Ribose-modified nucleosides as ligands for adenosine receptors: Synthesis, conformational analysis, and biological evaluation of 1′-C-Methyl adenosine analogues

Article abstract of DOI:10.1021/jm0102755

1′-C-Methyl analogues of adenosine and selective adenosine A₁ receptor agonists, such as N-[(1R)-1-methyl-2-phenylethyl]adenosine ((R)-PIA) and N⁶-cyclopentyladenosine, were synthesized to further investigate the subdomain that binds

Full text of DOI:10.1021/jm0102755

1196
J . Med. Chem. 2002, 45, 1196-1202
Ribose-Mod ified Nu cleosid es a s Liga n d s for Ad en osin e Recep tor s: Syn th esis,
Con for m a tion a l An a lysis, a n d Biologica l Eva lu a tion of 1′-C-Meth yl Ad en osin e
An a logu es
Loredana Cappellacci,† Grazia Barboni,† Micaela Palmieri,† Michela Pasqualini,† Mario Grifantini,†
Barbara Costa,‡ Claudia Martini,‡ and Palmarisa Franchetti*,†
Dipartimento di Scienze Chimiche, Universita` di Camerino, 62032 Camerino, Italy and Dipartimento di Psichiatria,
Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, 56126 Pisa, Italy
Received J une 19, 2001
1′-C-Methyl analogues of adenosine and selective adenosine A1 receptor agonists, such as
N-[(1R)-1-methyl-2-phenylethyl]adenosine ((R)-PIA) and N6-cyclopentyladenosine, were syn-
thesized to further investigate the subdomain that binds the ribose moiety. Binding affinities
of these new compounds at A1 and A2A receptors in rat brain membranes and at A3 in rat
testis membranes were determined and compared. It was found that the 1′-C-methyl
modification in adenosine resulted in a decrease of affinity, particularly at A1 and A2A receptors.
When this modification was combined with N6 substitutions with groups that induce high
potency and selectivity at A1 receptors, the high affinity was in part restored and the selectivity
was increased. The most potent compound proved to be the 1′-C-methyl analogue of (R)-PIA
with a Ki of 23 nM for the displacement of [3H]CHA binding from rat brain A1 receptors and
a >435-fold selectivity over A2A receptors. In functional assays, these compounds inhibited
forskolin-stimulated adenylate cyclase with IC50 values ranging from 0.065 to 3.4 µM, acting
as full agonists. Conformational analysis based on vicinal proton-proton J -coupling constants
and molecular mechanics calculations using the MM2 force field proved that the methyl group
on C1′ in adenosine has a pronounced impact on the furanose conformation by driving its
conformational equilibrium toward the north, γ+, syn form.
In tr od u ction
structure of adenosine itself. Substitution at N6 or C2
may enhance affinity and may impart A1/A2/A3 selectiv-
ity.1,13 Other modifications of the adenine moiety usually
lead to inactive compounds or weakly active com-
pounds.13
Adenosine is an endogenous nucleoside with an effect
in multiple physiological processes. Its different actions
are mediated by binding to extracellular receptors
coupled to guanyl nucleotide binding proteins (G-
proteins). The potential of adenosine receptors as drug
targets was reviewed.1 Four adenosine receptor sub-
types have now been characterized and cloned as fol-
lows: A1, A2A, A2B, and A3. The activation of A1 and A3
receptors causes the inhibition of adenylyl cyclase,
activation of phospholipase C, activation of potassium
channels, and inhibition of calcium channels influx. The
activation of A2A and A2B receptors stimulates adenylyl
cyclase via G-protein coupling.1 The A2B receptors also
couple to a phospholipase in human mast cells and may
be important in the mediation of allergic reactions.2
Adenosine receptors are widely distributed throughout
the body, and studies on their structure-activity rela-
tionships have been recently reviewed.1,3 Adenosine
receptor agonists are being studied for their potential
use as various agents: antiarrhythmic,4 antinocicep-
tive,5 antilipolytic (A1 subtype),6 cerebroprotective and
cardioprotective (A1 and A3 subtypes),7 hypotensive,8
Structure-activity relationship studies have pointed
out that the ribose recognition domain of adenosine and
its analogues contributes strongly to affinity at the
adenosine receptor subtypes. So, the removal or inver-
sion of 2′- and 3′-hydroxyl groups were generally not
well-tolerated by the binding site. However, the amide
substitution at the 5′-position as in NECA (5′-N-ethyl-
carboxamidoadenosine) provided increased potency at
A2A receptors. Substitution of the hydrogen atoms of the
ribose ring by a methyl group in adenosine and adeno-
sine analogues afforded compounds with various affinity
and selectivity. Replacement of the 4′-hydrogen by a
methyl group in adenosine was poorly tolerated; yet,
when combined with other favorable modifications, the
potency and selectivity at A3 receptors may be main-
tained.13 The 3′-C-methyl adenosine analogue was found
to bind weakly with A1 selectivity.13
We have reported that the introduction of a methyl
group at the 2′-C position in adenosine resulted in a
decrease of the affinity, particularly at A2A and A3
receptors.14 However, when this modification was com-
bined with N6 substitutions with groups that induce
high potency and selectivity at A1 receptors, the high
affinity was retained and the selectivity was increased.
To further investigate the subdomain that binds the
ribose moiety, in the present study, we report on the
antipsychotic (A2A subtype),9 antiinflammatory (A1, A2A
,
A
2B, and A3 subtypes),10 anticancer (A3),11 and as agents
for the treatment of cystic fibrosis (A2B).12 All known
adenosine agonists are closely related to the chemical
* To whom correspondence should be addressed. Tel: (+39)737-
402228. Fax: (+39)737-637345. E-mail: palmarisa.franchetti@unicam.it.
† Universita` di Camerino.
‡ Universita` di Pisa.
10.1021/jm0102755 CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/13/2002

Ribose-Modified Nucleosides as Ligands
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1197
Sch em e 1a
a
Reagents: (i) NaH, BnBr, THF; (ii) CH3Li, Et2O; (iii) Ac2O, DMAP, pyridine; (iv) 1.8 M EtAlCl2, MeCN; (v) liquid ammonia or RNH2,
EtOH, ∆; (vi) HCOONH4, 10% Pd/C, MeOH, reflux.
synthesis of a series of 1′-C-methyl analogues of ad-
enosine and A1 selective adenosine receptor agonists
(1a -d ).
conformation. Compounds 1a -d proved to be very
unstable in aqueous solutions at pH < 7.
We also attempted another route for the synthesis of
title compounds using the isopropylidene protecting
group instead of the benzylidene one. However, all
attempts of deprotection of isopropylidene derivatives
12a and 12b, prepared by the amination of 6-chloro-
9H-(6-O-benzyl-1-deoxy-3,4-O-isopropylidene-D-psico-
furanosyl)purine (10) followed by debenzylation, with
several Bronsted and Lewis acid catalysts under differ-
ent conditions, resulted in base cleavage.
Ch em istr y
The synthesis of 1′-C-methyladenosine (1a , 1′-Me-
Ado), N6-cyclopentyl-1′-C-methyladenosine (1b, 1′-Me-
CPA), N6-[(1R)-1-methyl-2-phenylethyl]-1′-C-methylad-
enosine (1c, 1′-Me-(R)-PIA), and N6-[(1S)-1-methyl-2-
phenylethyl]-1′-C-methyladenosine (1d , 1′-Me-(S)-PIA)
was carried out as shown in Scheme 1, using 2,3-O-
benzylidene-D-ribono-1,4-lactone (2)15 as the starting
compound. The reaction of endo-5-O-benzyl-2,3-O-ben-
zylidene-D-ribono-1,4-lactone (3), prepared by the ben-
zylation of 2, with methyllithium in diethyl ether, gave
4 as a mixture of R and â anomers. The reaction of 4
with acetic anhydride in pyridine in the presence of
4-(dimethylamino)pyridine afforded the acetoxy deriva-
tive 2-O-acetyl-6-O-benzyl-3,4-O-benzylidene-1-deoxy-
D-psicofuranose (5, anomeric mixture), which was coupled
with 6-chloropurine (6) in dry acetonitrile in the pres-
ence of ethylaluminum dichloride to give 6-chloropurine
ribonucleosides 7 (6-chloro-9H-(6-O-benzyl-3,4-O-ben-
zylidene-1-deoxy-D-psicofuranosyl)purine, â/R, 9:1 ratio).
Amination at the 6-position of derivative 7 with am-
monia, cyclopentylamine, l-amphetamine, or d-amphet-
amine yielded compounds 8a -d and the R anomers 9a -
d . Deprotection of the purified â anomer by catalytic
transfer hydrogenation with ammonium formate in
methanol in the presence of 10% Pd/C gave 1a -d . The
assignment of the â anomeric structure of compounds
8a -d was performed by nuclear Overhauser enhance-
ment (NOE) experiments. In fact, it was found that
when the methyl group hydrogens of 8a -d were irradi-
ated, a NOE effect was observed at the hydrogen atom
of the benzylidene protecting group confirming that the
1′-C-methyl group and the benzylidene one have a cis
Con for m a tion a l An a lysis
Proton nuclear magnetic resonance (1H NMR) data
and NOE experiments were employed to determine the
predominant conformation of the 1′-C-methyl derivative
of adenosine and adenosine analogues in solution. The
relevant H-8 enhancement observed in compounds
1a -d when the methyl group hydrogen atoms of these
nucleosides were irradiated supports a spatial arrang-
ment where H-8 and the methyl group are proximate,
as would be the case in the syn conformer. However,
the observation of a NOE effect also at H-2 when the
methyl group hydrogen atoms of compounds 1a -d were
irradiated, even though inferior to that observed at H-8,
and the appearance of the 5′-hydroxyl resonance as a
triplet16 led us to conclude that a syn conformation in
the low range predominates in solutions of these com-
pounds.
Further information concerning the solution confor-
mation was obtained by the coupling constant values.
Conformational analysis was based on the vicinal
proton-proton J -coupling constants J 2′3′ and J 3′4′ using
D2O as solvent. J 2′3′ values of 5.1-5.2 Hz and J 3′4′ values
of 7.0-7.1 Hz were found, very similar to those reported
for 1′-methyl-â-D-ribofuranosylthymine in the north type
puckered conformation.17 Thus, it can be concluded that

1198 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Cappellacci et al.
F igu r e 1. Molecular mechanics (MM2 force field) optimized structures for 1a (north, γ+, syn), 2′-C-methyladenosine (north, γ+,
anti), and 3′-C-methyladenosine (south, γ+, syn).
Ta ble 1. Affinity of 1′-C-Methyl-adenosine Derivatives in Radioligand Assays at Rat Brain A1 and A2A Receptors and at Rat Testis
A3 Receptorsa-c
Ki (nM) or % displacement at 10-5
M
a
b
c
compd
A1
A2A
A3
A2A/A1
A3/A1
A3/A2A
1a (1′-Me-Ado)
1b (1′-Me-CPA)
1c (1′-Me-(R)-PIA)
1d (1′-Me-(S)-PIA)
2′-Me-Ado
7700 ( 500
100 ( 10
23 ( 2
270 ( 20
740 ( 60
51 000 ( 8300
3%d
7%
0%
8%
0%
19%
>1.30
>100
>435
>37
>1.30
52
29
23
>13.5
>202
5170 ( 420
670 ( 30
6160 ( 480
0%
<0.52
<0.07
<0.62
>2.9
3400 ( 750
4.6
3′-Me-Ado
4′-Me-Ado
12%d
NDe
>202
0%d
NDf
2′-Me-CPA
9.5 ( 0.8
4.7 ( 0.6
50 ( 6
0.3 ( 0.02
0.6 ( 0.02
11 ( 4
7200 ( 930
7400 ( 110
17%
380 ( 80
750 ( 80
1800 ( 570
210 ( 40
1080 ( 260
10 000 ( 2500
260 ( 7
758
1574
>200
1266
1250
164
22.1
230
200
87
88
22
0.029
0.15
<1
0.07
0.07
0.13
2′-Me-(R)-PIA
2′-Me-(S)-PIA
CPA
(R)-PIA
(S)-PIA
53 ( 4g
240 ( 12g
a
b
Displacement of specific [3H]CHA binding in rat brain cortical membranes. Displacement of [3H]CGS21680 binding in rat brain
striatal membranes. c Displacement of [3H](R)-PIA binding in the presence of 150 nM DPCPX in rat testis membranes. Ref 13. e Ref
d
13: percent of displacement of [125I]ABMECA binding in membranes of CHO cells stably transfected with the human A3-cDNA ) 9%.
f Ref 13: percent of displacement of [125I]ABMECA binding in membranes of CHO cells stably transfected with the human A3-cDNA )
g
8%. Ref 19. The values are expressed as Ki ( SEM of three determinations or percent of inhibition of specific radioligand binding at 10
µM compound concentration.
the methyl group on C1′ in 1a -d has a pronounced
impact on the furanose conformation by driving the
conformational equilibrium toward the north form. The
north conformation appears to correspond with the
pseudoequatorial location of the Me group, which is
sterically favored. Figure 1 shows a molecular mechan-
ics (MM2 force field) optimized structure for north, γ+,
syn 1a as compared to that of north, γ+, anti 2′-C-
methyladenosine and south, γ+, syn 3′-C-methyladen-
osine.
enosine) as radioligands.18 Affinity for A3 receptors was
determined in competition assays for the receptor of rat
testis membranes using [3H](R)-PIA in the presence of
the A1 selective antagonists DPCPX (8-cyclopentyl-1,3-
dipropylxanthine).19 The data are summarized in Table
1 in comparison with the nonmethylated analogues and
2′-, 3′-, and 4′-C-methyladenosine.
1′-Me-Ado showed an affinity for A1 receptors (Ki )
7.7 µM), 10-fold lower than that of 2′-Me-Ado and 6.6-
fold superior to that of 3′-C-methyl isomer. At A2A
receptors, the 1′-Me-Ado and its analogues (1a -d ) were
found to have poor or no affinity, similar to the 3′- and
4′-substituted isomers. At A3 receptors, 1′-Me-Ado as
well as 2′-, 3′-, and 4′-Me-Ado showed no affinity.
Biologica l Eva lu a tion a n d Discu ssion
Compound 1a and its N6-substituted derivatives were
tested in radioligand binding assays to determine their
affinities toward adenosine A1, A2A, and A3 receptors.
Affinities for A1 and A2A receptors were determined in
competition assays in rat brain membranes (A1) and rat
brain striatum (A2A) using, respectively, [3H]CHA (N6-
cyclohexyladenosine) and [3H]CGS21680 (2-[4-(2-car-
boxyethyl)phenyl]ethyl-amino-5′-N-ethylcarboxamidoad-
As compared with the adenosine affinity for A1
receptors (10 nM),20 the substitution of hydrogen atoms
of the furanose ring by a methyl group is poorly
tolerated at this receptor subtype, in particular at the
3′- and 4′-position, while a similar substitution at the
1′- and 2′-position brings about a less marked reduction

Ribose-Modified Nucleosides as Ligands
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1199
N6-substituted analogues could depend on the confor-
mations of those molecules. We previously reported that
the high affinity and selectivity for A1 receptors of 2′-
C-methyl-ribosyl analogues of adenosine may be related
to their preferential conformation in solution, which was
determined to be anti with a north (3T2) puckered
furanose ring form, while the poor activity of 3′-C-
methyladenosine might be explained by its marked
preference for the south (2T3) syn conformation, with
pseudoequatorial location of the purine ring (Figure 1).14
We found that 1′-Me-Ado and its N6-substituted ana-
logues have a marked preference for the N-puckered
conformation with the purine ring in the low-range syn
conformation with pseudoaxial location. The preference
of these compounds for the syn conformation of the
purine ring was further confirmed by the fact that 1′-
Me-Ado was neither a substrate nor an inhibitor of
adenosine deaminase.21 So, the greater affinity of 1′- and
2′-C-methyladenosine for the A1 receptor, as compared
to that of 3′-C-methyladenosine, might be explained by
their preference for the N-puckered conformation of the
modified ribose ring. The anti conformation of the
purine ring favors the binding to this receptor subtype,
as proved by the greater affinity of N6-substituted 2′-
methyl analogues of adenosine as compared to that of
their 1′-C-methyl analogues.
In conclusion, the 1′-C-methyl modification in adeno-
sine resulted in a decrease of the affinity, which was
more marked at A1 and A2A receptors. When such
modification was combined with N6 substitutions with
groups that induce high potency and selectivity at A1
receptors, the high affinity was in part retained and the
selectivity increased. The good affinity and selectivity
for A1 receptors of 1′-C-methyl-ribosyl analogues of
N6-substituted adenosine derivatives may be related to
the preferential conformation in solution of the modified
ribose moiety whose puckered furanose ring form was
determined to be north (3T2), similar to that of their 2′-
C-methyl analogues. The lower potency of 1′-methyl-
N6-substituted derivatives as compared to that of the
2′-methyl analogues might be explained by their marked
preference to adopt a syn conformation about the
glycosyl bond, which is not optimal for binding at A1
receptors.
F igu r e 2. Inhibition of adenylyl cyclase in rat cortical
membranes. The assay was carried out as described in
Biological Methods in the presence of 100 µM forskolin. Each
data point is shown as mean ( SEM of at least three
independent experiments. Concentration-dependent effects on
adenylyl cyclase by 1′-Me-CPA (9), 1′-Me-(R)-PIA ([), 1′-Me-
(S)-PIA (1), 1′-Me-Ado (2), and CPA (0).
Ta ble 2. Inhibition of Adenylyl Cyclase Activity in Rat
Cortical Membranes by 1′-C-Methyl-adenosine and Its
Derivatives
compd
CPA
1′-Me-Ado
1′-Me-CPA
1′-Me-(R)-PIA
1′-Me-(S)-PIA
IC50 (nM)a
% maximal inhibition
12 ( 0.7
3440 ( 230
357 ( 23
65 ( 7
15.5 ( 2.6
22.0 ( 1.7
17.4 ( 0.6
23.7 ( 1.8
29.0 ( 3.0
140 ( 10
a
IC50 values were obtained from nonlinear curve fitting of data
using the GraphPad computer program. The maximal inhibitory
effects were at 10 µM for CPA and 100 µM for the other
compounds. All values are the mean ( SEM of three independent
experiments.
of affinity. At A3, a similar substitution was tolerated
only in the case of N6-substituted analogues of 1′-Me-
Ado. The order of selectivity of 1′-Me-Ado was A1 (Ki )
7.7 µM) > A3 (19%) g A2A (7%); for comparison, the order
of selectivity of adenosine was estimated to be A1 (0.01
µM) > A2A (0.03 µM) > A3 (1.0 µM).20 1′-C-Methyl-
substituted analogues of the A1 selective agonists CPA,
(R)-PIA, and (S)-PIA were slightly less potent at A1
receptors than the 2′-C-substituted parent compounds
with Ki values in the submicromolar range. The most
potent compound proved to be 1′-Me-(R)-PIA with an
affinity of 23 nM and a selectivity of >435-fold for A1
vs A2A and 29-fold for A1 vs A3. 1′-Me-CPA was 4.3-fold
less potent than 1′-Me-(R)-PIA and less selective for A1
vs A2A but more selective for A1 vs A3. The stereoselec-
tivity characteristic of the N6 regions of A1 receptors,
similar to that observed for (R)-PIA and (S)-PIA and
their 2′-C-methyl analogues, was maintained.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Bu¨chi
apparatus and are uncorrected. Elemental analyses were
determined on an EA 1108 CHNS-O (Fisons Instruments)
analyzer. Thin-layer chromatography was run on silica gel 60
F254 plates (Merck); silica gel 60 (70-230 and 230-400 mesh,
Merck) for column chromatography was used. 1H NMR and
13C NMR spectra were determined with a Varian VXR-300
spectrometer at 300 and 75 MHz, respectively. The chemical
shift values are expressed in δ values (parts per million)
relative to tetramethylsilane as an internal standard. All
exchangeable protons were confirmed by the addition of D2O.
Stationary NOE experiments were run on degassed solutions
at 25 °C. A presaturation delay of 1 s was used, during which
the decoupler low power was set at 20 dB attenuation. Mass
spectroscopy was carried out on an HP 1100 series instrument.
All measurements were performed in the positive ion mode
using an atmospheric pressure electrospray ionization.
5-O-Ben zyl-2,3-O-ben zylid en e-D-r ibon o-1,4-la cton e (3).
To a cooled (0 °C) solution of 2 (4.0 g, 16.93 mmol) in anhydrous
tetrahydrofuran (THF, 50 mL), a 60% NaH dispersion (812
mg, 20.29 mmol) was added portionwise over 15 min. Benzyl
Compounds 1a -d were also tested in a functional
assay at A1 receptors in rat cortical membranes for the
ability to inhibit forskolin-stimulated adenylyl cyclase.
The efficacy of these compounds was compared with that
obtained for CPA (Figure 2). Compounds 1a -d ap-
peared to be full agonists, with an inhibition of cyclase
with IC50 values ranging from 0.065 to 3.4 µM (Table
2). Statistical analysis of the results revealed that there
were significant differences between the maximal inhi-
bition of adenylyl cyclase activity values obtained for
1′-Me-(S)-PIA and CPA (p < 0.05).
The affinity of the adenosine receptors for 1a and its

1200 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Cappellacci et al.
bromide (2.41 mL, 20.29 mmol) was then added dropwise at
the same temperature. The reaction mixture was allowed to
warm slowly to room temperature and then was stirred
overnight. Ice was added slowly, and the mixture was then
poured into ice-water. After the mixture was extracted with
CH2Cl2 (3 × 40 mL), the organic layer was washed with 10%
brine and water, dried over anhydrous Na2SO4, and evaporated
to dryness. The residue was purified by flash chromatography
on silica gel eluting with petroleum ether/ethyl acetate (5:1)
1H, H-5′), 5.0 (d, J ) 6.2 Hz, 1H, H-4′), 5.20 (dd, J ) 4.6, 6.0
Hz, 1H, H-3′), 5.55 (s, 1H, CHC6H5), 7.15-7.40 (m, 12H, arom.,
NH2), 8.10 (s, 1H, H-2), 8.28 (s, 1H, H-8). Anal. (C25H25N5O4)
C, H, N.
N6-Cyclop en t yl-9H -(6-O-b en zyl-3,4-O-b en zylid en e-1-
d eoxy-â-D-p sicofu r a n osyl)a d en in e (8b) a n d N6-Cyclop en -
tyl-9H-(6-O-ben zyl-3,4-O-ben zylid en e-1-d eoxy-r-D-p sico-
fu r a n osyl)a d en in e (9b). A mixture of 7 (375 mg, 0.78 mmol)
in anhydrous EtOH (10 mL) and cyclopentylamine (0.46 mL,
4.68 mmol) under a nitrogen atmosphere was refluxed for 1.5
h. The mixture was concentrated in vacuo, and the residue
was purified by flash chromatography on silica gel (n-hex-
anes-EtOAc, 70:30). As the first eluate, 8b was separated as
1
to give 3 as an oil (2.7 g, 49%). H NMR (dimethyl sulfoxide
(DMSO)-d6): δ 3.70 (dd, J ) 2.1, 10.8 Hz, 1H, H-5), 3.80 (dd,
J ) 2.6, 10.8 Hz, 1H, H-5), 4.56 (s, 2H, CH2C6H5), 4.91 (d, J )
5.7 Hz, 1H, H-2), 4.95 (t, J ) 2.3 Hz, 1H, H-4), 5.02 (s, J ) 5.7
Hz, 1H, H-3), 6.0 (s, 1H, CH(C6H5)), 7.30-7.45 (m, 10H, arom).
Anal. (C19H18O5) C, H, N.
1
a foam (313 mg, 76%). H NMR (DMSO-d6): δ 1.53-1.70 (m,
6H, cyclopentyl), 1.80 (s, 3H, H-1′), 1.95 (m, 2H, cyclopentyl),
3.55 (m, 2H, H-6′), 4.40 (s, 2H, CH2C6H5), 4.60 (m, 1H, NHCH),
4.65 (m, 1H, H-5′), 5.02 (dd, J ) 2.5, 5.9 Hz, 1H, H-4′), 5.72
(d, J ) 6.5 Hz, 1H, H-3′), 6.0 (s, 1H, CHC6H5), 7.05-7.25 (2m,
5H, CHC6H5), 7.40-7.60 (2m, 5H, CH2C6H5), 7.70 (d, J ) 7.9
Hz, 1H, NH), 8.20 (s, 2H, H-2, H-8). Anal. (C30H33N5O4) C, H,
N.
2-O-Acetyl-6-O-ben zyl-3,4-O-ben zyliden e-1-deoxy-D-psi-
cofu r a n ose (5). To a stirred solution of 1.4 M methyllithium
in diethyl ether (7.8 mL) at -78 °C under a nitrogen atmo-
sphere was added dropwise a solution of 3 (2.54 g, 7.80 mmol)
in anhydrous diethyl ether (70 mL). The mixture was reacted
for 45 min and then warmed to 0 °C and treated with 10%
aqueous NH4Cl (70 mL). After the mixture was extracted with
diethyl ether (3 × 100 mL), the combined organic layers were
washed with ice-cold water (2 × 70 mL) and dried over
anhydrous Na2SO4. Filtration and concentration under reduced
pressure yielded an oil (4), which was used for the next step
without purification. The oil was dried with anhydrous pyri-
dine (3 × 10 mL) and was dissolved in pyridine (20 mL), and
then, acetic anhydride (0.68 mL, 7.20 mmol) and 4-(dimethyl-
amino)pyridine (42 mg, 0.34 mmol) were added. The reaction
mixture was stirred at room temperature for 48 h, and then,
ice-cold water (60 mL) was added. The aqueous phase was
extracted with CHCl3 (3 × 40 mL), and the combined organic
layers were washed with a cold, saturated NaHCO3 solution
and with ice-cold water (3 × 40 mL) and dried over anhydrous
Na2SO4. Evaporation to dryness yielded an oily residue that
was purified by flash chromatography on silica gel (n-hex-
Αs the second eluate, 9b was obtained as a foam (25 mg,
1
6%). H NMR (DMSO-d6): δ 1.65, 1.90 (2m, 8H, cyclopentyl),
2.05 (s, 3H, H-1′), 3.72 (d, J ) 4.8 Hz, 2H, H-6′), 4.22 (t, J )
6.3 Hz, 1H, H-5′), 4.55 (m, 1H, NHCH), 4.65 (s, 2H, CH2C6H5),
4.80 (m, 1H, H-4′), 5.05 (s, 1H, H-3′), 5.80 (s, 1H, CHC6H5),
7.15-7.45 (m, 10H, CHC6H5, CH2C6H5), 7.55 (d, J ) 8.4 Hz,
1H, NH), 8.10 (s, 1H, H-2), 8.18 (s, 1H, H-8). Anal. (C30H33N5O4)
C, H, N.
N6-[(1R)-1-Met h yl-2-p h en ylet h yl]-9H -(6-O-b en zyl-3,4-
O-ben zylid en e-1-d eoxy-â-D-p sicofu r a n osyl)a d en in e (8c)
a n d N6-[(1R)-1-Meth yl-2-p h en yleth yl]-9H-(6-O-ben zyl-3,4-
O-ben zylid en e-1-d eoxy-r-D-p sicofu r a n osyl)a d en in e (9c).
Compound 7 (410 mg, 0.86 mmol) was treated with l-amphet-
amine as described for 8b (reaction time 6 h). The crude
product was chromatographed on a silica gel column (CHCl3-
EtOAc, 95:5) to give 8c as the first eluate (50%) and 9c as the
second eluate (8.0%), both as a foam.
1
anes-EtOAc, 90:10) to give 5 as a clear oil (1.40 g, 47%). H
8c: 1H NMR (DMSO-d6): δ 1.20 (d, J ) 6.6 Hz, 3H, CHCH3),
1.80 (s, 3H, H-1′), 2.75, 3.0 (2m, 2H, CH2C6H5), 3.47 (dd, J )
7.6, 10.8 Hz, 1H, H-6′), 3.60 (dd, J ) 4.8, 10.6 Hz, 1H, H-6′),
4.40 (s, 2H, CH2C6H5) 4.65 (m, 2H, H-5′, CHCH3), 5.05 (dd, J
) 2.5, 5.9 Hz, 1H, H-4′), 5.56 (d, J ) 5.9 Hz, 1H, H-3′), 6.15 (s,
1H, CHC6H5), 7.20 (m, 5H, CH2C6H5), 7.40-7.55 (2m, 5H,
CHC6H5), 7.70 (d, J ) 8.0 Hz, 1H, NH), 8.20 (s, 2H, H-2, H-8).
Anal. (C34H35N5O4) C, H, N.
9c: 1H NMR (DMSO-d6): δ 1.20 (d, J ) 6.8 Hz, 3H, CHCH3),
2.0 (s, 3H, H-1′), 2.70 (dd, J ) 6.8, 13.4 Hz, 1H, CH2C6H5), 3.0
(dd, J ) 7.0, 13.6 Hz, 1H, CH2C6H5), 3.95 (m, 2H, H-6′), 4.50
(m, 1H, CHCH3), 4.60 (s, 2H, CH2C6H5) 4.75 (q, J ) 4.6 Hz,
2H, H-5′), 5.0 (d, J ) 5.8 Hz, 1H, H-4′), 5.20 (dd, J ) 4.0, 5.8
Hz, 1H, H-3′), 5.52 (s, 1H, CHC6H5), 7.25 (m, 10H, CH2C6H5,
CHC6H5), 7.80 (d, J ) 8.0 Hz, 1H, NH), 8.15 (s, 1H, H-2), 8.20
(s, 1H, H-8). Anal. (C34H35N5O4) C, H, N.
N6-[(1S)-1-Meth yl-2-p h en yleth yl]-9H-(6-O-ben zyl-3,4-O-
ben zyliden e-1-deoxy-â-D-psicofu r an osyl)aden in e (8d ) an d
N6-[(1S)-1-Met h yl-2-p h en ylet h yl]-9H -(6-O-b en zyl-3,4-O-
b en zylid en e-1-d eoxy-r-D-p sicofu r a n osyl)a d en in e (9d ).
Compounds 8d and 9d were obtained from 7 (360 mg, 0.75
mmol) with d-amphetamine as described above (reaction time
15 h). Chromatography on a silica gel column (CHCl3-EtOAc,
95:5) gave 8d as the first eluate (69%) as a foam. Compound
9d was also obtained as a foam (5%).
8d : 1H NMR (DMSO-d6): δ 1.20 (d, J ) 6.2 Hz, 3H,
CHCH3),1.80 (s, 3H, H-1′), 2.75 (dd, J ) 6.6, 13.5 Hz, 1H,
CH2C6H5), 3.05 (dd, J ) 7.5, 13.4 Hz, 1H, CH2C6H5), 3.50 (m,
2H, H-6′), 4.42 (s, 2H, CH2C6H5) 4.65 (m, 2H, H-5′, CHCH3),
5.0 (dd, J ) 2.6, 5.9 Hz, 1H, H-4′), 5.80 (d, J ) 6.0 Hz, 1H,
H-3′), 6.15 (s, 1H, CHC6H5), 7.25 (m, 5H, CH2C6H5), 7.40-7.55
(2m, 5H, CHC6H5), 7.70 (d, J ) 7.6 Hz, 1H, NH), 8.20 (s, 2H,
H-2, H-8). Anal. (C34H35N5O4) C, H, N.
NMR (DMSO-d6): δ 1.68 (s, 3H, H-1), 1.92 (s, 3H, OCOCH3),
3.56 (d, J ) 6.9 Hz, 2H, H-6), 4.40 (t, J ) 7.0 Hz, 1H, H-5),
4.57 (s, 2H, CH2C6H5), 4.84 (d, J ) 6.2 Hz, 1H, H-4), 4.93 (d,
J ) 6.2 Hz, 1H, H-3), 5.85 (s, 1H, CHC6H5), 7.30-7.50 (2m,
10H, arom). Anal. (C22H24O6) C, H, N.
6-Ch lor o-9H-(6-O-ben zyl-3,4-O-ben zylid en e-1-d eoxy-D-
p sicofu r a n osyl)p u r in e (7). To a stirred mixture of 5 (1.35
g, 3.51 mmol) and 6 (1.18 g, 7.65 mmol) in anhydrous
acetonitrile (35 mL) was added dropwise a 1.8 M solution of
EtAlCl2 in toluene (1.8 mL). The reaction mixture was stirred
at room temperature for 2 h and then poured into an ice-cold
mixture of saturated NaHCO3 solution (140 mL) and CH2Cl2
(240 mL). After the mixture was stirred (10 min), the resulting
solution was filtered through a pad of Celite and the sep-
arated organic layer was washed with a saturated solution of
NaHCO3 (120 mL) and brine (2 × 120 mL), dried over an-
hydrous Na2SO4, and concentrated under reduced pressure.
The oily residue was purified by flash chromatography on silica
gel (n-hexanes-EtOAc, 75:25) to give 7 as a foam containing
the inseparable R,â anomers (85% yield).
9H -(6-O-Ben zyl-3,4-O-b en zylid en e-1-d eoxy-â-D-p sico-
fu r a n osyl)a d en in e (8a ) a n d 9H-(6-O-Ben zyl-3,4-O-ben -
zylid en e-1-d eoxy-r-D-p sicofu r a n osyl)a d en in e (9a). A mix-
ture of 7 (285 mg, 0.59 mmol) and liquid ammonia (20 mL)
was reacted in a Parr bomb at 60 °C for 5 h. Ammonia was
removed, and the residue was purified by flash chromatogra-
phy on silica gel (CHCl3-MeOH, 97.3) to give 8a and 9a in a
9:1 ratio (76% overall yield).
8a : 1H NMR (DMSO-d6): δ 1.80 (s, 3H, H-1′), 3.50 (m, 2H,
H-6′), 4.42 (s, 2H, CH2C6H5), 4.65 (m, 1H, H-5′), 5.10 (dd, J )
2.5, 5.9 Hz, 1H, H-4′), 5.57 (d, J ) 5.9 Hz, 1H, H-3′), 6.15 (s,
1H, CHC6H5), 7.30 (m, 7H, CHC6H5, NH2), 7.40-7.60 (2m,
5H, CH2C6H5), 8.15 (s, 1H, H-2), 8.22 (s, 1H, H-8). Anal.
(C25H25N5O4) C, H, N.
9d : 1H NMR (DMSO-d6): δ 1.20 (d, J ) 6.6 Hz, 3H,
CHCH3), 2.0 (s, 3H, H-1′), 2.70 (dd, J ) 6.6, 13.2 Hz, 1H,
CH2C6H5), 3.05 (dd, J ) 7.2, 13.4 Hz, 1H, CH2C6H5), 3.90 (m,
9a : 1H NMR (DMSO-d6): δ 2.05 (s, 3H, H-1′), 3.75 (d, J )
4.8 Hz, 2H, H-6′), 4.65 (s, 2H, CH2C6H5), 4.78 (q, J ) 4.4 Hz,

Ribose-Modified Nucleosides as Ligands
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1201
2H, H-6′), 4.60 (m, 1H, CHCH3), 4.62 (s, 2H, CH2C6H5), 4.75
(q, 1H, H-5′), 5.0 (d, J ) 5.9 Hz, 1H, H-4′), 5.20 (dd, J ) 4.2,
5.8 Hz, 1H, H-3′), 5.55 (s, 1H, CHC6H5), 7.10-7.40 (m, 10H,
CH2C6H5, CHC6H5), 7.62 (d, J ) 7.7 Hz, 1H, NH), 8.15 (s, 1H,
H-2), 8.25 (s, 1H, H-8). Anal. (C34H35N5O4) C, H, N.
138.8 (C6H5), 139.7 (C-8), 147.1 (C-4), 152.1 (C-2), 154.1 (C-6).
MS: m/z 400.2 [MH]+, 422.2 [M + Na+]. Anal. (C20H25N5O4)
C, H, N.
6-Ch lor o-9H-(6-O-ben zyl-1-deoxy-3,4-O-isopr opyliden e-
D-p sicofu r a n osyl)p u r in e (10). The title compound was
prepared from 2-O-acetyl-6-O-benzyl-1-deoxy-3,4-O-isopropyl-
idene-D-psicofuranose22 (1.2 g, 3.4 mmol) as described for 7
(reaction time 1.5 h). After work up, 10 as an inseparable R,â
mixture was obtained (78% yield).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
1a -d . Compounds 1a -d were obtained from 8a -d (0.85
mmol) in MeOH (30 mL) by treatment with 10% Pd/C in the
presence of ammonium formate (6.8 mmol) under reflux. After
the compounds were cooled at room temperature, the mixture
was filtered and the filtrate was evaporated to dryness and
purified by chromatography on silica gel to give the desired
compounds.
9H-(6-O-Ben zyl-1-d eoxy-3,4-O-isop r op ylid en e-â-D-p si-
cofu r a n osyl)a d en in e (11a ). Compound 11a was obtained
from 10 (600 mg, 1.39 mmol) as described for 8a (reaction time
7 h) and was purified by chromatography on a silica gel column
(CHCl3-MeOH, 98:2) (foam , 71%). 1H NMR (DMSO-d6): δ
1.35, 1.53 (2s, 6H, C(CH3)2), 1.65 (s, 3H, H-1′), 3.42 (dd, J )
4.9, 10.4 Hz, 1H, H-6′), 3.52 (dd, J ) 3.2,10.5 Hz, 1H, H-6′),
4.38 (s, 2H, CH2C6H5), 4.51 (t, J ) 5.1 Hz, 1H, H-5′), 4.80 (dd,
J ) 1.5, 6.0 Hz, 1H, H-4′), 5.51 (d, J ) 6.0 Hz, 1H, H-3′), 7.10,
7.25 (2m, 7H, CH2C6H5, NH2), 8.15 (s, 1H, H-2), 8.20 (s, 1H,
H-8). Anal. (C21H25N5O4) C, H, N.
N6-Cyclop en t yl-9H -(6-O-b en zyl-1-d eoxy-3,4-O-isop r o-
p ylid en e-â-D-p sicofu r a n osyl)a d en in e (11b). The title com-
pound was obtained from 10 (500 mg, 1.16 mmol) as described
for 8b and was purified by flash chromatography on silica gel
(CH2Cl2-EtOH, 99.2:0.8) (foam, 75%). 1H NMR (CDCl3): δ
1.40, 1.60 (2s, 6H, C(CH3)2), 1.53-1.70 (m, 6H, cyclopentyl),
1.75 (s, 3H, H-1′), 2.10 (m, 2H, cyclopentyl), 3.45 (dd, J ) 5.3,
10.5 Hz, 1H, H-6′), 3.55 (dd, J ) 3.7, 10.4 Hz, 1H, H-6′), 4.35
(q, J ) 11.9 Hz, 2H, CH2C6H5), 4.51 (m, 1H, H-5′), 4.55 (dd, J
) 1.5, 6.1 Hz, 1H, H-4′), 4.60 (m, 1H, NHCH), 5.60 (d, br d, J
) 6.1 Hz, 2H, H-3′, NH), 7.0, 7.18 (2m, 5H, CH2C6H5), 8.0 (s,
1H, H-2), 8.40 (s, 1H, H-8). Anal. (C26H33N5O4) C, H, N.
9H-(1-Deoxy-3,4-O-isop r op ylid en e-â-D-p sicofu r a n osyl)-
a d en in e (12a ). The title compound was obtained from 11a
(350 mg, 0.85 mmol) as described for 1a (reaction time 1 h).
Chromatography on a silica gel column (CHCl3-MeOH, 92:8)
gave 12a as a foam (81%).1H NMR (DMSO-d6): δ 1.33, 1.53
(2s, 6H, C(CH3)2), 1.67 (s, 3H, H-1′), 3.38 (m, 2H, H-6′), 4.28
(t, J ) 6.6 Hz, 1H, H-5′), 4.76 (dd, J ) 1.8, 6.2 Hz, 1H, H-4′),
5.08 (t, J ) 5.1 Hz, 1H, OH), 5.50 (d, J ) 6.2 Hz, 1H, H-3′),
7.25 (br s, 2H, NH2), 8.15 (s, 1H, H-2), 8.22 (s, 1H, H-8). Anal.
(C14H19N5O4) C, H, N.
9H-(1-Deoxy-â-D-p sicofu r a n osyl)a d en in e (1a ). The title
compound was synthesized from 8a (reaction time 1 h) and
chromatographed on a silica gel column (CHCl3-MeOH, 85:
15) to give a white solid (57%); mp >230 °C dec. 1H NMR
(DMSO-d6): δ 1.72 (s, 3H, H-1′), 3.50 (m, 2H, H-6′), 3.75-4.0
(m, 2H, H-5′, H-4′), 4.60 (t, J ) 4.7 Hz, 1H, H-3′), 5.0 (d, J )
6.5 Hz, 1H, OH), 5.22 (t, J ) 5.5 Hz, 1H, OH), 5.70 (d, J ) 5.0
Hz, 1H, OH), 7.25 (br s, 2H, NH2), 8.12 (s, 1H, H-2), 8.40 (s,
1H, H-8). 13C NMR (DMSO-d6): δ 22.6 (C-1′), 60.4 (C-6′), 69.3
(C-4′), 74.6 (C-3′), 83.7 (C-5′), 97.2 (C-2′), 120.4 (C-5), 139.1
(C-8), 148.0 (C-4), 152.1 (C-2), 156.4 (C-6). MS: m/z 282.1
[MH]+, 304.1 [M + Na+], 320.1 [M + K+]. Anal. (C11H15N5O4)
C, H, N.
N6-Cyclop en t yl-9H -(1-d eoxy-â-D-p sicofu r a n osyl)a d e-
n in e (1b). Compound 1b was synthesized from 8b (reaction
time 1.5 h). Purification by chromatography on a silica gel
column (CHCl3-MeOH, 95:5) gave 1b as a white solid (60%);
mp 175-177 °C. 1H NMR (DMSO-d6): δ 1.60 (m, 6H, cyclo-
pentyl), 1.75 (s, 3H, H-1′), 1.90 (m, 2H, cyclopentyl), 3.55 (m,
2H, H-6′), 3.70-4.0 (m, 2H, H-5′, H-4′), 4.55 (m, 1H, NHCH),
4.60 (t, J ) 4.6 Hz, 1H, H-3′), 4.98 (d, J ) 6.3 Hz, 1H, OH),
5.20 (t, J ) 6.3 Hz, 1H, OH), 5.70 (d, J ) 5.0 Hz, 1H, OH),
7.65 (d, J ) 8.6 Hz, 1H, NH), 8.20 (s, 1H, H-2), 8.40 (s, 1H,
H-8). 13C NMR (DMSO-d6): δ 22.6 (C-1′), 23.7 (2C, cyclopentyl),
29.9 (2C, cyclopentyl), 32.7 (cyclopentyl), 60.4 (C-6′), 69.3
(C-4′), 74.6 (C-3′), 83.7 (C-5′), 97.2 (C-2′), 120.5 (C-5), 138.7
(C-8), 148.2 (C-4), 152.0 (C-2), 154.6 (C-6). MS: m/z 350.3
[MH]+, 372.2 [M + Na+], 388.2 [M + K+]. Anal. (C16H23N5O4)
C, H, N.
N6-[(1R)-1-Meth yl-2-p h en yleth yl]-9H-(1-d eoxy-â-D-p si-
cofu r a n osyl)a d en in e (1c). Compound 1c was synthesized
from 8c (reaction time 2.5 h) and chromatographed on a silica
gel column (CHCl3-MeOH, 90:10) (white solid, 52%); mp 128-
130 °C. 1H NMR (DMSO-d6): δ 1.20 (d, J ) 6.5 Hz, 3H,
CHCH3), 1.75 (s, 3H, H-1′), 2.75 (dd, J ) 6.7, 13.1 Hz, 1H,
CH2C6H5), 3.0 (dd, J ) 7.6, 13.4 Hz, 1H, CH2C6H5), 3.55 (m,
2H, H-6′), 3.70-4.0 (m, 3H, H-5′, H-4′, CHCH3), 4.60 (t, J )
4.7 Hz, 1H, H-3′), 5.0 (d, J ) 6.2 Hz, 1H, OH), 5.20 (t, J ) 5.5
Hz, 1H, OH), 5.70 (d, J ) 5.1 Hz, 1H, OH), 7.15, 7.25 (2m,
5H, CH2C6H5), 7.65 (d, J ) 8.3 Hz, 1H, NH), 8.15 (s, 1H, H-2),
8.40 (s, 1H, H-8). 13C NMR (DMSO-d6): δ 20.4 (C-1′), 22.6
(CHCH3), 41.9 (CH2C6H5), 47.2 (CHNH), 60.3 (C-6′), 69.2
(C-4′), 74.4 (C-3′), 83.6 (C-5′), 97.2 (C-2′), 120.4 (C-5), 126.2
(C6H5), 128.4 (2C, C6H5), 129.3 (2C, C6H5), 138.8 (C6H5), 139.7
(C-8), 147.1 (C-4), 152.1 (C-2), 154.1 (C-6). MS: m/z 400.2
[MH]+, 422.2 [M + Na+]. Anal. (C20H25N5O4) C, H, N.
N6-[(1S)-1-Meth yl-2-p h en yleth yl]-9H-(1-d eoxy-â-D-p si-
cofu r a n osyl)a d en in e (1d ). The title compound was obtained
from 8d (reaction time 3.5 h) and purified by chromatography
on a silica gel column (CHCl3-MeOH, 90:10) (white solid,
50%); mp 128-130 °C. 1H NMR (DMSO-d6): δ 1.20 (d, J )
6.3 Hz, 3H, CHCH3),1.75 (s, 3H, H-1′), 2.75, 3.0 (2m, 2H,
CH2C6H5), 3.55 (m, 2H, H-6′), 3.70-4.0 (m, 3H, H-5′, H-4′,
CHCH3), 4.60 (t, J ) 4.7 Hz, 1H, H-3′), 5.0 (d, J ) 6.2 Hz, 1H,
OH), 5.20 (t, J ) 5.1 Hz, 1H, OH), 5.70 (d, J ) 4.8 Hz, 1H,
OH), 7.15, 7.25 (2m, 5H, CH2C6H5), 7.65 (d, J ) 8.4 Hz, 1H,
NH), 8.15 (s, 1H, H-2), 8.40 (s, 1H, H-8). 13C NMR (DMSO-
d6): δ 20.4 (C-1′), 22.6 (CHCH3), 41.9 (CH2C6H5), 47.2 (CHNH),
60.3 (C-6′), 69.2 (C-4′), 74.4 (C-3′), 83.6 (C-5′), 97.2 (C-2′),
120.4 (C-5), 126.2 (2C, C6H5), 128.4 (2C, C6H5), 129.3 (C6H5),
N6-Cyclop en tyl-9H-(1-d eoxy-3,4-O-isop r op ylid en e-â-D-
p sicofu r a n osyl)a d en in e (12b). The title compound was
prepared from 11b (350 mg, 0.73 mmol), (reaction time 1.5 h)
and was purified by chromatography on a silica gel column
(CH2Cl2-EtOH, 98:2) (foam, 70%). 1H NMR (DMSO-d6): δ
1.33, 1.53 (2s, 6H, C(CH3)2), 1.55-1.70 (m, 6H, cyclopentyl),
1.67 (s, 3H, H-1′), 1.90 (m, 2H, cyclopentyl), 3.40 (m, 2H, H-6′),
4.28 (t, J ) 6.6 Hz, 1H, H-5′), 4.53 (m, 1H, NHCH), 4.76 (dd,
J ) 1.8, 6.2 Hz, 1H, H-4′), 5.08 (t, J ) 5.1 Hz, 1H, OH), 5.50
(d, J ) 6.2 Hz, 1H, H-3′), 7.65 (d, J ) 8.6 Hz, 1H, NH), 8.20
(s, 1H, H-2), 8.22 (s, 1H, H-8). Anal. (C19H27N5O4) C, H, N.
Biologica l Met h od s. Ma t er ia ls. [3H]-(R)-PIA (37 Ci/
mmol), [3H]cAMP (adenosine 3′,5′-cyclic monophosphate, 25
Ci/mmol), and [R-32P]ATP (adenosine 5′-triphosphate, 30-40
Ci/mmol) were from Amersham Corp., while [3H]CHA (sp. act.
32.5 Ci/mmol) and [3H]CGS21680 (37.5 Ci/mmol) were pur-
chased from NEN Life Science Products, Inc. CPA, (R)-PIA,
NECA, and other agents were purchased from RBI. Forskolin
and guanosine 5′-triphosphate were from Sigma-Aldrich Srl.
Myokinase, creatine kinase, and adenosine deaminase were
obtained from Boehringer-Mannheim (Mannheim, Germany).
All other reagents were from standard commercial sources and
of the highest grade commercially available.
Recep tor Bin d in g Assa y. Rat membranes of the cerebral
cortex, cerebral striatum, and testis were prepared as previ-
ously described.14 Rat cortical membranes were suspended in
10 volumes of ice-cold buffer A (1 mM ethylenediaminetet-
raacetic acid (EDTA), 5 mM MgCl2, and 50 mM Tris/HCl, pH
7.7) and homogenized, and binding of [3H]CHA to A1 receptors
was measured in triplicate, as previously described.18a Rat
striatal membranes were suspended in 20 volumes of buffer
B (10 mM MgCl2, 50 mM Tris/HCl, pH 7.4) and homogenized,

1202 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Cappellacci et al.
and binding of [3H]CGS21680 to A2a receptors was performed
as previously described.18b Rat testis membranes were sus-
pended in 15 volumes of buffer C (1 mM EDTA, 10 mM MgCl2,
and 50 mM Tris/HCl, pH 7.4) and homogenized, and binding
of [3H](R)-PIA to A3 adenosine receptors was measured in the
presence of DPCPX (150 nM) to block A1 adenosine receptors,
as previously described.19 Compounds were dissolved in DMSO
and then diluted with assay buffer, and final DMSO concen-
trations never exceeded 1%. At least six different concentra-
tions of each compound were used. IC50 values, computer-
generated using a nonlinear regression formula on a computer
program (GraphPad, San Diego, CCA), were converted to Ki
values, knowing the Kd values of radioligands in these different
tissues and using the Cheng and Prusoff equation.23
Ad en ylyl Cycla se Assa y. The cerebral cortex was obtained
from male Sprague-Dawley rats sacrificed by cervical disloca-
tion. Fresh tissue was used for membrane preparation, per-
formed as previously described.14 Adenylyl cyclase activity was
measured by monitoring the conversion of [R-32P]ATP to [R-32P]-
cAMP, using a previously reported method.23 The method
involved the addition of [R-32P]ATP to membranes in the
presence of forskolin to stimulate adenylyl cyclase and papav-
erine as a phosphodiesterase inhibitor. The assay was per-
formed as previously described.14 Compounds tested as inhibi-
tors of forskolin-stimulated adenylyl cyclase activity were
dissolved in DMSO and then diluted with 50 mM N-(2-
hydroxyethyl)piperazine-N′-ethanesulfonic acid/NaOH buffer,
pH 7.4, so that the final DMSO concentration never exceeded
1%. IC50 values were calculated using a nonlinear regression
analysis (GraphPad).24
J .; Linden, J .; Abraham, W. M. Pharmacology of 2-cyclohexyl-
methylidene-hydrazinoadenosine (WRC-0470), a novel, short-
acting adenosine A2A receptor agonist that produces selective
coronary vasodilation. Drug Dev. Res. 1997, 40, 313-324.
(9) Dixon, D. A.; Fenix, L. A.; Kim, D. M.; Raffa, R. B. Indirect
modulation of dopamine D2 receptors as potential pharmaco-
therapy for schizophrenia: I. Adenosine agonists. Ann. Phar-
macother. 1999, 33, 480-488.
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