Synthetic procedure for the preparation of novel potent and selective A3 adenosine receptor radioligands

Article abstract of DOI:10.1081/NCN-100002428

2-Phenylethynyladenosine and its N⁶-methyl derivative were synthesized and evaluated in binding assays at human adenosine receptors stably transfected on CHO cells. Results showed that the N⁶-methyl-2-phenylethynyladenosine is endowe

Full text of DOI:10.1081/NCN-100002428

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SYNTHETIC PROCEDURE FOR THE PREPARATION
OF NOVEL POTENT AND SELECTIVE A ADENOSINE
3
RECEPTOR RADIOLIGANDS
a
a
a
a
b
R. Volpini , S. Costanzi , C. Lambertucci , S. Vittori & G. Cristalli
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: R. Volpini , S. Costanzi , C. Lambertucci , S. Vittori & G. Cristalli (2001) SYNTHETIC PROCEDURE FOR
THE PREPARATION OF NOVEL POTENT AND SELECTIVE A ADENOSINE RECEPTOR RADIOLIGANDS, Nucleosides, Nucleotides
3
and Nucleic Acids, 20:4-7, 775-779, DOI: 10.1081/NCN-100002428
To link to this article: http://dx.doi.org/10.1081/NCN-100002428
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 775–779 (2001)
SYNTHETIC PROCEDURE FOR THE
PREPARATION OF NOVEL POTENT AND
SELECTIVE A ADENOSINE RECEPTOR
3
RADIOLIGANDS
∗
R. Volpini, S. Costanzi, C. Lambertucci, S. Vittori, and G. Cristalli
Dipartimento di Scienze Chimiche, University of Camerino,
I-62032 Camerino, Italy
ABSTRACT
6
2
-Phenylethynyladenosine and its N -methyl derivative were synthesized and
evaluated in binding assays at human adenosine receptors stably transfected
6
on CHO cells. Results showed that the N -methyl-2-phenylethynyladenosine
is endowed with very high affinity and selectivity at A3 receptor subtype.
6
Hence, an alternative procedure for the synthesis of tritiated N -methyl-2-
phenylethynyladenosine was set up to introduce tritiated methylamine in the
final step.
INTRODUCTION
There is evidence that the purine nucleoside adenosine (Ado) specifically
modulates neurotrasmission through the activation of four receptor subtypes
denominated: A , A , A and A (1). Over the last few years many efforts
1
2A
2B
3
have been directed toward discovery of potent and selective adenosine agonists. To
ꢀ
this purpose we synthesized a number of adenosine-5 -N-ethyluronamide (NECA)
derivatives substituted at the C-2-position with various (ar)alkynyl chains (2,3).
Binding studies at human recombinant adenosine receptors (AdoRs) showed that
the 2-alkynyl derivatives of NECA possess high affinity at the A receptor subtype.
3
∗
Corresponding author.
7
75
Copyright ꢁC 2001 by Marcel Dekker, Inc.
www.dekker.com

ORDER
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7
76
VOLPINI ET AL.
Moreover, the presence of different alkynes modulated the affinity of such deriva-
tives at the other subtypes (A , A , and A ). In particular, the 2-phenylethynyl-
1
2A
2B
NECA (PENECA) was found to possess high affinity combined with good selec-
tivity for A receptors (4) (K A = 6.2 nM; selectivity A /A = 90, and A /A =
3
i
3
1
3
2A
3
ꢀ
1
00). Aimed at investigating the role of the ethylcarboxamido group in 5 position
and to simplify the structure of this molecule we synthesized the corresponding
adenosine anologue 2-phenylethynyladenosine (PEAdo, 4 (5), Scheme 1). Further-
more, a methyl group was introduced on the amine in 6 position of PEAdo to obtain
5, since the presence of a substituent on the 6-amino group of adenosine and NECA
drives the agonist affinity toward A and A receptors. The choice of a small sub-
1
3
stituent was due to the fact that the presence of a sterically hindered substituent,
like an arylcarbamoyl group, decreases the A receptor affinity of 2-alkynylNECA
3
derivatives (6,7).
Preliminary binding studies at human AdoRs, stably transfected on CHO
cells (8), showed that the 2-PEAdo (4) possesses good and comparable affinity at
AdoRs in comparison to PENECA. The most exciting results were obtained with
PEAdo (5), a compound endowed with high affinity and very high selectivity for A3
receptors. (K A = 3.4 nM; selectivity A /A = 500 and A /A = 2500). These
i
3
1
3
1
2A
findings prompted us to set up a different synthetic procedure for the synthesis of
a new A radioligand.
3
R
R
Cl
N
NH
N
NH
N
N
N
N
N
N
N
N
N
N
I
I
R-NH
2
AcO
HO
(a)
HO
O
O
O
4
5
R = H
R = CH3
1
AcO
OAc
HO
OH
R = H
R = CH3
HO
OH
2
3
CH I
CH -NH3
3
2 2
(
a)
iC H11ONO
5
R
I
N
N
N
N
N
N
N
N
AcO
6
7
R = H
R =
HO
O
O
7
AcO
OAc
8
HO
OH
3 2 3
(a): (Ph P)PdCl ,CuI, Et
N
Scheme 1.

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ADENOSINE RECEPTOR RADIOLIGANDS
777
CHEMISTRY
6
The synthesis of 2-PEAdo (5) (4) and N -methyl-2-phenylethynyladenosine
5) was carried out starting from 6-chloro-2-iodo-9-(2,3,5-tri-O-acetyl-β-D-ribofu-
(
ranosyl)-9H-purine (1) (9). Treatment of 1 with liquid ammonia or methylamine
gave the derivatives 2 (9) or 3, respectively. The alkyne in 2-position was intro-
duced by reacting 2 or 3 with phenylethyne using a modification of the palladium
6
catalyzed cross-coupling reaction (10) to give PEAdo (4) and N -methylPEAdo (5)
(Scheme 1).
Aimed at finding a convenient synthetic procedure for the preparation of radi-
6
olabelled N -methyl-2-phenylethynyladenosine we coupled 1 with phenylethyne,
using the cross-coupling reaction conditions, to obtain 6. The 6-chlorine atom of
6
could be easily substituted by tritiated methylamine to give the corresponding
radioligand of 5. Unfortunately, reaction of 1 with phenylethyne did not give 6
but the disubstituted derivative 7. An alternative synthetic route was designed to
avoid the undesired disubstitution. Hence, 2-PEAdo (4) was used as the starting
material. Reaction of this compound with diiodomethane and isopentylnitrite gave
the versatile synthon 2- phenylethynyl-6-iodoadenosine (8) from which the desired
compound (5) was obtained by treatment with methylamine (Scheme 1). This pro-
cedure allows the introduction in the final step and with high yield of a tritiated
methylamine, leading to potent and selective A radioligand.
3
CONCLUSION
6
The N -methylPEAdo is the firstly reported adenosine derivative endowed
with very high affinity and selectivity for A adenosine receptor subtype.
3
The synthetic procedure to prepare the 6-iodo-2-phenylethynylAdo (8) could
be a general synthetic method to obtain A adenosine receptor radioligands, since
3
the presence of an iodine atom at the C-6-position of 2-alkynyladenosines allows
the introduction of tritiated amines in the final step.
EXPERIMENTAL
Melting points were determined with a B u¨ chi apparatus and are uncorrected.
H NMR spectra were obtained with Varian VXR 300 MHz spectrometer; δ in ppm,
1
J in Hz. All exchangeable protons were confirmed by addition of D O. TLC were
2
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 were deter-
vm ai lnu ee ds .on Carlo Erba model 1106 analyser and are within ± 0.4% of theoretical
6
2
-Iodo-N -methyl-9-(β-D-ribofuranosyl)adenine (3). To compound 1
(1.86 mmol) methylamine (10 mL) was added and the reaction mixture was

ORDER
REPRINTS
7
78
VOLPINI ET AL.
◦
allowed to stand at −20 C for 1 h. The exceeding amine was evaporated and the
residue was chromatographed on a silica gel column eluting with CHCl -MeOH
3
1
6
(
(
1
8
93:7 ) to give 3 (634 mg; 69%) as amorphous solid; H NMR (DMSO-d ) δ 2.92
ꢀ
ꢀ
d, 3H, J = 4.1 Hz, NH-CH ), 3.61 (m, 2H, CH -5 ), 3.95 (m, 1H, H-4 ), 4.14 (m,
3
ꢀ
2
ꢀ
ꢀ
H, H-3 ), 4.53 (m, 1H, H-2 ), 5.82 (d, 1H, J = 6.1 Hz, H-1 ), 8.14 (d, 1H, NH),
.31 (s, 1H, H-8). Anal. (C H IN O ) C, H, N.
1
1
14
3
4
Cross-coupling reaction for the synthesis of 5 and 7. To a solution of 3 or
1
(0.51 mmol) in dry DMF (15 mL), and Et N (2.3 mL) under an atmosphere of
3
N were added bis(triphenylphosphine)palladium dichloride (8.1 mg, 0.012 mmol)
2
and CuI (0.51 mg, 0.003 mmol). The phenylethyne (3.1 mmol) was added and the
reaction mixture was stirred under an atmosphere of N at room temperature for
2
the time reported for each compound. The solvent was removed in vacuo and the
residue was chromatographed on a silica gel column eluting with a suitable mixture
of solvents to give 5 or 7 as amorphous solids:
6
N -Methyl-2-(phenylethyn-1-yl)-9-(β-D-ribofuranosyl)adenine(5). The
reaction of 3 with phenylethyne for 16 h, followed by chromatography on a
silica gel column eluting with CHCl -MeOH (92:8), gave 5 (120 mg; 84%), af-
3
ter crystallization from EtOH.
The same compound was also obtained by reacting 8 (0.15 mmol) with methy-
◦
lamine (2 mL) at 5 C for 5 h. After removing the exceeded methylamine the residue
was chromatographed on flash silica gel column eluted with CHCl -cC H :MeOH
3
6
12
◦
1
(
(
4
2
87:10:3) to give 5 (42 mg; 73%); mp 229–231 C; H NMR (Me SO-d ) δ 2.99
2 6
ꢀ ꢀ ꢀ
br s, 3H, NH-C H ), 3.64 (m, 2H, CH -5 ), 3.98 (m, 1H, H-4 ), 4.15 (m, 1H, H-3 ),
3 2
ꢀ
ꢀ
.57 (m, 1H, H-2 ), 5.93 (d, 1H, J = 6.2 Hz, H-1 ), 7.47 (m, 3H, H-Ph), 7.65 (m,
H, H-Ph), 8.07 (m, 1H, NH), 8.47 (s, 1H, H-8). Anal. (C H N O ) C, H, N.
1
9
19
5
4
2
,6-(Diphenylethyn-1-yl)-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine
7). The reaction of 1 with phenylethyne for 24 h, followed by chromatography
on a silica gel column eluted with CHCl -cC H -MeCN (52:40:8) gave 7 (293
(
3
6
12
1
mg; 51%) as amorphous solid; H NMR (DMSO-d ) δ 2.04 (s, 3H, COCH ), 2.08
6
3
ꢀ ꢀ
s, 3H, COCH ), 2.15 (s, 3H, COCH ), 4.39 (m, 3H, CH -5 and H-4 ), 5.74 (m,
3 3 2
(
1
7
ꢀ
ꢀ
ꢀ
H, H-3 ), 6.00 (m, 1H, H-2 ), 6.40 (d, 1H, J = 5.3 Hz, H-1 ), 7.57 (m, 6H, H-Ph),
.74 (m, 6H, H-Ph), 8.99 (s, 1H, H-2). Anal. (C H N O ) C, H, N.
3
2
26
4
7
6-Iodo-2-(phenylethyn-1-yl)-9-(β-D-ribofuranosyl)adenine (8). To a so-
lution of 4 (0.27 mmol) in dry DMF (2 mL) diiodomethane (3.61 mL) and isopentyl-
◦
ꢀ
nitrite (1.12 mL) were added and the mixture was heated at 60 C for 30 . The solvent
was evaporated and the residue was chromatographed on a silica gel column eluted
with CHCl -cC H -MeOH (83:10:7) to give 7 (120 mg; 33%) as amorphous solid;
3
6
12
1
ꢀ
ꢀ
H NMR (DMSO-d ) δ 3.68 (m, 2H, CH -5 ), 4.01 (m, 2H, H-4 ), 4.20 (m, 1H,
H-3 ), 4.55 (m, 1H, H-2 ), 6.03 (d, 1H, J = 5.2 Hz, H-1 ), 7.53 (m, 3H, H-Ph), 7.73
m, 2H, H-Ph), 9.00 (s, 1H, H-8). Anal. (C H IN O ) C, H, N.
6
2
ꢀ
ꢀ
ꢀ
(
1
8
15
4
4

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REPRINTS
ADENOSINE RECEPTOR RADIOLIGANDS
779
ACKNOWLEDGMENTS
Supported by EC Contract No BMH4-98-3474 and by a grant from the Uni-
versity of Camerino (Fondo di Ricerca di Ateneo).
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