Structure-based optimization of pyrazolo[3,4-d]pyrimidines as Abl inhibitors and antiproliferative agents toward human leukemia cell lines

Article abstract of DOI:10.1021/jm701240c

Results from molecular docking calculations and Grid mapping laid the foundations for a structure-based optimization approach to improve the biological properties of pyrazolo-pyrimidine derivatives in terms of inhibition of Abl enzymatic activity and anti

Full text of DOI:10.1021/jm701240c

1252
J. Med. Chem. 2008, 51, 1252–1259
Structure-Based Optimization of Pyrazolo[3,4-d]pyrimidines as Abl Inhibitors and
Antiproliferative Agents toward Human Leukemia Cell Lines
Fabrizio Manetti,† Chiara Brullo,‡ Matteo Magnani,† Francesca Mosci,§ Beatrice Chelli,| Emmanuele Crespan,
Silvia Schenone,*,‡ Antonella Naldini,§ Olga Bruno,‡ Maria Letizia Trincavelli,| Giovanni Maga, Fabio Carraro,§
Claudia Martini,| Francesco Bondavalli,‡ and Maurizio Botta†
Dipartimento Farmaco Chimico Tecnologico, UniVersità degli Studi di Siena, Via Alcide de Gasperi 2, I-53100, Siena, Italy, Dipartimento di
Scienze Farmaceutiche, UniVersità degli Studi di GenoVa, Viale Benedetto XV 3, I-16132, GenoVa, Italy, Dipartimento di Fisiologia, Sezione di
Neuroimmunofisiologia, UniVersità degli Studi di Siena, Via Aldo Moro, I-53100, Siena, Italy, Dipartimento di Psichiatria, Neurobiologia,
Farmacologia e Biotecnologie, UniVersità di Pisa, Via Bonanno 6, I-56126, Italy, and Istituto di Genetica Molecolare, IGM-CNR,
Via Abbiategrasso 207, I-27100, PaVia, Italy
ReceiVed October 1, 2007
Results from molecular docking calculations and Grid mapping laid the foundations for a structure-based
optimization approach to improve the biological properties of pyrazolo-pyrimidine derivatives in terms of
inhibition of Abl enzymatic activity and antiproliferative properties toward human leukemia cells. Insertion
of halogen substituents with various substitution patterns, suggested by simulations, led to a significant
improvement of leukemia cell growth inhibition and to an increase up to 1 order of magnitude of the affinity
toward Abl.
Introduction
phase, 1 is highly effective toward CML, with excellent and
durable hematological and cytological responses in the majority
of patients. Nevertheless, primary refractoriness and acquired
resistance to this drug are observed frequently in patients in
the accelerated phase or blast crisis. The two main mechanisms
of resistance are the overexpression of Bcr-Abl, mainly due to
gene amplification,7 and, more frequently, mutations in the
kinase domain of Bcr-Abl.8
Chronic myelogenous leukemia (CMLa ) is a disease char-
acterized by the presence of the Philadelphia chromosome
(discovered in 1960 by Nowell and Hungerford)1 and results
from a reciprocal translocation between chromosomes 9 and
22.2 This translocation fuses the breakpoint cluster region (Bcr)
and the Abelson kinase (Abl) genes, forming the Bcr-Abl
oncogene3 that encodes the constitutively active cytoplasmatic
tyrosine kinase (TK) Bcr-Abl, present in >90% of CML.4 Bcr-
Abl causes hyperproliferation of the stem cells and the
consequent pathology of the disease that progresses through
three phases (chronic proliferative phase, accelerated phase, and
blast crisis phase), becoming more resistant to treatment in each
successive phase. The last phase is also characterized by the
presence of genomic instability and is ultimately fatal.5
The finding that Bcr-Abl is the cause of the leukemic
phenotype and that the tyrosine kinase activity of Abl is
fundamental for Bcr-Abl-mediated transformation made this
kinase an important target for the development of specific
therapies. In 1996, Novartis reported the first potent ATP-
competitive Abl kinase inhibitor, 1 (Chart 1).6 It was approved
by the Food and Drug Administration (FDA) in 2001 and
became, within a few years of its discovery, the first line therapy,
generally well tolerated for the treatment of CML. In the chronic
Second generation Abl inhibitors were synthesized through
rational drug design approaches to overcome resistance to 1.9
Recently, Novartis disclosed 2 (Chart 1), structurally related to
1, which showed promising results in preclinical studies and
was 10–25-fold more potent compared to 1 in cellular assays.
Compound 2 also inhibited the growth of cell lines expressing
Bcr-Abl mutants resistant to 1.10 On the other hand, unlike 1
and its derivatives, 3 (Chart 1) is a substrate binding site Abl
inhibitor that inhibits wild type kinase 10-fold more potently
than 1, and it also has activity against kinase domain mutations
resistant to 1.11
A different potential therapeutic approach for CML is based
on the study of Bcr-Abl/Src dual inhibitors,12–14 taking into
consideration that the activity of Src is elevated in the presence
of the Bcr-Abl oncoprotein. Compound 4 (Chart 1) is an ATP-
competitive thiazole-carboxamide derivative, 100–300-fold more
active than 1 and orally active in CML animal models.15,16a
This compound was recently approved by the FDA with the
name of Sprycel for the treatment of all phases of CML in adults
with resistance or intolerance to prior therapy, including 1.17
Moreover, very recently 4 was tested in vitro and in vivo against
the pediatric preclinical testing program with promising
results.16b Another promising molecule (5, Chart 1) is a purine
compound synthesized with the help of mutagenesis studies and
molecular modeling simulations, which inhibits both native and
1-resistant mutations. Unfortunately, this compound shows
nonspecific cytotoxicity.18 Moreover, 6 (Chart 1) is a potent
and selective dual Bcr-Abl/Lyn (a Src TK family member)
inhibitor. Although structurally similar to 1, 6 is 25–55-fold
more potent, and it is also active on the Bcr-Abl mutated form,
but not on the T315I mutation.19,20 Finally, 7 (Chart 1) was
* To whom correspondence should be addressed. Telephone: 0039 010
3538866. Fax: 0039 010 3538358. E-mail: schensil@unige.it.
†
Dipartimento Farmaco Chimico Tecnologico, Università degli Studi
di Siena.
‡
Dipartimento di Scienze Farmaceutiche, Università degli Studi di
Genova.
§
Dipartimento di Fisiologia, Sezione di Neuroimmunofisiologia, Uni-
versità degli Studi di Siena.
|
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
nologie, Università di Pisa.
Istituto di Genetica Molecolare, IGM-CNR.
Abbreviations: CML, chronic myelogenous leukemia; Bcr, breakpoint
a
cluster region; TK, tyrosine kinase; Abl, Abelson kinase; FDA, Food and
Drug Administration; MIFs, molecular interaction fields; STAT-5, signal
transducer and activator of transcription 5; HRI, hydrophobic region I; HRII,
hydrophobic region II.
10.1021/jm701240c CCC: $40.75
2008 American Chemical Society
Published on Web 02/08/2008

Pyrazolo[3,4-d]pyrimidines as Abl Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1253
Chart 1. Schematic Representation of Current Available Inhibitors of Abl (1-7) and PP2 (8, Used as Reference Compound in
Enzymatic and Cellular Assays Reported in the Paper)
reported very recently as a small molecule inhibitor of the
Aurora kinase, which also inhibits Abl at low nanomolar
concentrations.21
(in an attempt to fulfil the additional space available within both
HRI and HRII), while keeping the pyrazolo-pyrimidine core
intact. As an example, Figure 1A shows the binding mode of a
compound of the original series, namely 21 (Ki ) 0.4 µM toward
isolated Abl, Table 1), as determined by docking experiments.
Regions of minimum interaction energy (i.e., the most favorable
interaction points) for probe Cl (organic chlorine atom, magenta)
and F (organic fluorine atom, cyan) are also displayed. The
binding mode is characterized by the location of the side chains
at N1 and C4 within the HRI and HRII, respectively. Moreover,
N5 and the NH moiety of the inhibitor are engaged in hydrogen
bond contacts with the backbone NH and the carbonyl group
of Met318, respectively. Additional information is provided by
MIFs which show the presence of minima for Cl and F probes
located within both of the hydrophobic pockets surrounding the
phenyl moieties of inhibitors. This finding suggests the synthesis
of a set of derivatives bearing halogen substituents with different
substitution patterns on one or both of the aromatic rings of the
inhibitors. Accordingly, compounds 9-20 were synthesized as
reported in Scheme 1.
In this context, we have recently published a series of
pyrazolo[3,4-d]pyrimidine derivatives endowed with inhibitory
activity toward the c-Abl enzyme.22 Continuing our efforts in
this research field, we describe in this paper the rational design
and the synthesis of second generation derivatives showing a
significant improvement of their activity profile against both
isolated Abl and leukemia cells, in comparison to the parent
compounds.
Docking simulations on the original series of pyrazolo-
pyrimidines22 indicated two different binding modes of the
inhibitors within the ATP binding pocket of Abl, depending on
the presence or not of an alkylthio substituent at the C6 position
of the heterocyclic nucleus. However, in both cases, the
occupancy of the two main hydrophobic regions (hereafter
referred to as hydrophobic regions I and II and reported as HRI
and HRII) of the ATP binding pocket seemed to play a crucial
role in determining affinity toward Abl. In addition to this
finding, a detailed analysis of the binding site was performed
with the aim of identifying additional regions of favorable
interactions between inhibitors and the protein not exploited by
the first series of ligands. For this purpose, molecular interaction
fields (MIFs) were calculated for the binding site by means of
the software Grid.23 Briefly, Grid inserts the macromolecular
target under investigation (the binding site region of Abl) into
a grid and then computes, at each node of the grid, the
interaction energies between several probes (atoms or groups
of atoms representing various chemical functionalities found
in the inhibitor structures) and the target itself. The set of energy
values calculated for a specific probe constitutes the MIFs of
the probe-target system.
Chemistry. The starting compounds 22a-c, prepared from
the appropriate hydrazine and ethyl(ethoxymethylene)cyan-
acetate, were reacted with formamide for 8 h at 190 °C to give
the corresponding pyrazolo[3,4-d]pyrimidinones 23a-c in high
yields. Treatment of the latter with an excess of the Vilsmeier
complex (POCl3/DMF, 1:1) for 12 h at reflux in CHCl3 led to
the formation of the halogenated derivatives 24a-c. The C4
chlorine atom was in turn substituted with the appropriate
benzylamine, in anhydrous toluene at room temperature (rt), to
afford the final compounds 9-20 in yields ranging between 65%
and 80% (Table 1).
Results and Discussion
Details derived from both docking studies and Grid analysis
were then combined together, thus suggesting structural modi-
fications that should allow the location of a certain inhibitor
substituent in a region of favorable interaction with the protein.
Based on this structural information, we chose to introduce
substituents on both of the phenyl rings at positions N1 and C4
The new compounds were then submitted to biological tests
to evaluate their affinity toward the Abl enzyme in a cell-
free assay and their antiproliferative activity in cellular assays
on three human leukemia cell lines (namely, K-562, MEG-
01, and KU-812), in comparison to 8 (PP2, Chart 1), chosen

1254 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Manetti et al.
Figure 1. Graphical representation of the binding mode of pyrazolo[3,4-d]pyrimidines within the ATP binding pocket of c-Abl (mesh representation
of the surface). Regions of minimum energy as derived from molecular interaction field calculations are also displayed for probes Cl (magenta
spheres) and F (cyan spheres). (A) Binding mode of 21 (green). (B) Comparison of the binding modes of 21 (green, Ki ) 0.4 µM toward isolated
Abl) and 15 (orange, Ki ) 0.02 µM toward isolated Abl). The fluorine substituents of 15 are both at close contact with regions of minimum energy
(cyan spheres) for the F probe, suggesting profitable interactions between ligand and target.
Table 1. Structure and Inhibitory Activity of Compounds 9–21 toward Isolated Abl Kinase and Human Leukemia K-562, MEG-01, and KU-812 Cell
Lines
IC50 (µM)
compd
R1
R2
Caco-2a
Ki (µM)b
K-562c
MEG-01d
KU-812c
8 (PP2)
9
10
11
12
13
14
15
16
0.50 ( 0.20
2.0 ( 0.1
25 ( 3
114 ( 13
46 ( 6
79 ( 5
26 ( 4
58 ( 7
35 ( 3
36 ( 2
12 ( 2
34 ( 5
9 ( 1
17 ( 1
8.4 ( 0.4
14 ( 2
45 ( 3
NHCH2(p-F-Ph)
NHCH2(m-F-Ph)
NHCH2(o-F-Ph)
NHBn
NHCH2(p-F-Ph)
NHCH2(m-F-Ph)
NHCH2(o-F-Ph)
NHCH2(m-Cl-Ph)
NHCH2(o-Cl-Ph)
NHCH2(p-F-Ph)
NHCH2(p-Cl-Ph)
NHCH2(m-Cl-Ph)
NHBn
H
H
H
F
F
F
F
F
F
Cl
Cl
Cl
H
0.72
0.72
0.71
0.80
0.53
0.55
0.55
0.84
0.91
0.69
1.16
0.94
1.06
0.04 ( 0.01
0.73 ( 0.05
0.44 ( 0.06
0.20 ( 0.03
0.10 ( 0.02
0.02 ( 0.01
0.12 ( 0.01
0.20 ( 0.04
0.08 ( 0.04
8.5 ( 1.5
11 ( 5
16 ( 2
12 ( 3
12 ( 3
15 ( 1
14 ( 2
38 ( 2
8 ( 1
17
18
19
18 ( 1
5.5 ( 1.4
9.7 ( 1.5
5.0 ( 1.5
63 ( 5
37 ( 5
36 ( 2
48 ( 4
20
0.08 ( 0.01
0.40 ( 0.06
21e
a Caco-2 refers to the Caco2 permeation model (as implemented in Volsurf).24 Values represent a score ranging from -1 to +1, corresponding to the
following permeability values: -1 corresponds to a permeability <4 × 10-6 cm/s; 0 corresponds to a permeability between 4 × 10-6 and 8 × 10-6 cm/s;
1 corresponds to a permeability >8 × 10-6 cm/s. b Ki values toward isolated Abl calculated according to the following equation: Ki ) ID50/{E0 + [E0(Km(ATP)
/
S0)]}/E0, where E0 and S0 are the enzyme and the ATP concentrations (0.005 and 0.012 µM, respectively). c IC50 values are means ( SD of five experiments,
each performed in triplicate. d IC50 values are means ( standard error of three experiments, each performed in duplicate. e Reported elsewhere.26
Scheme 1a
a Reagents: (a) Formamide, 190 °C, 8 h; (b) POCl3/DMF, CHCl3, reflux, 12 h; (c) benzylamines, anhydrous toluene, rt, 48 h.
as the reference compound. As a result, cellular activity
underwent a significant improvement (Table 1), and the
enzymatic affinity ameliorated up to 1 order of magnitude,
in comparison with biological data of the corresponding
nonhalogenated parent compounds. As expected by previous
docking studies, halogenated derivatives adopted the binding
mode found for 6-unsubstituted pyrazolo-pyrimidines, while
halogen substituents were in most cases located in regions

Pyrazolo[3,4-d]pyrimidines as Abl Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1255
of space where Grid analysis found profitable interactions
between halogen atoms and the target. As an example, a
comparison between the binding mode of 15 (the most active
compound toward Abl, among the pyrazolo-pyrimidines
synthesized by us) and 21 (Figure 1B) showed the same
orientation within the ATP binding pocket of Abl, with
fluorine substituents of 15 that lie in regions of space
corresponding to points of minimum energy found by Grid
for the F probe. Such additional contacts allowed us to
account for the higher affinity of 15 (0.02 µM) with respect
to its congeneric analogues 14 and 13 (showing Ki values of
0.10 and 0.20 µM, respectively) with a different substitution
pattern on the phenyl ring at C4. In fact, their m-F and p-F
substituents at R1 (Table 1) were accommodated into portions
of HRII where no minima for the F probe were found.
Similarly, 17 and 16 showed a reduced affinity for Abl
because their o- and m-Cl substituents at R1, respectively,
are unable to profitably interact with minima for the Cl probe
within HRII. Moreover, also 12, due to the lack of any
substituent on the phenyl ring of the C4 benzylamino moiety,
was unable to make additional profitable contacts with HRII
and, consequently, showed a reduced affinity (0.44 µM).
cellular activity. Similarly, 12 and 16 showed permeability
values among the highest of the whole set, which could account
for the fact that they were two of the most active compounds
in cellular assays, despite their relatively low enzymatic potency
(0.44 and 0.12 µM, respectively).
On the basis of experimental results, 18 was selected as a
hit to perform additional biological assays aimed at better
understanding the antiproliferative activity of this compound
toward leukemia cells. In particular, to check if the antipro-
liferative effect of 18 toward each cell line was dependent
on the inhibition of Bcr-Abl activity, we evaluated the
phosphorylation of both Bcr-Abl and its downstream substrate
STAT-5 (Bcr-Abl is known to phosphorylate STAT-5, a
transcription factor directly activating Bcl-xL, which works
as an apoptosis inhibitor at the mitochondrial level) in
addition to the phosphorylation of Src. As a result, on K-562
cells, 18 markedly reduced Src phosphorylation with respect
to control and showed an activity also better than that of
PP2 (Figure 2A and D, left). Moreover, Bcr-Abl phospho-
rylation was potently inhibited (significantly better than PP2)
by 18 (Figure 2A and B, left), which was also able to reduce
STAT-5 phosphorylation at a higher extension with respect
to PP2 (Figure 2A and C, left). In a similar way, Bcr-Abl
phosphorylation of KU-812 cells was strongly and equally
inhibited by both 18 and PP2 (Figure 2A and B, right).
Moreover, 18 was also able to greatly reduce phosphorylation
of both STAT-5 (Figure 2A and C, right) and Src (Figure
2A and D, right) better than the reference compound PP2.
In summary, immunoblot analysis of lysates from compound-
treated cells with specific antiphospho antibodies revealed a
reduction in phosphorylation of all of the targets. Taken
together, reduction of the phosphorylation levels of both Bcr-
Abl and STAT-5 strongly suggested that effects mediated
by 18 on proliferation and apoptosis of leukemia cells are a
consequence of the reduction of Bcr-Abl kinase activity.
By changing the F substituent (at R2) of 16 into a Cl to give
20, affinity toward Abl underwent only a slight variation from
0.12 to 0.08 µM, respectively. An analysis of the location of
MIF maps showed that this result was probably due to the fact
that the interaction pathway of 16 and 20 was very similar. In
fact, no changes occurred within HRII, where the m-Cl-
benzylamino group of both compounds is located. On the other
molecular edge, the F substituent at R2 of 16, interacting with
a F minimum within HRI, was replaced by a Cl group of 20
that, in turn, was able to contact a Cl minimum in the same
region of HRI, leading to an unchanged interaction pathway.
Similarly, when transforming 13 (0.20 µM) into 18 (0.08 µM),
a 2.5-fold difference in affinity was found. Compounds 9 (2.0
µM) and 11 (0.73 µM), lacking the F substitution at the phenyl
ring on N1, showed a marked reduction in affinity toward Abl,
at least 1 order of magnitude lower with respect to the
corresponding fluoro derivatives 13 (0.2 µM) and 15 (0.02 µM),
respectively. The affinity of 10 (0.04 µM), which was better
than that of the corresponding F analogue 14 (0.10 µM), was
an exception to this trend, and it was impossible to be
rationalized on the sole basis of computational results.
Moreover, inhibition of STAT-5 phosphorylation by means
of 18 and involvement of STAT-5 in apoptosis control via
Bcl-xL led to the suggestion to investigate the expression of
Bax/Bcl-xL mRNA in the leukemia cells under study. For
this purpose, it is well-known that the ratio between Bax
mRNA and Bcl-xL mRNA expression may be used as a direct
index of the induction of the apoptotic process (the proapo-
ptotic action of Bax is antagonized by Bcl-xL) and could
help in explaining the molecular mechanism of apoptosis
induction. Accordingly, upon incubation of K-562 and KU-
812 cells for 72 h in the presence of 18 (Figure 3B and C),
a marked increase in the Bax/Bcl-xL ratio was evidenced,
confirming induction of leukemia cells toward apoptosis.
Moreover, exposure of K-562 cells to 18 for 3 h also strongly
affected the ratio between Bax mRNA and Bcl-xL mRNA
expression (Figure 3A).
The new compounds were found to have an antiproliferative
activity toward three human leukemia cell lines in the range
from low micromolar to micromolar concentration (Table 1),
and all of the cell lines seemed to be equally sensitive to the
treatment with the inhibitors. However, 18 showed the best
biological profile in terms of antiproliferative properties and
affinity toward Abl, although 15 had a 4-fold better affinity (0.02
µM of 15 versus 0.08 µM of 18).
It is important to point out that an analysis of biological data
revealed some discrepancies between enzymatic and cellular
activity. As an example, compound 15, showing the best
inhibitory activity in the enzymatic assay (0.02 µM), was about
3–4-fold less potent than 18 (showing a comparable activity in
the enzymatic assay, 0.08 µM) in cell-based assays. In an
attempt to rationalize these data, compounds were projected on
an in silico permeability model (a mathematical model describ-
ing the ability of compounds to permeate Caco2 cells) imple-
mented within the Volsurf software.24 Results showed that, in
principle, compounds were well absorbed through cell mem-
branes, with several differences. In fact, 15 exhibited a lower
permeability in comparison to 18, accounting for its lower
In conclusion, the previous knowledge of the binding mode
of pyrazolo[3,4-d]pyrimidines into the ATP binding site of
Abl (predicted on the basis of docking studies) was combined
with a detailed analysis of the binding pocket (performed
with the software Grid) to provide suggestions for the
synthesis of novel derivatives with improved affinity toward
the macromolecular target. Results of cellular and enzymatic
assays performed on the new compounds demonstrated that
structural modifications led to an overall improvement of the
inhibitory activities with respect to the corresponding deha-
logenated parent compounds. In particular, 18 emerged as a
hit with an antiproliferative activity toward Bcr-Abl-positive
human leukemia cells significantly ameliorated and with an

1256 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Manetti et al.
Figure 2. Compound 18 reduces phosphorylation of Bcr-Abl, STAT-5, and Src. (A) Effects of 18 on the phosphorylation of phospho Bcr-Abl,
phospho STAT-5, and phospho Src in K-562 and KU-812 cells, in comparison to PP2 used as the reference compound. The quantification is shown
in panels B, C, and D. Results are expressed as the mean ( SEM of three independent experiments. Asterisks indicate statistically significant (p
< 0.05) differences between the inhibition of phophorylation due to treatment with 18 and PP2 versus untreated cell lines, as determined by using
the Student’s t test and the Bonferroni’s correction.
affinity toward isolated Abl up to more than 1 order of
magnitude higher with respect to previous compounds. At
the molecular level, it was able to inhibit phosphorylation
of both Bcr-Abl and its downstream target STAT-5, thus
interfering in the apoptotic process regulated by the balance
between Bax and Bcl-xL. On the basis of these experimental
findings, identification of 18 allows a step forward in the
design of new antileukemia agents.
Experimental Details
A. Chemistry. Starting materials were purchased from Aldrich-
Italia (Milan, Italy).

Pyrazolo[3,4-d]pyrimidines as Abl Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1257
Figure 3. Compound 18 induces a proapoptic response on CML cell lines. (A) Exposure to 18 for 3 h affects the ratio of Bax/Bcl-xL mRNA
expression in K-562. The expression of Bax mRNA is also significantly enhanched after a 72 h exposure to the compound in (B) K-562 and
(C) KU-812 cells, respectively. The means ( SEM of four independent experiments are presented. Bax and Bcl-xL mRNA expression in
compound-treated cells was determined by qRT-PCR. All values were expressed as fold increase relative to the expression of ꢀ-Actin.
Asterisks indicate statistically significant (p < 0.05) differences between the medium and the investigated compound.
Melting points were determined with a Büchi 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)
charcoal, followed by precipitation with glacial acetic acid. The
solid was filtered and recrystallized from absolute ethanol to give
compounds 23b or 23c as white solids.
4. 1-[2-(4-Fluorophenyl)-2-hydroxyethyl]-1,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one (23b). Yield 73%, mp 281–282
1
relative to TMS as internal standard, with J in Hz. H patterns
1
°C (dec). H NMR: δ 4.22–4.35 and 4.39–4.54 (2m, 2H, CH2N),
are described using the following abbreviations: s ) singlet, d
) doublet, t ) triplet, q ) quartet, m ) multiplet, and br )
broad. All compounds were tested for purity by TLC (Merck,
Silica gel 60 F254, CHCl3 as the eluant). Analyses for C, H, and
N were within (0.3% of the theoretical values.
5.02–5.17 (m, 1H, CHO), 5.72 (br s, 1H, OH, disappears with D2O),
7.03–7.19 and 7.24–7.38 (2m, 4H Ar), 8.02 (s, 1H, H-3), 8.07 (s,
1H, H-6), 12.13 (br s, 1H, NH, disappears with D2O). IR (cm -1):
3387 (NH), 3168–2900 (OH), 1737 (CO). Anal. (C13H11N4O2F)
C, H, N.
Synthesis and analytical data of intermediates 22a, 23a, and 24a
were already reported.25
5. General Procedure for the Synthesis of 4-Chloro-1-[2-
chloro-2-(4-halophenyl)ethyl]-1H-pyrazolo[3,4-d]pyrim-
idines (24b,c). The Vilsmeier complex, previously prepared from
POCl3 (15.33 g, 100 mmol) and anhydrous dimethylformamide
(DMF) (7.31 g, 100 mmol) was added to a suspension of compound
23b or 23c (10 mmol) in CHCl3 (50 mL). The mixture was refluxed
for 12 h. After cooling at rt, the mixture 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 products 24b or 24c as white solids.
1. General Procedure for the Synthesis of Ethyl 5-Amino-
1-[2-(4-halophenyl)-2-hydroxyethyl]-1H-pyrazole-4-carboxy-
lates (22b,c). The appropriate hydrazine (20 mmol) was added to
a solution of ethyl(ethoxymethylene)cyanacetate (3.38 g, 20 mmol)
in anhydrous toluene (20 mL) and the mixture was heated at 80 °C
for 8 h. The solution was concentrated under reduced pressure to
half of the volume and allowed to cool to rt. The yellow pale solid
was filtered and recrystallized from toluene to afford 22b,c as white
solids.
2. Ethyl 5-Amino-1-[2-(4-fluorophenyl)-2-hydroxyethyl]-1H-
pyrazole-4-carboxylate (22b). Yield 70%, mp 163–164 °C. 1H
NMR: δ 1.33 (t, J ) 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 (m, 4H Ar), 7.55 (s, 1H, H-3). IR (cm-1): 3448,
3446 (NH2), 3300–3000 (OH), 1685 (CO). Anal. (C14H16N3O3F)
C, H, N.
6. 4-Chloro-1-[2-chloro-2-(4-fluorophenyl)ethyl]-1H-pyra-
zolo[3,4-d]pyrimidine (24b). Yield 75%, mp 128–129 °C. 1H
NMR: δ 4.73–4.88 and 4.92–5.08 (2m, 2H, CH2N), 5.38–5.54 (m,
1H, CHCl), 6.84–7.06 and 7.18–7.45 (2m, 4H Ar), 8.10 (s, 1H,
H-3), 8.68 (s, 1H, H-6). Anal. (C13H9N4Cl2F) C, H, N.
7. General Procedure for the Synthesis of 4-Benzylamino
Substituted 1-(2-Chloro-2-phenylethyl)- and 1-[2-Chloro-2-(4-
halophenyl)ethyl]-1H-pyrazolo[3,4-d]pyrimidines (9–20). To a
solution of 24a-c (10 mmol) in anhydrous toluene (10 mL) was
added the appropriate benzylamine (40 mmol), and the mixture was
stirred at rt for 48 h. Then, the organic phase was washed with
H2O (2 × 10 mL), dried (MgSO4), and concentrated under reduced
3. General Procedure for the Synthesis of 1-[2-(4-Halophe-
nyl)-2-hydroxyethyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-
4-ones (23b,c). A suspension of 22b or 22c (10 mmol) in
formamide (10 g, 333 mmol) was heated at 190 °C for 8 h and
then poured in H2O (300 mL). The crude product was filtered and
purified by dissolving in 2 M NaOH (100 mL) and boiling with

1258 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Manetti et al.
pressure. The crude oil was crystallized by adding a 1:1 mixture
of diethyl ether and petroleum ether (bp 40–60 °C).
Acknowledgment. Financial support provided by the Italian
MIUR (PRIN 2004_059221 and PRIN 2006_030948) and by
the Fondazione Monte dei Paschi di Siena is gratefully
acknowledged. Prof. Gabriele Cruciani (University of Perugia,
Italy) is also acknowledged for kindly providing us with the
programs Grid and VolSurf. We thank Dr. Giovanni Gaviraghi
(Sienabiotech S.p.A., Siena, Italy) for helpful discussions.
8. 1-(2-Chloro-2-phenylethyl)-N-(4-fluorobenzyl)-1H-pyra-
zolo[3,4-d]pyrimidin-4-amine (9). White solid, yield 69%, mp
168–169 °C. 1H NMR: δ 4.62–5.01 (m, 4H, CH2N + CH2Ar),
5.40–5.54 (m, 1H, CHCl), 6.90–7.44 (m, 9H Ar), 7.80 (s, 1H, H-3),
8.33 (s, 1H, H-6). IR (cm-1): 3247 (NH). Anal. (C20H17N5ClF) C,
H, N.
B. Biology. Compound 8 (PP2), used as the reference com-
pound, was purchased from Calbiochem (San Diego, CA).
1. Cell-free Assay with Recombinant Abl. Recombinant hu-
man Abl was purchased from Upstate Biotechnology (Waltham,
MA) and used to investigate the mechanism of kinase inhibition,
as previously reported.22
Supporting Information Available: Details on synthesis,
biological assays, molecular modeling, and a table of the elemental
analysis data of the new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
2. Western Blot Analysis. The inhibitory effect of compounds
toward the phosphorylation of Bcr-Abl (Tyr245) and STAT-5
(Tyr694) was tested using a PathScan Multiplex Western Detection
Kit (Cell Signaling Technology, Beverly, MA). This kit was used
to assay the inhibition of multiple proteins on one membrane
without stripping and reprobing. The inhibitory effect toward the
phosphorylation of Src (Tyr416) was assessed using specific
antibodies (Cell Signaling Technology). K-562 and KU-812 were
cultured at a concentration of 2.5 × 105 cells/mL and challenged
with the compound (50 µM) for 3 h. Later, cells were harvested
and lysed in an appropriate buffer containing 1% Triton X-100.
Proteins were quantitated by the BCA method (Pierce, Rockford,
IL). Equal amounts of total cellular protein were resolved by SDS-
polyacrylamide gel electrophoresis, transferred to nitrocellulose
filters, and subjected to immunoblot. Nonsaturated, immunoreactive
bands were detected with a CCD camera gel documentation system
(ChemiDocXRS, Bio-Rad Laboratories, Hercules, CA) and then
quantitated with Quantity One analysis software (Bio-Rad Labo-
ratories). The results were expressed as the mean ( SEM of three
independent experiments. Statistical analyses were performed using
Student’s t test and the Bonferroni’s correction.
3. mRNA Expression of Apoptotic Genes. The mRNA expres-
sion of apoptotic genes was performed using qRT-PCR. K-562 and
KU-812 cells were cultured at a concentration of 2.5 × 105 cells/
mL and challenged with the compound for 3 h and 72 h. Later, the
cells were harvested and lysed in an appropriate buffer (RNA wiz,
Ambion, Austin, CA). The extract was processed for the extraction
of mRNA. For qRT-PCR analysis, Bcl-xL (antiapoptotic gene) and
bax (proapoptotic gene) expression in K-562 and KU-812 cells was
determined using a MJ MiniOpticon Cycler (Bio-Rad Laboratories).
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 Labora-
tories) and specific primers from GenBank. Data were quantitatively
analyzed on an MJ OpticonMonitor detection system (Bio-Rad
Laboratories). All values were expressed as fold increase relative
to the expression of ꢀ-Actin.
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