Antiproliferative activity of new 1-aryl-4-amino-1H-pyrazolo[3,4-d] pyrimidine derivatives toward the human epidermoid carcinoma A431 cell line

Article abstract of DOI:10.1016/j.ejmech.2004.07.010

Synthesis and biological evaluation of a new class of 1-aryl-4-amino-1H- pyrazolo[3,4-d]pyrimidine derivatives are reported. A preliminary cellular assay system using the tumor cell line A431 responding to epidermal growth factor (EGF) for its growth, sho

Full text of DOI:10.1016/j.ejmech.2004.07.010

European Journal of Medicinal Chemistry 39 (2004) 939–946
www.elsevier.com/locate/ejmech
Original article
Antiproliferative activity of new
1-aryl-4-amino-1H-pyrazolo[3,4-d]pyrimidine derivatives toward
the human epidermoid carcinoma A431 cell line
Silvia Schenone a,*, Olga Bruno a, Francesco Bondavalli a, Angelo Ranise a, Luisa Mosti a,
Giulia Menozzi a, Paola Fossa a, Sandra Donnini b, Annalisa Santoro b, Marina Ziche b,
Fabrizio Manetti c, Maurizio Botta c
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, Viale Benedetto XV, 16132, Genova, Italy
b Dipartimento di Biologia Molecolare, Sezione di Farmacologia, Università degli Studi di Siena, Via Aldo Moro, 53100 Siena, Italy
c Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via Aldo Moro, 53100 Siena, Italy
Received 10 March 2004; received in revised form 1 June 2004; accepted 21 July 2004
Available online 07 October 2004
Abstract
Synthesis and biological evaluation of a new class of 1-aryl-4-amino-1H-pyrazolo[3,4-d]pyrimidine derivatives are reported.A preliminary
cellular assay system using the tumor cell line A431 responding to epidermal growth factor (EGF) for its growth, shows that the new
compounds are potent inhibitors of cell growth. The inhibition of tumor cell proliferation is not associated with blockage of EGF receptor
(EGFR), but substantially due to the interference with the signalling pathway at the level of Src tyrosine kinase and at the level of the
downstream effector signal mitogen activated protein kinases (MAPKs), ERK1-2.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Pyrazolo-pyrimidine; Antiproliferative activity; Tyrosine kinase; A431 cells
1. Introduction
PTKs can be divided into two classes: the transmembrane
growth factor receptor TK and the cytoplasmatic TK. In
particular, the cytoplasmatic protein c-Src is a ubiquitous
nonreceptor TK which is overexpressed or activated in diffe-
rent types of tumors [6], osteoporosis [7], and stroke [8].
Selective inhibition of c-Src function represents an important
field of research for treatment of these diseases. As an exam-
ple, Src phosphorylation inhibition may stop uncontrolled
tumor cell growth and play a crucial role in the treatment of
cancer. In addition to Src, a family of enzymes that has
attracted a significant amount of interest has been the growth
factor tyrosine kinase receptors, specially the EGF receptor
[9].
During the past decade, many tyrosine kinase inhibitors
have been described and reviewed in the literature [1,2,10–
13], some of them belonging to the pyrazolo[3,4-d]-
pyrimidine class. As an example, approaches aimed at iden-
tifying ATP-competitive small molecules led Traxler and
coworkers to find pyrazolo[3,4-d]pyrimidines showing po-
tent inhibitory activity toward EGFR tyrosine kinase [1,17],
The search for new anticancer chemotherapeutic agents
continues to be an active area of research at many pharma-
ceutical companies [1–4]. In the last 10 years, there has been
a growth in the field of research around inhibitors of tyrosine
kinases as potential cancer therapeutics. Determining the
role that these key signalling enzymes play in the prolifera-
tion and spread of cancer has been critical to the identifica-
tion of new biological targets with new mechanisms of ac-
tion. Protein tyrosine kinases (PTKs) utilize ATP to
phosphorylate specific tyrosine residues within the sequence
of functional proteins, thus mediating the transmission of
mitogenic signals and numerous other cellular events [5],
including cell proliferation, migration, differentiation, meta-
bolism and immune response.
* Corresponding author. Tel.: +39-010-353-8866;
fax: +39-010-353-8358.
E-mail address: schensil@unige.it (S. Schenone).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.07.010

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S. Schenone et al. / European Journal of Medicinal Chemistry 39 (2004) 939–946
while Hanke et al. [14], Shokat [15] and Hirst [16] reported
pyrazolo[3,4-d]pyrimidines as selective inhibitors of Src fa-
mily. Moreover, purine derivatives such as olomoucine and
roscovitine, structurally related to the pyrazolo[3,4-
d]pyrimidine compounds, exhibited a moderate activity but
good selectivity toward several CDKs kinases [31]. To sum-
marize, small variations on the chemical structure (either the
scaffold or the all around substituents) might shift the inhibi-
tory activity of a compound from EGFR to Src, to CDK
serine-threonine kinase.
On the basis of this knowledge, we decided to synthesize
new 4-aminopyrazolo[3,4-d]pyrimidines, structurally rela-
ted to the above mentioned inhibitors, but possessing a
2-chloro-2-phenylethyl or a stryryl chain on the N1 of the
pyrazole ring [18]. The antiproliferative activity of such
compounds was assessed on the human epidermoid carci-
noma A431 cell line, in comparison to AG-1478 (Chart 1),
chosen as the reference compound. Moreover, to study at the
molecular level the mechanism of action of the above com-
pounds, their influence on signalling molecules (namely, Src
and ERK1-2) involved in the EGF-induced cell proliferation,
was also investigated.
2. Chemistry
1H-Pyrazolo[3,4-d]pyrimidines 1a–l and 2a–f were syn-
thesized as follows (Scheme 1). The starting product was the
ethyl ester of 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-
pyrazole carboxylic acid 3, prepared following our published
procedure [19].
Reaction of 3 with formamide in excess and heating at
200 °C for 8 h afforded the pyrazolo[3,4-d]pyrimidinone 4
which was purified by dissolving the crude in NaOH 2 M,
boiling with coal, followed by precipitation with acetic acid.
The chlorination with Vilsmeier complex (POCl3:
DMF/1:1) in CHCl3 at reflux for 8 h afforded the dihaloge-
nated derivative 5, bearing a chlorine atom in position 4 of
the pyrimidine ring and on the side chain on the N1 of the
pyrazole ring. Product 5 was purified by a simple filtration on
Florisil® column.
Regioselective substitution of the C4 chlorine of com-
pounds 5 with an excess of various primary and secondary
amines, in toluene at rt, afforded the desired products 1a–l in
good yield.
Chart 1. Structure of the reference compounds cited ou used in this study.
Scheme 1
.

S. Schenone et al. / European Journal of Medicinal Chemistry 39 (2004) 939–946
941
Table 1
It is interesting to point out that the chlorine atom at the
side chain of all of the new compounds has never been
substituted by the amino group. This finding was proved by
the 1H NMR chemical shifts of the CH2CH side chain pro-
tons, which resonates unchanged at the same position and
with the same pattern.
Inhibitory effect of compounds 1a–l and 2a–d on the growth of A431 cells
Treatment of 4 at reflux with POCl3 afforded the 1-styryl
derivative 6.
Compound 2a–f were prepared by reaction of 6 with the
proper amine, in toluene at rt.
No.
R
R′
Percent
inhibition
EC50
relative
to the reference
compound
AG1478
53
3. Pharmacology
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
2a
2b
2c
2d
2e
2f
NHcyclopropyl
NHC3H7
CH2CHClPh
CH2CHClPh
CH2CHClPh
CH2CHClPh
CH2CHClPh
CH2CHClPh
CH2CHClPh
69
78
26
20
71
41
48
15
70
59
50
4.3
1.4
46
62
59
41
78
To study their cellular mechanism of action and specifi-
city, compounds 1a–l and 2a–f were tested in assay systems
using a cell line responding to EGF for its growth. Inhibition
of EGF-stimulated cell growth was measured in the human
epidermoid carcinoma A431 cells [20], known to overex-
press both EGFR and Src.
The growth inhibitory activity of the compounds studied
was compared with the activity of the selective EGFR inhi-
bitor AG1478 [21] at the concentration of 50 nM. The inhi-
bitory effect exerted by the compounds on cell proliferation
when tested at 50 nM, is reported in Table 1.
46
1-pyrrolidinyl
1-piperidinyl
4-morpholinyl
NH(CH2)2OC2H5
NHcyclohexyl
86
128
46
66
63
1-hexahydroazepinyl CH2CHClPh
107
49
NH(CH2)2Ph
NEt2
CH2CHClPh
CH2CHClPh
CH2CHClPh
CH2CHClPh
CH=CHPh
CH=CHPh
CH=CHPh
43
NHC4H9
57
NHCH2Ph
112
146
63
NHcyclopropyl
NH(CH2)2OC2H5
NHcyclohexyl
51
4. Results and discussion
1-hexahydroazepinyl CH=CHPh
58
NHCH2Ph
CH=CHPh
CH=CHPh
64
NH(CH2)2Ph
46
A431 cells appeared to be sensitive to the activity of these
new compounds. In fact, only four compounds (namely 1b,
1e, 1i, 1j and 2f) showed an inhibition activity toward the cell
growth lower than 50%, while five molecules (namely 1c, 1d,
1h, 1l and 2a) were reported to have activity higher than 86%
when compared with the 100% inhibitory activity of the
reference compound, AG1478. Regarding compounds 1, an
attempt to rationalize the relationships between the structural
features of the C4 substituent and their inhibitory activity
suggested that a better inhibition of the A431 cells growth
was found for compounds bearing large substituents at that
position. As an example, enlargement of the pyrrolidino ring
of 1c (86% inhibition) to a piperidino and 1-hexahydro-
azepino ring led to compounds 1d and 1h with higher activity
(128% and 107%, respectively). Moreover, transformation of
the piperidino ring of 1d to a morpholino moiety (1e) caused
a significant decrease in activity, from 128% to 46%, respec-
tively. Substitution of the morpholino ring with an ethoxy-
ethylamino side chain led to 1f with enhanced activity (66%)
with respect to the parent compound. Introduction of an
aromatic moiety at C4, such as a benzylamino group, was
favorable for activity (1l, 112%), while increasing the size of
this group into a phenylethylamino side chain produced a
drop in activity (1i, 49%). Smaller alkylamino side chains at
C4 (namely, cyclopropylamino, propylamino, butylamino,
and diethylamino) led to compounds 1a, 1b, 1k, and 1j,
respectively, with low activity, ranging from 43% to 57%.
MTT cell proliferation assay for A431 cell line in the presence with 50 nM
test or reference compounds. Cell growth was determined at 48 h. EGF was
not added to A431 cell line since its receptor is constitutively activated.
Results are expressed as percent values with respect to the reference inhibi-
tor AG1478, whose inhibition activity was set to 100, and were calculated
from six data taken from two separate experiments. EC50 values were
calculated from series separate assays, each performed in quadruplicate.
Substituent at the 1-position of the bicyclic nucleus is
worthy of further consideration. Compound 2a, bearing a
cyclopropylamino moiety at C4 and an unsaturated aryl
chain (i.e., styryl moiety) at N1 was characterized by an
inhibitory profile much more interesting with respect to the
corresponding phenyl chloroethyl derivative 1a. In fact, 2a
showed an inhibitory activity value of 146% (the highest
inhibitory activity found among all the new compounds)
toward A431 cells growth with respect to the reference com-
pound, while 1a possessed only 53% toward the same target.
Moreover, 2b–d showed inhibitory activity lower than the
corresponding phenylethyl derivatives 1f–h. In particular,
while activity of 1f and 2b were comparable (66% versus
63%), the introduction of bulkier side chains at C4 led to
more pronounced loss of activity, when compared to the
corresponding phenylethyl compounds. In fact, activity of 1g
was found to be 63% versus a 51% determined for 2c.
Finally, a further enlargement of the cycle at C4 from a
cyclohexyl to a 1-hexahydroazepinyl ring led to a deeper
decrease in activity, from 107% (1h) to 58% (2d). Similarly,

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S. Schenone et al. / European Journal of Medicinal Chemistry 39 (2004) 939–946
an aromatic substituent at C4 led to compounds 2e and 2f
with lower activity (64% and 46%, respectively) with respect
to the corresponding saturated derivatives 1l and 1i (112%
and 49%, respectively). These findings suggested that a pla-
nar system such as the styryl moiety, combined with a small
secondary amino group, should be more profitable, with
respect to saturated alkyl chains at N1 and bulkier amino
groups at C4, to interact with molecular targets leading to the
inhibition of A431 cells proliferation [22]. This result was
not in agreement with what found for compounds 1a–l where
large substituents at C4 produced an enhancement in activity.
To prove this speculation, more compounds with a N1 styryl
chain have been planned to be synthesized and tested.
Moreover, to further investigate their biological proper-
ties, EC50 values were determined for compounds 1 and 2. In
detail, the best results were obtained for compounds 1d, 1h,
1l, and 2a, showing an EC50 of 20, 15, 4.3, and 1.4 µM,
respectively, in comparison to a value of 2.9 µM found for the
reference compound AG1478. This result confirmed com-
pound 2a as characterized by the best biological profile with
respect to the remaining new pyrazolo-pyrimidine derivati-
ves.
The above experimental evidence prompted us to test the
new compounds for their inhibitory properties on EGF tyro-
sine kinase. In fact, (1) a relevant structural similarity was
shared by our compounds and those reported by Traxler et al.
[17] (i.e., the pyrazolo[3,4-d]pyrimidine nucleus; the hydro-
gen bond donor-acceptor motif represented by the amino
group at the C4 and the endocyclic N5 nitrogen atom, respec-
tively; the anilido group at the pyrimidine ring of several
compounds); (2) while compounds 1d and 1h showed a
remarkable inhibitory activity toward the growth of A431
cells, the activity of 1l (4.3 µM) and 2a (1.4 µM) was com-
parable to or higher than that found for the reference com-
pound AG1478 (2.9 µM).
reference compound. As a result, although PP2 was found to
be more efficient than 1l and 2a, the two compounds were
able to inhibit Src phosphorylation with almost the same
efficacy of PP2 (Fig. 1).
Moreover, we also investigated signalling molecules in-
volved in the EGF-induced cell proliferation. In further de-
tail, compound 11 showed an interesting 50% inhibition
(with respect to the reference compound staurosporine) on
the activity of the human Src kinase receptor, measured by
fluorescence polarization, according to the procedure repor-
ted by Parker [26]. Moreover, two compounds showing the
higher inhibitory activity toward the growth ofA431 cells (11
and 2a) were tested (at the concentration of 50 nM) for their
effects on the phosphorylation activity of the downstream
extracellular receptor kinases 1-2 (ERK1-2) belonging to the
family of the tyrosine-threonine mitogen activated protein
kinases (MAPKs) and playing a crucial role in the intracel-
lular signalling regulating the cellular growth process media-
ted by EGF.
As a result (Fig. 2), both 1l and 2a completely inhibited
ERK1-2 activity induced by EGF and measured as ERK1-2
phosphorylation [27]. It is worth to note that, at the same
nanomolar concentration, the selective MEK inhibitor PD
98059 [28] did not affect EGF-induced ERK1-2 phospho-
rylation. A 100 µM concentration of PD 98059 was required
to show an inhibition of ERK1-2 phosphorylation compara-
ble to that induced by a 50 nM concentration of 1l. In
summary, compounds 1l and 2a were found to inhibit the
tyrosine-threonine MEK kinase activity responsible for ERK
phosphorylation. In particular compound 1l emerged as a
potent inhibitor of EGFR dependent tumor cell growth (e.g.
A431) and its inhibitory effect appears to be not related to the
inhibition of protein tyrosine kinase EGFR, but associated
with both the Src and MAPK pathways.
The influence of such compounds on the activity of the
human EGF tyrosine kinase receptor was evaluated by
measuring the phosphorylation of poly GAT (Glu, Ala, Tyr)
using a purified A431 cell membrane preparation as the
enzyme source [23]. As a result, all the newly synthesized
compounds were inactive (data not shown). Compound 1l,
taken as an example, showed only a 2% inhibition of the
control enzyme activity, with respect to the reference com-
pound, PD 153035, known as an EGFR inhibitor similar to
AG1478 and erlotinib [24,25]. This result suggested that the
antiproliferative activity of the studied compounds did not
involve inhibition of EGFR.
Fig. 1
. Phospho-Src inhibition by compounds 1l and 2a toward A431 cells.
The figure is a representative gel of three similar experiments.
On the other hand, considering that the A431 cell line is
known to overexpress Src, we studied the influence of 1l and
2a (chosen on the basis of their high A431 antiproliferative
activity) on the phosphorylation of Src, through an in vitro
assay on A431 cell lysates. To determine whether such com-
pounds were able to inhibit the phosphorylation of Src at the
level of Tyr416, we used a phosphospecific anti-Src (Tyr416)
antibody. On the other hand, PP2, previously identified as a
Src-selective tyrosine kinase inhibitor [14], was used as the
Fig. 2. Effect of compounds 1l and 2a on ERK1/2 activity in the A431 cell
line. Western blot for phospho-ERK 1/2 (phospho-p44/p42) and total
p44/p42 in A431 cell line exposed for 1 h to compound 1l or 2a at 50 nM or
PD 98059 at 100 µM and then stimulated for 10 min with 50 ng/ml EGF. A
representative gel out of three is shown.

S. Schenone et al. / European Journal of Medicinal Chemistry 39 (2004) 939–946
943
Finally, it is interesting to point out that compound 1l and
2a did not show cell toxicity up to 10 µM concentration.
In conclusion, the new pyrazolo-pyrimidines showed an
interesting antiproliferative effect toward A431 cells, mainly
due to the interference with ERK1-2, and to a partial inhibi-
tion of the Src phosphorylation. As a consequence, the mole-
cules described in this paper could represent a useful tool to
discover new hit compounds to be studied as anticancer
agents [29]. For this purpose, while additional efforts are
ongoing in our laboratory to gain further insight on the
relationships between structure and activity of such com-
pounds, biological tests for the evaluation of their activity
and selectivity on a panel of tumor cell lines have been also
planned and results will be reported in due time.
The mixture was refluxed for 7 h. The solution was
washed with H2O (2 × 20 ml), dried (MgSO4), filtered and
concentrated under reduced pressure. The crude oil was puri-
fied by column chromatography (Florisil®, 100 Mesh), using
CH2Cl2 as eluant, to afford the pure product 5 (2.2 g, 75%) as
a white solid, mp 105–106 °C. H NMR d 4.78–4.97 and
5.02–5.18 (2dd, 2H, CH2), 5.50–5.59 (m, 1H, CHCl), 7.25–
7.48 (m, 5H Ar), 8.18 (s, 1H, H-3), 8.75 (s, 1H, H-6).
1
5.1.3. General procedure for 1-(2-chloro-2-phenylethyl)-
1H-pyrazolo[3,4-d]pyrimidines 4-amino substituted 1a–l
To a solution of 5 (2.93 g, 10 mmol) in anhydrous toluene
(20 ml), the proper amine (40 mmol) was added, and the
reaction mixture was stirred at room temperature for 36 h.
After it was extracted with H2O (10 ml), the organic phase
was dried under reduced pressure; the oil residue crystallized
by adding absolute ethanol (10 ml) to give products 1a–l.
1a. White solid, mp 194–195 °C, yield 90%. 1H NMR: d
0.73–0.87 and 0.98–1.10 (2m, 4H, 2CH2), 2.88–3.02 (m, 1H,
CH), 4.71–4.83 and 4.95–5.08 (2dd, 2H, CH2N), 5.53–5.62
(m, 1H, CHCl), 6.68 (br s, 1H, NH, disappears with D2O),
7.25–7.52 (m, 5HAr), 8.19 (s, 1H, H-3), 8.32 (s, 1H, H-6). IR
cm–1: 3410 (NH).
5. Experimental protocols
5.1. Chemistry
Starting materials were purchased from Aldrich-Italia
(Milan).
Melting points were determined with a Büchi B 540 appa-
ratus and are uncorrected. IR spectra were recorded in KBr
with a Perkin-Elmer 398 spectrophotometer. 1H NMR spec-
tra were recorded in CDCl3 solution on a Varian Gemini 200
(200 MHz) instrument, chemical shifts (d) are reported in
ppm relative to TMS as internal standard; J in Hz. 1H patterns
are described using the following abbreviations: s = singlet,
d = doublet, dd = double doublet, t = triplet, q = quartet,
sext = sextet, m = multiplet, br = broad.
All compounds were tested for purity by TLC (Merk,
Silica gel 60 F254, CHCl3 as eluant).
Analyses for C, H, N were within 0.3% of the theoretical
value.
1b. White solid, mp 142–143 °C, yield 88%. 1H NMR: d
1.04 (t, J = 7.0, 3H, CH3), 1.76 (sx, J = 6.0, 2H, CH2), 3.58 (q,
J = 6.0, 2H, CH2Nprop), 4.68–4.82 and 4.92–5.06 (2dd, 2H,
CH2N), 5.53–5.62 (m, 2H, CHCl), 5.90–6.20 (br s, 1H, NH,
disappears with D2O), 7.25–7.52 (m, 5H Ar), 7.90 (s, 1H,
H-3), 8.35 (s, 1H, H-6). IR cm–1: 3415 (NH).
1c. White solid, mp 161–162 °C, yield 75%. 1H NMR: d
1.95–2.25 (m, 4H, 2CH2), 3.78 (t, 4H, 2CH2Npyrr), 4.68–
4.82 and 4.95–5.06 (2dd, 2H, CH2N), 5.53–5.62 (m, 1H,
CHCl), 7.22–7.52 (m, 5H Ar), 7.92 (s, 1H, H-3), 8.36 (s, 1H,
H-6).
1d. White solid, mp 148–149 °C, yield 72%. 1H NMR: d
1.62–1.85 (m, 6H, 3CH2), 3.95–4.07 (m, 4H, 2CH2Npip),
4.67–4.82 and 4.92–5.08 (2dd, 2H, CH2N), 5.53–5.63 (m,
1H, CHCl), 7.25–7.52 (m, 5H Ar), 7.94 (s, 1H, H-3), 8.35 (s,
1H, H-6).
5.1.1. 1-(2-Hydroxy-2-phenylethyl)-1,5-dihydro-4H-pyra-
zolo[3,4-d]pyrimidin-4-one 4
A suspension of 3 (2.75 g, 10 mmol) in formamide (10 g,
333 mmol) was heated at 190 °C for 8 h and then poured in
H2O (300 ml). The crude was filtered and purified by dissol-
ving in 2 M NaOH (100 ml), boiling with coal, followed by
precipitation with glacial acetic acid. The solid was filtered
and recrystallized from absolute ethanol to give 4 (1.79 g,
70%) as a white solid, mp 270–271 °C. 1H NMR: d 4.25–4.55
(m, 2H, CH2), 5.05–5.15 (m, 1H, CHO), 5.65 (d, 1H, OH,
disappears with D2O), 7.25–7.45 (m, 5H Ar), 8.05 (s, 1H,
H-3), 8.10 (s, 1H, H-6), 12.15 (br s, 1H, NH, disappears with
D2O). IR cm–1: 3400 (NH), 3245–2500 (OH), 1740 (C=O).
1e. White solid, mp 132–133 °C, yield 70%. 1H NMR: d
3.79–3.89 and 3.91–4.04 (2m, 8H, 4CH2 morph), 4.71–
4.83 and 4.94–5.08 (2dd, 2H, CH2N), 5.53–5.62 (m, 1H,
CHCl), 7.24–7.52 (m, 5H Ar), 7.94 (s, 1H, H-3), 8.37 (s, 1H,
H-6).
1f. White solid, mp 119–120 °C, yield 65%. 1H NMR: d
1.24 (t, J = 7.0, 3H, CH3), 3.56 (q, J = 7.0, 2H, CH3CH2O),
3.68 (t, 2H, CH2O), 3.83 (q, 2H, CH2NH), 4.71–4.83 and
4.95–5.06 (2dd, 2H, CH2N), 5.52–5.60 (m, 1H, CHCl), 5.80
(br s, 1H, NH, disappears with D2O), 7.26–7.50 (m, 5H Ar),
7.91 (s, 1H, H-3), 8.39 (s, 1H, H-6). IR cm–1: 3439 (NH).
1g. White solid, mp 157–158 °C, yield 71%. 1H NMR: d
1.19–1.58, 1.62–1.95 and 2.04–2.20 (3m, 10H, 5CH2), 3.95–
4.15 (m, 1H, CHNH), 4.69–4.81 and 4.91–5.08 (2dd, 2H,
CH2N), 5.51–5.61 (m, 1H, CHCl), 7.12–7.62 (m, 5H Ar),
7.86 (s, 1H, H-3), 8.38 (s, 1H, H-6), 8.79 (br s, 1H, NH,
disappears with D2O). IR cm–1: 3403 (NH).
5.1.2. 4-Chloro-1-(2-chloro-2-phenylethyl)-1H-pyrazolo-
[3,4-d]pyrimidine 5
The Vilsmeier complex, previously prepared from POCl3
(6.13 g, 40 mmol) and anhydrous dimethylformamide
(DMF) (2.92 g, 40 mmol) was added to a suspension of 4
(2.56 g, 10 mmol) in CHCl3 (20 ml).

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S. Schenone et al. / European Journal of Medicinal Chemistry 39 (2004) 939–946
1h. White solid, mp 134–135 °C, yield 55%. 1H NMR: d
2b .White solid: mp 115–116 °C, yield 84%. 1H NMR: d
1.23 (t, J = 7.0, 3H, CH3), 3.56 (q, J = 7.0, 2H, CH3CH2O),
3.70 (t, 2H, CH2O), 3.83 (q, 2H, CH2NH), 6.08 (br s, 1H,
NH, disappears with D2O), 7.20–7.55 (m, 6H, 5HAr + CH=),
8.01 (s, 1H, H-3), 8.02 (d, Jtrans = 14.6, 1H, CH=), 8.44 (s,
1H, H-6). IR cm–1: 3440 (NH), 1662 (C=C).
1.55–1.78 and 1.80–2.03 (2m, 8H, 4CH2), 3.75–4.04 (m, 4H,
2CH2N), 4.68–4.82 and 4.93–5.08 (2dd, 2H, CH2N), 5.52–
5.65 (m, 1H, CHCl), 7.27–7.57 (m, 5HAr), 7.88 (s, 1H, H-3),
8.36 (s, 1H, H-6).
1
1i. White solid, mp 102–103 °C, yield 81%. H NMR: d
3.02 (t, J = 7.0, 2H, CH2), 3.91 (q, J = 7.0, 2H, CH2NH),
4.70–4.85 and 4.95–5.10 (2dd, 2H, CH2N), 5.51–5.64 (m,
2H, CHCl + NH, 1H disappears with D2O), 7.22–7.55 (m,
10H Ar), 7.87 (s, 1H, H-3), 8.40 (s, 1H, H-6). IR cm–1: 3420
(NH).
2c. White solid: mp 187–188 °C, yield 80%. 1H NMR: d
1.12–1.58, 1.60–1.93, 2.08–2.22 (3m, 11H cycloexyl), 4.08
(br s, 1H, NH, disappears with D2O), 7.20–7.58 (m, 6H, 5H
Ar + CH=), 7.99 (s, 1H, H-3), 8.00 (d, Jtrans = 14.4, 1H,
CH=), 8.41 (s, 1H, H-6). IR cm–1: 3405 (NH), 1658 (C=C).
2d. White solid, mp 121–122 °C, yield 72%. 1H NMR: d
1.53–1.73 (m, 4H, 2CH2), 1.82–2.08 (m, 4H, 2CH2), 3.80–
4.08 (m, 4H, 2CH2N), 7.20–7.62 (m, 6H, 5HAr + CH=), 8.05
(s, 1H, H-3), 8.07 (d, Jtrans = 14.4, 1H, CH=), 8.43 (s, 1H,
H-6). IR cm–1: 1655 (C=C).
1j. White solid, mp 161–162 °C, yield 85%. 1H NMR: d
1.30 (t, J = 7.0, 6H, 2CH3), 3.74 (q, J = 7.0, 4H, 2CH2),
4.68–4.83 and 4.85–5.06 (2dd, 2H, CH2N), 5.52–5.63 (m,
1H, CHCl), 7.15–7.58 (m, 5H Ar), 7.85 (s, 1H, H-3), 8.36 (s,
1H, H-6).
2e. White solid, mp 184–185 °C, yield 80%. 1H NMR: d
4.86 (d, 2H, CH2), 5.80–5.90 (br s, 1H, NH, disappears with
D2O), 7.20–7.59 (m, 6H, 5H Ar + CH=), 7.99 (s, 1H, H-3),
8.03 (d, Jtrans = 14.4, 1H, CH=), 8.45 (s, 1H, H-6). IR cm–1:
3430 (NH), 1655 (C=C).
1k. White solid, mp 93–94 °C, yield 80%. 1H NMR: d 0.98
(t, J = 7.0, 3H, CH3), 1.35–1.59 (sx, J = 7.0, 2H, CH2),
1.61–1.79 (quintet, J = 7.0, 2H, CH2), 3.60 (q, J = 7.0, 2H,
CH2NH), 4.70–4.81 and 4.92–5.08 (2dd, 2H, CH2N), 5.48–
5.63 (m, 2H, CHCl + NH, 1H disappears with D2O), 7.25–
7.55 (m, 5H Ar), 7.90 (s, 1H, H-3), 8.35 (s, 1H, H-6). IR
cm–1: 3415 (NH).
2f. White solid, mp 152–153 °C, yield 78%. 1H NMR: d
3.04 (t, J = 7.0, 2H, CH2Ar), 3.93 (q, J = 7.0, 2H, CH2N),
5.50–5.71(br s, 1H, NH, disappears with D2O), 7.24–7.60
(m, 6H, 5H Ar + CH=), 8.01 (s, 1H, H-3), 8.05 (d,
Jtrans = 14.0, 1H, CH=), 8.45 (s, 1H, H-6). IR cm–1: 3440
(NH), 1660 (C=C).
1l. White solid, mp 158–160 °C, yield 82%. 1H NMR: d
4.69–4.80 and 4.93–5.07 (2dd, 2H, CH2N), 4.82–4.88 (m,
2H, CH2NH), 5.51–5.59 (m, 2H, CHCl), 7.25–7.51 (m, 10H
Ar), 7.85 (s, 1H, H-3), 8.39 (s, 1H, H-6). IR cm–1: 3425
(NH).
6. Pharmacology
5.1.4. 4-Chloro-1-(2-phenylvinyl)-1H-pyrazolo[3,4-d]pyri-
midine 6
6.1. Reagents
POCl3 (14 g, 91 mmol) was added to 4 (2.56 g, 10 mmol),
the mixture was refluxed for 12 h and then cooled to room
temperature
Cell culture reagents and materials were from Sigma Che-
mical. FCS was from Hyclone. Human recombinant EGF
were from Calbiochem-Novabiochem. Vybrant MTT cell
proliferation assay kit was from Molecular Probe.
The excess of POCl3 was removed by distillation under
reduced pressure. H2O (20 ml) was carefully added to the
residue and the suspension was extracted with CHCl3 (3 ×
20 ml). The organic solution was washed with H2O (10 ml),
dried (MgSO4), filtered and concentrated under reduced
pressure. The crude brown oil was purified by column chro-
matography (Florisil® 100–200 Mesh), using CHCl3 as the
eluant to afford the pure product 6 (1.66 g, 65%) as a white
solid: mp 139–140 °C. 1H NMR: d 7.25–7.61 (m, 6H, 5H Ar
+ CH=), 8.04 (d, Jtrans = 14.6, 1H, CH=), 8.29 (s, 1H, H-3),
8.85 (s, 1H, H-6). IR cm–1 1658 (C=C).
6.2. Cytotoxicity
The cytotoxic effect of compound was evaluated by trypan
blue exclusion. Cells were stimulated for 1 h at 37 °C with
the increasing concentrations (10 nM–10 µM) of the test
compound in 1% FCS medium. Cells were then stained with
0.4% trypan blue in phosphate buffer saline (PBS) for 5 min.
The number of dead and living cells was counted at the
microscope in a blind manner. The percentage of dead cells
over the total number of cells was calculated.
5.1.5. General procedure for 1-(2-phenylvinyl)-
1H-pyrazolo[3,4-d]pyrimidines-4-amino substituted 2a–d
Compound 2a–d were prepared according to the synthetic
sequence described for compounds 1 starting from product 6
2a. White solid: mp 220–221 °C, yield 85%. 1H NMR: d
0.77–0.87 (m, 2H, CH2), 1.00–1.11 (m, 2H, CH2), 2.92–3.06
(m, 1H, CH cyclopr.), 6.35 (br. s, 1H, NH, disappears with
D2O), 7.20–7.58 (m, 6H, 5HAr + CH=), 8.07 (d, Jtrans = 14.6,
1H, CH=), 8.30 (s, 1H, H-3), 8.38 (s, 1H, H-6). IR cm–1:
3615 (NH), 1655 (C=C).
6.3. Cell culture
The human epidermoid carcinoma A431 cells were obtai-
ned from American Type Culture Collection (Rockville,
MD). A431 cells were maintained in culture in DMEM
supplemented with 4500 m/l glucose and 10% FCS. Cells
were split 1:5 twice a week.

S. Schenone et al. / European Journal of Medicinal Chemistry 39 (2004) 939–946
945
6.4. MTT cell proliferation assay
Research (AIRC) (M.Z.), and MIUR (Cofin 2001, grant
2001068248_005) (M.Z.) are gratefully acknowledged. M.
B. thanks the Merck Research Laboratories for the 2002
Academic Development Program Chemistry Award.
Cell proliferation was quantified by Vybrant MTT cell
proliferation assay kit (Molecular Probe). A431 cells (2.5 ×
103) were seeded in 96 multiwell plate with 10% serum for
5 h, stabilised for 16 h and then exposed to tested compounds
(50 nM) for 48 h. The medium was then removed and the
cells were incubated for 4 h with fresh medium in the pre-
sence of 1.2 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide). Live cells reduce MTT to a
strongly pigmented formazan product.After solubilization in
DMSO, the absorbance of the formazan was measured with a
microplate absorbance reader (Tecan) at 540 nm. The growth
inhibitory activity of the compounds studied was compared
with the activity of the selective EGFR inhibitor AG1478 at
the concentration of 50 nM.
References
[1] P. Traxler, P. Furet, Pharmacol. Ther. 82 (1999) 195–206 (references
therein cited).
[2] A.J. Bridges, Chem. Rev. 101 (2001) 2541–2572.
[3] R.M. Myers, N.N. Setzer, A.P. Spada, P.E. Persons, C.Q. Ly,
M.P. Maguire, A.L. Zulli, D.L. Cheney, A. Zilberstein, S.E. Johnson,
C.F. Franks, K.J. Mitchell, Bioorg. Med. Chem. Lett. 7 (1997) 421–
424.
[4] J.D. Wang, K. Miller, D.H. Boschelli, F. Ye, B. Wu, M.B. Floyd,
D.W. Powell, A. Wissner, J.M. Weber, F. Boschelli, Bioorg. Med.
Chem. Lett. 10 (2000) 2477–2480.
[5] D. Fabbro, S. Ruetz, E. Buchdunger, S.W. Cowan-Jacob, G. Fendrich,
J. Liebetanz, J. Mestan, T. O’Reilly, P. Traxler, B. Chaudhuri, H. Fretz,
J. Zimmermann, T. Meyer, G. Caravatti, P. Furet, P.W. Manley, Phar-
macol. Ther. 93 (2002) 79–98.
6.5. Western blotting for Src phosphorylation activity
Cells were cultured at a concentration of 203 cells/ml in
the presence of 10 µM of the compound 1l, 2a or the refe-
rence compound PP2, for 3 h at 37 °C. EGF (0.1 µM, Cell
Signaling Technology, MA, USA) was added 10 min before
lysis. Cell lysates (40 µg protein) were subjected to SDS-
PAGE and electrophoretically transferred to a polyvinyli-
dene fluoride membrane. Immunoblotting was performed
using the specific antibody. The membranes were visualized
with luminescent substrates.
[6] J.D. Bjorge,A. Jakymiw, D.J. Fujita, Oncogene 19 (2000) 5620–5635.
[7] E. Altmann, M. Missbach, J. Green, M. Susa, H.A. Wagenknecht,
L. Widler, Bioorg. Med. Chem. Lett. 11 (2001) 853–856 (references
cited therein).
[8] R. Paul, Z.G. Zhang, B.P. Eliceiri, Q. Jiang, A.D. Boccia, R.L. Zhang,
M. Chopp, D.A. Cheresh, Nat. Med. 7 (2001) 222–227.
[9] S. Schenone, O. Bruno, F. Bondavalli, A. Ranise, L. Mosti,
G. Menozzi, P. Fossa, F. Manetti, L. Morbidelli, L. Trincavelli,
C. Martini, A. Lucacchini, Eur. J. Med. Chem. 39 (2004) 153–160.
[10] P. Traxler, N. Lydon, Drugs Future 20 (1995) 1261–1274.
[11] A. Levitzki, A. Gazit, Science 267 (1995) 1782–1788.
[12] T.R. Burke, B. Ye, X. Yan, S. Wang, Z. Jia, L. Chen, Z.Y. Zhang,
D. Barford, Biochemistry 35 (1996) 15989–15996.
6.6. Western blotting for ERK1-2 kinase activity
A431 Cells (3 × 106) were seeded in a 6 cm plate with 10%
serum overnight, starved for 24 h, pretreated with 50 nM of
compounds 1l and 2a for 1 h and then stimulated with
50 ng/ml EGF for 10 min at 37 °C. As positive control,
100 µM PD 98059, a selective inhibitor of MEKK kinase
activity, the kinase upstream to ERK1-2, was used. Proteins
were extracted and 20 µg of lysate for each experimental
point were subjected to 10% SDS-polyacrilammide gel elec-
trophoresis and transferred to a PDVF membrane. Western
blot analysis was performed as described [30]. The mouse
anti-phosphop42/p44 antibody 1:1000 (Cell Signaling Tech-
nology) or rabbit anti-p42/p44 (1:1000) (Cell Signaling
Technology) for the determination of corresponding kinase
levels were incubate with the membranes for 18 h. Blots were
incubated with HRP-goat anti-mouse antibody or HRP-goat
anti-rabbit antibody, respectively (Upstate Biotechnology)
and the bands visualized with an enhanced chemilumines-
cence system (Amersham Pharmacia Biotech). Band intensi-
ties were measured using the ScioImage sotware package.
[13] S. Madhusudan, A.L. Harris, Curr. Opin. Pharmacol. 2 (2002) 403–
414.
[14] J.H. Hanke, J.P. Gardner, R.L. Dow, P.S. Changelian, W.H. Brissette,
E.J. Weringer, B.A. Pollok, P.A. Connelly, J. Biol. Chem. 271 (1996)
695–701.
[15] A.C. Bishop, C. Kung, K. Shah, L. Witucki, K.M. Shokat, Y. Liu, J.
Am. Chem. Soc. 121 (1999) 627–631.
[16] A.F. Burchat, D.J. Calderwood, M.M. Friedmann, G.C. Hirst, B. Li,
P. Rafferty, K. Ritter, B.S. Skinner, Bioorg. Med. Chem. Lett. 12
(2002) 1687–1690.
[17] P. Traxler, G. Bold, J. Frei, M. Lang, N. Lyndon, H. Mett, E. Buch-
dunger, T. Meyer, M. Mueller, P. Furet, J. Med. Chem. 40 (1997)
3601–3686.
[18] This particular chain was fundamental for the A1 adenosine receptor
inhibitory activity of our compounds (see Ref. [19]) and, based on the
consideration that similar inhibitors acting on theATP site of enzymes
are also adenosine receptor antagonists (G. Schulte, B.B. Fredholm,
Cell. Signal. 14 (2002) 109–113), we thought it could be also accom-
modated into a hydrophobic pocket of the ATP binding site described
by Traxler and coworkers.
[19] F. Bondavalli, M. Botta, O. Bruno, A. Ciacci, F. Corelli, P. Fossa,
F. Manetti, A. Lucacchini, C. Martini, G. Menozzi, L. Mosti, A. Ran-
ise, S. Schenone, A. Tafi, M.L. Trincavelli, J. Med. Chem. 45 (2002)
4875–4887.
[20] G. Carpenter, L. King, S.-J. Cohen, Biol. Chem. 254 (1979) 4884–
4891.
Acknowledgements
Financial support from Italian MIUR (Cofin 2002, grant
2002038577_002) (M.B.), the Italian Association for Cancer
[21] W. Liu,A.A.Akhand, M. Kato, I.Yokoyama, T. Miyata, K. Kurokawa,
K. Uchida, I. Nakashima, J. Cell Sci. 112 (1999) 2409–2417.

946
S. Schenone et al. / European Journal of Medicinal Chemistry 39 (2004) 939–946
[22] We have hypothesized that the extended planar system of the styryl
derivatives combined with small substituents at C4, represented an
optimal combination of structural features allowing for profitable
interactions with the receptor counterpart. However, since the mecha-
nism of action of the new compounds is still unclear at the molecular
level, we cannot further speculate on the reason(s) causing a better
activity for the styryl derivatives with respect to the 2-chloro-2-phenyl
compounds, as (for example) a different binding mode (or a different
orientation within the same binding site) proposed for pyrrolo[2,3-
d]pyrimidine derivatives. For a reference, see A. Gangjee, J. Yang,
M.A. Ihnat, S. Kamat, Bioorg. Med. Chem. 11 (2003) 5155–5170, and
references cited therein.
[25] A.J. Bridges, H. Zhou, D.R. Cody, G.W. Rewcastle, A. McMichael,
H.D. Showalter, D.W. Fry,A.J. Kraker, W.A. Denny, J. Med. Chem. 39
(1996) 267–276.
[26] G.J. Parker, T.L. Law, F.J. Lenoch, R.E. Bolger, J. Biomol. Screen. 5
(2000) 77–88.
[27] J. Albanell, J. Codony-Servat, F. Rojo, J.M. Del Campo, S. Sauleda,
J. Anido, G. Raspall, J. Girald, J. Rosello, R.I. Nicholson, J. Mendel-
sohn, J. Baselga, Cancer Res. 61 (2001) 6500–6510.
[28] T.K. Means, R.P. Pavlovich, D. Roca, M.W. Vermeulen, M.J. Fenton,
J. Leukoc. Biol. 67 (2000) 885–893.
[29] F. Bondavalli, M. Botta, F. Manetti, S. Donnini, S. Schenone,
M. Ziche, 4-Substituted 1-(2-chloro-2-phenylethyl)-1H-pirazolo[3,4-
d]pyrimidine derivatives and their application, May 28, 2003 Italian
Patent: IT RM2003000264.
[23] S.R. Wedge, D.J. Ogilvie, M. Dukes, J. Kendrew, J.O. Curwen,
L.F. Hennequin, A.P. Thomas, E.S.E. Stokes, B. Curry, G.H. Rich-
mond, P.F. Wadsworth, Cancer Res. 60 (2000) 970–975.
[30] Q. Qiu, F. Dudouit, S. Matheson, F. Brahimi, R. Banerjee,
P. McNamee, B. Jean-Claude, Cancer Chemother. Pharmacol. 51
(2003) 1–10.
[24] J. Stamos, M.X. Sliwkowski, C.J. Eigenbrot, Biol. Chem. 277 (2002)
46265–46272.
[31] T.M. Sielecki, J.F. Boylan, P.A. Benfield, G.L. Trainor, J. Med. Chem
43 (2000) 1–18.