New NO donors with antithrombotic and vasodilating activities, part 22: Nitrosation products of hexamethylenetetramine

Article abstract of DOI:10.1002/(SICI)1521-4184(19997)332:7<255::AID-ARDP255>3.0.CO;2-R

Two nitrosation products of hexamethylenetetramine, namely 1,3,5- trinitrosohexahydro-1,3,5-triazine (1) and 3,7-dinitroso-1,3,5,7- tetrazabicyclo[3.3.1]nonane (2), were synthesized. It is shown that both compounds in vitro a 37 °C (1 h, pH 7.4) form nitric oxide at a rate of 3.1% (1) or 1.3% (2), respectively. In rats (60 mg/kg p.o.) both compound inhibit thrombus formation in arterioles (1: 20%; 2: 16%) and venules (1. 18%; 2. 9%). Compound 2 does not influence the blood pressure in spontaneously hypertensive rats.

Full text of DOI:10.1002/(SICI)1521-4184(19997)332:7<255::AID-ARDP255>3.0.CO;2-R

Notes
New NO Donors with Antithrombotic and Vasodilating Activities, Part 22
Nitrosation Products of Hexamethylenetetramine
Martin Herpel and Klaus Rehse*
Institut für Pharmazie I, Freie Universität Berlin, Königin-Luise-Str. 2+4, D-14195 Berlin, Germany
Key Words: Nitric oxide; antithrombotic properties; trinitrosotriazine; dinitrosotetrazabicyclononane
Summary
Chemistry
Both compounds exist as E/Z isomers as described by
Farminer and Webb .
The ratio of these isomers seems to depend from the syn-
Two nitrosation products of hexamethylenetetramine, namely
1,3,5-trinitrosohexahydro-1,3,5-triazine (1) and 3,7-dinitroso-
1,3,5,7-tetrazabicyclo[3.3.1]nonane (2), were synthesized. It is
shown that both compounds in vitro at 37 °C (1 h, pH 7.4) form
nitric oxide at a rate of 3.1% (1) or 1.3% (2), respectively. In rats
(60 mg/kg p.o.) both compound inhibit thrombus formation in
arterioles (1: 20%; 2: 16%) and venules (1: 18%; 2: 9%). Com-
pound 2 does not influence the blood pressure in spontaneously
hypertensive rats.
[8]
thetic procedure. The above composition refers to the classi-
[1,2]
cal synthesis
. With the more recent procedure of
[9]
[8]
Bachmann and Deno which was used by Farminer 1E:1Z
is 1:2 and 2E:2Z is 2:3.
Biology
[10]
Introduction
a) Born Test
The isolation of nitrosation products of hexamethylene-
tetramine (urotropine) dates back to 1888. When this com-
pound reacts with an excess of nitrous acid 1,3,5-trinitroso-
hexahydro-1,3,5-triazine (1) is obtained . If on the other
hand an excess of urotropine is maintained during the whole
In the Born test (inducer collagen) no antiplatelet activity
could be observed. This indicates that at 37 °C no nitric oxide
can be released by platelet rich blood plasma from 1 or 2,
respectively.
[1]
reaction time 3,7-dinitroso-1,3,5,7-tetraazabicyclo[3.3.1]-
nonane is formed (2) .
Both compounds have gained vast attention as explosives
and foaming agents. To our knowledge a therapeutical appli-
cation has not been considered. The reason might be the fear
[11]
b) Antithrombotic Activities
ments
and Blood Pressure Experi-
[2]
[12]
The influence of the test compounds on the formation of
thrombi was assayed in a laser thrombosis model. The title
compounds were administered orally to rats (60 mg/kg). Af-
ter 2 h the formation of thrombi in mesenteric arterioles and
venules by the beam of an argon laser via a microscope
(35 mW, 50 ms) was observed. The number of exposures
(“shots”) necessary to form a thrombus of defined size is
counted. From the average shot number the percentage of
inhibition of thrombosis is calculated. The results are com-
piled in the first columns of Table 1.
that such nitrosamines possess carcinogenic activities. This
[3, 4]
indeed has been reported for 1 when
a long term appli-
cation (~1 year) was investigated. For 2 however neither
[5, 6]
[7]
carcinogenic
nor embryotoxic effects were seen . This
prompted us to consider 1 and 2 as potential antithrombotic
and/or antihypertensive drugs.
Table 1. Antithrombotic properties of 1 and 2 2 h after p.o. administration
of 60 mg/kg to rats and blood pressure lowering effects in hypertensive rats
at the same dose after the times indicated. Statistics: Wilcoxon, Man,
Whitney U-test; n.s. = not significant.
——————————————————————————————
Compound
Inhibition of thrombus formation Decrease in blood
arterioles
venules
pressure after 2 h
% ± sx p≤
% ± sx p≤
% ± sx [%]p≤
——————————————————————————————
1
20 ± 1 0.002 18 ± 1 0.002
4 ± 2 0.01
1 ± 1 n.s.
2
16 ± 1 0.002
43 ± 12 0.01
9 ± 1 0.002
15 ± 7 0.2
molsidomine
(10 mg/kg)
15 ± 3 0.01
Scheme 1: Stereochemistry of 1 and 2.
——————————————————————————————
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999 0365-6233/99/0707/0255 $17.50 +.50/0

256
Herpel and Rehse
Table 2. NO formation (chemiluminescence) and N2O formation (gas chromatography) from 1, 2, and SIN 1 as a function of the solvent (37 °C) and its pH
after 1 h. The values are in % ± S; n.d. = not detected; n = 5.
—————————————————————————————————————————————————————————————–
Cpd.
Detected
species
Mere
compound
Phosphate
buffer pH 7.4
Ethanol
Phosphate
buffer/ethanol 1: 1
Ethanol/HCl
0.1 N 1:1
—————————————————————————————————————————————————————————————–
1
NO
n.d.
3.15 ±0.15
1.9 ±0.17
6.8 ±0.30
4.0 ± 0.27
N2O
0.05±0.003
0.05 ±0.005
0.05±0.003
0.04 ±0.001
0.05 ±0.003
2
NO
n.d.
n.d.
1.3 ±0.02
1.1 ±0.08
1.1 ±0.08
0.05 ±0.004
0.03 ±0.002
N2O
0.08 ±0.006
n.d.
0.06 ±0.003
SIN 1
NO
n.d.
2.73 ±0.20
0.16±0.006
0.89 ±0.05
0.11 ±0.006
N2O
0.01±0.0007
0.03 ±0.003
n.d.
0.008±0.0006
n.d.
—————————————————————————————————————————————————————————————–
The pattern of pharmacological activities of NO donors also true for the other reaction conditions. To compare the
comprises a more or less pronounced decrease in blood compounds in the dissolved state a mixture of phosphate
pressure. We therefore assayed compounds 1 and 2 in spon- buffer and ethanol (1:1) was used. Hereby the generation of
•
taneously hypertensive rats (SHR). Briefly the conscious rat NO is enhanced in 1, remains constant in 2 and is decreased
in SIN 1. An acidic medium, i.e. ethanol/0.1N HCl (1:1), was
applied to approach the conditions in the stomach which
might be important for the passage after oral administration.
Here only 1 shows NO formation at a rate of 4%. This
decomposition of 1, however, will not much influence its
bioavailability. The results in summary show that from all
compounds NO can be liberated at 37 °C at a similar rate so
that 1 and 2 can be classified as NO donors.
is warmed up (10 ± 5 min) in a perspex tube to 37 °C. The rat
tail which is out of the tube is surrounded by a pressure cuff
and more distally by a piezo pulse wave transducer.
The measurement begins when the correct opening of the
tail arteries is indicated by a light signal for constant pulse.
The pressure in the cuff is raised until the pulsations cease.
Then the pressure is decreased continuously until the pulse
returns. The arterial blood pressure can be determined di-
rectly from the chart of the recorder. The results of the above
experiments are summarized in Table 1.
Both compounds quite obviously inhibit thrombus forma-
tion. The antithrombotic effect of 1 is slightly stronger than
that of 2. Surprisingly with 1 the inhibition in arterioles and
venules is equal. Normally it is more difficult to inhibit
thrombus formation in venules because the flow velocity is
slower and thrombus formation easier.
Experimental Part
1,3,5-Trinitroso-hexahydro-1,3,5-triazine[1] (1)
1,3,5,7-Tetrazatricyclo[3.3.1.13,7]decane (hexamethylenetetramine)
(2.0 g, 14.3 mmol) was dissolved in water (80 ml), cooled with ice, and cold
dilute (10%) hydrochloric acid (30 ml) added. The solution is mixed imme-
diately with sodium nitrite (5.0 g, 70 mmol) dissolved in a little water. After
a few min, yellow crystals form at the surface. They are filtered off under
suction and recrystallized from ethanol. Yellow crystals, mp 100 °C
(ref.[1]105 °C), yield 20%.– Anal. C3H6N6O3.
The lowering of the blood pressure by 1 is marginal while
[13]
2 lacks any antihypertensive effect. This again
shows that
it is possible in NO donors to separate the antithrombotic
properties from hypotonous effects. The reason might be that
endothelial and/or smooth muscle cells are not able to trans-
form 2 to nitric oxide while the metabolic potential of liver
cells is sufficient for this process so that blood platelets can
still be influenced.
3,7-Dinitroso-1,3,5,7-tetrazabicyclo[3.3.1]nonane[2] (2)
A concentrated solution of 1,3,5,7-tetrazatricyclo[3.3.1.13,7]decane
(15.0 g, 110 mmol) and sodium nitrite (7.6 g) is prepared and cooled with
ice. Dilute nitric acid is then added until a permanent smell of nitrous acid is
noted. After a few min crystals precipitate. After several additional min they
are filtered off under suction, washed with water, and recrystallized from
ethanol. Crystals, mp 202 °C (ref.[2] 207 °C), yield 8.5%.– Anal.
C5H10N6O2.
•
–
In Vitro Formation of NO and NO from 1 and 2
The formation of nitric oxide was determined by a chemi-
[14]
luminescence method . The quantification of nitrosohy-
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2
Arch. Pharm. Pharm. Med. Chem. 332, 255–257 (1999)

NO Donors with Antithrombotic, Vasodilating Activities
257
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Received: August 6, 1998 [FP325]
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