Benzoyl and/or benzyl substituted 1,2,3-triazoles as potassium channel activators. VIII

Article abstract of DOI:10.1016/j.ejmech.2005.01.010

This paper reports the preparation of new benzoyl and/or benzyl substituted 1,2,3-triazole derivatives and their pharmacological evaluation as potential BK channel openers, as a part of a research program which hypothesized a pharmacophoric structure cont

Full text of DOI:10.1016/j.ejmech.2005.01.010

European Journal of Medicinal Chemistry 40 (2005) 521–528
www.elsevier.com/locate/ejmech
Original article
Benzoyl and/or benzyl substituted 1,2,3-triazoles as
potassium channel activators. VIII
Vincenzo Calderone b, Irene Giorgi a, Oreste Livi a,*, Enrica Martinotti b, Elisabetta Mantuano a,
Alma Martelli b, Antonio Nardi a
a Dipartimento di Scienze Farmaceutiche, Facoltà di Farmacia, Università di Pisa, via Bonanno 6, I-56126 Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Facoltà di Farmacia, Università di Pisa, via Bonanno 6, I-56126 Pisa, Italy
Received 4 August 2004; received in revised form 9 December 2004; accepted 24 January 2005
Available online 03 March 2005
Abstract
This paper reports the preparation of new benzoyl and/or benzyl substituted 1,2,3-triazole derivatives and their pharmacological evaluation
as potential BK channel openers, as a part of a research program which hypothesized a pharmacophoric structure containing the 1,2,3-triazole
ring. The synthetic procedures consist essentially with the 1,3-dipolar cycloaddition of aryl or benzyl azides to the asymmetric alkyne ben-
zoylacetylene to give the wished 4-benzoyl-1,2,3-triazole isomers in larger amount. The pharmacological results show that the 1-(2-
hydroxybenzyl)-4-benzyl-1H-1,2,3-triazole possesses high vasorelaxing activity involving the opening of the BK channels. Therefore the
structure–activity relationships concerning this pharmacophoric structure confirm the usefulness of a phenolic function in the ortho position of
the aromatic ring and would suggest a 1,2,3-triazole model bearing benzyl substituents. In addition such substituents appear more flexible and
able to take different conformations with respect to phenyl groups which have higher trend to coplanar conformations.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Potassium channels; Potassium channel openers; BK-activators; 1,2,3-Triazoles; Vasodilator activity
1. Introduction
The large conductance calcium activated potassium chan-
nels (also known as BK or MAXI-K channels) are a subtype
of the large family of potassium channels and represent a
potential therapeutic target for the synthesis of new drugs.
The activation of these channels allows a concentration-
depending flow of potassium ions to the extracellular phase
and consequent membrane hyperpolarization and reduction
of the cellular excitability [1]. Therefore, compounds able to
open selectively the BK channels, afford a new therapeutic
approach for several pathological conditions involving cell
hyperexcitability, such as asthma, urge incontinence and blad-
der spasm, gastric hypermotility, hypertension, coronary
artery spasm, psychoses [1,2].
Following our research plain, addressed to synthesize 1,2,3-
triazole derivatives as potential BK channel activators, belong-
ing to the general formula A (Fig. 1), a previous paper [3]
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. B: 1,5-diaryl-1,2,3-triazole derivatives.
C: reference compound NS1619. D: 1,4- and/or 2,4-disubstituted-1,2,3-
triazole derivatives. E: 1,2,3-triazole derivatives with a secondary or tertiary
alcoholic function.
* Corresponding author. Tel.: +39 050 221 9551; fax: +39 050 221 9605.
E-mail address: livi@farm.unipi.it (O. Livi).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.01.010

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V. Calderone et al. / European Journal of Medicinal Chemistry 40 (2005) 521–528
reported 1,5-diarylsubstituted 1,2,3-triazole derivatives (B,
Fig. 1), whose heterocyclic ring represents the spacer between
the two phenyl rings; these compounds possessed a moderate
vasorelaxing activity in addition, the structure–activity rela-
tionships indicated that a phenolic function in the ortho posi-
tion of the aromatic ring was useful for the pharmacological
activity, whilst a primary amino function strongly decreased
it. Among these compounds, the 1-(2′-hydroxy-5′-
nitrophenyl)-5-phenyl-1H-1,2,3-triazole derivative showed a
good activity, exhibiting a lower efficacy parameter but a
potency index comparable to that of NS1619 (C, Fig. 1), a
BK-opener [4] chosen as reference compound.
2. Chemistry
The 2-methoxy-phenylazide (1a) [7] (Fig. 2) reacted with
benzoylacetylene (2) [8] in ethanol under reflux, to give the
expected mixture of the triazole isomers 4-benzoyl-substituted
3a and 5-benzoyl-substituted 4a in 74% yield. The gas-
chromatographic analysis showed a quantitative ratio
3a/4a = 4:1 and a crystallization from ethyl acetate allowed
the isolation (58% yield) and characterization of the isomer
3a. Similarly the 2-methoxy-5-chloro-phenylazide (1b) [9]
(Fig. 2) reacted with benzoylacetylene (2) [8], under the same
experimental conditions, to give the expected mixture of iso-
mers 3b and 4b in 86% yield and in a quantitative ratio 6:1
(by gas-chromatographic analysis). In this case also crystal-
lization from ethyl acetate provided the isomer 3b in 61%
yield.
The structure of the 4-benzoyl-substituted isomers 3a and
3b was assigned upon the basis of previously acquired con-
siderations for analogous reactions [10,11] and confirmed by
analytical, physico-chemical and spectroscopic methods.
In fact, as known [12], the addition of azides to asymmetri-
cal alkynes is regioselective, undergoing mesomeric and steric
effects which, in this case, promoted formation of the 4-
benzoyl-substituted isomer [10,11]. The gas-chromatographic
analysis of the reaction mixtures confirmed the theoric hypoth-
esis, indicating for 3a/4a a quantitative ratio 4:1, with
tR = 13.52 and tR = 7.88 min, respectively, and for 3b/4b a
ratio 6:1 with tR = 11.12 and tR = 14.77 min, respectively.
In addition, the 1,2,3-triazole, Refs. [12,13] shows that in
the 1H-NMR spectra of the isomer couples bearing substitu-
ents in the four and five positions, the triazole hydrogen in
the five position resonates at fields lower than that of the tria-
zole hydrogen in the four position. Examination of the
1H-NMR spectra of the reaction mixtures containing the iso-
mer couples 3a, 4a and 3b, 4b, shows clearly the presence of
singlet signals attributable to the triazole hydrogens, in a quan-
titative ratio 4:1 and 6:1, respectively, at chemical shift val-
ues 9.20 and 8.36 ppm for the couple 3a, 4a and 9.23 and
8.40 ppm for the couple 3b, 4b. Therefore, the isomers present
A further study about 1,4- and/or 2,4-disubstituted-1,2,3-
triazole derivatives, with/without a methylenic spacer between
the heterocyclic ring and one of the phenyl substituents [5]
(D, Fig. 1), suggested that a partially increased molecular flex-
ibility, induced by the benzyl substituent, appeared as a del-
eterious requirement uneffective for the pharmacological
activity. In any case the structure–activity relationships con-
firmed the importance of a phenolic function in the ortho posi-
tion of the phenyl substituent and the negative influence of an
amino function in the same position, suggesting the presence
of a hydrogen bond donor with appropriate acid properties
[6]. Also the introduction of a secondary or tertiary alcoholic
function, as a neutral hydrogen bond donor placed adjoining
to the four position of the 1,2,3-triazole ring [5] (E, Fig. 1),
caused a loss of activity.
In order to give a deeper explanation of the hypotheses of
structure–activity relationships, coming from the previous
studies, in this third paper concerning 1,2,3-triazole deriva-
tives, related to the general pharmacoforic structure A, we
synthesized and tested new compounds bearing further spac-
ers between the heterocyclic ring and one or both the phenyl
substituents. These spacers consist of carbonyl and a methyl-
enic groups, which confer to the structure a different molecu-
lar geometry. In particular, the planar carbonyl function, can
induce coplanar conformations of the two bound rings,
because of electronic resonance effects.
Fig. 2. a: D, EtOH; b: BBr3; c: Wolff-Kishner reduction; d: HONH2·HCl, pyridine, EtOH.

V. Calderone et al. / European Journal of Medicinal Chemistry 40 (2005) 521–528
523
Fig. 3. a: D, K2CO3, Me2CO; b: D, EtOH; c: BBr3; d: Wolff-Kishner reduction.
in larger amount with chemical shifts 9.20 and 9.23 ppm cor-
respond to the triazole compounds 4-benzoyl-substituted 3a
and 3b.
analysis). The mixture was fractionated by flash-
chromatography through silica gel and the triazole isomer
compounds were isolated in 31% and 24% yield, respec-
tively. The structure assignment to the two isomers was made
easy by the obtainment of the same isomer 9 by the 1,3-
dipolar cycloaddition reaction of 2-methoxy-benzylazide
(11a) [15] to benzoylacetylene (2) [8] (Fig. 3). This reaction
The demethylation reactions of the methoxy group of 3a
and 3b, carried out with boron tribromide in dichloromethane
at −78 °C, gave the 1-(2-hydroxyphenyl)-triazole derivatives
6a and 6b in 69% and 79% yield, respectively. The chemical
reduction of the ketone function of the benzoyl substituent of
3a and 3b, by the previously employed [10] Wolff-Kishner
reaction, caused at the same time the cleavage of the meth-
oxy substituent, to give the 4-benzyl-1-(2-hydroxyphenyl)-
triazole derivatives 5a and 5b in 77% and 71% yield, respec-
tively. Finally the benzoyltriazole 6b was converted to the
corresponding oxime 7b by the usual treatment with hydroxy-
lamine hydrochloride in ethanol in the presence of pyridine.
Under such a condition, compound 7b appeared to consist
with the expected mixture of the geometrical isomers sin/anti,
showed
a
quasi-regiospecific course, providing the
4-benzoyltriazole isomer 9 (tR = 16.49 min) in 9:1 ratio
regarding to the 5-benzoyl-substituted isomer. In this case
the structure was unequivocally assigned upon the basis of
1
the previous considerations (literature, Rf, tR, H-NMR)
reported for the characterization of 3a and 3b, obtained by
the same reaction. Compound 9 was easily isolated in high
yield (81%) by crystallization of the reaction product from
methanol.
Similarly the new 2-methoxy-5-chloro-benzylazide (11b),
by reaction with benzoylacetylene (2) [8] (Fig. 3), gave the
expected mixture of the 4- and 5-substituted triazole isomers
in a quantitative ratio 12:1, from which the 4-benzoyl-
substituted isomer 9b (tR = 15.29 min) was isolated in 75%
yield by crystallization from ethyl acetate to petroleum ether.
The 1-(substituted-benzyl)-4-benzoyl-1,2,3-triazoles 9 and
9b, treated with boron tribromide under the previously
employed experimental conditions, provided the correspond-
ing phenol derivatives 12a and 12b in 62% and 33% yield,
respectively. The same 1-(substituted-benzyl)-4-benzoyl-
1,2,3-triazoles 9 and 9b, underwent a Wolff-Kishner reduc-
tion under the experimental conditions employed for the
1
as shown by the melting range (195–230 °C) and H-NMR
spectrum with undoubled signals at 12.18 and 11.51 ppm for
the oxime proton, 9.18 and 8.61 ppm for the H-5 triazole
hydrogen and 11.02 and 10.96 ppm for the hydroxyl of the
phenolic function.
The nucleophilic substitution reaction of 2-methoxybenzyl
chloride with 4-benzoyl-1,2,3-triazole (8) [14] (Fig. 3), car-
ried out in refluxing acetone in the presence of anhydrous
potassium carbonate for 24 h, gave the mixture of the expected
triazole isomers 1-N-benzyl-substituted 9 (tR = 16.57 min)
and 2-N-benzyl-substituted 10 (tR = 9.95 min) in 71% total
yield, with a quantitative ratio 1:1 (by gas-chromatographic

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V. Calderone et al. / European Journal of Medicinal Chemistry 40 (2005) 521–528
Table 1
Both 5b and 14a exhibited TEA-sensitive effects, suggest-
ing that the vasorelaxing activity involves the activation of
K+ channels. Moreover, compound 14a was also tested in the
presence of IbTX (Iberiotoxin), selective blocker of BK chan-
nels. The toxin antagonized the effects of 14a, indicating that
BK channels probably are activated by this compound.
The analysis of the possible structure–activity relation-
ships seems to indicate that the presence of a carbonyl spacer
between the aromatic ring and position 4 of the triazole het-
erocycle is likely to be a deleterious requirement, and that a
methylene spacer is preferable, as clearly suggested by a direct
comparison of the couples of analogues 5b vs. 6b and 14a,
14b vs. 12a, 12b. The replacement of the carbonyl oxygen
atom of 6b with a hydroxyimino group (7b) seems to exert a
dramatic impact.
Experimental results: efficacy (Emax %) and potency (pIC50) values of the
tested compounds
Emax
%
PIC50
5a
5b
6a
6b
Ineffective
70 13
NC
6.75 0.08
NC
Ineffective
54 12
6.19 0.09
NC
7b
Ineffective
Ineffective
Ineffective
12a
12b
14a
14b
NS 1619
NC
NC
95
98
97
4
2
2
7.56 0.06
5.11 0.19
5.20 0.08
NC indicates that the parameter could not be calculated because of the low
efficacy (<50%).
The direct comparison of the most effective 1-N-benzyl
substituted compound 14a vs. its 1-N-phenyl substituted ana-
logue 5a seems to indicate that a more flexible conformation
due to the benzyl group is more favorable than a phenyl sub-
stituent, which probably leads to a coplanar conformation.
analogous triazole derivatives 3a and 3b, to give the expected
1-(substituted-benzyl)-4-benzyl 1,2,3-triazoles 13a and 13b
in good yield (59% and 55%, respectively). In this case, dif-
ferently from what experimented with the reduction of 3a
and 3b, only the ketone function was reduced whilst the cleav-
age of the ether function of the methoxyl did not occur. There-
fore the methoxy substituent of 13a and 13b had to be dem-
ethylated by the usual reaction with boron tribromide to give
the corresponding 2-hydroxybenzyltriazole derivatives 14a
and 14b in 48% and 37% yield, respectively.
Compounds showing
a direct link between the
2-hydroxyphenyl group and the 1 nitrogen of the triazole moi-
ety are potentiated by the presence of a chlorine atom in posi-
tion 4 of such an aromatic ring (5b vs. 5a and 6b vs. 6a). On
the contrary, this structural requirement seems to have a det-
rimental impact on the 1-N-benzyl substituted triazoles (14b
vs. 14a). To date, the state of the art about our studies on
diaryl-triazole derivatives does not allow us to recognize
whether the influences of the chlorine atom are due to elec-
tronic factors or to aspects concerning a steric hindrance.
The future synthetic efforts will be addressed to the devel-
opment of derivatives closely analogues to 14a, in order to
clarify the electronic and/or steric influences of different sub-
stituents on the two benzyl groups.
3. Pharmacology
The vasodilating effect of the novel potential BK-openers
on vascular contractile function was studied in vitro using
isolated rat aortic rings precontracted with KCl 20 mM (see
later for the pharmacological details).
4. Results and discussion
5. Experimental protocols
Since a vasorelaxing activity on endothelium-denuded rat
aortic rings is a pharmacodynamic property of well-
characterized BK-activators [16], this experimental model has
been chosen for a preliminary screening of the target com-
pounds 5a, 5b, 6a, 6b, 7b, 12a, 12b, 14a and 14b.
Compounds 5a, 6a, 7b, 12a and 12b showed weak or none
vasorelaxing efficacy, while 6b exhibited a moderate, but sig-
nificant (>50%) vasodilator efficacy, with a potency index in
the micromolar range (Table 1).
5.1. Chemistry
Melting points were determined on a Kofler hot-stage and
are uncorrected. IR spectra in nujol mulls were recorded on a
Mattson Genesis series FTIR spectrometer. 1H-NMR spectra
were recorded with a Varian Gemini 200 spectrometer in
DMSO-d6 or CDCl3, in d units, using TMS as internal stan-
dard. Mass spectra were performed with a Hewlett Packard
GC/MS System 5988 A gas-chromatographic analyses were
performed on a Shimadzu Mod. GC-17 A gas chromatogra-
phy with a flame ionization detector, using a stationary phase
SPB-5 [poly (5% diphenyl/95% dimethylsiloxane)] column
(15 m × 0.53 mm × 0.5 µm film thickness). TLC data were
obtained with Merck silica gel 60 F254 aluminum sheets, using
the elution mixtures reported for the flash-chromatographies.
Flash-column chromatographies were performed using Merck
Kieselgel 60 (230–400 mesh). Elemental analyses (C, H, N)
were within 0.4% of the theoretical values and were per-
The non-selective K+ channel blocker tetraethylammo-
nium chloride (TEA) antagonized the effects of 6b, indicat-
ing a possible role of these ion channels in the mechanism of
action. The efficacy parameters reached an appreciable level
for compound 5b, which showed an interesting potency (about
40-fold higher then that of NS1619), and were almost full for
compounds 14a (which exhibited a potency about 200-fold
higher than that of NS1619) and 14b (with a potency param-
eter comparable with that of the reference benzimidazolone;
Table 1).

V. Calderone et al. / European Journal of Medicinal Chemistry 40 (2005) 521–528
525
formed on a Carlo Erba ElementalAnalyzer Mod. 1106 appa-
ratus. Petroleum ether corresponds to the fraction boiling at
40–60 °C.
1H-NMR (DMSO-d6): d 4.07 (s, 2H, CH2), 6.92–7.59 (m,
9H, aromatics); 8.24 (s, 1H, H5 triazole); 10.47 (s, 1H, OH,
exchangeable).
Anal. for C15H13N3O. Calc. %: C, 71.70; H, 5.21; N, 16.72.
Found %: C, 71.66; H, 5.21; N, 16.77.
5.1.1. 1-(2-Methoxyphenyl)-4-benzoyl-1H-1,2,3-triazole
(3a)
5.1.4. 1-(2-Hydroxy-5-chlorophenyl)-4-benzyl-1H-1,2,3-
triazole (5b)
A mixture of 2-methoxy-phenylazide (1a) (0.50 g,
3.35 mmol) and benzoylacetylene (2) (0.48 g, 3.69 mmol) in
EtOH (10 ml) was stirred and heated under reflux for 22 h.
After cooling, the dark solution was evaporated in vacuo and
the resulting yellow solid (0.69 g, yield 74%; m.p. 80–84 °C)
consisted of a mixture of the title compound 3a and the
5-substituted isomer 4a in a 4:1 ratio, respectively (by gas-
chromatographic analysis). This mixture was separated by
crystallization from EtOAc to achieve the isomer 3a as pale
yellow needles: 0.54 g, yield 58%, m.p. 99–101 °C, IR
1648 cm–1 (CO). Mass (m/z): 279 (M+); 105 (100%).
1H-NMR (DMSO-d6): d 3.88 (s, 3H, OCH3), 7.19–8.30
(m, 9H, aromatics), 9.20 (s, 1H, H5 triazole).
A mixture of benzoyltriazole 3b (0.60 g, 1.92 mmol), 98%
hydrazine hydrate (0.6 ml, 11.52 mmol) and diethylene gly-
col (15 ml) was heated at 100 °C for 1 h. After cooling, the
yellow solution was added of KOH pellets (0.77 g) and the
resulting suspension was warmed on a boiling water-bath until
most of the pellets were dissolved. The flask was provided
with an air-condenser and an internal thermometer then it was
heated at 190 °C for 4 h. After cooling, the solution was
poured, drop by drop, into H2O (100 ml) then acidified (pH
6) with 37% HCl. The gray solid precipitated was collected
by filtration (0.39 g, yield 71%) and purified by crystalliza-
tion from MeOH: 0.32 g, yield 58%, m.p. 176–179 °C.
1H-NMR (DMSO-d6): d 4.08 (s, 2H, CH2), 7.08–7.69 (m,
8H, aromatics), 8.30 (s, 1H, H5 triazole), 10.85 (s, 1H, OH,
exchangeable).
1H-NMR (CDCl3): d 3.95 (s, 3H, OCH3), 7.12–8.51 (m,
9H, aromatics), 8.87 (s, 1H, H5 triazole).
Anal. for C16H13N3O2. Calc. %: C, 68.81; H, 4.69; N,
15.05. Found %: C, 69.12; H, 4.87; N, 15.23.
Anal. for C15H12ClN3O. Calc. %: C, 63.05; H, 4.23; N,
14.71. Found %: C, 63.3; H, 4.20; N, 14.81.
5.1.2. 1-(2-Methoxy-5-chlorophenyl)-4-benzoyl-1H-1,2,3-
triazole (3b)
5.1.5. 1-(2-Hydroxyphenyl)-4-benzoyl-1H-1,2,3-triazole
(6a)
A mixture of 2-methoxy-5-chlorophenylazide (1b) (1.24 g,
6.78 mmol) and benzoylacetylene (2) (0.97 g, 7.46 mmol) in
EtOH (25 ml) was stirred and heated under reflux for 24 h
After cooling, the dark solution was evaporated in vacuo and
the resulting yellow solid (1.83 g, yield 86%) consisted of
the title compound 3b and the 5-substituted isomer 4b in a
6:1 ratio, respectively (by gas-chromatographic analysis). This
mixture was separated by crystallization from EtOAc to give
the isomer 3b as pale brown plates: 1.30 g, yield 61%, m.p.
133–135 °C. Mass (m/z): 279 (M+); 105 (100%).
To a stirred solution of 3a (0.40 g, 1.43 mmol) in 20 ml of
anhydrous CH2Cl2, cooled at –78 °C and under a nitrogen
flow, a solution of BBr3 (1.0 ml, 10.53 mmol) in 5 ml of anhy-
drous CH2Cl2 was added dropwise. Stirring was continued
for 1 h, then the solution was left at room temperature over-
night.Afterwards, the mixture was cooled again in an ice-salt
bath and the reagent was decomposed by treatment with
MeOH (7 ml) followed by H2O (7 ml). The organic layer was
washed with H2O, dried (MgSO4) and evaporated in vacuo,
to give the title compound as a pale brown solid (0.32 g, yield
84%), which purified by crystallization from EtOAc appeared
as shiny thin plates: 0.26 g, 69%, m.p. 187–189 °C. Mass
(m/z): 265 (M+); 77 (100%).
1H-NMR (DMSO-d6): d 3.88 (s, 3H, OCH3), 7.40–8.28
(m, 8H, aromatics), 9.23 (s, 1H, H5 triazole).
Anal. for C16H12ClN3O2. Calc. %: C, 61.25; H, 3.86; N,
13.39. Found %: C, 61.28; H, 4.14; N, 13.31.
1H-NMR (DMSO-d6): d 7.00–8.30 (m, 9H, aromatics);
9.17 (s, 1H, H5 triazole); 10.74 (s, 1H, OH, exchangeable).
Anal. for C15H11N3O2. Calc. %: C, 67.92; H, 4.18; N,
15.84. Found %: C, 68.28; H, 4.20; N, 16.13.
5.1.3. 1-(2-Hydroxyphenyl)-4-benzyl-1H-1,2,3-triazole (5a)
A mixture of benzoyltriazole 3a (1.0 g, 3.58 mmol), 98%
hydrazine hydrate (1.0 ml, 21.48 mmol) and diethylene gly-
col (15 ml) was heated at 100 °C for 1 h. After cooling, 1.4 g
of KOH pellets were added and the suspension was warmed
on a boiling water-bath until most of the pellets were dis-
solved. The flask was provided with an air-condenser and an
internal thermometer then it was heated at 190 °C for 4 h.
After cooling, the solution was poured, drop by drop, into
H2O (200 ml) then acidified (pH 6) with 37% HCl. The white
solid precipitated was collected by filtration (0.83 g, yield
92%) and purified by crystallization from EtOAc: 0.69 g, yield
77%, m.p. 138–141 °C; IR 3401 cm–1 (OH), 1229 cm–1 (C-O).
Mass (m/z): 250 (M+); 118 (100%).
5.1.6. 1-(2-Hydroxy-5-chlorophenyl)-4-benzoyl-1H-1,2,3-
triazole (6b)
To a stirred solution of 3b (0.90 g, 2.87 mmol) in 40 ml of
anhydrous CH2Cl2, cooled at –78 °C and under a nitrogen
flow, a solution of BBr3 (2.0 ml, 21.06 mmol) in 10 ml of
anhydrous CH2Cl2 was added dropwise. Stirring was contin-
ued for 2 h, then the solution was left at room temperature for
2 h. Afterwards, the mixture was cooled again in an ice-salt
bath and the reagent was decomposed by treatment with
MeOH (14 ml) followed by H2O (14 ml). The organic layer

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V. Calderone et al. / European Journal of Medicinal Chemistry 40 (2005) 521–528
was washed with H2O, then extracted with 10% NaOH.Acidi-
fication of the alkaline extract provided a yellow precipitate
which was collected by filtration (0.79 g, yield: 92%). This
solid was purified by crystallization from a mixture of
MeOH/H2O, to give 6b as pale yellow needles: 0.68 g, yield
79%, m.p. 210–212 °C.
5.1.9. 1-(2-Methoxybenzyl)-4-benzoyl-1H-1,2,3-triazole (9)
A mixture of 2-methoxy-benzylazide (11a) (1.0 g,
6.13 mmol) and benzoylacetylene (2) (0.48 g, 3.69 mmol) in
EtOH (30 ml) was stirred and heated under reflux for 24 h.
After cooling, the solution was evaporated in vacuo to give a
residue consisting of a mixture of the triazole derivative 9
and its 5-substituted isomer in a 9:1 ratio (by G.C. analysis).
The title compound was obtained by crystallization from
MeOH, as a yellow solid: 1.46 g, yield 81%, m.p. 83–85 °C.
Mass (m/z): 293 (M+); 91 (100%).
1H-NMR (DMSO-d6): d 7.14–8.29 (m, 8H, aromatics);
9.20 (s, 1H, H5 triazole); 11.08 (s, 1H, OH, exchangeable).
Anal. for C15H10ClN3O2. Calc. %: C, 60.11; H, 3.36; N,
14.02. Found %: C, 60.31; H, 3.49; N, 13.86.
5.1.10. 2-Methoxy-5-chloro-benzylazide (11b)
5.1.7. [1-(5-Chloro-2-hydroxy-phenyl)-1H-1,2,3triazol-4-
yl]-phenyl-ketone oxime (7b)
A mixture of 5-chloro-2-methoxy-benzylchloride (0.90 g,
4.71 mmol), EtOH (15 ml), H2O (1.0 ml) and NaN3 (0.40 g,
6.15 mmol) was heated under reflux for 24 h. After cooling,
the inorganic residue was filtered off and the resulting solu-
tion was concentrated, diluted with H2O and extracted with
CHCl3. The combined organic extracts were dried (MgSO4)
and evaporated to give an oily residue (0.926 g) which was
dissolved in EtOAc and purified by filtration through a short
column of silica gel. Evaporation of the solvent left the title
azide as a yellow oil, which was directly utilized: 0.766 g,
yield 83%, IR 2102 cm–1 (N3).
A mixture of 6b (0.30 g, 1.05 mmol), NH2OH~HCl (0.50 g,
7.19 mmol) and pyridine (1.0 ml) in EtOH (15 ml) was heated
under reflux for 1 h. Evaporation in vacuo of the solution
gave a yellow oil which solidified after addition of few drops
of H2O. The white solid was purified by crystallization from
EtOH/H2O, to give the title compound as white plates:
0.215 g, yield 65%, m.p. 195–230 °C.
1H-NMR (DMSO-d6): d 7.10–7.79 (m, 8H, aromatics),
8.61 and 9.18 (s, 1H, H5 triazole), 10.96 and 11.02 (broads,
1H, OH exchangeable), 11.51 and 12.18 (s, 1H, OH oxime,
exchangeable).
Anal. for C15H11ClN4O2. Calc. %: C, 57.24; H, 3.52; N,
17.80. Found %: C, 57.14; H, 3.64; N, 17.92.
1H-NMR (DMSO-d6): d 3.81 (s, 3H, OCH3), 4.37 (s, 2H,
CH2), 7.06–7.42 (m, 3H, aromatics).
5.1.11. 1-(2-Methoxy-5-chlorobenzyl)-4-benzoyl-1H-1,2,3-
triazole (9b)
5.1.8. 1-(2-Methoxybenzyl)-4-benzoyl-1H-1,2,3-triazole (9)
and 2-(2-methoxybenzyl)-4-benzoyl-2H-1,2,3-triazole (10)
To a solution of 4-benzoyl-1,2,3-triazole (8) (1.0 g,
5.77 mmol) and 2-methoxy-benzylchloride (0.83 ml,
6.0 mmol) in anhydrous acetone (35 ml), anhydrous K2CO3
(2.0 g, 14.47 mmol) was added and the suspension was heated
under reflux for 22 h. The reaction mixture was concentrated
in vacuo, the oily residue was dissolved in CHCl3 and the
solution was washed with H2O. The combined organic layers
were dried (MgSO4) and evaporated in vacuo to give a semi-
solid residue (1.21 g, yield 71%), consisting of a mixture of
the title compounds. The gas-chromatographic analysis indi-
cated a 1:1 ratio of the two triazole isomers. Their separation
was achieved by flash-chromatography through a silica gel
column, eluting with a 1:4 mixture of EtOAc/petroleum ether.
At first compound 10 was obtained, followed from the iso-
mer 9.
A mixture of 2-methoxy-5-chloro-benzylazide (11b)
(0.65 g, 3.31 mmol) and benzoylacetylene (2) (0.43 g,
3.31 mmol) in EtOH (20 ml) was stirred and heated under
reflux for 24 h. After cooling, the solution was evaporated in
vacuo to give a residue consisting with a mixture of the tria-
zole derivative 9b and its 5-substituted isomer. The title com-
pound was obtained by crystallization from EtOAc/petroleum
ether, as a yellow solid: 0.81 g, yield 75%, m.p. 117–120 °C.
1H-NMR (DMSO-d6): d 3.83 (s, 3H, OCH3), 5.65 (s, 2H,
CH2), 7.08–8.24 (m, 8H, aromatics), 8.88 (s, 1H, H5 triaz-
ole).
Anal. for C17H14ClN3O2. Calc. %: C, 62.30; H, 4.31; N,
12.82. Found %: C, 61.94; H, 4.50; N, 12.65.
5.1.12. 1-(2-Hydroxybenzyl)-4-benzoyl-1H-1,2,3-triazole
(12a)
9: 0.53 g, yield 31%, m.p. 83–85 °C, TLC: Rf: 0.28.
1H-NMR (DMSO-d6): d 3.80 (s, 3H, OCH3), 5.63 (s, 2H,
CH2), 6.93–8.24 (m, 9H, aromatics), 8.81 (s, 1H, H5 triaz-
ole).
Anal. for C17H15ClN3O2. Calc. %: C, 69.61; H, 5.15; N,
14.33. Found %: C, 69.66; H, 5.38; N, 14.38.
10: 0.40 g, yield 24%, m.p. 76–78 °C, TLC: Rf: 0.50.
1H-NMR (DMSO-d6): d 3.80 (s, 3H, OCH3), 5.74 (s, 2H,
CH2), 6.95–8.16 (m, 9H, aromatics), 8.40 (s, 1H, H4 triaz-
ole).
Anal. for C17H15ClN3O2. Calc. %: C, 69.61; H, 5.15; N,
14.33. Found %: C, 69.58; H, 5.43; N, 14.58.
To a stirred solution of 9 (0.53 g, 1.81 mmol) in 35 ml of
anhydrous CH2Cl2, cooled at –78 °C and under a nitrogen
flow, a solution of BBr3 (1.4 ml, 14.31 mmol) in 8 ml of anhy-
drous CH2Cl2 was added dropwise. Stirring was continued
for 1 h, then the solution was left at –20 °C overnight. After-
wards, the mixture was cooled in an ice-salt bath and the
reagent was decomposed by treatment with MeOH (10 ml)
followed by H2O (15 ml). The organic layer was washed with
H2O, then extracted with 10% NaOH. Acidification of the
alkaline extract provided 12a as a white precipitate which
was extracted with CHCl3. The combined organic layers were
then dried (MgSO4) and evaporated in vacuo to give a yellow

V. Calderone et al. / European Journal of Medicinal Chemistry 40 (2005) 521–528
527
solid residue which was purified by crystallization from
EtOAc/petroleum ether. The title compound appeared as pale
yellow needles: 0.312 g, yield 62%, m.p. 148–150 °C. Mass
(m/z): 279 (M+); 107 (100%).
nal thermometer, was then heated at 190 °C for 4 h. After
cooling, the solution was poured, drop by drop, into H2O
(150 ml) and the title compound precipitated as a gray solid
which was collected by filtration and purified by crystalliza-
tion from EtOAc/petroleum ether: 0.53 g, yield 55%, m.p.
146–147 °C.
1H-NMR (DMSO-d6): d 5.62 (s, 2H, CH2), 6.81–8.24 (m,
9H, aromatics), 8.78 (s, 1H, H5 triazole), 9.98 (s, 1H, OH,
exchangeable).
1H-NMR (DMSO-d6): d 3.79 (s, 3H, OCH3), 3.98 (s, 2H,
CH2 in the four position), 5.47 (s, 2H, CH2 in the one posi-
tion), 7.05–7.41 (m, 8H, aromatics), 7.83 (s, 1H, H5 triazole).
Anal for C17H16ClN3O. Calc. %: C, 65.07; H, 5.14; N,
13.39. Found %: C, 65.40; H, 5.01; N, 13.18.
Anal. for C16H13N3O2. Calc. %: C, 68.81; H, 4.69; N,
15.05. Found %: C, 68.68; H, 4.80; N, 14.98.
5.1.13. 1-(2-Hydroxy-5-chlorobenzyl)-4-benzoyl-1H-1,2,3-
triazole (12b)
To a stirred solution of 9b (0.49 g, 1.50 mmol) in 30 ml of
anhydrous CH2Cl2, cooled at –78 °C and under a nitrogen
flow, a solution of BBr3 (1.35 ml, 14.31 mmol) in 8 ml of
anhydrous CH2Cl2 was added dropwise. The reaction was
worked up as for the preparation of 12a. Acidification of the
alkaline extract provided 12b as a white precipitate which
was collected by filtration (0.34 g) and crystallized from
MeOH/H2O: 0.154 g, yield 33%; m.p. 273–276 °C.
1H-NMR (DMSO-d6): d 5.68 (s, 2H, CH2), 7.15–8.19 (m,
8H, aromatics), 7.98 (s, 1H, H5 triazole), 10.78 (s, 1H, OH,
exchangeable).
5.1.16. 1-(2-Hydroxybenzyl)-4-benzyl-1H-1,2,3-triazole
(14a)
To a stirred solution of 13a (0.27 g, 0.966 mmol) in 30 ml
of anhydrous CH2Cl2, cooled at –78 °C and under a nitrogen
flow, a solution of BBr3 (0.8 ml, 8.46 mmol) in 8 ml of anhy-
drous CH2Cl2 was added dropwise. Stirring was continued
for 1 h, then the solution was left at –20 °C overnight. After-
wards, the mixture was cooled again in an ice-salt bath and
the reagent was decomposed by treatment with MeOH (5 ml)
followed by H2O (7 ml). The organic layer was separated and
the aqueous phase was washed several times with CH2Cl2.
The combined organic layers were dried (MgSO4) and evapo-
rated in vacuo to give an oily residue which, by refluxing
with a few ml of hexan, provided a solid residue which was
collected by filtration (0.17 g, yield 63%). This latter was
purified by crystallization from MeOH/H2O, to give the title
compound as colorless plates: 0.13 g, yield 48%; m.p. 112–
115 °C.
Anal. for C16H12ClN3O2. Calc. %: C, 61.25; H, 3.86; N,
13.39. Found %: C, 61.44; H, 3.79; N, 13.56.
5.1.14. 1-(2-Methoxybenzyl)-4-benzyl-1H-1,2,3-triazole
(13a)
A mixture of benzoyltriazole 9 (0.70 g, 2.39 mmol), 98%
hydrazine hydrate (0.70 ml, 14.34 mmol) and diethylene gly-
col (25 ml) was heated at 100 °C for 1 h. After cooling, the
solution was added of KOH pellets (1.30 g) and the resulting
suspension was warmed on a boiling water-bath until most of
the pellets were dissolved. The flask was provided with an
air-condenser and an internal thermometer then it was heated
at 190 °C for 4 h. After cooling, the solution was poured,
drop by drop, into H2O (130 ml) and the resulting cloudy
solution was left at room temperature overnight. The precipi-
tate was collected by filtration, washed with H2O and crys-
tallized from EtOAc/petroleum ether to give the title com-
pound as gray and transparent needles: 0.395 g, yield 59%,
m.p. 89–91 °C.
1H-NMR (DMSO-d6): d 3.97 (s, 2H, CH2 in the four posi-
tion), 5.43 (s, 2H, CH2 in the one position), 6.73–7.33 (m,
9H, aromatics), 7.73 (s, 1H, H5 triazole), 9.87 (s, 1H, OH,
exchangeable).
Anal. for C16H15N3O. Calc. %: C, 72.43; H, 5.70; N, 15.84.
Found %: C, 72.52; H, 5.69; N, 15.95.
5.1.17. 1-(2-Hydroxy-5-chlorobenzyl)-4-benzyl-1H-1,2,3-
triazole (14b)
To a stirred solution of 13b (0.50 g, 1.59 mmol) in 30 ml
of anhydrous CH2Cl2, cooled at –78 °C and under a nitrogen
flow, a solution of BBr3 (1.35 ml, 14.31 mmol) in 8 ml of
anhydrous CH2Cl2 was added dropwise. Stirring was contin-
ued for 1 h, then the solution was left at room temperature
overnight. Afterwards, the mixture was cooled again in an
ice-salt bath and the reagent was decomposed by treatment
with MeOH (10 ml) followed by H2O (15 ml). The organic
layer was washed with H2O, then extracted with 10% NaOH.
Acidification of the alkaline extract provided 14b as a white
precipitate which was collected by filtration and purified by
crystallization from MeOH/H2O: 0.177 g, yield 37%; m.p.
265–266 °C.
1H-NMR (DMSO-d6): d 3.80 (s, 3H, OCH3), 3.97 (s, 2H,
CH2 in the four position), 5.47 (s, 2H, CH2 in the one posi-
tion), 6.88–7.37 (m, 9H, aromatics), 7.77 (s, 1H, H5 triazole).
Anal. for C17H17N3O. Calc. %: C, 73.10; H, 6.13; N, 15.04.
Found %: C, 73.28; H, 6.16; N, 14.82.
5.1.15. 1-(2-Methoxy-5-chlorobenzyl)-4-benzyl-1H-1,2,3-
triazole (13b)
A mixture of benzoyltriazole 9b (1.00 g, 3.05 mmol), 98%
hydrazine hydrate (1.0 ml) and diethylene glycol (25 ml) was
heated at 100 °C for 1 h.After cooling, the solution was added
of KOH pellets (1.50 g) and the resulting mixture was warmed
on a boiling water-bath until most of the pellets were dis-
solved. The flask, provided with an air-condenser and an inter-
1H-NMR (DMSO-d6): d 3.87 (s, 2H, CH2 in the four posi-
tion), 5.45 (s, 2H, CH2 in the one position), 7.15–7.68 (m,
8H, aromatics), 8.11 (s, 1H, H triazole), 10.72 (s, 1H, OH,
exchangeable).

528
V. Calderone et al. / European Journal of Medicinal Chemistry 40 (2005) 521–528
Anal. for C16H14ClN3O. Calc. %: C, 64.11; H, 4.71; N,
14.02. Found %: C, 64.20; H, 4.69; N, 14.11.
diately before the pharmacological experimental procedures.
Previous experiments showed a complete ineffectiveness of
the vehicles.
5.1.18. Pharmacology
The vasorelaxing efficacy was evaluated as maximal
vasorelaxing response, expressed as a percentage (%) of the
contractile tone induced by KCl 20 mM. When the limit con-
centration 0.1 mM (the highest concentration, which could
be administered) of the tested compounds did not reach the
maximal effect, the parameter of efficacy represented the
vasorelaxing response, expressed as a percentage (%) of the
contractile tone induced by KCl 20 mM, evoked by this limit
concentration. The parameter of potency was expressed as
pIC50, calculated as negative Logarithm of the molar concen-
tration of the tested compounds, evoking a half reduction of
the contractile tone induced by KCl 20 mM. Those com-
pounds showing an efficacy parameter lower than 50% were
considered uneffective. The parameters of efficacy and
potency were expressed as mean standard error, for
5–10 experiments. Student’s t-test was selected as a statisti-
cal analysis, P < 0.05 was considered representative of a sig-
nificant statistical difference. Experimental data were ana-
lyzed by a computer fitting procedure (software: Graph Pad
Prism 3.0).
All the experimental procedures were carried out follow-
ing the guidelines of the European Community Council Direc-
tive 86-609.
A possible vasodilator mechanism of action was investi-
gated by testing the effects of the compounds on isolated tho-
racic aortic rings of male normotensive Wistar rats (250–
350 g). After a light ether anesthesia, the rats were sacrificed
by cervical dislocation and bleeding. The aortae were imme-
diately excised and freed of extraneous tissues. The endothe-
lial layer was removed by gently rubbing the intimal surface
of the vessels with a hypodermic needle. Five millimeters
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
1.05; NaH2PO4 0.41; NaHCO3 11.9; glucose 5.5), thermo-
stated 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
with a unirecord microdynamometer (Buxco Electronics).
After an equilibration period of 60 min, the endothelial
removal was confirmed by the administration of acetylcho-
line (ACh) (10 µM) to KCl (20 mM)-precontracted vascular
rings.A relaxation < 10% of the KCl-induced contraction was
considered representative of an acceptable lack of the endot-
helial layer, while the organs, showing a relaxation ≥ 10%
(i.e. significant presence of the endothelium), were dis-
carded. From 30 to 40 min after the confirmation of the endot-
helium removal, the aortic preparations were contracted by
KCl (20 mM) and when the contraction reached a stable pla-
teau, threefold increasing concentrations of the tested com-
pounds 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 min.
In other sets of experiments, the non-selective potassium
channel blocker TEA (10 mM) or the BK-selective blocker
Iberiotoxin (IbTX, 100 nM) were added, after the KCl
(20 mM)-induced contraction, followed by the administra-
tion of selected compounds.
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 mM)
in EtOH 95% and further diluted in bidistilled water, KCl
was dissolved (2 M) in Tyrode solution, TEA (Sigma) was
dissolved (10 mM) in Tyrode solution, IbTX (Sigma) was
dissolved (100 nM) in bidistilled water. All the synthesized
derivatives (5a, 5b; 6a, 6b; 7b; 12a, 12b; 14a, 14b) were
dissolved (10 mM) in DMSO; they all were further diluted in
Tyrode solution.All the solutions were freshly prepared imme-
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