Pyrazol-3-propanoic acid derivatives as novel inhibitors of leukotriene biosynthesis in human neutrophils

Article abstract of DOI:10.1016/j.ejmech.2011.08.009

We recently presented that compounds 4a-b moderately inhibited leukotriene (LT) formation in human neutrophils. For structural derivatization of 4a-b, novel thirty-six title compounds were synthesized and led to more potent inhibition of LT biosynthesis in activated human neutrophils exemplified by compounds 15, 27-30, 32-37, 41, 42 with IC₅₀ values in the range of 1.6-3.5 μM. Moreover, compounds 32, 35, 42, 43 and 44 showed a substantial inhibition of platelet COX-1 activity with IC₅₀ of 2.5, 0.041, 0.3, 0.9 and 0.014 μM, respectively, leading up to dual acting inhibitors. On the basis of their high potency in cellular environment, these straightforward pyrazole-3-propanoic acid derivatives may possess potential in the design of more potent compounds for intervention with inflammatory and allergic diseases.

Full text of DOI:10.1016/j.ejmech.2011.08.009

European Journal of Medicinal Chemistry 46 (2011) 5021e5033
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Pyrazol-3-propanoic acid derivatives as novel inhibitors of leukotriene
biosynthesis in human neutrophils
,
,
1
a
b
,
,
Burcu Çalıs¸ kan a 1, Susann Luderer b , Yagmur Özkan , Oliver Werz 2, Erden Banoglu a
*
ꢀ
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, 06330 Etiler, Ankara, Turkey
b Department of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, D-72076 Tuebingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently presented that compounds 4aeb moderately inhibited leukotriene (LT) formation in human
neutrophils. For structural derivatization of 4aeb, novel thirty-six title compounds were synthesized and
led to more potent inhibition of LT biosynthesis in activated human neutrophils exemplified by
Received 26 May 2011
Received in revised form
2 August 2011
Accepted 5 August 2011
Available online 11 August 2011
compounds 15, 27e30, 32e37, 41, 42 with IC50 values in the range of 1.6e3.5
32, 35, 42, 43 and 44 showed a substantial inhibition of platelet COX-1 activity with IC50 of 2.5, 0.041, 0.3,
0.9 and 0.014 M, respectively, leading up to dual acting inhibitors. On the basis of their high potency in
mM. Moreover, compounds
m
cellular environment, these straightforward pyrazole-3-propanoic acid derivatives may possess potential
in the design of more potent compounds for intervention with inflammatory and allergic diseases.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Diarylpyrazole
5-Lipoxygenase
Cyclooxygenase
Anti-leukotriene
1. Introduction
activate GPCRs such as CysLT1 and CysLT2 causing bronchocon-
striction, airway edema and mucus secretion [1]. Therefore, inhi-
It has been well established that leukotrienes (LTs) formed from
arachidonic acid (AA) by the 5-lipoxygenase (5-LO) pathway play
important roles in various biological processes such as broncho-
constriction, increased vascular permeability, and vasoconstriction
of coronary arteries [1]. 5-LO is the key enzyme in the conversion of
AA to LTA4 in two successive steps. First, oxygenation of AA forms
bition of LT formation represent valuable target for the therapy of
inflammatory ailments like asthma, allergic rhinitis, atherosclerosis
as well as certain types of cancers [5,6]. Today, while cysteinyl-LT
receptor antagonists (i.e. montelukast and zafirlukast) are
successfully applied for asthma therapy, the development of LT
biosynthesis inhibitors is little advanced [7e10] and thus far
zileuton is the only 5-LO inhibitor currently in clinical use [11]
(Fig. 1).
5(S)-hydroperoxy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic
acid
(5-
HPETE), and then, dehydration leads to the unstable epoxide LTA4
which is subsequently converted to either LTB4 by LTA4 hydrolase or
to cysteinyl-LTs (LTC4, D4 and E4) through conjugation with gluta-
thione catalyzed by LTC4 synthases [2]. In intact cells, the 5-LO-
mediated formation of LTA4 from AA also requires a concomitant
involvement of the 5-LO-activating protein (FLAP) that transfers AA
to 5-LO for efficient metabolism [3]. LTB4 acts as a high affinity
ligand for G protein-coupled receptors (GPCR), namely BLT1 and
BLT2 which stimulates the inflammatory response as well as
neutrophil and eosinophil chemotaxis [4]. The cysteinyl LTs also
Recently, we have presented that 1,5-diarylpyrazol-3-propanoic
acids (4aeb in Fig. 1) inhibit LTB4 biosynthesis in human neutro-
phils with IC50 values of 12e14 mM [12]. In order to obtain more
potent derivatives, we aimed at modifying structural features of
4aeb. In particular, the structural changes in the acidic part of
compounds under study did yield substantial improvement of LTB4
biosynthesis inhibition in comparison with the previously prepared
derivatives 4aeb. We hereby demonstrate that preparing certain
amides and esters of 4aeb may lead to potent inhibition of LT
biosynthesis in intact cells without interfering the 5-LO activity
since none of the compounds resulted in inhibition of 5-LO enzyme
in cell-free systems. Our results present a novel pharmacophore for
suppression of LT formation in intact cells, essentially lacking
inhibitory effects on other important enzymes within the AA
cascade such as 5/12/15-LO, COX-1/2, and microsomal prosta-
glandin E2 synthase (mPGES-1).
* Corresponding author. Tel.: þ90 312 2023240; fax: þ90 312 2235018.
E-mail address: banoglu@gazi.edu.tr (E. Banoglu).
1
Both authors contributed equally.
Present address: Chair of Pharmaceutical/Medicinal Chemistry, Institute of
2
Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena,
Germany.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.009

5022
B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
2. Results and discussion
2.1. Chemistry
The target compounds were prepared according to the reaction
sequence shown in Fig. 2. The synthesis of 6-phenyl-4,6-
dioxohexanoic acid derivatives (1aec) was achieved by condensa-
tion of the commercially available acetophenones with succinic
anhydride in the presence of a strong base as published previously
[12]. The regioselective synthesis of 1,5-diarylpyrazole derivatives
(4aec to 6aec) was conveniently accomplished by condensation of
the requisite diketone with the hydrochloride salt of the arylhy-
drazine derivative (4e6) in methanol/triethylamine with slight
modifications of the previously published procedures [12e14]. For
the quinoline derivatives (7aec), the reaction was carried out with
2-hydrazinoquinoline and diketones in the absence of triethyl-
amine. Nucleophilic substitution reaction of 6-chloropyridazine
derivatives (4aec) with sodium methoxide in methanol yielded
6-methoxypyridazines (8aec). The first series of amide derivatives
(9e11, 15e17, 22e24) were obtained by coupling of acids (4ae4c
and 6a) with various amines using ethyl chloroformate as the
carboxyl group activator in the presence of triethylamine. The same
Fig. 1. Structure of zileuton, the lead compounds (4aeb) and general structure of the
synthesized compounds.
Fig. 2. Synthesis of ester and amide derivatives of 1,5-diaryl-pyrazol-3-propanoic acids (9e44). Reagents and conditions: (a) Et3N, MeOH, reflux; (b) i. NaOCH3, MeOH, reflux ii. Hþ
(c) Amine derivatives, ethyl chloroformate, Et3N, CH2Cl2, rt/Amine or Phenol derivatives, EDC, DMAP, CH2Cl2, rt; (d) tert-butylbenzylpiperazine, EDC, DMAP, CH2Cl2, rt.
;

B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
5023
acids also coupled with various phenols under the activation of N-
ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride
First, the modifications of 1,5-diarylpyrazole-3-propanoic acid
structure were introduced basically to obtain compounds with
different physicochemical characteristics while varying the
substituent at the phenyl ring of C5-pyrazole and keeping the
chloropyridazine ring at the N1-pyrazole (compounds 9e26 in
Table 1). Among the tested compounds, only the ester derivative
formed with i-propylphenol (12) as well as the amide derivatives of
tert-butylbenzylpiperazine (15) and of ethyl piperidinecarboxylate
(23) showed potent inhibition of 5-LO product synthesis in the cell-
based assay. Interestingly, the other amide and ester analogs failed
in inhibition of 5-LO product synthesis. For example, linking the
carboxyl group with piperidinecarboxamide (22) instead of more
lipophilic ethyl piperidinecarboxylate (23) was detrimental for
inhibitory activity. With regard to piperazine amides, while the
lipophilic bulky tert-butylbenzyl substituent was well tolerated
(15), the activity with more polar piperazine derivatives (9, 11, 16,
24) was not evident. For the ester analogs, the most potent inhibitor
was the i-propylphenyl derivative (12) and keeping small lipophilic
p-substituents such as tert-butyl (18) or cyclo-pentyl (25) in the
phenol moiety still governed inhibition of cellular 5-LO product
formation. However, the replacement of i-propyl by electron
withdrawing substituents (trifluoromethoxy (14), thiophene (19))
or by heterocyclic ring systems (imidazole (21) and pyrrole (26))
generally diminished the inhibitory activity. Since the compounds
12 and 15 were the only derivatives that elicited more than 50%
(EDC) in the presence of 4-(dimethylamino)pyridine (DMAP) in
CH2Cl2 to afford the ester derivatives (12e14, 18e21, 25, 26 and
42e44). The second series of amide derivatives (27e41) were
synthesized by the reaction of acid derivatives (4e8aec) with
corresponding amines also using EDC and DMAP in CH2Cl2.
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 high resolution mass spectrometry (HRMS), IR and
1H-NMR spectral data.
Our strategy to optimize the activity in our series was concen-
trated on three distinct areas of the lead molecules, 3-(1-(6-
chloropyridazin-3-yl)-5-(4-methyl- or 4-fluorophenyl)-1H-pyr-
azol-3-yl)propanoic acid (4aeb), to deduce structure-activity
relationships (SARs). The areas that we addressed were (i) modi-
fication of the propanoic acid side-chain, (ii) replacement of the
chloropyridazin of the central pyrazole with different aryl groups,
and (iii) incorporation of different p-substituents on the C5-phenyl
of the central pyrazole ring. Together, by incorporation of distinct
chemical functionalities by preparing amide and ester derivatives
of 4aeb, we first initiated the synthesis of pyrazole derivatives
(9e26) as anti-LT agents (Table 1).
2.2. Analysis of inhibition of LT synthesis and structure-activity
relationships
inhibition at 10 mM, the IC50 values were further calculated from
concentration-response curves and determined as 8 and 1.6
respectively.
mM,
For LT biosynthesis, AA must be released from membrane
phospholipids by cPLA2 which is then converted to LTA4 by the
action of 5-LO aided by FLAP [2]. The activation of 5-LO occurs as
a result of Ca2þ mobilization and/or 5-LO phosphorylation by
protein kinases, eventually leading to an association of 5-LO with
membranes in close vicinity to FLAP [2]. For a thorough analysis of
the inhibitory potential of the test compounds, two different test
systems were applied, that is, a cell-based assay using human
neutrophils and a cell-free assay using human recombinant 5-LO. In
the cell-based assay various mechanisms aside from direct inter-
ference with 5-LO enzyme may cause suppression of LT formation
such as inhibition of AA release, antagonism of FLAP, suppression of
5-LO-activating kinases or of Ca2þ mobilization, and inhibition of 5-
LO membrane association [2].
It should be noted that, regardless of each and every modifica-
tion, none of the compounds demonstrated substantially inhibitory
activity against 5-LO activity in a cell-free assay, whereas the
reference compound BWA4C efficiently suppressed 5-LO activity
under the same conditions as expected. This suggests that the
inhibition of 5-LO product formation in intact neutrophils by the
active test compounds did not result from a direct interaction with
the 5-LO enzyme.
On the basis of the results from the initial screening and also
considering the piperazine moiety as a privileged structure with
drug-like properties, compound 15 was selected for further modi-
fications. The chloropyridazine core in 15 was systematically
modified with different aromatic counterparts such as chlor-
ophenyl (29e31), phenyl (32e34), quinoline (35e37) and
methoxypyridazine (38e40) while varying the p-substituent on the
phenyl moiety of C5-pyrazole (Table 1). While replacement of
chloropyridazine in 15 with a phenyl ring (33) slightly reduced the
potency, the phenyl substitution in 32 and 34 versus respective
chloropyridazine derivatives (27e28) did not change the efficiency
as seen from the calculated IC50 values. When the lipophilicity was
increased by incorporation of chlorophenyl (29e31) or quinoline
(35e37), the compounds retained the inhibitory activity although
efficiency was slightly reduced compared to the corresponding
chloropyridazine derivatives (15, 27 and 28). Finally, substituting
the chloropyridazine moiety by methoxypyridazine (38e40)
caused a decrease in the potency. Note that all these structural
modifications also failed to generate compounds that inhibited the
activity of 5-LO in the cell-free assay (Table 1). Detailed
concentration-response studies of active derivatives in the cell-
based assay (using neutrophils challenged with A23187 plus
The initial screening of 9e26 for their ability to inhibit 5-LO
product formation in isolated human neutrophils was performed
at a concentration of 10 m
M. Neutrophils were challenged with Ca2þ
ionophore A23187 that causes substantial formation of 5-LO
products as a result of elevated intracellular Ca2þ concentration
and thus release of AA as substrate for 5-LO [15,16]. However,
diminished 5-LO product formation may thus not necessarily result
from the inhibition of 5-LO but could also arise from the inhibition
of cPLA2 leading to suppressed levels of AA. Therefore, neutrophils
were supplemented with exogenous AA (20 mM) to circumvent the
necessity of cPLA2ꢀ mediated endogenous AA supply. In addition,
compounds were also tested in a cell-free system using superna-
tants of lysates of Escherichia coli expressing human recombinant 5-
LO (or ATP-affinity purified 5-LO), thereby assessing the direct
inhibition of crude enzymatic activity of 5-LO [17]. The iron ligand-
type 5-LO inhibitor N-(3-phenoxycinnamyl)-acetohydroxamic acid
(BWA4C) [18] which is closely related to the clinically developed
zileuton [19], was used as reference compound. BWA4C suppressed
5-LO product synthesis in the cell-based assay with IC50 value of
20
conclusion, among all compounds, the tert-butylbenzylpiperazine
amide derivative with vicinal 6-chloropyridazine- and 4-
fluorophenyl-substitution about a central pyrazole was the most
efficient one (15, IC50 ¼ 1.6 M) with a potency close to the clinical
drug zileuton (IC50 ¼ 0.8e1 M) under the same assay conditions
[19]. Moreover, the IC50 values of all other closely related
mM AA), revealed IC50 values in the range of 1.6e10 mM. In
a
0.06 ꢁ 0.01
m
m
M, while the IC50 value in the cell-free assay was
M, respectively. Zileuton is known to result in inhi-
0.038 ꢁ 0.04
m
bition of 5-LO product synthesis in PMNL assay with IC50 value of
m
about 0.8e1 mM [19,20].

5024
B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
Table 1
Inhibition of 5-LO product formation by test compounds (9e44) in (A) intact neutrophils and in (B) a cell-free assaya at 10
LO activity versus vehicle (0.3% DMSO), n ¼ 3e5 *p < 0.05, **p < 0.01.
m
M. Data are given as mean ꢁ SEM of remaining % 5-
Cl
R1
N
O
N
N
R
N
O
N
N
N
N
N
O
R1
C
N
R1
R
R
H3C
9-26
27-40
41-44
Compd.
R
R1
A
B
IC50
ND
[m
M]b
N
N
CH2
N
9
dCH3
dCH3
dCH3
86.0 ꢁ 11.5
105.6 ꢁ 16.4
90.0 ꢁ 11.0
93.6 ꢁ 6.9
O
H
N
O
10
11
106.2 ꢁ 6.4
96.3 ꢁ 5.0
ND
ND
N
O
N
N
S
CH2CH3
O
12
dCH3
46.4 ꢁ 9.3
92.7 ꢁ 12.7
8.0
O
13
14
dCH3
dCH3
86.5 ꢁ 4.2
95.0 ꢁ 10.9
115.8 ꢁ 6.5
ND
ND
O
O
O
CF3
70.5 ꢁ 12.3
OCF3
15
16
dF
dF
23.4 ꢁ 4.6
95.0 ꢁ 5.4
55.3 ꢁ 10.5
90.8 ꢁ 9.6
1.6
ND
N
N
N
N
CH2
C
O
O
H
N
17
18
dF
dF
88.3 ꢁ 7.7
91.6 ꢁ 14.9
92.7 ꢁ 4.3
ND
ND
OCH3
67.3 ꢁ 15.8
O
S
19
20
21
22
dF
90.8 ꢁ 6.6
90.1 ꢁ 7.7
111.4 ꢁ 4.4
104.3 ꢁ 6.3
83.4 ꢁ 24.9
86.5 ꢁ 8.7
ND
ND
ND
ND
O
O
O
dF
O(CH2)6CH3
N
dF
71.7 ꢁ 10.9
102.9 ꢁ 17.3
N
O
C
dCF3
N
NH2
OCH2CH3
O
O
C
23
24
dCF3
dCF3
62.0 ꢁ 7.9
72.6 ꢁ 9.2
15.0
ND
N
N
N
C
O
81.8 ꢁ 10.1
125.5 ꢁ 22.7
25
26
27
dCF3
dCF3
dCH3
77.2 ꢁ 6.9
94.1 ꢁ 11.3
14.6 ꢁ 0.5
92.0 ꢁ 6.3
90.8 ꢁ 14.9
107.4 ꢁ 25.0
ND
ND
2.8
O
O
N
Cl
N
N

B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
5025
M]b
Table 1 (continued )
Compd.
R
R1
A
B
IC50 [m
Cl
28
dCF3
17.4 ꢁ 8.1
88.9 ꢁ 28.4
2.9
N
N
29
30
31
32
33
34
35
dCH3
dF
27.7 ꢁ 8.4
13.2 ꢁ 3.4
43.9 ꢁ 9.9
13.5 ꢁ 5.8
3.7 ꢁ 2.2
111.3 ꢁ 23.6
143.3 ꢁ 3.5
137.8 ꢁ 37.5
85.6 ꢁ 27.8
78.5 ꢁ 20.4
109.3 ꢁ 25.2
51.5 ꢁ 6.1
2.9
2.6
8.0
2.7
2.5
2.9
3.5
Cl
Cl
Cl
dCF3
dCH3
dF
dCF3
dCH3
12.7 ꢁ 4.4
16.4 ꢁ 3.1
N
N
N
36
37
dF
10.4 ꢁ 5.2
114.1 ꢁ 31.6
87.3 ꢁ 24.8
2.8
3.0
3.3
dCF3
44.5 ꢁ 16.3
H3CO
H3CO
H3CO
38
39
40
41
dCH3
dF
27.1 ꢁ 4.6
50.1 ꢁ 14.1
36.2 ꢁ 15.4
6.7 ꢁ 3.0
86.1 ꢁ 8.2
69.2 ꢁ 3.5
N
N
N
N
> 10
N
N
dCF3
56.1 ꢁ 21.8
102.5 ꢁ 11.6
8
O
C
2.4
N
OCH2CH3
42
43
20.4 ꢁ 0.9
25.8 ꢁ 0.9
95.3 ꢁ 6.2
69.9 ꢁ 9.9
2.0
4.0
O
O
C
N
OCH2CH3
N
N
44
65.4 ꢁ 6.6
79.6 ꢁ 12.3
>10
O
a
Values for 5-LO product formation in intact human neutrophils challenged with 2.5
M arachidonic acid (B) as percentage of control at 10
M leading to 13.0 ꢁ 3.8% and 31.5 ꢁ 7.9% remaining 5-LO activity in the cell-based
m
M ionophore plus 20
m
M arachidonic acid (A) and under cell-free conditions
(supernatants of lysates of E. coli expressing human recombinant 5-LO incubated with 20
m
mM inhibitor concentrations are
expressed as mean ꢁ SE. BWA4C was used as reference compound at a concentration of 0.3
m
and the cell-free assay, respectively.
b
IC50 values for LT formation of selected compounds were obtained in intact neutrophils challenged with 2.5
m
M ionophore plus 20 M arachidonic acid.
m

5026
B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
Table 2
Comparison of the selectivity of some test compounds for related enzymes in the arachidonic acid cascade. Compounds are tested at 10e30
m
M concentrations and data are
given as mean ꢁ SEM of % remaining activity compared to vehicle (IC50 values,
mM, are given in parentheses).
Compd.
PMNLa
Cell-freeb,c
COX-1d
COX-2d
mPGES-1e
12-LOf 15-LOf
12
46.4 ꢁ 9.3 (8)
92.7 ꢁ 12.7b
51.1 ꢁ 3.9 (>10)
99.9 ꢁ 17.9 (>10)
84.2 ꢁ 9.2 (>10)
78.6 ꢁ 21.5 (>10)
97.0 ꢁ 11.7 (>10)
79.0 ꢁ 29.2 (>10)
86.3 ꢁ 9.1 (>10)
111.1 ꢁ 26.0 (>10)
66.3 ꢁ 9.1 (>10)
97.0 (n ¼ 1) (>10)
91.4 ꢁ 4.6 (>10)
75.6 ꢁ 7.0 (>10)
103.0 ꢁ 6.1 (>10)
84.6 ꢁ 2.5 (>10)
75.0 ꢁ 4.2 (>10)
82.6 ꢁ 3.5 (>10)
108.2 ꢁ 7.2 (>10)
84.0 ꢁ 4.1 (>10)
88.0 ꢁ 4.4 (>10)
77.1 ꢁ 29.2 (>10)
102.9 ꢁ 13.4 (>10)
90.4 ꢁ 25.4 (>10)
118.4 ꢁ 32.4 (>10)
110.6 ꢁ 23.7 (>10)
98.0 ꢁ 6.1 (>10)
67.2 ꢁ 0.2 (>10)
91.9 ꢁ 27.6 (>10)
67.8 ꢁ 15.9 (>10)
n.d.
83.4 ꢁ 15.9c (>10)
55.3 ꢁ 10.5b
15
23
32
35
41
42
43
44
23.4 ꢁ 4.6 (1.6)
62.0 ꢁ 7.9 (>10)
13.5 ꢁ 5.8 (2.7)
16.4 ꢁ 3.1 (3.5)
6.7 ꢁ 3.0 (2.4)
20.4 ꢁ 0.9 (2.0)
25.8 ꢁ 0.9 (4.0)
65.4 ꢁ 6.6 (>10)
82.9 ꢁ 3.2 (>10)
102.2 ꢁ 24.4 (>10)
21.0 ꢁ 2.9 (2.5)
8.2 ꢁ 1.0 (0.041)
57.1 ꢁ 12.1 (>10)
10.6 ꢁ 1.3 (0.3)
15.9 (n ¼ 1) (0.9)
175 ꢁ 43 (>10)
71 ꢁ 45 (>10)
261 ꢁ 49 (>10)
145.0 ꢁ 22 (>10)
196 ꢁ 30 (>10)
124 ꢁ 22 (>10)
149.0 ꢁ 18 (>10)
92.0 ꢁ 14 (>10)
102.8 ꢁ 11.2c (>10)
72.6 ꢁ 9.2b
84 ꢁ 13.8c (>10)
85.6 ꢁ 27.8b
62.9 ꢁ 17.9c (>10)
51.5 ꢁ 6.1b
115.1 ꢁ 10.5c (>10)
102.5 ꢁ 11.6b
79.5 ꢁ 13.1c (>10)
95.3 ꢁ 6.2b
92.6 ꢁ 18.0c (>10)
69.9 ꢁ 9.9b
55.9 (n ¼ 1)c (>10)
79.6 ꢁ 12.3b
16.9 at 1 mM (0.014)
86.1 ꢁ 12.9c (>10)
a
Values for 5-LO product formation at 10
m
M in intact human neutrophils.
under cell-free conditions with supernatants of lysates of E. coli expressing human recombinant 5-LO.
under cell free conditions with purified 5-LO using 20 M AA as substrate.
COX residual activity at 10 M [% 12-HHT of control].
mPGES-1 residual activity at 10 M [% PGE2 of control].
15- and 12-LO residual activity at 30
M, 15-H(P)ETE was analyzed as a product of 15-LO and 12-H(P)ETE as a product of 12-LO. n.d. ¼ not done.
b
c
d
e
f
m
m
m
m
derivatives were also found in a narrow and comparable range (IC50
of between 1.6 and 3.5 M except for 12, 23, 31, 39, 40, 43 and 44).
inflammatory reactions [1]. The clinical efficacy of the inhibition of
LT biosynthesis has been proven by the 5-LO iron-ligand inhibitor
zileuton, which is currently the only approved antiasthmatic agent
that inhibits LT biosynthesis by direct interference with 5-LO.
However, zileuton carries certain disadvantages including druge-
drug interactions, frequent dosing and hepatotoxic side-effects
[24]. Moreover, the utilization of anti-LT compounds in treating
the inflammatory component of cardiovascular disease comprising
atherosclerosis, myocardial infarction and stroke has only been
seriously conceived in the past few years [25]. In addition, LO and
COX metabolic pathways are becoming visible as key regulators of
cell proliferation and neo-angiogenesis and inhibitors of both
pathways are being investigated as potential anticancer drugs
[26,27].
m
Since amidation of the carboxylic acid side chain with ethyl
piperidinecarboxylate (23) and esterification with i-propylphenol
(12) in chloropyridazine series might contribute to the efficiency
for inhibition of 5-LO product synthesis in intact neutrophils, we
next evaluated the respective amide and ester analogs of phenyl
and quinoline substituted core structures (6a and 7a) in place of
chloropyridazine. As shown in Table 1, phenyl substituted
compounds (41e42) also significantly suppressed 5-LO product
synthesis at 10 mM, whereas quinoline substitution (43e44) was
not very effective. Apparently, combination of N1-phenyl and C5-p-
tolyl substitution at a central pyrazole and amidification with ethyl
piperidinecarboxylate or esterification with i-propylphenol even
increased the potency leading to 41 (IC50 ¼ 2.4
(IC50 ¼ 2.0 M) with respect to corresponding chloropyridazine
derivatives (23, IC50 ¼ 15 M and 12, IC50 ¼ 8 M).
m
M) and 42
Our results clearly show that derivatization of 1,5-diarylpyrazol-
3-propanoic acid scaffold into lipophilic amides and esters, exem-
plified in this work, act as suppressors of LT biosynthesis in intact
cells without remarkable direct inhibition of the 5-LO enzyme in
cell-free assays, exemplified by 15, 27e30, 32e37, 41e42 with IC50
m
m
m
Many researchers over the last decades have reported on
compounds with dual action that inhibit both 5-LO and other
enzymes involved in AA cascade [21e23]. Therefore, nine selected
compounds were also tested for enzyme selectivity for related
enzymes in the AA cascade such as COXs, mPGES-1, 12-LO and 15-
LO (Table 2). As a result, none of the compounds were found to
values in the range of 1.6e3.5 mM. More detailed analysis revealed
no or minor inhibition of the related 12/15-LOs, COX enzymes or
mPGES-1 by the chloropyridazine derivatives. Substitution of
chloropyridazine with more lipophilic phenyl or quinoline rings in
analogous compounds resulted in the potent inhibition of COX-1
activity in platelets besides inhibition of LT biosynthesis thereby
acting as dual inhibitors. Taken together, we present a novel
pharmacophore for interfering with cellular LT formation, and the
lead compounds may possess potential for developing novel
molecules for treatment of diseases requiring anti-LT therapy.
inhibit 12- or 15-LO up to 30
12-HETE or 15-HETE in the cell-based assay. In addition, none of
them demonstrated inhibition of mPGES-1 activity at 10
measured in microsomal preparations of IL-1 -stimulated A549
cells. The activity of COX-2 in IL-1 -stimulated A549 cells was not
mM as measured by the formation of
mM
b
b
significantly influenced at this concentration as well. However,
incorporation of phenyl (32 and 42) or quinoline (35, 43 and 44) in
place of chloropyridazine ring caused a potent inhibition of platelet
4. Experimental section
COX-1 activity with IC50 values of 2.5, 0.3, 0.041, 0.9 and 0.014
mM,
respectively, in which the two of them were more potent than the
4.1. Chemistry
used control inhibitor indomethacin (IC50 ¼ 0.07
mM) [12].
1H and 13C NMR spectra were recorded in CDCl3or DMSO-d6 on
a Varian Mercury 400 MHz High Performance Digital FT-NMR
spectrometer using tetramethylsilane as the internal standard at
the NMR facility of Faculty of Pharmacy, Ankara University. All
3. Conclusions
LTs are important mediators derived from AA metabolism by the
5-LO pathway and have been shown to mediate various
chemical shifts were recorded as
d (ppm). High resolution mass

B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
5027
spectra data (HRMS) were collected in-house using a Waters LCT
4.2.4. 3-[5-(4-Methylphenyl)-1-phenyl-1H-pyrazol-3-yl]propanoic
acid (6a)
Premier XE Mass Spectrometer (high sensitivity orthogonal accel-
eration time-of-flight instrument) operating in either ESI (þ) or ESI
(ꢀ) methods, also coupled to an AQUITY Ultra Performance Liquid
Chromatography system (Waters Corporation, Milford, MA, USA).
Melting points were determined with an SMP-II Digital Melting
Point Apparatus and are uncorrected (Schorpp Geaetetechnik,
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 CombiflashÒRf
automated flash chromatography system with RediSep columns
(Teledyne-Isco, Lincoln, NE, USA) using hexane-ethyl acetate or
dichloromethane-methanol solvent gradients. The purity of the
final compounds was determined to be >97% by UPLC with UV
detector. Compounds 1aec, 4, 4aec and 8aec were synthesized as
described previously [12]. 2-Hydrazinoquinoline (7) was synthe-
sized by the reaction of 2-chloroquinoline with hydrazine
hydrate [28].
Prepared from 1a and 6, purified by recrystallization from
hexane:ethyl acetate; yield 71%; Mp 145.5e146.4 ꢃC; IR (ATR):
1704 cmꢀ1 1H NMR (DMSO-d6):
; d 2.28 (s, 3H), 2.65 (t, 2H,
J ¼ 7.6 Hz), 2.87 (t, 2H, J ¼ 7.6 Hz), 6.45 (s, 1H), 7.08 (d, 2H,
J ¼ 8.4 Hz), 7.14 (d, 2H, J ¼ 8.0 Hz), 7.22 (m, 2H), 7.30e7.40 (m, 3H);
HRMS (m/z): [M þ H]þcalcd for C19H19N2O2, 307.1447; found,
307.1433.
4.2.5. 3-[5-(4-Fluorophenyl)-1-phenyl-1H-pyrazol-3-yl]propanoic
acid (6b)
Prepared from 1b and 6, purified by recrystallization from
hexane:ethyl acetate; yield 51%; Mp 142.3e142.8 ꢃC; IR (ATR):
1717 cmꢀ1; 1H NMR (DMSO-d6):
d
2.63 (t, 2H, J ¼ 7.6 Hz), 2.88 (t, 2H,
J ¼ 7.6 Hz), 6.50 (s, 1H), 7.17e7.26 (m, 6H), 7.33e7.41 (m, 3H); HRMS
(m/z): [M þ H]þcalcd for C18H16FN2O2, 311.1196; found, 311.1183.
4.2.6. 3-{1-Phenyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-3-yl}
propanoic acid (6c)
Prepared from 1c and 6, purified by flash chromatography using
4.2. General procedure for the synthesis of 5e7aec
CH2Cl2:methanol (95:5); yield 48%; Mp 105.5e106.3 ꢃC; IR (ATR):
1718 cmꢀ1; 1H NMR (DMSO-d6):
d
2.67 (t, 2H, J ¼ 7.6 Hz), 2.90 (t, 2H,
To
a
solution of 6-aryl-4,6-diketohexanoic acid (1aec)
J ¼ 7.6 Hz), 6.65 (s, 1H), 7.25 (m, 2H), 7.37e7.44 (m, 5H), 7.72 (d, 2H,
J ¼ 8.8 Hz), 12.1 (bs, 1H); HRMS (m/z): [M þ H]þcalcd for
C19H16F3N2O2, 361.1164; found, 361.1165.
(2.1 mmol) in methanol (15 ml) containing Et3N (4.2 mmol), 4-
chlorophenylhydrazine hydrochloride (5), phenylhydrazine
hydrochloride (6) or 2-hydrazinoquinoline (7) (2.1 mmol) was
added and heated at reflux for 4 h (in the case of 7, the reaction was
carried out in the absence of Et3N). The resulting solution was
evaporated in vacuo, taken up in ethyl acetate and washed with 1N
HCl (30 ml) and water (30 ml). The organic phase was extracted
with 5% NaHCO3 (3 ꢂ 30 ml) and aqueous phase containing sodium
salt of the product was acidified with 6N HCl. The precipitate was
collected by filtration and washed with water. The crude product
was purified by flash chromatography or by recrystallization from
the appropriate solvent.
4.2.7. 3-[5-(4-Methylphenyl)-1-quinolin-2-yl-1H-pyrazol-3-yl]
propanoic acid (7a)
Prepared from 1a and 7, purified by recrystallization from hex-
ane:ethyl acetate, yield 43.5%; Mp 128e128.5 ꢃC; IR (ATR):
1703 cmꢀ1; 1H NMR (CDCl3):
d
2.34 (s, 3H,), 2.87 (t, 2H, J ¼ 7.6 Hz),
3.12 (t, 2H, J ¼ 7.6 Hz), 6.38 (s, 1H), 7.08 (d, 2H, J ¼ 8 Hz), 7.19 (d, 2H,
J ¼ 8 Hz), 7.49 (m, 2H), 7.65 (t, 1H J ¼ 7.2), 7.77 (d, 1H, J ¼ 8 Hz) 7.85
(d, 1H, J ¼ 8.4 Hz), 8.11 (d, 1H, J ¼ 8.4 Hz), 10.92 (bs, 1H); 13C NMR
(CDCl3):
d 21.57, 23.65, 33.82, 108.60, 117.52, 126.87, 127.20, 127.65,
128.39, 129.06, 129.12, 129.40, 130.30, 138.44, 138.54, 145.30,
146.86, 151.19, 153.35, 178.49. HRMS (m/z): [M þ H]þcalcd for
C22H20N3O2, 358.1556; found, 358.1539.
4.2.1. 3-[1-(4-Chlorophenyl)-5-(4-methylphenyl)-1H-pyrazol-3-yl]
propanoic acid (5a)
Prepared from 1a and 5, purified by flash chromatography using
CH2Cl2:methanol (95:5); yield 44%; Mp 138.5e139.5 ꢃC; IR (ATR):
4.2.8. 3-[5-(4-Fluorophenyl)-1-quinolin-2-yl-1H-pyrazol-3-yl]
propanoic acid (7b)
1718 cmꢀ1 1H NMR (DMSO-d6):
; d 2.30 (s, 3H), 2.64 (t, 2H,
J ¼ 7.6 Hz), 2.86 (t, 2H, J ¼ 7.6 Hz), 6.46 (s, 1H), 7.10 (d, 2H,
J ¼ 8.0 Hz), 7.18 (d, 2H, J ¼ 8.0 Hz), 7.23 (m, 2H), 7.45 (m, 2H), 12.1
(bs, 1H); HRMS (m/z): [M þ H]þcalcd for C19H18ClN2O2, 341.1057;
found, 341.1073.
Prepared from 1b and 7, purified by recrystallization from
hexane:ethyl acetate, yield 26%; Mp 159e160.3 ꢃC; IR (ATR):
1696 cmꢀ1
;
1H NMR (CDCl3):
d
2.71 (t, 2H, J ¼ 7.4 Hz), 2.93 (t, 2H,
J ¼ 7.4 Hz), 6.58 (s, 1H), 7.19 (m, 2H), 7.37 (m, 2H), 7.44 (d, 1H,
J ¼ 8.0 Hz), 7.57 (m,1H), 7.69 (m, 1H), 7.92 (d,1H, J ¼ 8.8 Hz), 8.00 (d,
1H, J ¼ 7.2), 8.51 (d, 1H, J ¼ 8.8 Hz); HRMS (m/z): [M þ H]þcalcd for
C21H17FN3O2, 362.1305; found, 362.1295.
4.2.2. 3-[1-(4-Chlorophenyl)-5-(4-fluorophenyl)-1H-pyrazol-3-yl]
propanoic acid (5b)
Prepared from 1b and 5, purified by recrystallization from
hexane:ethyl acetate; yield 53%; Mp 126e127 ꢃC; IR (ATR):
4.2.9. 3-{1-Quinolin-2-yl-5-[4-(trifluoromethyl)phenyl]-1H-
pyrazol-3-yl}propanoic acid (7c)
1719 cmꢀ1; 1H NMR (DMSO-d6):
d
2.61 (t, 2H, J ¼ 7.4 Hz), 2.83 (t, 2H,
J ¼ 7.4 Hz), 6.48 (s,1H), 7.17e7.24 (m, 6H), 7.42 (m, 2H),12.1 (bs,1H);
HRMS (m/z): [MeH]ꢀcalcd for C18H13ClFN2O2, 343.0650; found,
343.0663.
Prepared from 1c and 7, purified by flash chromatography using
CH2Cl2:methanol (98:2); yield 48.5%; Mp 166.2e167.2 ꢃC; IR (ATR):
1689 cmꢀ1; 1H NMR (DMSO-d6):
J ¼ 7.4 Hz), 6.71 (s, 1H), 7.34 (d, 1H, J ¼ 8.4 Hz), 7.55e7.72 (m, 6H),
8.00 (m, 2H), 8.54 (d,1H, J ¼ 8.8 Hz); HRMS (m/z): [M þ H]þcalcd for
C22H17F3N3O2, 412.1273; found, 412.1266.
d
2.73 (t, 2H, J ¼ 7.4 Hz), 2.96 (t, 2H,
4.2.3. 3-{1-(4-Chlorophenyl)-5-[4-(trifluoromethyl)phenyl]-1H-
pyrazol-3-yl}propanoic acid (5c)
Prepared from 1c and 5, purified by recrystallization from hex-
ane:ethyl acetate; yield 30%; Mp 168e169.3 ꢃC; IR (ATR):
4.3. General procedure for the synthesis of 9e26
1714 cmꢀ1; 1H NMR (DMSO-d6):
d
2.66 (t, 2H, J ¼ 7.6 Hz), 2.89 (t, 2H,
J ¼ 7.6 Hz), 6.66 (s, 1H), 7.27 (d, 2H, J ¼ 8.4 Hz), 7.44 (d, 2H,
J ¼ 8.4 Hz), 7.48 (d, 2H, J ¼ 8.4 Hz), 7.75 (d, 2H, J ¼ 8.4 Hz), 12.1 (bs,
1H); HRMS (m/z): [M þ H]þcalcd for C19H15ClF3N2O2, 395.0774;
found, 395.0768.
Synthesis of amide derivatives (Procedure 1) Compounds 4aec
(1.27 mmol), Et3N (1.4 mmol) and ethyl chloroformate (1.27 mmol)
in CH2Cl2 (10 ml) was stirred at ꢀ5 ꢃC for 1 h. The solution of the
appropriate amine derivative (1.4 mmol) and triethylamine

5028
B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
(1.4 mmol) in CH2Cl2 (5 ml) was slowly added to the reaction
mixture, and stirred at room temperature overnight. It was diluted
with CH2Cl2 and washed with 1N HCl (3 ꢂ 25 ml), 5% NaHCO3
solution (3 ꢂ 25 ml) and brine. After drying over Na2SO4, solvent
was evaporated off under reduced pressure to dryness. The crude
product was purified by flash chromatography or by recrystalliza-
tion from the appropriate solvent.
Synthesis of ester derivatives (Procedure 2) To a mixture of the
appropriate acid derivative (4aec) (1 mmol), a phenol derivative
(1.1 mmol) and DMAP (0.2 mmol) in CH2Cl2 (10 ml), EDC (1.1 mmol)
was added, and stirred at room temperature overnight (DCC was
used instead of EDC for compounds 18 and 19). The reaction
mixture was diluted with CH2Cl2 and washed with 0.5 N HCl
(3 ꢂ 20 ml), 1% NaOH solution (3 ꢂ 20 ml) and brine. After drying
over Na2SO4, solvent was evaporated off under reduced pressure
and the crude product was purified by flash chromatography.
4.3.5. 4-[4-(Trifluoromethyl)phenoxy]phenyl 3-[1-(6-
chloropyridazin-3-yl)-5-(4-methylphenyl)-1H-pyrazol-3-yl]
propanoate (13)
Prepared from 4a and 4-[4-(trifluoromethyl)phenoxy]phenol
using the procedure 2. Flash chromatography CH2Cl2:MeOH (97:3);
yield 49%; Mp 142.3e142.3 ꢃC; IR (ATR): 1758 cmꢀ1 1H NMR
;
(CDCl3):
d
2.36 (s, 3H), 3.06 (t, 2H, J ¼ 7.6 Hz), 3.19 (t, 2H, J ¼ 7.2 Hz),
6.42 (s, 1H), 7.03e7.21 (m, 10H), 7.53e7.58 (m, 3H), 7.88 (d, 1H,
J ¼ 9.2 Hz); HRMS (m/z): [M þ H]þcalcd for C30H23ClF3N4O3,
579.1411; found, 579.1439.
4.3.6. 4-(Trifluoromethoxy)phenyl 3-[1-(6-chloropyridazin-3-yl)-5-
(4-methylphenyl)-1H-pyrazol-3-yl]propanoate (14)
Prepared from 4a and 4-(trifluoromethoxy)phenol using the
procedure 2. Flash chromatography CH2Cl2:MeOH (96:4); yield
45%; Mp 131.3e132.3 ꢃC; IR (ATR): 1758 cmꢀ1 1H NMR (CDCl3):
;
d
2.36 (s, 3H), 3.06 (t, 2H, J ¼ 7.0 Hz), 3.18 (t, 2H, J ¼ 7.4 Hz), 6.41 (s,
4.3.1. 3-Chloro-6-(5-(4-methylphenyl)-3-{3-oxo-3-[4-
(tetrahydrofuran-2-ylmethyl)piperazin-1-yl]propyl}-1H-pyrazol-1-
yl)pyridazine (9)
1H), 7.10e7.20 (m, 8H), 7.54 (d, 1H, J ¼ 9.2 Hz), 7.86 (d, 1H, J ¼ 8.8);
HRMS (m/z): [M þ H]þcalcd for C24H19ClF3N4O3,503.1098; found,
503.1102.
Prepared from 4a and 1-(tetrahydrofuran-2-yl)methyl)pipera-
zine using the procedure 1. Flash chromatography CH2Cl2:MeOH
4.3.7. 3-[3-{3-[4-(4-tert-Butylbenzyl)piperazin-1-yl]-3-
oxopropyl}-5-(4-fluorophenyl)-1H-pyrazol-1-yl]-6-
chloropyridazine (15)
(95:5); yield 68%; Mp 128e129 ꢃC; IR (ATR): 1649 cmꢀ1 1H NMR
;
(CDCl3):
d 1.47 (m, 1H), 1.87 (m, 2H), 1.99 (m, 1H), 2.35 (s, 3H),
2.38e2.55 (m, 6H), 2.78 (t, 2H, J ¼ 7.6 Hz), 3.08 (t, 2H, J ¼ 7.6 Hz),
3.53 (t, 2H), 3.64e3.89 (m, 4H), 4.02 (m, 1H), 6.38 (s, 1H), 7.17 (m,
4H), 7.53 (d, 1H, J ¼ 9.2 Hz), 7.83 (d, 1H, J ¼ 9.6 Hz); HRMS (m/z):
[M þ H]þcalcd for C26H32ClN6O2, 495.2275; found, 495.2275.
Prepared from 4b and 1-(4-t-butylbenzyl)piperazine using the
procedure 1. Flash chromatography CH2Cl2:MeOH (95:5); yield
56%; Mp 158.5e159.5 ꢃC; IR (ATR): 1636 cmꢀ1 1H NMR (CDCl3):
;
d
1.32 (s, 9H), 2.42 (m, 4H), 2.77 (t, 2H, J ¼ 7.6 Hz), 3.08 (t, 2H,
J ¼ 7.6 Hz), 3.47 (s,2H), 3.50 (t, 2H), 3.65 (t, 2H), 6.38 (s,1H), 7.04 (dd,
2H, J ¼ 8.4 and 8.8 Hz), 7.22 (d, 2H, J ¼ 8.4 Hz), 7.29 (dd, 2H,
J ¼ 8.8 Hz and 5.2 Hz), 7.34 (d, 2H, J ¼ 8.4 Hz), 7.56 (d,1H, J ¼ 8.8 Hz),
4.3.2. 3-[1-(6-Chloropyridazin-3-yl)-5-(4-methylphenyl)-1H-
pyrazol-3-yl]-N-(6-morpholin-4-ylpyridin-3-yl)propanamide (10)
Prepared from 4a and 6-(morpholin-4-yl)pyridin-3-amine using
the procedure 1. Flash chromatography CH2Cl2:MeOH (95:5), after
recrystallized from ethanol; yield 26.7%; Mp 242e243 ꢃC; IR (ATR):
7.95 (d,1H, J ¼ 9.2 Hz); 13C NMR (CDCl3):
d 24.02, 31.58, 32.27, 34.70,
41.95, 45.70, 52.99, 53.35, 62.76, 110.89, 115.56 (2JCF ¼ 21 Hz),
124.25, 125.43, 127.10 (4JCF
¼
3 Hz), 129.04, 130.19, 130.96
1686 cmꢀ1; 1H NMR (CDCl3):
d
2.35 (s, 3H), 2.83 (t, 2H, J ¼ 6.8 Hz),
(3JCF ¼ 8.4 Hz), 134.66, 144.87, 150.44, 154.76, 155.31, 155.63, 163.04
3.15 (t, 2H, J ¼ 6.8 Hz), 3.43 (t, 4H, J ¼ 4.6 Hz), 3.81 (t, 4H, J ¼ 4.6 Hz),
6.38 (s, 1H), 6.61 (d, 1H, J ¼ 8.8 Hz), 7.15 (s, 4H), 7.51 (d, 1H,
J ¼ 8.8 Hz), 7.72 (m, 2H), 7.90 (d, 1H, J ¼ 8.8 Hz), 8.13 (s, 1H); HRMS
(m/z): [M þ H]þcalcd for C26H27ClN7O2, 504.1915; found, 504.1892.
(JCF
¼
247 Hz), 170.39. HRMS (m/z): [M
þ
H]þcalcd for
C31H35ClFN6O, 561.2545; found, 561.2518.
4.3.8. 3-Chloro-6-(5-(4-fluorophenyl)-3-{3-oxo-3-[4-
(tetrahydrofuran-2-ylcarbonyl)piperazin-1-yl]propyl}-1H-pyrazol-
1-yl)pyridazine (16)
4.3.3. 3-Chloro-6-[3-{3-[4-(ethylsulfonyl)piperazin-1-yl]-3-
oxopropyl}-5-(4-methylphenyl)-1H-pyrazol-1-yl]pyridazine (11)
Prepared from 4a and 1-(ethylsulfonyl)piperazine using the
procedure 1. Flash chromatography CH2Cl2:MeOH (97.5:2.5); yield
Prepared from 4b and 1-((tetrahydrofuran-2-yl)carbonyl)
piperazine using the procedure 1. Flash chromatography
CH2Cl2:MeOH (95:5); yield 60.7%; Mp 162e164 ꢃC; IR (ATR):
40.7%; Mp 161e162 ꢃC; IR (ATR): 1643 cmꢀ1
;
1H NMR (CDCl3):
1636 cmꢀ1 1H NMR (CDCl3):
; d 1.90e2.34 (m, 4H), 2.82 (t, 2H,
d
1.36 (t, 3H, J ¼ 7.4 Hz), 2.36 (s, 3H), 2.81 (t, 2H, J ¼ 7.6 Hz), 2.94 (q,
J ¼ 7.6 Hz), 3.10 (t, 2H, J ¼ 7.6 Hz), 3.42e3.93 (m, 10H), 4.59 (m, 1H),
6.39 (s, 1H), 7.04 (m, 2H), 7.29 (m, 2H), 7.57 (d, 1H, J ¼ 8.8 Hz), 7.92
(m, 1H); HRMS (m/z): [M þ H]þcalcd for C25H27ClFN6O3, 513.1817;
found, 513.1807.
2H, J ¼ 7.4 Hz), 3.10 (t, 2H, J ¼ 7.4 Hz), 3.27 (t, 4H), 3.60 (t, 2H), 3.74
(t, 2H), 6.39 (s, 1H), 7.15 (s, 4H), 7.53 (d, 1H, J ¼ 8.8 Hz), 7.75 (d, 1H,
J ¼ 9.2 Hz); 13C NMR (CDCl3):
d 7.97, 21.53, 23.99, 32.22, 41.92, 44.45,
45.63, 45.92, 46.09, 110.16, 124.73, 127.60, 128.83, 129.40, 130.00,
139.01, 146.01, 154.94, 155.18, 155.37, 170.75. HRMS (m/z):
[M þ H]þcalcd for C23H28ClN6O3S, 503.1632; found, 503.1631.
4.3.9. 3-[1-(6-Chloropyridazin-3-yl)-5-(4-fluorophenyl)-1H-
pyrazol-3-yl]-N-(4-methoxyphenyl)propanamide (17)
Prepared from 4b and 4-methoxyaniline using the procedure 1.
4.3.4. 4-Isopropylphenyl 3-[1-(6-chloropyridazin-3-yl)-5-(4-
methylphenyl)-1H-pyrazol-3-yl]propanoate (12)
Recrystallized from methanol; yield 47%; Mp 149e150 ꢃC; IR (ATR):
1650 cmꢀ1
;
1H NMR (CDCl3):
d
2.80 (t, 2H, J ¼ 7.0 Hz), 3.15 (t, 2H,
Prepared from 4a and 4-isopropylphenol using the procedure 2.
Flash chromatography CH2Cl2:MeOH (99:1); yield 70.6%; Mp
J ¼ 7.4 Hz), 3.78 (s, 3H), 6.38 (s, 1H), 6.84 (m, 2H), 7.03 (m, 2H), 7.27
(m, 2H), 7.40 (m, 2H), 7.53 (d, 1H, J ¼ 8.8 Hz), 7.59 (s, 1H), 7.86 (d, 1H,
J ¼ 9.2 Hz); HRMS (m/z): [M þ Na]þcalcd for C23H19ClFN5O2Na,
474.1109; found, 474.1094.
160e160.7 ꢃC; IR (ATR): 1750 cmꢀ1; 1H NMR (CDCl3):
d 1.23 (d, 6H,
J ¼ 6.8 Hz), 2.36 (s, 3H), 2.90 (m, 1H), 3.03 (t, 2H, J ¼ 7.2 Hz), 3.17 (t,
2H, J ¼ 7.4 Hz), 6.41 (s, 1H), 6.99 (m, 2H), 7.14e7.22 (m, 6H), 7.54 (d,
1H, J ¼ 9.6 Hz), 7.90 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3):
d
21.56,
4.3.10. 4-tert-Butylphenyl 3-[1-(6-chloropyridazin-3-yl)-5-(4-
fluorophenyl)-1H-pyrazol-3-yl]propanoate (18)
23.83, 24.26, 33.56, 33.83, 110.12, 121.37, 124.49, 127.56, 127.87,
128.93, 129.28, 130.10, 138.87, 146.16, 146.60, 148.81, 154.49, 154.78,
155.45, 171.77. HRMS (m/z): [M þ Na]þcalcd for C26H25ClN4O2Na,
483.1564; found, 483.1552.
Prepared from 4b and 4-t-butylphenol using the procedure 2.
Flash chromatography CH2Cl2:MeOH (95:5); yield 52.6%; Mp
168e169.5 ꢃC; IR (ATR): 1752 cmꢀ1; 1H NMR (CDCl3):
d 1.31 (s, 9H),

B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
5029
3.04 (t, 2H, J ¼ 7 Hz), 3.17 (t, 2H, J ¼ 7.4 Hz), 6.41 (s, 1H), 6.99 (d, 2H,
J ¼ 8.4 Hz), 7.05 (m, 2H), 7.32 (m, 2H), 7.37 (d, 2H, J ¼ 8.4 Hz), 7.57 (d,
1H, J ¼ 9.2 Hz), 8.01 (d, 1H, J ¼ 9.6 Hz); HRMS (m/z): [M þ H]þcalcd
for C26H25ClFN4O2, 479.1650; found, 479.1661.
44%; Mp 151e152 ꢃC; IR (ATR): 1619 cmꢀ1; 1H NMR (CDCl3):
d
2.85
(t, 2H, J ¼ 7.4 Hz), 3.13 (t, 2H, J ¼ 7.4 Hz), 3.60e3.82 (m, 8H), 6.46 (s,
1H), 6.51 (dd, 1H, J ¼ 3.6 Hz and 2 Hz), 7.07 (d, 1H, J ¼ 3.2 Hz), 7.44
(d, 2H, J ¼ 8.0 Hz), 7.50 (s, 1H), 7.60 (m, 3H), 8.02 (d, 1H, J ¼ 8.8 Hz);
HRMS (m/z): [M þ H]þcalcd for C26H23ClF3N6O3, 559.1472; found,
559.1498.
4.3.11. 4-(3-Thienyl)phenyl 3-[1-(6-chloropyridazin-3-yl)-5-(4-
fluorophenyl)-1H-pyrazol-3-yl]propanoate (19)
Prepared from 4b and 4-(thiophen-3-yl)phenol using the
procedure 2. Flash chromatography CH2Cl2:MeOH (95:5); yield
4.3.17. 4-Cyclopentylphenyl 3-{1-(6-chloropyridazin-3-yl)-5-[4-
(trifluoromethyl)phenyl]-1H-pyrazol-3-yl}propanoate (25)
41.5%; Mp 187e188 ꢃC; IR (ATR): 1743 cmꢀ1; 1H NMR (CDCl3):
d
3.07
Prepared from 4c and 4-(cyclopentyl)phenol using the proce-
dure 2. Flash chromatography CH2Cl2:MeOH (80:20); yield 43.5%;
(t, 2H, J ¼ 7.2 Hz), 3.19 (t, 2H, J ¼ 7.2 Hz), 6.42 (s, 1H), 7.05 (dd, 2H,
J ¼ 8.8 Hz and 8.4 Hz), 7.11 (d, 2H, J ¼ 8.4 Hz), 7.30e7.41 (m, 5H),
7.56e7.59 (m, 3H), 8.00 (d, 1H, J ¼ 9.2 Hz); HRMS (m/z):
[M þ Na]þcalcd for C26H18ClFN4O2SNa, 527.0721; found, 527.0720.
Mp 157e158 ꢃC; IR (ATR): 1759 cmꢀ1; 1H NMR (CDCl3):
d 1.52e2.06
(m, 9H), 3.04 (t, 2H, J ¼ 7.2 Hz), 3.18 (t, 2H, J ¼ 7.2 Hz), 6.47 (s, 1H),
6.97 (d, 2H, J ¼ 8.4 Hz), 7.22 (d, 2H, J ¼ 8.4 Hz), 7.45 (d, 2H,
J ¼ 8.4 Hz), 7.61 (m, 3H), 8.08 (d, 1H, J ¼ 9.2 Hz); HRMS (m/z):
[M þ Na]þcalcd for C28H24ClF3N4O2Na, 563.1438; found, 563.1432.
4.3.12. 4-(Heptyloxy)phenyl 3-[1-(6-chloropyridazin-3-yl)-5-(4-
fluorophenyl)-1H-pyrazol-3-yl]propanoate (20)
Prepared from 4b and 4-heptyloxyphenol using the procedure 2.
Recrystallized from ethyl acꢀe1tate-hexane; yield 64.4%; Mp
4.3.18. 4-(1H-Pyrrol-1-yl)phenyl 3-{1-(6-chloropyridazin-3-yl)-5-
[4-(trifluoromethyl)phenyl]-1H-pyrazol-3-yl}propanoate (26)
Prepared from 4c and 4-(1H-pyrrol-1-yl)phenol using the
procedure 2. Flash chromatography CH2Cl2:MeOH (96:4); yield 39%;
136e137 ꢃC; IR (ATR): 1746 cm
; d 0.89 (t, 3H,
1H NMR (CDCl3):
J ¼ 6.6 Hz), 1.30e1.46 (m, 8H), 1.76 (m, 2H), 3.02 (t, 2H, J ¼ 7.2 Hz),
3.17 (t, 2H, J ¼ 7.2 Hz), 3.92 (t, 2H, J ¼ 6.6 Hz), 6.40 (s, 1H), 6.86 (d,
2H, J ¼ 8.8 Hz), 6.97 (d, 2H, J ¼ 8.8 Hz), 7.05 (dd, 2H, J ¼ 8.8 Hz and
8.4 Hz), 7.32 (dd, 2H, J ¼ 8.4 Hz and 5.2 Hz), 7.57 (d, 1H, J ¼ 8.8 Hz),
8.00 (d, 1H, J ¼ 9.6 Hz); HRMS (m/z): [M þ H]þcalcd for
C29H31ClFN4O3, 537.2069; found, 537.2048.
Mp 131e132 ꢃC; IR (ATR): 1749 cmꢀ1; 1H NMR (CDCl3):
d 3.08 (t, 2H,
J ¼ 7 Hz), 3.20 (t, 2H, J ¼ 7 Hz), 6.35 (dd, 2H, J ¼ 2.4 and 2.0 Hz), 6.48 (s,
1H), 7.04 (dd, 2H, J ¼ 2.4 and 2.0 Hz), 7.14 (d, 2H, J ¼ 8.8 Hz), 7.38 (d,
2H, J ¼ 8.8 Hz), 7.46 (d, 2H, J ¼ 8.4 Hz), 7.61 (d,1H, J ¼ 9.2 Hz), 7.62 (d,
2H, J ¼ 8.4 Hz), 8.07 (d, 1H, J ¼ 9.2 Hz); HRMS (m/z): [M þ H]þcalcd
for C27H20ClF3N5O2, 538.1258; found, 538.1253.
4.3.13. 4-(1H-Imidazol-1-yl)phenyl 3-[1-(6-chloropyridazin-3-yl)-
5-(4-fluorophenyl)-1H-pyrazol-3-yl]propanoate (21)
4.3.19. 3-[3-{3-[4-(4-tert-Butylbenzyl)piperazin-1-yl]-3-
oxopropyl}-5-(4-methylphenyl)-1H-pyrazol-1-yl]-6-
chloropyridazine (27)
Prepared from 4b and 4-(1H-imidazol-1-yl)phenol using the
procedure 2. Flash chromatography CH2Cl2:MeOH (95:5); yield
60.9%; Mp 112e113 ꢃC; IR (ATR): 1752 cmꢀ1; 1H NMR (CDCl3):
d
3.09
To a mixture of 4a (1 mmol), 1-(4-t-butylbenzyl)piperazine
(1.1 mmol) and DMAP (0.2 mmol) in CH2Cl2 (10 ml), EDC (1.1 mmol)
was added and stirred at room temperature overnight. The reaction
mixture was diluted with CH2Cl2 and washed with 1N HCl
(3 ꢂ 25 ml), 5% NaHCO3 solution (3 ꢂ 25 ml) and water. After drying
over Na2SO4 the solvent was evaporated off under reduced pressure
to dryness. The crude product was purified by flash chromatog-
raphy using CH2Cl2:MeOH (95:5); Yield 67.7%; Mp 148.5e151.5 ꢃC;
(t, 2H, J ¼ 7.2 Hz), 3.20 (t, 2H, J ¼ 7.2 Hz), 6.42 (s, 1H), 7.06 (dd, 2H,
J ¼ 8.8 Hz and 8.4 Hz), 7.20e7.23 (m, 3H), 7.25 (m, 1H), 7.32 (dd, 2H,
J ¼ 8.8 Hz and 5.2 Hz), 7.39 (m, 2H), 7.58 (d, 1H, J ¼ 9.2 Hz), 7.81 (s,
1H), 7.98 (d,1H, J ¼ 8.8 Hz); HRMS (m/z): [M þ H]þcalcd for
C25H19ClFN6O2, 489.1242; found, 489.1223.
4.3.14. 1-(3-{1-(6-Chloropyridazin-3-yl)-5-[4-(trifluoromethyl)
phenyl]-1H-pyrazol-3-yl}propanoyl)piperidine-4-carboxamide (22)
Prepared from 4c and piperidin-4-carboxamide using the
procedure 1. Recrystallized from methanol; Yield 51%; Mp
IR (ATR): 1634 cmꢀ1 1H NMR (CDCl3):
; d 1.32 (s, 9H), 2.35 (s, 3H),
2.40 (m, 4H), 2.77 (t, 2H), 3.08 (t, 2H), 3.47 (s, 2H), 3.49 (t, 2H), 3.65
(t, 2H), 6.38 (s, 1H), 7.14 (d, 2H, J ¼ 8.0 Hz), 7.18 (d, 2H, J ¼ 8.0 Hz),
7.22 (d, 2H, J ¼ 8.4 Hz), 7.34 (d, 2H, J ¼ 8.4 Hz), 7.53 (d, 1H,
J ¼ 8.8 Hz), 7.82 (d, 1H, J ¼ 9.2 Hz); HRMS (m/z): [M þ H]þcalcd for
C32H38ClN6O, 557.2796; found, 557.2799.
The same procedure was applied to convert 4a,c to 27, 28; 5aec
to 29e31; 6aec to 32e34; 7aec to 35e37 and 8aec to 38e40,
respectively.
156e157 ꢃC; IR (ATR): 1649, 1620 cmꢀ1; 1H NMR (CDCl3):
d 1.66 (m,
2H), 1.90 (m, 2H), 2.41 (m, 1H), 2.67e3.16 (m, 6H), 3.98 (d, 1H), 4.65
(m,1H), 5.50 (m, 2H), 6.46 (s, 1H), 7.43 (d, 2H, J ¼ 8.0 Hz), 7.61 (d, 2H,
J ¼ 8.0 Hz), 7.64 (d,1H, J ¼ 8.8 Hz), 8.05 (d,1H, J ¼ 8.8 Hz); HRMS (m/
z): [M þ H]þcalcd for C23H23ClF3N6O2, 507.1523; found, 507.1518.
4.3.15. Ethyl 1-(3-{1-(6-chloropyridazin-3-yl)-5-[4-
(trifluoromethyl)phenyl]-1H-pyrazol-3-yl}propanoyl)piperidine-4-
carboxylate (23)
Prepared from 4c and ethyl piperidin-4-carboxylate using the
procedure 1. Flash chromatography CH2Cl2:MeOH (98:2); yield
54.3%; Mp 121e122 ꢃC; IR (ATR): 1733,1652 cmꢀ1; 1H NMR (CDCl3):
4.3.20. 3-{3-{3-[4-(4-tert-Butylbenzyl)piperazin-1-yl]-3-
oxopropyl}-5-[4-(trifluoromethyl) phenyl]-1H-pyrazol-1-yl}-6-
chloropyridazine (28)
Prepared from 4c and purified by flash chromatography using
CH2Cl2:MeOH (95:5); yield 68%; Mp 155.5e156 ꢃC; IR (ATR):
d
1.26 (t, 3H, J ¼ 7.0 Hz), 1.66 (m, 2H), 1.94 (m, 2H), 2.54 (m, 1H),
1651 cmꢀ1; 1H NMR (CDCl3):
d 1.31 (s, 9H), 2.43 (s, 4H), 2.78 (t, 2H,
2.75e3.17 (m, 6H), 3.90 (m, 1H), 4.15 (q, 2H, J ¼ 7.0 Hz), 4.47 (m, 1H),
6.46 (s, 1H), 7.44 (d, 2H, J ¼ 7.6 Hz), 7.61 (m, 3H), 8.05 (d, 1H,
J ¼ 8.8 Hz); HRMS (m/z): [M þ H]þcalcd for C25H26ClF3N5O3,
536.1676; found, 536.1674.
J ¼ 7.6 Hz), 3.09 (t, 2H, J ¼ 7.6 Hz), 3.48 (m, 4H), 3.66 (m, 2H), 6.45 (s,
1H), 7.23 (d, 2H, J ¼ 8.0 Hz), 7.34 (d, 2H, J ¼ 8.0 Hz), 7.44 (d, 2H,
J ¼ 8.0 Hz), 7.59 (d, 1H, J ¼ 8.8), 7.61 (2H, d, J ¼ 8.0 Hz), 8.03 (d, 1H,
J ¼ 9.6 Hz); HRMS (m/z): [M þ H]þcalcd for C32H35ClF3N6O,
611.2513; found, 611.2510.
4.3.16. 3-Chloro-6-{3-{3-[4-(2-furoyl)piperazin-1-yl]-3-
oxopropyl}-5-[4-(trifluoromethyl) phenyl]-1H-pyrazol-1-yl}
pyridazine (24)
Prepared from 4c and 1-((furan-2-yl)carbonyl)piperazine using
the procedure 1. Flash chromatography CH2Cl2:MeOH (95:5); yield
4.3.21. 1-(4-tert-Butylbenzyl)-4-{3-[1-(4-chlorophenyl)-5-(4-
methylphenyl)-1H-pyrazol-3-yl]propanoyl}piperazine (29)
Prepared from 5a and purified by flash chromatography
CH2Cl2:MeOH (95:5); yield 78%; Mp 246e247 ꢃC; IR (ATR):

5030
B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
1643 cmꢀ1
;
1H NMR (CDCl3):
d
1.32 (s, 9H), 2.35 (s, 3H), 2.40 (m,
1643 cmꢀ1; 1H NMR (CDCl3):
d 1.31 (s, 9H), 2.35 (s, 3H), 2.41 (m, 4H),
4H), 2.77 (m, 2H), 3.06 (m, 2H), 3.45 (s, 2H), 3.56 (m, 2H), 3.64 (m,
2H), 6.32 (s, 1H), 7.07e7.13 (m, 4H), 7.19e7.23 (m, 4H), 7.28 (m, 2H),
7.34 (m, 2H); HRMS (m/z): [M þ H]þcalcd for C34H40ClN4O,
555.2891; found, 555.2871.
2.82 (m, 2H), 3.13 (m, 2H), 3.45 (s, 2H), 3.51 (m, 2H), 3.66 (m, 2H),
6.41 (s, 1H), 7.09 (d, 2H, J ¼ 8.4 Hz), 7.19e7.22 (m, 4H), 7.32 (d, 2H,
J ¼ 8.0 Hz), 7.44 (d, 1H, J ¼ 8.4 Hz), 7.51 (m, 1H), 7.67 (m, 1H), 7.80 (d,
1H, J ¼ 7.2 Hz), 7.88 (d, 1H, J ¼ 8.8 Hz), 8.11 (d, 1H, J ¼ 8.4 Hz); 13C
NMR (CDCl3):
d 21.53, 24.31, 31.61, 32.89, 34.70, 41.91, 45.77, 53.03,
4.3.22. 1-(4-tert-Butylbenzyl)-4-{3-[1-(4-chlorophenyl)-5-(4-
fluorophenyl)-1H-pyrazol-3-yl]propanoyl}piperazine (30)
53.41, 62.76, 108.97, 117.65, 125.42, 126.78, 127.12, 127.59, 128.48,
128.98,129.06,129.08,129.46,130.20,134.74,138.31, 138.34,145.06,
146.95, 150.37, 151.35, 154.19, 170.85. HRMS (m/z): [M þ H]þcalcd
for C37H42N5O, 572.3389; found, 572.3394.
Prepared from 5b and purified by flash chromatography
CH2Cl2:MeOH (95:5); yield 64%; Mp 245e246 ꢃC; IR (ATR):
1643 cmꢀ1; 1H NMR (CDCl3):
d 1.31 (s, 9H), 2.39 (m, 4H), 2.77 (t, 2H,
J ¼ 7.6), 3.06 (t, 2H, J ¼ 7.6), 3.46 (s, 2H), 3.50 (m, 2H), 3.65 (m, 2H),
4.3.28. 2-[3-{3-[4-(4-tert-Butylbenzyl)piperazin-1-yl]-3-
6.34(s,1H), 7.01 (m, 2H), 7.16e7.23 (m, 6H), 7.30 (m, 2H), 7.34 (m, 2H);
oxopropyl}-5-(4-fluorophenyl)-1H-pyrazol-1-yl]quinoline (36)
Prepared from 7b and purified by flash chromatography
CH2Cl2:MeOH (95:5); yield 58.5%; Mp 223e224 ꢃC; IR (ATR):
13C NMR (CDCl3):
d 24.08, 31.60, 32.74, 34.71, 41.93, 45.75, 53.02,
53.40, 62.78, 107.85, 115.93 (2JCF ¼ 22 Hz), 125.43, 126.40, 126.73
(4JCF ¼ 3.8 Hz), 129.03, 129.32, 130.69 (3JCF ¼ 7.6 Hz), 133.12, 134.71,
138.68,142.85,150.42,153.38,162.83 (JCF ¼ 247 Hz),170.74. HRMS (m/
z): [M þ H]þcalcd for C33H37ClFN4O, 559.2640; found, 559.2660.
1642 cmꢀ1 1H NMR (CDCl3):
; d 1.31 (s, 9H), 2.40 (m, 4H), 2.81 (m,
2H), 3.13 (m, 2H), 3.45 (s, 2H), 3.51 (m, 2H), 3.66 (m, 2H), 6.40 (s,
1H), 7.00 (dd, 2H, J ¼ 8.4 Hz and J ¼ 8.8 Hz), 7.21 (d, 2H, J ¼ 8.4 Hz),
7.28e7.34 (m, 4H), 7.51 (m, 1H), 7.63 (m, 2H), 7.73 (d, 1H, J ¼ 8.0 Hz),
7.80 (d, 1H, J ¼ 8.0 Hz), 8.17 (d, 1H, J ¼ 9.2 Hz); HRMS (m/z):
[M þ H]þcalcd for C36H39FN5O, 576.3139; found, 576.3158.
4.3.23. 1-(4-tert-Butylbenzyl)-4-(3-{1-(4-chlorophenyl)-5-[4-
(trifluoromethyl)phenyl]-1H-pyrazol-3-yl}propanoyl)piperazine
(31)
Prepared from 5c and purified by flash chromatography
4.3.29. 2-{3-{3-[4-(4-tert-Butylbenzyl)piperazin-1-yl]-3-
oxopropyl}-5-[4-(trifluoromethyl) phenyl]-1H-pyrazol-1-yl}
quinoline (37)
CH2Cl2:MeOH (90:10); yield 65%; Mp 272e273 ꢃC; IR (ATR):
1643 cmꢀ1; 1H NMR (CDCl3):
d 1.31 (s, 9H), 2.40 (m, 4H), 2.78 (t, 2H,
J ¼ 7.6), 3.08 (t, 2H, J ¼ 7.6), 3.46 (s, 2H), 3.50 (m, 2H), 3.65 (m, 2H),
6.43 (s, 1H), 7.18e7.23 (m, 4H), 7.30e7.35 (m, 6H), 7.57 (d,
2H,J ¼ 8.0); HRMS (m/z): [M þ H]þcalcd for C34H37ClF3N4O,
609.2608; found, 609.2621.
Prepared from 7c and purified by flash chromatography
CH2Cl2:MeOH (95:5); yield 24%; Mp 163e164 ꢃC; IR (ATR):
1643 cmꢀ1; 1H NMR (CDCl3):
d 1.31 (s, 9H), 2.41 (m, 4H), 2.82 (t, 2H,
J ¼ 7.6 Hz), 3.14 (t, 2H, J ¼ 7.6 Hz), 3.46 (s, 2H), 3.51 (m, 2H), 3.67 (m,
2H), 6.47 (s, 1H), 7.21 (d, 2H, J ¼ 8.4 Hz), 7.33 (d, 2H, J ¼ 8.4 Hz), 7.44
(d, 2H, J ¼ 8.4 Hz), 7.51 (m,1H), 7.57 (d, 2H, J ¼ 8.0 Hz), 7.61e7.64 (m,
2H), 7.75 (d, 1H, J ¼ 8.8 Hz), 7.80 (d, 1H, J ¼ 8.0 Hz), 8.20 (d, 1H,
J ¼ 8.8 Hz); HRMS (m/z): [M þ H]þcalcd for C37H39F3N5O, 626.3107;
found, 626.3129.
4.3.24. 1-(4-tert-Butylbenzyl)-4-{3-[5-(4-methylphenyl)-1-phenyl-
1H-pyrazol-3-yl]propanoyl}piperazine (32)
Prepared from 6a and purified by flash chromatography
CH2Cl2:MeOH (80:20); yield 27%; Mp 223e225 ꢃC; IR (ATR):
1642 cmꢀ1; 1H NMR (CDCl3):
d 1.30 (s, 9H), 2.34 (m, 3H), 2.40 (m, 4H),
2.79 (t, 2H, J ¼ 7.6 Hz), 3.06 (t, 2H, J ¼ 7.6 Hz), 3.45 (s, 2H), 3.51 (m, 2H),
4.3.30. 3-[3-{3-[4-(4-tert-Butylbenzyl)piperazin-1-yl]-3-
oxopropyl}-5-(4-methylphenyl)-1H-pyrazol-1-yl]-6-
methoxypyridazine (38)
3.63 (m, 2H), 6.32 (s, 1H), 7.00 (m, 2H), 7.10e7.37 (m, 11H); 13C NMR
(CDCl3):
d 21.46, 24.00, 31.36, 31.52, 35.01, 38.63, 42.63, 51.10, 51.91,
60.91, 107.18, 125.50, 126.56, 127.61, 127.64, 128.67, 129.25, 129.41,
131.39, 138.49, 140.48, 144.19, 152.20, 153.86, 171.23. HRMS (m/z):
[M þ H]þcalcd for C34H41N4O, 521.3280; found, 521.3270.
Prepared from 8a and purified by flash chromatography
CH2Cl2:MeOH (90:10); yield 48%; Mp 121e122 ꢃC; IR (ATR):
1641 cmꢀ1 1H NMR (CDCl3):
; d 1.31 (s, 9H), 2.34 (s, 3H), 2.40 (m,
4H), 2.77 (m, 2H), 3.07 (t, 2H), 3.46 (s, 2H), 3.49 (m, 2H), 3.65 (m,
2H), 4.10 (s, 3H), 6.36 (s, 1H), 7.02 (d, 1H, J ¼ 8.8 Hz) 7.11 (d, 2H,
J ¼ 8.0 Hz), 7.16 (d,2H, J ¼ 8.0 Hz), 7.22 (d, 2H, J ¼ 8.4 Hz), 7.33 (d, 2H,
J ¼ 8.0 Hz), 7.59 (d,1H, J ¼ 9.6 Hz); HRMS (m/z): [M þ H]þcalcd for
C33H41N6O2,553.3291; found, 553.3271.
4.3.25. 1-(4-tert-Butylbenzyl)-4-{3-[5-(4-fluorophenyl)-1-phenyl-
1H-pyrazol-3-yl]propanoyl}piperazine (33)
Prepared from 6b and purified by flash chromatography
CH2Cl2:MeOH (97:3); yield 68%; Mp 224e225 ꢃC; IR (ATR):
1643 cmꢀ1; 1H NMR (CDCl3):
d 1.32 (s, 9H), 2.39 (m, 4H), 2.78 (t, 2H,
J ¼ 7.6 Hz), 3.08 (t, 2H, J ¼ 7.6 Hz), 3.46 (s, 2H), 3.50 (m, 2H), 3.65 (m,
2H), 6.34 (s, 1H), 6.98 (m, 2H), 7.16e7.35 (m, 11H); HRMS (m/z):
[M þ H]þcalcd for C33H38FN4O, 525.3030; found, 525.3041.
4.3.31. 3-[3-{3-[4-(4-tert-Butylbenzyl)piperazin-1-yl]-3-
oxopropyl}-5-(4-fluorophenyl)-1H-pyrazol-1-yl]-6-
methoxypyridazine (39)
Prepared from 8b and purified by flash chromatography
4.3.26. 1-(4-tert-Butylbenzyl)-4-(3-{1-phenyl-5-[4-
(trifluoromethyl)phenyl]-1H-pyrazol-3-yl}propanoyl)piperazine
(34)
CH2Cl2:MeOH (95:5); yield 65%; Mp 151e152 ꢃC; IR (ATR):
1648 cmꢀ1 1H NMR (CDCl3):
; d 1.31 (s, 9H), 2.40 (m, 4H), 2.77 (m,
2H), 3.07 (t, 2H), 3.47 (s, 2H), 3.50 (m, 2H), 3.65 (m, 2H), 4.09 (s, 3H),
6.36 (s, 1H), 7.01 (m, 2H), 7.06 (d, 1H, J ¼ 8.8 Hz), 7.22 (d, 2H,
J ¼ 8.4 Hz), 7.26 (m, 2H), 7.34 (d, 2H, J ¼ 8.4 Hz), 7.72 (d, 1H,
J ¼ 9.6 Hz); HRMS (m/z): [M þ H]þcalcd for C32H38FN6O2, 557.3040;
found, 557.3014.
Prepared from 6c and purified by flash chromatography
CH2Cl2:MeOH (90:10); yield 61%; Mp 264e265 ꢃC; IR (ATR):
1643 cmꢀ1 1H NMR (CDCl3):
; d 1.31 (s, 9H), 2.51 (m, 4H), 2.80 (m,
2H), 3.09 (m, 2H), 3.5e3.62 (m, 6H), 6.43 (s, 1H), 7.24e7.38 (m,11H),
7.54 (d, 2H, J ¼ 8.4 Hz); HRMS (m/z): [M þ H]þcalcd for
C34H38F3N4O, 575.2998; found, 575.2973.
4.3.32. 3-{3-{3-[4-(4-tert-Butylbenzyl)piperazin-1-yl]-3-
oxopropyl}-5-[4-(trifluoromethyl) phenyl]-1H-pyrazol-1-yl}-6-
methoxypyridazine (40)
4.3.27. 2-[3-{3-[4-(4-tert-Butylbenzyl)piperazin-1-yl]-3-
oxopropyl}-5-(4-methylphenyl)-1H-pyrazol-1-yl]quinoline (35)
Prepared from 7a and purified by flash chromatography
CH2Cl2:MeOH (95:5); yield 83.7%; Mp 166e167 ꢃC; IR (ATR):
Prepared from 8c and purified by flash chromatography
CH2Cl2:MeOH (95:5); yield 77%; Mp 124e125 ꢃC; IR (ATR):
1646 cmꢀ1 1H NMR (CDCl3):
; d 1.32 (s, 9H), 2.42 (m, 4H), 2.78 (t,

B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
5031
2H,J ¼ 7.6 Hz), 3.09 (t, 2H, J ¼ 7.6 Hz), 3.47 (s, 2H) 3.50 (m, 2H), 3.66
4.4.1. Cell Preparation
(m, 2H), 4.09 (s, 3H), 6.45 (s, 1H), 7.10 (d, 1H, J ¼ 9.2 Hz), 7.22 (d, 2H,
J ¼ 8.0), 7.34 (d, 2H, J ¼ 8.4 Hz), 7.40 (d, 2H, J ¼ 8.0 Hz), 7.58 (d, 2H,
J ¼ 8.8 Hz), 7.82 (d, 1H, J ¼ 9.2 Hz). HRMS (m/z): [M þ H]þcalcd for
C33H38F3N6O2, 607.3008; found, 607.3029.
Human neutrophils were freshly isolated from leukocyte
concentrates obtained at the Blood Center of the University
Hospital Tuebingen (Germany). In brief, venous blood was taken
from healthy adult donors and leukocyte concentrates were
prepared by centrifugation at 4000g for 20 min at RT. Neutrophils
were immediately isolated by dextran sedimentation, centrifuga-
tion on Nycoprepcushions (PAA Laboratories, Linz, Austria), and
hypotonic lysis of erythrocytes as described [29]. Cells were finally
resuspended in phosphate-buffered saline pH 7.4 (PBS) containing
1 mg/mL glucose and 1 mM CaCl2 (PGC buffer) (purity >96e97%).
4.3.33. Ethyl 1-{3-[5-(4-methylphenyl)-1-phenyl-1H-pyrazol-3-yl]
propanoyl}piperidine-4-carboxylate (41)
Prepared from 6a and ethyl piperidin-4-carboxylate using the
same procedure for compound 27; purified by flash chromatog-
raphy CH2Cl2:MeOH (95:5); yield 38%; yellow oil; IR (ATR): 1639,
1726 cmꢀ1; 1H NMR (CDCl3):
d
1.25 (t, 3H, J ¼ 7.2 Hz), 1.62 (m, 2H),
1.92 (m, 2H), 2.33 (s, 3H), 2.51 (m, 1H), 2.80 (m, 3H), 3.10 (m, 3H),
3.92 (m, 1H), 4.13 (q, 2H, J ¼ 7.2 Hz), 4.45 (m, 1H), 6.34 (s, 1H), 7.09
(s, 5H), 7.27e7.34 (m, 4H); HRMS (m/z): [M þ H]þcalcd for
C27H32N3O3, 446.2444; found, 446.2448.
4.4.2. Expression of human recombinant 5-LO in E. coli and
preparation of high speed supernatants
E. coli MV1190 was transformed with pT3-5LO plasmid and
recombinant 5-LO protein was expressed at 30 ꢃC as described [29].
Cells were lysed by incubation in 50 mM triethanolamine/HCl pH
4.3.34. 4-Isopropylphenyl 3-[5-(4-methylphenyl)-1-phenyl-1H-
pyrazol-3-yl]propanoate (42)
8.0, 5 mM EDTA, soybean trypsin inhibitor (60 mg/mL), 1 mM
phenylmethylsulfonylfluoride (PMSF), and lysozyme (1 mg/mL),
homogenized by sonication (3 ꢂ 15 s), and centrifuged at 40,000 g
for 20 min at 4 ꢃC. The resulting 40,000 g supernatant was imme-
diately used for 5-LO activity assays.
Prepared from 6a and 4-isopropylphenol using the same
procedure for compound 27; Flash chromatography hexaneꢀ-e1thyl
acetate (80:20); yield 86%; Mp 74e75 ꢃC; IR (ATR): 1753 cm
;
1H
NMR (DMSO-d6):
d
1.18 (d, 6H, J ¼ 6.8 Hz), 2.29 (s, 3H), 2.89 (m, 1H),
3.01 (m, 4H), 6.52 (s, 1H), 6.99 (d, 2H, J ¼ 8.4 Hz), 7.10 (d, 2H,
4.4.3. Determination of 5-LO, 12-LO and 15-LO product formation in
intact cells
J ¼ 8.4 Hz), 7.16 (d, 2H, J ¼ 8.0 Hz), 7.24 (m, 4H), 7.24 (m, 3H). 13C
NMR (CDCl3):
d
21.42, 23.77, 24.59, 33.44, 33.56, 107.28, 122.15,
For assays of intact cells, 5 ꢂ 106 freshly isolated neutrophils
were resuspended in 1 mL PGC buffer. After preincubation with the
test compounds for 15 min at RT, 5-LO product formation was
125.64, 127.82, 127.98, 128.87, 129.66, 129.85, 138.43, 140.56, 143.86,
146.46, 149.19, 151.95, 172.08. HRMS (m/z): [M þ H]þ calculated for
C28H29N2O2, 425.2229; found, 425.2209.
started by addition of 2.5
After 10 min at 37 ꢃC, the reaction was stopped with 1 mL of
methanol and 30 L of 1 N HCl, 200 ng of prostaglandin B1, and
500 L of PBS were added. Formed 5-LO metabolites were extracted
mM ionophore A23187 plus 20 mM AA.
4.3.35. Ethyl 1-{3-[5-(4-methylphenyl)-1-quinolin-2-yl-1H-
pyrazol-3-yl]propanoyl}piperidine-4-carboxylate (43)
m
m
Prepared from 7a and ethyl piperidin-4-carboxylate using the
same procedure for compound 27; Flash chromatography
CH2Cl2:MeOH (97:3); yield 78%; Mp 109e110 ꢃC; IR (ATR): 1638,
and analyzed by HPLC as described [30]. 5-LO product formation is
expressed as ng of 5-LO products per 106 cells, which includes LTB4
and its all-trans isomers, and 5(S)-hydro(pero)xy-6-trans-8,11,14-
cis-eicosatetraenoic acid (5(S)-H(p)ETE). Cysteinyl LTs C4, D4, and E4
were not detected, and oxidation products of LTB4 were not
determined. 15-H(P)ETE was analyzed as product of 15-LO and 12-
H(P)ETE as product of 12-LO.
1718 cmꢀ1; 1H NMR (CDCl3):
d
1.24 (t, 3H, J ¼ 7.2 Hz), 1.65 (m, 2H),
1.92 (m, 2H), 2.35 (s, 3H), 2.51 (m, 1H), 2.83 (m, 3H), 3.12 (m, 3H),
3.90 (m, 1H), 4.13 (q, 2H, J ¼ 7.2 Hz), 4.47 (m, 1H), 6.40 (s, 1H), 7.09
(d, 2H, J ¼ 8.0 Hz), 7.19 (d, 2H, J ¼ 8.0 Hz), 7.44 (d, 1H, J ¼ 8.4 Hz),
7.52 (m, 1H), 7.67 (m, 1H), 7.79 (d, 1H, J ¼ 8.0 Hz), 7.89 (d, 1H,
J ¼ 8.4 Hz), 8.11 (d, 1H, J ¼ 8.4 Hz); 13C NMR (CDCl3):
d
14.40,
4.4.4. Determination of 5-LO product formation in cell-free systems
21.50, 24.29, 28.11, 28.69, 32.99, 41.27, 45.02, 60.79, 108.92, 117.65,
126.76, 127.10, 127.58, 128.46, 128.96, 129.06, 129.44, 130.18,
138.29, 138.34, 145.05, 146.94, 151.32, 154.19, 170.76, 174.42.
HRMS (m/z): [M þ H]þcalcd for C30H33N4O3, 497.2553; found,
497.2530.
For determination of the activity of 5-LO in 40,000 g superna-
tants, aliquots of the supernatants (approximately 20
mL, corre-
sponding to 4 mL E. coli cell culture) were added to 1 mL of a 5-LO
reaction mix (PBS, pH 7.4 containing 1 mM EDTA). Samples were
pre-incubated with the test compounds for 10 min at 4 ꢃC, samples
were prewarmed for 30 s at 37 ꢃC, and 2 mM CaCl2 and 20
mM AA
4.3.36. 4-Isopropylphenyl 3-[5-(4-methylphenyl)-1-quinolin-2-yl-
1H-pyrazol-3-yl]propanoate (44)
were added to start 5-LO product formation. The reaction was
stopped after 10 min at 37 ꢃC by addition of 1 mL ofice-cold
methanol, and the formed metabolites were analyzed as
described for intact cells.
Prepared from 7a and 4-isopropylphenol using the same
procedure for compound 27; Flash chromatography hexane-ethyl
acetate (85:15); yield 92%; Mp 81e82 ꢃC; IR (ATR): 1755 cmꢀ1
;
1H NMR (CDCl3):
d
1.23 (d, 6H, J ¼ 6.8 Hz), 2.36 (s, 3H), 2.90 (m,
4.4.5. Induction of mPGES-1 expression in A549 cells, isolation of
Microsomes, and determination of mPGES-1 inhibition
Preparation of A549 cells and determination of mPGES-1
activity was performed as described previously [23]. In brief,
A549 cells were incubated for 16 h at 37 ꢃC and 5% CO2. Subse-
quently, the culture medium was replaced by fresh DMEM/High
1H), 3.06 (t, 2H J ¼ 7.6 Hz), 3.23 (t, 2H, J ¼ 7.6 Hz), 6.43 (s, 1H), 7.02
(m, 2H), 7.10 (m, 2H), 7.21 (m, 4H), 7.52 (m, 2H), 7.67 (m, 1H), 7.80
(d, 1H, J ¼ 8.4 Hz), 7.86 (d, 1H, J ¼ 8.0 Hz), 8.14 (d, 1H, J ¼ 8.4 Hz);
HRMS (m/z): [M þ H]þ calculated for C31H30N3O2, 476.2338; found,
476.2322.
glucose (4.5 g/l) medium containing fetal calf serum (2%, v/v); IL-1
b
4.4. Biological assays
(1 ng/ml) was added, and cells were incubated for another 72 h.
Thereafter, cells were detached with trypsin/EDTA, washed with
PBS, and frozen in liquid nitrogen. Ice-cold homogenisation buffer
BWA4C, Arachidonic acid, Ca2þ-ionophore A23187 and all other
fine chemicals were from Sigma (Deisenhofen, Germany), unless
stated otherwise. HPLC solvents were from Merck (Darmstadt,
Germany).
(0.1
methanesulfonylfluoride, 60
ml leupeptin, 2.5 mM glutathione, and 250 mM sucrose) was
M
potassium phosphate buffer pH 7.4, 1 mM phenyl-
m
g/ml soybean trypsin inhibitor, 1 g/
m

5032
B. Çalıs¸kan et al. / European Journal of Medicinal Chemistry 46 (2011) 5021e5033
added, and after 15 min, cells were resuspended and sonicated on
ice (3 ꢂ 20 s). The homogenate was subjected to differential
centrifugation at 10,000 ꢂ g for 10 min and 174,000 ꢂ g for 1 h at
Acknowledgments
This study was supported by The Scientific and Technological
Research Council of Turkey (TÜBITAK No. 108S210).
ꢃC. The pellet (microsomal fraction) was resuspended in 1 ml
_
4
homogenization buffer, and the total protein concentration was
determined by Coomassie protein assay. Microsomal membranes
were diluted in potassium phosphate buffer (0.1 M, pH 7.4) con-
taining 2.5 mM glutathione to give a final concentration of 50 ìg/ml.
Test compounds or vehicle (1% DMSO) were added, and after
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