Synthesis of 11C-labelled bis(phenylalkyl)amines and their in vitro and in vivo binding properties in rodent and monkey brains

Article abstract of DOI:10.1248/bpb.27.531

Two new ¹¹C-labelled ligands, N-(3-(4-hydroxyphenyl)propyl)-3- (4-methoxyphenyl)propylamine ([¹¹C]2) and N-(3-(4-hydroxyphenyl) butyl)-3-(4-methoxyphenyl)butylamine ([¹¹C]3) were designed based on bis(phenylalkyl)-amines (1) which have been reported as polyamine site antagonists with high-selectivity for NR1A/2B NMDA receptors, and radiolabelling of the corresponding phenol precursors with [¹¹C]methyl iodide was readily accomplished. The in vitro inhibition experiments using rat brain slices showed that [¹¹C]2 and [¹¹C]3 share the binding sites with spermine and/or ifenprodil but not with CP-101,606, a highly potent NR2B-selective NMDA antagonist, and that divalent cations such as Zn ²⁺ produced significant inhibition of both [¹¹C]2 and [¹¹C]3 bindings. Intravenous injection of [¹¹C]3 in mice showed almost homogenous distribution throughout the brain. Attempts to block the tracer uptake of [¹¹C]3 by pre-injection with the unlabelled 3 or spermine in rats were unsuccessful, but a small decrease in the cerebral uptake of [¹¹C]3 by co-treatment with the unlabelled 3 was observed in a monkey PET study. The present findings indicate that none of these ¹¹C-labelled analogues have potential for PET study of binding sites on the N-methly-D-aspartate (NMDA) receptors.

Full text of DOI:10.1248/bpb.27.531

April 2004
Biol. Pharm. Bull. 27(4) 531—537 (2004)
531
Synthesis of 11C-Labelled Bis(phenylalkyl)amines and Their in Vitro and
in Vivo Binding Properties in Rodent and Monkey Brains
Shigeki SASAKI,a,c Fumie KUROSAKI,a Terushi HARADAHIRA,b,c Fumihiko YAMAMOTO,a,c Jun MAEDA,b
,a,c
Takashi OKAUCHI,b Kazutoshi SUZUKI,b Tetsuya SUHARA,b,c and Minoru MAEDA
*
a Graduate School of Pharmaceutical Sciences, Kyushu University; 3–1–1 Maidashi, Higashi-ku, Fukuoka 812–8582,
c
Japan: b National Institute of Radiological Sciences; 4–9–1 Anagawa, Inage-ku, Chiba 263–8555, Japan: and CREST,
Japan Science and Technology Corporation; 4–1–8 Honmachi, Kawaguchi, Saitama 332–0012, Japan.
Received December 15, 2003; accepted January 24, 2004
Two new 11C-labelled ligands, N-(3-(4-hydroxyphenyl)propyl)-3-(4-methoxyphenyl)propylamine ([11C]2) and
N-(3-(4-hydroxyphenyl)butyl)-3-(4-methoxyphenyl)butylamine ([11C]3) were designed based on bis(phenylalkyl)-
amines (1) which have been reported as polyamine site antagonists with high-selectivity for NR1A/2B NMDA
receptors, and radiolabelling of the corresponding phenol precursors with [11C]methyl iodide was readily accom-
plished. The in vitro inhibition experiments using rat brain slices showed that [11C]2 and [11C]3 share the binding
sites with spermine and/or ifenprodil but not with CP-101,606, a highly potent NR2B-selective NMDA antago-
nist, and that divalent cations such as Zn2؉ produced significant inhibition of both [11C]2 and [11C]3 bindings.
Intravenous injection of [11C]3 in mice showed almost homogenous distribution throughout the brain. Attempts
to block the tracer uptake of [11C]3 by pre-injection with the unlabelled 3 or spermine in rats were unsuccessful,
but a small decrease in the cerebral uptake of [11C]3 by co-treatment with the unlabelled 3 was observed in a
monkey PET study. The present findings indicate that none of these 11C-labelled analogues have potential for
PET study of binding sites on the N-methly-D-aspartate (NMDA) receptors.
Key words N-methly-D-aspartate (NMDA) receptor antagonist; 11C-labelling; brain distribution; inhibition experiment;
bis(phenylalkyl)amine; polyamine binding site
bis(phenylalkyl)amine, bis(phenylpropyl)amine ([11C]2) and
bis(phenylbutyl)amine ([11C]3), and examined their in vitro
and in vivo binding properties in rodent and monkey brains.
The N-methyl-D-aspartate (NMDA) class ionotropic recep-
tors for glutamate play a central role in excitatory neurotrans-
mission in the central nervous system (CNS). Some CNS
disorders such as ischemic, hypoxic, hypoglycemic, and trau-
matic insults, epilepsy, AIDS, dementia, and Huntington’s
and Alzheimer’s diseases are accompanied by disorders of
the NMDA receptors.1—8) For non-invasive diagnoses of
NMDA receptor dysfunctions by PET (positron emission to-
mography), a number of radioactive ligands labelled with
short-lived 18F (t1/2ϭ109.7 min) or 11C (t1/2ϭ20.3 min) have
been investigated. However, efforts have been limited by
poor brain entry or high levels of nonspecifc binding.9) Na-
tive NMDA receptors are present as complexes containing at
least one NR1 subunit and one or more of the four distinct
NR2 subunits (NR2A-D), with a number of recognition sites
for endo- and exogeneous ligands that modulate neuronal re-
sponses.10,11) Recently, a series of bis(phenylalkyl)amines (1),
structural analogues of ifenprodil, were reported as
polyamine site antagonists with high-selectivity for NR2B
containing NMDA receptors, as exemplified by compound
(1) (Fig. 1, mϭ5, nϭ2, IC50ϭ8 nM for the NR1A/2B subunit
combination).12—15) There are at least three independent
polyamine-binding sites on the NMDA receptors—the
glycine-dependent modulatory site, the glycine-independent
modulatory site, and the voltage-dependent ion channel
site.16—22) The bis(phenylalkyl)amines have been shown to
bind to the glycine-independent polyamine modulatory site,
which is absolutely required for the NR2B subunit.23) It has
been reported that the primary determinants of potency of
the bis(phenylalkyl)amines (1) at the NR1A/2B NMDA re-
ceptors are (1) the phenolic OH group; (2) the distance be-
tween the two aromatic rings; and (3) the nitrogen atom.15)
Thus, we designed mono [11C]methoxyl derivatives of 1. In
this study, we synthesized two 11C-labelled analogues of
MATERIALS AND METHODS
Radioactivity was quantified with a IGC-3R Curiemeter
(Aloka). High-pressure liquid chromatography (HPLC) was
done using a JASCO HPLC system for radioactive runs. Ef-
fluent radioactivity from the HPLC was determined using a
NaI(Tl) scintillation detector system. Carbon-11 was gener-
ated by the 14N(p,a)11C nuclear reaction using a CYPRIS-
HM-18 Cyclotron (Sumitomo Heavy Industries, Ltd). All an-
imal studies were carried out in compliance with the Animal
Ethical Committee of the National Institute of Radiological
Sciences and Japanese law. The preparation of [11C]CH3I and
subsequent 11C-methylations were carried out automatically
by using a synthetic apparatus for 11C-labelled compounds
developed by Suzuki et al.24) 1H-NMR spectra were obtained
on a JNM GX-270 spectrometer with TMS as an internal
standard, and IR spectra were recorded with a JASCO IR Re-
port 100 spectrometer. Low-resolution FAB mass spectra
Fig. 1. Structure of Bis(phenylalkyl)amines 1—3
To whom correspondence should be addressed. e-mail: maeda@phar.kyushu-u.ac.jp
© 2004 Pharmaceutical Society of Japan

532
Vol. 27, No. 4
were obtained on a JEOL JMS-D300 spectrometer, and high-
resolution FAB (HR-FAB-MS) mass spectra were obtained
on a JEOL NMS-SX 102-SX spectrometer. Column chro-
matography was done on a Merck Kieselgel 60 (70—230
mesh) or Fuji gel FL-60D. Thin-layer chromatography (TLC)
was carried out on Merck Kieselgel 60 F254 plates. Melting
points were determined on a Yanagimoto melting point appa-
ratus and are uncorrected. Elemental analyses were per-
formed by the staff of the micro-analysis section of Kyushu
University.
All chemicals were used without further purification un-
less otherwise stated. Three polyamine (spermine, spermi-
dine, putrescine) hydrochlorides, L-glutamic acid hydrochlo-
ride, glycine, ifenprodil tartrate, 5,7-dichlorokynurenic acid
(DCKA), CGS-19755, 8-OH-DPAT hydrobromide, prazosin
hydrochloride and ketanserin tartrate, were purchased from
Sigma Chemical Co. 1,3-Di-o-tolylguanidine (DTG) was
purchased from Aldrich Chemical Co.
Jϭ7.6 Hz), 3.10 (4H, t, Jϭ7.8 Hz), 3.79 (6H, s), 6.81 (4H, d,
Jϭ8.9 Hz), 7.03 (4H, d, Jϭ8.9 Hz), 7.26 (2H, d, Jϭ7.9 Hz),
7.62 (2H, d, Jϭ8.3 Hz); Anal. Calcd for C27H33NO4S: C,
69.35; H, 7.11; N, 3.00. Found. C, 69.32; H, 7.14; N, 3.01.
Synthesis of N,N-Bis(3-(4-methoxyphenyl)butyl)-p-tolu-
enesulfonamide (7) A solution of p-toluenesulfonamide
(1 g, 5.88 mmol) and 60% sodium hydride oil suspension
(484 mg, 12.1 mmol) in anhydrous DMF (35 ml) was heated
at 40—45 °C for 1 h, followed by the addition of 5 (3.5 g,
10.5 mmol) in three portions over 2 h at 50—55 °C. After the
reaction at 50—55 °C for 1 h, the reaction mixture was
worked up and purified as described above to give 7 as a col-
1
orless oil (1.91 g, 74%): H-NMR (CDCl3) d: 1.47—1.53
(8H, m), 2.40 (3H, s), 2.51 (4H, t, Jϭ6.9 Hz), 3.07 (4H, t,
Jϭ6.9 Hz), 3.78 (6H, s), 6.81 (4H, d, Jϭ8.6 Hz), 7.05 (4H, d,
Jϭ8.6 Hz), 7.25 (2H, d, Jϭ7.6 Hz), 7.64 (2H, d, Jϭ8.3 Hz);
FAB Mass (m/z): 496 (MϩH)ϩ.
Synthesis of N,N-Bis(3-(4-hydroxyphenyl)propyl)amine
Hydrobromide (8) A solution of 6 (500 mg, 1.07 mmol) in
AcOEt (10 ml) was added to a solution of phenol (10 g,
107 mmol) in 30% HBr/AcOH (194 ml). The reaction mix-
ture was stirred at room temperature for 18 h, then cooled in
an ice bath, followed by the addition of water (200 ml). The
aqueous layer was washed with Et2O, and concentrated in
vacuo. The residue was purified by chromatography on a sil-
ica gel column (chloroform : methanolϭ9 : 1) to give 8 as a
Synthesis of 1-p-Toluenesulfonyloxy-3-(4-methoxyphenyl)-
propane (4) p-Toluenesulfonyl chloride (6.3 g, 33 mmol)
was added to a solution of 3-(4-methoxyphenyl)propanol
(5 g, 30 mmol) in anhydrous pyridine (75 ml) at 0 °C. The re-
action mixture was stirred at 4 °C for 16 h, then added to a
solution of conc. HCl and ice (1/1, 100 ml). The resulting so-
lution was extracted with Et2O (300 ml). The separated or-
ganic layers were washed with brine, dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by chromatography on a silica gel column
(hexane : AcOEtϭ5 : 1) to give 4 as a white solid (9.0 g,
1
brown powder (244.8 mg, 63%): mp 165—168 °C; H-NMR
(CD3OD) d: 1.91 (4H, quintet, Jϭ7.7 Hz), 2.59 (4H, t,
Jϭ7.4 Hz), 2.94 (4H, t, Jϭ7.9 Hz), 6.70 (4H, d, Jϭ8.6 Hz),
7.01 (4H, d, Jϭ8.2 Hz); IR (neat) cmϪ1: 3425; HR-FAB-MS
(m/z); Calcd for C18H24NO2 (MϩH): 286.1807. Found.
286.1828.
1
94%): mp 38—39 °C; H-NMR (CDCl3) d: 1.88—1.96 (2H,
m), 2.44 (3H, s), 2.58 (2H, t, Jϭ7.4 Hz), 3.76 (3H, s), 4.01
(2H, t, Jϭ6.1 Hz), 6.77 (2H, d, Jϭ8.9 Hz), 6.97 (2H, d,
Jϭ8.6 Hz), 7.33 (2H, d, Jϭ7.9 Hz), 7.78 (2H, d, Jϭ8.6 Hz);
IR (neat) cmϪ1: 1355; Anal. Calcd for C17H20O4S: C, 63.73;
H, 6.29. Found. C, 63.86; H, 6.34.
Synthesis of N,N-Bis(3-(4-hydroxyphenyl)butyl)amine
Hydrobromide (9) A solution of 7 (500 mg, 1.00 mmol) in
AcOEt (10 ml) was added to a solution of phenol (10 g,
107 mmol) in 30% HBr/AcOH (194 ml). The reaction mix-
ture was stirred at room temperature for 18 h, worked up and
the crude product was purified as described above to give 9
Synthesis of 1-p-Toluenesulfonyloxy-3-(4-methoxyphenyl)-
butane (5) p-Toluenesulfonyl chloride (6.1 g, 32 mmol)
was added to a solution of 4-(4-methoxyphenyl)butanol
(4.7 ml, 27 mmol) in anhydrous pyridine (75 ml) at 0 °C. The
reaction mixture was stirred at 4 °C for 18 h. The work-up
and purification were carried out as described above to pro-
1
as a brown powder (156.6 mg, 40%): mp 213—217 °C; H-
NMR (CD3OD) d: 1.62—1.65 (8H, m), 2.56 (4H, t,
Jϭ6.9 Hz), 2.92 (4H, t, Jϭ7.6 Hz), 6.69 (4H, d, Jϭ8.6 Hz),
7.00 (4H, d, Jϭ8.6 Hz); IR (neat) cmϪ1: 2400—3500; HR-
FAB-MS (m/z); Calcd for C20H28NO2 (MϩH): 314.2120.
Found. 314.2101.
1
duce 5 as a colorless oil (7.3 g, 81%): H-NMR (CDCl3) d:
1.59—1.68 (4H, m), 2.44 (3H, s), 2.50 (2H, t, Jϭ7.1 Hz),
3.78 (3H, s), 4.03 (2H, t, Jϭ6.1 Hz), 6.80 (2H, d, Jϭ8.9 Hz),
7.01 (2H, d, Jϭ8.6 Hz), 7.33 (2H, d, Jϭ7.9 Hz), 7.78 (2H, d,
Jϭ8.3 Hz); IR (neat) cmϪ1: 1350; FAB Mass (m/z): 334
(M)ϩ.
Synthesis of N-(3-(4-Hydroxyphenyl)propyl)-3-(4-me-
thoxyphenyl)propylamine (2) The hydrobromide (8) was
added to a saturated aqueous solution of NaHCO3, and the
corresponding free amine was extracted with ether. The or-
ganic layers were washed with brine, dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. Sodium hydride
(60% suspension in oil, 2.7 mg, 0.068 mmol) was added to a
solution of the above free amine (white powder, 9.5 mg,
0.033 mmol) in anhydrous DMF (1 ml), and the mixture was
stirred for 5 min, followed by the addition of methyl iodide
(2.1 ml, 0.033 mmol). After the reaction mixture was stirred
for 20 min, it was quenched by the addition of 10% aqueous
acetic acid, and neutralized with saturated aqueous NaHCO3,
then extracted with Et2O. The separated organic layers were
washed with brine, dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by
HPLC (column: nacalai tesque COSMOSIL 5C18-MS,
Synthesis of N,N-Bis(3-(4-methoxyphenyl)propyl)-p-tolu-
enesulfonamide (6) A solution of p-toluenesulfonamide
(1 g, 6.1 mmol) and 60% sodium hydride oil suspension
(488 mg, 12.2 mmol) in anhydrous DMF (35 ml) was heated
at 40—45 °C for 1 h, followed by the addition of 4 (3.5 g,
10.9 mmol) in three portions over 2 h at 50—55 °C, and the
reaction mixture was stirred at 50—55 °C for 1 h. The reac-
tion mixture was diluted with water (35 ml), and extracted
with toluene. The separated organic layers were washed with
brine, dried over anhydrous Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by chromatography
on a silica gel column (hexane : AcOEtϭ5 : 1) to give 6 as a
1
white solid (1.68 g, 66%): mp 67—68 °C; H-NMR (CDCl3)
d: 1.78 (4H, quintet, Jϭ7.7 Hz), 2.41 (3H, s), 2.49 (4H, t,

April 2004
533
recorded as photostimulated luminescence (PSL) values per
mm2 which are proportional to the radioactivity of the mea-
sured sample,26) and was expressed as region-to-cerebellum
ratios calculated by dividing the PSL values in each brain re-
gion by the cerebellum PSL value on the same slice. Back-
ground PSL values were always subtracted from the total
PSL values on ROI. Nonradioactive 2 or 3 (100 mM) were si-
multaneously incubated with the corresponding radioligand
to determine their nonspecific binding.
10ϫ250 mm, 0.05% trifluoroacetic acid in methanol :
H2Oϭ3 : 2, UV monitor 254 nm, flow rate 4.0 ml/min) to give
2 as a yellow oil (7.3 mg, 74%). 1H-NMR (CD3OD) d:
1.87—1.93 (4H, m), 2.61 (4H, dd, Jϭ7.8 Hz), 2.92 (4H, t,
Jϭ8.1 Hz), 3.75 (3H, s), 6.71 (2H, d, Jϭ8.9 Hz), 6.84 (2H, d,
Jϭ8.9 Hz), 7.01 (2H, d, Jϭ8.6 Hz), 7.11 (2H, d, Jϭ8.9 Hz);
HR-FAB-MS (m/z); Calcd for C19H26NO2 (MϩH): 300.1964.
Found. 300.1954.
Synthesis of N-(3-(4-hydroxyphenyl)butyl)-3-(4-me-
thoxyphenyl)butylamine (3) The free amine form was ob-
tained from 9 as described above. Monomethylation of the
resulting white powder (7.6 mg, 0.024 mmol) and purification
were carried out as described above to give 3 as a yellow oil
Drug Inhibition for in Vitro Binding The rat brain
sagittal sections were incubated at 25 °C for 40 min in a
50 mM Tris–HCl buffer containing [11C]2 or [11C]3 (ca.
18 MBq, 1—2 nM) without or with drugs (10, 100 mM, or
1 mM). Following the incubation, the sections were rinsed,
dried and apposed to the imaging plates for 60 min in the
same manner as described above. A region of interest (ROI)
on the slices was placed on the frontal cortex and the ra-
dioactivity in this region was expressed as photostimulated
luminescence (PSL) values per mm2. The PSL data on the
ROI in the presence of drugs were divided by the corre-
sponding control PSL data in the absence of drugs. Back-
ground PSL values were subtracted from the total PSL values
on ROI. The obtained % control values (defined as 100% for
the 11C-ligand binding activity in the absence of drugs) were
averaged in at least triplicate sections from three animals.
In Vivo Brain Distribution [11C]3 (in 0.2 ml water, ca.
55 MBq/ml) was injected via a tail vein into ddY mice (36—
38 g). The animals were killed at 10, 30 and 50 min by de-
capitation. Their whole brains were rapidly removed and dis-
sected into the cerebellum, cerebral cortex, hippocampus,
and striatum. A sample of blood was also taken. Radioactiv-
ity in each sample was measured with a Packard gamma-
counter and corrected for decay. The results were expressed
as the percentage of injected dose per gram of tissue weight
(% dose/g). In separate experiments, the effects of pretreat-
ment of spermine and unlabelled 3 on the brain uptake of
male Sprague–Dawley rats (10—12 weeks old) were investi-
gated. The drugs (7 mmol/kg) were injected intravenously
10 min before injection of [11C]3 and the rats were killed at
30 min after the injection of [11C]3. The brain was removed,
dissected and regional radioactivity was determined as de-
scribed above. The control rats were treated with a water so-
lution under identical conditions.
In Vivo Brain Distribution in Monkey An awake-be-
having Rhesus monkey (male, 5 kg) was chaired and the head
was fixed with a specifically designed head-hold device. All
animal PET scans were performed with a high-resolution
SHR-7700 PET camera (Hamamatsu Photonics, Shizuoka,
Japan) in the same manner as described in the literature.27)
After transmission scans for attenuation correction, a dy-
namic emission scan in 2D acquisition mode was performed
for 90 min (1 minϫ10 scans, 2 minϫ20 scans, 4 minϫ
10 scans) following the injection of [11C]3 (303 MBq, in 2 ml
saline) via the crural vein as a single bolus. The emission
scan image was reconstructed with a Hanning filter of 4 mm
and circular ROIs with a 5-mm diameter were placed over
the frontal and occipital cortexes, striatum, and cerebellum.
The ROI values were expressed in kBq/ml, normalized to the
injected radioactivity of 185 MBq, and plotted against time .
A mixture of unlabelled 3 (6 mmol/kg) and [11C]3 (230 MBq)
in saline (3 ml) was injected into the same monkey to deter-
1
(2.5 mg, 32%). H-NMR (CD3OD) d: 1.57—1.65 (8H, m),
2.61 (4H, t, Jϭ6.8 Hz), 2.90—2.95 (4H, m), 3.73 (3H, s),
6.69 (2H, d, Jϭ8.6 Hz), 6.82 (2H, d, Jϭ8.6 Hz), 6.99 (2H, d,
Jϭ8.6 Hz), 7.09 (2H, d, Jϭ8.6 Hz); HR-FAB-MS (m/z);
Calcd for C21H30NO2 (MϩH): 328.2277. Found. 328.2290.
Radiosynthesis of N-(3-(4-Hydroxyphenyl)propyl)-3-(4-
[11C]methoxyphenyl)propylamine ([11C]2) and N-(3-(4-
Hydroxyphenyl)butyl)-3-(4-[11C]methoxyphenyl)butyl-
amine ([11C]3) The free base (0.9 mg) of 8 or 9 in anhy-
drous DMF (300 ml) was reacted with [11C]CH3I in the pres-
ence of four equivalents of NaH in anhydrous DMF (1 ml) at
30 °C for 3 min. After the HPLC mobile phase (500 ml)
(MeOH : 0.05%CF3COOHϭ45 : 55 for [11C]2, or MeOH :
0.05%CF3COOHϭ48 : 52 for [11C]3) had been added, the
reaction mixture was transferred onto a reverse phase
HPLC (column: Waters m-Bondapak C18, 7.8ϫ300 mm)
and eluted with the corresponding mobile phases at a flow
rate of 3 ml/min. The radioactive peaks corresponding to the
desired products (tRϭca. 10 min for [11C]2, or tRϭca. 9.5 min
for [11C]3) were collected in a flask, respectively. After the
solvents were removed under reduced pressure, distilled
water or isotonic saline was added to the flask and used for in
vitro or in vivo experiments. An aliquot was assayed for ra-
dioactivity and checked by HPLC (column: Waters m-Bonda-
pak C18, 3.9ϫ300 mm). The mobile phases were
MeOH : 0.05%CF3COOHϭ45 : 55 at 0.7 ml/min for [11C]2
and MeOH : 0.05%CF3COOHϭ50 : 50 at 0.8 ml/min for
[11C]3, respectively. The identity of the products was
achieved by HPLC co-injection with authentic 2 or 3. Spe-
cific radioactivity at the end of synthesis was calculated by
UV spectroscopy. The averages 1.2 GBq of [11C]2 and
1.6 GBq of [11C]3 were obtained in a total synthesis of 20—
25 min from the end of bombardment after a 10 min proton
bombardment at a beam current of 15 mA, respectively.
In Vitro Binding on Rat Brain Slices Autoradiography
on rat brain slices was performed essentially as has been pre-
viously described.25) Brain sagittal cryosections (20 mm) ob-
tained from male Sprague–Dawley rats (10—12 weeks old)
were pre-incubated in a 50 mM Tris–HCl buffer (pHϭ7.4) at
25 °C for 30 min, followed by incubation in the same buffer
containing [11C]2 or [11C]3 (ca. 18 MBq, 1—2 nM) for 40 min
at 25 °C. The sections were washed three times for 2 min
each with a cold incubation buffer, dipped into cold water,
dried under a stream of warm air, and placed in contact with
11C-sensitive imaging plates (BAS-SR 127, Fujiphoto Film)
for 60 min to analyze their radioactivity of distribution on the
slices with a BAS 3000 imaging analyzer (Fujiphoto Film).
Radioactivity in regions of interest (ROI) on the slices was

534
Vol. 27, No. 4
Fig. 2. Region to Cerebellum Ratios of in Vitro Bindings of [11C]2 and
[11C]3 on Rat Brain Slices
The ratios were obtained by dividing the PSL value in each brain region by the cere-
bellar PSL value on the same slice and expressed as meanϮS.D. (nϭ29 for [11C]2,
nϭ25 for [11C]3).
Chart 1. Synthesis of Bis(hydroxyphenylalkyl)amine (8, 9) and Their
Methylated Derivatives (2, 3)
mine specific binding. The time activity curves in the ROIs
were obtained for each scan of the brain.
RESULTS AND DISCUSSION
The synthesis was started with the tosylate (4 or 5), which
was reacted with p-toluenesulfonamide in the presence of
NaH in DMF to produce a dialkylated product (6 or 7), as
described in Chart 1. The methyl protecting groups of 6 and
7 were cleaved by treatment with HBr/AcOH in the presence
of phenol to give the diphenols (8, 9), respectively. After
neutralization with NaHCO3, the free amines of the diphenol
compounds (8, 9) were used for the synthesis of 2 and 3, re-
spectively, as well as for the 11C-methylation.
Fig. 3. Effects of Unlabelled Bis(phenylalkyl)amines (2 or 3, 100 mM) on
the in Vitro Binding of [11C]2 or [11C]3 to the Rat Brain Slices
The values (meanϮS.D.) were obtained by at least triplicate slices from three rats.
The radiosynthesis of [11C]2 and [11C]3 proved straightfor-
ward using standard O-methylation on the corresponding
diphenol precursors (8, 9) with [11C]CH3I in the presence of
four equivalents of NaH in DMF. This can be attributed to
the fact that the phenoxide group generated under basic con-
ditions is a stronger nucleophile than the secondary amino
group. Purification by reversed phase HPLC afforded [11C]2
and [11C]3 with more than 98% radiochemical purity, free
from starting precursors. The specific radioactivity was esti-
mated by UV spectroscopy to be around 75 GBq/mmol at the
end of synthesis. Although the reaction conditions have not
been optimized, the averages 1.2 GBq of [11C]2 and 1.6 GBq
of [11C]3 after a 10 min proton bombardment at a beam cur-
rent of 15 mA were obtained at end of synthesis, respectively.
In vitro bindings were initially examined by incubating the
rat brain slices in a Tris HCl buffer containing [11C]2 or
[11C]3. As shown in Fig. 2, the ratios regions to cerebellum, a
region free of the NR2B subunit, for the two radioligands
were almost unity in all brain regions tested (cerebral cortex,
hippocampus, striatum, and thalamus), thus demonstrating
almost homogeneous distribution throughout the brain. Non-
specific bindings of [11C]2 and [11C]3 were about 50% and
60% of total binding, respectively, in all brain regions, as de-
termined by the presence of the corresponding 100 mM non-
radioactive ligands (Fig. 3). No significant difference in the
bindings was observed between [11C]2 and [11C]3.
NMDA receptors, by various endogenous ligands related to
NMDA receptors and other receptor ligands. The results of
the inhibitory effects (% of control binding) are summarized
in Tables 1 and 2. Among the ligands examined, ifenprodil
and spermine inhibited the in vitro bindings with potencies
similar to those of non-radioactive 2 or 3 at the same 100 mM
concentration. The highly potent NR2B-selective NMDA an-
tagonist, CP-101,606, inhibited the binding of [11C]2 and
[11C]3 by only 20 and 6%, respectively, at a 10 mM concentra-
tion. It is known that ifenprodil shows equal affinities to sev-
eral other CNS receptors such as s-binding sites and a1-
adrenergic receptors.28,29) The bindings of [11C]2 and [11C]3
to s-binding sites and a1-adrenergic receptors, however,
were negligible by the fact that 100 mM s- and a-antagonists
did not show significant inhibition for either the [11C]2 or
[11C]3 bindings. Among the polyamines, spermine showed
the most potent inhibition, followed by spermidine, while
putrescine showed only 10—15% inhibition even at a 1 mM
concentration. These orders of inhibitory effects by poly-
amines are similar to those reported for the inhibition of
[3H]CP-101,606 and [3H]ifenprodil.30—33) Although the high-
affinity ifenprodil site is supposed to be located on the NR2B
subunit, a low-affinity ifenprodil site has also been proposed,
through which ifenprodil acts as a weak open-channel
blocker.34) Divalent cations also inhibited the [11C]2 and
[11C]3 bindings in the order of Zn2ϩϾϾCa2ϩϾMg2ϩ at their
physiological concentrations. The inhibitory effects of
polyamines and divalent cations have commonly been ob-
The in vitro bindings of the 11C-labelled ligands were fur-
ther characterized by examining the inhibition in the frontal
cortex area of the rat brain slices, which are rich in the

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535
Table 1. Effects of Various Ligands on in Vitro [11C]2 Binding to Rat Table 2. Effects of Various Ligands on in Vitro [11C]3 Binding to Rat
Brain Slices Brain Slices
% control binding
in frontal cortexa)
% control binding
in frontal cortexa)
Ligands
Ligands
10 mM
100 mM
1 mM
10 mM
100 mM
1 mM
Unlabelled compd
Polyamine site
Agonists
2
69.3
47.7
Unlabelled compd
Polyamine site
Agonists
3
79.5
63.3
Spermine
64.1
89.5
98.8
71.1
59
43.2
46.3
84.6
Spermine
61.4
88.3
105.4
99.2
49
67.6
88.3
Spermidine
Putrescine
CP-101,606
Ifenprodil
Spermidine
Putrescine
CP-101,606
Ifenprodil
Antagonists
81.1
67.2
Antagonists
94.1
83.1
71.9
Glutamate site
Agonist
Antagonist
Glycine site
Agonist
Glutamate site
Agonist
Antagonist
Glycine site
Agonist
Glutamate
CGS-19755
102.2
100.8
Glutamate
CGS-19755
102.8
103.7
96
114.5
100.4
Glycine
DCKA
Glycine
DCKA
Antagonist
Divalent catons
108.4
Antagonist
Divalent cations
MgCl2
CaCl2
Zn(OAc)2
93.3
91.6
45.1
65.2
63.2
26.7
MgCl2
CaCl2
Zn(OAc)2
94.9
96.3
69.7
76.4
66.3
33.8
Ion channel site
Antagonist
Others receptors
a1
Ion channel site
Antagonist
Other receptors
a1
MK-801
88.3
MK-801
104.3
Prazosin
DTG
8-OH-DPAT
Ketanserin
99.4
90.4
58.9
68.8
Prazosin
DTG
8-OH-DPAT
Ketanserin
94.2
101.7
97.9
s1, 2
s1, 2
5-HT1a
5-HT2
5-HT1a
5-HT2
93.8
a) Average values obtained by at least triplicate from three S.D. rats.
a) Average values obtained by at least triplicate from three S.D. rats.
Table 3. Biodistribution of [11C]3 in Mouse Brain and Blood
served in the in vitro binding of ifenprodil-like NR2B-selec-
tive drugs.10,25,35) Glutamate, glycine , glutamate site antago-
nist CGS-19755, glycine site antagonist DCKA and channel
site antagonist MK-801, had no inhibitory effect on either
[11C]2 or [11C]3 binding even at high concentrations. The
serotonergic receptor ligands, 8-OH-DPAT and ketanserin,
showed 30—40% inhibition toward the binding of [11C]2 at
concentrations of 100 mM, whereas no inhibition of [11C]3
was observed with these ligands at the same concentrations,
suggesting that the binding of [11C]2 was also occurring at
the serotonin recognition sites. As shown in Figs. 2 and 3,
taking into account the bindings of the radioligands to the
cerebellum, a region free of the NR2B subunit, it seems
likely that these radioligands bind to the spermine- and/or
ifenprodil-binding sites unrelated to the NR2B subunit. Our
efforts to determine the IC50 value from inhibition studies
using rat brain slices have not been successful because of
high nonspecific binding fractions, but bis(phenylalkyl)-
amine 2 and 3 are estimated to have moderate affinities to the
spermine- and/or ifenprodil-binding sites.
% dose/g (meanϮS.D.)a)
Tissue
10 min
30 min
50 min
Blood
Cerebral cortex
Striatum
Hippocampus
Cerebellum
0.74Ϯ0.12
0.89Ϯ0.15
0.86Ϯ0.19
0.74Ϯ0.12
0.87Ϯ0.16
0.61Ϯ0.05
1.07Ϯ0.05
0.95Ϯ0.05
0.95Ϯ0.01
0.93Ϯ0.01
0.68Ϯ0.06
1.11Ϯ0.15
1.07Ϯ0.18
0.90Ϯ0.13
0.95Ϯ0.13
a) Average of three mice.
spermine or with non-radioactive 3 on brain uptake using
rats. As shown in Fig. 4, pre-treatment with non-radioactive
3 (7 mmol/kg) caused a statistically significant increase of ra-
dioactivity in the cerebral cortex and hippocampus compared
with the control group. The reasons for this observation are
uncertain in the present study. However, it is likely that the
binding of [11C]3 in peripheral binding sites or to plasma
proteins might be prevented, resulting in an increased avail-
ability for uptake in the brain. Thus, this pre-treatment with
unlabelled 3 suggests that the uptake observed is nonspecific
in nature. Pre-treatment with spermine (7 mmol/kg) did not
produce any change of uptake in the brain compared to the
control. This finding is in contrast to the in vitro inhibition of
[11C]3 binding on brain slices. The insensitivity of a blocking
effect by exogenously administered spermine is probably due
to the low penetration of spermine across the blood-brain
barrier,36) and it is also feasible that a high concentration of
endogenous polyamines, such as spermine, in the central ner-
vous system would behave as an endogenous inhibitor for
[11C]3 binding in vivo. Recently it has been proven that the
From these in vitro results, [11C]3 was chosen for further
evaluation under in vivo conditions because of its greater se-
lectivity for the spermine- and/or ifenprodil-binding sites
than [11C]2. The brain regional distribution of [11C]3 after in-
travenous injection was investigated using mice. As shown in
Table 3, [11C]3 showed an initial moderate brain uptake
(0.8—0.9% dose/g) and increased slightly with time. The ra-
dioactivity levels of blood were lower than those in the brain.
No significant difference in the regional brain distribution
was observed, similar to the in vitro brain distribution.
Furthermore, we evaluated the effect of pre-treatment with

536
Vol. 27, No. 4
glycine-site antagonist, [11C]L-703,717, is susceptible to in-
hibition by the endogenous amino acids glycine and D-serine
in its in vivo binding.37) Thus ,we could not prove that the
brain accumulation of [11C]3 was due to the binding of the
ligand to spermine- and/or ifenprodil-binding sites.
slightly until the end of the experiments. No significant re-
gional differences in uptake of radioactivity were observed,
similar to what we obtained using mouse and rat brains, as
described above. When unlabelled 3 (6 mmol/kg) was co-in-
jected with [11C]3, a slight decrease in brain radioactivity
that was most apparent at later time-points, was induced as
shown in Fig. 5, seemingly indicating that there is a small
fraction of displaceable binding in these regions of monkey
brain. Additional experiments are, however, necessary to
more precisely reveal the specific binding sites of [11C]3 in
vivo.
In vivo brain distribution of [11C]3 was further assessed by
a conscious Rhesus monkey in conjunction with PET. Figure
5 shows the normalized time activity curves in the frontal
and occipital cortex, and cerebellum for 90 min of PET scan-
ning after intravenous injection of [11C]3. The radioactivity
reached maximal values at 20—30 min and then decreased
The new radiolabelled ligands, 11C-labelled bis(phenyl-
propyl)amine ([11C]2) and bis(phenylbutyl)amine ([11C]3),
were designed based on bis(phenylalkyl)amines (1) which
have been reported as polyamine site antagonists with high
selectivity for NR1A/2B NMDA receptors. However, in vitro
competitive experiments show that [11C]2 and [11C]3 share
the binding sites with spermine and/or ifenprodil but not
with CP-101,606, a highly potent NR2B-selective NMDA
antagonist, and that [11C]2 has, additionally, appreciable
affinity for the serotonergic receptors. Polyamines such as
spermine and spermidine are present in cells at high concen-
trations and the NMDA receptors contain multiple binding
sites for them. Ifenprodil binds at two sites on NMDA recep-
tors, together with three binding sites on other proteins. Such
multiplicity of binding sites of spermine and ifenprodil may
be responsible for high ratio of nonspecific binding of [11C]3
in vivo. In addition, endogenous divalent cations, as well as
endogenous polyamines, seem likely to affect the in vivo
[11C]3 binding. Both [11C]2 and [11C]3 are therefore not suit-
able radioligands for PET imaging of brain binding sites on
Fig. 4. Effects of Pretreatment with Spermine and Unlabelled 3 in the
Brain Uptake of [11C]3 in Rats
Spermine (7 mmol/kg) or unlabelled 3 (7 mmol/kg) was injected intravenously 10 min
before injection of [11C]3. Rats were killed at 30 min after injection of [11C]3. Signifi-
cant difference (pϽ0.05) versus the corresponding value of the control by two way
ANOVA followed by Turkey’s test.
Fig. 5. Time–Activity Curves in the Cerebellum, Frontal Cortex and Occipital Cortex of Monkey Brain Obtained by PET
Each point was normalized to the injected dose of 185 MBq. Control (closed circles) and co-treatment (open circles) with 6 mmol/kg of unlabelled 3.

April 2004
537
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