 128.67, 129.36, 131.18, 134.17, 134.57, 143.19, 143.73, 144.06,
145.49, 148.20, 152.66, 159.28, 165.63. HRMS (m/z): [M þ H]þ calcd
for C20H14Cl2N5O3: 442.0474; found 442.0463; Anal. Calcd for
128.32, 128.67, 130.52, 133.58, 143.87, 149.91, 156.76, 160.77, 160.91.
HRMS (m/z): [M þ H]þ calcd for C20H21ClN5O3S: 446.1054; found
446.1033.
C
20H13Cl2N5O3: C, 54.32; H, 2.96; N, 15.84; found C, 54.07; H, 2.92;
N, 15.67.
4.1.25. (5-(4-Chlorophenyl)-1-(6-(methylsulfonyl)pyridazin-3-yl)-
1H-pyrazol-3-yl)(morpholino)methanone (27)
4.1.21. Naphthalen-2-yl 5-(4-chlorophenyl)-1-(6-methoxypyridazin-
3-yl)-1H-pyrazole-3-carboxylate (23)
Prepared from 25 and morpholine using procedure 2. Flash
chromatography hexane:EtOAc; yield 86%; Mp 248e249 ꢀC. 1H
Prepared from 2 and naphthalen-2-ol using the procedure 2.
NMR (DMSO-d6): d 3.39 (3H, s), 3.74 (2H, t), 3.76e3.84 (4H, m), 4.04
Flash chromatography CH2Cl2:MeOH; yield 44%; Mp 216e217 ꢀC. IR
(2H, t), 6.93 (1H, s), 7.28 (2H, d, J ¼ 8.4 Hz), 7.38 (2H, d, J ¼ 8.4 Hz),
(ATR): 3152, 3058, 2977, 1729, 1091 cmꢁ1. 1H NMR (CDCl3):
d 4.14
8.26 (1H, d, J ¼ 8.8 Hz), 8.29 (1H, d, J ¼ 8.8 Hz). 13C NMR (DMSO-d6):
(3H, s), 7.17 (1H, d, J ¼ 9.6 Hz), 7.22 (1H, s), 7.34 (2H, m), 7.37 (2H,
d 41.37, 43.02, 47.87, 66.78, 67.16, 113.02, 125.79, 128.41, 129.13,
m), 7.40 (1H, dd, J1 ¼ 8.8 Hz, J2 ¼ 2.4 Hz), 7.47e7.54 (2H, m), 7.74
129.32, 131.30, 134.42, 144.72, 150.06, 157.52, 161.57, 161.78. HRMS
(1H, d, J ¼ 2.4 Hz), 7.84e7.93 (3H, m), 7.95 (1H, d, J ¼ 9.6 Hz). 13
C
(m/z): [M
448.0846.
þ
H]þ calcd for C19H19ClN5O4S: 448.0846; found
NMR (DMSO-d6):
d
55.81, 111.99, 119.37, 121.00, 122.13, 126.71,
127.50, 128.26, 128.44, 128.59, 129.35, 130.25, 131.13, 131.86, 134.04,
134.47, 144.39, 145.24, 148.49, 152.73, 160.70, 165.57. HRMS (m/z):
[M þ H]þ calcd for C25H18ClN4O3: 457.1067; found 457.1060; Anal.
Calcd for C25H17ClN4O3: C, 65.72; H, 3.75; N, 12.26; found C, 65.22;
H, 3.73; N, 12.15.
4.1.26. 5-(4-Chlorophenyl)-1-(6-oxo-1,6-dihydropyridazin-3-yl)-
1H-pyrazole-3-carboxylic acid (28)
A solution of compound 5 (0.003 mol) and sodium acetate
(0.006 mol) in 50 ml glacial acetic acid was refluxed for 10 h. After
cooling, it was poured into ice-water (100 ml) and the precipitate
formed was filtered off, washed with water, dried and recrystal-
lized from ethanol to obtain methyl 5-(4-chlorophenyl)-1-(6-oxo-
4.1.22. 3-Methylbutyl 5-(4-chlorophenyl)-1-(6-methoxypyridazin-
3-yl)-1H-pyrazole-3-carboxylate (24)
Prepared from 2 and isoamylalcohol using the procedure 2.
1,6-dihydropyridazin-3-yl)-1H-pyrazole-3-carboxylate
(yield
Flash chromatography CH2Cl2:MeOH; yield 79%; Mp 149e150 ꢀC. IR
91%), which was subsequently saponified (0.003 mol) with
lithium hydroxide (0.008 mol) in THF/water (1:1) mixture at 70 ꢀC
for 3 h. After acidification, the precipitate was filtered and
recrystallized from ethanol to furnish 28. Yield 85%; Mp 280e
(ATR): 3131, 3072, 2971, 1706, 1090 cmꢁ1 1H NMR (DMSO-d6):
.
d
0.97 (6H, d, J ¼ 6.4 Hz), 1.51e1.91 (3H, m), 4.12 (3H, s), 4.43 (2H, t,
J ¼ 6.8 Hz), 7.01 (1H, s), 7.14 (1H, d, J ¼ 9.6 Hz), 7.25 (2H, d,
J ¼ 8.4 Hz), 7.33 (2H, d, J ¼ 8.4 Hz), 7.91 (1H, d, J ¼ 9.2 Hz). 13C NMR
281 ꢀC. 1H NMR (DMSO-d6):
(2H, d, J ¼ 8.4 Hz), 7.51 (2H, d, J ¼ 8.4 Hz), 7.82 (1H, d, J ¼ 10.0 Hz),
13.4 (1H, s); HRMS (m/z): [M þ H]þ calcd for C14H10ClN4O3:
317.0441; found 317.0446.
d
7.12e7.15 (2H, d, J ¼ 10.0 Hz), 7.44
(DMSO-d6):
d
22.99, 25.26, 37.57, 55.77, 63.88, 111.20, 120.94,
128.53, 128.73, 129.27, 131.07, 134.33, 144.85, 145.27, 152.75, 161.90,
165.47. HRMS (m/z): [M þ H]þ calcd for C20H22ClN4O3: 401.1380;
found 401.1380; Anal. Calcd for C20H21ClN4O3: C, 59.92; H, 5.28; N,
13.98; found C, 59.25; H, 5.46; N, 13.70.
4.1.27. 6-(5-(4-Chlorophenyl)-3-(piperidine-1-carbonyl)-1H-
pyrazol-1-yl)pyridazin-3(2H)-one (29)
4.1.23. Synthesis of 5-(4-chlorophenyl)-1-(6-(methylsulfonyl)
pyridazin-3-yl)-1H-pyrazole-3-carboxylic acid (25)
Prepared from 28 and piperidine using procedure 2. Flash
chromatography CH2Cl2:MeOH; yield 55%; Mp 228e229 ꢀC. IR
Compound 5 (0.003 mol) was mixed with sodium thiometh-
oxide (0.009 mol) in THF (35 ml) under argon atmosphere and
refluxed for 3 h. After that, sodium hydroxide (0.0033 mol) and
25 ml water were added and refluxed for another 2 h. The solution
was poured into iced-water and acidified with HCl, the precipitate
was filtered to furnish the crude intermediate 5-(4-chlorophenyl)-
1-(6-(methylthio)pyridazin-3-yl)-1H-pyrazole-3-carboxylic acid,
(ATR): 3155, 3032, 2952, 1680, 1600 cmꢁ1. 1H NMR (CDCl3):
d 1.70
(6H, m), 3.80 (4H, m), 6.80 (1H, s), 7.06 (1H, d, J ¼ 10.0 Hz), 7.26 (2H,
d, J ¼ 8.8 Hz), 7.37 (2H, d, J ¼ 8.4 Hz), 7.72 (1H, d, J ¼ 10.0 Hz), 10.54
(1H, s). 13C NMR (DMSO-d6):
d 24.85, 25.89, 26.98, 43.88, 48.47,
111.40, 128.19, 129.13, 130.23, 131.01, 132.66, 135.49, 141.94, 143.85,
149.49, 159.94, 162.06. HRMS (m/z): [M
H]þ calcd for
19H19ClN5O2: 384.1227; found 384.1222.
þ
C

S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
51
4.1.28. 6-(5-(4-Chlorophenyl)-3-(morpholine-4-carbonyl)-1H-
pyrazol-1-yl)pyridazin-3(2H)-one (30)
39, which was used in the synthesis of 40. Yield 63%; 1H NMR
(CDCl3):
d 2.80 (3H, s), 3.98 (3H, s), 6.77 (1H, s), 7.68 (1H, d,
Prepared from 28 and morpholine using procedure 2. Flash
J ¼ 8.0 Hz), 8.27 (1H, d, J ¼ 8.0 Hz). 13C NMR (CDCl3):
d 15.16, 52.54,
chromatography CH2Cl2:MeOH; yield 61%; Mp 249e250 ꢀC. IR
111.93, 123.73, 130.72, 143.97, 145.69, 155.55, 156.04, 162.45. HRMS
(m/z): [M
(ATR): 3185, 3112, 2966, 1678, 1617 cmꢁ1. 1H NMR (CDCl3):
d
3.73e
þ
H]þ calcd for C10H10ClN4O2: 253.0492; found
4.08 (8H, m), 6.90 (1H, s), 7.07 (1H, d, J ¼ 10.0 Hz), 7.25 (2H, d,
253.0496). 39 (0.006 mol) and sodium methoxide (0.018 mol) in
methanol were refluxed for 2 h. After evaporating the solvent to
one-third, the water was added and refluxed for another hour and
acidified with HCl. The yellow precipitate was recrystallized from
acetoneewater mixture to furnish 40 as a white solid. Yield 89%;
J ¼ 8.8 Hz), 7.38 (2H, d, J ¼ 8.8 Hz), 7.65 (1H, d, J ¼ 10.0 Hz), 10.70
(1H, s). 13C NMR (DMSO-d6):
d 43.01, 47.85, 66.80, 67.16, 110.99,
128.57, 129.41, 130.93, 131.86, 132.93, 134.34, 141.46, 143.60, 148.46,
160.54, 161.77. HRMS (m/z): [M þ H]þ calcd for C18H17ClN5O3:
386.1026; found 386.1020.
Mp 211e211.5 ꢀC. 1H NMR (CDCl3):
d 4.10 (3H, s), 6.78 (1H, s), 7.50
(1H, d, J ¼ 9.6 Hz), 8.07 (1H, d, J ¼ 9.2 Hz); HRMS (m/z): [M þ H]þ
4.1.29. Synthesis of (3-(4-chlorophenyl)-1H-pyrazol-5-yl)(piperidin-
1-yl)methanone (33)
calcd for C10H11N4O3: 235.0831; found 235.0832.
After hydrolysis of the ester, thionyl chloride (0.025 mol) and
catalytic amount of DMF were added to the solution of acid de-
rivative (0.01 mol) in CH2Cl2 under argon atmosphere and refluxed
for 3 h. Then, to the reaction mixture, diisopropylethylamine
(0.015 mol) and piperidine (0.01 mol) were added and stirred
overnight at room temperature. After the reaction was complete,
the solvent was evaporated and the residue was purified by flash
chromatography using CH2Cl2:MeOH solvent gradient. Yield 59%;
Mp 223e224 ꢀC. IR (ATR): 3098, 2938, 1578 cmꢁ1. 1H NMR (CDCl3):
4.1.34. (1-(6-Methoxypyridazin-3-yl)-5-methyl-1H-pyrazol-3-
yl)(piperidin-1-yl)methanone (41)
Prepared from 40 and piperidine using procedure 2. Flash
chromatography CH2Cl2:MeOH; yield 53%; Mp 98.4e99.1 ꢀC. 1H
NMR (CDCl3):
d 1.65 (6H, m), 2.73 (3H, s), 3.74 (4H, m), 4.17 (3H, s),
6.50 (1H, s), 7.12 (1H, d, J ¼ 9.6 Hz), 8.03 (1H, d, J ¼ 9.6 Hz). 13C NMR
(DMSO-d6):
d 14.19, 24,77, 26.09, 27.02, 43.31, 48.10, 55.57, 110.60,
121.14, 126.14, 141.82, 148.67, 153.47, 162.21, 164.66. HRMS (m/z):
[M þ H]þ calcd for C15H20N5O2: 302.1617; found 302.1611.
d
1.69 (6H, m), 3.76 (4H, m), 6.73 (1H, s), 7.37 (2H, d, J ¼ 8.8 Hz), 7.70
(2H, d, J ¼ 8.8 Hz). 13C NMR (DMSO-d6 þ NaH þ 3 drops D2O):
4.1.35. (1-(6-Methoxypyridazin-3-yl)-5-methyl-1H-pyrazol-3-
yl)(morpholino) methanone (42)
d
25.10, 25.90, 26.64, 43.10, 48.53, 104.28, 126.57, 128.76, 129.48,
136.18, 147.70, 148.40, 165.86. HRMS (m/z): [M þ H]þ calcd for
Prepared from 40 and morpholine using procedure 2. Flash
C
15H17ClN3O: 290.1060; found 290.1053.
chromatography CH2Cl2:MeOH; yield 46%; Mp 104.5e105.5 ꢀC. 1H
NMR (CDCl3):
(3H, s), 6.66 (1H, s), 7.48 (1H, d, J ¼ 9.2 Hz), 8.06 (1H, d, J ¼ 9.6 Hz).
13C NMR (DMSO-d6):
14.14, 42.98, 47.82, 55.60, 66.83, 67.19,111.18,
d 2.60 (3H, s), 3.60e3.64 (6H, m), 3.91 (2H, t), 4.08
4.1.30. (3-(4-Chlorophenyl)-1H-pyrazol-5-yl)(morpholino)
methanone (34)
d
Prepared from 31 acid and morpholine as described for 33. Flash
121.14, 126.30, 141.98, 148.12, 153.43, 162.14, 164.75. HRMS (m/z):
[M þ H]þ calcd for C14H18N5O3: 304.1410; found 304.1412.
chromatography CH2Cl2:MeOH; yield 59%; Mp 248e248.5 ꢀC. IR
(ATR): 3132, 2976, 1591 cmꢁ1. 1H NMR (CDCl3):
d 3.76 (4H, m), 3.88
(4H, m), 6.80 (1H, s), 7.40 (2H, d, J ¼ 8.0 Hz), 7.64 (2H, d, J ¼ 8.0 Hz).
4.1.36. 3-(5-(4-Chlorophenyl)-1-(6-methoxypyridazin-3-yl)-1H-
pyrazol-3-yl)-1-(piperidin-1-yl)propan-1-one (45)
13C NMR (DMSO-d6 þ NaH þ 3 drops D2O):
d 43.39, 45.72, 66.61,
67.33, 105.02, 126.48, 128.69, 129.31, 136.41, 147.80, 148.47, 165.35.
HRMS (m/z): [M þ H]þ calcd for C14H15ClN3O2: 292.0853; found
292.0843.
Prepared from 44 and piperidine using procedure 2. Flash
chromatography CH2Cl2:MeOH; yield 89%; Mp 130e131 ꢀC. 1H NMR
(CDCl3):
d
1.53e1.63 (6H, m), 2.77 (2H, t, J ¼ 7.8 Hz), 3.08 (2H, t,
J ¼ 7.8 Hz), 3.43 (2H, t, J ¼ 5.4 Hz), 3.58 (2H, t, J ¼ 5.4 Hz), 4.09 (3H,
s), 6.39 (1H, s), 7.07 (1H, d, J ¼ 9.2 Hz), 7.22 (2H, d, J ¼ 8.4 Hz), 7.29
(2H, d, J ¼ 8.8 Hz), 7.75 (1H, d, J ¼ 9.6 Hz). 13C NMR (DMSO-d6):
4.1.31. (3-(4-Chlorophenyl)-1-methyl-1H-pyrazol-5-yl)(piperidin-
1-yl)methanone (35)
Prepared from 32 acid and piperidine as described for 33. Flash
d 24.20, 24.77, 26.02, 26.72, 32.11, 42.70, 46.51, 55.52,109.50,120.69,
chromatography CH2Cl2:MeOH; yield 47%; Mp 119.5e121 ꢀC. 1H
127.70, 129.09, 130.19, 130.70, 133.55, 143.65, 153.01, 154.77, 164.69,
169.98. HRMS (m/z): [M þ H]þ calcd for C22H25ClN5O2: 426.1697;
found 426.1679.
NMR (CDCl3): d 1.65 (6H, m), 3.60 (4H, m), 3.98 (3H, s), 6.53 (1H, s),
7.35 (2H, d, J ¼ 8.4 Hz), 7.70 (2H, d, J ¼ 8.8 Hz). 13C NMR (DMSO-d6):
d
24.61, 25.94, 26.77, 38.45, 43.03, 48.48, 103.81, 127.51, 129.39,
132.23, 132.87, 138.30, 148.19, 160.41. HRMS (m/z): [M þ H]þ calcd
4.1.37. 3-(5-(4-Chlorophenyl)-1-(6-methoxypyridazin-3-yl)-1H-
pyrazol-3-yl)-1-morpholinopropan-1-one (46)
for C16H19ClN3O: 304.1217; found 304.1220.
Prepared from 44 and morpholine using procedure 2. Flash
4.1.32. (3-(4-Chlorophenyl)-1-methyl-1H-pyrazol-5-yl)(morpholino)
methanone (36)
chromatography CH2Cl2:MeOH; yield 68%; Mp 127e128 ꢀC. 1H
NMR (CDCl3):
d
2.78 (2H, t, J ¼ 7.2 Hz), 3.09 (2H, t, J ¼ 6.4 Hz), 3.50
Prepared from 32 acid and morpholine as described for 33. Flash
(2H, m), 3.66 (6H, m), 4.10 (3H, s), 6.39 (1H, s), 7.07 (1H, d,
chromatography CH2Cl2:MeOH; yield 81%; Mp 158e159 ꢀC. 1H
J ¼ 9.2 Hz), 7.22 (2H, d, J ¼ 8.8 Hz), 7.30 (2H, d, J ¼ 8.4 Hz), 7.72 (1H,
NMR (CDCl3):
d
3.70 (8H, m), 4.10 (3H, s), 6.55 (1H, s), 7.36 (2H, d,
d, J ¼ 9.2 Hz). 13C NMR (DMSO-d6):
d 23.98, 31.87, 42.22, 46.02,
J ¼ 8.0 Hz), 7.70 (2H, d, J ¼ 8.0 Hz). 13C NMR (CDCl3):
d
38.66, 48.06,
55.50, 66.79, 109.47, 120.69, 127.68, 129.09, 130.15, 130.69, 133.56,
143.66, 152.98, 155.62, 164.69, 170.66. HRMS (m/z): [M þ H]þ calcd
for C21H23ClN5O3: 428.1489; found 428.1496.
55.44, 66.83, 72.30, 104.64, 127.51, 129.40, 132.15, 132.90, 137.41,
148.13, 160.66. HRMS (m/z): [M þ H]þ calcd for C15H17ClN3O2:
306.1009; found 306.1010.
4.1.38. 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-1H-pyrazole-3-
carboxylic acid (49)
Compound 48 [32] (0.003 mol) and lithium hydroxide
(0.008 mol) in THF/water mixture (1:1) were refluxed for 4 h. After
cooling, the reaction mixture was diluted with water, acidified and
the precipitate was filtered and recrystallized from ethanol. Yield
4.1.33. 1-(6-Methoxypyridazin-3-yl)-5-methyl-1H-pyrazole-3-
carboxylic acid (40)
Compound 38 (0.003 mol) and 4 (0.0032 mol) were dissolved in
methanol and HCl (0.003 mol) was added. The reaction mixture
was stirred at rt for 6 h. The precipitate formed was filtered to give

52
S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
95%; Mp 92e93 ꢀC. 1H NMR (DMSO-d6):
J ¼ 8.4), 7.07 (1H, s), 7.23e7.26 (4H, m), 7.42 (2H, d, J ¼ 8.4 Hz).
HRMS (m/z): [M þ H]þ calcd for C17H14ClN2O3: 329.0693; found
329.0702.
d
3.77 (3H, s), 6.99 (2H, d,
4.1.44. (4-(4-Chlorophenyl)-5-(4-methoxyphenyl)isoxazol-3-
yl)(morpholino)methanone (60)
Prepared from 58 and morpholine using procedure 2. Flash
chromatography hexane:EtOAc; yield 63%; Mp 169e170 ꢀC. 1H NMR
(CDCl3): d 3.41e3.46 (4H, m), 3.64e3.66 (2H, m), 3.70e3.73 (2H, m),
4.1.39. (5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-1H-pyrazol-3-
yl)(piperidin-1-yl)methanone (50)
3.83 (3H, s), 6.89 (2H, d, J ¼ 9.2 Hz), 7.32 (2H, d, J ¼ 8.4 Hz), 7.38 (2H,
d, J ¼ 8.8 Hz), 7.49 (2H, d, J ¼ 9.2 Hz). 13C NMR (DMSO-d6):
d 42.46,
Prepared from 49 and piperidine using procedure 2. Flash
47.41, 56.07, 66.50, 66.88, 112.95, 115.40, 119.24, 128.18, 129.35,
129.89, 131.82, 134.01, 158.73, 159.63, 161.75, 165.99. HRMS (m/z):
[M þ H]þ calcd for C21H20ClN2O4: 399.1112; found 399.1126.
chromatography CH2Cl2:MeOH; yield 77%; Mp 118e119 ꢀC. 1H NMR
(CDCl3): d 1.60e1.68 (6H, m), 3.73 (2H, m), 3.82 (3H, s), 3.93 (2H, m),
6.83 (1H, s), 6.86 (2H, d, J ¼ 9.2 Hz), 7.15 (2H, d, J ¼ 8.4 Hz), 7.18 (2H,
d, J ¼ 8.8 Hz), 7.27 (2H, d, J ¼ 8.4 Hz). 13C NMR (DMSO-d6):
d
24.83,
4.1.45. (5-(4-Chlorophenyl)-1-(6-methoxypyridazin-3-yl)-1H-
pyrazol-3-yl)(pyrrolidin-1-yl)methanone (61)
26.14, 27.10, 43.44, 48.10, 56.13, 109.71, 115.06, 127.69, 128.93,
129.37, 130.97, 132.86, 134.05, 142.63, 147.87, 159.64, 162.20. HRMS
(m/z): [M þ H]þ calcd for C22H23ClN3O2: 396.1479; found 396.1462.
Prepared from 6 and pyrrolidine using procedure 2. Flash
chromatography CH2Cl2:MeOH; yield 75%; Mp 185e186 ꢀC. 1H NMR
(CDCl3):
d
1.95 (4H, m), 3.70 (2H, t, J ¼ 6.8 Hz), 3.98 (2H, t,
4.1.40. (5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-1H-pyrazol-3-
yl)(morpholino) methanone (51)
J ¼ 6.4 Hz), 4.12 (3H, s), 7.01 (1H, s), 7.10 (1H, d, J ¼ 9.2 Hz), 7.25 (2H,
d, J ¼ 8.8 Hz), 7.32 (2H, d, J ¼ 8.8 Hz), 7.75 (1H, d, J ¼ 9.6 Hz). 13
C
Prepared from 49 and morpholine using procedure 2. Flash
NMR (DMSO-d6): d 24.11, 26.74, 47.26, 49.01, 55.71, 111.27, 120.97,
chromatography CH2Cl2:MeOH; yield 56%; Mp 142e143 ꢀC. 1H NMR
128.20, 129.20, 131.04, 134.08, 143.72, 149.99, 152.86, 160.74, 165.21.
HRMS (m/z): [M þ H]þ calcd for C19H19ClN5O2: 384.1227; found
384.1212.
(CDCl3):
d 3.73e3.80 (6H, m), 3.83 (3H, s), 4.18e4.20 (2H, m), 6.88
(2H, d, J ¼ 9.2 Hz), 6.93 (1H, s), 7.15 (2H, d, J ¼ 8.4 Hz), 7.18 (2H, d,
J ¼ 8.4 Hz), 7.28 (2H, d, J ¼ 8.4 Hz). 13C NMR (DMSO-d6):
d 43.06,
47.87, 56.15, 66.88, 67.21, 110.23, 115.08, 127.76, 128.82, 129.39,
130.99, 132.79, 134.12, 142.79, 147.32, 159.73, 162.16. HRMS (m/z):
[M þ H]þ calcd for C21H21ClN3O3: 398.1271; found 398.1278.
4.1.46. (5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-1H-pyrazol-3-
yl)(pyrrolidin-1-yl) methanone (62)
Prepared from 49 and pyrrolidine using procedure 2. Flash
chromatography CH2Cl2:MeOH; yield 88%; Mp 154.5e155 ꢀC (lit.
4.1.41. Ethyl 4-(4-chlorophenyl)-5-(4-methoxyphenyl)isoxazole-3-
carboxylate (57)
Mp 152 ꢀC) [33]. 1H NMR (CDCl3):
d 1.94 (4H, m), 3.69 (2H, t,
J ¼ 6.8 Hz), 3.83 (3H, s), 4.01 (2H, t, J ¼ 6.4 Hz), 6.87 (2H, d,
4-Chlorophenylboronic acid (2 mmol) was added to a mixture of
56 (1.7 mmol) and sodium bicarbonate (5.1 mol) in DMF (15 ml).
Afteraddition of water (5 ml), the resulting suspensionwas degassed
by evacuation-nitrogen purge (three cycles) and then bubbled with
nitrogen gas for 10 min. Dichlorobis(triphenylphosphine) palladiu-
m(II) (0.085 mmol) was added, and the reaction mixture was heated
under nitrogen atmosphere at 80 ꢀC for 3 h. The mixture was allowed
to cool, water (100 ml) was added, and extracted with EtOAc
(3 ꢂ 50 ml), and filtered through a Celite pad. The organic phase was
washed with water (2 ꢂ 100 ml) and with aqueous sodium chloride
solution (100 ml) and then dried over anhydrous Na2SO4 and
filtered. The filtrate was evaporated in vacuo, and the crude product
was purified by automated flash chromatography (hexaneeEtOAc).
J ¼ 8.8 Hz), 7.00 (1H, s), 7.16 (2H, d, J ¼ 8.4 Hz), 7.20 (2H, d,
J ¼ 8.8 Hz), 7.28 (2H, d, J ¼ 8.4 Hz). 13C NMR (DMSO-d6):
d 23.30,
25.97, 46.40, 48.22, 55.35, 109.19, 114.29, 126.82, 128.17, 128.60,
130.19, 132.19, 133.27, 141.82, 147.88, 158.87, 160.38. HRMS (m/z):
[M þ H]þ calcd for C21H21ClN3O2: 382.1322; found 382.1321.
4.2. Antiplatelet activity
Test compounds were investigated by the turbidimetric Born
method for their antiplatelet activities [27]. Platelet-reach plasma
(PRP) and platelet-poor plasma (PPP) were obtained at the Blood
Center of the Gazi University Hospital Ankara (Turkey) or prepared
in house from the venous blood of healthy volunteers. In brief,
freshly drawn venous human citrated (3.2% sodium citrate, 9:1)
blood from healthy subjects, who had not taken any nonsteroidal
anti-inflammatory or other drugs with antiplatelet activity for
fifteen days, was centrifuged at 800 rpm or at 3500 rpm (Digicen
21R, Orto Alresa, Madrid, Spain) to obtain platelet-rich plasma
(PRP) or platelet-poor plasma (PPP), respectively. Platelet numbers
were determined with a cell counter (Swelab Alfa, Boule Medical
AB, Stockholm, Sweden) and adjusted to 3.8 ꢂ 108 platelets/ml with
PPP. Platelet aggregation was measured turbidimetrically with the
APACT aggregometer (Automated Platelet Aggregation and Coagu-
lation Tracer, Biochemica GmbH, Flacht, Germany) with software
APACT professional version 1.1.
Yield 65%. Mp 125e126 ꢀC. 1H NMR (DMSO-d6):
d 1.15 (3H, t,
J ¼ 7.2 Hz), 3.77 (3H, s), 4.23 (2H, q, J ¼ 7.2 Hz), 7.02 (2H, d, J ¼ 8.4 Hz),
7.41 (4H, d, J ¼ 8.4 Hz), 7.52 (2H, d, J ¼ 8.0 Hz). HRMS (m/z): [M þ H]þ
calcd for C19H17ClNO4: 358.0846; found 358.0840.
4.1.42. 4-(4-Chlorophenyl)-5-(4-methoxyphenyl)isoxazole-3-
carboxylic acid (58)
Prepared from 57 as described for 49. Yield 80%. Mp 157e159 ꢀC.
1H NMR (DMSO-d6):
d
3.77 (3H, s), 7.01 (2H, d, J ¼ 8.4 Hz), 7.39 (4H,
d, J ¼ 8.0 Hz), 7.51 (2H, d, J ¼ 8.0 Hz). HRMS (m/z): [M þ H]þ calcd
for C17H13ClNO4: 330.0533; found 330.0526.
4.1.43. (4-(4-Chlorophenyl)-5-(4-methoxyphenyl)isoxazol-3-
yl)(piperidin-1-yl) methanone (59)
The test compound (or the standard inhibitor) is dissolved in
dimethylsulfoxide (DMSO). To eliminate solvent effects, the final
Prepared from 58 and piperidine using procedure 2. Flash
concentration of DMSO was fixed at <1%. Then 0.5
solution is given to 199 L PRP in the test cuvette and incubated
3 min at 37.4 ꢀC. Then, 10
L of the inducer (final concentrations of
the inducers: arachidonic acid, 700 M; ADP, 10 M; collagen, 2 g/
ml) is added and the change in light transmission was recorded. The
percentage of aggregation is determined as the ratio of heights of
the aggregation curves with and without the test compound. Each
curve is corrected automatically for the light absorption of platelet-
poor plasma (PPP) of the same donor. Percent platelet aggregation
mL of the test
chromatography hexane:EtOAc; yield 41%; Mp 132e133 ꢀC. 1H NMR
m
m
(CDCl3):
d
1.30e1.33 (2H, m), 1.53e1.59 (4H, m), 3.29 (2H, t,
J ¼ 5.4 Hz), 3.63 (2H, t, J ¼ 5.4 Hz), 3.83 (3H, s), 6.89 (2H, d,
m
m
m
J ¼ 9.2 Hz), 7.31e7.37 (4H, m), 7.50 (2H, d, J ¼ 9.2 Hz). 13C NMR
(DMSO-d6): d 24.30, 25.80, 26.60, 42.64, 47.88, 56.06, 112.64, 115.37,
119.29, 128.28, 129.38, 129.85, 131.71, 133.93, 159.35, 161.71, 165.78.
HRMS (m/z): [M þ H]þ calcd for C22H22ClN2O3: 397.1319; found
397.1318.

S. Levent et al. / European Journal of Medicinal Chemistry 64 (2013) 42e53
53
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This investigation was supported by a Scientific Research Grant
110S106 awarded by TUBITAK (The Scientific and Technological
Research Council of Turkey). The authors report no conflicts of
interest.
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