Carbon-11 labelling of MADAM in two different positions: a highly selective PET radioligand for the serotonin transporter

Article abstract of DOI:10.1002/jlcr.523

Imaging by scintigraphy the serotonin transporter (5-HTT) in the living human brain would be of great value in research on the pathophysiology and treatment of neuropsychiatric disorders such as depression. For that reason, and in order to obtain a select

Full text of DOI:10.1002/jlcr.523

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 1013–1023.
DOI: 10.1002/jlcr.523
Research Article
Carbon-11 labelling of MADAM in two
different positions: a highly selective PET
radioligand for the serotonin transporter
Jari Tarkiainen1, Johnny Vercouillie2, Patrick Emond2, Johan Sandell1,
Jukka hiltunen3, Yves Frangin2, Denis Guilloteau2 and
Christer Halldin1,*
1 Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section,
Karolinska Hospital, S-17176 Stockholm, Sweden
2
!
INSERM U316, Universite Francois Rabelais, F-37200 Tours, France
,
3 MAP Medical Technologies Oy, FIN-00251 Helsinki, Finland
Summary
Imaging by scintigraphy the serotonin transporter (5-HTT) in the living human
brain would be of great value in research on the pathophysiology and
treatment of neuropsychiatric disorders such as depression. For that reason,
and in order to obtain a selective radiotracer applicable to PET, we report here
the carbon-11 labelling of a selective 5-HTT radioligand: N, N-dimethyl-2-(2-
amino-4-methylphenylthio)benzylamine or MADAM in two different posi-
tions: [p-11C-methyl]MADAM and [N-11C-methyl]MADAM. The synthesis of
Bu3Sn-ADAM and N-dimethyl-MADAM is described. [p-11C-methyl]MA-
DAM was obtained by a Stille coupling reaction between Bu3Sn-ADAM and
[11C]methyl iodide using palladium (0) as a catalyst without (Ia) or with copper
chloride as a co-catalyst (Ib). [N-11C-methyl]MADAM was obtained by an
N-methylation reaction between N-demethyl-MADAM and [11C]methyl iodide
*Correspondence to: C. Halldin, Karolinska Institutet, Department of Clinical Neuroscience,
Psychiatry Section, Karolinska Hospital S-17176 Stockholm, Sweden.
Contract/grant sponsor: Swedish Medical Research Council; Contract/grant number; 12983-01A
Contract/grant sponsor: Karolinska Institutet
Contract/grant sponsor: INSERM-MFR
Contract grant sponsor: The Region Centre (France)
Contract/grant sponsor: European program; Contract/grant number: COST B12
Copyright # 2001 John Wiley & Sons, Ltd.
Received 14 August 2001
Revised 3 September 2001
Accepted 12 September 2001

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J. TARKIAINEN ET AL.
(II). The carbon-11 incorporation yield in [p-11C-methyl]MADAM was
10–30% (Ia and Ib) and in [N-11C-methyl]MADAM was 75-80% (II). The
final product in each case was obtained in 30 min total synthesis time, including
HPLC purification and with>99% radiochemical purity. Copyright # 2001
John Wiley & Sons, Ltd.
Key Words: [11C]MADAM; serotonin transporter; 5-HTT; carbon-11; PET
Introduction
The role of the serotonin transporter (5-HTT) system in psychiatric
diseases such as depression1 and in neurodegenerative disorders such as
Alzheimer’s and Parkinson’s diseases2,3 can be significant. This
transporter modulates extracellular serotonin levels and acts as a site
of action of many antidepressant drugs. It would be of a great value to
explore the 5-HTT in human brain either by positron emission
tomography (PET) or by single-photon emission computed tomography
(SPECT) with appropriate radioligands.
Although several compounds have been synthesized possessing high
affinity for the 5-HTT in vitro none of these are optimal in PET and
SPECT due to a lack of selectivity for 5-HTT over the dopamine
transporter (DAT) and the norepinephrine transporter (NET). This is
the case for [11C](+)McN5652 and certain cocaine derivatives.4–9
Concerning [11C]citalopram and [123I]5-iodo-6-nitroquipazine they
present unsuitable pharmacokinetic properties and a poor signal to
noise ratio.10–12
Recently, Oya et al.13 described a 403U76 derivative named ADAM
(Figure 1), which exhibits in vitro high affinity and selectivity for
5-HTT (Ki5-HTT=0.013 Æ 0.003 nM, KiDAT=840 Æ 100 nM, KiNET
=
699 Æ 80 nM) and is an excellent SPECT tracer for visualisation of
5-HTT. In view of these results we synthesized [11C]ADAM for PET
N
N
NH2
NH2
S
S
H3C
I
ADAM
MADAM 6
Figure 1. Structures of ADAM and MADAM (6)
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CARBON-11 LABELLING OF MADAM
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examination.14 However, the relatively slow binding kinetics obtained
by [11C]ADAM is not optimal for a PET radioligand.15 In order to
improve its pharmacokinetic properties several radiolabelled analogs
of ADAM have been reported including the 11C-methoxy (DAPP)
and 11C-cyano (DASB) derivatives16 that have demonstrated potential
for PET visualisation of 5-HTT in the human brain. We have
synthesized N,N-dimethyl-2-(2-amino-4-methylphenylthio)benzylamine
(MADAM) which exhibits high affinity and selectivity for 5-HTT
(Ki5-HTT=1.65 Æ 0.1 nM, KiDAT=>10 mM, KiNET=325 Æ 108 nM).
Labelled with carbon-11, MADAM has potential as a PET radiotracer
for visualization of the serotonin transporter in the human brain.
Here, we report the 11C-labelling of MADAM (Figure 1, 6) in two
different positions: in the methyl group of the phenyl ring, p-[11C]
methyl and of the tertiary amino moiety, N-[11C]methyl. Authentic
MADAM compound and the required precursors were synthesized.
[11C]Methyl iodide was used in both case precursors subsequently to
react in a Stille coupling reaction either with the Bu3Sn-ADAM
precursor using a palladium (0) catalyst with or without copper chloride
as co-catalyst or to react in an N-methylation with the N-demethyl-
MADAM precursor.
Results and discussion
Chemistry
The N-demethyl-MADAM precursor (3) and the authentic MADAM
(6) were prepared as outlined in Figure 2 by direct coupling of 4-bromo-
3-nitrotoluene with N-methyl-2-thiobenzamide and N,N-dimethyl-2-
thiobenzamide, respectively.14 The amide function was reduced using
B2H6 in THF, and the nitro group was reduced with SnCl2,
hydrochloric acid and methanol, giving N-demethyl-MADAM (3) and
MADAM (6). The synthesis of the stannylate precursor of MADAM
(7) (N,N-dimethyl-2-(2-amino-4-tri-n-butyltinphenylthio)benzylamine)
was carried out as previously described.13
Radiochemistry
The preparation of [p-11C-methyl]MADAM (8) was done by a Stille
coupling reaction between the tin precursor (7) and [11C]methyl iodide
Copyright # 2001 John Wiley & Sons, Ltd.
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J. TARKIAINEN ET AL.
R
R
N
O
N
O
NO2
NO2
Br
HS
S
DMF
K2CO3
+
H3C
H3C
R = H, 1 (76%)
R = CH3, 4 (94%)
R
N
R
N
NO2
NH2
B2H6
THF
S
SnCl2
S
HCl/MeOH
H3C
H3C
R = H, 2 (54%)
R = CH3, 5 (85%)
R = H, 3 (51%)
R = CH3, 6 (80%)
Figure 2. Synthesis of the N-desmethyl-MADAM (3) and MADAM (6)
in DMF. Our attempts using either palladium (0) catalyst with
(o-Tol)3P (Ia, 17) or copper(I) salts as co-catalyst along with palladium
(0) catalyst, potassium carbonate and (o-Tol)3P (Ib, 18) gave relatively
low yields (Figure 3). The incorporation yield of C-11 was in both cases
in the range of 10–30%. The preparation of [N-11C-methyl]MADAM
(9) was based on an N-methylation reaction of its N-demethyl precursor
(3) using [11C]methyl iodide in DMF (Figure 3). The radiochemical yield
of C-11 at EOS was in the range of 75–80% using 0.7 mg of precursor 3.
[11C]MADAM was obtained after HPLC purification (Figure 4) with
R2
N
R2
N
I
a: Pd(dba)3, (o-Tol)3P,
120 C, 7 min
NH2
NH2
S
S
Ib: Ia + CuCl, K2CO3
[
11C]CH3I, DMF
R1
R1
II: 90 C, 2 min
R1 = Bu3Sn, R2 = CH3, 7 (Ia and Ib)
R1 = CH3, R2 = H, 3 (II)
R1 = 11CH3, R2 = CH3, 8
R1 = CH3, R = 11CH3, 9
Figure 3. Syntheses (Ia, Ib and II) of [p-11C-methyl]MADAM (8) and [N-
11C-methyl]MADAM (9) from their precursors 3 and 7, respectively
Copyright # 2001 John Wiley & Sons, Ltd.
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CARBON-11 LABELLING OF MADAM
1017
high radiochemical purity (>99%) and specific radioactivity between 12
and 18 GBq/mmol at EOS.
Experimental
Chemistry
NMR spectra were recorded on a Bruker DPX Avance 200 spectro-
meter (200 MHz for 1H, 50.3 MHz for 13C). CDCl3 was used as solvent;
chemical shifts are expressed in ppm relative to TMS as an internal
standard. Mass spectra were obtained on a CG-MS Hewlett Packard
8
A
9
B
9
C
2
4
6
8
10
Time (min)
Figure 4. Radiochemical purity control by analytical HPLC. (A) Crude reaction
mixture before HPLC-purification from reaction (Ia). (B) Crude reaction
mixture before HPLC-purification from reaction (II). (C) After purification from
reaction (II)
Copyright # 2001 John Wiley & Sons, Ltd.
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J. TARKIAINEN ET AL.
5989A spectrometer (electronic impact at 70 eV). The thin-layer
chromatographic (TLC) analyses were performed using Merck 60F-
254 silica gel plates. Flash chromatography was used for routine
purification of reaction products using silica gel (230–400 Mesh).
Visualisation was accomplished under UV or in an iodine chamber.
All chemicals and solvents were of commercial quality and were purified
following standard procedures.
N-methyl-2-(4-methyl-2-nitrophenylthio)benzamide (1). A mixture of
4-bromo-3-nitrotoluene (5g, 23.1 mmol), N-methyl-2-thiobenzamide
(3.86 mg, 23.1 mmol) and K2CO3 (3.2 g, 23.1 mmol) in DMF (60 ml)
was stirred at 808C for 6 h. The solution was then poured into cold
water (120 ml) and extracted with CHCl3 (2 Â 60 ml). The combined
organic fractions were dried, filtered and evaporated to give a brown oil.
After flash-chromatography (EtOAc), 5.287 g of pure compound 1 was
obtained (76%).
1H NMR: d=2.38 (s, 3 H), 2.84 (d, 3 H), 6.54 (m, 1 H), 6.84 (d, 1 H,
3
4
3J=8.3 Hz), 7.22 (dd, 1 H, J=8.4 Hz, J=1.4 Hz), 7.47–7.60 (m, 3 H);
7.78 (dd, 1 H, 3J=6.7 Hz, 4J=2.1 Hz), 8.00 (d, 1 H, 4J=1.4 Hz).
13C NMR: d=20.4, 26.5, 125.6, 129.0, 129.6, 129.8, 130.1, 131.1, 133.4,
134.8, 136.5, 136.6, 140.9, 146.0, 168.0. MS (EI): (m=z)=302 (41), 245
(64), 209 (76), 208 (89), 197 (97), 180 (100), 166 (80), 165 (65), 58 (91).
N-methyl-2-(4-methyl-2-nitrophenylthio)benzylamine (2). Compound 1
(5g, 15.8 mmol). was dissolved in anhydrous THF (41 ml) and a
solution of 1 M borane complex in distilled THF (41 ml) was added at
08C under a nitrogen atmosphere. The reaction mixture was refluxed for
5h and then stirred at room temperature for 17 h. The reaction was
cooled and treated with concentrated hydrochloric acid (72 ml), warmed
at 708C for 3 h and concentrated in vacuum. The residue was dissolved
in CHCl3 and the precipitate filtered. The solid was diluted in water,
basified with NaOH and extracted with CHCl3. The organic phases
were dried and the solvent evaporated to give the crude product. After
flash-chromatography (EtOAc/MeOH/Et3N: 8/1/1) compound 2 was
obtained in 54% yield.
1H NMR: d=1.63 (s, 1 H), 2.40 (s, 3 H), 2.43 (s, 3 H), 3.85(s, 2 H),
3
6.63 (d, 1 H, J=8.3 Hz), 7.18 (dd, 1 H, J=8.3 Hz, J=1.9 Hz), 7.39
3
4
3
3
(td, 1 H, J=7.4 Hz, J=1.4 Hz), 7.49–7.65(m, 2 H), 8.11 (d, 1 H,
4J=1.9 Hz). 13C NMR: d=20.2, 35.7, 53.3, 125.7, 127.5, 128.3, 129.6,
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129.8, 130.3, 134.5, 135.0, 135.3, 137.1, 144.3, 144.6. MS (EI):
(m=z)=253 (20), 211 (32), 150 (22), 118 (31), 77 (29), 44 (100), 42 (81).
N-methyl-2-(2-amino-4-methylphenylthio)benzylamine (3). Compound
2 (350 mg, 1.21 mmol). was dissolved in 9.7 ml of methanol and 4.8 ml of
concentrated HCl. The mixture was cooled at 108C and SnCl2 (965mg,
5.1 mmol) was added dropwise and stirred at room temperature
overnight. The solution was basified with concentrated NaOH, ethyl-
acetate added and the organic layer was extracted (2 times). Combined
organic layers were dried (Na2SO4) and the solvent was evaporated. The
crude material was purified by flash-chromatography (EtOAc/MeOH/
Et3N: 8/1/1) to afford the pure compound 3 in 60% yield.
1H NMR: d=1.66 (s, 1 H), 2.35(s, 3 H), 2.55(s, 3 H), 3.96 (s, 2 H),
4.26 (s, 2 H), 6.60–6.68 (m, 2 H), 6.83–6.88 (m, 1 H), 7.07–7.18 (m, 2 H),
7.29–7.39 (m, 2 H).
13C NMR: d=21.4, 36.0, 53.9, 110.7, 115.9, 119.8, 125.1, 126.2, 127.7,
129.2, 136.2, 137.0, 137.3, 141.4, 148.7. MS (EI): (m=z)=258 (36),
212 (23), 151 (55), 150 (38), 120 (100), 44 (48), 42 (37).
N,N-dimethyl-2-(4-methyl-2-nitrophenylthio)benzamide (4). A solution
of 4-bromo-3-nitrotoluene (3.04 g, 14 mmol), K2CO3 (1.94 g, 14 mmol),
N,N-dimethyl-2-thiobenzamide (4.00 g, 22 mmol) in DMF (35ml) was
heated at 1208C overnight. After cooling at room temperature, the
reaction mixture was poured into cold water and extracted with EtOAc.
The solvent was then removed and the residue purified by flash
chromatography (EtOAc). Compound 4 was obtained as a yellow solid
in 94% yield.
1H NMR: d=2.37 (s, 3 H), 2.88 (s, 3 H), 3.07 (s, 3 H), 6.89 (d, 1 H,
3
4
3J=8.4 Hz), 7.22 (dd, 1 H, J=8.4 Hz, J=2.0 Hz), 7.42–7.62 (m, 4 H),
8.20 (d, 1 H, J=2.0 Hz). 13C NMR: d=20.9, 34.9, 38.9, 125.9, 127.9,
128.6, 130.2, 130.6, 130.9, 134.3, 135.0, 136.5, 137.3, 143.5, 145.8, 169.5.
4
MS: m=z=316 (15), 270 (61); 226 (100), 197 (66), 72 (72), 44 (43).
N,N-dimethyl-2-(4-methyl-2-nitrophenylthio)benzylamine (5). To
a
solution of compound 4 (3.7 g, 11.7 mmol) in THF (29 ml) under a
nitrogen atmosphere was added dropwise diborane-THF (1 M, 29 ml,
29 mmol) at 08C. The mixture was heated to reflux for 5h, stirred at
room temperature overnight and quenched with concentrated HCl. The
residue was then dissolved in water, basified with NaOH and extracted
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J. TARKIAINEN ET AL.
with CHCl3. After evaporation of the solvent, the crude product was
purified by flash chromatography (EtOAc/Et3N: 9/1) in 85% yield.
1H NMR: d=2.23 (s, 6 H), 2.40 (s, 3 H), 3.56 (s, 2 H), 6.63 (d, 1 H,
3J=8.3 Hz,), 7.16 (dd, 1 H, 3J=8.3 Hz, 4J=2.0 Hz), 7.38 (td, 1 H,
3
3J=7.4 Hz, 4J=1.5Hz), 7.50 (td, 1 H, J=7.4 Hz, 4J=1.5Hz), 7.56 (dd,
3
1 H, J=7.4 Hz, J=1.4 Hz), 7.69 (dd, 1 H, J=7.4 Hz, J=1.4 Hz),
4
3
4
4
8.08 (d, 1 H, J=2.0 Hz). 13C NMR: d=20.3, 45.2 (2C), 61.0, 125.7,
128.1, 128.2, 130.1, 130.3, 130.6, 134.3, 135.2, 135.3, 136.8, 143.5, 145.0.
MS: m=z=302 (12), 285 (42), 211 (75), 194 (21), 58 (100), 42 (25).
N,N-dimethyl-2-(2-amino-4-methylphenylthio)benzylamine (6). To a
solution of compound 5 (2.00 g, 6.61 mmol), concentrated HCl (24 ml)
and MeOH (49 ml) was added SnCl2 (4.9 g, 25.84 mmol) at below 108C.
The reaction mixture was stirred at room temperature overnight, treated
with water (120 ml), basified with NaOH and extracted with EtOAc.
After evaporation of the solvent, the residue was purified by flash
chromatography (EtOAc/petroleum ether/Et3N: 5/4.5/0.5) in 80%
1
yield. H NMR: d=2.34 (s, 6 H), 2.36 (s, 3 H), 3.62 (s, 2 H), 4.44
3
(s large, 2 H), 6.60–6.65(m, 2 H), 6.91 (dd, 1 H, J=7.3 Hz, 4J=1.6 Hz),
3
7.07–7.16 (m, 2 H), 7.27–7.31 (m, 1 H), 7.41 (d, 1 H, J=8.3 Hz). 13C
NMR: d=22.0, 45.8 (2C), 62.8, 112.2, 116.4, 120.0, 125.5, 127.6, 128.4,
130.6, 136.9, 138.0, 138.2, 141.7, 149.6. MS: m=z =272 (M+, 24), 165
(100), 164 (57), 150 (48), 134 (44), 132 (32), 58 (57), 44 (40).
Radiochemistry
All chemicals were obtained from commercial sources and were of
analytical grade. 11CO2 was produced batchwise using the Scanditronic
MC 16 cyclotron at the Karolinska Hospital/Institute by bombardment
of a nitrogen gas target with 16 MeV protons in the 14N(p,a)11C
reaction. Carbon-11 labelled methyl iodide was synthesised from 11CO2
utilising a one-pot reaction set-up similar to that reported previously.9
Semi-preparative reversed-phase HPLC was performed using a
Waters m-Bondapak C-18 column (300 Â 7.8 mm, 10 mm) and an UV-
detector (wavelength=254 nm) in series with a GM-tube for radiation
detection. [p-methyl-11C]MADAM (8, Ia) was purified by HPLC using
50
50
acetonitrile and 0.1 M ammonium formate ( ) as the mobile phase with
a flow rate of 6 ml/min. [p-methyl-11C]MADAM (8, Ib) was not purified
by semi-preparative system because of discouraging radiochemical
yields according to analytical HPLC. [N-methyl-11C]MADAM (9) was
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CARBON-11 LABELLING OF MADAM
1021
35
65
purified by HPLC using acetonitrile and 0.01 M ammonium formate ( )
as the mobile phase with a flow rate of 8 ml/min. The radiochemical
purity of the products was determined by reversed phase HPLC with
a Waters m-Bondapak C-18 column (300 Â 3.9 mm, 10 mm) and a UV-
detector (wavelength=234 nm) in series with a Beckman b-flow
radiodetector for radiation detection. Acetonitrile and 0.01 M
H3PO4 (3700) were used as the mobile phase with a flow rate of 2 ml/
min. The chemical identity of [p-methyl-11C]- and [N-methyl-11C]
MADAM (8 and 9) was determined by co-injection of unlabelled
MADAM (6).
Preparation (Ia) of [p-methyl-11C]MADAM (8). Catalysts, tris(diben-
zylidene-acetone)dipalladium(0)[Pd2(dba)3, 0.9 mg,. 1 mmol] and
tri(o-tolyl)phosphine [(o-Tol)3P, 1.2 mg, 4 mmol] in 350 ml of N,N-
dimethylformamide (DMF), were stirred in a N2 purged dry septum
equipped 1.0 ml vial for 10 min. [11C]Methyl iodide was trapped in the
solution at room temperature. After trapping the reaction mixture was
transferred to a septum equipped 1.0 ml vessel containing 2 mg
(3.7 mmol) of precursor 7. The reaction vessel was heated at 1208C for
7 min. The mobile phase (600 ml) was added prior to injection into the
semi-preparative HPLC column. The product fraction was collected
after the retention time of 11 min and the mobile phase was evaporated.
To the residue was added 8 ml of sterile physiological phosphate
buffer (pH=7.4) solution which was then filtered through a Millipore
filter (0.22 mm) yielding a solution which was sterile and free from
pyrogens.
Preparation (Ib) of [p-methyl-11C]MADAM (8). Catalysts, Pd2(dba)3
(1.7 mg, 1.9 mmol). and (o-Tol)3P (2.1 mg, 6.9 mmol) in 200 ml of DMF,
were stirred in a N2 purged dry septum equipped 1.0 ml vial for 10 min.
The mixture of tin precursor 7 (2.0 mg, 3.7 mmol), CuCl (1.8 mg,
18.2 mmol) and K2CO3 (2.1 mg, 15.2 mmol) was prepared in a dry septum
equipped 1.0 ml vial without DMF and flushed carefully with N2 gas for
10 min. [11C]Methyl iodide was trapped at room temperature in the
solution containing Pd-catalyst etc. DMF (100 ml) was added just before
transferring the trapping solution into the mixture of tin precursor 7 etc.
After the addition of the trapping solution the vial was shaken
vigorously and heated at 1208C for 7 min. A sample was taken and
analysed by HPLC, giving discouraging result.
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J. TARKIAINEN ET AL.
Preparation (II) of [N-methyl-11C]MADAM (9). [11C]Methyl iodide
was trapped at room temperature in a reaction vessel (1.0 ml) containing
the precursor [0.7 mg of the N-demethyl-MADAM (3)] and DMF (300
ml). The vessel was sealed and heated at 908C for 2 min. The mobile
phase (600 ml) was added prior to injection into the semi-preparative
HPLC column. The radioactive fraction containing the methylated
radioligand was collected after the retention time of 15min. After
evaporation of the mobile phase the residue was dissolved in 8 ml of
sterile physiological phosphate buffer (pH=7.4) solution and filtered
through a Millipore filter (0.22 mm), yielding a solution which was sterile
and free from pyrogens.
Acknowledgements
.
The authors would like to thank Mr Goran Printz for assistance with
the radionuclide production, Mr Arsalan Amir and Miss Vilar Marie-
Paule for technical assistance. Grants from the Swedish Medical
Research Council (12983-01A), Karolinska Institutet, INSERM-
!
MFR, the Region Centre (France) and European program (COST
B12) supported this work. We thank SAVIT (Prof. P. Dubois) for
chemical analysis.
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