New NO-donors with antithrombotic and vasodilating activities, Part 21. Pseudonitrosites and other azodioxides with vicinal electron acceptors.

Article abstract of DOI:10.1002/(SICI)1521-4184(199803)331:3<111::AID-ARDP111>3.0.CO;2-Z

Twelve vicinally substituted nitro-nitroso compounds (pseudonitrosites) were synthesized, nine of them for the first time. In the solid state the dimeric azodioxides are present. In the class of the pseudonitrosites 2a-h, all compounds exhibited comparatively strong antiplatelet activity in vitro (Born test, collagen). Four of them showed an IC50 below 10 microM, 2a being the most active substance with an IC50 = 2.1 microM. When administered orally to rats (60 mg/kg) small antithrombotic effects were observed. The pseudonitrosite 6d was the most active compound (18% inhibition in arterioles). The in vitro decomposition of 2a at 37 degrees C gave NO and N2O, indicating that the above pharmacological effects were mediated by an NO-dependent mechanism. The replacement of the nitro group in the pseudonitrosite partial structure by other electron acceptors i.e. acetyl, carboxyl, or acetyloxy groups leads to inactive (10a) or less active compounds (10b, e).

Full text of DOI:10.1002/(SICI)1521-4184(199803)331:3<111::AID-ARDP111>3.0.CO;2-Z

NO-Donors with Antithrombotic, Vasodilating Activities
111
New NO-Donors with Antithrombotic and Vasodilating Activities, Part 21
Pseudonitrosites and Other Azodioxides with Vicinal Electron
Acceptors✩
1)
Klaus Rehse* and Martin Herpel
Institut für Pharmazie I, Freie Universität Berlin, Königin-Luise-Str. 2+4, D-14195 Berlin, Germany
Key Words: Vicinal nitro-nitroso compounds; antiplatelet activities; antithrombotic activities; NO formation; N O formation
2
Summary
Chemistry
The type 2 azodioxides (see Fig. 1)which are trivially called
pseudonitrosites were obtained according to Klamann et al.
Twelve vicinally substituted nitro-nitroso compounds (pseudo-
nitrosites) were synthesized, nine of them for the first time. In the
solid state the dimeric azodioxides are present. In the class of the
pseudonitrosites 2a–h, all compounds exhibited comparatively
strong antiplatelet activity in vitro (Born test, collagen). Four of
them showed an IC50 below 10 µM, 2a being the most active
substance with an IC50 = 2.1 µM. When administered orally to rats
(60 mg/kg) small antithrombotic effects were observed. The
pseudonitrosite 6d was the most active compound (18% inhibition
in arterioles). The in vitro decomposition of 2a at 37 °C gave NO
and N2O, indicating that the above pharmacological effects were
mediated by an NO-dependent mechanism. The replacement of the
nitro group in the pseudonitrosite partial structure by other electron
acceptorsi.e. acetyl, carboxyl, or acetyloxygroups leads to inactive
(10a) or less active compounds (10b, c).
[4]
by reaction of alkenes with dinitrogen trioxide. This reagent
forms spontaneously when a mixture of nitric oxide and air
is passed through the solution of the alkene. Six of the
pseudonitrosites (2c–h) were prepared for the first time. The
1
substituents R were chosen on the basis of previous experi-
ence concerning suitable lipophilia and (platelet) membrane
affinity. The compounds form colorless crystals and hence
are type 2 azodioxides. This is backed up by the IR spectra
where the valence vibration for the NO group is found be-
–1
tween 1200–1209 cm , i.e. in the N-O single bond region.
[4a]
This confirms simultaneously that the E-isomer is present
.
When dissolved in chloroform the compounds form colorless
solutions. In the NMR spectra a single set of signals is seen.
Both observations indicate that only the dimers 2 and not the
monomeric form 3 are present. The azodioxide structure is so
stable that in the (+)-FAB mass spectrum the pseudomolecu-
Introduction
+
Recently we reported on antithrombotic azodioxides (I)
lar ion [M+H] is seen for the dimer, e.g. 2e = 293 with high
which were activated by electron acceptors geminal to the
intensity (50%). The nitromethylene group gives rise to a
prochiral moiety so that an AMX spin system with the vicinal
proton (2a–c) is observed (see Fig. 2).
[1,2]
nitroso group
. The rationale was that the acceptor X
should decrease the electron density of the C-NO bond and
thereby make those compounds prone to the release of nitric
oxide. With participation of the adjacent activated methylene
group the expulsion of nitrosohydrogen which is the reduced
congener of NO, should become favorable.
In 2d–h a typical AMXY pattern is seen. In acetone at
2
20 °C the pseudonitrosites are time dependently rearranged
to the nitrooximes 4. This reaction follows a first order
kinetic. After 6 h only 40% of the pseudonitrosite 2c are left.
1
This can be followed easily by the H-NMR where at 5.83
ppm the singlet for the methylene group of 4c is observed.
The small peak at 5.62 ppm shows that a small portion of the
Z-oxime (6 h: ≈1%) has formed, too. The 2-nitro-1-phenyl-
ethanone oxime 4a was isolated in pure state to test its activity
for comparison with the vinylogous nitrooxime FK 409 (v.i.)
[5]
for which the release of nitric oxide has been reported
.
When reacting methylacrylic acid esters (5) with N O the
2
3
[6]
azodioxides 6 are obtained . Formally these are pseudoni-
trosites with an additional carboxylic acid ester partial struc-
ture. This electron-withdrawing group was incorporated in
order to activate further the azodioxide for the release of NO.
In the solid state 6a–d form colorless crystals indicating the
azodioxide structure. In the (+)-FAB mass spectrum the
pseudomolecular ion is recorded with high intensity (e.g. 6a:
The success of this idea prompted us to investigate whether
an electron acceptor vicinal to the nitroso group (II) could
also ”activate” azodioxides to release the above NO species.
[3]
We were then further encouraged by the report of Wieland
who had already observed the formation of dinitrogen oxide
1
2
from styrene pseudonitrosite (II: X = NO ; R = ph; R = H;
2
+
see also compound 2a of this paper). Compounds 2a–c were
designed according to the suggestions of Topliss.
[M+H] = 353 (65)). Chloroform solutions of these com-
pounds are colorless. When gently warmed (50 °C) they
1
become blue i.e. the monomeric 7 are formed. The H-NMR
———
spectrum shows that a mixture of 6 and 7 is present. For
example, the ratio 6a/7a is 8:1.
1) Part of the PhD thesis M. Herpel, FU Berlin 1997.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1998
0365-6233/98/0303/0111 $17.50 +.50/0

112
Rehse and Herpel
1
and the H-NMR spectrum shows that the equilibrium is
dominated by the type 11 monomers.
Biology
a) Inhibition of blood platelet aggregation (in vitro, Born test)
The platelet aggregation experiments were carried out as
[9]
usual . In short platelet rich plasma (PRP) was prepared
from freshly drawn citrated human blood by mild centrifuga-
tion. Platelet aggregation was induced by collagen. The en-
suing increase in light transmission is recorded in an Elvi
aggregometer (control). Then the PRP is incubated with
different concentrations of the test compound and the influ-
ence of collagen on the light transmission is recorded again.
The concentration which inhibits the platelet aggregation
half-maximally (IC ) is determined graphically from the
50
percentage of inhibition at various concentrations of the test
compound. The results are compiled in the fifth column of
Table 1.
In the class of the pseudonitrosites 2a–h all compounds
exhibited rather strong antiplatelet activity. Four of them
Fig. 1: Synthesis of pseudonitrosites and other azodioxides with vicinal
electron acceptors
showed an IC below 10 µM, 2a being the most active
50
substance with an IC = 2.1 µM. Neither a decrease (2b) nor
50
1
an increase (2c) of electron density in the aromatic ring of R
could further improve this result. In the series of aliphatic
substituents peak activity was found for a pentyl group (2e).
This agrees with earlier observations indicating that this
group has a good affinity for the platelet membrane. The
arylalkyl derivatives 2g and 2h also exhibited antiplatelet
effects at almost the same concentration (≈ 9 µM). The
1
rearrangement product of 2a, i.e. 4a (R = ph) showed no
antiplatelet activity. This contrasts with FK 409, which is a
vinylogous nitrooxime for which an IC = 4 µM has been
50
[5a]
reported
.
In the pseudonitrosites with an additional carboxylic ester
group (6a–d) the antiplatelet effects covered a wide range
from4.2–38 µM. The more lipophilic esters6c and6d showed
the best results.
The replacement of the nitro group in X was not favorable.
The compounds were either inactive (10a) or showed poor
antiplatelet effects (10b, c).
b) Inhibition of thrombus formation
Fig. 2: Rearrangement of 2c to 4c in acetone at 20°C. Part of the 1H-NMR
spectrum (δ = 5–6 ppm) which shows the signals of the methylene groups.
The influence of the test compounds on the formation of
[10]
thrombi was assayed as usual in a laser thrombosis model
.
The title compounds were administered orally to rats
(60 mg/kg). After 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
In the type 10 azodioxides the electron-withdrawing nitro
group is replaced by other electron acceptors i.e. by an acetyl,
carboxyl, or acetyloxy group. These compounds were ob-
tained from the hydroxylamino derivatives 9 by oxidation
with bromine. Starting material were type 8 olefins for 10a
and 10b. Compound 9c was obtained from 2-methyl-2-nitro-
[7]
[11]
of inhibition of thrombosis is calculated . The results are
compiled in the last columns of Table 1.
propanol by acetylation and subsequent reduction with
[8]
Zn/NH Cl
.
In the row of the pseudonitrosites 2a–h five of eight com-
pounds showed small antithrombotic activities in the same
order of magnitude, i.e. ≈10% inhibition of thrombus forma-
tion in arterioles and 5% in venules. No correlation with the
in vitro results (Born test) could be observed. This suggests
4
The compounds 10a–c form colorless crystals. The infrared
–1
absorption between 1246 and 1259 cm supports the azodi-
oxide structure. When dissolved in chloroform immediately
1
a mixture of 10 and 11 is observed ( H-NMR). The solutions
are slightly blue. After 150 min the blue color is much deeper that the results are dominated by parameters such as stability
Arch. Pharm. Pharm. Med. Chem. 331, 111–117 (1998)

NO-Donors with Antithrombotic, Vasodilating Activities
113
[1]
in the gastrointestinal tract or different affinity for enzymes properties . This result suggests that the endothelium at
involved in the metabolism of the nitric oxide species. least of SHR is not able to convert pseudonitrosites to nitric
1
In the group of compounds with R being an carboxylic acid oxide.
ester the antithrombotic effects range from none (6a) to
medium in 6d which is the most active compound tested.
The type 10 derivatives showed no antithrombotic activity
Decomposition Experiments
at all. This might be due to the high stability of these com-
pounds. At least in CDCl solution after 150 min nearly no
decomposition was seen ( H-NMR). For 6b this was as well
3
1
From the pattern of antiplatelet and antithrombotic activi-
ties (see Table 1) we assumed that these pharmacological
effects are mediated by nitric oxide or its reduced congener
nitrosohydrogen. In principle the formation of both from the
pseudonitrosite 2a has already been demonstrated by
Wieland and Jonkman et al. . However highly unphysi-
ological conditions were used. Wieland observed the forma-
tion of dinitrogen oxide in concentrated sulfuric acid.
Jonkman et al. investigated the formation of NO at 60 °C in
styrene. We tried to approach more physiological conditions,
namely aqueous media and 37 °C as reaction temperature.
For our decomposition experiments we selected the
pseudonitrosite 2a which was the most active compound in
true in D O/NaOD solution. Compound 4a showed no an-
2
tithrombotic effect.
c) Influence on blood pressure
[3]
[12]
The pattern of pharmacological activities of NO donors
comprises more or less pronounced decrease in blood pres-
[11a, 11b]
[16]
sure
. We therefore assayed as usual
compounds
2a, 4a, 10a, and 10b which had been rather effective in the
thrombosis model in spontaneously hypertensive rats (SHR).
We did not observe a significant decrease in blood pressure
(2 h after p.o. administration of 60 mg/kg). Type 6 com-
pounds which are very unstable could notbe testedas wewere
unable to obtain a sufficient amount for the blood pressure
experiments. The pseudonitrosites are to our knowledge the
second class of NO donors where the desired antithrombotic
vitro (IC = 2.1 µM). Because of its poor solubility in phos-
50
phate buffer the decomposition experiments were also carried
out in ethanol. The results are summarized in Table 2. For
comparison the data for the well-known NO donor SIN 1 are
effect could be separated from the unwanted hypotonous given in Table 3.
Table 1: In vitro (Born test) antiplatelet and in vivo antithrombotic properties of pseudonitrosites 2a–h and other azodioxides with vicinal electron ac-
ceptors after p.o. adminstration to rats (60 mg/kg). Man Whitney U-test[12]. n.s.= not significant. In the Born test the deviation from the mean is about
10%. The IC50 of acetylsalicylic acid is 175 µM.
————————————————————————————————————————————————————————————
Com-
pound
X
R1
R2
Born test
IC50
Inhibition of thrombus formation
venules
arterioles
p ≥
[µM]
% ± sx
% ± sx
p ≥
————————————————————————————————————————————————————————————
2a
2b
2c
NO2
ph
H
2.1
10.5
19.5
18.0
9.4
10 ± 1
7 ± 1
8 ± 1
0.002
0.01
0.01
5 ± 1
5 ± 2
7 ± 1
0.02
0.05
0.01
NO2
4-Cl-ph
4-CH3O-ph
C3H7
H
NO2
H
2d
2e
NO2
H
n.s.
n.s.
n.s.
NO2
C5H11
H
11 ± 1
7 ± 2
0.002
0.02
2f
NO2
C7H15
H
15.0
8.7
4 ± 1
0.05
2g
NO2
CH2-ph
(CH2)2-ph
COOCH3
COOC2H5
COOC4H9
COOCyclhex
CH3
H
n.s.
n.s.
n.s.
n.s.
n.s.
2h
6a
NO2
H
9.6
NO2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
38.0
24.0
4.2
n.s.
6b
6c
NO2
13 ± 1
4 ± 2
0.002
0.2
9 ± 1
9 ± 1
0.002
0.002
NO2
n.s.
6d
10a
10b
10c
NO2
10.0
62.5
39.0
46.0
18 ± 1
0.002
COCH3
COOH
OCOCH3
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
CH3
CH3
————————————————————————————————————————————————————————————
Arch. Pharm. Pharm. Med. Chem. 331, 111–117 (1998)

114
Rehse and Herpel
Table 2: NO formation (chemiluminescence) and N2O formation (gas chro-
matography) of the pseudonitrosite 2a as a function of the solvent (T = 37 °C)
and its pH after 1 h.
Crystals are formed. They are filtered off under suction and washed with a
little cold petroleum ether.
——————————————————————————————
(E)-1,1′-Azobis-(2-nitro-1-phenyl-ethane) N,N′-dioxide (2a)
Solvent
NO-formation
N2O-formation
[% ± SD]
[% ± SD]
From 10.4 g styrene (0.1 mol) in 100 ml toluene at 60 °C[4]. Colorless
crystals, mp 113 °C (ref.[4] 112 °C), yield 2.1 g (11%).– Anal. C16H16N4O6.–
IR (KBr): ν = 1561 cm–1 (NO2); 1201 (NO dim.).– 1H-NMR ([D6] acetone):
δ = 5.04 (dd, J = 16.0/2.9 Hz, 2H, CH2-NO2, M-part), 5.43 (dd, J = 16.0/11.1
Hz, 2H, CH2-NO2, A-part), 6.97 (dd, J = 11.1/2.9 Hz, 2H, H-C-NO, X-part),
7.46 (m, 6H, ph-3-H, 4-H, 5-H), 7.53 (m, 4H, ph-2-H and 6-H).– 13C-NMR
([D6] acetone): δ = 68.7 (H-C-NO), 74.2 (CH2-NO2), 128.7 (ph-C-3 and
C-5), 129.9 (ph-C-2 and C-6), 130.4 (ph-C-4), 131.2 (ph-C-1).– MS ([+]-
FAB, acetone/m-nitrobenzyl alcohol): m/z (%) = 361 (0.7) [(M+H)+].
——————————————————————————————
none
not detected
< 0.03
(n = 5)
1.01 ± 0.10
(n = 5)
3.81 ± 1.62
(n = 5)
0.1 M phosphate buffer pH 7.4 not detected
(suspension)
Ethanol
0.18 ± 0.01
(n = 5)
——————————————————————————————
Table 3: NO formation (chemiluminescence) and N2O formation (gas chro-
matography) of the NO donor SIN 1 as a function of the solvent (T = 37 °C)
and its pH after 1 h.
(E)-1,1′-Azobis-[2-nitro-1-(4-chlorophenyl)-ethane] N,N′-dioxide (2b)
From 5.0 g 4-chlorostyrene (36.2 mmol). Colorless crystals, mp 118 °C
(ref.[14] 104 °C), yield 1.2 g (15%).– Anal. C16H14Cl2N4O6.– IR (KBr): ν =
1561 cm–1 (NO2); 1208 (NO dim.).– 1H-NMR ([D6] acetone): δ = 5.09 (m,
2H, CH2-NO2, M-part), 5.47 (m, 2H, CH2-NO2, A-part), 6.99 (dd, J =
11.0/2.7 Hz, 2H, H-C-NO, X-part), 7.51 (d, J = 8.5 Hz, 4H, ph-3-H and 5-H),
7.58 (d, J = 8.5 Hz, 4H, ph-2-H and 6-H) all diastereomer A; 5.09 (m, 2H,
CH2-NO2, M-part), 5.47 (m, 2H, CH2-NO2, A-part), 6.90 (dd, J = 11.0/2.7
Hz, 2H, H-C-NO, X-part), 7.25 (d, J = 8.4 Hz, 4H, ph-3-H and 5-H), 7.39
(d, J = 8.4 Hz, 4H, ph-2-H and 6-H) diastereomer B. Ratio A/B = 55:45.–
13C-NMR ([D6] acetone): δ = 67.5 (H-C-NO), 73.3 (CH2-NO2), 128.8
(ph-C-1), 129.5, 130.2, 135.6 (ph-C-4) all diastereomer A; 67.9 (H-C-NO),
73.0 (CH2-NO2), 128.8 (ph-C-1), 129.4, 130.1, 135.6 (ph-C-4) diastereomer
B.– MS ([+]-FAB, acetone/m-nitrobenzyl alcohol): m/z (%) = 429 (2)
[(M+H)+], 184 (59) [mon+-NO], 138 (100) [C8H7Cl+].
——————————————————————————————
Solvent
NO-formation
N2O-formation
[% ± SD]
[% ± SD]
——————————————————————————————
none
not detected
0.01 ± 0.001
(n = 5)
0.1 M phosphate buffer 2.7 ± 0.2
0.03 ± 0.003
(n = 5)
pH 7.4
(n = 5)
Ethanol
0.9 ± 0.05
(n = 5)
not detected
——————————————————————————————
In ethanol only small amounts of nitric oxide (0.18%) could
be detected. In the solid state and in suspension (phosphate
puffer) no decomposition to nitric oxide was seen. In contrast
considerable amounts of NO were released from SIN 1 espe-
cially in phosphate buffer. On the other hand large amounts
(E)-1,1′-Azobis-[2-nitro-1-(4-methoxyphenyl)-ethane] N,N′-dioxide (2c)
From 5.0 g 4-methoxystyrene (37.3 mmol). Colorless crystals, mp 109 °C,
yield 1.0 g (13%).– Anal. C18H20N4O8.– IR (KBr): ν = 1563 cm–1 (NO2);
1200 (NODim).– 1H-NMR ([D6] acetone): δ = 3.83 (s, 6H, OCH3), 5.00 (dd,
J = 15.9/2.9 Hz, 2H, CH2-NO2, M-part), 5.44 (dd, J = 15.9/11.1 Hz, 2H,
CH2-NO2, A-part), 6.90 (dd, J = 11.1/2.9 Hz, 2H, H-C-NO, X-part), 6.99 (d,
J = 8.7 Hz, 4H, ph-3-H and 5-H), 7.46 (d, J = 8.7 Hz, 4H, ph-2-H and 6-H).–
13C-NMR ([D6] acetone): δ = 55.2 (OCH3), 67.4 (H-C-NO), 73.7 (CH2-
NO2), 114.6(ph-C-3andC-5), 122.4(ph-C-1), 129.7(ph-C-2andC-6), 161.1
(ph-C-4).– MS ([+]-FAB, acetone/m-nitrobenzyl alcohol): m/z (%) = 421
(0.2) [(M+H)+], 180 (40) [mon+-NO], 134 (76) [C9H10O+].
of N O in phosphate buffer (1%) and ethanol (3.8%) have
2
been generated indicating that nitrosohydrogen primarily had
been formed (2 HNO → H N O → N O + H O). Obviously
2
2
2
2
2
the release of nitrosohydrogen is facilitated by the strong
electron withdrawing nitro group in vicinal position to the NO
moiety. SIN 1 did form nearly no N O under these conditions.
2
The results suggest that the observed antiplatelet and an-
tithrombotic effects (Table 1) might be mediated by an NO
dependent mechanism. The low IC of 2a in the Born test
50
•
–
(E)-2,2′-Azobis-(1-nitro-pentane) N,N′-dioxide (2d)
indicates that the release of NO and/or NO is supported by
the metabolic capacity of the PRP. Preferentially and primar-
ily 2a functions as releaser of nitrosohydrogen which how-
ever under aerobic conditions can be oxidized to nitric oxide.
From 7.0 g 1-pentene (0.1 mol). Colorless crystals, mp 78 °C, yield 0.5 g
(3%).– Anal. C10H20N4O6.– IR (KBr): ν = 1562 cm–1 (NO2); 1204 (NO
dim.).– 1H-NMR ([D6] acetone): δ = 0.94 (t, J = 7.3, 6H, CH3), 1.42 (m, 4H,
CH2-CH3), 1.81 (m, 4H, CH2-C2H5), 5.01 (dd, J = 15.6/2.9, 2H, CH2-NO2,
M-part), 5.17 (dd, J = 15.5/10.5, 2H, CH2-NO2, A-part), 6.07 (m, 2H,
H-C-NO, X-part).– 13C-NMR ([D6] acetone): δ = 13.4 (CH3), 18.2 (CH2-
CH3), 30.5 (CH2-C2H5), 64.1 (H-C-NO), 73.6 (CH2-NO2).– MS ([+]-FAB,
aceton/m-nitrobenzyl alcohol): m/z (%) = 293 (50) [(M+H)+], 116 (49)
[mon+-NO], 69 (100), 41 (66).
Experimental Part
1)
Chemistry
Mp (uncorr.), Lindström.– Elemental analysis: Elementar Vario EL.– IR:
ATI Mattson Genenis Serie FTIR.– UV/VIS: Kontron Instruments UVIKON
930.– NMR: Bruker AC 300 and DPX 400.– EI-MS: Varian MAT CH7 A
and Kratos MS 25 RF.– FAB-MS: Varian MAT CH 5-DF.
(E)-2,2′-Azobis-(1-nitro-heptane) N,N′-dioxide (2e)
From 9.8 g 1-heptene (0.1 mol). Colorless crystals, mp 71 °C, yield 0.7 g
(4%).– Anal. C14H28N4O6.– IR (KBr): ν = 1558 cm–1 (NO2); 1206 (NO
dim.).– 1H-NMR ([D6] acetone): δ = 0.89 (t, J = 6.9, 6H, CH3), 1.30–1.42
(m, 12H, (CH2)3-CH3), 1.80–1.86 (m, 4H, CH2-C4H9), 5.01 (dd, J = 15.6/2.8,
2H, CH2-NO2, M-part), 5.18 (dd, J = 15.6/10.5, 2H, CH2-NO2, A-part), 6.07
(m, 2H, H-C-NO, X-part).– 13C-NMR ([D6] acetone): δ = 13.6 (CH3), 22.4,
24.5, 28.5, 31.5, 64.3 (H-C-NO), 73.6 (CH2-NO2).– MS ([+]-FAB, ace-
tone/m-nitrobenzyl alcohol): m/z (%) = 349 (23) [(M+H)+], 144 (33) [mon+-
NO], 55 (100), 41 (43).
General procedure for the synthesis of 2a–h[4]
Into a stirred solution of 0.1 mol alkene in 80 ml diethyl ether/petroleum
ether (1+1) a mixture of nitric oxide and air is introduced at 0–5 °C for
30 min. Then the solution is stirred for 30 min and kept at –16 °C for 24 h.
———
1) The full set of data is given in the PhD thesis of M. Herpel, FU Berlin 1997.
Arch. Pharm. Pharm. Med. Chem. 331, 111–117 (1998)

NO-Donors with Antithrombotic, Vasodilating Activities
115
(E)-2,2′-Azobis-(1-nitro-nonane) N,N′-dioxide (2f)
(E)-2,2′-Azobis-(2-methyl-3-nitro-propanoic acid methyl ester) N,N′-dioxide
(6a)
From 12.6 g 1-nonene (0.1 mol). Colorless crystals, mp 73 °C, yield 0.4 g
(2%).– Anal. C18H36N4O6.– IR (KBr): ν = 1560 cm–1 (NO2); 1201 (NO
dim.).– 1H-NMR ([D6] acetone): δ = 0.89 (t, J = 7.0 Hz, 6H, CH3), 1.29–1.42
(m, 20 H, (CH2)5-CH3), 1.80–1.86 (m, 4H, CH2-C6H13), 5.01 (dd, J =
15.6/2.9, 2H, CH2-NO2, M-part), 5.18 (dd, J = 15.6/10.6, 2H, CH2-NO2,
A-part), 6.07 (m, 2H, H-C-NO, X-part).–13C-NMR (CDCl3): δ = 14.0 (CH3),
22.5, 24.9, 28.5, 28.8, 29.0, 31.6, 63.9 (H-C-NO), 72.9 (CH2-NO2).– MS
([+]-FAB, acetone/m-nitrobenzyl alcohol): m/z (%) = 405 (59) [(M+H)+],
172 (24) [mon+-NO], 69 (97), 55 (100), 41 (99).
From 5.0 g methyl methacrylate (50.0 mmol). Colorless crystals, mp 98 °C
(blue) (ref.[6] 100–110 °C), yield 0.5 g (6%).– Anal. C10H16N4O10.– IR
(KBr): ν = 1752 cm–1 (CO); 1561 (NO2).– UV/VIS (CHCl3): λmax = 655
nm.– 1H-NMR ([D6] acetone): δ = 1.83/1.87 (s, 6H, CH3), 3.80/3.83 (s, 6H,
OCH3), 5.22 (m, 2H, CH2-NO2, B-part), 5.48 (m, 2H, CH2-NO2, A-part) all
dimer; 1.45 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 5.22 (m, 1H, CH2-NO2,
B-part), 5.48 (m, 1H, CH2-NO2, A-part) monomer. Ratio dim/mon = 8:1.–
MS ([+]-FAB, acetone/m-nitrobenzyl alcohol): m/z (%) = 353 (65)
[(M+H)+], 246 (14), 206 (18) [C5H8N3O6+], 178 (69), 146 (96) [mon+-NO],
101 (100), 69 (86), 41 (76), 30 (71) [NO+].
2,2′-Azobis-(1-nitro-3-phenyl-propane) N,N′-dioxide (2g)
(E)-2,2′-Azobis-(2-methyl-3-nitro-propanoic acid ethyl ester) N,N′-dioxide
(6b)
From 11.8 g 3-phenyl-propene (0.1 mol). Colorless crystals, mp 125 °C,
yield 0.5 g (3%).– Anal. C18H20N4O6.– IR (KBr): ν = 1552 cm–1 (NO2).–
1H-NMR ([D6] acetone): δ = 2.92 (dd, J = 13.8/7.8 Hz, 2H, CH2-ph, B′-part),
3.07 (m, 2H, CH2-ph, A′-part), 4.84 (dd, J = 15.6/2.8 Hz, 2H, CH2-NO2,
M-part), 5.13 (dd, J = 15.5/10.1, 2H, CH2-NO2, A-part), 6.15 (m, 2H,
H-C-NO, X-part), 7.26–7.35 (m, 10 H, ph) all diastereomer A; 3.07 (dd, J =
13.9/7.9 Hz, 2H, CH2-ph, B′-part), 3.21 (dd, J = 13.9/5.3 Hz, 2H, CH2-ph,
A′-part), 4.89 (dd, J = 15.7/2.8 Hz, 2H, CH2-NO2, M-part), 5.15 (dd, J =
15.7/10.5, 2H, CH2-NO2, A-part), 6.15 (m, 2H, H-C-NO, X-part), 7.26–7.35
(m, 10 H, ph) diastereomer B. Ratio A/B = 1:5.– 13C-NMR ([D6] acetone):
δ = 34.6 (CH2-ph), 65.9 (H-C-NO), 73.2 (CH2-NO2), 128.4 (ph-C-4), 129.6,
130.4, 135.4 (ph-C-1) all diastereomer A; 34.0 (CH2-ph), 66.5 (H-C-NO),
73.1 (CH2-NO2), 128.4 (ph-C-4), 129.7, 130.3, 135.4 (ph-C-1) diastereomer
B.– MS ([+]-FAB, acetone/m-nitrobenzyl alcohol): m/z (%) = 389 (19)
[(M+H)+], 164 (39) [mon+-NO], 117 (69), 91 (100) [C7H7+].
From 5.7 g ethyl methacrylate (50.0 mmol). Colorless crystals, mp 63 °C
(blue), yield 0.5 g (5%).– Anal. C12H20N4O10.– IR (KBr): ν = 1743 cm–1
(CO); 1560 (NO2).– UV/VIS (CHCl3): λmax = 657 nm.– 1H-NMR ([D6]
acetone): δ = 1.24–1.33 (m, 6H, CH2-CH3), 1.83/1.87 (s, 6H, CH3), 4.15–
4.41 (m, 4H, OCH2), 5.18–5.27 (m, 2H, CH2-NO2, B-part), 5.39–5.50 (m,
2H, CH2-NO2, A-part) all dimer; 1.24–1.33 (m, 3H, CH2-CH3), 1.45 (s, 3H,
CH3), 4.15–4.41 (m, 2H, OCH2), 5.18–5.27 (m, 1H, CH2-NO2, B-part),
5.39–5.50 (m, 1H, CH2-NO2, A-part) monomer. Ratio dim/mon = 6:1.– MS
([+]-FAB, acetone/m-nitrobenzyl alcohol): m/z (%) = 381 (37) [(M+H)+],
220 (13) [C6H10N3O6+], 192 (44), 160 (74) [mon+-NO], 115 (100), 87 (40),
69 (93), 30 (67), 29 (84) [C2H5+].
(E)-2,2′-Azobis-(2-methyl-3-nitro-propanoic acid butyl ester) N,N′-dioxide
(6c)
2,2′-Azobis-(1-nitro-4-phenyl-butane) N,N′-dioxide (2h)
From 7.1 g butyl methacrylate (50.0 mmol). Colorless crystals, mp 42 °C
(blue), yield 0.6 g (5%).– Anal. C16H28N4O10.– IR (KBr): ν = 1750 cm–1
(CO); 1561 (NO2).– UV/VIS (CHCl3): λmax = 655 nm.– 1H-NMR ([D6]
acetone): δ = 0.87–0.96 (m, 6H, CH2-CH3), 1.36–1.49 (m, 4H, CH2-CH3),
1.65–1.74 (m, 4H, CH2-C2H5), 1.83/1.87 (s, 6H, CH3), 4.15–4.34 (m, 4H,
OCH2), 5.17–5.28 (m, 2H, CH2-NO2, B-part), 5.37–5.49 (m, 2H, CH2-NO2,
A-part) all dimer; 0.87–0.96 (m, 3H, CH2-CH3), 1.36–1.49 (m, 3H, CH2-CH3
and CH3), 1.65–1.74 (m, 2H, CH2-C2H5), 4.15–4.34 (m, 2H, OCH2), 5.17–
5.28 (m, 1H, CH2-NO2, B-part), 5.37–5.49 (m, 1H, CH2-NO2, A-part)
monomer. Ratio dim/mon = 7:1.– MS ([+]-FAB, acetone/m-nitrobenzyl
alcohol): m/z (%) = 437 (14) [(M+H)+], 248 (6) [C8H14N3O6+], 220 (19), 188
(34) [mon+-NO], 143 (54), 114 (33), 87 (53), 57 (100) [C4H9+], 41 (84),
29 (64).
From 13.2 g 4-phenyl-1-butene (0.1 mol). Colorless crystals, mp 121 °C,
yield 0.4 g (2%).– Anal. C20H24N4O6.– IR (KBr): ν = 1558 cm–1 (NO2).–
1H-NMR ([D6] acetone): δ = 2.17 (m, 4H, CH2-CH2-ph), 2.74 (m, 4H,
CH2-ph), 5.11 (dd, J = 15.6/2.8 Hz, 2H, CH2-NO2, M-part), 5.28 (dd, J =
15.6/10.6, 2H, CH2-NO2, A-part), 6.19 (m, 2H, H-C-NO, X-part), 7.18–7.31
(m, 10 H, ph).– 13C-NMR ([D6] acetone): δ = 31.0 and 31.5 (CH2-CH2-ph),
64.8 (H-C-NO), 74.0 (CH2-NO2), 127.1 (ph-C-4), 129.2, 129.3, 141.4 (ph-
C-1).– MS ([+]-FAB, acetone/m-nitrobenzyl alcohol): m/z (%) = 417 (14)
[(M+H)+], 208 (6) [mon+], 178 (7) [mon+-NO], 91 (100) [C7H7+].
2-Nitro-1-phenyl-ethan-1-on-oxim (4a)
1.0 g (2.8 mmol) 2a is refluxed in 20 ml ethanol for 1 h. After cooling the
solvent is removed in vacuo. The remaining oil in kept at 5 °C until crystals
have formed. They are filtered off under suction and recrystallized. Colorless
crystals, mp 88 °C, yield 0.3 g (31%).– Anal. C8H8N2O3.– IR (KBr): ν =
3231 (OH) cm–1; 2899; 1633; 1560 (NO2); 1447; 1380; 1284; 1075; 981;
(E)-2,2′-Azobis-(2-methyl-3-nitro-propanoic acid cyclohexyl ester) N,N′-di-
oxide (6d)
From 8.4 g cyclohexyl methacrylate (50.0 mmol). Colorless crystals, mp
70 °C (blue), yield 0.5 g (4%).– Anal. C20H32N4O10.– IR (KBr): ν = 1735
cm–1 (CO); 1562 (NO2).– UV/VIS (CHCl3): λmax = 657 nm.– 1H-NMR ([D6]
acetone): δ = 1.27–1.56 (m, 20 H, cyclohexyl-2-H, 3-H, 4-H, 5-H, 6-H), 1.87
(s, 6H, CH3), 4.82–4.91 (m, 2H, cyclohexyl-1-H), 5.16–5.49 (m, 4H, CH2-
NO2) all dimer; 1.27–1.56 (m, 10 H, cyclohexyl-2-H, 3-H, 4-H, 5-H, 6-H),
1.45 (s, 3H, CH3), 4.98 (m, 1H, cyclohexyl-1-H), 5.16–5.49 (m, 2H, CH2-
NO2) monomer. Ratio dim/mon = 4:1.– MS ([+]-FAB, acetone/m-nitroben-
zyl alcohol): m/z (%) = 489 (2) [(M+H)+], 325 (30), 214 (6) [mon+-NO], 132
(22), 83 (100) [C6H11+], 69 (30), 55 (90), 41 (92).
1
935; 762; 689.– H-NMR/400 MHz ([D6]DMSO): δ (ppm) = 5.81 (s, 2H,
CH2-NO2), 7.39 (br, s, 3H, ph-3H, 4-H and 5-H), 7.64 (br, s, 2H, ph-2-H and
6-H), 12.24 (s, 1H, =NOH, exchangeable).– 13C-NMR/100 MHz
([D6]DMSO): δ (ppm) = 68.7 (CH2-NO2), 125,7 (ph-C-3 and C-5), 128.6
(ph-C-2 and C-6), 129.3 (ph-C-4), 133.9 (ph-C-1), 147.2 (C=NOH).– MS
([+]-FAB, DMSO/glycerol): m/z (%) = 181 (100) [(M+H)+], 135 (51) [M+-
NO2], 117 (25), 103 (26), 79 (57).
General procedure for the synthesis of 6a-d[6]
A stirred solution of 50 mmol methylacrylic acid ester in 10 ml diethyl
ether is treated with 10 ml 40% H2SO4. The mixture is cooled to 0 °C and
5 g solid NaNO2 are added over a period of 15 min while maintaining the
temperature below 5 °C. After 30 min the diethyl ether phase is separated,
washed three times with 10 ml H2O and dried over Na2SO4. Then the diethyl
ether is removed in vacuo. The residue is dissolved in little diethyl ether/pe-
troleum ether and kept at –16 °C for 2 weeks. Crystals are formed. They are
quickly filtered off under suction off and washed with cold petroleum ether.
(E)-2,2′-Azobis-(2-methyl-pentan-4-one) N,N′-dioxide (10a)
To a stirred solution of 2.0 g (15.2 mmol) 4-hydroxylamino-4-methylpen-
tan-2-one (9a) in 40 ml H2O bromine is added until no decolorization of
bromine is observed. Then the reaction mixture is extracted three times with
40 ml diethyl ether. The collected extracts are washed with H2O, dried over
Na2SO4 and the diethyl ether is removed in vacuo. The residue is purified on
a clay plate and washed with little cold petroleum ether.
Arch. Pharm. Pharm. Med. Chem. 331, 111–117 (1998)

116
Rehse and Herpel
Colorless crystals, mp 73 °C (ref.[16] 75–76 °C), yield 0.5 g (25%).– Anal.
of nitrous oxide present in the vials. Hereby calibration values between
4.13–12.39 µmol N2O are obtained. Each standard deviation (σ = 0.4–2.3%
rel.) was obtained from 4 experiments.
C12H22N2O4.– IR (KBr): ν = 1716 cm–1 (CO); 1257 (NO dim.).– UV/VIS
(CHCl3): λmax (“ε“) = 666 (16.2) nm.– 1H-NMR (CDCl3): δ = 1.59 (s, 12H,
CH3), 2.15 (s, 6H, COCH3), 3.25 (s, 4H, CH2) all dimer; 1.31 (s, 6H, CH3),
2.15 (s, 3H, COCH3), 3.05 (s, 2H, CH2) monomer. Ratio dim/mon = 2:1.–
13C-NMR (CDCl3): δ = 24.9 (CH3), 30.8 (COCH3), 51.1 (CH2), 76.5
(C-NO), 204.5 (COCH3) all dimer; 21.8 (CH3), 31.6 (COCH3), 49.9 (CH2),
96.5 (C-NO), 205.2(COCH3) monomer.– MS ([+]-FAB, CHCl3/m-nitroben-
zyl alcohol): m/z (%) = 259 (9) [(M+H)+], 161 (25), 99 (60) [mon+-NO], 43
(100) [CH3CO+].
Determination of N2O from decomposition experiments
A sample of about 3 µmol 2a was weighed exactly into a headspace vial.
The vial was sealed gas tight. By means of two cannulas the vial was flushed
with argon (100 ml min–1) for 20 min. The cannulas were removed and 3 ml
of the solvent (see Table 2) which was also flushed with argon was injected
with a gas tight GC-syringe through the septum into the headspace vial. The
vial was placed in an incubator at 37 °C for 1h. Then the vial was placed in
the headspace sampler and assayed for N2O. The peak at tR = 4.5 min was
matched with the calibration curve and the percentage of N2O formation was
calculated.
(E)-3,3′-Azobis-(3-methyl-butanoic acid) N,N′-dioxide (10b)
To a stirred solution of 2.0 g (15.0 mmol) 3-hydroxylamino-3-methyl-
butyric acid (9b) in 20 ml 20% HCl bromine is added until no decolorization
of bromine is observed. Then the reaction mixture is extracted three times
with 20 ml diethyl ether. The diethyl ether phase is washed with H2O and
extracted three times with 20 ml 20% NaOH. The collected aqueous NaOH
extracts are acidified with 20% HCl (pH ≈ 2) and extracted three times with
20 ml diethyl ether. The extracts are washed with little H2O and dried over
Na2SO4. The diethyl ether is removed in vacuo and the residue is purified on
a clay plate.
NO determination
Device for NO determination
Head space vials: Perkin Elmer, volume 22 ml. NO/NOx
Analyzer: Antechnika AC 30 M, Karlsruhe.
Light blue crystals, mp 110 °C, yield 0.6 g (30%).– Anal. C10H18N2O6.–
IR (KBr): ν = 1702 cm–1 (CO); 1547 (NO mon.); 1246 (NO dim.).– UV/VIS
(CH3OH): λmax (“ε“) = 666 (17.5) nm.– 1H-NMR ([D6] DMSO): δ = 1.51
(s, 12H, CH3), 3.03 (s, 4H, CH2), 12.22 (s, 2H, D2O exchange, COOH) all
dimer; 1.18 (s, 6H, CH3), 3.08 (s, 2H, CH2), 12.22 (s, 1H, D2O exchange,
COOH) monomer. Ratio dim/mon = 3:1.– 13C-NMR ([D6] DMSO): δ = 23.5
(CH3), 41.2 (CH2), 75.5 (C-NO), 170.8 (COOH) all dimer; 20.7 (CH3), 40.9
(CH2), 96.1 (C-NO), 171.3(COOH) monomer.– MS ([+]-FAB, DMSO/glyc-
erol): m/z (%) = 263 (8) [(M+H)+], 163 (17), 101 (66) [mon+-NO], 83 (43),
59 (100) [C2H3O2+], 43 (28).
Generation of the calibration curve
The calibration curve was generated as described in ref.[17]
.
Determination of NO from decomposition experiments
The samples were prepared as described above (N2O determination) and
assayed for NO by the chemiluminescence method (described in detail in
ref.[17]).
(E)-2,2′-Azobis-[1-acetoxy-2-methyl-propane] N,N′-dioxide (10c)
References
From 10.0 g 2-methyl-2-nitro-propan-1-ol (84.0 mmol)[8]. Instead of
K2Cr2O7 bromine was used for oxidation. Colorless crystals, mp 63 °C
(ref.[8]67–69 °C), yield 0.9 g (7%).– Anal. C12H22N2O6.–IR (KBr): ν = 1742
cm–1 (CO); 1259 (NO dim.).– UV/VIS (CHCl3): λmax (“ε“) = 675 (22.2)
nm.– 1H-NMR (CDCl3): δ = 1.60 (s, 12H, CH3), 2.07 (s, 6H, OCOCH3), 4.59
(s, 4H, CH2) all dimer; 1.15 (s, 6H, CH3), 1.98 (s, 3H, OCOCH3), 4.80 (s,
2H, CH2) monomer. Ratio dim/mon = 1:3.– 13C-NMR (CDCl3): δ = 20.7
(OCOCH3), 21.3 (CH3), 65.9 (CH2), 77.8 (C-NO), 170.3 (OCOCH3) all
dimer; 18.2 (CH3), 20.5 (OCOCH3), 66.8 (CH2), 97.3 (C-NO), 170.5
(OCOCH3) monomer.– MS ([+]-FAB, CHCl3/m-nitrobenzyl alcohol): m/z
(%) = 291 (7) [(M+H)+], 115 (100) [mon+-NO], 43 (97) [CH3CO+].
✩
Dedicated to Prof. Dr. P. Weyerstahl on the occasion of his 65th
birthday.
[1] K. Rehse, M. Herpel, Arch. Pharm. Pharm. Med. Chem. 1998, 331, 79.
[2] K. Rehse, M. Herpel, Arch. Pharm. Pharm. Med. Chem.1998, 331,
104.
[3] H. Wieland, Justus Liebigs Ann. Chem. 1903, 329, 225–268.
[4] D. Klamann, W. Koser, P. Weyerstahl, M. Fligge, Ber. Dtsch. Chem.
Ges. 1965, 98, 1831–1836.
[4a] W. Lüttke, Z. Elektrochem. 1959, 63, 614–623.
Biology
[5] J. Decout, B. Roy, M. Fontecave et al., Bioorg. Med. Chem. Lett. 1995,
5, 973–978.
Born test[9] and thrombosis experiments[10] were carried out as usual.
[5a] Y. Kita, S. Fukuyama, Y. Hirasawa, Jpn. J. Pharmacol. 1995, 69,
69–74.
N2O determination
[6] S. Raganathan, S. K. Kar, Tetrahedron. 1975, 31, 1391–1398.
[7] C. Harries, R. Gley, Ber. Dtsch. Chem. Ges. 1898, 31, 1808.
[8] J. R. Schwartz, J. Am. Chem. Soc. 1957, 20, 4353–4355.
Device for N2O determination[16]
Head space vials: Perkin Elmer, volume 22 ml.
Gas chromatograph: Perkin Elmer Autosystem with headspace sampler
HS 40, Column: Stainless steel, 12 feet, inner diameter 2 mm, filled with
[9] K. Rehse, M. Kämpfe, K.-J. Schleifer, Arch. Pharm. (Weinheim) 1993,
326, 483–487.
Porapak Q (Supelco, Deisenhofen), 80–100 mesh. N2 flow: 10 ml min–1
;
electron capture detector range 1, attenuation 64; make up gas: argon/meth-
ane (95+5), 60 ml min–1. Inlet temp. 120 °C, detector temp 350 °C, oven
temp. 50 °C for 5 min, then rise in temp. 15 °C min–1 up to 110 °C. This
temp. is held for 2 min. Integrator Perkin Elmer Nelson 1020 (calibration
curve cubic and origin).
[10] K. Rehse, A. Kesselhut, V. Schein, M. Kämpfe, B. Rose, E. Unsöld,
Arch. Pharm. (Weinheim) 1991, 324, 301–305.
[11] K. Rehse, T. Ciborski, Arch. Pharm. (Weinheim) 1995, 328, 71–75.
[11a] K. Rehse, K.-J. Schleifer, T. Ciborski, H. Bohn, Arch. Pharm. (Wein-
heim) 1993, 326, 791–797.
Generation of the calibration curve[16]
[11b] K. Rehse, D. Piechocki, M. Schober, H. Scheffler, N. Reitner, E.
A gas sampling bulb (volume 123 ml) is flushed with pure nitrous oxide.
The molar amount of N2O is corrected for p and T. By means of a gas tight
syringe 100–300 µl in steps of 20 µl are transferred to a headspace sampler
and analyzed. The peak area is determined and correlated with the amount
Unsöld, Arch. Pharm. Pharm. Med. Chem. 1996, 329, 511–513.
[12] L. Sachs, Angewandte Statistik, Springer Verlag, Berlin–Heidelberg–
New York 1992, p. 230.
Arch. Pharm. Pharm. Med. Chem. 331, 111–117 (1998)

NO-Donors with Antithrombotic, Vasodilating Activities
117
[13] L. Jonkman, H. Muller, J. Kommandeur, J. Am. Chem. Soc. 1971, 93,
[16] K. Rehse, M. Herpel. D. Piechocki, Arch. Pharm. Pharm. Med. Chem.
1996, 329, 83–86.
5833–5838.
[14] J. Tuaillon, R. Perrot, Helv. Chim. Acta 1978, 61, 558–566.
[17] H. J. Duchstein, S. Riederer, Arch. Pharm. (Weinheim) 1995, 328,
317–324.
[15] C. Harries, L. Jablonsky, Ber. Dtsch. Chem. Ges. 1898, 31, 549–550.
Received: December 4, 1997 [FP265]
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