Synthesis and biodistribution of [11C]R116301, a promising PET ligand for central NK1 receptors

Article abstract of DOI:10.1016/j.bmc.2004.12.019

N1-(2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl) [carbonyl-¹¹C]benzoyl]hexahydro-4-pyridinyl}piperazino)acetamide ([¹¹C]R116301) was prepared and evaluated as a potential positron emission tomography (PET) li

Full text of DOI:10.1016/j.bmc.2004.12.019

Bioorganic & Medicinal Chemistry 13 (2005) 1579–1586
Synthesis and biodistribution of [11C]R116301, a promising
PET ligand for central NK1 receptors
M. Van der Mey,a,* C. G. M. Janssen,b F. E. Janssens,b M. Jurzak,b, X. Langlois,b
F. M. Sommen,b B. Verreet,b A. D. Windhorst,a J. E. Leysenc and J. D. M. Herscheida
aVU University Medical Center, Department of Nuclear Medicine and PET Research, Location Radionuclide Center,
De Boelelaan 1085c, 1081 HV Amsterdam, The Netherlands
bJohnson & Johnson Pharmaceutical Research & Development, Turnhoutseweg 30, 2340 Beerse, Belgium
cOnderzoekinstituut Neurowetenschappen/Research Institute Neurosciences, Vrije Universiteit, c/o Dept. of Anatomy,
Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
Received 2 July 2004; revised 3 December 2004; accepted 9 December 2004
Available online 11 January 2005
Abstract—N1-(2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)[carbonyl-11C]benzoyl]hexahydro-4-pyridinyl}-
piperazino)acetamide ([11C]R116301) was prepared and evaluated as a potential positron emission tomography (PET) ligand for
investigation of central neurokinin(1) (NK1) receptors. 1-Bromo-3,5-di(trifluoromethyl)benzene was converted in three steps into
3,5-di(trifluoromethyl)[carbonyl-11C]benzoyl chloride, which was reacted with N1-(2,6-dimethylphenyl)-2-{4-[(2R,4S)-2-benzylhexa-
hydro-4-pyridinyl]piperazino}acetamide providing [11C]R116301 in 45–57% decay-corrected radiochemical yield. The total synthesis
time, from end of bombardment (EOB) to the formulated product, was 35 min. Specific activity (SA) was 82–172 GBq/lmol (n = 10)
at the end of synthesis. N1-([4-3H]-2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)benzoyl]hexahydro-4-
pyridinyl}piperazino)acetamide ([3H]R116301) was also synthesized (SA: 467 GBq/mmol). The Bmax for [3H]R116301 measured
in vitro on Chinese hamster ovary cell membranes stably transfected with the human NK1 receptor was 19.10 1.02 pmol/mg pro-
tein with an apparent dissociation constant of 0.08 0.01 nM. Ex vivo, in vivo and in vitro autoradiography studies with
[3H]R116301 in gerbils demonstrated a preferential accumulation of the radioactivity in the striatum, olfactory tubercule, olfactory
bulb and locus coeruleus. In vivo, the biodistribution of [11C]R116301 in gerbils revealed that the highest initial uptake is in the lung,
followed by the liver and kidney. In the brain, maximum accumulation was found in the olfactory tubercules (1.10 0.08 injected
dose (ID)/g 20 min post injection (p.i.)) and the nucleus accumbens (1.00 0.12 ID/g 10 min p.i.). Tissue/cerebellum concentration
ratios for striatum and nucleus accumbens increased with time due to rapid uptake followed by a slow wash out (1.29 and 1.64,
respectively, 30 min p.i.). Atissue to cerebellum ratio of 1.33 and 1.62 was also observed for olfactory bulb and olfactory tubercules,
respectively (20 min p.i.). In summary, [11C]R116301 appears to be a promising radioligand suitable for the visualization of NK1
receptors in vivo using PET.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
tential analgesic compounds, recent clinical and preclin-
ical research has provided evidence that NK1 receptor
antagonists might be important new tools in anxiolytic
and antidepressant therapy.1,2 If confirmed by further
controlled clinical studies, this will represent a mecha-
nism of action distinct from all existing antidepressant
agents. An NK1 antagonist, aprepitant, is in clinical
use as anti-emetic agent for chemo- and radiotherapy-
induced emesis.3
Selective, nonpeptide antagonists for tachykinin recep-
tors first became available 10 years ago. Of the three
known tachykinin receptors, drug development focused
most intensively on the substance P-preferring receptor,
neurokinin(1) (NK1). Although originally studied as po-
Keywords: [11C]R116301; NK1 Antagonist; Autoradiography; PET.
N1-(2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-
di(trifluoromethyl)benzoyl]hexahydro-4-pyridinyl}pipe-
razino)acetamide (R116301, Fig. 1), a centrally active
NK1 antagonist, has been evaluated extensively in
*
Corresponding author. Tel.: +31 20 444 9720; fax: +31 20 444
9121; e-mail: mmeij@rnc.vu.nl
Present address: CADS Biomedical Research, Merck KGaA, Darms-
tadt, Germany.
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.019

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M. Van der Mey et al. / Bioorg. Med. Chem. 13 (2005) 1579–1586
several pharmacological assays in various species,
in vitro as well as in vivo.4–6 It shows high affinity and
selectivity for the NK1 receptor. R116301 exhibits
subnanomolar affinity for the human NK1 receptor
(Ki: 0.45 nM) and over 200-fold selectivity towards
NK2 and NK3 receptors. Since tachykinin receptors
are known to exhibit strong species dependence in phar-
macology, the NK1 affinity of R116301 was investigated
on tissue membrane preparations from forebrains of
guinea pigs, gerbils, ferrets and rats. Relative to its affin-
ity for the human NK1 receptor, R116301 showed about
10 times lower affinity for the gerbil, ferret and guinea
pig NK1 receptors (Ki: 6.4, 8.3 and 13 nM, respectively)
and 200 times lower affinity for the rat NK1 receptor (Ki:
98 nM). This shows that the rat is not a suitable species
for investigation of the biological properties of
R116301. In vivo, the compound inhibited substance
P-induced peripheral effects (skin reactions and plasma
extravasation in guinea pigs) and a central effect
(thumping in gerbils) at low doses (0.08-0.16 mg/kg, sub-
cutaneous or intraperitoneal injection), reflecting its
high potency as an NK1 receptor antagonist and excel-
lent brain disposition.
therapeutic and side-effects of the drugs. For these rea-
sons N1-(2,6-dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-
[3,5-di(trifluoromethyl)[carbonyl-11C]benzoyl]hexahydro-
4-pyridinyl}piperazino)acetamide ([11C]R116301) was
developed.7 PET experiments with [18F]SPA-RQ in hu-
mans have already confirmed the usefulness of labelled
antagonists for the quantification of NK1 receptor occu-
pancy with various doses of NK1 receptor antagonists.8
[11C]GR205171 was used successfully for the in vivo char-
acterization of NK1 receptor binding in rhesus monkey.9
This paper describes the synthesis and pre-clinical
evaluation of [11C]R116301. The biodistribution of
[11C]R116301 was investigated in gerbils. Furthermore,
R116301 was used for saturation radioligand binding
analysis and N1-([4-3H]-2,6-dimethylphenyl)-2-(4-{(2R,
4S)-2-benzyl-1-[3,5-di(trifluoromethyl)benzoyl]hexahydro-
4-pyridinyl}piperazino)acetamide ([3H]R116301) was
prepared for examining ex vivo, in vivo and in vitro
autoradiography on the gerbil brain.
2. Results and discussion
2.1. Radiosynthesis
H
CF3
N
Asynthesis route was recognized by which a 11C isotope
could be introduced converting 1-bromo-3,5-di(trifluo-
romethyl)benzene in three steps into 3,5-di(trifluoro-
methyl)[carbonyl-11C]benzoyl chloride ([11C]4), which
was then reacted with N1-(2,6-dimethylphenyl)-2-{4-
[(2R,4S)-2-benzylhexahydro-4-pyridinyl]piperazino}acet-
amide (5) providing [11C]R116301 as illustrated in
Scheme 1.10
N
N
O
N
F3C
O
R116301
Figure 1. Molecular structure of R116301.
Positron emission tomography (PET) imaging is a tech-
nique that can be used to investigate the occupancy of
the central receptors by new drugs in function of dose
and time, in humans in a noninvasive manner. The
receptor occupancy can be evaluated in relation to the
2.1.1. 3,5-Di(trifluoromethyl)[carboxy-11C]benzoicaidc
([11C]3). Synthesis of benzoic acid [11C]3 via a Grignard
reaction was carried out as follows (Scheme 1). First, a
1 M stock-solution of 3,5-di(trifluoromethyl)phenyl-
magnesium bromide (1) in diethyl ether was prepared
CF3
CF3
CF3
CF3
iii
i
ii
F3C
Br
F3C
R1
F3C
11CO2H
F3C
11COCl
R1 = MgBr (1) or Li (2)
3
4
H
N
CF3
CF3
N
N
iv
O
N
F3C
11COCl
F3C
11C
O
H
N
4
N
N
O
HN
[
11C]R116301
5
Scheme 1. Preparation of [11C]R116301. Reaction conditions: (i) 1; Mg, diethyl ether 2; n-BuLi, diethyl ether; (ii) 1. [11C]CO2, 2. HCl, diethyl ether;
(iii) oxalyl chloride, DCM; (iv) 5, DMF, TEA.

M. Van der Mey et al. / Bioorg. Med. Chem. 13 (2005) 1579–1586
1581
Table 1. Influence of temperature and time on the synthesis of
11C]R116301
Entry
from 1-bromo-3,5-di(trifluoromethyl)benzene and mag-
nesium. Next, Grignard reagent 1 was reacted with
[11C]CO2 and subsequently treated with 1 M HCl in
diethyl ether affording compound [11C]3. The decay-cor-
rected radiochemical yield of compound [11C]3 was
26 2% (n = 4) at a reaction temperature of 20 ꢁC. A
similar reaction was performed with commercially avail-
able phenylmagnesium bromide (1 M in tetrahydro-
furan (THF)) for comparison. Under the same condi-
tions, [carboxy-11C]benzoic acid was obtained in
74 5% yield (n = 4). This large difference in yield is
probably caused by diminished reactivity of Grignard
reagent 1 in comparison to phenylmagnesium bromide
due to the two electron withdrawing trifluoromethyl
moieties.
[
T (ꢁC)
Time (min)
[
11C]R116301a (%)
1
2
3
4
5
6
7
8
20
0
1
1
32
46
25
20
17
15
59
76
4
5
4
3
4
3
6
6
0 ! 60
0 ! 60
0 ! 80
0 ! 80
ꢀ10
5
10
5
10
1
ꢀ20
1
a Conversion of benzoyl chloride [11C]4 into [11C]R116301 (n P 4).
Solvent: DMF.
as summarized in Table 1. Changing the temperature
from 20 to 0 ꢁC led to a considerable increase in the
[11C]R116301 yield (compare entries 1 and 2, Table 1).
Additional heating of the reaction mixture from 0 to
60 and 80 ꢁC proved to be unfavourable due to product
decomposition (entry 2 vs entries 3–6, Table 1). Com-
parison of entries 1, 2, 7 and 8 reveals that the optimal
temperature was ꢀ20 ꢁC. At this temperature,
[11C]R116301 was obtained in a yield of 74 6% start-
ing from benzoyl chloride [11C]4.
Because of the low yields, compound [11C]3 was pre-
pared via an alternative synthesis route (Scheme 1). At
a temperature of ꢀ65 ꢁC, 1-bromo-3,5-di(trifluoro-
methyl)benzene was converted into the corresponding
lithium salt 2 with n-butyllithium.11 Next, [11C]CO2 was
passed through the solution containing derivative 2,
which after the addition of 1 M HCl in diethyl ether,
gave compound [11C]3 in almost quantitative decay-
corrected radiochemical yield (97 2%, n = 10).
2.1.2. 3,5-Di(trifluoromethyl)[carbonyl-11C]benzoyl chlo-
ride ([11C]4). Compound [11C]3 was transformed into
benzoyl chloride [11C]4 by treatment with oxalyl
chloride in dichloromethane (DCM). The conversion
yield was >84% (n = 10).
After purification by semipreparative high performance
liquid chromatography (HPLC) and isolation by
solid phase extraction, [11C]R116301 was produced in
an overall decay-corrected radiochemical yield of 45–
57% (n = 10). The total synthesis time, from EOB to
formulated product, was 35 min. Specific activity was
82–172 GBq/lmol (n = 10) at the end of synthesis.
After evaporation of the excess oxalyl chloride at 65 ꢁC,
benzoyl chloride [11C]4 was distilled into another vial
containing N,N-dimethylformamide (DMF). Unfortu-
nately, on average only 60% of the total amount of
benzoyl chloride [11C]4 was collected in the second vial
(n = 5). It was therefore decided to focus on developing
a one-pot procedure for the synthesis of [11C]R116301.
2.1.4. [3H]R116301. [3H]R116301 was obtained via
bromo-tritium exchange from N1-(4-bromo-2,6-dimeth-
ylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluorometh-
yl)benzoyl]-4-piperidyl}piperazino)acetamide4 (6) as
shown in Scheme 2. After purification the resulting
[3H]R116301 solution had a radiochemical purity of
98.3%, a specific activity of 467 GBq/mmol and a radio-
chemical concentration of 15.6 MBq/mL.
2.1.3. [11C]R116301. First, the influence of the solvent on
the formation of [11C]R116301 was examined. Asolu-
tion of N1-(2,6-dimethylphenyl)-2-{4-[(2R,4S)-2-benzyl-
hexahydro-4-pyridinyl]piperazino}acetamide (5) in
DCM, THF or DMF including triethylamine (TEA)
was added to benzoyl chloride [11C]4 at 20 ꢁC. DMF
was found to be the most suitable solvent resulting in
a conversion of 32 4% (n = 4). The conversions ob-
tained with DCM and THF were 0% and 10 2%,
respectively (n = 2).
2.2. Saturation binding
The binding of [3H]R116301 to membranes of Chinese
hamster ovary cells stably transfected with the human
NK1 receptor was saturable and the specific binding dis-
played a typical hyperbolic saturation curve (Fig. 2).
Analysis of the binding data of four independent exper-
iments using iterative nonlinear curve fitting indicated
that the [3H]R116301 binding was best fitted to a single
Next, compound 5, dissolved in DMF and TEA, was
added to benzoyl chloride [11C]4 at various temperatures
H
H
CF3
CF3
N
N
N
N
N
N
i
O
O
Br
3H
N
N
F3C
F3C
O
O
6
[3H]R116301
Scheme 2. Preparation of [3H]R116301. Reaction conditions: (i) tritium gas, 10% Pd/C, THF, TEA, 1 h.

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M. Van der Mey et al. / Bioorg. Med. Chem. 13 (2005) 1579–1586
30
20
10
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0
Free radioligand concentration (nM)
200
150
100
50
0
0
5
10
15
20
Bound (pmol/mg protein)
Figure 3. Ex vivo, in vivo and in vitro autoradiographic characteriza-
tion of R116301. (A) NK1 receptor occupancy by R116301 in gerbil
striatum 1 h after subcutaneous administration measured ex vivo with
[3H]-[Sar9, Met(O2)11]-substance P. (B) In vivo distribution of
[3H]R116301 in gerbil brain 1 h after intravenous administration of
370 kBq. STR: Striatum, OB: olfactory bulb, LC: locus coeruleus, CX:
cortex. (C) In vitro assessment of the specificity of [3H]R116301
binding in gerbil brain coronal sections at the level of the striatum and
olfactory tubercule. Nonspecific binding was measured in presence of
10 lM substance P.
Figure 2. Saturation analysis of the in vitro binding of [3H]R116301 to
membranes of Chinese hamster ovary cells stably expressing the
human NK1 receptor. The total (h), specific (d) and nonspecific
binding (n) are expressed as a function of the concentration of
radiolabelled drug added to the system. Below a Scatchard plot is
shown in which the ratio of specifically bound and free drug as a
function of the amount of specifically bound drug. Graphs of one
representative experiment are shown, the experiment was repeated
independently four times, binding parameters were derived by iterative
nonlinear curve fitting on the separate saturation curves and averaged;
mean values SEM (n = 4) are: Kd: 0.8 0.01 nM, Bmax
19.10 1.02 pmol/mg protein.
:
The ED50 value (95% confidence limit) generated from
the curve was 0.07 mg/kg (0.05–0.10). Figure 3B shows
that, after intravenous administration, [3H]R116301
is distributed preferentially to NK1 receptor rich areas
such as the striatum, olfactory bulb and locus coeruleus.
The specificity of [3H]R116301 binding to NK1 receptors
is illustrated in Figure 3C showing that addition of
10 lM substance P in the incubation medium prevented
totally the specific binding of [3H]R116301 to gerbil stri-
atum. The results demonstrate that [3H]R116301 gives
good brain penetration and that the binding in NK1 rich
areas is reversible. These are important features of a po-
tential PET ligand upon labelling with 11C. Although
Megens et al.6 describe that R116301 reaches its maxi-
mal effect 5 min after intravenous (iv) injection, the
gerbils in these experiments were sacrificed 60 min after
the iv administration of [3H]R116301 to diminish the
amount of nonspecific binding as much as possible.
Due to the short half-life of 11C this is less feasible for
[11C]R116301.
population of sites with Bmax = 19.10 1.02 pmol/mg
protein and a dissociation equilibrium constant of
Kd = 0.08 0.01 nM (n = 4). These results, together
with an extensive pharmacological evaluation reported
by Megens et al.,6 show that R116301 is an NK1 antag-
onist showing, saturable and selective binding to the
NK1 receptor. In in vitro and in behavioural studies,
mutual antagonism between R116301 and substance P
was observed.
2.3. Ex vivo, in vivo and in vitro autoradiography
Individual values and mean curve illustrating the occu-
pancy of NK1 receptors by R116301 in the gerbil stria-
tum are shown in Figure 3A. Occupancy of NK1
receptors by R116301 was detectable for dosages
P0.04 mg/kg and was total at dosages P0.63 mg/kg.

M. Van der Mey et al. / Bioorg. Med. Chem. 13 (2005) 1579–1586
1583
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
2.4. Biodistribution experiments in gerbils
5 min
10 min
20 min
30 min
Figure 4 summarizes the distribution of [11C]R116301 in
the blood, brain and selected peripheral organs 5, 10, 20
and 30 min after the injection. An additional time point
of 60 min was also considered. However, since the max-
imum uptake was already observed at 10 min post injec-
tion (p.i.), blood values are very low at 30 min p.i., and
the short half-life of 11C it was concluded that the
60 min time point would not contribute any useful data.
The uptake of [11C]R116301 is rapid, in most tissues it
reaches its maximum concentration at 10 min after tra-
cer injection. The accumulation of radioactivity was
highest in the lungs, followed by the liver and kidney.
The maximum brain uptake was observed in the olfac-
tory tubercules (1.10 0.08 injected dose (ID)/g 20 min
p.i.) and the nucleus accumbens (1.00 0.12 ID/g
10 min p.i.). To determine the target-to-nontarget ratios
in the brain, the amount of radioactivity in selected
brain regions were compared to that in the cerebellum,
an area devoid of NK1 receptors. As a result of rapid
uptake in the striatum and nucleus accumbens and slow
clearance from these tissues compared to the cerebellum,
the target-to-nontarget ratios increased with time (Fig.
5), which is a strong indication of selective binding to
NK1 receptors. At 30 min after injection, tissue/cerebel-
lum ratios of 1.29 0.02 and 1.64 0.07 were observed
for the striatum and nucleus accumbens, respectively.
The tissue/cerebellum ratios found for the olfactory bulb
and olfactory tubercules reached their corresponding
Olfact. bulb
Olfact. tubercules Nucl. accumbens
Striatum
Figure 5. Tissue to cerebellum ratio of [11C]R116301 in gerbil olfactory
bulb, olfactory tubercules, nucleus accumbens and striatum. Data are
means SEM (n = 4).
maximum values of 1.35 0.17 and 1.66 0.16 at
20 min p.i. (Fig. 5). In comparison, a similar degree of
uptake was displayed in the remainder of the dissected
brain areas and cerebellum throughout the time course
of the experiment (Fig. 4). Atissue/cerebellum ratio of
approximately 1 with no apparent maximum is indica-
tive of nonspecific binding.
The results indicate that [11C]R116301 is specifically re-
tained in the areas of high NK1 receptor density, that is,
the striatum, olfactory bulb, tuberculum olfactorium
and nucleus accumbens, in agreement with the areas of
high NK1 receptor density detected in radioligand auto-
radiography on brain sections, where specificity of
[3H]R116301 binding is demonstrated by displacement
with unlabelled substance P.
11
5 min
10 min
20 min
10
9
3. Conclusion
30 min
8
7
6
5
4
3
2
1
0
In summary, [11C]R116301 was synthesized in high
radiochemical yield, purity and specific activity by
means of a one-pot procedure including 4 (radio)chem-
ical steps. The ex vivo, in vivo and in vitro studies dem-
onstrated that labelled R116301 has high affinity for the
NK1 receptor, sufficiently enters the gerbil brain and
accumulates particularly in striatum, olfactory tuber-
cules, nucleus accumbens, olfactory bulb and locus coe-
ruleus, which are demonstrated to be areas of high NK1
receptor density. [11C]R116301 appears to be a promis-
ing radioligand suitable for the visualization of NK1
receptors in vivo using PET and will be evaluated as
such in human volunteers.
Blood
Heart
Lungs
Liver
Spleen
Kidney
5 min
1.25
1.00
0.75
0.50
0.25
0.00
10 min
20 min
30 min
4. Materials and methods
4.1. Chemistry
R116301 and the precursors for radiolabelling, com-
pound 5 and 6, were kindly provided by Johnson and
Johnson Pharmaceutical Research and Development
(Beerse, Belgium). DMF, TEA, n-butyllithium (1.6 M
solution in hexane), phenylmagnesium bromide (1.0 M
in THF), 1-bromo-3,5-di(trifluoromethyl)benzene, dieth-
yl ether, hydrogen chloride (1.0 and 2.0 M solution in
Figure 4. Tissue distribution of radioactivity after intravenous injec-
tion of [11C]R116301 in gerbils expressed as % ID/g. Data are
means SEM (n = 4).

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M. Van der Mey et al. / Bioorg. Med. Chem. 13 (2005) 1579–1586
diethyl ether) and oxalyl chloride (2.0 M solution in
DCM) were obtained from Sigma–Aldrich Chemie BV
(Zwijndrecht, The Netherlands). Diisopropylamine
(DIPA) was purchased from Merck-Schuchardt.
Ammonium acetate, acetonitrile (ACN) and methanol
(MeOH) were purchased from JT Baker Chemicals
B.K. (Deventer, The Netherlands). 10% Pd/C was ob-
tained from Merck and Co. (Darmstadt, Germany).
Diethyl ether was freshly distilled from LiAlH4 before
cal yield found for [carboxy-11C]benzoic acid was
74 5% (n = 4, decay-corrected).
4.1.1.2. Method B. The commercial solvent (hexane)
from 1.6 M n-butyllithium (10 lL, 160 lmol) was evap-
orated by bubbling helium through the solution at room
temperature (rt). Diethyl ether (300 lL) was added and
the reaction vial was cooled to ꢀ65 ꢁC. Subsequently,
a solution of 1-bromo-3,5-di(trifluoromethyl)benzene
(30 lL, 174 lmol) in diethyl ether (50 lL) was added.
After 5 min at ꢀ65 ꢁC, [11C]CO2, carried by a stream
of helium with a flow of 10 mL/min, was bubbled into
the mixture. After 2.5 min, the reaction was quenched
by the addition of a hydrogen chloride solution in
diethyl ether (160 lL, 160 lmol). The reaction mixture
was analyzed by HPLC as described above. Compound
[11C]3 was synthesized in a decay-corrected radiochemi-
cal yield of 97 2% (n = 10).
˚
use. DMF and TEAwere stored over 4 A molecular
sieves. All other chemicals and solvents were used as
received unless otherwise stated. Reactions were
performed under anhydrous conditions under
a
He-atmosphere. [11C]Carbon dioxide was produced by
the 14N(p,a)11C nuclear reaction using an IBACyclone
18/9. Semipreparative HPLC was performed with a
Jasco PU-1587 preparative 50 mL/min pump (Maars-
sen, The Netherlands), an in-line Jasco PU-1575 ultravi-
olet (UV) detector (wavelength 265 nm) and
a
homemade flow-through radioactivity detector. Jasco-
Borwin 1.5 HSS 1500 software was applied for data
acquisition and processing. Radioactivity was quantified
with a VDC-405 dose calibrator (Veenstra Instruments,
Joure, The Netherlands). HPLC for analysis and quality
control was performed with a LKB/Pharmacia 2249
pump, an in-line LKB/Pharmacia VWM 2141 UV detec-
tor (wavelength 265 nm), a flow-through NaI(Tl) crystal
scintillation detection system and Gina-Star version 2.12
(Raytest) for data acquisition and processing. All reac-
tions were carried out in homemade, remotely con-
trolled devices.12 In the case of tritium labelled
R116301, preparative HPLC was performed with only
on-line UV detection. For analytical HPLC, radioactiv-
ity detection was performed on a Berthold Radioactivity
Monitor LB 506 C system with a flow-through cell of
0.500 mL. The eluate was mixed with Pico-Fluor TM
30 (Packard, used as a scintillation cocktail) in an
LKB ultrograd mixing unit. Radioactivity of known ali-
quots was measured by liquid scintillation counting
(Packard Tri-Carb 4530), using Ultima Gold (Packard)
as a scintillation cocktail.
4.1.2. Radiosynthesis of 3,5-di(trifluoromethyl)[car-
bonyl-11C]benzoyl chloride ([11C]4). After quenching the
reaction mixture (see Method B), oxalyl chloride in
DCM (160 lL, 320 lmol) was added and the mixture
was heated to 50 ꢁC for 3 min in a closed vial. The reac-
tion mixture was cooled to 20 ꢁC in order to reduce the
pressure in the reaction vial before opening the exhaust,
and then concentrated to dryness under a stream of he-
lium gas by heating at 65 ꢁC. Subsequently, the reaction
vial was cooled to 20 ꢁC and ACN was added (1 mL).
The resulting solution was analyzed by HPLC (column:
Chrompack C18, 10 lm, 250 · 4.6 mm; flow: 1.0 mL/
min; eluent: MeOH/H2O/trifluoroacetic acid 80/20/0.1
(v/v/v); k = 254 nm). Retention times for compound
[11C]3 and benzoyl chloride [11C]4 were 5.20 and
6.43 min, respectively. Analogue [11C]3 was transformed
into benzoyl chloride [11C]4 for >84% (n = 10).
4.1.3. Radiosynthesis of [11C]R116301. Following the
preparation of benzoyl chloride [11C]4, a solution of
compound 5 (2–3 mg, 4.8–7.1 lmol) in DCM, THF or
DMF (200 lL) and TEA(40 lL) was added to the con-
centrated reaction mixture (see Table 1). Next, the
resulting solution was diluted with HPLC solvent
(1.5 mL). The reaction mixture was analyzed by HPLC
(column: Kromasil C18, 10 lm, 250 · 4.6 mm; flow:
1.0 mL/min; eluent: MeOH/H2O/DIPA80/20/0.2 (v/v/
v); k = 265 nm). The maximum conversion of benzoyl
chloride [11C]4 into [11C]R116301 (76 6%; Rt
[11C]R116301 = 11.87 min) was observed by adding
compound 5 at ꢀ20 ꢁC.
4.1.1. Radiosynthesis of 3,5-di(trifluoromethyl)-
[carboxy-11C]benzoicacid ([ 11C]3)
4.1.1.1. Method A. [11C]CO2, carried by a stream of
helium with a flow of 10 mL/min, was bubbled into a
solution of 3,5-di(trifluoromethyl)phenylmagnesium
bromide (1; 500 lL, 1 M)13 in diethyl ether. After
4 min, the reaction was quenched by the addition of a
hydrogen chloride solution in diethyl ether (500 lL,
1.0 M). An aliquot was checked by analytical HPLC
(column: Biorad Aminex ion exclusion HPX-87H,
300 · 7.8 mm; flow: 1.0 mL/min; eluent: ACN/water 1/
9 (v/v); k = 254 nm). Two radioactive peaks correspond-
ing to [11C]CO2 and compound [11C]3 were observed
(Rt = 6.0 min and Rt = 15.3 min, respectively). The
radiochemical yield found for compound [11C]3 was
26 2% (n = 4, decay-corrected).
Using the optimal reaction conditions for the synthe-
sis of [11C]R116301, the final mixture diluted with
HLPC eluent (1.5 mL) was purified by semipreparative
HPLC (column: Kromasil C18, 10 lm, 250 · 21 mm;
flow: 10 mL/min; eluent: MeOH/H2O/DIPA88/12/0.2
(v/v/v),
k = 265 nm).
The
fraction
containing
[11C]R116301 (Rt = 11.76 min) was collected in 50 mL
of 50 mM sterile NaOH solution and the desired prod-
uct was isolated by solid phase extraction using a Sep-
Pak tC18 plus. After washing with 20 mL of water, the
tC18 cartridge was sequentially eluted with 1.3 mL eth-
anol and 1.5 mL NaCl/NaH2PO4 solution. The resulting
Asimilar experiment was performed with phenylmagne-
sium bromide (0.5 mL, 1.0 M) for comparison. Both
[11C]CO2 and [carboxy-11C]benzoic acid (Rt = 17.3 min)
were detected in the reaction mixtures. The radiochemi-

M. Van der Mey et al. / Bioorg. Med. Chem. 13 (2005) 1579–1586
1585
solution was filtered through a DLL filter into 12 mL of
sterile NaCl/NaH2PO4 solution. The decay-corrected
radiochemical [11C]R116301 yield was 45–57% (n = 10)
and chemical and radiochemical purities were >96% as
determined by analytical HPLC as described above.
Specific activity was between 82 and 172 GBq/lmol
(n = 10) at the end of synthesis. The overall synthesis
time, from EOB to formulated product, was 35 min.
in 5 mM TrisHCl, pH 7.4 using an Ultra Turrax homo-
genizer (Janke and Kunkel IKALabortechnik, Staufen
im Breisgau, Germany) and recentrifuged at 29,900g
for 20 min at 4 ꢁC. The final pellet was suspended in
50 mM TrisHCl, pH 7.40. Protein concentration was
determined following the Bradford procedure. The
membranes were divided in 1 mL aliquots and stored
at ꢀ70 ꢁC.
4.1.4. Radiosynthesis of [3H]R116301. Amixture of com-
pound 6 dihydrochloride hydrate (1.25 mg, 1.51 lmol),
TEA(5 lL) and 10% Pd/C (5 mg) in dry THF
(0.75 mL) was reacted with tritium gas (230 GBq) for
1 h at rt in a closed vessel. Labile tritium was removed,
the residue was dissolved in a small amount of EtOH
and filtered to remove the catalyst. The solution was
concentrated under reduced pressure, the residue was
dissolved in DMF (200 lL) and purified by HPLC (col-
umn: Kromasil KR 100-10 RP18, 300 · 4.6 mm; flow:
2 mL/min; eluent: ACN/water/DIPA 70/30/0.2 (v/v/v),
k = 220 nm). The combined product fractions were con-
centrated in vacuo, coevaporated with EtOH (20 mL)
and diluted with EtOH to a final volume of 30.0 mL.
4.2.2. [3H]R116301 binding. Membranes were thawed on
ice and suspended in 50 mM TrisHCl, pH 7.40, supple-
mented with 1 mM ethylenebis(oxyethylenenitrilo)tetra-
acetic acid (EGTA), 2 mM MgCl2 and 0.1% bovine
serum albumin (BSA). Membranes were incubated with
[3H]R116301 in a final concentration ranging from 0.1
to 4 nM (R116301 was tritiated by Janssen Pharmaceu-
tica, specific activity was 467 GBq/mmol). Nonspecific
binding was determined as that remaining in the pres-
ence of 1 lM R164461 (N,N-dimethyl-(5-{[(2R,3S)-2-
({(1R)-1-[3,5-di(trifluoromethyl)phenyl]ethyl}oxy)-3-
phenyl-1,4-oxazinan-4-yl]methyl}-2H-1,2,3-triazol-4-yl)-
methanamine
hydrochloride).
Asolution
of
[3H]R116301, at a concentration of 50 times the final
incubation concentration, was made in dimethyl sulfox-
ide (DMSO). Part of this solution (10 lL) was added to
40 lL buffer. Incubation was started by addition of
400 lL of the membrane suspension and 50 lL of the
latter [3H]R116301 solution or blank. The protein con-
centration in the 0.5 mL final incubation volume was
5.2 lg. Incubation was run for 30 min in 25 ꢁC. Reac-
tion was stopped by addition of 5 mL ice-cold 50 mM
TrisHCl pH 7.40 and followed by rapid filtration over
GF/B glass fibre filters presoaked in 0.1% polyethylene
imine (for at least 1 h) using a 40-well multividor. The
filters were washed twice with ice-cold 50 mM TrisHCl
pH 7.40 to remove nonbound radioactivity and placed
in plastic mini-vials. Ultima Gold MV scintillation fluid
(3 mL) was added to each vial. Vials were vigorously
shaken and stored for at least 8 h at rt. Thereafter radio-
activity was counted in a Packard Tri-Carb 1600 liquid
scintillation counter. The counts were expressed in disin-
tegrations per minute (DPM) referring to Packard stan-
dard quench curves and internal standards. Saturation
binding curves were calculated by computerized nonlin-
ear curve fitting (GraphPadPrism 3 software), using the
equation of a rectangular hyperbola. The equilibrium
dissociation constant (Kd) and the maximal number of
binding sites (Bmax) were derived (see Fig. 2).
The specific activity (a) and radiochemical purity (b)
of the [3H]R116301 solution were determined by HPLC
((a) column: Hypersil ODS, 5 lm, 300 · 4.6 mm; flow:
1.0 mL/min; eluent: water/DIPA100/0.2 (v/v) to
MeOH/DIPA100/0.2 (v/v) in 30 min, followed by
10 min at the latter conditions; k = 264 nm; (b) column:
Kromasil KR 100-10 RP18, 300 · 4.6 mm; flow: 2 mL/
min; eluent: 1 M ammonium acetate/MeOH/ACN 10/
45/45 (v/v/v), k = 220 nm). The specific activity was
467 GBq/mmol and the radiochemical purity 98.3%.
The radioactivity concentration of the [3H]R116301
solution was 15.6 MBq/mL with a total yield of
468 MBq [3H]R116301 (66% radiochemical yield) as
determined by liquid scintillation counting.
4.2. [3H]R116301 binding studies
4.2.1. Cell culture and membrane preparation. Chinese
hamster ovary cells were stably transfected with the hu-
man neurokinin 1 receptor. The cells were maintained in
DulbeccoÕs modified Eagles medium (DMEM)/Nutrient
mixture HamÕs F12 (ratio 1:1) supplemented with 10%
heat inactivated fetal calf serum and an antibiotic solu-
tion containing 100 IU/mL penicillin, 100 lg/mL strep-
tomycin sulfate, 110 lg/mL pyruvic acid and 300 lg/
mL L-glutamine. Once a week, the cells are cultured in
a selective medium containing 800 lg/mL gentisic acid.
The cells were grown in 145 mm petri-dishes until 80–
90% confluency. Twenty-four hours before cell collec-
tion for membrane preparation, cells were stimulated
by adding 5 mM Na-butyrate to the medium. At the
day of membrane preparation, the medium was removed
and the cells were washed with phosphate buffered sal-
ine. The cells were scraped off the plates in 5 mL
50 mM tris(hydroxymethyl)aminomethane hydrochlo-
ride (TrisHCl), pH 7.40 using a rubber scraper. They
were pelleted by centrifugation at 23,500g for 10 min
at 4 ꢁC in a Sorvall-RC 5B centrifuge (DuPont Instru-
ments, Meyvis, Belgium). The pellets were homogenized
4.3. NK1 receptor occupancy by R116301 in gerbil
brain
Male Mongolian gerbils (40–60 g) were treated by sub-
cutaneous injections of vehicle or R116301 at six dos-
ages ranging from 0.0025 to 2.5 mg/kg body weight
(dosages: 0.0025, 0.01, 0.04, 0.16, 0.63, 2.5 mg/kg; 3–6
animals per dose). The animals were decapitated 1 h
after drug administration. Brains were immediately re-
moved from the skull and rapidly frozen in dry-ice
cooled 2-methylbutane (ꢀ40 ꢁC). Twenty micrometres
thick coronal sections were cut using a Leica CM 3050
cryostat microtome (van Hopplynus, Belgium) and
thaw-mounted on silanized microscope slides (Star

1586
M. Van der Mey et al. / Bioorg. Med. Chem. 13 (2005) 1579–1586
Frost, Knittel Gla¨ser, Germany). The sections were
stored at ꢀ20 ꢁC until use.
tion. Ablood sample was taken by cardiac puncture
and selected tissues were rapidly dissected and weighed.
The radioactivity was measured using a LKB Wallac
1282 compugamma CS and the results expressed as per-
centage of injected dose per gram of tissue (% ID/g). The
results are summarized in Figure 4. All animal studies
were performed with the approval of the animal experi-
ment ethical committee of the Vrije Universiteit.
Occupancy of NK1 receptors was measured in the stria-
tum of each individual gerbil. After thawing, sections
were dried under a cold stream of air and then incubated
at rt for 10 min with 3 nM [3H]-[Sar9, Met(O2)11]-sub-
stance P (NEN Life Science) in TrisHCl buffer
(50 mM, pH 7.4) containing 3 mM MnCl2, 0.3% (w/v)
BSA, 40 lg/mL bacitracin, 2 lg/mL chymostatin, 4 lg/
mL leupeptin. Nonspecific binding was measured on
adjacent sections in the presence of 1 lM substance P.
To stop the incubation, the slides were washed
(4 · 1 min) in TrisHCl buffer, pH 7.4 at 4 ꢁC followed
by a rapid dip in cold distilled water and drying under
a stream of cold air.
Acknowledgements
The authors acknowledge the help provided by Willy
Verluyten (Johnson and Johnson Pharmaceutical Re-
search and Development) for purification and analyses
of [3H]R116301. Jef Vermeire and Ilse Lenaerts (John-
son and Johnson Pharmaceutical Research and
Development) are thanked for their assistance during
[11C]R116301 biodistribution experiments. Peter van
Leuffen and co-workers (BV Cyclotron VU) are grate-
fully acknowledged for the [11C]CO2 production.
Quantitative autoradiography analysis was performed
after 2 h acquisition with the b-imager (Biospace, Paris)
according to a previously published method.5 The ED50
value (dose of drug producing 50% of NK1 receptor
occupancy) was calculated by nonlinear regression anal-
ysis, using the GraphPad Prism program (San Diego,
Ca).
References and notes
4.4. In vivo and in vitro autoradiography of [3H]116301
in gerbil brain
1. Kent, J. M.; Mathew, S. J.; Gorman, J. M. Biol.
Psychiatry 2002, 52, 1008.
2. Kramer, M. S.; Cutler, N.; Feighner, J.; Shrivastava, R.;
Carman, J.; Sramek, J. J.; Reines, S. A.; Liu, G.; Snavely,
D.; Wyatt-Knowles, E.; Hale, J. J.; Mills, S. G.; MacCoss,
M.; Swain, C. J.; Harrison, T.; Hill, R. G.; Hefti, F.;
Scolnick, E. M.; Cascieri, M. A.; Chicchi, G. G.; Sadow-
ski, S.; Williams, A. R.; Hewson, L.; Smith, D.; Carlson,
E. J.; Hargreaves, R. J.; Rupniak, N. M. J. Science 1998,
281, 1640.
3. De Wit, R.; Herrstedt, J.; Rapoport, B.; Carides, A. D.;
Guoguang-Ma, J.; Elmer, M.; Schmidt, C.; Evans, J. K.;
Horgan, K. J. Eur. J. Cancer 2004, 40, 403.
4. Janssens, F. E.; Sommen, F. M.; Surleraux, D. L.;
Leenaerts, J. E.; Van Roosbroeck, Y. E. WO 97/16440,
1997.
5. Langlois, X.; te Riele, P.; Wintmolders, C.; Leysen, J. E.;
Jurzak, M. J. Pharmacol. Exp. Ther. 2001, 299, 712.
6. Megens, A. A. H. P.; Ashton, D.; Vermeire, J. C. A.;
Vermote, P. C. M.; Hens, K. A.; Hillen, L. C.; Fransen, J.
F.; Mahieu, M.; Heylen, L.; Leysen, J. E.; Jurzak, M. R.;
Janssens, F. J. Pharmacol. Exp. Ther. 2002, 302,
696.
7. Van der Mey, M.; Leysen, J. E.; Windhorst, A. D.;
Herscheid, J. D. M. J. Labelled Compd. Radiopharm. 2001,
44, S277–S279.
In vivo [3H]R116301 (467 GBq/mmol) autoradiography
was performed according to the following procedure.
[3H]R116301 (370 kBq) was injected intravenously in
male Mongolian gerbils (40–60 g). One hour following
injection, the gerbils were sacrificed by decapitation,
the brains were removed and rapidly frozen in dry-ice
cooled 2-methylbutane (ꢀ40 ꢁC). Sagital sections,
20 lm thick, were cut using a Leica CM 3050 cryostat
microtome (van Hopplynus, Belgium) and thaw-
mounted on silanized microscope slides (Star Frost,
Knittel Gla¨ser, Germany). After application of copper
strips to the back surface of the slides, the slides were
placed in the b-imager (Biospace, Paris) for 64 h.
To assess the specificity of [3H]R116301 binding in
brain, in vitro autoradiography experiments were
performed according to the following procedure. After
a 15 min pre-incubation, cryostat-cut sections of gerbil
brain were incubated for 90 min at rt in buffer contain-
ing 50 mM TrisHCl (pH 7.4), 0.3% (w/v) BSA, 5 mM
MnCl2 and 3 nM [3H]R116301. Nonspecific binding
was determined by addition of 10 lM substance P to
the incubation medium. After incubation, the excess of
radioligand was washed off (3 · 10 min) in incubation
buffer at 4 ꢁC followed by a rapid dip in cold distilled
water. After washing, the slides were dried under a
stream of cold air and exposed to Hyperfilm for 6 weeks.
8. Hargreaves, R. J. J. Clin. Psychiatry 2002, 63(Suppl. 11),
18.
9. Bergstro¨m, M.; Fasth, K.-J.; Kilpatrick, G.; Ward, P.;
Cable, K. M.; Wipperman, M. D.; Sutherland, D. R.;
˚
Langstro¨m, B. Neuropharmacology 2000, 39, 664.
10. Mathews, W. B.; Burns, H. D.; Dannals, R. F.; Ravert, H.
T.; Naylor, E. M. J. Labelled Compd. Radiopharm. 1995,
36, 729.
11. Porwisiak, J.; Schlosser, M. Chem. Ber. 1996, 129, 233.
12. Windhorst, A. D.; Linden, T.; De Nooij, A.; Keus, J. F.;
Buijs, F. L.; Schollema, P. E.; Van Rooij, L. F.; Herscheid,
J. D. M. J. Labelled Compd. Radiopharm. 2001, 44, S1052–
S1054.
4.5. Biodistribution study in gerbils
[11C]R116301 (82–172 GBq/lmol) was injected intrave-
nously into male Mongolian gerbils (75–100 g). Under
diethyl ether anaesthesia the gerbils were sacrificed by
cervical dislocation at 5, 10, 20 and 30 min post injec-
13. Corrie, J. E. T. Tetrahedron 1998, 54, 5407.