Design, synthesis, and binding of homologated truncated 4′-thioadenosine derivatives at the human A3 adenosine receptors

Article abstract of DOI:10.1016/j.bmc.2010.08.018

We synthesized homologated truncated 4′-thioadenosine analogues 3 in which a methylene (CH₂) group was inserted in place of the glycosidic bond of a potent and selective A₃ adenosine receptor antagonist 2. The analogues were designed

Full text of DOI:10.1016/j.bmc.2010.08.018

Bioorganic & Medicinal Chemistry 18 (2010) 7015–7021
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and binding of homologated truncated 40-thioadenosine
derivatives at the human A3 adenosine receptors
Hyuk Woo Lee a, Hea Ok Kim b, Won Jun Choi a,c, Sun Choi b, Jin Hee Lee b, Seul-gi Park b, Lena Yoo d,
Kenneth A. Jacobson d, Lak Shin Jeong a,b,
*
a Department of Bioinspired Science, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
b Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
c College of Pharmacy, Dongguk University, Kyungki-do 410-774, Republic of Korea
d Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive Diseases and Kidney Diseases, National Institutes of Health, Bethesda,
MD 20892-0810, USA
a r t i c l e i n f o
a b s t r a c t
We synthesized homologated truncated 40-thioadenosine analogues 3 in which a methylene (CH2) group
was inserted in place of the glycosidic bond of a potent and selective A3 adenosine receptor antagonist 2.
The analogues were designed to induce maximum binding interaction in the binding site of the A3 aden-
osine receptor. However, all homologated nucleosides were devoid of binding affinity at all subtypes of
adenosine receptors, indicating that free rotation through the single bond allowed the compound to
adopt an indefinite number of conformations, disrupting the favorable binding interaction essential for
receptor recognition.
Article history:
Received 8 July 2010
Revised 6 August 2010
Accepted 7 August 2010
Available online 14 August 2010
Keywords:
Homologation
A3 adenosine receptor
Binding affinity
Truncated 40-thioadenosine
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
cently discovered to be good templates for the design of A3 AR
ligands.4,5 Compound 14,5 was found to be a very potent A3 AR
Adenosine, an endogenous modulator controls many physiolog-
ical functions through binding to the adenosine receptors (ARs)
which consist of four subtypes, A1, A2A, A2B, and A3.1 Binding of aden-
osine to the ARs changes the levels of the second messengers such as
diacylglycerol (DAG) and cyclic 30,50-monophosphate (cAMP) for
signal transduction.1 Therefore, adenosine receptors are regarded
as promising therapeutic targets for the treatment of many types
of diseases related to these signal transduction pathways.2
agonist (Ki = 0.38 nM) and showed very potent in vivo antitumor
activity in several types of tumor xenograft models ( Fig. 1).
A mechanistic study revealed that compound 1 induced cell-cycle
arrest by lowering levels of c-myc and cyclin D1 in a concentra-
tion- and time-dependent manner at lower concentrations and in-
duced apoptosis through PARP cleavage at higher concentrations.6
Compound 1 also inhibited the Wnt signaling pathway at a 10 nM
concentration.6
Among four subtypes of ARs, A3 AR is most recently identified
receptor and many efforts have been made to modulate A3 AR for
the treatment of several diseases such as cancer, cardiac ischemia,
asthma, glaucoma, and inflammation.3 40-Thionucloesides were re-
A molecular modeling study indicated that the NH of the meth-
ylamide of 1 serves as a hydrogen bonding donor required for the
receptor activation.7 On the basis of these findings, it was hypoth-
esized that the truncated analogue 2 derived from compound 1 in
which a hydrogen bonding donor NH, essential for the A3AR ago-
nist activity, was removed, could function as an A3 AR antagonist.
Compound 2 turned out to be a potent and selective antagonist
at the human (Ki = 4.16 nM) as well as rat (Ki = 3.89 nM) A3 AR.8
Introduction of a C–C single bond in a structure increases the
free rotation. The increase of the free rotation allows the molecule
to adopt many conformations, making it possible to induce maxi-
mum binding interaction in the binding site of the receptor. On
the basis of this hypothesis, we designed and synthesized the
homologated structure 3 in which a methylene (CH2) group was
inserted in place of the glycosidic bond of a potent and selective
Abbreviations: AR, adenosine receptor; CCPA, 2-chloro-N6-cyclopentyladeno-
sine; CHO, Chinese hamster ovary; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-50-N-
methylcarbamoyladenosine;
[
125I]I-AB-MECA,
[
125I]N6-(4-aminobenzyl)-50-N-
methylcarboxamidoadenosine; NECA, 50-N-ethylcarboxamidoadenosine; CPA, N6-
cyclopentyladenosine; CGS 21680, 2-[4-(2-carboxyethyl)phenethylamino]-50-N-
ethylcarboxamido-adenosine; PARP, poly (ADP-ribose) polymerase.
* Corresponding author. Address: Department of Bioinspired Science and Labo-
ratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, 11-
1 Seodaemun-gu, Daehyun-dong, Seoul 120-750, Republic of Korea. Tel.: +82 2 3277
3466; fax: +82 2 3277 2851.
E-mail address: lakjeong@ewha.ac.kr (L.S. Jeong).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.018

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H. W. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7015–7021
HN
HN
N
NHR
N
I
N
I
N
N
N
N
N
N
N
MeHN
O
N
Cl
N
Cl
S
S
S
X
HO
OH
HO
OH
HO
OH
1 (A3 AR agonist, Ki = 0.38 nM)
2 (A3 AR antagonist, Ki = 4.16 nM)
3 (R = 3-halobenzyl, X = H or Cl)
Figure 1. The rationale for the design of the target nucleoside 3.
A3 AR antagonist 2 with the expectation of maximum binding
interaction in the binding site of the receptor, as potential A3 AR
antagonists. Herein, we report the synthesis of the homologated
isomers. The structural assignment of N9-isomer and N7-isomer
was accomplished by the comparison of UV and 13C NMR data re-
ported in literatures.8,9 Removal of the isopropylidene group of 10
using 2 N HCl gave the 2-H analogue 12. Treatment of the 6-chlo-
ropurine derivative 12 with 3-halobenzyl amines yielded the N6-
(3-halobenzyl)amine derivatives 3a–d. Similarly, the 2,6-dichloro-
purine derivative 11 was converted to the 2-Cl analogues 3e–h via
compound 13.
truncated 40-thioadenosine analogues 3 from
D-mannose and the
results of binding assays at the human ARs.
2. Results and discussion
All AR binding experiments were performed using adherent
mammalian CHO (Chinese hamster ovary) cells stably transfected
with cDNA encoding the appropriate human ARs.10 Binding was
carried out using 1 nM [3H]CCPA, 10 nM [3H]CGS 21680, or 0.5
nM [125I]I-AB-MECA as radioligands for A1, A2A, and A3 ARs, respec-
tively. Values are expressed as mean sem, n = 3–5 (outliers elim-
inated). If a percentage is given, it represents the percent inhibition
Our synthetic strategy was to synthesize the homologated gly-
cosyl donor and then to condense it with a purine base. The homol-
ogated glycosyl donor 7 was first synthesized from
shown in Scheme 1. -Mannose was converted to the diol 4 accord-
ing to our previously published procedure.8
-Mannose was
D-mannose as
D
D
converted to the diacetonide 4 by treating with 2,2-dimethoxypro-
pane and acetone under acidic conditions. Reduction of 4 with so-
dium borohydride followed by mesylation of the resulting diol
afforded the dimesylate 5. Treatment of 5 with anhydrous sodium
sulfide in DMF at 80 °C yielded the thiosugar 6, which was hydro-
lyzed with 60% acetic acid to give diol 6. Oxidative cleavage of the
diol 6 with 1.2 equiv of Pb(OAc)4 produced the aldehyde 7, but use
of excess Pb(OAc)4 gave the anomeric acetate, resulting from the
oxidative decarboxylation.8 Reduction of the aldehyde 7 with
NaBH4 afforded the primary alcohol 8. Treatment of 8 with phos-
phorous oxychloride yielded the glycosyl donor 9, which was
directly used for the condensation with purine bases.
at a fixed concentration of 10 lM. As shown in Table 1, all homol-
ogated compounds synthesized in this study bound weakly for
determination of affinities at three subtypes of ARs. This result
indicated that the homologation of the rotatable methylene (CH2)
between 10 and N9 positions disrupted favorable binding interac-
tions at the ARs.
Since A2A is the only AR subtype whose X-ray crystal structure is
available (PDB code: 3EML),11 compounds 2 and 3h were docked
into the A2A AR structure, considering the flexibility of the binding
site residues (Fig. 2).
The docking results of compounds 2 and 3h were significantly
different as shown in Figure 2. Compound 2 (Ki = 341 nM for
hA2A) showed a binding mode that was consistent with previous
docking.7 Its N6-amino group formed an H-bond with Glu169,
and the 20- and 30-OH groups were involved in H-bonding with
The final nucleosides 3a–d and 3e–h were synthesized from di-
rect SN2 displacement of the chloride 9 with 6-chloropurine and
2,6-dichloropurine anions, as shown in Scheme 2. Condensation
of 9 with 6-chloropurine and 2,6-dichloropurine in the presence
of NaH in DMF afforded the desired N9-isomers, 6-chloropurine
derivative 10 and the 2,6-dichloropurine derivative 11, respec-
tively without concomitant formation of the corresponding N7-
Ser277 and His278. In addition, the adenine ring made the
stacking with Phe168 (Fig. 2A).
p–p
O
O
b,c
O
a
O
O
O
O
O
O
D-Mannose
OMs
OH
OMs
95%
86%
4
5
d,e
63%
O
OH
OH
Cl
OH
O
g
f
h
S
S
S
S
91%
from 6
O
O
O
O
O
O
O
9
8
7
6
Scheme 1. Reagents and conditions: (a) 2,2-dimethoxypropane, camphosulfonic acid, acetone, rt; (b) NaBH4, EtOH, rt; (c) MsCl, Et3N, CH2Cl2, rt; (d) Na2S, DMF, 80 °C; (e) 60%
AcOH, rt; (f) Pb(OAc)4, EtOAc, 0 °C; (g) NaBH4, EtOH, 0 °C; (h) POCl3, CH3CN, rt.

H. W. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7015–7021
7017
HN
N
N
Cl
N
N
Cl
N
N
X
N
N
Cl
N
S
c
a
b
N
N
S
N
S
S
R
94%
O
O
OH OH
OH OH
12
O
O
3a (X = F) (75%)
3b (X = Cl) (83%)
3c (X = Br) (78%)
3d (X = I) (75%)
9
10 (R = H) (67%)
11 (R = Cl) (72%)
b
92%
HN
N
Cl
N
X
N
N
N
N
c
N
N
S
OH OH
S
Cl
Cl
OH OH
13
3e (X = F) (82%)
3f (X = Cl) (77%)
3g (X = Br) (73%)
3h (X = I) (79%)
Scheme 2. Reagents and conditions: (a) NaH, 6-chloropurine or 2,6-dichloropurine, DMF, rt; (b) 2 N HCl, THF, rt; (c) 3-halobenzylamine, Et3N, EtOH.
Table 1
Inhibition of radioligand binding by known A3AR antagonist 2 and homologated 40-thioadenosine derivatives 3a–d and 3e–h at three
subtypes of ARsa
HN
N
N
N
X
N
S
R
HO
OH
Compounds
Ki (hA1AR)
Ki (hA2AAR)
Ki (hA3AR)
nM or % displ. at 10
l
M
nM or % displ. at 10
lM
nM or % displ. at 10 lM
3a (R = H, X = F)
3b (R = H, X = Cl)
3c (R = H, X = Br)
3d (R = H, X = I)
3e (R = Cl, X = F)
3f (R = Cl, X = Cl)
3g (R = Cl, X = Br)
3h (R = Cl, X = I)
2
0.3 0.3%
3.3 3.3%
6.6 3.8%
9.6 4.7%
7.9 4.0%
3.2 1.9%
23.6 3.4%
16.4 4.6%
2490 940
14.0 5.6%
13.0 8.2%
25.8 6.3%
18.2 7.1%
12.0 12.0%
11.0 6.5%
2.0 1.2%
8.7 6.0%
8.9 1.8%
6.1 3.2%
13.9 5.5%
17.6 4.6%
13.6 2.9%
21.0 4.7%
10.0 5.0%
4.16 0.50
18.4 4.3%
341 75
a
All AR experiments were performed using adherent mammalian cells stably transfected with cDNA encoding the human ARs (CHO
cells for A1AR and A3AR, HEK-293 cells for A2AAR). Percent inhibition of binding at the human A3AR was determined at 10 lM. Binding
was carried out using radioligand [3H]R-PIA at the human A1AR, [3H]CGS 21680 at the human A2AAR, or [125I]I-AB-MECA at the human
A3AR. Values from the present study are expressed as mean sem, n = 3–5.
In contrast with compound 2, compound 3h showed various
binding modes, losing important interactions ( Fig. 2B). It may
retain or lose the H-bonding interaction with Glu169 and/or the
binding region are known to be critical for nucleoside recognition
in the ARs.7
p–p stacking with Phe168. Furthermore, it is more likely to lose
3. Conclusions
H-bonding of the OH groups in the thio-ribose with Ser277/
His278. This might be due to the rotatable extra methylene group
(between the thio-ribose and the adenine ring), which might ex-
plain why compound 3h lost its binding affinity at the A2A AR.
Structural and conformational factors in the hydrophilic ribose
In this study, the synthesis of homologated truncated 40-thioad-
enosine derivatives from D-mannose and assay of their binding
affinities at the ARs were accomplished. The CH2 homologation in-
serted in place of the glycosyl bond was introduced to allow max-

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H. W. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7015–7021
HN
HN
N
I
N
N
N
I
N
N
N
N
Cl
S
S
Cl
HO
OH
3h
HO
OH
2
Figure 2. Predicted binding modes of compounds 2 and 3h in the A2A AR crystal structure. (A) Compound 2 (in magenta) interacts with the binding site residues. (B)
Compound 3h (in orange) shows various binding modes. The key residues are displayed in capped-stick with carbon atoms in white. Hydrogen bonds are drawn in black
dashed lines and non-polar hydrogens are undisplayed for clarity.
imum binding interaction through the single bond rotation, but the
rotatable extra methylene group resulted in losing important inter-
actions as illustrated in various predicted binding modes.
cated the absence of starting material. The reaction mixture was
filtered and the filtrate was diluted with EtOAc. The organic layer
was washed with saturated aqueous NaHCO3 solution, dried over
anhydrous MgSO4, and evaporated to give the aldehyde 7, which
was used for next step without further purification. To a stirred
solution of aldehyde 7 (5.6 g, 30.0 mmol) in EtOH (70 mL) was
carefully added sodium borohydride (1.3 g, 33.6 mmol) in several
portions at 0 °C and the reaction mixture was stirred for 30 min
at the same temperature and neutralized with glacial AcOH. After
the removal of the solvent, the mixture was partitioned between
EtOAc (150 mL) and brine (100 mL). The organic layer was dried
over anhydrous MgSO4, filtered, and evaporated. The resulting res-
idue was purified by silica gel column chromatography (hexane/
EtOAc = 2:1) to give 8 (5.2 g, 91%) as a colorless syrup: 1H NMR
(CDCl3) d 1.27 (s, 3H, CH3), 1.47 (s, 3H, CH3), 2.68 (br s, 1H, OH),
2.84 (dd, 1H, J = 2.0, 12.6 Hz, CHH), 3.06 (dd, 1H, J = 5.2, 13.0 Hz,
CHH), 3.38 (m, 1H, CH), 3.56 (dd, 2H, J = 2.8, 8.8 Hz, HOCH2), 4.66
(dd, 1H, J = 1.6, 5.6 Hz, OCH), 4.86 (td, 1H, J = 1.6, 4.8 Hz, OCH);
13C NMR (CDCl3) d 24.75, 26.66, 37.42, 56.62, 63.32, 83.70, 85.94,
111.21; FAB-MS m/z 191 (M+H+).
4. Experimental section
4.1. General methods
1H NMR spectra (CDCl3, CD3OD, or DMSO-d6) were recorded on
Varian Unity Inova 400 MHz. Chemical shifts were reported in ppm
units with TMS as the internal standard. 13C NMR spectra (CDCl3,
CD3OD, or DMSO-d6) were recorded on Varian Unity Inova
100 MHz. Optical rotations were determined on Jasco III in metha-
nol. UV spectra were recorded on U-3000 made by Histachi in
methanol. Elemental analyses were measured on EA1110. The
crude products were purified using a silica gel 60 (230–400 mesh,
Merck). Reagents were purchased from Aldrich Chemical Com-
pany. All the anhydrous solvents were distilled over CaH2 or P2O5
or Na/benzophenone prior to the reaction.
4.2. Chemical synthesis
4.2.2. 4-Chloromethyl-2,2-dimethyl-tetrahydro-thieno[3,4-
d][1,3]dioxole (9)
To a stirred solution of 8 (8.5 g, 40.7 mmol) in anhydrous aceto-
nitrile (100 mL) was added POCl3 (4.47 mL, 48.9 mmol) at 0 °C and
the reaction mixture was stirred for 30 min at room temperature.
The reaction mixture was dried in vacuo and co-evaporated with
4.2.1. (2,2-Dimethyl-tetrahydro-thieno[3,4-d][1,3]dioxol-4-yl)-
methanol (6)
To a stirred solution of 68 (20 g, 90.8 mmol) in ethyl acetate
(500 mL) was added Pb(OAc)4 (48.3 g, 109 mmol) at 0 °C and the
reaction mixture was stirred for 10 min at which time TLC indi-

H. W. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7015–7021
7019
toluene twice to give 9, which was directly used for next step with-
out further purification: 1H NMR (CDCl3) d 1.27 (s, 3H, CH3), 1.46 (s,
3H, CH3), 2.88 (m, 2H, CH2), 3.08 (dd, 1H, J = 4.8, 13.2 Hz, CH), 3.44
(m, 1H, CHHCl), 3.65 (dd, 1H, J = 4.0, 8.2 Hz, CHHCl), 4.81 (dd, 1H,
J = 0.8, 5.6 Hz, OCH), 4.89 (m, 1H, OCH); 13C NMR (CDCl3) d 24.73,
26.54, 37.87, 45.72, 55.32, 83.58, 86.18, 111.31.
1H NMR (DMSO-d6) d 2.61 (dd, 1H, J = 4.0, 10.8 Hz, CHH), 2.92 (dd,
1H, J = 4.4, 10.8 Hz, CHH), 3.67(m, 1H, CHHN), 3.80 (dd, 1H, J = 3.2,
6.6 Hz, CHHN), 4.11 (m, 1H, CHOH), 4.37 (dd, 1H, J = 7.2, 14.0 Hz,
CHOH), 4.55 (dd, 1H, J = 6.8, 14.2 Hz, CHOH), 5.04 (br s, 2H, OH),
8.72 (s, 1H, H-8); 13C NMR (DMSO-d6) d 33.19, 47.52, 73.58,
77.74, 130.40, 147.75, 148.65, 149.57, 153.53, 153.75.
4.2.5. General procedure for the N6-substitution reaction
4.2.3. General procedure for the condensation
To a solution of 6-chloropurine (21.69 mmol) and 2,6-dichloro-
purine (21.69 mmol) in a solution of anhydrous DMF (50 mL) were
added NaH (21.69 mmol) and the mixture was stirred at room
temperature until the solution became clear. A solution of 9
(18.07 mmol) in anhydrous DMF (10 mL) was added to the result-
ing solution at room temperature and stirred overnight at room
temperature. The reaction mixture was diluted with EtOAc
(100 mL) and washed with water several times, dried with anhy-
drous MgSO4 and evaporated. The residue was purified by flash sil-
ica gel column chromatography (hexane/EtOAc = 2:1) to give the
condensed products 10 and 11, respectively.
To
a
stirred solution of 6-chloropurine derivative 12
(0.45 mmol) or 2,6-dichloropurine derivative 13 (0.45 mmol) and
an appropriate amine hydrochloride salts or free amines
(0.90 mmol) in EtOH (10 mL) was added Et3N (1.35 mmol) and
the solution was stirred overnight at room temperature. After
removing the solvent under reduced pressure, the residue was
purified by flash silica gel column chromatography (CH2Cl2/
EtOAc/MeOH = 10:10:1) to give the N6-substituted amine deriva-
tives 3a–h.
4.2.5.1. N6-(3-Fluorobenzylamino)-9-ylmethyl-(4-thio-b-
D-ribo-
furanosyl)adenine (3a). Yield = 75%; white solid; mp 117–
4.2.3.1. 6-Chloro-9-(2,2-dimethyl-tetrahydro-thieno[3,4-d][1,3]
dioxol-4-ylmethyl)-9H-purine (10). Yield = 67%; white foam; UV
120 °C; UV (MeOH) kmax 274 nm (pH 7); ½a D25
ꢀ
+53.3° (c 0.30,
MeOH); 1H NMR (DMSO-d6) d 2.61 (dd, 1H, J = 5.2, 10.8 Hz, CHH),
2.88 (dd, 1H, J = 5.2, 10.4 Hz, CHH), 3.68 (m, 1H, CH), 3.78 (m, 1H,
NHCHH), 4.14 (m, 1H, NHCHH), 4.19 (dd, 1H, J = 7.6, 14.0 Hz,
CHHN), 4.47 (dd, 1H, J = 6.0, 14.2 Hz, CHHN), 4.66 (br s, 2H,
CHOH ꢁ 2), 5.02 (d, 1H, J = 4.8 Hz, OH), 5.09 (d, 1H, J = 5.6 Hz,
OH), 7.10 (t, 1H, J = 8.0 Hz, NBn-H), 7.36 (d, 1H, J = 7.6 Hz, NBn-
H), 7.58 (d, 1H, J = 7.6 Hz, NBn-H), 7.73 (s, 1H, NBn-H), 8.16 (s,
1H, H-8), 8.21 (s, 1H, H-2), 8.36 (br s, 1H, NHBn). 13C NMR
(DMSO-d6) d 32.82, 42.26, 46.81, 48.77, 73.56, 76.88, 94.70,
118.94, 126.69, 130.45, 135.32, 135.76, 141.04, 142.94, 148.94,
152.34, 154.17; FAB-MS m/z 376 (M+H+); Anal. Calcd for
C17H18FN5O2S: C, 54.39; H, 4.83; N, 18.65; S, 8.54. Found: C,
54.32; H, 4.96; N, 18.77; S, 8.62.
(MeOH) kmax 265 nm (pH 7); ½a D25
ꢀ
+128.0° (c 0.25, MeOH); FAB-MS
m/z 327 (M+H+); 1H NMR (CDCl3) d 1.19 (s, 3H, CH3), 1.38 (s, 3H,
CH3), 2.88 (m, 2H, CH2), 3.72 (td, 1H, J = 1.2, 2.4 Hz, CH), 4.26 (dd,
1H, J = 8.0, 16.0 Hz, CHHN), 4.59 (dd, 1H, J = 6.8, 14.0 Hz, CHHN),
4.66 (dd, 1H, J = 2.0, 5.0 Hz, OCH), 4.79 (m, 1H, OCH), 8.28 (s, 1H,
H-8), 8.67 (s, 1H, H-2); 13C NMR (CDCl3) d 24.70, 26.49, 36.99,
46.07, 54.13, 83.20, 86.02, 111.88, 131.18, 145.53, 151.07, 151.84,
152.16; Anal. Calcd for C13H15ClN4O2S: C, 47.78; H, 4.63; N,
17.14; S, 9.81. Found: C, 47.82; H, 4.65; N, 17.20; S, 9.85.
4.2.3.2.
2,6-Dichloro-9-(2,2-dimethyl-tetrahydro-thieno[3,4-
d][1,3]dioxol-4-ylmethyl)-9H-purine (11). Yield = 72%; white
foam; UV (MeOH) kmax 275 nm (pH 7); ½a D25
ꢀ
+173.3° (c 0.30,
MeOH); FAB-MS m/z 361 (M+); 1H NMR (CDCl3) d 1.27 (s, 3H,
CH3), 1.47 (s, 3H, CH3), 2.98 (m, 2H, CH2), 3.74 (td, 1H, J = 2.0,
6.2 Hz, CH), 4.21 (dd, 1H, J = 8.8, 14.2 Hz, CHHN), 4.47 (dd, 1H,
J = 6.8, 14.2 Hz, CHHN), 4.69 (dd, 1H, J = 2.0, 5.6 Hz, OCH), 4.89
(m, 1H, OCH), 8.23 (s, 1H, H-8); 13C NMR (CDCl3) d 24.82, 26.62,
37.04, 46.18, 54.13, 83.32, 86.22, 112.20, 146.13, 145.53, 152.14,
153.32, 153.37; Anal. Calcd for C13H14Cl2N4O2S: C, 43.22; H, 3.91;
N, 15.51; S, 8.88. Found: C, 43.29; H, 3.96; N, 15.63; S, 8.82.
4.2.5.2. N6-(3-Chlorobenzylamino)-9-ylmethyl-(4-thio-b-
D-ribo-
furanosyl)adenine (3b). Yield = 83%; white solid; mp 143–
147 °C; UV (MeOH) kmax 275 nm (pH 7); ½a D25
ꢀ
+71.8° (c 0.32,
MeOH); 1H NMR (DMSO-d6) d 2.61 (dd, 1H, J = 5.2, 10.4 Hz, CHH),
2.87 (dd, 1H, J = 5.2, 10.4 Hz, CHH), 3.68 (m, 1H, CH), 3.78 (m, 1H,
NHCHH), 4.16 (m, 1H, NHCHH), 4.19 (dd, 1H, J = 7.6, 14.0 Hz,
CHHN), 4.46 (dd, 1H, J = 8.0, 14.0 Hz, CHHN), 4.69 (br s, 2H,
CHOH ꢁ 2), 5.01 (d, 1H, J = 4.8 Hz, OH), 5.08 (d, 1H, J = 5.6 Hz,
OH), 7.25 (t, 1H, J = 8.0 Hz, NBn-H), 7.35 (d, 1H, J = 7.6 Hz, NBn-
H), 7.39 (d, 1H, J = 7.6 Hz, NBn-H), 7.54 (s, 1H, NBn-H), 8.16 (s,
1H, H-8), 8.22 (s, 1H, H-2), 8.38 (br s, 1H, NHBn); 13C NMR
(DMSO-d6) d 32.81, 42.40, 46.81, 48.76, 73.56, 76.83, 118.93,
121.52, 126.28, 129.46, 129.86, 130.42, 141.06, 143.09, 148.97,
152.34, 154.18; FAB-MS m/z 392 (M+H+); Anal. Calcd for
4.2.4. General procedure for the removal of the isopropylidene
group
To a stirred solution of 10 (12.4 mmol) and 11 (12.4 mmol) in
THF (50 mL) was added 2 N HCl at room temperature and the mix-
ture was stirred for 24 h at same temperature. The mixture was
neutralized with NH4OH and evaporated. The residue was purified
by flash silica gel column chromatography (CH2Cl2/EtOAc/
MeOH = 10:10:1) to give the products, 12 and 13, respectively.
C17H18ClN5O2S: C, 52.10; H, 4.63; N, 17.87; S, 8.18. Found: C,
52.33; H, 4.64; N, 17.90; S, 8.16.
4.2.5.3. N6-(3-Bromobenzylamino)-9-ylmethyl-(4-thio-b-
D-ribo-
4.2.4.1. 2-(6-Chloro-purin-9-ylmethyl)-tetrahydro-thiophene-
furanosyl)adenine (3c). Yield = 78%; white solid; mp 134–
3,4-diol (12). Yield = 94%; white foam; UV (MeOH) kmax 265 nm
139 °C; UV (MeOH) kmax 270 nm (pH 7); ½a D25
ꢀ
+72.0° (c 0.25,
(pH 7); FAB-MS m/z 287 (M+H+); ½a D25
ꢀ
+53.1° (c 0.65, MeOH); 1H
MeOH); 1H NMR (DMSO-d6) d 2.62 (dd, 1H, J = 5.2, 10.6 Hz, CHH),
2.87 (dd, 1H, J = 4.8, 10.2 Hz, CHH), 3.67 (m, 1H, CH), 3.78 (m,
1H, NHCHH), 4.14 (m, 1H, NHCHH), 4.19 (dd, 1H, J = 7.6, 14.2 Hz,
CHHN), 4.47 (dd, 1H, J = 6.4, 14.0 Hz, CHHN), 4.70 (br s, 2H,
CHOH ꢁ 2), 5.02 (d, 1H, J = 5.2 Hz, OH), 5.08 (d, 1H, J = 5.6 Hz,
OH), 7.31 (m, 3H, NBn-H), 7.39 (s, 1H, NBn-H), 8.16 (s, 1H, H-8),
8.21 (s, 1H, H-2), 8.37 (br s, 1H, NHBn). 13C NMR (DMSO-d6) d
32.18, 42.45, 46.81, 48.77, 73.56, 76.84, 118.96, 125.87, 126.56,
126.95, 130.10, 132.86, 141.06, 142.83, 148.99, 152.34, 154.21;
FAB-MS m/z 436 (M+); Anal. Calcd for C17H18BrN5O2S: C, 46.80;
NMR (DMSO-d6) d 2.62 (dd, 1H, J = 4.0, 10.0 Hz, CHH), 2.90 (dd,
1H, J = 4.0, 10.0 Hz, CHH), 3.70 (m, 1H, CH), 3.80 (br s, 1H, CHHN),
4.12 (br s, 1H, CHHN), 4.40 (dd, 1H, J = 7.2, 13.6 Hz, CHOH), 4.60
(dd, 1H, J = 7.2, 13.6 Hz, CHOH), 5.03 (br s, 2H, OH), 8.70 (s, 1H,
H-8), 8.78 (s, 1H, H-2); 13C NMR (DMSO-d6) d 33.04, 47.31, 48.00,
73.54, 77.40, 130.68, 147.60, 148.95, 151.51, 151.96.
4.2.4.2.
2-(2,6-Dichloro-purin-9-ylmethyl)-tetrahydro-thio-
phene-3,4-diol (13). Yield = 92%; white foam; UV (MeOH) kmax
275 nm (pH 7); FAB-MS m/z 321 (M+); ½a D25
ꢀ
+64.3° (c 0.60, MeOH);

7020
H. W. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7015–7021
H, 4.16; N, 16.05; S, 7.35. Found: C, 46.89; H, 4.21; N, 16.07; S,
7.41.
4.2.5.8. 2-Chloro-N6-(3-iodobenzylamino)-9-ylmethyl-(4-thio-b-
D
-ribofuranosyl)adenine (3h). Yield = 79%; white solid; mp 174–
180 °C; UV (MeOH) kmax 274 nm (pH 7); ½a D25
ꢀ
+136.4° (c 0.33,
MeOH); 1H NMR (DMSO-d6) d 2.62 (dd, 1H, J = 4.8, 10.6 Hz, CHH),
2.89 (dd, 1H, J = 4.8, 10.4 Hz, CHH), 3.65 (m, 1H, CH), 3.78 (m, 1H,
NHCHH), 4.13–4.20 (m, 2H, NHCHH, CHHN), 4.44 (dd, 1H, J = 6.0,
14.0 Hz, CHHN), 4.64 (br d, 2H, J = 4.8 Hz, CHOH ꢁ 2), 5.04 (d, 1H,
J = 4.8 Hz, OH), 5.09 (d, 1H, J = 5.6 Hz, OH), 7.27–7.40 (m, 4H,
NBn-H), 8.17 (s, 1H, H-8), 8.84 (br s, 1H, NHBn); 13C NMR
(DMSO-d6) d 32.94, 42.71, 47.07, 48.36, 73.60, 77.17, 118.07,
126.04, 126.81, 127.22, 130.19, 132.93, 141.69, 141.84, 149.98,
152.96, 154.75; FAB-MS m/z 518 (M+H+); Anal. Calcd for C17H17ClI-
N5O2S: C, 39.43; H, 3.31; N, 13.53; S, 6.19. Found: C, 39.52; H, 3.35;
N, 13.58; S, 6.22.
4.2.5.4.
N6-(3-Iodobenzylamino)-9-ylmethyl-(4-thio-b-
D-ribo-
furanosyl)adenine (3d). Yield = 75%; white solid; mp 172–
175 °C; UV (MeOH) kmax 272 nm (pH 7); ½a D25
ꢀ
+68.5° (c 0.35,
MeOH); 1H NMR (DMSO-d6) d 2.61 (dd, 1H, J = 5.2, 10.4 Hz, CHH),
2.87 (dd, 1H, J = 5.2, 10.6 Hz, CHH), 3.67 (m, 1H, CH), 3.78 (m, 1H,
NHCHH), 4.14 (m, 1H, NHCHH), 4.19 (dd, 1H, J = 7.6, 18.0 Hz,
CHHN), 4.46 (dd, 1H, J = 6.4, 14.0 Hz, CHHN), 4.71 (br s, 2H,
CHOH ꢁ 2), 5.02 (d, 1H, J = 4.8 Hz, OH), 5.08 (d, 1H, J = 5.6 Hz,
OH), 7.00–7.19 (m, 3H, NBn-H), 7.32 (m, 1H, NBn-H), 8.16 (s, 1H,
H-8), 8.21 (s, 1H, H-2), 8.36 (br s, 1H, NHBn); 13C NMR (DMSO-
d6) d 32.81, 42.51, 46.80, 48.76, 73.56, 76.83, 118.94, 123.15,
130.08, 130.16, 141.03, 143.28, 148.98, 152.34, 154.25, 160.95,
163.37; FAB-MS m/z 484 (M+H+); Anal. Calcd for C17H18IN5O2S: C,
42.25; H, 3.75; N, 14.49; S, 6.63. Found: C, 42.34; H, 3.78; N,
14.51; S, 6.65.
4.3. Molecular modeling study
Ligand structures were generated with Concord and energy
minimized using MMFF94s force field and MMFF94 charge until
the rms of Powell gradient was 0.05 kcal molꢂ1 Aꢂ1 in SYBYL
8.1.1 (Tripos Inc., St. Louis, MO, USA). The X-ray crystal structure
of human A2A AR (PDB code: 3EML) was prepared using Biopoly-
mer Structure Preparation Tool in SYBYL. The docking study was
performed using GOLD v.4.1.2 (Cambridge Crystallographic Data
Centre, Cambridge, UK), which employs a genetic algorithm (GA)
and allows for full ligand flexibility and partial protein flexibility.
The binding site was defined as the 9 Å around the co-crystallized
ligand (ZM-241385). The side chains of the eight residues (i.e.,
Thr88, Phe168, Glu169, Trp246, Leu249, Asn253, Ser277, and
His278) in the crystal ligand binding site were set to be flexible
with ‘crystal mode’. GoldScore scoring function was used and other
parameters were set as suggested by the GOLD authors except the
number of GA runs as 30. All computation calculations were under-
taken on IntelÒ Xeon™ Quad-core workstation with Linux Cent OS
release 4.6.
4.2.5.5. 2-Chloro-N6-(3-fluorobenzylamino)-9-ylmethyl-(4-thio-
b-D-ribofuranosyl)adenine (3e). Yield = 82%; white solid; mp
164–167 °C; UV (MeOH) kmax 275 nm (pH 7); ½a D25
ꢀ
+48.0° (c 0.50,
MeOH); 1H NMR (DMSO-d6) d 2.61 (dd, 1H, J = 4.8, 10.8 Hz, CHH),
2.89 (dd, 1H, J = 4.8, 10.4 Hz, CHH), 3.65 (m, 1H, CH), 3.78 (m, 1H,
NHCHH), 4.16 (m, 2H, NHCHH, CHHN), 4.43 (dd, 1H, J = 6.4,
14.0 Hz, CHHN), 4.60 (br d, 2H, J = 5.2 Hz, CHOH ꢁ 2), 5.03 (d, 1H,
J = 4.8 Hz, OH), 5.09 (d, 1H, J = 6.0 Hz, OH), 7.12 (t, 1H, J = 7.6 Hz,
NBn-H), 7.36 (d, 1H, J = 7.6 Hz, NBn-H), 7.59 (d, 1H, J = 8.0 Hz,
NBn-H), 7.74 (s, 1H, NBn-H), 8.17 (s, 1H, H-8), 8.83 (br s, 1H,
NHBn); 13C NMR (DMSO-d6) d 32.93, 42.54, 47.06, 48.36, 73.58,
77.14, 94.73, 118.06, 126.87, 130.52, 135.56, 136.08, 141.67,
141.94, 149.95, 152.95, 154.70; FAB-MS m/z 410 (M+H+); Anal.
Calcd for C17H17ClFN5O2S: C, 49.82; H, 4.18; N, 17.09; S, 7.82.
Found: C, 49.94; H, 4.25; N, 17.12; S, 7.85.
4.2.5.6.
thio-b-
2-Chloro-N6-(3-chlorobenzylamino)-9-ylmethyl-(4-
-ribofuranosyl)adenine (3f). Yield = 77%; white solid;
4.4. Binding assay
D
mp 154–158 °C; UV (MeOH) kmax 275 nm (pH 7); ½a D25
ꢀ
+77.5° (c
Human A1 and A2A adenosine receptors: For binding to human A1
0.40, MeOH); 1H NMR (DMSO-d6) d 2.62 (dd, 1H, J = 4.8, 10.6 Hz,
CHH), 2.90 (dd, 1H, J = 4.8, 10.8 Hz, CHH), 3.65 (m, 1H, CH), 3.78
(m, 1H, NHCHH), 4.11–4.20 (m, 2H, NHCHH, CHHN), 4.44 (dd, 1H,
J = 6.0, 14.0 Hz, CHHN), 4.63 (br d, 2H, J = 6.5 Hz, CHOH ꢁ 2), 5.04
(d, 1H, J = 4.8 Hz, OH), 5.09 (d, 1H, J = 5.6 Hz, OH), 7.27 (t, 1H,
J = 7.6 Hz, NBn-H), 7.35 (d, 1H, J = 7.6 Hz, NBn-H), 7.42 (d, 1H,
J = 7.6 Hz, NBn-H), 7.55 (s, 1H, NBn-H), 8.17 (s, 1H, H-8), 8.84 (br
s, 1H, NHBn); 13C NMR (DMSO-d6) d 32.93, 42.65, 47.06, 48.36,
73.59, 77.15, 118.07, 121.56, 126.45, 129.71, 130.14, 130.51,
141.69, 142.09, 149.97, 152.95, 154.72; FAB-MS m/z 426 (M+);
Anal. Calcd for C17H17Cl2N5O2S: C, 47.89; H, 4.02; N, 16.43; S,
7.52. Found: C, 47.92; H, 4.05; N, 16.50; S, 7.55.
receptors, [3H]PIA (1 nM) was incubated with membranes (40
lg/
tube) from CHO cells stably expressing human A1 receptors at
25 °C for 60 min in 50 mM Tris–HCl buffer (pH 7.4; MgCl2,
10 mM) in a total assay volume of 200
was determined using 10 M of CPA. For human A2A receptor bind-
ing, membranes (20 g/tube) from HEK-293 cells stably expressing
human A2AARs were incubated with 15 nM [3H]CGS21680 at 25 °C
for 60 min in 200 L 50 mM Tris–HCl, pH 7.4, containing 10 mM
lL. Nonspecific binding
l
l
l
MgCl2. Reaction was terminated by filtration with GF/B filters.
Human A3 adenosine receptor: For competitive binding assay,
each tube contained 100
tein) from CHO cells stably expressing the human A3AR, 50
125I]I-AB-MECA (0.5 nM), and 50
L of increasing concentrations
l
L suspension of membranes (20
lg pro-
l
L of
[
l
4.2.5.7.
thio-b-
N6-(3-Bromobenzylamino)-2-chloro-9-ylmethyl-(4-
-ribofuranosyl)adenine (3g). Yield = 73%; white solid;
of the nucleoside derivative in Tris–HCl buffer (50 mM, pH 7.4)
containing 10 mM MgCl2, 1 mM EDTA. Nonspecific binding was
determined using 10 lM of Cl-IB-MECA in the buffer. The mixtures
were incubated at 25 °C for 60 min. Binding reactions were termi-
nated 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 buf-
D
mp 165–169 °C; UV (MeOH) kmax 274 nm (pH 7); ½a D25
ꢀ
+70.0° (c
0.50, MeOH); 1H NMR (DMSO-d6) d 2.62 (dd, 1H, J = 4.4, 10.6 Hz,
CHH), 2.89 (dd, 1H, J = 5.2, 10.6 Hz, CHH), 3.66 (m, 1H, CH), 3.79
(m, 1H, NHCHH), 4.13–4.21 (m, 2H, NHCHH, CHHN), 4.44 (dd, 1H,
J = 6.4, 13.8 Hz, CHHN), 4.65 (br d, 2H, J = 5.2 Hz, CHOH ꢁ 2), 5.04
(d, 1H, J = 4.8 Hz, OH), 5.10 (d, 1H, J = 5.6 Hz, OH), 7.02–7.30 (m,
4H, NBn-H), 8.17 (s, 1H, H-8), 8.84 (br s, 1H, NHBn); 13C NMR
(DMSO-d6) d 32.95, 42.76, 47.08, 48.38, 73.62, 77.18, 118.01,
123.32, 130.21, 130.29, 141.67, 142.32, 149.98, 152.99, 154.81,
160.96, 163.37; FAB-MS m/z 472 (M+H+); Anal. Calcd for
fer. Radioactivity was determined in a Beckman 5500B c-counter.
For binding at all three subtypes, Ki values are expressed as
mean sem, n = 3–5 (outliers eliminated), and normalized against
a non-specific binder, 50-N-ethylcarboxamidoadenosine (NECA,
10
cific radioligand binding at 10
NECA assigned as 100%, is given.
l
M).9 Alternately, for weak binding a percent inhibition of spe-
C17H17BrClN5O2S: C, 43.37; H, 3.64; N, 14.88; S, 6.81. Found: C,
l
M, relative to inhibition by 10 lM
43.40; H, 3.70; N, 14.90; S, 6.85.

H. W. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7015–7021
7021
6. Kim, S.-J.; Min, H.-Y.; Chung, H.-J.; Park, E.-J.; Hong, J.-Y.; Kang, Y.-J.; Shin, D.-H.;
Jeong, L. S.; Lee, S. K. Cancer Lett. 2008, 264, 309.
Acknowledgments
7. Kim, S.-K.; Gao, Z.-G.; Jeong, L. S.; Jacobson, K. A. J. Mol. Graphics Modell. 2006,
25, 562.
This work was supported by the grant from the Korea Research
Foundation (NRF-2008-314-E00304) and the NIDDK Intramural
Research Program. H.W.L. acknowledges the research professor fel-
lowship from the Ewha Womans University (2009).
8. (a) Jeong, L. S.; Choe, S. A.; Gunaga, P.; Kim, H. O.; Lee, H. W.; Lee, S. K.; Tosh, D.
K.; Patel, A.; Palaniappan, K. K.; Gao, Z.-G.; Jacobson, K. A.; Moon, H. R. J. Med.
Chem. 2007, 50, 3159; (b) Jeong, L. S.; Pal, S.; Choe, S. A.; Choi, W. J.; Jacobson, K.
A.; Gao, Z.-G.; Klutz, A. M.; Hou, X.; Kim, H. O.; Lee, H. W.; Tosh, D. K.; Moon, H.
R. J. Med. Chem. 2008, 51, 6609.
9. Paquette, L. A.; Dong, S. J. Org. Chem. 2005, 70, 5655.
References and notes
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