Synthesis, in silico, in vitro, and in vivo investigation of 5-[ 11C]methoxy-substituted sunitinib, a tyrosine kinase inhibitor of VEGFR-2

Article abstract of DOI:10.1016/j.ejmech.2012.10.020

Sunitinib (SU11248) is a highly potent tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR). Radiolabeled inhibitors of receptor tyrosine kinases (RTKs) might be useful tools for monitoring RTKs levels in tumor tissue giving valuable information for anti-angiogenic therapy. Herein we report the synthesis of 5-methoxy-sunitinib 5 and its ¹¹C-radiolabeled analog [¹¹C]-5. The non-radioactive reference compound 5 was prepared by Knoevenagel condensation of 5-methoxy-2-oxindole with the corresponding substituted 5-formyl-1H-pyrrole. A binding constant (K_d) of 20 nM for 5 was determined by competition binding assay against VEGFR-2. In addition, the binding mode of sunitinib and its 5-methoxy substituted derivative was studied by flexible docking simulations. These studies revealed that the substitution of the fluorine at position 5 of the oxindole scaffold by a methoxy group did not affect the inhibitor orientation, but affected the electrostatic and van der Waals interactions of the ligand with residues near the DFG motif of VEGFR-2. 5-[¹¹C]methoxy-sunitinib ([¹¹C]-5) was synthesized by reaction of the desmethyl precursor with [¹¹C]CH₃I in the presence of DMF and NaOH in 17 ± 3% decay-corrected radiochemical yield at a specific activity of 162-205 GBq/μmol (EOS). In vivo stability studies of [¹¹C]-5 in rat blood showed that more than 70% of the injected compound was in blood stream, 60 min after administration.

Full text of DOI:10.1016/j.ejmech.2012.10.020

European Journal of Medicinal Chemistry 58 (2012) 272e280
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in silico, in vitro, and in vivo investigation of 5-[11C]methoxy-substituted
sunitinib, a tyrosine kinase inhibitor of VEGFR-2
Julio Caballero a, Camila Muñoz a, Jans H. Alzate-Morales a, Susana Cunha b, Lurdes Gano b,
,
Ralf Bergmann c, Joerg Steinbach c, Torsten Kniess c
*
a Centro de Bioinformática y Simulación Molecular, Universidad de Talca, 2 Norte 685, Casilla 721, Talca, Chile
b Unidade de Ciências Químicas e Radiofarmacêuticas, IST/ITN, Instituto Superior Técnico, Universidade Técnica de Lisboa, Estrada Nacional 10, 2686-953 Sacavém, Portugal
c Institute of Radiopharmacy, Helmholtz-Zentrum Dresden-Rossendorf e.V., POB 510119, D-01314 Dresden, Germany
a r t i c l e i n f o
a b s t r a c t
SunitinibÒ (SU11248) is a highly potent tyrosine kinase inhibitor targeting vascular endothelial growth
factor receptor (VEGFR). Radiolabeled inhibitors of receptor tyrosine kinases (RTKs) might be useful tools
for monitoring RTKs levels in tumor tissue giving valuable information for anti-angiogenic therapy.
Herein we report the synthesis of 5-methoxy-sunitinib 5 and its 11C-radiolabeled analog [11C]-5. The
non-radioactive reference compound 5 was prepared by Knoevenagel condensation of 5-methoxy-2-
oxindole with the corresponding substituted 5-formyl-1H-pyrrole. A binding constant (Kd) of 20 nM
for 5 was determined by competition binding assay against VEGFR-2. In addition, the binding mode of
sunitinibÒ and its 5-methoxy substituted derivative was studied by flexible docking simulations. These
studies revealed that the substitution of the fluorine at position 5 of the oxindole scaffold by a methoxy
group did not affect the inhibitor orientation, but affected the electrostatic and van der Waals interac-
tions of the ligand with residues near the DFG motif of VEGFR-2. 5-[11C]methoxy-sunitinib ([11C]-5) was
synthesized by reaction of the desmethyl precursor with [11C]CH3I in the presence of DMF and NaOH in
Article history:
Received 12 March 2012
Received in revised form
5 October 2012
Accepted 11 October 2012
Available online 23 October 2012
Keywords:
SunitinibÒ
VEGFR
Docking
Molecular dynamics
MM-GBSA
Carbon-11
Radiolabeling
17 ꢀ 3% decay-corrected radiochemical yield at a specific activity of 162e205 GBq/
mmol (EOS). In vivo
stability studies of [11C]-5 in rat blood showed that more than 70% of the injected compound was in
blood stream, 60 min after administration.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
correlate with increased metastatic invasion [3]. Among the VEGF
receptor kinases types VEGFR-2 is believed to be the key transducer
Angiogenesis is a well regulated physiological process involving
the growth of new blood vessels from pre-existing vascular and
capillary networks in vital mechanisms like wound healing.
However it is also a transitional step in the development of tumors
because cancer cells are generally limited to a diameter of 2e3 mm
without additional blood supply, so tumors often promote local
angiogenesis to support their growth and nutrition. A number of
pro- and anti-angiogenic factors shift the micro milieu of the tumor
cell in the favor of vessel development and consequently markers of
increased angiogenesis have been recognized as effective targets
for therapeutics as well as novel means of imaging of cancer [1,2].
The vascular endothelial growth factor (VEGF) is a key regulator
of neovascularization and an elevated level of VEGF is known to
for angiogenic and mitogenic properties [4]. Anti-angiogenic ther-
apies focus on targeted inhibition of overexpressed growth factors
with the aim of suppressing tumor growth. This inhibition has been
attempted by either blocking the extracellular ligand binding
domain of the receptor with antibodies, affibodies or peptides or by
inhibition of the intracellular tyrosine kinase at the adenosine
triphosphate (ATP) binding site with small molecule inhibitors [5].
Such targeted tumor therapies are accompanied with a more
sensitive need for dose optimization and monitoring of the thera-
peutic response. Direct non-invasive molecular imaging of tumor
vascularization and angiogenic processes in vivo would facilitate
the selection of patients and help to evaluate the efficacy of anti-
angiogenic therapies, due to the fact that anti-angiogenic agents
are generally cytostatic and do necessarily cause large reductions in
tumor volume on a short time scale even when they are effective
[6]. Radionuclide-based imaging technologies, like positron emis-
sion tomography (PET) and single photon emission computed
* Corresponding author.
E-mail address: t.kniess@hzdr.de (T. Kniess).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.020

J. Caballero et al. / European Journal of Medicinal Chemistry 58 (2012) 272e280
273
tomography (SPECT) are progressively affecting the clinical diag-
nosis and treatment of cancer. These techniques are based on the
concept that diagnostic radiotracers will accumulate in specific
tissues altered in a diseased situation due to interaction at molec-
ular level [7]. Direct non-invasive molecular imaging and reliable
quantitative method to determine in vivo the levels of VEGFR
expression would help to evaluate the efficacy of anti-angiogenic
therapies and facilitate the selection of patients for a customized
VEGFR-targeted cancer treatment. Consequently radiolabeled
peptide markers (RGD), radiolabeled antibodies (bevacizumab) and
radiolabeled small molecule receptor tyrosine kinase inhibitors
have been developed as radioactive probes for imaging anti-
angiogenic therapies; for detailed information on this field
a number of excellent reviews may be considered [5,7e9].
Radiotracers targeting VEGFR tyrosine kinase would provide
useful information about location and density of these receptors by
molecular imaging and would assist in the therapyof VEGFR positive
tumors. Consequently known VEGFR inhibitors like vandetanibÒ,
Ki8751, sorafenibÒ and brivanibÒ as well as diaryl-substituted mal-
eimide derivatives have been selected for radiolabeling preferably
with the positron emitters carbon-11 and fluorine-18 [10e16]. The
multi-targeted kinase inhibitor sunitinibÒ inhibits VEGFR-2 in the
low nanomolar range and was approved as anti-angiogenic drug for
treatment of renal cell carcinoma and gastrointestinal stromal drugs
in 2006 (Fig. 1). The radiosynthesis of the fluorine-18 labeled
sunitinibÒ has been described; however no radiopharmacological
data were published up to now [17]. Our laboratory has contributed
to this scientific area by designing and synthesizing an 18F-labeled
analog of SU5202, a VEGFR inhibitor in the micromolar range [18,19]
and recently with a 125I-labeled sunitinib derivative [20].
as of the corresponding desmethyl precursor 4 for 11C-radiolabeling
is outlined at Scheme 1. The key starting compound 5-hydroxy-
oxindole 1 is not commercially available and its synthesis was
hitherto described in the literature via a laborious 5-step synthesis
starting from p-anisidine [21]. To circumvent this drawback
hypervalent iodine reagents have been used for the oxidation of
phenols and anilines and recently the convenient synthesis of
5-hydroxy-oxindole
1 in a single step from oxindole with
phenyliodine(III)-bis(trifluoroacetate) PIFA was reported [22].
Following this procedure the desired 5-hydroxy-oxindole 1 was
obtained in 70% chemical yield in high purity after purification by
column chromatography. Selective O-methylation of 1 was ach-
ieved by using dimethyl sulfate in the presence of potassium
hydroxide as described by Laksmaiah et al. [23] to afford
5-methoxy-oxindole 2. However during this reaction several by-
products were formed affording 2 in a chemical yield that did not
exceed 35%, after column chromatography purification. Finally,
each oxindole derivative 1 and 2 reacted with N-[2-(diethylamino)
ethyl]-2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxamide 3 [24] via
Knoevenagel condensation, in the presence of ethanol and piperi-
dine. In this way the 5-hydroxy-substituted sunitinib 4 and its
5-methoxy substituted derivative
5 were obtained as pure
compounds and no further purification was required.
3. Docking simulations
Rigid docking simulations were performed by using Glide [25].
Characteristics of Glide docking method have been described in
previous reports [26]. The previously reported model of VEGFR-2
derived from the X-ray crystal structure (accession code in
Protein Data Bank (PDB): 3ewh) with the activation loop modeled
was used as the protein receptor in docking experiments [19]. The
structures of sunitinibÒ and compound 5 were sketched by using
Maestro software [27]. The docking poses for each ligand were
analyzed by examining their relative total energy score. The more
energetically favorable conformation was selected as the best pose.
Molecular dynamics (MD) simulations of sunitinibÒ and
compound 5 inside VEGFR-2 active site were studied using the
OPLS-AA force field in explicit solvent with the SPC water model
(OPLS-AA/SPC) [28], within the Desmond program [29] for MD
simulations. The initial coordinates for the MD calculations were
taken from the docking experiments. The SPC water molecules
were then added (the dimensions of each orthorhombic water box
Owed to the chemical structure of sunitinibÒ that gives less
possibility for further substitution with [18F]fluoride we aimed in
our ongoing studies to design a methoxy-substituted derivative
near to the sunitinibÒ lead structure and to develop a carbon-11
labeled radiotracer for imaging VEGFR-2.
The present paper describes the synthesis of 5-methoxy-
substituted sunitinibÒ (Fig. 1), its ability to inhibit VEGFR-2
assessment both in vitro and in vivo in cell models as well as the
radiosynthesis of the 11C-radiolabeled analog and its preliminary
in vivo stability evaluation. Studies on the binding modes of both
ligands inside the active site of VEGFR-2 were also carried out by
docking simulations and are reported herein.
ꢀ
ꢀ
ꢀ
2. Chemistry
were 84 A ꢁ 79 A ꢁ 65 A approximately, which ensured the whole
surfaces of the complexes to be covered by solvent model) and the
systems were neutralized by adding chloride counter ions to
balance the net charges of the systems. Before equilibration and
The synthetic strategy for preparation of the non-radioactive
reference compound, 5-methoxy-substituted sunitinib 5 as well
N
O
N
O
N
N
H
H
N
N
H
F
O
H
H3C
O
N
O
N
H
H
sunitinib
5-methoxy-sunitinib
Fig. 1. Structures of sunitinibÒ and 5-methoxy-sunitinib.

274
J. Caballero et al. / European Journal of Medicinal Chemistry 58 (2012) 272e280
N
O
N
O
N
H
H
N
H
1. PIFA
2.Me2SO4
N
H
O
N
H
O
O
3
R
R
O
O
O
N
H
N
H
N
H
1: R=H
2: R=CH3
4: R=H
5: R=CH3
Scheme 1. Synthesis of labeling precursor 4 and reference compound 5.
long production MD simulations, the systems were minimized and
pre-equilibrated using the default relaxation routine implemented
in Desmond. For this, the program ran six steps composed of
minimizations and short (12 and 24 ps) MD simulations to relax the
model system before performing the final long simulations. After
that, a first 5 ns long equilibration MD simulation was performed on
each complex system. To check whether the equilibrated MD
trajectory was stable, RMSD values of side-chain heavy atoms with
respect to the initial coordinates were tested. Then, a 20 ns long
production MD simulation was performed. The OPLS-2005 [28]
force field was used, along with module MacroModel [30] to
provide and check the necessary force field parameters for the
ligands. When MacroModel performs an energy calculation, the
program checks the quality of each parameter in use. All, bond,
angle, torsional and improper checked parameters were listed as
high and medium quality force field parameters for all ligands
studied. During MD simulations, the equations of motion were
integrated with a 2-fs time step in the NVT ensemble. The SHAKE
algorithm was applied to all hydrogen atoms; the VDW cutoff was
the curve image for 5-methoxy-substituted sunitinib 5, the amount
of kinase measured by qPCR (y-axis) is plotted against the
concentration of 5 in nM in log 10 scale (x-axis). In a duplicated
experimental determination a Kd value of 20 nM for 5-methoxy-
substituted sunitinib 5 for the inhibition of VEGFR-2 was found.
This Kd value is adequately low to justify classification of 5 as an
inhibitor of VEGFR-2, as for the lead compound Sunitinib a Ki value
of 9 nM is published [33].
4.2. Inhibition of cell proliferation
The potency of 5 to inhibit cell proliferation was assessed by the
colorimetric MTT assay in two VEGFR expressing cell lines, primary
endothelial HAEC and cancer HT29. This assay measures the
amount of tetrazolium dye (MTT) reduced by a mitochondrial
dehydrogenase located in the inner mitochondrial membrane of
viable cells that is involved in the oxidative phosphorylation. Thus,
cell viability is proportional to the reduction of MTT. The prolifer-
ation inhibitory activity of the compound was evaluated by deter-
mination of the IC50, the concentration needed to inhibit cell
proliferation by 50%, determined through doseeresponse curves
achieved from percentage of cell proliferation plotted against the
corresponding compound concentration. IC50 values are presented
in Table 1, in comparison with the values found for sunitinibÒ in the
same experimental conditions.
Analysis of these results shows the compounds potency to
inhibit the tyrosine kinase activity as well as its ability to cross the
cell membrane. As expected, the results indicated that 5-methoxy-
sunitinib is able to cross the cell membrane and to inhibit the cell
propagation. Nevertheless the IC50 values analysis in comparison
with the parent compound shows that its potency as inhibitor is
lower than that of sunitinibÒ. On the other hand the ability to
inhibit the cell proliferation also depends on the cell line. In general
ꢀ
set to 9 A. The temperature was maintained at 300 K, employing the
NoséeHoover thermostat method with a relaxation time of 1 ps.
Long-range electrostatic forces were taken into account by means
of the particle-mesh Ewald (PME) approach. Data were collected
every 1 ps during the MD runs. Visualization of proteineligand
complexes and MD trajectory analysis were carried out with the
VMD software package [31].
4. Results and discussion
4.1. Inhibition of tyrosine kinase activity
Due to the lack of information concerning the inhibitory prop-
erties of the new synthesized 5-methoxy-substituted sunitinib 5
the compound was submitted to a comprehensive test system for
screening compounds against large number of human kinases,
KINOMEscanÔ. This system is based on a competition binding
assay that quantitatively measures the ability of a compound to
compete with an immobilized, active-site directed ligand. The assay
is performed by combining three components: DNA-tagged kinase;
immobilized ligand and the test compound. The ability of the test
compound to compete with the immobilized ligand is measured via
quantitative PCR of the DNA tag [32].
For testing 5-methoxy-substituted sunitinib 5 against VEGFR-2
an 11-point 3-fold serial dilution of 5 was prepared in 100%
DMSO at 100 ꢁ final test concentration and subsequently diluted to
1ꢁ in the assay (final DMSO concentration ¼ 2.5%). The binding
constant (Kd) was calculated with a standard doseeresponse curve
using the Hill equation, curves were fitted using a non-linear least
square fit with the LevenbergeMarquardt algorithm. Fig. 2 displays
Fig. 2. Curve of inhibition of 5-methoxy-sunitinib 5 against VEGFR-2.

J. Caballero et al. / European Journal of Medicinal Chemistry 58 (2012) 272e280
275
Table 1
binding site and was almost planar with respect to the oxindole
scaffold, while the {[2-(diethylamino)ethyl]amino}carbonyl group
was exposed to the solvent, and had a high mobility for both
systems.
Inhibition of cell proliferation.
Compound
Inhibition of cell proliferation IC50
HAEC
(mM)
HT29
We estimated the binding-free energy (DGcalc_bind) of each
5
54.8 ꢀ 0.06
0.10 ꢀ 0.07
7.88 ꢀ 0.06
0.33 ꢀ 0.02
ligand using Prime molecular mechanics-generalized Born surface
area (MM-GBSA) method [27,35]. The calculations were performed
on each complex system using twenty snapshots from the MD
simulations. The following equation (1) was used:
Sunitinib
these findings, in both cell lines, are in agreement with the VEGFR-2
competition binding studies.
D
Gcalc bind
¼
D
EMM
þ
D
Gsolv ꢂ T
D
S
(1)
4.3. Docking results
In (1)
DEMM is the change of the gas phase MM energy upon
binding, and includes Einternal (bond, angle, and dihedral energies),
Eelect (electrostatic),and Evdw (vanderWaals)energies. Gsolv isthe
change of the solvation free energy upon binding, and includes the
electrostatic solvation free energy GsolvGB (polar contribution
calculated using generalized Born model), and the non-electrostatic
solvation component GsolvSA (nonpolar contribution estimated by
solvent accessible surface area). Finally, T S is the change of the
conformational entropy upon binding; this termwas calculated using
normal-mode analysis RRHO contained in MacroModel module [30].
The averaged energy values obtained from MM-GBSA calculations are
reported in Table 2. The experimental binding-free energy values
D
Our docking methodology consists of two steps: first, the
complexes were obtained by rigid docking, and then the movement
of the complexes was studied by MD simulations. The orientations
obtained by docking experiments were compared with the X-ray
crystallographic structure of the complex between KIT and
sunitinibÒ (accession code in PDB: 3g0e) [34]. Active sites of KITand
VEGFR-2 are very similar; in this sense, we expected that oxindole
scaffold should be oriented inside VEGFR-2’s active site in a similar
manner with respect to the orientation in KIT. Therefore, the
orientations obtained by docking experiments carried out inside
VEGFR-2’s active site were compared with the X-ray crystallo-
graphic structure of complex between KIT and the oxindole scaffold
of sunitinibÒ. The values of the root-mean square deviations
(RMSDs) for the docked structures of sunitinibÒ and its 5-methoxy-
derivative 5 with respect to the X-ray crystal inhibitor structure
(considering the 2,4-dimethyl-5-[(Z)-(2-oxo-1,2-dihydro-3H-indol-
3-ylidene)methyl]-1H-pyrrole-3-carboxamide moiety in KIT) were
D
D
D
D
D
D
(
D
Gexp_bind) derived from Ki values are ꢂ10.964 and ꢂ10.491 kcal/mol
for the complexes of sunitinibÒ and 5-methoxy-sunitinib 5 respec-
tively. Thedifferencebetweenthesevaluesis0.473kcal/molonly. This
is a small difference, and small differences are hard to calculate
precisely using computational methods. The calculated binding-free
energy value (DGcalc_bind) showed that both complexes have similar
binding affinities (the difference in the DGcalc_bind value between both
inhibitors was 0.346 kcal/mol).
ꢀ
0.288 and 0.879 A, respectively. According to this, we can conclude
that the docked structures showed the same orientation for
inhibitors in VEGFR-2 with respect to the known orientation of the
oxindole scaffold in KIT. Furthermore, the change of the fluoro- by
the methoxy-substituent at 5-position of the oxindole scaffold does
not affect the orientation of the inhibitor. Thus, compound 5 keeps
the interactions of a potent VEGFR-2 inhibitor.
According to the energy components of the binding free ener-
gies (Table 2), the major favorable contributors to ligand binding
are VDW and electrostatic terms, whereas polar solvation (
and entropy terms oppose binding. Nonpolar solvation terms
GsolvSA) contribute slightly unfavorably for the complex. If we
examine the contributions to each binding energy, the term Eelect
suggests a difference in the binding affinity. The better binding of
sunitinibÒ gains over 4.697 kcal/mol of
Eelect value compared with
5-methoxy-sunitinib 5, which influences the more favorable
binding-free energy value for sunitinibÒ. However, the term
Evdw
compensates for this difference in the binding affinity, since
DGsolvGB)
(D
D
Both inhibitors (sunitinibÒ and 5-methoxy-sunitinib 5) adopted
the same binding mode; this is not surprising because both
compounds contain the same scaffold and similar substituents. In
both compounds: i) the oxindole scaffold is surrounded by residues
Phe1047, Val848, Ala866, Lys868, Val899, Glu917, Phe918, Cys919,
and Leu840, and it forms hydrogen bond (HB) interactions between
the NH of the oxindole and the backbone carbonyl group of Glu917,
and between the carbonyl of the oxindole and the backbone NH of
Cys919, ii) 3,5-dimethyl-1H-pyrrol-2-yl group is at the entrance of
the pocket and is surrounded by residues Leu840, Phe1047, Phe918,
Gly922, and Asn923, iii) the group {[2-(diethylamino)ethyl]amino}
carbonyl is exposed to the solvent media, and iv) the groups at
position 5 of oxindole scaffold (F or OCH3), which are responsible of
the different activities between both inhibitors, are surrounded by
residues Lys868, Cys1045, Asp1046, and Phe1047 (DFG motif).
We also used explicit-water MD calculations to analyze the
movement of the systems and the effect of the solvent in the
formation of the complexes. Throughout the MD simulations, the
studied compounds were in the expected orientations. In both
simulations, the NH of the oxindole group established a stable HB
interaction with the backbone carbonyl group of Glu917, and the
carbonyl group of the oxindole scaffold established a stable HB
interaction with the backbone NH of Cys919 (Fig. 3). An intra-
molecular HB formed between the oxygen of the oxindole and the
NH of the 3,5-dimethyl-1H-pyrrol-2-yl group was also stable
during the MD simulations. The 3,5-dimethyl-1H-pyrrol-2-yl group
did not form HBs with the residues at the entrance of the VEGFR-2
D
D
5-methoxy-sunitinib 5 has a more favorable value of
DEvdw
(4.024 kcal/mol) compared with sunitinibÒ.
This suggests that sunitinibÒ establishes better electrostatic
interactions and 5-methoxy-sunitinib 5 established better VDW
interactions with VEGFR-2 residues. We analyzed the MD trajec-
tories to explain this paradigm. We focused on the residues located
in the surroundings of the groups at position 5 of oxindole scaffold
(F or OCH3), which are responsible of the different activities
between both inhibitors. We identified that several water mole-
cules occupy a position between the fluoro-group of sunitinibÒ and
the side chain of Asp1046 (DFG motif) during the MD simulation of
the complex VEGFR-2/sunitinibÒ (Fig. 3A). The presence of these
water molecules suggests that a water-mediated HB bridge con-
necting the fluorine of sunitinibÒ with the oxygens of the carbox-
ylate of Asp1046 can be formed. The HB acceptor capability of
halogens has been recognized in recent literature; however, current
molecular mechanics (MM) simulations, which rely primarily upon
empirical force fields that in general do not explicitly account for
electronic polarization, appear to be not appropriate for describing
the involvement of halogens in HBs [36,37]. In this sense, the
presence of halogen-mediated HBs cannot be confirmed using MM
methods. On the other hand, we identified that several water

276
J. Caballero et al. / European Journal of Medicinal Chemistry 58 (2012) 272e280
Fig. 3. Proposed docked structures of VEGFR-2/inhibitor complexes after rigid docking and MD simulation: (A) VEGFR-2/sunitinibÒ, (B) VEGFR-2/compound 5; water molecules in
the surroundings of the groups at position 5 of oxindole scaffold (F or OCH3) are represented with a space-filling representation.
molecules occupy a position between the NHþ3 side-chain group of
Lys868 and the side-chain of Asp1046 during the MD simulation of
the complex VEGFR-2/compound 5 (Fig. 3B). These water molecules
were located close to the methyl group of the OCH3 at position 5 of
oxindole scaffold of 5-methoxy-sunitinib 5, which could lead to
unfavorable interactions. This analysis suggests that the fluoro-
group has more favorable electrostatic interactions with the polar
groups of the residues of VEGFR-2, which can explain the more
making its VDW interactions with the above-mentioned residues
much more compact. This fact explains the more favorable value of
the DEvdw value of 5-methoxy-sunitinib 5.
4.4. Radiochemistry
The radiosynthesis of 5-[11C]methoxy-sunitinib [11C]-5 was
performed in
a remotely controlled TracerLabFxC gas phase
favorable value of the
D
Eelect value of sunitinibÒ. During the MD
synthesizer (GE) via O-methylation reaction of the corresponding
desmethyl precursor 4 with [11C]CH3I as the methylation reagent
(Scheme 2). The synthesis started from [11C]CH4 produced in the
cyclotron by irradiation of nitrogen containing 10% hydrogen gas in
an aluminum target as described by Buckley et al. [38]. The [11C]CH4
dynamics, the methoxy-group at position 5 of oxindole scaffold of
5-methoxy-sunitinib 5 established favorable VDW interactions
with the hydrophobic residues Ala1050, Val848, and Cys1045
(Fig. 3B). The methoxy-group is bulkier than the fluoro-group,
Table 2
Calculated binding free energies for VEGFR2eligand complexes using MM-GBSA for the snapshots of MD simulations.
Complex
DEinternal
DEelect
DEvdw
D
GsolvGB
D
GsolvSA
T
DS
DGbind
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
SunitinibeVEGFR2
5eVEGFR2
Difference (absolute values)
1.333 ꢀ 1.153
1.523 ꢀ 0.884
0.190
ꢂ20.845 ꢀ 1.435
ꢂ16.147 ꢀ 2.656
4.697
ꢂ48.620 ꢀ 1.888
ꢂ52.644 ꢀ 2.211
4.024
18.441 ꢀ 3.199
19.859 ꢀ 2.315
1.418
2.636 ꢀ 1.316
2.314 ꢀ 0.806
0.322
ꢂ16.781 ꢀ 0.910
ꢂ17.793 ꢀ 0.897
1.012
ꢂ26.956 ꢀ 3.107
ꢂ27.302 ꢀ 2.437
0.346

J. Caballero et al. / European Journal of Medicinal Chemistry 58 (2012) 272e280
277
N
O
N
O
N
H
N
H
N
H
N
H
O
H131C
HO
[11C]CH3I
O
O
N
H
N
H
[11C]-5
4
Scheme 2. Radiosynthesis of 5-[11C]methoxy-sunitinib ([11C]-5).
was trapped on a carbosphereÒ trap at ꢂ140 ꢃC and after release
converted to [11C]CH3I by a gas phase iodination at 720 ꢃC [39].
Then, the desmethyl precursor 4, dissolved in DMF, reacted with
injection, the radiotracer [11C]-5 was eluted with retention time
about 14 min and three other less lipophilic radioactive metabolites
were detected with Rt ¼ 4 min, 12.5 min and 13.5 min respectively
(Fig. 5). However an amount of more than 70% of the intact radio-
tracer detectable 60 min post injection suggests a relatively high
in vivo metabolic stability of [11C]-5.
[
11C]CH3I in the presence of aqueous NaOH at 80 ꢃC for 3 min: The
reaction mixture was analyzed by radio-HPLC for characterization.
From the chromatogram analysis it was evident that aside from the
desired radiotracer
[ [ a third
11C]-5 and unreacted 11C]CH3I
11C-methylated product was formed in radiochemical yields
ranging between 17 and 41%. Taking into consideration the chem-
ical structure of sunitinibÒ that comprises a pyrrole and an oxindole
moiety both bearing acidic hydrogens, an N-methylation reaction
was expected to occur. A set of experiments were carried out in
which different amounts of base were used in order to successfully
decrease the formation of the side-product. Finally, reaction of
1.5 equiv of NaOH with 1.0 equiv of labeling precursor increased the
formation of the desired radiotracer [11C]-5 in the labeling mixture
to a 40% yield .The labeled raw material was purified by semi-
preparative HPLC using as eluent a mixture of acetonitrile/0.02 M
Na2HPO4 ¼ 65/35 (v/v) to accomplish an efficient separation of
5. Conclusion
The search for radiolabeled probes toward VEGFR expression and
for in vivo monitoring processes involved in tumor proliferation and
angiogenesis, suggested us the development of a radiolabeled
derivative of sunitinibÒ. We focused on radiolabeling with the posi-
tron emitting isotope carbon-11, therefore a 5-methoxy-substituted
sunitinibÒ derivative was synthesized as non-radioactive reference
and a 5-hydroxyl-substituted sunitinibÒ was developed as labeling
precursor. The novel 5-methoxy-sunitinib 5 was identified as
a VEGFR-2inhibitor with a Kd value of 20 nM. The ability of 5 to inhibit
cell proliferation was assessed by the colorimetric MTT assay in two
VEGFRexpressing cell lines and revealed that the compound is able to
cross the cell membrane and inhibit the cell propagation. However in
comparison to sunitinibÒ it turned out that the potency of 5 to act as
inhibitor is lower and is cell line-dependent.
[
11C]-5 from the labeling precursor and the radiolabeled side-
product. Due to the alkaline analytical conditions of the mobile
phase a semi-preparative PRP-1 column was used. The fraction of
[
11C]-5 eluted at Rt ¼ 8e9 min was collected and separated by solid
phase extraction. After reconstitution with E153 solution the
radiotracer was suitable for further animal experiments. Under
these conditions [11C]-5 was obtained in 17 ꢀ 3% decay-corrected
Docking simulations were performed to describe the binding of
the methoxy-substituted compound 5 to VEGFR-2 and to compare
it with sunitinibÒ. Both inhibitors are orientated in a similar
manner; groups at position 5 of the oxindole scaffold could interact
with residues near the DFG motif of VEGFR-2. The presence of the
radiochemical yield at a specific activity of 162e205 GBq/mmol at
the end of synthesis. The identity of the radiotracer was confirmed
by co-injection with the non-radioactive reference compound 5 in
analytical HPLC system. The radiochemical purity of the final
product was higher than 96% and the obtained specific activity
equals in average a total amount of 0.3 mg/mL of non-radioactive 5
in the final product solution.
fluoro-substituent at position
5 of the oxindole scaffold in
sunitinibÒ favors electrostatic interactions with polar VEGFR-2
residues. When the fluorine is substituted by a methoxy-group,
electrostatic interactions become unfavorable, but more compact
VDW interactions are established with hydrophobic VEGFR-2
residues. As a result, both inhibitors are predicted with similar
4.5. In vitro stability studies
The metabolism of 5-[11C]methoxy-sunitinib
[
11C]-5 was
investigated in vivo by analysis of arterial blood plasma samples of
rats taken at different time points after injection of the radiotracer.
The plasma was separated by blood centrifugation, treated with
methanol to precipitate plasmatic proteins and analyzed by radio-
HPLC. The amount of the intact radiotracer [11C]-5, in blood
samples, expressed as % of total radioactivity, over post injection
time for two separate experiments is depicted in Fig. 4. As can be
observed in Fig. 4, [11C]-5 exhibits relatively high stability in blood
up to 60 min since a major radiochemical species with retention
time similar to that of the injected compound (72% and 73%,
respectively) could be detected in blood plasma. On the other hand
analysis of plasma samples taken 10 min after injection demon-
strated that 18e28% of the injected compound has been already
metabolized and no relevant change was found for the following
50 min. In HPLC analyses of plasma samples taken 1 h after
Fig. 4. Metabolic stability of 5-[11C]methoxy-sunitinib [11C]-5 in vivo expressed as % of
total radioactivity (n ¼ 2).

278
J. Caballero et al. / European Journal of Medicinal Chemistry 58 (2012) 272e280
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
0
5
10
15
20
25
30
retention time [min]
14000
12000
10000
8000
6000
4000
2000
0
0
5
10
15
20
25
30
retention time [min]
Fig. 5. Analytical radio HPLC of [11C]-5 from plasma; top: 3 min past injection; bottom: 60 min past injection.
binding-free energy values, which is in agreement with the in vitro
binding assay.
conducted using MERCK silica gel (mesh size 230e400 ASTM).
Thin-layer chromatography (TLC) was performed on MERCK silica
gel F-254 aluminum plates with visualization under UV (254 nm).
The11C-radiolabeledanalog[11C]-5wasobtainedin17ꢀ3%decay-
corrected radiochemical yield at a specific activity of 162e205 GBq/
m
mol at the end of synthesis, radiochemical purity of [11C]-5 excee-
6.1.1. 5-Hydroxy-2-oxindole 1
ded 96%. In vivo stability studies of the radiotracer in rat blood
revealed that the compound is satisfactory stable, hence 60 min after
injection more than 70% of the original compound could be detected.
In summary, these preliminary pharmacological data suggest
that 5-methoxy-sunitinib 5 is an inhibitor of VEGFR-2 albeit
showing a slightly lower potency as the lead compound and the
corresponding radiotracer [11C]-5 has provided evidence of meta-
bolic in vivo stability. Thus, these results indicate that this
compound can be a promising imaging agent of VEGFR-2 tyrosine
kinase expressing cells and further investigations to evaluate its
potential as radiotracer for angiogenesis and carcinogenesis are
worthwhile.
Compound 1 was prepared as described in Itoh et al. [22]:
799 mg (6.0 mmol) of oxindole were dissolved at room tempera-
ture in 40 mL of waterefree dichloromethane 3.1 g (7.2 mmol) of
phenyliodine(III)-bis(trifluoroacetate) PIFA was added in portions,
followed by 4.6 mL of trifluoroacetic acid. The mixture was stirred
at RT overnight and the dichloromethane was evaporated. 60 mL of
5% sodium bicarbonate solution and 50 mL of ethyl acetate were
added to the residue and the organic layer was separated by
a separatory funnel. The aqueous layer was extracted with ethyl
acetate (5 ꢁ 50 mL) the combined organic layer was dried with
Na2SO4, filtered and evaporated under reduced pressure. The crude
product was purified by column chromatography (silica gel,
dichloromethane/methanol ¼ 9:1 (v/v)) and afforded 627 mg (70%)
of 1 as light brown crystals. Mp: 266e268 ꢃC (lit: 260e263 ꢃC), 1H
6. Experimental
NMR (
d
, ppm, DMSO-d6): 3.36 (s, 2H, H-3); 6.55 (d, 1H, 2J ¼ 8.6 Hz,
H-6); 6.60 (d, 1H, 2J ¼ 8.7 Hz, H-7); 6.66 (s, 1-H, H-4); 8.93 (s, 1H,
OH); 10.47 (s, 1H, NH); ESI-MS (ES-): m/z ¼ 148 [M ꢂ H].
6.1. Chemistry
All commercial reagents and solvents were used without further
purification unless otherwise specified. Melting points were
determined with an apparatus GalenÔIII (Cambridge Instruments)
and are uncorrected. Nuclear magnetic resonance spectra were
recorded on a Unity 400 MHz spectrometer (Varian). 1H NMR
chemical shifts were given in ppm and were referenced with the
residual solvent resonances relative to tetramethylsilane (TMS).
Mass spectra were obtained with a Quattro/LC mass spectrometer
(Micromass) by electrospray ionization. Flash chromatography was
6.1.2. 5-Methoxy-2-oxindole 2
The compoundwas prepared as described at Laksmaiah et al. [23]:
179 mg (1.2 mmol) of 1 was dissolved in 1.3 mL of 1.0 M KOH solution
and cooled to 0 ꢃC. Dimethyl sulfate (124
mL,1.3 mmol) was added and
stirring was continued for 2 h, after dilution with 15 mL water the
mixture was extracted with ethyl acetate (3 ꢁ 15 mL). The combined
organic layer was dried with Na2SO4, filtered and evaporated under
reduced pressure. After column chromatographic purification (silica

J. Caballero et al. / European Journal of Medicinal Chemistry 58 (2012) 272e280
279
gel, dichloromethane/methanol ¼ 95:5 v/v) 69 mg (35%) 2 was ob-
tained ascrystals. Mp: 144e148 ꢃC (lit:148e151 ꢃC), ESI-MS(ESþ):m/
z ¼ 164 [M þ H].
for 3 h in the presence of 0.5 mg mLꢂ1 MTT (3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide, Sigma) in PBS (Gibco, Invi-
trogen, UK) at 37 ꢃC. The MTT solution was removed and 200
mL per
well of DMSO was added. After thorough mixing, absorbance of the
wells was read in an ELISA reader at test and reference wavelengths
of 570 nm. The mean of the optical density of different replicates of
the same sample and the percentage of each value was calculated
(mean of the OD of various replicates/OD of the control). The
percentage of the optical density against compound concentration
was plotted on a semilog chart and the IC50 from the doseeresponse
curve was determined. Three cell proliferation assays were per-
formed for each of the three compounds.
6.1.3. 5-[5-Hydroxy-2-oxo-1,2-dihydro-indol-(3-Z)-ylidenemethyl]-
2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl)-
amide 4
Ina25mLflask210mg(1.4mmol)1and445mg(1.68mmol)N-[2-
(diethylamino)ethyl]-2,4-dimethyl-5-formyl-1H-pyrrole-3-carbox-
amide 3 [24]were dissolved in 10 mL of ethyl alcohol and five drops of
piperidine were added. After 4 h of refluxing 4 was formed as a red
precipitate that after cooling was filtered of and washed with small
amounts of ethyl alcohol and petrol ether. The collected red crystals
were dried under vacuum and yielded 340 mg (61%) of 4. Mp: 262e
6.2.2. Metabolite analysis
266 ꢃC, 1H NMR (
d
, ppm, DMSO-d6): 0.92 (t, 6H, 2 ꢁ CH3), 2.34 (s,
Male Wistar-Unilever rats (n ¼ 2; body weight 150 ꢀ 12 g)
were anesthetized with desflurane (9e10% v/v, 30% oxygen/air).
The threshold value for breathing frequency was 65 breaths/min.
Animals were put in supine position and placed on a heating pad
to maintain body temperature. The spontaneously breathing rats
were heparinized with 100 units/kg heparin (Heparin-Natrium
25.000-ratiopharmÒ, ratiopharm GmbH, Germany) by subcuta-
neous injection to prevent blood clotting on intravascular cathe-
ters. After local anesthesia with lignocain (1%; XylocitinÒ loc, mibe,
Jena, Germany) into the right groin, a catheter (0.8 mm Umbilical
Vessel Catheter, Tyco Healthcare, Tullamore, Ireland) was intro-
duced into the right femoral artery for arterial blood sampling. A
second needle catheter (35 G) was placed into a tail vein and was
used for [11C]-5 radiotracer injection (39 MBq in 0.5 mL of E153/
10% ethanol, infusion 1 mL/min). Arterial blood samples were
taken 1.5, 10, 30 and 60 min after injection. Arterial plasma was
separated by centrifugation (11.000 g ꢁ 3 min) followed by
precipitation of the proteins with methanol (2 volumes to 1
volume plasma) followed by 5 min storage at ꢂ60 ꢃC. The clear
supernatant separated by centrifugation was used for analysis. The
radio-HPLC system (Agilent 1100 series) applied for metabolite
analysis was equipped with UV detection (254 nm) and an
external radiochemical detector (RAMONA, Raytest GmbH,
3H, CH3/pyrrole), 2.38 (s, 3H, CH3/pyrrole), 2.46 (m, 6H, 3 ꢁ CH2), 3.21 (q,
2H, CH2), 6.51 (m,1H, CH), 6.60 (d,1H, CH), 7.12 (d,1H, CH), 7.34 (t,1H,
CONH), 7.44 (s, 1H, CHvinyl), 8,89 (s, 1H, OH), 10.55 (s, 1H, NHoxindole),
13.67 (s,1H, NHpyrrole),13C NMR (
d, ppm, DMSO-d6): 10.45 (CH3),11.83
(CH3), 13.52 (CH3), 36.95 (CH2), 46.42 (CH2), 51.59 (CH2), 105.89,
109.73, 113.26, 115.87, 120.22, 123.01, 125.05, 126.44, 128.58, 131.07,
135.34, 152.39 (12 ꢁ C]C), 164.57 (C]O), 169.33 (C]O), ESI-MS
(ESþ): m/z ¼ 397.43 (M þ H).
6.1.4. 5-[5-Methoxy-2-oxo-1,2-dihydro-indol-(3-Z)-
ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-
diethylamino-ethyl)-amide 5
In a 10 mL flask 94 mg (0.58 mmol) 2 and 171 mg (0.64 mmol) 3
[24] were dissolved in 3.0 mL of ethyl alcohol, four drops of
piperidine were added and the mixture was refluxed for 4 h. The
solution was allowed to cool and stored in a refrigerator overnight.
The orange crystals that have precipitated were filtered and washed
with small amounts of ethyl alcohol and petrol ether and yielded
after drying under vacuum 107 mg (45%) of 5. Mp: 220e222 ꢃC, 1H
NMR (
d
, ppm, DMSO-d6): 0.99 (t, 6H, 2 ꢁ CH3), 2.45 (s, 3H, CH3/
pyrrole), 2.46 (s, 3H, CH3/pyrrole), 2.52 (m, 6H, 3 ꢁ CH2), 3.29 (q, 2H,
CH2), 3.79 (s, 3H, CH3O), 6.71 (m, 1H, CH), 6.78 (d, 1H, CH), 7.42 (t,
1H, CONH), 7.50 (d, 1H, CH), 7.68 (s, 1H, CHvinyl), 10.72 (s, 1H,
Germany). Analysis was performed on
a Zorbax C18 300SB
NHoxindole), 13.76 (s, 1H, NHpyrrole), 13C NMR (
d, ppm, DMSO-d6):
(250 9.4 mm; m) column with an eluent system C
ꢁ
4
m
10.63 (CH3), 11.84 (CH3), 13.23 (CH3), 36.90 (CH2), 46.43 (CH2), 51.56
(CH2), 55.56 (OCH3), 104.44, 109.80, 112.54, 115.60, 120.27, 13.64,
125.62, 126.40, 129.00, 132.19, 135.65, 154.81 (12 ꢁ C]C), 164.52
(C]O), 169.42 (C]O), ESI-MS (ESþ): m/z ¼ 411.42 (M þ H).
(water þ 0.1% TFA) and D (acetonitrile þ 0.1% TFA) in the following
gradient: 5 min 95% C, 10 min to 95% D, 5 min at 95% D and 5 min
to 95%C at a flow rate of 3 mL/min.
6.3. Radiochemistry
6.2. Biology
[
11C]CH4 was produced by the 14N(p, 11C reaction in a CYCLONE
a)
6.2.1. Inhibition of cell proliferation by MTT assay
18/9 cyclotron (IBA) by irradiation of nitrogen gas containing 10%
hydrogen gas in an aluminum target. Radiosynthesis and semi-
preparative purification was performed in an automated nucleo-
philic synthesizer TracerLabFxC (GE) that was modified in terms of
direct trapping of [11C]CH4. Semi-preparative HPLC purifications
6.2.1.1. Cell lines. Cellular studies were carried out in human aortic
endothelial cells (HAECs) and in a VEGFR-expressing cell line
(HT29) from a human colorectal adenocarcinoma. Primary endo-
thelial cells were cultured in Cell Growth Medium MV2 (Promocell)
supplemented with 1% penicillin/streptomycin and routinely
passaged and only cells between passage 4 and 7 were used in the
assays. Tumor cells were grown in McCoy’s 5A medium (Gibco)
supplemented with 10% fetal bovine serum and 1% penicillin/
streptomycin under a humidified 5% CO2 atmosphere at 37 ꢃC. Cells
were subcultured every 2 or 3 days.
were carried out with a PRP1 column (250 ꢁ 10 mm, 5
mm, Ham-
ilton) using an isocratic eluent of acetonitrile/0.02 M Na2HPO4 ¼ 65/
35 (v/v) by an S1122 HPLC-pump (Sykam) with a flow rate of 4 mL/
min. The product was monitored by a K-2001 filter photometer
(Knauer) at 254 nm and by a gamma-detector integrated in the
synthesizer module. Analytical HPLC investigation was performed
with a C18 column (250 ꢁ 4 mm, 5
mm, Nucleodur Isis, Machereye
6.2.1.2. MTT assay. The cells were seeded in a 96-well plate at
a density of 7500 cells per 200 mL per well and incubated for 24 h for
Nagel) using a gradient eluent of acetonitrile (A) and water con-
taining 0.1% TFA (B) by an L2500 pump (MERCK, Hitachi) with a flow
rate of 1.0 mL/min. The gradient was as follows: 0.0 mine7.0 min
(10% Ae95% A); 7.0 mine9.0 min (95% Ae100% A); 9.0 mine
10 min (100% Ae10% A). The products were monitored by an UV
detector L4500 (Merck, Hitachi) at 276 nm and by a gamma scin-
tillation detector GABI (Raytest).
attachment to the wells. The day after seeding, exponentially
growing cells were incubated with various concentrations of the
compounds (5 and sunitinibÒ) (ranging from 1 nM to 100
mM in 4
replicates) for 72 h. Controls consisted of wells without any
compound. The medium was removed and the cells were incubated

280
J. Caballero et al. / European Journal of Medicinal Chemistry 58 (2012) 272e280
6.3.1. 5-[5-[11C]methoxy-2-oxo-1,2-dihydro-indol-(3-Z)-
ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-
diethylamino-ethyl)-amide [11C]-5
[16] O. Ilovich, H. Billauer, S. Dotan, E. Mishani, Labeled 3-aryl-4-indolylmaleimide
derivatives and their potential as angiogenic PET biomarkers, Bioorg. Med.
Chem. 18 (2010) 612e620.
[17] J.Q. Wang, K.D. Miller, G.W. Sledge, Q.H. Zheng, Synthesis of [18F]SU11248,
a new potential PET tracer for imaging cancer tyrosine kinase, Bioorg. Med.
Chem. Lett. 15 (2005) 4380e4384.
Labeling precursor 4 (1.0 mg, 2.5
mmol) dissolved in 250 mL DMF
and 40 L 0.1 M aqueous NaOH was placed in the reacting vessel of
m
[18] T. Kniess, R. Bergmann, M. Kuchar, J. Steinbach, F. Wuest, Synthesis and radio-
pharmacological investigation of 3-[40-[18F]fluorobenzylidene]-indolin-2-one
as possible tyrosine kinase inhibitor, Bioorg. Med. Chem. 17 (2009) 7733e7742.
[19] C. Muñoz, F. Adasme, J.H. Alzate-Morales, A. Vergara-Jaque, T. Kniess,
J. Caballero, Study of differences in the VEGFR-2 inhibitory activities between
semaxanib and SU5205 using 3D-QSAR, docking, and molecular dynamics
simulations, J. Mol. Graph. Model. 32 (2012) 39e48.
[20] M. Kuchar, M.C. Oliveira, L. Gano, I. Santos, T. Kniess, Radioiodinated sunitinib
as a potential radiotracer for imaging angiogenesiseradiosynthesis and first
radiopharmacological evaluation of 5-[125I]iodo-sunitinib, Bioorg. Med. Chem.
Lett. (2012). http://dx.doi.org/10.1016/j.bmcl.2012.02.068.
the synthesizer unit. [11C]CH3I was transferred in a stream of
helium into the reaction vessel at a temperature of ꢂ20 ꢃC. After
completion of the transfer, the reaction vessel was sealed and
heated at 80 ꢃC for 3 min. The reactor was cooled to 40 ꢃC, 1 mL of
acetonitrile was added and the mixture was transferred onto
a semi-preparative PRP 1 column. The product eluting between 8
and 9 min was separated and diluted with 30 mL of water and
passed through an SPE-cartridge (Lichrolut, RP18, 500 mg). The
product was eluted from the cartridge with 0.75 mL of ethanol and
reconstituted with 6.5 mL of E153 solution. In a typical experiment
970 MBq of compound [11C]-5 could be obtained within 24 min
after EOB starting from 13,500 MBq of [11C]CH4 (17% decay cor-
rected radiochemical yield based upon [11C]CH4). The specific
[21] E. Giovannini, P. Portmann, Sur quelques derives de l’oxindole et de l’isatine.
II. Sur les amino-, hydroxy- et méthoxy-dérivés substitués en position 5 et 6,
Helv. Chim. Acta 31 (1948) 1381e1391.
[22] N. Itoh, T. Sakamoto, E. Miyazawa, Y. Kikugawa, Introduction of a hydroxyl
group at the para position and N-iodophenylation of N-arylamides using
phenyliodine(III)bis(trifluoroacetate), J. Org. Chem. 67 (2002) 7424e7428.
[23] G. Lakshmaiah, T. Kawabata, M. Shang, K. Fuji, Total synthesis of (ꢂ)-horsfiline
via asymmetric nitroolefination, J. Org. Chem. 64 (1999) 1699e1704.
[24] L. Sun, C. Liang, S. Shirazian, Y. Zhou, T. Miller, J. Cui, J.Y. Fukuda, J.Y. Chu,
A. Nematalla, X. Wang, H. Chen, A. Sistla, T.C. Luu, F. Tang, J. Wei, C. Tang,
Discovery of 5-[5-fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-
dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide, a novel
tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived
growth factor receptor tyrosine kinase, J. Med. Chem. 46 (2003) 1116e1119.
[25] R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz,
M.P. Repasky, E.H. Knoll, M. Shelley, J.K. Perry, D.E. Shaw, P. Francis, P.S. Shenkin,
Glide: a new approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy, J. Med. Chem. 47 (2004) 1739e1749.
[26] J.H. Alzate-Morales, A. Vergara-Jaque, J. Caballero, Computational study on the
interaction of N1 substituted pyrazole derivatives with B-raf kinase: an
unusual water wire hydrogen-bond network and novel interactions at the
entrance of the active site, J. Chem. Inf. Model. 50 (2010) 1101e1112.
[27] Maestro, Version 9.0, Schrödinger, LLC, New York, NY, 2007.
[28] G.A. Kaminski, R.A. Friesner, J. Tirado-Rives, W.L. Jorgensen, Evaluation and
reparametrization of the OPLS-AA force field for proteins via comparison with
accurate quantum chemical calculations on peptides, J. Phys. Chem. B 105
(2001) 6474e6487.
activity was determined to be 198 GBq/mmol at the end of synthesis.
Acknowledgments
The authors want to thank Lars Ruddigkeit, Department of
Chemistry and Biochemistry at the University of Bern (Switzerland)
for helpful discussions. The authors wish to thank S. Preusche for
radioisotope production and A. Suhr and P. Wecke for expert
technical assistance.
References
[1] J. Folkman, Tumor angiogenesis: therapeutic implications, N. Engl. J. Med. 285
(1971) 1182e1186.
[29] K.J. Bowers, E. Chow, H. Xu, R.O. Dror, M.P. Eastwood, B.A. Gregersen,
J.L. Klepeis, I. Kolossvary, M.A. Moraes, F.D. Sacerdoti, J.K. Salmon, Y. Shan,
D.E. Shaw, in: Proceedings of the 2006 ACM/IEEE Conference on Super-
computing, ACM, Tampa, Florida, 2006, p. 84.
[30] MacroModel, Version 9.5, Schrödinger, LLC, New York, NY, 2007.
[31] W. Humphrey, A. Dalke, K. Schulten, VMD: visual molecular dynamics, J. Mol.
Graph. 14 (1996) 33e38.
[32] M.A. Fabian, W.H. Biggs, D.K. Treiber, C.E. Atteridge, M.D. Azimioara,
M.G. Benedetti, T.A. Carter, P. Cireci, P.T. Edeen, M. Floyd, J.M. Ford, M. Galvin,
J.L. Gerlach, R.M. Grotzfeld, S. Herrgard, D.E. Insko, M.A. Insko, A.G. Lai,
J.M. Lelias, S.A. Metha, Z.V. Milanov, A.M. Velasco, L.M. Wodicka, H.K. Patel,
P.P. Zarrinkar, D.J. Lockhart, A small molecule-kinase interaction map for
clinical kinase inhibitors, Nat. Biotechnol. 23 (2005) 329e336.
[2] W.P. Leenders, B. Küsters, R.M. de Waal, Vessel co-option: how tumors obtain
blood supply in the absence of sprouting angiogenesis, Endothelium 9 (2002)
83e87.
[3] N. Ferrera, Vascular endothelial growth factor: basis science and clinical
progress, Endocr. Rev. 25 (2004) 581e611.
[4] M. Shibuya, L. Claesson-Welsh, Signal transduction by VEGF receptors in
regulation of angiogenesis and lymphagogenesis, Exp. Cell. Res. 312 (2006)
549e560.
[5] J.W. Hicks, H.F. Van Brocklin, A.A. Wilsom, S. Houle, N. Vasdev, Radiolabeled
small molecule protein kinase inhibitors for imaging with PET or SPECT,
Molecules 15 (2010) 8260e8278.
[6] M.H. Michalski, X. Chen, Molecular imaging in cancer treatment, Eur. J. Nucl.
Med. Mol. Imaging 38 (2011) 358e377.
[33] D.B. Mendel, D.A. Laird, X. Xin, S.G. Louie, J.G. Christensen, G. Li, R.E. Schreck,
T.J. Abrams, T.J. Ngai, L.B. Lee, L.J. Murray, J. Carver, E. Chan, K.G. Moss,
J.Ö. Haznedar, J. Sukbuntherng, R.A. Blake, L. Sun, C. Tang, T. Miller, S. Shirazian,
G. McMahon, J.M. Cherrington, In vivo antitumor activity of SU11248, a novel
tyrosine kinase inhibitor targeting vascular endothelial growth factor and
platelet-derived growth factor receptors: determination of a pharmacokinetic/
pharmacodynamics relationship, Clin. Cancer Res. 9 (2003) 327e337.
[34] K.S. Gajiwala, J.C. Wu, J. Christensen, G.D. Deshmukh, W. Diehl, J.P. DiNitto,
J.M. English, M.J. Greig, Y.A. He, S.L. Jacques, E.A. Lunney, M. McTigue,
D. Molina, T. Quenzer, P.A. Wells, X. Yu, Y. Zhang, A. Zou, M.R. Emmett,
A.G. Marshall, H.-M. Zhang, G.D. Demetri, KIT kinase mutants show unique
mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal
stromal tumor patients, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 1542e1547.
[35] P.A. Kollman, I. Massova, C. Reyes, B. Kuhn, S. Huo, L. Chong, M. Lee, T. Lee,
Y. Duan, W. Wang, O. Donini, P. Cieplak, J. Srinivasan, D.A. Case,
T.E. Cheatham 3rd, Calculating structures and free energies of complex
molecules: combining molecular mechanics and continuum models, Acc.
Chem. Res. 33 (2000) 889e997.
[7] V. Tolmachev, S. Stone-Elander, A. Orlova, Radiolabelled receptor-tyrosine-
kinase targeting drugs for patient stratification and monitoring of therapy
response: prospects and pitfalls, Lancet Oncol. 11 (2010) 992e1000.
[8] R. Haubner, H.J. Wester, W.A. Weber, M. Schwaiger, Radiotracer-based strat-
egies to image angiogenesis, Q. J. Nucl. Med. 47 (2003) 189e199.
[9] R. Haubner, H.J. Wester, Radiolabeled tracers for imagingtumor angiogenesis and
evaluation of anti-angiogenic therapies, Curr. Pharm. Des. 10 (2004) 1439e1455.
[10] E. Samen, J.O. Thorell, L. Lu, T. Tegnebratt, L. Holmgren, S. Stone-Elander,
Synthesis and preclinical evaluation of [11C]PAO as PET imaging tracer for
VEGFR-2, Eur. J. Nucl. Med. Mol. Imaging 36 (2009) 1283e1295.
[11] O. Ilovich, O. Jacobson, Y. Aviv, A. Litchi, R. Chisin, E. Mishani, Formation of
fluorine-18 labeled diaryl ureas e labeled VEGFR-2/PDGFR dual inhibitors as
molecular imaging agents for angiogénesis, Bioorg. Med. Chem. 16 (2008)
4242e4251.
[12] O. Ilovich, O. Aberg, B. Langsröm, E. Mishani, Rhodium-mediated
[
11C]
carbonylation:
a
library of N-phenyl-N0-{4-(4-quinolyloxy)-phenyl}-[11C]-
urea derivatives as potential PET angiogenic probes, J. Label. Compd. Radio-
pharm. 52 (2009) 151e157.
[36] P. Zhou, J. Lv, J. Zou, F. Tian, Z. Shang, Halogenewaterehydrogen bridges in
biomolecules, J. Struct. Biol. 169 (2010) 172e182.
[13] A. Chiharu, O. Masanao, K. Katsushi, F. Masayuki, K. Koichi, Y. Tomoteru, Y. Joji,
K. Kazunori, H. Akiko, F. Toshimitsu, Z. Ming-Rong, Efficient radiosynthesis of
[
11C]sorafenib using 11C]phosgene as
[ a labeling agent, J. Label. Compd.
[37] Y. Lu, Y. Wang, Z. Xu, X. Yan, X. Luo, H. Jiang, W. Zhu, C-X/H Contacts in
biomolecular systems: how they contribute to protein-ligand binding affinity,
J. Phys. Chem. B 113 (2009) 12615e12621.
Radiopharm. 54-S1 (2011) S96.
[14] A.J. Poot, B. Van der Wildt, M. Stigter-van Walsum, R.C. Schuit, M. Rongen,
G.A.M.S. Van Dongen, A.D. Windhorst, Two approaches for the synthesis of
[38] K.R. Buckley, J. Huser, S. Jivan, K.S. Chun, T.J. Ruth, 11C-methane production in
small volume, high pressure gas targets, Radiochem. Acta 88 (2000) 201e205.
[39] P. Larsen, J. Ulin, K. Dahlstrom, A new method for production of 11C-labelled
methyl iodide from 11C-methane, J. Label. Compd. Radiopharm. 37 (1995)
73e75.
[
11C]sorafenib, a tyrosine kinase inhibitor PET tracer to be used in cancer
therapy, J. Label. Compd. Radiopharm. 54-S1 (2011) S85.
[15] D. Dischino, T. Tran, D. Donelly, S. Bonascorsi, P. Chow, R. Roache, D. Kukral,
J. Kim, W. Hayes, J. Label. Compd. Radiopharm. 54-S1 (2011) S444.