Identification of a novel pyrazolo[3,4-d]pyrimidine able to inhibit cell proliferation of a human osteogenic sarcoma in vitro and in a xenograft model in mice

Article abstract of DOI:10.1021/jm061449r

New pyrazolo[3,4-d]pyrimidines were synthesized and found to inhibit Src phosphorylation in a cell-free assay. Some of them significantly reduced the growth of human osteogenic sarcoma (SaOS-2) cells. The best compound, in terms of inhibitory properties t

Full text of DOI:10.1021/jm061449r

J. Med. Chem. 2007, 50, 5579-5588
5579
Identification of a Novel Pyrazolo[3,4-d]pyrimidine Able To Inhibit Cell Proliferation of a
Human Osteogenic Sarcoma in Vitro and in a Xenograft Model in Mice†
Fabrizio Manetti,‡,¶ Annalisa Santucci,§,¶ Giada A. Locatelli,# Giovanni Maga,# Adriano Spreafico, Tommaso Serchi,§
Maurizio Orlandini,§ Giulia Bernardini,§ Nicola P. Caradonna,| Andrea Spallarossa,X Chiara Brullo,X Silvia Schenone,*,X
Olga Bruno,X Angelo Ranise,X Francesco Bondavalli,X Oskar Hoffmann,4 Mauro Bologna,3 Adriano Angelucci,3 and
Maurizio Botta‡
Dipartimento Farmaco Chimico Tecnologico, UniVersita` degli Studi di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy; Dipartimento di
Biologia Molecolare, UniVersita` degli Studi di Siena, Via Fiorentina 1, I-53100 Siena, Italy; Istituto di Genetica Molecolare, IGM-CNR, Via
Abbiategrasso 207, I-27100 PaVia, Italy; Dipartimento di Medicina Clinica e Scienze Immunologiche, Policlinico Le Scotte, UniVersita` degli
Studi di Siena, I-53100 Siena, Italy; Siena Biotech, Via Fiorentina 1, I-53100 Siena, Italy; Dipartimento di Scienze Farmaceutiche, UniVersita`
degli Studi di GenoVa, Viale Benedetto XV 3, I-16132 GenoVa, Italy; Department of Pharmacology and Toxicology, UniVersity of Vienna,
Althanstrasse 14, A-1090 Vienna, Austria; and Dipartimento di Biologia di Base ed Applicata, UniVersita` degli Studi dell’Aquila, Via Vetoio,
Loc. Coppito, I-67010 Coppito L’Aquila, Italy
ReceiVed December 20, 2006
New pyrazolo[3,4-d]pyrimidines were synthesized and found to inhibit Src phosphorylation in a cell-free
assay. Some of them significantly reduced the growth of human osteogenic sarcoma (SaOS-2) cells. The
best compound, in terms of inhibitory properties toward both Src and SaOS-2 cells, was further investigated
and found to reduce bone resorption when used to treat mouse osteoclasts, without interfering with normal
osteoblast growth. Moreover, its metabolic stability prompted its study on a human SaOS-2 xenograft tumor
model in nude mice, where the compound reduced significantly both the volume and weight of the tumor.
These experimental findings make the new compound an interesting hit in the field of bone-related diseases.
Introduction
cell death of malignant osteoblasts (such as the osteosarcoma
SaOS-2 cell line)6 and contributes, in combination with paxillin,
to the high metastatic potential of the human osteosarcoma
HuO9 cell line.7 All of these experimental data make Src an
attractive and promising therapeutic target for the treatment of
bone-related diseases such as osteoporosis,8-10 osteosarcoma,11
and cancer-induced bone metastasis.7
Osteoblasts and osteoclasts are involved in the dynamic and
highly regulated processes of bone remodeling, through a variety
of cellular signaling pathways involving protein kinases. Among
the latter, the tyrosine kinase Src has been demonstrated to play
a crucial regulatory role in both osteoblasts and osteoclasts. In
particular, Src has been implicated as a negative regulator of
osteoblast functional activity, on the basis of the fact that
reduction of Src expression stimulates osteoblast differentiation
and bone formation.1 Conversely, Src is also a mediator of anti-
apoptotic signaling in osteoblasts, induced by various factors.
As examples, Wnt proteins prevent apoptosis, prolonging the
survival of osteoblasts through the activation of the Src signaling
cascade.2 Moreover, anti-apoptotic effects of vitamin D3
analogues on the same cells are blocked by Src inhibitors such
as 4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo[3,4-d]py-
rimidine (PP1).3 On the other hand, disruption of the src gene
leads to osteopetrosis in mice,4 due to the inability of osteoclasts
to form ruffled borders and resorb bone during one of the final
stages of their maturation (positive regulatory role of Src).5 In
addition, Src is also involved in signaling pathways leading to
Recent literature reports a plethora of Src inhibitors originated
from a variety of molecular scaffolds that have demonstrated
potency up to the nanomolar range in enzymatic assays and
high efficacy in animal models of bone disease. Such com-
pounds are classified into SH2-SH3 domain inhibitors and
kinase domain inhibitors. The first ones block protein-protein
interactions between Src and other proteins involved in the
signaling pathways. As an example, blocking the SH2 domain
with high affinity might attenuate the excessive signal trans-
duction in bone resorption diseases, such as osteoporosis and
related bone diseases.12 On the other hand, many of the Src
kinase domain inhibitors belong to 6-6 and 5-6 bicyclic cores,
such as quinolines,13 quinazolines,14 pyrrolo[2,3-d]pyrimidine,15
and pyrazolo[3,4-d]pyrimidine.16 In this context, we have
recently described several pyrazolo[3,4-d]pyrimidines endowed
with in vitro antiproliferative and pro-apoptotic activity toward
epidermoid (A431) and breast cancer (8701-BC) cell lines,
acting through the inhibition of c-Src phosphorylation.17 In
particular, compound 1 (Table 1) showed a submicromolar
activity toward isolated Src and an inhibitory activity about
2-fold better than that of the reference compound 4-amino-5-
(4-chlorophenyl)-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine (PP2)
against both A431 and 8701-BC cells.17 Continuing our efforts
in the field of Src inhibitors, 1 was chosen as the parent
compound to design and synthesize new pyrazolo[3,4-d]-
pyrimidines (2-25) that, on the basis of our previous findings,
were expected to interfere with Src activity. Accordingly, they
were evaluated in a cell-free Src assay. Moreover, due to the
* Author to whom correspondence should be addressed (telephone 0039
010 3538866; fax 0039 010 3538358; e-mail schensil@unige.it).
† Dedicated to Professor Fulvio Gualtieri on the occasion of his 70th
birthday.
‡ Dipartimento Farmaco Chimico Tecnologico, Universita` degli Studi
di Siena.
§ Dipartimento di Biologia Molecolare, Universita` degli Studi di Siena.
¶ These authors contributed equally to this work.
# Istituto di Genetica Molecolare, IGM-CNR.
Dipartimento di Medicina Clinica e Scienze Immunologiche, Policlinico
Le Scotte, Universita` degli Studi di Siena.
| Siena Biotech.
X Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di
Genova.
4 Department of Pharmacology and Toxicology, University of Vienna.
3 Dipartimento di Biologia di Base ed Applicata, Universita` degli Studi
dell’Aquila.
10.1021/jm061449r CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/11/2007

5580 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Manetti et al.
Table 1. Structure, Physicochemical Properties, and Biological Data of Test Compounds
compd
R
R1
R2
mp
yield
Kia
CV (%)b after 24 and 48 h
1c,d
2
3
4
5
6
7
8
9
MeS
MeS
MeS
MeS
MeS
MeS
MeS
MeS
EtS
MeS
MeS
MeS
MeS
MeS
MeS
MeS
MeS
MeS
MeS
MeS
MeS
EtS
C6H5(CH2)2
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
Cl
F
H
H
H
H
H
H
H
F
73-74
76
73
70
68
70
71
62
71
82
81
80
75
72
71
68
74
74
55
43
40
60
55
52
62
45
0.7 ( 0.1
13 ( 3
102.7 ( 1.9; 99.5 ( 0.9
101.1 ( 1.1; 100.8 ( 2.1
102.9 ( 1.3; 100.5 ( 0.5
80.6 ( 0.7; 39.3 ( 2.0
85.2 ( 0.7; 75.4 ( 1.3
101.9 ( 1.9; 96.3 ( 1.1
101.0 ( 3.0; 49.8 ( 2.1
99.9 ( 0.9; 89.4 ( 2.3
107.3 ( 3.5; 22.2 ( 1.3
92.8 ( 1.3; 26.8 ( 1.2
101.7 ( 0.9; 102.1 ( 1.2
113.4 ( 1.5; 54.9 ( 2.4
104.1 ( 1.3; 99.7 ( 0.4
106.2 ( 3.0; 71.3 ( 2.1
102.1 ( 1.4; 99.2 ( 1.0
52.2 ( 2.4; 24.4 ( 2.5
62.7 ( 0.7; 44.4 ( 2.5
17.1 ( 1.3; 15.1 ( 0.7
94.8 ( 2.7; 84.3 ( 1.5
100.1 ( 1.1; 84.7 ( 0.3
100.1 ( 1.2; 94.4 ( 0.7
76.5 ( 1.9; 44.3 ( 1.8
77.8 ( 3.0; 68.4 ( 1.9
104.1 ( 0.4; 101.3 ( 1.5
63.1 ( 1.1; 55.4 ( 2.0
36.8 ( 1.7; 36.4 ( 1.6
m-F-C6H4(CH2)2
o-Cl-C6H4(CH2)2
m-Cl-C6H4(CH2)2
p-Cl-C6H4(CH2)2
p-Me-C6H4(CH2)2
p-MeO-C6H4(CH2)2
C6H5(CH2)2
111-112
99-100
4.0 ( 1.7
6.0 ( 2.1
25 ( 4
122-123
99-100
99-100
60-61
15 ( 2
4.0 ( 1.3
2.8 ( 1.2
7.5 ( 2.7
3.7 ( 0.9
3.1 ( 1.4
24 ( 5
127-129
99-100
C6H5(CH2)2
C6H5CH2
10c
11
12
13
14
15
16
17
18d
19
20
21
22d
23
24
25
PP2
142-143
158-159
126-127
133-134
113-114
136-137
130-131
152-153
102-103
212-213
250 (dec)
169-170
216-217
214-215
202-203
224-225
o-Cl-C6H4CH2
p-Cl-C6H4CH2
o-F-C6H4CH2
m-F-C6H4CH2
p-F-C6H4CH2
C6H5CH2
21 ( 4
5.2 (1.1
4.6 ( 1.7
3.0 ( 1.0
35 ( 8
C6H5CH2
m-Cl-C6H4
0.6 ( 0.1
1.4 ( 0.3
1.8 ( 0.2
1.2 ( 0.3
0.5 ( 0.1
1.2 ( 0.4
3.8 ( 1.7
4.1 ( 0.8
0.5 ( 0.1
m-F-C6H4
m-Br-C6H4
C6H5
m-Cl-C6H4
PrS
H
MeS
m-Cl-C6H4
m-Cl-C6H4
m-Br-C6H4
a Ki (expressed as a µM concentration) toward recombinant human Src was calculated according to the following equation: 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. b Antiproliferative activity of test
compounds (12.5 µM) toward SaOS-2 cells (MTT assay) after 24 and 48 h of treatment, expressed as cellular viability (CV, %) with respect to control
(100%). Values are means ( SD of three independent experiments performed in duplicates. c Compounds reported elsewhere.17 d Although 18 and 22 have
comparable Ki values for Src inhibition, they show significantly different activities in the cell proliferation assay. Because the replacement of the MeS group
with an EtS moiety is expected to have not a great effect on either the solubility (calculated solubilities are -6.53 and -6.82, respectively; Table 2) or the
metabolic stability, a reason of the discrepancy in Ki values and cell proliferation activity may be that 22 is able to inhibit Akt less (by 68%) than what 18
does (quantitative inhibition). However, the biological behavior of 22 deserves further investigations. Similarly, the biological properties of 1 are not completely
understood, yet. In fact, such a compound, with a 0.7 µM Ki toward Src, was previously demonstrated to have inhibitory properties toward several cell lines
presumably via Src-dependent mechanisms (see ref 17), whereas it is completely unable to inhibit SaOS-2 proliferation.
role of Src in osteoblast and osteoclast growth, some compounds
were also tested for their inhibitory properties toward human
osteoblasts (both normal and osteosarcoma SaOS-2 cell lines)
and osteoclasts. As a result, one compound (18) emerged for
its marked ability to reduce the viability of SaOS-2 cells in vitro,
without affecting normal osteoblasts. It also showed metabolic
stability in a Tier 1 metabolic stability screen. These experi-
mental findings prompted us to test it on a human SaOS-2
xenograft tumor in nude mice, resulting in a significant reduction
of both the volume and weight of the tumor.
raphy on a Florisil column. Finally, reaction of 31a-c with an
excess of various amines in toluene at rt afforded compounds
1-17 in yields ranging from 60 to 80%. The aniline derivatives
18-23 and 25 were obtained by reaction between 31a-e and
the appropriate amine in ethanol at reflux for 3 h.
The 6-unsubstituted derivative 24 was obtained by reaction
of 32, synthesized following our reported procedure,19 and
m-chloroaniline in ethanol at reflux for 3 h (Scheme 2).
Biology
Inhibition of SaOS-2 Proliferation. The new compounds
were evaluated for their ability to inhibit proliferation of the
human osteosarcoma SaOS-2 cell line through a preliminary
MTT assay (25 µM test dose, 24 and 48 h treatments), using
PP2 as the reference compound (Table 1).20 As a result, although
several compounds (namely, 16, 17, 18, and 25) were able to
reduce cell viability upon a 24 h treatment, the sole compound
18 dramatically reduced cell proliferation to about 17% after
24 h and to about 15% after 48 h, with respect to control. In
comparison, percent of cell growth allowed by PP2 is about 37
and 36% after 24 and 48 h, respectively. Moreover, IC50 values
were determined on the basis of seven test concentrations in
the range between 1 and 100 µM. Results showed a dose-
dependent inhibitory activity, with IC50 values of 8.1 and 12.6
µM for PP2 and 18, respectively.
Chemistry
Compounds 1-23 and 25 were prepared as reported in
Scheme 1, according to procedures previously described.17 The
starting products were the 2-hydrazinoethanols 26a-c, obtained
by starting from the appropriate phenyloxirane, following a
reported procedure.18 Reaction of 26a-c with ethyl(ethoxy-
methylene)cyanoacetate afforded the ethyl esters of 5-amino-
1H-pyrazole-4-carboxylic acids 27a-c, which were in turn
treated with benzoyl isothiocyanate in THF at reflux for 12 h
to give 28a-c. These intermediates were cyclized to the
pyrazolo[3,4-d]pyrimidinones 29a-c by treatment with 2 M
NaOH at 100 °C for 10 min, followed by acidification with
acetic acid. Alkylation of the thio group at position 6 with the
appropriate alkyl iodide in THF at reflux afforded the 6-alkylthio
derivatives 30a-e, which were in turn treated with the Vilsmeier
complex (POCl3:DMF, 4 equiv) in CHCl3 to obtain the
dihalogenated compounds 31a-e, bearing a chlorine atom both
at position 4 of the pyrimidine nucleus and at the N1 side chain.
These compounds were purified in good yield by chromatog-
Inhibition of Src Activity in a Cell-Free Assay. Because
pyrazolo[3,4-d]pyrimidines were previously found to inhibit Src
activity,17 we tested the new compounds on a cell-free assay to
assess their ability to influence the enzymatic activity of Src.
Biological data reported in Table 1 showed that compounds were

Inhibition of Cell Proliferation of Human Osteogenic Sarcoma
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5581
Scheme 1
Scheme 2
Figure 1. PP2 and 18 abolish Src-Tyr416 phosphorylation. Compounds
were used at their corresponding IC50. Cells were treated for 2 h prior
to their lysis. Specific Src phosphorylation was evaluated when whole-
cell lysates were immunoprobed with anti-total Src, anti-non-phospho-
Src-Y416, and anti-phospho-Src-Y416 antibodies. Results are repre-
sentative of three independent experiments.
competitive with the ATP substrate but not with a peptide used
as an Src substrate.21 In detail, the introduction of a substituent
on the phenyl ring of the C4 side chain (compounds 2-7) led
to a significant drop in affinity with respect to the parent
compound 1. In a similar way, transforming the N1 side chain
(8) and increasing the bulkiness of the substituent at C6 from
a methylthio to an ethylthio group (9) were detrimental for
affinity, despite these compounds retaining a micromolar Ki
value. Shortening the C4 phenylethylamino side chain to a
benzylamino group (10) and introducing substituents at the
benzyl moiety (11-15) led to a lower affinity. Activity in the
micromolar range was also found for derivatives of 10, in which
an additional halogen was introduced at the N1 side chain (16
and 17). On the contrary, the anilino derivative 18 was
characterized by one of the best affinity values toward Src (0.6
µM), comparable to that of the parent compound 1. On this
basis, to evaluate the influence of the molecular portions of 18
in Src inhibition, several derivatives were synthesized and tested.
When the chlorine group at the anilino moiety was changed
into F, Br, and H (19, 20, and 21, respectively), activity
underwent only a slight decrease (1.4, 1.8, and 1.2 µM,
respectively). Similarly, 22 and 23, bearing a bulkier substituent
at position 6, showed activities of 0.5 and 1.2 µM (comparable
to that of 18), respectively, whereas the C6-unsubstituted
derivative 24 had a 4-fold lower activity (3.8 µM). Insertion of
an additional halogen substituent on the N1 side chain led to
25 with a 2-fold decrease in affinity, in comparison to 20.
Inhibition of Src Phosphorylation. To determine whether
the new compounds were able to inhibit the phosphorylation
of Src at the level of Tyr416, we used specific anti-Src
antibodies toward total Src and toward Src with phosphorylated
Tyr416. Treatment with both PP2 and 18 (chosen as the hit
compound, on the basis of its highest activity in both enzymatic
and cellular assays) led to a marked reduction (77 and 62%,
respectively) of Tyr416 phosphorylation (Figure 1), suggesting
that inhibition of SaOS-2 cell proliferation was modulated, at
least in part, by Src.

5582 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Manetti et al.
Table 2. Log P, Solubility, and Membrane Permeability of Test
Compounds
this hypothesis is represented by compound 1, which showed
an activity toward the isolated Src similar to that of 18 (0.7 vs
0.6 µM, respectively), whereas its activity on the cell-based
assay was not significant. The metabolic stability of 1 was
assessed by a Tier 1 profiling screen by incubation of 1 µM
compound with recombinant human cytochrome P450 3A4 with
and without the cofactor NADPH for 1 h at 37 °C. Following
protein precipitation and filtration, the amount of remaining
compound was determined by UPLC-MS and found to be 10%,
allowing us to classify 1 as a metabolically instable compound.
compd
log Pa
solubilityb
Caco2c
1
2
3
4
5
6
7
8
5.55 (5.46)
5.76
6.22
6.22
6.22
6.04
5.54
6.22
5.90
5.23
5.90
5.90
5.44
5.44
5.44
5.90
5.44
6.05 (6.01)
5.60
6.14 (6.13)
5.39
6.40 (6.41)
6.93
4.99
-6.78
-6.23
-6.79
-6.74
-6.82
-6.62
-6.48
-6.66
-6.47
-6.29
-6.52
-6.88
-6.14
-6.15
-6.10
-6.45
-5.77
-6.53
-5.94
-6.85
-6.28
-6.82
-6.97
-5.76
-6.65
1.00
0.77
1.17
1.08
1.11
1.03
0.81
1.01
1.07
0.98
0.95
1.00
0.81
0.79
0.80
0.96
0.78
0.97
0.83
1.03
1.03
1.07
1.07
0.88
0.89
9
Moreover, contradictions in biological data could also depend
on the fact that cellular tests reflect the interference of a
compound in steps not shown in assays aimed at evaluating
the inhibition of Src activity. In other words, the enzymatic assay
reports only the effect of compounds in inhibiting Src activity,
whereas biological data referring to the antiproliferative activity
could also depend on other events, in addition to Src inhibition.
In this context, the micromolar inhibition of Src activity
mediated by all of the new compounds opposed to the wide
variability of cellular data also suggested that Src could not be
the sole or the major target of the pyrazolo-pyrimidines. To
check this hypothesis and to shed light on the mechanism of
action of 18 at the molecular level, we preliminarily tested it
toward a panel of 17 tyrosine kinases.24 As a result, a 10 µM
concentration of 18 was able to completely and selectively
inhibit the enzymatic activity of Akt, which is known to be
involved in a Src-dependent pathway found in human osteosa-
rcoma SaOS-2 cells.25 Moreover, activity of HER-1 and Ret
was also reduced by 42%, whereas a decrease of <30% was
found for the remaining kinases. On the other hand, three
additional compounds (namely, 1, 20, and 22), inactive in the
cell-based assay, were also tested for their activity toward the
kinase panel. As a result, 1 reduced activity of B-Raf-V599E
by 30%, whereas percent inhibition was lower or null for the
other kinases. The activity of B-Raf-V599E was slightly reduced
(by 15%) also by 22, which also partially inhibited Akt (by
68%). No significant inhibition toward the remaining kinases
was found. Finally, 20 showed a 17% reduction of the HER-1
activity, without influencing the remaining kinases.
This result supported the hypothesis that inhibition of SaOS-2
growth upon treatment with 18 may result from a combination
of targeting either Src or Akt.
To check if the best compound (18) was worthy of further in
vitro and in vivo biological investigations, its metabolic stability
was assessed. Upon submission of 18 to a Tier 1 screen (see
above), the remaining amount of the compound was 33% of
the original amount used in this assay, leading to the classifica-
tion of 18 as metabolically stable.
Cytotoxicity. Because cytotoxicity may occur as an undesir-
able side effect of compounds with a potential therapeutic
application, we evaluated viability with the Trypan blue assay.
As reported in Figure 2, PP2 was found to be more cytotoxic
than 18, causing 42% reduced viability. On the contrary, 18
exerted only a minor cytotoxic effect on SaOS-2 (13% reduced
viability), as also evidenced by a TUNEL assay assessing a pro-
apoptotic effect of 18 (Figure 3).
Inhibition of Normal Osteoblasts. Because 18 showed an
interesting inhibitory activity toward osteosarcoma cells, and
with the aim of checking if such a compound deserves to be
further studied as a possible lead against osteosarcoma cell lines,
we planned to test it toward human normal osteoblasts. Cells
were treated with the test compound (25 µM) for 48 h, following
a MTT assay. Osteoblast growth was moderately reduced by
18 (20%), different from PP2, which showed a 50% inhibitory
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
6.34
a Calculated by means of the AlogP98 method.30 In parentheses are
experimental values. b Solubility refers to the thermodynamic solubility
model (as implemented in Volsurf).22 Values are expressed as log[solubility],
in mol/L. The overall range is -8 < log[solubility] < +2, where lower
values indicate poor solubility and higher values indicate highly soluble
compounds. c Caco2 refers to the Caco2 permeation model (as implemented
in Volsurf).22 Values represent a score ranging from -1 to +1, correspond-
ing to the following permeability values: -1 corresponds to a permeability
of <4 × 10-6 cm/s, 0 corresponds to a permeability between 4 × 10-6 and
8 × 10-6 cm/s, and 1 corresponds to a permeability of >8 × 10-6 cm/s.
Metabolic Stability and Alternative Targets of 18. It is
worth noting that biological data revealed several inconsistencies
between cellular and enzymatic assays. In fact, essentially all
of the compounds were active in the enzymatic assay within a
relatively narrow range (from low to sub-micromolar concentra-
tions). On the other hand, their activity against osteosarcoma
cells varied widely, and MTT experiments demonstrated that a
25 µM concentration of the test compound was in many cases
unable to interfere with the growth of SaOS-2 cells (remaining
cellular viability values at 100%). Disagreement between
enzymatic and cellular data allowed us to formulate several
hypotheses about how compounds inhibit SaOS-2 cell growth.
In particular, although compounds were able to interact directly
with Src and reduce its enzymatic activity, it appeared that
physicochemical properties (such as solubility, membrane
permeability, log P) that, in turn, influence pharmacokinetic
parameters in part did not allow compounds to reach Src itself
within cells. A possible explanation could be a low solubility
of the pyrazolo-pyrimidine derivatives in the assay conditions,
which in part prevented compounds from entering cells. To
check this hypothesis, the solubility profiles of such compounds
were calculated by means of the software Volsurf,22 resulting
in a general low solubility (Table 2), whereas application of a
Caco2 permeation model showed that compounds are well
absorbed through cell membranes. Moreover, upon penetration
into the cells, several events may occurr (such as an increase in
drug efflux by activating the expression of the MDR1 gene,
which encodes for the P-glycoprotein)23 that lead to a partial
or marked inactivation of the compounds. Alternatively, the lack
of activity of some compounds toward SaOS-2 cells may also
depend on their metabolic instability. An example supporting

Inhibition of Cell Proliferation of Human Osteogenic Sarcoma
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5583
Figure 2. Cytotoxicity of PP2 and 18 toward SaOS-2 cells evaluated
by the Trypan blue assay. Cells were treated with compounds at their
corresponding IC50 for 24 h. Data are expressed as percentage of live
cells with respect to control (vehicle). Values are means ( SEM of
three independent experiments performed in duplicates. Error bars
indicate SDs from the mean values.
Figure 4. Effect of 18 and PP2 on bone resorption (mouse osteoclasts).
Figure 3. TUNEL assay of SaOS-2 cells after treatment with PP2
and 18 at 3, 12.5, and 25 µM for 48 h. Experiments were performed in
triplicate. Standard deviations are shown as bars.
replicates. Despite such a limitation, this assay is widely
recognized as the gold standard to assess the ability of
compounds to inhibit bone resorption by osteoclasts.27
activity. It is very important to note that, at the same concentra-
tion, the new pyrazolo-pyrimidine showed an antiproliferative
activity toward SaOS-2 cells of about 85%, suggesting that it
is highly selective in interfering with the proliferation of
abnormal osteoblasts. A time course experiment was also
performed to evaluate the pro-apoptotic activity of 18, further
supporting the hypothesis that the test compound is able to
specifically inhibit cell proliferation and to induce apoptosis in
transformed cells with respect to normal osteoblasts.26
Inhibition of Osteoclasts. Considering that Src-defective
osteoclasts showed abnormal organization of cytoskeletal ele-
ments necessary for bone resorption, 18 was also tested for its
ability to influence osteoclast-resorptive activity, in comparison
to PP2 (Figure 4). Compound 18 was able to abrogate bone
resorption in mouse osteoclasts at 10 and 1 µM concentrations,
whereas percent inhibition was lower but significant also for
reduced test dose (up to 1 nM).
It is very important to note that there is a significant difference
in bone resorption for the control (DMSO) in the two experi-
ments reported in Figure 4. In fact, when compound 18 was
tested, bone resorption in the control group was about 20%,
whereas only a 3% resorption was found in the control group
when PP2 was assayed. The method used to measure bone
resorption is based on the growth in vitro of osteoclasts that
then resorb bone. However, this cell-based assay is usually
characterized by a significant variability, mainly due to the fact
that the basic amount of bone resorption can vary among
Experimental in Vivo Model. Experimental data collected
for 18 are representative of a very interesting biological profile,
in particular in terms of selectivity toward malignant cells and
metabolic stability. Prompted by such results, we tested the
activity of 18 on a human SaOS-2 xenograft tumor model in
nude mice. In detail, SaOS-2 cells were injected sc in nude mice.
The SaOS xenograft showed an appreciable growth starting from
the fifth day after cell inoculation and doubled its volume in
about 15 days. To evaluate the effect of 18 in such a preclinical
experimental model of osteosarcoma, a treatment protocol was
applied via the oral administration once daily of a suspension
of the compound in cremphor vehicle. Interestingly, no ap-
preciable sign of distress or loss of weight in mice was
evidenced (data not shown). As a result, the administration of
18 (100 mg/kg) determined a significant reduction (about 37%
with respect to control, at the endpoint) in xenograft growth,
measured both as volume and as weight reduction (Figure 5).
Also, the administration of a 50 mg/kg dose determined a
smaller (about 15%) but significant reduction in tumor volume
with respect to control (Figure 5).
To evaluate the inhibition of Src activation in vivo, xenograft
tissues were processed for histologic evaluation of phospho-
Src expression (Figure 6). Tumor cells both in the control group
and in the treated group express c-Src (panels A and C), and
its localization was prevalently detected in the cytoplasm of
cells situated at the periphery of tumor mass. When the same

5584 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Manetti et al.
Figure 5. Inhibition of SaOS-2 xenograft growth. (a) Twelve mice were inoculated sc with tumor cells and were divided in three groups, each of
four mice, receiving a daily oral administration of 18 (50 or 100 mg/kg) in cremphor vehicle. Tumor volumes were calculated every 5 days, and
means ( SD were recorded. At the endpoint (26th day after cell injection), mice were sacrificed and tumors were measured and weighed. #, P <
0.05 according to Student’s t test. (b) Example images of tumor excised from mice of each group at the endpoint.
Figure 6. Immunohistologic analysis of xenografts. Tumor tissue sections were stained for the presence of total c-Src (A, C) and of phosphospecific-
Src (pY418) (B, D). Representative images of tumor tissues from control xenografts and from 100 mg/kg 18-treated group are shown in the left and
right panels, respectively (magnification ×300). The brown staining indicates the presence of the antigen, and nuclei are stained with hematoxylin.
tissues were analyzed for the presence of activated phospho-
Src, only a faint staining in the 100 mg/kg inhibitor-treated
tumors was detected, with respect to the control tumor sections
that, on the contrary, expressed high levels of the phospho-Src
antigen (panels B and D).
Molecular Docking Calculations. Because 18 was found to
inhibit Src phosphorylation through an ATP-competitive mech-
anism, it was docked into Src (entry 1y57 of the Protein Data
Bank),28 and the resulting complex was optimized by application
of a computational protocol previously described.17 Compound
18 (Figure 7) showed an orientation into the Src kinase domain
similar to that found for congeneric compounds bearing an
alkylthio substituent at C6.17 In fact, previous simulations on
pyrazolo-pyrimidine derivatives showed that the presence or the
absence of an alkylthio substituent at position 6 of the molecular
scaffold markedly influenced the binding mode of inhibitors

Inhibition of Cell Proliferation of Human Osteogenic Sarcoma
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5585
osteoblast SaOS-2 cells, with a very slight interference in the
growth of normal osteoblasts. On the other hand, 18 markedly
reduced bone resorption in mouse osteoclasts. Moreover, it was
also able to inhibit the activity of Akt (known to be involved
in a Src-dependent signaling pathway in osteosarcoma cells),
but at the moment, we are still unable to totally exclude the
involvement of other protein kinases in signaling pathways
regulating osteoblasts and osteoclast cells. Finally, the metabolic
stability of 18 prompted us to test it in vivo in mice; we found
a significant decrease in xenograft growth measured as both
volume and weight reduction, with respect to control. Additional
experiments are ongoing for a further in vitro and in vivo
characterization of 18 as a possible lead compound in the field
of therapeutical tools for bone diseases.
Experimental Section
Figure 7. Graphical representation of the binding mode of 18 (cyan)
into the ATP binding site of the active form of Src, in comparison to
PP2 (orange). Only a few residues are displayed; nonpolar hydrogens
are omitted, and hydrogen bonds are represented by black dotted lines.
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
to TMS as internal standard, with J in hertz. 1H 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 F254, CHCl3 as the eluant).
within the ATP binding site. In detail, compounds unsubstituted
at position 6 (such as 24) showed an interaction pathway
comparable to that of PP2, in terms of orientation, hydrogen
bond pattern, and contacts with the hydrophobic regions I and
II. On the other hand, the presence of an alkylthio substituent
at position 6, as in 18, induced a reorientation of the heterocyclic
nucleus, leading to a different interaction pathway. In particular,
the m-chlorophenylamino group was accommodated into the
hydrophobic region I, whereas the N1 chain was partially
inserted within the adenine region and directed toward the
hydrophobic pocket II. The methylthio group was located into
a hydrophobic region and was involved in favorable interactions
with Ala390. As expected, in contrast to PP2, this compound
was not engaged in any hydrogen bond. Interestingly, removal
of the hydrogen bond network was not associated with loss of
activity. In fact, despite the different orientation and the absence
of hydrogen bonds, 18 showed an inhibitory activity toward
Src comparable to that of PP2, suggesting that hydrogen bond
contacts were not essential for activity and their stabilizing
contribution could be replaced by additional hydrophobic
interactions involving the alkylthio side chain. This result clearly
suggested that lipophilic interactions resulting from full oc-
cupancy of hydrophobic regions I and II seemed to play a crucial
role in the affinity of ligands toward Src.
Similarly to 18, compounds 19-23 and 25, bearing an anilino
group at C4 in addition to the C6 alkylthio substituent, showed
their C4 and N1 side chains within the hydrophobic regions I
and II, respectively, resulting in a full and profitable occupancy
of the two pockets. On the other hand, as previously described,17
molecules bearing a larger substituent at C4 (benzylamino or
phenylethylamino, such as 1-17) assumed a different pose
within the binding pocket, with C4 and N1 side chains in an
opposite location with respect to 18 and other anilino analogues.
As a general trend, this different accommodation was associated
with less profitable interactions with amino acid residues and
with a decreased affinity toward the target. In this context,
compound 1 can be considered as an exception, showing one
of the most interesting affinity values despite a binding mode
common to less active compounds.
Analyses for C, H, N and S were within (0.3% of the theoretical
value.
Mass spectral (MS) data were obtained using an Agilent 1100
LC/MSD VL system (G1946C) with a 0.4 mL/min flow rate using
a binary solvent system of 95:5 methyl alcohol/water. UV detection
was monitored at 254 nm. Mass spectra were acquired in positive
mode scanning over the mass range of 50-1500. The following
ion source parameters were used: drying gas flow, 9 mL/min;
nebulizer pressure, 40 psig; drying gas temperature, 350 °C.
Synthesis and experimental data of compounds 1, 10, 26a, 27a,
28a, 29a, 30a, 30d, 31a, 31d, and 32 were already reported by
us.17,19
1-(4-Halophenyl)-2-hydrazinoethanols (26b,c). To hydrazine
monohydrate (30 mL, 0.6 mol), heated at 100 °C was added the
appropriate phenyloxirane (0.17 mmol). The solution was heated
for 30 min at 100 °C, and the excess of hydrazine was removed
under reduced pressure. The crude was purified by bulb to bulb
distillation to obtain the pure products as pale yellow oils.
1
26b: yield, 85%; bp, 175-180 °C (0.6 mmHg); H NMR, δ
2.62-2.87 (m, 2H, CH2N), 3.55-3.87 (m, 4H, NH2NH + OH,
disappears with D2O), 4.85-4.92 (m, 1H, CHO), 6.95-7.29 (m,
4H Ar); IR, 3323, 3280, 3250 (NH2 + NH), 3400-3000 (OH)
cm-1. Anal. (C8H11N2FO) C, H, N.
1
26c: yield, 80%; bp, 170-175 °C (0.6 mmHg); H NMR, δ
2.65-2.88 (m, 2H, CH2N), 3.56-3.90 (m, 4H, NH2NH + OH,
disappears with D2O), 4.88-4.92 (m, 1H, CHO), 7.00-7.32 (m,
4H Ar); IR, 3320, 3290, 3250 (NH2 + NH), 3350-2950 (OH)
cm-1. Anal. (C8H11N2ClO) C, H, N.
Ethyl 5-Amino-1-[2-(4-halophenyl)-2-hydroxyethyl]-1H-pyra-
zole-4-carboxylates (27b,c). The starting hydrazine 26b or 26c (20
mmol) was added to a solution of ethyl(ethoxymethylene)cyanoac-
etate (3.38 g, 20 mmol) in anhydrous toluene (20 mL), and the
mixture was heated at 80 °C for 8 h. The solution was then
concentrated under reduced pressure to half of the volume and
allowed to cool to room temperature. The pale yellow solids were
filtered and recrystallized from toluene to afford 27b and 27c as
white solids.
Conclusions
1
27b: yield, 70%; mp, 163-164 °C; H NMR, δ 1.33 (t, J )
The new pyrazolo-pyrimidines reported here showed inhibi-
tory properties toward Src phosphorylation, and one of them
(18) proved to be an antiproliferative agent against malignant
7.0, 3H, CH3), 3.73 (br s, 1H, OH, disappears with D2O), 3.90-
4.15 (m, 2H, CH2N), 4.29 (q, J ) 7.0, 2H, CH2O), 5.01-5.18 (m,
1H, CHO), 5.36 (br s, 2H, NH2, disappears with D2O), 7.03-7.40

5586 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Manetti et al.
(2 m, 4H Ar), 7.55 (s, 1H, H-3); IR, 3448, 3346 (NH2), 3300-
3000 (OH), 1685 (CO) cm-1. Anal. (C14H16N3FO3) C, H, N.
27c: yield, 75%; mp, 168-169 °C; 1H NMR, δ 1.38 (t, J ) 7.0,
3H, CH3), 3.56 (br s, 1H, OH, disappears with D2O), 3.91-4.19
(m, 2H, CH2N), 4.28 (q, J ) 7.0, 2H, CH2O), 5.05-5.18 (m, 1H,
CHO), 5.33 (br s, 2H, NH2, disappears with D2O), 7.25-7.46 (m,
4H Ar), 7.59 (s, 1H, H-3); IR, 3412, 3291 (NH2), 3219-3100 (OH),
1689 (CO) cm-1. Anal. (C14H16N3ClO3) C, H, N.
from POCl3 (6.13 g, 40 mmol) and anhydrous DMF (2.92 g, 40
mmol), was added to a suspension of 30b,c,e (10 mmol) in CHCl3
(20 mL), and 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 (Florisil 100-200 mesh), using diethyl
ether as the eluant, to afford the pure product 31b,c,e as white solids.
1
31b: yield, 70%; mp, 136-137 °C; H NMR, δ 2.65 (s, 3H,
Ethyl
5-{[(Benzoylamino)carbonothioyl]amino}-1-[2-(4-
CH3S), 4.74-5.03 (m, 2H, CH2N), 5.42-5.54 (m, 1H, CHCl),
6.96-7.08 and 7.29-7.44 (2 m, 4H Ar), 8.03 (s, 1H, H-3). Anal.
(C14H11N4Cl2F S) C, H, N, S.
halophenyl)-2-hydroxyethyl]-1H-pyrazole-4-carboxylates (28b,c).
A suspension of 27b or 27c (10 mmol) and benzoyl isothiocyanate
(1.7 g, 11 mmol) in anhydrous THF (20 mL) was refluxed for 12
h. The solvent was evaporated under reduced pressure, and 28b
was crystallized by adding diethyl ether (30 mL) as a white solid,
whereas 28c was used as crude oil for the next reaction.
1
31c: yield, 60%; mp, 142-143 °C; H NMR, δ 2.65 (s, 3H,
CH3S), 4.74-5.03 (m, 2H, CH2N), 5.42-5.53 (m, 1H, CHCl),
7.25-7.42 (m, 4H Ar), 8.03 (s, 1H, H-3). Anal. (C14H11N4Cl3S)
C, H, N, S.
1
1
28b: yield, 85%; mp, 129-130 °C; H NMR, δ 1.31 (t, J )
31e: yield, 65%; mp, 83-84 °C; H NMR, δ 1.05 (t, J ) 7.2,
7.0, 3H, CH3), 4.10-4.38 (m, 5H, 2CH2 + OH, 1H disappears
with D2O), 5.25-5.38 (m, 1H, CHO), 7.00-7.90 (m, 9H Ar), 8.05
(s, 1H, H-3), 9.36 (s, 1H, NH, disappears with D2O), 12.16 (s, 1H,
NH, disappears with D2O); IR, 3444, 3261 (NH), 3190-2940 (OH),
1683 (CO) cm-1. Anal. (C22H21N4FO4S) C, H, N, S.
1-[2-(4-Halophenyl)-2-hydroxyethyl]-6-thioxo-1,5,6,7-tetrahy-
dro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (29b,c). A solution of
28b or 28c (10 mmol) in 2 M NaOH (40 mL) was refluxed for 10
min and then diluted with H2O (40 mL) and acidified with glacial
acetic acid. After 12 h of standing in a refrigerator, a solid
crystallized that was filtered and recrystallized from absolute ethanol
to give 29b and 29c as white solids.
3H, CH3), 1.77 (sext, J ) 7.2, 2H, CH3CH2), 3.11 (t, J ) 7.2, 2H,
CH2S), 4.64-4.95 (m, 2H, CH2N), 5.32-5.46 (m, 1H, CHCl),
7.07-7.38 (m, 5H Ar), 7.95 (s, 1H, H-3). Anal. (C16H16N4Cl2S)
C, H, N, S.
General Procedure for the 4-Amino-1H-pyrazolo[3,4-d]py-
rimidines (2-9, 11-17). To a solution of 31a, 31b, 31c, or 31d
(5 mmol) in anhydrous toluene (20 mL) was added the proper amine
(20 mmol). The reaction mixture was stirred at room temperature
for 36 h and then extracted with H2O (2 × 20 mL). The organic
phase was dried (MgSO4), filtered, and concentrated under reduced
pressure. The oil residue was crystallized by adding diethyl ether
(10 mL) to give the final products as white solids.
29b: yield, 75%; mp, 252-253 °C; 1H NMR, δ 4.15-4.28 and
4.52-4.60 (2 m, 2H, CH2N), 4.88-5.00 (m, 1H, CHO), 5.69 (s,
1H, OH, disappears with D2O), 7.12-7.29 and 7.40-7.52 (2 m,
4H Ar), 7.98 (s, 1H, H-3), 12.20 (s, 1H, NH, disappears with D2O),
13.36 (s, 1H, NH, disappears with D2O); IR, 3315, 3200 (NH),
3320-2500 (OH), 1670 (CO) cm-1. Anal. (C13H11N4FO2S) C, H,
N, S.
2: yield, 73%; mp, 111-112 °C; 1H NMR, δ 2.49 (s, 3H, CH3S),
2.89 (t, J ) 7.0, 2H, CH2Ar), 3.75 (q, J ) 7.0, 2H, CH2NH), 4.58-
4.90 (m, 2H, CH2N), 5.14 (br s, 1H, NH, disappears with D2O),
5.34-5.51 (m, 1H, CHCl), 6.77-7.43 (m, 9H Ar), 7.64 (s, 1H,
H-3); IR, 3248 (NH) cm-1; MS (ESI), m/z 444.2 [M + H]+, 464.2
[M + Na]+. Anal. (C22H21N5ClFS) C, H, N, S.
General Procedure for the 4-Anilino-1H-pyrazolo[3,4-d]-
pyrimidines (18-25). To a solution of 31a, 31b, 31d, and 31e (5
mmol) in absolute ethanol (20 mL) was added the appropriate
aniline (5 mmol), and the reaction mixture was stirred at reflux for
3 h. After cooling, a solid precipitated that was filtered, washed
with H2O, and recrystallized from absolute ethanol (10 mL) to give
the final products as white solids. Compound 24 was prepared with
the same procedure starting from 32.
1
29c: yield, 70%; mp, 249-250 °C; H NMR, δ 4.13-4.65 (2
m, 2H, CH2N), 4.84-5.00 (m, 1H, CHO), 5.71 (br s, 1H, OH,
disappears with D2O), 7.30-7.51 (m, 4H Ar), 7.97 (s, 1H, H-3),
12.19 (s, 1H, NH, disappears with D2O), 13.38 (s, 1H, NH,
disappears with D2O); IR, 3390, 3220 (NH), 3100-2700 (OH),
1675 (CO) cm-1. Anal. (C13H11N4ClO2S) C, H, N, S.
1-Substituted-6-(alkyllthio)-1,5-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-ones (30b,c,e). A solution of 29a-c (10 mmol) and
the appropriate alkyl iodide (50 mmol) in anhydrous THF (20 mL)
was refluxed for 12 h. The solvent and the excess of alkyl iodide
were removed by distillation under reduced pressure. The residue
oil was crystallized by adding CHCl3 (10 mL) and was purified by
recrystallization with absolute ethanol to give 30b,c as white solids.
Compound 30e was purified by column chromatography (silica gel),
using a mixture of CHCl3/CH3OH (8:2) as eluant.
18: yield, 55%; mp, 102-103 °C;
1H NMR, δ 2.52 (s, 3H,
CH
3S), 4.58-4.90 (m, 2H, CH2N), 5.34-5.44 (m, 1H, CHCl),
7.08-7.36 (m, 9H Ar), 7.57 (s, 1H, H-3); IR, 3277 (NH) cm-1
;
MS (ESI), m/z 430.2 [M + H]+
Cl2S) C, H, N, S.
, 452.2 [M + Na] . Anal. (C20H17N5
-
+
Biology. Human Osteosarcoma Cell Culture. The human
osteosarcoma cell line SaOS-2 was obtained from ATCC (HTB-
85) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)
(Sigma-Aldrich, St. Louis, MO) supplemented with 10% (v/v) fetal
calf serum (FCS) (Sigma), at 37 °C, 2 mM L-glutamine (Sigma-
Aldrich), penicillin (100 units/mL), and streptomycin (100 µg/mL),
at 37 °C, in a humidified atmosphere of 7% CO2/93% air.
Treatments. c-Src inhibitors were synthesized as described,
dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) at various
concentrations, and diluted in cell culture medium prior to use. PP2
(Calbiochem, Darmstadt, Germany) was parallely used as a
reference compound. Controls were carried out with DMSO
concentrations corresponding to the higher doses of the test
compounds. The final DMSO concentration did not exceed 0.2%
(v/v) and did not affect the parameters analyzed. Cells were treated
when at confluence.
Cell Viability. Cell proliferation was quantified by using the
MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide]) cell proliferation assay kit (Molecular Probe). Cells (about
50000, estimated with Thoma chamber) were seeded in a 96-
multiwell plate with 10% (v/v) FCS, 2 mM L-glutamine, penicillin
(100 units/mL), and streptomycin (100 µg/mL), grown for 48 h
(confluence), and then exposed to test compounds (12.5 and 25
µM) for 24 or 48 h. The medium was then removed, and the cells
1
30b: yield, 72%; mp, 217-218 °C; H NMR, δ 2.51 (s, 3H,
CH3S), 4.24-4.50 (m, 2H, CH2N), 4.55 (br s, 1H, OH, disappears
with D2O), 5.00-5.15 (m, 1H, CHO), 7.02-7.30 (m, 4H Ar), 7.96
(s, 1H, H-3), 12.33 (s, 1H, NH, disappears with D2O); IR, 3418
(NH), 3120-2850 (OH), 1668 (CO) cm-1. Anal. (C14H13N4FO2S)
C, H, N, S.
1
30c: yield, 70%; mp, 210-211 °C; H NMR, δ 2.84 (s, 3H,
CH3S), 4.22-4.48 (m, 2H, CH2N), 4.94-5.16 (m, 1H, CHO), 5.76
(d, 1H, OH, disappears with D2O), 7.07-7.22 (m, 4H Ar), 7.94 (s,
1H, H-3), 12.32 (s, 1H, NH, disappears with D2O); IR, 3427 (NH),
3120-2850 (OH), 1667 (CO) cm-1. Anal. (C14H13N4ClO2S) C, H,
N, S.
30e: yield, 68%; mp, 165-166 °C; 1H NMR, δ 0.91 (t, J ) 7.2,
3H, CH3), 1.61 (sext, J ) 7.2, 2H, CH3CH2), 3.01 (t, J ) 7.2, 2H,
CH2S), 4.14-4.38 (m, 2H, CH2N), 4.92-5.05 (m, 1H, CHO), 5.76
(d, 1H, OH, disappears with D2O), 7.08-7.18 (m, 5H Ar), 7.87 (s,
1H, H-3), 12.20 (br s, 1H, NH, disappears with D2O); IR, 3346
(NH), 3130-2900 (OH), 1692 (CO) cm-1. Anal. (C16H18N4O2S)
C, H, N, S.
1-Substituted-[4-chloro-6-(alkylthio)-1H-pyrazolo[3,4-d]pyri-
midines (31b,c,e). The Vilsmeier complex, previously prepared

Inhibition of Cell Proliferation of Human Osteogenic Sarcoma
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5587
were incubated for 4 h with fresh medium in the presence of 1.2
mM MTT. Living cells reduce MTT to a strongly pigmented
formazan crystals. After solubilization in DMSO, the absorbance
of the formazan was measured with a microplate absorbance reader
at 540 nm. The growth inhibitory activity of the compounds studied
was compared with the activity of PP2, used at the same
concentrations of compounds.
Cytotoxicity. The cytotoxic effect of compounds was evaluated
by Trypan blue exclusion test. SaOS-2 cells plated in 24-well
multiplates and grown in DMEM supplemented with 10% (v/v)
FCS, 2 mM L-glutamine, penicillin (100 units/mL), and strepto-
mycin (100 µg/mL). Cells were treated with PP2 and 18 at their
respective IC50 concentrations (8 and 12.6 µM, respectively). IC50
values were determined on SaOS-2 cells after a 48 h of treatment
at variable concentrations of the inhibitors (1, 3, 5, 12.5, 25, 50,
and 100 µM). At the end of the treatment, cells were washed with
PBS, detached by treatment with 0.05% (w/v) trypsin/0.02% (w/
v) EDTA for 5 min at 37 °C and collected, stained with 0.05 mM
Trypan blue solution in buffer saline (PBS), and observed by
conventional light microscopy in a blind manner, using the Thoma
chamber. The number of Trypan blue-positive cells was calculated
and expressed as a percentage of the total number of cells of the
negative control.
4 million cells to each culture plate. The culture plates were then
incubated overnight, giving the osteoblasts time to attach to the
bone surface.
Mice used for the isolation of bone of bone marrow cells were
anesthetized with ether and killed by cervical dislocation (1 mouse
for each 48-well plate). The femora were put in a Petri dish on ice
containing sterile PBS, and the muscles were removed under sterile
conditions. The ends of the bones were then cut off with a scalpel.
The bone marrow was isolated from the contents of the bone with
R-MEM + 10% FCS, using a sterile syringe and a small needle.
The bone marrow and medium were then transferred to a 50 mL
centrifuge tube and centrifuged for 5 min at 1500 rpm at 4 °C. The
supernatant was removed and the pellet suspended in fresh medium,
the amount of medium depending on the total number of wells.
The medium in the wells of the culture was removed, and 400
µL of bone marrow cell suspension was added to each well. In
addition, 100 µL of medium with 1 × 10-5
M 1.25-(OH)2-vitamin
D3 and 1 × 10-3 M PGE2 was added to each well to reach an end
concentration of 1 × 10-9 M 1.25-(OH)2-vitamin-D3 and 1 × 10-6
M PGE2. The well plates were then incubated. After 48 h, the
medium was changed and, after spending another 24 h in the
incubator, the test substances (18 and PP2) were added. The well
plates were incubated for 48 h.
TUNEL Assay. The DNA strand breaks in the apoptotic cells
were evaluated by the TUNEL in situ cell death detection kit
according to the manufacturer’s instruction (Roche Applied Science,
Mannheim, Germany). Briefly, cells were fixed in 4% buffered
paraformaldehyde (pH 7.4), washed (3 × 5 min) in PBS, blocked
for 10 min with 3% H2O2 in methanol, subjected to permeabilization
with 0.1% Triton X-100 in 0.1% (w/v) sodium citrate for 2 min,
and incubated first for 10 min with an equilibration buffer and then
with the reaction buffer containing the TdT enzyme for 60 min at
37 °C. Cells can be observed in a drop of PBS under a fluorescence
microscope at this state, using an excitation wavelength in the range
of 450-500 nm and detection in the range of 515-565 nm. Cells
were treated with a solution of Convert-POD for 30 min in
humidified atmosphere at 37 °C and washed three times with PBS.
DAB substrate was added for 10 min and then, after three more
washes with PBS, cells were mounted under a glass coverslip and
analyzed under light microscope. For quantitative analysis, apoptotic
and total cells were counted and their ratio was computed and
represented in graph form.
Phosphorylation Assays. For phospho-SrcY416 detection, 10-
20 µg of cell culture protein lysates, diluted in reducing buffer,
were resolved by 12% SDS-PAGE and electrotransferred onto
nitrocellulose for detection with specific antibodies. All antibodies
employed were purchased from Cell Signaling Technology (Dan-
vers, MA). Proteins were probed with primary antibodies overnight
at 4 °C, followed by secondary antibodies for 1 h at rt. Detection
was obtained by ImmunoStar HRP (Bio-Rad, Hercules, CA). Band
areas were analyzed by scanning densitometry, using ImageScanner
(Amersham Bioscience, Little Chalfont, U.K.) and Melanie II and
PDQuest software (Bio-Rad). Band areas of phospho-SrcY416 were
normalized toward those of total Src as an internal control.
Effects of Compounds on Bone Resorption (Mouse OC).
Preparation of Bone. Preisolated bone from cattle was cut to slices
with a thickness of 300-350 µm using a low-speed saw with a
diamond blade. The slices were then cut to small equal squares
(about 0.5 × 0.5 cm) with a scalpel. Cleaning of bone pieces was
done by first washing three times with water and once with 70%
ethanol in an ultrasound bath for 5 min. The pieces were then stored
in 100% ethanol overnight. Sterilization was carried out using UV
light in a laminar airflow workbench (LAF) for 15 min on each
side. The sterilized bone pieces were finally fixed with sterile
silicone grease to the wells of a 48-well culture plate (four pieces
for each well) and preincubated in R-MEM + 10% heat-inactivated
FCS for a minimum of 2 h.
Staining of Bone Slices. Staining with toluidine blue makes it
possible to see the resorption areas under a microscope.29 To wash
off the medium, residual silicone, and the layer of cells, the bone
slices were placed in polypropylene tubes and cleaned with 70%
isopropanol for 15 min in an ultrasound bath. The slices were
stained with a 1% solution of toluidine blue for 4 min, before the
superfluous dye was rinsed off with bidistilled water.
The stained slices of bone were examined under a light
microscope, using a 10× magnifying objective lens. Digitalized
images were acquired, and the areas of the resorption lacunae could
be measured. The data were copied to Microsoft Excel, in which
the final calculations were made according to the following: (1)
total number of pits ) sum of all pits in five photographs from
each piece of bone; (2) total area ) sum of the areas of all pits in
the five photographs from each piece of bone; (3) average pit area
) total area of pits/total number of pits; (4) % resorption ) total
area of pits × 100/6000000, where the value of 6 million was
obtained by knowing that one field of vision equals 1.2 million
µm2 (because five fields of vision were photographed, the total
number was 6 million); (5) % inibiton ) [100 - (% resorption ×
100)]/mean of % resorption of controls.
Metabolic Stability. Compound 18 was incubated at 1 µM with
recombinant human cytochrome P450 3A4 with and without the
cofactor NADPH for 1 h at 37 °C. Following precipitation of protein
and filtration, percent remaining parent compound was detemined
using UPLC-MS. Compounds with a percent remaining lower than
20% are classified as unstable, whereas compounds with percent
remaining higher than 20% are classified as stable.
Animals and Experimental in Vivo Model. Male CD1 nude
mice (Charles River, Milan, Italy) were maintained under the
guidelines established by our Institution [University of L’Aquila,
Medical School and Science and Technology School Board
Regulations, complying with the Italian government regulation (n.
116, January 27, 1992) for the use of laboratory animals]. Before
any invasive manipulation, mice were anesthetized with a mixture
of ketamine (25 mg/mL)/xylazine (5 mg/mL). Xenografts were
obtained by injecting sc 106 SAOS cells in 100 µL of 12 mg/mL
Matrigel (Becton Dickinson, Franklin Lakes, NJ). Tumor growth
was monitored daily by measuring the average tumor diameter. The
volume of the tumor was expressed in cubic millimeters, according
to the formula 4πr3/3.
Xenograft Histology. Subcutaneous tumors were fixed in 4%
formaldehyde in 0.1 M phosphate buffer, pH 7.2, and embedded
in paraffin. Slide-mounted tissue sections (4 µm thick) were
deparaffinized in xylene and hydrated serially in 100, 95, and 80%
ethanol. Endogenous peroxidases were quenced in 3% H2O2 in PBS
for 1 h, and then slides were incubated with anti-human c-Src
antibody or anti-human Src (pY418) phosphospecific antibody
Preparation of the Coculture. Osteoblasts and bone marrow
cells from mice were cocultured to obtain osteoclasts from
precursors by fusion and differentiation. Osteoblasts were quickly
thawed at 37 °C in a water bath before the seeding of approximately

5588 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Manetti et al.
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Allen, J.; Lambert-van der Brempt, C.; Costello, G. Discovery of a
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(17) (a) Carraro, F.; Naldini, A.; Pucci, A.; Locatelli, G. A.; Maga, G.;
Schenone, S.; Bruno, O.; Ranise, A.; Bondavalli, F.; Brullo, C.; Fossa,
P.; Menozzi, G.; Mosti, L.; Modugno, M.; Tintori, C.; Manetti, F.;
Botta, M. 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. J. Med. Chem. 2006, 49, 1549-
1561. (b) Angelucci, A.; Schenone, S.; Gravina, G. L.; Muzi, P.;
Festuccia, C.; Vicentini, C.; Botta, M.; Bologna, M. Pyrazolo[3,4-
d]pyrimidines c-Src inhibitors reduce epidermal growth factor-
induced migration in prostate cancer cells. Eur. J. Cancer 2006, 42,
2838-2845.
(18) Bondavalli, F.; Botta, M.; Bruno, O.; Ciacci, A.; Corelli, F.; Fossa,
P.; Lucacchini, A.; Manetti, F.; Martini, C.; Menozzi, G.; Mosti, L.;
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(19) Schenone, S.; Bruno, O.; Ranise, A.; Mosti, L.; Menozzi, G.; Fossa,
P.; Donnini, S.; Santoro, A.; Ziche, M.; Manetti, F.; Botta, M.
Antiproliferative activity of new 1-aryl-4-amino-1H-pyrazolo[3,4-d]-
pyrimidine derivatives toward the human epidermoid carcinoma A431
cell line. Eur. J. Med. Chem. 2004, 39, 939-964.
(20) Although recent literature reports examples of compounds that could
be used as comparators to validate enzymatic and cell models, PP2
was used as the reference compound for three main reasons: (i)
because it was identified as a potent and selective inhibitor of Src
tyrosine kinases (ref. 16), PP2 remains an appropriate reference
compound when the activity toward Src is to be determined; (ii) a
great number of literature reports have been published on PP2 during
the past decade, giving us exhaustive information on its biological
profile toward Src and other kinases; (iii) PP2 is a commercially
and easily available compound.
(Biosource, Camarillo, CA) for 1 h at rt. Sections were washed
three times in PBS, and antibody binding was revealed using the
Sigma fast 3,3′-diaminobenzidine tablet set (Sigma, St. Louis, MO).
Acknowledgment. We gratefully acknowledge financial
support provided by the Italian MIUR (PRIN 2004059221), the
Fondazione Monte dei Paschi di Siena, and AIRC (cod. 1111).
We are indebted to Dr. Doriano Fabbro (Novartis Institutes for
Biomedical Research, Basel, Switzerland) for assays on the
tyrosine kinase panel. We also acknowledge Prof. Gabriele
Cruciani (University of Perugia, Italy) for kindly providing us
with the program VolSurf. We thank Dr. Giovanni Gaviraghi
(Sienabiotech, Siena, Italy) for helpful discussion and Antonella
Nardi (University of Siena) for LC-MS analysis.
Supporting Information Available: Spectroscopic and el-
emental analysis data of some compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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