New 5-substituted-1-(2-hydroxybenzoyl)-benzotriazoles, potassium channel activators. IV.

Article abstract of DOI:10.1016/S0014-827X(01)01146-6

This paper reports the synthesis of a series of new 5-substituted-1-(2-hydroxybenzoyl)-benzotriazoles, which have been tested for their activity as possible activators of potassium channels. In rat aortic rings, the 'opened' derivatives 1a-f, intermediates of synthesis, showed vasorelaxing properties, with appreciable values of potency. However, the most remarkable effects were recorded for the 2-hydroxybenzoylbenzotriazoles 3a-f, which showed full vasorelaxing efficacy and high potency values. The introduction of a 2-hydroxybenzyl substituent in the 1 position of the benzotriazole ring (compound 7) strongly decreased the activity, showing the importance of the electron-acceptor carbonyl function. The best compound, 3b, was further investigated, in order to evaluate the possible mechanism of action involved in the vasodilator activity. In the vascular model, different potassium channel blockers inhibited the effects of the compound, and an increase of the levels of membrane depolarisation induced a significant reduction of the recorded responses. Compound 3b was also tested in a model of isolated rat heart, retroperfused through the aorta and submitted to a global ischemia/reperfusion cycle. In such an experimental condition, 3b showed an interesting cardioprotective activity. All the above observations are in agreement with the hypothesis of a mechanism linked to the activation of potassium channels.

Full text of DOI:10.1016/S0014-827X(01)01146-6

www.elsevier.com/locate/farmac
Il Farmaco 56 (2001) 827–834
New 5-substituted-1-(2-hydroxybenzoyl)-benzotriazoles, potassium
channel activators. IV
Giuliana Biagi a, Irene Giorgi a, Oreste Livi a,*, Valerio Scartoni a, Pier Luigi Barili b,
Vincenzo Calderone c, Enrica Martinotti c
a Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Pisa, 6ia Bonanno 6, I-56126 Pisa, Italy
b Dipartimento di Bioorganica e Biofarmacia, Uni6ersita` di Pisa, 6ia Bonanno 33, I-56126 Pisa, Italy
c Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Uni6ersita` di Pisa, 6ia Bonanno 6, I-56126 Pisa, Italy
Received 20 January 2001; accepted 12 June 2001
Abstract
This paper reports the synthesis of a series of new 5-substituted-1-(2-hydroxybenzoyl)-benzotriazoles, which have been tested for
their activity as possible activators of potassium channels. In rat aortic rings, the ‘opened’ derivatives 1a–f, intermediates of
synthesis, showed vasorelaxing properties, with appreciable values of potency. However, the most remarkable effects were
recorded for the 2-hydroxybenzoylbenzotriazoles 3a–f, which showed full vasorelaxing efficacy and high potency values. The
introduction of a 2-hydroxybenzyl substituent in the 1 position of the benzotriazole ring (compound 7) strongly decreased the
activity, showing the importance of the electron-acceptor carbonyl function. The best compound, 3b, was further investigated, in
order to evaluate the possible mechanism of action involved in the vasodilator activity. In the vascular model, different potassium
channel blockers inhibited the effects of the compound, and an increase of the levels of membrane depolarisation induced a
significant reduction of the recorded responses. Compound 3b was also tested in a model of isolated rat heart, retroperfused
through the aorta and submitted to a global ischemia/reperfusion cycle. In such an experimental condition, 3b showed an
interesting cardioprotective activity. All the above observations are in agreement with the hypothesis of a mechanism linked to the
activation of potassium channels. © 2001 Elsevier Science S.A. All rights reserved.
Keywords: Salicylanilides; Benzotriazoles; Vasodilator activity; Cardioprotection; Potassium channels
reference compound NS 1619. However, the triazolyl-
benzotriazole derivatives showed higher vasodilator ac-
1. Introduction
tivity and potency than those of the corresponding
benzimidazolone derivatives, indicating the replacement
of the benzimidazolone heterocycle with the benzotria-
zole nucleus as an encouraging choice, for the develop-
ment of new potential potassium channel openers. A
further paper [3] concerning some modifications of the
structure and/or substituents of the above mentioned
benzimidazolone and benzotriazole derivatives showed
that the removal of the benzotriazole or benzimida-
zolone heterocycles and their replacement by different
moieties, such as the benzotriazinone ring, decreased
the activity of the compounds. In this work, the investi-
gation of new potential potassium channel activators
focused on the preparation of a series of 5-substituted-
1-(2-hydroxybenzoyl)-benzotriazoles with structural
In a previous paper [1], we had reported the synthesis
and pharmacological evaluation, as potential potassium
channel activators, of a series of 1,2,3-triazolyl-benzimi-
dazolone (A) and 1,2,3-triazolyl-benzotriazole (B)
derivatives (Fig. 1). These compounds had been studied
for their structural relationships with the benzimida-
zolone derivatives NS 004 and NS 1619 (Fig. 1), effec-
tive activators of the high conductance calcium
activated potassium channels (BKCa) [2]. None of the
triazolyl-benzimidazolones and triazolyl-benzotriazoles
showed any improvement, when compared with the
* Corresponding author.
E-mail address: livi@farm.unipi.it (O. Livi).
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01146-6

828
G. Biagi et al. / Il Farmaco 56 (2001) 827–834
Fig. 1. Comparison between the reference benzimidazolones NS 004 and NS 1619, and the previously synthesised triazolylbenzimidazolones (A)
and triazolylbenzotriazoles (B).
characteristics indicated by the previous experimental
evidence as important requirements for such a pharma-
cological activity.
the structural flexibility, but removed a hydrogen bond
acceptor site.
Thus, the benzotriazole was reacted with 2-nitroben-
zyl chloride in acetone solution in the presence of
anhydrous potassium carbonate to give in 82% yield the
expected mixture of the two isomeric benzotriazoles 4
and 5, substituted in the 1 and 2 positions, respectively.
2. Chemistry
The pharmacological evaluation indicated that the
activity of the benzotriazole ring was higher than that
of the benzimidazolone one [1,3]; therefore, a new
structural change involved the triazolyl-benzotriazole
derivatives and consisted of the replacement of the
1,2,3-triazole ring with a 2-hydroxybenzoyl substituent,
as reported in Scheme 1. This change combined the
adoption of a 2-hydroxyphenyl substituent, which is
present in the reference compounds NS 004 and NS
1619, with the estrangement of the same substituent
from the bicyclic ring and with the shift of the carbonyl
function out of the benzimidazolone ring. Thus, the
whole structure acquired greater flexibility because the
two molecular parts were connected by a bridge con-
sisting of the carbonyl function. In addition, the car-
bonyl placed in this position could behave as a full
hydrogen bond acceptor, whilst the behaviour was dif-
ferent in the benzimidazolone ring because a tautomeric
equilibrium might be present.
Scheme 1.
The salicylic acid chloride [4], obtained from the
acetylsalicylic acid sodium salt and thionyl chloride,
was reacted with the appropriate ortho-nitroaniline in
benzene solution to give the corresponding N-(4-substi-
tuted-2-nitrophenyl)-salicylamides 1a–f in good yields.
Reduction of the nitro group by catalytic hydrogena-
tion at room temperature and pressure provided the
salicyl-(2-amino-anilides) 2a–f, which were cyclised to
the expected 5-substituted-1-(2-hydroxybenzoyl)-benzo-
triazoles 3a–f, by treatment with nitrous acid in 50%
acetic acid solution (Scheme 1).
A structural change, useful to evaluate the impor-
tance of the carbonyl function, was achieved by the
replacement of the 2-hydroxybenzoyl substituent with a
2-hydroxybenzyl group, as reported in Scheme 2. In
fact, the introduction of a methylenic group to form a
bridge between the two parts of the molecule preserved
Scheme 2.

G. Biagi et al. / Il Farmaco 56 (2001) 827–834
829
Table 1
Physicochemical properties of compounds 1–3
Comp.
Yield (%)
Crystallisation solvent
M.p. (°C)
Mass
Elemental analysis
M+
Base
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
84
79
88
95
70
85
89
96
77
67
72
93
52
96
92
87
81
83
MeOH
MeOH
AcOEt
MeOH
MeOH
MeOH/H2O
MeOH
EtOH/H2O
MeOH/H2O
EtOH/H2O
MeOH
MeOH/H2O
MeOH
154–157
184–186
189–193
166–167
190–192
178–180
138–140
156–162
160–163
153–156
195–197
172–174
63–66
258
272
292
276
326
334
228
242
262
246
296
304
239
253
273
257
307
315
121
121
121
121
121
214
121
121
142
126
121
65
121
121
121
121
121
121
C13H10N2O4
C14H12N2O4
C13H9N2O4Cl
C13H9N2O4F
C14H9N2O4F3
C19H14N2O4
C13H12N2O2
C14H14N2O2
C13H11N2O2Cl
C13H11N2O2F
C14H11N2O2F3
C19H16N2O2
C13H9N3O2
MeOH
MeOH
MeOH
MeOH
88–90
133–135
93–95
110–112
105–107
C14H11N3O2
C13H8N3O2Cl
C13H8N3O2F
C14H8N3O2F3
C19H13N3O2
MeOH/H2O
Table 2
1H NMR data (l, ppm, J, Hz) and 13C NMR data (l, ppm) for compounds 1e, 2c, and 3f in DMSO-d6
Comp.
H-3
H-5
H-6
J3,5
J5,6
H-3%
H-4%
H-5%
H-6%
1H NMR
1e
2c
3f
8.41
6.82
8.54
8.11
6.60
8.14
8.90
7.25
8.30
2.5
2.4
2.2
8.8
8.4
8.4
7.03
6.95
7.03
7.46
6.97
6.93
6.97
7.98
7.43
8.00
a
a
13C NMR
Comp.
1e b
C-1
C-2
C-3
C-4
C-5
C-6
CꢀO
C-1%
C-2%
C-3%
C-4%
C-5%
C-6%
137.51
130.23
129.53
136.55
144.34
145.93
122.58
114.28
113.66
122.49
120.80
120.22
130.70
115.06
116.78
123.86
127.61
127.51
164.14
166.92
158.08
117.46
116.12
116.94
156.37
158.74
155.63
116.54
116.73
115.99
133.98
133.25
133.06
119.18
118.34
118.39
130.70
128.60
129.76
2c
3f c
a Signal submerged by phenyl resonances (from 7.86 to 7.44 l).
b Other signals: 125.50 (CF3).
c Other signals: 138.49, 128.64, 128.53, and 126.82 (phenyl).
The flash-chromatography of the mixture on silica gel
provided 13% of the isomer 2-(2-nitrobenzyl)-benzotria-
zole (5) followed by the 1-(2-nitrobenzyl)substituted
isomer 4 in 48% yield.
The nitrobenzyl derivative 4 was reduced by catalytic
hydrogenation to the corresponding amino derivative 6,
which was converted to the hydroxy derivative 7 by
diazotisation in 10% sulphuric acid and thermal decom-
position of the diazonium salt.
The structures of all the new compounds were as-
signed on the basis of the well-known reaction mecha-
nisms [5] and our previous evidence [1,3,6] and were
confirmed by analytical and spectroscopic methods
(Table 1). The 1H and 13C NMR spectra of some
significant compounds are reported in Table 2. In par-
ticular, the structures of compounds 4 and 5 were
assigned on the basis of the UV and 13C NMR data.
The UV spectra showed the bathochromic shift of the
benzenoid band (1Lb in the Platt notation [7]) described
for the 2-substituted-benzotriazoles with respect to the
1-substituted ones [8]: the experimental values are 253
nm for 4 and 273 nm for 5. The 13C NMR data led to
the same conclusions; in fact, the equivalence of the
three couples of carbons, 3a:7a, 4:7 and 5:6 in the
spectrum of 5 decreases the total number of the signals
to 10, whilst in the spectrum of 4 the signals are 13. In
addition, downfield shifts of $11 ppm for C-7a, of
$8 ppm for CH2 and C-7 and of $2.5 ppm for C-5 in
the spectrum of the 1-substituted compound 4, due to
the aminic nature of the 1-nitrogen, with respect to the
iminic nature of the same nitrogen in the 2-substituted
compound 5, are observed. This difference can establish

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G. Biagi et al. / Il Farmaco 56 (2001) 827–834
a good method for the structural attribution to the 1
and 2 substituted benzotriazole isomers, also in the
presence of a second substituent on the benzotriazole
phenyl ring. In this case, the second substituent causes
loss of symmetry to the 2-substituted benzotriazole
isomer, invalidating the above-mentioned assignment
criterion.
Besides, the role of the heart potassium channel in
the protection of the myocardium against the ischemia-
induced injury has been well assessed [12]. Therefore,
also the model of rat isolated and perfused heart,
submitted to appropriate ischemia/reperfusion cycles,
has been chosen as a pharmacological test.
3. Pharmacology
4. Results and discussion
Large conductance calcium-activated potassium
channels (BKCa) are involved in the modulation of
vascular smooth muscle tone in different circulatory
districts, such as the rabbit pulmonary artery [9], the rat
portal vein [10] and the rat aorta [11]. Indeed, the
membrane hyperpolarisation induced by an opening of
outward potassium-channels causes the inactivation of
voltage-operated calcium channels, the lowering of the
concentration of free intracellular calcium and conse-
quently a vasorelaxing effect. Thus, the functional eval-
uation of a vasorelaxing activity of the compounds
tested on isolated rat aortae can be considered a reli-
able preliminary screening method, to detect a possible
potassium channel opening effect.
The pharmacological study was aimed at investigat-
ing the potential vasorelaxing properties of the benzo-
triazole derivatives (3a–f and 7), representing the target
compounds of the whole synthetic work; however, it
seemed interesting to screen also the 2-nitro-salicy-
lanilides (1a–f), because of their close similarity with
the 5-(2%-nitroanilino)-1,2,3-triazoles, ‘opened’ structure
molecules which showed profiles of possible potassium
channel activators [13].
As indicated in Table 3, all the tested compounds
possessed full vasorelaxing efficacy. The order of mag-
nitude of the potency parameters for the ‘opened’ com-
pounds 1a–f was interesting and almost comparable
with that exhibited by the reference compound. The
potency values observed for the salicylbenzotriazoles
3a–f was very satisfactory; indeed, they were all higher
than that of NS 1619. The replacement of the carbonyl
group with a methylene bridge led to a significant
decrease of potency, as revealed by the comparison
between compound 7 (possessing the methylene bridge)
and its analogue 3a (possessing the carbonyl bridge).
The best compound, 3b, underwent further experi-
mental protocols to understand both its possible mech-
anism of action and to evaluate its possible protective
effects in the ischemic myocardium.
In the vascular model, the responses induced by 3b
on aortic smooth muscle were reduced by increased
levels of depolarisation. The consequent hypothesis of a
potassium channel opening mechanism was further
strengthened by the antagonism exerted by TEA,
quinine and 4-aminopyridine (4-AP), compounds able
to induce a significant rightward shift of the concentra-
tion–vasorelaxing response curves. The substantial in-
effectiveness of glibenclamide to antagonise the effects
of 3b seemed to exclude the involvement of the ATP-
sensitive potassium channel type (these results are sum-
marised in Table 4).
Table 3
Vasorelaxing potency and efficacy exhibited by the tested compounds
in aortic preparations pre-contracted by KCl 20 mM
Comp.
pIC50
Efficacy (%)
1a
1b
1c
1d
1e
1f
3a
3b
3c
3d
3e
3f
5.5190.034
5.6090.015
5.1690.029
4.9690.053
5.4690.052
5.2390.092
5.8190.047
6.8490.098
5.6190.056
5.5290.030
5.6790.089
5.4890.036
4.7190.015
5.3090.068
100
100
100
100
100
100
100
100
100
100
100
100
100
100
7
NS 1619
Table 4
Potency and efficacy parameters recorded for the selected compound
3b in control condition, in the presence of increased membrane
depolarisation (60 mM KCl) or after the administration of several
potassium channel blockers
In control hearts, the residual inotropic tension de-
veloped after an ischemia/reperfusion cycle dramati-
cally decreased (1498% of the pre-ischemic inotropic
tension). Compound 3b exhibited cardio-protective
properties against the ischemic injury: the hearts
reached levels of post-ischemic inotropic tension of
45910 and 62911% (of the pre-ischemic inotropic
tension), in the organs treated with 0.1 and 1 mM of 3b,
respectively. These values were almost equivalent to
Treatment
pIC50
Efficacy (%)
Control
60 mM KCl
+1 mM TEA
+200 mM Quinine
+3 mM 4-AP
+1 mM Glibenclamide
6.8490.098
5.6390.016
6.4590.014
5.6090.017
5.3890.019
6.7890.082
100
6596
100
9593
9792
100

G. Biagi et al. / Il Farmaco 56 (2001) 827–834
831
47912 and 56913%, recorded in the hearts treated
with 0.1 and 1 mM, respectively, of the reference cardio-
protective drug aprikalim.
5.1.2. N-(2-Aminophenyl)-salicylamide (2a),
N-(4-fluoro-2-aminophenyl)-salicylamide (2d),
N-(4-trifluoromethyl-2-aminophenyl)-salicylamide (2e)
and N-(2-aminobiphenyl)-salicylamide (2f)
Ten percent of Pd/C (30–35 mg) was added to a
solution of 1.0 mmol of the suitable nitro derivative
(1a, 1d, 1e or 1f) in 60 ml of AcOH and the mixture
was hydrogenated at room temperature and pressure.
The catalyst was filtered off, washed with AcOH and
the filtrate was evaporated in vacuo. The crude residue
was treated with 5% NaOH, paper filtered and the
filtrate acidified to give the title compounds (Table 1).
5. Experimental protocols
5.1. Chemistry
Melting points were determined on a Kofler hot-
stage and are uncorrected. IR spectra in Nujol mulls
were recorded in a Mattson Genesis series FTIR spec-
trometer. UV spectra were obtained in a Varian Cary 1
5.1.3. N-(4-Methyl-2-aminophenyl)-salicylamide (2b)
Ten percent of Pd/C (150 mg) was added to a
solution of 0.850 g (3.12 mmol) of the nitro derivative
1b in 500 ml of MeOH and the mixture was hydro-
genated at room temperature and pressure. The catalyst
was filtered off, washed with boiling MeOH and the
filtrate was evaporated in vacuo to give the title com-
pound (Table 1).
UV–Vis spectrophotometer. H and 13C NMR spectra
1
were recorded with a Bruker AC 200 spectrometer
(compounds 1e, 2c, 3f, 4 and 5) and with a Varian
Gemini 2000 spectrometer (other compounds) in
DMSO-d6 in l units, using TMS as an internal stan-
dard. Mass spectra were performed with a Hewlett
Packard MS/System 5988. Elemental analyses (C, H,
N) were within 90.4% of the theoretical values and
were performed in a Carlo Erba Elemental Analyser
Mod. 1106 apparatus. TLC data were obtained with a
Riedel de Haen, 37360 DC-Karten F254, 0.2 mm, elut-
5.1.4. N-(4-Chloro-2-aminophenyl)-salicylamide (2c)
Wet Ni-Raney (100 mg) was added to a solution of
0.500 g (1.70 mmol) of the nitro derivative 1c in 100 ml
of MeOH and the mixture was hydrogenated and
worked up as described for the preparation of 2a (Table
1).
ing with
a 1:3 AcOEt–petroleum ether mixture.
Petroleum ether corresponds to the fraction boiling at
40–60 °C.
5.1.5. 5-Substituted-1-(2-hydroxybenzoyl)-
benzotriazoles 3a–f
5.1.1. N-(4-Substituted-2-nitrophenyl)-salicylamides
1a–f
To an ice-cooled ($5 °C) and stirred solution of 2.0
mmol of the appropriate amino derivative (2a, 2b, 2c,
2d, 2e or 2f) in 20–30 ml of 50% AcOH, 0.165 g (2.40
mmol) of NaNO2 was added portionwise. The ice-bath
was removed and stirring was continued for 2 h. The
suspension obtained was further diluted with H2O and
the precipitate, consisting of the title compounds, was
collected by filtration and washed with H2O (Table 1).
To 6 ml of SOCl2, ice-cooled and stirred, 1.6 g (7.92
mmol) of sodium acetylsalicylate was added. After 20
min the ice-bath was removed and stirring was contin-
ued at room temperature for 2 h. The solvent was
evaporated in vacuo and the gelatinous residue, consist-
ing of the acid chloride, was dissolved in 40 ml of
anhydrous benzene. The appropriate 2-nitroaniline
(7.92 mmol) (2-nitroaniline, 4-methyl-2-nitroaniline, 4-
chloro-2-nitroaniline, 4-fluoro-2-nitroaniline, 4-trifluo-
romethyl-2-nitroaniline or 2-nitrobiphenylamine [14])
was added to the solution and the mixture was heated
under reflux for 16 h. The solvent was evaporated and
the residue was dissolved again in CHCl3. This solution
was extracted with 4% NaHCO3, 5% HCl and 10%
NaOH. By acidification of the aqueous alkaline extract
the title compounds were precipitated, which were col-
lected by filtration and washed with H2O (Table 1).
During the extraction with 10% NaOH a variable
amount of the title compounds could precipitate as a
sodium salt; in this case the precipitate was collected by
filtration, suspended or dissolved in H2O and acidified
to give the desired compound. The remaining product,
as a salt soluble in NaOH, was isolated in the usual
manner.
5.1.6. 1-(2-Nitrobenzyl)-benzotriazole (4) and
2-(2-nitrobenzyl)-benzotriazole (5)
Anhydrous K2CO3 ($2.0 g) was added to a solution
of benzotriazole (1.00 g, 8.40 mmol) and 2-nitrobenzyl
chloride (1.45 g, 8.45 mmol) in 40 ml of anhydrous
acetone and the mixture was heated under reflux for 20
h. The solvent was evaporated in vacuo and the residue
was treated with H2O and extracted with CHCl3. The
organic layer was dried and evaporated to give a solid
white residue (2.09 g, yield 82%), consisting of a mix-
ture of the two isomers 4 and 5 (TLC analysis: Rf=
0.34 and 0.54, respectively). The mixture was separated
by flash-chromatography through silica gel, eluting
with a 2:5 mixture of AcOEt–petroleum ether (40–
60 °C), at first the isomer 5 and then the isomer 4.

832
G. Biagi et al. / Il Farmaco 56 (2001) 827–834
4: 1.227 g, yield 48%, m.p. 114–116 °C from MeOH.
The pharmacological study was performed with the
following vascular procedure. Furthermore, a selected
compound was also tested as cardioprotective agent in
an ischemia/reperfusion protocol on isolated rat heart.
Anal. C13H10N4O2 (C, H, N). MS; m/z: 254 [M+], 106
1
[100]. UV (ethanol): umax 216 nm (log m 3.73) La band;
umax 253 nm (log m 3.70) 1Lb band. 1H NMR (DMSO, l,
ppm): 6.35 (2H, s, CH2); 6.84 (1H, H-6%); 7.42 (1H,
H-6); 7.55 (1H, H-5); 7.59 (1H, H-4%); 7.65 (1H, H-5%);
7.85 (1H, H-4); 8.09 (1H, H-7); 8.17 (1H, H-3%). 13C
NMR (DMSO, l, ppm): 48.4 (CH2); 110.7 (C-7); 119.3
(C-4); 124.2 (C-5); 125.2 (C-3%); 127.7 (C-6); 129.3 (C-
4%); 129.5 (C-6%); 130.9 (C-1%); 133.2 (C-7a); 134.3 (C-5%);
145.2 (C-3a); 147.5 (C-2%).
5.2.1. Vascular protocol
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 imme-
diately 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), ther-
mostated 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 micro-
dynamometer (Basile mod. 7050).
5: 0.324 g, yield 13%, m.p. 86–88 °C from MeOH;
Anal. C13H10N4O2 (C, H, N). UV (ethanol): umax 216
1
1
nm (log m 3.78) La band; umax 273 nm (log m 3.80) Lb
1
band. H NMR (DMSO, l, ppm): 6.36 (2H, s, CH2);
7.29 (1H, H-6%); 7.43 (2H, H-5 and H-6); 7.65 (1H,
H-4%); 7.74 (1H, H-5%); 7.91 (2H, H-4 and H-7); 8.16
(1H, H-3%). 13C NMR (DMSO, l, ppm): 56.6 (CH2);
117.9 (C-4 and C-7); 125.1 (C-3%); 126.7 (C-5 and C-6);
129.5 (C-1%); 130.4 (C-4%); 131.6 (C-6%); 134.3 (C-5%);
143.8 (C-3a and C-7a); 147.9 (C-2%).
5.1.7. 1-(2-Aminobenzyl)-benzotriazole (6)
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-
induced contraction was considered representative of
an acceptable lack of the endothelial layer, while the
organs, showing a relaxation ]20% (i.e. significant
presence of the endothelium), were not used in the
experimental procedures. Thirty to forty minutes after
confirmation of the endothelium removal, the aortic
preparations were contracted by treatment with a single
concentration of KCl (20 mM) and when the contrac-
tion reached a stable plateau, threefold increasing con-
centrations of the compounds (10 nM–1 mM) were
added cumulatively. In parallel sets of experiments, to
investigate the influence of a higher level of depolarisa-
tion on the responses evoked by the compound tested,
the aortic preparations were contracted by 60 mM KCl.
Then, threefold increasing concentrations of the com-
pounds (10 nM–1 mM) were added cumulatively.
Preliminary experiments showed that both the KCl
(20 and 60 mM)-induced contractions remained con-
stant in a stable tonic state for at least 40 min.
Ten percent of Pd/C (0.100 g) was added to a solu-
tion of 1.20 g (4.72 mmol) of the nitro isomer 4 in 200
ml of MeOH and the mixture was hydrogenated at
room temperature and pressure. The catalyst was
filtered off, washed with MeOH and the combined
filtrate was evaporated in vacuo to give the title com-
pound: 0.986 g, yield 93%, m.p. 104–106 °C from H2O.
Anal. C13H12N4 (C, H, N). IR (cm−1): 3434 and 3351
(NH2). MS; m/z: 224 [M+], 106 [100].
5.1.8. 1-(2-Hydroxybenzyl)-benzotriazole (7)
To a stirred solution of 0.500 g (2.23 mmol) of the
amino derivative 6 in 10 ml of 75% H2SO4, a solution
of NaNO2 (0.185 g, 2.68 mmol) in 5 ml of H2O was
added dropwise. After 30 min the solution was gradu-
ally heated up to 90–95 °C to complete the evolution
of N2 gas (begun at temperature \60 °C). The solu-
tion assumed a red colour and the brown oil that
separated was extracted with CHCl3. The chloroform
layer was extracted with 10% NaOH and the alkaline
solution was paper filtered and acidified (pH$3) to
precipitate the title compound which was collected by
filtration and washed with H2O: 0.302 g, yield 60%,
m.p. 165–167 °C from H2O. Anal. C13H11N3O (C, H,
N). IR (cm−1): 3376 (OH). MS; m/z: 225 [M+], 107
[100].
In other sets of experiments, the potassium channel
blockers tetraethylammonium chloride (TEA, 1 mM),
quinine hydrochloride (200 mM), 4-aminopyridine (4-
AP, 3 mM) or glybenclamide (1 mM) were added,
before the KCl (20 mM)-induced contraction, followed
by the administration of selected compounds.
5.2. Pharmacology
Norepinephrine hydrochloride, acetylcholine chlo-
ride, TEA, quinine hydrochloride, 4-AP and KCl were
dissolved in bi-distilled water. Glibenclamide was dis-
solved by sonication in aqueous NaOH (0.1 N). All the
All the experimental procedures were carried out
following the guidelines of the European Community
Council Directive 86-609.

G. Biagi et al. / Il Farmaco 56 (2001) 827–834
833
other synthesised derivatives and the reference com-
pound NS 1619 (RBI) were dissolved (10 mM) in
aqueous NaOH (0.1 N). All compounds were dissolved
at the concentration of 1 mM and further dilutions
were performed in bi-distilled water. All solutions were
prepared immediately before the pharmacological ex-
perimental procedures. Previous experiments showed a
complete ineffectiveness of the administration of the
vehicle.
contractile responses evoked by the two different con-
centrations of KCl, since the concentration 20 mM
could substantially induce a maximal effect, in endothe-
lium denuded aortic rings.
The parameter of potency of the vasorelaxing effects
was expressed as pIC50, representing the negative loga-
rithm of the vasodilator molar concentration determin-
ing a half reduction of the contractile tone induced by
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.
5.2.2. Cardiac protocol
Normotensive male Wistar rats (250–350 g) were
killed by cervical dislocation, under light ether anaes-
thesia. Immediately, the heart (connected with a por-
tion of thoracic aorta) was excised from the
mediastinum and rapidly perfused through a retrograde
cannulation of the aorta. The pressure of the perfusion
flow was constantly kept at 90 mmHg, to ensure the
opening of the aortic valve. The perfusion solution was
Tyrode buffer (composition 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 and richly bubbled with O2.
The inotropic tension developed by the sponta-
neously beating hearts was recorded by means of an
isometric transducer (Basile mod. 7005), connected with
a unirecord microdynamometer (Basile mod. 7050).
After 1 h of equilibration time (pre-ischemic period),
the perfusion was suspended and the hearts underwent
a 30 min period of global ischemia. Then, the organs
were re-perfused for 30 min. The hearts, showing sig-
nificant arrhythmic episodes during the equilibration
time, were discarded.
Together with the control group of heart (receiving
the standard Tyrode solution), a second group received
the selected synthesised compound (1 or 0.1 mM, dis-
solved in the perfusion Tyrode solution). A third group
of hearts underwent the administration of the reference
drug aprikalim (1 or 0.1 mM, dissolved in the perfusion
Tyrode solution), a KATP potassium channel opener,
possessing well-established cardioprotective properties
[15].
The statistical comparison of experimental data was
performed by the two-tailed Student’s t-test and
ANOVA. A value of PB0.05 was considered as repre-
sentative of significant differences.
5.2.3.2. Cardiac protocol. The inotropic tension devel-
oped by the hearts at the end of the re-perfusion period
was measured and expressed as a percentage of the
inotropic tension developed by the hearts at the end of
the pre-ischemic period. The values of percentage are
reported as mean9SEM.
The statistical comparison of experimental data was
performed by the two-tailed Student’s t-test and
ANOVA. A value of PB0.05 was considered as repre-
sentative of significant differences.
References
[1] B. Baragatti, G. Biagi, V. Calderone, I. Giorgi, O. Livi, E.
Martinotti, V. Scartoni, Triazolyl-benzimidazolones and tria-
zolyl-benzotriazoles: new potential potassium channel activators.
II, Eur. J. Med. Chem. 35 (2000) 949–955.
[2] S.P. Olesen, E. Munch, P. Moldt, J. Drejer, Selective activation
of Ca(2+)-dependent K+ channels by novel benzimidazolone,
Eur. J. Pharmacol. 251 (1994) 53–59.
[3] G. Biagi, V. Calderone, I. Giorgi, O. Livi, V. Scartoni, B.
Baragatti, E. Martinotti, Some structural changes on triazolyl-
benzotriazoles and triazolyl-benzimidazolones as potential potas-
sium channel activators. III, Farmaco 56 (2001) 841–849.
[4] E. Kopetschni, L. Karczag, Uber die darstellung von salicyl-
saure-chlorid, Berichte 47 (1914) 235–237.
[5] G.P. Ellis (Ed.), Synthesis of Fused Heterocycles, Part 2, Wiley,
New York, 1992.
5.2.3. Data analysis
[6] G. Biagi, I. Giorgi, O. Livi, V. Scartoni, S. Velo, P.L. Barili,
New 4-(benzotriazol-1-yl)-1,2,3-triazole derivatives, J. Hetero-
cycl. Chem. 33 (1996) 1847–1853.
[7] J.R. Platt, Classification of spectra of cata-condensed hydrocar-
bons, J. Chem. Phys. 17 (1949) 484–491.
[8] F.R. Benson, The High Nitrogen Compounds, Wiley, New
York, 1984, 308 pp.
[9] C. Vandier, P. Bonnet, Synergistic action of NS-004 and internal
Ca2+ concentration in modulating pulmonary artery K+ chan-
nels, Eur. J. Pharmacol. 295 (1996) 53–60.
5.2.3.1. Vascular protocol. The efficacy of the vasorelax-
ing responses was expressed as a maximal relaxant
effect (Emax), calculated as a percentage of the contrac-
tile tone developed by the smooth muscle preparation,
after the depolarising stimulus induced by KCl 20 mM.
The above parameters were calculated by means of
non-linear regression analysis of the sigmoidal concen-
tration–response curves (computer program: GRAPH-
PAD PRISM), and were expressed as the mean of six
experiments. Previous experiments demonstrated an al-
most complete quantitative equivalence between the
[10] G. Edwards, A. Niederste-Hollenberg, J. Schneider, T. Noack,
A.H. Weston, Ion channel modulation by NS 1619, the putative
BKCa channel opener, in vascular smooth muscle, Br. J. Phar-
macol. 113 (1994) 1538–1547.

834
G. Biagi et al. / Il Farmaco 56 (2001) 827–834
[11] C.A. Sargent, G.J. Grover, M.J. Antonaccio, J.R. McCullough,
The cardioprotective, vasorelaxant and electrophysiological
profile of the large conductance calcium-activated potassium
channel opener NS-004, J. Pharmacol. Exp. Ther. 266 (1993)
1422–1429.
[12] D. Escande, I. Cavero, Potassium channel openers in the heart,
in: D. Escande, N. Standen (Eds.), K+ Channels in Cardiovas-
cular Medicine, Springer, Paris, 1993, pp. 225–244.
[13] G. Biagi, V. Calderone, I. Giorgi, O. Livi, V. Scartoni, B.
Baragatti, E. Martinotti, 5-(4%-Substituted-2%-nitroanilino)-1,2,3-
triazoles as new potential potassium channel activators. I, Eur. J.
Med. Chem. 35 (2000) 715–720.
[14] F. Fichter, A. Sulzberger, Ueber das phenyl-benzochinon und
einige derivate des byphenyls, Berichte 37 (1904) 878–881.
[15] D. Escande, I. Cavero, K+ channel openers and ‘natural’ cardio-
protection, Trends Pharmacol. Sci. 13 (1992) 269–272.