Some structural changes on triazolyl-benzotriazoles and triazolyl-benzimidazolones as potential potassium channel activators. III

Article abstract of DOI:10.1016/S0014-827X(01)01148-X

This paper reports the synthesis and pharmacological evaluation of some compounds, obtained by structural modifications of 1,2,3-triazolyl-benzotriazoles and 1,2,3-triazolyl-benzimidazolones, which had shown activity as potential activators of the big-con

Full text of DOI:10.1016/S0014-827X(01)01148-X

www.elsevier.com/locate/farmac
Il Farmaco 56 (2001) 841–849
Some structural changes on triazolyl-benzotriazoles and
triazolyl-benzimidazolones as potential potassium channel
activators. III
Giuliana Biagi a, Vincenzo Calderone a, Irene Giorgi a, Oreste Livi a,*,
Valerio Scartoni a, Barbara Baragatti b, Enrica Martinotti b
a Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Pisa, 6ia Bonanno 6, 56126 Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Uni6ersita` di Pisa, 6ia Bonanno 6, 56126 Pisa, Italy
Received 20 January 2001; accepted 12 June 2001
Abstract
This paper reports the synthesis and pharmacological evaluation of some compounds, obtained by structural modifications of
1,2,3-triazolyl-benzotriazoles and 1,2,3-triazolyl-benzimidazolones, which had shown activity as potential activators of the
big-conductance calcium-activated potassium channels (BKCa). Changes have concerned the introduction of a hinderer substituent
in the 5-position of the benzimidazolone (4a, b) and benzotriazole (5a, b) rings, opening of the benzimidazolone ring (7) and
substitution of the 1,2,3-triazole ring with a 2-hydroxyphenyl ring (10). Furthermore a series of 3-aryl-benzotriazin-4-one
derivatives (13a–e) has been studied, which appears as a modification and/or combination of the benzimidazolone and
benzotriazole rings. Only compound 10 shows interesting activity, while the other structural modifications either do not increase
(compounds 4 and 5) or reduce (compounds 7 and 13) the pharmacological activity. However, these results provide useful
information about structure–activity relationships. © 2001 Elsevier Science S.A. All rights reserved.
Keywords: 1,2,3-Triazoles; Benzimidazolones; Benzotriazoles; Benzotriazin-4-ones; Potassium channels
1. Introduction
these derivatives possessed a good vasodilator activity,
probably induced by the potassium channel activation.
The triazolyl-benzotriazole derivatives showed an effec-
tiveness generally higher than that of the corresponding
triazolyl-benzimidazolones and the activity of some com-
pounds was comparable to that of NS 1619 tested as
standard.
On the basis of these results, we took into consideration
the introduction of some structural changes on the
triazolyl-benzimidazolone (A) or triazolyl-benzotriazole
(B) derivatives, in order to increase their effectiveness
and/or potency towards potassium channels and to
deduce some useful structure–activity relationships.
Thus, a methyl substituent in the 5-position of the
benzimidazolone or benzotriazole ring [1] appeared more
effective than the other experimented substituents (Cl, F,
CF3, OMe); therefore the first structural change consisted
in an increase of both the steric hindrance and the
lipophilicity of this substituent, by the introduction of two
new substituents such as a sec-butyl and a phenyl group.
In a previous paper [1] we reported the synthesis and
pharmacological evaluation as potassium channel activa-
tors, of a series of 1-triazolyl-benzimidazolones (A) and
of the corresponding series of 1-triazolyl-benzotriazoles
(B), bearing a substituent (Me, OMe, Cl, F and CF3) in
the 5-position (Fig. 1). These compounds had been
prepared for their clear structural correlation with the
benzimidazolone derivatives NS 004 and NS 1619 (Fig.
1), two known activators of the big conductance calcium-
activated potassium channel subtype (BKCa) [2], consid-
ered an interesting target for therapeutic approaches in
several cardiovascular, central nervous system and res-
piratory pathologies.
The pharmacological evaluation carried out on isolated
vascular smooth muscle preparations had shown that
* 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)01148-X

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G. Biagi et al. / Il Farmaco 56 (2001) 841–849
In addition, a formal opening of the benzimidazolone
substituted derivatives 4a and 5a were obtained in good
yield. The benzimidazolone compound 4a was prepared
by the reaction of 3a with phosgene in pyridine solu-
tion, whilst the analogous benzotriazole compound 5a
by the diazotisation reaction of 3a. For the synthesis of
the corresponding 5-phenylsubstituted derivatives 4b
and 5b, at first the new 2-nitro-biphenylazide (1b) was
prepared by diazotisation of 2-nitro-biphenylamine [4]
and treatment of the diazonium salt with sodium azide.
The 1,3-dipolar cycloaddition reaction of 1b to cyan-
acetamide provided the expected 5-(2-nitrobiphenyl-
amino)-1,2,3-triazole derivative 2b, which was reduced
to the corresponding 5-(2-aminobiphenylamino)-1,2,3-
triazole derivative 3b, by catalytic hydrogenation. Start-
ing from 3b, as described above for 3a, the
5-phenyl-benzimidazolone derivative 4b was obtained
by the reaction with phosgene, whilst the 5-phenyl-ben-
zotriazole 5b was obtained by a diazotisation reaction.
The 4-carboxamido-5-amino-1,2,3-triazole (6) [5], ob-
tained in low yield from azidoformate and cyan-
ring led to the design of N-phenyl-N%-triazolyl urea
moiety; while, the change of the 1,2,3-triazole sub-
stituent in the 1-position of the benzotriazole ring with
a 2-hydroxyphenyl substituent furnished a 1-(2%-hy-
droxy)-phenyl-benzotriazole, closely related to the ref-
erence compounds NS 004 and NS 1619.
Finally, the replacement of the benzotriazole and
benzimidazolone heterocycles by a benzotriazinone nu-
cleus represented a structural change, possessing a par-
tial combination of the two fundamental structures
(benzotriazole and benzimidazolone) by an enlargement
of the heterocyclic ring.
2. Chemistry
Starting from the 4-carboxamido-5-(4-sec-butyl-2-
aminoanilino)-1,2,3-triazole (3a) (Scheme 1), previously
described in the literature [3], the desired 5-sec-butyl-
Fig. 1. Comparison and previously prepared compounds.
Scheme 1.

G. Biagi et al. / Il Farmaco 56 (2001) 841–849
843
lytic hydrogenation. The thermic decomposition of 9
diazonium salt in aqueous sulfuric acid provided the
expected phenolic derivative 10 in 28% yield. The same
compound was also obtained in 54% yield by a direct
reaction between 2-hydroxyphenylazide [8] and benzine
[7].
Scheme 2.
In Scheme 4 the synthesis of a series of 3-arylsubsti-
tuted-benzotriazin-4-one derivatives 13a–e is reported.
The synthesis was carried out according to a procedure
described in the literature [9,10].
Thus the appropriate 2-nitrobenzoyl chloride in
refluxing benzene solution reacted with the suitable
aniline to give the expected anilides 11a–e in good
yields. These nitroanilides were reduced to the corre-
sponding aminoanilides 12a–e, by catalytic hydrogena-
tion at room temperature and pressure. Finally, by a
diazotisation reaction, the aminoanilides 12a–e under-
went an intramolecular cyclisation to give the expected
3-aryl-benzotriazin-4-ones 13a–e in high yield.
The structures of all the new compounds were as-
signed on the basis of the well-known reaction mecha-
nisms [9–12], our previous evidence [1,3,13,14] and
were confirmed by analytical and spectroscopic meth-
ods. 1H NMR spectra of the compounds tested are
reported in Table 3.
Scheme 3.
acetamide, was reacted with meta-trifluoromethyl-
phenylisocyanate in refluxing acetone (Scheme 2). The
expected asymmetrical disubstituted urea 7 was isolated
in 31% yield together with the symmetrical N,N%-(meta-
trifluoromethyl-phenyl)-urea as a by-product.
The 1-(2-hydroxyphenyl)-benzotriazole (10) was pre-
pared by the following two routes (Scheme 3). The
2-nitrophenylazide [6] reacted with benzine, obtained in
situ from anthranilic acid and isoamyl nitrite [7], to give
the 1-(2-nitrophenyl)-benzotriazole (8) which was con-
verted to the corresponding aminoderivative 9 by cata-
3. Pharmacology
Large conductance calcium-activated potassium
channels (BKCa) play an essential function in modulat-
ing the vascular smooth muscle tone, as experimentally
shown in different circulatory districts, such as the
Scheme 4.

844
G. Biagi et al. / Il Farmaco 56 (2001) 841–849
Table 1
Physico-chemical properties of compounds 4, 5, 7–10
Comp.
Yield (%)
Crystal solvent
M.p. (°C)
Analysis
Mass m/z
M+
Base
4a
4b
5a
5b
7
8
9
10
39
53
66
72
31
48
DMF–H2O
AcOEt
MeOH
MeOH–H2O
EtOH–H2O
i-PrOH
203–205
150–153
140–143
190–196
\350
120–121
130–132
196–198
C14H16N6O2
C16H12N6O2
C13H15N7O
C15H11N7O
C11H9N6O2F3
C12H8N4O2
300
320
161
210
44
234
161
50
229 (M+−Bu)
305
314
240
210
211
90
MeOH
EtOH–H2O
C
12H10N4
39
154
a
C12H9N3O
a The yields are 28 and 54% with the procedures A and B, respectively.
Table 2
Physico-chemical properties of compounds 11–13a–e
Comp.
Yield (%)
Crystal solvent
M.p. (°C)
Analysis
Mass m/z
M+
Base
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
81
62
81
60
84
95
93
98
90
95
95
96
93
87
93
MeOH
MeOH
154–156
262–265
161–162
215–216
163–164
131–134
234–236
133–135
136–138
150–153
189–190
165–175 dec
184–187
205–207
176–179
C14H12N2O4
C14H9N2O5Cl
272
320
276
292
276
242
290
246
262
246
253
301
257
273
257
122
150
92
108
92
120
120
92
154
92
105
121
77
245
77
MeOH–H2O
EtOH–H2O
MeOH–H2O
MeOH–H2O
MeOH–H2O
MeOH–H2O
MeOH–H2O
MeOH–H2O
EtOH
C13H9N2O3Cl
C13H9N2O4Cl
C13H9N2O3Cl
C14H14N2O2
C14H11N2O3Cl
C13H11N2OCl
C13H11N2O2Cl
C13H11N2OCl
C14H11N3O2
C14H8N3O3Cl
C13H8N3OCl
EtOH–H2O
MeOH
EtOH
C
13H8N3O2Cl
MeOH
C13H8N3OCl
rabbit pulmonary artery [15], the rat portal vein [16]
and the rat aorta [17]. Indeed, the membrane hyperpo-
larisation induced by an opening of outward potassium
channels is a crucial factor which determines the inacti-
vation of voltage-operated calcium channels, the lower-
ing of the concentration of free intracellular calcium
and consequently a vasorelaxing effect. Thus, the func-
tional evaluation of a vasorelaxing activity of the com-
pounds tested on isolated rat aortae was chosen as a
preliminary screening method, to unmask a possible
potassium channel opening effect.
zoles 5a, b consisted of an almost full abolition of the
contractile tone induced by the depolarising stimulus
(administration of 20 mM KCl), with potency orders of
magnitude a little lower than that recorded for the
reference compound NS 1619. Also the benzimida-
zolone 4b induced an almost full vasorelaxation, but
with a lower level of potency. Compound 4a showed a
very low efficacy (:50%) that made it impossible to
calculate the potency value. The differences of activity
between the two etherocyclic rings agreed with the
previous results [1]. A preliminary investigation of the
potential potassium channel opening mechanism of ac-
tion was also performed on the isolated vessels. It is
widely known that drugs, acting through potassium
channel activation, undergo a dramatic reduction of
potency and efficacy, when tested under experimental
conditions of increased membrane depolarisation. This
‘depolarisation-sensitive’ vasorelaxing activity is a typi-
cal and characteristic profile of potassium channel
openers, among the several classes of vasodilators [18].
4. Results and discussion
In agreement with the previous experimental obser-
vations about the pharmacological properties of tria-
zolyl-benzimidazolones and triazolyl-benzotriazoles [1],
compounds 4a, b and 5a, b showed vasorelaxing effects
(Table 4). The biological responses to the benzotria-

G. Biagi et al. / Il Farmaco 56 (2001) 841–849
845
Table 3
existence has already been suggested by the literature
[17]. It is interesting to observe that the potency values
of sec-butyl 5a and phenyl 5b substituted benzotriazoles
were almost comparable; furthermore, they were not
significantly different from that previously recorded for
the methyl-substituted benzotriazole (pIC50=4.869
0.21; Emax=100) [1].
The evaluation of the pharmacological properties of
the asymmetrical disubstituted urea 7 suggested that the
formal opening of the benzimidazolone ring led to a
substantial decrease of activity.
Compound 10, bearing the replacement of the tria-
zolyl group in the 1-position of the benzotriazole moi-
ety with a 2-hydroxyphenyl substituent, showed good
vasorelaxing properties with full efficacy and satisfac-
tory potency. Indeed, 10 was almost tenfold more po-
tent than the analogous triazolyl-benzotriazole (with a
hydrogen atom in the 5-position) previously tested
(pIC50=3.9590.059; Emax=100) [1] and its activity
was significantly reduced both by high levels of mem-
brane depolarisation (60 mM KCl) and by TEA, indi-
cating the possibly pharmacodynamic profile of
potassium channel openers (Table 4).
1H NMR data (l, ppm) in DMSO-d6 for the compounds tested
4a
4b
5a
0.80–2.78 (m, 9H, sec-Bu), 7.10–8.14 (m, 5H,
benzimidazolone+NH2), 10.1 (NH)
6.97–8.10 (m, 10H, Ph+benzimidazolone+NH2), 8.8 and
10.7 (NH)
0.72–2.95 (m, 9H, sec-Bu), 7.47–8.24 (m, 3H,
benzotriazole), 7.25 (NH2), 9.4 (NH)
7.03–8.20 (m, 10H, Ph+benzotriazole+NH2), 12.2 (NH)
7.43 (dd, 1H, 4-H), 7.53 (d, 1H, 2-H), 8.06 (d, 1H, 5-H),
9.68, 10.5, 15.2 (NH and NH2)
7.00–7.28 (m, 2H, substituent), 7.41–7.68 (m, 5H,
benzotriazole+substit.), 8.17 (m, 1H, benzotriazole), 10.4
(OH)
6.90–7.24 (m, 3H, substituent), 7.90–8.33 (m, 4H,
benzotriazine), 2.28 (s, 3H, CH3), 9.7 (OH)
7.76–8.38 (m, 7H, benzotriazine+substit.), 13.3 (COOH)
7.53–7.68 (m, 5H, Ph), 8.01 (dd, 1H, 6-H), 8.31 (d, 1H,
5-H), 8.42 (d, 1H, 8-H)
6.92–7.10 (m, 2H, substituent), 7.32–7.46 (m, 2H,
substit.), 8.00 (dd, 1H, 6-H), 8.29 (d, 1H, 5-H), 8.41 (d,
1H, 8-H), 10.0 (OH)
7.51–7.69 (m, 5H, Ph), 8.17 (dd, 1H, 7-H), 8.29 (d, 1H,
5-H), 8.31 (d, 1H, 8-H)
5b
7
10
13a
13b
13c
13d
13e
Therefore, compounds 4a, b and 5a, b were also tested
on isolated aortae, whose contractile tone was induced
by a higher level of membrane depolarisation (due to
the administration of 60 mM KCl). Under these condi-
tions, significant decreases of potency and efficacy
could be observed, fitting the profile of response ex-
pected for a potassium channel opener. Moreover, the
responses induced by compounds 5a, b were markedly
antagonised by the potassium channel blocker te-
traethylammonium chloride (TEA, Table 4). As previ-
ously observed, the reference compound NS 1619 did
not show any significant decrease of activity, when
administered to preparations pre-contracted by 60 mM
KCl. However, this anomalous behaviour is probably
due to different ancillary mechanisms of actions, whose
On the contrary, all the benzotriazinone compounds
13a–e failed to induce a significant vasorelaxing re-
sponse, indicating a possible strong incompatibility be-
tween this heterocyclic ring and the required
pharmacophoric model, well represented by the benzo-
triazole nucleus.
5. Experimental
5.1. Chemistry
Melting points (m.p.) were determined on a Kofler
hot-stage and are uncorrected. IR spectra in Nujol
Table 4
Pharmacological effects of the tested compounds
Comp.
20 mM KCl
pIC50
60 mM KCl
pIC50
20 mM KCl+1 mM TEA
Eff. (%)
Eff. (%)
pIC50
Eff. (%)
4a
4b
5a
5b
7
10
13a–e
NC
5493
100
8391
100
6791
100
NC
2095
7592
7392
8193
4691
9092
not tested
not tested
4.6390.061
4.5190.070
not tested
4.2690.16
5.0190.064
5.0690.084
4.1090.056
4.7590.036
ineffective
3.2490.021
4.4190.050
3.7690.055
NC
9693
9394
4.0790.043
4.2690.056
9593
NS 1619
5.3790.14
100
5.4190.11
100
not tested
The values of potency (pIC50) are expressed as the mean9SEM. The efficacy parameter indicates the maximal vasorelaxation, as a percentage
of the contractile tone. The values of potency and efficacy, under a standard membrane depolarisation (20 mM KCl), under increased
depolarisation (60 mM KCl), and in the presence of TEA are shown. The potency parameters of compounds possessing a low efficacy (:50%
or lower) could not be calculated (NC).

846
G. Biagi et al. / Il Farmaco 56 (2001) 841–849
mulls were recorded in a Mattson Genesis series FTIR
spectrometer. H NMR spectra were recorded with a
compound: 0.168 g, yield 92%; m.p. 181–185 °C from
MeOH–H2O. IR (w, cm−1): 3341 and 3189 (NH2); 1662
(CO). MS; m/z: 294 [M+], 44 [base peak]. Anal.
(C15H14N6O) C, H, N.
1
Varian Gemini 2000 spectrometer in DMSO-d6 in l
units, using TMS as an internal standard. Mass spectra
were performed with a Hewlett Packard MS/System
5988. Elemental analyses (C, H, N) were within 90.4%
of theoretical values and were performed in a Carlo
Erba Elemental Analyser Mod. 1106 apparatus. TLC
data were obtained with Riedel de Haen, 37360 DC-
Karten F254, 0.2 mm, eluting with a ethyl acetate–
petroleum ether 1:3 mixture. Petroleum ether
corresponds to the fraction boiling at 40–60 °C.
5.1.4. 1-(5-Carboxamido-1,2,3-triazol-4-yl)-5-sec-
butyl-benzimidazolone (4a) and 1-(5-carboxamido-
1,2,3-triazol-4-yl)-5-phenyl-benzimidazolone (4b)
To an ice-cooled (0–5 °C) and stirred solution of 1.0
mmol of the appropriate compound 3a or 3b in 6 ml of
anhydrous pyridine, a solution of 20% phosgene in
toluene (0.8 ml, $1.5 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 3) with 10% HCl, caused
precipitation of the title compounds which were col-
lected by filtration after 2–3 h of further stirring (Table
1).
5.1.1. 2-Nitro-biphenylazide (1b)
To an ice-cooled and stirred solution of 2-nitro-
biphenylamine [4] (2.00 g, 9.34 mmol) in 100 ml of 75%
H2SO4, a solution of NaNO2 (0.84 g, 12.2 mmol) in 10
ml of H2O was added. After 30 min, a solution of
NaN3 (0.79 g, 12.2 mmol) in 10 ml of H2O was added
drop by drop to the clear solution and stirring was
continued for 2 h. The reaction mixture was diluted
with H2O, the acidity was reduced (pH 3–4) by addi-
tion of solid NaOH and after 1 h of stirring, 1b was
collected by filtration: 1.42 g, yield 63%; m.p. 102–
104 °C from MeOH–H2O. IR (w, cm−1): 2129 (N3);
1531 and 1352 (NO2). MS; m/z: 212 [M+−N2], 102
[base peak]. Anal. (C12H8N4O2) C, H, N.
5.1.5. 1-(5-Carboxamido-1,2,3-triazol-4-yl)-5-sec-
butyl-benzotriazole (5a)
To an ice-cooled (0–5 °C) and stirred solution of 3a
(0.275 g, 1.0 mmol) in 20 ml of 18% HCl, a solution of
NaNO2 (0.084 g, 1.2 mmol) in 10 ml of H2O was added
drop by drop. After 20 min, the ice-bath was removed
and stirring continued at room temperature for 2 h.
The precipitate obtained, consisting of the title com-
pound, was collected by filtration and washed with H2O
(Table 1).
5.1.2. 4-Carboxamido-5-(2-nitro-biphenylamino)-1,2,3-
triazole (2b)
To a stirred solution of sodium ethoxide (0.124 g,
5.39 mmol of Na) in 70 ml of absolute ethanol, 0.454 g
(5.40 mmol) of cyanacetamide was added. After 20 min,
the suspension was cooled in an ice-bath (−10 °C) and
1.00 g (4.16 mmol) of 1b in 250 ml of absolute ethanol
was added drop by drop keeping the temperature
B0 °C. After 1 h the ice-bath was removed and stir-
ring continued at room temperature for 24 h. The
solvent was evaporated under reduced pressure, H2O
and 8–10 ml of 10% NaOH were added and the
mixture was heated under reflux for 20–30 min. After
cooling, the precipitated 2-nitro-biphenylamine (0.73 g)
was filtered off and the filtrate was acidified (pH 1–2)
to give 2b as an orange solid which was collected by
filtration: 0.337 g, yield 25%; m.p. 162–166 °C from
MeOH–H2O. IR (w, cm−1): 3220 (NH); 1666 (CO);
1528 and 1376 (NO2). MS; m/z: 324 [M+], 221 [base
peak]. Anal. (C15H12N6O3) C, H, N.
5.1.6. 1-(5-Carboxamido-1,2,3-triazol-4-yl)-5-phenyl-
benzotriazole (5b)
To an ice-cooled (0–5 °C) and stirred solution of 3b
(0.294 g, 1.0 mmol) in 20 ml of 50% H2SO4, a solution
of NaNO2 (0.084 g, 1.2 mmol) in 10 ml of H2O was
added drop by drop. After 20 min, the ice-bath was
removed and stirring continued at room temperature
for 3 h. Solid NaHCO3 was added to decrease the
solution acidity (pH 3–4) and the precipitate obtained,
consisting of the title compound, was collected by
filtration and washed with H2O (Table 1).
5.1.7. N-(3-Trifluoromethylphenyl)-N%-(5-
carboxamido-1,2,3-triazol-4-yl)-urea (7)
To a solution of 3-trifluoromethyl-phenylisocyanate
(0.630 g, 3.40 mmol) in 10 ml of anhydrous acetone,
0.432 g (3.40 mmol) of 6 was added and the mixture
was refluxed for 10 h. The solvent was evaporated in
vacuo and the solid residue was stirred with 10%
NaOH ($20 ml). The insoluble material consisting of
di-(3-trifluoromethyl-phenyl)-urea was filtered off and
the filtrate was acidified (pH 3) to precipitate 7 which
was collected by filtration (Table 1).
5.1.3. 4-Carboxamido-5-(2-amino-biphenylamino)-
1,2,3-triazole (3b)
To a solution of 2b (0.200 g, 0.62 mmol) in 100 ml of
MeOH, 10% Pd/C (0.020 g) was added 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

G. Biagi et al. / Il Farmaco 56 (2001) 841–849
847
5.1.8. 1-(2-Nitrophenyl)-benzotriazole (8)
5.1.11. 2-Nitrobenzoic acid-(2-hydroxy-5-methyl-
anilide) (11a) and 2-nitro-4-chloro-benzoic
acid-(2-hydroxy-anilide) (11d)
A solution of 2-nitrophenylazide (1.64 g, 10.0 mmol)
and isoamyl nitrite (1.6 ml, 12.0 mmol) in 50 ml of
CHCl3 was heated under reflux for 2 h. A solution of
anthranilic acid (1.51 g, 11.0 mmol) in 15 ml of anhy-
drous acetone was slowly added drop by drop to the
boiling solution (:2 h). The solvent was evaporated in
vacuo and the tarry residue was extracted three times
with 50 ml portions of boiling petroleum ether to
remove unreacted azide (:3 h). The treatment of the
new residue with 10–15 ml of isopropanol caused pre-
cipitation of the title compound as an orange solid
which was collected by filtration (Table 1).
A suspension of 10.0 mmol of the appropriate dried
acid (2-nitrobenzoic or 2-nitro-4-chlorobenzoic) in 5 ml
of SOCl2 was heated under reflux for 2 h. The solvent
was evaporated in vacuo and the residue, consisting of
the acid chloride, was dissolved in 30 ml of anhydrous
benzene. Et3N (5 ml) and 12 mmol of the suitable
phenolamine (2-hydroxy-5-methyl-aniline or 2-hydroxy-
aniline, respectively) were added and the mixture was
refluxed for 4 h. The solution was extracted with 10%
HCl, 5% NaHCO3 and 10% NaOH. The alkaline ex-
tract was paper filtered then acidified to precipitate the
title compounds which were collected by filtration and
washed with H2O (Table 2).
5.1.9. 1-(2-Aminophenyl)-benzotriazole (9)
To a solution of 8 (0.350 g, 1.5 mmol) in 30 ml of
MeOH, 0.035 g of 10% Pd/C was added and the
mixture was hydrogenated at room temperature and
pressure. The catalyst was filtered off and the filtrate
was evaporated to give the title compound (Table 1).
5.1.12. 2-Nitrobenzoic
acid-(2-carboxy-5-chloro-anilide) (11b)
A suspension of 0.835 g (5.0 mmol) of dried 2-ni-
trobenzoic acid in 4 ml of SOCl2 was heated under
reflux for 2 h. The solvent was evaporated in vacuo and
the residue, consisting of the acid chloride, was dis-
solved in 30 ml of anhydrous benzene. Et3N (6 ml) and
0.943 g (5.5 mmol) of 2-carboxy-5-chloro-aniline were
added and the mixture was refluxed for 16 h. After one
night the precipitate was collected by filtration, stirred
for 1 h with 10% HCl and the insoluble material,
consisting of 11b, was collected and washed with H2O
(Table 2).
5.1.10. 1-(2-Hydroxyphenyl)-benzotriazole (10)
5.1.10.1. Procedure A. To a cooled (0–5 °C) and stirred
solution of 9 (0.100 g, 0.5 mmol) in 10 ml of 40%
H2SO4, a solution of NaNO2 (0.041 g, 0.6 mmol) in 2–3
ml of H2O was added drop by drop (:15 min). After
20 min of stirring the mixture was paper filtered and the
filtrate was slowly heated up to 80 °C. When the
evolution of N2 gas ceased, the solution was extracted
with CHCl3 which in turn was extracted with 5%
NaOH solution. Acidification (pH:4) of the alkaline
layer with 36% HCl provided a precipitate which was
isolated by extraction with CHCl3. Evaporation of the
solvent gave the title compound (Table 1).
5.1.13. 2-Nitro-4-chloro-benzanilide (11c) and
2-nitro-5-chloro-benzanilide (11e)
A suspension of 2.02 g (10.0 mmol) of the suitable
nitroacid (2-nitro-4-chlorobenzoic or 2-nitro-5-chloro-
benzoic) in 10 ml of SOCl2 was heated under reflux for
2 h. The solvent was evaporated in vacuo and the
residue, consisting of the respective acid chloride, was
dissolved in 40 ml of anhydrous benzene. Aniline (4.5
ml, 49.4 mmol) was added and the mixture was refluxed
for 4 h. After one night the precipitate was collected by
filtration and worked up as described for the prepara-
tion of 11b (Table 2).
5.1.10.2. Procedure B. A solution of 2-hydroxyphenyl-
azide (3.50 g, 26.0 mmol) and isoamyl nitrite (4.2 ml,
31.0 mmol) in 100 ml of CHCl3 was heated under reflux
for 2 h. A solution of anthranilic acid (3.92 g, 28.6
mmol) in 40 ml of anhydrous acetone was slowly added
drop by drop to the boiling solution (:2 h). The
solvent was evaporated in vacuo and the tarry residue
was extracted three times with 50 ml portions of boiling
petroleum ether to remove unreacted azide (:2 h). The
new residue was afterwards extracted five times with
ether portions under reflux (300 ml, :3 h). The black
residue was filtered off and the combined filtrates were
evaporated to give a viscous reddish liquid which was
dissolved in :30 ml EtOAc. This solution was filtered
through a silica gel column and the filtrate was evapo-
rated to give a semisolid residue which was triturated
with 5–6 ml of ether. The title compound separated as
a pale yellow solid which was collected by filtration
(Table 1).
5.1.14. 2-Aminobenzoic acid-(2-hydroxy-5-methyl-
anilide) (12a) and 2-aminobenzoic acid-(2-carboxy-5-
chloro-anilide) (12b)
To a solution of 1.80 mmol of 11a or 11b in 100 ml
and 300 ml, respectively, of MeOH, $0.100 g of wet
Raney Ni was added and the mixture was hydrogenated
at room temperature and pressure. The catalyst was
filtered off, washed with hot MeOH and the filtrate was
evaporated to give the title compounds (Table 2).

848
G. Biagi et al. / Il Farmaco 56 (2001) 841–849
5.1.15. 2-Amino-4-chloro-benzanilide (12c),
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 compounds 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.
2-amino-4-chloro-benzoic acid-(2-hydroxy-anilide)
(12d) and 2-amino-5-chloro-benzanilide (12e)
To a solution of 2.50 mmol of the appropriate ni-
troderivative (11c, 11d or 11e) in 60–70 ml of MeOH,
$0.100 g of wet Raney Ni was added. The mixture was
then hydrogenated and worked up as described for the
preparation of 12a (Table 2).
5.1.16. 3-(2-Hydroxy-5-methyl-phenyl)-benzotriazin-
4-one (13a) and
3-(2-hydroxy-phenyl)-7-chloro-benzotriazin-4-one (13d)
To an ice-cooled (0–5 °C) and stirred suspension of
2.00 mmol of the suitable 2-amino-benzanilide (12a or
12d) in 50 ml of 10% HCl, a solution of 0.145 g (2.1
mmol) of NaNO2 in 6 ml of H2O was added drop by
drop. After 4 h the precipitate newly formed, consisting
of the title compounds, was collected by filtration and
washed with H2O (Table 2).
In other sets of experiments, the potassium channel
blocker tetraethylammonium chloride (1 mM) was
added, before the KCl (20 mM)-induced contraction,
followed by the administration of selected compounds.
Compounds 13c and 13e were dissolved (1 mM) in
ethanol and further diluted in bi-distilled water.
Norepinephrine hydrochloride (Sigma), acetylcholine
chloride (Sigma) and KCl were dissolved in bi-distilled
water. All the other 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-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.
5.1.17. 3-(2-Carboxy-5-methyl-phenyl)-benzotriazin-
4-one (13b), 3-phenyl-7-chloro-benzotriazin-4-one (13c)
and 3-phenyl-6-chloro-benzotriazin-4-one (13e)
To an ice-cooled (0–5° C) and stirred suspension of
2.00 mmol of the suitable 2-amino-benzanilide (12b, 12c
or 12e) in 16 ml of 18% HCl, a solution of 5.0, 3.4 and
3.6 mmol, respectively, of NaNO2 in 12 ml of H2O was
added drop by drop. The reaction was then worked up
as described for the preparation of 13a (Table 2).
5.2. Pharmacology
All the experimental procedures were carried out
following the guidelines of the European Community
Council Directive 86-609.
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).
5.2.1. Data analysis
The efficacy of the vasorelaxing responses was ex-
pressed as a maximal relaxant effect (Emax), calculated
as a percentage of the contractile tone developed by the
smooth muscle preparation, after the depolarising stim-
ulus induced by 20 mM KCl. The above parameters
were calculated by means of non-linear regression anal-
ysis of the sigmoidal concentration–response curves
(computer program: GRAPHPAD PRISM), and were ex-
pressed as the mean of six experiments. Previous exper-
iments demonstrated an almost complete quantitative
equivalence between the contractile responses evoked
by the two different concentrations of KCl, since the
concentration 20 mM could substantially induce a max-
imal effect, in endothelium 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-
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-

G. Biagi et al. / Il Farmaco 56 (2001) 841–849
849
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sigmoidal concentration–response curves (computer
program: GRAPHPAD PRISM), and were expressed as the
mean9SEM of four to six experiments.
The statistical comparison of experimental data was
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