Design, radiosynthesis, and biodistribution of a new potent and selective ligand for in vivo imaging of the adenosine A2A receptor system using positron emission tomography [2]

Full text of DOI:10.1021/jm0009843

J . Med. Chem. 2000, 43, 4359-4362
4359
radioisotopes: (E)-8-(3-chlorostyryl)-1,3-dimethyl-7-[11C]-
methylxanthine ([11C]CSC),7 (E)-8-(3,4-dimethoxystyryl)-
1,3-dipropyl-7-[11C]methylxanthine ([11C]KF17837),8
(E)-8-(3,4,5-trimethoxystyryl)-1,3-dimethyl-7-[11C]meth-
ylxanthine ([11C]KF18446),9 and (E)-1,3-diallyl-7-
[11C]methyl-8-(3,4,5-trimethoxystyryl)xanthine ([11C]-
KF19631).9 [11C]CSC is highly selective, but its affinity
for adenosine A2A receptors is relatively low (Ki ) 54
nM). [11C]KF17837 has higher affinity (Ki ) 1 nM) for
adenosine A2A receptors, but a low striatum-to-cerebel-
lum ratio was found when tested in vivo with PET.10,11
Among the 11C-labeled ligands for adenosine A2A recep-
tors prepared to date, the most suitable ligand for PET
application appears to be [11C]KF18446, which shows
good in vivo selectivity and specificity for the target
tissues.9 However, the above compounds are xanthine
derivatives and are subject to photoisomerization, which
is a specific drawback of this class of molecules.
In recent years efforts have been made to synthesize
novel non-xanthine compounds with antagonistic prop-
erties toward the adenosine A2A receptor subtype. In
particular, the synthesis of a large group of pyrazolo-
[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines behaving as po-
tent and selective A2A antagonists has recently been
reported.12-14 In general, this class of compounds shows
a good binding profile in terms of in vitro affinity and
receptor selectivity. The two prototypes of this family,
SCH58261 (1) and SCH63390 (2), together with their
4-hydroxyphenyl derivatives (3, 4), proved to be the best
derivatives in terms of both affinity and selectivity for
human A2A adenosine receptors.
Design , Ra d iosyn th esis, a n d
Biod istr ibu tion of a New P oten t a n d
Selective Liga n d for in Vivo Im a gin g of
th e Ad en osin e A2A Recep tor System
Usin g P ositr on Em ission Tom ogr a p h y
Sergio Todde,† Rosa Maria Moresco,†
Pasquale Simonelli,† Pier Giovanni Baraldi,‡
Barbara Cacciari,‡ Giampiero Spalluto,§
Katia Varani,| Angela Monopoli, Mario Matarrese,†
Assunta Carpinelli,† Fulvio Magni,†
Marzia Galli Kienle,† and Ferruccio Fazio*,†
CNR-INB, University of Milano-Bicocca, Scientific Institute
H San Raffaele, Via Olgettina 60, I-20132 Milano, Italy,
Dipartimento di Scienze Farmaceutiche and Dipartimento
di Medicina Clinica e Sperimentale-Sezione di
Farmacologia, Universita` degli Studi di Ferrara,
Via Fossato di Mortara 17-19, I-44100 Ferrara, Italy,
Dipartimento di Scienze Farmaceutiche, Universita` degli
Studi di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy,
and Schering-Plough Research Institute, San Raffaele
Scientific Park, Via Olgettina 58, I-20132 Milano, Italy
Received May 16, 2000
In tr od u ction . Adenosine is an endogenous modula-
tor of neurotransmission in both the central and pe-
ripheral nervous systems. This neuromodulator inter-
acts with four different G-protein-coupled receptors
1
classified as A1, A2A, A2B, and A3 located on cell
membranes. In particular, the A2A adenosine receptor
subtype, which is positively coupled with adenylyl
cyclase, is highly expressed in the striatum,2 where it
is functionally linked to dopamine D2 receptors. Experi-
mental evidence indicates that in striato-pallidal neu-
rons the administration of A2A adenosine receptor
agonists decreases the affinity of D2 dopamine receptor
agonists.3,4 These findings suggest that adenosine A2A
receptor antagonists may be useful in the treatment of
Parkinson’s disease.
Positron emission tomography (PET) enables the in
vivo study of several physiological and neurochemical
variables using methods originally developed for quan-
titative autoradiography.5 Radioligands suitable for PET
studies should fulfill several criteria, such as receptor
affinity in the nanomolar range, high selectivity for the
target receptor subtype, minimal metabolism in tissues,
permeability across the blood-brain barrier (BBB), and
low nonspecific binding.6 Owing to the short half-life of
positron-emitting radioisotopes, ligand design should
also guarantee an easy and fast chemical approach to
the radiosynthesis of the labeled form and a high specific
radioactivity of the final product. To date, several
xanthine derivatives with antagonist activity for A2A
receptors have been labeled with positron-emitting
* To whom correspondence should be addressed. Phone: +39 02
2153056. Fax: +30 02 26415202. E-mail: fazio@mednuc.hsr.it.
† University of Milano-Bicocca.
‡ Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di
Ferrara.
Out of this family, we selected for the development
of a new PET ligand the derivative 5-amino-7-(3-(4-
methoxyphenyl)propyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-
triazolo[1,5-c]pyrimidine (SCH442416, 5), whose chemi-
§ Universita` degli Studi di Trieste.
| Dipartimento di Medicina Clinica e Sperimentale-Sezione di
Farmacologia, Universita` degli Studi di Ferrara.
Schering-Plough Research Institute.
10.1021/jm0009843 CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/28/2000

4360 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Communications to the Editor
F igu r e 1. Structure and biological profile of selected compound 5.
Sch em e 1a
The in vitro binding profile of compound 5 was
evaluated on rat cerebral membranes and on CHO
human cells transfected with A1 adenosine receptors
and [3H]DPCPX as a radioligandand, on HEK-293 cells
transfected with A2A, A2B, and A3 adenosine receptors
using [3H]SCH58261, [3H]DPCPX, and [125I]AB-MECA,
respectively. Compound 5 showed high affinity and
selectivity for both human and rat adenosine A2A
receptor subtypes. In particular, for the human A2A
adenosine receptor subtypes we found an in vitro
affinity in the picomolar range, indicating that com-
pound 5 is one of the most potent antagonists ever
reported.
The synthesis of [11C]SCH442416 (7) was carried out
via the direct alkylation of compound 4 with [11C]CH3I.
The labeled methyl iodide 8 was prepared starting from
[11C]CO2 (obtained by irradiating a mixture of 14N2 and
1% O2 with a cyclotron proton beam of 11 MeV) which,
after reduction with lithium aluminum hydride and
subsequent hydrolysis with HI, afforded [11C]CH3I with
high yield (80-90% decay corrected) (Scheme 3).15
a
(i) See Baraldi et al. J . Med. Chem. 1998, 41, 2126-2133.
cal structure allows an easy introduction of a methyl
group by direct O-alkylation of the phenolic function
with [11C]CH3I under alkaline conditions (Figure 1).
The aim of this study was to use [11C]SCH442416 as
a new ligand for the in vivo imaging of A2A receptors
using PET. This report describes the in vitro binding
profile of “cold” SCH442416, the radiosynthesis of
[11C]SCH442416, and its in vivo evaluation in rats.
Resu lts a n d Discu ssion . Compound 5 was prepared
following the general synthetic strategy for the synthe-
sis of the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines
previously reported,14 starting from the 3-(4-methoxy-
phenyl)propyl hydrazine 6 (Scheme 1).
The reaction of (ethoxymethylene)malononitrile with
6 afforded the pyrazole 11, which was then transformed
to the corresponding imidate 12 by refluxing in triethyl
orthoformate. The imidate was reacted with 2-furoic
acid hydrazide in refluxing 2-methoxyethanol to provide
the pyrazolo[4,3-e]pyrimidine intermediate 13 which,
without any purification, was converted through a
thermally induced cyclization in diphenyl ether to the
derivative 14 in good yield. Treatment of 14 with dilute
hydrochloric acid at reflux temperature induced pyr-
imidine ring opening to furnish the 5-amino-4-(1H-1,2,4-
triazol-5-yl)pyrazole 15 in quantitative yield. This de-
rivative was converted into the final compound 5 by
reaction with an excess of cyanamide in 1-methyl-2-
pyrrolidone at 140 °C. Purification procedures yielded
5 as a white solid (Scheme 2).
Compound 7 was prepared by dissolving 4 in di-
methylformamide, followed by the addition of 5 M
NaOH as a base; 8 was bubbled into the reaction vessel
in a helium stream at room temperature, and the
mixture was allowed to react at 90 °C for 4 min. The
final product 7 was purified by preparative HPLC and
solid-phase extraction (SPE) techniques. The method
proved reliable, and the overall synthesis lasted 40 min,
thus minimizing the loss of activity due to decay.16 The
average radiochemical yield, calculated from the start-
ing [11C]CO2 and decay corrected, was 28.7 ( 7.2% (n
) 18); the specific activity of the final [11C]SCH442416
ranged 400-3390 mCi/µmol, with a mean of 1490 ( 978
mCi/µmol (n ) 18). This variability was probably due
to the different quality of the lithium aluminum hydride/
tetrahydrofuran solution, which is known to be a major
source of “cold” CO2 contamination. 7 was consistently
obtained with radiochemical purity >96% and chemical
purity >95%.
Biodistribution studies indicated that [11C]SCH442416
preferentially accumulates not only in adrenal glands
and kidneys, where A2A receptors are highly expressed,

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4361
Sch em e 2a
a
Reagents: (i) 2-furoic hydrazide, MeO(CH2)2OH; (ii) Ph2O, 260 °C, flash chromatography; (iii) HCl, reflux; (iv) NH2CN, 1-methyl-2-
pyrrolidone, pTsOH, 140 °C.
Sch em e 3a
a
Reagents: (i) LAH, THF; (ii) HI (57%), 145 °C; (iii) compound 4, DMF, NaOH, 90 °C.
Ta ble 1. Biodistribution of [11C]SCH442416 in Rats
Ta ble 2. Biodistribution of [11C]SCH442416 in Rat Brain
% ID/g
% ID/g
organs
5 min
15 min
30 min
60 min
90 min
brain areas
pituitary
5 min
15 min
30 min
60 min
90 min
blood
plasma
heart
lung
0.13 ( 0.01 0.09 ( 0.02 0.06 ( 0.01 0.03 ( 0.02 0.05 ( 0.01
0.19 ( 0.01 0.13 ( 0.02 0.08 ( 0.01 0.07 ( 0.01 0.07 ( 0.01
0.26 (0.03 0.15 ( 0.02 0.09 ( 0.03 0.04 ( 0.01 0.04 ( 0.01
0.38 ( 0.11 0.52 ( 0.21 0.20 ( 0.13 0.37 ( 0.28 0.23 ( 0.18
0.18 ( 0.03 0.06 ( 0.01 0.05 ( 0.01 0.02 ( 0.01 0.03 ( 0.01
hypothalamus 0.16 ( 0.01 0.07 ( 0.01 0.04 ( 0.01 0.02 ( 0.01 0.02 ( 0.01
hippocampus 0.17 ( 0.02 0.09 ( 0.02 0.04 ( 0.01 0.02 ( 0.00 0.02 ( 0.00
striatum
thalamus
pons
cerebellum
anti-CTX
post-CTX
0.43 ( 0.19 0.46 ( 0.10 0.20 ( 0.03 0.06 ( 0.01 0.04 ( 0.02
0.20 ( 0.04 0.10 ( 0.02 0.05 ( 0.02 0.02 ( 0.00 0.02 ( 0.01
0.20 ( 0.04 0.11 ( 0.02 0.06 ( 0.01 0.02 ( 0.00 0.02 ( 0.01
0.19 ( 0.04 0.10 ( 0.02 0.05 ( 0.01 0.02 ( 0.00 0.02 ( 0.01
0.18 ( 0.03 0.12 ( 0.03 0.06 ( 0.02 0.02 ( 0.00 0.02 ( 0.00
0.19 ( 0.03 0.10 ( 0.02 0.05 ( 0.02 0.02 ( 0.01 0.02 ( 0.01
trachea 0.17 ( 0.03 0.12 ( 0.02 0.09 ( 0.05 0.04 ( 0.01 0.03 ( 0.01
liver 0.94 ( 0.17 0.74 ( 0.14 0.52 ( 0.06 0.36 ( 0.03 0.37 ( 0.08
adrenal 0.85 ( 0.33 0.44 ( 0.10 0.25 ( 0.09 0.13 ( 0.00 0.14 ( 0.03
kidney
spleen
testes
0.69 ( 0.09 0.49 ( 0.09 0.24 ( 0.06 0.13 ( 0.02 0.11 ( 0.04
0.18 ( 0.03 0.12 ( 0.01 0.07 ( 0.01 0.06 ( 0.01 0.05 ( 0.01
0.12 ( 0.02 0.13 ( 0.02 0.23 ( 0.27 0.03 ( 0.00 0.03 ( 0.00
intestine 0.37 ( 0.13 0.28 ( 0.07 0.25 ( 0.10 0.10 ( 0.02 0.16 ( 0.07
muscle 0.15 ( 0.04 0.11 ( 0.01 0.05 ( 0.01 0.04 ( 0.01 0.02 ( 0.01
the maximum uptake was reached between 5 and 15
min after injection. In agreement with the known
regional distribution of A2A adenosine receptors within
the brain, 7 highly accumulated in the striatum, whereas
in the remaining brain regions examined the tracer
retention was definitely lower and fairly homogeneous
(Table 2).
At the time of maximum uptake, the radioactivity
concentration ratio between striatum (which is rich in
A2A adenosine receptors) and cerebellum (where the
expression of A2A adenosine receptors is low or negli-
gible) was 4.6 ( 0.17. A similar distribution was
observed on coronal brain slices obtained with a phos-
phor-imaging technique, indicating that 7 is mainly
retained at the level of basal ganglia (Figure 2).
HPLC chromatograms of plasma extracts showed the
presence of three main radioactive compounds: one
but also in lung and liver, reaching its maximum uptake
5 min after injection. Tracer accumulation was lower
in the heart, where adenosine receptors are mainly
represented by the A1 subtype (Table 1). In the remain-
ing organs radioactivity concentration ranged between
0.1% and 0.2% ID/g. When injected in rats with an
intravenous pulse, 7 was rapidly cleared from plasma,
at 15 min after tracer injection, radioactivity concentra-
tion being 0.13 ( 0.020% ID/g. The tracer preferentially
distributed in plasma, as indicated by its plasma-to-
blood ratio which was always >1 during the experimen-
tal time.
Our results clearly showed that [11C]SCH442416
permeates the BBB and accumulates in the brain where

4362 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Communications to the Editor
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails. This information is available free of charge via the
Internet at http://pubs.acs.org.
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F igu r e 3. Time course of radioactive metabolites and parent
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more lipophilic compound (tR ) 9.5 min) that corre-
sponds to unmetabolized 7, as confirmed by the HPLC
chromatogram of the cold SCH442416 used as a refer-
ence standard, and two more hydrophilic compounds (tR
) 4.5 and 2.5 min, respectively) probably attributable
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slow, and the plasma concentration of the injected tracer
accounted for more than 40% of total plasma activity
after 60 min (Figure 3).
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At the time of the higher cerebral uptake (15 min
after injection), HPLC chromatograms of acetonitrile
brain extracts demonstrated the presence of only one
metabolite with a retention time similar to that of the
first plasma metabolite (tR ) 2 min) which represented
only 6% of the total extractable activity in the cerebel-
lum and less than 1% in the striatum.
Con clu sion s. In conclusion, the regional distribution
in brain and periphery, the good signal-to-noise ratio
observed between 5 and 15 min after injection, and the
low occurrence of radioactive metabolites all suggest
that [11C]SCH442416 is applicable as the first non-
xanthine ligand suitable for the in vivo imaging of A2A
adenosine receptors using PET. In addition, the data
obtained from the binding experiments showed a higher
affinity of the title compound for human A2A adenosine
receptors vs rat receptors (0.048 nM vs 0.5 nM). Due to
the lower Ki values found in human cell lines, a higher
striatum-to-cerebellum ratio should be expected for the
in vivo measurement of the A2A receptor in human brain
using PET.
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