Synthesis, biological evaluation and docking studies of 4-amino substituted 1H-pyrazolo[3,4-d]pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2008.01.034

The synthesis of new 4-amino substituted pyrazolo[3,4-d]pyrimidines along with their activity in cell-free enzymatic assays on Src and Abl tyrosine kinases is reported. Some compounds emerged as good dual inhibitors of the two enzymes, showed antiprolifer

Full text of DOI:10.1016/j.ejmech.2008.01.034

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European Journal of Medicinal Chemistry 43 (2008) 2665e2676
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Original article
Synthesis, biological evaluation and docking studies of 4-amino
substituted 1H-pyrazolo[3,4-d]pyrimidines
a
a
a
Silvia Schenone a, , Chiara Brullo , Olga Bruno , Francesco Bondavalli ,
Luisa Mosti a, Giovanni Maga b, Emmanuele Crespan b, Fabio Carraro c,
Fabrizio Manetti d, Cristina Tintori d, Maurizio Botta d
*
a Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Genova,
Viale Benedetto XV 3, I-16132 Genova, Italy
b Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, I-27100 Pavia, Italy
c Dipartimento di Fisiologia, Universita` di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy
d Dipartimento Farmaco Chimico Tecnologico, Universita` di Siena, Via Aldo Moro, I-53100 Siena, Italy
Received 26 November 2007; received in revised form 22 January 2008; accepted 23 January 2008
Available online 6 February 2008
Abstract
The synthesis of new 4-amino substituted pyrazolo[3,4-d]pyrimidines along with their activity in cell-free enzymatic assays on Src and
Abl tyrosine kinases is reported. Some compounds emerged as good dual inhibitors of the two enzymes, showed antiproliferative effects on
two BcreAbl positive leukemia cell lines K-562 and KU-812, and induced apoptosis, as demonstrated by the PARP assay. Docking studies
have been also performed to analyze the binding mode of compounds under study and to identify the structural determinants of their interaction
with both Src and Abl.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Abl; Src; Pyrazolo-pyrimidines; Chronic myeloid leukemia; Docking
1. Introduction
High level or overexpression of Src in tumors is correlated
with poor prognosis.
c-Src is a non-receptor tyrosine kinase that plays important
roles in several signal transduction pathways and is involved in
development and progression of human solid malignancies, in-
cluding colon, breast, pancreatic and lung cancers [1e3]. Src
kinase activity is also linked with tumor metastatic growth, be-
ing involved in the disruption of focal adhesions that ensure
the interaction between the cell and the extracellular matrix,
and of adherens junctions that permit the adhesion of the cells
to each other and are critical for cell migration [4,5]. Transfec-
tion of cancer cells with activated c-Src leads to disorganized
and less effective focal adhesion structures, with reduced cell
clustering, promoting the release of cells from the matrix [6].
Besides having a role in solid tumors, Src is also involved in
the progression of haematological malignancies, in particular
chronic myeloid leukemia (CML) and acute myeloid leukemia
(AML). These diseases are characterized by the presence of the
Philadelphia chromosome, derived from a reciprocal transloca-
tion between chromosomes 9 and 22 [7]; the translocation fuses
the breakpoint cluster region (Bcr) and the Abl genes, forming
the BcreAbl oncogene which encodes a constitutively active
cytoplasmatic tyrosine kinase (TK) BcreAbl. This deregulated
enzyme causes hyperproliferation of the leukemic cells and the
consequent pathology of the disease. The finding that BcreAbl
is the cause of the leukemic phenotype and that the tyrosine ki-
nase activity of Abl is fundamental for BcreAbl-mediated
transformation made this kinase an important target for the de-
velopment of specific therapies. In the recent past, advances in
the selective inhibition of BcreAbl kinase activity led to the
* Corresponding author. Tel.: þ39 010 3538866; fax: þ39 010 3538358.
E-mail address: schensil@unige.it (S. Schenone).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.034

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S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
development of Imatinib mesylate (Gleevec) that now repre-
sents the first line treatment for CML. Unfortunately, either
overexpression or mutations of BcreAbl confer resistance to
Gleevec. As a consequence, there is a growing interest in the
development of second generation small molecule inhibitors,
able to treat Gleevec resistant CML [8e11].
It has been recently demonstrated that Src kinase activity
remains active following Imatinib inhibition of BcreAbl in
leukemic cells [12], and that simultaneous inhibition of both
BcreAbl and c-Src kinases results in long-term survival of
mice with AML [13].
These reasons, together with the striking similarity between
the catalytically active states of the c-Src and Abl kinases,
prompted medicinal chemists, helped by computational
methods, to synthesize dual Src/Abl inhibitors that might be
active against Philadelphia positive forms of leukemia [14].
Actually, structurally different compounds identified as c-Src
inhibitors were subsequently also described as Abl inhibitors
[15]. The most recent and important dual Src/Abl inhibitors
include the pyrido-pyrimidine PD166326 1 [16], the purine
AP23846 2, by Ariad Pharmaceuticals [17], the anilinoquina-
zoline AZD0530 3, by AstraZeneca [18], the quinolinecarbo-
nitrile SKI-606 (bosutinib) 4 by Wyeth [19e21] and its recent
furyl derivatives [22], the benzotriazine 5 [23]; finally the thi-
azole-carboxamide BMS-354825 6 (dasatinib), by Bristol-
Myers Squibb [24e26], was approved by the FDA in 2006
for the treatment of CML [27] (Fig. 1).
In this context, in continuing our efforts to find new anti-
cancer agents, we have recently synthesized several pyra-
zolo[3,4-d]pyrimidines that showed inhibition properties
toward Src in a cell-free assay, as well as antiproliferative
activity toward the epidermoid (A431) and breast cancer
(BC-8701) cell lines, blocking Src phosphorylation and in-
ducing apoptosis. In particular, compound 7a (Table 1)
showed a submicromolar activity toward isolated Src and
an inhibitory activity about two-fold higher than that of the
O
O
O
P
Cl
HN
N
O
O
Cl
N
NH
N
N
Cl
N
N
HO
N
O
N
H
N
N
O
N
N
2 AP23846
3 AZD0530
1 PD166326
Cl
Cl
O
Cl
HN
N
N
O
N
N
CN
O
Cl
N
H
N
O
N
N
5
4 SKI-606
bosutinib
Cl
H
N
N
N
N
S
N
H
O
N
Cl
N
OH
6 BMS-354825
dasatinib
Fig. 1. Structures of Abl/Src dual inhibitors.

S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
2667
Table 1
Structure and inhibitory activity of compounds 7aem, 8aeq, 9aeh and 10aec toward isolated Abl and Src kinases
R1
N
N
N
N
R
R2
Compd.
R
R1
R2
Src enzyme
Kia (mM)
Abl enzyme
Kib (mM)
7ac
7bc
7cc
7dc
7ec
7fc
7gc
7hc
7ic
7lc
7m
8a
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SC2H5
SC3H7
SC3H7
SC3H7
SC3H7
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
N(CH3)2
N(CH3)2
N(CH3)2
NHCH2CH2C6H5
NHCH2C6H5
NHC3H7
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H4-pF
CH2eCHCleC6H4-pF
CH2eCHCleC6H4-pF
CH2eCHCleC6H4-pF
CH2eCHCleC6H4-pF
CH2eCHCleC6H4-pF
CH2eCHCleC6H4-pF
CH2eCHCleC6H4-pCl
CH2eCHCleC6H4-pCl
CH2eCHCleC6H4-pCl
CH2eCHCleC6H4-pCl
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHBreC6H5
CH2eCHBreC6H5
CH2eCHBreC6H5
CH2eCHBreC6H5
CH2eCHBreC6H5
CH2eCHBreC6H5
CH2eCHBreC6H5
CH2eCHBreC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
CH2eCHCleC6H5
0.7 ꢀ 0.2
3.7 ꢀ 0.9
2.9 ꢀ 0.8
1.7 ꢀ 0.4
NA
7.3 ꢀ 1.2
0.3 ꢀ 0.1
4.8 ꢀ 0.6
1.2 ꢀ 0.3
1.5 ꢀ 0.5
NHC4H9
NH(CH2)2OC2H5
1-Pyrrolidinyl
1-Piperidinyl
4-Morpholinyl
N(C2H5)2
ND
NA
2.4 ꢀ 0.7
6.5 ꢀ 1.0
0.5 ꢀ 0.1
1.2 ꢀ 0.3
1.4 ꢀ 0.3
10.2 ꢀ 1.8
2.61 ꢀ 1.2
5.02 ꢀ 0.9
3.32 ꢀ 1.0
0.31 ꢀ 0.07
0.30 ꢀ 0.06
3.25 ꢀ 0.8
0.21 ꢀ 0.02
1.32 ꢀ 0.4
1.53 ꢀ 0.6
1.62 ꢀ 0.5
3.21 ꢀ 0.7
5.28 ꢀ 0.5
3.60 ꢀ 0.4
2.56 ꢀ 0.7
5.37 ꢀ 0.9
0.51 ꢀ 0.1
2.55 ꢀ 0.5
0.81 ꢀ 0.3
5.66 ꢀ 0.9
1.63 ꢀ 0.4
0.32 ꢀ 0.03
2.57 ꢀ 0.7
0.22 ꢀ 0.01
2.80 ꢀ 0.4
4.10 ꢀ 0.5
3.63 ꢀ 0.4
4.22 ꢀ 0.5
0.50 ꢀ 0.1
31.0 ꢀ 2.2
NA
NA
0.40 ꢀ 0.05
NHC6H5
0.40 ꢀ 0.07
0.40 ꢀ 0.1
0.40 ꢀ 0.1
0.57 ꢀ 0.1
0.11 ꢀ 0.02
0.97 ꢀ 0.3
0.22 ꢀ 0.03
0.34 ꢀ 0.02
0.20 ꢀ 0.04
0.22 ꢀ 0.04
0.40 ꢀ 0.03
0.39 ꢀ 0.06
0.30 ꢀ 0.1
0.50 ꢀ 0.05
0.33 ꢀ 0.1
0.25 ꢀ 0.04
0.52 ꢀ 0.08
0.28 ꢀ 0.01
0.10 ꢀ 0.01
0.88 ꢀ 0.2
0.32 ꢀ 0.04
0.55 ꢀ 0.04
1.40 ꢀ 0.3
0.88 ꢀ 0.4
0.97 ꢀ 0.3
0.19 ꢀ 0.03
0.27 ꢀ 0.03
0.32 ꢀ 0.04
0.28 ꢀ 0.02
0.16 ꢀ 0.01
0.52 ꢀ 0.2
0.013 ꢀ 0.005
NHC6H4-mF
NHCH2CH2C6H4-oF
NHC3H7
8b
8c
NHC4H9
8d
8e
4-Morpholinyl
NHCH2C6H4-pF
NHCH2C6H4-oF
NHCH2CH2C6H5
NHC6H4-mF
NHC3H7
8f
8g
8h
8i
8j
8k
NHC4H9
N(C2H5)2
8l
4-Morpholinyl
N(C2H5)2
NHC3H7
8m
8n
8o
8p
NHC4H9
N(C2H5)2
8q
9a
NHCH2C6H5
NHC3H7
NHC4H9
9b
9c
NH(CH2)2OC2H5
1-Pyrrolidinyl
1-Piperidinyl
4-Morpholinyl
NHCH2C6H5
NHCH2CH2C6H5
NHC3H7
9d
9e
9f
9g
9h
10a
10b
10c
PP2
Imatinib
a
4-Morpholinyl
NHCH2C6H5
Ki values toward isolated Src calculated according to the following equation: Ki ¼ (ID50 ꢁ E0/2)/{E0 ꢁ [S0/Km(ATP) ꢁ 1]/E0}, where E0 and S0 are the enzyme
and the ATP concentrations (0.0125 and 0.0160 mM, respectively). NA ¼ not active (ID50 >2 mM). ND ¼ not determined.
b
Ki values toward isolated Abl calculated according to the following equation: Ki ¼ ID50/{E0þ[E0(Km(ATP)/S0)]}/E0, where E0 and S0 are the enzyme and the
ATP concentrations (0.005 and 0.012 mM, respectively). NA ¼ not active (ID50 >2 mM). ND ¼ not determined.
c
Structures and inhibitory activity of previously published compounds [29,30].
reference compound 4-amino-5-(4-chlorophenyl)-7-(t-butyl)-
pyrazolo[3,4-d]pyrimidine (PP2) [28,29]. Successively, based
on the fact that compounds acting as c-Src inhibitors often
also showed activity toward BcreAbl, we tested a set of
our pyrazolo[3,4-d]pyrimidine derivatives on Abl isolated
enzyme and on a panel of leukemia cell lines: the studied
compounds were able to inhibit BcreAbl and c-Src
phosphorylation, induced apoptosis and, as the activation of
Src and Abl is an important step in the progression of leuke-
mia cells (in particular, CML) [30], reduced cell
proliferation.
We are reporting here the synthesis of a new series of
4-amino substituted pyrazolo[3,4-d]pyrimidines bearing a
2-chloro-2-phenylethylamino N1 side chain, present in the

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S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
most active compounds previously reported by us. We de-
cided to insert a halogen atom (F or Cl) on the N1 phenyl
ring side chain in a number of compounds (8bel), to verify
the possible influence of such substitution on the activity.
Moreover in derivatives 9aeh the chlorine atom on the C2
of side chain was replaced with a bromine. Thio-methyl,
-ethyl and -propyl groups at C6 have been maintained, the
only exception being derivatives 10aec, where a dimethyla-
mino group has been inserted at C6, using a different chem-
ical approach. The newly synthesized compounds have been
tested in cell-free enzymatic assays against c-Src and Abl
and the most active derivatives of the series have been also
tested for their antiproliferative effects on BcreAbl-express-
ing cells, namely the CML K-562 cells and the basophilic
leukemia KU-812 cells.
of 2-hydrazino-1-phenylethanol and ethoxymethylenemalono-
nitrile in ethanol at reflux as previously reported [33]. Interme-
diate 15 was reacted with N,N-dimethylphosgeniminium
chloride in dichloroethane at reflux for 2 h to give the corre-
sponding dimethylcarbamimidic chloride 16, then cyclized
with anhydrous hydrochloric acid to the 4-chloro-1-(2-
chloro-2-phenylethyl)-N,N-dimethyl-1H-pyrazolo[3,4-d]pyri-
midin-6-amine 17 in satisfactory yield (65%). C4 chlorine
atom was substituted by reaction with the appropriate amines
in anhydrous toluene at room temperature for 24 h to give the
desired compounds 10aec (Scheme 3).
3. Biology
The affinity toward c-Src and Abl TKs of the newly synthe-
sized compounds was evaluated in cell-free assays. Enzymatic
data are reported in Table 1 along with those of the reference
compounds PP2 and Imatinib. Three of the most interesting
molecules were then tested to assess their effects on the pro-
liferation of two BcreAbl positive leukemia cell lines, namely
K-562 and KU-812, and their activity was compared to that of
PP2, chosen as reference compound. In fact, PP2 inhibits ei-
ther Src [34] or Abl TK [35] with similar potency, thus result-
ing an appropriate reference when the activity toward both
kinases is to be compared. Moreover, a huge amount of liter-
ature has been recently published on PP2, giving us exhaustive
information on its biological profile toward both TKs; finally,
PP2 is an easily available commercial compound. Assays on
proapoptotic activity of selected compounds have been per-
formed as well.
2. Chemistry
The synthesis of derivatives 8aeq, 9aeh and 10aec are
depicted in Schemes 1, 2 and 3, respectively.
Compounds 8aeq were prepared starting from 1-
substituted-4-chloro-6-(alkylthio)-1H-pyrazolo[3,4-d]pyrimi-
dines 11aee, obtained in four steps from the appropriate ethyl
5-amino-1-[2-phenyl or 2-(4-halophenyl)-2-hydroxyethyl]-
1H-pyrazole-4-carboxylates, following our reported procedure
[31,32]. Regioselective substitution of the C4 chlorine atom
with an excess of various amines in anhydrous toluene at
room temperature afforded the desired compounds 8aeq in
high yields (Scheme 1).
Treatment of intermediate 12 [31] with phosphorous tribro-
mide and pyridine in anhydrous toluene and anhydrous DMF
afforded the 1-(2-bromo-2-phenylethyl)-6-(methylthio)-1,5-
dihydro-4H-pyrazolo[3,4ed]pyrimidin-4-one 13, that was in
turn treated with the Vilsmeier complex (POCl3:DMF 1:1,
15 equiv) in CHCl3 to obtain the C4 chlorinated derivative
14. This compound was purified in good yield by chromatog-
raphy on Florisil column. Finally, reaction of 14 with an ex-
cess of various amines in anhydrous toluene at room
temperature afforded compounds 9aeh (Scheme 2).
4. Results and discussion
4.1. Enzymatic assays
Compounds 8aeq, 9aeh, and 10aec showed interesting
dual Src/Abl activity, with a general improvement in the affin-
ity toward both the enzymes as compared with that previously
reported by us [29,30], even if generally resulted more active
toward Abl than c-Src when tested in cell-free assays (Table 1).
In more detail, they possessed Ki values lower than 0.5 mM for
Abl, the only exceptions being 8d, 9a, 9d, 9e and 9f, whose Ki
The starting product for the synthesis of dimethylamino de-
rivatives 10aec is 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-
pyrazole-4-carbonitrile 15, obtained in good yield by reaction
Cl
R1
N
N
N
N
(a)
Cl
N
N
R
N
Cl
N
R
11a R = SCH3, Y = H
11b R = SC2H5, Y = H
11c R = SC3H7, Y = H
11d R = SCH3, Y = F
11e R = SCH3,Y = Cl
8a-q
Y
Y
Scheme 1. a) Amines, toluene, r.t.

S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
2669
O
O
HN
HN
N
N
(a)
N
(b)
OH
N
Br
S
N
N
S
CH3
CH3
12
13
Cl
R1
N
N
N
(c)
N
N
Br
Br
N
N
N
S
S
CH3
CH3
14
9a-h
Scheme 2. (a) PBr3, Py, toluene, DMF, r.t.; (b) POCl3/DMF, CHCl3, reflux; (c) amines, toluene, r.t.
values ranged between 0.88 and 1.40 mM. The most active
derivatives were 8c, 8e, 8g, 8h, 8n, 8q, 9g and 10c, with Ki
values of 0.11, 0.22, 0.20, 0.22, 0.25, 0.10, 0.19 and
0.16 mM, respectively, but also 8f, 8k, 8m, 8p, 9b, 9h, 10a
and 10b showed interesting potency with Ki values lower
than 0.35 mM.
Regarding Abl inhibition, the most important structural fea-
ture was represented by the amino-substituent on C4; in fact,
a benzylamino group in such position conferred a higher activ-
ity (see 8e, 8q, 9g and 10c), independently from the C6 and
the N1 side chain. The meta-fluoro anilino derivative 8h and
the phenylethylamino derivatives 8g and 9h (Ki values of
0.22, 0.20 and 0.27 mM, respectively) showed a good potency,
pointing out the importance of a phenyl-substituted side chain
at C4. Short aliphatic amino or cyclo-amino groups generally
induced a drop in the activity, with the notable exception of the
butylamino-derivative 8c, possessing a Ki value of 0.11 mM;
this compound was one of the most potent compounds of
this series, together with 10b (0.28 mM), bearing a morpholino
substituent group at C4. As a general trend, the 2-bromine
atom of the 2-halo-2-phenylethyl chain at N1 is preferable
to chlorine; moreover, among the 2-chloro-2-phenylethyl de-
rivatives, a para-fluoro or para-chloro phenyl substitution at
the N-1 side chain was present in a number of active com-
pounds, namely 8c, 8e, 8g, 8h and 8k. Overall, the new deriv-
atives bearing a halogen substitution on the phenyl ring of the
N1 side chain were more active on Abl when compared to
those unsubstituted and previously reported by us [30]. As
an example, the C4 butylamino-1-(2-chloro-2-phenylethyl)-
6-methylthio derivative 7d possessed a Ki of 1.2 mM, while
NC
NC
N
OH
N
N
N
(b)
(a)
OH
N
H2N
H3C
N
Cl
CH3
15
16
Cl
N
R1
N
N
N
(c)
N
H3C
H3C
N
Cl
N
CH3
N
Cl
N
N
CH3
17
10a-c
Scheme 3. a) N,N-Dimethylphosgeniminium chloride, ClCH2CH2Cl, reflux; (b) HCl (g), ClCH2CH2Cl, r.t.; (c) amines, toluene, r.t.

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S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
the corresponding para-fluoro 8c and para-chloro 8j showed
Ki values of 0.11 and 0.39 mM, respectively; the same behav-
iour was detected in the n-propyl derivatives, for which the ac-
tivity increased by an order of magnitude (4.8 mM for the
unsubstituted derivative 7c and 0.57 and 0.40 mM for the
para-fluoro derivative 8b and for the para-chloro 8i derivative,
respectively).
The same compounds, besides showing interesting antipro-
liferative activity, were also able to inhibit BcreAbl phosphor-
ylation with an activity comparable to PP2 on both cell lines
(data not shown).
4.3. Proapoptotic activity
As regards the activity toward c-Src, 8e, 8f, 8h, 8q, 9e and
9g emerged as the most potent compounds of the series, with
Ki values of 0.31, 0.30, 0.21, 0.51, 0.32 and 0.22 mM, respec-
tively; noteworthy, also in this case the importance of a benzy-
lamino or anilino-group on C4 (the only exception being the
piperidino-derivative 9e) and of the 2-chloro-2-(4-fluorophe-
nyl)ethyl chain at N1 was confirmed.
Also the C-6 substitution seemed to play a fundamental role
for Src activity: in fact, the activity decreased with the length-
ening of the S-alkyl group from methyl to ethyl or propyl, with
the only exception of the benzylamino derivative 8q, endowed
with a Ki value of 0.51 mM toward c-Src.
A similar trend was observed for derivatives 10aec, bear-
ing at C6 a bulkier dimethylamino group, which showed
a drop in activity when compared with the corresponding
thio-methyl compounds.
Derivative 9g has been also tested on a panel of TKs show-
ing selectivity for Src and Abl [36].
Given the antiproliferative activity showed by compounds
8c, 8q and 9g on both K-562 and KU-812 cell lines, the
same agents were subsequently tested for their proapoptotic
activity on a poly-ADP-Ribose-Polymerase (PARP) assay
[29]. Compounds 8c, 8q and 9g potently induced apoptosis
in KU-812 cell line, with cleaved/uncleaved PARP ratio higher
than that of PP2 (39, 33, 35 and 20% for 8c, 8q, 9g and PP2,
respectively). Also on K-562 cell line 8c, 8q and 9g showed
significant proapoptotic activity (24, 22, 25 and 23% for 8c,
8q, 9g and PP2, respectively), though lower in comparison
to that showed on KU-812 cell line. On the basis of the ability
of 9g to induce apoptosis in both cell lines, we also investi-
gated the expression of Bax/Bcl-xL mRNA in KU-812 cells
treated with this compound.
It is known that molecular mechanisms of apoptosis involve
change in expression of distinct genes. As an example, the
ratio between proapoptotic Bax and antiapoptotic Bcl-2 or
Bcl-xL genes is a critical determinant to induce cells toward
apoptosis [37]. Death signals induce an increase in expression
of the Bax mRNA message, leading Bax itself to gain proa-
poptotic activity [38]. On the other hand, the proapoptotic
action of Bax is antagonized by Bcl-2 and BcL-xL, which
are both able to inhibit the release of cytochrome C from
mitochondria. In this context, BcreAbl participates in mainte-
nance of an antiapoptotic environment through the regulation
of Bcl-2 antiapoptotic members [39]. For the above reasons,
the ratio between Bax mRNA and Bcl-xL mRNA expression
may be used as a direct index of the induction of the apoptotic
process and could be useful in explaining the molecular mech-
anism of apoptosis induction. Incubation of KU-812 cells for
72 h in the presence of the inhibitor 9g led to an increase in
the Bax/Bcl-xL ratio (that was 100 for the medium control
and about 280 in the presence of 50 mM of 9g).
4.2. Inhibition of proliferation
Compounds that showed lower Ki values on Abl isolated
enzyme, namely 8c, 8q and 9g, were also evaluated for their
inhibitory activity on two leukemia cell lines, using PP2 as
a reference compound. Results of antiproliferative assays
(Table 2) showed that the selected compounds also possessed
an interesting cellular activity, in particular toward the K-562
cell line, with IC50 values comparable with those of the refer-
ence compound. However, it is important to note that the im-
proved enzymatic potency of pyrazolo-pyrimidine derivatives
with respect to PP2 had no marked effect on the cellular activ-
ity. Experimental and theoretical pharmacokinetic data regard-
ing pyrazolo-pyrimidines similar to those reported herein
suggested that for some of the studied compounds the scarce
activity in cellular assay may be due to their metabolic insta-
bility or, alternatively, to their low solubility in the assay con-
ditions, which in part would prevent compounds from entering
cells [32].
4.4. Modeling studies
Docking studies have been performed on the novel pyra-
zolo-pyrimidines to explore the structural features responsible
for their activity toward both c-Src and Abl kinases. Likewise
previous molecules of the same series, compounds 8aeq, 9ae
h and 10aec were shown to act as ATP competitive inhibitors
[29,30]; they were therefore subjected to focused docking ex-
periments within the ATP binding site of the two target en-
zymes. Computational analysis was performed by using
a recently deposited crystallographic structure of Src (PDB en-
try: 1YOL) [40]; it involved the same procedures previously
applied by our research group to address an analogous task
[29,30,41] and provided very similar results, as expected.
In fact, for all derivatives reported herein we identified the
binding mode that we have already described for other
Table 2
Inhibitory activity of compounds 8c, 8q and 9g on human leukemia K-562 and
KU-812 cell lines
a
IC50 (mM)
K-562
KU-812
PP2
8c
8q
9g
a
24.8 ꢀ 0.9
29.2 ꢀ 1.5
27.3 ꢀ 1.0
25.0 ꢀ 1.3
45.2 ꢀ 2.9
37.5 ꢀ 4.1
67.1 ꢀ 1.8
35.9 ꢀ 2.0
IC50 values are means ꢀ SD of five experiments, each performed in
triplicate.

S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
2671
6-substituted pyrazolo-pyrimidines of the same family. In de-
tail, in both enzymes compounds 8aeq, 9aeh and 10aec lo-
cated their pyrazolo-pyrimidine nucleus within the adenine
region of the ATP binding site, directing the side chains at
C4 and N1 toward two hydrophobic regions, labeled as hydro-
phobic regions I and II, and the alkylthio substituent toward
the external region of the binding pocket, though still involved
in hydrophobic contacts. However, whereas the hydrophobic
region I in Src was occupied by C4 substituents, the same re-
gion in Abl hosted the N1 side chain. Conversely, the hydro-
phobic pockets II of c-Src and Abl were contacted by
substituents at N1 and C4, respectively. Finally, some differ-
ences also involved the location of the alkylthio groups, which
occupied an additional small hydrophobic region in c-Src
while sharing the hydrophobic region II with the side chain
at N1 in the case of Abl. Concerning the hydrogen bonds,
two interactions were identified between the studied molecules
and the Src binding site (the second hydrogen bond was ob-
served only for compounds bearing a secondary amino group,
which could act as hydrogen bond donor, at C4) whereas only
one interaction characterized the binding with Abl. As an ex-
ample, Fig. 2 reports a schematic representation of the binding
models proposed for 8h, one of the most interesting com-
pounds as dual inhibitor, for both c-Src and Abl.
particular, such compounds showed good enzymatic activity
toward Abl, suggesting that some of them could be effective
agents in the chemotherapy armamentarium against CML.
However, at the moment we cannot exclude that their anti-
proliferative activity could in part derive from targeting
other tyrosine kinases, and further experiments are therefore
ongoing to better investigate the mechanism of action of
such compounds.
6. Experimental protocols
6.1. Chemistry
Starting materials were purchased from Aldrich-Italia (Mi-
lan, Italy). Melting points were determined with a Buchi 530
¨
apparatus and are uncorrected. IR spectra were measured in
KBr with a PerkineElmer 398 spectrophotometer.
spectra were recorded in a (CD3)2SO solution on a Varian
Gemini 200 (200 MHz) instrument. Chemical shifts are re-
ported as d (ppm) relative to TMS as internal standard, J in hertz.
1H patterns are described using the following abbreviations:
s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, quint ¼ quintet,
sx ¼ sextet, m ¼ multiplet, br ¼ broad.
1H NMR
All compounds were tested for purity by TLC (Merck,
Silica gel 60 F254, CHCl3 as the eluant).
Analyses for C, H, N and S were within ꢀ0.3% of the the-
5. Conclusion
oretical value.
The inhibition properties of some new pyrazolo[3,4-d]py-
rimidines toward Abl and c-Src in cell-free assays along
with their antiproliferative activity toward two human leuke-
mia cell lines are reported, demonstrating that some com-
pounds (8e, 8f, 8h, 8q and 9g) are interesting dual
inhibitors of Abl/Src and could be important hits in the field
of antileukemic agents, as they probably affect a pathway
that is pivotal for the growth of transformed cells. In
6.1.1. 1-(2-Bromo-2-phenylethyl)-6-(methylthio)-
1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one 13
To a mixture of 1-(2-hydroxy-2-phenylethyl)-6-(methylthio)-
1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one 12 (3.02 g,
10 mmol) [31] in anhydrous dimethylformamide (DMF)
(20 mL) was added dropwise at 0 ꢂC a solution of PBr3
(3.27 g, 12.1 mmol), pyridine (0.5 mL) and anhydrous toluene
Fig. 2. Schematic representation of the binding mode of compound 8h into the ATP binding site of (a) Src: 8h establishes two hydrogen bonds (black dashed lines)
with Src, the first between the amino group at C4 and the side chain of Thr340, and the second between the N2 and the NH backbone of Met343 and (b) Abl: 8h is
engaged in a single hydrogen bond involving the C4 amino group and the carbonyl backbone of Met318. For the sake of clarity, only a few residues among those
interacting with the compound are displayed. The figure shows that the location of side chains at N1 and C4 is inverted in the two enzymes, and that the alkylthio
moiety occupies a slightly different position within the two binding pockets.

2672
S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
(5 mL). The mixture was stirred at room temperature for 60 h.
Toluene was removed under reduced pressure, then the mix-
ture was poured into cold water; standing in a refrigerator
for 12 h, a white solid was obtained, then filtered, washed
with H2O and purified by column chromatography (silicagel,
100e200 mesh) using as the eluant a mixture of CHCl3/
CH3OH (9:1), to afford the pure product 13 (2.37 g, 65%) as
a white solid, mp 216e217 ꢂC. 1H NMR: d 2.61 (s, 3H,
SCH3), 4.86e5.00 (m, 2H, CH2N), 5.64e5.73 (m, 1H,
CHBr), 7.25e7.56 (m, 5H Ar), 7.99 (s, 1H, H-3), 12.35 (br
s, 1H, NH disappears with D2O). IR cmꢁ1: 3100e2800
(NH), 1671 (CO). Anal. C14H13N4OBrS (C, H, N, S).
mesh), using a mixture of diethyl ether/petroleum ether (bp
40e60 ꢂC) (1:1) as the eluant, to afford the pure product 17
1
(2.18 g, 65%) as a white solid, mp 118e119 ꢂC. H NMR:
d 3.25 (s, 6H, 2CH3), 4.61e4.84 (m, 2H, CH2N), 5.38e5.67
(m, 1H, CHCl), 7.17e7.56 (m, 5H Ar), 7.82 (s, 1H, H-3).
Anal. C15H15N5Cl2 (C, H, N).
6.1.5. General procedure for 4-amino-1H-
pyrazolo[3,4-d]pyrimidines 8aeq, 9aeh, 10aec
To a solution of 11aee, 14, 17 (5 mmol) in anhydrous tol-
uene (20 mL), the proper amine (20 mmol) was added. The re-
action mixture was stirred at room temperature for 24e36 h
and then extracted with H2O (2 ꢃ 20 mL). The organic phase
was dried (MgSO4), filtered and concentrated under reduced
pressure. The oily residue was crystallized by adding diethyl
ether (10 mL) to give the final products as white solids.
Compound 8a. White solid, yield 81%, mp 111e112 ꢂC. 1H
NMR: d 2.49 (s, 3H, SCH3), 2.93 (t, J ¼ 6.8, 2H, CH2Ar), 3.74
(q, J ¼ 6.8, 2H, CH2NH), 4.58e4.86 (m, 2H, CH2N), 5.38e
5.52 (m, 1H, CHCl), 6.86e7.43 (m, 9H Ar), 7.63 (s, 1H, H-
3). IR cmꢁ1: 3371 (NH). Anal. C22H21N5ClFS (C, H, N, S).
Compound 8b. White solid, yield 60%, mp 108e109 ꢂC. 1H
NMR: d 1.03 (t, J ¼ 7.2, 3H, CH3), 1.74 (sx, J ¼ 7.2, 2H,
CH2CH3), 2.59 (s, 3H, SCH3), 3.56 (q, J ¼ 7.2, 2H,
CH2NH), 4.72e4.96 (m, 2H, CH2N), 5.26 (br s, 1H, NH, dis-
appears with D2O), 5.48e5.59 (m, 1H, CHCl), 6.94e7.08 and
7.26e7.46 (2m, 4H Ar), 7.78 (s, 1H, H-3). IR cmꢁ1: 3241
(NH). Anal. C17H19N5ClFS (C, H, N, S).
6.1.2. 1-(2-Bromo-2-phenylethyl)-4-chloro-6-
(methylthio)-1H-pyrazolo[3,4-d]pyrimidine 14
The Vilsmeier complex, previously prepared from POCl3
(23 g, 150 mmol) and anhydrous DMF (10.97 g, 150 mmol)
was added to a suspension of 1-(2-bromo-2-phenylethyl)-6-
(methylthio)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one
13 (3.65 g, 10 mmol) in CHCl3 (50 mL). The mixture was re-
fluxed for 12 h. After cooling, the solution was washed with
H2O (2 ꢃ 20 mL), dried (MgSO4), filtered, and concentrated
under reduced pressure. The crude oil was purified by column
chromatography (Florisil, 100e200 mesh) using diethyl ether
as the eluant, and afforded the pure product 14, which crystal-
lized on standing in a refrigerator by adding a mixture of di-
ethyl ether/petroleum ether (bp 40e60 ꢂC) (1:1) as a light
1
yellow solid (3.57 g, 93%), mp 89e90 ꢂC. H NMR: d 2.66
(s, 3H, SCH3), 4.78e5.16 (m, 2H, CH2N), 5.47e5.62 (m,
1H, CHBr), 7.25e7.47 (m, 5H Ar), 8.02 (s, 1H, H-3). Anal.
C14H12N4ClBrS (C, H, N, S).
Compound 8c. White solid, yield 65%, mp 108e109 ꢂC. 1H
NMR: d 0.99 (t, J ¼ 7.2, 3H, CH3), 1.47 (sx, J ¼ 7.2, 2H,
CH2CH3), 1.67 (quint, J ¼ 7.2, 2H, CH2CH2CH3), 2.60 (s,
3H, SCH3), 3.60 (q, J ¼ 7.2, 2H, CH2NH), 4.70e4.94 (m,
2H, CH2N), 5.47e5.58 (m, 1H, CHCl), 6.94e7.06 and
7.36e7.44 (2m, 4H Ar), 7.77 (s, 1H, H-3). IR cmꢁ1: 3239
(NH). Anal. C18H21N5ClFS (C, H, N, S).
Compound 8d. White solid, yield 70%, mp 147e148 ꢂC. 1H
NMR: d 2.58 (s, 3H, SCH3), 3.81e3.88 and 3.90e4.00 (2m,
8H, 4CH2 morph), 4.72e4.96 (m, 2H, CH2N), 5.46e5.59
(m, 1H, CHCl), 6.95e7.09 and 7.34e7.46 (2m, 4H Ar),
7.82 (s, 1H, H-3). Anal. C18H19N5OClFS (C, H, N, S).
Compound 8e. White solid, yield 85%, mp 151e152 ꢂC. 1H
6.1.3. N0-[4-Cyano-1-(2-hydroxy-2-phenylethyl)-
1H-pyrazol-5-yl]-N,N-dimethylcarbamimidic chloride 16
To a solution of 5-amino-1-(2-hydroxy-2-phenylethyl)-1H-
pyrazole-4-carbonitrile 15 (2.28 g, 10 mmol) in 1,2-dichloro-
ethane (50 mL), N,N-dimethylphosgeniminium chloride
(6.5 g, 40 mmol) was added and the solution was refluxed
for 2 h. The solvent was removed by distillation under reduced
pressure, the oily residue was crystallized by adding diethyl
ether (10 mL). The yellow solid obtained was purified by re-
crystallization with absolute ethanol to afford the pure product
NMR:
d 2.51 (s, 3H, SCH3), 4.60e4.86 (m, 4H,
1
16 (2.22 g, 70%) as a pale yellow solid, mp 123e124 ꢂC. H
CH2N þ CH2Ar), 5.35e5.50 (m, 1H, CHCl), 5.55 (br s, 1H,
NH, disappears with D2O), 6.82e7.04 and 7.11e7.57 (2m,
8H Ar), 7.63 (s, 1H, H-3). IR cmꢁ1: 3294 (NH). Anal.
C21H18N5ClF2S (C, H, N, S).
NMR: d 3.26 (s, 6H, 2CH3), 4.36e4.62 (m, 2H, CH2N),
5.26e5.37 (m, 1H, CHO), 7.24e7.38 (m, 5H Ar), 7.67 (s,
1H, H-3). IR cmꢁ1: 3443 (OH), 2224 (C^N). Anal.
C15H16N5ClO (C, H, N).
Compound 8f. White solid, yield 80%, mp 151e152 ꢂC. 1H
NMR:
d 2.55 (s, 3H, SCH3), 4.60e4.86 (m, 4H,
6.1.4. 4-Chloro-1-(2-chloro-2-phenylethyl)-N,N-
dimethyl-1H-pyrazolo[3,4-d]pyrimidin-6-amine 17
CH2N þ CH2Ar), 5.32e5.47 (m, 1H, CHCl), 6.86e7.36 (m,
8H Ar), 7.74 (s, 1H, H-3). IR cmꢁ1: 3278 (NH). Anal.
C21H18N5ClF2S (C, H, N, S).
Compound 8g. White solid, yield 61%, mp 121e122 ꢂC. 1H
NMR: d 2.61 (s, 3H, SCH3), 3.01 (t, J ¼ 7.2, 2H, CH2Ar), 3.87
(q, J ¼ 7.2, 2H, CH2NH), 4.70e4.89 (m, 2H, CH2N), 5.36 (br
s, 1H, NH disappears with D2O), 5.46e5.59 (m, 1H, CHCl),
6.92e7.05 and 7.18e7.45 (2m, 9H Ar), 7.72 (s, 1H, H-3).
IR cmꢁ1: 3252 (NH). Anal. C22H21N5ClFS (C, H, N, S).
In a solution of N0-[4-cyano-1-(2-hydroxy-2-phenylethyl)-
1H-pyrazol-5-yl]-N,N-dimethylcarbamimidic chloride (3.17 g,
10 mmol) in 1,2-dichloroethane (50 mL), anhydrous hydro-
chloric acid was bubbled for 4 h and the solution was stirred
at room temperature for 2 days. The solvent was removed
by distillation under reduced pressure to afford a crude that
was purified by column chromatography (silica gel, 100

S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
2673
Compound 8h. White solid, yield 55%, mp 250e251 ꢂC.
1H NMR: d 2.56 (s, 3H, SCH3), 4.64e4.87 (m, 2H, CH2N),
5.36e5.50 (m, 1H, CHCl), 6.82e7.53 (m, 9H, 8Ar þ H-3).
IR cmꢁ1: 3098 (NH). Anal. C20H16N5ClF2S (C, H, N, S).
Compound 8i. White solid, yield 65%, mp 124e125 ꢂC. 1H
NMR: d 1.04 (t, J ¼ 7.4, 3H, CH3), 1.73 (sx, J ¼ 7.4, 2H,
CH2CH3), 2.59 (s, 3H, SCH3), 3.56 (q, J ¼ 7.4, 2H,
CH2NH), 4.74e4.90 (m, 2H, CH2N), 5.46e5.58 (m, 1H,
CH2N þ CH2Ar), 5.38e5.51 (m, 1H, CHCl), 7.06e7.38 (m,
10H Ar), 7.65 (s, 1H, H-3). IR cmꢁ1: 3250 (NH). Anal.
C23H24N5ClS (C, H, N, S).
Compound 9a. White solid, yield 50%, mp 123e124 ꢂC. 1H
NMR: d 1.02 (t, J ¼ 7.2, 3H, CH3), 1.71 (sx, J ¼ 7.2, 2H,
CH2CH3), 2.61 (s, 3H, SCH3), 3.54 (q, J ¼ 7.2, 2H,
CH2NH), 4.84e5.09 (m, 2H, CH2N), 5.40 (br s, 1H, NH dis-
appears with D2O), 5.57e5.67 (m, 1H, CHBr), 7.20e7.54 (m,
5H Ar), 7.77 (s, 1H, H-3). IR cmꢁ1: 3239 (NH). Anal.
C17H20N5BrS (C, H, N, S).
CHCl), 7.21e7.43 (m, 4H Ar), 7.77 (s, 1H, H-3). IR cmꢁ1
:
3269 (NH). Anal. C17H19N5Cl2S (C, H, N, S).
Compound 8j. White solid, yield 77%, mp 119e120 ꢂC. 1H
NMR: d 0.97 (t, J ¼ 7.4, 3H, CH3), 1.47 (sx, J ¼ 7.4, 2H,
CH2CH3), 1.65 (quint, J ¼ 7.4, 2H, CH2CH2CH3), 2.59 (s,
3H, SCH3), 3.59 (q, J ¼ 7.4, 2H, CH2NH), 4.64e4.93 (m,
2H, CH2N), 5.31 (br s, 1H, NH disappears with D2O), 5.42e
5.58 (m, 1H, CHCl), 7.11e7.54 (m, 4H Ar), 7.77 (s, 1H, H-
3). IR cmꢁ1: 3246 (NH). Anal. C18H21N5Cl2S (C, H, N, S).
Compound 9b. White solid, yield 66%, mp 97e98 ꢂC. H
1
NMR: d 0.98 (t, J ¼ 7.4, 3H, CH3), 1.37e1.45 and 1.61e
1.82 (2m, 4H, 2CH2 butylam.), 2.61 (s, 3H, SCH3), 3.59 (q,
J ¼ 7.4, 2H, CH2NH), 4.87e5.09 (m, 2H, CH2N), 5.30 (br s,
1H, NH disappears with D2O), 5.57e5.68 (m, 1H, CHBr),
7.20e7.55 (m, 5H Ar), 7.77 (s, 1H, H-3). IR cmꢁ1: 3240
(NH). Anal. C18H22N5BrS (C, H, N, S).
Compound 9c. White solid, yield 61%, mp 126e127 ꢂC. 1H
NMR: d 1.24 (t, J ¼ 7.0, 3H, CH3), 2.61 (s, 3H, SCH3), 3.55
(q, J ¼ 7.0, 2H, CH2CH3), 3.67 (t, J ¼ 4.6, 2H, CH2CH2O),
3.79 (q, J ¼ 4.6, 2H, CH2NH), 4.81e5.08 (m, 2H, CH2N),
5.56e5.68 (m, 1H, CHBr), 5.76 (br s, 1H, NH disappears
with D2O), 7.24e7.53 (m, 5H Ar), 7.77 (s, 1H, H-3). IR
cmꢁ1: 3311 (NH). Anal. C18H22N5OBrS (C, H, N, S).
Compound 9d. White solid, yield 60%, mp 149e150 ꢂC. 1H
NMR: d 1.93e2.25 (m, 4H, 2CH2 pyrr.), 2.62 (s, 3H, SCH3),
3.67e3.87 (m, 4H, 2CH2N pyrr.), 4.85e5.08 (m, 2H, CH2N),
5.60e5.70 (m, 1H, CHBr), 7.15e7.57 (m, 5H Ar), 7.79 (s, 1H,
H-3). Anal. C18H20N5BrS (C, H, N, S).
1
Compound 8k. White solid, yield 66%, mp 67e68 ꢂC. H
NMR: d 1.22 (t, J ¼ 7.0, 6H, 2CH3 diethylam.), 2.50 (s, 3H,
SCH3), 3.65 (q, J ¼ 7.0, 4H, 2CH2 diethylam.), 4.60e4.87
(m, 2H, CH2N), 5.43e5.54 (m, 1H, CHCl), 6.86e7.02 and
7.22e7.41 (2m, 4H Ar), 7.64 (s, 1H, H-3). Anal.
C18H21N5Cl2S (C, H, N, S).
Compound 8l. White solid, yield 79%, mp 109e110 ꢂC. 1H
NMR: d 2.59 (s, 3H, SCH3), 3.80e3.88 and 3.90e4.00 (2m,
8H, 4CH2 morph), 4.72e4.96 (m, 2H, CH2N), 5.47e5.58
(m, 1H, CHCl), 7.24e7.44 (m, 4H Ar), 7.82 (s, 1H, H-3).
Anal. C18H19N5OCl2S (C, H, N, S).
1
Compound 8m. White solid, yield 77%, mp 83e84 ꢂC. H
NMR: d 1.22 (t, J ¼ 7.0, 6H, 2CH3 diethylam.), 1.38 (t,
J ¼ 7.4, 3H, CH3CH2S), 3.10 (q, J ¼ 7.4, 2H, SCH2), 3.64
(q, J ¼ 7.0, 4H, 2CH2 diethylam.), 4.57e4.92 (m, 2H,
CH2N), 5.43e5.57 (m, 1H, CHCl), 7.14e7.46 (m, 5H Ar),
7.66 (s, 1H, H-3). Anal. C19H24N5ClS (C, H, N, S).
Compound 9e. White solid, yield 90%, mp 109e110 ꢂC. 1H
NMR: d 1.47e1.79 (m, 6H, 3CH2 pip.), 2.60 (s, 3H, SCH3),
3.81e3.95 (m, 4H, 2CH2N pip.), 4.85e5.09 (m, 2H, CH2N),
5.61e5.72 (m, 1H, CHBr), 7.25e7.53 (m, 5H Ar), 7.82 (s,
1H, H-3). Anal. C19H22N5BrS (C, H, N, S).
1
1
Compound 8n. White solid, yield 34%, mp 97e98 ꢂC. H
Compound 9f. White solid, yield 90%, mp 98e99 ꢂC. H
NMR: d 0.88e1.09 (m, 6H, 2CH3), 1.54e1.84 (m, 4H,
2CH2), 3.08 (t, J ¼ 7.0, 2H, SCH2), 3.48 (q, J ¼ 6.4, 2H,
CH2NH), 4.60e4.88 (m, 2H, CH2N), 5.16 (br s, 1H, NH dis-
appears with D2O), 5.40e5.52 (m, 1H, CHCl), 7.16e7.40 (m,
5H Ar), 7.70 (s, 1H, H-3). IR cmꢁ1: 3371 (NH). Anal.
C19H24N5ClS (C, H, N, S).
NMR: d 2.61 (s, 3H, SCH3), 3.80e3.89 and 3.90e3.99 (2m,
8H, 4CH2 morph), 4.80e5.10 (m, 2H, CH2N), 5.55e5.70
(m, 1H, CHBr), 7.26e7.52 (m, 5H Ar), 7.82 (s, 1H, H-3).
Anal. C18H20N5OBrS (C, H, N, S).
Compound 9g. White solid, yield 70%, mp 137e138 ꢂC. 1H
NMR:
d 2.61 (s, 3H, SCH3), 4.73e5.07 (m, 4H,
1
Compound 8o. White solid, yield 49%, mp 77e78 ꢂC. H
CH2N þ CH2Ar), 5.53e5.66 (m, 1H, CHBr), 7.20e7.53 (m,
10H Ar), 7.72 (s, 1H, H-3). IR cmꢁ1: 3243 (NH). Anal.
C21H20N5BrS (C, H, N, S).
NMR: d 0.90 (t, J ¼ 7.4, 3H, CH3), 1.02 (t, J ¼ 7.4, 3H,
CH3), 1.26e1.43 and 1.49e1.63 and 1.65e1.83 (3m, 6H,
3CH2), 3.07 (t, J ¼ 7.4, 2H, SCH2), 3.48 (q, J ¼ 7.4, 2H,
CH2NH), 4.61e4.88 (m, 2H, CH2N), 5.38e5.53 (m, 1H,
Compound 9h. White solid, yield 73%, mp 112e113 ꢂC.
1H NMR: d 2.63 (s, 3H, SCH3), 3.00 (t, J ¼ 7.0, 2H,
CH2Ar), 3.87 (q, J ¼ 7.0, 2H, CH2NH), 4.86e5.10 (m, 2H,
CH2N), 5.30 (br s, 1H, NH disappears with D2O), 5.57e5.68
(m, 1H, CHBr), 7.21e7.52 (m, 10H Ar), 7.73 (s, 1H, H-3).
IR cmꢁ1: 3240 (NH). Anal. C22H22N5BrS (C, H, N, S).
Compound 10a. White solid, yield 51%, mp 112e113 ꢂC.
1H NMR: d 1.03 (t, J ¼ 7.4, 3H, CH3 prop.), 1.70 (sx,
J ¼ 7.4, 2H, CH2CH3), 3.22 (s, 6H, 2NCH3), 3.52 (q,
J ¼ 7.4, 2H, CH2NH), 4.62e4.88 (m, 2H, CH2N), 5.06 (br s,
1H, NH, disappears with D2O), 5.54e5.66 (m, 1H, CHCl),
7.21e7.51 (m, 5H Ar), 7.64 (s, 1H, H-3). IR cmꢁ1: 3365
(NH). Anal. C18H23N6Cl (C, H, N).
CHCl), 7.14e7.41 (m, 5H Ar), 7.70 (s, 1H, H-3). IR cmꢁ1
:
3275 (NH). Anal. C20H26N5ClS (C, H, N, S).
1
Compound 8p. White solid, yield 81%, mp 80e81 ꢂC. H
NMR: d 0.94e1.06 and 1.10e1.30 (2m, 9H, 3CH3), 1.75
(sx, J ¼ 7.2, 2H, SCH2CH2CH3), 3.06 (t, J ¼ 7.2, 2H,
SCH2), 3.52e3.73 (m, 4H, 2CH2N diethylam.), 4.56e4.91
(m, 2H, CH2N), 5.42e5.56 (m, 1H, CHCl), 7.16e7.43 (m,
5H Ar), 7.65 (s, 1H, H-3). Anal. C20H26N5ClS (C, H, N, S).
Compound 8q. White solid, yield 68%, mp 136e137 ꢂC. 1H
NMR: d 1.01 (t, J ¼ 7.0, 3H, CH3), 1.73 (sx, J ¼ 7.0, 2H,
CH2CH3), 3.08 (t, J ¼ 7.0, 2H, SCH2), 4.56e4.87 (m, 4H,

2674
S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
Compound 10b. White solid, yield 79%, mp 104e105 ꢂC.
0.5% FCS and antibiotics (100 U mLꢁ1 penicillin and
100 mg mLꢁ1 streptomycin) at 37 ꢂC in 5% CO2. Later the
spent medium was removed and the culture was refreshed
with new medium or medium containing different concentra-
tions (0e100 mM) of the studied compounds. After 3 days
(control cultures did not reach confluence), the antiprolifera-
tive effect of the compounds was determined by 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
proliferation assay. Briefly, cells were treated with an MTT so-
lution (10 mL, 5 mg mLꢁ1) and, 4 h later, acid propan-2-ol
(100 mL, 0.04 M HCl in propan-2-ol) was 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 (Mi-
crosoft). Results are reported as mean ꢀ SD of five experi-
ments, each performed in triplicate.
1H NMR: d 3.21 (s, 6H, 2NCH3), 3.80e3.93 (m, 8H, 4CH2
morph), 4.66e4.88 (m, 2H, CH2N), 5.57e5.68 (m, 1H,
CHCl), 7.26e7.52 (m, 5H Ar), 7.70 (s, 1H, H-3). Anal.
C19H23N6OCl (C, H, N).
1
Compound 10c. White solid, yield 79%, mp 70e71 ꢂC. H
NMR:
d
3.24 (s, 6H, 2CH3), 4.62e4.87 (m, 4H,
CH2N þ CH2Ar), 5.24 (br s, 1H, NH, disappears with D2O),
5.54e5.66 (m, 1H, CHCl), 7.25e7.50 (m, 10H Ar), 7.61 (s,
1H, H-3). IR cmꢁ1: 3359 (NH). Anal. C22H23N6Cl (C, H, N).
6.2. Biology
PP2 (AG 1879, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyr-
azolo[3,4-d]pyrimidine) and Imatinib mesylate, used as the
reference compounds, were obtained from Calbiochem (San
Diego, CA) and Novartis Pharma AG (Basel, Switzerland),
respectively.
6.2.3. Proapoptotic activity
6.2.1. Enzymatic assays with recombinant Src
Proapoptotic effect of selected pyrazolo[3,4-d]pyrimidines
was tested using a poly-ADP-Ribose-Polymerase (PARP) as-
say (Roche Dignostic, Milan, Italy). KU-812 cell line was cul-
tured at a concentration of 2 ꢃ 105 cells mLꢁ1 and challenged
with the compound (50 mM). After 72 h, cells were harvested
and lysed in an appropriate buffer containing 1% Triton X-
100. Immunoblot analysis was performed using PARP-specific
antibodies to both the uncleaved (113 kDa) and cleaved
(89 kDa) forms of PARP. Quantification of cleaved PARP
over uncleaved PARP expression was achieved with Sigma
Gel analysis software and the results were expressed as the
mean of three independent experiments. Statistical analyses
were performed using Student’s t-test and the Bonferroni’s
correction.
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), accord-
ing to the manufacturer’s protocol. Ki (expressed as a mM con-
centration) toward recombinant human Src was calculated
according to the following equation: Ki ¼ (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 done in triplicate and
mean values were used for the interpolation. Curve fitting
was performed with the program GraphPad Prism.
Cell-free assay with recombinant Abl. Recombinant human
Abl was purchased from Upstate Biotechnology (Waltham,
MA) and used to investigate the mechanism of kinase inhibi-
tion, as previously reported [30]. In detail, activity was mea-
sured in a filter-binding assay using an Abl specific peptide
substrate (Abltide, Upstate Biotechnology) in the presence of
0.012 mM [g-32P]ATP, 50 mM peptide, and 0.005 mM c-Abl.
The apparent affinity (Km) of Abl for the peptide and ATP sub-
strates was determined separately (1.5 and 10 mM, respec-
tively). Each experiment was done in triplicate and mean
values were used for the interpolation. Curve fitting was per-
formed with the program GraphPad Prism. Ki values toward
isolated Abl were calculated according to the following equa-
tion: Ki ¼ ID50/{E0þ[E0(Km(ATP)/S0)]}/E0, where E0 and S0 are
the enzyme and the ATP concentrations (0.005 and 0.012 mM,
respectively).
6.2.4. mRNA expression of apoptotic genes
The mRNA expression of apoptotic genes was performed
using qRT-PCR. KU-812 cells were treated as reported
above, and lysed in an appropriate buffer (OMNI-zol for
RNAeDNAeProtein extraction Kit, Eurocione, Devon,
UK). The extract was processed for the extraction of
mRNA. For qRT-PCR analysis, Bcl-xL (antiapoptotic gene)
and Bax (proapoptotic gene) expression in KU-812 cells
was determined using an MJ MiniOpticon Cycler (Bio-Rad
Laboratories, Hercules, CA). First strand cDNA synthesis
was performed using iScript cDNA Synthesis Kit (Bio-Rad
Laboratories). qRT-PCR was performed using iTaq SYBR
Green Supermix with ROX (Bio-Rad Laboratories) and spe-
cific primers from GenBank. Data were quantitatively ana-
lyzed on an MJ OpticonMonitor detection system (Bio-Rad
Laboratories). The values were expressed as fold increase rel-
ative to the expression of b-actin.
6.2.2. Cell culture
Human CML K-562 cells in blast crisis [42] and human
CML KU-812 [43] cell line in myeloid blast crisis were ob-
tained from the American Type Culture Collection and were
grown in RPMI 1640 medium (BioWhittaker, Vallensbaek,
DK) containing 10% fetal calf serum (FCS) (BioWhittaker).
The cultures were free of mycoplasma. For the proliferation
assay, cell lines (2 ꢃ 104 cells mLꢁ1) were incubated overnight
in 100 mL RPMI 640 culture medium, supplemented with
6.3. Docking studies
Consistent with the previous works [29,30,41], docking
studies were performed on the newly synthesized compounds
within the ATP binding site of c-Src and Abl kinases using

S. Schenone et al. / European Journal of Medicinal Chemistry 43 (2008) 2665e2676
2675
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the software packages Autodock 3.0.5 [44] and Gold 3.0.1
[45e47], respectively. Structures of inhibitors were repre-
sented using MacroModel 8.5 [48] and minimized with the
Amber force field using the PolakeRibiere conjugated gradi-
[10] T. O’Hare, A.S. Corbin, B.J. Druker, Curr. Opin. Genet. Dev. 16 (2006)
92e99.
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Natl. Acad. Sci. U.S.A. 103 (2006) 16870e16875.
˚
ent method (0.001 kJ/mol A convergence or 10 000 iterations).
To remove unfavourable contacts, a preliminary structure op-
timization was performed with MacroModel 8.5 on the X-ray
crystallographic structures of c-Src (PDB entry: 1YOL) [40]
and c-Abl (PDB entry: 1M52) [49] through the all-atom Am-
ber* 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
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F. Corelli, M. Botta, J. Med. Chem. 49 (2006) 3278e3286.
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Y. Wang, R. Sundaramoorthi, P. Sliz, D.R. Veach, W.G. Bornmann,
B. Clarkson, D.C. Dalgarno, T.K. Sawyer, G.Q. Daley, Proc. Natl.
Acad. Sci. U.S.A. 103 (2006) 9244e9249.
˚
set to 0.01 kJ/mol A.
With respect to Autodock simulations, the Lamarckian ge-
netic algorithm (LGA) was employed to explore the possible
orientations/conformations of the inhibitors into the binding
site. For each of the 100 independent runs, a maximum num-
ber of 1 500 000 operations were performed with a population
size of 200 individuals. The binding pocket was inserted into
a grid box centred on the bound ligand CGP77675 and enclos-
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C. Lambert-van der Brempt, R. Morgentin, R.A. Norman, A. Olivier,
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L. Otterbein, P.A. Ple, N. Warin, G. Costello, J. Med. Chem. 49 (2006)
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[19] D.H. Boschelli, F. Ye, D.Y. Wang, M. Dutia, S.L. Johnson, B. Wu,
K. Miller, D.W. Powell, D. Yaczko, M. Young, M. Tischler, K. Arndt,
C. Discafani, C. Etienne, J. Gibbons, J. Grod, J. Lucas, J.M. Weber,
F. Boschelli, J. Med. Chem. 44 (2001) 3965e3977.
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ing residues lying within about 8 A from the ligand itself.
[20] J.M. Golas, K. Arndt, C. Etienne, J. Lucas, D. Nardin, J. Gibbons,
P. Frost, F. Ye, D.H. Boschelli, F. Boschelli, Cancer Res. 63 (2003)
375e381.
As regards Gold calculations, the ChemScore was chosen
as fitness function. For each of the 50 independent genetic al-
gorithm runs, a maximum number of 100 000 GA operations
were performed on a set of five groups with a population
size of 200 individuals. The remaining GA parameters were
kept to their default values. Hydrophobic fitting points were
[21] M. Puttini, A.M. Coluccia, F. Boschelli, L. Cleris, E. Marchesi,
A. Donella-Deana, S. Ahmed, S. Radaelli, R. Piazza, V. Magistroni,
F. Andreoni, L. Scapozza, F. Formelli, C. Gambacorti-Passerini, Cancer
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F. Boschelli, J. Med. Chem. 49 (2006) 7868e7876.
˚
calculated on the target for a 10 A radius around the co-
crystallized ligand PD173955.
[23] G. Noronha, K. Barrett, A. Boccia, T. Brodhag, J. Cao, C.P. Chow,
E. Dneprovskaia, J. Doukas, R. Fine, X. Gong, C. Gritzen, H. Gu,
E. Hanna, J.D. Hood, S. Hu, X. Kang, J. Key, B. Klebansky,
A. Kousba, G. Li, D. Lohse, C.C. Mak, A. McPherson, M.S.S. Palanki,
V.P. Pathak, J. Renick, F. Shi, R. Soll, U. Splittgerber, S. Stoughton,
S. Tang, S. Yee, B. Zeng, N. Zhao, H. Zhu, Bioorg. Med. Chem. Lett.
17 (2007) 602e608.
˚
Finally, results differing less than 1.5 A in ligandeall atom
RMSD were clustered together.
For each inhibitor, the first ranked solution as well as the
lowest energy conformation of the most populated cluster,
were selected for further analysis.
[24] N.P. Shah, C. Tran, F.Y. Lee, P. Chen, D. Norris, C.L. Sawyers, Science
305 (2004) 399e402.
Acknowledgements
[25] L.J. Lombardo, F.Y. Lee, P. Chen, D. Norris, J.C. Barrish, K. Behnia,
S. Castaneda, L.A. Cornelius, J. Das, A.M. Doweyko, C. Fairchild,
J.T. Hunt, I. Inigo, K. Johnston, A. Kamath, D. Kan, H. Klei,
P. Marathe, S. Pang, R. Peterson, S. Pitt, G.L. Schieven, R.J. Schmidt,
J. Tokarski, M.L. Wen, J. Wityak, R.M. Borzilleri, J. Med. Chem. 47
(2004) 6658e6661.
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F. Giles, M. Talpaz, J. Cortes, Blood 109 (2007) 497e499.
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treatment for a rare type of leukemia, http://www.fda.gov/bbs/topics/
NEWS/2006/NEW01400.htlm (accessed 2006).
Financial support provided by the Italian MIUR (PRIN
2006030948) and by the Fondazione Monte dei Paschi di
Siena is gratefully acknowledged. We thank Dr. Giovanni
Gaviraghi (Sienabiotech S.p.A.) for helpful discussion. We
are indebted to Dr. Doriano Fabbro (Novartis Institutes for
Biomedical Research, Basel, Switzerland) for assays on the ty-
rosine kinase panel.
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F. Bondavalli, C. Brullo, P. Fossa, G. Menozzi, L. Mosti, F. Manetti,
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