Identification of potent c-Src inhibitors strongly affecting the proliferation of human neuroblastoma cells

Article abstract of DOI:10.1016/j.bmcl.2011.07.079

Neuroblastoma (NB) represents the most common extracranial paediatric solid tumor for which no specific FDA-approved treatment is currently available. The tyrosine kinase c-Src has been reported to play an important role in the differentiation, cell-adhes

Full text of DOI:10.1016/j.bmcl.2011.07.079

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5928–5933
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of potent c-Src inhibitors strongly affecting the proliferation
of human neuroblastoma cells
Marco Radi a, Chiara Brullo b, Emmanuele Crespan c, Cristina Tintori a, Francesca Musumeci b,
a
a
c
Mariangela Biava d, Silvia Schenone b, , Elena Dreassi , Claudio Zamperini , Giovanni Maga ,
⇑
Dafne Pagano c, Adriano Angelucci e, Mauro Bologna e, Maurizio Botta a,f,
⇑
a Dipartimento Farmaco Chimico Tecnologico, University of Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy
b Dipartimento di Scienze Farmaceutiche, University of Genova, Viale Benedetto XV 3, I-16132 Genova, Italy
c Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, I-27100 Pavia, Italy
d Istituto Pasteur Fondazione Cenci-Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università ‘La Sapienza’, piazzale Aldo Moro 5, I-00185 Roma, Italy
e Department of Experimental Medicine, University of L’Aquila, via Vetoio, 67100 Coppito, L’Aquila, Italy
f Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Suite 333,
1900 N 12th Street, Philadelphia, PA 19122, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Neuroblastoma (NB) represents the most common extracranial paediatric solid tumor for which no spe-
cific FDA-approved treatment is currently available. The tyrosine kinase c-Src has been reported to play
an important role in the differentiation, cell-adhesion and survival of NB cells. Starting from dual Src/Abl
inhibitors previously found active in NB cell lines (1–3), small modification of the original structures
almost abolished the Abl activity with a contemporary improvement of affinity and specificity for c-
Src. Among the synthesized compounds, the most potent c-Src inhibitor (10a) showed a very interesting
antiproliferative activity in SH-SY5Y cells with an IC50 of 80 nM and a favourable ADME profile. A 3D SAR
analysis was also attempted and may guide the design of more potent c-Src inhibitors as potential agents
for NB treatment.
Received 30 May 2011
Revised 21 July 2011
Accepted 22 July 2011
Available online 29 July 2011
Keywords:
Neuroblastoma
c-Src
Bcr-Abl
Kinase
Ó 2011 Elsevier Ltd. All rights reserved.
Pyrazolo[3,4-d]pyrimidines
Neuroblastoma (NB) is the most common extracranial solid
cancer in childhood whose outcome has modestly improved in
the last years. Although treatment of early stage NB patients has
shown favourable outcomes, long-term survival for stage 4 pa-
tients is still less than 40%.1 Treatment options used in the man-
agement of NB include surgery, chemotherapy and radiotherapy.2
However, due to the high toxicity of the conventional anticancer
agents used, the development of targeted therapies characterized
by less pronounced toxic effects is a compelling priority in paedi-
atric oncology. Tyrosine kinases have been associated with onco-
genic transformation in many human tumors and represent
today the target of less toxic anti-tumor treatments.3 Among the
many tyrosine kinases targeted for the treatment of human can-
cers, cellular-Src (c-Src) is a fundamental enzyme for cell differen-
tiation, replication, growth, adhesion, motility, angiogenesis and
bone metabolism being involved in transmitting extracellular sig-
nals across the cell membrane to distant locations in the cytoplasm
and in the nucleus.4–6
Hyperactivation of c-Src causes therefore an aberrant cell
activity that can contribute to cancer development.7,8 High levels
of c-Src have been detected in several cancers and are generally cor-
related to a poor prognosis.9 Interestingly, c-Src has been reported
to play an important role in the differentiation, cell-adhesion and
survival of NB cells10 were it resulted hyperactivated, rather than
overexpressed.10 Moreover, c-Src was hypothesized to play an
oncogenic role in the progression of aggressive NB forms.11 The
inhibition of such kinase as an effective approach to treat NB has
been recently reported: the well known c-Src inhibitor PP2 proved
in fact to reduce cell survival/proliferation and to promote aggrega-
tion in NB cell lines.12 In addition, also the dual Src/Abl inhibitor
Dasatinib proved to be effective in reducing NB growth both
in vitro and in vivo.13 Due to the high homology between the active
forms of c-Src and Abl kinases14 and considering also the advanta-
ges connected with the use of multiple kinase inhibitors in
overcoming drug resistance,15 a few dual Src/Abl inhibitors have
been advanced in clinical trials for the treatment of solid tumors.
They include Dasatinib, the quinazoline Saracatinib (AZD-0530)
and the quinoline Bosutinib (SKI-606).7 As part of our efforts to
⇑
Corresponding authors. Tel.: +39 010 3538866; fax: +39 010 3538358
(S. Schenone); tel./fax: +39 0577 234306 (M. Botta).
E-mail addresses: schensil@unige.it (S. Schenone), botta.maurizio@gmail.com
(M. Botta).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.079

M. Radi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5928–5933
5929
Cl
O
O
N
EtO
a)
HN
N
N
N
H
N
N
NH2
N
N
N
N
H
N
H2N
S
N
N
O
N
N
Cl
OH
5a R1 = H
11a R1 = H
R1
N
Dasatinib
5b R1 = CH3 R1
11b R1 = CH3
PP2
b)
NHR
N
NHR2
N
Cl
N
N
1; R = -CH2C6H5
N
c)
2; R = -CH2CH2C6H5
3; R = -C6H5-mCl
N
H3CS
N
N
N
N
N
N
Cl
Figure 1. Dual Src/Abl inhibitors active in neuroblastoma cell lines.
12a R1 = H
10i-o
R1
R1
12b R1 = CH3
10i : R1 = H; R2= -CH2C6H4-m
10j : R1 = H; R2 = -C6H4-mCl
10k : R1 = CH3; R2 = -CH2C6H5
F
identify new antitumor agents,16 we have developed a family of
pyrazolo[3,4-d]pyrimidines active as dual Src/Abl inhibitors which
proved to act as potent antiproliferative agents on different cancer
cell lines. These compounds also showed in vivo activity reducing
the weight of tumor mass in mouse models.17–20 Selected members
of this family (compounds 1–3, Fig. 1), characterized by a C6
methylthio group on the pyrazolo[3,4-d]pyrimidine scaffold,
displayed a promising antiproliferative activity in SH-SY5Y cell
cultures of human neuroblastoma:21 Treatment with the most
10l : R1 = CH3; R2 = -CH2C6H4-oF
10m : R1 = CH3; R2 = -CH2C6H4-m
F
F
10n : R1 = CH3; R2 = -CH2C6H4-p
10o : R1 = CH3; R2 = -C6H4-mCl
Scheme 2. Reagents and conditions: (a) formamide, 190 °C, 4–8 h; (b) POCl3/DMF,
CHCl3, reflux, 8–12 h; (c) Method A: R2NH2, toluene, rt, 48 h (for 10i and 10k–n);
Method B: R2NH2, EtOH, reflux, 4–5 h (for 10j and 10o).
active compound 3 (72 h, 10 lM) led to a 75% growth reduction,
cell cycle arrest, induced apoptosis and decreased adhesion/
invasiveness.
were also synthesized since similar compounds have been recently
reported as interesting antiproliferative agents.22
In order to get further insight on the potentiality of such pyraz-
olo[3,4-d]pyrimidines for the treatment of NB, we decided to syn-
The synthesis of compounds 10a–h is reported in Scheme 1.
(2-Phenylethyl)hydrazine 4a23 and (2-phenylpropyl)hydrazine
4b (prepared from 1-bromo-2-phenylpropane, see Supplemen-
tary data) were reacted with ethyl(ethoxymethylene)cyanoace-
tate at reflux to afford the ethyl 5-amino-1H-pyrazole-4-
carboxylates 5a–b. The latter intermediates were treated with
thesize
a
small collection of closely related analogues
characterized by the presence of a C6 methylthio group (common
to 1–3) and to explore the role played by different functional
groups in N1 and C4 for the biological activity. As a comparison,
a
few C6-unsubstituted pyrazolo[3,4-d]pyrimidine derivatives
O
O
O
EtO
HN
HN
C6H5
EtO
HN
c)
N
b)
N
a)
N
N
H2N
N
N
CHCH2NHNH2
R1
S
N
H
S
R1
R1
R1
4a R1 = H
4b R1 = CH3
O
7a R1 = H,
6a R1 = H,
5a R1 = H,
7b R1 = CH3
6b R1 = CH3
5b R1 = CH3
d)
NHR2
Cl
N
10a : R1 = H; R2 = -C6H4-mCl
10b : R1 = CH3; R2 = -CH2C6H5
10c : R1 = CH3; R2 = -CH2C6H4-o
O
e)
f)
N
HN
H3CS
N
N
N
F
F
N
N
10d : R1 = CH3; R2 = -CH2C6H4-p
H3CS
N
N
N
N
H3CS
10e : R1 = CH3; R2 = -CH2C6H4-oCl
10f : R1 = CH3; R2 = -C6H5
R1
R1
R1
9a R1 = H,
8a R1 = H,
8b R1 = CH3
10g : R1 = CH3; R2 = -C6H4-mCl
10h : R1 = CH3; R2 = -C6H4-mBr
10a-h
9b R1 = CH3
Scheme 1. Reagents and conditions: (a) ethyl(ethoxymethylene)cyanoacetate, EtOH or toluene, reflux, 5–8 h; (b) benzoyl isothiocyanate, THF, reflux, 12 h; (c) 1 M NaOH,
H2O, 100 °C, 10 min, then AcOH; (d) CH3I, THF reflux, 18 h; (e) POCl3/DMF, CHCl3, reflux, 8 h; (f) Method A: R2NH2, toluene, rt, 48 h (for 10b–e); Method B: R2NH2, EtOH, reflux,
3–5 h (for 10a and 10f–h).

5930
M. Radi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5928–5933
benzoyl isothiocyanate in anhydrous THF at reflux for 12 h to
give intermediates 6a–b. These compounds were then cyclized
to the pyrazolo[3,4-d]pyrimidinones 7a–b by treatment with
1 M NaOH at 100 °C for 10 min, followed by acidification with
acetic acid. Alkylation of the thiocarbonyl group at position C6
with iodomethane in anhydrous THF at reflux afforded the 6-
methylthio derivatives 8a–b, in turn treated with the Vilsmeier
complex (POCl3:DMF, 1:1, 20 equiv) in CHCl3 to obtain com-
pounds 9a–b, bearing a chlorine atom in C4. Finally, reaction
of 9b with an excess of appropriate amines in toluene at room
temperature afforded compounds 10b–e in yields ranging from
55% to 75%. Differently, compounds 10a, 10f–h were obtained
reacting 9a,b with the suitable anilines in absolute ethanol at
reflux for 3–5 h. For the synthesis of the C6-unsubstituted
compounds 10i–o, 5-amino-1H-pyrazole-4-carboxylates 5a–b
were treated with an excess of formamide at 190 °C to give
the corresponding pyrazolo[3,4-d]pyrimidinones 11a–b in good
yields (Scheme 2). Treatment of the latter compounds with an
excess of the Vilsmeier complex (POCl3/DMF, 1:1) for 8–12 h at
reflux in CHCl3 led to the formation of the halogenated deriva-
tives 12a–b. Finally, reaction of 12a,b with an excess of the
appropriate amines in toluene at room temperature afforded
compounds 10i, 10k–n, in yields ranging from 55% to 78%. Dif-
ferently, the aniline derivatives 10j and 10o were obtained by
reaction of 12a–b with 3-chloroaniline in absolute ethanol at re-
flux for 4–5 h.
The synthesized compounds 10 were initially tested in a cell-
free assay to evaluate their affinity toward Src and Abl kinases in
comparison with the progenitor derivatives 1–3; Ki values are
reported in Table 1. While the C6-unsubstituted compounds
10i–o showed poor or no activity against both Src and Abl, the
most interesting results were obtained from the 6-methylthio
derivatives 10a–h. As we recently observed, an inverse relation-
ship between the bulkiness of the C4-substituent (NHR2) and the
Src affinity was found for the C6-substituted series (10a–h):16a
the most active derivatives (10a, 10f–h) were all characterized
by a substituted aniline moiety in C4 regardless of the N1 sub-
stituent (phenylethyl or 2-phenyl-propyl, respectively). However,
it was interesting to note that small modification on the N1
chain (changing R1 from Cl to H or Me) significantly affected
the activity profile of both series: (i) within the C6-unsubstituted
series (10i–o), changing R1 from Cl to H (10i,j) or Me (10k–o)
decreased or abolished the activity against both Src and Abl
(activity values for R1 = Cl are reported in brackets within
Table 1); (ii) within the C6-substituted series (10a–h), changing
R1 from Cl to H (10a) or Me (10b–h) almost abolished the
Abl activity with a contemporary improvement of the Src inhibi-
tion. In summary, starting from the dual Src/Abl inhibitors 1–3,
small modification on the N1-chain of the C6-substituted series
led to compounds endowed with a nanomolar activity against
Src and no longer inhibiting the Abl kinase (e.g., compare data
for 3 and 10a,g in Table 1). Recently, FAK kinase has also
been proposed as
a druggable target for the treatment of
neuroblastoma.24 However, compounds 10a, 10f–h proved to
be completely inactive against FAK and weakly active against
other selected tyrosine kinases (see Table S1, Supplementary
data).
In the attempt to understand how the functionalization in
N1 could affect the dual Src/Abl activity profile, compounds
10a–o were submitted to molecular modeling simulations on
both kinases. As previously reported for the pyrazolo[3,4-
d]pyrimidines characterized by R1 = Cl,16a docking studies on
10a–o within the ATP binding sites of c-Src and Abl allowed
to propose three different binding modes, henceforth referred
to as binding mode I, II and III (Fig. 2A–D). Within the Abl
binding pocket, two different binding modes (BM) were pre-
dicted depending on the functionalization in C6 (BM-I or II
for R3 = H or SCH3, respectively). The common feature of these
binding modes is represented by the presence of the N1 substi-
tuent within the hydrophobic region I (HY-I) and this is re-
flected in a common loss of activity changing R1 from Cl to
Me. Also for Src, two BM with a completely different orienta-
tion were predicted depending on the functionalization in C6
(BM-I or III for R3 = H or SCH3, respectively). In this case, chang-
ing R1 from Cl to Me was unfavourable only for the compounds
characterized by the BM-I while the C6-substituted derivatives
characterized by the BM-III showed a considerable improvement
of the affinity with the only exception represented by 10b (see
Table 1
Structure and inhibitory activity of compounds 1–3 and 10a–o toward isolated c-Src and Abl kinases
NHR2
N
N
R3
N
N
R1
Binding
mode
Binding
mode
Kia
(lM)
Kia
(lM)
Compd R1
R2
R3
SCH3 3.7
–CH2CH2C6H5 SCH3 0.7
c-Src
Abl
c-Src Abl Compd R1
R2
R3
c-Src
Abl
c-Src Abl
1
2
3
10a
10b
10c
10d
10e
10f
Cl
Cl
Cl
H
–CH2C6H5
0.26
7.3
0.6
III
III
III
III
III
III
III
II
II
II
II
II
II
II
II
II
10g
10h
10i
10j
10k
10l
10m
10n
10o
CH3 –C6H4-mCl
CH3 –C6H4-mBr
H
H
SCH3 0.025 (0.7)
SCH3 0.018 (1.76) 1.07 (0.064) III
NA (0.6)
III
II
II
I
–C6H4-mCl
–C6H4-mCl
SCH3 0.7
–CH2C6H4-mF
–C6H4-mCl
H
H
H
H
H
H
H
0.51 (0.081) 4.92 (0.04)
0.48 (1.71)
NA (3)
NA (0.151)
NA (0.081)
NA (0.5)
I
I
I
I
I
I
I
SCH3 0.09 (0.7)b NAc (0.6)
NA (0.24)
NA (0.37)
NA (0.73)
NA (0.04)
NA (2)
I
I
I
I
I
I
CH3 –CH2C6H5
CH3 –CH2C6H4-oF
CH3 –CH2C6H4-pF
SCH3 NA (3.7)
SCH3 2.16 (21.2) 1.0 (0.08)
SCH3 2.81 (4.6) 1.86 (0.1)
NA (0.26)
CH3 –CH2C6H5
CH3 –CH2C6H4-oF
CH3 –CH2C6H4-mF
CH3 –CH2C6H4-pF
CH3 –C6H4-mCl
CH3 –CH2C6H4-oCl SCH3 2.0 (NA)
CH3 –C6H5
0.77 (0.089) III
SCH3 0.025 (1.6) NA (n.t.)d
III
7.14 (1.71)
NA (0.24)
Activities of the corresponding R1 chlorinated analogues are reported in parenthesis. Predicted binding modes within c-Src and Abl are also reported
a
Ki values are means of at least two experiments and were calculated according to the following equation: Ki = [ID50/(1+S1/Km ATP)]/(1+S2/Km pep) where S1 and S2 are the
M each).
Activities of the corresponding R1 chlorinated analogues is reported in parenthesis.
ATP and the peptide substrate concentrations (50
l
b
c
NA: Not Active at 100
n.t.: Not tested.
lM (the highest concentration tested).
d

M. Radi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5928–5933
5931
Figure 2. Binding mode of compounds 10o (green) and 10g (red) within the ATP binding site of Abl (A, C) and c-Src (B, D). A and B are referred as binding mode I, C as binding
mode II and D as binding mode III. E) CPCA pseudofield plot showing field differences between c-Src (magenta) and Abl (light blue) for the GRID C3 probe (yellow polyhedra).
As an example, the binding mode of the compound 10g within the ATP binding site of c-Src is reported. The 6-methylthio of compound 10g is located in the region where the
interaction between an alkyl group and the enzyme is more favourable in c-Src than in Abl.
Table 1). This behaviour seems to suggest the presence of a
selectivity pocket that could be exploited by compounds 10a–
h to discriminate between Src and Abl. To support this hypoth-
esis, the ATP binding pockets of Src and Abl were compared by
means of the GRID/CPCA approach (see Supplementary data for
details).25 Starting from several X-ray structures of both kinases,
a multivariate description of the binding sites was performed
using the program GRID. The Molecular Interaction Fields (MIFs)
Figure 3. Antitumor in vitro effect of selected compounds. Neuroblastoma cells SH-SY5Y and normal fibroblast Hs27 were treated with increasing concentrations (from 0.01
to 10 M) of PP2, 10a, 10f, 10g, and 10h. After 72 h the number of viable cells was measured and expressed as percentage respect to untreated control (100%). The IC50
concentration for each compound is reported in the table. In the graph each bar indicates the mean SD of at least three distinct experiments.
l

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M. Radi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5928–5933
Table 2
Acknowledgments
In vitro ADME properties of selected compounds
a
Compd PAMPA-BBB assay
Water solubility
(g/mL)
Metabolic
stability (%)
The present work was supported by the National Interest
b
c
Pe  10À6 (cm/s)
Research Projects (PRIN_2008_5HR5JK). We thank Professor
Gabriele Cruciani for assistance with Grid/Golpe calculations
(Molecular Discovery Ltd). E.C. was the recipient of an FIRC
Fellowship. M.B. whish to thank Professor Massimo Valoti and Dr.
Stefania Dragoni for the assistance on metabolic stability assays.
10a
10h
3
3.43
1.48
5.66
0.12
<0.01
0.046
99
95
91
a
Parallel Artificial Membrane Permeability Assay (PAMPA) was evaluated using
porcine polar brain lipid.
b
Effective permeability was calculated as reported in Ref. 27.
Expressed as percentage of unmodified parent drug.
Supplementary data
c
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.079.
were analyzed by means of the consensus principal component
analysis (CPCA). Figure 2E shows the pseudofield differences for
the methyl probe between Abl and c-Src. The large yellow con-
tour indicates a region where interaction between the C3-probe
and the enzyme is more favourable in c-Src than in Abl. This
area of selectivity is mainly due to the different folding of the
P-loop of Abl and c-Src. In particular, since Tyr253 partially
occupies the ribose pocket in Abl enzyme, the 6-methylthio-
pyrazolo[3,4-d]pyrimidines cannot assume in Abl the binding
mode III, otherwise the 6-methylthio would clash with it. Con-
versely, the P-loop in c-Src is totally open allowing the place-
ment of 6-substituted-pyrazolo[3,4-d]pyrimidines as described
above. As a support for these findings, the same selectivity site
has been also proposed by Wang et al. while studying the
activity profile of N9-arenethenyl purines.26
We then investigated the ability of the four most active der-
ivates (10a, 10f–h) to inhibit the proliferation of neuroblastoma
SH-SY5Y cells in comparison with the reference compound PP2.
As shown in Figure 3, although with different efficacy rate, all
the selected compounds reduced the cell growth rate in a time
and concentration-dependent manner and their antiproliferative
capacities were more marked with respect to PP2. In particular,
the strongest effect on SH-SY5Y was obtained by 10a that
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showed an IC50 value at 72 h of 0.08
worthy IC50 values were observed also for 10f–h. All these IC50
were lower than that calculated for PP2 (6.10 M). The antipro-
lM. Higher but still note-
l
15. Metz, J. T.; Johnson, E. F.; Soni, N. B.; Merta, P. J.; Kifle, L.; Hajduk, P. J. Nat. Chem.
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liferative effect was evident from 48 h after treatment, and
immediate toxic/necrotic events were not observed. Moreover
the proliferation assay of non transformed human diploid fibro-
blasts Hs27, according to the same treatment protocol, demon-
strated that compounds 10a, 10f–h had a comparable or even
better safe profile with respect to PP2. A significant retardation
in proliferative rate of Hs27 was observed only from about
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10
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Finally, in vitro ADME properties (effective permeability in BBB
model, water solubility, microsomal stability) for compounds 10a
and 10h were also evaluated in comparison with the progenitor
derivative 3 (Table 2; see Supplementary data for details). Com-
pound 10a was characterized by the best metabolic stability and
water solubility while showing a BBB-permeation comparable to
that of 3. These results could account for the better activity showed
by 10a in cell assays.
In summary, the present work reports the identification of
potent c-Src inhibitors able to inhibit the growth of SH-SY5Y
NB cell lines. While the dual Src/Abl inhibitors 1–3 have previ-
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ness at 10 lM, compounds 10a, 10f–h herein identified showed
an improved antiproliferative activity in SH-SY5Y treated cells
with 10a showing a very interesting IC50 of 80 nM after 72 h.
These results can be considered as an advanced starting point
to investigate the structure–activity relationship of these new
pyrazolo[3,4-d]pyrimidines in order to develop new selective c-
Src inhibitors as potential drugs for the treatment of
neuroblastoma.

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