Synthesis and biological evaluation of S-[11C]methylated mercaptoimidazole piperazinyl derivatives as potential radioligands for imaging 5-HT1A receptors by positron emission tomography (PET)

Article abstract of DOI:10.1002/jlcr.924

The novel 2-mercaptoimidazole derivatives, 1-[4-((2-methoxyphenyl)-1- piperazinyl)-butyl]-2-mercaptoimidazole (3) and methyl[4-((2-methoxyphenyl)-1- piperazinyl))butyl] (2-mercapto-1-methylimidazol-5-yl)methanamide (8), were efficiently labelled with

Full text of DOI:10.1002/jlcr.924

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 301–315.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.924
Research Article
Synthesis and biological evaluation of
S-[11C]methylated mercaptoimidazole piperazinyl
derivatives as potential radioligands for imaging
5-HT1A receptors by positron emission tomography
(PET)
1
Raquel Garcia , Catarina Xavier , Antonio Paulo *, Isabel Santos , Torsten
1
1,
1
!
2
Kniess , R. Bergmann and Frank Wust
2
2
.
1
! !
Departamento de Quımica, ITN, Estrada Nacional 10, 2686-953 Sacavem Codex,
Portugal
2 Institut of Bioinorganic and Radiopharmaceutical Chemistry, Research Center
Rossendorf, PF 51 01 19, D-01314 Dresden, Germany
Summary
The novel 2-mercaptoimidazole derivatives, 1-[4-((2-methoxyphenyl)-1-piperazinyl)-
butyl]-2-mercaptoimidazole (3) and methyl[4-((2-methoxyphenyl)-1-piperazinyl))bu-
tyl] (2-mercapto-1-methylimidazol-5-yl)methanamide (8), were efficiently labelled with
11C through methylation of the thioketone function with [11C]methyl iodide. The
resulting radioligands 1-[4-((2-methoxyphenyl)-1-piperazinyl))butyl]-2-thio[11C]-
methylimidazole ([11C]9) and methyl[4-((2-methoxyphenyl)-1-piperazinyl))butyl] (2-
thio[11C]methyl-1-methylimidazol-5-yl)-methanamide ([11C]10) were synthesized in
radiochemical yields of 20–30% (decay-corrected, related to [11C]CO2) at a specific
radioactivity of 0.2–0.4 Ci/mmol within 40–45 min including HPLC-purification. The
radiochemical purity exceeded 99%. The reference compounds 9 and 10 were tested in
a competitive receptor binding assay to determine their affinity toward the 5-HT1A
receptor.
Both
compounds
exhibit
excellent
sub-nanomolar
affinities
(IC50=0.576 Æ 0.008 nM (9); IC50=0.86 Æ 0.02 nM (10)) for the 5-HT1A receptor
while displaying a high selectivity towards the 5-HT2A subtype of receptors
(IC50>480 nM). By contrast, compound 9 also shows substantial binding for the
alpha1-adrenergic receptor (IC50=3.00 Æ 0.02 nM) when compared with compound
10 (IC50=54.5 Æ 0.6 nM). Preliminary biodistribution studies in rats showed an initial
!
*Correspondence to: Dr A. Paulo, Departamento de Quımica, ITN, Estrada Nacional 10, 2686-953
!
Sacavem Codex, Portugal. E-mail: apaulo@itn.mces.pt
Contract/grant sponsor: FCT; contract/grant number: POCTI/QUI/42939/2001
Contract/grant sponsor: National Foundation for Science and Technology
Copyright # 2005 John Wiley & Sons, Ltd.
Received 21 July 2004
Revised 10 December 2004
Accepted 12 December 2004

302
R. GARCIA ET AL.
brain uptake of 1.14 Æ 0.11 and 0.37 Æ 0.04% ID/g after 5 min, which decreased to
0.18 Æ 0.04 and 0.16 Æ 0.01% ID/g after 60 min for compounds [11C]9 and [11C]10,
respectively. For both compounds, the cerebellum and rest of the brain uptake are
very similar at the different time points. Unlike [11C]9, the radioligand [11C]10 has
significant uptake and retention in the adrenal glands. Due to their washout from the
brain compounds [11C]9 and [11C]10 seem not to be good candidates as radioligands
for imaging 5-HT1A receptors by PET. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: carbon-11; mercaptoimidazoles; piperazines; 5-HT1A receptors; positron
emission tomography
Introduction
Central 5-hydroxytryptamine (5-HT1A) receptors are implicated in the
pathophysiology of major neuropsychiatric disorders such as schizophrenia,
anxiety and depression. The functional imaging of 5-HT1A receptors by
positron emission tomography (PET) represents an innovative approach for
the non-invasive assessment of their role in the development of those
neuropsychiatric disorders. Moreover, PET imaging is also a useful tool for
evaluating the action of new 5-HT1A receptor targeting drugs, allowing the
design of more efficient therapeutic strategies.1,2
The identification of WAY-100635 as a potent and selective antagonist for
the 5-HT1A receptor was accompanied by numerous attempts to label the
compound with the short-lived positron emitter 11C (t1/2=20.4 min) to study
5-HT1A receptors in the human brain (Scheme 1).
Originally, the radiolabelling of WAY-100635 with 11C was accomplished
via O-methylation with [11C]methyl iodide of the corresponding desmethyl
precursor.3 However, the resulting radioligand [O-methyl-11C]WAY-100635
undergoes hepatic metabolism, which involves cleavage of the amide moiety
and results in the formation of a lipophilic radioactive metabolite. This
metabolite is accumulated in the brain, and it displays high affinity for both
5-HT1A and alpha1-adrenergic receptors. These facts complicate an exact
quantification of 5-HT1A brain receptors based on [O-methyl-11C]WAY-
100635.4,5 In order to overcome this drawback, [carbonyl-11C]WAY-100635
O
11C
O
O11CH3
N
OCH3
N
N
N
N
N
N
N
[O-methyl-11C]WAY-100635
[carbonyl-11C]WAY-100635
Scheme 1. 11C-labelled 5-HT1A receptor ligands
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SYNTHESIS AND BIOLOGICAL EVALUATION OF
303
was synthesized and evaluated.6 Here the compound is labelled in the carbonyl
position with 11C. Although suffering from hepatic metabolism, [carbo-
nyl-11C]WAY-100635 is transformed into polar radioactive cyclohexanecar-
boxylic acid fragments which are unable to cross the blood brain barrier.4,5
Thus, [carbonyl-11C]WAY-100635 can be considered to be an effective
radioligand for the mapping of 5-HT1A receptors in the human brain.7–10
However, [carbonyl-11C]WAY-100635 was prepared via a laborious 11C
acylation with [carbonyl-11C]cyclohexanecarbonyl chloride. Consequently, the
search for alternative radioligands which can be prepared more easily while
showing improved metabolic resistance is still a relevant issue.4,5,11–15
Several studies highlighted the tolerance of the WAY-100635 lead
structure to replacement of the pyridinyl amide group by other substituents,
including bulky metallic fragments, such as [TcO]3+, [TcN]2+ and fac-
[Tc(CO)3]+, anchored by adequate bifunctional chelators.13,16–18 Recently,
our group introduced the novel organometallic building blocks
fac-[M{k3-R(m-H)B(tim Me)2}(CO)3] (R=H, alkyl, aryl; timMe=1-methyl-2-
mercaptoimidazolyl; M=Re, 99mTc), which are anchored by tripodal ligands
of the bis(mercaptoimidazolyl)borate type.19,20 These building blocks were
functionalized with piperazinyl fragments, which were incorporated at the 5-
position of the azole ring.21 Some of the resulting functionalized complexes
displayed excellent sub-nanomolar affinities for 5-HT1A receptors, and their
biodistribution is currently under evaluation.22 Based on these findings, we
expected that mercaptoimidazoles bearing piperazinyl moieties would also
retain, by themselves, good affinity and selectivity for 5-HT1A receptors.
Benefiting from the easy alkylation of thione functions with [11C]methyl
iodide, we decided to evaluate the usefulness of 11C-labelled mercaptoimida-
zoles as radioligands for mapping brain 5-HT1A receptors by means of PET.
Herein, we report on the radiosynthesis and biological evaluation of two
S-11C-methylated mercaptoimidazole piperazinyl derivatives ([11C]9 and
[11C]10). Attachment of the 1-(2-methoxyphenyl)piperazinyl part of WAY-
100635 to the azole moiety was accomplished via a butyl spacer (Scheme 2).
11CH3
S
N
N CH3
11CH3
N
S
OMe
N
OMe
N
O
N
N
N
N
CH3
[
11C]9
[
11C]10
Scheme 2. Novel S-11C-methylated mercaptoimidazole piperazinyl derivatives
[11C]9 and [11C]10
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R. GARCIA ET AL.
The biological evaluation included the determination of 5-HT1A receptor
binding affinities of compounds 9 and 10, as well as preliminary biodistribu-
tion studies of [11C]9 and [11C]10 in male Wistar rats.
Results and discussion
Chemistry
Coupling of the 1-(2-methoxyphenyl)piperazinyl moiety to the 2-mercaptoi-
midazole ring was carried out at positions 1 or 5 of the azole, and involved the
synthesis of the mercaptoimidazole precursors 1-[4-((2-methoxyphenyl)-1-
piperazinyl))butyl]-2-mercaptoimidazole (3) and methyl[4-((2-methoxyphe-
nyl)-1-piperazinyl)butyl] (2-mercapto-1-methylimidazol-5-yl)methanamide (8)
as depicted in Schemes 3 and 4, respectively. In both cases, we started from
4-[(2-methoxyphenyl)-1-piperazinyl]butylamine (1), which was synthesized
according to literature methods.23
The synthesis of the labelling precursor 3 was accomplished in 50% yield by
cyclization of a diethyl acetal derivative (2) with potassium thiocyanate under
acidic conditions, which is a common method for the synthesis of
2-mercaptoimidazoles (Scheme 3).24,25
Amino acetal 2 has been obtained by alkylation of compound 1 with
bromoacetaldehyde diethyl acetal. The reaction was performed using an excess
of primary amine 1 to avoid overalkylation.26
As depicted in Scheme 4, the labelling precursor 8 was obtained by a multi-
step synthesis, starting from [(2-mercapto-1-methylimidazol-5-yl)methyloxy]-
succimide (7), which has been synthesized by methods reported in the
OMe
N
OMe
N
H
N
BrCH2CH(OC2H5)2
NH2
N
N
i
(C2H5O)2CH
1
2
ii
CH3
S
S
OMe
OMe
N
iii
N
N
N
N H
N
N
N
9
3
Scheme 3. Synthesis of the labelling precursor 3 and reference compound 9: (i)
K2CO3, acetonitrile, reflux, 48 h; (ii) KSCN, HCl 2 N, ethanol, reflux, overnight;
(iii) NaOH, CH3I, ethanol, rt, 3 h
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SYNTHESIS AND BIOLOGICAL EVALUATION OF
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OMe
N
OMe
H
i
NH2
N
CF3
N
N
N
O
1
4
ii
OMe
N
OMe
CH3
N
iii
NHCH3
CF3
N
N
N
O
5
6
S
H3C
O
N
N H
iv
O
O
N
O
7
CH3
S
S
H3C
O
N
N
H
H3C
O
N
N
MeO
MeO
N
v
N
N
N
N
N
H3C
H3C
8
10
Scheme 4. Synthesis of labelling precursor 8 and reference compound 10: (i)
CF3COOC2H5, dichloromethane, À88C to rt, 4 h; (ii) NaH, (CH3)2SO4, DMF,
rt, overnight; (iii) K2CO3, methanol/water (30:1 v/v), reflux, 2 h; (iv) DMF, rt,
24 h; (v) NaOH, CH3I, ethanol, rt, overnight
literature.27 Direct coupling of activated ester 7 with compound 6 provided
labelling precursor 8 in 33% yield. The moderate to low yield can be explained
by the low solubility of the activated ester 7 and to the low reactivity of the
secondary amine 6. Secondary amine 6 was prepared by protection of the
primary amine group as a trifluoroacetylamide followed by mono methylation
with dimethylsulfate and subsequent removal of the amine protecting group
under moderate basic conditions.28 The overall chemical yield for the three
steps was 68%.
Reference compounds 9 and 10 were synthesized in a straightforward way
by the reaction of compounds 3 and 8 with methyl iodide (Schemes 3 and 4).
Compounds 9 and 10 were used to identify the radioligands [11C]9 and [11C]10,
and they were used in the competitive binding assays to determine their affinity
toward the 5-HT1A receptor.
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R. GARCIA ET AL.
11CH3
N
S
OMe
N
S
11C]CH3I
EtOH, 80 C, 6 min
OMe
N
[
N
N
N
N H
N
[
11C]9
3
11CH3
S
S
H3C
O
N
N
H3C
N
N H
MeO
N
MeO
N
[
11C]CH3I
O
N
N
N
EtOH, 80 ºC, 6 min
N
H3C
H3C
8
[
11C]10
Scheme 5. Radiosynthesis of [11C]9 and [11C]10
Radiochemistry
Efficient S-[11C]-methylation reactions of thioketones have already been
reported.29 However, to the best of our knowledge, the labelling of
2-mercaptoimidazoles with [11C]methyl iodide has not been applied yet.
S-Methylation of labelling precursors 3 and 8 with [11C]methyl iodide was
easily accomplished in ethanol at 808C for 6 min using NaOH as a base, giving
the desired radioligands 1-[4-((2-methoxyphenyl)-1-piperazinyl))butyl]-2-
thio[11C]methylimidazole ([11C]9) and methyl[4-((2-methoxyphenyl)-1-pipera-
zinyl)butyl] (2-thio[11C]methyl-1-methyl-imidazol-5-yl)methanamide ([11C]10)
(Scheme 5).
The radioligands [11C]9 and [11C]10 were purified by semi-preparative
HPLC using a Kromasil-100 C18 column and isocratic elution with CH3CN/
0.2% aqueous NEt3 (70:30, v/v) at a flow rate of 3 ml/min. Under these
conditions, radioligands [11C]9 and [11C]10 were easily separated from the
excess of 2-mercaptoimidazole precursors 3 and 8 and very low amounts
(55%) of non-reacted [11C]methyl iodide. Both radiotracers were obtained
chemically and radiochemically pure (>99%), as checked by analytical
HPLC. The identity of the radioligands [11C]9 and [11C]10 was confirmed by
comparison of their HPLC profiles with those of reference compounds 9
and 10.
Typically, the radioligands [11C]9 and [11C]10 were obtained with
radiochemical yields of 20–30% (decay-corrected, related to [11C]CO2) at a
specific radioactivity of 0.2–0.4 Ci/mmol, within 40–45 min including HPLC
separation.
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SYNTHESIS AND BIOLOGICAL EVALUATION OF
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Table 1. Inhibition constants (IC50) and log P values of 9, 10 and WAY-100635
IC50 (nM)
5-HT1A
5-HT2A
alpha-1
[3H]8-OH-DPAT
rat hippocampus
homogenate
[3H]Ketanserin
rat cortex
homogenate
[3H]prazosin
rat cortex
homogenate
Compound
Log Pa
9
10
0.576 Æ 0.008
0.86 Æ 0.02
6.0b
1050 Æ 16
480 Æ 10
3.00 Æ 0.02
54.5 Æ 0.6
4.2
2.4
WAY 100635
4.7b
a Calculated log P values have been calculated based on ACDLabs predictions.
b Literature value.11
In vitro binding assays
Reference compounds 9 and 10 were subjected to in vitro competitive receptor
binding assay using rat brain homogenates as the source for the receptor
proteins to determine their IC50 values for the 5-HT1A, 5-HT2A and alpha1-
adrenergic receptors. The IC50 values obtained for both compounds and
WAY-100635 along with log P values are depicted in Table 1.
Compounds 9 and 10 exhibit excellent sub-nanomolar affinities for the
5-HT1A receptors, comparable to that found for WAY-100635.11 Moreover,
both compounds display good selectivities against 5-HT2A receptors, with
5-HT2A/5-HT1A ratios of 1553 and 558 for 9 and 10, respectively. By contrast,
much lower selectivities were found to the alpha1-adrenergic receptor. This
finding is consistent with reports in the literature on compounds bearing a
2-methoxyarylpiperazine moiety showing substantial affinity toward alpha1-
adrenergic receptor.23 However, compound 9 displays an 18-fold higher
affinity to the alpha1-adrenergic receptor when compared with compound 10,
being 3 and 54 nM, respectively. This difference in the binding affinity and
consequently in the selectivity is likely to be influenced by the position at which
the arylpiperazine pharmacophore is coupled to the 2-mercaptoimidazole ring
and/or the presence of the tertiary amide function in the molecule.
Biodistribution studies
Preliminary biodistribution studies of [11C]9 and [11C]10 were performed in
male Wistar rats to evaluate brain uptake and brain activity retention. Table 2
summarizes the biodistribution data obtained for [11C]9 and [11C]10, expressed
in percent injected dose per gram tissue (% ID/g).
Both compounds were able to cross the BBB, showing an initial brain
uptake of 1.14 Æ 0.11% ID/g ([11C]9) and 0.37 Æ 0.11% ID/g ([11C]10) after
5 min. The initial higher brain uptake of compound [11C]9 seems to correlate
with a higher lipophilicity of compound [11C]9 when compared to compound
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Table 2. Biodistribution of [11C]9 and [11C]10 in Wistar rats (mean of % ID/g
tissue Æ SD, n=4)
% ID/g
[11C]9
[11C]10
Organ
5 min
60 min
5 min
60 min
Blood
0.11 Æ 0.02
0.82 Æ 0.13
1.14 Æ 0.11
4.16 Æ 0.51
1.48 Æ 0.26
3.77 Æ 1.03
2.85 Æ 0.43
0.49 Æ 0.08
1.68 Æ 0.19
0.80 Æ 0.18
0.10 Æ 0.03
0.14 Æ 0.03
0.18 Æ 0.04
5.85 Æ 0.84
0.92 Æ 0.19
1.11 Æ 0.16
0.81 Æ 0.16
0.19 Æ 0.03
0.48 Æ 0.06
1.37 Æ 0.35
0.12 Æ 0.03
0.29 Æ 0.04
0.37 Æ 0.04
3.34 Æ 0.57
1.27 Æ 0.17
7.95 Æ 3.09
2.56 Æ 0.32
0.41 Æ 0.08
1.14 Æ 0.17
1.33 Æ 0.28
0.04 Æ 0.00
0.14 Æ 0.01
0.16 Æ 0.01
1.86 Æ 0.13
0.51 Æ 0.07
7.32 Æ 0.75
1.09 Æ 0.15
0.10 Æ 0.01
0.33 Æ 0.07
2.75 Æ 0.72
Cerebellum
Rest of brain
Pancreas
Spleen
Adrenals
Kidneys
Heart
Lung
Liver
[11C]10 (Table 1). However, a washout from the brain was observed for both
compounds, reaching 0.18 Æ 0.04 and 0.16 Æ 0.01% ID/g after 60 min. The
blood clearance is fast, and after 5 min the brain/blood ratio is 10 for
compound [11C]9 and 3 for compound [11C]10. However, blood activity of
compound [11C]9 is comparable at 5 and 60 min, being 0.11% ID/g and 0.10%
ID, respectively. This phenomenon could be explained by a high proportion of
protein binding of compound [11C]9 due to its higher lipophilicity compared to
compound [11C]10. Despite displaying excellent in vitro binding affinities for
5-HT1A receptors compounds [11C]9 and [11C]10 show comparable uptake in
the cerebellum and in the rest of the brain after 60 min post-injection (p.i). This
finding suggests that the found brain uptake is mainly related to high non-
specific binding of both compounds. Moreover, the low specific activity of
compounds [11C]9 and [11C]10 may also lead to a saturation of specific binding
sites.
Remarkably, compound [11C]10 showed a substantial uptake in the adrenals
(7.95 Æ 3.09% ID/g, at 5 min p.i.) with almost no washout (7.32 Æ 0.75% ID/
g, at 60 min p.i.). A possible explanation for this uptake could be the moderate
affinity (IC50=54.5 Æ 0.6 nM) of [11C]10 for alpha1-adrenergic receptors,
which are active in adrenal glands. However, a much lower retention of
activity was observed in other organs bearing adrenergic receptors, like heart,
pancreas or spleen. Moreover, radioligand [11C]9, having a much higher
affinity (IC50=3.00 Æ 0.02 nM) for the adrenergic receptors, showed a lower
adrenal uptake (3.77 Æ 1.03% ID/g, at 5 min p.i.) with a significant washout
(1.11 Æ 0.16% ID/g, at 60 min p.i.). Apparently, these data do not support a
relationship between the adrenal uptake of compound [11C]10 and its affinity
for the alpha1-adrenergic receptor. Adrenal glands being a receptor-rich
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SYNTHESIS AND BIOLOGICAL EVALUATION OF
309
organ, we cannot exclude the involvement of other type of receptors in the
high uptake of [11C]10 by the adrenals.
In summary, we have shown that 2-mercaptoimidazole derivatives bearing
2-methoxypiperazinyl pharmacophores are promptly labelled with [11C]methyl
iodide by S-alkylation of the thioketone function, giving radioligands [11C]9
and [11C]10 in sufficient radiochemical yields and high radiochemical purity. In
spite of their excellent in vitro affinity for 5-HT1A receptor and good
selectivities against the 5-HT2A receptor, compounds [11C]9 and [11C]10 are
not promising radiotracers for PET imaging of CNS receptors, due to their
fast washout from the brain. Surprisingly, [11C]10 showed a remarkable and
prolonged uptake in the adrenals, promoting further studies to clarify this
phenomenon. This should include, namely, the determination of the affinity
toward other receptors occurring in adrenals, studies on metabolism, as well as
some selected blocking experiments to verify specific uptake.
Experimental section
Dichloromethane, ethanol and dimethylformamide were dried and distilled
according to described procedures. The compounds 4-[(2-methoxyphenyl)-1-
piperazinyl]butylamine (1) and [(2-mercapto-1-methylimidazol-5-yl)methylox-
y]succimide (7) were synthesized as described elsewhere.23,27 All other solvents
1
and chemicals were used as purchased. H and 13C NMR spectra were
recorded on a Varian Unity 300 MHz spectrometer; 1H and 13C chemical shifts
are given in ppm and were referenced with the residual solvent resonances
relative to Me4Si. Mass spectra were obtained on a Quattro/LC mass
spectrometer (MICROMASS) by electrospray ionization.
[11C]CO2 was produced by the 14N(p,a)11C reaction on a IBA CYCLONE
18/9 cyclotron. The synthesis of [11C]methyl iodide was done by procedures
described in the literature,30 and involved reduction of [11C]CO2 with LiAlH4,
followed by hydrolysis and iodination with hydroiodic acid. All radiochemical
syntheses including semi-preparative HPLC-separation were run on the fully
automated synthesis module (PET tracer synthesizer, Nuclear Interface
.
GmbH, Munster, Germany). Analytical HPLC was performed using a JASCO
HPLC system (JASCO, HPLC-pump PU-1580) equipped with a UV-vis
detector (JASCO UV-1575) and a gamma detector (Raytest Gabi).
The animal experiments were carried out in accordance with the guidelines
on the use of living animals in scientific investigations and on the German law,
and followed the principles of laboratory animal care.
Chemistry
1-(Acetaldehydediethyacetal)-4-((2-methoxyphenyl)-1-piperazinyl)butylamine
(2). To a solution of compound 1 (1.5 g, 5.7 mmol) in acetonitrile was added
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R. GARCIA ET AL.
bromoacetaldehyde diethyl acetal (0.43 ml, 2.9 mmol) and 1.57 g (11.4 mmol)
of K2CO3, and the resulting suspension was refluxed during 48 h. After cooling
to room temperature and filtration, the solvent was removed under vacuum to
give an oily residue. Column chromatography of this residue on silica gel with
CH2Cl2/methanol (95:5) afforded compound 2 (567 mg, 1.50 mmol, Z=26%)
as an orange oil.
1H NMR (300 MHz, CDCl3) d: 1.17 (6H, t, –OCH2CH3), 1.57 (m, 4H,
CH2), 2.42 (t, 2H, CH2), 2.64–2.72 (m, 6H, CH2+NCH2), 3.07 (d, 2H, CH2),
3.40 (br, 4H, NCH2), 3.48–3.58 (m, 2H, OCH2CH3), 3.65–3.75 (m, 2H,
OCH2CH3), 3.81 (s, 3H, –OCH3), 4.64 (t, 1H, CH), 6.80–6.99 (m, 4H, Ar).
1-[4-((2-Methoxyphenyl)-1-piperazinyl)butyl]-2-mercaptoimidazole (3). To
an ethanolic solution of 2 (755 mg, 2 mmol) was added 3 ml of 2 N
HCl and solid KSCN (400 mg, 4.12 mmol), and the reaction mixture
was refluxed overnight. After cooling to room temperature, a white insoluble
solid was removed by filtration. To the supernatant was added a
saturated NaHCO3 solution, followed by extraction with CHCl3. The
solvent from the organic phase was removed, yielding a yellow-brown
solid. Column chromatography of this solid on silica gel with
methanol afforded compound 3 (360 mg, 1 mmol, Z=50%) as a yellowish
solid.
1H NMR (300 MHz, CD3OD) d: 1.58 (q, 2H, CH2), 1.82 (q, 2H, CH2),
2.47(t, 2H, CH2), 2.64 (br, 4H, NCH2), 3.05 (br, 4H, NCH2), 3.84 (s, 3H,
–OCH3), 4.06 (t, 2H, CH2), 6.85 (d, 1H, CH– Simz), 6.86–7.00 (m, 4 H, Ar),
7.05 (d, 1H, CH– Simz). 13C NMR (75.37 MHz, CD3OD) d: 24.3, 28.2, 47.6,
51.5, 54.3, 55.9, 59.1, 112.7, 115.7, 119.4, 119.8, 122.5, 124.7, 142.2, 153.9,
161.0. LRMS(ESI positive) (m/z): 347 [M+H]+.
4-[((2-Methoxyphenyl)-1-piperazinyl)]butyltrifluoroacetamide (4). To a
solution of compound 1 (612 mg, 2.33 mmol) in CH2Cl2 at À88C was added
dropwise a solution of CF3COOC2H5 (305 ml, 2.56 mmol) in CH2Cl2. After
complete addition, the mixture was stirred at À88C for 2 h and at room
temperature for 1.5 h. Removal of the solvent yielded compound 4 as a yellow
oil (800 mg, Z=96%).
1H NMR (300 MHz, CDCl3) d: 1.68 (m, 4H, CH2), 2.47 (t, 2H, CH2), 2.66
(br, 4H, NCH2), 3.08 (br, 4H, NCH2), 3.37 (t, 2H, CH2), 3.84 (s, 3H, OCH3),
6.80–7.02 (m, 4H, Ar), 8.35 (br, 1H, NH).
1-Methyl-4-[(2-methoxyphenyl)-1-piperazinyl]butylamine (6). To a suspen-
sion of NaH (64 mg, 2.67 mmol) in dry DMF at 08C was added dropwise a
solution of 4 (800 mg, 2.2 mmol) in DMF, and after complete addition the
mixture was stirred at room temperature for 1 h. Then, a solution of
(CH3)2SO4 (253 ml, 2.67 mmol) in DMF was added dropwise at 08C, and the
reaction mixture was stirred overnight at room temperature. Removal of
solvent under vacuum gave crude methyl[4-((2-methoxyphenyl)-1-piperazi-
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nyl))butyl]trifluoroacetamide (5), which has been used in the next step without
further purification.
To a solution of 5 in MeOH (30 ml) and water (1 ml) was added an excess of
K2CO3 (1:10 molar ratio), and the mixture was refluxed for 2 h. After cooling
to room temperature and filtration, the solvent was removed under vacuum.
The crude was dissolved in water and the pH adjusted to about pH=14 with
40% NaOH. The aqueous solution was extracted four times with chloroform
and the combined organic phases were dried over MgSO4. Removal of the
solvent gave 6 as a yellow oil (416 mg, 1.50 mmol, Z=68%).
Compound 5, 1H NMR (300 MHz, CDCl3) d: 1.50–1.72 (m, 4H, CH2), 2.41
(m, 2H, CH2), 2.65 (br, 4H, CH2N), 3.09 (br, 7H, CH2N and CH3N), 3.47 (t,
2H, CH2), 3.84 (s, 3H, OCH3), 6.83–7.01 (m, 4H, Ar).
1
Compound 6, H NMR (300 MHz, CDCl3) d: 1.50–1.57 (m, 4H, CH2),
2.38–2.45 (m, 5H, m, CH3N+CH2), 2.56–2.63 (m, 6H, CH2N+CH2), 3.09
(br, 4H, CH2N), 3.84 (s, 3H, OCH3), 6.82–6.99 (m, 4H, Ar).
Methyl[4-((2-methoxyphenyl)-1-piperazinyl)butyl](2-mercapto-1-methyli-
midazol-5-yl)methanamide (8). To a suspension of compound 7 (976 mg,
3.8 mmol) in DMF at 08C was added dropwise a DMF solution of 6 (1.060 g,
3.8 mmol). The reaction mixture was allowed to warm to room temperature,
and was stirred for 24 h. After this time, the solvent was removed under
vacuum. The crude residue was purified by column chromatography (silica
gel 60, eluent 15% MeOH in CH2Cl2). Removal of the solvent from the
collected fractions, followed by washing with aqueous 10% NaHCO3 and
prolonged drying under vacuum, gave compound 8 (532 mg, Z=33%) as a
yellow solid.
1H NMR (300 MHz, CD3CN) d: 1.46 (m, 2H, CH2), 1.60 (m, 2H, CH2), 2.34
(tr 2H, CH2), 2.51 (br, 4H, NCH2), 2.90–3.10 (br, 4+3H, NCH2+NCH3),
3.45 (tr 2H, CH2), 3.51 (s, 3H, N-CH3), 3.78 (s, 3H, –OCH3), 6.83–6.97 (m,
4H+1H, Ar+CH– Simz). 13C NMR (75.37 MHz, CDCl3) d: 23.3, 24.9, 32.6,
36.3, 47.6, 49.9, 52.8, 54.9, 57.4, 110.6, 115.2, 117.7, 120.5, 122.6, 124.0, 140.5,
151.7, 159.6, 162.4. LRMS (ESI-positive) (m/z): 418 [M+H]+.
1-[4-((2-Methoxyphenyl)-1-piperazinyl)butyl]-2-thiomethylimidazole (9).
Solid NaOH (12 mg, 0.29 mmol) was added to an ethanolic solution of
compound 3 (100 mg, 0.29 mmol), followed by dropwise addition of an
ethanolic solution of CH3I (45 mg, 0.32 mmol). After complete addition, the
reaction mixture was stirred at room temperature for 3 h. Removal of the
volatiles gave a yellow residue, which was dissolved in CH2Cl2. After washing
with H2O, the organic phase was dried over MgSO4, filtered and the solvent
removed under vacuum, affording compound 9 (83 mg, 0.23 mmol, Z=80%)
in the form of a yellow solid.
1H NMR (300 MHz, CD3CN) d: 1.54 (q, 2H, CH2), 1.80 (q, 2H, CH2), 2.43
(t, 2H, CH2), 2.51 (s, 3H, –SCH3), 2.62 (br, 4H, NCH2), 3.04 (br, 4H, NCH2),
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3.83 (s, 3H, –OCH3), 4.05 (t, 2H, CH2), 6.82–7.02 (m+d, 4H+1H, Ar+CH–
Simz), 7.19 (d. 1H, CH– Simz). 13C NMR (75.37 MHz, CD3CN) d: 17.1, 24.4,
29.7, 47.4, 51.5, 54.2, 55.9, 59.0, 112.7, 119.4, 122.2, 122.8, 124.7, 129.5, 142.1,
143.5, 153.8. LRMS (ESI-positive) (m/z): 361 [M+H]+.
Methyl[4-((2-methoxyphenyl)-1-piperazinyl)butyl](2-thiomethyl-1-methyli-
midazol-5-yl)methanamide (10). Compound 10 has been synthesized as
described above for 9, but the reaction was allowed to proceed overnight.
Starting from 100 mg (0.24 mmol) of compound 8, were obtained 28 mg
(Z=27%) of 10 in the form of a colourless oil, after purification by column
chromatography (silica gel 60, eluent 5% MeOH in CH2Cl2).
1H NMR (300 MHz, CDCl3) d: 1.53 (m, 2H, CH2), 1.64 (m, 2H, CH2), 2.42
(t, 2H, CH2), 3.08 (br, 7H, NCH3+CH2N), 3.51 (t, 2H, CH2), 6.64 (s, 3H,
CH3), 3.82 (s, 3H, OCH3), 6.80–6.97 (m, 4H, Ar), 7.20 (s, 1H, CH). 13C NMR
(75.37 MHz, CDCl3) d: 15.1, 23.8, 25.6, 32.4, 36.3, 47.7, 50.4, 53.3, 55.3, 58.0,
111.0, 118.1, 120.9, 122.9, 127.0, 131.6, 141.1, 147.5, 152.1, 161.6. LRMS (ESI-
positive) (m/z): 432 [M+H]+.
Radiosynthesis
1-[4-((2-Methoxyphenyl)-1-piperazinyl)butyl]-2-thio[11C]methylimidazole
([11C]9). Methylation with [11C]methyl iodide was carried out by distillation
of approximately 100 mCi [11C]CH3I into a second reaction vessel containing
1.30 mg (0.0036 mmol) of the mercaptoimidazole precursor 3 in 0.6 ml of
ethanol and 20 ml of 5 N NaOH. The reaction mixture was heated for 6 min at
808C. After cooling to room temperature and dilution with 1 ml of acetonitrile,
the reaction mixture was injected into a Kromasil-100 C18 semi-preparative
HPLC column (300 mm  8 mm, 7 mM). Isocratic elution with CH3CN: 0.2%
aqueous NEt3 (70:30, v/v) at 3 ml/min flow rate allowed the purification of
radioligand [11C]9. The effluent from the column containing [11C]9 was
collected in 50 ml of distilled water, and separated by solid-phase extraction by
a SEP-Pak C-18 cartridge (Millipore). Elution of the cartridge with 1 ml of
ethanol, allowed the recovering of approximately 30 mCi of radioligand [11C]9,
which was collected into a vial containing 5.0 ml of saline.
The final solution of radioligand [11C]9 was assayed for total radioactivity
and its radiochemical purity evaluated in an analytical HPLC Phenomenex
Luna C-18 (250 Â 4.6 mm) column, using CH3CN: 0.2% aqueous NEt3 (70:30,
v/v) as eluent, at 1.0 ml/min flow rate. In these chromatographic conditions,
retention times were 4.35 min for [11C]9 and 3.08 min for the mercaptoimida-
zole precursor 3, while 11CH3I is eluted at 5.23 min. The identity of the
radiolabelled product [11C]9 was confirmed by HPLC in comparison with the
isolated ‘cold’ congener 9. Compound [11C]9 was obtained in approximate
30% yield (decay-corrected, related to [11C]CO2), with a radiochemical purity
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SYNTHESIS AND BIOLOGICAL EVALUATION OF
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better than 99% and with a specific activity of 0.2–0.4 Ci/mmol at the end-of-
synthesis.
Methyl[4-((2-methoxyphenyl)-1-piperazinyl)butyl]-(2-thio[11C]methyl-1-
methylimidazol-5-yl-)methanamide ([11C]10). The radiosynthesis and purifi-
cation of [11C]10 has been performed using the procedures described above for
[11C]9, starting from 1.12 mg (0.0027 mmol) of precursor 8. Compound
([11C]10) was obtained in approximate 20% yield (decay-corrected, related to
[11C]CO2), with a radiochemical purity better than 99% and with a specific
activity at the end-of-synthesis comparable to [11C]9. The identity of [11C]10
was confirmed by HPLC comparison with the isolated ‘cold’ congener 10.
Using the chromatographic conditions described above, the retention times
were 3.75 min for [11C]10 and 2.82 min for the mercaptoimidazole precursor 8.
Receptor binding assays in rat brain homogenates
The receptor binding assays were performed as described elsewhere.23 For the
5-HT1A receptor binding assay [3H]8-OH-DPAT (NEN, 4.6 TBq/mmol) as
specific radioligand and rat hippocampus homogenate were used. Rat cortex
homogenates were used for the 5-HT2A receptor binding assay with
[3H]ketanserin (NEN, 2.3 TBq/mmol) as specific radioligand. [3H]prazosin
(NEN, 2.5 TBq/mmol) was used as specific radioligand to measure the alpha1-
adrenergic receptor binding.
The incubation step of binding assays was terminated by rapid filtration
through GF/B glass fiber filters (Whatman) using a 30-port Brandell Cell
Harvester. The filters were rapidly washed with 4 ml portions of ice-cold
buffer, transferred into 4 ml scintillation liquid (Ultima Gold, Packard) and
analysed for radioactivity by scintillation counting (Beckmann-LS 600-LL).
Biodistribution studies in rats
Groups of four male Wistar rats, 5–6 weeks old, were injected with [11C]9 or
[11C]10 (0.5 ml, 1.13 mCi) into the tail vein under ether anaesthesia. The rats
were sacrificed by heart puncture at 5 and 60 min post-injection. The organs of
interest were removed and weighted. The radioactivity of the different organs
was measured in a COBRA II counter (CANBERRA-PACKARD), and the
percentage of the injected dose per organ (% ID) and dose per gram organ
were calculated (% ID/g).
Acknowledgements
This work has been partially supported by the FCT (POCTI/QUI/42939/
*
2001). Catarina Xavier and Raquel Garcia would like to thank the Fundac-ao
-
#
para a Ciencia e Tecnologia (National Foundation for Science and
Technology) for a BIC research grant and a PhD research grant, respectively.
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