1-Methyl and 1-(2-hydroxyalkyl)-5-(3-alkyl/cycloalkyl/phenyl/naphthylureido)-1H-pyrazole-4-carboxylic acid ethyl esters as potent human neutrophil chemotaxis inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.03.035

In this paper we report the synthesis and the chemotaxis inhibitory activity of a number of 1H-pyrazole-4-carboxylic acid ethyl esters 2 functionalized in N1 with a methyl group or different hydroxyalkyl chains and in position 5 with a series of 3-substituted urea groups. These compounds were designed as development of previous pyrazole-urea derivatives that resulted potent IL8-induced neutrophil chemotaxis inhibitors in vitro. Most of the new compounds revealed a potent inhibition of both IL8- and fMLP-OMe-stimulated neutrophil chemotaxis. The most active compounds in the fMLP-OMe induced chemotaxis test showed IC₅₀ in the range 0.19 nM-2 μM; but we observed a very strong inhibition in the IL8-induced chemotaxis test, having the most active compounds IC₅₀ at pM concentrations. In vivo compounds 2e and 2f, although to a lesser extent, at 50 mg/kg os decreased granulocyte infiltration in zymosan-induced peritonitis in mice.

Full text of DOI:10.1016/j.bmc.2009.03.035

Bioorganic & Medicinal Chemistry 17 (2009) 3379–3387
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1-Methyl and 1-(2-hydroxyalkyl)-5-(3-alkyl/cycloalkyl/phenyl/naphthylureido)-
1H-pyrazole-4-carboxylic acid ethyl esters as potent human
neutrophil chemotaxis inhibitors
a
a
a
b
Olga Bruno a, , Chiara Brullo , Francesco Bondavalli , Silvia Schenone , Susanna Spisani ,
Maria Sofia Falzarano b, Katia Varani c, Elisabetta Barocelli d, Vigilio Ballabeni d, Carmine Giorgio d,
Massimiliano Tognolini d
*
a Dipartimento di Scienze Farmaceutiche, Università di Genova, Viale Benedetto XV, 3, 16132 Genova, Italy
b Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Ferrara, via Luigi Borsari, 46, 44100 Ferrara, Italy
c Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Ferrara, via Fossato di Mortara 17/19, 44100 Ferrara, Italy
d Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Università degli Studi di Parma, V.le GP Usberti, 27/A, 43100 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper we report the synthesis and the chemotaxis inhibitory activity of a number of 1H-pyrazole-
4-carboxylic acid ethyl esters 2 functionalized in N1 with a methyl group or different hydroxyalkyl chains
and in position 5 with a series of 3-substituted urea groups. These compounds were designed as devel-
opment of previous pyrazole-urea derivatives that resulted potent IL8-induced neutrophil chemotaxis
inhibitors in vitro. Most of the new compounds revealed a potent inhibition of both IL8- and fMLP-
OMe-stimulated neutrophil chemotaxis. The most active compounds in the fMLP-OMe induced chemo-
Received 14 November 2008
Revised 13 March 2009
Accepted 20 March 2009
Available online 25 March 2009
Keywords:
taxis test showed IC50 in the range 0.19 nM–2 lM; but we observed a very strong inhibition in the
1-(2-Hydroxyalkyl)-5-(3-alkyl/benzyl/-
phenyl/naphthylureido)-1H-pyrazole-4-
carboxylic acid ethyl esters
Neutrophil chemotaxis inhibitors
IL8/CXCL8 (Interleukin 8)
fMLP-OMe
IL8-induced chemotaxis test, having the most active compounds IC50 at pM concentrations. In vivo com-
pounds 2e and 2f, although to a lesser extent, at 50 mg/kg os decreased granulocyte infiltration in zymo-
san-induced peritonitis in mice.
Ó 2009 Elsevier Ltd. All rights reserved.
N-Formyl-methionyl-leucyl-phenylalanine
methyl ester
Zymosan-induced peritonitis
1. Introduction
In the last years the search for new small molecules able to
interfere with numerous and different mechanisms involved in
Neutrophils are the main cells infiltrated in the course of acute
inflammation. Their activation consists in subsequent events: mar-
gination, diapedesis and migration (chemotaxis) to site of inflam-
mation where they take part in the phagocytosis of foreign
particles and reactive free radicals production.1,2 Chemotaxis is a
very complex multi-step process induced by various chemoattrac-
tans as N-formyl-methionyl-leucyl-phenylalanine (fMLP),3,4 plate-
let activating factor (PAF),5 leukotriene B4 (LTB4),6 C5a
anaphylotoxin,7 and different chemokines such as the CC chemo-
kine MIP 1b (macrophage inhibition factor), or the CXC chemokine
interleukine 8 (IL8, also named CXCL8).8
neutrophil upregulation greatly engaged many medicinal chemis-
try researchers.
In this context, we recently reported many pyrazoles (com-
pounds 1, Fig. 1) which resulted active as IL8-induced chemotaxis
inhibitors in vitro.9 These compounds, obtained by functionaliza-
tion with a urea moiety of the 5-amino-1-(2-hydroxy-2-phenyl-
ethyl)-1H-pyrazole-4-carboxylic acid ethyl ester, blocked the IL8-
When the control of cellular recruitment breaks down, both
acute and chronic autoimmune inflammatory disorders (e.g., asth-
ma, rheumatoid arthritis, multiple sclerosis) will ensue.
* Corresponding author. Tel.: +39 010 353 8367; fax: +39 010 353 8358.
E-mail address: obruno@unige.it (O. Bruno).
Figure 1. Chemical structure of compounds 1.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.035

3380
O. Bruno et al. / Bioorg. Med. Chem. 17 (2009) 3379–3387
induced neutrophil chemotaxis, while they did not block fMLP-
mediated chemotaxis. The most active compounds were 3-ben-
zyl-, 3-(4-benzylpiperazinyl)-, 3-phenyl- and 3-isopropyl-ureido
derivatives, which showed an IC50 of 10, 14, 45 and 55 nM, respec-
tively. They were inactive in the binding assays on CXCR1 and
CXCR2 (IL8 receptors), whereas they inhibited the phosphorylation
of PTKs (Protein Tyrosine Kinases) in the 50–70 kDa region. More-
over, in the presence of the same derivatives, we observed a com-
plete block of F-actin rise and pseudopod formation. The results of
a preliminary in vivo study of these compounds and some SAR
(structure–activity relationship) considerations suggested a rela-
tion between anti-chemotaxis activity and molecular lipophilicity.
To acquire further insight into the SAR of the pyrazolyl-ureas
we synthesized a new series of 4-carboxyethyl-pyrazole deriva-
tives (compounds 2a–u, Fig. 2) substituted in N1 with more or less
lipophilic chains, keeping constant the urea moieties in position 5
of the pyrazole scaffold which have given the best results in a pre-
vious series. Among compounds 1 the 3-fluorophenyl derivative
showed a good anti-inflammatory activity in vivo, therefore we
prepared now also some naphthyl-ureido derivatives as fluoro-
phenyl bioisosteres and we tested some of the most active com-
pounds for their anti-inflammatory activity in intraperitoneal
zymosan-induced peritonitis in mice.
esters 4a–e. Title compounds (2a–u) were then synthesized start-
ing from compounds 4a–e following Method A or Method B
(Scheme 1).
In the Method A the pyrazoles 4a–d were preliminarily treated
with phosgene in anhydrous THF and, later, with suitable amines
yielding compounds 2b–d, i–k, p–u. In the Method B compounds
4b–e reacted with a little excess of proper isocyanate in anhydrous
toluene, at reflux for 6 h, to give derivatives 2a, 2e–h and 2l–o. IR
and 1H NMR spectral data confirmed the urea structure and ex-
cluded the involvement of the hydroxy group.
3. Biological results
Release of the cytoplasmic enzyme LDH was used as an indica-
tor of cell viability. In none of the experiments described below
(stimulated, unstimulated or compounds-treated neutrophils)
was the percentage of total LDH release >3% (data not shown).
All compounds were tested in in vitro chemotaxis assays, in hu-
man neutrophils stimulated by 10 nM fMLP-OMe and 1 nM IL8 fol-
lowing the methods already reported.10 Molecules were added to
neutrophils 10 min before the incubation step for chemotaxis.
The data of antagonism (percentage of activity) were obtained by
comparing the chemotactic index (C.I.), in the absence and in the
presence of derivatives.
2. Chemistry
In Figures 3 and 4 we reported the dose–response curves of the
most active compounds toward the fMLP-OMe and IL8-induced
chemotaxis, respectively. The potency of the novel antagonists
was calculated through IC50 values, which represent the concentra-
tion able to inhibit the 50% of the fMLP-OMe- or IL8-induced che-
motaxis. The experimental data represented by the percentage of
the chemotaxis reduction were analysed through the computer
The condensation of ethyl (ethoxymethylene)cyanoacetate with
commercial hydrazines (namely, methylhydrazine 3a) or with
hydrazines 3b–e, in its turn obtained from proper oxyranes with
hydrate hydrazine, gives the intermediates 5-amino-1-methyl- or
5-amino-1-(2-hydroxyalkyl)-1H-pyrazole-4-carboxylic acid ethyl
O
O
O
R'N
N
N
N
H
R
HO
CH
HO
CH
HO
CH
HO
CH
2
2
2
2
R =
R =
R =
R =
NR’
R = CH
3
C H
CH
C H
C H
3 7
6
5
3
2
5
NH
2b
2i
2p
2s
2t
2c
2d
2e
2j
2k
2l
2q
2r
H
C NH
2
2u
H
C
F
N
N
2
NH
NH
F
2f
2m
2g
2h
2n
2o
F
NH
HN
2a
Figure 2. Chemical structure of compounds 2a–u.

O. Bruno et al. / Bioorg. Med. Chem. 17 (2009) 3379–3387
3381
Scheme 1. Reagents and conditions: (a) 15 min, 70–100 °C; (b) (ethoxymethylene)cyanoacetate, anhydrous toluene or absolute ethanol, reflux, 8–12 h. Method A: (1)
anhydrous THF cooled at 0 °C, anhydrous CH3COONa, phosgene (20% in toluene), reflux, 4 h; (2) anhydrous THF, amines added at 0 °C, rt overnight. Method B: isocyanate,
anhydrous toluene, reflux, 6 h.
(Table 1). In addition, IC50 values were compared with positive
(cyclosporine A, CSA) and negative (DMSO, blank) controls.
The most active compounds toward chemotaxis (2c, 2d, 2e, 2f,
2h, 2k, 2m and 2q) were selected to evaluate their anti-inflamma-
tory effect in a mice model of zymosan-induced peritonitis. The
study showed that compounds 2e and 2f (50 mg/kg os) were able
to reduce granulocyte infiltration in the peritoneal exudates of
34 5% (P < 0.05), and 21 4%, respectively. The reference drug
Dexamethasone (3 mg/kg os) reduced infiltration of 35 5%
(P < 0.05). Data for active compounds are reported in Table 2.
4. Discussion
All tested compounds inhibited the fMLP-OMe- and the IL8-in-
Figure 3. Dose responses curve of compounds 2a, 2h, 2m, 2n, 2q, 2r in neutrophil
duced chemotaxis at very low concentrations. With respect to the
fMLP-OMe-stimulated chemotaxis. Data are expressed as a percentage of the C.I.
previous compounds (1) we remark a notable increase of activity
(Chemotactic Index). SEs are within 10% of the mean value.
for all the new pyrazolyl-ureas, particularly for those having a
hydroxypropyl chain in N1 (see compounds 2b–h). In fact, the
most active compounds in the series 1 had IC50 ranging from 10
to 33 nM toward IL8-induced chemotaxis, while in this new series
the most active compounds (2b–h and 2k) showed IC50 inferior to
1 pM concentration; anyway, all the other compounds were very
active, their IC50 ranging from 1.6 pM (compound 2l) to 9.6 nM
(compound 2o).
In addition, while compounds 1 were completely inactive in the
fMLP-OMe-induced chemotaxis, most part of compounds
showed relevant inhibitory activity, the IC50 ranging from
0.19 nM (compound 2m) to 2 M (compound 2k); only few com-
pounds (2d,f,g,i,j) showed low inhibition percent at 10 M concen-
tration, thereafter their IC50 values were not calculated.
2
l
l
We can also remark that the inhibitory activity toward the IL8-in-
duced chemotaxis is more than thousand times greater than exhib-
ited toward fMLP-OMe-induced cell recruitment. Interestingly,
some of the most active compounds in the IL8-induced chemotaxis
test (2d–g, 2i–k) were the less active in the fMLP-OMe-induced chal-
lenge. This evidence prompted us to hypothesize that compounds 2
differently affect chemotaxis activated by fMLP-OMe or IL8.
Figure 4. Dose responses curve of compounds 2b, 2c, 2d, 2e, 2f, 2h, 2k in
neutrophil IL8-stimulated chemotaxis. Data are expressed as a percentage of the C.I.
(Chemotactic Index). SEs are within 10% of the mean value.
analysis by using the non-linear regression curve fitting computer
program Graph Pad Prism (Graph Pad, San Diego, CA, USA)

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O. Bruno et al. / Bioorg. Med. Chem. 17 (2009) 3379–3387
Table 1
Potency expressed as IC50 values of the novel compounds 2a–u in the chemotaxis inhibition of human neutrophils
O
O
O
R'N
N
N
N
R
H
Compound
R
NR0
Inhibition of fMLP-OMe-induced
neutrophil migration IC50 (nM)a
Inhibition of IL8-induced neutrophil
migration IC50 (pM)a
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
CH2–CHOH–C6H5
CH2–CHOH–CH3
CH2–CHOH–CH3
CH2–CHOH–CH3
CH2–CHOH–CH3
CH2–CHOH–CH3
CH2–CHOH–CH3
CH2–CHOH–CH3
CH2–CHOH–C2H5
CH2–CHOH–C2H5
CH2–CHOH–C2H5
CH2–CHOH–C2H5
CH2–CHOH–C2H5
CH2–CHOH–C2H5
CH2–CHOH–C2H5
CH2–CHOH–C3H7
CH2–CHOH–C3H7
CH2–CHOH–C3H7
CH3
a
-Naphthylamino
7.8 0.52
90 9.3
15 1.1
7.60 1.0
0.82 0.07
0.54 0.06
0.99 0.08
0.55 0.01
0.70 0.01
200 60
0.97 0.06
4.2 0.7
7.4 0.2
0.08 0.003
1.6 0.2
360 60
1,900 130
9,600 1,000
1,000 50
3.2 0.1
Isopropylamino
Benzylamino
N-Benzylpiperazino
o-F-anilino
m-F-anilino
p-F-anilino
>10
lM (63%)
100 10.3
>10
>10
l
l
M (60%)
M (55%)
a
-Naphthylamino
9.8 0.90
Isopropylamino
Benzylamino
N-Benzylpiperazino
o-F-anilino
m-F-anilino
p-F-anilino
>10
>10
l
l
M (52%)
M (54%)
2000 200
120 12
0.19 0.01
3.2 0.40
12 1.2
a
-Naphthylamino
Isopropylamino
Benzylamino
N-Benzylpiperazino
Isopropylamino
Benzylamino
20 2.2
0.43 0.05
1.1 0.10
77 7.7
90
4
2s
2t
2u
CSA
DMSO
1.8 0.5
1300 110
54.6 0.5
CH3
CH3
18 1.6
N-Benzylpiperazino
150 14.2
0.047 0.006
>10,000
a
The values are the means SEM of six independent experiments. IC50 values obtained from functional experiments are the concentration of tested compounds that
inhibits the 50% of the maximal effect; the percentage in the parentheses represents the % of the reduction in neutrophil migration by the novel compounds at 10
concentration.
lM
Table 2
In vivo anti-inflammatory activity of compounds 2e and 2f on zymosan-induced peritonitis in mice
Relative composition
Treatments
Protein conc. (
l
g/mL)
Cells (millions/cavity)
Granulocytes (millions/cavity)
Granulo cytes (%)
Mono/lympho cytes (%)
Saline
Zymosan
0.63 0.07
2.65 0.18
1.75 0.13**
2.62 0.27
2.68 0.39
5.5 0.8
1.8 0.3
33
86
81
3
1
2
67
14
20
3
1
2
31.6 2.3
21.8 1.8**
24.3 2.1*
28.1 3.1
27.2 2.3
17.6 1.6**
18.0 2.2**
21.6 2.2
Zymosan + Dexamethasone 3 mg/kg os
Zymosan + 2e 50 mg/kg os
Zymosan + 2f 50 mg/kg os
74 4**
77 2**
26 4**
23 3**
*p < 0.05 **p < 0.01 by student’s t-test compared with zymosan treated mice. All the data of zymosan group are significantly different (p < 0.001) when compared with saline
group. Animals received the compounds under study or dexamethasone 1 h before intraperitoneal injection of 1 mg zymosan. Peritoneal exudates were collected 4 h later and
analyzed. Data for inactive compounds (2c, 2d, 2h, 2k, 2m, 2q) are not reported.
As concerns the structure–activity relationship we can under-
line that the presence of hydroxypropylchain in N1 gave the most
active compounds in IL8-induced chemotaxis test (2c,d,e,f,h): its
very strong activity seems independent of the urea moiety substit-
uents. In this sub-group compound 2g (NR0 = p-F-anilino) is the
sole exception having an IC50 of 200 pM.
The increase of chain length and lipophilicity causes, in general, a
proportional decrease of chemotaxis inhibition. Compound 2k
(hydroxybutyl chain in N1 and NR0 = N-benzylpiperazino) is an
exception being the most active with an IC50 of 0.08 pM. Also the
presence of a simple methyl group in N1 (see compounds 2s–u) is
detrimental for the action. No clear correlation has been found from
the urea substituents and activity, except for the fluoroanilino deriv-
atives which were always active in the order ortho > meta > para (see
compounds 2e–g and 2l–n). Moreover, it is worthy to note that the
introduction of a a-naphthyl-ureido group gave active compounds,
also in the presence of hydroxyphenylethyl chain (incidentally, the
chain present in the previous compounds 1) in N1 (compound 2a).
In the fMLP-OMe-induced chemotaxis compounds 2m (hydrox-
ybutyl chain, NR0 = m-F-anilino) and 2q (hydroxypentyl chain,
NR0 = benzylamino) were the most active agents with IC50 of 0.19
and 0.43 nM, respectively. The other results pointed out only the
detrimental effect of hydroxypropyl chain in N1, while it is difficult
to correlate the activity with the urea substituents.
To study the in vivo anti-inflammatory effect of the most attrac-
tive compounds we chose the zymosan-induced peritonitis test, an
experimental model of inflammation characterized by an early
phase (4 h) with a considerable infiltration of granulocytes at the
inflammation site and a late phase with mononuclear and lympho-
cyte number increase during progression of the process (24 h). The

O. Bruno et al. / Bioorg. Med. Chem. 17 (2009) 3379–3387
3383
analysis of mice peritoneal exudates taken 4 h after zymosan injec-
tion revealed a significant increase of protein concentration, cells
and granulocyte infiltration in comparison with saline-treated ani-
mals. Dexamethasone (3 mg/kg os) decreases the cell number, and
in particular granulocyte infiltration, due to the inhibition of che-
mokine production whereas it is known that NSAIDs like indo-
methacin (10 mg/kg os) are completely inactive because of the
poor participation of arachidonic acid metabolites in this process.11
(Table 2)
In the previous study on group 1 compounds, the 3-fluoroanili-
no derivative evidenced a good anti-inflammatory activity, causing
a 33% inhibition of granulocyte recruitment at 100 mg/kg os. In the
present work, most of the tested compounds (2c, 2d, 2h, 2k, 2m,
2q) did not exhibit anti-inflammatory activity leaving unmodified
protein content, cells and granulocytes present in the peritoneal
cavity lavage of zymosan-treated mice. Also compounds 2e and
2f (ortho- and meta-fluoroanilino derivatives) (50 mg/kg os) did
not modify pleural fluid protein content at variance with dexa-
methasone, but they reduced relative and absolute granulocyte
infiltration. In particular compound 2e showed a higher potency
than compound 2f.
cellular signalling triggered by different chemotaxines, is of great
importance in models of inflammation and these considerations
represent the rational base and stimulate further studies for the
design of new anti-inflammatory agents.
6. Experimental protocols
6.1. Chemistry
6.1.1. General
Chiminord and Aldrich Chemical, Milan, Italy purchased all
chemicals. Solvents were reagent grade. THF was dried by filtration
on neutral Al2O3 column and stored on molecular sieves (5 Å 1/
1600 pellets). Toluene was stored in the presence of sodium. Un-
less otherwise stated, all commercial reagents were used without
further purification. Organic solutions were dried over anhydrous
magnesium sulphate.
Aluminium backed silica gel plates (Merck DC-Alufolien Kiesel-
gel 60 F254) were used in thin-layer chromatography (TLC) for rou-
tine monitoring the course of reactions. Detection of spots was
made by UV light. Merck silica gel, 230–400 mesh, was used for
chromatography.
Melting points are not ‘corrected’ and were measured with a
Büchi 540 instrument. IR spectra were recorded with a Perkin–El-
mer 398 spectrophotometer. 1H NMR spectra were recorded on a
Varian Gemini 200 (200 MHz) instrument; chemical shifts are re-
ported as d (ppm) relative to tetramethylsilane (TMS) as internal
standard; signals were characterized as s (singlet), d (doublet), t
(triplet), q (quartet), sept (septet) m (multiplet), br s (broad signal);
J in Hertz. Elemental analyses, indicated by the symbols of the ele-
ments were within 0.4% of the theoretical values and were deter-
mined with an Elemental Analyzer EA 1110 (Fison-Instruments,
Milan, Italy).
5. Conclusions
The structural modification of previously reported pyrazolyl-ur-
eas 1 gave a new series of 1-methyl- and 1-(2-hydroxyalkyl)-5-(3-
alkyl/cycloalkyl/phenyl/naphthylureido)-1H-pyrazole-4-carbox-
ylic acid ethyl esters 2a–u that resulted potent inhibitors of human
neutrophil chemotaxis both fMLP-OMe- and IL8-induced. Particu-
larly, compounds having a hydroxypropyl chain in N1 showed a
very strong inhibitory activity toward IL8. Among them, the 5-[3-
(2-fluorophenyl)ureido]- and 5-[3-(3-fluorophenyl)ureido]-deriva-
tives (2e and to a lesser extent 2f) reduced granulocyte migration
in mice peritoneal exudates caused by intraperitoneal zymosan
application.
The SAR study evidenced a good correlation between chemo-
taxis inhibitory activity and length and lipophilicity of N1 chain,
while the in vitro activity not strictly depends on the nature of urea
group. Only the 2-fluoroanilino and 3-fluoroanilino derivatives
showed anti-inflammatory activity in vivo, in the mouse model
of zymosan-induced peritonitis, confirming the results of the pre-
vious compounds 1.
6.1.2. Syntheses of 1-hydrazino-alkan-2-ols 3b–e
1-Hydrazinopropan-2-ol 3b and 1-hydrazinobutan-2-ol 3c
were synthesized following the method already reported.17 2-
Hydrazino-2-phenylethanol 3e was synthesized following the
method already reported.18
6.1.2.1. Synthesis of 1-hydrazinopentan-2-ol 3d. To hydrazine
hydrate (30 mL, 0.6 mol) heated at 60–70 °C, 1,2-epoxypentane
(14.64 g, 0.17 mol) was added drop wise and the mixture was
heated at 90–95 °C for 1 h. Then, the excess of hydrazine hydrate
was removed under reduced pressure and the crude was distilled
bulb to bulb to give a viscous oil that becomes wax solid.
Yield 82%, bp 120 °C/0.6 mmHg.
Members of the CXC and FPR (formyl peptide) receptor fami-
lies all induce multiple neutrophil functions, including cell migra-
tion, granule secretion and superoxide anion generation. As all
belong to the pertussis toxin-sensitive GPCR superfamily, it has
been generally accepted that they possess many signalling simi-
larities. However, we have previously reported that neutrophils
chemotaxis, activated by fMLP and pure chemoattractants, is al-
most insensitive to any variation of Ca++ concentration and this
could be related to its poor efficacy in enhancing cAMP level.12,13
Moreover, more recently, it has been demonstrated that the che-
motactic response is associated with specific PKC b1 isoform
translocation and p38 mitogen-activated protein kinase phos-
phorylation by two independent pathways.14 Heit and co-work-
6.1.3. General procedure for 5-amino-1-(2-hydroxyalkyl)-1H-
pyrazole-4-carboxylic acid ethyl esters 4b,c,d
To a solution of the proper 1-hydrazino-alcohol (3b, 3c, 3d)
(20 mmol) in anhydrous toluene (for 3b) or in absolute ethanol
(for 3c and 3d) (20 mL), ethyl(ethoxymethylene)cyanoacetate
(3.38 g, 20 mmol) was added and the mixture was heated at 70–
80 °C for 8 h. After cooling, the toluene was washed with H2O
(20 mL), dried (MgSO4) and concentrated under reduced pressure
(compound 4b). Whereas, the ethanol solutions were concentrated
under reduced pressure and cooled to room temperature to give
yellow solids (compounds 4c and 4d). The crudes were purified
by flash-chromatography using a mixture of CH2Cl2/CH3OH (8:2)
as eluent and then were crystallized by adding diethyl ether. The
solids obtained were recrystallized from absolute ethanol.
ers15 proposed
a
model in which the end target
chemoattractants (such as fMLP) functions primarily by stimulat-
ing p38 MAPK, whereas intermediary chemoattractants (such as
IL8) primarily function via the PI3 K/Akt (protein kinase B) path-
way, and a hierarchy between the two pathways exists. The same
signalling hierarchy exists with regard to NADPH-oxidase activa-
tion by formylated peptide and IL8.16
Results of the in vitro screening suggested a possible interfer-
ence of our compounds in different mechanisms of neutrophil
recruitment. Therefore, a more complete understanding of intra-
6.1.3.1.
5-Amino-1-(2-hydroxypropyl)-1H-pyrazole-4-carbox-
ylic acid ethyl ester 4b. Yield 61%, mp 66–68 °C. 1H NMR (CDCl3):
d 1.26 (d, J = 6.4, 3H, CH3CH), 1.35 (t, J = 7.0, 3H, CH3CH2), 3.13 (br s,

3384
O. Bruno et al. / Bioorg. Med. Chem. 17 (2009) 3379–3387
1H, OH, disappearswith D2O), 3.78–4.42 (m, 5H, CH2O + CH2N + CH),
5.44 (br s, 2H, NH2, disappears with D2O), 7.61 (s, 1H, H3). IR
(KBr): cmꢀ1 3426, 3390, 3313 (NH2 + OH), 1697 (CO).
Anal. Calcd for (C9H15N3O3): C, 50.69; H, 7.09; N, 19.71. Found:
C, 50.79; H, 7.06; N, 19.88.
tion mixture was heated at reflux for 4 h. After removal in vacuo
of the excess phosgene, the suitable amine (25 mmol), solved in
anhydrous THF, was added drop wise, at 0 °C; the mixture was stir-
red at room temperature overnight. After removal of the volatiles
in vacuo, the crude was suspended in water (20 mL), extracted
twice with CH2Cl2 (20 mL) and dried (MgSO4). The solvent was
evaporated under reduced pressure to give yellow oils, which were
purified by silica gel column chromatography using diethyl ether
as eluant. The white solids obtained were recrystallized from abso-
lute ethanol.
6.1.3.2. 5-Amino-1-(2-hydroxybutyl)-1H-pyrazole-4-carboxylic
acid ethyl ester 4c. Yield 61%, mp 90–91 °C. 1H NMR (DMSO-d6):
d 0.88 (t, J = 7.4, 3H, CH3), 1.23 (t, J = 7.0, 3H, CH3CH2O), 1.24–
1.43 (m, 2H, CH2CH), 3.62–3.77 (m, 1H, CH), 3.85 (d, J = 5.6, 2H,
CH2N), 4.16 (q, J = 7.4, 2H, CH2O), 4.98 (d, J = 5.2, 1H, OH, disap-
pears with D2O), 6.09 (br s, 2H, NH2, disappears with D2O), 7.45
(s, 1H, H3). IR (KBr): cmꢀ1 3433, 3343 (NH2), 3209 (OH), 1689 (CO).
Anal. Calcd for (C10H17N3O3): C, 52.85; H: 7.54; N, 18.49. Found:
C, 52.42; H, 7.60; N, 18.66.
6.1.5.1.
1-(2-Hydroxypropyl)-5-(3-isopropylureido)-1H-pyra-
zole-4-carboxylic acid ethyl ester 2b. Yield 58%, mp 93–94 °C.
1H NMR (CDCl3): d 1.19 (d, J = 6.6, 6H, 2CH3 isop.), 1.30–1.46 (m,
6H, 2CH3), 3.72–3.90 (m, 1H, CHNH), 3.99–4.13 (m, 2H, CH2N),
4.29 (q, J = 7.2, 2H, CH2O), 4.55–4.70 (m, 1H, disappears with
D2O), 4.76–4.90 (m, 1H, CHOH), 5.61–5.69 (m, 2H, disappears with
D2O), 7.62 (s, 1H, H3). IR (KBr): cmꢀ1 3456, 3357, 3322 (NH, OH),
1693 (COOEt), 1665 (CONH).
6.1.3.3.
5-Amino-1-(2-hydroxypentyl)-1H-pyrazole-4-carbox-
ylic acid ethyl ester 4d. Yield 72%, mp 84–86 °C. 1H NMR (CDCl3):
d 0.95 (t, J = 7.0, 3H, CH3), 1.37 (t, J = 7.0, 3H, CH3CH2O), 1.38–1.57
(m, 4H, 2CH2), 3.19 (br s, 1H, OH, disappears with D2O), 3.75–3.91
(m, 1H, CH), 3.86–4.08 (m, 2H, CH2N), 4.25 (q, J = 7.0, 2H, CH2O),
5.46 (br s, 2H, NH2, disappears with D2O), 7.59 (s, 1H, H3). IR
(KBr): cmꢀ1 3405, 3291, 3215 (NH2 + OH), 1690 (CO).
Anal. Calcd for (C13H22N4O4): C, 52.34; H, 7.43; N, 18.78. Found:
C, 52.44; H, 7.26; N, 18.92.
6.1.5.2. 1-(2-Hydroxypropyl)-5-(3-benzylureido)-1H-pyrazole-
4-carboxylic acid ethyl ester 2c. Yield 52%, mp 72–73 °C. 1H
NMR (CDCl3): d 1.20–1.35 (m, 6H, 2CH3), 3.96–4.07 (m, 2H,
CH2N), 4.19 (q, J = 7.0, 2H, CH2O), 4.30 (d, J = 5.8, 2H, CH2Ar),
4.74–4.90 (m, 1H, CHOH), 5.10–5.21 (m, 1H, disappears with
D2O), 5.41–5.76 (m, 2H, disappear with D2O), 7.12–7.35 (m, 5H,
Ar), 7.54 (s, 1H, H3). IR (KBr): cmꢀ1 3429, 3327, 3229 (NH, OH),
1703 (COOEt) 1688 (CONH).
Anal. Calcd for (C11H19N3O3): C, 54.76; H, 7.94; N, 17.41. Found:
C, 54.74; H, 7.92; N, 17.44.
6.1.3.4. 5-Amino-1-methyl-1H-pyrazole-4-carboxylic acid ethyl
ester 4a. Compound 4a was prepared by condensation of 1-
methyl-hydrazine 3a with ethyl (ethoxymethylene)cyanoacetate
in anhydrous toluene, as previously reported.19
Anal. Calcd for (C17H22N4O4): C, 58.95; H, 6.40; N, 16.17. Found:
C, 59.04; H, 6.69; N, 15.95.
6.1.3.5. 5-Amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-
carboxylic acid ethyl ester 4e. Compound 4e was prepared by
condensation of 2-hydrazino-1-phenylethanol 3e with ethyl (eth-
oxymethylene)cyanoacetate in anhydrous toluene, as previously
reported.20
6.1.5.3.
1-(2-Hydroxypropyl)-5-[3-(4-benzyl)piperazinylurei-
do]-1H-pyrazole-4-carboxylic acid ethyl ester 2d. Yield 58%,
mp 129–130 °C. 1H NMR (CDCl3): d 1.29–1.41 (m 6H, 2CH3),
2.34–2.50 (m, 4H, 2CH2N piperaz.), 3.42–3.57 (m, 6H, 2CH2N pipe-
raz + CH2Ar), 3.92–4.18 (m, 2H, CH2N piraz.), 4.28 (q, J = 7.2, 2H,
CH2O), 4.73–4.88 (m, 1H, CHOH), 5.61–5.69 (m, 2H, disappears
with D2O), 7.23–7.36 (m, 5H, Ar), 7.60 (s, 1H, H3). IR (KBr): cmꢀ1
3402, 3302, 3225 (NH, OH), 1700–1680 (COOEt + CONH).
Anal. Calcd for (C21H29N5O4): C, 60.71; H, 7.04; N, 16.86. Found:
C, 60.93; H, 7.26; N, 16.96.
6.1.4. Synthesis of 1-(2-hydroxy-2-phenylethyl)-5-[3-(1-naph-
thyl)ureido]-1H-pyrazole-4-carboxylic acid ethyl ester 2a
(Method A)
To a suspension of 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-
pyrazole-4-carboxylic acid ethyl ester 4e (1.38 g, 5 mmol) in anhy-
drous toluene (10 mL), a-naphthylisocyanate (1.02 g, 6 mmol) was
added and the reaction mixture was refluxed for 6 h. After cooling,
the solvent was evaporated under reduced pressure and the crude
was solved in CH2Cl2 (10 mL), washed with H2O (2 ꢁ 10 mL) and
with 1 M HCl (10 mL), dried (MgSO4) and concentrated under re-
duced pressure. The crude crystallized as a white solid by adding
a mixture of diethyl ether/ petroleum ether (bp 40–60 °C) (1:1).
Yield 52%, mp 181–183 °C. 1H NMR (CDCl3): d 1.26 (t, J = 7.2, 3H,
CH3), 1.61 (br s, 2H, disappears with D2O) 4.06–4.28 (m, 4H,
CH2O + CH2N), 5.93–6.04 (m, 1H, CHOH), 7.14–7.90 (m, 14H,
12HAr + H3 + 1H which disappears with D2O).
6.1.5.4. 1-(2-Hydroxybutyl)-5-(3-isopropylureido)-1H-pyrazole-
4-carboxylic acid ethyl ester 2i. Yield 51%, mp 102–103 °C. 1H
NMR (CDCl3): d 0.97 (t, J = 7.4, 3H, CH3), 1.19 (d, J = 6.6, 6H, 2CH3
isop.), 1.35 (t, J = 7.0, 3H, CH3CH2O), 1.57–1.89 (m, 2H, CH2CH),
3.75–3.93 (m, 1H, CHNH), 4.07–4.14 (m, 2H, CH2N), 4.28 (q,
J = 7.0, 2H, CH2O), 4.52–4.74 (m, 2H, CHOH + 1H which disappears
with D2O), 5.72–5.85 (m, 2H, disappears with D2O), 7.60 (s, 1H, H3).
IR (KBr): cmꢀ1 3416, 3352, 3307 (NH, OH), 1700–1680
(COOEt + CONH).
IR (KBr): cmꢀ1 3447, 3393, 3328 (NH, OH), 1710 (COOEt), 1676
(CONH).
Anal. Calcd for (C14H24N4O4): C, 53.83; H, 7.74; N, 17.94. Found:
C, 53.86; H, 7.68; N, 17.93.
Anal. Calcd for (C25H24N4O4): C, 67.54; H, 5.45; N, 12.61. Found:
C, 67.40; H, 5.74; N, 12.63.
6.1.5.5. 1-(2-Hydroxybutyl)-5-(3-benzylureido)-1H-pyrazole-4-
carboxylic acid ethyl ester 2j. Yield 68%, mp 99–101 °C. 1H NMR
(CDCl3): d 0.89 (t, J = 7.4, 3H, CH3), 1.26 (t, J = 7.2, 3H, CH3CH2O),
1.53–1.80 (m, 2H, CH2CH), 4.00 (d, J = 5.4, 2H, CH2N), 4.14 (q,
J = 7.2, 2H, CH2O), 4.30 (d, J = 6.0, 2H, CH2Ar), 4.53–4.66 (m, 1H,
CH), 5.20–5.30 (m, 1H, disappears with D2O), 5.49–5.80 (m, 2H, dis-
appears with D2O), 7.14–7.33 (m, 5H, Ar), 7.56 (s, 1H, H3). IR
(KBr): cmꢀ1 3424, 3298, 3320(NH, OH), 1698(COOEt), 1674(CONH).
Anal. Calcd for (C18H24N4O4): C, 59.99; H, 6.71; N, 15.55. Found:
C, 60.32; H, 6.67; N, 15.49.
6.1.5. General procedure for 1-(2-hydroxyalkyl)-5-(3-alkyl/
cycloalkylureido)-1H-pyrazole-4-carboxylic acid ethyl esters
2b–d, 2i–k and for 1-methyl-5-(3-alkyl/cycloalkylureido)-1H-
pyrazole-4-carboxylic acid ethyl esters 2p–u (Method B)
To a mixture of the suitable 5-amino-1H-pyrazole-4-carboxylic
acid ethyl esters (4a–d) (10 mmol) and anhydrous CH3COONa
(2.0 g, 24 mmol) in anhydrous THF (30 mL), cooled at 0 °C, phos-
gene (20% in toluene, 8 mL) was added drop wise; then, the reac-

O. Bruno et al. / Bioorg. Med. Chem. 17 (2009) 3379–3387
3385
6.1.5.6. 1-(2-Hydroxybutyl)-5-[3-(4-benzyl)piperazinylureido)-
1H-pyrazole-4-carboxylic acid ethyl ester 2k. Yield 47%, mp
117–118 °C. 1H NMR (CDCl3): d 0.97 (t, J = 7.4, 3H, CH3), 1.35 (t,
J = 7.0, 3H, CH3CH2O), 1.60–1.92 (m, 2H, CH2CH), 2.33–2.51 (m,
4H, 2CH2N piperaz.), 3.43–3.60 (m, 6H, 2CH2N piperaz. + CH2Ar),
4.02–4.10 (m, 2H, CH2N piraz.), 4.27 (q, J = 7.0, 2H, CH2O), 4.50–
4.63 (m, 1H, CHOH), 5.78–5.92 (m, 2H, disappears with D2O),
7.21–7.41 (m, 5H, Ar), 7.59 (s, 1H, H3). IR (KBr): cmꢀ13418, 3331,
3231 (NH, OH), 1700–1660 (COOEt + CONH).
Anal. Calcd for (C20H22N4O4): C, 62.82; H, 5.80; N, 14.65. Found:
C, 62.69; H, 5.96; N, 14.36.
6.1.6.2. 1-(2-Hydroxybutyl)-5-[3-(1-naphthyl)ureido]-1H-pyr-
azole-4-carboxylic acid ethyl ester 2o. Yield 60%, mp 121–
123 °C. 1H NMR (DMSO-d6): d 0.88 (t, J = 7.4, 3H, CH3), 1.16 (t,
J = 7.2, 3H, CH3CH2O), 1.40–1.73 (m, 2H, CH2CH), 3.98–4.18 (m,
4H, CH2O + CH2N), 4.90–5.05 (m, 1H, CHOH), 6.10–6.23 (m, 2H,
disappears with D2O), 7.28–7.93 (m, 8H, 7HAr + H3), 9.34 (br s,
1H, disappears with D2O). IR (KBr): cmꢀ1 3450, 3394, 3323 (NH,
OH), 1711 (COOEt), 1673 (CONH).
Anal. Calcd for (C22H31N5O4): C, 61.52; H, 7.27; N, 16.31. Found:
C, 61.58; H, 7.29; N, 16.12.
Anal. Calcd for (C21H24N4O4): C, 63.62; H, 6.10; N, 14.13. Found:
C, 63.46; H, 5.98; N, 13.79.
6.1.5.7. 1-(2-Hydroxypentyl)-5-(3-isopropylureido)-1H-pyra-
zole-4-carboxylic acid ethyl ester 2p. Yield 49%, mp 70–71 °C.
1H NMR (CDCl3): d 0.91 (t, J = 7.2, 3H, CH3), 1.19 (d, J = 6.6, 6H,
2CH3 isop.), 1.35 (t, J = 7.2, 3H, CH3CH2O), 1.40–1.60 (m, 4H,
2CH2), 3.73–3.90 (m, 1H, CHNH), 4.01–4.11 (m, 2H, CH2N), 4.28
(q, J = 7.2, 2H, CH2O), 4.58–4.74 (m, 2H, CHOH + 1H which disap-
pears with D2O), 5.69–5.83 (m, 2H, disappears with D2O), 7.60 (s,
1H, H3). IR (KBr): cmꢀ1 3420, 3350, 3312 (NH, OH), 1693–1680
(COOEt + CONH).
6.1.7. General procedure for 1-(2-hydroxyalkyl)-5-[3-(2/3/4-
fluorophenyl)ureido]-1H-pyrazole-4-carboxylic acid ethyl
esters 2e–g, 2l–n (Method A)
A mixture of the suitable 5-amino-1-(2-hydroxyalkyl)-1H-pyra-
zole-4-carboxylic acid ethyl esters (4b or 4c) (2.2 mmol) and the
proper fluoro-phenylisocyanate (0.41 g, 3 mmol) in anhydrous tol-
uene (30 mL) was refluxed for 6 h. After cooling, the solution was
washed with 3 M HCl (2 ꢁ 20 mL), with water (20 mL), dried
(MgSO4) and evaporated under reduced pressure. The crude crys-
tallized by adding a solution of diethyl ether/petroleum ether
(p.eb. 50–60 °C) 1/1. The white solids obtained were recrystallized
from absolute ethanol.
Anal. Calcd for (C15H26N4O4): C, 55.20; H, 8.03; N, 17.17. Found:
C, 55.29; H, 8.17; N. 17.18.
6.1.5.8. 1-(2-Hydroxypentyl)-5-(3-benzylureido)-1H-pyrazole-
4-carboxylic acid ethyl ester 2q. Yield 50%, mp 111–112 °C. 1H
NMR (CDCl3): d 0.82 (t, J = 7.2, 3H, CH3), 1.26 (t, J = 7.2, 3H,
CH3CH2O), 1.31–1.69 (m, 4H, 2CH2), 3.99 (d, J = 5.4, 2H, CH2N),
4.18 (q, J = 7.2, 2H, CH2O), 4.30 (d, J = 5.8, 2H, CH2Ar), 4.58–4.75
(m, 1H, CHOH), 5.18–5.29 (m, 1H, disappears with D2O), 5.46–
5.80 (m, 2H, disappears with D2O), 7.13–7.34 (m, 5H, Ar), 7.51
(s, 1H, H3). IR (KBr): cmꢀ1 3446, 3370, 3331 (NH, OH), 1724 (COO-
Et) 1694 (CONH).
6.1.7.1. 1-(2-Hydroxypropyl)-5-[3-(2-fluorophenyl)ureido]-1H-
pyrazole-4-carboxylic acid ethyl ester 2e. Yield 34%, mp 131–
132 °C. 1H NMR (CDCl3): d 1.27 (t, J = 7.0, 3H, CH3CH2), 1.35 (d,
J = 6.6, 3H, CH3CH), 3.92–4.10 (m, 2H, CH2N), 4.20 (q, J = 7.0, 2H,
CH2O), 4.93–5.03 (m, 1H, CHOH), 5.36–5.55 (m, 2H, disappears
with D2O), 6.79–8.02 (m, 6H, 4HAr + H3 + 1H which disappears
with D2O). IR (KBr): cmꢀ1 3425, 3335, 3235 (NH, OH), 1711 (COO-
Et), 1689 (CONH).
Anal. Calcd for (C19H26N4O4): C: 60.95; H, 7.00; N, 14.96. Found:
C, 60.87; H, 6.93; N, 15.09.
Anal. Calcd for (C16H19N4O4F): C, 54.85; H, 5.47; N, 15.99.
Found: C, 54.66; H, 5.63; N, 15.93.
6.1.5.9. 1-(2-Hydroxypentyl)-5-[3-(4-benzyl)piperazinylurei-
do)-1H-pyrazole-4-carboxylic acid ethyl ester 2r. Yield 68%,
mp 128–129 °C. 1H NMR (CDCl3): d 0.91 (t, J = 7.2, 3H, CH3), 1.35 (t,
J = 7.2, 3H, CH3CH2O), 1.40–1.81 (m, 4H, 2CH2), 2.37–2.51 (m, 4H,
2CH2N piperaz.), 3.44–3.59 (m, 6H, 2CH2N piperaz. + CH2Ar), 4.01–
4.16 (m, 2H, CH2N piraz.), 4.28 (q, J = 7.2, 2H, CH2O), 4.55–4.70 (m,
1H, CHOH), 5.80–5.89 (m, 2H, disappears with D2O), 7.27–7.41 (m,
5H, Ar), 7.60 (s, 1H, H3). IR (KBr): cmꢀ1 3402, 3307, 3227 (NH, OH),
1700–1680 (COOEt + CONH).
6.1.7.2. 1-(2-Hydroxypropyl)-5-[3-(3-fluorophenyl)ureido]-1H-
pyrazole-4-carboxylic acid ethyl ester 2f. Yield 48%, mp 146–
147 °C. 1H NMR (CDCl3): d 1.20–1.36 (m, 6H, 2CH3), 3.90–4.07
(m, 2H, CH2N), 4.20 (q, J = 7.2, 2H, CH2O), 4.84–5.03 (m, 1H, CHOH),
5.35–5.52 (m, 2H, disappears with D2O), 6.65–7.30 (m, 5H,
4HAr + 1H which disappears with D2O), 7.55 (s, 1H, H3). IR
(KBr): cmꢀ1 3427, 3336, 3266 (NH, OH), 1712 (COOEt), 1689
(CONH).
Anal. Calcd for (C23H33N5O4): C, 62.28; H, 7.50; N, 15.79 Found:
C, 62.04; H, 7.34; N, 15.70.
Anal. Calcd for (C16H19N4O4F): C, 54.85; H, 5.47; N, 15.99.
Found: C, 54.69; H, 5.50; N, 15.72.
6.1.6. General procedure for 1-(2-hydroxyalkyl)-5-[3-(1-naph-
thyl)ureido]-1H-pyrazole-4-carboxylic acid ethyl esters 2h, 2o
(Method A)
To a suspension of the suitable 5-amino-1-(2-hydroxyalkyl)-
1H-pyrazole-4-carboxylic acid ethyl esters (4b or 4c) (2.2 mmol)
6.1.7.3. 1-(2-Hydroxypropyl)-5-[3-(4-fluorophenyl)ureido]-1H-
pyrazole-4-carboxylic acid ethyl ester 2g. Yield 50%, mp 175–
177 °C. 1H NMR (CDCl3): d 1.14–1.36 (m, 6H, 2CH3), 3.89–4.08
(m, 2H, CH2N), 4.19 (q, J = 7.2, 2H, CH2O), 4.82–5.00 (m, 1H, CHOH),
5.36–5.54 (m, 2H, disappears with D2O), 6.84–7.32 (m, 5H,
4HAr + 1H which disappears with D2O), 7.54 (s, 1H, H3). IR
(KBr): cmꢀ1 3413, 3321, 3215 (NH, OH), 1709 (COOEt), 1682
(CONH).
in anhydrous toluene (10 mL),
a-naphthylisocyanate (0.51 g,
3 mmol) was added and the reaction mixture was refluxed for
6 h. After cooling, the white solids obtained were filtered and
recrystallized from absolute ethanol.
Anal. Calcd for (C16H19N4O4F): C, 54.85; H, 5.47; N, 15.99.
Found: C, 54.96; H, 5.37; N, 15.77.
6.1.6.1. 1-(2-Hydroxypropyl)-5-[3-(1-naphthyl)ureido]-1H-pyr-
azole-4-carboxylic acid ethyl ester 2h. Yield 92%, mp 124–
126 °C. 1H NMR (CDCl3): d 1.26 (t, J = 7.0, 3H, CH3CH2), 1.35 (d,
J = 6.4, 3H, CH3CH), 3.90–4.10 (m, 2H, CH2N), 4.18 (q, J = 7.0, 2H,
CH2O), 4.86–5.08 (m, 1H, CHOH), 5.20–5.60 (m, 2H, disappears
with D2O), 6.96–7.92 (m, 9H, 7HAr + H3 + 1H which disappears
with D2O). IR (KBr): cmꢀ1 3414, 3322, 3224 (NH, OH), 1709 (COO-
Et), 1682 (CONH).
6.1.7.4. 1-(2-Hydroxybutyl)-5-[3-(2-fluorophenyl)ureido]-1H-
pyrazole-4-carboxylic acid ethyl ester 2l. Yield 42%, mp 99–
100 °C. 1H NMR (CDCl3): d 0.95 (t, J = 7.6, 3H, CH3), 1.27 (t,
J = 7.0, 3H, CH3CH2O), 1.56–1.86 (m, 2H, CH2CH), 4.07 (d, J = 5.8,
2H, CH2N), 4.20 (q, J = 7.0, 2H, CH2O), 4.63–4.80 (m, 1H, CHOH),
5.45–5.69 (m, 2H, disappears with D2O), 6.82–8.06 (m, 6H,

3386
O. Bruno et al. / Bioorg. Med. Chem. 17 (2009) 3379–3387
4HAr + H3 + 1H which disappears with D2O). IR (KBr): cmꢀ1 3460,
3350, 3211 (NH, OH), 1706 (COOEt), 1681 (CONH).
Anal. Calcd for (C17H21N4O4F): C, 56.04; H, 5.81; N, 15.38.
Found: C, 55.99; H, 5.87; N, 15.48.
Data were expressed in terms of chemotactic index (C.I), which
is the ratio: [(migration toward test attractant-migration toward
the buffer)/migration toward the buffer]. The C.I. of fMLP-OMe is
1.2 at 10ꢀ9 M, while the value of IL8 is 1.02 at 10ꢀ8 M.
Neutrophils were preincubated with tested compounds 10 min
before the functional test.
6.1.7.5.
1-(2-Hydroxybutyl)-5-[3-(3-fluorophenyl)ureido]-1H-
pyrazole-4-carboxylic acid ethyl ester 2m. Yield 49%, mp 128–
129 °C. 1H NMR (CDCl3): d 0.91 (t, J = 7.6, 3H, CH3), 1.26 (t, J = 7.2,
3H, CH3CH2O), 1.52–1.84 (m, 2H, CH2CH), 4.03 (d, J = 5.6, 2H,
CH2N), 4.19 (q, J = 7.2, 2H, CH2O), 4.63–4.77 (m, 1H, CHOH),
5.46–5.63 (m, 2H, disappears with D2O), 6.64–7.38 (m, 5H,
4HAr + 1H which disappears with D2O), 7.54 (s, 1H, H3). IR
(KBr): cmꢀ1 3458, 3347, 3247 (NH, OH), 1707 (COOEt), 1680
(CONH).
The antagonism was measured as C.I. in the absence (100%) and
in the presence of the tested compounds at different concentra-
tions, following the methods already reported.10
IC50 values were calculated from the sigmoid dose–response
curve by a non-linear regression analyses (Graph Pad Prism, San
Diego, USA).
6.2.5. Measurement of cell viability
Anal. Calcd for (C17H21N4O4F): C, 56.04; H, 5.81; N, 15.38.
Found: C, 56.04; H, 5.67; N, 15.50.
In order to assess possible cytotoxic effects of the tested com-
pounds, the cytoplasmatic marker enzyme, lactate dehydrogenase
(LDH), was determined by measuring the rate of oxidation of
NADH. The absorbance change was followed at 340 nm.22
6.1.7.6.
1-(2-Hydroxybutyl)-5-[3-(4-fluorophenyl)ureido]-1H-
pyrazole-4-carboxylic acid ethyl ester 2n. Yield 50%, mp 130–
131 °C. 1H NMR (CDCl3): d 0.93 (t, J = 7.6, 3H, CH3), 1.27 (t, J = 7.2,
3H, CH3CH2O), 1.53–1.86 (m, 2H, CH2CH), 4.06 (d, J = 5.8, 2H,
CH2N), 4.19 (q, J = 7.2, 2H, CH2O), 4.59–4.76 (m, 1H, CHOH),
5.41–5.77 (m, 2H, disappears with D2O), 6.80–7.41 (m, 5H,
4HAr + 1H which disappears with D2O), 7.54 (s, 1H, H3). IR
(KBr): cmꢀ1 3467, 3362, 3252 (NH, OH), 1710 (COOEt), 1682
(CONH).
6.2.6. Induction of peritonitis, cell count, cell composition and
determination of protein concentration in the peritoneal
exudate
The experiments were performed using male Swiss mice (25–
30 g) fasted 16 h before the experiment, but with free access to
water. Mice were randomly assigned to groups of 12 animals orally
treated with vehicle or the compounds under examination (50 mg/
kg) 1 h before the induction of peritonitis. Dexamethasone (3 mg/
kg) was used as reference drug. Peritonitis was induced following
a modification of Thurmond’s method.23 Briefly, 5 mg/mL zymosan
or phosphate-buffered-saline (PBS) was injected into the perito-
neal space of mice (final volume 0.2 mL). After 4 h, the animals
were euthanized; the peritoneal cavities were washed with 3 mL
of PBS containing 3 mM EDTA and the volume collected with auto-
matic pipettes. Total leukocyte counts were performed in a Neu-
bauer chamber by means of optical microscopy after diluting a
sample of the peritoneal fluid with Türk solution (1:10). Differen-
tial cell counts were performed using a light microscope. Their
chromatic characteristics and the shape of the nucleus relative to
the cytoplasm easily differentiated neutrophils.
Anal. Calcd for (C17H21N4O4F): C, 56.04; H, 5.81; N, 15.38.
Found: C, 55.91; H, 6.08; N, 15.28.
6.2. Biological methods
6.2.1. Neutrophils preparation
Cells were obtained from the blood of healthy subjects, and hu-
man peripheral blood neutrophils were purified by using the stan-
dard techniques of dextran (Pharmacia, Uppsala, Sweden)
sedimentation, centrifugation on Ficoll–Paque (Pharmacia), and
hypotonic lysis of contaminating red cells. Cells were washed twice
and resuspended in Krebs–Ringer phosphate (KRPG), pH 7.4, at a
final concentration of 50 ꢁ 106 cells/mL and kept at room temper-
ature until used. Neutrophils were 98–100% viable, as determined
using the Trypan blue exclusion test. Local Ethics Committee ap-
proved the study and informed consent was obtained from all
participants.
Protein content was spectrophotometrically determined fol-
lowing the bicinchonate method with a commercial kit (Pierce,
BCA protein assay kit). Experiments were carried out in accor-
dance to the Italian law (DL 116/92) and approved by ‘Ministry
of Health’.
6.2.2. Preparation of chemoattractants and tested compounds
fMLP-OMe and tested compounds (10ꢀ2 M) were solved in
dimethylsulfoxide (DMSO), while IL8 (10ꢀ5 M) was solved in
water. Before the use, all the solutions were diluted in KRPG con-
taining 1 mg/mL of bovine serum albumin (BSA; Orha Behring-
werke, Germany) to obtain the final concentrations ranging from
10ꢀ15 to 10ꢀ5 M. At the concentrations used, DMSO did not inter-
fere with any of the biological assays performed.
Acknowledgements
The authors wish to thank Dr. R. Raggio, Mr. O. Gagliardo, F.
Tuberoni for spectral recording, Dr. G. Domenichini for skilful
assistance in in vivo experiments, and Banca del Sangue di Ferrara
for providing fresh blood. Financial support from University of
Genoa and ‘Fondazione Cassa di Risparmio di Ferrara’ are gratefully
acknowledged.
6.2.3. Random locomotion
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