Synthesis and biological evaluation of N-pyrazolyl-N′-alkyl/benzyl/ phenylureas: A new class of potent inhibitors of interleukin 8-induced neutrophil chemotaxis

Article abstract of DOI:10.1021/jm0704402

Neutrophils chemotaxis is a complex multistep process that, if upregulated, causes acute inflammation and a number of autoimmune diseases. We report here the synthesis of a new N-(4-substituted)pyrazolyl-N′-alkyl/benzyl/ phenylureas that are potent inhibi

Full text of DOI:10.1021/jm0704402

3618
J. Med. Chem. 2007, 50, 3618-3626
Synthesis and Biological Evaluation of N-Pyrazolyl-N′-alkyl/benzyl/phenylureas: a New Class of
Potent Inhibitors of Interleukin 8-Induced Neutrophil Chemotaxis
Olga Bruno,*,† Chiara Brullo,† Francesco Bondavalli,† Silvia Schenone,† Angelo Ranise,† Nicoletta Arduino,‡
Maria B. Bertolotto,‡ Fabrizio Montecucco,‡ Luciano Ottonello,‡ Franco Dallegri,‡ Massimiliano Tognolini,§ Vigilio Ballabeni,§
Simona Bertoni,§ and Elisabetta Barocelli§
Dipartimento di Scienze Farmaceutiche, UniVersity of Genoa, V.le Benedetto XV, 3-Genoa, Italy, Dipartimento di Medicina Interna, UniVersity
of Genoa, V.le Benedetto XV, 6-Genoa, Italy, and Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, UniVersita` degli
Studi di Parma, V.le GP Usberti, 27/A-Parma, Italy
ReceiVed April 13, 2007
Neutrophils chemotaxis is a complex multistep process that, if upregulated, causes acute inflammation and
a number of autoimmune diseases. We report here the synthesis of a new N-(4-substituted)pyrazolyl-N′-
alkyl/benzyl/phenylureas that are potent inhibitors of interleukin-8 (IL8)-induced neutrophil chemotaxis.
The first series of compounds, obtained by functionalization with a urea moiety of the 5-amino-1-(2-hydroxy-
2-phenylethyl)-1H-pyrazole-4-carboxylic acid ethyl ester 3, blocked the IL8-induced neutrophil chemotaxis,
while they did not block N-formylmethionylleucylphenylalanine-mediated chemotaxis. The most active
compounds, 3-benzyl- (4d), 3-(4-benzylpiperazinyl)- (4i), 3-phenyl- (4k) and 3-isopropylureido (4a)
derivatives, showed an IC50 of 10, 14, 45, and 55 nM, respectively. Several different molecules were then
synthesized to obtain more information for SAR study. Compounds 4a, 4d, and 4k 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. Moreover, in the presence of the same derivatives, we
observed a complete block of F-actin rise and pseudopod formation.
Introduction
specific G-protein-coupled receptors, which are expressed on
target cells.11-16
Inflammation is the first response of the immune system to
infection or irritation.1 Generally, the inflammation results in
connective tissue scarring. Nevertheless, if the injurious agent
continues or the control of cellular recruitment breaks down,
both acute and chronic autoimmune inflammatory disorders
(such as asthma, rheumatoid arthritis, multiple sclerosis, and
inflammatory bowel disease) will ensue.
Neutrophils are the main cells infiltrated in the course of acute
inflammation. Their activation consists of various events:
margination (rolling and adhesion to vessel walls), diapedesis
(transmigration across the endothelial barrier), chemotaxis
(migration to site of inflammation) and phagocytosis of foreign
particles, and reactive free radicals production.2,3
Chemotaxis is a multistep process4 induced by various
chemoattractants such as fMLP,a,5,6 PAF,7 LTB4,8 C5a ana-
phylotoxin9 and different chemokines such as the CC chemokine
MIP 1â or the CXC chemokine IL8 (also named CXCL8).10
Both IL8 and fMLP responses are induced by activation of
Downstream of heterotrimeric G-proteins, a number of
intracellular signaling systems are activated including (a)
stimulation of PLC, followed by PKC activation and increasing
in cytosolic calcium; (b) activation of the enzyme PI3K,
resulting in increase of PIP3 and PIP2; and (c) activation of
small GTP-binding proteins of the Rho family and of the MAPK
cascade, as well as activation of PTKs and protein phos-
phatases.17
This complex system results in the rapid development of
ruffles on the cell body and in the rise of polymerized F-actin
in the ruffles. Then, the cells develop a polarized shape with
formation of a contracted tail in the rear and F-actin-rich ruffles
at the front.18-20 Newly polymerized actin filaments are enriched
in the leading edge of a migrating cell,21,22 and the increase in
filamentous actin causes pseudopod formation.23-25 The block
of actin polymerization abolishes chemotaxis.26-29
The mechanism of F-actin polymerization is not yet com-
pletely understood;30 it depends on PI3K activation, particularly
on phosphorylation of PI3Kγ31,32 and PI3Kδ33 isoforms, and
PIP3 production; moreover, the activation of Akt/PKB is a major
downstream regulator of PI3K-dependent chemotaxis.4,34,35
Recently, two distinct pathways of actin polymerization were
reported. The PI3K-dependent signaling depends on PKC-ú and
Akt; in contrast, the PI3K-independent signaling depends on
ROCK kinase, Src family tyrosine kinases, and NADPH, and
it is modulated by cAMP. These distinct pathways are used by
the different chemotactic receptors.36,37
* Corresponding author. Tel: +39 010 353 8367. Fax: +39 010 353
8358. E-mail: obruno@unige.it.
† Dipartimento di Scienze Farmaceutiche, University of Genoa.
‡ Dipartimento di Medicina Interna, University of Genoa.
§ Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche
Applicate, Universita` degli Studi di Parma.
a Abbreviations: IL8/CXCL8, interleukin 8; Akt/PKB, protein kinase
B; BSA, bovine serum albumin; CCR1/CCR2/CCR3, CC chemokine
receptors; CXCR1, CXCR2, interleukin 8 receptors; ERK1/2, extracellular
signal-regulated kinase 1/2; Fgr, fetal growth restriction; fMLP, N-
formylmethionylleucylphenylalanine; HBSS, Hank’s balanced salt solution;
HcK, hematopoietic cell kinase; JNK1/2, c-Jun N-terminal kinase 1/2; LTB4,
leukotriene B4; MAPK, mitogen-activated protein kinase; MIP1b, mac-
rophage inflammatory protein 1b; PAF, platelet activating factor; PBS,
Dulbecco’s phosphate-buffered saline; PI3K, phosphoinositide 3-kinase;
PIP2, phosphatidylinositol 3,4-biphosphate; PIP3, phosphatidylinositol 3,4,5-
triphosphate; PLC, phospholipase C; PKC, protein kinase C; PTKs, protein
kinases; pyk2, proline-rich tyrosine kinase 2; Rho, Ras homology gene;
ROCK, RhoA/Rho-kinase; SRC, sarcoma; Zap-70, ú-associated protein 70.
Recently, many academic medicinal chemists and pharma-
ceutical research divisions have been greatly involved in the
search for new molecules able to interfere with the neutrophil
upregulation.
SB 225002 [N-(2-hydroxy-4-nitrophenyl)-N′-(2-bromophe-
nyl)urea] (Figure 1) was first reported as a potent and selective
10.1021/jm0704402 CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/04/2007

Inhibitors of Neutrophil Chemotaxis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3619
Table 1. Chemical Structures of Title Compounds and Chemotaxis
Inhibition Valuesa
Figure 1. Structures of some CXC receptors inhibitors.
inhibn of IL8-induced
neutrophil migration,
compd
R
NR′R′′
IC50 (µM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
COOC2H5 NHCH(CH3)2
COOC2H5 1-cyclopropylamino
COOC2H5 NH(CH2)2OCH2CH3
COOC2H5 NHCH2C6H5
COOC2H5 NHCH2CH2C6H5
COOC2H5 1-pyrrolidinyl
COOC2H5 1-piperidinyl
COOC2H5 1-(4-methylpiperazinyl)
COOC2H5 1-(4-benzylpiperazinyl)
COOC2H5 4-morpholinyl
COOC2H5 NHC6H5
0.055 ( 0.021
5.52 ( 2.313
1.00 ( 0.14
0.010 ( 0.005
0.61 ( 0.25
4.95 ( 1.669
3.090 ( 1.341
0.865 ( 0.453
0.014 ( 0.007
0.95 ( 0.341
0.045 ( 0.007
0.251 ( 0.035
0.825 ( 0.021
0.035 ( 0.021
0.225 ( 0.007
0.033 ( 0.006
0.036 ( 0.002
0.033 ( 0.001
1.560 ( 0.19
0.345 ( 0.11
0.424 ( 0.013
0.092 ( 0.008
0.398 ( 0.009
0.445 ( 0.027
1.20 ( 0.05
Figure 2. Structures of some p38Ks inhibitors.
4j
4k
5a
5b
5c
5d
6a
6b
6c
6d
6e
6f
COOH
H
CN
NHCH(CH3)2
NHCH(CH3)2
NHCH(CH3)2
NHC6H5
CN
COOC2H5 NHC6H4F(o)
COOC2H5 NHC6H4F(m)
COOC2H5 NHC6H4F(p)
Figure 3. Structures of some PI3Ks inhibitors.
nonpeptide IL8 inhibitor binding to CXCR2.38 It was followed
by many small molecules belonging to different chemical series,
acting at CXC receptors,39,40 some of which also showed
interesting pharmacological activities in vivo (see as example
N-(3-(aminosulfonyl)-4-chloro-2-hydroxyphenyl)-N′-(2,3-dichlo-
rophenyl)urea 1, Figure 1).41,42
The inhibition of intracellular chemokine-activated signaling
is another possible strategy. In this context, several potent
p38MAPK inhibitors (see as example SB203580 or VX-745 in
Figure 2)43 were efficacious in disease models, including
inflammation,44 arthritis,45 and pulmonary injury.46
Now, the goal of researchers is to identify selective inhibitors
for the PI3K isoforms.47 LY294002 (see Figure 3) was the first
synthetic molecule known to inhibit PI3Ks. Currently, the PI3K
inhibitors can be divided into two classes: derivatives of
LY294002 and unrelated structures, which are the major part
(see for example the late reported compounds 2 in Figure 3).48
For a long time we have been carrying out the synthesis of
new heterocycle derivatives as potential anti-inflammatory
agents.49-53 Now we have focused our interest on the identifica-
tion of new molecules able to impact neutrophil activation and
migration.54
In pursuing the search for new small molecules active in this
field, we functionalized with a urea moiety the 5-amino-1-(2-
hydroxy-2-phenylethyl)-1H-pyrazole-4-carboxylic acid ethyl
ester 3, already used as a crucial intermediate,55 and we obtained
derivatives 4a-k (Table 1).
COOC2H5 NHC6H4Cl(m)
COOC2H5 NHC6H4Cl(p)
COOC2H5 NHC6H4Br(m)
COOC2H5 NHC6H4Br(p)
COOC2H5 NHC6H4CH3(m)
COOC2H5 NHC6H4CH3(p)
COOC2H5 NHC6H4OCH3(o)
COOC2H5 NHC6H4OCH3(m)
COOC2H5 NHCH2C6H4F(o)
COOC2H5 NHCH2C6H4F(m)
COOC2H5 NHCH2C6H4F(p)
COOC2H5 NHCH2C6H4Cl(o)
COOC2H5 NHCH2C6H4Cl(m)
COOC2H5 NHCH2C6H4Cl(p)
COOC2H5 NHCH2C6H4Br(o)
COOC2H5 NHCH2C6H4Br(m)
COOC2H5 NHCH2C6H4Br(p)
6g
6h
6i
6j
6k
7a
7b
7c
7d
7e
7f
7g
7h
7i
1.40 ( 0.20
1.487 ( 0.344
0.318 ( 0.053
0.212 ( 0.033
1.244 ( 0.255
0.570 ( 0.080
0.935 ( 0.167
1.777 ( 0.086
0.198 ( 0.024
3.886 ( 0.039
a Neutrophils chemotaxis was evaluated in a Boyden chamber assays,
as described in the Experimental Section, after stimulation with 10-8
M
IL8 or 10-9 M fMLP, in the absence and presence of tested compounds.
The net migration was determined by subtracting spontaneous migration,
i.e., the distance travelled by neutrophils in the absence of the stimulus,
from the distance travelled by neutrophils toward the stimulus. The
concentration of each compound giving 50% inhibition (IC50) of net
migration was obtained from nonlinear regression analysis, and the results
reported are the mean of at least three experiments. No inhibitory activity
was observed in fMLP-induced chemotaxis.
anoacetate in anhydrous toluene, as previously reported.55
Starting from this intermediate, we synthesized most of the title
compounds following method A or method B (see Scheme 1).
The former is a one-pot reaction that needs a preliminary
treatment with phosgene in anhydrous THF at reflux; later, the
suitable amines were added to the mixture to give compounds
4a-j and 7a-i. In the second way (method B), compound 3
was refluxed in anhydrous toluene with a little excess of the
appropriate phenyl isocyanate to yield compounds 4k and 6a-
To evaluate the role of the substituent in position 4 of the
pyrazole moiety, in compounds 5a-d we replaced the 4-car-
boxyethyl group with other ones, maintaining as ureido sub-
stituents the aliphatic (isopropyl) or the aromatic (phenyl) group.
Finally, to investigate the influence of electron-donor or
electron-drawing substituents in the phenyl- and benzylurea
moiety, we synthesized new 4-carboxyethyl derivatives (com-
pounds 6a-k and 7a-i).
1
k. IR and H NMR spectral data confirmed the urea structure
and excluded the involvement of the 2-hydroxy-2-phenylethyl
group. On the other hand, we already evidenced this occurrence
in analogous reactions of compound 3 with different electro-
philes.56,57
Starting from compound 4a we obtained in good yield
compound 5a by a simple basic hydrolysis of the 4-carboxyethyl
Chemistry
5-Amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-car-
boxylic acid ethyl ester 3 was prepared by condensation of
2-hydrazino-1-phenylethanol with ethyl ethoxymethylenecy-

3620 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Scheme 1. Synthesis of Compounds 4a-k, 6a-k, and 7a-ia
Bruno et al.
a Reagents: (a) anhyd toluene, reflux, 8 h. Method A, step1: COCl2, anhyd AcONa, anhyd THF, reflux, 4 h; step 2: excess of primary or secondary
amines, anhyd THF, rt, 12 h. Method B: suitable phenyl isocyanate, anhyd toluene, reflux, 6 h.
Scheme 2. Synthesis of Compounds 5a,ba
a Reagents: (a) Step 1: NaOH, EtOH, reflux, 2 h. Step 2: H3O+. (b) Heating at 165 °C.
Scheme 3. Synthesis of Compounds 8, 5c, and 5da
a Reagents: (a) ethanol, reflux, 8 h. (b) Step1: COCl2, anhyd AcONa, anhyd THF, reflux, 4 h. Step 2: excess of isopropylamine, anhyd THF, rt, 12 h.
(c) Phenyl isocyanate, anhyd toluene, reflux, 6 h.
group (Scheme 2). The following decarboxylation at high
temperature gives compound 5b. For compounds 5c and 5d,
we prepared the new intermediate, 5-amino-1-(2-hydroxy-2-
phenylethyl)-1H-pyrazole-4-carbonitrile 8, by condensation of
2-hydrazino-1-phenylethanol with ethoxymethylenemalononitrile
in absolute ethanol (Scheme 3). Then, method A or method B
was applied to obtain compound 5c or 5d, respectively.
It has been reported that the phosphotyrosine content of
several proteins in the 60-70, 100, and 120 kDa regions rapidly
increases in IL8-stimulated neutrophils.58 Thus, we tested the
most active compounds, 4a, 4d, and 4k, in an immunoblotting
test of IL8-induced tyrosine phosphorylation. The study showed
a certain ability to reduce the phosphorylation in the 60-70
and 50 kDa regions (see Figures 4 and 5).
Consequently, we submitted the same compounds to a binding
assay on a series of tyrosine (unspecific SRC, Fgr, Hck, Zap-
70) kinases having a molecular weight in the cited regions; in
addition, we tested them on some serine-threonine (p38R, p38â2,
p38δ, p38γ, ERK1, ERK2, JNK1, JNK2, pKB/Akt1) kinases,
selected as among the most involved in the neutrophils action,
to confirm a kinase inhibition and a possible selectivity.
Biological Studies
All synthesized compounds were tested to evaluate the
inhibition of IL8- and fMLP-induced neutrophil migration. To
establish the role of the ureido group in the chemotaxis inhibi-
tion, we tested, in the same assays, the intermediate 5-amino
pyrazole 3 also.
The interesting results obtained in the functional IL8-induced
chemotactic test, and the nearly complete inability to interfere
in the fMLP-induced chemotaxis, prompted us to examine the
possible interaction of our compounds with the IL8 receptors
(CXCR1 and CXCR2). The negative response of the binding
assays suggested a likely interference in the intracellular signal
transduction pathways. As reported in the introduction section,
this is a very complex sequence of reactions in which a series
of serine-threonine or tyrosine protein kinases are involved.
In addition, we also determined the content of F-actin in IL8-
stimulated neutrophils, in the absence and presence of com-
pounds 4a, 4d, and 4k. An additional test of Akt phosphorylation
was performed to verify the involvement of the same compounds
in the PI3K-dependent actin polymerization.
Finally, some of the most active compounds (4a, 4d, 4h, 4i,
4k, 6a, 6b, and 6c) were preliminarly tested in vivo in a mouse
model of zymosan-induced peritonitis.

Inhibitors of Neutrophil Chemotaxis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3621
3, devoid of ureido function, was completely unable to block
the neutrophil migration. Moreover, the presence of an aromatic
moiety (phenyl, benzyl, or 4-benzylpiperazinyl) gave very potent
compounds, but the 3-isopropylureido derivative 4a also showed
a very strong activity.
Hydrolysis or complete removal of the 4-carboxyethyl group
was detrimental for the chemotaxis inhibition. In fact, in the
3-isopropylureido derivatives, IC50 increases from 55 nM
(compound 4a) to 250 and 825 nM (compounds 5a and 5b,
respectively). The substitution of 4-carboxyethyl with a 4-cyano
group did not give clear information: in the 3-isopropylureido
derivative (5c), the activity slightly increases (IC50 being 35
nM versus 55 nM for the analogue 4a); in contrast, in the
3-phenylureido derivative (5d) the potency decreases (IC50 being
225 nM versus 45 nM for the analogue 4k). Thus, we suppose
that a substituent in position 4 of the pyrazole is needed, but
further investigations are required to clarify which is the better
one.
Electron-donor substituents in the phenylureido moiety
strongly decreased the activity: in fact, compounds 6h-k
showed IC50 values 10-30-fold higher than the analogue 4k.
Of the electron-withdrawing substituents (Br, Cl, F) only
fluorine, whether in the ortho, meta, or para position, slightly
increased the activity compared to the unsubstituted 4k (see
compounds 6a-c). These data suggested that a detrimental steric
hindrance nullifies the favorable electronic effect.
Finally, in the last series of 3-benzylureido derivatives, all
the substitutions were detrimental for the activity, compounds
7a-i having an IC50 ranging from 0.2 to 3.8 µM, about 20-
40-fold higher than the unsubstituted 4d (one of the most active
compounds previously synthesized).
Some compounds (4a, 4d, 4k) selected among the most active
in the migration test were submitted to a binding assay on
CXCR1, CXCR2, CCR1, CCR2, and CCR3, but they were
completely inactive (data not shown).
Stimulation of neutrophils with 10-7 M IL8 very rapidly
increased the phosphotyrosine content of proteins in the 60-
70 and 50 kDa regions; moreover, the immunoblotting test also
evidenced a strong stimulation in the 116 KDa region (see line
1 in Figure 4), corresponding to proline-rich tyrosine kinase
pyk2, already reported as one of the possible targets of
neutrophil chemotaxis inhibitors.59 Interestingly, the pretreatment
Figure 4. (a) Effect of compounds 4a, 4d, and 4k on the tyrosine
phosphorylation in human neutrophils stimulated with IL8 for 0.5 min.
The three compounds reduce tyrosine phosphorylation (lines 2 and 3)
but are ineffective on the phosphorylation of 100-120 kDa proteins
(line 1). (b) To verify equal protein amount in the different samples,
nitrocellulose membrane was stained with Ponceau S.
Results
No compounds, at the used concentrations, showed cytotoxic
activity toward neutrophils, the percentage of viable cells never
being lower than 90%.
All tested compounds, except the 5-aminopyrazole 3, blocked
the IL8-induced neutrophil chemotaxis, while they were inactive
toward fMLP. In the first series, the most active were the
3-benzyl-, 3-(4-benzylpiperazinyl)-, 3-phenyl-, and 3-isopropy-
lureido derivatives (4d, 4i, 4k, and 4a) with an IC50 of 10, 14,
45, and 55 nM, respectively (Table 1). On the other hand,
compounds 4e and 4j also showed a very interesting activity,
having an IC50 of 0.61 and 0.95 µM, respectively, while
compounds 4b, 4c, 4f, and 4g were the least active, with IC50
ranging from 1.00 to 5.52 µM.
These preliminary data allowed us to underline the crucial
role of the urea group and their substituents. First, compound
Figure 5. Densitometric analysis of Western blot for tyrosine phosphorylation in human neutrophils stimulated with IL8 for 0.5 min. Densitometric
analysis was carried out using the slab shown in Figure 4. Each panel shows analysis of the lines 1, 2, and 3 of the slab, respectively.

3622 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Bruno et al.
Figure 6. Determination of F-actin content in neutrophils stimulated with IL8 in the absence and presence of compounds 4a, 4d, and 4k. F-actin
was stained with alexa-Fluor 488-conjugated phalloidin. The content of F-actin was determined by fluorescence microscopy as described in the
Experimental Section. The figures are representative of three independent experiments.
Table 2. Effect of Compound 6b on Cell and Granulocyte Recruitment
Induced by Zymosan in Mousea
no.
peritoneal
cells
no.
peritoneal
%
%
%
granulocytes mono- lympho- granulo-
compounds
vehicle
(% inhibn) (% inhibn)
cytes
cytes
cytes
9
10
25
28
66
62
6b
29*
36*
33*
(100 mg/kg os)
dexamethasone
(1 mg/kg os)
62**
14
44
42
a Animals received the compound under study (100 mg/kg os) or
dexamethasone (1 mg/kg os) 1 h before intraperitoneal injection of zymosan
(1 mg/mouse). Peritoneal fluid was collected and examined microscopically
4 h later. *p < 0.05, **p < 0.01 by Student’s t-test compared to vehicle-
treated mice.
Figure 7. Determination of F-actin content in neutrophils IL8
stimulated in the absence and presence of compounds 4a, 4d, and 4k.
Analysis of the slides reported in Figure 6. The mean fluorescence
density was determined from a linear measurement of individual cell
fluorescence. Results are shown as the mean ( SD of the fluorescence
densities expressed as area percentage of each field for each individual
slide.
the binding test on Akt enzyme, confirming the absence of
activity toward this serine-threonine-kinases-dependent pathway.
Concerning the in vivo assay, most of the tested compounds
(4a, 4d, 4h, 4i, 4k, 6c, 6a) did not modify the inflammatory
response induced by zymosan intraperitoneal injection. Protein
content of peritoneal cavity lavage was similar in vehicle- or
compound-treated mice. In contrast, treatment with compound
6b caused a significant decrease of cells and granulocytes
present in the peritoneal cavity compared to vehicle-treated mice
(Table 2).
with compounds 4a, 4d, and 4k reduced the tyrosine phospho-
rylation in the 60-70 and 50 kDa regions (see image in Figure
4 and densitometric analysis in Figure 5). In particular, line 2
evidenced 53%, 63%, and 81% reduction for compound 4a, 4d,
and 4k, respectively; line 3 showed 20%, 70%, and 71%
reduction for compound 4a, 4d, and 4k, respectively. In contrast,
in line 1 we observed a slight increase of phosphorylation;
however, we cannot clearly relate this behavior to the chemo-
taxis inhibition. Moreover, all the binding tests on the tyrosine
kinases having MW ranging from 50 to 70 kDa (unspecific SRC,
Fgr, Hck, Zap-70) as well as those on the serine-threonine
kinases (p38R, p38â2, p38δ, p38γ, ERK1, ERK2, JNK1, JNK2,
Akt1) were negative.
Conclusions
In conclusion, we reported the synthesis of a large series of
N-pyrazolyl-N′-substituted ureas that strongly inhibited IL8-
induced neutrophils recruitment. The most active compounds
were the 3-benzyl-, 3-(4-benzylpiperazinyl)-, 3-phenyl-, 3-(fluo-
ro)phenyl, and the 3-isopropylureido derivatives, having a
carboxyethyl group in position 4 of the pyrazole moiety. These
compounds were inactive toward the CXC receptors.
The involvement of the synthesized compounds in the
complex intracellular mechanisms of neutrophil recruitment was
confirmed by the determination of F-actin content: in fact, a
remarkable decrease was noted in the presence of tested
compounds. The negative test on Akt phosphorylation, con-
Compounds 4a, 4d, and 4k also reduced the content of
polymerized actin in IL8-stimulated neutrophils (as shown in
Figures 6 and 7) 97%, 69%, and 97%, respectively.
However, the Western blot on Akt phosphorylation showed
a complete inactivity of tested compounds (data not shown).
On the other hand, compounds 4a, 4d, and 4k were inactive in

Inhibitors of Neutrophil Chemotaxis
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3623
Synthesis and Analytical Data of 1-(2-Hydroxy-2-phenyl-
ethyl)-5-(3-isopropylureido)-1H-pyrazole-4-carboxylic Acid 5a.
To a solution of 1-(2-hydroxy-2-phenylethyl)-5-(3-isopropylureido)-
1H-pyrazole-4-carboxylic acid ethyl ester 4a (1.8 g, 5 mmol) in
95% ethanol (9 mL) was added 2 M NaOH (9 mL) and the mixture
was heated at reflux for 2 h. After removal of ethanol in vacuo,
the crude was dissolved in water (20 mL) and then the basic solution
was washed with CHCl3 (20 mL) and made acid with 6 N HCl to
afford a white solid, which was filtered and recrystallized by
firmed by the negative result of the binding assay on Akt,
indicates that our compounds inhibit the actin polymerization
by a PI3K-independent mechanism.
The inhibition of tyrosine phosphorylation in the 50-70 kDa
region, evidenced by the immunoblotting test, suggests that our
compounds act toward one or more of the numerous kinases
involved in these complex cascade-activation processes. How-
ever, the screening of a large number of kinases enzyme assays
showed negative results; thus, the exact molecular target is not
yet known.
In the preliminary in vivo test, only the 3-fluorophenyl
derivative 6b showed a good inhibition of neutrophils chemo-
taxis. It is worth nothing that the unsubstituted analogue 4k,
which showed a comparable activity in vitro, was inactive in
vivo. Consequently, we assume that a fluorine in the phenyl
ring is important to achieve activity in vivo.
These results prompted us to pursue the synthetic work, to
obtain new derivatives that are more potent in vivo, and to
plan in-depth biological analyses to clarify the mechanism of
action.
absolute ethanol. Yield: 91%. Mp: 163-164 °C. IR (KBr) cm-1
:
1
3449, 3400, 3324, 3232 (OH, NH), 1697 (CO), 1655 (CON). H
NMR (DMSO-d6): δ 0.97 (d, J ) 4.0, 3H, CH3CH), 0.99 (d, J )
4.0, 3H, CH3CH), 3.88-4.39 (m, 2H, CH2N), 4.83-5.01 (m, 1H,
CHNH), 5.64-5.75 (m, 1H, disappears with D2O), 5.84-6.00 (m,
1H, CHOH), 6.05-6.20 (m, 2H, disappears with D2O), 7.02-7.50
(m, 6H, 5Ar + H-3), 11.75 (s, 1H, COOH, disappears with D2O).
Anal. (C16H20N4O4) C, H, N.
Synthesis and Analytical Data of 2-[5-(3-Isopropylureido)-
pyrazol-1-yl]-1-phenylethan-1-ol 5b. Compound 5a (1.30 g, 4
mmol) was heated at 165 °C until complete development of CO2;
the residue was cooled to room temperature and purified by Florisil
(100-200 mesh) chromatography using CH2Cl2 as eluent. The
white solid obtained was recrystallized from absolute ethanol.
Yield: 17%. Mp: 136-137 °C. IR (KBr) cm-1: 3392, 3320, 3215
Experimental Section
1
(OH, NH), 1711 (CON). H NMR (DMSO-d6): δ 1.05-1.18 (m,
6H, 2CH3), 3.28-3.46 (m, 2H, disappears with D2O), 3.46-3.84
(m, 1H, CHNH), 4.27-4.60 (m, 2H, CH2N), 4.90-4.95 (m, 1H,
disappears with D2O), 5.48 (d, J ) 4.0, 1H, H-4), 5.90-6.00 (m,
1H, CHOH), 6.21 (d, J ) 4.0, 1H, H-3), 7.23-7.40 (m, 5H, Ar).
Anal. (C15H20N4O2) C, H, N.
Chemical Materials and Methods. All chemicals were obtained
from Sigma-Aldrich s.r.l. (Milan, Italy).
Melting points are not corrected and were measured with a Bu¨chi
540 instrument. IR spectra were recorded with a Perkin-Elmer 398
1
spectrophotometer. H NMR spectra were recorded on a Varian
Synthesis and Analytical Data of 5-Amino-1-(2-hydroxy-2-
phenylethyl)-1H-pyrazole-4-carbonitrile 8. To a solution of
2-hydrazino-1-phenylethanol (6.08 g, 40 mmol) in absolute ethanol
(50 mL) was added ethoxymethylenemalononitrile (4.88 g, 40
mmol) and the reaction mixture was refluxed for 6 h. The solvent
was concentrated to 50% of the initial volume and cooled. The
yellow solid obtained was filtered and recrystallized from absolute
ethanol. Yield: 63%. Mp: 180-181 °C. IR (KBr) cm-1: 3438,
3339, 3199 (OH, NH2), 2223 (CN). 1H NMR (CDCl3): δ 3.15 (br
s, 1H, OH, disappears with D2O), 3.98-4.27 (m, 2H, CH2N), 4.80
(br s, 2H, NH2, disappears with D2O), 5.08-5.18 (m, 1H, CHOH),
7.26-7.45 (m, 5H, Ar), 7.46 (s, 1H, H-3). Anal. (C12H12N4O) C,
H, N.
Synthesis and Analytical Data of 1-(2-Hydroxy-2-phenyl-
ethyl)-5-(3-isopropylureido)-1H-pyrazole-4-carbonitrile 5c. To
a mixture of 8 (2.3 g, 10 mmol) and anhydrous CH3COONa (2.0
g, 24 mmol) in anhydrous THF (30 mL), cooled at 0 °C, was added
phosgene (20% in toluene, 8 mL) dropwise, and the reaction mixture
was refluxed for 4 h. The excess phosgene was removed under
reduced pressure and isopropylamine (1.48 g, 25 mmol), solved in
anhydrous THF (5 mL), was added dropwise, at 0 °C. The mixture
reaction was stirred at room temperature overnight. After removal
of the volatiles in vacuo, the crude was suspended in water (20
mL) and extracted twice with CH2Cl2 (20 mL), and the organic
phase was dried (MgSO4). The solvent was evaporated under
reduced pressure to afford a yellow oil, which was purified by
Florisil (100-200 mesh) chromatography using diethyl ether as
eluent. Yield: 57%. Mp: 170 °C. IR (KBr) cm-1: 3306, 3229,
3194 (OH, NH), 2229 (CN), 1683 (CO). 1H NMR (CDCl3): δ 1.08
and 1.12 (dd, J ) 5.6, 6H, CH3), 2.60-3.00 (m, 2H, 2NH,
disappears with D2O), 3.75-3.94 (m, 1H, CHNH), 4.04-4.14 (m,
2H, CH2N), 4.96-5.08 (m, 1H, CHOH), 6.06 (d, J ) 6.0, 1H, OH,
disappears with D2O), 7.18-7.37 (m, 5H, Ar), 7.62 (s, 1H, H-3).
Anal. (C16H19N5O2) C, H, N.
Gemini 200 (200 MHz) instrument; chemical shifts are reported
as δ (ppm) relative to tetramethylsilane (TMS) as internal standard;
signals were characterized as s (singlet), d (doublet), t (triplet), q
(quartet), sept (septet), m (multiplet), or br s (broad signal); J are
reported in hertz.
All compounds were tested for purity by TLC (Kieselgel 60F254
DC-Alufolien, E. Merck, Darmstadt, Germany).
Elemental analyses, indicated by the symbols of the elements,
were within (0.4% of the theoretical values and were determined
with an EA 1110 elemental analyzer (Fison-Instruments, Milan,
Italy).
General Procedure for 1-(2-Hydroxy-2-phenylethyl)-5-(3-
alkyl/cycloalkylureido)-1H-pyrazole-4-carboxylic Acid Ethyl
Esters 4a-j and 1-(2-Hydroxy-2-phenylethyl)-5-(3-benzylure-
ido)-1H-pyrazole-4-carboxylic Acid Ethyl Esters 7a-i (Method
A). To a mixture of ethyl 5-amino-1-(2-hydroxy-2-phenylethyl)-
1H-pyrazole-4-carboxylate 3 (2.7 g, 10 mmol) and anhydrous CH3-
COONa (2.0 g, 24 mmol) in anhydrous THF (30 mL), at 0 °C,
was added dropwise phosgene (20% in toluene, 8 mL) and then
the reaction 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 dropwise at 0 °C; the mixture was
stirred 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
crystallized with diethyl ether. All products were purified by
recrystallization from absolute ethanol.
Analytical data for compounds 4a-j and 7a-i are available as
Supporting Information.
General Procedure for 1-(2-Hydroxy-2-phenylethyl)-5-(3-
phenylureido)-1H-pyrazole-4-carboxylic Acid Ethyl Esters 4k
and 6a-k (Method B). A mixture of 5-amino-1-(2-hydroxy-2-
phenylethyl)-1H-pyrazole-4-carboxylic acid ethyl ester 3 (2.7 g, 10
mmol) and the suitable phenyl isocyanate (11 mmol) in anhydrous
toluene (30 mL) was refluxed for 6 h. After cooling, the white
solids obtained were filtered and recrystallized from absolute
ethanol.
Synthesis and Analytical Data of 1-(2-Hydroxy-2-phenyl-
ethyl)-5-(3-phenylureido)-1H-pyrazole-4-carbonitrile 5d. A solu-
tion of 8 (2.3 g, 10 mmol) and phenyl isocyanate (1.2 g, 11 mmol)
in anhydrous toluene (30 mL) was refluxed for 6 h. After cooling,
the white solid obtained was filtered and recrystallized from absolute
ethanol. Yield: 78%. Mp: 214-215 °C. IR (KBr) cm-1: 3385,
3283, 3202 (NH, OH), 2226 (CN), 1715 (CON). 1H NMR (DMSO-
Analytical data for compounds 4k and 6a-k are available as
Supporting Information.

3624 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Bruno et al.
d6): 3.36 (d, J ) 1.8, 1H, OH, disappears with D2O), 4.18-4.50
(m, 2H, CH2N), 6.05-6.17 (m, 1H, CHOH), 6.65 (br s, 1H, NH,
disappears with D2O), 6.90-7.50 (m, 10H, Ar), 7.54 (s, 1H, H-3),
9.73 (br s, 1H, NH, disappears with D2O). Anal. (C19H17N5O2) C,
H, N.
Biological Materials and Methods. Hank’s balanced salt
solution without phenol red (HBSS, ICN Biomed, Milan, Italy)
mixed with Dulbecco’s phosphate-buffered saline (PBS, ICN
Biomed) (HBSS:PBS ) 3:1) containing 1 mg/mL bovine serum
albumin (BSA, Sigma, Milan, Italy) was used as incubation
medium. fMLP, IL8, ethidium bromide, and fluorescein diacetate
were from Sigma Chemical, St. Louis, MO.
Neutrophilic Polymorphonuclear Leukocyte Preparation.
Heparinized venous blood (10 U/mL heparin) was obtained from
healthy volunteers (20-37 yr old) after informed consent. Neu-
trophilic polymorphonuclear leukocytes (neutrophils) were prepared
by dextran sedimentation, followed by centrifugation (400g, 30 min)
on a Ficoll-Hypaque density gradient, as previously described.60
Contaminating erythrocytes were removed by hypotonic lysis.
Neutrophils resuspended in incubation medium were >97% pure,
as determined by morphologic analysis of Giemsa-stained cyto-
preparation.
stimulated with 10-7 M IL8 for 0.5 min. The reactions were stopped
by transferring 100 mL of each sample in an equivalent volume of
sample buffer (Tris-HCl, pH 6.8, 2 mmol/L Na3VO4, 10-3 M PMSF,
10 µg/mL aprotinin, 10 µg/mL leupeptin, 8% sodium dodecyl
sulfate (SDS), 10% mercaptoethanol, 17.5% glycerol, and 0.1%
bromophenol blue) prewarmed to 95 °C. The samples were
denatured by boiling for 10 min and then were loaded onto 7.5%-
20% SDS polyacrylamide gel. After the electrophoresis, the proteins
were transferred on nitrocellulose membrane at 4 °C for 45 min at
240 mA. The blots were blocked with 5% non-fat dry milk and
washed in Tris-buffered saline/Tween 20 (TBS-T, containing 10
mM Tris-base pH 7.4, 154 mM NaCl, and 0.05% Tween 20). The
membranes were stained with µg/mL anti-phosphotyrosine mAb
clone 4G10 (Upstate) (overnight incubation at 4 °C). After washing
with TBS-T, the membranes were incubated with anti-mouse
horseradish peroxidase-linked anti-IgG Ab. The membranes were
washed again under the same conditions, and antibody complexes
were visualized by the enhanced chemiluminescence (ECL) detec-
tion system (Amersham, Little Chalfont, U.K.) according to the
manufacturer’s instructions.
IL8-Induced Akt Phosphorylation in Human Neutrophils.
Western blot analysis was carried out as previously described.64
After purification, neutrophils (1 × 107cells/mL) were pretreated
with compounds 4a (0.06 µM), 4d (0.01 µM), and 4k (0.05 µM),
for 30 min, and then they were incubated in air at 37 °C in the
absence or presence of 10 nM IL8 for 5 min. The reaction was
stopped on ice, and the cells were centrifugated at 4 °C. After
removing the supernatants, the pellets were lysed in 400 µL of NP40
buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM NaF, 1%
NP40, 10 µg/mL glycerol, 10 µg/mL aprotinin, 10 µg/mL leupeptin,
1 mM PMSF, 0.5 mM Na3VO4) and incubated for 30 min at 4 °C.
The samples were spun at 13 000g for 10 min at 4 °C. Protein
content was determined with BCA protein assay (Pierce) using
bovine serum albumin as standard. The cytoplasmic extracts were
boiled in loading buffer (62.5 mM Tris-HCl pH 6.8, 0.75% SDS,
3.75% 2-mercaptoethanol, 8.75% glycerol, and 0.025% bromophe-
nol blue) and then resolved by SDS-polyacrilamide electrophoresis
(5% stacking gel and 12% running gel). After the electrophoresis,
the proteins (40 g each sample) were transferred on nitrocellulose
membrane at 4 °C for 45 min at 240 mA. The blots were blocked
with 5% non-fat dry milk and washed in Tris-buffered saline/Tween
20 (TBS-T, containing 10 mM Tris-base pH 7.4, 154 mM NaCl,
and 0.05% Tween 20). The membranes were stained with µg/mL
anti-human phosphorylated Akt mAb (Santa Cruz Biotechnology)
(overnight incubation at 4 °C). After washing with TBS-T, the
membranes were incubated with anti-rabbit horseradish peroxidase-
linked anti-IgG Ab. The membranes were washed again under the
same conditions, and antibody complexes were visualized by the
enhanced chemiluminescence (ECL) detection system (Amersham,
Little Chalfont, U.K.) according to the manufacturer’s instructions.
Densitometric and Statistical Analysis. Images of blots were
analyzed by scanning densitometry quantified with the image
analyzer system (Syngene). Data were expressed as mean ( SEM.
One-way ANOVA with Bonferroni’s post-test was performed using
GraphPad InStat version 3.05 for Windows XP, GraphPad Software
(San Diego, CA). Differences between treatment groups were
judged to be statistically significant at p < 0.05.
Assessment of Neutrophil Viability. Neutrophils (2 × 106/mL)
were incubated for 2 h in tissue culture tubes (17 × 100 mm,
Falcon, Becton Dickinson) in incubation medium at 37 °C in a
CO2 atmosphere (0.5 mL final volume), with appropriate doses of
compounds 3, 4a-k, 5a-d, 6a-k, and 7a-i. Cell viability,
measured as integrity of membrane, was assessed by an ethidium
bromide-fluorescein diacetate test according to Dankberg61 as
previously described by Ottonello.62 Briefly, cells (4 × 104/100
mL) harvested from culture tubes were mixed with 50 mL of
staining solution (2 mg/mL fluorescein diacetate, 4 µg/mL ethidium
bromide) in HBSS and incubated for 10 min at room temperature.
Thereafter, a drop of cell suspension was placed on a slide, sealed
with a coverslip, and analyzed under UV light in a dark field
illumination. Neutrophils with intact membrane (i.e., viable cells)
appeared as green fluorescent cells, whereas neutrophils with
damage and ethidium bromide-permeable membrane (i.e., necrotic
cells) displayed a fluorescent red nucleus.
Boyden Chamber Migration Assay. Neutrophil locomotion was
studied by means of the leading front method, as previously
described by Corcione.63 Neutrophils were preincubated in the
absence or presence of appropriate doses of compounds 3, 4a-k,
5a-d, 6a-k, and 7a-i, for 15 min at room temperature. Tests
were conducted in duplicate, using blind well chambers (Neuro-
Probe, Gathersburg, MD) with a 3 µm pore size cellulose ester
filter (Millipore, Milan, Italy) separating the upper from the lower
compartment of the chambers. Then cells (4 × 105), in the absence
or presence of various amounts of tested compounds, were placed
in the upper compartment of the chambers. Chemoattractants, 10-9
M IL8 or 10-8 M fMLP, were placed in the lower compartment of
the chambers. Experiments were also carried out without chemoat-
tractants in the lower compartment (spontaneous migration). After
incubation at 37 °C for 45 min, the filters were removed, fixed in
ethanol, stained with Harris hematoxylin, dehydrated, cleared with
xylene, and mounted in Eukitt (Kindler, GmbH). Then, the distance
(mm) travelled by the leading front of cells was measured × 400
magnification. Five randomly chosen fields were read for each filter.
The net migration was determined by subtracting spontaneous
migration, i.e., the distance travelled by neutrophils in the absence
of the stimulus, from the distance travelled by neutrophils toward
the stimulus. The concentration of each compound giving 50%
inhibition (IC50) of net migration was obtained from nonlinear
regression analysis with SPSS for Windows version 6.0, Wacker
Drive, Chicago, IL.
Determination of the Total Cellular F-Actin. After the isolation
from blood from young healthy volunteers, neutrophils were tested
in the presence or absence of compounds 4a, 4d, and 4k. After the
incubation, neutrophils were washed three times and then stimulated
for 30 min with control medium or 100 nM IL8. After this
treatment, neutrophil slides were prepared by cytospin centrifuge,
and F-actin was stained with alexa-Fluor 488-conjugated phalloidin.
The content of F-actin was determined by fluorescence microscopy
(Nikon Optiphot-2, Nikon, Melville, NY). Image capturing was
performed with a Hamamatsu color-chilled 3 CCD camera. All
images were captured using identical camera settings (time of
exposure, brightness, contrast and sharpness) and an appropriated
white balance set according to the fluorescence filter and acquired
and analyzed by Image-Pro Plus 4.0 (from Media Cybernetics).
IL8-Induced Tyrosine Phosphorylation in Human Neutro-
phils. Western blot analysis was carried out as previously de-
scribed.64 Neutrophils were incubated at 2.5 × 107 cells/mL with
10-3 M diisopropylfluorophosphate for 30 min at room temperature.
The cells were then incubated with or without compounds 4a (0.06
µM), 4d (0.01 µM), and 4k (0.05 µM) for 30 min and subsequently

Inhibitors of Neutrophil Chemotaxis
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