1,2,3-Triazol-carboxanilides and 1,2,3-triazol-(N-benzyl)-carboxamides as BK-potassium channel activators. XII

Article abstract of DOI:10.1016/j.ejmech.2008.02.032

The chemical structures of many synthetic activators of large-conductance calcium-activated potassium channels (BK channels) satisfy a simple pharmacophore model, consisting of two appropriately substituted phenyl rings connected by a linker of a heterogeneous nature. In this paper, a series of new compounds with modifications of the linker portion of the above pharmacophore are described. In particular, in these new derivatives, the linker portion is represented by a 1,2,3-triazole-carboxamide group, which can be viewed as a combination of two different kinds of linker, independently used in previous series of BK-openers: the amide function and the 1,2,3-triazole ring. The overall finding of this study indicated that the triazole-carboxamide derivatives were generally poorly effective and that this structural modification of the linker is deleterious for activity on BK channels. Therefore, it can be hypothesized that the increase of the steric hindrance of the linker and/or the increase of the distance between the two aromatic portions are negative for the interaction with the biological target.

Full text of DOI:10.1016/j.ejmech.2008.02.032

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European Journal of Medicinal Chemistry 43 (2008) 2618e2626
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Laboratory note
1,2,3-Triazol-carboxanilides and 1,2,3-triazol-(N-benzyl)-carboxamides
as BK-potassium channel activators. XII
b
b
b
Vincenzo Calderone a, , Francesca Lidia Fiamingo , Gabriella Amato , Irene Giorgi ,
*
Oreste Livi b, Alma Martelli a, Enrica Martinotti a
a Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, via Bonanno 6, I-56126 Pisa, Italy
b Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, via Bonanno 6, I-56126 Pisa, Italy
Received 27 July 2007; received in revised form 28 February 2008; accepted 29 February 2008
Available online 7 March 2008
Abstract
The chemical structures of many synthetic activators of large-conductance calcium-activated potassium channels (BK channels) satisfy
a simple pharmacophore model, consisting of two appropriately substituted phenyl rings connected by a linker of a heterogeneous nature. In
this paper, a series of new compounds with modifications of the linker portion of the above pharmacophore are described. In particular, in these
new derivatives, the linker portion is represented by a 1,2,3-triazole-carboxamide group, which can be viewed as a combination of two different
kinds of linker, independently used in previous series of BK-openers: the amide function and the 1,2,3-triazole ring. The overall finding of this
study indicated that the triazole-carboxamide derivatives were generally poorly effective and that this structural modification of the linker is
deleterious for activity on BK channels. Therefore, it can be hypothesized that the increase of the steric hindrance of the linker and/or the
increase of the distance between the two aromatic portions are negative for the interaction with the biological target.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: BK-potassium channel; Triazole-carboxamide; Triazole-carboxanilide; BK-activator
1. Introduction
plasmalemma, membrane hyperpolarization and reduction of
cellular excitability. Conversely, the availability of exogenous
BK-activators can represent a pharmacological tool for the
clinical management of pathological states, related to a cell
hyperexcitability, such as asthma, urge incontinence and blad-
der spasm, gastric hypermotility, neurological and psychiatric
disorders [1e3].
With regard to the cardiovascular system, BK channels
ensure the predominant component of the outward Kþ current
in vascular smooth muscle cells, playing fundamental roles in
the modulation of the muscular tone of vessels [4,5]. Conse-
quently, the vasorelaxing effects of exogenous BK-openers
can be a rational basis for treatment of hypertension and/or
other diseases related to an impaired contractility of vessels
(for example, coronary vasospasm) [1,2].
Large-conductance, calcium-activated potassium channels
(BKCa or BK channels) are distributed in both excitable and
non-excitable cells and are involved in many cellular functions
such as action potential repolarization; neuronal excitability;
neurotransmitter release; hormone secretion; tuning of
cochlear hair cells; innate immunity; and modulation of the
tone of vascular, airway, uterine, gastrointestinal, and urinary
bladder smooth muscle tissues [1e3].
The physiological activation of BK channels, induced
mainly by two triggering signals, such as the rise of the intra-
cellular free calcium ions and membrane depolarization,
ensures massive flow of potassium ions (with a single channel
conductance of 150e300 pS) to the extracellular side of the
Our research program about potential activators of BK
channels considered heterocyclic compounds [6e9] which re-
ferred to the benzimidazolone derivatives NS 004 and NS1619
* Corresponding author. Tel.: þ39 50 2219589.
E-mail address: calderone@farm.unipi.it (V. Calderone).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.032

V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 2618e2626
2619
(Fig. 1A) reported in the literature [10] as pioneer BK-openers.
A
B
HO
OH
Thereafter, based on an hypothetical simple pharmacophore
(Fig. 1B) consisting of two appropriately substituted phenyl
rings connected by a linker, 1,2,3-triazole derivatives [11e13]
and benzanilides [14e16] were synthesized and tested. In
these compounds, the heterocyclic ring and the amide func-
tion, respectively, represented the linker.
O
Heterocycle
O
Cl
Cl
N
H
NH
Heterocycle
C
D
R2
In particular, after a first paper [14] which had shown a BK
channel-induced vasorelaxing activity of N-(2-hydroxy-5-
chlorophenyl)-2-methoxy-5-chlorobenzamide higher than
NS1619, research on new compounds bearing the amide linker
was extended, changing the benzanilide structure [15] by the
introduction of a 5- or 6-membered aromatic heterocyclic sub-
stituent in the acid moiety of the amide, keeping unaltered the
basic moiety as 2-hydroxy-5-chloro-anilide (Fig. 2A). In addi-
tion, an aromatic or aliphatic 6- or 5-membered heterocyclic
substituent was introduced in the basic moiety of the amide,
keeping unaltered the acid moiety as 2-hydroxy-5-chloroben-
zoyl (Fig. 2B). The pharmacological results indicated that
the presence of nitrogen heterocycles on the acid side of the
amide linker seems to be a negative requirement, while furan
and thiophene rings were tolerated. On the contrary, the intro-
duction of unsaturated heterocyclic rings (pyridine and thia-
zole) on the basic side of the amide linker, led to biological
activity, but the presence of aliphatic heterocycles lowered
the pharmacological effect. The importance of the presence
of a phenol function as an H-bond donor was confirmed.
Further structural changes of the benzanilide pharmaco-
phore concerned a deepening of the structureeactivity rela-
tionships of benzanilide derivatives previously studied as BK
channel activators [16] (Fig. 2C). In that paper, several substi-
tutions on the phenyl rings of the reference benzanilides were
reported, which possessed particular and specific properties
from a mesomeric and/or steric point of view. The pharmaco-
logical results indicated that several compounds exhibited
vasorelaxing effects which could not be attributed to the
activation of BK channels, while two derivatives showed
a clear profile of BK-activators with a vasodilator activity
comparable to or slightly lower than that recorded for the
reference benzimidazolone NS1619.
O
(CH2)n
R1
R1
NH
R2
(CH2)n
NH
R3
O
n = 0 , 1.
R3
R4
Fig. 2. Chemical structures of previously synthesized compounds (AeD).
In this new paper, our research program on potential activa-
tors of BK channels has been developed with further changes
on the linker portion of the usual pharmacophore. For this
purpose, on the basis of our previous results [13,14], the
1,2,3-triazole ring [13] and the amide function [14] were com-
bined to give a triazole-carboxamide linker, by the synthesis
and pharmacological evaluation of a new series of 1,2,3-tria-
zol-carboxanilides and 1,2,3-triazol-(N-benzyl)-carboxamides
corresponding to the general formula reported in Fig. 3.
2. Chemistry
Scheme 1 reports the preparation of a series of 1,2,3-tria-
zol-carboxanilides bearing in the 1 position of the triazole
ring an ortho-substituted benzyl group to give the structure
the flexibility useful for the biological activity [13]. The ani-
lino substituent of the 1,2,3-triazol-4-carboxanilide bears
a phenolic hydroxyl, a well-known requirement useful for
the biological activity, in the ortho position of the phenyl
ring, in its turn bearing a chlorine substituent (compounds
7aed) or a methyl substituent (compounds 8aed).
Thus, starting from the suitable azides (2-chlorobenzyl- [18],
2-fluorobenzyl-[19], 2-trifluoromethylbenzyl-[20]or2-methox-
ybenzyl- [13]), the expected 1,2,3-triazolesters 2aed were
A third structural modification led to the introduction of
methylenic spacers between the amide linker and the two aro-
matic rings, to evaluate the biological effect caused by this
lengthening (Fig. 2D). The pharmacological results suggested
that these structural modifications were generally deleterious
[17].
d in satisfactory yields by 1,3-dipolar cycloaddition
obtaine
reaction to ethyl acetoacetate, carried out in dimethyl sulph-
oxide in the presence of anhydrous potassium carbonate.
Alkaline hydrolysis provided the corresponding carboxylic
acids 3aed which were converted to the respective acyl
chlorides 4aed, by heating with thionyl chloride. These
H
F C
N
OH
3
R
1
O
LINKER
N
R1
OH
N
R2
N
N
CH2
CH3
A
B
R
2
EWG
R3
NH
(CH2)n
O
F C
3
n = 0 : Triazolcarbxanilides ; n = 1 : Triazol-N-benzylamide
Fig. 1. Chemical structure of NS1619 (A). Pharmacophore model of BK-
activators (B). R1 and R2 represent substituents of various nature. EWG is
an electron withdrawing group.
Fig. 3. General formula of the new series of 1,2,3-triazol-carboxanilides and
1,2,3-triazol-(N-benzyl)-carboxamides.

2620
V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 2618e2626
COOEt
COOH
The presence of a methoxy group on the benzyl substituent
of 12d, 13d and 14d allowed to obtain a phenolic hydroxyl on
the acid component of the 1,2,3-triazolamide (compounds 15,
16, 19), via cleavage of the ether function with boron tribro-
mide. In addition, the presence of a methoxyl on the anilino
substituent of 14a, b and d allowed the obtainment of a pheno-
lic function on the basic component of the 1,2,3-triazolanilide
(compounds 17e19).
Clearly, two phenolfunctionsare present on the derivative19.
Finally, Scheme 3 reports the preparation of a series of
1,2,3-triazolbenzylamides obtained according to the usual re-
action sequence, starting from the acyl chlorides 4a, b and d.
Each acyl chloride was reacted with benzylamine (20),
2-chlorobenzylamine (21) or 2-methoxybenzylamine (22) in
toluene under reflux, in the presence of TEA, to give the
expected 1-(2-substituted-benzyl)-4-carboxybenzylamido-5-
methyl-1H-1,2,3-triazoles 23a, b, d, 24a, b, d and 25a, b, d,
respectively, in good yield. Also these derivatives have substit-
uents which are different for mesomeric and steric properties;
in addition they have a methylene bridge on the nitrogen atom
of the amido linker, which lends more flexibility to the whole
molecule.
N
N
N
N
N
COOEt
3
N
N
CH
3
CH
3
R
O
+
CH
3
R
R
1a-d
2a-d
3a-d
a : R = Cl ; b : R = F ; c : R = CF
d : R = OCH3
3
COCl
N
N
N
CH
3
R
4a-d
NH
H N
2
2
Cl
OH
Cl
HO
CH3
6
5
CH
3
Also in this case, the presence of a methoxyl in the ortho
position of the benzyl substituent on the triazole nitrogen of
the compounds 23d, 24d and 25d allowed the introduction
of a phenol function, generally useful for the biological
activity.
CONH
N
CONH
N
N
N
N
HO
N
CH
3
HO
CH
3
8a-d
7a-d
The demethylation reaction, carried out in the usual manner
with boron tribromide, gave the derivatives 26, 27 and 30.
Similarly, the cleavage of the ether function of the 2-
methoxybenzylamides 25a, b and d allowed the introduction
of a hydroxyl function on the benzylamido component too,
with obtainment of the compounds 28e30. Clearly, two
phenol functions are present on the derivative 30.
R
R
Scheme 1. Synthetic route for compounds 7aed and 8aed.
last compounds were not isolated, but, after the evaporation
of the reagent, were dissolved in anhydrous toluene and to
the solution obtained an equimolar amount of the suitable an-
iline and triethylamine (TEA) in toluene solution were
added, then the mixture was heated under reflux for several
hours. The 2-hydroxy-5-chloro-aniline (5) provided deriva-
tives 7aed, while the 2-hydroxy-5-methyl-aniline (6) gave
the analogous derivatives 8aed.
Scheme 2 reported the preparation of another series of 1,2,
3-triazol-anilides, obtained by a similar regiospecific 1,3-dipo-
lar cycloaddition reaction of the suitable azide (1a, b or d) to
the selected acetoacetanilides, N-(phenyl)-acetoacetamide
(9), N-(4-chlorophenyl)-acetoacetamide (10) or N-(2-methox-
yphenyl)-acetoacetamide (11).
The structures of all the newly prepared compounds were
confirmed by analytical and spectroscopic data (Tables 1 and 2).
3. Pharmacology
As a preliminary indication about a possible BK-activating
mechanism of action, the vasodilating effect of the new
compounds was studied in vitro on isolated rat aortic rings
precontracted with KCl 20 mM.
4. Results and discussion
By this single-step route, triazolamides bearing substituents
with different mesomeric and steric properties, both on the
acid component and on the basic one of the amide, were
made available.
Thus 2-chlorobenzylazide (1a) reacted with the acetoaceta-
nilides 9e11 to give the corresponding triazol-carboxanilides
12a, 13a and 14a, while 2-fluorobenzylazide (1b) reacted
with the same acetoacetanilides to give the analogous deriva-
tives 12b, 13b and 14b. Similarly 2-methoxybenzylazide (1d)
reacted to give the corresponding derivatives 12d, 13d and
14d.
In this work new potential BK-activators have been
projected and synthesized on the basis of the general pharma-
cophoric model, reported in Fig. 1. In particular, in these new
derivatives, the linker portion of the pharmacophore is
represented by a 1,2,3-triazole-carboxamide group, which
can be viewed as a combination of two different kinds of
linker, independently used in other series of BK-openers.
(a) The amide function, which can be considered a simple and
satisfactory linker portion, present in previous series of
derivatives of ours [14e17].

V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 2618e2626
2621
(b) The 1,2,3-triazole ring, also used in previous work [11e13]
of ours and belonging to a wider ‘‘family’’ of heterocyclic
linkers [1].
that the presence of a fluorine in the structure exerted a nega-
tive impact, as emerged from the lowered efficacy recorded in
compounds 18 and 29. In order to obtain more detailed infor-
mation about the mechanism of action, compound 15, exhibit-
ing the highest levels of potency and efficacy in this series,
was also tested in the presence of tetraethylammonium chlo-
ride (10 mM). The presence of this potassium channel blocker
antagonized the vasorelaxing responses evoked by 15, indicat-
ing the probable involvement of BK-activation.
In conclusion, the overall finding of this study indicated
that the triazole-carboxamide derivatives showed a poor activ-
ity and that this structural modification of the linker is delete-
rious for activity on BK channels. Therefore, in agreement
with earlier results [16,17], it can be confirmed that the
increase of the steric hindrance of the linker and/or the
increase of the distance between the two aromatic portions
is deleterious for the interaction with the biological target.
As a preliminary indication about a possible BK-activating
property, all the target triazole-carboxamide derivatives of this
series were tested by functional pharmacological tests, in order
to evaluate a vasorelaxing effect. The results are summarized
in Table 3. Compounds 7c, and 8aec resulted almost ineffec-
tive and all these compounds are characterised by the presence
of the phenyl hydroxyl group on the aromatic ring directly
bound to the amidic nitrogen. This same structural character-
istic is also present in 7a, b, d and 8d, as well as in compounds
17 and 18, which globally exhibited modest levels of vasore-
laxing efficacy (indeed, only compound 17 showed an efficacy
slightly higher than 50%). Although many derivatives showing
a phenol hydroxyl group on the benzyl bound to the triazole
ring were also devoid of high levels of efficacy, it is notewor-
thy that this structural characteristic is shared by only the three
derivatives (15, 19 and 30) which exhibited high levels of vas-
orelaxing efficacy, although with potency parameters signifi-
cantly lower than that of the reference compound NS1619.
This remark seems to indicate that the insertion of the hydroxy
group (widely recognized as a fundamental requirement for
the interaction with BK channels) in the aromatic ring at the
side of the triazole moiety of the linker is preferable. As re-
gards the influence of halogen atoms, it is particularly evident
5. Experimental
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, in d units, using TMS as internal standard. TLC
N3
a : R = Cl
b : R = F
1
d : R = OCH3
R
___________________________________________________________
+
+
+
2'-Methoxyacetoacetanilide
Acetoacetanilide
4'-Chloroacetoacetanilide
9
10
11
Cl
O
O
O
N
N
N
N
H
N
H
N
H
N
N
N
OCH3
N
N
N
R
R
R
13a,b,d
14a,b,d
12a,b,d
Cl
O
O
O
N
N
N
N
H
N
H
N
H
N
N
N
OH
N
N
N
17: R = Cl
18: R = F
19: R = OH
R
HO
HO
15
16
Scheme 2. Synthetic route for compounds 15e19.

2622
V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 2618e2626
COCl
N
N
a : R = Cl
N
CH3
b : R = F
4
d : R = OCH3
R
+
+
+
2'-Methoxybenzylamine
2'-Chlorobenzylamine
Benzylamine
20
21
22
O
O
O
N
N
NH
NH
N
NH
N
N
N
N
N
N
CH3
CH3
CH3
OCH3
Cl
23 a,b,d
25 a,b,d
24 a,b,d
R
R
R
O
O
O
N
NH
N
NH
N
NH
N
N
N
N
N
N
CH3
CH3
CH3
OH
Cl
26
27
28: R=Cl
29: R=F
OH
R
OH
30: R=OH
Scheme 3. Synthetic route for compounds 26e30.
was performed on precoated silica gel F254 plates (Merck).
Elemental analyses (C, H, N) were within ꢀ0.4% of the theo-
retical values and were performed on a Carlo Erba Elemental
Analyzer Mod. 1106 apparatus.
was heated under reflux for 3e4 h. The reaction mixture
was concentrated, diluted with H2O and acidified (pH y 2)
to precipitate the title compounds which were collected by
filtration and purified by crystallization (Table 1).
5.1.1. 1-(2-Substituted-benzyl)-4-carbethoxy-
5-methyl-1H-1,2,3-triazoles (2aed)
5.1.3. N-(2-Hydroxy-5-chlorophenyl)-1-(2-substituted-
benzyl)-5-methyl-1H-1,2,3-triazol-4-carboxamides (7aed)
A solution of 3.0 mmol of the suitable carboxytriazole (3a, b,
c or d) in 8 mL of SOCl2 was heated under reflux for 1 h. The
reagent was distilled and the residue, consisting of the corre-
sponding acyl chloride 4a, b, c or d, was dissolved in 20 mL
of anhydrous toluene. This solution was added dropwise to
a solution of 2-hydroxy-5-chloroaniline 5 (0.430 g, 3.0 mmol)
and TEA (1 mL, 7 mmol) in 15e20 mL of anhydrous toluene
and the mixture was refluxed for 18e20 h. For the isolation of
7a and b, the toluene solution, after cooling, separated a crystal-
line solid which was collected by filtration and treated with H2O
to dissolve the probably present TEA hydrochloride.
To a solution of 5.0 mmol of the appropriate azide (1a, b, c
or d) and ethyl acetoacetate (0.715 g, 5.5 mmol) in 8e10 mL
of anhydrous DMSO, 5e6 g of finely powdered K2CO3 was
added and the suspension was stirred at 35e40 ꢁC for 48 h.
The reaction mixture was diluted with H2O to precipitate the
title compounds, stirring was continued for 1 h then the solid
compounds were collected by filtration, washed with H2O and
purified by crystallization (Table 1).
5.1.2. 1-(2-Substituted-benzyl)-4-carboxy-
5-methyl-1H-1,2,3-triazoles (3aed)
A solution of 7.0 mmol of the appropriate triazolester
(2a, b, c or d) in 10 mL of 10% NaOH and 10 mL of EtOH
The insoluble material consisted of a first portion of 7a or
b. The toluene filtrate was evaporated in vacuo, the residue

V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 2618e2626
2623
Table 1
Physico-chemical properties
Compound
Yield (%)
Crystall. solvent
M.p. (ꢁC)
Analyses (C, H, N)
IR, n (cmꢂ1
)
2a
2b
77
85
75
70
82
90
91
88
65
62
65
70
67
60
70
67
75
73
78
75
69
73
78
72
68
60
55
60
58
55
75
72
75
70
74
73
65
70
75
50
54
52
65
62
EtOAc/hexane
Cyclohexane
CHCl3/hexane
e
90e92
53e55
C13H14N3O2Cl
C13H14N3O2F
C14H14N3O2F3
1704 (CO)
1714 (CO)
1706 (CO)
1726 (CO)
1704 (CO)
1690 (CO)
1703 (CO)
1682 (CO)
2c
2d
107e108
Oil
3a
3b
Iso-PrOH
EtOAc/hexane
MeOH
196e197
178e179
203e205
188e190
256e258
242e244
217e220
244e245
243e245
204e205
197e200
207e208
142e144
134e137
168e170
146e148
138e140
163e165
127e129
113e115
132e134
173e175
216e218
188e190
216e218
237e239
130e132
128e130
163e165
152e154
137e139
130e132
153e155
115e117
145e147
197e200
218e220
170e172
190e192
223e225
C11H10N3O2Cl
C11H10N3O2F
C12H10N3O2F3
C12H13N3O3
3c
3d
MeOH
7a
7b
MeOH/H2O
MeOH/H2O
MeOH/H2O
MeOH
C17H14N4O2Cl2
C17H14N4O2ClF
C18H14N4O2ClF3
C18H17N4O3Cl
C18H17N4O2Cl
C18H17N4O2F
C19H17N4O2F3
C19H20N4O3
1671 (CO); 3100b (OH); 3383 (NH)
1673 (CO); 3250b (OH); 3384 (NH)
1669 (CO); 3230b (OH); 3353 (NH)
1669 (CO); 3150b (OH); 3385 (NH)
1664 (CO); 3280b (OH); 3395 (NH)
1652 (CO); 3300b (OH); 3398 (NH)
1680 (CO); 3300b (OH); 3396 (NH)
1675 (CO); 3200b (OH); 3392 (NH)
1670 (CO); 3288 (NH)
7c
7d
8a
8b
MeOH
MeOH/H2O
MeOH/H2O
MeOH/H2O
MeOH
8c
8d
12a
12b
12d
13a
13b
13d
14a
14b
14d
15
C17H15N4OCl
C17H15N4OF
C18H18N4O2
MeOH
MeOH
MeOH
1666 (CO); 3315 (NH)
1668 (CO); 3330 (NH)
C17H14N4OCl2
C17H14N4OClF
C18H17N4O2Cl
C18H17N4O2Cl
C18H17N4O2F
C19H20N4O3
1672 (CO); 3251 (NH)
1672 (CO); 3365 (NH)
MeOH/H2O
MeOH
MeOH
1681 (CO); 3375 (NH)
1682 (CO); 3370 (NH)
MeOH
MeOH
1672 (CO); 3351 (NH)
1675 (CO); 3357 (NH)
MeOH/H2O
MeOH/H2O
MeOH/H2O
MeOH/H2O
MeOH/H2O
MeOH/H2O
MeOH/H2O
MeOH
C17H16N4O2
1651 (CO); 3130b (OH); 3360 (NH)
1666 (CO); 3370b (NH, OH)
1690 (CO); 3040b (OH); 3395 (NH)
1685 (CO); 3100b (OH); 3399 (NH)
1635 (CO); 3160b (OH); 3374 (NH)
1648 (CO); 3330 (NH)
16
17
C17H15N4O2Cl
C17H15N4O2Cl
C17H15N4O2F
C17H16N4O3
18
19
23a
23b
23d
24a
24b
24d
25a
25b
25d
26
C18H17N4OCl
C18H17N4OF
C19H20N4O2
1654 (CO); 3322 (NH)
1649 (CO); 3340 (NH)
MeOH
MeOH
C18H16N4OCl2
C18H16N4OClF
C19H19N4O2Cl
C19H19N4O2Cl
C19H19N4O2F
C19H20N4O2
1660 (CO); 3348 (NH)
1655 (CO); 3298 (NH)
MeOH
MeOH
1665 (CO); 3314 (NH)
1660(CO); 3344 (NH)
MeOH/H2O
MeOH
MeOH
1669 (CO); 3317 (NH)
1665 (CO); 3365 (NH)
C18H18N4O2
1640 (CO); 3200b (OH); 3298 (NH)
1638 (CO); 3300b (NH, OH)
1628 (CO); 3180b (OH); 3283 (NH)
1634 (CO); 3150b (OH); 3293 (NH)
1634 (CO); 3290b (NH, OH)
27
28
MeOH
MeOH
C18H17N4O2Cl
C18H17N4O2Cl
C18H17N4O2F
C18H18N4O3
29
30
MeOH
MeOH/H2O
was dissolved in CHCl3 and the new solution, after washing
with 5% NaHCO3 and 10% HCl, was dried (MgSO4) and
evaporated to give a further amount of 7a or b. The combined
fractions were purified by crystallization (Table 1). For the
isolation of 7c and d, the toluene solution was evaporated
and worked up as the previous filtrate (Table 1).
6 (0.369 g, 3.0 mmol) and TEA (1 mL, 7 mmol) in 20 mL
of anhydrous toluene and the mixture was refluxed for
18e20 h. For the isolation of the title compounds 8aed,
the toluene solution, after cooling, separated a crystalline
solid. The reaction mixtures were worked up as described
for 7a or b (Table 1).
5.1.4. N-(2-Hydroxy-5-methylphenyl)-1-(2-substituted-
5.1.5. N-(Phenyl)-1-(2-substituted-benzyl)-
benzyl)-5-methyl-1H-1,2,3-triazol-4-carboxamides (8aed)
A solution of 3.0 mmol of the suitable carboxytriazole
(3a, b, c or d) in 8 mL of SOCl2 was heated under reflux
for 1 h. The reagent was distilled and the residue, consisting
of the corresponding acyl chloride 4a, b, c or d, was dis-
solved in 20 mL of anhydrous toluene. This solution was
added dropwise to a solution of 2-hydroxy-5-methylaniline
5-methyl-1H-1,2,3-triazol-4-carboxamides (12a, b, d)
To a solution of 6.0 mmol of the appropriate azide (1a, b or
d) and acetoacetanilide (1.06 g, 6.0 mmol) in 12 mL of anhy-
drous DMSO, 6e7 g of finely powdered anhydrous K2CO3
was added and the suspension was stirred at 100 ꢁC for
48 h. The reaction mixture was diluted with H2O to precipitate
the title compounds, stirring was continued for 1 h then the

2624
V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 2618e2626
Table 2
1H NMR in DMSO-d6, ppm from TMS
2a
2b
1.30 (t, 3H, CH3); 2.50 (s, 3H, CH3); 4.30 (q, 2H, OCH2); 5.69 (s, 2H, CH2); 6.96, 7.36, 7.53 (d, 1H, m, 2H, d, 1H, Ar).
1.29 (t, 3H, CH3); 2.51 (s, 3H, CH3); 4.29 (q, 2H, OCH2); 5.67 (s, 2H, CH2); 7.13e7.46 (m, 4H, Ar).
1.32 (t, 3H, CH3); 2.45 (s, 3H, CH3); 4.32 (q, 2H, OCH2); 5.79 (s, 2H, CH2); 6.82, 7.60, 7.83 (d, 1H, m, 2H, d, 1H, Ar).
1.30 (t, 3H, CH3); 2.50 (s, 3H, CH3); 4.30 (q, 2H, OCH2); 5.69 (s, 2H, CH2); 6.96, 7.36, 7.53 (d, 1H, m, 2H, d, 1H, Ar).
2.48 (s, 3H, CH3); 5.68 (s, 2H, CH2); 6.98, 7.37, 7.53 (d, 1H, m, 2H, d, 1H, Ar).
2c
2d
3a
3b
2.49 (s, 3H, CH3); 5.65 (s, 2H, CH2); 7.12e7.48 (m, 4H, Ar); 13.0b (COOH).
2.47 (s, 3H, CH3); 5.79 (s, 2H, CH2); 6.73, 7.49, 7.76 (d, 1H, m, 2H, d, 1H, Ar).
3c
3d
2.47 (s, 3H, CH3); 3.79 (s, 3H, OCH3); 5.48 (s, 2H, CH2); 6.91, 7.04, 7.32 (d, 2H, d, 1H, m, 1H, Ar); 12.8 (COOH).
2.57 (s, 3H, CH3); 5.73 (s, 2H, CH2); 6.95, 7.37, 7.54, 8.30 (m, 3H, m, 2H, d, 1H, s, 1H, Ar); 9.5 (NH); 10.7 (OH).
2.59 (s, 3H, CH3); 5.71 (s, 2H, CH2); 6.97, 7.18e7.46, 8.30 (m, 2H, m, 4H, s, 1H, Ar); 9.5 (NH); 10.7 (OH).
2.52 (s, 3H, CH3); 5.84 (s, 2H, CH2); 6.85e8.20 (m, 7H, Ar); 9.8 (NH); 10.6b (OH).
7a
7b
7c
7d
2.57 (s, 3H, CH3); 3.81 (s, 3H, OCH3); 5.54 (s, 2H, CH2); 6.89e7.37, 8.30 (m, 6H, s, 1H, Ar); 9.5 (NH); 10.6 (OH).
2.22 (s, 3H, CH3); 2.56 (s, 3H, CH3); 5.71 (s, 2H, CH2); 6.76, 7.00, 7.37, 7.53, 8.07 (m, 2H, d, 1H, m, 2H, d, 1H, s, 1H, Ar); 9.5 (NH); 10.0 (OH).
2.21 (s, 3H, CH3); 2.57 (s, 3H, CH3); 5.70 (s, 2H, CH2); 6.78, 7.18e7.48, 8.06 (m, 2H, m, 4H, s, 1H, Ar); 9.5 (NH); 10.0 (OH).
2.22 (s, 3H, CH3); 5.82(s, 2H, CH2); 6.80, 7.61, 7.83, 8.08 (m, 3H, m, 2H, d, 1H, s, 1H, Ar); 9.5 (NH); 10.0 (OH).
2.22 (s, 3H, CH3); 2.56 (s, 3H, CH3); 3.81 (s, 3H, OCH3); 5.53 (s, 2H, CH2); 6.70e7.36, 8.08 (m, 6H, s, 1H, Ar); 9.5 (NH); 10.0 (OH).
2.54 (s, 3H, CH3); 5.74 (s, 2H, CH2); 6.95, 7.08, 7.28e7.44, 7.54, 7.83 (d, 1H, t, 1H, m, 4H, d, 1H, d, 2H, Ar); 10.4 (NH).
2.56 (s, 3H, CH3); 5.71 (s, 2H, CH2); 7.04e7.48, 7.83 (m, 7H, d, 2H, Ar); 10.4 (NH).
2.54 (s, 3H, CH3); 3.82 (s, 3H, OCH3); 5.54 (s, 2H, CH2); 6.93, 7.07, 7.32, 7.83 (d, 2H, m, 2H, m, 3H, d, 2H, Ar); 10.3 (NH).
2.54 (s, 3H, CH3); 5.73 (s, 2H, CH2); 6.98, 7.36, 7.52, 7.88, (d, 1H, m, 4H, d, 1H, d, 2H, Ar); 10.6 (NH).
2.56 (s, 3H, CH3); 5.71 (s, 2H, CH2); 7.17e7.50, 7.88 (m, 6H, d, 2H, Ar); 10.5 (NH).
8a
8b
8c
8d
12a
12b
12d
13a
13b
13d
14a
14b
14d
15
2.54 (s, 3H, CH3); 3.82 (s, 3H, OCH3); 5.54 (s, 2H, CH2); 6.94, 7.07, 7.35, 7.88 (d, 2H, d, 1H, m, 3H, d, 2H, Ar); 10.5 (NH).
2.56 (s, 3H, CH3); 3.91 (s, 3H, OCH3); 5.72 (s, 2H, CH2); 6.92e7.13, 7.37, 7.53, 8.27 (m, 4H, m, 2H, d, 1H, d, 1H, Ar); 9.5 (NH).
2.57 (s, 3H, CH3); 3.90 (s, 3H, OCH3); 5.70 (s, 2H, CH2); 6.91e7.47, 8.28 (m, 7H, d, 1H, Ar); 9.5 (NH).
2.57 (s, 3H, CH3); 3.82 (s, 3H, OCH3); 3.91 (s, 3H, OCH3); 5.54 (s, 2H, CH2); 6.94e7.36, 8.29 (m, 7H, d, 1H, Ar); 9.5 (NH).
2.55 (s, 3H, CH3); 5.51 (s, 2H, CH2); 6.71e7.36, 7.83 (m, 7H, d, 2H, Ar); 9.9 (NH); 10.3 (OH).
16
17
2.54 (s, 3H, CH3); 5.51 (s, 2H, CH2); 6.72e7.20, 7.37, 7.88 (m, 4H, d, 2H, d, 2H, Ar); 9.9 (NH);10.5 (OH).
2.58 (s, 3H, CH3); 5.70 (s, 2H, CH2); 6.76e6.96, 7.18, 7.49, 8.20 (m, 3H, m, 4H, d, 1H, Ar); 9.5 (NH); 10.2 (OH).
2.56 (s, 3H, CH3); 5.71 (s, 2H, CH2); 6.76e7.58 (m, 7H, Ar); 8.21 (d, 1H, Ar); 9.5 (NH); 10.2 (OH).
2.57 (s, 3H, CH3); 5.50 (s, 2H, CH2); 6.73e7.21, 8.20 (m, 7H, d, 1H, Ar); 9.5 (NH); 10.0b (OH); 11.3b (OH).
2.49 (s, 3H, CH3); 4.43 (d, 2H, CH2); 5.68 (s, 2H, CH2); 6.94, 7.18e7.43, 7.50 (d, 1H, m, 7H, d, 1H, Ar); 9.0 (t, NH).
2.50 (s, 3H, CH3); 4.42 (d, 2H, CH2); 5.65 (s, 2H, CH2); 7.12e7.47 (m, 9H, Ar); 9.0 (t, NH).
18
19
23a
23b
23d
24a
24b
24d
25a
25b
25d
26
2.49 (s, 3H, CH3); 3.81 (s, 3H, OCH3); 4.43 (d, 2H, CH2); 5.49 (s, 2H, CH2); 6.88e7.37 (m, 9H, Ar); 9.0 (t, NH).
2.49 (s, 3H, CH3); 4.51 (d, 2H, CH2); 5.69 (s, 2H, CH2); 6.99, 7.28e7.53 (d, 1H, m, 7H, Ar); 9.0 (t, NH).
2.51 (s, 3H, CH3); 4.51 (d, 2H, CH2); 5.67 (s, 2H, CH2); 7.16e7.47 (m, 8H, Ar); 9.0 (t, NH).
2.49 (s, 3H, CH3); 3.81 (s, 3H, OCH3); 4.51 (d, 2H, CH2); 5.50 (s, 2H, CH2); 6.93, 7.05, 7.24e7.46 (d, 2H, d, 1H, m, 5H, Ar); 9.0 (t, NH).
2.50 (s, 3H, CH3); 3.83 (s, 3H, OCH3);4.44 (d, 2H, CH2); 5.69 (s, 2H, CH2); 6.86e7.57 (m, 8H, Ar); 8.7 (t, NH).
2.50 (s, 3H, CH3); 3.81 (s, 3H, OCH3); 4.41 (d, 2H, CH2); 5.65 (s, 2H, CH2); 6.83e7.47 (m, 8H, Ar); 8.7 (t, NH).
2.49 (s, 3H, CH3); 3.82 (s, 6H, OCH3); 4.42 (d, 2H, CH2); 5.50 (s, 2H, CH2); 6.86e7.37 (m, 8H, Ar); 8.7 (t, NH).
2.49 (s, 3H, CH3); 4.42 (d, 2H, CH2); 5.46 (s, 2H, CH2); 6.71e7.32 (m, 9H, Ar); 8.9 (t, NH); 10.0b (OH).
2.49 (s, 3H, CH3); 4.50 (d, 2H, CH2); 5.47 (s, 2H, CH2); 6.70e7.46 (m, 8H, Ar); 8.9 (t, NH); 9.8b (OH).
2.49 (s, 3H, CH3); 4.39 (d, 2H, CH2); 5.68 (s, 2H, CH2); 6.70e7.54 (m, 8H, Ar); 8.7 (t, NH); 9.6 (OH).
2.50 (s, 3H, CH3); 4.39 (d, 2H, CH2); 5.66 (s, 2H, CH2); 6.69e7.47 (m, 8H, Ar); 8.7 (t, NH); 9.6 (OH).
2.48 (s, 3H, CH3); 4.38 (d, 2H, CH2); 5.46 (s, 2H, CH2); 6.68e7.16 (m, 8H, Ar); 8.7 (t, NH); 9.9 (b, 2H, OH).
27
28
29
30
solid compounds were collected by filtration, washed with
H2O and purified by crystallization (Table 1).
12 mL of anhydrous DMSO, 6e7 g of finely powdered anhy-
drous K2CO3 was added and the suspension was stirred at
100 ꢁC for 48 h. The reaction mixture was diluted with H2O
and worked up as described above (Table 1).
5.1.6. N-(4-Chlorophenyl)-1-(2-substituted-benzyl)-
5-methyl-1H-1,2,3-triazol-4-carboxamides (13a, b, d)
To a solution of 6.0 mmol of the appropriate azide (1a, b or
d) and 4-chloro-acetoacetanilide (1.27 g, 6.0 mmol) in 12 mL
of anhydrous DMSO, 6e7 g of finely powdered anhydrous
K2CO3 was added and the suspension was stirred at 100 ꢁC
for 48 h. The reaction mixture was diluted with H2O and
worked up as described above (Table 1).
5.1.8. 1-(2-Substituted-benzyl)-5-methyl-
1H-1,2,3-triazol-4-carboxanilides (15e19)
To a solution of 2.0 mmol of the suitable methoxy deriva-
tive (12d, 13d, 14a, b or d) in 100e120 mL of anhydrous
CH2Cl2, cooled at ꢂ30 ꢁC, a solution of BBr3 (z2 mL,
z20 mmol; z3 mL, z30 mmol for 14d) in 10e12 mL of an-
hydrous CH2Cl2 was added drop by drop, under stirring. After
1 h at this temperature, the reaction mixture was left at ꢂ20 ꢁC
overnight. The excess of the reagent was decomposed by the
cautious addition of MeOH (10 mL) and H2O (40 mL). The
organic phase, after washing with H2O, was extracted with
5.1.7. N-(2-Methoxyphenyl)-1-(2-substituted-benzyl)-
5-methyl-1H-1,2,3-triazol-4-carboxamides (14a, b, d)
To a solution of 6.0 mmol of the appropriate azide (1a, b or
d) and 4-methoxy-acetoacetanilide (0.910 g, 6.0 mmol) in

V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 2618e2626
2625
Table 3
Parameters of vasorelaxing efficacy (Emax %) and potency (pIC50) exhibited by
the synthesized compounds and by the reference drug NS1619
5.1.10. N-(Benzyl)-1-(2-hydroxybenzyl)-5-methyl-1H-1,2,3-
triazol-4-carboxamide (26) and N-(2-chloro-benzyl)-
1-(2-hydroxy-benzyl)-5-methyl-1H-1,2,3-triazol-4-
carboxamide (27); N-(2-hydroxy-benzyl)-
Compound
Emax (%)
pIC50
7a
7b
39 ꢀ 13a
25 ꢀ 2a
Ineffective
37 ꢀ 6a
Ineffective
Ineffective
Ineffective
43 ꢀ 8a
89 ꢀ 2
NC
NC
1-(2-substituted-benzyl)-5-methyl-1H-1,2,3-triazol-4-
carboxamides (28e30)
7c
7d
e
NC
To a solution of 1.6 mmol of the suitable methoxy deriva-
tive (23d, 24d, 25a, b or d) in 100e120 mL of anhydrous
CH2Cl2, cooled at ꢂ30 ꢁC, a solution of BBr3 (z1.6 mL,
z16 mmol; z2 mL, z20 mmol for 25d) in 6e8 mL of anhy-
drous CH2Cl2 was added drop by drop, under stirring. After
1 h at this temperature, the reaction mixture was left at
ꢂ20 ꢁC overnight, then worked up as described for the prepa-
ration of 15e19. Compounds 26e28 and 29 were isolated by
acidification of the alkaline extract, collected by filtration and
purified by crystallization (Table 1). For the isolation of the
dihydroxyderivative 30, the alkaline extract appeared as a sus-
pension caused by the partial precipitation of the sodium salt,
but the compound was similarly isolated by direct acidification
(Table 1).
8a
8b
e
e
e
NC
8c
8d
15
15 þ TEAC
4.75 ꢀ 0.03a
37 ꢀ 4b
53 ꢀ 8a
52 ꢀ 2a
35 ꢀ 2a
81 ꢀ 9
NC
NC
16
17
NC
NC
18
19
4.69 ꢀ 0.04a
26
27
48 ꢀ 4a
51 ꢀ 3a
49 ꢀ 2a
24 ꢀ 3a
76 ꢀ 5
NC
NC
28
29
NC
NC
30
NS1619
4.66 ꢀ 0.03a
94 ꢀ 4
5.29 ꢀ 0.03
NC indicates that the potency value could not be calculated because of the low
efficacy (ꢄ55%).
5.2. Pharmacology
a
Significantly different from the corresponding value exhibited by NS1619.
Significantly different from the corresponding value recorded in the
absence of tetraethylammonium chloride (TEAC, 10 mM).
b
All the experimental procedures were carried out follow-
ing the guidelines of the European Community Council Di-
rective 86-609. A possible vasodilator mechanism of action
was investigated by testing the effects of the compounds on
isolated thoracic aortic rings of male normotensive Wistar
rats (250e350 g). After light ether anaesthesia, the rats
were sacrificed by cervical dislocation and bleeding. The aor-
tae were immediately excised and freed of extraneous tissues.
The endothelial layer was removed by gently rubbing the in-
tima surface of the vessels with a hypodermic needle. Five
millimeter 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; glu-
cose 5.5), thermostated at 37 ꢁC and continuously gassed
with a mixture of O2 (95%) and CO2 (5%). Changes in ten-
sion were recorded by means of an acquisition data software
(BioPac MP 100).
10%NaOH. Compounds 15, 16 and 19 precipitated by acidifi-
cation (36% HCl) of the alkaline extract, were then collected
by filtration and purified by crystallization (Table 1). For the
isolation of 17 and 18, the treatment with 10% NaOH caused
precipitation of the respective sodium salt which was collected
by filtration, suspended in H2O and acidified. A further small
amount of the compound precipitated from the alkaline filtrate
by acidification (Table 1).
5.1.9. N-(Benzyl)-1-(2-substituted-benzyl)-
5-methyl-1H-1,2,3-triazol-4-carboxamides (23a, b, d);
N-(2-chlorobenzyl)-1-(2-substituted-benzyl)-
5-methyl-1H-1,2,3-triazol-4-carboxamides (24a, b, d)
and N-(2-methoxybenzyl)-1-(2-substituted-benzyl)-
5-methyl-1H-1,2,3-triazol-4-carboxamides (25a, b, d)
A solution of the suitable acyl chloride (4a, b or d), pre-
pared from 3.0 mmol of the corresponding carboxytriazole
(3a, b or d) in 20 mL of anhydrous toluene (see compounds
7aed), was added dropwise to a solution of 3.0 mmol of the
appropriate benzylamine [benzylamine (20), 2-chlorobenzyl-
amine (21) or 2-methoxybenzylamine (22)] and TEA
(1 mL, 7 mmol) in 20 mL of the same solvent and the mix-
ture was refluxed for 18e20 h. The reaction mixture was
evaporated in vacuo and the residue was treated with H2O
to dissolve the TEA hydrochloride and extracted with
CHCl3. The organic solution, after washing with 10% HCl
and 10% NaOH, was dried (MgSO4) and evaporated to
give the title compounds which were purified by crystalliza-
tion (Table 1).
After an equilibration period of 60 min, endothelial re-
moval was confirmed by the administration of acetylcholine
(Ach, 10 mM) to KCl (20 mM)-precontracted vascular rings.
A relaxation <10% of the KCl-induced contraction was con-
sidered representative of an acceptable lack of the endothelial
layer, while the organs showing a relaxation ꢃ10% (i.e. sig-
nificant presence of endothelium) were discarded. From 30 to
40 min after confirmation of the endothelium removal, the
aortic preparations were contracted by treatment with a single
concentration of KCl (20 mM) and, when the contraction
reached a stable plateau, 3-fold increasing concentrations
(10 nMe30 mM) of the tested compounds or of the reference
drug NS1619 (a well-known BK-activator) were added cumu-
latively. For the selected compound 15, the same protocol
was also carried out in the presence of the potassium channel

2626
V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 2618e2626
blocker tetraethylammonium chloride (TEAC; 10 mM),
which was incubated for 20 min before the addition of KCl
20 mM. Each compound was tested in 5e10 experiments.
Preliminary experiments showed that the KCl (20 mM)-in-
duced contractions remained in a stable tonic state for at least
40 min. The reference drug NS1619 (Sigma) was dissolved
(10 mM) in EtOH 95% and further diluted in Tyrode
solution. Acetylcholine chloride (Sigma) was dissolved
(100 mM) in EtOH 95% and further diluted in bidistilled wa-
ter, whereas KCl was dissolved in Tyrode solution. TEAC
was diluted in bidistilled water. All the synthesized deriva-
tives were dissolved (10 mM) in DMSO, and then diluted
in Tyrode solution. All the solutions were freshly prepared
immediately before the pharmacological experimental proce-
dures. Previous experiments showed complete ineffectiveness
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 limit concentration 30 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 percent-
age (%) of the contractile tone induced by KCl 20 mM,
evoked by this limit concentration. The parameter of potency
was expressed as pIC50, calculated as a negative Logarithm
of the molar concentration of the compounds tested, evoking
a half reduction of the contractile tone induced by KCl
20 mM. The pIC50 value could not be calculated for those
compounds that showed an efficacy value <55%. Compounds
exhibiting an efficacy level <20% were considered as
ineffective. The parameters of efficacy and potency were
expressed as mean ꢀ standard error, for 5e10 experiments.
Student’s t-test was selected as a statistical analysis,
P < 0.05 was considered representative of a significant
statistical difference. Experimental data were analysed
Appendix. Supplementary data
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.ejmech.2008.02.032.
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Prism 3.0).