Development of Alkyne-Containing Pyrazolopyrimidines to Overcome Drug Resistance of Bcr-Abl Kinase

Article abstract of DOI:10.1021/acs.jmedchem.5b01125

Despite the success of imatinib at inhibiting Bcr-Abl and treating chronic myelogenous leukemia (CML), resistance to the therapy occurs over time in patients. In particular, the resistance to imatinib caused by the gatekeeper mutation T315I in Bcr-Abl remains a challenge in the clinic. Inspired by the successful development of ponatinib to curb drug resistance, we hypothesize that the incorporation of an alkyne linker in other heterocyclic scaffolds can also achieve potent inhibition of Bcr-Abl^T315I by allowing for simultaneous occupancy of both the active site and the allosteric pocket in the Abl kinase domain. Herein, we describe the design, synthesis, and characterization of a series of alkyne-containing pyrazolopyrimidines as Bcr-Abl inhibitors. Our results demonstrate that some alkyne-containing pyrazolopyrimidines potently inhibit not only Abl^T315I in vitro but also Bcr-Abl^T315I in cells. These pyrazolopyrimidines can serve as lead compounds for future development of novel targeted therapy to overcome drug resistance of CML.

Full text of DOI:10.1021/acs.jmedchem.5b01125

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Article
Development of Alkyne-Containing Pyrazolopyrimidines
to Overcome Drug Resistance of Bcr-Abl Kinase
Chao Zhang, Alvin kung, xu liu, G. K. Surya Prakash, and Brock Malinoski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01125 • Publication Date (Web): 12 Nov 2015
Downloaded from http://pubs.acs.org on November 17, 2015
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Development of AlkyneꢀContaining
Pyrazolopyrimidines to Overcome Drug
Resistance of BcrꢀAbl Kinase
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†
†
†
†
*,†,‡
Xu Liu, Alvin Kung, Brock Malinoski, G. K. Surya Prakash, Chao Zhang
†
Loker Hydrocarbon Research Institute & Department of Chemistry, University of
Southern California, University Park, Los Angeles, CA 90089, USA
‡
USC Norris Comprehensive Cancer Center, University of Southern California, Los
Angeles, CA 90089, USA
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ABSTRACT:
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Despite the success of imatinib at inhibiting BcrꢀAbl and treating chronic myelogenous
leukemia (CML), resistance to the therapy occurs over time in the patients. In particular,
the resistance to imatinib caused by the gatekeeper mutation T315I in BcrꢀAbl remains a
challenge in the clinic. Inspired by the successful development of ponatinib to curb drug
resistance, we hypothesize that the incorporation of an alkyne linker in other heterocyclic
scaffolds can also achieve potent inhibition of BcrꢀAblT315I by allowing for simultaneous
occupancy of both the active site and the allosteric pocket in the Abl kinase domain.
Herein, we describe the design, synthesis, and characterization of a series of
alkyneꢀcontaining pyrazolopyrimidines as BcrꢀAbl inhibitors. Our results demonstrate that
some alkyneꢀcontaining pyrazolopyrimidines potently inhibit not only AblT315I in vitro, but
also BcrꢀAblT315I in cells. These pyrazolopyrimidines can serve as lead compounds for
future development of novel targeted therapy to overcome drug resistance of CML.
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INTRODUCTION
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Chronic myelogenous leukemia (CML) is a type of cancer characterized by an
uncontrolled growth of myeloid cells, a type of white blood cells.1 Initially discovered in
1840s, CML was probably the first type of leukemia that was clearly documented.2 It was
then discovered in 1960 that the majority of CML cells harbored a reciprocal translocation
between chromosome 9 and chromosome 22, which produces a short chromosome known
as the Philadelphia (Ph) chromosome.3ꢀ4 This interꢀchromosomal translocation results in
the fusion of two genes, breakpoint cluster region protein (BCR) and Abelson murine
leukemia viral oncogene homolog 1 (ABL), normally located in chromosome 22 and 9,
respectively. 1, 5
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Abl encodes a tyrosine protein kinase that is known to catalyze the transfer of phosphoryl
groups to tyrosine residues in substrate proteins.6 Cellular Abl (cꢀAbl) is typically located
in cytoplasm and its catalytic activity is highly regulated in cells.6 The BcrꢀAbl fusion,
however, is thought to induce constitutive activation of Abl activity and transform cells.
BcrꢀAbl has been found to be the driver of 95% chronic myelogenous leukemia (CML),
and is one of the most well established oncogenes.7 The fact that BcrꢀAbl is the sole driver
of most CML cases highlights the oncogenic kinase as an ideal drug target. Accordingly,
pharmaceutical companies initiated programs to discover inhibitors of BcrꢀAbl as CML
therapy since 1990s.8ꢀ10 Imatinib, a smallꢀmolecule inhibitor of BcrꢀAbl developed by
Novartis, was approved by FDA in 2001 for the treatment of CML.11ꢀ13 This drug is the
first soꢀcalled “targeted therapy” that can treat cancer by targeting a genetic lesion specific
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to the cancer cells.14 Moreover, crystal structures from the Kuriyan lab revealed that
imatinib binds not only to the active site but also to a hydrophobic allosteric pocket that
was only exposed upon an outward shift of the conserved DFG motif (Figure 1A).15ꢀ16 The
discovery of imatinib paves the way for the development of numerous additional type II
inhibitors for a variety of protein kinases. Subsequently, some of these type II inhibitors
such as sorafenib were also approved for clinical use.17ꢀ18 The design of type II inhibitors
has been rationalized for maximizing the benefits in terms of improving selectivity of
protein of interest.19ꢀ20 Imatinib utilizes a phenyl linker between the adenine pocket binder
and the allosteric pocket binder (Figure 1A).15, 21 Although the drug initially induces 67%
positive therapeutic responses in CML patients, resistance is often observed within 6
months.22ꢀ23 It has been established that the drug resistance observed in the clinic is largely
due to mutations within the Abl kinase domain.22ꢀ23 One particular mutation occurring at
the gatekeeper position, T315I, accounts for approximately 20% of the resistance observed
in the clinic.24ꢀ25 This mutation was found to diminish the binding affinity of imatinib to
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Abl by over 100 fold due to a direct steric clash between imatinib and I315 (Figure 1B).23,
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Extensive efforts were invested in the identification of novel inhibitors that can
overcome imatinibꢀresistant mutants of Abl in the pharmaceutical industry. Out of these
efforts, ponatinib was successfully developed by Ariad Pharmaceuticals to target T315I
BcrꢀAbl (Figure 1C).27ꢀ29 Ponatinib contains an alkyne linker between the diarylamide side
chain and the imidazo[1,2ꢀb]pyridazine core.28ꢀ29 As revealed by the crystal structure, the
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slim alkyne linker is crucial to the success of ponatinib by avoiding steric clash with a
bulky isoleucine residue at the gatekeeper position.30 Nevertheless, ponatinib’s sale was
suspended by FDA ten months after its approval for “the risk of lifeꢀthreatening blood clots
and severe narrowing of blood vessels”.31 Although the suspension was partially lifted in
December 2013, ponatinib prescription has to include a “Black Box Warning” and a "Risk
Evaluation and Mitigation Strategy" for concerns over risks of its usage. Thus, there is still
an urgent need to develop improved therapy with reduced side effects to combat CML.
We hypothesize that the introduction of an alkyne linker at proper positions on
alternative heterocyclic scaffolds can also lead to compounds that inhibit T315I BcrꢀAbl
by avoiding steric clash with I315 (Figure 1D). We have thus designed, synthesized, and
characterized a series of alkyneꢀcontaining pyrazolo[3,4ꢀd]pyrimidines for the inhibition
of Abl kinases. Our results show that some pyrazolopyrimidines can potently inhibit both
the catalytic activity of AblT315I in vitro, and the activity of BcrꢀAblT315I in Ba/F3 cells.
Furthermore, our pyrazolopyrimidines have different kinase inhibition profiles from
ponatinib. These pyrazolopyrimidines can serve as lead compounds for the development of
improved therapy for overcoming drug resistance of CML.
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RESULTS
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We chose the pyrazolo[3,4ꢀd]pyrimidine as the scaffold because it is an isosteric mimic
of adenine, which can bind to the adenineꢀbinding pocket of numerous kinases, including
Abl with moderate affinity.32ꢀ33 The C3 position of pyrazolopyrimidine was chosen to
tether alkynyl substituents due to its similar position and substitution angle to the C3
position of ponatinib (Figure 1). Initially, a series of pyrazolopyrimidines 2 ꢀ 13 that
contain a phenylacetylenyl group at the 3 position was synthesized. They contain diverse
substituents at different positions on the phenyl ring. The synthesis exploited a common
starting material, 3ꢀiodoꢀ1ꢀisopropylꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine (1), which
was prepared based on previously described procedures.34 The Sonogashira coupling of 1
with various alkynes yielded 2 ꢀ 11 (Scheme 1). Treatment of 10 and 11 with BBr3
allowed for the removal of the methyl group to give the corresponding phenol
compounds 12 and 13. The Heck coupling of 3ꢀvinylphenol with 1 yielded 14 that
contains an alkene linker instead of alkyne (Scheme 1).
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After the synthesis of pyrazolopyrimidines 2 – 14, their inhibitory activity against AblWT
and AblT315I was evaluated using the phosphocellulose paper disk assay in vitro.35 Imatinib
and ponatinib were included in the assay as controls. As shown in Table 1, imatinib
potently inhibited the enzymatic activity of AblWT (IC50 = 170 nM), whereas its inhibition
of AblT315I was rather poor (IC50 = 74 µM). This translates into over 300ꢀfold increase in
the IC50 value of imatinib due to the T315I mutation, which is in agreement with previous
reports.23, 26 In contrast, ponatinib potently inhibited both AblWT and AblT315I with low
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nanomolar IC50 values (Table 1). Most of the alkyneꢀcontaining pyrazolopyrimidines 2 ꢀ
13 inhibited AblWT and AblT315I with similar potency. While the alkeneꢀcontaining analog
14 inhibits AblWT potently, its inhibition of AblT315I is ~5 fold less potent. These results are
consistent with our hypothesis that an alkyne linker can avoid steric clash with an
isoleucine residue at the gatekeeper position in Abl. Among 2 ꢀ 14, the most effective
inhibitor of AblT315I is 3, which contains a metaꢀhydroxyl group on the phenyl ring. 3
inhibits AblWT and AblT315I with equal potency (IC50 = 8 nM). Nonetheless, its potency is
lower than that of ponatinib. Noticing 3 and ponatinib are type I and type II inhibitors,
respectively, we reasoned that the higher potency of ponatinib may stem from its additional
binding to the allosteric pocket in Abl. We thus attached a 3ꢀtrifluoromethylphenyl group,
a wellꢀestablished binder of the allosteric pocket, via an amide linker (in forward or reverse
orientation) to the meta position of the phenyl ring (Scheme 1).19 The resulting 15 and 16
inhibited both AblWT and AblT315I potently, with IC50 values less than 2 nM (Table 1). Such
potency is comparable to that of ponatinib (Table 1). These results suggest that the
conversion of the alkyneꢀcontaining pyrazolopyrimidines from type I inhibitors to type II
improved potency presumably due to additional binding to the allosteric pocket.
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Having demonstrated potent inhibition of Abl by the alkyneꢀcontaining
pyrazolopyrimidines in vitro, we subsequently examined their inhibition of BcrꢀAbl
activity in cells. Ba/F3 cells transformed with BcrꢀAbl are routinely used to determine
inhibition of BcrꢀAbl activity in cells because their growth is dependent on the oncogenic
Abl activity in the absence of the cytokine interleukin 3.36 Similar to the in vitro results,
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imatinib inhibits the growth of BcrꢀAblWT cells at high nanomolar concentrations but not
that of the T315I mutant. In comparison, ponatinib inhibits both forms with high potency
(Table 2). Despite their potency spanning three orders of magnitude, our alkyneꢀcontaining
pyrazolopyrimidines inhibited BcrꢀAblWT and BcrꢀAblT315I cells with similar potency
(Figure 2). These results suggest that the alkyne linker enable potent inhibition of
BcrꢀAblT315I in cells by alleviating steric clash with the bulky gatekeeper residue. Among
these pyrazolopyrimidines, 15 and 16 afforded the most potent growth inhibition of Ba/F3
cells expressing BcrꢀAbl, demonstrating the superior potency of these two type II
inhibitors in cells (Figure 2). To confirm that the observed antiꢀproliferative effects are due
to inhibition of BcrꢀAbl rather than other cellular targets, we conducted a counterꢀscreen of
the two most potent pyrazolopyrimidines against parental Ba/F3 cells that do not express
BcrꢀAbl. 15 and 16 do not inhibit the proliferation of parental Ba/F3 cells at concentrations
below 1 µM (Table S1, Supporting Information). The over 100ꢀfold window between IC50
values against parental Ba/F3 cells and those expressing BcrꢀAbl suggests that
antiꢀproliferative effects of these two pyrazolopyrimidines are due to onꢀtarget inhibition
of BcrꢀAbl.
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In addition to examining the effects of our pyrazolopyrimidines on viability of Ba/F3
cells expressing BcrꢀAbl, we investigated their effects on phosphoꢀsignal transduction in
cells. Specifically, four most potent pyrazolopyrimidines (3, 14, 15 & 16) were selected
along with imatinib and ponatinib and evaluated for their effects on phosphorylation of
BcrꢀAbl and STAT5 (Figure 3).37ꢀ40 Since BcrꢀAbl activity has been established to lead to
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phosphorylation of Abl at Tyr412 and a downstream transcription factor STAT5 at Tyr694,
phosphorylation levels at these sites are routinely used as markers of BcrꢀAbl activity in
cells. Immunoꢀblots with antiꢀpAbl and antiꢀpSTAT5 revealed that 15 and 16 at a
concentration of 500 nM abolished phosphorylation of Abl and STAT5 in a similar manner
to ponatinib in both BcrꢀAblWT and BcrꢀAblT315I Ba/F3 cells (Figure 3). In contrast, 3 and
14 only partially inhibited the phosphorylation of Abl and STAT5 at 500 nM, reflecting
their lower potency at inhibiting BcrꢀAbl in cells (Figure 3). There is little effect of the
inhibitors on the total protein levels of Abl or STAT5, consistent with the notion that they
exert effects through inhibiting phosphorylation rather than changing protein stability. To
evaluate the potency of 15 and 16 on BcrꢀAblWT and BcrꢀAblT315I in cells, we determined
their EC50 by Western blot analysis. 15 and 16 potently and effectively suppress the
autophosphorylation of BcrꢀAblWT (1 nM and 3 nM, respectively) and BcrꢀAblT315I (20 nM
and 25 nM, respectively) in a doseꢀdependent manner (Figure 4).
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To understand the binding mode of the alkyneꢀcontaining pyrazolopyrimidines, we
conducted in silico docking. The two most potent Abl inhibitors, 15 and 16, were
individually docked into AblT315I using AutoDock Vina. The docking models suggest that
15 and 16 can occupy the allosteric pocket and are thus type II inhibitors (Figure 5).
Furthermore, these models indicate no steric clash between the bulky gatekeeper residue
I315 and the alkyne linker in the pyrazolopyrimidine, which is in good agreement with the
potent inhibition against AblT315I that we observed.
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We determined the kinase inhibition profiles of 15, 16 and ponatinib using the
SelectScreen Kinase Profiling Services offered by Life Technologies. A diverse set of 66
kinases was selected to include common oncogenic mutants of Abl and to cover main
kinase subfamilies (Table S2, Supporting Information). The profiling data show that
ponatinib inhibited more kinases than 15 and 16 when all drugs were used at the same
concentration of 50 nM. Despite the apparent potency difference between 15 and
ponatinib, these two inhibitors share many targets in common. For example, 8 out of the 9
kinases potently inhibited (defined as >80% inhibition at 50 nM) by 15 are also potently
inhibited by ponatinib. Furthermore, 15 affords moderate inhibition (40ꢀ80% inhibition) of
many of the additional kinases that are potently inhibited by ponatinib, such as CSF1R and
Yes1. Beyond the shared targets, ponatinib and 15 inhibited a few kinases with distinct
potency. For example, Blk, MUSK and TrkA are potently inhibited by ponatinib but not
significantly affected by 15. The two pyrazolopyrimidine isomers 15 and 16 share very
similar inhibition profiles to each other although the latter is generally less potent. These
results suggest that the direction of the amide linker, the only structural difference between
these two pyrazolopyrimidines, alters inhibitor potency but not inhibition profiles.
We had the pharmacokinetic (PK) parameters of our most potent BcrꢀAbl inhibitor 15
determined at the Shanghai Medicilon Inc. Plasma levels of 15 in male SD rats were
monitored after a single intravenous (IV) administration of the drug at the dose of 2
mg/kg (Figure S1, Supporting Information). The key PK parameters of 15 were
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summarized in Table 3. The halfꢀlife (t1/2) of 15 was calculated to be about 1.1 h.
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Discussions
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In this study, we developed novel pyrazolopyrimidineꢀbased inhibitors to overcome drug
resistance caused by the T315I mutation in the BcrꢀAbl kinase. An alkyne linker was
featured in our inhibitor design to avoid steric clash with the isoleucine gatekeeper residue
in BcrꢀAblT315I. The comparable potency of these alkyneꢀcontaining pyrazolopyrimidines
against BcrꢀAblWT and BcrꢀAblT315I suggests that the slim alkyne linker has fulfilled our
design goal. Our best pyrazolopyrimidines are a few fold less potent than ponatinib at
inhibiting BcrꢀAblT315I in cells, which may be due to their suboptimal solubility and cell
membrane permeability. Further chemical elaboration, such as installing the methyl group
on the phenyl ring near the alkyne linker and the piperazine group at the end of the
hydrophobic moiety, will likely improve the efficacy as well as selectivity at inhibiting
BcrꢀAbl in cells.
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Our profiling results show that the pyrazolopyrimidine 15 shares a number of kinase
targets with ponatinib. Beyond these common targets, a few kinases including Blk,
MUSK and TrkA are potently inhibited by ponatinib but not significantly affected by 15.
This suggests that changing the heterocycle which occupies the adenineꢀbinding pocket
can alter kinase inhibition profile. Whether this difference in inhibition profiles can
translate into better therapeutic effects remains to be tested. While the PK parameters of
15 is not excellent, it is likely that these parameters can be improved through further
chemical modifications, such as installing groups to block drug metabolism or to enhance
solubility.
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Although structurally similar alkyneꢀcontaining pyrazolopyrimidines were recently
reported, those pyrazolopyrimidines were only characterized for their inhibition of Src,
an experimental drug target in triple negative breast cancer.41 Our data show that these
pyrazolopyrimidines inhibit not only Abl and Src but also a number of other kinases. It
will be interesting to see if the polyꢀpharmacology feature of these alkyneꢀcontaining
pyrazolopyrimidines can be exploited in disease settings such as CML. In another study,
the imidazopyridazine core of ponatinib was replaced with a pyrazolopyridine ring to
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yield a small molecule GZD824 that potently inhibits BcrꢀAblT315I 26
While the
.
pyrazolopyridine is an isostere of the pyrozolopyrimidine, the position of alkyne
substitution differs between our study and theirs. The alkyne substituent was attached to
the 5 position of the pyrazolopyridine in GZD824 instead of the 3 position of the
pyrazolopyrimidine that we focus on here. In addition, modification beyond swapping the
core with the pyrazolopyridine was not attempted at all in the study reporting GZD824.26
Together with these prior studies, our study strongly supports the notion that the use of an
alkyne linker can be a general strategy for developing inhibitors of protein kinases
containing a bulky gatekeeper residue. Specifically, our study raises the interesting
possibility that modifying the additional known scaffolds targeting BcrꢀAbl, such as
recently developed dasatinib, bosutinib and tozasertib, with an alkyne linker can lead to
potent inhibitors of BcrꢀAblT315I. This study is currently ongoing in our laboratory.
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Chemistry. H NMR spectra were acquired on a 400 MHz Varian spectrometer and C
NMR spectra were acquired on a 500 MHz Varian spectrometer. Highꢀresolution mass
was determined using Synapt G2ꢀSi ESI/LCꢀMS. All the target molecules (2 – 16) were
found to be >95% pure based on HPLC analysis (see pages 24ꢀ31 of Supporting
Information).
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3-Iodo-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1) was synthesized based
on a known procedure.34
General
Procedure
for
the
cross
coupling
reaction
between
3ꢀiodoꢀ1ꢀisopropylꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine (1) and alkyne. Solid
components including 1 (0.33 mmol), tetrakis(triphenylphosphine)palladium (0.066
mmol), CuI (0.165 mmol) were first added to flask (50 mL). This was followed by addition
of anhydrous THF (10 mL), selected alkyne (1.32 mmol), and diisopropylamine (3.3
mmol). The reaction flask was covered by aluminum foil and the mixture was stirred for 17
h at room temperature. The reaction mixture was concentrated at reduced pressure and the
crude product was purified by flash column chromatography (Hexanes/ethyl acetate).
1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2). This
1
compound was synthesized using ethynylbenzene (yield = 73%). H NMR (400 MHz,
DMSO) δ 8.25 (s, 1H), 7.75 ꢀ 7.71 (m, 2H), 7.48 ꢀ 7.45 (m, 3H), 5.04 (sep, 1H), 1.47 (d, J =
8Hz, 6H). 13C NMR (125 MHz, DMSO) δ 157.60, 156.04, 152.09, 131.66, 129.23, 128.49,
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124.85, 121.21, 92.76, 81.05, 48.58, 21.57 ppm. HRMS (ESI) calculated for C16H16N5
[M+H]+ is 278.1406, found 278.1407.
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3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)phenol
(3).
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This compound was synthesized using 3ꢀethynylphenol (yield = 68%). H NMR (400
MHz, DMSO) δ 9.76 (s, 1H), 8.24 (s, 1H), 7.26 (t, J = 6 Hz, 1H), 7.14 (d, J = 4 Hz, 1H),
7.07 (s, 1H), 6.88 (d, J = 4 Hz, 1H), 5.04 (sep, 1H), 1.47 (d, J = 4 Hz, 6H). 13C NMR (125
MHz, DMSO) δ 157.56, 157.07, 155.99, 152.01, 129.61, 124.82, 122.41, 121.95, 117.94,
116.71, 100.46, 92.92, 80.41, 48.53, 21.51 ppm. HRMS (ESI) calculated for C16H16N5O
[M+H]+ is 294.1355, found 294.1355.
3-((3-Aminophenyl)ethynyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(4). This compound was synthesized using 3ꢀethynylaniline (yield = 66%). 1H NMR (400
MHz, DMSO) δ 8.24 (s, 1H), 7.09 (t, J = 6Hz, 1H), 6.83 (s, 1H), 6.825 (d, J = 4Hz, 1H),
6.655 (d, J = 4Hz, 1H), 5.30 (s, 2H), 5.03 (sep, 1H), 1.47 (d, J = 4 Hz, 6H). 13C NMR (125
MHz, DMSO) δ 157.82, 156.20, 152.19, 148.82, 129.22, 125.22, 121.42, 119.16, 116.32,
115.25, 100.68, 94.09, 79.87, 48.73, 21.74 ppm. HRMS (ESI) calculated for C16H17N6
[M+H]+ is 293.1515, found 293.1511.
3-((3-Fluorophenyl)ethynyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
1
(5). This compound was synthesized using 1ꢀethynylꢀ3ꢀfluorobenzene (yield = 50%). H
NMR (400 MHz, DMSO) δ 8.25 (s, 1H), 7.70 ꢀ 7.30 (m, 4H), 5.04 (sep, 1H), 1.47 (d, J =
6.8 Hz, 6H). 13C NMR (125 MHz, DMSO) δ 162.55, 160.60, 157.48, 156.04, 152.12,
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130.53, 128.04, 124.42, 123.23, 118.22, 116.37, 100.46, 91.25, 81.88, 21.53 ppm. HRMS
(ESI) calculated for C16H15FN5 [M+H]+ is 296.1311, found 296.1315.
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3-((3-Chlorophenyl)ethynyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
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(6). This compound was synthesized using 1ꢀchloroꢀ3ꢀethynylbenzene (yield = 75%). H
NMR (400 MHz, DMSO) δ 8.24 (s, 1H), 7.91 (t, J = 2, 1H), 7.69 (dt, J = 7.6, 1.3 Hz, 1H),
7.56 ꢀ 7.46 (m, 2H), 5.04 (sep, 1H), 1.47 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz, DMSO)
δ 157.45, 156.00, 152,09, 132.97, 131.10, 130.31, 130.25, 129.17, 124.38, 123.22, 100.44,
91.01, 82.17, 48.58, 21.50 ppm. HRMS (ESI) calculated for C16H15ClN5 [M+H]+ is
312.1016, found 312.1012.
1-Isopropyl-3-(m-tolylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (7). This
1
compound was synthesized using 1ꢀethynylꢀ3ꢀmethylbenzene (yield = 70%). H NMR
(400 MHz, DMSO) δ 8.25 (s, 1H), 7.57 (s, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.35 (t, J = 7.6 Hz,
13
1H), 7.29 (d, J = 7.6 Hz, 1H), 5.04 (sep, 1H), 2.35 (s, 3H), 1.47 (d, J = 6.8 Hz, 6H). C
NMR (125 MHz, DMSO) δ 157.57, 156.01, 152.06, 137.82, 131.98, 129.97, 128.73,
128.38, 124.86, 121.02, 100.45, 92.92, 80.75, 48.54, 21.55, 20.56 ppm. HRMS (ESI)
calculated for C17H18N5 [M+H]+ is 292.1562, found 292.1560.
1-Isopropyl-3-((3-(trifluoromethyl)phenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4
-amine (8). This compound was synthesized using 1ꢀethynylꢀ3ꢀ(trifluoromethyl)benzene
(yield = 45%). 1H NMR (400 MHz, DMSO) δ 8.25 (s, 1H), 8.19 (s, 1H), 8.03 (d, J = 8 Hz,
1H), 7.826 (d, J = 8 Hz, 1H), 7.70 (t, J = 8 Hz, 1H), 5.05 (sep, 1H), 1.47 (d, J = 6.8 Hz, 6H).
13C NMR (125 MHz, DMSO) δ 157.22, 155.80, 151.91, 135.31, 129.35, 128.07, 128.04,
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125.32, 124.10, 122.24, 100.21, 90.66, 82.25, 54.47, 48.38, 21.29 ppm. HRMS (ESI)
calculated for C17H15F3N5 [M+H]+ is 346.1280, found 346.1271.
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1-Isopropyl-3-((3-methoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(9). This compound was synthesized using 1ꢀethynylꢀ3ꢀmethoxybenzene (yield = 75%). 1H
NMR (400 MHz, DMSO) δ 8.24 (s, 1H), 7.38 ꢀ 7.26 (m, 3H), 7.05 ꢀ 7.01 (m, 1H), 5.04
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(sep, 1H), 3.81 (s, 3H), 1.47 (d, J = 8Hz, 6H). C NMR (125 MHz, DMSO) δ 160.35,
158.96, 157.44, 153.50, 131.06, 126.18, 125.46, 123.64, 117.88, 117.10, 101.85, 94.07,
82.27, 56.57, 49.98, 22.97 ppm. HRMS (ESI) calculated for C17H18N5O [M+H]+ is
308.1511, found 308.1509.
1-Isopropyl-3-((4-methoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(10). This compound was synthesized using 1ꢀethynylꢀ4ꢀmethoxybenzene (yield = 54%).
1H NMR (400 MHz, DMSO) δ 8.34 (s, 1H), 7.53 (d, J = 8.8Hz, 2H), 6.92 (d, J = 9.2 Hz,
2H), 6.22 (s, 2H), 5.15(sep, 1H), 3.85 (s, 3H), 1.59 (d, 6H). 13C NMR (125 MHz, DMSO) δ
161.56, 157.87, 155.41, 153.00, 134.38, 127.68, 115.27, 114.30, 109.99, 95.59, 80.40,
56.36, 50.61, 22.95 ppm. HRMS (ESI) calculated for C17H18N5O [M+H]+ is 308.1511,
found 308.1505.
1-Isopropyl-3-((2-methoxyphenyl)ethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(11). This compound was synthesized using 1ꢀethynylꢀ4ꢀmethoxybenzene (yield = 46%).
1H NMR (400 MHz, DMSO) δ 8.26 (s, 1H), 7.59 (dd, J = 7.6, 1.2 Hz, 1H), 7.47 (td, J = 8.8,
1.6 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 7.05 (td, J = 7.2, 0.8 Hz, 1H), 5.02 (sep, 1H), 3.94 (s,
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3H), 1.47 (d, J = 6.8 Hz, 6H). C NMR (125 MHz, DMSO) δ 159.92, 157.66, 156.31,
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152.00, 131.86, 131.04, 124.99, 120.75, 111.22, 109.72, 101.18, 90.88, 85.49, 55.62,
48.57, 21.58 ppm. HRMS (ESI) calculated for C17H18N5O [M+H]+ is 308.1511, found
308.1509.
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4-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)phenol (12).
To a stirred solution of 10 (40 mg, 0.13 mmol) in 1 mL of dry dichloromethane was added
at ꢀ78 °C a solution of 1M BBr3 in dichloromethane (0.39 mL, 0.39 mmol). After 2 h the
mixture was warmed to 25 °C. The reaction was monitored by TLC; after completion, the
mixture was quenched with saturated sodium bicarbonate, extracted by EtOAc, washed
with water and brine. The crude product was purified by flash column chromatography
1
(DCM/MeOH) to give a white solid (26.7 mg, 0.09 mmol, 70%). H NMR (400 MHz,
DMSO) δ 10.02 (s, 1H), 8.22 (s, 1H), 7.54 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 5.02
(sep, 1H), 1.46 (d, J = 6.4 Hz, 6H) 13C NMR (125 MHz, DMSO) δ 158.63, 157.78, 156.14,
152.16, 133.56, 125.53, 115.67, 111.39, 100.48, 93.76, 79.41, 48.60, 21.73 ppm. HRMS
(ESI) calculated for C16H16N5O [M+H]+ is 294.1355, found 294.1355.
2-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)phenol (13).
To a stirred solution of 11 (40 mg, 0.13 mmol) in 1 mL of dry dichloromethane was added
at ꢀ78 °C a solution of 1M BBr3 in dichloromethane (0.39 mL, 0.39 mmol). After 2 h the
reaction mixture was warmed to 25 °C. The progress of the reaction was monitored by
TLC; after completion, the mixture was quenched with saturated sodium bicarbonate,
extracted by EtOAc, washed with water and brine. The crude product was purified by flash
column chromatography (DCM/MeOH) to give a white solid (23.3 mg, 0.09 mmol, 61%).
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1H NMR (400 MHz, DMSO) δ 10.68 (s, 1H), 8.24 (s, 1H), 7.48 (dd, J = 7.6 Hz, 1H),
7.31ꢀ7.26 (m, 1H), 6.99 (d, J = 8Hz, 1H), 6.88 (t, J = 7.6Hz, 1H), 5.02 (sep, 1H), 1.46 (d, J
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= 6.8 Hz, 6H) C NMR (125 MHz, DMSO) δ 159.03, 157.77, 152.05, 131.73, 130.74,
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125.31, 119.25, 115.27, 108.42, 101.00, 91.51, 84.91, 48.49, 21.61 ppm. HRMS (ESI)
calculated for C16H16N5O [M+H]+ is 294.1355, found 294.1356.
(E)-3-(2-(4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)vinyl)phenol
(14). A mixture of 3ꢀvinylphenol, 14a (100 mg, 0.83 mmol, Supporting Information), 1
(252.3 mg, 0.83 mmol), triethanolamine (124.2 mg, 0.83 mmol)) and Pd(OAc)2 (8.3 mg,
0.037 mmol) was stirred under nitrogen at 100 °C for 24 h. The reaction mixture was
cooled to 25 °C, quenched by the addition of dil. aq. hydrochloric acid (2M, 10 mL), and
extracted with ether (3 × 100 mL). The organic phases were dried with sodium sulfate, the
solvents evaporated, and the crude product was purified by flash column chromatography
1
(hexane/ethyl acetate) to give a white solid (48.2 mg, 0.16 mmol, 54%). H NMR (400
MHz, DMSO) δ 9.40 (s, 1H), 8.15 (s, 1H), 7.58 (d, J = 16 Hz, 1H), 7.51 (s, 2H), 7.35 (d, J
= 16 Hz, 1H), 7.21 ꢀ 7.15 (m, 3H), 6.74 ꢀ 6.71 (m, 1H), 5.01 (sep, 1H), 1.48 (d, J = 6.8Hz,
6H) 13C NMR (125 MHz, DMSO) δ 158.09, 157.50, 155.24, 140.23, 137.85, 131.24,
129.37, 118.41, 118.35, 115.16, 113.80, 98.15, 48.16, 21.75 ppm. HRMS (ESI) calculated
for C16H18N5O [M+H]+ is 296.1511, found 296.1507.
N-(3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)phenyl)-3-(
trifluoromethyl)benzamide (15). To a solution of 4 (100 mg, 0.34 mmol) in
dichloromethane (2 mL) were added triethylamine (5.2 mg, 0.05 mmol) and
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3ꢀ(trifluoromethyl)benzoyl chloride (70.9 mg, 0.34 mmol). The reaction mixture was
stirred at room temperature overnight, then extracted with DCM. The organic layer was
washed with H2O and dried over sodium sulfate. The solvent was removed under reduced
pressure. The residue was purified by flash column chromatography (hexane/ethyl acetate)
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to give a yellowish solid (77.9 mg, 0.167 mmol, 49%). H NMR (400 MHz, DMSO) δ
10.61 (s, 1H), 8.25 (s, 1H), 8.32 ꢀ 7.46 (m, 8H), 5.05 (sep, 1H), 1.48 (d, J = 6.8Hz, 6H) 13C
NMR (125 MHz, DMSO) δ 174.58, 165.14, 158.71, 157.15, 153.20, 139.92, 136.43,
132.83, 130.76, 130.11, 130.02, 128.34, 125.82, 125.17, 124.40, 122.60, 122.50, 109.59,
101.60, 93.67, 82.13, 49.72, 22.66 ppm. HRMS (ESI) calculated for C24H20F3N6O [M+H]+
is 465.1651, found 465.1643.
3-((4-Amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)-N-(3-(triflu
oromethyl)phenyl)benzamide (16). Solid components were first added to flask: 1 (88 mg,
0.29 mmol), tetrakis(triphenylphosphine)palladium (67 mg, 0.058 mmol), CuI (27.6 mg,
0.145 mmol), followed by addition of anhydrous THF (10 ml), 16b (335.5 mg, 1.16 mmol,
Supporting Information) and diisopropylamine (293.45 mg, 2.9 mmol). The reaction flask
was covered in foil and the mixture was stirred for 17 h at room temperature. The reaction
mixture was concentrated under reduced pressure. The compound was purified by flash
column chromatography (CH2Cl2/MeOH) to give a light yellowish solid (60.7 mg, 0.13
mmol, 45%). 1H NMR (400 MHz, DMSO) δ 10.66 (s, 1H), 8.35 (s, 1H), 7.47 – 8.24 (m,
8H) 5.04 (sep, 1H), 1.45 (d, 6H) 13C NMR (125 MHz, DMSO) δ 164.84, 157.60, 156.08,
152.16, 139.64, 134.81, 134.63, 130.73, 129.79, 128.83, 128.52, 124.62, 123.65, 121.58,
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120.00, 119.97, 116.27, 116.24, 116.21, 91.91, 81.87, 48.63, 21.59 ppm. HRMS (ESI)
calculated for C24H20F3N6O [M+H]+ is 465.1651, found 465.1644.
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Protein Expression and Purification. The kinase domain of human cꢀAbl (residues
227ꢀ515) in modified pETꢀN6 vector (Plexxikon) was expressed.42 Briefly, E. coli BL21
(DE3) coꢀtransformed with Abl (pETꢀN6) was grown at 37 °C, 30induced with IPTG (0.5
mM) between OD600 of 0.6 ꢀ 0.8 units, and then grown at 18 °C overnight. The cells were
harvested by centrifugation at 4,300 rpm, reꢀsuspended in lysis buffer (50 mM Tris, 500
mM NaCl, 25 mM imidazole, 5% glycerol, pH 7.5), and lysed by sonication (Q500
Sonicator, Qsonica). After the cell lysate was centrifuged at 10,200 × g for 1 hour at 4 °C,
the supernatant was incubated with NiꢀNTA beads. Then the resins were washed with wash
buffer 4 times before proceeding to elution of the protein with elution buffer. The wash
buffer contains 20 mM Tris, 500 mM NaCl, and 25 mM imidazole at pH 7.5 while the
elution buffer is identical except a higher concentration (300 mM) of imidazole. The
elution fraction was collected and analyzed by SDSꢀPAGE gel for purity determination.
Chicken cꢀSrc (residues 251ꢀ533) was prepared as previously described.43ꢀ44 In brief, the
protein was coꢀexpressed with fullꢀlength YopH phosphatase in E. coli BL21 (DE3) cells.
The protein was initially purified by NiꢀNTA beads. The 6× Hisꢀtag was then removed by
TEV cleavage. Protein purification was followed with anion exchange chromatography
and sizeꢀexclusion chromatography. Protein in 50 mM Tris (pH 8.0), 100 mM NaCl, 5%
Glycerol, 1 mM DTT was concentrated to 10 mg/mL and flash frozen for storage at −80°C.
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In vitro Kinase Assay. The kinase activity was measured by quantifying the amount of
32P transfer from [γꢀ32P]–ATP to the peptide substrate. The assay was carried out in 50 mM
Tris (pH 8.0), 10 mM MgCl2, 5.3 nM [γꢀ32P]– ATP (PerkinElmer), 1 mg/mL BSA, 0.1 mM
peptide substrate (EAIYAAPFAKKK, Genscript) along with different concentrations of
inhibitors (2 ꢀ 16) at 25°C in 30 ꢁL reaction. The kinase reaction was initiated by the
addition of [γꢀ32P]–ATP and was allowed to proceed for 30 minutes. The reaction was
transferred onto phosphocellulose filter disc paper (Whatman P81/GE Healthcare),
quenched with 10% aq. acetic acid, washed with 0.5% aq. phosphoric acid (3 times), and
rinsed with acetone. The dried phosphocellulose filter was added scintillation fluid
(BetaMaxꢀES, MP Biomedicals), and counted on a scintillation counter (Beckman
LC6500). The data was analyzed with GraphPad Prism5 to calculate the IC50.
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Cells and Reagents. Ba/F3 cells expressing BcrꢀAblWT and BcrꢀAblT315I were obtained
from Dr. Shah laboratory (UCSF, San Francisco, CA, USA). The cells were maintained in
RPMI 1640 (Corning) supplemented with 10% fetal bovine serum (v/v, HyClone,
ThermoScientific) and 1X penicillin, strepatmycin, glutamine (GE Healthcare) at 37°C
and 5.0% CO2 conditions. Imatinib and ponatinib were purchased from Selleckchem.
Dimethyl sulfoxide (DMSO) was purchased from SigmaꢀAldrich (St. Louis, MO, USA).
The following antibodies: antiꢀAbl, antiꢀphosphoꢀAbl (Y412), antiꢀphosphoꢀSTAT5
(Tyr694), and antiꢀSTAT5, were purchased from Cell Signaling (Boston, MA, USA).
Antiꢀactin was purchased from Sigma Aldrich (St. Louis, MO, USA). Antiꢀmouse
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secondary antibody was purchased from Jackson ImmunoResearch Laboratories (West
Grove, PA, USA) while antiꢀrabbit secondary antibody from Abcam (Cambridge).
Western Blot Analysis. Ba/F3 cells expressing BcrꢀAbl were treated with vehicle
(DMSO) or drug at desired concentration for 3 hours. The cells were lysed, collected, and
normalized using BCA protein assay kit (BioꢀRad) before being diluted in 1x SDS loading
buffer (BioꢀRad). The samples were resolved on 4ꢀ20% SDSꢀpage gel (BioꢀRad) and
then transferred onto nitrocellulose membrane (BioꢀRad). Standard immunoblotting
protocols were used. Briefly, all western blots were blocked with 5% nonꢀfat milk in TBST
(10 mM Tris, 150 mM NaCl, 0.1% Tweenꢀ20, pH 8.0) for 1 hour at room temperature. The
primary antibodies were diluted to appropriate concentration and incubated overnight at
4°C Subsequently the blots were then probed with horseradish peroxidase
(HRP)ꢀconjugated secondary antibodies (1:10,000) for 1 hour at room temperature and
then developed using enhanced chemiluminescence (ECL, BioꢀRad) and visualized by
ChemiDoc XRS+ molecular imager (BioꢀRad).
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MTT Cell Proliferation Assay. Ba/F3 cells were plated into 96ꢀwell plates (2×104
cells/well) and incubated with imatinib, ponatinib, or a pyrazolopyrimidine at indicated
concentrations for 48 h. Subsequently, the cell viability was measured by adding MTT
(Calbiochem) to all wells and incubated for 1 h. Upon completion, the reaction was
quenched with formazanꢀsolubilization reagent (50% DMF (v/v), 20% SDS w/v in H2O)
and the absorbance was measured at 540 nm using SpectraFluoro Plus plate reader (Tecan)
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to determine the cell viability. Each assay was performed in triplicates and the IC50 values
were calculated using Graphpad Prism with a nonlinear regression method.
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Figure 1. Schematic representations illustrating binding of the different inhibitors to the
Abl kinase. (A) As a type II inhibitor, imatinib binds in both the active site and the
allosteric pocket in BcrꢀAblWT; (B) T315I mutation induces steric clash to imatinib and
causes drug resistance; (C) an alkyne linker allows ponatinib to bypass the bulky
gatekeeper residue I315 and occupy both pockets; (D) alkyneꢀcontaining
pyrazolo[3,4ꢀd]pyrimidines were designed to bypass I315 and bind both pockets in a
similar manner to ponatinib.
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Figure 2. Effects of the inhibitors on the viability of Ba/F3 cells expressing WT or T315I
BcrꢀAbl. Cell viability in the presence of varying concentrations of (A) imatinib, (B)
ponatinib, (C) 15, or (D) 16 was measured using the MTT assay to generate doseꢀresponse
curves (GraphPad Prism).
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Figure 3. Pyrazolopyrimidines inhibit the activity of both (A) BcrꢀAblWT and (B)
BcrꢀAblT315I in Ba/F3 cells as ponatinib does. Ba/F3 cells expressing BcrꢀAblWT or
BcrꢀAblT315I were cultured at a density of 8 x 104/mL and treated for 3 hr at a final
concentration of 500 nM for each inhibitor. The cell lysates were resolved in PAGE gel,
transferred to nitrocellulose membrane, and probed with antibodies for Abl, pY412ꢀAbl,
STAT5, pY694ꢀSTAT5 and βꢀactin sequentially.
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Figure 4. Pyrazolopyrimidines inhibits kinase activity of both (A) BcrꢀAblWT and (B)
BcrꢀAblT315I in Ba/F3 cells in a doseꢀdependent manner. Ba/F3 cells expressing BcrꢀAblWT
or BcrꢀAblT315I were cultured at a density of 8 x 104/mL and treated for 3 hr at indicated
concentration for 15 and 16. The cell lysates were resolved in PAGE gel, transferred to
nitrocellulose membrane, and probed with antibodies for Abl, pY412ꢀAbl, and βꢀactin
sequentially.
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Figure 5. Docking models of AblT315I in complex with 15 (A) and 16 (B). 15 and 16 were
docked into AblT315I using AutoDock Vina. A crystal structure of AblT315I in complex with
a type II inhibitor (PDB entry: 3OY3) was used for docking. The images were generated
using PyMol. 30
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Scheme 1. Synthesis of Pyrazolo[3,4ꢀd]pyrimidines 2 - 16
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Reagents and conditions: (a) alkyne, THF, Pd(PPh3)4, CuI, N2, r.t., 18 h; (b) BBr3, dry
DCM, ꢀ78 °C, 2 h; (c) Pd(OAc)2, 3ꢀvinylphenol, N2, 100 °C, 24 h; (d)
3ꢀ(trifluoromethyl)benzoyl chloride, TEA, DCM, r.t., 12 h.
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