New substituted 9-alkylpurines as adenosine receptor ligands

Article abstract of DOI:10.1016/S0968-0896(98)00007-8

In the present study an investigation of the structure-activity relationships in 9-ethylpurine derivatives, aimed at preparing A₁, A(2A), A(2B), and A₃ selective adenosine receptor antagonists, was undertaken. Our synthetic approach was to introduce various substituents (amino, alkoxy and alkynyl groups) into the 2-, 6-, or 8-positions of the purine ring. The starting compounds for each series of derivatives were respectively: 2-iodo-9-ethyladenine (9), obtained from 2-amino-6-chloropurine (5); 9-ethyl-6-iodo-9H-purine (11), 8-bromo-9-ethyl-adenine (3) and 8-bromo-9-ethyl-6-iodo-9H-purine (13), obtained from 9-ethyl-adenine (2). The synthesized compounds were tested in in vitro radioligand binding assays at A₁, A(2A), and A₃ human adenosine receptor subtypes. Due to the lack of a suitable radioligand the affinity of the 9-ethyladenine derivatives at A(2B) adenosine receptors was determined in adenylyl cyclase experiments. In general, the series of 9-ethylpurine derivatives exhibited a similar pharmacological profile at A₁ and A(2A) receptors whereas some differences were found for the A₃ and the A(2B) subtypes. 8-Bromo-9-ethyladenine (3) showed higher affinity for all receptors in comparison to the parent compound 2, and the highest affinity in the series for the A(2A) and A(2B) subtypes (K(i)=0.052 and 0.84μM, respectively). Analyzing the different substituents, a phenethoxy group in 2-position (10a) gave the highest A(2A) versus A(2B) selectivity (near 400-fold), whereas a phenethylamino group in 2- and 6-position (10b and 12b, respectively) improved the affinity at A(2B) receptors, compared to the parent compound 2. The presence of a hexynyl substituent in 8-position led to a compound with good affinity at the A₃ receptor (4d, K(i)=0.62μM), whereas (ar)alkynyl groups are detrimental for the potency at the A(2B) subtype. These differences give raise to the hope that further modifications will result in the development of currently unavailable leads with good affinity and selectivity for A(2B) adenosine receptors. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00007-8

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 523±533
New Substituted 9-Alkylpurines as Adenosine Receptor Ligands
Emidio Camaioni,a Stefano Costanzi,a Sauro Vittori,a Rosaria Volpini,a
Karl-Norbert Klotzb and Gloria Cristallia,*
a
Á
Dipartimento di Scienze Chimiche, Universita di Camerino, 62032 Camerino, Italy
È
bInstitut fur Pharmakologie, Universitat of Wurzburg, D-97078 Wurzburg, Germany
È
È
È
Received 4 September 1997; accepted 4 November 1997
AbstractÐIn the present study an investigation of the structure±activity relationships in 9-ethylpurine derivatives,
aimed at preparing A1, A2A, A2B, and A3 selective adenosine receptor antagonists, was undertaken. Our synthetic
approach was to introduce various substituents (amino, alkoxy and alkynyl groups) into the 2-, 6-, or 8-positions of the
purine ring. The starting compounds for each series of derivatives were respectively: 2-iodo-9-ethyladenine (9),
obtained from 2-amino-6-chloropurine (5); 9-ethyl-6-iodo-9H-purine (11), 8-bromo-9-ethyl-adenine (3) and 8-bromo-9-
ethyl-6-iodo-9H-purine (13), obtained from 9-ethyl-adenine (2). The synthesized compounds were tested in in vitro
radioligand binding assays at A1, A2A, and A3 human adenosine receptor subtypes. Due to the lack of a suitable
radioligand the anity of the 9-ethyladenine derivatives at A2B adenosine receptors was determined in adenylyl cyclase
experiments. In general, the series of 9-ethylpurine derivatives exhibited a similar pharmacological pro®le at A1 and
A2A receptors whereas some di€erences were found for the A3 and the A2B subtypes. 8-Bromo-9-ethyladenine (3)
showed higher anity for all receptors in comparison to the parent compound 2, and the highest anity in the series
for the A2A and A2B subtypes (Ki=0.052 and 0.84 mM, respectively). Analyzing the di€erent substituents, a phenethoxy
group in 2-position (10a) gave the highest A2A versus A2B selectivity (near 400-fold), whereas a phenethylamino group
in 2- and 6-position (10b and 12b, respectively) improved the anity at A2B receptors, compared to the parent com-
pound 2. The presence of a hexynyl substituent in 8-position led to a compound with good anity at the A3 receptor
(4d, Ki=0.62 mM), whereas (ar)alkynyl groups are detrimental for the potency at the A2B subtype. These di€erences
give raise to the hope that further modi®cations will result in the development of currently unavailable leads with good
anity and selectivity for A2B adenosine receptors. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
selectivity for A1 and A2A subtypes.4±6 The in vivo use
of these compounds is however hampered by their poor
solubility: hence considerable e€orts have been devoted
to the discovery of non-xanthine antagonists7 to be used
as pharmacological probes and potential therapeutic
agents.8 More recently, it has been demonstrated that
the cloned rat A3 receptor subtype is quite insensitive to
xanthines.9a However Linden et al. found that certain
xanthines bind appreciably to sheep and human A3
receptors but generally with less anity than at A1 and
A2A receptors in a variety of species.9b,c Furthermore,
no selective high anity ligands or radioligands are
currently available for the A2B subtype.
The purine nucleoside adenosine speci®cally modulates
neurotransmission through the interaction with four
surface receptors designated as A1, A2A, A2B and A3 on
the basis of biological experiments and receptor clon-
ing.1,2
The ®rst compounds that were identi®ed as adenosine
receptor antagonists were the naturally occurring xan-
thines, ca€eine and theophylline.3 These xanthines are
non-selective and of moderate potency. Substitution at
appropriate sites of xanthine may enhance anity and
A ribose or ribose equivalent at N-9 is present in all
currently known adenosine receptor agonists. Replace-
ment of ribose with a methyl group led to a relatively
Key words: Adenosine receptors; adenosine antagonists; purine
derivatives.
*Corresponding author. E-mail: cristall@camserv.unicam.it
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00007-8

524
E. Camaioni et al./Bioorg. Med. Chem. 6 (1998) 523±533
In the present study an investigation on the structure-
activity relationships of 9-ethyladenine derivatives,
aimed at ®nding selective adenosine receptor antago-
nists, was undertaken. This molecule was selected on the
basis that preliminary studies showed 9-ethyladenine to
be the most potent ligand at A1 and A2A receceptor
subtypes in a series of 9-alkyladenines.14
The synthetic program included the introduction of
phenethylamino, phenethoxy, phenylbutynyl and hexyn-
yl substituents in the 2-, 6-, or 8-positions of 9-ethyl-
adenine, respectively. Furthermore,
a
few 6,8-
disubstituted compounds bearing a bromine atom in
position 8 were prepared. These substituents have been
selected on the basis of previous ®ndings on adenosine
receptor agonist anity.
Chemistry
9-Ethyladenine (2)15 was selected as the parent com-
pound and the synthesis of its derivatives, substituted in
turn in 8-, 2- or 6-position with amino, alkoxy and
alkynyl groups was designed as reported in Schemes 1±
3. The starting compounds for each series of derivatives
were respectively: 8-bromo-9-ethyladenine (3),16 9-ethyl-
2-iodoadenine (9), 9-ethyl-6-iodo-9H-purine (11), and 8-
bromo-9-ethyl-6-iodo-9H-purine (13).
Scheme 1.
unselective antagonist and adenine itself is a weak ade-
nosine antagonist.10 As in the case of adenosine deriva-
tives, alkyl and cycloalkyl substituents in N6 increase
the anity of 9-methyladenine for the A1 receptors.11,12
Moreover, the presence of a 3-iodobenzyl group at the
6-amino position of 9-methyladenine resulted in com-
pounds with properties indicating an A3 versus A1
selectivity. However, a 9-methyl adenine substituted in
the 2-position with a phenylethoxy group has been
reported not to discriminate between A2 receptors in the
coronary vessels and A1 receptors in the atria of guinea
pig.13
The commercially available adenine (1) was reacted with
ethyl iodide in dry dimethylformamide, employing
potassium carbonate as base, to give 9-ethyladenine (2)
in 60% yield. The yield of 2 is higher than that reported
earlier (40%), obtained by using sodium hydride as
base.15 The reaction of 9-ethyladenine with N-bromo-
Scheme 2.

E. Camaioni et al./Bioorg. Med. Chem. 6 (1998) 523±533
525
an unambiguous chemical proof reported below. The
iodide in 2-position was introduced by the classical di-
azotization/halogenation procedure using iso-pentyl
nitrite as the nitrosating agent. From 6-chloro-9-ethyl-2-
iodo-9H-purine (8) the desired 9-ethyl-2-iodoadenine (9)
was obtained in 65% yield by treatment with liquid
ammonia at room temperature in a sealed tube. It is well
documented that the selective substitution of the halo-
gen in 6-position can be easily achieved, since nucleo-
philic displacements at this position are more feasible at
room temperature than those at 2-position. However,
catalytic deiodination of 9 was carried out to give 9-
ethyladenine (2). This reaction con®rmed both the
assignment of the alkylation site of 2-amino-6-chloro-9-
ethyl-9H-purine (6) and, at the same time, the selective
substitution of the chlorine atom in 6-position of com-
pound 8 (Scheme 2).
Starting from 2, 9-ethyl-6-iodopurine (11) was obtained
in 45% yield by modi®cation of the method previously
reported.15 Attempt to obtain 8-bromo-9-ethyl-6-iodo-
9H-purine (13) from 11 by treating it with N-bromo-
succinimide or bromine in dry DMF at 60 ꢀC was
unsuccessful. Alternatively, the desired compound was
obtained from the 6-amino derivative 3, by introducing
the iodide in 6-position through the classical diazotiza-
tion/halogenation procedure using iso-pentyl nitrite as
the nitrosating agent.
Scheme 3.
succinimide gave the 8-bromo-9-ethyladenine (3) in 55%
yield (Scheme 1).
Reaction of the commercially available 2-amino-6-
chloropurine (5) with ethyl iodide gave the desired N-9
derivative 6, together with the 7-alkylated isomer 7, in
an about 3:1 ratio. The isomers were isolated in 67%
and 20% yields, respectively. The isomeric structure was
assigned on the basis of 1H NMR and UV spectra,
according to data reported in the literature for similar
alkylated 2-amino-6-chloropurines,17 and by means of
Substitution of halogens (iodine or bromine) in com-
pounds 3, 9, 11, and 13 with di€erent groups was car-
ried out following the general methods A±C, described
in the experimental section, and according to the reac-
tion conditions reported in Table 1. Brie¯y:
Table 1. Preparation of compounds in Schemes 1±3
Compd
Method
Base
t (ꢀC)
Time (h)
Chromatography solventa
Yield (%)
4a
A
B
C
C
A
B
C
C
A
B
C
C
A
B
C
C
K2CO3
85
100
rt
16
36
60
60
3
CHCl3:cC6H12:MeOH(80:13:7); TLC
CHCl3:MeOH(95:5)
35
70
75
73
84
80
62
83
60
60
70
82
65
45
64
45
4b
4c
Et3N
Et3N
CHCl3:cC6H12:MeOH (86:10:4)
CHCl3:MeOH (99:1);f
4d
rt
10a
10b
10c
10d
12a
12b
12c
12d
14a
14b
14c
14d
NaOH
85
130
rt
EtOAc:cC6H12:MeOH (60:33:7)
EtOAc:cC6H12:MeOH (60:36:4)
CHCl3:cC6H12:MeOH (86:10:4)
CH2Cl2:MeOH (97:3)
24
24
20
16
6
Et3N
Et3N
rt
NaOH
85
60
rt
CH3CN:H2O:MeOH(40:40:20); rp
CHCl3:MeOH (99:1);f
Et3N
Et3N
K2CO3
Et3N
Et3N
Et3N
3
CHCl3:MeOH (98:2)
rt
3
CHCl3:MeOH (98:2); TLC
cC6H12:EtOAc:MeOH (50:49:1); TLC
CHCl3:MeOH (99:1); TLC
CHCl3; TLC
rt
72
16
16
20
60
rt
rt
CHCl3; TLC
aTLC: preparative thin-layer chromatography; f: ¯ash chromatography; rp: reverse-phase (C18).

526
E. Camaioni et al./Bioorg. Med. Chem. 6 (1998) 523±533
Method A: The alkoxy derivatives 4a, 10a, 12a, and 14a
were obtained by reaction of the suitable synthon with
2-phenethyl alcohol in dry acetonitrile in the presence of
K2CO3 or NaOH at the temperature and for the time
reported in Table 1.
The same modi®cations in 6-position (12a±d) did not
have major e€ects on the anity at the four subtypes,
with the exception of the phenethylamino group in
compound 12b, which improved the anity for the A2B
and A3 receptors to Ki=8.13 and 10.8 mM, respectively.
The combined presence of a phenethylamino group in 6-
and a bromine in the 8-position led to compound 14b
with even higher anity for the A2B and A3 receptors
(Ki=2.21 and 2.44 mM, respectively).
Method B: The amino derivatives 4b, 10b, 12b, and 14b
were obtained by reacting the suitable synthon with 2-
phenethylamine at the temperature and for the time
reported in Table 1.
Analyzing the di€erent substituents, a phenethoxy
group in 2-position (10a) gave the highest A2A versus
A2B selectivity (near 400-fold). On the other hand, a
phenethylamino group in 2- or 6-position (10b and 12b,
respectively) was the most e€ective in improving the
anity at A2B receptors, compared to the parent com-
pound 2. The presence of a hexynyl substituent in 2-
or 8-position (10d and 4d, respectively) led to com-
pounds with good anity at A3 receptor, whereas
(ar)alkynyl groups are detrimental for the potency at
A2B subtype.
Method C: The introduction of the alkynyl chain to give
compounds 4c and d, 10c and d, 12c and d, and 14c and
d was carried out by modi®cation of the classical cross-
coupling reaction in the presence of CuI, Et3N and
(Ph3P)2PdCl2 at room temperature for the time reported
in Table 1.
Biological activity
All the synthesized compounds were tested in radio-
ligand competition at membranes from stably trans-
fected CHO cells expressing the human A1, A2A, and
A3 adenosine receptor subtypes,18 using DPCPX and
theophylline as reference compounds. [3H]DPCPX19 on
A1, [3H]CGS 2168020 on A2A, and [125I]ABMECA21 on
A3 have been used as radioligands. Due to the lack
of a suitable radioligand the anity (KB, Ki) of the
9-ethyladenine derivatives at A2B adenosine receptors
was determined by inhibition of NECA-stimulated
adenylyl cyclase activity. The results are reported in
Table 2.
It is worthwhile to note that in both the N6-substituted
series, 12a±d and 14a±d, the most active compounds
bear the phenethylamino group. Replacing the 6-amino
by alkoxy or alkynyl groups greatly diminished anity
at the four adenosine receptor subtypes, suggesting that
a N±H group is required to provide a H-bond donor
for a good interaction with all the adenosine recep-
tors.
In conclusion, most compounds with higher anity at
A2A receptors were also more potent at A1 versus A3
subtype, with the exception of 8-hexynyl-9-ethyladenine
(4d), which was the most potent compound at A3
receptors (Ki=0.62 mM), with a fourfold selectivity for
this subtype versus A1.
All the compounds tested showed higher anity at A2A
receptor in comparison with other subtypes except for 12d
and 14a. This ®nding is in agreement with preliminary
results indicating that the presence of an ethyl group
in 9-position increased the A2A versus A1 selectivity.14
In general, the series of 9-ethylpurine derivatives exhib-
ited a similar pharmacological pro®le at A1 and A2A
receptors (Figure 1(a)) whereas some di€erences in the
pro®le were found for the A3 and the A2B subtypes, as
shown in Figure 1(b). In fact the diagram in Figure 1(b)
clearly illustrates the A2B versus A3 selectivity of com-
pound 3 and conversely the A3 versus A2B selectivity of
compound 4d. On the other hand, compounds 10b, 12b,
and 14b exhibit no preference for A2B or A3 receptors.
8-Bromo-9-ethyladenine (3) showed higher anity for
all receptors in comparison to the parent compound 2,
and the highest anity in the series for the A2A and A2B
subtypes (Ki=0.052 and 0.84 mM, respectively). The 8-
phenethoxy-, 8-phenethylamino-, and 8-(ar)alkynyl-
compounds 4a±d showed in general decreased potency
at all receptors in comparison with compound 3, with
the exception of the alkynyl derivatives 4c and 4d which
showed higher anity at the A3 subtype (Ki=3.20 and
0.62 mM, respectively versus 27.8 mM). It is worthwhile to
note that the hexynyl derivative 4d exibited the higher
anity at the A3 subtype in the series. The introduction of
the same substituents in 2-position of compound 2 (10a±
d) enhanced the anity for all receptors in comparison
with 9-ethyladenine itself. The 2-phenethoxy derivative
10a resulted to be the most potent in the series in respect
to the A1 receptor suptype (Ki=0.17 mM).
The di€erences detected in this study will serve as a
basis for further modi®cations, including a selection
of di€erent 9-(ar)alkylpurine derivatives, for the devel-
opment of new ligands with currently unavailable
anity and selectivity for the A2B subtype. This will
help to identify relevant in vivo functions of A2B
adenosine receptors, in particular in cardiovascular
regulation.

E. Camaioni et al./Bioorg. Med. Chem. 6 (1998) 523±533
527
Table 2. Anity of 9-ethylpurine derivatives in radioligand binding and NECA-stimulated adenylyl cyclase assays at membranes
from stably transfected CHO cells expressing human adenosine receptor subtypes
Compd
Substituent
Y
Binding (Ki, mM)a
A2A A2B
X
H
Z
A1
A3
2
NH2
NH2
H
7.44
2.20
>100
>100
(4.22±13.12)
0.28
(1.40±3.53)
0.052
3
H
Br
0.84
27.8
(0.25±0.32)
0.89
(0.024±0.113)
0.15
(0.63±1.12)
7.59
(22.3±34.7)
36.2
4a
H
NH2
O-(CH2)2-C6H5
(0.59±1.33)
(0.096±0.23)
3.01
(4.35±13.25)
(16.9±77.6)
49.4
4b
H
NH2
NH-(CH2)2-C6H5 14.2
(5.99±33.7)
CꢁC-(CH2)2-C6H5 4.60
(2.62±8.06)
CꢁC-(CH2)3-CH3 2.34
(1.40±3.89)
0.17
>100
>100
22.4
(1.48±6.11)
1.62
(26.0±93.7)
3.20
4c
H
NH2
(0.75±3.46)
0.44
(2.19±4.65)
0.62
4d
H
NH2
(0.22±0.87)
0.12
(11.1±45.1)
45.8
(0.34±1.14)
7.15
10a
10b
10c
10d
12a
12b
12c
12d
14a
14b
14c
14d
O-(CH2)2-C6H5
NH2
H
H
(0.13±0.23)
0.33
(0.07±0.22)
0.15
(29.8±70.5)
2.41
(2.95±17.3)
3.15
NH-(CH2)2-C6H5
NH2
(0.21±0.51)
0.21
(0.11±0.21)
0.15
(1.44±4.03)
(2.44±4.06)
4.05
CꢁC-(CH2)2-C6H5
NH2
H
>100
(0.12±0.39)
0.55
(0.091±0.23)
0.42
(2.93±5.62)
2.30
CꢁC-(CH2)3-CH3
NH2
H
12.5
(0.24±1.23)
19.2
(0.26±0.69)
17.1
(5.89±26.7)
(1.08±4.90)
22.1
H
H
H
H
H
H
H
H
O-(CH2)2-C6H5
NH-(CH2)2-C6H5
CꢁC-(CH2)2-C6H5
CꢁC-(CH2)3-CH3
O-(CH2)2-C6H5
NH-(CH2)2-C6H5
CꢁC-(CH2)2-C6H5
CꢁC-(CH2)3-CH3
H
>100
(7.32±50.5)
8.73
(7.37±39.7)
1.34
(17.8±27.6)
10.8
H
8.13
(3.79±20.1)
14.1
(0.67±2.68)
4.90
(5.98±11.06)
74.9
(8.66±13.4)
39.3
H
(11.1±17.8)
12.1
(4.27±5.62)
17.6
(42.7±131.7)
(31.1±49.6)
39.2
H
>100
(8.75±16.7)
11.7
(8.03±38.6)
19.3
(13.8±111.3)
60.9
Br
Br
Br
Br
>100
(9.41±14.5)
1.54
(13.9±26.9)
0.28
(29.9±124.2)
2.44
2.21
(1.46±3.35)
>100
(1.30±1.81)
16.7
(0.12±0.68)
10.8
(1.52±3.90)
44.7
(9.66±28.8)
23.6
(5.29±21.9)
6.09
(20.4±97.3)
19.0
>100
1.00
(10.7±52.1)
0.0039
1.94±19.1
0.13
(12.6±29.6)
4.00
DPCPXb
(0.0035±0.0042) (0.064±0.26)
(0.60±1.70)
40.0
(2.60±6.00)
86.0
Theophyllineb
6.8
1.70
(4.10±11.3)
(1.00±2.90)
(36.0±44.0)
(74.0±101.0)
aReceptor binding anity at A1, A2A, and A3 receptors was determined using [3H]DPCPX,19 [3H]CGS 21680,20 and [125I]ABMECA21
as radioligands, respectively. Anity of the 9-ethylpurine derivatives at A2B adenosine receptors was determined in adenylyl cyclase
experiments. Data are geometrical means from at least three separate experiments; 95% con®dence limits in parenthesis.
bSee Ref. 25.

528
E. Camaioni et al./Bioorg. Med. Chem. 6 (1998) 523±533
Experimental
exchangeable protons were con®rmed by addition of
D2O. TLC was carried out on precoated TLC plates
with silica gel 60 F-254 (Merck). For column chroma-
tography, silica gel 60 (Merck) was used. Micro-
analytical results are indicated by atomic symbols and
are within ‹0.4% of theoretical values.
Chemistry
Melting points were determined with a Buchi apparatus
and are uncorrected. 1H NMR spectra were obtained
with Varian VX 300 and 200 MHz spectrometers. All
(a)
10
19.2
A1
8
A2A
A3
A2B
6
4
2
0
2
3
10a 10b 10c 10d 4a 4b 4c 4d 12a 12b 12c 12d 14a 14b 14c 14d
(b)
2
1.5
1
A3
A2B
0.5
0
2
3
10a 10b 10c 10d 4a 4b 4c 4d 12a 12b 12c 12d 14a 14b 14c 14d
Figure 1. (a) Anity of 9-ethylpurine derivatives at adenosine receptors. The association constants (Ka) of 9-ethylpurine derivatives
are shown as an indication of their anity to adenosine receptor subtypes. Values given as 1/mM and were calculated from Ki-values
in Table 2. (b) Comparison of A2B and A3 adenosine receptor binding of 9-ethylpurine derivatives. Association constants (Ka) of
9-ethylpurine derivatives at A2B and A3 receptors are derived from Ki-values in Table 2. For further details see text.

E. Camaioni et al./Bioorg. Med. Chem. 6 (1998) 523±533
529
General method A
(Me2SO-d6) d 1.41 (t, 3H, CH2CH3), 4.29 (q, 2H,
CH2CH3), 7.19 (br s, 2H, NH2), 8.13 (s, 1H, H-2) 8.16
(s, 1H, H-8). Anal. calcd for C7H9N5: C, 51.52, H, 5.56,
N, 42.92; found: C, 51.37, H, 5.82, N, 42.73.
To a suspension of iodo or bromo 9-ethyl-9H-purine
derivative (1 mmol) and dry K2CO3 (2 mmol) or NaOH
(5 mmol) in dry CH3CN (25 mL) was added 2-phenethyl
alcohol (5 mL, 40 mmol). This mixture was heated at
85 ꢀC for the time reported in the Table 1. The solvent
was removed under reduced pressure and the residue
was neutralized with 2 N HCl and extracted with
CHCl3. The organic layer was dried (Na2SO4) and con-
centrated in vacuo. The residue was chromatographed
on a silica gel column eluting with a suitable mixture of
solvents (Table 1) to give the desired derivatives as
chromatographically pure solids.
8-Bromo-9-ethyladenine (3). To a solution of compound
2 (1.6 g, 9.8 mmol) in dry DMF (25 mL) was added N-
bromosuccinimide (3.5 g, 19.6 mmol) and the reaction
mixture was stirred at room temperature for 20 h. The
solvent was removed in vacuo and the residue was ¯ash
chromatographed on a silica gel column using a gra-
dient from CHCl3 to CHCl3:MeOH (98:2) to give com-
pound 3 (1.42 g, 60%) as a chromatographically pure
solid: mp 218±220 ꢀC; 1H NMR (Me2SO-d6) d 1.33 (t,
3H, J=7.3 Hz, CH2CH3), 4.17 (q, 2H, J=7.1 Hz,
CH2CH3), 7.39 (br s, 2H, NH2), 8.15 (s, 1H, H-2). Anal.
calcd for C7H8BrN5: C, 34.73, H, 3.33, N, 28.93; found:
C, 35.02, H, 3.41, N, 28.67.
General method B
To iodo or bromo 9-ethyl-9H-purine derivative
(1 mmol) was added 2-phenethylamine (5 mL) and the
mixture was heated at the temperature and for the time
listed in Table 1. In the case of compound 14b, 2-phen-
ethylamine was added in equimolar amount in dry
CH3CN (20 mL) and triethylamine (2 mL). The solvent
was removed in vacuo and the residue was chromato-
graphed on a silica gel column eluting with a suitable
mixture of solvents (Table 1) to give the desired deriva-
tives as chromatographically pure solids.
9-Ethyl-8-(2-phenylethyloxy)-9H-adenine (4a). This
compound was synthesized following general method A:
mp 176±178 ꢀC; 1H NMR (Me2SO-d6) d 1.17 (t, 3H,
J=7.0 Hz, CH2CH3), 3.13 (t, 2H, J=6.6 Hz, CH2Ph),
3.89 (q, 2H, J=7.0 Hz, CH2CH3) 4.68 (t, 2H, J=6.5 Hz,
CH2O), 6.78 (br s, 2H, NH2), 7.30 (br m, 5H, H-Ph),
8.03 (s, 1H, H-2). Anal. calcd for C15H17N5O: C, 63.59,
H, 6.05, N, 24.72; found: C, 63.88, H, 6.20, N, 24.61.
9-Ethyl-8-(2-phenylethylamino)-9H-adenine (4b). This
compound was synthesized following general method B:
mp 213±215 ꢀC; 1H NMR (Me2SO-d6) d 1.17 (t, 3H,
J=7.0 Hz, CH2CH3), 2.96 (ps t, 2H, J=7.7 Hz,
CH2CH2NH), 3.58 (q, 2H, J=7.6 Hz, CH2NH) 3.97 (q,
2H, CH2CH3), 6.40 (s, 2H, NH2), 6.85 (m, 1H, NH),
7.28 (br m, 5H, H-Ph), 7.92 (s, 1H, H-2). Anal. calcd for
C15H18N6: C, 63.81, H, 6.43, N, 29.76; found: C, 64.03,
H, 6.67, N, 29.48.
General method C
To a solution of iodo or bromo 9-ethyl-9H-purine deri-
vative (0.84 mmol), dry DMF (10 mL), dry CH3CN
(25 mL), and Et3N (3.4 mL) under an atmosphere of N2
was added bis(triphenylphosphine)palladium dichloride
(12 mg) and CuI (0.84 mg). 4-Phenyl-1-butyne or 1-hex-
yne (4.2 mmol) was added and the reaction mixture was
stirred under an atmosphere of N2 at room temperature
for the time reported in Table 1. In the case of com-
pounds 14c and 14d, the terminal alkyne was added in
equimolar amount. The solvent was removed in vacuo
and the residue was chromatographed on a silica gel
column eluting with a suitable mixture of solvents
(Table 1) to give the desired derivatives as chromato-
graphically pure solids.
9-Ethyl-8-(4-phenyl-1-butyn-1-yl)-9H-adenine (4c). This
compound was synthesized following general method C:
mp 180±182 ꢀC; 1H NMR (Me2SO-d6) d 1.22 (t, 3H,
J=7.2 Hz, CH2CH3), 2.94 (s, 4H, CꢁCCH2CH2), 4.04
(q, 2H, J=7.0 Hz, CH2CH3), 7.34 (br m, 7H, H-Ph and
NH2), 8.15 (s, 1H, H-2). Anal. calcd for C17H17N5: C,
70.08, H, 5.88, N, 24.04; found: C, 70.39, H, 6.01, N,
23.89.
9-Ethyladenine (2). To a solution of adenine (1) (3.0 g,
22.2 mmol) in dry DMF (10 mL) was added iodoethane
(2.3 mL, 28.8 mmol) and dried K2CO3 (3.5 g,
25.3 mmol), and the suspension was stirred at room
temperature for 16 h. The reaction mixture was ®ltered
to remove the insoluble material and the ®ltrate was
coevaporated three times with toluene, and then con-
centrated to a residue which was ¯ash chromatographed
on a silica gel column. Elution with CH2Cl2:MeOH
(97:3) gave compound 2 (2.2 g, 60%) as a white solid:
mp 185±187 ꢀC [lit.15 mp 192±193 ꢀC]; 1H NMR
9-Ethyl-8-(1-hexyn-1-yl)-9H-adenine (4d). This com-
pound was synthesized following general method C: mp
173±175 ꢀC; 1H NMR (Me2SO-d6) d 0.95 (t, 3H,
J=7.1 Hz, CH2CH3), 1.35 (t, 3H, J=7.3 Hz,
NCH2CH3), 1.38±1.61 (br m, 4H, CH2CH2CH3), 2.60
(t, 2H, J=6.8 Hz, CꢁCCH2), 4.20 (q, 2H, J=7.1 Hz,
NCH2CH3), 7.39 (br s, 2H, NH2) 8.19 (s, 1H, H-2).
Anal. calcd for C13H17N5: C, 64.17, H, 7.04, N, 28.78;
found: C, 64.28, H, 7.27, N, 28.51.

530
E. Camaioni et al./Bioorg. Med. Chem. 6 (1998) 523±533
2-Amino-6-chloro-9-ethyl-9H-purine (6) and 2-amino-6-
chloro-7-ethyl-7H-purine (7). To a solution of 2-amino-
6-chloropurine (5) (2.5g, 14.7 mmol) in dry DMF (20 mL)
under an atmosphere of N2 was added dried K2CO3
(3.0 g, 21.7 mmol) and iodoethane (1.5 mL, 18.7 mmol)
and the suspension was stirred at room temperature for
16 h. The reaction mixture was ®ltered to remove the
insoluble material and the ®ltrate was coevaporated
three times with toluene, and then concentrated to a
residue which was ¯ash chromatographed on a silica gel
column. Elution with CHCl3 gave 2-amino-6-chloro-9-
ethyl-9H-purine (6) (1.95 g, 67%), and then elution with
CHCl3:MeOH (98:2) gave 2-amino-6-chloro-7-ethyl-
7H-purine (7) (0.59 g , 20%) as white solids.
9-Ethyl-2-(2-phenylethyloxy)-9H-adenine (10a). This
compound was synthesized following general method A:
mp 181±183 ꢀC; 1H NMR (Me2SO-d6) d 1.38 (t, 3H,
J=7.2 Hz, CH2CH3), 3.02 (t, 2H, J=6.9 Hz, CH2-Ph),
4.08 (q, 2H, J=7.3 Hz, CH2CH3) 4.42 (t, 2H, J=7.0 Hz,
CH2O), 7.21 (s, 2H, NH2), 7.32 (br m, 5H, H-Ph), 7.96
(s, 1H, H-8). Anal. calcd for C15H17N5O: C, 63.59, H,
6.05, N, 24.72; found: C, 63.81, H, 6.17, N, 24.40.
9-Ethyl-2-(2-phenylethylamino)-9H-adenine (10b). This
compound was synthesized following general method B:
mp 139±141 ꢀC; 1H NMR (Me2SO-d6) d 1.37 (t, 3H,
CH2CH3), 2.84 (q, 2H, CH2CH2NH), 3.44 (m, 2H,
CH2NH) 4.01 (q, 2H, CH2CH3), 6.23 (t, 1H, NH), 6.62
(s, 2H, NH2), 7.24 (br m, 5H, H-Ph), 7.71 (s, 1H, H-8).
Anal. calcd for C15H18N6: C, 63.81, H, 6.43, N, 29.76;
found: C, 63.89, H, 6.49, N, 29.54.
Compound 6: Mp 151±153 ꢀC; UV lmax (MeOH)
221 nm (" 23700), 246 (" 4800), 307 (" 5800), (pH 1)
1
216 nm (" 24700), 240 (" 5800), 321 (" 5400); H NMR
(Me2SO-d6) d 1.34 (t, 3H, CH2CH3), 4.05 (q, 2H,
CH2CH3), 6.90 (s, 2H, NH2), 8.14 (s, 1H, H-8). Anal.
calcd for C7H8ClN5: C, 42.54, H, 4.08, N, 35.44; found:
C, 42.71, H, 4.25, N, 35.27.
9-Ethyl-2-(4-phenyl-1-butyn-1-yl)-9H-adenine (10c). This
compound was synthesized following general method C:
mp 117±120 ꢀC; 1H NMR (Me2SO-d6) d 1.39 (t, 3H,
J=7.3 Hz, NCH2CH3), 2.72 (ps t, 2H, CꢁCCH2), 2.88
(ps t, 2H, CH2Ph) 4.14 (q, 2H, J=7.3 Hz, NCH2), 7.32
(br m, 7H, H-Ph and NH2), 8.20 (s, 1H, H-8). Anal.
calcd for C17H17N5: C, 70.08, H, 5.88, N, 24.04; found:
C, 70.18, H, 5.92, N, 23.77.
Compound 7: Mp 185 ꢀC (dec); UV lmax (MeOH)
221 nm (" 18800), 253 (sh), 321 (" 4400), (pH 1) 216 nm
1
(" 22900), 240 (sh), 322 (" 5300); H NMR (Me2SO-d6)
d 1.38 (t, 3H, CH2CH3), 4.30 (q, 2H, CH2CH3), 6.61 (s,
2H, NH2), 8.38 (s, 1H, H-8).Anal. calcd for C7H8ClN5:
C, 42.54, H, 4.08, N, 35.44; found: C, 42.77, H, 4.28, N,
35.15.
9-Ethyl-2-(1-hexyn-1-yl)-9H-adenine (10d). This com-
pound was synthesized following general method C: mp
165±167 ꢀC; 1H NMR (Me2SO-d6) d 0.89 (t, 3H,
J=7.2 Hz, CH2CH3), 1.35 (t, 3H, J=7.2 Hz,
NCH2CH3), 1.41 and 1.49 (m, 2H each, CH2), 2.38 (t,
2H, J=7.2 Hz, CꢁCCH2), 4.10 (q, 2H, J=7.2 Hz,
NCH2), 7.24 (s, 2H, NH2) 8.15 (s, 1H, H-8). Anal. calcd
for C13H17N5: C, 64.17, H, 7.04, N, 28.78; found: C,
64.23, H, 7.16, N, 28.52.
6-Chloro-9-ethyl-2-iodo-9H-purine (8).
A mixture of
compound 6 (2.9 g, 14.7 mmol) in dry THF (75 mL),
diiodomethane (12.6 mL), and iso-pentyl nitrite (6.3 mL)
was heated at 85 ꢀC for 1 h. The solvent was removed
under reduced pressure (oil pump, 60 ꢀC) and the resi-
due was puri®ed by ¯ash chromatography on a silica gel
column eluting with CHCl3:n-C6H14 (70:30) to give
compound 8 (2.8 g, 62%) as a pure solid: mp 117±
9-Ethyl-6-(2-phenylethyloxy)-9H-purine
(12a).
This
compound was synthesized following general method A:
mp 61±63 ꢀC; 1H NMR (Me2SO-d6) d 1.43 (t, 3H,
J=7.3 Hz, CH2CH3), 3.14 (t, 2H, J=7.0 Hz, CH2-Ph),
4.26 (q, 2H, J=7.3 Hz, CH2CH3), 4.76 (t, 2H,
J=7.0 Hz, CH2O), 7.12±7.40 (br m, 5H, H-Ph), 8.41 (s,
1H, H-8), 8.52 (s, 1H, H-2). Anal. calcd for C15H16N4O:
C, 67.15, H, 6.01, N, 20.88; found: C, 67.33, H, 6.22, N,
20.67.
119 ꢀC; H NMR (Me2SO-d6) d 1.42 (t, 3H, CH2CH3),
1
4.24 (q, 2H, CH2CH3), 8.65 (s, 1H, H-8). Anal. calcd for
C7H6ClIN4: C, 27.25, H, 1.96, N, 18.16; found: C,
27.38, H, 2.06, N, 17.98.
9-Ethyl-2-iodo-9H-adenine (9). A solution of compound
8 (3.0 g, 9.7 mmol) in liquid ammonia (20 mL) was
sealed in a stainless steel tube and set aside at room
temperature for 24 h. The solvent was removed in vacuo
and the residue was puri®ed by ¯ash chromatography
on a silica gel column eluting with a gradient of
CH2Cl2:MeOH from (99.5:0.5) to (99:1) to give com-
pound 9 (1.85 g, 66%) as a chromatographically pure
9-Ethyl-6-(2-phenylethylamino)-9H-purine (12b). This
compound was synthesized following general method B:
mp 56±56 ꢀC; 1H NMR (Me2SO-d6) d 1.40 (t, 3H,
J=7.3 Hz, CH2CH3), 2.92 (t, 2H, J=7.2 Hz, CH2-Ph),
3.71 (br m, 2H, CH2NH) 4.18 (q, 2H, J=7.3 Hz,
CH2CH3), 7.27 (br m, 5H, H-Ph), 7.74 (br s, 1H, NH),
8.16 (s, 1H, H-8), 8.22 (s, 1H, H-2). Anal. calcd for
C15H17N5: C, 67.39, H, 6.41, N, 26.20; found: C, 67.51,
H, 6.47, N, 25.93.
solid: mp 235±237 ꢀC; H NMR (Me2SO-d6) d 1.35 (t,
1
3H, CH2CH3), 4.09 (q, 2H, CH2CH3), 7.59 (s, 2H, NH2),
8.07 (s, 1H, H-8). Anal. calcd for C7H8IN5: C, 29.08, H,
2.79, N, 24.23; found: C, 29.39, H, 2.95, N, 23.95.

E. Camaioni et al./Bioorg. Med. Chem. 6 (1998) 523±533
531
9-Ethyl-6-(4-phenyl-1-butyn-1-yl)-9H-purine (12c). This
compound was synthesized following general method C:
mp 110±112 ꢀC; 1H NMR (Me2SO-d6) d 1.46 (t, 3H,
J=7.3 Hz, CH2CH3), 2.94 (ps t, 4H, CꢁCCH2CH2),
4.30 (q, 2H, J=7.3 Hz, CH2CH3), 7.34 (br m, 5H, H-
Ph), 8.67 (s, 1H, H-8), 8.84 (s, 1H, H-2). Anal. calcd for
C17H16N4: C, 73.89, H, 5.84, N, 20.27; found: C, 73.96,
H, 6.02, N, 19.88.
m, 5H, H-Ph), 8.85 (s, 1H, H-2). Anal. calcd for
C17H15BrN4: C, 57.48, H, 4.26, N, 15.77; found: C,
57.81, H, 4.38, N, 15.61.
8-Bromo-9-ethyl-6-(1-hexyn-1-yl)-9H-purine (14d). This
compound was synthesized following general method C
to give a chromatographically pure oil; 1H NMR
(Me2SO-d6) d 0.95 (t, 3H, J=7.3 Hz, CH2CH3), 1.37 (t,
3H, J=7.3 Hz, NCH2CH3), 1.45±1.60 (br m, 4H,
CH2CH2CH3), 2.63 (t, 2H, J=6.9 Hz, CꢁCCH2), 4.29
(q, 2H, J=7.2 Hz, NCH2), 8.85 (s, 1H, H-2). Anal. calcd
for C13H15BrN4: C, 58.83, H, 4.92, N, 18.24; found: C,
59.06, H, 5.27, N, 17.97.
9-Ethyl-6-(1-hexyn-1-yl)-9H-purine (12d). This com-
pound was synthesized following general method C to
give a chromatographically pure oil; 1H NMR (Me2SO-
d6) d 0.94 (t, 3H, J=7.1 Hz, CH2CH3), 1.45 (t, 3H,
J=7.2 Hz, NCH2CH3), 1.38±1.61 (br m, 4H,
CH2CH2CH3), 2.62 (t, 2H, J=6.7 Hz, CꢁCCH2), 4.30
(q, 2H, J=7.2 Hz, NCH2CH3), 8.66 (s, 1H, H-8), 8.85
(s, 1H, H-2). Anal. calcd for C13H16N4: C, 68.39, H,
7.06, N, 24.54; found: C, 68.23, H, 7.25, N, 24.32.
Biological studies
[3H]DPCPX, [3H]NECA, [3H]CGS21680, and [32P]ATP
were from Du Pont NEN, Dreieich, Germany, and
[
125I]AB-MECA from Amersham Buchler, Braunsch-
8-Bromo-9-ethyl-6-iodo-9H-purine (13). A mixture of
compound 3 (0.31 g, 1.3 mmol) in dry CH3CN (25 mL),
CH2I2 (8 mL), and iso-pentyl nitrite (0.8 mL, 5.9 mmol)
was heated at 85 ꢀC for 1 h. The solvent was removed
under reduced pressure (oil pump, 60 ꢀC) and the resi-
due was puri®ed by chromatography on a silica gel
column eluting with CHCl3 to give compound 13
weig, Germany. The 96-well microplate ®ltration system
(MultiScreen MAFC) was obtained from Millipore,
Eschborn, Germany. All other materials were from
sources as described earlier.6,22 Of all substances under
investigation 10 mM stock solutions in DMSO were
prepared which were diluted with binding bu€er to
0.4 mM. All other concentrations needed were prepared
by further dilution of the 0.4 mM solution with binding
bu€er.
(0.33 g, 72%) as a pure solid: mp 178±180 ꢀC; H NMR
1
(Me2SO-d6) d 1.37 (t, 3H, J=7.3 Hz, CH2CH3), 4.27 (q,
2H, J=7.3 Hz, CH2CH3), 8.64 (s, 1H, H-2). Anal. calcd
for C7H6BrN4: C, 23.82, H, 1.71, N, 15.87; found: C,
24.14, H, 1.95, N, 15.63.
Cells and membranes
CHO cells were stably transfected with all the human
adenosine receptor subtypes as described for b-adren-
ergic receptors.18 After growing cells on Petri dishes
(é 140 mm) they were washed and frozen in the
dishes until preparation of membranes. Details have
been published elsewhere. Crude membranes were pre-
pared by thawing frozen cells followed by scraping
them o€ the Petri dishes in hypotonic bu€er (5 mM
Tris/HCl, 2 mM EDTA, pH 7.4). The cell suspension
was homogenized (Ultra±Turrax, 2Â15 sec at full
speed) and the homogenate was spun for 10 min at
1,000 g. The supernatant was then centrifuged for
40 min at 50,000 g. The membrane pellet was resus-
pended in 50 mM Tris/HCl bu€er pH 7.4 (for A3
adenosine receptors: 50 mM Tris/HCl, 10 mM MgCl2,
1 mM EDTA, pH 8.25), frozen in liquid nitrogen at a
protein concentration of 1±3 mg/mL and stored at
80 ꢀC.
8-Bromo-9-ethyl-6-(2-phenylethyloxy)-9H-purine (14a).
This compound was synthesized following general
method A: mp 102±104 ꢀC; 1H NMR (Me2SO-d6) d 1.21
(t, 3H, J=7.3 Hz, CH2CH3), 3.17 (t, 2H, J=6.4 Hz,
CH2-Ph), 3.98 (q, 2H, J=7.2 Hz, CH2CH3) 4.81 (t, 2H,
J=6.5 Hz, CH2O), 7.31 (br m, 5H, H-Ph), 8.43 (s, 1H,
H-2). Anal. calcd for C15H15BrN4O: C, 51.89, H, 4.35,
N, 16.14; found: C, 52.16, H, 4.56, N, 15.89.
8-Bromo-9-ethyl-6-(2-phenylethylamino)-9H-purine (14b).
This compound was synthesized following general
method B: mp 114±117 ꢀC; 1H NMR (Me2SO-d6) d 1.33
(t, 3H, J=7.3 Hz, CH2CH3), 2.92 (t, 2H, J=7.3 Hz,
CH2-Ph), 3.72 (br m, 2H, CH2NH) 4.18 (q, 2H,
J=7.1 Hz, CH2CH3), 7.28 (br m, 5H, H-Ph), 8.02 (m,
1H, NH), 8.23 (s, 1H, H-2). Anal. calcd for
C15H16BrN5: C, 52.04, H, 4.36, N, 20.23; found: C,
52.35, H, 4.91, N, 20.02.
Radioligand binding
8-Bromo-9-ethyl-6-(4-phenyl-1-butyn-1-yl)-9H-purine (14c).
This compound was synthesized following general
method C: mp 105±106 ꢀC; 1H NMR (Me2SO-d6) d 1.37
(t, 3H, J=7.2 Hz, NCH2CH3), 2.94 (m, 4H,
CꢁCCH2CH2), 4.29 (q, 2H, J=7.2 Hz, NCH2), 7.33 (br
Dissociation constants (Ki-values) of the 9-ethyladenine
derivatives were determined in radioligand competition
experiments. Ki-values were calculated from IC50-values
according to Cheng and Pruso€.23

532
E. Camaioni et al./Bioorg. Med. Chem. 6 (1998) 523±533
For A1 adenosine receptors [3H]DPCPX was used as an
antagonist radioligand. Binding was carried out as
described previously23 in a total volume of 200 mL in 96-
well microplate ®ltration system (Millipore MultiScreen
MAFC) containing the compound to be tested at dif-
ferent concentrations, 1 nM [3H]DPCPX, 0.2 U/mL
adenosine deaminase and 20 mg of membrane protein in
50 mM Tris/HCl pH 7.4. Samples were incubated for
3 h at 25 ꢀC, ®ltered and washed three times with 200 mL
of ice-cold binding bu€er. After addition of 20 mL of
scintillator to the dried ®lterplates samples were counted
in a Wallac MicroBeta counter.
acknowledged. The authors are involved in the con-
certed-action ADEURO (Biomed 1).
References
1. Muller, C. E.; Scior, T. Pharm. Acta Helv. 1993, 68, 77.
2. (a) Daly, J. W.; Jacobson, K. A. In Adenosine and Adenine
Nucleotides: from Molecular Biology to Integrative Physiology;
Belardinelli, L.; Pelleg, A., Eds.; Kluwer Academic: Boston,
1995; p 157. (b) Fredholm, B.; Abbracchio, M. P.; Burnstock,
G.; Daly, J. W.; Harden, T. K.; Jacobson, K. A. Le€, P.; Wil-
liams, M. Pharmacol. Rev. 1994, 46, 143.
3. Daly, J. W.; Butts-Lamb, P.; Padgett, W. Cell. Mol. Neuro-
biol. 1983, 3, 67.
Conditions for A2A adenosine receptor binding with
[3H]CGS21680 or [3H]NECA (30 nM) as radioligand
were essentially the same as for A1 receptor binding.
Samples with a protein concentration of 50±80 mg were
incubated for 2 h at 25 ꢀC and ®ltered individually as
described for conventional radioligand binding.6
4. Jacobson, K. A.; van Galen, P. J. M.; Williams, M. J. Med.
Chem. 1992, 35, 407.
5. Belardinelli, L.; Shryock, J. C.; Zhang, Y.; Scammells, P. J.;
Olsson, R.; Dennis, D.; Milner, P.; P®ster, J.; Baker, S. P. J.
Pharmacol. Exp. Ther. 1995, 275, 1167.
6. Lohse, M. J.; Klotz, K-N.; Lindenborn-Fotinos J.; Red-
dington M.; Schwabe U.; Olsson R. A. Naunyn-Schmiedeberg's
Arch. Pharmacol. 1987, 336, 204.
A3 adenosine receptor binding was also performed by
incubation and ®ltration of single samples with 125I-AB-
MECA (0.2±0.3 nM) at conditions adopted from Ji et
al.24 As a binding bu€er 50 mM Tris/HCl, 1 mM
EDTA, 10 mM MgCl2, pH 8.25 was used and samples
were incubated for 2 h at 25 ꢀC.
7. (a) Siddiqi, S. M.; Ji, X. D.; Olah, M. E.; Melman, N.; Olah,
M. E.; Jain, R.; Evans, P.; Glashofer, M.; Padgett, W. L.;
Cohen, L. A.; Daly, J. W.; Bellamkonda, Y.; Stiles, G. L.;
Jacobson, K. A. Nucleosides Nucleotides 1996, 15, 693. (b)
Baraldi, P. G.; Cacciari, B.; Spalluto, G.; Borioni, A.; Viziano,
M.; Dionisotti, S.; Ongini, E. Curr. Med. Chem. 1995, 2, 707.
Adenylyl cyclase activity
8. (a) Ongini, E.; Fredholm, B. B. TiPS 1996, 17, 364. (b)
Erfurth, A.; Schmauss, M. Fortschr. Neurol. Psychiatr. 1995,
63, 93.
Due to the lack of a suitable radioligand the anity (Ki,
KB) of the 9-ethyladenine derivatives at A2B adenosine
receptors was determined in adenylyl cyclase experi-
ments. The procedure was carried out as previously
published with minor modi®cations.25 About
100,000 cpm of [a-32P]ATP were used and EGTA and
NaCl were omitted from the incubation mixture. IC50-
values for the concentration-dependent inhibition of
NECA-stimulated (5 mM) adenylyl cyclase were deter-
mined and converted to Ki-values according to Cheng
and Pruso€.23 For this conversion the actual EC50-value
of NECA (2.4 mM) was used instead of the KD. In
selected experiments the concentration-dependent shift
of NECA-stimulated adenylyl cyclase caused by the
9-ethyladenine derivatives was used as a second method
to determine the dissociation constants by Schild plots.
Comparison of the methods con®rmed that the two
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Acknowledgements
This work was supported by grants from MURST and
University of Camerino. The expert technical assistance
of Ms. Sonja Kachler (Universitat of Wurzburg) and
Mrs. Maurizio Brandi, Franco Lupidi and Gianni
Rafaiani (Universita di Camerino) is gratefully
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