2-Triazole-substituted adenosines: A new class of selective A3 adenosine receptor agonists, partial agonists, and antagonists

Article abstract of DOI:10.1021/jm0608208

"Click chemistry" was explored to synthesize two series of 2-(1,2,3-triazolyl)adenosine derivatives (1-14). Binding affinity at the human A₁, A_2A, and A₃ARs (adenosine receptors) and relative efficacy at the A₃A

Full text of DOI:10.1021/jm0608208

J. Med. Chem. 2006, 49, 7373-7383
7373
2-Triazole-Substituted Adenosines: A New Class of Selective A3 Adenosine Receptor Agonists,
Partial Agonists, and Antagonists
Liesbet Cosyn,† Krishnan K. Palaniappan,‡ Soo-Kyung Kim,‡ Heng T. Duong,‡ Zhan-Guo Gao,‡ Kenneth A. Jacobson,*,‡ and
Serge Van Calenbergh*,†
Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences (FFW), Ghent UniVersity, Harelbekestraat 72, B-9000 Ghent,
Belgium, and Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and DigestiVe and Kidney
Diseases, National Institutes of Health, Bethesda, Maryland 20892
ReceiVed July 13, 2006
“Click chemistry” was explored to synthesize two series of 2-(1,2,3-triazolyl)adenosine derivatives (1-14).
Binding affinity at the human A1, A2A, and A3ARs (adenosine receptors) and relative efficacy at the A3AR
were determined. Some triazol-1-yl analogues showed A3AR affinity in the low nanomolar range, a high
ratio of A3/A2A selectivity, and a moderate-to-high A3/A1 ratio. The 1,2,3-triazol-4-yl regiomers typically
showed decreased A3AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce
relative A3AR efficacy. Thus, several 5′-OH derivatives appeared to be selective A3AR antagonists, i.e., 10,
with 260-fold binding selectivity in comparison to the A1AR and displaying a characteristic docking mode
in an A3AR model. The corresponding 5′-ethyluronamide analogues generally showed increased A3AR affinity
and behaved as full agonists, i.e., 17, with 910-fold A3/A1 selectivity. Thus, N6-substituted 2-(1,2,3-triazolyl)-
adenosine analogues constitute a novel class of highly potent and selective nucleoside-based A3AR antagonists,
partial agonists, and agonists.
Introduction
However, the A3AR, more than other AR subtypes, is
amenable to the design of nucleoside-based antagonists. The
efficacy of nucleoside derivatives in activation of the A3AR is
particularly sensitive to molecular substitution of the ligand.19
A wide range of adenosine derivatives have been shown to
antagonize this receptor, including the highly potent A1AR
agonist 2-chloro-N6-cyclopentyladenosine. N6-Benzyl groups are
associated with reduced A3AR efficacy, leading to partial
agonists and antagonists. However, many of the nucleosides so
far demonstrated to be antagonists of the A3AR are not highly
subtype-selective.20 Nucleoside-based A3AR antagonists main-
taining an intact ribose moiety were reported by Volpini et al.,21
with a series of 8-alkynyladenosine derivatives that exhibited
A3AR selectivity, but suffered from weak A3AR affinity. A
spirolactam derivative, in which the 5′-alkyluronamide group
was cyclized onto the 4′ carbon, was found to potently and
selectively antagonize the A3AR.15,19 An advantage of nucleo-
side-based A3AR antagonists over other heterocyclic antagonists
is the ability to achieve high affinity at murine species.
Recently, researchers from CV Therapeutics described a series
of 2-pyrazolyladenosine analogues.22 Several representative
compounds containing an N6-methyl substituent proved to
display high affinity and selectivity for the A3AR. This study
confirms the former finding23 that introduction of a methyl group
into the N6 position increases the affinity for the human A3AR
and enhances the selectivity versus A1 and A2AARs. On the
basis of these results, we explored the versatile “click chemistry”
approach24 to synthesize two series of N6-methyl-2-(1,2,3-
triazolyl)adenosine derivatives and evaluated their affinity,
selectivity, and efficacy at the A3AR. Although a number of
1,2,3-triazole nucleoside derivatives have been described,25 most
involve base replacement with 1,2,3-triazole or introduction of
1,2,3-triazole at C8 or at the sugar moiety.
Adenosine receptors (AR) are G-protein-coupled receptors
and consist of four subtypes classified as A1, A2A, A2B, and A3.
Among the four AR subtypes, the A3AR is the most recently
identified.1 The distribution of A3AR is species-dependent, and
in humans this subtype occurs in the lungs, liver, heart, kidneys,
and brain.2-4 Activation of this receptor subtype inhibits
adenylyl cyclase activity, increases phosphatidylinositol-specific
phospholipase C activity, and stimulates Ca2+ mobilization.3
Adenosine A3 receptor stimulation induces cardioprotection
through the activation of KATP channels4 and is also involved
in neuroprotection, suggesting the possibility of using A3AR
agonists to treat cardial and cerebral ischemia.5 A3AR agonists
also exhibit systemic anticancer and chemoprotective effects.6
A3AR modulators have been proposed as antiinflammatory and
antiasthmatic drugs.7,8 Selective A3AR antagonists promise to
be useful in the regulation of cell growth8,9 and as cerebropro-
tective agents.10,11 They also seem to enhance anticancer
treatment by counteracting P-glycoprotein efflux in multidrug
resistance.12 A3AR antagonists are also proposed as potential
therapeutics for the treatment of glaucoma; application of A3-
AR antagonists externally to the eye substantially lowers
intraocular pressure in mice and monkeys.13-15
Although diverse in structure, most AR antagonists share
some common structural features. In general, they are planar,
aromatic, or π-electron-rich and nitrogen-containing hetero-
cycles. Additionally, most AR antagonists lack the ribose
moiety, which seems essential for agonist activity.16 Various
heterocyclic classes have been identified as promising leads for
A3AR antagonists, among them 1,4-dihydropyridines, pyridines,
deazaadenines, pyrazolopyrimidines, adenines, and 1,2,4-tria-
zolo[4,3-a]quinoxalin-1-ones.4,7,17,18
* To whom correspondence should be addressed. For K.A.J.: phone,
301-496-9024; fax, 301-480-8422; e-mail, kajacobs@helix.nih.gov. For
S.V.C.: phone, +32(0)9 264 81 24; fax, +32(0)9 264 81 46; e-mail,
serge.vancalenbergh@ugent.be.
Results and Discussion
Chemistry. The synthesis of the 1,2,3-triazol-1-yladenosine
derivatives is depicted in Scheme 1. 2-Iodo-N6-methyladenosine
2223 was prepared by reacting 2126 with 2.0 M CH3NH2 in THF.
† Ghent University.
‡ National Institutes of Health.
10.1021/jm0608208 CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/15/2006

7374 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Cosyn et al.
Scheme 1. Synthesis of 1,2,3-Triazol-1-yl Analogues of
N6-Methyladenosine 1-11a
Scheme 3. Synthesis of 1,2,3-Triazol-4-yl Analogues of
N6-Methyladenosine 12-14a
a Reagents and conditions: (a) CH3NH2 in THF, 2 days; (b) CuSO4‚5H2O,
sodium ascorbate, L-proline, Na2CO3, NaN3, H2O/tBuOH 1:1, 60 °C; (c)
CuSO4‚5H2O, sodium ascorbate, alkyne, H2O/tBuOH 3:1, room temp.
Scheme 2. Azido/Tetrazole Tautomerism of a 2-Substituted
Adenosine Derivative 23
a Reagents and conditions: (a) trimethylsilylacetylene, CuI, (Ph3P)3PdCl2,
DMF; (b) 7 N NH3 in MeOH, 0 °C; (c) CuSO4‚5H2O, sodium ascorbate,
alkyne, H2O/tBuOH 3:1, room temp.
Scheme 4. Synthesis of 1,2,3-Triazol-1-yl Analogues of
N6-Methyladenosine-5′-N-ethyluronamide 15-19a
Since the reaction conditions27 for a Cu(I)-catalyzed nucleophilic
substitution are very similar to those used in the “click” variant
of Huisgen’s 1,3-dipolar cycloaddition, we initially attempted
to perform a one-pot conversion of 22 to the desired 1,4-
disubstituted 1,2,3-triazoles. The 2-azido derivative 23 was
isolated as the main reaction product, and only a minor amount
of the appropriate triazole was formed. This event forced us to
perform the reaction in two steps. First the azido intermediate
1
23 was prepared in 66% yield from 22. H and 13C NMR in
DMSO-d6 proved the presence of a tautomeric fused tetrazole
form (17%) of the 2-azidoadenosine derivative 23, due to a
spontaneous cyclization (Scheme 2). Such azido/tetrazole tau-
tomerism has been previously reported for 2-azidoadenine and
for 2-azidoadenosine.28,29 Next we applied a Cu(I)-catalyzed 1,3-
cycloaddition reaction of azide 23 with the appropriate alkyne
to generate the triazole analogues 1-11 (Scheme 1).30 Generally,
the use of a water/butanol mixture as a solvent for the 1,3-
cycloaddition allowed simple isolation of the desired com-
pounds, which precipitated from the reaction medium.
a Reagents and conditions: (a) KMnO4, KOH, room temp, 20 h; (b)
p-nitrophenol, EDCI, DMF, room temp; (c) ethylamine; (d) 80% TFA/H2O;
(e) CuSO4‚5H2O, sodium ascorbate, L-proline, Na2CO3, NaN3, H2O/tBuOH
1:1, 60 °C; (f) CuSO4‚5H2O, sodium ascorbate, alkyne, H2O/tBuOH 1:1,
room temp.
Similarly, the 1,2,3-triazol-4-yl analogues 12-14 (Scheme
3) were prepared by a Cu+-catalyzed Huisgen 1,3-dipolar
cycloaddition reaction of 2-ethynyl-N6-methyladenosine (25)
with the appropriate azide.
31 with the appropriate alkyne to generate the triazole analogues
15-19 (Scheme 4).
2-Azido-N6-(5-chloro-2-methoxybenzyl)-2-(4-cyclopentyl-
methyl-1,2,3-triazol-1-yl)-9-(â-D-ribofuranosyl)adenine (20) was
prepared in three steps starting from intermediate 21, as depicted
in Scheme 5.
The synthesis of the 5′-N-ethylcarbamoyl 2-(1,2,3)-triazol-
1-yladenosine analogues 15-19 was carried out starting from
2-iodo-9-(2′,3′-O-isopropylidene-â-D-ribofuranosyl)-N6-methyl-
adenine (26). After permanganate oxidation, carboxylic acid 27
was converted into its p-nitrophenyl ester 28, which upon
treatment with ethylamine gave uronamide 29. Deprotection with
80% trifluoroacetic acid yielded 5′-ethylcarbamoyl-N6-methyl-
2-iodoadenosine (30).30 The conversion of this 2-iodo derivative
into the azido intermediate 31 was performed in 79% yield.
The presence of a tautomeric fused tetrazole form (20%) of the
2-azidoadenosine derivative 31, due to a spontaneous cycliza-
tion, was here also observed in the NMR spectrum. Finally we
applied the Cu(I)-catalyzed 1,3-cycloaddition reaction of azide
Biological Evaluation. The binding affinities of the newly
synthesized adenosine derivatives were measured at the hA1,
hA2A, and hA3ARs expressed in CHO (Chinese hamster ovary)
cells as previously described,20 and their relative efficacy in the
activation of the A3AR was determined (Table 1). The binding
affinity of more potent compounds at the ARs was evaluated
with full competition curves, while the weaker compounds at
the hA1 and hA2A ARs were measured at a fixed concentration
of 10 µM. Several compounds showed affinity for the A3AR in
the low nanomolar range, a very high ratio of A3/A2A selectivity,
and a moderate-to-high A3/A1 selectivity ratio. A functional

2-Triazole-Substituted Adenosines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7375
Scheme 5. Synthesis of Compound 20, 2-(4-Cyclopentylmethyl-
1,2,3-triazol-1-yl)-N6-(2-chloro-5-methoxybenzyl)adenosinea
unsubstituted, 4-butyl, 4-pyridin-2-yl, and 4-benzyl substituted
1,2,3-triazol-1-yl combinations (15, 16, 17, and 18, in com-
parison to 1, 3, 7, and 9, respectively). Curiously, one 5′-
uronamide, compound 19, exhibited decreased A3AR affinity
(Ki ) 11.5 nM) compared to its potent 5′-OH analogue 10 (Ki
) 1.3 nM).
Replacement of the N6-methyl substituent of the 2-azido
precursor 23 by a sterically demanding 2-chloro-5-methoxy-
benzyl group yielded 33, which manifested very high A3AR
affinity (Ki ) 1.4 nM). A similar replacement of the N6-methyl
group of an analogue 10, also having a bulky 2-position
substituent, to yield 20 reduced A3AR affinity but not ap-
preciably. This was in accordance with previous observations
that a simultaneous substitution at the 6 and 2 positions did not
improve A3AR affinity.22,33 Thus, the effects of substitution at
the 2 and N6 positions were not independent; however, it was
possible to retain considerable A3AR selectivity (46-fold in
compound 20). This was not representative of findings in a
previous study in which double substitution greatly diminished
the affinity and selectivity at the human A3AR.22
Whereas some previously synthesized 2-substituted adenosine
derivatives22,23 displayed selective A3AR agonist activity, all
2-triazol-1-yl-N6-methyladenosine analogues synthesized with
an unmodified ribose moiety (1-11 and 20) behaved as
antagonists or weak partial agonists. Similar findings were
reported for 2-ester derivatives of adenosine, in which a
combination of 2 and N6 substitution reduced efficacy.34 Direct
ring substitution at the 4-position of the 1,2,3-triazole with alkyl
or aryl groups resulted in weak partial agonists (1, 2, 4-8), but
subtle changes of structure resulted in a loss of efficacy, e.g.,
the 4-butyl derivative 3. 2-Triazol-1-yl-N6-methyladenosine
analogues with a methylene spacer between the 1,2,3-triazole
moiety and a ring system yielded full A3AR antagonists (9-
11, 20), since they bound to the receptor but did not activate it.
The 2-triazol-4-yl-N6-methyladenosine derivatives (12-14) also
behaved as full A3AR antagonists. Thus, the 5′-OH derivatives
3, 9-14, and 20 appeared to be A3AR antagonists with the
following order of decreasing selectivity for the A3AR in
comparison to the A1AR: 4-cyclopentylmethyl-N6-methyl 10
(260-fold) > 4-butyl-N6-methyl 3 (72-fold), 4-cyclohexylmethyl-
N6-methyl 11 (67-fold) > 4-cyclopentylmethyl-N6-(5-chloro-
2-methoxybenzyl) 20.
a Reagents and conditions: (a) 2-chloro-5-methoxybenzylammonium
chloride, Et3N, EtOH, reflux; (b) CuSO4‚5H2O, sodium ascorbate, L-proline,
Na2CO3, NaN3, H2O/tBuOH 1:1, 60 °C; (c) CuSO4‚5H2O, sodium ascorbate,
alkyne, H2O/tBuOH 1:1, room temp.
assay of A3AR activation consisted of the ability of a single,
high concentration of the nucleoside (10 µM) to inhibit
forskolin-stimulated adenylyl cyclase measured by the method
of Nordstet and Fredholm,32 in comparison to the full agonist
NECA (10 µM). Cl-IB-MECA (2-chloro-N6-(3-iodobenzyl)-5′-
N-methylcarboxamidoadenosine) was also a full agonist (100%)
in this assay.19 The range of efficacies observed depended on
the nature of the groups at the 2 and N6 positions.
The 2-azido precursor 23 showed high binding affinity at the
A3AR (Ki ) 10.8 nM) and modest selectivity in comparison to
the A1AR. The 1,2,3-triazol-1-yl derivatives obtained by 1,3-
dipolar cycloaddition of azide 23 with acetylene (1), butyne (2),
and hexyne (3) maintained high affinity for the A3AR and
increased selectivity. They displayed Ki values of 10.4, 13.8,
and 11.7 nM, respectively. Also, aromatic triazole substituents
(6, 7, 9) resulted in similar Ki values of about 10 nM and even
greater selectivity. Introducing nitrogen or oxygen including
substituents at position 4 of the 1,2,3-triazole ring (4, 5, and 8)
reduced the A3AR affinity. Among the investigated analogues,
the 4-cyclopentylmethyl derivative 10 exhibited the highest
affinity for the A3AR (Ki ) 1.3 nM) and 260-fold selectivity in
comparison to the A1AR. Replacement of the cyclopentyl ring
with a phenyl (9) or cyclohexyl (11) moiety adversely affected
A3AR affinity. Remarkably, the 1,2,3-triazol-4-yl regiomers
(12-14) showed decreased affinity for the A3AR in comparison
to similar 1,2,3-triazol-1-yl regiomers. In particular, a compari-
son of homologous compounds 12 and 9 indicated a 6-fold loss
of affinity at the A3AR for the 4-yl isomer, approximately the
same affinity at the A1AR, and no significant measurable gain
in affinity at the A2AAR.
Interestingly, the 5′-N-ethyluronamide modification was able
to reestablish the A3AR agonist activity in analogues with
sterically bulky substitution at the 2 position. This is consistent
with previous findings that similar 5′-uronamides overcome the
efficacy-reducing activity of substitution at the adenine 2 and
N6 positions but not at the ribose 3′ position.19,35,36 Indeed, all
5′-N-ethyluronamide analogues studied here (15-19) proved
to be full agonists at the A3AR. Among them are highly selective
A3AR agonists, N6-methyladenosine-5′-N-ethyluronamide 2-(1,2,3-
triazol-1-yl) derivatives: pyridin-2-yl 17 (910-fold) > unsub-
stituted 15 (280-fold) > benzyl 18 (180-fold). The 2-azido-N6-
methyl precursors 23 and 31 also showed full agonist activity,
whereas azide 33 having a bulky N6 group showed partial
agonist activity.
Selected potent agonists in this series were measured in a
functional assay of the human A2BAR. At 10 µM, compounds
3-7, 15, and 16 did not significantly stimulate adenylyl cyclase
in human A2BAR-expressing CHO cells (<10% of the effect
of 10 µM NECA, as a full agonist). Compounds 2, 8-14, 17,
19, and 23 at 10 µM stimulated adenylyl cyclase by <50%.
Compounds 18 and 31 produced approximately 50% stimulation
at 10 µM. Thus, selectivity for the A3AR was demonstrated;
Replacement of the ribose 4′-hydroxymethyl moiety of the
2-azido derivative 23 by a 5′-N-ethyluronamide did not ap-
preciably affect affinity at any of the AR subtypes. However, a
similar substitution in the 2-(1,2,3-triazol-1-yl)-substituted series
provided a modest (2- to 5-fold) increase in A3AR affinity and
small or no changes in A1AR affinity, as demonstrated for the

7376 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Cosyn et al.
Table 1. Binding Affinities of Adenosine Derivatives at Human A1, A2A, and A3ARs Expressed in CHO Cells and Relative Efficacy at the A3ARa
Ki (nM) or % inhibition (in parentheses) at 10 µM
% efficacyb
hA3
R1
R2
R3
hA1
hA2A
hA3
1
2
3
4
5
6
7
8
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
C2H5NHCO
C2H5NHCO
C2H5NHCO
C2H5NHCO
C2H5NHCO
CH2OH
CH2OH
C2H5NHCO
CH2OH
H
ethyl
butyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
1000 ( 30
2920 ( 910
848 ( 76
1270 ( 260
3800 ( 600
(36)
(13 ( 3)
10.4 ( 0.2
13.8 ( 3.3
11.7 ( 3.1
45.0 ( 4.4
117 ( 25
14.9 ( 1.7
10.3 ( 1.5
25.2 ( 2.6
9.5 ( 0.7
1.3 ( 0.4
21.3 ( 8.1
53.9 ( 6.6
86.1 ( 3.8
81.1 ( 5.0
2.1 ( 0.1
5.6 ( 0.2
1.8 ( 0.6
2.8 ( 1.3
11.5 ( 1.4
18 ( 11
41 ( 6
23c
(18)
(23)
(14)
(6)
(5)
(40)
(14)
(20)
(39)
(16 ( 1)
(21 ( 5)
(43 ( 10)
(39 ( 10)
(18 ( 3)
(43 ( 1)
(45 ( 12)
(33 ( 2)
(36 ( 7)
6000
3 ( 8
2-hydroxyethyl
dimethylaminomethyl
phenyl
pyridin-2-yl
4-propoxyphenyl
benzyl
cyclopentylmethyl
cyclohexylmethyl
benzyl
3-methoxybenzyl
3-Cl-benzyl
H
butyl
pyridin-2-yl
benzyl
cyclopentylmethyl
cyclopentylmethyl
25c
8c
14 ( 8
11 ( 4
31c
1970 ( 210
(49)
9
589 ( 55
335 ( 13
1430 ( 60
770 ( 210
957 ( 65
956 ( 6
590 ( 70
750 ( 110
1640 ( 90
510 ( 50
1250 ( 150
830 ( 40
230 ( 10
429 ( 55
60 ( 10
-1 ( 5
-5 ( 7
2 ( 5
2 ( 3
-1 ( 3
0 ( 5
102 ( 5
89 ( 3
90 ( 7
86 ( 5
83c
10d
11
12
13
14
15d
16
17d
18d
19
20
23
31
33
2-Cl-5-MeO-Bn
CH3
CH3
-6 ( 3
84 ( 9
112c
(23)
(18 ( 3)
1800 ( 500
10.8 ( 3.1
11.4 ( 4.2
1.4 ( 0.1
2-Cl-5-MeO-Bn
44 ( 5
a All binding experiments were performed using cells stably transfected with cDNA encoding one of the human ARs. Binding at human A1, A2A, and
A3ARs in this study was carried out as described in Experimental Section using [3H]CCPA, [3H]CGS 21680, or [125I]IAB-MECA as a radioligand. Values
from the present study are expressed as Ki values (mean ( SEM, n ) 3, unless otherwise noted) or as percent displacement of radioligand. b % activation
at 10 µM, relative to cyclic AMP inhibitory effect of 10 µM NECA ()100%). Cl-IB-MECA was also a full agonist (100%) in this assay. c n ) 2. d 10, LC
153; 15, LC 260; 17, LC 257; 18, LC 259.
these nucleosides that activate the A3AR at low nanomolar
concentrations activated the A2BAR only at substantial micro-
molar concentrations.
purine N1 atom and the side chain of N250 (6.55). The 2′-OH
group of the ribose moiety formed a H-bond with the side chain
of Q167 (EL2), and the 3′-OH group formed an intramolecular
H-bond with the 5′-OH group. Unlike the previously reported
docking models of N6-substituted adenosines,37 here the 5′-OH
group H-bonded with the side chain of H272 (7.43) and the
backbone carbonyl group of S271 (7.42). The cyclopentyl
moiety interacted with aliphatic hydrophobic residues, M177
(5.38) and V178 (5.39), through a hydrophobic interaction and
were situated in proximity to F168 (EL2) F182 (5.43), consistent
with the optimized binding affinity of compound 10.
A comparison of the docking modes of Cl-IB-MECA and
compound 10 in the putative binding domain showed consider-
able overlap of the ribose rings and of the adenine moieties,
although in compound 10 both were situated a little closer to
extracellular loop 2 (Figure 1B). Previously, it was noted that
5′-uronamide analogues, typically of derivatives having bulky
N6-subsitituents, generally gain affinity in comparison to the
corresponding 5′-hydroxyl analogue. Here, the 5′-uronamide
analogue 19 (agonist) of the most potent 5′-hydroxyl analogue
10 (antagonist) displayed a lower binding affinity, which could
be explained by the shift of the ribose position in adenosine
analogue having a bulky 2-(4-cyclopentylmethyl-1,2,3-triazol-
1-yl) substituent in comparison to those having N6 bulky
substituents. The binding of the cyclopentylmethyl group in 10
was directed more toward the upper part of TM5, partially
overlapping with the binding site of the 3-iodophenyl ring in
Cl-IB-MECA. Curiously, other closely related triazolo deriva-
tives displayed a higher potency of the 5′-uronamide analogues;
thus, compound 10 must bind to the receptor in a very distinct
On the basis of previous findings, it is predicted that only
the analogues containing the substituted N6-benzyl group (5-
chloro-2-methoxy), i.e., full agonist 20 and partial agonist 33,
would be expected to bind in the nanomolar range to the rat
A3AR. Small alkyl groups at the N6 position, such as methyl
and ethyl, although conducive to high affinity at the human A3-
AR, led to negligible affinity at the rat homologue of the
receptor. Selected compounds were measured in binding to the
rat A3AR expressed in CHO cell membranes using [125I]I-AB-
MECA. The Ki values determined were as follows: compounds
5, 7, and 8, Ki > 10 µM; compound 9, Ki ) 1.79 µM; compound
10, Ki ) 0.312 µM.
Molecular Modeling. To explain the structural basis for the
high binding affinity of the nucleoside 2-(4-cyclopentylmethyl-
1,2,3-triazol-1-yl)-N6-methyl-9-(â-D-ribofuranosyl)adenine 10 at
hA3AR, we performed a computational study of ligand docking
in a previously derived A3AR model based on the high-
resolution structure of bovine rhodopsin.37,38 Various bound
conformations of the C2-substituent and ø1 angles for the
adenine ring were generated for an energetically favorable
binding location and orientation, and the resulting conformations
were compared energetically in the putative binding site.
The result of docking 10 in the putative binding site of the
A3AR is shown in Figure 1A. The purine ring was surrounded
by a hydrophobic pocket, defined by L91 (3.33) and L246
(6.51). In addition, the H-bonds formed between the exocyclic
amine and the hydroxyl group of S247 (6.52) and between the

2-Triazole-Substituted Adenosines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7377
Conclusions
Several 2-(1,2,3-triazol-1-yl)-N6-methyl-substituted adenosine
derivatives described in the present study displayed A3AR
affinities in the low nanomolar range, showed very high A3/
A2A, and a moderate to high A3/A1 selectivity. Contrary to what
we expected, the 2-triazole analogues with an unmodified ribose
moiety (1-14) showed antagonist or weak partial agonist
activity at the A3AR. A 2-(4-cyclopentylmethyl-(1,2,3-triazol-
1-yl))-N6-methyl derivative 10 was 260-fold selective in binding
in comparison to the A1AR. The binding of the 4-cyclopentyl-
methyl group in 10, in distinction to the binding of closely
related bulky groups pendent on the triazole ring, was directed
more toward the upper part of TM5 partially overlapping with
the binding site of the 3-iodophenyl ring in Cl-IB-MECA. The
5′-N-ethyluronamide modification was dominant over the ef-
ficacy reducing effects at the 2 position and was capable of
fully re-establishing the A3AR agonist activity, resulting in
highly potent and selective A3AR agonists 15-19. The most
selective agonist derivative was compound 17, 9-(5-ethylcar-
bamoyl-â-D-ribofuranosyl)-N6-methyl-2-(4-pyridin-2-yl-1,2,3-
triazol-1-yl)adenine, which was 910-fold selective in binding
to the A3AR in comparison to the A1AR. The retention of high
human A3AR affinity in compound 20 was not typical of
previous findings that double bulky substitution at the 2 and
N6 positions tended to reduce A3AR affinity markedly. Thus,
the 2-triazol-1-yl-N6-methyladenosine analogues 1-11 constitute
a novel class of highly potent and selective nucleoside-based
A3AR partial agonists and antagonists (all of which maintain
an intact ribose in the molecular structure) and agonists. Since
the reported analogues show excellent affinity for the A3AR
and span the full intrinsic activity range, they might be useful
as pharmacological tools or as leads for further optimization.
Experimental Section
All reagents were from standard commercial sources and of
analytic grade, except for the benzylic azides, which were prepared
by treating the corresponding benzylic bromides with NaN3 in DMF.
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-anisaldehyde spray. Column chroma-
tography was performed on ICN silica gel (63-200 µm, 60 Å, ICN
Biochemicals, Eschwege, Germany). NMR spectra were obtained
with a Varian Mercury 300 MHz spectrometer. Chemical shifts
are given in ppm (δ) relative to the residual solvent signals, which
Figure 1. (A) Docking complexes of compound 10, 2-(4-cyclopen-
tylmethyl-1,2,3-triazol-1-yl)-N6-methyladenosine. (B) Superimposition
of Cl-IB-MECA in red and compound 10 in color by atom type.
Residues that were within 5 Å to the ligand in this putative binding
site were L91 (3.33), T94 (3.36), H95 (3.37), Q167 (EL2), F168 (EL2),
M172 (EL2), S181 (5.42), M177 (5.38), V178 (5.39), F182 (5.43),
W243 (6.48), L246 (6.51), S247 (6.52), N250 (6.55), C251 (6.56), I268
(7.39), S271 (7.42), and H272 (7.43). The ligand is represented by a
ball-and-stick model. The H-bonds are indicated with yellow dots. By
use of the MOLCAD ribbon surface program, the A3AR is shown in a
ribbon model with different colors for each TM (TM1, red; TM2,
orange; TM3, yellow; TM4, green; TM5, cyan; TM6, blue; TM7,
purple; H8, violet).
1
in the case of DMSO-d6 were 2.54 ppm for H and 40.5 ppm for
13C. Structural assignment was confirmed with COSY and DEPT.
All signals assigned to hydroxyl groups were exchangeable with
D2O. Exact mass measurements were performed on a quadrupole/
orthogonal-acceleration time-of-flight (Q/oaTOF) tandem mass
spectrometer (qToF 2, Micromass, Manchester, U.K.) equipped with
a standard electrospray ionization (ESI) interface. Samples were
infused in a 2-propanol/water (1:1) mixture at 3 µL/min. For the
compounds that precipitated from the reaction medium, the yields
were calculated from the amount obtained after filtration and are
lower than the actual yields, since in most cases a considerable
amount remained in solution.
N6-Methyl-9-(â-D-ribofuranosyl)-2-(1,2,3-triazol-1-yl)ade-
nine (1). In a pressure tube was added 23 (165 mg, 0.51 mmol),
trimethylsilylacetylene (292 µL, 2.05 mmol), and 4 mL of DMF.
The mixture was stirred at 105 °C for 15 h. After solvent
evaporation, the yellowish residue was dissolved in 2 mL of a 1.0
M solution of tetrabutylammonium fluoride in THF and stirred for
5 h. The reaction was monitored by NMR. After evaporation of
the solvent, the residue was dissolved in ethyl acetate. Water was
added, and the triazole product was precipitated in the water layer.
After overnight cooling and filtration, the precipitate was further
purified on a silica gel column (CH2Cl2/MeOH, 92:8) and yielded
manner. There was a subtle difference in orientation between
the 2-cyclopentyl group and bulkier groups like benzyl (9) or
cyclohexylmethyl (11), which were associated with unfavorable
van der Waals interactions and resulted in a decrease of binding
affinity of 7- and 16-fold, respectively. In addition, the same
preferred ø1 angles of the energetically favorable bound
conformation, common to Cl-IB-MECA and compound 10, were
consistent with the empirical finding that the combination of
bulky N6 and C2 substituents was unfavorable for A3AR
selectivity because of competitive interaction of these bulky
substituents. Thus, the modeling has demonstrated that the
human A3AR preference of 2-(4-cyclopentylmethyl-1,2,3-tria-
zol-1-yl) derivatives in the 5′-OH series might be explained by
optimal van der Waals interactions.

7378 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Cosyn et al.
1
compound 1 as a white solid (82 mg, 46%). H NMR (300 MHz,
(app t, 1H, J ) 5.6 Hz, 5′-OH), 5.24 (d, 1H, J ) 5.0 Hz, 3′-OH),
5.50 (d, 1H, J ) 6.2 Hz, 2′-OH), 5.93 (d, 1H, J ) 6.2 Hz, H-1′),
8.31 (d, 1H, J ) 4.7 Hz, N6-H), 8.43 (s, 1H, H-8), 8.54 (s, 1H,
H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 27.83 (N6-CH3), 29.70
(CH2), 60.85 (CH2-OH), 62.13 (C-5′), 71.15 (C-3′), 74.35 (C-2′),
86.43 (C-4′), 87.81 (C-1′), 119.56 (C-5), 122.02 (C-5′′), 140.49
(C-8), 145.49, 149.88, and 149.65 (C-2, C-4, and C-4′′), 156.05
(C-6). HRMS (ESI-MS) C15H21N8O5 [M + H]+: 393.1630 found,
393.1634 calcd. Anal. (C15H20N8O5‚H2O) C, H, N.
DMSO-d6): δ 3.06 (d, 3H, J ) 4.5 Hz, N6-CH3), 3.54-3.61 (m,
1H, H-5′A), 3.65-3.72 (m, 1H, H-5′B), 3.96 (dd, 1H, J ) 3.8 and
7.9 Hz, H-4′), 4.20 (dd, 1H, J ) 4.7 and 8.2 Hz, H-3′), 4.65 (app
q, J ) 5.9 Hz, H-2′), 4.98 (t, 1H, J ) 5.6 Hz, 5′-OH), 5.23 (d, 1H,
J ) 5.0 Hz, 3′-OH), 5.48 (d, 1H, J ) 6.2 Hz, 2′-OH), 5.95 (d, 1H,
J ) 5.9 Hz, H-1′), 7.92 (d, 1H, J ) 1.2 Hz, H-4′′), 8.37 (d, 1H, J
) 4.6 Hz, N6-H), 8.46 (s, 1H, H-8), 8.82 (br s, 1H, H-5′′). 13C
NMR (300 MHz, DMSO-d6): δ 27.84 (N6-CH3), 62.19 (C-5′), 71.13
(C-3′), 74.30 (C-2′), 86.42 (C-4′), 88.01 (C-1′), 119.75 (C-5), 124.67
(C-5′′), 134.25 (C-4′′), 141.13 (C-8), 149.56 and 149.85 (C-2 and
C-4), 156.082 (C-6). HRMS (ESI-MS) C13H17N8O4 [M + H]+:
349.1367 found; 349.1372 calcd. Anal. (C13H16N8O4‚1/2H2O) C, H,
N.
2-(4-Ethyl-1,2,3-triazol-1-yl)-N6-methyl-9-(â-D-ribofuranosyl)-
adenine (2). Compound 23 (70 mg, 0.217 mmol), sodium ascorbate
(8.6 mg, 0.043 mmol), and CuSO4‚5H2O (2.2 mg, 0.009 mmol)
were suspended in 20 mL of H2O/tBuOH (3:1). The mixture
was saturated with 1-butyne and stirred for 4 days at room
temperature in a Parr apparatus. Purification on a preparative TLC
plate (CH2Cl2/MeOH, 90:10) resulted in compound 2 as a white
2-(4-Dimethylaminomethyl-1,2,3-triazol-1-yl)-N6-methyl-9-(â-
D-ribofuranosyl)adenine (5). The reaction of 23 (70 mg, 0.217
mmol) with 1-dimethylamino-2-propyne (47 µL, 0.435 mmol) gave
compound 5 without precipitation. The volatiles were removed
under reduced pressure, and the residue was purified on a silica
gel column (80:20 CH2Cl2/MeOH + 1% 7 N NH3 in MeOH).
Compound 5 was obtained as a white solid in 67% yield. 1H NMR
(300 MHz, DMSO-d6): δ 2.20 (s, 6H, 2 × CH3), 3.05 (d, 3H, J )
4.4 Hz, N6-CH3), 3.54-3.62 (m, 3H, H-5′A and CH2), 3.65-3.72
(m, 1H, H-5′B), 3.95 (dd, 1H, J ) 4.1 and 7.6 Hz, H-4′), 4.18 (dd,
1H, J ) 5.0 and 8.2 Hz, H-3′), 4.62 (app q, 1H, J ) 5.4 Hz, H-2′),
4.98 (t, 1H, J ) 5.6 Hz, 5′-OH), 5.23 (d, 1H, J ) 5.0 Hz, 3′-OH),
5.49 (d, 1H, J ) 6.2 Hz, 2′-OH), 5.95 (d, 1H, J ) 6.2 Hz, H-1′),
8.38 (d, 1H, J ) 4.4 Hz, N6-H), 8.45 (s, 1H, H-8), 8.64 (s, 1H,
H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 27.85 (N6-CH3), 45.18
(N(CH3)2), 53.85 (CH2), 62.10 (C-5′), 71.12 (C-3′), 74.38 (C-2′),
86.41 (C-4′), 87.84 (C-1′), 119.64 (C-5), 123.328 (C-5′′), 140.97
(C-8), 144.41, 149.59, 149.79 (C-2, C-4, and C-4′′), 156.05 (C-6).
HRMS (ESI-MS) C16H24N9O4 [M + H]+: 406.1944 found,
406.1951 calcd. Anal. (C16H23N9O4) C, H, N. N calcd, 31.09; found,
30.41.
1
solid in 40% yield. H NMR (300 MHz, DMSO-d6): δ 1.27 (t,
3H, J ) 7.62 Hz, CH3), 2.75 (q, 2H, J ) 7.6 Hz, CH2), 3.05 (d,
3H, J ) 4.4 Hz, N6-CH3), 3.51-3.59 (m, 1H, H-5′A), 3.61-3.70
(m, 1H, H-5′B), 3.94 (dd, 1H, J ) 4.0 and 7.6 Hz, H-4′), 4.15 (dd,
1H, J ) 4.4 and 7.9 Hz, H-3′), 4.60 (app q, J ) 5.6 Hz, H-2′),
4.97 (t, 1H, J ) 5.6 Hz, 5′-OH), 5.22 (d, 1H, J ) 4.7 Hz, 3′-OH),
5.47 (d, 1H, J ) 6.2 Hz, 2′-OH), 5.93 (d, 1H, J ) 6.2 Hz, H-1′),
8.34 (d, 1H, J ) 4.4 Hz, N6-H), 8.45 (s, 1H, H-8), 8.55 (s, 1H,
H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 14.32 (CH3), 19.08
(CH2), 27.84 (N6-CH3), 62.13 (C-5′), 71.15 (C-3′), 74.34 (C-2′),
86.43 (C-4′), 87.84 (C-1′), 119.57 (C-5), 120.962 (C-5′′), 140.92
(C-8), 149.31, 149.63, 149.88 (C-2, C-4, and C-4′′), 156.05 (C-6).
HRMS (ESI-MS) C15H21N8O4 [M + H]+: 377.1682 found;
377.1685 calcd. Anal. (C15H20N8O4) C, H, N.
N6-Methyl-2-(4-phenyl-1,2,3-triazol-1-yl)-9-(â-D-ribofurano-
syl)adenine (6). The reaction of 23 (70 mg, 0.217 mmol) with
phenylacetylene (48 µL, 0.435 mmol) yielded compound 6 (54%)
1
as a white solid. H NMR (300 MHz, DMSO-d6): δ 3.11 (d, 3H,
J ) 4.5 Hz, N6-CH3), 3.55-3.63 (m, 1H, H-5′A), 3.67-3.74 (m,
1H, H-5′B), 3.97 (dd, J ) 3.6 and 7.2 Hz, H-4′), 4.21 (dd, J ) 4.8
and 8.1 Hz, H-3′), 4.66 (app q, J ) 5.4 Hz, H-2′), 5.03 (app t, 1H,
J ) 5.6 Hz, 5′-OH), 5.29 (d, 1H, J ) 5.0 Hz, 3′-OH), 5.54 (d, 1H,
J ) 6.2 Hz, 2′-OH), 5.99 (d, 1H, J ) 5.9 Hz, H-1′), 7.4 (t, 1H, J
) 7.3 Hz, Ph), 7.50 (t, 2H, J ) 7.5 Hz, Ph), 8.06 (d, 2H, J ) 7.3
Hz, Ph), 8.44 (d, 1H, J ) 4.5 Hz, N6-H), 8.49 (s, 1H, H-8), 9.31
(s, 1H, H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 27.92 (N6-CH3),
62.16 (C-5′), 71.17 (C-3′), 74,39 (C-2′), 86.48 (C-4′), 87.94 (C-
1′), 119.85 (C-5), 120.68 (C-5′′), 126.27, 128.95, 129.61, and 130.85
(Ph), 141.082 (C-8), 147.02 and 149.77 (C-2, C-4, and C-4′′),
156.10 (C-6). HRMS (ESI-MS) C19H21N8O4 [M + H]+: 425.1689
found, 425.1685 calcd. Anal. (C19H20N8O4) C, H, N.
General Procedure for the Synthesis of 4′′-Substituted
2-(1,2,3-Triazol-1-yl)adenosine Derivatives 3-11. Compound 23
(70 mg, 0.217 mmol), sodium ascorbate (8.6 mg, 0.043 mmol),
and CuSO4‚5H2O (2.2 mg, 0.009 mmol) were suspended in 2 mL
of H2O/tBuOH (3:1). The appropriate alkyne (2 equiv) was
subsequently added, and the mixture was stirred overnight at room
temperature. The 2-triazol-1-yl compounds (generally) precipitated
from this reaction medium and were isolated by filtration with water.
2-(4-Butyl-1,2,3-triazol-1-yl)-N6-methyl-9-(â-D-ribofuranosyl)-
adenine (3). The reaction of 23 (70 mg, 0.217 mmol) with 1-hexyne
1
(50 µL, 0.435 mmol) gave compound 3 in 59% yield. H NMR
(300 MHz, DMSO-d6): δ 0.93 (t, 3H, J ) 7.3 Hz, CH3), 1.38 (m,
2H, CH2), 1.66 (m, 2H, CH2), 2.71 (t, 2H, J ) 7.2 Hz, C4′′-CH2),
3.05 (d, 3H, J ) 4.0 Hz, N6-CH3), 3.52-3.62 (m, 1H, H-5′A),
3.62-3.72 (m, 1H, H-5′B), 3.95 (dd, 1H, J ) 3.6 and 7.2 Hz, H-4′),
4.18 (dd, 1H, J ) 4.8 and 8.1 Hz, H-3′), 4.62 (app q, 1H, J ) 5.7
Hz, H-2′), 5.01 (t, 1H, J ) 5.2 Hz, 5′-OH), 5.29 (d, 1H, J ) 4.0
Hz, 3′-OH), 5.54 (d, 1H, J ) 5.6 Hz, 2′-OH), 5.94 (d, 1H, J ) 5.9
Hz, H-1′), 8.36 (d, 1H, J ) 4.1 Hz, N6-H), 8.45 (s, 1H, H-8), 8.56
(s, 1H, H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 14.37 (CH3),
22.37 (CH2), 25.21 (CH2), 27.83 (N6-CH3), 31.68 (CH2), 62.14 (C-
5′), 71.15 (C-3′), 74.36 (C-2′), 86.43 (C-4′), 87.88 (C-1′), 119.56
(C-5), 121.33 (C-5′′), 140.96 (C-8), 147.88 and 149.90 (C-2, C-4
and C-4′′), 156.07 (C-6). HRMS (ESI-MS) C17H25N8O4 [M + H]+:
405.1992 found, 405.1998 calcd. Anal. (C17H24N8O4) C, H, N.
2-[4-(2-Hydroxyethyl)-1,2,3-triazol-1-yl]-N6-methyl-9-(â-D-ri-
bofuranosyl)adenine (4). The reaction of 23 (70 mg, 0.217 mmol)
with 3-butyn-1-ol (33 µL, 0.435 mmol) afforded compound 4
without precipitation. After solvent evaporation, the mixture was
purified on a silica gel column (90:10 CH2Cl2/MeOH + 1% 7 N
NH3 in MeOH), yielding compound 11 as a white solid in 68%
yield. 1H NMR (300 MHz, DMSO-d6): δ 2.85 (t, 2H, J ) 6.9 Hz,
CH2), 3.03 (d, 3H, J ) 4.7 Hz, N6-CH3), 3.52-3.59 (m, 1H, H-5′A),
3.63-3.72 (m, 3H, H-5′B and CH2), 3.94 (dd, 1H, J ) 3.5 and 7.3
Hz, H-4′), 4.16 (dd, 1H, J ) 4.8 and 8.1 Hz, H-3′), 4.59 (app q,
1H, J ) 5.4 Hz, H-2′), 4.74 (t, 1H, J ) 5.8 Hz, CH2-OH), 4.97
N6-Methyl-2-[4-pyridin-2-yl-1,2,3-triazol-1-yl]-9-(â-D-ribo-
furanosyl)adenine (7). The reaction of 23 (70 mg, 0.217 mmol)
with 2-ethynylpyridine (44 µL, 0.435 mmol) afforded compound
1
7 as a white solid in 55% yield. H NMR (300 MHz, DMSO-d6):
δ 3.09 (d, 3H, J ) 4.1 Hz, N6-CH3), 3.57-3.66 (m, 1H, H-5′A),
3.67-3.76 (m, 1H, H-5′B), 3.97 (dd, 1H, J ) 3.9 and 7.5 Hz, H-4′),
4.19 (dd, 1H, J ) 4.8 and 8.1 Hz, H-3′), 4.64 (app q, 1H, J ) 6.0
Hz, H-2′), 4.99 (t, 1H, J ) 5.6 Hz, 5′-OH), 5.26 (d, 1H, J ) 5.0
Hz, 3′-OH), 5.54 (d, 1H, J ) 6.2 Hz, 2′-OH), 5.99 (d, 1H, J ) 5.9
Hz, H-1′), 7.42 (m, 1H, pyridin-2-yl) , 7.96 (m, 1H, pyridin-2-yl),
8.16 (d, 1H, J ) 7.3 Hz, pyridin-2-yl), 8.42 (d, 1H, J ) 4.1 Hz,
1H, N6-H), 8.49 (s, 1H, H-8), 8.68 (d, 1H, J ) 4.1 Hz, pyridin-2-
yl), 9.16 (s, 1H, H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 27.92
(N6-CH3), 62.13 (C-5′), 71.14 (C-3′), 74.49 (C-2′), 86.47 (C-4′),
88.16 (C-1′), 119.92 (C-5), 120.53 (C-5′′), 121.07 (pyridin-2-yl),
122.25 (pyridin-2-yl), 137.79 (pyridin-2-yl), 141.28 (C-8), 148.47,
150.45 and 150.56 (C-2, C-4, C-4′′, and pyridin-2-yl), 156.28 (C-
6). HRMS (ESI-MS) C18H19N9O4Na [M + Na]+: 448.1458 found,
448.1457 calcd. Anal. (C18H19N9O4) C, H, N.
N6-Methyl-2-[4-(4-propoxyphenyl)-1,2,3-triazol-1-yl]-9-(â-D-
ribofuranosyl)adenine (8). The reaction of 23 (70 mg, 0.217 mmol)
with 1-eth-1-ynyl-4-propoxybenzene (57 µL, 0.435 mmol) afforded
1
compound 8 as a white solid in 36% yield. H NMR (300 MHz,
DMSO-d6): δ 0.99 (t, 3H, J ) 7.3 Hz, CH3), 1.71-1.78 (m, 2H,

2-Triazole-Substituted Adenosines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7379
CH2), 3.10 (d, 3H, J ) 4.1 Hz, N6-CH3), 3.54-3.62 (m, 1H, H-5′A),
3.64-3.72 (m, 1H, H-5′B), 3.98 (m, 3H, H-4′ and CH2), 4.18 (dd,
1H, J ) 4.8 and 8.1 Hz, H-3′), 4.64 (app q, 1H, J ) 5.4 Hz, H-2′),
4.99 (t, 1H, J ) 5, 7 Hz, 5′-OH), 5.25 (d, 1H, J ) 4.1 Hz, 3′-OH),
5.50 (d, 1H, J ) 5.9 Hz, 2′-OH), 5.97 (d, 1H, J ) 6.2 Hz, H-1′),
7.03 (d, 2H, J ) 8.8 Hz, Ph), 7.94 (d, 2H, J ) 8.8 Hz, Ph), 8.36
(d, 1H, J ) 4.1 Hz, 1H, N6-H), 8.45 (s, 1H, H-8), 9.15 (s, 1H,
H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 11.08 (CH3), 22.73
(CH2), 27.90 (N6-CH3), 62.16 (C-5′), 69.71 (OCH2), 71.18 (C-3′),
74.38 (C-2′), 86.46 (C-4′), 87.91 (C-1′), 115.50 (Ph), 119.60 (C-
5), 123.28 (C-5′′), 127.66 (Ph), 140.01 (C-8), 147.01, 149.82, 150.45
(C-4, C-2, and C-4′′), 156.10 (C-6). HRMS (ESI-MS) C22H27N8O5
[M + H]+: 483.2109 found, 483.2104 calcd. Anal. (C22H26N8O5)
C, H, N.
N6-Methyl-2-(4-benzyl-1,2,3-triazol-1-yl)-9-(â-D-ribofurano-
syl)adenine (9). The reaction of 23 (70 mg, 0.217 mmol) with
3-phenyl-1-propyne (54 µL, 0.435 mmol) gave compound 9 as a
white solid in 43% yield. 1H NMR (300 MHz, DMSO-d6): δ 3.03
(d, 3H, J ) 3.8 Hz, N6-CH3), 3.53-3.62 (m, 1H, H-5′A), 3.64-
3.71 (m, 1H, H-5′B), 3.95 (dd, 1H, J ) 3.7 and 7.2 Hz, H-4′), 4.11
(s, 2H, CH2), 4.17 (dd, 1H, J ) 4.8 and 8.1 Hz, H-3′), 4.61 (app
q, 1H, J ) 5.7 Hz, H-2′), 4.97 (app t, 1H, J ) 5.3 Hz, 5′-OH),
5.22 (d, 1H, J ) 4.7 Hz, 3′-OH), 5.47 (d, 1H, J ) 5.9 Hz, 2′-OH),
5.94 (d, 1H, J ) 5.9 Hz, H-1′), 7.23 (m, 1H, Ph), 7.32 (d, 4H, J )
4.4 Hz, Ph), 8.34 (d, 1H, J ) 3.8 Hz, N6-H), 8.45 (s, 1H, H-8),
8.59 (s, 1H, H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 25.32 (N6-
CH3), 32.51 (CH2), 62.11 (C-5′), 71.13 (C-3′), 74.43 (C-2′), 86.41
(C-4′), 87.96 (C-1′), 121.42 (C-5), 121.97 (C-5′′), 126.91, 129.12,
129.21, and 140.02 (Ph), 140.88 (C-8), 147.42, 149.58, and 149.81
(C-4, C-2, and C-4′′), 156.09 (C-6). HRMS (ESI-MS) C20H23N8O4
[M + H]+: 439.1846 found, 439.1842 calcd. Anal. (C20H22N8O4)
C, H, N.
(100 mg, 0.32 mmol), sodium ascorbate (13 mg, 0.06 mmol mmol),
and CuSO4‚5H2O (3 mg, 0. 013 mmol) were suspended in 30 mL
of H2O/tBuOH (3:1). The appropriate azide (2 equiv) was subse-
quently added, and the mixture was stirred overnight at room
temperature. The 2-triazol-4-yl compounds (generally) precipitated
from this reaction medium and were isolated by filtration with water.
2-(1-Benzyl-1,2,3-triazol-4-yl)-N6-methyl-9-(â-D-ribofurano-
syl)adenine (12). The reaction of 25 (100 mg, 0.32 mmol) with 85
mg (0.64 mmol) of benzylazide gave compound 12 in 78% yield
1
(110 mg). H NMR (300 MHz, DMSO-d6): δ 3.03 (br s, 3H, N6-
CH3), 3.52-3.58 (m, 1H, H-5′A), 3.60-3.66 (m, 1H, H-5′B), 3.92
(app d, H-4′, J ) 2.9 Hz, H-4′), 4.15 (dd, 1H, J ) 4.7 and 7.6 Hz,
H-3′), 4.60 (app q, 1H, J ) 5.9 Hz, H-2′), 5.08 (t, 1H, J ) 5.4 Hz,
5′-OH), 5.19 (d, 1H, J ) 3.5 Hz, 3′-OH), 5.44 (d, 1H, J ) 5.6 Hz,
2′-OH), 5.68 (s, 2H, CH2), 5.97 (d, 6.2 Hz, H-1′), 7.38 (br s, 5H,
Ph), 7.82 (br s, 1H, N6-H), 8.36 (s, 1H, H-8), 8.66 (s, 1H, H-5′′).
13C NMR (300 MHz, DMSO-d6): δ 27.54 (N6-CH3), 53.62 (CH2),
62.29 (C-5′), 71.30 (C-3′), 74.30 (C-2′), 86.41 (C-4′), 87.77 (C-
1′), 119.52 (C-5), 126.41 (C-5′′), 128.63, 128.86, 129.48 and 136.71
(Ph), 140.27 (C-8), 148.22, 153.56, 153.72 (C-2, C-4, and C-4′),
155.69 (C-6). HRMS (ESI-MS) C20H23N8O4 [M + H]+: 439.1834
found, 439.1842 calcd. Anal. (C20H22N8O4) C, H, N.
2-[1-(3-Methoxybenzyl)-1,2,3-triazol-4-yl)-N6-methyl-9-(â-D-
ribofuranosyl)adenine (13). The reaction of 25 (100 mg, 0.32
mmol) with 104 mg (0.64 mmol) of 3-methoxybenzylazide gave
compound 13 in 80% yield (120 mg). 1H NMR (300 MHz, DMSO-
d6): δ 3.03 (br s, 3H, N6-CH3), 3.52-3.60 (m, 1H, H-5′A), 3.64-
3.72 (m, 1H, H-5′B), 3.95 (app d, H-4′, J ) 2.9 Hz, H-4′), 4.15
(dd, 1H, J ) 4.7 and 7.6 Hz, H-3′), 4.60 (app q, 1H, J ) 6.4 Hz,
H-2′), 5.07 (t, 1H, J ) 5.1 Hz, 5′-OH), 5.21 (d, 1H, J ) 4.2 Hz,
3′-OH), 5.46 (d, 1H, J ) 6.3 Hz, 2′-OH), 5.62 (s, 2H, CH2), 5.95
(d, 1H, J ) 6.6 Hz, H-1′), 6.91 (m, 3H, Ph), 7.29 (t, 1H, J ) 7.9
Hz, Ph), 7.79 (br s, 1H, N6-H), 8.34 (s, 1H, H-8), 8.63 (s, 1H, H-5′′).
13C NMR (300 MHz, DMSO-d6): δ 27.59 (N6-CH3), 53.45 (CH2),
55.82 (OCH3), 62.30 (C-5′), 71.33 (C-3′), 74.25 (C-2′), 86.42 (C-
4′), 87.70 (C-1′), 114.44 and 114.20 (Ph), 119.51 (C-5), 120.72
(Ph), 130.65 (C-5′′), 138.21 (C-8), 148.22, 153.56, 153.64 (C-2,
C-4, and C-4′), 155.69 (C-6), 160.14 (Ph). HRMS (ESI-MS)
C21H25N8O5 [M + H]+: 469.1938 found, 469.1947 calcd. Anal.
(C21H24N8O5) C, H, N.
2-(4-Cyclopentylmethyl-1,2,3-triazol-1-yl)-N6-methyl-9-(â-D-
ribofuranosyl)adenine (10). The reaction of 23 (70 mg, 0.217
mmol) with 3-cyclopentyl-1-propyne (57 µL, 0.435 mmol) yielded
compound 10 (32%) as a white solid. 1H NMR (300 MHz, DMSO-
d6): δ 1.23-1.28 (m, 2H, cyclopentyl), 1.48-1.62 (m, 4H,
cyclopentyl), 1.71-1.75 (m, 2H, cyclopentyl), 2.19-2.25 (m, 1H,
cyclopentyl), 2.72 (d, 2H, J ) 7.3 Hz, CH2-cyclopentyl), 3.05 (d,
3H, J ) 3.9 Hz, N6-CH3), 3.53-3.61 (m, 1H, H-5′A), 3.63-3.72
(m, 1H, H-5′B), 3.96 (dd, 1H, J ) 3.6 and 7.2 Hz, H-4′), 4.19 (dd,
1H, J ) 4.8 and 8.1 Hz, H-3′), 4.62 (app q, 1H, J ) 5.9 Hz, H-2′),
4.98 (t, 1H, J ) 5.3 Hz, 5′-OH), 5.23 (d, 1H, J ) 4.7 Hz, 3′-OH),
5.49 (d, 1H, J ) 6.5 Hz, 2′-OH), 5.95 (d, 1H, J ) 5.9 Hz, H-1′),
8.34 (d, 1H, J ) 3.9 Hz, N6-H), 8.45 (s, 1H, H-8), 8.55 (s, 1H,
H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 25.37 (cyclopentyl),
27.83 (N6-CH3), 31.56 (CH2), 32.55 (cyclopentyl), 62.15 (C-5′),
71.15 (C-3′), 74.34 (C-2′), 86.43 (C-4′), 87.87 (C-1′), 119.60 (C-
5), 121.58 (C-5′′), 140.92 (C-8), 147.33, 149.89 (C-4, C-2, and
C4′′), 156.07 (C-6). HRMS (ESI-MS) C19H27N8O4 [M + H]+:
431.2153 found, 431.2155 calcd. Anal. (C19H26N8O4) C, H, N.
2-(4-Cyclohexylmethyl-1,2,3-triazol-1-yl)-N6-methyl-9-(â-D-ri-
bofuranosyl)adenine (11). The reaction of 23 (70 mg, 0.217 mmol)
with cyclohexyl-1-propyne (63 µL, 0.435 mmol) gave compound
2-[1-(3-Chlorobenzyl)-1,2,3-triazol-4-yl)-N6-methyl-9-(â-D-ri-
bofuranosyl)adenine (14). The reaction of 25 (100 mg, 0.32 mmol)
with 107 mg (0.64 mmol) of 3-chlorobenzylazide gave compound
1
14 in 73% yield (110 mg). H NMR (300 MHz, DMSO-d6): δ
3.04 (br s, 3H, N6-CH3), 3.53-3.60 (m, 1H, H-5′A), 3.64-3.71
(m, 1H, H-5′B), 3.96 (app d, H-4′, J ) 2.9 Hz, H-4′), 4.17 (dd,
1H, J ) 4.7 and 7.6 Hz, H-3′), 4.64 (app q, 1H, J ) 5.8 Hz, H-2′),
5.09 (t, 1H, J ) 5.3 Hz, 5′-OH), 5.20 (d, 1H, J ) 4.4 Hz, 3′-OH),
5.45 (d, 1H, J ) 6.2 Hz, 2′-OH), 5.97 (d, 1H, J ) 6.2 Hz, H-1′),
7.31-7.36 (m, 1H, Ph), 4.41-7.47 (m, 3H, Ph), 7.83 (br s, 1H,
N6-H), 8.37 (s, 1H, H-8), 8.72 (s, 1H, H-5′′). 13C NMR (300 MHz,
DMSO-d6): δ 27.54 (N6-CH3), 52.80 (CH2), 62.31 (C-5′), 71.33
(C-3′), 74.27 (C-2′), 86.43 (C-4′), 87.69 (C-1′), 119.56 (C-5), 126.63
(C-5′′), 127.53, 128.53, 128.84, 131.42, 133.98, and 139.15 (Ph),
140.36 (C-8), 148.22, 150.03, and 153.43 (C-2, C-4, and C-4′),
155.78 (C-6). HRMS (ESI-MS) C20H22N8O4Cl [M + H]+: 473.1452
found, 473.1452 calcd. Anal. (C20H21N8O4Cl) C, H, N.
1
11 in 82% yield. H NMR (300 MHz, DMSO-d6): δ 0.86-1.28
(br m, 6H, cyclohexyl), 1.54-1.72 (br m, 5H, cylcohexyl), 2.58
(d, 2H, J ) 6.9 Hz, CH2), 3.03 (d, 3H, J ) 3.9 Hz, N6-CH3), 3.51-
3.59 (m, 1H, H-5′A), 3.63-3.70 (m, 1H, H-5′B), 3.94 (dd, 1H, J
) 4.2 and 7.5 Hz, H-4′), 4.16 (dd, 1H, J ) 4.8 and 8.1 Hz, H-3′),
4.59 (app q, 1H, J ) 6.3 Hz, H-2′), 4.97 (t, 1H, J ) 5.5 Hz, 5′-
OH), 5.22 (d, 1H, J ) 4.8 Hz, 3′-OH), 5.47 (d, 1H, J ) 6.3 Hz,
2′-OH), 5.93 (d, 1H, J ) 6.0 Hz, H-1′), 8.30 (d, 1H, J ) 3.9 Hz,
N6-H), 8.43 (s, 1H, H-8), 8.51 (s, 1H, H-5′′). 13C NMR (300 MHz,
DMSO-d6): δ 26.31 (cyclohexyl), 26.66 (cyclohexyl), 27.84 (N6-
CH3), 33.16 (cyclohexyl), 38.22 (CH2), 62.14 (C-5′), 71.15 (C-3′),
74.35 (C-2′), 86.44 (C-4′), 87.86 (C-1′), 119.60 (C-5), 121.93 (C-
5′′), 140.92 (C-8), 146.45, 149.92 (C-4, C-2, and C-4′′), 153.51
(C-6). HRMS (ESI-MS) C20H29N8O4 [M + H]+: 445.2305 found,
445.2311 calcd. Anal. (C20H28N8O4) C, H, N.
9-(5-Ethylcarbamoyl-â-D-ribofuranosyl)-N6-methyl-2-(1,2,3-
triazol-1-yl)adenine (15). In a pressure tube was added 31 (110
mg, 0.30 mmol), trimethylsilylacetylene (259 µL, 1.81 mmol), and
4 mL of DMF. The mixture was stirred at 105 °C for 15 h. Solvent
evaporation yielded a yellowish solid that was dissolved 6 mL of
a 1.0 solution of tetrabutylammonium fluoride in THF and stirred
for 5 h. After solvent evaporation, the residue was dissolved in
ethyl acetate. Water was added, and the triazole precipitated in the
water layer. After overnight cooling and filtration, the precipitate
was further purified on a silica gel column (CH2Cl2/MeOH, 93:7)
and yielded compound 15 as a white solid (49 mg, 42%). 1H NMR
(300 MHz, DMSO-d6): δ 0.90 (t, 3H, CH3), 3.05-3.21 (m, 2H,
N-CH2), 3.05 (d, 3H, J ) 4.2 Hz, N6-CH3), 4.26 (m, 1H, H-3′),
4.33 (d, 1H, J ) 2.1 Hz, H-4′), 5.61 (d, 1H, J ) 6.2 Hz, 3′-OH),
General Procedure for the Synthesis of 4′′-Substituted
2-(1,2,3-Triazol-4-yl)adenosine Derivatives 12-14. Compound 25

7380 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Cosyn et al.
5.71 (d, 1H, J ) 4.7 Hz, 2′-OH), 6.04 (d, 1H, J ) 7.2 Hz, H-1′),
7.91 (d, 1H, J ) 1.2 Hz, H-4′′), 8.07 (t, 1H, J ) 5.3 Hz, NHCO),
8.41 (d, 1H, J ) 4.2 Hz, N6-H), 8.54 (s, 1H, H-8), 8.82 (br s, 1H,
H-5′′). 13CMR (300 MHz, DMSO-d6): δ 15.16 (CH3), 27.85 (N6-
CH3), 34.07 (CH2), 73.64 (C-2′ and C-3′), 84.98 (C-4′), 88.02 (C-
1′), 119.91 (C-5), 124.70 (C-5′′), 134.29 (C-4′′), 141.59 (C-8),
149.69 and 149.90 (C-2 and C-4), 156.14 (C-6), 169.73 (CdO).
HRMS (ESI-MS) C15H20N9O4 [M + H]+: 390.1676 found,
390.1683 calcd. Anal. (C15H19N9O4) C, H, N.
General Procedure for the Synthesis of 4′′-Substituted
2-(1,2,3-Triazol-1-yl)adenosine Derivatives 16-18. To a mixture
of 31 (100 mg, 0.28 mmol), CuI (5 mg, 0.03 mmol), and
triethylamine (40 µL, 0.28 mmol) in water/acetonitrile (1:1), the
appropriate alkyne (2 equiv) was added. The mixture was stirred
for 5 days at room temperature. The reaction was monitored by 1H
NMR. The product was precipitated with water and cooled
overnight. After filtration, the yellowish solid was purified on a
silica gel column (CH2Cl2/MeOH, 90:10) to obtain the 1,2,3-triazol-
1-yladenosine derivative as a white solid.
84.98 (C-4′), 87.87 (C-1′), 119.91 (C-5), 122.11 (C-5′′), 126.93,
129.13, 129.22, and 140.07 (Ph), 140.48 (C-8), 147.05, 149.87 (C-
4, C-2, and C-4′′), 156.12 (C-6), 169.71 (CdO). HRMS (ESI-MS)
C22H26N9O4 [M + H]+: 480.2098 found, 480.2107 calcd. Anal.
(C22H25N9O4) C, H, N.
2-(4-Cyclopentylmethyl-1,2,3-triazol-1-yl)-9-(5-ethylcarbam-
oyl-â-D-ribofuranosyl)-N6-methyladenine (19). Compound 31 (60
mg, 0.17 mmol), sodium ascorbate (13 mg, 0.066 mmol) and
CuSO4‚5H2O (3.5 mg, 0.013 mmol) were suspended in 4 mL of
tBuOH/H2O (1:1). 3-Cyclopentyl-1-propyne (58 µL, 0.44 mmol)
was subsequently added, and the mixture was stirred for 2 days at
room temperature. The 2-triazol-1-yl compound precipitated from
the reaction medium. Water was added, and the mixture was cooled
overnight. The precipitate was filtered off and washed with water
1
and hexane to obtain 19 as a white solid in 33% yield. H NMR
(300 MHz, DMSO-d6): δ 0.89 (t, 3H, CH3), 1.19-1.28 (m, 2H,
cyclopentyl), 1.45-1.75 (m, 6H, cyclopentyl), 2.15-2.25 (m, 1H,
CH, cyclopentyl), 2.72 (d, 2H, J ) 7.2 Hz, CH2), 3.06 (d, 3H, J )
4.5 Hz, N6-CH3), 3.09-3.22 (m, 2H, N-CH2), 4.24 (dt, 1H, J )
1.5 and 4.8 Hz, H-3′), 4.33 (d, 1H, J ) 2.1 Hz, H-4′), 4.71-7.76
(app q, 1H, J ) 6.6 Hz, H-2′), 5.61 (d, 1H, J ) 6.3 Hz, 2′-OH),
5.71 (d, 1H, J ) 4.8 Hz, 3′-OH), 6.03 (d, 1H, J ) 6.9 Hz, H-1′),
8.10 (t, 1H, J ) 5.7 Hz, NHCO), 8.40 (d, 1H, J ) 5.1 Hz, N6-H),
8.54 (s, 1H, H-8), 8.56 (s, 1H, H-5′′). 13C NMR (300 MHz, DMSO-
d6): δ 15.17 (CH3), 25.36 (cyclopentyl), 27.85 (N6-CH3), 31.53
(cyclopentyl), 31.51 (CH2), 34.07 (CH2), 73.63 and 73.56 (C-2′
and C-3′), 84.99 (C-4′), 87.93 (C-1′), 119.77 (C-5), 121.69 (C-5′′),
141.51 (C-8), 147.48 and 149.92 (C-2, C-4, and C-4′′), 156.10 (C-
6), 169.74 (CdO). HRMS (ESI-MS) C21H30N9O4 [M + H]+:
472.2415 found, 472.2420 calcd. Anal. (C21H29N9O4) C, H, N.
N6-(5-Chloro-2-methoxybenzyl)-2-(4-cyclopentylmethyl-1,2,3-
triazol-1-yl)-9-(â-D-ribofuranosyl)adenine (20). Compound 33
(100 mg, 0.22 mmol), sodium ascorbate (17 mg, 0.086 mmol), and
CuSO4‚5H2O (3.5 mg, 0.017 mmol) were suspended in 4 mL of
H2O/tBuOH (1:1). 3-Cyclopentyl-1-propyne (29 µL, 0.44 mmol)
was subsequently added, and the mixture was stirred for 2 days at
room temperature. The 2-triazol-1-yl compound precipitated from
the reaction medium. Water was added, and the mixture was cooled
overnight. The precipitate was filtered off and washed with water
2-(4-Butyl-1,2,3-triazol-1-yl)-9-(5-ethylcarbamoyl-â-D-ribo-
furanosyl)-N6-methyladenine (16). The reaction of compound 31
(100 mg, 0.28 mmol) with 1-hexyne (64µL, 0.56 mmol) gave 65
1
mg (53%) of compound 16 as a white solid. H NMR (300 MHz,
DMSO-d6) δ 0.88-0.95 (m, 6H, 2 × CH3), 1.31-1.43 (m, 2H,
CH2), 1.61-1.71 (m, 2H, CH2), 2.72 (t, 2H, J ) 7.8 Hz, C4′′-
CH2), 3.05-3.22 (m, 2H, N-CH2), 3.05 (d, 3H, J ) 4.1 Hz, N6-
CH3), 4.25 (m, 1H, H-3′), 4.33 (d, 1H, J ) 2.1 Hz, H-4′), 4.73 (m,
1H, H-2′), 5.59 (d, 1H, J ) 6.3 Hz, 3′-OH), 5.69 (d, 1H, J ) 4.5
Hz, 2′-OH), 6.04 (d, 1H, J ) 6.9 Hz, H-1′), 8.09 (t, 1H, J ) 5.4
Hz), 8.37 (d, 1H, J ) 4.1 Hz, N6-H), 8.53 (s, 1H, H-8), 8.56 (s,
1H, H-5′′). 13C NMR (300 MHz, DMSO-d6): 14.36 (CH3), 15.17
(CH3), 22.35 (CH2), 25.21 (CH2), 27.84 (N6-CH3), 31.70 (CH2),
34.07 (CH2), 73.60 and 73.64 (C-2′ and C-3′), 84.99 (C-4′′), 87.91
(C-1′), 119.79 (C-5), 121.45 (C-5′′), 141.49 (C-8), 147.97, 149.72,
and 149.93 (C-2, C-4, and C-4′′), 156.11 (C-6), 169.73 (CdO).
HRMS (ESI-MS) C19H28N9O4 [M + H]+: 446.2256 found,
446.2264 calcd. Anal. (C19H27N9O4) C, H, N.
9-(5-Ethylcarbamoyl-â-D-ribofuranosyl)-N6-methyl-2-(4-py-
ridin-2-yl-1,2,3-triazol-1-yl)adenine (17). The reaction of com-
pound 31 (50 mg, 0.14 mmol) with 2-ethynylpyridine (56 µL, 0.56
1
and hexane to obtain 20 as a white solid in 59% yield. H NMR
1
(300 MHz, DMSO-d6): δ 1.23-1.28 (m, 2H, cyclopentyl), 1.48-
1.61 (m, 4H, cyclopentyl), 1.66-1.73 (m, 2H, cyclopentyl), 2.15-
2.25 (m, 1H, cyclopentyl), 2.72 (d, 2H, J ) 7.2 Hz, CH2-
cyclopentyl), 3.56-3.61 (m, 1H, H-5′A), 3.66-3.71 (m, 1H,
H-5′B), 3.86 (s, 3H, OCH3), 3.97 (m, 1H, H-4′), 4.20 (m, 1H, H-3′),
4.64 (m, 1H, H-2′), 4.73 (br s, 2H, N6-CH2), 4.96 (t, 1H, J ) 6.0
Hz, 5′-OH), 5.22 (d, 1H, J ) 4.8 Hz, 3′-OH), 5.48 (d, 1H, J ) 5.7
Hz, 2′-OH), 5.95 (d, 1H, J ) 6.3 Hz, H-1′), 7.04 (d, 1H, J ) 9.0
Hz, Ph), 7.25-7.29 (m, 2H, Ph), 8.40 (s, 1H, H-8), 8.81 (s, 1H,
H-5′′), 8.81 (br s, 1H, N6-H). 13C NMR (300 MHz, DMSO-d6): δ
25.36 (cyclopentyl), 31.54 (CH2), 32.54 (cyclopentyl), 38.86 (CH2),
56.57 (OCH3), 62.13 (C-5′), 71.15 (C-3′), 74.37 (C-2′), 86.46 (C-
4′), 88.07 (C-1′), 113.15 (Ph), 119.58 (C-5), 121.37 (C-5′′), 124.66,
128.23, and 129.93 (Ph), 141.37 (C-8), 147.40, 149.66, and 150.12
(C-2, C-4, and C-4′′), 155.51 (Ph), 156.33 (C-6). HRMS (ESI-MS)
C26H32N8O5Cl [M + H]+: 571.2184 found, 571.2184 calcd. Anal.
(C26H31N8O5Cl) C, H, N.
mmol) gave 35 mg (54%) of compound 17 as a white solid. H
NMR (300 MHz, DMSO-d6): δ 0.93 (t, 3H, J ) 7.2 Hz, CH3),
3.09-3.21 (m, 2H, N-CH2), 3.10 (d, 3H, J ) 4.2 Hz, N6-CH3),
4.25 (m, 1H, H-3′), 4.35 (d, 1H, J ) 2.1 Hz, H-4′), 4.74 (m, 1H,
H-2′), 5.68 (d, 1H, J ) 6.6 Hz, 2′-OH), 5.75 (d, 1H, J ) 4.5 Hz,
3′-OH), 6.08 (d, 1H, J ) 6.6 Hz, H-1′), 7.41 (m, 1H, pyridin-2-
yl), 7.96 (m, 1H, pyridin-2-yl), 8.10 (t, 1H, J ) 6.0 Hz, NHCO),
8.16 (d, 1H, J ) 8.1 Hz, pyridin-2-yl), 8.48 (d, 1H, J ) 4.1 Hz,
1H, N6-H), 8.60 (s, 1H, H-8), 8.67 (d, H, J ) 5.1 Hz, pyridin-2-
yl), 9.17 (s, 1H, H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 15.18
(CH3), 27.94 (N6-CH3), 34.11 (N-CH2), 73.69 and 73.78 (C-2′ and
C-3′), 84.95 (C-4′), 87.98 (C-1′), 120.04 (C-5), 120.65 (C-5′′),
122.20 and 124.11 (pyridin-2-yl), 138.027 (pyridin-2-yl), 141.64
(C-8), 147.95, 149,64, 149.67, and 149.98 (C-2, C-4, C-4′′, and
pyridin-2-yl), 150.49 (pyridin-2-yl), 156.13 (C-6), 169.76 (CdO).
HRMS (ESI-MS) C20H23N10O4 [M + H]+: 467.1899 found,
467.1903 calcd. Anal. (C20H22N10O4) C, H, N. N calcd, 30.03;
found, 29.55.
2-Azido-N6-methyl-9-(â-D-ribofuranosyl)adenine (23). Sodium
ascorbate (19.4 mg, 0.098 mmol) and CuSO4‚5H2O (12.2 mg, 0.049
mmol) were added to a mixture of 22 (200 mg, 0.491 mmol),
sodium azide (38,3 mg, 0.589 mmol), L-proline (11,3 mg, 0.098
mmol), and sodium carbonate (10.4 mg, 0.098 mmol) in 10 mL of
H2O/tBuOH (1:1). The mixture was stirred overnight at 65 °C and
9-(5-Ethylcarbamoyl-â-D-ribofuranosyl)-N6-methyl-2-(4-ben-
zyl-1,2,3-triazol-1-yl)adenine (18). The reaction of compound 31
(70 mg, 0.19 mmol) with 3-phenyl-1-propyne (49µL, 0.56 mmol)
1
gave 35 mg (38%) of compound 18 as a white solid. H NMR
(300 MHz, DMSO-d6): δ 0.87 (t, 3H, J ) 7.5 Hz, CH3), 3.03-
3.20 (m, 2H, N-CH2), 3.03 (d, 3H, 4.5 Hz, N6-CH3), 4.11 (s, 2H,
CH-Ph), 4.24 (m, 1H, H-3′), 4.32 (d, 1H, J ) 2.1 Hz, H4′), 4.72
(m, 1H, H-2′), 5.59 (d, 1H, J ) 6.6 Hz, 3′-OH), 5.69 (d, 1H, J )
4.5 Hz, 2′-OH), 6.03 (d, 1H, J ) 6.9 Hz, H1′), 7.22 (m, 1H, Ph),
7.32 (d, 4H, J ) 4.2 Hz, Ph), 8.06 (t, 1H, J ) 5.7 Hz, NHCO),
8.38 (d, 1H, J ) 4.1 Hz, N6-H), 8.54 (s, 1H, H-8), 8.60 (s, 1H,
H-5′′). 13C NMR (300 MHz, DMSO-d6): δ 15.16 (CH3), 27.84
(N6-CH3), 31.72 (CH2), 34.07 (N-CH2), 73.64 (C-2′ and C-3′),
1
was monitored by H NMR. Then 50 mL of dilute NH4OH was
added and the crude mixture extracted with ethyl acetate (3 × 60
mL). The organic layer was washed with brine (60 mL), dried over
MgSO4, and purified on a silica gel column (CH2Cl2/MeOH, 95:5)
to afford compound 23 as a slightly yellow solid in 66% yield. 1H
NMR (300 MHz, DMSO-d6): δ 2.91 (d, 3H, J ) 4.4 Hz, N6-CH3),
3.48-3.55 (m, 1H, H-5′A), 3.58-3.66 (m, 1H, H-5′B), 3.90 (dd,
1H, J ) 3.8 and 7.3 Hz, H-4′), 4.10 (dd, 1H, J ) 4.7 and 9.7 Hz,

2-Triazole-Substituted Adenosines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7381
H3′), 4.53 (app q, 1H, J ) 5.9 Hz, H-2′), 5.04 (dd, 1H, J ) 5.2
and 6.2 Hz, 5′-OH), 5.19 (d, 1H, J ) 5.0 Hz, 3′OH), 5.43 (d, 1H,
J ) 6.2 Hz, 2′-OH), 5.78 (d, 1H, J ) 6.2 Hz, H-1′), 8.12 (d, J )
4.4 Hz, N6-H), 8.27 (s, 1H, H-8). Small peaks from 1/6 tetrazole
tautomeric form: δ 3.15 (d, 3H, J ) 5.0 Hz, N6-CH3), 3.95 (d,
H-4′), 4.15 (d, H-3′), 5.51 (d, 2′-OH), 5.94 (d, H-1′), 8.51 (s, H-8).
13C NMR (300 MHz, DMSO-d6): δ 27.54 (N6-CH3), 62.21 (C-5′),
71.17 (C-3′), 74.12 (C-2′), 86.35 (C-4′), 88.01 (C-1′), 118.07 (C-
5), 139.99 (C-8), 156.06 and 156.20 (C-2 and C-6). Small peaks
from 1/6 tetrazole tautomeric form: δ 31.89 (N6-CH3), 61.79 (C-
5′), 70.77 (C-3′), 74.37 (C-4′), 112.30 (C-12), 142.91 (C-11), 147.60
(C-6). HRMS (ESI-MS) C11H15N8O4 [M + H]+: 323.1208 found,
323.1216 calcd. Anal. (C11H14N8O4) C, H, N.
purified by silica gel chromatography (CH2Cl2/MeOH, 96:4).
Compound 30 was obtained as a white solid in 78% yield (840
mg). 1H NMR (300 MHz, DMSO-d6): δ 1.05 (t, 3H, J ) 7.2 Hz,
CH3), 2.91 (d, 3H, J ) 4.4 Hz, N6-CH3), 3.19-3.29 (m, 2H,
N-CH2), 4.16 (dt, 1H, J ) 2.1 and 4.4 Hz, H-3′), 4.31 (d, 1H, J )
2.1 Hz, H-4′), 4.58 (app q, 1H, J ) 5.6 Hz, H-2′), 5.59 (d, 1H, J
) 6.5 Hz, 2′-OH), 5.71 (d, 1H, J ) 4.7 Hz, 3′-OH), 5.92 (d, 1H,
J ) 7.1 Hz, H-1′), 8.12 (t, 1H, J ) 5.5 Hz, NHCO), 8.19 (d, 1H,
J ) 4.4 Hz, N6-H), 8.38 (s, 1H, H-8). HRMS (ESI-MS) C13H18N6O4I
[M + H]+: 449.0429 found, 449.0436 calcd.
2-Azido-9-(5-ethylcarbamoyl-â-D-ribofuranosyl)-N6-methyl-
adenine (31). Sodium ascorbate (69 mg, 0.34 mmol) and CuSO4‚
5H2O (5.6 mg, 0.17 mmol) were added to a mixture of 30 (780
mg, 1.74 mmol), sodium azide (226 mg, 3.48 mmol), L-proline (40
mg, 0.35 mmol), and sodium carbonate (37 mg, 0.35 mmol) in 20
mL of H2O/tBuOH (1:1). The mixture was stirred for 2 days at 65
N6-Methyl-9-(â-D-ribofuranosyl)-2-[2-trimethylsilylethyn-1-
yl]adenine (24). Compound 22 (500 mg, 1.23 mmol), CuI (12 mg,
0.062 mmol), and (Ph3P)3PCl2 were dissolved in 9 mL of DMF.
Triethylamine (205 µL, 1.47 mmol) and trimethylsilylacetylene (210
mg, 1.47 mmol) were added, and the reaction mixture was stirred
overnight. After solvent evaporation, the residue was dissolved in
CH2Cl2 and filtered through a pad of Celite. Purification on a silica
gel column (CH2Cl2/MeOH, 95:5) yielded 305 mg (66%) of
1
°C and monitored by H NMR. An amount of 100 mL of dilute
NH4OH was added, and the crude mixture was extracted with ethyl
acetate (5 × 150 mL) and washed with brine (150 mL). The organic
layer was dried over MgSO4 and purified on a silica gel column
(CH2Cl2/MeOH, 96:4) to afford compound 31 as a white solid in
1
1
compound 23. H NMR (300 MHz, DMSO-d6): δ 0.00 (9H, s,
79% yield (500 mg). H NMR (300 MHz, DMSO-d6): δ 1.06 (t,
(CH3)3Si), 2.7 (3H, N6-CH3), 3.26-3.34 (m, 1H, H-5′A), 3.37-
3.44 (m, 1H, H-5′B), 3.68 (dd, 1H, J ) 3.5 Hz, H-4′), 3.86 (dd,
1H, J ) 3.4 and 8.2 Hz, H-3′), 4.23 (app q, 1H, J ) 5.9 Hz, H-2′),
4.87 (t, 1H, J ) 5.0, 5′-OH), 4.94 (d, 1H, J ) 4.99 Hz, 3′-OH),
5.21 (d, 1H, J ) 6.2 Hz, 2′-OH), 5.63 (d, 1H, J ) 6.2 Hz, H-1′),
7.69 (br s, 1H, N6-H), 8.19 (s, 1H, H-8). HRMS (ESI-MS)
C16H24N5O4Si: [M + H]+: 378.1586 found; 378.1597 calcd.
2-Ethynyl-N6-methyl-9-(â-D-ribofuranosyl)purine (25). An
amount of 300 mg (0.8 mmol) of compound 24 was dissolved in
7 N ammonia in methanol and stirred for 2 h at 0 °C. After solvent
evaporation, the residue was purified by silica gel chromatography
(CH2Cl2/MeOH, 95:5) to obtain 160 mg (65%) of derivative 25.
1H NMR (300 MHz, DMSO-d6): δ 2.91 (s, 3H, N6-CH3), 3.48-
3.56 (m, 1H, H-5′A), 3.61-3.68 (m, 1H, H-5′B), 3.92 (m, 1H, H-4′),
4.02 (s, 1H, CtCH), 4.11 (dd, 1H, J ) 5.0 and 8.2 Hz, H-3′), 4.53
(app q, 1H, J ) 5.9 Hz, H-2′), 5.15 (m, 2H, 3′-OH and 5′-OH),
5.44 (d, 1H, J ) 6.15 Hz, 2′-OH), 5.84 (d, 1H, J ) 6.16 Hz, H-1′),
7.95 (br s, 1H, N6-H), 8.40 (s, 1H, H-8). HRMS (ESI-MS)
C13H16N5O4 [ M + H]+: 306.1197 found; 306.1202 calcd.
1-Deoxy-1-(6-methylamino-2-iodo-9H-purin-9-yl)-2,3-O-iso-
propylidene-â-D-ribufuranuronic Acid (27). To a stirred solution
of 3.8 g (8.5 mmol) of 26 in 560 mL of H2O were added 1.4 g of
KOH and, dropwise, a solution of 4.03 g (25.5 mmol) of KMnO4
in 110 mL of H2O. The mixture was stirred in the dark for 20 h,
cooled to 0 °C, and quenched with 30 mL of 7% H2O2. The mixture
was filtered through Celite. The filtrate was concentrated in vacuo
and then acidified to pH 4 with 3 N HCl. The resulting precipitate
was filtered off and successively washed with water and ether to
3H, J ) 7.2 Hz, CH3), 2.96 (d, 3H, J ) 4.0 Hz, N6-CH3), 3.16-
3.29 (m, 2H, N-CH2), 4.14 (dt, 1H, J ) 1.8 and 4.8 Hz, H-3′),
4.29 (d, 1h, J ) 1.8, H-4′), 4.53-4.59 (app q, 1H, J ) 6.3 Hz,
H-2′), 5.52 (d, 1H, J ) 6.3 Hz, 2′-OH), 5.68 (d, 1H, J ) 4.8 Hz,
3′-OH), 5.90 (d, 1H, J ) 7.2 Hz, H-1′), 8.20 (d, 1H, J ) 4.0 Hz,
N6-H), 8.35 (s, 1H, H-8), 8.49 (t, 1H, J ) 6.0 Hz, NHCO). Small
peaks from 1/5 tetrazole tautomeric form: δ 1.08-1.12 (t, 3H, J
) 7.2 Hz, CH3), 4.34 (d, 1H, J ) 2.1 Hz, H-4′), 4.67-4.73 (app
q, 1H, J ) 7.2 Hz, H-2′), 5.54 (d, 1H, J ) 4.5 Hz, 2′-OH) 6.00 (d,
1H, J ) 7.2 Hz, H-1′). 13C NMR (300 MHz, DMSO-d6): δ 15.48
(CH3), 27.52 (N6-CH3), 33.94 (N-CH2), 73.06 and 73.75 (C-2′ and
C-3′), 85.15 (C-4′), 88.09 (C-1′), 118.36 (C-5), 140.69 (C-8),
156.032 and 156.143 (C-2 and C-4), 169.79 (C-6). HRMS (ESI-
MS) C13H18N9O4 [M + H]+: 364.1473 found; 364.1481 calcd.
Anal. (C13H17N9O4) C, H, N.
N6-(5-Chloro-2-methoxybenzyl)-2-iodo-9-(â-D-ribofuranosyl)-
adenine (32). Compound 21 (1 g, 1.86 mmol) was dissolved in
EtOH (30 mL). 5-Chloro-2-methoxybenzylammonium chloride (580
mg, 2.79 mmol) and Et3N (392 µL, 2.79 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 2′-hydroxyl group. The volatiles
were removed under reduced pressure, and the residue was purified
by silica gel column (CH2Cl2/MeOH, 97:3). The product, compound
32, was realized in 80% yield. 1H NMR (300 MHz, DMSO-d6): δ
3.51-3.58 (m, 1H, H-5′A), 3.63-3.68 (m, 1H, H-5′B), 3.85 (s,
3H, OCH3), 3.94 (m, 1H, H-4′), 4.13 (m, 1H, H-3′), 4.52-4.59
(m, 3H, N6-CH2 and H-2′), 5.03 (t, 1H, J ) 5.6 Hz, 5′-OH), 5.21
(d, 1H, J ) 5.0 Hz, 3′-OH), 5.48 (d, 1H, J ) 5.8 Hz, 2′-OH), 5.83
(d, 1H, J ) 6.2 Hz, H-1′), 7.03 (d, 1H, J ) 8.8 Hz, Ph), 7.16 (d,
1H, J ) 2.7 Hz, Ph), 7.29 (dd, 1H, J ) 2.7 and 8.8 Hz, Ph), 8.35
(s, 1H, H-8), 8.62 (br s, 1H, N6-H). HRMS (ESI-MS) C18H20N5O5-
ICl [M + H]+: 548.0204 found; 548.0199 calcd.
1
give 2.98 g (76%) of 27 as a white solid. H NMR (300 MHz,
DMSO-d6): δ 1.36 (s, 3H, CH3), 1.51 (s, 3H, CH3), 2.89 (d, 3H,
J ) 3.3 Hz, N6-CH3), 4.68 (d, 1H, J ) 1.8 Hz, H-4′), 5.40 (d, 1H,
J ) 6.0 Hz, H-2′), 5.47 (dd, 1H, J ) 6.0 and 1.8 Hz, H-3′), 6.28
(s, 1H, H-1′), 8.08 (d, 1H, J ) 3.3 Hz, N6-H), 8.16 (s, 1H, H-8).
HRMS (ESI-MS) C14H16N5O5I [M + H]+: 462.0273 found;
462.0276 calcd.
9-(5-Ethylcarbamoyl-â-D-ribofuranosyl)-2-iodo-N6-methylad-
enine (30). p-Nitrophenol (402 mg, 2.89 mmol) and 1-[3-(dim-
ethylamino)propyl]-3-ethylcarbodiimide hydrochloride (506 mg,
2.65 mmol) were added to a solution of 27 (1.11 g, 2.41 mmol) in
10 mL of dry DMF. The reaction mixture was stirred for 3 h at
room temperature and cooled to 0 °C, and 1.6 mL (24.1 mmol) of
ethylamine was added. The solution turned yellow immediately and
was further stirred for 1 h at room temperature. After evaporation
of the volatiles, the residue was partitioned between ethyl acetate
(3 × 100 mL) and H2O (100 mL). The organic layer was washed
with brine (100 mL), dried over MgSO4, and concentrated to
dryness. The residue was dissolved in 80% aqueous TFA (20 mL)
and stirred for 2 h at room temperature. The mixture was
concentrated in vacuo, coevaporated several times with EtOH, and
2-Azido-N6-(5-chloro-2-methoxybenzyl)-9-(â-D-ribofuranosy-
l)adenine (33). Sodium ascorbate (14 mg, 0.073 mmol) and CuSO4‚
5H2O (9 mg, 0.037 mmol) were added to a mixture of 32 (200 mg,
0.365 mmol), sodium azide (47 mg, 0.73 mmol), L-proline (8 mg,
0.073 mmol), and sodium carbonate (8 mg, 0.073 mmol) in 4 mL
of H2O/tBuOH (1:1). The mixture was stirred for 2 days at 65 °C
1
and monitored by H NMR. An amount of 10 mL of dilute NH4-
OH was added, and the crude mixture was extracted with ethyl
acetate (5 × 15 mL) and washed with brine (15 mL). The organic
layer was dried over MgSO4 and purified on a silica gel column
(CH2Cl2/MeOH, 97:3) to afford compound 33 as a white solid in
82% yield. 1H NMR (300 MHz, DMSO-d6): δ 3.51-3.56 (m, 1H,
H5′-A), 3.60-3.66 (m, 1H, H5′-B), 3.83 (s, 3H, OCH3), 3.93 (m,
1H, H 4′), 4.13 (m, 1H, H-3′), 4.56-4.62 (m, 3H, N6-CH2 and
H-2′), 5.03 (t, 1H, J ) 5.4 Hz, 5′-OH), 5.17 (d, 1H, J ) 4.8 Hz,
3′-OH), 5.41 (d, 1H, J ) 6.3 Hz, 2′-OH), 5.81 (d, 1H, J ) 6.0 Hz,

7382 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Cosyn et al.
H-1′), 7.02 (d, 1H, J ) 8.7 Hz, Ph), 7.15 (d, 1H, J ) 3.0 Hz, Ph),
7.28 (dd, 1H, J ) 2.7 and 8.7 Hz, Ph), 8.34 (s, 1H, H-8), 8.63 (br
s, 1H, N6-H). 13C NMR (300 MHz, DMSO-d6): δ 38.59 (CH2),
56.49 (OCH3), 62.19 (C-5′), 71.15 (C-3′), 74.13 (C-2′), 86.36 (C-
4′), 88.13 (C-1′), 113.02 (Ph), 119.58 (C-5), 124.29 (Ph), 128.10
(Ph), 129.93 (Ph), 140.43 (C-8), 150.69 (C-4), 155.51 (Ph and C-2),
156.33 (C-6). HRMS (ESI-MS) C18H20N8O5Cl [M + H]+: 463.1248
found, 463.1245 calcd. Anal. (C18H19N8O5Cl) C, H, N.
Cell Culture and Membrane Preparation. CHO cells express-
ing recombinant human ARs or the rat A3AR were cultured in
DMEM (Dulbecco’s modified Eagle’s medium) and F12 (1:1)
supplemented with 10% fetal bovine serum, 100 units/mL penicillin,
100 µg/mL streptomycin, and 2 µmol/mL glutamine. After harvest
and homogenization, the cells were centrifuged at 500g for 10 min.
The pellet was resuspended in 50 mM Tris-HCl buffer (pH 7.4)
containing 10 mM MgCl2 and 1 mM EDTA. The suspension was
homogenized with an electric homogenizer for 10 s and was then
recentrifuged at 20000g for 20 min at 4 °C. The resulting pellets
were resuspended in buffer containing 3 units/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.39
Radioligand Binding Studies. For the A3AR binding experi-
ments, the procedures were similar to those previously described.19
Briefly, each tube contained 100 µL of membrane suspension, 50
µL of [125I]I-AB-MECA ([125I]N6-(4-amino-3-iodobenzyl)adenosine-
5′-N-methyluronamide, final concentration of 0.5 nM), and 50 µL
of increasing concentrations of compounds in Tris-HCl buffer (50
mM, pH 7.4) containing 10 mM MgCl2 and 1 mM EDTA.
Nonspecific binding was determined using 10 µM NECA (adenos-
ine-5′-N-ethyluronamide). 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 γ-counter. The binding of [3H]CCPA (2-chloro-
N6-cyclopentyladenosine) to the recombinant hA1AR and the
binding of [3H]CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-
5′-N-ethylcarboxamidoadenosine) to the recombinant hA2AAR was
performed as previously described.20,44
Sketch Molecule of SYBYL 7.1 (Tripos Inc., 1699 South Hanley
Road, St. Louis, MO 63144). A grid search was performed in which
flexible bonds were rotated by 0° and 180° for t1 (C5-C6-N6-
C
Me) at the N6 position, t2 (4′O-4′C-5′C-5′OH) at the 5′-position,
and t3 (N3-C2-N1′-N2′) and by 60°, 180°, and -60° for t4 (N3′-
C4′-CMe-CCyc) and t5 (C4′-CMe-CCyc-CCyc) at the C2 position.
The low-energy conformers from the grid search were reoptimized,
removing all torsional constraints. Merck molecular force field
(MMFF)41 and charges were applied with the use of distance-
dependent dielectric constants and the conjugate gradient method
until the gradient reached 0.05 kcal‚mol-1‚Å-1. After the low-energy
conformers from the result of the grid search were clustered, the
representative ones from all groups were reoptimized by semiem-
pirical molecular orbital calculations with the PM3 method in the
MOPAC 6.0 package.42
A human A3AR model (PDB code 1OEA) constructed by
homology to the X-ray structure of bovine rhodopsin with 2.8 Å
resolution (PDB code 1F88)38 was used for the docking study. All
atom types were assigned by the Amber7_FF99 force field.43 Amber
charges for protein and MMFF charges for ligand were calculated.
The starting geometry of the ligand conformation was chosen from
the human A3AR complex model with Cl-IB-MECA,19 which was
already validated by point mutation. The ribose binding position
was fixed, using an atom-by-atom fitting method for the carbon
atoms of the ribose ring. To determine the binding region of the
2-(4-cyclopentylmethyl-1,2,3-triazole) moiety at the adenine 2
position, the flexible bond defining a ø1 (O-C1′-N9-C4) angle
was searched while docked within the putative binding cavity
through various low-energy conformers with diverse t1-t5 angles,
rotating by -60°, -110°, and -160°, assuming an anti conforma-
tion. Several conformations without any steric bump were selected
for further optimization. The initial structures of all complexes were
optimized using the Amber force field with a fixed dielectric
constant of 4.0 and a terminating gradient of 0.05 kcal‚mol-1‚Å-1
.
Acknowledgment. This research was supported in part by
the Intramural Research Program of the NIH, National Institute
of Diabetes and Digestive and Kidney Diseases.
Supporting Information Available: Elemental analysis data
for compounds 1-20, 23, 31, and 33. This material is available
free of charge via the Internet at http://pubs.acs.org.
Cyclic AMP Accumulation Assay. Intracellular levels of 3′,5′-
cyclic AMP were measured by the competitive protein binding
method.32 CHO cells expressing recombinant human40 ARs were
harvested by trypsinization. After resuspension in the medium, cells
were plated in 24-well plates in 0.5 mL of medium/well. After 24
h the medium was removed and cells were washed three times with
1 mL/well of DMEM containing 50 mM N-2-hydroxyethylpipera-
zine-N′-2-ethanesulfonic acid, pH 7.4. Cells were then treated with
agonists and/or test compounds in the presence of rolipram (10
µM) and adenosine deaminase (3 units/mL) and incubated at 37
°C. For the A3AR, after 45 min forskolin (10 µM) 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 µL/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 µL of the cell lysate,
and 30 µL 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 scintillation spectrometry. Calcula-
tion of the relative maximal efficacy at the A3AR was determined
at a fixed concentration of the nucleoside analogue (10 µM) and
expressed as a relative percent of the effect of 10 µM NECA
determined in each experiment, which typically reached ∼50%
inhibition of the forskolin stimulated cyclase.
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