Synthesis and radiopharmacological investigation of 3-[4′-[18F]fluorobenzylidene]indolin-2-one as possible tyrosine kinase inhibitor

Article abstract of DOI:10.1016/j.bmc.2009.09.038

The radiosynthesis and radiopharmacological evaluation of 3-[4′-[¹⁸F]fluorobenzylidene]indolin-2-one, a derivative of tyrosine kinase inhibitor SU5416, is described. The radiosynthesis was accomplished by Knoevenagel condensation of 4-[^18<

Full text of DOI:10.1016/j.bmc.2009.09.038

Bioorganic & Medicinal Chemistry 17 (2009) 7732–7742
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and radiopharmacological investigation of 3-[40-[18F]fluorobenzylid-
ene]indolin-2-one as possible tyrosine kinase inhibitor
a
a
a
b
Torsten Kniess a, , Ralf Bergmann , Manuela Kuchar , Jörg Steinbach , Frank Wuest
*
a Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf e.V., PO Box 510119, D-01314 Dresden, Germany
b Department of Oncologic Imaging, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2
a r t i c l e i n f o
a b s t r a c t
The radiosynthesis and radiopharmacological evaluation of 3-[40-[18F]fluorobenzylidene]indolin-2-one, a
derivative of tyrosine kinase inhibitor SU5416, is described. The radiosynthesis was accomplished by
Knoevenagel condensation of 4-[18F]fluorobenzaldehyde with oxindole in a remotely controlled synthesis
module. The reaction conditions were optimized through screening the influence of different bases on the
radiochemical yield. The radiotracer was obtained after a two-step labelling procedure in 4% decay-cor-
Article history:
Received 12 June 2009
Revised 15 September 2009
Accepted 21 September 2009
Available online 25 September 2009
rected radiochemical yield at a specific activity of 48–61 GBq/
purity after semi-preparative HPLC purification exceeded 98%.
lmol within 90 min. The radiochemical
Keywords:
Positron emission tomography (PET)
4-[18F]fluorobenzaldehyde
Tyrosine kinase inhibitor
Knoevenagel condensation
SU5416
The biodistribution was studied in Wistar rats. After distribution the radiotracer was rapidly accumulated
in the adrenals, liver and kidneys, however, it was cleared from these and the most other organs. Only the
adipose tissue remained the activity over 60 min. Unexpected high transient uptake was observed in the
brain, pancreas, heart and lung. The fast clearance of 3-[40-[18F]fluorobenzylidene]indolin-2-one was
caused by excretion, approximately one half each was renal and biliary excreted and the other part cleared
by metabolic processes. In arterial blood plasma two more polar metabolites were found by radio-HPLC.
After 20 min post-injection, only 12% of intact radiotracer has been detected. Consequently, in small animal
PET studies with FaDu tumour bearing mice no specific uptake in the tumours could be observed.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
potent anti-cancer agents have been approved or are currently
studied in clinical trials.3,4
Receptor tyrosine kinases (RTKs) are membrane-spanning cell
surface proteins positioned in key crossroads within the cellular
communication network. As master switches, RTKs play critical
roles in the transduction of extracellular signals to the cytoplasm.1
Moreover, RTKs have been found to play an important role in tu-
mour angiogenesis through signal regulation for proliferation,
migration and differentiation between tumour and endothelial
cells.2 Activated forms of these enzymes can result in enhanced tu-
mour cell proliferation and growth, induction of anti-apoptopic ef-
fects, and angiogenesis and metastasis.3 Many human cancers
derive from epithelial tissues, and RTKs are often over-expressed
in these tumours. This finding led to the development of several
selective tyrosine kinase inhibitors with potencies in the nano-
and subnanomolar range. Small molecule tyrosine kinase inhibi-
tors like Imatinib mesylate (GleevecÒ), Gefitinib (IressaÒ), Erlotinib
(TarcevaÒ), VatalinibÒ (PTK 787) and SorafinibÒ (BAY43-9006) as
The growing number of anti-angiogenic therapies is accompa-
nied with an increasing need for imaging tumour neovasculariza-
tion, and monitoring anti-angiogenic therapies. Positron emission
tomography (PET) is a powerful in vivo diagnostic molecular imag-
ing technique applying tracer concentrations in the picomolar
range to allow visualization of specific targets on the molecular le-
vel.5,6 Direct noninvasive molecular imaging of angiogenesis would
allow patient selection for anti-angiogenesis therapy, and assess-
ment of the efficacy of angiogenesis targeted therapies.6 Overex-
pression of tyrosine kinases in various human cancers represents
an attractive basis for the development of small molecule tyrosine
kinase inhibitors labelled with the short-lived positron emitters
carbon-11
( ( =
11C, t1/2 = 20.4 min) and fluorine-18 18F, t1/2
109.8 min) as molecular probes for imaging RTK expression
in vivo. Recently, anilinoquinazoline-based tyrosine kinase inhibi-
tor gefitinib (IressaÒ) was used as a lead structure for the develop-
ment of various 11C-labelled7–11 and 18F-labelled radiotracers.12–15
However, the majority of these reports describe mainly radiochem-
istry and in vitro experiments on inhibition of autophosphoryla-
tion. Only one report deals with the study of tumour uptake
in vivo in xenografted tumour model rats involving a radiolabelled
* Corresponding author. Tel.: +49 351 260 2760; fax: +49 351 260 2915.
E-mail address: t.kniess@fzd.de (T. Kniess).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.038

T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
7733
tyrosine kinase inhibitor.11 More recently, the synthesis and radio-
pharmacological evaluation of the 11C-labelled tyrosine kinase
inhibitor GleevecÒ was studied by means of PET in baboons.16
Another class of small lipophilic RTK inhibitors are based on an
oxindole scaffold. Prominent examples are SU5416 (SemaxinibÒ)
and SU11248 (SutentÒ), which have entered preclinical and multi-
center clinical studies as inhibitors of vascular endothelial growth
factor receptor (VEGFR) and for anti-angiogenic and antitumour
activity3 (Fig. 1).
hyde and Knoevenagel condensation was performed in an auto-
mated nucleophilic fluorination module (Nuclear Interface,
Münster, Germany). Semi-preparative HPLC purifications were car-
ried out with a Nucleosil 100-7 C18 column (250 ꢀ 16 mm, 7
lm,
Macherey-Nagel) using an isocratic eluent (acetonitrile/
water = 1:1) with a flow rate of 8 mL/min. The product was moni-
tored by UV detector UV2075 (Jasco) and by radioactivity detector
integrated in the synthesizer module. Analytical HPLC analysis
were carried out with a Discovery C18 column (15 ꢀ 4 mm,
SU11248 has been approved as anticancer agent for treatment of
kidney cancer and gastrointestinal stromal tumours, whereas ran-
domized phase III studies of SU5416 in patients with metastatic
colorectal cancer failed for no showing survival benefits and reveal-
ing toxic side effects.17 However, oxindole SU5416 exhibits high
5 lm, Suppelco) using an isocratic eluent (acetonitrile/
water = 40:60) by a gradient pump L2500 (MERCK, HITACHI) with
a flow rate of 1 mL/min. The products were monitored by an UV
detector L4500 (MERCH, HITACHI) at 254 nm and by gamma-
detection with a scintillation detector GABI (X-RAYTEST).
affinity towards VEGF-RTKs (IC50 = 0.06 l l
M;18 IC50 <0.8 M24), and
demonstrated anti-angiogenic activity in mice tumour xenografts.
Thus, compound SU5416 holds promise as molecular probe for
imaging RTKs expression in tumour by means of PET. Synthesis of
18F-labelled SU11248 starting from the corresponding nitro-pre-
cursor was recently described. However, no radiopharmacological
data have been reported.19
The aim of the present study is the development of a general
synthesis approach for the preparation of 18F-labelled benzylidene
oxindoles as potential radiotracers for PET imaging. The radiosyn-
thesis of compounds containing the characteristic benzylidene
oxindole scaffold as found in SU5416 was achieved via Knoevena-
gel condensation with the readily available 18F labelling precursor
4-[18F]fluorobenzaldehyde. The novel radiotracer was used in
radiopharmacological studies involving biodistribution in mice,
metabolic stability assessment in vivo and small animal PET stud-
ies in FaDu tumour-bearing mice.
2.2. Synthesis of reference compounds and precursors
2.2.1. 3-[(20,40-Dimethylpyrrol-50-yl)methylidene]5-fluoro-
indolin-2-one 2
Hundred and fifty milligrams (1 mmol) of 5-fluoro-oxindole 1
and 148 mg (1.2 mmol) of 3,5-dimethylpyrrol-2-carbaldehyde
were refluxed in 2 mL of ethanol with 2 drops of piperidine for
4–5 h. The product was collected by filtration and purified by col-
umn chromatography (silica gel, ethyl acetate/petrol ether = 1:1).
Yield: 79 mg orange crystals (31%), mp: 271–273 °C; 1H NMR (d,
ppm, DMSO-d6): 2.30 (s, 3H, CH3); 2.32 (s, 3H, CH3); 6.02 (s, 1H,
Hpyrrol); 6.82 (d, 1H, 7-H); 6.88 (t, 1H, 6-H); 7.63 (s, 1H, Hvinyl);
7.66 (d, 1H, 4-H); 10,77 (s, 1H, NHoxinol); ESI-MS (ES+): m/z = 257
(M+H).
2.2.2. 3-[(20,40-Dimethylpyrrol-50-yl)methylidene]5-nitro-
indolin-2-one 5
The compound was prepared from 5-nitro-oxindole 3 and
3,5.dimethylpyrrol-2-carbaldehyde in 72% yield as previously
described.20
2. Materials and methods
2.1. Materials
2.2.3. 3-[(20,40-Dimethylpyrrol-50-yl)methylidene]5-
dimethylamino-indolin-2-one 6
All commercial reagents and solvents were used without fur-
ther purification unless otherwise specified. Nuclear magnetic res-
Three hundred and fifty two milligrams (2 mmol) of 5-dimeth-
ylamino-oxindole 421 and 295 mg (2.4 mmol) of 3,5-dimethylpyr-
rol-2-carbaldehyde were refluxed in 4 mL of ethanol with 5
drops of piperidine for 6 h. The solvent was evaporated and the res-
idue purified by column chromatography (silica gel, ethyl acetate/
petrol ether = 1:1). Yield: 519 mg dark crystals (92%), mp: 260–
267 °C, 1H NMR (d, ppm, DMSO-d6): 2.31 (2s, 6H, 2 ꢀ CH3pyrrol);
2.86 (s, 6H, 2 ꢀ CH3); 5.97 (s, 1H, Hpyrrol); 6.54 (d, 1H, 6-H); 6.70
(d, 1H, 7-H); 7.22 (s, 1H, 4-H); 7.55 (s, 1H, Hvinyl); 10.45 (s, 1H,
NHoxindole); ESI-MS (ES+): m/z = 282 (M+H).
onance spectra were recorded on
a Varian Unity 400 MHz
spectrometer. 1H NMR chemical shifts were given in ppm and were
referenced with the residual solvent resonances relative to tetra-
methylsilane (TMS). Mass spectra were obtained on a Quattro/LC
mass spectrometer (MICROMASS) by electrospray ionization.
Flash chromatography was conducted using MERCK silica gel
(mesh size 230–400 ASTM). Thin-layer chromatography (TLC)
was performed on Merck silica gel F-254 aluminium plates with
visualization under UV (254 nm).
No-carrier-added aqueous [18F]fluoride ion was produced in a
IBA CYCLONE 18/9 cyclotron by irradiation of [18O]H2O via the
18O(p,n)18F nuclear reaction. Synthesis of 4-[18F]fluorobenzalde-
2.2.4. 5-Trimethylammonium-3-[(20,40-dimethylpyrrol-50-
yl)methylidene]indolin-2-one-trifluoromethan-sulfonate 7
Three hundred milligrams (1.06 mmol) of 6 were dissolved in
10 mL of dichloromethane and 140 lL (1.3 mmol) of ethyl-trifluoro-
H
N
O
methanesulfonate were added at rt. After stirringfor 24 h theprecip-
itate was collected by filtration and washed with 2 mL of
dichloromethane. Yield: 360 mg brown precipitate (76%), mp:
>280 °C, subl., 1H NMR (d, ppm, DMSO-d6): 2.35 (2s, 6H, 2 ꢀ
CH3pyrrol); 3.62 (s, 9H, 3 ꢀ CH3); 6.09 (s, 1H, Hpyrrol); 7.00 (d, 1H, 7-
H); 7.61 (d, 1H, 6-H); 7.77 (s, 1H, Hvinyl); 8.28 (s, 1H, 4-H); 11.11 (s,
1H, NHoxindole); ESI-MS (ES+): m/z = 296 (M+).
N
N
N
F
H
H
O
O
N
N
2.2.5. 3-[40-Fluorobenzylidene]indolin-2-one 10 (SU5205)
H
H
The synthesis was carried out with 133 mg (1 mmol) oxindole
and 129 lL (1.2 mmol) of 4-fluorobenzaldehyde following the pro-
cedure in 2.2.1., the product was purified by column chromatogra-
SU11248 (Sutent)
SU5416 (Semaxinib)
Figure 1. Structures of the tyrosine kinase inhibitors SU5416 and SU11248.
phy (silica gel, trichloromethane/methanol 95:5). Yield: 103 mg

7734
T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
yellow crystals (43%), mp: 185–189 °C; 1H NMR (d, ppm, DMSO-
d6): 6.82 (m, 2H, Hoxindole); 7.23 (t, 1H, Hoxindole); 7.36 (m, 2H,
Hbenzyl); 7.49 (d, 1H, Hoxindole); 7.60 (s, 1H, Hvinyl); 7.77 (d, 2H,
Hbenzyl); 10.62 (s. 1H, NH); ESI-MS (ES+): m/z = 261 (M+Na).
tridge (250 mg, Waters). The cartridge was dried in a stream of
nitrogen for 5 min and the 4-[18F]fluorobenzaldehyde was recov-
ered by eluting the cartridge with 3.0 mL of ethanol. The eluent
solution was directed in a separate reacting vial containing
10 mg of oxindole and 30 lL of base. The sealed vial was heated
2.2.6. 3-[40-Nitrobenzylidene]indoline-2-one 11
at 90 °C for performing Knoevenagel condensation, after 20 min
the mixture was cooled and conducted to semi-preparative HPLC;
by using an isocratic eluent (water/acetonitrile = 1:1). The radio-
tracer 14 was eluted between 20 and 22 min at a flow rate of
8 mL/min. The product 14 was separated from the eluent by means
of solid phase extraction (Lichrolut RP18, 500 mg, Merck), removed
from the cartridge with 0.7 mL of ethanol and formulated for bio-
logical investigation with 6.5 mL of 0.9% sodium chloride solution.
In this way the radiotracer 14 was synthesized in 90 min synthesis
time in 4% total decay corrected yield from [18F]fluoride in radio-
The synthesis was carried out using 133 mg (1 mmol) oxindole
and 181 mg (1.2 mmol) of 4-nitrobenzaldehyde following the pro-
cedure in 2.2.1., the product was collected by filtration, and
washed with 3 mL ethyl acetate. Yield: 217 mg yellow crystals
(82%), mp: 245–250 °C; 1H NMR (d, ppm, DMSO-d6): 6.84 (m, 2H,
Hoxindole); 7.26 (t, 1H, Hoxindole); 7.40 (d, 1H, Hoxindole); 7.67 (s, 1H,
Hvinyl); 7.94 (d, 2H, Hbenzyl); 8.34 (d, 2H, Hbenzyl); 10.71 (s, 1H,
NH); ESI-MS (ES+): m/z = 289 (M+Na).
2.2.7. 3-[40-Dimethylaminobenzylidene]indoline-2-one 12
The synthesis was carried out with 266 mg (2 mmol) oxindole
and 358 mg (2.4 mmol) of 4-dimethylaminobenzaldehyde follow-
ing the procedure in 2.2.1., the product was collected by filtration
and washed with ethyl acetate/petrol ether = 1:1. Yield: 504 mg
brown crystals (95%), mp: 225–232 °C; 1H NMR (d, ppm, DMSO-
d6): 3.03 (s, 6H, 2 ꢀ CH3); 6.77 (m, 3H, Hbenzyl/Hoxindole); 6.93 (t,
1H, Hoxindole); 7.11 (t, 1H, Hoxindole); 7.60 (d, 1H, Hoxindole); 7.63 (s,
1H, Hvinyl); 8.43 (d, 2H, Hbenzyl), 10.45 (s, 1H, NH); ESI-MS (ES+):
m/z = 287 (M+Na).
chemical purity of 98% and a specific activity of 48–61 GBq/lmol.
2.4. Distribution coefficient
The octanol/buffer distribution coefficient (log Doct 7.4) at pH 7.4
was measured using radiolabelled 14 in phosphate buffer (0.1 M)
and an equal volume of water-saturated n-octanol in separation
funnel. After vortexing for 1 min, the mixture was fixed, and the
two phases were allowed to separate. Aliquots of the separated
phases were assayed for tracer activity by the gamma well counter.
Samples were analyzed in duplicate and re-extracted three times
to ensure stability of the log Doct 7.4 value.
2.2.8. 3-[40-Trimethylammoniumbenzylidene]indoline-2-one-
trifluoromethansulfonate 13
Two hundred and sixty four milligrams (1 mmol) of 12 were
2.5. Stability studies in vitro
dissolved at 10 mL of dichloromethane and 131 lL (1.2 mmol) of
ethyl-trifluoromethanesulfonate were added at rt. After stirring
for 24 h the precipitate was collected by filtration and washed with
ethyl acetate/petrol ether 1:1. Yield: 392 mg yellow crystals (92%);
mp: 218–223 °C, 1H NMR (d, ppm, DMSO-d6): 3.63 (s, 9H, CH3);
6.83 (d, 1H, Hoxindole); 7.01 (t, 1H, Hoxindole); 7.25 (t, 1H, Hoxindole);
7.71 (s, 1H, Hoxindole); 7.88 (s, 1H, Hvinyl), 8.03 (d, 2H, Hbenzyl);
8.50 (d, 2H, Hbenzyl); 10.69 (s, 1H, NH); ESI-MS (ES+): m/z = 279
(M+).
The in vitro stability of radiotracer 14 was evaluated by radio-
HPLC analysis at several time points after incubation with samples
of blood and plasma. Briefly 0.5 MBq of radiotracer have been
added to 400 lL of blood sample taken from Wistar rats and incu-
bated in a thermo-mixer at 37 °C at 600 rpm for 5, 30 and 60 min.
Plasma was separated by centrifugation (3 min, 13,000g) followed
by precipitation of plasma proteins with acetonitrile/water/trifluo-
roacetic acid (50:45:5) in a 1:2 ratio. The clear supernatant sepa-
rated by second centrifugation (3 min, 13,000g) was used for
analysis. For in vitro stability in rat plasma the blood cells have
been removed by centrifugation before radiotracer addition and
incubation.
2.3. Radiochemical synthesis
2.3.1. Labelling experiments with [18F]fluoride
No-carrier-added aqueous [18F]fluoride ion was produced in a
IBA CYCLONE 18/9 cyclotron by irradiation of [18O]H2O via the
18O(p,n)18F nuclear reaction. The aqueous [18F]fluoride (300–
500 MBq) was fixed on an anion exchange cartridge (QMA plus,
Waters) and resolubilized by a solution of KryptofixÒ 222 and
K2CO3 in a conical vial. Removal of water was accomplished by aze-
otropic distillation with acetonitrile in a stream of nitrogen. Finally
the dried [18F]KF was dissolved in an appropriate volume of anhy-
drous solvent (acetonitrile, DMF, DMSO) and heated in a sealed vial
in presence of 7–15 mg of the appropriate precursors 5, 6, 11 or 13
in an oil bath for 20 min at the indicated temperatures. After cool-
ing the mixture was diluted with 2 mL of water and the products
were monitored by radio-TLC (silicagel on aluminium, ethyl ace-
tate/petrol ether 1:1) and analytical radio-HPLC (RP18 column,
acetonitrile/water = 40:60).
Wistar-Unilever rats were anesthetized with Desflurane (7.0–
12.0% v/v). The guide value for breathing frequency was 65
breaths/min. Animals were put in the supine position and placed
on a heating pad to maintain body temperature. The spontaneous
breathing rats were heparinized with 100 units/kg heparin (Hepa-
rin-Natrium 25,000-ratiopharmÒ, ratiopharm GmbH, Germany) by
a subcutaneous injection to prevent blood clotting on intravascular
catheters. After local anaesthesia by injection of Lignocain 1%
(XylocitinÒ loc, mibe, Jena, Germany) into the right groin, catheters
were introduced into the left arteria carotis (0.8 mm Umbilical Ves-
sel Catheter, Tyco Healthcare, Tullamore, Ireland) for arterial blood
sampling, and a second catheter into the left vena jugularis vein
was used for radiotracer injection. For stability studies in vivo
the radiotracer 14 was injected intravenously into male Wistar rats
(30 MBq) and arterial blood samples were taken 1, 3, 5, 10, 20, 30
and 60 min after injection. Plasma was separated by centrifugation
followed by precipitation and removal of plasma proteins as above
described.
2.3.2. Radiosynthesis of 3-[40-[18F]fluorobenzylidene]indolin-2-
one 14 via Knoevenagel condensation
Synthesis of 4-[18F]fluorobenzaldehyde was performed by
reacting [18F]fluoride with 4-trimethylammoniumbenzaldehyde
triflate22 in an automated synthesizer module. Briefly 15 mg pre-
cursor dissolved at 1.0 mL of acetonitrile were heated with dried
The radio-HPLC system (Agilent 1100 series) applied for metab-
olite analysis was equipped with UV detection (254 nm) and an
external radiochemical detector (Canberra-Packard, Radiomatic
Flo-one Beta 150TR). Analysis was performed on a ZorbaxIII 300
[
18F]KF at 90 °C for 10 min. After cooling 11 mL of water was added
SB-C18 (250 ꢀ 9.4 mm; 5
lm) column with an eluent system C
(water + 0.05%TFA) and D (acetonitrile + 0.04%TFA) in a gradient
and the mixture conducted on a pre-conditioned HLB-plus car-

T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
7735
15 min 50%D to 90%D and 5 min at 90%D at a flow rate of 2 mL/
min.
anol) of approximately 11 MBq 14 was infused over 1 min with a
Harvard Apparatus 44 syringe pump (Harvard Apparatus, Hollis-
ton, Ma, USA) using a needle catheter into a tail vein. Data acquisi-
tion was performed in 3D list mode. Emission data were collected
continuously. The list mode data were sorted into sinograms with
32 frames (15 ꢀ 10 s, 5 ꢀ 30 s, 5 ꢀ 60 s, 4 ꢀ 300 s, 3 ꢀ 600 s). The
data were decay, scatter and attenuation corrected. The frames
were reconstructed by a Ordered Subset Expectation Maximization
applied to 3D sinograms (OSEM3D) with 14 subsets, 15 OSEM3D
2.6. Biodistribution studies in Wistar rats
The animal research committee of the Regierungspräsidium
Dresden approved the animal facilities and the experiments
according to institutional guidelines and the German animal wel-
fare regulations. The experimental procedure used conforms to
the European Convention for the Protection of Vertebrate Animals
used for Experimental and other Scientific Purposes (ETS No. 123),
to the Deutsches Tierschutzgesetz, and to the Guide for the Care
and Use of Laboratory Animals published by the US National Insti-
tutes of Health (DHEW Publication No. (NIH) 82-23, Revised
1996, Office of Science and Health Reports, DRR/NIH, Bethesda,
MD 20205). The Wistar rats (Wistar Unilever, HsdCpb: Wu, Harlan
Winkelmann GmbH, Borchen, Germany, 138 16 g body weight)
were housed under standard conditions with free access to stan-
dard food and tap water. The biodistribution of 14 was studied in
5 male rats at 5 min and 60 min after tracer injection. The animals
were anesthetized with Desflurane (Suprane, Baxter Healthcare
Corporation Deerfield, IL, USA) (7.0–10.0% v/v in 30% oxygen) and
iterations, 25 maximum
a posteriori (MAP) iterations, and
1.8 mm resolution using the FastMAP algorithm. The pixel size
was 0.07 by 0.07 by 0.12 cm, and the resolution in the centre of
field of view was 1.8 mm. No correction for partial volume effects
was applied. The image volume data were converted to Siemens
ECAT7 format for further processing. The image files were then
processed using the ROVER software (ABX GmbH, Radeberg, Ger-
many). Masks for defining three-dimensional regions of interest
(ROI) were set and the ROI’s were defined by thresholding and
ROI time activity curves (TAC) were for the subsequent data anal-
ysis. The ROI data and TAC were further analyzed using R (R is
available as Free Software under the terms of the Free Software
Foundation’s GNU General Public License in source code form)
and especially developed program packages (Jörg van den Hoff,
Forschungszentrum Dresden-Rossendorf, Dresden, Germany). The
standardized uptake values (SUVPET, SUVPET = (activity/mL tissue)/
(injected activity/body weight), mL/g)) were calculated in ROI.
1.7 MBq 14 aliquots were administered in 500 lL electrolyte solu-
tion E153 (Serumwerk Bernburg AG, Bernburg, Germany) with 10%
ethanol and into a tail vein. After recovery from anaesthesia rats
were again anaesthetized at 5 or 60 min after tracer injection,
respectively. Blood was withdrawn by heart puncture, and the ani-
mals were euthanized. Organs and tissues were removed, dried,
weighted, and the radioactivity was measured in a cross calibrated
well counter (WIZARD, Automatic Gamma Counter, Perkin Elmer,
Waltham, Ma, USA) or activimeter (Activimeter Isomed 2000;
MCD Nuklear Medizintechnik, Dresden, Germany). The data were
decay corrected and normalized to the amount of injected activity
calculated from the activity of injection syringes before and after
injection and expressed as percentage of injected activity (%ID)
3. Results and discussion
3.1. Synthesis of labelling precursors and reference compounds
The fluorinated non-radioactive reference compound 2 was eas-
ily obtained by the reaction of commercially available 5-fluoro-
oxindole 1 with 3,5-dimethylpyrrol-2-carbaldehyde and piperidine
as the base in ethanol under reflux. This special type of aldol con-
densation is also referred to as Knoevenagel reaction (Fig. 2).
The introduction of the isotope 18F into the aromatic core may
be usually accomplished via nucleophilic substitution of a nitro
or trimethylammonium leaving group with [18F]fluoride in aprotic
solvents.23 For precursor synthesis it seems to be reasonable at first
introducing the leaving group to the oxindole and completing the
precursor by Knoevenagel condensation with the aldehyde in a
second step (Fig. 2). In this straightforward way the nitro-precur-
sor 5 was obtained from the corresponding 5-nitro-oxindole 3 in
72% yield.20 Introduction of the N,N-dimethylamino group to the
oxindole was achieved by electrophilic aromatic substitution with
N-chloro-dimethylamine, a reaction that provided 5-dimethyl-
amino-oxindole 4 in high yield of 92%.21 Compound 4 has been re-
acted with the aldehyde to the dimethylamino-compound 6, after
subsequent quaternization with ethyl-trifluoromethanesulfonate
the corresponding trimethylammonium-precursor 7 was obtained.
For developing an alternative approach for labelling the lead
drug SU5416 with 18F we chose the 4-position of the 3,5-dimethyl-
pyrrol-2-carbaldehyde and synthesized a congruent nitro-precur-
sor 15 starting from the nitro-substituted pyrrole20 (Fig. 3).
However all efforts of substitution with a nucleophile especially
with [18F]fluoride to get the 18F-labelled SU5416 derivative 16
failed due to the electron rich character of the five-membered
heterocycle.
or standardized uptake values in biodistribution studies (SUVbio
,
SUVbio = (tissue activity/tissue weight)/(total given activity/rat
body weight)). Values are quoted as mean standard deviation
(mean SD) for a group of animals.
2.7. Small animal PET studies on rats and mice with
xenografted FaDu tumours
Positron emission tomography experiments were performed on
a established human tumour cell lines HT-29 (ECACC 91072201), a
colorectal adenocarcinoma cell line and a hypopharyngeal squa-
mous cell carcinoma line (FaDu), kept in high passage by the Amer-
ican Type Culture Collection ATCC HTB-43 (Rockville, MD). In nude
mice, FaDu grows as a poorly differentiated, nonkeratinizing carci-
noma. Following a standardized protocol, small tumour chunks
were transplanted sc into the right hind leg of the recipient mice.
When the tumours reached a diameter of about of 7–9 mm, imag-
ing studies were performed. The animals were anesthetized
through inhalation of Desflurane (7–10% Suprane) in 30% oxygen/
air (gas flow, 1 L/min). Mice were positioned and immobilized
prone with their medial axis parallel to the axial axis of the scanner
and their thorax, abdomen, and hind legs (organs of interest: liver
and tumour) in the centre of the field of view of the microPETÒ P4
(Siemens preclinical solutions, Knoxville, TN, USA) scanner. For
attenuation correction, a 10 min transmission scan was obtained
using a rotating 57Co point source before tracer injection and col-
lection of the emission scans. The radioactivity of the injection
solution in 1 mL syringe was measured in the well counter cross-
calibrated with the scanner. The emission scan of 60-min PET
acquisition was started and with a delay of 30 s the infusion of
the radiotracer was initiated. A solution of 0.5 mL (E153, 10% eth-
To overcome this drawback we decided replacing the pyrrole by
a phenyl ring, where a labelling in para-position with 18F should be
more facilitated. This strategy was forced by the fact that for 4-
fluorophenyl-substituted oxindole 10 (SU5205) was found an inhi-
bition of ligand-induced endothelial mitogenesis for VEGF an IC50
value of 5.1 lM, what is by factor 7 higher than that for SU5416

7736
T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
O
N
H
N
R
R
H
H
O
O
N
H
N
H
i)
1: R=F
2: R=F
3: R=NO2
5: R=NO2
4: R=NMe2
6: R=NMe2
ii)
7: R=NMe3TfO
Figure 2. Synthesis of reference compound and precursors for labelling of SU5416, (i) EtOH, piperidine, reflux; (ii) CF3SO3CH3, CH2Cl2, rt.
18F
NO2
[
18F]fluoride
N
H
N
H
O
O
N
N
H
i)
H
15
16
[
18F]fluoride
N
18F
N
R
H
H
O
O
N
H
N
H
i)
5: R=NO2
7: R=NMe3TfO
8
Figure 3. Routes for labelling of SU5416 with [18F]fluoride on the oxindole and pyrrole core, (i) Kryptofix222, DMF, acetonitrile, DMSO.
(IC50 <0.8
l
M) determined with the same assay.24 It was expected
of acetonitrile, DMF or DMSO at temperatures in the range be-
tween 80 and 140 °C. Each reaction was examined after 20 min
by radio-TLC and analytical HPLC for radiolabelled products. The
non-radioactive compound 2 served as reference for UV detection
by HPLC and TLC. Summarizing all of the labelling attempts it must
be realized that in no cases the desired radiotracer 8 has been
formed. In a row of experiments using trimethylammonium-pre-
cursor 7 and acetonitrile as solvent, a new radiolabelled product
was detected in up to 12% yield, but its retention time on HPLC
was dissenting with that from the standard 2. The structure of this
from its eluation profile on HPLC more hydrophilic radiolabelled
product could not be identified. This findings were much more dis-
appointing because the successful radiolabelling of another 5-ni-
that this modification will result a slight drop in tyrosine kinase
inhibition activity, however we proceeded on the assumption that
a radiolabelled 4-[18F]fluorophenyl-substituted oxindole 10 could
have adequate properties for imaging processes related with
VEGFR. Table 1 displays the experimental results of in vitro RTK as-
says for SU5416 und 10 (SU5205) as found in literature.24
Compound 10 (SU5205) was easily prepared from oxindole 9 and
4-fluorobenzaldehyde by Knoevenagel condensation (Fig. 4). As cor-
responding labelling precursors the 4-nitro-phenyl substituted
oxindole 11 and a 4-trimethylammonium substituted derivative
13 have been synthesized from 9 and 4-nitro-benzaldehyde and 4-
dimethylaminobenzaldehyde, respectively, as depicted in Figure 4.
tro-substituted oxindole (SU11248) with
[
18F]fluoride was
3.2. Labelling experiments with [18F]fluoride
recently described.19
To use an alternative labelling position besides the oxindole
ring and after unsuccessful efforts of labelling a 3-nitro-substituted
pyrrol precursor20 with [18F]fluoride we concentrated on the phe-
nyl-substituted oxindole derivative 10 (SU5205) as surrogate for
SU 5416. For labelling 5–10 mg of precursors 11 and 13, respec-
tively, were heated with dried [18F]KF/Kryptofix222 at various
temperatures and reaction times. The results of the labelling exper-
iments are shown in Table 2, the yield of the radiolabelled product
14 was quantified by gamma detection on analytical HPLC, the
retention time of UV-absorption of the non-radioactive compound
10 served as reference.
A large scale of labelling experiments with the precursors 5 and
7 was set up by heating 7.5–15 mg of precursor with the dried
[
18F]KF/Kryptofix222 complex in solvent volumes of 0.5–1.0 mL
Table 1
Inhibition activity towards particular RTKs and inhibition of ligand-induced endo-
thelial cell mitogenesis of SU5416 and compound 10 (SU5205)24
FLK-1, IC50
(l
M)
VEGF, IC50 (lM)
SU5416
10 (SU5205)
0.11
9.6
<0.8
5.1

T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
7737
F
O
F
H
O
O
N
H
i)
N
9
H
18F
10
O
ii) or iii)
H
i)
18F
R
[18F]fluoride
v)
O
O
N
H
N
H
11: R=NO2
12: R=NMe2
13: R=NMe3TfO
14
iv)
Figure 4. Synthesis of precursors and reference compounds and radiosynthesis of 3-[4-[18F]fluorobenzylidenyl]indolin-2-one 14, (i) EtOH, piperidine, reflux; (ii) 4-nitro-
benzaldehyde, EtOH, piperidine, reflux; (iii) 4-dimethylamino-benzaldehyde, EtOH, reflux; (iv) CF3SO3CH3, CH2Cl2, rt; (v) Kryptofix222, K2CO3, DMF, acetonitrile.
Table 2
Yields and conditions of radiosynthesis of 3-[40-[18F]fluorobenzylidene]indolin-2-one 14 starting from precursors 11 and 13 by radiolabelling with [18F]fluoride
Run
Precursor
Mass (mg)
Solvent
Reaction time (min)
Temperature (°C)
Yield of 14
1
2
3
4
5
6
7
8
9
11
11
11
13
13
13
13
13
13
13
13
13
7.5
7.5
7.5
10
10
10
10
10
10
10
10
5.0
DMF
DMF
DMF
DMF
10
10
10
20
20
20
20
20
20
10
40
20
120
140
160
100
120
120
80
100
120
100
100
100
—
1.2
1.6
5.2
5.4
1.8
2.6
8.4
6.4
5.0
4.0
0.6
DMF
DMSO
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
10
11
12
By interpretation of the results in Table 2 it becomes clear vis-
ible, that the nitro-substituted precursor 11 gave only poor yield of
1.6% in labelling with [18F]fluoride, even at high temperatures of
160 °C (runs 1–3). The trimethylammonium-substituted precursor
13 showed under standard conditions in DMSO and DMF also low
but in relation to 11 increased yields up to 5.4%. The solvent of
choice for labelling precursor 13 was found to be acetonitrile
whereby a distinct dependence of labelling yield from the reaction
temperature was determined. The optimum reaction temperature
was found to be 100 °C, where a maximum yield of 8.4% was
achieved (runs 7–9). Bisection or doubling of reaction time did
not gave an improvement (runs 10 and 11), as well as decline of
precursor amount to 5 mg gave only 0.6% product (run 12).
From this background was decided to enter an alternative syn-
thetic route covering the synthesis of 4-[18F]fluorobenzaldehyde as
labelling block and performing a Knoevenagel condensation with
oxindole in an additional step. This approach was described earlier
for the synthesis of [18F]fluorophenylalanine by reaction of 4-
[
18F]fluorobenzaldehyde with 2-phenyl-5-oxazolone.26 A prerequi-
site for this pre-labelling method is the availability of 4-[18F]fluoro-
benzaldehyde what is produced in most published synthetic
procedures with DMSO as solvent.22,25,26 However we found by
HPLC investigation the chemical and radiochemical purity of 4-
[
18F]fluorobenzaldehyde not sufficient for further reaction. In con-
trast when heating 4-trimethylammonium-benzaldehyde trifluo-
romethanesulfonate in acetonitrile at 90 °C and separating the
product via solid phase extraction, after elution with ethanol 4-
3.3. Labelling experiments with 4-[18F]fluorobenzaldehyde by
Knoevenagel condensation
[
18F]fluorobenzaldehyde with radiochemical purity >91% was ob-
tained containing only traces of non-radioactive by-products. For
solid phase extraction HLB-plus cartridges (Waters) turned out to
be the most appropriate compared to other commercial RP18
SPE-cartridges. After washing the cartridge with water for removal
of [18F]fluoride and residual precursor the 4-[18F]fluorobenzalde-
hyde was recovered by eluation with 3 mL of ethanol and con-
ducted to a second reacting vessel, containing the oxindole 9 and
We found the labelling yield of 8.4% for 14 starting from precur-
sor 13 in general dissatisfying compared with the radiosynthesis of
4-[18F]fluorobenzaldehyde where starting with a 4-trimethylam-
monium-benzaldehyde trifluoromethanesulfonate precursor label-
ling yields up to 80% could be achieved.22,25

7738
T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
a base. For optimization of the Knoevenagel condensation with 4-
18F]fluorobenzaldehyde a number of bases under distinct reaction
found out that a prolongation of the reaction time to 40 min did
not result in an insignificant improvement of yield (run 9). Finally
we ascertained that the Knoevenagel condensation of oxindole and
4-[18F]fluorobenzaldehyde with diethylamine was the best choice
(48% yield) and was selected it for all further labelling experiments
(run 3).
[
conditions has been tested, the results are presented in Table 3.
In Table 3 the bases used are arranged with increasing pKa and
it seems that basicity has an influence on the progress of reaction.
Best results could be obtained by using amine bases piperidine
(18–29%) or diethylamine (48%). Application of stronger amine
bases (DABCO, N-ethyl-diisopropyl amine) or weaker bases
(ammonium acetate, 2,6-di-tert-butyl pyridine) resulted in lower
or no product formation. Phosphazane as very strong base yielded
26% of desired product 14 along with formation of large amounts
of non-identified side products. Additional there seems to be an
optimum in the amount of oxindole 9 used, so by starting with
5 mg only 8% yield was obtained, utilization of 10 mg raised the
yield to 18%, but further improvement with 15 mg of 9 was not
achieved (runs 4–6).
By increasing the reaction temperature to 100 °C an enhanced
yield of 29% was obtained, on the other hand this resulted in a very
high pressure in the reactor together with uncontrolled release of
the solvent ethanol (run 7). The similar high yield of 28% of 14
could be achieved by utilization of 50 lL of piperidine at 90 °C
but in this case more than 30% of radioactive by-products were ob-
3.4. Biodistribution
The distribution of radioactivity in selected tissues and organs
was studied in normal Wistar rats at different time points (1, 5,
10, 30 and 60 min) after injection of 3-[40-[18F]fluorobenzylid-
ene]indolin-2-one 14. The results are depicted in Table 4 (SUVBiodis
)
and Table 5 (%ID).
The radiotracer was rapidly cleared from the blood with a start-
ing radioactivity concentration (SUVBiodis) of 0.83 0.16 reaching
0.15 0.02 at 60 min p.i. The adipose tissue was the only organ,
which remained the activity (1.15 0.25 (60 min)) after a fast up-
take (0.70 0.02 (1 min)). The organs with the highest starting
activity concentration such as adrenals, lung, kidneys, liver, brain,
heart and pancreas (4.95 0.80, 4.26 0.41, 3.36 0.16,
2.98 0.46, 2.84 0.15, 2.51 0.05, 2.40 0.26) show over
60 min a clearance. The slowest clearance was observed in the
served what turned out as not feasible (run 8). In addition we
Table 3
Yields and conditions of radiosynthesis of 3-[40-[18F]fluorobenzylidene]indolin-2-one 14 via Knoevenagel condensation of 4-[18F]fluorobenzaldehyde with oxindole
Run
Mass of oxindole (mg)
Time (min)
Base
pKa
Temperature (°C)
Yield of 14 (%)
a
1
2
3
4
5
6
7
8
9
10
10
10
5.0
10
15
10
10
10
10
10
10
20
20
20
20
20
20
20
20
40
20
20
20
CH3COONH4
2.3
3.6
10
11
11
11
11
11
11
12
18
40
90
90
90
90
90
90
100
90
90
90
90
90
—
2,6-Di-tbutyl-pyridineb
Diethylamineb
Piperidineb
14
48
8
18
19
29
28e
26
7
Piperidineb
Piperidineb
Piperidineb
Piperidinec
Piperidineb
10
11
12
N-Ethyl-diisopropyl-amineb
DABCOd
3
Phosphazane-baseb
26e
a
b
c
25 mg.
30
50
l
l
L.
L.
d
e
10 mg.
>30% of radiolabelled by-products
Table 4
Radioactivity concentration in rat organs and tissues after single intravenous application of 14 as mean SD as SUVBiodis
Time p.i. (min)
1 min
SD
5 min
SD
10 min
SD
30 min
60 min
Mean
n
Mean
n
Mean
n
Mean
SD
n
Mean
SD
n
Blood
0.83
1.42
0.67
2.84
2.40
0.98
4.95
3.36
0.70
0.82
2.52
4.26
1.10
1.55
1.92
2.98
0.89
0.00
0.16
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0.56
2.29
0.92
1.67
1.66
0.79
5.71
2.64
1.21
0.83
1.33
1.30
0.82
0.99
2.43
3.74
0.64
0.41
0.04
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0.47
1.83
0.74
0.97
1.13
0.52
3.20
2.27
1.26
0.69
0.88
0.85
0.53
0.75
1.91
3.03
0.51
0.00
0.03
0.33
0.12
0.13
0.16
0.01
0.31
0.21
0.05
0.05
0.00
0.07
0.04
0.10
0.33
0.20
0.01
0.00
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0.27
0.98
0.48
0.41
0.55
0.28
1.39
1.69
1.17
0.45
0.44
0.47
0.26
0.40
1.07
1.68
0.28
0.00
0.02
0.32
0.02
0.09
0.11
0.05
0.42
0.20
0.07
0.11
0.09
0.10
0.04
0.14
0.22
0.42
0.02
0.00
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0.15
0.63
0.19
0.18
0.30
0.13
1.11
0.99
1.15
0.19
0.20
0.27
0.13
0.16
0.46
1.30
0.19
0.19
0.02
0.10
0.03
0.04
0.07
0.03
0.39
0.07
0.25
0.03
0.04
0.04
0.02
0.02
0.09
0.11
0.01
0.01
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Brown adipose tissue
Hair and skin
Brain
Pancreas
Spleen
Adrenals
Kidney
Adipose tissue
Muscle
Heart
Lung
Thymus
Thyroid
0.40
0.03
0.15
0.26
0.07
0.80
0.16
0.02
0.34
0.05
0.41
0.07
0.21
0.23
0.46
0.16
0.00
0.39
0.12
0.15
0.12
0.06
1.55
0.28
0.28
0.05
0.10
0.08
0.10
0.09
0.28
0.27
0.07
0.32
Harderian glands
Liver
Femur
Testes

T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
7739
Table 5
Radioactivity amount in rat organs after single intravenous application of 14 as mean SD as %ID
Time p.i. (min)
1 min
SD
5 min
SD
10 min
SD
30 min
SD
60 min
SD
mean
n
mean
n
mean
n
mean
n
mean
n
Brain
Pancreas
Spleen
Adrenals
Kidney
Heart
3.09
0.56
0.31
0.15
3.13
1.01
2.68
0.50
0.09
0.29
17.16
0.44
0.00
8.84
0.71
3.97
0.27
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1.87
0.38
0.23
0.17
2.51
0.54
1.02
0.34
0.08
0.34
18.40
0.35
0.38
13.96
0.94
2.39
0.32
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
1.09
0.28
0.14
0.10
2.28
0.35
0.66
0.23
0.05
0.25
17.84
0.32
0.00
0.00
0.00
6.98
0.16
0.10
0.01
0.00
0.35
0.02
0.00
0.03
0.02
0.01
1.11
0.00
0.29
0.43
0.28
0.58
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0.44
0.11
0.08
0.04
1.45
0.18
0.33
0.08
0.03
0.14
8.95
0.13
0.00
0.00
0.00
11.23
0.06
0.02
0.01
0.00
0.04
0.03
0.02
0.01
0.00
0.02
1.87
0.01
0.03
4.63
0.00
4.39
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0.20
0.07
0.04
0.04
0.91
0.08
0.19
0.05
0.01
0.06
5.98
0.11
0.24
30.16
0.60
43.53
0.05
0.01
0.02
0.02
0.11
0.02
0.02
0.01
0.00
0.02
0.61
0.01
0.02
2.22
0.40
5.45
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0.03
0.00
0.01
0.11
0.17
0.01
0.20
0.01
0.03
2.15
0.08
0.00
0.00
0.00
2.46
0.03
0.04
0.06
0.42
0.04
0.08
0.11
0.02
0.09
1.53
0.08
0.64
2.27
0.69
1.14
Lung
Thymus
Thyroid
Harderian glands
Liver
Femur
Testes
Intestine
Stomach
Urine calculated
adrenals, lung, kidney, liver, brain, heart and pancreas (% activity at
60 min in comparison to 5 min: 95, 46, 37, 35, 30, 27, 26). Organs
with the highest tissue to blood ration at 5 min that extracted the
activity from the blood were adrenals, liver, kidney, Harderian
glands, brown adipose tissue, brain, pancreas, heart and lung
(10.2, 6.7, 4.7, 4.4, 4.1, 3.0, 3.0 tissue/blood at 5 min). This situation
qualitatively did not alter after 60 min, the highest tissue to blood
ratios remained in liver, adipose tissue, adrenals, brown adipose
tissue Harderian glands, pancreas, lung heart (8.9, 7.9, 7.6, 6.8,
4.3, 3.2, 2.1, 1.8, 1.4 tissue/blood at 60 min).
3.5. Stability investigations
The metabolism of 3-[40-[18F]fluorobenzylidene]indolin-2-one
14 was investigated in vitro by treatment in samples of blood, plas-
ma and as well as in vivo by analyzing arterial blood plasma sam-
ples at different time points. The amount of intact component over
time in plasma and in blood samples in vitro as well as in blood
samples in vivo is depicted in Figure 5.
As clear visible at Figure 5 the radiotracer 14 was almost stable
over 60 min in blood and in blood plasma showing 84% and 96% of
original compound, respectively. In contrast, in vivo arterial blood
samples indicated that the tracer with a retention time of about
11 min was metabolized rapidly into two more polar metabolites
with 6 and 9 min retention time, respectively. The biliary elimi-
nated metabolite (tR 5.7 min) was detected in the intestine.
Interestingly in the brain was found also one metabolite to-
gether with the original tracer, what means that both radioactive
compounds have passed the blood brain barrier. In summary
1 min after radiotracer injection 70%, at 3 min 54%, at 5 min 40%,
at 10 min 18% and at 20 min 12% of the intact radiotracer could
be detected; 60 min past injection the original compound was
practically no more present. The time course of the original com-
pound in the plasma could be described by a two-phase elimina-
The elimination of the 3-[40-[18F]fluorobenzylidene]indolin-2-
one 14 reflects in the time course of the 18F-activity amount in
the body (Table 5, Fig. 6). After 5 min were 18.40 1.53%ID and
13.96 2.27%ID in the intestine and liver, but only 2.51 0.42%ID
and 2.39 1.14%ID in the kidneys and urine. However, after
60 min were 43.53 5.45%ID and 30.16 2.22%ID in the urine
and intestine. The liver and kidneys contained at this time
5.98 0.61%ID and 0.91 0.11%ID, respectively. Activity amount
in the carcass was 17.18 2.98%ID at 60 min.
Taken together, the dominating property of 3-[40-[18F]fluoro-
benzylidene]indolin-2-one 14 is the high lipophilicity with a log D
of 3.39 determined by the shake flask technique in n-octanol/phos-
phate buffer at pH 7.4 causing that directly after injection, when
the most activity in the blood was existing as the original com-
pound (see Section 3.5) the radiotracer was fast distributed into
the adipose tissue increasing from 1 to 5 min and maintained at
this level over the observation time. Also a high uptake was ob-
served in well perfused tissues like adrenals, liver, lung and brain.
However, the decrease of original compound in the blood plasma
shifted the equilibrium, so that the non-bound activity in the tissue
was released and eliminated.
100
90
80
70
The high distribution coefficient of 3.39 did not prevent the
brain uptake through the blood brain barrier even if it is known
that an optimal log P value for BBB penetration should be 1.5–
2.7.27 This demonstrates that 14 is not a substrate of the multidrug
resistant system. All these effects are evidences for a high extrac-
tion of the radiotracer.
The heart uptake is in ranges achieved by PET-tracers for imag-
ing myocardial transporters like 4-[18F]fluorometaraminol,28 an
amino-alcohol bearing an 4-fluoro-phenyl-substitutent too, but
in disadvantage to 4[18F]FMR] the radiotracer 14 displays a very
high uptake of the lungs. Another question is, as a result of the
low metabolic stability of 14 as discussed in Section 3.5., whether
the uptake of radioactivity in brain and heart derives from the ori-
ginal radiotracer and not from one of its metabolites.
60
plasma in vitro
50
blood in vitro
40
blood in vivo
30
20
10
0
0
10
20
30
40
50
60
time in min
Figure 5. Metabolic stability of radiotracer 14 in vitro and in vivo expressed as % of
total activity.

7740
T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
Figure 6. 18F-activity distributions in the excreting and eliminating organs of Wistar rats (mean SD, data from Table 4).
Figure 7. Transversal (A), coronal (B) and saggital (C) sections from a PET study with 14 in a Wistar rat after 1, 5 and 45 min p.i.
tion with half lives of 8 and 267 s. These somehow surprising find-
ings pushed us to gather more information on the structure of the
metabolites and we compared the retention time with that of
known reference substances. It was found that the retention time
of one radio-metabolite was high conform with fluorobenzene, so
it is likely that the radiotracer 14 is transformed in vivo to
18F]fluorobenzene al least. The other metabolite at 6 min could
be represent a derivative of 14 which was hydroxylated at the 5
position of the benzene ring like it was shown for the lead com-
pound SU5416.29
[

T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
7741
3.6. Small animal PET studies
the radionuclide into the oxindole core as well into the pyrrole core
of SU5416 was unsuccessful, we developed 3-[40-fluorobenzylid-
ene]indolin-2-one 10 (SU5205) as surrogate for SU5416 by substi-
tution of the dimethyl-pyrrol moiety by a 4-fluoro-phenyl ring. The
radiolabelled analogue 3-[40-[18F]fluorobenzylidene]indolin-2-one
14 could be prepared in a two-step procedure by Knoevenagel con-
densation of oxindole and 4-[18F]fluorobenzaldehyde in a remote
controlled synthesizer unit, in the course of this the reaction con-
ditions and the influence of different bases has been optimized.
The radiopharmacological investigation of the radiotracer 14 re-
sulted in the findings that the compound, although stable in vitro is
metabolized in vivo very rapidly, what means that 20 min past
injection, only 12% of the original tracer could be detected in arte-
rial blood samples. Biodistribution and PET studies showed that
the radiotracer enters the brain in spite of the high lipophilicity
and the heart, what is displayed in a brain uptake of 2.84% and
heart uptake of 2.52% of standardized activity concentration,
respectively.
Small animal PET imaging with 3-[40-[18F]fluorobenzylid-
ene]indolin-2-one 14 was performed with Wistar rats, HT-29 and
FaDu tumour bearing mice after single intravenous application.
The results confirm the findings obtained in the biodistribution
profile, showing radioactivity in brain, lung, heart and liver
(Fig. 7). After 1 h radioactivity was observed in the olfactory bulb,
the Harderian glands and the brown adipose tissue. The 18F-activ-
ity distribution in the mice was comparable (Fig. 8). However, in
the tumour almost no radioactivity uptake was detected at
45 min p.i. One reason for that behaviour is certainly the resulting
low area under the time–activity curve of the blood as well as the
low metabolic in vivo stability of the radiotracer in mice. Figure 9
displays exemplarily the time–activity curves of 14 of the brain,
blood, liver, and tumour in FaDu bearing mice, showing that the
activity has been practically cleared from the large organs.
The small animal PET investigations on HT-29 and FaDu tumour
bearing mice did not indicate any uptake of radioactivity in the tu-
mour what is not surprising in respect to the in vivo instability of
the substance. The lack of stability and the only moderate IC50
affinity towards VEGF-RTKs makes compound 14 not a suitable
4. Summary and concluding remarks
The aim of the present work was the labelling of an inhibitor of
tyrosine kinase with the oxindole scaffold of SU5416 with the pos-
itron emitting isotope fluorine-18. Because of the introduction of
Figure 8. Transversal (A), coronal (B) and saggital (C) sections from a PET study with 14 in a FaDu tumour bearing mouse after 1, 5 and 45 min p.i.

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T. Kniess et al. / Bioorg. Med. Chem. 17 (2009) 7732–7742
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The authors wish to thank Stephan Preusche for radioisotope
production and Regina Herrlich and Andrea Suhr for assistance at
the radiopharmacological investigations.
References and notes
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