N6-alkyl-2-alkynyl derivatives of adenosine as potent and selective agonists at the human adenosine A3 receptor and a starting point for searching A2B ligands

Article abstract of DOI:10.1021/jm0109762

A series of N⁶-alkyl-2-alkynyl derivatives of adenosine (Ado) have been synthesized and evaluated for their affinity at human A₁, A₂, and A₃ receptors and for their potency at A_2B adenosine receptor s

Full text of DOI:10.1021/jm0109762

J . Med. Chem. 2002, 45, 3271-3279
3271
N6-Alk yl-2-a lk yn yl Der iva tives of Ad en osin e a s P oten t a n d Selective Agon ists a t
th e Hu m a n Ad en osin e A3 Recep tor a n d a Sta r tin g P oin t for Sea r ch in g A2B
Liga n d s
Rosaria Volpini,† Stefano Costanzi,† Catia Lambertucci,† Sara Taffi,† Sauro Vittori,† Karl-Norbert Klotz,‡ and
Gloria Cristalli*,†
Dipartimento di Scienze Chimiche, Universita` di Camerino, Via S. Agostino, 1, 62032 Camerino, Italy, and
Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Wu¨rzburg, D-97078 Wu¨rzburg, Germany
Received J uly 4, 2001
A series of N6-alkyl-2-alkynyl derivatives of adenosine (Ado) have been synthesized and
evaluated for their affinity at human A1, A2A, and A3 receptors and for their potency at A2B
adenosine receptor subtypes. The corresponding 2-(1-alkynyl) derivatives of 5′-N-ethylcarboxa-
midoadenosine (NECA) and Ado are used as reference compounds. Binding studies demon-
strated that the activities of 2-alkynylAdos were slightly increased for the adenosine A1 receptor
and slightly decreased for both A3 and A2B subtypes compared to those of their corresponding
NECA derivatives, whereas the A2A receptor affinities of the two series of nucleosides were
similar. The presence of a methyl group on N6 of the 2-alkynyladenosines, inducing an increase
in affinity at the human A3 receptor and a decrease at the other subtypes, resulted in an increase
in A3 selectivity. In particular, 2-phenylethynyl-N6-methylAdo (8b) showed an A3 affinity in
the low nanomolar range (Ki(A3) ) 3.4 nM), with a A1/A3 and A2A/A3 selectivity of about 500
and 2500, respectively. These findings motivated us to search for the preparation of new selective
radioligands for the A3 subtype; hence, a procedure to introduce a tritiated alkylamino group
in these molecules was carried out. As far as the potency at the A2B receptor, the type of
2-alkynyl chain and the presence of the ethylcarboxamido group on the sugar seem to be very
important; in fact, the (S)-2-phenylhydroxypropynylNECA [(S)-PHPNECA, 1e, EC50(A2B) )
0.22 µM] proved to be one of the most potent A2B agonist reported so far. On the other hand,
the (S)-2-phenylhydroxypropynyl-N6-ethylAdo (9e, EC50(A2B) ) 0.73 µM) showed a significantly
increase of potency at the A2B subtype in comparison with the N6-methyl, N6-isopropyl, and
the unsubstituted adenosine derivatives, although it resulted in being less potent than (S)-
PHPNECA (1e, EC50(A2B) ) 0.22 µM). These observations suggest that the introduction of an
ethyl group in the N6-position and an ethylcarboxamido substituent in the 4′-position of (S)-
2-phenylhydroxypropynyladenosine could lead to a compound endowed with high potency at
the A2B receptor.
In tr od u ction
adenosine A3 receptor agonists has also been shown to
have protective actions in the brain, in contrast to acute
high-dose administration, which can cause toxicity.9,10
More recently, it has been suggested that adenosine
exerts a cytostatic effect on the proliferation of the Nb2-
11C rat lymphoma cell line mainly through binding to
its A3 receptor.11
Adenosine and natural adenosine derivatives are
involved in the regulation of many aspects of cellular
metabolism, and purinergic mechanism is an expanding
therapeutic target for the discovery of novel drugs.1,2
However, to date only adenosine itself is approved for
human use, although over the past years much effort
has been directed toward the discovery of potent and
selective adenosine agonists.
Most actions of adenosine are mediated by four
extracellular receptors termed A1, A2A, A2B, and A3.3,4
In particular, the physiological role of the A3 receptor
subtype is not clearly understood owing to its recent
characterization and a lack of truly selective ligands for
in vivo studies. There are reports that agonists acting
via the adenosine A3 receptor have cardioprotective
effects,5-7 although it was proven that the A3 receptor
serves a different cardioprotective function compared to
the adenosine A1 receptor.8 Chronic administration of
In a search for selective adenosine receptor ligands,
it has been found that 2-hexynyladenosine-5′-N-ethyl-
uronamide (HENECA, 1a ) displays high affinity at rat
A2A receptors combined with a good A2A vs A1 selectivity
(Figure 1).12-15 Moreover, HENECA was also found to
possess good affinity for the rat A3 receptor subtype.16
Furthermore, several 2-alkynyl derivatives of NECA
were tested in membranes of CHO cells that express
the human A3, and in particular, HENECA, 2-phenyl-
ethynylNECA (PENECA, 1b), and 2-phenylhydroxy-
propynylNECA (PHPNECA, 1c) showed high affinity
and different degrees of selectivity for the human
adenosine A3 receptor.17 The ethyl substituent of the
carboxamido group in the 5′-position of HENECA (1a )
has been replaced with a cyclopentyl or benzyl group.
The introduction of a cyclopentyl ring led to a compound
* To whom correspondence should be addressed. Phone: 39-0737-
402252. Fax: 39-0737-402295. E-mail gloria.cristalli@unicam.it.
† Universita` di Camerinoo.
‡ Universita¨t Wu¨rzburg.
10.1021/jm0109762 CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/18/2002

3272 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Volpini et al.
Sch em e 1
F igu r e 1. Some 2-alkynyl-N6-ethylcarboxamidoadenosines
that resulted in potent ligands for A3 adenosine receptors.
that showed an 8-fold decrease of affinity at all human
receptor subtypes compared with HENECA; when a
benzyl group was introduced in the same position, the
corresponding derivative showed a more pronounced
loss in affinity, demonstrating that a bulky substituent
is less tolerated at the 5′-position.18 Furthermore, NECA
derivatives bearing arylcarbamoyl substituents on N6
were found to possess high affinity and moderate
selectivity for the rat A3 receptor.19
In an attempt to potentiate the affinity and selectivity
at A3 receptors through a trisubstitution, we synthesized
NECA derivatives bearing arylcarbamoyl substituents
on N6 and (ar)alkynyl chains in the 2-position. However,
these trisubstituted derivatives showed a reduced af-
finity for both the A1 and A3 subtypes (Ki in the high
nanomolar range), together with loss of A2 activity,
resulting in compounds with good A2A/A1 and A2A/A3
selectivity.20
molecules was carried out to obtain a new selective
radioligand for the A3 subtype (Schemes 2 and 3).
These observations prompted us to investigate the
effect of small substituents in the N6-position of such
molecules.
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of 2-alkynyladenosines
(7a -e) and 2-alkynyl-N6-alkyladenosines (8a -e, 9a -
e, and 10a -e) was carried out starting from 9-(2,3,5,-
tri-O-acetyl-â-D-ribofuranosyl)-6-chloro-2-iodopurine (2).25
Reaction of 2 with liquid ammonia at room temperature
or with the suitable alkylamine at -20 °C for 30 min
and then at room temperature for 1 h gave compounds
321 and 4-6, respectively, obtained by selective substi-
tution of the chlorine atom in the 6-position and
contemporary deprotection of the sugar (Scheme 1).
Substitution of the iodine in the 2-position of 2-iodo-
adenosine (3)25 or of 2-iodo-N6-methyladenosine (4-6)
with five different alkynyl chains [commercially avail-
able 1-hexyne, ethynylbenzene, (R,S)-1-phenyl-2-pro-
pyn-1-ol, (R)-1-phenyl-2-propyn-1-ol, and (S)-1-phenyl-
2-propyn-1-ol] was carried out by a modification of the
palladium-catalyzed cross-coupling reaction12 to give
compounds 7-10a -e.
Taking into account both the differences in synthesis
(2-alkynylAdo derivatives can be obtained from com-
mercially available guanosine through five steps, whereas
for the synthesis of the corresponding NECA deriva-
tives, 10 steps are needed) and the fact that 2-alkynyl-
NECAs have affinity for adenosine receptors similar to
that of the corresponding adenosine derivatives,12 we
synthesized 2-alkynyl- (hexynyl-, phenylethynyl-, (R,S)-
phenylhydroxypropynyl-) adenosines bearing on N6
a
methyl (8a -c), an ethyl (9a -c), and an isopropyl (10a -
c). As reference compounds, the corresponding 2-alkyn-
yladenosines [HEAdo (7a ),12,21 PEAdo (7b), and PHPAdo
(7c)] were prepared.22 In the case of PHPAdo, both the
racemic mixture of nucleosides and the (R) and (S)
diastereomers were synthesized, leading to 7c, 7d , and
7e.23 (During the preparation of this manuscript, the
synthesis of 7d and 7e was reported by IJ zerman et
al.,24 with some differences in the method, yields, and
1H NMR spectrum attributions). The (R) (8d -10d ) and
(S) (8e-10e) diastereomers of N6-methyl-, N6-ethyl-,
and N6-isopropylPHPAdo (8c-10c) were also prepared
(Scheme 1).
Furthermore, to find a convenient synthetic procedure
for the preparation of radiolabeled N6-methyl-2-alkyn-
yladenosines, we carried out an attempt to couple 2
selectively with the suitable alkyne, using the cross-
coupling reaction conditions to obtain monosubstituted
2-alkynyl-6-chloro-9-(2,3,5,-tri-O-acetyl-â-D-ribofurano-
syl)purines. The 6-chlorine atom of these intermediates
Finally, a procedure to study the possibilities for
introduction of a tritiated alkylamino group in these

Alkynyl Derivatives of Adenosine
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3273
Sch em e 2
Sch em e 3
Unfortunately, reaction of 2 with the desired alkynes
in general failed to give the monosubstituted nucleosides
in satisfactory yield. In particular, the results of such
reactions are reported in Scheme 2. Coupling of 2 with
1-hexyne afforded a mixture of the monosubstituted
compound 6-chloro-2-hexynyl-9-(2,3,5,-tri-O-acetyl-â-D-
ribofuranosyl)purine (11) and the disubstituted com-
pound 12 in 31% and 45% yields, respectively. The
alkynylation site of 11 was confirmed by a chemical
proof. In fact, reaction of 11 with liquid ammonia at
room temperature for 24 h gave 2-hexynyladenosine
(HEAdo, 7a ) in 50% yield. In the case of phenylethyne,
the coupling reaction gave only the disubstituted nu-
cleoside 13 in 51% yield. Coupling of 2 with (R,S)-1-
phenyl-2-propyn-1-ol resulted in a monosubstituted
derivative in 60% yield, which was identified as the
phenylketopropenyl analogue 14 due to a tautomeric
rearrangement on the side chain. The structure of (E)-
6-chloro-2-(3-keto-3-phenyl-1-propen-1-yl)-9-(2,3,5,-tri-
O-acetyl-â-D-ribofuranosyl)purine (14) was confirmed by
1
1H NMR. The H NMR spectrum of 14 displayed two
doublets of the (E) double bond protons at δ 7.75 and
8.35 with a coupling constant of 15.8 Hz, while signals
corresponding to the protons of the CHOH group were
missing. On the other hand, the CHOH protons were
1
clearly identified in the H NMR spectra of the phenyl-
hydroxypropynyl derivatives 7c-e, 8c-e, 9c, 10c, and
15e, where conversely the double bond protons are
missing.
An alternative synthetic route, depicted in Scheme
3, was designed to avoid the undesired disubstitution.
Hence, the 2-alkynyladenosines 7a , 7b, and 7e were
used as the starting material and reaction of these
could have been easily substituted by tritiated methyl-
amine to give the corresponding radioligands of 8a -e.

3274 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Volpini et al.
Ta ble 1. Affinities of the Adenosine Derivatives 7a -e, 8a -e, 9a -e, and 10a -e in Radioligand Binding Assays at Human A1, A2A
,
and A3 Adenosine Receptors
compd
R
R′
Ki(A1),a nM
60
Ki(A2A),b nM
Ki(A3),c nM
A1/A3
25
A2A/A3
3
1a , HENECA
6.4
2.4
(50-72)
(3.8-11)
(2.0-2.9)
7a , HEAdo
H
CH3(CH2)3
CH3(CH2)3
CH3(CH2)3
CH3(CH2)3
18
5.7
4.7
4
297
60
4
1
1118
116
20
(9.4-36)
327
(255-419)
138
(119-158)
43
(5.2-6.2)
1,230
(453-3310)
267
(201-355)
196
(4.4-5.2)
1.1
(0.71-1.80)
2.3
(1.0-5.2)
9.7
8a
CH3
9a
C2H5
10a
CH(CH3)2
(34-56)
(132-293)
(9.4-10)
1b, PENECA
560
620
6.2
90
24
497
240
25
6
100
23
(480-650)
391
(284-556)
1,690
(1020-2790)
1,176
(849-1630)
429
(300-1300)
363
(285-462)
8,530
(7400-9850)
4,450
(3460-5720)
1,100
(5.1-7.5)
16
(13-19)
3.4
(2.0-5.8)
4.9
(3.1-7.9)
17
7b, PEAdo
H
C6H5
C6H5
C6H5
C6H5
8b
CH3
2509
908
65
9b
C2H5
CH(CH3)2
10b
(386-478)
2.7
(783-1550)
3.1
(8.0-34)
0.42
1c, (R,S)-PHPNECA
7
(1.7-4.1)
(2.4-3.9)
(0.17-1.0)
7c, (R,S)-PHPAdo
H
(R,S)-C6H5CH(OH)
(R,S)-C6H5CH(OH)
(R,S)-C6H5CH(OH)
(R,S)-C6H5CH(OH)
0.67
7.0
3.3
0.2
11
3
2
(0.55-0.80)
8.4
(6.8-10)
2.7
(2.4-2.9)
1.5
(0.96-2.2)
1.9
(1.8-2.1)
0.44
(0.38-0.52)
7.1
(5.4-9.5)
2.2
(2.0-2.4)
1.1
(3.7-13)
273
(216-346)
94
(72-123)
96
(65-142)
39
(25-59)
29
(19-45)
1,560
(989-2460)
1,181
(864-1614)
324
(2.3-4.8)
0.76
(0.60-0.96)
0.97
(0.58-1.6)
2.3
(1.6-3.4)
5.5
(3.6-8.5)
5.0
(3.2-7.7)
1.2
(0.71-2.1)
1.4
(0.70-2.9)
4.9
8c
CH3
359
97
9c
C2H5
CH(CH3)2
10c
0.7
0.3
0.1
6
42
1d , (R)-PHPNECA
7
7d , (R)-PHPAdo
H
(R)-C6H5CH(OH)
(R)-C6H5CH(OH)
(R)-C6H5CH(OH)
(R)-C6H5CH(OH)
6
8d
CH3
1300
843
66
9d
C2H5
CH(CH3)2
2
10d
0.2
3
(0.79-1.4)
2.1
(165-636)
2.0
(2.3-10.4)
0.75
1e, (S)-PHPNECA
3
(1.2-3.7)
(1.2-3.5)
(0.52-1.1)
7e, (S)-PHPAdo
H
(S)-C6H5CH(OH)
(S)-C6H5CH(OH)
(S)-C6H5CH(OH)
(S)-C6H5CH(OH)
0.67
1.8
1.4
0.5
18
17
1
1
(0.47-0.96)
9.6
(7.6-12)
3.4
(2.7-4.2)
1.7
(1.1-3.0)
163
(154-173)
64
(34-120)
43
(0.78-2.4)
0.53
(0.46-0.61)
0.20
(0.14-0.28)
1.5
8e
CH3
307
320
29
9e
C2H5
CH(CH3)2
10e
(1.4-2.0)
(24-79)
(1.0-2.0)
a
Displacement of specific [3H]CCPA binding in CHO cells stably transfected with human recombinant A1 adenosine receptor, expressed
as Ki (nM). Displacement of specific [3H]NECA binding in CHO cells stably transfected with human recombinant A2A adenosine receptor,
b
expressed as Ki (nM). c Displacement of specific [3H]NECA binding in CHO cells stably transfected with human recombinant A3 adenosine
receptor, expressed as Ki (nM).
compounds with diiodomethane and isopentylnitrite
gave the versatile synthons 2-alkynyl-6-iodoadenosine
15a , 15b, and 15e. Treatment of them with methyl-
amine at 0 °C for 5 h afforded the desired compounds
8a , 8b, and 8e. This procedure allows the introduction
in the final step at low temperature and with high yield
of a tritiated methylamine.
P h a r m a cology. The 2-alkynyl derivatives of adeno-
sine and of N6-alkyladenosines 7a -e, 8a -e, 9a -e, and
10a -e were evaluated at the human recombinant
adenosine receptors, stably transfected into Chinese
hamster ovary (CHO) cells, utilizing radioligand binding
studies (A1, A2A, A3) or adenylate cyclase activity assays
(A2B),26 and the results are reported in Tables 1 and 2.

Alkynyl Derivatives of Adenosine
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3275
Ta ble 2. Effects of the Adenosine Derivatives 7a -e, 8a -e,
9a -e, and 10a -e on Adenylyl Cyclase Activity at Human A2B
Adenosine Receptor
subtype is diminished (EC50 ) 100 vs 6.1 µM) as shown
in Table 2. A similar profile is obtained by comparing
PENECA (1b), PHPNECAs 1c-e, and the correspond-
ing adenosine derivatives PEAdo (7b), (R,S)-PHPAdo
(7c), (R)-PHPAdo (7d ), and (S)-PHPAdo (7e).
The presence of a methyl group on N6 of 2-alkynyl-
adenosine derivatives always decreased the affinity for
the A1 and A2A receptors and correspondingly increased
A3 affinity, leading to a significant improvement of A3
selectivity in comparison with all the other subtypes.
In particular, the N6-methyl derivative of PEAdo (8b)
showed low affinity for A1 and A2A subtypes (Ki(A1) )
1690 nM and Ki(A2A) ) 8530 nM) and much higher
affinity at the A3 receptor (Ki(A3) ) 3.4 nM), resulting
in an A1/A3 and A2A/A3 selectivity ratio of about 500 and
2500, respectively.
The compounds obtained when the N6-methyl group
is replaced by an ethyl (9a -c) or an isopropyl group
(10a -c) maintained high affinity for the A3 receptor but
showed in most cases decreased A1/A3 and A2A/A3
selectivity.
The potency of the synthesized compounds at the A2B
receptor has been evaluated, and the results are shown
in Table 2. All compounds resulted in full agonists in
stimulating adenylate cyclase activity in comparison
with NECA.
In addition to the consideration that the NECA
derivatives 1a -e are always more potent than the
corresponding adenosine nucleosides, it is clear that the
influence of the alkynyl chain in the 2-position is
determinant for the activity of these molecules at the
A2B receptor subtype. In fact, most of the 2-hydroxy-
phenylpropynyl derivatives showed EC50 in the low
micromolar range (see all c-e derivatives), the most
potent being the (S)-PHPNECA (1e)27 with an EC50 in
the nanomolar range (EC50 ) 0.22 µM).
a
compd
R
R′
EC50
6.1
,
µM
1a
(4.0-9.3)
7a
8a
9a
10a
1b
7b
8b
9b
10b
1c
H
CH3
CH3(CH2)3
CH3(CH2)3
CH3(CH2)3
CH3(CH2)3
100
100
100
100
C2H5
CH(CH3)2
>100
H
CH3
C6H5
C6H5
C6H5
C6H5
>100
100
C2H5
CH(CH3)2
>100
>100
1.1
(0.47-2.6)
7c
H
(R,S)-C6H5CH(OH)
(R,S)-C6H5CH(OH)
(R,S)-C6H5CH(OH)
(R,S)-C6H5CH(OH)
2.4
(1.5-3.7)
2.7
(1.3-5.6)
8c
CH3
9c
C2H5
CH(CH3)2
1.7
(0.97-3.0)
6.1
(4.3-8.5)
10c
1d
7d
2.4
(1.5-3.8)
6.2
(2.9-13)
H
(R)-C6H5CH(OH)
8d
9d
CH3
(R)-C6H5CH(OH)
(R)-C6H5CH(OH)
100
9.1
C2H5
(6.9-12)
10d
1e
CH(CH3)2
(R)-C6H5CH(OH)
8.9
(4.5-18)
0.22
(0.22-0.23)
On the other hand, the contemporary presence of a
hydroxyphenylpropynyl group in the 2-position and of
an ethyl substituent on N6 increased the potency at the
A2B adenosine receptor subtype (Table 2). In fact, the
EC50 of the N6-ethyl derivative of (S)-PHPAdo (9e) was
0.73 µM, and this compound resulted in being slightly
more potent than the corresponding N6-methyl and N6-
7e
H
(S)-C6H5CH(OH)
(S)-C6H5CH(OH)
(S)-C6H5CH(OH)
(S)-C6H5CH(OH)
0.92
(0.71-1.2)
2.6
(1.7-4.0)
8e
CH3
9e
C2H5
CH(CH3)2
0.73
(0.61-0.89)
0.96
(0.50-1.9)
10e
isopropyl derivatives (8e, EC50 ) 2.6 µM; 10e, EC50
)
0.96 µM) and more potent than the unsubstituted (S)-
PHPAdo (7e, EC50 ) 0.92 µM).
a
Measurement of receptor-stimulated adenylyl cyclase activity
in CHO cells stably transfected with human recombinant A2B
adenosine receptor, expressed as EC50 (µM).
Con clu sion s
HENECA (1a ),12 PENECA (1b),15 (R,S)-PHPNECA
(1c),14 (R)-PHPNECA (1d ),27 and (S)-PHPNECA (1e)27
have been used as reference compounds.
From this study, it is possible to conclude that
removing the carboxamido group of the 2-alkynyl de-
rivatives of NECA 1a -e led to the adenosine derivatives
7a -e, which in general maintain high affinity at all
adenosine receptor subtypes. In particular, the activities
of 2-alkynylAdos were slightly higher at A1 and lower
at A3 and A2B subtypes than those of the corresponding
NECA derivatives. The affinities at the A2A subtype are
similar for the two series of nucleosides.
Introduction of a methyl group on N6 of the 2-alkyn-
yladenosines brought about an increase in affinity at
the human A3 receptor and a corresponding decrease
at the other subtypes, resulting in a relevant increase
in A3 selectivity (Figure 2). In particular, 2-phenylethyn-
yl-N6-methylAdo (8b) showed an A3 affinity in the low
The results of binding and cyclase studies showed that
removing the N-ethylcarboxamido group of HENECA
(1a ) and of all other NECA derivatives 1b-e, reported
in Figure 1, led to adenosine derivatives that maintain
an affinity comparable to that of the parent compounds
for the A1, A2A, and A3 human adenosine receptor
subtypes. In particular, 2-hexynyladenosine (HEAdo,
7a ) showed slightly higher affinity at the A1 receptor
than the corresponding NECA derivative 1a (Ki ) 18
vs 60 nM), very similar affinity at the A2A subtype (Ki
) 5.7 vs 6.4 nM), and slightly lower affinity at the A3
receptor (Ki ) 4.7 vs 2.4 nM). The potency at the A2B

3276 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Volpini et al.
F igu r e 2. 2-Phenylhydroxypropynyladenosine derivatives. In
all cases the (S) diastereomers are more active than the
corresponding (R) diastereomers at A2A, A2B, and A3 receptors;
however, they are slightly less active at A1 receptors.
F igu r e 3. 2-Alkynyladenosines and the corresponding N6-
nanomolar range and an A1/A3 and A2A/A3 selectivity
ratio of about 500 and 2500, respectively.
methyl derivatives. Comparison of the activities at A1, A2A, A2B
,
and A3 receptors reveals that A3 receptors have a preference
for the N6-methyl derivatives. These compounds, which show
reduced activities at the other receptor subtypes in comparison
with the N6-unsubstituted derivatives, are endowed with high
A3 selectivity.
mp 180-182 °C (dec); 1H NMR (DMSO-d6) δ 2.92 (d, 3H, J )
4.1 Hz, NHCH3), 3.61 (m, 2H, CH2-5′), 3.95 (m, 1H, H-4′), 4.14
(m, 1H, H-3′), 4.53 (m, 1H, H-2′), 5.82 (d, 1H, J ) 6.1 Hz, H-1′),
8.14 (m, 1H, NH), 8.31 (s, 1H, H-8). Anal. (C11H14IN5O4) C, H,
N.
On the other hand, the presence of an ethyl group in
N6 has significantly increased the potency at the A2B
subtype in comparison with the N6-methyl, N6-isopropyl,
and the unsustituted adenosine derivatives. However,
for the potency at the A2B receptor, it seems to be more
important than the type of alkynyl chain in the 2-posi-
tion and the presence of the ribose carboxyamido group,
the (S)-2-phenylhydroxypropynylNECA27 [(S)-PHPN-
ECA, 1c, EC50(A2B) ) 0.22 µM] being one of the most
potent A2B agonist reported so far.
N6-Eth yl-2-iod oa d en osin e (5). Reaction of 2 with ethyl-
amine at -20 °C for 30 min and then at room temperature for
1 h, followed by silica gel flash chromatography elution with
CHCl3-CH3OH (97:3), gave 5 as a white solid in 76% yield:
mp 95-97 °C (dec); 1H NMR (DMSO-d6) δ 1.18 (t, 3H, J ) 7.0
Hz, NHCH2CH3 ), 3.57 (m, 4H, NHCH2CH3 and CH2-5′), 3.95
(m, 1H, H-4′), 4.13 (m, 1H, H-3′), 4.52 (m, 1H, H-2′), 5.82 (d,
1H, J ) 6.3 Hz, H-1′), 8.22 (m, 1H, NH), 8.31 (s, 1H, H-8′).
Anal. (C12H16IN5O4) C, H, N.
The combination of an ethyl group in N6-position and
an ethylcarboxamido substituent in the 4′-position of
(S)-2-hydroxypropynyladenosine could lead to a com-
pound endowed with high potency at the A2B receptor.
Finally, it is worthwhile to note that the four human
adenosine receptors showed a different behavior toward
the diastereomers of all the phenylhydroxypropynyl
derivatives 1d , 1e, 7d , 7e, 8d , and 8e. In fact, the A1
subtype had a slight preference for the (R) diastereomer
whereas the other subtypes showed a significant prefer-
ence for the compounds in the (S) configuration, as
clearly illustrated by Figure 3.
2-Iod o-N6-isop r op yla d en osin e (6). Reaction of 2 with
ethylamine at -20 °C for 30 min and then at room tempera-
ture for 16 h, followed by silica gel flash chromatography with
elution with CHCl3-CH3OH (98:2), gave 6 as a white solid in
1
72% yield: mp 100-102 °C (dec); H NMR (DMSO-d6) δ 1.21
(d, 6H, J ) 6.6 Hz, NCH(CH3)2), 3.61 (m, 2H, CH2-5′), 3.95
(m, 1H, H-4′), 4.13 (m, 1H, H-3′), 4.33 (m, 1H, NCH(CH3)2),
4.53 (m, 1H, H-2′), 5.83 (d, 1H, J ) 6.2 Hz, H-1′), 8.10 (m, 1H,
NH), 8.32 (s, 1H, H-8). Anal. (C13H18IN5O4) C, H, N.
Exp er im en ta l Section
Ch em istr y. Melting points were determined with a Bu¨chi
apparatus and are uncorrected. 1H NMR spectra were obtained
with a Varian VXR 300 MHz spectrometer; δ is in ppm, J is
in Hz. All exchangeable protons were confirmed by addition
of D2O. TLC experiments were carried out on precoated TLC
plates with silica gel 60 F-254 (Merck). For column chroma-
tography, silica gel 60 (Merck) was used. Elemental analyses
were determined on a Fisons model EA 1108 analyzer and are
within (0.4% of theoretical values.
Gen er a l P r oced u r e for th e Syn th esis of 2-Alk yn yla d -
en osin es (7a -e) a n d 2-Alk yn yl-N6-a lk yla d en osin es (8a -
e, 9a -e, a n d 10a -e). To a solution of 200 mg (0.51 mmol) of
2-iodoadenosine (3)25 or 2-iodo-N6-alkyladenosine (4-6) in 8
mL of dry DMF and 2.3 mL of triethylamine under an
atmosphere of N2 8 mg (0.011 mmol) of bis(triphenylphos-
phine)palladium dichloride and 0.51 mg (0.003 mmol) of CuI
were added. The appropriate terminal alkyne (3.1 mmol) was
added, and the reaction mixture was stirred at room temper-
ature for the time reported below. After evaporation in vacuo,
the residual oil was purified by silica gel column chromatog-
raphy with elution with a suitable mixture of solvents.
2-P h en yleth yn yla d en osin e (7b). Reaction of 3 with eth-
ynylbenzene for 3 h, followed by silica gel column chromatog-
raphy with elution with CHCl3-CH3OH (90:10), gave 7b as a
white solid in 78% yield (CHCl3-CH3CN): mp 215-216 °C;
1H NMR (DMSO-d6) δ 3.65 (m, 2H, CH2-5′), 3.99 (m, 1H, H-4′),
4.15 (m, 1H, H-3′), 4.57 (m, 1H, H-2′), 5.92 (d, 1H, J ) 6.2 Hz,
H-1′), 7.48 (m, 3H, H-Ph), 7.62 (m, 4H, H-Ph and NH2), 8.47
(s, 1H, H-8). Anal. (C18H17N5O4) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 2-Iod oa d en o-
sin e (3) a n d N6-Alk yl-2-iod oa d en osin es (4-6). To 1.86
mmol (1.003 g) of 9-(2,3,5-tri-O-acetyl-â-D-ribofuranosyl)-6-
chloro-2-iodopurine (2)25 8 mL of the suitable amine was added.
The mixture was set aside at room temperature or at -20 °C
for the time reported below. The residual amine was evapo-
rated, and the residue was chromatographed on a silica gel
column, eluting with a suitable mixture of solvents to give
compounds 3-6.
2-Iod o-N6-m eth yla d en osin e (4). Reaction of 2 with me-
thylamine at -20 °C for 30 min and then at room temperature
for 1 h, followed by silica gel chromatography with elution with
CHCl3-CH3OH (93:7), gave 4 as a white solid in 84% yield:
(R ,S )-2-(3-H yd r oxy-3-p h e n yl-1-p r op yn -1-yl)a d e n os-
in e (7c). Reaction of 3 with (R,S)-1-phenyl-2-propyn-1-ol for

Alkynyl Derivatives of Adenosine
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3277
6 h, followed by silica gel column chromatography with elution
with CHCl3-CH3OH (85:15), gave 7c as a white solid in 65%
yield (EtOH): mp 183-185 °C; 1H NMR (DMSO-d6) δ 3.63 (m,
2H, CH2-5′), 3.96 (m, 1H, H-4′), 4.14 (m, 1H, H-3′), 4.52 (m,
1H, H-2′), 5.61 (d, 1H, J ) 6.2 Hz, CHOH), 5.88 (d, 1H, J )
5.8 Hz, H-1′), 6.28 (d, 1H, J ) 6.0 Hz, CHOH), 7.46 (m, 7H,
H-Ph and NH2), 8.45 (s, 1H, H-8). Anal. (C19H19N5O5) C, H,
N.
(R)-2-(3-Hydr oxy-3-ph en yl-1-pr opyn -1-yl)aden osin e (7d).
Reaction of 3 with (S)-1-phenyl-2-propyn-1-ol for 6 h, followed
by silica gel column chromatography with elution with CHCl3-
CH3OH-cC6H12 (77:13:10), gave 7d as a white solid in 55%
yield (EtOH): mp 183-185 °C; 1H NMR (DMSO-d6) δ 3.64 (m,
2H, CH2-5′), 3.96 (m, 1H, H-4′), 4.13 (m, 1H, H-3′), 4.51 (m,
1H, H-2′), 5.61 (d, 1H, J ) 6.0 Hz, CHOH), 5.89 (d, 1H, J )
5.9 Hz, H-1′), 6.28 (d, 1H, J ) 6.0 Hz, CHOH), 7.46 (m, 7H,
H-Ph and NH2), 8.45 (s, 1H, H-8). Anal. (C19H19N5O5) C, H,
N.
(S)-2-(3-Hydr oxy-3-ph en yl-1-pr opyn -1-yl)aden osin e (7e).
Reaction of 3 with (R)-1-phenyl-2-propyn-1-ol for 6 h, followed
by silica gel column chromatography with elution with CHCl3-
CH3OH (88:12), gave 7e as a white solid in 67% yield (MeOH-
EtOH): mp 143-145 °C (dec); 1H NMR (DMSO-d6) δ 3.64 (m,
2H, CH2-5′), 3.96 (m, 1H, H-4′), 4.15 (m, 1H, H-3′), 4.53 (m,
1H, H-2′), 5.62 (d, 1H, J ) 6.1 Hz, CHOH), 5.88 (d, 1H, J )
5.9 Hz, H-1′), 6.28 (d, 1H, J ) 5.9 Hz, CHOH), 7.46 (m, 7H,
H-Ph and NH2), 8.45 (s, 1H, H-8). Anal. (C19H19N5O5) C, H,
N.
2-(1-Hexyn -1-yl)-N6-m eth yla d en osin e (8a ). Reaction of
4 with 1-hexyne for 16 h, followed by silica gel column
chromatography with elution with CHCl3-CH3OH (92:8), gave
8a as a white solid in 78% yield (EtOH): mp 152-155 °C; 1H
NMR (DMSO-d6) δ 0.93 (t, 3H, J ) 6.9 Hz, CH3), 1.50 (m, 4H,
(CH2)2CH3), 2.44 (t, 2H, J ) 6.6 Hz, CH2CtC), 2.94 (br s, 3H,
NHCH3), 3.63 (m, 2H, CH2-5′), 3.96 (m, 1H, H-4′), 4.13 (m,
1H, H-3′), 4.54 (m, 1H, H-2′), 5.88 (d, 1H, J ) 5.9 Hz, H-1′),
7.89 (m, 1H, NH), 8.40 (s, 1H, H-8). Anal. (C17H23N5O4) C, H,
N.
d6) δ 2.94 (m, 3H, NHCH3), 3.64 (m, 2H, CH2-5′), 3.96 (m, 1H,
H-4′), 4.14 (m, 1H, H-3′), 4.51 (m, 1H, H-2′), 5.64 (d, 1H, J )
5.9 Hz, CHOH), 5.88 (d, 1H, J ) 5.9 Hz, H-1′), 6.31 (d, 1H, J
) 5.9 Hz, CHOH), 7.40 (m, 3H, H-Ph), 7.57 (m, 2H, H-Ph),
7.96 (m, 1H, NH), 8.44 (s, 1H, H-8). Anal. (C20H21N5O5) C, H,
N.
N6-Eth yl-2-(1-h exyn -1-yl)a d en osin e (9a ). Reaction of 5
with 1-hexyne for 24 h, followed by silica gel column chroma-
tography with elution with CHCl3-CH3OH (92:8), gave 9a as
a white solid in 72% yield (EtOH): mp 187-189 °C; 1H NMR
(DMSO-d6) δ 0.93 (t, 3H, J ) 7.0 Hz, (CH2)2CH3), 1.18 (t, 3H,
J ) 14 Hz NCH2CH3), 1.51 (m, 4H, (CH2)2CH3), 2.46 (m, 2H,
CH2CtC), 3.61 (m, 4H, CH2-5′ and NCH2CH3), 3.96 (m, 1H,
H-4′), 4.13 (m, 1H, H-3′), 4.54 (m, 1H, H-2′), 5.87 (d, 1H, J )
6.1 Hz, H-1′), 7.94 (m, 1H, NH), 8.40 (s, 1H, H-8). Anal.
(C18H25N5O4) C, H, N.
N6-Eth yl-2-p h en yleth yn yla d en osin e (9b). Reaction of 5
with ethynylbenzene for 4 h, followed by silica gel column
chromatography with elution with CHCl3-CH3OH (92:8), gave
9b as a white solid in 83% yield (EtOH): mp 195-199 °C; 1H
NMR (DMSO-d6) δ 1.21 (t, 3H, J ) 14.2 Hz, NHCH2CH3), 3.59
(m, 4H, NHCH2 and CH2-5′), 3.98 (m, 1H, H-4′), 4.15 (m, 1H,
H-3′), 4.57 (m, 1H, H-2′), 5.92 (d, 1H, J ) 6.2 Hz, H-1′), 7.49
(m, 3H, H-Ph), 7.64 (m, 2H, H-Ph), 8.06 (m, 1H, NH), 8.46
(s, 1H, H-8). Anal. (C20H21N5O4) C, H, N.
(R,S)-N6-E t h yl-2-(3-h yd r oxy-3-p h en yl-1-p r op yn -1-yl)-
a d en osin e (9c). Reaction of 5 with (R,S)-1-phenyl-2-propyn-
1-ol for 6 h, followed by silica gel column chromatography with
elution with CHCl3-CH3OH (92:8), gave 9c as a white solid
in 69% yield (EtOH): mp 95-98 °C; 1H NMR (DMSO-d6) δ
1.18 (t, 3H, J ) 7.0 Hz, NHCH2CH3), 3.57 (m, 4H, NHCH2-
CH3 and CH2-5′), 3.97 (m, 1H, H-4′), 4.14 (m, 1H, H-3′), 4.52
(m, 1H, H-2′), 5.64 (d, J ) 5.9 Hz, CHOH), 5.89 (d, 1H, J )
6.0 Hz, H-1′), 6.31 (d, 1H, J ) 5.9 Hz, CHOH), 7.40 (m, 3H,
H-Ph), 7.58 (m, 2H, H-Ph), 8.02 (m, 1H, NH), 8.45 (s, 1H,
H-8). Anal. (C21H23N5O5) C, H, N.
(R)-N6-Eth yl-2-(3-h yd r oxy-3-p h en yl-1-p r op yn -1-yl)a d -
en osin e (9d ). Reaction of 5 with (S)-1-phenyl-2-propyn-1-ol
for 16 h, followed by silica gel column chromatography with
elution with CHCl3-cC6H12-CH3OH (60:35:5), gave 9d as a
N6-Meth yl-2-p h en yleth yn yla d en osin e (8b). Reaction of
4 with ethynylbenzene for 16 h, followed by silica gel column
chromatography with elution with CHCl3-CH3OH (92:8), gave
8b as a white solid in 84% yield (EtOH): mp 229-231 °C; 1H
NMR (DMSO-d6) δ 2.99 (br s, 3H, NHCH3), 3.64 (m, 2H, CH2-
5′), 3.98 (m, 1H, H-4′), 4.15 (m, 1H, H-3′), 4.57 (m, 1H, H-2′),
5.93 (d, 1H, J ) 6.2 Hz, H-1′), 7.47 (m, 3H, H-Ph), 7.65 (m,
2H, H-Ph), 8.07 (m, 1H, NH), 8.47 (s, 1H, H-8). Anal.
(C19H19N5O4) C, H, N.
1
white solid in 47% yield (MeOH): mp 122-125 °C; H NMR
(DMSO-d6) δ 1.17 (t, 3H, J ) 7.0 Hz, NHCH2CH3), 3.57 (m,
4H, NHCH2CH3 and CH2-5′), 3.95 (m, 1H, H-4′), 4.13 (m, 1H,
H-3′), 4.50 (m, 1H, H-2′), 5.64 (d, 1H, J ) 5.6 Hz, CHOH), 5.87
(d, 1H, J ) 5.8 Hz, H-1′), 6.31 (d, 1H, J ) 5.8 Hz, CHOH),
7.42 (m, 3H, H-Ph), 7.57 (m, 2H, H-Ph), 8.00 (m, 1H, NH),
8.44 (s, 1H, H-8). Anal. (C21H23N5O5) C, H, N.
(R,S)-2-(3-Hyd r oxy-3-p h en yl-1-p r op yn -1-yl)-N6-m eth yl-
a d en osin e (8c). Reaction of 4 with (R,S)-1-phenyl-2-propyn-
1-ol for 6 h, followed by silica gel column chromatography with
elution with CHCl3-cC6H12-CH3OH (75:15:10), gave 8c as a
(S)-N6-Eth yl-2-(3-h yd r oxy-3-p h en yl-1-p r op yn -1-yl)a d -
en osin e (9e). Reaction of 5 with (R)-1-phenyl-2-propyn-1-ol
for 7 h, followed by silica gel column chromatography with
elution with CHCl3-CH3OH (93:7), gave 9e as a white solid
in 25% yield (MeOH): mp 102-106 °C; 1H NMR (DMSO-d6) δ
1.17 (t, 3H, J ) 7.0 Hz, NHCH2CH3), 3.56 (m, 4H, NHCH2-
CH3 and CH2-5′), 3.95 (m, 1H, H-4′), 4.12 (m, 1H, H-3′), 4.49
(m, 1H, H-2′), 5.63 (d, 1H, J ) 6.0 Hz, CHOH), 5.87 (d, 1H, J
) 5.8 Hz, H-1′), 6.31 (d, 1H, J ) 5.8 Hz, CHOH), 7.41 (m, 3H,
H-Ph), 7.56 (m, 2H, H-Ph), 7.98 (m, 1H, NH), 8.43 (s, 1H,
H-8). Anal. (C21H23N5O5) C, H, N.
1
white solid in 73% yield (EtOH): mp 115 °C (dec); H NMR
(DMSO-d6) δ 2.94 (br s, 3H, NHCH3), 3.63 (m, 2H, CH2-5′),
3.95 (m, 1H, H-4′), 4.13 (m, 1H, H-3′), 4.50 (m, 1H, H-2′), 5.64
(d, 1H, J ) 5.8 Hz, CHOH), 5.88 (d, 1H, J ) 5.7 Hz, H-1′),
6.31 (d, 1H, J ) 5.9 Hz, CHOH), 7.40 (m, 3H, H-Ph), 7.57
(m, 2H, H-Ph), 7.95 (m, 1H, NH), 8.44 (s, 1H, H-8). Anal.
(C20H21N5O5) C, H, N.
(R)-2-(3-H yd r oxy-3-p h en yl-1-p r op yn -1-yl)-N6-m et h yl-
a d en osin e (8d ). Reaction of 4 with (S)-1-phenyl-2-propyn-1-
ol for 6 h, followed by silica gel column chromatography with
elution with CHCl3-CH3OH (90:10), gave 8d as a white solid
in 62% yield (EtOEt): mp 127 °C (dec); 1H NMR (DMSO-d6) δ
2.94 (br s, 3H, NHCH3), 3.63 (m, 2H, CH2-5′), 3.95 (m, 1H,
H-4′), 4.13 (br s, 1H, H-3′), 4.50 (m, 1H, H-2′), 5.64 (d, 1H, J
) 5.9 Hz, CHOH), 5.89 (d, 1H, J ) 5.9 Hz, H-1′), 6.31 (d, 1H,
J ) 6.0 Hz, CHOH), 7.40 (m, 3H, H-Ph), 7.57 (d, 2H, J ) 6.7
Hz, H-Ph), 7.97 (m, 1H, NH), 8.44 (s, 1H, H-8). Anal.
(C20H21N5O5) C, H, N.
(S)-2-(3-H yd r oxy-3-p h en yl-1-p r op yn -1-yl)-N6-m et h yl-
a d en osin e (8e). Reaction of 4 with (R)-1-phenyl-2-propyn-1-
ol for 6 h, followed by silica gel column chromatography with
elution with CHCl3-CH3OH (90:10), gave 8e as a white solid
in 60% yield (EtOEt): mp 126-128 °C (dec); 1H NMR (DMSO-
2-(1-Hexyn -1-yl)-N6-isop r op yla d en osin e (10a ). Reaction
of 6 with 1-hexyne for 12 h, followed by silica gel column
chromatography with elution with CHCl3-CH3OH (92:8), gave
10a as a white solid in 74% yield (EtOH): mp 193-195 °C;
1H NMR (DMSO-d6) δ 0.93 (t, 3H, J ) 7.0 Hz, (CH2)2CH3),
1.21 (d, 6H, J ) 6.6 Hz, CH(CH3)2), 1.49 (m, 4H, (CH2)2CH3),
2.44 (t, 2H, J ) 13.6 Hz, CH2CtC), 3.61 (m, 4H, CH2-5′), 3.96
(m, 1H, H-4′), 4.12 (t, 1H, J ) 7.9 Hz, H-3′), 4.53 (m, 2H, H-2′
and CH(CH3)2), 5.87 (d, 1H, J ) 6.1 Hz, H-1′), 7.74 (d, 1H, J
) 8.8 Hz, NH), 8.39 (s, 1H, H-8). Anal. (C19H27N5O4) C, H, N.
2-P h en yleth yn yl-N6-isop r op yla d en osin e (10b). Reac-
tion of 6 with ethynylbenzene for 4 h, followed by silica gel
column chromatography with elution with CHCl3-CH3OH (92:
8), gave 10b as a white solid in 65% yield (EtOH): mp 192-
195 °C; 1H NMR (DMSO-d6) δ 1.24 (d, 6H, J ) 6.9, CH(CH3)2),
3.63 (m, 2H, CH2-5′), 3.98 (m, 1H, H-4′), 4.14 (m, 1H, H-3′),

3278 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Volpini et al.
4.55 (m, 2H, H-2′ and CH(CH3)2), 5.91 (d, 1H, J ) 5.9 Hz, H-1′),
7.49 (m, 3H, H-Ph), 7.63 (m, 2H, H-Ph), 7.92 (m, 1H, NH),
8.47 (s, 1H, H-8). Anal. (C21H23N5O4) C, H, N.
3H, COCH3), 2.08 (s, 3H, COCH3), 2.15 (s, 3H, COCH3), 4.39
(m, 3H, CH2-5′ and H-4′), 5.74 (m, 1H, H-3′), 6.00 (m, 1H, H-2′),
6.40 (d, 1H, J ) 5.3 Hz, H-1′), 7.57 (m, 6H, H-Ph), 7.74 (m,
6H, H-Ph), 8.99 (s, 1H, H-8). Anal. (C32H26N4O7) C, H, N.
(E)-6-Ch lor o-2-(3-k eto-3-p h en yl-1-p r op en -1-yl)-9-(2,3,5,-
tr i-O-a cetyl-â-D-r ibofu r a n osyl)p u r in e (14). Reaction of 2
with (R,S)-1-phenyl-2-propyn-1-ol for 48 h, followed by chro-
(R,S)-2-(3-Hyd r oxy-3-p h en yl-1-p r op yn -1-yl)-N6-isop r o-
p yla d en osin e (10c). Reaction of 6 with (R,S)-1-phenyl-2-
propyn-1-ol for 6 h, followed by silica gel column chromatog-
raphy with elution with CHCl3-CH3OH (92:8), gave 10c as a
white solid in 74% yield (EtOH): mp 115-120 °C (dec); 1H
NMR (DMSO-d6) δ 1.21 (d, 6H, J ) 6.6 Hz, NHCH(CH3)2), 3.57
(m, 2H, CH2-5′), 3.95 (m, 1H, H-4′), 4.13 (m, 1H, H-3′), 4.38
(m, 1H, NHCH(CH3)2), 4.50 (m, 1H, H-2′), 5.63 (d, J ) 5.9 Hz,
CHOH), 5.88 (d, 1H, J ) 5.9 Hz, H-1′), 6.29 (d, 1H, J ) 6.2
Hz, CHOH), 7.40 (m, 3H, H-Ph), 7.56 (m, 2H, H-Ph), 7.80
(d, 1H, J ) 8.4 Hz, NH), 8.44 (s, 1H, H-8). Anal. (C22H25N5O5)
C, H, N.
matography on a silica gel column with elution with cC6H12
-
EtOAc (75:25), gave 14 (60%) as an amorphous solid: 1H NMR
(DMSO-d6) δ 1.89 (s, 3H, COCH3), 2.12 (s, 3H, COCH3), 2.14
(s, 3H, COCH3), 4.39 (m, 3H, CH2-5′ and H-4′), 5.92 (m, 1H,
H-3′), 6.15 (m, 1H, H-2′), 6.43 (d, J ) 3.0 Hz, 1H, H-1′), 7.75
(m, 4H, H-Ph and dCH), 8.14 (d, J ) 7.5, 2-H, H-Ph), 8.35
(d, J ) 15.8 Hz, 1H, dCH), 8.95 (s, 1H, H-8). Anal. (C25H23
-
ClN4O8) C, H, N.
(R)-2-(3-Hyd r oxy-3-p h en yl-1-p r op yn -1-yl)-N6-isop r op -
yla d en osin e (10d ). Reaction of 6 with (S)-1-phenyl-2-propyn-
1-ol for 16 h, followed by silica gel column chromatography
with elution with CHCl3-cC6H12-CH3OH (75:15:10), gave 10d
as a white solid in 61% yield (MeOH): mp 205 °C (dec); 1H
NMR (DMSO-d6) δ 1.19 (d, 6H, J ) 6.6 Hz, NHCH(CH3)2), 3.60
(m, 2H, CH2-5′), 3.94 (m, 1H, H-4′), 4.11 (m, 1H, H-3′), 4.47
(m, 2H, H-2′ and NHCH(CH3)2), 5.61 (d, 1H, J ) 6.2 Hz,
CHOH), 5.87 (d, 1H, J ) 6.0 Hz, H-1′), 6.29 (d, 1H, J ) 6.0
Hz, CHOH), 7.38 (m, 3H, H-Ph), 7.55 (m, 2H, H-Ph), 7.81
(d, 1H, J ) 8.8 Hz, NH), 8.42 (s, 1H, H-8). Anal. (C22H25N5O5)
C, H, N.
(S)-2-(3-Hyd r oxy-3-p h en yl-1-p r op yn -1-yl)-N6-isop r op -
yla d en osin e (10e). Reaction of 6 with (R)-1-phenyl-2-propyn-
1-ol for 16 h, followed by silica gel column chromatography
with elution with CHCl3-cC6H12-CH3OH (75:15:10), gave 10e
as a white solid in 41% yield (MeOH): mp 96-98 °C (dec); 1H
NMR (DMSO-d6) δ 1.20 (d, 6H, J ) 6.6 Hz, NHCH(CH3)2), 3.60
(m, 2H, CH2-5′), 3.95 (m, 1H, H-4′), 4.12 (m, 1H, H-3′), 4.49
(m, 2H, H-2′ and NHCH(CH3)2), 5.63 (d, 1H, J ) 6.0 Hz,
CHOH), 5.87 (d, 1H, J ) 5.8 Hz, H-1′), 6.30 (d, 1H, J ) 6.0
Hz, CHOH), 7.39 (m, 3H, H-Ph), 7.56 (m, 2H, H-Ph), 7.83
(d, 1H, J ) 8.8 Hz, NH), 8.44 (s, 1H, H-8). Anal. (C22H25N5O5)
C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-Alk yn yl-
6-c h lo r o -9-(2,3,5-t r i-O -a c e t y l-â-D -r ib o fu r a n o s y l)p u -
r in es. To a solution of 2 (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 suitable 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,
eluting with a suitable mixture of solvents to give compounds
11-14 as amorphous solids.
6-Ch lor o-2-(h exyn -1-yl)-9-(2,3,5-t r i-O-a cet yl-â-D-r ib o-
fu r a n osyl)p u r in e (11) a n d 2,6-(Dih exyn -1-yl)-9-(2,3,5-tr i-
O-a cetyl-â-D-r ibofu r a n osyl)p u r in e (12). Reaction of 2 with
1-hexyne for 16 h, followed by chromatography on a silica gel
column with elution with CHCl3-cC6H12-MeCN (50:45:5),
gave 11 (31%) and 12 (45%) as amorphous solids.
Gen er a l P r oced u r e for th e Syn th esis of 6-Iod o-2-
a lk yn ylp u r in es 15a , 15b, a n d 15e. To a solution of the
suitable 2-alkynyladenosine (0.27 mmol) in dry DMF (2 mL)
diiodomethane (3.61 mL) and isopentylnitrite (1.12 mL) were
added, and the mixture was heated at 60 °C for 30 min. The
solvent was evaporated, and the residue was chromatographed
on a silica gel column with elution with a suitable mixture of
solvents to give compounds 15a , 15b, and 15e.
2-(Hexyn -1-yl)-6-iodo-9-(â-D-r ibofu r an osyl)pu r in e (15a).
Reaction of 7a according to the general procedure and puri-
fication with elution with CHCl3-cC6H12-MeOH (83:10:7)
gave 15a as an amorphous solid in 38% yield: 1H NMR
(DMSO-d6) δ 0.95 (t, 3H, J ) 7.1 Hz, (CH2)2CH3), 1.54 (m, 4H,
(CH2)2CH3), 2.52 (m, 2H, CH2CtC), 3.68 (m, 2H, CH2-5′), 3.99
(m, 2H, H-4′), 4.18 (m, 1H, H-3′), 4.52 (m, 1H, H-2′), 5.97 (d,
1H, J ) 5.1 Hz, H-1′), 8.94 (s, 1H, H-8). Anal. (C16H19IN4O4)
C, H, N.
6-Iod o-2-(p h en ylet h yn -1-yl)-9-(â-D-r ib ofu r a n osyl)p u -
r in e (15b). Reaction of 7b according to the general procedure
and purification with elution with CHCl3-cC6H12-MeOH (83:
10:7) gave 15b as an amorphous solid in 37% yield: 1H NMR
(DMSO-d6) δ 3.68 (m, 2H, CH2-5′), 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. (C18H15IN4O4) C, H, N.
(S)-2-(3-Hyd r oxy-3-p h en yl-1-p r op yn -1-yl)-6-iod o-9-(â-D-
r ibofu r a n osyl)p u r in e (15e). Reaction of 7e according to the
general procedure and purification with elution with CHCl3-
MeOH-cC6H12 (82:10:8) gave 15e as an amorphous solid in
28% yield: 1H NMR (DMSO-d6) δ 3.68 (m, 2H, CH2-5′), 4.00
(m, 1H, H-4′), 4.18 (m, 1H, H-3′), 4.51 (m, 1H, H-2′), 5.71 (d,
1H, J ) 6.0 Hz, CHOH), 5.98 (d, 1H, J ) 4.5 Hz, H-1′), 6.28
(d, 1H, J ) 6.0 Hz, CHOH), 7.42 (m, 3H, H-Ph), 7.58 (m, 2H,
H-Ph), 8.98 (s, 1H, H-8). Anal. (C19H17IN4O5) C, H, N.
Biologica l Stu d ies. Cloning of the human adenosine
receptors, stable transfection of cells, cell culture, membrane
preparation, radioligand binding, and determination of ade-
nylyl cyclase activity have been fully described elsewhere.26
Briefly, all human subtypes were stably transfected into
Chinese hamster ovary (CHO) cells in order to be able to study
their pharmacological profile in an identical cellular back-
ground utilizing radioligand binding studies (A1, A2A, A3) or
adenylyl cyclase activity assays (A2B).
11: 1H NMR (DMSO-d6) δ 0.94 (t, 3H, J ) 7.1 Hz, CH3),
1.55 (m, 4H, (CH2)2CH3), 2.04 (s, 3H, COCH3), 2.06 (s, 3H,
COCH3), 2.14 (s, 3H, COCH3), 2.52 (t, 2H, J ) 6.6 Hz, CH2Ct
C), 4.39 (m, 3H, CH2-5′ and H-4′), 5.63 (m, 1H, H-3′), 6.00 (m,
1H, H-2′), 6.34 (d, 1H, J ) 5.4 Hz, H-1′), 8.94 (s, 1H, H-8).
Anal. (C22H25ClN4O7) C, H, N.
Receptor binding affinity was determined using [3H]CCPA
as radioligand at A1 receptors, whereas [3H]NECA was used
at A2A and A3 subtypes. Because of the lack of a suitable
radioligand, the relative potency of agonists at A2B adenosine
receptors was determined in adenylyl cyclase experiments.
12: 1H NMR (DMSO-d6) δ 0.94 (m, 6H, J ) 7.1 Hz, 2CH3),
1.53 (m, 8H, 2(CH2)2CH3), 2.04 (s, 3H, COCH3), 2.05 (s, 3H,
COCH3), 2.14 (s, 3H, COCH3), 2.53 and 2.64 (m, 2H each,
CH2CtC), 4.35 (m, 3H, CH2-5′ and H-4′), 5.63 (m, 1H, H-3′),
5.94 (m, 1H, H-2′), 6.31 (d, 1H, J ) 5.43 Hz, H-1′), 8.96 (s, 1H,
H-8). Anal. (C28H34N4O7) C, H, N.
Ack n ow led gm en t. This work was supported by EC
Contract BMH4-98-3474 and by a grant from the
Ministry of Research (Grant COFIN, 2001). We thank
M. Brandi, F. Lupidi, and G. Rafaiani for technical
assistance.
2,6-(Dip h en yleth yn -1-yl)-9-(2,3,5-tr i-O-a cetyl-â-D-r ibo-
fu r a n osyl)p u r in e (13). Reaction of 2 with ethynylbenzene
for 16 h, followed by chromatography on a silica gel column
with elution with CHCl3-cC6H12-MeCN (52:40:8), gave 13
(51%) as an amorphous solid: 1H NMR (DMSO-d6) δ 2.04 (s,
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