1,4- and 2,4-substituted-1,2,3-triazoles as potential potassium channel activators. VII

Article abstract of DOI:10.1016/j.farmac.2005.03.004

New 1,4- and 2,4-substituted 1,2,3-triazole derivatives were synthesized and tested as potential BK_Ca channel openers, as a part of a research program, which hypothesizes a pharmacophoric structure containing the 1,2,3-triazole ring. The struct

Full text of DOI:10.1016/j.farmac.2005.03.004

Il Farmaco 60 (2005) 367–375
http://france.elsevier.com/direct/FARMAC/
Original article
1,4- and 2,4-substituted-1,2,3-triazoles as potential
potassium channel activators. VII
Vincenzo Calderone b, Irene Giorgi a, Oreste Livi a,*, Enrica Martinotti b,
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 29 September 2004; received in revised form 28 February 2005; accepted 5 March 2005
Available online 28 April 2005
Abstract
New 1,4- and 2,4-substituted 1,2,3-triazole derivatives were synthesized and tested as potential BKCa channel openers, as a part of a
research program, which hypothesizes a pharmacophoric structure containing the 1,2,3-triazole ring. The structure–activity relationships were
studied introducing some structural changes concerning molecular geometry and the presence of a hydrogen bond donor as a primary amino
group and a phenolic or alcoholic hydroxy function. The compounds were prepared by nucleophilic substitution on the 1,2,3-triazole ring and
by 1,3-dipolar cycloaddition of azides to selected alkynes and to phenylacetone. The new compounds tested on rat aortic rings did not exhibit
any significant vasorelaxing activity.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Potassium channels; Potassium channel openers; BK-activators; 1,2,3-Triazoles; Vasodilator activity
1. Introduction
As a development of a larger study [1–5], we have under-
taken a research program regarding synthesis and pharmaco-
logical evaluation of new 1,2,3-triazole derivatives, corre-
sponding to the general formula A (Fig. 1), as potential
potassium channel (BKCa) activators. In Fig. 1 the reference
benzimidazolones NS004 and NS1619 (B) as well as their
1,2,4-triazol-3-one active analog C [6] are also reported.
The open structure A, consisting of two substituted aro-
matic rings bonded to a heterocyclic linker, represents a phar-
macophoric pattern for effective BK-activators [6–9]. The
activation of potassium channels allows the concentration-
dependent passage of potassium ions to the extracellular phase
and, consequently, it causes membrane hyperpolarization and
reduction of cellular excitability [10]. Therefore, compounds
able to open selectively the BK channels, afford a new thera-
peutic approach for several pathological conditions such as
Fig. 1. General formula A: R1, R2, R3, R4 = hydrogen bond donors and/or
asthma, urge incontinence and bladder spasm, gastric hyper-
motility, hypertension, coronary artery spasm, psychoses [11].
acceptors and/or electron withdrawing groups. (X) and/or (Y): when
present = CH2, CHOH, CO groups. Comparison benzimidazolones B: NS004
(R = Cl) and NS1619 (R = CF3), their deannulated and structurally-related
active analog C. General formula of 4,5-diaryl-1,2,3-triazoles (D), where
EWG indicates withdrawing groups.
* Corresponding author. Tel.: +39 050 221 9551; fax: +39 050 221 9605.
E-mail address: livi@farm.unipi.it (O. Livi).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.03.004

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V. Calderone et al. / Il Farmaco 60 (2005) 367–375
In the previous paper of this research program [12], 1,5-
decomposition. Thus, we prepared the 4-phenyl-1,2,3-
triazole (1) (Scheme 1), which had been reported in the lit-
erature by several synthetic procedures [15]. We made use of
the 1,3-cycloaddition reaction between sodium azide and phe-
nylacetylene [16] and, lengthening the reaction time, the prod-
uct yield was improved. The 4-phenyltriazole (1) underwent
a nucleophilic substitution reaction with 2-nitrobenzyl chlo-
ride in acetone in the presence of anhydrous potassium car-
bonate (Scheme 1) to give a mixture of the 1,2,3-triazole iso-
mers 1-(2-nitrobenzyl)-substituted (2) and 2-(2-nitrobenzyl)-
substituted (3) in 63% yield and in a 1:1.7 ratio respectively
(by gas-chromatographic analysis). This mixture was frac-
tionated by flash-chromatography through silica gel and elu-
tion with a 1:2 mixture of EtOAc/petroleum ether provided
the isomer 3 (44% yield) followed from the isomer 2 (16%
yield).
disubstituted-1,2,3-triazole derivatives D (Fig. 1) were stud-
ied. Only the 1-(2′-hydroxy-5′-nitrophenyl)-5-phenyl-1H-
1,2,3-triazole showed appreciable effectiveness, exhibiting a
lower efficacy parameter but a potency index comparable to
that of the reference BKCa opener NS1619 [13]. In this paper
new 1,2,3-triazole derivatives bearing aryl substituents in posi-
tions 1,4- or 2,4 respectively, with/without one methylene as
a spacer group, are taken into consideration, to further evalu-
ate the pharmacological effect of the 1,2,3-triazole ring as a
heterocyclic linker between two aromatic or cycloalkyl sub-
stituents. These substituents are placed one on the nitrogen
atom in the position 1 or 2, the other in the position 4 or 5 of
the triazole ring respectively, so that two unsubstituted nitro-
gen atoms of the heterocyclic nucleus could act as hydrogen
bond acceptors.
The attribution of the structure to the isomers 2 and 3 was
performed by evaluation of their physical and spectroscopic
properties and indirectly by a chemical demonstration. In fact
from the nucleophilic substitution reaction, only two of the
three expected isomers were isolated and upon the basis of
steric hindrance considerations, the more probable isomers
appeared to be those substituted on the 1,2,3-triazole ring in
position 1 or 2 respectively. Therefore, as a chemical demon-
stration, we synthesized the mixture of 1-(2-nitrobenzyl)-
triazole isomer (2) and 3-(2-nitrobenzyl)-triazole isomer (5)
(Scheme 1), by 1,3-dipolar cycloaddition reaction of 2-nitro-
benzyl azide (4) [17] to phenylacetylene.
2. Chemistry
At first we undertook the preparation of new 1,2,3-triazole
derivatives bearing a phenyl substituent in the 4 position and
a benzyl substituent on one of the nitrogen atoms of the ring,
in order to introduce a methylenic bridge as a further spacer
group between the two aromatic rings. This spacer supplies
better molecular flexibility, which was shown to be a valid
structural feature in designing BKCa-openers [14]. In addi-
tion the nitro group on the benzyl substituent was selected
planning to convert it into a hydroxyl function via reduction
to amine, formation of the diazonium salt and its hydrolytic
This reaction is not regiospecific and provided the mixture
of the isomers 2 and 5 in a ≈1:1 ratio, in high yield. The reac-
Scheme 1. (a) K2CO3, Me2CO; (b) H2, Pd/C; (c) D, toluene.

V. Calderone et al. / Il Farmaco 60 (2005) 367–375
369
tion had been reported in the literature [18] and the Jordan
authors also described the separation of the two isomers by
preparative chromatography, although the physico-chemical
properties of the two compounds are practically identical.
Really the Rf values of the TLC analysis almost overlap as
well as the chemical shift values of the H5-triazole and
H4-triazole in the proton NMR spectra in CDCl3. Neverthe-
less, examination of the same isomer mixture prepared by us,
showed that 1- and 3-substituted 1,2,3-triazole compounds 2
and 5 have different retention time (tR) in gas-chroma-
tographic analysis, i.e. 14.18 min (amount 51%) and 8.82 min
(amount 44%) respectively. Since the previously obtained iso-
mers 2 and 3, probably substituted on the nitrogen in the 1 or
2 position of the triazole ring, show tR values corresponding
to 14.38 min (amount 37%) and 8.28 min (amount 62%)
respectively, this result allows us to assign the 1-substituted
structure, corresponding to isomer 2, to the compound with
tR 14.38 min. In addition the gas-chromatographic analysis
of the isomer mixture coming from the nucleophilic substi-
tution reaction shows, together with the peaks of compounds
2 and 3, also the presence of a third peak (1.6%) with tR
8.70 min, reasonably attributable to the triazole isomer sub-
stituted on position 3 of the ring, sterically hindered, which
was not isolated. Therefore, the attribution of the 2-substituted
structure to the isomer 3 could be deduced. A further confir-
mation of the previous structure attribution comes from the
proton spectra in DMSO-d6 of the isomers 2 and 3. In fact the
H5-triazole values are easily identified at 8.62 d and 8.35 d
respectively, whilst in CDCl3 the values overlap at 7.98 d. As
known from the literature [19], the chemical shift value of
the H5-triazole resonates at fields lower than that of the
H4-triazole. Furthermore the UV spectra of isomers 2 and 3
show kmax values at 248 nm and 256 nm respectively, indi-
cating that the 2-substituted isomer 3 shifts towards greater
k. This agrees with the literature, which suggests a batochro-
mic shift for the 2-substituted 1,2,3-triazole derivatives [20].
An attempt to prepare the 1-(2-nitrobenzyl)-5-phenyl-1H-
1,2,3-triazole isomer (5), by ionic cycloaddition between
2-nitrobenzylazide (4) [17] and ethyl benzoylacetate to obtain
the corresponding 4-carbethoxy-5-phenyl triazolester, which
by hydrolysis and decarboxylation should give the expected
compound 5, failed. In fact, the ethyl benzoylacetate, under
the basic experimental conditions required for the reaction,
underwent the characteristic acid cleavage of the b-ketoesters.
The nitrogroup of 2-(2-nitrobenzyl)-4-phenyl-2H-1,2,3-
triazole (3) was reduced with stannous chloride or better by
catalytic hydrogenation, to give the expected 2-benzylamino-
triazole derivative (6). Some attempts at converting 6 to the
corresponding phenol derivative failed. However, we thought
it interesting to evaluate the pharmacological effect of the
amino function, as a hydrogen bond donor, but with basic
properties reversed to those of a phenol function, whose acid
characteristics appear to have useful role for BK-channel
activity [21]. The benzylamino derivative 6 showed a low
vasorelaxing activity on rat aortic rings, so that, in consider-
ation of the difficulty to obtain the corresponding phenol
derivative by this route, the 1-(2-nitrobenzyl)-triazole isomer
(2) was not treated.
Such a synthetic limitation was overcome by an analogs
nucleophilic substitution reaction between 2-methoxybenzyl
chloride and 4-phenyl-1,2,3-triazole (1) [16] (Scheme 2). The
reaction was carried out in the usual manner, in acetone in
the presence of anhydrous potassium carbonate, to give a
2:1 mixture (by gas-chromatographic analysis) of the triaz-
ole isomers 7 and 8 respectively. This mixture was fraction-
ated by flash-chromatography through silica gel, obtaining
the 2-substituted triazole isomer 8 in 21% yield, followed by
the 1-substituted triazole isomer 7 in 41% yield. It is worth
noting that this nucleophilic substitution reaction on the 1,2,3-
triazole ring with 2-methoxybenzyl chloride shows a course
different from that of the previous reaction with 2-nitrobenzyl
chloride, confirming the involvement of mesomeric besides
steric factors. In fact in the first case the isomeric ratio
1-substituted triazole 2/2-substituted triazole 3 was 1:2, whilst
in this case the isomeric ratio 7/8 was reversed to 2:1. Clearly
the physico-chemical and spectroscopic properties of the two
isomers were the same and for a comparison with the previ-
ous analogs substitution reaction they directed us towards a
correct structure attribution.
So it was observed that the 2-substituted triazole isomer 8
had a Rf 0.75 greater and a tR 6.07 min lower than the
1-substituted triazole isomer 7, with values of Rf 0.45 and tR
10.88 min, respectively; the chemical shift values of the
1
H5-triazole in the H-NMR spectra were 8.26 d and 8.50 d
respectively. In addition, as previously reported, gas-chromato-
graphic analysis of the original reaction mixture showed the
presence of a third peak with tR 7.33 min (4.0%), assignable
to the 3-substituted triazole isomer 10 (Scheme 2), which had
not been isolated.
On the other hand the thermic cycloaddition reaction
between 2-methoxybenzylazide (9) [22] (Scheme 2) and phe-
nylacetylene provided a mixture of the expected isomers
1-substituted triazole 7 and 3-substituted triazole 10 in a ratio
1.3:1 (by gas-chromatographic analysis), with tR 10.86 min
and tR 7.53 min, respectively, to confirm unequivocally the
structures assigned. The methoxy group of 7 was cleft by treat-
ment with boron tribromide in CH2Cl2 at –78 °C to give the
corresponding phenol derivative 11 in 56% yield. A modified
structure without the methylenic spacer, but bearing the appro-
priate phenol function in the ortho position of the phenyl sub-
stituent and with a chlorine atom as a further substituent, was
obtained by a regiospecific cycloaddition reaction (Scheme
3). Thus the 2-methoxy-5-chloro-phenylazide (12) [23]
reacted with phenylacetone in ethanol in the presence of
sodium ethoxide, to give in moderate yield the 1-(2-methoxy-
5-chlorophenyl)-4-phenyl-5-methyl-1H-1,2,3-triazole (13),
which was converted to the corresponding 2-hydroxy deriva-
tive 14 in the usual manner, by treatment with boron tribro-
mide.
Finally a further substantial structural modification con-
sisted with the change of position of the hydrogen bond donor
function, which was introduced as an alcoholic hydroxyl in a
position adjoining to the 4 position of the 1,2,3-triazole ring.

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V. Calderone et al. / Il Farmaco 60 (2005) 367–375
Scheme 2. (a) K2CO3, Me2CO, (b) BBr3; (c) D, toluene.
Scheme 3. (a) EtONa, EtOH; (b) BBr3.
The reaction between 2-methoxy-phenylazide (15) [24] and
a-ethynyl-benzyl alcohol (Scheme 4) led to the expected mix-
ture (71% yield) of 4- and 5-substituted triazole isomers in
2.4:1 ratio respectively. Fractional crystallization of the mix-
ture allowed isolation of the 4-hydroxybenzyl substituted iso-
mer 16, in 25% yield. In this compound the secondary alco-
holic function represents a spacer inserted between the
heterocyclic ring and the phenyl and it contributes flexibility
to the molecular structure and to the hydrogen bond donor.
Similarly a tertiary alcoholic function bonded to a cy-
cloalkyl substituent, more engaged and sterically hindered
regarding the previous analog compound 16, was introduced
adjoining the 4 position of the 1,2,3-triazole ring (Scheme 5).
Thus the 4-fluoro-benzylazide (17) [25] and 2-methoxy-
benzylazide (9) [22] were reacted with 1-ethynyl-
cyclohexanol to give the corresponding 1,2,3-triazole deriva-
tives bearing substituents in the 1-position with different
mesomeric properties and with more structural flexibility
compared to compound 16. The 4-fluoro-benzylazide (17)
[25] provided the usual mixture (76% yield) of the 4- and
5-cyclohexyl substituted triazole isomers in 2:1 ratio respec-
tively. The 1-(4-fluorobenzyl)-4-(1-hydroxycyclohexyl)-1H-
1,2,3-triazole isomer (18) was present in a larger amount and
was isolated in 36% yield by fractional crystallization. The
2-methoxy-benzylazide (9) [22] provided the expected mix-
ture of the 4- and 5-substituted triazole isomers in 1.7:1 ratio

V. Calderone et al. / Il Farmaco 60 (2005) 367–375
371
Scheme 4. (a) D, DMSO 90 °C.
aortic rings. The ineffectiveness of compounds 11 and 14 can-
not be easily explained only by the lack of a carbonyl group
in the heterocyclic spacer, because also some other 1,2,3-
triazole derivatives, previously described by us, do not show
this structural pattern, but possess vasorelaxing effects involv-
ing the activation of potassium channels [12].
In order to perform a deeper investigation on the structural
requirements for the BK activating properties of diaryl-1,2,3-
triazole and to define a suitable pharmacofore model, the
development of new compounds modified in the position of
the aryl groups and/or in their substituents borne by the aro-
matic rings is needed.
Scheme 5. (a) D, toluene.
respectively. The 1-(2-methoxybenzyl)-4-(1-hydroxycyclo-
hexyl)-1H-1,2,3-triazole isomer (19) was isolated in 35.5%
yield by crystallization from ethyl acetate.
4. Experimental part
Structures of the new compounds 16, 18 and 19, coming
from regioselective cycloaddition reactions, were assigned
upon the basis of analytical and spectroscopic data as well as
other chemical considerations, as above reported for analo-
gous problems.
4.1. Chemistry
Melting points (m.p.) were determined on a Kofler hot-
stage and are uncorrected. IR spectra in nujol mulls were
recorded on a Mattson Genesis series FTIR spectrometer. UV
spectra were obtained on a Perkin–Elmer Lambda 15 UV–
1
3. Biological results and discussion
Vis spectrophotometer in EtOH. H-NMR spectra were
recorded with a Varian Gemini 200 spectrometer in DMSO-
d6, in d units, using TMS as internal standard. 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 Chromatography 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-
formed on a Carlo Erba ElementalAnalyzer Mod. 1106 appa-
ratus. Petroleum ether corresponds to the fraction boiling at
40–60 °C.
The target compounds 2, 3, 6, 11, 16, 18 and 19 can be
viewed as the structural development of previously prepared
1,5-diaryl-1,2,3-triazoles showing, in some cases, a vasore-
laxing activity probably due to the activation of potassium
channels. Hence, they were submitted to a functional test,
targeted to evaluate their possible vasodilator effects on KCl
20 mM precontracted rat aortic rings. Compounds 2, 3, 6, 16,
18 and 19 seem to differ from the casual structural features of
the most common diarylheterocyclic BK-openers, described
to date [7,8,26–28]. Typically these BK-activators show a
hydroxy group in the 2 position of one of their aryl rings and
probably this hydroxy group may be crucial for the pharma-
cological properties of such molecules. Therefore, it was not
unexpected that compounds 2, 3, 6, 16, 18 and 19, lacking
this requirement, were devoid of any vasorelaxing effect and,
consequently, of any BK-channel activating property.As con-
cerns compounds 11 and 14, their structures are relatively
similar to those exibited by some recent compounds, such as
1-(2-hydroxy-4-chloro)-phenyl-3-phenyl-1,2,4-(4H)-triazol-
5-one, which showed smooth muscle relaxant activity on a
rat bladder, due to the activation of BK channels [7]. Never-
theless 11 and 14 did not exibit any vasorelaxing effect on rat
4.1.1. 4-Phenyl-1,2,3-triazole (1)
A mixture of phenylacetylene (5.0 ml, 45.57 mmol) and
NaN3 (16.0 g, 246 mmol) in 100 ml of anhydrous DMSO
was heated at 120 °C under vigorous stirring for 11 days.
Starvation of NaN3 during the reaction led to its decomposi-
tion as already observed in the literature [29]. The reaction

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V. Calderone et al. / Il Farmaco 60 (2005) 367–375
mixture was poured into crushed ice (≈1 l), acidified (pH 3–4)
with conc. HCl and stirred at room temperature for 30 min.
The yellow precipitated solid was collected by filtration and
repeatedly washed with H2O (3.73 g). This solid was dis-
solved in 10% NaOH and the red solution was washed with
CH2Cl2 (3 × 60 ml). The aqueous layer was then acidified
(pH 3–4) and the title compound precipitated as pale yellow
needles which were collected by filtration and dried: 3.39 g,
yield 51%, m.p. 147–148 °C.
genated at room temperature and pressure. The catalyst
was filtered off, washed with hot EtOH and the filtrate was
evaporated under reduced pressure. The residue (0.280 g,
yield 68%) consisted of 6.
4.1.4. 1-(2-Methoxybenzyl)-4-phenyl-1H-1,2,3-triazole (7)
and 2-(2-methoxybenzyl)-4-phenyl-2H-1,2,3-triazole (8)
To a solution of 4-phenyl-1,2,3-triazole (1) (1.00 g,
6.89 mmol) and 2-methoxybenzyl chloride (1.0 ml, 7.0 mmol)
in 40 ml of anhydrous acetone, anhydrous K2CO3 (2.0 g) was
added and the mixture was heated under reflux for 22 h. The
reaction mixture was concentrated in vacuo and the residue
was treated with H2O and extracted with CHCl3. The com-
bined organic layers were dried (MgSO4) and evaporated to
give a semisolid residue (1.42 g) consisting essentially of a
mixture of the title compounds 7 and 8 in a 2:1 ratio respec-
tively (by gas-chromatographic analysis). This mixture was
fractionated by flash-chromatography through a silica gel col-
umn, eluting with a mixture 1:3 of EtOAc/petroleum ether to
give the 2-substituted isomer 8 followed by the 1-substituted
isomer 7.
4.1.2. 1-(2-Nitrobenzyl)-4-phenyl-1H-1,2,3-triazole (2)
and 2-(2-nitrobenzyl)-4-phenyl-2H-1,2,3-triazole (3)
To a solution of 4-phenyl-1,2,3-triazole (1) (1.00 g,
6.89 mmol) and 2-nitrobenzyl chloride (1.20 g, 6.99 mmol)
in 40 ml of anhydrous acetone, anhydrous K2CO3 (2.0 g) was
added and the mixture was heated under reflux for 22 h. The
reaction mixture was concentrated in vacuo and the residue
was treated with H2O and extracted with CHCl3. The com-
bined organic layers were dried (MgSO4) and evaporated to
give a semisolid residue consisting of a mixture of the title
compounds 2 and 3: 1.21 g, yield 63%. Gas-chromatographic
analysis indicated a relative ratio 1:2 of the two isomeric com-
pounds 2 and 3 respectively. This mixture was fractionated
by flash-chromatography through a silica gel column. Elu-
tion with a mixture 1:2 of EtOAc/petroleum ether provided
the 2-substituted isomer 3 followed by the 1-substituted iso-
mer 2.
7: 0.743 g, yield 41%; m.p. 93–96 °C from MeOH. GC:
tR = 10.88 min. TLC: Rf = 0.45. 1H-NMR (DMSO-d6): d 3.84
(s, 3H, OCH3); 5.57 (s, 2H, CH2); 6.90–7.89 (m, 9H, aromat-
ics); 8.50 (s, 1H, H triazole). Mass (m/z): 265 (M+); 91
(100%).
Anal. for C16H15N3O: C, H, N.
2: 0.190 g, yield 16%; m.p. 143–145 °C from MeOH. GC:
tR = 14.38 min. TLC: Rf = 0.83. 1H-NMR (DMSO-d6): d 6.02
(s, 2H, CH2); 7.13–8.19 (m, 9H, aromatics); 8.62 (s, 1H, H
triazole). UV (1.4 × 10–4 conc. in EtOH): kmax 248 nm; log e
3.745. Mass (m/z): 280 (M+); 116 (100%).
8: 0.390 g, yield 21%; m.p. 61–64 °C from MeOH/H2O.
GC: tR = 6.07 min. TLC: Rf = 0.75. 1H-NMR (DMSO-d6): d
3.81 (s, 3H, OCH3); 5.63 (s, 2H, CH2); 6.87–7.87 (m, 9H,
aromatics); 8.26 (s, 1H, H triazole). Mass (m/z): 265 (M+);
91 (100%).
Anal. for C16H15N3O: C, H, N.
Anal. for C15H12N4O2: C, H, N.
3: 0.520 g, yield 44%; m.p. 130–132 °C from MeOH. GC:
tR = 8.28 min. TLC: Rf = 068. 1H-NMR (DMSO-d6): d 6.08
(s, 2H, CH2); 7.11–8.17 (m, 9H, aromatics); 8.35 (s, 1H, H
triazole). UV (1.4 × 10–4 conc. in EtOH): kmax 256 nm; log e
3.792. Mass (m/z): 280 (M+); 145 (100%).
4.1.5. 1-(2-Hydroxybenzyl)-4-phenyl-1H-1,2,3-triazole (11)
To a cooled (–78 °C) and stirred solution of 7 (0.300 g,
1.13 mmol) in 50 ml of anhydrous CH2Cl2, under a nitrogen
flow, a solution of BBr3 (1.5 ml, 15.8 mmol) in 8 ml of anhy-
drous CH2Cl2 was slowly added dropwise. Stirring was con-
tinued for 2 h, then the solution was left at –20 °C for 24 h.
After 1 h at room temperature, the mixture was cooled in an
ice–salt bath and the reagent was decomposed by treating with
MeOH (10 ml) followed by H2O (10 ml). The organic layer
was washed with H2O, then extracted with 10% NaOH.Acidi-
fication of the alkaline extract provided 9 as a white precipi-
tate which was collected by filtration: 0.160 g, yield 56%;
Anal. for C15H12N4O2: C, H, N.
4.1.3. 2-(2-Aminobenzyl)-4-phenyl-2H-1,2,3-triazole (6)
A)A mixture of 2-(2-nitrobenzyl)-4-phenyl-2H-1,2,3-triazole
(3) (0.400 g, 1.43 mmol) and SnCl2 2H2O (0.966 g,
4.28 mmol) in 30 ml of 24% HCl was heated under reflux
for 4 h. The suspension obtained was filtered and the fil-
trate was treated with 10% NaOH until complete dissolu-
tion of the precipitated tin hydroxides. The new suspen-
sion was stirred for 3–4 h, then the white precipitate was
collected by filtration and washed with H2O: 0.123 g, yield
34%; m.p. 102–104 °C from MeOH/H2O. IR (m): 3430 and
1
m.p. 209–211 °C from EtOAc/petroleum ether. H-NMR
(DMSO-d6): d 5.53 (s, 2H, CH2); 6.75–7.87 (m, 9H, aromat-
ics); 8.49 (s, 1H, H triazole); 9.92 (s, 1H, OH).
Anal. for C15H13N3O: C, H, N.
1
3340 cm–1 (NH2). H-NMR (DMSO-d6): d 5.21 (s, 2H,
4.1.6. 1-(2-Methoxy-5-chlorophenyl)-4-phenyl-5-methyl-
1H-1,2,3-triazole (13)
To a stirred solution of EtONa (from 0.171 g, 7.45 mmol
of sodium in 10 ml of absolute EtOH), a solution of
2-methoxy-5-chlorophenylazide (12) (1.36 g, 7.45 mmol) and
NH2); 5.54 (s, 2H, CH2); 6.49–7.86 (m, 9H, aromatics);
8.28 (s, 1H, H triazole). Mass (m/z): 250 (M+); 106 (100%).
Anal. for C15H14N4: C, H, N.
B)To a solution of 3 (0.460 g, 1.64 mmol) in 20 ml of EtOH,
0.020 g of 5% Pd/C was added and the mixture was hydro-

V. Calderone et al. / Il Farmaco 60 (2005) 367–375
373
phenylacetone (1.00 g, 7.45 mmol) in 15 ml of absolute EtOH
was added drop by drop and the mixture was heated at 50 °C
for 4 h. The reaction mixture was evaporated in vacuo and
the black oil residue was extracted with boiling petroleum
ether (25 ml) to remove the unreacted azide. The new residue
was then extracted with boiling EtOAc, which left a unchar-
acterized black semisolid. The combined organic layers were
concentrated and purified by filtration through a silica gel col-
umn, eluting with EtOAc. Evaporation of the solvent gave
the title compound as a white solid: 0.825 g, yield 37%; m.p.
99–102 °C from EtOAc/petroleum ether. 1H-NMR (DMSO-
d6): d 2.30 (s, 3H, CH3); 3.83 (s, 3H, OCH3); 7.35–7.79 (m,
8H, aromatics).
in 25 ml of toluene was heated under reflux for 24 h. The
solvent was evaporated in vacuo and a semisolid residue, con-
sisting with the expected mixture of the 1,2,3-triazole iso-
mers, was obtained: 0.839 g, yield 76%; relative ratio of the
two isomers 1:2 by gas-chromatographic analysis (tR 8.48 and
8.99 min respectively). This residue was refluxed with 20 ml
of petroleum ether, which, after cooling, was decanted off.
The new residue, as a white crystalline solid (0.650 g, yield
59%), was dissolved in EtOAc. The solution was paper fil-
tered and cooled at –20 °C to give the title compound as big
colorless dipyramidal crystals: 0.394 g, yield 36%, m.p. 119–
122 °C from EtOAc; GC: tR = 8.48 min. 1H-NMR (DMSO-
d6): d 1.19–2.00 (m, 10 H, cyclohexyl); 4.84 (s, 1 H, OH);
5.53 (s, 2 H, CH2); 7.16–7.43 (AA′BB′, 4 H, aromatics); 7.92
(s, 1 H, H5 triazole). Mass (m/z): 275 (M+); 109 (100%).
Anal. for C15H18N3OF: C, H, N.
Anal. for C16H14N3OCl: C, H, N.
4.1.7. 1-(2-Hydroxy-5-chlorophenyl)-4-phenyl-5-methyl-
1H-1,2,3-triazole (14)
To a stirred solution of 13 (0.300 g, 1.0 mmol) in 25 ml of
anhydrous CH2Cl2, cooled at –78 °C and under a nitrogen
flow, a solution of BBr3 (1.0 ml, 10.6 mmol) in 7 ml of anhy-
drous CH2Cl2 was slowly added dropwise. The cooling bath
was removed and stirring was continued at room temperature
for 15 h. To the reaction mixture, cooled in an ice-salt bath,
was added MeOH (10 ml) followed by H2O (15 ml) to destroy
the reagent, then the organic layer was extracted with 10%
NaOH. The combined aqueous extracts were paper filtered
then acidified with conc. HCl to precipitate the title com-
pound as a white solid, which was collected by filtration:
0.231 g, yield 81%; m.p. 287–290 °C from DMF/H2O.
1H-NMR (DMSO-d6): d 2.32 (s, 3H, CH3); 7.14–7.80 (m,
8H, aromatics); 10.81 (s, 1H, OH).
4.1.10. 1-(2-Methoxybenzyl)-4-(1-hydroxycyclohexyl)-1H-
1,2,3-triazole (19)
A solution of 2-methoxy-benzylazide (9) (2.0 g,
12.25 mmol) and 1-ethynyl-cyclohexanol (1.53 g,
12.32 mmol) in 60 ml of toluene was heated under reflux for
48 h. The solvent was evaporated in vacuo and the yellow oil
residue, consisting with the expected mixture of the 1,2,3-
triazole isomers, underwent a gas-chromatographic analysis:
relative ratio of the two isomers 1:1.7 with tR = 10.93 and
11.93 min respectively. This residue was refluxed with 20 ml
of petroleum ether, which, after cooling, was decanted off.
The new residue, as a white crystalline solid (2.350 g, yield
67%), was crystallized from EtOAc to give the title com-
pound as white plates: 1.250 g, yield 35. %, m.p. 120–122 °C
Anal. for C15H12N3OCl: C, H, N.
1
from EtOAc; GC: tR = 10.98 min. H-NMR (DMSO-d6): d
4.1.8. 1-(2-Methoxyphenyl)-4-(␣-hydroxybenzyl)-1H-1,2,3-
triazole (16)
1.32–1.85 (m, 10 H, cyclohexyl); 3.83 (s, 3 H, OCH3); 4.64
(s, 1 H, OH); 5.48 (s, 2 H, CH2); 6.90–7.38 (m, 4 H, aromat-
ics); 7.76 (s, 1 H, H5 triazole). Mass (m/z): 287 (M+); 121
(100%).
A stirred solution of 2-methoxy-phenylazide (15) (0.770 g,
5.16 mmol) and a-ethynylbenzyl alcohol (1.1 ml, 8.9 mmol)
in 18 ml of DMSO was heated at 90 °C for 4 h. After cooling,
the brown solution was diluted with H2O (60 ml) and the
mixture was extracted with Et2O (3 × 90 ml). The combined
organic layers were washed with H2O (4 × 100 ml), dried
(MgSO4) and evaporated to give the expected mixture of the
1,2,3-triazole isomers as a dark oil: 1.030 g, yield 71%; rela-
tive ratio of the two isomers 1:2.4 by gas-chromatographic
analysis (tR 9.19 and 11.47 min respectively). The mixture
was dissolved in EtOAc and cooled at –20 °C to give the title
compound as pale yellow needles which were collected by
filtration: 0.360 g, yield 25%, m.p. 124–125 °C from EtOAc;
Anal. for C16H21N3O2: C, H, N.
4.2. Pharmacology
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
investigated by testing the effects of the compounds on iso-
lated thoracic aortic rings of male normotensive Wistar rats
(250–350 g). After a light ether anaesthesia, the rats were
sacrificed by cervical dislocation and bleeding. The aortae
were immediately excised and freed of extraneous tissues.
The endothelial layer was removed by gently rubbing the inti-
mal 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 (composi-
tion 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),
thermostated at 37 °C and continuously gassed with a mix-
ture of O2 (95%) and CO2 (5%). Changes in tension were
1
GC: tR = 11.48 min. H-NMR (DMSO-d6): d 3.84 (s, 3 H,
OCH3); 5.89 (d, 1 H, CH); 6.09 (d, 1 H, OH); 7.07–7.62 (m,
9 H, aromatics); 8.21 (s, 1 H, H5 triazole). Mass (m/z): 281
(M+); 77 (100%).
Anal. for C16H15N3O2: C, H, N.
4.1.9. 1-(4-Fluorobenzyl)-4-(1-hydroxycyclohexyl)-1H-
1,2,3-triazole (18)
A solution of 4-fluoro-benzylazide (17) (0.606 g,
4.00 mmol) and 1-ethynyl-cyclohexanol (0.512 g, 4.12 mmol)

374
V. Calderone et al. / Il Farmaco 60 (2005) 367–375
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After an equilibration period of 60 min, the endothelial
integrity was confirmed by the administration of acetylcho-
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solutions were freshly prepared immediately before the phar-
macological experimental procedures. Previous experiments
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