Nucleosides and nucleotides. 177. 9-(6,7-Dideoxy-β-d-allo-hept-5- ynofuranosyl)adenine: A selective and potent ligand for P3 purinoceptor- like protein [4]

Full text of DOI:10.1021/jm9802822

2676
J . Med. Chem. 1998, 41, 2676-2678
Ta ble 1. P3 Purinoceptor-like Protein (P3LP) Binding Activity
of Various 5′-Modified Adenosine Analoguesa
Nu cleosid es a n d Nu cleotid es. 177.
9-(6,7-Did eoxy-â-D-a llo-h ep t-5-
yn ofu r a n osyl)a d en in e: A Selective a n d
P oten t Liga n d for P 3 P u r in ocep tor -lik e
P r otein 1
compds
Ki (nM)
compd
Ki (nM)
1
2
3
4
5
6
7
8
37 ( 1.2
51 ( 12
9
10
11
17a
17b
18a
18b
1100 ( 520
280 ( 38
64 ( 12
64 ( 20
21000 ( 7300
84 ( 4.1
19 ( 8.9
450 ( 140
100 ( 57
5600 ( 1700
Akira Matsuda,*,† Haruyo Kosaki,† Yoshiko Saitoh,‡
Yuichi Yoshimura,† Noriaki Minakawa,† and
Hiroyasu Nakata‡
81 ( 2.5
54 ( 4.5
70 ( 25
P3LP binding activity to [3H]NECA (40 nM) was determined
as described previously.6 P3LP was partially purified by hydroxyl-
apatite chromatography from a CHAPS-solubilized preparation of
rat brain membranes. A1, A2A, A2B, and A3 adenosine receptors
and adenotin were not present in this preparation. Ki values were
expressed as mean ( SEM (n ) 3).
a
Graduate School of Pharmaceutical Sciences,
Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, J apan, and Department of Molecular
and Cellular Neurobiology, Tokyo Metropolitan Institute for
Neuroscience, Fuchu, Tokyo 183-8526, J apan
Received May 5, 1998
Adenine nucleosides and nucleotides induce a large
variety of physiological responses in various tissues and
cells via specific receptors, i.e., purinoceptors, on the cell
surface membranes.2,3 Purinoceptors have been clas-
sified into types P1 and P2 based on their pharmacologi-
cal properties. P1 purinoceptors, which are usually
called adenosine receptors, are selective to adenosine
and its analogues, while P2 purinoceptors are specific
to adenine nucleotides such as ATP and their analogues.
P1 purinoceptors are subclassified into A1, A2A, A2B, and
A3 adenosine receptors. P2 purinoceptors are also
subclassified. Although the presence of such purinocep-
tor subtypes explains most of the functions of purines,
it may be necessary to identify additional subtypes to
fully explain the functional roles of adenosine and
adenine nucleotides in various tissues or cells.
The non-P1 and non-P2 muscle relaxant effect of ATP
in rabbit thoracic aorta has recently been attributed to
a P3 purinoceptor which is activated by either adenosine
or ATP.4,5 Saitoh and Nakata purified a new [3H]-5′-
N-ethylcarboxamidoadenosine (NECA) binding protein
from rat brain membranes.6 The ligand specificity of
this protein was demonstrated to follow the order NECA
> adenosine > inosine > ATP > N6-cyclopentyladeno-
sine > 2-chloroadenosine, and most xanthines were
inactive. This result is very similar to the ranking of
potency toward the muscle receptor reported by Chinel-
lato et al. (NECA > adenosine > ATP),4,5 but is slightly
different from the relative order of potency in adrenergic
nerves of the rat caudal artery (2-chloroadenosine >
ATP > adenosine).7 Therefore, this [3H]NECA binding
protein from the rat brain membranes was thought to
be a subtype of P3 purinoceptor or P3 purinoceptor-like
protein (P3LP). Since the physiological roles of P3LP
and P3 purinoceptor have not been fully elucidated, we
need a specific ligand to obtain further information
regarding this receptor. In this communication, we
describe the structural requirements of various 5′-
modified adenosine analogues with regard to P3LP
binding and present a new specific ligand, 9-(6,7-
dideoxy-â-D-allo-hept-5-ynofuranosyl)adenine (17a ).
F igu r e 1. Structures of compounds 1-11.
P3LP binding assays were carried out using P3LP that
was partially purified from rat brain membranes by [3H]-
NECA (40 nM) in the presence of various concentrations
of adenosine analogues.6 Ki values were calculated
using Graph Pad software (ISI Software), and the
results are summarized in Table 1. Since P3LP binds
tightly to NECA, we examined several NECA analogues
with regard to P3LP binding. NECA (1), 5′-N-methyl-
carboxamidoadenosine (2), and 5′-carboxamidoadeno-
sine (3) were found to be good ligands, with Ki values
of 37, 51, and 64 nM, respectively. Compounds with a
longer alkyl group at the carboxamide nitrogen showed
slightly better activity. However, 5′-N,N-dimethyl-
carboxamidoadenosine (4) did not bind well (Ki ) 21
µM). Therefore, the terminal bulky alkyl group at the
carboxamide nitrogen is believed to be well-tolerated
and the NH group may play an important role in
binding to P3LP. To obtain further information regard-
ing the binding region around the 5′-position of adeno-
sine, we tested other 5′-deoxy-5′-substituted analogues
of adenosine which did not have an acidic proton.
Compounds 5, 6, 7, 8, and 11 (Figure 1) also showed
potent inhibitory activities, with Ki values in the nano-
molar range. However, compounds 9 and 10 were less
effective inhibitors than the other compounds. There-
fore, some bulky substituents can be well accommodated
at the 5′-position in P3LP. To further clarify whether
an acidic proton of the substituent plays an important
role and since, among the compounds tested thus far,
NECA binds most potently to P3LP, we designed 17a
and its diastereomer 17b and their congeners 18a ,b.
Ch em istr y
* To whom reprint requests should be addressed. Phone: +81-11-
706-3228. Fax: +81-11-706-4980. E-mail: matuda@pharm.hokudai.ac.jp.
† Hokkaido University.
The synthesis of the target compounds was straight-
‡ Tokyo Metropolitan Institute for Neuroscience.
forward, as shown in Scheme 1. Treatment of N6-
S0022-2623(98)00282-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/30/1998

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2677
Ta ble 2. Affinities of Ligands to Adenosine A1, A2A, A2B, and A3 Receptors and P3 Purinoceptor-like Protein (P3LP)a
Ki (nM)
c
c
c
d
compd
P3LPb
A1
A2a
A2b
A3
17a
17b
NECA
19 ( 8.9
450 ( 140
37 ( 1.2
33% inhibn at 10 µM
21% inhibn at 10 µM
6.3e
40% inhibn at 100 µM
40% inhibn at 100 µM
10e
no inhibn at 100 µM
no inhibn at 100 µM
2600e
no inhibn at 10 µM
no inhibn at 10 µM
110f
a
b
Ki values were expressed as mean ( SEM (n ) 3). See Table 1 for the binding assay. c The activities of A1 and A2A adenosine receptors
were measured by binding [3H]DPCPX (2 nM) and [3H]CGS 21680 (5.9 nM) to rat brain membranes, respectively. A2B adenosine receptor
activity was determined by measuring cAMP production in VA-13 cells.10 The activity of A3 adenosine receptor was measured by binding
d
[
125I]AB-MECA (0.15 nM) to RBL-2H3 cell membranes in the presence of 100 nM DPCPX and 100 µM AppNHp (A) and in the presence
of 100 µM DPCPX, 100 µM AppNHp, and 10 nM IB-MECA (B). Specific A3 adenosine receptor activity was calculated from the difference
between the binding activity in (A) and (B). e Data taken from ref 10. f Data taken from ref 12.
Sch em e 1
binding was found to be 17a , with a Ki value of 19 nM,
while its 5′ diastereomer 17b was 24-fold less effective
(Table 1). A similar tendency for stereoselective recog-
nition at the 5′-position by P3LP was also found in 18a ,b
although 18a and 18b were 5-fold and 63-fold less
effective than 17a , respectively. Therefore, P3LP rec-
ognizes not only the stereochemistry of the hydroxyl
group at the 5′-position but also the bulkiness of the
substituents. Again, an acidic proton of the substituent
plays an important role.
We next compared the receptor selectivity for selected
nucleosides 17a ,b and NECA against P1 purinoceptor
subtypes, such as adenosine A1, A2A, A2B, and A3
receptors, since 17a ,b are just adenosine analogues. As
shown in Table 2, 17a showed almost no inhibitory
activity against A2A, A2B, and A3 receptors, although it
inhibited only slightly [3H]DPCPX binding against
adenosine A1 receptor (33% inhibition at 10 µM).
Compound 17b was also a selective ligand for P3LP, but
much less potent for P3LP binding than 17a . NECA
showed a high affinity to P3LP, but was a nonselective
adenosine receptor agonist. Therefore, 17a is the first
selective and potent ligand for P3LP.
In summary, we have identified the first selective and
potent ligand for P3 purinoceptor-like protein. Further
studies will be needed to elucidate the structure-
activity relationships of adenosine analogues against P3-
LP in greater detail, particularly with regard to the
pharmacological activity of 17a .
benzoyl-2′,3′-O-isopropylideneadenosine-5′-aldehyde (12)
with TMSCtCMgBr (3 equiv, prepared from TMSCt
CH and EtMgBr) at -20 °C in THF gave a diastereo-
mixture of 13 (R:S ) about 2:1, determined by its 1H
NMR) in 70% yield, which was partially separable by
silica gel column chromatography. However, the ster-
eochemistry at the 5′-position of 13 could not be
determined at this stage. A mixture of less polar 13
with a amall amount of the more polar one was treated
with NH3 in MeOH (saturated at 0 °C) for 26 h at room
temperature to give crystalline 15a in 74% yield. X-ray
structural analysis of 15a 8 showed that 15a has an R
configuration at the 5′-position. Compound 15a was
further treated with aqueous 90% trifluoroacetic acid
to give 17a 9 in 79% yield. 9-(6,7-Dideoxy-R-L-talo-hept-
5-ynofuranosyl)adenine (17b) was also obtained by the
same sequential deprotection of more polar 13. To
further study the structure-activity relationship, 9-(7-
C-butyl-6,7-dideoxy-â-D-allo-hept-5-ynofuranosyl)ade-
nine (18a ) and its diastereomer 18b were synthesized
in a similar way starting from 12, as shown in Scheme
1.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data of 15a and the synthetic methods described
Scheme 1 (10 pages). Ordering information is given on any
current masthead page.
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Biologica l Eva lu a tion a n d Discu ssion
The most potent compound in the series of 5′-modified
adenosine analogues in P3 purinoceptor-like protein

2678 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Communications to the Editor
(7) Shinozuka, K.; Bjur, R. A.; Westfall, D. P. Characterization of
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(8) C15H17N5O4, M ) 331.33, orthorhombic, P212121, a ) 10.068(2)
Å, b ) 21.536(4) Å, c ) 7.444(1) Å, V ) 1613.9 Å3, Dcalcd ) 1.363
g cm-1. A total of 1367 independent reflections were collected
and used for the structure analysis. The final R value was 0.034.
(9) Data: mp 242-243 °C; LRMS (EI) m/z 291 (M+); 1H NMR
(DMSO-d6 + D2O) 8.27 (s, 1 H, H-2), 8.11 (s, 1 H, H-8), 5.88 (d,
1 H, H-1′, J 1′,2′ ) 7.3 Hz), 4.63 (dd, 1 H, H-2′, J 2′,1′ ) 7.3, J 2′,3′
)
4.6 Hz), 4.45 (dd, 1 H, H-5′, J 5′,4′ ) 4.0, J 5′,CtCH ) 2.0 Hz), 4.19
(dd, 1 H, H-3′, J 3′,2′ ) 4.6, J 3′,4′ ) 1.3 Hz), 3.96 (dd, 1 H, H-4′,
J 4′,5′ ) 4.0, J 4′,3′ ) 1.3 Hz), 3.35 (d, 1 H, CtCH, J CtCH,5′ ) 2.0
Hz). Anal. (C12H13N5O4‚0.25H2O) C, H, N.
J M9802822