Pyrazolo[3,4-d]pyrimidines Endowed with Antiproliferative Activity on Ductal Infiltrating Carcinoma Cells

Article abstract of DOI:10.1021/jm034257u

Novel 1,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (better than the reference compound) of Src phosphorylation of the breast cancer cells 8701-BC

Full text of DOI:10.1021/jm034257u

© Copyright 2004 by the American Chemical Society
Volume 47, Number 7
March 25, 2004
Letters
progressive loss of the stationary epithelial phenotype
and the acquisition of a mesenchimal-like phenotype,
correlated with the ability to migrate and invade sur-
rounding and distant tissues.3 Ductal infiltrating car-
cinoma (DIC) of the breast is the most common and
potentially aggressive form of cancer.4 The 8701-BC cell
line5 was established from a primary DIC, that is, before
the clonal selection of the metastatic process. This cell
line maintains a number of properties in culture that
are typical of the mammary tumor cells and is known
to overexpress Src tyrosine kinase (TK).6 Such TK is
the prototype member of the nonreceptor Src family of
protein tyrosine kinases (PTK).7 Src is activated follow-
ing engagement of many different classes of cellular
receptors and participates as a convergence point in
different signaling pathways.8,9 In this regard, Src is a
critical component of the signaling cascades initiated
by TK-linked receptors, such as the epidermal growth
factor receptor (EGFR) and G-protein-coupled receptors,
and is directly associated with, and may regulate
signaling via, the EGFR and HER-2/neu PTK recep-
tor,10,11 both of which are involved in cancer. Finally,
Src overexpression and activation have been correlated
with a large number of growth-regulatory processes
where Src participates. The process of activation of Src
is mediated by the phosporylation of the tyrosine 416.
On this basis, inhibitors of Src phosphorylation process
may halt uncontrolled tumor cell growth and play an
important role as new therapeutic agents for the treat-
ment of cancer.
P yr a zolo[3,4-d ]p yr im id in es En d ow ed
w ith An tip r olifer a tive Activity on Du cta l
In filtr a tin g Ca r cin om a Cells
Fabio Carraro,† Annalisa Pucci,† Antonella Naldini,†
Silvia Schenone,*,‡ Olga Bruno,‡ Angelo Ranise,‡
Francesco Bondavalli,‡ Chiara Brullo,‡ Paola Fossa,‡
Giulia Menozzi,‡ Luisa Mosti,‡ Fabrizio Manetti,§ and
Maurizio Botta§
Dipartimento di Fisiologia, Unita` di Fisiologia Cellulare e
Molecolare, Universita` degli Studi di Siena, Via Aldo Moro,
I-53100 Siena, Italy, Dipartimento di Scienze
Farmaceutiche, Universita` degli Studi di Genova,
Viale Benedetto XV, I-16132, Genova, Italy, and
Dipartimento Farmaco Chimico Tecnologico, Universita`
degli Studi di Siena, Via Aldo Moro, I-53100 Siena, Italy
Received December 15, 2003
Abstr a ct: Novel 1,4,6-trisubstituted pyrazolo[3,4-d]pyrim-
idines are reported with preliminary in vitro activity data
indicating that several of them are potent inhibitors (better
than the reference compound) of Src phosphorylation of the
breast cancer cells 8701-BC, known to overexpress Src. The
ability of such compounds to significantly reduce 8701-BC cell
proliferation suggests that this scaffold could be a promising
lead for the development of antitumoral agents able to block
Src phosphorylation of breast cancer cells.
Over the past 3 decades, breast cancer has been the
most common malignancy among Western women, and
the incidence rates of first primary breast cancer have
been increasing over time.1 Meanwhile, the survival
rates of breast cancer patients have increased substan-
tially, but the incidence rates of second primary breast
cancer are still very high especially among women with
a first primary that was either lobular or medullary.2
The major cellular changes involved in the conversion
of normal to malignant breast epithelial cells are the
In light of the morbidity and invasiveness of breast
cancer, there remains a deep ongoing interest in the
identification of potent compounds with specific inhibi-
tory activity against breast cancer cells. Hence, in the
course of our drug discovery programs, we were in-
trigued by the potency and selectivity of PP1 and PP2
(1-(tert-butyl)-3-(4-methylphenyl)-4-aminopyrazolo[3,4-
d]pyrimidine and 1-(tert-butyl)-3-(4-chlorophenyl)-4-
aminopyrazolo[3,4-d]pyrimidine, respectively) toward
the Src family tyrosine kinase.12-14 This experimental
evidence, combined with our previous experience in the
synthesis of such bicyclic compounds and their ana-
* To whom correspondence should be addressed. Phone: 0039 010
3538866. Fax: 0039 010 3538358. E-mail: schensil@unige.it.
† Unita` di Fisiologia Cellulare e Molecolare.
‡ Universita` degli Studi di Genova.
§ Dipartimento Farmaco Chimico Tecnologico.
10.1021/jm034257u CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/21/2004

1596 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
Letters
Ta ble 1. Activity Data (Expressed as IC50 Values) of the New
Pyrazolopyrimidines toward 8701-BC Cells
corresponding to about 50%) were characterized by
activity values ranging between 31.2 (1f) and 64.2 µM
(1b), comparable or higher to that of PP2 (61.8 µM,
Figure 1).
A summary of activity data suggested the following.
(1) The most active compounds belonged to subclasses
1 and 2, bearing both an alkylthio substituent at C6
and a chlorophenylethyl side chain at N1. However, the
styryl derivative bearing a morpholino ring at C4 (4b)
was found to be more active than the congeneric
compounds 1b and 2b. On the other hand, 4a was found
to be inactive, similar to the corresponding piperidino
derivative belonging to the subclass 2 (namely, 2a ). (2)
A butyl chain was found as the optimal substituent
within the ethylthio subclass, while only the phenylethyl
chain led to a better result among the thiomethyl
derivatives 1. (3) Both the deletion of the thioalkyl chain
at C6 and the transformation of the chlorine atom of
the N1 chlorophenylethyl side chain with the corre-
sponding hydroxy group led to 3 with lower or insig-
nificant activity.
compd
R1
R2
IC50 (µM)a
PP2
1a
1b
1c
1d
1e
1f
2a
2b
2c
3a
3b
3c
4a
4b
4c
4d
61.8 ( 4.4
67.5 ( 1.2
64.2 ( 1.8
59.8 ( 3.1
63.8 ( 2.1
53.2 ( 1.2
31.2 ( 0.5
NA
79.8 ( 3.0
38.8 ( 0.5
NA
NA
NA
1-piperidinyl
4-morpholinyl
NH(CH2)2OC2H5
NHC4H9
NHCH2C6H5
NH(CH2)2C6H5
1-piperidinyl
4-morpholinyl
NHC4H9
4-morpholinyl
NHC4H9
1-piperidinyl
1-piperidinyl
4-morpholinyl
NHC4H9
Me
Me
Me
Me
Me
Me
Et
Et
Et
NA
58.8 ( 0.7
80.5 ( 6.8
93.7 ( 0.6
To further investigate the antiproliferative activity of
these compounds, the inhibitory effects of 1a , 1e, 1f, and
2c toward the phosphorylation of Src (Tyr416) were
measured on cells treated with 100 nM epithelial growth
factor (EGF). Immunoblot analysis, performed using
phospho-specific antibodies to Src (Tyr416), clearly
showed an inhibition of the Src activation-phosphoryl-
ation of 8701-BC cells exposed to the studied com-
pounds, with respect to the control. In particular, 1a ,
1e, and 1f were found to inhibit Src phosphorylation
better than the reference compound PP2 (Figure 2). In
contrast, although 2c retained appreciable inhibitory
activity, it was found to be less potent than PP2. The
occurrence, at the same time, of a reduction of Src
phosphorylation and an inhibition of cell proliferation
led us to assume that our compounds were able to
inhibit proliferation through inhibition of Src phos-
phorylation, since the latter mediates a cascade leading
to the stimulation of cell proliferation and mobility.
Moreover, independent experiments have shown that
all these compounds do not interfere with the phos-
phorylation of EGF receptor TK.20
1-hexahydroazepinyl
a
IC50 values were determined on exponentially growing 8701-
BC cells incubated with each of the compounds (0-100 µM) for
72 h using the MTT assay.19 The IC50 values are the mean ( SEM
of three independent experiments performed in duplicate. NA )
not active at 100 µM.
logues,15 led us to design and synthesize a new class of
pyrazolo[3,4-d]pyrimidines (1-4). They were submitted
to preliminary in vitro biological studies demonstrating
that they are potent inhibitors of the breast cancer cell
line 8701-BC via inhibition of Src phosphorylation, even
when compared with the reference compound PP2. In
addition, on the basis of the experimental evidence that
Src kinase has been found to be active in many tu-
mors,16-18 its inhibitors could become useful anticancer
agents for many types of cancer. As a consequence, the
pyrazolopyrimidine derivatives disclosed here could
represent a profitable tool to inhibit cancer cell growth
through the blocking of the Src phosphorylation mech-
anism.
Cell P r olifer a tion Exp er im en ts a n d Im m u n oblot
An a lyses. The antiproliferative effect of the new pyr-
azolopyrimidines was evaluated on the 8701-BC breast
cancer cell line (kindly provided by Prof. I. Pucci-
Minafra, University of Palermo, Italy) using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) cell proliferation assay,19 in comparison with the
activity of PP2, chosen as the reference compound (Table
1). The mechanism of action of the new compounds was
not investigated at the molecular level yet. Conse-
quently, it is unclear if the studied compounds inter-
acted with the phosphorylation of Src through a mech-
anism similar to or different from that of PP2, known
as a potent and selective inhibitor of the Src family.
However, the structural similarity between PP2 and our
compounds led to the suggestion that the last could
share with PP2 the same mechanism of Src phos-
phorylation inhibition. Following this hypothesis, the
activity of the new compounds was compared to that of
PP2. Such a comparison showed that seven of the newly
synthesized compounds (namely, 1b-f, 2c, and 4b,
The proapoptic activity of 1a , 1e, 1f, and 2c was also
tested using a poly-ADP-ribose-polymerase (PARP) as-
say (Roche Diagnostics, Milano, Italy). Immunoblot
analysis of uncleaved and cleaved PARP after a 3 h
treatment with the specified compound indicated a
significant increase of the cleaved PARP after treatment
with 1a and 2c, similar to that found for the reference
compound PP2 (Figure 3). On the other hand, for 1e
and 1f, the increase of the cleaved fraction of PARP was
less intense and the amount of the uncleaved protein
was lower in comparison to that found in the experi-
ments with 1a and 1c. Enhancement of the cleaved
PARP level demonstrated that cells were induced in
apoptosis. In particular, 1a and 2c showed a pro-
apoptotic effect toward 8701-BC comparable to that
found for the reference compound and more relevant
with respect to that of 1e and 1f, even if they showed a
better inhibitory activity on Src. This may be interpreted
as a more pronounced cytostatic effect rather than a
proapoptic activity. However, it should be also consid-

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7 1597
F igu r e 1. Inhibitory effects of 1a , 1e, 1f, and 2c on 8701-BC cells measured by the MTT assay. Values are the mean ( SEM of
three independent experiments performed in duplicate.
F igu r e 2. Phospho-Src inhibition by 1a , 1e, 1f, and 2c toward 8701-BC cells. Immunoblot analysis was performed using phospho-
specific antibodies to Src (Tyr416). Filters were additionally reprobed with specific non-phospho anti-Src antibodies after
stripping: (lane 1) cell control; (lane 2) cells treated for 15 min with EGF (100 nM); (lane 3-7) cells challenged for 3 h in the
presence of 10 µM of the specified compound and then treated for 15 min with EGF (100 nM). Results are representative of three
independent experiments.
most potent and selective Src inhibitors known (PP2),
we think that such compounds deserve great consider-
ation. As a consequence, since, to the best of our
knowledge, these are the first reported compounds with
such an antiproliferative activity toward the 8701-BC
cell line, 1f and 2c have been chosen as leads for more
F igu r e 3. Detection of cleaved PARP in 8701-BC cell extracts.
focused experiments in the field of anticancer agents
Immunoblot analysis was performed using anti-PARP specific
acting via Src phosphorylation inhibition.21 Neverthe-
antibodies. An antirabbit secondary antibody conjugated with
less, it is important to point out that further investiga-
tions are required to evaluate the selectivity of the new
compounds toward different cancer cell lines. In addi-
tion, studies aimed at identifying at the molecular level
the mechanism of action of such compounds are cur-
rently ongoing. Results of such experiments will be
reported in due time.
peroxidase and a peroxidase substrate revealed the presence
of PARP cleavage products in apoptotic cells. The antibody
recognized both the uncleaved PARP (113 kDa) and the larger
cleaved fragment (85 kDa): (lane 1) control cells; (lanes 2-6)
cells treated for 3 h with 10 µM of the specified compound.
Results are representative of three independent experiments.
ered that the time of cellular exposure to the compounds
may be relevant for apoptotic induction.
Ack n ow led gm en t. Financial support provided by
the Italian MIUR (Cofin 2002, Grant 2002038577_002)
is gratefully acknowledged. M.B. thanks the Merck
Research Laboratories for the 2002 ADP Chemistry
Award.
In conclusion, considering the morbidity of breast
cancer and the characteristics of the cell line chosen for
this study, along with the antiproliferative activity
exerted on these cells by the reported compounds
(particularly 1f and 2c) when compared with one of the

1598 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 7
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J M034257U