Conversion of A3 adenosine receptor agonists into selective antagonists by modification of the 5′-ribofuran-uronamide moiety

Article abstract of DOI:10.1016/j.bmcl.2005.10.054

The highly selective agonists of the A₃ adenosine receptor (AR), Cl-IB-MECA (2-chloro-N⁶-(3-iodobenzyl)-5′-N- methylcarboxamidoadenosine), and its 4′-thio analogue, were successfully converted into selective antagonists simply by app

Full text of DOI:10.1016/j.bmcl.2005.10.054

Bioorganic & Medicinal Chemistry Letters 16 (2006) 596–601
Conversion of A3 adenosine receptor agonists into selective
antagonists by modification of the 50-ribofuran-uronamide moiety
Zhan-Guo Gao,a Bhalchandra V. Joshi,a Athena M. Klutz,a Soo-Kyung Kim,a
Hyuk Woo Lee,b Hea Ok Kim,b Lak Shin Jeongb and Kenneth A. Jacobsona,*
aMolecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
bLaboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
Received 30 September 2005; revised 14 October 2005; accepted 14 October 2005
Available online 10 November 2005
Abstract—The highly selective agonists of the A3 adenosine receptor (AR), Cl-IB-MECA (2-chloro-N6-(3-iodobenzyl)-50-N-meth-
ylcarboxamidoadenosine), and its 40-thio analogue, were successfully converted into selective antagonists simply by appending a
second N-methyl group on the 50-uronamide position. The 2-chloro-50-(N,N-dimethyl)uronamido analogues bound to, but did
not activate, the human A3AR, with Ki values of 29 nM (40-O) and 15 nM (40-S), showing >100-fold selectivity over A1, A2A
,
and A2BARs. Competitive antagonism was demonstrated by Schild analysis. The 2-(dimethylamino)-50-(N,N-dimethyl)uronamido
substitution also retained A3AR selectivity but lowered affinity.
Ó 2005 Elsevier Ltd. All rights reserved.
Antagonists of the A3 adenosine receptor (AR) are of
potential use for clinical targets, including the treatment
of glaucoma, allergic conditions, and inflammation.1
Potent and selective antagonists for the human A3AR
have recently been synthesized.2–6 These human A3AR
antagonists were found to be weak or ineffective at the
rat A3AR7,8 and were unsuitable for evaluation in small
animal models or for further development as drugs.
Thus, A3AR antagonists of which the affinity and selec-
tivity are independent of species are sought as drug can-
didates. In previous studies, it was found that
antagonists derived from adenosine analogues, in con-
trast to nonpurine heterocyclic antagonists, could be
species-independent, potent, and selective A3AR
antagonists.9
Among the four subtypes of receptors for adenosine 1,
the intrinsic efficacy of nucleosides acting at the A3 sub-
type is known to be especially sensitive to structural
changes. For example, substitution by a benzyl or a 3-
iodobenzyl group at the N6 position of adenosine was
demonstrated to increase affinity but decrease efficacy
at the human A3AR, in the inhibition of forskolin-stim-
ulated adenylyl cyclase.9–11 Additional substitution by a
2-Cl substituent further increased affinity and decreased
efficacy.9,10 As a result, N6-(3-iodobenzyl)adenosine 2
was a partial agonist achieving only 46% of the maximal
effect, while 2-chloro-N6-(3-iodobenzyl)adenosine 3 was
a potent antagonist for the A3AR, albeit nonselective. A
50-methyluronamide substitution of 3 restored its effica-
cy and rendered it selective for the A3AR. Thus, Cl-IB-
MECA 4 and its 40-thio analogue LJ568 5 are selective
agonists for the A3AR.9,12,13 Here, we report that 4
and 5 were successfully converted into selective antago-
nists by appending an additional methyl group on the
50-uronamide nitrogen (see Chart 1). The finding that
removing H-binding ability in the region of the 50-uro-
namide lowers efficacy is consistent with expectations
derived from rhodopsin-based, dynamic molecular mod-
eling and ligand docking.14
Abbreviations: AR, adenosine receptor; CGS21680, 2-[p-(2-carboxy-
ethyl)phenylethylamino]-50-N-ethylcarboxamido-adenosine;
CHO,
Chinese hamster ovary; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-50-
N-methylcarboxamidoadenosine; CPA, N6-cyclopentyladenosine;
DMEM, DulbeccoÕs modified EagleÕs medium; I-AB-MECA, N6-(4-
amino-3-iodobenzyl)-50-N-methylcarboxamidoadenosine; NECA, 50-
N-ethylcarboxamidoadenosine; PIA, N6-(phenylisopropyl)adenosine;
PTLC, preparative thin layer chromatography.
Keywords: Nucleoside; G protein-coupled receptor; Adenylyl cyclase;
Molecular modeling; Radioligand binding.
*
Synthetic routes to N,N-dimethylamide derivatives 6–8
are shown in Scheme 1. The 50-ester group was amino-
Corresponding author. Tel.: +301 496 9024; fax: +301 480 8422;
e-mail: kajacobs@helix.nih.gov
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.10.054

Z.-G. Gao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 596–601
597
NH2
NH
NH
NH
I
I
I
N
N
N
N
N
N
N
N
N
N
N
N
R
Cl
CH3NHCO
R
N
O
N
O
N
X
N
X
HOCH2
HOCH2
(CH3)2NCO
HO OH
HO OH
HO OH
HO OH
1 Adenosine, full agonist 2 R = H MRS541, partial agonist 4 X = O Cl-IB-MECA, full agonist
6 X = O, R = Cl, antagonist
7 X = S, R = Cl, antagonist
3 R = Cl MRS542, antagonist
5 X = S LJ-568, full agonist
8 X = O, R = N(CH3)2, antagonist
Chart 1.
HN
HN
N
N
O
N
N
I
MeO
Me
Me
O
N
N
O
I
R
N
O
OAc
N
N
Cl
O
MeO
ref. 25
O
AcO
OAc
HO
OH
a,b,c
c
AcO
OAc
9
25%
6 R = Cl
10
HN
N
8 R = N(CH ) 42%
3 2
HN
N
BzO
N
N
S
N
N
OAc
d,e
I
I
O
Me
Me
O
ref. 12
N
Cl
O
O
N
60%
Cl
HO
N
S
S
11
7
12
TBSO
OTBS
HO
OH
Scheme 1. Reagents and conditions: (a) K2CO3, MeOH, rt; (b) HOAc; (c) 40% aq. Me2NH, rt; (d) Me2NH, EDC, HOBT, DIPEA; (e) TBAF, THF.
lyzed with dimethylamine, which also led to a side prod-
uct substituted at the 2-position.15 The 40-S analogue
was prepared from an acetoxy intermediate,16 similar
to a reported series of derivatives.12
rat A3AR. The Ki values were 286 31 and
321 74 nM for 6 and 7, respectively.
In functional assays consisting of measuring inhibition
of forskolin-stimulated production of 30,50-cyclic-adeno-
sine monophosphate (cAMP) in intact CHO cells heter-
ologously expressing ARs,18 single concentration
determinations (Table 1) indicated that A3AR agonism
was absent in compounds 6–8. In contrast, the concen-
tration–response curve for compound 4 indicates full
agonism, as previously reported, with an EC50 of
1.2 nM at the human A3AR (Fig. 1A). A Schild analy-
sis19 indicated that 6 concentration-dependently antago-
nized the A3 agonist 4 to inhibit forskolin-stimulated
cAMP accumulation in CHO cells stably expressing
the human A3AR with a KB value of 48 nM (Figs. 1B
and C). In contrast, 6, at a concentration of 1 lM,
had no significant effect on cAMP accumulation inhibit-
ed by the A1 agonist CPA (N6-cyclopentyladenosine) in
CHO cells expressing the human A1AR under the simi-
lar conditions (data not shown).
Binding assays were carried out using standard radio-
ligands in Chinese hamster ovary (CHO) cells stably
expressing a human AR subtype.17 The binding affin-
ity at the human A3AR of the 2-Cl antagonist deriv-
atives 6 and 7 was shown to be 29 and 15 nM,
respectively (Table 1). Thus, the compounds were
demonstrated to be over 100-fold selective in binding
to the human A3AR in comparison to other AR sub-
types. In addition, the 2-(dimethylamino) substitution
in 8 resulted in A3AR selectivity but with lower
affinity.
To probe species differences, the affinity of 6 and 7 was
also measured at the rat A3AR. Although the affinity de-
creased with respect to the affinity at the human A3AR,
these two compounds showed moderate affinity at the

598
Z.-G. Gao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 596–601
Table 1. Potency of a series of adenosine derivatives for four subtypes of human ARs and the efficacy at the A3AR
Compound
Potency (Ki or EC50, nM)
Efficacyc
A3 (%)
100
d
d
e
d
A1
A2A
A2B
A3
1a
2b
3b
4b
5f
6g
7g
8
310e
7.4 1.7
700e
24,000
ꢀ10,000
>10,000
>110,000
ND
290e
132 22
197 34
5.8 0.4
1.8 0.1
1.4 0.3
0.38 0.07
29.0 4.9
15.5 3.1
315 19
46
0
8
16.8 2.2
222 22
193 46
5360 2470
223 36
100
114
0
9
5870 930
6220 640
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
0
>10,000
0
All experiments were done on CHO cells stably expressing one of four subtypes of human ARs. The binding affinity for A1, A2A, and A3ARs was
expressed as Ki values and was determined by using agonist radioligands ([3H]R-PIA), ([3H]CGS21680), and [125I]I-AB-MECA, respectively. The
potency at the A2BAR was expressed as EC50 values and was determined by stimulation of cAMP production in AR-transfected CHO cells. The
efficacy at A3ARs was determined by inhibition of forskolin-stimulated cAMP production in AR-transfected CHO cells, as described in the text.
Data are expressed as means standard error.
ND, not determined.
a Values from Ref. 24.
b Values from Refs. 9,21.
c At a concentration of 10 lM, in comparison to the maximal effect of a full agonist NECA at 10 lM.
d Ki in binding, unless noted.
e cAMP assay.
f Ki values from Ref. 13.
g 6, MRS3771; 7, LJ-1256.
A
B
100
50
0
Control
100
75
50
25
0
+ 6 0.1 µM
+ 6 1.0
+ 6 10 µM
µM
4
7
6
-12 -11 -10 -9
-8
-7
-6
-5 -4
-11 -10
-9
-8
-7
-6
-5
-4
log [Agonist], M
log [4], M
D
C
2500
2
1
0
2000
1500
1000
500
0
KB=48 nM
NECA
7
6
-11
-10
-9
-8
-7
-6
-5
-8
-7
-6
-5
-4
log [agonist], M
log [6], M
Figure 1. (A) Inhibition by nucleoside derivatives of forskolin-stimulated cAMP accumulation in CHO cells stably expressing the human A3AR.
Cl-IB-MECA 4 concentration-dependently inhibited with an EC50 of 1.2 0.3 nM (n = 3), while 6 and 7 alone had no effect. (B) Right-shift of the
concentration–response curves and (C) Schild analysis for the concentration-dependent inhibition by 6 of the effect of Cl-IB-MECA 4 on
forskolin-stimulated cAMP accumulation in CHO cells stably expressing the human A3AR. (D) A3AR-induced changes in intracellular [Ca2+] in
A3AR-expressing CHO cells (proportional to relative fluorescence units, RFU) upon activation by nucleosides. In control CHO cells, there was no
response to NECA.
Similar results were obtained in an assay of A3AR-in-
duced changes in intracellular [Ca2+].20 The nonselective
AR agonist NECA (50-N-ethyl-carboxamidoadenosine)
activated the human A3AR expressed in CHO cells to
induce a concentration-dependent rise in intracellular
[Ca2+] with a potency corresponding to an EC50 of
58 16 nM (n = 4), while 6 and 7 alone did not induce
calcium transients (Fig. 1D). Compound 8 also did not
induce a Ca2+ response (data not shown). Increasing
concentrations of 6 right-shifted the activation curves
for NECA (not shown), and a KB value for 6 of
20 nM was determined. The A3AR antagonist 7 similar-

Z.-G. Gao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 596–601
599
ly shifted the NECA-induced Ca2+ response to the right
(not shown).
verted to an antagonist by cyclization of the 50-
uronamide moiety into a spirolactam, resulting in a
selective A3AR antagonist of moderate affinity for both
human and rat A3ARs.9 Another advantage of nucleo-
side A3AR antagonists over other heterocycles7 is in-
creased aqueous compatibility. For example, the
C log P values of 6 and 7 are 1.69 and 1.73, respectively,
in comparison to 6.86 for MRS1191, a dihydropyridine
antagonist of the A3AR.7 Here, two additional selective
antagonists with reasonable affinity for both human and
rat A3ARs were introduced, which increased the diversi-
ty of rat A3AR antagonists.
In this study, two selective A3AR agonists, Cl-IB-
MECA and its 40-thio analogue, have been successfully
transformed into antagonists selective for the A3AR
by appending an additional N-methyl group on the 50-
uronamide position. Other related N,N-dialkyl substitu-
tion in the 40-thio nucleoside series of adenosine agonists
had more complex changes in affinity and efficacy.12
Thus, it appears that the 50-(N,N-dimethyl)uronamido
group especially tends to preserve affinity and selectivity
in N6-3-iodobenzyladenosine derivatives, while entirely
abolishing activation of the human A3AR. There was
also an interdependence of the effects of an N,N-dimeth-
yl moiety and the N6-substituent, since the N6-methyl
It is clear that more potent and selective antagonists
are needed for eventual therapeutic purposes. The new-
ly synthesized A3AR antagonists could be evaluated in
models of a number of disorders related to the A3AR,
such as glaucoma and inflammation. Currently, exist-
ing nucleoside analogues should be good templates
for further modification and development of potent
and selective antagonists for the A3ARs in diverse
species.
analogue corresponding to 7 was a partial agonist.12
A
further advantage of the N6-benzyl substitution of 6
and 7 over many other smaller or larger groups is that
the high A3AR affinity tends to be retained in murine
species,21 which was confirmed in the present study.
Molecular modeling of the A3AR indicated that flexibil-
ity of the adenosine derivative in the 50-region correlated
with putative conformational changes of the receptor
associated with activation. Although there is no global
conformational model of the activated state of the
receptor, local conformational changes have been pro-
posed. One such change is the rotation, anticlockwise
from the extracellular perspective of the receptor, of
the conserved W243 in TM6(6.48).9,21 We have pro-
posed that both flexibility of the 50-uronamide and its
ability to make and break multiple H-bonds as this
conformational change occurs are needed for receptor
activation. The low efficacy of 50-thioether derivatives
is also consistent with the need for H-bonding in this
region in order to activate the A3AR.22
Acknowledgments
B.V. Joshi thanks Gilead Sciences (Foster City, CA) for
financial support. This research was supported in part
by the Intramural Research Program of the NIH,
National Institute of Diabetes and Digestive and Kidney
Diseases, and by the Korea Health R&D Project, Min-
istry of Health & Welfare, Korea (03-PJ2-PG4-BD02-
0001). We thank Prof. Mortimer Civan (University of
Pennsylvania) for helpful discussion.
References and notes
The present findings are consistent, in that we have re-
moved the H-bond-donating ability of the 50-uronamide
with a relatively subtle structural alteration, resulting in
the loss of ability to activate the A3AR. From a compar-
ison of the A2A and A3AR models,23 the main difference
in the docking complex was the preference of the v an-
gle; the A3AR preferred nucleosides bound in an anti-
form (185°), while the A2AAR preferred a high-anti con-
formation (approximately ꢁ70°) about the glycosidic
bond. Another difference was the binding at the 50-posi-
tion. In the A2AAR, the 50-NH formed a H-bond with
an important T88 residue in TM3(3.36), but the model
of the A3AR complex featured a stronger interaction
proposed between the 50-carbonyl group and S271 in
TM7(7.42). Accordingly, the 50-dimethylamides dis-
played a dramatically diminished binding affinity at
the A2AAR.
1. Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz,
K. N.; Linden, J. Pharmacol. Rev. 2001, 53, 527.
2. Kim, Y. C.; Ji, X. D.; Jacobson, K. A. J. Med. Chem.
1996, 39, 4142.
3. van Muijlwijk-Koezen, J. E.; Timmerman, H.; Link, R.;
van der Goot, H.; IJzerman, A. P. J. Med. Chem. 1998, 41,
3994.
4. Baraldi, P. G.; Cacciari, B.; Moro, S.; Spalluto, G.;
Pastorin, G.; Da Ros, T.; Klotz, K.-N.; Varani, K.; Gessi,
S.; Borea, P. A. J. Med. Chem. 2002, 45, 770.
5. Maconi, A.; Pastorin, G.; Da Ros, T.; Spalluto, G.; Gao,
Z. G.; Jacobson, K. A.; Baraldi, P. G.; Cacciari, B.;
Varani, K.; Moro, S.; Borea, P. A. J. Med. Chem. 2002,
45, 3579.
6. Okamura, T.; Kurogi, Y.; Hashimoto, K.; Sato, S.;
Nishikawa, H.; Kiryu, K.; Nagao, Y. Bioorg. Med. Chem.
Lett. 2004, 14, 3775.
7. Moro, S.; Spalluto, G.; Gao, Z. G.; Jacobson, K. A. Med.
Res. Rev. (in press).
8. Yang, H.; Avila, M. Y.; Peterson-Yantorno, K.; Coca-
Prados, M.; Stone, R. A.; Jacobson, K. A.; Civan, M. M.
Current Eye Res. 2005, 30, 747–754.
9. Gao, Z. G.; Kim, S. K.; Biadatti, T.; Chen, W.; Lee, K.;
Barak, D.; Kim, S. G.; Johnson, C. R.; Jacobson, K. A.
J. Med. Chem. 2002, 45, 4471.
A3AR antagonists are potentially useful therapeutically
for a number of disorders.1,8,24 However, the A3 antag-
onists have not been widely used in animal models due
to their extremely weak potency in murine species.8
Thus, A3AR antagonists independent of species are of
high priority to be developed. For this reason, a nucleo-
side analogue IB-MECA (2-H analogue of 4) was con-
10. Gao, Z. G.; Blaustein, J. B.; Gross, A. S.; Melman, N.;
Jacobson, K. A. Biochem. Pharmacol. 2003, 65, 1675.

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Z.-G. Gao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 596–601
11. Gao, Z. G.; Jeong, L. S.; Moon, H. R.; Kim, H. O.; Choi,
W. J.; Shin, D. H.; Elhalem, E.; Comin, M. J.; Melman,
N.; Mamedova, L.; Gross, A. S.; Rodriguez, J. B.;
Jacobson, K. A. Biochem. Pharmacol. 2004, 67, 893.
12. Jeong, L. S.; Lee, H. W.; Jacobson, K. A.; Kim, H. O.;
Shin, D. H.; Lee, J. A.; Gao, Z. G.; Lu, C.; Duong, H. T.;
Gunaga, P.; Lee, S. K.; Jin, D. Z.; Chun, M. W.; Moon,
H. R. J. Med. Chem. (in press).
13. Jeong, L. S.; Jin, D. Z.; Kim, H. O.; Shin, D. H.; Moon,
H. R.; Gunaga, P.; Chun, M. W.; Kim, Y.-C.; Melman,
N.; Gao, Z.-G.; Jacobson, K. A. J. Med. Chem. 2003, 46,
3775.
UV (MeOH) kmax 274 nm (pH 7); 1H NMR (DMSO-d6) d
2.95 (s, 3 H, N-CH3), 3.04 (s, 3H, N-CH3), 4.30 (d, 1 H,
J = 4.6 Hz, 2-H), 4.52 (br dd, 1 H, J = 4.6, 8.4 Hz, 3-H),
4.58 (m, 1 H, 4-H), 4.65 (d, 2 H, J = 5.7 Hz, N-CH2), 5.59
(d, 1 H, J = 5.5 Hz, exchangeable with D2O, OH), 5.86 (d,
1 H, J = 5.1 Hz, exchangeable with D2O, OH), 5.91 (d, 1
H, J = 5.4 Hz, 5-H), 7.17 (t, 1 H, J = 7.8 Hz, 50-H), 7.40
(d, 1 H, J = 7.6 Hz, 60-H), 7.65 (d, 1 H, J = 7.8 Hz, 40-H),
7.78 (s, 1 H, 20-H), 8.52 (s, 1 H, H-8), 9.00 (br t, 1 H,
J = 6.1 Hz, exchangeable with D2O, NH); 13C NMR
(CD3OD) d 36.6, 38.2, 44.5, 64.6, 77.2, 80.7, 95.0, 119.7,
128.3, 131.5, 137.5, 138.0, 141.5, 142.8, 145.2, 151.7, 155.8,
156.5, 172.7; FAB-MS m/z 575 (M++1); Anal. (C19H20ClI-
N6O3S) C, H, N, S.
14. Jacobson, K. A.; Kim, S. K.; Costanzi, S.; Gao, Z. G.
Mol. Interventions 2004, 4, 337.
15. (2S,3S,4R,5R)-5-[2-Chloro-6-(3-iodo-benzylamino)-purin-
17. The CHO cells stably expressing recombinant ARs were
cultured in DMEM and F12 (1:1) supplemented with 10%
fetal bovine serum, 100 U/ml penicillin, 100 lg/mL strep-
tomycin, 2 lmol/ml glutamine, and 800 lg/ml geneticin.
After harvest and homogenization, cells were centrifuged
at 500g for 10 min, and the pellet was re-suspended in
50 mM Tris–HCl buffer (pH 7.4) containing 10 mM
MgCl2, 1 mM EDTA. The suspension was homogenized
with an electric homogenizer for 10 s and was then re-
centrifuged at 20,000g for 20 min at 4 °C. The resultant
pellets were resuspended in buffer in the presence of 3 U/
ml adenosine deaminase, and the suspension was stored at
ꢁ80 °C until the binding experiments. The protein con-
centration was measured as described [Bradford, M. M.
Anal. Biochem.; 1976, 72, 248]. For A3AR binding assays,
each tube contained 100 ll of membrane suspension, 50 ll
9-yl]-3,4-dihydroxy-tetrahydrofuran-2-carboxylic
acid
dimethylamide (6). The methyl ester 10 (0.031 g,
0.05 mmol) was dissolved in MeOH (5 mL), potassium
carbonate (0.014 g, 0.1 mmol) was added, and the mixture
was stirred at room temperature for 10 min. Acetic acid
(0.2 mL) was added to neutralize the base, and the
resulting diol was treated in situ with aqueous dimethyl-
amine (0.5 mL, 40%) and further stirred for 1 h. The
reaction mixture was concentrated under reduced pressure
and subjected to preparative thin layer chromatography
by using chloroform/methanol (9:1) as solvent to afford
the dimethylamide 6 as a colorless solid (0.0072 g, 26%).
1H NMR (CDCl3) d 2.97 (s, 3 H, N-CH3), 3.14 (s, 3 H,
N-CH3), 3.52–3.80 (m, 4 H), 4.45–4.85 (m, 3 H), 5.05 (d, 1
H, J = 5.5 Hz), 6.06 (d, 1 H, J = 5.1 Hz), 7.17 (t, 1 H,
J = 7.8 Hz, 50-H), 7.65–7.82 (m, 3 H), 8.21 (s, 1 H);
TOFMS m/z 559.0353 (M+H+) (calculated for C19H21
N6O4ClI+) 559.0358. (2S,3S,4R,5R)-5-[2-dimethylamino-
6-(3-iodo-benzylamino)-purin-9-yl]-3,4-dihydroxy-tetrahy-
drofuran-2-carboxylic acid dimethylamide (8). Aqueous
dimethylamine (0.5 mL, 40%) was added to methyl ester
10 (0.016 g, 0.025 mmol) and, the resulting reaction
mixture was stirred at room temperature for 6 h. The
mixture was concentrated under reduced pressure and
purified by preparative thin layer chromatography by
using chloroform/methanol (9:1) as solvent to afford the
[
125I]I-AB-MECA (final concentration 0.5 nM), and 50 ll
of increasing concentrations of compounds in Tris–HCl
buffer (50 mM, pH 7.4) containing 10 mM MgCl2. Non-
specific 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 (Brandell, Gaithersburg, MD). Filters were
washed three times with ice-cold buffer. Radioactivity
was determined in a Beckman 5500B c-counter. The
binding of [3H]R-PIA to A1ARs and the binding of
[3H]CGS21680 to A2AARs were as previously described.10
IC50 values were converted to Ki values as described
[Cheng Y.-C., Prusoff W. H. Biochem. Pharmacol.; 1973,
22, 3099]. [125I]N6-(4-amino-3-iodo-benzyl)adenosine-50-
N-methyluronamide ([125I]I-AB-MECA; 2000 Ci/mmol),
[3H]R-PIA (R-N6-[phenylisopropyl]adenosine, 34 Ci/
mmol), [3H]CGS21680 (2-[p-(2-carboxyethyl)phenyleth-
ylamino]-50-N-ethylcarboxamido-adenosine, 47 Ci/mmol),
and [3H]cAMP (40 Ci/mmol) were from Amersham
Pharmacia Biotech (Buckinghamshire, UK). NECA,
CGS21680, CPA, and R-PIA were purchased from
Sigma-RBI (St. Louis, MO). Other chemicals were from
standard commercial sources and of analytical grade.
18. Intracellular cAMP levels were measured with a compet-
itive protein binding method [Nordstedt, C.; Fredholm, B.
B. Anal. Biochem.; 1990, 189, 231]. CHO cells expressing
one of four subtypes of recombinant ARs were harvested
by trypsinization. After resuspension in medium, cells
were planted in 24-well plates in 0.5 ml medium. After
24 h, the medium was removed, and cells were washed
three times with 0.5 ml DMEM, containing 50 mM Hepes,
pH 7.4. Cells were then treated with agonists and/or test
compounds in the presence of rolipram (10 lM) and
adenosine deaminase (3 U/mL). After 45 min, forskolin
(10 lM) was added to the medium, and incubation was
continued for an additional 15 min. The reaction was
terminated by removing the medium, and cells were lysed
upon the addition of 200 lL of 0.1 M ice-cold HCl. The
1
dimethylamide 8 as a colorless solid (0.0058 g, 42%). H
NMR (CDCl3) d 2.90–3.23 (m, 12 H), 3.79 (bs, 3 H), 4.42–
4.95 (m, 4 H), 6.11 (d, 1 H, J = 5.1 Hz), 6.22 (bs, 1 H) 7.14
(t, 1 H, J = 7.5 Hz, 50-H), 7.40 (d, 1 H, J = 7.6 Hz, 60-H),
7.65 (d, 1 H, J = 7.8 Hz, 40-H), 7.78 (s, 1 H, 20-H), 8.22
(s, 1 H, H-8); TOFMS m/z 568.1162 (M+H+) (calculated
for C21H27N7O4I+) 568.1169.
16. (2S,3S,4R,5R)-5-[2-Chloro-6-(3-iodo-benzylamino)-purin-
9-yl]-3,4-dihydroxy-tetrahydro-thiophene-2-carboxylic acid
dimethylamide (7). To
a solution of 12 (483.0 mg,
0.622 mmol), N-(3-dimethylaminopropyl)-N0-ethylcarbo-
diimide hydrochloride (EDC, 179 mg, 0.933 mmol), 1-
hydroxybenzotriazole (HOBt, 126 mg, 0.933 mmol), and
dimethylamine–HCl (76 mg, 0.933 mmol) in CH2Cl2
(20 mL) was added N,N-diisopropylethylamine (DIPEA,
0.325 mL, 1.87 mmol), and the mixture was stirred at
room temperature for 12 h. The reaction mixture was
evaporated, and the residue was purified by a silica gel
column chromatography (hexane/EtOAc = 10:1–5:1) to
give the silyl-protected amide intermediate as a white
foam. To a stirred solution of the silyl amide (414 mg,
0.516 mmol) in THF (10 mL) was added tetrabutylammo-
nium fluoride (1.29 mL, 1.29 mmol, 1 M THF solution)
and the reaction mixture was stirred at room temperature
for 1 h. The solvent was evaporated and the resulting
residue was purified by silica gel column chromatography
(CH2Cl2/MeOH = 10:1) to give 7 (214 mg, 64%): white
solid; mp 186.1–186.3 °C; [a]D20 ꢁ12.4 (c 0.10, MeOH);

Z.-G. Gao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 596–601
601
cell lysate was resuspended and stored at ꢁ20 °C. For
determination of cAMP production, protein kinase A was
incubated with [3H]cAMP (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 or 50 lL cAMP
solution (0–16 pmol/200 lL for standard curve). Bound
radioactivity was separated by rapid filtration through
Whatman GF/C filters and washed once with cold buffer.
Bound radioactivity was measured by liquid scintillation
spectrometry.
using dilutions of various compounds in HanksÕ buffer.
For antagonist studies, both agonist and antagonist were
added to the sample plate. Samples were run in duplicate
using a Molecular Devices Flexstation I at room temper-
ature. Cell fluorescence (Excitation = 485 nm; Emis-
sion = 525 nm) was monitored following exposure to
compound. Increases in intracellular calcium are reported
as the maximum fluorescence value after exposure minus
the basal fluorescence value before exposure.
21. Tchilibon, S.; Joshi, B. V.; Kim, S. K.; Duong, H. T.;
Gao, Z. G.; Jacobson, K. A. J. Med. Chem. 2005, 48,
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20. CHO cells stably expressing human A3ARs were grown
overnight in 100 ll media in 96-well flat-bottomed plates
at 37 °C at 5% CO2 to reach approx. 90% confluency. The
calcium assay kit (Molecular Devices) was used as directed
with no washing of cells and with probenecid added to the
loading dye at a final concentration of 2.5 mM to increase
dye retention. Cells were loaded with 50 ll dye with
probenecid to each well and incubated for 60 minutes at
room temperature. The compound plate was prepared
22. van Tilburg, E. W.; von Frijtag Drabbe Kunzel, J.; de
¨
Groote, M.; IJzerman, A. P. J. Med. Chem. 2002, 45, 420.
23. Kim, S. K.; Gao, Z.-G.; Van Rompaey, P.; Gross, A. S.;
Chen, A.; Van Calenbergh, S.; Jacobson, K. A. J. Med.
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24. Fredholm, B. B.; Irenius, E.; Kull, B.; Schulte, G.
Biochem. Pharmacol. 2001, 61, 443.
25. Kim, H. O.; Hawes, C.; Towers, P.; Jacobson, K. A.
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