Design, synthesis and biological evaluation of new pyrazolyl-ureas and imidazopyrazolecarboxamides able to interfere with MAPK and PI3K upstream signaling involved in the angiogenesis

Article abstract of DOI:10.1016/j.ejmech.2017.03.066

Taking into account the structure activity relationship information given by our previous studies, we designed and synthesized a small library of pyrazolylureas and imidazopyrazolecarboxamides fluorinated on urea moiety and differently decorated on pyrazo

Full text of DOI:10.1016/j.ejmech.2017.03.066

Accepted Manuscript
Design, synthesis and biological evaluation of new pyrazolyl-ureas and
imidazopyrazolecarboxamides able to interfere with MAPK and PI3K upstream
signaling involved in the angiogenesis
Elda Meta, Chiara Brullo, Adama Sidibe, Beat A. Imhof, Olga Bruno
PII:
S0223-5234(17)30233-7
10.1016/j.ejmech.2017.03.066
EJMECH 9326
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 December 2016
Revised Date: 9 March 2017
Accepted Date: 26 March 2017
Please cite this article as: E. Meta, C. Brullo, A. Sidibe, B.A. Imhof, O. Bruno, Design, synthesis and
biological evaluation of new pyrazolyl-ureas and imidazopyrazolecarboxamides able to interfere with
MAPK and PI3K upstream signaling involved in the angiogenesis, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.066.
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Graphical abstract
Design, synthesis and biological evaluation of new pyrazolyl-ureas and
imidazopyrazolecarboxamides able to interfere with MAPK and PI3K upstream
signaling involved in the angiogenesis
Elda Meta, Chiara Brullo, Adama Sidibe, Beat A. Imhof and Olga Bruno

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of new pyrazolyl-ureas and
imidazopyrazolecarboxamides able to interfere with MAPK and PI3K upstream signaling
involved in the angiogenesis
Elda Meta a, Chiara Brullo a*, Adama Sidibe b, Beat A. Imhof b and Olga Bruno a
a
Department of Pharmacy, Medicinal Chemistry Section, University of Genoa, Viale Benedetto
XV, 3 -16132 Genoa, Italy
b Department of Pathology and Immunology, University of Geneva, Rue Michel-Servet 1-
CH - 1211 Geneva, Switzerland
Key words
p38MAPK phosphorylation; ERK1/2 phosphorylation; Akt phosphorylation; migration;
pyrazolylureas; imidazopyrazole derivatives.
Abstract
Taking into account the structure activity relationship information given by our previous studies, we
designed and synthesized a small library of pyrazolylureas and imidazopyrazolecarboxamides
fluorinated on urea moiety and differently decorated on pyrazole nucleus. All compounds were
preliminary screened by Western blotting technique to evaluate their activity on MAPK and PI3K
pathways by monitoring ERK1/2, p38MAPK and Akt phosphorylation, and also screened with a
wound healing assay to assess their capacity in inhibiting endothelial cell migration, using human
umbilical vein endothelial cells stimulated with VEGF. Pyrazoles and imidazopyrazoles did not
show the same activity profile. SAR consideration showed that specific substituents and their
position in pyrazole nucleus, as well as the type of substituent on the phenylurea moiety play a
pivotal role in determining increase or decrease of kinases phosphorylation. On the other hand the
loss of flexibility in imidazopyrazole derivatives is responsible for activity potentiation. Screening
of the compound library for inhibition of endothelial cell migration, a function required for
angiogenesis, showed significant activity for compound 3. This compound might interfere with cell
migration by modulating the activity of different upstream target kinases. Therefore, compound 3
represents a potential inhibitor of angiogenesis. Furthermore, it may be used as a tool to identify
unknown mediators of endothelial migration and thereby unveiling new therapeutic targets for
controlling pathological angiogenesis in diseases such as cancers.

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Abbreviations
Human umbilical vein endothelial cells, HUVECs; bone marrow-derived cells, BMDCs; basic
fibroblast growth factor, bFGF; epithelial-mesenchymal transition, EMT; extracellular response
kinases 1 and 2, ERK1/2; formyl-methyl-leucyl-phenylalanine peptide, fMLP; inteleukine-8, IL-8,
CXCL8; matrix metallo-proteinases 2, MMP-2; mitogen activated protein kinases, MAPKs;
phosphatidylinositol 3-kinase, PI3K; protein kinases B, Akt/PKB; structure activity relationship,
SAR; transforming growth factor-β, TGF-β; vascular endothelial growth factor, VEGF.
1. Introduction
Angiogenesis consists in the formation of new blood vessels by sprouting from pre-existing ones;
consequently it is vital for physiological processes during development and in the adult during
wound healing, tissue growth and repair.
Pathological angiogenesis is required for the outcome of several diseases including cancers [1,2].
Indeed tumor cell hyperproliferation generates hypoxic areas inducing angiogenesis to rebuild
oxygen and nutrient supply [3].
Physiological and pathological angiogenesis share similar mechanisms, one of them is the
involvement of vascular endothelial growth factor (VEGF) and it’s receptors (VEGFR-1 and 2).
Expression of VEGF is induced by hypoxia and is up-regulated in solid tumors [4, 5].
The VEGF/VEGFR axis induces key events such as endothelial cell sprouting, migration,
proliferation and survival; VEGF induces these functions through activation of several
serine/threonine kinases in the phosphatidylinositol 3-kinase (PI3K) signaling pathway including
protein kinases B (Akt/PKB) and the mitogen-activated protein kinases (MAPK) signaling
pathways such as extracellular response kinases 1 and 2 (ERK1/2) and p38MAPK. Finding new
inhibitors that could affect the function of these kinases in endothelial cells would help to develop
new potential therapeutic agents against pathological angiogenesis in solid cancers. Moreover,
activation of ERK1/2 and p38MAPK was found crucial for other processes related to poor
prognosis in cancers including epithelial to mesenchymal transition (EMT) (a typical process of the
tumor cells), metastasis and recruitment of bone marrow-derived cells [6, 7]. This indicates that
new drugs interfering with these mediator activities may target different components of the tumor
microenvironment.
During our studies aimed at developing new anti-inflammatory drugs, we previously designed and
synthesized different compound libraries able to inhibit the neutrophils chemotaxis by interfering
with ERK1/2, Akt and p38MAPK phosphorylation after stimulation by Interleukine-8 (IL-8) or
formyl-methyl-leucyl-phenylalanine peptide (fMLP) [8–13]. In particular, we demonstrated that our

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pyrazolyl-urea (compounds I, Fig.1) differentially acted on ERK1/2, PI3K/Akt and p38MAPK
phosphorylation depending on which chemoattractant (IL-8 or fMLP) was used [10]. A series of 4-
carboxyethyl-pyrazole derivatives (compounds II, Fig. 1), bearing in N1 hydroxyalkyl chains
characterized by different length, showed a notable increase of inhibitory activity, with respect to
the previous compounds, inhibiting both fMLP-OMe- (a synthetic derivative of fMLP) and IL-8-
induced chemotaxis at very low concentration (pM-nM range) [9]. We also reported the inhibitory
effects of a little series of imidazo[1,2-b]pyrazole derivatives bearing in position 7 an acid, an ester
or different amides substituents (compounds III, Fig. 1); implemented blocking effects was then
evidenced by a library of more complex molecules containing the peculiar substituted urea moiety
(typical of compounds I and II) fused with the more rigid structure of imidazopyrazole scaffold
(compounds IV, Fig. 1) [11, 12]. All imidazopyrazoles inhibited fMLP-OMe or IL8-induced
neutrophil chemotaxis in a dose-dependent manner with IC50 values in the low nanomolar range.
Further investigation evidenced that our inhibitors were able to significantly decrease PKC and
p38MAPK phosphorylation after fMLP-OMe or IL-8 stimulation [13]. Notably, in the pyrazole
series the 3-fluorophenylurea derivatives in vivo showed the most efficient chemotaxis inhibition
[8,9].
Fig. 1. General structure of compounds I, II, III and IV.
Given these potent inhibitory actions of our library compounds on MAPK activation and
subsequent neutrophil chemotaxis, we aimed at designing and synthesizing new series of
pyrazolylureas (compounds 1–16, Fig. 2) and imidazopyrazolecarboxamides (compounds 17–27,
Fig. 2), that could interfere with MAPK and PI3K signaling pathways in endothelial cells during
angiogenesis.
Taking into account the structure activity relationship (SAR) information given by previous studies,
we functionalized the urea moiety in all new derivatives with a phenyl ring, bearing in turn

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fluorinated substituents in different positions. Then, we combined old and new decorations, their
type and position, both on the pyrazole and imidazopyrazole scaffold, to increase the molecular
diversity.
In detail, the carboxyethyl substituent in position 4, which was typical for the previous compounds I
and II, was maintained in 1–8; the same hydroxyalkyl chains already present in compounds II were
inserted in N1 (compounds 1–4) while the hydroxyphenylethyl group was oxidized (compounds 5–
8) in order to modulate the lipophilicity of the molecules. As concerns the phenylurea moiety, we
inserted in compound 1–4 a trifluoromethyl substituent in position 3, while compounds 5–8 were
decorated with different fluorinated substituents in all positions. In compounds 9–16, differently
than in compounds I and II, a carboxyethyl or t-butyl substituent was inserted on position 3 of the
pyrazole scaffold (position never investigated for our previous compounds). In addition we
designed the new imidazopyrazoles 17–19 by maintaining in position 7 the most efficient
substituents of previous IV (carboxyethyl, amide, piperidine-amide) while in the urea moiety we
inserted the new 3-trifluoromethylphenyl substituent. Finally, compounds 20–27 were designed as
more rigid analogues of compounds 9–16.
Fig. 2. General structures of compounds 1–27.
All compounds were preliminary screened using Western blotting in order to evaluate their activity
on the phosphorylation of ERK1/2, p38MAPK and Akt in human umbilical vein endothelial cells
(HUVEC) stimulated with VEGF, and in a wound healing assay to evaluate the activity on
endothelial cell migration upon VEGF stimulation. We report in this paper the synthesis, Western
blot analyses and SAR considerations regarding the newly designed compounds 1–27, as well as
their activity on endothelial cell migration.

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2. Results and discussion
2.1 Chemistry
To obtain compounds 1–4 we condensed ethyl ethoxymethylenecyanoacetate with the hydrazino-
hydroxyalkyl derivatives 28a–d, which were in turn prepared by reaction of the appropriate oxirane
with hydrate hydrazine, as previously reported [9]. The obtained intermediate 4-carboxyethyl-5-
amino-pyrazoles 29a–d were then treated with 3-trifluoromethylphenylisocyanate in anhydrous
toluene affording the desired ethyl 3-(trifluoromethyl)phenylureido-pyrazole-4-carboxylates 1–4
(Scheme 1).
Scheme 1. Synthesis of compounds 1–4. Reagents and conditions: (a) Hydrate hydrazine, 100 °C, 15 min, 70–82%; (b)
Ethyl ethoxymethylenecyanoacetate, an. toluene, 70–80 °C, 8 h, 61–72%; (c) 3-trifluoromethyl-phenylisocyanate, an.
toluene, reflux, 5h, 41–58%.
To prepare compounds 5–8 the hydroxy group of 4-carboxyethyl-5-amino-pyrazole 29d was
oxidized with sulphur trioxide pyridine complex in DMSO in the presence of triethylamine (TEA)
affording the keton 30 (scheme 2), which was then reacted with a little excess of the suitable
phenylisocyanate in anhydrous toluene at reflux giving the desired urea derivatives.
Scheme 2. Synthesis of compouds 5–8. Reagents and conditions: (a) sulphur trioxide pyridine complex, DMSO, TEA,
20 °C, 30 min, 82%; (b) substituted phenylisocyanate, an. toluene, reflux, 12h, 32–55%.
Compounds 9–12 and 13–16 were synthesized as reported in scheme 3. The key intermediate for
the preparation of the pyrazole scaffold is the potassium salt of 1-cyano-3-ethoxy-3-oxoprop-1-en-
2-olate 31 that was prepared following a literature procedure [14] by reacting acetonitrile with
diethyloxalate in the presence of potassium tert-butoxide. The use of 18-Crown-6 as a catalyst

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provided a strong increase in reaction yield in respect to the literature method. The obtained salt 31
was then condensed with the 2-hydrazino-1-phenylethanol 28d in absolute ethanol in the presence
of catalytic amount of glacial acetic acid yielding the ethyl 5-amino-1-(2-hydroxy-2-phenylethyl)-
1H-pyrazole-3-carboxylate 32. The same intermediate 28d was reacted with the 4,4-dimethyl-3-
oxopentanenitrile to obtain the 2-(5-amino-3-(tert-butyl)-1H-pyrazol-1-yl)-1-phenylethanol 33. The
reaction of 32 or 33 with a little excess of the suitable phenylisocyanate in anhydrous toluene at
reflux gave the urea derivatives 9–12 and 13–16, respectively.
Scheme 3. Synthesis of compounds 9–16. Reagents and conditions: (a) an THF, 18-Crown-6, 0–60 °C, 30 min, 98%;
(b) 31, abs. ethanol, conc. acetic acid, reflux 6 h, 59%; (c) 4,4-dimethyl-3-oxopentanenitrile, an. ethanol, conc. acetic
acid, reflux, 10 h, 83%; (d) substituted phenylisocyanate, an. toluene, reflux, 6–12 h, 37–89%.
As previously reported [11], by dehydration of the 4-carboxyethyl-5-amino-pyrazole 29d, we
obtained the ethyl 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-7-carboxylate 34 (scheme 4).
The carboxyethyl function of 34 was hydrolyzed to give carboxylic derivative 35 which was then
reacted with an excess of piperidine in the presence of diphenylphosphorylazide (DPPA) to obtain
the amide derivative 36, as previously reported [11]. The treatment of 34 or 36 with a little excess
of 3-trifluoromethyl-phenylisocyanate in anhydrous toluene at reflux afforded compounds 17 and
19, respectively (Scheme 4).

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Scheme 4. Synthesis of compounds 17 and 19. Reagents and conditions: (a) conc. H2SO4, rt, 15 min, then NH4OH,
80%; (b) 3-(trifluoromethyl)phenylisocyanate, an. DMF, reflux, 18 h, 36%; (c) 2M NaOH , EtOH, reflux, 4 h, 79%; (d)
piperidine excess, an. DMF, DPPA, TEA, 30–60 °C, 12 h, 52%.
By condensation of 2-hydrazino-1-phenylethanol 28d with ethoxymethylenemalononitrile we
obtained the 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carbonitrile 37, which was
hydrolized to the amide derivative 38; the latter was cyclized to the imidazopyrazole derivative 39
as previously reported [11], and finally we treated 39 with 3-trifluoromethylphenylisocyanate to
give compound 18 (Scheme 5).
O
NC
H2N
O
O
H2N
O
H2N
NH2
N
N
H2N
HO
H2N
HO
HN
N
N
N
N
a
b
c
d
HN
N
N
N
HO
N
H
F3C
28d
37
38
39
18
Scheme 5. Synthesis of compounds 18. Reagents and conditions: (a) Ethoxymethylenemalononitrile, abs. EtOH, 70–
80 °C, 6 h. 63%; (b) 2M NaOH , EtOH, reflux, 4 h, 65%; (c) conc. H2SO4, rt, 15 min, then NH4OH, 41%; (d) 3-
(trifluoromethyl)phenylisocyanate, an. DMF, reflux, 18 h, 28%.
Starting from the above reported 3-substituted pyrazoles 32 and 33, we obtained, by water
elimination in the presence of concentrated sulfuric acid, the imidazopyrazoles 40 and 41,
respectively, which were treated with a little excess of the suitable substituted phenyl isocyanate in
anhydrous toluene at reflux giving the imidazopyrazole-1-carboxamides 20–23 and 24–27,
respectively (Scheme 6).

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Scheme 6. Synthesis of compounds 20–27. Reagents and conditions: (a) conc. H2SO4, rt, 15 min., then NH4OH, 78–
80%; (b) substituted phenylisocyanate, an. toluene, reflux, 12–18 h, 27-83%.
2.2 Analysis of the compounds effects on MAPK and PI3K signaling pathways in HUVEC
The newly synthesized compounds were tested for their effects on the activation of MAPK and
PI3K signaling pathways by monitoring p38MAPK, ERK1/2 and Akt phosphorylation induced by
VEGF in HUVEC. Therefore, the compounds (1–27) were dissolved in DMSO and diluted in M199
medium to obtain a final solution at 20µM concentration. Confluent HUVEC were starved for at
least 4 h to reduce baseline kinase activities due to the serum in the culture medium. The cells were
then incubated for 10 min with fresh medium containing the compounds or control DMSO and then
stimulated with VEGF (50 ng/ml) and the indicated compound for 20 min. Then protein extracts
were prepared and analyzed for phosphorylation of p38MAPK, ERK1/2 and Akt by Western
blotting.
2.2.1 Compounds activities monitored with p38MAPK phosphorylation and SAR considerations
The activity of the compounds on p38MAPK phosphorylation is shown in Fig. 3. The most
representative appearing bands and the relative densitometric analyses are shown. The results are
expressed as percentage of control of the same blot (VEGF and DMSO only). Data are
representative of at least two independent experiments. All new compounds (1–27) were able to
interfere with the p38MAPK phosphorylation. Some of them decreased phosphorylation, others
increased it. In particular, the 4-substituted pyrazoles (1–8) strongly increase phosphorylation, the
most active being 3 (+ 180%) and 4 (+ 150%). The oxidation of the hydroxyalkyl chains (5–8) did
not cause a relevant difference in phosphorylation increasing, therefore suggesting that neither the
hydroxyl function nor the chiral center are important for the activity. Conversely the 3-substituted

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pyrazoles (9–16) generally showed a decrease, the most active being 11 (- 81%), 12 (- 61%), 13 (-
74%) and 15 (- 70%). Exceptions were observed for compounds 9 and 14 that strongly increased
p38MAPK phosphorylation (+ 97% and + 199% respectively).
These results demonstrate that in VEGF-stimulated HUVEC, the 4-substitutions in pyrazole mainly
interfered with the MAPK signaling pathways leading to increase of p38MAPK phosphorylation,
whereas the 3-substitution in pyrazoles led to a blockade of p38MAPK phosphorylation as clearly
evidenced by comparing the isomers 4 and 12.
As concerns the more rigid imidazopyrazole derivatives (17–27) the Western blot revealed a
behavior similar to that of pyrazole derivatives. In particular, all 7-substituted derivatives (17–19)
strongly increased the p38MAPK phosphorylation (+ 200–300%), more than the flexible analogues
4-substituted pyrazoles This indicates that in the 7-substituted imidazopyrazoles the structure
rigidity may stabilize and improve the increasing effect, as it is clearly evidenced by comparing 17
with 4.
The 6-substituted imidazopyrazoles (20–26), as their analogs 3-substituted pyrazoles, showed a
moderate phosphorylation decrease. In this case the imidazopyrazole rigidity rendered them less
potent toward decreasing p38MAPK phosphorylation. The only exception was compound 27 that
showed a big increase (+ 140%).

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Fig. 3. Representative Western blotting analyses on p38MAPK phosphorylation. HUVEC were preincubated with the
compounds for 10 min and then stimulated with both VEGF and the compounds for 20 min. DMSO was used as control.
The proteins extracted and analyzed by western blotting, representative band of at least two independent experiments
are reported (A). The densiometric analyses were done with ImageJ (B). Tubulin was used as the protein loading
control. All compounds affected p38MAPK phosphorylation.
2.2.2 Compounds activities monitored with ERK1/2 phosphorylation and SAR considerations
The activity of the compounds (1–27) on the ERK 1/2 phosphorylation was measured in the same
manner as for p38MAPK (Fig 4). Also in these experiments most of the compounds were able to
interfere with the protein kinases phosphorylation. The pyrazole series 1–4 globally decreased
ERK1/2 phosphorylation showing higher effect with longer chains at N1 of the pyrazoles.
Exceptionally, compound 2 was inactive on ERK1/2 phosphorylation. Also the series 9–16 showed
a general decrease of ERK1/2 phosphorylation. In these pyrazole series, compounds 3, 4, 9 and 15
showed the highest decrease (- 59%, - 70%, - 89%, - 67%, respectively).
The oxidation of the hydroxyalkyl chain did not cause a clear influence on this kinase activity, as
two compounds (6 and 7) increased ERK1/2 phosphorylation, one (8) decreased it, while one (5)
was inactive.
In the imidazopyrazole series, particularly in compounds 17–19, the activity seems strictly to rely
on the nature of the substituent in position 7. Indeed, compound 17, bearing a 7-carboxyethyl group,
showed slight decrease of ERK phosphorylation (- 26%), while the 7-piperidine amide 19 increased
ERK phosphorylation (+ 83%). No activity was observed with the 7-amide derivative 18. Of note,
in general the 3-trifluoromethylphenylureas caused a stronger decrease of ERK phosphorylation,
the most active being 23 (- 75%), with the only exception of 19 that strongly increased it.
SAR considerations on ERK and p38 phosphorylation are different showing that our derivatives
may interfere at different upstream levels in the MAPK signaling pathways.

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Fig. 4. Representative Western blotting analyses on ERK1/2 phosphorylation. HUVEC were preincubated with the
compounds for 10 min and then stimulated with both VEGF and the compounds for 20 min. DMSO was used as control.
The proteins extracted and analyzed by western blotting, representative band of at least two independent experiments
are reported (A). The densiometric analyses were done with ImageJ (B). Tubulin was used as the protein loading
control. Most of the compounds affected ERK1/2 phosphorylation.
2.2.3 Compounds activities monitored with Akt phosphorylation and SAR considerations
All compounds were also analyzed for their interference with PI3K signaling pathway in HUVEC
upon VEGF stimulation. Therefore we analyzed their effect on Akt phosphorylation (Fig. 5). In the
first pyrazole series (1–4) a moderate decrease of Akt phosphorylation was observed. Compounds 3
and 4 showed the highest effect (-29% and -37% respectively). This effect seemed to slightly
correlate with the length of the chain in N1, as seen also for ERK1/2. The oxidation of the alcoholic
group caused an inversion of the activity as clearly seen by comparing compound 4 (decrease) with
its oxidized analogue 8 (increase). Interestingly, among this series (5–8) compounds 6 and 7,
bearing a 3-fluorophenyl and 4-fluorophenyl urea moiety respectively, showed the highest Akt
phosphorylation increase (+ 63% and + 96%). It is to note that the 2-fluorophenyl urea derivative 5,

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as well as all the others 2-fluorophenyl urea derivatives in the other series (9, 13, 20 and 24), did
not affect Akt phosphorylation.
The 3-carboxyethyl substituted pyrazoles 9–12 showed a poor decrease of Akt phosphorylation
therefore indicating that the movement of the carboxyethyl group from position 4 (compounds 1–4)
to position 3 (compounds 9–12) did not affect type of activity neither potency. Exception was the 3-
fluorophenylurea derivative 10, which showed strong increase (+ 51%). By introducing in position
3 a tert-butyl instead of a carboxyethyl group (series 13–16), we obtained more potent inhibitors of
Akt phosphorylation, being compound 15 the most active (- 83%). Interestingly, within this series
compound 14, which is a 3-fluorophenylurea derivative as 10, is another exception. Indeed, 14
showed an increase of Akt phosphorylation, which was also stronger than that of the carboxyethyl
derivative 10. These results demonstrated that the 3-fluorophenylurea group determines an
inversion of activity (from decrease to increase) towards Akt phosphorylation, while the tert-butyl
substituent increments anyway the potency in respect with the carboxyethyl one.
In the imidazopyrazole series 17–23 we observed an increase of Akt phosphorylation. In particular
by comparing the 3-carboxyethylpyrazole derivatives 9–12 with the analogue 6-carboxyethyl
imidazopyrazoles 20–23, we may suppose that the scaffold rigidity is responsible for the activity
inversion (from decrease to increase) and for the implemented potency of the 3-fluorophenylurea
derivative 21 in respect with 10. Finally, the 6-tert-butyl derivatives 24–27 showed a different
behavior in respect to the 6-carboxyethyl series 20–23 but there is not a clear structure activity
relationship.

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Fig. 5. Representative Western blotting analyses on Akt phosphorylation. HUVEC were preincubated with the indicated
compound for 10 min and then stimulated with both VEGF and the compounds for 20 min. DMSO was used as control.
The proteins extracted and analyzed by western blotting, representative band of at least two independent experiments
are reported (A). The densiometric analyses were done with ImageJ (B). Tubulin was used as the protein loading
control. Most of the compounds affected Akt phosphorylation.
2.3 Effect of compounds 1–27 on HUVEC migration in the wound healing assay.
As most of the compounds affected MAPK and PI3K signaling pathways likely through targeting
different mediators, we tested their effect on endothelial cell (HUVEC) migration, a key function
required for angiogenesis.
All compounds were screened using the wound healing assay upon VEGF stimulation.
Therefore, confluent HUVEC were injured with a sterile tip to create a scratch that they can heal by
migrating toward the scratched area. The cells were washed with PBS and stimulated with VEGF in
the presence of the indicated compound. The wounds were imaged just after VEGF addition and 15
h later with the ImageXpress microscope.
Only compound 3 showed a significant inhibitory effect on the HUVEC migration (Fig. 6, data are
relative to the control set at 100 %). The compound 1 and 4 also showed trends toward inhibition of

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endothelial cell migration, though not significant. Having the same pyrazole core and presenting
similar modulatory profiles on p38MAPK, ERK1/2 and AKT phosphorylation, this inhibitory effect
indicates that these molecular entities may be blockers of angiogenesis. Other compounds though
strongly altered the phosphorylation of p38MAPK, ERK1/2 and AKT in endothelial cells
stimulated by VEGF. For example, compound 15 that did not block endothelial cell migration and
other derivatives even showed trends toward promotion of this cellular process. This suggests that
the relationship between activation of these mediators of MAPK and PI3K signaling pathways in
endothelial cells and subsequent migration of the cells is more complex than expected. This
definitely demonstrated that the strict inhibition or activation of p38MAPK, ERK and AKT is not
sufficient to block endothelial cell migration in contrast to other cells such as neutrophils. This also
suggested that modulation of the activation patterns of other different mediators of MAPK and
AKT may be a better strategy.
*
VEGF+compound#
Figure 6. Representative migration analysis of compound 1–27 on wound healing assay. Confluent HUVEC were
injured with a sterile tip to create a scratch and stimulated with both VEGF and the compounds, DMSO was used as
control. The wounds were imaged just after VEGF addition and 15h later with the ImageXpress microscope. The area
of the wound at time 0 and after 15 h was calculated with ImageJ. The reported data are relatively to the control set at
100%.
3. Conclusions
We report here design and synthesis of a new small library of pyrazole-ureas and
imidazopyrazolecarboxamides, bearing a characteristic fluorophenyl substituent and different
chemical substitution on pyrazole moiey never investigated in our previous studies.
To verify a possible action of these new derivatives on the MAPK and PI3K intracellular pathways
we preliminarily screened them by monitoring ERK1/2, p38MAPK and Akt phosphorylation by
Western blotting in human umbilical vein endothelial cells (HUVEC) stimulated with VEGF.

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In general, all tested compounds showed a good ability in interfering with the phosphorylation of
the studied kinases. However, the type of action (stimulation or inhibition of kinases
phosphorylation) was different by considering each kinases on its own.
SAR considerations evidenced a pivotal role of the carboxyethyl position both in pyrazole and
imidazopyrazole scaffold to interfere with the MAPK signaling pathways, leading to increase or
decrease p38MAPK phosphorylation. In addition, in some imidazopyrazoles derivatives it is
possible to observe an increase of potency clearly related to the flexibility loss in respect to the
pyrazole analogues.
In the ERK1/2 both the pyrazoles and the imidazopyrazoles generally showed a decrease of
phosphorylation, with few exceptions. A clear role in phosphorylation decrease was determined by
length of chains at N1 on the pyrazoles 1–4, while in the imidazopyrazole series 17–19 the activity
seems strictly to rely on the nature of the substituent in position 7. Of note, in general the 3-
trifluoromethylphenylureas caused a stronger decrease of ERK phosphorylation.
In Akt phosphorylation main pyrazoles showed a poor decrease of activity. Interstingly, results for
compounds 10 and 14 demonstrated that the 3-fluorophenylurea group determines an inversion of
activity (from decrease to increase) towards Akt phosphorylation, while the tert-butyl substituent
increments anyway the potency in respect with the carboxyethyl one. Increasing activity on Akt
phosphorylation, particularly evident in the 3-fluorophenylurea derivative 21, was also determined
by flexibility reduction in the imidazopyrazole derivatives.
In conclusion, the differently substituted forms of the pyrazoles and imidazopyrazoles did not show
neither the same trends toward increasing or inhibiting p38MAPK, ERK1/2 and Akt
phosphorylation, nor the same structure-activity relationship.
This may be explained partially by the crosstalk between different signaling pathways.
We could also speculate that the compound might interfere with different numbers of target kinases
upstream of the monitored protein phosphorylation. However, altogether our results definitely
showed that some structural features such as: 1) the type of substituent (carboxyethyl or ter-butyl)
and their position (3 or 4) on the pyrazole; 2) the type of substitution on phenylurea moiety; 3) the
flexibility loss in the imidazopyrazole derivatives in respect with pyrazoles ones, serve like a switch
between increasing or decreasing the phosphorylation levels of p38MAPK, ERK1/2 and Akt and
also determine the activity potency.
Despite their activities on the three kinases, most of the compounds did not inhibit endothelial cell
migration; in fact the only one that showed a significant inhibition was compound 3. Thus, in a next
follow-up study it will be interesting to investigate its action on pathological angiogenesis and

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identify the primary target kinases. This will be of fundamental importance also for identification of
eventual unknown mediators of angiogenesis, thereby unveiling new therapeutic targets for
controlling pathological angiogenesis in diseases such as cancer.
AKNOWLEDGEMENTS
We thank Dr. M. Anzaldi, R. Raggio, Mr. O. Gagliardo, and F. Tuberoni for spectral recording and
Mr. V. Ruocco for the informatics support to calculations.
This work has been supported by the University of Genoa (PRA 2013; PRA 2014), and grants from
the Swiss National Science Foundation (310030-153456) and Oncosuisse (KFS-2914-02-2012) to
BAI. The authors thank the Swiss Confederation for the Swiss Government Excellence Scholarship
grant to EM.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AUTHORS INFORMATION
*Corresponding author: Chiara Brullo
Department of Pharmacy-Section of Medicinal Chemistry - School of Medical and Pharmaceutical
Sciences – University of Genoa
Viale Benedetto XV, 3- 16132 Genoa, Italy
Phone: +39 010 353 8368
Fax: +39 010 353 8358
E-mail: chiara.brullo@unige.it
4. Experimental
4.1 Chemistry
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, Darmstad, Germany),
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, 230–400 mesh, was used for
chromatography.
Melting points are not “corrected” and were measured with a Buchi M-560 instrument. IR spectra
1
were recorded with a Perkin-Elmer 398 spectrophotometer. H NMR spectra were recorded on a
Varian Gemini 200 (200 MHz) instrument, chemical shifts are reported as δ (ppm) relative to

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tetramethylsilane (TMS) as internal standard; signals were characterized as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), br s (broad signal); J in Hz. Elemental analyses were determined
with an elemental analyzer EA 1110 (Fison-Instruments, Milan, Italy) and the purity of all
synthesized compounds was >95%.
4.1.1 General procedure for the synthesis of ethyl 1-(2-hydroxyalkyl)-5-(3-(3-
(trifluoromethyl)phenyl)ureido)-1H-pyrazole-4-carboxylates 1–4.
A mixture of the suitable 5-amino-1H-pyrazole-4-carboxylic acid ethyl esters (29a–d) (10 mmol)
and 3-(trifluoromethyl)phenyl isocyanate (2 g, 11 mmol) in anhydrous toluene (70 mL) was
refluxed for 6 h. After cooling to room temperature, the solution was washed with 3 M HCl (2 x 20
mL), with water (10 mL), dried (MgSO4) and evaporated under reduced pressure. The crude
crystallized by adding a solution of diethyl ether/petroleum ether (b.p. 50–60 °C) (1:1). The white
solids obtained were recrystallized from absolute ethanol.
4.1.1.1
Ethyl
1-(2-hydroxypropyl)-5-(3-(3-(trifluoromethyl)phenyl)ureido)-1H-pyrazole-4-
1
carboxylate 1. White solid. Yield 41%; mp 160–161 °C. H-NMR (DMSO-d6): δ 1.24 (t, J = 7.0,
6H, 2CH3), 3.30–3.42 (m, 2H, CH2N), 4.08–4.22 (m, 3H, CH2O e CHOH), 5.20 (br s, 1H, OH
disappears with D2O), 6.32 (m, 2H, 2NH, disappear with D2O), 7.30–7.70 (m, 4H, Ar), 7.80 (s, 1H,
H3). IR (KBr): cm-1 3430, 3340, 3265 (NH + OH), 1713 (COOEt), 1689 (CONH). Anal.
(C17H19F3N4O4) calcd for C, H, N.
4.1.2 Synthesis of ethyl 5-amino-1-(2-oxo-2-phenylethyl)-1H-pyrazole-4-carboxylate 30.
A solution of sulphur trioxide pyridine complex (4.77 g, 30 mmol) in DMSO (20 mL) was added
dropwise to a solution of ethyl 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-4-carboxylate
(29d) (2.75 g, 10 mmol) in DMSO (10 mL) and TEA (9 mL). The reaction mixture was stirred at
20–25 °C for 30 min. Then, 1M HCl solution was added until pH 5–6 and the reaction mixture was
poured into water. The solid obtained was filtered and recrystallized from absolute ethanol. White
1
solid. Yield: 82%; mp: 138–139 °C. H-NMR (CDCl3): δ 1.33 (t, J = 7.1, 3H, CH3), 4.27 (q, J =
7.1, 2H, CH2O), 5.10–5.20 (m, 2H, NH2, disappears with D2O), 5.48 (s, 2H, CH2N), 7.45–7.75 (m,
4H, Ar), 8.02 (m, 1H, H3). IR (KBr): cm-1 3425, 3332 (NH2), 1688 (CO), 1668 (COOEt). Anal.
(C14H15N3O3) calcd for C, H, N.
4.1.3 General procedure for the synthesis of ethyl 1-(2-oxo-2-phenylethyl)-5-(3-phenylureido)-1H-
pyrazole-4-carboxylates 5–8.
A mixture of ethyl 5-amino-1-(2-oxo-2-phenylethyl)-1H-pyrazole-4-carboxylate (30) (2.73 g, 10
mmol) and the proper phenyl isocyanates (11 mmol) in anhydrous toluene (70 mL) was refluxed for
12 h. After cooling to room temperature, the solution was extracted with diethyl ether, washed with

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3 M HCl (2 x 20 mL), with water (10 mL), dried (MgSO4) and evaporated under reduced pressure.
The crude crystallized by adding a solution of diethyl ether/petroleum ether (b.p. 50–60 °C) (1:1).
The white solids obtained were recrystallized from absolute ethanol.
4.1.3.1 Ethyl 5-(3-(2-fluorophenyl)ureido)-1-(2-oxo-2-phenylethyl)-1H-pyrazole-4-carboxylate 5.
White solid. Yield 32%; mp: 159–161 °C. 1H-NMR (CDCl3): δ 1.43 (t, J = 7.0, 3H, CH3), 4.30 (q, J
= 7.0, 2H, CH2O), 5.90 (s, 2H, CH2N), 6.90–8.40 (m, 12H, 9Ar + H3 + 2NH, 2H disappear with
D2O). IR (CHCl3): cm-1 3020 (NH), 1720–1650 (COOEt + CONH + CO). Anal. (C21H19FN4O4)
calcd for C, H, N.
4.1.4 Synthesis of potassium 1-cyano-3-ethoxy-3-oxoprop-1-en-2-olate 31.
Diethyl oxalate (0.44 g, 2.9 mmol) was slowly added at 0 °C to a solution of potassium t-butoxide
(0.34 g, 3 mmol) in an. THF (27 mL) in the presence of 18-Crown-6 (0.63 g, 2.4 mmol). The
mixture was heated until 60 °C, acetonitrile (0.12 g, 3 mmol) was slowly added and the mixture was
heated at 60 °C for 30 min. After cooling to room temperature a yellow solid was obtained, which
was collected by filtration and used in the following step as crude. Yield: 98%. (let. 78% [14]).
4.1.5 Synthesis of ethyl 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-3-carboxylate 32.
A mixture of 2-hydrazino-1-phenylethanol (28d) (1.52 g, 10 mmol), potassium 1-cyano-3-ethoxy-3-
oxoprop-1-en-2-olate (1.79 g, 10 mmol) and glacial acetic acid (1 mL) in absolute ethanol (10 mL)
was heated at reflux for 6 h. After cooling to room temperature, the solvent was evaporated, and the
residue was dissolved in water and extracted with ethyl acetate (3 x 20 mL). The combined organic
phases were then washed with NaHCO3 sat. solution (20 mL), brine (20 mL) and twice with water
(2 x 20 mL), dried (MgSO4) and evaporated under reduced pressure. The crude was then purified by
Silicagel column chromatography using diethyl ether as the eluent. White solid. Yield 59%, mp
1
121–122 °C. H-NMR (CDCl3): δ 1.37 (t, J = 7.0, 3H, CH3), 3.48 (br s, 3H, NH2 + OH, 1H
disappears with D2O), 4.10–4.43 (m, 4H, CH2N + CH2O), 5.07–5.20 (m, 1H, CHOH), 6.03 (s, 1H,
H4), 7.24–7.45 (m, 5H, Ar). IR (KBr): cm-1 3484 (OH), 3386, 3306 (NH2), 1727 (CO). Anal.
(C14H17N3O3) calcd. for C, H, N.
4.1.6 General procedure for the synthesis of ethyl 1-(2-hydroxy-2-phenylethyl)-5-(3-phenylureido)-
1H-pyrazole-3-carboxylates 9–12.
A mixture of ethyl 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-3-carboxylate (32) (2.7 g, 10
mmol) and the suitable phenyl isocyanate (11 mmol) in anhydrous toluene (30 mL) was refluxed for
6 h. After cooling to room temperature, the white solid obtained was filtered and recrystallized from
absolute ethanol.

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4.1.6.1 Ethyl 5-(3-(2-fluorophenyl)ureido)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-3-carboxylate
9. White solid. Yield 65%; mp 91–92 °C. 1H-NMR (CDCl3): δ 1.21 (t, J = 7.0, 3H, CH3), 3.20 (br
s, 1H, OH, disappears with D2O), 4.05–4.60 (m, 4H, CH2N + CH2O), 4.95–5.10 (m, 1H, CHOH),
6.78–7.32 (m, 9H, Ar), 7.63–8.70 (m, 3H, H4 + 2NH, 2H disappear with D2O). IR (CHCl3): cm-1
3420–3050 (OH + NH), 1735–1690 (COOEt + CONH). Anal. (C21H21FN4O4) calcd for C, H, N.
4.1.7 Synthesis of 2-(5-amino-3-tert-butyl-1H-pyrazol-1-yl)-1-phenylethanol 33.
4,4-dimethyl-3-oxopentanenitrile (1.25 g, 10 mmol) and concentrated acetic acid (0.20 mL) were
added to a solution of 2-hydrazino-1-phenylethanol (28d) (1.52 g, 10 mmol) in absolute ethanol (20
mL) and the reaction mixture was refluxed for 10 h. The solvent was evaporated under reduced
pressure and a crude solid crystallized by adding a solution of diethyl ether/petroleum ether (b.p
1
50–60°C) 1/1. Yellow solid. Yield: 83%; mp: 131–133 °C. H-NMR (DMSO-d6): δ 1.20 (s, 9H,
3CH3), 3.80–4.00 (m, 2H, CH2N), 4.80–5.00 (3H, CHOH + NH2, 2H disappear with D2O), 5.20 (s,
1H, H4), 5.85 (br s, 1H, OH, disappears with D2O), 7.20–7.40 (m, 5H, Ar). IR (CHCl3): cm-1 3500–
3000 (NH2 + OH). Anal. (C15H21N3O) calcd for C, H, N.
4.1.8 General procedure for the synthesis of 1-(3-(tert-butyl)-1-(2-hydroxy-2-phenylethyl)-1H-
pyrzol-5-yl)-3-phenylureas 13–16.
A mixture of 2-(5-amino-3-tert-butyl-1H-pyrazol-1-yl)-1-phenylethanol (33) (2.59 g, 10 mmol)
and the suitable phenyl isocyanate (11 mmol) in anhydrous toluene (100 mL) was refluxed for 12 h.
The solvent was evaporated under reduced pressure and the crude was dissolved in DCM (50 mL),
washed with 3M HCl (2 x 20 mL), water (10 mL), dried (MgSO4) and evaporated under reduced
pressure. The solid obtained was crystallized from DCM, or, if necessary, purified by
chromatography on Silica gel using a mixture of DCM/CH3OH (8:2) as eluent.
4.1.8.1 1-(3-(tert-butyl)-1-(2-hydroxy-2-phenylethyl)-1H-pyrazol-5-yl)-3-(2-fluorophenyl)urea 13.
White solid. Yield: 37%; mp: 96–99 °C. 1H-NMR (CDCl3): δ 1.26 (s, 9H, 3CH3), 4.08–4.39 (m, 2H,
CH2N), 5.03–5.19 (m, 1H, CHOH), 6.17 (s, 1H, H4), 6.90–7.60 and 8.03–8.20 (2m, 11H, 9Ar +
2NH, 2H disappear with D2O). IR (CHCl3): cm-1 3400–3000 (NH + OH), 1708 (CONH). Anal.
(C22H25FN4O2) calcd for C, H, N.
4.1.9 General procedure for the synthesis of the compounds 17–19.
3-(trifluoromethyl)phenyl isocyanate (1.12 g , 0.83 mL, 6 mmol) was added dropwise to a solution
of the proper ethyl 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole (34, 36, 39) (5mmol) in
anhydrous DMF (5 mL) and the reaction mixture was refluxed for 18 h. After cooling to room
temperature, the mixture was poured into an ice-water bath (50 mL) and 1M HCl solution was
added until pH 5. The solid precipitated was filtered, dried on air and the purity was verified by

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TLC. The crude was purified by chromatography on Silica gel using diethyl ether as eluent or
recrystallized from absolute ethanol.
4.1.9.1 Ethyl 2-phenyl-1-((3-(trifluoromethyl)phenyl)carbamoyl)-2,3-dihydro-1H-imidazo[1,2-
1
b]pyrazole-7-carboxylate 17. White solid. Yield: 36%; mp: 153–154 °C. H-NMR (CDCl3): δ 1.42
(t, J = 5.4, 3H, CH3), 4.20 (near t, 1H, H3), 4.42 (q, J = 5.4, 2H, CH2), 4.81 (near t, 1H, H2), 6.50
(near t, 1H, H3), 7.20–7.80 (m, 9H, Ar), 7.91 (s, 1H, H6), 11.60 (s, 1H, NH, disappears with D2O).
IR (CHCl3): cm-1 3214 (NH), 1710–1690 (COOEt + CONH). Anal. (C22H19F3N4O3) calcd for C, H,
N.
4.1.9.2
2-Phenyl-N1-(3-(trifluoromethyl)phenyl)-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-1,7-
dicarboxamide 18. White solid. Yield: 28%; mp: 226–227 °C. 1H-NMR (DMSO-d6): δ 3.72 (near t,
1H, H3), 4.58 (near t, 1H, H2), 5.40 (near t, 1H, H3), 7.05–7.42 (m, 10H, 9Ar + NH, 1H disappears
with D2O), 7.62 (s,1H, H6). IR (KBr): cm-1 3450, 3223, 2871 (NH2 + NH), 1666 (CONH2), 1609
(CONH). Anal. (C20H16F3N5O2) calcd for C, H, N.
4.1.9.3
2-Phenyl-7-(piperidine-1-carbonyl)-N-(3-(trifluoromethyl)phenyl)-2,3-dihydro-1H-
1
imidazo[1,2-b]pyrazole-1-carboxamide 19. White solid. Yield: 50%; mp: 159–160 °C. H-NMR
(CDCl3): δ 1.45–1.80 (m, 6H, 3CH2-pip), 3.71–3.93 (m, 4H, 2CH2N-pip), 4.18 (near t, 1H, H3),
4.78 (near t, 1H, H2), 6.35 (near t, 1H, H3), 7.20–7.55 (m, 9H, Ar), 7.90 (s, 1H, H6), 10.90 (s, 1H,
NH, disappears with D2O). IR (CHCl3): cm-1 3020 (NH), 1692 (CONH), 1576 (CON-pip). Anal.
(C25H24F3N5O2) calcd for C, H, N.
4.1.10 Synthesis of ethyl 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-6-carboxylate 40.
Ethyl 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-pyrazole-3-carboxylate (32) (0.27 g, 1 mmol) was
dissolved in concentrated sulfuric acid (2 mL) at 0 °C and the mixture was stored at room
temperature for 15 min. Then, ice-water (50 mL) was added and the solution was made neutral with
NH4OH solution. The yellow solid obtained was filtered, washed with water and recrystallized from
1
95% ethanol. White solid. Yield: 80%; mp 128–129 °C. H-NMR (CDCl3): δ 1.41 (t, J = 7.2, 3H,
CH3), 4.03 (near t, 1H, H3), 4.40 (q, J = 7.2, 2H, CH2O), 4.61 (near t, 1H, H2), 4.79 (br s, 1H, NH,
disappears with D2O), 5.38 (near t, 1H, H3), 5.98 (s, 1H, H7), 7.27–7.52 (m, 5H, Ar).
IR (KBr): cm-1 3350–3050 (NH), 1720 (CO). Anal. (C14H15N3O2) calcd for C, H, N.
4.1.11 General procedure for the synthesis of ethyl 1-carbamoyl-2-phenyl-2,3-dihydro-1H-
imidazo[1,2-b]pyrazole-6-carboxylates 20–23.
A mixture of ethyl 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-6-carboxylate 40 (2.57 g, 10
mmol) and the suitable phenyl isocyanate (11 mmol) in an. DMF (30 mL) was refluxed for 18 h.
After cooling to room temperature the mixture was poured into ice-water (100 mL) and 1M HCl
solution was added until pH 5. The precipitated solid was filtered and dissolved in DCM (30 mL).

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The solution was washed with 3 M HCl (2 x 20 mL), with water (10 mL), dried (MgSO4) and
evaporated under reduced pressure to give a crude that crystallized by adding diethyl ether.
4.1.11.1 ethyl 1-((2-fluorophenyl)carbamoyl)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-6-
carboxylate 20. White solid. Yield: 64%; mp 192–195 °C. 1H-NMR (CDCl3): δ 1.41 (t, J = 7.2, 3H,
CH3), 4.31 (near t, 1H, H3), 4.40 (q, J = 7.2, 2H, CH2O), 4.91 (near t, 1H, H2), 5.88 (near t, 1H,
H3), 6.60 (s, 1H, H7), 6.63–8.20 (m, 10H, 9Ar + NH, 1H disappears with D2O). IR (CHCl3): cm-1
3395 (NH), 1720–1690 (COOEt + CONH). Anal. (C21H19FN4O3) calcd for C, H, N.
4.1.11 Synthesis of 6-tert-butyl-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole 41.
2-(5-amino-3-tert-butyl-1H-pyrazol-1-yl)-1-phenylethanol 33 (0.26 g, 1 mmol) was dissolved in
concentrated sulfuric acid (2 mL) at 0 °C and the mixture was stored at room temperature for 15
min. Then, ice-water (50 mL) was added and the solution was made neutral with NH4OH solution;
the yellow solid obtained was filtered, washed with water and recrystallized from 95% ethanol.
1
Yellow solid. Yield: 78%; mp 110–112°C. H-NMR (CDCl3): δ 1.31 (s, 9H, 3CH3), 3.95 (near t,
1H, H3), 4.25 (br s, 1H, NH, disappears with D2O), 4.52 (near t, 1H, H2), 5.22–5.40 (m, 2H, H3 +
H7), 7.22–7.48 (m, 5H, Ar). IR (CHCl3): cm-1 3393 (NH). Anal. (C15H19N3) calcd for C, H, N.
4.1.12 General procedure for the synthesis of 6-(tert-butyl)-N,2-diphenyl-2,3-dihydro-1H-
imidazo[1,2-b]pyrazole-1-carboxamides 24–27.
A mixture of 6-tert-butyl-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole 41 (2.41 g, 10 mmol)
and the suitable phenylisocyanate (11 mmol) in an. DMF (30 mL) was refluxed for 12 h. After
cooling to room temperature, the mixture was poured into ice-water (100 mL) and 1 M HCl solution
was added until pH 5. The precipitated solid was filtered and recrystallized from diethyl
ether/petroleum ether (b.p. 50–60°C) (1:1) or purified by chromatography on Silica gel, using
diethyl ether as eluent.
4.1.12.1
6-(tert-butyl)-N-(2-fluorophenyl)-2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole-1-
1
carboxamide 24. White solid. Yield: 64%; mp: 140–143 °C. H-NMR (CDCl3): δ 1.39 (s, 9H,
3CH3), 4.23 (near t, 1H, H3), 4.83 (near t, 1H, H2), 5.84 (near t, 1H, H3), 5.99 (s, 1H, H7), 6.92–
7.53 (m, 9H, Ar), 8.18 (br s, 1H, NH disappears with D2O). IR (CHCl3): cm-1 3020 (NH), 1695
(CONH). Anal. (C22H23FN4O) calcd for C, H, N.
Analytical data for compounds 2–4, 6–8, 10–12, 14–16, 21–23, 25–27 are available as Supporting
Information.

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4.2 Biology
4.2.1 Western blotting
HUVEC were seeded in a 6 well plate pre-coated with 0.2% of gelatin and Collagen G at 0.1mg/mL
in PBS. The cells were cultured in M199 (GIBCO) supplemented with 10% fetal calf serum, 1%
Endothelial Cell Growth Supplement (EmdMillipore), 0.1mg/mL of heparin sodium, 0.1µM of
hydrocortisone (Sigma), 1% antibiotics-glutamine mixture, 0.1% of vitamin C for 48 h to become
confluent. Confluent HUVEC were starved for at least 4 h with medium alone before stimulation.
After starvation, the cells were pre-incubated with the indicated compound at 20 µM for 10 min at
37°C and then stimulated with both VEGF at 50 ng/mL and the compound at 20 µM for 20 min. As
the compounds were dissolved in dimethylsulfoxyde (DMSO), incubation with DMSO was used as
control to the compounds. Treated cells were washed and lysed for protein extraction. Protein
concentration was determined with the MicroBCATM Protein Assay Kit (ThermoScientific). Equal
amounts of proteins (30 µg) were subjected to a gel electrophoresis and then transferred to
nitrocellulose blotting membranes. Membranes were blocked with a blocking buffer made of PBS
containing 5% not-fat dry milk and 0.05% of Tween 20. Membranes were incubated overnight at
4 °C with primary antibodies diluted in the blocking buffer. The following primary antibodies were
used in this study: rabbit anti-phospho-p38MAPK (pp38MAPK) (Cell signaling) used at 1:1000
dilution, rabbit anti-phospho-ERK1/2 (pERK1/2) (Cell signaling) at 1:1000 dilution, rabbit anti-
phospho-Akt (pAkt) (Cell signaling) at 1:1000 dilution and mouse 〈-tubuline antibody (Millipore)
at 1:4000 dilution. Membranes were washed three times for 5 minutes with PBS-Tween 20 0.05%
and incubated at room temperature for 1 h with the adequate secondary antibody. Horseradish
peroxidase (HRP)-coupled goat anti-rabbit antibody was used at dilution of 1:10000 and the HRP-
Goat anti-mouse antibody at 1:3000 dilution. Both secondary antibodies were from Jackson
ImmunoResearch. Membranes were washed and the signals detected using the enhanced
chemioluminescence system (Advansta, WesternBrightTM Sirius). Protein band intensities were
quantified with ImageJ and the tubuline intensity was used to ensure equal loaded protein amounts.
The results were expressed relative to condition of treatment with VEGF and DMSO serving as
control in the same blot and set at 100%. Data are representative of at least two independent
experiments.
4.2.2 Wound healing assay
HUVEC were seeded in a 96-well plate for 48 h to become confluent. Once confluent cells were
stained with a green tracker, the 5-chloromethylfluorescein diacetate (CMFDA) at 1:5000 (stock
solution 10 mM) and the cell monolayers were injured with a sterile tip to create a scratch. Each
well was washed with PBS to remove detached cells and incubated with both VEGF at 50 ng/mL

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and the indicated compound at 20 µM. The wounds were imaged just after VEGF addition and 15 h
later with the ImageXpress microscope. The wound area were analysed with ImageJ to measure the
colonized area in µm [colonized area = (area T 0 h – area T 15 h)], which was then represented as a
percentage of migrated cells respect to the control set at 100%. Data are representative of at least
three independent experiments done in triplicate.
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ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of new pyrazolyl-ureas and imidazopyrazol-
carboxamides able to interfere with p38MAPK and other kinases involved in tumor cell
migration
Elda Meta, Chiara Brullo, Adama Sidibe, Beat A. Imhof, Olga Bruno
Highlights
• New of pyrazolylureas and imidazopyrazolylcarboxamides are reported.
• Activity on the phosphorylation of ERK1/2, p38MAPK and Akt in HUVEC cells was
investigated.
• SAR consideration revelead a different trend in increasing or inhibiting p38MAPK, ERK1/2
and Akt phosphorylation.
• Specific substituents in pyrazole nucleus differently interfere with VEGF-induced signaling.
• Carboxyethyl subtituent on the pyrazole serve like a switch between increasing or decreasing
the kinase phosphorylation levels.
• New compounds might interfere on angiogenesis and with different upstream target kinases
on the MAPK and PI3K pathways.
• Compound 3 showed a significant inhibitory effect on endothelial cell migration, a key
function for angiogenesis.