New pyrazolo[3,4-d]pyrimidines endowed with A431 antiproliferative activity and inhibitory properties of Src phosphorylation

Article abstract of DOI:10.1016/j.bmcl.2004.03.013

New 4-aminopyrazolo[3,4-d]pyrimidines bearing various substituents at the position 1 and 6, were synthesized. The new compounds showed antiproliferative activity toward A431 cells, were found to be inhibitors of Src phosphorylation, and induced apoptotic

Full text of DOI:10.1016/j.bmcl.2004.03.013

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2511–2517
New pyrazolo[3,4-d]pyrimidines endowed with A431
antiproliferative activity and inhibitory properties of Src
phosphorylation
S. Schenone,a,* O. Bruno,a A. Ranise,a F. Bondavalli,a C. Brullo,a P. Fossa,a L. Mosti,a
G. Menozzi,a F. Carraro,b A. Naldini,b C. Bernini,b F. Manettic and M. Bottac
aDipartimento di Scienze Farmaceutiche, Universitꢀa di Genova, Viale Benedetto XV, I-16132 Genova, Italy
bDipartimento di Fisiologia, Universitꢀa di Siena, Via Aldo Moro, I-53100 Siena, Italy
cDipartimento Farmaco Chimico Tecnologico, Universitꢀa di Siena, Via Aldo Moro, I-53100 Siena, Italy
Received 24 September 2003; revised 23 February 2004; accepted 1 March 2004
Abstract—New 4-aminopyrazolo[3,4-d]pyrimidines bearing various substituents at the position 1 and 6, were synthesized. The new
compounds showed antiproliferative activity toward A431 cells, were found to be inhibitors of Src phosphorylation, and induced
apoptotic cell death. In particular, 2h was a better inhibitor of Src phosphorylation than the reference compound PP2.
Ó 2004 Elsevier Ltd. All rights reserved.
The understanding of the fundamental biology of cancer
increased dramatically in recent years1 and has strongly
impacted on both experimental and gradually also on
clinical tumor therapy. We now believe that the future
of tumor therapy is in the development of molecularly
targeted agents that specifically block key mechanisms
involved in development and progression of specific
types of cancer.2 Due to their critical role in tumor
development and progression, molecules capable of
inhibiting protein kinases are of central interest in tar-
geted cancer therapy. In particular, tyrosine kinases
(TKs) are enzymes that catalyze the specific phosphor-
ylation of tyrosine residues on proteins.
Src is a critical component of the signaling cascades
initiated by tyrosine kinase-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 receptor PTKs,10;11 both of which are involved in
cancer. Finally, Src overexpression and activation has
been correlated with a large number of growth-regula-
tory processes in which Src participates. In particular,
the Src protein is overexpressed in many tumors such as
colon, breast, gastric, and prostatic tumors and plays a
key role in controlling their proliferation and invasive-
ness.12;13
Protein tyrosine kinases (PTKs) participate in a wide
variety of cellular activities including proliferation,
secretion, adhesion, and responses to mitogens and
stress.3;4 The TK Src is the prototype member of the
nonreceptor Src family of PTKs that include c-Yes, Fyn,
c-Fgr, Lyn, Lck, Hck, Clk, Yrk, and c-Src.5 In partic-
ular, the nonreceptor pp60c-Src PTK transduces signals
that control the above cellular processes.6–8 Src is acti-
vated following engagement of many different classes of
cellular receptors and participates as a convergence
point in different signaling pathways.3;4;9 In this regard,
On the basis of these considerations, inhibitors of Src
phosphorylation process may stop uncontrolled tumor
cell growth and play an important role as new thera-
peutic agents for the treatment of cancer.
During the past decade, examples of pyrazolo[3,4-d]-
pyrimidines active as TKs inhibitors have been reported
in the literature by different authors. Many of these
compounds were highly active, and some of them have
been largely used as standard or reference compounds
and as a tool to set up in vitro assays and to prove their
functionality. However, only a few demonstrated in vivo
activity. As an example, approaches aimed at identifying
ATP-competitive small molecules led to the character-
ization of derivatives 1 (Fig. 1) as potent inhibitors of
* Corresponding author. Tel.: +39-0103538866; fax: +39-0103538358;
e-mail: schensil@unige.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.013

2512
S. Schenone et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2511–2517
R
R
NH2
Cl
HN
N
HN
N
NH
N
N
N
N
N
N
N
N
R
N
H
N
R1
N
H
Olomoucine:
1
PP1: R = Me
PP2: R = Cl
R = CH2CH2OH, R1 = Me
Roscovitine:
(EGFR TK inhibitors)
(c-Src inhibitors)
R = CH(CH2OH)CH2CH3,
R
1 = CH(CH3)2
(CDKs inhibitors)
Figure 1.
EGFR TK,14 while PP1 and PP2 were described as very
strong and selective inhibitors of the c-Src family of
kinases.15 Unfortunately, attempts to improve the bio-
logical profile of the latter compounds have so far met
little success. Following these studies, some other
inhibitors, possessing different chemical structures and
interesting c-Src inhibitory activity, have been recently
reported. In particular, pyrrolo[2,3-d]pyrimidines have
been described as selective and potent inhibitors of c-Src
with inhibition potency of about 10 nM and 20-fold
selectivity toward a panel of other TKs.16 Other efforts
directed to c-Src kinase inhibitors included substituted
5,10-dihydropyrimido[4,5-b]quinolines, showing an in
vitro c-Src inhibition activity in the nanomolar range.17
NaOH at 100 °C for 10 min, followed by acidification
with acetic acid (80% yield). Alkylation of the 6-thio
group with methyl or ethyl iodide in THF at reflux
afforded the 6-alkylthio derivatives 6, that were in turn
treated with the Vilsmeier complex (POCl3/DMF,
10 equiv) in CHCl3 to obtain the dihalogenated com-
pounds 7, bearing the chlorine atom in both the position
4 of the pyrimidine nucleus and on the side chain at N1.
Compounds 7 were purified in good yield by chroma-
tography on Florisilâ column. Finally, regioselective
substitution of the C4 chlorine atom with an excess of
various amines afforded the desired compounds 2a–n in
yields ranging between 60% and 80%. Notably, the
chlorine atom on the N1 side chain has never been
substituted by the amine in spite of its benzylic position,
1
Finally, well known compounds as olomoucine and
roscovitine (Fig. 1), representative of purine derivatives,
strictly correlated with the pyrazolo-pyrimidine com-
pounds, exhibited a moderate inhibitory activity but
good selectivity for a panel of CDKs kinases,18 that have
recently raised considerable interest as the principal
regulators of the cell division cycle.
as shown by the H NMR chemical shifts of the CH2–
CH side chain, which give an ABX complex pattern,
similar to that of the starting material.
Compounds 2v–y have been obtained by treatment of
the corresponding chlorophenylethyl derivatives 2m, 2h,
2e, and 2f, respectively, with a 4 M solution of NaOH in
EtOH/water (1:8) at reflux for 4 h.
To summarize, a careful observation of compounds
depicted in Figure 1 reveals that small variations on the
chemical structure (either the scaffold or the all around
substituents) might shift the inhibitory activity of a
compound from EGFR to c-Src, to CDK serine threo-
nine kinase.
Reaction of 3 with formamide in excess at 190 °C for 8 h
afforded the pyrazolo[3,4-d]pyrimidinone 8, which was
purified by dissolving the crude in 2 M NaOH, boiling
with charcoal, followed by precipitation with acetic acid.
Compound 10 was prepared in a yield of 44% following
21
the Beal and Veliz procedure by treatment of 8 with a
ꢁ
All these experimental evidences prompted us to syn-
thesize a new family of compounds 2 (Scheme 1, Table
1), bearing a pyrazolo[3,4-d]pyrimidine scaffold with
substituents at the position 1 (namely, a 2-chloro-2-
phenylethyl, or a 2-hydroxy-2-phenylethyl, or a styryl
side chain), 4 (linear and cyclic, aliphatic and aromatic
amines), and 6 (a alkylthio group),19 to be tested as
possible inhibitors of c-Src and, consequently, as anti-
proliferative agents.
mixture of HMPT/NBS in acetonitrile at )20 °C fol-
lowed by addition of LiBr and refluxing. It is interesting
to point out that the secondary OH on the side chain
remained unaltered by this procedure, as shown by its
1H NMR spectrum. Treatment of 10 with propylamine
or butylamine gave the desired compounds 2o and 2p.
Reaction of 8 at reflux with POCl3 afforded the 1-styryl
derivative 9, which was reacted with an excess of various
amines in toluene to give 2q–u in good yield.
Compounds 2a–n were prepared starting from the
ethyl ester of 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-
pyrazole-4-carboxylic acid (3), in turn obtained follow-
ing our reported procedure.20 Reaction of 3 with
benzoyl isothiocyanate in THF at reflux for 8 h yielded
the intermediate 4, which was cyclized to the pyraz-
olo[3,4-d]pyrimidine derivative 5 by treatment with 1 M
The new pyrazolo[3,4-d]pyrimidine derivatives 2a–y
were studied in an assay system with the aim of evalu-
ating their antiproliferative properties on the human
epidermoid carcinoma A431 cells,22 known to over-
express both EGFR and Src. Results are reported in

S. Schenone et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2511–2517
2513
R1
Br
N
Amines
2o R1 = NHPr
2p R1 = NHBu
N
N
N
N
N
N
N
CH2CHC6H5
OH
CH2CHC6H5
OH
2o,p
10
HMPT/NBS/LiBr
CH3CN
Cl
O
R1
C2H5OOC
H2N
HCONH2
POCl3
N
N
HN
N
Amines
N
N
N
N
reflux
N
N
N
N
N
N
CH=CHC6H5
CH2CHC6H5
OH
CH=CHC6H5
CH2CHC6H5
OH
3
9
2q-u
8
2q R1 = NHPr
C6H5
C
O
N=C=S
2r R1 = NHBu
2s R1 = 1-pyrrolidino
2t R1 = 1-piperidino
2u R1 = 4-morpholino
THF, reflux
O
O
C2H5OOC
N
HN
N
N
1) NaOH, 100°C
2) CH3COOH
HN
RI, THF
CH2CHC6H5
N
HN
HN
N
RS
N
N
S
N
H
OH
S
CH2CHC6H5
OH
CH2CHC6H5
OH
6
O
5
POCl3/DMF
CHCl3
4
2a R = CH3, R1 = NHPr
2b R = CH3, R1 = NHBu
2c R = CH3, R1 = N(Et2)
2d R = CH3, R1 = 1-pyrrolidino
2e R = CH3, R1 = 1-piperidino
2f R = CH3, R1 = 4-morpholino
2g R = CH3, R1 = NH(CH2)2OEt
2h R = CH3, R1 = NHBn
2i R = C2H5, R1 = NHPr
2j R = C2H5, R1 = NHBu
2k R = C2H5, R1 = 1-pyrrolidino
2l R = C2H5, R1 = 1-piperidino
2m R = C2H5, R1 = 4-morpholino
2n R = C2H5, R1 = NHBn
R1
N
Cl
Amines
N
N
N
N
RS
N
N
RS
N
CH2CHC6H5
Cl
CH2CHC6H5
Cl
7
2a-n
NaOH
EtOH
reflux
R1
2v R = C2H5, R1 = 4-morpholino
2w R = CH3, R1 = NHBn
2x R = CH3, R1 = 1-piperidino
2y R = CH3, R1 = 4-morpholino
N
2 v-y
N
N
RS
N
CH=CHC6H5
Scheme 1.
Table 1. A431 cells appeared to be sensitive to the
activity of these new compounds, some of them showing
an inhibition of cell growth similar or higher to that of
PP2 (Fig. 2), known as a very potent and selective
inhibitor of Src and chosen as the reference compound.
Analysis of the antiproliferative activity toward A431
cells led us to rule out general considerations on the
relationships between biological data and structural
properties of such compounds.
chains), cyclic amino moieties (such as 1-pyrrolidino,
1-piperidino, and 4-morpholino rings), and aromatic
amines (such as a benzylamino substituent) at the
position 4 led to compounds with antiproliferative
activity comparable or higher to that of the reference
compound PP2, found to be 32 lM. In particular,
elongation of the propyl chain of 2a (32 lM) to a butyl
group, afforded compound 2b with slightly enhanced
activity (27 lM). On the other hand, when the bulkiness
of the amino side chain was increased as for compound
2c, antiproliferative activity underwent a significant
decrease (70 lM). Moreover, compounds 2d–f bearing a
1-pyrrolidino, a 1-piperidino, and a 4-morpholino sub-
stituent at the position 4, were found to have an activity
of 32, 38, and 24 lM, respectively. The morpholino
derivative was also found to be the most active com-
pound among all the pyrazolo-pyrimidines presented in
this paper. Transformation of the morpholino ring into
Derivatives bearing a methylthio group at the position 6
were found more active than the corresponding ethyl-
thio counterparts, suggesting that the size of the alkyl
group at this position was an important element to
influence antiproliferative activity.
Among the methylthio compounds, both linear primary
alkylamino groups (such as propyl- and butylamino

2514
S. Schenone et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2511–2517
Table 1. Physicochemical and antiproliferative activity of compounds 2a–y toward A431 cells
Compound
R
R1
Mp (°C)
Yield (%)
IC50 (lM)a
2a
2b
Me
Me
Me
Me
Me
Me
Me
Me
Et
NHPr
NHBu
N(Et2)
126–127
106–107
91–92
76
60
80
60
65
75
78
81
71
61
70
81
80
76
65
70
75
45
53
60
75
65
72
90
88
32 0.5
27 0.5
70 1.4
32 0.8
38 0.8
24 0.4
32 0.6
25 0.7
79 1.8
40 0.4
89 1.7
176 1.9
42 0.7
124 2.5
91 1.4
83 1.8
61 1.0
91 2.0
63 1.0
69 1.2
59 1.0
50 0.8
40 0.6
54 0.7
36 0.6
20 0.4
32 0.7
2c
2d
1-Pyrrolidino
1-Piperidino
4-Morpholino
NH(CH2)2OEt
NHBn
151–152
94–95
2e
2f
2g
116–117
115–116
142–143
72–73
2h
2i
NHPr
NHBu
2j
Et
Et
102–103
131–132
92–93
2k
2l
1-Pyrrolidino
1-Piperidino
4-Morpholino
NHBn
Et
Et
2m
2n
130–131
165–166
104–105
125–126
132–133
106–107
151–152
94–95
Et
2o
2p
NHPr
NHBu
2q
2r
NHPr
NHBu
2s
1-Pyrrolidino
1-Piperidino
4-Morpholino
4-Morpholino
NHBn
2t
2u
116–117
155–156
185–186
155–156
161–162
2v
Et
2w
2x
2y
AG1478b
PP2b
Me
Me
Me
1-Piperidino
4-Morpholino
a IC50 values are means SEM of series separate assays, each performed in quadruplicate.
b To test our system, we have used the well known antiproliferative agent AG1478. As a result, the IC50 of such compound toward A431 cells
(20 lM), was comparable with that reported in the literature.31 However, since the studied compounds were found to inhibit Src phosphorylation,
we discussed their structure–activity relationships in comparison to the activity of PP2, known as a very potent and selective inhibitor of Src
phosphorylation.
the corresponding opened counterpart yielded the eth-
oxyethylamino derivative 2g with an antiproliferative
activity of 32 lM. Finally, introduction of an arylamino
moiety at the same position led to 2h characterized by an
activity of 25 lM, comparable to that of the corre-
sponding morpholino derivative 2f.
ence, at the same time, of a benzylamino chain at C4 and
an ethylthio group at C6 (both of them being the
bulkiest substituents added to these positions) could led
to steric clashes with the corresponding receptor coun-
terparts, in turn avoiding profitable interactions.
The substituent at N1 has been varied to evaluate its
influence on the ability of the compounds to inhibit the
proliferation of the A431 cells. In particular, when the
2-chloro-2-phenylethyl chain was replaced by a styryl
group, keeping fixed the methylthio group at C6, a slight
decrease of activity was observed. In fact, compounds
2w–y showed an activity (40, 54, and 36 lM, respec-
tively) lower with respect to the corresponding chloro-
phenylethyl derivatives 2h, 2e, and 2f. Similarly, the
ethylthio analogue of 2y (2v), bearing a morpholino ring
at C4 and a styryl group at N1, was characterized by an
activity of 50 lM, lower than that of the corresponding
chlorophenylethyl derivative 2m (42 lM). These experi-
mental results evidenced that the optimal combination
for substituents at N1 and C6 is represented by a
2-chloro-2-phenylethyl chain and a methylthio group,
respectively.
Regarding the ethylthio compounds, antiproliferative
activity followed a trend similar to that found for the
corresponding methylthio derivatives, with IC50 values
significantly higher. In fact, while activity of the methyl-
thio compounds ranged from 24 to 70 lM, the corre-
sponding ethylthio derivatives showed activity spanning
from 40 to 176 lM. In particular, in agreement with
what found for 2a and 2b, the butyl chain of 2j was
preferred to the propyl group of 2i, being 40 and 79 lM
the activity values of these compounds toward A431
cells. It is also important to note that 2j showed the best
activity among the ethylthio derivatives, similarly to 2b
that was one of the most active compounds among the
methylthio counterparts. Moreover, activity of 2k–m
(89, 176, and 42 lM, respectively) followed the same
trend of the corresponding methylthio compounds, with
the antiproliferative activity enhanced in the order
4-morpholino > 1-pyrrolidino > 1-piperidino. Finally, an
unexpected low activity (124 lM) was measured for 2n,
about 5-fold decreased in comparison to the methylthio
counterpart 2h (25 lM). This loss of antiproliferative
activity could be rationalized by the fact that the pres-
To further investigate the structure–activity relation-
ships of these pyrazolo-pyrimidines, two additional
subclasses were designed and synthesized, both of them
lacking the C6 substituent, and bearing a 2-hydroxy-2-
phenylethyl or styryl side chain at N1. Regarding the

S. Schenone et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2511–2517
2515
pylamino chain (with the only exception of compounds
2q and 2r), while the diethylamino substituent led to a
more pronounced lowering of activity. Moreover, cyclic
amino substituents were also profitable for activity, with
a 4-morpholino ring preferred to pyrrolidino and pip-
eridino groups. Finally, when at the same time a C4
benzylamino chain and a C6 methylthio group were
added to the pyrazolo-pyrimidine nucleus, compounds
with interesting biological profiles were obtained
(namely, 2h and 2w). On the contrary, increasing the size
of the alkylthio group of 2h to a ethylthio substituent
yielded 2n with a very low antiproliferative activity. (2)
Regarding the influence of N1 and C6 substitutions,
biological data showed that a methylthio group was
preferred if the 2-chloro-2-phenylethyl side chain was
attached to N1. When, at the same time, the 2-chloro-2-
phenylethyl group was transformed into a 2-hydroxy-2-
phenylethyl chain and the alkylthio substituent was
removed, the compounds obtained (2o and 2p) showed
antiproliferative activity significantly lower (91 and
83 lM) to the corresponding parent compounds (2a, 2b,
and 2i, 2j). Similarly, compounds without any C6 sub-
stitution and bearing both a styryl chain at N1 and a
heterocycle at C4 (2s and 2t) showed a higher activity
with respect to the C6 ethylthio analogues 2k and 2l
(with the exception of 2u that was found less active than
2m), but a significantly lower antiproliferative activity in
comparison to the corresponding C6 methylthio com-
pounds 2d–f. Moreover, 2q and 2r were less active than
the corresponding C6 alkylthio parents, with the
exception of compound 2i (79 lM) found to be less ac-
tive than 2q. (3) A comparison between the 2-hydroxy-2-
phenylethyl derivatives 2o, 2p and the styryl compounds
2q and 2r, suggested that the antiproliferative activity of
the latter increased in the order propyl > butyl, differ-
ently from that found in all the other subclasses.
Figure 2. Inhibitory effects of compounds 2f, 2h, and 2j on A431 cells
measured by the MTT assay.30 Values are means SEM of three
independent experiments performed in duplicates.
To better understand the reason of the reported anti-
proliferative activity of these new compounds, their
inhibitory property to human EGF TK receptor has
been evaluated by measuring the EGF receptor and the
phospho-EGF Receptor (Tyr1045) using a specific
antibody (Cell Signaling Technology, MA, USA) that
detects EGF receptor only when phosphorylated at
Tyr1045 (a Src-independent phosphorylation site).
Compounds 2a–y demonstrated no effect on the phos-
phorylation (data not shown), consequently the anti-
proliferative activity was not mediated by inhibition of
the EGF receptor. However, considering that A431 cells
are known to overexpress Src, we studied the behavior
of 2f, 2h, and 2j (found to possess the best A431 anti-
proliferative activity in the two subseries of methylthio
and ethylthio derivatives, respectively) on the Src
phosphorylation.
hydroxy derivatives, they showed the lowest antiprolif-
erative activity in comparison to the corresponding
congeneric compounds (i.e., propylamino and butyl-
amino analogues) belonging to the other subclasses,
with the exception of 2p that was found to have an
activity of 83 lM, slightly higher than the corresponding
styryl derivative 2r (91 lM). Moreover, among the styryl
compounds lacking a C6 substitution, the activity values
of the alkylamino derivatives depended from the length
of the alkyl chain in the order propyl > butyl, differently
to that found in the other three subclasses. In fact,
activity of 2q and 2r was found to be 61 and 91 lM,
respectively. Finally, insertion of a heterocyclic amino
moiety at C4, such as 1-pyrrolidino, 1-piperidino, and
4-morpholino, led to 2s–u, respectively, whose activity
values followed the same trend found for the corre-
sponding 2-chloro-2-phenylethyl derivatives 2d–f and
2k–m with an increase in activity in the order 4-mor-
pholino > 1-pyrrolidino > 1-piperidino.
Phosphorylation of Src Tyr416 is a crucial step in the
mechanism of the enzyme activation.23 A431 cells have
been well characterized in respect to EGF-dependent
signal transduction. In more details, these cells express
high levels of EGF receptor on their surface and pro-
liferate in response to autocrine secretion of trans-
forming growth factor-a,24 but they also require
phosphorylation of Src to proliferate. To determine
With the aim of summarizing these experimental find-
ings, one can say that (1) the length and the bulkiness of
the alkylamino side chain at the position 4 influenced
activity, being a butylamino group preferred to a pro-

2516
S. Schenone et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2511–2517
Figure 4. Detection of cleaved PARP in A431 cell extracts. Immuno-
blot analysis was performed using anti-PARP specific antibodies. An
anti-rabbit secondary antibody conjugated with peroxidase and a
peroxidase substrate revealed the presence of PARP cleavage products
in apoptotic cells. The antibody recognized both the uncleaved PARP
(116 kDa) and the larger cleaved fragment (85 kD). Cells were treated
for 3 h with 10 lM of the specified compound. Results are represen-
tative of three independent experiments.
Figure 3. Phospho-Src inhibition by compounds 2f, 2h, and 2j toward
A431 cells. Cells were cultured at a concentration of 203 cells/mL in the
presence of 10 lM of the specific compound for 3 h at 37 °C. EGF
(0.1 lM, Cell Signaling Technology, MA, USA) was added 10 min
before lysis. Cell lysates (40 lg protein) were subjected to SDS-PAGE
and electrophoretically transferred to a polyvinylidene fluoride mem-
brane. Immunoblotting was performed using the specific antibody. The
membranes were visualized with luminescent substrates. The figure is a
representative gel of three similar experiments.
inhibition of Src phosphorylation, induced apoptotic
cell death probably as a consequence of the inhibition of
the tyrosine kinase positive signaling pathways. This is
in agreement with the increasing evidence that tyrosine
kinase oncogenes generate strong viability signals (e.g.,
stem cell factor) and that turning off a kinase leads to
rapid apoptosis. This effect has already been
nicely demonstrated for BCR/ABL both in vitro and
in vivo.27;28
whether these pyrazolo-pyrimidine compounds were
able to inhibit the phosphorylation of Src Tyr416, we
used a phosphospecific anti-Src (Tyr416) antibody (Cell
Signaling Technology, MA, USA). PP2, used as a ref-
erence compound, has been identified as a Src-selective
tyrosine kinase inhibitor and has been used extensively
to investigate signaling pathways involving Src kinases,
including events downstream of the stem cell factor
(SCF) receptor c-Kit.15d As a result, we found that
compounds 2f, 2h, and 2j were able to inhibit Src
phosphorylation with almost the same efficacy of PP2
(Fig. 3, Table 2).
As a consequence, the molecules described in this paper
could represent a useful tool to discover new hit com-
pounds to be studied as anticancer agents.29 For this
purpose, while additional efforts are ongoing in our
laboratory to gather further insight on the structure–
activity relationships of such compounds, biological
tests aimed at evaluating the activity of these com-
pounds on tumor cell lines have been also planned and
will be reported in due time.
Moreover, prompted by the ability of such three com-
pounds in reducing Src phosphorylation, we also tested
their pro apoptic activity on a Poly-ADP-Ribose-Poly-
merase (PARP) assay. PARP is a 116 kDa zinc-depen-
dent protein that binds specifically at DNA strand
breaks produced by various genotoxic agents.25 PARP is
also a substrate for certain caspases (e.g., caspase 3 and
7) activated during early stages of apoptosis. These
proteases cleave PARP to fragments of approximately
85 and 24 kDa. Detection of the 85 kDa PARP fragment
with anti-PARP thus serves as an early marker of
apoptosis.26
Acknowledgements
Financial support from Italian MIUR (Cofin 2002, prot.
2002038577_002) is gratefully acknowledged. One of us
(M.B.) thanks the Merck Research Laboratories for the
2002 Academic Development Program (ADP) Chemis-
try Award.
Treatment of A431 cells with 2j and 2h (10 lM) caused
proteolytic cleavage of PARP, with accumulation of the
85 kDa species and reduction of the full-size 116 kDa
protein (Fig. 4), similarly to the effect of PP2. PARP
cleavage was apparent within 3 h of treatment with 2j
and 2h, while 2f showed no appreciable effect. This fact
may be due to the short time of exposure of the A431
cells to the compound.
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Table 2. Inhibitory effects of compounds 2f, 2j, and 2h on phospho-Src after EGF stimulation of A431 cells measured by immunoblot
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