Pyrazolo[3,4-d]pyrimidines as potent antiproliferative and proapoptotic agents toward A431 and 8701-BC cells in culture via inhibition of c-Src phosphorylation

Article abstract of DOI:10.1021/jm050603r

We report here the synthesis of new pyrazolo[3,4-d]pyrimidine derivatives along with their biological properties as inhibitors of isolated Src and cell line proliferation (A431 and 8701-BC cells). Such compounds block the growth of cancer cells by interfe

Full text of DOI:10.1021/jm050603r

J. Med. Chem. 2006, 49, 1549-1561
1549
Pyrazolo[3,4-d]pyrimidines as Potent Antiproliferative and Proapoptotic Agents toward A431
and 8701-BC Cells in Culture via Inhibition of c-Src Phosphorylation
Fabio Carraro,† Antonella Naldini,† Annalisa Pucci,† Giada A. Locatelli,‡ Giovanni Maga,‡ Silvia Schenone,*,§ Olga Bruno,§
Angelo Ranise,§ Francesco Bondavalli,§ Chiara Brullo,§ Paola Fossa,§ Giulia Menozzi,§ Luisa Mosti,§ Michele Modugno,|
Cristina Tintori,| Fabrizio Manetti,| and Maurizio Botta|
Dipartimento di Fisiologia, Sezione di Neuroimmunofisiologia, UniVersita` degli Studi di Siena, Via Aldo Moro, I-53100, Siena, Italy, Istituto di
Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, I-27100, PaVia, 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 Alcide de Gasperi 2, I-53100, Siena, Italy
ReceiVed June 24, 2005
We report here the synthesis of new pyrazolo[3,4-d]pyrimidine derivatives along with their biological
properties as inhibitors of isolated Src and cell line proliferation (A431 and 8701-BC cells). Such compounds
block the growth of cancer cells by interfering with the phosphorylation of Src, and they act as proapoptotic
agents through the inhibition of the anti apoptotic gene BCL2. Several of them were found to be more
active than the reference compound (1-(tert-butyl)-3-(4-chlorophenyl)-4-aminopyrazolo[3,4-d]pyrimidine,
PP2) in inhibiting cell proliferation and in inducing apoptosis, and as active as PP2 in the inhibition of the
phosphorylation of isolated Src. Moreover, molecular modeling simulations have been performed to
hypothesize the way, at the molecular level, by which the inhibitors were able to act as antiproliferative
agents.
Introduction
chemical classes. As examples, quinoline derivatives exhibit IC50
values up to the subnanomolar range in enzymatic assays and
in the nanomolar range in Src-dependent cell proliferation
assays.16 Additional classes of important Src inhibitors are
quinazoline17 and pyrimidine derivatives. Among the latter, a
huge number of pyrrolo-18 and pyrazolo-pyrimidines19 (such as
1-(tert-butyl)-3-(4-chlorophenyl)-4-aminopyrazolo[3,4-d]pyri-
midine, PP2) have been described.
The understanding of the fundamental biology of cancer
increased dramatically in recent years1 and has strongly impacted
on experimental and gradually also on clinical tumor therapy.
We now believe that the future of tumor therapy will be
represented by the development of molecularly targeted agents
that specifically interfere with the key mechanisms involved in
development and progression of specific types of cancer.2 Due
to their critical role in tumor development and progression,
compounds able to inhibit protein kinases are of great interest
in targeted cancer therapy. Protein kinases nearly control all
aspects of cellular signal transmission under both physiological
and pathological conditions.1 In particular, protein-tyrosine
phosphorylation catalyzed by protein tyrosine kinases (PTKs)
has been implicated in the intracellular signaling cascade that
acts immediately downstream of cell surface receptors to elicit
a variety of cellular functions.2 Much attention has been paid
to the understanding of the structure and function of the
cytoplasmic type PTKs c-Src tyrosine kinase.3-7 The roles
played by c-Src in signaling changes in the steps of cellular
adhesion and motility, together with its ability to promote
proliferation, are well established.8-10 Moreover, there is
experimental evidence that Src kinase is active in many
tumors.11-15 On this basis, inhibitors of the Src phosphorylation
process may halt uncontrolled tumor cell growth and play an
important role as new therapeutic agents for the treatment of
cancer.
In this context, within our efforts to find new anticancer
agents, we have recently reported a series of pyrazolo[3,4-d]-
pyrimidine derivatives, found to be able to inhibit c-Src
phosphorylation. Preliminary in vitro biological studies dem-
onstrated that such compounds have a potent antiproliferative
effect on the human epidermoid carcinoma A431 cells,20 known
to overexpress both EGFR and Src. This finding, together with
the knowledge that the breast cancer cell line 8701-BC also
overexpresses Src,21 led us to assess the antiproliferative
properties of the new compounds also toward 8701-BC cells.22
Biological data showed that several of the reported compounds
were inhibitors of 8701-BC cells23 with noteworthy potency.
Results on both A431 and 8701-BC cells prompted us to
design and synthesize additional members of the pyrazolo-
pyrimidine structural class and to test them toward the same
human epidermoid and human breast cancer cell lines, as well
as toward isolated Src. Several of the new compounds 1-7
reported in this paper were found to inhibit Src phosphorylation
in the micro- and sub-micromolar range, similarly to the
reference compound PP2. They also selectively block cell cycle
progression and promote apoptosis. Moreover, we demonstrated
that the induction of apoptosis in 8701-BC cells was mediated
by the inhibition of BCL2 expression. As a consequence of all
these biological findings, the pyrazolo-pyrimidine derivatives
disclosed here could represent a profitable tool to inhibit cancer
cell growth through blocking the Src phosphorylation mecha-
nism and inducing apoptosis.
Although there is no approved drug acting as Src inhibitor,
a plethora of compounds, potent and selective toward this kinase
family, are continuously designed and synthesized. Many of
them are ATP-competitive inhibitors belonging to different
* To whom correspondence should be addressed. Tel: 0039 010 3538866.
Fax: 0039 010 3538358. E-mail: schensil@unige.it.
† Dipartimento di Fisiologia, Universita` degli Studi di Siena.
‡ Istituto di Genetica Molecolare, IGM-CNR.
§ Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di
Genova.
Chemistry. Compounds 1 and 2 (Scheme 1) were prepared
starting from the ethyl ester of 5-amino-1-(2-hydroxy-2-phe-
nylethyl)-1H-pyrazole-4-carboxylic acid 8, obtained following
| Dipartimento Farmaco Chimico Tecnologico, Universita` degli Studi
di Siena.
10.1021/jm050603r CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/09/2006

1550 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Carraro et al.
Scheme 1
our reported procedure.24 Reaction of 8 with benzoyl isothio-
cyanate in THF at reflux for 12 h yielded the intermediate 9,
which was cyclized to the pyrazolo[3,4-d]pyrimidine 10 by
treatment with 1 M NaOH at 100 °C for 10 min, followed by
acidification with acetic acid (80% yield). Alkylation of the thio
group in position 6 with methyl or ethyl iodide in THF at reflux
afforded the 6-thioalkyl derivatives 11, that were in turn treated
with the Vilsmeier complex (POCl3:DMF, 4 equiv) in CHCl3
to obtain the dihalogenated compounds 12 bearing the chlorine
atom both at the position 4 of the pyrimidine nucleus and the
N1 side chain. Compounds 12 were purified in good yield by
chromatography on silica gel column. Finally, regioselective
substitution of the C4 chlorine atom with an excess of various
amines afforded the desired compounds 1 and 2 in yields ranging
between 60 and 80%. Notably, the chlorine atom at the side
chain has never been substituted by the amine despite its
benzylic position, as shown by the 1H NMR chemical shifts of
the CH2-CH side chain, which give an ABX complex pattern
which it is similar to that of the starting material.
Reaction of 13 at reflux with POCl3 afforded the 1-styryl
derivative 14 which was reacted with an excess of various
amines in toluene to give 5a-k in good yield.
Compound 15 was prepared in a yield of 44% following the
Beal and Ve´liz25 procedure by treatment of 13 with a mixture
of HMPT/NBS in acetonitrile at -20 °C followed by addition
of LiBr and refluxing. It is interesting to point out that the se-
condary OH on the side chain remained unaltered by this proce-
dure, as shown by its 1H NMR spectrum. Treatment of 15 with
the appropriate amines gave the desired compounds 6a-g.
A number of thiomethyl derivatives 1 and thioethyl deriva-
tives 2 were treated with an excess of DBU (1,8-diazabicyclo-
[5,4,0]undec-7-ene) at 90 °C for 4 h to give the corresponding
styryl derivatives 3a-e and 4a,b in high yield (Scheme 2).
Reaction of the pyrimidinone 13 in DMF with a solution of
phosphorus tribromide, pyridine and toluene at room temperature
for 3 days led to the intermediate 16 (60% yield), bearing a
bromine atom on the N1 side chain. Compound 16 was in turn
chlorinated at C4 by treatment with the Vilsmeier complex, to
afford compound 17 (80% yield) which was reacted with an
excess of various amines (following the same conditions
described above) to obtain derivatives 7a-k in yield ranging
between 60 and 80%. Also in this case the bromine atom on
Reaction of 8 with an excess of formamide at 190 °C for 8
h afforded the pyrazolo[3,4-d]pyrimidinone 13 which was
purified by dissolving the crude in 2 M NaOH, boiling with
charcoal, followed by precipitation with acetic acid (yield 70%,
mp 271-272 °C).

Pyrazolo[3,4-d]pyrimidine Inhibitors of c-Src Phosphorylation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1551
Scheme 2
the N1 side chain was never sustituted by the amino group
(Scheme 2).
found to have an antiproliferative activity toward 8701-BC cells
comparable or higher than that of the reference compound (62
µM), with the exception of 1c, a 4-diethylamino derivative,
showing an activity of 87 µM. Moreover, with the exception
of compounds 3 and 4 (without no significant activity toward
this cell line), compounds with activity higher than that of the
reference compound were found among all the other subclasses.
Compound 1k, bearing a N1 phenylethyl side chain was
characterized by the best biological profile, being the most active
compound toward A431 cells and one of the most active
compounds, together with 2a, toward 8701-BC cells. It showed
a very good activity toward the isolated Src (0.7 µM) compa-
rable to that of PP2. Taken together, biological data showed
that many of the new pyrazolo-pyrimidines showed a compa-
rable activity toward both the cell lines, while A431 cells were
more sensitive to the treatment with PP2, in comparison to the
8701-BC cell line. With the aim of better understanding the
reason(s) of the antiproliferative activity of these new com-
pounds, some of them were selected (on the basis of their
biological profile in terms of activity toward both A431 and
8701-BC cells) to be submitted to further assays.
Inhibition of Src Phosphorylation. In particular, the inhibi-
tory effect toward the phosphorylation of Src Tyr419 (a crucial
step in the mechanism of the enzyme activation)27 was first
studied on cells treated with 100 nM EGF. Such a treatment is
usually necessary to enhance Src phosphorylation that is
generally very low. Regarding 8701-BC cells, they normally
produce VEGF and EGF, and consequently Src is phosphory-
lated even in the 8701-BC control group (21%, Figure 1, right)
(numerical values represented the percent ratio between phos-
phorylated and non phosphorylated Src). However, also for
8701-BC cells, an enhancement of Src phosphorylation (found
to rise to 51% upon EGF treatment, Figure 1, right) is required
to better appreciate the antiproliferative activity of the studied
compounds.
Results and Discussion
Compounds 1-7 were evaluated for their ability to inhibit
proliferation of both A431 and 8701-BC cells, as well as to
interfere with Src phosphorylation. Moreover, the activity of
such compounds in a cell-free assay (isolated Src) is also
reported in Table 1.
To test how A431 and 8701-BC cells grown in our laboratory
responded to well-known antiproliferative agents, 1-(tert-butyl)-
3-(4-chlorophenyl)-4-aminopyrazolo[3,4-d]pyrimidine (PP2) was
used as a reference compound, due to its potency and selectivity
in inhibiting phosphorylation of the Src family tyrosine kinase
members.19,26 As a result, IC50 of such compound toward A431
cells (32 µM) was comparable with that reported in the
literature,19 while inhibitory activity against 8701-BC cells was
about 2-fold lower (62 µM).
Cytotoxicity. The cytotoxic effect of compound was evalu-
ated by trypan blue exclusion. Cells were stimulated for 1 h at
37 °C with increasing concentrations (10 nM to 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. All the compounds
reported in Table 1 showed no citotoxicity (at the tested
concentrations) toward both the A431 and 8701-BC cell lines.
Antiproliferative Activity. Inspection of biological data
(inhibition of proliferation) suggested that compounds 1, bearing
a chlorophenylethyl side chain at N1 and a methylthio group at
the position 6 on the bicyclic nucleus, were characterized by
the best profile of activity toward A431 cells. In fact, nine of
them (75% of compounds belonging to the subclass of com-
pounds 1) showed an activity comparable or higher with respect
to the reference compound PP2 (32 µM). Moreover, among all
the novel pyrazolo-pyrimidines presented in the paper, 1k was
the most active compound toward A431 cells.
To determine whether the new pyrazolo-pyrimidine com-
pounds were able to inhibit the phosphorylation of Src at the
level of Tyr419, we used a phosphospecific anti-Src (Tyr419)
antibody (Cell Signaling Technology, Danvers, MA), with PP2
as the reference compound. Stimulation of A431 cells with EGF
significantly increased phosphorylation of Src from 26% (Figure
The new compounds were also evaluated for their ability to
inhibit proliferation of the breast cancer cell line 8701-BC, using
the same reference compound (PP2). All compounds 1 were

1552 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Carraro et al.
Table 1. Structure and Physicochemical Properties of Compounds 1-7 and Their Antiproliferative Activity toward A431 and 8701-BC Cell Lines
IC50 (µM)c
a
compd
R
R1
R2
mp (°C)
yield (%)
Ki (µM)b
8701-BC
A431
1a
1b
1c
1d
1e
1f
1g
1h
1i
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SEt
SEt
SEt
SEt
SEt
SEt
SEt
SMe
SMe
SMe
SMe
SMe
SEt
SEt
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NHPr
NHBu
N(Et)2
NH(CH2)2OEt
1-pyrrolidinyl
1-piperidinyl
4-morpholinyl
1-hexahydroazepinyl
NHcyclohexyl
NHBn
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
C
C
C
C
C
C
C
D
D
D
D
D
D
D
D
D
D
D
126-127
106-107
91-92
115-116
151-152
94-95
116-117
99-100
138-139
142-143
73-74
164-165
74-75
100-101
131-132
99-100
130-131
165-166
112-113
185-186
121-122
155-156
161-162
160-161
155-156
147-148
189-190
220-221
145-146
115-116
159-160
157-158
172-173
187-188
190-191
184-185
152-153
172-173
168-169
169-170
125-126
179-180
132-133
149-150
136-137
162-163
150-151
95-96
75
45
80
68
53
60
75
60
72
81
76
65
75
45
65
65
80
85
72
70
65
67
75
65
75
65
86
74
64
84
95
80
85
80
72
81
78
74
68
58
80
75
77
62
60
70
65
70
60
65
68
67
70
64
80
2.9 ( 0.8
1.7 ( 0.4
0.5 ( 0.1
NA
64.1 ( 1.7
63.8 ( 2.1
86.6 ( 1.2
59.8 ( 3.1
56.4 ( 1.0
67.5 ( 1.2
64.2 ( 1.8
54.4 ( 0.9
35.5 ( 1.5
53.2 ( 1.2
31.2 ( 0.5
69.9 ( 6.7
29.5 ( 0.9
38.8 ( 0.5
71.7 ( 1.9
NA
79.8 ( 3.0
43.3 ( 1.2
96.5 ( 4.1
82.4 ( 2.3
95.1 ( 1.9
NA
81.7 ( 2.4
94.2 ( 1.4
87.2 ( 2.5
NA
56.8 ( 2.4
68.0 ( 1.8
80.5 ( 6.8
43.1 ( 1.3
50.5 ( 0.7
NA
58.8 ( 0.7
49.8 ( 0.7
93.7 ( 0.6
65.1 ( 1.2
65.1 ( 2.1
NA
NA
NA
NA
NA
45.8 ( 1.5
64.5 ( 5.1
NA
89.0 ( 1.7
78.6 ( 1.5
37.4 ( 0.9
89.8 ( 3.2
NA
NA
NA
NA
63.8 ( 1.8
43.5 ( 0.8
61.8 ( 4.4
32.1 ( 0.5
27.5 ( 0.5
70.2 ( 1.4
31.6 ( 0.6
32.3 ( 0.8
38.4 ( 0.8
24.3 ( 0.4
62.0 ( 1.2
29.8 ( 1.4
24.8 ( 0.7
18.2 ( 0.9
27.6 ( 1.0
79.1 ( 1.8
40.4 ( 0.4
88.9 ( 1.7
NA
ND
2.4 ( 0.7
6.5 ( 1.0
ND
ND
1j
1k
1l
3.7 ( 0.9
0.7 ( 0.2
NA
0.7 ( 0.1
0.6 ( 0.2
ND
8.6 ( 1.3
3.6 ( 0.9
8.2 ( 1.4
NA
NA
NA
NA
NA
NA
NA
23 ( 6
ND
9.2 ( 1.4
NA
ND
NA
NA
NA
12 ( 2.5
NA
2.4 ( 0.4
NA
NA
NA
2.4 ( 0.6
NA
NA
2.4 ( 0.9
NA
0.9 ( 0.2
ND
2.8 ( 0.8
2.9 ( 1.0
2.7 ( 0.5
9.6 ( 2.2
1.6 ( 0.5
ND
ND
ND
3.3 ( 0.5
0.5 ( 0.1
NH(CH2)2Ph
4-(2,6-dimethyl)-morpholinyl
NHPr
2a
2b
2c
2d
2e
2f
2g
3a
3b
3c
3d
3e
4a
4b
5a
5b
5c
5d
5e
5f
NHBu
1-pyrrolidinyl
1-piperidinyl
4-morpholinyl
NHBn
4-(2,6-dimethyl)-morpholinyl
NHBn
NH(CH2)2Ph
1-piperidinyl
4-morpholinyl
4-(2,6-dimethyl)-morpholinyl
4-morpholinyl
4-(2,6-dimethyl)-morpholinyl
NHPr
42.3 ( 0.7
NA
50.7 ( 1.3
39.9 ( 0.6
53.9 ( 1.7
49.8 ( 0.7
35.7 ( 0.6
74.4 ( 2.8
49.8 ( 0.8
42.0 ( 1.0
61.3 ( 1.0
81.1 ( 2.2
91.4 ( 2.0
49.4 ( 2.7
62.7 ( 1.0
69.1 ( 1.2
59.1 ( 1.0
NA
NHcyclopropyl
NHBu
NH(CH2)2OEt
1-pyrrolidinyl
1-piperidinyl
4-morpholinyl
NHcyclohexyl
hexahydroazepinyl
NHBn
NH(CH2)2Ph
NHPr
1-piperidinyl
NHBn
5 g
5h
5i
60.9 ( 2.1
NA
NA
5j
5k
6a
6b
6c
6d
6e
6f
6 g
7a
7b
7c
7d
7e
7f
91.2 ( 1.4
86.4 ( 1.6
81.7 ( 3.2
82.9 ( 1.8
84.2 ( 3.7
77.1 ( 2.9
91.7 ( 2.4
60.5 ( 2.7
49.6 ( 2.4
45.9 ( 2.1
33.6 ( 2.0
40.1 ( 2.1
78.8 ( 3.2
35.1 ( 0.7
19.2 ( 0.9
28.2 ( 1.9
57.6 ( 3.1
41.7 ( 1.7
32.2 ( 0.7
NHBu
4-morpholinyl
NH(CH2)2Ph
NHCH2C6H4-p-F
NHPr
NHcyclopropyl
NHiPr
H
H
H
H
H
H
H
H
H
H
H
H
NHBu
NH(CH2)2OEt
1-pyrrolidinyl
1-piperidinyl
4-morpholinyl
NHcyclohexyl
1-hexahydroazepinyl
NHBn
109-110
127-128
144-145
111-112
154-155
124-125
157-158
7 g
7h
7i
7j
7k
PP2d
a A ) 2-chloro-2-phenylethyl, B ) styryl, C ) 2-hydroxy-2-phenylethyl, D ) 2-bromo-2-phenylethyl. b Calculated from eq 2, where E0 ) 0.0125 µM
and S0 ) 0.0160 µM. NA ) Not active (ID50 > 2 mM). ND ) Not determined. c IC50 values are means ( SEM of series separate assays, each performed
in quadruplicate. NA ) not active at 100 µM. d Since PP2 is a noncompetitive inhibitor, ID50 ) Ki.
2, control) to 55% (Figure 2, control + EGF). PP2 strongly
inhibited the stimulated cells (21% phospho/non phospho),
similarly to that found for 2b, 5a, and 5i. Interestingly,
compounds 1f, 1j, and 1k showed an inhibitory activity (8%
ratio) more pronounced with respect to PP2. Finally, 1g and 2e
were characterized by a slight, nonsignificant reduction of Src
phosphorylation, even if they showed high antiproliferative
activity (24 µM, better than PP2, and 42 µM, respectively).
Similar experiments were performed to evaluate the inhibitory
properties of compounds 1f, 1j, 1k, 2b, 2e, and 6e toward the
phosphorylation of Src on the 8701-BC cell line (Figure 1). As
previously found for A431 cells, EGF stimulation induced a

Pyrazolo[3,4-d]pyrimidine Inhibitors of c-Src Phosphorylation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1553
Figure 1. Effect of compounds 1f, 1j, 1k, 2b, 2e, and 6e on the phosphorylation of Src in 8701-BC cells, in comparison to PP2 (reference
compound). 8701-BC cells were cultured at a concentration of 2 × 104 cells/mL. (left) Lane 1: cell control; lane 2: cells treated for 5 min with
EGF (100 nM); lane 3-8: cells challenged for 3 h in the presence of 10 µM of the tested compound and then treated for 5 min with EGF (100
nM); lane 9 cells challenged for 3 h in the presence of 10 µM of PP2 and then treated for 5 min with EGF (100 nM). After 3 h, cell lysates were
obtained and analyzed. Immunoblot analysis was performed using phospho-specific antibodies to Src (Tyr419). Filters were additionally reprobed
with specific non phospho anti-Src antibodies after stripping. Results are representative of three independent experiments. (right) Quantification of
phospho and non phospho Src expression was achieved with Sigma Gel analysis software and the results represented the percent of the phospho
Scr over non phospho Src. The means ( SEM of three independent experiments are presented. A double asterisk (**) indicates a significant (p <
0.05) effect of EGF treatment (control vs control + EGF). Single asterisks (*) indicate statistically significant differences between control + EGF
and 8701-BC cells treated with specific compound + EGF. The statistical analyses were performed using Student’s t test and the Bonferroni’s
correction.
Figure 2. Effect of compounds 1f, 1g, 1j, 1k, 2b, 2e, 5a, and 5i on the phosphorylation of Src in A431 cells, in comparison to PP2 (reference
compound). A431 were cultured at a concentration of 2 × 104 cells/mL. (left) Lane 1: cell control; lane 2: cells treated for 5 min with EGF (100
nM); lane 3: cells challenged for 3 h in the presence of 10 µM PP2 and then treated for 5 min with EGF (100 nM); lane 4-11: cells challenged
for 3 h in the presence of 10 µM of the tested compound and then treated for 5 min with EGF (100 nM). After 3 h, cell lysates were obtained and
analyzed. Immunoblot analysis was performed using phospho-specific antibodies to Src (Tyr419). Filters were additionally reprobed with specific
non phospho anti-Src antibodies after stripping. Results are representative of three independent experiments. (right) Quantification of phospho and
non phospho Src expression was achieved with Sigma Gel analysis software, and results represented the percent of the phospho Scr over non
phospho Src. The means ( SEM of three independent experiments are presented. A double asterisk (**) indicates a significant (p < 0.05) effect
of EGF treatment (control vs control + EGF). Single asterisks indicate statistically significant differences between control + EGF and A431 cells
treated with specific compound + EGF. Statistical analyses were performed using Student’s t test and the Bonferroni’s correction.
marked increase of Src phosphorylation. On the other hand, PP2
showed a reduced ability to inhibit phosphorylation (about 40%
ratio), while all the compounds tested, with the exception of
6e, were characterized by inhibitory properties better than that
of the reference compound. Similarly to that found for A341
cells, compounds 1f, 1j, and 1k showed high inhibition of
phosphorylation with a phospho/non phospho ratio lower than
20%.
Studies on Isolated Src. The mechanism of cell growth
inhibition was further investigated in a cell-free assay and it
was found to be competitive with the ATP substrate but not
with a peptide used as a Src substrate.28 Several of the
compounds tested showed inhibitory activity toward the isolate
Src in the micro- and sub-micromolar range. It is worth
mentioning that some of them showed an activity in cellular
assays higher than that in the in vitro inhibition assay (Table
1). There are, at least, two possible explanations. A first one
relates to the artificial conditions of the enzymatic assay. In
fact, it is well-known that in vivo the activity of Src is modulated
by its cellular context, as well as by the different substrates.29
Upon binding to a substrate, Src is subjected to extensive
conformational changes. Moreover, the presence of specific
phosphorylated residues in the substrate plays an important role
in attracting Src and stabilizing its binding. In our in vitro
system, we used an artificial substrate, namely a short peptide.
It is possible, then, that the catalytic efficiency, as well as the
conformation of the enzyme, were suboptimal under our in vitro
conditions, with respect to the physiological context of an intact
cell. This is likely reflected by the rather low affinity (Km ) 30
µM) displayed by the recombinant enzyme for the peptidic
substrate and could influence also the affinity of the enzyme
for our inhibitors. Indeed, the activity of the reference compound
PP2 was found to be 0.5 µM, whereas data in the literature
report a lower activity. Another possible explanation for the
higher activities detected in cell-based assays is the possibility
that our compounds target also other kinases. In fact, preliminary

1554 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Carraro et al.
Figure 3. Cyclin A inhibition. 8701-BC cells were cultured at a concentration of 2 × 104 cells/mL in the absence and in the presence of 10 µM
of the tested compound for 3 h at 37 °C, in comparison to PP2 used as the reference compound. Cell lysates from control and treated 8701-BC cells
were obtained and analyzed by RNAse protection assay. L32 was used as a housekeeping gene. Quantification of cyclin A expression was achieved
with Sigma Gel analysis software, and the results, representing the percentage of inhibition over control, are presented (right).
Figure 4. Proapoptotic effect of compounds 1f, 1g, 1j, 1k, 2b, 2e, 5a, and 5i measured by PARP cleavage, in comparison to PP2 (reference
compound). A431 cells were cultured at a concentration of 2 × 104 cells/mL. (left) Lane 1: cell control; lane 2: cells challenged for 3 h with 10
µM PP2; lane 3-10: cells challenged for 3 h in the presence of 10 µM of the tested compound. After 3 h, cell lysates were obtained and analyzed.
Immunoblot analysis was performed using PARP-specific antibodies to both the uncleaved (113 kDa) and cleaved (89 kDa) form of parp. (right)
Quantification of PARP expression was achieved with Sigma Gel analysis software, and the results, representing the percent of cleaved over
uncleaved PARP, are expressed as means ( SEM of three independent experiments. Asterisks indicate statistically significant differences (p <
0.05) between control and A431 cells treated with the tested compound. Statistical analyses were performed using Student’s t test and the Bonferroni’s
correction.
experiments showed that several of the new compounds were
active also against other nonreceptor, or cytoplasmic, tyrosine
kinases and demonstrate activity toward malignant cells ex-
pressing c-kit.30 Nevertheless, we are still unable to rule out if
such compounds, in addition to be competitive for ATP, could
or not act through a mixed-mode of Src inhibition or interact
with an allosteric site on Src.31
Studies on Cyclins. We were also interested in investigating
wether the inhibition of c-Src could result in an alteration of
the cyclins. Cell cycle progression through the G1 phase is
regulated by the cyclin dependent kinase (CDK) 4 and CDK2
activities under the control of cyclins D (namely, D1, D2 and
D3) and E, while cyclins A and B are considered important
mitotic agents and their expression is necessary for cells to enter
S phase and progress from G2 into mitosis.32 Using the
ribonuclease protection assay (RPA) technique, we demonstrated
that the inhibition of Src was sufficient to reduce the cyclin
mRNA expression on 8701-BC cell line. In particular, com-
pounds 1k, 2b, and PP2 were able to reduce cyclin A expression
(Figure 3).
8701-BC cell lines, we also tested their proapoptotic activity
on a Poly-ADP-Ribose-Polymerase (PARP) assay (Figures 4
and 5). PARP is a 113 kDa zinc-dependent protein that binds
specifically at DNA strand breaks produced by various genotoxic
agents.33 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
89 and 24 kDa. Thus, detection of the 89 kDa PARP fragment
with specific anti-PARP antibodies represents an early marker
for apoptosis.34 Figure 4 shows that compounds 1k and 2b
potently induced apoptosis in A431 cells (48% of cleaved/
uncleaved PARP ratio), while 1j and 2e showed lower, but
significant, proapoptotic activity (32 and 21% ratio, respec-
tively). All the remaining compounds, including PP2, were
characterized by an unsignificant proapoptotic activity, com-
parable to that found in the control (6 to 12% ratio). As for
8701-BC cells is concerned, the cleaved/uncleaved PARP ratio
of the control was about 23% (Figure 5), while proapoptotic
activity of all the tested inhibitors was comparable each other.
In fact, while 2b was the most interesting compound (43%),
the proapoptotic activity of the remaining compounds was found
to be in the range between 28% (1j) and 38% (PP2).
Proapoptotic Activity. Prompted by the ability of such
compounds in reducing Src phosphorylation of both A431 and

Pyrazolo[3,4-d]pyrimidine Inhibitors of c-Src Phosphorylation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1555
Figure 5. Proapoptotic effect of the compounds 1f, 1j, 1k, 2b, and 6e measured by PARP cleavage in 8701-BC cells, in comparison to PP2
(reference compound). 8701-BC cells were cultured at a concentration of 2 × 104 cells/mL. (left) Lane 1: cell control; lane 2: cells challenged for
3 h with 10 µM of PP2; lane 3-7: cells challenged for 3 h in the presence of 10 µM of the tested compound. After 3 h, cell lysates were obtained
and analyzed. Immunoblot analysis was performed using PARP-specific antibodies to both the uncleaved (113 kDa) and cleaved (89 kDa) form of
parp. (right) Quantification of PARP expression was achieved with Sigma Gel analysis software and the results, representing the percent of cleaved
over uncleaved PARP, are expressed as means ( SEM of three independent experiments. Asterisks indicate statistically significant differences (p
< 0.05) between control and 8701-BC cells treated with specific compound. The statistical analyses were performed using Student’s t test and the
Bonferroni’s correction.
detail, in the first step, the three-dimensional structure of c-Src
in an active conformation (entry 1Y57 of the Brookhaven
Protein Data Bank)36 was relaxed through an energy minimiza-
tion routine (all-atom Amber* force field, Polack-Ribiere
conjugate gradient method, continuum solvation with water,
extended cutoffs, convergence at 0.01 kJ/mol‚Å) to avoid any
steric bump eventually occurred in the X-ray structure. Next,
the software Autodock3.037 was chosen as the tool to perform
docking simulations of PP2 onto the Src structure. As a result,
within the first and most populated cluster of orientations/
conformations ranked by the software, PP2 showed an orienta-
tion with the network of hydrogen bond contacts (involving
Met341 and Glu339) and hydrophobic interactions reported in
a theoretical model previously published,38 and also in agreement
Figure 6. Inhibition of the anti apoptotic gene BCL2. 8701-BC cells
were cultured at a concentration of 2 × 104 cells/mL in the absence
and in the presence of 10 µM of the tested compound for 3 h at 37 °C,
in comparison to PP2, used as the reference compound. Cell lysates
from control and treated 8701-BC cells were obtained and analyzed
by RNAse protection assay. L32 was used as a housekeeping gene.
Quantification of BCL2 expression was achieved with Sigma Gel
analysis software and the results, representing the percentage of
inhibition over control, are presented.
with the binding orientation and interactions of PP2 onto the
Lck structure39 (the template structure usually used to model
by homology the structure of Src in its active form). Taken
together, these results led us to hypothesize that the computa-
tional approach can be considered as a reliable modeling
procedure to be applied for finding the orientations and
interactions of ligands inside the kinase domain of Src. In
addition, to further assess the reliability of Autodock results,
we have performed additional simulations by means of the
software MacroModel.40 In detail, we have applied a docking
protocol that allows for rotation and translation of the inhibitor
within the kinase domain of Src. Moreover, all the rotatable
bonds of the inhibitor were submitted to a statistical confor-
mational search according to a procedure previously reported.41
As a result, the best conformational model was very similar to
the first orientation found by Autodock, with slight differences
in the orientation of both N1 and C4 side chains (the root-mean-
square deviation calculated on all the heavy atoms was found
to be 0.32 Å).
Once the reliability of the docking procedure was tested, the
interaction pathway between inhibitors and Src was simulated
applying the same protocol used for PP2. Simulations allowed
for the identification of two different binding modes, depending
on the presence or on the absence of an alkylthio substituent at
position 6 of the pyrazolo-pyrimidine ring.
In general, compounds unsubstituted at position 6 showed
an interaction pathway similar to that of PP2 (Figure 7). In
particular, both N5 and the amino group at C4 are involved in
two hydrogen bonds with the NH and the carbonyl backbone
groups of Met341, respectively, similarly to that found for PP2
Studies on BCL2. Finally, on the basis of the ability of some
of these compounds to induce apoptosis in both cell lines, we
also investigated the expression of BCL2 mRNA by RPA. BCL2
is an integral membrane 26 kDa protein which, when overex-
pressed, especially in tumor cell lines, blocks apoptosis.35 The
inhibition of the anti apoptotic gene BCL2 (Figure 6) could help
in explaining the molecular mechanism of apoptosis induction.
Molecular Docking Simulations. Molecular modeling simu-
lations have been performed to hypothesize the way by which
the inhibitors bind Src and to gain insight on the peculiar
chemical features that influenced their activity toward the
isolated enzyme. In detail, the new pyrazolo-pyrimidines were
found to inhibit Src phosphorylation on both A431 and 8701-
BC cell lines, as well as to be ATP-competitive inhibitors in a
cell-free assay (isolated Src). Based on this experimental
evidence, molecular docking simulations followed by optimiza-
tion of the resulting complexes between Src and its inhibitors
have been performed. To this aim, the reliability of the docking
protocol was first checked by simulation of the interactions
between PP2 and Src and comparison of the modeled complexes
with the experimental and theoretical data already available. In

1556 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Carraro et al.
responsible for a reorientation of the heterocyclic nucleus that
approached the hydrophobic region I, also occupied by C4
substituents. On the other hand, N1 substituents were partially
inserted into the adenine region and directed toward the
hydrophobic pocket II. Methyl- or ethylthio groups were located
into an additional hydrophobic region and were involved in
favorable interactions with Leu393 and Ala403. Differently from
PP2 and 6-unsubstituted compounds, the structure of 6-alkylthio
derivatives was not engaged in any hydrogen bond. Interestingly,
the lack of the hydrogen bond network was in general not
associated with inactivity, as shown in Table 1. In fact, despite
the different orientation and the absence of any hydrogen bond,
several compounds bearing an alkylthio substituent at position
6 were endowed with an inhibitory activity comparable to C6
unsubstituted compounds. As an example, 1c was the most
active compound of the whole set. Thus, docking studies
suggested that hydrogen bond contacts were not essential for
the activity of 6-alkylthio pyrazolo-pyrimidine derivatives. These
findings were also in agreement with the fact that several
6-unsubstituted compounds, unable to establish a hydrogen bond
donor-acceptor network due to the lack of a NH group at C4
(i.e., 7f and 7g), maintained a good inhibitory activity. Alter-
natively, one might suppose that 6-alkylthio derivatives coun-
terbalanced the loss of hydrogen bond interactions with addi-
tional hydrophobic interactions involving the alkylthio side
chains. In this context, computational studies showed that
lipophilic interactions resulting from full occupancy of hydro-
phobic regions I and II seemed to play a crucial role for the
activity of ligands. On this basis, the lack of activity that affected
most compounds with a hydroxy side chain at N1 could be
accounted by the unsatisfactory filling of the hydrophobic pocket
I, due to the presence of a hydrophilic hydroxy group on the
N1 side chain.
Finally, it is worth noting that compounds with a phenyl-
ethylamino substituent at position 4 showed a peculiar behavior,
being located with a common orientation, independent from the
presence of an alkylthio substituent at position 6 (Figure 7). In
fact, such compounds disposed their pyrazolo-pyrimidine nucleus
in the adenine region, the phenylethylamino moiety in the
hydrophobic region I, and the C4 side chain in the hydrophobic
region II. The alkylthio substituent, when present, lay in the
adenine region, without altering the orientation of the hetero-
cyclic ring.
Figure 7. Graphical representation of the binding mode of the new
compounds into the ATP binding site of the active form of Src, in
comparison to the orientation of PP2 (green). For sake of clarity, only
a few residues are displayed, nonpolar hydrogen atoms are omitted,
and hydrogen bond interactions are represented by black dashed lines.
(A) The best conformer of 7a (orange) is able to make a network of
hydrogen bonds with the receptor counterpart, similar to that of PP2.
(B) Compound 1c (magenta) in its best orientation lacks the network
of hydrogen bond with amino acid residues. (C) The orientation of 6f
(cyan) into the Src binding site is the same found for all compounds
bearing a phenylethylamino side chain at C4.
Conclusions
The synthesis of a series of pyrazolo[3,4-d]pyrimidine deriva-
tives along with their inhibition properties toward Src in a cell-
free assay, as well as their antiproliferative activity toward the
A431 and BC-8701 cell lines, was reported, demonstrating that
several compounds were more active than PP2, chosen as the
reference compound. In detail, the studied compounds were
found to block Src phosphorylation, induce apoptosis, and
reduce cell proliferation, the activation of Src being an important
step in the progression of cancer. Biological data reported here
when correlated to the structural properties of the compounds
allowed the identification of key elements for the synthesis of
new compounds with enhanced capacity to inhibit Src phos-
phorylation. In fact, an N1 chlorophenyl ethyl side chain in
combination with a 6-methylthio group seemed to be the optimal
combination of chemical features for antiproliferative activity.
On the other hand, substituents at the position 4 of the
heterocyclic core appeared to be only responsible for the fine-
tuning of the antiproliferative activity. The new compounds may
play an important role in the field of anticancer agents, because
that interacted with Met341 and Glu339. Moreover, the N1 side
chain of the new compounds, superposed to the p-chlorophenyl
group of PP2, was located into the hydrophobic pocket I,
interacting with Thr338, Lys295, Val281, Met314, Val323,
Ala293, and Phe405. On the other hand, substituents at C4,
similarly to the tert-butyl moiety of PP2, are accommodated
within the hydrophobic pocket II, showing favorable lipophilic
contacts with Gly344, Tyr340, and Leu273. Finally, the
heterocyclic cores shared a common location within the adenine
region of the ATP binding site.
In the alternative binding mode, compounds bearing an
alkylthio substituent at position 6, although located within the
kinase domain, showed a different orientation with respect to
6-unsubstituted compounds described above and PP2 (Figure
7). In particular, the alkylthio substituent seemed to be

Pyrazolo[3,4-d]pyrimidine Inhibitors of c-Src Phosphorylation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1557
1H, CHO), 7.26-7.47 (m, 5H Ar), 8.10 (s, 1H, H-3). IR cm-1 3470
(NH), 3450-3140 (OH), 1670 (CO). Anal. (C15H16N4O2S) C, H,
N, S.
they affect a selective pathway that is crucial for the growth of
transformed cells. In particular, such compounds showed an
important in vitro activity toward 8701-BC cells, suggesting
that some of them could be effective agents in the chemotherapy
armamentarium against breast cancer (for example, in the
multimodality therapies, targeting multiple biological mecha-
nisms)42,43 and could satisfy the need of new molecules to be
used as therapeutical tools for many patients eventually devel-
oping resistant cells in the course of their disease.
4-Chloro-1-(2-chloro-2-phenylethyl)-6-(methylthio)-1H-pyra-
zolo[3,4-d]pyrimidine (12a). The Vilsmeier complex, previously
prepared from POCl3 (6.13 g, 40 mmol) and anhydrous dimethyl-
formamide (DMF) (2.92 g, 40 mmol) was added to a suspension
of 11a (3.02 g, 10 mmol) in CHCl3 (20 mL). The mixture was
refluxed for 4 h. The solution was washed with H2O (2 × 20 mL),
dried (MgSO4), filtered, and concentrated under reduced pressure.
The crude oil was purified by column chromatography (silica gel,
100 mesh), using a mixture of diethyl ether/petroleum ether (bp
40-60 °C) (1:1) as the eluant, to afford the pure product 12a (2.2
Experimental Section
Chemistry. Starting materials were purchased from Aldrich-Italia
(Milan, Italy). Melting points were determined with a Bu¨chi 530
apparatus and are uncorrected. IR spectra were measured in KBr
with a Perkin-Elmer 398 spectrophotometer. 1H NMR spectra were
recorded in a (CD3)2SO solution on a Varian Gemini 200 (200
MHz) instrument. Chemical shifts are reported as δ (ppm) relative
1
g, 65%) as a white solid, mp 95-96 °C. H NMR: δ 2.62 (s, 3H,
CH3S), 4.77-5.05 (m, 2H, CH2N), 5.45-5.56 (m, 1H, CHCl),
7.29-7.46 (m, 5H Ar), 8.02 (s, 1H, H-3). Anal. (C14H12N4SCl2)
C, H, N, S.
4-Chloro-1-(2-chloro-2-phenylethyl)-6-(ethylthio)-1H-pyrazolo-
[3,4-d]pyrimidine (12b). Compound 12b was prepared according
to the synthetic sequence described for compound 12a, starting from
11b, to afford the pure product 12b (2.12 g, 60%) as a white solid,
1
to TMS as internal standard, J in Hz. H patterns are described
using the following abbreviations: s ) singlet, d ) doublet, t )
triplet, q ) quartet, sx ) sextect, m ) multiplet, br ) broad. All
compounds were tested for purity by TLC (Merk, Silica gel 60
1
mp 89-90 °C. H NMR: δ 1.49 (t, J ) 7.2, 3H, CH3), 3.23 (q, J
) 7.2, 2H, CH2S), 4.75-5.03 (m, 2H, CH2N), 5.43-5.57 (m, 1H,
CHCl), 7.28-7.47 (m, 5H Ar), 8.04 (s, 1H, H-3). Anal. (C15H14N4-
SCl2) C, H, N, S.
F
254, CHCl3 as eluant). Analyses for C, H, N were within (0.3%
of the theoretical value.
Ethyl 5-{[(Benzoylamino)carbonothioyl]amino}-1-(2-hydroxy-
2-phenylethyl)-1H-pyrazole-4-carboxylate (9). A suspension of
8 (2.7 g, 10 mmol) and benzoyl isothiocyanate (1.7 g, 11 mmol) in
anhydrous tetrahydrofuran (THF) (20 mL) was refluxed for 12 h.
The solvent was evaporated under reduced pressure; the oily residue
crystallized by adding diethyl ether (30 mL) to afford the pure
General Procedure for Compounds 1a-l and 2a-g. To a
solution of 12a or 12b (10 mmol) in anhydrous toluene (20 mL)
was added the appropriate amine (40 mmol), and the reaction
mixture was stirred at room temperature for 24 h. The mixture was
washed with H2O (2 × 10 mL), and the organic phase was dried
(MgSO4), filtered and evaporated under reduced pressure; the oily
residue crystallized by adding petroleum ether (bp 40-60 °C) (10
mL), to give the final products 1a-l and 2a-g.
1
product 9 (4.07 g, 93%) as a white solid; mp 171-172 °C. H
NMR: δ 1.29 (t, J ) 7.0, 3H, CH3), 3.97-4.20 (m, 5H, 2CH2 +
OH, 1H disappears with D2O), 4.58-4.68 (m, 1H, CHO), 7.05-
7.98 (m, 10H Ar), 8.02 (s, 1H, H-3), 8.70 (s, 1H, NH, disappears
with D2O), 12.05 (s, 1H, NH, disappears with D2O). IR cm-1: 3221
(NH), 3190-2940 (OH), 1708 and 1671 (2 CO). Anal. (C22H22-
N4O4S) C, H, N, S.
1-(2-Hydroxy-2-phenylethyl)-1,5-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one (13). A suspension of 8 (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 product was filtered and
purified by dissolving in 2 M NaOH (100 mL) and boiling with
charcoal, followed by precipitation with glacial acetic acid. The
solid was filtered and recrystallized from absolute ethanol to give
1-(2-Hydroxy-2-phenylethyl)-6-thioxo-1,5,6,7-tetrahydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (10). A solution of 9 (4.38 g, 10
mmol) in 2 M NaOH (40 mL) was refluxed for 10 min and
successively diluted with H2O (40 mL). The solution was acidified
with glacial acetic acid. After being stored in a refrigerator for 12
h, a solid crystallized and was filtered and recrystallized from
absolute ethanol to give 10 (2.3 g, 80%) as a white solid; mp 264-
1
13 (1.79 g, 70%) as a white solid: mp 270-271 °C. H NMR: δ
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
(C13H12N4O2) C, H, N.
: 3400 (NH), 3245-2900 (OH), 1740 (CO). Anal.
1
265 °C. H NMR: δ 4.15-4.30 and 4.55-4.72 (2m, 2H, CH2N),
4.85-5.00 (m, 1H, CHO), 5.66 (br s, 1H, OH, disappears with
D2O), 7.20-7.51 (m, 5H Ar), 8.02 (s, 1H, H-3), 12.20 (s, 1H, NH,
disappears with D2O), 13.40 (s, 1H, NH, disappears with D2O). IR
cm-1: 3362 (NH), 3242-2973 (OH), 1681 (CO). Anal. (C13H12-
N4O2S) C, H, N, S.
4-Chloro-1-(2-phenylvinyl)-1H-pyrazolo[3,4-d]pyrimidine (14).
POCl3 (14 g, 91 mmol) was added to 13 (2.56 g, 10 mmol) and
the mixture was refluxed for 12 h and then cooled to room
temperature. 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 chromatography (Florisil 100-200
mesh), using CHCl3 as the eluant to afford the pure product 14
1-(2-Hydroxy-2-phenylethyl)-6-(methylthio)-1,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (11a). A solution of 10 (2.88 g,
10 mmol) and methyl iodide (7.10 g, 50 mmol) in anhydrous THF
(20 mL) was refluxed for 12 h. The solvent and the excess of methyl
iodide were removed by distillation under reduced pressure; the
oily residue crystallized by adding CHCl3 (10 mL) and was purified
by recrystallization with absolute ethanol to give 11a (2.17 g, 72%)
as a white solid; mp 208-209 °C. 1H NMR: δ 2.51 (s, 3H, CH3S),
4.27-4.50 (m, 2H, CH2N), 5.04-5.18 (m, 1H, CHO), 5.68 (d, 1H,
OH, disappears with D2O), 7.20-7.42 (m, 5H Ar), 7.97 (s, 1H,
H-3). IR cm-1: 3544 (NH), 3450-3100 (OH), 1678 (CO). Anal.
(C14H14N4O2S) C, H, N, S.
1-(2-Hydroxy-2-phenylethyl)-6-(ethylthio)-1,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (11b). Compound 11b was pre-
pared according to the synthetic sequence described for compound
11a, by using ethyl iodide as alkylating agent. In this case, the
crude product was purified by column chromatography (silica gel,
100 mesh) using a mixture of CHCl3:MeOH (9:1) as the eluant, to
afford the pure product 11b (2.05 g, 65%) as a light yellow solid:
mp 199-200 °C. 1H NMR: δ 1.45 (t, J ) 7.4, 3H, CH3), 3.22 (q,
J ) 7.4, 2H, CH2S), 4.44-4.68 (m, 2H, CH2N), 5.21-5.30 (m,
1
(1.66 g, 65%) as a white solid: mp 139-140 °C. H NMR: δ
7.25-7.61 (m, 6H, 5H Ar + CHd), 8.04 (d, Jtrans ) 14.6, 1H,
CHd), 8.29 (s, 1H, H-3), 8.85 (s, 1H, H-6). IR cm-1: 1658 (Cd
C). Anal. (C13H9N4Cl) C, H, N.
General Procedure for Compounds 5a-k. Compounds 5a-k
were prepared according to the synthetic sequence described for
compounds 1 and 2 starting from the intermediate 14.
2-(4-Bromo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-1-phenyletha-
nol (15). To a cold (-20 °C) suspension of 13 (2.56 g, 10 mmol)
and NBS (7.12 g, 40 mmol) in dry acetonitrile (20 mL) was added
hexamethylphosphorus triamide (HMPT) (6.53 g, 40 mmol) drop-
wise. After addition, the cold bath was removed and the reaction
mixture stirred at room temperature for 0.5 h. LiBr (3.44 g, 40
mmol) was added, and the mixture was heated at 70 °C for 5 h.
The mixture was evaporated under reduced pressure. The dark oil

1558 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Carraro et al.
was purified by column chromatography (silica gel, 100 mesh) using
the classical Cheng-Prusoff relationship was not applicable.
Instead, Ki values were calculated according to eq 2: Ki ) (ID50
- E0/2)/{E0 - [S0/Km - 1]/E0}, where S0 is the concentration of
the competing substrate (ATP), and E0 is the concentration of the
enzyme. Each experiment was in triplicate and mean values were
used for the interpolation. Curve fitting was performed with the
program GraphPad Prism.
CHCl3 as the eluant, to afford the pure product 15 (1.40 g, 44%)
1
as a white solid: mp 146-147 °C. H NMR: δ 3.46-3.53 (br s,
1H, OH, disappears with D2O), 4.57-4.79 (m, 2H, CH2N), 5.18-
5.29 (m, 1H, CHO), 7.30-7.52 (m, 5H Ar), 8.01 (s, 1H, H-3),
8.48 (s, 1H, H-6). IR cm-1: 3240-2500 (OH). Anal. (C13H11N4-
OBr) C, H, N.
Cell Culture. Human epidermoid carcinoma A431 cells44 were
obtained from the American Type Culture Collection and were
grown in Dulbecco’s modified Eagle’s medium containing 10%
fetal calf serum. The highly metastatic 8701-BC breast cancer cell
line45 (kindly provided by Prof. I. Pucci-Minafra, University of
Palermo, Italy) was maintained as monolayer cultures in RPMI 1640
medium (BioWhittaker, Vallensbaek, DK) containing 10% fetal calf
serum. 8701-BC cell line deserves particular attention, since it 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.21 The cultures
were free of Mycoplasma. For proliferation assay cells line (2 ×
104 cells/mL) were incubated in 100 µL of RPMI 1640 culture
medium (BioWhittaker, Vallensbaek, DK), supplemented with 10%
fetal calf serum (FCS; BioWhittaker, Vallensbaek, DK) and
antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), at
37 °C in 5% CO2 for 8 h, to allow adhesion. After adhesion, the
medium was replaced with 100 µL of medium supplemented with
0.5% FCS. After an overnight incubation, the spent medium was
then removed, and the cultures were refreshed with new medium
(100 µL of RPMI 1640 with 10% FCS) or medium along with
different concentrations (0-100 µM) of the studied compounds.
After 3 days (control cultures did not reach confluence), the
antiproliferative effect of the compounds was determined by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) pro-
liferation assay.46 Briefly, cells were treated with 10 µL of the MTT
solution (5 mg/mL). Four h later, 100 µL of acid propan-2-ol (0.04
M HCl in propan-2-ol) were added to dissolve the formazan product.
The microplates were read using an ELISA plate reader at 570 nm
with a reference wavelength of 630 nm. Data analysis for IC50
calculations was performed with the LSW Data Analysis Package
plug-in for Excel (Microsoft). Result are reported as mean ( SEM
of five experiments performed in triplicate.
General Procedure for Compounds 6a-g. Compounds 6a-g
were prepared according to the synthetic sequence described for
compound 1, 2 and 5 starting from the intermediate 15.
General Procedure for Compounds 3a-e and 4a,b. DBU (5
g, 33.44 mmol) was added to 1j, 1k, 1f, 1g, 1l, 2e, and 2g (10
mmol). The mixture was heated at 90 °C for 8 h. Absolute ethanol
(5 mL) was added to give the crudes (3a-e and 4a,b, respectively),
which were then recrystallized from absolute ethanol.
1-(2-Bromo-2-phenylethyl)-1,5-dihydro-4H-pyrazolo[3,4-d]py-
rimidin-4-one (16). To a suspension of 13 (2.56 g, 10 mmol), in
anhydrous DMF (20 mL), cooled at 0° C, was added a solution of
phoshorous tribromide (3.27 g, 12,1 mmol), pyridine (0.5 mL) and
toluene (5 mL) previously prepared, dropwise. The mixture was
stirred at r.t. for 3 days. The solvent was removed under reduced
pressure, and the residue was poured in water and ice (200 mL).
The solid was filtered and recrystallized from absolute ethanol, to
give the pure product 16 (1.91 g; 60%), as a white solid; mp 230-
1
231 °C. H NMR: δ 4.80-4.97 and 5.01-5.19 (2m, 2H, CH2N),
5.68-5.72 (m, 1H, CHBr), 7.30-7.65 (m, 5H Ar), 7.97 (s, 1H,
H-3), 8.13 (s, 1H, H-6), 12.25 (br s, 1H, NH, disappears with D2O).
IR cm-1: 3440 (NH), 1665 (CdO). Anal. (C13H11N4BrO) C, H,
N.
1-(2-Bromo-2-phenylethyl)-4-chloro-1H-pyrazolo[3,4-d]pyri-
midine (17). The Vilsmeier complex, previously prepared from
POCl3 (15.3 g, 100 mmol) and anhydrous dimethylformamide
(DMF) (7.3 g, 100 mmol) was added to a suspension of 16 (3.19
g, 10 mmol) in CHCl3 (50 mL). The mixture was refluxed for 12
h. The solution was washed with H2O (2 × 20 mL), dried (MgSO4),
filtered, and concentrated under reduced pressure. The crude oil
was purified by column chromatography (Florisil, 100 mesh), using
Et2O as eluant, to afford the pure product 17 (2.63 g, 78%) as a
1
white solid, mp 114-115 °C. H NMR: δ 4.95-5.10 and 5.18-
5.38 (2m, 2H, CH2N), 5.58-5.78 (m, 1H, CHBr), 7.25-7.70 (m,
5H Ar), 8.17 (s, 1H, H-3), 8.77 (s, 1H, H-6). Anal. (C13H10N4-
BrCl) C, H, N.
Western Blot. The inhibitory effects of compounds toward the
phosphorylation of Src (Tyr419) were evaluated using immunoblot
analyses. Cell lines were cultured at a concentration of 2 × 104
cells/mL, challenged with the compounds (10 µM) for 3 h, and
then treated with 100 nM EGF (Cell Signaling Technology). Five
minutes later, cells were harvested and lysed in an appropriate buffer
containing 1% Triton X-100. Proteins were quantitated by the BCA
method (Pierce, Rockford, IL). Equal amounts of total cellular
protein were resolved by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose filters, and subjected to immunoblot
using phospho-specific antibodies against Src (Y419) (Cell Signal-
ing Technology). Filters were additionally reprobed with specific
non phospho anti-Src antibodies (Cell Signaling Technology) after
stripping. Quantification of phospho and non phospho Src expres-
sion was achieved with Sigma Gel analysis software, and results
represented the percent of the phospho Scr over non phospho Src.
The means ( SEM of three independent experiments are presented.
A double asterisk (**) in the right portion of Figures 1, 2, 4, and
5 indicates a significant (p < 0.05) effect of EGF treatment (control
vs control + EGF). Single asterisks indicate statistically significant
differences between control + EGF and cell lines treated with
specific compound + EGF. Statistical analyses were performed
using Student’s t test and the Bonferroni’s correction. The proapo-
ptotic activity of some of the compounds was also tested, using a
Poly-ADP-Ribose-Polymerase (PARP) assay (Roche Diagnostics,
Milano, Italy). Cell lines were cultured at a concentration of 2 ×
104 cells/mL and challenged with the compounds (10 µM). Three
hours later, cells were harvested and lysed as reported above.
Immunoblot analysis was performed using PARP-specific antibodies
to both the uncleaved (113 kDa) and cleaved (89 kDa) form of
General Procedure for the 4-Amino-Substituted 1-(2-Bromo-
2-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidines (7a-k). To a so-
lution of 17 (1.69 g, 5 mmol) in anhydrous toluene (20 mL) was
added the proper amine (20 mmol), and the reaction mixture was
stirred at room temperature for 36 h. After it was extracted with
H2O (2 × 20 mL), the organic phase was dried (MgSO4), filtered,
and concentrated under reduced pressure; the oily residue crystal-
lized by adding Et2O (10 mL) to give products 7a-k.
Biology. Enzymatic Assay. Recombinant human Src was
purchased from Upstate (Lake Placid, NY). Activity was measured
in a filter-binding assay using a commercial kit (Src Assay Kit,
Upstate), according to the manufacturer’s protocol, using 150 µM
of the specific Src peptide substrate (KVEKIGEGTYGVVYK) and
in the presence of 0.125 pmol of Src and 0.160 pmol of [γ-32P]-
ATP. The apparent affinity (Km) values of the Src preparation used
for its peptide and ATP substrates were determined separately and
found to be 30 µM and 5 µM, respectively.
Kinetic Analysis. Dose-response curves where generated by
fitting the data by computer simulation to eq 1: E(%) ) Emax/(1 +
[I]/ID50), where E(%) is the fraction of the enzyme activity measured
in the presence of the inhibitor, Emax is the activity in the absence
of the inhibitor, [I] is the inhibitor concentration, and ID50 is the
inhibitor concentration at which E(%) ) 0.5Emax
.
The ID50 were converted to Ki according to a competitive
mechanism with respect to the substrate ATP. The second substrate
of the reaction (the peptide) was kept at saturating concentrations
(4-fold higher its Km). Since (i) the ATP concentration was limiting,
i.e., [ATP] , Km(ATP), and (ii) the enzyme concentration was not
negligible with respect to the ATP concentration, i.e., [E] g [ATP],

Pyrazolo[3,4-d]pyrimidine Inhibitors of c-Src Phosphorylation
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1559
Table 2.
outlev 1
diagnostic output level
rmstol 1.5
cluster_tolerance/A
extnrg 1000.0
external grid energy
e0max 0.0 10000
ga_pop_size 200
ga_num_evals 1500000
ga_num_generations 50000
ga_elitism 1
ga_mutation_rate 0.02
ga_crossover_rate 0.8
ga_window_size 10
ga_cauchy_alpha 0.0
ga_cauchy_beta 1.0
set_ga
sw_max_its 300
sw_max_succ 4
sw_max_fail 4
sw_rho 1.0
sw_lb_rho 0.01
ls_search_freq 0.06
set_psw1
max initial ernergy; max number of retries
number of individuals in population
maximum number of energy evaluations
maximum number of generations
number of top individuals to survive to next generation
rate of gene mutation
rate of crossover
Alpha parameter of Cauchy distribution
Beta parameter Cauchy distribution
set the above parameters for GA or LGA
iterations of Solis & Wets local search
consecutive successes before changing rho
consecutive failures before changing rho
size of local search space to sample
lower bound on rho
probability of performing local search on individual
set the above pseudo-Solis & Wets parameters
do this many hybrid GA-LS runs
ga_run 250
analysis
perform a ranked cluster analysis
PARP. Quantification of PARP expression was achieved with
Sigma Gel analysis software, and the results, representing the
percent of cleaved over uncleaved PARP, are expressed as means
( SEM of three independent experiments. Asterisks indicate
statistically significant differences (p < 0.05) between control and
cells lines treated with the tested compound. Statistical analyses
were performed using Student’s t test and the Bonferroni’s
correction.
(0.001 kJ/mol‚Å convergence or 10 000 iterations). To be exported
to Autodock, the minimized structures were converted into the mol2
file format using Babel software. Molecular docking and dynamics
calculations were performed with MacroModel software.
To remove unfavorable contacts, a preliminary structure opti-
mization was performed on the X-ray crystallographic structure of
the active conformation of c-Src (entry 1y57 of the Brookhaven
Protein Data Bank, 1.9 Å resolution)36 through the all-atom Amber*
force field and Polak-Ribiere conjugate gradient method. A
continuum solvation method, with water as the solvent, was also
applied. Extended cutoffs were used and convergence was set to
0.01 kJ/mol‚Å. The output structure of Src was imported in
Autodock Tools and prepared for docking simulations. To prepare
the input structures for Autodock calculations, the Src structure
was further manipulated by removing nonpolar hydrogens, while
Kollman united-atom partial charges and solvent parameters were
added. Similarly to the protein, the structure of the inhibitors was
also prepared by deleting their nonpolar hydrogen atoms and adding
Gasteiger atomic charges. Finally, the rigid root and rotatable bonds
were defined using AutoDockTools.
During the next step, several atom probes (characterized by the
same stereoelectronic properties as the atoms constituting the
inhibitor) were moved on the grid nodes, while the interaction
energy between the probe and the inhibitor was calculated at each
node. In such a way, grid maps can be generated for each atom
probe, describing its interactions with the inhibitor. Autogrid 3.0,
as implemented in the Autodock 3.0 software package, was used
to generate grid maps. The Lamarckian genetic algorithm (LGA)37
was employed to generate orientations/conformations of the ligand
within the binding site. Parameters for blind docking runs are shown
in Table 2 (imported from a docking parameter file), on the basis
of literature suggestions.48The choice of the best conformation was
based on the assumption that, although for high throughput
screening protocols, only the first ranked conformation should be
considered (that is, the conformation characterized by the lowest
estimated free energy of binding);49 in other cases, the lowest energy
conformation of the most populated cluster should be also taken
into account.50
Ribonuclease Protection Assay (RPA). The mRNA expression
of cyclins and BCL2 gene were performed using RPA. mRNA
expression was determined using panels of commercially available
probes (Multiprobe Ribonuclease Protection Assay, Pharmingen,
San Diego, CA) as previously described.47 8701-BC were treated
as reported above, but the cells were challenged with the compounds
(10 µM) for 3 h for cyclins expression and for 72 h for BCL2 gene
expression. Next, the cells were harvested and lysed in an
appropriate buffer (OMNIzol for RNA-DNA-Protein Extraction Kit,
Euroclone, Devon, UK). The extracts were processed for the
extraction of mRNA. The RNA probes were synthesized according
to the suggested procedure (MAXIscript, Ambion, Austin, TX),
using T7 phage polymerase and a biotin RNA labeling mixture
(Boehringer, Mannheim, Germany). Briefly, an equal amount of
mRNA extracted from the cell lysates and appropriate RNA probes
were hybridized, according to the suggested procedure (Direct
Protect, Lysate Ribonuclease Protection Assay Kit, Ambion). The
products of RNase protection assay were then separated for analysis
on a denaturing polyacrylamide gel (5% acrylamide/8 M urea) by
running the gel at 200 V for 1 h. Gels were then transferred to a
positively charged nylon membrane, using a semidry transfer unit
at 90 mA for 1 h (Hoefer Pharmacia Biotech, San Francisco, CA).
The transcripts were then immobilized by UV cross-linking (bio-
link, Euroclone). Nonisotopic detection was performed with the
BrightStar BioDetect kit (Ambion), following the manufacturer’s
instructions. The identity and quantity of each mRNA species in
the original sample was then determined from the signal intensities
given by the appropriately sized protected probe fragment bands.
Antisense GAPDH and L32 (housekeeping genes) probe transcripts
were synthesized and used in a series of parallel reactions, to
normalize the amounts of RNA present in the lysates (protected
nucleotide size for L32: 112 nt). Gel electrophoretic autoradio-
graphs were then quantitated by Sigma Gel analysis software (Jandel
Scientific, San Rafael, CA).
The reliability of the docking protocol was first tested by
simulations of the binding mode of PP2, and comparison of the
modeled complex with the available three-dimensional structure
derived by X-ray crystallography of the complex between PP2 and
Lck. The program successfully reproduced the X-ray coordinates
of the inhibitor binding conformation, as well as the features of
pharmacophoric models reported in the literature.38
Computational Details. All calculations and graphical manipu-
lations were performed on Silicon Graphics computers (Origin 300
server and Octane workstations) using the software packages
Autodock 3.0.537 and MacroModel 8.5.40 Structures of inhibitors
were represented using MacroModel and minimized with the Amber
force field using the Polak-Ribiere conjugated gradient method
Acknowledgment. Financial support provided by the Italian
MIUR (Cofin 2004-prot. 2004059221_004) and by the Fondazi-

1560 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Carraro et al.
breast cancer. Epidemiol. Biomarkers PreV. 1999, 8, 855-861).
Among this tumor type, ductal infiltrating carcinoma (DIC) of the
breast is the most common and potentially aggressive form of cancer
(Donegan, W. L. Tumor-related prognostic factors for breast cancer.
CA Cancer J. Clin. 1997, 47, 28-51), for the treatment of which
there is no approved drug, yet.
one Monte dei Paschi di Siena is gratefully acknowledged. We
thank Dr. Giovanni Gaviraghi (Sienabiotech S.p.A.) for helpful
discussion. The "Centro Universitario per l′Informatica e la
Telematica" of the University of Siena is also acknowledged.
(23) Carraro, F.; Pucci, A.; Naldini, A.; Schenone, S.; Bruno, O.; Ranise,
A.; Bondavalli, F.; Brullo, C.; Fossa, P.; Menozzi, M.; Mosti, L.;
Manetti, F.; Botta, M. Pyrazolo[3,4-d]pyrimidines Endowed with
Antiproliferative Activity on Ductal Infiltrating Carcinoma Cells. J.
Med. Chem. 2004, 47, 1595-1598.
Supporting Information Available: Experimental details of
the synthesis, spectral data, and elemental analysis data of some
representative compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(24) Bondavalli, F.; Botta, M.; Bruno, O.; Ciacci, A.; Corelli, F.; Fossa,
P.; Lucacchini, A.; Manetti, F.; Martini, C.; Menozzi, G.; Mosti, L.;
Ranise, A.; Schenone, S.; Tafi, A.; Trincavelli, M. L. Synthesis,
molecular modeling studies, and pharmacological activity of selective
A1 receptor antagonists. J. Med. Chem. 2002, 45, 4875-4887.
(25) Ve´liz, E. A.; Beal, P. A. C6 substitution of inosine using hexam-
ethylphosphorous triamide in conjunction with carbon tetrahalide or
N-halosuccinimide. Tetrahedron Lett. 1999, 41, 1695-1697.
(26) Liu, Y.; Bishop, A.; Witucki, L.; Kraybill, B.; Shimizu, E.; Tsien,
J.; Ubersax, J.; Blethrow, J.; Morgan, D. O.; Shokat, K. M. Structural
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