Design and synthesis of 3′-ureidoadenosine-5′-uronamides: Effects of the 3′-ureido group on binding to the A3 adenosine receptor

Article abstract of DOI:10.1016/j.bmcl.2004.07.042

Novel 3′-ureidoadenosine analogues, 3 and 4 were synthesized from 1,2:5,6-di-O-isopropylidene-d-glucose in order to cause stronger hydrogen bonding than the corresponding 3′-aminoadenosine derivatives at the A ₃ adenosine receptor. On the basis

Full text of DOI:10.1016/j.bmcl.2004.07.042

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4851–4854
Design and synthesis of 30-ureidoadenosine-50-uronamides:
effects of the 30-ureido group on binding
to the A3 adenosine receptor
Lak Shin Jeong,a,* Myong Jung Kim,b Hea Ok Kim,a,c Zhan-Guo Gao,c Soo-Kyung Kim,c
Kenneth A. Jacobsonc and Moon Woo Chunb
aLaboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, South Korea
bResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea
cLaboratory of Bioorganic Chemistry, Molecular Recognition Section, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, MD 20982, USA
Received 18 June 2004; revised 21 July 2004; accepted 21 July 2004
Available online 13 August 2004
Abstract—On the basis of high binding affinity at the A3 adenosine receptor of 30-aminoadenosine derivatives with hydrogen bond-
ing donor ability, novel 30-ureidoadenosine analogues were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose in order to lead
to stronger hydrogen bonding than the corresponding 30-aminoadenosine derivatives. However, the synthesized 30-ureidoadenosine
analogues were totally devoid of binding affinity, because 30-urea moiety caused steric and electrostatic repulsions at the binding site
of the A3 adenosine receptor, leading to conformational distortion.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
A number of N6- and/or 2-substituted adenosine deriva-
tives have been synthesized and evaluated for A3 adeno-
sine receptor agonistic activity.9–11
Adenosine is an endogenous material and regulates
many physiological functions through the four subtypes
of adenosine receptors (A1, A2A, A2B, and A3),1 among
which the A3 adenosine receptor is the most recently
identified subtype.2
Among these compounds, N6-(3-iodobenzyl)-50-N-
methylcarbamoyladenosine (1a, IB-MECA) and 2-
chloro-N6-(3-iodobenzyl)-50-N-methylcarbamoyladeno-
sine (1b, Cl-IB-MECA) were found to be highly selective
full agonists with high binding affinities (Ki = 1.8 0.7,
1.4 0.3nM, respectively), at the human A3 adenosine
receptor (Fig. 1).11,12 Fishman et al. have reported that
IB-MECA (1a) exhibited anticancer activity by down
regulating the Wnt signaling pathway.3 This compound
is now undergoing Phase II clinical trials as an antican-
cer agent. The 2-chloro analogue of IB-MECA (1a), Cl-
IB-MECA (1b) also exhibited high binding affinity to
the A3 adenosine receptor, but due to its in vivo toxicity,
is being widely used as a pharmacological tool, instead
of being developed as a clinically useful agent.12,13 Re-
cently, on the basis of high binding affinity and selectiv-
ity of Cl-IB-MECA, we have reported the synthesis of
the 30-fluoro analogue of Cl-IB-MECA to determine if
the 30-hydroxyl group of Cl-IB-MECA acts as a hydro-
gen bonding donor or acceptor upon binding to the
binding site of the A3 adenosine receptor. From this
The A3 adenosine receptor is negatively coupled to
adenylyl cyclase and positively coupled to phospholi-
pase C, resulting in the increase of the Ca2+ level.1 The
selective agonism of A3 adenosine receptors has been
associated with anticancer activity,3 cardioprotective
activity,4,5 and cerebroprotective activity,6,7 while selec-
tive antagonists8 for A3 adenosine receptors are of
potential clinical use for inflammation and asthma.
Thus, the A3 adenosine receptors have been promising
targets for the development of clinically useful agents.
Keywords: A3 adenosine receptor; 30-Ureidoadenosine derivatives;
Hydrogen bonding donor; Binding affinity; Steric effects.
*
Corresponding author. Tel.: +82-2-3277-3466; fax.: +82-2-3277-
2851; e-mail: lakjeong@ewha.ac.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.042

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L. S. Jeong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4851–4854
Scheme 1. Reagents and conditions: (a) Tf2O, pyridine, 0°C, 1h; (b)
NaN3, DMF, rt, 48h; (c) (i) 75% AcOH, 55°C, 1.5h; (ii) NaIO4,
RuCl3ÆH2O, CCl4/CH3CN/H2O, rt, 4h; (d) (i) (COCl)2, DMF, CH2Cl2,
rt, 16h; (ii) 2M CH3NH2, CH2Cl2, 0°C, 3h; (e) (i) 85% HCO2H, 60°C,
1.5h; (ii) Ac2O, pyridine, rt, 16h.
(2/2/3) produced acid derivative. Without purification,
acid derivative was treated with oxalyl chloride to give
the activated ester, which was converted to methyl
amide 7 by treating with methylamine in methylene
chloride. Hydrolysis of the 1,2-acetonide group in 7
using 85% formic acid followed by acetylation of the
resulting diol with acetic anhydride in pyridine afforded
the key intermediate 8.15
Figure 1. The rationale for the design of the desired nucleosides.
study, we concluded that the 30-hydroxyl group might
act as hydrogen bonding donor, but not acceptor.14 This
result was independently confirmed with 30-aminoaden-
osine analogues, among which CP-608039 (2)15 was re-
ported to be a highly selective agonist of the human
A3 adenosine receptor, indicating that the 30-amino
group might play a key role as hydrogen bonding donor
in the binding site of the human A3 adenosine receptor.
The glycosyl donor 8 was utilized for the synthesis of the
target nucleosides, 3 and 4, as shown in Scheme 2. Con-
densation of 8 with silylated 6-chloropurine and 2,6-
dichloropurine in the presence of TMSOTf as a Lewis
acid catalyst afforded the protected nucleosides 9a15
and 9b, respectively. 6-Chloropurine derivative 9a was
treated with methylamine and 3-iodobenzylamine to
give the N6-substituted nucleosides, 10a and 10b, respec-
tively. The acetyl protecting groups of 9a and 9b were
removed in the process and replaced with TBS ethers
in 12a and 12b, respectively, because of the facile migra-
tion of a 20-acetyl group to the 30-ureido group. Reduc-
tion of azido group of 12a and 12b was achieved using
triphenylphosphine and ammonium hydroxide in aque-
ous solution to yield amino derivatives, 14a and 14b,
respectively.
Thus, on the basis of these results, it was very interesting
to design 30-ureidoadenosine analogues, 3 and 4 since
the 30-ureido moiety may form stronger hydrogen bonds
in the binding site than the corresponding 30-amino- or
30-hydroxy-substituted nucleosides (Fig. 1). It is also
of interest to find out whether the larger 30-ureido group
can be tolerated in the active site in comparison with
smaller substituents that is the 30-hydroxy or 30-amino
groups. Herein, we wish to report the efficient synthesis
of novel 30-ureidoadenosine derivatives and their effects
on binding to the A3 adenosine receptor.
For the introduction of a urea moiety at the 30-position,
the amino derivatives, 14a and 14b were treated with
chloroacetyl isocyanate in DMF to yield 30-chloroacetyl
urea derivatives, 16a and 16b, which were smoothly con-
verted to the 30-ureido derivatives, 18a and 18b, respec-
tively, by the treatment with sodium methoxide.16 As
mentioned above, a 20-acetyl or benzoyl group would
easily migrate to the 30-ureido group upon treating 30-
chloroacetyl urea derivatives, 16a and 16b with sodium
methoxide, giving N-acetyl- or N-benzoylureido deriva-
tives as sole products. This migration was prevented by
using a TBS group as a protecting group. Finally,
removal of the TBS group of 18a and 18b using tetra-
n-butylammonium fluoride in THF afforded the final
30-ureidoadenosine derivatives, 3a and 3b, respectively.
Using a similar strategy, 2,6-dichloropurine derivative
9b was converted to other 2-chloro-3-ureidoadenosine
derivatives, 4a17 and 4b, respectively.
2. Results and discussion
2.1. Synthesis
The target nucleosides 3 and 4 were synthesized starting
from 1,2:5,6-di-O-isopropylidene-D-glucose via the
glycosyl donor, 3-deoxy-3-azidosugar 8, which was
synthesized by the known procedure15 (Scheme 1).
1,2:5,6-Di-O-isopropylidene-D-glucose (5) was treated
with trifluoromethanesulfonic anhydride in pyridine to
give the triflate, which reacted with sodium azide in
DMF at room temperature to afford azido sugar 6.
Selective hydrolysis of 5,6-acetonide in 6 using 75%
aqueous acetic acid followed by the treatment of the
resulting diol with NaIO4/RuCl3 in CCl4/CH3CN/H2O

L. S. Jeong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4851–4854
4853
Scheme 2. Reagents and conditions: (a) silylated 6-chloropurine or 2,6-dichloropurine, TMSOTf, C2H4Cl2, 0–60°C, 2h; (b) MeNH2, 1,4-dioxane, rt,
4h or 3-iodobezylamine hydrochloride, Et3N, EtOH, 50°C, 18h, then NaOMe, MeOH, rt, 2h; (c) TBSCl, imidazole, DMF, rt, 24h; (d) Ph3P,
NH4OH/H2O, THF, rt, 18h; (e) chloroacetyl isocyanate, DMF, 0°C, 3h; (f) NaOMe, MeOH, rt, 18h; (g) TBAF, THF, rt, 4h.
3. Binding affinity at the adenosine receptors
adenosine receptor, a receptor docking study10 of N6-
methyl-30-ureidoadenosine-50-uronamide (3a) was per-
formed. From the result of a conformational search
using MOPAC PM3 calculation, the lowest energy con-
former of 3a displayed intramolecular hydrogen bond-
ing between the 30-carbonyl oxygen of ureido group
and the 50-amino group, and between the 30-amino
and 20-hydroxyl groups.
The synthesized nucleosides, 3a, 3b, 4a, and 4b were sub-
jected to competitive radioligand binding assays.18 All
of A3 adenosine receptor experiments were performed
using adherent CHO cells stably transfected with cDNA
encoding the human A3 adenosine receptor using [125I]I-
AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-50-N-
methyl-uronamide) (1.0nM) as radioligand.18,19 Binding
at A1 and A2A adenosine receptors was carried out using
[3H]R-PIA (R-N6-[2-phenylisopropyl]adenosine) (2.0nM,
recombinant human A1 AR) or [3H]CGS21680 (2-
[p-(carboxyethyl)phenylethylamino]-50-N-ethylcarbox-
amidoadenosine) (10nM, recombinant human A2A AR)
as radioligands.18
When the lowest energy conformer of 3a was superim-
posed onto the NECA hA3 adenosine receptor com-
plex10 to establish the ribose binding position, there
was steric repulsion in the starting geometry between
the 30-substituent and the side chain of H272 (7.43) of
the receptor and electrostatic repulsion between the 30-
carbonyl and backbone carbonyl oxygen atom of S271
(7.42) of the receptor. This docking model showing ster-
ic and electrostatic interference indicated conforma-
tional distortion of the ribose moiety of 3a, possibly
explaining the lack of binding affinity of 3a. Thus, if a
less sterically bulky group having strong hydrogen
bonding ability, such as hydroxylamine, is introduced,
binding affinity to the A3 adenosine receptor might be
restored.
None of the tested compounds displayed measurable
binding affinities to A1 and A2A adenosine receptors,
while they exhibited at best very weak affinity at the
A3 adenosine receptor (Table 1).
To explain the structural basis for the lack of interaction
of 30-ureidoadenosine analogues with the human A3
Table 1. Binding affinity of the final nucleosides at human A1, A2A
and A3 adenosine receptorsa
,
4. Conclusions
Compound number
Ki (nM) or % inhibition at 10lMb
We have accomplished the synthesis of novel 30-ureido-
adenosine derivatives, starting from 1,2:5,6-di-O-ispro-
pylidene-D-glucose. The key 30-urea moiety was
introduced upon reacting the 30-amino derivative with
chloroacetyl urea followed by treating with sodium
methoxide. Although we did not discover new, potent
A3 adenosine receptor agonists, using molecular mode-
ling we revealed unfavorable steric and electrostatic
interactions likely to occur upon binding of the 30-
ureido derivatives in the agonist binding site of the A3
hA1
hA2A
hA3
IB-MECA (1a)
Cl-IB-MECA (1b)
51.2 5.1
222 22
<3%
2910 580
5360 2470
<3%
1.8 0.7
1.4 0.3
<3%
3a
3b
4a
4b
<3%
<3%
<3%
<3%
9%
8%
<3%
3%
24%
a Values from this study are means SEM, N = 3.
b Binding using [3H]R-PIA (A1 AR), [3H]CGS21680 (A2A AR), or
125I]I-AB-MECA (A3 AR), unless noted.
[

4854
L. S. Jeong et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4851–4854
15. DeNinno, M. P.; Masamune, H.; Chenard, L. K.; DiRico,
K.; 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.
adenosine receptor, which will provide valuable infor-
mation about the identification of binding site of the
A3 adenosine receptor.
16. To a stirred solution of 14a (250mg, 0.59mmol) in
anhydrous DMF (10mL) was added chloroacetyl isocya-
nate (0.055mL, 0.65mmol) at 0°C. After being stirred for
2h at 0°C, the reaction mixture was evaporated and the
residue was purified by silica gel column chromatography
(Hex/EtOAc = 1/4) to give 16a (300mg, 94%) as an oil: 1H
NMR (300MHz, CDCl3): d 8.92 (d, 1H, J = 5.1Hz), 8.53
(d, 1H, J = 4.6Hz), 8.47 (s, 1H), 7.96 (s, 1H), 6.71 (br s,
1H), 6.51 (br s, 1H), 6.26 (d, 1H, J = 4.2Hz), 5.96 (d, 1H,
J = 5.7Hz), 5.01 (t, 1H, J = 7.9Hz), 4.74–4.81 (m, 2H),
4.13 (s, 2H), 3.29 (br s, 3H), 2.98 (d, 1H, J = 4.7Hz), 0.86
(s, 9H), 0.00 (s, 3H), ꢀ0.20 (s, 3H); IR (KBr): 3296, 2953,
1703, 1626, 1536, 1237, 1155, 840, 756cmꢀ1; FAB-MS m/z:
542 [M+H]+ Anal. Calcd for C21H33ClN8O5Si: C, 46.62;
H, 6.15; N, 20.71. Found: C, 46.63; H, 6.03; N, 20.32. To a
stirred solution of 16a (300mg, 0.55mmol) in MeOH
(10mL) was added 28% NaOMe (0.27mL) at rt and the
reaction mixture was stirred for 18h at rt and evaporated.
The residue was purified by silica gel column chromato-
graphy (CH2Cl2/MeOH = 7/1) to give 18a (200mg, 78%)
as a foam: 1H NMR (300MHz, CDCl3): d 8.69 (br s, 1H),
8.37 (s, 1H), 7.79 (s, 1H), 5.95 (br s, 1H), 5.78 (d, 1H,
J = 7.2), 5.37 (br s, 1H), 4.95 (t, 1H, J = 7.4Hz), 4.74 (d,
1H, J = 2.1Hz), 4.05 (m, 1H), 3.23 (br s, 3H), 2.95 (d, 1H,
J = 5.0Hz), 0.78 (s, 9H), ꢀ0.13 (s, 3H), ꢀ0.32 (s, 3H); IR
(KBr): 3301, 2933, 1668, 1625, 1377, 1252, 1116, 840,
755cmꢀ1; FAB-MS m/z: 465 [M+H]+. Anal. Calcd for
C19H32N8O4Si: C, 49.12; H, 6.94; N, 24.12. Found: C,
48.88; H, 7.04; N, 23.92.
Acknowledgements
This research was supported by a grant from Korea
Research Foundation (KRF-2003-042-E20111).
References and notes
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17. 1H NMR (300MHz, DMSO-d6): d 8.70 (s, 1H), 8.36 (d,
1H, J = 4.8Hz), 8.27 (d, 1H, J = 4.6Hz), 6.32 (d, 1H,
J = 4.2Hz), 6.24 (d, 1H, J = 7.0Hz), 5.94 (d, 1H,
J = 2.6Hz), 5.76 (s, 2H), 4.35–4.41 (m, 2H), 4.23 (d, 1H,
J = 6.1Hz), 2.90 (d, 1H, J = 4.5Hz), 2.64 (d, 1H,
J = 4.6Hz); IR (KBr): 3423, 1666, 1630, 1534, 1356,
1306, 1084, 936, 636cmꢀ1; FAB-MS m/z: 385 [M+H]+.
Anal. Calcd for C13H17ClN8O4: C, 40.58; H, 4.45; N,
29.12. Found: C, 40.59; H, 4.32; N, 29.11.
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