Synthesis of new 2-phenyladenines and 2-phenylpteridines and biological evaluation as adenosine receptor ligands

Article abstract of DOI:10.1002/ardp.200600168

Synthesis and biological assays of a series of 2-phenylpteridine derivatives are described to compare their affinities to adenosine receptors with those of the corresponding adenines, purposely prepared, and 8-azaadenines previously described. This study

Full text of DOI:10.1002/ardp.200600168

Arch. Pharm. Chem. Life Sci. 2007, 340, 81–87
I. Giorgi et al.
81
Full Paper
Synthesis of New 2-Phenyladenines and 2-Phenylpteridines
and Biological Evaluation as Adenosine Receptor Ligands
Irene Giorgi1, Giuliana Biagi1, Oreste Livi1, Michele Leonardi1, Valerio Scartoni1, and
Daniele Pietra2
1 Dipartimento di Scienze Farmaceutiche, Universitꢀ di Pisa, Pisa, Italy
2 Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universitꢀ di Pisa, Pisa, Italy
Synthesis and biological assays of a series of 2-phenylpteridine derivatives are described to com-
pare their affinities to adenosine receptors with those of the corresponding adenines, purposely
prepared, and 8-azaadenines previously described. This study demonstrates that the enlarge-
ment of the five-membered ring of the adenine nucleus to a six-membered one is a modification
that does not allow the molecules to maintain high activity towards adenosine receptors; in
fact, pteridine derivatives did not show themselves to be good adenosine receptor ligands. On
the contrary, N6-cycloalkyl- or N6-alkyl-2-phenyladenines showed a very high affinity and selecti-
vity for A1 adenosine receptors. We demonstrate also that the 9-benzyl substituent is crucial for
conferring high affinity for A3 receptors to molecules having a 2-phenyladenine-like nucleus.
Keywords: A1 Adenosine receptors / A3 Adenosine receptors / 2-Phenyladenines / 2-Phenylpteridines /
Received: October 16, 2006; accepted: November 24, 2006
DOI 10.1002/ardp.200600168
chronotropic, dromotropic and inotropic effects. In the
kidney, activation of A1 receptors causes vasoconstric-
tion, inhibition of renine secretion, diuresis, natriuresis,
and others effects [1]. Extracellular adenosine and adeno-
sine A2A and A3 receptors serve as anti-inflammatory sig-
nals and sensors of excessive inflammatory tissue
damage [2]. A2A receptors play a role in mediating pain
via peripheral sites, inhibiting platelet aggregation and
regulating blood pressure [3]. A2A receptors are also criti-
cally important for the motor stimulant effects of caf-
feine [3, 4] and regulate the inflammatory response [5].
Adenosine A3 receptors have shown important roles in
physiological systems, including protective actions
against myocardial and brain ischemia [6–9], antiproli-
ferative effects [10–12] and airway inflammation [13, 14]
implicating a potential drug target for ischemia, cancer
and asthma.
In the past, we have synthesized and assayed a large
number of ligands of A1, A2A and A3 adenosine receptors,
some of these demonstrated a very good affinity and
selectivity for A1 and A3 subtypes. During our research,
we have studied some modifications of the purine
nucleus to demonstrate the importance of the kind of
atoms present in the various positions of this nucleus. In
Introduction
Adenosine is constitutively present at low levels in the
extracellular space but, in metabolically stressful condi-
tions, such as in injury, ischemia and inflammation, its
extracellular concentration dramatically increases. Ade-
nosine, acting mostly at its receptors, is a powerful sig-
nalling molecule that participates in the regulation of a
wide variety of physiological and pathophysiological pro-
cesses [1].
Four adenosine receptors were identified and cloned:
A1, A2A, A2B and A3 [1]. A1 receptors are widely distributed
in the central nervous system and in peripheral tissue
and mediate diverse biological effects. For example the
role of adenosine as a neuroprotective agent during
hypoxia and ischemic conditions seems to be mediated
by the A1 receptors; these receptors have been implicated
in sedative, anticonvulsant and anxiolytic effects. A1
receptors mediate cardiac depression through negative
Correspondence: Prof. Irene Giorgi, Dipartimento di Scienze Farmaceu-
tiche, Universitꢀ di Pisa, via Bonanno 6, I-56126 Pisa, Italy
E-mail: igiorgi@farm.unipi.it
Fax: +39 050 2219605
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

82
I. Giorgi et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 81–87
Figure 2. Structure of Brimstone butterfly pigments.
Figure 1. Purine-like nuclei, synthesized as adenosine receptor
ligands.
Fig. 1 are shown the purine-like nuclei that have been
synthesized and assayed by us. In a recent paper, we
demonstrated bioisosterism of the 8-azaadenine (nucleus
A, Fig. 1) and adenine nucleus [15]; instead, other modifi-
cations (nucleus B, C, D, Fig. 1) caused a decrease of affi-
nity of the compounds with respect to the corresponding
adenines. In particular, among all the inactive 1,2,3-tria-
zolo[4,5-d]pyridazines (nucleus B, Fig. 1) [16–18] synthe-
sized, only the 3-benzyl-4-(m-tolylamino)-1,2,3-tria-
zolo[4,5-d]pyridazine-7-one showed good affinity towards
A1 receptors [18] and no 3-benzyl-5-phenyl-7-alkylami-
nothiazolo[4,5-d]pyrimidine-2(3H)-thiones [19] (nucleus
C, Fig. 1) were a good ligand. Regarding 3-deaza-8-azaade-
nines (nucleus D, Fig. 1), this nucleus can be considered
bioisoster of adenine, but also this modification caused a
general lowering of affinity with respect to the corre-
sponding adenines and 8-azaadenines [20].
The pteridine nucleus can also be viewed as a modifica-
tion of the adenine nucleus: the carbon atom in position
8 is substituted by an ethylene group (see Fig. 1). In the
past, other authors also have considered pteridines struc-
turally similar to purines [21], so we decided to evaluate
how this modification could affect the activity of some
active adenosine receptor ligands synthesized now or in
the past.
Figure 3. Pteridine ring system.
nitroxide synthase, dimethylsulfoxide reductase or
xanthine oxidase [25].
In this paper we describe the synthesis and biological
assays of a series of 2-phenylpteridine derivatives, we
compare their affinities toward adenosine receptors with
those of the corresponding adenines, purposely pre-
pared, and 8-azaadenines previously described.
Results and Discussion
Chemistry
Compounds 4–8, 10–12 and 14–15 were synthesized
starting from 4,5-diamino-6-chloro-2-phenylpyrimidine 2
[15] (Scheme 1), obtained from 5-amino-4,6-dichloro-2-
The pteridine nucleus is a very important bicyclic
nucleus present in many natural compounds and is stud-
ied from the chemical and biological points of view. The
natural pteridine pigments can be regarded as the origin
of pteridine chemistry since, more than 100 years ago,
Hopkins focused his attention for the first time on these
compounds [22] on English Brimstone butterfly pigments
(Fig. 2). The components were isolated and structurally
identified by Purrmann [23] in 1940 and the new ring sys-
tem was termed pteridine (Fig. 3) by Wieland [24].
Another group of natural pteridine pigments is present
in the eyes of Drosophila melanogaster and in the dorsal
skin of frogs. More natural substances contain the pteri-
dine nucleus in their molecular structure, such as folic
acid, riboflavine, and the cofactor of some enzymes e. g.
Reagents: i: NH3, 1108C, 24 h; ii: Glyoxal, EtOH/AcOH/H2O, reflux, 1 h; iii: Suitable
amine, HMDS, 1108C, 6 h; iv: Triethylorthoformate, acetic anhydride, 1208C, 90 min;
v: Suitable amine, absolute ethanol, 908C, 9 h; vi: Suitable isocyanate, CH3CN, reflux,
3 h.
Scheme 1. Synthetic pathway of compounds 10–12 and 14–
15.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 81–87
Synthesis of 2-Phenyladenines and 2-Phenylpteridines
83
Table 1. Biological results. The Ki values are the mean € SEM
of four separate assays, each performed in triplicate.
Reagents: i: stirring 1 h, then 2-methylpyridine, reflux, 30 min; ii: THF, Na2S2O4,
NaOH ; iii: glyoxal, EtOH/AcOH/H2O, reflux, 1 h; iv: suitable isocyanate, CH3CN,
reflux, 3 h.
Scheme 2. Synthetic pathway of compounds 19–20.
phenylpyrimidine 1 [26] by a known synthetic pathway
[15]. The diamino derivative 2 was cyclised with glyoxal
(40 wt.% solution in water) in ethanol and glacial acetic
acid: the compound formed was 2-phenyl-4-hydroxy-pter-
idine 3 [27] owing to displacement of the chlorine atom
by a hydroxyl group. Treatment of 3 with 1,1,1,3,3,3-hex-
amethyldisilazane (HMDS) and the suitable amine in the
presence of ammonium sulphate as a Lewis catalyst, gave
compounds 4–8.
The chloro-purine 9 was obtained by cyclisation of the
diamino derivative 2 with triethylorthoformate and
acetic anhydride at reflux temperature as reported in the
literature [15]. Displacement of the chlorine atom of 9
with suitable amines in absolute ethanol gave derivatives
10–12; the same reaction with gaseous ammonia in abso-
lute ethanol gave 2-phenyladenine 13 [28], which was
converted in the 2-phenyl-N6-(substituted)-phenylcarba-
moyladenines 14 and 15 by treatment with the corre-
sponding isocyanates and triethylamine in fresh distilled
THF at reflux temperature.
the percentage of inhibition resulted a60% at 10 lM,
compounds were considered inactive.
Adenine derivatives 10–12 showed a very high affinity
A faster route (Scheme 2) was used to obtain the pheny-
lureidopteridine derivatives 19 and 20. The reaction of towards A1 receptors and much lower or no affinity for
A2A and A3, so they can be considered selective for A1 sub-
type; also compound 10, showing an interesting Ki value
(84.1 nM) for A2A receptors, has a selectivity A2A/A1 of 15.
These compounds were more active than the correspond-
ing 8-azaadenine derivatives synthesized and assayed in
the past [15, 32] (see compounds III-V in Table 2). Among
benzamidine chloride with a slight excess of silver salt of
isonitrosomalononitrile gave 4,6-diamino-5-nitroso-2-
phenylpyrimidine 16 as described in the literature [29,
30]. Compound 16 was treated with sodium hydrosul-
phite to give, by reduction of the nitroso group, 4,5,6-tria-
mino-2-phenylpyrimidine 17 [30, 31]. Cyclisation of 17
with aqueous glyoxal furnished 18 [31] which reacted pteridine derivatives, only compound 4 showed good
activity towards A1 receptors (Ki = 66 l 5 nM), compounds
5–7 showed low activity (in the range of micromolar),
and compound 8 was completely inactive for all the
receptor subtypes. These results demonstrated that the
activity of pteridine compounds towards A1 receptors is
dramatically lower with respect to the corresponding
adenine derivatives (10–12, I, II) and also of the corre-
with the suitable isocyanates to give the 1-substituted
phenyl-3-(2-phenyl-pteridin-4-yl)-ureas 19 and 20.
Biological assays
All new compounds obtained were tested in radioligand
binding assays for affinity towards A1, A2A, and A3 adeno-
sine receptors. The results are reported in Table 1; when
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

84
I. Giorgi et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 81–87
Table 2. N6-substituted-2-phenyladenines synthesised and
assayed in the past.
tors, the enlargement of the five-membered ring of ade-
nine nucleus to a six-membered one is a modification
that does not allow the molecules to maintain high activ-
ity towards these receptors. On the contrary N6-
cycloalkyl- or N6-alkyl-2-phenyladenines showed a very
high affinity and selectivity for A1 adenosine receptors.
The affinity values are comparable with those of the 9-
benzylsubstituted analogous ones (N6-cyclopentyl-9-ben-
zyl-2-phenyladenine; Ki value of 12.2 nM, N6-cyclohexyl-9-
benzyl-2-phenyladenine; Ki value of 9.4 nM) [15], demon-
strating that the 9-benzyl substituent is not very impor-
tant for the binding of these molecules to A1 receptors.
On the contrary, comparing biological results of com-
pounds 14 and 15 with the active compounds synthesized
in the past (see Fig. 4) [33], we demonstrate that the 9-ben-
zyl substituent is crucial to confer high affinity for A3
receptors to molecules having a 2-phenyladenine-like
nucleus.
This research has been supported by the Italian MIUR (Minis-
tero Istruzione Universitꢀ Ricerca).
Figure 4. Chemical structure of active 8-azaadenines
Experimental
sponding 8-azaadenine derivatives (Table 2, compounds
III–VII).
Chemistry
Compounds 14, 15, 19 and 20 were synthesized and
assayed on the basis of results recently obtained regard-
ing the high affinity of some 9-benzyl-2-phenyl-N6-substi-
tuted-phenyl-carbamoyl-8-azaadenines for A3 adenosine
receptors (Fig. 4) [33]. Regarding compounds 14 and 15,
we observed that the insertion of a phenylureic function
on the 6-position of 2-phenylpurine leads to molecules
that bind adenosine receptors very weakly. The main
structural difference between these molecules and the
active 8-azaadenines shown in Fig. 4 is the absence of the
benzyl group in the 9-position; this fact should be the rea-
son for the lack of activity of 14 and 15. Also in the case of
compounds 19 and 20, we can see low percentages of
inhibition. For these pteridine derivatives the low activ-
ity could be due to two reasons: the absence of the substi-
tuent in the 8-position of 2-phenylpteridine (correspond-
ing to the 9-position of 8-azaadenines) and the change of
the nucleus structure, from 8-azaadenine to pteridine.
Melting points were determined on a Kofler hot-stage apparatus
(C. Reichert, Vienna, Austria) and are uncorrected. IR spectra in
Nujol mulls were recorded on a Mattson Genesis series FTIR spec-
trometer (Mattson Instruments, USA). 1H-NMR spectra were
recorded on a Bruker AC 200 spectrometer (Bruker Bioscience,
Billerica, MA, USA) in d units from TMS as an internal standard;
the compounds were dissolved in DMSO-d6. Mass spectra data
were obtained with a Hewlett-Packard GC/MS system 5988 (Hew-
lett Packard, Palo Alto, CA, USA). TLC was performed on pre-
coated silica gel F254 plates (Merck, Darmstadt, Germany). Micro-
analyses (C H N) were carried out on a Carlo Erba elemental ana-
lyser (Model 1106; Carlo Erba, Milan, Italy) and were within
l 0.4% of the theoretical values.
4,5-Diamino-6-chloro-2-phenylpyrimidine 2
Starting from 1 [26], compound 2 was obtained as described in
the literature [15].
2-Phenyl-4-hydroxypteridine 3
A mixture of 2 (0.50 g, 2.27 mmol), glyoxal (40 wt.% solution in
water) (5 mL), ethanol (5 mL), water (2.5 mL), and glacial acetic
acid (1.5 mL) was stirred at reflux for 1 h. After cooling, the mix-
ture was diluted with water and extracted with chloroform. The
organic layer was dried over MgSO4, filtered and evaporated to
yield a crude product, which was crystallized from ethanol to
give 3 [27] (0.256 g, 50.5%) as a white solid: m. p. 280–2818C; 1H-
NMR: d = 13.08 (s, 1H, exch), 9.05 (d, J = 2 Hz, 1H, arom), 8.82 (d,
J = 2 Hz, 1H, arom), 8.22 (m, 2H, arom), 7.62 (m, 3H, arom).
Conclusions
This study demonstrates that, as pteridine derivatives
cannot be considered good ligands of adenosine recep-
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 81–87
Synthesis of 2-Phenyladenines and 2-Phenylpteridines
85
8.31 (m, 2H, arom), 7.57 (m, 3H, arom), 4.27 (m, 2H, NH-CH +
exch), 1.68 (m, 10H, (CH2)5).
General procedure to obtain compounds 4–8
A mixture of 3, (0.250 g, 1.12 mmol), HMDS (0.538 g, 3.36 mmol),
ammonium sulphate (5 mg) and the suitable amine (4.0 mmol)
was heated at 1008C for 6 h in a steel bomb; after cooling, the
mixture was diluted with methanol, evaporated at reduced pres-
sure to yield a crude product that was dissolved in HCl 10%
(25 mL) and extracted with chloroform. The organic layer was
dried over MgSO4, filtered, evaporated and the residue was flash-
chromatographed on silica gel using ethyl ether/petroleum
ether 40–608 (85:15).
N6-n-Butyl-2-phenyladenine 12
was synthesized from 9 (0.100 g, 0.43 mmol) using the proce-
dure described for 4 and was obtained as a white solid (0.040 g,
35%): m. p. 222–2238C; 1H-NMR: d = 12.92 (s, 1H, exch), 8.36 (m,
2H, arom), 8.08 (s, 1H, C-H), 7.71 (m, 1H, exch), 7.45 (m, 3H,
arom), 3.63 (m, 2H, NH-CH2), 1.67 (m, 2H, CH2), 1.41 (m, 2H, CH2),
0.92 (m, 3H, CH3).
4-Cyclopentylamino-2-phenylpteridine 4
0.152 g, 46%. 1H-NMR: d = 9.08 (d, J = 2 Hz, 1H, arom), 8.85 (d, J = 2
Hz, 1H, arom ), 8.40 (m, 2H, arom), 7.62 (m, 3H, arom), 4.73 (m, 1
H, exch), 4.38 (m, 1H, NH-CH), 1.56 (m, 8H, (CH2)4).
2-Phenyladenine 13
Starting from 9, compound 13 was obtained as described in the
literature [28].
4-Cyclohexylamino-2-phenylpteridine 5
N6-(4-Fluorophenylcarbamoyl)-2-phenyladenine 14
To a solution of 13 (0.040 g, 0.119 mmol) and triethylamine
(0.019 g, 0.11 mmol) was added dropwise 4-fluorophenylisocya-
nate (0.019 g, 0.14 mmol) and heated at reflux under stirring for
1 h. After cooling, the solid precipitate was filtered and crystall-
ized from ethanol to give 14 as a white solid: m. p. 196–1978C;
1H-NMR: d = 10.92 (s, 1H, exch), 10.28 (s, 1H, exch), 8.67 (s, 1H,
arom), 8.38 (m, 2H, arom), 7.31 (m, 8H, arom + exch). MS: m/z
348 [M+].
0.227 g; 66.5%: m. p. 145–1478C; 1H-NMR: d = 9.08 (d, J = 2 Hz, 1H,
arom), 8.87 (d, J = 2 Hz, 1H, arom), 8.40 (m, 2H, arom), 7.62 (m,
3H, arom), 4.73 (m, 1 H, exch), 4.38 (m, 1H, C-H), 1.56 (m, 10H,
(CH2)5).
4-n-Butylamino-2-phenylpteridine 6
0.162 g, 52%: 1H-NMR: d = 9.08 (d, J = 2 Hz, 1H, arom), 8.78 (d, J = 2
Hz, 1H, arom), 8.51 (m, 2H, arom), 7.56 (m, 3H, arom), 3.71 (m,
3H, N-CH2 + exch), 1.72 (m, 2H, CH2), 1.39 (m, 2H, CH2), 0.952 (m,
3H, CH3).
N6-(4-Trifluoromethylphenylcarbamoyl)-2-phenyladenine
15
4-[(R)-(1-Phenyl-ethyl)]amino-2-phenyl-pteridine 7
0.194 g, 53%: 1H-NMR: d = 9.28 (d, J = 7.8 Hz, 1H, exch), 9.08 (d, J =
2 Hz, 1H, arom), 8.80 (d, J = 2 Hz, 1H, arom), 8.47 (m, 2H, arom),
7.41 (m, 8H, arom), 5.71 (m, 1H, CH-CH3), 1.69 (d, J = 6.8 Hz, 3H,
CH3).
was synthesized from 13 (0.040 g, 0.10 mmol) using the proce-
dure described for 14 to give pure 15 (0.020 g, 54.5%) as a white
solid: m. p. 198–1998C; 1H-NMR: d = 11.09 (s, 1H, exch), 10.51 (s,
1H, exch), 8.68 (s, 1H, arom), 8.38 (m, 2H, arom), 7.47 (m, 8H,
arom + exch). MS: m/z 398 [M+].
4-[(S)-(1-Phenyl-ethyl)]amino-2-phenyl-pteridine 8
0.274 g, 75%: 1H-NMR: d = 9.33 (d, J = 7.8 Hz, 1H, exch), 9.09 (d, J =
2 Hz, 1H, arom), 8.82 (d, J = 2 Hz, 1H, arom), 8.51 (m, 2H, arom),
7.41 (m, 8H, arom), 5.72 (m, 1H, CH-CH3), 1.69 (d, J = 6.8 Hz, 3H,
CH3).
4,6-diamino-5-nitroso-2-phenylpyrimidine 16
Compound 16 was obtained as described in the literature [29].
4,5,6-Triamino-2-phenylpyrimidine 17
To an iced and stirred suspension of 1.07 g (5 mmol) of 16 in
10 mL of THF, a solution of Na2S2O4 dihydrate (3 g) in 20 mL of
1M NaOH was added in small portion (the temperature of reac-
tion must remain under 308C), then the stirring was maintained
for 20 h. The organic phase was separated and evaporated at
reduced pressure to give pure 17 (0.8 g, 80%).
6-Chloro-2-phenylpurine 9
Starting from 2, compound 9 was obtained as described in the
literature [15].
N6-Cyclopentyl-2-phenyladenine 10
A mixture of 9 (0.100 g, 0.43 mmol) and cyclopentylamine
(0.53 g, 6.2 mmol) in absolute ethanol (3 mL) was heated at 908C
for 12 h in a well-stopped pyrex tube. After cooling, the solvent
was evaporated, chloroform was added (10 mL), the solution was
washed with HCl 10% and then evaporated to give 10 (0.093 g,
2-Phenylpteridine 18
Compound 18 was obtained from 16 as described in the litera-
ture [31].
1
78%) as a white solid: m. p. 238–2408C; H-NMR: d = 9.17 (br s,
1H, exch), 8.85 (s, 1H, arom), 8.33 (m, 2H, arom), 7.55 (m, 3H,
arom), 4.70 (m, 2H, NH-CH + exch), 1.89 (m, 8H, (CH2)4).
4-(4-Fluorophenylureido)-2-phenylpteridine 19
was synthesized from 18 (0.040 g, 0.19 mmol), using the proce-
dure described for 14, to give pure 19 as a with solid (0.056 g,
N6-Cyclohexyl-2-phenyladenine 11
1
82%): m. p. 219-2208C; H-NMR: d = 10.99 (s, 1H, exch), 10.26 (s,
was synthesized from 9 (0.100 g, 0.43 mmol) using the proce-
dure described for 4 and was obtained as a white solid (0.065 g,
52%): m. p. 235–2368C; 1H-NMR: d = 8.82 (m, 2H, arom + exch),
1H, N-H), 9.28 (d, J = 1.8 Hz, 1H, arom), 9.00 (d, J = 1.8 Hz, 1H,
arom), 8.52 (m, 2H, arom), 7.65 (m, 5H, arom), 7.23 (m, 2H,
arom).
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

86
I. Giorgi et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 81–87
4-(4-Trifluoromethylphenylureido)-2-phenylpteridine 20
was synthesized from 18 (0.040 g, 0.19 mmol), using the proce-
dure described for 14, and was obtained as a white solid (0.045 g,
56%): m. p. 226–2278C; 1H-NMR: d = 11.14 (s, 1H, exch), 10.45 (s,
1H, exch), 9.29 (d, J = 1.7 Hz, 1H, arom), 9.01 (d, J = 1.7 Hz, 1H,
arom), 8.56 (m, 2H, arom), 7.75 (m, 7H, arom).
A3 adenosine receptor binding assay
CHO cells, stably transfected by Dr K. N. Klotz with human A3
adenosine receptors were maintained in Dulbecco's modified
Eagle's medium with F12 nutrient mixture without nucleotides,
supplemented with 10% fetal calf serum, L-glutamine (2 nM),
penicillin (100 units/mL), streptomycin (100 lg/mL) in a humidi-
fied athmosphere of 5% CO2/95% air at 378C, essentially as pre-
viously described [35]. Cell cultures were split two or three times
a week in a ratio between 1:5 and 1:20 and used for binding
experiments at subconfluency.
At removal, the cells were harvested by centrifugation at
500 g. The crude membranes were prepared as described by
Olah et al. [36]. [125I]AB-MECA binding assay to transfected cell
membranes was carried out as previously described [36]. In brief,
the cell membranes (80 lg per assay) were incubated in 100 lL of
50 mM Tris, 10 mM MgCl2 and 1 mM EDTA (pH 8.12) which con-
tained [125I]AB-MECA at a concentration of 1 nM (Kd 0.6 nM) and
adenosine deaminase (2 units/mL). Non-specific binding was
defined by 50 lM R-PIA. Assays were incubated to equilibrium
for 1 h at 258C and rapidly filtered on Whatman GF/C filters.
In all competition experiments the compounds were routi-
nely dissolved in DMSO and added to the assay mixture, final
DMSO concentrations never exceeded 1%. At least six different
concentrations spanning three orders of magnitude, adjusted
approximately for IC50 of each compound, were used. IC50 values,
computer-generated using a nonlinear formula on a computer
program (GraphPad, San Diego, CA, USA), were converted to Ki
values, knowing the Kd values of radioligands and using the
Cheng and Prusoff equation [37].
Biological assays
[
125I]-ABMECA were purchased from Amersham Biosciences,
[3H]CHA and [3H]CGS 21680 were obtained from NEN Life Science
Products, Inc. (Kꢀln, Germany). Adenosine deaminase (ADA) was
obtained from Boehringer-Mannheim (Mannheim, Germany).
Cell culture media and fetal calf serum were obtained from Bio-
Wittaker (Walkersville, MD, USA). (-)-N6-(2-phenylisopropyl) ade-
nosine (R-PIA), and other agents were purchased from Sigma-
Aldrich, srl (Milan, Italy).
A1 receptor-binding assay
Bovine cerebral cortex was homogenised in ice-cold 0.32 M
sucrose-containing protease inhibitors, as previously described
[34]. The homogenate was centrifuged at 1000 g for 10 min at
48C and the supernatant again centrifuged at 48000 g for
15 min at 48C. The final pellet was dispersed in 50 mM Tris-HCl,
pH 7.7 fresh buffer, incubated with adenosine deaminase
(2 units mL– 1) to remove endogenous adenosine at 378C for
60 min, and then recentrifuged at 48000 g for 15 min at 48C.
The pellet was suspended in buffer and used in the binding
assay. The [3H]CHA binding assay was performed in triplicate by
incubating aliquots of the membrane fraction (0.2–0.3 mg of
protein) at 258C for 45 min in 50 mM Tris-HCl, pH 7.7, contain-
ing 2 mM MgCl2, with approximately 1.2 nM [3H]CHA (Kd
0.5 nM). Nonspecific binding was defined in the presence of
50 mM R-PIA. Binding reactions were terminated by filtration
through Whatman GF/C filters under reduced pressure. Filters
were washed three times with 5 mL of ice-cold buffer and placed
in scintillation vials. Radioactivity was counted in a 4 mL Beck-
man Ready-Protein scintillation cocktail (Beckman Ready Pro-
tein, Fullerton, CA, USA) in a liquid scintillation counter.
References
[1] B. B. Fredholm, A. P. Ijzerman, K. A. Jacobson, K. N. Klotz,
J. Linden, Pharmacol Rev. 2001, 53, 527–552.
[2] M. V. Sitkovsky, D. Lukshev, S. Apasov, H. Kojima, et al.,
Annu. Rev. Immunol. 2004, 22, 657–682.
[3] C. Ledent, J. M. Vaugeois, S. N. Schiffmann, T. Pedrazzini,
et al., Nature 1997, 388, 674–678.
[4] M. El Yacoubi, C. Ledent, J. F. Menard, M. Parmentier, et
al., Br. J. Pharmacol. 2000, 129, 1465–1473.
A2A receptor binding assay
[5] A. Ohta, M. Sitkovsky, Nature 2001, 414, 916–920.
Bovine striatum was homogenised in 20 volumes of ice-cold
50 mM Tris-HCl, pH 7.5, containing 10 mM MgCl2 and protease
inhibitors. The membrane homogenate was centrifuged at
48000 g for 10 min at 48C. The resulting pellet was resuspended
in buffer containing 2 units/mL of adenosine deaminase and
incubated at 378C for 30 min. The membrane homogenate was
centrifuged, and the final pellet frozen at –808C. Routine assays
were performed in triplicate by incubation of an aliquot of stria-
tal membranes (0.2–0.3 mg of protein) in cold 50 mM Tris-HCl,
pH 7.5, containing 10 mM MgCl2 with approximately 5 nM
[3H]CGS 21680 (Kd 16 nM) in a final volume of 0.5 mL. Incubation
was carried out for 90 min at 258C. Non-specific binding was
defined in the presence of 50 lM CGS 21680. Binding reactions
were terminated by filtration through Whatman GF/C filters
under reduced pressure. Filters were washed three times with
5 mL of ice-cold buffer and placed in scintillation vials. Radioac-
tivity was counted in a 4 mL Beckman Ready-Protein scintilla-
tion cocktail in a scintillation counter.
[6] K. Mubagwa, W. Flameng, Cardiovasc. Res. 2001, 52, 25–
39.
[7] B. T. Liang, K. A. Jacobson, Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 6995–6999.
[8] W. R. Tracey, W. Magee, H. Masamune, S. P. Kennedy, et
al., Cardiovasc. Res. 1997, 33, 410–415.
[9] D. K. J. E. von Lubitz, Eur J. Pharmacol. 1999, 371, 85–102.
[10] P. Fishman, L. Madi, S. Bar-Yehuda, I. Cohn, Anticancer
Drugs 2002, 13, 437–443.
[11] P. Fishman, S. Bar-Yehuda, G. Ohana, S. Pathak, et al., Eur.
J. Cancer 2000, 36, 1452–1458.
[12] P. Fishman, S. Bar-Yehuda, F. Barer, L. Madi, et al., Exp.
Cell Res. 2001, 269, 230–236.
[13] M. Fan, W. Qin, S. Mustafa, Am. J. Physiol. Lung Cell Mol.
Physiol. 2003, 284, L1012–1019.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 81–87
Synthesis of 2-Phenyladenines and 2-Phenylpteridines
87
[14] C. I. Ezeamuzie, Biochem. Pharmacol. 2001, 61, 1551–1559.
[27] J. Clark, P. N. T. Murdoch, D. L. Roberts, J. Chem. Soc. Per-
kin Trans I 1969, 1408–1412.
[15] A. M. Bianucci, G. Biagi, A. Coi, O. Livi, et al., Drug Dev.
Res. 2001, 54, 52–65.
[28] W. Traube, L. Herrmann, Chem Ber. 1904, 37, 2267–2272.
[29] E. C. Taylor, O. Vogl, C. C. Cheng, J. Am. Chem. Soc. 1959,
81, 2442–2448.
[16] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, Farmaco 1994, 49,
175–181.
[30] L. G. Frohlich, P. Kotsonis, H. Traub, S. Taghavi–Mogha-
[17] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, Farmaco 1994, 49,
dam, et al., J. Med. Chem. 1999, 42, 4108–4121.
357–362.
[31] R. M. Evans, P. G. Jones, P. J. Palmer, F. F. Stephens, J.
Chem. Soc. 1956, 4106–4113.
[18] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, et al., Farmaco 1995,
50, 99–105.
[32] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, et al., Farmaco 1995,
50, 659–667.
[19] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, Farmaco 1995, 50,
579–586.
[33] G. Biagi, A. M. Bianucci, A. Coi, B. Costa, et al., Bioorg.
Med. Chem. 2005, 13, 4679–4693.
[20] G. Biagi, I. Giorgi, O. Livi, A. Nardi, et al., Eur. J. Med.
Chem. 2003, 38, 983–990.
[34] C. Martini, M. G. Poli, A. Lucacchini, Bull. Mol. Biol. Med.
1986, 11, 1–10.
[21] M. E. Hawkins, W. Pfleiderer, F. M. Balis, D. Porter, J. R.
Knutson, Anal. Biochem. 1997, 244, 86–95.
[35] M. E. Olah, C. Gallo-Rodriguez, K. A. Jacobson, G. L. Stiles,
[22] F. G. Hopkins, Nature 1889, 40, 335.
Mol. Pharmacol 1994, 45, 978–982.
[23] R. Purrmann, Ann. 1940, 544, 182–190.
[36] V. Calotta, D. Catarzi, F. Varano, G. Filacchioni, et al.,
Arch. Pharm. (Weinheim) 2004, 337, 35–41.
[24] H. Wieland, C. Schopf, Ber. Deut. Chem. Ges. 1925, 58,
2178–2183.
[37] Y. C. Cheng, W. H. Prusoff, Biochem. Pharmacol. 1973, 22,
3099–3108.
[25] W. Pfleiderer, Adv. Exp. Med. Biol. 1993, 338, 1–16.
[26] H. W. van Meeteren, H. C. van der Plas, Recl. Trav. Chim.-
Pays-Bas, 1968, 87, 1089–1098.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com