Formation of fluorine-18 labeled diaryl ureas-labeled VEGFR-2/PDGFR dual inhibitors as molecular imaging agents for angiogenesis

Article abstract of DOI:10.1016/j.bmc.2008.02.081

Urea subunits are common components of various pharmaceuticals' core structure. Since in most cases the design and development of PET biomarkers is based on approved or potential drugs, there is a growing need for a general labeling methodology of urea-containing pharmacophores. As a part of research in the field of molecular imaging of angiogenic processes, we synthesized several highly potent VEGFR-2/PDGFR dual inhibitors as potential PET biomarkers. The structure of these inhibitors is based on the N-phenyl-N′-{4-(4-quinolyloxy)phenyl}urea skeleton. A representative inhibitor was successfully labeled with fluorine-18 by a three-step process. Initially, a two-step radiosynthesis of 4-[¹⁸F]fluoro-aniline from 1,4-dinitrobenzene (60 min, EOB decay corrected yield: 63%) was performed. At the third and final step, the 4-[¹⁸F]fluoro-aniline synthon reacted for 30 min at room temperature with 4-(2-fluoro-4-isocyanato-phenoxy)-6,7-dimethoxy-quinoline to give complete conversion of the labeled synthon to 1-[4-(6,7-dimethoxy-quinolin-4-yloxy)-3-fluoro-phenyl]-3-(4-[¹⁸F]fluoro-phenyl)-urea. The desired labeled product was obtained after total radiosynthesis time of 3 h including HPLC purification with 46 ± 1% EOB decay corrected radiochemical yield, 99% radiochemical purity, 99% chemical purity, and a specific activity of 400 ± 37 GBq/mmol (n = 5).

Full text of DOI:10.1016/j.bmc.2008.02.081

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4242–4251
Formation of fluorine-18 labeled diaryl ureas—labeled
VEGFR-2/PDGFR dual inhibitors as molecular
imaging agents for angiogenesis
O. Ilovich, O. Jacobson, Y. Aviv, A. Litchi, R. Chisin and E. Mishani*
Department of Medical Biophysics and Nuclear Medicine, Hadassah Hebrew University Hospital, Jerusalem 91120, Israel
Received 27 December 2007; revised 17 February 2008; accepted 26 February 2008
Available online 29 February 2008
Abstract—Urea subunits are common components of various pharmaceuticals’ core structure. Since in most cases the design and
development of PET biomarkers is based on approved or potential drugs, there is a growing need for a general labeling methodology
of urea-containing pharmacophores. As a part of research in the field of molecular imaging of angiogenic processes, we synthesized
several highly potent VEGFR-2/PDGFR dual inhibitors as potential PET biomarkers. The structure of these inhibitors is based on
the N-phenyl-N0-{4-(4-quinolyloxy)phenyl}urea skeleton. A representative inhibitor was successfully labeled with fluorine-18 by a
three-step process. Initially, a two-step radiosynthesis of 4-[18F]fluoro-aniline from 1,4-dinitrobenzene (60 min, EOB decay corrected
yield: 63%) was performed. At the third and final step, the 4-[18F]fluoro-aniline synthon reacted for 30 min at room temperature with
4-(2-fluoro-4-isocyanato-phenoxy)-6,7-dimethoxy-quinoline to give complete conversion of the labeled synthon to 1-[4-(6,7-dime-
thoxy-quinolin-4-yloxy)-3-fluoro-phenyl]-3-(4-[18F]fluoro-phenyl)-urea.
The desired labeled product was obtained after total radiosynthesis time of 3 h including HPLC purification with 46 1% EOB
decay corrected radiochemical yield, 99% radiochemical purity, 99% chemical purity, and a specific activity of 400 37 GBq/mmol
(n = 5).
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
progress, fueled by specific radiolabeled biomarkers.
With this approach, careful patient selection combined
with extensive therapy monitoring provides an optimal
platform for in vivo drug efficacy evaluation. Moreover,
pharmacodynamic interactions, pharmacokinetics and
target quantity and occupancy can be measured in hu-
mans by PET. Development of a general synthetic route
for the labeling of aryl-ureas moieties with positron
emitting isotopes will broaden the use of PET and spe-
cifically, accelerate the development of potential drugs
containing this moiety, allowing for more effective ther-
apeutic monitoring.
Urea subunits are common components of lead pharma-
ceuticals. Recently, urea moieties have been used as part
of colchicine-binding site antagonists1 and polycyclic
imides with antimicrobial activity,2 while diaryl ureas
specifically have emerged in multitarget tyrosine kinase
inhibitors,3 p38 MAPK inhibitors,4 insulin-like growth
factor I receptor (IGF-1R) inhibitors5 and as combined
vascular endothelial growth factor receptor 2 and plate-
let-derived growth factor receptor (VEGFR-2/PDGFR)
inhibitors.6 Most of the above-mentioned pharmaco-
phores were designed as highly selective targeted thera-
pies. In order to evaluate and monitor efficiency of
targeted therapies, a sensitive and quantitative molecu-
lar imaging modality is needed.7,8 This may be achieved
by means of Positron Emission Tomography (PET) cou-
pled with selective labeled biomarkers. In the last dec-
ade, the field of PET imaging has witnessed great
Cyclic ureas have been labeled with 11C by the produc-
tion of [11C]phosgene, which further reacts with free
amines to give the desired urea product. There are two
major drawbacks of using [11C]phosgene as a labeling
agent. The first is the multi-step process of [11C]phos-
gene production which takes about one half-life of 11C
and predominantly involves the use of the toxic chlorine
gas. The second drawback is the highly reactive nature
of phosgene which makes the synthesis of asymmetric
diaryl ureas extremely difficult, thus limiting this
Keywords: VEGF; Fluorine-18; PET; Angiogenesis.
*
Corresponding author. Tel.: +972 2 677 7931; fax: +972 2 642
1203; e-mail: eyalmi@ekmd.huji.ac.il
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.081

O. Ilovich et al. / Bioorg. Med. Chem. 16 (2008) 4242–4251
4243
approach to cyclization reactions of diamino or hydrox-
yamino precursor molecules.9–12 Another approach for
[11C]urea production is the rhodium(I)-promoted car-
bonylation reaction using phenyl azide and [11C]carbon
monoxide developed by Doi et al.13 This approach al-
lows the formation of a wide spectrum of diaryl ureas
since an azide and an amine are used rather than two
amines. Since the half-life of 11C is relatively short
(20.4 min), PET studies are limited to a short time win-
dow following injection of the radiolabeled biomarker.
In order to counter this time restraint, labeling with
longer half-life isotopes such as 18F and 124I
(109.8 min and 4.2 days, respectively) is required.
temperature. The reaction was monitored using IR via
absorption at 2285 cmꢀ1 which is associated with isocy-
anate formation. Upon completion of the reaction, all
volatiles were evaporated to give a yellow solid which
was used as such for radiosynthesis.
The isopropenyl carbamate precursor (9) was prepared
by reaction of 7 with isopropenyl chloroformate in
THF as described before.17 This precursor proved to
be stable enough for column chromatography purifica-
tion and characterization.
Reaction of 7 with N,N0-carbonyldiimidazole (CDI) was
attempted under different conditions,18–22 but unfortu-
nately the carbamoyl imidazole intermediate could not
be isolated. When 4-fluoroaniline was added to the
above-mentioned reaction mixtures, formation of 1
was evident by mass spectrometry analysis. At the out-
set, our primary reason of choosing the carbamoyl imid-
azole intermediate over the isocyanate precursor was to
afford precursor purification and isolation; however, this
procedure turned out to be unfeasible, and thus this ap-
proach was no longer explored in radiosynthesis.
To the best of our knowledge, there is no report on fluo-
rine-18 labeling of a diaryl urea and only one report has
surfaced on the labeling of an urea-containing molecule
with fluorine-18.14 In this particular report, the dialkyl
urea was formed in a 5 h, four-step radiosynthesis route
using diphosgene to yield the desired product with a
radiochemical yield of 12% (decay corrected).
One approach to label diaryl ureas is first to produce 18F
labeled anilines.15,16 This radiolabeled synthon can fur-
ther react with a precursor carrying a reactive functional
group to yield the labeled diaryl urea. In order to eval-
uate the feasibility of this approach, we focused on the
VEGFR-2/PDGFR dual inhibitors which contain a dia-
ryl urea moiety. Three potential precursors with differ-
ent reactive functional groups were chosen: isocyanate,
isopropenyl carbamate, and carbamoyl imidazoles
(Scheme 1).
2.2. Radiochemistry
2.2.1. 4-[18F]fluoroaniline ([18F]-11). The labeling of the
4-[18F]fluoro-aniline was described earlier.23 Briefly,
1,4-dinitrobenzene was first 18F-fluorinated using a
[18F]KF/Kryptofix complex in DMF and then the result-
ing 4-[18F]fluoro-nitrobenzene ([18F]-10) was reduced by
means of raney-nickel and hydrazine hydrate (Scheme
4). This labeled intermediate was purified by an Oasis
cartridge and eluted from the cartridge with various sol-
vents such as DCM, DCE, and toluene in order to afford
several conditions for the next step. The synthesis to fur-
nish the desired labeled product lasted approximately
60 min. This labeled intermediate was meant to assess
the feasibility of using the different precursor molecules,
namely the isocyanate and isopropenyl carbamate, to
furnish [18F]-1.
2. Results and discussion
2.1. Chemistry
The synthesis of two new (1 and 2) and one published (3)
derivatives of VEGFR-2/PDGFR dual inhibitors
(Scheme 2) was performed based on the chemical course
described by Kubo et al.6 with several modifications.
The first step was the cyclization of 2-amino-4,5-dime-
thoxyacetophenone in dimethoxyethane at room tem-
perature to give 6,7-dimethoxy-4-quinolone (4, yield:
95%). This quinolone was first chlorinated using oxalyl
chloride in DCM at reflux (5) rather than phosphoryl
chloride and then reacted with (3-fluoro-4-hydroxy-phe-
nyl)-carbamic acid tert-butyl ester and DMAP in DMF
at 145 ꢁC to give the protected phenoxyquinoline (6,
50%) which was de-protected with HCl in dioxane to
give the free amine (7, 66%). It should be noted that
both reaction of 5 with nitrophenol followed by nitro
reduction and a direct reaction of 5 with an aminophe-
nol in the presence of NaH either did not produce 7
or furnished the desired molecule accompanied with a
large quantity of side products. In the last step, reaction
of 7 with triphosgene followed by the addition of the
corresponding fluoroaniline in dry DCM yielded the ti-
tle compounds, 1–3 (50–65%) (Scheme 3).
2.2.2. Carbamoyl imidazoles. The first approach for
labeling one of the title compounds involved the CDI-
mediated urea synthesis. CDI is a phosgene equivalent
which is commercially available and readily handled as
a crystalline solid. Usually, the carbamoyl imidazole
intermediate is formed in situ and further reacted with-
out purification. In some cases, this intermediate can be
isolated18 and this could be an advantage over the highly
reactive isocyanates which are extremely hard to purify.
As mentioned above, precursor isolation was not possi-
ble and this route was no longer explored in
radiosynthesis.
2.2.3. Isopropenyl carbamate. The second method chosen
for the radiosynthesis of unsymmetrical ureas was via
the isopropenyl carbamate intermediate. Although car-
bamates have the advantage of being sufficiently stable
for purification and analysis, most carbamates form
urea bonds via lengthy equilibrium reactions. Since
PET radiochemistry often deals with short-lived iso-
topes, the use of carbamates, which require long reac-
The isocyanate precursor (8) for radiosynthesis was pre-
pared by reacting 7 with triphosgene in DCM at room

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O. Ilovich et al. / Bioorg. Med. Chem. 16 (2008) 4242–4251
F
NCO
O
O
O
F
NH
2
N
O
8
O
O
H
N
F
O
N
O
7
O
N
O
O
CDI
9
N
H
N
N
F
O
O
O
O
N
Scheme 1. Candidate precursors for labeling diaryl urea.
R
1
H
N
H
F
N
O
O
R
2
O
Compound 1 - R1=H R2=F
Compound 2 - R1=F R2=H
Compound 3 - R1=F R2=F
O
N
Scheme 2. VEGFR-2/PDGFR dual inhibitors.
tion times, may be inapplicable. However, isopropenyl
carbamates, formed by reaction with isopropenyl chlo-
roformate, are unique in this family since they react with
amines rapidly and irreversibly to give unsymmetrical
ureas in high yield and purity.17
[18F]-11 to [18F]-1 reached a maximum EOB decay cor-
rected radiochemical yield of 10% (n = 3).
2.2.4. Isocyanate. The final method is the commonly
used isocyanate-mediated urea formation. While isocy-
anates react with amines rapidly, under mild condi-
tions, their formation requires the use of phosgene
or one of its analogs (diphosgene or triphosgene). A
major drawback in this approach is the use of phos-
gene which is a toxic gas and while both analogs
are less toxic, they spontaneously release phos-
gene.24–26 Furthermore, the most common way of
purifying the reactive isocyanates is via distillation,
which is a challenging undertaking when dealing with
high molecular weight derivatives requiring distillation
at high temperatures.
The synthesis and purification of the carbamate precursor
(9) were performed as described by Gallou et al.17 Accord-
ing to this report, different isopropenyl carbamates may
require entirely different conditions for urea formation.
In our case, in non-radioactive experiments, compound
1 formation was obtained at reaction temperatures above
145 ꢁC. Under these temperatures, some precursor degra-
dation was also evident in mass spectroscopy analysis.
When these conditions were applied at the tracer level,
extensive degradation was observed while conversion of

O. Ilovich et al. / Bioorg. Med. Chem. 16 (2008) 4242–4251
4245
O
O
Cl
N
MeO
MeO
MeO
MeO
MeO
MeO
b
a
NH
2
N
H
5
4
F
NHBoc
F
NH
2
O
N
O
c
d
MeO
MeO
MeO
MeO
N
7
6
R
1
H
N
H
N
F
O
R
O
2
e
MeO
MeO
N
1-3
Scheme 3. Synthetic routes for the production of VEGFR-2/PDGFR dual inhibitors. Reagents: (a) NaOMe, ethyl formate; (b) oxalyl chloride,
DCM; (c) (3-fluoro-4-hydroxy-phenyl)-carbamic acid tert-butyl ester, DMF, DMAP; (d) HCl/dioxane; (e) triphosgene followed by a reaction with a
corresponding aniline.
18F
18F
O2N
a
b
NO2
NH2
NO2
[
18F]-10
[
18F]-11
H
N
H
N
O
O
N
18F
c
O
O
[
18F]-1
Scheme 4. Radiosynthesis of [18F]-1. Reagents and conditions: (a) [18F]KF/Kryptofix complex, DMF, 120 ꢁC; (b) raney-Ni/hydrazine hydrate,
EtOH:H2O, 57 ꢁC; (c) isocyanate precursor, DCM, rt, 30 min.
Although triphosgene was successfully used for the syn-
thesis of our non-labeled title compounds, the isocya-
nates were used without any purification and therefore
several hurdles might evolve at the tracer level transfor-
mation. At the tracer level, since the concentration of
the impurities is, in most cases, higher than that of the
radioactive intermediate, it may lead to the formation
of numerous side products.
transformed into isocyanates and then reacted with
the counter amine to give the desired [18F]-1.
Although both options were attempted, only one suc-
ceeded. When [18F]-11 was reacted with either a com-
mercially available solution of phosgene in toluene or
triphosgene in DCM, HPLC revealed that a multiple
number of radioactive moieties had formed after
10 min at room temperature. None of these moieties
reacted with 7 (n = 2). We suspect that since the 4-
[18F]fluoro-aniline synthon ([18F]-11) was eluted from
the Oasis cartridge with a significant amount of 1,4-
With the triphosgene, there are two possible ap-
proaches. Either the [18F]-11 or compound 7 can be

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O. Ilovich et al. / Bioorg. Med. Chem. 16 (2008) 4242–4251
diaminobenzene, there was already an urea forming
reaction before the addition of 7.
the formation of [18F]-1 was not detected by analytical
HPLC. Although it is possible to separate [18F]-11
from all the other non-radioactive species using HPLC
purification prior to transformation into labeled isocy-
anates, this procedure is not customary practice in the
In order to overcome this problem, triphosgene was
added to a solution of [18F]-11 and 7 in DCM, but
Figure 1. HPLC chromatograms of analytical injections of 1, [18F]-1 and a co-injection of the two. From top to bottom: UV chromatogram of 1,
Radio chromatogram of [18F]-1, A co-injection of 1 and [18F]-1 showing both UV and radio chromatograms. A difference of ꢁ0.5 min exists between
the UV and radio peaks since the solvent passes through UV detector and only then through the radioactivity detector.

O. Ilovich et al. / Bioorg. Med. Chem. 16 (2008) 4242–4251
4247
Table 2. Extracted fraction and extracted intact [18F]-1 from ex vivo
blood stability experiments
realm of radiochemical transformation with short-
lived isotopes, and thus one last alternative was
attempted.
Incubation
(min)
Extracted
fraction (%)
Intact tracer
(HPLC, TLC) (%)
The non-labeled isocyanate precursor 8 formed as de-
scribed in Section 2.1 was reacted with [18F]-11 in dry
DCM to give complete conversion of the latter to
[18F]-1 after 30 min at room temperature (Fig. 1). The
reaction proceeded efficiently and no other radioactive
moieties were detected by HPLC (Scheme 4). [18F]-1
was purified using semipreparative HPLC giving an
overall 46% radiochemical yield decay corrected to
EOB, a specific activity of 400 37 GBq/mmol and a
99% radiochemical purity (n = 5).
0
30
60–68
74–77
70–72
79–80
99
99
99
99
60
120
2.4. Ex vivo blood stability
[18F]-1 was evaluated ex vivo for its stability in human
blood as described in the past32 with several modifica-
tions. After incubation in blood samples, the tracer
was extracted at different time points (0, 30, 60, and
120 min). The extracted radioactivity fraction was mea-
sured, and formation of metabolites was evaluated using
both HPLC (Table 2 and Fig. 2) and TLC [data not
shown]. The extracted fraction was in the range of 68–
79% (n = 2, in duplicates) of the total radioactivity. Both
HPLC and TLC revealed that 99% of the extracted
radioactivity corresponded to the intact tracer at all time
points.
2.3. Biology
Several inhibitors of both vascular endothelial growth
factor receptor 2 (VEGFR-2) and platelet-derived growth
factor receptor (PDGFR) were synthesized, based on the
N-phenyl-N0-{4-(4-quinolyloxy)phenyl}urea chemical
core structure. This family of molecules was selected
due to its high specificity toward these receptors, known
to be upregulated in angiogenic processes.27 The three
derivatives (1–3) were synthesized with varying fluorine
positioning on the aniline ring for structure–activity rela-
tionship evaluation of receptor phosphorylation inhibi-
tion (Scheme 2).
3. Conclusion
In conclusion, out of the three approaches, only one
proved to be valuable in our attempt to develop a gen-
eral method for labeling diaryl ureas. The isocyanate
group reacted rapidly with a n.c.a fluorine-18 labeled
aniline to give high yields of diaryl urea moiety under
mild conditions and short reaction times. This successful
approach was used to label the high affinity and selective
VEGFR-2/PDGFR inhibitor [18F]-1 for the potential of
being used as PET imaging agents in angiogenesis. [18F]-
1 was found to be highly stable in ex vivo blood tests.
Evaluation of median inhibitory concentration (IC50)
values for inhibition of receptor phosphorylation was
carried out using porcine aortic endothelial (PAE/
KDR) and NIH/PDGFR cells, expressing VEGFR-2
and PDGFRb, respectively.28,29 Inhibitory selectivity
with respect to the tyrosine kinase receptors EGFR
and HER2 was determined using DHER14 and
CSH12 cells, which express EGFR and a HER1-
HER2 chimera, respectively.30–32 Selectivity toward the
IGF-1R was assessed by ELISA.33 Although published
results reported varying VERGFR-2/PDGFR selectivity
as a result of fluorine positioning on the different phenyl
rings,6 we observed minor influence of fluorine position-
ing on VERGFR-2/PDGFR selectivity in whole cell
assays. All three compounds exhibited high autophos-
phorylation inhibition with IC50 values of around 5–
15 nm for both VEGFR-2 and PDGFRb, in addition
to having very high selectivity in comparison to other
members of the receptor tyrosine kinase (RTK) super-
family (Table 1).
4. Experimental
4.1. General methods
All operations with air- and moisture-sensitive com-
pounds were performed by the Schlenk techniques under
argon atmosphere. All solvents were of analytical grade
or better. THF was distilled over sodium/benzophenone;
other solvents were purchased as anhydrous. 1H and 19
F
NMR spectra were recorded on 300 MHz spectrometers
1
in DMSO-d6. 1H signals are reported in ppm. H NMR
signals are referenced to the residual proton (2.50 ppm
for DMSO-d6) of a deuterated solvent and for 19F
NMR spectra; the signal of CFCl3 (0 ppm) was used
as an internal reference. Mass spectra were obtained
on a spectrometer equipped with CI, EI, and FAB
probes and on a spectrometer equipped with ESI probe.
HRMS results were obtained on MALDI-TOF and ESI
mass spectrometers. IR spectra were recorded on a
FTIR spectrometer. Reaction progress was monitored
by TLC (SiO2) and visualized using UV light. Flash
chromatography was carried out on SiO2 (0.04–
Table 1. IC50 values (nM) obtained in whole cell assays for the three
tested compounds
Compound PDGFRb VEGFRb EGFRc Her-2c IGF-1Ra,b
1
2
3
2–5
2–5
2–5
8–20
4–10
n.d.
>500
>500
>500
>500
>500
>500
>2000
>2000
>2000
n.d., not determined.
a IGF-1R values were obtained in cell-free assays.
b n = 3.
c n = 2.

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O. Ilovich et al. / Bioorg. Med. Chem. 16 (2008) 4242–4251
Figure 2. HPLC chromatograms of the radioactivity extracted from blood sample at 0, 60, and 120 min.
0.063 mm). HPLC was performed on a system with a
variable wavelength detector operating at 254 nm and
a radioactivity detector with a NaI crystal.
a reversed-phase system using a Waters lBondapak C-
18 column (10 lm, 125A, 300 · 3.9 mm) and 55%
CH3CN/45% acetate buffer 0.1 M/pH 3.8 as solvents at
a flow rate of 1 mL/min. Fluorine-18 was produced on
an IBA 18/9 cyclotron by irradiation of 2 mL water tar-
get (97%-enriched [18O]water) by the [18O(p,n)18F] nucle-
ar reaction and was transferred to the appropriate hot
cell.
Thin layer chromatography (TLC) for blood stability
experiments was preformed using normal phase TLC
plates (LK6DF, Whatman) and 1:9 methanol/dichloro-
methane as eluent. TLC plates were dried and exposed
for 1.5 h to phosphor imager plates (BAS-IP MS 2040
Fuji Photo Film Co., LTD) for visualization of radioac-
tive bands. The plates were scanned with a BAS reader
3.1-version scanner, and analyzed with TINA 2.10 g
software. Two HPLC systems were used: (A) a re-
versed-phase system employing a Bishoff 100 C-18 col-
umn (7 lm, 250 · 10 mm) and 45% CH3CN/55%
acetate buffer 0.1 M/pH 3.8 as eluent, at a flow rate of
4.8 mL/min. Fractions of 4.0 mL were collected. (B)
Analysis of formulated radiotracer was performed on
4.2. Chemistry
4.2.1. 6,7-Dimethoxy-4-quinolone (4).6 To a solution of
2-amino-4,5-dimethoxyacetophenone (10.0 g, 51 mmol)
in dimethoxyethane (250 mL), NaOMe (8.3 g,
154 mmol) was added and the mixture stirred at room
temperature for 70 min. Ethyl formate (21 mL,
261 mmol) was then added and stirring continued for
2 h. Reaction was stopped by adding water (10 mL).

O. Ilovich et al. / Bioorg. Med. Chem. 16 (2008) 4242–4251
4249
The solution was neutralized with 10% aq HCl solution
forming a white precipitate. The resulting solid was col-
lected by filtration and washed with water, dioxane, and
AcOEt. The solid was dried under vacuum to obtain
10.0 g (95%) of 4. 1H NMR (DMSO-d6): d 7.75 (d,
J = 7.5 Hz, 1H), 7.41 (s, 1H), 6.95 (s, 1H), 5.91 (d,
J = 7.5 Hz, 1H), 3.83 (s, 3H), 3.8 (s, 3H). MS (ESI, m/
z): 206 (M+1).
ature for 2 h and evaporated to dryness. The solid ob-
tained was redissolved in dry DCM (10 mL) and a
solution of 2-fluoroaniline (0.12 mL, 1.3 mmol) and
Et3N (0.18 mL, 1.3 mmol) in dry DCM (10 mL) were
added in one portion. After stirring at room tempera-
ture overnight, the solvent was evaporated and the prod-
uct purified using flash column chromatography with
50% EtOAc:Hexane to give 185 mg of the title com-
1
pound as a white solid (65%). Mp > 250 ꢁC. H NMR
4.2.2. 4-Chloro-6,7-dimethoxy-quinoline34 (5). 6,7-Dime-
thoxy-4-quinolone (4, 2 g, 10 mmol) and oxalyl chloride
(4.4 mL, 50 mmol) were dissolved in DCM (40 mL) and
DMF (0.2 mL) was added. The mixture was refluxed
overnight, the volatiles were distilled out, and the purity
of the product, 4-chloro-6,7-dimethoxy-quinoline, was
determined using a reversed-phase C-18 analytical
HPLC column (94% purity). The compound was kept
at 0 ꢁC, and used without any further purification for
(DMSO-d6): d 9.38 (s, 1H), 8.64 (s, 1H), 8.47 (d,
J = 5.1 Hz, 1H), 8.11 (t, J = 8 Hz, 1H), 7.75 (d,
J = 14 Hz, 1H) 7.52 (s, 2H), 7.39 (t, J = 9.5 Hz, 1H) 7–
7.29 (m, 5H), 6.46 (d, J = 5.1 Hz, 1H), 3.93 (s, 6H).
19F NMR (DMSO-d6): d ꢀ128.89 (dd, J1 = 13.5 Hz,
J2 = 9 Hz, 1F), ꢀ130.45 (m, 1F). HRMS 452.1404
(M+1).
4.2.6. 1-[4-(6,7-Dimethoxy-quinolin-4-yloxy)-3-fluoro-
phenyl]-3-(4-fluoro-phenyl)-urea (1). A similar procedure
as for 2 was used with 4-fluoroaniline to give a white so-
lid which was purified using flash column chromatogra-
phy with 100% EtOAc to give 140 mg of the title
compound as a white solid (50%). Mp > 250 ꢁC. 1H
NMR (DMSO-d6): d 9.08 (s, 1H), 8.88 (s, 1H), 8.46
(dd, J1 = 6.6 Hz, J2 = 1.5 Hz, 1H), 7.73 (d,
J = 13.5 Hz, 1H), 7.34–7.51 (m, 5H), 7.24 (d,
J = 8.4 Hz, 1H) 7.12 (t, J = 7.8 Hz, 2H), 6.43 (d,
J = 5.4 Hz, 1H), 3.93 (s, 6H). 19F NMR (DMSO-d6): d
ꢀ129.13 (dd, J1 = 14.1 Hz, J2 = 8.7 Hz, 1F), ꢀ121.92
(m, 1F). HRMS 452.1439 (M+1).
1
the next step. Mp 130–131 ꢁC. H NMR (DMSO-d6):
d 8.68 (d, J = 5.1 Hz, 1H), 7.66 (d, J = 4.8 Hz, 1H),
7.49 (s, 1H), 7.4 (s, 1H), 3.99 (s, 3H), 3.98 (s. 3H). MS
(ESI, m/z): 224 (M+1).
4.2.3. [4-(6,7-Dimethoxy-quinolin-4-yloxy)-3-fluoro-phe-
nyl]-carbamic acid tert-butyl ester (6). Compound 5
(4.33 g, 19.4 mmol) and (3-fluoro-4-hydroxy-phenyl)-
carbamic acid tert-butyl ester (7.22 g, 31.7 mmol) and
DMAP (4 g, 31.7 mmol) were dissolved in DMF
(150 mL) and stirred at 145 ꢁC for 5 h. The DMF
was evaporated under reduced pressure and replaced
with DCM, washed with brine, dried over Na2SO4,
and evaporated to give a brown oil. Purification was
achieved using flash column chromatography with
40% EtOAc:Hexane as eluent to give 4.13 g (51%) of
yellow oil. 1H NMR (DMSO-d6): d 9.73 (s, 1H),
8.44 (d, J = 5.1 Hz, 1H), 8.432 (s, 1H), 7.93 (s, 1H),
7.64 (d, J = 12.3 Hz, 1H), 7.5 (s, 1H), 7.28-7.4 (m,
2H), 3.92 (s, 6H), 1.48 (s, 9H). MS (ESI, m/z): 415
(M+1).
4.2.7. 1-[4-(6,7-Dimethoxy-quinolin-4-yloxy)-3-fluoro-phe-
nyl]-3-(2,4-difluoro-phenyl)-urea6 (3). A similar proce-
dure as for 2 was used with 2,4-difluoroaniline to give
a white solid which was purified using flash column
chromatography with 100% EtOAc to give 152 mg of
the title compound as a white solid (50%). Mp 205–
1
206 ꢁC. H NMR (DMSO-d6): d 9.34 (s, 1H), 8.6 (s,
1H), 8.46 (d, J = 5.1 Hz, 1H), 8.03 (m, 1H), 7.73 (dd,
J1 = 10.8 Hz, J2 = 2.4 Hz, 1H) 7.51 (s, 1H), 7.28–7.42
(m, 3H), 7.22 (d, J = 9.3 Hz, 1H), 7.305 (t, J = 9.3 Hz,
1H) 6.43 (d, J = 5.4 Hz, 1H) 3.93 (s, 6H). 19F NMR
(DMSO-d6): d ꢀ128.92 (dd, J1 = 12.9 Hz, J2 = 9.9 Hz,
1F), ꢀ125.21 (m, 1F), ꢀ118.39 (m, 1F). HRMS
470.1350 (M+1).
4.2.4. 4-(6,7-Dimethoxy-quinolin-4-yloxy)-3-fluoro-phen-
ylamine6 (7). Compound 6 (4.13 g, 9.98 mmol) was dis-
solved in HCl in dioxane (2 M, 300 mL) and stirred at
room temperature for 5 h. A white sediment formed.
The solution was diluted with ether and filtered to give
the HCl salt as a white paste. This paste was first dis-
solved in water and washed with DCM and then by
increasing the pH, the free base sediments were collected
by filtration. To ascertain product purity, the solid was
cleaned using a short silica plug with EtOAc as eluent
to give 2.07 g (66%) of the title compound. Mp 196–
197 ꢁC. 1H NMR (DMSO-d6): d 8.43 (d, J = 6.3 Hz,
1H), 7.48 (s, 1H), 7.35 (s, 1H), 7.05 (t, J = 8.7 Hz,
1H), 6.35–6.55 (m, 3H), 5.48 (s, 2H), 3.91 (s, 6H). MS
(ESI, m/z): 315 (M+1).
4.2.8. 4-(2-Fluoro-4-isocyanato-phenoxy)-6,7-dimethoxy-
quinoline (8). A solution of triphosgene (12 mg, 41 lmol)
in dry DCM (2 mL) was cooled to 0 ꢁC and a solution of
compound 7 (20 mg, 64 lmol) and Et3N (17 lL, 12 mg,
122 lmol) in dry DCM (4 mL) was added slowly over a
period of 1 h. The solution was then stirred at room
temperature for 3 h and a sample taken under nitrogen
for IR evaluation. Volatiles were evaporated under
nitrogen to dryness to give a yellow solid which was kept
at 4 ꢁC under nitrogen until further use. IR (NaCl)
4.2.5. 1-[4-(6,7-Dimethoxy-quinolin-4-yloxy)-3-fluoro-phe-
nyl]-3-(2-fluoro-phenyl)-urea (2). A solution of triphos-
gene (190 mg, 0.65 mmol) in dry DCM (10 mL) was
cooled to 0 ꢁC and a solution of compound 7 (200 mg,
0.64 mmol) and Et3N (180 lL, 1.3 mmol) in dry DCM
(10 mL) were added slowly over a period of 1 h under
nitrogen. The solution was then stirred at room temper-
2285 cmꢀ1
.
4.2.9. [4-(6,7-Dimethoxy-quinolin-4-yloxy)-3-fluoro-phe-
nyl]-carbamic acid isopropenyl ester (9). Compound 7
(100 mg, 0.32 mmol) and N-methylmorpholine (40 lL,
0.38 mmol) were dissolved in THF (8 mL) and the solu-
tion was cooled in an ice bath. Isopropenyl chlorofor-

4250
O. Ilovich et al. / Bioorg. Med. Chem. 16 (2008) 4242–4251
mate (40 lL, 0.38 mmol) was added dropwise over
30 min and the reaction stirred at room temperature
for 4 h. Reaction was quenched by adding water, ex-
tracted with EtOAc and the organic fractions washed
with brine, dried over Na2SO4 and evaporated to give
a yellow solid. Purification was performed by flash col-
umn chromatography with 60% EtOAc:Hexane as elu-
ent to give 94 mg of the title compound as a white
solid (74%) 1H NMR (DMSO-d6): d 10.31 (s, 1H),
8.45 (d, J = 5.4 Hz, 1H), 7.61 (dd, J1 = 10.5 Hz,
J2 = 2.4 Hz, 1H), 7.5 (s, 1H), 7.31–7.45 (m, 3H), 6.41
(d, J = 4.2 Hz, 1H), 4.77 (d, J = 8.1 Hz, 2H), 3.92 (s,
6H), 1.94 (s, 3H). MS (ESI, m/z): 399 (M+1).
perature under nitrogen for 30 min and then the DCM
was evaporated and replaced with CH3CN/H2O (1:1)
(1 mL). The product was purified using HPLC system
B, generating [18]F-U8 at rt = 24 min. The fractions col-
lected were diluted with water, loaded onto a C-18 Sep-
Pak, dried and eluted with EtOH (1 mL). A 50 lL sam-
ple was injected into an analytical HPLC using system A
for purity and specific activity analysis. The labeled
compound was obtained with a 74% radiochemical
yield, a specific activity of 400 37 GBq/mmol and a
99% radiochemical purity (n = 5). Co-injection of [18]F-
1 and 1 standard (system A) followed to ensure of the
compound’s identity (Fig. 1).
4.2.10. 4-[18F]fluoro-nitrobenzene23 ([18F]-10). An [18O]H2O/
[18Fꢀ] (26 GBq) mixture was trapped and transferred to a
reactor through an ion exchange column (preactivated with
0.8 mL of EtOH and 3 mL of HPLC water) by elution with
0.5 mL of potassium carbonate (2.76 mg/mL). Following
the addition of Kryptofix 2.2.2 solution (6 mg, 16 lmol in
1 mL CH3CN), azeotropic removal of water and acetoni-
trile was achieved by heating the reactor to 100 ꢁC under
a stream of nitrogen for 3 min and under vacuum for an-
other 1 min. 1,4-Dinitrobenzene (2.2 mg, 13 lmol) dis-
solved in DMF (600 lL) was added, the reactor
temperature was increased to 120 ꢁC and the reaction mix-
ture was stirred for 20 min. After cooling the mixture to
30 ꢁC, 7 mL of water was added to the reactor, and the
mixture was loaded on a C-18 cartridge (Waters Sep-Pak;
preactivated with 5 mL of EtOH and 10 mL of sterile
water). The product, 4-[18F]fluoro-nitrobenzene, was
washed with another 10 mL of water and eluted with
2 mL of EtOH into a collection vial after a total radiosyn-
thesis time of 40 min and was obtained with an 82% radio-
chemical yield. The product was analyzed by HPLC using
system A (rt = 7.4 min) and compared to a standard
material.
4.3. Biology
4.3.1. Inhibition of VEGFR-2 phosphorylation in PAE/
KDR cells. PAE/KDR cells (1 · 105 cells/well) were
grown in 6-well plates for 24 h, reaching 80–90% conflu-
ence. Cells were incubated with inhibitors 1–3 at increas-
ing concentrations (0.05% DMSO, 0.1% EtOH) for 1 h.
Following incubation of the tested compound, cells were
supplemented with Na3VO4 (100 lM, 5 min) (Sigma) to
inhibit endogenous phosphatase activity, and stimulated
for 5 min at 37 ꢁC with 50 ng/mL VEGF-A165 (CytoLab
Ltd, Israel). Following a wash with ice-cold PBS con-
taining 100 lM Na3VO4, cell lysates were prepared in
boiling Laemmli buffer. The extent of VEGFR-2 phos-
phorylation was evaluated by Western blot analysis as
described in the past.32
4.3.2. Selectivity assay. CSH12, DHER14, and NIH/
PDGFR cells, expressing either the HER1-HER2 chi-
mera, EGFR or PDGFRb, respectively, were used for
the determination of inhibitory selectivity. Cells
(7.5 · 104) were grown in 6-well plates (Nalge Nunc
Int.) for 24 h and then incubated in 0.25% FCS-contain-
ing medium for an additional 24 h to ꢁ90% confluence.
Duplicate sets of cells were treated with the tested com-
pound at varying concentrations for 1 h. The final con-
centration of the vehicle in the medium was 0.05%
DMSO, 0.1% EtOH. After removal of the inhibitor
from the medium, PBS wash (·2) and addition of
0.25% serum-containing medium to the wells, the cells
were stimulated with either 20 ng/mL hEGF (Sigma)
for 5 min (CSH12 and DHER14 cells) or 50 ng/mL hu-
man PDGFbb (Sigma) for 10 min (NIH/PDGFR cells)
at 37 ꢁC. Following stimulation with the growth factor,
the cells were washed with cold PBS. Cell lysate prepa-
ration and Western blot analysis were described in the
past.32
4.2.11. 4-[18F]fluoro-aniline23 ([18F]-11). Hydrazine
monohydrate (150 lL) was added to a reactor con-
taining 200 lL of EtOH/H2O (4:1) and raney (Ra)-
Ni (400 lL) at 57 ꢁC, and then [18F]-10 in EtOH/
H2O (2.5 mL, 4:1) was added dropwise to the reactor.
Synthesis continued at 57 ꢁC for 7 min, another por-
tion of hydrazine monohydrate (150 lL) was added
to the reactor and synthesis continued for an addi-
tional 5 min. The product was diluted with 10 mL of
water, passed through an Oasis HLB plus cartridge
(Waters; preactivated with 5 mL of MeOH and
10 mL of sterile water) and dried under argon for
20 min to yield 4-[18F]fluoro-aniline with a 76% decay
corrected radiochemical yield. The product was
eluted with our solvent of choice (2 mL), dried over
Na2SO4, filtered via 0.45 lm filter (Whatman,
Puradisc), and analyzed by HPLC using system A
4.3.3. Cell-free assay. An evaluation of the IGF-1R
autophosphorylation IC50 values was obtained by
means of an ELISA screen, performed as depicted ear-
lier.35 For each compound, at least three independent
assays that gave similar results were considered. Each
assay was carried out using duplicate samples.
(rt = 5.8 min) and compared to
material.
a
standard
4.2.12. 1-[4-(6,7-Dimethoxy-quinolin-4-yloxy)-3-fluoro-
phenyl]-3-(4-[18F]fluoro-phenyl)-urea ([18]F-1). To the iso-
cyanate precursor described above dry (sodium distilled)
Et3N (30 lL, 0.2 mmol) and [18F]-11 in dry DCM
(2 mL) were added. Reaction was stirred at room tem-
4.3.4. In vitro blood stability. Fresh human blood sam-
ples were collected in a VacutainerTM containing EDTA
(K3, BD, USA), divided into 1 mL aliquots in glass-vials
and incubated for 5 min at 37 ꢁC with gentle shaking.

O. Ilovich et al. / Bioorg. Med. Chem. 16 (2008) 4242–4251
4251
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Acknowledgment
This research was supported by the Israel Science Foun-
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