Synthesis and biological evaluation of a fluorine-18 derivative of dasatinib

Article abstract of DOI:10.1021/jm070342g

Tyrosine kinases often play pivotal roles in the pathogenesis of cancer and are good candidates for therapeutic intervention and targeted molecular imaging. The precursor synthesis, radiosynthesis, and biological characterization of a fluorine-18 analog o

Full text of DOI:10.1021/jm070342g

J. Med. Chem. 2007, 50, 5853-5857
5853
Synthesis and Biological Evaluation of a Fluorine-18 Derivative of Dasatinib
Darren R. Veach,†,‡ Mohammad Namavari,‡,§ Nagavarakishore Pillarsetty,†,‡ Elmer B. Santos,‡ Tatiana Beresten-Kochetkov,†
Caryl Lambek,† Blesida J. Punzalan,‡ Christophe Antczak,†,# Peter M. Smith-Jones,†,‡ Hakim Djaballah,†,# Bayard Clarkson,† and
Steven M. Larson*,†,‡
Department of Molecular Pharmacology and Chemistry and Department of Radiology, Memorial Sloan-Kettering Cancer Center,
1275 York AVenue, New York, New York 10021
ReceiVed March 23, 2007
Tyrosine kinases often play pivotal roles in the pathogenesis of cancer and are good candidates for therapeutic
intervention and targeted molecular imaging. The precursor synthesis, radiosynthesis, and biological
characterization of a fluorine-18 analog of dasatinib, a multitargeted kinase inhibitor, are reported. Compound
5 potently inhibits Abl, Src, and Kit kinases and inhibits K562 and M07e/p210bcr-abl human leukemic cell
growth. Using positron emission tomography, we visualized K562 tumor xenografts in mice with [18F]-5.
Introduction
malignancies, mutated in a few examples, and is often associated
with increased motility, invasiveness, or metastasis in cancer.6
The abundance, activation, and disregulation of Bcr-Abl and
Src in cancer have made these kinases attractive targets for drug
development and, more recently, molecular imaging.
A central focus of modern medicine is to develop care that
is highly individualized to each patient. An important facet of
this has been kinase inhibitor therapy, and signal transduction
modulation in general is revolutionizing disease treatment.
Another key aspect of customized care is obtaining a detailed
disease profile through noninvasive medical imaging techniques
such as positron emission tomography (PETa) and using this to
assess disease status and determine the optimal course of
treatment. Molecular and functional imaging with radiotracers
such as [18F]-fluoro-2-deoxy-D-glucose (FDG) and [18F]-3′-
deoxy-3′-fluorothymidine (FLT) are particularly useful as sur-
rogate markers in cancer diagnosis and management.1,2 How-
ever, more sophisticated tumoral information is necessary to
predict response or observe the onset of drug resistance.
Radiolabeled small molecule imaging modalities that are
matched to a given kinase inhibitor and are capable of querying
a specific molecular target are one solution, potentially. Here,
we describe the synthesis, in vitro, and preliminary in vivo
micro-PET imaging of one such kinase-directed probe, a
fluorine-18 derivative of dasatinib.
Our focus is the development of PET radiotracers targeting
Abl, Src, Kit, and other kinases relevant to cancer. Normal Abl
and Src kinases are expressed in a variety of tissues and are
tightly regulated and inactive most of the time. Both have many
functions and associations in vivo, but generally, Src regulates
cell adhesion and motility, while Abl is involved in cytoskeletal
reorganization3 and cell death signaling.4 In some leukemias, a
reciprocal t(9;22) translocation between the ABL and BCR
genes forms the Philadelphia chromosome (Ph), whose mutant
gene product, Bcr-Abl, is a constitutively activated tyrosine
kinase. Bcr-Abl causes chronic myelogenous leukemia (CML)
and some types of acute lymphoblastic leukemia (ALL).5 Src
tyrosine kinase is activated and overexpressed in numerous
Imatinib (1), a Bcr-Abl tyrosine kinase inhibitor, is one of
the most well-known molecularly targeted therapeutics and has
revolutionized treatment of CML.7,8 Imatinib is also approved
for gastrointestinal stromal tumor (GIST) therapy and acts via
inhibition of c-Kit receptor tyrosine kinase.9 While imatinib has
been a major breakthrough, resistance to kinase inhibitor therapy
arises from a number of mechanisms including kinase-domain
point mutations (pre-existing or acquired), upregulation of Bcr-
Abl, activation of alternate, compensatory kinase pathways (Src
family), and drug transporters.10 These issues have fueled the
development of a number of next-generation Bcr-Abl inhibi-
tors.11,12 Dasatinib (BMS-354825, 2) is a high affinity dual Src/
Abl and c-Kit inhibitor recently approved for all categories of
imatinib-refractory CML and Ph+ ALL.13,14 Dasatinib is
effective in many imatinib resistant Bcr-Abl kinase domain
mutants, but the “gatekeeper” mutants like T315I or F317L
remain problematic.14
* To whom correspondence should be addressed. Phone: (212) 639-
7373. Fax: (212) 717-3263. E-mail: larsonS@mskcc.org.
† Department of Molecular Pharmacology and Chemistry.
‡ Department of Radiology.
§ Current address: Multimodality Molecular Imaging Lab, Department
of Radiology, Stanford University, School of Medicine, Stanford, CA 94305.
# High Throughput Screening Facility, Memorial Sloan-Kettering Cancer
Center, New York, NY 10021.
14C- and 3H-labeled (beta-emitting) radiotracers are produced
routinely in drug development, but kinase inhibitors bearing
positron-emitting isotopes are much less developed. No kinase
inhibitor-based imaging probe exists yet for routine use in
humans, though the number of promising kinase-targeted PET
radiotracers is growing rapidly. Thus far, the majority of effort
a Abbreviations: ALL, acute lymphoblastic leukemia; CML, chronic
myelogenous leukemia; FDG, [18F]-fluoro-2-deoxy-D-glucose; FLT, [18F]-
3′-deoxy-3′-fluorothymidine; GIST, gastrointestinal stromal tumor; PET,
positron emission tomography; Ph, Philadelphia chromosome.
10.1021/jm070342g CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/23/2007

5854 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Brief Articles
Scheme 1. Synthesis of an Unlabeled (19F) Fluorinated
Derivative of Dasatiniba
Figure 1. Cavity depth Connolly surface rendering of 5 docked into
Abl kinase domain.
has been by vanBrocklin, Mishani, and others on quinazoline-
type small-molecule probes for EGFR tyrosine kinase, which
is overexpressed in some cancers.15-17 More recently, Wang et
al. reported the synthesis of [11C]-gefitinib18 and [18F]-suni-
tinib.19
At first, perhaps hematopoietic disorders, like CML, may not
seem like logical imaging targets due to their peripheral nature,
but imaging hyperproliferation in bone marrow has been known
for some time.20,21 Recently, [18F]-FLT PET was used to clearly
distinguish bone marrow in patients with myeloproliferative
disorders from normal.22 [11C]-AG957 was the first example
of a Bcr-Abl-targeted radiotracer specifically developed for PET,
but this tracer suffers from inherent chemical instability and
weak target binding relative to newer inhibitors.23 Our group
has reported an [124I]-pyridopyrimidinone derivative,24 which
binds tightly to Bcr-Abl, among other kinases, but does not
possess an ideal logP. Recently, the Fowler group reported [11C]-
imatinib, which was imaged in baboon.25 Generally, 18F is more
convenient for PET imaging studies, as it has a 110-minute half-
life, unlike 11C (T1/2 ) 20 min).
When considering the structure of dasatinib, the hydroxy-
ethylpiperazinyl moiety was ideal for derivatization based on
binding orientation. Chemically, the most straightforward ap-
proach conveniently was at the same site; N-alkylation of the
unsubstituted piperazine with a simple fluorine-containing group
or activated precursor for fluoride displacement.26 Thus, we
endeavored to construct an analog of dasatinib bearing an [18F]
fluoroethyl substituent. N-(2-Fluoroethyl)piperazines are not
widely reported, but Katzenellenbogen and Welch described a
successful synthesis of activated ethylpiperazines and subsequent
displacement with 18F.27 Sterically, hydroxyl-to-fluoro substitu-
tion is tolerated well, so long as the hydroxyl is not involved in
critical H-bonding. Prior experience with pyridopyrimidinone
Src/Abl inhibitors28 and molecular docking studies into the Abl
crystal structure predicted that the dasatinib pharmacophore
would share much of the same binding characteristics in which
an arene sits deep within the catalytic pocket and the substi-
tutents on N4 of the piperazine would protrude from a solvent
accessible hole in the kinase catalytic domain (Figure 1).
a Reagents and conditions: (a) piperazine, diisopropyl-ethylamine,
dioxane, reflux, 12 h, 78%; (b) 1-bromo-2-fluoroethane, Na2CO3, KI,
CH3CN, 60 °C, 4 h, 84%.
process in which a two-carbon synthon containing two leaving
groups was displaced with F-18 first, then reacted with
piperazine 4. A one-step radiosynthesis would be ideal, however,
we feared that intramolecular cyclization would be a problematic
competing reaction in a precursor containing a X-CH2CH2-
NR2 systemsa piperazine beta to a leaving group that is
significantly reactive with fluoride ion.
In this study, it was found that either trifluoromethane-
sulfonate or p-toluenesulfonate precursors worked. 2-Bromoethyl
triflate, 6, has been used to install a [18F]-fluoroethyl moiety
on piperazines before and is easily obtained by triflation of
2-bromoethanol and triflic anhydride.27 Precursor 6 was treated
with [18F]-KF/Kryptofix 2.2.2 to form [18F]-1-bromo-2-fluoro-
ethane, 7. The decay-corrected radiochemical yield of the
N-alkylation step was 25.1 ( 5.8%. Overall, a 6.6 ( 2.3% yield
was obtained from starting fluoride. The specific activity was
29.7 ( 9.7 mCi/µmol (n ) 3). These conditions were optimized
and it was found that [18F]-7 could be distilled rapidly prior to
the alkylation of 4, which improved yields of [18F]-5 somewhat
to 9.8 ( 5.0%. The average specific activity improved consider-
ably to 2560 mCi/µmol (n ) 11, range 108-7350 mCi/µmol).
The low yield could be a result of complications due to
hydrolysis or volatility of 6 or 7. To address this problem, we
examined a less labile, less volatile alternative. [18F]-2-fluoro-
ethyl tosylate, 8, is generated in situ in a similar fashion from
ethylene glycol ditosylate.29 The decay-corrected radiochemical
yield of [18F]-5 from the tosylate, 8, was somewhat better over
two steps (23%) but with much lower specific activity of 3-6
mCi/µmole (n ) 3; Scheme 2). The total time of preparation
(radiosynthesis and chromatography and formulation) ranged
from 120 to 130 min (125 ( 5 min). Compound 5 has a
favorable log D(o/w) of 2.1 ( 0.6 and is highly protein bound in
serum (98.5 ( 1.0%) and 1% BSA (99.0 ( 0.3%).
Results and Discussion
Synthesis. The synthesis of both 18F radiotracer and 19F
reference analogs began with chloropyrimidine 3, an intermedi-
ate that was synthesized according to the literature.13 An SNAr
displacement with piperazine gave compound 4 in good yield
(78%). The 2-fluoroethyl reference compound 5 was obtained
by alkylation of 4 with 1-bromo-2-fluoroethane in the presence
of Na2CO3 and catalytic KI (Scheme 1).
To determine whether compound 5 retains a kinase inhibition
profile that is similar to dasatinib, we characterized the inhibition
of kinase activity. In vitro and cellular assays demonstrated that
Radiosynthesis. We envisioned that the best way to arrive
at the [18F]-N-2-fluoroethyl-labeled compound was a two-step

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5855
Scheme 2. Two Radiosynthetic Routes to an [18F] Derivative of
Dasatiniba
a Reagents and conditions: (a) [18F]-KF/Kryptofix 2.2.2, K2CO3, o-
dichlorobenzene, 105 °C, 10 min; (b) [18F]-KF/Kryptofix 2.2.2, K2CO3,
CH3CN, 110 °C, 10 min; (c) NaI, CsCO3, 1:1 DMF/CH3CN, 140 °C, 40
min; (d) DMSO, 160 °C, 30 min.
Figure 2. Inhibitory activity of 5 on 21 kinases at 10 nM.
Table 1. Compound 5 Has Kinase and Cellular Inhibition
Characteristics That Are Similar to Dasatiniba
dasatinib IC50
(nM)
cmpd 5 IC50
(nM)
Abl protein
Src protein
K562 cells
R10 neg cells
(M07e/p210bcr-abl
M07e cellsb
4.2 ( 0.4
1.5 ( 1.1
1.0 ( 0.2
0.07 ( 0.02
9.1 ( 0.8
3.5 ( 2.2
1.1 ( 0.2
0.10 ( 0.02
)
1.2 ( 0.8
1.1 ( 0.3
a Mean of three experiments in each. b Grown in 50 ng/mL SCF (Kit
ligand).
fluorinated analog 5 has inhibitory activity that closely parallels
that of dasatinib. Compound 5 inhibits Abl and Src kinase
activity at roughly half the potency of dasatinib in our assays
(Table 1).
Compound 5 is equipotent with dasatinib in inhibiting
proliferation of cells dependent on Bcr-Abl for growth. K562
growth was inhibited at an IC50 of 1.1 nM and M07e/p210bcr-abl
cells at 0.10 nM. Also, Kit ligand dependent growth of the
parental M07e line was inhibited with an IC50 of 1.1 nM. This
result correlates well with the strong inhibition of Kit kinase as
seen in the kinase panel.
Figure 3. MicroPET imaging of a K562 xenograft in a mouse with
[18F]-5 from 60 to 75 min.
Kinase inhibition by 5 at 10 nM was examined in a panel of
21 kinases, which includes many relevant members for malig-
nancies of interest (Figure 2). As expected, the pattern of kinase
binding data for dasatinib30 is very similar to the kinase
inhibition profile of compound 5 (see Table S1). Abl, Src, and
Kit are inhibited at >97% at 10 nM, which corresponds to IC50
values of <2 nM. Furthermore, 5 potently inhibited Tec kinase
and two representative ephrin receptor tyrosine kinases, EphA2
and EphB4. Recently, Tec and Btk kinases were found to be
major targets of dasatinib by chemical proteomics.31 While
inhibiting the ephrin receptors may be a double-edged sword
for therapeutics due to tumor-suppressor signaling,32 they are
upregulated in a variety of cancers33 and hold promise in
molecular imaging.34
have shown this selective uptake is possible with a related
kinase-targeted radiotracer in Bcr-Abl overexpressing K562
cells.24
In preliminary in vivo studies in mice, 5 has not shown
toxicity at a dose of 0.1 mg/kg. HPLC analysis of plasma
samples revealed that [18F]-5 was metabolized significantly over
a 2 h time course (see Table S2).
In Vivo PET Imaging. Preliminary in vivo results have been
obtained in three athymic mice bearing subcutaneous K562
(CML) tumor xenografts in the right shoulder. [18F]-5 (14 ( 1
MBq, 375 ( 25 µCi) was injected through the tail vein and the
subjects were imaged in a microPET scanner. Figure 3 shows
the microPET scan of one representative mouse 60 min after
injection; the exposure time was 15 min. Tracer activity was
evident within the tumor xenograft (white arrow) and was
determined to be 1.1% of the injected dose by ROI (region of
interest) analysis. The coronal image shows [18F]-5 activity in
the tumor, blood pool activity in the head (H), physiologic
excretion into the liver and gastrointestinal (GI) tract as well as
into the kidneys and bladder (B). The transaxial image was taken
While we are particularly interested in potent Src/Abl binding
radiotracers, it is obvious that this pharmacophore is not selective
and interacts with a number of kinases.30 A tumor overexpress-
ing a particular kinase, such as Bcr-Abl or Src, can however
selectively uptake a high-affinity probe in the presence of
surrounding tissues that have negligible kinase expression. We

5856 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Brief Articles
N-(2-Chloro-6-methylphenyl)-2-(6-(4-(2-fluoroethyl)piperazin-
1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide, 5.
Piperazine 4 (50 mg, 0.11 mmol), 1-bromo-2-fluoroethane (21 µL,
0.27 mmol), K2CO3 (78 mg, 0.56 mmol), NaI (2 mg, 0.01 mmol),
and 5 mL of CH3CN were added to a 10 mL screw-top tube under
argon. The vial was sealed and stirred 2 h at 60 °C. Another 21 µL
(0.27 mmol) of 1-bromo-2-fluoroethane was added, and the mixture
was stirred another 2 h. The reaction mixture was partitioned
between 30 mL of EtOAc and 30 mL water. The organic layer
was washed with water and brine, dried over MgSO4, and
concentrated to find a yellow oil. Purification by gradient flash
chromatography (SiO2, 0% to 10% 7 N NH3 in MeOH/CH2Cl2)
yielded 46 mg (84%) of compound 5, a white powder: 1H NMR
(DMSO-d6) δ 11.44 (s, 1H), 9.85 (s, 1H), 8.20 (s, 1H), 7.39 (d,
1H, J ) 7.0 Hz), 7.27 - 7.24 (m, 2H), 6.04 (s, 1H), 4.62 - 4.48
(dt, 4H, J ) 47.8, 4.8 Hz), 3.51 (m, 4H), 2.69 - 2.60 (m, 2H, dt,
at the level of the known palpable tumor as shown in the coronal
section (broken line). The intensity of the radiotracer activity
is color-graded as depicted by the colored scale.
There was no significant uptake observed in bone, suggesting
that [18F]-5 did not undergo rapid metabolic defluorination.
Instead, the compound appears to be cleared via the hepatobil-
iary route predominantly. This result correlates with the
distribution of dasatinib in mice.
The question remains whether uptake is a function of
expression of Bcr-Abl. The expression of Bcr-Abl in K562 can
be modulated using siRNAs;35 further experiments using this
approach are underway to determine the correlation between
the expression of Bcr-Abl and tumor uptake.
Conclusions
4H, J ) 28.8, 4.8 Hz), 2.68 (m, 4H) 2.39 (s, 3H), 2.22 (s, 3H); 13
C
NMR (DMSO-d6) δ 165.1, 162.5, 162.4, 159.9, 156.9, 140.8, 138.8,
133.5, 132.4, 129.0, 128.1, 127.0, 125.7, 82.6, 81.7 (d, J ) 164
Hz), 57.5 (d, J ) 19 Hz), 52.4 (2), 43.5 (2), 25.5, 18.3; 19F NMR
(DMSO-d6) δ -217; FTIR (ATR) νmax 3202, 2945, 1622, 1576,
1504, 1413, 1394, 1290, 1188, 768; MS-ESI m/z 490 [M + H]+;
HRMS (FAB+) calcd for C22H25ClFN7OS, 489.1514; found,
489.1519; HPLC tR ) 5.6 min (Phenomenex Gemini C18 250 ×
4.6 mm, 50% 20 mM pH 4.1 KH2PO4/50% CH3CN, 1 mL/min, λ
) 254 nm).
2-Bromoethyltriflate, 6. 2-Bromoethyltriflate, 6, was produced
as in Chi et al., distilled, aliquated, sealed under argon, and stored
at -20 °C.27 1H NMR (CDCl3) δ 4.75 (t, 2H, J ) 6.4 Hz), 3.61 (t,
2H, J ) 6.4 Hz); 13C NMR (CDCl3) δ 118.5 (q, J ) 320 Hz),
74.2, 26.1; 19F NMR (CDCl3) δ -75.0.
Radiosynthesis. [18F]-N-(2-Chloro-6-methylphenyl)-2-(6-(4-(2-
fluoroethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thia-
zole-5-carboxamide, [18F]-5. The radiosynthesis of [18F]-5 was
performed via two methods from 2-bromoethyltriflate, 6 (method
A) or ethylene glycol ditosylate (method B; Supporting Informa-
tion). Routine production of [18F]-5 currently uses method A.
Method A. The QMA cartridge containing cyclotron-produced
[18F] fluoride ion was eluted with a solution containing 420 µL of
H2O and 120 µL of 0.25 M K2CO3 into a 10 mL Reacti-vial
containing 15 mg of Kryptofix [2.2.2] (4,7,13,16,21,24-hexaoxa-
1,10-diazabicyclo[8.8.8]hexacosane) in 1.0 mL of CH3CN. Water
was removed azeotropically with CH3CN (3 × 1.0 mL) at 100-
105 °C. The Reacti-vial was cooled to 0 °C, and to the anhydrous
[18F] KF/K2CO3 complexed with Kryptofix was added a solution
of 2-bromoethyltriflate, 6, (0.054 mmole) in o-dichlorobenzene (500
µL) and heated to 105 °C for 10 min. The [18F]-1-bromo-2-
fluoroethane ([18F]-7) formed was distilled at 120 °C by bubbling
a stream of argon (100 mL/min) into another Reacti-Vial maintained
at -25 °C, containing a solution of piperazine precursor 4 (6.5
mg, 14.6 µM), NaI (9.0 mg, 60 µM), and Cs2CO3 (5 mg, 15.3 µM)
in 500 µL of 1:1 CH3CN:DMF. The activity in the receiving vial
was measured periodically to follow the distillation procedure (5
min). The Reacti-Vial was fitted with a new, unpierced septum to
minimize loss of [18F]-7 at high temperature. The solution was
heated to 120 °C for 40 min, cooled, diluted with 1.2 mL of 1:4
CH3CN/50 mM pH 5.5 NaOAc and passed through a 13 mm
syringe filter (0.25 µm). This solution was injected onto a C18
semipreparative HPLC column and eluted under gradient conditions;
80%A (50 mM pH 5.5 NaOAc)/20% B (CH3CN) to 20% A/80%
B. [18F]-5 eluted at 15.3 min, which was well resolved from
precursor 4 (tR ) 13.4 min). For intravenous administration, the
product-containing fraction was stripped of solvent by rotary
evaporation, formulated in 5% BSA in saline to the proper dosage
and sterile filtered. The radiochemical purity of the final formulation
was confirmed using analytical HPLC. Coelution with nonradioac-
tive 19F reference compound 5 confirmed the identity of the
radiotracer. To measure radiochemical and chemical purity (>99%),
[18F]-5 was reinjected from the semi-prep HPLC product peak on
analytical HPLC (product tR ) 13.2 min, isocratic 60% 50 mM
We report the radiosynthesis, biological evaluation, and
preliminary in vivo micro-PET imaging of a fluorine-18
radiotracer based on a potent, multitargeted kinase inhibitor,
dasatinib, which is approved for the treatment of imatinib-
resistant CML and Ph+ ALL. Compound 5 has similar target
selectivity to dasatinib in vitro and retains strong antitumor
potency. Radiosynthesis of [18F]-5 was accomplished in a two-
step approach by radiofluorination of either 2-bromoethyltriflate
or ethylene glycol ditosylate and subsequent alkylation of
piperazine precursor 4. Production runs of [18F]-5 from 2-bro-
moethyltriflate had an average specific activity of 2560 mCi/
µmol (n ) 11) in 125 ( 5 min after end-of-bombardment. Probe
[18F]-5 had significant K562 tumor uptake in mice and, thus, is
being investigated further as a molecularly targeted PET imaging
probe with in vivo models of systemic CML, GIST, and other
malignancies involving Abl, Src, and Kit.
Possibly the most exciting potential of kinase-targeted probes
like [18F]-5 is to visualize tumor characteristics on a molecular
level, noninvasively, such as the existence or emergence of drug-
resistant leukemia in bone marrow. Established proliferative
imaging modalities like [18F]-FLT or [18F]-FDG are valuable
but cannot give the same information about the molecular
changes occurring during disease progression or the emergence
of resistance. Our ultimate goal is to determine whether [18F]-5
is useful in identifying malignancies that might respond to
dasatinib treatment and thereby use PET imaging with [18F]-5
as a prognostic tool for kinase inhibitor therapy.
Experimental Section
Chemistry. Dasatinib, 2. Dasatinib, 2, was synthesized accord-
ing to the procedure of Lombardo, et al.13
N-(2-Chloro-6-methylphenyl)-2-(2-methyl-6-(piperazin-1-yl)-
pyrimidin-4-ylamino)thiazole-5-carboxamide, 4. 2-(6-Chloro-2-
methylpyrimidin-4-ylamino)-N-(2-chloro-6-methylphenyl)thiazole-
5-carboxamide, 313 (1.00 g, 2.54 mmol), piperazine (2.19 g, 25.4
mmol), and N,N-diisopropylethylamine (0.84 mL, 5.07 mmol) were
dissolved in 30 mL of dry 1,4-dioxane and refluxed overnight. The
solvent was stripped and the residue was triturated several times
with DI water/MeOH, MeOH/ether, and ether. The white solid was
1
dried under high vacuum to give precursor 4 (0.88 g, 78%). H
NMR (DMSO-d6) δ 9.85 (s, 1H), 8.20 (s, 1H), 7.39 (dd, 1H, J )
7.5, 1.5 Hz), 7.29 - 7.22 (m, 2H), 6.01 (s, 1H), 3.43 (m, 4H), 2.73
(m, 4H), 2.39 (s, 3H), 2.23 (s, 3H); 13C NMR (DMSO-d6) δ 165.1,
162.6, 162.5, 159.9, 156.9, 140.8, 138.8, 133.5, 132.4, 129.0, 128.1,
127.0, 125.6, 82.4, 45.3 (2), 44.8 (2), 25.6, 18.3; FTIR (ATR) νmax
3190, 2950, 1619, 1571, 1506, 1410, 1294, 1205, 1185, 769; MS-
ESI m/z 445 [M + H]+; HRMS (FAB+) calcd for C20H22ClN7OS,
443.1295; found, 443.1303; HPLC tR ) 2.6 min (Phenomenex
Gemini C18 250 × 4.6 mm, 50% 20 mM pH 4.1 KH2PO4/50%
CH3CN, 1 mL/min, λ ) 254 nm).

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5857
(16) Johnstrom, P.; Fredriksson, A.; Thorell, J. O.; Stone-Elander, S.
Synthesis of [methoxy-C-11]PD153035, a selective EGF receptor
tyrosine kinase inhibitor. J. Labelled Compd. Radiopharm. 1998, 41,
623-629.
(17) Lim, J. K.; Negash, K.; Hanrahan, S. M.; VanBrocklin, H. F.
Synthesis of 4-(3′-[I-125]iodoanilino)-6,7-dialkoxyquinazolines: Ra-
diolabeled epidermal growth factor receptor tyrosine kinase inhibitors.
J. Labelled Compd. Radiopharm. 2000, 43, 1183-1191.
(18) Wang, J. Q.; Gao, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q. H.
Synthesis of [11C]Iressa as a new potential PET cancer imaging agent
for epidermal growth factor receptor tyrosine kinase. Bioorg. Med.
Chem. Lett. 2006, 16, 4102-4106.
(19) Wang, J. Q.; Miller, K. D.; Sledge, G. W.; Zheng, Q. H. Synthesis
of [18F]SU11248, a new potential PET tracer for imaging cancer
tyrosine kinase. Bioorg. Med. Chem. Lett. 2005, 15, 4380-4384.
(20) Larson, S. M.; Nelp, W. B. The radiocolloid bone marrow scan in
malignant disease. J. Surg. Oncol. 1971, 3, 685-697.
(21) Shreeve, W. W. Use of isotopes in the diagnosis of hematopoietic
disorders. Exp. Hematol. 2007, 35, 173-179.
(22) Agool, A.; Schot, B. W.; Jager, P. L.; Vellenga, E. 18F-FLT PET in
hematologic disorders: A novel technique to analyze the bone
marrow compartment. J. Nucl. Med. 2006, 47, 1592-1598.
(23) Ackermann, U.; Tochon-Danguy, H. J.; Nerrie, M.; Nice, E. C.;
Sachinidis, J. I.; Scott, A. M. Synthesis, C-11 labeling and biological
properties of derivatives of the tyrphostin AG957. Nucl. Med. Biol.
2005, 32, 323-328.
(24) Veach, D. R.; Namavari, M.; Beresten, T.; Balatoni, J.; Minchenko,
M.; Djaballah, H.; Finn, R. D.; Clarkson, B.; Gelovani, J. G.;
Bornmann, W. G.; Larson, S. M. Synthesis and in vitro examination
of [I-124]-, [I-125]- and [I-131]-2-(4-iodophenylamino) pyrido[2,3-
d]pyrimidin-7-one radiolabeled Abl kinase inhibitors. Nucl. Med. Biol.
2005, 32, 313-321.
(25) Kil, K.; Ding, Y.; Lin, K.; Alexoff, D.; Kim, S. W.; Shea, C.; Xu,
Y.; Muench, L.; Fowler, J. S. Synthesis and positron emission
tomography studies of carbon-11-labeled imatinib (Gleevec). Nucl.
Med. Biol. 2007, 34, 153-163.
(26) Tokarski, J. S.; Newitt, J. A.; Chang, C. Y. J.; Cheng, J. D.; Wittekind,
M.; Kiefer, S. E.; Kish, K.; Lee, F. Y. F.; Borzillerri, R.; Lombardo,
L. J.; Xie, D. L.; Zhang, Y. Q.; 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, 5790-5797.
(27) Chi, D. Y.; Kilbourn, M. R.; Katzenellenbogen, J. A.; Welch, M. J.
A rapid and efficient method for the fluoroalkylation of amines and
amidessdevelopment of a method suitable for incorporation of the
short-lived positron emitting radionuclide F-18. J. Org. Chem. 1987,
52, 658-664.
(28) Nagar, B.; Hantschel, O.; Young, M. A.; Scheffzek, K.; Veach, D.;
Bornmann, V.; Clarkson, B.; Superti-Furga, G.; Kuriyan, J. Structural
basis for the autoinhibition of c-Abl tyrosine kinase. Cell 2003, 112,
859-871.
(29) Block, D.; Coenen, H. H.; Stocklin, G. The nca nucleophilic F-18
fluorination of 1,N-disubstituted alkanes as fluoroalkylation agents.
J. Labelled Compd. Radiopharm. 1987, 24, 1029-1042.
(30) 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.; 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, 11011-11016.
(31) Hantschel, O.; Rix, U.; Schmidt, U.; Burckstummer, T.; Kneidinger,
M.; Schutze, G.; Colinge, J.; Bennett, K. L.; Ellmeier, W.; Valent,
P.; Superti-Furga, G. The Btk tyrosine kinase is a major target of
the Bcr-Abl inhibitor dasatinib. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 13283-13288.
(32) Wang, J. Y. Eph tumour suppression: The dark side of Gleevec.
Nat. Cell Biol. 2006, 8, 785-786.
(33) Noren, N. K.; Pasquale, E. B. Paradoxes of the EphB4 receptor in
cancer. Cancer Res. 2007, 67, 3994-3997.
pH 5.5 NaOAc/40% CH3CN, 1.0 mL/min). An example of this
chromatogram appears in the Supporting Information (Figure S2).
Total time of radiosynthesis was 120 ( 5 minutes from EOB. The
decay-corrected radiochemical yields (n ) 3) were 25.1 ( 5.8%
from [18F]-1-bromo-2-fluoroethane and 6.6 ( 2.3% overall from
starting [18F]-fluoride. The specific activity ranged from 108-7350
mCi/µmol (average 2560 mCi/µmol, n ) 11).
Acknowledgment. The authors thank the U.S. Department
of Energy for support under Contract Nos. DE-FG02-86ER60407
and ER63693 and the National Institutes of Health under
Contract Nos. P50 CA86438 and P30 CA008748. Technical
services provided by the MSKCC Small-Animal Imaging Core
Facility, supported in part by NIH Small-Animal Imaging
Research Program (SAIRP) Grant No. R24 CA83084 and NIH
Center Grant No. P30 CA08748, are gratefully acknowledged.
D.R.V. and B.C. also thank the MeadWestvaco Corp. for their
continuing support and the Kirin Brewery Co., Ltd. (Tokyo,
Japan) for rhGM-CSF and rhSCF.
Supporting Information Available: Experimental details of
general synthesis, molecular modeling, protein binding, log Do/w
determination, kinase assays, cell proliferation assays, metabolite
analysis, and mouse PET imaging. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Hicks, R. J. The role of PET in monitoring therapy. Cancer Imaging
2005, 5, 51-57.
(2) Shields, A. F. Positron emission tomography measurement of tumor
metabolism and growth: Its expanding role in oncology. Mol.
Imaging Biol. 2006, 8, 141-150.
(3) Hernandez, S. E.; Krishnaswami, M.; Miller, A. L.; Koleske, A. J.
How do Abl family kinases regulate cell shape and movement?
Trends Cell Biol. 2004, 14, 36-44.
(4) Wang, J. Y. Regulation of cell death by the Abl tyrosine kinase.
Oncogene 2000, 19, 5643-5650.
(5) Wong, S.; Witte, O. N. The BCR-ABL story: Bench to bedside and
back. Annu. ReV. Immunol. 2004, 22, 247-306.
(6) Chen, T.; George, J. A.; Taylor, C. C. Src tyrosine kinase as a
chemotherapeutic target: Is there a clinical case? Anticancer Drugs
2006, 17, 123-131.
(7) Deininger, M.; Buchdunger, E.; Druker, B. J. The development of
imatinib as a therapeutic agent for chronic myeloid leukemia. Blood
2005, 105, 2640-2653.
(8) Zimmermann, J.; Buchdunger, E.; Mett, H.; Meyer, T.; Lydon, N.
B. Potent and selective inhibitors of the Abl-kinase: Phenylamino-
pyrimidine (PAP) derivatives. Bioorg. Med. Chem. Lett. 1997, 7,
187-192.
(9) Hornick, J. L.; Fletcher, C. D. The role of KIT in the management
of patients with gastrointestinal stromal tumors. Hum. Pathol. 2007,
38, 679-687.
(10) Melo, J. V.; Chuah, C. Resistance to imatinib mesylate in chronic
myeloid leukaemia. Cancer Lett. 2007, 249, 121-132.
(11) Tauchi, T.; Ohyashiki, K. The second generation of BCR-ABL
tyrosine kinase inhibitors. Int. J. Hematol. 2006, 83, 294-300.
(12) Weisberg, E.; Manley, P. W.; Cowan-Jacob, S. W.; Hochhaus, A.;
Griffin, J. D. Second generation inhibitors of BCR-ABL for the
treatment of imatinib-resistant chronic myeloid leukaemia. Nat. ReV.
Cancer 2007, 7, 345-356.
(13) Lombardo, L. J.; Lee, F. Y.; Chen, P.; Norris, D.; Barrish, J. C.;
Behnia, K.; Castaneda, S.; Cornelius, L. A. M.; Das, J.; Doweyko,
A. M.; Fairchild, C.; Hunt, J. T.; Inigo, I.; Johnston, K.; Kamath,
A.; Kan, D.; Klei, H.; Marathe, P.; Pang, S. H.; Peterson, R.; Pitt,
S.; Schieven, G. L.; Schmidt, R. J.; Tokarski, J.; Wen, M. L.; Wityak,
J.; Borzilleri, R. M. Discovery of N-(2-chloro-6-methylphenyl)-2-
(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylami-
no)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase
inhibitor with potent antitumor activity in preclinical assays. J. Med.
Chem. 2004, 47, 6658-6661.
(14) Shah, N. P.; Tran, C.; Lee, F. Y.; Chen, P.; Norris, D.; Sawyers, C.
L. Overriding imatinib resistance with a novel ABL kinase inhibitor.
Science 2004, 305, 399-401.
(15) Abourbeh, G.; Dissoki, S.; Jacobson, O.; Litchi, A.; Ben Daniel, R.;
Laki, D.; Levitzki, A.; Mishani, E. Evaluation of radiolabeled ML04,
a putative irreversible inhibitor of epidermal growth factor receptor,
as a bioprobe for PET imaging of EGFR-overexpressing tumors. Nucl.
Med. Biol. 2007, 34, 55-70.
(34) Cai, W.; Ebrahimnejad, A.; Chen, K.; Cao, Q.; Li, Z.-B.;
Tice, D. A.; Chen, X. Quantitative radioimmunoPET imaging
of EphA2 in tumor-bearing mice. Eur. J. Nucl. Med. Mol. Imaging
2007, (published online Aug 3, 2007) doi:10.1007/s00259-007-
0503-5.
(35) Baker, B. E.; Kestler, D. P.; Ichiki, A. T. Effects of siRNAs in
combination with Gleevec on K-562 cell proliferation and Bcr-Abl
expression. J. Biomed. Sci. 2006, 13, 499-507.
JM070342G