2-(N-Acyl) and 2-N-acyl-N6-substituted analogues of adenosine and their affinity at the human adenosine receptors

Article abstract of DOI:10.1016/j.bmcl.2004.01.011

A series of 2-(N-acyl) and 2-(N-acyl)-N⁶-alkyladenosine analogues have been synthesized from the intermediate 2-amino-6-chloroadenosine derivatives (2b and 7) and evaluated for their affinity at the human A ₁, A_2A, and A₃ receptors. We found that 2-(N-acyl) derivatives of adenosine showed relatively low affinity at A _2A and A₃ receptors, while the N⁶-cyclopentyl substituent in 4h and 4i induced high potency [A₁ (K _i)=20.7 and 31.8 nM respectively] at the A₁ receptor and resulted therefore in increased selectivity for this subtype. The general synthetic methods and their binding studies are presented herein.

Full text of DOI:10.1016/j.bmcl.2004.01.011

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1495–1498
2-(N-Acyl) and 2-N-acyl-N6-substituted analogues of adenosine
and their affinity at the human adenosine receptors
Prakash G. Jagtap,a,* Zhiyu Chen,a Csaba Szaboa and Karl-Norbert Klotzb
aInotek Pharmaceuticals Corporation, 100 Cummings Center, Suite 419E, Beverly, MA 01915, USA
bInstitut fur Pharmakologie und Toxikologie, Universitat Wurzburg, D-97078 Wurzburg, Germany
¨
¨
¨
¨
Received 1 October 2003; revised 18 December 2003; accepted 9 January 2004
Abstract—A series of 2-(N-acyl) and 2-(N-acyl)-N6-alkyladenosine analogues have been synthesized from the intermediate 2-amino-
6-chloroadenosine derivatives (2b and 7) and evaluated for their affinity at the human A1, A2A, and A3 receptors. We found that
2-(N-acyl) derivatives of adenosine showed relatively low affinity at A2A and A3 receptors, while the N6-cyclopentyl substituent in
4h and 4i induced high potency [A1 (Ki)=20.7 and 31.8 nM respectively] at the A1 receptor and resulted therefore in increased
selectivity for this subtype. The general synthetic methods and their binding studies are presented herein.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Adenosine mediates its physiological effects through
four G protein-coupled receptors named A1, A2A, A2B
,
and A3.1 A large number of agonists with high affinity
at all but the A2B subtype have been developed over the
years. Many compounds originally thought to be selective
for the A1 or A2A subtypes later turned out to be also
potent agonists at the recently discovered A3 receptor.
Here we study the potential of 2-(N-acyl) adenosine
derivatives to serve as leads for novel A1 selective
agonists.
Figure 1. 2-(N-Substituted)-adenosine derivatives.
Agonists substituted at C-2 position are usually known
for their selectivity at adenosine A2A receptor.2,3
Various modifications at the C-2 position of adenosine
have been performed by several research groups using
C–C bond formation2aꢀc or by nucleophilic replacement
of C-2 halogen atom with amines,2d alcohols2e or thiols.2f
Condensation reaction of appropriately modified bases
and protected sugar derivatives have also been applied
to prepare C-2 modified adenosines.3 Such functional
groups at the C-2 position or combination of modifi-
cations at C-2 and N6 positions have been shown to
result in different activity profiles and selectivity at the
four known adenosine receptor subtypes.2,3 The striking
difference in selectivity between 2-N-modified adenosines
1a and b (A2 agonists)2d,e and 1c and its N6-cyclopentyl
derivative (A1 agonists)4 suggest the proper combi-
nation of substituent at C-2 and N6 position yields
selective agonists for the A1 receptor (Fig. 1).4 In this
communication we report the synthesis of novel adeno-
sine derivatives carrying the N-acyl side chain at the C-2
position and their evaluation at the human adenosine
receptors.
2. Results and discussion
Although there are several reports on the 2-N sub-
stituted adenosine derivatives in the literature,2d,e,4 the
2-N-acyl analogues of adenosine have not been eval-
uated for their affinity at any known adenosine receptor.
* Corresponding author. Tel.: +1-978-232-9660x295; fax: +1-978-
232-8975; e-mail: pjagtap@inotekcorp.com
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.011

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P. G. Jagtap et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1495–1498
The main objective of current work is to develop a general
synthetic method which will give an easy access to 2-(N-
acyl)adenosines compared to the known literature
methods.5,6 The synthetic routes to obtain 2-(N-acyl)
substituted (4a–f), 2,6-disubstituted derivatives of adeno-
sine (4g–i) and 50-carboxamide analogue 12 from the
intermediates 2b and 7 are depicted in Schemes 1 and 2.
Treatment of 20,30,50-tri-O-acetoxyguanosine (2a) with
phosphorous oxychloride7 and tetraethyl ammonium
chloride in dry acetonitrile and dry N,N-dimethylaniline
at 100 ꢁC gave the intermediate 2-amino-6-chloro-
adenosine (2b), which was acylated using various acid
chlorides in the presence of pyridine and 4-dimethyl-
amino-pyridine (DMAP) in methylene chloride to furnish
2-(N-acyl)-6-chloroadenosines (3a–f). Nucleophilic dis-
placement of the chlorine atom and cleavage of tri-O-
acetyl groups using 2M solution of ammonia in ethanol
at 90–100 ꢁC in a sealed tube furnished nucleosides 4a–f.
Similarly, reaction of 3c with ethylamine in ethanol
(2M) provided 2-[N-(2-cyclohexylacetyl)]-N6-ethyl ade-
nosine (4g). N6-Cyclopentyl derivatives 4h–i were pre-
pared from 3b and e by reacting with cyclopentyl amine
in ethanol. Minor amount of amide cleavage product 2-
aminoadenosine (4j) was also isolated when the com-
pounds 3a–f were heated with amines in ethanol for a
longer time or >100 ꢁC.
Scheme 1. Reagents and conditions: (i) POCl3, Et4NCl, N,N-dimethylaniline, dry MeCN, 100 ꢁC, 10 min, 2b=95%; (ii) DMAP, R1COCl, pyridine,
methylene chloride, rt (60 ꢁC for 3a), overnight; (iii) amine, EtOH, sealed tube, 90–100 ꢁC, 12–24 h.
Scheme 2. Reagents and conditions: (i) Ac2O, DMAP, Et3N, MeCN, 8 h, 6a=87%, 6b=9%; (ii) POCl3, DMA, 15 min, 7=79%; (iii) DMAP,
pyridine, cyclopentyl-CH2CH2COCl, CH2Cl2, 8=87%; (iv) NH3–EtOH (2N), 90 ꢁC, 24 h, sealed tube, 9a=33%, 9b=50%; (v) BAIB, TEMPO,
MeCN–H2O (1:1), rt, 24 h, 10=49%; (vi) EDAC, CH2Cl2, DMF, EtNH2 in THF, rt, 24 h, 11=35%; (vii) TFA–H2O (1:4), rt, 1 h, 12=60%.

P. G. Jagtap et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1495–1498
1497
Synthesis of 50-carboxamide derivative 12 is shown in
Scheme 2. O-Acetylation8 of the commercially available
nucleoside (5) was carried out using acetic anhydride,
DMAP and triethylamine in acetonitrile to give 20,30-O-
isopropylidene-50-O-acetylguanosine (6a) as a major
product and minor amount of 20,30-O-isopropylidene-2-
N-50-O-diacetylguanosine (6b). The next two steps
chlorination and acylation for the synthesis of 7 and 8
are similar to that of Scheme 1. Although amination
reaction of 20,30-O-isopropylidene-50-O-acetyl-2-[N-(2-
cyclopentyl-propanoyl)]-6-chloroadenosine (8) with
ammonia in ethanol was carried out using analogous
conditions described above, it produced mixture of 9a
and amide cleaved product 9b which were isolated by
silica gel column chromatography. Oxidation of 9a with
[bis(acetoxy)-iodo] benzene (BAIB) and 2,2,6,6-tetra-
methyl-1-piperidinyloxy (TEMPO) in MeCN–H2O pro-
vided 50-carboxylic acid 10 in 49% yield.9 Coupling
reaction of ethylamine and acid 10 using EDAC and
triethylamine gave amide (11) which on treatment with
TFA–water (1:4) gave 2-[N-(2-cyclopentylpropanoyl)]-
NECA (12).
affinity. The number of methylene groups (1–3) and the
size of the cycloalkyl (hexyl or pentyl) appears to play a
minor role as for each of the compounds 4c, e and f
virtually identical Ki-values at the A1, A2A, and A3 sub-
types, respectively, were measured. Replacement of the
terminal cyclopentyl in the 2-substituent of 4e for a
phenyl ring (4b) results in an about 2-fold decrease of
affinity at all three receptor subtypes. An 6-fold increase
in A1 affinity was achieved by the introduction of a double
bond in the 2-substituent of 4b with a resulting 58-fold A1
selectivity compared to A2A. Although the resulting
compound 4a is 3-fold more potent at A3 compared to 4b
it also shows a 12-fold A1 selectivity versus A3.
In addition to the novel 2-(N-acyl) subs0tituents simul-
taneous modification of the N6- and the 5 -position were
probed. First, the effect of the introduction of an N6-
ethyl group in compound 4c was tested. The resulting
compound 4g shows little change in the A1 Ki-value, an
about three-fold loss of A2A affinity and an increased A3
affinity. These changes are comparable to the effects
of an N6-ethyl group in a series of 2-alkynyl derivatives
of adenosine described recently by Volpini et al.2b Of all the
compounds investigated in this study 4g is the only one
with a submicromolar A3 affinity. All other compounds
have strikingly similar A3 Ki-values in the range of
about 1–3 mM.
3. Pharmacology
The affinity of the novel compounds at A1, A2A, and A3
receptors was determined in binding studies according to
the methods described earlier.10 In order to get additional
information about the relative A2B affinity, activation of
adenylyl cyclase in membranes form CHO cells with
stably transfected human A2B receptors was measured
as described.10 All compounds exhibit very low potency
at this receptor subtype as at 100 mM they showed less
than 20% of the maximal NECA-signal (data not
shown). The binding data are summarized in Table 1.
Since the data showed some degree of selectivity for the
A1 subtype for most of the compounds presented in this
study, the Ki value of the highly potent prototypical A1
agonist 2-chloro-N6-cyclopentyladenosine (CCPA) is
included in Table 1 as reference.
N6-Substitution with a cyclopentyl ring like in CCPA
has long been known to increase the A1 affinity of
adenosine derivatives. Such an effect may not necessarily
be observed with concomitant substitution of the purine
ring of adenosine. The N6-cyclopentyl substituted com-
pound 4h is the most potent A1 agonist in this study
with a Ki-value of 20.7 nM. It is over 20-fold more
potent than the corresponding unsubstituted compound
4b and 25-fold less potent than prototypical A1 agonist
CCPA. That evidence, although limited, suggests the
2-chloro or other 2-halo groups contribute significantly
to the affinity of 2-halo-N6-cycloalkyl adenosines for the
A1 receptor.11,12 A 8-fold increase in A1 affinity was
found for compound 4i compared to 4e with the
unsubstituted N6-position. Compound 4h showed 60-fold
selectivity versus the A3 subtype which matches the
selectivity of CCPA. The selectivity of 4h and 4i
Compound 4d with a 2-N-cyclohexanoyl substituent
exhibits the lowest A1 affinity whereas all compounds
with the cyclohexyl separated from the carbonyl by at
least one methylene group (4c,f) show increased A1
Table 1. Affinities of the adenosine analogues 4a–i and 12 in radioligand binding assays at human A1, A2A and A3 adenosine receptors
Compd
Ki(A1)a (nM)
Ki(A2A
)
b (nM)
Ki(A3)c (nM)
A2a/A1
A3/A1
CCPAd
4a
4b
4c
4d
4e
4f
4g
0.83 (0.55–1.25)
70.7 (39.1–128)
432 (335–556)
342 (264–444)
1620 (1360–1940)
252 (228–278)
733 (423–1270)
207 (159–270)
20.7 (12.4–34.5)
31.8 (23.9–42.3)
5450 (3750–7940)
2270 (1950–2660)
4100 (2630–6390)
4570 (3540–5900)
2410 (1870–3100)
1780 (1430–2230)
2000 (1070–3760)
1940 (1590–2360)
7750 (5000–12,000)
5660 (3780–8480)
2180 (1860–2560)
10,800 (8910–13,000)
42.3 (32.1–55.8)
900 (739–1100)
2710 (2260–3270)
1230 (860–1,780)
2760 (2600–2930)
1040 (936–1140)
1060 (652–1710)
181 (130–252)
2730
58
11
7
1.1
8
3
37
273
69
2
50
13
6
4
1.7
4
1.4
0.9
60
44
0.2
4h
4i
12
1250 (963–1630)
1410 (1040–1920)
1010 (533–1900)
a Displacement of specific [3H]CCPA binding in CHO cells stably transfected with human recombinant A1 adenosine receptor, expressed as Ki (nM).
b Displacement of specific [3H]NECA binding in CHO cells stably transfected with human recombinant A2A adenosine receptor, expressed as Ki (nM).
c Displacement of specific [3H]NECA binding in HEK cells stably transfected with human recombinant A3 adenosine receptor, expressed as Ki (nM).
d Data from ref 10. All data are geometric means with 95% confidence intervals in parantheses.

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P. G. Jagtap et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1495–1498
compared to A2A is 273 and 69-fold, respectively. In a
recently published series of 2-alkynyl derivatives of
adenosine it was shown that a 50-N-carboxamido modi-
fication has only a minor effect on the affinity at A1,
A2A, and A3 receptors compared to0 the unmodified
ribose.2b Compound 12 bears such a 5 -modified ribose
and exhibits an unchanged affinity at A3, however, the
affinity was 22-fold and 5-fold reduced at A1 and A2A
respectively, versus compound 4e with the intact ribose.
2. (a) Matsuda, A.; Shinozaki, M.; Yamaguchi, T.; Homma,
H.; Nomoto, R.; Miyasaka, T.; Watanable, Y.; Abiru, T.
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Lambertucci, C.; Taffi, S.; Vittori, S.; Klotz, K.-N.; Cris-
talli, G. J. Med. Chem. 2002, 45, 3271. (c) Zablocki, J. A.;
Palle, V. P.; Elzein, E. O. US Patent 6,214,807 B1. (d)
Francis, J. E.; Webb, R. L.; Ghai, G. R.; Hutchison, A. J.;
Moskal, M. A.; deJasus, R.; Yokoyama, R.; Rovinski,
S. L.; Contardo, N.; Dotson, R.; Barclay, B.; Stone, G. A.;
Jarvis, F. J. J. Med. Chem. 1991, 34, 2570. (e) Ueeda, M.;
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4. Conclusion
3. (a) Lau, J.; Lars, J. S. US Patent 5,589,467, 1996. (b)
Mantell, S. J.; Monaghan, S. M.; Stephenson, P. T. US
Patent 6,525,032 B2, 2003. (c) Keeling, S. E.; Albinson,
F. D.; Ayres, B. E.; Butchers, P. R.; Chambers, C. L.;
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499.
To summarize, we synthesized a series of 2-N-acyl
analogues of adenosine and evaluated their ability in
radioligand binding assays at human A1, A2a and A3
adenosine receptors. The compounds we present in this
study show various degrees of potency at the human A1
adenosine receptor and weaker potency than the CCPA.
However, the most potent compound 4h, which is 25-
fold weaker than CCPA, binds with a Ki-value of 20.7
nM and is 273-fold selective versus A2A and also
showed 60-fold selectivity versus the A3 subtype, which
is slightly better than the selectivity of CCPA as one of
the most potent and selective A1 agonists known. Overall,
we present novel 2-substituted adenosine derivatives with
high affinity at the A1 receptor and marked selectivity for
this adenosine receptor subtype. We hope to use these
findings to design novel A1 selective agonists.
7. (a) Robins, M. J.; Uznanski, B. Can. J. Chem. 1981, 59,
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Acknowledgements
8. van Tilburg, E. W.; von Frijtag Drabbe Kunzel, J.; de
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B.; Fredholm, B. B.; Lohse, M. J. Naunyn-Schmiedeberg’s
Arch. Pharmacol. 1998, 357, 1.
The expert technical assistance of Ms. Sonja Kachler
and Mr. Nico Falgner is greatly appreciated. This work
was supported by a grant from the National Institutes
of Health (R44AI46167).
11. Hutchinson, S. A.; Baker, S. P.; Scammells, P. J. Bioorg.
Med. Chem. 2002, 10, 115.
References and notes
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