Design, synthesis, and biological activity of N6-substituted-4′-thioadenosines at the human A3 adenosine receptor

Article abstract of DOI:10.1016/j.bmc.2006.03.030

A large series of N⁶-substituted-4′-thioadenosines were synthesized starting from d-gulonic-γ-lactone, and structure-activity relationships were studied at the human A₃ and other subtypes of adenosine receptors (ARs). 2-Chloro-substi

Full text of DOI:10.1016/j.bmc.2006.03.030

Bioorganic & Medicinal Chemistry 14 (2006) 4718–4730
6
Design, synthesis, and biological activity of N -substituted-4 -
0
thioadenosines at the human A adenosine receptor
3
a,
b
a
a
c
*
Lak Shin Jeong, Hyuk Woo Lee, Hea Ok Kim, Ji Young Jung, Zhan-Guo Gao,
c
c
c
a
Heng T. Duong, Srikar Rao, Kenneth A. Jacobson, Dae Hong Shin,
Jeong A Lee, Prashantha Gunaga, Sang Kook Lee, Dong Zhe Jin,
a
a
a
b
b
d
Moon Woo Chun and Hyung Ryong Moon
a
Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
b
College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes,
c
and Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD 20892, USA
d
Pusan National University, Busan 609-753, Republic of Korea
Received 22 February 2006; revised 15 March 2006; accepted 16 March 2006
Available online 5 April 2006
6
0
Abstract—A large series of N -substituted-4 -thioadenosines were synthesized starting from D-gulonic-c-lactone, and structure–ac-
tivity relationships were studied at the human A
analogues were compared. 2-Chloro-N -methyl-4 -thioadenosine 19b was a highly potent and selective agonist (K
3
and other subtypes of adenosine receptors (ARs). 2-Chloro-substituted and 2-H
= 0.8 ± 0.1 nM in
6
0
i
3
binding) at the A AR, and displayed the same relative efficacy in receptor activation as a known full agonist, Cl-IB-MECA. Most of
6
N -substituted-4 -thioadenosines were less potent in binding than the corresponding N -substituted-adenosines or N -substituted-4 -
0
6
6
0
0
6
thioadenosine-5 -uronamides. N -(3-Iodobenzyl) derivative 19g was demonstrated to be an A
ing a K value of 3.2 nM.
2006 Elsevier Ltd. All rights reserved.
3
AR-selective partial agonist display-
i
ꢀ
1
1
1
. Introduction
agonists of this receptor are 2 (Cl-IB-MECA) and its
0
12
0
4 -thio analogue 3 (LJ-529). Adenosine-5 -uronamide
derivatives 1–3 and other highly selective A AR ago-
Extracellular adenosine acts as a signaling molecule by
activating four subtypes (A , A , A , and A ) of aden-
osine receptors (ARs). The structure–activity relation-
ships of adenosine derivatives acting at the four
subtypes have been explored in detail. Several selective
3
1
3
nists, such as CP-608039, contain the potency- and
0
1
2A
2B
3
1
efficacy-enhancing 5 -alkyluronamide moiety.
6
The exploration of N -substitution of adenine-9-riboside
A AR agonists are currently in clinical trials for the treat-
3
ment of cancer, pulmonary diseases, pain, and other
2
diseases.
derivatives, for example, 4–7, has provided new leads
4–16
1
into the design of A AR-selective agonists.
Binding
affinity and relative efficacy at the human A AR of a
3
3
6
wide range of N -substituted adenosine derivatives were
Agonists acting at the A AR are associated with cardio-
3
studied in intact Chinese hamster ovary (CHO) cells
1
4
protective, cerebroprotective, and cytostatic proper-
–7
stably expressing A and other ARs. At the human
3
3
6
A AR, small groups such as N -methyladenosine (4)
ties.
was 1 (IB-MECA), which is currently in clinical trials
The first selective A AR agonist to be reported
3
3
6
enhanced affinity, although N -methyl analogues are
6
nearly inactive at the rat A AR. N -Cyclopropylmethyl-
for the treatment of colon carcinoma and rheumatoid
8
3
–10
arthritis (Chart 1).
Among the most selective
adenosine (5) was equipotent to 4 as a full agonist at the
human A AR but demonstrated significantly enhanced
3
6
affinity at the A AR. The N -(3-iodobenzyl) derivatives,
1
Keywords: G protein-coupled receptor; Nucleoside; Radioligand bind-
ing; Purines; Partial agonist; Antagonist.
known to enhance the affinity at both the rat and human
0
*
Corresponding author. Tel.: +82 2 3277 3466; fax: +82 2 3277
851; e-mail: lakjeong@ewha.ac.kr
A AR and contained in compound 6 and 5 -uronamide
3
2
derivatives 1–3, also led to a reduction in efficacy in the
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.030

L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
4719
Chart 1. Binding affinity of the adenosine derivatives at human adenosine receptors.ꢀ
0
acterization of a large series of 4 -thioadenosine deriv-
atives (Fig. 1).
2. Results and discussion
2.1. Chemistry
0
Synthesis of the desired 4 -thioadenosine derivatives
started from D-gulonic-c-lactone (8), as shown in
Scheme 1.
0
Figure 1. Structures of the target 4 -thionucleosides.
D-Gulonic-c-lactone (8) was converted to the glycosyl
1
2
donor 9 according to the reported procedure developed
by our laboratory. Direct condensation of 9 with 6-chlo-
ropurine or 2,6-dichloropurine in the presence of
TMSOTf and triethylamine in a solution of acetonitrile
and 1,2-dichloroethane afforded the b-anomers 10 or 12
with minor formation of a-anomers 11 or 13, respective-
0
6
-OH series. Other bulky substitutions at the N -position
5
of the adenine moiety, including other benzyl substitu-
tions, 2,2-diphenylethyl, and various cycloalkyl groups,
were found to decrease the maximum agonist efficacy at
1
2,17
6
the A AR.
However, N -(trans-2-phenylcyclopro-
pyl)adenosine (7) exhibited full agonism at the human
3
3
ly. The initially formed N -isomer was smoothly convert-
9
ed to the N -isomer upon heating to 80 ꢁC, as reported by
Chu and co-workers. Anomeric configurations of 10
A AR and greatly enhanced affinity and selectivity.
3
19
A 2-chloro substituent was demonstrated to further de-
crease the A AR efficacy in combination with N -
1
and 11 were readily assigned by H NMR NOE experi-
6
3
0
ments. Irradiation of 1 -H of compound 10 gave NOE
0
1
8
substitution.
on its 4 -H, indicating b-anomer, while no NOE was
observed on the same experiment in the case of com-
pound 11, resulting in a-anomer. The same pattern was
observed in the case of 12 and 13. Removal of the benzoyl
group of 10 and 12 was achieved using methylmagnesium
iodide in THF to give 14 and 15, respectively. Treatment
with sodium methoxide or methanolic ammonia, typical-
ly used for the deprotection of a benzoyl group, resulted
in the formation of 6-methoxy or 6-aminopurine deriva-
tive. Treatment of 14 and 15 with 80% aqueous acetic
acid at 70 ꢁC gave the triols 16 and 17, respectively.
While the structure–activity relationships (SARs) have
0
0
been well explored within the 4 -oxo series of 5 -OH
derivatives with respect to both affinity and relative
0
efficacy, the SAR within the 4 -thio series is not yet
well developed. Although there is a bioisosteric ratio-
nale for the replacement of the ring oxygen with sul-
fur, there may be differences in the effects on
potency, selectivity, and efficacy. Thus, in the search
for improved A AR agonists or partial agonists, herein
3
we describe the synthesis and in vitro biological char-
6
Substitution of N -positions of 16 and 17 with various
primary and secondary amines for the synthesis of the
final nucleosides 18a–v and 19a–n, respectively, is shown
in Scheme 2. The amines used in this substitution were
ꢀ
K
i
values (nM) in binding to A
1 3
A2A, and A adenosine receptors
(human, unless noted as rat, r).

4720
L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
Scheme 1. Reagents and conditions: (a) 6-Chloropurine or 2,6-dichloropurine, TMSOTf, ET
MeMgI in ether, THF, rt, 4 h; (c) 80% AcOH, 70 ꢁC, 12 h.
3 3 2 2
N, CH CN-ClCH CH Cl, rt to 80 ꢁC; (b) 3.0 M
0
alkyl-, cycloalkyl-, arylalkyl-, and cyclic-amines. In to-
tal, 36 final nucleoside derivatives were prepared as
shown in Scheme 2.
4 -thioadenosine series (X = H), compound 18h
showed binding
(K = 1.9 ± 0.4 nM) at the human A AR with selec-
the
highest
affinity
i
3
tivity over the human A1 and A2AARs by 24- and
300-fold, respectively. The primary amine-substituted
2
.2. Biological activity
6
6
0
N -alkyl or N -cycloalkyl-4 -thioadenosine derivatives
(18b–18f) exhibited moderate binding affinity at the
human A AR, while the derivatives (18s–18v) substi-
All AR experiments were performed using adherent
Chinese hamster ovary (CHO) cells stably transfected
with cDNA encoding the human ARs. Binding at the
human A AR in this study was carried out using [ I]I-
AB-MECA (N -(4-amino-3-iodobenzyl)adenosine-5 -N-
ethylcarboxamidoadenosine) as a radioligand. In cases
of weak binding, the percent inhibition of radioligand
3
2
0
tuted with secondary amines, such as piperidine,
piperazine, and morpholine, were totally devoid of
activity at the human ARs, indicating that, similar
1
25
3
6
0
0
to 4 -oxoadenosine derivatives, at least one hydrogen
atom at the N -position is essential for hydrogen
6
binding to the human A AR was determined at
3
bonding in the binding site of the AR. In the 3-hal-
ogen-substituted benzylamine series 18g–18l, the larg-
er halogen atom, the higher binding affinity was
observed. In the arylalkyl series, monophenyl-substi-
tuted alkylamines such as 18n–18p showed high affin-
1
human A AR (inhibition of adenylate cyclase in
0 lM. Furthermore, the percent activation of the
3
comparison to the full agonist 2) was determined at
3
1
N -(2-phenylisopropyl)adenosine) or A2AAR (using
0 lM. Binding at the human A AR (using [ H]R-PIA,
1
6
ity at the human A AR, while diphenyl-substituted
3
3
[ H]CGS2 1680, (2-[p-(2-carboxyethyl)phenylethylami-
0
no]-5 -N-ethylcarboxamido-adenosine)) was carried out
alkylamines such as 18q and 18r lost their binding
at the human A AR possibly due to the steric
3
as described in Section 3.1.
repulsion at the binding site despite a favorable
hydrophobic interaction. In comparison with the
6
As shown in Table 1, a variety of N -alkyl, cyclo-
alkyl, and arylalkyl substituents in 4 -thioadenosine
derivatives have produced nanomolar binding affinity
0
0
4 -oxo series, 4–7, the corresponding 4 -thio
derivatives 18b, 18f, 18h, and 18p were found to
exhibit lower binding affinity at the human A AR.
0
3
at the human A AR subtype, among which 19b
3
1
2
0
(
affinity (K = 0.8 ± 0.1 nM) at this AR subtype.
R = CH NH, X = Cl) exhibited the highest binding
In the case of 2-chloro-4 -thioadenosine series (X = Cl),
the order of compounds showing high binding affinity at
3
i
Compound 19b was selective for the human A AR
3
the A AR is as follows: 19b (R = CH NH, K = 0.8 ±
i
3
3
versus the human A AR by 790-fold and showed
1
0.1) > 19m (R = trans-2-phenyl-cyclopropyl-NH, K =
i
almost no measurable binding affinity at the human
A2AAR (4% inhibition at 10 lM). Within the
1.9 ± 0.4) > 19g (R = 3-iodobenzyl-NH, K = 3.2 ± 0.9)
i
> 19j (R = phenethyl-NH, K = 4.40 ± 0.33) ꢀ 19k (R =
i

L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
4721
Scheme 2.
6
human A AR. From this study, 2-chloro-N -methyl-
3
K = 4.9 ± 1.3). Compounds with bulky substituents such
-fluoro-phenethyl-NH, K = 4.7 ± 1.6) ꢀ 19a (R = NH ,
i
2
3
0
4 -thioadenosine (19b) was discovered as the most po-
tent and selective full agonist (K = 0.8 ± 0.1 nM) at
i
as 19h (R = a-naphthylmethyl-NH), 19l (R = 1,2-diphe-
nylethyl-NH), and 19n (R = 3,3-diphenylpropyl-NH) lost
their binding to the A AR, while substituted-benzylamine
i
6
the human A AR. Most of N -substituted-4 -thioadeno-
0
3
sines were less potent than the corresponding
3
6
N -substituted-adenosines or N -substituted-4 -thioade-
6
0
analogues, such as 19d–19f with the exception of 19g, were
found to show moderate binding affinity at the human
A AR. Among compounds (18h, 18i, 18n, 18p, 19a, 19b,
3
0
nosine-5 -uronamides. It was also revealed that, similar
0
to 4 -oxoadenosine derivatives, at least one hydrogen
atom at the N -position is essential for hydrogen
6
19g, 19j, 19k, and 19m) showing high binding affinity at
the human A AR, compounds 18p and 19m substituted
bonding in the binding site of the AR, and bulky
substituents at the N -position reduced binding affinity.
3
6
with N -(trans-2-phenylcyclopropyl)amino group and
6
6
9b substituted with a N -methylamino group were found
1
to be full agonists at the human A AR.
These findings may provide good insights into the
identification of the binding interaction between
nucleoside derivatives and ARs.
3
Other compounds exhibited partial agonism at the
human A AR. For example, N -benzyl 19d and
6
3.1. Experimental section
3
6
N -(2-ethyloxybenzyl) 19f derivatives were partial
1
agonists with >100-fold selectivity for the A AR.
3
H NMR spectra (CDCl , CD OD, or DMSO-d ) were
6
3
3
Higher affinity (3.2 nM) was observed for the
A AR-selective partial agonist, N -(3-iodobenzyl)
derivative 19g.
recorded on Varian Unity Inova 400 MHz. Chemical
shifts were reported in ppm units with TMS as the inter-
nal standard. C NMR spectra (CDCl , CD OD, or
3 3
6
3
1
3
DMSO-d ) were recorded on Varian Unity Inova
6
100 MHz. Optical rotations were determined on Jasco
3
. Conclusions
III in methanol. UV spectra were recorded on U-3000
made by Histachi in methanol. Elementary analyses
were measured on EA1110. The crude products were
purified using a silica gel 60 (230–400 mesh, Merck).
We have established structure–activity relationships of a
6
large series of N -substituted-4 -thioadenosines at the
0

4722
L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
0
Table 1. Potency of 4 -thioadenosine derivatives at human A
a
cells
1
, A2A, and A
3
ARs and maximal agonist effects at human A
3
ARs expressed in CHO
R
N
N
N
N
X
HO
S
OH OH
a
a
a
Compound
R
X
K
i
(hA
1
AR) nM
K
i
(hA2AAR) nM
K
i
(hA
3
AR) nM
% Activation
b
or % displ. at 10 lM or % displ. at 10 lM or % displ. at 10 lM (hA
3
AR) at 10 lM
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
8a
8b
8c
8d
8e
8f
NH
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5240 ± 740
359 ± 69
22.5 ± 0.3
15.6 ± 4.0
0%
5740 ± 980
26%
40%
445 ± 54
10.3 ± 0.7
45.2 ± 5.8
48.0 ± 4.9
65.3 ± 3.6
22.9 ± 0.8
155 ± 33
1.9 ± 0.4
6.7 ± 0.4
13.9 ± 5.7
57.6 ± 12.9
32.7 ± 6.7
42.2 ± 13.0
5.6 ± 1.1
11.3 ± 0.6
6.6 ± 2.9
1080 ± 70
1650 ± 150
21%
0
CH –NH
3
60 ± 11
78 ± 6
91 ± 2
99 ± 4
96 ± 4
87 ± 7
60 ± 4
62 ± 3
48 ± 9
63 ± 11
29 ± 7
72 ± 4
86 ± 5
89 ± 9
114 ± 8
54 ± 3
0 ± 3
Cyclopropyl-NH
Cyclobutyl-NH
4950 ± 160
28%
23%
3-Methyl-butyl-NH
Cyclopropyl-methyl-NH
Benzyl-NH
7%
8g
8h
8i
597 ± 8
45.9 ± 2.1
91.8 ± 6.0
179 ± 19
651 ± 110
430 ± 37
129 ± 23
82.6 ± 6.8
83.4 ± 6.3
136 ± 13
504 ± 77
1950 ± 40
25%
2110 ± 740
575 ± 75
995 ± 206
3610 ± 1110
13%
3-Iodo-benzyl-NH
3-Chloro-benzyl-NH
3-Methyl-benzyl-NH
3-Fluoro-benzyl-NH
3-(Trifluoro-methyl)-benzyl-NH
Naphth-1-yl-methyl-NH
2-Phenethyl-NH
8j
8k
8l
42%
8m
8n
8o
8p
8q
8r
8s
8t
206 ± 15
507 ± 31
774 ± 86
2330 ± 700
1500 ± 400
911 ± 209
21%
3-Fluoro-phenethyl-NH
trans-2-Phenyl-cyclopropyl-NH
1,2-Diphenyl-ethyl-NH
3,3-Diphenyl-propyl-NH
Piperidine
0
4-Benzyl-piperidine
4-(4-Fluoro-benzyl)-piperazine
Morpholine
0%
0%
5%
26%
10%
22%
0
8u
8v
9a
9b
9c
9d
9e
9f
0
26%
19%
2440 ± 310
4%
16%
0
c
NH
CH
2
Cl 47%
Cl
4.9 ± 1.3
0.8 ± 0.1
94.4 ± 29.2
18.2 ± 2.6
48.9 ± 16.6
17.2 ± 2.2
3.2 ± 0.9
268 ± 185
50.4 ± 26.5
4.40 ± 0.33
4.7 ± 1.6
1300 ± 610
1.9 ± 0.4
720 ± 193
64 ± 18
96 ± 5
68 ± 3
63 ± 4
62 ± 4
60 ± 2
32 ± 7
45 ± 5
112 ± 0
81 ± 9
71 ± 7
38 ± 5
102 ± 4
0
3
–NH
629 ± 168
Cyclopentyl-NH
Benzyl-NH
Cl 32.0 ± 6.5
Cl 2280 ± 180
1410 ± 140
47%
2-Methyl-benzyl-NH
2-Ethyloxy-benzyl-NH
Cl 853 ± 133
Cl 2570 ± 630
3280 ± 170
4630 ± 510
1190 ± 290
1340 ± 160
34%
c
9g
9h
9i
3-Iodo-benzyl-NH
Cl
Cl
Cl
Cl
Cl
554 ± 64
581 ± 36
474 ± 199
139 ± 71
294 ± 33
a-Naphthylmethyl-NH
Fluoren-9-yl-methyl-NH
2-Phenethyl-NH
9j
1460 ± 20
1650 ± 110
3910 ± 690
1930 ± 320
3790 ± 320
9k
9l
3-Fluoro-phenethyl-NH
1,2-Diphenylethyl
Cl 3320 ± 770
856 ± 155
Cl 38%
9m
9n
trans-2-Phenyl-cyclopropyl-NH Cl
3,3-Diphenyl-propyl-NH
a
All AR experiments were performed using adherent CHO cells stably transfected with cDNA encoding the human ARs. Percent activation of the
3
human A
3
AR was determined at 10 lM. Binding at human A
1
and A2AARs in this study was carried out as described in Methods using [ H]R-PIA
3
or [ H]CGS 21680 as a radioligand. Values from the present study are expressed as means ± SEM, n = 3–5.
b
c
3
Percent activity at 10 lM, relative to 10 lM Cl-IB-MECA (A ).
20
Binding affinity values from Gao et al.
Reagents were purchased from Aldrich Chemical
Company. All the anhydrous solvents were distilled over
CaH2 or P O or Na/benzophenone prior to the
the mixture was stirred at room temperature until the
solution became clear. A solution of sulfoxide 28
(10.85 mmol) in dry 1,2-dichloroethane (10 mL) was
added to the resulting solution in one shot at room tem-
2
5
reaction.
perature. An additional amount of Et N (21.69 mmol)
3
3
3
.2. Chemical synthesis
was added to the reaction mixture to initiate the Pum-
merer reaction. The reaction mixture was stirred under
reflux at 80 ꢁC for 4 days, during which time the initially
formed N-3 isomer was converted to N-9 isomer. The
reaction mixture was partitioned between EtOAc and
.2.1. General procedure for the condensation. To a sus-
pension of 6-chloropurine (21.69 mmol) or 2,6-dichlo-
ropurine (21.69 mmol) in a solution of dry CH CN
3
(20 mL) and 1,2-dichloroethane (10 mL) were added
Et N (21.69 mmol) and TMSOTf (43.38 mmol), and
aqueous saturated NaHCO solution, and the organic
3
layer was washed with brine, dried (MgSO ), filtered,
4
3

L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
4723
and evaporated. The residue was purified by flash silica
gel column chromatography (hexane/EtOAc = 5:1) to
give the condensed product.
3.7. 2,6-Dichloro-9-[(2,3-O-isopropylidene)-4-thio-b-D-
ribofuranosyl]purine (15)
Yield = 65%; yellowish syrup; UV (MeOH) k
274 nm
max
25
1
3
.3. 6-Chloro-9-[(5-O-benzoyl-2,3-O-isopropylidene)-4-
(pH 7); ½aꢁ ꢂ5.81 (c 0.86, MeOH); H NMR (CDCl ) d
D
1.36 (s, 3H, CH ), 1.66 (s, 3H, CH ), 3.93–3.97 (m, 1H,
3
thio-b-D-ribofuranosyl]purine (10)
3 3
4
-H), 4.07–4.14 (m, 2H, 5-H), 5.01 (dd, 1H, J = 1.2,
+
Yield = 53%; white foam; FAB-MS m/z 447 (M +1);
4.8 Hz, 3-H), 5.20 (dd, 1 H, J = 3.2, 5.2 Hz, 2-H), 6.71
(d, 1H, J = 3.2 Hz, 1-H), 8.60 (s, 1H, H-8); Anal. Calcd
for C H Cl N O S: C, 41.39; H, 3.74; N, 14.85; S,
25
UV (MeOH) k
274 nm (pH 7); ½aꢁ 12.35 (c 1.00,
max
D
1
MeOH); H NMR (CDCl ) d 1.39 (s, 3H, CH ), 1.66
3
3
13 14
2
4
3
(
s, 3H, CH ), 4.11 (td, 1H, J = 2.8, 6.4 Hz, 4-H), 4.53
8.50. Found: C, 41.33; H, 3.56; N, 14.45; S, 8.21.
3
(
dd, 1H, J = 6.4, 12.0 Hz, 5-H ), 4.74 (dd, 1H, J = 6.8,
a
1
5
2.0 Hz, 5-H ), 5.15 (dd, 1H, J = 1.6, 5.4 Hz, 3-H),
b
.45 (dd, 1H, J = 2.0, 5.6 Hz, 2-H), 6.07 (d, 1H,
3.8. General procedure for the removal of the isopropyl-
idene group
J = 2.0 Hz, 1-H), 7.35–7.91 (m, 5 H, Ph), 8.39 (s, 1H,
1
3
H-8), 8.80 (s, 1H, H-2); C NMR (CDCl ) d 25.20,
A solution of 14 (0.19 mmol) or 15 (0.19 mmol) in 80%
aqueous AcOH solution (6.36 mL) was stirred under
reflux at 70 ꢁC for 12 h. After removing the solvent
under reduced pressure, the residue was purified by flash
silica gel column chromatography (CH Cl /MeOH =
3
2
1
1
4
1
7.28, 54.09, 65.70, 68.00, 85.85, 89.23, 112.64, 128.39,
29.39, 129.40, 129.64, 133.27, 152.30, 153.32, 154.79,
66.01; Anal. Calcd for C H ClN O S: C, 53.75; H,
2
0
19
4
4
.29; N, 12.54; S, 7.17. Found: C, 53.77; H, 4.69; N,
2.43; S, 7.55.
2
2
10:1) to give 16 or 17, respectively.
3.9. 6-Chloro-9-(4-thio-b-D-ribofuranosyl)purine (16)
Yield = 60%; white solid; UV (MeOH) k 270 nm (pH
3
4
.4. 2,6-Dichloro-9-[(5-O-Benzoyl-2,3-O-isopropylidene)-
-thio-b-D-ribofuranosyl]purine (12)
max
+
25
D
25
D
1
Yield = 54%; white foam; FAB-MS m/z 482 (M +1); ½aꢁ
ꢂ1.46 (c 1.03, MeOH); UV (MeOH) k
7); ½aꢁ ꢂ8.43 (c 1.00, MeOH); H NMR (CD OD) d
3
269 nm (pH 7);
H NMR (CDCl ) d 1.39 (s, 3H, CH ), 1.64 (s, 3H, CH ),
3.53 (dd, 1H, J = 4.8, 9.6 Hz, 4-H), 3.84–3.90 (m, 2H,
5-H), 4.27 (t, 1H, J = 3.6 Hz, 3-H), 4.65 (dd, 1H,
J = 3.6, 4.8 Hz, 2-H), 5.98 (d, 1H, J = 4.8 Hz, 1-H),
8.84 (s, 1H, H-8), 8.98 (s, 1H, H-2); Anal. Calcd for
C H ClN O S: C, 39.67; H, 3.66; N, 11.71; S, 10.59.
max
1
3
3
3
4
1
5
.11 (td, 1H, J = 2.7, 6.8 Hz, 4-H), 4.57 (dd, 1H, J = 6.6,
1.4 Hz, 5-H ), 4.75 (dd, 1H, J = 7.1, 11.4 Hz, 5-H ),
.19 (dd, 1H, J = 2.7, 5.4 Hz, 3-H), 5.40 (dd, 1H,
a
b
1
0
11
4
3
J = 1.9, 5.4 Hz, 2-H), 6.10 (d, 1H, J = 1.9 Hz, 1-H),
7
Found: C, 39.98; H, 3.23; N, 11.31; S, 10.33.
1
.37–7.97 (m, 5 H, Ph), 8.38 (s, 1H, H-8); C NMR
3
(
8
1
CD OD) d 25.44, 27.54, 54.17, 65.59, 68.30, 85.84,
9.14, 113.34, 128.64, 129.35, 129.74, 133.65, 144.94,
52.39, 152.47, 153.32, 166.17. Anal. Calcd for
3.10. 2,6-Dichloro-9-(4-thio-b-D-ribofuranosyl)purine
(17)
3
C H Cl N O S: C, 49.90; H, 3.77; N, 11.64; S, 6.66.
4
Found: C, 49.53; H, 3.42; N, 11.93; S, 6.36.
Yield = 55%; yellowish solid; UV (MeOH) k
275 nm
max
2
0
18
2
4
25
1
(pH 7); ½aꢁ ꢂ0.50 (c 1.00, MeOH); H NMR (CD OD)
D
3
d 3.54 (dd, 1H, J = 5.2, 9.6 Hz, 4-H), 3.87–3.97 (m, 2H,
5-H), 4.33 (t, 1H, J = 3.6 Hz, 3-H), 4.71 (dd, 1H, J = 3.6,
3
.5. General procedure for the debenzoylation
5.2 Hz, 2-H), 6.02 (d, 1H, J = 5.2 Hz, 1-H), 8.97 (s, 1H,
H-8); Anal. Calcd for C H Cl N O S: C, 35.62; H,
2.99; N, 16.62; S, 9.51. Found: C, 35.23; H, 3.32; N,
16.22; S, 9.50.
To a stirred solution of 10 (11.73 mmol) or 12
11.73 mmol) in THF (125 mL) was added methylmag-
1
0
10
2
4
3
(
nesium iodide (MeMgI) (33.8 mmol, 3.0 M solution in
diethyl ether) at 0 ꢁC and the mixture was stirred for 4 h
at room temperature. The mixture was partitioned be-
tween EtOAc and water, dried, filtered and evaporated.
The residue was purified by flash silica gel column
chromatography (hexane/EtOAc = 1:1) to give the
debenzoylated products, 14 or 15, respectively.
6
3.11. General procedure for the N -substitution reaction
To a stirred solution of 6-chloropurine or 2,6-dichloropu-
rine derivative (0.298 mmol) and an appropriate amine-
sÆHCl or free amines (0.446 mmol) in EtOH (6 mL) was
added Et N (0.894 mmol) and the solution was stirred
3
3
furanosyl]purine (14)
.6. 6-Chloro-9-[(2,3-O-isopropylidene)-4-thio-b-D-ribo-
overnight at room temperature. After removing the sol-
vent under reduced pressure, the residue was purified by
flash silica gel column chromatography (CH Cl /MeOH =
2
2
6
Yield = 62%; white solid; UV (MeOH) k
274 nm (pH
15:1) to give the N -substituted amine derivatives.
max
2
5
1
7
1
); ½aꢁ ꢂ13.54 (c 1.00, MeOH); H NMR (CDCl ) d
D
.39 (s, 3H, CH ), 1.68 (s, 3H, CH ), 3.95 (m, 1H, 4-
3
3.12. 6-Amino-9-(4-thio-b-D-ribofuranosyl)purine (18a)
Yield = 58%; white solid; mp 125.4–126.3 ꢁC; FAB-MS
3 3
H), 4.13–4.16 (m, 2H, 5-H), 5.04 (dd, 1H, J = 4.6,
.2 Hz, 3-H), 5.21 (dd, 1H, J = 4.2, 3.2 Hz, 2-H), 6.74
d, 1H, J = 3.2 Hz, 1-H), 8.62 (s, 1H, H-8), 8.82 (s,
H, H-2); Anal. Calcd for C H ClN O S: C, 45.55;
1
(
1
+
25
m/z 284 (M ); ½aꢁ ꢂ47.1 (c 0.05, MeOH); UV (MeOH)
D
1
k
1H, 4-H), 3.64 (m, 1H, 5-H ), 3.79 (m, 1H, H -5), 4.20
260 nm (pH 7); H NMR (DMSO-d ) d 3.32 (m,
max 6
1
3
15
4
3
H, 4.41; N, 16.34; S, 9.35. Found: C, 44.98; H, 4.21;
N, 16.12; S, 9.30.
a b
(br dd, 1H, J = 3.7, 7.4 Hz, 3-H), 4.66 (m, 1H, 2-H),

4724
L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
5.21 (t, 1H, J = 5.5 Hz, exchangeable with D O,
HOCH ), 5.35 (d, 1H, J = 4.6 Hz, exchangeable with
3.16. 6-(3-Methylbutyl)amino-9-(4-thio-b-D-ribofurano-
syl)purine (18e)
2
2
D O, OH), 5.60 (d, 1H, J = 6.4 Hz, exchangeable with
2
D O, OH), 5.85 (d, 1H, J = 6.4 Hz, 1-H), 7.28 (br s,
2
Yield = 61%; white solid; mp 176.3–177.5 ꢁC; FAB-MS
+
25
2
8
4
4
H, exchangeable with D O, NH ), 8.14 (s, 1H, H-8),
2
m/z 354 (M ); ½aꢁ ꢂ72.5 (c 0.05, MeOH); UV (MeOH)
2
D
1
.45 (s, 1H, H-2); Anal. Calcd for C H N O S: C,
3
k
H, J = 3.6 Hz, CH(CH ) ), 1.48 (m, 2H), 1.63 (m, 1H),
267 nm (pH 7); H NMR (DMSO-d ) d 0.89 (d, 6
max 6
1
0
13
5
2.40; H, 4.63; N, 24.72; S, 11.32. Found: C, 42.56; H,
.34; N, 24.44; S, 11.03.
3 2
3.33 (t, 2H, NHCH ), 3.39 (m, 1H, 4-H), 3.64 (m, 1H,
2
5
6.4 Hz, 3-H), 4.66 (m, 1H, 2-H), 5.20 (t, 1H,
-H ), 3.81 (m, 1H, 5-H ), 4.20 (dd, 1H, J = 3.2,
a b
3
(
.13. 6-Methylamino-9-(4-thio-b-D-ribofuranosyl)purine
18b)
J = 5.5 Hz, exchangeable with D O, HOCH ), 5.32 (d,
2
2
1
1H, J = 6.4 Hz, exchangeable with D O, OH), 5.86 (d,
H, J = 4.6 Hz, exchangeable with D O, OH), 5.55 (d,
2
Yield = 64%; white solid; mp 178.0–180.0 ꢁC; FAB-MS
2
+
25
m/z 298 (M ); ½aꢁ ꢂ56.6 (c 0.05, MeOH); UV (MeOH)
1H, J = 6.4 Hz, 1-H), 7.79 (br s, 1H, exchangeable with
D O, NH-isoamyl), 8.21 (s, 1H, H-8), 8.43 (s, 1H, H-2);
D
1
k
J = 3.6 Hz, NHCH ), 3.20 (m, 1H, 4-H), 3.63 (m, 1H, 5-
266 nm (pH 7); H NMR (DMSO-d ) d 2.95 (d, 3H,
max
6
2
Anal. Calcd for C H N O S: C, 50.97; H, 6.56; N,
15 23
3
5
3
H ), 3.80 (m, 1H, 5-H ), 4.20 (dd, 1H, J = 3.7, 7.4 Hz, 3-
a
19.81; S, 9.07. Found: C, 51.28; H, 6.34; N, 19.41; S,
8.98.
b
H), 4.67 (m, 1H, 2-H), 5.22 (t, 1H, J = 5.5 Hz, exchange-
able with D O, HOCH ), 5.35 (d, 1H, J = 4.6 Hz,
exchangeable with D O, OH), 5.57 (d, 1H, J = 5.6 Hz,
2
2
3.17. 6-(Cyclopropylmethyl)amino-9-(4-thio-b-D-ribofur-
anosyl)purine (18f)
2
exchangeable with D O, OH), 5.86 (d, 1H, J = 6.8 Hz,
2
1
-H), 7.76 (br s, 1H, exchangeable with D O, NHCH ),
2 3
8.24 (s, 1H, H-8), 8.44 (s, 1H, H-2); Anal. Calcd for
C H N O S: C, 44.44; H, 5.08; N, 23.55; S, 10.78.
Found: C, 44.42; H, 4.87; N, 23.56; S, 10.88.
Yield = 72%; white solid; mp 214.5–216.4 ꢁC; FAB-MS
+
25
m/z 338 (M ); ½aꢁ ꢂ26.3 (c 0.05, MeOH); UV (MeOH)
1
1
15
5
3
D
1
k
270 nm (pH 7); H NMR (DMSO-d ) d 0.26 (m,
max 6
2
1H, cyclopropyl), 3.34 (t, 2H, NHCH ), 3.38 (m, 1H,
H, cyclopropyl), 0.40 (m, 2H, cyclopropyl), 1.14 (m,
3.14. 6-Cyclopropylamino-9-(4-thio-b-D-ribofurano-
syl)purine (18c)
2
4-H), 3.63 (m, 1H, 5-H ), 3.79 (m, 1H, 5-H ), 4.20
a
b
(dd, 1 H, J = 3.6, 7.2 Hz, 3-H), 4.67 (m, 1H, 2-H),
5.22 (t, 1H, J = 5.5 Hz, exchangeable with D O,
HOCH ), 5.34 (d, 1H, J = 4.6 Hz, exchangeable with
2
D O, OH), 5.56 (d, 1H, J = 6.4 Hz, exchangeable with
2
D O, OH), 5.86 (d, 1H, J = 6.4 Hz, 1-H), 7.90 (br s,
2
Yield = 61%; white solid; mp 255.3–258.0 ꢁC; FAB-MS
2
+
25
m/z 324 (M ); ½aꢁ ꢂ74.0 (c 0.05, MeOH); UV (MeOH)
D
1
k
2
1
270 nm (pH 7); H NMR (DMSO-d ) d 0.61 (m,
max
6
H, cyclopropyl), 0.72 (m, 2H, cyclopropyl), 3.04 (m,
H, cyclopropyl), 3.34 (m, 1H, 4-H), 3.63 (m, 1H, 5-
1H, exchangeable with D O, NH-cyclopropyl), 8.21 (s,
2
H ), 3.79 (m, 1H, 5-H ), 4.20 (br dd, 1H, J = 3.2,
b
1H, H-8), 8.45 (s, 1H, H-2); Anal. Calcd for
C H N O S: C, 49.84; H, 5.68; N, 20.76; S, 9.50.
Found: C, 49.56; H, 5.86; N, 20.36; S, 9.13.
a
6
J = 5.5 Hz, exchangeable with D O, HOCH ), 5.35 (d,
.4 Hz, 3-H), 4.67 (m, 1H, 2-H), 5.22 (t, 1H,
1
4
19
5
3
2
2
1
1
1
H, J = 4.6 Hz, exchangeable with D O, OH), 5.57 (d,
2
H, J = 6.2 Hz, exchangeable with D O, OH), 5.86 (d,
2
H, J = 6.4 Hz, 1-H), 7.95 (br s, 1H, exchangeable with
3.18. 6-Benzylamino-9-(4-thio-b-D-ribofuranosyl)purine
(18g)
D O, NH-cyclopropyl), 8.25 (s, 1H, H-8), 8.45 (s, 1H,
2
H-2); Anal. Calcd for C H N O S: C, 48.29; H, 5.30;
1
N, 21.66; S, 9.92. Found: C, 48.69; H, 5.67; N, 21.25;
S, 9.73.
Yield = 63%; white solid; mp 168.8–170.7 ꢁC; FAB-MS
3
17
5
3
+
25
m/z 374 (M ); ½aꢁ ꢂ58.4 (c 0.05, MeOH); UV (MeOH)
D
1
k
270 nm (pH 7); H NMR (DMSO-d ) d 3.33 (m,
6
max
1
H, 4-H), 3.63 (m, 1H, 5-H ), 3.79 (m, 1H, 5-H ), 4.21
a b
3.15. 6-Cyclobutylamino-9-(4-thio-b-D-ribofuranosyl)-
purine (18d)
(br, 1H, J = 3.2, 6.4 Hz, 3-H), 4.69 (br s, 3H, PhCH2
and 2-H), 5.21 (t, 1H, J = 5.6 Hz, exchangeable with
D O, HOCH ), 5.34 (d, 1H, J = 4.6 Hz, exchangeable
2
2
Yield = 54%; white solid; mp 181.3–183.1 ꢁC; FAB-MS
with D O, OH), 5.57 (d, 1H, J = 5.8 Hz, exchangeable
2
with D O, OH), 5.87 (d, 1H, J = 6.4 Hz, 1-H), 7.27–
2
+
25
m/z 338 (M ); ½aꢁ ꢂ63.2 (c 0.05, MeOH); UV (MeOH)
D
1
k
272 nm (pH 7); H NMR (DMSO-d ) d 1.61 (m,
7.33 (m, 5 H, Ph), 8.22 (s, 1H, H-8), 8.49 (br s, 1H,
exchangeable with D O, NH) 8.50 (s, 1H, H-2); Anal.
max
6
2
(
H, cyclobutyl), 2.09–2.24 (m, 4H, cyclobutyl), 3.20
m, 1H, cyclopropyl), 3.39 (m, 1H, 4-H), 3.62 (m, 1H,
2
Calcd for C H N O S: C, 54.68; H, 5.13; N, 18.75;
1
7
19
5
3
5
4
-H ), 3.78 (m, 1H, 5-H ), 4.19 (dd, 1H, J = 2.4,
a
.8 Hz, 3-H), 4.65 (m, 1H, 2-H), 5.20 (t, 1H,
S, 8.59. Found: C, 54.69; H, 5.11; N, 18.35; S, 8.99.
b
J = 5.8 Hz, exchangeable with D O, HOCH ), 5.33 (d,
2
3.19. 6-(3-Iodobenzyl)amino-9-(4-thio-b-D-ribofurano-
syl)purine (18h)
2
1
1
1
H, J = 4.6 Hz, exchangeable with D O, OH), 5.56 (d,
2
H, J = 5.8 Hz, exchangeable with D O, OH), 5.86 (d,
2
H, J = 4.8 Hz, 1-H), 8.08 (br s, 1H, exchangeable with
Yield = 78%; white solid; mp 194.3–196.0 ꢁC; FAB-MS
+
25
D O, NH-cyclobutyl), 8.20 (s, 1H, H-8), 8.46 (s, 1H, H-
2
m/z 500 (M ); ½aꢁ ꢂ54.0 (c 0.05, MeOH); UV (MeOH)
D
1
2
2
9
); Anal. Calcd for C H N O S: C, 49.84; H, 5.68; N,
1
0.76; S, 9.50. Found: C, 49.55; H, 5.99; N, 20.43; S,
.41.
k
1H, 4-H), 3.62 (m, 1H, 5-H ), 3.80 (m, 1H, 5-H ), 4.21
268 nm (pH 7); H NMR (DMSO-d ) d 3.33 (m,
max 6
4
19
5
3
a b
(dd, 1 H, J = 3.2, 6.4 Hz, 3-H), 4.69 (br m, 3H, PhCH2

L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
4725
and 2-H), 5.21 (t, 1H, J = 5.5 Hz, exchangeable with
D O, HOCH ), 5.33 (d, 1H, J = 4.6 Hz, exchangeable
with D O, OH), 5.55 (d, 1H, J = 6.3 Hz, exchangeable
3.23. 6-(3-Trifluoromethylbenzyl)amino-9-(4-thio-b-D-
ribofuranosyl)purine (18l)
2
2
2
with D O, OH), 5.88 (d, 1H, J = 6.4 Hz, 1-H), 7.11 (t,
2
Yield = 58%; white solid; mp 124.6–125.8 ꢁC; FAB-MS
+
25
0
0
1
7
H, J = 7.8 Hz, 5 -H), 7.37 (d, 1H, J = 7.6 Hz, 6 -H),
0
m/z 442 (M ); ½aꢁ ꢂ51.6 (c 0.05, MeOH); UV (MeOH)
D
1
0
.59 (d, 1H, J = 7.8 Hz, 4 -H), 7.72 (s, 1H, 2 -H), 8.22
k
1H, 4-H), 3.61 (m, 1H, 5-H ), 3.79 (m, 1H, 5-H ), 4.21
266 nm (pH 7); H NMR (DMSO-d ) d 3.34 (m,
max 6
(
NH) 8.49 (s, 1H, H-2); Anal. Calcd for C H IN O S:
s, 1H, H-8), 8.45 (br s, 1H, exchangeable with D O,
2
a b
(dd, 1H, J = 3.4, 6.8 Hz, 3-H), 4.79 (br s, 3H, PhCH2
and 2-H), 5.21 (t, 1H, J = 5.6 Hz, exchangeable with
D O, HOCH ), 5.34 (d, 1H, J = 4.6 Hz, exchangeable
1
7
18
5
3
C, 40.89; H, 3.63; N, 14.03; S, 6.42. Found: C, 40.78; H,
3
.43; N, 14.01; S, 6.13.
2
2
with D O, OH), 5.55 (d, 1H, J = 5.8 Hz, exchangeable
2
3
syl)purine (18i)
.20. 6-(3-Chlorobenzyl)amino-9-(4-thio-b-D-ribofurano-
with D O, OH), 5.87 (d, 1H, J = 6.4 Hz, 1-H), 7.52–
2
0
7.66 (m, 3H, Ph), 7.72 (s, 1H, 2 -H), 8.22 (s, 1H, H-8),
8.50 (s, 1H, H-2), 8.51 (br s, 1H, exchangeable with
D O, NH); Anal. Calcd for C H F N O S: C, 48.98;
Yield = 70%; white solid; mp 183.5–185.0 ꢁC; FAB-MS
2
18 18
3
5
3
+
25
D
1
m/z 408 (M ); ½aꢁ ꢂ67.6 (c 0.05, MeOH); UV (MeOH)
H, 4.11; N, 15.86; S, 7.26. Found: C, 49.01; H, 3.98;
N, 15.44; S, 7.25.
k
268 nm (pH 7); H NMR (DMSO-d ) d 3.34 (m,
6
max
1
H, 4-H), 3.63 (m, 1H, 5-H ), 3.80 (m, 1H, 5-H ), 4.21
a b
(
dd, 1 H, J = 3.6, 7.2 Hz, 3-H), 4.68 (br s, 3H, PhCH2
3.24. 6-(1-Naphthylmethyl)amino-9-(4-thio-b-D-ribofur-
anosyl)purine (18m)
and 2-H), 5.20 (t, 1H, J = 5.5 Hz, exchangeable with
D O, HOCH ), 5.33 (d, 1H, J = 4.6 Hz, exchangeable
with D O, OH), 5.56 (d, 1H, J = 6.4 Hz, exchangeable
with D O, OH), 5.86 (d, 1H, J = 6.4 Hz, 1-H), 7.26–
2
7
2
2
Yield = 71%; white solid; mp 167.8–169.0 ꢁC; FAB-MS
2
+
25
m/z 424 (M ); ½aꢁ ꢂ48.5 (c 0.05, MeOH); UV (MeOH)
D
1
0
.30 (m, 3H, Ph), 7.38 (s, 1 H, 2 -H), 8.22 (s, 1H, H-
), 8.48 (br s, 1H, exchangeable with D O, NH), 8.50
k
1H, 4-H), 3.60 (m, 1H, 5-H ), 3.79 (m, 1H, 5-H ), 4.21
272 nm (pH 7); H NMR (DMSO-d ) d 3.33 (m,
max 6
8
2
a b
(
5
4
s, 1H, H-2); Anal. Calcd for C H ClN O S: C,
1
0.06; H, 4.45; N, 17.17; S, 7.86. Found: C, 49.98; H,
.16; N, 16.99; S, 7.99.
(br dd, 1H, J = 3.7, 7.4 Hz, 3-H), 4.69 (m, 1H, 2-H),
5.18 (br s, 2H, naphthalenyl-CH ), 5.21 (t, 1H,
7
18
5
3
2
J = 5.5 Hz, exchangeable with D O, HOCH ), 5.34 (br,
2
2
1H, exchangeable with D O, OH), 5.55 (br, 1H,
exchangeable with D O, OH), 5.88 (d, 1H, J = 6.2 Hz,
2
3.21. 6-(3-Methylbenzyl)amino-9-(4-thio-b-D-ribofurano-
syl)purine (18j)
2
1-H), 7.42–7.94 (m, 7 H, Ph), 8.22 (s, 1H, H-8), 8.47
(br s, 1H, exchangeable with D O, NH), 8.49 (s, 1H,
2
Yield = 67%; white solid; mp 151.3–513.0 ꢁC; FAB-MS
H-2); Anal. Calcd for C H N O S: C, 59.56; H, 5.00;
21 21 5 3
N, 16.54; S, 7.57. Found: C, 59.17; H, 5.40; N, 16.13;
S, 7.17.
+
25
m/z 388 (M ); ½aꢁ ꢂ75.1 (c 0.05, MeOH); UV (MeOH)
D
1
k
PhCH ), 3.34 (m, 1H, 4-H), 3.63 (m, 1H, 5-H ), 3.79 (m,
267 nm (pH 7); H NMR (DMSO-d ) d 2.26 (s, 3H,
6
max
3
a
1
H, 5-H ), 4.20 (br dd, 1H, J = 3.7, 7.4 Hz, 3-H), 4.67
b
3.25. 6-Phenethylamino-9-(4-thio-b-D-ribofuranosyl)-
purine (18n)
(br s, 3H, PhCH and 2-H), 5.21 (t, 1H, J = 5.5 Hz,
exchangeable with D O, HOCH ), 5.33 (d, 1H,
2
2
2
J = 4.6 Hz, exchangeable with D O, OH), 5.56 (d, 1H,
2
J = 6.3 Hz, exchangeable with D O, OH), 5.87 (d, 1H,
2
Yield = 66%; white solid; mp 150.3–152.4 ꢁC; FAB-MS
+
25
m/z 388 (M ); ½aꢁ ꢂ65.5 (c 0.05, MeOH); UV (MeOH)
D
1
J = 6.4 Hz, 1-H), 7.01–7.19 (m, 4H, Ph), 8.21 (s, 1H,
H-8), 8.37 (br s, 1H, exchangeable with D O, NH)
k
J = 7.6 Hz, PhCH ), 3.34 (m, 1H, 4-H), 3.64 (m, 1H, 5-
268 nm (pH 7); H NMR (DMSO-d ) d 2.92 (t, 2H,
max 6
2
2
8
5
5
.47 (s, 1H, H-2); Anal. Calcd for C H N O S: C,
3
5.80; H, 5.46; N, 18.07; S, 8.28. Found: C, 55.76; H,
.67; N, 18.47; S, 8.23.
H ), 3.70 (br s, 2H, NH–CH ), 3.79 (m, 1H, 5-H ), 4.21
b
1
8
21
5
a
2
(dd, 1 H, J = 3.6, 6.2 Hz, 3-H), 4.66 (m, 1H, 2-H), 5.21
(t, 1H, J = 5.8 Hz, exchangeable with D O, HOCH ),
2
2
5.34 (d, 1H, J = 5.2 Hz, exchangeable with D O, OH),
5.55 (d, 1H, J = 6.4 Hz, exchangeable with D O, OH),
2
3.22. 6-(3-Fluorobenzyl)amino-9-(4-thio-b-D-ribofurano-
syl)purine (18k)
2
5.87 (d, 1H, J = 6.4 Hz, 1-H), 7.18–7.31 (m, 5 H, Ph),
7.89 (br s, 1H, exchangeable with D O, NH), 8.25 (s,
2
Yield = 61%; white solid; mp 161.7–162.8 ꢁC; FAB-MS
1H, H-8), 8.45 (s, 1H, H-2); Anal. Calcd for
C H N O S: C, 55.80; H, 5.46; N, 18.07; S, 8.28.
Found: C, 55.94; H, 5.15; N, 17.99; S, 8.09.
+
25
m/z 392 (M ); ½aꢁ ꢂ66.6 (c 0.05, MeOH); UV (MeOH)
D
1
18 21
5
3
k
271 nm (pH 7); H NMR (DMSO-d ) d 3.34 (m,
max
6
1
H, 4-H), 3.62 (m, 1H, 5-H ), 3.79 (m, 1H, 5-H ), 4.21
a b
(
(
br s, 1H, 3-H), 4.70 (br s, 3H, PhCH and 2-H), 5.21
2
3.26. 6-(3-Fluorophenethyl)amino-9-(4-thio-b-D-ribofur-
anosyl)purine (18o)
t, 1H, J = 4.8 Hz, exchangeable with D O, HOCH ),
2
2
5
1
1
1
8
5
4
.34 (br, 1H, exchangeable with D O, OH), 5.55 (d,
2
H, J = 6.3 Hz, exchangeable with D O, OH), 5.88 (d,
2
Yield = 63%; white solid; mp 186.3–188.0 ꢁC; FAB-MS
+
25
H, J = 6.8 Hz, 1-H), 7.02–7.35 (m, 4H, Ph), 8.22 (s,
H, H-8), 8.49 (br s, 1H, exchangeable with D O, NH)
m/z 406 (M ); ½aꢁ ꢂ45.8 (c 0.05, MeOH); UV (MeOH)
D
1
k
J = 8.0 Hz, PhCH ), 3.33 (m, 1H, 4-H), 3.61 (m, 1H, 5-
270 nm (pH 7); H NMR (DMSO-d ) d 2.95 (t, 2H,
max 6
2
.50 (s, 1H, H-2); Anal. Calcd for C H FN O S: C,
1
7
18
5
3
2
2.17; H, 4.64; N, 17.89; S, 8.19. Found: C, 52.46; H,
.99; N, 17.56; S, 7.98.
H ), 3.72 (br s, 2H, NH–CH ), 3.79 (m, 1H, 5-H ), 4.20
a
2
b
(dd, 1 H, J = 3.2, 6.4 Hz, 3-H), 4.66 (m, 1H, 2-H), 5.20

4726
L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
(
t, 1H, J = 5.6 Hz, exchangeable with D O, HOCH ),
2
H-8), 8.44 (s, 1H, H-2); Anal. Calcd for C H N O S:
25 27 5 3
2
5
5
5
7
1
.34 (d, 1H, J = 4.6 Hz, exchangeable with D O, OH),
2
.55 (d, 1H, J = 6.3 Hz, exchangeable with D O, OH),
2
C, 62.87; H, 5.70; N, 14.66; S, 6.71. Found: C, 62.56;
H, 5.55; N, 14.26; S, 6.67.
.87 (d, 1H, J = 6.4 Hz, 1-H), 6.99–7.34 (m, 4H, Ph),
.93 (br s, 1H, exchangeable with D O, NH), 8.25 (s,
H, H-8), 8.45 (s, 1H, H-2); Anal. Calcd for
3.30. 6-(Piperidin-1-yl)amino-9-(4-thio-b-D-ribofurano-
syl)purine (18s)
2
C H FN O S: C, 53.32; H, 4.97; N, 17.27; S, 7.91.
1
8
20
5
3
Found: C, 52.98; H, 5.08; N, 16.99; S, 7.51.
Yield = 85%; white solid; mp 124.9–125.8 ꢁC; FAB-MS
+
25
m/z 352 (M ); ½aꢁ ꢂ74.6 (c 0.05, MeOH); UV (MeOH)
D
1
3.27. 6-(trans-2-Phenyl-cyclopropyl)amino-9-(4-thio-b-D-
ribofuranosyl)purine (18p)
k
(m, 6 H, piperidinyl), 3.38 (m, 1H, 4 -H), 3.61 (m, 1H,
279 nm (pH 7); H NMR (DMSO-d ) d 1.57–1.68
max
6
0
5
-H ), 3.79 (m, 1H, 5-H ), 4.07 (t, 1H, J = 8.6 Hz),
a b
Yield = 74%; white solid; mp 201.2–203.0 ꢁC; FAB-MS
4.19 (br dd, 5 H, piperidyl-N(CH2)2 and 3-H), 4.63
(m, 1H, 2-H), 5.19 (t, 1H, J = 5.5 Hz, exchangeable with
D O, HOCH ), 5.31 (d, 1H, J = 4.6 Hz, exchangeable
+
25
m/z 400 (M ); ½aꢁ ꢂ62.1 (c 0.05, MeOH); UV
D
1
(
MeOH) k
272 nm (pH 7); H NMR (DMSO-d )
max 6
2
2
d 1.23–1.44 (m, 2H, cyclopropyl CH ), 2.10 (m, 1H,
with D O, OH), 5.56 (d, 1H, J = 6.3 Hz, exchangeable
2
with D O, OH), 5.88 (d, 1H, J = 6.4 Hz, 1-H), 8.22 (s,
2
2
cyclopropyl CH–Ph), 3.20 (br s, 1H, NH-cyclopropyl
CH) 3.35 (m, 1H, 4-H), 3.61 (m, 1H, 5-H ), 3.79 (m,
1H, H-8), 8.47 (s, 1H, H-2); Anal. Calcd for
C H N O S: C, 51.27; H, 6.02; N, 19.93; S, 9.12.
Found: C, 51.56; H, 6.42; N, 19.67; S, 9.02.
a
1
4
H, 5-H ), 4.20 (br dd, 1H, J = 3.6, 7.2 Hz, 3-H),
b
.66 (m, 1H, 2-H), 5.20 (t, 1H, J = 5.8 Hz, exchange-
15 21
5
3
able with D O, HOCH ), 5.32 (d, 1H, J = 4.6 Hz,
2
2
exchangeable with D O, OH), 5.54 (d, 1H,
2
J = 6.4 Hz, exchangeable with D O, OH), 5.88 (d,
2
3.31. 6-(4-Benzylpiperidin-1-yl)amino-9-(4-thio-b-D-ribo-
furanosyl)purine (18t)
1
H, J = 6.4 Hz, 1-H), 7.16–7.30 (m, 5 H, Ph), 8.23
br s, 1H, exchangeable with D O, NH), 8.26 (s, 1H,
(
H-8), 8.47 (s, 1H, H-2); Anal. Calcd for C H N O S:
Yield = 82%; white solid; mp 194.3–196.0 ꢁC; FAB-MS
2
+
25
m/z 442 (M ); ½aꢁ ꢂ63.3 (c 0.05, MeOH); UV (MeOH)
1
9
21
5
3
D
1
C, 57.13; H, 5.30; N, 17.53; S, 8.03. Found: C, 57.45;
H, 5.56; N, 17.14; S, 7.98.
k
2H, piperidinyl), 1.69 (m, 2H, piperidinyl), 1.88 (m,
280 nm (pH 7); H NMR (DMSO-d ) d 1.13 (m,
max 6
1
H, BnCH), 3.08 (br m, 4H, N(CH ) ), 3.38 (m, 1H,
2
2
3
.28. 6-(1,2-Diphenylethyl)amino-9-(4-thio-b-D-ribofur-
anosyl)purine (18q)
4-H), 3.60 (m, 1 H, 5-H ), 3.77 (m, 1H, 5-H ), 4.18 (br
a b
dd, 1H, J = 3.7, 7.4 Hz, 3-H), 4.62 (m, 1H, 2-H), 5.19
t, 1 H, J = 5.5 Hz, exchangeable with D O, HOCH ),
5.31 (d, 1H, J = 4.6 Hz, exchangeable with D O, OH),
(
2
2
Yield = 68%; white solid; mp 112.7–114.0 ꢁC; FAB-MS
2
+
25
m/z 464 (M ); ½aꢁ ꢂ57.7 (c 0.05, MeOH); UV (MeOH)
5.33 (br s, 2H, PhCH ), 5.55 (d, 1H, J = 6.3 Hz,
2
exchangeable with D O, OH), 5.88 (d, 1H, J = 6.4 Hz,
2
D
1
k
271 nm (pH 7); H NMR (DMSO-d ) d 3.05–3.10
max
6
(
m, 2H, NHCHCH ), 3.35 (dd, 1H, J = 4.8 Hz, 4-H),
.60 (m, 1H, 5-H ), 3.77 (m, 1H, 5-H ), 4.18 (br dd,
a b
1-H), 7.17–7.30 (m, 5 H, Ph), 8.22 (s, 1H, H-8), 8.47
(s, 1H, H-2); Anal. Calcd for C H N O S: C, 59.84;
H, 6.16; N, 15.86; S, 7.26. Found: C, 59.98; H, 6.02;
N, 15.99; S, 7.05.
2
3
1
1
5
5
22 27
5
3
H, J = 3.6, 7.2 Hz, 3-H), 4.63 (m, 1H, 2-H), 5.23 (t,
H, J = 5.5 Hz, exchangeable with D O, HOCH ),
.33 (d, 1H, J = 4.6 Hz, exchangeable with D O, OH),
2
2
2
.60 (br s, 1H, NH–CH), 5.63 (d, 1H, J = 6.3 Hz,
3.32. 6-[4-(4-Fluorobenzyl)-piperazin-1-yl]amino-9-(4-
thio-b-D-ribofuranosyl)purine (18u)
exchangeable with D O, OH), 5.80 (d, 1H, J = 6.4 Hz,
2
1
8
-H), 7.10–7.52 (m, 10 H, 2· Ph), 8.11 (s, 1H, H-8),
.46 (s, 1H, H-2), 8.47 (br s, 1H, exchangeable with
Yield = 79%; white solid; mp 218.3–220.0 ꢁC; FAB-MS
+
25
D O, NH); Anal. Calcd for C H N O S: C, 62.19;
2
H, 5.44; N, 15.11; S, 6.92. Found: C, 62.18; H, 5.57;
N, 15.01; S, 6.88.
m/z 460 (M ); ½aꢁ ꢂ82.36 (c 0.05, MeOH); UV
24 25
5
3
D
1
(MeOH) k
3.28–3.35 (br m, 8 H, piperazinyl), 3.38 (m, 1H, 4-H),
282 nm (pH 7); H NMR (DMSO-d ) d
max 6
3
1H, J = 3.6, 7.2 Hz, 3-H), 4.37 (br s, 2H, PhCH ), 4.64
.61 (m, 1H, 5-H ), 3.78 (m, 1H, 5-H ), 4.19 (br dd,
a
b
3
anosyl)purine (18r)
.29. 6-(3,3-Diphenylpropyl)amino-9-(4-thio-b-D-ribofur-
2
(m, 1H, 2-H), 5.20 (t, 1 H, J = 4.8 Hz, exchangeable with
D O, HOCH ), 5.33 (d, 1H, J = 4.6 Hz, exchangeable
with D O, OH), 5.58 (d, 1H, J = 6.4 Hz, exchangeable
2
2
Yield = 79%; white solid; mp 138.5–139.7 ꢁC; FAB-MS
2
+
25
m/z 478 (M ); ½aꢁ ꢂ66.8 (c 0.05, MeOH); UV (MeOH)
with D O, OH), 5.90 (d, 1H, J = 6.4 Hz, 1-H), 7.03–
2
D
1
k
2
3
1
269 nm (pH 7); H NMR (DMSO-d ) d 2.35 (dd,
7.10 (m, 5 H, Ph), 8.28 (s, 1H, H-8), 8.54 (s, 1H, H-2);
Anal. Calcd for C H FN O S: C, 54.77; H, 5.47; N,
max
6
H, J = 14.4 Hz, 7.2 Hz, NHCH CH ), 3.31–3.38 (m,
H, 4-H and NH–CH ), 3.61 (m, 1H, 5-H ), 3.79 (m,
H, 5-H ), 4.07 (t, 1 H, J = 8.6 Hz), 4.19 (dd, 1H,
b
2
2
21 25
6
3
18.25; S, 6.96. Found: C, 54.39; H, 5.25; N, 18.56; S,
6.76.
2
a
J = 3.6, 7.2 Hz, 3-H), 4.65 (m, 1H, 2-H), 5.20 (t, 1H,
J = 5.6 Hz, exchangeable with D O, HOCH ), 5.32 (d,
3.33. 6-(Morpholin-1-yl)amino-9-(4-thio-b-D-ribofurano-
syl)purine (18v)
2
2
1
1
1
H, J = 4.6 Hz, exchangeable with D O, OH), 5.52 (d,
2
H, J = 6.3 Hz, exchangeable with D O, OH), 5.86 (d,
2
H, J = 6.4 Hz, 1-H), 7.14–7.34 (m, 10 H, 2· Ph), 7.89
Yield = 68%; white solid; mp 144.5–146.0 ꢁC; FAB-MS
+
25
(
br s, 1H, exchangeable with D O, NH), 8.17 (s, 1H,
m/z 354 (M ); ½aꢁ ꢂ90.7 (c 0.05, MeOH); UV (MeOH)
2
D

L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
4727
1
kmax 282 nm (pH 7); H NMR (DMSO-d ) d 3.39 (m,
3.37. 6-Benzylamino-2-chloro-9-(4-thio-b-D-ribofurano-
syl)purine (19d)
6
1H, 4-H), 3.60 (m, 1H, 5-H ), 3.63 (br s, 4H, morpholi-
nyl), 3.79 (m, 1H, 5-H ), 4.19 (br, 5 H, morpholine-H
a
b
and 3-H), 4.63 (m, 1 H, 2-H), 5.20 (t, 1H, J = 5.5 Hz,
exchangeable with D O, HOCH ), 5.34 (d, 1H,
J = 4.6 Hz, exchangeable with D O, OH), 5.57 (d, 1H,
Yield = 75%; white solid; mp 135.0–140.0 ꢁC; UV
+
25
D
(MeOH) k
max
274 nm; MS (FAB) m/z 408.0 (M ); ½aꢁ
2
2
1
ꢂ3.51 (c 0.97, MeOH); H NMR (CD OD) d 3.52 (dd,
2
3
J = 6.3 Hz, exchangeable with D O, OH), 5.89 (d, 1H,
2
1H, J = 4.8 Hz ,4-H), 3.79 (br t, 2H, NH–CH ), 3.85–
2
J = 6.4 Hz, 1-H), 8.27 (s, 1H, H-8), 8.53 (s, 1H, H-2);
Anal. Calcd for C H N O S: C, 47.58; H, 5.42; N,
3.96 (m, 2H, 5-H), 4.29 (t, 1H, J = 4.4 Hz, 3.6 Hz, 3-
H), 4.66 (dd, 1H, J = 3.6, 5.4 Hz, 2-H), 4.75 (br s, 2H,
NHCH ), 5.88 (d, 1H, J = 5.6 Hz, 1-H), 7.23-7.39 (m,
14
19
5
4
1
9
9.82; S, 9.07. Found: C, 47.78; H, 5.23; N, 19.98; S,
.35.
2
5 H, Ph), 8.40 (s, 1H, H-8); Anal. Calcd for
C H ClN O S: C, 50.06, H, 4.45, N, 17.17, S, 7.86.
Found: C, 50.23, H, 4.05, N, 17.35, S, 7.65.
1
7
18
5
3
3.34. 2-Chloro-6-amino-9-(4-thio-b-D-ribofuranosyl)-
purine (19a)
3.38. 2-Chloro-6-(2-methylbenzyl)amino-9-(4-thio-b-D-
Yield = 53%; white solid; mp 220.1–222.2 ꢁC; FAB-MS
ribofuranosyl)purine (19e)
+
25
m/z 318 (M ); ½aꢁ ꢂ24.6 (c 0.07, MeOH); UV (MeOH)
D
1
k
264 nm (pH 7); H NMR (DMSO-d ) d 3.30 (br m,
Yield = 72%; white solid; mp 186.3–189.3 ꢁC; UV
max
6
+
25
D
1
H, 4-H), 3.62 (m, 1H, 5-H ), 3.79 (m, 1H, 5-H ), 4.20
(MeOH) k
max
274 nm; MS (FAB) m/z 422.0 (M ); ½aꢁ
a
b
1
(
5
br dd, 1H, J = 3.7, 7.4 Hz, 3-H), 4.61 (m, 1H, 2-H),
.16 (t, 1H, J = 5.5 Hz, exchangeable with D O,
HOCH ), 5.34 (d, 1H, J = 4.6 Hz, exchangeable with
ꢂ3.78 (c 0.98, MeOH); H NMR (CD OD) d 2.37 (d,
3
3H, J = 10.8 Hz, 2-CH –Ph–), 3.52 (dd, 1H, J = 4.8,
3
2
5.2 Hz, 4-H), 3.83–3.95 (dd, 2H, J = 4.8, 11.6 Hz, 5-
H), 4.29 (t, 1H, J = 3.6, 4.8 Hz, 3-H), 4.66 (dd, 1H,
J = 3.6, 5.2 Hz, 2-H), 4.74 (br s, 2H, NH–CH ), 5.87
2
D O, OH), 5.58 (d, 1H, J = 6.3 Hz, exchangeable with
2
D O, OH), 5.77 (d, 1H, J = 6.8 Hz, 1-H), 7.81 (br s,
2
2
2
Anal. Calcd for C H ClN O S: C, 37.80; H, 3.81; N,
H, exchangeable with D O, NH ), 8.48 (s, 1H, H-8);
(d, 1H, J = 5.2 Hz, 1-H), 7.13–7.32 (m, 5 H, Ph), 8.40
(s, 1H, H-8); Anal. Calcd for C H ClN O S: C,
2
2
1
0
12
5
3
18 20
5
3
2
1
2.04; S, 10.09. Found: C, 37.92; H, 3.75; N, 22.24; S,
0.41.
51.24, H, 4.78, N, 16.60, S, 7.60. Found: C, 51.57, H,
5.04, N, 16.26, S, 7.29.
3.35. 2-Chloro-6-methylamino-9-(4-thio-b-D-ribofurano-
syl)purine (19b)
3.39. 2-Chloro-6-(2-ethoxybenzyl)amino-9-(4-thio-b-D-
ribofuranosyl)purine (19f)
Yield = 52%; white solid; mp 239.3–241.3 ꢁC; FAB-MS
Yield = 86%; white solid; mp 147.9–150.4 ꢁC; UV
+
25
+
276 nm; MS (FAB) m/z 452.1 (M );
m/z 332 (M ); ½aꢁ ꢂ30.6 (c 0.1, MeOH); UV (MeOH)
(MeOH) k
D
1
max
25
1
k
3
1
7
271 nm (pH 7); H NMR (DMSO-d ) d 2.92 (d,
½aꢁ ꢂ3.20 (c 1.03, MeOH); H NMR (CD OD) d
max
6
D
1.40 (t, 3H, J = 6.0, 7.2 Hz, OCH CH ), 3.52 (dd,
3
H, J = 4.3 Hz, N-CH ), 3.30 (m, 1H, 4-H), 3.62 (m,
H, 5-H ), 3.79 (m, 1H, 5-H ), 4.20 (dd, 1H, J = 3.6,
.9 Hz, 3-H), 4.61 (m, 1H, 2-H), 5.16 (t, 1H,
3
2 3
1H, J = 4.4, 5.2 Hz, 4-H), 3.85–3.96 (m, 2H, 5-H),
4.10 (q, 2H, J = 6.4, 7.6 Hz, OCH CH ), 4.29 (t, 1H,
J = 3.6, 4.8 Hz, 3-H), 4.58 (br s, 1H, NH–CH ), 4.65
a
b
2
3
J = 5.5 Hz, exchangeable with D O, HOCH ), 5.34 (d,
2
2
2
1
1
1
H, J = 4.6 Hz, exchangeable with D O, OH), 5.58 (d,
2
H, J = 6.3 Hz, exchangeable with D O, OH), 5.77 (d,
2
H, J = 6.8 Hz, 1-H), 8.26 (br q, 1H, exchangeable with
(t, 1H, J = 4.0, 5.2 Hz, 2-H), 5.87 (d, 1H, J = 5.2 Hz,
1-H), 6.86–7.32 (m, 4H, Ph), 8.41 (s, 1H, H-8); Anal.
Calcd for C H ClN O S: C, 50.50, H, 4.91, N,
1
9
22
5
3
D O, NH), 8.47 (s, 1H, H-8); Anal. Calcd for
2
15.50, S, 7.10. Found: C, 50.85, H, 4.96, N, 15.63, S,
6.90.
C H ClN O S: C, 39.82; H, 4.25; N, 21.11; S, 9.66.
1
1
14
5
3
Found: C, 39.97; H, 4.42; N, 21.20; S, 9.77.
3.40. 2-Chloro-6-(3-iodobenzyl)amino-9-(4-thio-b-D-ribo-
3.36. 2-Chloro-6-cyclopentylamino-9-(4-thio-b-D-ribofur-
anosyl)purine (19c)
furanosyl)purine (19g)
Yield = 53%; white solid; mp 112.5–114.3 ꢁC; FAB-MS
+
25
Yield = 84%; white solid; mp 187.5–188.6 ꢁC; FAB-MS
m/z 534 (M ); ½aꢁ ꢂ28.3 (c 0.1, MeOH); UV (MeOH)
D
1
+
25
m/z 386 (M ); ½aꢁ ꢂ31.4 (c 0.05, MeOH); UV (MeOH)
k
1H, 4-H), 3.63 (m, 1H, 5-H ), 3.78 (m, 1H, 5-H ), 4.20
273 nm (pH 7); H NMR (DMSO-d ) d 3.29 (m,
max 6
D
1
k
266 nm (pH 7); H NMR (DMSO-d ) d 1.34–1.75
max
6
a b
(
1
br m, 8 H, cyclopropyl), 3.30 (m, 1H, 4-H), 3.62 (m,
H, 5-H ), 3.76 (m, 1H, 5-H ), 4.21 (br dd, 1H,
(br d, 1H, J = 3.4 Hz, 3-H), 4.61 (m, 3H, 2-H and N-
CH ), 5.17 (t, 1H, J = 5.5 Hz, exchangeable with D O,
HOCH ), 5.34 (d, 1H, J = 4.7 Hz, exchangeable with
D O, OH), 5.58 (d, 1H, J = 6.1 Hz, exchangeable with
2
D O, OH), 5.78 (d, 1H, J = 6.8 Hz, 1-H), 7.13 (t, 1H,
2
a
b
2
2
J = 3.7, 6.8 Hz, 3-H), 4.44 (m, 1H, NH–CH), 4.63 (m,
H, 2-H), 5.14 (t, 1H, J = 5.5 Hz, exchangeable with
D O, HOCH ), 5.33 (d, 1H,J = 4.6 Hz, exchangeable
2
1
2
2
0
0
with D O, OH), 5.57 (d, 1H, J = 6.0 Hz, exchangeable
2
with D O, OH), 5.78 (d, 1H, J = 6.4 Hz, 1-H), 8.20 (br
2
s, 1H, exchangeable with D O, NH), 8.45 (s, 1H, H-8);
2
Anal. Calcd for C H ClN O S: C, 46.69; H, 5.22; N,
1
J = 7.7 Hz, 5 -H), 7.34 (d, 1H, J = 7.5 Hz, 6 -H), 7.59
0
0
(d, 1H, J = 7.8 Hz, 4 -H), 7.74 (s, 1H, 2 -H), 8.53 (s,
1H, H-8), 8.92 (br t, 1H, J = 5.8 Hz, exchangeable with
D O, NH); Anal. Calcd for C H ClIN O S: C, 38.25;
5
20
5
3
2
17 17
5
3
1
7
8.15; S, 8.31. Found: C, 46.78; H, 5.45; N, 18.45; S,
.99.
H, 3.21; N, 13.12; S, 6.01. Found: C, 38.47; H, 3.42; N,
13.42; S, 6.20.

4728
L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
3
ribofuranosyl)purine (19h)
.41. 2-Chloro-6-(2-naphthylmethyl)amino-9-(4-thio-b-D-
3.94 (m, 2H, 5-H), 4.26 (t, 1H, J = 4.8 Hz, 3-H), 4.61
(d, 1H, J = 4.0 Hz, 2-H), 5.60 (br s, 1H, NH–CH),
.83 (d, 1H, J = 5.6 Hz, 1-H), 7.07–7.42 (m, 10 H, 2·
Ph), 8.40 (s, 1H, H-8); Anal. Calcd for C H ClN O S:
C, 57.88, H, 4.86, N, 14.06, S, 6.44. Found: C, 57.79, H,
4.46, N, 14.58, S, 6.45.
5
Yield = 86%; white solid; mp 145.4–150.9 ꢁC; UV
2
4
24
5
3
+
25
D
(
MeOH) k
280 nm; MS (FAB) m/z 458 (M ); ½aꢁ
max
1
ꢂ4.15 (c 1.06, MeOH); H NMR (CD OD) d 3.52 (dd,
3
1
(
5
H, J = 4.8, 5.2 Hz, 4-H), 3.85–3.95 (m, 2H, 5-H), 4.28
t, 1H, J = 4.0, 4.4 Hz, 3-H), 4.65 (dd, 1H, J = 3.6,
.2 Hz, 2-H), 5.20 (br s, 2H, NH–CH ), 5.86 (d, 1H,
3.46. 2-Chloro-6-(trans-2-phenyl-cyclopropyl)amino-9-(4-
thio-b-D-ribofuranosyl)purine (19m)
2
J = 5.2 Hz, 1-H), 7.41–8.14 (m, 7 H, naphthalenyl H),
8
5
4
.42 (s, 1H, H-8); Anal. Calcd for C H ClN O S: C,
3
5.08, H, 4.40, N, 15.29, S, 7.00. Found: C, 55.03, H,
.80, N, 15.53, S, 7.31.
Yield = 79%; white solid; mp 181.8–183.8 ꢁC; UV
2
1
20
5
+
25
D
d
(MeOH) k
275 nm; MS (FAB) m/z 434.1 (M ); ½aꢁ
max
1
ꢂ2.12 (c 0.99, MeOH);
H NMR (CD OD)
3
1
.28–1.39 (m, 2H, cyclopropyl CH ), 2.16–2.21 (m,
2
3.42. 2-Chloro-6-(fluoren-9-yl)amino-9-(4-thio-b-D-ribo-
furanosyl)purine (19i)
1H, cyclopropyl CH-Ph), 3.11 (br s, 1H, NH-cyclopro-
pyl CH), 3.53 (dd, 1H, J = 4.8, 5.2 Hz, 4-H), 3.86–3.96
(m, 2H, 5-H), 4.30 (t, 1H, J = 4.4 Hz, 3-H), 4.66 (dd,
Yield = 85%; white solid; mp 139.2–146.1 ꢁC; UV
1H, J = 3.8, 5.4 Hz, 2-H), 5.89 (d, 1H, J = 5.6 Hz,
1-H), 7.28–7.29 (m, 5 H, Ph), 8.42 (s, 1H, H-8); Anal.
Calcd for C H ClN O S: C, 52.59, H, 4.65, N, 16.14,
+
273 nm; MS (FAB) m/z 429.4 (M -H O-
(
MeOH) k
max
2
2
5
1
Cl); ½aꢁ 5.60 (c 1.00, MeOH); H NMR (CD OD) d
D
.55 (dd, 1H, J = 4.8 Hz, 4-H), 3.86–3.97 (m, 2H, 5-
3
19 20
5
3
3
H), 4.29 (t, 1H, J = 4.0 Hz, 3-H), 4.68 (t, 1H, J = 3.6,
S, 7.39. Found: C, 52.91, H, 4.65, N, 16.20, S, 7.63.
5
1
1
.2 Hz, 2-H), 5.90 (d, 1H, J = 5.6 Hz, 1-H), 6.60 (s,
H, NH–CH), 7.27-7.79 (m, 8 H, fluorenyl H), 8.34 (s,
H, H-8); Anal. Calcd for C H ClN O S: C, 57.32,
3.47. 2-Chloro-6-(3,3-diphenylpropyl)amino-9-(4-thio-b-
D-ribofuranosyl)purine (19n)
2
3
20
5
3
H, 4.18, N, 14.53, S, 6.65. Found: C, 57.44, H, 4.53,
N, 14.31, S, 6. 68.
Yield = 82%; white solid; mp 113.9–116.8 ꢁC; UV
+
25
D
(MeOH) k
273 nm; MS (FAB) m/z 512 (M ); ½aꢁ
max
1
ꢂ2.57 (c 1.05, MeOH); H NMR (CD OD) d 2.43 (dd,
3
3.43. 2-Chloro-6-phenethylamino-9-(4-thio-b-D-ribofur-
anosyl)purine (19j)
2H, J = 7.2, 14.4 Hz, NHCH CH ), 3.48–3.54 (m, 3H,
2
2
4-H and NH–CH ), 3.85–3.96 (dd, 2H, J = 4.8,
2
1
1.6 Hz, 5-H), 4.09 (pseudo t, 1H, J = 7.2, 8.4 Hz,
CH CH CH), 4.29 (pseudo t, 1H, J = 3.6, 4.4 Hz, 3-
Yield = 85%; white solid; mp 131.2–135.3 ꢁC; UV
2
2
+
273 nm; MS (FAB) m/z 422.1 (M +Na);
(
MeOH) k
H), 4.65 (pseudo t, 1H, J = 4.0, 4.8 Hz, 2-H), 5.86 (d,
1H, J = 5.2 Hz, 1-H), 7.12–7.31 (m, 10 H, 2· Ph), 8.38
(s, 1H, H-8); Anal. Calcd for C H ClN O S: C,
max
2
D
5
1
½
aꢁ ꢂ3.18 (c 1.07, MeOH); H NMR (CD OD) d 2.97
3
(
t, 2H, J = 7.6 Hz, CH –Ph), 3.52 (dd, 1H,J = 4.8 Hz,
2
25 26
5
3
4
-H), 3.79 (br t, 2H, NH–CH ), 3.79-3.96 (m, 2H, 5-
2
58.64, H, 5.12, N, 13.68, S, 6.26. Found: C, 58.38, H,
5.40, N, 13.46, S, 6.92.
H), 4.30 (t, 1H, J = 4.4 Hz, 3-H), 4.65 (dd, 1H,
J = 3.6, 5.2 Hz, 2-H), 5.87 (d, 1H, J = 5.6 Hz, 1-H),
7
for C H ClN O S: C, 51.24, H, 4.78, N, 16.60, S,
.16–7.27 (m, 5H, Ph), 8.39 (s, 1H, H-8); Anal. Calcd
3.48. Biological assays
1
8
20
5
3
7
.60. Found: C, 51.30, H, 4.84, N, 16.47, S,7.84.
3.48.1. Cell culture and membrane preparation. Chinese
hamster ovary (CHO) cells expressing the recombinant
human A , A , or A AR were cultured in DMEM
(Dulbecco’s modified Eagle’s medium) and F12 (1:1)
supplemented with 10% fetal bovine serum, 100 U/mL
penicillin, 100 lg/mL streptomycin, and 2 lmol/mL glu-
tamine. Cells were harvested by trypsinization. After
homogenization and suspension, cells were centrifuged
at 500g for 10 min, and the pellet was resuspended in
50 mM Tris–HCl buffer (pH 7.4) containing 10 mM
3
ribofuranosyl)purine (19k)
.44. 2-Chloro-6-(3-fluorophenethyl)amino-9-(4-thio-b-D-
1
2A
3
Yield = 83%; white solid; mp 126.1–130.0 ꢁC; UV
+
25
D
(
MeOH) k
272 nm; MS (FAB) m/z 440 (M ); ½aꢁ
max
1
ꢂ3.86 (c 1.01, MeOH); H NMR (CD OD) d 2.99 (t,
3
2
5
2
H, J = 7.6 Hz, CH –Ph), 3.52 (dd, 1H, J = 4.8,
2
.2 Hz, 4-H), 3.81 (br t, 2H, NH–CH ), 3.85–3.96 (m,
2
H, 5-H), 4.29 (t, 1H, J = 4.4 Hz, 3-H), 4.65 (dd, 1H,
MgCl . The suspension was homogenized with an elec-
2
J = 3.8, 5.4 Hz, 2-H), 5.87 (d, 1H, J = 5.2 Hz, 1-H),
.89–7.30 (m, 4H, Ph), 8.39 (s, 1H, H-8); Anal. Calcd
for C H ClN O S: C, 49.15, H, 4.35, N, 15.92, S,
tric homogenizer for 10 s and was then re-centrifuged
at 20,000g for 20 min at 4 ꢁC. The resultant pellets were
resuspended in buffer containing 3 U/mL adenosine
deaminase, and the suspension was stored at ꢂ80 ꢁC un-
til the binding experiments. The protein concentration
6
1
8
19
5
3
7
.29. Found: C, 49.18, H, 4.31, N, 15.53, S, 7.01.
2
1
3.45. 2-Chloro-6-(1,2-diphenylethyl)amino-9-(4-thio-b-D-
ribofuranosyl)purine (19l)
was measured using the Bradford assay.
3
.48.2. Binding assays at the human A and A ARs. For
1
2A
Yield = 85%; white solid; mp 79.3–80.8 ꢁC; UV (MeOH)
binding to the human A AR, 50 lL of increasing con-
centrations of a test ligand and 50 lL of [ H]R-PIA
(2 nM) were incubated with membranes (40 lg/tube)
1
+
25
3
k
275 nm; MS (FAB) m/z 498 (M ); ½aꢁ ꢂ2.06 (c
max
D
1
0.97, MeOH); H NMR (CD OD) d 3.16–3.28 (m, 2
H, NHCHCH ), 3.51 (dd, 1H, J = 4.8 Hz, 4-H), 3.84–
3
from CHO cells stably expressing the human A AR at
2
1

L. S. Jeong et al. / Bioorg. Med. Chem. 14 (2006) 4718–4730
4729
2
5 ꢁC for 60 min in 50 mM Tris–HCl buffer (pH 7.4;
converted to K values using the Cheng-Prusoff equa-
i
tion. Data were expressed as means ± standard error.
2
3
MgCl , 10 mM) in a total assay volume of 200 lL. Non-
specific binding was determined using 10 lM of N -
2
6
cyclopentyladenosine. For human A2AAR binding,
membranes (20 lg/tube) from HEK-293 cells stably
expressing the human A2AAR were incubated at 25 ꢁC
Acknowledgments
This research was supported in part by the Intramural
Research Program of the NIH, National Institute of
Diabetes and Digestive and Kidney Diseases, by the
Grant of the Korea Science and Engineering Founda-
tion (R01-2005-000-10162-0), and by Pusan National
University Research Grant, 2003 (H.R. Moon).
for 60 min with a final concentration of 15 nM
3
[
H]CGS21680 in a mixture containing 50 lL of increas-
ing concentrations of a test ligand and 200 lL of 50 mM
0
Tris–HCl, pH 7.4, containing 10 mM MgCl . N-5 -Eth-
2
yluronamidoadenosine (10 lM) was used to define non-
specific binding. The reaction was terminated by
filtration with GF/B filters.
References and notes
3
.48.3. Binding assay at the human A AR. Each tube in
3
the competitive binding assay contained 100 ll mem-
1
25
1. Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz,
K. N.; Linden, J. Pharmacol. Rev. 2001, 53, 527–552.
brane suspension (20 lg protein), 50 ll [ I]I-AB-
MECA (1.0 nM), and 50 ll of increasing concentrations
of the test ligands in Tris–HCl buffer (50 mM, pH 8.0)
2
. Yan, L.; Burbiel, J. C.; Maass, A.; M u¨ ller, C. E. Expert
Opin. Emerging Drugs 2003, 8, 537–576.
containing 10 mM MgCl , 1 mM EDTA. Nonspecific
2
3. Liang, B. T.; Jacobson, K. A. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 6995–6999.
0
binding was determined using 10 lM of 5 -N-ethylcarb-
oxamidoadenosine in the buffer. The mixtures were
incubated at 25 ꢁC for 60 min. Binding reactions were
terminated by filtration through Whatman GF/B filters
under reduced pressure using a MT-24 cell harvester
(Brandell, Gaithersburgh, MD, USA). Filters were
washed three times with 9 mL ice-cold buffer. Radioac-
4. Jacobson, K. A. Trends Pharmacol. Sci. 1998, 19, 184–191.
5. Yao, Y.; Sei, Y.; Abbracchio, M. P.; Kim, Y.-C.;
Jacobson, K. A. Biochem. Biophys. Res. Commun. 1997,
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32, 317–322.
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001, 59, 76–82.
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Valle, L.; Khalili, K. Oncogene 2002, 21, 4060–4064.
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guez, C.; Olah, M. E.; Stiles, G. L.; Daly, J. W. FEBS Lett.
6
7
8
2
tivity was determined in a Beckman 5500B c-counter.
3
.49. Cyclic AMP accumulation assay
1
993, 336, 57–60.
Intracellular cyclic AMP levels were measured with a
2
9. Madi, L.; Ochaion, A.; Rath-Wolfson, L.; Bar-Yehuda,
S.; Erlanger, A.; Ohana, G.; Harish, A.; Merimski, O.;
Barer, F.; Fishman, P. Clin. Cancer Res. 2004, 10, 4472–
2
competitive protein binding method. CHO cells that
expressed recombinant human A ARs were harvested
3
4
479.
by trypsinization. After centrifugation and resuspension
in medium, cells were plated in 24-well plates in 0.5 mL
medium. After 24 h, the medium was removed, and cells
1
0. Baharav, E.; Bar-Yehuda, S.; Madi, L.; Silberman, D.;
Rath-Wolfson, L.; Halpren, M.; Ochaion, A.; Weinberger,
A.; Fishman, P. J. Rheumatol. 2005, 32, 469–476.
were washed three times with 1 mL DMEM, containing
0
1
1. Kim, H. O.; Ji, X-d; Siddiqi, S. M.; Olah, M. E.; Stiles, G.
L.; Jacobson, K. A. J. Med. Chem. 1994, 37, 3614–3621.
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5
0 mM N-(2-hydroxyethyl)piperazine-N -(2-ethanesulf-
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agonists and/or test compounds in the presence of roli-
pram (10 lM) and adenosine deaminase (3 U/mL).
After 45 min forskolin (10 lM) was added to the medi-
um, and incubation was continued an additional
1
1
5 min. The reaction was terminated upon removal of
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 ꢂ20 ꢁC. For determination
of cyclic AMP production, protein kinase A (PKA)
2
006, 49, 273–281.
1
3. Tracey, W. R.; Magee, W. P.; Oleynek, J. J.; Hill, R. J.;
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3
was incubated with [ H]cyclic AMP (2 nM) in
K HPO /EDTA buffer (K HPO , 150 mM; EDTA,
1
2
4
2
4
1
0 mM), 20 lL of the cell lysate, and 30 lL of 0.1 M
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00 lL for standard curve). Bound radioactivity was
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Varani, K.; Borea, P. A.; Spalluto, G. Med. Res. Rev.
2
2
000, 20, 103–128.
separated by rapid filtration through Whatman GF/C
filters and washed once with cold buffer. Bound radioac-
tivity was measured by liquid scintillation spectrometry.
1
1
1
6. Klotz, K.-N.; Hessling, J.; Hegler, J.; Owman, B.; Kull, B.;
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3
.50. Statistical analysis
Binding and functional parameters were calculated using
Prism 5.0 software (GraphPAD, San Diego, CA, USA).
IC50 values obtained from competition curves were

4
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2