Triazolyl-benzimidazolones and triazolyl-benzotriazoles: New potential potassium channel activators. II

Article abstract of DOI:10.1016/S0223-5234(00)01170-3

This paper reports the synthesis and pharmacological evaluation of a series of 5-substituted-triazolyl-benzotriazoles (2a-f) and the corresponding series of 5-substituted-triazolyl-benzimidazolones (6a-f), as potential activators of the big-conductance calcium-activated potassium channels (BK(Ca)). The synthesis and structure demonstration of the stock compounds of the two series have been described in our previous works, as well as the common starting compounds 4-carboxamido-5-(4-substituted-2-amino-anilino)-1,2,3-triazoles (1a-f). The triazolyl-benzotriazoles were obtained by diazotization, while the triazolyl-benzimida-zolones were obtained by thermal intramolecular cyclization of ethoxycarbonylamino derivatives or directly with phosgene. Benzimidazolone compounds generally showed little effect whilst the compounds with a benzotriazole ring showed full efficacy, with vasorelaxing properties and potency parameters a little lower than that of the reference compound NS 1619. These effects were significantly reduced by an increased membrane depolarization. This depolarization-sensitive response is in agreement with the pharmacodynamic hypothesis of activation of potassium channels. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)01170-3

Eur. J. Med. Chem. 35 (2000) 949–955
´
© 2000 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
Short communication
PII: S0223-5234(00)01170-3/SCO
Triazolyl–benzimidazolones and triazolyl–benzotriazoles: new potential
potassium channel activators. II
Barbara Baragattib, Giuliana Biagia, Vincenzo Calderonea, Irene Giorgia, Oreste Livia*,
Enrica Martinottib, Valerio Scartonia
aDipartimento di Scienze Farmaceutiche, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
bDipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
Received 3 January 2000; revised 25 April 2000; accepted 26 April 2000
Abstract – This paper reports the synthesis and pharmacological evaluation of a series of 5-substituted-triazolyl–benzotriazoles
(2a–f) and the corresponding series of 5-substituted-triazolyl–benzimidazolones (6a–f), as potential activators of the big-conduc-
tance calcium-activated potassium channels (BKCa). The synthesis and structure demonstration of the stock compounds of the two
series have been described in our previous works, as well as the common starting compounds 4-carboxamido-5-(4-substituted-2-
amino-anilino)-1,2,3-triazoles (1a–f). The triazolyl–benzotriazoles were obtained by diazotization, while the triazolyl–benzimida-
zolones were obtained by thermal intramolecular cyclization of ethoxycarbonylamino derivatives or directly with phosgene.
Benzimidazolone compounds generally showed little effect whilst the compounds with a benzotriazole ring showed full efficacy, with
vasorelaxing properties and potency parameters a little lower than that of the reference compound NS 1619. These effects were
significantly reduced by an increased membrane depolarization. This depolarization-sensitive response is in agreement with the
´
pharmacodynamic hypothesis of activation of potassium channels. © 2000 Editions scientifiques et me´dicales Elsevier SAS
1,2,3-triazoles / benzotriazoles / benzimidazolones / potassium channel activators
1. Introduction
(A), whose structures might be correlated to that of
NS 1619 (figure 1).
Potassium channel activators have been indicated
as emerging drugs for the therapy of several cardio-
vascular, respiratory or CNS diseases [1]. The benzim-
idazolone derivative NS 1619 [1-(2-hydroxy-5-tri-
fluoromethylphenyl)-5-trifluoromethyl-benzimidazo-
lone] [2] is one of the best known activators of the
big-conductance calcium-activated potassium channel
subtype (BKCa), which is a potentially important
pharmacological target for the above therapeutic ap-
proaches. Therefore, this molecule has been viewed as
a pilot structure to project and develop original hete-
rocyclic moieties, with a possible pharmacodynamic
profile of BKCa-openers.
Such open derivatives showed a high vasodilator
activity as a probable consequence of their action on
the K-channels.
As a continuation of this work, because the 5-(2-ni-
troanilino)-1,2,3-triazole derivatives represent com-
mon key intermediates, we report the synthesis and
pharmacological evaluation, towards the BKCa chan-
nels, of the corresponding 5-substituted triazolyl ben-
zimidazolones (B) and 5-substituted triazolyl
benzotriazoles (C), whose structures appear more
closely correlated to that of NS 1619 (figure 1).
In a previous paper [3] we reported synthesis and
pharmacological activity toward potassium channels,
of some 5-(2-nitroanilino)-1,2,3-triazole derivatives
2. Chemistry
The preparation of the new triazolyl–benzotriazole
derivatives 2c–f and triazolyl–benzimidazolone
derivatives 6b–f (table I) is reported in figure 2.
* Correspondence and reprints.

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B. Baragatti et al. / European Journal of Medicinal Chemistry 35 (2000) 949–955
Figure 1. General reference structures.
The starting compounds 4-carboxamido-5-anilino-
1,2,3-triazoles 1a–f, useful for the synthesis of the two
series of derivatives 2 and 6, were described in the
previously mentioned work [3].
The preparation and the chemical and spectro-
scopic structure demonstration of the stock com-
pounds belonging to the two series are reported in our
previous papers: triazolyl–benzotriazoles (2a: RꢀH;
Figure 2. Synthetic routes for the preparation of compounds 2a–f and 6a–f.

B. Baragatti et al. / European Journal of Medicinal Chemistry 35 (2000) 949–955
951
2b: RꢀCH3) [4] and triazolyl–benzimidazolone 6a
(RꢀH) [5].
The new 5-substituted-1-(4-carboxamido-1,2,3-tria-
zol-5-yl)-benzotriazole derivatives 2c–f were obtained
by diazotization of the corresponding triazole deriva-
tives 1c–f, bearing the ortho-phenylendiamino moiety
appropriate for conversion into benzotriazole.
The new 5-substituted-1-(4-carboxamido-1,2,3-tria-
zol-5-yl)-benzimidazolone derivatives 6b–f were ob-
tained by different synthetic routes. Thus, according
to the previously employed procedure [5], the 5-
anilino-triazole intermediates 1b and 1c were reacted
with 1.2 equiv of ethyl chloroformate in pyridine
solution, to give the corresponding ethoxycarbony-
lamino derivatives 3b and 3c in moderate yield. When
heating 3b and 3c in boiling DMF, the expected
benzimidazolone derivatives 6b and 6c were obtained
in good yield, by intramolecular cyclization.
The 5-fluoro-benzimidazolone derivative 6e was
prepared by an alternative procedure, analogous to
the previous one [5]: the 4-carboxamido-5-(4-fluoro-2-
nitroanilino)-1,2,3-triazole [3] was heated in an excess
of ethyl chloroformate to give the ethoxycarbony-
lamino derivative 4e in high yield, which was con-
verted to the corresponding amino derivative 5e by
catalytic hydrogenation at room temperature and
pressure. Heating 5e in boiling DMF, the expected
benzimidazolone derivative 6e was obtained.
Figure 3. Previously prepared compounds.
The other benzimidazolone derivatives 6d,f to-
gether with the previously described 6a [5], were ob-
tained by a new direct procedure, starting from
the suitable 4-carboxamido-5-(4-substituted-2-amino-
anilino)-1,2,3-triazoles 1a,d,f in pyridine solution at
0 °C, by reaction with a solution of 20% phosgene in
toluene.
For pharmacological purposes, the triazolyl–ben-
zotriazole derivatives 7a and 7b (figure 3) were also
prepared as previously described [4], starting from 2a,
by alkaline hydrolysis and decarboxylation.
The structures of all the new compounds were
assigned on the basis of known reaction mechanisms
[6, 7] and our previous evidence [3–5] and were confi-
rmed by analytical and spectroscopic methods.
1H-NMR data for compounds 2 and 6 are reported
in table II.
Table I. Physico-chemical properties.
Compound
R
Yield (%)
Crystall. solvent
M.p. (° C)
Analysis C, H, N
Mass m/z
M+
Base
2a
2b
2c
2d
2e
2f
H
[4]
[4]
273–275
278–282
283–285
273–277
CH3
OCH3
Cl
F
CF3
72
74
78
59
MeOHꢁH2O
MeOHꢁH2O
EtOH
C10H9N7O2
C9H6N7OCl
C9H6N7OF
C10H6N7OF3
259
263
247
297
44
137
121
44
EtOH
3b
3c
CH3
OCH3
60
33
AcOEt
AcOEt
165–167
208–210
C13H16N6O3
C13H16N6O4
320
44
4e
5e
F
F
83
95
AcOEt
MeOH
201–203
142–144
C12H11N6O5F
C12H13N6O3F
338
308
44
136
6a
6b
6c
6d
6e
6f
H
[5]
CH3
OCH3
Cl
F
CF3
76
75
61
56
58
AcOEt
AcOEt
AcOEt/Pet. Eth.
AcOEt/Pet. Eth.
DMF/H2O
295–300
253–256
318–322
\350
C11H10N6O2
C11H10N6O3
C10H7N6O2Cl
C10H7N6O2F
C11H7N6OF3
258
44
278
262
312
44
246
44
287–290

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B. Baragatti et al. / European Journal of Medicinal Chemistry 35 (2000) 949–955
Table II. 1H-NMR data (l, ppm) for compounds 2 and 6.
Compound
4-H
6-H
7-H
5-subst.
NH and NH2
2a
2b
2c
2d
2e
2f
6a
6b
6c
6d
6e
6f
8.19
7.95
7.62
8.38
8.07
8.74
7.04
6.90
6.83
8.07
7.57
7.62
7.63
7.46
7.28
7.69
7.56
7.87
7.04
6.89
6.94
7.37
6.95
7.50
7.65
7.54
7.58
7.69
7.68
7.96
6.79
7.64
8.08
8.32
7.10
8.35
7.53
2.50
3.88
–
–
–
7.04
2.30
3.65
–
–
–
8.1, 7.8
8.1, 7.7
8.1, 7.8
8.1, 7.8
8.2, 7.7
8.2, 7.8
11.0, 7.7, 7.4
11.2, 7.6, 7.4
11.5, 8.4, 7.7
10.7, 8.8, 7.8, 7.6
11.0, 8.8, 8.1, 7.7
10.8, 8.5, 7.6
3. Pharmacology
ence on potency and efficacy. The benzotriazoles 7a,b
did not show any vasorelaxing properties, therefore
the carboxamido group should appear as a useful
structural requirement.
The triazolyl–benzimidazolone compounds (6a–f),
albeit more similar to the reference molecule than the
benzotriazoles, did not reach the same vasoactive
properties. They showed partial vasorelaxing efficacy,
as they were unable to cause a complete reversion of
the KCl 20 mM-induced contractile tone. This partial
efficacy (often about 50%) did not permit a satisfac-
tory calculation of the potency parameter (IC50) (table
III).
Finally, a further investigation of the possible
potassium channel opening mechanism of action was
also performed in the isolated vessels. It is widely
reported that drugs, acting through potassium chan-
nel activation, possess the highest degree of ‘depolari-
sation-sensitive’ inhibition of the functional
responses, among the several classes of vasodilators
[11]. Therefore, the compounds were also tested on
isolated aortae, whose contractile tone was induced
by a higher level of membrane depolarisation (due to
the administration of KCl 80 mM). In such an exper-
imental condition, significant decreases of potency
and efficacy values were observed, fitting the profile
of response expected for a potassium channel opener.
The reference compound NS1619 did not show any
significant decrease of activity, when administered to
preparations pre-contracted by KCl 80 mM. This
anomalous behaviour could be due to different ancil-
lary mechanisms of action, whose existence has al-
ready been suggested by the literature [10]. Further-
more, the vasorelaxing effect of compound 2b (con-
Pharmacological studies, performed in different cir-
culatory districts, such as the rabbit pulmonary artery
[8], the rat portal vein [9] and the rat aorta [10]
demonstrated that high conductance calcium-acti-
vated potassium channels (BKCa) play an important
role in modulating the vascular smooth muscle tone.
Indeed, potassium-channel activation determines a
membrane hyperpolarisation, followed by a lowering
of the concentration of free intracellular calcium and
consequently by miorelaxation. Thus, the functional
evaluation of a vasorelaxing activity of the com-
pounds tested was chosen as a preliminary screening
method, to unmask a possible potassium channel
opening effect.
4. Results and discussion
All the compounds tested belonging to the tria-
zolyl–benzotriazole heterocyclic series (2a–f) showed
vasorelaxing effects, consisting of a complete aboli-
tion of the contractile tone induced by the administra-
tion of KCl 20 mM. The potency orders of magnitude
were slightly lower than those recorded for the refer-
ence compound NS1619 (table III). Preliminary ob-
servations of the structure-activity relationships
suggested that the absence of any substituent in the 5
position of the benzene ring (2a) led to a decrease of
potency (but not of efficacy). The experimental data
seemed to suggest a possible correlation between the
recorded potencies and the steric hindrance in this
position. The methoxy group showed a negative influ-

B. Baragatti et al. / European Journal of Medicinal Chemistry 35 (2000) 949–955
953
trol efficacy %=100; control pIC50 =4.8690.21) re-
sulted strongly inhibited by the non-selective potas-
sium channel antagonists 1 mM tetraethylammonium
chloride (efficacy %=3495; pIC50 not calculable)
and 3 mM 4-aminopyridine (efficacy %=2491;
pIC50 not calculable), whereas the KATP-selective
blocking sulphanylurea glybenclamide did not pro-
duce any significant inhibition (control efficacy %=
100; pIC50 =4.6390.17). The above experimental
data suggested a possible involvement of the calcium-
activated potassium channel in the vasodilator prop-
erties of the tested compounds.
collected by filtration and washed with H2O. The com-
pounds were purified by crystallization (table I). 1H-
NMR data are reported in table II.
5.4. 4-Carboxamido-5-(2-ethoxycarbonylamino-4-
substituted-anilino)-1,2,3-triazoles (3b,c)
To an ice-cooled and stirred solution of 0.500 g of 1b
[3] (2.15 mmol) or 1c [3] (2.0 mmol) in 10 mL of
anhydrous pyridine, 1.2 equiv of ethyl chloroformate
were added. The solution was heated at 50 °C for 4 h
and, after 20 h at room temperature, it was acidified
with 18% HCl and extracted with CHCl3. The organic
layer was washed with 10% HCl, H2O, dried (MgSO4)
and evaporated to give the title compounds, which were
5. Experimental protocols
1
purified by crystallization (table I). H-NMR data (l):
3b: 1.34 (t, 3H, CH3); 2.24 (s, 3H, CH3); 4.37 (q, 2H,
CH2); 6.92 (m, 1H, 5%-H); 7.19 (m, 1H, 3%-H); 7.44 (m,
1H, 6%-H); 5.7, 6.0, 7.1, 7.5 (5H, NH and NH2). 3c: 1.36
(t, 3H, CH3); 3.65 (s, 3H, OCH3); 4.42 (q, 2H, CH2);
6.60 (m, 1H, 5%-H); 6.78 (m, 1H, 3%-H); 8.02 (m, 1H,
6%-H); 5.5, 6.3, 7.4 (5H, NH and NH2).
5.1. Chemistry
Melting points were determined on a Kofler hot-stage
and are uncorrected. IR spectra in Nujol mulls were
recorded on a Mattson Genesis series FTIR spectrome-
1
ter. H-NMR spectra were recorded with a spectrometer
Varian Gemini 2000 in DMSO-d6 in d units, using TMS
as the internal standard. Mass spectra were performed
with a Hewlett Packard MS/system 5988. Elemental
analyses (C, H, N) were within 90.4% of the theoreti-
cal values and were performed on a Carlo Erba elemen-
tal analyzer Mod. 1106 apparatus. TLC data were
obtained with Riedel de Haen, 37360 DC-Karten F254,
0.2 mm, eluting with a 1:3 AcOEt/petroleum ether mix-
ture. Petroleum ether corresponds to fraction boiling at
40–60 °C.
5.5. 4-Carboxamido-5-[N-(ethoxycarbonyl),
N-(4-fluoro-2-nitrophenyl)]-amino-1,2,3-triazole (4e)
A suspension of 4-carboxamido-5-(4-fluoro-2-nitro-
anilino)-1,2,3-triazole [3] (0.750 g, 2.82 mmol) in 10 mL
of ethyl chloroformate was heated under reflux for 24 h.
After cooling, the solution was poured into water-
crushed ice, then neutralised (pH 6–7) with portions of
solid NaHCO3. After 3 h of stirring, the precipitate
consisting of 4e was collected by filtration, washed with
1
H2O and purified by crystallization (table I). H-NMR
5.2. 4-Carboxamido-5-(4-substituted-2-aminoanilino)-
1,2,3-triazoles (1a–f)
data (l): 1.38 (t, 3H, CH3); 4.55 (q, 2H, CH2); 7.85 (m,
1H, 6%-H); 8.09 (m, 1H, 5%-H); 8.64 (m, 1H, 3%-H); 8.1,
8.5, 11.6 (3H, NH2 and NH).
The preparation of the title compounds is reported in
the literature [3].
5.6. 4-Carboxamido-5-[N-(ethoxycarbonyl),
5.3. 5-Substituted-1-(4-carboxamido-1,2,3-triazol-5-yl)-
benzotriazoles (2c–f)
N-(4-fluoro-2-aminophenyl)]-amino-1,2,3-triazole (5e)
To a solution of 4e (0.300 g, 1.0 mmol) in 150 mL of
MeOH was added 0.030 g of 10% Pd/C and the mixture
was hydrogenated at room temperature and pressure.
The catalyst was filtered off, washed with hot MeOH
and the filtrate was evaporated in vacuo, to give the title
compound, which was purified by crystallization (table
To an ice-cooled (0–5 °C) and stirred solution of 2.0
mmol (0.500 g) of the appropriate 4-carboxamido-5-(4-
substituted-2-aminoanilino)-1,2,3-triazole (1c, 1d, 1e or
1f) in 15 mL of 18% HCl, a solution of NaNO2 (0.165 g,
2.4 mmol) in 10 mL of H2O was added drop by drop.
After 20 min, the ice-bath was removed and the stirring
was continued at room temperature for 1 h. The precip-
itate obtained, consisting of the title compounds, was
1
I). H-NMR data (l): 1.36 (t, 3H, CH3); 4.47 (q, 2H,
CH2); 6.43 (m, 1H, 6%-H); 6.58 (m, 1H, 5%-H); 7.48 (m,
1H, 3%-H); 5.1, 7.9, 8.0, 8.3 (5H, NH2 and NH).

954
B. Baragatti et al. / European Journal of Medicinal Chemistry 35 (2000) 949–955
5.6.1. 5-Substituted-1-(4-carboxamido-1,2,3-triazol-
5-yl)-benzimidazolones (6b,c,e)
5.7. Pharmacology
All the experimental procedures were carried out fol-
lowing the guidelines of the European Community
Council Directive 86-609.
A solution of 0.500 g (1.60 mmol) of the appropriate
ethoxycarbonyl derivative 3b, 3c or 5e in 10–15 mL of
DMF was heated under reflux for 4–5 h. The reaction
mixture was concentrated in vacuo (1/2–1/3 of the
original volume) and H2O was added to precipitate the
title compounds, which were collected by filtration and
To determine a possible vasodilator mechanism of
action, the compounds were tested on isolated thoracic
aortae of male normotensive Wistar rats (250–350 g).
The rats were killed by cervical dislocation under light
ether anaesthesia and bled. The aortae were immediately
excised, freed of extraneous tissues and the endothelium
was removed by gently rubbing the intimal surface of
the vessels. Aortic rings were suspended, under a
preload of 2 g, in 10 mL organ baths, containing Tyrode
solution (composition of saline in mM: NaCl 136.8; KCl
2.95; CaCl2 1.80; MgSO4·7H2O 1.05; NaH2PO4 0.41;
NaHCO3 11.9; Glucose 5.5), thermostated at 37 °C and
continuously bubbled with a mixture of O2 (95%) and
CO2 (5%). Changes in tension were recorded by means
of an isometric transducer (Basile mod. 7005), connected
with a unirecord microdynamometer (Basile mod. 7050).
After an equilibration period of 60 min, the endothe-
lial integrity was confirmed by acetylcholine (ACh) (55
mM)-induced relaxation of norepinephrine (NE, 1 mM)-
precontracted tissues. A relaxation B20% of the NE-in-
duced contraction was considered representative of an
acceptable lack of the endothelial layer, while the or-
gans, showing a relaxation ]20% (i.e. significant pres-
ence of the endothelium), were not used in the
experimental procedures. Thirty to 40 min after confir-
mation of the endothelium removal, the aortic prepara-
tions were contracted by treatment with a single
concentration of KCl (20 mM) and when the contrac-
tion reached a stable plateau, 3-fold increasing concen-
trations of the compounds (10 nM–1 mM) were added
cumulatively. In parallel sets of experiments, to investi-
gate the influence of a higher level of depolarisation on
the responses evoked by the compound tested, the aortic
preparations were contracted by KCl 80 mM. Then,
3-fold increasing concentrations of the compounds (10
nM–1 mM) were added cumulatively.
1
crystallised from the appropriate solvent (table I). H-
NMR data are reported in table II.
5.6.2. 5-Substituted-1-(4-carboxamido-1,2,3-triazol-
5-yl)-benzimidazolones (6a,d, f)
To an ice-cooled (0–5 °C) and stirred solution of 1.0
mmol of the appropriate compound 1a [4], 1d [3] or 1f
[3] in 8 mL of anhydrous pyridine, a solution of 20%
phosgene in toluene (0.8 mL, 1.6 mmol) was added. The
reaction mixture was stirred for 2 h, then the ice-bath
was removed and stirring continued for 20 h. Dilution
with H2O and acidification (pH 2–3) with 10% HCl
caused precipitation of the title compounds, which were
collected by filtration and crystallised from the appro-
1
priate solvent (table I). H-NMR data are reported in
table II.
Table III. Pharmacological effects of the compounds testeda.
Compound
pIC50
Efficacy %
Dep.
2a
3.9590.059
4.8690.21
3.8190.049
4.2690.090
4.0390.036
4.4390.11
100
100
70
100
100
100
ineffective
71
ineffective
53
+
+
+
+
+
+
2b
2c
2d
2e
2f
6a
6b
4.0390.17
+
6c
b
b
b
6d
+
+
+
−
6e
48
55
100
6f
NS 1619
5.3090.29
Preliminary experiments showed that both the KCl
(20 and 80 mM)-induced contractions remained con-
stant in a stable tonic state for at least 40 min.
In other sets of experiments, the non-selective potas-
sium channel blocker tetraethylammonium chloride (1
mM), the non-selective potassium channel blocker 4-
aminopyridine (3 mM) or the ATP-sensitive potassium
channel selective blocker glybenclamide (1 mM) were
added, before the KCl (20 mM)-induced contraction,
followed by the administration of selected compounds.
a The values of potency (pIC50) are expressed as the mean9
SEM. The efficacy parameter indicates the maximal vasorelax-
ation, as a % of the contractile tone induced by KCl 20 mM.
The potency parameters of compounds possessing a low effi-
cacy (50% or lower) could not be calculated. The effectiveness
(+) or the ineffectiveness (−) of the increased depolarisation
(KCl 80 mM) to reduce the activity of the compounds, are
also shown (Dep.).
b The pIC50 values could not be calculated because of the low
efficacy %.

B. Baragatti et al. / European Journal of Medicinal Chemistry 35 (2000) 949–955
955
Norepinephrine hydrochloride (Sigma), acetylcholine
chloride (Sigma), tetraethylammonium chloride
the contractile agent. The above parameters were calcu-
lated by means of non-linear regression analysis of the
sigmoidal concentration–response curves (computer
program: GraphPad Prism), and were expressed as the
mean9SEM of six experiments.
The statistical comparison of experimental data was
performed by the two-tailed Student t-test and Anova.
A value of PB0.05 was considered as representative of
significant differences.
(Aldrich), 4-aminopyridine (Sigma) and KCl were dis-
solved in bi-distilled water. Glybenclamide (1 mM) was
dissolved by sonication in an aqueous solution of NaOH
(0.1 N) and further diluted in bi-distilled water. All the
synthesised derivatives and the reference compound NS
1619 (RBI) were dissolved (10 mM) in aqueous NaOH
(0.1 N). All further dilutions were performed in bi-dis-
tilled water. All solutions were prepared immediately
before the pharmacological experimental procedures.
Previous experiments showed a complete ineffectiveness
of the administration of the vehicle.
References
[1] Cook N.S., Potassium channels: structure, classification, func-
tion and therapeutic potential. Ellis Horwood Limited,
Southampton, UK, 1990.
5.7.1. Data analysis
The efficacy of the vasorelaxing responses was ex-
pressed as a maximal relaxant effect (Emax), calculated
as a % of the contractile tone developed by the smooth
muscle preparation, after the depolarising stimulus in-
duced by KCl 20 mM. The above parameters were
calculated by means of non-linear regression analysis of
the sigmoidal concentration-response curves (computer
program: GraphPad Prism), and were expressed as the
mean of six experiments. Previous experiments demon-
strated an almost complete quantitative equivalence be-
tween the contractile responses evoked by the two
different concentrations of KCl, since the concentration
20 mM could substantially induce a maximal effect, in
endothelium denuded aortic rings.
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