First no-carrier-added radioselenation of an adenosine-A1 receptor ligand

Article abstract of DOI:10.1002/jlcr.829

The precursor synthesis and the no-carrier-added (n.c.a.) radiosynthesis of the adenosine-A₁ receptor ligand 5′-(methyl[ ⁷⁵Se]seleno)-N⁶-cyclopentyladenosine ([⁷⁵Se]1) are described in this report. A method was developed starting from elemental n.c.a. selenium-75, followed by a three-step polymer-supported radioselenation and deprotection which gave the radioligand with a radiochemical yield of 30%, a radiochemical purity of > 99% and a specific radioactivity of > 300 GBq/mmol (8 Ci/mmol). Preparation time was 40 min. The nonradioactive compound 5′-(methylseleno)-N⁶-cyclopentyladenosine (1) was pharmacologically evaluated in vitro and showed high affinity and selectivity for the adenosine-A₁ receptor. These preliminary results suggest that this compound could be a useful radioligand for the noninvasive imaging of the brain adenosine-A₁ receptors using positron emission tomography (PET) when labelled with the positron emitter selenium-73 (half-life: 7.1 h). Copyright

Full text of DOI:10.1002/jlcr.829

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 415–427.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.829
Research Article
First no-carrier-added radioselenation of an adenosine-
A1 receptor ligand
Till Blumy, Johannes Ermert*, Walter Wutz, Dirk Bier and Heinz H. Coenen
Institut fu.r Nuklearchemie, Forschungszentrum Ju.lich GmbH, Ju.lich D-52425, Germany
Summary
The precursor synthesis and the no-carrier-added (n.c.a.) radiosynthesis of the
adenosine-A1 receptor ligand 50-(methyl[75Se]seleno)-N6-cyclopentyladenosine ([75Se]1)
are described in this report. A method was developed starting from elemental n.c.a.
selenium-75, followed by a three-step polymer-supported radioselenation and
deprotection which gave the radioligand with a radiochemical yield of 30%, a
radiochemical purity of >99% and a specific radioactivity of >300 GBq/mmol (8 Ci/
mmol). Preparation time was 40 min. The nonradioactive compound 50-(methylsele-
no)-N6-cyclopentyladenosine (1) was pharmacologically evaluated in vitro and showed
high affinity and selectivity for the adenosine-A1 receptor. These preliminary results
suggest that this compound could be a useful radioligand for the noninvasive imaging
of the brain adenosine-A1 receptors using positron emission tomography (PET) when
labelled with the positron emitter selenium-73 (half-life: 7.1 h). Copyright # 2004
John Wiley & Sons, Ltd.
Key Words: adenosine-A1 receptor ligand; n.c.a. radioselenation; selenium-75;
positron emission tomography
Introduction
Purinergic receptors can be subdivided into P1 and P2 receptors. So far, four P1
receptors, adenosine-A1, -A2A, -A2B and -A3, have been defined pharmaco-
logically and cloned. All adenosine receptors are G-protein-coupled, activated
by adenosine and antagonized by xanthines.1 Adenosine-A1 receptors are
widely distributed in the central nervous system, and have been extensively
characterized in brain where they are expressed in high density (0.5–1 pmol/mg
membrane protein). In the periphery, they are found in the heart, in adipose
tissue and in the kidneys.
.
*Correspondence to: J. Ermert, Institut fur Nuklearchemie, Forschungszentrum Julich GmbH, Julich
D-52425, Germany. E-mail: j.ermert@fz-juelich.de
.
.
y Present address: School of Chemistry, University of St Andrews, St Andrews KY16 9ST, United Kingdom.
Copyright # 2004 John Wiley & Sons, Ltd.
Received 19 June 2003
Revised 20 October 2003
Accepted 4 December 2003

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T. BLUM ET AL.
Ligands for the adenosine-A1 receptor subtype have a broad therapeutic
potential because of its wide organ and tissue distribution.2 Agonists for the
adenosine-A1 receptors could, for example, be useful as sedatives and in the
diagnosis of diseases of coronary arteries.3 However, severe cardiovascular
side effects can be expected, caused by the strong hypotensive effects of the
adenosine agonists.4 These side effects are major drawbacks in the therapeutic
use of adenosine receptor agonists. Partial agonists might have less
pronounced cardiovascular effects and may act more selectively. Another
advantage of partial agonists would be that they probably induce less receptor
downregulation and desensitization.5
Previously, van der Wenden et al.6 have synthesized such partial agonists by
substituting the 50-position of N6-cyclopentyladenosine. Studies showed that
among other 50-(methylseleno)-N6-cyclopentyladenosine (1) (Scheme 1)
proved to be selective for the adenosine-A1 receptors, displaying an affinity
in the nanomolar range.
Scheme 1. 50-(Methylseleno)-N6-cyclopentyladenosine (1)
Since all adenosine-A1 receptor ligands, which are under evaluation as PET
ligands at present, are labelled with carbon-117 and one with fluorine-18,8,9 the
aim of this study was to obtain a much longer-lived radioligand. Therefore,
compound 1 was selected for labelling with the positron emitter selenium-73.
In order to investigate the general feasibility of an appropriate radioselenation
method at the no-carrier-added (n.c.a.) level selenium-75 was used for initial
tracer development and in vitro evaluation.
Results and discussion
Preparation of the standard and precursor
The standard compound 50-(methylseleno)-N6-cyclopentyladenosine (1) and
the precursor for radioselenation 50-bromo-20,30-isopropylidenedioxy-N6-
cyclopentyladenosine (5) were synthesized in five and three steps, respectively,
starting from 6-chloropurine riboside (2) (Scheme 2).
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FIRST NO-CARRIER ADDED RADIOSELENATION
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Scheme 2. Synthesis of 50-(methylseleno)-N6-cyclopentyladenosine (1) Reaction
conditions: (i) cyclopentylamine, EtOH, reflux; (ii) sulfuric acid (conc.), acetone,
08C; (iii) triphenylphosphine, carbon tetrabromide, CH2Cl2, r.t.; (iv) dimethyldi-
selenide, sodium borohydride, EtOH, r.t.; (v) acetic acid, reflux
N6-Cyclopentyladenosine (3) was obtained by reacting 2 with cyclopentyl-
amine in ethanol to substitute the chlorine atom.10 To obtain a selectively
reactive 50-group, the cis vicinal 20- and 30-hydroxyl groups of N6-
cyclopentyladenosine were selectively protected with an isopropylidene moiety
by sulfuric acid-catalyzed reaction in acetone,11 yielding 20,30-isopropylidene-
dioxy-N6-cyclopentyladenosine (4). Subsequently, by reaction with carbon
tetrabromide and triphenylphosphine in CH2Cl2, the 50-hydroxyl group of 4
was replaced by a bromine atom as leaving group,12 resulting 50-bromo-20,
30-isopropylidenedioxy-N6-cyclopentyladenosine (5). This compound was
quite unstable and therefore had to be used directly after purification. The
reaction between 5 and methyl selenolate, which was obtained by reductive
cleavage of dimethyldiselenide with sodium borohydride,13 yielded 50-
(methylseleno)-20,30-isopropylidenedioxy-N6-cyclopentyladenosine (6).
Deprotecting the 20- and 30-position of 6 with acetic acid14 gave the final
product 50-(methylseleno)-N6-cyclopentyladenosine (1) with an overall yield of
about 20% (based on 2).
Radiosynthesis of n.c.a. 50-(methyl[75Se]seleno)-N6-cyclopentyladenosine
Since the specific activity of a potential radioligand for in vivo studies is a
crucial factor and should be as high as possible in order to avoid a saturation
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T. BLUM ET AL.
of the receptor system by the compound administered, an appropriate n.c.a.
labelling method is required.
For convenience, the development and optimization of the radiosynthesis
was performed using the longer-lived selenium-75 (T1/2=120.4 days). It has
been demonstrated15 that selenium-75 is a suitable model-nuclide for selenium-
73 and that radiosyntheses developed via selenium-75 may serve as well for the
preparation of 73Se-labelled compounds without any necessity of modifying
the reaction conditions.
A previously developed n.c.a. radioselenation strategy15 was tested for the
preparation of n.c.a. 50-(methyl[75Se]seleno)-N6-cyclopentyladenosine ([75Se]1).
Starting from n.c.a. elemental selenium-75, cyclohexyl isocyanide and
aminomethylated polystyrene, the corresponding polymer-bound [75Se]sele-
nourea ([75Se]7resin) was synthesized in benzene at 808C within 10 min with a
radiochemical yield (RCY) of 80 ꢀ 5% (based on n.c.a. 75Se0) (Scheme 3). The
Scheme 3. Polymer-supported radiosynthesis of methyl [75Se]selenouronium salt
([75Se]8resin). Reaction conditions: (i) cyclohexyl isocyanide, aminomethylated
polystyrene, benzene, 808C; (ii) washing with benzene; (iii) methyl triflate,
benzene, r.t.; (iv) separation via washing with benzene and THF
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FIRST NO-CARRIER ADDED RADIOSELENATION
419
intermediate [75Se]7resin was purified by washing with benzene. The RCY was
determined as activity bound on the resin of purified [75Se]7resin, since it is
likely that 75Se was present on the polymer in no other form than in the
corresponding selenourea group.
Subsequent alkylation in benzene via methyl triflate yielded the correspond-
ing methyl [75Se]selenouronium salt ([75Se]8resin) within 5 min with an RCY of
92 ꢀ 3% (activity bound on the purified resin, based on [75Se]7resin) (Scheme
3). Purification of [75Se]8resin was carried out by repeated washing with
benzene and tetrahydrofuran (THF) in order to get rid of surplus alkyl triflate
and to determine the RCY of the purified [75Se]8resin
.
Hydrolysis of [75Se]8resin by means of tetrabutylammonium hydroxide
(TBAH) and alkylation of the resulting methyl [75Se]selenolate with 50-bromo-
20,30-isopropylidenedioxy-N6-cyclopentyladenosine (5) gave 50-(methyl[75Se]se-
leno)-20,30-isopropylidenedioxy-N6-cyclopentyladenosine ([75Se]6) with an
RCY of about 70% (based on [75Se]8resin) under optimized reaction conditions
(Scheme 4). Subsequent deprotection of [75Se]6 using hydrochloric acid at
808C yielded the final n.c.a. product [75Se]1 within 5 min and with an RCY of
55% (based on [75Se]6). Thus, 50-(methyl[75Se]seleno)-N6-cyclopentyladenosine
was obtained at the n.c.a. level with an overall RCY of approximately 30%
(based on 75Se0), with a radiochemical purity of >99% and within a total
reaction time of 40 min.
With the detection limit of 1 nmol found here and the starting activity
used, the specific activity of n.c.a. [75Se]1 was determined to be more
than 300 GBq/mmol (8 Ci/mmol). However, with a theoretical maximal
specific activity (As,max) of 37 TBq/mmol (1 Ci/mmol) for carrier-free
selenium-75, the actual specific activity will amount to a value in the
range between these two limits. Provided production of higher starting
activities would not increase the dilution with stable selenium, i.e. the
amount of carrier will still remain below 1 nmol, one can suggest
that the actual specific activity would increase with the same factor
as the starting activity. In case of the positron emitter selenium-73
the theoretical As,max amounts to 16 TBq/mmol (400 Ci/mmol). Therefore,
one can suggest that an even higher specific activity will be obtained if the
starting activity is in the GBq-range. Thus, a specific activity appears feasible,
which is sufficient to perform in vivo receptor studies with 73Se-labelled ligands
using PET.
Radioligand binding studies
Results of radioligand binding studies for 1 in the literature6 are promising
regarding selectivity for the adenosine-A1 receptor, since they showed a 150
fold better affinity for the A1 vs the A2A receptor, which is probably due to the
presence of the N6-cyclopentyl substituent. Although the Ki value of 1 for the
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T. BLUM ET AL.
Scheme 4. Radiosynthesis of 50-(methyl[75Se]seleno)-N6-cyclopentyladenosine
([75Se]1). Reaction conditions: (v) TBAH, 50-bromo-20,30-isopropylidenedioxy-
N6-cyclopentyladenosine (5), THF, 708C; (vi) hydrochloric acid, 808C
adenosine-A1 receptor was not very satisfactory with only 76 nM; the Ki value
for the A2A receptor amounted to 11 mM. These literature data were obtained
using membranes of rat brain cortex with [3H]DPCPX as the radioligand for
the A1 receptor and using rat striatal membranes with [3H]CGS21680 as the
radioligand for the A2 receptor, respectively.
Nevertheless, 1 seems to be a very attractive selective ligand for the A1
receptor. Therefore, radioligand binding studies were carried out here also
under different conditions (cf. Experimental). Its affinity for the adenosine-A1
receptor was determined by competition with [3H]CPFPX16 on pig cortical
membranes. The Ki value of 1 for the adenosine-A1 receptor was 0.9 nM
(0.8–1.1 nM, 95% confidence interval). On the other hand, 1 had a Ki value of
1.3 mM (0.3–5.5 mM, 95% confidence interval) for the adenosine-A2A receptor
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FIRST NO-CARRIER ADDED RADIOSELENATION
421
measured by displacement of [3H]CGS21680 from pig striatal membranes.
These results suggest, that 50-(methylseleno)-N6-cyclopentyladenosine (1) is
selective for the adenosine-A1 vs the -A2A receptor with a 1500 fold better
affinity. The differences of this study and literature data can be explained by
the altered conditions of the radioligand binding studies, i.e. by a more
selective radioligand in case of the A1 receptor study and by tissues of different
animals in the studies. Therefore, the results of the biological experiments
require further evaluation and make a study on cloned human receptors
advisable.
Conclusion
In conclusion, n.c.a. 50-(methyl[75Se]seleno)-N6-cyclopentyladenosine ([75Se]1)
was be synthesized via a polymer-supported radioselenation strategy with an
overall radiochemical yield of 30% within 40 min. Furthermore, it was
demonstrated, that 1 bound to the pig adenosine-A1 receptor with a Ki of
0.9 nM. The Ki at the pig adenosine-A2A receptor was 1300 nM, thus giving a
selectivity of >1500-fold. These results make the use of this radioligand in its
selenium-73 labelled form promising as a PET ligand.
Experimental
Material and methods
All chemicals and solvents were purchased from Aldrich (Germany), Fluka
(Switzerland) and Merck (Germany). They were reagent grade or better and
used without further purification. [3H]CPFPX was prepared in house, as
reported16 and New England Nuclear provided [3H]CGS 21680. Amino-
methylated polystyrene was obtained from Novabiochem (Germany). All
selenocompounds were prepared under a slight positive pressure of argon.
Flash chromatography was done with Fluka silica gel 60 (220–440 mesh).
Mixtures of elution solvents are given as V/V ratios.
Melting point determinations employed a Mettler FP 61 apparatus. Melting
points (m.p.) are uncorrected. Analytical thin-layer chromatography (TLC)
was performed with precoated silica gel plates (5 ꢁ 7.5 cm plates Type F254S
,
Merck, Germany). Visualization of TLC slides was by UV. 1H and 13C nuclear
magnetic resonance (NMR) spectra were recorded at 200 and 50 MHz,
respectively, on a Bruker DPX Avance 200 spectrometer with samples
dissolved in d6-DMSO or CDCl3. 13C NMR were routinely run with broad
band decoupling. All chemical shifts are reported in d ppm using the signals of
the solvent as a reference. Mass spectra (MS) were obtained using a
Thermoquest Automass Multi mass spectrometer using the electron spray
ionization method. Reported values for the mass of selenium containing
compounds base on selenium-80.
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T. BLUM ET AL.
Analytical radio-HPLC was performed on a system consisting of a Knauer
pump 6400 and a Knauer UV/vis photometer 3060 with a detector wavelength
of 220 nm. Sample injection was accomplished by a Rheodyne-Injector block
7125. For measurement of radioactivity the outlet of the UV detector was
connected to a NaI(Tl) well-type scintillation detector (EG&G ACE MateTM
)
and the recorded data were processed by the software system Raytest Winnie
(Raytest, Germany). HPLC of aliquots of labelled products and standards
was performed using a Lichrosorb RP Select B (250 ꢁ 4 mm) column
(CS-Chromatographie Service GmbH, Germany) and a mobile phase
consisting of MeOH/H2O in various concentrations (given as V/V ratios) at
a flow rate of 1.0 ml/min. Radio-TLC was performed on Merck silica gel plates
with the solvent system CH2Cl2/MeOH in various concentrations (given as
V/V ratios). The developed TL-chromatograms were measured for radio-
activity on an Instant ImagerTM (Packard, USA).
N6-Cyclopentyladenosine (3) [from10]
A mixture of 6-chloropurine ribonucleoside (5.7 g, 20 mmol), cyclopentyla-
mine (11.8 ml, 120 mmol) and EtOH (100 ml) was refluxed for 15 h, and it was
evaporated in vacuo to dryness. The residue was triturated with a small
amount of EtOH, and colorless crystals of N6-cyclopentyladenosine were
1
obtained. Yield: 4.56 g (68%) (lit.10: 75%); m.p.: 78.88C (lit.: 77–818C); H-
NMR (d6-DMSO): d 8.36 (s, 1H, H8), 8.21 (s, 1H, H2), 7.80 (d, 1H, N6H),
5.92–5.86 (d, 1H, H10), 5.51–5.44 (m, 2H, OH-20/OH-30), 5.25–5.19 (m, 1H,
OH-50), 4.72–4.59 (m, 1H, H20), 4.56 (bs, 1H, HCN6), 4.19–4.14 (m, 1H, H30),
4.01–3.96 (m, 1H, H40), 3.67 (m, 2H, H50), 1.78 (m, 8H, Hcyclopentyl); (m, 8 H,
Hcyclopentyl); 13C-NMR: d 155.3 (C4/C6), 153.1 (C2), 144.9 (C8), 140.4 (C5),
88.8 (C10), 86.8 (C40), 74.3 (C20), 71.5 (C30), 62.6 (C50), 56.9 (C1cyclopentyl), 33.0
(C2/5cyclopentyl), 24.3 (C3/4cyclopentyl); MS: m/e 336 (M+1, 100%); Rf: 0.68
(CH2Cl2/MeOH 1:4).
20,30-Isopropylidenedioxy-N6-cyclopentyladenosine (4) [from11]
N6-Cyclopentyladenosine (3) (4.36 g, 13 mmol) dissolved in acetone (50 ml)
was added dropwise to a stirred mixture of acetone (50 ml) and concentrated
sulfuric acid (1.3 ml, 26 mmol) at 08C. After complete addition, the mixture
was stirred at r.t. for 90 min. The solution was cooled again to 08C and
neutralized with NaOHaq (20 ml, 20%). The mixture was concentrated and
partitioned between H2O and CH2Cl2. The aqueous layer was extracted
several times with CH2Cl2. The combined organic layers were dried (Na2SO4),
and the solvent was evaporated. The residue was purified via flash
chromatography (CH2Cl2/MeOH 100:2) to give 20,30-isopropylidenedioxy-
N6-cyclopentyladenosine as colorless crystals after evaporation of the eluent.
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Yield: 3.79 g (78%); m.p.: 968C; 1H-NMR (d6-DMSO): d 8.36 (s, 1H, H8), 8.23
(s, 1H, H2), 7.81 (d, 1H, N6H), 6.18–6.14 (d, 1H, H10), 5.38–5.26 (m, 2H, H20/
OH-50), 5.01–4.97 (m, 1H, H30), 4.56 (bs, 1H, HCN6), 4.14–4.24 (m, 1H, H40),
3.57–3.53 (m, 2H, H50), 1.97–1.56 (m+s, 11H, Hcyclopentyl/CH3,isopropylidene),
1.34 (s, 3H, CH3,isopropylidene); 13C-NMR: d 155.8 (C4/C6), 153.4 (C2), 144.9
(C8), 140.2 (C5), 113.9 (Cq,isopropylidene), 90.6 (C10), 87.3 (C40), 84.1 (C20), 82.3
(C30), 62.5 (C50), 57.0 (C1cyclopentyl), 33.0 (C2/5cyclopentyl), 27.9 (CH3,isopropyli-
dene), 26.0 (CH3,isopropylidene), 24.3 (C3/4cyclopentyl); MS: m/e 375 (M, 100%), 204
(35); Rf: 0.35 (CH2Cl2/MeOH 25:1).
50-Bromo-20,30-isopropylidenedioxy-N6-cyclopentyladenosine (5) [from12]
Triphenylphosphine (2.36 g, 9 mmol) in dry CH2Cl2 (20 ml) was added
dropwise to a stirred solution of 4 (3.38 g, 9 mmol) and CBr4 (5 g, 15 mmol)
in dry CH2Cl2 (20 ml) at r.t. After 15 h the solvent was evaporated and the
residue was purified via flash chromatography (CH2Cl2/MeOH 100:2) to give
50-bromo-20,30-isopropylidenedioxy-N6-cyclopentyladenosine as an oil after
evaporation of the eluent. Yield: 2.09 g (53%); 1H-NMR (d6-CDCl3): d 8.36 (s,
1H, H8), 7.86 (s, 1H, H2), 6.11 (d, 1H, H10), 5.91–5.87 (d, 1H, N6H), 5.51–5.48
(m, 1H, H20), 5.20–5.16 (m, 1H, H30), 4.75–4.35 (bs, 1H, HCN6), 4.52–4.49 (m,
1H, H40), 3.72–3.40 (m, 2H, H50), 2.17–1.50 (m+s, 11H, Hcyclopentyl
/
CH3,isopropylidene), 1.39 (s, 3H, CH3,isopropylidene); 13C-NMR: d 155.0 (C4/C6),
153.7 (C2), 144.8 (C8), 132.1 (C5), 114.8 (Cq,isopropylidene), 91.6 (C10), 87.2
(C40), 84.6 (C20), 84.0 (C30), 54.4 (C50), 52.8 (C1cyclopentyl), 32.3 (C2/5cyclopentyl),
27.4 (CH3,isopropylidene), 25.7 (CH3,isopropylidene), 24.1 (C3/4cyclopentyl); MS: m/e
440 (M, 100%); Rf: 0.81 (CH2Cl2/MeOH 25:1).
50-(Methylseleno)-20,30-isopropylidenedioxy-N6-cyclopentyladenosine (6) [fol-
lowing13]
50-Bromo-20,30-isopropylidenedioxy-N6-cyclopentyladenosine
(5)
(1.75 g,
4 mmol), dissolved in EtOH (20 ml), was added dropwise to a stirred mixture
of dimethyl diselenide (1.07 ml, 8 mmol) and sodium borohydride (2 g) in
EtOH (20 ml). After complete addition, the mixture was stirred at r.t. for
30 min. The solution was concentrated and partitioned between H2O and
CH2Cl2. The aqueous layer was extracted several times with CH2Cl2. The
combined organic layers were dried (Na2SO4), and the solvent was evaporated.
The residue was purified via flash chromatography (CH2Cl2/MeOH 100:2) to
give 50-(methylseleno)-20,30-isopropylidenedioxy-N6-cyclopentyladenosine as
1
oil after evaporation of the eluent. Yield: 1.5 g (83%); H-NMR (d6-DMSO):
d 8.35 (s, 1 H, H8), 8.25 (s, 1 H, H2), 7.76 (d, 1 H, N6H), 6.20 (d, 1 H, H10),
5.56–5.52 (m, 1 H, H20), 5.04–5.00 (m, 1 H, H30), 4.56 (bs, 1 H, HCN6),
4.37–4.29 (m, 1 H, H40), 2.87–2.72 (m, 2 H, H50), 1.74–1.55 (m+2 s, 14 H,
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T. BLUM ET AL.
Hcyclopentyl/SeCH3/CH3,isopropylidene), 1.35 (s, 3 H, CH3,isopropylidene); 13C-NMR:
d 155.6 (C4/C6), 153.5 (C2), 144.9 (C8), 140.5 (C5), 114.1 (Cq,isopropylidene),
90.1 (C10), 87.2 (C40), 84.7 (C20), 84.2 (C30), 58.1 (C1cyclopentyl), 33.0 (C2/
5cyclopentyl), 27.8 (C50), 27.5 (CH3,isopropylidene), 26.0 (CH3,isopropylidene), 24.3
(C3/4cyclopentyl), 5.0 (SeCH3); MS: m/e 454 (M, 100%), 360 (97); k0: 4.22
(MeOH/H2O: 7/3); Rf: 0.35 (CH2Cl2/MeOH 25:1).
50-(Methylseleno)-N6-cyclopentyladenosine (1) [from14]
A solution of 6 (1.36 g, 3 mmol) in acetic acid (60 ml, 65%) was refluxed for
4 h. The solvent was evaporated and the residue was purified via flash
chromatography (CH2Cl2/MeOH 100:2) to give 50-(methylseleno)-N6-cyclo-
pentyladenosine6 as a colorless solid. The product recrystallized from EtOH.
1
Yield: 1.08 g (87%); m.p.: 71.38C (lit.6: 66–728C); H-NMR (d6-DMSO): d
8.37 (s, 1H, H8), 8.24 (s, 1H, H2), 7.69 (d, 1H, N6H), 6.18 (d, 1H, H10),
5.54–5.52 (m, 1H, OH-20), 5.42–5.36 (m, 1H, OH-30), 4.80–4.78 (m, 1H, H20),
4.62–4.39 (bs, 1H, HCN6), 4.24–4.11 (m, 2H, H30/H40), 2.99–2.80 (m, 2H,
H50), 1.96–1.23 (m+s, 11H, Hcyclopentyl/SeCH3); 13C-NMR: d 155.2 (C4/C6),
153.4 (C2), 145.0 (C8), 140.3 (C5), 88.2 (C10), 85.1 (C40), 74.1 (C20), 73.7 (C30),
56.9 (C1cyclopentyl), 33.1 (C2/5cyclopentyl), 28.2 (C50), 24.3 (C3/4cyclopentyl), 5.4
(SeCH3); MS: m/e 414 (M+1, 100%); k0: 2.07 (MeOH/H2O: 6/4); Rf: 0.22
(CH2Cl2/MeOH 25:1).
Production of [75Se]selenium
N.c.a. [75Se]selenium was produced using 20 MeV protons at the compact
cyclotron CV-28 of the Forschungszentrum Julich GmbH via the 75As
.
(p,n)75Se reaction on a solid Cu3As target. After thermochromatographic
separation n.c.a. selenium-75 is available in its oxidized form as [75Se]SeO32ꢂ in
hydrochloric acid. It was then reduced with sulfur dioxide to obtain elemental
n.c.a. selenium-75, which was extracted into benzene following procedures
earlier described in detail.17,18
The radiosynthesis described below was conducted under argon in a conical
5 ml reaction vessel equipped with a magnetic stirring bar and a teflon rubber
septum.
N.c.a. 50-(methyl[75Se]seleno)-N6-cyclopentyladenosine
The labelled adenosine-derivative was prepared as reported previously15 for
[75Se]selenoethers in general and is briefly described here, starting from
elemental n.c.a. selenium-75. The formation of polymer-supported n.c.a. N-
cyclohexyl [75Se]selenourea-N0-methyl polystyrene ([75Se]7resin) was performed
at 808C within 10 min after adding cyclohexyl isocyanide (10 ml, 0.08 mmol)
and aminomethylated polystyrene (40 mg) to a solution of n.c.a. 75Se0 in
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FIRST NO-CARRIER ADDED RADIOSELENATION
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benzene (0.5 ml, typically containing 370 kBq (10 mCi)). [75Se]7resin was
purified by washing with benzene (2 ml) before use in the following
reaction step.
Alkylation of n.c.a. [75Se]7resin with trifluoromethanesulfonic acid methyl
ester (11 ml, 0.1 mmol) in benzene (0.5 ml) gave the corresponding methyl
[75Se]selenouronium salt ([75Se]8resin) at r.t. within 5 min, which was purified
by washing the resin with benzene (2 ml) and THF (2 ml). Hydrolysis
with tetrabutylammonium hydroxide (0.06 mmol) in 0.6 ml THF in the
presence of 50-bromo-20,30-isopropylidenedioxy-N6-cyclopentyladenosine (5)
(44 mg, 0.1 mmol) resulted in 50-(methyl[75Se]seleno)-20,30-isopropylidene-
dioxy-N6-cyclopentyladenosine ([75Se]6) at 708C within 5 min. After deprotec-
tion using hydrochloric acid (0.3 ml, 1 N) at 808C, 50-(methyl[75Se]seleno)-N6-
cyclopentyladenosine ([75Se]1) was obtained within 5 min and analyzed by
radio-HPLC and radio-TLC. ([75Se]1) was purified and separated by radio-
HPLC (k0=2.07; MeOH/H2O 60:40).
Radioligand binding assays
Corpora striata (for A2AAR assays) and frontal cortices (for A1AR assays)
were dissected from pig brain and the tissue was homogenized for 1 min in 20
volumes of ice-cold 50 mM tris-HCl buffer, pH 7.4 containing 10 mM MgCl2,
soybean trypsin inhibitor (20 mg/ml), Bacitracin (200 mg/ml), and Benzamidine
HCl (160 mg/ml) by means of an Ultra Turrax at 20 000 rpm. The homogenate
was centrifuged at 48 000 ꢁ g for 10 min at 48C (Beckmann Optima L, SW41Ti
rotor). The pellet was suspended in 20 volumes of tris-HCl, pH 7.4, containing
adenosine deaminase (2 U/ml) and trypsin inhibitor (20 mg/ml), and then
incubated for 30 min at 378C. After centrifugation at 48 000 ꢁ g for 10 min at
48C the resulting pellet was diluted in 20 volumes of 50 mM tris-HCl, pH 7.4,
containing 10 mM MgCl2. Aliquots of the homogenate (1 ml) were stored
at –808C. The assays were performed in triplicate by incubating of 20 ml
aliquots of the membrane fractions (87 mg protein/assay for A1-assays, 68 mg
protein/assay for A2A-assays) in tris-HCl, pH 7.4, containing adenosine
deaminase (2 U/ml). Cortical homogenates were used for A1AR- and striatal
homogenates for A2AAR-assays. Incubation was carried out at 208C for
60 min in a total assay volume of 200 ml. In A2AAR competition experiments
[3H]CGS21680 (A2AA receptor radioligand, KD=26 nM) was used in a
concentration of 5 nM. In A1AR competition experiments [3H]CPFPX (A1A
receptor radioligand, KD=0.62 nM) was used in a concentration of 2 nM.
Centrifugation at 48 000 ꢁ g for 6 min at 88C separated bound from free
ligand. Supernatants were discarded, the pellets washed with 1 ml ice cold
buffer and dissolved by incubating in SolvableTM (500 ml, Canberra-Packard)
for 120 min at 508C. Aliquots of 450 ml were placed in scintillation vials
with scintillation cocktail (10 ml, Ultima Gold XR, Canberra-Packard).
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 415–427

426
T. BLUM ET AL.
Radioactivity was measured in a liquid scintillation analyzer Tri-carb 2300Tr
(Canberra-Packard). Protein estimation was performed with a commercial
assay (Bio-Rad DC Protein Assay) after solubilization in 15% NH4OH
containing 2% SDS (v,w); human serum albumin served as a standard. Ki was
calculated by a computer-assisted curve-fitting program (GraphPad Prism,
version 3.0) as exemplified in Scheme 5 for the adenosine A1 receptor.
100
80
60
40
20
0
-10
-9
-8
-7
-6
ligand log c
Scheme 5. Binding inhibition of [3H]CPFPX by 50-(methyseleno)-N6-cyclopen-
tyladenosine (1) in homogenates of pig frontal cortices
Acknowledgements
The authors thank S. Spellerberg for his helpful assistance in preparation of
n.c.a. selenium-75 and Dr M. Holschbach and S. Mahbubfar for recording the
NMR and mass spectra.
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