Synthesis and biological evaluation of new phenidone analogues as potential dual cyclooxygenase (COX-1 and COX-2) and human lipoxygenase (5-LOX) inhibitors

Article abstract of DOI:10.1016/j.farmac.2004.09.002

A new series of potential human 5-LOX inhibitors structurally related to the 1-phenyl-3-pyrazolidinone (phenidone, 2) has been synthesized and the activity against COX-1, COX-2, and human 5-LOX enzymes has been evaluated. In contrast with literature data, we observed that phenidone resulted to be inactive against human 5-LOX, while retains its activity against cyclooxygenases in a micromolar range. The present results suggest that the substitution of the amino function at the 4-position is detrimental in terms of activity toward COX-1 and COX-2, while the presence of a double bond at the 4,5-position does not alter the biological profile against COX. The absence of activity vs. human 5-LOX strongly suggests a re-consideration of phenidone and its analogs as 5-LOX inhibitors in humans.

Full text of DOI:10.1016/j.farmac.2004.09.002

IL FARMACO 60 (2005) 7–13
http://france.elsevier.com/direct/FARMAC/
Original article
Synthesis and biological evaluation of new phenidone analogues as
potential dual cyclooxygenase (COX-1 and COX-2) and human
lipoxygenase (5-LOX) inhibitors
Claudia Cusan a, Giampiero Spalluto a, Maurizio Prato a, Michael Adams b, Antje Bodensieck b,
Rudolf Bauer b, Aurelia Tubaro c, Paolo Bernardi d, Tatiana Da Ros a,*
a Dipartimento di Scienze Farmaceutiche, Università di Trieste, Piazzale Europa 1, I-34127, Trieste, Italy
b Department of Pharmacognosy, Institute of Pharmaceutical Sciences, Karl-Franzens-Universitaet Graz Universitaetsplatz 4 A-8010 Graz, Austria
c Dipartimento di Economia e Merceologia delle Risorse Naturali e della Produzione, Università di Trieste, Via Valerio 6, I-34127, Trieste, Italy
d Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Viale G. Colombo 3, I-35121 Padova, Italy
Received 26 July 2004; accepted 26 September 2004
Available online 24 December 2004
Abstract
A new series of potential human 5-LOX inhibitors structurally related to the 1-phenyl-3-pyrazolidinone (phenidone, 2) has been synthe-
sized and the activity against COX-1, COX-2, and human 5-LOX enzymes has been evaluated. In contrast with literature data, we observed
that phenidone resulted to be inactive against human 5-LOX, while retains its activity against cyclooxygenases in a micromolar range. The
present results suggest that the substitution of the amino function at the 4-position is detrimental in terms of activity toward COX-1 and
COX-2, while the presence of a double bond at the 4,5-position does not alter the biological profile against COX. The absence of activity vs.
human 5-LOX strongly suggests a re-consideration of phenidone and its analogs as 5-LOX inhibitors in humans.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Cyclooxygenase; Lipoxygenase; Dual inhibitors; Phenidone derivatives
1. Introduction
death through the activation of mitochondrial mechanisms
such as by opening the transition pore [11–13]. These studies
have been supported by several experimental details, in fact
simultaneous administration of COX-2 and 5-LOX inhibi-
tors induces apoptosis in human prostate tumor cell line (PC3)
and mouse liver tumor cells (MH1C1) [14].
A better approach for increasing arachidonic acid levels
was based on the administration of a compound capable to
inhibit both COX and LOX. This approach has been con-
firmed by using a dual inhibitor derived from diclofenamic
acid (1), which inhibits both COX and LOX at concentra-
tions ranging from 0.1 to 0.5 µM [15], but its use in therapy is
limited by the high toxicity (5–10 µM), which induces mito-
chondrial death (Fig. 1).
Arachidonic acid is an important fatty acid present in cell
membranes, being a precursor of several metabolites (leucot-
rienes, prostaglandines, prostacyclines), which are involved
in several patho-physiological processes such as bron-
cospasm and inflammation [1–4]. It is well known that the
formation of these metabolites is catalyzed by cyclooxyge-
nases (COX-1 and COX-2) and lipoxygenases (5-, 8-, 12-
and 15-LOX), in fact their block is the most common approach
for anti-inflammatory therapy [5–10].
Very recently, it has been demonstrated that the simulta-
neous inhibition of COX and LOX, which produces increased
levels of arachidonic acid, is capable to induce tumor cell
For this reason, the search for novel dual (COX and LOX)
inhibitors, possessing low toxicity, represents an attractive tool
for cancer chemotherapy. With the aim to discover new dual
inhibitors, we selected phenidone (2) as lead compound. In
fact, it is widely reported that this compound is a potent rat
* Corresponding author. Dipartimento di Scienze Farmaceutiche, Univer-
sità di Trieste, Piazzale Europa 1, 34127 Trieste, Italy. Tel.: +39-040-
5583110; fax: +39-040-52572.
E-mail address: daros@units.it (T. Da Ros).
0014-827X/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.09.002

8
C. Cusan et al. / IL FARMACO 60 (2005) 7–13
ous steric and lipophilic properties, while the presence of a
double bond confers more rigidity to the system, permitting
to analyze new structural requirements indispensable for activ-
ity.
2. Chemistry
The designed compounds 4a–i have been prepared by acy-
lation under standard conditions (dioxane reflux) of amine 5
with the appropriate acyl chlorides. Amine 5 was prepared
by the known procedure [20–22] briefly summarized in
Scheme 2.
Oxidation with potassium dichromate in acetic acid [20],
or FeCl3 [22] of commercially available phenidone (2)
afforded the corresponding dihydroderivate (6) which, after
treatment with concentrated nitric acid at 0 °C, gave nitro
derivative (7) [21]. The latter was then reduced with hydro-
gen in the presence of Pd/C to yield the desired amine (5)
[21].
Amine 5 was coupled with the appropriate acyl chloride
(8a–i) (when not commercially available, the acyl chlorides
were prepared from the corresponding acid by treatment with
thionyl chloride) to afford the final compounds 4a–i. When
the acyl chloride of succinic acid monomethyl ester was used,
the compound of both N- and O-acylation (9) was also iso-
lated (Scheme 3).
Fig. 1
.
5-LOX inhibitor both in vitro and in vivo (0.1–0.5 µM) [16,17]
and a quite potent COX-1 and COX-2 (7 µM tested in a mix-
ture of enzymes) inhibitor [15,18,19]. Several modifications
on this lead compound permitted to obtain a quite detailed
structure–activity relationship profile, demonstrating that lipo-
philic substituents at the 4-position (compound 3) could sig-
nificantly increase 5-LOX inhibition potency (IC50 = 60 nM)
[16] (Figs. 2 and 3).
Taking into account these experimental observations, we
decided to investigate a new series of phenidone analogues
of general formula 4 (Scheme 1).
In particular, the introduction of an amino function at the
4-position on the pyrazoline ring (5) [20–22] could be an ideal
appendage for linking different substituents possessing vari-
3. Results and discussion
All the synthesized compounds (4a–i, 5, 9) have been
tested in inhibition assays against purified COX-1, COX-2,
and human 5-LOX enzymes, using phenidone (2), indometa-
cine (COX inhibitor), and zileuton (5-LOX inhibitor) as inter-
nal controls.
In Table 1 are reported only the compounds showing sig-
nificant activity in COX-1 and COX-2 inhibition assays (4i,
5).As clearly indicated, the not substituted derivative (5) pre-
sented considerable activity against both COX-1 and COX-
2 enzymes, comparable to the reference compound (2), giv-
ing 43% of inhibition toward COX-2 with a selectivity ratio
COX-1/COX-2 of 0.27, quite similar to that of the reference
compound (2). In contrast, the introduction of acyl groups at
the 4-position seems to be detrimental in terms of activity.
Only compound 4i, which bears a bulky and highly lipo-
Figs. 2 and 3
.
Scheme 1
.
Scheme 2.

C. Cusan et al. / IL FARMACO 60 (2005) 7–13
9
Table 2
Estimation of IC50 values in inhibition assay against COX-1 and COX-2 of
selected compounds
Compound
COX-1a
COX-2b
COX-1/COX-2
(IC50, µM)
20.2 2.3
36.7 4.2
(IC50, µM)
9.2 0.8
13.4 2.3
Phenidone, 2
5
2.2
2.7
a COX-1 is from ram seminal vesicles (Cayman Chemical Company, Ann
Arbor, MI, USA).
b COX-2 is from sheep placental cotyledones (Cayman Chemical Com-
pany, Ann Arbor, MI, USA). n = 2–4 experiments in duplicate. Values are
reported as absolute percent inhibition of enzyme and standard error of the
mean (S.E.M).
prisingly, while the reference compound zilutron showed
inhibitory activity, phenidone (2), in contrast to literature data,
proved to be ineffective (7% inhibition at 10 µM concentra-
tion). These results seem to suggest that this total inactivity
could not be attributed to the structural modifications per-
formed on the phenidone (2) nucleus, but to the choice of the
lead compound. The discrepancy with literature data
[16,17,19] strongly supports a significant difference in the
sequence homology between murine and human 5-LOX.
Scheme 3
.
Table 1
4. Conclusions
Inhibition percentage of compounds 4i, 5 against COX-1, COX-2, and 5-LOX
at 10 µM concentrationa
The present work reports a new investigation of pheni-
done (2) and its structurally related analogs as dual inhibitors
for COX and human 5-LOX enzymes. Only not substituted
derivative (5) showed significant activity against both COX-
1 and COX-2, while resulted to be inactive against human
5-LOX.
Compound
Phenidone, 2
Indometacine
Zileuton
4i
COX-1b
COX-2c
80.2 12
5-LOXd
0
20.3
50
4
7
50
3
ND
ND
8.3
12
ND
50
0
4
2
3.2 0.8
43.2
5
1
6
0
Nevertheless, the inactivity against human 5-LOX of this
compound could be attributed not only to the kind of substi-
tutions, but, most probably, to the choice of phenidone (2) as
a lead compound. In fact, while phenidone and its derivative
proved to be potent rat 5-LOX inhibitors, we found it totally
inactive in human 5-LOX inhibition assay, suggesting that
phenidone (2) could not be considered a 5-LOX inhibitor in
human. Nevertheless, further studies are in course in our labo-
ratories to support these preliminary experimental observa-
tions.
ND: not determined.
a All the compounds not included in the table resulted to be completely
inactive.
b COX-1 is from ram seminal vesicles (Cayman Chemical Company, Ann
Arbor, MI, USA).
c COX-2 is from sheep placental cotyledones (Cayman Chemical Com-
pany, Ann Arbor, MI, USA).
d For the test with 5-LOX, we have used the leukocytes suspension from
human blood of volunteers. n = 2–4 experiments in duplicate. Values are
reported as absolute percent inhibition of enzyme and standard error of the
mean (S.E.M).
philic substituent at the 4-position, displays poor activity
(3–8% inhibition at 10 µM). These results seems to suggest
that substituents at the 4-position on the pyrazoline nucleus
highly decrease the COX-1 and COX-2 inhibition, while the
presence of the double bond does not influence significantly
the activity with respect to the reference compound (2). To
further validate these experimental data, the exact IC50 val-
ues against COX-1 and COX-2 enzymes for compound (5)
were measured (Table 2).
As reported, derivative (5) showed IC50 values very simi-
lar to that of the reference compound (2) with a slight
improved COX-1/COX-2 selectivity (2.7 vs. 2.2).
Instead, unexpected data were obtained in the evaluation
of the human 5-LOX inhibition. In fact, all the synthesized
compounds resulted to be inactive against 5-LOX, but sur-
5. Experimental section
5.1. Chemistry
5.1.1. General remarks
Reactions were routinely monitored by thin-layer chroma-
tography (TLC) on silica gel (precoated F254 Merck plates)
and products visualized with iodine or aqueous potassium
permanganate. Infrared spectra (IR) were measured on a Jasco
FT-IR instrument using NaCl cells (oils) or KBr powder
(DRIFT system). UV-spectra were recorded on a Perkin-
Elmer Lambda 20 spectrophotometer. 1H and 13C NMR were
determined in DMSO-d6 solutions, unless otherwise noted,

10
C. Cusan et al. / IL FARMACO 60 (2005) 7–13
at 200 and 50 MHz, respectively, with a Varian Gemini
200 spectrometer, peak positions are given in parts per mil-
lion (d) downfield, and J values are given in Hertz. Melting
points were determined on a Buchi-Tottoli instrument and
are uncorrected. Electrospray mass spectra were recorded on
a Perkin-Elmer PE SCIEX API 1 spectrometer, and com-
pounds were dissolved in methanol/tetrahydrofuran 4/1,
unless otherwise noted. EI-MS spectra were recorded on a
VG7070 H spectrometer using electron energy 70 eV. Chro-
matography was performed with Merck 60–200 mesh silica
gel. All products reported showed IR and NMR spectra in
agreement with the assigned structures. Organic solutions
were dried over anhydrous magnesium sulfate.All final com-
pounds showed a purity >99% checked by HPLC using
reverse phase column (Luna 5 µ, C18(2) 250 × 4.60 mm),
eluting with a mixture of MeCN (0.35 ml/min) and H2O
(0.15 ml/min).
5.1.2.3. Heptanoic acid (3-oxo-1-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-amide (4c). C16H21N3O2 (287 MW); yield:
64%; (chromatography eluent: CH2Cl2/ethyl acetate, 8:2);
white solid, m.p.: 166–168 °C; IR-DRIFT: cm–1 3283, 2923,
2853, 2399, 1863, 1651, 1600, 1507, 1461, 1233, 1195, 1117,
1055, 966, 938, 892, 830, 775, 750, 689, 587, 503, 449, 416;
1H NMR: d 10.75 (bs, 1H), 9.62 (bs, 1H), 8.37 (s, 1H), 7.61
(d, 2H, J = 8.0 Hz), 7.39 (t, 2H, J = 7.6 Hz), 7.13 (t, 1H,
J = 7.3 Hz), 2.31 (t, 2H, J = 7.3 Hz), 1.55 (m, 2H), 1.35–1.17
(m, 6H), 0.85 (t, 3H, J = 6.2 Hz); 13C NMR: d 170.61, 154.09,
139.72, 129.31, 124.08, 119.07, 116.16, 109.64, 35.01, 31.05,
28.35, 25.25, 22.02, 13.97; EI-MS: m/z 287 (M+, 17%), 175
(M+—C4H9CO, 100%), 130 (C6H13CONH+, 12%), 104
+
(PhN2 , 52%), 77 (Ph+, 78%); UV-Vis (EtOH): kmax 194, 201,
295.
5.1.2.4. Decanoic acid (3-oxo-1-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-amide (4d). C19H27N3O2 (329 MW); yield:
59%; (chromatography eluent: petroleum ether/ethyl acetate,
1:1); white solid, m.p.: 159–161 °C; IR-DRIFT: cm–1 3283,
2917, 2848, 2279, 1776, 1654, 1601, 1528, 1465, 1372, 1233,
1099, 1055, 918, 810, 747, 687, 607, 437; 1H NMR: d 10.73
(bs, 1H), 9.62 (bs, 1H), 8.37 (s, 1H), 7.61 (d, 2H, J = 8.0 Hz),
7.39 (t, 2H, J = 7.7 Hz), 7.13 (t, 1H, J = 7.4 Hz), 2.31 (t, 2H,
J = 7.4 Hz), 1.64–1.46 (m, 2H), 1.34–1.14 (m, 12H), 0.84 (t,
3H, J = 6.7 Hz); 13C NMR: d 170.62, 154.07, 139.73, 129.32,
124.09, 119.06, 116.15, 109.64, 35.01, 31.30, 28.93, 28.83,
28.71, 25.29, 22.13, 13.99; EI-MS: m/z 329 (M+, 50%), 218
(M+—C8H17, 15%), 202 (M+—C9H19, 11%), 176
5.1.2. General procedure for the preparation of phenidone
analogues 4a–i, 9
To a solution of amine (5) (2 mmol) in dry dioxane (10 ml)
the appropriate acyl chloride (1 mmol) dissolved in dry diox-
ane (10 ml) was added. The resulting mixture was stirred over-
night, heating to reflux. The solvent was then removed under
reduced pressure and the crude material was purified by flash
chromatography to give the final product as a white solid,
which was crystallized by diethyl ether/ethanol.
5.1.2.1. N-(3-Oxo-1-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-
propionamide (4a). C16H13N3O2 (279 MW); yield: 32%;
(chromatography eluent: petroleum ether/ethyl acetate, 1:1);
white solid, m.p.: 236–238 °C; IR-DRIFT: cm–1 3263, 3167,
3066, 2538, 2019, 1639, 1544, 1406, 1332, 1208, 1069, 952,
(M+—C9H19CO, 100%), 104 (PhN2 , 15%), 77 (Ph+, 12%);
+
UV-Vis (EtOH): kmax 296, 199.
5.1.2.5. N-(3-Oxo-1-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-
benzamide (4e). C16H13N3O2 (279 MW); yield: 31%; (chro-
matography eluent: petroleum ether/ethyl acetate, 1:1); white
solid, m.p.: 236–238 °C; IR-DRIFT: cm–1 3263, 3167, 3066,
2538, 2019, 1639, 1544, 1406, 1332, 1208, 1069, 952, 904,
806, 753, 696, 512, 426; 1H NMR: d 10.49 (bs, 1H), 8.90 (bs,
1H), 8.16 (d, 2H, J = 7.3 Hz), 7.94–7.37 (m, 8H), 7.13 (t, 1H,
J = 7.5 Hz); 13C NMR: d 164.77, 155.16, 139.68, 133.65,
131.62, 129.39, 128.37, 127.58, 124.44, 121.21, 116.37,
109.01; ES-MS: m/z 279 (M+), 302 (M + Na+)+, 318 (M +
1
904, 806, 753, 696, 512, 426; H NMR: d 10.49 (bs, 1H),
8.90 (bs, 1H), 8.16 (d, 2H, J = 7.3 Hz), 7.94–7.37 (m, 8H),
7.13 (t, 1H, J = 7.5 Hz); 13C NMR: d 164.77, 155.16, 139.68,
133.65, 131.62, 129.39, 128.37, 127.58, 124.44, 121.21,
116.37, 109.01; ES-MS (MeOH–THF): m/z 279 (M+), 302
(M + Na+)+, 318 (M + K+)+; EI-MS: m/z 279 (M+, 33%), 105
+
(PhN2 , 100%), 77 (Ph+, 78%); UV-Vis (EtOH): kmax 199,
203, 220.
+
5.1.2.2. Pentanoic acid (3-oxo-1-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-amide (4b). C14H17N3O2 (259 MW); yield:
56%; (chromatography eluent: CH2Cl2/ethyl acetate, 8:2);
white solid, m.p.: 173–174 °C; IR-DRIFT: cm–1 3287, 2957,
2867, 2407, 1939, 1863, 1727, 1651, 1601, 1508, 1450, 1409,
1384, 1289, 1226, 1196, 1107, 1075, 1055, 965, 938, 891,
831, 778, 747, 687; 1H NMR: d 10.75 (bs, 1H), 9.63 (bs, 1H),
8.37 (s, 1H), 7.61 (d, 2H, J = 7.9 Hz), 7.40 (t, 2H, J = 7.3 Hz),
7.13 (t, 1H, J = 7.3 Hz), 2.32 (t, 2H, J = 7.3 Hz), 1.54 (m,
2H,), 1.29 (m, 2H,), 0.88 (t, 3H, J = 7.3 Hz); 13C NMR: d
170.63, 154.13, 139.74, 129.33, 124.09, 119.12, 116.15,
109.65, 34.74, 27.43, 21.83, 13.78; EI-MS: m/z 259 (M+,
K+)+; EI-MS: m/z 279 (M+, 33%), 105 (PhN2 , 100%), 77
(Ph+, 78%); UV-Vis (EtOH): kmax 199, 203, 220.
5.1.2.6. N-(3-Oxo-1-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-
phenyl-acetamide (4f). C17H15N3O2 (293 MW); yield: 42%;
(chromatography eluent: CH2Cl2/ethyl acetate, 9:1); white
solid, m.p.: 224–226 °C; IR-DRIFT: cm–1 3280, 3033, 1664,
1571, 1509, 1408, 1318, 1199, 1067, 959, 904, 813, 691, 562,
488, 429; 1H NMR: d 10.82 (bs, 1H), 9.92 (bs, 1H), 8.39 (s,
1H), 7.60 (d, 2H, J = 7.4 Hz), 7.52–7.18 (m, 7H), 7.12 (t, 1H,
J = 7.3 Hz), 3.66 (s, 2H); 13C NMR: d 168.29, 154.09, 139.72,
136.07, 129.34, 129.03, 128.18, 126.40, 124.16, 119.20,
116.19, 109.47, 41.89; EI-MS: m/z 293 (M+, 100%), 175
+
27%), 175 (M+—C4H9CO, 100%), 104 (PhN2 , 48%), 77
+
+
+
+
(Ph+, 60%), 57 (C4H9 , 50%), 41 (C3H7 , 49%); UV-Vis
(M+—PhCH2CO, 78%), 104 (PhN2 , 32%), 91 (PhCH2 ,
(EtOH): kmax 198, 295.
88%), 77 (Ph+, 52%); UV-Vis (EtOH): kmax 295, 193.

C. Cusan et al. / IL FARMACO 60 (2005) 7–13
11
5.1.2.7. 2-(2-Bromo-phenyl)-N-(3-oxo-1-phenyl-2,3-dihydro-
1H-pyrazol-4-yl)-acetamide (4g). C17H14BrN3O2 (372 MW);
yield: 61%; (chromatography eluent: CH2Cl2/ethyl acetate,
9:1); white solid, m.p.: 218–219 °C; IR-DRIFT: cm–1 3266,
3025, 1655, 1565, 1511, 1414, 1320, 1205, 1063, 956, 814,
754, 687, 565, 500, 431; 1H NMR: d 10.81 (bs, 1H), 9.96 (bs,
1H), 8.40 (s, 1H), 7.59 (d, 2H, J = 8.0 Hz), 7.45–7.26 (m,
4H), 7.32–7.05 (m, 3H), 3.86 (s, 2H); 13C NMR: d 166.55,
153.55, 139.23, 135.28, 131.69, 131.48, 128.86, 128.15,
127.02, 123.97, 123.64, 118.60, 115.66, 109.07, 41.53;
EI-MS: m/z 371 (M+, 16%), 291 (M+—Br, 25%), 176
3H), 2.96 (t, 4H, J = 6.5 Hz), 2.71 (t, 4H, J = 7.0 Hz), 2.61
(m, 4H); 13C NMR: d 172.71, 172.68, 1689.90, 168.82,
146.52, 139.12, 129.52, 125.93, 120.11, 117.43, 113.57,
51.65, 51.39, 29.80, 28.58, 28.49, 28.29; ES-MS: m/z 404
(M+), 427 (M + Na+), 443 (M + K+); UV-Vis (EtOH): kmax
193, 200, 277.
5.2. Biology
5.2.1. COX-1 and COX-2 inhibition assays
The assays were carried out in a 96 microwell titre plate
(Bibby Sterilin, Staffordshire, UK), as elsewhere described
[23], and using purified COX-1 from ram seminal vesicles
and purified COX-2 from sheep placental cotyledones (both:
Cayman Chemical Company,AnnArbor, MI, USA). The incu-
bation mixture contained 180 µl 0.1 M TRIS/HCl buffer (pH
8.0) (Roth, Karlsruhe, Germany), 5 µM hematin (porcine,
ICN,Aurora, OH, USA), 18 mM epinephrin-hydrogentartrate
(Fluka, Buchs, Switzerland), 0.2 U of enzyme preparation
and 50 µM Na2EDTA (only COX-2 assay, Titriplex III, Merck,
Darmstadt, Germany).After adding 10 µl of synthesized com-
pound dissolved in EtOH p.a. or DMSO for compound 4i
(final concentration 10 µM) or 10 µl of EtOH p.a. in control
wells, the system was pre-incubated for 5 min at room tem-
perature.
+
(M+—BrPhCH2CO, 100%), 169 (BrPhCH2 , 14%), 130
+
(PhCH2CON+, 53%), 104 (PhN2 , 50%), 77 (Ph+, 98%);
UV-Vis (EtOH): kmax 295, 202, 193.
5.1.2.8. N-(3-Oxo-1-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-
(2,4,6-tribromo-phenyl)-acetamide (4h). C17H12Br3N3O2
(530 MW); yield: 15%; (chromatography eluent:
CH2Cl2/ethyl acetate, 9:1); white solid, m.p.: >300 °C;
IR-DRIFT: cm–1 3258, 3070, 2624, 1661, 1597, 1565, 1534,
1436, 1412, 1338, 1240, 1188, 1152, 1111, 1056, 965, 920,
1
856, 752, 729, 671; H NMR: d 10.85 (bs, 1H), 10.07 (bs,
1H), 8.39 (s, 1H), 7.95 (s, 2H), 7.59 (d, 2H, J = 8.3 Hz), 7.38
(t, 2H, J = 7.5 Hz), 7.12 (t, 1H, J = 7.9 Hz); 13C NMR: d
165.02, 153.92, 139.75, 134.95, 133.85, 129.32, 126.49,
124.14, 120.88, 119.04, 116.19, 109.48, 42.59; EI-MS: m/z
The addition of 5 µM arachidonic acid (10 µl) (Cayman
Chemical Company) triggered the enzymatic conversion of
arachidonic acid to PGE2 metabolite. After incubation for
20 min at 37 °C in darkness, the reaction is stopped by addi-
tion of 10% HCOOH (10 µl).
530 (M+, 3%), 451 (M+—Br, 16%), 330 (Br3PhCH2 , 5%),
+
294 (M+—3Br, 3%), 248 (Br2PhCH2+, 3%), 175
(M+—Br3PhCH2CO, 68%), 104 (PheN2 , 47%), 77 (Ph+,
+
100%); UV-Vis (EtOH): kmax 334, 296, 213.
5.1.2.9. N-(3-Oxo-1-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-
succinamic acid methyl ester (4i). C14H15N3O4 (289 MW);
yield: 42%; (chromatography eluent: CH2Cl2/ethyl acetate,
8:2); white solid, m.p.: 187–189 °C; IR-DRIFT: cm–1 3345,
3244, 3153, 3106, 2925, 2852, 1776, 1716, 1683, 1595, 1559,
1503, 1438, 1388, 1322, 1253, 1169, 1122, 1053, 1001, 964,
5.2.2. Determination of PGE2 by enzyme immunoassay
After dilution of the incubation mixture by EIA buffer, the
concentration of PGE2 was determined utilizing a competi-
tive enzyme immunoassay PGE2-EIA-kit (R&D Systems,
Minneapolis, MN, USA), used according to the company’s
instruction.
1
946, 883, 853, 794, 758, 684, 645; H NMR: d 10.74 (bs,
The EIA evaluation was performed with an ELISA reader
“rainbow” (TECAN) and determined as elsewhere described
[23]. Inhibition was inferred from the reduction of PGE2 for-
mation in comparison to a blank run without inhibitor. The
positive controls were indomethacin (ICN) and NS-398 (Cay-
man Chemical Company).
1H), 9.74 (bs, 1H), 8.38 (s, 1H), 7.61 (d, 2H, J = 8.3 Hz),
7.39 (t, 2H, J = 8.1 Hz), 7.12 (t, 1H, J = 7.3 Hz), 3.63 (s, 3H),
2.61 (m, 4H); 13C NMR: d 172.71, 169.00, 153.98, 139.73,
129.33, 124.09, 119.03, 116.14, 109.52, 51.34, 29.68,
28.63; EI-MS: m/z 289 (M+, 2%), 257 (M+—OCH3,
100%), 176 (M+—COC2H4COOCH3, 50%), 130
+
+
(NH2COC2H4COOCH3 , 20%), 104 (PhN2 , 17%), 77 (Ph+,
28%), 55 (C2H4CO+, 40%); UV-Vis (EtOH): kmax 201, 292.
5.3. 5-LOX inhibition assays
5.3.1. Isolation of human neutrophile granulocytes
5.1.2.10. Succinic acid 4-(3-methoxycarbonyl-propionyl-
amino)-1-phenyl-1H-pyrazol-3-yl ester methyl ester
(9). C19H23N3O7 (405 MW); yield: 32%; (chromatography
eluent: CH2Cl2/ethyl acetate, 7:3); white solid, m.p.: 137–
139 °C; IR-DRIFT: cm–1 3286, 3158, 2925, 2575, 1734, 1657,
1623, 1580, 1521, 1440, 1412, 1359, 1325, 1203, 1168, 1062,
1003, 982, 957, 918, 848, 808, 755, 719, 691;1H NMR: d
9.76 (bs, 1H), 8.67 (bs, 1H), 7.73 (d, 2H, J = 8.3 Hz), 7.46 (t,
2H, J = 8.1 Hz), 7.27 (t, 1H, J = 7.3 Hz), 3.63 (s, 3H), 3.59 (s,
Human blood (30 ml) from volunteers is collected with
Vacutainer™ system with preanalytical citric acid solution
to prevent clogging. The blood is immediately transferred to
a falcon tube containing 20 ml of Dextran solution (1.9 g
NaCl, 12.0 g Dextran T-500, H2O to 200.0 ml) and incubated
for 60 min at 4 °C; then it is centrifuged at 1600 rpm at 4 °C
for 10 min. Leukocytes precipitated forming a pellet while
thrombocytes and plasma remain in the supernatant, which is

12
C. Cusan et al. / IL FARMACO 60 (2005) 7–13
discarded. The pellet is then suspended in 10 ml of washing
buffer (1.48 g CaCl2·2 H2O p.a., 0.2 g anhydrous D-glucose,
0.04 g MgCl2·6 H2O, 0.08 g KCl, 3.5 g TRIS p.a., H2O to
200.0 ml, pH 7.6). After centrifugation at 1400 rpm at 4 °C
for 10 min and removal of the supernatant, the resulting pel-
let is suspended in 10 ml of hypotonic lysis buffer (0.17 g
NH4Cl, 0.2 g TRIS, H2O to 100.0 ml, pH 7.2) and gently
shaken for 5 min at room temperature to destroy remaining
erythrocytes. The suspension is centrifuged at 1400 rpm at
4 °C for 5 min. The pellet is dissolved in 10 ml of washing
buffer and then centrifuged at 1400 rpm at 4 °C for 15 min.
The resulting pellet, which now mainly contains neutrophile
granulocytes, is suspended in 2 ml of TRIS buffer (5.25 g
TRIS p.a., 2.7 g NaCl, H2O to 300.0 ml, pH 7.4).
inferred from the reduction of LTB4 formation in comparison
to a blank run without inhibitor. The positive control was
zilutron.
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C. Cusan et al. / IL FARMACO 60 (2005) 7–13
13
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