Synthesis and in vivo evaluation of (S)-6-(4-fluorophenoxy)-3-((1-[ 11C]methylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one, a potential PET tracer for growth hormone secretagogue receptor (GHSR)

Article abstract of DOI:10.1016/j.bmc.2011.02.021

The peptide hormone ghrelin mediates through action on its receptor, the growth hormone secretagogue receptor (GHSR), and is known to play an important role in a variety of metabolic functions including appetite stimulation, weight gain, and suppression o

Full text of DOI:10.1016/j.bmc.2011.02.021

Bioorganic & Medicinal Chemistry 19 (2011) 2368–2372
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vivo evaluation of (S)-6-(4-fluorophenoxy)-3-((1-[11C]meth-
ylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one, a potential PET
tracer for growth hormone secretagogue receptor (GHSR)
⇑
Rachel Potter, Andrew G. Horti , Hayden T. Ravert, Daniel P. Holt, Paige Finley, Ursula Scheffel,
Robert F. Dannals, Richard L. Wahl
Division of Nuclear Medicine, Department of Radiology, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287-0816, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The peptide hormone ghrelin mediates through action on its receptor, the growth hormone secretagogue
receptor (GHSR), and is known to play an important role in a variety of metabolic functions including
appetite stimulation, weight gain, and suppression of insulin secretion. In light of the fact that obesity
is one of the major health problems plaguing the modern society, the ghrelin signaling system continues
to remain an important and attractive pharmacological target for the treatment of obesity. In vivo imag-
ing of the GHSR could shed light on the mechanism by which ghrelin affects feeding behavior and thus
offers a new therapeutic perspective for the development of effective treatments. Recently, a series of
piperidine-substituted quinazolinone derivatives was reported to be selective and potent GHSR antago-
nists with high binding affinities. Described herein is the synthesis, in vitro, and in vivo evaluation of (S)-
6-(4-fluorophenoxy)-3-((1-[11C]methylpiperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one ([11C]1), a
potential PET radioligand for imaging GHSR.
Received 13 December 2010
Revised 4 February 2011
Accepted 11 February 2011
Available online 16 February 2011
Keywords:
PET
Ghrelin
11C
Growth hormone secretagogue receptor
GHSR
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
physiological role in appetite regulation but also aid the develop-
ment of new pharmacological treatment for obesity and related
Obesity and obesity related morbidities represent one of the
most prevalent, chronic, and fatal epidemics of the 21st century.
While currently available therapies for obesity focus on weight
reduction, treatments designed to specifically modulate the funda-
mental physiologic regulatory mechanism that controls food in-
take and energy balance may represent an attractive approach
for the development of anti-obesity drugs.
conditions. A recent study presented several fluorine-bearing ana-
logs of ghrelin that were synthesized as potential PET probes,12 but
no GHSR in vivo imaging data have been published so far.
In this study we report the radiosynthesis of (S)-6-(4-fluoro-
phenoxy)-3-((1-[11C]methylpiperidin-3-yl)methyl)-2-o-tolylqui-
nazolin-4(3H)-one ([11C]1) (Scheme 2) and its in vitro and in vivo
evaluation as a potential PET radioligand for imaging GHSR.
Recently, there has been some evidence that ghrelin is a con-
tributing factor in the development of diet-induced obesity.1–3
Ghrelin, a 28 amino acid acylated peptide hormone, is an endoge-
nous ligand for the human growth hormone secretagogue receptor
(GHSR). First discovered in the stomach it is now known to be ex-
pressed in the hypothalamus and pituitary and in the peripheral
tissues such as heart, lungs, spleen, adrenal, and thyroid gland.4–7
While a substantial amount of data highlights the presence of
ghrelin in the CNS8–11 and its role in appetite stimulation through
a putative interaction with the receptor GHSR localized in the
hypothalamus, the exact mechanism of action has yet to be deter-
mined. Monitoring the GHSR receptor in vivo and non-invasively
with positron emission tomography (PET) would not only greatly
improve our understanding of this receptor and its complex
2. Results and discussion
The discovery of the orexigenic role of ghrelin1–3 has stimulated
a substantial effort in synthesizing ghrelin antagonists that may
interfere with body weight regulation. Industrial and academic
medicinal chemists have developed a number of small molecule
ghrelin antagonists.13 Recently, Bayer Pharmaceuticals disclosed
a series of quinazolinone derivatives as orally available ghrelin
receptor antagonists for treatment of diabetes and obesity.14 Two
members of the series, racemic 6-(4-fluorophenoxy)-3-(piperidin-
3-yl)methyl)-2-o-tolylquniazolin-4(3H)-one (2) and its N-isopro-
pyl derivative exhibit high GHSR binding affinity (Ki = 0.9 and
0.47 nM, respectively).14 (S)-Stereoisomers of this class appeared
to exhibit better binding affinities than the corresponding (R)-iso-
mers.14 We hypothesized that the (S)-stereoisomer of N-methyl
derivative of 2 (compound 1, Scheme 1) should also manifest good
⇑
Corresponding author. Tel.: +1 410 614 5130.
E-mail address: ahorti1@jhmi.edu (A.G. Horti).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.021

R. Potter et al. / Bioorg. Med. Chem. 19 (2011) 2368–2372
2369
N
NH
O
O
i
O
N
O
N
N
F
N
F
1
2
Scheme 1. Syntheses of compound 1. Reagents and conditions: (i) HCOOH, H2CO (aq), reflux, 72%.
11CH3
Table 1
Brain regional distribution data for [11C]1 in CD1 mice
N
Brain region
10 min
30 min
60 min
90 min
O
N
i, ii
O
Cerebellum
Hypothalamus
Hippocampus
Cortex
0.63 0.06
0.56 0.08
0.60 0.13
0.63 0.09
0.54 0.07
0.56 0.09
0.58 0.08
0.59 0.09
0.60 0.06
0.57 0.06
0.59 0.08
0.74 0.12
0.71 0.08
0.65 0.12
0.62 0.02
0.59 0.10
0.86 0.20
0.65 0.12
0.66 0.15
0.54 0.08
N
2
F
Rest of brain
[
11C]1
Data are the mean of %injected dose/g tissue SD (n = 3).
Scheme 2. Radiosynthesis of [11C]1. Reagents and conditions: (i) DMF, [11C]CH3OTf,
80 °C; (ii) HPLC purification.
which is too small for the mouse dissection, and it was not studied
in our work. Accumulation of [11C]1 radioactivity was relatively
low in all brain regions known to have a low density of GHSR
including cortex, cerebellum, and hippocampus. By contrast, in
the mouse hypothalamus there was a gradual increase of accumu-
lation of [11C]1 radioactivity over the 90 min of the study (Table 1).
The hypothalamus-to-cerebellum ratio increased steadily over the
observation period reaching value of 1.6.
GHSR binding affinity. Compound 1 is a small molecule with a
molecular weight of 457.5 Da, which is within the conventional
range (up to 500 Da) for passive blood–brain transport.15 The cal-
culated physical–chemical properties of
1 (brain/blood ratio
log BB = 0.45) suggest that 1 can penetrate the blood–brain barrier
and its P-glycoprotein efflux probability (P-gl = 0.2) value is rela-
tively low,16 and, thus, the radiolabeled [11C]1 may be suitable
for PET imaging of cerebral GHSR.
A tracer dose (approximately 0.5 nmol/kg) of [11C]1 injected
intravenously into CD1 mice (n = 30) produced no observed phar-
macological effects.
2.1. Chemistry
To test our hypothesis we synthesized 1 (Scheme 1) by reduc-
tive methylation of the desmethyl precursor 2.14 The structure of
1 was confirmed by NMR and high-resolution mass-spectroscopy
analysis.
2.3. Blockade study
A blocking dose of GHSR antagonist 2 significantly inhibited
[
11C]1 binding in the hypothalamus at 90 min after administration
In vitro inhibition binding assays demonstrated that 1 binds at
GHSR with a binding affinity Ki value of 22 nM.
of the radioligand suggesting that the binding in this region is spe-
cific and it is mediated by GHSR. As expected, the blockade in the
rest of the brain was minimal (Fig. 1).
Radiosynthesis of [11C]1 was performed by treating precursor 2
in DMF solution with [11C]methyl triflate at 80 °C (Scheme 2). The
radiochemical purity of [11C]1 after purification by HPLC and solid
phase extraction was determined to be 99%. The final product con-
tained only trace amount of impurities (acetonitrile <12 ppm by GC
The regional brain distribution and blockade results suggest
that [11C]1 is a radioligand with moderate specific cerebral uptake
and relatively high non-specific binding. The low specific signal can
be explained by the very low value of the GHSR density (Bmax, see
above). The high non-specific binding can be attributed to the high
lipophilicity of the radioligand [11C]1 (log D7.4 = 4.1 measured with
the ACD log PDB program16) that is borderline for the conventional
range for CNS radioligands (log D = 0–4). It would appear that a
better GHSR radioligand may be needed for monkey and human
brain PET studies. These future GHSR radioligands will likely re-
analysis; precursor <0.1 lg/batch by HPLC analysis). The total syn-
thesis time was 35 min from the end-of-bombardment (EOB) with
a non-decay-corrected radiochemical yield of 25% and specific
radioactivity at the end of synthesis (EOS) of 8300 750 mCi/lmol
(n = 4).
The identity of the radiotracer [11C]1 was confirmed by co-
injection with non-radioactive reference compound 1 onto an ana-
lytical HPLC system (HPLC traces in Supplementary data). The final
product [11C]1 was formulated as a sterile solution in saline with
8% alcohol.
quire binding affinity in the picomolar range and
lipophilicity.
a lower
2.4. Body organs study
2.2. In vivo experiments
Knowing that the GHSR is widely distributed in the body and
that there are available data on the ghrelin mRNA expression in
the body tissues,6 it was possible to determine if the radioactivity
distribution of [11C]1 matched the mRNA expression in the various
body organs. Seven organs of CD1 mice with wide range of ghrelin
expression were analyzed in dissection studies and the time-
uptake curves for [11C]1 in these organs were generated (Fig. 2).
The lungs, liver and adrenal glands were the organs with the great-
est uptake of radioactivity (Fig. 2, Table 2). A blocking dose of GHSR
2.2.1. Baseline experiments
The only known brain region with an elevated density of GHSR
is the hypothalamus with a Bmax value of 56 fmol/mg protein or
0.7 fmol/mg tissue in humans17,18 and rats,19 respectively. The
radioactivity accumulation for [11C]1 was determined as %ID/g tis-
sue in five regions of the CD1 mouse brain after intravenous injec-
tion of the radioligand (Table 1). GHSR is also present in pituitary,

2370
R. Potter et al. / Bioorg. Med. Chem. 19 (2011) 2368–2372
20
18
16
14
12
10
8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
6
4
2
0
Control
Block
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05 1.E+06
Figure 1. Comparison of regional brain uptake of
after injection in control and blocking experiments with
[
11C]1 in mice at 90 min
(1 mg/kg). Black
Ghrelin mRNA copy number/total mg RNA
2
bars—hypothalamus, white bars—the rest of brain. Data are mean SD (n = 5).
⁄P = 0.04, significantly different from controls; ⁄⁄P >0.05, insignificantly different
from controls (ANOVA single-factor analysis).
Figure 3. Correlation of distribution of [11C]1 radioactivity in CD1 mouse organs
(mean %ID/g tissue SD (n = 5)) with post-mortem ghrelin mRNA expression in
human subjects.6
3. Materials and methods
3.1. Chemistry
LUN
HRT
LIV
ADR
STOM
MUS
TEST
35
30
25
20
15
10
5
All chemicals were purchased from Sigma–Aldrich (Milwaukee,
WI) and used without further purification. Column flash
chromatography was carried out using E. Merck silica gel 60F
(230–400 mesh). Analytical thin layer chromatography (TLC) was
performed on plastic sheets coated with Silica Gel 60 F254
(0.25 mm thickness, Macherey-Nagel). 1H NMR (nuclear magnetic
resonance) spectra were recorded with a Varian-400 MHz in CDCl3.
The spectra were acquired at 293 K. The chemical shifts were
recorded on the ppm scale and were referenced to the internal
standard trimethylsilane (TMS) at dH 0 ppm. Coupling constant (J)
values were given in Hertz. Multiplicity was defined by s (singlet),
d (doublet), t (triplet), and m (multiplet).
0
0
20
40
60
80
100
Time, min
3.1.1. (S)-6-(4-Fluorophenoxy)-3-((1-methylpiperidin-3-
yl)methyl)-2-o-tolylquniazolin-4(3H)-one (1) (Scheme 1)
Figure 2. Time-uptake curves of [11C]1 in various body organs. Data are mean SD
(n = 5). Symbols: LUN—lungs, LIV—liver, ADR—adrenal gland, STOM—stomach,
HRT—heart, MUS—muscle, TEST—testis.
(S)-6-(4-Fluorophenoxy)-3-(piperidin-3-ylmethyl)-2-o-tolyl-
quinazolin-4(3H)-one (2)14 (188 mg, 0.423 mmol) was heated at re-
flux in a 4 mL mixture of formaldehyde (37%)/formic acid (98%)
(4:3) for 4 h under a nitrogen atmosphere. After cooling the reac-
tion mixture was concentrated in vacuo. The residue was taken
up in 10 mL of saturated aqueous sodium carbonate, and extracted
with dichloromethane (3 Â 5 mL). The combined organic layer was
washed with aqueous sodium carbonate (5 mL), dried over sodium
sulfate and concentrated in vacuo. The crude compound was then
purified by column chromatography on silica gel using hexane/
ethyl acetate/methanol (50:45:5) as eluent to yield 0.112 mg
(58%) as pale yellow solid, mp = 76–78 °C: 1H NMR (400 MHz,
CDCl3) d ppm 7.74 (d, J = 2.93 Hz, 1H), 7.72 (d, J = 2.19 Hz, 1H),
7.45 (dd, J = 8.79, 2.93 Hz, 1H), 7.37–7.42 (m, 2H), 7.29–7.35 (m,
2H), 7.07–7.10 (m, 4H), 4.12–4.19 (dd, J = 13.19, 7.33 Hz, 1H),
3.40–3.45 (m, 1H), 2.59 (t, J = 11.73 Hz, 2H), 2.37 (d, J = 12.46 Hz,
1H), 2.21 (s, 3H), 2.14 (d, J = 14.60 Hz, 3H), 1.70–1.88 (m, 2H),
1.37–1.60 (m, 2H), 1.19–1.25 (m, 1H), 0.75–0.92 (m, 1H); 13C
NMR (400 MHz, CDCl3) d 162.1, 160.7, 157.0, 154.5, 151.9, 142.9,
135.6, 134.4, 130.9, 129.9, 129.3, 128.0, 126.2, 125.6, 121.2, 116.9,
112.8, 59.7, 55.9, 48.0, 46.9, 36.5, 27.7, 24.6, 19.2; HRMS (TOF-
ESI) calcd for C28H28FN3O2 (MH+): 458.2238. Found: 458.2241.
Table 2
Whole body distribution data for
experiments with 2 (1 mg/kg)
[
11C]1 in CD1 mice, baseline and blocking
Baseline
Block*
10 min
30 min
90 min
90 min
Blood
Heart
Lung
Adrenals
Testis
Muscle
Liver
2.14 0.13
5.11 0.30
26.44 1.32
15.57 1.76
1.07 0.01
3.79 0.68
18.63 1.29
1.87 0.31
3.91 0.55
24.02 8.55
10.77 2.65
1.39 0.20
3.02 0.45
14.60 1.89
2.01 0.29
2.94 0.33
14.74 3.46
7.87 1.22
1.96 0.14
2.09 0.47
13.46 0.97
2.01 0.53
3.74 0.57
16.28 3.65
10.02 1.80
2.25 0.30
2.22 0.79
14.77 1.02
Data are mean of %injected dose/g tissue SD (n = 5).
*
P >0.05, insignificantly different from the 90 min baseline (ANOVA single-factor
analysis).
antagonist 2 did not demonstrate a significant effect on the [11C]1
accumulation in the blood, heart, lungs, adrenal glands, testes,
muscle, and liver at 90 min after administration of the radioligand.
This finding can be explained either by the absence of specific
binding or by a receptor reserve that is too large6 for the blocker
3.2. Radiochemistry
The semi-preparative high performance liquid chromatography
(HPLC) system consisted of a Waters model 610 pump, a Valco
injector, a Varian Prostar 325 LC detector set to 254 nm, a Bioscan
dose. The distribution of
[
11C]1 radioactivity correlated well
(R2 = 0.58) with the expression of ghrelin RNA6 (Fig. 3).

R. Potter et al. / Bioorg. Med. Chem. 19 (2011) 2368–2372
2371
Flow-Count PMT radioactivity detector. Analytical HPLC was per-
formed using a Varian Prostar 210 pump with a Prostar 410 Auto-
sampler, a Varian Prostar 325 LC detector set to 254 nm, a Bioscan
Flow-Count PMT radioactivity detector. Gas chromatography (GC)
was performed with a Varian 3800 GC and a FID detector. The
GC column is a Phenomenex ZB-WAX 30 m column, 0.25 mm ID,
radioactivity content was counted along with the tissue samples.
The percent of injected dose per gram of tissue (%ID/g tissue)
was calculated. All animal protocols were approved by the Animal
Care and Use Committee of the Johns Hopkins University.
3.4.2. Blocking dissection studies in mice
0.25
l
m film. All HPLC and GC were recorded and analyzed with
In vivo GHSR blocking studies were performed by subcutaneous
administration of 1 mg/kg of 2 followed 15 min later by an intrave-
nous injection of the radiotracer (0.1 mCi in 0.2 mL saline; specific
Varian Galaxie Chromatography Data System software (version
1.9.302.952). A dose calibrator (Capintec 15R) was used for all
radioactivity measurements. [11C]Methyl iodide was prepared
from 11CO2 using a Tracerlab FX MeI module (General Electric)
and a PETtrace biomedical cyclotron (General Electric).
radioactivity ꢀ4000 mCi/
lmol). Blocker 2 was dissolved in a vehi-
cle solution (saline/alcohol 9:1) and administered in a volume of
0.1 mL. Control animals were injected with 0.1 mL of the vehicle
solution. Ninety minutes after administration of the tracer, brain
tissues were harvested, and the radioactivity content was deter-
mined. There were five animals in each, baseline and blockade
group.
3.2.1. Synthesis of (S)-6-(4-fluorophenoxy)-3-((1-[11C]methyl-
piperidin-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one ([11C]1)
Precursor 214 (approx. 1 mg) was dissolved in 100
lL of anhy-
drous DMF and capped in a small V-vial. [11C]Methyl triflate was
swept by argon flow into the vial. After the radioactivity reached
a plateau, the vial was assayed in the dose calibrator and then
4. Conclusion
In summary, (S)-6-(4-fluorophenoxy)-3-((1-[11C]methylpiperi-
din-3-yl)methyl)-2-o-tolylquinazolin-4(3H)-one ([11C]1), a radioli-
gand with GHSR binding affinity Ki = 22 nM, has been synthesized.
In the mouse brain, [11C]1 specifically accumulated in the hypo-
thalamus, a region with elevated density of GHSR, whereas the up-
take of [11C]1 in all other brain regions with low densities of GHSR
was non-specific. The whole body distribution of [11C]1 in CD1
mice matched well the expression of ghrelin mRNA in various body
regions. However, blockade of [11C]1 with compound 2 in the
whole body did not demonstrate a significant difference the distri-
bution of radioactivity when compared to the baseline whole body
study.
heated at 80 °C for 5 min. Water (200 lL) was added and the solu-
tion was injected onto the semi-preparative HPLC column (Waters
XBridge C-18 column, 10 Â 150 mm; mobile phase: 410:590:1
v/v/v CH3CN/0.1 M ammonium formate/triethylamine, 12 mL/min).
The retention time of normethyl precursor was 3.2 min. The prod-
uct peak, having retention time of 7.9 min, was collected in 50 mL
of HPLC water. The water solution was transferred through a
Waters C-18 Oasis HLB Plus LP, 30 mg cartridge. The product was
eluted with 1 mL ethanol into a vial and diluted with 10 mL of sal-
ine. The final product [11C]1 was then analyzed by analytical HPLC
(Waters XBridge C-18, 4.6 Â 100 mm, mobile phase: 410:590:1
v/v/v CH3CN/0.1 M aqueous ammonium formate/triethylamine,
2.5 mL/min, Rt = 5.3 min) to determine the identity, radiochemical
purity and the specific radioactivity at the end of synthesis.
The results of cerebral distribution of [11C]1 in CD1 mice sug-
gest that the radioligand imaging properties (specific bind-
ing = 38%) may not be sufficient for further brain studies in
monkey and human subjects. Future development of cerebral
GHSR radioligands should target a compound with picomolar bind-
ing affinity and moderate lipophilicity.
3.3. In vitro binding assay
The in vitro inhibition binding assay of 1 was performed com-
mercially by Ricerca Biosciences, (Concord, OH) under conditions
similar to those previously published.17,20 This assay measures
binding of [125I]ghrelin (human) to human GHSR. CHO-K1 cells sta-
bly transfected with a plasmid encoding human GHS receptors are
used to prepare membranes in modified HEPES pH 7.4 buffer using
standard techniques. A 0.25 mg aliquot of membrane was incu-
bated with 0.03 nM [125I]ghrelin (human) for 60 min at 25 °C.
Non-specific binding was estimated in the presence of 0.1 mM
ghrelin (human). Radioactivity trapped onto the filters was assessed
using liquid scintillation spectrometry. The assays were done
in duplicate at multiple concentrations of the test compounds.
Binding assay results were analyzed using a one-site competition
model, and IC50 curves were determined by a non-linear, least
squares regression analysis using MathIQ™ (ID Business Solutions
Ltd, UK). The Ki value was calculated using the Cheng–Prusoff
equation.21
Acknowledgments
The authors would like to thank Ms. Judy Buchanan for editorial
assistance. This research was supported in part by the Division of
Nuclear Medicine of Johns Hopkins University School of Medicine
and by NIH Grants MH079017 and DA020777 (A.G.H.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.02.021.
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