Structure-activity relationships of 2-chloro-N6-substituted-4′-thioadenosine-5′-N,N-dialkyluronamides as human A3 adenosine receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.01.070

On the basis of potent and selective A₃ adenosine receptor (AR) antagonist, 2-chloro-N⁶-(3-iodobenzyl)-4′-thioadenosine-5′-N,N-dimethyluronamide, structure-activity relationships were studied for a series of 5′-N,N-dialkyluronamide d

Full text of DOI:10.1016/j.bmcl.2008.01.070

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1612–1616
Structure–activity relationships of 2-chloro-N6-substituted-
40-thioadenosine-50-N,N-dialkyluronamides
as human A3 adenosine receptor antagonists
Lak Shin Jeong,a,* Hyuk Woo Lee,a Hea Ok Kim,a Dilip K. Tosh,a Shantanu Pal,a
Won Jun Choi,a Zhan-Guo Gao,b Amit R. Patel,b Wanda Williams,c
Kenneth A. Jacobsonb and Hee-Doo Kimd
aLaboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
bMolecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institutes of Health, Bethesda, MD 20892, USA
cChemical Biology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD 20892, USA
dCollege of Pharmacy, Sookmyung Women’s University, Seoul 140-742, Republic of Korea
Received 26 November 2007; revised 14 January 2008; accepted 16 January 2008
Available online 19 January 2008
Abstract—On the basis of potent and selective A3 adenosine receptor (AR) antagonist, 2-chloro-N6-(3-iodobenzyl)-40-thioadeno-
sine-50-N,N-dimethyluronamide, structure–activity relationships were studied for a series of 50-N,N-dialkyluronamide derivatives,
synthesized from D-gulonic c-lactone. From this study, it was revealed that removal of the hydrogen bond-donating ability of
the 50-uronamide was essential for the pure A3AR antagonism. 50-N,N-Dimethyluronamide derivatives exhibited higher binding
affinity than larger 50-N,N-dialkyl or 50-N,N-cycloalkylamide derivatives, indicating that steric factors are crucial in binding to
the human A3AR. A N6-(3-bromobenzyl) derivative 6c (Ki = 9.32 nM) exhibited the highest binding affinity at the human A3AR
with very low binding affinities to other AR subtypes.
Ó 2008 Elsevier Ltd. All rights reserved.
Adenosine (1) regulates many physiological functions
through four subtypes (A1, A2a, A2b, and A3) of adeno-
sine receptors (AR).1 Among these subtypes, activation
of the A3AR inhibits adenylate cyclase to lower the level
of cAMP.1 Also, activation of the A3AR stimulates
phospholipase C (PLC) through the b, c subunit of
the G protein, to increase levels of inositol 1,4,5-tris-
phosphate (IP3). Control of A3AR is thought to be rel-
evant to experimental treatment modalities for a variety
of disorders related to these signal transduction
pathways, such as cancer, cerebral or cardiac ischemia,
glaucoma, allergic conditions, and inflammation.1,2
on the N6- and/or 40-hydroxymethyl group of 1 resulted
in 2 (Cl-IB-MECA) as a potent and selective human
A3AR full agonist4 (Chart 1). Bioisosteric replacement
of the furanose oxygen of 2 with a sulfur atom produced
another potent and selective human A3AR full agonist 3
(LJ-529).5 However, until recently it has been difficult to
discover potent and selective human A3AR full antago-
nists with a nucleoside skeleton because of the tendency
of derivatives of adenosine (1) to act as full agonists.
Thus, most of the potent and selective antagonists for
the human A3AR belong to the category of nonpurine
heterocyclic compounds,6–10 which act competitively
with adenosine derivatives at the orthosteric binding site
of the A3AR. However, these nonpurine heterocyclic
compounds showed weak binding affinity at the rat
A3AR11,12 and were unsuitable for efficacy evaluation
in small animal models and thus not optimal for further
drug development. Therefore, it has been highly desir-
able to discover potent and selective A3AR full antago-
nists of which the affinity and selectivity are independent
of species. Since it was reported that antagonists with a
nucleoside skeleton13 could be species-independent, po-
On the basis of the structure of adenosine (1), worldwide
efforts have been made to discover potent and selective
human A3AR agonists and antagonists.2,3 Modification
Keywords: 40-Thionucleoside; A3 adenosine receptor antagonist; Con-
formational change; Radioligand binding.
*
Corresponding author. Tel.: +82 2 3277 3466; fax: +82 2 3277
2851; e-mail: lakjeong@ewha.ac.kr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.070

L. S. Jeong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1612–1616
1613
NH2
N
HN
N
N
N
N
N
N
I
O
N
Cl
HO
MeHN
O
X
HO
OH
HO
OH
2. X=O, Cl-IB-MECA, full agonist
3. X=S, LJ-529, full agonist
1. Adenosine, full agonist
NHR1
HN
N
N
N
N
N
R2
N
I
O
SAR
N
Cl
Me
Me
O
N
R3
N
Cl
S
N
X
HO
OH
HO
OH
6a~r
4. X=O, full antagonist
5. X=S, full antagonist
(R1 = alkyl, cyloalkyl, or arylalkyl;
R2, R3 = alkyl or cycloalkyl)
Chart 1.
Cl
tent, and selective A3AR full antagonists, we searched
for novel nucleoside analogues, among which appending
an additional methyl group to the 50-uronamide nitro-
gen of compounds 2 and 3 converted these A3AR full
agonists into the potent and selective A3AR full antago-
nists 4 and 5.14 40-Thionucleoside analogue 5 was found
to be a more potent and selective antagonist than the
corresponding 40-oxonucleoside analogue 4.14 Thus, on
the basis of these findings, we modified the N6- and 40-
hydroxymethyl groups of 40-thionucleoside analogue 5
in order to search for potent and selective A3AR full
antagonists. Herein, we report the structure–activity
relationship (SAR) study of 2-chloro-N6-substituted-40-
thioadenosine-50-N,N-dialkylamides at the human
A3AR.
O
S
N
N
N
BzO
O
a
54%
N
Cl
O
BzO
S
O
O
HO
HO
OH OH
O
O
7
8
9
NHR1
NHR1
N
N
N
N
N
N
N
N
Cl
c,d,e
BzO
b
Cl
S
HO
S
O
O
TBSO
OTBS
Synthesis of the key intermediates 11a–j started from D-
gulonic c-lactone (7), as shown in Scheme 1.
10a (R1 = 3-iodobenzyl) (90%)
11a (R1 = 3-iodobenzyl) (80%)
11b (R1 = 3-fluorobenzyl) (71%)
11c (R1 = 3-chlorobenzyl) (85%)
11d (R1 = 3-bromobenzyl) (80%)
11e (R1 = dimethyl) (71%)*
10b (R1 = 3-fluorobenzyl) (91%)
10c (R1 = 3-chlorobenzyl) (93%)
10d (R1 = 3-bromobenzyl) (96%)
10e (R1 = dimethyl) (88%)*
D-Gulonic c-lactone (7) was converted to the glycosyl
donor 8, using our previously published procedure.5,15
Pummerer-type condensation of 8 with 2,6-dichloropu-
rine in the presence of TMSOTf and Et3N afforded the
desired nucleoside 95,15 as a single diastereomer.
10f (R1 = 2-methoxymethyl) (87%)
10g (R1 = cyclopropyl) (84%)
11f (R1 = 2-methoxymethyl) (69%)
11g (R1 = cyclopropyl) (74%)
11h (R1 = cyclopropylmethyl) (80%)
11i (R1 = cyclobutyl) (83%)
10h (R1 = cyclopropylmethyl) (93%)
10i (R1 = cyclobutyl) (89%)
10j (R1 = cyclopentyl) (85%)
11j (R1 = cyclopentyl) (76%)
Treatment of 2,6-dichloropurine derivative 9 with vari-
ous alkyl or arylalkyl amines gave N6-substituted ana-
logues 10a–j. Because of the difficulty in removing the
isopropylidene group at the final step, the isopropyli-
dene intermediates 10a–j were converted to the TBS
derivatives by treatment with 80% acetic acid followed
by reprotection with TBSOTf. The resulting compounds
were treated with sodium methoxide to give the key
intermediates 11a–j.
Scheme 1. Reagents and conditions: (a) 2,6-dichloropurine, TMSOTf,
Et3N, CH3CN–ClCH2CH2Cl, rt to 83 °C; (b) R1NH2, Et3N, EtOH, rt;
(c) 80% AcOH, 70 °C; (d) TBSOTf, pyridine, 50 °C; (e) NaOMe,
MeOH. *N6 (CH3)2.
of 11a–j were oxidized by treatment with PDC in
DMF to the carboxylic acids 12a–j, which were con-
verted to various tertiary amides 6a–r16 by coupling
the acids 12a–j with various dialkyl or cycloalkyl amines
in the presence of EDC and HOBt.
Conversion of 11a–j to the target nucleosides 6a–r is
illustrated in Scheme 2. The primary hydroxyl groups

1614
L. S. Jeong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1612–1616
NHR1
NHR1
NHR1
N
1) R2R3NH, EDC,
HOBt, DIPEA,
CH2Cl2
N
N
N
N
N
N
N
N
N
N
R2
PDC
DMF
O
Cl
O
Cl
N
Cl
HO
N
R3
HO
S
2) TBAF, THF
S
S
TBSO
OTBS
HO
OH
TBSO
OTBS
11a (R1 = 3-iodobenzyl)
11b (R1 = 3-fluorobenzyl)
11c (R1 = 3-chlorobenzyl)
11d (R1 = 3-bromobenzyl)
11e (R1 = dimethyl)*
12a (R1 = 3-iodobenzyl)
12b (R1 = 3-fluorobenzyl)
12c (R1 = 3-chlorobenzyl)
12d (R1 = 3-bromobenzyl)
12e (R1 = dimethyl)*
5 (R1 = 3-iodobenzyl, R2 = R3 = Me) (64%)
6a (R1 = 3-fluorobenzyl, R2 = R3 = Me) (58%)
6b (R1 = 3-chlorobenzyl, R2 = R3 = Me) (60%)
6c (R1 = 3-bromobenzyl, R2 = R3 = Me) (67%)
6d (R1 = dimethyl, R2 = R3 = Me) (51%)*
11f (R1 = 2-methoxymethyl)
11g (R1 = cyclopropyl)
11h (R1 = cyclopropylmethyl)
11i (R1 = cyclobutyl)
12f (R1 = 2-methoxymethyl)
12g (R1 = cyclopropyl)
12h (R1 = cyclopropylmethyl)
12i (R1 = cyclobutyl)
6e (R1 = 2-methoxyethyl, R2 = R3 = Me (48%)
6f (R1 = cyclopropyl, R2 = R3 = Me) (68%)
6g (R1 = cyclopropylmethyl, R2 = R3 = Me) (59%)
6h (R1 = cyclobutyl, R2 = R3 = Me) (66%)
11j (R1 = cyclopentyl)
12j (R1 = cyclopentyl)
6i (R1 = cyclopentyl, R2 = R3 = Me) (66%)
6j (R1 = 3-iodobenzyl, R2 = Me, R3 = propyl) (48%)
6k (R1 = 3-iodobenzyl, R2 = Me, R3 = CH2CH2OH) (45%)
6l (R1 = 3-iodobenzyl, R2 = Et, R3 = phenyl) (50%)
6m (R1 = 3-iodobenzyl, R2 = R3 = piperidine) (63%)
6n (R1 = 3-iodobenzyl, R2 = R3 = 4-methylpiperzine) (42%)
6o (R1 = 3-iodobenzyl, R2 = R3 = azetidine) (55%)
6p (R1 = 3-iodobenzyl, R2 = R3 = pyrrolidine) (61%)
6q (R1 = 3-iodobenzyl, R2 = R3 = 4-hydroxypiperidine) (48%)
6r (R1 = 3-iodobenzyl, R2 = R3 = thiomorpholine) (64%)
Scheme 2. *N6 (CH3)2.
Binding assays were carried out using standard radioli-
gands in Chinese hamster ovary (CHO) cells stably
expressing a human AR subtype,14,17 and binding affin-
ities of the final nucleosides 6a–r at the three subtypes of
human ARs are shown in Table 1. In general, 50-N,N-
dimethylamide derivatives exhibited higher binding
affinity than larger 50-N,N-dialkyl or 50-N,N-cyclo-
alkylamide derivatives indicating that steric factors are
crucial in binding to human A3AR. The effects on the
binding affinity of various N6-substituted analogues also
having a 50-N,N-dimethyl group at the 50-uronamide po-
sition were examined. The N6-(3-halobenzyl) series gen-
erally showed better binding affinity at the human
A3AR than the N6-dialkyl or N6-cycloalkyl series. With-
in the N6-(3-halobenzyl) series, the rank order of binding
affinity at the human A3AR was 3-bromobenzyl > 3-
iodobenzyl > 3-chlorobenzyl > 3-fluorobenzyl. This re-
sult indicated that the human A3AR prefers bromo as
an optimal size of 3-halo substitution, and larger (I) or
smaller (F) substitution reduced binding affinity. Among
compounds tested, compound 6c (Ki = 9.32 nM)
exhibited the highest binding affinity at the human
A3AR with very low binding affinities to other AR
subtypes.
nists, while the concentration–response curve for Cl-
IB-MECA (2) indicated full agonism, as previously re-
ported,4 with an EC50 of 1.2 nM at the human A3AR.
To probe species differences, the affinity of N,N-50-dim-
ethylamide derivative 5 was also measured at the rat
A3AR. Although the affinity decreased with respect to
the affinity at the human A3AR, this compound showed
moderate affinity (Ki = 321 74 nM) at the rat A3AR.14
Molecular modeling of the A3AR indicated that the
hydrogen bond-donating ability of the 50-uronamide is
responsible for the conformational change needed for
receptor activation.18,19 Because the synthesized 50-
N,N-dialkyl derivatives which removed the hydrogen
bond-donating ability of the 50-uronamide could not
lead to the conformational change, resulting in the loss
of ability to activate the A3AR, the 50-N,N-dialkyl series
of compounds act as pure antagonists.
In summary, on the basis of potent and selective human
A3AR antagonism of the dimethyl derivative 5, we
carried out structure–activity relationship studies of
2-chloro-N6-substituted-40-thioadenosine-50-N,N-dial-
kylamides. From this study, we identified compound 6c
as a highly potent and selective human A3AR antago-
nist, in which removal of the hydrogen bond-donating
ability of the 50-uronamide was essential for the pure
A3AR antagonism. 50-N,N-dimethyluronamide deriva-
tives generally exhibited higher binding affinity than lar-
The functional efficacy of compounds 514 and 6c showed
that high binding affinity at human A3AR was deter-
mined by inhibition of forskolin-stimulated cAMP pro-
duction in AR-transfected CHO cells and measured at a
concentration of 10 lM, in comparison to the maximal
effect of a full agonist, N-ethyl-50-N-ethylcarboxamido-
adenosine (NECA), at 10 lM. In these functional as-
says, A3AR agonism was absent in these compounds,
indicating that these analogues are pure A3AR antago-
ger
50-N,N-dialkyl
or
50-N,N-dicycloalkylamide
derivatives, indicating that steric factors in the 50 region
are important in binding of nucleosides to the human
A3AR. The newly synthesized A3AR antagonists stud-
ied here could be evaluated in models of a number of

L. S. Jeong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1612–1616
1615
Table 1. Potency of 2-chloro-40-thioadenosine-50-dialkylamide derivatives in binding at human A1, A2A, and A3ARs expressed in CHO cells
NHR1
R2
N
N
N
N
O
Cl
N
R3
S
OH OH
Compound No. (R1,R2,R3)
Ki (nM SEM) or % inhibition at 10 lMa
hA1ARb
hA2AARb
hA3ARb
1.4 0.3
2c
222 22
193 46
5360 2470
223 36
>10,000
>10,000
(1%)
3c
0.38 0.07
29.0 4.9
15.5 3.1
121 11
21.3 2.1
9.32 3.20
136
4d
5870 930
6220 640
(16 4%)
(10 8%)
(37 2%)
(8 2%)
5d (3-iodobenzyl, Me, Me)
6a (3-fluorobenzyl, Me, Me)
6b (3-chlorobenzyl, Me, Me)
6c (3-bromobenzyl, Me, Me)
6d (Me2, Me, Me)e
(ꢀ1%)
(7%)
(ꢀ2%)
(1%)
6e (2-methoxyethyl, Me, Me)
6f (cyclopropyl, Me, Me)
6g (cyclopropylmethyl, Me, Me)
6h (cyclobutyl, Me, Me)
6i (cyclopentyl, Me, Me)
6j (3-iodobenzyl, Me, Propyl)
6k (3-iodobenzyl, Me, CH2CH2OH)
6l (3-iodobenzyl, Et, phenyl)
6m (3-iodobenzyl, piperidine)
6n (3-iodobenzyl, 4-methylpiperazine)
6o (3-iodobenzyl, azetidine)
6p (3-iodobenzyl, pyrrolidine)
6q (3-iodobenzyl, 4-hydroxypiperidine)
6r (3-iodobenzyl, thiomorpholine)
(ꢀ7 7%)
(7 16%)
404 120
345 152
(18 21%)
(50 1%)
425
(ꢀ8 4%)
(0 6%)
(10%)
109 16
30.7 9.4
115 50
837
(4 27%)
(5%)
(13%)
727
237
6
126 17
398
565
(18 5%)
(27 5%)
(19 5%)
(8 6%)
(8%)
(7%)
(ꢀ7%)
(12%)
(14%)
667
43.4 2.6
117 31
1530
(20 3%)
(16 7%)
(19 9%)
(10%)
(15%)
867
a All experiments were done on CHO cells stably expressing one of three subtypes of human ARs. The binding affinity for A1, A2A, and A3ARs was
expressed as Ki values and was determined by using agonist radioligands ([3H]CCPA), ([3H]CGS21680), [125I]I-AB-MECA, respectively. Values in
parentheses are for weak binding, corresponding to an IC50 P 10 lM. Data are expressed as means standard error.
b Ki in binding, unless noted.
c Ki values from Ref. 5.
d Ki values from Ref. 14.
e N6 (CH3)2.
4. Kim, H. O.; Ji, X.-d.; Siddiqi, S. M.; Olah, M. E.; Stiles,
G. L.; Jacobson, K. A. J. Med. Chem. 1994, 37, 3614.
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Moon, H. R.; Gunaga, P.; Chun, M. W.; Kim, Y.-C.;
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2003, 46, 3775; (b) Jeong, L. S.; Lee, H. W.; Jacobson, K.
A.; Kim, H. O.; Shin, D. H.; Lee, J. A.; Gao, Z. G.; Lu,
C.; Duong, H. T.; Gunaga, P.; Lee, S. K.; Jin, D. Z.;
Chun, M. W.; Moon, H. R. J. Med. Chem. 2006, 49, 273.
6. Kim, Y. C.; Ji, X. D.; Jacobson, K. A. J. Med. Chem.
1996, 39, 4142.
disorders related to the A3AR, such as glaucoma,
inflammation, and asthma.
Acknowledgments
This research was supported by the National Core Re-
search Center (NCRC) program (No. R15-2006-020)
of the Ministry of Science and Technology (MOST)
and the Korea Science and Engineering Foundation
(KOSEF) and by the National Institutes of Health,
NIDDK Intramural Research Program.
7. van Muijlwijk-Koezen, J. E.; Timmerman, H.; Link, R.;
van der Goot, H.; IJzerman, A. P. J. Med. Chem. 1998, 41,
3994.
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Pastorin, G.; Da Ros, T.; Klotz, K.-N.; Varani, K.; Gessi,
S.; Borea, P. A. J. Med. Chem. 2002, 45, 770.
References and notes
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16. General procedure for the synthesis of 6a–r: To a stirred
solution of per mmol of 40-hydroxymethyl analogues
(11a–j) in dry DMF (10 mL) was added pyridinium
dichromate (10.0 equiv), and the reaction mixture was
stirred at rt for 20 h. After being poured into water
(50 mL/mmol), the reaction mixture was stirred at rt for
1 h. The precipitate was filtered, and the filter cake was
washed with excess water and dried under high vacuum to
give brownish solid, which was used in the next step
without further purification. To a solution of per mmol of
acid derivatives (12a–j), EDC (1.5 equiv), HOBt (1.5
equiv), and appropriate amine (1.5 equiv) in CH2Cl2
(20 mL) was added DIPEA (3.0 equiv), and the mixture
was stirred at rt for 12 h. The reaction mixture was
evaporated, and the residue was purified by a silica gel
column chromatography (hexane/EtOAc = 10:1–5:1) to
give silyl amides as a white foam. To a stirred solution
of per mmol of silyl amides in THF (5 mL) was added
TBAF (2.5 equiv) and the reaction mixture was stirred at
rt for 1 h. The solvent was evaporated and the resulting
residue was purified by silica gel column chromatography
(CH2Cl2/EtOAc/MeOH = 20:20:1) to give 6a–r.
Compound 6c: yield 67%; white solid; mp 180.4–182.0 °C;
20
½aꢁD ꢀ 16:5 (c 0.10, MeOH); UV (MeOH) kmax 274 nm
1
(pH 7); H NMR (CD3OD) d 2.94 (s, 3 H, N–CH3), 3.03
(s, 3 H, N–CH3), 4.41 (d, 1H, J = 4.6 Hz, 40-H), 4.57 (br
dd, 1H, J = 4.6, 8.4 Hz, 30-H), 4.64 (m, 1H, 20-H), 4.73 (d,
2H, J = 5.7 Hz, N–CH2), 5.96 (d, 1H, J = 5.4 Hz, 10-H),
7.22–7.56 (m, 4H, aromatic H), 8.52 (s, 1H, H-8); 13C
NMR (CD3OD) d 36.6, 38.2, 44.6, 54.3, 64.6, 77.2, 80.7,
127.4, 128.8, 130.0, 131.1, 134.9, 141.4, 142.8, 143.1, 151.7,
155.8, 156.4, 172.8; FAB-MS m/z 528 (M++H); Anal.
(C19H20BrClN6O3S) C, H, N, S.
17. (a) Gao, Z.-G.; Blaustein, J. B.; Gross, A. S.; Melman, N.;
Jacobson, K. A. Biochem. Pharmacol. 2003, 65, 1675; (b)
Gao, Z.-G.; Jeong, L. S.; Moon, H. R.; Kim, H. O.; Choi,
W. J.; Shin, D. H.; Elhalem, E.; Comin, M. J.; Melman,
N.; Mamedova, L.; Gross, A. S.; Rodriguez, J. B.;
Jacobson, K. A. Biochem. Pharmacol. 2004, 67, 893.
18. van Tilburg, E. W.; von Frijtag Drabbe Kunzel, J.; de
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Groote, M.; IJzerman, A. P. J. Med. Chem. 2002, 45, 420.
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1225.