Synthesis and adenosine receptor affinity and potency of 8-alkynyl derivatives of adenosine

Article abstract of DOI:10.1081/NCN-100002509

Adenosine derivatives bearing different (ar)alkynyl chains at the 8-position were synthesized and tested at human adenosine receptors. Binding studies showed that all compounds possess affinity for the A₃ subtype in the high nM range. Moreover, guanosine 5′-O-(3-[³⁵S]thio)triphosphate binding assay indicated that the 8-alkynyl adenosines behaved as antagonists of NECA at A₃ receptors.

Full text of DOI:10.1081/NCN-100002509

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SYNTHESIS AND ADENOSINE RECEPTOR AFFINITY AND
POTENCY OF 8-ALKYNYL DERIVATIVES OF ADENOSINE
C. Lambertucci a , S. Costanzi a , S. Vittori a , R. Volpini a & G. Cristalli b
a Dipartimento di Scienze Chimiche, University of Camerino, Camerino, I-62032, Italy
b Dipartimento di Scienze Chimiche, University of Camerino, Camerino, I-62032, Italy
Published online: 07 Feb 2007.
To cite this article: C. Lambertucci , S. Costanzi , S. Vittori , R. Volpini & G. Cristalli (2001): SYNTHESIS AND ADENOSINE
RECEPTOR AFFINITY AND POTENCY OF 8-ALKYNYL DERIVATIVES OF ADENOSINE, Nucleosides, Nucleotides and Nucleic Acids,
20:4-7, 1153-1157
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1153–1157 (2001)
SYNTHESIS AND ADENOSINE RECEPTOR
AFFINITY AND POTENCY OF 8-ALKYNYL
DERIVATIVES OF ADENOSINE
C. Lambertucci, S. Costanzi, S. Vittori, R. Volpini, and G. Cristalli∗
Dipartimento di Scienze Chimiche, University of Camerino,
I-62032 Camerino, Italy
ABSTRACT
Adenosinederivativesbearingdifferent(ar)alkynylchainsatthe8-positionwere
synthesized and tested at human adenosine receptors. Binding studies showed
that all compounds possess affinity for the A3 subtype in the high nM range.
Moreover, guanosine 5ꢀ-O-(3-[35S]thio)triphosphate binding assay indicated
that the 8-alkynyl adenosines behaved as antagonists of NECA at A3 receptors.
Adenosine (Ado) is a signalling molecule that mediates diverse biologi-
cal effects via interaction with four different cell surface receptors termed A1,
A2A, A2B and A3 (1). In recent years we demonstrated that C-2-substituted 5ꢀ-N-
ethylcarboxamidoadenosine (NECA) are potent and selective ligands for adenosine
receptor subtypes (2,3). In particular compounds bearing a hexynyl, phenylethynyl,
and phenylhydroxypropynyl chains at the 2-position of NECA possess high affin-
ity for A3 receptors combined, in same cases, with good selectivity (4). In order
to investigate the role of the carboxamido group at the 5ꢀ-position and to simplify
the structure of these molecules, we synthesized adenosine derivatives bearing the
above mentioned alkynyl chains at the C-2-position (5). Binding studies at hu-
man adenosine receptors showed that the 2-alkynyl derivatives of Ado possess
good and comparable affinity at all receptor subtype in comparison to the corre-
sponding NECA derivatives (5). These findings prompted us at investigating the
effect of such substituent in a different position of adenosine; to this purpose we
∗ Corresponding author.
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C
Copyright 2001 by Marcel Dekker, Inc.
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LAMBERTUCCI ET AL.
synthesized adenosine derivatives bearing in C-8-position the three above men-
tioned alkynyl chains. All the synthesized compounds were evaluated at human
recombinant adenosine receptors, stably transfected into Chinese Hamster Ovary
(CHO) cells, utilizing radioligand bindings (A1, A2A, A3) or adenylyl cyclase ac-
tivity assays (A2B) (6). Preliminary results showed that in all cases the introduction
of an alkynyl chain at the C-8 position of Ado is detrimental for the affinity and
potency at A1, A2A, and A2B receptors, while is more tolerated by the A3 receptor
(Ki A3 in the high nM range).
Furthermore, the intrinsic activity of the C-8-alkynylAdos were determined
in guanosine 5ꢀ-O-(3-[35S]thio)triphosphate ([35S]GTPγ S) binding assay in mem-
branes of CHO cells which express the human A3 receptor subtype. NECA (1 µM)
was used as reference full agonist and its stimulation of [35S]GTPγ S binding to
the G protein was set to 100% (7). Surprisingly, the C-8-alkynylAdos behaved as
A3 adenosine antagonists. In fact they did not stimulate basal GTPγ S binding, but
inhibited to various extent NECA-stimulated binding.
CHEMISTRY
The synthesis of 8-substituted adenosines (2a–d) was accomplished starting
from commercial 8-bromoadenosine (8-BrAdo, 1). Substitution of the bromine in
the 8-position of 1 with the three different alkynyl chains (commercially available
hexyne, phenylethyne or phenylhydroxypropyne) was carried out by a modifica-
tion of the palladium catalyzed cross-coupling reaction (2) described in the gen-
eral method (Scheme 1). Reaction of 1 with phenylhydroxypropyne did not give
the corresponding 8-phenylhydroxypropyne (2d) but a product deriving from a
tautomeric rearrangement on the side chain (2c). The structure of (Z)-8-(3-keto-
3-phenyl-1-propen-1-yl)-9-(β-D-ribofuranosyl)adenine (2c) was confirmed by 1H
NMR and 13C NMR. In fact the 1H NMR displayed two doublets of the (Z) dou-
ble bond protons at δ 7.87 and 8.22 with a coupling constant of 15.2 Hz, while
the 13C NMR showed a carbonyl group signal at 188.4 ppm. The same reaction,
performed on the acetylated 8-bromoAdo (3) (8) as reported in Scheme 1, was
successful and gave compound 4 which was deprotected with methanolic ammonia
to obtain the desired 8-phenylhydroxy propynylAdo (2d), although in very low
yield.
EXPERIMENTAL
Melting points were determined with a Bu¨chi apparatus and are uncorrected.
1H and 13C NMR spectra were obtained with Varian VXR 300 MHz spectrometer;
δ in ppm, J in Hz. All exchangeable protons were confirmed by addition of D2O.
TLC were carried out on pre-coated TLC plates with silica gel 60 F-254 (Merck).
For column chromatography, silica gel 60 (Merck) was used. Elemental analyses

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8-ALKYNYL DERIVATIVES OF ADENOSINE
1155
Scheme 1.
were determined on Carlo Erba model 1106 analyser and are within 0.4% of
theoretical values.
General method for the synthesis of 8-(ar)alkynyladenosines 2a–c, and 4.
To a solution of 1 or 3 (0.51 mmol) in dry DMF (15 mL), and Et3N (2.3 mL) under
an atmosphere of N2 were added bis(triphenylphosphine)palladium dichloride (8.1
mg, 0.012 mmol) and CuI (0.51 mg, 0.003 mmol). The appropriate terminal alkyne
(3.1 mmol) was added and the reaction mixture was stirred under an atmosphere of
N2 at room temperature for the time reported for each compound. The solvent was
removed in vacuo and the residue was chromatographed on a silica gel column or
TLC plates eluting with a suitable mixture of solvents to give the desired derivatives
2a–c, or 4 as amorphous solids.
8-(1-Hexyn-1-yl)-9-(β-D-ribofuranosyl)adenine (2a). The reaction of 1
with 1-hexyne for 16 h, followed by chromatography on a silica gel column eluted
with CHCl3-MeOH (92:8), gave 2a (138 mg; 69%); 1H NMR (Me2SO-d6) δ 0.94
(t, J = 7.1 Hz, 3H, CH3), 1.53 (m, 4H, (CH2)2-CH3), 2.59 (t, J = 6.7 Hz, 2H,

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LAMBERTUCCI ET AL.
CH2-C≡C), 3.62 (m, 2H, CH2-5ꢀ), 3.99 (m, 1H, H-4ꢀ), 4.22 (m, 1H, H-3ꢀ), 5.03 (m,
1H, H-2ꢀ), 5.95 (d, J = 6.9 Hz, 1H, H-1ꢀ), 7.50 (bs, 2H, NH2), 8.15 (s, 1H, H-8).
Anal. calcd. for C16H21N5O4 (347.37): C 55.32, H 6.09, N 20.16; found: C 55.25,
H 6.00, N 20.28.
8-(Phenylethyn-1-yl)-9-(β-D-ribofuranosyl)adenine (2b). The reaction
of 1 with phenylethyne for 48 h, followed by chromatography on a silica gel column
eluted with CHCl3-MeOH (92:8), gave 2b (153 mg; 72%); 1H NMR (Me2SO-d6)
δ 3.64 (m, 2H, CH2-5ꢀ), 4.03 (m, 1H, H-4ꢀ), 4.23 (m, 1H, H-3ꢀ), 5.04 (m, 1H, H-2ꢀ),
6.07 (d, J = 6.7 Hz, 1H, H-1ꢀ), 7.55 (m, 3H, H-Ph), 7.66 (m, 4H, H-Ph and NH2),
8.20 (s, 1H, H-8). Anal. calcd. for C18H17N5O4 (367.36): C 58.85, H 4.66, N 19.06;
found: C 55.50, H 4.56, N 19.16.
(Z)-8-(3-Keto-3-phenyl-1-propen-1-yl)-9-(β-D-ribofuranosyl)adenine
(2c). The reaction of 1 with 3-hydroxy-3-phenyl-1-propyne for 4 days, followed
by chromatography on a silica gel TLC plate eluted with CHCl3-MeOH (85:15),
gave 2c (92 mg; 45%); 1H NMR (Me2SO-d6) δ 3.64 (m, 2H, CH2-5ꢀ), 4.04 (m, 1H,
H-4ꢀ), 4.20 (m, 1H, H-3ꢀ), 4.89 (m, 1H, H-2ꢀ), 6.10 (d, J = 6.6 Hz, 1H, H-1ꢀ), 7.69
(m, 5H, H-Ph and NH2), 7.87 (d, J = 15.2 Hz, 1H, CH), 8.08 (d,J = 7.0, 2-H,
−
H-Ph), 8.18 (s, 1H, H-2). 8.22 (d, J = 15.2 Hz, 1H, CH),13 C NMR (Me2SO-d6)
δ 61.9 (C-5ꢀ), 70.6 (C-3ꢀ), 72.3 (C-2ꢀ), 86.6 (C-4ꢀ), 87.8 (C-1ꢀ), 119.5 (C-5), 128.0
(C = C), 128.2 (C-Ph), 128.4 (C-Ph), 128.6 (C-Ph), 129.1 (C-Ph), 133.7 (C = C),
136.9 (C-Ph), 145.6 (C-8), 150.0 (C-4), 153.1 (C-2), 156.2 (C-6), 188.4 (CO). Anal.
calcd. for C19H19N5O5 (397.38): C 57.43, H 4.82, N 17.62; found: C 57.38, H 4.77,
N 17.82.
8-(3-Hydroxy-3-phenyl-1-propyn-1-yl)-9-(2,3,5-tri-O-acetyll-β-D-ribo-
furanosyl)adenine(4). Thereactionof3(8)with3-hydroxy-3-phenyl-1-propyne
for 3 days, followed by chromatography on a silica gel column eluted with CHCl3-
MeOH (96:4), gave 4 (34 mg; 13%); 1H NMR (Me2SO-d6) δ 1.96 (s, 3H, COCH3),
2.04 (s, 3H, COCH3), 2.12 (s, 3H, COCH3), 4.17 (m, 1H, CH2-5ꢀ), 4.40 (m, 2H,
CH2-5ꢀ and H-4ꢀ), 5.75 (m, 2H, H-1ꢀ and H-3ꢀ), 6.17 (m, 2H, H-2ꢀ and CH-OH),
7.43 (m, 3H, H-Ph), 7.59 (m, 4H, H-Ph and NH2), 8.22 (s, 1H, H-2). Anal. calcd.
for C25H25N5O8 (523.49): C 57.36, H 4.81, N 13.38; found: C 57.33, H 4.75,
N 13.52.
8-(3-Hydroxy-3-phenyl-1-propyn-1-yl)-9-(β-D-ribofuranosyl)adenine
(2d). A mixture of 4 (0.07 mmol) and methanolic ammonia (3 mL) was stirred at
room temperature for 30 min. The reaction mixture was evaporated and
the residue was chromatographed on a silica gel TLC plate eluted with CHCl3-
MeOH (80:20) to give 2d (6 mg; 23%); 1H NMR (Me2SO-d6) δ 3.61 (m, 2H, CH2-
5ꢀ), 3.98 (m, 1H, H-4ꢀ), 4.21 (m, 1H, H-3ꢀ), 5.03 (m, 1H, H-2ꢀ), 5.77 (d, J = 5.7 Hz,
1H, CH-OH), 5.99 (d, J = 6.7 Hz, 1H, H-1ꢀ), 7.44 (m, 2H, H-Ph), 7.62 (m, 5H,

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H-Ph and NH2), 8.17 (s, 1H, H-2). Anal. calcd. for C19H19N5O5 (397.38): C 57.43,
H 4.82, N 17.62; found: C 57.23, H 4.79, N 17.82.
ACKNOWLEDGMENT
Supported by EC Contract No BMH4-98-3474 and by a grant from the
University of Camerino (Fondo di Ricerca di Ateneo).
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