In vivo Positron Emission Tomography (PET) imaging of Mesenchymal - Epithelial Transition (MET) receptor

Article abstract of DOI:10.1021/jm900803q

We report the radiosynthesis and evaluation of 3-[3,5-dimethyl-4-(4-[ ¹¹C]methylpiperazinecarbonyl)-1H-pyrrol-2-ylmethylene]-2-oxo-2, 3-dihydro-1H-indole-5-sulfonic acid (3-chlorophenyl)methylamide, termed [ ¹¹C]SU11274 ([¹¹C]14) for in vivo imaging of mesenchymal - epithelial transition (MET) receptor by positron emission tomography (PET). Following the synthesis of the precursor (13) that was achieved in 10 steps with a total yield of 9.7%, [¹¹C]14 was obtained through radiomethylation in a range of 5-10% radiochemical yield and over 95% radiochemical purity. For in vivo PET studies, two human lung cancer xenograft models were established using MET-positive NCI-H1975 and MET-negative NCI-H520 cell lines. Quantitative [¹¹C]14-PET studies showed that the tumor uptake of [ ¹¹C]14 in the NCI-H1975 xenografts was significantly higher than that in the NCI-H520 xenografts, which is consistent with their corresponding immunohistochemical tissue staining patterns of MET receptors from the same animals. These studies demonstrated that [¹¹C]14-PET is an appropriate imaging marker for quantification of MET receptor in vivo, which can facilitate efficacy evaluation in the clinical development of MET-targeted cancer therapeutics. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900803q

J. Med. Chem. 2010, 53, 139–146 139
DOI: 10.1021/jm900803q
In Vivo Positron Emission Tomography (PET) Imaging of Mesenchymal-Epithelial Transition (MET)
Receptor
Chunying Wu,† Zhe Tang,‡ Weiwen Fan,‡ Wenxia Zhu,† Changning Wang,† Edurado Somoza,† Norbert Owino,†
Ruoshi Li,† Patrick C. Ma,‡ and Yanming Wang*,†
†Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Case Western Reserve University,
Cleveland, Ohio 44106 and ‡Division of Hematology/Oncology, Department of Medicine, Case Western Reserve University,
University Hospitals Case Medical Center, and Case Comprehensive Cancer Center, Cleveland, Ohio 44106
Received June 4, 2009
We report the radiosynthesis and evaluation of 3-[3,5-dimethyl-4-(4-[11C]methylpiperazinecarbonyl)-
1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (3-chlorophenyl)methylamide,
termed [11C]SU11274 ([11C]14) for in vivo imaging of mesenchymal-epithelial transition (MET)
receptor by positron emission tomography (PET). Following the synthesis of the precursor (13) that
was achieved in 10 steps with a total yield of 9.7%, [11C]14 was obtained through radiomethylation
in a range of 5-10% radiochemical yield and over 95% radiochemical purity. For in vivo PET studies,
two human lung cancer xenograft models were established using MET-positive NCI-H1975 and
MET-negative NCI-H520 cell lines. Quantitative [11C]14-PET studies showed that the tumor uptake
of [11C]14 in the NCI-H1975 xenografts was significantly higher than that in the NCI-H520 xenografts,
which is consistent with their corresponding immunohistochemical tissue staining patterns of MET
receptors from the same animals. These studies demonstrated that [11C]14-PET is an appropriate
imaging marker for quantification of MET receptor in vivo, which can facilitate efficacy evaluation in
the clinical development of MET-targeted cancer therapeutics.
1. Introduction
provides spatiallyand temporally staticinformation about the
expression of MET, it is often not feasible in clinical trials,
especially in lung tumors. Therefore, there is an urgent need to
develop noninvasive molecular imaging techniques to assay
and quantify the putative molecular targets such as MET in
human subjects. For this reason, several imaging modalities
have been explored for in vivo imaging of MET expression.
Magnetic resonance imaging (MRI) together with Doppler
ultrasound were first used to measure blood oxygenation level,
which was found to correlate with MET activity in vivo.18,19 An
anti-MET contrast agent was also evaluated on the basis of a
superparamagnetic iron oxide for MRI imaging of MET
expression.20 In addition, PET and single photon emission
computed tomography (SPECT) were used in combination
with anti-Met monoclonal antibodies that were radiolabeled
with either zirconium-89 for PET or iodine-125 for SPECT.21,22
To date, several small molecule MET inhibitors have been
developed.11,17,23-34 Among them, 3-[3,5-dimethyl-4-(4-[11C]-
methylpiperazinecarbonyl)-1H-pyrrol-2-ylmethylene]-2-oxo-
2,3-dihydro-1H-indole-5-sulfonic acid (3-chlorophenyl)-
methylamide, 14 (SU11274), is a specific, reversible MET
inhibitor that induces apoptosis in cells transformed by onco-
genic MET and translocated promoter region-MET tyrosine
kinase.11 Compound 14 inhibits the catalytic activity of MET
with an IC50 of 20 nM and a selectivity at least 1 order of
magnitude higher than that for a variety of other tyrosine
kinases.11,35 Inhibition of the MET kinase activity by 14 leads
to time- and dose-dependent reduction of cell growth by
inducing G1 cell cycle arrest and apoptosis. In addition, 14
possesses a structure that permits direct radiolabeling with
Cancer therapy for humans has entered into a new era of
molecular medicine.1 To date, a series of small molecule
inhibitors such as imatinib,2 gefitinib, erlotinib, lapatinib,3
sunitinib,4 and sorafenib5 have been approved by the U.S.
Food and Drug Administration for cancer therapies that
target various receptor tyrosine kinases (RTKsa). An impor-
tant RTK that plays a prominent role in regulating invasive
cellular oncogenic signaling is mesenchymal-epithelial transi-
tion (MET) receptor.6,7 Upon binding of its natural ligand hepa-
tocyte growth factor/scatter factor, MET is phosphorylated at a
number of juxtamembrane and cytoplasmic kinase phospho-
epitopes, resulting in signal transduction and regulation of multi-
tudes of cellular mitogenic and motogenic processes such as
cell growth, proliferation, and survival.6,8,9 Overexpression or
mutation of MET results in activation of oncogenic signaling
pathways and leads to up-regulation of diverse tumor cell
functions.7,10 Mounting evidence has validated MET receptor
as a novel target for anticancer therapy.11-17
For efficacy evaluation of various MET-targeted anti-
cancer therapies currently under development, it is critical to
develop an imaging tool that permits detection and quantifi-
cation of MET expression in vivo. Although invasive biopsy
*To whom correspondence should be addressed. Phone: þ1-216-844-3288.
Fax: þ1-216-844-8062. E-mail: yanming.wang@case.edu.
a Abbreviations: EGFR, epidermal growth factor receptor; MET,
mesenchymal-epithelial transition; MRI, magnetic resonance imaging;
PET, positron emission tomography; RTK, receptor tyrosine kinases;
SMP, small-molecule probe; SPECT, single photon emission computed
tomography.
r
2009 American Chemical Society
Published on Web 12/07/2009
pubs.acs.org/jmc

140 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Wu et al.
pyrrole aldehyde 11 and oxindole sulfonamide 3 in the
presence of pyrrolidine yielded 4-(5-{5-[3-chlorophenyl)-
methyl sulfomyl]-2-oxodihydroindol-3-yllidenemethyl}-2,4-
dimethyl-1H-pyrrole-3-carbonyl)piperazine-1-carboxylic acid
tert-butyl ester (12). Further decarboxylation of 12 with
hydrochloric acid in ethyl acetate afforded the precursor
3-[3,5-dimethyl-4-(piperazine-1-carbonyl)-1H-pyrrol-2-yl-
methylene]-2-oxo-2,3-dihyro-1H-indole-5-sulfonic acid (3-chloro-
phenyl)methylamide hydrochloride (13) for 11C radiolabel-
ing of 14.40,41
2.2. Radiosynthesis of [11C]14. The radiosynthesis of
[11C]14 was accomplished through direct methylation of 13
with [11C]methyl iodide in the presence of potassium carbo-
nate (see Scheme 2). After reaction for 10 min at 140 °C, the
resulting mixture was subjected to a purification procedure
using solid-phase extraction on C-18 Sep-Pak cartridge prior
to semipreparative HPLC purification. The overall radio-
chemical yield of [11C]14 was 5-10% decay corrected to the
activity of [11C]methyl iodide trapped. [11C]14 was obtained
with radiochemical purity of over 98% and a specific activity
of 0.3-0.5 Ci/mmol at the end of synthesis. The radio-
chemical identity of [11C]14 was verified by co-injection with
the nonradioactive cold standard which was commercially
purchased from Sigma-Aldrich (St. Louis, MO).
2.3. Preparation of Human Lung Cancer Xenografts and in
Vivo Micro-PET/MRI Studies. For in vivo imaging studies,
murine xenograft tumor models were established from two
different human lung cancer cell lines, NCI-H1975 (high
MET expression) and NCI-H520 (negligible MET ex-
pression).42 The human lung cancer xenografts were estab-
lished as described in the Experimental Section. For each
imaging study, a pair of mice was used and placed on the
same bed under anesthesia. After initial localization scans,
[11C]14 (500 μCi, 18.5 MBq) was then administrated through
tail vein injection into each of the two mice. Dynamic micro-
PET imaging was performed immediately for 90 min of
duration. The mice were kept on the same bed and trans-
ferred to the 9.4 T MRI scanner. T2-weighted MRI images
were acquired for 30 min. Following micro-PET and MRI
scans, images from both modalities were coregistered for
quantitative measurements of [11C]14 concentration in each
of the tumor regions. The radioactivity concentrations were
determined as standard uptake value (SUV) in each region of
interest through 90 min of the PET scan (Figure 2).
2.4. Correlation of MET Expression by ex Vivo Bioassay.
Following imaging studies, the tumor tissues were harvested
for immunohistochemical staining for correlation with the
[11C]14-PET imaging results. Thus, immunoblotting and the
respective H&E staining of both tumor xenografts were first
conducted for comparison of MET expression. As shown in
Figure 3, NCI-H1975 cells express high levels of both MET and
epidermal growth factor receptor (EGFR), which are phos-
phorylated under serum-stimulated conditions. On the other
hand, NCI-H520 expresses negligible levels of MET and
EGFR. The two cell lines were used to establish tumor
xenografts in nude mice. In addition, immunohistochemical
analysis of MET expression was conducted (Figure 4). In NCI-
H520 xenograft, negligible MET expression was observed (0 to
1þ) (Figure 4a,b). On the other hand, MET expression was
seen at a much higher level (3þ) in the NCI-H1975 xenograft
(Figure 4c,d), and significant membranous localization of the
MET staining is evident in this cell line as well (Figure 4d,
yellow arrows). These results are in concordance with the cell
line expression data under cell culture conditions.
Figure 1. [11C]14-PET imaging of lung cancer xenografts. Coronal
micro-PET images of nude mouse pair xenografted with NCI-H520
(animal on the left) and NCI-H1975 (animal on the right). Mice
were injected with 18.5 MBq of [11C]14 via the tail vein, and dynamic
scanning was performed for 90 min thereafter as detailed in the
Experimental Section. Image planes have been chosen for illustra-
tion where both the left and right tumors are visible. The locations of
the lung cancer tumor xenografts are highlighted by yellow arrows.
positron-emitting carbon-11 that will not cause any alterna-
tion of the authentic structure. Thus, the binding properties
and pharmacokinetics of 14 will be preserved. These binding
affinity and specificity as well as its flexibility for radiolabeling
make 14 a promising small-molecule probe (SMP) for PET
imaging of MET expression. Compared to immuno-PET
imaging, SMP-PET imaging holds several advantages: (1)
SMPs can be directly radiolabeled with a clearly defined
structure so that the pharmacokinetics, toxicity, and meta-
bolism can be precisely determined; (2) SMP PET provides a
more quantitative capacity than immuno-PET; (3) the struc-
tures of SMP can be readily modified for optimal imaging
outcomes. In this study we report the first multistep synthetic
approach to the production of [11C]14. We also investigated
thepotentialof[11C]14for PETimaging of MET expressionin
vivo in human lung cancer xenografts (Figure 1).
2. Results
2.1. Synthesis of the Labeling Precursor. The synthesis of
the [11C]14 labeling precursor is described in Scheme 1. The
2-oxo-2,3-dihydro-1H-indole-5-sulfonic acid (3) was first
prepared starting from the commercially available 1,3-dihy-
droindolin-2-one (1). Sulfonation of 1 yielded 2-oxo-2,3-di-
hydro-1H-indole-5-sulfonic acid (2), which underwent ami-
dation of the sulfonyl chloride group to yield the indole
sulfonamide (3).36,37 The oxime (5) was then prepared from
tert-butyl acetoacetate (4) based on previously reported
procedures.38,39 Knorr reaction of 5 with ethyl 3-oxobuty-
rate (6) in the presence of zinc dust gave 3,5-dimethyl-1H-
pyrrole-2,4-dicarboxylic acid 2-tert-butyl ester 4-ethyl ester
(7) in quantitative yield. The decarboxylation of the pyrrole 7
under acidic conditions (hydrochloric acid/ethanol) yielded
2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (8),
which underwent Vilsmeier formylation to give the key
intermediate 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic
acid ethyl ether (9) for the aldol condensation of the
oxindole and pyrrole aldehyde moieties. Basic hydrolysis
of 9 yielded 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic
acid (10). Amidation of 10 with piperazine-1-carboxylic
acid tert-butyl ester in the presence of 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride and hydroxy-
benzotriazole yielded the functionalized pyrrole aldehyde
4-(5-formyl-2,4-dimethyl-1H-pyrrole-3-carbony)piperazine-
1-carboxylic acid tert-butyl ester (11). Coupling of the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 141
Scheme 1. Synthesis of the 11C Radiolabeling Precursor 13
3. Discussion
developedconsistsof the followingthreesteps:(1) preparation
of the oxindole core; (2) preparation of functionalized pyrrole
aldehyde unit; (3) aldol condensation of the oxindole and
pyrrole aldehyde units to furnish the indolinone.36 Starting
from commercially available 2-oxindole, the total synthesis
of 13 was achieved in 10 steps with a combined yield of 9.7%.
By use of this synthetic route, large quantities of 13 can be
obtained, which can be stored as the HCl salt for long periods
without decomposition.
The radiosynthesis of [11C]14 was achieved through direct
radiomethylation using [11C]methyl iodide, which was prepared
through reduction of [11C]CO2 generated by an on-site cyclo-
tron. Although there are a total of five nitrogens present in 13,
only the unsubstituted nitrogen in the unconjugated piperazine
As a linear compound, 14 consists of three functionalized
heterocyclic components: oxindole, pyrrole, and piperazine.
The methyl group on the nucleophilic nitrogen of the piper-
azine ring is uniquely suited as a radiolabeling site. This is due
to the fact that the N-methyl group can be readily replaced by
a [11C]methyl group through direct radiomethylation with
[11C]methyl iodide without causing any structural alternation.
For this reason, we designed 13 with no methyl group on the
piperazine ring as the radiolabeling precursor, which is other-
wise identical to 14, the final product. Because of this struc-
tural variation, the chemical synthesis of13 warrants design of
a new synthetic approach that is different from the previously
reported approach to 14. The synthetic approach we have

142 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Scheme 2. Radiosynthesis of [11C]14
Wu et al.
ring can be methylated by [11C]methyl iodide in the presence of
potassium carbonate. The radiomethylation was carried out at
elevated temperature (140 °C) in DMF for 10 min. The [11C]14
product was purified by HPLC with a radiochemical purity of
>95% and a radiochemical yield of 5-10% after decay
correction.
histochemistry bioassay results, suggesting that [11C]14-PET is
a valid imaging marker for in vivo quantification of MET
expression. These results justify further development of [11C]14
and its analogues as molecular imaging probes for efficacy
evaluation of MET-targeted cancer therapy.
4. Experimental Section
The in vivo imaging studies were carried out in two lung
cancer xenograft mouse models, which were prepared with
NCI-H520 and NCI-H1975 lung adenocarcinama cells.
H1975 cells express high level of wild-type MET without
genomic amplification. In contrast, MET expression in
H520 cells is negligible. Thus, use of both xenografts allowed
us to determine the specificity of [11C]14-PET imaging in vivo.
As shown in Figure 2, time-radioactivity curves were deter-
mined in both xenografttumor models, which showed that the
uptake of the [11C]14 in the NCI-H1975 xenograft was
significantly higher than in the NCI-H520 xenograft (p =
0.000 19). Compared to NCI-H520 xenograft mice, the NCI-
H1975 xenograft mice exhibited an increased tumor uptake of
[11C]14 throughout the entire measurement period of 90 min.
At 10 min postinjection of [11C]14, the radioactivity concen-
tration in NCI-H1975 mice was 1.35-fold higher than that in
NCI-H520 mice. Moreover, at 65 min postinjection, the
radioactivity concentration ratio of H1975/H520 reached a
statistically significant and enhanced ratio of 2.1 and peaked
at 2.6 at 80 min postinjection. Hence, our results suggest that
[11C]14-PET is a specific imagingmarker thatcan differentiate
and quantify MET expression in vivo.
Immediately following completion of the micro-PET ima-
ging, the tumor xenografts were dissected and MET expression
in tumor tissues of both xenograft mouse models was evalu-
ated through immunoblotting and immunohistochemical
staining using antihuman MET antibody (Figures 3 and 4).
The results confirmed that NCI-H1975 xenograft tumor cells
expressed a readily detectable high level of MET receptor,
with both membranous and cytoplasm localization discern-
ible through standard immunohistochemistry using antihu-
man MET antibody. On the other hand, NCI-H520 xenograft
tumor cells showed negligible MET expression level and
specifically no membranous MET localization was seen.
These results are consistent with the imaging data and thus
demonstrate that [11C]14-PET is a promising imaging marker
of MET expression.
4.1. General Remarkers. All chemicals were purchased from
Sigma-Aldrich (St. Louis, MO) and used without further puri-
fication unless otherwise specified. 1H NMR spectra were
recorded on a Bruker AMX 400 MHz spectrometer using
5 mm NMR tubes (Wilmad 528-PP) in CDCl3 or DMSO-d6
(Sigma-Aldrich or Cambridge Isotopes) solutions at room
temperature. Chemical shifts are reported as δ values relative
to internal tetramethylsilane (TMS). HR-ESIMS were recorded
under the electron spray ionization (ESI) condition. 13C NMR
spectra were recorded on an Oxford AS 100 MHz spectrometer
using 5 mm NMR tubes (Wilmad 528-PP) in CDCl3 or DMSO-
d6 (Sigma-Aldrich or Cambridge Isotopes). Six-week-old female
Ncr-nu (Nude) mice were purchased from Charles River Labo-
ratories (Wilmington, MA) and hosted in a pathogen-free
animal facility at Case Western Reserve University, Cleveland,
OH. Analytical radio-TLC was performed on silica gel F-254
aluminum plates, with visualization under UV (254 nm). Che-
mical and radiochemical purity was determined by an Agilent
1100 series high pressure liquid chromatography (HPLC) sys-
tem equipped with UV and Bioscan flow count detectors. The
purities of all the intermediates and final product were >95%.
Micro-PET imaging was conducted under an R4 micro-PET
system (Siemens Medical Solutions, Knoxville, TN). MRI
imaging was performed on a Bruker Biospec 9.4 T MRI scanner
to produce high resolution images as roadmaps for micro-PET
imaging.
4.2. Synthesis of 2-Oxo-2,3-dihydro-1H-indole-5-sulfonic Acid
(2). Chlorosulfonic acid (27 mL, 406 mmol) was slowly added to
a solution of 1,3 dihydroindolin-2-one (1, 13.3 g, 100 mmol) at
room temperature. The reaction mixture was stirred at room
temperature for 1.5 h followed by stirring for another 1 h at
68 °C in a water bath. The reaction mixture was then cooled to
room temperature and poured into water. The solid precipitate
was filtered, washed with water, and dried to yield 2-oxo-2,3-
dihydro-1H-indole-5-sulfonic acid (2, 11.0 g, 50% yield), which
was used without further purification.
4.3. Synthesis of 2-Oxo-2,3-dihydro-1H-indole-5-sulfonic Acid
(3). To a solution of compound 2 (4.2 g, 18.2 mmol) dissolved in
dry ethanol (10 mL), 3-chlorophenylamine (3.0 g, 21 mmol) was
added. The resulting solution was stirred at room temperature
for 12 h. The reaction mixture was then concentrated and recrys-
tallized from dichloromethane to give 2-oxo-2,3-dihydro-1H-
indole-5-sulfonic acid (3, 4.3 g 70% yield). 1H NMR (400 MHz,
DMSO-d6): 10.89 (br, s, NH), 6.92-7.16 (m, 7H, aromatic H),
3.53 (s, 2H, CH2), 3.08 (s, 3H, CH3).
In summary, we successfully developed an efficient radio-
chemical synthesis of [11C]14. Micro-PET studies demonstrated
that the tumor uptake of [11C]14 is consistent with the levels of
MET expression in the xenograft tumor models. The [11C]14-
PET imaging results correlated well with the MET immuno-

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 143
Figure 2. Pharmacokinetic profile of [11C]14-PET imaging of lung
cancer xenografts. The average radioactivity-time curve in terms of
SUV during the time period of [11C]14-PET imaging of each of the
lung cancer xenografts was plotted as illustrated. The radiotracer
uptake level in NCI-H1975 xenograft is higher than that in the
control xenograft NCI-H520 (p = 0.000 19). Regions of interest were
manually drawn by creating a volume of interest in the tumor region
based on MR images. p < 0.05 indicates statistical significance.
Figure 4. Immunohistochemical analysis of MET receptor expres-
sion in tumor xenograft of human lung cancer (NCI-H1975 and
H520) in vivo. The expression of MET receptor in the lung cancer
xenografts established was evaluated using immunohistochemistry
of the xenograft formalin-fixed paraffin-embedded sections using
anti-MET primary antibody as described in the Experimental
Section.
(100 mL) in an oil bath, zinc dust (50 g 0.76 mol) was added
portionwise. The mixture was heated to 60 °C. At this tempera-
ture, the crude tert-butyl 2-hydroximinooxobutyrate precipitated
slowly. The temperature was then increased to 75 °C for 1 h, and
then the reaction mixture was poured into water (40 mL). The
precipitate was collected by filtration to yield 3,5-dimethyl-1H-
pyrrole-2,4-dicarboxylic acid 2-tert-butyl ester-4-ethyl ester (7) in
quantitative yield as a white solid. 1H NMR (400 MHz, CDCl3):
9.47 (br, s, 1H, NH), 4.29 (q, 2H, CH2), 2.52, 2.54 (2s, 6H, 2CH3),
1.58 (s, 9H, 3CH3), 1.36 (t, 3H, CH3). 13C NMR (100 MHz,
CD3SO): 165.8, 161.6, 138.7, 130.2, 119.4, 113.6, 81.4, 59.6, 28.7,
14.7, 14.6, 12.3. Retention time: 5.98 min (HPLC C-18 column,
acetonitrile/DMGA (5 mM, pH 7.4) = 80:20 (v:v)).
4.6. Synthesis of 2,4-Dimethyl-1H-pyrrole-3-carboxylic Acid
Ester (8). A solution of the product 7 (18 g, 67 mmol) dissolved
in ethanol (78 mL) was stirred vigorously as hydrochloric acid
(10 N, 3.6 mL) was slowly added at 20 °C. Then the reaction
mixture was heated to 67 °C for 1 h, cooled to 5 °C, and poured
into ice water. The solid was collected by filtration and washed
with water to yield 2,4-dimethyl-1H-pyrrole-3-carboxylic acid
Figure 3. Murine xenograft models of human NCI-H1975 and
NCI-H520 lung cancer cell lines. Expression of MET and EGFR
in the NCI-H1975 and NCI-H520 human lung cancer cell lines is
shown here. The two lung cancer cell lines were cultured in regular
growth media in cell culture, with the cell lysates collected for
SDS-PAGE and immunoblotting using phospho-MET and total
MET antibodies as described in the Experimental Section. Immu-
noblotting using antiphospho-EGFR and anti-EGFR antibodies
was also performed for comparison. Actin antibody was used as a
protein loading control. H&E staining of the xenografts is also
shown here (40X).
1
ester (8, 10 g, 89% yield). H NMR (400 MHz, CDCl3): 10.48
(br, s, 1H, NH), 6.35 (s, 1H, CH), 4.10 (q, 2H, CH2), 2.35, 2.15
(2s, 2CH3), 1.21 (t, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
166.0, 135.9, 120.1, 115.4, 110.2, 58.9, 15.1, 14.2, 13.3. Reten-
tion time: 3.42 min (HPLC C-18 column, acetonitrile/DMGA
(5 mM, pH 7.4) = 80:20 (v:v), flow rate 1.0 mL/min).
4.7. Synthesis of 5-Formyl-2,4-dimethyl-1H-pyrrole-3-carbo-
xylic Acid Ethyl Ester (9). Phosphorus oxychloride (2.8 mL,
30 mmol) was added dropwise to a solution of dimethylform-
amide (2.3 mL, 30 mmol) in dry dichloromethane (50 mL) in an
ice bath. After 30 min compound 8 (3.5 g, 21 mmol) was slowly
added. The mixture was heated at 40 °C for 2 h, cooled to 10 °C,
followed by addition of ice-water (10 mL) and hydrochloric
acid (10 N, 10 mL) with vigorous stirring. The mixture was
allowed to stand at room temperature for 12 h until the layers
were separated. The aqueous layer was adjusted to pH 12-13
with potassium hydroxide (10 N), while the temperature was
maintained at 55 °C. The reaction mixture was then cooled to
10 °C in an ice bath and stirred for another hour. The solid
formed was collected by filtration and washed with water to
yield 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid ethyl
4.4. Synthesis of tert-Butyl 2-Hydroximino-3-oxobutyrate (5).
tert-Butyl 3-oxobutanoate (40 mL, 241 mmol) was dissolved in
acetic acid (80 mL) in a round-bottomed flask, and the mixture
was cooled in an ice bath to about 10 °C. Sodium nitrite (18 g,
261 mmol) was added over 1 h while keeping the temperature
under 15 °C. The cold bath was removed and the mixture was
allowed to stir for 3.5 h at room temperature to give a crude
solution of tert-butyl 2-hydroximino-3-oxobutyrate (5) which
was used in the next step without further purification.
4.5. Synthesis of 3,5-Dimethyl-1H-pyrrole-2,4-dicarboxylic
Acid 2-tert-Butyl Ester 4-Ethyl Ester (7). To a stirred mixture
of ethyl 3-oxobutyrate (6, 10 mL, 79 mmol) and acetic acid
1
ester (9, 3.7 g, 91%). H NMR (400 MHz, CDCl3): 10.79 (s,
1H, NH), 9.60 (s, 1H, CHO), 4.31 (q, 2H, CH2), 2.56, 2.60

144 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Wu et al.
(2s, 2CH3), 1.38 (t, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
177.6, 165.3, 144.4, 136.9, 128.5, 114.4, 59.9, 14.6, 14.5, 10.9.
Retention time: 3.35 min (HPLC C-18 column, acetonitrile/
DMGA (5 mM, pH 7.4) = 80:20 (v:v), flow rate 1.0 mL/min).
4.8. Synthesis of 5-Formyl-2,4-dimethyl-1H-pyrrole-3-carbo-
xylic Acid (10). A mixture of compound 9 (3.0 g, 15.4 mmol),
potassium hydroxide pellet (1.2 g, 16.5 mmol), methanol (8 mL),
and water (50 mL) was heated at 100 °C for 2 h and subsequently
cooled to 8 °C in an ice bath. The reaction product was extracted
with dichloromethane. The aqueous portion was acidified to pH
4 with hydrochloric acid (10 N). The product was collected by
filtration, washed with water, and dried over MgSO4 to yield
5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (10, 2.4 g,
94% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6):
12.07 (s, 1H, NH), 9.60 (s, 1H, CHO), 3.33 (s, 1H, COOH), 2.39,
δ 2.43 (2s, 2CH3). 13C NMR (100 MHz, DMSO-d6): 178.4,
166.6, 143.4, 134.7, 128.8, 114.0, 14.3, 11.1. Retention time: 2.33 min
(HPLC, C-18 column, acetonitrile/DMGA (5 mM, pH 7.4) =
80:20 (v:v), flow rate 1.0 mL/min).
4.9. Synthesis of 4-(5-Formyl-2,4-dimethyl-1H-pyrrole-3-car-
bonyl)piperazine-1-carboxylic Acid tert-Butyl Ester (11). A solu-
tion of compound 10 (2.0 g, 12 mmol) dissolved in dimethyl-
formamide (40 mL) was stirred at room temperature while
N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochlo-
ride (3.5 g, 18 mmol), hydroxybenzotriazole hydrate (2.4 g,
18 mmol), triethylamine (15 mL), and 1-Boc-piperazine (2.8 g,
15 mmol) were added. The reaction mixture was stirred at room
temperature for 12 h and then diluted with water (30 mL), brine
(20 mL), saturated sodium carbonate (30 mL). The pH of the
resultant mixture was adjusted to 10 with sodium hydroxide
(10 N) and extracted with 10% methanol in dichloromethane.
The organic extract was dried over anhydrous sodium sulfate
and rotary-evaporated to dryness. The dry product was treated
with toluene (19 mL) and rotary-evaporated again to dryness.
The residue was triturated with hexane/diethyl ether, 3:1 v/v
(15 mL), then washed with ethyl acetate to give 4-(5-formyl-2,4-
dimethyl-1H-pyrrole-3-carbonyl)piperazine-1-carboxylic acid
tert-butyl ester (11, 2.5 g, 63%). 1H NMR (400 MHz, CDCl3):
11.86 (s, 1H, NH), 9.48 (s, 1H, CHO), 3.30 (br, s, 8H, piperazy
ring), 2.18, 2.13 (2s, 2CH3), 1.36 (s, 9H, 3CH3). 13C NMR
(150 MHz, CD3SO): 177.8, 166.0, 161.7, 154.4, 136.4, 128.6,
119.3, 79.8, 45.2, 28.6, 21.9, 12.4, 9.9. Retention time: 2.92 min
(HPLC C-18 column, acetonitrile/DMGA (5 mM, pH 7.4) =
80:20 (v:v), flow rate 1.0 mL/min).
2,4-dimethyl-1H-pyrrole-3-carbonyl)piperazine-1-carboxylic acid
tert-butyl ester (12, 150 mg, 0.22 mmol) was suspended in 5 mL
of ethyl acetate, and 5 mL of hydrochloric acid/ethyl acetate (4 M)
was added at ice-cold temperature. After the solution was allowed
to stand for 1 h at room temperature, the solvent was evaporated
under vacuum, yielding 3-[3,5-dimethyl-4-(piperazine-1-carbonyl)-
1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihyro-1H-indole-5-sulfo-
nic acid (3-chlorophenyl)methylamide hydrochloride (13) as a
1
yellow solid (100 mg, 74%). H NMR (400 MHz, DMSO-d6)
2.30, 2.43 (2 ꢀ s, 2 ꢀ 3H, 2 ꢀ CH3); 3.18 (s, 3H, CH3); 3.40 (m,
8H, piperazyl-H); 7.00-7.70 (m, 7H, aromatic-H and 1H,
olefinic-H). 13C NMR (100 MHz, DMSO-d6) 3.08 (s, 3H,
CH3); 3.53 (s, 2H, CH2); 6.92-7.16 (m, 7H, H-aromatic);
10.89 (br, s, NH). HR-ESIMS: m/z calcd for C27H29N5O4SClþ
(M - Cl), 554.16233; found 554.16248. Retention time: 6.08 min
(HPLC C-18 column, acetonitrile/DMGA (5 mM, pH 7.4) =
80:20 (v:v)).
4.12. Radiosynthesis of [11C]14. The cyclotron-made
[11C]carbon dioxide was converted to [11C]methyl iodide by
reduction with lithium aluminum hydride and hydroiodide.43
The labeled methyl iodide [11C]CH3I was concurrently distilled
and trapped in a dry 5 mL conical reaction vial cooled in an ice
bath which was previously filled with a solution of 2 mg of 13
and 10 mg of potassium carbonate dissolved in 0.3 mL dimethyl-
formamide. Trapping of [11C]methyl iodide was monitored by
measuring the activity in the isotope calibrator until the maxi-
mal value was attained. The reaction mixture was sealed and
heated at 140 °C for 10 min in a heating block, cooled to room
temperature, and diluted with water. The radiolabeled reaction
mixture was passed through a Waters C-18 Sep-Pak cartridge
previous conditioned with ethanol and then water to remove the
nonorganic impurities. The Sep-Pak cartridge was washed with
10 mL of water and dried with a rapid air bolus, and the
radiolabeled product was eluted with ethanol, loaded onto a
preparative HPLC (Luna 5 μm C18 250mm ꢀ 10 mm) column,
and eluted with acetonitrile and water (flow rate 3 mL/min
(8:2 v/v)). The retention time was 6.08 min (HPLC C-18 column,
acetonitrile/DMGA (5 mM, pH 7.4) = 80:20 (v:v)). The radio-
chemical purity of [11C]14 was >95% as determined by radio-
HPLC, and the total synthesis time was 30-40 min from the end
of bombardment (EOB).
4.13. Lung Cancer Cell Culture and in Vivo Nude Mouse
Xenograft Model. Lung cancer xenografts with NCI-H520 and
NCI-H1975 lung adenocarcinoma cells were established for this
imaging study. Both the NCI-H520 and NCI-H1975 cell lines
were obtained from American Type Culture Collection (ATCC,
Manassas, VA) and grown in RPMI-1640 media (Hyclone,
Logan, UT) supplemented with 10% (v/v) fetal bovine serum
under standard cell culture conditions. Six-week-old female
Ncr-nu (Nude) mice were purchased from Charles River La-
boratories (Wilmington, MA) and hosted in the pathogen-free
animal facility at Case Western Reserve University. In vivo
animal studies were performed according to institution-ap-
proved protocols and guidelines. Xenografts of the NCI-
H1975 and NCI-H520 lung cancer cells were established by
intradermally injecting 3 ꢀ 106 viable cells in RPMI 1640 media
into the flank/leg region of nude mice to produce subcutaneous
tumors.42 Body weight was recorded for each animal twice
weekly to monitor potential toxicities. Tumor xenografts were
subsequently dissected and harvested at the end of the experi-
ments after completion of the imaging studies and formalin-
fixed and stained with hematoxylin and eosin (H&E) using
standard techniques.
4.10. Synthesis of 4-(5-{5-[3-Chlorophenyl)methylsulfomyl]-
2-oxodihydroindol-3-yllidenemethyl}-2,4-dimethyl-1H-pyrrole-
3-carbonyl)piperazine-1-carboxylic Acid tert-Butyl Ester (12).
A mixture of compounds 3 (1.0 g, 2.9 mmol) and 11 (1.2 g,
3.6 mmol) and pyrrolidine (3 mL) was dissolved in ethanol and
heated to 90 °C for 5 h. After the reaction mixture was cooled
to room temperature, the precipitate was filtered, washed with
ethanol, and dried to yield 4-(5-{5-[3-chlorophenyl)methyl-
sulfomyl]-2-oxodihydroindol-3-yllidenemethyl}-2,4-dimethyl-
1H-pyrrole-3-carbonyl)piperazine-1-carboxylic acid tert-butyl
1
ester (12, 1.5 g, 78% yield). H NMR (400 MHz, DMSO): δ
7.65-6.95 (m, 7H, aromatic-H and 1H, olefinic-H), 3.30 (m, 8H,
piperazyl-H), 3.15 (s, 3H, CH3), 2.29, 2.35 (2s, 2CH3), 1.47
(s, 9H, 3CH3). 13C NMR (100 MHz, DMSO; 100 MHz,
CD3SO): δ 170.3, 166.0, 154.5, 143.6, 142.4, 136.1, 133.5,
131.2, 131.0, 128.8, 127.5, 127.0, 127.0, δ126.7, 126.6, 126.6,
125.3, 121.0, 118.7, 113.5, 110.0, 79.9, 28.7, 13.2, 10.9. Retention
time: 5.10 min (HPLC C-18 column, acetonitrile/DMGA
(5 mM, pH 7.4) = 80:20 (v:v), flow rate 1.0 mL/min). HR-
ESIMS: m/z calcd for C32H37N5O6SClþ (M þ Hþ) 654.214 76,
found 654.214 99; m/z calcd for C32H37N5O6SClþ (M þ Naþ)
676.196 70, found 676.196 97.
4.14. Micro-PET/MRI imaging. A pair of xenograft model
mice was placed on a plastic bed that was suitable for both PET
and MRI scanning following anesthesia with 2.0% isoflurane
delivered in oxygen gas with a nosecone. The micro-PET
experiment was performed on a Concord R4 micro-PET scan-
ner (Siemens Medical Solutions, Knoxville, TN). Transmis-
sion scans were acquired for 10 min by using a 57Co source
4.11. Synthesis of 3-[3,5-Dimethyl-4-(piperazine-1-carbonyl)-
1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihyro-1H-indole-5-sulfonic Acid
(3-Chlorophenyl)methylamide Hydrochloride (13). 4-(5-{5-[3-Chloro-
phenyl)methylsulfomyl]-2-oxo-dihydroindol-3-yllidenemethyl}-

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 145
which was subsequently used for attenuation correction. [11C]14
(18.5 MBq) was then injected into the tail vein. The image
acquisition started immediately after the injection. The dynamic
imaging was collected for 90 min in a list mode. PET images were
reconstructed into a 128 ꢀ 128 matrix with 0.8 mm slice
thickness. Twelve frames were acquired within the scan duration
starting from 10 min (frames 0-5) to 5 min (6-11) during the
experiment.
Following micro-PET imaging, the animals were immediately
transferred to a Bruker Biospec 9.4 T MRI scanner under
anesthesia to produce high resolution images as roadmaps for
micro-PET imaging. The entire animal bed was moved to
minimize movement of the animals between the MRI and
PET scans. After initial localization scans, the two mice were
simultaneously scanned using T2-weighted imaging. Each ani-
mal’s respiration rate was monitored, and the animal’s core
body temperature was also maintained at 37 ( 2 °C throughout
the scanning procedure. High-resolution anatomic MR images
of the tumor region were acquired using contiguous multislice
2D spin echo and 3D gradient echo techniques.
4.15. Quantitative Image Analysis. Following micro-PET and
MRI imaging, we conducted quantitative image analysis in
order to evaluate the in vivo pharmacokinetic profiles of
[11C]14 in xenograft tumor tissues. The micro-PET images were
registered with MR images to ensure the consistency of the
anatomical localization of [11C]14 in tumor tissue. Volumes of
interest (VOI) of the xenografts were then defined and deli-
neated in 3D on MR images. The coregistered images were used
for quantitative image analysis to determine [11C]14 uptake and
retention associated with tumor tissues. To generate a radio-
activity versus time curve, the mean radioactivity in the VOI was
calculated, corrected for 11C decay, plotted, and expressed in
terms of standard uptake volume [(μCi/cm3)/(μCi/g)] as a func-
tion of time.
and 13C NMR. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Sun, S.; Schiller, J. H.; Spinola, M.; Minna, J. D. New molecularly
targeted therapies for lung cancer. J. Clin. Invest. 2007, 117, 2740–
2750.
(2) Savage, D. G.; Antman, K. H. Imatinib mesylate;a new oral
targeted therapy. N. Engl. J. Med. 2002, 346, 683–693.
(3) Ryan, Q.; Ibrahim, A.; Cohen, M. H.; Johnson, J.; Ko, C. W.;
Sridhara, R.; Justice, R.; Pazdur, R. FDA drug approval summary:
lapatinib in combination with capecitabine for previously treated
metastatic breast cancer that overexpresses HER-2. Oncologist
2008, 13, 1114–1119.
(4) Goodman, V. L.; Rock, E. P.; Dagher, R.; Ramchandani, R. P.;
Abraham, S.; Gobburu, J. V.; Booth, B. P.; Verbois, S. L.; Morse,
D. E.; Liang, C. Y.; Chidambaram, N.; Jiang, J. X.; Tang, S.;
Mahjoob, K.; Justice, R.; Pazdur, R. Approval summary: sunitinib
for the treatment of imatinib refractory or intolerant gastrointest-
inal stromal tumors and advanced renal cell carcinoma. Clin.
Cancer Res. 2007, 13, 1367–1373.
(5) Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas, J.;
Smith, R. A.; Schwartz, B.; Simantov, R.; Kelley, S. Discovery
and development of sorafenib: a multikinase inhibitor for treating
cancer. Nat. Rev. Drug Discovery 2006, 5, 835–844.
(6) Birchmeier, C.; Birchmeier, W.; Gherardi, E.; Vande Woude, G. F.
Met, metastasis, motility and more. Nat. Rev. Mol. Cell Biol. 2003,
4, 915–925.
(7) Ma, P. C.; Maulik, G.; Christensen, J.; Salgia, R. c-Met: structure,
functions and potential for therapeutic inhibition. Cancer Meta-
stasis Rev 2003, 22, 309–325.
(8) Birchmeier, C.; Gherardi, E. Developmental roles of HGF/SF and
its receptor, the c-Met tyrosine kinase. Trends Cell Biol 1998, 8,
404–410.
(9) Rosario, M.; Birchmeier, W. How to make tubes: signaling by the
Met receptor tyrosine kinase. Trends Cell Biol 2003, 13, 328–335.
(10) Dietrich, S.; Uppalapati, R.; Seiwert, T. Y.; Ma, P. C. Role of
c-MET in upper aerodigestive malignancies;from biology to
novel therapies. J. Environ. Pathol., Toxicol. Oncol. 2005, 24,
149–162.
4.16. Ex Vivo Bioassay of MET Expression. For the in vitro
cellular protein expression analysis, the lung cancer cell lines
were cultured under regular growth conditions to exponential
growth phase. Cellular proteins were extracted from whole cells
as previously described.16 Immunoblotting was performed as
previously described16,44 using the following primary antibodies
as indicated: p-MET [Y1234/1235] (Cell Signaling Technology,
Danvers, MA), MET (C-12, Santa Cruz Biotechnology, Santa
Cruz, CA), p-EGFR[Y1068] (Cell Signaling), EGFR (Santa
Cruz Biotechnology), and actin (Santa Cruz Biotechnology).
Following MRI and micro-PET studies, the animals bearing the
lung cancer xenografts were sacrificed in accordance with
institutional guidelines, and the xenograft tumors were dissected
and harvested under standard techniques. The xenografts were
then formalin-fixed in 5% formalin for 16 h, processed for
hematoxylin and eosin (H&E) staining as well as immunohisto-
chemical staining using rabbit polyclonal primary antibody
against total MET (C-12, Santa Cruz Biotechnology) at 1:100
dilution as previously described.16 Isotype matched rabbit IgG
was used as negative control treatment as well. The immuno-
histochemistry was performed using the Tissue Procurement
and Histology Core Facility of the Case Comprehensive Cancer
Center.
(11) Sattler, M.; Pride, Y. B.; Ma, P.; Gramlich, J. L.; Chu, S. C.;
Quinnan, L. A.; Shirazian, S.; Liang, C.; Podar, K.; Christensen,
J. G.; Salgia, R. A novel small molecule met inhibitor induces
apoptosis in cells transformed by the oncogenic TPR-MET tyro-
sine kinase. Cancer Res. 2003, 63, 5462–5469.
(12) Corso, S.; Comoglio, P. M.; Giordano, S. Cancer therapy: can the
challenge be MET? Trends Mol. Med. 2005, 11, 284–292.
(13) Eder, J. P.; Vande Woude, G. F.; Boerner, S. A.; LoRusso, P. M.
Novel therapeutic inhibitors of the c-Met signaling pathway in
cancer. Clin. Cancer Res. 2009, 15, 2207–2214.
(14) Knudsen, B. S.; Vande Woude, G. Showering c-MET-dependent
cancers with drugs. Curr. Opin. Genet. Dev. 2008, 18, 87–96.
(15) Ma, P. C.; Tretiakova, M. S.; Nallasura, V.; Jagadeeswaran, R.;
Husain, A. N.; Salgia, R. Downstream signalling and specific
inhibition of c-MET/HGF pathway in small cell lung cancer:
implications for tumour invasion. Br. J. Cancer 2007, 97, 368–377.
(16) Ma, P. C.; Jagadeeswaran, R.; Jagadeesh, S.; Tretiakova, M. S.;
Nallasura, V.; Fox, E. A.; Hansen, M.; Schaefer, E.; Naoki,
K.; Lader, A.; Richards, W.; Sugarbaker, D.; Husain, A. N.;
Christensen, J. G.; Salgia, R. Functional expression and mutations
of c-Met and its therapeutic inhibition with SU11274 and small
interfering RNA in non-small cell lung cancer. Cancer Res. 2005,
65, 1479–1488.
(17) Ma, P. C.; Schaefer, E.; Christensen, J. G.; Salgia, R. A selective
small molecule c-MET inhibitor, PHA665752, cooperates with
rapamycin. Clin. Cancer Res. 2005, 11, 2312–2319.
(18) Shaharabany, M.; Abramovitch, R.; Kushnir, T.; Tsarfaty, G.;
Ravid-Megido, M.; Horev, J.; Ron, R.; Itzchak, Y.; Tsarfaty, I. In
vivo molecular imaging of met tyrosine kinase growth factor
receptor activity in normal organs and breast tumors. Cancer
Res. 2001, 61, 4873–4878.
(19) Tsarfaty, G.; Stein, G. Y.; Moshitch-Moshkovitz, S.; Kaufman,
D. W.; Cao, B.; Resau, J. H.; Vande Woude, G. F.; Tsarfaty,
I. HGF/SF increases tumor blood volume: a novel tool for the
in vivo functional molecular imaging of Met. Neoplasia 2006, 8,
344–352.
(20) Towner, R. A.; Smith, N.; Tesiram, Y. A.; Abbott, A.; Saunders,
D.; Blindauer, R.; Herlea, O.; Silasi-Mansat, R.; Lupu, F. In vivo
detection of c-MET expression in a rat hepatocarcinogenesis model
using molecularly targeted magnetic resonance imaging. Mol.
Imaging 2007, 6, 18–29.
Acknowledgment. We thank Dr. George Bakale for proof-
reading the manuscript. This work is supported by Case
Center for Imaging Research and Case Comprehensive Can-
cer Center. P.C.M. is supported by NIH/National Cancer
Institute;K08 Mentored Clinical Scientist Career Develop-
ment Award (Grant 5K08CA102545-04) and the Sol Siegal
Lung Cancer Research in Honor of Molly Siegal Grant
Program.
Supporting Information Available: Table listing the analytical
data for compounds 7-13; spectra of HPLC, HRMS, 1HNMR,

146 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Wu et al.
(21) Perk, L. R.; Stigter-van Walsum, M.; Visser, G. W.; Kloet,
R. W.; Vosjan, M. J.; Leemans, C. R.; Giaccone, G.; Albano, R.;
Comoglio, P. M.; van Dongen, G. A. Quantitative PET imaging
of Met-expressing human cancer xenografts with 89Zr-labelled
monoclonal antibody DN30. Eur. J. Nucl. Med. Mol. Imaging
2008, 35, 1857–1867.
(22) Hay, R. V.; Cao, B.; Skinner, R. S.; Su, Y.; Zhao, P.; Gustafson,
M. F.; Qian, C. N.; Teh, B. T.; Knudsen, B. S.; Resau, J. H.; Shen,
S.; Waters, D. J.; Gross, M. D.; Vande Woude, G. F. Nuclear
imaging of Met-expressing human and canine cancer xenografts
with radiolabeled monoclonal antibodies (MetSeek). Clin. Cancer
Res. 2005, 11, 7064s–7069s.
(23) Schroeder, G. M.; An, Y.; Cai, Z. W.; Chen, X. T.; Clark, C.;
Cornelius, L. A.; Dai, J.; Gullo-Brown, J.; Gupta, A.; Henley, B.;
Hunt, J. T.; Jeyaseelan, R.; Kamath, A.; Kim, K.; Lippy, J.;
Lombardo, L. J.; Manne, V.; Oppenheimer, S.; Sack, J. S.;
Schmidt, R. J.; Shen, G.; Stefanski, K.; Tokarski, J. S.; Trainor,
G. L.; Wautlet, B. S.; Wei, D.; Williams, D. K.; Zhang, Y.;
Fargnoli, J.; Borzilleri, R. M. Discovery ofN-(4-(2-amino-3-chloro-
pyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-
oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selec-
tive and orally efficacious inhibitor of the Met kinase superfamily.
J. Med. Chem. 2009, 52, 1251–1254.
(24) Zhang, Y.; Kaplan-Lefko, P. J.; Rex, K.; Yang, Y.; Moriguchi, J.;
Osgood, T.; Mattson, B.; Coxon, A.; Reese, M.; Kim, T. S.; Lin, J.;
Chen, A.; Burgess, T. L.; Dussault, I. Identification of a novel
recepteur d’origine nantais/c-met small-molecule kinase inhibitor
with antitumor activity in vivo. Cancer Res. 2008, 68, 6680–6687.
(25) Liu, L.; Siegmund, A.; Xi, N.; Kaplan-Lefko, P.; Rex, K.; Chen, A.;
Lin, J.; Moriguchi, J.; Berry, L.; Huang, L.; Teffera, Y.; Yang, Y.;
Zhang, Y.; Bellon, S. F.; Lee, M.; Shimanovich, R.; Bak, A.;
Dominguez, C.; Norman, M. H.; Harmange, J. C.; Dussault, I.;
Kim, T. S. Discovery of a potent, selective, and orally bioavailable
c-Met inhibitor: 1-(2-hydroxy-2-methylpropyl)-N-(5-(7-methoxy-
quinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihy-
dro-1H-pyrazole-4-carboxamide (AMG 458). J. Med. Chem. 2008,
51, 3688–3691.
(32) Smolen, G. A.; Sordella, R.; Muir, B.; Mohapatra, G.; Barmettler,
A.; Archibald, H.; Kim, W. J.; Okimoto, R. A.; Bell, D. W.; Sgroi,
D. C.; Christensen, J. G.; Settleman, J.; Haber, D. A. Amplification
of MET may identify a subset of cancers with extreme sensitivity
to the selective tyrosine kinase inhibitor PHA-665752. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 2316–2321.
(33) Wang, S. Y.; Chen, B.; Zhan, Y. Q.; Xu, W. X.; Li, C. Y.; Yang,
R. F.; Zheng, H.; Yue, P. B.; Larsen, S. H.; Sun, H. B.; Yang, X.
SU5416 is a potent inhibitor of hepatocyte growth factor receptor
(c-Met) and blocks HGF-induced invasiveness of human HepG2
hepatoma cells. J. Hepatol. 2004, 41, 267–273.
(34) Christensen, J. G.; Schreck, R.; Burrows, J.; Kuruganti, P.; Chan,
E.; Le, P.; Chen, J.; Wang, X.; Ruslim, L.; Blake, R.; Lipson, K. E.;
Ramphal, J.; Do, S.; Cui, J. J.; Cherrington, J. M.; Mendel, D. B. A
selective small molecule inhibitor of c-Met kinase inhibits c-Met-
dependent phenotypes in vitro and exhibits cytoreductive antitu-
mor activity in vivo. Cancer Res. 2003, 63, 7345–7355.
(35) Christensen, J. G.; Burrows, J.; Salgia, R. c-Met as a target for
human cancer and characterization of inhibitors for therapeutic
intervention. Cancer Lett. 2005, 225, 1–26.
(36) Kopka, K.; Faust, A.; Keul, P.; Wagner, S.; Breyholz, H. J.;
Holtke, C.; Schober, O.; Schafers, M.; Levkau, B. 5-Pyrrolidinyl-
sulfonyl isatins as a potential tool for the molecular imaging of
caspases in apoptosis. J. Med. Chem. 2006, 49, 6704–6715.
(37) Li, H. H.; Zheng, X. H.; Tan, J. Z.; Chen, L. L.; Liu, H.; Luo, X.
M.; Shen, X.; Lin, L. P.; Chen, K. X.; Ding, J.; Jiang, H. L. Design,
synthesis, antitumor evaluations and molecular modeling studies
of novel 3,5-substituted indolin-2-one derivatives. Acta Pharmacol.
Sin. 2007, 28, 140–152.
(38) Sun, L.; Liang, C.; Shirazian, S.; Zhou, Y.; Miller, T.; Cui, J.;
Fukuda, J. Y.; Chu, J. Y.; Nematalla, A.; Wang, X.; Chen, H.;
Sistla, A.; Luu, T. C.; Tang, F.; Wei, J.; Tang, C. 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.
2003, 46, 1116–1119.
(26) Yang, Y.; Wislez, M.; Fujimoto, N.; Prudkin, L.; Izzo, J. G.; Uno,
F.; Ji, L.; Hanna, A. E.; Langley, R. R.; Liu, D.; Johnson, F. M.;
Wistuba, I.; Kurie, J. M. A selective small molecule inhibitor of
c-Met, PHA-665752, reverses lung premalignancy induced by
mutant K-ras. Mol. Cancer Ther. 2008, 7, 952–960.
(27) Chattopadhyay, C.; El-Naggar, A. K.; Williams, M. D.; Clayman,
G. L. Small molecule c-MET inhibitor PHA665752: effect on cell
growth and motility in papillary thyroid carcinoma. Head Neck
2008, 30, 991–1000.
(28) Tseng, J. R.; Kang, K. W.; Dandekar, M.; Yaghoubi, S.; Lee, J. H.;
Christensen, J. G.; Muir, S.; Vincent, P. W.; Michaud, N. R.;
Gambhir, S. S. Preclinical efficacy of the c-Met inhibitor CE-
355621 in a U87 MG mouse xenograft model evaluated by 18F-
FDG small-animal PET. J. Nucl. Med. 2008, 49, 129–134.
(29) Koon, E. C.; Ma, P. C.; Salgia, R.; Welch, W. R.; Christensen,
J. G.; Berkowitz, R. S.; Mok, S. C. Effect of a c-Met-specific, ATP-
competitive small-molecule inhibitor SU11274 on human ovarian
carcinoma cell growth, motility, and invasion. Int. J. Gynecol.
Cancer 2008, 18, 976–984.
(30) Zou, H. Y.; Li, Q.; Lee, J. H.; Arango, M. E.; McDonnell, S. R.;
Yamazaki, S.; Koudriakova, T. B.; Alton, G.; Cui, J. J.; Kung,
P. P.; Nambu, M. D.; Los, G.; Bender, S. L.; Mroczkowski, B.;
Christensen, J. G. An orally available small-molecule inhibitor of
c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy
through antiproliferative and antiangiogenic mechanisms. Cancer
Res. 2007, 67, 4408–4417.
(39) Bell, I. M.; Stirdivant, S. M.; Ahern, J.; Culberson, J. C.; Darke,
P. L.; Dinsmore, C. J.; Drakas, R. A.; Gallicchio, S. N.; Graham,
S. L.; Heimbrook, D. C.; Hall, D. L.; Hua, J.; Kett, N. R.; Kim,
A. S.; Kornienko, M.; Kuo, L. C.; Munshi, S. K.; Quigley, A. G.;
Reid, J. C.; Trotter, B. W.; Waxman, L. H.; Williams, T. M.;
Zartman, C. B. Biochemical and structural characterization of a
novel class of inhibitors of the type 1 insulin-like growth factor and
insulin receptor kinases. Biochemistry 2005, 44, 9430–9440.
(40) Manley, J. M.; Kalman, M. J.; Conway, B. G.; Ball, C. C.;
Havens, J. L.; Vaidyanathan, R. Early amidation approach to
3-[(4-amido)pyrrol-2-yl]-2-indolinones. J. Org. Chem. 2003, 68,
6447–6450.
(41) Sun, L.; Cui, J.; Liang, C.; Zhou, Y.; Nematalla, A.; Wang, X.;
Chen, H.; Tang, C.; Wei, J. Rational design of 4,5-disubstituted-
5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones as a novel class of
inhibitors of epidermal growth factor receptor (EGF-R) and Her2-
(p185(erbB)) tyrosine kinases. Bioorg. Med. Chem. Lett. 2002, 12,
2153–2157.
(42) Tang, Z.; Du, R.; Jiang, S.; Wu, C.; Barkauskas, D. S.; Richey, J.;
Molter, J.; Lam, M.; Flask, C.; Gerson, S.; Dowlati, A.; Liu, L.;
Lee, Z.; Halmos, B.; Wang, Y.; Kern, J. A.; Ma, P. C. Dual MET-
EGFR combinatorial inhibition against T790M-EGFR-mediated
erlotinib-resistant lung cancer. Br. J. Cancer 2008, 99, 911–922.
(43) Langstrom, B.; Lundqvist, H. The preparation of 11C-methyl
iodide and its use in the synthesis of 11C-methyl-L-methionine.
Int. J. Appl. Radiat. Isot. 1976, 27, 357–363.
(31) Puri, N.; Khramtsov, A.; Ahmed, S.; Nallasura, V.; Hetzel, J. T.;
Jagadeeswaran, R.; Karczmar, G.; Salgia, R. A selective small
molecule inhibitor of c-Met, PHA665752, inhibits tumorigenicity
and angiogenesis in mouse lung cancer xenografts. Cancer Res.
2007, 67, 3529–3534.
(44) Ma, P. C.; Tretiakova, M. S.; MacKinnon, A. C.; Ramnath,
N.; Johnson, C.; Dietrich, S.; Seiwert, T.; Christensen, J. G.;
Jagadeeswaran, R.; Krausz, T.; Vokes, E. E.; Husain, A. N.; Salgia,
R. Expression and mutational analysis of MET in human solid
cancers. Genes, Chromosomes Cancer 2008, 47, 1025–1037.