NO donors, part 8 [1]: Synthesis and vasodilating activities of substituted benzylnitrates compared to cyclohexylmethylnitrate and GTN

Article abstract of DOI:10.1016/S0223-5234(03)00079-5

A series of substituted benzylnitrates (1) and the formally but not chemically similar cyclohexylmethylnitrate (CHMN) have been synthesised. Vasodilating activities were measured on endothelium-intact and N^G-nitro-L-arginine (L-NNA)-blocked porcine right coronary arteries, precontracted with prostaglandin F_2α (PGF_2α). Glyceroltrinitrate (GTN) was used as reference. In intact coronary arteries the vasodilating activities of all benzylnitrates are lower compared with GTN, but higher compared with CHMN. However, blocking the function of the endothelium by L-NNA, the activity of all benzylnitrates increased, whereas that of CHMN and GTN remained nearly unaffected. Under these conditions, the mononitrates 4-nitro-benzylnitrate (1c) and 4-nitrooxymethyl-benzonitrile (1h) even showed higher vasodilator activities than the trinitrate GTN and in general, vasorelaxation by the benzylnitrates as defined by the concentrations for half maximal effects (EC₅₀ values) was found to be 2-3 orders of magnitude higher than that induced by CHMN. The study demonstrates that the in vitro activities of organic nitrates do not correlate with the number of nitrate groups within the molecule nor to the lipophilicity of the molecules. Instead, vasodilator activity is highly sensitive to the structure and the type of the substituents in the molecular carrier of the nitrate group.

Full text of DOI:10.1016/S0223-5234(03)00079-5

European Journal of Medicinal Chemistry 38 (2003) 581ꢀ586
/
www.elsevier.com/locate/ejmech
Original article
NO donors, part 8 [1]: synthesis and vasodilating activities of
substituted benzylnitrates compared to cyclohexylmethylnitrate and
GTN
Christoph Weßler a, Alexander Homann b, Uwe Fricke b, Jochen Lehmann a,*
a Institut fur Pharmazie, Friedrich-Schiller-Universitat Jena, Philosophenweg 14, 07743 Jena, Germany
¨
¨
b Institut fur Pharmakologie, Klinikum der Universitat zu Koln, Gleueler Str. 24, 50931 Koln, Germany
¨
¨
¨
¨
Received 31 October 2002; received in revised form 17 March 2003; accepted 17 March 2003
Abstract
A series of substituted benzylnitrates (1) and the formally but not chemically similar cyclohexylmethylnitrate (CHMN) have been
synthesised. Vasodilating activities were measured on endothelium-intact and NG-nitro-
-arginine ( -NNA)-blocked porcine right
L
L
coronary arteries, precontracted with prostaglandin F2a (PGF2a). Glyceroltrinitrate (GTN) was used as reference. In intact coronary
arteries the vasodilating activities of all benzylnitrates are lower compared with GTN, but higher compared with CHMN. However,
blocking the function of the endothelium by L-NNA, the activity of all benzylnitrates increased, whereas that of CHMN and GTN
remained nearly unaffected. Under these conditions, the mononitrates 4-nitro-benzylnitrate (1c) and 4-nitrooxymethyl-benzonitrile
(1h) even showed higher vasodilator activities than the trinitrate GTN and in general, vasorelaxation by the benzylnitrates as defined
by the concentrations for half maximal effects (EC50 values) was found to be 2ꢀ3 orders of magnitude higher than that induced by
/
CHMN. The study demonstrates that the in vitro activities of organic nitrates do not correlate with the number of nitrate groups
within the molecule nor to the lipophilicity of the molecules. Instead, vasodilator activity is highly sensitive to the structure and the
type of the substituents in the molecular carrier of the nitrate group.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Benzylnitrates; Organic nitrates; GTN; Vasodilator potency; Nitric oxide
1. Introduction
residue might be influenced*
/
probably differently*by
/
the chemistry of its molecular carrier and more structu-
rally different nitrates should be pharmacologically
examined.
Organic nitrates have been used for the treatment of
angina pectoris, acute myocardial infarction and acute
as well as chronic congestive heart failure for over a
century. Vasodilating activity and preload reduction,
caused by these nitrates, are mainly attributed to the
release of nitric oxide (NO), which is considered to be an
enzymatic metabolic reduction process [2,3]. In contrast
to that, recently evidence was found against NO being
the active principle of glyceroltrinitrate (GTN) [4],
which might revive the idea of a ‘‘nitrate receptor’’. In
both cases, the vasodilator activity of an organic nitrate
Bearing this in mind, we synthesised a set of
substituted benzylnitrates (1) and the formally but not
chemically similar cyclohexylmethylnitrate (CHMN).
Radicals may be intermediates in the degradation of
organic nitrates and benzyl radicals are formed more
easily than the cyclohexylmethyl radical. In addition,
benzylnitrate (1a), but not CHMN, is known to undergo
an internal redox reaction under elevated temperature
yielding benzaldehyde and inorganic nitrite [5,6]. In
order to evaluate the influence of the substituted benzyl
structures on their vasodilating potency, we investigated
the action of 1aꢀ
/
i, CHMN and GTN as reference on
L
-arginine ( -NNA)-
* Corresponding author. Present address: Lehrstuhl fur
¨
endothelium-intact and NG-nitro-
L
Pharmazeutische/Medizinische Chemie, Philosophenweg 14, 07743
Jena, Germany.
E-mail address: j.lehmann@uni-jena.de (J. Lehmann).
blocked porcine right coronary arteries, precontracted
with prostaglandin F2a (PGF2a).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00079-5

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C. Weßler et al. / European Journal of Medicinal Chemistry 38 (2003) 581ꢀ586
/
2. Chemistry
7.9 min) and peak areas were identical before and after
storing under these simulated physiological conditions.
All of the organic nitrates 1aꢀi and CHMN were
/
prepared in satisfactory yield by treating the appropriate
bromides with silver nitrate in dried acetonitrile (Fig. 1).
If not available commercially, the benzylbromides 2
were obtained by bromination of the corresponding
methylbenzenes with N-bromosuccimide (NBS). 4-Bro-
momethyl-benzaldehyde (2d) was synthesised by che-
moselective reduction of 4-bromomethyl-benzonitrile
(2c) with diisobutylaluminiumhydride (DIBAL-H) in
chlorobenzene. The disubstituted benzylbromide 2e was
prepared by nitration of 5d with KNO3 dissolved in
conc. H2SO4.
3. Pharmacology and discussion
All benzylnitrates as well as CHMN showed a dose-
dependent vasorelaxation of porcine coronary arteries
after precontraction with 50 mM PGF2a. Fig. 1 shows
some relaxation curves, Table 1 gives the results of all
compounds. GTN (pD2ꢁ
organic nitrate in intact porcine coronary arteries,
CHMN displayed the lowest activity (pD2ꢁ3.80)
/6.46) being the most active
/
(Fig. 2). In between the benzylnitrates covering the full
range of two orders of magnitude of pD2 from 3.88 to
5.95.
2.1. Stability
In L-NNA-blocked vessels, CHMN remains the
weakest vasodilator. While pD2 values of CHMN and
GTN changed only marginally, the benzylnitrates
showed little (e.g., 1e) or very significant (e.g., 1c)
increases of vasodilator activities (Fig. 3). In vessels
with blocked endothelium, 4-cyano- (1c) and 4-nitro-
substituted (1h) benzylnitrates showed an even higher
vascular activity than GTN.
The influence of the endothelial function on the
vasorelaxation by the organic nitrates can be expressed
as the ratio (Q) of the concentrations for half maximal
The stability of all organic nitrates was checked under
simulated physiological conditions by dissolving 10 mg
of the substances in a small amount of DMSO, then
filling up to 10 mL with phosphate-buffer pH 7.4 and
storing for 24 h at 37 8C. Before and after that period,
GC-analyses using an Optima-5 column and an FID-
detector were performed with 1 mL of the corresponding
solutions. All substances could be detected without
decomposition. Retention times (ranging from 4.2 to
effects of the respective compounds in L-NNA-blocked
coronary arteries versus endothelium-intact vessels. A
rather strong influence of the endothelial function was
observed with the benzylnitrates 1h, 1f and 1c (Table 1,
Fig. 3).
Our results give some indication for a high vasoactiv-
ity of benzylmononitrates as a new group of nitrovaso-
dilators which are nearly equipotent to the trinitrate
GTN. Differences in the efficacy of the compounds
studied may be deduced from the different substituents.
Rising the lipophilic properties of the compounds seems
to decrease slightly rather than increase efficacy, how-
ever, the overall correlation of lipophilicity of the
compounds studied (cf. Table 1) and the half maximal
effects (given as pD2 values, see Table 1) was not
significant (Fig. 4). But up to now, any reliable
correlations between electronic or steric parameters of
the substituents and vasoactivity of the compounds
could not yet be recognised. The higher efficacy of the
benzylnitrates in
L-NNA-blocked coronary arteries
versus endothelium-intact vessels confirms former ob-
servations on nitrovasodilators, supporting a down-
regulation of the soluble guanylcyclase as well as
cGMP-dependent protein kinase by endogenous NO
[7ꢀ
Moreover, CHMN causes the lowest activity in
coronary arteries with intact as well as -NNA-blocked
endothelium, obviously the aromatic ring system gen-
erally enhances the liberation of NO or increases the
/
9].
L
Fig. 1. Syntheses of nitrates and intermediates: (i) DIBAL, chlor-
obenzene, 0 8C; (ii) KNO3, H2SO4, CHCl3; (iii) AgNO3, acetonitrile,
r.t.

C. Weßler et al. / European Journal of Medicinal Chemistry 38 (2003) 581ꢀ
/586
583
Table 1
Lipophilicity (log P) of and vasorelaxation (given as pD2 values in ꢂ
porcine coronary arteries and after blockade by -NNA
/
log mol lꢂ1) by the benzylnitrates studied in PGF2a-precontracted intact
L
a
Compound
R
R1
log P Endothelium intact (increasing pD2)
n
Compound Endothelium blocked by
(increasing pD2)
L
-NNA
n
Q
CHMN
ꢀ
/
ꢀ
/
ꢀ
/
3.809
4.450 3.889
2.057 4.089
2.367 4.529
2.240 4.859
/
0.03 b
0.05
8
7
7
8
7
CHMN
1i
3.859
5.029
5.409
5.459
5.699
/
0.12 b
0.29
0.15
0.09
0.12
8
8
8
8
8
ꢀ
/
1f
1c
1g
1i
t-C4H9
CN
CO2H
H
H
H
H
/
/
1.04
1.19
1.41
1.03
/
0.38
1g
1f
1d
/
/
0.08 b
/
b
/0.09
/
1h
1b
NO2
CO2C2H5
H
H
2.367 4.909
3.122 5.169
2.624 5.369
1.977 5.519
1.904 5.959
6.469
/
0.07
0.09
0.29
0.01
0.11
0.07
8
8
8
8
8
8
1a
1b
5.869
5.899
6.249
6.339
6.599
6.749
/
0.14
0.22
0.06
0.13
0.31
0.06
8
8
8
8
8
8
1.09
1.14
1.05
0.98
1.34
1.65
H
/
/
1a
H
/
1e
/
1d
1e
CHO
CHO
H
/
GTN
1h
/
H
/
/
GTN
ꢀ/
NO2
ꢀ/
/
1c
/
Mean values and S.E.M. of n individual experiments are given.
a
Ratio of pD2 values of blocked to intact endothelium.
Extrapolated values.
b
Elmer 1420 or an FT-IR-spectrophotometer Paragon
1000 from PerkinꢀElmer, respectively, using KBr pellets
/
1
for solids and NaCl plates for liquid substances. H-
nuclear magnetic resonance (1H-NMR) spectra were
determined on a Bruker WH 90 (90 MHz) or a Bruker
AC 200 (200 MHz) spectrometer, respectively, using
CDCl3 as the solvent. Chemical shifts are reported in d
(ppm) relative to tetramethylsilane as the internal
standard. Elemental analysis indicated by the symbols
of the elements were carried out with an Elementar
Vario EL and were within 0.4% of the calculated values.
In general, organic nitrates are potential explosives.
Due to the high CH:NO ratio, benzylmononitrates can
be considered as relatively ‘‘safe’’ nitrates and the liquid
compounds could be distilled without significant decom-
position using the given conditions.
4.1.1. Ethyl 4-bromomethyl-benzoate (2b)
To a solution of 12.3 g (0.075 mol) ethyl 4-methyl-
benzoate in 75 mL of CCl4 were added 13.3 g (0.075
mol) of NBS and 0.9 g of dibenzoylperoxide and the
mixture was heated under reflux for 5 h. After cooling,
the filtrate of the mixture was evaporated and the
remaining oil was crystallised by trituration with Et2O.
The resulting solid was recrystallised twice from n-
Fig. 2. Vasodilator activity of benzylnitrate (1a) [m] on isolated
porcine right coronary arteries in comparison with CHMN [%]. GTN
[j] was used as reference.
affinity of the intact nitrate molecules to a putative
nitrate receptor.
hexane. 10.25 g (56%) colourless crystals; m.p. 39ꢀ
([9], 41 8C); IR (cmꢂ1): 1730 (CꢀO); 1H-NMR (90
MHz): dꢁ1.33 (t, Jꢁ7.1 Hz, 3H, CH3), 4.32 (q, Jꢁ7.1
Hz, OꢁCH2), 4.78 (s, 2H, CH2ꢁBr), 7.56 (d, Jꢁ8.3 Hz,
aromat. H-3, H-5), 7.95 (d, Jꢁ8.3 Hz, aromat. H-2, H-
/
40 8C
/
/
/
/
/
/
/
4. Experimental protocols
/
6). Anal. (C10H12BrO2): C, H, N.
4.1. Chemistry
Melting points were determined in open capillary
tubes on Gallenkamp melting point apparatus and are
4.1.2. 4-Bromomethyl-benzaldehyde (2d)
To a stirred solution of 6.0 g (30.6 mmol) of 4-
bromomethyl-benzonitrile (2c) in 60 mL of dry chlor-
not corrected. IR spectra were recorded on a Perkinꢀ
/

584
C. Weßler et al. / European Journal of Medicinal Chemistry 38 (2003) 581ꢀ586
/
Fig. 3. Vasorelaxation by increasing concentrations of the benzylnitrates 1c [%/I] and 1e [m/k] in comparison with GTN [j/I] in dependence on
endothelial function (intact [%mj] versus -NNA-blocked vessels [IkI]).
L
8.10 (dd, Jꢁ
/
7.9/2.6 Hz, 1H, aromat. H-6), 8.51 (d, Jꢁ
/
2.6 Hz, 1H, aromat. H-2), 10.08 (s, 1H, CHO). Anal.
(C8H6BrNO3): C, H, N.
4.1.4. General procedure for preparation of
benzylnitrates
A solution of benzylbromide 2 (15 mmol) in 30 mL of
dry acetonitrile was added dropwise to a solution of 3.0
g (17.5 mmol) of AgNO3 in 30 mL of acetonitrile and
stirred at r.t. for 24 h. The filtrate of the mixture was
poured into 800 mL of icewater. Precipitated solids were
separated, dried in vacuo and recrystallised. Oils were
extracted twice with 50 mL of CH2Cl2. The organic
extracts were dried over Na2SO4, evaporated and the
oily residue was distilled in vacuo.
Fig. 4. Correlation between lipophilicity (log P) and the concentra-
tions for half maximal effects (expressed as pD2 values) of the organic
nitrates in isolated porcine right coronary arteries.
obenzene, 50 mL of a 1 M solution of DIBAL-H in n-
hexane was added over a period of 20 min at 0 8C. After
stirring for 2.5 h at 0 8C the solution was diluted with
100 mL of CHCl3, treated with 150 mL of an aqueous
HCl solution (10%) and extracted into CHCl3. The
organic extracts were dried over Na2SO4 and evaporated
to give an oil which was crystallised from Et2O and
recrystallised from n-hexane. 4.1 g (68%) pale yellow
4.1.5. Benzylnitrate (1a)
Colourless oil; 1.9 g (82%); b.p. 42 8C at 0.5 mbar
([11], b.p. 101ꢀ
1279 (Nꢀ
O); 1H-NMR (90 MHz): dꢁ
CH2ꢁONO2), 7.42 (s, 5H, aromat. H).
/
104 8C at 12 Torr); IR (cmꢂ1): 1633 and
/
/
5.41 (s, 2H,
/
crystals; m.p. 96 8C ([10], 94ꢀ
/
96 8C); IR (cmꢂ1): 1690
O); H-NMR (90 MHz): dꢁ4.50 (s, 2H, CH2ꢁBr),
7.53 (d, Jꢁ8.4 Hz, 2H, aromat. H-3, H-5), 7.85 (d, Jꢁ
1
(Cꢀ
/
/
/
4.1.6. Ethyl 4-nitrooxymethyl-benzoate (1b)
Colourless crystals; 2.9 g (68%); m.p. 37ꢀ
/
/
/
38 8C; IR
1
8.4 Hz, 2H, aromat. H-2, H-6), 10.01 (s, 1H, CHO).
(cmꢂ1): 1720 (Cꢀ
(90 MHz): dꢁ1.39 (t, Jꢁ
Jꢁ6.5 Hz, 2H, OꢁCH2), 5.43 (s, 2H, CH2ꢁ
(d, Jꢁ7.8 Hz, 2H, aromat. H-3, H-5), 8.10 (d, Jꢁ
/
O) 1640 and 1270 (Nꢀ
6.5 Hz, 3H, CH3), 4.38 (q,
ONO2), 7.42
7.8
/
O); H-NMR
/
/
/
/
/
4.1.3. 4-Bromomethyl-3-nitrobenzaldehyde (2e)
/
/
To a solution of 1.4 g (0.014 mol) of KNO3 in 15 mL
of conc. H2SO4, a solution of 2.5 g (0.0125 mol) 4-
bromomethyl-benzaldehyde (2d) in 10 mL of CHCl3 was
added dropwise at 0 8C. After stirring for 2 h at room
temperature (r.t.), the mixture was poured into 500 mL
of icewater. The precipitated crude product was sepa-
rated by filtration, dried in vacuo and recrystallised
Hz, 2H, aromat. H-2, H-6). Anal. (C10H11NO5): C, H,
N.
4.1.7. 4-Nitrooxymethyl-benzonitrile (1c)
White needles; 2.1 g (79%); m.p. 31 8C; IR (cmꢂ1):
2232 (Cꢂ
dꢁ5.48 (s, 2H, CH2ꢁ
aromat. H-3, H-5), 7.70 (d, Jꢁ
H-6). Anal. (C8H6N2O3): C, H, N.
/
N) 1636 and 1282 (Nꢀ
ONO2), 7.50 (d, Jꢁ
7.8 Hz, 2H, aromat. H-2,
/
O); 1H-NMR (90 MHz):
from Et2O to give 2e as a yellow solid; 2.2 g (73%); IR
(cmꢂ1): 1702 (Cꢀ
/
/
/7.8 Hz, 2H,
1
/
O); H-NMR (200 MHz): dꢁ
/
4.86 (s,
7.9 Hz, 1H, aromat. H-5),
/
2H, CH2ꢁ
/
Br), 7.76 (d, Jꢁ
/

C. Weßler et al. / European Journal of Medicinal Chemistry 38 (2003) 581ꢀ
/586
585
4.1.8. 4-Nitrooxymethyl-benzaldehyde (1d)
Yellow needles; 1.2 g (45%); m.p. 43 8C ([10], 41 8C);
4.1.14. Cyclohexylmethylnitrate
CHMN was prepared from cyclohexylmethylbromide
(3) as described for the benzylnitrates; colourless fluid;
1.7 g (72%); b.p. 66 8C at 2.7 mbar; IR (cmꢂ1): 1635 and
IR (cmꢂ1): 1705 (Cꢀ
/
O) 1650 and 1270 (Nꢀ
5.48 (s, 2H, CH2ꢁONO2), 7.52 (d,
7.6 Hz, 2H, aromat. H-3, H-5), 7.93 (d, Jꢁ7.6 Hz,
/
O); 1H-
NMR (90 MHz): dꢁ
/
/
Jꢁ
/
/
1280 (Nꢀ
/
O); 1H-NMR (90 MHz): dꢁ
/
0.76ꢀ
/
2.15 (m,
2H, aromat. H-2, H-6), 10.02 (s, 1H, CHO). Anal.
(C8H7NO4): C, H, N.
11H, ring protons), 4.25 (d, Jꢁ
/
6.5, CH2ONO2). Anal.
(C7H13NO3): C, H, N.
4.2. Pharmacology
4.1.9. 3-Nitro-4-nitrooxymethyl-benzaldehyde (1e)
White needles; 2.7 g (81%); m.p. 62ꢀ64 8C; IR
/
4.2.1. Vasodilator activity
(cmꢂ1): 1711 (Cꢀ
(200 MHz): dꢁ5.96 (s, 2H, CH2ꢁ
7.9 Hz, 1H, aromat. H-5), 8.21 (dd, Jꢁ
aromat. H-6), 8.66 (d, Jꢁ2.6 Hz, 2H, aromat. H-2),
/
O) 1644 and 1284 (Nꢀ
ONO2), 7.81 (d, Jꢁ
7.9/2.6 Hz, 1H,
/
O); H-NMR
1
Vasodilator activity was tested at a temperature of
37 8C on isolated right coronary arteries from porcine
hearts which were obtained from a local slaughterhouse.
Proximal parts of the arteries were rapidly dissected
from the fresh hearts. Transport of the arteries was
performed in a carbogen (95% O2, 5% CO2)-saturated
/
/
/
/
/
10.10 (s, 1H, CHO). Anal. (C8H6N2O6): C, H, N.
KrebsꢀHenseleit solution of the following composition
/
4.1.10. 4-tert-Butyl-benzylnitrate (1f)
Colourless fluid; 2.4 g (77%); b.p. 56 8C at 0.4 mbar;
(in mmol Lꢂ1): Naꢃ, 143.1; Kꢃ, 5.9; Mg2ꢃ, 16.0;
Ca2ꢃ, 1.6; Clꢂ, 126.0; HCO3ꢂ, 25.0; H2PO4ꢂ, 1.2;
SO24ꢂ, 1.2; and glucose, 5.1; being equilibrated with
carbogen to attain a pH of 7.4. The prepared arteries
IR (cmꢂ1): 1633 and 1279 (Nꢀ
/
O); 1H-NMR (90 MHz):
dꢁ
/
5.38 (s, 2H, CH2ꢁ
/
ONO2), 7.29 (d, Jꢁ
/
9.0 Hz, 2H,
aromat. H-3, H-5), 7.45 (d, Jꢁ
/9.0 Hz, 2H, arom. H-2,
were cut into ring segments (3ꢀ4 mm), fixed between
/
H-6); Anal. (C11H15NO3): C, H, N.
two stainless steel hooks as previously described [14] and
incubated in a 10 mL organ bath containing equilibrated
4.1.11. 4-Nitrooxymethyl-benzoic acid (1g)
White powder; 2.45 g (83%); m.p. 164 8C ([12],
KrebsꢀHenseleit solution. Resting tension was 2 g.
/
Vascular tone was measured isotonically using a strain
gauge and was recorded after amplification (Fleck,
Mainz, Germany) on a digital printing recorder (Lins-
seis, Selb, Germany). After an equilibration period of 45
min, during which the tissues were frequently washed,
the arterial rings were contracted by 50 mmol Lꢂ1
PGF2a. After stabilisation, each compound studied was
added in a cumulative manner at 30-min intervals to
allow steady state of drug action. Each individual
165 8C); IR (cmꢂ1): 1680 (Cꢀ
/
O) 1640 and 1280 (Nꢀ
O); 1H-NMR (90 MHz): dꢁ
5.69 (s, 2H, CH2ꢁ
ONO2), 7.57 (d, Jꢁ8.1 Hz, 2H, aromat. H-3, H-5),
7.99 (d, Jꢁ8.1 Hz, 2H, aromat. H-2, H-6).
/
/
/
/
/
4.1.12. 4-Nitro-benzylnitrate (1h)
Colourless crystals; 2.4 g (82%); m.p. 66 8C ([13],
1
O); H-NMR
68.2 8C); IR (cmꢂ1): 1650 and 1281 (Nꢀ
/
concentrationꢀeffect curve was followed by the addition
/
(90 MHz): dꢁ
/
5.72 (s, 2H, CH2ꢁ
/
ONO2); 7.70 (d, Jꢁ
/8.7
of 0.2 mmol Lꢂ1 papaverine-HCl for maximal relaxa-
tion. For elimination of endothelial NO-synthase ex-
Hz, 2H, aromat. H-2, H-6), 8.28 (d, Jꢁ
/
8.7 Hz, 2H,
aromat. H-3, H-5). Anal. (C7H6N2O5): C, H, N.
posure to
-NNA-blocked vessels).
The contraction of the coronary arteries was calcu-
lated from the changes in their diameter using the
equation Wꢁmgs, with Wꢁwork, mꢁtension, gꢁ
9.81 m sꢂ2, sꢁ
change in diameter, and is given in mJ.
L
-NNA (50 mmol lꢂ1) was allowed for 15 min
(
L
4.1.13. 4-Nitrooxymethyl-benzaldehydeethylenacetale
(1i)
A solution of 1.0 g (5.5 mmol) of 4-nitrooxymethyl-
benzaldehyde (1d), 0.41 g (6.6 mmol) of ethylenglykole
and 0.1 g of p-toloulsulfonic acid in 20 mL of CHCl3
was refluxed for 48 h with continuous removal of water
by a Dean-Stark apparatus. The solution was washed
with an aqueous NaOH solution (10%) and the organic
layer was dried over Na2SO4 and evaporated. The
residue was recrystallised from MeOH to give a pale
/
/
/
/
/
Vasorelaxation by the drugs studied is expressed as
percentage of the maximal contractile response achieved
with PGF2a at the beginning of the experiments. The
concentrations for half maximal inhibition of PGF2a-
induced vasoconstriction (EC50 values) were calculated
from the individual concentrationꢀeffect curves by logit
/
yellow solid; 2.26 g (70%); m.p. 42 8C; IR (cmꢂ1): 1638
transformation as proposed by Hafner et al. [15]. pD2
values represent the negative logarithm of the concen-
tration of the respective nitrates required for half
maximal relaxation. All data were analysed by standard
statistical methods (mean value and standard error of
the mean, regression analysis, Student’s t-test).
1
and 1270 (Nꢀ
(m, 4H, 2x Oꢁ
1H, OꢁCHꢁO), 7.38 (d, Jꢁ
/
O); H-NMR (90 MHz): dꢁ
/
3.96ꢀ
ONO2), 5.81 (s,
7.6 Hz, 2H, aromat. 3-H, 5-
/
4.15
/
CH2), 5.41 (s, 2H, CH2ꢁ
/
/
/
/
H), 7.54 (d, 7.6 Hz, 2H, aromat. H-2, H-6). Anal.
(C10H11NO5): C, H, N.

586
C. Weßler et al. / European Journal of Medicinal Chemistry 38 (2003) 581ꢀ
/586
[4] A. Kleschyov, M. August, A. Mulsch, Th. Munzel, Nitric Oxide
¨ ¨
4.2.2. Materials
(2000) 413.
[5] R. Boschan, R.T. Merrow, Chem. Rev. 55 (1955) 485ꢀ
[6] S.J. Cristol, J.E. Leffler, J. Am. Chem. Soc. 76 (1954) 4468ꢀ
GTN was purchased from Merck, Darmstadt, Ger-
many, PGF2a (Minprostin† F2a Amp) was a generous
gift from Pharmacia & Upjohn, Erlangen (Germany). L-
NNA was purchased from Sigma-Aldrich Chemie,
Steinheim (Germany) and papaverine-HCl from Serva,
Heidelberg (Germany).
/
510.
/
4469.
[7] G. Kojda, J.K. Beck, W. Meyer, E. Noack, Br. J. Pharmacol. 112
(1994) 533ꢀ540.
/
[8] T. Yamashita, S. Kawashima, Y. Ohashi, M. Ozaki, Y. Rikitake,
N. Inoue, K. Hirata, H. Akita, M. Yokoyama, Hypertension 36
All benzylnitrates were dissolved in DMSO, Merck,
Darmstadt (Germany), all other chemicals in bidistil-
lated water.
(2000) 97ꢀ
[9] E. Snyder, J. Am. Chem. Soc. 80 (1958) 4622ꢀ
[10] J. Lehmann, R. Kahlich, C. Meyer zum Gottesberge, U. Fricke,
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/
102.
/
4624.
/
/
884.
[12] S. Endres, A. Hacker, E. Noack, G. Kojda, J. Lehmann, Eur. J.
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