Development of spin-labeled probes for adenosine receptors

Article abstract of DOI:10.1021/jm049513x

Functionalized xanthine derivatives bearing a nitroxide moiety at the 3- or 8-position were synthesized as electron paramagnetic resonance (EPR) probes. The 8-cyclopentyl-1-propylxanthine derivative 4, spin-labeled at N3 by substitution with a nitroxide-bearing dihydropyrrole moiety, was a potent and selective A₁ adenosine receptor antagonist (K_i for A ₁ 5.5 nM, 1600-fold selectivity vs A_2A, > 200-fold vs A₂B, and 310-fold vs A₃ adenosine receptors). 8-(1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)-1,3-dipropylxanthine 10 (K_i for A₁ 8.2 nM) was similarly potent and selective, while 8-(1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl)-1,3-dipropylxanthine 11 (K_i for A₁ 160 nM) exhibited significantly lower affinity for A₁ adenosine receptors. 8-[4-(((1-Oxyl-2,2,6,6- tetramethylpiperidin-4-yl)amino)-2-oxoethoxy)phenyl]-1-propylxanthine 14, a 3-unsubstituted xanthine derivative, was found to be a potent A_2B adenosine receptor antagonist (K_i for A_2B 48 nM) but also exhibited high affinity for A₁ receptors (K_i for A ₁ 15.7 nM). An X-ray structure of compound 10 was obtained, confirming the proposed structure. The novel spin-labeled A₁- selective or A₁/A_2B-nonselective adenosine receptor antagonists may become useful probes for biophysicochemical investigations of adenosine receptors in their membrane environment.

Full text of DOI:10.1021/jm049513x

2108
J. Med. Chem. 2005, 48, 2108-2114
Development of Spin-Labeled Probes for Adenosine Receptors†
Janez Ilasˇ,‡,§ Slavko Pecˇar,§,| Jo¨rg Hockemeyer,‡ Harald Euler, Armin Kirfel, and Christa E. Mu¨ller*,‡
Pharmaceutical Institute, Pharmaceutical Chemistry Poppelsdorf, University of Bonn, Kreuzbergweg 26,
D-53113 Bonn, Germany, Faculty of Pharmacy, University of Ljubljana, Asˇkercˇeva 7, SI-1000 Ljubljana, Slovenia,
J. Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia, and Mineralogisch-Petrologisches Institut, University of Bonn,
Poppelsdorfer Schloss, D-53115 Bonn, Germany
Received June 21, 2004
Functionalized xanthine derivatives bearing a nitroxide moiety at the 3- or 8-position were
synthesized as electron paramagnetic resonance (EPR) probes. The 8-cyclopentyl-1-propyl-
xanthine derivative 4, spin-labeled at N3 by substitution with a nitroxide-bearing dihydropyrrole
moiety, was a potent and selective A1 adenosine receptor antagonist (Ki for A1 5.5 nM, 1600-
fold selectivity vs A2A, >200-fold vs A2B, and 310-fold vs A3 adenosine receptors). 8-(1-Oxyl-
2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)-1,3-dipropylxanthine 10 (Ki for A1 8.2 nM) was
similarly potent and selective, while 8-(1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl)-1,3-dipropyl-
xanthine 11 (Ki for A1 160 nM) exhibited significantly lower affinity for A1 adenosine receptors.
8-[4-(((1-Oxyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)-2-oxoethoxy)phenyl]-1-propylxanthine
14, a 3-unsubstituted xanthine derivative, was found to be a potent A2B adenosine receptor
antagonist (Ki for A2B 48 nM) but also exhibited high affinity for A1 receptors (Ki for A1 15.7
nM). An X-ray structure of compound 10 was obtained, confirming the proposed structure.
The novel spin-labeled A1-selective or A1/A2B-nonselective adenosine receptor antagonists may
become useful probes for biophysicochemical investigations of adenosine receptors in their
membrane environment.
Introduction
Functionalized receptor ligands have been extensively
used for elucidating those processes, including radiola-
beled compounds,9 ligands bearing photoaffinity labels,10
and fluorescence-labeled probes.11,12 To the best of our
knowledge, spin-labeled ligands as electron paramag-
netic resonance (EPR) probes have not been developed
for ARs so far.
Stable free radicals of the nitroxide type can be
utilized as spin labels and spin probes in electron
paramagnetic resonance (EPR) spectroscopy. The fine
structure of the resulting spectra provides information
about the environment of the radical moiety and allows
a description of the dynamical, structural, and redox
properties of its surroundings.13 For this reason spin-
labeling can also contribute to the elucidation of the
structure, the dynamics, and thus the function of
biomolecules such as proteins and nucleic acids. The
microgeography of many active sites of enzymes could
be described by EPR spectroscopy long before their
X-ray structures were obtained.14-17
The successful application of spin-labeling methods
in enzyme and receptor studies strongly depends on a
properly designed spin probe molecule. High biological
activity and/or binding affinity of the paramagnetic
compound are key requirements.
Nitroxide radicals are paramagnetic compounds.18 By
steric hindrance it is possible to reduce their reactivity.
Shielding can be achieved by inserting the nitroxide
moiety into a 2,2,5,5- or 2,2,6,6-tetramethylated five-
or six-membered nonaromatic ring system (e.g., piperi-
dine, pyrrolidine, or even dihydropyrrole). These stable
radicals can be isolated in pure form and stored for up
to several years without any decomposition. In addition,
they can be functionalized in order to provide bifunc-
Adenosine is a building block of many biologically
relevant molecules such as ATP and nucleic acids. In
addition, it has important regulatory functions mediated
through adenosine receptors (ARs).1,2 So far four ARs
(A1, A2A, A2B, and A3) have been characterized and
cloned from several mammalian species. A1 and A2A
receptors are so-called “high-affinity” subtypes, acti-
vated by adenosine in nanomolar concentrations, while
A2B and A3 receptors (“low-affinity” subtypes) are usu-
ally activated by adenosine only at micromolar con-
centrations.1-4 ARs are important new drug targets. All
ARs are G protein-coupled receptors (GPCR), and being
membrane proteins they are not easily amenable to
crystallization. Therefore an exact structure elucidation
through X-ray diffraction has not been achieved yet. The
three-dimensional architecture of only two related
membrane proteins, bacteriorhodopsin5 and (bovine)
rhodopsin,6 has been revealed. These proteins have been
used as templates for the modeling of GPCRs. To
characterize the adenosine binding site and receptor
structure, different indirect approaches have been used,
including chemical modification of histidine residues
and site-directed mutagenesis.7,8 However, many char-
acteristics of ARs still have to be revealed, such as the
exact process of ligand interaction with the cell mem-
branes and the processes and mechanisms of receptor
ligand recognition and signal transduction.
† Dedicated to the memory of Dr. Paul Janssen.
* Address correspondence to this author: phone +49-228-73-2301;
fax +49-228-73-2567; e-mail christa.mueller@uni-bonn.de.
‡ Pharmaceutical Institute, Pharmaceutical Chemistry Poppelsdorf,
University of Bonn.
§ University of Ljubljana.
| J. Stefan Institute.
Mineralogisch-Petrologisches Institut, University of Bonn.
10.1021/jm049513x CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/11/2004

Spin-Labeled Probes for Adenosine Receptors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2109
Scheme 1a
a Reagents, conditions, and yields: (a) K2CO3, DMF, rt, 16 h (55% 3); (b) THF/HMDS, pressure tube, microwave (MW) 100 W, 140 °C,
20 min (28% 4).
Scheme 2a
a Reagents, conditions, and yields: (a) EDC, methanol, rt 2 days (67% 8, 32% 9); (b) 1 M NaOH/dioxane (1:1), reflux 45 min (75% 10,
72 % 11).
tional compounds that can serve to introduce the radical
functionality into a diamagnetic compound. The result-
ing spin-labeled probes can be very powerful molecular
tools to study the interaction of ligands with receptors
or enzymes by EPR spectroscopy.13
Our goal was to develop EPR probes for (a) the A1
AR, which is the best investigated AR subtype, and (b)
for the A2B AR, still the most “enigmatic” AR subtype.19
Xanthine derivatives are the most intensively investi-
gated class of AR antagonists. Structure-activity rela-
tionships are well-known. The A1 AR receptor tolerates
both bulky substituents such as cyclopentyl, phenyl, and
3-noradamantyl in the 8-position and large substituents
(e.g., phenethyl) in the 3-position.4 These positions may
therefore be suitable for substitution with bulky nitrox-
ide moieties without significant influence on binding
affinity.
On the other hand, A2B-selective AR antagonists have
been described in a series of 3-unsubstituted xanthines
bearing a para-substituted 8-phenyl residue.20 To obtain
spin-labeled probes for A2B ARs, we therefore planned
to attach a nitroxide-bearing moiety to 1-propyl-8-
phenylxanthine via a spacer group. The synthesis and
physicochemical and biological characterization of four
new spin-labeled xanthine derivatives is described, two
of which exhibit high affinity and selectivity for A1 ARs
and one of which is a potent but nonselective A1/A2B
receptor ligand.
carbodiimide hydrochloride (EDC) to yield compound
1.22 The N1-atom of the uracil derivative 1 could easily
be alkylated in DMF in the presence of potassium
carbonate with the spin-labeled alkyl bromide 223 at
room temperature, yielding the spin-labeled uracil
derivative 3. Ring closure reactions under elimination
of water to yield the final xanthine derivatives are
usually performed either in aqueous alkaline solution,
or by heating with the nonpolar 1,1,1,3,3,3-hexamethyl-
disilazane (HMDS) as a solvent and condensing agent.
If polar reactants are used, conventional heating with
HMDS (bp 126 °C) often requires long reaction times
and high reaction temperatures.24 For the ring closure
reaction of 3 to the corresponding spin-labeled xanthine
derivative 4, a mixture of HMDS and tetrahydrofuran
(THF) in a microwave-assisted reaction (100 W, 140 °C,
20 min) was found to give the best results, with a yield
of 28% (Scheme 1).
Xanthine derivatives 10 and 11, spin-labeled in the
8-position, were prepared starting from 5,6-diamino-1,3-
di-n-propyluracil 7. Coupling of 7 with carboxylic acid
derivatives of stable nitroxide radicals 523,25,26 and 6,27
respectively, in methanol at room temperature with
EDC as a condensing agent led directly to the uracil
derivatives 8 and 9. After a reaction time of 2 days
under argon, 8 could be obtained in good and 9 in
moderate yields (Scheme 2).
The ring closure reaction of 8 and 9 to their corre-
sponding xanthine derivatives 10 and 11 resulted in
better yields when carried out in a mixture of aqueous
sodium hydroxide solution and dioxane (1:1) under
reflux conditions as compared to the condensation with
HMDS. After a reaction time of only 45 min, highly pure
10 and 11 could be obtained in yields of more than 70%
Synthesis
1-Propyl-8-cyclopentylxanthine derivative 4, labeled
in the 3-position, was prepared by condensation of 3-n-
propyl-5,6-diaminouracil21 and cyclopentyl carboxylic
acid in the presence of (3-dimethylaminopropyl)ethyl-

2110 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Ilasˇ et al.
Scheme 3a
a Reagents, conditions, and yield: (a) EDC, DMAP, DMF/CH2Cl2, rt, 6 days, (14% 14).
(Scheme 2). In the case of 10, crystallization from
dichloromethane/diethyl ether provided sufficiently good
crystals for X-ray analysis.
1,8-Disubstituted xanthine derivative 14, spin-labeled
at the 8-residue, was synthesized starting from xanthine
derivative 12.20 The carboxylic group of xanthine de-
rivative 12 was initially transferred to the correspond-
ing acid chloride, which was subsequently condensed
with the spin-labeled amine 13.18,28 However, this
reaction sequence resulted in only low yields of the
desired compound 14 (<5%) associated with the forma-
tion of a wide range of side products. Coupling of 12
and 13 in the presence of EDC and 4-(N,N-dimethyl-
amino)pyridine (DMAP) resulted in better yields of 14
of up to 14% after a reaction time of 6 days with a 2-fold
excess of the amine 13 (Scheme 3).20 Compound 14 was
poorly soluble in usual organic solvents, with the
exception of dimethylformamide (DMF) and dimethyl
sulfoxide (DMSO). Due to its low solubility, 14 was
isolated from the reaction mixture by extraction with
large quantities of ethyl acetate.
Figure 1. Structure of the spin-labeled xanthine derivative
10 in the crystal. Atoms are drawn by spheres of arbitrary
radius.
Analytical Characterization of Spin-Labeled
Ligands. For the new compounds 4, 10, 11, and 14,
EPR spectra were recorded to prove the presence of the
nitroxide group in the molecules and to determine the
purity of the compounds. The EPR spectra of freshly
prepared 1 mM solutions in dichloromethane, recorded
at room temperature, showed the expected three-line
spectra with three hyperfine lines for the spin-labeled
compounds. 14N Hyperfine coupling constants (aN) in the
range of 14.76-15.93 G could be extracted for 4, 10, 11,
and 14. Further characterization of the final spin-
labeled compounds was achieved by thin-layer chroma-
tography, infrared spectroscopy, and mass spectrometry
(electrospray ionization mode). Elemental analysis and
melting points were also determined. An X-ray structure
of compound 10 provided further evidence for the
proposed structure (Figure 1).
X-ray Structure Analysis of Compound 10. Since
radicals cannot be investigated by standard NMR
techniques, structural data gained by X-ray analysis
were required. These data will also be valuable for the
interpretation of data obtained by biophysicochemical
investigations and subsequent molecular modeling stud-
ies of receptor-ligand complexes.
The crystal structure of 10 exhibits one molecule with
normal intramolecular bond lengths per asymmetric
unit. The four molecules per unit cell form two dimers
that are held together by van der Waals forces. Each
dimer consists of two molecules related by inversion
symmetry and bonded via the hydrogen bonds N7-H7‚‚‚
O2 (corresponding to N7-H‚‚‚OdC6 according to the
numbering in Scheme 2: N7-H7 ) 0.917(4) Å, H7‚‚‚
O2 ) 1.877(9) Å, N7-H7-O2 ) 174.40(1)°; see Figure
1). The planar central parts of all dimers are oriented
perpendicular to both (100) and alternatingly to [012]
and [0-12], respectively. The xanthine ring system is
almost planar with an rms value of 0.025 Å for the atom
deviations from the least-squares plane defined by N1,
C2, N3, C4, C5, C6, N7, C8, N9, O1, O2, and H7. The
plane of the pyrroline-1-oxyl ring, defined by C10, C11,
C12, N13, C14, O3, and H11 (rms ) 0.035 Å), is tilted
against the main plane by 14.3(3)°. Detailed crystal data
are given in the Supporting Information.
Biological Activity
The spin-labeled xanthine derivatives were investi-
gated in radioligand binding studies at A1, A2A, A2B, and
A3 adenosine receptors by use of [3H]-2-chloro-N6-
cyclopentyladenosine ([3H]CCPA), [3H]-(E)-3-(3-hydroxy-
propyl)-8-[2-(3-methoxphenyl)vinyl]-7-methyl-1-prop-2-
ynyl-3,7-dihydropurine-2,6-dione ([3H]MSX-2), [3H]-8-
[[4-(2-hydroxyethylamino)-2-oxoethoxy]phenyl]-1-
propylxanthine ([3H]PSB-298), and [3H]-2-phenyl-8-
ethyl-4-methyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-
i]purin-5-one ([3H]PSB-11), respectively, as A1, A2A, A2B
,
and A3 radioligands. Native brain tissues were used for
A1 (cortex) and A2A (striatum) assays. Human recom-
binant receptors were used for A2B and A3 receptor
assays. It had been shown that species differences for
A1 and A2A receptors between rat and human receptors
are moderate; therefore, we assumed that the rat data
for these subtypes would be a good estimate for the
human receptors as well.4
The results of the radioligand binding studies at ARs
are presented in Tables1 and 2 and compared with the
data of non-spin-labeled analogues. Compound 4 showed
high affinity for the A1 AR and at the same time it was
selective for A1 vs A2A ARs by 1600-fold. Its A1 affinity

Spin-Labeled Probes for Adenosine Receptors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2111
Table 1. Adenosine Receptor Affinities and A1 Selectivities of 3- and 8-Spin-Labeled Xanthine Derivatives in Comparison with
Analogous Standard Compounds
Ki ( SEM (nM)
A1 affinity,
A2A affinity,
A2B affinity,
A3 affinity,
compd
R1
R3
R8
rat vs [3H]CCPA rat vs [3H]MSX-2 human vs [3H]PSB-298
human vs [3H]PSB-11
3- or 8-Spin-Labeled Xanthines
4
10
11
propyl nitroxide-
bearing moiety
cyclopentyl
5.47 ( 0.92
8.23 ( 1.30
160 ( 6
8780 ( 1340
3800 ( 500
6750 ( 750
>1000 (23 ( 2% inha)
3100 ( 1400
1700 ( 200
propyl propyl
nitroxide-
∼10 000 (47 ( 5% inha)
bearing moiety
nitroxide-
propyl propyl
>10 000 (25 ( 0% inh.a) ∼10 000 (45 ( 5% inh.a)
bearing moiety
1,3,8-Trialkylated Xanthines for Comparison
15
16
propyl benzyl
propyl propyl
cyclopentyl
cyclopentyl
8.7b
0.9
511c
470
ndd
51e
54.6
795
a inh ) percent inhibition of radioligand binding at the indicated concentration (1 or 10 µM). b Versus [3H]R-PIA. c Versus [3H]NECA.
d Not determined. e Versus [3H]ZM-241385.
Table 2. Adenosine Receptor Affinities of A2B-Selective Xanthine Derivatives and Their Spin-Labeled Analogue 14
Ki ( SEM (nM)
A1 affinity,
A2A affinity,
A2B affinity,
A3 affinity,
compd
14
R′
rat vs [3H]CCPA
rat vs [3H]MSX-2
human vs [3H]PSB-298
human vs [3H]PSB-11
Spin-Labeled Xanthine
1270 ( 530
propyl
15.7 ( 0.7
48 ( 0.7
350 ( 100
Structurally Related Xanthines for Comparison20
17
18
19
propyl
butyl
butyl
24
41
18
365
479
290
10a
5.3a
5.5a
ndb
676
ndb
a Versus [3H]ZM-241380. b Not determined.
was similar to that of the related 3-benzyl-5-cyclopentyl-
1-propylxanthine (15; Ki for A1 ) 8.7 nM),29 showing
that the replacement of the benzyl group by a 1-oxyl-
2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-ylmethyl
group had no significant effect on the affinity. In
comparison with DPCPX (16),20 which bears a propyl
group in the 3-position, the larger 1-oxyl-2,2,5,5-tetra-
methyl-2,5-dihydro-1H-pyrrol-3-ylmethyl-substituted de-
rivative 4 was about 6-fold less potent at A1 ARs, but
similarly or even more selective.
1,3-Dipropylxanthines spin-labeled in position 8 with
either a 1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyr-
rol-3-yl group (10) or a 1-oxyl-2,2,6,6-tetramethylpip-
eridin-4-yl group (11) were not as potent as their non-
spin-labeled analogue DPCPX (16). The pyrrolyl nitroxide
moiety led to a 9-fold decrease in affinity to A1 ARs of
compound 10 in comparison with xanthine derivative
16, which is provided with a cyclopentyl group in
position 8.
Compound 11 with a larger piperidinyl nitroxide
residue was 19-fold less potent than compound 10
(pyrrolyl nitroxide group) and 177-fold less potent than
DCPCX. It is evident that the 1-oxyl-2,2,6,6-tetrameth-
ylpiperidin-4-yl group is already too bulky and the
volume of the piperidine ring in this region of 11 is too
large to fit well into the A1 receptor ligand binding site.
The polarity of the nitroxide group may also be unfavor-
able for binding to the lipophilic pocket of the receptor.
Compound 10 was found to be a potent and highly
selective spin-labeled A1 antagonist (462-fold vs A2A,
377-fold vs A2B, >1000-fold vs A3), while 11 exhibited
only moderate affinity (Ki for A1 ) 160 nM) but was also
quite selective (>40-fold) versus all other AR subtypes
(Table 1).

2112 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Ilasˇ et al.
IR (KBr) 3320 (NH2), 2969, 2871, 1700, 1634 cm-1; MS (ESI
mode) 434.5 (M + H)+; Rf (ethyl acetate) ) 0.35.
Compound 14 was designed as an A2B antagonist. As
a matter of fact, 14 showed reasonably high affinity to
A2B ARs (Ki ) 48 nM); however, it had even somewhat
more affinity toward A1 ARs (Ki ) 16 nM) and is
therefore a mixed A1/A2B ligand (Table 2). Compound
14 exhibited good selectivity versus A2A (81-fold) and
moderate selectivity versus A3 ARs (7-22-fold). In
comparison with structurally related xanthine deriva-
tives (17, 18, and 19),20 spin-labeled compound 14 was
somewhat weaker at A2B and more potent at A1 ARs,
resulting in a loss of A2B selectivity (see Table 2).
8-Cyclopentyl-3-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-
1H-pyrrol-3-ylmethyl)-1-propylxanthine (4). Compound 3
(200 mg, 0.46 mmol) was suspended in 3 mL of THF in a 10
mL pressure tube. Finely ground ammonium sulfate (0.05 g)
and 2 mL (10 mmol) of HMDS were added, forming a viscous
yellow suspension. After microwaving (100 W, 140 °C, 20 min),
2 mL of methanol was added to the warm (50 °C) mixture.
The solvent was removed under reduced pressure and the
residue was purified by column chromatography (petrol ether/
diethyl ether ) 3:1), yielding 54 mg (28%) of a pale-red oil:
IR (KBr) 3168 (NH), 2968, 2872, 1712, 1652, 1598 cm-1; MS
(ESI mode) 415.3 (M + H)+; aN ) 14.95 G. Anal. (C22H32N5O3)
H; C, calcd 63.75, found, 62.09; N, calcd 16.89, found 15.73. Rf
(ethyl acetate) ) 0.79.
Reaction of 5,6-Diamino-1,3-dipropyluracil (7) with
Spin-Labeled Carboxylic Acids 5 and 6. To a mixture of
0.45 g (2.0 mmol) of 5,6-diamino-1,3-dipropyluracil 7 and 2.05
mmol of 523,25,26 or, respectively, 6 in methanol (10 mL) was
added EDC (0.4 g, 2.1 mmol). The mixture was stirred for 2
days under argon and protected from light. The solvent was
removed under reduced pressure and the residue was purified
by column chromatography (eluent dichloromethane/methanol,
92:8). The product was obtained as a yellow oil 8 or a red oil
9, respectively.
Conclusions
The synthesis of four spin-labeled xanthine deriva-
tives has been achieved. Two of the compounds (4 and
10), bearing spin-labels in different positions exhibit
high affinity in the low nanomolar concentration range
for A1 ARs and are highly selective versus all other AR
subtypes (.100-fold). One compound (14) was a potent
mixed A1/A2B ligand. The new compounds should be
suitable probes for electron paramagnetic resonance
studies of A1 or A2B ARs, respectively. Such probes will
allow biophysical studies that may contribute to the
understanding of the membrane receptor structures and
their interaction with ligands.
6-Amino-5-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-
pyrrol-3-yl)carboxamido-3-propyluracil (8). Yellow oil
(yield 67%): IR (KBr) 3329 (NH2), 2972, 2934, 2875, 1702, 1622
cm-1; MS (ESI mode) 393.4 (M + H)+; Rf (eluent dichlo-
romethane/methanol, 9:1) ) 0.32.
Experimental Section
6-Amino-5-(1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl)-
carboxamido-3-propyluracil (9). Red oil (yield 32%): IR
(KBr) 3210 (NH2), 2972, 2933, 2877, 1644 cm-1; MS (ESI mode)
409.4 (M + H)+; Rf (eluent dichloromethane/methanol, 9:1) )
0.38.
Preparation of 1,3-Dipropylxanthines with a Spin-
Labeled Substituent in the 8-Position (10, 11). Compound
8 or 9, respectively (0.7 mol), was dissolved in a mixture of 1
M aqueous NaOH solution (8 mL) and dioxane (8 mL) and
subsequently heated under reflux for 45 min. The mixture was
cooled to room temperature, acidified with ca. 10 mL of a 1 M
aqueous HCl solution, and left overnight to complete the
precipitation. The product was filtered under reduced pressure,
washed with water, and dried at 70 °C.
8-(1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-
yl)-1,3-dipropylxanthine (10). Yellow crystals (yield 75%):
mp 188 °C; IR (KBr) 3148 (NH), 2969, 2933, 2872, 1702, 1651,
1597 cm-1; MS (ESI mode) 375.4 (M + H)+; aN ) 14.76 G. Anal.
(C19H28N5O3) C; H, calcd 7.54, found 7.48; N, calcd 18.70, found
18.36. Rf (ethyl acetate) ) 0.63.
Crystal Structure Data. Yellow single crystals of 10 (FW
) 374.46 for C19H28N5O3) were obtained from a dichloro-
methane/diethyl ether (1:1) solution.
For a crystal with dimensions of 0.35 × 0.3 × 0.2 mm, the
structure was established by X-ray single-crystal diffractom-
etry (Figure 1). Diffraction data were collected with a Rigaku
AFC6R four-circle single-crystal diffractometer, equipped with
a graphite monochromator, using Mo KR radiation from a
rotating anode generator. Compound 10 crystallizes in the
monoclinic space group P21/c (no. 14). Unit cell dimensions
were determined from 25 reflections (7.7° < θ < 11.9°): a )
13.051(2) Å, b ) 17.505(3) Å, c ) 9.188(1) Å, â ) 103.35(1)°, V
) 2042.3(5) Å3, Z ) 4, Dc ) 1.218 g/cm3, F(000) ) 804.
Intensities of 7482 reflections with 1.98° < θ < 20.04° were
measured by ω scans. The data were corrected for the
variations of three standard reflections monitored every 50
reflections. After data reduction, the structure was solved by
direct methods with SHELXS-97 and refined on F2 with
SHELXL-97. The non-hydrogen atoms were refined with
anisotropic displacement parameters, all hydrogen atoms
located by difference Fourier calculations with isotropic dis-
placement parameters. The refinement on a total of 1917
independent reflections varying 357 parameters converged at
All commercially available reagents were obtained from
various producers (Acros, Aldrich, Fluka, Merck, Sigma) and
used without further purification. Solvents were used without
additional purification or drying, unless otherwise noted. The
reactions were monitored by thin-layer chromatography (TLC)
on aluminum sheets with silica gel 60 F245 (Merck). Column
chromatography was carried out with silica gel 0.060-0.200
mm, pore diameter ca. 6 nm. EPR spectra were recorded at
room temperature on an EPR spectrometer (Bruker ESP 300)
at the Institute Jozˇef Stefan, Ljubljana, Slovenia. Mass spectra
were recorded on an API 2000 (Applied Biosystems, Darm-
stadt, Germany) mass spectrometer at the Institute of Phar-
maceutical Chemistry Poppelsdorf, University of Bonn, Ger-
many, or on a Varian-MAT 311 A mass spectrometer at the
Institute Jozˇef Stefan, Ljubljana, Slovenia. IR spectra were
recorded on a Perkin-Elmer 1600 FTIR spectrometer at the
Institute of Pharmaceutical Chemistry Poppelsdorf, University
of Bonn, Germany. Elemental analyses were performed by the
Institute of Pharmaceutical Chemistry Endenich, University
of Bonn, Germany. The melting points were determined on a
Wepa Apotec (Wepa, Ho¨hr-Grenzhausen, Germany) capillary
melting point apparatus. The room-temperature X-ray single-
crystal analysis was carried out on an AFC6R single-crystal
diffractometer (Rigaku) by the Mineralogical-Petrological
Institute, University of Bonn, Germany.
Syntheses of Stable Nitroxide Radicals. 1-Oxyl-3-car-
boxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole (5),18 1-oxyl-
4-carboxyl-2,2,6,6-tetramethylpiperidine (6)27 and 1-oxyl-4-
amino-2,2,6,6-tetramethylpiperidine (13)18,28 were prepared in
multistep syntheses from commercially available 2,2,6,6-tetra-
methylpiperidin-4-one as described.18,27,28 1-Oxyl-3-bromo-
methyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole (2) was pre-
pared from 1-oxyl-3-carboxyl-2,2,5,5-tetramethyl-2,5-dihydro-
1H-pyrrole (5) as described.23,25,26
6-Amino-1-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-
pyrrol-3-ylmethyl)-5-cyclopentanoylamido-3-propyl-
uracil (3). Compounds 122 (320 mg, 1.15 mmol), 223 (280 mg,
1.20 mmol, 1.05 equiv), and anhydrous K2CO3 (240 mg, 1.75
mmol, 1.5 equiv) were suspended in dry DMF (13 mL). The
suspension was stirred overnight at room temperature. The
solvent was removed under reduced pressure and the residue
was purified on a silica gel column with ethyl acetate/
ethyldimethylamine (98:2), yielding 238 mg (55%) of a red oil:

Spin-Labeled Probes for Adenosine Receptors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2113
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R1 ) 0.0635 and wR2 ) 0.0891 and for 1424 reflections with I
> 2σ(I) at R1 ) 0.0364, wR2 ) 0.0805, GoF ) 1.047. Final
residual electron density features were between -0.12 and
+0.11 e-/Å3.
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8-(1-Oxyl-2,2,6,6-tetramethylpiperidin-4-yl)-1,3-dipro-
pylxanthine (11). Red crystals could be obtained by recrys-
tallization from CH2Cl2/diethyl ether (ca. 1:1): yield 72%, mp
192 °C; IR (KBr) 3193 (NH), 2972, 2938, 2875, 1703, 1661 cm-1
;
MS (ESI mode) 391.3 (M + H)+; aN ) 15.93 G; Anal.
(C20H32N5O3) H; C, calcd 61.52, found 61.22; N, calcd 17.93,
found 17.66. Rf (ethyl acetate) ) 0.57.
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8-[4-(((1-Oxyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)-
2-oxoethoxy)phenyl]-1-propylxanthine (14). A solution of
1220 (0.51 g, 1.48 mmol), 1318,28 (0.51 g, 2.98 mmol, 2.0 equiv),
EDC (0.63 g, 3.28 mmol, 2.2 equiv), and DMAP (0.1 g, 0.82
mmol, 0.55 equiv) in a mixture of DMF (160 mL) and CH2Cl2
(80 mL) was stirred for 6 days at room temperature under
argon. The mixture was evaporated to dryness under reduced
pressure, and the residue was divided into three portions. Each
portion was dissolved in ethyl acetate (350 mL). The solution
was washed 5 times with 5% citric acid (70 mL) and then 5
times with a saturated solution of NaHCO3 (70 mL). The ethyl
acetate extracts were combined and dried over MgSO4. The
solvent was removed under reduced pressure, yielding 105 mg
(14%) of a pale red powder, mp > 350 °C; IR (KBr) 2969, 2929,
1716 (CdO), 1640 (CdN), 1055 cm-1; MS (ESI mode) 498.5
(M + H)+; aN ) 15.93 G. Anal. (C25H33N6O5) C; H, calcd 6.68,
found 6.85; N, calcd 16.89, found 16.60. Rf (ethyl acetate) )
0.38.
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thesis, Pharmaceutical Institute, University of Bonn (Germany),
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chim. Biophys. Acta 1995, 1249, 155-160.
Biological Studies. Radioligand competition assays were
performed as recently described,24,30 with rat brain cortical
membrane preparations as a source of A1, rat brain striatal
membrane preparations as a source for A2A ARs, and mem-
brane preparations containing human A2B or human A3
adenosine receptors stably expressed in CHO or HEK cells as
a source for A2B and A3 receptors, respectively. Radioligands
were obtained from the following sources: [3H]CCPA was from
NEN Life Sciences (54.9 Ci/mmol), and [3H]MSX-2 (85 Ci/
mmol), [3H]PSB-11 (53 Ci/mmol), and [3H]PSB-298 (124 Ci/
mmol) were custom-labeled by Amersham. The precursors
were synthesized in our laboratory as previously described.31-35
(16) Sˇentjurc, M.; Pecˇar, S.; Stojan, J.; Marchot, P.; Radicˇ, Z.; Grubicˇ,
Z. Electron paramagnetic resonace reveals altered active center
gorge of acetylcholinesterase after binding to fasculin to the
peripheral site. Biochim. Biophys. Acta 1999, 38856, 349-358.
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Medicinal chemistry and pharmacology of A2B adenosine recep-
tors. Curr. Top. Med. Chem. 2003, 3, 427-443.
(20) Hayallah, A. M.; Sandoval-Ramirez, J.; Reith, U.; Preiss, B.;
Schumacher, B.; Daly, J. W.; Mu¨ller, C. E. 1,8-Distributed
Xanthine Derivatives: Synthesis of Potent A2B Adenosine Re-
ceptor Antagonists. J. Med. Chem. 2002, 45, 1500-1510.
(21) Mu¨ller, C. E. General Synthesis and Properties of 1-Monosub-
stituted Xanthines. Synthesis 1993, 1, 125-128.
(22) Weyler, S.; Hayallah, A. M.; Mu¨ller, C. E. Versatile, convenient
synthesis of pyrimido[1,2,3-cd]purinediones. Tetrahedron 2003,
59, 47-54.
(23) Hankovszky, H. O.; Hideg, K.; Lex, L. Nitroxyls; VII. Synthesis
and Reactions of Highly Reactive 1-Oxyl-2,2,5,5-tetramethyl-2,5-
dihydropyrrol-3-ylmethyl Sulfonates. Synthesis 1980, 11, 914-
916.
Acknowledgment. We thank Dr. Joachim C. Bur-
biel for many valuable contributions as well as Ms.
Sonja Hinz and Mr. Dieter Baumert for performing the
radioligand binding assays. J.I. was supported by the
European Commission within the ERASMUS program.
C.E.M. is grateful for the support by the Fonds der
Chemischen Industrie and the BMBF.
Supporting Information Available: Details on the X-ray
analysis of compound 10. This material is available free of
charge via the Internet at http://pubs.acs.org.
(24) Hockemeyer, J.; Burbiel, J. C.; Mu¨ller, C. M. Multigram-Scale
Syntheses, Stability, and Photoreactions of A2A Adenosine
Receptor Antagonists with 8-Styrylxanthine Structure: Poten-
tial Drugs for Parkinson’s Disease. J. Org. Chem. 2004, 69,
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a Useful Spin Labeled Probe, 1-Oxyl-4-carboxyl-2,2,6,6-tetram-
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