Synthesis and biological evaluation of a new class of acyl derivatives of 3-amino-1-phenyl-4,5-dihydro-1H-pyrazol-5-one as potential dual cyclooxygenase (COX-1 and COX-2) and human lipoxygenase (5-LOX) inhibitors

Article abstract of DOI:10.1016/j.farmac.2004.12.009

A series of acyl derivatives of 3-amino-1-phenyl-4,5-dihydro-1H-pyrazol-5- one as potential human 5-LOX and COX 1 and COX-2 inhibitors structurally related to the 1-phenyl-3-pyrazolidinone (phenidone, 1) have been synthesized and the activity against COX-

Full text of DOI:10.1016/j.farmac.2004.12.009

Il Farmaco 60 (2005) 327–332
http://france.elsevier.com/direct/FARMAC/
Synthesis and biological evaluation of a new class of acyl derivatives
of 3-amino-1-phenyl-4,5-dihydro-1H-pyrazol-5-one 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 Institute of Pharmaceutical Sciences, Department of Pharmacognosy, 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 11 November 2004; received in revised form 23 December 2004; accepted 27 December 2004
Available online 26 February 2005
Abstract
A series of acyl derivatives of 3-amino-1-phenyl-4,5-dihydro-1H-pyrazol-5-one as potential human 5-LOX and COX 1 and COX-
2 inhibitors structurally related to the 1-phenyl-3-pyrazolidinone (phenidone, 1) have been synthesized and the activity against COX-1,
COX-2 and human 5-LOX enzymes has been evaluated. All the derivatives showed poor activity against enzymes. These data, together with
our previous studies, indicated that phenidone and related compounds are not suitable as human 5-LOX inhibitors and that pyrazoline nucleus
should not be considered a good scaffold for inhibitors of human 5-LOX enzyme, suggesting the necessity to revisit the proposed mechanism
of action of phenidone (1) in human models.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Cyclooxygenase; Lipoxygenase; Dual inhibitors; Phenidone derivatives
1. Introduction
enteric tract and the kidney [2–5]. As a result, a new strategy
has been considered: the simultaneous inhibition of 5-LOX
and COX enzymes [6]. The simultaneous inhibition of COX
and LOX produces increased levels of arachidonic acid, and
this phenomenon induces tumor cell death through the acti-
vation of mitochondrial mechanisms such as by opening the
transition pore [7–9]. In fact, simultaneous administration of
COX-2 and 5-LOX inhibitors induces apoptosis in human
prostate tumor cell line (PC3) and mouse liver tumor cells
(MH1C1), permitting to consider these derivatives a new
appealing approach to cancer treatment.
Arachidonic acid (AA) is a polyunsaturated fatty acid
stored in cell membranes. After its release from phospholip-
ids, it is metabolized by two enzymatic families, the cyclooxy-
genases (COX-1, -2 and -3) and the lipoxygenases (5-, 8-,
12- and 15-LOX). These enzymes convert the arachidonic
acid to prostaglandines, prostacyclines, and leucotrienes,
which are involved in several physiological processes as
inflammation and cancer development [1].
In the last decades, many COX inhibitors have been widely
used for the treatment of inflammatory diseases. Moreover,
COX inhibition leads to an up-regulation of the 5-LOX path-
way, leading various adverse effects, especially in the gastro-
Recently, various chemical families of 5-LOX inhibitors
have been designed, and they have been classified depending
on their characteristics and mechanism of action. One of these
chemical classes includes antioxidant agents, generally small
lipophilic aromatic molecules, such as pyrazole derivatives
[6]. Phenidone (1) could be considered the prototype of this
* Corresponding author. Tel.: +39 040 5583110; fax: +39 040 52572.
E-mail address: daros@units.it (T. Da Ros).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.12.009

328
C. Cusan et al. / Il Farmaco 60 (2005) 327–332
family, and its inhibiting action of rat 5-LOX is well described
[10–13].
2. Results and discussion
The designed compounds 5a–i have been synthesized by
acylation under standard conditions (dioxane, reflux) of amine
3 with the appropriate acyl chlorides (when not commer-
cially available, the acyl chlorides were prepared from the
corresponding acid by treatment with thionyl chloride). Com-
pound 5l was obtained by hydrolysis of compound 5i by 1 M
NaOH in THF (Scheme 1).
All the compounds (3, 5a–l) have been tested in inhibition
assays of purified COX-1, COX-2 and human 5-LOX en-
zymes, using phenidone (1), amine 4, indometacine (COX
inhibitor), and Zileuton (5-LOX inhibitor) as internal con-
trols.
In our previous paper, we have described the activity of
phenidone (1) and some structurally related derivatives (2a–i)
in COX-1, COX-2 and human 5-LOX inhibition (Fig. 1) [14].
We have preliminarily observed that phenidone and some
structural related compounds could not be considered human
5-LOX inhibitors, but they still retain a quite good COX-
2 selective inhibition [14].
Nevertheless, due to the low number of previously reported
compounds, it is not still clear if this biological profile should
be attributed to the substitutions on the pyrazoline nucleus or
if the heterocycle itself could be considered responsible for
the inactivity versus the human 5-LOX. Moreover, if this
behavior should be confirmed, the mechanism of action of
phenidone and derivatives proposed for the rat 5-LOX inhi-
bition, based on redox process, would not be applicable in
the human model.
The compounds showing significant activity in inhibition
assay (3, 5h) are reported in Table 1. As clearly indicated, the
derivatives do not display any significant activity versus
human 5-LOX enzyme. As previously described, only the
To have more data for speculating on this biological pro-
file, we decided to synthesize and to study a new series of
compounds, and we selected the 3-amino-1-phenyl-4,5-
dihydro-pyrazol-5-one as lead compound (3), which is an iso-
mer of amine 4 used in our previous work (Fig. 2).
Amine 3 has been chosen because of its significant inhib-
iting activity versus enzymes involved in the arachidonic acid
pathway, as reported in the literature (15-LOX [IC50 = 6 µM],
COX [IC50 = 43 µM] and thromboxane synthetase
[IC50 = 71 µM]) [15], and, moreover, it is commercially avail-
able.
We prepared a new series of derivatives (see general for-
mula 5, Fig. 2) by acylation of amine 3, using a variety of
substituents with different steric and lipophilic properties, as
already described for amine 4.
Scheme 1. Synthesis of compounds 5a-l
Table 1
Percentage of inhibition of reference compounds and derivatives 3 and 5h
against COX-1, COX-2 and 5-LOX at 10 µM concentration a
Compound
COX-1 b
COX-2 c
5-LOX d
(% inhibition
at 10 µM)
(% inhibition
at 10 µM)
80.2 12
(% inhibition
at 10 µM)
Phenidone, 1
Amine 4
Indometacine
Zileuton
3
20.3
12
4
0
1
7
43.2
50
6
0
50
3
ND
ND
ND
50
0
4
Fig. 1
compounds.
. Phenidone structure and general formula of the previously reported
11.4 1.8
1.5 0.3
0.9 0.6
0
5h
0
ND: not determined. Values are reported as absolute percent inhibition of
enzyme and standard error of the mean (SEM).
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).
Fig. 2
5.
.
Structures of compounds 3 and 4 and general formula of compound
d For the test with 5-LOX, we have used the leukocytes suspension from
human blood of volunteers. n = 2–4 experiments in duplicate.

C. Cusan et al. / Il Farmaco 60 (2005) 327–332
329
unmodified derivative 3 presents a COX-1 inhibition activity
4.2. General procedure for the preparation of compounds
5a–l
comparable to the reference compound 4. In fact, the inhibi-
tion percentage of 3 toward COX-1 is 11%, while the activity
against COX-2 is significantly diminished with respect to ref-
erence compounds 1 and 4.
These results suggest that the shift of the double bond is
detrimental in terms of activity versus COX-2 while a slight
retention of action against COX-1 is conserved with respect
to amine 4. Also, in this case, there are not improvements in
the inhibition of human 5-LOX.
To a solution of amine (3) (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
(compound 5i), or by crystallization in diethyl ether/ethanol.
The compound 5l was obtained by basic hydrolysis (NaOH
1 M, 4 ml) of compound 5i (0.1 mmol), in THF (4 ml). The
solvent was removed under reduced pressure, then HCl (1 M,
4 ml) was added to the crude material. Derivative 5i was crys-
tallized by ethanol.
3. Conclusions
The present work reports a new investigation of pyrazo-
line analogs as potential dual inhibitors for COX and 5-LOX
enzymes. All the derivatives showed poor activity against
enzymes. Only unsubstituted derivative 3 showed poor activ-
ity against COX-1 while resulted to be totally inactive against
COX-2 and human 5-LOX.
These data suggest that phenidone and related compounds
are not suitable as human 5-LOX inhibitors and that pyrazo-
line nucleus should not be considered a good scaffold for
inhibitors of the arachidonic acid pathway, in particular for
the human 5-LOX enzyme. In addition, we could speculate
about the proposed mechanism of action of phenidone (1),
which most probably should be better clarified considering
human 5-LOX.
N-(5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-propio-
namide (5a)
C12H13N3O2 (231 MW); yield: 43%; white solid, m.p.:
217–219 °C; IR-DRIFT: cm–1 4416, 4313, 4089, 2977, 2674,
2556, 2389, 2150, 1965, 1879, 1711, 1633, 1499, 1359, 1177,
1123, 1071, 1021, 938, 701, 577, 506, 427; 1H NMR: d 10.35
(s, 1H), 7.68 (d, 2H, J = 7.6 Hz), 7.41 (t, 2H, J = 6.8 Hz), 7.18
(t, 1H, J = 7.4 Hz), 5.95 (s, 2H), 2.28 (q, 2H, J = 7.0 Hz), 1.02
(t, 3H, J = 7.6 Hz); 13C NMR: d 171.23, 168.02, 152.00,
147.00, 138.66, 128.76, 124.80, 120.22, 117.67, 80.57, 28.74,
9.58; EI-MS: m/z 231 (M+, 14%), 174 (M+—COC2H5, 60%),
+
91 (PhN+, 18%), 77 (Ph+, 63%), 57 (COC2H5 , 100%);
UV–Vis (EtOH): kmax 260, 199.
Pentanoic acid (5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazol-
3-yl)-amide (5b)
C14H17N3O2 (259 MW); yield: 67%; white solid, m.p.:
188–190 °C; IR-DRIFT: cm–1 3164, 3066, 2951, 2868, 1684,
1632, 1594, 1498, 1448, 1412, 1345, 1266, 1199, 1162, 1102,
973, 902, 754, 689, 642; 1H NMR: d 10.35 (s, 1H), 7.68 (d,
2H, J = 8.0 Hz), 7.41 (t, 2H, J = 7.7 Hz), 7.19 (t, 1H,
J = 7.3 Hz), 5.94 (s, 2H), 2.26 (t, 2H, J = 7.3), 1.52 (quin, 2H,
4. Experimental section
4.1. Chemistry
General Remarks: Reactions were routinely monitored by
thin-layer chromatography (TLC) on silica gel (precoated F254
Merck plates) and products visualized with iodine or aque-
ous potassium permanganate. Infrared spectra (IR) were mea-
sured on a Jasco FT-IR instrument using NaCl cells (oils) or
KBr powder (DRIFT system). UV-spectra were recorded on
J = 7.4 Hz), 1.33–1.21 (m, 2H), 0.87 (t, 3H, J = 7.3 Hz); 13
C
NMR: d 171.66, 170.55, 152.04, 147.00, 138.70, 128.79,
124.84, 120.28, 117.70, 80.62, 35.26, 27.17, 21.78, 13.76;
EI-MS: m/z 259 (M+, 25%), 174 (M+—COC4H9, 100%), 77
(Ph+, 38%), 57 (C4H9 , 46%); UV–Vis (EtOH): kmax 259,
+
a Perkin-Elmer Lambda 20 spectrophotometer. H and 13C
1
206, 198.
NMR were determined in DMSO-d6 solutions, unless other-
wise noted, at 200 and 50 MHz, respectively, with a Varian
Gemini 200 spectrometer, peak positions are given in parts
per million (d) downfield, and J values are given in Hz. All
the compounds have been crystallized by ethanol. Melting
points were determined on a Buchi-Tottoli instrument and
are uncorrected. EI-MS spectra were recorded on aVG7070 H
spectrometer using electron energy 70 eV. Chromatography
was performed with Merck 60-200 mesh silica gel. All prod-
ucts reported showed IR and NMR spectra in agreement with
the assigned structures. Organic solutions were dried over
anhydrous magnesium sulfate. All final compounds showed
a purity >99% checked by HPLC using reverse phase col-
umn (Luna 5 µ, C18(2) 250 × 4.60 mm), eluting with a mix-
ture of CH3CN (0.35 ml/min) and H2O (0.15 ml/min).
Heptanoic acid (5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazol-
3-yl)-amide (5c)
C16H21N3O2 (287 MW); yield: 63%; white solid, m.p.:
188–190 °C; IR-DRIFT: cm–1 3163, 3065, 2951, 2851, 1684,
1634, 1592, 1497, 1453, 1411, 1345, 1199, 1159, 1114, 976,
899, 754, 688, 644, 581, 505, 415; 1H NMR: d 10.35 (s, 1H),
7.69 (d, 2H, J = 8.0 Hz), 7.41 (t, 2H, J = 8.0 Hz), 7.19 (t, 1H,
J = 7.4 Hz), 5.94 (s, 2H), 2.26 (t, 2H, J = 7.1 Hz), 1.53 (quint,
2H, J = 7.1 Hz), 1.34–1.18 (m, 6H), 0.85 (t, 3H, J = 6.6 Hz);
13C NMR: d 170.53, 166.75, 152.18, 147.06, 138.70, 128.75,
124.76, 120.21, 117.75, 80.63, 35.54, 31.02, 28.29, 24.97,
21.99, 13.96; EI-MS: m/z 287 (M+, 55%), 174
+
(M+—COC6H13, 100%), 113 (COC6H13+, 12%), 85 (C6H13
,
18%), 77 (Ph+, 80%), 57 (C4H9 , 30%); UV–Vis (EtOH):
+
kmax 260, 201, 193.

330
C. Cusan et al. / Il Farmaco 60 (2005) 327–332
Decanoic acid (5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazol-
N-(5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-2-(2,4,6-
3-yl)-amide (5d)
tribromo-phenyl)-acetamide (5 h)
C17H12Br3N3O2 (530 MW); yield: 18%; white solid, m.p.:
255–257 °C; IR-DRIFT: cm–1 3248, 3072, 2934, 1768, 1659,
1572, 1536, 1503, 1482, 1437, 1413, 1389, 1327, 1203, 1143,
1108, 1017, 964, 922, 860, 816, 758, 731, 694; 1H NMR: d
10.85 (s, 1H), 8.03–7.97 (m, 3H), 7.49–7.33 (m, 4H), 6.60 (s,
2H), 4.08 (s, 2H); 13C NMR: d 165.87, 164.58, 149.63,
142.63, 136.96, 134.46, 134.33, 133.91, 132.17, 129.34,
127.09, 126.53, 126.12, 122.22, 122.15, 121.13, 88.72, 43.03,
41.55; EI-MS: m/z 530 (M+, 6%), 356 (Br3PhCH2CO+, 18%),
328 (Br3PhCH2+, 12%), 249 (Br2PhCH2+, 40%), 174
(M+—Br3PhCH2CO 100%), 77 (Ph+, 31%); UV–Vis (EtOH):
kmax 265, 225, 213, 194.
C19H27N3O2 (329 MW); yield: 52%; white solid, m.p.:
168–170 °C; IR-DRIFT: cm–1 3162, 3068, 2920, 2854, 2637,
1687, 1634, 1494, 1409, 1349, 1201, 1159, 1115, 971, 895,
749, 687, 647, 573, 503; 1H NMR: d 10.31 (s, 1H), 7.66 (d,
2H, J = 8.1 Hz), 7.38 (t, 2H, J = 7.9 Hz), 7.16 (t, 1H,
J = 7.3 Hz), 5.92 (s, 2H), 2.23 (t, 2H, J = 7.2 Hz), 1.62–1.41
(m, 2H), 1.31–1.15 (m, 12H), 0.82 (t, 3H, J = 5.9 Hz); 13C
NMR: d 170.55, 168.02, 152.03, 147.00, 138.68, 128.78,
124.83, 120.26, 117.69, 80.61, 35.54, 31.30, 28.90, 28.80,
28.70, 28.63, 25.02, 22.13, 13.99; EI-MS: m/z 329 (M+, 80%),
174 (M+—COC9H19, 100%), 155 (COC9H19+, 8%), 85
+
(C6H13+, 22%), 77 (Ph+, 82%), 55 (C4H7 , 80%); UV–Vis
N-(5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-succi-
namic acid methyl ester (5i)
(EtOH): kmax 261, 204.
N-(5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-benza-
mide (5e) [16]
C14H15N3O4 (289 MW); yield: 61% (chromatography elu-
ent: petroleum ether/ethyl acetate, 7:3); white solid, m.p.: 199–
200 °C; IR-DRIFT: cm–1 3166, 3080, 2967, 1717, 1681, 1627,
1500, 1438, 1351, 1228, 1160, 1061, 994, 958, 912, 844, 760,
712, 653, 585, 504, 479; 1H NMR: d 10.47 (s, 1H), 7.69 (d,
2H, J = 7.5 Hz), 7.42 (t, 2H, J = 7.7 Hz), 7.20 (t, 1H,
J = 7.4 Hz), 5.91 (s, 2H), 3.58 (s, 3H), 2.56 (s, 4H); 13C NMR:
d 172.70, 169.09, 152.07, 146.87, 138.67, 128.81, 124.87,
120.29, 117.71, 80.53, 51.36, 30.13, 28.35; EI-MS: m/z
289 (M+, 17%), 258 (M+—OCH3, 100%), 202
(M+—C2H4COOCH3, 8%), 176 (M+—C2OCH2COOCH3,
82%), 91 (PhN+, 67%), 77 (Ph+, 88%), 56 (C2H4CO+, 85%);
UV–Vis (EtOH): kmax 260, 202.
C16H13N3O2 (279 MW); yield: 75%; white solid, m.p.:
209–220 °C; IR-DRIFT: cm–1 4316, 4064, 3937, 3311, 3067,
2973, 2724, 2542, 2291, 2144, 1963, 1681, 1615, 1478, 1350,
1257, 1199, 1112, 1024, 920, 803, 758, 699, 622, 503, 412;
1H NMR: d 10.87 (s, 1H), 8.00 (d, 2H, J = 6.5 Hz), 7.73 (d,
2H, J = 8.7 Hz), 7.60–7.36 (m, 5H), 7.20 (t, 1H, J = 7.3 Hz),
6.11 (s, 2H); 13C NMR: d 169.24, 165.42, 152.78, 147.67,
139.24, 138.72, 134.48, 133.29, 132.45, 129.61, 129.55,
129.24, 129.08, 128.78, 128.41, 125.99, 125.13, 121.42,
118.84, 82.93; EI-MS: m/z 279 (M+, 20%), 105 (PhCO+,
100%), 77 (Ph+, 58%); UV–Vis (EtOH): kmax 274, 200.
N-(5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-2-
phenyl-acetamide (5f)
N-(5-Oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-succi-
namic acid (5 l)
C13H13N3O4 (275 MW); yield: 92%; white solid, m.p.:
C17H15N3O2 (293 MW); yield: 63%; white solid, m.p.:
221–223 °C; IR-DRIFT: cm–1 3068, 2965, 1675, 1625, 1493,
1441, 1353, 1257, 1200, 1127, 1031, 989, 898, 807, 728, 690,
242–243 °C; IR-DRIFT: cm–1 3168, 3086, 2980, 1696, 1627,
1
1495, 1431, 1341, 1163, 919, 758, 684, 625, 576, 514; H
NMR: d 11.97 (bs, 1H), 10.44 (s, 1H), 7.68 (d, 2H,
J = 12.1 Hz), 7.40 (t, 2H, J = 7.1 Hz), 7.18 (t, 1H, J = 7.7 Hz),
5.92 (s, 2H), 2.51 (s, 4H); 13C NMR: d 173.75, 169.42, 152.22,
147.02, 138.73, 128.84, 124.88, 120.30, 117.78, 80.62, 30.35,
28.70; EI-MS: m/z 257 (M+—H2O, 88%), 202
(M+—C2H4COOH, 12%), 174 (M+—COC2H4COOH, 30%),
1
580, 512, 423; H NMR: d 10.69 (s, 1H), 7.68 (d, 2H,
J = 8.0 Hz), 7.41 (t, 2H, J = 8.0 Hz), 7.39-7.06 (m, 6H), 5.92
(s, 2H), 3.66 (s, 2H); 13C NMR: d 168.39, 152.12, 146.93,
138.65, 135.92, 129.09, 128.84, 128.22, 126.45, 124.95,
120.34, 117.73, 80.53, 42.59; EI-MS: m/z 293 (M+, 53%),
202 (M+—CH2Ph, 7%), 174 (M+—COCH2Ph, 100%), 91
+
158 (M+—NH2COC2H4COOH, 14%), 105 (PhN2 , 30%), 91
+
(PhCH2 , 80%), 77 (Ph+, 23%); UV–Vis (EtOH): kmax 262,
(PhN+, 78%), 77 (Ph+, 80%), 56 (C2H4CO+, 100%); UV–Vis
201, 195.
(EtOH): kmax 259, 202.
2-(2-Bromo-phenyl)-N-(5-Oxo-1-phenyl-4,5-dihydro-1H-
pyrazol-3-yl)-acetamide (5 g)
4.3. Biology
C17H14BrN3O2 (372 MW); yield: 53%; white solid, m.p.:
191–193 °C; IR-DRIFT: cm–1 3066, 2964, 1947, 1682, 1628,
1491, 1434, 1350, 1234, 1139, 1026, 985, 901, 791, 745, 692,
4.3.1. 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
[22], 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 (por-
cine, ICN, Aurora, Ohio, USA), 18 mM epinephrin-
hydrogentartrate (Fluka, Buchs, Switzerland), 0.2 U of
1
577, 501, 430; H NMR: d 10.73 (s, 1H), 7.69 (d, 2H,
J = 8.1 Hz), 7.59 (d, 1H, J = 8.0 Hz), 7.51–7.31 (m, 4H), 7.20
(t, 2H, J = 7.3), 5.91 (s, 2H), 3.81 (s, 2H); 13C NMR: d 167.16,
152.16, 146.91, 138.67, 135.63, 132.22, 132.17, 128.83,
128.69, 127.51, 124.92, 124.51, 120.32, 80.53, 42.51; EI-MS:
m/z 372 (M+, 8%), 174 (M+—COCH2PhBr, 100%), 170
(BrCH2Ph+, 16%), 77 (Ph+, 27%); UV–Vis (EtOH): kmax 261,
209, 206, 196.

C. Cusan et al. / Il Farmaco 60 (2005) 327–332
331
enzyme preparation and 50 µM Na2EDTA (only COX-
2 assay, Titriplex III, Merck, Darmstadt, Germany).After add-
ing 10 µl of synthesized compound dissolved in EtOH p.a. or
DMSO for compound 5h (final concentration 10 µM) or 10 µl
of EtOH p.a. in control wells, the system was preincubated
for 5 min at room temperature.
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).
microscope at 1000× magnification. Dead cells appear larger
and dark because of the absorption of Trypan blue solution,
whereas vital cells remain smaller, lighter and more granu-
lose. The vitality of the cells must be over 90%.
Leukocytes concentration was determined using cell
diluted suspension distributed into a Neubauer chamber
(Assistant, Germany) and a light microscope with 100× mag-
nification.
For the bioassay, the cell concentration must be
5000 cells/µL with TRIS buffer.
4.5. The 5-LOX bioassay
4.3.2. 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.
The EIA evaluation was performed with an ELISA reader
″rainbow″ (TECAN) and determined as elsewhere described
[22]. 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).
The assay was performed in the 96 microwell titre plate
(Bibby Sterilin, Staffordshire, UK). The incubation mixture
consists of 225 µl of leukocytes suspension (5000 cells/µL),
10 µl CaCl2 2 mM, 10 µl eicosatetraenoic acid 10 µM as
12-LOX pathway inhibitor, 5 µl of synthesized compound dis-
solved in EtOH p.a. (DMSO for compound 5h) to the final
concentration of 10 µM or 5 µl of EtOH p.a. in control wells.
The addition of Ca Ionophore A23187 (10 µl, 17 µM) and
5 µl of 120 µM arachidonic acid (Cayman Chemical Com-
pany) triggers the enzymatic conversion of arachidonic acid
to LTB4 metabolite. After incubation for 10 min at 37 °C in
darkness, the reaction is stopped by addition of 20 µl 10%
HCOOH. The microplate is centrifuged for 15 min at
1400 rpm to separate the free LTB4 from cellular particles.
The supernatant is then diluted 50-fold with TRIS buffer.
4.4. 5-LOX inhibition assays
4.4.1. Isolation of human neutrophile granulocytes
Human blood (30 ml) from volunteers is collected with
VacutainerTM 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
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).
4.6. Determination of LTB4 by enzyme immunoassay
The concentration of LTB4 was measured utilizing a com-
petitive enzyme immunoassay LTB4-EIA-kit (Cayman
Chemical Company, Ann Arbor, USA) used according to the
company’s instruction.
The EIA evaluation was performed with a photometric
ELISA plate reader (TECAN) at k = 412 nm. Inhibition was
inferred from the reduction of LTB4 formation in comparison
to a blank run without inhibitor. The positive control was
Zileuton.
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