N-Aryl-2-phenyl-2,3-dihydro-imidazo[1,2-b]pyrazole-1-carboxamides 7-substituted strongly inhibiting both fMLP-OMe- and IL-8-induced human neutrophil chemotaxis

Article abstract of DOI:10.1016/j.ejmech.2011.11.031

Anomalous activation of neutrophil recruitment is one of the causes of many inflammatory diseases. The chemoattractants N-formyl-methionyl-leucyl- phenylalanine (fMLP), and interleukine 8 (IL8) play a pivotal role in neutrophil chemotaxis regulation in th

Full text of DOI:10.1016/j.ejmech.2011.11.031

European Journal of Medicinal Chemistry 47 (2012) 573e579
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
N-Aryl-2-phenyl-2,3-dihydro-imidazo[1,2-b]pyrazole-1-carboxamides
7-substituted strongly inhibiting both fMLP-OMe- and IL-8-induced human
neutrophil chemotaxis
,
,
,
Chiara Brullo a 1, Susanna Spisani b 1, Rita Selvatici c, Olga Bruno a
*
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, viale Benedetto XV, 3, 16132 Genova, Italy
b Dipartimento di Biochimica e Biologia Molecolare, Sezione di Biologia Molecolare, Università degli Studi di Ferrara, via Luigi Borsari, 46, 44121 Ferrara, Italy
c Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Genetica Medica, Università degli Studi di Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Anomalous activation of neutrophil recruitment is one of the causes of many inflammatory diseases. The
Received 20 April 2011
Received in revised form
17 November 2011
Accepted 17 November 2011
Available online 25 November 2011
chemoattractants N-formyl-methionyl-leucyl-phenylalanine (fMLP), and interleukine
8 (IL8) play
a pivotal role in neutrophil chemotaxis regulation in the latter and early stages, respectively, probably by
two independent mechanisms. We reported here synthesis and biological evaluation of new N-aryl-2-
phenyl-2,3-dihydro-imidazo[1,2-b]pyrazole-1-carboxamides 7-substituted which were designed as
possible multi-target antiinflammatory agents. Many of the title compounds showed a good inhibition, in
the nano molar range, of human neutrophil chemotaxis selectively acting toward fMLP-OMe (methyl-
ester of fMLP) or IL8 stimulus; whereas, two compounds showed an interesting dual activity inhibiting
both fMLP-OMe and IL8-induced chemotaxis at nano molar concentration.
Keywords:
N-Aryl-2-phenyl-2,3-dihydro-imidazo[1,2-
b]pyrazole-1-carboxamides
IL8
Ó 2011 Elsevier Masson SAS. All rights reserved.
fMLP-OMe
Chemotaxis
Human neutrophils
Inflammation
1. Introduction
inflamed site. Each step of this patho-physiological event is regu-
lated by downstream signalling molecules, including phosphati-
In the last decade the over-regulation of neutrophil recruitment
mechanisms has been clearly indicated as one of the cause of many
inflammatory diseases.
As an example one of the most characteristic changes of
inflammation in cystic fibrosis (CF) is a large infiltration by
neutrophils into the lung parenchyma [1].
dylinositol 3-kinase (PI3K), G-protein-coupled receptor kinases
(GPKs), phospholipase C, that produce the second messengers
responsible for elevation of intracellular free calcium, protein
kinase C (PKC) and mitogen activated protein kinases (MAPKs)
activation [2e4]. Recently, it has been demonstrated that the fMLP-
OMe chemotactic response is associated with specific PKC b1 iso-
The chemoattractants more involved in neutrophil chemotaxis
are N-formyl-methionyl-leucyl-phenylalanine (fMLP) and inter-
leukine 8 (IL8 also named CXCL8) which bind on specific G-protein-
coupled receptors (FPR and CXCR8, respectively). While fMLP plays
a pivotal role in inflammation consequent to bacterial infections,
IL8 mediates the response of neutrophils to physical injury.
Neutrophil recruitment is a complex process including rolling,
adhesion, extravasation, actin polymerization and movement to
form translocation and p38 MAPK phosphorylation [5,6].
Heit and co-workers [7] proposed a model in which the end
target chemoattractants (such as fMLP), that is of importance
during the latter phase of the tracking process, function primarily
by stimulating p38 MAPK, whereas intermediary (intermediate-
type) chemoattractants (such as IL8), as one of importance in the
early stage of neutrophil recruitment, primarily function via the
PI3K/Akt (protein kinase B) pathway. Moreover, it has been
demonstrated that a hierarchy between the two pathways exists.
The same signalling hierarchy exists with regard to NADPH-oxidase
activation by both formylated peptide and IL8 [8].
* Corresponding author. Tel.: þ39 010 3538367; fax: þ39 010 3538358.
E-mail address: obruno@unige.it (O. Bruno).
In the search for new antiinflammatory agents interfering with
human neutrophil migration we recently reported many pyrazolyl-
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.031

574
C. Brullo et al. / European Journal of Medicinal Chemistry 47 (2012) 573e579
EtOOC
1a NR' = isopropylamino
1b NR' = benzylamino
1c NR' = N-benzylpiperazino
1d NR' = anilino
O
N
N
N
R'N
H
OH
1e NR' = oF-anilino
1f NR' = mF-anilino
1g NR' = pF-anilino
R
O
1h NR' = 1-naphthylamino
1
Ar
N
N
H
N
N
R
3
2a R = H
N
2b R = COOH
HN
N
2c R = COOEt
2d R = CONH
2e R = CO-cyclopropylamino,
2f R = CO-piperidino
2
2
Fig. 1. General molecular structure of compounds 1, 2 and 3.
Fig. 2. Doseeresponse curve of compounds 3a, 3b, 3c, 3h, 3i, 3j, 3l, 3m, 3r, CSA
(positive control) and DMSO (negative control) in human neutrophil fMLP-OMe-
stimulated chemotaxis. Data are expressed as a percentage of the C.I.. SEs are within
10% of the mean value.
urea derivatives (compounds 1, Fig. 1) able to block IL8-induced
chemotaxis at nM concentrations [9].
The most active compounds were the 3-benzyl-, 3-(4-
benzylpiperazinyl)-, 3-phenyl-, 3-fluorophenyl, and the 3-
isopropylureido derivatives, having a carboxyethyl group in posi-
tion 4 of the pyrazole moiety. These compounds were inactive
toward the CXC receptors, while their involvement in the complex
intracellular mechanisms of neutrophil recruitment was confirmed
by the inhibition of tyrosine phosphorylation in the 50e70 kDa
region and by a remarkable decrease of F-actin polymerization.
At the same time, we synthesized new interesting 2-phenyl-2,3-
dihydro-1H-imidazo[1,2-b]pyrazoles 7-substituted (compounds 2,
Fig. 1) which, in a preliminary study, resulted in inhibiting at nM
concentration the fMLP-OMe-induced chemotaxis [10]. The most
active compounds are reported in Fig. 1. They were unable to
displace [3H]-fMLP from its specific binding site and were found to
be ineffective as antagonists in superoxide anion production, as
well as in granule enzyme release. For that reason they could be
considered as “pure” fMLP-induced chemotaxis inhibitors [11].
A more complete understanding of intracellular signalling trig-
gered by different chemotaxins should be of great importance in
models of inflammation. In this context the development of new
molecules able to discriminate between the different activation
pathways of chemotaxis is of great interest for this search area.
On the other hand, in the next ten years emerged a new drug
discovery paradigm which consider the development of “multiple
targeted drugs” more profitable than the classic “one target-one-
disease” approach [12,13]. The “polypharmacology” paradigm is
aimed to increase the in vivo efficacy by synergic actions and to
reduce the side effects by a decreasing of dose administration.
NR2
O
NR2
O
O
O
O
O
HN
N
Ar
Ar
N
N
N
N
H
N
N
H
N
N
e
N
9a, NR'2 = cyclopropylamino, 71%
9b, NR'2 = piperidino, 52%
19-45%
23-43%
3o-r
3f - j
O
f
e
O
O
OH
O
O
N
O
H2N
HO
N
N
N
N
N
Ar
HN
N
HN
N
HN
N
N
H
N
N
e
a
c
d
b
OH
NHNH2
70%
80%
79%
74%
51-59%
g
4
5
6
7
8
3a - e
NC
NH2
NH2
NH2
O
O
O
O
N
b
H2N
HO
h
e
N
N
N
N
Ar
H2N
HO
N
N
HN
N
N
H
N
63%
65%
41%
10-63%
3k-n
10
12
11
Scheme 1. Reagents and conditions: (a) (Ethoxymethylene) cyanoacetate, anhydrous toluene, 70e80 ꢀC, 8 h; (b) conc. H2SO4, r.t., 15 min; (c) 2M NaOH solution, 120 ꢀC, 4 h; (d)
heating at 190 ꢀC to complete CO2 evolution; (e) anhydrous N,N-DMF, arylisocyanate, 100 ꢀC, 12e18 h; (f) anhydrous N,N-DMF, excess cyclopropylamine or piperidine, anhydrous
Et3N, DPPA, 30e60 ꢀC, 12 h g) (ethoxymethylene)malononitrile, absolute ethanol, reflux, 6 h; (h) 2M ethanol/water (50%) NaOH solution, reflux, 2 h.

C. Brullo et al. / European Journal of Medicinal Chemistry 47 (2012) 573e579
575
Compounds 3 have been tested in in vitro chemotaxis assays, in
human neutrophils stimulated by both fMLP-OMe and IL8 in order
to obtain more structure activity relationship (SAR) information
about their ability to interact with different chemoattractant
pathways.
2. Chemistry
Compounds 3aer were prepared reacting the intermediates 6,
8, 9a, 9b, and 12 with the suitable arylisocyanates in anhydrous
N,N-DMF, as reported in Scheme 1.
The 2-hydrazino-1-phenylethanol (4) is the key starting product
to obtain the above intermediates. Compound (5), obtained by
reaction of 4 with ethyl (ethoxymethylene) cyanoacetate [14], was
treated with concentrated sulphuric acid at 0 ꢀC to give the 2-
phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylic acid
ethyl ester (6). Alkaline hydrolysis to the carboxylic acid 7, and
subsequent decarboxylation give 2-phenyl-2,3-dihydro-1H-imi-
dazo[1,2-b]pyrazole (8) [10]. Acid derivative 7 was treated with an
excess of the suitable amine (cyclopropilamine or piperidine) in the
presence of anhydrous triethylamine and diphenylphosphorylazide
(DPPA) to afford compounds 9aeb. This method gave only the
amide derivatives without carboxy-azide formation and subse-
quent Curtius rearrangement, as we have already reported [10]. By
condensation of 2-hydrazino-1-phenylethanol (4) with ethox-
ymethylenemalononitrile in absolute ethanol we obtained the
5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbonitrile
(10) [10] which was then hydrolysed in alkaline ethanol/water
solution to 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-
carboxamide (11). Finally, 2-phenyl-2,3-dihydro-1H-imidazo[1,2-
b]pyrazole-7-carboxamide (12) was prepared by the same cycliza-
tion procedure used for 6. It is to note that all our attempts to obtain
compounds 3 by direct cyclization of the pyrazolyleureas 1 were
unsuccessful.
Fig. 3. Doseeresponse curve of compounds 3a, 3b, 3c, 3h, 3i, 3j, 3l, 3m, 3r, CSA
(positive control) and DMSO (negative control) in human neutrophil IL8-stimulated
chemotaxis. Data are expressed as a percentage of the C.I.. SEs are within 10% of the
mean value.
Multitarget therapies have been already tried by combining
different therapeutic mechanism with drugs cocktails or with
multicomponent formulations. A more recent strategy is to develop
a single molecule able to simultaneously interact with different
pharmacological targets.
Taking into account the above considerations we designed
a new series of N-aryl-2-phenyl-2,3-dihydro-imidazo[1,2-b]pyr-
azole-1-carboxamides 7-substituted (compounds 3, Fig. 1)
including in the same structure both the rigid scaffold of fMLP-OMe
(2) and the peculiar urea moieties of the most active IL8-induced
chemotaxis inhibitors (1). The substituents which were peculiar
of the most active compounds 2 were maintained in position 7.
Table 1
Structure and potency expressed as IC50 values of the compounds 3aer in the chemotaxis inhibition of human neutrophils.
R
O
Ar
N
N
H
N
N
Comp.
R
Ar
Inhibition of fMLP-OMe-induced
Inhibition of IL8-induced
chemotaxis IC50 ꢁ SEM (nM)
chemotaxis IC50 ꢁ SEM (nM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
H
H
H
H
C6H5
4.8 ꢁ 0.43
5.4 ꢁ 0.60
12.1 ꢁ 1
95 ꢁ 9
>1000
>1000
>1000
>1000
>1000
>1000
7.5 ꢁ 0.7
111 ꢁ 10
>1000
206 ꢁ 18
6.2 ꢁ 0.7
1.2 ꢁ 0.1
484 ꢁ 45
>1000
(o)-F-C6H4
(m)-F-C6H4
(p)-F-C6H4
H
a
-naphthyl
>1000
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
CONH2
CONH2
CONH2
CONH2
C6H5
98 ꢁ 9.1
116 ꢁ 10
9.2 ꢁ 0.88
10.3 ꢁ 0.92
13.8 ꢁ 1.2
>1000
3.9 ꢁ 0.40
11.4 ꢁ 1.1
110 ꢁ 10
123 ꢁ 13
138 ꢁ 14
980 ꢁ 90
5.5 ꢁ 0.63
(o)-F-C6H4
(m)-F-C6H4
(p)-F-C6H4
a
-naphthyl
(o)-F-C6H4
(m)-F-C6H4
(p)-F-C6H4
a-naphthyl
C6H5
(m)-F-C6H4
C6H5
(m)-F-C6H4
COeNHecycloprop
COeNHecycloprop
CO-Pip
13.2 ꢁ 1.1
>1000
>1000
1.2 ꢁ 0.1
CO-Pip

576
C. Brullo et al. / European Journal of Medicinal Chemistry 47 (2012) 573e579
3. Pharmacology
scaffold is essential to inhibit fMLP-OMe-induced chemotaxis.
Biological data suggest that the lack of substituents in position 7
directs the action toward fMLP-OMe signal pathway rather than IL8
one. However, in the 7-substituted compounds it is possible to
selectively modulate the activity toward the two different ways by
changing the steric hindrance of the carbamoyl moiety.
On the other hand, compounds 3l and 3r, strongly inhibiting
either fMLP-OMe- and IL8- induced chemotaxis, demonstrated
that these new class of arylcarbamoyl-imidazo-pyrazoles can
be properly defined as multi-target neutrophil chemotaxis
inhibitors.
Indeed, it is well know that fMLP stimulation is prevalent in the
latter phase of the tracking process, while IL8 acts in the early stage
of neutrophil recruitment. It has been demonstrated that they have
two different pathways of action and that a hierarchy between the
two pathways exists. Actually, these new imidazopyrazoles are able
to interfere with two different steps of neutrophils recruitment.
Preliminary Western blotting experiments with the compounds 31
in neutrophils activated with either fMLP or with IL8, do not show
AKT phosphorylation, suggesting that PI3K/Akt pathway is not
activated. A further careful investigation about its mechanism of
action, particularly in the PI3K/Akt, MAPK and PKC pathways which
are the primarily involved in the neutrophil recruitment cascade,
has been planned.
Finally, we reported here new imidazoepyrazoles which are the
first example of compounds able to inhibit two different chemo-
taxis mechanisms. This good versatility represents the most
important characteristic of these new molecules, particularly as
concerns its pharmacological application. In fact, the more selective
ones could be very useful as pharmacological tools to clearly
understand the intracellular mechanism of neutrophil recruitment.
Whereas, the dual active compounds are a main starting point for
the development of new multi-targeted antiinflammatory drugs.
Release of the cytoplasmic enzyme LDH was used as an indicator
of cell viability. In none of the experiments described below was the
percentage of total LDH release >3% (data not shown).
All compounds were tested in in vitro chemotaxis assays, in
human neutrophils stimulated by 10 nM fMLP-OMe and 1 nM IL8
following 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, in the absence and
in the presence of derivatives.
In Fig. 2 (graphic 1) and Fig. 3 (graphic 2) we reported the
doseeresponse curves of the most active compounds towards the
fMLP-OMe and IL8-induced chemotaxis, respectively. The antago-
nist concentrations inhibiting the fMLP-OMe- or IL-8-induced
chemotaxis by 50% (IC50) were obtained by the computer analysis
of inhibition curves (Table 1). Data where compared with positive
(cyclosporine A, CSA) and negative (dimethylsulfoxide, DMSO,
blank) controls.
4. Results and discussion
The introduction in the rigid scaffold of compounds 2 of peculiar
urea moieties of compounds 1 gave new substituted 2,3-dihydro-
imidazopyrazoles 3 able to inhibit both fMLP-OMe and IL8-induced
neutrophil chemotaxis. In particular 3aee, which were unsub-
stituted in position 7, were selective toward fMLP-OMe triggered off
mechanism; 3hej, 3l, 3m and 3r (bearing a carboxyethyl or a car-
boxamide in 7 position) were the most active having IC50 values in
the nanomolar range.
Compounds 3o and 3p, having in position 7 a cyclopropylamide
group and an arylcarbamoyl substituents in 1, differently than the
analogue unsubstituted in 1 position compound 2e, were inactive.
Most of the active compounds (3b, 3c, 3h, 3i, 3l, 3m, and 3r) have
a fluoroanilino-carbamoyl substituent in position 1.
6. Experimental Protocols
As concerns IL8-induced chemotaxis compounds 3g, 3k, 3l, 3o
and 3r (bearing a carboxyethyl or a carboxamide in 7 position) were
strongly active having IC50 in the nanomolar range. All the other
compounds were very poor or inactive. The fluoroanilino-
carbamoyl substitution in position 1 (see 3g, 3h, 3l, 3r) gave the
most active compounds in the IL8-induced chemotaxis, as well; it is
noteworthy that the previous compounds 1 showed the same
behaviour and the 3-fluorophenyl derivative 1b showed a good
inhibition of neutrophils chemotaxis in a preliminary in vivo test
(mouse model of zymosan-induced peritonitis) [9].
Finally, compounds 3l and 3r (both having an amide substituent
in position 7 and a m-fluoroanilino group in the urea moiety)
showed an interesting dual activity profile being active at nano-
molar concentration in both fMLP-OMe and IL8-induced
chemotaxis.
6.1. Chemistry
6.1.1. General
Chiminord and Aldrich Chemical, Milan, Italy purchased all
chemicals. Solvents were reagent grade. Unless otherwise stated,
all commercial reagents were used without further purification.
Aluminium backed silica gel plates (Merck DC-Alufolien Kieselgel
60 F254) were used in thin-layer chromatography (TLC) for
routine monitoring the course of reactions. Detection of spots was
made by UV light. Merck silica gel, 230e400 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-
Elmer 398 spectrophotometer. 1H NMR and 13C NMR spectra
were recorded on a Varian Gemini 200 (200 MHz) instrument;
chemical shifts are reported as d (ppm) relative to tetramethylsilane
5. Conclusion
(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 Hz. Elemental analyses, indi-
cated by the symbols of the elements, were within ꢁ0.4% of the
theoretical values and were determined with an Elemental Analy-
ser EA 1110 (Fison-Instruments, Milan, Italy).
In conclusion, we designed and synthesized new 1-arylcarba-
moyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazoles in order to obtain
by a unique structure both the inhibition of IL8- and fMLP-OMe-
induced chemotaxis (activities which were already showed by
our previous pyrazolyleureas 1 and 1-unsubstituted 2,3-dihydro-
1H-imidazo[1,2-b]pyrazoles 2, respectively). Most of the new
synthesized compounds were chemotaxis inhibitors, particularly
toward fMLP-OMe chemoattractant, at nM concentration. Since the
6.1.2. General procedure for compounds 3aer
To the starting product (8 for compounds 3aee,
6 for
compounds 3fej, 12 for compounds 3ken and 9a,b for compounds
3oer) (5 mmol) solved in anhydrous N,N-dimethylformamide
(DMF) (5 mL) the proper arylisocyanate (6 mmol) was added and
the reaction mixture was stirred at 100 ꢀC for 12 h (compounds
previously synthesized analogue pyrazolyleureas
completely inactive as fMLP-OMe-induced chemotaxis inhibitors,
we can conclude that the more rigid dihydro-imidazo-pyrazolyl
1
were

C. Brullo et al. / European Journal of Medicinal Chemistry 47 (2012) 573e579
577
3aej) or 18 h (compounds 3ker). After cooling the mixture was
poured into an ice-water bath and 1 M HCl solution was added until
pH 2. The solid precipitated was filtered, dried on air and the purity
was verified by TLC. When it needed the final product was purified
by flash chromatography (Silica gel, CHCl3 or diethyl ether as
eluents). The crude solids were recrystallized from absolute
ethanol.
6.1.2.7. Ethyl
dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylate 3g. Yield 23%,
m.p. 122e123 ꢀC 1H NMR (CDCl3):
4.16 (near q, 1 H, H3), 4.34 (q, J ¼ 7.2, 2 H, CH3CH2), 4.78 (near t, 1 H,
H2), 6.48 (near q, 1 H, H3), 6.98e7.80 (m, 9 H, Ar), 7.91 (s, 1 H, H6),
10.83 (s, 1 H, NH, slowly disappears with D2O). 13C NMR (DMSO):
1-{[(2-fluorophenyl)amino]carbonyl}-2-phenyl-2,3-
d
1.38 (t, J ¼ 7.2, 3 H, CH3CH2),
d
13.65, 39.13, 51.90, 60.61, 66.01, 96.90, 115.06, 115.45, 123.95,
124.02, 124.54, 125.35, 127.76, 128.51, 140.24, 144.44, 144.74, 150.39,
163.84. IR (KBr): cmꢂ1 3242 (NH), 1681 (CONH þ COOEt). Anal.
(C21H19FN4O3) C, H, N.
6.1.2.1. N-Phenyl-2-phenyl-2,3-dihydro-imidazo[1,2-b]pyrazole-1-car-
boxamide 3a. Yield 53%, m.p. 155 ꢀC. 1H NMR (CDCl3):
d 4.22 (near q,
1 H, H3), 4.81 (near t, 1 H, H2), 5.85 (near q, 1 H, H3), 6.03 (d, J ¼ 2.0,
1 H, H7), 6.53 (s,1 H, NH, slowly disappears with D2O), 6.98e7.50 (m,
6.1.2.8. Ethyl
dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylate 3h. Yield 40%,
m.p. 123e124 ꢀC 1H NMR (CDCl3):
1-{[(3-fluorophenyl)amino]carbonyl}-2-phenyl-2,3-
10 H, Ar), 7.54 (d, J ¼ 2.0, 1 H, H6). 13C NMR (DMSO):
d 39.07, 53.56,
63.57, 87.57, 119.53, 122.61, 125.18, 127.79, 128.15, 128.56, 138.35,
140.18, 143.25, 145.25, 149.47. IR (KBr): cmꢂ1 3357 (NH), 1680
(CONH). Anal. (C18H16N4O) C, H, N.
d
1.42 (t, J ¼ 7.2, 3 H, CH3CH2),
4.16 (near q, 1 H, H3), 4.42 (q, J ¼ 7.2, 2 H, CH3CH2), 4.78 (near t, 1H,
H2), 6.48 (near q, 1 H, H3), 6.62e6.80 and 7.10e7.50 (2 m, 9 H, Ar),
7.91 (s, 1 H, H6), 11.44 (s, 1H, NH, slowly disappears with D2O). 13
C
6.1.2.2. N-(2-Fluorophenyl)-2-phenyl-2,3-dihydro-imidazo[1,2-b]pyr-
NMR (DMSO): d 13.60, 39.15, 51.95, 60.59, 66.12, 96.96, 114.36,
azole-1-carboxamide 3b. Yield 59%, m.p. 149e150 ꢀC 1H NMR
125.22, 127.51, 128.60, 140.70, 140.91, 141.15, 143.02, 143.38, 150.38,
163.86. IR (KBr): cmꢂ1 3259 (NH), 1680 (CONH þ COOEt). Anal.
(C21H19FN4O3) C, H, N.
(CDCl3):
d 4.25 (near q, 1 H, H3), 4.83 (near t, 1 H, H2), 5.87 (near q,
1 H, H3), 6.03 (d, J ¼ 2.0, 1 H, H7), 6.82 (s, 1 H, NH, slowly disappears
with D2O), 6.85e7.46 (m, 8 H, Ar), 7.49 (d, J ¼ 2.0, 1 H, H6), 8.09 (t,
J ¼ 8.0, 1 H, Ar). 13C NMR (DMSO):
d
(39.13, 53.57, 63.88, 87.43,
6.1.2.9. Ethyl
dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylate 3i. Yield 36%,
m.p. 149 ꢀC. 1H NMR (CDCl3):
1-{[(4-fluorophenyl)amino]carbonyl}-2-phenyl-2,3-
114.96, 115.35, 123.85, 125.07, 125.44, 125.60, 127.81, 128.54, 139.95,
143.25, 144.97, 149.44, 152.12, 157.01). IR (KBr): cmꢂ1 3387 (NH),
1679 (CONH). Anal. (C18H15FN4O) C, H, N.
d
1.41 (t, J ¼ 7.2, 3 H, CH3CH2), 4.16
(near q, 1 H, H3), 4.40 (q, J ¼ 7.2, 2 H, CH3CH2), 4.78 (near t, 1 H, H2),
6.46 (near q, 1 H, H3), 6.85e7.05 (m, 2 H Ar), 7.15e7.60 (m, 7 H, Ar),
6.1.2.3. N-(3-Fluorophenyl)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]
7.91 (s, 1 H, H6), 11.29 (s, 1 H, NH, slowly disappears with D2O). 13
C
pyrazole-1-carboxamide 3c. Yield 50%, m.p. 138e139 ꢀC 1H NMR
NMR (DMSO): d 13.59, 39.17, 51.95, 60.55, 66.11, 96.95, 114.70,
(CDCl3):
d
4.21 (near q, 1 H, H3), 4.79 (near t, 1 H, H2), 5.84 (near q,
115.10, 120.41, 120.60, 125.31, 127.59, 128.53, 135.49, 140.81, 143.25,
143.42, 150.60, 163.84. IR (KBr): cmꢂ1 3264 (NH), 1683 (COOEt),
1672 (CONH). Anal. (C21H19FN4O3) C, H, N.
1 H, H3), 6.01 (d, J ¼ 2.0, 1 H, H7), 6.62 (s, 1 H, NH, slowly disappears
with D2O), 6.69e7.48 (m, 9 H Ar), 7.53 (d, J ¼ 2.0, 1 H, H6). 13C NMR
(DMSO):
d (39.10, 51.71, 65.20, 99.69, 114.40, 125.32, 127.71, 128.52,
140.72, 140.80, 141.15, 142.98, 143.45, 150.40). IR (KBr): cmꢂ1 3336
(NH), 1674 (CONH). Anal. (C18H15FN4O) C, H, N.
6.1.2.10. Ethyl
dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylate 3j. Yield 27%,
m.p. 131e132 ꢀC. 1H NMR (CDCl3):
1-[(1-Naphthylamino)carbonyl]-2-phenyl-2,3-
d
1.33 (t, J ¼ 7.2, 3 H, CH3CH2),
6.1.2.4. N-(4-Fluorophenyl)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]
4.15e4.35 (m, 3 H, CH3CH2 þ H3), 4.84 (near t, 1 H, H2), 6.52 (near q,
pyrazole-1-carboxamide 3d. Yield 59%, m.p. 155e156 ꢀC 1H NMR
1 H, H3), 7.20e7.92 (m, 12 H, Ar), 7.97 (s, 1 H, H6), 11.18 (s, 1 H, NH,
(CDCl3):
d
4.21 (near q, 1 H, H3), 4.80 (near t, 1 H, H2), 5.85 (near q,
slowly disappears with D2O). 13C NMR (DMSO):
d 14.09, 39.12,
1 H, H3), 5.99 (d, J ¼ 2.0, 1 H, H7), 6.52 (s, 1 H, NH, slowly disappears
52.97, 58.44, 64.67, 91.33, 126.42, 127.58, 128.20, 140.31, 143.08,
154.61, 161.99. IR (KBr): cmꢂ1 3171 (NH), 1680 (CONH þ COOEt).
Anal. (C25H22N4O3) C, H, N.
with D2O), 6.80e7.49 (m, 9 H, Ar), 7.52 (d, J ¼ 2.0, 1 H, H6). 13C NMR
(DMSO):
d (39.13, 53.57, 63.59, 87.55, 114.48, 114.92, 121.43, 121.59,
125.17, 127.75, 128.54, 134.66, 140.17, 143.21, 145.15, 149.55, 155.25,
160.01). IR (KBr): cmꢂ1 3340 (NH),1681 (CONH). Anal. (C18H15FN4O)
C, H, N.
6.1.2.11. N1-(2-Fluorophenyl)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-
b]pyrazole-1,7-dicarboxamide 3k. Yield 42%, m.p. 221 ꢀC 1H NMR
(CDCl3):
d 1.78 (s, 2 H, NH2 disappears with D2O), 4.18 (near q, 1 H,
6.1.2.5. N-(1-Naphthyl)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyr-
H3), 4.82 (near t, 1 H, H2), 6.41 (near t, 1 H, H3), 7.00e7.70 (m, 9 H,
azole-1-carboxamide 3e. Yield 51%, m.p. 124e125 ꢀC 1H NMR
Ar), 7.75 (s, 1 H, H6), 10.85 (s, 1 H, NH, slowly disappears with D2O).
(CDCl3):
d
4.24 (near q, 1 H, H3), 4.81 (near t, 1 H, H2), 5.84 (near q,
13C NMR (DMSO):
d 39.10, 51.32, 64.80, 99.65, 115.06, 115.45, 123.95,
1 H, H3), 6.09 (d, J ¼ 2.0, 1 H, H7), 6.70e6.85 (m, 2 H Ar), 7.15e7.83
124.02, 124.54, 125.35, 127.76, 128.51, 140.24, 144.44, 144.74, 150.39,
165.72. IR (KBr): cmꢂ1 3345, 2773 (NH2), 3195 (NH), 1680 (CONH),
1642 (CONH2). Anal. (C19H16FN5O2) C, H, N.
(3m, 12 H, 10 Ar þ H6 þ NH, 1 H disappears with D2O). 13C NMR
(DMSO):
d 39.10, 53.57, 63.78, 87.22, 126.40, 127.59, 128.23, 140.34,
143.05, 154.55). IR (KBr): cmꢂ1 3399 (NH), 1686 (CONH). Anal.
(C22H18N4O *0,5 (C2H5OH) C, H, N.
6.1.2.12. N1-(3-Fluorophenyl)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-
b]pyrazole-1,7-dicarboxamide 3l. Yield 63%, m.p. 206 ꢀC 1H NMR
6.1.2.6. Ethyl 1-{[phenylamino]carbonyl}-2-phenyl-2,3-dihydro-1H-
(DMSO-d6): d 4.02 (near q, 1 H, H3), 4.87 (near t, 1 H, H2), 6.40 (near
imidazo[1,2-b]pyrazole-7-carboxylate
3f. Yield
43%,
m.p.
t, 1 H, H3), 6.52e7.50 (m, 9 H, 7Ar þ NH2 which disappears with
141e142 ꢀC 1H NMR (CDCl3):
d
1.41 (t, J ¼ 7.0, 3 H, CH3CH2), 4.15
D2O), 7.92 (s, 1 H, H6), 8.00e8.35 (m, 2 H, Ar), 12.95 (s, 1 H, NH,
(near q, 1 H, H3), 4.32 (q, J ¼ 7.0, 2 H, CH3CH2), 4.79 (near t, 1 H, H2),
6.50 (near q, 1 H, H3), 6.95e7.78 (m, 10 H, Ar), 7.91 (s, 1 H, H6), 11.23
slowly disappears with D2O). 13C NMR (DMSO):
d 39.10, 51.31, 64.83,
99.69, 114.38, 125.20, 127.61, 128.49, 140.67, 140.89, 141.11, 142.92,
143.38, 150.36, 165.74. IR (KBr): cmꢂ1 3344, 2798 (NH2), 3193 (NH),
1650 (CONH2). Anal. (C19H16FN5O2).
(s, 1 H, NH, slowly disappears with D2O). 13C NMR (DMSO):
d (13.65,
39.17, 51.90, 60.61, 66.01, 96.90, 119.63, 122.71, 125.28, 127.80,
128.25, 128.56, 138.41, 140.20, 143.27, 145.35, 149.57, 163.84). IR
(KBr): cmꢂ1 3260 (NH), 1697 (COOEt), 1682 (CONH). Anal.
(C21H20N4O3) C, H, N.
6.1.2.13. N1-(4-Fluorophenyl)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-
b]pyrazole-1,7-dicarboxamide 3m. Yield 41%, m.p. 224 ꢀC 1H NMR

578
C. Brullo et al. / European Journal of Medicinal Chemistry 47 (2012) 573e579
(DMSO-d6):
d
4.03 (near q, 1 H, H3), 4.84 (near t, 1 H, H2), 6.38 (near
6.2. Biological assays
t, 1 H, H3), 7.00e7.55 (m, 9 H, 7 H Ar þ NH2 which disappears with
D2O), 7.90 (s, 1 H, H6), 8.00e8.35 (m, 2 H, Ar), 12.70 s, 1 H, NH,
6.2.1. Neutrophils preparation
slowly disappears with D2O. 13C NMR (DMSO):
d
39.12, 51.33, 64.77,
Cells were obtained from the blood of healthy subjects, and
human peripheral blood neutrophils were purified by using the
standard techniques of dextran (Pharmacia, Uppsala, Sweden) sedi-
mentation, centrifugation on FicollePaque (Pharmacia), and hypo-
tonic lysis of contaminating red cells. Cells were washed twice and
resuspended in KrebseRinger phosphate (KRPG), pH 7.4, at a final
concentration of 50 ꢃ 106 cells/mL and kept at room temperature
until used. Neutrophils were 98e100% viable, as determined using
the Trypan blue exclusion test. Local Ethics Committee approved the
study and informed consent was obtained from all participants.
99.67, 114.68, 115.12, 120.38, 120.54, 125.20, 127.57, 128.47, 135.47,
140.79, 143.12, 143.37, 150.57, 165.70. IR (KBr): cmꢂ1 3352, 2843
(NH2), 3201 (NH), 1671 (CONH), 1642 (CONH2). Anal. (C19H16FN5O2)
C, H, N.
6.1.2.14. N1-1-Naphthyl-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyr-
azole-1,7-dicarboxamide 3n. Yield 10%, m.p. 228e229 ꢀC 1H NMR
(DMSO-d6): d 1.55 (br s, 2 H, NH2 disappears with D2O), 4.08 (near q,
1 H, H3), 4.91 (near t, 1 H, H2), 6.43 (near q, 1 H, H3), 7.20e8.12 (m,
12 H, Ar), 8.20 (s, 1H, H6), 12.41 (s, 1 H, NH, slowly disappears with
D2O). 13C NMR (DMSO):
d
39.10, 51.31, 64.83, 99.68, 126.40, 127.36,
6.2.2. Preparation of chemoattractants and tested compounds
fMLP-OMe (Sigma, St. Louis, MO, USA) and tested compounds
(10ꢂ2 M) were solved in DMSO, while CXCL8 (10ꢂ5 M) was solved in
water. Before the use, all the solutions were diluted in KRPG to
obtain the final concentrations wished from the experimental
protocol. At the concentrations used, DMSO did not interfere with
any of the biological assays performed.
128.10, 140.44, 143.28, 154.42, 165.74. IR (KBr): cmꢂ1 3334, 2800
(NH2), 3197 (NH),1665 (CONH),1643 (CONH2). Anal. (C23H19N5O2) C,
H, N.
6.1.2.15. N7-Cyclopropyl-N1,2-diphenyl-2,3-dihydro-1H-imidazo[1,2-
b]pyrazole-1,7-dicarboxamide 3o. Yield 19%, m.p. 228e229 ꢀC 1H
NMR (CDCl3): d 0.60e0.78 and 0.89e0.95 (2 m, 4 H, 2 CH2-cyclop.),
2.89 (m,1 H, CH-cyclop.), 4.14 (near q,1 H, H3), 4.79 (near t,1 H, H2),
6.65 (near q, 1 H, H3), 6.98e7.90 (m, 12 H, 10 Ar þ H6 þ NH which
disappears with D2O), 11.55 (s, 1 H, NH, slowly disappears with
6.2.3. Random locomotion
Random locomotion was performed with 48-well micro-
chemotaxis chamber (Bio Probe, Milan, Italy) and migration into
the filter was evaluated by the leading-front method [15], esti-
mating the distance in micrometers which the leading edge of the
D2O). 13C NMR (DMSO):
d 5.25, 5.41, 22.53, 39.17, 51.27, 64.77, 99.91,
118.78, 122.40,125.22, 127.58,128.37, 128.47, 139.08, 140.77, 142.69,
142.93, 150.61, 164.80. IR (KBr): cmꢂ1 3308 (NH), 2686 (NH-cy-
clop.), 1668 (CONH-Ph), 1636 (CONH-cyclop.). Anal. (C22H21N5O2)
C, H, N.
cell migrated. The actual control random movement is 35 ꢁ 3
mm SE
of six separate experiments performed in duplicate.
6.2.4. Chemotaxis
6.1.2.16. N7-Cyclopropyl-N1-(3-fluorophenyl)-2-diphenyl-2,3-dihyd-
ro-1H-imidazo[1,2-b]pyrazole-1,7-dicarboxamide 3p. Yield 30%,
Directional movement or chemotaxis was measured with the
same chamber used for the random locomotion, adding fMLP-OMe
10 nM or CXCL8 1 nM in the lower compartment.
m.p. 235e236 ꢀC 1H NMR (CDCl3):
d 0.50e0.60 and 0.70e0.80 (2 m,
4 H, 2 CH2-cyclop.), 2.80e3.00 (m, 1 H, CH-cyclop.), 4.02 (near q,
1 H, H3), 4.82 (near t, 1 H, H2), 6.38 (near q, 1 H, H3), 6.75e6.90 (m,
2 H, 1 H Ar þ NH), 7.15e7.50 (m, 7 H, Ar), 8.00 (s, 1 H, H6), 8.70 (d,
1 H, Ar), 12.65 (s, 1 H, NH slowly disappears with D2O). 13C NMR
Each tested compound was added in the upper compartment of
the chemotaxis chamber, diluted at concentrations ranging from
10ꢂ12 to 10ꢂ6M with KRPG containing 1 mg/mL of bovine serum
albumin (BSA; Orha Behringwerke, Germany).
(DMSO):
d
5.20, 5.41, 22.60, 39.20, 51.35, 64.50, 99.90, 114.41,
Neutrophils were preincubated with tested compounds 10 min
before the functional test. 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].
125.25, 127.72, 128.55, 140.80, 140.95, 141.21, 142.98, 143.45, 150.42,
164.85. IR (KBr): cmꢂ1 3311 (NH), 2654 (NH-cyclop.), 1668 (CONH-
Ph), 1636 (CONH-cyclop). Anal. (C22H20FN5O2) C, H, N.
6.1.2.17. N7-Piperidinyl-N1,2-diphenyl-2,3-dihydro-1H-imidazo[1,2-
The C.I. of fMLP-OMe is 1.2 at 10ꢂ8 M, while the value of CXCL8 is
b]pyrazole-1,7-dicarboxamide 3q. Yield 22%, m.p. 224e225 ꢀC 1H
1.02 at 10ꢂ9 M.
NMR (CDCl3):
d
1.45e1.75 (m, 6 H, 3 CH2-pip.), 3.55e3.91 (m, 4 H, 2
The antagonism was measured as C.I. in the absence (100%) and
in the presence of the tested compounds at different concentra-
tions. The values are means of six separate experiments performed
in duplicate. Standard errors are within 10% of the mean value [16].
IC50 values were calculated from the sigmoid doseeresponse
curve by a non-linear regression analyses (Graph Pad Prism, San
Diego, USA).
CH2N-pip.), 4.15 (near q, 1 H, H3), 4.76 (near t, 1 H, H2), 6.32 (near
q, 1 H, H3), 6.92e7.09 (m, 1 H, Ar), 7.20e7.61 (m, 10 H, 9Ar þ H6)
10.41 (s, 1 H, NH, slowly disappears with D2O). 13C NMR (DMSO):
d
23.50, 39.15, 52.35, 65.07, 98.80, 104.93, 105.40, 107.45, 108.75,
118.76, 122.46, 125.30, 127.57, 128.40, 128.50, 139.28, 140.85,
142.70, 142.97, 150.75, 195.60. IR (KBr): cmꢂ1 3251 (NH), 1686
(CONH-Ph), 1624 (CON-pip.). Anal. (C24H25N5O2) C, H, N.
6.2.5. Measure of cell viability
6.1.2.18. N7-Piperidinyl-N1-(3-fluorophenyl)-2-diphenyl-2,3-dihydro-
1H-imidazo[1,2-b]pyrazole-1,7-dicarboxamide 3r. Yield 45%, m.p.
In order to assess possible cytotoxic effects of the tested
compounds, the cytoplasmic marker enzyme, lactate dehydroge-
nase (LDH), was determined by measuring the rate of oxidation of
NADH. The absorbance change was followed at 340 nm [17].
192e193 ꢀC 1H NMR (DMSO-d6):
d 1.35e1.65 (m, 6H, 3 CH2-pip),
3.40e3.80 (m, 4H, 2 CH2-N-pip), 4.07 (near q, 1 H, H3), 4.83 (near t,
1 H, H2), 6.21 (near q, 1 H, H3), 6.73e6.88 (m, 1H Ar), 7.15e7.42 (m,
8H, Ar), 7.59 (s, 1H, H6), 10.46 (s, 1H, NH, disappears with D2O). 13
C
Acknowledgements
NMR (DMSO):
d 23.49, 39.12, 52.24, 65.03, 98.72, 104.92, 105.44,
107.58, 108.70, 114.10, 115.38, 125.29, 127.70, 128.49, 129.89, 140.30,
140.54, 142.36, 142.62, 149.55, 163.57, 172.31, 195.46. IR (KBr): cmꢂ1
3264 (NH), 1692 (CONH), 1612 (CON-pip). Anal. (C24H24FN5O2)
C, H, N.
This work was supported by Grants from University of Genoa
and University of Ferrara; Fondazione CARIGE, Genova; Associa-
zione Emma e Ernesto Rulfo per la Genetica Medica, Parma; Fon-
dazione Cassa di Risparmio di Ferrara, Italy.

C. Brullo et al. / European Journal of Medicinal Chemistry 47 (2012) 573e579
579
[9] O. Bruno, C. Brullo, F. Bondavalli, S. Schenone, A. Ranise, N. Arduino,
M.B. Bertolotto, F. Montecucco, L. Ottonello, F. Dallegri, M. Tognolini,
V. Ballabeni, S. Bertoni, E. Barocelli, Synthesis and biological evaluation of N-
pyrazolyl-N0-alkyl/benzyl/phenylureas: a new class of potent inhibitors of
interleukin 8-induced neutrophil chemotaxis, J. Med. Chem. 50 (2007)
3618e3626.
The authors wish to thank Dr. R. Raggio, M. Anzaldi, Mr. O.
Gagliardo and F. Tuberoni for spectral recording; Dr. Katia Varani
University of Ferrara, for help in IC50 calculation and Banca del
Sangue di Ferrara for providing fresh blood.
[10] E. Fabbri, S. Spisani, L. Barbin, C. Biondi, M. Buzzi, S. Traniello, G. Pagani
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