600
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);