N6-substituted D-4′-thioadenosine-5′-methyluronamides: Potent and selective agonists at the human A3 adenosine receptor

Article abstract of DOI:10.1021/jm034098e

4′-Thio analogues 3-5 of Cl-IB-MECA (2) (K_i = 1.0 ± 0.2 nM at the human A₃ adenosine receptor) were synthesized from D-gulono-γ-lactone via 4-thioribosyl acetate 14 as the key intermediate. All synthesized 4′-thionucleosides exhibite

Full text of DOI:10.1021/jm034098e

J . Med. Chem. 2003, 46, 3775-3777
3775
N6-Su bstitu ted
D-4′-Th ioa d en osin e-5′-m eth ylu r on a m id es:
P oten t a n d Selective Agon ists a t th e
Hu m a n A3 Ad en osin e Recep tor
Lak Shin J eong,*,† Dong Zhe J in,‡ Hea Ok Kim,†
Dae Hong Shin,† Hyung Ryong Moon,†
Prashantha Gunaga,† Moon Woo Chun,‡
Yong-Chul Kim,§ Neli Melman,# Zhan-Guo Gao,# and
Kenneth A. J acobson#
Laboratory of Medicinal Chemistry, College of Pharmacy,
Ewha Womans University, Seoul 120-750, Korea,
College of Pharmacy, Seoul National University,
Seoul 151-742, Korea, Department of Life Science, Kwangju
Institute of Science & Technology, Gwangju 500-712, Korea,
and Molecular Recognition Section, Laboratory of
Bioorganic Chemistry, National Institute of Diabetes and
Digestive and Kidney Disease, National Institutes of Health,
Bethesda, Maryland 20892
Received May 10, 2003
F igu r e 1. Rationale for the design of the target 4′-thionucleo-
sides.
Abstr a ct: 4′-Thio analogues 3-5 of Cl-IB-MECA (2) (Ki ) 1.0
( 0.2 nM at the human A3 adenosine receptor) were synthe-
sized from D-gulono-γ-lactone via 4-thioribosyl acetate 14 as
the key intermediate. All synthesized 4′-thionucleosides ex-
hibited higher binding affinity to the human A3 adenosine
receptor than Cl-IB-MECA, among which 4 showed the most
potent binding affinity (Ki ) 0.28 ( 0.09 nM). 4 was also
selective for A3 vs human A1 and human A2A receptors by 4800-
and 36000-fold, respectively.
IB-MECA (1) was found to be a highly potent rat A3
agonist (Ki ) 1.1 nM), which is 50-fold selective for rat
brain A3 versus either A1 or A2 receptors.12 Introduction
of chlorine at the 2-position of IB-MECA, resulting in
the formation of Cl-IB-MECA (2) dramatically increased
binding affinity and selectivity.12 2 has been reported
to display a Ki value of 0.33 nM and showed 2500- and
1400-fold rat A3 receptor selectivity versus A1 and A2A
receptors, respectively. 2 is now being used extensively
as a pharmacological tool for studying A3 receptors.13
Therefore, on the basis of the high binding affinity
and selectivity of 2 on A3 adenosine receptors, we
designed and synthesized the 4′-thio analogue 3 of
Cl-IB-MECA and its related analogues 4 and 5, since a
sulfur atom may serve as a bioisostere of an oxygen
atom (Figure 1). The N6-methyl group was introduced
because this substituent is associated with enhanced
affinity at the human (but not rat) A3 adenosine
receptor.14 A new ligand, thio-Cl-IB-MECA (3), and its
derivatives 4 and 5 were efficiently synthesized from
D-gulono-γ-lactone using a highly efficient synthetic
route to prepare the 4-thioribose derivative. The syn-
thesized N6-substituted 4′-thionucleoside derivatives 3,
4, and 5 consistently exhibited subnanomolar affinity
and high selectivity for human A3 receptors versus
human A1 and A2A receptors. Such agonists may be
useful as pharmacological tools and also are of interest
for the development as therapeutic agents.
In tr od u ction . Adenosine is known to be involved in
regulation of many physiological functions through
specific adenosine receptors, which are expressed on the
surface of nearly all cells.1 Four subtypes of adenosine
receptors have now been cloned, characterized pharma-
cologically, and classified as A1, A2A, A2B, and A3. Among
these, the A3 adenosine receptor was the most recently
identified and cloned from a rat brain cDNA library and
later from sheep and human brain.2-4 This receptor is
a G-protein-coupled receptor, and its activation inhibits
adenylate cyclase and stimulates phospholipase C,
leading to protein kinase C activation and histamine
release from rat mast cells.5 Since the A3 adenosine
receptor is closely related to several diseases such as
cardiac ischemia,6 cerebral ischemia,7 and inflamma-
tion,8,9 it has been a promising target for the develop-
ment of new therapeutic agents. Thus, A3 adenosine
receptor agonists are under consideration in the treat-
ment of cardiac and cerebral ischemia and cancer,10
while A3 adenosine receptor antagonists have been
suggested to be useful as a potential treatment for
glaucoma and asthma.11
Resu lts a n d Discu ssion . Ch em istr y. The synthetic
strategy to the desired nucleosides was to synthesize
the glycosyl donor and then to condense with nucleo-
bases. The synthesis of the glycosyl donor 14, starting
from D-gulono-γ-lactone (6), is shown in Scheme 1.
Starting material 6 was converted to the diacetonide
7 under the standard reaction conditions. Reduction of
7 with NaBH4 followed by mesylation of the resulting
diol afforded the dimesylate 8. Cyclization of 8 with
anhydrous sodium sulfide smoothly proceeded upon
A number of ligands have been synthesized and tested
for binding affinity at the rat, sheep, and human A3
versus A1 and A2A receptors. Among these ligands,
* To whom correspondence should be addressed. Phone: 82-2-3277-
3466. Fax: 82-2-3277-2851. E-mail: lakjeong@ewha.ac.kr.
† Ewha Womans University.
‡ Seoul National University.
§ Kwangju Institute of Science & Technology.
#
National Institute of Diabetes and Digestive and Kidney Disease.
10.1021/jm034098e CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/06/2003

3776 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Sch em e 1. Synthesis of the Glycosyl Donora
Letters
Sch em e 2. Synthesis of the Target Nucleosidesa
a
Reagents: (a) CH3COCH3, H2SO4, CuSO4, room temp; (b)
NaBH4, MeOH; (c) MsCl, Et3N, CH2Cl2; (d) Na2S, DMF, heat; (e)
30% AcOH; (f) Pb(OAc)4, EtOAc; (g) NaBH4, MeOH; (h) BzCl,
pyridine; (i) mCPBA, CH2Cl2; (j)Ac2O.
a
Reagents: (a) silylated 2,6-dichloropurine, TMSOTf; (b) RNH2;
(c) BzCl, pyridine; (d) 80% AcOH; (e) TBSOTf, pyridine; (f) NaOMe,
MeOH; (g) (i) PDC, DMF, (ii) K2CO3, (CH3O)2SO2; (h) 2 N MeNH2,
THF; (i) n-Bu4NF, THF.
heating in DMF to yield the thiosugar 9 in 94% yield.
Selective hydrolysis of the 5,6-acetonide in the presence
of 2,3-acetonide was achieved using 30% aqueous acetic
acid to give diol 10 in 90% yield, based on recovered
starting material. Oxidative cleavage of 10 with lead
tetraacetate followed by reduction of the resulting alde-
hyde with NaBH4 afforded 11, which was treated with
benzoyl chloride to give the benzoate 12. Oxidation of
12 with mCPBA followed by heating of the sulfoxide 13
with acetic anhydride produced the key intermediate
14.
with 2 N methylamine, and the final desilylation yielded
the final nucleosides 3, 4, and 5, respectively.
Biologica l Activity. The synthesized analogues were
tested in radioligand binding assays15 using rat cortical
A1 receptors or striatal A2A receptors or in Chinese
hamster ovary (CHO) cells stably expressing the recom-
binant receptors (human A1, A2A, or A3). Radioligands
for A1, A2A, and A3 receptors were the selective agonists
[3H]-N6-(R)-phenylisopropyladenosine (PIA) (A1), [3H]-
CGS21680 (A2A), and [125I]I-AB-MECA [N6-(4-amino-3-
iodobenzyl)adenosine-5′-(N-methyluronamide)] (A3), re-
spectively.
As shown in Table 1, all synthesized 4′-thionucleo-
sides exhibited high binding affinity to the human A3
receptor in the nanomolar range, while very low binding
affinities to A1 and A2A receptors were observed. Among
them, 4 displayed a Ki value of 0.28 nM at the A3
receptor and had the highest affinity among the syn-
thesized compounds. It was selective for A3 vs human
A1 and human A2A receptors by 4800- and 36000-fold,
respectively. 3 and 5 with 3-iodobenzylamino and amino
substituents at the N6 position also exhibited higher
binding affinity to the A3 receptor (Ki ) 0.38 and 0.40
nM, respectively) than Cl-IB-MECA. To our knowledge,
it is the first example of a 4′-thionucleoside exhibiting
highly potent and selective binding affinity to the A3
adenosine receptor. Compounds 3-5 were demonstrated
The glycosyl donor 14 was utilized for the synthesis
of 2-chloro N6-substituted 4′-thiopurine nucleosides 3-5
as shown in Scheme 2. The glycosyl donor 14 was
condensed with silylated 2,6-dichloropurine in the pres-
ence of TMSOTf to give the â-anomer 15 (60%) and its
R-anomer in 9:1 ratio. Anomeric configurations were
1
easily assigned using results from H NOE experiments.
The â-anomer 15 was converted to 16, 17a ,b, and 18a ,b
by treating with 3-iodobenzylamine, methylamine, and
ammonia, respectively. For the synthesis of the urona-
mide derivatives, compounds 16, 17a , and 18a were
treated with 80% aqueous acetic acid to give the diol
derivatives, which were protected as disilyl ethers and
then debenzoylated to afford the 4′-hydroxymethyl ana-
logues 19, 20, and 21, respectively. Oxidation of 19, 20,
and 21 with PDC in DMF followed by esterification with
potassium carbonate and dimethyl sulfate in acetone,
conversion of the 5′-esters to 5′-uronamides by treating
Ta ble 1. Affinities of 4′-Thionucleoside Analogues in Radioligand Binding Assays at A1, A2A, and A3 Receptors (Human or Rat, As
Indicated)a
Ki (nM) or, if indicated, % displacement
compd
hA1
rA1
hA2A
rA2A
hA3
Cl-IB-MECA (2)
1240 ( 320
193 ( 46
1330 ( 242
89.2 ( 11.7
820 ( 570
140 ( 43
198 ( 14
294 ( 115
5360 ( 2470
223 ( 36
20% (10 µM)
158 ( 29
470 ( 365
348 ( 110
6340 ( 90
<10% (1 µM)
1.0 ( 0.2
3
4
5
0.38 ( 0.07
0.28 ( 0.09
0.40 ( 0.06
a
All human AR experiments were performed using adherent CHO cells stably transfected with cDNA encoding the appropriate receptor.
Binding at rat A1 and A2A ARs was carried out using rat brain homogenates. Radioligands: A1 ([3H]-N6-(R)-phenylisopropyladenosine),
A2A ([3H]CGS21680), A3 ([125I]iodo-AB-MECA).

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3777
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to be full agonists in an assay of human A3 receptor
mediated inhibition of cyclic AMP in transfected CHO
cells.16 The IC50 values were 0.21 ( 0.4 nM (4), 0.38 (
0.6 nM (3), and 1.0 ( 0.3 nM (5).
In conclusion, we have designed and synthesized
novel 4′-thioanalogues of Cl-IB-MECA as the A3 receptor
ligands on the basis of the bioisosteric rationale. We
have found novel 2-chloro-N6-methyl-4′-thioadenosine-
5′-methyluronamide (4) as a highly potent and selective
agonist at the human A3 adenosine receptor.
This result is of great significance in that there are
few highly selective A3 agonists. The selectivity of
IB-MECA is evident in some pharmacological systems
but not in others.17 Cl-IB-MECA is selective; however,
it has associated toxicity in vivo. The finding that the
4′-thio modification is associated with high potency and
selectivity significantly expands the possibilities to
design additional A3 agonists, which may potentially be
useful as in vivo tools. Thorough investigation of the
structure-activity relationship of this series and a
molecular modeling study are in progress in our labora-
tory and will be reported in due course.
Ack n ow led gm en t. This research was supported by
a grant from the Korea Science and Engineering Foun-
dation (Grant KOSEF 2002-2-21500-001-3).
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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