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New NO-Donors with Antithrombotic and Vasodilating Activities Part 15 Nitrolic Acids.

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Nitrolic Acids
New NO-Donors with Antithrombotic and Vasodilating Activities, Part 15
Nitrolic Acids *
Klaus Rehse", Martin Herpel, and Daniela Piechocki
Institut fiir Pharmazie, Freie Universitat Berlin, Konigin-Luise-Str. 2+4, D-14195 Berlin, Germany
Key Words: nitrolic acid; antithrombotic properties; blood pressure; N20 formation; NOx formation
NOH
Summary
Five 1 -0ximin0- 1-nitro compounds (nitrolic acids) were prepared
and tested for antithrombotic and blood pressure lowering activities. 1-Nitro-ethanone l-oxime (1, ethylnitrolic acid) 2 h after oral
adminisbrationto rats inhibitedthrombusformationby a laserbeam
in msenteric arterioles of rats by 69% and by 46% in venules
(60mg/kg). "be blood pressure of S H R rats even at this high dose
only was decreased by 10%suggesting that antithrombotic and
blood pnssure effects can be dissociated. The in virro decomposition of 1 at 37 "C nearly exclusively gave N f l and acetic acid
indicating that HNO primarily had been formed.
(4)
cti3-c
1
JNoH
cicooczn5
\
___)
N32
cn3-c
\
c
I
0-
N
02N
Introduction
The synthesis of the first nitrolic acid (1) was reported by
the famous chemist Victor Meyer in 1875. Since then nitrolic
acids remained a forgotten class of chemical compounds and
no pharmacological activities have been found hitherto. We
supposed that these compounds could function as nitric oxide
donors. We therefore prepared and investigated the compounds shown in Scheme 1.
Scheme 1: Synthetic routes to alkylnitrolic acids (1 -4) and a derivative (5).
group so that the proton signal at 2.34 ppm has to be attributed
to this grou
V. MeyerEi already stated that ethylnitrolic acid in the solid
state is prone to decomposition even at room temperature, in
spite of having a sharp melting point (80 "C). The time course
of degradation was unpredictable. As reaction products
Meyer identified NO2 and acetic acid. From the pattern of
Chemistry
antithrombotic and blood pressure lowering activities we
In general alkylnitrolic acids can be prepared from o-ni- guessed that NO rather than NO2 should be formed so that
troalkanes with nitrite"] (e.g. 1 and 2 in eq. 1). More specially NO2 should be a secondary reaction product with oxygen. In
a-carboxyalkylnitrolic acids are obtained from cyclic secon- order to test this hypothesis we carried out some decomposidary alcohols like cyclohexanol with nitric acid (see eq. 2)r21. tion experiments under controlled conditions (37 "C, 96 h) in
The third method comprises the solvolysis of activated cyclic head space vials in an argon atmosphere. The decomposition
pseudonitrols with hydroxylamine (eq. 3)[31.In order to avoid experiments were carried out in the solid state as in solution
the dissociation of ethylnitrolic acid in the gut, thereby im- 1 was much more stable. After 24 h at 37 "C in Hepes buffer
proving the absorption, we tried to synthesize the O-ethoxy- (argon atmosphere) only 0.1 % of 1 had decomposed to N20.
carbonyl derivative of 1. Instead of the desired product we We analyzed the volatile reaction products by GC using a
isolated only the dimer 5. Obviously the monomeric ester is Porapak Q column. At a retention time of 4.5 min we found
so reactive that nucleophilic substitution by the oxyimino a strong peak suggesting that either NO2 or N2O had formed
in about 75 k 7 % (0,n = 4) yield. To differentiate between
group of a second molecule of 1 occurs immediately.
The chemical structure of 5 is thoroughly backed by its these two reaction products we transferred the gas phase
spectroscopic data. Thus in the +FAB-mass spectrum the formed in the reaction vials by a gas tight connection into a
molecular ion [M + HI+ is found at m/z = 234 with 21% high resolution mass spectrometer. The results are compiled
relative intensity. The base peak m/z = 43 indicates that a in Table 1.
CH3-CO fragment is present. The intense peak at m/z = 46
Obviously almost no NO2 (m/z = 46) could be detected.
suggests that the compound bears a nitro substituent. The Instead a strong peak at m/z = 44 was observed. By means of
'H-NMR spectrum shows two singlets for methyl groups at precise mass determination we could show that 90% of the
2.34 or 2.63 ppm, respectively. The 13C-'H-COSY spectrum peak intensity consisted of N20. The rest was C02 possibly
shows that the first signal is correlated with a carbon atom at formed from the other reaction product name1 acetic acid of
15.3 ppm while the rotons at 2.63 ppm belong to a carbon which the formation could be determined by H-NMR specatom at 12.6 ppm. P3C-additivity
suggest that the troscopy: The signal of the methyl group of 1 is found at 2.47
downfield carbon atom belongs to the CHyC(OR)=N methyl ppm while acetic acid forms a singlet at 2.09 ppm (both in
Y
Arch. Pharm. Phann. Med. Chem.
0VCH Verlagsgesellschaft mbH, D-6945 1 Weinheim, 1996
0365-6233/96/0202-0083$5.00 + .25/0
84
Rehse, Herpel, and Piechock
Table 1: Mass spectrometric analysis of volatile reaction products obtained by thermal decomposition of 1 in an argon atmosphere after 96 h at 37 "C. Relative
intensities given in %, base peak = 100%.The time after connection of the reaction vial to the mass spectrometer is indicated. The changing intensities between
time 0 - 86 min reflect the changing partial pressure of the volatile components when argon is pumped off successively.
d
z
time
[min]
28
30
32
40
44
44
46
ion+'
H2O
background I00
NZ
NO
0 2
Ar
N20
COZ
NO2
CH3COOH
12
-
1
-
-
-
-
-
2
18
60
41
-
16
100
0.6
0.05
-
-
24
<I
97
<1
33
100
2.3
0.2
-
-
55
<1
100
<I
48
99
1.9
0.2
-
86
I00
98
21
15
4.2
0.4
0.1
0
1.4
CDC13). At d z = 30 a peak of small intensity could be
identified as NO' by precise mass determination. This corresponds to chemiluminescence measurements whereby only
trace amounts of NO could be detected. We do interpret these
results as a special case of the Nef reaction[91 according to
Scheme 2. As first step we propose the thermally induced
rearrangement to the hypothetical nitrite la, followed by the
formation of the nitrosoketone l b and the elimination of
nitrosohydrogen. By hydrolysis of 1b acetic acid and another
molecule of nitrosohydrogen is formed.
0
1
(4)
2 HNO + 0,
la
--
No' + NO+;
lb
H,O
Scheme 2: Formation of NzO, NO, and NO2 from nitrolic acids
The reaction will take place even when humidity is excluded because the amount of water necessary for hydrolysis
is generated by the reaction of HNO to N2O (see eq. 3). The
observation (of Meyer) that - in the presence of oxygen NO2' is formed, can easily be understood from eq. 4. The view
that HNO is the first reaction product is further backed by
Meyer's observation' that in concentrated sulfuric acid
solely N 2 0 and acetic acid are formed.
Biological Experiments
Antithrombotic Propertie5
Firstly the compounds were assayed by the Born testc5]for
their antiplatelet activities. Collagen was used to induce the
platelet aggregation. The concentrations giving semi-maxi-
~
2.9
mum inhibition of aggregation (IC50 [ymol/L]) were as follows: 1 (25),5 (15), others (> 62.5) [ASA: 1751. Therefore 1
and 5 in vitro have to be classified as poor antiplatelet agents
while the other compounds have to be regarded as inactive.
As all NO donors are prodrugs of which the most important
(nitroglycerol, molsidomine) are as well inactive in vitro, we
submitted 1 - 5 to an in vivo thrombosis modelL6].
Briefly the formation of thrombi in mesenteric vessels of
rats is induced by the beam of an argon laser via a microscope
(35 mW, 50 ms). 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 compiled in Table 2. It is obvious that all
compounds exhibit strong (1) or moderate antithrombotic
effects. An inhibition of 69% in arterioles approximates the
maximum effect which can be measured in this hard thrombosis model. This effect is superior to other antiplatelet drugs
like acetylsalicylic acid, pentoxiphylline or ti~lopidine[~]
and
nearly reaches the effect of molsidomine, which however
cannot be applied therapeutically in such high doses for its
strong blood pressure decreasing properties. The effect of 1
Table 2: Antithrombotic effects of nitrolic acids as a function of dose and
time after p.0. administration to male Whistar rats. Results are given as
percentage of inhibition of thrombus formation'71Statistics: Man-Whitney
U test. n.s. = not significant
dose
[mgkl
time
Ihl
% inhib. k SEM
arterioles P 5
%
' inhib. k SEM
venules
P5
1
60
2
69 5 1
0.002
46k 1
0.002
1
30
2
34k2
0.002
16f4
0.002
1
60
4
33f2
0.002
15+1
0.002
1
60
6
O f 1
2
60
2
31 + 2
0.002
24f2
0.002
3
60
2
24+ 1
0.002
14f 1
0.002
4
60
2
11 f 1
0.002
12f 1
0.002
5
60
2
14+ 1
0.002
11
*1
0.002
ASA
60
2
481- 10
0.002
20f5
0.002
Comp.
I5.S.
1+1
n.s.
An.h. Phum. Pharm. Mrd. Chem 329,8346 (1996)
85
Ni trolic Acids
in venules had as well to be classified as strong. The antithrombotic properties in either microvessels are time and
dose dependent. With 30 mgkg p.0. of 1 still a very considerable antithrombotic activity especially in arterioles is observed. With 60 mgkg 4 h after administration a similar effect
is obtained. After 6 h no more activity of 1 is seen. Compound
2 in a dose of 60 mgkg had the same effect as 1 in a 30 mgkg
dose while 3 - 5 were less active.
I -Nitro-ethanone I-Oxime ( I )
Mp 80 "C (.ref."' 81-82 "C).- 'H-NMR ([DslDMSO): 6 = 2.30 (s, 3H,
CH3), 12.93 (s, lH, OH), 'H-NMR (CDC13): 6 = 2.47 (s, 3H, CH3), 9.5 (s,
lH, OH).
I-Nitro-propunone 1-0xime (2)
Mp 60 "C (ref.'*] 60 "C).- 'H-NMR ([DhlDMSO): 6 = 1.09 (1. J = 7.5 Hz,
3H, CH3), 2.76 (4, J = 7.5 Hz, 2H, CHz), 12.94 (s, lH, OH).
Influence on Blood Pressure
6-Hydroxyimino-6-nitro-hexanoic
Acid (3)
The antithrombotic effects probably are due to an antiplatelet effect mediated by a rise in c-GMP in the platelet via
NO formation presumably in the liver. The NO should enter
there immediately the platelets and of course the red blood
cells.
The decrease in blood pressure by NO-donors however is
due to the formation of NO (or EDRF) from the donors
compounds in endothelial cells and diffusion (or transport) to
the subendothelial smooth
It is
possible that
after diffusion of the intact NO donor the release of NO there
occurs directly. We therefore investigated the effect of 1 on
the blood pressure in SHR rats.
The K%ults are summarized in
3. They indicate that
even at the highest dose of60 mgkg only a very mild decrease
in blood pressure of about 10% is measured after 4 or 6 h,
respectively. This effect just is significant. No significant
effect on the heart rate was observed. We interpret this finding
in the sense that 1 is poorly metabolized in the endothelium.
The results suggest that it should be possible to dissociate the
antithrombotic effect Of NO donors from their blood pressure
lowering properties.
Mp 77 "C (ref."' 74-75 "C).- 'H-NMR ([DhIDMSO): 6 = 1.54 (m, 4H,
3,4-CH2), 2.24 (t, J = 7 Hz, 2H, 2-CH2), 2.80 (t, J = 7 Hz, 2H, 5-CHz).
6-Hydroxyimino-6-nitro-hexunohydroxamic Acid (4)
Mp 138 "C (ref.13' 136 "C).- 'H-NMR ([D6]DMSO): 6 = 1.50 (br. s, 4H,
3,4-CH2), 1.96 (br. s, 2H, 2-CH2), 2.78 (hr. s, 2H, 5-CH2), 8.68 (s, IH, D20
exchange, NH-OH), 10.34 (s, lH, Dz0 exchange, NH-OH), 12.95 (s, 1H,
D2O exchange, =NOH).
[ [ [ I - [ [ ( l . N j t r o e t h ~ l i d e ~ ) i ~ i n o ] o x y ] e t h y l i ~ e ~ ] i ~ i n o ] - o x yAcid
].~eth~~oi~
Ethyl Ester (5)
1.04 g (10 mmol) nitroethanone oxime are dissolved in ether (absol.). The
solution is cooled with ice and 1.0 g (10 mmol) triethylamine added. Now
1.1 g (10 mmol) ethyl chloroformate is slowly added dropwise. The suspension forming is stirred for 3 h. The precipitate is sucked off, washed with
water, and dried.
Crystals, mP 155 "C, yield 10 %.- Anal. C7HiiN306.- IR (KBr): 1768
cm-' ( o - c o - o ) , 1649 (C=N), 1557 ( N o d 1233 (co-o-).- 'H-NMR
(CDC13): 6 = 1.37 (t, J = 7 Hz, 3H, CHzCHj), 2.34 (s, 3H, 0-C- CH3), 2.63
(s, 3H, 02N-C- CH3), 4.33 (q, J = 7 Hz, CH2-CH3).- I3C-NMR (CDCb): 6
= 12.6 (cH~), 14.3 (cH~-cH~),15.3 (cH~), 65.1 (cH~), 153.3 (N=c-o),
160.0 (N=c.N), 163.7 ( ~ 0 1 . -MS: +FAB (DMSO/m-nitrobenzylic alcohol)
m/z (%) = 234 (21) [(M + H)'], 46 (78) [N02+],43 (100) [CH3CO+].
Biology
Table 3: Blood pressure lowering effect of 1 (60 mgkg p.0) 2 - 6h after
administration to SHR rats. Values given as percent of control at time zero
(100%). Statistics: Man-Whitney U test. n.s. = not significant
time
2h
4h
6h
% f s (control)
98.8 f 6.6
98.5 f 5.7
94.7 k 6.5
% k s (with 1)
90.0 f 2.8
89.2 f 7.2
90.7 f 6.9
P12
0.02
0.05
n.s.
minoxidil2 mgkg
78.0 f 5.1
80.2 f 5.5
80.6 f 5.4
Acknowledgement
We thank E. Christman-Osterreich (GC), U. Ostwald (MS), N. Reitner,
and H. Scheffler (animal experiments) for their skillful assistance.
Experimental Part
Chemistry
'H- and I3C-NMR: Bruker AC 300.- MS: Varian MAT 71 1.- FAB-MS:
Varian MAT CH5DF.- GC: Perkin-Elmer Autosystem with headspace sampler HS 40. Column: Stainless steel, 12 feet, inner diameter 2 mm, filled with
Porapak Q, 8G100 mesh. N2 flow: 10 ml min-'. Retention time 4.5 min.
Electron capture detector.- IR: Perkin-Elmer 1420.- Mp: Mettler FP-1.Element analysis: Elementar, Vario EL.
Arch. P h a m Pharm. Med. Chem. 329,8346 (19%)
B~~ test[5];md thrombosis experimentsi6]were carried out as usual,
Blood Pressure Measurements in Conscious SHR Ruts
Principle and Device
All experiments were carried out with spontaneously hypertensive inbred
rats (SHR) from Mollegaard breeding centre. The systolic blood pressure
rises with the age of the rat from 29 kPa (220 mm Hg, 3 months) to 40 kPa
(300 mm Hg, 5 months). The weight of the rats also rises with age from 250
to 350g. The heartrate is 35(!-450pulses/min. Water andAltromii@standard
diet were given ad libitum.
The systolic blood pressure and heart rate were determined with the system
BMN 1701 (Fohr Medical Instruments GmbH, Egelsbach, Germany) of
which the measuring range had been extended to 47 !@a (360 mm Hg). The
complete setup comprises a sphygmomanometer-monitor, warming up unit
for four rats, a two channel recorder, and a heated water bath with pump.
Briefly the rat is warmed up (10 f 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 risen until the pulsations have ceased. Then the
pressure is decreased continuously until the pulse returns. As pressure and
pulse are recorded simultaneously the arterial blood pressure can he determined directly from the chart of the recorder. The heart rate is recorded
digitally.
Procedure
In the morning blood pressure and heart rate of each SHR rat are measured
three times. The difference between the highest and lowest value in the same
rat must not exceed 20 mm Hg (trained animals!). Now groups of 8 - 10
animals with similar blood pressure are formed. The test compound in a dose
86
Rehse, Herpel, and Piechocki
of 60 mg/kg in 1 ml test suspension or emulsion is administered orally to six
rats with the least intra- and interindividal differences in blood pressure. The
blood pressure again is recorded 2h, 4h, 6h, and 24 h after administration of
the test substance. By means of a computer program the results can be
expressed numerically and graphically in absolute values as well as percent
of control. For statistical evaluation the Wilcoxon-Man-Whitney U test is
used18'.
Mass Spectrometric Measurements
The decomposition apparatus for mass spectrometric investigations is
outlined below:
,ound
LV
one
Decomposition Experiments
mass spectrometei
(MS)
Device for N20 determination
Head space vials: Perkin Elmer, volume 22 mL
Gas chromatograph: Perkin Elmer Autosystem with head space sampler
HS40, Column: Stainless steel, 12 feet, inner diameter 2 mm, filled with
Porapak Q (Supelco, Deisenhofen), 80 - 100 mesh. N2 flow: 10 ml min-';
electron capture detector range 1, attenuation 64; make up gas: argodmethane (95 %/5 %), 60 mL mn-'. Inlet temp. 120 "C, detector temp 350 "C,
oven temp. 50 "C for 5 min, then rise in temp 15 min-' up to 110 "C. This
temp. is kept for 2 min. Integrator Perkin Elmer Nelson 1020 (calibration
curve cubic and origin).
About 3 mg 1 are weighed exactly into the dec. vial (tap closed). The vial
is closed gas tightly with a rubber stopper. By means of two cannulas which
are inserted through the stopper the vial is flushed with argon (100 mumin)
for 20 min. The cannulas are removed and the vial kept at 37 "C. for 96 h. It
is then connected via a vacuum tap (closed) with the high resolution mass
spectrometer (MS) (Varian MAT 71 1). Firstly the gas between the vac. tap
and the MS is evacuated via the MS. Then the vac. tap is opened a little and
the region between the two taps is evacuated. When a vacuum of
Pa is
reached the underground is recorded. Then the vacuum tap is closed and the
tap of the reaction vial opened a little. The tap is closed again and the vacuum
tap opened carefully and the mass spectra recorded. This procedure is
repeated several times. After the times indicated in Table 1 the corresponding
peak intensities were found.
Generution of the Calibration Curve
A gas sampling bulb (volume 123 mL) is flushed with pure nitrous oxide.
The molar amount of N20 is corrected for p and T. By means of a gas tight
syringe 100 - 300 p1 in steps of 20 p1 are transferred to a head space vial
which is filled with pure nitrogen. The head space vials are placed in the head
space sampler and analyzed. The peak area is determined and correlated with
the amount of nitrous oxide present in the vials. Hereby calibration values
between 4.13 - 12.39 pmol N20 are obtained. Each standard deviation (o =
0.4 - 2.3 % rel.) was obtained from 4 experiments.
References
Dedicated to Prof. Dr. Dr. E. Mutschler on the occasion of his 65th
birthday.
V. Meyer, Jusrus Liebigs Ann. Chem. 1875,175,88-140.
H. C. Godt, J. F. Quinn, J. Am. Chem. Soc. 1956, 78, 1461-1464.
F. Minisci, A. Quilico, 0rg. Prep. Proced. 1969, 1, 5-10.
E. Pretsch, T. Clerc, I. Seibl, W. Simon, Strukturaufilarung organischer Verbindungen,3rd Edit., Ch. 10, Springer-Verlag 1990.
Determination of N20 ,from Decomposition Experiments
A sample of about 1 mg (10 pmol) 1 was weighed exactly into a head space
vial. The vial was sealed gas tight1 By means of two cannulas the vial was
flushed with nitrogen (100 ml min ) for 20 min. The cannulas were removed
and the vial placed in an incubator at 37 "C for 96 h. The vial was placed in
the head space sampler and assayed for N20. The peak at R = 4.5 min was
matched with the calibration curve. The standard deviation owas 7 c/a rel.
determined from four experiments.
-7.
K. Rehse, U. Siemann,Arch. Pharm. (Weinheim),1981,314,627430.
K. Rehse, A. Kesselhut, V. Schein, M. Kampfe, B. Rose, E. Unsold,
Arch. Pharm. (Weinheim),1991,324,301-305.
K. Rehse, T. Ciborski, Arch. Pharm. (Weinheim),1995,328, 71-75.
L. Sachs, Angewandte Statistik, Springer-Verlag, Berlin 1984, p. 230.
I. U. Nef, Justus Liebigs Ann. Chem. 1894,280, 263-291.
Received: October 6, 1995 [FP063]
Arch. Phann Pharm. Med. Chem. 329,83-86(19%)
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