Design, synthesis and biological evaluation of pyrazole derivatives as potential multi-kinase inhibitors in hepatocellular carcinoma

Article abstract of DOI:10.1016/j.ejmech.2011.12.031

We described the optimization, by molecular modelling, of small pyrazole derivatives, as kinase inhibitors, obtained through a 1,3-dipolar cycloaddition between nitrile imines and functionalized acetylenes. The two compounds, selected as potential agents

Full text of DOI:10.1016/j.ejmech.2011.12.031

European Journal of Medicinal Chemistry 48 (2012) 391e401
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and biological evaluation of pyrazole derivatives as potential
multi-kinase inhibitors in hepatocellular carcinoma
Elena Strocchi a, Francesca Fornari b, Manuela Minguzzi b, Laura Gramantieri b, Maddalena Milazzo b,
Valentina Rebuttini a, Simone Breviglieri a, Carlo Maurizio Camaggi a, Erica Locatelli a, Luigi Bolondi b,
,
Mauro Comes-Franchini a
*
a Department of Organic Chemistry “A. Mangini”, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy
b Department of Clinical Medicine and Center for Applied Biomedical Research, S. Orsola-Malpighi Universitary Hospital, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We described the optimization, by molecular modelling, of small pyrazole derivatives, as kinase inhib-
itors, obtained through a 1,3-dipolar cycloaddition between nitrile imines and functionalized acetylenes.
The two compounds, selected as potential agents active against hepatocellular carcinoma (HCC) were
then evaluated in vitro for their biological activity on HCC-derived cell lines. The compounds show
a promising inhibitory growth efficacy (IC50 50e100 mM) in SNU449 cell line, as well as block of cell cycle
progression and induction of apoptosis, and can be considered as lead compounds for further SAR
Received 24 May 2011
Received in revised form
7 October 2011
Accepted 20 December 2011
Available online 29 December 2011
developments.
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
Pyrazole
Dipolar cycloaddition
Hepatocellular carcinoma
Molecular docking
Kinase inhibitors
1. Introduction
activates the serine/threonine kinase AKT. It is reported that about
20% of HCC patients had elevated levels of AKT phosphorylation
Hepatocellular carcinoma (HCC) accounts for 80e90% of
primary liver cancers and it is the third cause of death from cancer
worldwide [1]. The high lethality of HCC is partly due to its resis-
tance to traditional chemotherapics. Despite the survival advantage
achieved by Sorafenib, effective treatments for advanced HCC are
still needed [2]. It was recently reported that combination of
specific AKT inhibitors with Sorafenib restored the sensitivity of
resistant cells to apoptotic cell death [3].
on Ser-473 [9]. Moreover, levels of phosphorylated mTOR and p70
S6 kinase resulted increased in 15% and 45% of HCC cases,
respectively [10].
Recent evidences showed that inhibition of the PI3K/AKT/mTOR
pathway with a Rapamacyn analogue, Everolimus, slowed tumour
growth and increased survival in a HCC xenograft model, suggest-
ing that the activation of this pathway exerts a crucial role in
hepatocarcinogenesis [7].
In the recent years the involvement of different molecular
alterations that arise during the development and progression of
HCC have been reported [4]. In particular, aberrant activation of
different cellular pathways such as IGF, Ras, PI3K/Akt/mTOR and
Wnt signalling has been identified in HCC [5,6]. The serine/thre-
In an attempt to search for novel antitumour compounds, we
focused our attention to multi-kinase inhibitor molecules and
we investigated their antiproliferative activity on HCC-derived
cell lines.
Indeed, it has been reported that the 3,5-pyrazole ring is an
interesting scaffold for the design of kinase inhibitors targeting the
ATP pocket of protein kinases [11e14].
onine kinase AKT acts as
a regulator of important cellular
processes, such as cell cycle, cell survival and cell growth. In HCC,
over expression of this pathway is associated with poor prognosis
[7], invasion and metastasis [8]. The binding of growth factors (e.g.
IGF and EGF) to their receptors activates PI3K, which in turn
Owing to its high synthetic efficiency and high regio- and stereo
selectivity, the 1,3-dipolar cycloaddition (1,3-DC) of 1,3-dipoles
with
p-electronic-deficient systems has emerged as a popular
way for the obtainment of heterocycles [15]. In particular, nitrogen-
containing heterocycles have attracted widespread attention in the
field of synthetic organic chemistry as well as in medicinal chem-
istry [16]. Among them, pyrazoles are synthetic targets of utmost
* Corresponding author. Tel.: þ390512093626; fax: þ390512093654.
E-mail address: mauro.comesfranchini@unibo.it (M. Comes-Franchini).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.031

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E. Strocchi et al. / European Journal of Medicinal Chemistry 48 (2012) 391e401
importance in the pharmaceutically industry, since such a five-
membered heterocyclic moieties bear the core structure of
numerous drugs [17].
Two general methods are known for the synthesis of pyrazoles.
The first method involves the standard reaction of hydrazines with
1,3-difunctional substrates such as 1,3-dicarbonyl compounds [18],
ranking docked binding energy was considered to be the “best”
docking result.
Satisfactory docking results were observed for the two
compounds then indicated as Target A and Target B, having the
following structures in Fig. 1.
Both compounds, nicely fitted inside the ATP site pocket of the
examined kinases, as indicated from binding free energy reported
in Table 1 and from analysis of ligand binding pose inside the ATP
pocket sites.
Better docking results seem to be observed to AKT2 with both
compounds; the best poses of Target A and Target B into AKT2
ynones [19] or b-aminoacrolein [20]. In addition, the 1,3-dipolar
cycloaddition (1,3-DC) between alkynes and nitrile imines
provides a direct access to pyrazoles [21], but regioisomeric
mixtures of pyrazoles are frequently obtained.
Following our interest in 1,3-DC [22] and in the synthesis of
kinase inhibitors [23] we have recently developed a method for the
region-controlled synthesis of pyrazoles based on the 1,3-DC of
nitrile imines with a class of functionalized acetylenes as the pro-
pionamides [24].
binding site had an estimated binding free energy [
D
G] of ꢀ9.99,
and ꢀ9.65 kcal/mol respectively. Nevertheless, both compounds
show similar results with all the examined kinases.
Target A, characterized by a C-5-carboxamide with an amino-
phenyl substituent and a C-3-dimethylphenyl substituent, exhibits
slight better values of binding free energies to AKT2, PI3K and CDK2
kinases (Table 1), while Target B shows better binding affinity to
the remaining kinases.
2. Results and discussion
2.1. Preliminary molecular docking
The best poses, obtained after docking to different kinases PDB
structures, suggest that the binding conformation of Target A is
similar to that reported for Target B in all kinases, and that are
typical of aspecific ATP competitors. The binding poses of Target A
and Target B into ATP binding site of AKT2 and GSK-3b are shown
in Fig. 2.
Our approach towards the synthesis of substituted pyrazoles as
multi-kinase inhibitors involved a computer-assisted strategy in
the molecular design. Docking simulations were performed for lead
optimization and to characterize the binding pose and affinity of
putative lead candidates to the ATP binding site of several kinases
involved in HCC pathways (AKT, PI3K, GSK-3
b
, CDK2, AurA, MAPK,
In all the cases, the substituted heterocyclic scaffold fitted and
entered into the ATP binding site with good binding affinity.
Target B could form two or three hydrogen-bonds into the
binding site of the selected kinases, showing interactions between
the terminal aminogroup of phenylamines with a carboxyl of GLU
EGFR, IGFR). 3,5-disubstituted pyrazoles were found to have the
minimum requirements for tight binding at the ATP active site of
the kinase domain. Therefore, we started from a preliminary
docking evaluation of some pyrazole derivatives obtainable
through 1,3-DC.
For the subsequent development, structures substituted in
position 5 of the pyrazole moiety through a carboxamide group
and with an aryl groups in position 3 were explored. We evaluated
two series of compounds with starting structures containing
a phenylamine group at position 1 (which could either improve
the water solubility of inhibitors and form hydrogen-bonds within
the ATP cleft), a carboxamide-R group at position 5 and at postion
3: (1) carboxyl group or (2) dimethylphenyl moiety. Subsequent
derived structures were characterized as follow: (1) aminophenyl
(AKT2: GLU230 and Glu200) or ASP residues (GSK-3
between carbonyl of C5-carboxamide and NH3þ of LYS residue
(GSK-3 : LYS85), or between NH of C5-carboxamide and OH of THR
b: ASP133), and
b
(AKT2: THR-292) respectively.
Target A could form two hydrogen-bonds into the binding
site of the selected kinases, showing interactions between the
terminal aminogroup of phenylamines with a carboxyl of GLU
or ASP residues (AKT2: GLU200 and GLU230; GSK-3b: ASP133)
or carbonyl in the backbones of PRO residues (GSK-3
PRO136).
b:
connected to carboxamide at position
5
of pyrazole and
The examined compounds revealed a common binding mode
in the hydrophobic cleft also through a complementary apolar
interaction surface. Pyrazole ring or the hydrophobic aryl moie-
ties fit tightly into the ATP binding site of the different kinases
through piepi interactions with the aromatic ring of PHE or TYR
residues.
carboxamide-dimethylphenyl at position 3; (2) aminophenyl
connected to carboxamide at position 5 and dimethylphenyl at
position 3.
The selected target proteins for the docking evaluation of the
proposed compounds were retrieved from the Protein Data Bank
(http://www.rcsb.org/pdb/) and were: AKT2 (PDB ID: 2jdr), GSK-3
b
Pi-cation interactions between the amino-substituted phenyl
ring, connected through an amide linkage e to the pyrazole ring (in
position 5) and the protonated aminogroup of LYS residues, are
present for both compounds docked with the selected kinases.
The two above described structures, with more promising
docking results, were therefore suggested for the synthesis and
testing as multi-kinase inhibitors.
(PDB ID: 1pyx), PI3K (PDB ID: 2v4l), CDK2 (PDB ID: 2j9m), MAPK
(PDB ID ID: 2pzy), IGFR (PDB ID: 2zm3), EGFR (PDB ID: 2rpg), AurA
(PDB ID: 2x6d).
Binding modes and binding affinities of the evaluated
compounds within the ATP binding site of all the selected kinases
were calculated using the Autogrid 4.0 and Autodock 4.1 programs
[25]. The ADT 1.6.1 package was used for visualization of the results
(see experimental section for the preparation of the molecules,
preparation of the target macromolecules and for the procedure of
molecular docking).
O
N
CH3
CH3
CH3
CH3
NH
H
N
H
N
2.2. Analysis of the binding mode
N
NH2
N
NH2
N
O
O
We analyzed the location and orientation of the evaluated
compounds and the interactions into the binding site of the
selected kinases above indicated. The predicted binding free energy
(that includes intermolecular energy and torsional free energy) was
used as the criterion for ranking. The conformation with the lowest
Target B
Target A
NH2
NH2
Fig. 1. Chemical structures for Target A and Target B.

E. Strocchi et al. / European Journal of Medicinal Chemistry 48 (2012) 391e401
393
Table 1
The reaction with 2,3-dimethylbenzoylchloride (obtained in
quantitative yield from the carboxylic acid) in THF in the presence
of Et3N gave the corresponding benzoylhydrazine in 75% yield.
The subsequent reaction of 2,3-dimethyl-N0-(4-nitrophenyl)ben-
zohydrazide with triphenylphosphine-CCl4 in CH3CN [17] gave the
hydrazonoyl chlorides 2 in 86% yield. The C-carboxymethyl-N-aryl
hydrazonoyl chloride 3 was prepared staring from N-p-Boc-phe-
nylendiamine dissolved in MeOH that was first converted to its
diazonium salt with NaNO2/HCl and then by treatment with
Docking results, expressed in term of binding free energy (DG, kcal/mol).
Target kinases
AKT2 GSK-3
b
PI3K
EGFR
IGFR
CDK2 AurA
MAPK
Target A ꢀ9.99 ꢀ8.80
Target B ꢀ9.65 ꢀ9.23
ꢀ9.33 ꢀ9.19 ꢀ8.24 ꢀ9.67 ꢀ8.49 ꢀ8.53
ꢀ8.99 ꢀ9.46 ꢀ8.24 ꢀ9.61 ꢀ8.84 ꢀ9.02
2.3. Synthesis
methyl-2-chloroacetoacetate in MeOH gave
(Scheme 1).
3 in 55% yield
For the synthesis of tert-butyl (4-propiolamidophenyl)carba-
mate 1, commercially available propiolic acid was reacted in diethyl
ether with N-p-Boc-phenylendiamine in the presence of dicyclo-
hexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) in
THF for 24 h giving 1 in 72% yield. (E)-2,3-dimethyl-N0-(4-
nitrophenyl)benzohydrazonoyl chloride 2 and (Z)-methyl-2-(2-(4-
((tert-butoxycarbonyl)amino)phenyl) hydrazono)-2-chloroacetate
3 have been prepared as already reported by us [23]. The C-aryl-
N-aryl hydrazonoyl chloride 2 was obtained starting from the cor-
responding p-nitrophenyl hydrazines.
We next examined the conditions required for the cycloaddition
of 1 with 2 and 3. The cycloadditions were carried out in dry
dioxane at 80 ꢁC for 24 h using 2.5 equivalents of Ag2CO3 until
complete conversion of 1, which was always used in stoichiometric
amount with respect to the dipoles (Scheme 2). The regioisomeric
ratio was always calculated by 1H NMR analysis of the crude
mixtures using diagnostic signals. The identification of the 5-
pyrazoles and the 4-pyrazoles was based on the 1H NMR signals
taking advantage from the CH signal on the C5 for the 4-substituted
pyrazole which resonates at about 8.0 ppm as reported with X-ray
analysis by us [24c].
As reported in Scheme 2, the 1,3-DC of 1 with 2 gave 5-
substituted pyrazole 4 as the only regioisomer in 48% yield. Then
we carried out the reduction of the nitro group with Pd/C (10% W/
W) and ammonium formate in 60% yield and quantitatively the Boc
removal with HCl/EtOAc giving 5 (Target A) as the dichlorhydrate
salt.
In the case of 3, its cycloaddition with 1 gave 6 in 64% yield as
a mixture 80:20 of regioisomers 5-substituted/4-substituted with
the 5-substituted being the major (Scheme 3). Despite several
attempts it was not possible to isolate the minor isomer, because
after several run on preparative TLC plates degradation was
observed. Synthetic elaboration was therefore performed to isolate
the target compound. Saponification with NaOH gave the corre-
sponding carboxylic acid in 40% yield that was reacted in THF with
N,N0-carbonyldiimidazole (CDI) and 2,3-dimethylaniline at room
temperature for 3 h. An overall 48% yield for these two last steps
was obtained for the pyrazole 7 (Target B).
2.4. In vitro kinase inhibition assay
Based on the docking results, the two compounds showed
a potential affinity with several kinases suggesting a multi-kinase
activity.
Both compounds were preliminarily tested against a panel of 20
human protein kinases [ALK, CDK2/cyclinA, CDK2/cyclinE, Flt3,
GSK3-
PKB , PKB
(KinaseProfiler service; Millipore UK Ltd, UK) in the presence of
a
, GSK3-
b
, IGF-1R, IKK
b, JAK2, JAK3, MAPK1, MAPK2, mTOR,
a
b, Src, PI3K(p110a/b/d)] to assess their kinase inhibition
3
mM compound and 10
m
M ATP. Results were presented as
O
Cl
Cl
O
H
N
N
N
N H
N H
NO2
Fig. 2. The image display the low energy conformations of Target A and Target B. On
the top (A): best docking binding poses of Target A (red) and Target B (blue) into the
ATP binding site of AKT2. On the bottom (B): best docking binding poses of Target A
O
NHBoc
3
2
1
(red) and Target B (blue) into binding site of GSK-3b. The aminoacidic residues of
NHBoc
binding pockets closed to molecules and involved in H-bonds (shown in green), piepi
and piecation interactions are indicated. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
Scheme 1. Structures of dipolarophile 1 and precursors of the 1,3-dipoles 2 and 3.

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E. Strocchi et al. / European Journal of Medicinal Chemistry 48 (2012) 391e401
H
N
H
N
N
N
N
H2N
N
BocHN
O
O
a
b, c
1 + 2
5
4
(Target A)
NH2
NO2
Scheme 2. 1,3-Dipolar cycloaddition of 1 with 2 and synthetic elaboration of the cycloadduct 4 for the synthesis of 5 (Target A). Reagents and conditions: (a) Ag2CO3, 1,4-dioxane,
80 ꢁC, 24 h, 35%. (b) Pd-C, NH4HCO2, MeOH, 0 ꢁC-1 h to rt overnight, 60%. (c) HCl 3 M in EtOAc, rt, 8 h, 100%.
percentage of inhibition compared to control; details of assay
conditions used can be found at the website www.millipore.com.
PI3K/AKT pathway is often deregulated in hepatocellular carcinoma
and its involvement in HCC development and progression is well
known [8], we investigated the biological effect of the two inhibitors
on this molecular pathway. Firstly, we analyzed by Western blot the
protein expression level of the serineethreonine protein kinase AKT
in five HCC-derived cell lines. Indeed, AKT is one of the major
downstream target of the kinase PI3K and its phosphorylation plays
a key role in promoting cell proliferation and cell survival by the
inactivation of Cyclin-Dependent Kinase (CDK) inhibitors and pro-
apoptotic genes such as BAD [26]. We analysed both the total and
the phosphorylated isoforms of AKT and we observed very different
levels of its phosphorylation in the tested HCC cell lines (Fig. 4).
In particular, two cell lines, SNU449 and SNU475, displayed
a very high extent of the phosphorylated AKT isoform (P-AKT). High
phosphorylation levels in SNU449 and SNU475 cells let us to
hypothesize a constitutive activation of the PI3K pathway in these
cell lines. In the light of these data, we chose SNU449 cells to
investigate the inhibitory effect of the two molecules on PI3K/AKT
pathway, because the very high P-AKT expression levels in SNU475
cells could mask the inhibitory activity of our molecules or
underestimate their true effect.
At 3
mM concentration all the evaluated kinases were not
inhibited by both compounds. So, the suggested concentrations for
further in vitro biological activity started from 50 mM.
2.5. Antiproliferative effect and cytotoxicity
To define the biological activity, in terms of cell proliferation
inhibition and cytotoxicity, the compounds, 5 and 7, were tested in
SNU449 cells at 50, 100 and 150 mM.
Evaluating the number of viable cells at 48 h after treatment,
a decrease of total cell number was observed with both molecules
at the concentration of 50
mM; in particular the effect on cell
proliferation results more evident for 5 than 7, comparing the
number of viable cells to the control (Ctrl) cells. At 72 h of treat-
ment, both compounds strongly reduced growth and exerted an
evident cytotoxic effect. Analysing the percentage of viable cells
after 5 and 7 treatment at the same concentrations, we could
establish the IC50 for both compounds (Fig. 3). Compound 7, as
previously observed, showed a slower effect on the proliferation
Initially, we studied the regulation of AKT phosphorylation
following treatment with 7 and 5 compounds at three different
concentrations (25e50e100
mM) in SNU449 cells. A decrease of P-
and the IC50 value is 150
Otherwise, the effect of 5 is stronger also at 48 h, where the IC50
value is less the 100 M and at 72 h is less than 50 M. Both the
mM at 48 h and around 50 mM at 72 h.
m
m
AKT levels was observed with the two highest concentrations of
tested molecules at both 48 and 72 h of treatment (Fig. 5AeB).
Since no alteration was observed at the lowest concentration,
we chose the intermediate one to analyse if molecular changes
could occur downstream of AKT pathway following the adminis-
inhibitors after 72 h of treatment have an IC50 less than 50 mM.
2.6. Effect of 7 and 5 on PI3K/AKT pathway
Among the ATP-dependent kinases tested, our in silico
(computational docking) analysis identified phosphoinositide-3-
kinase (PI3K) and serine/threonine-protein kinase B (AKT) as
hypothetical molecular targets of 7 and 5 kinase inhibitors. Since
tration of 7 and 5 inhibitors. The 100
mM concentration was
excluded to perform the subsequent analysis as it induced, espe-
cially with 5 molecule at 72 h, evident cell death and we supposed
O
O
N
H
H
N
OMe
H
N
N
N
N
H2N
N
BocHN
O
O
a
b, c
1 + 3
7
6
(Target B)
NH2
NHBoc
Scheme 3. 1,3-Dipolar cycloaddition of 1 with 3 and synthetic elaboration of the cycloadduct 6 for the synthesis of 7 (Target B). Reagents and conditions: (a) Ag2CO3, 1,4-dioxane,
80 ꢁC, 24 h, 64%. (b) NaOH, MeOH, rt, overnight, 40%. (c) N,N-carbonyldiimidazole, THF, 2,3-dimethylaniline, rt, 2 h, 48%. HCl 3 M in EtOAc, rt, 8 h, 100%.

E. Strocchi et al. / European Journal of Medicinal Chemistry 48 (2012) 391e401
395
Fig. 5. P-AKT expression following treatments with 7 and 5. Western blot analysis of P-
AKT levels in SNU449 cells treated with 7 and 5 for 48 h (A) and 72 h (B). ß-actin was
used as housekeeping gene. Numbers represent the ratio between P-AKT and
expression. Ctrl represent cells treated with DMSO.
b-actin
These findings demonstrated that the two tested molecules
deregulate the PI3K/AKT pathway with a consistent decrease of the
AKT kinase activity resulting in a down-regulation of the phos-
phorylation of its downstream target genes.
2.7. Cell cycle regulation by 7 and 5 molecules in SNU449 cells
To further characterize the biological role of the tested mole-
cules, we analysed the cell cycle distribution following their
administration in HCC cells. SNU449 cell line was treated with 7
and 5 at three different concentrations (25e50e100 mM) for 48 and
Fig. 3. Inhibitory activity of 5 and 7 on SNU449 HCC cell line after 48 h and 72 h of
incubation with different compound concentrations.
72 h. Cytofluorimetric analysis showed an inhibitory activity of cell
cycle progression with an increase of the G1 phase cell population
and a correspondent decrease of the S phase. In particular, treat-
ment with the lowest concentration of 5 (25 mM) determined a G1
that early regulation of molecular pathways could be hidden in this
specific setting.
To investigate the functional activity of AKT kinase after treat-
ment with the two inhibitors, the phosphorylation levels of both
the direct and indirect AKT downstream effectors, glycogen syn-
phase arrest at both 48 and 72 h. Regarding 7, it exerted a cell cycle
regulation at a higher concentration and a longer incubation time
than 5, especially it arrested cells in the G1 phase at a concentration
of 50
mM following 72 h of treatment. At the highest concentration
tested (100
m
M), both the two molecules played a role in cell cycle
thase kinase 3 beta (GSK-3
analysed in SNU449 cells. A decrease of the inhibitory phosphor-
ylation (Ser-9) of GSK-3 was observed at 48 and 72 h with both
molecules. In line with this finding a decrease of -catenin protein
b) and ribosomal protein S6 (RpS6), was
regulation of SNU449 cells, with a marked increase of the G1 phase
and a decrease of the S phase both at 48 and 72 h (Fig. 7A).
Subsequently, we evaluated by Western blot which cell cycle
modulators could be involved in the arrest of SNU449 cells at the
G1 checkpoint following treatment with the two molecules. To this
aim, we tested the treatment with the intermediate concentration
b
b
levels were displayed in the same experimental settings. As
regarding the PI3K/AKT/mTOR pathway, a decrease of the phos-
phorylated isoform of RpS6 was shown with both molecules and
especially following administration of inhibitor 5 at 72 h. On the
contrary, any inhibition of ERK1/2 phosphorylated isoform was
revealed following treatment with 7 and 5 molecules (Fig. 6).
(50 mM) at 72 h, when both molecules exerted an inhibitory effect
on cell cycle progression.
In agreement with FACS analysis, a decrease of cyclin D1
(CCND1) protein level was obtained after treatment with 7 and 5
compounds. In addition, an up-regulation of the cell cycle inhibitor
CDKN1A/p21 was observed for both tested molecules, whereas no
changes were shown for CDKN1B/p27. To deeply investigate which
molecular pathways could be involved in cell cycle modulation, we
analysed the protein expression level of the principal CDKN1A/p21
transcriptional regulator, p53. In accordance with CDKN1A/p21 up-
regulation, an increase of p53 expression levels was observed
following treatment with 7 and 5 compounds in SNU449 cells
(Fig. 7B).
In order to try to explain the increase of p53 protein levels
following treatment with 7 and 5, we analysed the phosphorylation
levels of the principal p53 inhibitor, MDM2. In particular, we
Fig. 4. Analysis of AKT in HCC-derived cell lines. Western blot analysis of AKT and P-
AKT expression levels in HCC-derived cell lines. ß-actin was used as housekeeping
gene. Numbers represent the ratio between AKT or P-AKT and b-actin expression.

396
E. Strocchi et al. / European Journal of Medicinal Chemistry 48 (2012) 391e401
Fig. 6. Analysis of AKT targets following treatments with 7 and 5. Western blot analysis of P-AKT, P-RpS6, P-GSK-3
b,
b-catenin and P-ERK172 levels in SNU449 cells treated with
50 mM of 7 and 5 compounds for 48 and 72 h -actin was used as housekeeping gene. Numbers represent the ratio between phosphorylated proteins and
b-actin expression. Ctrl
represent cells treated with DMSO.
investigated the phosphorylation levels of Ser-166 which is directly
phosphorylated by the AKT kinase. This phosphorylation deter-
mines MDM2 activation, allowing its entry into the nucleus where
it targets p53 for degradation. Western blot analysis showed
a decrease of phosphorylated-MDM2 isoform in SNU449 cells
treated with both inhibitory compounds, whereas the levels of total
MDM2 remained unchanged (Fig. 7B).
Taken together, these findings demonstrated that the two
inhibitors play an active role in cell cycle modulation of HCC cells, at
least in part through the down-regulation of MDM2 phosphoryla-
tion and an activation of p53 transcriptional activity and the inhi-
3. Conclusions
Thepreliminarydockinginvestigation fora rationaledevelopment
of our 3,5-pyrazole derivatives with potential affinity and selectivity
toward different kinases, suggested that a potential effective inhibi-
tory molecule to the ATP kinase binding site could be attained by the
following structural features: (1) C-3 hydrophobic aryl moiety; (2)
pyrazole moiety with amino-phenyl substituent at N1 and lateral
aminophenyl substituents in the 5eCOeNH-R. The common binding
mode of the two derivatives into the ATP binding site through two
possible relevant hydrogen-bonding interactions and a complemen-
tary apolar interaction surface could explain the inhibitory activity of
our two compounds in HCC cell lines. The introduction of a carbox-
amide group in position 3 of 7, connected with the dimethyl-phenyl
group, did not improve the inhibitory activity.
bition of WNT/b-catenin pathway.
2.8. Apoptotic cell death is triggered by treatment with 7 and 5
compounds in SNU449 cells
The binding pose of 5 and 7 e obtained with docking results e
within ATP binding site of the evaluated kinases, suggest that the
two compounds are aspecific ATP competitors. In vitro study
confirmed inhibitory effect - at micromolar concentrations - against
the HCC-derived cell line SNU449, after an incubation of 48 and 72 h.
The more favourable logP partition coefficent of 5 (logP equal to
4.47) in comparison with that of 7 (logP equal to 3.27) could explain
the better in vitro results due to more lipophilic characteristic, sug-
gesting a more easily entrance of the compound inside the cells.
Here we demonstrated that 7 and 5 exert an inhibitory activity
on the PI3K/AKT/mTOR pathway resulting in a down-regulation of
the phosphorylated isoform of both direct and indirect down-
After 72 h of treatment with the two molecules at 50e100 mM,
SNU449 cells displayed a decreased growth rate and an increased
cell death (Fig. 8A).
To characterize if programmed cell death, apoptosis, was
responsible for decreased SNU449 cell number following 7 and 5
administration, a FACS-Annexin V assay was performed. This assay
showed an increase of apoptosis following treatment with 5
inhibitor at both 50 and 100
mM concentrations. Regarding 7
molecule, a trend toward increased apoptotic cell death was dis-
played, even if it did not reach the statistical significance. Following
treatment with the two compounds, the low and almost unchanged
Propidium Iodide (PI) incorporation confirmed that apoptotic cell
death is an early event in SNU449 cells at the tested concentrations
(Fig. 8B).
stream targets such GSK-3
b, ribosomal subunit S6 and MDM2. The
unselective action of the two compounds against multiple kinases
was ruled out by testing ERK1/2 phosphorylation, whose levels did
not change after treatment with both 7 and 5.

E. Strocchi et al. / European Journal of Medicinal Chemistry 48 (2012) 391e401
397
Fig. 7. Compounds 5 and 7 affect cell cycle. (A) Analysis of cell cycle variations induced by different concentrations of 5 and 7 in SNU449 cells for 48 and 72 h. Controls (Ctrl)
represent cells treated with DMSO. Columns, average values; bars, SD. *means p < 0.05. (B) Western blot analysis of cell cycle regulators, p53, MDM2 and P-MDM2 in SNU449 cells
treated with 50 mM of 7 and 5 for 72 h -actin was used as housekeeping gene. Numbers represent the protein expression ratio between the analyzed genes and b-actin.
Moreover, the tested compounds determined a block of cell
cycle in the G1 phase, with a correspondent decrease of the S and
G2/M phases. In accordance with a decrease of AKT kinase activity,
observed in SNU449 cells treated with 7 and 5 molecules, a down-
regulation of phosphorylation levels of MDM2 protein at Ser-166
was also reported. We suggest that a decrease of phosphorylated-
MDM2 isoform is responsible for p53 increased expression which,
in turn, transcriptionally activates its target gene, CDKN1A/p21 [27]
and that the block of cell cycle observed in SNU449 cells treated
with the two molecules might be ascribed, at least in part, to the
activation of CDKN1A/p21. To further characterise the effects of 7
and 5, cyclin D1 protein levels were investigated and their decrease
in treated cells proved a direct effect of the two compounds on cell
analysis which demonstrated that both compounds, in particular 5,
triggered apoptosis in HCC-derived cells.
Therefore, this synthetic protocol will offer a general entry to
3,5-disubstituted pyrazoles having in position 3 both aromatic and
amidic functional group, as a new strategy to inhibit the PI3K/AKT/
mTOR pathway in HCC which might improve the drug response to
molecularly-targeted therapy agents. These compounds could
therefore be considered as leads for SAR development.
4. Experimental
4.1. Material and methods
proliferation. This might be related with the activation of GSK-3
b
,
1H NMR and 13C NMR spectra were recorded using CDCl3 or
which in turn phosphorylates
uitination and degradation.
b
-catenin leading to its ubiq-
CD3OD or DMSO-d6 solutions at 300, 400 and 600 MHz for 1H and
75.46, 100.6 and 150.92 MHz for 13C. Chemical shifts (
d) are re-
Microscopical view of SNU449 cells following 72 h of treatment
with 7 and 5 showed both a decrease of cell proliferation and cell
death, suggesting a contemporaneous cytostatic and cytotoxic effect.
The nature of cell death was investigated by a cytofluorimetric
ported in ppm relative to CHCl3 (
d
¼ 7.26 for 1H and
d
¼ 77.0 for 13C).
J values are given in Hz. 1H NMR and 13C NMR spectral assignments
were made by DEPT, gCOSY and gHSQC experiments. IR spectra
were recorded in solvent as specified. Mass spectra (MS) were

398
E. Strocchi et al. / European Journal of Medicinal Chemistry 48 (2012) 391e401
Fig. 8. Compounds 5 and 7 trigger apoptosis. (A) Microscopic view of SNU449 cells treated with 100
mM of 5 and 7 or vehicle molecule (Ctrl). Original magnification, X10. (B) FACS-
Annexin V analysis of SNU449 cells treated with different concentrations of 7 and 5 for 72 h. Columns, average values; bars, SD. *means p < 0.05.
obtained with an electrospray ionization source (ESI-MS). All the
ESIMS spectra were performed using MeOH as the solvent. High
Resolution Mass Spectra (HRMS) were recorded on a micromass
LCT spectrometer using electrospray (ESþ) ionisation techniques.
Reactions were conducted in oven-dried (120 ꢁC) glassware under
a positive Ar atmosphere. Transfer of anhydrous solvents or
mixtures was accomplished with oven-dried syringes/septum
techniques. THF was distilled from sodium/benzophenone just
prior to use and stored under Ar. Toluene was distilled from sodium.
Et2O was distilled from phosphorus pentoxide. CH2Cl2 was passed
through basic alumina and distilled from CaH2 prior to use. Other
solvents were purified by standard procedures. Light petroleum
ether refers to the fraction with bp 40e60 ꢁC. The reactions were
monitored by TLC performed on silica gel plates (Baker-flex IB2-F).
Column chromatography was performed with Merck silica gel 60
(70e230 mesh). Preperative TLC was carried out on glass plates
using a 1 mm layer of Merck silica gel 60 Pf 254. All chemicals were
used as obtained or purified as needed.
(26 mmol) of propiolic acid and 13 mL of diethyl ether were added.
The temperature was lowered to ꢁC, 5.4 g (26 mmol) of DCC were
slowly added followed by 37 mg (0.3 mmol) of DMAP. N-p-Boc-
phenylendiamine (5.4 g, 26 mmol) in 25 mL of THF was slowly
added. The reaction was stirred for 24 h at 35 ꢁC. The mixture was
filtered over celite and the THF was removed in vacuo, EtOAc (10 mL)
was added, the organic phase was dried over Na2SO4 and the crude
was purified by column chromatography on silica gel (petroleum
ether:EtOAc 2:1) giving 4.87 g (72%) of 1 as a yellow solid. M.p.:
174e176 ꢁC. MS (EI, m/z) 260 (Mþ). 1H NMR (CDCl3, 400 MHz)
d: 1.52
(s, 9H, CH3), 2.92 (s, 1H, CH), 6.48 (brs, 1H, NH), 7.52 (brs, 1H, NH),
7.32 (d, J ¼ 8,8 Hz, 2H, CH), 7.44 (d, J ¼ 8,8 Hz, 2H, CH).
(E)-2,3-Dimethyl-N0-(4-nitrophenyl)benzohydrazonoyl chloride
2: In a two-neck round-bottom flask, under stirring and nitrogen,
3.0 g (20 mmol) of 2,3-dimethylbenzoic acid were added and the
mixture was refluxed with 3.62 mL (50 mmol) of SOCl2. After 8 h
the excess of SOCl2 was removed at the rotavapor and under
vacuum to give the 3,3-dimethyl benzoyl chloride as a yellow oil in
quantitative yield that was used further without purification.
In a two-neck round-bottom flask, under stirring and nitrogen,
4.2. Chemistry
were added 307 mg (2 mmol) of p-nitrophenyl hydrazine, 227 mL
Tert-butyl-(4-propiolamidophenyl)carbamate 1: In a two-neck
round-bottom flask, under stirring and nitrogen, 1.60 mL
(2 mmol) of triethylamine and 5 mL of THF. The temperature was
lowered to 0 ꢁC, 2,3-dimethylbenzoylchloride (2.0 mmol) was

E. Strocchi et al. / European Journal of Medicinal Chemistry 48 (2012) 391e401
399
slowly added in 15 mL of THF and the reaction mixture was stirred
at room temperature for 4 h. THF was removed under reduced
pressure, EtOAc was added (10 mL) and the organic layer was
washed with water 5 times (5 mL each). The organic phase was
dried over Na2SO4 and the crude was crystallized with EtOH giving
428 mg (75%) of 2,3-dimethyl-N0-(4-nitrophenyl)benzohydrazide
as an orange solid. M. p. ¼ 275e277 ꢁC. MS (ESI) 308 (Mþ þ Na), 284
(60%) of tert-butyl (4-(1-(4-aminophenyl)-3-(2,3-dimethylphenyl)-
1H-pyrazole-5-carboxamido)phenyl)carbamate as a white sticky
solid. MS (ESI) 498 (M þ 1), 520 (Mþ þ Na), 536 (M þ K). 1H NMR
(CD3OD, 300 MHz) d: 1.51 (s, 9H, CH3), 2.34 (s, 3H, CH3), 2.38 (s, 3H,
CH3), 6.76 (d, J ¼ 8.7 Hz, 2H, CH), 6.95 (s, 1H, CH), 7.16 (m, 2H, CH),
7.25 (d, J ¼ 8.7 Hz, 2H, CH), 7.34 (m, 3H, CH), 7.46 (d, J ¼ 9 Hz, 2H,
CH), 7.90 (s, 1H, NH), 8.92 (brs, 1H, NH). 13C NMR (CD3OD, 75 MHz)
(Mþ-1). 1H NMR (CD3OD, 400 MHz)
d
: 2.55 (s, 3H), 2.57 (s, 3H), 6.92
d: 17.2, 20.8, 28.7, 80.8, 109.8, 115.8, 119.9, 122.2, 126.3, 126.9, 128.6,
(d, J ¼ 9.0 Hz, 2H), 7.09 (m, 3H), 8.14 (d, J ¼ 9.0 Hz, 2H).
130.8, 131.4, 133.7, 133.8, 135.92, 137.3, 138.4, 139.1, 149.3, 153.3,
155.1, 160.2.
The above reported 2,3-dimethyl-N0-(4-nitrophenyl) benzohy-
drazide (571 mg, 2 mmol) was dissolved in acetonitrile (3.0 mL),
N,1-bis(4-aminophenyl)-3-(2,3-dimethylphenyl)-1H-pyrazole-
5-carboxamide 5: Starting from 324 mg (0.65 mmol) of tert-butyl
(4-(1-(4-aminophenyl)-3-(2,3-dimethylphenyl)-1H-pyrazole-5-
carboxamido)phenyl)carbamate. Compound 5 was obtained in
100% yield as a white solid. Characterization was carried out after
basification with conc. ammonia and extraction with EtOAc. M.p.:
243e246 ꢁC. MS (ESI). 398 (Mþ1), 420 (MþþNa). 1H NMR (CD3OD,
then 656 mg (2.50 mmol) of triphenylphosphine and 242
ml
(2.50 mmol) of CCl4 were added. After stirring for 24 h at room
temperature the crude was purified on column chromatography on
silica gel (petroleum ether: EtOAc 3:1) giving 425 mgþ(70%) of 2 as
a yellow solid. M. p. ¼ 210e211 ꢁC. MS (ESI): 284 (M -1). 1H NMR
(CDCl3, 300 MHz) d: 2.36 (s, 3H, CH3), 2.40 (s, 3H, CH3), 7.15 (d,
J ¼ Hz, 2H, CH), 7.19 (d, J ¼ 9 Hz, 1H, CH), 7.25 (d, J ¼ 7 Hz, 1H, CH),
400 MHz) d: 2.35 (s, 3H, CH3), 2.41 (s, 3H, CH3), 7.19 (m, 2H, CH),
7.38 (d, J ¼ 9 Hz, 1H, CH), 8.20 (d, J ¼ 9 Hz, 2H, CH), 8.38 (s, 1H, NH).
7.26 (s, 1H, CH), 7.37 (d, J ¼ 7.6 Hz, 1H, CH), 7.41 (d, J ¼ 8.8 Hz, 2H,
13C NMR (CDCl3, 100 MHz)
d: 16.9, 20.5, 112.5, 125.5, 126.0, 127.36,
CH), 7.57 (d, J ¼ 8.4 Hz, 2H, CH), 7.64 (s, 1H, NH), 7.73 (d, J ¼ 8.8 Hz),
128.3, 131.5, 134.8, 135.3, 137.9, 141.2, 148.3.
7.85 (d, J ¼ 8.8 Hz, 2H, CH). 13C NMR (CD3OD, 100 MHz)
d: 17.2, 20.7,
(Z)-methyl-2-(2-(4-((tert-butoxycarbonyl)amino)phenyl) hydrazono)-
2-chloacetate 3: Prepared according to reference 24a. Starting
from N-p-Boc-phenylendiamine (3.12 g, 15 mmol) to give 2.7 g
(55%) of 3 as a yellow sticky oil that was used further without
purification. ESI-MS: 350 [Mþ þ Na]. 1H NMR (CDCl3, 400 MHz)
111.8, 123.0, 124.7, 124.9, 126.6, 127.8, 128.7, 131.5, 131.5, 133.3, 136.1,
138.6, 140.2, 141.9, 154.7, 159.9. Anal. Calcd for C24H23N5O: C,
75.52; H, 5.83; N, 17.62. Found: C, 75.48; H, 5.85; N, 17.63.
4.2.2.2. SynthesisofN5,1-bis(4-aminophenyl)-N3-(2,3-dimethylphenyl)-
1H-pyrazole-3,5-dicarboxamide 7. Methyl 1-(4-((tert-butoxycarbonyl)
d
: 8.31 (s, 1H), 7.35 (d, J ¼ 22 Hz, 2H), 7.17 (d, J ¼ 22 Hz, 2H),
6.42 (s, 1H), 3.92 (s, 3H), 1.51 (s, 9H).
amino)phenyl)-5-((4-((tert-butoxycarbonyl)
moyl)-1H-pyrazole-3-carboxylate 6: Starting from
amino)phenyl)carba-
(645 mg,
3
4.2.1. General procedure for the 1,3-DC of 1 with 2 and 3
To a solution of 1 (1 mmol) and the precursor of the 1,3-dipoles 2
or 3 (1 mmol) in 1,4-dioxane (4.4 mL) was added Ag2CO3 (0.69 g,
2.5 mmol) and the reaction mixture was stirred at 80 ꢁC for 24 h.
The reaction was filtered over celite and the solvent was removed
under vacuum. Purification and characterization was carried out as
reported below.
2.0 mmol),1 (520 mg, 2.0 mmol). The cycloaddition gave a ratio 4/1 in
favour of the major isomer. Purified on column chromatography on
silica gel (petroleum ether: EtOAc 1:1) as a yellow oil. Yield: 64%. ESI-
MS: 574 [Mþ þ Na]. 1H NMR (CDCl3, 300 MHz)
d: 1.50 (s, 9H), 1.51 (s,
9H), 3.95 (s, 3H), 6.50 (s, 1H), 6.68 (s, 1H), 7.36e7.40 (m, 2H),
7.45e7.50 (m, 2H), 7.52 (s, 1H), 7.67 (d, J ¼ 30 Hz, 2H), 7.71 (d,
J ¼ 30 Hz, 2H), 8.63 (s,1H). 1H NMR (CDCl3, 300 MHz)
d: 28.5 (br), 80.0
(br), 120.1e123.2 (Ar CH), 133., 133.6, 135.1, 135.4 (Ar C(IV)), 152.5,
152.7, 160.3, 162.6. Minor isomer methyl 1-(4-((tert-butoxycarbonyl)
amino)phenyl)-4-((4-((tert-butoxycarbonyl)amino)phenyl)carba-
moyl)-1H-pyrazole-3-carboxylate. Attempted purification failed to
produce pure compound, only a 1H NMR could be extrapolated but
some impurities, derived from degradation on plates, were always
4.2.2. General procedure for the Boc deprotection
The Boc deprotection was performed starting from 0.5 mmol of
product dissolved in EtOAc (5 mL) and adding 0.5 mL of 3 M HCl in
EtOAc. After 2e8 h at room temperature (following by TLC), the
solvent was evaporated to yield pure products as chlorohydrate,
then the free amine was obtained after basification with conc.
Ammonia and extraction with EtOAc.
present. 1H NMR (CDCl3, 300 MHz)
d: 1.50 (s, 9H), 1.51 (s, 9H), 4.07 (s,
3H), 6.59 (s, 1H), 6.85 (s, 1H), 7.36e7.45 (m, 2H), 7.60e7.65 (m, 2H),
7.70e7.75 (m, 2H), 8.65 (s, 1H).
4.2.2.1. Synthesis of N,1-bis(4-aminophenyl)-3-(2,3-dimethylphenyl)-
1H-pyrazole-5-carboxamide 5. Tert-butyl (4-(3-(2,3-dimethylphenyl)-
1-(4-nitrophenyl)-1H-pyrazole-5-carboxamido)phenyl)carbamate 4:
Starting from 2 (421 mg, 1.4 mmol), 1 (361 mg, 1.4 mmol). Purification
on column chromatography on silica gel (petroleum ether:EtOAc 3:1)
gave 4 as a yellow sticky oil. Yield: 35%. MS (ESI) 550 (Mþ þ Na). 1H
In a two-neck round-bottom flask, under stirring and nitrogen,
compound 6 (1.33 g, 2.41 mmol) was dissolved in 16 mL of MeOH and
4 mL of a 2 M solution of NaOH were added. The reaction was stirred
overnight at room temperature and then methanol was eliminated.
Et2O was added (5 mL) and the organic layer was washed twice with
water (5 mL). The pH was adjusted to 4 with HCl conc. The acidic layer
was extracted twice with EtOAc (5 mL) and once with chloroform
(5 mL). The combined organic phases were dried over Na2SO4,
filtered, concentrated and dried under vacuum giving 1-(4-((tert-
butoxycarbonyl)amino)phenyl)-5-((4-((tert-butoxycarbonyl) amino)
phenyl)carbamoyl)-1H-pyrazole-3-carboxylic acid as a brown solid
(40%) that was used without further purification. ESI-MS: 560
[Mþ þ Na].
NMR (CDCl3, 300 MHz) d: 1.52 (s, 9H, CH3), 2.36 (s, 3H, CH3), 2.40 (s,
3H, CH3), 6.51 (br s, 1H, NH), 6.96 (s, 1H, CH), 7.20 (m, 3H, CH), 7.36 (d,
J ¼ 8.1 Hz, 2H, CH), 7.50 (d, J ¼ 8.7 Hz, 2H, CH), 7.78 (d, J ¼ 9 Hz, 2H,
CH), 7.87 (brs, 1H, NH), 8.31 (d, J ¼ 9 Hz, 2H, CH). 13C NMR (CDCl3,
75 MHz) d: 17.3, 20.9, 28.5, 80.9, 110.6, 119.2, 121.1, 124.4, 125.1, 125.7,
127.6, 130.5, 131.6, 131.9, 135.0, 135.7, 137.4, 137.8, 144.6, 146.7, 152.7,
154.0, 157.2.
In a two-neck round-bottom flask, under stirring and nitrogen, 4
(458 mg, 0.9 mmol) was dissolved in MeOH (9 mL). The tempera-
ture was lowered to ꢁC, then 86.8 mg of Pd/C (10% w/w) and
ammonium formate (547 mg, 9 mmol) were added. The mixture
was stirred 1 h at ꢁC and then overnight at room temperature. The
crude was filtered over celite and purified on column chromatog-
raphy on silica gel (petroleum ether: EtOAc 2:1) giving 269 mg
In a two-neck round-bottom flask, under stirring and nitrogen,
1-(4-((tert-butoxycarbonyl)amino)phenyl)-5-((4-((tert-butoxy-
carbonyl)amino)phenyl)carbamoyl)-1H-pyrazole-3-carboxylic acid
(490 mg, 0.91 mmol) was dissolved in THF (4 mL). Slowly, N-
N-carbonyldiimidazole (200 mg, 1.25 mmol) was added and
the reaction was stirred at room temperature for 1 h 2,3-
dimethylaniline (223 mL, 1.25 mmol) in 1.5 mL of THF was added

400
E. Strocchi et al. / European Journal of Medicinal Chemistry 48 (2012) 391e401
and the mixture was stirred for 2 h. The solvent was removed under
reduced pressure, EtOAc (10 mL) was added and the organic layer
was washed with HCl 1 M (5 mL). The organic phase was dried over
Na2SO4 filtered, concentrated and dried under vacuum giving
a crude that was purified on column chromatography on silica gel
(petroleum ether: EtOAc 2:1) as a yellow oil giving the corre-
sponding amide (48%) as a sticky yellow solid. ESI-MS: 663
4.4. Biological assays
4.4.1. Cell culture and treatments
HepG2 cell line was cultured with Minimum Essential Medium
(Eagle) while SNU398, SNU182, SNU449 and SNU475 cell lines were
cultured with RPMI 1640; both media were supplemented with 10%
foetal bovine serum (FBS) and antibiotics. Cells were treated at
different concentrations (range 25e150 mM) of the two compounds
for 24, 48 and 72 h and cells were collected for Western blot and
FACS analysis.
[Mþ þ Na]. 1H NMR (CDCl3, 300 MHz)
d: 1.51 (s. 9H), 1.53 (s, 9H),
2.20 (s, 3H), 2.29 (s, 3H), 6.45 (s, 1H), 6.67 (s, 1H), 6.98e7.10 (m, 2H),
7.39e7.49 (m, 9H), 7.60e7.70 (m, 2H), 8.26 (s, 1H), 8.66 (s, 1H).
Following the general procedure for the Boc removal. Starting
from the above reported amide (107 mg, 0.17 mmol). N5,1-bis(4-
aminophenyl)-N3-(2,3-dimethylphenyl)-1H-pyrazole-3,5-
4.4.2. Cytotoxicity assay
Exclusion test of cell viability was performed using trypan blue
(TB) viable cell counts protocol (Shapiro, H.M. 1988 [30]). Pyrazoles
were dissolved in dimethylsulphoxide (SigmaeAldrich, Saint Louis,
MO) at 10 mM stock concentration, then diluted. SNU449 cells were
treated with pyrazoles or DMSO (as control) at different concen-
trations for 48 and 72 h and in triplicate. Then 0.4% TB solution was
added to each well following the standard protocol procedure. Cell
countings were performed using a Burker chamber. Triplicate
values were measured. Inhibitory growth of compounds was re-
ported as mean percentage of viable cells after 48 and 72 h of
incubation.
dicarboxamide 7 was isolated as a white solid. Yield: 100%. M. p.:
>300 ꢁC dec. Characterization was carried out after basification
with conc. ammonia and extraction with EtOAc. ESI-MS: 463
[Mþ þ Na]. 1H NMR (DMSO-d6, 400 MHz)
d: 2.15 (s, 3H), 2.32 (s, 3H),
7, 14e7.18 (m, 1H), 7.26e7.30 (m, 1H), 7.35e7.38 (m, 2H), 7.41e7.45
(m, 2H), 7.62e7.65 (m, 2H), 7.72 (s, 1H), 7.84e7.90 (m, 2H), 9.97 (s,
1H), 10.85 (s, 1H). 13C NMR (DMSO-d6, 100 MHz)
d: 14.8, 20.8, 110.6,
121.8, 124.3, 124.8, 125.9, 127.3, 128.2, 132.8, 136.3, 136.8, 137.6,
138.7, 138.9, 147.0, 157.7, 160.0. Anal. Calcd for C25H24N6O2: C,
68.17; H, 5.49; N, 19.08. Found: C, 68.14; H, 5.46; N, 19.95.
4.4.3. Western blot
4.3. Molecular docking
HCC-derived cell lines were assayed with anti-AKT, anti-phos-
pho-AKT (Ser-473), anti-phospho-S6 Ribosomal Protein (Ser-240/
The following operating procedures were followed for the
development of the 3,5-pyrazole derivatives: (a) geometry opti-
mization of the proposed ligands; (b) virtual docking of the
proposed pyrazole derivatives to calculate their predicted binding
energy to the active site of several kinases.
244), anti-phospho-GSK-3b (Ser-9), anti-phospho-MDM2 (Ser-
166), ant-phospho ERK1/2 (Cell Signalling, Danvers, MA), anti-p27
(clone 57, BD Biosciences, Franklin Lakes, NJ), anti-p53 (clone DO-
7, Dakocytomation, England, UK), anti-cyclin D1 (clone DCS-6,
Novocastra, Newcastle upon Tyne, UK) anti-p21 (clone F-5), anti-
MDM2 (clone N-20),anti-b-catenin (clone E-5) and anti-b-actin
4.3.1. Preparation of molecules
(Santa Cruz Biotecnology, Santa Cruz, CA) antibodies. Digital images
were acquired and quantified with Fluor-S MultiImager (Quantity-
one,Bio-R Hercules, CA), as previously reported [31].
Before starting the docking calculations, ligand geometry opti-
mization was done by semi-empirical AM1 [28] of the minimum-
energy conformation using Gaussian 3 [29].
4.4.4. Flow cytometry analysis
4.3.2. Preparation of target macromolecule
For flow cytometric analysis of DNA content, 2.5 ꢂ 104 SNU449
cells in exponential growth were treated at different concentra-
tions of the tested compounds for 48 and 72 h. After this incubation
time, cells were fixed with ice-cold ethanol (70%) for 16 h at ꢀ20 ꢁC,
and treated as previously described [32] and analysed by using the
FACSAria cell sorter (BD Biosciences). Detection of apoptotic cells
was performed following 72 h of treatment by using Annexin V/
Propidium Iodide detection kit (Bender MedSystems, Vienna,
Austria) according to the manufacturer’s instructions and then
analysed by flow cytometry.
The target proteins AKT2 (PDB: 2jdr), GSK3b (PDB: 1pyx), PI3K
(PDB: 2v4l), CDK2 (PDB: 2j9m), AurA (PDB: 2x6d), MAPKJ (PDB:
2pzy), EGFR (PDB: 2rpg), IGFR (PDB: 2zm3), were retrieved from
the Protein Data Bank (http://www.rcsb.org/pdb/). The proteins
were prepared with Maestro 9.1 software (Schrödinger, LLC, New
York, NY, 2010) deleting waters, optimizing H-Bond assignments
and deleting original ligands.
4.3.3. Procedure for molecular docking
Docking calculations were carried out using AutoDock 4.1 [25]
and Autogrid 4.0 on a dual-xeon T7400 Dell workstation. Grids
(one grid for each atom type in the ligand, plus an electrostatic and
a desolvation map) were centred on the binding site and were
chosen to be large enough (70 ꢂ 70 ꢂ 70 Aꢁ) to allow the ligand to
rotate freely, even in its most fully-extended conformation.
Docking was performed using the AutoDock empirical free
energy function and the Lamarckian genetic algorithm with local
search. Lamarckian genetic algorithms can handle ligands with
more degrees of freedom than the simulated annealing method.
One hundred fifty docking runs with 2.500.000 energy evalua-
tions for each run were performed for each molecule and all the
evaluated kinases. Cluster analysis (RMS tolerance equal to 0.5 Å)
was then carried out on the docked results. Inhibitors were
compared according to the cluster with lowest docked energy
found. The inhibition constants e Ki e were calculated from the
docked energy.
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