Carbon-11 labeled indolylpropylamine analog as a new potential PET agent for imaging of the serotonin transporter

Article abstract of DOI:10.1016/j.bmc.2008.05.006

The synthesis and structure-activity relationship of a new class of indole derivatives with low-nanomolar affinity for the SERT and high selectivity versus the 5-HT1A receptor were recently reported. Based on their chemical structure, four new indolylpropylamine derivatives which contain atoms to afford future labeling with PET isotopes, were synthesized and evaluated as SERT ligands. The chemistry of these novel derivatives, their biological evaluation, the general method of preparing the precursor indole for labeling, and the C-11 labeling of the most promising indole derivative, are described herein.

Full text of DOI:10.1016/j.bmc.2008.05.006

Bioorganic & Medicinal Chemistry 16 (2008) 6364–6370
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Carbon-11 labeled indolylpropylamine analog as a new potential PET agent
for imaging of the serotonin transporter
R. Ben-Daniel a, W. Deuther-Conrad b, M. Scheunemann b, J. Steinbach b, P. Brust b, E. Mishani a,
*
a Department of Medical Biophysics and Nuclear Medicine, Hadassah-Hebrew University Hospital, Jerusalem 91120, Israel
b Institute of Interdisciplinary Isotope Research Permoserstrasse, Leipzig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure–activity relationship of a new class of indole derivatives with low-nanomolar
affinity for the SERT and high selectivity versus the 5-HT1A receptor were recently reported. Based on
their chemical structure, four new indolylpropylamine derivatives which contain atoms to afford future
labeling with PET isotopes, were synthesized and evaluated as SERT ligands. The chemistry of these novel
derivatives, their biological evaluation, the general method of preparing the precursor indole for labeling,
and the C-11 labeling of the most promising indole derivative, are described herein.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 13 January 2008
Revised 29 April 2008
Accepted 5 May 2008
Available online 7 May 2008
Keywords:
PET
Carbon-11
Serotonin transporter
Indolylpropylamine
1. Introduction
ping of human brain SERT by PET could be very valuable for under-
standing alterations of the serotonergic system and might prove
Serotonergic neurotransmission is known to modulate numer-
ous physiological and behavioral functions in the central nervous
system (CNS).1 It is involved in ‘integrating’ emotion, cognition,
motor function, and pain as well as circadian rhythm and neuroen-
docrine functions. The serotonin (5-hydroxytryptamine, 5-HT)
transporter (SERT; 5-HTT), located on presynaptic nerve endings,
terminates the synaptic actions of serotonin and recycles it into
the presynaptic neurotransmitter pool. Deficits in serotonergic
transmission have been implicated in several neurological and psy-
chiatric disorders such as depression, drug addiction, eating disor-
ders, schizophrenia and obsessive compulsive disorder (OCD).2,3
Depression affects about 100 million people worldwide, and has
been ranked by the World Health Organization (WHO) as the lead-
ing cause of disability and premature death, which results in high
utilization of health services and decreased work productivity.4
Based on the implication of SERT in the pathogenesis and therapy
of neuropsychiatric diseases and the possibility to use SERT as a
marker of the integrity of the serotonergic system, considerable ef-
forts have been invested in recent years in the design, synthesis,
radiolabeling, and biological and pharmacological characterization
of radiolabeled biomarkers (radiotracers) for non-invasive quanti-
tative molecular imaging of SERT using nuclear medicine modali-
ties such as Single Photon Emission Computed Tomography
(SPECT) and Positron Emission Tomography (PET).5,1 In vivo map-
useful for the monitoring of antidepressant therapy.6 Several puta-
tive SPECT and PET radiotracers for SERT have been suggested and
reported, but none proved to be the ultimate agent for the imaging
of the transporter. The first generation of SERT radiotracers, such as
C-11 labeled cyanoimipramine, citalopram, and sertraline was
characterized by various shortcomings, such as poor signal-to-
noise ratios,7–9 and lack of selectivity for the SERT over the dopa-
mine transporter (DAT)10 or the norepinephrine transporter
(NET).11 Until recently, the most widely used PET radiotracer for
the in vivo investigation of highly expressed midbrain and tha-
lamic SERT has been [11C]McN5652.12–14 It has been used for quan-
tification of SERT in the normal human brain,15–18 and in patients
intoxicated with methylenedioxymethamphetamine (MDMA),19,20
with social phobia,21 major depression and bipolar disorder.22
However, its rather high nonspecific binding in the brain and the
resulting weak signal-to-noise ratio precludes imaging of SERT in
regions of moderate or low densities such as the limbic and neo-
cortical regions. Introducing F-18 into McN5652 could only par-
tially improve its imaging properties.23–25 In the series of aryl-
nortropane compounds, radiolabeled b-CIT derivatives have been
developed and used in SPECT and PET.26–28 These SERT imaging
agents have been tested in normal subjects29–31 and in subjects
with neurologic32 and psychiatric disorders33–35 however, they
have not proven to be effective as expected in part because of their
considerable and undesirable affinity for the DAT. More recently, a
new series of PET radiotracers, based on the substituted diarylsul-
fide class of compounds, such as [11C]DASB, [11C]MADAM, and
* Corresponding author. Tel.: +972 2 677 7931; fax: +972 2 642 1203.
E-mail address: eyalmi@ekmd.huji.ac.il (E. Mishani).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.006

R. Ben-Daniel et al. / Bioorg. Med. Chem. 16 (2008) 6364–6370
6365
[
11C]AFM have emerged.36–48 All of these compounds have been
2.2.3. 3-(5-Iodo-1H-indole-3-yl) propanol (1c)
shown to be selective SERT radiotracers with varying specific-to-
nonspecific binding ratios in vivo and diverse brain kinetics. The
path toward the development of a useful radiotracer for in vivo
imaging of SERT continues its course. The search for fluorine-18 la-
beled radiotracers in order to reliably determine to what extent the
serotonergic system is affected in neuropsychiatric disorders re-
mains a challenge.
1H NMR (DMSO-d6) d 10.934 (s, NH), 7.822 (s, 1H), 7.191 (m,
1H), 7.162 (s, 2H), 4.427 (s, OH), 3.429 (m, 2H), 2.669 (m, 2H),
1.774 (t, J = 6.3 Hz, 2H). GCMS, m/z 287, 36% yield.
2.2.4. 3-(3-Bromo-propyl)-5-fluoro-1H-indole (2a)49
To a solution of triphenylphosphine (0.025 mol) in 80 mL of dry
acetonitrile, a solution of 1a (0.02 mol) in 75 mL acetonitrile was
Given the growing importance of the above-mentioned brain
diseases for public health and the closely connected socio-eco-
nomic impact of neuropsychiatric disorders, there is indeed a
pressing need to develop a more suitable PET radiotracer for SERT
in terms of low nonspecific binding, absence of radiolabeled
metabolites that cross the blood brain barrier (BBB), measurable
free fraction in plasma, and optimal affinity and selectivity. Based
on a new class of indole derivatives which hold low-nanomolar
affinity for the SERT and high selectivity versus the 5-HT1A recep-
tor,49 we synthesized four new indolylpropylamine derivatives
which contain atoms to afford future labeling with PET isotopes.
The chemistry of these novel derivatives, their biological evalua-
tion, the general method of preparing the precursor indole for
labeling, and the C-11 labeling of the most promising indole deriv-
ative are described herein.
added dropwise. Then
a solution of carbon tetrabromide
(0.027 mol) in 25 mL acetonitrile was added dropwise. The reac-
tion mixture is allowed to stir at rt for 3 h. Evaporation and purifi-
cation on silica-gel column (50% hexanes:50% CH2Cl2) yielded 4.5 g
(80%) of 2a. 1H NMR (DMSO-d6) d 11.02 (s 1H), 7.55 (s,1H), 7.23 (d,
J = 1.2 Hz, 1H), 7.08 (m, 2H), 3.42 (t, J = 1.4 Hz, 2H), 2.89 (t,
J = 7.1 Hz, 2H), 2.112 (m, 2H). GCMS, m/z 241.
2.2.5. 3-(3-Bromo-propyl)-5-bromo-1H-indole (2b)
1H NMR (DMSO-d6) d 11.036 (s, NH), 7.684 (s, 1H), 7.309 (d,
J = 0.6 Hz, 1H), 7.197 (s, 1H), 7.158 (d, J = 6.6 Hz, 1H), 3.536 (t,
J = 0.9 Hz, 2H), 2.794 (t, J = 7.2 Hz, 2H), 2.125 (m, 2H). GCMS, m/z
203.
2.2.6. 3-(3-Iodo-propyl)-5-bromo-1H-indole (2c)
1H NMR (DMSO-d6) d 11.006 (s, NH), 7.856 (s,1H), 7.308 (d,
J = 8.1 Hz, 1H), 7.201 (m, 2H), 3.539 (t, J = 3.9 Hz, 2H), 2.783 (t,
J = 6.6 Hz, 2H), 2.119 (m, 2H). MS, m/z 349.
2. Materials and methods
2.1. General methods
2.2.7. 6-Methoxy-1,2,3,4-tetrahydroisoquinoline (3a)50
All chemicals were purchased from Sigma–Aldrich, Fisher Scien-
tific, Merck, or J.T. Baker. Chemicals were used as supplied, excluding
THF, whichwasrefluxedoversodiumandbenzophenoneandfreshly
distilled prior to use. Mass spectra were obtained on a LCQ DUO,
Thermo Quest Finnigan spectrometer, equipped with ESI probe
and GC Mass spectroscopy was performed in EI mode on a Thermo
Quest, Finnigan Trace MS spectrometer at the Hadassah-Hebrew
University Mass Spectroscopy Facility. HRMS results were obtained
on MALDI-TOF and ESI mass spectrometers at the Schulich Faculty of
Chemistry, Technion - Israel Institute of Technology. 1H NMR spectra
were obtained on a Bruker AMX 300 MHz instrument. Radiosynthe-
ses were carried out with a [11C]-CH3I module (GE, Munster, Ger-
many). Specific radioactivity was determined by HPLC, using cold
mass calibration curves. [11C]-CO2 was produced by the 14N(p,
a)11C nuclear reaction on nitrogen containing 1% oxygen, using an
18/9 IBA-cyclotron. Bombardment was carried out for 15–30 min
with a 26 lA beam of 16 MeV protons. At the end of bombardment,
the target gas was delivered and trapped by a cryogenic trap in the
3-2-(3-Methoxy-phenyl)-ethylamine(0.013 mol) was dissolved
in 1 N HCl to obtain pH = 0.4. Then, aqueous formaldehyde (39%,
1.88 mL, 25 mmol) was added. The mixture was stirred at room
temperature for 24 h. The solution was basified with sodium
hydrogen carbonate, extracted with ethyl acetate (3 Â 50 mL) and
evaporated. Purification of the two isomers; 6-methoxy- and 8-
methoxy-1,2,3,4-tetrahydroisoquinoline with silica-gel column
yielded 0.7 g (35%) of 3a. 1H NMR (DMSO-d6) d 9.05 (s, NH), 7.11
(d, J = 2.7 Hz, 1H), 6.92 (s, 2H) 4.08 (s, 2H), 3.88 (s, 3H), 3.28 (s,
2H), 3.05 (t, J = 5.0 Hz, 2H). MS (m/z) 163 (MH+).
2.2.8. 5-Methoxy-1,2,3,4-tetrahydroisoquinoline (3b)50
2-(2-Methoxy-phenyl)-ethylamine (0.013 mmol) was dissolved
in 1N HCl to obtain pH = 0.4. Then, aqueous formaldehyde
(1.88 mL, 39%, 25 mmol) was added. The mixture was stirred at
room temperature for 24 h. The solution was basified with sodium
hydrogen carbonate, extracted with ethyl acetate (3 Â 50 mL) and
evaporated. Purification with silica-gel column yielded 0.8 g
[
11C]-CH3I module.
l
(38%) of 3b. H NMR (DMSO-d6) d 9.12 (s, NH), 7.10 (d, J = 2.7 Hz,
1H), 6.81 (s, 2H) 4.2 (s, 2H), 3.85 (s, 3H), 3.35(s, 2H), 3.1 (t,
2.2. Chemistry
J = 5.0 Hz, 2H). MS (m/z) 163 (MH+).
2.2.1. 3-(5-Fluoro-1H-indole-3-yl) propanol (1a)49
To a solution of 4-fluoro phenylhydrazine (6.92 mmol) in aque-
ous H2SO4 (4%, 10 mL), dihydropyran (630 lL) was added drop-
wise. The mixture was refluxed for 2 h. After cooling it was
extracted with ethyl acetate (2 Â 100 mL). The organic phase was
washed with water and evaporated. Further purification on silica
gel (1% MeOH: 99% CH2Cl2) yielded 300 mg (43%) of 1a. 1H NMR
(DMSO-d6) d 10.843 (s, NH), 7.312 (m, 1H), 7.282 (d, J = 4.5 Hz,
1H), 7.205 (s, 2H), 4.474 (s, OH), 3.393 (t, J = 5.1 Hz, 2H), 2.67 (m,
2H), 1.765 (t, J = 1.2 Hz, 2H). GCMS, m/z 179.
2.2.9. 2-[3-(5-Fluoro-1H-indol-3-yl)-propyl]-6-methoxy-
1,2,3,4- tetrahydro-isoquinoline (4)50
A mixture of 3-(3-bromo-propyl)-5-fluoro-1H-indole (0.0039
mol) and 6-methoxy-1,2,3,4-tetrahydro-isoquinoline (0.0039 mol)
and triethylamine (0.8 mL, 0.0058 mol) in DMSO (10 mL) was stir-
red at 100 °C for 24 h. The reaction mixture was cooled and ex-
tracted with ethyl acetate (3Â 50 mL). The organic phase was
washed with water (200 mL), dried with magnesium sulfate, and
evaporated. Purification on silica gel (2% MeOH: 98% CH2Cl2)
yielded 0.26 g (20%) of 4. 1H NMR (DMSO-d6) d 10.858 (s, NH),
7.315 (m, 3H), 6.949 (m, 2H), 6.679 (m, 2H), 3.677 (s, 3H), 3.439
(s, 2H), 2.762 (t, J = 5.1 Hz, 2H), 2.664 (t, J = 6.6 Hz, 2H), 2.598 (t,
J = 5.4 Hz, 2H) 2.476 (m, 2H), 1.859 (t, J = 7.2 Hz, 2H). MS (m/z)
338 (MH+), HRMS (C21H24N2OF): 339.1870.
2.2.2. 3-(5-Bromo-1H-indole-3-yl) propanol (1b)
1H NMR (DMSO-d6) d 10.962 (s, NH), 7.679 (s, 1H), 7.32 (d,
J = 3 Hz, 1H), 7.205 (s, 2H), 4.501 (s, OH), 3.497 (m, 2H), 2.7157
(m, 2H), 1.822 (t, J = 8.1 Hz, 2H). GCMS, m/z 239. 44% yield.

6366
R. Ben-Daniel et al. / Bioorg. Med. Chem. 16 (2008) 6364–6370
2.2.10. 2-[3-(5-Fluoro-1H-indol-3-yl)-propyl]-5-methoxy-
1,2,3,4-tetrahydro-isoquinoline (5)49
2.3. Biological assay
1H NMR (DMSO-d6) d 10.858 (s, NH), 7.315 (m, 3H), 6.949 (m,
2H), 6.679 (m, 2H), 3.677 (s, 3H), 3.439 (s, 2H), 2.762 (t,
J = 5.1 Hz, 2H), 2.664 (t, J = 6.6 Hz, 2H), 2.598 (t, J = 5.4 Hz, 2H)
2.476 (m, 2H), 1.859 (t, J = 7.2 Hz, 2H). MS (m/z) 338 (MH+). HRMS
(C21H24N2OF): 339.1873.
2.3.1. Cell culture
HEK293 cells stably expressing human monoamine reuptake
transporters were obtained from R. Blakely (hSERT-HEK293; Van-
derbilt University, Nashville, USA), H. Boenisch (hNET-HEK293;
University of Bonn, Germany), and A. Storch (hDAT-HEK293; Tech-
nical University Dresden, Germany). The transfected cells, main-
tained at 37 °C in a 5% CO2 humidified atmosphere, were grown
in complete medium (Dulbecco’s modified Eagle’s medium, 10% fe-
tal calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 lg/
mL streptomycin) supplemented with 250 lg/mL G418. The cells
were grown to 95% confluency in 175 cm2 cell culture flasks. Med-
ium was aspirated, and cells were washed with phosphate-buf-
fered saline and collected by scraping. The cells were sedimented
by low-speed centrifugation at 800 rpm, resuspended in ice-cold
buffer (50 mM Tris–HCl, pH 7.4) for cell disruption, and stored at
À25 °C. The protein concentrations of the membrane suspensions
were determined with the BCA assay.
2.2.11. 2-[3-(5-Bromo-1H-indol-3-yl)-propyl]-6-methoxy-
1,2,3,4-tetrahydro-isoquinoline (6)
1H NMR (DMSO-d6) d 10.992 (s, NH), 7.666 (s, 1H), 7.298 (d,
J = 1.8 Hz, 1H), 7.184 (m, 2H), 6.952 (d, J = 8.4 Hz, 1H), 6.677 (m,
2H), 3.675 (s, 3H), 3.45(s, 2H), 3.350 (t, J = 5.1 Hz, 2H), 2.784 (t,
J = 5.3 Hz, 2H), 2.653 (t, J = 5.4 Hz, 2H) 2.581 (m, 2H), 1.851(t,
J = 7.2 Hz, 2H). MS (m/z) 398 (MH+). HRMS(C21H24N2OBr):
399.1070.
2.2.12. 2-[3-(5-Bromo-1H-indol-3-yl)-propyl]-5-methoxy-
1,2,3,4- tetrahydro-isoquinoline (7)
1H NMR (DMSO-d6) d 10.968 (s, NH), 7.663 (s, 1H), 7.293 (d,
J = 2.1 Hz, 1H), 7.180 (m, 2H), 6.949 (d, J = 8.4 Hz, 1H), 6.669 (m,
2H), 3.677 (s, 3H), 3.443 (s, 2H), 3.152 (t, J = 5.3 Hz, 2H), 2.774 (t,
J = 5.3 Hz, 2H), 2.703 (t, J = 5.4 Hz, 2H) 2.653 (m, 2H), 1.851 (t,
J = 7.1 Hz, 2H). MS (m/z) 398 (MH+), HRMS(C21H24N2OBr):
399.1072.
2.3.2. Animals
Female SPRD rats (approximately 200 g., 10–12 weeks old;
Medizinisch-Experimentelles Zentrum, University Leipzig) were
anesthetized by carbon dioxide inhalation and killed by cervical
dislocation. Brains were immediately excised and dissected on
ice. The cortices were collected, homogenized in ice-cold 50 mM
Tris–HCl (pH 7.4 at 4 °C) with a Potter Elvehjem homogenizer,
and centrifuged (20.000g, 15 min, 4 °C). The membrane pellet
was washed two times by rehomogenization and centrifugation
as before. The final membrane pellet was dissolved with 50 mM
Tris–HCl (pH 7.4 at 4 °C) at 1 g original wet weight/10 mL buffer,
and stored in aliquots at À25 °C.
2.2.13. 2-[3-(5-Iodo-1H-indol-3-yl)-propyl]-6-methoxy-1,2,3,4-
tetrahydro isoquinoline (8)
1H NMR (DMSO-d6) d 10.969 (s, NH), 7.844 (s, 1H), 7.274 (m, 1H,
J = 1.8 Hz), 7.195(m, 2H, J = 2.7 Hz), 6.931 (d, 3H, J = 3.0 Hz), 3.650
(s, 3H), 3.45(s, 2H), 3.350 (t, J = 5.1 Hz, 2H), 2.794 (t, J = 7.5 Hz,
2H), 2.643 (t, J = 6.6 Hz, 2H) 2.584 (m, J = 6.3 Hz, 2H), 1.831(t,
J = 7.4 Hz, 2H). MS (m/z) 446 (MH+), HRMS(C21H24N2OI): 447.0933.
2.3.3. In vitro hSERT, hNET, hDAT, and rHT1A binding assays
For hSERT, hNET, and hDAT, the IC50 values of the test com-
pounds were determined on preparations of HEK293 cells express-
ing the respective transporter protein and [3H]paroxetine (SERT;
PerkinElmer Life Sciences; AS = 706 GBq/mmol), [3H]citalopram
(SERT; Amersham GE Healthcare; AS = 3121 GBq/mmol), [3H]nisox-
etine (NET; Perkin-Elmer Life Sciences; AS = 2960 GBq/mmol), and
[3H]WIN35,428 (DAT; PerkinElmer Life Sciences; AS = 3145 GBq/
mmol) as specific radioligands. The r5HT1A affinity was determined
on rat cortical membrane homogenate and [3H]-8-OH-DPAT
(Amersham GE Healthcare; AS = 8399 GBq/mmol) as specific
radioligand.
For competitive binding experiments, membrane suspensions
were thawed, diluted with the respective assay buffer (hSERT
and hNET: 50 mM Tris–HCl, pH 7.4, 120 mM NaCl, and 5 mM
KCl; hDAT: 50 mM Tris–HCl, pH 7.4, and 100 mM NaCl;
r5HT1A: 50 mM Tris–HCl, pH 7.4, 4 mM CaCl2, 0.1% ascorbic acid)
and homogenized by a 27-gauge needle. The particular mem-
brane preparation (50–80 lg protein/assay tube) was incubated
with the respective radioligand (ꢀworking concentrations:
1 nM [3H]paroxetine, 0.5 nM [3H]citalopram, 0.5 nM [3H]nisoxe-
tine, 0.5 nM [3H]WIN35,428, 0.3 nM [3H]-8-OH-DPAT) and 6–12
concentrations of the test compounds in glass tubes. The test
compounds (10 mM stock solutions in DMSO) were diluted with
the respective assay buffer. Nonspecific binding was determined
with 300 lM clomipramin (hSERT), 100 lM protriptyline (hNET),
10 lM GBR12909 (hDAT), or 10 lM serotonin hydrochloride
(r5HT1A). The tubes were gently mixed and incubated at 21 °C
for 60 min (hSERT, hNET, and r5HT1A), or on ice for 120 min
(hDAT), rapidly filtered through Whatman GF/B glass-fibre filters,
and washed four times with ice-cold 50 mM Tris–HCl, pH 7.4.
Filter-bound radioactivity was determined by scintillation
counting.
2.2.14. 2-[3-(5-Iodo-1H-indol-3-yl)-propyl]-5-methoxy-1,2,3,4-
tetrahydro-isoquinoline (9)
1H NMR (DMSO-d6) d 10.945 (s, NH), 7.834 (s, 1H), 7.294 (m,
1H), 7.187 (m, 2H), 6.739 (d, J = 8.1 Hz, 3H), 3.736 (s, 3H), 3.481
(s, 2H), 3.314 (t, J = 1.8 Hz, 2H), 2.691 (t, J = 7.5 Hz, 4H), 2.481 (t,
J = 6.5 Hz, 2H), 1.848 (t, J = 7.4 Hz, 2H). LCMS, m/z (relative inten-
sity) MS (m/z) 446 (MH+), HRMS(C21H24N2OI): 447.0928.
2.2.15. 6-Hydroxy-1,2,3,4-tetrahydroisoquinoline (10)
Compound 3a (0.003 mol) was dissolved in hydrobromic acid
(conc., 15 mL) and refluxed for 3 h. The reaction mixture was
cooled and basified with sodium bicarbonate, extracted with ethyl
acetate (2 Â 50 mL), dried with MgSO4, and evaporated. Crystalli-
l
zation from MeOH gave 0.045 g (10%) of 10. H NMR (DMSO-d6) d
6.812 (d, J = 7.5 Hz, 1H), 6.501 (d, J = 6.2 Hz,1H), 6.379 (d,
J = 3.6 Hz, 1H), 3.729 (s, 2H), 2.881 (t, J = 1.4 Hz, 2H), 2.419 (t,
J = 1.2 Hz, 2H). MS (m/z) 149 (MH+).
2.2.16. 2-[3-(5-Fluoro-1H-indol-3-yl)-propyl]-6-hydroxy-
1,2,3,4-tetrahydro-isoquinoline (11)
A mixture of 3-(3-bromo-propyl)-5-fluoro-1H-indole (0.0039 mol),
6-hydroxy-1,2,3,4-tetrahydro-isoquinoline
(0.0039 mol),
and
triethylamine (0.8 mL, 0.0058 mol) in DMSO (10 mL) was stirred at
100 °C for 24 h. The reaction mixture was cooled and extracted with
ethyl acetate (3 Â 50 mL). The organic phase was washed with water
(200 mL), dried with magnesium sulfate, and evaporated.
Purification on silica gel (2% MeOH: 98% CH2Cl2) yielded 0.65 g
(48%) of 11. 1H NMR (DMSO-d6) d 10.858 (s, NH), 9.089 (s, OH),
7.323 (m, J = 4.8 Hz, 3H), 6.907 (m, J = 8.1 Hz, 2H), 6.521 (m,
J = 2.1 Hz, 2H), 3.160 (s, 2H), 2.711 (m, J = 4.5 Hz, 3H), 2.601 (t,
J = 5.1 Hz, 2H), 2.486 (t, J = 1.5 Hz, 2H), 1.977 (t, J = 1.8 Hz, 3H). MS
(m/z) 324 (MH+).

R. Ben-Daniel et al. / Bioorg. Med. Chem. 16 (2008) 6364–6370
6367
Saturation binding experiments of [3H]paroxetine, [3H]citalo-
NH
R
1
pram, [3H]nisoxetine, and [3H]WIN35,428 were performed by
homologous competition according to the described protocols.
N
R1= 5,6 Methoxy
2.3.4. Data analysis
R
2
All test compounds were assayed in at least three independent
experiments, each conducted in triplicate. The binding parameters
IC50, KD, and Bmax were estimated using iterative nonlinear curve
fitting. The KD of [3H]-8-OH-DPAT was taken from literature
(KD = 3.1 nM on rat cortex; Newman-Tancredi et al., Int. J. Neuro-
psychopharmacol. 8, 2005, 1–16). The Cheng–Prusoff equation
was applied to calculate Ki values from estimated IC50 values.
R2= F, Br, I
4: F-6 Methoxy
6: Br-6 Methoxy
8: I-6 Methoxy
5: F-5 Methoxy
7: Br-5 Methoxy
9: I- 5 Methoxy
Figure 1. Chemical structure of the SERT ligands used for PET radiotracer design.
2.4. Radiochemistry
The tetrahydroisoquinoline analogs, 3a–b, were prepared by Pic-
tet-Spengler cyclization of methoxy phenylethylamine with form-
aldehyde in acidic solution with a 10% yield. This procedure
simplifies the multi-step synthesis via the chloroformate interme-
diate as described by Meagher et al.49 In the case of 2-methoxy
phenylethylamine, the cyclization with formaldehyde yielded only
the 5-methoxy tetrahydroisoquinoline product, whereas, in the
case of 3-methoxy phenylethylamine, cyclization yielded a mix-
ture of 6- and 8-methoxy tetrahydroisoquinoline products. The
8-methoxy tetrahydroisoquinoline was obtained as the minor
product, probably due to steric hindrance. A coupling reaction of
methoxy-1,2,3,4-tetrahydroisoquinoline and 3-(3-bromo-propyl)-
5-R-1H-indole yields the six final compounds 4–9. The yield and
selectivity of the final step are rather low (20%).
Carbon-11 MeI was prepared according to well-documented
procedures. Briefly, [11C]CO2 (37 GBq, 1000 mCi) was trapped at
À160 °C. The temperature of the cooling trap was increased to
À50 °C, and the activity was transferred by a stream of argon
(40 mL/min) to a reactor containing 300 lL of 0.25 N LiAlH4 in
THF at À50 °C. After 90 s, the solvent was removed under reduced
pressure. In this manner, more than 80% of the activity was recov-
ered. The reactor temperature was increased to 160 °C, HI was
added, and [11C]MeI was distilled (argon flow of 15 mL/min)
through a NaOH column to a second reactor, containing the precur-
sor, 11, in 600 lL of dry DMF and 30 lL NaOH 5 N at À20 °C. After
1 min of distillation, an average of 550 30 mCi (n = 4) was
trapped in the second reactor. The reactor was sealed and heated
to 80 °C for 5 min. The mixture was cooled to 40 °C, 600 lL
CH3CN/H2O (1:1) was added, and the crude product
(250 20 mCi) was automatically injected to HPLC [Bischoff Nucle-
osil 100-7 C18 reverse phase preparative column (7 lm, 250Â
16 mm, 55% ACN: 45% Acetate buffer, pH 3.8, flow rate of 10 ml/
min]. The labeled compound was collected in a flask containing
85 mL of water. The solution was passed through a C-18 cartridge
(Waters Sep-Pak Plus, pre-activated with 10 mL EtOH and 20 mL of
water). The product was eluted with 0.75 mL of EtOH, followed by
4.25 mL of saline, and collected into the product vial after a total
radiosynthesis time of 50 min, with total activity of 20 6 mCi
and radiochemical yield of 15 3% decay corrected EOB (n = 4).
Identification of the products and determination of chemical purity
were obtained by reversed-phase HPLC C-18 analytical column in
comparison with the standard and by co-injection.
The synthesis of the precursor 11 for the radiolabeling is de-
scribed in Scheme 2. The first step is demethylation with HBr conc.
at 120 °C to obtain 10 in 10% yield, followed by coupling reaction in
DMSO in the presence of triethylamine as base in 20% yield.
3.2. Affinity data
For [3H]paroxetine, [3H]citalopram, [3H]nisoxetine, and [3H]
WIN35,428 the following KD values were obtained on the human
monoamine transporters hSERT, hNET, and hDAT by homologous
competition experiments: 0.69 nM [3H]paroxetine, 4.46 nM
[3H]citalopram, 6.77 nM [3H]nisoxetine, and 24.1 [3H]WIN35,428.
The values correspond to previously published data on these
monoamine transporters.51,52
The specific Ki values of the two reference compounds 4, 5 and
the four newly developed compounds6, 7, 8, 9 (Fig. 1) are given in
Table 1. While compounds 4, 5, and 6 possess high affinity in the
nanomolar range and 70- to 200-fold specificity for SERT over
DAT and NET, compounds 7, 8, and 9 are characterized by remark-
ably low SERT affinity and selectivity. Compounds 4 and 5 are par-
ticularly interesting in this series because they possess the highest
SERT selectivity versus the r5HT1A receptor. Furthermore, in con-
trast to the diphenyl-thioether MADAM, compounds 4 and 5 dis-
criminate significantly between the hSERT binding sites labeled
with either [3H]citalopram and [3H]paroxetine, respectively,
(p < 0.05, t test). This preliminary result may indicate the possibil-
ity to detect the integrity of SERT by investigating different parts of
the protein conformation.
3. Results and discussion
3.1. Chemistry
The chemical structure of indole derivatives and 5-HT serves as
a template for the development of new chemical entities that could
potentially target the 5-HT1A receptor and the SERT. In 2001, Mea-
gher et al.49 designed a new class of indole derivatives as a dual
antidepressant drug targeting the SERT and the 5-HT1A receptor.
The biological evaluation of these compounds indicated that some
of these derivatives had high affinity and selectivity toward the
SERT. Based on these results, we synthesized additional four new
indole derivatives and evaluated all six compounds as potential
selective SERT PET radiotracers. These compounds contain atoms
such as fluorine, bromine, and iodine on the indole moiety to afford
future labeling with PET isotopes (Fig. 1).
Due to its affinity and specificity profile, compound 4 was cho-
sen as the most promising candidate for radiolabeling with the PET
isotope carbon-11.
3.3. Radiochemistry
Scheme 1 shows the synthesis of the target molecules. The
hydroxypropyl indole intermediates 1a–c were obtained by reac-
tion of 4R phenylhydrazine and dihydropyran in 43, 44, and 37%
yields, respectively. The second step is bromination with carbon
tetrabromide and triphenylphosphine to obtain 2a–c in 80% yield.
Based on the biological assay, compound 4 was chosen for label-
ing with C-11 on the hydroxy position at the isoquinoline ring.
The radiosynthesis is outlined in Scheme 3. The radiolabeling
method was based on a C-11 methylation reaction on the phenolic

6368
R. Ben-Daniel et al. / Bioorg. Med. Chem. 16 (2008) 6364–6370
OH
Br
R
R
R
b
a
+
N
N
H
O
H
2a,b,c
1a: R= F
1b: R= Br
1c: R=I
NHNH2
MeO
MeO
NH2
(c)
+
NH
NH
NH
3a= 6-methoxy
OMe
OMe
OMe
NH2
(c)
3b= 5-methoxy
Br
R
d
NH
+
MeO
MeO
NH
N
N
H
3a,b
2a,b,c
4-9
R
4: F-6 Methoxy
6: Br-6 Methoxy
8: I-6 Methoxy
5: F-5 Methoxy
7: Br-5 Methoxy
9: I- 5 Methoxy
Scheme 1. Reagents and conditions: (a) H2SO4 (4%), 100 °C, 2 h; (b) CBr4, PPh3, CH2Cl2, 3 h; (c) HCHO (39%), 1 N HCl, rt, 24 h; (d) Et3N, DMSO, 100 °C, 24 h.
HO
MeO
(a)
NH
NH
3a
10
Br
HO
HO
F
NH
(b)
+
N
NH
N
H
11
2a
F
Scheme 2. Reagents and conditions: (a) HBr, 120 °C, 3 h; (b) Et3N, DMSO, 100 °C, 24 h.
Table 1
Binding affinities of reference and test compounds at hSERT, r5HT1A, hDAT, and hNET
Target/structure
Radioligand
hSERT; Ki (nM)
r5HT1A; Ki (nM)
hNET; Ki (nM)
hDAT; Ki (nM)
[3H]citalopram
[3H]paroxetine
[3H]-8-OH-DPAT
[3H]nisoxetine
[3H]WIN35,428
4
6
8
5
7
9
4.11 1.34
11.4 3.50
374 226
6.46 3.94
1910 468
213 51.1
0.42 0.13
4.38 1.17
0.73 0.37
25.3 14.2
13.3 7.22
1110 617
44.7 6.19
2980 1320
2660 1480
0.38 0.12
32.8 8.69
2.03 1.08
54.0 5.40
50.0 5.46
685 184
164 176
1990 157
1120 99.9
–
1680 (1810; 1560)
2770 1940
712 524
2880 (1990; 3770)
6600 2300
1810 (2490; 1120)
—
—
—
2490 (2620; 2360)
1030 (1290; 769)
3650 (1800; 5500)
1485 (1700; 1270)
3490 1820
1155 (1140; 1170)
—
Paroxetine
R/S-Citalopram
MADAM
—
—
—
—
IC50 values were obtained from triplicates in each experiment, and the calculated Ki values are given as single values (n = 1), means and single values (n = 2), or means SD
(n > 2).

R. Ben-Daniel et al. / Bioorg. Med. Chem. 16 (2008) 6364–6370
6369
HO
O
NH
H311C
NH
11CH3I
N
N
[11C]-4
11
F
F
Scheme 3. Reagents and conditions: DMF, NaOH 5 N, 80 °C.
16. Buck, A.; Gucker, P. M.; Schönbächler, R. D.; Argoni, M.; Kneifel, S.;
Vollenweider, F. X.; Ametamey, S. M.; Burger, C. J. Cereb. Blood Flow Metab.
2000, 20, 253–262.
17. Parsey, R. V.; Kegeles, L. S.; Hwang, D. R.; Simpson, N.; Abi-Dargham, A.;
Mawlawi, O.; Slifstein, M.; Van Heertum, R. L.; Mann, J. J.; Laruelle, M. J. Nucl.
Med. 2000, 41, 1465–1477.
18. Ikoma, Y.; Suhara, T.; Toyama, H.; Ichimiya, T.; Takano, A.; Sudo, Y.; Inoue, M.;
Yasuno, F.; Suzuki, K. J. Cereb. Blood Flow Metab. 2002, 22, 490–501.
19. McCann, U. D.; Szabo, Z.; Scheffel, U.; Dannals, R. F.; Ricaurte, G. A. Lancet 1998,
352, 1433–1437.
20. Ricaurte, G. A.; McCann, U. D.; Szabo, Z.; Scheffel, U. Toxicol. Lett. 2000, 112-113,
143–146.
21. Kent, J. M.; Coplan, J. D.; Lombardo, I.; Hwang, D. R.; Huang, Y.; Mawlawi, O.;
Van Heertum, R. L.; Slifstein, M.; Abi-Dargham, A.; Gorman, J. M.; Laruelle, M.
Psychopharmacology (Berl.) 2002, 164, 341–348.
22. Ichimiya, T.; Suhara, T.; Sudo, Y.; Okubo, Y.; Nakayama, K.; Nankai, M.; Inoue,
M.; Yasuno, F.; Takano, A.; Maeda, J.; Shibuya, H. Biol. Psychiatry 2002, 51, 715–
722.
23. Brust, P.; Hinz, R.; Kuwabara, H.; Hesse, S.; Zessin, J.; Pawelke, B.; Stephan, H.;
Bergmann, R.; Steinbach, J.; Sabri, O. Neuropsychopharmacology 2003, 28, 2010–
2019.
24. Brust, P.; Zessin, J.; Kuwabara, H.; Pawelke, B.; Kretzschmar, M.; Hinz, R.;
Bergman, J.; Eskola, O.; Solin, O.; Steinbach, J.; Johannsen, B. Synapse 2003, 47,
143–151.
25. Marjamäki, P.; Zessin, J.; Eskola, O.; Grönroos, T.; Haaparanta, M.; Bergman, J.;
Lehikoinen, P.; Forsback, S.; Brust, P.; Steinbach, J.; Solin, O. Synapse 2003, 47,
45–53.
26. Bergström, K. A.; Halldin, C.; Hall, H.; Lundkvist, C.; Ginovart, N.; Swahn, C. G.;
Farde, L. Eur. J. Nucl. Med. 1997, 24, 596–601.
27. Hiltunen, J.; Akerman, K. K.; Kuikka, J. T.; Bergström, K. A.; Halldin, C.; Nikula,
T.; Räsänen, P.; Tiihonen, J.; Vauhkonen, M.; Karhu, J.; Kupila, J.; Länsimies, E.;
Farde, L. Eur. J. Nucl.Med. 1998, 25, 19–23.
28. Kim, S. E.; Choi, J. Y.; Choe, Y. S.; Choi, Y.; Lee, W. Y. J. Nucl. Med. 2003, 44, 870–
876.
hydroxy group. C-11 methyliodide was produced using a commer-
cial module (GE) and was prepared according to well documented
procedures.53 C-11 methyliodide was then reacted with 5 mg of
precursor 11. The optimal reaction temperature was found to be
80 °C. After 5 min reaction, HPLC solvent was added and the crude
mixture was purified by a built-in HPLC to yield the final product
[
11C]-4. The product was collected into a flask containing 85 ml
of water in order to dilute the acetonitrile. Finally the product
was eluted through C-18 cartridge with ethanol/saline solution.
Total synthesis time was 50 min. HPLC analysis showed moderate
radiochemical yield (15 3% decay corrected EOB) and high chem-
ical purity. The high specific activity (0.423 Ci/lmol) is an impor-
tant factor for the development of PET biomarkers that target
low capacity systems.
4. Conclusion
Six indolylpropylamine derivatives were synthesized and
evaluated as SERT antagonists. The most promising compound, 2-
[3-(5-fluoro-1H-indol-3-yl)-propyl]-6-methoxy-1,2,3,4-tetrahydro-
isoquinoline was identified and successfully labeled with C-11. This
compound will be further evaluated in vivo using PET in animal
models.
Acknowledgments
29. van Dyck, C. H.; Malison, R. T.; Seibyl, J. P.; Laruelle, M.; Klumpp, H.; Zoghbi, S.
S.; Baldwin, R. M.; Innis, R. B. Neurobiol. Aging 2000, 21, 497–501.
30. Kuikka, J. T.; Tammela, L.; Bergström, K. A.; Karhunen, L.; Uusitupa, M.;
Tiihonen, J. Eur. J. Nucl. Med. 2001, 28, 911–913.
31. Staley, J. K.; Krishnan-Sarin, S.; Zoghbi, S.; Tamagnan, G.; Fujita, M.;
Seibyl, J. P.; Maciejewski, P. K.; Malley, S. O.; Innis, R. B. Synapse 2001,
41, 275–284.
The research is funded by the Joint German-Israeli Research
Program Ministry of Science and Technology (MOST, # 1888), Bun-
desministerium für Bildung und Forschung (BMBF).
References and notes
32. Hesse, S.; Barthel, H.; Hermann, W.; Murai, T.; Kluge, R.; Wagner, A.; Sabri, O.;
Eggers, B. J. Neural Transm. 2003, 110, 923–933.
33. Malison, R. T.; Price, L. H.; Berman, R.; van Dyck, C. H.; Pelton, G. H.;
Carpenter, L.; Sanacora, G.; Owens, M. J.; Nemeroff, C. B.; Rajeevan, N.;
Baldwin, R. M.; Seibyl, J. P.; Innis, R. B.; Charney, D. S. Biol. Psychiatry 1998,
44, 1090–1098.
34. Willeit, M.; Praschak-Rieder, N.; Neumeister, A.; Pirker, W.; Asenbaum, S.;
Vitouch, O.; Tauscher, J.; Hilger, E.; Stastny, J.; Brucke, T.; Kasper, S. Biol.
Psychiatry 2000, 47, 482–489.
35. Tauscher, J.; Pirker, W.; Willeit, M.; de Zwaan, M.; Bailer, U.; Neumeister, A.;
Asenbaum, S.; Lennkh, C.; Praschak-Rieder, N.; Brucke, T.; Kasper, S. Biol.
Psychiatry 2001, 49, 326–332.
1. Brust, P.; Hesse, S.; Müller, U.; Szabo, Z. Curr. Psychiatry Rev. 2006, 2, 111–149.
2. Meltzer, C. C.; Smith, G.; DeKosky, S. T.; Pollock, B. G.; Mathis, C. A.; Moore, R.
Y.; Kupfer, D. J.; CF r, d. R. Neuropsychopharmacology 1998, 18, 407–430.
3. Mann, J. J.; Huang, Y. Y.; Underwood, M. D.; Kassir, S. A.; Oppenheim, S.; Kelly,
T. M.; Dwork, A. J.; Arango, V. Arch. Gen. Psychiatry 2000, 57, 729–738.
4. Lopez, A.D.; Murray, C.C. 1990–2020. Nat. Med. 1998, 4, 1241–1243.
5. Brust, P.; Scheffel, U.; Szabo, Z. Idrugs 1999, 2, 129–145.
6. Meyer, J. H.; Wilson, A. A.; Ginovart, N.; Goulding, V.; Hussey, D.; Hood, K.;
Houle, S. Am. J. Psychiatry 2001, 158, 1843–1849.
7. Scheffel, U.; Dannals, R. F.; Suehiro, M.; Ricaurte, G. A.; Carroll, F. I.; Kuhar, M. J.;
Wagner, H. N. NIDA Res. Monogr. 1994, 138, 11–130.
8. Ichimiya, T.; Suhara, T.; Sudo, Y.; Okubo, Y.; Nakayama, K.; Nankai, M.; Inoue,
M.; Yasuno, F.; Takano, A.; Maeda, J.; Shibuya, H. Biol. Psychiatry 2002, 51, 715–
722.
9. Parsey, R. V.; Kegeles, L. S.; Hwang, D. R.; Simpson, N.; Abi-Dargham, A.;
Mawlawi, O.; Slifstein, M.; Van Heertum, R. L.; Mann, J. J.; Laruelle, M. J. Nucl.
Med. 2000, 41, 1465–1477.
36. Choi, S. R.; Hou, C.; Oya, S.; Mu, M.; Kung, M. P.; Siciliano, M.; Acton, P. D.; Kung,
H. F. Synapse 2000, 38, 403–412.
37. Houle, S.; Ginovart, N.; Hussey, D.; Meyer, J. H.; Wilson, A. A. Eur. J. Nucl. Med.
2000, 27, 1719–1722.
38. Oya, S.; Choi, S. R.; Hou, C.; Mu, M.; Kung, M. P.; Acton, P. D.; Siciliano, M.; Kung,
H. F. Nucl. Med. Biol. 2000, 27, 249–254.
10. Bergström, K. A.; Halldin, C.; Hall, H.; Lundkvist, C.; Ginovart, N.; Swahn, C. G.;
Farde, L. Eur. J. Nucl. Med. 1997, 24, 596–601.
39. Wilson, A. A.; Ginovart, N.; Schmidt, M.; Meyer, J. H.; Threlkeld, P. G.; Houle, S.
J. Med. Chem. 2000, 43, 3103–3110.
11. Szabo, Z.; Scheffel, U.; Suehiro, M.; Dannals, R. F.; Kim, S. E.; Ravert, H. T.;
Ricaurte, G. A.; Wagner, H. N. J. Cereb. Blood Flow Metab. 1995, 15, 798–805.
12. Suehiro, M.; Scheffel, U.; Dannals, R. F.; Ravert, H. T.; Ricaurte, G. A.; Wagner, H.
N. J. Nucl. Med. 1993, 34, 121–127.
40. Acton, P. D.; Choi, S. R.; Hou, C.; Plossl, K.; Kung, H. F. J. Nucl. Med. 2001, 42,
1556–1562.
41. Ginovart, N.; Wilson, A. A.; Meyer, J. H.; Hussey, D.; Houle, S. J. Cereb. Blood Flow
Metab. 2001, 21, 1342–1353.
13. Szabo, Z.; McCann, U. D.; Wilson, A. A.; Scheffel, U.; Owonikoko, T.; Mathews,
W. B.; Ravert, H. T.; Hilton, J.; Dannals, R. F.; Ricaurte, G. A. J. Nucl. Med. 2002,
43, 678–692.
14. Szabo, Z.; Owonikoko, T.; Peyrot, M.; Varga, J.; Mathews, W. B.; Ravert, H. T.;
Dannals, R. F.; Wand, G. Biol. Psychiatry 2004, 55, 766–771.
42. Meyer, J. H.; Wilson, A. A.; Ginovart, N.; Goulding, V.; Hussey, D.; Hood, K.;
Houle, S. Am. J. Psychiatry 2001, 158, 1843–1849.
43. Vercouillie, J.; Tarkiainen, J.; Halldin, C.; Emond, P.; Chalon, S.; Sandell, J.;
Langer, O.; Guilloteau, D. J. Labelled Compd. Radiopharm. 2001, 44, 113–
120.
15. Szabo, Z.; Scheffel, U.; Mathews, W. B.; Ravert, H. T.; Szabo, K.; Kraut, M.;
Palmon, S.; Ricaurte, G. A.; Dannals, R. F. J. Cereb. Blood Flow Metab. 1999, 19,
967–981.
44. Huang, Y.; Hwang, D. R.; Narendran, R.; Sudo, Y.; Chatterjee, R.; Bae, S. A.;
Mawlawi, O.; Kegeles, L. S.; Wilson, A. A.; Kung, H. F.; Laruelle, M. J. Cereb. Blood
Flow Metab. 2002, 22, 1377–1398.

6370
R. Ben-Daniel et al. / Bioorg. Med. Chem. 16 (2008) 6364–6370
45. Huang, Y.; Hwang, D. R.; Zhu, Z.; Bae, S. A.; Guo, N.; Sudo, Y.; Kegeles, L. S.;
Laruelle, M. Nucl. Med. Biol. 2002, 29, 741–751.
46. Chalon, S.; Tarkiainen, J.; Garreau, L.; Hall, H.; Emond, P.; Vercouillie, J.; Farde,
L.; Dasse, P.; Varnas, K.; Besnard, J. C.; Halldin, C.; Guilloteau, D. J. Pharmacol.
Exp. Ther. 2003, 304, 81–87.
47. Shiue, G. G.; Choi, S. R.; Fang, P.; Hou, C.; Acton, P. D.; Cardi, C.; Saffer, J. R.;
Greenberg, J. H.; Karp, J. S.; Kung, H. F.; Shiue, C. Y. J. Nucl. Med. 2003, 44, 1890–
1897.
49. Meagher, K. L.; Mewshaw, R. E.; Evrard, D. A.; Zhou, P.; Smith, D. L.; Scerni, R.;
Spangler, T.; Abulhawa, S.; Shi, X.; Schechter, L. E.; Andree, T. H. Bioorg. Med.
Chem. Lett. 2001, 11, 1885–1888.
50. Sall, J. D.; Grunewald, G. L. J. Med. Chem. 1987, 30, 2208–2216.
51. Chen, F.; Larsen, M. B.; Sánchez, C.; Wiborg, O. Eur. Neuropsychopharmacol.
2005, 152, 193–198.
52. Kim, D. I.; Deutsch, H. M.; Ye, X.; Schweri, M. M. J. Med. Chem. 2007, 5011,
2718–2731.
48. Huang, Y.; Narendran, R.; Bae, S. A.; Erritzoe, D.; Guo, N.; Zhu, Z.; Hwang, D. R.;
Laruelle, M. Nucl. Med. Biol. 2004, 31, 727–738.
53. Crouzel, C.; Langstrom, B.; Pike, V. W.; Coenen, H. H. J. Appl. Radiat. Isot. 1987,
38, 601–604.