2-Nitro analogues of adenosine and 1-deazaadenosine: Synthesis and binding studies at the adenosine A1, A(2A) and A3 receptor subtypes

Article abstract of DOI:10.1016/S0960-894X(00)00415-7

The influence of nitro substituents on the properties of adenosine and 1-deazaadenosine was studied. Combination of a nitro group at the 2-position with several N⁶ substituents such as cyclopentyl and m-iodobenzyl gave a series of analogues with good adenosine receptor affinity, showing directable selectivity for the A₁, A(2A) and A₃ adenosine receptor subtypes. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00415-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2141±2144
2-Nitro Analogues of Adenosine and 1-Deazaadenosine:
Synthesis and Binding Studies at the Adenosine A1, A2A
and A3 Receptor Subtypes
Martin J. Wanner,a Jacobien K. Von Frijtag Drabbe Kunzel,b Ad P. IJzermanb
and Gerrit-Jan Koomena,*
aLaboratory of Organic Chemistry, Institute of Molecular Chemistry, University of Amsterdam,
Nieuwe Achtergracht 129, NL-1018 WS Amsterdam, The Netherlands
bDivision of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University,
PO Box 9502, 2300 RA Leiden, The Netherlands
Received 4 May 2000; revised 18 July 2000; accepted 19 July 2000
AbstractÐThe in¯uence of nitro substituents on the properties of adenosine and 1-deazaadenosine was studied. Combination of a
nitro group at the 2-position with several N6 substituents such as cyclopentyl and m-iodobenzyl gave a series of analogues with
good adenosine receptor anity, showing directable selectivity for the A1, A2A and A3 adenosine receptor subtypes. # 2000
Elsevier Science Ltd. All rights reserved.
Regulation of adenosine receptor anity is an expand-
ing target for drug development.1,2 Due to its low in
vivo stability adenosine itself has only limited ther-
apeutic applications which requires the availability of
selective and metabolically stable ligands for the A1,
A2A A2B or A3 adenosine receptor subtypes. Modi®ca-
tion of adenosine is a subtle process, and only variation
of the adenosine 2, N6, and/or 50 position was shown to
produce selective agonists. From the numerous analo-
gues that have been prepared, it can be deduced that
substituents on the adenosine 2-position such as chlor-
ine, have a positive e€ect on receptor anity and
selectivity.3 5 To further explore this trend we have
chosen for a nitro-group at the adenosine 2-position.
Nitro-substituents in (hetero)aromatic ring systems are
widely used in pharmacology and in particular nitro
substituted imidazoles and furans have found a variety
of clinical applications.6 Previous to our work7,8 the
appearance of nitro substituents in purines, both from
synthetical and biological point of view, was limited to
only a few examples.9,10 In this study the e€ect of a
nitro-group at the (1-deaza)-adenosine 2-position in
combination with receptor selective N6 substituents on
the anity at the adenosine A1, A2A and A3 receptor
will be described.
Chemistry
Functionalization of adenosines at the 2-position by
direct aromatic substitution is not a general process.
For instance halogenation reactions take place exclu-
sively at the purine 8-position. To obtain C-2 sub-
stituted adenosines, 2,6-dichloropurines or guanosine
are commonly used as starting materials.10 14 Recently we
have shown that nitration of a number of 6-substituted
nucleosides using a mixture of tetrabutylammonium
nitrate (TBAN) and tri¯uoroacetic anhydride (TFAA),
eciently results in the formation of 2-nitropurine and
2-nitro-deazapurine nucleosides.7,8 In the present study
this nitration strategy was applied for the preparation of
a series of receptor ligands.
In a ®rst attempt towards a short synthetic route for 2-
nitroadenosine, penta-acetylated adenosine 1 was nitra-
ted with TBAN/TFAA and the acetate protecting
groups of the corresponding 2-nitro product 2 were
removed with potassium cyanide in methanol (Scheme 1).
In particular removal of the second N-acetyl group from
the 6-position was rather slow and 2-methoxyadenosine
3 was formed as the main product via nucleophilic sub-
stitution of the nitro group. This side reaction also
occurred during deprotection of nitrated tetra-acetyl
N6-cyclopentyladenosine 5, resulting in the formation of
2-methoxy-CPA 6.
*Corresponding author. Tel.: +31-20-5256933; fax: +31-20-5255670;
e-mail: gjk@org.chem.uva.nl
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00415-7

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M. J. Wanner et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2141±2144
nitrogen nucleophiles and reaction temperatures below
0 ꢀC the 6-aminated purines were obtained without
a€ecting the acetates or the nitro group. Removal of the
acetate protecting groups from the sugar part with
potassium cyanide in methanol gave adenosine analo-
gues 9b±9e.16 Shorter reaction times reduced competing
methanolysis of the nitro group, as was described in
Scheme 1, to a minor process.
For the preparation of the parent compound 2-nitro-
adenosine 9a, a three-step procedure was developed to
avoid the use of ammonia as a nucleophile.17 Substitu-
tion of 8 with sodium azide, subsequent reaction of the
azide with triphenylphosphine and acid hydrolysis of
the corresponding iminophosphorane 11 produced 2-
nitroadenosine 9a.16 For the synthesis of 2-nitro-1-dea-
zaadenosines 14a±c, 6-nitro-derivative 12 (purine num-
bering) was used (Scheme 3). This precursor for 1-
deazaadenosine was prepared via an ecient literature
procedure by electrophilic nitration of 1-deazapurine-3-
oxide followed by regioselective ribosylation.18 Intro-
duction of a second nitrogroup in 12 was accomplished
with TBAN/TFAA to give 2,6-dinitro-1-deazapurine
riboside 13.7 The 6-nitro substituent in this electron-
poor ring system displayed good leaving group proper-
ties and nucleophilic substitution with amines proceeded
exclusively at this position. 1-Deaza-adenosine analo-
gues 14b and 14c19 were obtained in good overall yield
from reaction with cyclopentylamine and m-iodobenzyl-
Scheme 1. (i) Acetic anhydride, DMAP, re¯ux (1: 79%; 4: 70%); (ii)
1.5 equiv TBAN/TFAA, DCM, 0 ꢀC (2: 55%; 5: 48%); (iii) 1.2 equiv
KCN, MeOH, 48 h, rt (3: 62%; 6: 73%).
An alternative synthetic procedure for 2-nitroadeno-
sines starts with tri-O-acetyl protected 6-chloropurine-
riboside 7 which is readily available from inosine
(Scheme 2).15 Crystalline 6-chloro-2-nitro derivative 8
was obtained in 65±71% overall yield based on inosine.8
Introduction of a nitro substituent in the purine ring
had a strong accelerating e€ect on substitution reactions
at the 6-position. With almost equimolar amounts of
Scheme 2. (i) 1.7 equiv TBAN/TFAA, DCM, 0 ꢀC, 65±71% (3 steps); (ii) 1.2equiv RNH2, TEA, DMF, 0 ꢀC; (iii) KCN, MeOH, 2 h, rt (9b: 42%; 9c:
53%; 9d: 46%; 9e: 56%, 2 steps); (iv) 1.0 equiv NaN3, DMF, 18ꢀC; (v) PPh3, DCM, rt; (vi) HOAc:H2O 3:1, 45ꢀC, 64%, 3 steps; (vii) KCN, methanol,
2 h, rt, 80%.
Scheme 3. (i) 1.5 equiv TBAN/TFAA, DCM, 0 ꢀC, 73%; (ii) 1.2 equiv RNH2, TEA, DMF, 6 h, rt; (iii) KCN, MeOH, 5 h, rt (14b: 60%; 14c: 68% (2
steps); (iv) 1.1 equiv NaN3, DMF, 0 ꢀC; (v) PPh3, DCM, rt; (vi) HOAc:H2O 3:1, 40 ꢀC, 51%, 3 steps; (vii) 0.3 equiv KCN, MeOH, 5 h, rt, 92%.

M. J. Wanner et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2141±2144
2143
amine respectively. Conversion of 13 into the parent
compound 2-nitro-1-deazaadenosine 14a19 was per-
formed in three steps via the azide 15 and the imino-
phosphorane 16, analogous to the sequence described
for 9a. X-ray analysis of 2-nitro-1-deazaadenosine tria-
cetate 177 con®rmed the regioselectivity of the SNAr
reaction.
The A3 receptor also seems to accommodate the nitro-
substituent very well. When 9d is compared to the ana-
logous compound lacking the nitro-group, an increase
in anity was observed. N6-Benzyladenosine was shown
to have a Ki value of 550 nM versus. 163 nM for the 2-
nitro substituted counterpart 9d. Introduction of the 3-
iodobenzyl enhanced both anity and selectivity for the
A3 receptor: 28 nM versus 12 nM.24 The highest anity
for the A3 receptor was obtained for 1-deaza-analogue
14c: Ki=9.8 nM. The corresponding material without
the 2-nitro substituent is not known to us. In conclu-
sion, introduction of the 2-nitro group, a substituent
with outspoken physico-chemical characteristics, a€ec-
ted receptor anities only marginally. Further work on
transformation reactions of the nitronucleosides into
new receptor ligands is currently underway.
Biological Evaluation
Next the anity of the 2-nitro adenosine derivatives for
the adenosine A1, A2A and A3 receptors was studied.
Receptor anities were determined by radioligand
binding studies according to previously reported protocols
and are given in nanomolar concentrations or as per-
centage displacement at a single concentration of
10 mM.20 22 The results of the binding studies (Table 1)
show that the reference agonist for the adenosine A1
receptor, N6-cyclopentyladenosine (CPA), which has
anities of 5.9 and 580 nM for adenosine A1 and A2A
receptors, respectively,23 can be compared to 2-nitro-
derivative 9c. Both ligands show selectivity for A1 rela-
tive to A2A and A3 receptors and the Ki value is in the
same range as for the reference compound CPA. Simi-
larly, 14b is the 2-nitro equivalent of 1-deaza-N6-cyclo-
pentyladenosine. In this case the two compounds are
more or less equipotent, since 1-deaza-N6-cyclopentyla-
denosine has Ki values of 100 nM and 10mM for adeno-
sine A1 and A2A receptors, respectively.3 From these
values it appears that the introduction of the 2-nitro sub-
stituent is fairly well tolerated by the A1 receptor.
References and Notes
1. Poulsen, S.-A.; Quinn, R. J. Bioorg. Med. Chem. 1998, 6,
619.
2. Siddiqui, S. M.; Jacobsen, K. A.; Esker, J. L.; Olah, M. E.;
Ji, X.; Melman, N.; Tiwari, K. N.; Secrist, J. A., III; Schneller,
S. W.; Cristalli, G.; Stiles, G. L.; Johnson, C. R.; IJzerman, A.
P. J. Med. Chem. 1995, 38, 1174.
3. Vittori, S.; Lorenzen, A.; Stannek, C.; Costanzi, S.; Vol-
pini, R.; IJzerman, A. P.; Von Frijtag Drabbe Kunzel, J. K.;
Cristalli, G. J. Med. Chem. 2000, 43, 250.
4. Ha, S. B.; Melman, N.; Jacobson, K. A.; Nair, V. Bioorg.
Med. Chem. Letters 1997, 7, 3085.
5. Knutsen, L. J. S.; Lau, J.; Petersen, H.; Thomsen, C.; Weis,
J. U.; Shalmi, M.; Judge, M. E.; Hansen, A. J.; Sheardown,
M. J. J. Med. Chem. 1999, 42, 3463.
6. Siim, B. G.; Denny, W. A.; Wilson, W. R. Oncology
Research 1997, 9, 357.
Table 1. Adenosine receptor anities (Ki valuesÆSEM) as deter-
7. Deghati, P. Y. F.; Bieraugel, H.; Wanner, M. J.; Koomen,
G.-J. Tetrahedron Lett. 2000, 41, 569.
mined in radioligand binding studies
8. Deghati, P. Y. F.; Wanner, M. J.; Koomen, G.-J. Tetra-
hedron Lett. 2000, 41, 1291.
9. Nitroinosine was obtained in low yield from diazotation of
guanosine: Shapiro, R. S.; Pohl, S. H. Biochemistry 1968, 7,
448.
10. A 6-nitro prodrug of F-ddI was formed as a side product
from a diazotation reaction: Driscoll, J. S.; Siddiqui, M. A.;
Ford, H., Jr.; Kelley, J. A.; Roth, J. S.; Mitsuya, H.; Tanaka,
M.; Marquez, V. E. J. Med. Chem. 1996, 39, 1619.
11. Robins, M. J.; Uznanski, B. Can. J. Chem. 1981, 59, 2601.
12. Nair, V.; Sells, T. B. Tetrahedron Lett. 1990, 31, 807.
13. Adah, S. A.; Nair, V. Tetrahedron 1997, 53, 6747.
14. Kumamoto, H.; Tanaka, H.; Tsukioka, R.; Ishida, Y.;
Nakamura, A.; Kimura, S.; Hayakawa, H.; Kato, K.; Miya-
saka, T. J. Org. Chem. 1999, 64, 7773.
Compounds
R
A1
(Ki, nM)a
A2A
(Ki, nM)b
A3
(Ki, nM)c
9a
9b
9c
9d
9e
14a
14b
14c
H
344Æ16
1160Æ570
47.1Æ3.4
1420Æ240
138Æ30
646Æ150
52.6Æ6.8
110Æ36
5.9
286Æ112
9%d
202Æ103
35.8Æ27.8
222Æ145
163Æ43
12.0Æ3.7
216Æ89
340Æ54
9.8Æ3.2
120
Methoxy
Cyclopentyl
Benzyl
3-I-Benzyl
H
15. Buck, I. M.; Reese, C. B. J. Chem. Soc. Perkin Trans. 1
1990, 293.
3510Æ1940
20%d
16. Selected data: 9a: mp 218±220 ꢀC; 1H NMR (d6-DMSO) d
8.67 (s, 1H, H8), 8.31 (bs, NH2), 5.92 (d, 1H, J=5.9 Hz, H10);
HRMS obs. mass 313.0895, calcd for C10H13O6N6 (M+1)
313.0901; 9b: yellow needles, mp 106±108 ꢀC; 1H NMR (d6-
DMSO) d 12.02 (bs, NH), 8.75 (s, 1H, H8), 5.96 (m, 1H, H10),
3.84 (s, 3H, CH3O); HRMS obs. mass 343.1002, calcd for
1440Æ790
437Æ147
52%d
Cyclopentyl
3-I-Benzyl
208Æ22
CPA
1-Deaza-CPA
3-I-Benzyladenosine
580
10100
340
100
79
Ð
28
C11H15O7N6 (M+1) 343.1002; 9c: mp 206±209 ꢀC; H NMR
1
(d6-DMSO) d 8.80 (bs, NH), 8.67 (s, 1H, H8), 5.92 (d, 1H,
J=5.6 Hz, H10), 5.1 and 4.5 (m, 1H, CHN); HRMS obs. mass
381.1532, calcd for C15H21O6N6 (M+1) 381.1507; 9d: mp
107±109 ꢀC; 1H NMR (d6-DMSO) d (two rotamers, ratio
80:20) 9.35 and 9.25 (m, NH), 8.70 (s, 1H, H8), 5.94 (d, 1H,
aDisplacement of [3H]DPCPX from rat cortical membranes.
bDisplacement of [3H]ZM241,385 from rat striatal membranes.
cDisplacement of [125I]-ABMECA from human A3 receptors expressed
on HEK293 cells.
d% Displacement at 10 mM.

2144
M. J. Wanner et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2141±2144
J=5.7 Hz, H10), 5.25 and 4.73 (m, 2H, CH2N); M+1 (FAB):
402; 9e: mp 105±107 ꢀC; 1H NMR (d6-DMSO) d (two rota-
mers, ratio 80:20) 9.35 and 9.25 (m, NH), 8.71 (s, 1H, H8),
5.94 (d, 1H, J=5.7 Hz, H10), 5.19 and 4.68 (br, 2H, CH2N);
M+1 (FAB): 529.1.
20. Adenosine A1 receptor anities were determined on rat
cortical membranes with [3H]DPCPX as radioligand accord-
ing to a protocol published previously: Pirovano, I. M.; IJzer-
man, A. P.; Van Galen, P. J. M.; Soudijn, W. Eur. J. Pharmacol.
1989, 172, 185.
17. With ammonia as a nucleophile partial aminolysis of the
acetates and substitution of the nitro-group take place.
18. Cristalli, G.; Franchetti, P.; Grifantini, M.; Vittori, S.;
Martelli, S.; Bordoni, T.; Geroni, C. J. Med. Chem. 1987, 30,
1686.
21. Adenosine A2A receptor anities were determined on rat
striatal membranes with [3H]ZM 241,385 as radioligand accord-
ing to a recently reported protocol: Gao, Z.-G.; Jiang, Q.;
Jacobson, K. A.; IJzerman, A. P. Biochem. Pharmacol. in press.
22. Adenosine A3 receptor anities were determined on
membranes prepared from HEK293 cells stably transfected
with human A3 receptor cDNA, with [125I]-ABMECA as
radioligand: Van Muijlwijk-Koezen, J. E.; Timmerman, H.;
Link, R.; Van der Goot, H.; IJzerman, A. P. J. Med. Chem.
1998, 41, 3987.
23. Van der Wenden, E. M.; Carnielli, M.; Roelen, H. C. P.
F.; Lorenzen, A.; Von Frijtag Drabbe Kunzel, J. K.; IJzer-
man, A. P. J. Med. Chem. 1998, 41, 102.
24. Van Tilburg, E. W.; Von Frijtag Drabbe Kunzel, J. K.; De
Groote, M.; Vollinga, R. C.; Lorenzen, A.; IJzerman, A. P. J.
Med. Chem. 1999, 42, 1393.
19. Selected data: 14a: mp 269±273 ꢀC; H NMR (d6-DMSO)
1
d 8.62 (s, 1H, H8), 7.40 (s, 1H, H1); 7.27 (bs, NH2), 5.96 (d,
1H, J=6.1 Hz, H10); HRMS obs. mass 312.0939, calcd for
C11H14O6N5 (M+1) 313.0955; 14b: mp 216±218 ꢀC; H NMR
1
(d6-DMSO) d 8.63 (s, 1H, H8); 7.60 (d, 1H, J=7.4 Hz, NH),
7.35 (s, 1H, H1), 5.97 (d, 1H, J=6.0 Hz, H10), 4.5±4.2 (m, 1H,
CHN); HRMS obs. mass 380.1568, calcd for C16H22O6N5
(M+1) 381.1573; 14c: mp 110±112 ꢀC; 1H NMR (d6-DMSO) d
8.68 (s, 1H, H8); 8.32 (br, NH), 7.32 (bs, 1H, H1), 5.98 (d, 1H,
J=6.0 Hz, H10), 4.8 (br, 2H, CH2N); HRMS obs. mass
528.0395, calcd for C18H19O6N5 I (M+1) 528.0367.