Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position

Article abstract of DOI:10.1016/j.bmc.2004.03.031

We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N⁶-substitutions known to enhance human A ₃AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A₁, A_2A, A_2B, and A₃ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA₃AR affinity and efficacy in the cases of N⁶-(3-iodobenzyl) and N ⁶-(trans-2-phenyl-1-cyclopropyl), for which a full A₃AR agonist was converted into a selective antagonist; the 2-cyano-N ⁶-methyl analogue was a full A₃AR agonist. The combination of N⁶-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A₁AR. The environment surrounding the 2-position within the putative A₃AR binding site was explored using rhodopsin-based homology modeling and ligand docking.

Full text of DOI:10.1016/j.bmc.2004.03.031

Bioorganic & Medicinal Chemistry 12 (2004) 2995–3007
Modulation of adenosine receptor affinity and intrinsic efficacy in
adenine nucleosides substituted at the 2-position
Michihiro Ohno,a Zhan-Guo Gao,a Philippe Van Rompaey,b Susanna Tchilibon,a
Soo-Kyung Kim,a Brian A. Harris,a Ariel S. Gross,a Heng T. Duong,a
Serge Van Calenberghb and Kenneth A. Jacobsona,*
aMolecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health (NIH), DHHS, Bldg. 8A, Rm. B1A-19, NIH, NIDDK, LBC, Bethesda,
MD 20892-0810, USA
bLaboratorium voor Medicinale Chemie (FFW), Harelbekestraat 72B-9000 Gent, Belgium
Received 19 December 2003; revised 1 March 2004; accepted 14 March 2004
Available online 20 April 2004
Abstract—We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents
at the 2-position of adenosine were combined with N6-substitutions known to enhance human A3AR affinity. Selectivity of binding
of the analogues and their functional effects on cAMP production were studied using recombinant human A1, A2A, A2B, and A3ARs.
Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano
group decreased hA3AR affinity and efficacy in the cases of N6-(3-iodobenzyl) and N6-(trans-2-phenyl-1-cyclopropyl), for which a
full A3AR agonist was converted into a selective antagonist; the 2-cyano-N6-methyl analogue was a full A3AR agonist. The
combination of N6-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A1AR.
The environment surrounding the 2-position within the putative A3AR binding site was explored using rhodopsin-based homology
modeling and ligand docking.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
separate from the structure–affinity relationship derived
from receptor binding experiments, was analyzed.7
Substitution with N6-benzyl groups, 2-chloro substitu-
tion of the adenine moiety, and conformational con-
straint of the ribose moiety all contribute to a reduction
of efficacy. In the present study, we evaluated the binding
affinity and functional properties of adenosine deriva-
tives modified at the N6-position to achieve high A3AR
affinity and at the 2-position with simple substitutions,
such as cyano, carbonyl, aminomethyl, and trifluoro-
methyl. Previously, the effects of such substitutions at
AR subtypes had not been fully evaluated.8
Receptors for the extracellular local modulator adeno-
sine consist of four subtypes: A1, A2A, A2B, and A3.1 The
selective activation of the A3AR (adenosine receptor) is
both cardioprotective and cerebroprotective in a variety
of ischemic models.2;3 The activation of this receptor
subtype has also been associated with a cytostatic,
anticancer effect in several tumor models.4 Thus, selec-
tive A3AR agonists have therapeutic potential.
Previous medicinal chemical studies demonstrated that
an adenosine derivative’s ability to activate the A3AR is
more structure sensitive than at other AR subtypes.5–7
Changes in various regions of the adenosine molecule
have been shown to reduce A3AR efficacy, which leads to
nucleoside analogues that are partial agonists or anta-
gonists. A structure–efficacy relationship at this subtype,
2. Results and discussion
2.1. Chemistry
Adenosine agonists 2–17 and 19–21 were prepared
(Schemes 1–3) to study the effects of 2-position substi-
tution in interactions with ARs. Key synthetic interme-
diates 24 and 25 contained the 2-iodo and 2-cyano group,
respectively, in combination with 6-chloro substitution.
Keywords: Purines; Cyclic AMP; Binding; Antagonists; Agonists;
GPCR; Molecular modeling.
* Corresponding author. Tel.: +301-496-9024; fax: +301-480-8422;
e-mail: kajacobs@helix.nih.gov
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.031

2996
M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
Cl
N
Cl
Cl
N
N
N
N
N
N
N
N
N
Pd2(dba)3
Ac2O
P(2-Fur)3
t-BuONO
N
NH2
DMF, Pyridine
N
NH2
I
HO
Zn(CN)2
CH2I2
69%
AcO
AcO
O
O
O
Tetramethylurea
89%
59%
HO
OH
AcO
OAc
AcO
OAc
22
23
24
Cl
N
NHMe
N
NHMe
N
N
N
N
N
N
N
NH2
1) MeNH2,THF
2) MeOH
N
CN
N
N
CN
AcO
BH3-THF
36%
HO
HO
O
O
O
66%
AcO
OAc
HO
OH
HO
OH
5
25
2
NHMe
MeNH2,THF,MeOH,
50oC
N
N
N
N
NH
NHMe
18%
HO
O
HO
OH
6
Scheme 1.
R1
R1
N
HN
N
HN
N
N
N
N
NaOH, H2O2
EtOH, H2O
N
N
CN
HO
O
R2
HO
O
1) RNH2-HCl,
i-Pr2NEt, DMF
2) column
HO
OH
Cl
Cl
HO
OH
16 : R1=
17 : R1=
Cl
R2=COOH
15 : R1=
19 : R1=
TMSCHN2
MeOH, H2O
MeO
MeO
3) K2CO3, MeOH
MeO
R2=COOMe
Ph
20 : R1=
21 : R1=
Ph
Ph
R2=COOH
R2=CONH2
64% for 15
20% for 19
I
HN
I
I
NH3Cl
HN
N
N
N
N
N
25
N
CN
AcO
NaOH
O
i-Pr2NEt2
DMF
N
N
R2
HO
O
87%
AcO
OAc
26
10 : R2=CN
HO
OH
12 : R2=COOMe
I
HN
N
1) I
N
NH2
N
H
(excess)
, T HF
N
N
I
HO
O
O
2) K2CO3, MeOH
76%
13
HO
OH
Scheme 2.
The introduction of the 2-cyano group was carried out by
the reaction of zinc cyanide on the corresponding 2-iodo
analogue with palladium chemistry (Scheme 1). We
chose the protected 2-amino nucleoside 23 as the

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
2997
Cl
NHR
N
Cl
N
N
N
N
N
N
N
1) Zn, CF2Br2, DMF
2) CuI, HMPA
N
I
AcO
N
O
N
CF3
RNH2
AcO
N
CF3
AcO
O
O
28 R=Me
77%
29% for 28
42% for 29
24
AcO
OAc
27
AcO
OAc
AcO
OAc
29 R=3-I-Bn
3-I-BnNH2-HCl
iPr2NEt, DMF
43% for 3
NaOH,
MeOH
86%
45% for 11
I
I
HN
N
NHR
N
HN
N
N
N
N
N
N
N
N
NaOH, MeOH
63%
N
I
N
CF3
HO
AcO
I
O
HO
O
O
3
R=Me
30
HO
OH
AcO
OAc
9
HO
HO
OH
11 R=3-I-Bn
Cl
N
NHMe
N
N
N
N
N
N
NH2
NH2
HO
N
MeNH2, H2O
94%
O
O
HO
OH
HO
OH
22
4
Scheme 3.
synthetic intermediate because of the simplicity of its
synthesis from (ꢀ)-2-amino-6-chloropurine riboside 22.
An attempted Sandmeyer-type cyanation (t-BuONO,
CuCN, MeCN, 65 ꢁC) on 23 resulted in decomposition
without isolation of the desired 2-CN compound. We
then applied Pd chemistry to this cyanation. The 2-amino
group of 23 was converted to 2-iodo using a neutral
purinyl radical, which was generated transiently from the
thermal homolysis of the 2-diazonium intermediate.9
Stille-type conditions (PdCl2(PPh3)2, Bu3SnCN) or use
of potassium cyanide (PdCl2(PPh3)2, KCN)10 on the iodo
derivative 24 resulted in decomposition. Only use of zinc
cyanide and a Pd(0) complex generated in situ from tris-
2-furylphosphine and Pd2(dba)3 (tris(dibenzylideneace-
tone)dipalladium(0) chloroform adduct)11 effected
cyanation of 24. Use of tetramethylurea as solvent gave a
higher yield than N-methyl-2-pyrrolidinone, which was
recommended in the literature.
2-methoxycarbonyl derivative 12. In methanol-free
hydrolysis conditions, the desired N6-(3-iodobenzyl)
derivative 10 was obtained. Adding excess 3-iodoben-
zylamine to 25 and heating resulted in amine addition at
the 2-cyano group to give the 2-(3-iodobenzylamide)
derivative 13 upon hydrolysis.
Trifluoromethylation was performed on the intermedi-
ate 24 by treatment with ‘CF3Cu’ species, which was
generated in situ from CF3ZnBr and CuI (Scheme 3).12;13
Intermediate 27 was coupled with methylamine or
3-iodobenzylamine, and the acetyl groups were hydro-
lyzed to give the final target compounds 3 and 11.
A 2-iodoadenosine derivative 9 was also synthesized
from 24 by treatment with 3-iodobenzylamine followed
by hydrolysis. A 2-amino-N6-methyl derivative 4 was
synthesized by treatment of 22 with methylamine with-
out the use of a protecting group. Each compound
obtained was purified by column chromatography,
preparative thin-layer chromatography, or HPLC.
Intermediate 25 was coupled with the selected amines
(methylamine, 5-chloro-2-methoxybenzylamine, or
trans-2-phenyl-1-cyclopropylamine) at room tempera-
ture, and the acetyl groups were hydrolyzed to give the
final target compounds 2, 15, and 19 (Schemes 1 and 2).
The 2-cyano group could be reduced with BH3 to give
the 2-aminomethyl derivative 5 (Scheme 1) and in other
cases was hydrolyzed to give two 2-carboxylic acid
derivatives 16 and 20 (Scheme 2). In the case of the N6-
methyl derivative 2, the 2-cyano group also reacted with
methylamine at elevated temperature and provided a
N-methyl-carboxyamidine (C(@NH)NHMe), which was
hydrolyzed to give 6. In the case of the 3-iodobenzyl-
amine derivative 26, the methanol solvent reacted with
the 2-cyano group and upon hydrolysis produced the
2.2. Biological activity
We measured the binding affinities of the adenosine
derivatives examined in this study (1–21) at hA1, hA2A
,
and hA3 (human) ARs and at the rA3AR (rat) and their
degree of activation at the four hAR subtypes (Table 1).
The efficacy of each of these adenosine derivatives in
activation of the hARs was evaluated at a fixed con-
centration of 10 lM. Four different N6-substitutions
were included: methyl, 3-iodobenzyl, 5-chloro-2-meth-
yloxybenzyl, and trans-2-phenyl-1-cyclopropyl. The

2998
M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
choice of these four N6-substituents was based on either
their prior use as groups that enhance hA3AR affinity or
selectivity6;7;14 or structural similarity to such a moiety.15
Stereoselectivity of the N6-functional group of adeno-
sine derivatives in receptor binding has been established
at A3ARs.7 Although the trans-2-phenyl-1-cyclopropyl
analogues 18–21 shown in Table 1 are diastereomeric
mixtures, it was previously established that the principal
biological activity at the A3AR for this series is associ-
ated with the (1S,2R) isomer.7 These N6-substitutions
were combined with various 2-modifications, including
chloro, iodo, cyano, trifluoromethyl, carboxylic acid,
amide, ester, or aminomethyl groups.
rather resulted in a reduction in the ability to activate the
receptor. For example, for N6-(5-chloro-2-methoxyben-
zyl), the efficacy of the 2-cyano analogue 15 was some-
what reduced compared to the 2-Hanalogue 14 of
similar A3AR affinity. Nevertheless, both were partial
agonists. Similarly, a loss of efficacy was seen for the
corresponding N6-(3-iodobenzyl) analogues, that is 10
compared with 7. The 2-cyano analogue 15 showed im-
proved hA3AR selectivity (A1AR/A3AR ¼ 23-fold,
A2AAR/A3AR ¼ 457-fold); 14 behaved as an A1AR
full agonist and an A3AR partial agonist. For a pair
of trans-2-phenyl-1-cyclopropyl derivatives, at the
hA3AR the addition of the 2-cyano group transformed a
selective full agonist 18 into a selective full antagonist 19.
The codependence of N6 and C-2 regions in AR binding
and activation was evident. With a small group at the N6
position (methyl), the affinity at the hA3AR was reduced
by some groups at C-2 (trifluoromethyl 3, amino 4,
aminomethyl 5, and N-methyl-carboxamidine 6) and
enhanced by another group, cyano 2. In the case of N6-
methyl derivatives, the 2-cyano, 2-trifluoromethyl, and
2-amino analogues 2–4 were full agonists, and 2 also
showed moderate hA3AR selectivity (A1AR/A3AR ¼ 20-
fold, A2AAR/A3AR > 1000-fold). Aminomethyl 5 and
N-methyl-carboxamidine 6 analogues, being positively
charged at physiological pH, decreased the affinity at
ARs significantly. Also, by virtue of a charged group, 5
and 6 may prove to be neoligands for engineered ARs.16
Other 2-substitutions had varied effects. Curiously,
when substituted with 2-COOMe, the high affinity of
these N6-substituted adenosine analogues at the hA3AR
was maintained or even increased (12 compared with 7
with N6-(3-iodobenzyl)). Also, 12 showed favorable
hA3AR selectivity (A1AR/A3AR ¼ 23-fold, A2AAR/
A3AR ¼ 130-fold). Compound 12 appeared to be an
A3AR antagonist and an A1AR weak, partial agonist.
Another 2-COOMe derivative, 17, was a highly selective
A3AR partial agonist.
At the hA1AR, our compounds showed variable effi-
cacy. In a previous report, 2-halo analogues showed full
agonist activity at the A1AR.17 In our results, 14, 18,
and 21 showed full agonism, and 9–11 were antagonists.
Compounds 2, 7, 8, 12, and 15 appeared to be partial
In contrast to N6-methyl, with a larger N6-substituent,
the 2-cyano substitution did not enhance the affinity, but
Table 1. Binding affinities and maximal agonist effects of adenosine derivatives (upon structural variation at N6 and C2 positions) at human A1, A2A
A2B, and A3ARs and at rat A3ARs expressed in CHO (Chinese hamster ovary) cellsa
,
#
N6-R1
C-2
Ki (hA1AR)a % Act.
or % displ.
(hA1AR)b
Ki
% Act.
% Act.
Ki
% Act.
(hA3AR)b
Ki
(hA2AAR)a (hA2AAR)b (hA2BAR)b (hA3AR)a
or % displ.
(rA3AR)a
1
2
3
4
5
6
CH3
CH3
CH3
CH3
CH3
CH3
H5970
CN
CF3
Æ 2030 38 Æ 10
17%
23%
9%
5 Æ 5
0
16 Æ 3
0
9.3 Æ 0.4
3.4 Æ 0.8
64.5 Æ 3.8
39.0 Æ 2.4
719 Æ 37
96 Æ 3
101 Æ 7
93 Æ 3
98 Æ 3
57 Æ 4
6390
69.8 Æ 4.4
4650 Æ 580
484 Æ 22
27%
60 Æ 7
4 Æ 3
>10,000
>10,000
>10,000
>10,000
ND
20 Æ 3
29 Æ 6
30 Æ 3
12 Æ 3
0
NH2
13 Æ 2
32 Æ 4
8 Æ 5
15%
25%
9%
11 Æ 2
4 Æ 2
13 Æ 8
CH2NH2
CH(@NH)–
NHCH3
H7.4
10%
2730 Æ 268 7 Æ 1
7
IB
IB
Æ 1.7
78 Æ 6
8 Æ 3
0
135 Æ 22
197 Æ 34
90 Æ 7
99 Æ 2
58 Æ 1
16 Æ 3
9 Æ 6
0
5.8 Æ 0.4
1.8 Æ 0.1
24.3 Æ 3.1
119 Æ 19
138 Æ 7
46 Æ 8
9.5 Æ 1.4c
2.7 Æ 1.2
ND
8
Cl
16.8 Æ 2.2
191 Æ 12
0
0
0
0
0
0
9
IB
IB
I
CN
1910 Æ 320 92 Æ 8
10
11
12
13
14
15
16
17
18
19
20
21
1750 Æ 290
612 Æ 131
73.1 Æ 22.9
1 Æ 1
0
25%
35%
32 Æ 5
61 Æ 3
91 Æ 9
256 Æ 30
257 Æ 19
IB
IB
CF3
CO2CH3
15 Æ 2
18 Æ 2
5 Æ 2
56 Æ 4
16 Æ 3
6 Æ 7
6 Æ 4
20 Æ 15
6 Æ 4
3 Æ 1
16 Æ 3
38 Æ 4
66 Æ 16
98 Æ 8
36 Æ 13
Æ 10
390 Æ 160
3.21 Æ 0.17
133 Æ 19
6.05 Æ 0.16
ND
ND
IB
CONH(3-IB) 500 Æ 160
3990 Æ 150 52 Æ 18
CMB
CMB
CMB
CMB
PC
H9.2
CN
Æ 0.5
63.2 Æ 16.9
33
399 Æ 6
93 Æ 6
1.31 Æ 0.16 53 Æ 3
2.76 Æ 0.51 29 Æ 6
1260 Æ 190 90 Æ 15
12.8 Æ 2.3
698 Æ 100
ND
COOH41%
CO2CH3
H124
9%
35%
31 Æ 6
74 Æ 3
211 Æ 36
0
6200 Æ 790
Æ 30
30 Æ 4
101 Æ 13
28 Æ 14
Æ 2
8.04 Æ 0.4
35 Æ 4
2530 Æ 720 88 Æ 23
5640 Æ 780 51 Æ 8
0.86 Æ 0.09 101 Æ 5
399 Æ 28
ND
ND
PC
PC
CN
COOH30%
CONH2
1750 Æ 280
6
8.7 Æ 2.1
1730 Æ 540 28 Æ 1
19.7 Æ 0.4 74 Æ 6
0
0%
41%
5 Æ 5
PC
295 Æ 25
98 Æ 7
94 Æ 4
ND
a All AR binding experiments were performed using adherent CHO cells stably transfected with cDNA encoding the human or rat ARs (unit: nM).
Radioligands: 2 nM [3H]R-PIA (A1), 15 nM [3H]CGS21680 (A2A), and 0.5 nM [125I]I-AB-MECA (A3). % displacement at 10 lM.
b Percent activity at 10 lM, relative to 10 lM CPA (A1), 10 lM NECA (A2A, A2B), or 10 lM Cl-IB-MECA (A3). Values are expressed as mean Æ sem,
n ¼ 3–5.
c Data from Kim et al.25 ND, not determined. IB, 3-iodobenzyl; CMB, 5-chloro-2-methyloxybenzyl; PC, trans-2-phenyl-1-cyclopropyl.

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
2999
agonists at the hA1AR. hA2AAR affinities were moder-
ate to weak, and there was no indication of partial
agonism. In a previous report, 2-halo substituents
reduced the efficacy at the A2AAR.17 At the hA2AAR,
2-Cl (8) and 2-I (9) analogues showed full agonism.
Compounds 7, 12, 14, 15, and 21 were also full A2AAR
agonists. At the hA2BAR, only 7 and 14 substantially
activated at 10 lM.
of point mutation in TMs (transmembrane helical
domains) 3, 6, 7, and EL2.6;19–21
The purine ring was surrounded by a hydrophobic
pocket, which was defined by L91 (3.33) and L246 (6.51).
In addition, H-bonds formed between the exocyclic
amine and the hydroxyl group of S247 (6.52), which was
also in proximity to N250 (6.55), and between the purine
N3 atom and the side chain of Q167 (EL2) (Fig. 1A). The
20-OHgroup of the ribose moiety formed an Hbond with
the backbone carbonyl group of I268 (7.39), and the 30-
OHgroup Hbonded with the backbone carbonyl group
of S271 (7.42) and with the side chain of H272 (7.43),
consistent with our A3 neoceptor model.19 The
The loss of A3AR affinity of 13, which was substituted
with a 2-CONHBn group, could be explained by the
bulkiness of this group. The less bulky 2-iodo analogue 9
was four-fold less potent in binding to the hA3AR than
the 2-H, while the corresponding methyl ester 12 was
twice as potent. Another electron withdrawing group, 2-
CF3, was similar to 2-CN in its effect on A3AR affinity (11
compared with 10) when N6-(3-iodobenzyl) was present.
However, with N6-methyl there was a 20-fold reduction (3
compared with 2). 2-COOH, which is also charged at
physiological pH, decreased the affinity at several ARs
significantly (16 compared with 14 and 20 compared with
18). However, 2-CONH2 21 retained A3AR affinity. Al-
though compound 21 fully activated the A1AR, it was
only a partial agonist at the A3AR.
The usefulness of the compounds may also be limited by
species differences. For example, with the N6-methyl
group, the affinity at the rA3AR was dramatically re-
duced (e.g., 1 and 2); this is similar to previous obser-
vations.7;18 Species differences at the A3ARs were also
observed for larger N6-substituent derivatives, for
example, 18. However, for substituted N6-benzyl deriv-
atives, binding affinity at the rA3AR tended to be pre-
served. For example, 12 displayed a Ki value of 6 nM at
the rA3AR, which was almost the same as its hA3AR
affinity. Compound 12 also displayed substantial A3AR
selectivity. Thus, 12 may prove to be an A3AR-selective
antagonist in the study of rat models. In general, for N6-
(3-iodobenzyl)–derivatized compounds, the affinity at
the rA3AR was no more than three-fold weaker than at
hA3AR, making these compounds attractive for testing
in nonprimate animals.
2.3. Molecular modeling
To locate energetically favorable binding orientations of
2-Hand 2-CN derivatives 1, 2, 7, 10, 14, and 15, the
previously reported Cl-IB-MECA hA3AR complex was
used as the starting geometry for the ribose-binding
position.6 For the optimal interaction, various t0 angles
for the N6-substituent and v1 angles for the adenine ring
were generated, and the resulting conformations were
compared energetically in the putative binding site.
The hA3AR docking result of the high-affinity nucleo-
side 15 is shown in Figure 1. Residues in the putative
ꢀ
binding site that were within 5-A proximity to the ligand
Figure 1. The complexes of the A3AR with 2-cyano ligands (A) the
overall binding site of 15 and (B) the 2-cyano binding site of 2 with
ꢀ
distances in A. All ligands are displayed as ball-and-stick models in
were L91 (3.33), T94 (3.36), H95 (3.37), Q167 (EL2),
F168 (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), I268 (7.39), S271 (7.42), and H272 (7.43).
Molecular modeling results were correlated with results
magenta color, and the side chains of the hA3AR are shown as stick
models. The Hbonding between the ligand and the hA 3AR is dis-
played in yellow. The A3AR is represented by a tube model, with a
different color for each TM (TM3 in yellow, TM5 in green, TM6 in
cyano, TM7 in purple).

3000
M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
50-hydroxyl group also formed an Hbond with T94
(3.36). The phenyl moiety showed an additional hydro-
phobic interaction with F168 (EL2), and the methyl of
the methoxy group was stabilized through hydrophobic
interaction with L91 (3.33) and I186 (5.47), consistent
with its higher binding affinity.
2-substitution with the cyano group and other small
groups has variable effects.
Although the SAR (structure–activity relationship) of the
many 2-substitutions of adenosine at ARs has been
thoroughly explored,23–26 the cyano, trifluoromethyl,
simple ester, and other groups included here have not
been among them. However, various 2-alkyne deriva-
tives,18;27 such as 2-hexynyladenosine-50-N-ethyluron-
amide, have been found to bind potently to both A2A and
A3ARs. The 2-iodo group was found previously to reduce
efficacy at the A2AAR and retain efficacy at the A1AR.17
In our study, 2-iodo analogue 9 showed full agonism at
the hA2AAR and antagonism at the hA1AR.
In comparison to N6-methyl adenosine 1, the more po-
tent 2-cyano-N6-methyl derivative 2 showed a more
favorable nonbonding van der Waals interaction be-
tween the cyano group and the side chains of L90 (3.32)
and L91 (3.33) and between the methyl group of the side
chain of T87 (3.29) and the aromatic ring of F168 of
EL2 (Fig. 1B). The distances between the N atom of the
cyano group and the closest carbon atoms of the sur-
ꢀ
rounding hydrophobic residues were 3.4–4.3 A. The C-2
substituent was also proximal to the hydrophilic residue
We have studied the effects of mainly sterically small
groups substituted at the 2-position on the affinity and
intrinsic efficacy at A1, A2A, A2B, and A3 receptors. The
previously reported compound 8 appears to be a mixed
A1/A3AR antagonist. Compound 12 appears to be an
A3AR antagonist and an A1AR weak, partial agonist.
Compound 14 behaves as an A1AR full agonist and an
A3AR partial agonist, and compound 17 is a highly
selective A3AR partial agonist. Compound 19 is a potent
A3AR antagonist, and the related compound 21 is an
A1AR full agonist and an A3AR partial agonist. Thus,
we have modulated both the AR affinity and efficacy
upon substitution at the 2-position in combination with
various N6-substituents. This has resulted in a collection
of adenosine analogues having widely differing phar-
macological activities, both quantitatively and qualita-
tively. The dependence of intrinsic efficacy at the A3AR
has recently been emphasized,5–7 and with this study we
extend the analysis to other AR subtypes.
ꢀ
Q167 (EL2), with a distance of only 3.9 A between the N
atom of the cyano group and the side chain carbonyl O
atom of Q167. There are some variable amino acids
surrounding the binding site of the 2-cyano group. The
amino acid corresponding to L90 (3.32) was Val in other
ARs. T87 (3.29) was Ala in other human AR subtypes
and Ser for the rA3AR. Q167 was a highly variable
residue among the different subtypes and species, cor-
responding to Glu for A1AR, Leu for A2A and A2BAR,
and His for rat A3AR. Thus, the hA3AR preference of
2-cyanoadenosine derivatives might be explained by an
optimal van der Waals interaction. Another possibility
is that a different binding preference for v1 angle at each
AR subtype would vary the residues of interaction with
the C-2 substituents.
The preferred t0 and v1 angles of the energetically favor-
able bound conformation, derived from hA3AR docking
of various compounds, depended on the particular N6
and C-2 substituents. In the lowest-energy complexes of 1
and 2, the t0 and v1 angles were ꢀ134.7ꢁ and ꢀ102.1ꢁ and
ꢀ162.7ꢁ and ꢀ97.3ꢁ, respectively. For larger N6 groups,
such as 3-I-benzyl, the preferred t0 and v1 angles were
ꢀ113.8ꢁ and ꢀ111.5ꢁ for 7 and ꢀ117.5ꢁ and ꢀ108.3ꢁ for
10. Compared to 2, the binding of the 2-cyano group in 10
was directed more toward the methyl group of T87 (3.29),
resulting in unfavorable van der Waals interactions,
consistent with decreased binding affinity. The difference
in 2-CN orientation between 2 and 10 was a consequence
of additional hydrophobic interaction of the phenyl ring
of the N6-substituent. Thus, the modeling has demon-
strated how the interactions at the N6 and C-2 regions of
the hA3AR might be interdependent.22
In conclusion, we have expanded the range of structures
suitable as nucleoside antagonists of the A3AR. Such
compounds include 12 and 16. A3AR antagonists are of
interest for treatment of glaucoma and possibly asthma.1
Partial A1AR agonists, such as 2, 12, and 15, may be of
interest for treatment of cardiac arrhythmias.26 Whether
the presence of A3AR agonism or antagonism in these
compounds might provide a therapeutic advantage is
unknown. Compound 2 as a selective hA3AR agonist
could be examined for activity as a cardioprotective and
cerebroprotective agent.2;3;28 These compounds may also
be useful as receptor probes for mutagenesis and for the
design of neoceptors (e.g., moderately potent ligands
that have either negative, for example, 16, or positive,
for example, 5, charges near the 2-position).16;19–21 The
expanded analysis of the SAR of nucleoside binding
at the A3AR may now be applied to other agonists
series, to probe the additivity with ribose ring modifi-
cations.6;7
3. Conclusions
In previous studies, it was demonstrated that 2-chloro
and other 2-substitutions of the adenine ring may
increase the affinity and decrease the efficacy of
the adenosine derivatives for the hA3AR.5–7 The 2-chloro
modification has been incorporated into many of
the more highly potent AR probes. At the A1ARs, 2-
chloro has also been observed to enhance affinity
of selective agents. Here we further demonstrated that the
4. Experimental materials
[
125I]N6-(4-Amino-3-iodobenzyl)adenosine-50-N-methyl-
uronamide ([125I]I-AB-MECA; 2000 Ci/mmol), N6-[(R)-
phenylisopropyl]adenosine ([3H]R-PIA; 34 Ci/mmol),
and [3H]cyclic AMP (40 Ci/mmol) were obtained from
Amersham Pharmacia Biotech (Piscataway, NJ). 2-[p-

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
3001
(2-carboxyethyl)phenyl-ethylamino]-50-N-ethylcarbox-
amido-adenosine ([3H]CGS21680; 47 Ci/mmol) was
supplied by Perkin–Elmer Life Sciences (Boston, MA).
Several N6-substituted adenosine derivatives 1, 7, 8, and
18 were prepared as reported (Ref. 7 and references
therein). All other chemicals were of analytical grade
from standard commercial sources.
307.1155, found 307.1158; HPLC (System B) 24.6 min
(99%) (System C), 9.9 min (99%).
4.3. (2R, 3S, 4S, 5R)-2-(6-Methylamino-2-trifluoromethyl-
purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (3)
To a solution of 28 (2.6 mg, 0.0055 mmol) in MeOH
(2.0 mL) was added 3 N NaOH(0.02 mL) and the mix-
ture was stirred at rt for 4 h. The solvent was removed
under reduced pressure and the residue was purified by
4.1. Chemistry
Nucleosides and synthetic reagents were purchased from
Sigma Chemical Co. (St. Louis, MO) and Aldrich
(Milwaukee, WI). HNMR spectra were obtained with
preparative TLC (silica gel, thickness 0.25 mm, solvent
1
AcOEt), to give 3 (0.8 mg, 43%). HNMR (CD OD) d
3
1
8.38 (s, 1H), 6.00 (d, 1H, J ¼ 6:0 Hz), 4,72 (t, 1H,
J ¼ 5:4 Hz), 4.34 (dd, 1H, J ¼ 3:3, 5.1 Hz), 4.14 (dd, 1H,
J ¼ 3:3, 6.3 Hz), 3.88 (dd, 1H, J ¼ 3:0, 12.3 Hz), 3.75
(dd, 1H, J ¼ 3:6, 12.3 Hz), 3.13 (br s, 3H); MS (m=e)
(positive-FAB) 350 (M þ H)þ, 372 (M þ Na)þ. HRMS
(positive-FAB) calcd for C12H15N5O4F3 (M þ H)þ
350.1076, found 350.1069; HPLC (System C) 11.8 min
(99%) (System E), 4.3 min (99%).
a Varian Gemini-300 spectrometer (300 MHz) with
D2O, CDCl3, CD3OD, and DMSO-d6 as a solvent.
Purity of the nucleosides was checked using a Hewlett–
Packard 1100 HPLC equipped with a Luna 5 l RP-
C18(2) analytical column (250 · 4.6 mm; Phenomenex,
Torrance, CA). System A: linear gradient solvent sys-
tem: H2O/CH3CN/AcOHfrom 90/10/0.05 to 50/50/0.05
in 20 min and 10/90/0.05 in 50 min, flow rate 0.5 mL/min.
System B: linear gradient solvent system: H2O/CH3CN/
AcOHfrom 90/10/0.05 to 50/50/0.05 in 40 min and 0/
100/0.05 in 60 min, flow rate 0.5 mL/min. System C:
linear gradient solvent system: CH3CN/TBAP from 5/95
to 80/20 in 20 min, flow rate 1.0 mL/min. System D:
linear gradient solvent system: H2O/CH3CN/AcOH
from 80/20/0.05 to 60/40/0.05 in 5 min and 30/70/0.05 in
20 min, flow rate 1.0 mL/min. System E: linear gradient
solvent system: H2O/CH3CN from 95/5 to 0/100 in
30 min, flow rate 1.0 mL/min. Peaks were detected by
UV absorption with a diode array detector. All deriva-
tives tested for biological activity showed P97% purity
in the HPLC systems. Low-resolution mass spectra were
measured with a Finnigan–Thermoquest LCQ with
APCI (Atmospheric Pressure Chemical Ionization)
interface. Vaporizer heater was 480 ꢁC interfaced with
HP1100 LC apparatus (System A). Low-resolution and
high-resolution FAB (fast atom bombardment) mass
spectrometry was performed with a JEOL SX102 spec-
trometer with 6-kV Xe atoms following desorption from
a glycerol matrix.
4.4. (2R, 3S, 4S, 5R)-2-(2-Amino-6-methylamino-purin-9-
yl)-5-hydroxy-methyl-tetrahydrofuran-3,4-diol (4)
A solution of 22 (19 mg, 0.063 mmol) in 30% methyl-
amine in H2O (2.0 mL) was stirred at 60 ꢁC for 7 h. The
solvent was removed under reduced pressure and the
residue was purified by C18 reverse-phase silica gel
chromatography (eluted by a linear gradient of H2O/
MeCN ¼ 100/0 to 0/100), to give 4 (18 mg, 94%). 1H
NMR (DMSO-d6) d 7.94 (s, 1H), 5.90 (d, 1H,
J ¼ 6:6 Hz), 4.77–4.72 (m, 1H), 4.41 (dd, 1H, J ¼ 3:0,
5.4 Hz), 4.27 (dd, 1H, J ¼ 3:0, 6.0 Hz), 3.91 (dd, 1H,
J ¼ 2:7, 12.9 Hz), 3.82 (dd, 1H, J ¼ 3:3, 12.9 Hz), 3.04
(s, 3H); MS (m=e) (positive-FAB) 297 (M þ H)þ. HRMS
(positive-FAB) calcd for C11H17N6O4 (M þ H)þ
297.1311, found 297.1317; HPLC (System C) 5.5 min
(99%) (System E), 3.3 min (99%).
4.5. (2R, 3S, 4S, 5R)-2-(2-Aminomethyl-6-methylamino-
purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (5)
4.2. (2R, 3S, 4S, 5R)-2-(2-Cyano-6-methylamino-purin-9-
yl)-5-hydroxy-methyl-tetrahydrofuran-3,4-diol (2)
To a solution of 2 (2.4 mg, 0.0079 mmol) in THF
(0.50 mL) was added 1.0 M BH3–THF complex THF
solution (1.5 mL), and the mixture was stirred at 60 ꢁC
for 20 h. MeOH(2.0 mL) was added to the reaction
mixture, and the solvent was removed under reduced
pressure. The residue was purified on an Amberlite CG-
50 column (eluted by a linear gradient of ammonium
bicarbonate 0–1.0 M) and HPLC (Luna 5 l RP-C18(2)
column, eluted by a linear gradient of H2O/MeCN/
AcOH ¼ 100/0/0.05 to 80/20/0.05 in 15 min), to give 5 as
acetic acid salt (1.1 mg, 36%). 1HNMR (D 2O) d 8.23 (s,
1H), 6.10 (d, 1H, J ¼ 3:9 Hz), 4.56 (m, 1H), 4.44 (m,
1H), 4.26 (m, 1H), 4.20–4.12 (br, 2H), 3.92–3.73 (m,
2H), 3.12 (br s, 3H) 1.90 (s, 3H); MS (m=e) (CI) 311
(M þ H)þ (positive-FAB), 311 (M þ H)þ. HRMS (posi-
tive-FAB) calcd for C12H19N6O4 (M þ H)þ 311.1468,
found 311.1464; HPLC (System B) 5.1 min (99%) (Sys-
tem C), 2.4 min (99%).
A solution of 2.0 M MeNH2 in THF (2.0 mL) was added
to a solution of 25 (42 mg, 0.096 mmol) in THF
(0.80 mL), and the mixture was stirred at rt (room
temperature) for 26 h. MeOH(2.0 mL) was added, and
the reaction mixture was stirred at rt for 3 h. The solvent
was removed under reduced pressure and the residue
was purified by preparative TLC (silica gel, thickness
0.25 mm, solvent: CHCl3/MeOH ¼ 10/1) to give 2
1
(19 mg, 66%). HNMR (DMSO- d6) d 8.62 (s, 1H), 8.48
(br, 1H), 5.90 (d, 1H, J ¼ 5:7 Hz), 5.52 (d, 1H,
J ¼ 6:1 Hz), 5.23 (d, 1H, J ¼ 5:1 Hz), 5.05 (t, 1H,
J ¼ 5:7 Hz), 4.52 (m, 1H), 4.15 (m, 1H), 3.95 (m, 1H),
3.75–3.60 (m, 1H), 3.60–3.50 (m, 1H), 2.97 (d, 3H,
J ¼ 4:2 Hz); MS (m=e) (CI) 307 (M þ H)þ, HRMS
(positive-FAB) calcd for C12H15N6O4 (M þ H)þ

3002
M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
4.6. (2R, 3S, 4S, 5R)-9-(3,4-Dihydroxy-5-hydroxymethyl-
tetrahydrofuran-2-yl)-6-methylamino-9H-purine-2-carb-
oxamidine (6)
(1.2 mg, 20%). 1HNMR (DMSO- d6) d 8.51 (s, 1H), 7.34
(br s, 1H), 7.68 (m, 1H), 7.60 (m, 1H), 7.35 (m, 1H), 7.12
(m, 1H), 5.95 (m, 1H), 4.65 (br, 2H), 4.51 (m, 1H), 4.13–
4.10 (m, 2H), 3.92 (m, 2H); MS (m=e) (CI) 509
To a solution of 25 (21 mg, 0.047 mmol) was added to a
2.0 M MeNH2 THF solution (2.0 mL), and the mixture
was stirred at rt for 2 h. MeOH(0.5 mL) was added, and
the reaction mixture was stirred at 50 ꢁC for 2 h. The
solvent was removed under reduced pressure and the
residue was purified on a short column (silica gel 2.0 mL,
eluent: CHCl3/MeOH ¼ 10/1) and an Amberlite CG-50
column (eluted by linear gradient of ammonium bicar-
bonate 0–1.0 M), to give 6 (2.8 mg, 18%). 1HNMR
(D2O) d 8.61 (s, 1H), 6.20 (d, 1H, J ¼ 6:9 Hz), 4.99 (t,
1H, J ¼ 6:5 Hz), 4.49 (m, 1H), 4.21 (m, 1H) 3.82 (d, 2H,
J ¼ 3:6 Hz), 3.17 (s, 3H), 2.87 (s, 3H); MS (m=e) (CI)
338 (M þ H)þ (positive-FAB), 338 (M þ H)þ. HRMS
(positive-FAB) calcd for C13H20N7O4 (M þ H)þ
338.1577, found 338.1571; HPLC (System B) 12.1 min
(99%) (System C), 2.1 min (99%).
(M þ H)þ,
HRMS
(positive-FAB)
calcd
for
C18H18N6O4I (M þ H)þ 509.0434, found 509.0429;
HPLC (System B) 49.2 min (99%) (System C), 16.1 min
(99%) (System D), 11.6 min (99%).
4.9. (2R, 3S, 4S, 5R)-2-(6-(3-Iodo-benzylamino)-2-triflu-
oromethyl-purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-
3,4-diol (11)
To a solution of 29 (9.2 mg, 0.014 mmol) in MeOH
(1.0 mL) was added potassium carbonate (18.5 mg,
0.13 mmol), and the mixture was stirred at rt for 4 h. The
solvent was removed under reduced pressure, and the
residue was purified by column chromatography (silica
gel, eluent: CHCl3/MeOH ¼ 10/1) and preparative TLC
(silica gel, thickness 0.25 mm, solvent AcOEt), to give 11
(3.4 mg, 45%). 1HNMR (CDCl 3) d 7.98 (s, 1H), 7.76 (s,
1H), 7.63 (d, 1H, J ¼ 8:1 Hz), 7.37 (d, 1H, J ¼ 7:2 Hz),
7.08 (t, 1H, J ¼ 7:8 Hz), 6.65 (br s, 1H), 5.84 (d, 1H, J ¼
6:9 Hz), 4.99 (m, 1H), 4.80 (m, 2H), 4.49 (d, 1H, J ¼
4:5 Hz), 4.35 (s, 1H), 3.95 (dd, 1H, J ¼ 1:8, 12.6 Hz),
3.77 (d, 1H, J ¼ 12:6 Hz); MS (m=e) (positive-FAB)
552 (M þ H)þ. HRMS (positive-FAB) calcd for
C18H18N5O4F3I (M þ H)þ 552.0356, found 552.0336;
HPLC (System C) 17.8 min (99%) (System E), 8.2 min
(99%).
4.7. (2R, 3S, 4S, 5R)-2-(2-Iodo-6-(3-iodo-benzylamino)-
purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (9)
To a solution of 24 (18.5 mg, 0.034 mmol) in DMF
(0.10 mL) was added 3-iodobenzylamine (25 mg) and
i-Pr2NEt (0.10 mL), and the mixture was stirred at rt for
20 h. The solvent was removed under reduced pressure
and the residue was purified by column chromatography
(silica gel, eluent: AcOEt/petroleum ether ¼ 2/1), to give
triacetate (21 mg). To a solution of this triacetate
(21 mg) in MeOH(1.0 mL) was added 3 N NaOH
(0.10 mL), and the mixture was stirred at rt for 3 h.
The solvent was removed under reduced pressure, and
the residue was purified by short-column chromatogra-
phy (silica gel, eluent: AcOEt) and preparative TLC
4.10. (2R, 3S, 4S, 5R)-2-(2-Methoxycarbonyl-6-(3-iodo-
benzylamino)-purin-9-yl)-5-hydroxymethyl-tetrahydro-
furan-3,4-diol (12)
(silica gel, thickness 0.25 mm, solvent AcOEt), to give
1
To a solution of 26 (10.5 mg, 0.017 mmol) in i-PrOH
(0.60 mL), MeOH(0.05 mL) and THF (0.20 mL) was
added 3 N aqueous NaOH(0.040 mL), and the mixture
was stirred at rt for 5 h. The solvent was removed under
reduced pressure, and the residue was purified by col-
umn chromatography (silica gel, eluent: CHCl3/
MeOH ¼ 10/1), to give 12 (1.3 mg, 15%). 1HNMR
(DMSO-d6) d 8.74 (br s, 1H), 8.56 (s, 1H), 7.82 (br s,
1H), 7.58 (m, 1H), 7.39 (m, 1H), 7.10 (m, 1H), 5.93 (d,
1H, J ¼ 6:0 Hz), 5.46 (d, 1H, J ¼ 6:3 Hz), 5.22 (d, 1H,
J ¼ 4:5 Hz), 5.03 (t, 1H, J ¼ 4:5 Hz), 4.65 (m, 1H), 4.58
(br, 2H), 4.12 (m, 1H), 3.94 (m, 1H), 3.85 (s, 3H), 3.65
(m, 2H); MS (m=e) (CI) 542 (M þ H)þ, HRMS (positive-
FAB) calcd for C19H21N5O6I (M þ H)þ 542.0537, found
542.0540; HPLC (System A) 28.6 min (99%) (System C),
14.5 min (99%).
9 (11 mg, 53%). HNMR (CD OD) d 8.15 (s, 1H), 7.79
3
(s, 1H), 7.60 (d, 1H, J ¼ 7:8 Hz), 7.39 (d, 1H,
J ¼ 7:8 Hz), 7.09 (t, 1H, J ¼ 7:8 Hz), 5.89 (d, 1H,
J ¼ 6:3 Hz), 4.65 (m, 1H), 4.30 (dd, 1H, J ¼ 3:3,
5.1 Hz), 4.13 (m, 1H), 3.88 (dd, 1H, J ¼ 2:7, 12.6 Hz),
3.74 (dd, 1H, J ¼ 3:3, 12.6 Hz); MS (m=e) (positive-
FAB) 610 (M þ H)þ. HRMS (positive-FAB) calcd for
C17H18N5O4I2 (M þ H)þ 609.9448, found 609.9424;
HPLC (System C) 16.0 min (99%) (System E), 6.9 min
(99%).
4.8. (2R, 3S, 4S, 5R)-2-(2-Cyano-6-(3-iodo-benzylamino)-
purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (10)
To a solution of 26 (7.4 mg, 0.012 mmol) in MeCN
(1.00 mL) and H2O (0.030 mL) was added 3 N aqueous
NaOH(0.030 mL), and the mixture was stirred at rt for
6 h. Aqueous citric acid (5%, 0.050 mL) was added to
neutralize the reaction and the solvent was removed
under reduced pressure. The residue was purified by
column chromatography (silica gel, eluent: AcOEt then
CHCl3/MeOH ¼ 5/1) first and then purified again by
HPLC (Luna 5 l RP-C18(2) column, eluted by a linear
gradient of H2O/MeCN/AcOH ¼ 70/30/0.05), to give 10
4.11. (2R, 3S, 4S, 5R)-9-(3,4-Dihydroxy-5-hydroxy-
methyl-tetrahydrofuran-2-yl)-6-(3-iodo-benzylamino)-
9H-purine-2-carboxylic acid 3-iodo-benzylamide (13)
To a solution of 25 (9 mg, 0.020 mmol) in THF
(1.50 mL) was added 3-iodobenzylamine (19 mg,
0.082 mmol), and the mixture was stirred at 50 ꢁC for

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
3003
21.5 h. MeOH(2.0 mL) and potassium carbonate
4.14. (2R, 3S, 4S, 5R)-9-(3,4-Dihydroxy-5-hydroxy-
methyl-tetrahydrofuran-2-yl)-6-(5-chloro-2-methoxy-
benzylamino)-9H-purine-2-carboxylic acid (16)
(24 mg, 0.174 mmol) were added, and stirring continued
at rt for 3 h. The solvent was removed under reduced
pressure and the residue was purified by preparative
TLC (silica gel, thickness 0.25 mm, solvent CHCl3/
MeOH ¼ 5/1), to give 13 (11 mg, 76%). 1HNMR
(DMSO-d6) d 9.05 (br s, 1H), 8.84 (s, 1H), 7.80 (d, 2H,
J ¼ 5:4 Hz), 7.58 (d, 2H, J ¼ 8:4 Hz), 7.39 (d, 2H,
J ¼ 8:7 Hz), 7.09 (t, 2H, J ¼ 8:0 Hz), 6.10 (d, 1H,
J ¼ 5:4 Hz), 5.49 (d, 1H, J ¼ 6:0 Hz), 5.24 (d, 1H,
J ¼ 4:2 Hz), 4.89 (s, 2H), 4.76 (s, 2H), 4.55 (m, 1H), 4.17
(m, 1H), 3.98 (m, 1H), 3.65–3.50 (m, 2H); MS (m=e)
(positive-FAB) 743 (M þ H)þ. HRMS (positive-FAB)
calcd for C25H25N6O5I2 742.9976, found 742.9972;
HPLC (System A) 24.6 min (99%) (System B), 35.0 min
(99%) (System C), 12.9 min (99%).
To a solution of 15 (3.1 mg, 0.0069 mmol) in EtOH
(0.50 mL) was added 3 N NaOH(0.100 mL) and 30%
H2O2 (0.040 mL), and the mixture was stirred at rt for
4 h. The solvent was removed under reduced pressure,
and the residue was purified by Sephadex column
chromatography (eluted by a linear gradient of ammo-
nium bicarbonate 0–0.5 M), to give 16 (1.1 mg, 33%). 1H
NMR (D2O) d 8.32 (s, 1H), 7.40 (d, 1H, J ¼ 3:0 Hz),
7.33 (dd, 1H, J ¼ 3:0, 9.0 Hz), 7.04 (d, 1H, J ¼ 9:0 Hz),
6.12 (d, 1H, J ¼ 5:7 Hz), 4.88 (m, 1H), 4.73 (s, 2H), 4.44
(m, 1H), 4.30 (m, 1H), 3.93 (dd, 1H, J ¼ 2:7, 12.9 Hz),
3.88 (s, 3H), 3.83 (dd, 1H, J ¼ 2:7, 12.9 Hz); MS (m=e)
(CI) 466, 468 (M þ H)þ (relative peak height ratio was
3:1) (negative-FAB), 464, 466 (M ꢀ H)þ (relative peak
height ratio was 3:1). HRMS (negative-FAB) calcd for
C19H19N5O7Cl (M ꢀ H)þ 464.0973, found 464.0969;
HPLC (System B) 36.0 min (99%) (System C), 13.1 min
(99%).
4.12. (2R, 3S, 4S, 5R)-9-(3,4-Dihydroxy-5-hydroxy-
methyl-tetrahydrofuran-2-yl)-6-(5-chloro-2-methoxy-
benzylamino)-9H-purine (14)
Compound 14 (240 mg, 0.57 mmol) was prepared in
one step19 from 6-chloropurine riboside (180 mg,
0.64 mmol), 5-chloro-2-methoxy-benzylamine hydro-
chloride29 (200 mg, 0.96 mmol) and triethylamine
(120 lL, 0.83 mmol) in 88% yield. 1HNMR (DMSO- d6)
d 8.41 (s, 1H), 8.35 (br s, 1H), 8.18 (s, 1H), 7.24 (dd,
1H, J ¼ 2:5, 8.7 Hz), 7.04 (s, 1H), 7.00 (d, 1H), 5.89
(d, 1H, J ¼ 5:9 Hz), 5.37 (t, 1H, J ¼ 5:6 Hz), 5.20 (d,
1H, J ¼ 4:7 Hz), 4.63 (br, 3H), 4.14 (d, 1H, J ¼ 2:9 Hz),
3.95 (d, 1H, J ¼ 2:9 Hz), 3.83 (s, 3H), 3.66 (m, 2H), 3.54
(m, 2H); HRMS (ESI) calcd for C18H21ClN5O5
4.15. (2R, 3S, 4S, 5R)-9-(3,4-Dihydroxy-5-hydroxy-
methyl-tetrahydrofuran-2-yl)-6-(5-chloro-2-methoxy-
benzylamino)-9H-purine-2-carboxylic acid methyl ester
(17)
To a solution of 16 (1.0 mg, 0.0021 mmol) in MeOH
(0.30 mL) and H2O (0.10 mL) was added (trimethyl-
silyl)diazomethane (0.100 mL, 2.0 M in ether), and
the mixture was stirred at rt for 5 h. The solvent
was removed under reduced pressure, and the residue
was purified by preparative TLC (silica gel, thick-
(M þ H)þ:
422.1231,
(C18H20ClN5O5) C, H , N.
found
422.1223;
Anal.
ness 0.25 mm, solvent CHCl3/MeOH ¼ 10/1), to give
1
17 (0.5 mg, 50%). HNMR (CD OD) d 8.41 (s, 1H),
3
4.13. (2R, 3S, 4S, 5R)-9-(3,4-Dihydroxy-5-hydroxy-
methyl-tetrahydrofuran-2-yl)-6-(5-chloro-2-methoxy-
benzylamino)-9H-purine-2-carbonitrile (15)
7.54 (br s, 1H), 7.21 (dd, 1H, J ¼ 3:0, 8.7 Hz), 6.95
(d, 1H, J ¼ 8:7 Hz), 6.01 (d, 1H, J ¼ 6:0 Hz), 4.70 (m,
1H), 4.58 (s, 2H), 4.33 (m, 1H), 4.16 (m, 1H), 3.98
(s, 3H), 3.88 (s, 3H), 3.75 (m, 1H), 3.55 (m, 1H); MS
(m=e) (CI) 480, 482 (M þ H)þ (relative peak height
ratio was 3/1). HRMS (positive-FAB) calcd for
C20H23N5O7Cl (M þ H)þ 480.1286, found 480.1279;
HPLC (System A) 25.5 min (99%) (System C), 14.1 min
(99%).
To a solution of triacetate 25 (6.2 mg, 0.014 mmol) in
DMF (0.10 mL) was added 5-chloro-2-methoxybenzyl-
amine hydrochloride (7.0 mg, 0.034 mmol) and i-Pr2NEt
(0.10 mL), and the mixture was stirred at rt for 24 h. The
solvent was removed under reduced pressure, and the
residue was purified by short-column chromatography
(silica gel, eluent: AcOEt). The obtained material was
dissolved in MeOH(1.0 mL) and added potassium
carbonate (15 mg, 0.108 mmol), and the mixture was
stirred at rt for 6 h. The solvent was removed under
reduced pressure, and the residue was purified by col-
umn chromatography (silica gel, eluent: CHCl3/
4.16. (2R, 3S, 4S, 5R)-9-(3,4-Dihydroxy-5-hydroxy-
methyl-tetrahydrofuran-2-yl)-6-(2-phenyl-cyclopropyl-
amino)-9H-purine-2-carbonitrile (19)
To a solution of 25 (8.1 mg, 0.019 mmol) in DMF
(0.10 mL) was added trans-2-phenyl cyclopropylamine
hydrochloride (10.0 mg, 0.059 mmol) and i-Pr2NEt
(0.10 mL), and the mixture was stirred at rt for 26 h. The
solvent was removed under reduced pressure, and the
residue was purified by short-column chromatography
(silica gel, eluent: AcOEt). The obtained material
(5.7 mg) was dissolved in MeCN (1.00 mL) and H2O
(0.030 mL), and the mixture was treated with 3 N
NaOHaq (0.030 mL) and was stirred at rt for 4 h. 5%
citric acid aq (0.050 mL) was added to neutralize the
1
MeOH ¼ 10/1 then 5/1), to give 15 (5.2 mg, 82%). H
NMR (DMSO-d6) d 8.49 (s, 1H), 7.28 (d, 1H,
J ¼ 2:7 Hz), 7.23 (dd, 1H, J ¼ 2:7, 8.5 Hz), 6.97 (d, 1H,
J ¼ 8:5 Hz), 6.01 (d, 1H), 4.65 (t, 1H, J ¼ 5:4 Hz), 4.32
(t, 1H, J ¼ 4:7 Hz), 4.13 (s, 2H), 3.88 (m, 3H), 3.63 (s,
3H); MS (m=e) (CI) 447, 449 (M þ H)þ (relative peak
height ratio was 3:1), HRMS (positive-FAB) calcd for
C19H20N6O5Cl (M þ H)þ 447.1184, found 447.1188;
HPLC (System A) 31.2 min (99%) (System C), 15.6 min
(99%).

3004
M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
reaction, and the solvent was removed under reduced
pressure. The residue was purified by column chroma-
tography (silica gel, eluent: AcOEt), to give 19 (1.2 mg,
5.75 (t, 1H, J ¼ 5:0 Hz), 5.19 (br s, 2H), 4.49–4.36 (m,
3H), 2.15 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H); MS (m=e)
(positive-FAB) 428, 430 (M þ H)þ (relative peak height
ratio was 3:1).
1
15%). HNMR (CDCl ) d 8.02 (s, 1H), 7.34–7.02 (m,
3
5H), 6.44 (br s, 1H), 5.85 (d, 1H, J ¼ 6:9 Hz), 4.95 (m,
1H), 4.70 (m, 1H), 4.52 (m, 1H), 4.35 (m, 1H), 3.99
(m, 1H), 3.83 (m, 1H), 3.44 (br, 1H), 3.15 (br, 1H), 2.87
(m, 1H), 2.22 (m, 1H), 0.88 (m, 2H); (DMSO-d6) d 8.93
(br s, 1H), 8.64 (s, 1H), 7.32–7.10 (m, 5H), 5.90 (d, 1H,
J ¼ 5:7 Hz), 5.52 (d, 1H, J ¼ 5:7 Hz), 5.23 (d, 1H,
J ¼ 5:4 Hz), 5.04 (t, 1H, J ¼ 5:1 Hz), 4.50 (m, 1H), 4.14
(m, 1H), 3.95 (m, 1H), 3.65 (m, 1H), 3.57 (m, 1H), 3.10
(m, 1H), 2.16 (m, 1H), 1.45–1.30 (m, 2H); MS (m=e) (CI)
409 (M þ H)þ, HRMS (positive-FAB) calcd for
C20H21N6O4 (M þ H)þ 409.1624, found 409.1627;
HPLC (System A) 30.2 min (99%) (System B), 46.7 min
(99%) (System C), 15.3 min (99%).
4.19. (2R, 3S, 4S, 5R)-2-(6-Chloro-2-iodo-purin-9-yl)-3,4-
diacetoxy-5-acetoxymethyl-tetrahydrofuran (24)
To a solution of 23 (206 mg, 0.48 mmol) in MeCN
(0.50 mL) and CH2I2 (2.0 mL) was added t-butyl nitrite
(0.200 mL, 2.22 mmol), and the mixture was stirred at
80 ꢁC for 3 h. The reaction mixture was directly purified
by silica gel column chromatography (eluent: chloro-
form then AcOEt/petroleum ether ¼ 2/1), to give 2-iodo
1
compound 24 (178 mg, 69%). HNMR (CDCl ) d 8.20
3
(s, 1H), 6.21 (d, 1H, J ¼ 5:4 Hz), 5.78 (t, 1H,
J ¼ 5:6 Hz), 5.59 (t, 1H, J ¼ 5:0 Hz), 4.48 (t, 1H,
J ¼ 3:9 Hz), 4.42 (m, 2H), 2.18 (s, 3H), 2.15 (s, 3H), 2.11
(s, 3H); MS (m=e) (positive-FAB) 539, 541 (M þ H)þ
(relative peak height ratio was 3:1).
4.17. (2R, 3S, 4S, 5R)-9-(3,4-Dihydroxy-5-hydroxy-
methyl-tetrahydrofuran-2-yl)-6-(2-phenyl-cyclopropyl-
amino)-9H-purine-2-carboxylic acid (20) and 9-(3,4-
dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-6-(2-
phenyl-cyclopropyl-amino)-9H-purine-2-carboxamide
(21)
4.20. (2R, 3S, 4S, 5R)-2-(6-Chloro-2-cyano-purin-9-yl)-
3,4-diacetoxy-5-acetoxymethyl-tetrahydrofuran (25)
To a solution of 19 (4.4 mg, 0.0108 mmol) in EtOH
(0.50 mL) was added 3 N NaOH(0.100 mL) and 30%
H2O2 (0.050 mL), and the mixture was stirred at rt for
28 h. The solvent was removed under reduced pressure,
and the residue was purified by Sephadex column
chromatography (eluted by a linear gradient of ammo-
nium bicarbonate 0–0.5 M), to give 20 (1.0 mg, 21%) and
21 (1.2 mg, 26%). For 20: 1HNMR (D 2O) d 8.31 (s, 1H),
7.45–7.32 (m, 5H), 6.13 (d, 1H, J ¼ 5:7 Hz), 5.05–5.00
(m, 1H), 4.43 (t, 1H, J ¼ 4:8 Hz), 4.30 (m, 1H), 3.96–
3.81 (m, 2H) 3.35 (m, 1H), 2.34 (m, 1H), 1.51–1.41 (m,
2H); MS (m=e) (CI) 428 (M þ H)þ. HRMS (positive-
FAB) calcd for C20H22N5O6 (M þ H)þ 428.1570, found
A solution of Pd2 (dba)3–CHCl3 (50 mg, 0.048 mmol)
and tri-2-furylphosphine (150 mg, 0.646 mmol) in
tetramethylurea (0.50 mL) was stirred at 80 ꢁC for
30 min. The solution was cooled to rt and treated with
24 (110 mg, 0.204 mmol) and Zn(CN)2 (125 mg,
1.06 mmol) in tetramethyl urea (3.50 mL) and stirred at
80 ꢁC for 22 h. The reaction mixture was passed through
a short column (SiO2 3.0 mL and eluent AcOEt), and the
eluted fraction was evaporated to dryness. The residue
was purified twice by silica gel column chromatography
(first column eluent: AcOEt/petroleum ether ¼ 2/1,
second column eluent: AcOEt/petroleum ether ¼ 1/1), to
give 2-cyano compound 25 (52 mg, 59%). 1H
NMR (CDCl3) d 8.51 (s, 1H), 6.28 (d, 1H, J ¼ 5:7 Hz),
5.77 (t, 1H, J ¼ 5:7 Hz), 5.54 (dd, 1H, J ¼ 5:7, 3.9 Hz),
4.53 (dd, 1H, J ¼ 3:9, 7.5 Hz), 4.44 (m, 2H), 2.19 (s,
3H) 2.17 (s, 3H), 2.11 (s, 3H); MS (m=e) (CI) 438
(M þ H)þ.
428.1571; HPLC (System B) 34.4 min (99%) (System C),
1
13.5 min (99%). For 21: HNMR (D O) d 8.36 (s, 1H),
2
7.43–7.23 (m, 5H), 6.14 (d, 1H, J ¼ 5:7 Hz), 5.00–4.95
(m, 1H), 4.44 (t, 1H, J ¼ 4:5 Hz), 4.29 (m, 1H), 3.96–
3.80 (m, 2H) 3.07 (m, 1H), 2.15 (m, 1H), 1.47 (m, 2H);
MS (m=e) (CI) 427 (M þ H)þ. HRMS (positive-FAB)
calcd for C20H23N6O5 (M þ H)þ 427.1730, found
427.1733; HPLC (System B) 35.0 min (99%) (System C),
12.1 min (99%).
4.21. (2R, 3S, 4S, 5R)-2-(2-Cyano-6-(3-iodo-benzyl-
amino)-purin-9-yl)-3,4-diacetoxy-5-acetoxymethyl-
tetrahydrofuran (26)
4.18. (2R, 3S, 4S, 5R)-2-(2-Amino-6-chloro-purin-9-yl)-
3,4-diacetoxy-5-acetoxymethyl-tetrahydrofuran (23)
To a solution of 25 (8.3 mg, 0.019 mmol) in DMF
(0.10 mL) was added 3-iodobenzylamine hydrochloride
(9.0 mg, 0.033 mmol) and i-Pr2NEt (0.10 mL), and the
mixture was stirred at rt for 25 h. The solvent
was removed under reduced pressure, and the residue
was purified by short-column chromatography (silica
gel, eluent: AcOEt), to give triacetate 26 (10.5 mg, 87%).
1HNMR (CDCl 3) d 8.06 (s, 1H), 7.73 (s, 1H), 7.64
(d, 1H, J ¼ 7:8 Hz), 7.36 (d, 1H, J ¼ 7:8 Hz), 7.09 (t,
1H, J ¼ 7:8 Hz), 6.36 (br s, 1H), 6.18 (d, 1H,
J ¼ 5:7 Hz), 5.78 (t, 1H, J ¼ 5:6 Hz), 5.57 (t, 1H,
J ¼ 5:1 Hz), 4.80 (br, 2H), 4.45 (m, 1H), 4.40 (m, 2H),
To a solution of (ꢀ)-2-amino-6-chloropurine riboside 22
(995 mg, 3.30 mmol) in DMF (15 mL) was added pyr-
idine (2.0 mL, 25 mmol) and acetic anhydride (2.0 mL,
21 mmol), and the mixture was stirred at 80 ꢁC for 2 h.
The solvent was removed under reduced pressure and
the residue was purified by silica gel column chroma-
tography (eluent: AcOEt/petroleum ether ¼ 1/1 then 1/
0), to give 23 (1.26 g, 89%). 1HNMR (CDCl 3) d 7.87 (s,
1H), 6.01 (d, 1H, J ¼ 4:8 Hz), 5.96 (t, 1H, J ¼ 5:2 Hz),

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
3005
2.17 (s, 3H) 2.16 (s, 3H), 2.10 (s, 3H); MS (m=e) (CI) 635
4.25. Biological assays
(M þ H)þ.
4.25.1. Cell culture and membrane preparation. CHO
cells expressing recombinant human and rat A3ARs
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 lg/mL
streptomycin, 2 lmol/mL glutamine, and 800 lg/mL
geneticin. After harvest and homogenization, the cells
were centrifuged at 500g for 10 min. The pellet was
resuspended in 50 mM Tris–HCl buffer (pH 8.0) con-
taining 10 mM MgCl2 and 1 mM EDTA. The suspen-
sion was homogenized with an electric homogenizer for
10 s, and was then recentrifuged at 20,000g for 20 min at
4 ꢁC. The resulting pellets were resuspended in buffer
containing 3 units/mL of adenosine deaminase, and the
suspension was stored at ꢀ80 ꢁC prior to the binding
experiments. The rat A3AR was expressed recombi-
nantly via transfection in CHO cells, and the procedure
was the same as for the human subtype. The protein
concentration was measured using the Bradford assay.30
4.22. (2R, 3S, 4S, 5R)-Acetic acid 4-acetoxy-2-acetoxy-
methyl-5-(6-chloro-2-trifluoromethyl-purin-9-yl)-
tetrahydrofuran-3-yl ester (27)
To a suspension of activated Zn powder (140 mg,
2.20 mmol) in DMF (0.50 mL) was added CF2Br2
(0.10 mL, 1.08 mmol), and the mixture was stirred at rt
for 2.5 h. HMPA (0.40 mL) was added to the reaction
mixture, and it was cooled to 0 ꢁC. CuI (76 mg) was
added to the reaction mixture. A solution of 24 (76 mg,
0.141 mmol) in DMF (0.50 mL) was added at 0 ꢁC. The
reaction mixture was stirred at 50 ꢁC for 26.5 h and
filtered. The solvent was removed under reduced pres-
sure and the residue was purified by column chroma-
tography (silica gel, solvent AcOEt/petroleum
ether ¼ 2/1), to give 27 (52 mg, 77%). 1HNMR (CDCl )
3
d 9.09 (br s, 1H), 6.34 (d, 1H, J ¼ 5:4 Hz), 5.84 (t, 1H,
J ¼ 5:4 Hz), 5.62 (t, 1H, J ¼ 5:0 Hz), 4.55 (m, 1H), 4.45
(m, 2H), 2.20 (s, 3H), 2.15 (s, 3H), 2.09 (s, 3H); MS
(m=e) (positive-FAB) 481 (M þ H)þ.
4.25.2. Binding assay. For the A3AR binding experi-
ments, the procedures used were similar to those previ-
ously described.6 Briefly, each tube contained 100 lL of
membrane suspension, 50 lL of [125I]I-AB-MECA (final
concentration 0.5 nM), and 50 lL of increasing concen-
trations of compounds in Tris–HCl buffer (50 mM,
pH7.4) containing 10 mM MgCl 2, 1 mM EDTA. Non-
specific binding was determined using 10 lM NECA (50-
N-ethyluronamidoadenosine). The mixtures were incu-
bated at 25 ꢁC for 60 min. Binding reactions were ter-
minated by filtration through Whatman GF/B filters
under reduced pressure using a MT-24 cell harvester
(Brandel, Gaithersburg, MD). Filters were washed three
times with ice-cold buffer. Radioactivity was determined
in a Beckman 5500B c-counter. The binding of [3H]R-
PIA to the recombinant hA1AR and the binding of
[3H]CGS21680 to the recombinant hA2AAR was per-
formed as previously described.7;31
4.23. (2R, 3S, 4S, 5R)-Acetic acid 4-acetoxy-2-acetoxy-
methyl-5-(6-methylamino-2-trifluoromethyl-purin-9-yl)-
tetrahydrofuran-3-yl ester (28)
A solution of 27 (8.8 mg, 0.019 mmol) in 2 N methyl-
amine in THF (2.0 mL) was stirred at rt for 20 h and at
70 ꢁC for 24 h. The solvent was removed under reduced
pressure, and the residue was purified by column chro-
matography (silica gel, eluent: CHCl3/MeOH ¼ 5/1) and
preparative TLC (silica gel, thickness 0.25 mm, solvent
AcOEt/petroleum ether ¼ 1/1), to give 28 (2.6 mg, 29%).
1HNMR (CDCl 3) d 7.98 (s, 1H), 6.16 (d, 1H,
J ¼ 4:8 Hz), 6.04 (br s, 1H), 5.83 (t, 1H, J ¼ 5:3 Hz),
5.72 (t, 1H, J ¼ 5:1 Hz), 4.50–4.35 (m, 3H), 3.22 (s, 3H),
2.17 (s, 3H), 2.10(s, 3H), 2.09 (s, 3H); MS (m=e) (posi-
tive-FAB) 476 (M þ H)þ, 498 (M þ Na)þ.
4.25.3. Cyclic AMP accumulation assay. Intracellular
cyclic AMP levels were measured with a competitive
protein binding method.32 CHO cells expressing
recombinant human33 and rat34 ARs were harvested by
trypsinization. After resuspension in the medium, cells
were plated in 24-well plates in 0.5 mL medium/well.
After 24 h, the medium was removed and cells were
washed three times with 1 mL/well of DMEM, con-
4.24. (2R, 3S, 4S, 5R)-Acetic acid 4-acetoxy-2-acetoxy-
methyl-5-(6-(3-iodo-benzylamino)-2-trifluoromethyl-pu-
rin-9-yl)-tetrahydrofuran-3-yl ester (29)
To a solution of 27 (16.2 mg, 0.034 mmol) in DMF
(0.40 mL) was added 3-iodobenzylamine hydrochloride
(30 mg, 0.111 mmol) and i-Pr2NEt (0.10 mL), and the
mixture was stirred at rt for 24 h. The solvent was
removed under reduced pressure, and the residue was
purified by column chromatography (silica gel, solvent
AcOEt) and preparative TLC (silica gel, thickness
0.25 mm, solvent AcOEt/petroleum ether ¼ 1/1), to give
29 (9.5 mg, 42%). 1HNMR (CDCl 3) d 8.00 (s, 1H), 7.78
(s, 1H), 7.63 (d, 1H, J ¼ 8:0 Hz), 7.38 (d, 1H,
J ¼ 8:0 Hz), 7.08 (t, 1H, J ¼ 8:0 Hz), 6.31 (br s, 1H),
6.16 (d, 1H, J ¼ 5:1 Hz), 5.84 (t, 1H, J ¼ 5:3 Hz), 5.71
(t, 1H, J ¼ 5:1 Hz), 4.80 (br, 2H), 4.50–4.30 (m, 3H),
2.16 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H); MS (m=e) (po-
sitive-FAB) 678 (M þ H)þ, 700 (M þ Na)þ.
taining
50 mM
N-2-hydroxyethylpiperazine-N0-2-
ethanesulfonic acid, pH7.4. Cells were then treated with
agonists and/or test compounds in the presence of roli-
pram (10 lM) and adenosine deaminase (3 units/mL)
and incubated at 37 ꢁC. For A2A and A2BARs, incuba-
tion was carried out for 1 h. For A1 and A3ARs, after
45 min forskolin (10 lM) was added to the medium, and
incubation was continued for an additional 15 min. The
reaction was terminated upon removal of the medium,
and the cells were lysed with 200 lL/well of 0.1 M ice-
cold HCl. The cell lysate was resuspended and stored at
ꢀ20 ꢁC. For determination of cyclic AMP production,

3006
M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
protein kinase A (PKA) was incubated with [3H]cyclic
AMP (2 nM) in K2HPO4/EDTA buffer (K2HPO4,
150 mM; EDTA, 10 mM), 20 lL of the cell lysate, and
30 lL 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
liquid scintillation spectrometry.
thank Dr. Tom Spande and Dr. Victor Livengood
(NIDDK) for mass spectral measurements.
References and notes
1. Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz,
K. N.; Linden, J. Pharmacol. Rev. 2001, 53, 527.
2. Liang, B. T.; Jacobson, K. A. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 6995.
3. von Lubitz, D. K.; Lin, R. C.; Popik, P.; Carter, M. F.;
Jacobson, K. A. Eur. J. Pharmacol. 1994, 263, 59.
4. Fishman, P.; Bar-Yehuda, S. Curr. Top. Med. Chem. 2003,
3, 463.
4.26. Statistical analysis
Binding and functional parameters were estimated with
GraphPAD Prism software (GraphPAD, San Diego,
CA). IC50 values obtained from competition curves were
converted to Ki values using the Cheng–Prusoff equa-
tion.35 Data were expressed as mean Æ standard error.
5. van Tilburg, E. W.; van der Klein, P. A.; von Frijtag
€
Drabbe Kunzel, J.; de Groote, M.; Stannek, C.; Lorenzen,
A.; IJzerman, A. P. J. Med. Chem. 2001, 44, 2966.
6. 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.
7. Gao, Z. G.; Blaustein, J.; Gross, A. S.; Melman, N.;
Jacobson, K. A. Biochem. Pharmacol. 2003, 65, 1675.
8. Bruns, R. F. Can. J. Physiol. Pharmacol. 1980, 58, 673.
9. Nair, V.; Fasbender, A. J. C-2 Tetrahedron 1993, 49,
2169.
10. Tnaji, K.; Higashino, T. Heterocycles 1990, 30, 435.
11. Gundersen, L.-L. Acta Chemica Scand. 1996, 50, 58.
12. Burton, D. J.; Wiemer, D. M. J. Am. Chem. Soc. 1985,
107, 5014.
13. Wiemer, D. M.; Burton, D. J. J. Am. Chem. Soc. 1986,
108, 832.
4.27. Molecular modeling
All calculations were performed on a Silicon Graphics
Octane workstation (300 MHz MIPS R12000 (IP30)
processor). All ligand structures were modified from the
lowest-energy conformation of Cl-IB-MECA6 with the
Sketch Molecule of SYBYL 6.9.36 In all cases, MMFF
force field37 and charge were applied using distance-
dependent dielectric constants and conjugate gradient
ꢀ1
ꢀ
A .
method until the gradient reached 0.05 kcal molꢀ1
14. Mogensen, J. P.; Roberts, S. M.; Bowler, A. N.; Thomsen,
C.; Knutsen, L. J. Bioorg. Med. Chem. Lett. 1998, 8,
1767.
15. DeNinno, M. P.; Masamune, H.; Chenard, L. K.; DiRico,
K. J.; Eller, C.; Etienne, J. B.; Tickner, J. E.; Kennedy,
S. P.; Knight, D. R.; Kong, J.; Oleynek, J. J.; Tracey, W.
R.; Hill, R. J. J. Med. Chem. 2003, 46, 353.
16. Jacobson, K. A.; Gao, Z. G.; Chen, A.; Barak, D.; Kim, S.
A.; Lee, K.; Link, A.; Van Rompaey, P.; Van Calenbergh,
S.; Liang, B. T. J. Med. Chem. 2001, 44, 4125.
17. Hutchinson, S. A.; Baker, S. P.; Scammells, P. J. Bioorg.
Med. Chem. 2002, 10, 1115.
18. Volpini, R.; Costanzi, S.; Lambertucci, C.; Taffi, S.;
Vittori, S.; Klotz, K. N.; Cristalli, G. J. Med. Chem.
2002, 45, 3271.
A hA3AR model (PDB code: 1o74) constructed by
homology to the X-ray structure of bovine rhodopsin
38
ꢀ
with 2.8-A resolution was used for the docking study.
Compounds 1, 2, 7, 10, 14, and 15 in Table 1 were
docked within the hA3AR model. The atom types of all
ligands were manually assigned with Amber all-atom
force field,39 and their charges were calculated before
docking. The starting geometry of ligand conformation
was chosen from the hA3AR complex model with Cl-IB-
MECA, which was already validated by point muta-
tion.6 The ribose-binding position of this series was
fixed, using an atom-by-atom fitting method for the
carbon atoms of the ribose ring. To determine the
binding region of N6 derivatives and C-2 substituents,
19. Kim, S.-K.; Gao, Z.-G.; Van Rompaey, P.; Gross, A. S.;
Chen, A.; Van Calenbergh, S.; Jacobson, K. A. J. Med.
Chem. 2003, 46, 4847.
20. Gao, Z.-G.; Kim, S.-K.; Gross, A. S.; Chen, A.; Blaustein,
J. B.; Jacobson, K. A. Mol. Pharmacol. 2003, 63, 1021.
0
the flexible bonds, v1 (O–C1 –N9–C4) and t0 (C5–C6–N6–
C) angles were variously searched in the putative bind-
ing cavity. v1 was rotated by ꢀ60ꢁ, ꢀ110ꢁ, and ꢀ160ꢁ
within the anti-conformational range and was rotated in
30ꢁ increments. Several conformations without any ste-
ric bump were selected for further optimization. The
initial structures of all complexes were optimized using
the Amber force field with fixed dielectric conꢀst1ant of 4.0
€
21. Gao, Z.-G.; Chen, A.; Barak, D.; Kim, S.-K.; Muller,
C. E.; Jacobson, K. A. J. Biol. Chem. 2002, 277, 19056.
22. The relative energy differences for the 1/hA3AR complex
according to various t0 and v1 angles from the six different
clusters was very small, that is within 2 kcal/mol energy,
except for one cluster, which had 3.6 kcal/mol higher
energy, whereas its 2-cyano analogue 2 displayed two
clusters showing a wider distribution of 10 kcal/mol (data
not shown). In addition, in the docking study of 2-cyano
analogues having larger N6-(3-I)benzyl and (5-Cl-2-
MeO)benzyl substituents, 10 and 15, most of the clusters
displayed a higher relative energy for six different com-
plexes. It was suggested the N6-methyladenosine intro-
duced more conformational flexibility in the N6 binding
site than the analogues having bulky N6-substituents, for
which there was more conformational rigidity due to the
additional binding of the phenyl ring and its substituent(s).
and terminating gradient of 0.1 kcal molꢀ1
A
.
ꢀ
Acknowledgements
M.O. thanks Toray Industries (Tokyo, Japan) for
financial support. We thank Prof. Joel Linden (Uni-
versity of Virginia, Charlottesville) for the gift of CHO
cells expressing the human A1AR and A2AAR. We

M. Ohno et al. / Bioorg. Med. Chem. 12 (2004) 2995–3007
3007
23. Thompson, R. D.; Secunda, S.; Daly, J. W.; Olsson, R. A.
J. Med. Chem. 1991, 34, 3388.
32. Nordstedt, C.; Fredholm, B. B. Anal. Biochem. 1990, 189,
231.
24. Daly, J. W.; Padgett, W. Biochem. Pharmacol. 1992, 43,
1089.
33. Salvatore, C. A.; Jacobson, M. A.; Taylor, H. E.; Linden,
J.; Johnson, R. G. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
10365.
34. Zhou, Q. Y.; Li, C.; Olah, M. E.; Johnson, R. A.; Stiles,
G. L.; Civelli, O. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
7432.
35. Cheng, Y.-C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
36. Sybyl Molecular Modeling System, version 6.9; Tripos
Inc., St. Louis, Missouri 63144, USA.
25. Kim, H. O.; Ji, X.-D.; Siddiqi, S. M.; Olah, M. E.; Stiles,
G. L.; Jacobson, K. A. J. Med. Chem. 1994, 37, 3614.
26. Zablocki, J.; Palle, V.; Blackburn, B.; Elzein, E.; Nudel-
man, G.; Gothe, S.; Gao, Z.; Li, Z.; Meyer, S.; Belard-
inelli, L. Nucleos. Nucleot. Nucleic Acids 2001, 20, 343.
27. Matsuda, A.; Shinozaki, M.; Yamaguchi, T.; Homma, H.;
Nomoto, R.; Miyasaka, T.; Watanabe, Y.; Abiru, T.
J. Med. Chem. 1992, 35, 241.
28. Tracey, W. R.; Magee, W. P.; Oleynek, J. J.; Hill, R. J.;
Smith, A. H.; Flynn, D. M.; Knight, D. R. Am. J. Physiol.
Heart Circ. Physiol. 2003, 285, H2780.
29. Yu, G.; Mason, H. J.; Wu, X.; Endo, M.; Douglas, J.;
Macor, J. E. Tetrahedron Lett. 2001, 42, 3247.
37. Halgren, T. A. J. Comput. Chem. 1999, 20, 730.
38. Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.;
Motoshima, H.; Fox, B. A.; Le Trong, I.; Teller, D. C.;
Okada, T.; Stenkamp, T. E.; Yamamoto, M.; Miyano, M.
Science 2000, 289, 739.
30. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
31. Hutchison, A. J.; Williams, M.; de-Jesus, R.; Yokoyama,
R.; Oei, H. H.; Ghai, G. R.; Webb, R. L.; Zoganas, H. C.;
Stone, G. A.; Jarvis, M. F. J. Med. Chem. 1990, 33, 1919.
39. Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.;
Merz, K. M., Jr.; Ferguson, D. M.; Spellmeyer, D. C.;
Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem.
Soc. 1995, 117, 5179.