Naturally occurring 2'-O-methylpurine nucleosides with hypotensive properties

Article abstract of DOI:10.1007/s000180050131

2'-O-Methylinosine (1) has been isolated for the first time and shown to be an intrinsic hypotensive principle. Its probable in vivo precursor. 2'-O-methyladenosine (3), showed stronger and even orally potent hypotensive activity. Resistance of the methyladenosine (3) against adenosine deaminase is thought to contribute to its long-lasting activity. The effect of both nucleosides (1 and 3) was not accompanied with any significant change in heart rate, which is often observed with adenosine.

Full text of DOI:10.1007/s000180050131

CMLS, Cell. Mol. Life Sci. 54 (1998) 125–128
1420-682X/98/020125-04 $ 1.50+0.20/0
© Birkha¨user Verlag, Basel, 1998
Research Articles
Naturally occurring 2%-O-methylpurine nucleosides with
hypotensive properties
T. Yamada, K. Kageyama, Y. Joh, J. Konishi and K. Ienaga*
Institute of Bio-Active Science, Nippon Zoki Pharm. Co. Ltd., Yashiro, Kato-gun, Hyogo 673-14 (Japan),
Fax +81 795 42 5332
Received 2 October 1997; accepted 28 October 1997
Abstract. 2%-O-Methylinosine (1) has been isolated for sine (3) against adenosine deaminase is thought to con-
the first time and shown to be an intrinsic hypotensive tribute to its long-lasting activity. The effect of both
principle. Its probable in vivo precursor, 2%-O-methyl- nucleosides (1 and 3) was not accompanied with any
adenosine (3), showed stronger and even orally potent significant change in heart rate, which is often observed
hypotensive activity. Resistance of the methyladeno- with adenosine.
Key words. Methyladenosine; methylinosine; hypotension; adenosine; adenosine deaminase.
In the course of a search for novel low molecular weight first applied onto a Bio-Gel P-2 column (H2O), and
bioactive metabolites induced by inflammation in ani-
mals, we previously isolated several bioactive hetero-
cyclic compounds from extracts of inflamed rabbit skin
tissues [1, 2]. In seeking compounds with a hypotensive
effect on spontaneously hypertensive rats (SHR), we
fractionatd the phenol extract [1] of the inflamed rabbit
skin tissues inoculated with vaccinia virus and obtained
2%-O-methylinosine (1) as one of the active principles. In
this paper, we report that both (1) and its precursor,
2%-O-methyladenosine (3), have unique hypotensive ac-
tivities.
active fraction was separated by high-performance liq-
uid chromatography (HPLC) column (ODS, 0.1% tri-
fluoro-acetic acid (TFA)-MeOH) to give a colourless
crystalline compound as an active principle, 2%-O-
1
methylinosine (1), melting point (mp) 180–181 °C; H
NMR (400 MHz, D2O) l 3.39 (3H, s), 3.85 (1H, dd,
J=3.42, 12.69), 3.94 (1H, dd, J=2.93, 12.69), 4.30
(1H, m), 4.52 (1H, t, J=5.85), 4.61 (1H, t, J=5.85),
6.19 (1H, d, J=5.85), 8.25 (1H, s), 8.39 (1H, s); SIMS
m/z 283 (MH+). Its structure was determined by com-
parison of its physical data with those of a synthetic
authentic specimen. Results of co-melting and co-
elution on HPLC of both isolated and synthetic speci-
mens showed that they are identical; HPLC conditions
were as follows: system, JASCO 800 series (JASCO,
Japan); column, inertsil ODS-2 (4.6×105 mm, GL
Science, Japan); column temp., room temp.; eluent, 10%
MeOH containing 0.1% TFA; flow rate, 0.8 ml/min;
detection, ultraviolet (UV, 250 nm) with 875-UV
(JASCO, Japan); retention time of the single peak of
2%-O-methylinosine, 8 min after injection.
Materials and methods
Isolation of 2%-O-methylinosine (1). In the following
fractionation, hypotensive effect on SHR was used as
an index to obtain an active component. After boiling
the aqueous phenol extract of inflamed rabbit skin
tissues [1], and evaporation, the resulting residue was
* Corresponding author.

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CMLS 54 (1998), Birkha¨user Verlag, CH-4010 Basel/Switzerland
Research Articles
Table 1. Effect of 2%-O-methylinosine and h-methyldopa on systolic blood pressure on SHR.
Drug
Dose
(mg/kg, i.p)
No. of
rats
Blood pressure (mmHg)
% Decrease
before adm.
2 h after adm.
Control
2%-O-Me Ino
h-Methyldopa
-
3
3
3
23397
24598
24295
23197
9797***
185912*
0.491.8
100
100
60.493.5+++
24.895.8+
Each value represents the mean value9SE.
***,* Significantly different from control at PB0.001 and PB0.05, respectively.
+++ +
,
Significantly different from the value observed before administration at PB0.001 and PB0.05, respectively (t test).
Preparation of 2%-O-methyladenosine (3). A modified 1H NMR (400 MHz, D2O) l 3.40 (3H, s), 3.81 (1H, dd,
method of Robins et al. was adapted [3]. A suspension J=3.42, 12.70), 3.94 (1H, dd, J=2.93, 12.70), 4.30
(1H, m), 4.52 (1H, t, J=5.85), 4.61 (1H, t, J=5.85),
6.19 (1H, d, J=5.85), 8.29 (1H, s), 8.42 (1H, s); SIMS
m/z 283 (MH+).
Experimental animals. Male spontaneously hyperten-
sive rats (SHR; 16 weeks old, Hoshino-Dohbutu,
Japan) were kept at 22–24 °C, 45–70% humidity, and
were fasted for 18 h before the experiment.
of adenosine (4) (37.5 nmol) in a solution of SnCl2 (2
mmol) in MeOH (2 l) was treated with excess diazo-
methane (200 mmol) in 1,2-dimethoxyethane. The sol-
vents were removed in vacuo, and the residue was
chromatographed on Dowex 1×8 (OH− form) with
60% EtOH as an eluent. The appropriate fractions were
collected and evaporated to dryness, and the residue
was recrystallized from EtOH to give pure 2%-O-methyl-
adenosine (3) in 34% yield; mp 205–206 °C (lit., 200–
202 °C [3]) (Found: C, 47.0; H, 5.4; N, 24.7.
C11H13N5O4 requires C, 47.0; H, 5.4; N, 24.9);
1H NMR (400 MHz, D2O) l 3.39 (3H, s), 3.86 (1H, dd,
J=2.90, 12.70), 3.93 (1H, dd, J=2.40, 12.70), 4.26
(1H, m), 4.40 (1H, t, J=5.90), 4.56 (1H, dd, J=5.90,
4.90), 6.01 (1H, d, J=5.90), 8.01 (1H, s), 8.20 (1H, s);
SIMS m/z 282 (MH+).
Preparation of 2%-O-methylinosine from 2%-O-methyl-
adenosine. Sodium nitrite (72 mmol) was added to a
solution of 3 (3.6 mmol) in 25% aqueous acetic acid (10
ml) maintained at 20 °C. After 5 h, the reaction mixture
was neutralized with solid sodium bicarbonate and ap-
plied to an Amberlite XAD-7 column. The column was
washed with water and eluted with 30% MeOH. The
eluate was evaporated to dryness, and the residue was
recrystallized from MeOH to give pure 2%-O-methyl-
inosine (1) in 72% yield; mp 180–181 °C (lit., 177–
180 °C [3]) (Found: C, 47.1; H, 5.1; N, 19.6.
C11H14N4O5 requires C, 46.8; H, 5.0; N, 19.9);
Measurement of systolic blood pressure. Systolic blood
pressure was measured by an indirect method using a
pressure cuff and ring-shaped pulse transducer (Narco-
Bio-Systems, Houston, TX, USA, PE-300). Pressure
changes and pulse waves were recorded before and 2 h
after administration of a sample, and heart rate was
determined from the pulse trace at the same time. Each
effect was compared with those of a saline control and
h-methyldopa as placebo. In each case, the difference in
pressure before and 2 h after administration was calcu-
lated as percentage decrease.
Adenosine deaminase assay. The stability of adenosine
(4) or 2%-O-methyladenosine (3) in the presence of
adenosine deaminase (EC 3.5.4.4.) was followed by
measuring the change in optical density at 265 nm
resulting from the conversion of 4 to 2, or 3 to 1 using
a spectrophotometer (Beckman, model DU-650, Fuller-
ton, CA, USA). The assay mixture in 2 ml contained 50
mM phosphate buffer (pH 7.5), 0.1 mM test sample,
and 0.02 U of adenosine deaminase at 25 °C. Deami-
nase inhibition was determined by observing the de-
crease in the rate of adenosine deamination.
Results and discussion
Fractionation of the extract of inflamed rabbit skin
tissues provided a colourless crystalline compound as
one of the hypotensive principles. This was identified as
2%-O-methylinosine (1); its presence in vivo had not
been previously demonstrated. 2%-O-Methylinosine (1)
exhibited a significant hypotensive effect in decreasing
blood pressure by about 40% when administered by
intraperitoneal (i.p.) injection at a dosage of 100 mg/kg,
and showed stronger activity than h-methyldopa at the

Research Articles
CMLS 54 (1998), Birkha¨user Verlag, CH-4010 Basel/Switzerland
127
Figure 1. Stability of adenosine and 2%-O-methyladenosine
against adenosine deaminase.
Figure 2. Dose-hypotensive effects of 2%-O-methyladenosine on
systolic blood pressure in SHR. Each point and vertical bar
represent the mean9SE. *Significantly different from control at
PB0.05.
same dosage (table 1). This nucleoside (1) was thought
most likely to be a metabolite of 2%-O-methyladenosine
(3), known as a component in RNA [4]. In fact, we have
found that 3 can be enzymically converted to 1 by
adenosine deaminase (see fig. 1), but its rate is much
slower than that for the conversion of adenosine (4) to
inosine (2).
Although adenosine (4) itself had been known to have
hypotensive effects, O-methylated adenosine (3) had
not previously been shown to have similar effects, nor
would they have been possible to predict. It had been
widely believed that the free 2%-OH group in the ribose
moiety of adenosine was necessary for its hypotensive
activity [5], and that O-methylation at the 2%-position
would cause inactivation. On the other hand, adenosine
(4) had been known to show much stronger hypotensive
activity than inosine (2) [6], and if 2%-O-methylinosine
(1) was hypotensive, then 2%-O-methyladenosine (3)
could be analogously hypotensive. In order to clarify
these points, we compared the hypotensive activities of
these two naturally occurring methylated purine nu-
cleosides (1 and 3) with each other and with their
unmethylated nucleosides (2 and 4).
hypotensive activities when they were injected via the
i.p. route (data not shown). However, as shown in table
2, neither methylinosine (1), inosine (2) nor adenosine
(4) showed hypotensive activity when administered
orally at dosages of 200 mg/kg. On the contrary, 2%-O-
methyladenosine (3) decreased blood pressure signifi-
cantly in a dose-dependent manner (fig. 2) 2 h after oral
administration, and this effect was more prominent
than with h-methyldopa under the same conditions. Far
from inactivation, our result indicated that 2%-O-
methylation enhanced the original activity of (1).
Furthermore, the hypotensive effects of both natural
methylpurine nucleosides (1 and 3), by i.p. injection of
1 and i.p. or administration per os (p.o.) of 3, was
not accompanied with any significant change in heart
rate.
The hypotensive action of adenosine (4) occurs through
several mechanisms, and it includes vasodilatation in
the peripheral vasculature and cardiac depression: the
former is probably mediated by A2 receptors and the
latter by A1 receptors [5]. However, extrinsic 4 is so
All four natural nucleosides (1–4) showed more or less short-lasting that it could not be orally active owing to
Table 2. Effect of 2%-O-methyladenosine, -inosine and h-methyldopa on systolic blood pressure on SHR.
Drugs
Dose
(mg/kg, p.o.)
No. of
rats
Blood pressure (mmHg)
% Decrease
before adm.
2 h after adm.
Control
Adenosine
Inosine
2%-O-Me Ado
2%-O-Me Ino
h-Methyldopa
-
3
3
3
3
3
3
22798
19199
19397
218921
23996
235914
236911
202912
20095
103921*
210912
18696
−4.091.8
−6.192.6
−3.791.2
53.297.3+
12.092.7
200
200
200
200
200
22.092.5++
Each value represents the mean value9SE.
* Significantly different from control at PB0.05.
+
Significantly different from the value observed before administration at PB0.05 (t test).

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CMLS 54 (1998), Birkha¨user Verlag, CH-4010 Basel/Switzerland
Research Articles
its rapid uptake into cells, deamination by adenosine 10−5 M. The mechanism of this inhibition will be the
deaminase and/or phosphorylation by adenosine kinase. subject of a future paper.
The reason 2%-O-methyladenosine (3) was orally hy-
potensive is not yet clear. Some stable A2-selective
adenosine agonists (e.g. CGS 21680) showed hypoten-
sive effects without affecting heart rate [7]. So there is a
possibility that 3 may act as an A2-selective adenosine
agonist. However, because the free 2%-OH group in the
ribose moiety of adenosine has been reported to be
essential for the manifestation of receptor affinity [5],
we are now evaluating the interaction of 3 with recep-
tor(s), although this possibility appears to be doubtful
at present. Resistance of methyladenosine (3) against
adenosine deaminase may well be partly responsible for
its potent and long-lasting activity, since methyl-
adenosine (3) was more stable to the action of
adenosine deaminase than adenosine, as shown in figure
1. As an alternative possibility, the potentiation of
endogenous adenosine by methyladenosine (3) may play
a more important role in its potent and long-lasting
activity. Methyladenosine (3) was a competitive in-
hibitor of adenosine deaminase, with Ki values of 1.3×
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3 Broom A. D. and Robins R. K. (1965) Direct preparation of
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4 Smith J. D. and Dunn D. B. (1959) An additional sugar
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(1980) Hypotensive effect of inosine and related compounds in
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