A type-ii kinase inhibitor capable of inhibiting the T315I "Gatekeeper" mutant of Bcr-Abl

Article abstract of DOI:10.1021/jm901808w

The second generation of Bcr-Abl inhibitors nilotinib, dasatinib, and bosutinib developed to override imatinib resistance are not active against the T315I "gatekeeper" mutation. Here we describe a type-II T315I inhibitor 2 (GNF-7), based upon a 3,4-dihydr

Full text of DOI:10.1021/jm901808w

J. Med. Chem. 2010, 53, 5439–5448 5439
DOI: 10.1021/jm901808w
A Type-II Kinase Inhibitor Capable of Inhibiting the T315I “Gatekeeper” Mutant of Bcr-Abl
Hwan Geun Choi,†, Pingda Ren,‡ Francisco Adrian,‡ Fangxian Sun,‡ Hyun Soo Lee,‡ Xia Wang,‡ Qiang Ding,‡
Guobao Zhang,‡ Yongping Xie,‡ Jianming Zhang,† Yi Liu,‡ Tove Tuntland,‡ Markus Warmuth,‡ Paul W. Manley,§
§
Jurgen Mestan, Nathanael S. Gray,*,† and Taebo Sim*,‡,
€
†Dana Farber Cancer Institute, Harvard Medical School, Department of Cancer Biology and Department of Biological Chemistry and
Molecular Pharmacology, 250 Longwood Avenue, Seeley G. Mudd Building 628A, Boston, Massachusetts 02115, ‡Genomics Institute of
the Novartis Research Foundation, Department of Chemistry, 10675 John Jay Hopkins Drive, San Diego, California 92121, §Novartis Institutes
for Biomedical Research, CH-4056 Basel, Switzerland, and Life Sciences Research Division, Korea Institute of Science and Technology,
39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791, Korea
Received December 8, 2009
The second generation of Bcr-Abl inhibitors nilotinib, dasatinib, and bosutinib developed to override
imatinib resistance are not active against the T315I “gatekeeper” mutation. Here we describe a type-II
T315I inhibitor 2 (GNF-7), based upon a 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffold
which is capable of potently inhibiting wild-type and T315I Bcr-Abl as well as other clinically relevant
Bcr-Abl mutants such as G250E, Q252H, Y253H, E255K, E255V, F317L, and M351T in biochemical
and cellular assays. In addition, compound 2 displayed significant in vivo efficacy against T315I-Bcr-
Abl without appreciable toxicity in a bioluminescent xenograft mouse model using a transformed
T315I-Bcr-Abl-Ba/F3 cell line that has a stable luciferase expression. Compound 2 is among the first
type-II inhibitors capable of inhibiting T315I to be described and will serve as a valuable lead to design
the third generation Bcr-Abl kinase inhibitors.
Introduction
mutations in the kinase domain. Extensive in vitro and clinical
work has elucidated a large number of mutations that confer
resistance to imatinib either by directly influencing the drug
binding site or by disfavoring the conformational rearrange-
mentsrequiredforimatinibtobind. Severalsecondgeneration
Bcr-Abl inhibitors have been developed including nilotinib
(AMN107),1 bosutinib (SKI-606),2 and dasatinib (BMS-
354825)3 that are capable of inhibiting most of the known Bcr-
Abl mutants with the exception of the so-called “gatekeeper”
mutation T315I.4
The successful development of the Bcr-Abla inhibitor im-
atinib for the treatment of chronic myelogenous leukemia
(CML) has provided the paradigm for the development of
a host of other small molecule inhibitors targeting kinases
whose activity becomes deregulated in cancer. One major
problem facing the development of selective protein kinase
inhibitors is the emergence of drug resistance caused by
*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.
Several small molecules capable of inhibiting the T315I
mutant in biochemical and cellular assays have been reported.
a Abbreviations: Axl, Axl receptor tyrosine kinase; ATP, adenosine
triphosphate; AUC, area under the concentration time curve; Bcr-Abl,
breakpoint cluster region abelson tyrosine kinase; BMX, BMX non-
receptor tyrosine kinase; CaMKIV, calcium/calmodulin-dependent pro-
tein kinase type IV; CDK, cyclin-dependent kinases; CHK2, checkpoint
homologue 2; CK2, casein kinase II; C-Kit, receptor tyrosine kinase for
the cytokine stem cell factor; CLs, clearance; Clast, last measured
concentration; Cmax, maximum concentration observed; m-CPBA, meta-
chloroperoxybenzoic acid; C-Raf, V-raf-1 murine leukemia viral onco-
gene homologue 1; Csk, C-terminal Src kinase; DCM, dichloromethane;
DDR, discoidin domain receptor tyrosine kinase; DFG, aspartic acid
phenylalanine glycine; DIEA, N,N-diisopropylethylamine; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; EGFR, epidermal
growth factor receptor; F, bioavailability; Fes, feline sarcoma oncogene;
FGFR1, fibroblast growth factor receptor 1; FGFR3, fibroblast growth
factor receptor 3; FLT3, FMS-like tyrosine kinase 3; FLT3-ITD, FLT3
internal tandem duplication; FT-NMR, Fourier transform nuclear
magnetic resonance; GSK3β, glycogen synthase kinase 3β; HATU,
2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluoro-
phosphate; IC50, half-maximal inhibitory concentration; IKKR, IκB
(inhibitor of κ B) kinase R; IKKβ, IκB kinase β; IR, insulin receptor;
Jak1,Janus kinase 1; JNK, c-Jun N-terminal kinase; LCK, lymphocyte-
specific protein tyrosine kinase; Lyn, v-yes-1 Yamaguchi sarcoma
viral related oncogene homologue; MAPK1, mitogen-activated protein
kinase 1; MAPKAP-K2, MAP kinase-activated protein kinase 2;
MEK1, MAP kinase kinase 1; MKK4, MAP kinase kinase 4; MKK6,
MAP kinase kinase 6; NPM-ALK, nucleophosmin-anaplastic lympho-
ma kinase; p70S6K, p70 S6 kinase; P-loop, phosphate-binding loop;
PAK2, p21 activated kinase 2; Pd/C, palladium on carbon or palladium
on charcoal; PDGFR, platelet derived growth factor receptor; PDK1,
phosphoinositide-dependent kinase-1; PEG, polyethylene glycol; PK,
pharmacokinetics; PKBR, AKT family protein kinase; PKCR, protein
kinase C R; PKD2, protein kinase D2; PO, peroral (orally); PP1, 4-amino-
5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; QD, quaque
die/once a day; Rf, retention factor; ROCK-II, Rho-associated coiled-
coil containing protein kinase 2; Rsk1, p90 ribosomal S6 kinase 1;
SAPK2, stress activated protein kinase 2; SAPK3, stress activated
protein kinase 3; SGK, serum/glucocorticoid regulated kinase; SCID,
severe combined immunodeficiency; Src, transforming sarcoma indu-
cing gene of Rous sarcoma virus; Syk, spleen tyrosine kinase; T/C,
treated versus control (untreated); T1/2, elimination half-life; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; Tlast, time of last measured;
Tmax, time to maximum concentration; TPR-Met, translocated promo-
ter region mesenchymal-epithelial transition factor; TrkC, neurotrophic
tyrosine kinase receptor type 3; TrkB, neurotrophic tyrosine kinase
receptor type 2; VEGFR2, vascular endothelial growth factor receptor
2; Vss, volume in steady state; UV, ultraviolet; ZAP-70, ζ-chain-
associated protein kinase 70.
r
2010 American Chemical Society
Published on Web 07/06/2010
pubs.acs.org/jmc

5440 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Choi et al.
(E)-N-Benzyl-2-cyano-3-(3,4-dihydroxyphenyl)acrylamide
(AG-490),5 an inhibitor of Jak2 which is a kinase implicated in
signal transduction downstream of Bcr-Abl, was shown to
induce apoptosis in Ba/F3-Bcr-Abl-T315I cell line.6 2-Cyclo-
pentyl-N-(4-(dipropylphosphoryl)phenyl)-9-ethyl-9H-purin-6-
amine (AP23846),7,8 originally developed as a Src kinase inhi-
bitor, inhibits T315I Bcr-Abl dependent cellular proliferation
(IC50 of 297 nM) but also inhibits parental Ba/F3 cell lines,
suggesting it possesses additional intracellular targets. N-(4-
(4-(5-Methyl-1H-pyrazol-3-ylamino)-6-(4-methylpiperazin-1-yl)-
pyrimidin-2-ylthio)phenyl)cyclopropanecarboxamide (VX-680,
MK-0457),9 originally developed as an aurora kinase inhibi-
tor, exhibitspotent enzymatic inhibition of T315I-Abl(IC50 of
30 nM) but only modestly inhibited cellular autophosphor-
ylation (IC50 of ca. 5 μM) of Ba/F3 transformed with T315I
Bcr-Abl.10 Another Aurora kinase inhibitor, danusertib (PHA-
739358),11 currently being investigated in a phase II clinical
trial for patients with relapsed chronic myelogeneous leuke-
mia, exhibited potent inhibition of T315I-Abl enzyme (IC50 of
5 nM). Crystallographic analysis of danusertib in complex
with T315I-Abl reveals12 that the isoleucine side chain of
T315I mutant does not cause a steric clash with danusertib
in contrast to imatinib. (E)-4-(2-(2-(6-(4-(2-Hydroxyethyl)pip-
erazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazol-5-yl)vinyl)-
phenol (TG101113),13 a thiazole-based inhibitor, also exhi-
bited good potency (IC50 of 66 nM) against T315I mutant
enzyme. Another reported class of Bcr-Abl inhibitor is ex-
emplified by (R,E)-2-(5-methoxy-2-((2,4,6-trimethoxystyryl-
sulfonyl)methyl)phenylamino)propanoic acid (ON012380),14
which is claimed to be a non-ATP-competitive Bcr-Abl inhi-
bitor, potently inhibits imatinib-resistant Bcr-Abl mutants
such as T315I in cellular and biochemical assays, with IC50
values below 10 nM. This compound appears to target sub-
strate binding site of Abl kinase domain, but numerous other
cellular kinases are inhibited by this compound. It should be
noted that most T315I inhibitors disclosed to date are cate-
gorized as type-I kinase inhibitors which bind exclusively to
the ATP binding site of kinase. Recently, a few compounds
from the type-II class that recognize the “DFG-out” con-
formation have been reported to inhibit T315I. These
include(E)-3-(2-(6-(cyclopropylamino)-9H-purin-9-yl)vinyl)-
4-methyl-N-(3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)-
phenyl)benzamide (AP24163)15 and DSA series compounds.16
This 9-(arenethenyl)purine analogue exhibited modest
potency (IC50 of 400 nM) against T315I in biochemical
and cellular assays and N-(3-(3-(4-(4-methoxyphenylamino)-
1,3,5-triazin-2-yl)pyridin-2-ylamino)-4-methylphenyl)-4-((4-
methylpiperazin-1-yl)methyl)benzamide (DSA8) showed great
potency (IC50 of 33 nM) on T315I enzyme along with moderate
antiproliferative activity (IC50 of 500 nM) evaluated using
T315I Bcr-Abl transformed Ba/F3 cells. A cocrystal structure
with wild-type and gatekeeper mutant of Src with a PP1-based
type-II inhibitor revealed how the inhibitor could leave ample
space for an enlarged gatekeeper residue.17 Most recently, it
has been disclosed that 3-(imidazo[1,2-b]pyridazin-3-ylethynyl)-
4-methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoro-
methyl)phenyl)benzamide (AP24534), an imidazo[1,2-b]pyri-
dazine-based multitargeted kinase inhibitor, possesses
potent inhibitory activity against T315I in cellular and in vivo
models.18
Figure 1. 3,4-Dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffold
for type-II T315I inhibitors.
which occupies the ATP binding cleft as well as an immedi-
ately adjacent hydrophobic pocket of Abl kinase domain. A
representative member of this class, 1 (GNF-6)19 was crystal-
lized with Abl and shown to bind in the type-II conformation.
The pyrimidopyrimidinone inhibitor described here, 2, is
capable of inhibiting wild-type and T315I Bcr-Abl activity in
biochemical and cellular assays and also inhibits other clini-
cally relevant Bcr-Abl mutants such as G250E, Q252H, Y253H,
E255K, E255 V, F317L, and M351T. We also demonstrate
that2 is capableofinhibitingT315I-Bcr-Abl dependent tumor
growth in a murine model of CML.1
Chemistry. The synthesis of compounds 1-4 commenced
from commercially available ethyl 4-chloro-2-(methylthio)-
pyrimidine-5-carboxylate. Scheme 1 shows an efficient synthetic
route for making 1-3, all of which bear 3-(trifluoromethyl)-
benzamide moeity in the “tail region”. The quantitative
displacement of chloride group on compound 5 with methy-
lamine proceeded readily at low reaction temperatures. The
reduction of ester compound 6 with LiAlH4 followed by
oxidation using MnO2 afforded the corresponding aldehyde
compound 8 in high yields. One-pot reductive amination
reaction using Na(CN)BH3 proceeded smoothly to provide
diamine derivative 10, which was subsequently subjected to
cyclic urea formation with triphosgene to give com-
pound 11. An elevated reaction temperature was required
to complete this cyclic urea formation. Exposure of the
sulfide group of compound 11 to m-CPBA resulted in the
corresponding sulfone compound 12, which could be trans-
formed into the desired amine compounds. Ammonia and
aniline were readily reacted with this sulfone compound 12
under simple thermal amination conditions to furnish 1 and 3,
respectively, in good (>70%) yields. The synthesis of 2 re-
quired the presence of trifluoroacetic acid and elevated react-
ion temperature to catalyze the coupling of 6-methylpyridin-
3-amine and sulfone compound 12.
Compound 4 contains 4-methylimidazole group on the
3-(trifluoromethyl)benzamide “tail” in common with niloti-
nib. Scheme 2 describes an effective synthetic route which
allows facile modification of 3-(trifluoromethyl)benzamide
“tail” as is exemplified by the synthesis of 4. Amide coupling
reaction of aniline derivative 16 and carboxylic acid deriva-
tive 17 derived from 3-fluoro-5-(trifluoromethyl)benzonitrile
was effected using HATU and DIEA in 89% yield.
Design Rationale for a Type-II T315I Bcr-Abl Inhibitor. An
examination of the cocrystal structures of imatinib,20 nilo-
tinib,1 and dasatinib21 with Abl demonstrate that all three
inhibitors form a critical hydrogen bond with the side-chain
hydroxyl group of T315I and would also require a significant
Results and Discussion
Here we describe a type-II T315I inhibitor (Figure 1), based
upon a 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffold

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5441
Scheme 1a
a Reagents and conditions: (a) methylamine, THF, 0 °C; (b) LiAlH4, THF, 0 °C; (c) MnO2, DCM; (d) Na(CN)BH3, AcOH, MeOH; (e) triphosgene,
DIEA, THF, 0 °C; (f) m-CPBA, DCM, 0 °C; (g) NH3 in 2-propanol or aniline, 1,4-dioxane, 120 °C.
Scheme 2a
a Reagents and conditions: (a) Na(CN)BH3, AcOH, MeOH; (b) triphosgene, DIEA, THF; (c) Pd/C, H2, MeOH; (d) HATU, DIEA, DMF; (e) m-CPBA,
DCM, 0 °C; (f) methylamine in THF, 1,4-dioxane, 120 °C.
rearrangement of their binding conformation to accommo-
date a larger residue at the gatekeeper position. Mutation of
this gatekeeper position appears to be a general theme for re-
sistance to kinase inhibitors.22-25 Pyridopyrimidinones such
as 20 (PD173955, 6-(2,6-dichlorophenyl)-8-methyl-2-[(3-methyl-
sulfanylphenyl)amino]pyrido[6,5-d]pyrimidin-7-one) were
originally developed as inhibitors of Src kinase26 and recep-
tor tyrosine kinase inhibitors such a EGFR, FGFR, PDGFR,
and c-Kit27 and were only later demonstrated to possess potent
cellular and enzymatic activity on wild-type and mutant forms
of Bcr-Abl.28 Co-crystal structures of 20 with Abl (PDB:
1m52) demonstrate that this compound binds to the ATP-bind-
ing site with the kinase otherwise assuming a conformation
normally utilized to bind ATP (type-I binding).20 Interestingly,
20 exhibitsapproximately30-foldmorepotent cellular activity
against Bcr-Abl relative to imatinib, presumably as a con-
sequence of possessing a much higher affinity to the ATP-
binding pocket.28b In contrast, imatinib only exhibits cellular
activity against Bcr-Abl and some receptor kinases, such as
KIT, PDGFR, and DDR1/2, but is known not to inhibit the
cellular activity of any Src-family kinases. The crystal struc-
ture of imatinib bound to the kinase domain of c-Abl20
demonstrated that the compound binds to a conformation
of the kinase where the activation loop is in a so-called “in-
active” conformation (type-II binding). This binding con-
formation allows the piperazine-benzamide “tail” moiety of

5442 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Choi et al.
Table 1. In Vitro Potency Profiling for 3,4-Dihydropyrimido[4,5-
d]pyrimidin-2(1H)-one on Bcr-Abl
compd code
1
2
3
4
Antiproliferative Activity on Native or Bcr-Abl Transformed Ba/F3
Cells (IC50, nM)
native Ba/F3
wtBcr-Abl
G250E
1799
<5
1370
<5
<5
<5
<5
<5
<5
11
2690
<5
12
1785
<5
<5
16
294
<5
Q252H
Y253H
E255K
19
<5
<5
<5
22
15
13
E255 V
T315I
F317L
<5
27
21
30
43
303 (243a)
<5
<5
<5
<5
6
<5
<5
<5
M351T
Enzymatic Activity (IC50, nM)
c-Abl
<5
ND
<5
1567
<5
133
136
122
61
135
206
<5
130
<5
<5
720
<5
<5
ND
G250E
E255 V
T315I
Figure 2. Superimposed structure of 20 (green sticks) bound to Abl
(pink ribbon PDB: 1m52) and nilotinib (yellow sticks) bound to
Abl (PDB: 3cs9). Hydrogen bonds are indicated by red hatched lines
to key amino acids (blue sticks).
M351T
<5
a Inhibitory potency (IC50, nM) against cellular kinase autopho-
sphorylation (Elisa), ND means not determined.
imatinib to access an additional hydrophobic pocket directly
adjacentto the ATP binding site. Bysuperimposing the bound
conformation of 20 and nilotinib as depicted in Figure 2, it
is clear that additional functionality can be appended to the
dichlorophenyl ring of 20 to access this adjacent hydropho-
bic pocket to create a new “hybrid” structure.19,29 We hypo-
thesized that the resulting hybrid compound might be able to
inhibit the T315I mutation due to enhanced affinity both to
the hinge-binding region and to the hydrophobic back-
pocket in addition to not forming a hydrogen bond to the
gatekeeper position.
Table 2. The Growth Inhibitory Potency (IC50, μM) of 2
cell line
IC50, μM
3.401
TPR-Met-Ba/F3
NPM-ALK-Ba/F3
JAK1-Ba/F3
JAK2-Ba/F3
JAK2-V617F-Ba/F3
JAK3-Ba/F3
FGFR3-Ba/F3
Flt3-Ba/F3
1.466
0.044
0.127
0.050
2.018
0.009
0.359
0.012
0.011
0.008
0.005
0.001
5.956
>10
Flt3-ITD-Ba/F3
PDGFR-Ba/F3
TrkC-Ba/F3
Colo205
In Vitro Potency. To assess the cellular activity of the com-
pounds, we tested them against wild-type and mutant Bcr-Abl
transformed Ba/F3 cells. Wild-type Ba/F3 cells require the pre-
senceofinterlukin-3(IL-3) forgrowthandsurvival, but Ba/F3
cells transformed by oncogenic kinase such as Bcr-Abl be-
comes capable of growing in the absence of IL-3, which pro-
vides a robust and commonly used assay for selective kinase
inhibition.30 The first hybrid compound we made, comp-
ound 1, exhibited an IC50 of less than 5 nM on wild-type
Bcr-AblandanIC50 of303nMonT315Iwhile exhibiting non-
specific inhibition of untransformed Ba/F3 cells with an IC50
of 1.7 μM (Table 1). Compound 1 also effectively inhibited
cellular kinase autophosphorylation of T315I-Bcr-Abl-Ba/
F3 with an IC50 of 243 nM, further demonstrating that the
antiproliferative activity against this mutant correlates with
direct inhibition of the T315I-Abl enzyme. A cocrystal struc-
ture19 of 1 with Abl revealed that the compound bound to a
conformation that was virtually indistinguishable from that
used by imatinib, which clearly validates the design stra-
tegy.31 We next evaluated how altering the structure in the
hinge binding region and hydrophobic back pocket might
change the potency of cellular inhibition. We replaced the
amino-pyrimidine hinge binding motif of 1 with the pheny-
laminopyrimidine motif, which is known to form a stronger
interaction with the kinase hinge residues as shown for 2 and
3. These modifications improved absolute cellular potency
against T315I as well as increase the ratio for selectivity
relative to untransformed Ba/F3 cells. In addition, these
modifications resulted in improved inhibitory potency on
SW620
U87
HEK293T
T315I enzyme as well as against T315I cellular autopho-
sphorylation. We next incorporated a 3-methylimidazole by
analogy to nilotinib, and the resulting compound 4 also
demonstrated excellent activity against wild-type and mu-
tant Bcr-Abl (Table 1). In particular, this methylimidazole of
4significantly contributes to excellent potency (IC50 of <5 nM)
of 4 against T315I enzyme.
Compound 2 displayed excellent growth inhibitory activ-
ity against human colon cancer cells (Colo205 and SW620),
while a noncancer cell line, HEK293T, was not particularly
sensitive to inhibition by 2 as described in Table 2. As dis-
cussed below, the large number of kinases inhibited by 2
would suggest that additional profiling of this inhibitor
against a larger panel of human cancer cell lines32 would
potentially reveal additional utility for this inhibitor.
Kinase Selectivity of 2. We next investigated the kinase
selectivity of 2 against a panel of 41 recombinant kinase en-
zymes and on a panel of Ba/F3 cell lines transformed with a
diverse set of tyrosine kinases as summarized in Tables 2 and
3. This analysis revealed that 2 is capable of potently inhi-
biting a large number of tyrosine and serine/threonine ki-
nases. Despite its broad kinase selectivity profile, compound

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5443
Table 3. Kinase Selectivity Profile of 2: Enzymatic Activity
(% Inhibition) of 2 at 10 μM
% inhibition at
10 μM
% inhibition at
10 μM
kinase
kinase
Aurora-A(h)
Axl(h)
Bmx(h)
31
69
98
100
0
MAPKAP-K2(h)
MEK1(h)
MKK4(m)
MKK6(h)
p70S6K(h)
PAK2(h)
0
58
98
100
92
45
62
0
c-RAF(h)
CaMKIV(h)
CDK1/cyclinB(h)
CHK2(h)
CK2(h)
3
12
6
PDGFRR(h)
PDK1(h)
CSK(h)
Fes(h)
100
98
100
69
59
69
49
89
97
99
99
68
PKBR(h)
7
PKCR(h)-His
PKCθ(h)
PKD2(h)
51
22
77
2
FGFR3(h)
Flt3(h)
GSK3ss(h)
IKKR(h)
IKKss(h)
IR(h)
ROCK-II(h)
Rsk1(h)
95
95
98
35
0
SAPK2a(h)
SAPK2b(h)
SAPK3(h)
SGK(h)
JNK1R1(h)
JNK2R2(h)
Lck(h)
Syk(h)
TrkB(h)
ZAP-70(h)
98
100
6
MAPK1(h)
Table 4. Pharmacokinetic Properties of 2 on Balb/C Mouse
PEG300 (100%),
solution
PEG300/water 1:1,
solution
formulation
dosing
5 mpk, intravenous
8.6
20 mpk, peroral
CLs (mL/min/kg)
Vss (L/kg)
AUC (h nM)
1.12
18527
11707
0.03
10
875.71
3616
3
3
C
Tmax (h)
max (nM)
Figure 3. Bioluminescent in vivo efficacy study (oral administra-
tion, once-daily dosing) for 2 using Ba/F3-T315I-Bcr-Abl cell line
that has stable luciferase expression. Mice were imaged at day 5 and
7 after 2 treatment.
Clast (nM)
T
15
24
last (h)
T/2 (h)
F (%)
24
3.8
3.2
36
(F = 36%) being observed following oral administration of a
single dose of 20 mg/kg. Compound 2 displayed significant in
vivo efficacy against T315I-Bcr-Abl in the bioluminescent
xenograft mouse model using a transformed T315I-Bcr-Abl-
Ba/F3 cell line that has stable luciferase expression. As illus-
trated in the Figure 3, light emission from mice that were
administered an oral dose of 10 or 20 mg/kg of 2 once per day
was significantly (T/C 38% and 23%, respectively) reduced
compared with that from untreated control mice, indicating
that 2 effectively inhibits tumor growth of T315I-Bcr-Abl-
Ba/F3 cells in mice at low doses (10 mg/kg). However,
appreciable (>10%) body weight loss was observed in mice
treated with doses of 2 of 20 mg/kg and above which is a
common symptom of in vivo toxicity. However, the 10 mg/kg
dosing group exhibited only a very small weight change as
plotted in the Figure 4. The significant body weight loss in
the 20 mg/kg group forced the dosing to be discontinued at
day 6. An acceptable therapeutic index of 2 would be anti-
cipated at around 10 mg/kg dose, as remarkable efficacy was
observed with little body weight at this dose.
2 exhibits some selectivity (4-100-fold) for T315I Bcr-Abl
(IC50 of 11 nM) relative to kinases such as TPR-Met, NPM-
ALK, JAK-3, Flt-3. Although the cocrystal structure of 1
with Abl demonstrates binding to the inactive conformation
(type-II), it is possible that an alternative binding conforma-
tion is exploited when binding to a subset of the potently
inhibited kinases. Further work will be required to inves-
tigate to what extent broad inhibition of mutant alleles of
Bcr-Abl can be achieved while still maintaining significant
selectivity relative to other kinases. Considering that the gate-
keeper residue is a crucial kinase selectivity determinant,24
this will likely present a significant challenge. For example, a
recently disclosed type-II inhibitor, imidazo[1,2-b]pyridazine
derivative18 currently undergoing phase I clinical trials, ex-
hibits high potency against T315I as well as several kinases
such as c-Src, Lyn, c-Kit, VEGFR2, FGFR1, and PDGFR.
In Vivo Efficacy and Toxicity of 2. To investigate whether 2
could inhibit wild-type and T315I Bcr-Abl at well tolerated
doses in vivo, we investigated the tumor efficacy using a luci-
ferase xenograft model. One shortcoming of the original 20
compound was a poor pharmacokinetic profile in mice. Com-
pound 2 exhibited excellent pharmacokinetic parameters in
mice as depicted in Table 4, with good systemic exposure
Conclusion
Our results demonstrate thatitispossibletodesign a type-II
inhibitor that can circumvent the T315I Bcr-Abl “gatekeeper”
mutation by bridging the ATP and adjacent hydrophobic
(AUC = 875.71 h nM, Cmax = 3.6 μM) along with reason-
3
able half-life (t1/2 = 3.2 h) and favorable oral bioavailability

5444 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Choi et al.
FT-NMR (400 MHz for 1H, and 100 MHz for 13C), or a Varian
Inova-600 (600 MHz for 1H) spectrometer. Chemical shifts are
reported relative tochloroform(δ = 7.26) for 1H NMRand chlo-
roform (δ = 77.2) for 13C NMR or dimethyl sulfoxide (δ = 2.50)
for 1H NMR and dimethyl sulfoxide (δ = 39.5) for 13C NMR.
Data are reported as (br = broad, s = singlet, d = doublet, t =
triplet, q=quartet, m =multiplet). Highresolutionmassspectra
were recorded on a 4.7 T IonSpec ESI-FTMS or a Micromass
LCT ESI-TOF mass spectrometer.
Ethyl 4-(Methylamino)-2-(methylthio)pyrimidine-5-carboxy-
late (6). To a solution of compound 5 (1.0 g, 4.3 mmol) in
THF (14.3 mL) was added 2.0 M methylamine solution in THF
(5.37 mL, 10.7 mmol) at 0 °C, and the reaction mixture was
stirred at room temperature for 1 h. The reaction mixture was
partitioned between ethyl acetate (50 mL) and saturated NaH-
CO3 (30 mL). The organic layer was washed with brine, dried
over MgSO4, filtered through a pad of celite, and concentrated
under reduced pressure. The resulting white solid title com-
pound (950 mg, 97% yield) was used for the next step without
Figure 4. Mice body weight change during bioluminescent in vivo
efficacy study (oral administration, once-daily dosing) for 2.
binding site using a linker segment that can accommodate
a larger gatekeeper residue. The ability of the compounds
to tolerate diverse gatekeeper amino acids results in com-
pounds with broad kinase selectivity profiles. This study
also demonstrates that type-II inhibitors as a class are not
necessarily more selective than type-I inhibitors. Compound
2 is likely to serve as a valuable starting point for developing
type-II inhibitors for a variety of kinases of therapeutic
interest.
1
further purification. Rf = 0.47 (1/4 ethyl acetate/hexane). H
NMR 600 MHz (CDCl3) δ 8.60 (s, 1H), 8.16 (bs, 1H), 4.32 (q,
J = 7.2 Hz, J = 13.8 Hz, 2H), 3.07 (d, J = 5.4 Hz, 3H), 2.54 (s,
3H), 1.36 (t, J = 7.2 Hz, 3H). 1H NMR 400 MHz (DMSO-d6) δ
8.49 (s, 1H), 8.23 (bs, 1H), 4.26 (q, J = 7.1 Hz, J = 14.1 Hz, 2H),
2.96 (d, J = 4.8 Hz, 3H), 2.48 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H).
13C NMR 100 MHz (DMSO-d6) δ 175.49, 166.22, 160.14,
158.13, 101.26. 61.01, 27.80, 14.53, 14.04. HRMS (ESI) m/z
[M þ Na]þ calcd 250.0626, found 250.0628.
Experimental Section
Detailed experimental procedures for Bcr-Abl kinase assay,
Ba/F3 cellproliferation assay, and phosphotyrosine analysis such
as autophosphorylation estimation are described in our previous
publications.19,33
1. In Vivo Efficacy Evaluation Using T315I-Bcr-Abl-Ba/F3
Orthotopic Xenograft Model. SCID beige female mice, 6-8
weeks old (n = 5 for each 2 treated or vehicle control group),
were injected via tail vein with 1 ꢀ 106 Ba/F3 cells coexpressing
Bcr-abl/T315I mutant and luciferase. Three days postinjection,
mice were orally dosed once daily with 10 or 20 mg/kg 2 for seven
days. At day 5 and 7, bioluminescence was quantified using a
Xenogen IVIS living imaging system (Caliper LifeSciences,
Hopkinton, MA).
2. Experimental Section of PK Study. Male Balb/c mice, 5-6
weeks old (20-25 g), were obtained from Jackson Laboratory
(Bar Harbor, ME). Compound 2 was dissolved in a 100%
PEG300 solution formulation (2.5 mg/mL) and dosed at 5 mg/kg
intravenously via the lateral vein (n = 3). The oral dose was pre-
pared in a 1:1 formulation of PEG300 and distilled water and
administered at 20 mg/kg via oral gavage (n = 3). Five blood
samples (50 μL) were serially drawn via retro orbital sinus within
24 h after dosing. Plasma concentrations of 2 were quantified
utilizing a liquid chromatography/mass spectrometry (LC/MS/
MS) assay. Pharmacokinetic parameters were calculated by
noncompartmental regression analysis using Winnonlin 4.0
software (Pharsight, Mountain View, CA).
(4-(Methylamino)-2-(methylthio)pyrimidin-5-yl)methanol (7).
To a solution of compound 6 (300 mg, 1.32 mmol) in THF
(6.6 mL) was added 2.0 M lithium aluminum hydride solution in
THF (0.79 mL, 1.58 mmol) at 0 °C. The reaction mixture was
stirred at room temperature for 2 h and treated with saturated
NH4Cl solution (2 mL). After stirred at room temperature for
30 min, the reaction mixture was filtered through a pad of celite.
The filtrate was partitioned between ethyl acetate (30 mL) and
water (20 mL). The organic layer was washed with brine, dried
over MgSO4, filtered through a pad of celite, and concentrated
under reduced pressure. The resulting white solid (210 mg, 86%
yield) was used for the next step without further purification.
Rf = 0.37 (100% ethyl acetate). 1H NMR 600 MHz (CDCl3) δ
7.61 (s, 1H), 5.90 (bs, 1H), 4.76 (s, 2H), 3.03 (d, J = 5.4 Hz, 3H),
2.51 (s, 3H). 1H NMR 400 MHz (DMSO-d6) δ 7.80 (s, 1H), 6.81
(bs, 1H), 5.05, (t, J = 5.4 Hz, 1H), 4.28 (d, J = 5.2 Hz, 2H), 2.84
(d, J = 4.6 Hz, 3H), 2.41 (s, 3H). 13C NMR 100 MHz (DMSO-
d6) δ 169.17, 160.45, 152.04, 113.90, 57.99, 27.71, 13.79. HRMS
(ESI) m/z [M þ Na]þ calcd 208.0521, found 208.0520.
4-(Methylamino)-2-(methylthio)pyrimidine-5-carbaldehyde (8).
To a solution of compound 7 (210 mg, 1.14 mmol) in dichlor-
omethane (3.8 mL) was added activated manganese(IV) oxide
(980 mg, 11.4 mmol) at room temperature and stirred for over-
night. The reaction mixture was filtered through a pad of celite
and concentrated under reduced pressure. The resulting crude
product was purified by flash silica gel chromatography with
ethyl acetate/hexane (1/9 to 1/4) to give (190 mg, 89% yield) of
the title product as a white solid. Rf = 0.29 (1/4 ethyl acetate/
3. Chemistry. Reactions were monitored by thin layer chro-
matography (TLC) with 0.25 mm E. Merck precoated silica gel
plates (60 F254). Visualization was accomplished with either
UV light, or by immersion in solutions of ninhydrin, p-anisal-
dehyde, or phosphomolybdic acid (PMA) followed by heating
on a hot plate for about 10 s. Purification of reaction products
was carried out by flash chromatography using Kieselgel 60 Art
9385 (230-400 mesh). The purity of all compounds was over
95% and was analyzed with Waters LCMS system (Waters 2998
photodiode array detector, Waters 3100 mass detector, Waters
SFO system fluidics organizer, Waters 2545 binary gradient
module, Waters Reagent Manager, Waters 2767 sample manager)
using SunFireTM C18 column (4.6 mm ꢀ 50 mm, 5 μm particle
size): solvent gradient = 60% (or 95%) A at 0 min, 1% A at
5 min; solvent A = 0.035% TFA in Water; solvent B = 0.035%
1
hexane). H NMR 600 MHz (CDCl3) δ 9.67 (s, 1H), 8.52 (bs,
1H), 8.27 (s, 1H), 3.10 (d, J = 5.4 Hz, 3H), 2.54 (s, 3H). 1H NMR
400 MHz (DMSO-d6) δ 9.75 (s, 1H), 8.62 (bs, 1H), 8.49 (s, 1H),
2.98 (d, J = 4.8 Hz, 3H), 2.49 (s, 3H). 13C NMR 100 MHz
(DMSO-d6) δ 192.11, 176.34, 163.80, 158.87, 109.95, 27.57,
14.12. LCMS m/z 184.2 [M þ H]þ.
N-(3-Amino-4-methylphenyl)-3-(trifluoromethyl)benzamide (9).
To a solution of 4-methyl-3-nitrobenzenamine (2.0 g, 13.15 mmol)
in dried THF (65 mL) were added DIEA (3.26 mL, 19.73 mmol)
and 3-(trifluoromethyl)benzoyl chloride (3.0 g, 14.46 mmol) at
0 °C. The reaction mixture stirred for 3 h at room temperature.
The reaction mixture was partitioned between ethyl acetate
(100 mL) and saturated NaHCO3 (50 mL). The organic layer
was washed with brine, dried over MgSO4, filtered through a
1
TFA in MeOH; flow rate: 3.0 (or 2.5) mL/min. H NMR and
13C NMR spectra were obtained using a Bruker 400 MHz

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5445
pad of celite, and concentrated under reduced pressure. The result-
ing white solid, N-(4-methyl-3-nitrophenyl)-3-(trifluoromethyl)-
benzamide (3.9 g, 91% yield) was used for the next step without
(DMSO-d6) δ 10.52 (s, 1H), 8.41 (s, 1H), 8.24 (m, 2H), 7.97 (d,
J = 7.6 Hz, 1H), 7.79 (m, 2H), 7.63 (dd, J = 1.9 Hz, J = 8.2 Hz,
1H), 7.31 (d, J = 6.3 Hz, 1H), 4.78 (d, J = 14.6 Hz, 1H), 4.59
1
further purification. Rf = 0.41 (1/4 ethyl acetate/hexane). H
(d, J = 14.6 Hz, 1H), 3.38 (s, 3H), 2.49 (s, 3H), 2.07 (s, 3H). 13
C
NMR 400 MHz (DMSO-d6) δ 10.75 (s, 1H), 8.51 (d, J = 1.6,
1H), 8.30 (s, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.00 (m, 2H), 7.80 (t,
J = 7.7 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 2.48 (s, 3H). 13C
NMR 100 MHz (DMSO-d6) δ 164.72, 148.90, 138.22, 135.56,
133.52, 132.37, 130.27, 129.86, 129.54, 128.95, 128.49, 125.39, 124.72,
116.14, 19.75. HRMS (ESI) m/z [M þ Na]þ calcd 347.0619, found
347.0621.
To a solution of N-(4-methyl-3-nitrophenyl)-3-(trifluoromethyl)-
benzamide (3.0 g, 9.2 mmol) in methanol (40 mL) was added
10% Pd/C (300 mg). After two vacuum/H2 cycles to replace air
inside the reaction flask with hydrogen, the reaction mixture was
stirred at room temperature under a hydrogen balloon for 4 h.
The reaction mixture was filtered through a pad of celite and
concentrated under reduced pressure. The title compound (2.4 g,
88% yield) was used for the next step without further purifica-
tion. Rf = 0.14 (1/4 ethyl acetate/hexane). 1H NMR 400 MHz
(DMSO-d6) δ 10.17 (s, 1H), 8.27 (s, 1H), 8.25 (d, J = 7.9 Hz,
1H), 7.91 (d, J = 7.8 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.15 (s,
1H), 6.87 (m, 2H), 4.89 (s, 2H), 2.04 (s, 3H). 13C NMR 100 MHz
(DMSO-d6) δ 164.02, 147.05, 137.70, 136.62, 132.18, 130.20,
130.00, 129.75, 129.43, 128.22, 124.62, 117.67, 109.39, 106.99,
17.45. HRMS (ESI) m/z [M þ H]þ calcd 317.0878, found
317.0879.
N-(4-Methyl-3-((4-(methylamino)-2-(methylthio)pyrimidin-5-yl)-
methylamino)phenyl)-3-(trifluoromethyl)benzamide (10). To a
solution of compound 8 (190 mg, 1.03 mmol) in methanol
(5.2 mL) were added acetic acid (0.12 mL, 2.06 mmol), com-
pound 9(304 mg, 1.03 mmol), and Na(CN)BH3 (323 mg, 5.15 mmol)
at room temperature and stirred for overnight. The reaction
mixture was partitioned between ethyl acetate (30 mL) and
saturated NaHCO3 (20 mL), and then the water layer was
extracted with ethyl acetate (10 mL ꢀ 3). The combined organic
layer was washed with brine, dried over MgSO4, filtered through
a pad of celite, and concentrated under reduced pressure. The
resulting crude product was purified by silica gel chromatogra-
phy with ethyl acetate/hexane (1/4 to 2/3) to give (334 mg, 63%
yield) of the title product as a white solid. Rf = 0.21 (2/3 ethyl
acetate/hexane). 1H NMR 400 MHz (CDCl3) δ 8.55 (s, 1H), 8.11
(s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.76 (s, 1H), 7.74 (d, J = 7.8 Hz,
1H), 7.54 (t, J = 7.8 Hz, 1H), 7.23 (s, 1H), 7.02 (d, J = 8.0 Hz,
1H), 6.97 (dd, J = 1.7 Hz, J = 7.9 Hz, 1H), 5.86 (m, 1H), 3.98 (s,
2H), 3.41 (bs, 1H), 3.01 (d, J = 4.85 Hz, 3H), 2.51 (s, 3H), 2.07 (s,
3H). 13C NMR 100 MHz (CDCl3) δ 171.17, 164.59, 161.14,
153.43, 145.99, 137.03, 135.79, 131.22, 130.89, 130.45, 129.25,
128.18, 124.14, 120.09, 110.69, 109.33, 103.89, 43.57, 27.60,
17.11, 14.02. HRMS (ESI) m/z [M þ Na]þ calcd 484.1395, found
484.1398.
N-(4-Methyl-3-(1-methyl-7-(methylthio)-2-oxo-1,2-dihydropyri-
mido[4,5-d]pyrimidin-3(4H)-yl)phenyl)-3-(trifluoromethyl)benzamide
(11). To a solution of compound 10 (334 mg, 0.73 mmol) in 1,4-
dioxane (3.65 mL) were added DIEA (0.36 mL, 2.19 mmol) and
triphosgene (70 mg, 0.24 mmol) at 0 °C and stirred at room
temperature for 1 h. The precipitate was filtered off and the
filtrate was stirred at 110 °C for 3 h. The reaction mixture was
cooled to room temperature and was partitioned between ethyl
acetate (20 mL) and saturated NaHCO3 (20 mL) solution. The
organic layer was washed with brine, dried over MgSO4, filtered
through a pad of celite, and concentrated under reduced pres-
sure. The resulting crude product was purified by silica gel
chromatography with ethyl acetate/hexane (1/4 to 2/3) to give
(230 mg, 65% yield) of the title product as a white solid. Rf = 0.25
(2/3 ethyl acetate/hexane). 1H NMR 600 MHz (CDCl3) δ 8.98 (s,
1H), 8.13 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 8.03 (s, 1H), 7.73 (m,
2H), 7.56 (t, J = 7.8 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.03 (d,
J = 8.4 Hz, 1H), 4.68 (d, J = 14.4 Hz, 1H), 4.42 (d, J = 14.4 Hz,
1H), 3.51 (s, 3H), 2.59 (s, 3H), 1.72 (s, 3H). 1H NMR 400 MHz
NMR 100 MHz (DMSO-d6) δ 170.34, 164.33, 156.87, 152.76,
152.23, 141.38, 138.06, 136.06, 132.29, 131.39, 131.25, 130.26,
128.66, 124.61, 120.38, 119.77, 108.31, 46.87, 28.53, 17.23,
14.07. HRMS (ESI) m/z [M þ Na]þ calcd 510.1187, found
510.1186.
N-(4-Methyl-3-(1-methyl-7-(methylsulfonyl)-2-oxo-1,2-dihydro-
pyrimido[4,5-d]pyrimidin-3(4H)-yl)phenyl)-3-(trifluoromethyl)-
benzamide (12). To a solution of compound 11 (230 mg,
0.47 mmol) in dichloromethane (2 mL) was added m-chloro-
perbenzoic acid (245 mg, 1.42 mmol) at 0 °C. The reaction
mixture was stirred for 30 min at 0 °C and then stirred for
additional 3 h at room temperature. The reaction mixture was
partitioned between dichloromethane (20 mL) and saturated
NaHCO3 (20 mL). The organic layer was washed with brine and
dried over MgSO4, filtered through a pad of celite, and con-
centrated under reduced pressure. The resulting white solid
(200 mg, 81% yield) was used for the next step without further
1
purification. Rf = 0.50 (4/1 ethyl acetate/hexane). H NMR
600 MHz (DMSO-d6) δ 10.52 (s, 1H), 8.57 (s, 1H), 8.28 (s, 1H),
8.24 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.84 (dd, J =
1.8 Hz, J = 9.6 Hz, 1H), 7.78 (t, J = 7.8 Hz, 1H), 7.62 (d, J =
8.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 4.93 (d, J = 15.6 Hz, 1H),
4.75 (d, J = 15.6 Hz, 1H), 3.39 (s, 3H), 2.47 (s, 3H), 2.12 (s, 3H).
1H NMR 400 MHz (CDCl3) δ 9.05 (s, 1H), 8.24 (s, 1H), 8.11 (s,
1H), 8.08 (d, J = 7.7 Hz, 1H), 7.72 (m, 2H), 7.56 (t, J = 7.7 Hz,
1H), 7.31 (d, J = 8.2 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 4.81 (d,
J = 15.8 Hz, 1H), 4.59 (d, J = 15.8 Hz, 1H), 3.47 (s, 3H), 3.33 (s,
3H), 1.67 (s, 3H). 13C NMR 100 MHz (CDCl3) δ 164.90, 164.19,
158.12, 152.38, 152.31, 139.21, 137.75, 135.27, 131.39, 131.23,
130.78, 130.13, 129.21, 128.24, 127.85, 124.25, 120.88, 119.52,
114.54, 47.07, 39.03, 29.00, 16.31. HRMS (ESI) m/z [M þ Na]þ
calcd 542.1086, found 542.1088.
N-(3-(7-Amino-1-methyl-2-oxo-1,2-dihydropyrimido[4,5-d]pyri-
midin-3(4H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide
(1). To a solution of compound 12 (100 mg, 019 mmol) in 1,4-
dioxane (1.0 mL) was added 2.0 M ammonia in 2-propanol
(0.97 mL, 1.93 mmol). The reactionmixture was stirred for 24 h at
120 °C in sealed reaction vessel. The reaction mixture was cooled
to room temperature and concentrated under reduced pressure.
The crude reaction mixture was partitioned between ethyl
acetate (10 mL), and the water layer was extracted with ethyl
acetate (5 mL ꢀ 3). The combined organic layer was washed
with brine, dried with MgSO4, filtered through a pad of celite,
and concentrated under reduced pressure. The resulting crude
product was purified by silica gel chromatography with MeOH/
CH2Cl2 (1/99 to 5/95) to give (68 mg, 77% yield) of the title
product as a white solid. Rf = 0.27 (5/95 MeOH/CH2Cl2). 1H
NMR 400 MHz (DMSO-d6) δ 10.52 (s, 1H), 8.29 (s, 1H), 8.26
(d, J = 7.9 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.92 (s, 1H), 7.78 (t,
J = 7.8 Hz, 1H), 7.74 (d, J = 2.1 Hz, 1H), 7.64 (dd, J = 2.0 Hz,
J = 8.3 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 6.57 (s, 2H), 4.60 (d, J =
13.8 Hz, 1H), 4.42 (d, J = 13.8 Hz, 1H), 3.23 (s, 3H), 2.07 (s, 3H).
13C NMR 100 MHz (DMSO-d6) δ 164.32, 163.22, 157.51,
153.94, 152.91, 141.80, 138.02, 136.07, 132.30, 131.38, 131.17,
130.25, 128.65, 124.61, 120.16, 119.80, 100.86, 47.08, 28.35,
17.27. HRMS (ESI) m/z [M þ Na]þ calcd 479.1419, found
479.1417.
N-(4-Methyl-3-(1-methyl-7-(6-methylpyridin-3-ylamino)-2-oxo-
1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)phenyl)-3-(trifluo-
romethyl)benzamide (2). To a solution of compound 12 (100 mg,
019 mmol) in 1,4-dioxane (1.0 mL) were added 6-methylpyridin-
3-amine (104 mg, 0.96 mmol) and trifluoroacetic acid (73 uL,
0.96 mmol). The reaction mixture was stirred for 48 h at 120 °C.
The reaction mixture was cooled to room temperature and was
concentrated. The crude reaction mixture was partitioned be-
tween ethyl acetate (20 mL) and saturated NaHCO3 (20 mL)

5446 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Choi et al.
solution. The organic layer was washed with brine, dried with
MgSO4, filtered through a pad of celite, and concentrated under
reduced pressure. The resulting crude product was purified by
silica gel chromatography with MeOH/CH2Cl2 (1/99 to 5/95) to
give (34 mg, 32% yield) of the title product as a white solid. Rf =
were added DIEA (0.31 mL, 1.88 mmol) and triphosgene
(65 mg, 0.22 mmol) at 0 °C and stirred at room temperature
for 1 h. The precipitate was filtered off and the filtrate was stirred
at 110 °C for 3 h. The reaction mixture was cooled to room
temperature and was partitioned between ethyl acetate (20 mL)
and saturated NaHCO3 (20 mL) solution. The organic layer was
washed with brine, dried over MgSO4, filtered through a pad of
celite, and concentrated under reduced pressure. The resulting
crude product was purified by silica gel chromatography with
ethyl acetate/hexane (1/4 to 2/3) to give (170 mg, 78% yield) of
the title product as a yellow solid. Rf = 0.39 (2/3 ethyl acetate/
hexane). 1H NMR 400 MHz (CDCl3) δ 8.16 (d, J = 2.2 Hz, 1H),
8.15 (dd, J = 2.3 Hz, J = 8.3 Hz, 1H), 8.10 (s, 1H), 7.49 (d, J =
8.3 Hz, 1H), 4.85 (d, J = 14.1 Hz, 1H), 4.52 (d, J = 14.6 Hz, 1H),
3.47 (s, 3H), 2.59 (s, 3H), 2.33 (s, 3H). 13C NMR 100 MHz
(CDCl3) δ 172.29, 156.49, 152.38, 151.97, 147.04, 144.22,
141.34, 132.00, 122.97, 122.35, 106.31, 47.16, 28.59, 18.17,
14.23. HRMS (ESI) m/z [M þ Na]þ calcd 368.0793, found
368.0794.
3-(5-Amino-2-methylphenyl)-1-methyl-7-(methylthio)-3,4-dihy-
dropyrimido[4,5-d]pyrimidin-2(1H)-one (16). To a solution of
compound 15 (150 mg, 0.43 mmol) in methanol (2 mL) was
added 10% Pd/C (15 mg). After two vacuum/H2 cycles to replace
air inside the reaction flask with hydrogen balloon, the reaction
mixture was stirred at room temperature for 6 h. The reaction
mixture was filtered through a pad of Celite and concentrated
under reduced pressure. The title product (130 mg, 94% yield)
was used for the next step without further purification. Rf = 0.31
(4/1 ethyl acetate/hexane). 1H NMR 400 MHz (CDCl3) δ 8.03 (s,
1H), 7.06 (d, J = 8.1 Hz, 1H), 6.62 (dd, J = 2.4 Hz, J = 8.1 Hz,
1H), 6.56 (d, J = 2.3 Hz, 1H), 4.66 (d, J = 14.6 Hz, 1H), 4.48 (d,
J = 14.6 Hz, 1H), 3.44 (s, 3H), 2.57 (s, 3H), 2.09 (s, 3H). 13C
NMR 100 MHz (CDCl3) δ 171.71, 156.82, 152.50, 151.67,
145.75, 141.05, 131.97, 124.93, 115.43, 113.56, 160.82, 47.19,
28.44, 16.64, 14.20. HRMS (ESI) m/z [M þ Na]þ calcd 338.1052,
found 338.1055.
3-(4-Methyl-1H-imidazol-1-yl)-5-(trifluoromethyl)benzoic Acid
(17). To a solution of 3-fluoro-5-(trifluoromethyl)benzonitrile
(5.0 g, 26.45 mmol) in N,N-dimethylacetamide (25 mL) was
added 4-methyl imidazole (6.5 g, 79.36 mmol). The reaction
mixture was stirred at 150 °C for 24 h. The reaction mixture was
cooled to room temperature. The crude reaction mixture was
partitioned between ethyl acetate (100 mL), and the water layer
was extracted with ethyl acetate (5 mL ꢀ 3). The combined
organic layer was washed with brine, dried with MgSO4, filtered
through a pad of celite, and concentrated under reduced pres-
sure. The resulting crude product was purified by silica gel
chromatography with ethyl acetate/hexane (1/3 to 2/1) to give
(4.8 g, 73% yield) 3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoromet-
hyl)benzonitrile as a white solid. Rf = 0.21 (3/2 ethyl acetate/
hexane). 1H NMR 400 MHz (DMSO-d6) δ 8.53 (s, 1H), 8.45 (s,
1H), 8.40 (s, 1H), 8.24 (s, 1H), 7.71 (s, 1H), 2.15 (s, 3H). 13C
NMR 100 MHz (DMSO-d6) δ 139.60, 138.64, 135.73, 132.38,
132.5, 127.22, 126.89, 121.07, 117.45, 114.55, 114.44, 14.00. HRMS
(ESI) m/z [M þ Na]þ calcd 274.0568, found 274.0569.
1
0.23 (5/95 MeOH/CH2Cl2). H NMR 600 MHz (DMSO-d6) δ
10.50 (s, 1H), 9.61 (s, 1H), 8.77 (d, J = 3.0 Hz, 1H), 8.28 (s, 1H),
8.25 (d, J = 8.4 Hz, 1H), 8.13 (s, 1H), 8.04 (dd, J = 2.4 Hz, J =
8.4 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.78 (m, 2H), 7.63 (dd,
J = 1.8 Hz, J = 8.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.16 (d, J =
9.0 Hz, 1H), 4.69 (d, J = 13.8 Hz, 1H), 4.52 (d, J = 13.8 Hz, 1H),
3.32 (s, 3H), 2.40 (s, 3H), 2.12 (s, 3H). 1H NMR 400 MHz
(CDCl3) δ 9.19 (s, 1H), 8.74 (d, J = 2.2 Hz, 1H), 8.17 (s, 1H),
8.13 (d, J = 7.7 Hz, 1H), 8.00 (dd, J = 2.3 Hz, J = 8.4 Hz, 1H),
7.94 (s, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.69 (s, 1H), 7.56 (m, 2H),
7.30 (d, J = 8.2 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.00 (d, J =
8.4 Hz, 1H), 4.64 (d, J = 14.1 Hz, 1H), 4.42 (d, J = 14.2 Hz, 1H),
3.51 (s, 3H), 2.54 (s, 3H), 1.62 (s, 3H). 13C NMR 100 MHz
(CDCl3) δ 164.23, 159.20, 157.48, 153.49, 152.88, 152.19,
140.43, 139.91, 137.72, 135.61, 133.71, 131.38, 131.02, 130.87,
129.05, 128.06, 127.23, 124.38, 123.05, 120.72, 120.03, 102.73,
47.29, 28.73, 23.61, 16.24. HRMS (ESI) m/z [M þ Na]þ calcd
570.1841, found 542.1839.
N-(4-Methyl-3-(1-methyl-2-oxo-7-(phenylamino)-1,2-dihydropyri-
mido[4,5-d]pyrimidin-3(4H)-yl)phenyl)-3-(trifluoromethyl)benzamide
(3). To a solution of compound 12 (100 mg, 019 mmol) in 1,4-
dioxane (0.5 mL) was added aniline (0.17 mL, 1.93 mmol). The
reaction mixture was stirred for 24 h at 130 °C. The reaction
mixture was cooled to room temperature and concentrated
under reduced pressure. The crude reaction mixture was parti-
tioned between ethyl acetate (10 mL), and the water layer was
extracted with ethyl acetate (5 mL ꢀ 3). The combined organic
layer was washed with brine, dried with MgSO4, filtered through
a pad of celite, and concentrated under reduced pressure. The
resulting crude product was purified by silica gel chromatogra-
phy with MeOH/CH2Cl2 (1/99 to 5/95) to give (72 mg, 70%
yield) of the title product as a white solid. Rf = 0.30 (5/95
1
MeOH/CH2Cl2). H NMR 400 MHz (DMSO-d6) δ 10.53 (s,
1H), 9.56 (s, 1H), 8.30 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H), 8.14 (s,
1H), 7.97 (d, J = 7.8 Hz, 1H), 7.78 (m, 4H), 7.65 (dd, J = 2.0 Hz,
J = 8.3 Hz, 1H), 7.29 (m, 3H), 6.93 (t, J = 7.4 Hz, 1H), 4.71 (d,
J = 14.1 Hz, 1H), 4.53 (d, J = 13.8 Hz, 1H), 3.26 (s, 3H), 2.13 (s,
3H). 13C NMR 100 MHz (DMSO-d6) δ 164.33, 159.46, 157.39,
153.65, 152.65, 141.67, 141.02, 138.06, 136.08, 132.30, 131.42,
131.22, 130.25, 128.95, 128.64, 124.62, 121.75, 120.26, 119.82,
119.25, 103.12, 47.12, 28.70, 17.27. HRMS (ESI) m/z [M þ Na]þ
calcd 555.1732, found 555.1730.
N-Methyl-5-((2-methyl-5-nitrophenylamino)methyl)-2-(methyl-
thio)pyrimidin-4-amine (14). To a solution of compound 8(500mg,
2.73 mmol) in methanol (10 mL) were added acetic acid (0.31 mL,
2.06 mmol), 2-methyl-5-nitrobenzenamine (415 mg, 2.73 mmol), and
Na(CN)BH3 (858 mg, 13.66 mmol) at room temperature and stirred
for overnight. The reaction mixture was partitioned between ethyl
acetate (50 mL) and saturated NaHCO3 (30 mL), and then the water
layer was extracted with ethyl acetate (30 mL ꢀ 3). The combined
organic layer was washed with brine, dried over MgSO4, filtered
through a pad of celite, and concentrated under reduced pressure.
The resulting crude product was purified by silica gel chromatogra-
phy with ethyl acetate/hexane (1/4 to 2/3) to give (310 mg, 36% yield)
of the title product as a yellow solid. Rf = 0.30 (2/3 ethyl acetate/
hexane). 1H NMR 400 MHz (DMSO-d6) δ 7.86 (s, 1H), 7.38 (dd,
J = 2.1 Hz, J = 8.6 Hz, 1H), 7.22 (d, J = 8.2 Hz, 1H), 7.18 (d, J =
2.0 Hz, 1H), 7.14 (m, 1H), 5.98 (m, 1H), 4.18 (d, J = 5.5 Hz, 2H),
2.88 (d, J = 4.5 Hz, 3H), 2.36 (s, 3H), 2.20 (s, 3H). 13C NMR 100
MHz (DMSO-d6) δ 169.21, 160.48, 152.97, 147.41, 147.12, 131.10,
130.72, 111.25, 109.93, 103.22, 27.81, 18.41, 13.78. HRMS (ESI) m/z
[M þ Na]þ calcd 342.1001, found 342.1003.
To a solution of 3-(4-methyl-1H-imidazol-1-yl)-5-(trifluoro-
methyl)benzonitrile (3 g, 11.95 mmol) in dioxane (30 mL) was
added 2 N NaOH solution (30 mL, 59.76 mmol). The reaction
mixture was stirred for 24 h under reflux condition. The reaction
mixture was cooled to room temperature, and the organic
solvent was removed under reduced pressure. To a reaction
mixture was added 6N HCl solution until white solid (pH =
4-5) precipitated. The produced white solid was filtered and
dried over nitrogen gas flow. The title compound (2.6 g, 80%
yield) was used for the next step without further purification. 1H
NMR 400 MHz (DMSO-d6) δ 8.51 (s, 1H), 8.35 (s, 1H), 8.40 (s,
1H), 8.29 (s, 1H), 8.07 (s, 1H), 7.74 (s, 1H), 2.17 (s, 3H). 13C
NMR 100 MHz (DMSO-d6) δ 165.81, 138.72, 138.39, 135.70,
134.41, 131.64, 131.31, 124.46, 123.59, 121.14, 115.0, 13.68. HRMS
(ESI) m/z [M þ H]þ calcd 271.0689, found 271.0691.
1-Methyl-3-(2-methyl-5-nitrophenyl)-7-(methylthio)-3,4-dihy-
dropyrimido[4,5-d]pyrimidin-2(1H)-one (15). To a solution of
compound 14 (200 mg, 0.63 mmol) in 1,4-dioxane (3.0 mL)

Article
3-(4-Methyl-1H-imidazol-1-yl)-N-(4-methyl-3-(1-methyl-7-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5447
119.10, 114.16, 47.38, 28.44, 28.20, 16.22, 13.64. HRMS (ESI)
(methylthio)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-
yl)phenyl)-5-(trifluoromethyl)benzamide (18). To a solution of
compound 16 (100 mg, 0.32 mmol) in dried DMF (1.5 mL) were
added compound 17 (86 mg, 0.32 mmol), HATU (362 mg,
0.95 mmol), and DIEA (0.26 mL, 1.58 mmol). The reaction mix-
ture was stirred for 24 h at room temperature. The reaction mix-
ture was partitioned between ethyl acetate (5 mL) and water
(5 mL), and then the water layer was extracted with ethyl acetate
(5 mL ꢀ 3). The combined organic layer was washed with brine,
dried over MgSO4, filtered through a pad of celite, and con-
centrated under reduced pressure. The resulting crude product
was purified by silica gel chromatography with MeOH/CH2Cl2
(1/95 to 5/95) to give (161 mg, 89% yield) of the title product as a
white solid. Rf = 0.28 (5/95 MeOH/CH2Cl2). 1H NMR 400 MHz
(CDCl3) δ 9.47 (s, 1H), 8.14 (m, 2H), 8.04 (s, 1H), 7.88 (s, 1H),
7.71 (s, 1H), 7.60 (s, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.10 (s, 1H),
7.02 (d, J = 8.4 Hz, 1H), 4.68 (d, J = 14.7 Hz, 1H), 4.46 (d, J =
14.7 Hz, 1H), 3.47 (s, 3H), 2.59 (s, 3H), 2.28 (s, 3H), 1.68 (s, 3H).
13C NMR 100 MHz (CDCl3) δ 172.11, 163.03, 156.32, 153.62,
152.04, 140.61, 139.78, 138.00, 137.64, 134.47, 132.73, 132.40,
131.16, 130.89, 124.49, 123.32, 122.64, 120.85, 120.01, 119.80,
114.15, 105.34, 47.21, 28.48, 16.36, 14.22, 13.60. HRMS (ESI)
m/z [M þ Na]þ calcd 590.1562, found 590.1563.
m/z [M þ Na]þ calcd 573.1950, found 573.1952.
Acknowledgment. We thank Sungjoon Kim, Xiang-ju Gu
for Ba/F3 cell proliferation assay, and acknowledge scientific
guidance from Peter G. Schultz.
References
(1) Weisberg, E.; Manley, P. W.; Breitenstein, W.; Bruggen, J.; Cowan-
Jacob, S. W.; Ray, A.; Huntly, B.; Fabbro, D.; Fendrich, G.; Hall-
Meyers, E.; Kung, A. L.; Mestan, J.; Daley, G. Q.; Callahan, L.;
Catley, L.; Cavazza, C.; Azam, M.; Neuberg, D.; Wright, R. D.;
Gilliland, D. G.; Griffin, J. D. Characterization of AMN107, a
selective inhibitor of native and mutant Bcr-Abl. Cancer Cell 2005,
7 (2), 129–141.
(2) Puttini, M.; Coluccia, A. M.; Boschelli, F.; Cleris, L.; Marchesi,
E.; Donella-Deana, A.; Ahmed, S.; Redaelli, S.; Piazza, R.;
Magistroni, V.; Andreoni, F.; Scapozza, L.; Formelli, F.; Gambacorti-
Passerini, C. In vitro and in vivo activity of SKI-606, a novel Src-
Abl inhibitor, against imatinib-resistant Bcr-Ablþ neoplastic cells.
Cancer Res. 2006, 66 (23), 11314–11322.
(3) Shah, N. P.; Tran, C.; Lee, F. Y.; Chen, P.; Norris, D.; Sawyers,
C. L. Overriding imatinib resistance with a novel ABL kinase
inhibitor. Science 2004, 305 (5682), 399–401.
(4) O’Hare, T.; Walters, D. K.; Deininger, M. W.; Druker, B. J.
AMN107: tightening the grip of imatinib. Cancer Cell 2005, 7 (2),
117–119.
(5) Miyamoto, N.; Sugita, K.; Goi, K.; Inukai, T.; Lijima, K.; Tezuka,
T.; Kojika, S.; Nakamura, M.; Kagami, K.; Nakazawa, S. The
JAK2 inhibitor AG490 predominantly abrogates the growth of
human B-precursor leukemic cells with 11q23 translocation or
Philadelphia chromosome. Leukemia 2001, 15 (11), 1758–1768.
(6) Samanta, A. K.; Lin, H.; Sun, T.; Kantarjian, H.; Arlinghaus, R. B.
Janus kinase 2: a critical target in chronic myelogenous leukemia.
Cancer Res. 2006, 66 (13), 6468–6472.
(7) Summy, J. M.; Trevino, J. G.; Lesslie, D. P.; Baker, C. H.;
Shakespeare, W. C.; Wang, Y.; Sundaramoorthi, R.; Metcalf,
C. A.; Keats, J. A.; Sawyer, T. K.; Gallick, G. E. AP23846, a novel
and highly potent Src family kinase inhibitor, reduces vascular
endothelial growth factor and interleukin-8 expression in human
solid tumor cell lines and abrogates downstream angiogenic pro-
cesses. Mol. Cancer Ther. 2005, 4 (12), 1900–1911.
(8) Azam, M; Nardi, V.; Shakespeare, W. C.; Metcalf, C. A., III;
Bohacek, R. S.; Wang, Y.; Sundaramoorthi, R.; Sliz, P.; Veach,
D. R.; Bornmann, W. G.; Clarkson, B.; Dalgarno, D. C.; Sawyer,
T. K.; Daley, G. Q. Activity of dual SRC-ABL inhibitors highlights
the role of BCR/ABL kinase dynamics in drug resistance. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103 (24), 9244–9249.
(9) Harrington, E. A.; Bebbington, D.; Moore, J.; Rasmussen, R. K.;
Ajose-Adeogun, A. O.; Nakayama, T.; Graham, J. A.; Demur, C.;
Hercend, T.; Diu-Hercend, A.; Su, M.; Golec, J. M.; Miller, K. M.
VX-680, a potent and selective small-molecule inhibitor of the
Aurora kinases, suppresses tumor growth in vivo. Nature Med.
2004, 10 (3), 262–267.
3-(4-Methyl-1H-imidazol-1-yl)-N-(4-methyl-3-(1-methyl-7-(met-
hylsulfonyl)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-
yl)phenyl)-5-(trifluoromethyl)benzamide (19). To a solution of
compound 18 (130 mg, 0.23 mmol) in dichloromethane (1 mL)
was added m-chloroperbenzoic acid (158 mg, 0.91 mmol) at 0 °C.
The reaction mixture was stirred for 30 min at 0 °C and then
stirred for 3 h at room temperature. The reaction mixture was
partitioned between dichloromethane (10 mL) and saturated
NaHCO3 (10 mL). The organic layer was washed with brine,
dried over MgSO4, filtered through a pad of celite, and concen-
trated under reduce pressure. The resulting white solid (122 mg,
88% yield) was used for the next step without further purifica-
tion. Rf = 0.27 (5/95 MeOH/CH2Cl2). 1H NMR 400 MHz
(DMSO-d6) δ 10.66 (s, 1H), 8.58 (s, 1H), 8.47 (s, 1H), 8.39 (s,
1H), 8.23 (s, 1H), 8.16 (s, 1H), 7.85 (m, 1H), 7.69 (s, 1H), 7.67
(dd, J = 1.9 Hz, J = 8.3 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 4.94
(d, J = 15.6 Hz, 1H), 4.77 (d, J = 15.6 Hz, 1H), 3.40 (s, 3H), 3.35
(s, 3H), 2.14 (s, 3H), 2.10 (s, 3H). 13C NMR 100 MHz (DMSO-
d6) δ 164.63, 163.56, 158.19, 153.02, 151.55, 141.01, 139.42,
138.21, 137.96, 137.84, 135.72, 131.63, 131.48, 131.38, 123.06,
122.33, 120.64, 119.83, 119.63, 115.94, 114.72, 47.14, 28.95,
17.20, 14.00.HRMS (ESI) m/z [M þ Na]þ calcd 622.1460, found
622.1463.
3-(4-Methyl-1H-imidazol-1-yl)-N-(4-methyl-3-(1-methyl-7-(met-
hylamino)-2-oxo-1,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-
phenyl)-5-(trifluoromethyl)benzamide (4). To a solution of com-
pound 19 (100 mg, 0.16 mmol) in 1,4-dioxane (2.0 mL) was added
2.0 M methylamine in THF (0.83 mL, 1.67 mmol). The reaction
mixture was stirred for 24 h at 120 °C in the sealed reaction vessel.
The reaction mixture was cooled to room temperature and
concentrated under reduced pressure. The crude reaction mixture
was partitioned between ethyl acetate (10 mL), and the water
layer was extracted with ethyl acetate (5 mL ꢀ 3). The combined
organic layer was washed with brine, dried over MgSO4, filtered
through a pad of celite, and concentrated under reduced pres-
sure. The resulting crude product was purified by silica gel chro-
matography with MeOH/CH2Cl2 (1/99 to 5/95) to give (75 mg,
82% yield) of the title product as a white solid. Rf = 0.23 (5/95
MeOH/CH2Cl2). 1H NMR 400 MHz (CDCl3) 9.49 (s, 1H), 8.15
(s, 2H), 7.89 (s, 1H), 7.83 (s, 1H), 7.69 (s, 1H), 7.49 (s, 1H), 7.46
(d, J = 7.9 Hz, 1H), 7.10 (s, 1H), 6.98 (d, J = 8.4 Hz, 1H), 5.18
(m, 1H), 4.56 (d, J = 13.8 Hz, 1H), 4.35 (d, J = 13.8 Hz, 1H),
3.36 (s, 3H), 3.03 (d, J = 5.0 Hz, 3H), 2.28 (s, 3H), 1.60 (s, 3H).
13C NMR 100 MHz (CDCl3) δ 163.01, 162.45, 157.13, 154.23,
153.09, 140.58, 140.07, 137.94, 137.83, 137.72, 134.51, 132.64,
132.31, 131.00, 130.73, 124.53, 123.38, 122.84, 120.19, 119.66,
(10) Carter, T. A.; Wodicka, L. M.; Shah, N. P.; Velasco, A. M.;
Fabian, M. A.; Treiber, D. K.; Milanov, Z. V.; Atteridge, C. E.;
Biggs, W. H., 3rd; Edeen, P. T.; Floyd, M.; Ford, J. M.; Grotzfeld,
R. M.; Herrgard, S.; Insko, D. E.; Mehta, S. A.; Patel, H. K.; Pao,
W.; Sawyers, C. L.; Varmus, H.; Zarrinkar, P. P.; Lockhart, D. J.
Inhibition of drug-resistant mutants of ABL, KIT, and EGF
receptor kinases. Proc. Natl. Acad. Sci U.S.A 2005, 102 (31),
11011–11016.
(11) Carpinelli, P.; Ceruti, R.; Giorgini, M. L.; Cappella, P.; Gianellini,
L.; Croci, V.; Degrassi, A.; Texido, G.; Rocchetti, M.; Vianello, P.;
Rusconi, L.; Storici, P.; Zugnoni, P.; Arrigoni, C.; Soncini, C.; Alli,
C.; Patton, V.; Marsiglio, A.; Ballinari, D.; Pesenti, E.; Fancelli, D.;
Moll, J. PHA-739358, a potent inhibitor of Aurora kinases with a
selective target inhibition profile relevant to cancer. Mol. Cancer
Ther. 2007, 6 (12), 3158–3168.
(12) Modugno, M.; Casale, E.; Soncini, C.; Rosettani, P.; Colombo, R.;
Lupi, R.; Rusconi, L.; Fancelli, D.; Carpinelli, P.; Cameron, A. D.;
Isacchi, A.; Moll, J. Crystal structure of the T315I Abl mutant in
complex with the aurora kinases inhibitor PHA-739358. Cancer
Res. 2007, 67 (17), 7987–7990.
(13) 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.; Palanki, M. S. S.; Pathak, V. P.; Renick, J.; Soll,
R.; Zeng, B. Inhibitors of ABL and the ABL-T315I mutation. Curr.
Top. Med. Chem. 2008, 8 (10), 905–921.

5448 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Choi et al.
(14) Gumireddy, K.; Baker, S. J.; Cosenza, S. C.; John, P.; Kang, A. D.;
Robell, K. A.; Reddy, M. V.; Reddy, E. P. A non-ATP-competitive
inhibitor of BCR-ABL overrides imatinib resistance. Proc. Natl.
Acad. Sci. U.S.A 2005, 102 (6), 1992–1997.
the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib
in idiopathic hypereosinophilic syndrome. N. Engl. J. Med. 2003,
348 (13), 1201–1214. (b) Blencke, S.; Zech, B.; Engkvist, O.; Greff, Z.;
Orfi, L.; Horvath, Z.; Keri, G.; Ullrich, A.; Daub, H. Characterization of
a conserved structural determinant controlling protein kinase sensitivity
to selective inhibitors. Chem. Biol. 2004, 11 (5), 691–701.
(15) Huang, W. S.; Zhu, X.; Wang, Y.; Azam, M.; Wen, D.;
Sundaramoorthi, R.; Thomas, R. M.; Liu, S.; Banda, G.; Lentini,
S. P.; Das, S.; Xu, Q.; Keats, J.; Wang, F.; Wardwell, S.; Ning, Y.;
Snodgrass, J. T.; Broudy, M. I.; Russian, K.; Daley, G. Q.; Iuliucci,
J.; Dalgarno, D. C.; Clackson, T.; Sawyer, T. K.; Shakespeare,
W. C. 9-(Arenethenyl)purines as Dual Src/Abl Kinase Inhibitors
Targeting the Inactive Conformation: Design, Synthesis, and
Biological Evaluation. J. Med. Chem. 2009, 52 (15), 4743–47565.
(16) Seeliger, M. A.; Ranjitkar, P.; Kasap, C.; Shan, Y.; Shaw, D. E.;
Shah, N. P.; Kuriyan, J.; Maly, D. J. Equally potent inhibition of
c-Src and Abl by compounds that recognize inactive kinase con-
formations. Cancer Res. 2009, 69 (6), 2384–2392.
(26) Kraker, A. J.; Hartl, B. G.; Amar, A. M.; Barvian, M. R.;
Showalter, H. D.; Moore, C. W. Biochemical and cellular effects
of c-Src kinase-selective pyrido[2,3-d]pyrimidine tyrosine kinase
inhibitors. Biochem. Pharmacol. 2000, 60 (7), 885–898.
(27) (a) Panek, R. L.; Lu, G. H.; Klutchko, S. R.; Batley, B. L.; Dahring,
T. K.; Hamby, J. M.; Hallak, H.; Doherty, A. M.; Keiser, J. A. In
vitro pharmacological characterization of PD 166285, a new
nanomolar potent and broadly active protein tyrosine kinase
inhibitor. J. Pharmacol. Exp. Ther. 1997, 283 (3), 1433–1444.
(b) Klutchko, S. R.; Hamby, J. M.; Boschelli, D. H.; Wu, Z.; Kraker,
A. J.; Amar, A. M.; Hartl, B. G.; Shen, C.; Klohs, W. D.; Steinkampf,
R. W.; Driscoll, D. L.; Nelson, J. M.; Elliott, W. L.; Roberts, B. J.;
Stoner, C. L.; Vincent, P. W.; Dykes, D. J.; Panek, R. L.; Lu, G. H.;
Major, T. C.; Dahring, T. K.; Hallak, H.; Bradford, L. A.; Showalter,
H. D.; Doherty, A. M. 2-Substituted aminopyrido[2,3-d]pyrimidin-
7(8H)-ones. Structure-activity relationships against selected tyrosine
kinases and in vitro and in vivo anticancer activity. J. Med. Chem.
1998, 41 (17), 3276–3292.
(28) (a) Dorsey, J. F.; Jove, R.; Kraker, A. J.; Wu, J. The pyrido[2,3-
d]pyrimidine derivative PD180970 inhibits p210Bcr-Abl tyrosine
kinase and induces apoptosis of K562 leukemic cells. Cancer Res.
2000, 60 (12), 3127–3131. (b) Wisniewski, D.; Lambek, C. L.; Liu, C.;
Strife, A.; Veach, D. R.; Nagar, B.; Young, M. A.; Schindler, T.;
Bornmann, W. G.; Bertino, J. R.; Kuriyan, J.; Clarkson, B. Character-
ization of potent inhibitors of the Bcr-Abl and the c-kit receptor tyrosine
kinases. Cancer Res. 2002, 62 (15), 4244–4255.
(17) Dar, A. C.; Lopez, M. S.; Shokat, K. M. Small molecule recogni-
tion of c-Src via the Imatinib-binding conformation. Chem. Biol.
2008, 15 (10), 1015–1022.
(18) O’Hare, T.; Shakespeare, W. C.; Zhu, X.; Eide, C. A.; Rivera,
V. M.; Wang, F.; Adrian, L. T.; Zhou, T.; Huang, W.-S.; Xu, Q.;
Metcalf Iii, C. A.; Tyner, J. W.; Loriaux, M. M.; Corbin, A. S.;
Wardwell, S.; Ning, Y.; Keats, J. A.; Wang, Y.; Sundaramoorthi,
R.; Thomas, M.; Zhou, D.; Snodgrass, J.; Commodore, L.; Sawyer,
T. K.; Dalgarno, D. C.; Deininger, M. W. N.; Druker, B. J.;
Clackson, T. AP24534, a Pan-BCR-ABL Inhibitor for Chronic
Myeloid Leukemia, Potently Inhibits the T315I Mutant and
Overcomes Mutation-Based Resistance. Cancer Cell 2009, 16 (5),
401–412.
(19) Okram, B.; Nagle, A.; Adrian, F. J.; Lee, C.; Ren, P.; Wang, X.;
Sim, T.; Xie, Y.; Xia, G.; Spraggon, G.; Warmuth, M.; Liu, Y.;
Gray, N. S. A general strategy for creating “inactive-conformation”
abl inhibitors. Chem. Biol. 2006, 13 (7), 779–786.
(29) Cowan-Jacob, S. W.; Guez, V.; Fendrich, G.; Griffin, J. D.;
Fabbro, D.; Furet, P.; Liebetanz, J.; Mestan, J.; Manley, P. W.
Imatinib (STI571) resistance in chronic myelogenous leukemia:
molecular basis of the underlying mechanisms and potential
strategies for treatment. Mini-Rev. Med. Chem. 2004, 4 (3),
285–299.
(20) Nagar, B.; Bornmann, W. G.; Pellicena, P.; Schindler, T.; Veach,
D. R.; Miller, W. T.; Clarkson, B.; Kuriyan, J. Crystal structures of
the kinase domain of c-Abl in complex with the small molecule
inhibitors PD173955 and imatinib (STI-571). Cancer Res. 2002, 62
(15), 4236–4243.
(21) Tokarski, J. S.; Newitt, J. A.; Chang, C. Y.; Cheng, J. D.; Wittekind,
M.; Kiefer, S. E.; Kish, K.; Lee, F. Y.; Borzillerri, R.; Lombardo,
L. J.; Xie, D.; Zhang, Y.; Klei, H. E. The structure of Dasatinib
(BMS-354825) bound to activated ABL kinase domain elucidates
its inhibitory activity against imatinib-resistant ABL mutants.
Cancer Res. 2006, 66 (11), 5790–5797.
(22) Pao, W.; Miller, V. A.; Politi, K. A.; Riely, G. J.; Somwar, R.;
Zakowski, M. F.; Kris, M. G.; Varmus, H. Acquired resistance of
lung adenocarcinomas to gefitinib or erlotinib is associated with a
second mutation in the EGFR kinase domain. PLoS Med. 2005, 2
(3), e73.
(23) Fabbro, D.; Ruetz, S.; Bodis, S.; Pruschy, M.; Csermak, K.; Man,
A.; Campochiaro, P.; Wood, J.; O’Reilly, T.; Meyer, T. PKC412-a
protein kinase inhibitor with a broad therapeutic potential.
Anti-Cancer Drug Des. 2000, 15 (1), 17–28.
(24) Cools, J.; Mentens, N.; Furet, P.; Fabbro, D.; Clark, J. J.; Griffin,
J. D.; Marynen, P.; Gilliland, D. G. Prediction of resistance to
small molecule FLT3 inhibitors: implications for molecularly
targeted therapy of acute leukemia. Cancer Res. 2004, 64 (18),
6385–6389.
(30) (a) Melnick, J. S.; Janes, J.; Kim, S.; Chang, J. Y.; Sipes, D. G.;
Gunderson, D.; Jarnes, L.; Matzen, J. T.; Garcia, M. E.; Hood,
T. L.; Beigi, R.; Xia, G.; Harig, R. A.; Asatryan, H.; Yan, S. F.;
Zhou, Y.; Gu, X. J.; Saadat, A.; Zhou, V.; King, F. J.; Shaw, C. M.;
Su, A. I.; Downs, R.; Gray, N. S.; Schultz, P. G.; Warmuth, M.;
Caldwell, J. S. An efficient rapid system for profiling the cellular
activities of molecular libraries. Proc. Natl. Acad. Sci. U.S.A 2006,
103 (9), 3153–3158. (b) Warmuth, M.; Kim, S.; Gu, X. J.; Xia, G.;
Adrian, F. Ba/F3 cells and their use in kinase drug discovery. Curr.
Opin. Oncol. 2007, 19 (1), 55–60.
(31) Liu, Y.; Gray, N. S. Rational design of inhibitors that bind to
inactive kinase conformations. Nature Chem. Biol. 2006, 2 (7),
358–364.
(32) McDermott, U.; Sharma, S. V.; Dowell, L.; Greninger, P.;
Montagut, C.; Lamb, J.; Archibald, H.; Raudales, R.; Tam, A.;
Lee, D.; Rothenberg, S. M.; Supko, J. G.; Sordella, R.; Ulkus,
L. E.; Iafrate, A. J.; Maheswaran, S.; Njauw, C. N.; Tsao, H.;
Drew, L.; Hanke, J. H.; Ma, X.-J.; Erlander, M. G.; Gray, N. S.;
Haber, D. A.; Settleman, J. Identification of genotype-correlated
sensitivity to selective kinase inhibitors by using high-throughput
tumor cell line profiling. Proc. Natl. Acad. Sci. U.S.A. 2007, 104
(50), 19936–19941.
(33) Adrian, F. J.; Ding, Q.; Sim, T.; Velentza, A.; Sloan, C.; Liu, Y.;
Zhang, G.; Hur, W.; Ding, S.; Manley, P.; Mestan, J.; Fabbro, D.;
Gray, N. S. Allosteric inhibitors of Bcr-abl-dependent cell prolifer-
ation. Nature Chem. Biol. 2006, 2 (2), 95–102.
(25) (a) Cools, J.; DeAngelo, D. J.; Gotlib, J.; Stover, E. H.; Legare,
R. D.; Cortes, J.; Kutok, J.; Clark, J.; Galinsky, I.; Griffin, J. D.;
Cross, N. C.; Tefferi, A.; Malone, J.; Alam, R.; Schrier, S. L.;
Schmid, J.; Rose, M.; Vandenberghe, P.; Verhoef, G.; Boogaerts,
M.; Wlodarska, I.; Kantarjian, H.; Marynen, P.; Coutre, S. E.;
Stone, R.; Gilliland, D. G. A tyrosine kinase created by fusion of