1,5-Diarylsubstituted 1,2,3-triazoles as potassium channel activators. VI

Article abstract of DOI:10.1016/j.farmac.2004.01.012

As part of our program toward designing potassium channel openers, synthesis of a novel series of 1,5-diphenylsubstituted 1,2,3-triazoles, as potential activators of the large-conductance calcium-activated potassium channels (BK), as well as their vasorel

Full text of DOI:10.1016/j.farmac.2004.01.012

IL FARMACO 59 (2004) 397–404
www.elsevier.com/locate/farmac
1,5-Diarylsubstituted 1,2,3-triazoles as potassium channel activators. VI
Giuliana Biagi a, Vincenzo Calderone b, Irene Giorgi a, Oreste Livi a,*, Enrica Martinotti b,
Alma Martelli b, Antonio Nardi a
a Dipartimento di Scienze Farmaceutiche, via Bonanno 6, 56126 Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Facoltà di Farmacia, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy
Received 26 July 2003; accepted 17 January 2004
Abstract
As part of our program toward designing potassium channel openers, synthesis of a novel series of 1,5-diphenylsubstituted 1,2,3-triazoles,
as potential activators of the large-conductance calcium-activated potassium channels (BK), as well as their vasorelaxant activity are
presented. The functional effect of these potential structurally novel BK-openers on vascular contractile function were studied in vitro, using
isolated rat aortic rings pre-contracted with KCl 20 mM. Among the target compounds, only 16 showed appreciable effectiveness, exhibiting
an efficacy parameter (57%) lower than that of NS1619 and a comparable potency index (pIC50: 5.58).
© 2004 Elsevier SAS. All rights reserved.
Keywords: Potassium channels; Potassium channel openers; BK-activators; 1,2,3-triazoles; Vasodilator activity
1. Introduction
sponding series of 5-substituted-triazolyl-benzotriazoles
were prepared [5]. Differently from the benzimidazolone
derivatives, the benzotriazole derivatives generally showed
full efficacy with vasorelaxing properties and potency pa-
rameters a little lower than that of the reference compound
NS 1619. Some structural modifications of the 1,2,3-
triazolyl-benzimidazolone and 1,2,3-triazolyl-benzotriazole
derivatives [5] did not increase activity, even if they provided
useful information about structure-activity relationships [6].
Finally, a series of new 5-substituted-1-(2-hydroxybenzoyl)-
benzotriazole derivatives [7] showed full vasorelaxing effi-
cacy and high potency values. The best compound in this
series, further investigated in order to evaluate the possible
mechanism of action involving BK channels, showed also an
interesting cardioprotective activity [7].
Large-conductance calcium-activated potassium channels
(also known as BK or BKCa or MAXI-K channels) are a
subtype of the large family of potassium channels and repre-
sent a potential therapeutic target for the synthesis of new
drugs. In fact, the activation of these channels, allowing the
concentration-depending passage of the potassium ions to
the extracellular phase, causes membrane hyperpolarization
and reduction of cellular excitability [1]. Therefore, com-
pounds able to activate selectively BK channels afford a new
therapeutic approach for several pathological conditions in-
volving cell hyperexcitability, such as asthma, urge inconti-
nence and bladder spasm, gastric hypermotility, hyperten-
sion, coronary artery spasm, psychoses [1,2].
Previously, we had synthesised and tested as potential
potassium channel activators a series of 5-(4’-substituted-2’-
nitro-anilino)-1,2,3-triazole derivatives [3] which showed a
vasorelaxant activity, involving potassium channel opening
and with a potency comparable to that of the reference
On the basis of this experience of ours focused on the
structural modifications of the prototypical NS004 and con-
sidering the chemical structure and pharmacophoric charac-
teristics of potent BK activators recently investigated, bear-
ing a disubstituted and five-membered terms heterocyclic
ring [8-11], we planned the synthesis and pharmacological
evaluation of new disubstituted monocyclic 1,2,3-triazole
derivatives, corresponding to the general structure A (Fig. 1).
In Figure 1, the comparison compounds NS004 and NS1619
(B) as well as their 1,2,4-triazol-3-one active analogue C [8]
are reported. This first paper reports the results obtained from
compound NS 1619 [4]. Afterwards,
5-substituted-triazolyl-benzimidazolones and the corre-
a series of
* To whom correspondence should be addressed: Prof. Oreste Livi,
Dipartimento di Scienze Farmaceutiche, Università di Pisa, via Bonanno 6,
56126 Pisa, Italy.
E-mail address: livi@farm.unipi.it (O. Livi).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.01.012

398
G. Biagi et al. / IL FARMACO 59 (2004) 397–404
N 3
N
H
N
4
5
R
2
(X)
O
N
R1
(Y)
N
1
OH
R2
R3
R4
R
B
A
1
H
2
3
N
H
N
N
N
O
5
N
4 N
F3C
OH
OH
EWG
CF3
EWG
Cl
D
C
General formula A. R1, R2, R3, R4 = hydrogen bond donors and / or acceptors and /
or electron withdrawing groups. (X) and / or (Y), when present = CH2, CHOH, CO groups.
Comparison benzimidazolones NS004 (R = Cl) and NS1619 (R = CF3) (B), their deannulated
and structurally-related active analogue C [8] and general formula of target 1,2,3-triazoles (D),
where EWG indicates electron withdrawing groups.
Fig. 1. General formula A. R1, R2, R3, R4 = hydrogen bond donors and/or acceptors and/or electron withdrawing groups. (X) and/or (Y), when present = CH2,
CHOH, CO groups. Comparison benzimidazolones NS004 (R = Cl) and NS1619 (R = CF3) (B), their deannulated and structurally-related active analogue C [8]
and general formula of target 1,2,3–triazoles (D), where EWG indicates electron withdrawing groups.
the study of new 1,5-disubstituted 1,2,3-triazole derivatives
D structurally-related to the compounds B and C (Fig. 1).
N3
COOEt
N
N
N
Cl
NO2
2. Chemistry
i
1
O2N
+
R
The 2-nitro-4-chloro-phenylazide 1 [12], chosen for the
chemical versatility of the nitro group, reacted with the
appropriate b-ketoester (2a or 2b) (Scheme 1) in absolute
ethanol in the presence of sodium ethoxide at -10°C, to give
the expected 1,3-dipolar cycloaddition products, the 1,2,3-
triazolesters 3a and 3b respectively, in moderate yield, to-
gether with the corresponding acids 4a [13] and 4b. These
acids were the only reaction products when the reaction
mixture was directly hydrolized without isolation of the
esters 3a and 3b and were easily obtained by mild alkaline
hydrolysis of the same esters. The carboxylic acids 4a [13]
and 4b, by refluxing in DMF, were easily decarboxylated to
the 1,5-disubstituted triazole derivatives 5a [13] and 5b.
Reduction of the nitro group of 5a with stannous chloride in
acidic medium gave the corresponding aminoderivative 6a,
previously described in the literature [13]. A similar chemi-
cal reduction or a reduction by catalytic hydrogenation of 5b,
involved also a reduction of the other nitrogroup to give the
diaminoderivative 6b in moderate yield. The presence of a
primary amino function in the ortho position of the phenyl
substituent was considered interesting in order to evaluate its
pharmacological effect, because it can act as a hydrogen
bond donor. In fact, lots of BKCa channel openers, taken as
comparison compounds [4,8-11], generally bear a hydroxy
R
CO
COOEt
CH2
3a, 3b
Cl
2a : R = H
2b : R = NO2
ii
COOH
N
N
N
O2N
R
4a, 4b
iii
Cl
H
H
N
N
N
N
N
iv
N
H2N
O2N
R
R
6a : R = H
6b : R = NH2
5a, 5b
Cl
Cl
(i) EtONa / EtOH, r.t.; (ii) 4% KOH; (iii) DMF, reflux;
(iv) H2/Pd/C or SnCl2/H+.
Scheme 1.

G. Biagi et al. / IL FARMACO 59 (2004) 397–404
399
COOH
COOEt
Cl
N3
O2N
N3
N
N
N
N
N
N
7
OCH3
12
OCH3
i
H3CO
H3CO
i
+
NO2
+
ii
13
O2N
CO
COOEt
CH2
8
Cl
NO2
CO
COOEt
CH2
2b
iv
ii
COOH
2a
N
N
COOEt
N
N
N
N
H3CO
H
N
H3CO
N
NO2
14
N
NO2
Cl
H3CO
11
NO2
iii
9
Cl
H
iii
N
N
H
H
N
N
N
N
N
N
iv
N
HO
HO
H3CO
NO2
10
Cl
15
16
NO2
NO2
(i) EtONa / EtOH, 50°C; (ii) DMF, reflux;
(iii) BBr3; (iv) EtOH, H+, reflux.
(i) EtONa / EtOH, r.t.; (ii) EtONa / EtOH, 50°C;
(iii) DMF, reflux; (iv) BBr3.
Scheme 2.
Scheme 3.
function in the same position. Anyhow we thought of con-
verting the amino to a hydroxy function, via diazotization
and hydrolytic decomposition of the diazonium salt. Unfor-
tunately, the treatment of 6a with sodium nitrite in sulfuric
acid solution at 0-5°C and then decomposition of the diazo-
nium salt either by heating or by stirring at room temperature
(25°C) did not provide the expected phenolic derivative. The
only product, isolated from the tarry reaction mixture in very
low yield was the 5-chloro-benzotriazole [14], formed by the
cleavage of the triazole ring owing to the intramolecular
addition of the diazonium salt. In order to prevent this incon-
venience, the 2-methoxy-5-chloro-phenylazide (7) [15] was
similarly reacted with ethyl p-nitrobenzoylacetate (2b)
(Scheme 2), under the usual experimental conditions. In this
case, the 1,3-dipolar cycloaddition reaction provided directly
the carboxylic acid 8 in 50% yield, which was, in its turn,
decarboxylated by heating in boiling DMF, to give the 1-(2’-
methoxy-5’-chloro)-5-(4’-nitrophenyl)-1H-1,2,3-triazole
(9) in 63 % yield. The treatment of the methoxy derivative 9
with boron tribromide in dichloromethane at -78°C until it
reached room temperature, caused the ether cleavage to give
the desired phenolic derivative 10 in 35 % yield. The triazo-
lester 11 was prepared (in moderate yield) by Fisher esterifi-
cation of 8.
analogous reaction sequence (Scheme 3). Thus the ionic
cycloaddition reaction of azide 12 to ethyl benzoylacetate
(2a), carried out under mild experimental conditions, al-
lowed isolation of the triazolester 13 in 36% yield. When the
reaction was carried out under stronger experimental condi-
tions, hydrolysis of the intermediate ester took place and the
reaction mixture provided directly the triazole acid 14 in 70%
yield. The acid was decarboxylated to 15 in the usual manner
and this was converted to 1-(2’-hydroxy-5’-nitro)-5-phenyl-
1H-1,2,3-triazole (16), by treatment with an excess of boron
tribromide under the above reported experimental condi-
tions.
In order to further change the arrangement of the electron
withdrawing substituents on the two aromatic rings, a new
synthetic procedure [17] was carried out to introduce a chlo-
rine atom on the phenyl ring in the 5 position of the triazole
(Scheme 4). Thus, the 2-methoxy-5-chloro-phenylazide (7)
[15] reacted with an equimolar amount of 4-chlorobenzoyl-
methylen-triphenylphosphorane (17) [18] in toluene (80°C)
for three days, to give the 1-(2’-methoxy-5’-chloro)-5-(4’-
chlorophenyl)-1H-1,2,3-triazole (18) in 68 % yield. This was
then converted to the corresponding phenol derivative 19, by
treatment with boron tribromide.
The structure of all the new compounds obtained was
assigned upon the basis of the mechanism of the well-known
reactions employed. In fact, the 1,3-dipolar cycloaddition of
azides to b-ketoesters is regiospecific [17] as well as the
addition to a-keto-phosphorus ylides [14] and the following
Similarly, the 2-methoxy-5-nitro-phenylazide (12) [16],
bearing in the 2 position the methoxy substituent convertible
to a hydroxy function and in the 5 position a different and
stronger electron withdrawing group, was employed in an

400
Cl
G. Biagi et al. / IL FARMACO 59 (2004) 397–404
the pharmacological study have been summarised in Table 3,
N3
H
N
N
where the values of the vasorelaxing potency and efficacy of
the reference compound NS1619 and of the target com-
pounds have been reported. In his Table, the pharmacological
parameters of compound 3b are also reported, as a unique
non-target compound disclosed with significant and tetra-
ethylammonium chloride (TEA)-sensitive vasorelaxant ac-
tivity. Noteworthy, from a pharmacological point of view, 3b
is the best of all compounds tested, showing a potency about
10-fold higher than that of NS1619 but a not-full vasorelax-
ing efficacy. Besides, among the target compounds (10, 16,
19), only 16 showed appreciable effectiveness, exhibiting an
efficacy parameter (57%) lower than than that of NS1619 and
N
7
OCH3
i
H3CO
+
Cl
Cl
17
CO
CH P(Ph)3
18
Cl
ii
H
N
N
N
19
Cl
HO
a comparable potency index (pIC50: 5.58 vs pIC50 (NS1619)
:
Cl
5.18). The vasorelaxing effects were significantly inhibited
by TEA, suggesting the involvement of potassium channels.
Both the low efficacy parameters of 10 and 19 and the
moderate parameter of 16 indicate that the viability of such
deannulation of NS004 and the bioisosteric replacement of
4,5-diphenyltriazol-3-one with 1,5-diphenyl-1,2,3-triazole
nucleus do not seem profitable and deserving of further
synthetic and pharmacological investigation. As far as the
low biological activity is concerned, one of the most impor-
tant pharmacophore requirements which is not conserved in
the 1,2,3-triazoles vs 1,2,4-triazol-3-ones seems to be the
hydrogen bond donor capability on the heterocycle ring,
supposing that the series of target compounds can interact at
the same site as 4,5-diphenyltriazol-3-ones on BK channels.
The paramount role of H-bond donors would be in agreement
with the observation that in the vicinal and diphenyl substi-
tuted heterocyclic series the presence of at least two potential
H-bond donor sites is crucial [8]. Again, a common general
pharmacophore in lots of BK-openers [1] involves an H-bond
donor moiety often placed on a cyclic or acyclic spacer
linking two properly substituted phenyl rings, one of which
fitted with a further potential H-bond donor such as a hy-
(i) benzene, reflux, three days; (ii) BBr3
Scheme 4.
reactions (decarboxylation, nitro reduction, ether cleavage),
involve functional groups placed in known positions. In addi-
tion the reaction accomplishment was confirmed by analyti-
cal and spectroscopic methods (Tables 1 and 2) .
3. Biological results and discussion
Target compounds 10, 16 and 19 underwent a functional
evaluation of their vasorelaxing properties in in vitro assays
as a preliminary screening test to point out their potential
activation of potassium channels. Then, compounds 3-6a,b,
8, 9, 11, 13-15, 18 were also tested because of their clear
chemical structural similarity with the target compounds and
in order to explore and unmask a possible and new
structurally-related chemical lead structure. The results of
Table 1
Chemical and physical properties of the prepared compounds
Comp.(a)
3a
3b
4b
5b
6b
8
9
10
11
13
Yield %
36
Crystall. Solvent
MeOH/H2O
M.p. (°C)
132-134
173-175
119-123
171-172
135-137
186-188
132-135
261-263
228-231
228-230
165-167
180-182
278-280
154-157
273-274
Analyses (C, H, N)
IR m cm-1
1724 (CO)
1732 (CO)
1716 (CO)
---------
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
17H13N4O4Cl
17H12N5O6Cl
15H8N5O6Cl
14H8N5O4Cl
14H12N5Cl
73
EtOH
(b)
82
81
(c)
EtOAc/Petr.Eth.
EtOH/H2O
EtOH
3340, 3213 (NH2)
1701 (CO)
--------
50
63
35
33
36
70
82
32
68
40
16H11N4O5Cl
15H11N4O3Cl
14H9N4O3Cl
18H15N4O5Cl
18H16N4O5
EtOH/H2O
EtOH
3448 (OH)
1724 (CO)
1728 (CO)
1715 (CO)
-------
EtOH
EtOH/H2O
EtOH
14
15
16
18
16H12N4O5
EtOH
15H12N4O3
EtOAc
14H10N4O3
(OH)
MeOH/H2O
EtOH/H2O
15H11N3OCl2
14H9N3OCl2
-------
19
3441 (OH)
(a) : compounds 4a, 5a and 6a are described in the literature [10].
(b) : purified by dissolution in 10% NaOH, filtration and precipitation by acidification.
(c) : procedure A: 55%; procedure B: 76%.

G. Biagi et al. / IL FARMACO 59 (2004) 397–404
401
Table 2
rather than a phenyl substituent on the heterocyclic ring
enhanced flexibility and dramatically augmented the biologi-
cal activity. In fact a better BK-opener activity had been
pointed out in other more flexible diphenylheterocyclic de-
rivatives [10,24,25].
1H-NMR spectra in DMSO-d6 (d)
3a
1.15 (t, 3H, CH3); 4.24 (q, 2H, CH2); 7.31-7.46 (m, 5H, Ar);
7.81-8.37 (m, 3H, Ar)
3b
4b
5b
6b
1.17 (t, 3H, CH3); 4.25 (q, 2H, CH2); 7.68-8.43 (m, 7H, Ar)
7.65-8.42 (m, 7H, Ar); 13.55 (bs, 1H, COOH)
7.59-8.49 (m, 7H, Ar); 8.45 (s, 1H, H4 triaz.)
5.37 (bs, 2H, NH2); 5.42 (bs, 2H, NH2); 5.46-6.62 (m, 3H, Ar);
6.68-6.99 (m, 4H, Ar); 7.94 (s, 1H, H4 triaz.)
3.49 (s, 3H, OCH3); 7.10-8.24 (m, 7H, Ar); 13.25 (bs, 1H,
COOH)
4. Experimental
8
9
4.1. Chemistry
3.43 (s, 3H, OCH3); 8.24-8.33 (m, 3H, Ar); 7.54-7.85 (m, 4H,
Ar); 8.34 (s, 1H, H4 triaz.)
All melting points were determined on a Kofler hot stage
and are uncorrected. IR spectra in Nujol mulls were recorded
10
11
6.98-8.25 (m, 7H, Ar); 8.33 (s, 1H, H4 triaz.); 10.55 (bs, 1H, OH)
1.26 (t, 3H, CH3); 3.50(s, 3H, OCH3); 4.23 (q, 2H, CH2); 7.10-
8.26 (m, 7H, Ar)
1
on a Mattson Genesis series FTIR spectrometer. H-NMR
spectra were recorded on aVarian Gemini 2000 spectrometer
in DMSO-d6 in d units, using TMS as an internal standard.
Elemental analyses (C, H, N) were within + 0.4 % of the
theoretical values and were performed in a Carlo Erba El-
emental Analyser Mod. 1106 apparatus. Petroleum ether
corresponds to the fraction boiling at 40-60°C.
13
14
15
1.16 (t, 3H, CH3); 3.64 (s, 3H, OCH3); 4.22 (q, 2H, CH2);
7.30-8.66 (m, 3H, Ar); 7.35 (s, 5H, Ph)
3.65 (s, 3H, OCH3); 7.35 (s, 5H,Ar); 8.36-8.57 (m, 3H,Ar); 13.10
(bs, 1H, COOH)
3.61 (s, 3H, OCH3); 7.30-7.43 (m, 5H, Ar); 8.45-8.55 (m, 3H,
Ar); 8.16 (s, 1H, H4 triaz.)
16
18
19
7.12-8.34 (m, 7H, Ar); 8.15 (s, 1H, H4 triaz.); 11.96 (bs, 1H, OH)
3.49 (s, 3H, OCH3); 7.21-7.77 (m, 7H, Ar); 8.17 (s, 1H, H4 triaz.)
6.95-7.65 (m, 7H, Ar); 8.16 (s, 1H, H4 triaz.); 10.52 (bs, 1H, OH)
4.1.1. Ethyl 1-(2’-nitro-4’-chlorophenyl)-5-phenyl-1H-1,2,3-
triazole-4-carboxylate (3a
)
To an ice-salt cooled and stirred solution of sodium ethox-
ide (from 0.460 g, 20.0 mmol of sodium) in 15 ml of absolute
EtOH, a solution of 2-nitro-4-chloro-phenylazide (1) (2.00 g,
10.0 mmol) and ethyl benzoylacetate (2a) (2.32 g,
12.0 mmol) in 50 ml of absolute EtOH was slowly added.
After 4 h, 80 ml of ice-water were added to the suspension
and the mixture was extracted with CHCl3. The combined
organic layers were washed (H2O), dried (MgSO4) and
evaporated in vacuo to give a solid residue (1.84 g) which
was extracted with boiling petroleum ether (15 ml × 3) to
dissolve the unreacted azide. The new residue, consisting of
the title compound, was purified by crystallisation (Table 1).
By acidification of the aqueous mother liquors, a little
amount of the corresponding acid 4a [13] precipitated.
Table 3
Experimental results: potency (pIC50) and efficacy
(Emax %) values of the tested compounds
Comp.
3b
10
16
19
Efficacy (Emax %)
56 16
pIC50
6.55 0.03
N.C.
24 14
57 11
5.58 0.25
N.C.
38
7
NS1619
100a
5.18 0.05
Compounds 3a, 4-6a,b, 8, 9, 11, 13-15, 18 are ineffective.
N.C. indicates that the parameter could not be calculated because of low
efficacy (<50%).
The symbol a indicates that a full vasorelaxing effect was reached in all the
experiments performed, hence the standard error could not be expressed.
droxy function. These useful observations have recently led
us to the synthesis and pharmacological evaluation of new
potent BK-opener benzanilides, bearing an amide moiety as
a new acyclic H-bond donor spacer unit between an ortho-
hydroxyphenyl group and a multi-substituted phenyl ring
[19].
As also follows from the literature, the other crucial struc-
tural requirements of the diphenyltriazolone derivatives [8],
as well as other synthetic [1,11] or natural [20] BK-openers,
such as an electron deficient phenyl ring and an ortho-
hydroxy-para-chlorophenyl substituent are conserved in the
target 1,2,3-triazole series.
4.1.2. Ethyl 1-(2’-nitro-4’-chlorophenyl)-5-(4’-nitro-phenyl)-
1H-1,2,3-triazole-4-carboxylate (3b
)
To an ice-salt cooled and stirred solution of sodium ethox-
ide (from 0.740 g, 32.2 mmol of sodium) in 15 ml of absolute
EtOH, a solution of 2-nitro-4-chloro-phenylazide (1) (3.12 g,
15.7 mmol) and ethyl 4-nitrobenzoylacetate (2b) (4.52 g,
19.0 mmol) in 85 ml of absolute EtOH was slowly added.
After 2 h the yellow solid precipitated was collected by
filtration and purified by crystallisation (Table 1).
4.1.3. 1-(2’-Nitro-4’-chlorophenyl)-5-(4’-nitrophenyl)-1H-
1,2,3-triazole-4-carboxylic acid (4b)
So, it follows that these latter pharmacophoric requisites
appear not to be sufficient for biological activity and to be
probably dependent on the chemical nature of their spacer
unit. Finally, another problem could be their poor structural
flexibility, as evidenced in some papers about small molecule
BK-openers [21,22] and on some of our 1,2,3-triazole deriva-
tives [23]. In these compounds the insertion of a benzyl
A solution of the triazolester 3b (0.300 g, 0.72 mmol) in
4% KOH solution (22 ml of EtOH and 3 ml of H2O) was
stirred at room temperature for 24 h. The reaction mixture
was then concentrated in vacuo at room temperature, diluted
with H2O (25 ml) and acidified with 10% HCl to precipitate
4b as a yellow solid which was collected by filtration
(Table 1).

402
G. Biagi et al. / IL FARMACO 59 (2004) 397–404
4.1.4. 1-(2’-Nitro-4’-chlorophenyl)-5-(4’-nitrophenyl)-1H-
1,2,3-triazole (5b)
anhydrous CH2Cl2 was added dropwise. Stirring was contin-
ued for 1 h, 10 ml of MeOH followed by 10 ml of H2O were
added dropwise, then the reaction mixture was extracted with
CH2Cl2. The combined extracts were washed with H2O,
dried (MgSO4) and evaporated to give a solid residue which
was treated with 10% NaOH. After filtration the alkaline
solution was acidified with 10% HCl to precipitate the title
compound which was collected by filtration (Table 1).
A solution of the triazole acid 4b (0.300 g, 0.77 mmol) in
1 ml of DMF was heated under reflux for 3 h. After cooling,
the dark solution was diluted with H2O and the fine precipi-
tate was extracted with CHCl3. The combined extracts were
repeatedly washed with H2O, then dried (MgSO4) and evapo-
rated in vacuo to give 5b as a solid residue (Table 1).
4.1.5. 1-(2’-Amino-4’-chlorophenyl)-5-(4’-aminophenyl)-
1H-1,2,3-triazole (6b)
4.1.9. Ethyl 1-(2’-Methoxy-5’-chlorophenyl)-5-(4’-nitro-
phenyl)-1H-1,2,3-triazole-4-carboxylate (11)
A) A mixture of dinitrotriazole 5b (0.345 g, 1.00 mmol)
and SnCl2 · 2 H2O (1.35 g, 5.98 mmol) in 14 ml of 36%
HCl and 7 ml of H2O was refluxed for 3.5 h. After
cooling, the reaction mixture was paper filtered and the
filtrate was treated with an excess of 10% NaOH to
precipitate the title compound 6b which was collected
by filtration and washed with H2O (Table 1).
B) A solution of the triazole acid 5b (0.210 g, 0.61 mmol)
in 45 ml of MeOH was hydrogenated at room tempera-
ture and pressure, in the presence of 5% Pd/C
(0.100 g). The catalyst was filtered off, washed with
boiling MeOH and the combined filtrates were evapo-
rated to give 6b (Table 1).
To a solution of triazole acid 8 (0.300 g, 0.800 mmol) in
20 ml of absolute EtOH, 2 drops of conc. H2SO4 were added
and the mixture was heated under reflux for 2 h. The reaction
mixture was evaporated in vacuo and the residue was dis-
solved in CHCl3. The chloroform solution was extracted with
4% NaOH, dried (MgSO4) and evaporated to give the title
compound (Table 1). From the alkaline extract, by acidifica-
tion, 20% of the starting acid 8 was recovered (Table 1).
4.1.10. Ethyl 1-(2’-Methoxy-5’-nitrophenyl)-5-phenyl-1H-
1,2,3-triazole-4-carboxylate (13)
To cooled (-10°C) and stirred solution of sodium ethoxide
(from 0.100 g, 4.35 mmol of sodium) in 10 ml of absolute
EtOH, a solution of 2-methoxy-5-nitro-phenylazide (12)
(0.600 g, 3.09 mmol) and ethyl benzoylacetate (2a) (0.710 g,
3.71mmol) in 30 ml of absolute EtOH was slowly added.
After 2 h the ice-bath was removed and the mixture stirred at
room temperature for 6 h. The reaction mixture was concen-
trated in vacuo at room temperature and the semisolid resi-
due was treated with crushed ice, then extracted with CHCl3.
The combined organic layers were dried (MgSO4) and
evaporated to give a yellow oil which, by trituration with a
small amount of boiling petroleum ether, left the title com-
pound as a white solid (Table 1).
4.1.6. 1-(2’-Methoxy-5’-chlorophenyl)-5-(4’-nitrophenyl)-
1H-1,2,3-triazole-4-carboxylic acid (8)
To a cooled (-10°C) and stirred solution of sodium ethox-
ide (from 0.160 g, 6.96 mmol of sodium) in 10 ml of absolute
EtOH, a solution of 2-methoxy-5-chloro-phenylazide (7)
(0.700 g, 3.81 mmol) and ethyl 4-nitro-benzoylacetate (2b)
(1.08 g, 4.58 mmol) in 30 ml of absolute EtOH was slowly
added. The ice-bath was removed and stirring was continued
at room temperature for 2.5 h then the reaction mixture was
heated at 50°C for 9 h. The suspension obtained was then
concentrated in vacuo and the remaining EtOH was decanted
and the residue treated with crushed ice. The resulting aque-
ous solution was washed with CHCl3 then acidified with
conc. HCl to precipitate 8 as a pale yellow solid which was
collected by filtration (Table 1).
4.1.11. 1-(2’-Methoxy-5’-nitrophenyl)-5-phenyl-1H-1,2,3-
triazole-4-carboxylic acid (14)
To a cooled (-10C) and stirred solution of sodium ethoxide
(from 0.550 g, 23.9 mmol of sodium) in 20 ml of absolute
EtOH, a solution of 2-methoxy-5-nitro-phenylazide (12)
(2.00 g, 10.3 mmol) and ethyl benzoylacetate (2a) (2.74 g,
14.2 mmol) in 95 ml of absolute EtOH was slowly added.
The ice-bath was removed and the mixture was heated at
60°C under stirring for 10 h. The reaction mixture was
concentrated in vacuo, the semisolid residue was treated with
H2O, then extracted with CHCl3. Acidification of the aque-
ous alkaline solution with 10% HCl gave acid 14 as a yellow
precipitate which was collected by filtration (Table 1).
4.1.7. 1-(2’-Methoxy-5’-chlorophenyl)-5-(4’-nitrophenyl)-
1H-1,2,3-triazole (9)
A solution of the triazole acid 8 (0.860 g, 2.29 mmol) in
6 ml of DMF was heated under reflux for 4 h. After cooling,
the dark solution was diluted with H2O to precipitate a brown
solid which was collected by filtration and dissolved in
CHCl3. The organic solution was washed with 10% NaOH,
dried (MgSO4) and evaporated in vacuo to give 9 as a yellow
solid residue (Table 1).
4.1.12. 1-(2’-Methoxy-5’-nitrophenyl)-5-phenyl-1H-1,2,3-
triazole (15)
A solution of the triazole acid 14 (0.650 g, 1.91 mmol) in
5 ml of DMF was heated under reflux for 4 h. After cooling,
the solution was diluted with H2O and the brown precipitate
was collected by filtration and washed with H2O. This solid
4.1.8. 1-(2’-Hydroxy-5’-chlorophenyl)-5-(4’-nitrophenyl)-
1H-1,2,3-triazole (10)
To a stirred solution of 9 (0.500 g, 1.52 mmol) in 35 ml of
anhydrous CH2Cl2, cooled at -10°C and under a nitrogen
flow, a solution of BBr3 (1.0 ml, 10.6 mmol) in 8 ml of

G. Biagi et al. / IL FARMACO 59 (2004) 397–404
403
was then treated with 10% NaOH, the mixture was paper
filtered and the filtrate was acidified to give 15 as a precipitate
which was collected by filtration (Table 1).
pound as a white solid which was collected by filtration
(Table 1).
4.2. Pharmacology
4.1.13. 1-(2’-Hydroxy-5’-nitrophenyl)-5-phenyl-1H-1,2,3-
triazole (16)
All the experimental procedures were carried out follow-
ing the guidelines of the European Community Council Di-
rective 86-609. To determine a possible vasodilator mecha-
nism of action, the compounds were tested on isolated
thoracic aortic rings of male normotensive Wistar rats (250-
350 g). The rats were sacrificed by cervical dislocation under
light ether anaesthesia and bled. The aortae were immedi-
ately excised and freed of extraneous tissues. The endothelial
layer was removed by gently rubbing the intimal surface of
the vessels with a hypodermic needle. Five mm wide aortic
rings were suspended, under a preload of 2 g, in 20 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 gassed with a mixture of O2 (95%)
and CO2 (5%). Changes in tension were recorded by means
of an isometric transducer (Grass FTO3), connected to a
unirecord microdynamometer (Buxco Electronics).
After an equilibration period of 60 minutes, the endothe-
lial removal was confirmed by the administration of acetyl-
choline (ACh) (10 µM) to KCl (20 mM)-precontracted vas-
cular rings. A relaxation < 10% of the KCl-induced
contraction was considered representative of an acceptable
lack of the endothelial layer, while the organs, showing a
relaxation ≥ 10% (i.e. significant presence of endothelium),
were discarded. From 30 to 40 minutes after confirmation of
the endothelium removal, the aortic preparations were con-
tracted by treatment with a single concentration of KCl
(20 mM) and when the contraction reached a stable plateau,
3-fold increasing concentrations of the tested compounds or
of the reference drug NS 1619 (a well-known BK-activator)
were added cumulatively. Preliminary experiments showed
that the KCl (20 mM)-induced contractions remained in a
stable tonic state for at least 40 minutes. In other sets of
experiments, the non-selective potassium channel blocker
TEA (10 mM) was added, after KCl (20 mM)-induced con-
traction, followed by the administration of selected com-
pounds. The reference drug NS 1619 (Sigma) was dissolved
(10 mM) in EtOH 95% and further diluted in Tyrode solu-
tion.Acetylcholine chloride (Sigma) was dissolved (100 nM)
in EtOH 95% and further diluted in bidistilled water whereas
KCl and TEA were both dissolved in Tyrode solution. Some
of the synthesised derivatives (4b, 10, 16, 19) were dissolved
(10 mM) in NaOH 0.1 N whereas all the other tested com-
pounds in DMSO (10 mM); they were all further diluted in
Tyrode solution. All the solutions were freshly prepared
immediately before pharmacological experimental proce-
dures. Previous experiments showed a complete ineffective-
ness of the vehicles. The vasorelaxing efficacy was evaluated
as maximal vasorelaxing response, expressed as a percentage
(%) of the contractile tone induced by KCl 20 mM. When the
To a stirred solution of 15 (0.400 g, 1.35 mmol) in 30 ml of
anhydrous CH2Cl2, cooled at -78°C and under a nitrogen
flow, a solution of BBr3 (0.90 ml, 9.50 mmol) in 7 ml of
anhydrous CH2Cl2 was added dropwise. The reaction mix-
ture was kept for a night at -20°C and 1 h at 4°C then 10 ml of
MeOH and 10 ml of H2O were added dropwise and basified
with 10% NaOH. The aqueous layer was acidified with 10%
HCl and extracted with CHCl3. The combined extracts were
dried (MgSO4) and evaporated in vacuo to give the title
compound as a solid residue (Table 1).
4.1.14. 4-Chlorobenzoyl-methylen-triphenylphosphorane
(17)
To a solution of x-bromo-acetophenone (5.00 g,
21.4 mmol) in 50 ml of anhydrous THF, triphenylphosphine
(5.62 g, 21.4 mmol) was added.After few minutes a fine solid
began to precipitate and after 15 min the suspension was
heated under reflux for 6 h. The reaction mixture was cooled
in an ice-salt bath, then the precipitate was collected by
filtration. This solid (10.15 g) was treated with 500 ml of
0.25% NaOH, the suspension was stirred for a night, then the
precipitate, consisting of 17, was collected by filtration and
washed with H2O: 8.25 g, yield 93 % ; m.p. 196-198°C (lit.
[18] 195-197°C).
4.1.15. 1-(2’-Methoxy-5’-chlorophenyl)-5-(4’-chlorophe-
nyl)-1H-1,2,3-triazole (18)
A
solution of 2-methoxy-5-chloro-phenylazide (7)
(0.370 g, 2.0 mmol) and a-keto-phosphorus ylide 17
(0.830 g, 2.0 mmol) in 25 ml of anhydrous toluene was
heated (80°C) for 3 days. The solvent was evaporated in
vacuo and the brown oil residue, triturated with petroleum
ether, provided a brown solid (0.590 g) which was dissolved
in CHCl3. This solution was washed with 10% HCl and H2O,
then dried (MgSO4) and evaporated to give a new solid
residue (0.500 g) which had to be purified by crystallisation
(Table 1).
4.1.16. 1-(2’-Hydroxy-5’-chlorophenyl)-5-(4’-chlorophe-
nyl)-1H-1,2,3-triazole (19)
To a stirred solution of 18 (0.320 g, 1.0 mmol) in 50 ml of
anhydrous CH2Cl2, cooled at -70°C and under a nitrogen
flow, a solution of BBr3 (1.0 ml, 10.6 mmol) in 7 ml of
anhydrous CH2Cl2 was added very slowly (1 h). After 3 h of
stirring the reaction mixture was kept at –20°C for a night.
The mixture was transferred into an ice-salt bath, 10 ml of
MeOH and 10 ml of H2O were added dropwise, stirred for
15 min, then the organic phase was separated, washed with
H2O and extracted with 5% NaOH. The combined alkaline
layers were acidified (10% HCl) to precipitate the title com-

404
G. Biagi et al. / IL FARMACO 59 (2004) 397–404
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which could be administered) of the tested compounds did
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(%) of the contractile tone induced by KCl 20 mM, evoked by
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