Synthesis and in vitro evaluation of [18F](R)-FEPAQ: A potential PET ligand for VEGFR2

Article abstract of DOI:10.1016/j.bmcl.2012.05.099

Synthesis and in vitro evaluation of [¹⁸F](R)-N-(4-bromo-2- fluorophenyl)-7-((1-(2-fluoroethyl)piperidin-3-yl)methoxy)-6-methoxyquinazolin- 4-amine ((R)-[¹⁸F]FEPAQ or [¹⁸F]1), a potential imaging agent for the VEGFR2, usin

Full text of DOI:10.1016/j.bmcl.2012.05.099

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5104–5107
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of [18F](R)-FEPAQ: A potential PET ligand
for VEGFR2
Jaya Prabhakaran a, , Victoria Arango a,b, Vattoly J. Majo a, Norman R. Simpson a,b, Suham A. Kassir b,
⇑
Mark D. Underwood a,b, Hanish Polavarapu a, Jeffrey N. Bruce c, Peter Canoll c, J. John Mann a,b,d
,
J. S. Dileep Kumar a,b
a Division of Molecular Imaging and Neuropathology, Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, USA
b New York State Psychiatric Institute, New York, USA
c Department of Neurology, Columbia University, New York, USA
d Department of Radiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
a r t i c l e i n f o
a b s t r a c t
Synthesis and in vitro evaluation of [18F](R)-N-(4-bromo-2-fluorophenyl)-7-((1-(2-fluoroethyl)piperidin-
3-yl)methoxy)-6-methoxyquinazolin-4-amine ((R)-[18F]FEPAQ or [18F]1), a potential imaging agent for
the VEGFR2, using phosphor image autoradiography are described. Synthesis of 2, the desfluoroethyl pre-
cursor for (R)-FEPAQ was achieved from t-butyl 3-(hydroxymethyl)piperidine-1-carboxylate (3) in five
steps and in 50% yield. [18F]1 was synthesized by reaction of sodium salt of compound 2 with [18F]fluoro-
ethyl tosylate in DMSO. The yield of [18F]1 was 20% (EOS based on [18F]FÀ) with >99% radiochemical pur-
Article history:
Received 2 April 2012
Revised 25 May 2012
Accepted 29 May 2012
Available online 7 June 2012
Keywords:
VEGFR2
Radiotracer
PET
Glioma
Bioimaging
ity and specific activity of 1–2 Ci/lmol (n = 10). The total synthesis time was 75 min. The radiotracer
selectively labeled VEGFR2 in slide-mounted sections of human brain and higher binding was found in
surgically removed human glioblastoma sections as demonstrated by in vitro phosphor imager studies.
These findings suggest [18F]1 may be a promising radiotracer for imaging VEGFR2 in brain using PET.
Ó 2012 Elsevier Ltd. All rights reserved.
A key protein in the regulation of angiogenesis and vasculogen-
esis is vascular endothelial growth factor (VEGF), which is overex-
pressed in virtually all human tumors.1–6 In addition to tumors,
VEGF exerts neuroprotective actions directly through the inhibi-
tion of programmed cell death (PCD) or apoptosis, and the stimu-
lation of neurogenesis.7–9 Apart from angiogenesis, VEGF is a
mediator of multiple processes enhancing blood brain barrier
(BBB) permeability for glucose, and antioxidant activation, which
indirectly results in neuroprotection.10 VEGF protects against neu-
ronal death from hypoxia and glucose deprivation.7,8,11,12 VEGF sig-
naling through VEGFR2 was also shown to be required for
antidepressants (fluoxetine, desipramine) to increase cell prolifer-
ation. Chronic antidepressant administration increases VEGF
expression in both neurons and endothelial cells in the hippocam-
pus.13–45 Thus, VEGF is a key component in the regulation of neu-
ron and vessel growth in brain and other pathologies. There are
excellent reviews that detail the therapeutic potential of VEGF
and its receptors in CNS.6,7,16,17 VEGF effects are mediated by a
family of receptor tyrosine kinases (TKs), including VEGFR-1 (Flt-
1), VEGFR-2 (KDR or Flk-1) and VEGFR-3 (Flt-4).7,18 Of these, VEG-
FR-2 appears to mediate almost all of the known cellular responses
to VEGF. Antagonism of the VEGF pathway results in inhibition of
angiogenesis and tumor growth in a number of tumor model sys-
tems.19–21 Although there has been significant advance in the
imaging of tumors, there is a lack of agents for imaging and quan-
tifying indices of angiogenesis. Inhibiting VEGF expression and
interfering with angiogenesis may be a useful treatment target
for tumors. It is reported that tumor perfusion changes dynami-
cally during anti-VEGF treatment and that specific molecular
markers in tumors and vasculature can be correlated with these
perfusion changes.22–25 Noninvasive imaging of VEGFR2 is of great
potential clinical importance, as it may identify factors contribut-
ing to tumor resistance. Positron Emission Tomography (PET)
imaging of VEGFR2 in tumor patients at baseline and in response
to chemotherapy treatment may also provide novel information
about the mechanisms that contribute to therapy resistance, and
allow more effective use of VEGF inhibition. The multimodality
molecular imaging of VEGF and VEGFR with radiotracers have re-
cently been reviewed.26–28 Efforts to image VEGF/VEGFR first fo-
cused VEGFR-specific uptake and the dynamic nature of the
receptor expression in tumors as well as biological responses on
tracing the distribution of the ligand by targeting with anti-VEGF
antibodies labeled with 124-I and 89-Zr.29–31 VEGFR-specific
uptake and the dynamic nature of the receptor expression in
tumors as well as biological responses to myocardial infarction
⇑
Corresponding author. Fax: +1 212 543 1054.
E-mail address: jp2155@columbia.edu (J. Prabhakaran).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.099

J. Prabhakaran et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5104–5107
5105
and ischemia have been successfully demonstrated using these
tracers.31–33 However, imaging the expression of a ligand in circu-
lation using long-lived antibodies as imaging probes requires wait-
ing days until the unbound tracer has cleared sufficiently from the
blood and the nonspecific uptake has declined sufficiently for good
target/nontarget contrast. Mutated-VEGF121 (VEGFDEE) has also
been developed to increase specificity for VEGFR2 and the results
obtained were promising for potential translation to clinical stud-
ies.34 However, these peptide ligands do not cross BBB and hence
the tracers are limited to imaging studies outside the brain. An
understanding of the potential therapeutic value associated with
binding to VEGFR2 in brain requires development of selective
non-peptide PET ligands. Therapeutic drugs that act by binding
to VEGFR2 can be evaluated and their therapeutic dose determined
by an occupancy study using such a specific PET tracer. A specific
PET tracer could also serve as a biological marker for angiogenesis
and for the evaluation of diseases in which changes in VEGF recep-
tor binding occurs. For example, VEGFR2 changes in tissue prolifer-
ation or angiogenesis can be quantified in vivo using PET. However,
there is currently no good radiotracer for the measurement of VEG-
FR2 binding expression in tumors and to monitor their role in angi-
ogenesis. Racemic [11C]PAQ was reported as the first VEGFR2
selective PET tracer which was proved specific for kidney tumors
in rodents.35 Radiolabeling of racemic [18F]sunitinib, [11]vandeta-
nib and [11C]chloro-vandetanib has been recently reported, how-
ever, biological evaluation of these tracers are not reported yet.36
All these radiotracers are 4- or 3-piperidinyl analogues of vandeta-
nib (rINN, Caprelsa ZD6474), the first VEGFR2 drug approved by
FDA.37,38 Moreover, it has been reported that the (R)-enantiomer
of vandetanib is 10-fold more active than (S)-enantiomer, also
there is a better selectivity of (R)-enantiomer with respect to EGFR
and VEGFR1.39 Herein we report the synthesis and evaluation of
(R)-enantiomer of a viable [F-18]analogue of PAQ [18F]fluoroethyl
PAQ ([18F]FEPAQ, [18F]1) as a potential candidate for VEGFR2 imag-
ing with PET.
pot, afforded 4-((4-bromo-2-fluorophenyl)amino)-6-methoxy qui-
nazolin-7-ol (7) in 70% yield.39 Compound 7 upon reaction with
(R)-tert-butyl 3-((tosyloxy)methyl)piperidine-1-carboxylate (4),
which in turn was obtained by the tosylation of commercial (R)-
tert-butyl 3-(hydroxymethyl)piperidine-1-carboxylate (3), re-
sulted in the isolation of BOC protected precursor using column
chromatography (2–3% methanol in methylene chloride). The
BOC protection was removed by treatment with trifluoro acetic
acid in methylene chloride (1:1) to afford the radiolabeling precur-
sor (R)-desfluoroethyl-PAQ (2) in 50% yield.35 The nonradioactive
standard (R)-FEPAQ (1) was obtained by reaction of compound 2
with fluoroethyl-bromide in 60% yield.48 The VEGFR2 binding
affinity (IC50) of compound 1 was found to be 40 nM based on VEG-
FR2 incubation mobility-shift KDR kinase assay using microfluidic
chip to measure the conversion of a fluorescent peptide substrate
to a phosphorylated product.49 The radiolabeling of compound 1
was achieved in two steps (Scheme 1).40 The radiolabeling was at-
tempted under several conditions and the optimal method we
adopted is present here. [18F]Fluoroethyl tosylate ([18F]FE-OTs) is
prepared first by reacting [18F]FÀ with ethylene glycol ditosylate
using a modified procedure reported elsewhere.41 The crude prod-
uct was purified using solid phase extraction using C-18 Sep-PakÒ
to yield 70% of [18F]FE-OTs. The [18F]FE-OTs obtained is azeotropi-
cally dried and reacted with freshly prepared sodium salt of 2 in
DMSO. The radiolabeled product was further purified using semi-
preparative HPLC to afford [18F]1 in 20 5% yield (n = 10). The
chemical identity of [18F]1 was confirmed by co-injecting with
nonradiolabeled (R)-1 by HPLC technique. The total time required
for the radiosynthesis was 75 min. The radiochemical purity of
[
18F]1 was found to be >99% and the specific activity in the ranges
of 1–2 Ci/ mol (EOB).
l
After synthesizing the radioligand in consistent yield and suffi-
cient specific activity, [18F]1 was tested in surgically removed hu-
man glioblastoma using phosphor image autoradiography to
determine its binding to VEGFR2 (Fig. 1).42 We choose glioblas-
toma model for the evaluation of [18F]1 because it is the most com-
mon brain malignancy, with high levels of VEGF and VEGFR2
expression when compared to other non-neural solid cancers.43–
45 Additionally, studies in animal models have shown that VEGFR2
inhibitors suppress the growth of gliomas in vivo and cause regres-
sion of blood vessels.46,47 Frozen brain sections were used for the
We synthesized (R)-fluoroethyl-PAQ ((R)-FEPAQ) and the corre-
sponding desfluoroethyl-PAQ with appropriate modifications of
literature methods (Scheme 1).35,39 In brief, reaction of 4-hydro-
xy-6-methoxyquinazolin-7-yl benzoate (5) with POCl3 and N,N-
diisopropylethyl amine and the coupling of the intermediate
choroquinoline formed with 4-bromo-2-fluoroaniline (6) in one
Boc
N
Boc
N
i
OH
OTs
3
4
HN
N
NH2
N
N
F
BzO
O
N
F
4, iv, v
ii, iii
N
F
O
+
HO
HN
Br
N
HN
Br
O
OH
Br
6
O
5
2
7
R
N
N
vi
2
N
F
O
HN
Br
O
R = F; 1
R = 18F, [18F]1
Scheme 1. Synthesis and radiosynthesis of (R)-FEPAQ and (R)-[18F]FEPAQ. Reagents and conditions: (i) (Ts)2O, THF, 6 h; (ii) POCl3, CH2Cl2; (iii) K2CO3, THF; (iv) 4, K2CO3, THF;
(v) TFA, CH2Cl2; (vi) fluoroethyl bromide/[18F]FEOTs, DMSO.

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J. Prabhakaran et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5104–5107
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Figure 1. Phosphor image of [18F]1 in glioma sections. T: total binding; NS1:
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40. Radiosynthesis of [18F]1: To an aqueous [18F]fluoride solution were added a
solution containing kryptofixÒ2.2.2. (11 mg, 0.03 mmol), potassium carbonate
(2 mg, 0.014 mmol) in 0.2 mL of 95:5 acetonitrile/water. The mixture was dried
18F]1 for 60 min at room temperature. Adjacent sections were
incubated with ZM-323881 or (R)-PAQ, the two known selective
VEGFR2 antagonists (1 M) to determine nonspecific binding. After
l
the incubation, sections were washed with ice-cold buffer and
slides were quickly dried under a stream of cold air and exposed
to a phosphor-imaging screen with high- and low-activity stan-
dards for 60 min. Screens are scanned with a Packard Cyclone
phosphor-imaging system and analyzed with OptiQuant Acquisi-
tion and Analysis software (Packard). Displacement of total binding
was observed with both VEGFR2 ligands as evident from Figure 1.
In summary, we successfully synthesized [18F]1, a potential
imaging agent for VEGFR2. The total time required for the radio-
synthesis was 75 min from EOS using [18F]FE-OTs in DMSO.
[
18F]1 was obtained in 20 5% yield (EOS) with excellent radio-
chemical purities and specific activity. Phosphor image studies
indicate that this newly developed [18F]1 ligand binds to VEGFR2
in surgically removed human glioma. The results obtained indicate
the feasibility of using [18F]1 as a potential imaging agent to visu-
alize VEGFR2 in brain using PET.
in
a stream of argon at 95 °C for 10 min, followed by azeotrope with
acetonitrile (3 Â 0.5 mL) for three times. To the dried kryptofixÒ2.2.2./[18F]
fluoride complex, 4 mg ethyleneglycol-1,2-ditosylate in 1 mL acetonitrile was
added and heated in a sealed vial for 10 min. At the end of the reaction, the
crude product was diluted with 10 mL DI water and passed through an
activated C-18 Sep-PakÒ. The C-18 Sep-PakÒ cartridge was further washed with
4 mL hexane and dried under argon. The Sep-PakÒ was then eluted with 2 mL
ether to obtain [18F]FEOTs in 70%. The ether solution was dried under argon,
added with 4 mg of freshly prepared sodium salt of (R)-2 in 0.25 mL DMSO and
heated for 20 min at 90 °C (The sodium salt was prepared by dissolving 4 mg of
Acknowledgments
This work was partially supported by Clinical and Translational
Science (CTSA) award, Columbia University Medical Center (to J.P.).
compound 2, dissolved in 0.5 mL of acetonitrile followed by addition of 2–4 lL
of 5 M NaOH solution. The mixture was vortexed and the solvent was removed
azeotropically at 90 °C under a stream of argon. The sodium salt thus obtained
was dissolved in 0.25 mL of DMSO for further reaction). The crude product was
diluted with 0.25 mL of acetonitrile and was directly injected into a semi
preparative RP-HPLC (Phenomenex C18, 10 Â 250 mm, 10) and eluted with
acetonitrile: 0.1 M ammonium formate solution (35:65) at a flow rate of
10 mL/min. The precursor eluted after 6–7 min during the HPLC analysis. The
References and notes
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product fraction with a retention time of 10–11 min based on c-detector was
collected, diluted with 50 mL of deionized water, and passed through a C-18
Sep-PakÒ cartridge. Reconstitution of the product in 1 mL of absolute ethanol
afforded [18F]1 (20 5% yield, based on [18F]FÀ at EOS). A portion of the ethanol
solution was analyzed by analytical RP-HPLC (Phenomenex, Prodigy ODS(3)
4.6 Â 250 mm, 5 m; mobile phase: acetonitrile/0.1 M ammonium formate;
40:60, flow rate: 2 mL/min, retention time: 7 min, wavelength: 254 nm) to
determine the specific activity and radiochemical purity. The ethanol solution
of [18F]1 was used for further studies.
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The frozen glioma tissues were sectioned (20 m) were collected onto glass
[
18F]1: The in vitro experiments will be
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l

J. Prabhakaran et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5104–5107
5107
slides and were used for the phosphor image studies. Four sections each for
total and nonspecific binding were assayed. The slides mounted sections were
brought to room temperature and were incubated with 50 mM Tris–HCl, 5 mM
of KCl and 150 mM of NaCl at pH 7.4 containing [18F]1 (0.1 nM) for 1 h. The
adjacent sections were incubated with 1 mM of ZM 323881ÁHCl or 1 mM of
(R)-PAQ for 1 h to determine non specific binding. After the incubation,
sections were washed with ice-cold buffer containing 50 mM Tris-buffer (pH
7.4) at 4 °C and briefly dipped in ice-cold water to remove salts. Slides were
quickly dried under a stream of cold air and exposed to a phosphor-imaging
screen (Packard, ST screen wrapped in Mylar film) with high- and low-activity
standards (American Radiolabeled Chemicals) for 60 min. Screens are scanned
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7.0 (s, 1H), 4.7 (m, 1H), 4.6 (m, 1H), 4.1 (m, 2H), 3.9 (s, 3H), 3.2 (m, 1H), 3.0–2.6
(m, 6H), 2.4–1.8 (m, 4H); HRMS (m/z calcd C23H25BrF2N4O2; 507.1214; found
507.1203); HPLC (Phenomenex, Prodigy ODS(3) 4.6 Â 250 mm, 5
l; mobile
phase: acetonitrile/0.1 M ammonium formate; 40:60, flow rate: 2 mL/min,
retention time: 7 min, wavelength: 254 and 270 nm, >99% purity.
49. KDR in vitro enzymatic binding assay was performed by Calipers Life Sciences
Discovery and Alliance services (http://www.caliperls.com/products/contract-
research/in-vitro/kinases/kdr-vegfr2-flk1-h.htm).
with
a Packard Cyclone phosphor-imaging system and analyzed with
OptiQuant Acquisition and, Analysis software (Packard).
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