Structure-activity relationships of truncated adenosine derivatives as highly potent and selective human A3 adenosine receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2009.03.034

On the basis of potent and selective binding affinity of truncated 4′-thioadenosine derivatives at the human A₃ adenosine receptor (AR), their bioisosteric 4′-oxo derivatives were designed and synthesized from commercially available 2,3-O-isopr

Full text of DOI:10.1016/j.bmc.2009.03.034

Bioorganic & Medicinal Chemistry 17 (2009) 3733–3738
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of truncated adenosine derivatives
as highly potent and selective human A3 adenosine receptor antagonists
Shantanu Pal a, Won Jun Choi a, Seung Ah Choe a, Cara L. Heller b, Zhan-Guo Gao b, Moshe Chinn b,
Kenneth A. Jacobson b, Xiyan Hou a, Sang Kook Lee a, Hea Ok Kim a, Lak Shin Jeong a,
*
a Department of Bioinspired Science and Division of Life and Pharmaceutical Sciences, Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University,
Seoul 120-750, Republic of Korea
b Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
On the basis of potent and selective binding affinity of truncated 40-thioadenosine derivatives at the
human A3 adenosine receptor (AR), their bioisosteric 40-oxo derivatives were designed and synthesized
from commercially available 2,3-O-isopropylidene-D-erythrono lactone. The derivatives tested in AR
Article history:
Received 14 February 2009
Revised 19 March 2009
Accepted 20 March 2009
Available online 25 March 2009
binding assays were substituted at the C2 and N6 positions. All synthesized nucleosides exhibited potent
and selective binding affinity at the human A3 AR. They were less potent than the corresponding 40-thio
analogues, but showed still selective to other subtypes. The 2-Cl series generally were better than the 2-H
series in view of binding affinity and selectivity. Among compounds tested, compound 5d (X = Cl, R = 3-
bromobenzyl) showed the highest binding affinity (Ki = 13.0 6.9 nM) at the hA3 AR with high selectivity
(at least 88-fold) in comparison to other AR subtypes. Like the corresponding truncated 40-thio series,
compound 5d antagonized the action of an agonist to inhibit forskolin-stimulated adenylate cyclase in
hA3 AR-expressing CHO cells. Although the 40-oxo series were less potent than the 40-thio series, this class
of human A3 AR antagonists is also regarded as another good template for the design of A3 AR antagonists
and for further drug development.
Keywords:
A3 Adenosine receptor
Antagonists
Truncated adenosine
Structure–activity relationships
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
agent. On the basis of a bioisosteric rationale, we designed and syn-
thesized the 40-thio analogue 212 of Cl-IB-MECA, which exhibited
Adenosine, an endogenous chemical messenger binds to four
subtypes (A1, A2A, A2B, and A3) of adenosine receptors (ARs) and
regulates many physiological functions in the cell.1 A3 AR activa-
tion stimulates phospholipase C (PLC), increasing the levels of ino-
sitol triphosphate (IP3) and diacylglycerol (DAG) and inhibits
adenylate cyclase, decreasing the level of cAMP. Since IP3, DAG,
and cAMP serve as second messengers important for cell signaling,
A3 AR has been promising targets for the treatment of several dis-
eases associated with cell signaling.2 Thus, A3 AR agonists have the
therapeutic potentials against cancer,3 cerebral ischemia,4 and
myocardiac ischemia,5 while A3 AR antagonists can be developed
as anti-inflammatory,6 anti-asthma,7 and anti-glaucoma agents.8
A number of purine and non-purine compounds have been
developed as potent and selective A3 AR ligands.9,10 Most of aden-
osine derivatives were found to be A3 AR agonists, among which
compound 1 (Cl-IB-MECA)11 showed high binding affinity (Ki for
hA3 = 1.4 nM) at the human (h) A3 AR with high selectivities to
other subtypes (Chart 1) and is under clinical trials as an anticancer
better binding affinity (Ki for hA3 = 0.38 nM) than compound 1.
On the other hand, nonpurine heterocyclic compounds13 have
been reported to be better templates than purine derivatives in
developing potent and selective hA3 AR antagonists. However,
these A3 AR antagonists were weak or ineffective in binding to
the rat A3 AR, indicating that they were not suitable for efficacy
evaluation in small animal models and thus as drug candidates.14
Thus, searching for potent and selective species-independent A3
AR antagonists has been an immediate goal. Because a few nucle-
oside derivatives have been reported to be species-independent A3
AR antagonists14, we began to search for novel potent and selec-
tive A3 AR antagonists, derived from nucleoside templates.
As a result, the truncated 40-thio analogue 315 removing a
hydrogen bonding donor ability of the NH in the 50-uronamide re-
quired for the activation of the A3 AR in compound 2 was found to
be one of the best A3 AR antagonists which bound selectively to the
A3 AR in a species-independent manner (Ki = 4.16 nM at the hA3 AR
and Ki = 3.9 nM at the rat A3 AR) (Chart 1). Thus, it is of interest to
design bioisosteric analogues of compound 3, such as the truncated
40-oxo derivatives 4 and 5, and to compare their binding affinities
at the hA3 AR. In this article, we report the synthesis of truncated
* Corresponding author. Tel.: +82 2 3277 3466; fax: +82 2 3277 2851.
E-mail address: lakjeong@ewha.ac.kr (L.S. Jeong).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.034

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S. Pal et al. / Bioorg. Med. Chem. 17 (2009) 3733–3738
HN
N
HN
I
I
N
N
N
N
N
MeHN
O
N
MeHN
O
Cl
N
Cl
S
O
OH OH
2 (Full A3 AR agonist)
OH OH
1 (Full A3 AR agonist)
Ki (nM)
hAR
Ki (nM)
hAR
A1 : 193
A2A : 223
A3 : 0.38
A1 : 220
A2A : 5,400
A3 : 1.4
NHR
HN
N
I
N
N
N
N
N
N
X
N
Cl
O
S
OH OH
OH OH
3 (Full A3 AR antagonist)
Ki (nM)
4 (X = H, R = Me or 3-halobenzyl)
5 (X = Cl, R = Me or 3-halobenzyl)
hAR
A1 : 2,490
A2A : 341
A3 : 4.16
Chart 1. Binding affinity (A1 AR, A2A AR, A3 AR) at human adenosine receptors.
adenosine derivatives 4 and 5 as potent and selective hA3 AR
antagonists.
meric assignment of 9 was easily achieved by 1H NOE experiments.
Strong NOE effect between H-8 and 20-H in compound 9 was
shown, indicating that it is b-anomer. Removals of the isopropyli-
dene groups in 9 and 10 under acidic conditions afforded 11 and
12, respectively.
Treatment of 11 with methylamine and 3-halobenzyl amines
yielded the N6-methylamino derivative 4a and the N6-(3-haloben-
zyl)amino derivatives 4b–e. Similarly, compound 12 was con-
verted to the 2-chloro-N6-methylamino derivative 5a and the 2-
chloro-N6-(3-halobenzyl)amino derivatives 5b–e.
2. Results and discussion
2.1. Chemistry
Our synthetic strategy to the final nucleosides was first to
synthesize the glycosyl donor and then condense with purine
bases. Synthesis of the glycosyl donor 716 from commercially avail-
able 2,3-O-isopropylidene-D-erythronolactone (6) is shown in
2.2. Biological activity
Scheme 1.
Reduction of 6 with DIBAL in toluene gave the lactol 716, which
was treated with acetic anhydride in pyridine to give the glycosyl
donor 8.
The glycosyl donor 8 was then condensed with silyalted 6-chlo-
ropurine and 2,6-dichloropurine to give 6-chloro derivative 9 and
2,6-dichloropurine derivative 10, respectively (Scheme 2).
During condensation reaction, the initially formed N-3 isomer
was smoothly converted to the N-9 isomer on refluxing. The ano-
Binding assays were carried out using adherent mammalian
cells stably transfected with cDNA encoding the appropriate
hARs (A1 and A3 in CHO cells and A2A in HEK-293 cells) and
standard radioligands (3.5 nM [3H]NECA for A1 AR, 10 nM
[3H]CGS21680 for A2A AR, and 0.5 nM [125I]I-AB-MECA for A3
AR).17,18 Table 1 shows that all synthesized compounds exhib-
ited potent and selective binding affinity at the hA3 AR,
although they were less potent than the corresponding 40-thio
analogues. In general, the 2-Cl series showed higher binding
affinity to the hA3 AR and better selectivity to other subtypes
(A1 and A2A) than the 2-H series. The similar trend was ob-
served in the corresponding 40-thio analogues. Within the 2-Cl
series, compound 5d (R = 3-bromobenzyl) showed the highest
binding affinity (Ki = 13 nM) at the hA3 AR with high selectivity
to other subtypes, A1 AR and A2A AR. The magnitude of the
binding affinity in the 3-halobenzyl series is decreased in the
following order: Br-Bn > I-Bn > Cl-Bn > F-Bn, indicating that the
b
O
O
O
a
OH
OAc
O
O
O
O
O
O
O
8
6
7
Scheme 1. Reagents and conditions: (a) DIBAL, toluene, ꢀ78 °C, 30 min; (b) Ac2O,
pyridine, rt, 3 h.

S. Pal et al. / Bioorg. Med. Chem. 17 (2009) 3733–3738
3735
Cl
N
3. Conclusions
N
N
N
On the basis of a bioisosteric rationale, novel truncated adeno-
sine derivatives were designed and synthesized as potent and
selective hA3 AR antagonists. All the synthesized nucleosides
showed high binding affinity at the hA3 AR with high selectivity
to the hA1 and hA2A ARs. Among compounds tested, compound
5d (X = Cl, R = 3-bromobenzyl) showed the highest binding affinity
(Ki = 13.0 6.9 nM) at the hA3 AR with high selectivity (at least 88-
fold) in comparison to other AR subtypes. Like the corresponding
truncated 40-thio analogue series, compound 5d was shown to be
an antagonist of the hA3 AR in an assay of adenylate cyclase.
Although the truncated 40-oxo series showed lower binding affinity
at the hA3 AR than the corresponding 40-thio series, this class of po-
tent hA3 AR antagonists is also regarded as another good template
for the design of A3 AR antagonists and for further drug
development.
a
O
OAc
X
O
O
O
O
O
8
9 (X = H)
10 (X = Cl)
b
Cl
NHR
N
N
N
N
N
N
X
N
H
c
N
O
O
X = H
4. Experimental
OH OH
OH OH
4a (R = Me)
4b (R = 3-F-Bn)
4c (R = 3-Cl-Bn)
4d (R = 3-Br-Bn)
4e (R = 3-I-Bn)
4.1. General methods
11 (X = H)
12 (X = Cl)
Melting points are uncorrected. 1H NMR (400 MHz) and 13C
NMR (100 MHz) spectra were measured in CDCl3, CD3OD or
DMSO-d6, and chemical shifts are reported in parts per million
(d) downfield from tetramethylsilane as internal standard. Column
chromatography was performed using Silica Gel 60 (230–400
mesh). Anhydrous solvents were purified by the standard
procedures.
c
X = Cl
NHR
N
N
N
Cl
N
O
4.2. Synthesis
OH OH
4.2.1. (3aR,4R,6aR)-2,2-Dimethyltetrahydrofuro[3,4-d][1,3]dioxol-
4-ol (7)
5a (R = Me)
5b (R = 3-F-Bn)
5c (R = 3-Cl-Bn)
5d (R = 3-Br-Bn)
5e (R = 3-I-Bn)
To a stirred suspension of 6 (8.0 g, 50.58 mmol) in toluene
(150 mL) was added DIBAL (61.1 mL, 61.1 mmol, 1 M solution in
toluene) at ꢀ78 °C and the reaction mixture was stirred at the
same temperature for 30 min and then quenched by the slow addi-
tion of methanol. The colloidal suspension was filtered on a Celite
pad and washed with EtOAc. The combined organic layers were
dried over MgSO4, filtered, and evaporated. The residue was puri-
fied by flash silica gel column chromatography (hexane/
EtOAc = 3:1) to give 7 (7.20 g, 89%) as a syrup, whose spectral data
were identical with those of authentic sample.16
Scheme 2. Reagents and conditions: (a) silylated6-chloropurine or 2,6-dichloro-
purine, TMSOTf, 1,2-dichloroethane, rt to 80 °C; (b) 2 N HCl, THF, rt, overnight; (c)
RNH2, ET3N, EtOH, rt, 1–3 days.
proper size of halogen is essential for optimal hydrophobic
interaction at the hA3 AR. The same trend was observed in
the 2-H series.
4.2.2. (3aR,4R,6aR)-2,2-Dimethyltetrahydrofuro[3,4-d][1,3]dioxol-
4-yl-acetate (8)
To a stirred solution of 716 (6.2 g, 38.7 mmol) in pyridine
(30 mL) was added acetic anhydride (4.39 mL, 46.44 mmol) at
0 °C and the reaction mixture was stirred for 3 h at rt and evapo-
rated. The residue was dissolved in EtOAc and washed with water.
The organic layer was dried over MgSO4 and evaporated. The resi-
due was purified by flash silica gel column chromatography (hex-
ane/EtOAc = 8:1) to give 8 (6.4 g, 82%) as a syrup: 1H NMR (CDCl3) d
6.16 (s, 1H), 4.86 (dd, 1H, J = 3.6, 6.0 Hz), 4.66 (d, 1H, J = 6.0 Hz),
4.12 (d, 1H, J = 6.4 Hz), 3.99 (dd, 1H, J = 3.6, 10.8 Hz), 2.05 (s, 3H),
1.48 (s, 3H), 1.33 (s, 3H).
One representative truncated 40-oxo analogue 5d was chosen
for testing in a functional assay consisting of 10 lM forskolin-stim-
ulated cyclic AMP production in CHO cells stably expressing the
hA3 AR. Compound 5d was shown to be an antagonist of the hA3
AR in this assay and did not activate the hA3 AR. Figure 1 shows
that the concentration–response curve for inhibition of adenylate
cyclase by the full agonist Cl-IB-MECA 1 was right shifted by
300 nM 5d. The EC50 value of 1 shifted from 1.14 to 11.9 nM in
the presence of the antagonist.
The binding affinity of compound 5d at the rat A3 AR was
also measured to determine if it shows species-independent
binding affinity at the A3 AR. This compound still showed high
and species-independent binding affinity (Ki = 82.8 22.9 nM) at
the rat A3 AR, although it was six times less potent than at
the human A3 AR. This result indicates that truncated 40-oxo
analogues are also suitable for evaluation in small animal
models or for further drug development as truncated 40-thio
analogues.
4.2.3. 6-Chloro-9-((3aR,4R,6aS)-2,2-dimethyltetrahydrothieno[3,4-
d][1,3]dioxol-4-yl)-9H-purine (9)
6-Chloropurine (2.29 g, 14.84 mmol), ammonium sulfate
(98 mg, 0.742 mmol), and HMDS (25 mL) were refluxed overnight
under inert and dry condition. The solution was evaporated under
high vacuum. The resulting solid was re-dissolved in 1,2-dichloro-
ethane (30 mL) and cooled in ice. To this mixture, a solution of 8

3736
S. Pal et al. / Bioorg. Med. Chem. 17 (2009) 3733–3738
Table 1
4.2.4. 2,6-Dichloro-9-((3aR,6R,6aR)-2,2-dimethyltetrahydrofuro[3,4-
d][1,3]dioxol-6-yl)-9H-purine (10)
Binding affinities of known A3 AR agonists, 1 and 2 and antagonist 3, and truncated 40-
adenosine derivatives 4a–e and 5a–e at three subtypes of hARs
Compound 8 (702 mg, 3.47 mmol) was condensed with 2,6-
dichloropurine to give compound 10 (793 mg, 69%) as a foam
according to the similar procedure used for the preparation of 9:
UV (MeOH) kmax 276.5 nm; 1H NMR (CDCl3) d 8.15 (s, 1H), 6.07
(s, 1H), 5.41 (d, 1H, J = 6.0 Hz), 5.29–5.26 (m, 1H), 4.31–4.25 (m,
NHR
N
N
N
X
N
2 H), 1.57 (s, 3 H), 1.41 (s, 3 H); ½a D25
ꢁ
ꢀ21.00 (c 0.10, DMSO);
Y
FAB-MS m/z 331 [M+H]+. Anal. Calcd for C12H12Cl2N4O3: C, 43.52;
H, 3.65; N, 16.92. Found: C, 43.08; H, 3.61; N, 16.70.
HO
OH
Affinity, Ki, nM SEM (or % inhibition)a
4.2.5. (2R,3R,4S)-2-(6-Chloro-9H-purin-9-yl)-tetrahydrothiophene-
3,4-diol (11)
Compound
hA1
hA2A
hA3
To a solution of 9 (300 mg, 1.01 mmol) in THF (5 mL) was added
2 N hydrochloric acid (2.5 mL) and the mixture was stirred at rt
overnight. The mixture was neutralized with 1 N NaOH solution,
and then carefully evaporated. The mixture was purified by a flash
silica gel column chromatography (CH2Cl2/MeOH = 20:1) to give 11
(165 mg, 64%) as a white solid: mp 165.5–166.7 °C; UV (MeOH)
kmax 264.0 nm; 1H NMR (DMSO-d6) d 8.95 (s, 1H), 8.82 (s, 1H),
6.01 (d, 1H, J = 6.4 Hz), 5.54 (d, 1H, J = 6.4 Hz), 5.29 (d, 1H,
J = 4 Hz), 4.83 (ddd, 1H, J = 4.8, 6.4, 10.8 Hz), 4.41 (dd, 1H, J = 3.2,
9.2 Hz), 4.33–4.29 (m, 1H), 3.86 (dd, 1H, J = 1.6, 9.2 Hz); 13C NMR
(DMSO-d6) d 151.8, 151.7, 149.4, 146.5, 131.5, 88.4, 74.6, 74.1,
1 (Cl-IB-MECA)
2 (thio-Cl-IB-MECA)
3 (X = Cl, Y = S, R = 3-iodobenzyl)
4a (X = H, Y = O, R = methyl)
4b (X = H, Y = O, R = 3-fluorobenzyl)
4c (X = H, Y = O, R = 3-chlorobenzyl)
4d (X = H, Y = O, R = 3-bromobenzyl)
4e (X = H, Y = O, R = 3-iodobenzyl)
5a (X = Cl, Y = O, R = methyl)
5b (X = Cl, Y = O, R = 3-fluorobenzyl)
5c (X = Cl, Y = O, R = 3-chlorobenzyl)
5d (X = Cl, Y = O, R = 3-bromobenzyl)
5e (X = Cl, Y = = O, R = 3-iodobenzyl)
220
193
2490
44 2%
5400
223
341
1.4
0.38
4.16
25 5%
6560 1560
3410 400
1400 120
997 125
15 5%
42 4%
3630 1080
30%
70.1 18.3
537 58
76.1 3.9
59.0 0.2
90.2 39.8
37.5 5.0
284 37
75.0 11.7
13.0 6.9
42.9 8.9
36 1%
3100 890%
1060 180
795 101
49 1%
43 1%
2480 590
1150 62
2230 700
43%
70.3; ½a 2D5:6
ꢁ
ꢀ95.71 (c 0.14, DMSO); FAB-MS m/z 257 [M+H]+. Anal.
a
All binding experiments were performed using adherent mammalian cells
stably transfected with cDNA encoding the appropriate hAR (A1 AR and A3 AR in
CHO cells and A2A AR in HEK-293 cells). Binding was carried out using 3.5 nM
[3H]NECA, 10 nM [3H]CGS21680, or 0.5 nM [125I]I-AB-MECA as radioligands for A1,
A2A, and A3 ARs, respectively. Values are expressed as mean sem, n = 3–4 (outliers
eliminated), and normalized against a non-specific binder, 50-N-ethylcarboxami-
Calcd for C9H9ClN4O3: C, 42.12; H, 3.53; N, 21.83. Found: C, 42.17;
H, 3.83; N, 20.43.
4.2.6. (2R,3R,4R)-2-(2,6-Dichloro-9H-purin-9-yl)tetrahydrofuran-
3,4-diol (12)
doadenosine (NECA, 10
inhibition at a fixed concentration of 10
l
M). If a percentage is given, it represents the percent
Compound 10 (683 mg, 2.06 mmol) was converted to 12
(344 mg, 57%) as a white solid according to the similar procedure
used for the preparation of 11: mp 122.7–123.4 °C; UV (MeOH)
kmax 276.5 nm; 1H NMR (DMSO-d6) d 8.98 (s, 1H), 5.96 (d, 1H,
J = 6.4 Hz), 5.57 (d, 1H, J = 6.0 Hz), 5.32 (d, 1H, J = 4.0 Hz), 4.74–
4.69 (m, 1H), 4.41 (dd, 1H, J = 3.6, 9.2 Hz), 4.32–4.29 (m, 1H),
3.87 (dd, 1H, J = 2.0, 9.6 Hz); 13C NMR (DMSO-d6) d 153.2, 151.2,
lM.
150.04, 147.1, 131.2, 88.4, 74.8, 74.1, 70.1; ½a D25
ꢀ68.09 (c 0.14,
ꢁ
DMSO); FAB-MS m/z 291 [M+H]+. Anal. Calcd for C9H8Cl2N4O3: C,
37.13; H, 2.77; N, 19.25. Found: C, 37.23; H, 3.11; N, 19.45.
4.3. General procedure for the synthesis of 4a–e and 5a–e
To a solution of 11 and 12 in EtOH (5 mL) was added appropri-
ate amines (1.5 equiv) at room temperature and the mixture was
stirred at room temperature for 1 d to 3 d and evaporated. The res-
idue was purified by a flash silica gel column chromatography
(CH2Cl2/MeOH = 20:1) to give 4a–e and 5a–e, respectively.
Figure 1. Right-shift of the concentration–response curves of the full agonist Cl-IB-
MECA 1 by 300 nM of antagonist 5d.
4.3.1. (2R,3R,4R)-Tetrahydro-2-(6-(methylamino)-9H-purin-9-
yl)-furan-3,4-diol (4a)
(1.5 g, 7.42 mmol) in 1,2-dichloroethane (10 mL) and TMSOTf
(2.69 mL, 14.84 mmol) were successively added and the mixture
was stirred at 0 °C for 30 min, at rt for 1 h, and then heated at
80 °C for 2 h. The mixture was cooled, diluted with CH2Cl2 and
washed with saturated NaHCO3 solution. The organic layer was
dried over MgSO4 and evaporated. The yellowish syrup was puri-
73% Yield; white solid; mp 208.5–209.0 °C; UV (MeOH) kmax
265.0 nm; 1H NMR (DMSO-d6) d 8.33 (s, 1H), 8.23 (s, 1H), 7.73
(br s, 1H), 5.85 (d, 1H, J = 6.4 Hz), 5.43 (d, 1H, J = 6.4 Hz), 5.19 (d,
1H, J = 4.0 Hz), 4.87–4.82 (m, 1H), 3.34 (dd, 1H, J = 4.0, 9.6 Hz),
4.27–4.25 (m, 1H), 3.79 (dd, 1H, J = 1.2, 9.2 Hz), 2.95 (br s, 3H);
13C NMR (DMSO-d6) d 155.0, 152.6, 148.5, 139.9, 119.9, 87.7,
fied by
a flash silica gel column chromatography (CH2Cl2/
74.2, 73.6, 70.3, 26.9; ½a D25:8
ꢀ115.45 (c 0.11, DMSO); FAB-MS m/z
ꢁ
MeOH = 50:1) to give 9 (1.5 g, 68%) as a white solid: mp 118.5–
119.2 °C; UV (MeOH) kmax 264.0 nm; 1H NMR (CD3OD) d 8.73 (s,
1H), 8.62 (s, 1H), 6.27 (s, 1H), 5.52 (d, 1H, J = 5.6 Hz), 5.31 (dd,
1H, J = 3.6, 6.0 Hz), 4.27 (dd, 1H, J = 3.6, 10.4 Hz), 4.19 (d, 1H,
J = 10.4 Hz), 1.54 (s, 3H), 1.38 (s, 3H); 13C NMR (CD3OD) d 153.2,
152.8, 151.7, 147.9, 132.9, 114.3, 93.1, 85.9, 82.8, 77.0, 26.8, 25.1;
252 [M+H]+. Anal. Calcd for C10H13N5O3: C, 47.81; H, 5.22; N,
27.87. Found: C, 47.50; H, 5.55; N, 27.66.
4.3.2. (2R,3R,4R)-2-(6-(3-Fluorobenzylamino)-9H-purin-9-
yl)tetrahydrofuran-3,4-diol (4b)
80% yield; white solid; mp 196.3–197.0 °C; UV (MeOH) kmax
268.0 nm; 1H NMR (DMSO-d6) d 8.42 (br s, 1H), 8.39 (s, 1H), 8.21
(s, 1H), 7.36–7.30 (m, 1H), 7.18–7.11 (m, 2 H), 7.03 (dt, 1H, J = 2.0,
½
a 2D5:8
ꢁ
ꢀ45.04 (c 0.333, DMSO); FAB-MS m/z 297 [M+H]+. Anal.
Calcd for C12H13ClN4O3: C, 48.58; H, 4.42; N, 18.88. Found: C,
49.26; H, 4.61; N, 18.70.

S. Pal et al. / Bioorg. Med. Chem. 17 (2009) 3733–3738
3737
8.4 Hz), 5.86 (d, 1H, J = 6.4 Hz), 5.43 (d, 1H, J = 6.4 Hz), 5.18 (d, 1H,
J = 4.0 Hz), 4.88–4.83 (m, 1H), 4.71 (br s, 2 H), 4.34 (dd, 1H, J = 3.6,
9.2 Hz), 4.28–4.25 (m, 1H), 3.79 (dd, 1H, J = 1.6, 9.2 Hz); 13C NMR
(DMSO-d6) d 163.4, 160.9, 154.3, 152.5, 148.9, 143.2, 140.3, 130.13
(d, J = 31.6 Hz), 123.1 (d, J = 8.0 Hz), 119.8, 113.5 (dd, J = 82.4,
1H), 7.39–7.33 (m, 1H), 7.18–7.13 (m, 2 H), 7.06 (dt, 1H, J = 2.0,
8.4 Hz), 5.81 (d, 1H, J = 6.8 Hz), 5.47 (d, 1H, J = 6.0 Hz), 5.22 (d,
1H, J = 4.0 Hz), 4.74–4.64 (m, 3 H), 4.41 (dd, 1H, J = 3.6, 9.2 Hz),
4.25 (br s, 1H), 3.80 (dd, 1H, J = 1.2, 9.6 Hz); 13C NMR (DMSO-d6)
d 163.4, 163.9, 154.9, 153.1, 149.9, 142.2 (d, J = 26.8 Hz), 140.7,
143.6 Hz), 87.8, 74.2, 73.7, 70.3, 42.5; ½a D25:7
ꢁ
ꢀ96.52 (c 0.115, DMSO);
130.3 (d, J = 34.8 Hz), 123.2, 118.8, 113.8 (dd, J = 82.4, 122.0 Hz),
FAB-MS m/z 346 [M+H]+. Anal. Calcd for C16H16FN5O3: C, 55.65; H,
87.5, 74.4, 73.7, 70.2, 42.7; [
a
]
-73.40 (c 0.094, DMSO); FAB-
23.2
D
4.67; N, 20.28. Found: C, 55.29; H, 4.75; N, 19.93.
MS m/z 380 [M+H]+. Anal. Calcd for C16H15ClFN5O3: C, 50.60; H,
3.98; N, 18.44. Found: C, 50.30; H, 4.01; N, 18.04.
4.3.3. (2R,3R,4R)-2-(6-(3-Chlorobenzylamino)-9H-purin-9-yl)-
tetrahydrofuran-3,4-diol (4c)
4.3.8. (2R,3R,4R)-2-(2-Chloro-6-(3-chlorobenzylamino)-9H-
purin-9-yl)-tetrahydrofuran-3,4-diol (5c)
83% Yield; white solid; mp 187.0–187.7 °C; UV (MeOH) kmax
267.0 nm; 1H NMR (DMSO-d6) d 8.43 (br s, 1H), 8.39 (s, 1H), 8.22
(s, 1H), 7.38 (s, 1H), 7.34–7.24 (m, 3 H), 5.85 (d, 1H, J = 6.4 Hz),
5.44 (d, 1H, J = 6.8 Hz), 5.20 (d, 1H, J = 3.6 Hz), 4.85 (dd, 1H,
J = 6.4, 10.8 Hz), 4.70 (s, 2H), 4.35 (dd, 1H, J = 3.6, 9.2 Hz), 4.28–
4.25 (m, 1H), 3.80 (dd, 1H, J = 1.6, 9.2 Hz); 13C NMR (DMSO-d6) d
154.3, 152.5, 148.9, 142.8, 140.4, 132.9, 130.2, 126.9, 126.6,
76% Yield; white solid; mp 196.4–197.0 °C; UV (MeOH) kmax
271.5 nm; 1H NMR (DMSO-d6) d 8.92 (t, 1H, J = 6.0 Hz), 8.43 (s,
1H), 7.39 (s, 1H), 7.37–7.28 (m, 3H), 5.80 (d, 1 H, J = 6.8 Hz), 5.47
(d, 1H, J = 6.4 Hz), 5.22 (d, 1H, J = 4.0 Hz), 4.73–4.64 (m, 3H), 4.32
(dd, 1H, J = 3.6, 9.2 Hz), 4.24 (br s, 1H), 3.79 (dd, 1H, J = 1.6,
9.2 Hz); 13C NMR (DMSO-d6) d 154.8, 153.1, 149.9, 141.8, 140.7,
132.9, 130.2, 127.1, 126.8, 125.9, 118.7, 87.5, 74.4, 73.7, 70.2,
125.8, 119.8, 87.8, 74.3, 73.7, 70.4, 42.4; ½a D25:8
ꢀ92.92 (c 0.099,
ꢁ
DMSO); FAB-MS m/z 362 [M+H]+. Anal. Calcd for C16H16ClN5O3:
42.7; ½a 2D2:9
ꢁ
ꢀ78.19 (c 0.133, DMSO); FAB-MS m/z 396 [M]+. Anal.
C, 53.12; H, 4.46; N, 19.36. Found: C, 53.12; H, 4.57; N, 18.96.
Calcd for C16H15Cl2N5O3: C, 48.50; H, 3.82; N, 17.68. Found: C,
48.56; H, 3.89; N, 17.12.
4.3.4. (2R,3R,4R)-2-(6-(3-Bromobenzylamino)-9H-purin-9-yl)-
tetrahydrofuran-3,4-diol (4d)
4.3.9. (2R,3R,4R)-2-(2-Chloro-6-(3-bromobenzylamino)-9H-
purin-9-yl)-tetrahydrofuran-3,4-diol (5d)
86% Yield; white solid; mp 174.0–174.9 °C; UV (MeOH) kmax
267.0 nm; 1H NMR (DMSO-d6) d 8.44 (br s, 1H), 8.39 (s, 1H), 8.21
(s, 1 H), 7.52 (s, 1H), 7.42 (dt, 1H, J = 2.0, 8.8 Hz), 7.36 (d, 1H,
J = 8.0 Hz), 7.25 (d, 1 H, J = 8.0 Hz), 5.86 (d, 1H, J = 6.4 Hz), 5.43
(d, 1 H, J = 6.4 Hz), 5.19 (d, 1H, J = 3.6 Hz), 4.88–4.82 (m, 1H),
4.69 (br s, 2 H), 4.35 (dd, 1H, J = 3.6, 9.2 Hz), 4.28–4.25 (m, 1H),
3.80 (dd, 1H, J = 1.6, 9.6 Hz); 13C NMR (DMSO-d6) d 154.3, 152.5,
81% Yield; white solid; mp 181.5–181.7 °C; UV (MeOH) kmax
274.5 nm; 1H NMR (DMSO-d6) d 8.92 (t, 1H, J = 6.0 Hz), 8.43 (s,
1H), 7.55 (s, 1H), 7.44 (d, 1H, J = 8.0 Hz), 7.35–7.33 (m, 1H), 7.30–
7.26 (m, 1H), 5.81 (d, 1H, J = 6.4 Hz), 5.47 (d, 1H, J = 6.4 Hz), 5.22
(d, 1H, J = 4.0 Hz), 4.69–4.66 (m, 1H), 4.62 (s, 2H), 4.32 (dd, 1H,
J = 3.6, 9.2 Hz), 4.25 (br s, 1H), 3.80 (dd, 1H, J = 1.6, 9.2 Hz); 13C
NMR (DMSO-d6) d 154.9, 153.2, 149.9, 142.1, 140.7, 130.6, 130.1,
148.9, 143.0, 140.4, 130.4, 129.8, 129.5, 126.2, 121.5, 119.8, 87.8,
74.2, 73.7, 70.3, 42.3; [
a]
-88.00 (c 0.15, DMSO); FAB-MS m/z
129.8, 126.4, 121.6, 118.8, 87.5, 74.4, 73.7, 70.2, 42.6; ½a D25
ꢁ
23.3
D
406 [M]+. Anal. Calcd for C16H16BrN5O3: C, 47.31; H, 3.97; N,
17.24. Found: C, 47.30; H, 3.94; N, 17.47.
ꢀ62.75 (c 0.10, DMSO); FAB-MS m/z 440 [M+H]+. Anal. Calcd for
C16H15ClBrN5O3: C, 43.61; H, 3.43; N, 15.89. Found: C, 43.92; H,
3.44; N, 16.05.
4.3.5. (2R,3R,4R)-2-(6-(3-Iodobenzylamino)-9H-purin-9-yl)-
tetrahydrofuran-3,4-diol (4e)
4.3.10. (2R,3R,4R)-2-(2-Chloro-6-(3-iodobenzylamino)-9H-
purin-9-yl)-tetrahydrofuran-3,4-diol (5e)
77% Yield; white solid; mp 202.8–203.5 °C; UV (MeOH) kmax
267.0 nm; 1H NMR (DMSO-d6) d 8.42 (br s, 1H), 8.39 (s, 1H), 8.21
(s, 1H), 7.71 (s, 1H), 7.57 (dt, 1H, J = 2.8, 8.0 Hz), 7.35 (d, 1H,
J = 7.6 Hz), 7.10 (d, 1H, J = 7.6 Hz), 5.86 (d, 1H, J = 6.4 Hz), 5.44 (d,
1H, J = 6.4 Hz), 5.19 (d, 1H, J = 3.6 Hz), 4.88–4.83 (m, 1H), 4.66 (br
s, 2 H), 3.35 (dd, 1H, J = 3.6, 9.2 Hz), 4.27–4.25 (m, 1H), 3.80 (dd,
1H, J = 1.6, 9.6 Hz); 13C NMR (DMSO-d6) d 154.3, 152.5, 148.9,
142.9, 140.37, 135.7, 135.3, 130.5, 126.6, 119.8, 94.7, 87.8, 74.2,
78% Yield; white solid; mp 195.5–195.8 °C; UV (MeOH) kmax
274.0 nm; 1H NMR (DMSO-d6) d 8.91 (t, 1H, J = 6.4 Hz), 8.44 (s,
1H), 7.75 (s, 1H), 7.61 (d, 1H, J = 8.0 Hz), 7.36 (d, 1H, J = 7.6 Hz),
7.13 (t, 1H, J = 4.0 Hz), 5.81 (d, 1H, J = 6.8 Hz), 5.47 (d, 1H,
J = 6.8 Hz), 5.23 (d, 1H, J = 4.0 Hz), 4.72 (dd, 1H, J = 6.4, 10.8 Hz),
4.61 (d, 1H, J = 6.0 Hz), 4.34 (dd, 1H, J = 3.6, 9.2 Hz), 3.81 (dd, 1H,
J = 1.2, 9.2 Hz); 13C NMR (DMSO-d6) d 154.8, 153.2, 149.9, 141.9,
140.7, 136.0, 135.6, 130.6, 126.8, 118.8, 94.8, 87.5, 74.4, 73.7,
73.7, 70.3, 42.2. ½a D25:6
ꢀ67.74 (c 0.124, DMSO); FAB-MS m/z 454
ꢁ
[M+H]+. Anal. Calcd for C16H16IN5O3: C, 42.40; H, 3.56; N, 15.45.
Found: C, 42.44; H, 3.59; N, 15.22.
70.2, 42.5; ½a 2D5
ꢁ
ꢀ68.07 (c 0.12, DMSO); FAB-MS m/z 488 [M+H]+.
Anal. Calcd for C16H15ClIN5O3: C, 39.41; H, 3.10; N, 14.36. Found:
C, 39.66; H, 3.08; N, 14.53.
4.3.6. (2R,3R,4R)-2-(2-Chloro-6-(methylamino)-9H-purin-9-yl)-
furan-3,4-diol (5a)
4.4. Pharmacological methods
85% Yield; white solid; mp 247 °C (dec); UV (MeOH) kmax
269.0 nm; 1H NMR (DMSO-d6)
d
8.38 (s, 1H), 8.25 (q, 1H,
[
125I]N6-(4-Amino-3-iodobenzyl)adenosine-50-N-methylurona-
J = 4.4 Hz), 5.79 (d, 1H, J = 6.8 Hz), 5.48 (d, 1H, J = 6.4 Hz), 5.22 (d,
1H, J = 4.0 Hz), 4.73–4.69 (m, 1H), 4.30 (dd, 1 H, J = 3.6, 9.2 Hz),
4.25–4.23 (m, 1H), 3.79 (dd, 1H, J = 1.6, 9.2 Hz), 2.92 (d, 3H,
J = 4.4 Hz); 13C NMR (DMSO-d6) d 155.5, 153.3, 149.4, 140.2,
mide (I-AB-MECA; 2200 Ci/mmol), and [3H]CGS21680 (2-[p-(2-car-
boxyethyl)phenyl-ethylamino]-50-N-ethylcarboxamido-adenosine)
(40.5 Ci/mmol) were purchased from Perkin–Elmer Life and Ana-
lytical Science (Boston, MA). [3H]NECA (18.64 Ci/mmol) was a cus-
tom synthesis product (Perkin Elmer). Test compounds were
prepared as 5 mM stock solutions in DMSO and stored frozen.
Cell Culture and Membrane Preparation: CHO (Chinese hamster
ovary) cells stably expressing the recombinant human A1 and A3,
and HEK-293 (Human Embryonic Kidney) cells stably expressing
human A2A were cultured in Dulbecco0s modified Eagle medium
(DMEM) and F12 (1:1) supplemented with 10% fetal bovine serum,
118.8, 87.4, 74.4, 73.6, 70.2, 27.1; ½a D23:5
ꢀ40.77 (c 0.13, DMSO);
ꢁ
FAB-MS m/z 286 [M+H]+. Anal. Calcd for C10H12ClN5O3: C, 42.04;
H, 4.23; N, 24.51. Found: C, 42.38; H, 4.14; N, 24.27.
4.3.7. (2R,3R,4R)-2-(2-Chloro-6-(3-fluorobenzylamino)-9H-
purin-9-yl)-tetrahydrofuran-3,4-diol (5b)
83% Yield; white solid; mp 187.0–187.9 °C; UV (MeOH) kmax
271.0 nm; 1H NMR (DMSO-d6) d 8.92 (t, 1H, J = 6.0 Hz), 8.43 (s,
100 units/mL penicillin, 100 lg/mL streptomycin, and 2 lmol/mL

3738
S. Pal et al. / Bioorg. Med. Chem. 17 (2009) 3733–3738
glutamine. In addition, 800
l
g/mL geneticin was added to the A2A
Cheng–Prusoff equation.21 Data were expressed as mean stan-
media, while 800
lg/mL hygromycin was added to the A1 and A3
dard error.
media. After harvesting, cells were homogenized and suspended
in PBS. Cells were then centrifuged at 240g for 5 min, and the pellet
was resuspended in 50 mM Tris–HCl buffer (pH 7.5) containing
10 mM MgCl2. The suspension was homogenized and was then ul-
tra-centrifuged at 14,330g for 30 min at 4 °C. The resultant pellets
were resuspended in Tris buffer, incubated with adenosine deam-
inase (3 units/mL) for 30 min at 37 °C. The suspension was homog-
enized with an electric homogenizer for 10 s, pipet into 1 mL vials
and then stored at ꢀ80 °C until the binding experiments. The pro-
tein concentration was measured using the BCA Protein Assay Kit
from Pierce Biotechnology, Inc. (Rockford, 1000 mL).19
Acknowledgements
This work was supported by the Korea Research Foundation
Grant (KRF-2008-E00304), the Grant from the World Class Univer-
sity (WCU) Project from Ministry of Education and Science, Korea
(R31-2008-000-10010-0), and the Intramural Research Program
of NIDDK, NIH, Bethesda, MD, USA.
References and notes
Binding assays: Into each tube in the binding assay was added
50
buffer (50 mM, pH 7.5) containing 10 mM MgCl2, 50
appropriate agonist radioligand, and finally 100 L of membrane
suspension. For A1 receptors (22 g of protein/tube) the radioli-
gand used was [3H]NECA (3.5 nM). For A2A receptors (20
g/tube)
the radioligand used was [3H]CGS21680 (10 nM). For A3 receptors
(21
g/tube) the radioligand used was 125I-AB-MECA (0.34 nM).
Nonspecific binding was determined using a final concentration
of 10
M 50-N-ethylcarboxamidoadenosine (NECA) diluted with
the buffer. The mixtures were incubated at 25 °C for 60 min in a
shaking water bath. Binding reactions were terminated by filtra-
tion through Brandel GF/B filters under a reduced pressure using
a M-24 cell harvester (Brandel, Gaithersburg, MD). Filters were
washed three times with 3 mL of 50 mM ice-cold Tris–HCl buffer
(pH 7.5). Filters for A1 and A2A AR binding were placed in scintilla-
tion vials containing 5 mL of Hydrofluor scintillation buffer and
counted using a Perkin Elmer Liquid Scintillation Analyzer (Tri-
Carb 2810TR). Filters for A3 AR binding were counted using a Pack-
l
L of increasing concentrations of the test ligand in Tris–HCl
1. Olah, M. E.; Stiles, G. L. Pharmacol. Ther. 2000, 85, 55.
2. Jacobson, K. A.; Gao, Z.-G. Nat. Rev. Drug Disc. 2006, 5, 247.
3. (a) Fishman, P.; Madi, L.; Bar-Yehuda, S.; Barer, F.; Del Valle, L.; Khalili, K.
Oncogene 2002, 21, 4060; (b) Lee, E. J.; Min, H. Y.; Chung, H. J.; Park, E. J.; Shin, D.
H.; Jeong, L. S.; Lee, S. K. Biochem. Pharmacol. 2005, 70, 918; (c) Chung, H.; Jung,
J.-Y.; Cho, S.-D.; Hong, K.-A.; Kim, H.-J.; Shin, D.-H.; Kim, H.; Kim, H. O.; Lee, H.
W.; Jeong, L. S.; Gong, K. Mol. Cancer Ther. 2006, 5, 685; (d) Kim, S. J.; Min, H. Y.
H.; Chung, J.; Park, E. J.; Hong, J. Y.; Kang, Y. J.; Shin, D. H.; Jeong, L. S.; Lee, S. K.
Cancer Lett. 2008, 264, 309.
4. von Lubitz, D. K. J. E.; Ye, W.; McClellan, J.; Lin, R. C.-S. Ann. N.Y. Acad. Sci. 1999,
890, 93.
5. Liang, B. T.; Jacobson, K. A. Proc. Natl. Acad. Sci. 1998, 95, 6995.
6. Sajjadi, F. G.; Takabayashi, K.; Foster, A. C.; Domingo, R. C.; Firestein, G. S.
J. Immunol. 1996, 156, 3435.
7. Meade, C. J.; Mierau, J.; Leon, I.; Ensinger, H. A. J. Pharmacol. Exp. Ther. 1996, 279,
1148.
8. Avila, M. Y.; Stone, R. A.; Civan, M. M. Invest. Opthalmol. Vis. Sci. 2002, 43,
3021.
9. Klotz, K.-N. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2000, 362, 382.
10. Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Merighi, S.; Varani, K.; Borea, P. A.;
Spalluto, G. Med. Res. Rev. 2000, 20, 103.
11. Kim, H. O.; Ji, X.-d.; Siddiqi, S. M.; Olah, M. E.; Stiles, G. L.; Jacobson, K. A. J. Med.
Chem. 1994, 37, 3614.
lL of the
l
l
l
l
l
ard Cobra II
GraphPad Prism for all assays.
c
-counter. The Ki values were determined using
12. (a) 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; (b) 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. 2006, 49, 273; (c) Jeong, L. S.; Lee, H. W.; Kim,
H. O.; Jung, J. Y.; Gao, Z.-G.; Duong, H. T.; Rao, S.; Jacobson, K. A.; Shin, D. H.; Lee,
J. A.; Gunaga, P.; Lee, S. K.; Jin, D. Z.; Chun, M. W. Bioorg. Med. Chem. 2006, 14,
4718.
13. Baraldi, P. G.; Tabrizi, M. A.; Romagnoli, R.; Fruttarolo, F.; Merighi, S.; Varani, K.;
Gessi, S.; Borea, P. A. Curr. Med. Chem. 2005, 12, 1319.
14. (a) Gao, Z. G.; Blaustein, J.; Gross, A. S.; Melman, N.; Jacobson, K. A. Biochem.
Pharmacol. 2003, 65, 1675; (b) Yang, H.; Avila, M. Y.; Peterson-Yantorno, K.;
Coca-Prados, M.; Stone, R. A.; Jacobson, K. A.; Civan, M. M. Curr. Eye Res. 2005,
30, 747.
Cyclic AMP accumulation assay: Intracellular cyclic AMP levels
were measured with a competitive protein binding method.20
CHO cells that expressed the recombinant hA3AR were harvested
by trypsinization. After centrifugation and resuspended in med-
ium, cells were planted in 24-well plates in 1.0 mL medium. After
24 h, the medium was removed and cells were washed three times
with 1 mL DMEM, containing 50 mM HEPES, pH 7.4. Cells were
then treated with the agonist NECA and/or test compound (e.g.,
5d) in the presence of rolipram (10
(3 units/mL). After 30 min forskolin (10
medium, and incubation was continued for an additional 15 min.
lM) and adenosine deaminase
15. Jeong, L. S.; Choe, S. A.; Gunaga, P.; Kim, H. O.; Lee, H. W.; Lee, S. K.; Tosh, D. K.;
Patel, A.; Palaniappan, K. K.; Gao, Z.-G.; Jacobson, K. A.; Moon, H. R. J. Med.
Chem. 2007, 50, 3159.
lM) was added to the
16. Choi, W. J.; Park, J. G.; Yoo, S. J.; Kim, H. O.; Moon, H. R.; Chun, M. W.; Jung, Y.
H.; Jeong, L. S. J. Org. Chem. 2001, 66, 6490.
17. (a) Klotz, K.-N.; Hessling, J.; Hegler, J.; Owman, C.; Kull, B.; Fredholm, B.
B.; Lohse, M. J. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1998, 357, 1; (b)
Gao, Z. G.; Mamedova, L.; Chen, P.; Jacobson, K. A. Biochem. Pharmacol.
2004, 68, 1985.
18. Kim, Y. C.; Ji, X.-d.; Melman, N.; Linden, J.; Jacobson, K. A. J. Med. Chem. 2000, 43,
1165.
19. Walsh, M. K.; Wang, X.; Weimer, B. C. J. Biochem. Biophys.. Methods 2001, 47,
221.
The reaction was terminated by removing the supernatant, and
cells were lysed upon the addition of 200 lL of 0.1 M ice-cold
HCl. The cell lysate was resuspended and stored at ꢀ20 °C. For
determination of cyclic AMP production, a cyclic AMP kit (Sigma)
was used.
4.4.1. Statistical analysis
Binding and functional parameters were calculated using Prism
4.0 software (GraphPAD, San Diego, CA, USA). IC50 values obtained
from competition curves were converted to Ki values using the
20. Nordstedt, C.; Fredholm, B. B. Anal. Biochem. 1990, 189, 231.
21. Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.