Expanding the diversity of allosteric Bcr-Abl inhibitors

Article abstract of DOI:10.1021/jm100555f

Inhibition of Bcr-Abl kinase activity by imatinib for the treatment of chronic myeloid leukemia (CML) currently serves as the paradigm for targeting dominant oncogenes with small molecules. We recently reported the discovery of GNF-2 (1) and GNF-5 (2) as selective non-ATP competitive inhibitors of cellular Bcr-Abl kinase activity that target the myristate binding site. Here, we used cell-based structure-activity relationships to guide the optimization and diversification of ligands that are capable of binding to the myristate binding site and rationalize the findings based upon an Abl-compound 1 cocrystal. We elucidate the structure-activity relationships required to obtain potent antiproliferative activity against Bcr-Abl transformed cells and report the discovery of new compounds (5g, 5h, 6a, 14d, and 21j-I) that display improved potency or pharmacological properties. This work demonstrates that a variety of structures can effectively target the Bcr-Abl myristate binding site and provides new leads for developing drugs that can target this binding site.

Full text of DOI:10.1021/jm100555f

6934 J. Med. Chem. 2010, 53, 6934–6946
DOI: 10.1021/jm100555f
Expanding the Diversity of Allosteric Bcr-Abl Inhibitors†
^
Xianming Deng,‡,§,ꢀ Barun Okram, ,ꢀ Qiang Ding,^,ꢀ Jianming Zhang,‡,§,ꢀ Yongmun Choi,‡,§ Francisco J. Adrian,
ꢀ
Amy Wojciechowski,‡,§ Guobao Zhang,^ Jianwei Che,^ Badry Bursulaya,^ Sandra W. Cowan-Jacob,# Gabriele Rummel,#
Taebo Sim,*,¥ and Nathanael S. Gray*,‡,§
‡Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,
§Department of Cancer Biology, Dana-Farber Cancer Institute, 250 Longwood Avenue, SGM 628, Boston, Massachusetts 02115,
Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037,
^Department of Biological Chemistry, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego,
California 92121, #Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland, and ¥Life/Health Division, Korea Institute of Science
and Technology, 39-1 Hawolgok-Dong, Wolsong-Gil5, Seongbuk-Gu, Seoul, 136-791, Korea. ꢀ These authors contributed equally to this work.
Received May 7, 2010
Inhibition of Bcr-Abl kinase activity by imatinib for the treatment of chronic myeloid leukemia (CML)
currently serves as the paradigm for targeting dominant oncogenes with small molecules. We recently
reported the discovery of GNF-2 (1) and GNF-5 (2) as selective non-ATP competitive inhibitors of
cellular Bcr-Abl kinase activity that target the myristate binding site. Here, we used cell-based
structure-activity relationships to guide the optimization and diversification of ligands that are capable
of binding to the myristate binding site and rationalize the findings based upon an Abl-compound 1
cocrystal. We elucidate the structure-activity relationships required to obtain potent antiproliferative
activity against Bcr-Abl transformed cells and report the discovery of new compounds (5g, 5h, 6a, 14d,
and 21j-I) that display improved potency or pharmacological properties. This work demonstrates that a
variety of structures can effectively target the Bcr-Abl myristate binding site and provides new leads for
developing drugs that can target this binding site.
Introduction
non-ATP competitive kinase inhibitors is that they can be
highly selectivefor a particularkinase because they can exploit
nonconserved kinase regulatory mechanisms. Indeed, 1 de-
monstrated exclusive cellular activity against Bcr-Abl trans-
formed cells (EC50 = 140 nM) and did not inhibit the activity
of 40 other tyrosine kinases in cellular assays or the biochem-
ical activityof a panel of 80 diverse kinases.8 Compound 1 was
demonstrated to bind to the myristate binding site located
near the C-terminus of the Abl kinase domain using protein
crystallography and NMR spectroscopy.9 Compound 2,9 the
hydroxylethylamide analogue of 1, with improved pharma-
cological properties is efficacious in Bcr-Abl-dependent xeno-
graft and bone marrow transplantation models against wild-
type Bcr-Abl and is capable of inhibiting T315I Bcr-Abl when
used in combination with the ATP competitive inhibitor
nilotinib. Binding of 1 or 2 to the myristate binding site induces
changes to the conformational dynamics of the ATP-site as
revealed by hydrogen-deuterium exchange studies, but the
precise conformation induced by myristate-site binding remains
to be elucidated.
Chronic myelogenous leukemia (CMLa) is a hematological
disorder caused by a chromosomal rearrangement that gen-
erates a fusion protein, Bcr-Abl, with deregulated tyrosine
kinase activity.1 Although clinical remission has been achieved
with the ATP-binding site targeting drug imatinib, many
patients relapse because of emergence of clones expressing
inhibitor-resistant forms of Bcr-Abl.2 To address these re-
lapses, two more potent ATP-site directed agents, nilotinib3,4
and dasatinib,5,6 have recently received clinical approval.
Although both compounds inhibit most of the mutations that
induce resistance to imatinib, neither compound is capable of
inhibiting the “gatekeeper” T315I mutation, which is situated
in the middle of the ATP-binding cleft.7
In an effort to find new pharmacological modalities of Bcr-
Abl inhibition, we performed a differential cytotoxicity screen
that resulted in the identification of 1, a non-ATP competitive
inhibitor of cellular Bcr-Abl activity.8 A major advantage of
†PDB code: 3K5V.
Compound 1 was developed by iterative structure-activity
guided optimization using Bcr-Abl transformed Ba/F3 cells.
The original“hit”compound, GNF-1(4a),8 was discovered by
screening a combinatorial library of 50000 heterocycles orig-
inally designed to target the ATP binding site. The library was
synthesized on solid phase using the IRORI nanokan system.10
The screen sought compounds that were differentially cyto-
toxic between murine 32D cells versus 32D cells transformed
with Bcr-Abl. Compounds 1 and 4a have a 4,6-disubstituted
pyrimidine core structure that has received some attention as
*To whom correspondence should be addressed. For T.S.: phone, 82-
2-958-6437; fax, 82-2-958-5189; e-mail, tbsim@kist.re.kr. For N.S.G.:
phone, 1-617-582-8590, fax, 1-617-582-8615; e-mail, nathanael_gray@
dfci.harvard.edu.
a Abbreviations: Akt, v-akt murine thymoma viral oncogene homo-
logue 1; ATP, adenosine triphosphate; Bcr-Abl, breakpoint cluster
region Abelson tyrosine kinase; CDK, cyclin-dependent kinases; DIEA,
N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; HATU,
2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate; mTor, mammalian target of rapamycin; PKC, protein
kinase C; SK1, sphingosine kinase 1; TFA, trifluoroacetic acid; THF,
tetrahydrofuran.
r
pubs.acs.org/jmc
Published on Web 09/09/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6935
Scheme 2a
Scheme 1a
a Reagents and conditions: (a) methyl 2-mercaptoacetate, K2CO3,
DMF, room temp to 90 ꢀC; (b) LiAlH4, 0 ꢀC ; (c) LiOH/THF; (d) primary
or secondary amine, HATU, DMF, room temp.
Scheme 3a
a Reagents and conditions: (a) 4-trifluoromethoxylaniline (1.1 equiv),
DIEA (1.1 equiv), EtOH, reflux; (b) primary or secondary amine, DIEA,
2-BuOH, reflux; (c) substituted phenylboronic acid (1.0 equiv), Pd-
(PPh3)4 (5%), Na2CO3 (4.0 equiv), CH3CN/H2O (v/v = 1/1), reflux;
(d) amine, HATU, DIEA, DMF, room temp; (e) substituted pyrazole-
boronic acid (1.0 equiv), Pd(PPh3)4 (5%), Na2CO3 (4.0 equiv), CH3CN/
H2O (v/v = 1/1), reflux; (f) Cs2CO3, R6Br (1.1 equiv); (g) heteroaro-
matic boronic acid (1.0 equiv), Pd(PPh3)4 (5%), Na2CO3 (4.0 equiv),
CH3CN/H2O (v/v = 1/1), reflux; (h) 2-morpholinoethanamine (1.0
equiv), DIEA (1.5 equiv), EtOH, reflux; (i) 4-(trifluoromethoxy)phenol,
NaH, dioxane, room temp to reflux.
an ATP-binding site directed scaffold11,12 but has not been as
extensively investigated as the corresponding 2,4-disubstitut-
ed pyrimidines.13,14 Here, we describe the structure-activity
relationships for the 4,6-disubsituted pyrimidine class of Bcr-Abl
myristate binding site-targeted ligands. Using established med-
icinal chemistry approaches such as introduction of ring con-
straints to reduce entropic penalties upon ligand binding, we
have successfully discovered additional heterocyclic ring systems
such as thieno[2,3-d]pyrimidines, pyrrolo[2,3-d]pyrimidines, and
pyrazolo[3,4-d]pyrimidines that can inhibit cellular Bcr-Abl
activity by binding to the myristate-binding site.
a Reagents and conditions: (a) primary iodo- or bromoalkane, NaH,
DMF, 0 ꢀC to room temp; (b) 4-(trifluoromethoxy)aniline, 2-BuOH, 120 ꢀC;
(c) methyl 3-bromopropanoate, NaH, THF; (d) 4-(trifluoromethoxy)-
aniline, 2-BuOH, 120 ꢀC; (e) Cs2CO3, THF/H2O; (f) R2NH2, HATU,
DIEA, DMF, room temp.
Results and Discussion
Scheme 4a
Synthesis. 4,6-Disubstituted pyrimidines were prepared
from 4,6-dichloropyrimidine by base-promoted amination
followed by palladium mediated coupling with boronic acids
or esters, anilines, or phenols (Scheme 1).15
Further elaboration of the pyrimidine 6-substituent was
accomplished by alkylation, sulfonamide or amide-bond
formation. 2,4-Disubstituted pyrimidines, 2,6-disubstituted
pyrazines, 2,4-disubstituted triazines, and 3,6-disubstituted
pyridazines were prepared using methodology similar to that
of 4,6-disubstituted pyrimidines preparation as described in
the Supporting Information in order to explore similar core
structures to the 4,6-disubstituted pyrimidines. Thieno[2,3-
d]pyrimidines substituted at the 6-position were synthesized
in three steps by condensation of methyl 2-mercaptoacetate
with 4-chloro-6-(4-(trifluoromethoxy)phenylamino)pyrimidine-
5-carbaldehyde under basic conditions, followed sequen-
tially by ester hydrolysis and amide bond formation using
HATU (Scheme 2).
a Reagents and conditions: (a) RNHNH2, Na2CO3, 2-BuOH, 60 ꢀC;
(b) NH2NH2, THF; (c) primary bromo- or chloroalkane, Cs2CO3 or
K2CO3, DMF.
moalkanes and sodium hydride, followed by thermal substitution
of the 6-chloro group with the corresponding anilines. Further
modification of the N9-substituent was accomplished by ester
hydrolysis and amide bond formation using HATU (Scheme 3).
Pyrrolo[2,3-d]pyrimidines were prepared by N9 alkylation on
4-chloro-7H-pyrrolo[2,3-d]pyrimidine with primary iodo- or bro-

6936 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Table 1. SAR of 4,6-Disubstituted Pyrimidine
Deng et al.
a Cytotoxicity (EC50, μM) on parental Ba/F3. b Antiproliferative activity (EC50, μM) on wtBcr-Abl-Ba/F3.
Pyrazolo[3,4-d]pyrimidines were prepared through two
different synthetic routes: either 4-chloro-6-(4-(trifluoro-
methoxy)phenylamino)pyrimidine-5-carbaldehyde was con-
densed with an appropriate alkylhydrazine or this condensa-
tion was performed using hydrazine followed by N1 alkylation
with a primary iodo- or bromoalkane under cesium or potas-
sium carbonate (Scheme 4). The regioisomeric products were
subsequently separated by mass-triggered high-pressure liquid
chromatography.
Bcr-Abl Structure-Activity Relationships for 4,6-Disubsti-
tuted Pyrimidines. Following the identification of 4a as a
screening “hit” that specifically inhibited Bcr-Abl trans-
formed cells with an EC50 of 1 μM, a systematic evaluation
of the structural features necessary to impart activity as a
cellular Bcr-Abl inhibitor was investigated. We initiated our
investigation by replacing the morpholinoethylamino sub-
stituent located at the pyrimidine C4-position with a range of
functionalities including primary and secondary amines,
anilines, phenols, and various aromatic and heteroaromatic
substituents (Table 1). Compounds containing a direct aro-
matic linkage such as 1, 5a, and 5c displayed the most potent
activity, while alkylamine (4b and 4c) derivatized com-
pounds displayed reduced activity. The advantage of direct
aromatic linkage at the pyrimidine C4-position (1) is evident
by the loss of activity observed for the corresponding ether
(4e) and amine-linked (4f) derivatives. In the aryl series,
optimal activity was achieved with hydrogen bond donating
or accepting functionalities (CONR1R2, SO2CH3, SO2NHR,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6937
Table 2. SAR of Pyrimidine C6-position
a Cytotoxicity (EC50, μM) on parental Ba/F3. b Antiproliferative activity (EC50, μM) on wtBcr-Abl-Ba/F3.
NH2) at the 3- or 4-position of the phenyl ring as shown by 1,
2, 5a, and 5d-h. Substitution with a 2-carboxamide (5b)
resulted in an inactive compound presumably as a result
of steric hindrance or due to unfavorable torsional angle
between the pyrimidine and 4-phenyl substituent. We next
investigated additional analogues of 1 focusing on replace-
ments of the C4-benzamide substitutent. Replacements of
the amide with sulfone (5e), primary amine (5f), sulfonamide
(5g), and homologated amides (5i-k) all resulted in com-
pounds with cellular potency comparable to that of 1 and 5c
(Table 1). These results suggested that this position maybe
faces the solvent-exposed region of the enzyme and would
provide a good location for modulating the physical and
pharmacological properties of the compound. Indeed the
hydroxyethylamide analogue 2 displayed similar cellular
Bcr-Abl inhibitory activity as well as favorable pharma-
cokinetic properties and was chosen for evaluation in murine
leukemia models.9
We also introduced sulfonamide and “reverse” sulfonamide
at the para position of the C6-phenyl. The unsubstituted
sulfonamide 5g (EC50 = 120 nM) and reverse methylsulfo-
namide 5h (EC50 = 40 nM) were obtained with improved
inhibitory activity. We installed a variety of heterocyclic ring
systems at C4 and discovered that unsubstituted (6a) and
alkyl substituted pyrazoles (6b, 6c, and 6e) displayed super-
ior potency compared to 4a (Table 1). Introduction of 5,6-
fused aromatic systems to the pyrimidine 4-position was also
tolerated with potent activity being observed for indoles
attached via the indole 4- or 5-position (7a, 7b) and azain-
doles attached via the azaindole 4-position (7c).
pyrimidine C6-position (Table 2). For example, replacement
of the aniline NH with an N-methyl substitution (8a) or
oxygen (8d) or direct phenyl ring connection (8e) resulted in a
complete loss of cellular activity, suggesting that this group
maybe responsible for a critical contact with the enzyme.
N-Methyl substitution was chosen as the modification to
prepare the negative control for both the affinity chromato-
graphy and NMR-binding studies.8,9 Similarly, regioiso-
meric m-trifluoromethoxyanilines (8c) or p-trifluoromethoxy-
lbenzylamino-substituted compounds (8g) were completely
inactive. To our surprise, replacement of the trifluoromethyl
group with a methyl group also resulted in a complete loss of
activity (8b). The only substitution that was tolerated at C6
was the conservative replacement of the trifluoromethoxy
with a trifluoromethyl group (8f).
We next investigated the consequences of varying the
positions of the nitrogens located on the central pyrimidine
core (Table 3). Here, we discovered a strict requirement for
the 4,6-pyrimidine; both of the 2,4-pyrimidine regioisomers
(9c and 9d), pyrazine (9e), pyridazine (9f), and triazine (9l)
were inactive. This result suggested that in the context of
compound 4a, the 4,6-substitution pattern is strictly required
to maintain cellular Bcr-Abl inhibitory activity.
We next focused our attention on using structure-activity
relationships to deduce the bioactive conformation of
improved leads such as 1. One interesting observation was
that the 4,6-disubstitution requirement for the central pyri-
midine was different from that of ATP-competitive kinase
inhibitors where in general the 2,4-pyrimidine is favored as a
motif that binds to the kinase hinge region. This is believed to
be a consequence of 2,4-pyrimidines being able to assume
the cis conformation with respect to the NH-C2 bond that is
required to form a bidentate H-bond to the kinase hinge region.
In sharp contrast to the range of functionality that was
tolerated at the pyrimidine C4-position, very few replace-
ments to the 4-trifluoromethoxyaniline were tolerated at the

6938 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Table 3. SAR of Central Pyrimidine Core
Deng et al.
a Cytotoxicity (EC50, μM) on parental Ba/F3. b Antiproliferative activity (EC50, μM) on wtBcr-Abl-Ba/F3.
For example, the classical ATP-competitive 2,4-pyrimidine
CDK inhibitors such as 3-[[4-[2-[(3-chlorophenyl)amino]-
pyrimidin-4-yl]pyridin-2-yl]amino]propan-1-ol (CGP60474)
bind to CDK2 hinge residues using a cis conformation.16
Considering that 1 was not able to function as an ATP-
competitive Bcr-Abl inhibitor, this led us to speculate that
the requirement for the 4,6-pyrimidine scaffold reflected a
need for these compounds to adopt a “trans” conformation
to be active as cellular Bcr-Abl inhibitors. To investigate this
hypothesis, we created a series of compounds that would be
structurally similar but exhibit different preferences for cis or
trans conformations due to potential steric interactions
between ortho position of the p-trifluoromethoxyaniline ring
and the pyrimidine C5 in the cis-conformation (Figure 1).
Biological evaluation of the resulting compounds demonstrated
that only the inhibitors with a preference for the trans-
conformation were able to inhibit cellular Bcr-Abl activity.
Reasoning that the enhanced cellular potency of com-
pound 5c might allow us to uncover subtleties regarding
SAR at C6 that would not be apparent in the less potent 4a,
we prepared further analogues at this position (Table 3). We
again observed that methylation of the aniline NH (9g)
resulted in a complete loss of cellular activity. Similar to
4a, substitution of the trifluoromethoxy group with fluoro
(9h) and methoxy (9i) groups resulted in a complete loss in
activity. Only removal of the oxygen of the trifluoromethoxy
group (9j) and introduction of a meta nitrogen (9k) resulted
in inhibitors that retained some activity.
Structural Biology of the Myristate Binding Site. The
myristate binding site is a cylindrical shaped cavity located

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6939
at the C-terminus of the Abl kinase domain. Before the
availability of the 1-Abl cocrystal (PDB code 3K5V),9 we
initially used the docking program Glide to compute poten-
tial binding conformations of 1 to Abl.17 The lowest energy
pose places 1 in an extended trans conformation with the
4-trifluoromethoxyaniline pointed toward the bottom of the
pocket. With the exception of the positioning of the benza-
mide, the Glide generated binding conformation for 1 was
the same as in the subsequently determined crystal structure
and could be used to rationalize the aforementioned structure-
activity relationships. Specifically it explains the strict
requirement for 4-substitution of the trifluoromethoxy group
and the preference for 4,6- versus 2,4-disubstitution which
favors the extended trans-conformation of the pyrimidine
and the tolerance for diverse substitution at the pyrimidine
6-positions which point out toward the solvent exposed
region. In the cocrystal (Figure 2), a water molecule forms
a hydrogen bond bridge between the aniline NH and the
main chain carbonyls of A433 (Abl 1a numbering) and E462
in the myristate binding site. This observation can explain
the loss of potency observed when the aniline NH is replaced
with oxygen (8d) or an N-methyl group (8a). Residues
making contact with 1 at the base of the pocket are L341
and A344 from RE, I432 from RF, V468 from RH, F493 from
RI, and I502 from RI0. The surface in the central part of the
pocket is formed by A337 from RE, C464 and P465 from the
Figure 1. SAR for analogues of 4a indicates that a trans conforma-
tion is favored.
Figure 2. Cocrystal structure of 1 with Abl kinase (PDB entry 3K5V). (A) Binding conformation of 1 (cyan) in the myristate binding site of Abl
(side chains from helix RE are green, RF orange, RI dark orange, R0I red). Hydrogen bonds to a water molecule (red sphere) are indicated by dashed
yellow lines. (B) Solvent accessible surface of the myristate binding pocket colored as in panel A viewed from the mouth of the binding pocket.

6940 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Deng et al.
start of RH, A433 from RF, and V506 from RI0. The fit in this
region is quite snug, explaining the intolerance for substitu-
tion to the trifluoromethylphenyl ring. There are fewer
interactions at the mouth of the pocket (Y435 from RF,
E462 from the loop before RH, and L510 at the end of RI0),
which is caused by the flexibility and hence slightly weaker
electron density of the benzamide part of 1. This explains the
diverse range of functionalities that can be incorporated into
this position. A mutagenesis screen resulted in the identifica-
tion of five mutations around the myristate binding pocket
(C464Y, P465S, V506L, F497L, E505K) that cause resis-
tance to 1 and 2.9 On the basis of the crystal structure, these
would be expected to cause resistance because of direct steric
hindrance to binding or more indirectly because of the
position of helix I.
Ring Constrained Analogues of 1. We were interested in
further optimizing the cellular potency of the 1 class of Bcr-
Abl kinase inhibitors and exploring their potential to target a
variety of Bcr-Abl mutants that are resistant to ATP-com-
petitive inhibitors such as imatinib, nilotinib, and dasatinib.
For example, 1 is active on wild-type Bcr-Abl, E255V, and
Y253H mutants but is inactive on the T315I “gatekeeper”
mutant which has proven to be the most recalcitrant to inhibit.
To create further analogues of 1, we designed conformation-
ally constrained compounds by introducing fused ring systems
(Figure 3). We initially explored the potential to fuse a five-
membered ring between the pyrimidine 5 and 6 positions to
create four classes of 6,5-fused heterocyclic ring systems:
6-substituted thieno[2,3-d]pyrimidines, 7-substituted pyrrolo-
[2,3-d]pyrimidines, and 1- and 2-substituted pyrazolo[3,4-
d]pyrimidines (Figure 3, constraint A). Collectively these scaf-
folds provide the possibility of directing one or two substitu-
ents from various positions of the five-membered ring system.
Substitution of the 6-position of the thieno[2,3-d]-
pyrimidines scaffold with a variety of carboxamides (Table 4)
Figure 3. Rationale for the evolution of structures from screening
hit 4a to 1, 2, and a variety of 6,5-fused heterocyclic ring systems.
Table 4. SAR of the Thieno[2,3-d]pyrimidines
a Cytotoxicity (EC50, μM) on parental Ba/F3. b Antiproliferative activity (EC50, μM) on wtBcr-Abl-Ba/F3.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6941
Table 5. SAR of 7-Substituted Pyrrolo[2,3-d]pyrimidines
a Cytotoxicity (EC50, μM) on parental Ba/F3. b Antiproliferative activity (EC50, μM) on wtBcr-Abl-Ba/F3.
resulted in the identification of several compounds with EC50
values of approximate 1 μM for the inhibition of cellular Bcr-
Abl activity. The optimal compound in this series was
obtained by introduction of an N-methylpiperazine as
exemplified by compound 14d, which possessed an EC50 of
48 nM (Table 4). Interestingly this compound was approxi-
mately 10-fold more potent than the compound lacking
N-methylation (14c).
Preparation of six 7-substituted pyrrolo[2,3-d]pyrimidines
resulted in the optimal activity being identified from mor-
pholinoethyl (16c) substituent (Table 5). Inhibitor 16c, which
can be viewed as the ring fused analogue of 4a, is approxi-
mately equipotent to 4a.
We prepared a 20-membered focused library for 1- and
2-substituted pyrazolo[3,4-d]pyrimidines (Table 6). The
potency of the 1- or 2-substituted regioisomers was typically
similar with the biggest difference being observed for the
benzyl substituted compound 21f where the 2-substituted
compound is completely inactive (Table 6). A variety of
compounds were discovered that possessed potency compar-
able to that of 1 such as alkyl substituted inhibitors 21a-f.
The most potent inhibitor in this series was tert-butyl sub-
stituted compound 21j-I which possessed an EC50 of 160 nM,
even though compound 21j-I inhibited the growth of
untransformed Ba/F3 cells to some degree (EC50 = 7.3 μM),
suggesting additional cellular targets for this compound
class.
Biological Profiling of Select 1 Analogues. We selected
eight compounds that possessed optimal Bcr-Abl potency
and that were representative of the different structure classes
and tested them in a panel of biochemical and cellular assays
to assess their mechanism of action relative to 1. Because
several of the compounds are structurally similar to ATP-
competitive kinase inhibitors, we sought to investigate their
potential to interact with diverse kinase ATP-binding sites.
This was assessed using KINOMEscan (Ambit Biosciences,
San Diego, CA), a high-throughput method for screening
kinase inhibitors against a panel of 353 kinases.18 Com-
pounds were screened at 10 μM; none of the compounds
displayed the ability to target the ATP-binding site of any
kinases in the panel. In order to demonstrate that these new
compounds indeed function as myristate-site ligands, we
tested their ability to compete for binding to Abl kinase
domain with a fluorescein-linked 1 analogue using a fluore-
scence polarization assay (Figure 4).19 By performing this
assay at a range of inhibitor concentrations, we could measure
a dissociation constant (Kd) for each compound (Figure 4B).
We demonstrated that all compounds were competitive with
respect to 1-FITC for binding to the myristate binding site.
In addition, there was a good correlation between the EC50
for inhibition of cellular Bcr-Abl activity and Kd determined
using the fluorescence polarization assay (Figure 4C). This
provides evidence that changes in cellular potency that we
observe for these analogues are likely to reflect the affinity
with which they bind to the myristate binding site rather than
differences in cell penetration and accumulation. All analo-
gues were also unable to inhibit the proliferation of two point
mutants located in the myristate binding site (A337N and
A344L) that we had previously8 demonstrated induced resis-
tance to 1. This provides evidence that the cellular site of action
of these analogues is also the Bcr-Abl myristate binding site.
Cumulatively, these results are consistent with these new
compounds exploiting the same mechanism of action as 1 to
inhibit Bcr-Abl dependent proliferation.
To ascertain the ability of these compounds to inhibit
clinically relevant Bcr-Abl mutants, they were tested against
T315I, E255K, and E505K mutants (Table 7).20
The results revealed that most of the compounds have
submicromolar potency against wild-type and E255K with
the exception of 14d, 16c, and 21a-I. As expected, all the
compounds are inactive on gate-keeper mutant T315I and
myristate bind site mutant E505K. As we have previously
reported that 2 is capable of synergizing with ATP-compe-
titive inhibitors such as nilotinib to inhibit the proliferation
of T315I Bcr-Abl, we tested the potential of selected com-
pounds to exhibit synergistic inhibition with the type-I ATP
competitive inhibitor dasatinib (Table 8).9 Interestingly the
compounds exhibited considerable differences in their ability

6942 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Table 6. SAR of 1- and 2-Substituted Pyrazolo[3,4-d]pyrimidines
Deng et al.
a Cytotoxicity (EC50, μM) on parental Ba/F3. b Antiproliferative activity (EC50, μM) on wtBcr-Abl-Ba/F3.
to combine with dasatinib to inhibit T315I Bcr-Abl-depen-
dent proliferation. In addition, the calculated combination
indices (CI)21 did not correlate with the ability of the com-
pounds to inhibit wild-type Bcr-Abl as single agents. Under-
standing the mechanistic basis for this will require a structure
of Abl ligated with both dasatinib and a myristate-site ligand.
(EC50 = 160 nM), which are still less potent than highly
optimized ATP-competitive ligands such as dasatinib (EC50
=
20 nM) and nilotinib (EC50 = 40 nM) under our assay con-
ditions. We demonstrate that a good correlation exists
between the binding affinity for the myristate binding site and
potency as a cellular Bcr-Abl inhibitor. Although further
optimization of the binding affinity 1 class of ligand may be
possible, because the mechanism of inhibition of Bcr-Abl is
allosteric, it maybe limited by something other than myristate
ligand affinity. We have also discovered that different myri-
state ligands display a differential ability to synergize with
dasatinib to inhibit the T315I Bcr-Abl mutant. For example,
potent wild-type inhibitor6a (EC50 = 90 nM) does notexhibit
synergy with dasatinib against T315I Bcr-Abl while the less
potent inhibitor 21a-I does. The mechanistic basis for this
effect is currently under investigation. The work presented
here demonstrates that a variety of promising lead com-
pounds can be identified that bind potently to the myristate
binding site and inhibit cellular Bcr-Abl activity. The ability of
these compounds to synergize with ATP-competitive inhibi-
tors to inhibit the growth of cells transformed with Bcr-Abl
mutants and likely to prevent or delay the emergence of
resistance mutations should encourage this new therapeutic
Conclusions
We have presented a comprehensive exploration of Bcr-Abl
myristate targeted ligands derived from 1/4a lead structures.
We demonstrate that the 4-trifluoromethoxyaniline is an
obligate structural feature for achieving submicromolar
potency as a cellular Bcr-Abl inhibitor but that the pyrimidine
ring can be elaborated to include a variety of other hetero-
cyclic ring systems. We identify several analogues with 5- to
10-fold improved potency against wild-type and some Bcr-
Abl mutants such as E255K relative to 1. The most potent
myristate-site targeted inhibitors we have discovered to date
are sulfonamide 5g (EC50 = 120 nM), reverse methylsulfo-
namide 5h (EC50 = 40 nM), pyrazole-compound 6a (EC50 =
90 nM), 6-methylpiperazine amide thieno[2,3-d]pyrimidine
14d (EC50 = 48 nM), and pyrazolo[3,4-d]pyrimidines 21j-I

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6943
Figure 4. Dissociation constants (Kd) for a series of 1 analogues correlate with EC50 for inhibition of cellular Bcr-Abl activity. (A) Chemical
structure of 1-FITC tracer. (B) Dissociation constants (Kd) were determined using a fluorescence polarization competition binding assay to
Abl kinase domain with 1 analogues and fluorescein-linked 1 tracer. (C) Graph showing the correlation between compound dissociation
constant (Kd) and its cellular EC50 value for Bcr-Abl inhibition.
Table 7. Cellular Activity of Select 1 Analogues against Some Clinically
Relevant Bcr-Abl Mutantsa
chemical shifts are reported in parts per million (ppm, δ) down-
field from tetramethylsilane (TMS). Coupling constants (J) are
reported in Hz. Spin multiplicities are described as s (singlet), brs
(broad singlet), t (triplet), q (quartet), and m (multiplet). Mass
spectra were obtained on a Waters Micromass ZQ instrument.
Preparative HPLC was performed on a Waters Symmetry C18
column (19 mm ꢀ 50 mm, 5 μM) using a gradient of 5-95%
acetonitrile in water containing 0.05% trifluoacetic acid (TFA)
over 8 min (10 min run time) at a flow rate of 30 mL/min. Purities
of assayed compounds were in all cases greater than 95%, as
determined by reverse-phase HPLC analysis.
cellular EC50 (μM)
compd
Wt-Bcr-Abl
T315I
E255K
E505K
Wt-Ba/F3
1
0.14
0.43
0.12
0.04
0.09
0.05
0.38
0.25
0.16
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.60
0.58
0.31
0.36
0.52
1.33
1.22
1.61
0.50
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2
5g
5h
6a
14d
16c
21a-I
21j-I
Cell Culture and Cell Proliferation Assay. Ba/F3.p210 cells
were obtained by transfecting the IL-3-dependent murine
hematopoietic Ba/F3 cell line with a pEYK vector containing
p210BCR-ABL and Bcr-Abl mutations.9,27 All cell lines were
cultured with 5% CO2 at 37 ꢀC in RPMI 1640 (Invitrogen) with
10% fetal bovine serum (FBS) and supplemented with 1%
L-glutamine. Parental Ba/F3 cells were similarly cultured with
10% WEHI-conditioned medium as a source of IL-3. Trans-
fected cell lines were cultured in media supplemented with 25 μg/
mL zeocin. The 48 h cell proliferation studies were obtained
using the CellTiter-Glo assay (Promega) as previous described.8
For drug combination studies, compounds were added
simultaneously at fixed ratios to cells, and cell viability was
determined by CellTiter-Glo assay (Promega) and expressed as
a function of growth affected (FA) drug-treated cells versus
control cells. Synergy was assessed by Calcusyn software
(Biosoft; Ferguson, MO, and Cambridge, U.K.), using the
Chou-Talalay method.21 The combination index CI indicates
the strength of the synergy effect. Values less than 1 indicate
synergy, whereas values greater than 1 indicate antagonism.
Procedure for the Synthesis of N4-(3-Morpholinopropyl)-N6-
(4-(trifluoromethoxy)phenyl)pyrimidine-4,6-diamine (4b). To a
stirred solution of 4,6-dichloro-pyrimidine (298.0 mg, 2.0 mmol)
and 4-trifluoromethoxyaniline (0.27 mL, 2.0 mmol) in 1.0 mL of
EtOH was added DIEA (0.35 mL, 2.0 mmol). The reaction
mixture was stirred for 16 h at 80 ꢀC. Then the mixture was
concentrated and purified by silica gel column chromatography
to give the title compound 3 (521 mg, 90% yield). MS (ESI) m/z
290 (M þ H)þ.
a Antiproliferative activity (EC50, μM) on select cell lines.
Table 8. Synergistic Effect of Select 1 Analogues with Dasatinib against
T315I Mutant
CI value at
compd (ratio
ED50
ED75
ED90
Dm
r
dasatinib þ 5g (1:1)
dasatinib þ 5h (1:1)
dasatinib þ 6a (1:1)
dasatinib þ 14c (1:1)
dasatinib þ 14d (1:1)
dasatinib þ 20a (1:1)
0.4973 0.27007 0.48539 0.99256 0.94707
0.61781 0.38123 0.25814 0.90849 0.55459
0.54179 0.71033 0.95607 0.77202 0.94716
0.75652 0.70471 0.65645 0.41015 0.90394
0.30119 0.13126 0.11157 0.96888 0.50907
0.40752 0.35515 0.47618 0.96398 2.23613
dasatinib þ 21a-I (1:1) 0.70277 0.36185 0.48377 1.30175 0.76679
modality to be explored clinically. As a variety of kinases such
as PKC,22 mTor,23 Arg,24 and SK125 and Akt26 have been
demonstrated toberegulated throughinter- orintramolecular
interactions with lipids, screening this compound collection
for allosteric inhibitors of lipid binding sites may prove
valuable.
Experimental Section
Chemistry. Unless otherwise noted, reagents and solvents
were obtained from commercial suppliers and were used without
further purification. 1H NMR spectra were recorded at 400 MHz
(Bruker XWIN-NMR) or 600 MHz (Varian AS600), and
To a stirred solution of compound 3 (29 mg, 0.10 mmol) and
DIEA (35 μL, 0.20 mmol) in 1 mL of sec-BuOH was added
3-morpholinopropan-1-amine (29 μL, 0.20 mmol) at room

6944 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Deng et al.
temperature. Then the resulting mixture was stirred at 120 ꢀC.
After the reaction was complete as monitored by reverse phase
analytical liquid chromatography-electrospray mass spectro-
metry (LC-MS), the solvent was removed and the title com-
pound was purified by reverse-phase preparative HPLC using
a water (0.05% TFA)/acetonitrile (0.05% TFA) gradient to
afford the title compound 4b as the TFA salt (30 mg, 59%). 1H
NMR (600 MHz, CD3OD) δ 8.26 (s, 1H), 7.50 (d, J = 7.2 Hz,
2H), 7.34 (d, J = 8.4 Hz, 2H), 5.92 (s, 1H), 4.03 (brs, 2H), 3.79
(brs, 2H), 3.47 (brs, 4H), 3.26-3.23 (m, 2H), 3.14 (brs, 2H),
2.12-2.08 (m, 2H). MS (ESI) m/z 398 (M þ H)þ. HRMS (ESI)
calcd for C18H22F3N5O2 397.1726, found 398.1797 (M þ H)þ.
Procedure for the Synthesis of N-(2-Hydroxyethyl)-3-(6-(4-
(trifluoromethoxy)phenylamino)pyrimidin-4-yl)benzamide (2). A
mixture of 6-chloro-N-(4-(trifluoromethoxy)phenyl)pyrimidin-
4-amine (200 mg, 0.69 mmol), 3-carboxyphenylboronic acid
(115 mg, 0.69 mmol), Pd(PPh3)4 (40 mg, 0.034 mmol), and
sodium carbonate (292 mg, 2.76 mmol) in acetonitrile/water
(v/v = 1/1, 10 mL) was heated to 90 ꢀC under an argon atmo-
sphere. After refluxing for 8 h, the hot reaction mixture was
filtered. The filtrate was cooled to room temperature and treated
with 6 N HCl (pH is around 4) to afford 3-(6-(4-(trifluoro-
methoxy)phenylamino)pyrimidin-4-yl)benzoic acid as a pale
yellow precipitate, which was collected and washed with water
and air-dried to give 202 mg (78% yield) of the title com-
pound. MS (ESI) m/z 376 (M þ H)þ.
LiOH (25.5 mg, 1.07 mmol) was dissolved in THF (4 mL) and H2O
(1 mL). The mixture was stirred at 60 ꢀC for 3 h. Acidification with
acetic acid resulted in a brown solid which was collected by
filtration. Compound 13 was then dried in vacuo and used for the
next step without any further purification (115 mg, 79% yield). 1H
NMR (400 MHz, DMSO-d6) δ10.13 (s, 1H), 8.97 (brs, 1H), 8.61 (s,
1H), 8.00 (d, J = 9.2 Hz, 2H), 7.42 (d, J = 9.2 Hz, 2H). MS (ESI)
m/z 356 (M þ H)þ.
4-(4-(Trifluoromethoxy)phenylamino)thieno[2,3-d]pyrimidine-
6-carboxylic acid (30 mg, 0.08 mmol) was mixed with DIEA
(30 μL, 0.172 mmol) and HATU (39 mg, 0.1 mmol) in 2 mL of
DMF at room temperature. 2-Aminoethanol (6.1 mg, 0.1 mmol)
was added to the reaction mixture 0.5 h later. After being stirred
at room temperature for 2 h, the reaction mixture was concen-
trated and purified by preparative HPLC to afford the title
compound 14a as a TFA salt (25 mg, 61% yield). 1H NMR (400
MHz, DMSO-d6) δ 10.10 (s, 1H), 9.26 (s, 1H), 8.82 (s, 1H), 8.21
(d, J = 8.8 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.34 (s, 1H), 3.02 (t,
J = 6.4 Hz, 2H), 2.65 (t, J = 6.4 Hz, 2H). MS m/z 399 (M þ H)þ.
Procedure for Synthesis of N-(4-(Trifluoromethoxy)phenyl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine (16a). To a stirred solution
of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (46.1 mg, 0.3 mmol)
and DIEA (78 μL, 0.45 mmol) in 2.0 mL of 2-BuOH was added
4-trifluoromethoxyaniline (79.7 mg, 0.45 mmol) at room tem-
perature. Then the mixture was heated to 120 ꢀC. After the
reaction was complete, as monitored by LC-MS, the solvent
was removed and purified by preparative HPLC to afford the
title compound 16a (66.6 mg, 60% yield). 1H NMR (400 MHz,
DMSO-d6) δ 11.42 (s, 1H), 9.58 (s, 1H), 8.52 (s, 1H), 7.76 (d, J =
9.2 Hz, 2H), 7.24 (d, J = 9.2 Hz, 2H), 7.39 (d, J = 6.2 Hz, 1H),
6.52 (dd, J = 3.2, 1.6 Hz, 1H). MS (ESI) m/z 295 (M þ H)þ.
Procedure for Synthesis of N-(3-Methoxypropyl)-3-(4-(4-
(trifluoromethoxy)phenylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-
propanamide (20a). To a stirred solution of 4-chloro-7H-pyrrolo-
[2,3-d]pyrimidine (1.0 g, 6.51 mmol) in DMF (30 mL) was added
NaH (60% in mineral oil) (391 mg, 9.76 mmol) at 0 ꢀC. After the
mixture was stirred for 30 min at room temperature, methyl
3-bromopropanoate (1.20 g, 7.18 mmol) was added and the
resulting mixture was stirred for 16 h at 40 ꢀC. Then the reaction
was quenched with saturated NH4Cl solution and extracted with
ethyl acetate. The organic extracts were washed with brine, dried
over Na2SO4, and concentrated. The residue was purified by silica
gel column chromatography to afford compound 17 (1.2 g, 77%
yield). MS m/z 240 (M þ H)þ.
To a solution of 3-(6-(4-(trifluoromethoxy)phenylamino)-
pyrimidin-4-yl)benzoic acid (56.3 mg, 0.15 mmol), ethanol-
amine (27 μL, 0.45 mmol), and diisopropylethylamine (53 μL,
0.30 mmol) in 1.50 mL of dimethylformamide was added 1-hydroxy-
1H-benzotriazole “HOBt” (30 mg, 0.22 mmol) at room tempera-
ture. Then the mixture was cooled to 0 ꢀC and (3-(dimethyla-
mino)propyl)ethylcarbodiimide hydrochloride “EDCI” (52 mg,
0.27 mmol) was added. And the mixture was warmed to room
temperature gradually. After the reaction was completed, as
monitored by LC-MS, the reaction mixture was poured into
water and extracted with ethyl acetate. The organic layer was
washed with brine, dried over anhydrous Na2SO4, concentrated,
and purified by silica gel column chromatography to afford 2 (50
mg, 80% yield). 1H NMR (600 MHz, DMSO-d6), δ 9.92 (s, 1H),
8.75 (s, 1H), 8.60 (t, J = 5.4 Hz, 1H), 8.51 (s, 1H), 8.17 (d, J =
7.8 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.84 (d, J = 9.0 Hz, 2H),
7.61 (t, J = 7.8 Hz, 1H), 7.34 (d, J = 9.0 Hz, 2H), 7.33 (s, 1H),
4.73 (t, J = 5.4 Hz, 1H), 3.54 (t, J = 6.0 Hz, 2H), 3.36 (t, J = 6.0
Hz, 2H). MS (ESI) m/z 419 (M þ H)þ.
To a solution of 3-(4-chloro-pyrrolo[2,3-d]pyrimidin-7-yl)-
propionic acid methyl ester (100 mg, 0.42 mmol) and DIEA
(0.11 mL, 0.63 mmol) in sec-BuOH (3 mL) was added 4-tri-
fluoromethoxyaniline (80 mg, 0.45 mmol). The resulting mixture
was stirred at 120 ꢀC for 2 h. Then the mixture was concentrated
and purified by silica gel column chromatography to afford
compound 18 (87.5 mg, 55% yield). MS m/z 381 (M þ H)þ.
To a solution of 3-[4-(4-trifluoromethoxyphenylamino)pyrrolo-
[2,3-d]pyrimidin-7-yl]propionic acid methyl ester (87.5 mg, 0.22
mmol) in 5 mL of mixed solvent THF/H2O (v/v = 3/1)) was
added Cs2CO3 (78 mg, 0.40 mmol). The resulting mixture was
stirred at 80 ꢀC for 2 h. The mixture was concentrated and
neutralized with acetic acid. The solid was filtered and dried to
yield compound 19 as a brown solid (62 mg, 77%). MS m/z 367
(M þ H)þ.
Procedure for Synthesis of Methyl 4-(4-(Trifluoromethoxy)-
phenylamino)thieno[2,3-d]pyrimidine-6-carboxylate (11). To a
stirred solution of 4,6-dichloropyrimidine-5-carbaldehyde (1.0 g,
5.65 mmol) in 10 mL of THF was added 4-trifluoromethoxyani-
line (0.72 mL, 5.36 mmol) at -10 ꢀC. After 10 min, the mixture
was stirred at room temperature for 5 h. Then the mixture was
diluted by ethyl acetate and filtered through Celite. The filtrate
was concentrated and purified by silica gel column chromatography
to give compound 10 (1.13 g, 63% yield). MS m/z 318 (M þ H)þ.
4-Chloro-6-(4-trifluoromethoxyphenylamino)pyrimidine-5-
carbaldehyde 10 (500 mg, 1.57 mmol) in DMF (2 mL) was added
to a suspension of K2CO3 (620 mg, 4.5 mmol) in DMF (1 mL) at
room temperature. After the mixture was stirred for 30 min,
methyl thioglucolate (210 mg, 1.9 mmol) was added slowly to
the reaction mixture. The resulting mixture was heated at 90 ꢀC
for 1 h. The reaction mixture was then cooled at room tempera-
ture and poured into ice-water. The precipitate was collected
and dried to afford the title compound 11 (400 mg, 69% yield).
1H NMR (600 MHz, CDCl3) δ 8.64 (s, 1H), 8.16 (s, 1H), 7.78 (d,
J = 9.0 Hz, 2H), 7.25 (d, J = 9.0 Hz, 2H), 3.92 (s, 3H). MS m/z
370 (M þ H)þ.
3-[4-(4-Trifluoromethoxyphenylamino)pyrrolo[2,3-d]pyrimidin-
7-yl]propionic acid (25 mg, 0.068 mmol) was mixed with DIEA
(0.030 mL, 0.172 mmol) and HATU (28.5 mg, 0.075 mmol) in
1.0 mL of DMF at room temperature. 3-Methoxypropan-1-
amine (12.1 mg, 0.14 mmol) was added into the reaction mixture
0.5 h later. After being stirred at room temperature for 2 h, the
mixture was concentrated and purified by preparative HPLC to
afford the title compound 20a (17.8 mg, 60% yield). 1H NMR
(600 MHz, CDCl3) δ 11.53 (s, 1H), 8.29 (s, 1H), 7.44 (d, J = 8.4
Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 3.0 Hz, 1H), 6.23
(s, 1H), 5.46 (d, J = 2.4 Hz, 1H), 4.56 (t, J = 6.0 Hz, 4H), 3.43
Procedure for the Synthesis of N-(2-Hydroxyethyl)-4-(4-(tri-
fluoromethoxy)phenylamino)thieno[2,3-d]pyrimidine-6-carboxamide
(14a). A mixture of methyl 4-(4-(trifluoromethoxy)phenylamino)-
thieno[2,3-d]pyrimidine-6-carboxylate (152 mg, 0.41 mmol) and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6945
(t, J = 6.0 Hz, 2H), 3.30 (s, 3H), 2.72 (t, J = 6.0 Hz, 2H), 1.70
(quintet, J = 6.0 Hz, 2H). MS (ESI) m/z 438 (M þ H)þ.
Procedure for Synthesis of 2-(4-(4-(Trifluoromethoxy)pheny-
lamino)-1H-pyrazolo-[3,4-d]pyrimidin-1-yl)ethanol (21a-I) and
2-(4-(4-(Trifluoromethoxy)phenylamino)-2H-pyrazolo[3,4-d]pyri-
midin-2-yl)ethanol (21a-II).28 To a solution of 4-chloro-6-(4-
trifluoromethoxyphenylamino)pyrimidine-5-carbaldehyde (50 mg,
0.16 mmol) in 2.0 mL of sec-BuOH was added Na2CO3 (30 mg,
0.28 mmol) and (2-hydroxyethyl)hydrazine (20 mg, 0.26 mmol).
The mixture was stirred at 60 ꢀC for 8 h. The reaction mixture
was partitioned in 50 mL of water and 50 mL of ethyl acetate.
The aqueous layer was extracted with ethyl acetate three times.
The combined organic extracts were washed with brine, dried
over anhydrous Na2SO4, and concentrated. The crude product
was purified by silica gel column chromatography to give title
compounds 21a-I (20 mg) and 21a-II (5.0 mg).
and the concentration of competing ligand required to displace
half of the bound 1-FITC ligand was determined (IC50).
Acknowledgment. We thank the members from Nathanael
S. Gray laboratory for experimental assistance and insightful
comments throughout this study. Some of the cellular and
biochemical assays were performed at the ICCB at Harvard
MedicalSchoolwithhelpfromAndrewDaab,SeanM.Johnston,
and Stewart Rudnicki. The Gray laboratory acknowledges the
generous financial support from Novartis and NIH Grant R01
CA130876-02 and useful discussions with Michael Eck, Markus
Warmuth, Paul Manley, Doriano Fabbro, and John Engen.
Supporting Information Available: Spectral data of 4a,c-e,
5a-k, 6a-f, 7a-d, 8a-g, 9a-l, 12, 14b-g, 16b-d, 20b, 21b-f,
21h-l, and 22; pharmacokinetic parameters of compounds 5g,
6a, 14d, and 21b-I; calculations of relative transformed energy
from trans to cis (4a, 9a, 9c, 9l); fluorescence polarization assay
results of additional compounds (5g, 7a, 12, 21a-I). This
material is available free of charge via the Internet at http://
pubs.acs.org.
21a-I: 1H NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1H), 8.45 (s,
1H), 8.32 (s, 1H), 7.98 (d, J = 9.6 Hz, 2H), 7.41 (d, J = 9.6 Hz,
2H), 4.40 (t, J = 6.0 Hz, 2H), 3.83 (t, J = 6.0 Hz, 2H). MS (ESI)
m/z 340 (M þ H)þ.
21a-II: 1H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 8.68
(s, 1H), 8.57 (s, 1H), 7.97 (d, J = 9.6 Hz, 2H), 7.45 (d, J = 9.6
Hz, 2H), 4.41 (t, J = 6.0 Hz, 2H), 3.85 (t, J = 6.0 Hz, 2H). MS
(ESI) m/z 340 (M þ H)þ.
References
Procedure for Synthesis of 1-(Pyridin-3-ylmethyl)-N-(4-(trifluo-
romethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (21g-I)
and 2-(Pyridin-3-ylmethyl)-N-(4-(trifluoromethoxy)phenyl)-2H-
pyrazolo[3,4-d]pyrimidin-4-amine (21g-II). To a stirred solution
of N-(4-(trifluoromethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-
4-amine (60 mg, 0.2 mmol) in 3.0 mL of DMF was added NaH
(60% in mineral oil) (32 mg, 0.8 mmol) at 0 ꢀC. After the mixture
was stirred for 1 h, 3-(bromomethyl)pyridine hydrobromide (48
mg, 0.19 mmol) was added. The resulting mixture was warmed
to room temperature gradually. After the reaction was com-
plete, as monitored by LC-MS, the mixture was poured into
ice-water and extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over anhydrous
Na2SO4, and concentrated. The crude product was purified by
preparative TLC to give the title compounds.
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21g-I: 1H NMR (600 MHz, CD3OD) δ 8.54 (s, 1H), 8.44 (d,
J = 4.8 Hz, 1H), 8.43 (s, 1H), 8.18 (brs, 1H), 7.89 (d, J = 9.0 Hz,
2H), 7.77 (dt, J = 1.8, 7.8 Hz, 1H), 7.38 (dd, J = 4.8, 7.8 Hz, 1H),
7.28 (d, J=8.4 Hz, 2H), 5.64(s, 2H). MS(ESI) m/z387 (M þ H)þ.
21g-II: 1H NMR (600 MHz, CD3OD) δ 8.62 (d, J = 1.8 Hz,
1H), 8.52 (dd, J = 1.2, 4.8 Hz, 1H), 8.47 (brs, 1H), 8.38 (s, 1H),
7.90 (d, J = 8.4 Hz, 2H), 7.86 (dt, J = 1.8, 7.8 Hz, 1H), 7.45
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