Imatinib analogs as potential agents for PET imaging of Bcr-Abl and c-KIT expression at a kinase level

Article abstract of DOI:10.1016/j.bmc.2013.10.040

We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT receptor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [ ¹⁸F]-labeled STI-571 was prepared with high specific activity (75 GBq/μmol) by nucleophilic displacement and an average radiochemical yield of 12%. [¹³¹I]-labeled STI-571 was prepared with high purity (>95%) and an average radiochemical yield of 23%. The uptake rates of [ ¹⁸F]-STI-571 in K562 cells expressing Abl and in U87WT cells overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71 (confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [¹⁸F]-STI-571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.

Full text of DOI:10.1016/j.bmc.2013.10.040

Bioorganic & Medicinal Chemistry 22 (2014) 623–632
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Imatinib analogs as potential agents for PET imaging of Bcr-Abl and
c-KIT expression at a kinase level
Zhenghong Peng a, David S. Maxwell a, Duoli Sun a, Basvoju A. Bhanu Prasad a, Ashutosh Pal b,
Shimei Wang b, Julius Balatoni b, Pradip Ghosh b, Seok T. Lim b, Andrei Volgin b, Aleksander Shavrin b,
Mian M. Alauddin b, Juri G. Gelovani b, William G. Bornmann a,
⇑
a Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA
b Department of Experimental Diagnostic Imaging, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized two series of imatinib mesylate (STI-571) analogs to develop a Bcr-Abl and c-KIT recep-
tor-specific labeling agent for positron emission tomography (PET) imaging to measure Bcr-Abl and c-KIT
expression levels in a mouse model. The methods of molecular modeling, synthesis of STI-571 and its
analogs, in vitro kinase assays, and radiolabeling are described. Molecular modeling revealed that these
analogs bind the same Bcr-Abl and c-KIT binding sites as those bound by STI-571. The analogs potently
inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, similarly to STI-571. [18F]-labeled STI-571 was
Received 21 August 2013
Revised 16 October 2013
Accepted 24 October 2013
Available online 6 November 2013
Keywords:
c-KIT
KIT
STI-571
PET
Bcr-Abl
Imatinib
Radiolabel
Analog
prepared with high specific activity (75 GBq/lmol) by nucleophilic displacement and an average radio-
chemical yield of 12%. [131I]-labeled STI-571 was prepared with high purity (>95%) and an average radio-
chemical yield of 23%. The uptake rates of [18F]-STI-571 in K562 cells expressing Abl and in U87WT cells
overexpressing c-KIT were significantly higher than those in the U87 cell and could be inhibited by STI-71
(confirming the specificity of uptake). PET scans of K562 and U87WT tumor-bearing mice with [18F]-STI-
571 as a contrast agent showed visible tumor uptake and tumor-to-non-target contrast.
Ó 2013 Published by Elsevier Ltd.
1. Introduction
selection of patients for clinical trials to assess drugs targeting
the c-KIT kinase. The serious clinical challenges for selection of
Activating mutations of KIT are associated with certain human
neoplasms, including those in the majority of patients with sys-
temic mast cell disorders as well as those in patients with semi-
noma, acute myelogenous leukemia (AML), gastrointestinal
stromal tumors (GISTs), and hypopigmentary disorders1 Improved
knowledge of the mechanisms causing pathological mast cell
growth will lead to the discovery of novel treatment options
including drugs targeting the mutated KIT protein. The small-mol-
ecule tyrosine kinase inhibitor imatinib mesylate (GleevecÒ, STI-
571) is a potent inhibitor of wild-type (WT) KIT and certain mutant
KIT isoforms and has become the standard of care for treating pa-
tients with metastatic GIST.2 However, distinct forms of tyrosine
kinase domain (TKD), juxtamembrane domain, exon 8, and internal
tandem duplication (ITD) mutations of c-KIT were observed in
about 46% of core binding factor leukemia (CBFL) patients, and ac-
quired KIT activation loop mutations can be associated with imati-
nib mesylate resistance in GIST.3
optimal setting(s) in which to test and monitor both the biological
efficacy and the therapeutic efficacy of these novel drugs include
(a) identification of patients whose disease would likely respond
well to therapy with c-KIT kinase inhibitors, (b) individualization
of the therapeutic dose(s) and therapeutic regimens, (c) proper
integration of these novel drugs into established cytotoxic thera-
peutic protocols, and (d) development of effective markers and
methods for noninvasive monitoring of the early efficacy of
therapy.
Noninvasive positron emission tomography (PET) imaging with
c-KIT kinase-specific radiolabeled agents could provide a better
assessment of the levels and heterogeneity of c-KIT expression
and activity in tumors in individual patients and could provide
selection criteria for inclusion of patients in redesigned clinical tri-
als. Also, the ability to monitor the levels of expression and activity
of c-KIT at the kinase level should provide a direct measure of bio-
logical drug efficacy (kinase inhibition) in tumors as opposed to
surrogate tissues (e.g., hair, skin, etc.) before any therapeutic effect
is expected.
The current lack of diagnostic assays and markers predictive of
sensitivity to c-KIT inhibitors limits the possibility of proper
The use of [18F]-fluoro-2-deoxy- -glucose ([18F]-FDG) in the
D
staging of and early prediction of response to STI-571 therapy in
patients with recurrent GISTs has been extensively studied by
⇑
Corresponding author. Tel.: +1 713 563 0081; fax: +1 713 563 1878.
E-mail address: wbornmann@mdanderson.org (W.G. Bornmann).
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.10.040

624
Z. Peng et al. / Bioorg. Med. Chem. 22 (2014) 623–632
Gayed et al.,4 who demonstrated that [18F]-FDG is superior to CT in
predicting early response to therapy. In addition, more recently,
Jager et al.5 as well as other investigators confirmed this finding.
Noteworthy is the observation that GIST shares immunohisto-
chemical, ultrastructural, and histogenetic similarities with the
interstitial cells of Cajal. Both GIST and the interstitial cells of Cajal
express c-KIT, the receptor tyrosine kinase that is the protein prod-
uct of the c-KIT proto-oncogene. It has also been found that c-KIT
from GIST cells has a high frequency of mutations that lead to con-
stitutive activation of the c-KIT tyrosine kinase in the absence of
stimulation by the kinase’s physiologic ligand (stem cell factor),
which in turn causes uncontrolled stimulation of downstream sig-
naling cascades with aberrant cellular proliferation and resistance
to apoptosis.3b While these studies are of the great clinical value,
they cannot truly identify before treatment those patients who will
be responsive to imatinib mesylate chemotherapy. Our long term
aim is to use kinase-specific [131I]- and [18F]-labeled STI-571 ana-
logs for PET imaging to help identify patients with GISTs that over-
express c-KIT and that would respond favorably to therapy with
STI-571. On PET, overexpression would correlate with high tumor
uptake and retention of labeled STI-571 analogs, and favorable tu-
mor response would be indicated by early decreases in tumor up-
take and retention of labeled analogs. Similarly, we aim to identify
GISTs with low c-KIT expression or activity as having low [131I]- or
-4-(3-pyridyl)-2-pyrimidineamine 6 in high yield. In order to syn-
thesize the STI-571 and its analogs, the coupling acid chloride
derivatives 10 were prepared in two simple steps. First, alkylation
of piperizine derivatives 8 with a-chloro-p-toluic acid 7 in reflux-
ing ethanol to produce the corresponding acid 9, which was subse-
quently activated with thionyl chloride to give the corresponding
acid chloride 10. It was noted that, during the acid chloride forma-
tion most of the primary alcohol in 10c was converted into chloride
and obtained as mixture. The acid chlorides were immediately
used for the next reaction without further purification as they
found to be less stable. Condensation of N-(2-methyl-5-aminophe-
nyl)-4-(3-pyridyl)-2-pyrimidine-amine 6 and acid chloride deriva-
tives 10 in pyridine yielded the STI-571 and its analogs (Scheme 1)
in good yields.
Furthermore, 4-iodobenzyl substituted STI derivative 11e and
4-n-tributyltinbenzyl substituted STI derivative 11f (Scheme 2)
were also synthesized. The salt 9e was prepared by alkylation of
9d with 4-iodobenzyl bromide 12, and in other hand, palladium
catalyzed transmetallation of 13 with tributyltin metal, then
reductive amination of the resultant stannyl benzaldehyde 14 with
piperazine benzoic acid 9d gave 9f as a salt.9 The salts 9e and 9f
were treated with thionyl chloride to obtain 10e and 10f, respec-
tively, which were then subjected to condensation with 6 in pyri-
dine to produce 11e and 11f, respectively in good yields
(Scheme 2).
[
18F]STI-571 analog uptake and retention and thus as being poten-
tially poor responders to STI-571 therapy.
In additions to the synthesis of STI-571 and its analogs, we
chose to make radiolabeled STI analogs. Converting the primary
alcohol of 11c into as a good leaving group then knocking it off
with radiolabeled nucleophiles would lead to the radiolabeled STI
analogs. However, all our efforts to synthesize the mesylate, tosyl-
ate or triflate derivative of 11c were unsuccessful due the internal
attack of the piperazine amine as a nucleophile onto the leaving
group to form highly unstable aziridinium salt which was then
decomposed to give back 11c (STI-OH) or 11d (STI-Cl).10 Since
the conversion of primary alcohol of 11c (STI-OH) into a good leav-
ing was ineffective, we chose 11d (STI-Cl) as a precursor for the
synthesis of radiolabeled STI analog.
As part of an ongoing program to develop new potent inhibitors
of c-KIT tyrosine kinases,6,7 we have developed a class of STI-571
analogs (4-(3-pyridyl)-2-pyrimidines) with significant c-KIT inhibi-
tion activities, and we aim to synthesize analogs of STI-571 radio-
labeled with 131I or 18F. The plan to modify imatinib with
radioactive fluorine was also pursued by Larson and colleagues,8
who used an 18F-radiolabeled derivative of imatinib to measure
Bcr-Abl expression. In this study, we aimed to measure both Bcr-
Abl and c-KIT expression levels in a mouse model
2. Results and discussions
2.1. Molecular modeling
2.3. Radiolabeling
STI-[18F] 11g was prepared by fluorination of the STI-Cl 11d
with n-Bu4N[18F] followed by HPLC purification (Scheme 3).
n-Bu4N[18F] was prepared in situ from n-Bu4NHCO3 and aqueous
H18F using 0.40 mL (1% solution, ꢁ7 mmol) of n-Bu4NHCO3 to elute
the activity from the ion exchange cartridge.11 After radiofluorina-
tion of the precursor 11d, the crude reaction mixture was passed
through a silica gel cartridge (900 mg; Alltech), and the crude
product was eluted with 30% MeOH in dichloromethane. Silica
gel holds the unreacted [18F] fluoride from the product and helps
to avoid overloading the HPLC column with high levels of free fluo-
ride during purification. The recovered 18F-labeled STI was purified
by semi preparative HPLC and was eluted at 11.6 min. using a 50%
MeCN and 50% water-containing 0.1% ammonium formate solvent
system with a flow of 4 mL/min. Ammonium formate (0.1%) in
water was used to improve the peak shape. Quality control analysis
of the final product STI-[18F] 11g was performed by analytical HPLC
using the same solvent system at a flow rate of 1 mL/min. Co-injec-
tion of the final product 11g with an authentic sample of STI-F 11b
showed that the radiolabeled compound and standard STI-F were
co-eluted at 6.7 min. The radiochemical yield of this synthesis
was 8–16% with an average of 12% in 12 runs (corrected for decay).
The radiochemical purity was >99%, with specific activity >74 GBq/
mmol. The synthesis time was 65–75 min from the end of
bombardment.
Our FlexX docking results for the highest ranked configuration
of each STI analog/c-KIT combination showed that STI-571 (11a),
STI-F (11b), and STI-I (11e) (Scheme 1) had similar scores
(ꢀ41.47, ꢀ38.54, ꢀ37.70, respectively) and nearly identical bind-
ing configurations. Scores for the same structures in Abl were
ꢀ37.09, ꢀ38.39, and ꢀ40.58. As the scores are an estimate of the
free energy of binding, more negative values are better. There is
no evidence to suggest that these modifications would cause any
unfavorable steric overlap. Therefore, the modified molecules
should have activities for c-KIT and Abl kinase similar to those of
STI-571.
2.2. Synthesis of STI-571 and its analogs
The synthesis of STI-571 and its analogs began with treatment
of 2-methyl-5-nitroaniline 1 with 65% nitric acid in ethanol
followed by the addition of cyanamide to give the corresponding
2-methyl-5-nitroaniline-guanidine nitrate salt 2 (Scheme 1). Pyri-
dine derivative 4 was synthesized by refluxing 3-acetylpyridine 3
with N,N-dimethylformamide dimethyl acetal under neat condi-
tions for about 18 h. The resulted compound 4 was subjected to
condensation reaction with nitrate salt 2 in presence of sodium
hydroxide in refluxing isopropanol to produce pyrimidineamine
derivative 5, which was subsequently hydrogenated with 10%
palladium on carbon to obtain N-(2-methyl-5-aminophenyl)
STI-[131I] 11h was prepared by treating STI-Sn 11f precursor
with a mixture of 30% hydrogen peroxide/glacial acetic acid (1:3)

Z. Peng et al. / Bioorg. Med. Chem. 22 (2014) 623–632
625
NO3
HN
NH3
1. 65% HNO3 in EtOH
2. 50% NH2CN in H2O
reflux 18 h
H2N
Me
NO2
HN
Me
NO2
H
N
H
N
N
NO2
N
NH2
N
N
N
N
NaOH
iPrOH
reflux
1
2
Me
Me
OMe
Me
Pd/H2
MeO
N
O
O
Me
Me
5
6
neat, reflux, 18 h
Me
N
Me
N
N
3
4
H
N
H
N
N
O
N
COCl
COOH
pyridine
24 h, rt
N
N
COOH
SOCl2
reflux
EtOH
reflux, 18 h
Me
H
N
+
Cl
Cl
N
R
N
N
Cl
R
Cl
R
H
H
N
Cl
N
N
H
11a STI-571 (R = Me)
R
H
7
8
9
10
8a R = Me
9a R = Me
10a R = Me
10b R = CH2CH2F
10c R = CH2CH2Cl & CH2CH2OH
11b R = CH2CH2F
11c R = CH2CH2OH
11d R = CH2CH2Cl
8b R = CH2CH2F
8c R = CH2CH2OH
8d R = Boc
9b R = CH2CH2F
9c R = CH2CH2OH
9d R = H
Scheme 1. Synthesis of STI-571 and its analogs.
COCl
STI-[131I] in crude form averaged 23.3% (19–27%, n = 3). The radio-
COOH
chemical purity of the final product was >95%.
9d
Br
3. Bcr-Abl kinase assay
EtOH
reflux, 18 h
SOCl2
reflux
H
H
Cl
H
Cl
H
N
N
N
N
The kinase assay was performed on STI-F (11b), and STI-I (11e),
STI-OH (11c) and STI-571 (11a). STI-571 inhibits Bcr-Abl with an
Cl
Cl
I
IC50 of 0.11
0.73 M, respectively. STI-OH was the least potent, with an IC50
of 7.08 M (Fig. 1).
lM; STI-F and STI-I IC50 values were 1.3 lM and
l
R
R
l
9e
10e
R = I
10f
R = Sn(n-Bu)3
R = I
12
9f
R = Sn(n-Bu)3
4. Free energy perturbation (FEP) calculation results
6
9d
Pyridine
24 h, rt
In order to better understand the observed experimental
difference in inhibitory effect of the compounds versus STI-571
(11a), we ran Monte Carlo/free energy perturbation (MC/FEP)
NaCNBH3
H
H
N
N
N
O
N
O
O
[Sn(n-Bu)3]2
Pd(PPh3)4
N
N
Me
H
N
H
N
N
O
N
Sn(n-Bu)3
R
Br
13
N
N
N
n-Bu4NHCO3
11e
11f
R = I
Me
14
R = Sn(n-Bu)3
H[18F]
11d
Scheme 2. Synthesis of STI-I and STI-Sn analogs.
N
11g
([18F]STI-571
F18
in the presence of [131I]iodide for 3 min at room temperature
(Scheme 3).12 The reaction was quenched with an aqueous solution
of saturated sodium metabisulfite and loaded onto a Sep-Pak C18
column. The column was washed with water, and the product
was eluted with ethanol. Crude STI-[131I] was purified by prepara-
tory HPLC, concentrated on a Sep-Pak C18 column, and isolated by
elution with ethanol. Radiochemical purity was determined by
radio-thin-layer chromatography using chloroform/MeOH (85:15)
on silica gel plates. STI-[131I] 11h was also characterized by
radio-HPLC on an analytical C8 column using acetonitrile/0.1%
ammonium formate in water (1:1) at a 1 mL/min flow rate, with
UV detection set at 265 nm. A mixture of standard STI-I 11e and
STI-[131I] 11h was found to co-elute. The radiochemical yield of
H
H
N
N
N
O
N
30% H2O2
N
Me
CH3COOH, [131I]
11f
N
I131
N
11h
([131I]STI-571
Scheme 3. Synthesis of radiolabeled [18F]STI-571 and [131I]STI-571 analogs.

626
Z. Peng et al. / Bioorg. Med. Chem. 22 (2014) 623–632
Table 1
Comparison of experimental and theoretically calculated DDGbind for STI-571 and
analogs
a
b
Compound
DDGbind (calcd)
DDGbind (exptl)
IC50 (nM)
11a
11b
11c
11e
R = ethyl
R = propyl
0
0
116.0
1378
7083
739.5
4.09 0.22
3.42 0.20
—
2.58 0.12
5.04 0.17
1.47
2.44
1.10
—
—
a
Calculated from DDGbind = RTln [IC50(compd)/IC50(11a)]. Rt = 0.59219 at 298 K;
values are listed in kcal/mol.
b
Determined by fit of IC50 curve to log(concd) versus normalized %phosphory-
lation with GraphPad Prism v5.04.
Figure 1. Kinetic inhibitory effect rates of STI-571 and its analogs (determined by
measuring phosphorylation rates in spectrophotometric assays of Bcr-Abl kinase
activity).
value, the DDGbind would give 2.53 0.21 kcal/mol and
3.20 0.23 kcal/mol for 2-hydroxyethyl and 2-fluoroethyl, respec-
tively. As shown in the IC50 plots, there is also error associated with
the biological experiments, which could also explain the differ-
ences observed between the theoretical and experimental values.
calculations for 11a, 11b, and 11c. These calculations were per-
formed in stages starting from the docked STI-571 structure. For
set 1, perturbations involved ethyl->methyl, propyl->2-hydroxy-
ethyl and propyl->ethyl. In set 2, perturbations were 2-hydroxy-
ethyl->2-fluoroethyl and propyl->2-hydroxyethyl. Shown in
Figure 2 is the structure with lowest average total system energy
out of those stored at the end of each 250 K configurations. The
majority of interactions were identical to the crystal structure.
One exception was the piperazine unit, which is nearby the back-
bone carbonyls of His 95/Arg 96 in the crystal, but directly hydro-
gen bonds with Asp115 in the simulation. Extending STI-571 by
one or two carbons on the piperazine was increasingly disfavored
as shown in Table 1. This is expected, since the additional methyl
groups are not interacting with a hydrophobic area in the protein.
Changing to 2-hydroxyethyl was favorable compared to the propyl
group by 1.62 0.11 kcal/mol, but overall unfavorable by
3.42 0.20 kcal/mol in comparison to 11a. This was most likely
due to the hydrogen bonding capability of the OH group, which ac-
cepts a hydrogen bond from Arg 96 in the lowest energy structure
(not shown). The experimental DDGbind value for this change was
2.44 kcal/mol. The calculated DDGbind for 2-hydroxyethyl to 2-flu-
oroethyl was 0.67 0.09 kcal/mol, but the experimental deter-
mined value was ꢀ0.97. With 2-fluoroethyl, there is a water
bridging the interaction to Asp115, which is also observed for
2-hydroxyethyl. Error in the theoretical values could be due to
incomplete sampling and another estimate may be determined
from comparing the set 1 and set 2 perturbations, which repeat
the propyl->2-hydroxyethyl calculations from a different starting
point. In that calculation, the DDGbind was ꢀ2.51 0.12 kcal/mol
as compared with ꢀ1.62 0.11 kcal/mol from set 1. Using that
5. In vitro studies
U87WT cells were produced by retroviral transduction of U87
cells with a wild-type human c-KIT receptor. Western blotting of
protein extracts from K562, U87, and U87WT cells was performed
to assess Bcr-Abl and c-KIT expression levels (Fig. 3). K562 cells ex-
pressed Bcr-Abl, and U87WT cells expressed c-KIT.
In vitro radiotracer accumulation, washout, and blocking stud-
ies performed in our laboratory showed that the binding of
[
18F]STI-571 diminished as excess STI-571 was added to the cell
cultures (Fig. 4A and B). The uptake and washout of [18F]STI-571
at 1 and 2 h in K562 cells revealed an increase in residual intracel-
lular radioactivity (Fig. 4C), which may be explained, at least in
part, by intracellular re-compartmentalization and entrapment of
the Abl kinase domain-[18F]STI-571 complex in the compartment
with poor exchange (i.e., proteasome). The uptake and washout
at 1 and 2 h in wild-type U87 cells showed no increase in residual
radioactivity (Fig. 4D). The results of uptake and washout studies
indicated that [18F]STI-571 and STI-571 have the same binding
target.
6. Small-animal PET studies
The rates of [18F]STI-571 uptake in K562 cells expressing Abl
and in U87WT cells overexpressing c-KIT were significantly higher
than that in wild-type U87 cells and were susceptible to inhibition
by cold STI-571 (confirming the specificity of uptake). A PET scan
Figure 2. (Left) Interaction of STI-571 and (right) 11b from MCPRO+ calculations. Only nearby residues and water with H-bonding interactions with the ligand are shown.
Images were created with PYMOL v1.5.0.4.26

Z. Peng et al. / Bioorg. Med. Chem. 22 (2014) 623–632
627
Figure 3. (A) Western blot of c-KIT expression in U87WT cells. (B and C) K562 cells expressed Bcr-Abl, Bcr, and Abl but not c-KIT.
100
A
B
6
5
4
3
2
75
50
25
0
-2
-1
0
1
2
0
30
60
90
120
150
Time (min)
Log contraꢀon (μM)
C 80
D
40
60
40
20
0
30
20
10
0
0
30
60
90
120 150 180 210
0
30
60
90
120 150 180 210
Time (min)
Time (min)
Figure 4. (A) [18F]STI-571 rapidly accumulates in K562 cells and then reaches a dynamic at 30 min. (B) The [18F]STI-571 equilibrium in K562 cells was progressively
decreased by exposure to increasing concentrations of STI-571. (C) Uptake and washout of [18F]STI-571 at 1 and 2 h in K562 cells. (D) Uptake and washout of [18F]STI-571 at 1
and 2 h in wild-type U87 cells.
imaging study of K562 and U87WT-bearing mice was undertaken
to compare [18F]FDG levels and the results were shown in Figure 5.
Tumors implanted in the right shoulder were visible in all mice;
the uptake of the tracer in the digestive tract was quite high. The
tumors were visualized at 60 and 120 min after injection. The large
amounts of radioactivity in the liver and the intestines were not
unexpected, because imatinib is eliminated mainly through biliary
excretion or hepatic metabolism, presumably as a result of catab-
olism and defluorination of [18F]STI-571 in mice, as evidenced by
extensive accumulation of [18F] in the bone. In contrast, [18F]FDG
was clearly visible in the mice at 45 min. [18F]STI-571 PET images
showed higher uptake of the radiotracer in c-KIT-overexpressing
U87WT tumors (right shoulder) than in U87 tumors (left shoulder),
which do not express c-KIT. In both studies, tumor-to-muscle and
c-KIT+ tumor-to-control ratios were the highest at 60 min after
18F]STI-571 administration.
[
7. Conclusion
In summary, we have synthesized a series of non-labeled STI-
571 analogs along with [18F] and [131I] radiolabeled versions of
two of them. The goal was to develop a labeling agent for positron
emission tomography (PET) imaging that could measure Bcr-Abl
and c-KIT kinase expression levels. We have shown that these ana-
logs inhibit the tyrosine kinase activity of Bcr-Abl and c-KIT, albeit
to a lesser degree than STI-571, which is further supported by our
FEP calculations. The uptake rates of [18F]-STI-571 in K562 cells
expressing Abl and in U87WT cells overexpressing c-KIT were

628
Z. Peng et al. / Bioorg. Med. Chem. 22 (2014) 623–632
Figure 5. (A) [18F]STI-571 PET images of Bcr-Abl expression in K562 tumors (top). PET images of [18F]FDG accumulation are provided for comparison. (B) [18F]STI-571 PET
images showing the higher uptake of the radiotracer in c-KIT overexpressing U87WT tumors (right shoulder) than in wild-type U87 tumors (down), which do not express c-
KIT.
significantly higher than those in the wild type U87 and dimin-
ished with excess of STI-571 confirming the specificity of uptake.
PET scans of K562 and U87WT tumor-bearing mice with [18F]-
STI-571 as a contrast agent showed visible tumor uptake and tu-
mor-to-non-target contrast. Thus, [18F]-STI-571 could be utilized
as a marker of sensitivity to Bcr-Abl and c-KIT inhibitors and assist
with the selection of patients that could most likely benefit from
drugs targeting the Bcr-Abl and c-KIT kinases. Further studies with
131I-labeled STI-571 are under way.
primary docking region selected for FlexX included all residues
within 6.5 Å of the STI-571. Ten configurations were requested
for each ligand, and formal charge assignment for the ligand was
template based.
8.1.2. Free energy perturbation (FEP) calculations
The free energy perturbation (FEP) calculations were completed
on a 4-quad core (16-core) 2.4 Ghz AMD Opteron system using the
RHEL 5.x OS and a 2-eight core (16-core) 3.0 Ghz AMD Opteron
system. The Suite 2012 from Schrödinger was utilized for all dock-
ing and Monte Carlo/free-energy perturbation (MC/FEP) calcula-
tions. The PDB structure 2hyy was downloaded from the Protein
Data Bank16 and read into Maestro17 with alternative positions
turned off, and all chains except chain A were removed. A basic
protein preparation was performed using program defaults, with
the addition of filling in missing side chains and deleting water
molecules beyond 5 Å from heteroatoms. The complete prepara-
tion of the protein portion of the model involved the following:
(1) basic protein preparation; (2) assignment of heteroatom states;
(3) H-bond assignment, including PROPKA; (4) deletion of waters
with less than three H-bonds to non-waters; (5) Impref ‘H-only’
minimization; and (6) Impref ‘Minimize All’ to root mean square
deviation of 0.5. The structure of STI-571 was drawn in Chem-
DrawUltra 12.0 on a PC, transferred to the Linux box and converted
to an SDF formatted file. A Ligprep18 calculation was performed on
the structure by using default criteria and Epik19 ionization. The
Glide Grid for docking was created with crystal ligand as basis
8. Materials and methods
8.1. Modeling
8.1.1. Docking of STI-571 and F- or I-labeled analogs
The docking calculations were completed on a 4-processer
R16000 SGI Tezro running Sybyl 7.214 and FlexX15 docking soft-
ware. The crystal structures of STI-571 bound to c-KIT (PDB code
1T46) and Abl (PDB code 1IEP) were obtained from the Protein
Data Bank.13 The structures were brought into Sybyl and all addi-
tional chains, waters, and ions were removed before docking. The
STI-571 ligand from 1IEP was extracted and modified with correct
atom types for the Tripos force field and protonated at the pipera-
zine nitrogen furthest from the phenyl group. A minimization was
completed using the Powell method and a gradient cutoff of
0.05 kcal/(mol Å). This structure was the basis for creating STI-F
and STI-I, which were also minimized in the same manner. The

Z. Peng et al. / Bioorg. Med. Chem. 22 (2014) 623–632
629
and no constraints were selected during the calculation. The STI-
8.2.2. 3-Dimethylamino-1-(3-(6-methyl-pyridyl))-2-propen-1-
one (4)23
571 structure was docked using the virtual screening workflow,20
which involved docking with Glide XP mode starting with three ex-
3-Acetylpyridine 3 (100 g, 0.19 mol) was added to dimethyl-
formamide dimethyl acetal (156.5 g, 1.27 mol), and the mixture
was reacted under reflux for 23 h. After the reaction mixture was
cooled to 0 °C, a mixture of diethyl ether and hexane (3:2, v/v)
(500 mL) was added, and the whole mixture was stirred for 4 h.
The resulting solid was filtered and washed with a mixture of
diethyl ether and hexane (500 mL, 3/2, v/v) to give 3-dimethyl-
21
tra conformations and rescoring with the Prime MMGBSA
DGbind.
Variations were selected in the Ligand Functional Group Mutation
by FEP panel for MCPRO+.22 Perturbations for set 1 involved ethyl-
>methyl, propyl->2-hydroxyethyl and propyl->ethyl. For set 2, the
starting structure was modified to contain a propyl group instead
of a methyl group. No optimization of the structure was done
and the residue was renumbered and renamed. Perturbations for
set 2 were 2-hydroxyethyl->2-fluroethyl and propyl->2-hydroxy-
ethyl. Simulations were completed with default parameters.
Briefly, this allowed for full ligand flexibility and protein flexibility
for residue side chains within 8 Å of ligand and those beyond 11 Å
were chopped. Solvation involved a 22 Å TIP3P water cap around
the isolated ligand and complex. Atom type parameters came from
the OPLS2005 force field. The MC/FEP protocol for MCPRO+ in-
volved running 10 M configurations of solvent only, 5 M of system
equilibration, and 10 M of system averaging at 25 °C.
amino-1-(3-pyridyl)-2-propen-1-one
4 (120 g, 85%). Rf = 0.46
(DCM/MeOH, 9:1). 1H and 13C NMR were in agreement with the re-
ported data.
8.2.3. N-(2-Methyl-5-nitrophenyl)-4-(3-pyridyl)-2-pyrimidine-
amine (5)23
Dimethylamino-1-(3-pyridyl)-2-propen-1-one 4 (25 g, 0.14 mol),
2-methyl-5-nitrophenyl-guanidine nitrate 2 (36 g, 0.14 mol), and
sodium hydroxide powder (6.5 g, 0.163 mol) were dissolved in iso-
propanol and reacted under reflux for 18 h. The reaction solution
was cooled to 0 °C, filtered, washed with isopropanol and MeOH,
and dried to give N-(2-methyl-5-nitrophenyl)-4-(3-pyridyl)-2-
pyrimidine-amine 5 (20 g). Rf = 0.6 (DCM/MeOH, 9:1). NMR was in
agreement with the reference.
8.2. Chemistry
All chemicals and solvents were obtained from Sigma–Aldrich
(Milwaukee, WI) or Fisher Scientific (Pittsburgh, PA) and used
without further purification. Analytical HPLC was performed on a
Varian Prostar system, with a Varian Microsorb-MW C18 column
8.2.4. N-(5-Amino-2-methylphenyl)-4-(3-pyridyl)-2-
pyrimidine-amine (6)23
(250 ꢂ 4.6 mm; 5
l
) using the following solvent system: A = H2O/
N-(2-Methyl-5-nitrophenyl)-4-(3-pyridyl)-2-pyrimidine-amine
5 (35 g, 0.114 mol) and stannous chloride dihydrate (128.5 g,
0.569 mol) were dissolved in a solvent mixture of ethyl acetate
and ethanol (250 mL, 10/1, v/v), and the reaction solution was re-
fluxed for 4 h. The solution was cooled to room temperature,
washed with 10% aqueous sodium hydroxide solution, and concen-
trated to give N-(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-
pyrimidine-amine 6 (35 g). Rf = 0.45 (DCM/MeOH, 9:1). NMR was
in agreement with the reference.
0.1% TFA, B = acetonitrile/0.1% TFA. The Varian PrepStar prepara-
tive system equipped with a Prep Microsorb–MW C18 column
(250 ꢂ 41.4 mm; 6
l; 60 Å) was used for preparative HPLC with
the same solvent system. Low resulution Mass spectra (ion spray,
a variation of electrospray) were acquired on an Applied Biosys-
tems Q-Trap 2000 LC–MS–MS system. High resolution Mss spectra
(ion spray) were acquired on an Agilent Time of Flight Accurate
Mass 6220A system with Agilent LC1200 HPLC at the front end.
UV was measured on a PerkinElmer Lambda 25 UV/vis spectrome-
ter. IR was measured on a PerkinElmer Spectrum One FT-IR spec-
trometer. 1H NMR, and 13C NMR spectra were recorded on a
8.2.5. 4-(4-Methylpiperazinomethyl)benzoic acid
dihydrochloride (9a)24
Bruker Biospin spectrometer with
a
B-ACS 60 autosampler
To a well-stirred suspension consisting of 17.1 g (0.10 mol) of
(600.13 MHz for 1H NMR and 150.92 MHz for 13C NMR). Chemical
shifts (d) were determined relative to d4-MeOH (referenced to
3.34 ppm (d) for 1H NMR and 49.86 ppm for 13C NMR). Proton–pro-
ton coupling constants (J) are given in hertz, and spectral splitting
patterns are designated as singlet (s), doublet (d), triplet (t), qua-
druplet (q), multiplet or overlapped (m), and broad (br). Flash
chromatography was performed using Merck silica gel 60 (mesh
size 230–400 ASTM) or a Teledyne Isco (Lincoln, NE) CombiFlash
Companion and SQ16x flash chromatography system with RediSep
columns (normal phase silica gel; mesh size 230–400 ASTM) and
Fisher Optima TM grade solvents. Thin-layer chromatography
(TLC) was performed on E. Merck (Darmstadt, Germany) silica gel
F-254 aluminum-backed plates with visualization under UV
(254 nm) and by staining with potassium permanganate or ceric
ammonium molybdate.
a-chloro-p-toluic acid 7 in 150 mL of absolute ethanol under a
nitrogen atmosphere at room temperature (ꢁ20 °C), a solution
consisting of 44.1 g (0.44 mol) of N-methylpiperazine 8a dissolved
in 50 mL of ethanol was added drop wise. The resulting reaction
mixture was refluxed for 16 h and then cooled to room tempera-
ture. The cooled reaction mixture was concentrated in vacuo, and
the residue was partitioned between 100 mL of diethyl ether and
100 mL of 3 N NaOH. The separated aqueous layer was then
washed three times with 100 mL of diethyl ether, cooled in an
ice-water bath and subsequently acidified with concentrated
hydrochloric acid. The resulting solids were filtered, air-dried, trit-
urated with 150 mL of boiling isopropyl alcohol, and stirred for
2 min. After the product was filtered while hot and dried, 9.4 g
(35%) of pure 4-(6-methylpiperazinomethyl)benzoic acid dihydro-
chloride 9a was obtained as the hemihydrate (mp 310–312 °C). 1H
NMR (D2O) d 8.04 (d, J = 8.21 Hz, 2H), 7.59 (d, J = 8.21 Hz, 2H), 3.50
(s, 2H), 3.63 (br, 8H), 2.97 (s, 3H); 13C NMR d 170.18, 133.13,
131.91, 130.90, 60.22, 50.61, 48.77, 43.25.
8.2.1. 2-Methyl-5-nitrophenyl-guanidine nitrate (2)23
2-Methyl-5-nitroaniline 1 (100 g, 0.657 mol) was dissolved in
ethanol (250 mL), and a 65% aqueous nitric acid solution (48 mL,
0.65 mol) was added thereto. When the exothermic reaction was
stopped, cyanamide (41.4 g) dissolved in water (41.4 g) was added.
The brown mixture was reacted under reflux for 24 h. The reaction
mixture was cooled to 0 °C, filtered, and washed with ethanol–
diethyl ether (1:1, v/v) to give 2-methyl-5-nitrophenyl-guanidine
nitrate 2 (98 g). Rf = 0.1 (DCM/MeOH/25% aq ammonia, 150:10:1).
MS: 195.2 (M+H); 1H NMR (DMSO-d6) 8.63–8.64 (br, 1H), 7.21–
7.27 (m, 1H), 6.72–6.76 (d, 1H), 6.59 (s, 3H), 1.43 (s, 3H).
8.2.6. 4-(4-(2-Fluoro-ethyl)-piperazinomethyl)benzoic acid
dihydrochloride (9b)
Synthesis of 9b followed as described for synthesis of 9a by
using N-(2-fluoro-ethyl)-piperazine 8b, compound 9b was ob-
tained as a solid. 1H NMR (D2O) d 8.05 (d, J = 8.21 Hz, 2H), 7.63
(d, J = 8.21 Hz, 2H), 4.85 (t, J = 48.0 Hz H-F coupling, H-H 1-3 cou-
pling 4.9 Hz, 2H), 4.54 (s, 2H), 3.71 (b, 10H). 13C NMR (151 MHz,
D2O) d 169.88, 132.84, 131.85, 131.64, 130.59, 78.57, 77.47,

630
Z. Peng et al. / Bioorg. Med. Chem. 22 (2014) 623–632
59.93, 56.64, (JC–F = 22 Hz, 1-3 coupling), 49.17, 48.80. (JC–F = 112
Hz, 1–2 coupling). 19F NMR (D2O) d ꢀ221.51 (1H decoupled),
ꢀ221.41 (1H coupled), ꢀ221.46, ꢀ221.49, ꢀ221.51, ꢀ221.54,
ꢀ221.58, ꢀ221.59, ꢀ221.63, ꢀ221.67. HRMS (C14H19FN2O2) calcd
267.1503 found 267.1504 [M+H].
thionyl chloride (194 g, 1.625 mol) was added to form a beige-
white suspension. The reaction mixture was refluxed for 24 h
and then cooled to room temperature (ꢁ20 °C). The resulting sus-
pension was filtered, and the recovered solids were washed with
diethyl ether and dried to 17.0 g (81%) of pure 4-(4-methylpiperaz-
inomethyl)benzoyl chloride dihydrochloride 10a. The material was
used for the next reaction without characterization.
8.2.7. 4-(4-(2-Hydroxy-ethyl-piperazinomethyl)benzoic acid
dihydrochloride (9c)
Synthesis of 9c followed as described for synthesis of 9a by
using N-(2-hydroxy-ethyl)-piperazine 8c, compound 9c was ob-
tained as a solid. 1H NMR (D2O) d 8.02 (d, J = 8.21 Hz, 2H), 7.57
(d, J = 8.21 Hz, 2H), 4.48 (s, 2H), 3.86 (m, 2H), 3.63 (br, 8H), 3.37
(m, 2H); 13C NMR d 169.85, 132.77, 131.84, 131.50, 130.49,
59.86, 54.85, 48.78, 48.17, 40.52. HRMS (C14H20N2O3) calcd
265.1547 found [M+H].
8.2.12. N-4-[3-(4-Methyl)-pyridyl]-2-pyrimidine amine
(11a)(STI-571)4
A
mixture of N-(2-methyl-5-aminophenyl)-4-pyridyl))-2-
pyrimidine-amine 6 (5 g, 18 mmol) and 4-(4-methylpiperazinom-
ethyl)benzoyl chloride dihydrochloride 10a (5 g, 20 mmol) was
stirred in 50 mL of anhydrous pyridine at 20 °C for 18 h. The reac-
tion mixture was concentrated in a vacuum. The residue was sub-
jected to silica gel chromatography using 5% MeOH (7 M ammonia)
in DCM to produce 11a in 5 g (60% yield). HRMS (C29H31N7O) calcd
494.2663, found 494.2653 [M+H].
8.2.8. 4-(Piperazinomethyl)benzoic acid dihydrochloride (9d)24
Synthesis of 9d followed as described for synthesis of 9a by
using N-Boc-piperazine 8d, compound 9d was obtained as a solid.
1H NMR (D2O) d 8.02 (d, J = 7.96 Hz, 2H), 7.51 (d, J = 8.21 Hz, 2H),
4.40 (s, 2H), 3.46 (br, 8H), 3.37 (m, 2H).
8.2.13. N-4-[3-(4-Methyl)-pyridyl]-2-pyrimidine amine (STI-571,
mesylate salt)25
The free base STI-571 11a was dissolved in 15 mL of ethanol and
1 equiv of methyl sulfonic acid was added. The solution was re-
acted at 45 °C for 2 h, and the solvent was evaporated to give the
mesylate salt. 1H NMR (D2O) d 8.63 (s, 1H), 8.20 (d, J = 5 Hz, 1H),
8.04 (d, J = 5 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.83 (s, 1H), 7.49 (d,
J = 7.5 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.05 (m, 1H), 6.96 (d,
J = 8.0 Hz, 1H), 6.85 (m, 2H), 3.58 (s, 2H), 3.0 (br, 8H), 2.80 (s,
3H), 2.75 (s, 3H). 13C NMR (151 MHz, D2O) d 167.18, 159.65,
158.69, 158.16, 144.00, 142.91, 140.61, 136.11, 135.68, 135.10,
132.07, 131.53, 131.07, 128.28, 127.22, 117.62, 108.47, 59.84,
50.45, 48.48, 42.89, 38.57, 16.61. HRMS (C29H31N7O) calcd
494.2663, found 494.2653 [M+H], 590.2542 [M+CH3SO3H].
8.2.9. 4-(4-(4-Iodobenzyl)-piperazinomethyl)benzoic acid
dihydrochloride (9e)
To a well-stirred suspension consisting of 5 g (17 mmol) of 4-
iodobenzyl bromide 12 in 50 mL of anhydrous ethanol under an ar-
gon atmosphere at room temperature, a solution of 7 g (17 mmol)
of compound 9d in 50 mL of ethanol was added drop wise; then,
0.7 mL triethylamine was added. The resulting reaction was re-
fluxed for 16 h and then cooled to room temperature. The cooled
reaction mixture was concentrated in vacuo and the resulting res-
idue was partitioned between 50 mL of diethyl ether and 50 mL of
3 N NaOH. The separated aqueous layer was then washed three
times with 50 mL of diethyl ether, cooled in an ice-water bath,
and acidified with concentrated hydrochloric acid. The resulting
solids were filtered and air-dried, titrated with 100 mL of boiling
isopropyl alcohol, and stirred for 2 min. After filtering and drying,
the material was used for the next reaction without characteriza-
tion. 1H NMR (600 MHz, D2O) d 8.02 (d, 2H), 7.83 (d, J = 8.1, 2H),
7.55 (d, J = 8.0, 2H), 7.19 (d, J = 8.1, 2H), 4.41 (s, 2H), 4.31 (s, 2H),
3.53 (s, 8H). 13C NMR (151 MHz, D2O) d 138.60, 132.88, 131.52,
131.39, 130.49, 130.45, 60.23, 59.93, 48.44, 48.19, 40.77. HRMS
(C19H21IN2O2) calcd 437.0720 found 437.0719 [M+H].
8.2.14. STI-F (11b)
Synthesis of 11b followed as described for synthesis of 11a. STI-
F was obtained as a solid. 1H NMR (CD3OD) d 10.17 (s, 1H), 9.28 (s,
1H), 8.98 (s, 1H), 8.69 (d, J = 3 Hz, 1H), 8.51 (dd, J = 4.8, 1.8 Hz, 1H),
8.48 (dd, J = 7.8, 1.8 Hz, 1H), 8.10 (s, 1H), 7.91 (d, J = 6.6, 2H), 7.52
(t, J = 7.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.431 (m, 3H), 7.21 (d,
J = 5.0 Hz, 1H), 4.52 (dt, JH-F = 48.0 Hz, JH-H = 4.9 Hz, 2H), 3.55 (s,
2H), 3.34 (b, 4H), 2.65 (dt, JH-F = 28.0 Hz, JH-H = 5 Hz, 2H), 2.50 (b,
4H), 2.23 (s, 3H); 13C NMR (CD3OD) d 165.71, 162.10, 161.68,
159.92, 151.84, 148.68, 138.28, 137.68, 134.88, 134.32, 132.71,
130.48, 129.13, 128.05, 124.23, 117.72, 117.24, 107.99, 82.72,
81.63, 61.98, 57.97, 57.85 (JC–F = 18 Hz, 1-3 coupling), 53.32,
52.97 (JC–F = 52.82 Hz, 1-2 coupling). 19F NMR (CD3OD) d ꢀ217.20
(1H decoupled), d (1H coupled), ꢀ217.05, ꢀ217.10, ꢀ217.14,
ꢀ217.15, ꢀ217.19, ꢀ217.22, ꢀ217.27, ꢀ217.32. HRMS (C30H32FN7-
O) calcd 526.2725, found 526.2719 [M+H].
8.2.10. 4-(4-(4-Tributylstannnylbenzyl)-
piperazinomethyl)benzoic acid dihydrochloride (9f)
Compound 9d (446 mg, 1 mmol) was dissolved in 20 mL of DMF
and cooled at 0 °C. 4-Tributylstannylbenzaldehyde 14 (395 mg,
1 mmol) dissolved in 2.5 mL of acetic acid was added drop wise
to the solution, followed by sodium cyanoborohydride (42 mg).
The reaction mixture was kept at room temperature for 18 h. The
solvent was dried, and the residue was subjected to silica gel chro-
matography using 10% MeOH (7 M ammonia)/DCM; 400 mg was
obtained 9f (70%). 1H NMR (DMSO) d 7.786 (t, J = 7.29 Hz, 4H),
7.16 (d, J = 8.21 Hz, 2H), 3.41 (s, 2H), 3.34 (s, 2H), 2.31 (br, 8H),
1.48 (m, 6H), 1.27 (m, 6H), 0.86 (t, 6H), 0.81 (t, 9H). 13C NMR
(75 MHz, CDCl3) d 142.75, 139.79, 136.60, 133.02, 129.96, 129.26,
129.08, 62.92, 62.58, 55.66, 53.35, 29.39, 27.52, 14.14, 9.82. HRMS
(C31H48N2O2Sn) calcd 601.2811, found 601.2821 [M+H].
8.2.15. STI-OH (11c) and STI-Cl (11d)
Syntheses of 11c and 11d were followed as described for syn-
thesis of 11a. STI-OH and STI-Cl were separated via silica gel chro-
matography using 5% MeOH (7 M ammonia) in DCM.
8.2.16. STI-OH (11c)
1H NMR (CD3OD) d 9.29 (d, 1H), 8.65 (dd, J = 6.5, 1.5 Hz, 1H),
8.60 (d, J = 8.0 Hz, 1H), 8.48 (d, J = 5 Hz, 1H), 8.22 (s, 1H), 7.93 (d,
J = 8.5 Hz, 2H), 7.56 (dd, J = 8.0, 1.5 Hz, 1H), 7.51 (t, 3H), 7.43 (t,
J = 8.0 Hz, 2H), 7.37 (d, J = 5.0 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H),
3.71 (t, J = 8.0 Hz, 2H), 3.63 (s, 2H), 3.58 (s, 2H), 2.70 (br, 8H),
2.57 (t, J = 8.0 Hz, 2H), 2.32 (s, 3H). 13C NMR (151 MHz, DMSO) d
165.28, 161.58, 161.15, 159.44, 151.33, 148.13, 142.03, 137.76,
8.2.11. 4-(4-Methylpiperazinomethyl)benzoyl chloride
dihydrochloride (10a)24
To 20 g (0.065 mol) of 4-(4-methylpiperazinomethyl)benzoic
acid dihydrochloride 9a under a nitrogen atmosphere, 119 mL of

Z. Peng et al. / Bioorg. Med. Chem. 22 (2014) 623–632
631
137.15, 134.42, 133.71, 132.19, 130.01, 128.62, 127.58, 127.52,
10 min later at 450 nm. The working volume was 100
l
L (300
lL
123.77, 117.19, 116.74, 107.50, 61.62, 60.20, 58.45, 53.12, 52.62,
17.60. HRMS (C30H33N7O2) calcd 524.2768, found 524.2761 [M+H].
for BSA). A wash (5 ꢂ 100
lL 20 mM TRIS(pH 7.4) was performed
after each step.
8.2.17. STI-Cl (11d)
10. Western blots
1H NMR (CD3OD) d 9.28 (s, 1H), 8.68 (dd, 1H), 8.51 (dd, J = 4.8,
1.8 Hz, 1H), 8.48 (dd, J = 7.8, 1.8 Hz, 1H), 8.11 (s, 1H), 7.91 (d,
J = 6.6, 2H), 7.52 (t, J = 7.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.431
(m, 3H), 7.21 (d, J =5.0 Hz, 1H), 3.67 (t, J = 8.0 Hz, 2H), 3.63 (s,
2H), 3.58 (s, 2H), 2.50 (br, 8H), 2.63 (t, J = 8.0 Hz, 2H), 2.23 (s,
3H). 13C NMR (126 MHz, DMSO) d 165.78, 162.07, 161.64, 159.96,
151.85, 148.65, 138.26, 137.65, 134.92, 134.25, 132.68, 130.52,
129.17, 128.05, 128.02, 124.28, 117.67, 117.22, 108.00, 61.99,
59.58, 52.95, 41.91, 40.41, 18.11. HRMS (C30H32ClN7O) calcd
542.2419, found 542.2426 [M+H].
U87 cells were plated at 2.5 ꢂ 105/well in 6-well plates in
RPMI-1640 medium supplemented with 10% FBS. After the cells
were allowed to attach for 24 h, they were treated with 1
lM
STI-F or imatinib for 6 h. For Western blotting, 50 g of total pro-
l
tein was separated by SDS–PAGE (Bio-Rad) and blotted onto nitro-
cellulose membranes (Bio-Rad Laboratories, Hercules, CA). The
antibodies were anti-phospho-c-KIT polyclonal antibody (Tyr703
and Tyr721) (Zymed Laboratories Inc., South San Francisco, CA),
anti-c-KIT monoclonal antibody E-1 (sc-17806) (Santa Cruz Bio-
technology, Dallas, TX); and anti-b-actin monoclonal antibody
(Sigma, St. Louis, MO).
8.2.18. STI-I (11e)
1H NMR (DMSO) d 10.16 (S,1H), 9.29 (d, 1H), 8.698 (S, 1H), 8.67
(dd, J = 6.5, 1.5 Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.48 (d, J = 5 Hz, 1H),
8.07 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.66 (dd, J = 8.0, 1.5 Hz, 2H),
7.52 (dd, J = 8, 1.5 Hz, 1H), 7.47 (d, J = 5 Hz, 1H), 7.43 (m, 3H),
7.20 (d, J = 8.50 Hz, 1H), 7.10 (d, J = 8.0 Hz, 2H), 3.53 (s, 2H), 2.24
(s, 2H), 2.70 (br, 8H), 2.22 (s, 3H). 13C NMR (126 MHz, DMSO) d
165.76, 162.06, 161.63, 159.96, 151.85, 148.64, 142.46, 138.60,
138.24, 137.64, 137.38, 134.92, 134.21, 132.68, 131.61, 130.51,
129.12, 128.08, 128.03, 124.28, 117.66, 117.21, 107.99, 93.12,
62.03, 61.73, 53.06, 52.97, 18.11. HRMS (C35H34IN7O) calcd
696.1942, found 696.1929 [M+H].
K562 cells were plated at 5 ꢂ 105/well. Two hours later, the
cells were treated for 6 h with 1 lM Sti-F. The antibodies were
anti-phospho-c-Abl (Tyr 412) monoclonal antibody 247C7 (Cell
Signaling Technology, Inc., Danvers, MA) and anti-Abl monoclonal
antibody (Sigma).
11. Small-animal PET studies
A dedicated small-animal PET scanner (microPET Focus 120;
Siemens Medical Solutions, Malvern, PA) was used for mouse
imaging. K562 human CML cells and U87 human glioblastoma cells
were obtained from the ATCC. U87WT cells were produced by ret-
roviral transduction of U87 cells with a wild-type human c-KIT
receptor. STI-571 was labeled with F-18 by nucleophilic substitu-
tion. Tumor cells were injected into dorsal shoulder areas of nude
8.2.19. STI-Sn (11f)
1H NMR (DMSO) d 10.16 (S, 1H), 9.29 (d, 1H), 8.97 (S, 1H), 8.67
(dd, J = 6.5, 1.5 Hz, 1H), 8.58 (d, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.48 (d,
J = 5 Hz, 3H), 8.10 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.45 (m, 6H), 7.22
(d, 2H), 3.53 (s, 2H), 3.42 (s, 2H), 2.70 (br, 8H), 2.22 (s, 3H), 1.48 (m,
6H), 1.27 (m, 6H), 0.86 (t, 6H), 0.81 (t, 9H). HRMS (C47H61N7OSn)
calcd 860.4032, found 860.4058 [M+H].
mice (107 cells/200
l
L in Matrigel). PET imaging was performed at
30, 60, and 90 min after the [18F]STI-571 injection (100
lCi/mouse
iv). Some animals were sacrificed at 60 or 90 min after injection,
for tissue sampling and autoradiography.
9. Kinase assay
Acknowledgements
Anti-phospho-Abltide (rabbit, polyclonal), anti-phosphotyro-
sine (rabbit, polyclonal), anti-rabbit (HRP conjugate) antibodies,
Abl, Abltide (biotin conjugate), and c-KIT, Poly(Glu4-Tyr) (biotin
conjugate) were obtained from Upstate Biotechnology, Lake Placid,
NY. All chemicals were obtained from Sigma-Aldrich, St. Louis, MO.
TMB (tetramethylbenzidine) was obtained from Pierce Biotechnol-
ogy, Rockford, IL. Ninety-six-well plates (streptavidin coated) were
obtained from Nunc, Roskilde, Denmark. A plate reader was ob-
tained from Tecan, Salzburg, Austria.
The authors would like to acknowledge the Cancer Center Sup-
port grant CA016672 for the support of the Translational Chemistry
Core Facility (TCCF) and NMR facility at the University of Texas M.
D. Anderson Cancer Center.
References and notes
1. Schittenhelm, M. M.; Shiraga, S.; Schroeder, A.; Corbin, A. S.; Griffith, D.; Lee, F.
Y.; Bokemeyer, C.; Deininger, M. W. N.; Druker, B. J.; Heinrich, M. C. Cancer Res.
2006, 66, 473.
Phosphorylation levels and anti-enzymatic activities of STI-571
and its derivatives were assayed as follows. Biotinylated peptides
(25 lM Abltide or 150 nM Poly(Glu4-Tyr)) in 100 mM sodium
2. Crossman, L. C.; O’Brien, S. G. Hematol. Oncol. Clin. North Am. 2004, 18, 605.
3. (a) Wilkins, R.; Janes, S. Clin. Lab. Haematol. 2004, 26, 73; (b) Silva, C. d.; Reid, R.
Pathol. Oncol. Res. 2003, 9, 13; (c) Potenza, L.; Luppi, M.; Riva, G.; Morselli, M.;
Ferrari, A.; Imovilli, A.; Giacobbi, F.; Temperani, P.; Donelli, A.; Narni, F.; Torelli,
G. Am. J. Hematol. 2006, 81, 45; (d) Finnegan, D.; Jones, F.; McMullin, M.
Hematol. Oncol. 2005, 23, 133; (e) Xavier, S.; Fagundes, E.; Hassan, R.; Bacchi, C.;
Conchon, M.; Tabak, D.; Spector, N.; Zalcberg, I. Leuk. Res. 2003, 27, 1063.
4. Gayed, I.; Vu, T.; Iyer, R.; Johnson, M.; Macapinlac, H.; Swanston, N.; Podoloff, D.
J. Nucl. Med. 2004, 45, 17.
5. Jager, P.; Gietema, J.; Graaf, V. D. Nucl. Med. Commun. 2004, 25, 433.
6. Wisniewski, D.; Lambek, C.; Liu, C.; Strife, A.; Veach, D.; Nagar, B.; Young, M.;
Schindler, T.; Bornmann, W. G.; Bertino, J.; Kuriyan, J.; Clarkson, B. Cancer Res.
2002, 62, 4244.
7. Nagar, B. B.; Bornmann, W. G.; Pellicena, P. Cancer Res. 2002, 62, 4236.
8. Glekas, A. P.; Pillarsetty, N. K.; Punzalan, B.; Khan, N.; Smith-Jones, P.; Larson, S.
M. J. Nucl. Med. 2011, 52, 1301.
9. Sessler, J. L.; Wangt, B.; Harriman, A. J. Am. Chem. Soc. 1995, 217, 704.
10. The highly unstable aziridinium ion 11d00 was readily underwent ring-opening
reaction with the readily available counter ions such as Cl or OH before the
bicarbonate buffer (pH 8.0) were incubated in 96-well plates for
2 h at room temperature. After that, nonspecific binding sites were
blocked by incubation with 3% BSA for 1 h. Phosphorylation was
initiated by adding of 10 ng of Abl or 25 ng of c-KIT in an assay buf-
fer (20 mM TRIS (pH 7.4), 10 mM MgCl2, 1 mM MnCl2, 0.2 mM ATP,
1 mM dithiothreitol, 25 mM glycerol phosphate, 1 mM sodium
orthovanadate, 5 mM EGTA). Different concentrations of drugs
were added simultaneously, and the mixtures were incubated for
30 min at 37 °C. Anti-phospho-Abltide or antiphosphotyrosine
and anti-rabbit antibodies (dilution 1:1000) were incubated, in
consecutive order, for 1 h at room temperature. A color reaction
was launched by adding TMB, and absorbance was measured

632
Z. Peng et al. / Bioorg. Med. Chem. 22 (2014) 623–632
attack of the external
products.
F
ion and hence resulted 11c or 11d as major
18. LigPrep, Version 2.5; Schrödinger LLC.: New York, NY, 2012.
19. Epik, Version 2.3; Schrödinger LLC.: New York, NY, 2012.
MsCl
or
F
N
N
(Tf)2O
or
HCl
F
N
N
N
11b
X
N
N
N
NsCl
or
TsCl
OH
OR
N
R = Ms, Tf, Ns, Ts
X = Cl, OH
11d"
11d
N
11d'
OR'
R = OH
11c
11d
R = Cl
20. Schrödinger Suite 2012 Virtual Screening Workflow; Glide version 5.7;
Schrödinger LLC.: New York, NY, 2012; LigPrep version 2.5; Schrödinger LLC.:
New York, NY, 2012; QikProp Version 3.4; Schrödinger LLC.: New York, NY,
2012, Schrödinger LLC.: New York, NY, 2012.
11. (a) Kim, D. W.; Jeong, H.-J.; Lim, S. T.; Sohn, M.-H. Nucl. Med. Mol. Imaging 2010,
44, 25; (b) Alauddin, M. M.; Conti, P. S.; Fissekis, J. D. J. Labelled Compd.
Radiopharm. 2002, 45, 583; (c) Alauddin, M. M.; Conti, P. S.; Fissekis, J. D. J.
Labelled Compd. Radiopharm. 2003, 46, 285.
21. Prime, Version 3.1; Schrödinger LLC.: New York, NY, 2012.
12. Zhang, M.-R.; Kumata, K.; Maeda, J.; Haradahira, T.; Noguchi, J.; Suhara, T.;
Halldin, C.; Suzuki, K. J. Med. Chem. 2007, 50, 848.
22. MCPRO+, Version 2.9; Schrödinger LLC.: New York, NY, 2012.
23. Kim, D.-Y.; Kim, J.-G.; Cho, D.-J.; Lee, G.-Y.; Kim, H.-Y.; Woo, S.-H.; Kim, Y.-S.;
Bae, W.-C.; Lee, S.-A.; Han, B.-C. US20040248918A1, 2004.
13. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
14. The Sybyl Software, Version 7.2; Tripos Inc: St Louis, MO, USA, 2006.
15. (a) Kramer, B.; Rarey, M.; Lengauer, T. Proteins 1999, 37, 228; (b) Rarey, M.;
Wefing, S.; Lengauer, T. J. Comput. Aided Mol. Des. 1996, 10, 41.
16. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank Nucleic Acids Res 2000, 28,
235.
24. Lombardino, J. G.; Niantic, C. US4623486, 1986.
25. (a) Anli, H.; Xing, L.; Zelikovitch, L.; Kaspi, J. Patent: US2006/149061 A1, 2006.;
(b) Fernández, A.; Sanguino, A.; Peng, Z.; Ozturk, E.; Chen, J.; Crespo, A.; Wulf,
S.; Shavrin, A.; Qin, C.; Ma, J.; Trent, J.; Lin, Y.; Han, H.-D.; Mangala, L. S.;
Bankson, J. A.; Gelovani, J.; Samarel, A.; Bornmann, W. G.; Sood, A. K.; Lopez-
Berestein, G. J. Clin. Invest. 2007, 117, 4044.
26. The PYMOL Molecular Graphics System, Version 1.5.0.4; Schrödinger LLC.
17. Maestro, Version 9.3; Schrödinger LLC.: New York, NY, 2012.