Design, synthesis, and biological evaluation of pyrazolo[3,4-d]pyrimidines active in vivo on the Bcr-Abl T315I mutant

Article abstract of DOI:10.1021/jm400233w

Starting from our in-house library of pyrazolo[3,4-d]pyrimidines, a cross-docking simulation was conducted on Bcr-Abl T315I mutant. Among the selected compounds (2a-e), the 4-bromo derivative 2b showed the best activity against the Bcr-Abl T315I mutant. Deeper computational studies highlighted the importance of the bromine atom in the para position of the N1 side chain phenyl ring for the interaction with the T315I mutant. A series of 4-bromo derivatives was thus synthesized and biologically evaluated. Compound 2j showed a good balance of different ADME properties, high activity in cell-free assays, and a submicromolar potency against T315I Bcr-Abl expressing cells. In addition, it was converted into a water-soluble formulation by liposome encapsulation, preserving a good activity on leukemic T315I cells and avoiding the use of DMSO as solubilizing agent. In vivo studies on mice inoculated with 32D-T315I cells and treated with 2j showed a more than 50% reduction in tumor volumes.

Full text of DOI:10.1021/jm400233w

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of
Pyrazolo[3,4‑d]pyrimidines Active in Vivo on the Bcr-Abl T315I
Mutant
Marco Radi,†,‡ Cristina Tintori,† Francesca Musumeci,§ Chiara Brullo,§ Claudio Zamperini,†
Elena Dreassi,† Anna Lucia Fallacara,†,∥ Giulia Vignaroli,† Emmanuele Crespan,⊥ Samantha Zanoli,⊥
Ilaria Laurenzana,# Irene Filippi,△ Giovanni Maga,⊥ Silvia Schenone,*,§ Adriano Angelucci,○
and Maurizio Botta*,†,◆
†Dipartimento di Biotecnologie, Chimica e Farmacia, Universita
̀
degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy
‡Dipartimento di Farmacia, Universita
§Dipartimento di Farmacia, Universita
̀
̀
degli Studi di Parma, Viale delle Scienze 27/A, 43124 Parma, Italy
degli Studi di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
∥Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita
̀
di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy
⊥Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
#Laboratory of Preclinical and Translational Research, IRCCS-Referral Cancer Center of Basilicata (CROB), Rionero in Vulture
(PZ), Italy
△Dipartimento di Medicina Molecolare e dello Sviluppo, Universita
○Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Universita
̀
degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy
dell’Aquila Via Vetoio, 67100 Coppito, L’Aquila, Italy
̀
◆Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology,
Temple University, BioLife Science Building, Suite 333, 1900 North 12th Street, Philadelphia, Pennsylvania 19122, United States
S
* Supporting Information
ABSTRACT: Starting from our in-house library of pyrazolo-
[3,4-d]pyrimidines, a cross-docking simulation was conducted
on Bcr-Abl T315I mutant. Among the selected compounds
(2a−e), the 4-bromo derivative 2b showed the best activity
against the Bcr-Abl T315I mutant. Deeper computational
studies highlighted the importance of the bromine atom in the
para position of the N1 side chain phenyl ring for the
interaction with the T315I mutant. A series of 4-bromo
derivatives was thus synthesized and biologically evaluated.
Compound 2j showed a good balance of different ADME
properties, high activity in cell-free assays, and a submicromo-
lar potency against T315I Bcr-Abl expressing cells. In addition,
it was converted into a water-soluble formulation by liposome
encapsulation, preserving a good activity on leukemic T315I cells and avoiding the use of DMSO as solubilizing agent. In vivo
studies on mice inoculated with 32D-T315I cells and treated with 2j showed a more than 50% reduction in tumor volumes.
resistant mutants but proved to be ineffective against the T315I
mutation of the gatekeeper residue, which accounts for 15−
20% of clinically observed mutations.6,7
Preclinical and clinical studies showed that ATP-competitive
dual Src/Bcr-Abl inhibitors bosutinib (SKI-606),8 bafetinib
(INNO-406, NS-187),9 3-[2-[2-cyclopentyl-6-(4-
dimethylphosphorylanilino)purin-9-yl]ethyl]phenol
(AP23464),10 and 6-(2,6-dichlorophenyl)-2-{[3-
(hydroxymethyl)phenyl]amino}-8-methylpyrido[2,3-d]-
pyrimidin-7(8H)-one (PD166326)11 inhibit Bcr-Abl more
INTRODUCTION
■
Chronic myeloid leukemia (CML) is a hematological
malignancy caused by the constitutively activated kinase Bcr-
Abl, resulting from the Philadelphia chromosome (Ph)
translocation.1 Since 2001, the Bcr-Abl tyrosine kinase (TK)
inhibitor imatinib (IM) has represented the first line therapy for
CML, being particularly active in the chronic phase of the
disease, with impressive response and survival rates.2 However,
many patients eventually develop resistance that is frequently
associated with mutations in the Bcr-Abl kinase domain.3 The
second-generation Bcr-Abl inhibitor nilotinib4 and the dual
Src/Bcr-Abl inhibitor dasatinib,5 both approved by FDA for
IM-resistant CML treatment, are active against many IM-
Received: February 14, 2013
Published: June 7, 2013
© 2013 American Chemical Society
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Figure 1. Selected second- and third-generation Bcr-Abl inhibitors.
efficiently than IM and overcome resistance caused by most
mutations, with the notable exception of T315I. Optimization
of CML treatment remains an area of active research, and much
effort has been devoted to the development of more effective
TK inhibitors such as ponatinib (AP24534).12 This compound
inhibits both native and mutant Bcr-Abl, including T315I,
acting as a pan-Bcr-Abl inhibitor also targeting other TKs.13
The T315I point mutation precludes drug binding to the Abl
catalytic domain by either direct steric hindrance or
stabilization of its active conformation, with the DFG-loop in
the “in” disposition (DGF-in).14 The development of new
drugs targeting the open and active conformation of the T315I
mutant is therefore a major challenge in CML therapy.15
Despite the past efforts to develop selective targeted
inhibitors for the treatment of cancer, the main strategy has
recently turned to find compounds acting on multiple targets in
order to face the drug resistance, often connected with the
activation of alternative signaling pathways.16 Furthermore, Abl
shares significant sequence homology and remarkable structural
resemblance with Src family members in their active state and
several ATP-competitive Src inhibitors targeting the active
conformation showed to be also potent Abl inhibitors.17a In this
context, our research group has deeply investigated a class of
pyrazolo[3,4-d]pyrimidine derivatives acting as dual inhibitors
of Src and Abl kinases. These compounds showed potent
antiproliferative and proapoptotic activity toward different
cancer cell lines depending on the nature and position of
substituents on the heterocyclic core.18−21
sensitive and imatinib resistant CML patients, these last bearing
the three most frequent Bcr-Abl mutations (including
T315I).22 We decided to get further insights into this type of
compounds with the aim of obtaining other active derivatives
and of evaluating their activity in vivo.
In the present work, we describe the rational development of
novel pyrazolo[3,4-d]pyrimidines able to inhibit the Bcr-Abl
T315I mutant both in vitro and in vivo. A combination of in
silico and in vitro studies highlighted the importance of a
bromine atom in the para position of the N1 side chain phenyl
ring for the interaction with the hydrophobic region I of the
T315I mutant. Following this computational study, we
synthesized a small collection of 4-bromo derivatives, which
led to the identification of compound 2j as the most promising
inhibitor showing significant reduction of tumor volume in
mice inoculated with 32D-T315I cells.
RESULTS AND DISCUSSION
■
Computational Studies on Bcr-Abl T315I Mutant. A
cross-docking approach was employed in this study for the in
silico screening of our in-house library of pyrazolo[3,4-
d]pyrimidines (consisting of about 300 structurally charac-
terized compounds with purity >98%) to select the most
promising potential binders of the Bcr-Abl T315I mutant for
biological investigation. Two crystal structures were used for
this purpose, the structure of the T315I mutant of Abl kinase
bound by the pyrrolo-pyridine inhibitor 3 (PPY-A) (Figure 1)
(1.95 Å resolution, PDB code 2Z60)23 and that of the T315I
mutant Abl kinase domain in complex with another pyrrolo-
pyridine 4 (SX7) (2.01 Å resolution, PDB code 3DK7).24 In
Recently, we identified two pyrazolo-pyrimidine based Bcr-
Abl inhibitors, namely 1a and 1b (Figure 1), which were
effective against CD34+ cells collected from both imatinib-
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contrast to other inhibitors cocrystallized with the T315I Abl
mutant, such as danusertib (PHA-739358),25 ponatinib
(AP24534),26 5-[(5-{[4-{[4-(2-hydroxyethyl)piperazin-1-yl]-
methyl}-3-(trifluoromethyl)phenyl]carbamoyl}-2-
methylphenyl)ethynyl]-1-methyl-1H-imidazole-2-carboxamide
(AP24589),27 and rebastinib (DCC2036),28 compounds 3 and
4 have a shape that more closely resembles that of our
pyrazolo[3,4-d]pyrimidine compounds, including 1a,b (Figure
1).
Compounds analyzed herein are already known dual Src/Abl
inhibitors with activity ranging from 0.02 to 100 μM. From a
structural point of view, these compounds are related to the Src
inhibitors PP1 and PP2 but bear a different substitution pattern
on the position 1, 3, 4, and 6 of the pyrazolo[3,4-d]pyrimidine
nucleus. In agreement with our previously reported simu-
lations,20 docking studies within the ATP binding site of T315I
Abl gave two different outcomes depending on the presence or
the absence of an alkylthio substituent at position 6 of the
pyrazolo-pyrimidine ring. Docking calculations on the C6-
substituted compounds within the ATP binding site of the
T315I Abl mutant did not converge to a reasonable solution,
while profitable contacts could be established by the same
compounds within the wild-type enzyme, in agreement with the
binding mode described elsewhere.29 Unfavorable binding
poses within the T315I Abl catalytic pocket were due to the
steric hindrance caused by the residue Ile315, which precludes
the access of the C6-substituted pyrazolo[3,4-d]pyrimidines, as
shown by our earlier results.20 Instead, a well-defined binding
mode was found for C6-unsubstituted compounds within the
two X-ray structures of T315I Abl mutant used for calculations
(Figure 2A). In detail, the C6-unsubstituted derivatives are
involved in two hydrogen bonds with the Met318 of the hinge
region by means of the atom N5 and the exocyclic amino group
at C4. Moreover, the N1 side chain is located within a
hydrophobic pocket formed by residues Lys271, Met290,
Phe382, Ile313, Ile315, Val299, Val256, and Tyr253 (hydro-
phobic region I), while substituents at C4 interact with Leu248,
Gly321, and Phe317 (hydrophobic region II). Because the
amino acid in position 315 belongs to the hydrophobic region I,
where the interactions between the C6-unsubstituted-pyrazolo-
pyrimidines and the protein are exclusively hydrophobic, its
mutation to isoleucine does not affect the correct placement of
the C6-unsubstituted derivatives within the binding site. As a
consequence, the binding mode proposed for the compounds
within the T315I Abl binding pocket is the same of that
previously found in the wild-type enzyme.29 Comparable results
were obtained using the two proteins 2Z60 and 3DK7 in terms
of both chemscore values and docking poses. Examination of
the results indicated that all the inhibitors showed chemscore
values ranging from 25 to 37 and from 26 to 38 in 2Z60 and
3DK7, respectively. However, slight differences between the
two X-ray structures were observed in the interaction of C6-
unsubstituted ligands with hydrophobic region I (Figure 2B).
While in 2Z60 the ligand adopted an extended conformation
with the N1-2-chloro-2-phenylethyl substituent immersed in
the deep pocket, in 3DK7, the N1-side chain was rotated by
∼50° with the terminal phenyl group exposed to the solvent.
The different placement of the substituent at N1 in the two
crystallographic structures was mainly due to the different
conformation adopted by the conserved DFG motif that affects
the shape of the pocket and consequently the pattern of
interactions between the N1 side chain and the protein.
Figure 2. (A) Schematic representation of the binding mode of C6-
unsubstituted pyrazolo[3,4-d]pyrimidines within the T315I Abl
mutant binding site. (B) Binding mode of compound 2b within
2Z60 (cyano) and 3DK7 (magenta) structures. Slight differences can
be observed in the orientation of the ligand within the hydrophobic
region I of the two structures.
According to the above-described docking results, the in-
house available C6-unsubstituted compounds 2a−e were
selected for in vitro evaluation against the isolated T315I Abl
together with compound 1b, used as a reference (Table 1).
These compounds were selected for testing according to the
predicted score values, the number of clusters obtained, the
binding poses observed, and their molecular structure in order
to evaluate the importance of the R2 substituent. Biological
results are reported in Table 1. It is noteworthy that compound
2b, in which R2 is a bromine atom, proved to be the most active
against the T315I Abl mutant (36 nM) with a comparable
activity against Abl wild-type (55 nM). To rationalize these
biological data, the binding free energies between Abl T315I
and ligands (1b and 2a−e) were calculated by employing the
molecular mechanics/generalized Born surface area (MM-
GBSA) scoring method on the docking poses (Table S2,
Supporting Information). Two interesting aspects emerged
from this analysis: (a) all compounds had a higher affinity for
protein 2Z60 (compared to 3DK7) giving more stable
complexes; (b) the ΔG values obtained with the use of 2Z60
structure better correlate with the experimental activities. In
fact, the lowest binding energy (−53.2 KJ/mol) was found for
compound 2b, the most active of the series. On the other hand,
no correlation between calculated and experimental data was
obtained by using the 3DK7 structure. Overall, these results
highlighted that 2Z60 was the most reliable structure for in
silico studies on our pyrazolo-pyrimidine inhibitors and thus we
decided to use only this one in the following calculations.
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Table 1. Enzymatic Activities, Cellular Activities, and ADME Properties
a
b
Ki (μM)
IC50 (μM)
met stab
Abl
Abl
solub H2O
human
c
d
e
compd
R1
R2
Cl
(WT) (T315I) c-Src
P210 parental T315I PAMPA Papp 10−6 cm/s (%MR)
(μg mL−1
)
(%)
1b
2a
2b
2c
2d
2e
2f
CH2C6H4-4F
C6H4-3F
0.08
0.732
0.055
0.1
0.16
2.430
0.036
23.6
0.8
2.7
>10
3.1
0.5
3.0
1.7
16.6 (64)
13.9 (5.4)
ND
<0.01
0.03
<0.01
ND
94.0
92.3
ND
ND
ND
ND
97.2
96.8
98.0
ND
96.4
ND
92.4
95.2
93.8
ND
ND
95.3
ND
ND
95.0
ND
ND
ND
ND
ND
ND
F
0.160
0.045
1.5
>10
>10
ND
ND
>10
>10
>10
>10
ND
>10
ND
>10
>10
ND
ND
ND
>10
ND
ND
ND
ND
ND
ND
ND
ND
ND
CH2C6H4-3F
CH2C6H4-3F
CH2C6H4-3F
C6H4-3Cl
Br
F
0.91
ND
ND
3.8
ND
ND
7.5
ND
H
0.04
16.7
0.08
ND
ND
F
0.18
2.69
1.4
ND
<0.01
0.04
<0.01
0.08
ND
C6H4-3Br
F
0.656
0.260
1.440
0.89
3.24
0.340
0.395
2.350
0.11
>10
7.2
3.3
6.8 (29.3)
3.20 (60)
7.5 (29.5)
ND
2g
2h
2i
C6H4-3Br
H
1.280
3.000
37.5
1.7
C6H4-3F
H
>10
ND
2.1
8.3
C6H5
H
ND
0.12
ND
4.5
2j
C6H5
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
CH3
I
0.610
1.308
0.021
0.005
0.140
3.342
1.100
2.530
2.719
NA
0.090
0.575
0.970
0.002
0.029
1.5
0.064
0.618
0.070
0.620
0.006
0.610
0.500
1.080
0.165
1.700
1.097
0.723
0.770
0.150
0.637
ND
6.64 (32.1)
ND
0.06
ND
2k
2l
CH2C6H5
ND
4.7
CH2C6H4-3Cl
CH2C6H4-2Cl
CH2C6H4-4Cl
CH2C6H3-2,5Cl
CH2C6H3-3,4Cl
CH2C6H4-4F
CH2C6H3-2,5F
CH2C6H3-3,4F
CH2C6H4-4Br
CH2C6H4-2Br
CH2C6H4-3Br
CH2C6H4-3OCH3
CH2C6H3-3,5CF3
C6H5
9.1 (23.3)
8.78 (21)
4.24 (45.8)
ND
<0.01
<0.01
<0.01
ND
2m
2n
2o
2p
2q
2r
0.8
0.22
ND
ND
ND
0.15
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.1
0.497
0.006
1.080
2.710
0.099
1.343
0.673
3.400
1.155
1.900
ND
ND
10.7 (21.8)
ND
<0.01
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2s
2t
ND
ND
3.000
NA
3.41 (50.7)
ND
<0.01
ND
2u
2v
2w
2x
2y
2z
1.264
4.750
1.810
0.060
0.050
ND
ND
ND
ND
ND
ND
ND
ND
C6H5
0.580
ND
ND
ND
a
b
c
Values are the mean of at least two experiments. IC50 values are means SEM of series separate assays, each performed in triplicate. PAMPA see
d
Experimental Section for details. Membrane retention (MR) expressed as percentage of compound unable to reach the acceptor compartment.
e
Expressed as percentage of unmodified parent drug.
Next, we focused our attention on compounds 2b, 2c, and
2d, which differ only for the R2 substituent on the N1 side
chain phenyl ring (Br, F, H, respectively). To study the
contribution of the bromo substituent to the activity of
compound 2b, a detailed analysis of the binding site of 2Z60
was performed with the aim of identifying regions of favorable
interactions between the protein and halogen atoms (Br, F, and
Cl, respectively). For this purpose, molecular interaction fields
(MIFs) were calculated for the binding site by means of the
software Grid.30 Docking results and Grid analysis were then
combined together. Figure 3 shows the computed binding
mode of compound 2b together with the most favorable
interaction point determined for the probe Br (organic bromine
atom, dark-red sphere) within the binding site of the 2Z60
protein. Interestingly, this minimum grid point is characterized
by a significant favorable energy (−10.42 kcal/mol) and is
located in proximity of the bromo substituent of the inhibitor
2b. In the same position, a point of profitable interaction was
also found by using the probe F. An energy of −4.34 higher
than that found with Br was associated with this minimum.
Interestingly, a minimum grid point for F with an energy of
−3.68 kcal/mol was also found around the meta position of
benzyl group at C4.
Concerning the minimum grid points calculated for probe Cl
(organic chlorine atom), the most profitable interaction (−8.43
kcal/mol) was identified near the aliphatic chlorine atom of the
N1-substituent. Moreover, a minimum grid point for Cl with an
energy of −5.28 kcal/mol was found around the meta position
of the N1 side chain phenyl ring while no minima were
identified in proximity of the para position. Overall, docking
results in combination with Grid analysis and MM-GBSA
rescoring were able to explain the better inhibitory activity
against T315I Abl mutant of compound 2b substituted with a
bromine atom in para position of the N1 side chain phenyl ring.
On this basis, we decided to synthesize a small library of
compounds bearing a 2-(4-bromophenyl)-2-chloroethyl N1
side chain and different amines at C4. Remarkably, a good
correlation was found between calculated free energy of binding
and experimental Ki toward the T315I mutant (Table S2,
Supporting Information). To quantitatively study the bromine
contribution to the affinity for T315I Abl mutant, Monte
Carlo/free-energy perturbation (MC/FEP) calculations were
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other side, the weaker binding affinity of the methyl derivative
2y could be due to both electronic and steric reasons.
Chemistry and ADME Properties. Compounds 2e−j and
2y,z, bearing an anilino group in C4, were obtained in yields
ranging from 57 to 71% by reacting the corresponding 4-chloro
derivatives 5a−e with an excess of the suitable aniline in
absolute ethanol at reflux for 3−5 h. On the other side, the
benzylamino derivatives 2k−x were obtained in good yield by
reacting the intermediate 5c with an excess of the more
nucleophilic benzylamines in anhydrous toluene at room
temperature for 48−72 h (Scheme 1).
a
Scheme 1
Figure 3. Binding mode of compound 2b (R enantiomer) within the
T315I Abl binding site. Compound locates the N1 side chain and the
C4-substituent in hydrophobic region I and II, respectively. It also
establishes two hydrogen bonds with Met318, involving the C4 amino
group and the N5 of the pyrazolo[3,4-d]pyrimidine nucleus. The most
favorable interaction point determined for probe Br is visualized as a
red sphere. It falls in the hydrophobic region I and exactly in the
region where the Br substituent of 2b takes place.
performed to obtain computed changes in the free energy of
binding for the presence of a bromine atom in the para position
of the N1 chain phenyl ring. The experimental data showed
that the addition of a bromo substituent in such a position is
crucial, transforming a poorly active compound into an active
one with a Ki of 0.090 μM (compare 2i with 2j, Table 1).
The MC/FEP calculations for the conversion of 2j into 2i
gave consistent results, favoring the binding of 2j to the T315I
a
Reagents and conditions: (i) anilines, ethanol, reflux, 3−5 h (method
A); benzylamines, toluene, rt, 48−72 h (method B).
The synthesis of intermediates 5a−c was previously
performed by us, using as starting products the suitable
phenyloxiranes and hydrazine monohydrate.17b The synthesis
of intermediates 5d,e was performed by us using a different
route because the corresponding oxiranes are not commercially
available. Allopurinol 6 was chlorinated with POCl3/DMF and
successively substituted on N1 with the suitable 2-bromoace-
tophenone, in the presence of TBAF as a base, affording
intermediates 8a,b. Reduction of these compounds with sodium
borohydride led to the corresponding alcohols 9a,b that were
finally chlorinated to 5d,e (Scheme 2).
The most interesting compounds identified by the biological
assays were then submitted to a thorough in vitro ADME study
to determine their aqueous solubility, parallel artificial
membrane permeability (PAMPA), and human liver micro-
somes (HLM) stability in order to early assess the absorption/
stability of these drug candidates (Table 1). Passive membrane
permeability was evaluated with the PAMPA assay, applying a
validated protocol recently developed by us for poorly water-
soluble pyrazolo[3,4-d]pyrimidines.32 Although the presence of
a cosolvent (DMSO) proved to slightly decrease the
penetration of the compounds, a good correlation was found
with the experimental data, and overall, this protocol gives
useful insights in ranking the intestinal absorption of lipophilic
compounds. Compound solubility was then evaluated following
the method developed by Avdeef et al., and results were
expressed in μg/mL.33 Metabolic stability was finally evaluated
by incubating the above-mentioned compounds with 5 μL of
man-pooled HLM for 1 h at 37 °C in order to simulate phase I
metabolism. The parent drugs and metabolites were sub-
sequently determined by LC-MS analysis.
mutant by 2
0.2 kcal/mol. Introduction of the para-Br
substituent is computed to be particularly favorable as it
projects into a hydrophobic region lined by the side chains of
Ile313, Ile315, Met290, Phe382, and Glu286. Conversely, the
introduction of a para fluorine into the N1-phenylethyl side
chain had only little effect on Ki passing from 3 to 2.4 μM
(compare 2h with 2a, Table 1). Accordingly, the replacement
of hydrogen by fluorine in that position was predicted to be
favorable (more negative free energy of binding) by only 0.42
0.1 kcal/mol.
These results confirmed the importance of the bromine atom
in para position of the N1 side chain phenyl ring for the
interaction with the hydrophobic region I in the T315I mutant.
In addition, taking into account the presence of the Glu286
residue in proximity of the para position of the N1 side chain
together with information obtained by SAR studies, we
hypothesized that a halogen bonding31 contribution could
occur in the stabilization of the inhibitors’ binding. Such
interaction becomes more favorable in progressing from F to Cl
to Br. An optimal distance of ca. 3.1 Å was found between the
bromine atom of compound 2j and the closest carboxyl oxygen
of Glu286 during Monte Carlo FEP simulations. To better
investigate this aspect, we synthesized and tested the 2j
analogues 2y and 2z, in which the bromine atom was replaced
with a methyl group or a iodine atom, respectively. In cell-free
assays, compounds 2y and 2z showed a lower affinity toward
T315I compared with the parent p-Br substituted compound
2j. Although the iodo-derivative 2z should give a halogen
bonding contribution in the binding to the T315I mutant, the
bulky nature of the iodine atom determines an unfavorable
binding mode that justifies the lower activity of 2z. On the
In general, the synthesized compounds showed values of
membrane permeation and metabolic stability comparable to
that of the reference 1b but, in most of the cases, they displayed
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a
Scheme 2
a
Reagents and conditions: (i) POCl3/DMF reflux, 12 h; (ii) TBAF 2M in THF, BrCH2COC6H4R2, THF, rt, 24 h; (iii) NaBH4, THF, 0 °C; (iv)
POCl3/DMF, reflux 5 h.
Figure 4. Cell death in response to treatment with selected inhibitors. (a) The percentage of dead cells was calculated considering the total number
of 32D-T315I cells treated for 48 h with 1 μM of representative compounds. The values of three different experiments were considered to calculate
the mean and the standard deviation. (b) Agarose gel electrophoretic analysis of genomic DNA extracted from 32D-T315I cells treated for 48 h with
0.1 and 1 μM 2j in comparison with untreated cells (control). (c) Percentage of CD34+ cells from bone marrow samples of CML (blu lines) and
T315I CML patients (red lines) after treatment with 2q (left) and 2j (right).
a lower membrane retention (Table 1, data in parentheses).
The ADME investigation highlighted good metabolic stability
(higher than 90%) and membrane permeability (ranging from
3.20 to 13.9 × 10−6 cm/s) for the most representative
compounds, while the water solubility was in the medium−low
range. A few compounds (2a, 2f, 2h, and 2j) presented
however a better water solubility with respect to the reference
compound 1b.
In Vitro Biological Activity. All the synthesized com-
pounds were initially tested in a cell-free assay to evaluate their
affinity toward Abl, Abl T315I mutant, and Src enzymes in
comparison with the reference compound 1b, which was
previously reported to inhibit Ba/F3 cells expressing the Bcr-
Abl T315I mutant.20 As can be appreciated from Table 1,
among the compounds selected by the virtual screening (2a−
e), derivative 2b showed to be a potent inhibitor of the T315I
mutant. This activity was linked to the presence of the bromine
atom in para position of the N1 side chain phenyl ring, as
hypothesized by our molecular modeling calculations and
further confirmed by comparing the activity data of 2b with
those of 2c,d. All para bromo derivatives herein synthesized,
with the only exceptions of 2s and 2u, maintained a dual Src/
Abl inhibitory profile. As suggested by the molecular modeling
studies, all compounds bearing a bromine atom in para position
of the N1 side chain phenyl ring (2b, 2j−x) maintained or
improved the activity against the T315I mutant. Among these
derivatives, compounds 2b, 2j, 2m, 2n, 2q, and 2t showed a
high affinity for Abl T315I, with a Ki in the nanomolar range
(36, 90, 2, 29, 6, and 99 nM, respectively) while the affinity for
the wild-type Abl was generally in a lower range (55, 610, 5,
140, 2530, and 3000 nM, respectively). Selected compounds
(2a,b, 2e−h, 2j, 2l−m, and 2q) were then evaluated for their in
vitro cytotoxic effect using the murine myeloid cell line 32D
expressing the nonmutated Bcr-Abl fusion protein (p210) or
the mutated form T315I. Cells were treated for 48 h with
increasing concentrations of the inhibitors (0.01−50 μM), and
IC50s were calculated by considering the number of vital cells
with respect to the control (Table 1). Notably, all the tested
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Figure 5. Liposomes formulation experiments. (a) TEM image of negative stain liposomes loaded with 2j; SUV (<200 nm) are clearly visible. (b)
Myeloid cells, 32D-p210, and 32D-T315I (p210 and T315I) were treated with 2j (0.1 and 1 μM) prepared in DMSO or encapsulated in liposomes
and cell viability was measured after 48 h of incubation. The values of three different experiments were considered to calculate the mean and the
standard deviation.
Figure 6. (a) Nude mice were inoculated sc with 32D-p210 or 32D-T315I cells, and tumor growth was monitored by measuring tumor mass width
and length. For each cell line inoculated, mice were divided in a control group and a 2j-treated group, with each group represented at least by five
mice. P < 0.01 according to Student’s t test. (b) At the end point of the experiment in (a), tumor masses were excised from mice inoculated with
32D-T315I cells. Representative tumor masses from each experimental group are shown. (c) Histological image taken from 32D-T315I graft treated
with 2j, showing a necrotic area within the tumor. A region containing apoptotic figures is evident in the boundary of the necrotic zone (white
arrow). In the inset, a magnification image illustrating the apoptotic figures is shown.
compounds showed a higher cytotoxic activity against IM-
resistant cells expressing T315I mutated Bcr-Abl compared to
IM-sensitive 32D-p210 cells, in partial accordance with the
enzymatic data. In 32D-T315I cells, IC50s ranged from 0.12 to
8.3 μM and compounds demonstrated different capacities in
inducing cell death. When 32D-T315I cells were treated for 48
h with 1 μM of the compounds, cell death was observed for all
compounds analyzed and the number of dead cells was higher
than 50% of the total only for 2e, 2g, and 2j (Figure 4a).
Apoptosis was the prevalent modality of death as indicated by
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the ordered fragmentation of genomic DNA (Figure 4b). In
addition, it was noteworthy that these 4-bromo derivatives did
not show cytotoxic effects when tested up to 10 μM
concentration in parental cell lines (see Table 1) which did
not express the chimeric Bcr-Abl protein. Furthermore,
compounds 2q and 2j were also tested on early hematopoietic
progenitors (CD34+) from Ph+ CML patients at diagnosis and
from Ph+ CML patients who developed resistance to Imatinib
mesylate in consequence of T315I Bcr-Abl mutation. Cells
were seeded at 4 × 105 cells/mL density and treated with 2q
and 2j and with DMSO vehicle control at concentrations of 1,
10, and 20 μM for 48 h. In Ph+ T315I mutant CML cells, the
percentage of viable cells after treatment with the two
compounds decreases with increasing compound concentra-
tion. By contrast, in Ph+ CML cells, no effects were observed
(Figure 4c).
Most of the compounds present therefore an activity profile
complementary to that of IM, dasatinib, and other known Abl
inhibitors, being inhibitors of cell lines expressing the T315I
Bcr-Abl construct with a moderate effect on IM-sensitive cells.
Among the most promising T315I inhibitors, compounds 2j
was selected for the in vivo studies because it showed a good
balance of different ADME properties, high activity in cell-free
assays, and interesting submicromolar potency against T315I
Bcr-Abl expressing cells at the same time. An important issue
for a potential in vivo application in cancer therapy of a novel
agent effective in vitro at relatively high dosage is represented
by the design of a suitable drug formulation, characterized by a
prolonged blood half-life and a good biocompatibility. The use
of DMSO as solubilizing agent for drugs with a lower solubility
would be not suitable for clinical settings. According to these
considerations, liposome formulation of 2j was prepared for in
vitro screening in order to check if the activity profile could be
improved by a further increase of water solubility. The small
unilamellar vesicle (SUV) liposomes were characterized by
negative stain transmission electron microscopy (TEM) for
vesicle morphology and size (Figure 5a). Entrapment efficiency
was calculated with HPLC-UV analysis showing a good
entrapment efficiency (>98%).
Liposome encapsulated 2j was tested on 32D-p210 and 32D-
T315I cells at concentrations 0.1 and 1 μM in comparison with
the DMSO dissolved 2j at the same concentration (Figure 5b).
The cytotoxic effect of liposome encapsulated 2j at both
concentrations was almost the same as that of 2j dissolved in
DMSO. This result is important because it shows that the
liposome formulation, which is completely water soluble, can
avoid the use of DMSO as a solubilizing agent to test our
poorly water-soluble pyrazolo-pyrimidines.
In Vivo Studies. The antitumor activity of 2j was finally
tested in vivo using a xenograft mouse model. Mice inoculated
with 32D-p210 or 32D-T315I cells were treated daily with 80
mg/kg 2j starting from the appearance of a visible tumor mass,
and the tumor volume was evaluated at regular intervals. 2j
caused a significant reduction in tumor volumes after 17 days of
treatment only in mice inoculated with 32D-T315I cells, with a
reduction of more than 50% in mean tumor volume compared
to placebo treated mice (Figure 6a, right graph). In mice
inoculated with 32D-p210 cells, we observed a reduction in
mean tumor volume after treatment with 2j, mainly in the first
week, but this difference was not statistically significant (Figure
6a, left graph). It is notable that 32D-p210 cells had a slower
growing rate in vivo with respect to 32D-T315I, and this aspect
further demonstrated the effectiveness of 2j in a very aggressive
tumor model. The antitumor effect of 2j was evident when
comparing tumor masses excised from mice at the end of the
experiment (Figure 6b). The tumors from mice inoculated with
32D-T315I were analyzed by routine histology methods and
revealed the presence of a significant amount of necrotic tissue
component in mice treated with 2j. In particular, in these
tumors, apoptotic zone flanking necrotic areas resulted and
were clearly visible (Figure 6c).
CONCLUSIONS
■
Starting from our internal collection of pyrazolo[3,4-d]-
pyrimidines, a cross-docking approach allowed identification
of a point modification, consisting in placing a bromine atom
on the para position of the N1 side chain phenyl ring as a key
feature to establish profitable interactions with the hydrophobic
region I in Abl T315I mutant. Results of the docking studies in
combination with Grid analysis, MM-GBSA rescoring, and
MC/FEP calculations were in fact able to explain the important
role played by such 4-Br derivatives in the binding to the T315I
Abl mutant. A small collection of pyrazolo[3,4-d]pyrimidines
(2j−x) bearing this 4-bromo substitution on the N1 side chain
phenyl ring was thus synthesized and tested in cell-free assays
against Abl, Src, and Abl T315I; as expected, 2j−x maintained
or improved the activity against the T315I mutant.
All 4-bromo derivatives herein synthesized, with the only
exceptions of 2s and 2u, maintained a dual Src/Abl inhibitory
profile. As suggested by the molecular modeling studies, all
compounds bearing a bromine atom in para position of the N1
side chain phenyl ring (2j−x) maintained or increased the
activity against the T315I mutant, compared to 2b, while the
same trend was not observed with other derivatives lacking this
4-Br substitution (2c−i).
A few compounds (2a, 2f, 2h, and 2j) showed superior
ADME properties, when compared with the reference
compound 1b, and were further investigated for their in vitro
cytotoxic effect using the murine myeloid cell line 32D
expressing the nonmutated Bcr-Abl fusion protein (p210) or
the mutated form T315I. An interesting submicromolar
potency against T315I Bcr-Abl expressing cells was found
without any cytotoxic effects when tested up to 10 μM
concentration in parental cell lines. Most of the compounds
present therefore an activity profile complementary to that of
IM, dasatinib, and other known Abl inhibitors, being better
inhibitors of cell lines expressing the T315I Bcr-Abl construct
with a moderate effect on IM-sensitive cells. Finally, in vivo
studies on mice inoculated with 32D-T315I cells and treated
with 2j showed a significant reduction (more than 50%) of
tumor volumes with respect to placebo treated mice. In
conclusion, compound 2j represents an improved pyrazolo[3,4-
d]pyrimidine analogue active in vivo on the Bcr-Abl T315I
mutant and therefore worthy of further investigation.
EXPERIMENTAL SECTION
■
Computer Modeling. Docking Studies. Docking studies of all
pyrazolo[3,4-d]pyrimidine derivatives were performed within the ATP
binding site of the T315I Abl mutant (PDB code 2Z60 and 3DK7)
using the software package Gold 4.1.34 Compounds were first
processed with the Schrodinger LigPrep tool to generate separate
files for all possible enantiomers and protonation states at
physiological pH. The protein structures were prepared using the
Protein Preparation Wizard in the Schrodinger software graphical user
interface Maestro (version 9.1).35 Chemscore was chosen as the fitness
function for docking calculation with the CHO_TYPE term set to
SPECIAL to enable the recognition of activated CH groups for
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hydrogen bonding. The genetic algorithm parameter settings were
employed using the search efficiency set at 100%, and 100 runs were
carried out for each ligand. The reliability of the docking protocol was
first checked by simulation of the interactions between 3 and 4 with
the two crystal structures of the Abl T315I mutant and comparison of
the modeled complexes with the X-ray data. As a results, compounds 3
and 4 showed in both proteins an orientation in agreement with their
binding orientation and interactions onto the relative X-ray structure.
Taken together, these results led us to hypothesize that the
computational approach can be considered as a reliable modeling
procedure to be applied for finding the orientations and interactions of
ligand inside the kinase domain of T315I Abl mutant.
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 the internal standard, J in Hz. 1H patterns are described using
the following abbreviations: s = singlet, m = multiplet, and br s = broad
singlet. TLC was carried out using Merck TLC plates silica gel 60
F254. Chromatographic purifications were performed on columns
packed with Merk 60 silica gel, 23−400 mesh, for flash technique.
Analyses for C, H, and N were within 0.3% of the theoretical value.
Mass spectra (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 methanol/water. UV detection was
monitored at 254 nm. MS were acquired in positive and negative
modes, scanning over the mass range 50−1500. The following ion
source parameters were used: drying gas flow, 9 mL/min; nebulizer
pressure, 40 psig; drying gas temperature, 350 °C. All target
compounds possessed a purity of ≥95% as verified by elemental
analyses by comparison with the theoretical values. Synthesis and
analytical data of intermediates 3a−c and compounds 2a−d were
previously reported by us.17b,41
General Procedure for the Synthesis of 2e−j and 2y,z. A solution
of the appropriate 4-chloro derivatives 5a−c17b,41 or 5d,e (1 mmol)
and the suitable aniline (2 mmol) in absolute ethanol was refluxed for
3−5 h. After cooling, the solvent was evaporated under reduced
pressure, and the crude was treated with water (20 mL) then extracted
with CH2Cl2 (20 mL); the organic phases were washed with water (20
mL), dried (MgSO4), and concentrated under reduced pressure. The
obtained oils were crystallized by adding a mixture of diethyl ether/
petroleum ether (bp 40−60 °C) (1:1) and standing in a refrigerator to
afford compounds 2e−j and 2y,z as white solids, which were
recrystallized from absolute ethanol.
MM-GBSA. To remove unfavorable contacts, protein−ligand
complexes were imported in Maestro 9.1 and minimized through
the OPLS-AA force field and Polak−Ribiere conjugate gradient
method (0.05 KJ/mol·Å convergence or 200 iterations). A continuum
solvation method, with water as the solvent, was applied. Next, to
rescore ligands, MM-GBSA calculations of free energy of binding were
performed using the program Prime in Maestro suite from
Schrodinger.35
̈
MIF Analysis. Computation of MIFs within the ATP binding pocket
of Abl T315I was carried out with the software Grid.30 Box dimensions
were defined so as to accommodate all the residues constituting the
binding site and the number of planes of grid points per Angstrom
(NPLA) parameter was set to 1. MIFs were computed for Br, Cl, and
F probes. To achieve the points of minimum of MIFs, Grid
calculations were performed for each probe separately and using the
export GRIDKONT option. The kont files corresponding to each MIF
were then processed by means of the minim and filmap programs
(both implemented in the Grid package) that extract all the points of
minimum of each MIF and retain only those falling under a user-
defined cutoff.
1-[2-Chloro-2-(4-fluorophenyl)ethyl]-N-(3-chlorophenyl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (2e). White solid (273 mg, 68%);
Free Energy Perturbation. MC/FEP36 calculations were performed
with the MCPRO37 program and followed standard protocols.38,39 Z-
Matrix for the protein−ligand complexes, and free ligands were
obtained with the molecular growing program BOMB36 starting from
the docking pose of compound 2j. A conformational search was then
carried out on the ligands using the BOSS37 program with the OPLS/
CM1A force field and GB/SA hydration. The resultant conformer with
the lowest-energy was used for FEP calculation. The unbound ligands
and complexes were solvated in TIP4P water spheres (“caps”) with a
25 Å radius; after the removal of water molecules in too close contact
with solute atoms, ca. 2000 and 1250 water molecules remained for the
unbound and bound MC simulations. All protein residues within ca.
15 Å of any ligand atom were included. A few remote side chains were
neutralized in order to maintain overall charge neutrality for each
system. The ligand and the protein side chains within 10 Å of any
ligand atoms were sampled during the MC simulations. The only
constraints were the bond lengths in side chains, and all backbone
atoms were frozen after a short conjugate-gradient minimization. The
energetics for the systems were evaluated with the OPLS-AA force
field for the protein and OPLS/CM1A for the ligand.40 As usual, the
CM1A atomic charges were scaled by 1.14 for neutral molecules.
Differences in free energies of binding were determined from the usual
thermodynamic cycle that requires conversion of one ligand to another
both free in water and bound to the protein. The FEP calculations
utilized 11 windows of simple overlap sampling. For the unbound
ligand, each window consisted of 40 M configurations of equilibration
and 60 M configurations for averaging. For the bound calculations,
each window covered 20 M configurations of solvent-only
equilibration, 40 M configurations of full equilibration, and 50 M
configurations of averaging. All MC simulations were run at 298 K.
Due to the fact that the biological evaluation of all the chiral
compounds reported here was carried out using racemic mixtures,
MC/FEP analysis was performed on both R- and S-enantiomers and
medium values of free energy of binding was calculated.
mp 186−187 °C. H NMR: δ 4.62−4.78 and 4.83−4.98 (2m, 2H,
1
CH2N), 5.38−5.49 (m, 1H, CHCl), 6.86−7.47 (m, 8H Ar), 7.57 (s,
1H, H-3), 8.37 (s, 1H, H-6). IR cm−1: 3168 (NH). MS: m/z 402 [M +
H]+. Anal. (C19H14N5Cl2F) C, H, N.
General Procedure for the Synthesis of 2k−x. To a solution of 1-
[2-(4-bromophenyl)-2-chloroethyl]-4-chloro-1H-pyrazolo[3,4-d]-
pyrimidine 5c41b (1 mmol, 372 mg) in anhydrous toluene (10 mL),
the suitable amine (4 mmol) was added and the mixture was stirred at
room temperature for 48−72 h. Then the organic phase was washed
with water (2 × 10 mL), dried (MgSO4), and concentrated under
reduced pressure. The crude oils were crystallized by adding a mixture
of diethyl ether/petroleum ether (bp 40−60 °C) (1:1) and standing in
a refrigerator to afford compounds 2k−x as white solids, which were
recrystallized from absolute ethanol.
N-Benzyl-1-[2-(4-bromophenyl)-2-chloroethyl]-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (2k). White solid (261 mg, 59%); mp 182−184
°C. 1H NMR: δ 4.68−4.77 and 4.84−4.89 (2m, 4H, CH2N + CH2Ar),
5.42−5.45 (m, 1H, CHCl), 7.19−7.39 (m, 10H, 9Ar + H-3), 7.88 (s,
1H, H-6). IR cm−1: 3210 (NH). MS: m/z 443 [M + H]+. Anal.
(C20H17N5BrCl) C, H, N.
Synthesis of 4-Chloro-1H-pyrazolo[3,4-d]pyrimidine (7). Allopur-
inol 6 (2 g, 14.7 mmol) was added to POCl3 freshly distilled (15 mL).
Dry DMF (3.4 mL) was added to this mixture, and the solution was
refluxed for 12 h. After cooling at room temperature, the reaction
mixture was poured in a hydrogen carbonate saturated aqueous
solution at 0 °C. The aqueous solution was extracted with EtOAc (50
mL). The organic phase was dried (Na2SO4) and concentrated under
reduced pressure to obtain a yellow solid (1.72 g, 76%) used without
further purification in the next step.
1H NMR: δ 8.32 (s, 1H, H-3), 8.93 (s, 1H, H-6), 12.23 (br s, 1H,
NH, disappears with D2O). MS: m/z 154 [M − H]−.
General Procedure for the Synthesis of 8a,b. Compound 7 (200
mg, 1.29 mmol) was dissolved in dry THF (10 mL) at room
temperature, and TBAF 2.0 M in THF solution (2.58 mmol) was
added, following the procedure described by Brik et al.42 The desired
2-bromoacetophenone (1.29 mmol) was added to the solution, and
the reaction mixture was stirred at room temperature for 24 h. The
reaction mixture was filtered on Celite pad then extracted with EtOAc
Chemistry. Starting materials were purchased from Aldrich-Italia
(Milan, Italy). Melting points were determined with a Buchi 530
̈
apparatus and are uncorrected. IR spectra were measured in KBr with
a Perkin-Elmer 398 spectrophotometer. 1H NMR spectra were
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(100 mL) and water. The organic phase was dried (Na2SO4) and
concentrated under reduced pressure. The crude was purified by flash
chromatography on silica gel (eluent CH2Cl2/MeOH 99:1).
2-(4-Chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-1-(4-
methylphenyl)ethanone (8a). Yellow oil (267 mg, 72%). 1H NMR: δ
2.28 (s, 3H, CH3), 5.86 (s, 2H, CH2N), 7.13−7.15 and 7.76−7.78
(2m, 4H Ar), 8.10 (s, 1H, H-3), 8.59 (s, 1H, H-6). MS: m/z 287 [M +
H]+. Anal. (C14H11N4ClO) C, H, N.
were tested in three different plates on different days. The sandwich
was incubated for 5 h at room temperature under gentle shaking. After
the incubation time, the sandwich plates were separated, and samples
taken from both receiver and donor sides were analyzed using LC-UV-
MS method. Permeability (Papp) for PAMPA, was calculated according
to the equation obtained from Wohnsland and Faller.43
LC-UV-MS Method. Chromatographic analysis were performed with
an Agilent 1100 LC/MSD VL system (G1946C) (Agilent
Technologies, Palo Alto, CA) constituted by a vacuum solvent
degassing unit, a binary high-pressure gradient pump, an 1100 series
UV detector, and an 1100 MSD model VL benchtop mass
spectrometer. Chromatographic separation was obtained using a
Varian Polaris 5 C18-A column (150 mm × 4.6 mm, 5 μm particle
size) and gradient elution: eluent A being ACN and eluent B
consisting of an aqueous solution of formic acid (0.1%). The analysis
started with 2% of eluent A, which was rapidly increased up to 70% in
10 min, then slowly increased up to 98% in 15 min. The flow rate was
1.0 mL/min, and injection volume was 20 μL. The Agilent 1100 series
mass spectra detection (MSD) single-quadrupole instrument was
equipped with the orthogonal spray API-ES (Agilent Technologies,
Palo Alto, CA). Nitrogen was used as nebulizing and drying gas. The
pressure of the nebulizing gas, the flow of the drying gas, the capillary
voltage, the fragmentor voltage, and the vaporization temperature were
set at 40 psi, 9 L/min, 3000 V, 70 V, and 350 °C, respectively. UV
detection was monitored at 280 nm. The LC-ESI-MS determination
was performed by operating the MSD in the positive ion mode.
Spectra were acquired over the scan range m/z 100−1500 using a step
size of 0.1 u.
Microsomal Stability Assay. Each compound in DMSO solution
was incubated at 37 °C for 60 min in 125 mM phosphate buffer (pH
7.4), 5 μL of human liver microsomal protein (0.2 mg/mL), in the
presence of a NADPH-generating system at final volume of 0.5 mL
(compound final concentration 100 μM).19 The reaction was stopped
by cooling in ice and adding 1.0 mL of acetonitrile. The reaction
mixtures were centrifuged, and the parent drug and metabolites were
subsequently determined by the above-mentioned LC-UV-MS
method.
2-(4-Chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-1-(4-iodophenyl)-
1
ethanone (8b). Light-brown oil (278 mg, 70%). H NMR: δ 5.88 (s,
2H, CH2N), 7.65−7.67 and 7.81−7.83 (2m, 4H Ar), 8.20 (s, 1H, H-
3), 8.70 (s, 1H, H-6). MS: m/z 399 [M + H]+. Anal. (C13H8N4ClIO)
C, H, N.
General Procedure for the Synthesis of 9a,b. NaBH4 (119 mg,
3.14 mmol) was added to a solution of 8a or 8b (1.57 mmol) in dry
THF at 0 °C. The reaction mixture was stirred until the starting
material disappeared. The reaction was quenched by adding water then
it was extracted with EtOAc (50 mL). The organic phase was dried
(Na2SO4) and concentrated under reduced pressure. The solid was
purified by flash chromatography on silica gel (eluent CH2Cl2/MeOH
99:1).
2-(4-Chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-1-(4-
methylphenyl)ethanol (9a). Yellow solid (290 mg, 64%); mp 85−87
1
°C. H NMR: δ 2.00 (s, 3H, CH3), 4.50−4.55 and 4.63−4.68 (2m,
2H, CH2N), 5.16−5.19 (m, 1H, CHOH), 7.03−7.05 and 7.19−7.21
(2m, 4H Ar), 8.02 (s, 1H, H-3), 8.55 (s, 1H, H-6). MS: m/z 289 [M +
H]+. Anal. (C14H13N4ClO) C, H, N.
2-(4-Chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-1-(4-iodophenyl)-
ethanol (9b). Yellow solid (388 mg, 62%); mp 90−92 °C. 1H NMR: δ
4.61−4.66 and 4.74−4.80 (2m, 2H, CH2N), 5.21−5.24 (m, 1H,
CHOH), 7.08−7.10 and 7.58−7.60 (2m, 4H Ar), 8.02 (s, 1H, H-3),
8.57 (s, 1H, H-6). MS: m/z 401 [M + H]+. Anal. (C13H10N4ClIO) C,
H, N.
General Procedure for the Synthesis of 5d,e. Compound 9a or 9b
(1.06 mmol) was added to POCl3 freshly distilled (10 mL). To this
mixture was added DMF (3.0 mL), and the solution was refluxed for 5
h. After cooling at room temperature, the reaction mixture was poured
in a hydrogen carbonate saturated aqueous solution at 0 °C. The
aqueous solution was extracted with EtOAc (20 mL). The organic
phase was dried (Na2SO4) and concentrated under reduced pressure.
The reaction crude was purified by flash chromatography on silica gel
(eluent Hex/EtOAc 95:5).
Water Solubility Assay. Each solid compound (1 mg) was added to
1 mL of water. The samples were shaked in a shaker bath at 20 °C for
24−36 h. The suspensions were filtered through a 0.45 μm nylon filter
(Acrodisc), and the solubilized compound determined by LC-UV-MS
assay. For each compound, the determination was performed in
triplicate.
4-Chloro-1-[2-chloro-2-(4-methylphenyl)ethyl]-1H-pyrazolo[3,4-
1
d]pyrimidine (5d). White solid (129 mg, 40%); mp 100−102 °C. H
Liposomal Formulation. Liposomal formulations (phosphatidyl-
choline/cholesterol 90:10 (w/w)) were prepared with thin layer
evaporation method. Compound 2j, lipid(s), and cholesterol were
dissolved in chloroform/ethanol 3:2 (v/v), and the organic solvent was
evaporated to get a dry powder. Proliposomes (powder) were
hydrated in pH 7.4 phosphate buffered saline (PBS) by vortex and
sonication to obtain SUV liposomes. Liposomes were characterized by
negative stain electron microscopy (TEM) for vesicle morphology and
size. Entrapment efficiency was calculated with HPLC-UV analysis.
Biological Studies. Enzymatic Assay on Isolated Src. Recombi-
nant human Src was purchased from Upstate (Lake Placid, NY).
Activity was measured in a filter-binding assay using a commercial kit
(Src Assay Kit, Upstate), according to the manufacturer’s protocol,
using 150 μM of the specific Src peptide substrate (KVEKIGEG-
TYGVVYK) and in the presence of 0.125 pmol of Src and 0.160 pmol
of [γ-32P]-ATP. The apparent affinity (Km) values of the Src
preparation used for its peptide and ATP substrates were determined
separately and found to be 30 and 5 μM, respectively.
Kinetic Analysis. Dose−response curves were generated by fitting
the data by computer simulation to eq 1: E(%) = Emax/(1 + [I]/ID50),
where E(%) is the fraction of the enzyme activity measured in the
presence of the inhibitor, Emax is the activity in the absence of the
inhibitor, [I] is the inhibitor concentration, and ID50 is the inhibitor
concentration at which E(%) = 0.5Emax. The ID50 were converted to Ki
according to a competitive mechanism with respect to the substrate
ATP. The second substrate of the reaction (the peptide) was kept at
saturating concentrations (4-fold higher its Km). Because (i) the ATP
NMR: δ 2.30 (s, 3H, CH3), 4.81−4.86 and 5.02−5.08 (2m, 2H,
CH2N), 5.48−5.52 (m, 1H, CHCl), 7.11−7.12 and 7.30−7.32 (2m,
4H Ar), 8.15 (s, 1H, H-3), 8.73 (s, 1H, H-6). MS: m/z 307 [M + H]+.
Anal. (C14H12N4Cl2) C, H, N.
4-Chloro-1-[2-chloro-2-(4-iodophenyl)ethyl]-1H-pyrazolo[3,4-d]-
1
pyrimidine (5e). Yellow solid (168 mg, 38%); mp 112−114 °C. H
NMR: δ 4.81−4.86 and 4.98−5.04 (2m, 2H, CH2N), 5.44−5.48 (m,
1H, CHCl), 7.15−7.17 and 7.64−7.66 (2m, 4H Ar), 8.15 (s, 1H, H-3),
8.74 (s, 1H, H-6). MS: m/z 419 [M + H]+. Anal. (C13H9N4Cl2I) C, H,
N.
ADME Assays. Chemicals. All solvents, reagents, L-α-phosphati-
dylcholine, and cholesterol were from Sigma-Aldrich Srl (Milan, Italy).
Dodecane was purchased from Fluka (Milan, Italy). Pooled Male
Donors 20 mg/mL HLM were from BD Gentest-Biosciences (San
Jose, California). Milli-Q quality water (Millipore, Milford, MA, USA)
was used. Hydrophobic filter plates (MultiScreen-IP, clear plates, 0.45
μm diameter pore size), 96-well microplates, and 96-well UV-
transparent microplates were obtained from Millipore (Bedford, MA,
USA).
Parallel Artificial Membrane Permeability Assay (PAMPA). Donor
solution (0.5 mM) were prepared by diluting 1 mM DMSO
compound stock solution using phosphate buffer (pH 7.4, 0.025 M).
Filters were coated with 5 μL of a 1% (w/v) dodecane solution of
phosphatidylcholine. Donor solution (150 μL) were added to each
well of the filter plate. To each well of the acceptor plate were added
300 μL of solution (50% DMSO in phosphate buffer). All compounds
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concentration was limiting, i.e., [ATP] ≪ Km(ATP), and (ii) the
enzyme concentration was not negligible with respect to the ATP
concentration, i.e., [E] ≥ [ATP], the classical Cheng−Prusoff
relationship was not applicable. Consequently, Ki values were
were anesthetized with a mixture of ketamine (25 mg/mL) and
xylazine (5 mg/mL). Tumor grafts were obtained by injecting sc 1
×106 32D cells in 100 μL of 12 mg/mL Matrigel (Becton Dickinson,
Franklin Lakes, NJ, USA). Tumor growth was monitored daily by
measuring the average tumor diameter. The tumor volume was
expressed in mm3 according to the formula (width)2 × length/2. For
in vivo administration, 2j was prepared as suspension in cremophor/
ethanol/water solution (1:1:10, v/v/v). Each mouse received daily oral
administration of cremophor vehicle or of 80 mg/kg 2j. At the end
point, tumor masses were excised from mice and processed for
histological evaluation. Briefly, tissues 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. Sections were
washed three times in PBS and stained with hematoxylin and eosin
stain method.
calculated according to eq 2: Ki = (ID50 − E0/2)/{E0 − [S0/Km
−
1]/E0}, where S0 is the concentration of the competing substrate
(ATP) and E0 is the concentration of the enzyme. Each experiment
was done in triplicate, and mean values were used for the interpolation.
Curve fitting was performed with the program GraphPad Prism.
Enzymatic assay on isolated Abl. Recombinant human Abl was
purchased from Upstate. Activity was measured in a filterbinding assay
using an Abl specific peptide substrate (Abtide, Upstate). Reaction
conditions were: 0.012 μM [γ-32P]ATP, 50 μM peptide, 0.022 μM c-
Abl. The apparent affinity (Km) values of the Abl preparation used for
its peptide, and ATP substrates were determined separately and found
to be 1.5 μM and 10 μM, respectively. Kinetic analysis was performed
as described for c-Src. Due to the noncompetitive mode of action and
to the fact that the enzyme concentration was higher than the ATP
substrate concentration, ID50 values were converted to Ki by eq 3: Ki =
ID50/{E0 + [E0(Km(ATP)/S0)]}/E0, where E0 and S0 are the enzyme and
the ATP concentrations, respectively. Each experiment was done in
triplicate, and mean values were used for the interpolation. Curve
fitting was performed with the program GraphPad Prism.
ASSOCIATED CONTENT
■
S
* Supporting Information
Analytical data of compounds 2f−j and 2l−z, a table with
elemental analysis for new synthesized compounds, and a table
with binding free energies of to the Abl T315I mutant. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Cell Proliferation/Vitality Assay. In vitro experiments were carried
out using the murine myeloblast-like cell lines 32D, 32D-p210, and
32D-T315I. These latter have been transfected with wild-type Bcr-Abl
gene or the mutated form T315I, respectively. These cells have been
kindly provided by Prof. M. A. Santucci (University of Bologna, Italy).
Cells were cultured in RPMI medium with 10% FCS. Culture medium
of parental cells 32D contained also 10 ng/mL IL-3. To determine
antiproliferative effect of these pyrazolo[3,4-d]pyrimidines, 32D cells
were seeded at 2 × 105 cells/mL density and treated with the
compounds at increasing concentrations from 0.01 to 10 μM. The
cultures were maintained at 37 °C in 5% v/v CO2 for 48 or 72 h. Cell
number and vitality were evaluated using the automatic cell counter
NucleoCounter (Chemometec, Denmark). Results from the Nucleo-
Counter represented either total or nonviable cell concentration,
depending on the sample preparation indicated by manufacturer. IC50
(drug concentration that determined the 50% of growth inhibition)
was calculated by Grafit 4 software using the best fitting sigmoid curve.
For apoptosis evaluation, DNA was extracted from cells by incubating
them for 1 h at 55 °C in 10% SDS and 20 mg/mL of proteinase K.
After isolation with phenol/chloroform/isoamyl alcohol, the genomic
DNA was loaded in 2% agarose gels containing ethidium bromide and
the presence of fragmented DNA was visualized by UV trans-
illuminator.
Apoptosis Assay on CD34+ from CML Patients. Bone marrow
samples from Ph+ CML patients at diagnosis (n = 2) and Ph+ T315I
mutant CML patients (n = 2) were collected after informed consent.
Mononuclear cells were isolated by density gradient centrifugation in
Ficoll-Hypaque and resuspended in RPMI medium with 10% FBS.
Cells were seeded at 4 × 105 cells/mL density and treated with 2q, 2j,
and DMSO vehicle control at increasing concentration from 1 to 20
μM for 48 h. After incubation time, cells were harvested and labeled
for 15 min with antibodies against human CD34 and CD45 (APC
Mouse Anti-Human CD34 and PerCP Mouse Anti-Human CD45, BD
Pharmingen). To determine apoptotic effects of compounds, cells
were washed with phosphate-buffered saline (PBS), resuspended in
annexin buffer, and labeled for 15 min with Annexin V-FITC/
propidium iodide (PI) (FITC Annexin V Apoptosis Detection Kit II,
BD Pharmingen). At least 150000 events per samples were analyzed
using a BD FACSCalibur flow cytometer. The expression levels were
calculated as mean fluorescence intensity ratio of the antibodies
divided by mean intensity of the controls.
AUTHOR INFORMATION
■
Corresponding Author
*For S.S.: phone, +39 010 3538866; fax, +39 010 3538358 E-
mail, schensil@unige.it. For M.B.: phone, +39 0577 234306;
fax, +39 0577 234306; E-mail, botta.maurizio@gmail.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partially supported by the Italian Association for
Cancer Research AIRC IG_4538 grant to G.M., and by
National Interest Research Projects (PRIN_2007_N7KYCY)
to G.M., M.B., and S.S. (PRIN_2008_5HR5JK). Financial
support has been also received from the Istituto Toscano
Tumori-ITT-Grant proposal 2010. S.S. was also supported by
the National Interest Research Project PRIN_2010_5YY2HL.
The University of Siena is also acknowledged for financial
support. We are grateful to Lead Discovery Siena Srl and Cost
Action CM1106 “Chemical Approaches to Targeting Drug
Resistance in Cancer Stem Cells”. We thank Prof. Pietro
Lupetti for liposomes morphological characterization. We are
indebted to Molecular Discovery for access to the GRID code.
ABBREVIATIONS USED
■
Ph, Philadelphia chromosome; TK, tyrosine kinase; IM,
imatinib; MM-GBSA, molecular mechanics/generalized born
surface area; MIF, molecular interaction field; FEP, free energy
perturbation; MC/FEP, Monte Carlo/free-energy perturbation;
HLM, human liver microsomes; NPLA, number of planes of
grid points per Angstrom; MSD, mass spectra detection; SUV,
small unilamellar vesicle; TEM, transmission electron micros-
copy; FBS, fetal bovine serum; APC, antigen-presenting cell;
FITC, fluorescein isothiocyanate; MR, membrane retention
Animals and Experimental in Vivo Model. Female CD1 nude mice
(Charles River, Milan, Italy) were maintained under the guidelines
established by host Institution (University of L’Aquila, Medical School
and Science and Technology School Board Regulations, complying
with the Italian government regulation no. 116, January 27, 1992, for
the use of laboratory animals). Before any invasive manipulation, mice
REFERENCES
■
(1) Hantschel, O.; Superti-Furga, G. Regulation of the c-Abl and Bcr-
Abl tyrosine kinases. Nat. Rev. Mol. Cell Biol. 2004, 5, 33−44.
5392
dx.doi.org/10.1021/jm400233w | J. Med. Chem. 2013, 56, 5382−5394

Journal of Medicinal Chemistry
Article
(2) Deininger, M.; Buchdunger, E.; Druker, B. J. The development of
imatinib as a therapeutic agent for chronic myeloid leukemia. Blood
2005, 105, 2640−2653.
(16) Apsel, B.; Blair, J. A.; Gonzalez, B.; Nazif, T. M.; Feldman, M. E.;
Aizenstein, B.; Hoffman, R.; Williams, R. L.; Shokat, K. M.; Knight, Z.
A. Targeted polypharmacology: discovery of dual inhibitors of tyrosine
and phosphoinositide kinases. Nature Chem. Biol. 2008, 4, 691−699.
(17) (a) Schenone, S.; Botta, M.; Radi, M.; Bruno, O. New insights
into small-molecule inhibitors of Bcr-Abl. Med. Res. Rev. 2011, 31, 1−
41. (b) Schenone, S.; Bruno, O.; Radi, M.; Botta, M. 4-Amino-
substituted pyrazolo[3,4-d]pyrimidines: synthesis and biological
properties. Mini-Rev. Org. Chem. 2009, 6, 220−233.
(18) Radi, M.; Brullo, C.; Crespan, E.; Tintori, C.; Musumeci, F.;
Biava, M.; Schenone, S.; Dreassi, E.; Zamperini, C.; Maga, G.; Pagano,
D.; Angelucci, A.; Bologna, M.; Botta, M. Identification of potent c-Src
inhibitors strongly affecting the proliferation of human neuroblastoma
cells. Bioorg. Med. Chem. Lett. 2011, 21, 5928−5933.
(19) Radi, M.; Dreassi, E.; Brullo, C.; Crespan, E.; Tintori, C.;
Bernardo, V.; Valoti, M.; Zamperini, C.; Daigl, H.; Musumeci, F.;
Carraro, F.; Naldini, A.; Filippi, I.; Maga, G.; Schenone, S.; Botta, M.
Design, synthesis, biological activity, and ADME properties of
pyrazolo[3,4-d]pyrimidines active in hypoxic human leukemia cells: a
lead optimization study. J. Med. Chem. 2011, 54, 2610−2626.
(20) Santucci, M. A.; Corradi, V.; Mancini, M.; Manetti, F.; Radi, M.;
Schenone, S.; Botta, M. C6-Unsubstituted pyrazolo[3,4-d]pyrimidines
are dual Src/Abl inhibitors effective against imatinib mesylate resistant
chronic myeloid leukemia cell lines. ChemMedChem 2009, 4, 118−126.
(21) 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.
(22) Santucci, M. A.; Mancini, M.; Corradi, V.; Iacobucci, I.;
Martinelli, G.; Botta, M.; Schenone, S. New SRC/ABL inhibitors for
chronic myeloid leukemia therapy show selectivity for T315I ABL
mutant CD34+ cells. Invest. New Drugs 2010, 28, 876−878.
(23) Zhou, T.; Parillon, L.; Li, F.; Wang, Y.; Keats, J.; Lamore, S.; Xu,
Q.; Shakespeare, W.; Dalgarno, D.; Zhu, X. Crystal structure of the
T315I mutant of AbI kinase. Chem. Biol. Drug. Des. 2007, 70, 171−
181.
(24) Bounaud, P. Y.; Gosberg, A.; Hendle, J.; Lewis, H. A.; Romero,
R.; Wilson, M. E.; Zhang, F. Crystal structure of mutant ABL kinase
domain in complex with small molecule fragment. Protein Data Bank
2009, DOI:10.2210/pdb3dk7/pdb.
(25) Modugno, M.; Casale, E.; Soncini, C.; Rosettani, P.; Colombo,
R.; Lupi, R.; Rusconi, L.; Fancelli, D.; Carpinelli, P.; Cameron, A. D.;
Isacchi, A.; Moll, J. Crystal structure of the T315I Abl mutant in
complex with the aurora kinases inhibitor PHA-739358. Cancer Res.
2007, 67, 7987−7990.
(26) O’Hare, T.; Shakespeare, W. C.; Zhu, X.; Eide, C. A.; Rivera, V.
M.; Wang, F.; Adrian, L. T.; Zhou, T.; Huang, W. S.; Xu, Q.; Metcalf,
C. A., III; Tyner, J. W.; Loriaux, M. M.; Corbin, A. S.; Wardwell, S.;
Ning, Y.; Keats, J. A.; Wang, Y.; Sundaramoorthi, R.; Thomas, M.;
Zhou, D.; Snodgrass, J.; Commodore, L.; Sawyer, T. K.; Dalgarno, D.
C.; Deininger, M. W.; Druker, B. J.; Clackson, T. AP24534, a pan-
BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the
T315I mutant and overcomes mutation-based resistance. Cancer Cell.
2009, 16, 401−412.
(27) Zhou, T.; Commodore, L.; Huang, W. S.; Wang, Y.; Thomas,
M.; Keats, J.; Xu, Q.; Rivera, V. M.; Shakespeare, W. C.; Clackson, T.;
Dalgarno, D. C.; Zhu, X. Structural mechanism of the Pan-BCR-ABL
inhibitor ponatinib (AP24534): lessons for overcoming kinase
inhibitor resistance. Chem. Biol. Drug. Des. 2011, 77, 1−11.
(28) Chan, W. W.; Wise, S. C.; Kaufman, M. D.; Ahn, Y. M.;
Ensinger, C. L.; Haack, T.; Hood, M. M.; Jones, J.; Lord, J. W.; Lu, W.
P.; Miller, D.; Patt, W. C.; Smith, B. D.; Petillo, P. A.; Rutkoski, T. J.;
Telikepalli, H.; Vogeti, L.; Yao, T.; Chun, L.; Clark, R.; Evangelista, P.;
Gavrilescu, L. C.; Lazarides, K.; Zaleskas, V. M.; Stewart, L. J.; Van
Etten, R. A.; Flynn, D. L. Conformational control inhibition of the
BCR-ABL1 tyrosine kinase, including the gatekeeper T315I mutant, by
(3) Jabbour, E.; Kantarjian, H. Chronic myeloid leukemia: 2012
update on diagnosis, monitoring, and management. Am. J. Hematol.
2012, 87, 1037−1045.
(4) Kantarjian, H.; Giles, F.; Wunderle, L.; Bhalla, K.; O’Brien, S.;
Wassmann, B.; Tanaka, C.; Manley, P.; Rae, P.; Mietlowski, W.;
Bochinski, K.; Hochhaus, A.; Griffin, J. D.; Hoelzer, D.; Albitar, M.;
Dugan, M.; Cortes, J.; Alland, L.; Ottmann, O. G. Nilotinib in
imatinib-resistant CML and Philadelphia chromosome-positive ALL.
N. Engl. J. Med. 2006, 354, 2542−2551.
(5) Shah, N. P.; Tran, C.; Lee, F. Y.; Chen, P.; Norris, D.; Sawyers, C.
L. Overriding imatinib resistance with a novel ABL kinase inhibitor.
Science 2004, 305, 399−401.
(6) O’Hare, T.; Eide, C. A.; Deininger, M. W. New Bcr-Abl inhibitors
in chronic myeloid leukemia: keeping resistance in check. Expert Opin.
Invest. Drugs 2008, 17, 865−878.
(7) Schenone, S.; Brullo, C.; Botta, M. New opportunities to treat the
T315I-Bcr-Abl mutant in chronic myeloid leukaemia: tyrosine kinase
inhibitors and molecules that act by alternative mechanisms. Curr.
Med. Chem. 2010, 17, 1220−1245.
(8) Golas, J. M.; Arndt, K.; Etienne, C.; Lucas, J.; Nardin, D.;
Gibbons, J.; Frost, P.; Ye, F.; Boschelli, D. H.; Boschelli, F. SKI-606, a
4-anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases,
is a potent antiproliferative agent against chronic myelogenous
leukemia cells in culture and causes regression of K562 xenografts in
nude mice. Cancer Res. 2003, 63, 375−381.
(9) Kimura, S.; Naito, H.; Segawa, H.; Kuroda, J.; Yuasa, T.; Sato, K.;
Yokota, A.; Kamitsuji, Y.; Kawata, E.; Ashihara, E.; Nakaya, Y.;
Naruoka, H.; Wakayama, T.; Nasu, K.; Asaki, T.; Niwa, T.;
Hirabayashi, K.; Maekawa, T. NS-187, a potent and selective dual
Bcr-Abl/Lyn tyrosine kinase inhibitor, is a novel agent for imatinib-
resistant leukemia. Blood 2005, 106, 3948−3954.
(10) O’Hare, T.; Pollock, R.; Stoffregen, E. P.; Keats, J. A.; Abdullah,
O. M.; Moseson, E. M.; Rivera, V. M.; Tang, H.; Metcalf, C. A., III;
Bohacek, R. S.; Wang, Y.; Sundaramoorthi, R.; Shakespeare, W. C.;
Dalgarno, D.; Clackson, T.; Sawyer, T. K.; Michael, W.; Deininger, M.
W.; Druker, B. J. Inhibition of wild-type and mutant Bcr-Abl by
AP23464, a potent ATP-based oncogenic protein kinase inhibitor:
implications for CML. Blood 2004, 104, 2532−2539.
(11) Huron, D. R.; Gorre, M. E.; Kraker, A. J.; Sawyers, C. L.; Rosen,
N.; Moasser, M. M. A novel pyridopyrimidine inhibitor of abl kinase is
a picomolar inhibitor of Bcr-abl-driven K562 cells and is effective
against STI571-resistant Bcr-Abl mutants. Clin. Cancer Res. 2003, 9,
1267−1273.
(12) Huang, W. S.; Metcalf, C. A.; Sundaramoorthi, R.; Wang, Y.;
Zou, D.; Thomas, R. M.; Zhu, X.; Cai, L.; Wen, D.; Liu, S.; Romero, J.;
Qi, J.; Chen, I.; Banda, G.; Lentini, S. P.; Das, S.; Xu, Q.; Keats, J.;
Wang, F.; Wardwell, S.; Ning, Y.; Snodgrass, J. T.; Broudy, M. I.;
Russian, K.; Zhou, T.; Commodore, L.; Narasimhan, N. I.;
Mohemmad, Q. K.; Iuliucci, J.; Rivera, V. M.; Dalgarno, D. C.;
Sawyer, T. K.; Clackson, T.; Shakespeare, W. C. Discovery of 3-[2-
(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpi-
perazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide
(AP24534), a potent, orally active pan-inhibitor of breakpoint cluster
region-abelson (BCR-ABL) kinase including the T315I gatekeeper
mutant. J. Med. Chem. 2010, 53, 4701−4719.
(13) Gozgit, J. M.; Wong, M. J.; Moran, L.; Wardwell, S.;
Mohemmad, Q. K.; Narasimhan, N. I.; Shakespeare, W. C.; Wang,
F.; Clackson, T.; Rivera, V. M. Ponatinib (AP24534), a multitargeted
pan-FGFR inhibitor with activity in multiple FGFR-amplified or
mutated cancer models. Mol. Cancer Ther. 2012, 11, 690−699.
(14) Azam, M.; Latek, R. R.; Daley, G. Q. Mechanisms of
autoinhibition and STI-571/imatinib resistance revealed by muta-
genesis of BCR-ABL. Cell 2003, 112, 831−843.
(15) Martinelli, G.; Soverini, S.; Rosti, G.; Baccarani, M. Dual
tyrosine kinase inhibitors in chronic myeloid leukemia. Leukemia 2005,
19, 1872−1879.
5393
dx.doi.org/10.1021/jm400233w | J. Med. Chem. 2013, 56, 5382−5394

Journal of Medicinal Chemistry
Article
the switch-control inhibitor DCC-2036. Cancer Cell 2011, 19, 556−
568.
(29) Manetti, F.; Pucci, A.; Magnani, M.; Locatelli, G. A.; Brullo, C.;
Naldini, A.; Schenone, S.; Maga, G.; Carraro, F.; Botta, M. Inhibition
of Bcr-Abl phosphorylation and induction of apoptosis by pyrazolo-
[3,4-d]pyrimidines in human leukemia cells. ChemMedChem 2007, 2,
343−353.
(30) Grid 22; Molecular Discovery Ltd: Pinner, Middlesex, U.K.,
2004.
(31) Jorgensen, W. L.; Schyman, P. Treatment of halogen bonding in
the OPLS-AA force field: application to potent anti-HIV agents. J.
Chem. Theory Comput. 2012, 8, 3895−3901.
(32) Dreassi, E.; Zizzari, A. T.; Mori, M.; Filippi, I.; Belfiore, A.;
Naldini, A.; Carraro, F.; Santucci, A.; Schenone, S.; Botta, M. 2-
Hydroxypropyl-β-cyclodextrin strongly improves water solubility and
anti-proliferative activity of pyrazolo[3,4-d]pyrimidines Src-Abl dual
inhibitors. Eur. J. Med. Chem. 2010, 45, 5958−5964.
(33) (a) Avdeef, A. High-throughput measurements of solubility
profiles. Pharmacokinetic optimization in drug research: biological,
physicochemical, and computational strategies. LogP2000, 2nd
Lipophilicity Symposium, Lausanne, Switzerland, March 5−9, 2000,
pp 305−325. (b) Bard, B.; Martel, S.; Carrupt, P. A. High throughput
UV method for the estimation of thermodynamic solubility and the
determination of the solubility in biorelevant media. Eur. J. Pharm. Sci.
2008, 33, 230−240.
(34) Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.;
Taylor, R. D. Improved protein−ligand docking using GOLD. Proteins
2003, 52, 609−623.
(35) Maestro, version 9.1; Schrodinger, LLC: Portland, OR, 2007;
̈
www.schrodinger.com.
(36) Jorgensen, W. L.; Thomas, L. T. Perspective on free-energy
perturbation calculations for chemical equilibria. J. Chem. Theory
Comput. 2008, 4, 869−876.
(37) Jorgensen, W. L.; Tirado-Rives, J. Molecular modeling of
organic and biomolecular systems using BOSS and MCPRO. J.
Comput. Chem. 2005, 26, 1689−1700.
(38) Zeevaart, J. G.; Wang, L.; Thakur, V. V.; Leung, C. S.; Tirado-
Rives, J.; Bailey, C. M.; Domaoal, R. A.; Anderson, K. S.; Jorgensen, W.
L. Optimization of azoles as anti-HIV agents guided by free-energy
calculations. J. Am. Chem. Soc. 2008, 130, 9492−9499.
(39) Leung, C. S.; Zeevaart, J. G.; Domaoal, R. A.; Bollini, M.;
Thakur, V. V.; Spasov, K.; Anderson, K. S.; Jorgensen, W. L. Eastern
extension of azoles as non-nucleoside inhibitors of HIV-1 reverse
transcriptase; cyano group alternatives. Bioorg. Med. Chem. Lett. 2010,
20, 2485−2488.
(40) Jorgensen, W. L.; Tirado-Rives, J. Potential energy functions for
atomic-level simulations of water, and organic and biomolecular
systems. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 6665−6670.
(41) (a) Manetti, F.; Brullo, C.; Magnani, M.; Mosci, F.; Chelli, B.;
Crespan, E.; Schenone, S.; Naldini, A.; Bruno, O.; Trincavelli, M. L.;
Maga, G.; Carraro, F.; Martini, C.; Bondavalli, F.; Botta, M. Structure-
based optimization of pyrazolo[3,4-d]pyrimidines as Abl inhibitors and
antiproliferative agents toward human leukemia cell lines. J. Med.
Chem. 2008, 51, 1252−1259. (b) Falchi, F.; Manetti, F.; Carraro, F.;
Naldini, A.; Maga, G.; Crespan, E.; Schenone, S.; Bruno, O.; Brullo, C.;
Botta, M. 3D QSAR models built on structure-based alignments of Abl
tyrosine kinase inhibitors. ChemMedChem 2009, 4, 976−987.
(c) Crespan, E.; Radi, M.; Zanoli, S.; Schenone, S.; Botta, M.; Maga,
G. Dual Src and Abl inhibitors target wild type Abl and the Abl T315I
Imatinib-resistant mutant with different mechanisms. Bioorg. Med.
Chem. 2010, 18, 3999−4008.
(42) Brik, A.; Wu, C.; Best, M. D.; Wong, C. Tetrabutylammonium
fluoride-assisted rapid N9-alkylation on purine ring: application to
combinatorial reactions in microtiter plates for the discovery of potent
sulfotransferase inhibitors in situ. Bioorg. Med. Chem. 2005, 13, 4622−
4626.
(43) Wohnsland, F.; Faller, B. High-throughput permeability pH
profile and high-throughput alkane/water log P with artificial
membranes. J. Med. Chem. 2001, 44, 923−930.
5394
dx.doi.org/10.1021/jm400233w | J. Med. Chem. 2013, 56, 5382−5394