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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.