9-(Arenethenyl)purines as dual Src/Abl kinase inhibitors targeting the inactive conformation: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1021/jm900166t

A novel series of potent dual Src/Abl kinase inhibitors based on a 9-(arenethenyl)purine core has been identified. Unlike traditional dual Src/Abl inhibitors targeting the active enzyme conformation, these inhibitors bind to the inactive, DFG-out conformation of both kinases. Extensive SAR studies led to the discovery of potent and orally bioavailable inhibitors, some of which demonstrated in vivo efficacy. Once-daily oral administration of inhibitor 9i (AP24226) significantly prolonged the survival of mice injected intravenously with wild type Bcr-Abl expressing Ba/F3 cells at a dose of 10 mg/kg. In a separate model, oral administration of 9i to mice bearing subcutaneous xenografts of Src Y527F expressing NIH 3T3 cells elicited dose-dependent tumor shrinkage with complete tumor regression observed at the highest dose. Notably, several inhibitors (e.g., 14a, AP24163) exhibited modest cellular potency (IC₅₀ = 300-400 nM) against the Bcr-Abl mutant T315I, a variant resistant to all currently marketed therapies for chronic myeloid leukemia. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900166t

J. Med. Chem. 2009, 52, 4743–4756 4743
DOI: 10.1021/jm900166t
9-(Arenethenyl)purines as Dual Src/Abl Kinase Inhibitors Targeting the Inactive Conformation:
Design, Synthesis, and Biological Evaluation
Wei-Sheng Huang,*,† Xiaotian Zhu,† Yihan Wang,† Mohammad Azam,‡ David Wen,† Raji Sundaramoorthi,†
R. Mathew Thomas,† Shuangying Liu,† Geetha Banda,† Scott P. Lentini,† Sasmita Das,† Qihong Xu,† Jeff Keats,†
Frank Wang,† Scott Wardwell,† Yaoyu Ning,† Joseph T. Snodgrass,† Marc I. Broudy,† Karin Russian,† George Q. Daley,‡
John Iuliucci,† David C. Dalgarno,† Tim Clackson,† Tomi K. Sawyer,† and William C. Shakespeare†
†ARIAD Pharmaceuticals, Inc., 26 Landsdowne Street, Cambridge, Massachusetts 02139, and ‡Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Division of Hematology/Oncology, Brigham and Women’s Hospital, Dana-Farber
Cancer Institute, and Division of Hematology/Oncology, The Children’s Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115
Received February 9, 2009
A novel series of potent dual Src/Abl kinase inhibitors based on a 9-(arenethenyl)purine core has been
identified. Unlike traditional dual Src/Abl inhibitors targeting the active enzyme conformation, these
inhibitors bind to the inactive, DFG-out conformation of both kinases. Extensive SAR studies led to the
discovery of potent and orally bioavailable inhibitors, some of which demonstrated in vivo efficacy.
Once-daily oral administration of inhibitor 9i (AP24226) significantly prolonged the survival of mice
injected intravenously with wild type Bcr-Abl expressing Ba/F3 cells at a dose of 10 mg/kg. In a separate
model, oral administration of 9i to mice bearing subcutaneous xenografts of Src Y527F expressing
NIH 3T3 cells elicited dose-dependent tumor shrinkage with complete tumor regression observed at
the highest dose. Notably, several inhibitors (e.g., 14a, AP24163) exhibited modest cellular potency
(IC50 = 300-400 nM) against the Bcr-Abl mutant T315I, a variant resistant to all currently marketed
therapies for chronic myeloid leukemia.
Introduction
inhibited with second-generation inhibitors dasatinib and
nilotinib, although neither compound inhibits the T315I
mutant, which represents ∼15-20% of all clinically observed
mutants. Currently, there are no approved pharmaceutical
therapies for CML patients harboring this mutation, and
development of T315I targeted inhibitors addresses a signifi-
cant unmet medical need.
Chronic myeloid leukemia (CMLa) is characterized by a
reciprocal translocation between chromosome 9 and 22,
resulting in the short Philadelphia (Ph) chromosome1 carrying
the Bcr-Abl (Breakpoint cluster region-Abelson leukemia)
oncogene.2 This oncogene encodes the chimeric Bcr-Abl
fusion protein, which incorporates an activated Abl tyrosine
kinase domain and is responsible for the initial phase of
CML.3 In 2001, imatinib, a potent Bcr-Abl inhibitor, was
approved for the treatment of CML.4 Despite its tremendous
clinical success, imatinib is relatively ineffective against
advanced phases of CML and Philadelphia chromosome-
positive acute lymphoblastic leukemia (Phþ ALL).5 Muta-
tions in the kinase domain of Bcr-Abl are the major mechan-
ism of acquired imatinib resistance.6 To date, at least 50
different point mutations that encode distinct single amino
acid substitutions have been identified in CML patients
who are resistant to imatinib. Most mutants are effectively
Although the most widely accepted mechanism for imatinib
resistance is the presence of kinase domain mutations, several
studies have provided a rationale for the use of dual Src/Abl
kinase inhibitors to overcome imatinib resistance.7 For ex-
ample, overexpression of Src family kinases (SFK) has re-
cently been implicated in Bcr-Abl mediated leukemogenesis,
particularly the induction of B-cell acute lymphoblastic leu-
kemia (B-ALL), and also in CML disease progression.8
Beyond CML, a recent study demonstrated endogenous Abl
kinases (c-Abl and Arg) are activated by deregulated EGFR
and Src kinases, driving invasion of aggressive breast cancer
cells9 and further highlighting thepotentialexpandedutility of
dual Src/Abl inhibitors to this solid tumor indication.10 More-
over, Src activation or overexpression itself has been demon-
strated in a variety of human tumor types, including colon,
breast, pancreas, lung, liver, brain, and bladder cancers.11
Taken together, dual Src/Abl kinase inhibitors, targeting
either kinase independently or simultaneously, possess signif-
icant therapeutic potential for the treatment of both solid and
hematologic malignancies. In 2006, dasatinib, a potent dual
Src/Abl inhibitor, was approved for the treatment of adults in
all phase of CML with resistance or intolerance to imatinib,12
and there are now multiple dual Src/Abl kinase inhibitors in
*To whom correspondence should be addressed. Phone: 617-621-
2341. Fax: 617-494-8144. E-mail: wei-sheng.huang@ariad.com.
a Abbreviations: CML, chronic myeloid leukemia; Bcr-Abl, break-
point cluster region-abelson; Phþ ALL, Philadelphia chromosome-
positive acute lymphoblastic leukemia; Src/Abl, sarcoma /abelson;
SFK, Src family kinases; B-ALL, B-cell acute lymphoblastic leukemia;
Arg, Abl-related gene; EGFR, epidermal growth factor receptor; Lck,
leukocyte-specific protein tyrosine kinase; c-Kit, cytokine receptor;
PDGF, platelet-derived growth factor; P-loop: phosphate binding loop;
ATP, adenosine-50-triphosphate; DFG, aspartic acid-phenylalanine-
glycine; VEGFR, vascular endothelial growth factor receptor; MAP,
mitogen-activated protein; KDR, kinase insert domain receptor; PK,
pharmacokinetic; WT, wild type.
r
2009 American Chemical Society
Published on Web 07/02/2009
pubs.acs.org/jmc

4744 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Huang et al.
Figure 1. Representative kinase inhibitors targeting the DFG-out conformation.
clinical trials for the treatment of breast cancer, pancreatic
cancer, and other solid tumors.13
Imatinib is a relatively selective kinase inhibitor. Prior to a
recent study demonstrating modest Lck inhibition (IC50=160
nM),14 imatinib was found to inhibit only three kinases: Bcr-
Abl, c-Kit, and the PDGF receptor.4 The high degree of
kinase selectivity exhibited by imatinib is, to a large extent,
attributed to its binding to the inactive conformation of Abl.
In this conformation, the glycine rich P-loop folds down over
Figure 2. Design of DFG-out dual Src/Abl inhibitors based on 1.
the ATP binding site and the activation loop adopts a con-
formation in which it occludes substrate binding and disrupts
the ATP binding site, blocking the catalytic activity of the
enzyme.15a This binding mode induces changes in the orienta-
tion of the conserved Asp-Phe-Gly(DFG)-motif atthe base of
the activation loop in which the phenylalanine residue moves
both proteins. Recently, we have reported progress on the
“DFG-in” targeted inhibitors highlighting an N-9 vinyl link-
age in a series of stable, synthetically accessible purine based
dual Src/Abl inhibitors.22 Herein we describe our work based
on the same template but targeting the “DFG-out” conforma-
tion of both proteins.
˚
more than 10 A from its position in the active conformation;
the inactive conformation is therefore also termed the “DFG-
out” conformation. The DFG-out conformation creates an
additional hydrophobic pocket adjacent to the ATP pocket.
The amino acids surrounding this pocket are less conserved
relative to those in the ATP binding pocket, which may
explain why inhibitors bound to this conformation of the
protein usually exhibit more focused kinase selectivity.15b The
chemical structure of imatinib and several other inhibitors
targeting the DFG-out conformation of Abl and other im-
portant kinases are depicted in Figure 1.
Previously, we have described 1 (AP23464, Figure 2) as a
purine-based, ATP-competitive, dual Src/Abl inhibitor that
potently inhibits Src, wildtype Bcr-Abl and a panel of Bcr-Abl
mutants (except T315I) with subnanomolar potencies.21 To
furtherprobe thepurinecoreasa template forpotent dual Src/
Abl inhibitors, alternate 2-atom linkers between the template
and a pendant hydrophobic substituent were explored. On the
basis of this concept, two parallel programs were established
targeting the “DFG-in” and “DFG-out” conformations of
Inhibitor Design
To transform 1 into a compound targeting the DFG-out
conformations of Src and Abl, we initially used Abl as the
primary target and proposed molecules 2 and 3a (Figure 2),
with the N9-hydroxyphenethyl moiety being replaced by a
privileged DFG-out targeting structural fragment, diary-
lamide.23 Crystal structures of several “DFG-out” bound
inhibitors have revealed that the amide functional group
forms crucial hydrogen bonds with the protein and that the
aryl groups fill adjacent hydrophobic pockets. Compound 3a
shares the 9-(arenethenyl)purine scaffold that was initially
designed for our “DFG-in” dual Src/Abl inhibitor series and
can also be viewed as conformationally restricted analogue of
2. To investigate the molecular interactions between these
inhibitors and Abl kinase, both compounds were docked into
an Abl model based on the DFG-out conformation to which
imatinib binds (PDB: 1iep). Side chains around the ligand site

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4745
were allowed to relax by using induced fit protocol in
the Schrodinger modeling package.24 The docked structure
reveals that inhibitor 2 forms four hydrogen bonds with the
protein. Two H-bonds are formed between the kinase hinge
region and N-7 of purine core and methylamino group at
C-1, respectively. The amide group of compound 2 forms two
additional H-bonds with the side chain of Glu286 in the RC-
helix and main chain of Asp381 in the DFG motif, respec-
tively. The trifluoromethylphenyl moiety (B ring) fills a
hydrophobic pocket that is only available in the DFG-out
conformation. Extensive van der Waals (vdw) interactions
are observed between the trifluoromethyl group and a small
pocket formed by residue Ile293, Leu298, Leu354, and
Val379. These vdw contacts between the trifluoromethyl
group and the protein have been highlighted in the design of
other DFG-out kinase inhibitors such as Abl inhibitors
nilotinib, NS-187 (Figure 1),17 and the dual Raf-KDR in-
hibitor sorafenib. Compound 3a is predicted to bind to Abl in
an identical mode to that of 2, except that the olefinic proton
(β to purine N-9) can potentially form a nonclassic hydrogen
bond with the OH group of the Thr315 gatekeeper residue
(Figure 3). Overall, inhibitors 2 and 3a were predicted to
interact with Abl in the DFG-out, inactive conformation,
analogously to imatinib and nilotinib. The cyclopentyl group
at purine C-2 in 1, which was orientated toward the ribose-
binding pocket of the ATP site,25 was not accommodated in a
DFG-out targeted inhibitor due to clashes with residues
located in the glycine-rich P-loop (such as Leu248 and Tyr
253). In fact, the docking model suggested that the space
therein was too tight to tolerate a group/atom larger than a
proton. As such, our DFG-out targeted dual Src/Abl inhibi-
tors would bear substituents at the 6,9-positions on the purine
template as opposed to the traditional 2,6,9-trisubstitution.
Figure 3. Model of designed inhibitor 3a bound to Abl kinase
Scheme 1. Preparation of 3-(Trifluoromethyl)benzamides 3a
Chemical Synthesis
a Reagents and conditions: (a) RNH2, THF, rt, 5 h; (b) 2.5 mol % Pd
(OAc)2, 5 mol % P(o-tol)3, 120 mol % (i-Pr)2NEt, DMF, 100 °C, 15 h;
(c) 3-CF3C6H4CO2H, EDCI, HOBt, THF, rt, overnight; (d) 2 equiv
ArNH2, 10 mol % Pd2(dba)3, 15 mol % PhP(c-hexyl)2, 1.5 equiv K3PO4,
μW, 150 °C, 5 or 10 min.
We postulated that inhibitor 2 could be easily accessed by
simple hydrogenation of 3a, and so the synthesis of the latter
compound became our primary objective. The synthetic
approach to 9-(arenethenyl)purine 3 described in the litera-
ture is based on the Horner-Wadsworth-Emmons (HWE)
reaction of N9-(phosphorylmethyl)purine and substituted
benzaldehydes.26 This method was employed in the initial
phase of our “DFG-in” inhibitor program but presented
several challenges in the preparation of “DFG-out” targeted
compounds because the corresponding elaborated benzalde-
hyde could only be made via a lengthy chemical transforma-
tion sequence. Other inconveniences included a multistep
synthesis of the requisite HWE reagent and the separation
of Z- andE-isomeric products. To circumvent these problems,
we have developed an efficient synthesis of 3 based on a Heck
reaction of 9-vinylpurines and aryl halides.27 As shown in
Scheme 1, 6-chloro-9-vinylpurine (4), prepared by direct
vinylation of commercially available 6-chloropurine with
excessive vinyl acetate in the presence of catalytic amount
of sulfuric acid and mercury acetate,28 underwent SNAr
displacement by aliphatic amines or palladium-catalyzed
N-arylation with aryl amines to yield 6-(aryl/alkyl)amino-9-
vinylpurines 5. Subsequent Heck reaction of 5 and iodoben-
zamide 6, which was prepared by a standard condensation of
requisite acid and aniline, furnished 3 in high yields and
exclusively as the E-isomers. Inhibitors 3a, 3b, 3g, and 3h
(Table 1) were prepared in this manner, and subsequent
hydrogenation of 3a furnished 2 quantitatively.
Inhibitors 3c, 3d, 3e, and 3f were synthesized in the reverse
order by first introducing the N9-arenethenyl moiety onto the
purine template followed by installation of the arylamino
group at C-6. Thus, Heck reaction of 4 with 6 generated a
common intermediate 7, which underwent palladium-
catalyzed N-arylation with various aryl amines to furnish
the targeted compounds.
As depicted in Scheme 2, the reverse amides 9 and the urea
analogue 11 were prepared from a similar Heck coupling of 5b
with iodobenzamides 8 and N,N0-diarylurea 10, respectively.
The precursors 8 were assembled by coupling 3-iodo-4-
methylbenzoyl chloride with a variety of aryl amines and 10
was made from the addition of 3-iodo-4-methylaniline to
3-(trifluoromethyl)phenyl isocyanate.
Scheme 3 illustrates the preparation of inhibitors that bear
further substitution on C-300 of the B ring. For the synthesis of
inhibitors 14, the imidazole rings were coupled via copper-
catalyzed N-arylations with 3-bromo-5-(trifluoromethyl)ani-
line in the presence of a bidentate ligand.29 For the prepara-
tion of 17, morpholine and 4-methylpiperazine were also
introduced in the first step of the reaction sequence but via
straightforward SNAr displacement. It is noteworthy that

4746 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Table 1. In Vitro Assay Results of Inhibitors 2 and 3 (IC50 in nM)
Huang et al.
Scheme 2. Preparation of Reverse Amides 9 and Urea 11a
trends observed from the enzymatic assays. Inhibition of
parental, nontransfected Ba/F3 cells was used as a control.
Additionally, pharmacokinetic properties for active com-
pounds were evaluated in rats to help guide series selection.
The prototype inhibitor 3a demonstrated kinase IC50s of 52
and 25 nM against Src and Abl, respectively, which validated
our initial inhibitor design and original docking model. More-
over, when 3a was tested in cells, it was remarkably potent
against both cell lines without any effect on the parental
control at concentrations up to 10 μM (Table 1). Reduction
of 3a to 2 resulted in decreased kinase and cellular activity,
suggesting the more rigid double bond may introduce bias
toward the preferred binding conformation. Interaction of the
olefinic proton with the threonine gatekeeper residue via a
pseudo hydrogen bond, as mentioned in our inhibitor design,
could also have contributed to the enhanced potency of 3a.
Preliminary pharmacokinetic assessment of compound 3a
suggested it was cleared rapidly after being dosed orally in
rats (vide infra). Previously, we have disclosed that the
enamine-like N9-(phenethenyl) moiety in the context of
DFG-in targeted dual Src/Abl inhibitors is stable in vivo.
For example, AP24149 (Figure 4, compound 10a from refer-
ence 22) demonstrated oral bioavailability of approxi-
mately 20% in rats. Therefore, we speculated that the
methylamino group at purine C-6 might be responsible for
the poor oral bioavailability because the N-phenyl 3-triflu-
oromethyl-benzamide moiety has been highlighted in other
orally bioavailable DFG-out targeted inhibitors.23 To test this
hypothesis, we substituted the methyl group with a cyclopro-
pyl ring (3b), which is metabolically more stable and therefore
less susceptible to rapid N-dealkylation. The preference for a
cyclopropyl group was also partially due to the small change
in molecular weight relative to 3a. Gratifyingly, compound 3b
displayed satisfactory pharmacokinetics with high exposure
and good oral bioavailability (F = 40%, Table 5) while
maintaining target kinase potency and cellular activity.
a Reagents and conditions: (a) (i) SOCl2, reflux, 1 h, (ii) ArNH2,
(i-Pr)2NEt, cat. DMAP, THF, rt, 2 h; (b) 2.5 mol % Pd(OAc)2, 5 mol %
P(o-tol)3, 120 mol %(i-Pr)2NEt, DMF, 100°C, 15h;(c)3-CF3C6H4NCO,
CH2Cl2, rt, 4 h.
protection of the free amino group was unnecessary under
both conditions. However, an excess of the nucleophiles was
required to drive the SNAr reaction to completion. Subse-
quent amide bond formation and Heck coupling furnished 14
and 17.
Structure-Activity Relationship (SAR)
In vitro biological evaluation of the inhibitors included
measuringtheirkinaseinhibitory activityagainstboth Src and
Abl and their cellular activity associated with Bcr-Abl inhibi-
tion using both K562 (a human derived CML cell line
expressing wild-type (WT) Bcr-Abl) and Ba/F3 cells trans-
fected with wild-type Bcr-Abl. In general, potency in both
cellular assays was comparable and consistent with SAR

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4747
Scheme 3. Preparation of Analogues 14 and 17 Bearing Additional B Ring Substitutionsa
a Reagents and conditions: (a) imidazole or 4-methylimidazole, 15 mol % 8-hydroxyquinoline, 15 mol % CuI, 110 mol % K2CO3, DMSO, 120 °C,
15 h; (b) 3-iodo-4-methylbenzoyl chloride, (i-Pr)2NEt, THF, rt, 2 h; (c) 2.5 mol % Pd(OAc)2, 5 mol % P(o-tol)3, 120 mol % (i-Pr)2NEt, DMF, 100 °C,
15 h; (d) 4 equiv morpholine or 4-methylpiperazine, 2 equiv CsOH H2O, DMSO, 120 °C, 4 h.
3
respectively, and inhibited the proliferation of both cell lines
in the single digit nM range. Additionally, this compound
demonstrated excellent selectivity relative to the control cell
line. Unfortunately, despite their increased kinase and cellular
potencies, inhibitors 3d, 3e, and 3g failed to demonstrate
satisfactory preliminary pharmacokinetics. Therefore, in
further optimization, we focused on inhibitors bearing a
cyclopropylamine at C-6 of purine template.
Next, we briefly explored the reversed amide (9) and ureido
(11) linkages connecting both pendent aryl rings at N-9 on the
Figure 4. Orally bioavailable “DFG-in” N9-(arenethenyl)purine.22
purine template (Table 2). Both linkers modeled well and were
predicted to form the requisite hydrogen bonds to the protein.
Compound 9a, which bears the same amide linkage as nilo-
tinib, was found to be slightly more potent than 3b that has a
“imatinib-like” amide. Urea 11 displayed a marked decrease
in potency against Src kinase but almost no change relative to
Abl, which may reflect different activation loop conforma-
tions adopted by the inactive forms of both Src and Abl.
Nevertheless, molecule 11 demonstrated poor cellular activity
in K562 cells (IC50 > 1 μM) and was not pursued further.
With the amide linkage optimized, we explored SAR
on the pendent B-ring in the DFG binding pocket. Simple
halogenation at either the ortho or the para position (9b and
9c) slightly reduced potency relative to 9a. Next we explored
the possibility of replacing the trifluoromethyl group with
reduced molecular weight and/or smaller hydrophobic sub-
stituents. tert-Butyl group substitution (9d) retained potency
relative to 9a, while the isopropyl analogue (9e) exhibited a 3-
6 fold decrease depending upon the enzyme. This could be
rationalized by the larger volume of the tert-butyl group
relative to isopropyl and the consequential increased van
der Waals contact in the well-defined hydrophobic pocket.
Nevertheless, the tert-butyl derivative (9d) had a higher
cLogP31 relative to 9a (6.76 vs 6.24, respectively), which we
viewed as undesirable. To counterbalance this, we explored
incorporating heteroatoms through several commercially avail-
able five-membered heteroaryls bearing tert-butyl substituents.
Encouraged by these data demonstrating that larger, lipo-
philic molecules like 3b can be orally bioavailable, we focused
on improving both the kinase and cellular potency through
further SAR exploration at C-6 on the purine template. As
shown in Table 1, several arylamines increased kinase activity
while an alkyl derivative (3h) demonstrated reduced potency.
This suggested the aryl rings might beengagedin hydrophobic
orπ stacking interactionswithresidueslining the hinge region.
An exception was pyridine-2-ylamine (3c), which reduced
kinase potency by about 50%. A tentative explanation for
this observation is that the lone pair electrons on the 2-pyridyl
N and those from N-1 on the purine template are repulsed,
disrupting the coplanarity that contributes to optimal bind-
ing. In the case of the 3-pyridyl (3d) or 4-pyridyl (3e) deriva-
tives, such electron repulsions do not exist. Instead, a
nonclassical hydrogen bond between N-1 on purine template
and the C-20 aromatic proton on the pyridine ring might help
stabilize the preferred coplanar conformation and hence,
increase the kinase activity of 3d and 3e.30 It is noteworthy
that both compounds show dramatic increases in cellular
potency with IC50s for inhibition of K562 cells in the single
digit nM range. Introduction of a (4-dimethylphosphinoyl)-
phenyl moiety, one of the key molecular recognition elements
responsible for the excellent potency observed with 1, greatly
improvedpotency inthis series as well. Compound3gpotently
inhibited Src and Abl with IC50s of 3.8 and 5.7 nM,

4748 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Table 2. In Vitro Assay Results of Inhibitors 9 and 11 (IC50 in nM)
Huang et al.
Gratifyingly, isoxazole analogue 9g (cLogP = 5.29) was
found to retain potency relative to 9a against both target
kinases. Despite this, both 9d and 9g exhibited less desirable
rat pharmacokinetics (vide infra) relative to 9a and this
series was abandoned. Further work to reduce the lipophili-
city of 9a through heteroatom incorporation ultimately led
to the discovery of 9i (cLogP=5.32), which not only main-
tained enzymatic and cell-based potency but also demon-
strated increased exposure and was essentially 100% orally
bioavailable.
The then-recent structural disclosure of nilotinib and its
increased potency relative to imatinib prompted us to make
several compounds based on this modified B ring (14a, 14b)
and, more generally, analogues in which alternative hetero-
cyclic rings were installed at the C-300 position (17) (Scheme 3
and Table 3). Inhibitors 14a and 14b retained Src and Abl
potency relative to 9i, while 17 displayed a slight decrease in
potency, but overall, the impact of substitutions meta to the
trifluoromethyl appeared to be minor.
activity. It has been reported that neither imatinib nor nilotinib
inhibits Src. The structural basis for Src inactivity of these potent
DFG-out Abl binders is still not fully understood.32 It is
generally believed that Abl can easily adopt a DFG-out con-
formation, whereas Src appears to pay a high intrinsic energetic
penalty to adopt such a binding mode, which makes it challen-
ging to design a potent DFG-out inhibitor against both Abl and
Src.33 To better understand why our DFG-out Abl inhibitors
also possess strong affinity for Src, we docked 9i into the DFG-
out conformation of Src kinase. Because of the lack of a DFG-
out Src/ligand cocrystal at the time and the very close resem-
blance of Lck and Src kinases, we built the DFG-out Src model
based on a published DFG-out structure of Lck (PDB: 2og8).34
As shown in this model (Figure 6), 9i makes two H-bonds
with the Src hinge region through purine N-7 and the
cyclopropyl amine. Two additional H-bonds are formed
from the amide group of 9i to the DFG backbone and
Glu310 of R helix C, respectively. Notice that the H-bonding
pattern in Src is essentially identical to that predicted in the
Abl model. Also, as in the Abl model, the methylphenyl
group of 9i binds to the selective hydrophobic pocket located
behind the gatekeeper residue, while the (trifluoromethyl)
pyridyl group binds to a hydrophobic pocket, which is
typically occupied by Phe405 of Src in the DFG-in confor-
mation. The similarity in H-bonding and van der Waals
(vdw) interactions of 9i with Src and Abl observed in these
models could explain the dual activities of 9i and other
related inhibitors in this chemical series against Src and Abl.
Understanding Dual Src/Abl Activities
Many inhibitors (e.g., dasatinib,12 bosutinib,13a and
AZD0530;13b Figure 5), initially designed and synthesized
for Src inhibition, were subsequently found to possess potent
Abl inhibitory activity as well. This is not unexpected because
of the high sequence homology between Src and Abl and their
similar active DFG-in conformations. However, DFG-out
Abl inhibitors do not necessarily possess potent Src inhibitory

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4749
Table 3. In Vitro Assay Results of Inhibitors 14 and 17 (IC50 in nM)
compd
Src kinase
Abl kinase
K562
WT Abl (Ba/F3)
parental (Ba/F3)
14a
14b
17a
17b
7.6 (n = 8)
8.0 (n = 1)
33 (n = 1)
41 (n = 1)
25 (n = 8)
13 (n = 1)
72 (n = 1)
24 (n = 1)
4.3 (n = 7)
5.4 (n = 3)
17 (n = 3)
14 (n = 3)
7.3 (n = 6)
4.2 (n = 3)
10 (n = 3)
11 (n = 3)
6455 (n = 6)
7120 (n = 2)
2525 (n = 1)
543 (n = 1)
the DFG-out conformation. Particularly, in this 9-(arenethe-
nyl)purine series of DFG-out inhibitors, it appears that the
Src inhibitors are more dependent on the formation of two
H-bonds to the hinge region to gain potency, whereas Abl
inhibitors bearing a single H-bond acceptor to the hinge
region can achieve the same degree of potency due to addi-
tional interactions with the Gly-rich loop.
Pharmacokinetics of Selected Compounds
Figure 5. Representative dual Src/Abl kinase inhibitors targeting
the “DFG-in” conformations.
During this program, potent inhibitors were continually
evaluated for their pharmacokinetic (PK) properties immedi-
ately following the assessment of their in vitro activity
(Table 5). We observed that the introduction of certain
functionalities consistently resulted in poor pharmacoki-
netics. Such examples included heteroarylamines (3d, 3e)
and certain 4-substituted anilines (e.g., 3g) at C-6 on the
purine core. To the contrary, compounds incorporating a
6-cyclopropylamine at the same position usually displayed
favorable PK profiles (3b, 9g, 9i, 14a). Both 9i and 14a
exhibited low rates of clearance and high oral exposure. As
a prerequisite to evaluating both compounds in in vivo
efficacy models, PK parameters for both were determined
orally in mice at three different doses; excellent dose propor-
tionality was observed for both compounds (data not shown).
Figure 6. Model of compound 9i bound to Abl (left) and Src
(right).
Although the overall binding modes are similar, structural
differences in loop conformations and ligand interactions are
present in the docking models of Abl and Src. The largest
difference resides in the conformation of the Gly-rich loops.
The Gly-rich loop in Abl appears to be more flexible than that
in Src based on a number of reported crystal structures of Abl
and Src;33,34 this loop is collapsed around bound inhibitors in
Abl and makes numerous contacts with them. In contrast, the
Gly-rich loop in Src adopts an extended conformation and
makes minimal contacts with the inhibitor, as seen in our
model and other reported crystal structures of Src (PDB: 3el7,
3el8) and its homologue Lck.33,34 The loop conformational
variation is contributed by the difference in amino acid
sequence of the Gly-rich loops, especially Phe 278 in Src and
Tyr253 in Abl, as well as their neighboring residues. The
additional interactions of the Gly-rich loop with inhibitors
make this class of compounds less sensitive to chemical
modifications in terms of binding affinity to Abl. One mod-
ification introduced to 9i was the removal of the C-6 sub-
stituent, leading to the loss of the hydrogen bond donor to
the hinge region, as exemplified by inhibitors 18a and 18b
(Table 4). Comparative kinase inhibition data indicated that
the modification had little effect on Abl potency but had a
much larger impact on Src. In fact, as shown in Table 4,
compound 18a is 10-fold less potent than 14a, while 18b is 45
times less potent than 9i in the Src kinase assay. Interestingly,
the same hydrogen bond donor to the hinge region is also
absent in imatinib and nilotinib, both of which have minimal
activities against Src. Taken together, structural differences in
Abl and Src do exist despite the overall similarity and these
differences can lead to Abl or Src specific inhibitors targeting
In Vivo Efficacy
The excellent in vitro kinase and cellular potency coupled
with desirable pharmacokinetics of inhibitor 9i prompted us
to evaluate its antitumor activity in vivo in two well-estab-
lished mouse efficacy models. In the first model, Ba/F3 cells
expressing wild-type Bcr-Abl were injected into the tail vein of
SCID mice. After 3 days, the animals were dosed orally with9i
(Figure 7) at 10 mg/kg once daily for 19 consecutive days. In
the controlgroup, median survivalwas approximately 20days
with noted splenomegaly at autopsy. In the 9i treated animals,
median survival was approximately 35 days, representing a
75% increase in overall survival relative to control. For
comparison, similar efficacy was observed in nilotinib treated
animals atan oraldoseof75mg/kg/day. In the secondefficacy
model, to evaluate Src inhibitory activity, subcutaneous
tumors were established with NIH 3T3 cells transfected
with constitutively activated Src Y527F. Daily oral adminis-
tration of 9i elicited dose-dependent tumor shrinkage with
complete tumor growth inhibition observed at the highest
dose (Figure 8).
Compound 14 Inhibits Bcr-Abl Mutant T315I
To determine whether any of our compounds possessed
activity against the refractory T315I mutant,6 we screened
several compounds in an Abl-T315I kinase assay and ulti-
mately in Ba/F3 cells transfected with Bcr-Abl T315I. Inter-
estingly, two inhibitors bearing imidazole appendages on the
B ring, 14a and 14b, demonstrated modest kinase inhibition
and inhibited the proliferation of such Ba/F3 cells with IC50s
between 300 and 400 nM (Table 6). Moreover, inhibition of

4750 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Huang et al.
Table 4. Impact of Additional H-Bond with Hinge on Potency (IC50 in nM)
compd
Src kinase
Abl kinase
WT Abl (Ba/F3)
14a
18a
9i
8
84
25
48
20
38
9
7
6
5
7
307
18b
Table 5. Mean Pharmacokinetic Parameters Following Intravenous (iv) or Oral Dosing (po) in CD Rats or Mice a
iv
po
compd
CL (L/h/ kg)
Vd (L/kg)
t1/2 (h)
AUC0-24 h (ng h/mL)
Cmax (ng/mL)
tmax (h)
F (%)
3
3a
3b
3d
3e
0.38
1.60
1.08
1.70
9.01
3.54
5.31
4.87
0.34
0.78
2.95
4.0
2.4
6.7
3.1
2.1
3.7
5.7
8.3
N/Ce
32151c
3311c
N/C
146c
297c
7540c
428459c
17249c
21195d
6927d
N/C
2613
387
N/C
1.0
2.0
6.0
1.0
1.0
1.0
2.0
2.0
2.0
1.0
N/C
40
0.19b
0.49b
0.96b
0.79b
1.79b
0.90b
0.04b
0.06b
11
483
63
N/C
0.8
3.5
45
3g
9d
9g
9i
100
1040
23525
1753
3097
1020
116
7
14a
9i
14a
a n = 3 animals per study. b Dosed iv in rats at 5 mg/kg as a solution in 50% DMA, 45% PEG-400, and 5% Tween-80. c Dosed po in rats at 15 mg/kg as
a solution 15% DMA, 15% TPGS, 5% Tween-80, 25% PEG-400, and 40% water. d Dosed po in mice at 10 mg/kg as a solution in 15% DMA, 15%
TPGS, 5% Tween-80, 25% PEG-400, and 40% water. e N/C = not calculated.
Figure 7. In vivo efficacy of 9i in a Ba/F3 WT Bcr-Abl survival
model.
Figure 8. In vivo efficacy of 9i in a Src Y527F mouse tumor model.
cell proliferation by 14a directly correlated with decreased
cellular phosphorylation of pBcr-Abl (data not shown,
see ref 35) and the IC50 for inhibition of the nontransfected
parental Ba/F3 control was greater than 6 μM. Inhibitors
bearing other ring systems (17) or without substitution at the
C-300 position (9i) were either less effective or completely
inactive against this mutation. Previously, we observed that
compoundsbearingthese substitutionsgave risetoonly minor
changes in potency against wild type Abl. The model of 14a
dockedinto T315I Abl kinase demonstrates thatthe 4-methyl-
imidazole group fills a shallow pocket formed by Glu228,
Glu282, Val289, Asp381, and Phe359 (Figure 9). This addi-
tional interaction with the protein appears to contribute

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4751
Table 6. Inhibition of Bcr-Abl Mutant T315I (IC50 in nM)
10 to 100 mg/kg. Inhibitor 14a (AP24163),36 an alternative
member of thisnovelseries, not only potently inhibitsbothSrc
and Abl but also displays clear cellular potency against a
number of clinically identified Bcr-Abl mutants including the
highly refractory T315I mutation. Its modest cellular potency
(IC50 = 420 nM) against T315I is encouraging given the
insensitivity of this mutant to all currently marketed first- and
second-generation Bcr-Abl inhibitors.
14a
14b
17a
17b
9i
T315I kinase
T315I (Ba/F3)
478
422
542
298
>5000
nd
1818
nd
14412
2746
Experimental Section
Chemistry. All reagents and solvents were used as received. 1H
NMR (300.1 MHz), 19F NMR (282.4 MHz), and 31P NMR
(121.5 MHz) spectra were recorded on a Bruker ARX300
spectrometer, using TMS as internal standard and CFCl3 or
85% H3PO4 as external standard, respectively. MS spectra were
recorded on a Waters Micromass ZQ spectrometer. Elemental
analyses were performed by Robertson Microlit Laboratories
in Madison, NJ. HPLC was performed on an Agilent 1100
HPLC system. The purity of all SAR compounds was deter-
mined to be g95% by reverse phase HPLC (C-18 column,
MeCN/H2O with 0.1% CF3CO2H as the mobile phase),
and certain compounds were further checked for their purity
via combustion analysis.
General Procedure for the Preparation of 6-(Alkylamino)-9-
vinylpurines 5; N-methyl-9-vinyl-9H-purin-6-amine (5a), Method
A. 6-Chloro-9-vinylpurine28(0.54 g, 3 mmol) was added to the
solution of methylamine (7.5 mL, 15 mmol, 2.0 M in THF) in
THF (5 mL), and the mixture was stirred for 5 h at rt. Water was
added to dissolve the white precipitate (methylamine hydro-
chloride). The aqueous layer was extracted with ethyl acetate,
and the combined organic layers were dried over sodium sulfate,
concentrated on a rotavap, and then subjected to silica gel
column chromatography (5% methanol/methylene chloride),
yielding the desired product as white solid in 75% yield. 1H
NMR (CD3OD): δ 3.11 (s, 3H), 5.18 (dd, J = 9.2, 1.3 Hz, 1H),
5.97 (dd, J = 16.0, 1.3 Hz, 1H), 7.28 (dd, J = 16.0, 9.2 Hz, 1H),
8.28 (s, 1H), 8.32 (s, 1H).
Figure 9. Model of compound 14a bound to Abl T315I.
significantly to its activity against T315I. It is noteworthy that
inhibitor 14a shares an identical structural fragment with
nilotinib but that the latter is completely ineffective against
Abl T315I. When nilotinib is docked into the model of Abl
kinase with the T315I mutation, there is an obvious steric
clash between the amino group on the pyrimidine ring and the
larger isoleucine residue. Such a clash is not observed between
the less sterically demanding double bond linkage in 14a,
which may explain its increased activityagainst this important
clinical mutation.
To further characterize the activity of 14a, we also deter-
mined the antiproliferation activity against a broader panel of
Ba/F3 cells expressing clinically relevant Bcr-Abl mutants:
L248R, G250E, Q252H, Y253H, and E255K. Relative to
T315I-expressing cells, these cell lines were more potently
inhibited with IC50s of 64, 63, 42, 44, and 24 nM, respectively,
which is comparable or superior to the potency of nilotinib
against the same mutations.16a
N-Cyclopropyl-9-vinyl-9H-purin-6-amine (5b). Method A;
1
yield: 92%. H NMR (CDCl3): δ 0.60 (m, 2H), 0.87 (m, 2H),
3.00 (m, 1H), 5.08 (dd, J = 9.1, 1.4 Hz, 1H), 5.80 (dd, J = 16.0,
1.4 Hz, 1H), 6.39 (br, 1H), 7.20 (dd, J = 16.0, 9.1 Hz, 1H), 7.94
(s, 1H), 8.45 (s, 1H).
N-(4-(Dimethylphosphoryl)phenyl)-9-vinyl-9H-purin-6-amine
(5g). A mixture of 6-chloro-9-vinylpurine (0.18 g, 1 mmol),
4-dimethylphosphinylaniline (0.17 g, 1 mmol), and pyridine
hydrochloride (0.11 g, 1 mmol) was dissolved in 2-ethoxyetha-
nol (2 mL). The resulting solution was heated at 160 °C for
10 min under microwave irradiation. The mixture was concen-
trated to dryness on a rotavap. EtOAc and aq NaHCO3 were
added. The aqueous layer was extracted with ethyl acetate, and
the combined organic layers were dried over sodium sulfate,
concentrated on a rotavap, and then subjected to silica gel
column chromatography (eluent: 10% methanol/methylene
Conclusion
Through our program of structure guided drug design, we
have identified a novel series of purine-based compounds that
potently inhibit Src and Abl by specifically targeting the
inactive conformation of both proteins. Extensive SAR
revealed structural features contributing to high potency.
Several key ligand-protein interactions responsible for po-
tency against both kinases include a hydrogen bond between
the hinge region and N-7 on the purine core, hydrogen bonds
between the amide group and the Glu residue in the RC-helix
and Asp residue in the DFG motif, respectively, and hydro-
phobic interaction between the trifluoromethylphenyl and the
proteins. The NH at C-6 on the purine template was identified
as critical for achieving high potency against Src through a
well-defined hydrogenbondwith a hinge residue. Compounds
bearing cyclopropylamine at C-6 on the purine core were
found to display highly desirable pharmacokinetics in general.
Once-daily oral administration of inhibitor 9i (AP24226)36 at
10 mg/kg significantly prolonged the survival of mice injected
intravenously with wild type Bcr-Abl expressing Ba/F3 cells,
withpotencycomparable tonilotinibwhen dosedat75mg/kg.
Furthermore, 9i also elicited dose-dependent tumor shrinkage
in a Src Y527F mouse tumor model atoral doses ranging from
1
chloride), yielding the desired product in 56% yield. H NMR
(CD3OD): δ 1.70 (d, J = 13.2 Hz, 3H), 1.81 (d, J = 13.3 Hz,
3H), 5.23 (d, J = 9.2 Hz, 1H), 6.07 (d, J = 16.0 Hz, 1H), 7.34
(dd, J = 16.0, 9.1 Hz, 1H), 7.33 (m, 2H), 7.79 (m, 2H), 8.46 (s,
1H), 8.50 (s, 1H).
N-(3-(N0,N0-Dimethylamino)propyl)-9-vinyl-9H-purin-6-amine (5h).
Method A; yield: 95%. 1H NMR (CDCl3): δ 2.00 (m, 2H), 2.44 (s,
6H), 2.67 (m, 2H), 3.80 (m, 2H), 5.15 (dd, J= 9.1, 1.4 Hz, 1H), 5.85 (dd,
J = 16.0, 1.4 Hz, 1H), 6.63 (br, 1H), 7.23 (dd, J = 16.0, 9.1 Hz, 1H),
7.97 (s, 1H), 8.41 (s, 1H).
N-(3-Iodo-4-methylphenyl)-3-(trifluoromethyl)benzamide (6).
A solution of 3-iodo-4-methylaniline (1.22 g, 5.25 mmol), 3-(tri-
fluoromethyl)benzoic acid (0.95 g, 5 mmol), HOBt (0.68 g,
5 mmol), and EDCI (1.05 g, 5.5 mmol) in THF (20 mL) was
stirred overnight at rt. Water was added, and the aqueous layer

4752 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Huang et al.
was extracted with dichloromethane. The combined organic
layers were dried over sodium sulfate, concentrated on a rota-
vap, and then subjected to silica gel column chromatography
(3:1 hexane/ethyl acetate), yielding the desired product as white
solid in 83% yield. 1H NMR (CD3OD): δ 2.40 (s, 3H), 7.27 (d,
J = 8.3 Hz, 1H), 7.62 (dd, J = 8.3, 2.2 Hz, 1H), 7.71 (dd, J =
7.8, 7.8 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 7.9 Hz,
1H), 8.25 (d, J = 7.9 Hz, 1H), 8.26 (dd, J = 7.9, 2.1 Hz, 1H).
General Procedure for the Heck Reaction of 9-Vinylpurines
and Aryl Iodides; (E)-N-(4-Methyl-3-(2-(6-(methylamino)-9H-
purin-9-yl)vinyl)phenyl)-3-(trifluoromethyl)-benzamide (3a), Me-
thod B. A solution of 9-vinylpurine 5a (1 mmol), aryl iodide 6
(1.1 mmol), Pd(OAc)2 (11.2 mg, 0.05 mmol), and P(o-tol)3 (30.4
mg, 0.1 mmol) in DMF (3 mL) was degassed with bubbling N2 for
10 min. (i-Pr)2NEt (0.52 mL, 3 mmol) was added under N2, The
resulting solution was stirred at 110 °C for 15 h. Water was added,
and the aqueous layer was extracted with EtOAc. The combined
organic layers were dried over Na2SO4, concentrated on a rota-
vap, and then subjected to silica gel column chromatography (5%
MeOH/CH2Cl2), yielding the desired product as white powder in
82% yield; mp 201 °C. 1H NMR (CDCl3): δ 2.42 (d, J = 7.9 Hz,
3H), 3.24 (s, 3H), 5.87 (s, 1H), 7.20 (d, J=8.2Hz, 1H), 7.48(d, J=
14.5 Hz, 1H), 7.52 (dd, J = 7.1, 2.0 Hz, 1H), 7.59 (d, J = 14.5 Hz,
1H), 7.65 (d, J= 7.8 Hz, 1H), 7.81 (d, J= 8.2 Hz, 1H), 7.84 (d, J=
1.9 Hz, 1H), 8.02 (s, 1H), 8.11 (d, J=7.8Hz, 1H), 8.18(s, 2H), 8.48
(s, 1H). 19F NMR (CDCl3): δ -61.92. MS [M þ H]þ 453.3.
(E)-N-(4-Methyl-3-(2-(6-(cyclopropylamino)-9H-purin-9-yl)vinyl)-
phenyl)-3-(trifluoromethyl)benzamide (3b). Method B: This com-
pound was made from 5b and 6 in 56% yield. 1H NMR (DMSO-
d6): δ 0.67 (m, 2H), 0.77 (m, 2H), 2.42 (s, 3H), 3.24 (m, 1H), 7.37
(d, J = 8.4 Hz, 1H), 7.68 (dd, J = 8.1, 1.9 Hz, 1H), 7.73 (s, 1H),
7.81 (d, J = 7.2 Hz, 1H), 7.82 (d, J = 14.5 Hz, 1H), 8.00 (d, J =
7.1 Hz, 1H), 8.07 (d, J = 2.0 Hz, 2H), 8.30 - 8.38 (m, 3H), 8.74
(s, 1H), 10.47 (s, 1H). 19F NMR (CDCl3): δ -57.17. MS [M þ H]þ
(E)-N-(3-(2-(6-Chloro-9H-purin-9-yl)vinyl)-4-methylphenyl)-
3-(trifluoromethyl)-benzamide (7). Method B: 29% yield. 1H
NMR (DMSO-d6): δ 2.42 (s, 3H), 7.28 (d, J = 8.3 Hz, 1H),
7.67 (dd, J = 8.2, 2.1 Hz, 1H), 7.77 (d, J = 14.7 Hz, 1H), 7.80
(dd, J = 7.9, 7.8 Hz, 1H), 7.92 (d, J = 14.7 Hz, 1H), 8.00 (d, J =
7.9 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H),
8.34 (s, 1H), 8.92 (s, 1H), 9.24 (s, 1H), 10.49 (s, 1H). MS [M -
H]þ 455.6.
General Procedure for the Preparation of 3 from Pd-Catalyzed
Amination of 7 with Arylamines; (E)-N-(4-Methyl-3-(2-(6-(pyr-
idine-2-yl-amino)-9H-purin-9-yl)vinyl)phenyl)-3-(trifluoromethyl)-
benzamide (3c), Method C. A mixture of compound 7 (46 mg,
0.1 mmol), 2-aminopyridine (22 mg, 0.2 mmol), dicyclohexylphe-
nylphosphine (11 mg, 0.03 mmol), Pd2(dba)3 (18 mg, 0.02 mmol),
and K3PO4 (32 mg, 0.15 mmol) was charged in a microwave
reaction tube and then degassed via three cycles of vacuum-Ar
refilling. Anhydrous DMF (1 mL) was added, and the resulting
solution was heated under microwave at 150 °C for 5 min. After
cooling to rt, the reaction mixture was filtered and subjected to a
silica gel column chromatography (5-10% methanol/methylene
chloride), giving the desired product as white solid in 36% yield.
1H NMR (CDCl3): δ 2.41 (s, 3H), 7.03 (dd, J = 6.1, 5.9 Hz, 1H),
7.19 (d, J = 6.8 Hz, 1H), 7.49 (s, 1H), 7.52 (d, J = 8.2 Hz, 1H),
7.61-7.69 (m, 2H), 7.76-7.83 (m, 2H), 7.89 (s, 1H), 8.13-8.19 (m,
4H), 8.34 (d, J = 3.7 Hz, 1H), 8.66 (s, 1H), 8.68 (m, 1H). 19FNMR
(CDCl3): δ -63.49. MS [M]þ 515.1.
(E)-N-(4-Methyl-3-(2-(6-(pyrid-3-yl-amino)-9H-purin-9-yl)vi-
nyl)phenyl)-3-(trifluoromethyl)-benzamide (3d). Method C: pur-
ified by reverse phase prep-HPLC (MeCN/H2O, 0.1% TFA) as
TFA salt. 1H NMR (DMSO-d6): δ 2.43 (s, 3H), 7.28 (d, J = 8.3
Hz, 1H), 7.66-7.70 (m, 2H), 7.74 (s, 1H), 7.79 (s, 1H), 7.83 (d,
J = 7.9 Hz, 1H), 7.87-7.94 (m, 1H), 8.00 (d, J = 8.0 Hz, 1H),
8.12 (d, J = 1.9 Hz, 1H), 8.30-8.35 (m, 2H), 8.42 (d, J = 5.0 Hz,
1H), 8.63 (s, 1H), 9.00 (s, 1H), 9.35 (d, J = 2.2 Hz, 1H), 10.49 (s,
1H), 10.61 (s, 1H). 19F NMR (CDCl3): δ -57.09, -70.31. MS
[M]þ 515.4.
479.1. Anal. calcd for (C25H21F3N6O 0.07CH2Cl2): C, H, N.
3
(E)-N-(3-(2-(6-(4-(Dimethylphosphoryl)phenylamino)-9H-purin-9-yl)-
vinyl)-4-methylphenyl)-3-(trifluoromethyl)benzamide (3g). Me-
thod B: This compound was made from 5g and 6 in 47% yield.
1H NMR (DMSO-d6): δ 1.65 (d, J = 13.2 Hz, 6H), 2.44 (s, 3H),
7.29 (d, J = 8.2 Hz, 1H), 7.69 (dd, J = 7.2, 1.9 Hz, 1H), 7.73-
7.87 (m, 3H), 7.78 (d, J = 14.8 Hz, 1H), 7.90 (d, J = 14.7 Hz,
1H), 8.00 (d, J = 7.8 Hz, 1H), 8.11 (m, 1H), 8.14 (d, J = 2.0 Hz,
1H), 8.17 (d, J = 2.0 Hz, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.36 (s,
1H), 8.60 (s, 1H), 8.97 (s, 1H), 10.29 (s, 1H), 10.49 (s, 1H). 19F
NMR (DMSO-d6): δ -57.17. 31P NMR (DMSO-d6): δ 36.77.
(E)-N-(4-Methyl-3-(2-(6-(pyrid-4-yl-amino)-9H-purin-9-yl)vi-
nyl)phenyl)-3-(trifluoromethyl)-benzamide (3e). Method C: pur-
ified by reverse phase prep-HPLC (MeCN/H2O, 0.1% TFA) as
TFA salt. 1H NMR (DMSO-d6): δ 2.44 (s, 3H), 7.30 (d, J = 8.2
Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.77 - 7.84 (m, 2H), 7.94 (d,
J = 14.8 Hz, 1H), 8.00 (d, J = 7.7 Hz, 1H), 8.16 (s, 1H), 8.31 (d,
J = 8.2 Hz, 1H), 8.35 (s, 1H), 8.58 (d, J = 6.9 Hz, 2H), 8.70 (d,
J = 6.9 Hz, 2H), 8.86 (s, 1H), 9.17 (s, 1H), 10.50 (s, 1H), 11.70
(s, 1H). 19F NMR (CDCl3): δ -57.09, -69.99. MS [M]þ 515.6.
(E)-N-(4-Methyl-3-(2-(6-(pyrimid-4-yl-amino)-9H-purin-9-yl)-vinyl)-
phenyl)-3-(trifluoromethyl)-benzamide (3f). Method C: purified
by reverse phase prep-HPLC (MeCN/H2O, 0.1% TFA) as TFA
salt. 1H NMR (DMSO-d6): δ 2.27 (s, 3H), 7.25 (d, J = 8.3 Hz,
1H), 7.69 (dd, J = 7.9, 1.9 Hz, 1H), 7.70 (d, J = 15.0 Hz, 1H),
7.76 (d, J = 15.5 Hz, 1H), 7.78 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H),
7.98 (d, J = 7.7 Hz, 1H), 8.05 (d, J = 1.9 Hz, 1H), 8.23 (d, J =
5.8 Hz, 1H), 8.32 (d, J = 8.1 Hz, 1H), 8.34 (d, J = 5.6 Hz, 1H),
8.36 (d, J = 6.2 Hz, 1H), 8.60 (s, 1H), 8.66 (s, 1H), 10.49 (s, 1H).
19F NMR (CDCl3): δ -57.16. MS [M]þ 516.7. Anal. calcd for
MS [M þ H]þ 591.4. Anal. calcd for (C30H26F3N6O2P 0.25
3
CH2Cl2): C, H, N.
(E)-N-(4-Methyl-3-(2-(6-(3-(N0,N0-dimethylamino)propyla-
mino)-9H-purin-9-yl)vinyl)phenyl)-3-(trifluoromethyl)benzamide (3h).
Method B: This compound was made from 5h and 6in 45% yield. 1H
NMR (CDCl3): δ 1.74 (tt, J = 6.6, 6.6 Hz, 2H), 2.16 (s, 6H), 2.32 (s,
3H), 2.34 (t, J = 6.7 Hz, 2H), 3.64 (m, 2H), 6.75 (t, J = 5.3 Hz, 1H),
7.00 (d, J = 8.3 Hz, 1H), 7.21 (d, J = 17.5 Hz, 1H), 7.34 (d, J = 17.6
Hz, 1H), 7.39 (dd, J=8.2, 2.1Hz, 1H), 7.46(d, J=7.8Hz, 1H), 7.59
- 7.65 (m, 2H), 7.85 (s, 1H), 7.94 (d, J = 2.9 Hz, 1H), 8.09 (s, 1H),
8.31 (s, 1H), 8.68 (s, 1H). 19FNMR(CDCl3): δ-63.45. MS [M þ H]þ
524.4. Anal. calcd for (C27H28F3N7O): C, H, N.
(C26H19F3N8O 0.6CF3CO2H): C, H, N.
3
General Procedure for the Preparation of 3-Iodo-4-methylben-
zamides 8; 3-Iodo-4-methyl-N-(4-(trifluoromethyl)pyridin-2-yl)
benzamide (8i), Method D. 3-Iodo-4-methylbenzoic acid
(2.62 g, 10 mmol) was refluxed in SOCl2 (10 mL) for 1 h. The
volatile components were removed on a rotavap, and the residue
was dissolved in benzene (10 mL), concentrated to dryness on a
rotavap, and further dried under vacuum. The resulting acyl
chloride was added to a solution of 2-amino-4-(trifluoromethyl)
pyridine (1.65 g, 10.2 mmol), N,N-diisopropylethylamine
(1.56 g, 12 mmol), and a catalytic amount of DMAP in THF
(20 mL). After stirring at rt for 2 h, the reaction was quenched
with water. EtOAc was added, and the layers were separated.
The combined organic layers were concentrated to dryness,
giving a mixture of desired product (major) and bis-acylated
N-(4-Methyl-3-(2-(6-(methylamino)-9H-purin-9-yl)ethyl)phenyl)-
3-(trifluoromethyl)-benzamide (2). To a solution of 3a (0.10 g) in
MeOH was added a catalytic amount of 10% palladium on carbon.
The resulting mixture was hydrogenated under 50 psi H2 for 48 h, at
which point HPLC indicated completion. The catalyst was re-
moved by filtration, and the filtrate was concentrated on a rotavap
and further dried under vacuum, giving the desired product in
quantitative yield. 1H NMR (CDCl3): δ 2.13 (d, J = 7.3 Hz, 3H),
3.21 (t, J = 7.1 Hz, 2H), 3.30 (s, 3H), 4.47 (t, J = 7.1 Hz, 2H), 7.17
(d, J = 8.2 Hz, 1H), 7.37 (d, J = 7.5 Hz, 1H), 7.48 (s, 1H), 7.55 (s,
1H), 7.65 (dd, J=7.8, 7.7Hz, 1H), 7.81(s, 1H), 7.85(d, J=7.5 Hz,
1H), 8.08 (d, J = 7.9 Hz, 1H), 8.13 (s, 1H), 8.33 (s, 1H). MS
[M þ H]þ 455.3.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4753
byproduct; the latter is slightly less polar (TLC) but difficult
to separate by column chromatography. The product mixture
was then dissolved in THF (30 mL) and stirred with 4 N aq
NaOH (30 mL) at 50 °C for 15 min. Extraction with EtOAc
followed by concentration of combined organic layers gave
the desired product, which was pure by NMR and used in
next step without purification. Yield: 95%. 1H NMR (DMSO-
d6): δ 2.45 (s, 3H), 7.48 (d, J = 8.1 Hz, 1H), 7.54 (dd, J = 5.1, 1.9
Hz, 1H), 7.97 (dd, J =7.9, 1.8 Hz, 1H), 8.49 (d, J =1.8 Hz,
1H), 8.51 (m, 1H), 8.68 (d, J =5.1 Hz, 1H), 11.33 (s, 1H). MS
[M þ H]þ 407.0.
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-(3-(iso-propyl)phenyl)benzamide (9e). Method B; purified
by reverse phase prep-HPLC (MeCN/H2O, 0.1% TFA) as TFA
salt. 1H NMR (DMSO-d6): δ 0.71 (m, 2H), 0.80 (m, 2H), 1.23 (d,
J = 6.9 Hz, 6H), 2.51 (s, 3H), 2.89 (septet, J = 6.9 Hz, 1H), 3.06
(m, 1H), 7.00 (d, J = 7.6 Hz, 1H), 7.28 (dt, J = 2.5, 7.8 Hz, 1H),
7.42 (d, J = 8.0 Hz, 1H), 7.65 (m, 2H), 7.83 (dd, J = 2.0, 1.7 Hz,
1H), 7.90(s, 1H), 7.92 (s, 1H), 8.24 (d, J = 1.5 Hz, 1H), 8.45 (s,
1H), 8.75 (s, 1H), 10.21 (s, 1H). MS [M þ H]þ 452.7.
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-(5-(tert-butyl)thiazo-2-yl)benzamide (9f). Method B. 1H
NMR (CDCl3): δ 0.70 (m, 2H), 0.89 (m, 2H), 1.31 (s, 9H),
2.53 (s, 3H), 3.09 (m, 1H), 6.01 (s, 1H), 6.61 (s, 1H), 7.38 (d, J =
8.0 Hz, 1H), 7.61 (d, J = 14.6 Hz, 1H), 7.78 (d, J = 14.9 Hz, 1H),
7.82 (dd, J = 8.0, 1.9 Hz, 1H), 8.06 (s, 1H), 8.17 (d, J = 1.6 Hz,
1H), 8.56 (s, 1H). Anal. calcd for (C25H27N7OS): C, H, N.
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-(5-(tert-butyl)oxazo-3-yl)benzamide (9g). Method B. 1H
NMR (CDCl3): δ 0.75 (m, 2H), 0.98 (m, 2H), 1.39 (s, 9H),
2.52 (s, 3H), 3.15 (m, 1H), 6.62 (br, 1H), 6.88 (s, 1H), 7.27-7.52
(m, 2H), 7.66 (m, 1H), 8.00 (m, 1H), 8.05 (d, J = 18.8 Hz, 1H),
8.49 (s, 1H), 9.31 (s, 1H).
N-(3-(tert-Butyl)phenyl)-3-iodo-4-methylbenzamide (8d). Meth-
od D. 1H NMR (DMSO-d6): δ1.29 (s, 9H), 2.45 (s, 3H), 7.14 (dtt, J
= 7.9, 1.8, 1.0 Hz, 1H), 7.09 (dd, J = 7.9, 7.9 Hz, 1H), 7.48 (d, J =
8.1 Hz, 1H), 7.66 (dtt, J = 8.0, 1.9, 1.0 Hz, 1H), 7.75 (dd, J = 1.9,
1.8 Hz, 1H), 7.91 (dd, J = 7.9, 1.8 Hz, 1H), 8.41 (d, J = 1.8 Hz,
1H), 10.16 (s, 1H).
N-(3-(iso-Propyl)phenyl)-3-iodo-4-methylbenzamide (8e). Meth-
od D. 1H NMR (DMSO-d6): δ 1.22 (d, J = 6.9 Hz, 6H), 2.44 (s,
3H), 2.88 (septet, J = 6.9 Hz, 1H), 6.99 (d, J = 7.7 Hz, 1H), 7.26
(dd, J = 7.8, 7.7 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.61 - 7.63 (m,
2H), 7.91 (dd, J = 7.9, 1.8 Hz, 1H), 8.41 (d, J = 1.8 Hz, 1H), 10.16
(s, 1H).
N-(5-(tert-Butyl)-1H-oxazo-3-yl)-3-iodo-4-methylbenzamide
(8g). Method D. 1H NMR (CDCl3): δ 1.39 (s, 9H), 2.52 (s, 3H),
6.84 (s, 1H), 7.37 (d, J = 7.9 Hz, 1H), 7.84 (dd, J = 7.9, 1.7 Hz,
1H), 8.40 (d, J = 1.7 Hz, 1H), 9.36 (s, 1H).
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-(1-methyl-3-(tert-butyl)pyrazo-5-yl)benzamide (9h). Meth-
od B; purified by reverse phase prep-HPLC (MeCN/H2O, 0.1%
TFA) as TFA salt. 1H NMR (DMSO-d6): δ 0.71 (m, 2H), 0.80
(m, 2H), 1.27 (s, 9H), 2.51 (s, 3H), 3.06 (m, 1H), 3.64 (s, 3H), 6.11
(s, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.82 (dd, J = 8.0, 1.6 Hz, 1H),
7.84 (d, J = 14.8 Hz, 1H), 7.89 (d, J = 10.4 Hz, 1H), 7.93 (d, J =
14.7 Hz, 1H), 8.26 (s, 1H), 8.44 (s, 1H), 8.78 (s, 1H), 10.29 (s,
N-(1-Methyl-3-(tert-butyl)-1H-pyrazo-5-yl)-3-iodo-4-methyl-
1
benzamide (8h). Method D. H NMR (DMSO-d6): δ 1.23 (s,
9H), 2.45 (s, 3H), 3.61 (s, 3H), 6.09 (s, 1H), 7.49 (d, J = 8.1 Hz,
1H), 7.89 (dd, J = 7.9, 1.7 Hz, 1H), 8.39 (d, J = 1.7 Hz, 1H),
10.24 (s, 1H).
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-methyl-
N-(3-(trifluoromethyl)phenyl)benzamide (9a). Method B. 1H
NMR (CDCl3): δ 0.71 (m, 2H), 0.97 (m, 2H), 2.50 (s, 3H), 3.13
(m, 1H), 7.34 (d, J = 7.9Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.48 (d,
J = 8.0 Hz, 1H), 7.55 (d, J = 16.0 Hz, 1H), 7.62 (s, 1H), 7.66 (s,
1H), 7.73 (dd, J = 7.9, 1.8 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 8.01
(s, 1H), 8.07 (m, 2H), 8.23 (s, 1H), 8.50 (s, 1H). Anal. calcd for
1H). MS [M þ H]þ 470.7. Anal. calcd for (C26H30N8O 2
3
CF3CO2H): C, H, N.
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-(4-(trifluoromethyl)pyridin-2-yl)benzamide (9i). Method B;
1
82% yield, white solid, mp 232 °C. H NMR (DMSO-d6, 300
MHz): δ 11.44 (s, 1H), 8.72 (s, 1H), 8.69 (s, 1H), 8.59 (s, 1H), 8.38
(m, 2H), 8.05 (m, 1H), 8.03 (d, 1H, J=14.7 Hz), 7.87 (d, 1H, J=7.9
Hz), 7.81 (d, 1H, J=14.7 Hz), 7.55 (d, 1H, J=4.1 Hz), 7.42 (d, 1H,
J=8.0 Hz), 3.09 (m, 1H), 2.43 (s, 3H), 0.74 (m, 2H), 0.66 (m, 2H).
MS [MþH]þ 480.0 Anal. calcd for (C24H20F3N7O): C, H, N.
N-(3-Iodo-4-methylphenyl)-N0-(3-(trifluoromethyl)phenyl)urea
(10). A mixture of 3-(trifluoromethyl)phenylisocyanate (1.03 g,
5.5 mmol) and 3-iodo-4-methylaniline (1.17 g, 5 mmol) in
CH2Cl2 (20 mL) was stirred at rt for 4 h. Filtration gave pure
product as white solid, 98% yield. 1H NMR (DMSO-d6): δ 2.31
(s, 3H), 7.22-7.33 (m, 3H), 7.48-7.59 (m, 2H), 8.00 (s, 1H), 8.09
(d, J = 2.0 Hz, 1H), 8.80 (s, 1H), 9.03 (s, 1H).
(C25H21F3N6O 0.02CH2Cl2): C, H, N.
3
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-(2-fluoro-5-(trifluoromethyl)phenyl)benzamide (9b). Meth-
od B; purified by reverse phase prep-HPLC (MeCN/H2O, 0.1%
TFA)as TFA salt. 1H NMR(DMSO-d6):δ 0.72(m, 2H), 0.82 (m,
2H), 2.51 (s, 3H), 2.98 (m, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.58 (dd,
J = 9.6, 9.2 Hz, 1H), 7.68 (m, 1H), 7.85 (dd, J = 7.9, 1.8 Hz, 1H),
7.90 (d, J = 10.0 Hz, 1H), 7.94(d, J = 14.7 Hz, 1H), 7.85 (dd, J =
7.9, 1.8 Hz, 1H), 8.11 (dd, J = 6.7, 1.9 Hz, 1H), 8.29 (d, J = 1.5
Hz, 1H), 8.46 (s, 1H), 8.80 (s, 1H), 10.43 (s, 1H). MS [M þ H]þ
(E)-1-(3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-methyl-
phenyl)-3-(3-(trifluoromethyl)phenyl)urea (11). Method B. 1H NMR
(CD3OD): δ 0.67 (m, 2H), 0.89 (m, 2H), 2.39 (s, 3H), 2.98 (m, 1H),
7.12-7.33 (m, 3H), 7.46 (m, 1H), 7.58-7.80 (m, 4H), 7.93 (s, 1H),
8.37 (s, 1H), 8.44 (s, 1H).
496.7. Anal. calcd for (C25H20F4N6O 1.7CF3CO2H): C, H, N.
3
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-(4-chloro-3-(trifluoromethyl)phenyl)benzamide (9c). Meth-
od B; purified by reverse phase prep-HPLC (MeCN/H2O, 0.1%
TFA) as TFA salt. 1H NMR (DMSO-d6): δ 0.71 (m, 2H), 0.81
(m, 2H), 2.51 (s, 3H), 3.04 (m, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.45
(d, J = 8.1 Hz, 1H), 7.85 (dd, J = 7.9, 1.8 Hz, 1H), 7.90 (d, J =
3.2 Hz, 2H), 8.15 (dd, J = 8.6, 2.4 Hz, 1H), 8.25 (d, J = 1.6 Hz,
1H), 8.38 (d, J = 2.5 Hz, 1H), 8.46 (s, 1H), 8.76 (s, 1H), 10.67 (s,
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-methyl-N-
(3-(4-methyl-1-imidazol-1-yl)-5-(trifluoromethyl)phenyl)benzamide
(14a). Method B. 1H NMR (DMSO-d6): δ 0.66 (m, 2H), 0.74 (m,
2H), 2.19 (s, 3H), 2.50 (s, 3H), 3.29 (m, 1H), 7.45 (d, J = 8.1 Hz,
1H), 7.50 (s, 1H), 7.74 (s, 1H), 7.86 (dd, J = 7.9, 1.6 Hz, 1H), 7.90
(d, J = 1.3 Hz, 2H), 8.16-8.21 (m, 2H), 8.27 (d, J = 1.3 Hz, 1H),
8.33-8.39(m, 2H),8.67(s, 1H),10.71(s,1H).MS[M þ H]þ 558.8.
1H). MS [M þ H]þ 512.7. Anal. calcd for (C25H20ClF3N6O 1.4
3
CF3CO2H): C, H, N.
Anal. calcd for (C29H25F3N8O 0.09CH2Cl2): C, H, N.
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-(3-(tert-butyl)phenyl)benzamide (9d). Method B; purified
by reverse phase prep-HPLC (MeCN/H2O, 0.1% TFA) as TFA
3
3-Iodo-4-methyl-N-[3-(imidazol-1-yl)-5-(trifluoromethyl)phenyl]-
benzamide (13b). Prepared via our previously reported procedure
for the synthesis of 13a.29 1H NMR (DMSO-d6): 2.47 (s, 3H), 7.28
(s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.84 (s, 2H), 7.95 (d, J = 7.9 Hz,
1H), 8.22 (s, 1H), 8.33 (s, 1H), 8.36 (s, 1H), 8.50 (s, 1H), 10.71 (s, 1H).
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-(3-(1H-imidazol-1-yl)-5-(trifluoromethyl)phenyl)benzamide
(14b). Method B. 1H NMR (DMSO-d6): 0.67 (m, 2H), 0.76
(m, 2H), 2.50 (s, 3H), 3.67 (m, 1H), 7.44 (d, J = 8.0 Hz, 2H),
1
salt. H NMR (DMSO-d6): δ 0.71 (m, 2H), 0.81 (m, 2H), 1.31
(s, 9H), 2.51 (s, 3H), 3.06 (m, 1H), 7.15 (m, 1H), 7.29 (dd, J =
7.9, 7.9 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.69 (m, 1H), 7.79
(dd, J = 1.9, 1.9 Hz, 1H), 7.84 (dd, J = 7.6, 1.7 Hz, 1H), 7.91 (d,
J = 3.8 Hz, 2H), 8.24 (d, J = 1.4 Hz, 1H), 8.45 (s, 1H), 8.75 (s,
1H), 10.21 (s, 1H). MS [M þ H]þ 466.7. Anal. calcd for
(C28H30N6O 1.2CF3CO2H): C, H, N.
3

4754 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Huang et al.
7.84-7.92 (m, 4H), 8.06 (d, J = 8.1 Hz, 2H), 8.26 (s, 1H), 8.29 (d,
J =16.8 Hz, 2H), 8.39 (s, 1H), 8.67 (s, 1H), 10.62 (s, 1H). MS
[M þ H]þ 545.1.
homogeneous time-resolved fluorescence resonance energy
transfer (TR-FRET) assay using purified human wild-type
Abl kinase (Panvera), Abl T315I (Upstate Biotechnology
Inc.), or partially purified human Src (Upstate Biotechnology
Inc.), and biotinylated phosphokinase substrate peptides: Bio-
tin-EKKMAAEAIYAAPFAK-NH2 (Quality Controlled Bio-
chemicals) for wild type Abl and Abl T315I, and Biotin-
KVEKIGEGTYGVVYK-NH2 (Pierce) for Src. The kinase
reactions were carried out in complete LANCE kinase buffer
(LKB) and consisted of 20 mM NaHEPES pH 7.4, 0.1 mg/mL
BSA, 1 mM ATP, 10 mM MgCl2, and 0.41 mM DTT in round-
bottom black 96-well Microfluor plates (Dynex Technologies
Inc.) preblocked with 1% BSA in PBS at 4 °C overnight. For the
kinase reaction, compound dilutions were incubated with wild
type Abl or Abl T315I (40 pM) or Src (165 pM) and their
substrate peptide (50 nM) 1 h for Abl T315I or 2 h for Abl at
room temperature, or 2 h at 37 °C for Src, in a total volume of
0.1 mL LKB. The kinase reaction was terminated by addition of
0.05 mL of a kill/detection solution containing 15 μM of a
potent ARIAD kinase inhibitor, 6 nM europium-labeled anti-
phosphotyrosine mAb PT66 and 60 nM allophycocyanin-
labeled streptavidin (LANCE reagents from Perkin-Elmer
Inc.). Assay plates were incubated at room temperature in the
dark for 20 min prior to reading fluorescence at 615 and 665 nm
on a Victor2 V plate reader (Perkin-Elmer). Positive and nega-
tive controls and a standard curve for phosphorylated peptide
were included on each plate. Data values were transferred to an
Excel spreadsheet and IC50s were calculated from the fluores-
cence at 665 nm by interpolation between the averaged duplicate
well data on 3-fold serial dilutions of test inhibitor.
Proliferation Assay. K562 human wild-type Bcr-Abl CML
cell line and murine pro-B Ba/F3 cell line stably transfected with
constructs expressing full-length wild-type Bcr-Abl or Bcr-Abl
with various kinase domain point mutations (T315I, L248R,
G250E, Q252H, Y253H, and E255K) were used in the cell
proliferation assays. The parental Ba/F3 cell line was used
as control. K562 cell line was obtained from the American
Type Culture Collection (ATCC) and maintained in Iscove’s
modified Dulbecco’s medium with 10% FBS. Ba/F3 cell lines
were kindly provided by Brian J. Druker (Howard Hughes
Medical Institute, Oregon Health and Science University,
Portland, OR) and were maintained in RPMI 1640 growth
medium with 10% FBS except the parental Ba/F3 cell line,
which was maintained in the same medium supplemented with
10 ng/mL IL-3. All cells were incubated at 37 °C in 5% CO2.
After the compounds were incubated with the cells for 3 days,
the number of viable cells in each well in 96-well plate was
measured using an MTS assay (Promega). MTS reagent was
added to all wells and then the plates were returned to the
incubator at 37 °C for 2 h. The absorbance in each well was then
measured at 490 nm using a Wallac Victor2V plate reader. The
IC50 was calculated by determining the concentration of com-
pound required to decrease the MTS signal by 50% in best-fit
curves using Microsoft XLfit software and comparing with
baseline, the DMSO control, as 0% inhibition.
5-(4-Morpholinyl)-3-(trifluoromethyl)benzenamine (15a). A
suspension of 3-bromo-5-(trifluoromethyl)aniline (3.0 g, 12.5
mmol), N-methylpiperazine (5.55 g, 50 mmol), and CsOH
monohydrate (4.20 g, 25 mmol) in DMSO (48 mL) was heated
at 120 °C in a sealed tube for 4 h. DMSO was then removed
under vacuum. The residue was suspended in CH2Cl2, and the
inorganic salts were filtered off. The filtrate was concentrated on
rotavap and then subjected to silica gel column chromatography
(5% MeOH/CH2Cl2), yielding the desired product as pale-
1
yellow oil. H NMR (CDCl3): δ 3.23 (m. 4H), 3.92 (m, 4H),
6.35 (s, 1H), 6.45 (s, 1H), 6.56 (s, 1H).
3-(4-Methyl-1-piperazinyl)-5-(trifluoromethyl)benzenamine (15b).
1H NMR (CDCl3): δ 2.34 (s, 3H), 2.65 (m, 4H), 3.30 (m, 4H), 3.79
(br, 2H), 6.31 (s, 1H), 6.39 (s, 1H), 6.51 (s, 1H).
3-Iodo-4-methyl-N-[3-(morpholin-4-yl)-5-(trifluoromethyl)phenyl]-
benzamide (16a). Method D. 1H NMR (DMSO-d6): 3.21 (m, 4H),
3.78 (m, 4H), 7.01 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.67 (s, 1H), 7.69
(s, 1H), 7.98 (d, J = 7.9 Hz, 1H), 8.41 (s, 1H), 10.31 (s, 1H).
3-Iodo-4-methyl-N-[3-(4-methylpiperazin-1-yl)-5-(trifluoromethyl)-
phenyl]benzamide (16b). Method D. 1H NMR (DMSO-d6): 2.23 (s,
3H), 2.46 (s, 3H), 2.50 (m, 4H), 3.27 (m, 4H), 6.97 (s, 1H), 7.54 (d, J=
8.0 Hz, 1H), 7.64 (s, 1H), 7.66 (s, 1H), 7.94 (d, J = 7.9 Hz, 1H), 8.33
(s, 1H), 10.29 (s, 1H).
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-[3-(morpholin-4-yl)-5-(trifluoromethyl)phenyl]benzamide (17a).
Method B. H NMR (DMSO-d6): 0.67 (m, 2H), 0.73 (m, 2H),
1
2.49 (s, 3H), 3.14 (m, 1H), 3.21 (t, J = 4.6 Hz, 2H), 3.77 (t, J = 4.6
Hz, 2H), 6.98 (s, 1H), 7.43 (d, J =8.0 Hz, 1H), 7.70(m, 2H), 7.83 (d,
J = 7.71, 1H), 7.91 (s, 1H), 7.93 (d, J = 15.5 Hz, 1H), 8.05 (d, J =
3.5 Hz, 1H), 8.24 (s, 1H), 8.39 (s, 1H), 8.66 (s, 1H), 10.39 (s, 1H). MS
[M þ H]þ 564.1. Anal. calcd for (C29H28F3N7O2): C, H, N.
(E)-3-(2-(6-(Cyclopropylamino)-9H-purin-9-yl)vinyl)-4-meth-
yl-N-[3-(4- methylpiperazin- 1-yl)-5-(trifluoromethyl)phenyl]ben-
1
zamide (17b). Method B. H NMR (DMSO-d6): 0.68 (m, 2H),
0.77 (m, 2H), 2.36 (s, 3H), 2.50 (s, 3H), 2.60 (m, 4H), 3.25
(m, 4H), 4.05 (m, 1H), 7.01 (s, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.86
(d, J = 14.8 Hz, 1H), 7.81 - 7.91 (m, 4H), 8.04 (br, 1H), 8.24
(s, 1H), 8.38 (s, 1H), 8.66 (s, 1H), 10.57 (s, 1H). MS [M þ H]þ
577.1.
(E)-3-(2-(Purin-9-yl)vinyl)-4-methyl-N-(3-(4-methyl-1-imida-
zol-1-yl)-5-(trifluoromethyl)phenyl)benzamide (18a). Method B.
1H NMR (DMSO-d6): 2.19 (s, 3H), 2.50 (s, 3H), 7.49 (d, J = 8.3
Hz, 1H), 7.50 (s, 1H), 7.75 (s, 1H), 7.89 (d, J = 7.9 Hz, 1H), 8.02
(d, J = 3.1 Hz, 2H), 8.19 (d, J = 17.1 Hz, 2H), 8.32 (d, J = 6.8
Hz, 2H), 9.11 (s, 2H), 9.29 (s, 1H), 10.72 (s, 1H). MS [M þ H]þ
503.9. Anal. calcd for (C26H20F3N7O): C, H, N.
(E)-3-(2-(Purin-9-yl)vinyl)-4-methyl-N-(4-(trifluoromethyl)pyri-
din-2-yl)benzamide (18b). Method B. 1H NMR (DMSO-d6): 2.73
(s, 1.5H), 2.89 (s, 1.5H), 7.43 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 4.3
Hz, 1H), 7.88-7.94 (m, 2H), 8.13 (d, J = 14.7 Hz, 1H), 8.40 (s,
1H), 8.59 (s, 1H), 8.70 (d, J = 4.7 Hz, 1H), 9.10 (s, 1H), 9.14 (s,
1H), 9.28 (s, 1H), 11.41 (s, 1H). MS [M þ H]þ 424.9. Anal. calcd
for (C21H15F3N6O 0.15CH2Cl2): C, H, N.
Pharmacokinetics (PK) and in Vivo Efficacy. All in vivo
experiments were conducted in accordance with ARIAD
Pharmaceuticals, Inc. Institutional Animal Care and Use
Committee (IACUC) guidelines. Unless otherwise stated,
all animals were obtained from Charles River Laboratories
(Wilmington, MA). Rats were housed two per cage and mice
were housed five per cage, were in micro isolator caging, and
were offered food and water ad libitum unless otherwise
stated.
PK Studies. Oral and intravenous pharmacokinetic testing was
performed on CD rats. For oral dosing, the rats were fasted
overnight and received food 2 h post dosing. Each rat was dosed
according to each individual body weight measured just prior
to dose administration. The dosing levels and volumes were set
at 15 mg/kg and 4 mL for po administration and 5 mg/kg and
3
Modeling Studies. The binding site model used for Abl dock-
ing was based on the X-ray coordinates of Abl bound with an
imatinib derivative, PDB-ID 1fpu. Protein side chains within a
˚
5 A radius of the bound ligand were allowed to relax to optimize
binding interactions using the Induced Fit protocol in the
Schrodinger package.24 The binding site model used for Src
docking was based on a homology model of Src built from the
crystal structure of Lck bound with a DFG-out inhibitor
(2og8)34 using Prime in the Schrodinger package. Docking
studies were performed with Glide in the Schrodinger modeling
package.24 The solution with the best docking score was chosen
and used for analysis.
Wild Type Abl, Abl T315I, and Src LANCE Assay. Inhibition
of wild type Abl, Abl T315I, or Src activity was measured in a

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4755
2 mL for iv administration. Blood was collected into microcentri-
fuge tubes containing a small amount of 5 mM EDTA from the
retro orbital plexus of each rat at prescribed time points, the tubes
were centrifuged, and plasma was isolated for LC-MS analysis.
As a prerequisite to evaluating in vivo efficacy, some com-
pounds were subjected to oral pharmacokinetic testing on mice.
The animals were not fasted and were dosed at a fixed volume of
10 mL and three different levels (10, 30, and 100 mg/kg) for the
purpose of observing dose response. Blood was collected from
three mice at each time point via exsanguination.
Bcr-Abl kinase domain mutations, drug resistance, and the road
to a cure for chronic myeloid leukemia. Blood 2007, 110, 2242–
2249. (d) O’Hare, T.; Eide, C. A.; Deininger, M. W. New Bcr-Abl
inhibitors in chronic myeloid leukemia: keeping resistance in
check. Exp. Opin. Invest. Drugs 2008, 17, 865–878. (e) Lee, F.;
Fandi, A.; Voi, M. Overcoming kinase resistance in chronic
myeloid leukemia. Int. J. Biochem. Cell Biol. 2008, 40, 334–343.
(f) Tanaka, R.; Kimura, S. Abl tyrosine kinase inhibitors for
overriding Bcr-Abl/ T315I: from the second to third generation.
Exp. Rev. Anticancer Ther. 2008, 8, 1387–1398. (g) Noronha, G.;
Cao, J.; Chow, C. P.; Dneprovskaia, E.; Fine, R. M.; Hood, J.;
Kang, X.; Klebansky, B.; Lohse, D.; Mak, C. C.; McPherson, A.;
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Inhibitors of ABL and the ABL- T315I mutation. Curr. Top.
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In Vivo Efficacy Studies. (1) Ba/F3 WT Bcr-Abl survival
model: Ba/F3 cell line expressing wild type Bcr-Abl was kindly
provided by Brian J. Druker (Howard Hughes Medical Insti-
tute, Oregon Health and Science University, Portland, OR).
Eight to ten week old female CB17 SCID mice were injected
intravenously with 1 ꢀ 106 cells per mouse on day zero and
randomly selected into groups (n=10). Treatments began on
day three after cell injection and were administered daily for up
to 21 consecutive days. Mice were observed twice daily during
the week and once daily during the weekend. Animals were
euthanized via CO2 asphyxiation when they became moribund
according to the company IACUC guidelines. Mice dead of
disease were verified showing splenomegaly at necropsy. Percent
survival refers to the percentage of mice remaining “on-study”
at the end of the day. (2) Src Y527F mouse tumor model: A c-Src
Y527F overexpressing cell line was established from NIH3T3
mouse fibroblasts transformed with a mutant chicken Src
(Y527F). It was kindly provided by Dr. David Shalloway at
Cornell University. Eight to ten week old female CD-1 nude
mice were injected subcutaneously with 2 ꢀ 106 cells per mouse
and were randomly selected into treatment groups (n=10) when
the average tumor size was ∼200 mm3 (day zero). Treatments
were administered orally for 21 consecutive days. Subcutaneous
tumors were measured 2 or 3 times weekly. Tumor volume
(in mm3) was calculated using the formula (L ꢀ W2)/2. When a
tumor reached 10% of the body weight of the host, the animal
was euthanized via CO2 asphyxiation.
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Supporting Information Available: Combustion analysis of
certain tested compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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