Synthesis of hypermodified adenosine derivatives as selective adenosine A3 receptor ligands

Article abstract of DOI:10.1016/j.bmc.2005.09.062

We investigated the A₃AR affinity and selectivity of a series of 2-substituted 3′-azido and 3′-amino adenosine derivatives as well as some 5′-uronamide derivatives thereof. All compounds showed high A ₃AR selectivity. While the 3′-az

Full text of DOI:10.1016/j.bmc.2005.09.062

Bioorganic & Medicinal Chemistry 14 (2006) 1403–1412
Synthesis of hypermodified adenosine derivatives
as selective adenosine A3 receptor ligands
Liesbet Cosyn,a Zhan-Guo Gao,b Philippe Van Rompaey,a Changrui Lu,b
Kenneth A. Jacobsonb,* and Serge Van Calenbergha,*
aLaboratory for Medicinal Chemistry (FFW), UGent, Harelbekestraat 72, B-9000, Belgium
bMolecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health (NIH), DHHS, Bldg. 8A, Rm. B1 A-19, NIH,
NIDDK, LBC, Bethesda, MD 20892-0810, USA
Received 4 July 2005; revised 21 September 2005; accepted 27 September 2005
Available online 2 November 2005
Abstract—We investigated the A3AR affinity and selectivity of a series of 2-substituted 30-azido and 30-amino adenosine derivatives
as well as some 50-uronamide derivatives thereof. All compounds showed high A3AR selectivity. While the 30-azides appeared to be
A3AR antagonists with moderate A3AR affinity, their 30-amino congeners exhibit significantly improved A3AR affinity and behave
as partial agonists. For both the 30-azides and the 30-amines, the 50-methylcarbamoyl modification improved the overall affinity.
Introduction of a 2-phenylethynyl substituent provided high affinity for the A3AR.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
receptor subtype and tissue to be of therapeutic value.12
Numerous structure–activity studies of adenosine deri-
vatives as receptor agonists conclude that selectivity
may be provided by specific substitutions of the adenine
ring.13,1 Substitution at the 8-position of the ring is not
well tolerated by any AR subtype.14,15 The nitrogen
atoms at positions 3 and 7 are required for high affinity
of adenosine at all subtypes.1 2-Alkynyl derivatives of
NECA possess high affinity at the A3 receptor subtype.
Moreover, the presence of 2-alkyne substituents en-
hanced the A3AR selectivity.16
Adenosine receptors (AR) belong to the family of G
protein-coupled receptors. They are subdivided into
four subtypes designated A1, A2A, A2B and A3, accord-
ing to the chronological discovery of the receptors.1 The
A3ARs are coupled to Gi proteins and, therefore, inhibit
adenylate cyclase leading to a decrease in intracellular
levels of cAMP.2 The selective activation of the A3AR
is both cardioprotective and cerebroprotective in a vari-
ety of ischaemic models.3,4 Selective A3AR antagonists
promise to be useful in the regulation of cell growth5,6
and as anti-asthmatic,7 cerebroprotective4,8 and anti-
inflammatory agents.9 A3AR antagonists appear to low-
er the intraocular pressure in mice and monkeys and are
proposed as new potential therapeutics for the treatment
of glaucoma.10,11
DeNinno et al. discovered that introduction of an amino
group at the 30 position improves the selectivity for the
human A3AR, while enhancing the water solubility.
The affinity drop caused by this 30-substitution could
be overcome by elaborating the N6-substituents.17 The
combination of a large N6-substitituent with a 2-alkynyl
group has proven to be unsuccessful because of the ste-
ric hindrance caused by the two large substituents,
reflected by a decrease in A3AR affinity.16 Therefore,
the present study investigated the effect of a 2-alkynyl
substituent in concert with a small N6-substituent on
the affinity and selectivity of a series of 30-azido and
30-amino adenosine derivatives. In addition, we evaluat-
ed the effect of the 50-methylcarbamoyl modification on
the overall affinity and efficacy of these compounds
(Fig. 1).
Adenosine receptors are ubiquitously distributed
throughout the body. As a consequence, ligands need
to be highly selective in their action with respect to
Keywords: Adenosine A3 receptor; Selectivity; Affinity; Ribose and
purine modifications.
*
Corresponding authors. Tel.: +32(0)9 264 81 24; fax: +32(0)9 264 81
46 (S.V.C.); tel.: +1 301 496 9024; fax: +1 301 480 8422 (K.A.J.);
e-mail addresses: kajacobs@helix.nih.gov; serge.vancalenbergh@
ugent.be
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.062

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L. Cosyn et al. / Bioorg. Med. Chem. 14 (2006) 1403–1412
Figure 1.
+
2. Results and discussion
Cu -catalysed Huisgen dipolar cycloaddition21 of the
30-azide with phenylacetylene. Consequently, another
strategy was used to gain access to compound 14
(Scheme 2), starting from 6-chloro-2-iodo-(9-tetrahy-
dropyran-2-yl)purine (16), obtained from the 2-unsub-
stituted analogue via a lithiation-mediated stannyl
transfer process followed by 2-tributylstannyl-iodine ex-
change.22 Sonogashira coupling of 17, followed by
deprotection, provided 19. Unfortunately, classical
2.1. Chemistry
3-Azido-3-deoxy-1,2-di-O-acetyl-a-D-ribofuronamide (10)
was prepared from the commercially available 1,2-O-
isopropylidene-a-D-xylofuranose as described by us pre-
viously.18 It was coupled under Vorbruggen conditions
¨
with silylated 2-amino-6-chloropurine to give 11 in
79% yield. Classical procedures allowed a straightfor-
ward conversion of 11 to 13. Triphenylphosphine reduc-
tion of the azido moiety yielded the corresponding
amine 7.
Vorbruggen coupling,23 as described for the synthesis
¨
of 11, did not give satisfying results. By using N,O-
bis(trimethylsilyl)acetamide (BSA) as silylating agent,24
9-(2-acetyl-3-azido-3-deoxy-5-methylcarbamoyl-b-D-ribo-
furanosyl)-N6-methyl-2-phenylethynyladenine (20) was
obtained in poor yield.
Based on the results of Cristalli et al.19 we have chosen
phenylethynyl as the most promising C2-substituent.
Reaction conditions used to perform a Sonogashira
coupling20 of 13 with phenylacetylene yielded the 30-(4-
phenyl-1,2,3-triazol-1-yl) derivative (15) of the 2-alkyny-
lated compound (Scheme 1). This result was due to a
During the course of this work it became clear that the
30-amino-analogues generally exhibit much better A3AR
affinities than their 30-azide precursors. Consequently,
we focussed only on the 30-amino derivatives for further
Scheme 1. Reagents and conditions: (a) i—HMDS, (NH4)2SO4, reflux, 20 h, ii—2-amino-6-chloropurine, TMSOTf; (b) isoamyl nitrite, I2, CuI,
CH2I2 in THF, reflux; (c) CH3NH3Cl, Et3N, EtOH, reflux; (d) Ph3P, H2O, THF, 2 days; (e) phenylacetylene, (PPh3)2PdCl2, CuI, Et3N, DMF.

L. Cosyn et al. / Bioorg. Med. Chem. 14 (2006) 1403–1412
1405
Scheme 2. Reagents and conditions: (a) CH3NH3Cl, DMAP, EtOH, reflux; (b) phenylacetylene (PPh3)2PdCl2, CuI, Et3N, DMF; (c) TFA, CH2Cl2;
(d) 10, BSA, TMSOTf, CH3CN reflux; (e) 7 N NH3 in MeOH; (f) Ph3P, H2O, THF, 2 days.
Scheme 3. Reagents and conditions: (a) 2-amino-6-chloropurine, BSA, TMSOTf, CH3CN, reflux; (b) isoamylnitrite, I2, CuI, CH2I2 in THF, reflux;
(c) i—CH3NH3Cl, Et3N, EtOH, reflux, ii—7 N NH3 in MeOH; (d) Na° in MeOH; (e) Ph3P, H2O, THF, 2 days; (f) alkyne, (PPh3)2PdCl2, CuI, Et3N,
DMF.
synthesis. This direction permitted us to reduce the 30-
azide to a 30-amine before the Sonogashira coupling,
avoiding the unwanted cycloaddition.
30-Amine 6 (Fig. 1) was prepared by catalytic hydroge-
nation of the 30-azide precursor which has been
described.18
9-(3-Amino-3-deoxy-b-D-ribofuranosyl)-N6-methyl-2-iodo-
purine (1) served as a suitable synthon for the synthesis
of the 2-alkynylated 30-amino-adenosines 3–5 (Scheme
3). It was obtained by coupling of sugar 21 with 2-ami-
no-6-chloropurine. Elaboration of the base moiety to
yield 25 was essentially accomplished as for 13. Stau-
dinger reduction allowed the unmasking of the amine
group. Finally, Sonogashira coupling on amine 1 pro-
vided the alkynylated analogues 3–5 in 80–82% yield.
2.2. Biological evaluation
For the adenosine derivatives prepared in this study
(1–9, 13, 14, 15, 25, 27 and 29) we measured both
the binding affinities at the hA1, hA2A and hA3AR
and their degrees of activation of the A3AR subtype.
The results are reported in Table 1. The ability of each
of these adenosine derivatives to compete for radioli-
gand binding at each of these hARs was evaluated at
a fixed concentration of 10 lM, and full competition
curves were determined at the A3AR. Six different 2-
substituents were included: H, I, NH2, Ph-C„C,
pMePh-C„C and nBu-C„C. The choice of the methyl
group as a small N6 substituent was based on the
results of Cristalli et al., who demonstrated that it
To continue the exploration of the 2-position, we
synthesized the 2-I and the 2-NH2 derivatives of the
50-OH and the 2-H and the 2-I derivatives of the
50-methylcarbamoyl 30-amino-N6-aminomethyl adeno-
sine analogues (Scheme 4).

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L. Cosyn et al. / Bioorg. Med. Chem. 14 (2006) 1403–1412
Scheme 4. Reagents and conditions: (a) i—CH3NH3Cl, Et3N, EtOH, reflux, ii—7 N NH3 in MeOH; (b) Na° in MeOH; (c) Ph3P, H2O, THF, 2 days.
Table 1. Binding affinities of adenine derivatives at human A1, A2A and A3ARs expressed in CHO cellsa
H
Me
N
N
N
N
N
R1
R2
O
R3 OH
Compound R1
R2
R3
% Inhibition (hA3AR)
(hA1AR) (%) (hA2AAR) (%)
Ki (nM)
% Activationd (hA3AR)
1
2
I
NH2
CH2OH
CH2OH
CH2OH
NH2
NH2
NH2
NH2
NH2
39
16
50
33
4
12
4
879 346
654 42
67
57
36
23
6
2
6
3
3
Ph-C„C
pMePh-C„C CH2OH
nBu-C„C
H
14
0
126
4
4
145 35
389 112
32.3 3.9
71.4 12.8
536 247
15.6 3.6
2530b
5
CH2OH
3
66 11
72 10
18 10
6
CONHMe NH2
CONHMe NH2
18
16
ꢀ14
9
12
24
10
9
7
I
NH2
8
CONHMe NH2
CONHMe NH2
92
67 11
3
9
Ph-C„C
I
13
14
15
25
27
2918
CONHMe N3
CONHMe N3
CONHMe 4-Ph-1,2,3-triazol-1-yl
16
31
14
85b,c
0
ꢀ8
5
8
Ph-C„C
Ph-C„C
16
2
78.9 12.4 ꢀ11
1820 770
6540 320
28,800b
0
I
CH2OH
CH2OH
N3
N3
27%
16
10
ꢀ3
2
4
NH2
H
49
12
0
CONHMe N3
1140 300
38
a All A3AR experiments were performed using adherent CHO cells stably transfected with cDNA encoding one of the human adenosine receptors.
Binding at human A1, A2A and A3ARs in this study was carried out as described in methods using [3H]PIA, [3H]CGS 21680 or [125I]AB-MECA as
radioligand. Values from the present study are expressed as Ki values (means SEM, n = 3, unless noted) or as percent displacement of radioligand
at 10 lM.
b n = 1.
c Ki (A1 AR) = 1850 nM.
d % Inhibition at 100 lM of forskolin-stimulated cAMP production at 10 lM, in CHO cells expressing the hA3AR, as a percentage of the response of
the full angonist CI-IB-MECA (n = 3).
increased the affinity for the human A3AR and signif-
icantly enhanced the A3AR selectivity.19
than those of the 30-azides. For all derivatives studied,
the 50-methylcarbamoyl modification, in general, en-
hanced the affinity at the A3AR in comparison to 50-
CH2OH. Except for 25, all evaluated compounds showed
a very high selectivity for the A3AR compared to the other
Results from the competition experiments showed that
the A3AR affinities of the 30-amines were much higher

L. Cosyn et al. / Bioorg. Med. Chem. 14 (2006) 1403–1412
1407
ARs. The most potent compound (9) displayed a Ki value
4. Experimental
4.1. Chemicals and solvents
of 16 nM at the A3AR. The C-2 substituent of this com-
pound, a phenylethynyl moiety, was previously shown
to enhance A3AR affinity and selectivity,19 and proved
to have a superior contribution to A3AR affinity than a
p-methyl-phenylethynyl (4) or a 1-hexynyl (5) moiety.
Furthermore, the 2-phenylethynyl modification appeared
to overcome the reduction of affinity caused by the
30-azide (cf. Ki = 78.9 nM for 14 vs 1140 nM for 29). Con-
sequently, this C-2 substituent was selected to be com-
bined with a 30-amino and a 50-methylcarbamoyl
modification.
All reagents were from standard commercial sources
and of analytic grade.
4.2. Chromatography
Precoated Merck silica gel F254 plates were used for
TLC and spots were examined under UV light at
254 nm and further visualized by sulfuric acid-anisalde-
hyde spray. Column chromatography was performed on
Uetikon silica (0.2–0.06 mm).
Previous studies showed that 30-amino derivatives exhibit
a decreased affinity at the A3AR compared to their 30-hy-
droxy analogues. The affinity reduction associated with
this 30 modification could be overcome by elaborating
the N6-substituents, for example with a substituted benzyl
group.17 The high affinity of compound 9 (Ki = 15.6 nM)
demonstrated that a 2-phenylethynyl modification in con-
cert with a small N6-substituent was likewise capable of
overcoming this reduction in affinity. Note that in our
experiments the affinity of derivative 6 for the A3AR
(Ki = 32.3 nM) was 4-fold higher than that reported by
DeNinno et al.15 The 2-I analogue 7 also showed appre-
ciable A3AR affinity (Ki = 71.4 nM). Conversely, the 2-
4.3. Instruments and analyses
NMR spectra were obtained with a Varian Mercury
300 MHz spectrometer. Chemical shifts are given in
ppm (d) relative to the residual solvent signal, in the
1
case of DMSO-d6 2.54 ppm for H and in the case of
1
CDCl3 7.26 ppm for H. All signals assigned to amino,
amide hydrogen and hydroxyl groups were exchange-
able with D2O. Exact mass measurements were per-
formed on a quadrupole/orthogonal-acceleration time-
of-flight (Q/oaTOF) tandem mass spectrometer (qToF
2, Micromass, Manchester, UK) equipped with a stan-
dard electrospray ionization (ESI) interface. Samples
were infused in a 2-propanol/water (1:1) mixture at
3 lL/min.
NH2 analogue
(Ki = 536 nM).
8 exhibited weak A3AR affinity
The results of the cyclic cAMP-assay (Table 1) indicated
that all 30-azides were A3AR antagonists, except for
compound 29 which showed partial agonist activity.
Also the 30-(4-phenyl-1,2,3-triazol-1-yl) derivative 15 ap-
peared to be an A3AR antagonist. All other compounds
were partial agonists, except for compound 8, which
manifested full agonist activity.
4.4. 9-(3-Amino-3-deoxy-b-D-ribofuranosyl)-2-iodo-N6-
methyladenine (1)
This compound was synthesized from 30 mg
(0.069 mmol) of 25 by the procedure described for
the synthesis of 7; yield: 26 mg (92%). 1H NMR
(300 MHz, DMSO-d6): d 1.66 (br s, 2H, NH2), 2.87
(d, 3H, J = 4 Hz, N6-CH3), 3.41 (app t, 1H,
J = 6.0 Hz, H30), 3.54–3.71 (m, 3H, H40 and H50A
and H50B), 4.10 (br s, 1H, 20-OH), 4.19 (dd, 1H,
J = 2.6 and 4.4 Hz, H20), 4.98 (br s, 1H, 50-OH), 5.82
(d, 1H, J = 2.6 Hz, H10), 8.11 (d, 1H, J = 4 Hz, N6H),
8.30 (s, 1H, H8); Exact Mass (ESI-MS, i-PrOH/H2O):
Calcd for C11H16N6O3I[M+H]+: 407.0330. Found:
407.0332.
3. Conclusions
The 2,30,50-trisubstituted and 2,30-disubstituted N6-
methyl adenosine derivatives described in the present
study were synthesized in good overall yields. All the
compounds had A3AR affinities in the low micromolar
or nanomolar range and showed very high A3AR selec-
tivity. The 30-azides appeared to be A3AR antagonists
with a moderate A3AR affinity. The 30-amino modifica-
tion significantly improved the A3AR affinity and result-
ed in partial A3AR agonists. For both the 30-azido and
the 30-amino derivatives, the 50-methylcarbamoyl modi-
fication improved the overall affinity. Curiously, the
presence of a 50-uronamide did not restore full A3AR
efficacy in 2-position derivatives, as was demonstrated
in the case of N6-substituents that reduced efficacy.26
The 2-phenylethynyl derivative 9 demonstrated high
A3AR receptor affinity with a Ki value of 15.6 nM and
>1000-fold selectivity. Previous studies revealed that
30-amines exhibit a decreased affinity compared to their
30-hydroxy analogues. This study demonstrated that
introduction of a 2-phenylethynyl substituent in concert
with the N6-methyl group is capable of overcoming this
affinity drop.
4.5. 9-(3-Amino-3-deoxy-b-D-ribofuranosyl)-2-amino-N6-
methyladenine (2)
This compound was synthesized from 27 (35 mg,
0.11 mmol) by the procedure described for the syn-
thesis of 7; yield: 30 mg (93%). 1H NMR
(300 MHz, DMSO-d6): d 1.70 (br s, 2H, 30-NH2),
2.86 (s, 3H, N6-CH3), 3.34 (app t, 1H, J = 6.0 Hz,
H30), 3.52–3.70 (m, 3H, H40 and H50A and
H50B), 4.16 (br s, 2H, 20-OH and H20), 5.16 (br
s, 1H, 50-OH), 5.74 (d, 1H, J = 2.8 Hz, H10), 5.83
(s, 2H, 2-NH2), 7.24 (br s, 1H, N6H), 7.91 (s,
1H, H8); Exact Mass (ESI-MS, i-PrOH/H2O): Calcd
for C12H18N7O3
296.1470.
[M+H]+:
296.1471.
Found:

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L. Cosyn et al. / Bioorg. Med. Chem. 14 (2006) 1403–1412
4.6. General procedure for the synthesis of alkynes 3, 4 and 5
from 1
for 10 min, H2O was added (270 lL, 15 lmol) and the
reaction mixture was stirred for 2 days. The residue ob-
tained after solvent evaporation was purified by column
chromatography (CH2Cl2/MeOH, 95:5) to yield 82% of
compound 7. 1H NMR (300 MHz, DMSO-d6): d 1.79 (s,
2H, 30-NH2), 2.69 (d, 3H, J = 4.7 Hz, CH3NHCO), 2.88
(d, 3H, J = 4.1 Hz, N6-CH3), 3.54 (t, 1H, J = 11.1 Hz,
H30), 4.10 (d, 1H, J = 6.2 Hz, H40), 4.30 (app t, 1H,
J = 8.8 Hz, H20), 5.91 (br s, 1H, 20-OH), 5.93 (d, 1H,
J = 3.81 Hz, H10), 8.03 (d, 1H, J = 4,7 Hz, NHCO),
8.13 (d, 1H, J = 4.1 Hz, N6H), 8.48 (s, 1H, H8); Exact
Mass (ESI-MS, i-PrOH/H2O): Calcd for C12H17ClI-
N7O3[M+H]+: 434.0439. Found: 434.0445.
Compound 1 (50 mg, 0.12 mmol) was dissolved in Et3N
(1.5 mL) and DMF (1 mL). After purging the solution
with N2, (PPh3)2PdCl2 (8.6 mg, 0.012 mmol) and CuI
(2.3 mg, 0.012 mmol) were added. The appropriate al-
kyne (2 equiv) was subsequently added dropwise and
the mixture was stirred at room temperature for 3 h.
The solvents were removed under reduced pressure,
the residue was taken up in ethyl acetate and the solu-
tion was filtered over a Celite pad. The residue remain-
ing after solvent evaporation was purified on silica gel
column (CH2Cl2/MeOH, 90:10).
4.11. 9-(3-Amino-3-deoxy-5-methylcarbamoyl-b-D-
ribofuranosyl)-2-amino-N6-methylpurine (8)
4.7. 9-(3-Amino-3-deoxy-b-D-ribofuranosyl)-N6-methyl-2-
phenylethynyladenine (3)
Compound 28 (40 mg, 0.12 mmol) and PPh3 (66 mg,
0.25 mmol) were dissolved in THF (2 mL). After stirring
for 10 min, H2O was added (310 lL, 17 mmol) and the
mixture was stirred for 2 days. The residue obtained after
solvent evaporation was purified by column chromatog-
raphy (CH2Cl2/MeOH, 80:20) to give compound 8 in
80% yield. 1H NMR (300 MHz, DMSO-d6): d 2.95 (br s,
2H, 30-NH2), 2.66 (d, 3H, J = 4.7 Hz, CH3NHCO), 2.86
(s, 3H, N6-CH3), 3.54 (app t, 1H, J = 5.4 Hz, H30), 4.05
(d, 1H, J = 5.6 Hz, H40), 4.37 (app t, 1H, J = 4.7 Hz,
H20), 5.82 (d, 1H, J = 4.1 Hz, H10), 5.88 (s, 2H, 2-NH2),
7.31 (s, 1H, N6H), 8.04 (s, 1H, H8), 8.27 (d, 1H,
J = 4.4 Hz, NHCO); Exact Mass (ESI-MS, i-PrOH/
H2O): Calcd for C12H19N8O3[M+H]+: 323.1580. Found:
323.1579.
The reaction of 1 (50 mg, 0.12 mmol) with phenylacety-
lene (27 lL, 0.24 mmol) gave compound 3 in 81% yield.
1H NMR (300 MHz, DMSO-d6) d 2.07 (br s, 2H, 30-
NH2), 2.94 (s, 3H, N6-CH3), 3.44 (app t, 1H, J = 5.3 Hz,
H30), 3.56–3.75 (m, 3H, H40 and H50A and H50B), 4.09
(br s, 1H, 20-OH), 4.24 (dd, 1H, J = 2.6 and 4.4 Hz,
H20), 5.11 (br s, 1H, 50-OH), 5.94 (d, 1H, J = 2.3 Hz,
H10), 7.46 (m, 3H, Ph), 7.61 (m, 2H, Ph), 7.95 (br s, 1H,
N6H), 8.47 (s, 1H, H8); Exact Mass (ESI-MS, i-PrOH/
H2O): Calcd for C19H21N6O3[M+H]+: 381.1675. Found:
381.1675.
4.8. 9-(3-Amino-3-deoxy-b-D-ribofuranosyl)-N6-methyl-2-
(4-methyl-phenyl)ethynyladenine (4)
The reaction of 1 (50 mg, 0.12 mmol) with 4-methyl-
phenylacetylene (31 lL, 0.24 mmol) gave compound 4
4.12. 9-(3-Amino-3-deoxy-5-methylcarbamoyl-b-D-
ribofuranosyl)-N6-methyl-2-phenylethynyladenine (9)
1
in 82% yield. H NMR (300 MHz, DMSO-d6) d 2.33
(s, 3H, CH3Ph), 2.94 (s, 3H, N6-CH3), 3.50–3.79 (m,
4H, H30, H40, H50A and H50B), 4.31 (dd, 1H, J = 2.4
and 4.5 Hz, H20), 5.15 (br s, 1H, 50-OH), 5.74 (s, 1H,
20-OH), 5.96 (d, 1H, J = 2.4 Hz, H10), 7.25 (m, 2H,
Ph), 7.50 (m, 2H, Ph), 7.95 (br s, 1H, N6H), 8.46 (s,
1H, H8); Exact Mass (ESI-MS, i-PrOH/H2O): Calcd
for C20H23N6O3[M+H]+: 395.1831. Found: 395.1823.
This compound was synthesized by the procedure de-
scribed for the synthesis of 7 from 20 mg (0.046 mmol)
of 14 in 96% yield (18 mg). 1H NMR (300 MHz,
DMSO-d6): d 1.78 (s, 2H, 30-NH2), 2.71 (d, 3H,
J = 4.4 Hz, CH3NHCO), 2.94 (s, 3H, N6-CH3), 4.33 (dd,
1H, J = 5.0 and 5.3 Hz, H30), 4.13 (d, 1H, J = 5.3 Hz,
H40), 4.36 (br s, 1H, H20), 5.94 (s, 1H, 20-OH), 6.02 (d,
1H, J = 4.40, H10), 7.46 (m, 3H, Ph), 7.61 (m, 2H, Ph),
8.04 (d, 1H, J = 4.4 Hz, N6H), 8.41 (d, 1H, J = 4.7 Hz,
NHCO), 8.62 (s, 1H, H8); Exact Mass (ESI-MS, i-
PrOH/H2O): Calcd for C20H22N7O3[M+H]+: 408.1784.
Found: 408.1787.
4.9. 9-(3-Amino-3-deoxy-b-D-ribofuranosyl)-N6-methyl-2-
(1-hexyn-1-yl)adenine (5)
The reaction of 1 (50mg, 0.12 mmol) with 1-hexyn (28 lL,
0.24 mmol) gave compound 5 in 80% yield. H NMR
1
(300 MHz, DMSO-d6) d 0.90 (t, 3H, J = 7.03 Hz,
CH2CH3), 1.37–1.54 (m, 4H, CH2CH2CH3), 2.41 (t, 2H,
J = 7.04 Hz, C„CCH2), 2.88 (s, 3H, N6-CH3), 3.48–
3.73 (m, 4H, H30, H40, H50A and H50B), 4.10–4.23 (m,
2H, H20 and 20-OH), 5.10 (br s, 1H, 50-OH), 5.90 (d,
1H, J = 2.5 Hz, H10), 7.83 (br s, 1H, N6H), 8.41 (s, 1H,
H8); Exact Mass (ESI-MS, i-PrOH/H2O): Calcd for
C17H25N6O3[M+H]+: 361.1988. Found: 361.1982.
4.13. 9-(2-O-Acetyl-3-azido-3-deoxy-5-methylcarbamoyl-
b-D-ribofuranosyl)-2-amino-6-chloropurine (11)
4.13.1. Silylation of the base. 2-Amino-6-chloropurine
(462 mg, 2.7 mmol) was treated with 1,1,1,3,3,3-hexam-
ethyldisilazane (HMDS, 40 mL) and (NH4)2SO4
(0.27 mmol, 36 mg) and refluxed for 20 h. The silylated
compound was concentrated and used without further
purification.
4.10. 9-(3-Amino-3-deoxy-5-methylcarbamoyl-b-D-
ribofuranosyl)-N6-methyl-2-iodoadenine (7)
4.13.2. Vorbru¨ggen coupling. 3-Azido-3-deoxy-1,2-di-O-
acetyl-a-D-ribofuronamide (10, 600 mg, 2.09 mmol) in
dry 1,2-dichloroethane (25 mL) was added to the silylated
Compound 13 (50 mg, 0.11 mmol) and PPh3 (57 mg,
0.21 mmol) were dissolved in THF (2 mL). After stirring

L. Cosyn et al. / Bioorg. Med. Chem. 14 (2006) 1403–1412
1409
2-amino-6-chloropurine (462 mg, 2.7 mmol). The solu-
4.16. 9-(3-Azido-3-deoxy-5-methylcarbamoyl-b-D-
ribofuranosyl)-N6-methyl-2-phenylethynyladenine (14)
tion was gently refluxed, and after 5 min TMSOTf
(417lL, 2.3 mmol) was added dropwise. After 4 h, the
mixture was cooled to room temperature, quenched with
a cold saturated NaHCO3 solution (80 mL) and extract-
ed with CH2Cl2 (40 mL). The organic layer was dried
with MgSO4, filtered and evaporated to dryness. The
residue was purified by column chromatography
(CH2Cl2/MeOH, 98:2) to give 650 mg (79%) of com-
A solution of compound 20 (30 mg, 0.06 mmol) and
10 mL of 7 N NH3 in methanol was kept at room temper-
ature for 2 h to allow deprotection of the 20-hydroxyl
group. The mixture was concentrated to dryness and puri-
fied on a silica gel column (CH2Cl2/MeOH, 97:3). A 91%
yield of compound 14 was obtained. 1H NMR (300 MHz,
DMSO-d6): d 2.73 (d, 3H, J = 3.5 Hz, CH3NHCO), 2.96
(s, 3H, N6-CH3), 4.33 (d, 1H, J = 2.6 Hz, H40), 4.51 (dd,
1H, J = 3.0 and 5.27 Hz, H30), 4.96 (app t, 1H,
J = 5.3 Hz, H20), 5.98 (d, 1H, J = 6.7 Hz, H10), 7.46 (m,
3H, Ph), 7.64 (m, 2H, Ph), 8.12 (s, 1H, N6H), 8.51 (s,
1H, H8), 8.53 (d, 1H, J = 4.4 Hz, NHCO); Exact Mass
(ESI-MS, i-PrOH/H2O): Calcd for C20H20N9O3[M+H]+:
434.1688. Found: 434.1686.
1
pound 11. H NMR (300 MHz, CDCl3): d 2.15 (s, 3H,
CH3CO), 2.90 (d, 3H, J = 5.0 Hz, CH3N), 4.53 (d, 1H,
J = 3.2 Hz, H40), 4.90 (dd, 1H, J = 3.5 and 5.0 Hz,
H30), 5.14 (s, 2H, NH2), 5.94 (t, 1H, J = 5.3 Hz, H20),
5.97 (d, 1H, J = 6.2 Hz, H10), 7.09 (br s, 1H, NHCO),
7.82 (s, 1H, H8); Exact Mass (ESI-MS, i-PrOH/H2O):
Calcd for C13H15ClN9O4[M+H]+: 396.0935. Found:
396.0932.
4.14. 9-(2-O-Acetyl-3-azido-3-deoxy-5-methylcarbamoyl-
b-D-ribofuranosyl)-6-chloro-2-iodopurine (12)
4.17. Attempted synthesis of compound 14
Attempted conversion of 13 to 14 using the procedure as
described for 3, 4 and 5 failed to give 14, but resulted in
the formation of triazole 15 as the sole reaction product.
1H NMR (300 MHz, DMSO-d6): d 2.78 (d, 3H,
J = 4.6 Hz, N6-CH3), 3.0 (br s, 3H, CH3NHCO), 5.15
(app q, 1H, J = 6.3 Hz, H20), 5.17 (d, 1H, J = 3.4 Hz,
H40), 5.61 (dd, 1H, J = 3.6 and 6.3 Hz, H30), 6.26 (d,
1H, J = 5.4 Hz, 20OH), 6.27 (d, 1H, J = 6.3 Hz, H10),
7.36 (t, 1H, J = 7.3 Hz, 400-Ph), 7.46–7.51 (m, 2H, 400-
Ph and 3H, C„CPh), 7.65 (br d, 2H, J = 6.8 Hz,
C„CPh), 7.90 (d, 2H, J = 7.6 Hz, 400-Ph), 8.59 (s, 1H,
H8), 8.71 (m, 2H, H500 and NHCO); Exact Mass (ESI-
MS, i-PrOH/H2O): Calcd for C28H25N9O3[M+H]+:
536.2158. Found: 536.2166.
Isoamylnitrite (681 lL, 4.98 mmol) was added to a
mixture of 11 (650 mg, 1.65 mmol), I2 (418 mg,
1.65 mmol), CH2I2 (1.37 mL, 16.5 mmol) and CuI
(330 mg, 1.72 mmol) in 15 mL THF. The mixture
was refluxed for 45 min and then cooled to room tem-
perature. Insoluble materials were removed by filtra-
tion, and the filtrate was concentrated to dryness.
The residue was purified by means of a silica gel col-
umn, which was washed with CH2Cl2 until the iodine
colour disappeared and then eluted with CH2Cl2/
MeOH, 98:2. Compound 12 was obtained in 79%
yield. 1H NMR (300 MHz, CDCl3): d 2.13 (s, 3H,
CH3CO), 3.06 (d, 3H, J = 4.98 Hz, CH3N), 4.56 (d,
1H, J = 2.9 Hz, H40), 4.80 (dd, 1H, J = 2.9 and
5.6 Hz, H30), 5.75 (dd, 1H, J = 5.9 and 7.0 Hz, H20),
6.06 (d, 1H, J = 7.3 Hz, H10), 7.09 (br s, 1H, NHCO),
8.11 (s, 1H, H8); Exact Mass (ESI-MS, i-PrOH/H2O):
Calcd for C13H13ClIN8O4[M+H]+: 506.9794. Found:
506.9822.
4.18. 2-Iodo-N6-methyl-(9-tetrahydropyran-2-yl)adenine
(17)
Methylammonium chloride (28 mg, 0.41 mmol) and
DMAP (67 mg, 0.54 mmol) were added to 100 mg
(0.27 mmol) of compound 16 in EtOH (6 mL), and the
solution was refluxed overnight. The mixture was con-
centrated to dryness and the residue was purified on a
silica gel column (pentane/ethyl acetate, 50:50). The title
compound was obtained in 81% yield. 1H NMR
(300 MHz, DMSO-d6): d 1.52 (m, 6H, H2A0 and
H2B0, H3A0 and H3B0, H4A0 and H4B0), 2.87 (s, 3H,
NH CH3), 3.68 (t, 1H, J = 11.4 Hz, H50A), 3.96 (app
d, 1H, J = 11.4 Hz, H50B), 5.43 (dd, 1H, J = 2.3 and
10.9 Hz, H10), 7.26 (br s, 1H, NHCH3), 7.87 (s, 1H,
H8); Exact Mass (ESI-MS, i-PrOH/H2O): Calcd for
C11H15N5O3I[M+H]+: 360.0323. Found: 360.0333.
4.15. 9-(3-Azido-3-deoxy-5-methylcarbamoyl-b-D-
ribofuranosyl)-N6-methyl-2-iodoadenine (13)
Compound 12 (460 mg, 0.91 mmol) was dissolved in
EtOH (10 mL). Methylammonium chloride (92 mg,
1.36 mmol) and Et3N (158 lL, 1.13 mmol) were added,
and the solution was refluxed overnight. The mixture
was concentrated to dryness, dissolved in 7 N NH3 in
methanol and stirred at room temperature for 2 h to
deprotect the 20-hydroxyl group. The volatiles were re-
moved under reduced pressure, and the residue was
purified by silica gel column (CH2Cl2/MeOH, 98:2).
1
The product, 13, was realized, in 77% yield. H NMR
4.19. N6-Methyl-2-phenylethynyl-tetrahydropyranyladenine
(18)
(300 MHz, DMSO-d6): d 2.71 (d, 3H, J = 4.7 Hz,
CH3NHCO), 2.89 (d, 3H, J = 4.4 Hz, N6-CH3), 4.33
(d, 1H, J = 3.5 Hz, H40), 4.47 (dd, 1H, J = 3.5 and
5.0 Hz, H30), 4.92 (dd, 1H, J = 5.6 and 11.4 Hz, H20),
5.89 (d, 1H, J = 6.5 Hz, H10), 6.33 (d, 1H, J = 5.6 Hz,
20-OH), 8.10 (d, 1H, J = 4,7 Hz, NHCO), 8.17 (d, 1H,
J = 4.7 Hz, N6H), 8.36 (s, 1H, H8); Exact Mass (ESI-
MS, i-PrOH/H2O): Calcd for C12H15IN9O3[M+H]+:
460.0344. Found: 460.0350.
Compound 17 (200 mg, 0.557 mmol) was dissolved in a
mixture of Et3N (3 mL) and DMF (1 mL) and the solu-
tion was purged with N2. (PPh3)2PdCl2 (39 mg,
0.056 mmol) and CuI (10.6 mg, 0.056 mmol) were added.
Phenyl acetylene (112 lL, 1.11 mmol) was subsequently
added dropwise, and the mixture was stirred at room
temperature for 3 h. The solvents were removed under

1410
L. Cosyn et al. / Bioorg. Med. Chem. 14 (2006) 1403–1412
reduced pressure, the residue was taken up in ethyl acetate
and the resulting solution was filtered over a pad of Celite.
After solvent evaporation, the residue was purified on a
silica gel column (pentane/ethyl acetate, 50:50) to give
compound 18 in 91% yield. 1H NMR (300 MHz, CDCl3):
d 1.18–2.25 (m, 6H, H2A0 and H2B0, H3A0 and H3B0,
H4A0 and H4B0), 3.21 (s, 3H, NH CH3), 3.72 (app t,
1H, J = 11.4 Hz, H50A), 3.96 (app d, 1H, J = 10.3 Hz,
H50B), 5.79 (d, 1H, J = 9.1 Hz, H10), 7.20 (s, 1H, H8),
7.30 (d, 3H, J = 5.3 Hz, H-Ph), 7.61 (m, 2H, H-Ph); Exact
Mass (ESI-MS, i-PrOH/H2O): Calcd for C19H20N5
O[M+H]+: 334.1667. Found: 334.1671.
CO3 and extracted with CH2Cl2. The organic layer was
washed with brine, filtered through a short Celite pad
and evaporated to dryness. The residue was purified by
column chromatography (CH2Cl2/MeOH, 99:1) to yield
1
200 mg (65%) of compound 22. H NMR (300 MHz,
CDCl3): d 2.19 (s, 3H, CH3CO), 2.41(s, 3H, CH3-Ph),
4.40 (m, 1H, H40), 4.52–4.58 (m, 1H, H50A0), 4.76–4.82
(m, 2H, H30 and H50B), 5.13 (br s, 2H, NH2), 5.94
(d, 1H, J = 3.8 Hz, H10), 5.80 (dd, 1H, J = 3.8 and
5.6 Hz, H20), 7.23 (d, 2H, J = 7.63 Hz, Ph), 7.79 (s,
1H, H8), 7.86 (d, 2H, J = 8.2 Hz, Ph); Exact Mass (ESI-
MS, i-PrOH/H2O): Calcd for C20H19N8O5Cl[M+H]+:
487.1245. Found: 487.1246.
4.20. N6-Methyl-2-phenylethynylpurine (19)
4.23. 9-(2-Acetyl-3-azido-3-deoxy-5-O-toluoyl-b-D-
ribofuranosyl)-6-chloro-2-iodopurine (23)
To a solution of 18 (170 mg, 0.510 mmol) in 10 mL of
CH2Cl2 was added slowly a solution of 0.78 mL TFA
(10.2 mmol) and 0.78 mL CH2Cl2. After stirring at room
temperature for 1 h, the solvent was evaporated, and the
residue was taken up in ethyl acetate, and the solution
was washed with 7% NaHCO3. After silica gel chroma-
tography (CH2Cl2/MeOH, 97:3), pure 19 was obtained
This compound was prepared by the procedure described
for the synthesis of 12 from 22 (200 mg, 0.41 mmol); yield:
200 mg (81%). 1H NMR (300 MHz, DMSO-d6): d 2.15 (s,
3H, CH3CO), 2.35 (s, 3H, CH3-Ph), 4.37 (dd, 1H, J = 4.1
and 7.6 Hz, H40), 4.52 (dd, 1H, J = 4.4 and 12.3 Hz,
H50A0), 4.65 (dd, 1H, J = 3.2 and 12.3 Hz, H50B), 4.96
(dd, 1H, J = 5.8 and 7.6 Hz, H30), 6.03 (dd, 1H, J = 2.6
and 5.28 Hz, H20), 6.29 (d, 1H, J = 2.9 Hz, H10), 7.24
(d, 2H, J = 7.9 Hz, Ph), 7.68 (d, 2H, J = 8.2 Hz, Ph),
8.74 (s, 1H, H8); Exact Mass (ESI-MS, i-PrOH/H2O):
Calcd for C20H17N7O5ClINa[M+Na]+: 619.9924. Found:
619.9920.
1
in a 72% yield. H NMR (300 MHz, DMSO): d 2.97
(s, 3H, NH CH3), 7.30 (m, 3H, H-Ph), 7.61 (m, 2H,
H-Ph), 7.88 (br s, 1H, NHCH3), 8.24 (s, 1H, H8); Exact
Calcd
Mass
(ESI-MS,
i-PrOH/H2O):
for
C14H12N5[M+H]+: 250.1092. Found: 250.1073.
4.21. 9-(2-Acetyl-3-azido-3-deoxy-5-methylcarbamoyl-b-
D-ribofuranosyl)-N6-methyl-2-phenylethynyladenine (20)
4.24. 9-(3-Azido-3-deoxy-2-hydroxyl-5-O-toluoyl-b-D-
ribofuranosyl)-2-iodo-N6-methyladenine (24)
To a mixture of 19 (150 mg, 0.602 mmol) and methyl 3-az-
(10)
ido-3-deoxy-1,2-di-O-acetyl-a-D-ribofuronamide
(207 mg, 0.722 mmol) in 3 mL CH3CN were successively
added 223 lL (0.903 mmol) of N,O-bis(trimethylsilyl)-
acetamide (BSA) and 131 lL (0.722 mmol) TMSOTf.
The suspension was refluxed for 10 h. After being cooled
to room temperature, the reaction was quenched with 7%
NaHCO3 and extracted with CH2Cl2. The organic layer
was washed with brine, filtered through ashort padof Cel-
ite and evaporated to dryness. The crude material was
purified by column chromatography (CH2Cl2/MeOH,
99:1), and compound 20 was obtained in 24.4% yield.
1H NMR (300 MHz, DMSO-d6): d 2.12 (s, 3H, CH3CO),
3.02 (d, 3H, J = 4.69 Hz, CH3NHCO), 3.25 (s, 3H, N6-
CH3), 4.58 (d, 1H, J = 2.6 Hz, H40), 4.78 (dd, 1H,
J = 2.1 and 5.3 Hz, H30), 5.78 (dd, 1H, J = 7.3 and
12.9 Hz, H20), 5.92 (s, 1H, N6H) 6.02 (d, 1H, J = 7.6 Hz,
H10), 7.40 (app d, 3H, J = 7.0 Hz H-Ph), 7.63 (app d,
2H, J = 9.1 Hz, H-Ph), 7.81 (s, 1H, H8), 8.83 (s, 1H,
NHCO); Exact Mass (ESI-MS, i-PrOH/H2O): Calcd for
C22H22N9O4[M+H]+: 476.1794. Found: 476.1800.
The title compound was prepared as described for the
synthesis of 13 from 23 (200 mg, 0.335 mmol); yield:
127 mg (69%). H NMR (300 MHz, DMSO-d6): d 2.36
1
(s, 3H, CH3-Ph), 2.87 (d, 3H, J = 3.5 Hz, N6-CH3),
4.29 (dd, J = 5.3 and 9.7 Hz, H40), 4.46–4.60 (m, 3H,
H30, H50A and H50B), 5.01 (t, 1H, J = 4.7 Hz, H20),
5.87 (d, 1H, J = 4.7 Hz, H10), 6.43 (d, 1H, J = 5.3 Hz,
20-OH), 7.29 (d, 2H, J = 8.2 Hz, Ph), 7.78 (d, 2H,
J = 8.2 Hz, Ph), 8.17 (d, 1H, J = 4.4 Hz, N6H), 8.21 (s,
1H, H8); Exact Mass (ESI-MS, i-PrOH/H2O): Calcd
for C19H20N8O4I [M+H]+: 551.0653. Found: 551.0649.
4.25. 9-(3-Azido-3-deoxy-b-D-ribofuranosyl)-2-iodo-N6-
methyl-adenine (25)
Ester 24 (127 mg, 0.23 mmol) was dissolved in 2.5 mL
MeOH. Na° (11.28 mg, 0.32 mmol) was added and the
mixture was stirred at room temperature for 1 h. The
reaction was quenched by adding
a mixture of
CH3COOH/H2O (9:1, v/v) to pH 7. The solution was
concentrated to dryness, and the residue was purified
by column chromatography (CH2Cl2/MeOH, 98:2) to
yield 100 mg (95%) of compound 25. 1H NMR
(300 MHz, DMSO-d6): d 2.88 (d, 3H, J = 4 Hz, N6-
CH3), 3.52–3.67 (m, 2H, H50A and H50B), 3.94 (dd,
1H, J = 7.26 and 3.81 Hz, H40), 4.28 (app t, 1H,
J = 4.5 Hz, H30), 4.89 (app t, 1H, J = 5.4 Hz, H20),
5.20 (br s, 1H, 50-OH), 5.80 (d, 1H, J = 6.16 Hz, H10),
6.25 (br s, 1H, 20-OH), 8.18 (d, 1H, J = 4 Hz, N6H),
8.28 (s, 1H, H8); Exact Mass (ESI-MS, i-PrOH/H2O):
4.22. 9-(2-Acetyl-3-azido-3-deoxy-5-O-toluoyl-b-D-
ribofuranosyl)-2-amino-6-chloropurine (22)
To a mixture of 2-amino-6-chloropurine (90 mg, 0.53
mmol) and 3-azido-3-deoxy-1,2-di-O-acetyl-5-O-toluo-
yl-a-D-ribofuranose (21) (240 mg, 0.64 mmol) in 3 mL
CH3CN were successively added 196 lL (0.79 mmol)
BSA and 115 lL (0.64 mmol) TMSOTf. The suspension
was heated at 80 °C for 3 h. After being cooled to room
temperature, the reaction was quenched with 7% NaH-

L. Cosyn et al. / Bioorg. Med. Chem. 14 (2006) 1403–1412
1411
Calcd for C11H14N8O3I [M+H]+: 433.0235. Found:
433.0237.
4.29. Biological assays
4.29.1. Cell culture and membrane preparation. CHO cells
expressing recombinant human A3ARs were cultured in
DMEM (DulbeccoÕs modified EagleÕs medium) and F12
(1:1) supplemented with 10% foetal bovine serum,
100 U/mL penicillin, 100 lg/mL streptomycin, 2 lmol/
mL glutamine and 800 lL geneticin. After harvest and
homogenization, the cells were centrifuged at for
10 min. The pellet was resuspended in 50 mM Tris–HCl
buffer (pH 8.0) containing 10 mM MgCl2 and 1 mM
EDTA. The suspension was homogenized with an electric
homogenizer for 10 s and was then recentrifuged at
20,000g for 20 min at 4 °C. The resulting pellets were
resuspended in buffer containing 3 U/mL of adenosine
deaminase, and the suspension was stored at ꢀ80 °C prior
to the binding experiments. The protein concentration
was measured using the Bradford assay.25
4.26. 9-(3-Azido-3-deoxy-5-O-toluoyl-b-D-ribofuranosyl)-
2-amino-N6-methyl-adenine (26)
Derivative 22 (100 mg, 0.21 mmol) was solubilized in
EtOH (5 mL). Methylammonium chloride (35 mg,
0.515 mmol) and Et3N (72 lL, 0.515 mmol) were added,
and the solution was refluxed overnight. The mixture
was concentrated to dryness, the residue redissolved in
methanolic NH3 and the solution stirred at room temper-
ature for 2 h to allow deprotection of the 20-hydroxyl
group. The mixture was concentrated to dryness and puri-
fied on a silica gel column (CH2Cl2/MeOH, 97:3). Com-
pound 26 was obtained in 68% yield. 1H NMR
(300 MHz, DMSO-d6): d 2.36 (s, 3H, CH3-Ph), 2.84 (br
s, 3H, N6-CH3), 4.23 (dd, J = 5.27 and 9.38 Hz, H40),
4.43 (dd, 1H, J = 5.6 and 11.6, H50A), 4.55 (m, 2H,
H3 and H50B), 4.48 (app t, 1H, 4.7 Hz, H20), 5.78
(d, 1H, J = 4.7 Hz, H10), 5.95 (br s, 2H, 2-NH2), 6.34
(d, 1H, J = 7.9 Hz, 20-OH), 7.28 (br s, 1H, N6H), 7.29
(d, 2H, J = 7.9 Hz, Ph), 7.79 (s, 1H, H8), 7.82 (d, 2H,
J = 8.2 Hz, Ph); Exact Mass (ESI-MS, i-PrOH/H2O):
Calcd for C19H22N9O4 [M+H]+: 440.1794. Found:
440.1789.
4.29.2. Radioligand binding studies. For the A3AR binding
experiments, the procedures were similar to those previ-
ously described.26 Briefly, each tube contained 100 lL
of membrane suspension, 50 lL [125I]I-AB-MECA (final
concentration 0.5 nM) and 50 lL of increasing concen-
trations of compounds in Tris–HCl buffer (50 mM, pH
7.4) containing 10 mM MgCl2, 1 mM EDTA. Non-spe-
cific binding was determined using 10 lM NECA. The
mixtures were incubated at 25 °C for 60 min. Binding
reactions were terminated by filtration through Whatman
GF/B filters under reduced pressure using a MT-24 cell
harvester (Brandel, Gaithersburg, MD). Filters were
washed three times with ice-cold buffer. Radioactivity
was determined in a Beckman 5500B c-counter. The bind-
ing of [3H]R-PIA to the recombinant hA1AR and the
binding of [3H]CGS21680 to the recombinant hA2AAR
were performed as previously described.27,28
4.27. 9-(3-Azido-3-deoxy-b-D-ribofuranosyl)-2-amino-N6-
methyladenine (27)
The title compound was synthesized from 26 (60 mg,
0.013 mmol) by the procedure described for the synthe-
sis of 25; yield: 40 mg (91%). 1H NMR (300 MHz,
DMSO-d6): d 2.85 (br s, 3H, N6-CH3), 3.48–3.64 (m,
2H, H50A and H50B), 3.89 (dd, 1H, J = 3.2 and
7.0 Hz, H40), 4.25 (dd, 1H, J = 3.2 and 5.6 Hz, H30),
4.88 (app t, 1H, J = 5.3 Hz, H20), 5.58 (t, J = 6.8 Hz,
1H, 50-OH), 5.71 (d, 1H, J = 6.2 Hz, H10), 5.85 (s, 2H,
NH2), 6.16 (s, 1H, 20-OH), 7.31 (br s, 1H, N6H), 7.89
(s, 1H, H8); Exact Mass (ESI-MS, i-PrOH/H2O): Calcd
for C11H16N9O3[M+H]+: 322.1376. Found: 322.1365.
4.29.3. Cyclic AMP accumulation assay. Intracellular cyc-
lic AMP levels were measured by the competitive protein
binding method.29 CHO cells expressing recombinant hu-
man30 ARs were harvested by trypsinization. After resus-
pension in the medium, cells were plated in 24-well plates
in 0.5 mL medium/well. After 24 h, the medium was re-
moved and cells were washed three times with 1 mL/well
DMEM containing 50 mM N-2-hydroxyethylpiper-
azine-N0-2-ethanesulfonic acid, pH 7.4. Cells were then
treated with agonists and/or test compounds in the pres-
ence of rolipram (10 lM) and adenosine deaminase
(3 U/mL) and incubated at 37 °C. For A3AR, after 45
min forskolin (10 lM) was added to the medium, and
incubation was continued for an additional 15 min. The
reaction was terminated upon removal of the medium,
and the cells were lysed with 200 lL/well of 0.1 M ice-cold
HCl. The cell lysate was resuspended and stored at
ꢀ20 °C. For determination of cyclic AMP production,
protein kinase A (PKA) was incubated with [3H]cyclic
AMP (2 nM) in K2HPO4/EDTA buffer (K2HPO4,
150 mM; EDTA, 10 mM), 20 lL of the cell lysate and
30 lL of 0.1 M HCl. Bound radioactivity was separated
by rapid filtration through Whatman GF/C filters under
reduced pressure and washed once with cold buffer.
Bound radioactivity was subsequently measured by scin-
tillation spectrometry.
4.28. 9-[3-Azido-3-deoxy-5-(methylcarbamoyl)-b-D-
ribofuranosyl]-2-amino-6-chloropurine (28)
Derivative 11 (120 mg, 0.30 mmol) was dissolved in EtOH
(6 mL). Methylammonium chloride (30 mg, 0.46 mmol)
and Et3N (53 lL, 0.38 mmol) were added, and the solu-
tion was refluxed overnight. The mixture was concentrat-
ed to dryness, the remaining solid dissolved in 7 N NH3 in
methanol and the solution stirred at room temperature
for 2 h to deprotect the 20-hydroxyl group. The mixture
was concentrated to dryness and the residue was purified
on a silica gel column (CH2Cl2/MeOH, 95:5). Compound
28 was obtained in 79% yield. 1H NMR (300 MHz,
DMSO-d6): d 2.68 (d, 3H, J = 4.7 Hz, CH3NHCO), 2.87
(s, 3H, N6-CH3), 4.24 (d, 1H, J = 2.9 Hz, H-40), 4.42
(dd, 1H, J = 2.9 and 5.3 Hz, H30), 4.97 (dd, 1H, J = 5.6
and 11.7 Hz, H20), 5.81 (d, 1H, J = 6.8 Hz, H10), 5.91 (s,
2H, NH2), 6.26 (d, 1H, J = 5.3 Hz, 20-OH), 7.31 (s, 1H,
N6H), 7.96 (s, 1H, H8), 8.35 (d, 1H, J = 4.7 Hz, NHCO);
Exact Mass (ESI-MS, i-PrOH/H2O): Calcd for
C12H17N10O3[M+H]+: 349.1484. Found: 349.1491.

1412
L. Cosyn et al. / Bioorg. Med. Chem. 14 (2006) 1403–1412
16. Volpini, R.; Constanzi, S.; Lambertucci, C.; Vittori, S.;
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