New amido derivatives as potential BKCa potassium channel activators. XI

Article abstract of DOI:10.1016/j.ejmech.2007.06.005

The vasorelaxing effects of exogenous activators of large-conductance calcium-activated potassium channels (BK channels) can furnish the pharmacological rational basis for the treatment of hypertension and/or other diseases related with an impaired contractility of vessels. Since in previous works some benzanilide derivatives showed BK channel-induced vasorelaxing activity, in this paper we have taken into consideration the introduction of methylene spacer(s) between the amide linker and one or both the aromatic substituents, to evaluate the pharmacological effect caused by these lengthenings and to obtain possible useful information about structure-activity relationships. Overall, the main findings of this work suggest that the introduction of one or two methylene group(s) in the amide linker exerts a negative influence on the BK-opening properties, which can be due to an excessive lengthening of the spacer between the two aromatic rings and/or to further degrees of conformational freedom.

Full text of DOI:10.1016/j.ejmech.2007.06.005

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 792e799
http://www.elsevier.com/locate/ejmech
Original article
New amido derivatives as potential BKCa potassium channel activators. XI
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 6 April 2007; received in revised form 13 June 2007; accepted 15 June 2007
Available online 5 July 2007
Abstract
The vasorelaxing effects of exogenous activators of large-conductance calcium-activated potassium channels (BK channels) can furnish the
pharmacological rational basis for the treatment of hypertension and/or other diseases related with an impaired contractility of vessels. Since in
previous works some benzanilide derivatives showed BK channel-induced vasorelaxing activity, in this paper we have taken into consideration
the introduction of methylene spacer(s) between the amide linker and one or both the aromatic substituents, to evaluate the pharmacological
effect caused by these lengthenings and to obtain possible useful information about structureeactivity relationships. Overall, the main findings
of this work suggest that the introduction of one or two methylene group(s) in the amide linker exerts a negative influence on the BK-opening
properties, which can be due to an excessive lengthening of the spacer between the two aromatic rings and/or to further degrees of conforma-
tional freedom.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: BK potassium channel; Benzanilide; Amide; BK-activator
1. Introduction
efflux, which leads to rapid hyperpolarization of the excitatory
membrane and reduces Ca2þ influx through voltage-dependent
Ca2þ channels.
Then, the availability of exogenous compounds able to
activate BK channels can guarantee an innovative pharmaco-
logical tool for the clinical management of many pathological
states, due to a cell hyperexcitability, such as asthma, urge
incontinence and bladder spasm, gastric hypermotility,
neurological and psychiatric disorders [1,2].
As concerns the cardiovascular system, it is now widely
accepted that BK channels ensure the predominant component
of the outward Kþ current in vascular smooth muscle cells,
accounting for fundamental function of such ion channels in
the modulation of the muscular tone of vessels [4,5]. Conse-
quently, the vasorelaxing effects of exogenous BK-openers
can furnish the pharmacological rational basis for the
treatment of hypertension and/or other diseases related with
an impaired contractility of vessels (for example, coronary
vasospasm) [1,2].
The large-conductance, calcium-activated potassium
channels (BK, also termed BKCa, Slo, or MaxiK) are distrib-
uted in both excitable and non-excitable cells. They are
involved in many cellular functions such as action potential
repolarization, neuronal excitability, neurotransmitter release,
hormone secretion, tuning of cochlear hair cells, innate immu-
nity, and modulation of the tone of vascular, airway, uterine,
gastrointestinal, and urinary bladder smooth muscle tissues
[1e3].
BKCa channels characteristically respond to two distinct
physiological stimuli, changes in membrane voltage and in
cytosolic Ca2þ concentration. The BK channels gate-open in
response to an increase in cytosolic Ca2þ concentration and
membrane depolarization, resulting in an increase of Kþ
* Corresponding author. Tel.: þ39 50 2219589.
E-mail address: calderone@farm.unipi.it (V. Calderone).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.06.005

V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 792e799
793
In a previous work [6], we could observe that the synthes-
ised 1-(20-hydroxybenzoyl)-5-methyl-benzotriazole, showing
structural analogies with the reference BK-openers NS 004
and NS 1619 and exhibiting vasorelaxing effects probably
due to the activation of vascular BK channels, was able to
confer a significant protection of the myocardial function, in
isolated rat hearts submitted to ischemia/reperfusion cycles.
This result (originally unexpected) can be now explained;
thanks to more recent experimental evidence showing that
the activation of cardiac calcium-activated potassium channels
could be involved in the cardioprotective mechanisms of ‘‘is-
chemic preconditioning’’ and that the administration of BK-
openers, such as NS 1619, could reduce the cardiac injury
following an ischemic event [7e9]. Of course, these reports
let us foresee a further potential use of BK-activators in
cardiovascular pharmacotherapy, as anti-ischemic drugs.
Our research program about potential activators of BK
channels, in the beginning considered heterocyclic compounds
[6,10e12] which referred to the benzimidazolone derivatives
NS 004 and NS 1619 reported in the literature [13] as BK-
openers. After regarding to an hypothetic simple pharmaco-
phore (Fig. 1A) which consisted of two appropriately
substituted phenyl rings connected by a linker, 1,2,3-triazole
derivatives [14e16] and benzanilides [17e19] were synthes-
ised and tested, in which the 1,2,3-triazole ring and the amide
function, respectively, represented the linker.
requirement, while furan and thiophene rings were well toler-
ated. On the contrary, the introduction of unsaturated heterocy-
clic rings (pyridine and thiazole) on the basic side of the amide
linker, led to satisfactory biological activity, but the presence of
aliphatic heterocycles lowered the pharmacological effect. The
presence of a phenolic function as a certain H-bond donor was
confirmed.
Another structural change of the benzanilide pharmaco-
phore concerned a further deepening of the structureeactivity
relationships of benzanilide derivatives previously studied as
BK channel activators [19]. In this paper were reported several
substitutions on the phenyl rings of the reference benzanilides,
which possessed particular and specific properties from
a mesomeric and/or steric point of view. The pharmacological
results indicated that several compounds exhibited vasorelax-
ing effects which could not be attributed to the activation of
BK channels, but two derivatives showed a clear profile
of BK-activators with a vasodilator activity comparable to or
slightly lower than that recorded for the reference benzimida-
zolone NS 1619.
In this paper we have taken into consideration the introduc-
tion of methylenic spacers between the amide linker and the
two aromatic substituents which form the benzanilide pharma-
cophore to evaluate the pharmacological effect caused by
these lengthenings.
In particular, after a first paper [17] which had shown a BK
channel-induced vasorelaxing activity of the N-(2-hydroxy-
5-chlorophenyl)-2-methoxy-5-chlorobenzamide(Fig. 1B)higher
than NS 1619, the research about new compounds bearing the
amide linker was advanced changing the benzanilide structure
[18] by the introduction of a five or six membered aromatic
heterocyclic substituent in the acid moiety of the amide, keep-
ing unaltered the basic moiety as 2-hydroxy-5-chloro-anilide
(Fig. 1C). In addition an aromatic or aliphatic six or five
membered heterocyclic substituent was introduced in the basic
moiety of the amide, keeping unaltered the acid moiety as
2-methoxy-5-chlorobenzoyl, which was then converted to the
corresponding 2-hydroxy derivative (Fig. 1D). The pharmaco-
logical results indicated that the presence of nitrogen heterocy-
cles on the acid side of the amide linker seems to be a negative
2. Chemistry
At first a methylenic bridgewas introduced in the acid moiety
of the benzanilide, by the preparation of a new series of phenyl-
acetanilides (Scheme 1). Compounds 3aeg were prepared by
reaction of 2-methoxy-phenylacetic acid chloride (1) with the
suitable substituted aniline (2aeg) in toluene in the presence
of triethylamine. The employed anilines had differentiated
substituents as 2-hydroxy-5-chloro- (2a), 2-hydroxy-5-methyl-
(2b), 2-methoxy-5-nitro- (2c), 2-methyl-4-nitro- (2d), 4-nitro-
(2e), 2-fluoro- (2f) together with the unsubstituted aniline
(2g). Thus the phenol function, which appears as one of the
potential requirement for the pharmacological activity, is
present in the ortho-position of the basic moiety of the
COCl
CONH Ar
HO
H2N
+
Ar
OCH3
O
OCH3
HO
OCH3
2a-g
1
LINKER
3a-g
OH
R1
N
H
HO
Cl
b : Ar =
a : Ar =
EWG
R2
Cl
A
B
CH3
Cl
CONH
Ar
CH3O
H3C
HO
O
Cl
O
OH
4c-g
Heterocycle
NO2
d : Ar =
c : Ar =
e : Ar =
NH
Heterocycle
NH
NO2
NO2 f : Ar =
Scheme 1. Synthetic route for compounds 3aeg and 4ceg.
F
Cl
OH
C
D
g : Ar =
Fig. 1. Pharmacophore model of BK-activators (A) and chemical structures of
previously synthesised compounds (BeD).

794
V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 792e799
phenylacetanilides 3a and 3b. In any case, in the acid moiety of
the phenylacetanilides, a methoxy substituent is present which
can be converted to a phenol function by action of boron tribro-
mide. Such a reaction allowed the preparation of the correspond-
ing derivatives 4ceg, bearing the hydroxy group on the acid
moiety of the amide.
COCl
CONH
OCH3
Ar
Ar
+
H2N-CH2-Ar
OCH3
7a-c
8a-c
1
a : Ar
c : Ar
b : Ar
=
=
CONH
CH3O
Compound 3c underwent monodemethylation of the
methoxy group on the acid moiety as confirmed by the
=
OH
O2N
1
comparison of its H NMR spectrum (OCH3 signals at 3.81
9a-c
and 3.99 ppm) with the spectrum of 4c (OCH3 signal at
4.02 ppm). This result agrees with the monodemethylation of
N-(2-methoxyphenyl)-2-methoxy-benzanilides in which the
methoxy group of the acid moiety, adjacent to the carbonyl
function, more easily forms a complex with boron tribromide
and undergoes demethylation [16,19,20].
Scheme 3. Synthetic route for compounds 8aec and 9aec.
7aec, to prepare an analogous little series of benzobenzyla-
mides (11aec) (Scheme 4). The new compounds 11aec
were obtained in the same manner by reaction of the suitable
benzylamine (7aec) with 2-methoxy-benzoic acid chloride
(10). The cleavage of the methoxy groups with boron tribro-
mide allowed the preparation of the corresponding phenol de-
rivatives 12aec. In this case also, the dimethoxy derivative
11b, under the employed experimental conditions, provided
the monohydroxy derivative 12b. According to the previous
results of demethylation reactions, this compound maintained
the methoxy group on the benzylamine moiety as shown by
the 1H NMR data and by the obtainment of 2-hydroxy-benzoic
acid from the alkaline hydrolysis of 11b.
As a further change of the acid portion, two new 2-trifluoro-
methyl-phenylacetanilides 6a and 6b (Scheme 2) were also
prepared and tested, obtained by reaction of 2-trifluoro-
methyl-phenylacetic acid chloride (5) with 2-hydroxy-
5-chloro-aniline (2a) and 2-fluoro-aniline (2f), respectively.
Then the methylenic bridge was introduced either in the
acid moiety or in the basic one of the benzanilide structure,
with the preparation of a limited series of phenylacetobenzyla-
mides (8aec), which show two spacers between the amide
linker and the substituted phenyls (Scheme 3). Compounds
8aec were obtained in the usual manner, by reaction of the
same 2-methoxy-phenylacetic acid chloride (1) with the suit-
able benzylamine (7aec). Therefore benzylamine (7a), 2-me-
thoxy-benzylamine (7b) and 2-nitro-benzylamine (7c) were
chosen for the lengthening of the basic moiety. In this case
also, the presence of a methoxy group on the acid moiety of
the three derivatives 8aec as well as on the basic moiety of
8b allowed the obtainment of the corresponding phenol
compounds 9aec, by cleavage of the ether function with
boron tribromide. It is worth noting that, under the employed
experimental conditions, compound 8b underwent the mono-
demethylation of the methoxy group on the acid moiety to
give the monohydroxy derivative 9b. The structure of 9b
was hypothesized by the comparison of the 1H NMR chemical
shift values of the methoxy group of the available derivatives
and unequivocally confirmed by the alkaline hydrolysis of 9b
which gave 2-hydroxy-phenylacetic acid and 2-methoxy-ben-
zylamine. This result also agrees with the monodemethylation
reported in the literature [16,19,20].
The structures of all the prepared compounds were con-
firmed 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) (see later for the pharmaco-
logical details).
4. Results and discussion
The vasorelaxing efficacy and potency of the tested
compounds and NS 1619 are summarised in Table 3. Many
of the synthesised compounds resulted ineffective or exhibited
low levels of vasorelaxing efficacy (<50%).
The first evidence emerging from the pharmacological
results seems to give a clear indication about the negative im-
pact exerted by the structural features showed by compounds
8, 9, 11 and 12.
At last the methylenic bridge was introduced only in the ba-
sic moiety of the benzanilide, using the same benzylamines
COCl
OCH3
CONH
OCH3
Ar
+
H2N-CH2-Ar
7a-c
11a-c
R1
10
R1
COCl
CONH
CF3
H N
a : Ar
c : Ar
b : Ar
=
+
=
=
2
CONH
OH
Ar
CF3
CH3O
R2
2a,f
R2
5
6a,b
12a-c
a: R1 = OH ; R2 = Cl. b:R1 = F ; R2 = H
O2N
Scheme 2. Synthetic route for compounds 6a and 6b.
Scheme 4. Synthetic route for compounds 11aec and 12aec.

V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 792e799
795
Table 1
Physico-chemical properties of compounds 3, 4, 6, 8, 9, 11 and 12
Compound
Yield (%)
Crystal solvent
M.p. (ꢀC)
Analyses
(C, H, N)
IR (cmꢁ1
)
Mass (m/z)
Mþ
Base
3a
54
51
70
76
78
73
82
54
55
58
59
63
80
85
85
66
60
58
62
55
57
62
80
63
65
50
MeOH/H2O
MeOH/EtOAc
MeOH/H2O
MeOH
174e176
156e158
167e170
153e155
142e144
104e106
103e105
172e174
145e147
198e200
145e147
153e155
173e175
135e137
78e80
C15H14ClNO3
C16H17NO3
C16H16N2O5
C16H16N2O4
1656 (CO); 3340 (NH); 3150 br (OH)
1649 (CO); 3341 (NH); 3130 br (OH)
1683 (CO); 3330 (NH)
291
271
148
107
3b
3c
3d
3e
1667 (CO); 3278 (NH)
1668 (CO); 3260 (NH)
300
287
259
142
218
148
MeOH
C15H14N2O4
3f
3g
MeOH/H2O
MeOH/H2O
MeOH/H2O
MeOH
C15H14FNO2
C15H15NO2
C14H12N2O5
1663 (CO); 3327 (NH)
1662 (CO); 3323 (NH)
4c
1662 (CO); 3310 (NH); 3156 (OH)
1669 (CO); 3430 br (NH, OH)
1669 (CO); 3406 br (NH, OH)
1736 (CO); 3253 br (NH, OH)
1681 (CO); 3345 (NH); 3198 (OH)
1671 (CO); 3375 (NH); 3160 br (OH)
1663 (CO); 3261 (NH)
4d
4e
C15H14N2O4
286
272
121
138
MeOH
MeOH/H2O
MeOH
C14H12N2O4
C14H12FNO2
[21]
4f
4g
6a
MeOH/H2O
MeOH/H2O
MeOH/H2O
EtOAc/hexane
MeOH/H2O
EtOAc/hexane
EtOAc/hexane
MeOH/H2O
EtOAc/hexane
EtOAc/hexane
EtOAc/hexane
MeOH/H2O
H2O
C15H11ClF3NO2
C15H11F4NO
[22]
6b
8a
1654 (CO); 3298 (NH)
1649 (CO); 3284 (NH)
1649 (CO); 3230 (NH)
255
255
8b
8c
97e100
102e104
98e100
110e112
160 (dec.)
85e86
C17H19NO3
C16H16N2O4
C15H15NO2
C16H17NO3
C15H14N2O4
[23]
9a
1613 (CO); 3305 (NH); 3390 br (OH)
1649 (CO); 3384 (NH); 3000 br (OH)
1627 (CO); 3334 (NH); 3000 br (OH)
1630 (CO); 3306 (NH)
9b
9c
271
286
252
193
11a
11b
11c
12a
12b
12c
72e74
84e85
C16H17NO3
1642 (CO); 3392 (NH)
1654 (CO); 3400 (NH)
271
227
136
91
C15H14N2O4
[24]
138e140
103e105
120e122
1640 (CO); 3354 (NH); 3120 br (OH)
1633 (CO); 3350 (NH); 3400 br (OH)
1624 (CO); 3351 (NH); 3399 (OH)
C15H15NO3
C14H12N2O4
MeOH/H2O
For the crystallization from EtOAc/hexane the solutions were cooled at ꢁ20 ꢀC.
Table 2
1H NMR spectra in DMSO-d6, d values
3a
3b
3c
3d
3e
3f
3.72 (s, 2H, CH2); 3.82 (s, 3H, OCH3); 6.82e7.10, 7.23e7.33, 8.07(m, m, s, 7H, Ar); 9.1(NH); 10.3 (OH)
2.15 (s, 3H, CH3); 3.66 (s, 2H, CH2); 3.80 (s, 3H, OCH3); 6.69, 6.88e7.06, 7.20e7.27, 7.68 (s, m, m, s, 7H, Ar); 9.0 (NH); 9.6 (OH)
3.75 (s, 2H, CH2); 3.81 (s, 3H, OCH3); 3.99 (s, 3H, OCH3); 6.88e7.07, 7.21e7.32, 7.99, 9.03 (m, m, d, s, 7H, Ar); 9.4 (NH)
2.35 (s, 3H, CH3); 3.75 (s, 2H, CH2); 3.79 (s, 3H, OCH3); 6.86e7.03, 7.22e7.30, 7.93e8.13 (m, m, m, 7H, Ar); 9.5 (NH)
3.70 (s, 2H, CH2); 3.75 (s, 3H, OCH3); 6.87e7.29, 7.85, 8.21 (m, d, d, 8H, Ar); 10.7 (NH).
3.35 (s, 2H, CH2); 3.98 (s, 3H, OCH3); 7.12e7.37, 7.60, 7.83, 8.13 (m, d, s, m, 8H, Ar); 10.2 (NH)
3.62 (s, 2H, CH2); 3.76 (s, 3H, OCH3); 6.86e7.09, 7.19e7.33, 7.60 (m, m, d, 9H, Ar); 10.2 (NH)
3g
4c
4d
4e
4f
2.75 (s, 2H, CH2); 4.02 (s, 3H, OCH3); 6.74e6.88, 7.08e7.27, 7.99, 9.05 (m, m, d, s, 7H, Ar); 9.5 s, 9.8 s (NH; OH)
2.34 (s, 3H, CH3); 3.72 (s, 2H, CH2); 6.73e7.20, 7.98e8.12 (m, m, 7H, Ar); 9.5 (NH); 9.9 (OH)
3.66 (s, 2H, CH2); 6.70e7.16, 7.85, 8.22 (m, m, d, 8H, Ar); 9.5 (NH); 10.7 (OH)
3.66 (s, 2H, CH2); 6.72e6.83, 7.02e7.29, 7.93 (m, m, m, 8H, Ar); 9.6, 9.7 (NH, OH)
3.59 (s, 2H, CH2); 6.71e6.83, 6.98e7.34, 7.60 (m, m, d, 9H, Ar); 9.5 (NH); 10.0 (OH)
4g
6a
6b
4.02 (s, 2H, CH2); 6.84e6.96, 7.42e7.73, 7.96 (m, m, s, 7H, Ar); 9.5 (NH); 10.2 (OH)
4.00 (s, 2H, CH2); 7.10e7.32, 7.47e7.88, (m, m, 8H, Ar); 10.0 (NH)
8a
3.45 (s, 2H, CH2); 3.74 (s, 3H, OCH3); 4.27 (d, 2H, CH2); 6.81e6.98, 7.14e7.36 (m, m, 9H, Ar); 8.3 t (NH)
3.61 (s, 2H, CH2); 3.71 (s, 3H, OCH3); 3.74 (s, 3H, OCH3); 4.39 (d, 2H, CH2); 6.80e6.99, 7.10e7.33 (m, m, 8H, Ar); 6.3 br (NH)
3.58 (s, 2H, CH2); 3.82 (s, 3H, OCH3); 4.61 (d, 2H, CH2); 6.88e6.99, 7.20e7.60, 8.03 (m, m, d, 8H, Ar); 6.7 br (NH)
8ba
8ca
9a
9b
9c
3.46 (s, 2H, CH2); 4.29 (d, 2H, CH2); 6.71e6.82, 7.01e7.11, 7.25 (m, m, m, 9H, Ar); 8.5 t (NH); 9.6 br (OH)
3.47 (s, 2H, CH2); 3.78 (s, 3H, OCH3); 4.24 (d, 2H, CH2); 6.70e7.20 (m, 8H, Ar); 8.2 (NH); 9.7 (OH)
3.48 (s, 2H, CH2); 4.55 (d, 2H, CH2); 6.71e6.83, 7.00e7.11, 7.50e7.74, 8.02 (m, m, m, d, 8H, Ar); 8.5 t (NH); 8.6 (OH)
11aa
11b
11c
3.88 (s, 3H, OCH3); 4.50 (d, 2H, CH2); 6.99e7.52, 7.74 (m, d, 9H, Ar); 8.7 t (NH).
3.86 (s, 3H, OCH3); 3.92 (s, 3H, OCH3); 4.47 (d, 2H, CH2); 6.88e7.31, 7.48, 7.81 (m, t, d, 8H, Ar); 8.6 t (NH)
3.93 (s, 3H, OCH3); 4.77 (d, 2H, CH2); 7.00e7.23, 7.46e7.84, 8.06 (m, m, d, 8H, Ar); 8.9 t (NH)
12a
12b
12c
a
4.52 (d, 2H, CH2); 6.87e6.94, 7.35, 7.90 (m, m, d, 9H, Ar); 9.4 t (NH); 12.5 (OH)
3.83 (s, 3H, OCH3); 4.48 (d, 2H, CH2); 6.84e7.03, 7.16e7.44, 7.94 (m, m, d, 8H, Ar); 9.2 t (NH); 12.4 (OH)
4.79 (d, 2H, CH2); 6.88e6.95, 7.37e8.08 (m, m, 8H, Ar); 9.3 t (NH); 12.1 (OH)
Registered in CDCl3.

796
V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 792e799
Table 3
Parameters of vasorelaxing efficacy (Emax in %) and potency (pIC50) exhibited
by the synthesised compounds and by the reference drug NS 1619
supported by the observation of the pharmacological profile
exhibited by the couples of analogues 4ce3c, 4de3d and
4ee3e.
Compound
Emax (%)
pIC50
On the contrary, the poor pharmacological effects of analo-
gous compounds, bearing a phenolic hydroxyl group at the
ortho-position of the benzene ring at the basic side of the amide
linker (3a, 3b, and 6a), suggest that this structural feature plays
a negligible role for the interaction with the biological target.
Furthermore, the presence of an aromatic nitro-group in the
benzene ring at the basic side of the amide linker seems to be
a further favourable requirement. Indeed, this group is present
in compounds 4cee (i.e., in the most effective compounds of
these series), while it is absent in compounds 4g and 4f, whose
vasorelaxing efficacy resulted markedly decreased. In previous
papers, we reported benzanilide derivatives which, in some
cases, showed a satisfactory profile of BK-openers. Therefore,
this work was aimed at evaluating the influence of structural
variation on the biological properties of such derivatives.
Overall, the main findings of this work suggest that the intro-
duction of one or two methylene group(s) in the amide linker
exerts a negative influence on the vasorelaxing responses,
which can be due to an excessive lengthening of the spacer
between the two aromatic rings and/or to the conferring of
further degrees of conformational freedom, leading to a re-
duced ability of a potential interaction with the ‘‘receptorial’’
site of the BK channel.
3a
3b
37 ꢂ 10a
61 ꢂ 5a
21 ꢂ 3a
Ineffective
29 ꢂ 2a
28 ꢂ 13a
Ineffective
82 ꢂ 2
NC
4.61 ꢂ 0.03a
3c
3d
NC
e
NC
3e
3f
3g
NC
e
4c
4d þ IbTX 100 nM
4.74 ꢂ 0.02a
84 ꢂ 2, 32 ꢂ 8b
79 ꢂ 5
4.70 ꢂ 0.02a, NC
4e
4.64 ꢂ 0.03a
4f
4g
43 ꢂ 4a
40 ꢂ 4a
50 ꢂ 16a
55 ꢂ 4a
25 ꢂ 2a
22 ꢂ 2a
NT
NC
NC
6a
6b
NC
4.56 ꢂ 0.05a
8a
8b
NC
NC
8c
9a
33 ꢂ 3a
40 ꢂ 16a
40 ꢂ 4a
23 ꢂ 2a
16 ꢂ 3a
29 ꢂ 2a
47 ꢂ 4a
NT
NC
NC
NC
NC
NC
NC
NC
9b
9c
11a
11b
11c
12a
12b
12c
NS 1619
47 ꢂ 4a
NC
5.36 ꢂ 0.04
91 ꢂ 3
NT: not tested; NC indicates that the potency value could not be calculated
because of the low efficacy (ꢃ50%).
5. Experimental section
a
Significantly different from the corresponding value exhibited by NS 1619.
Significantly different from the corresponding value recorded in the
absence of IbTX.
b
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 Trace GC Q plus,
thermo quest Finnigan. Elemental analyses (C, H, N) were
within ꢂ0.4% of the theoretical values and were performed
on a Carlo Erba Elemental Analyzer Mod. 1106 apparatus.
In particular, these compounds, all characterised by very
low levels of vasorelaxing efficacy, show the presence of
a methylenic bridge at the basic side of the amide linker,
which probably represents the main cause for their poor phar-
macological activity. On the other hand, some of these mole-
cules (in particular, compounds 8 and 9) exhibit also the
presence of a further methylene spacer, at the acidic side of
the amide, but this structural characteristic is likely to be
less responsible for their poor efficacy. In fact, this last struc-
tural feature is also present in compounds 3, 4 and 6. Among
these phenyl acetanilide derivatives 4cee showed appreciable
levels of vasorelaxing efficacy, although with modest potency
parameters, significantly lower than that of NS 1619
(P < 0.05). Furthermore, the selective BK-blocker iberiotoxin
(IbTX; 100 nM) caused a significant (P < 0.05) decrease of
the vasorelaxing efficacy of compound 4d, indicating that
the activation of these potassium channels plays a major role
in the vasodilator activity.
5.1.1. N-(Substituted-phenyl)-2-methoxy-
phenylacetamides (3aeg)
A solution of 2-methoxy-phenylacetic acid (0.830 g,
5.0 mmol) in 10 mL of SOCl2 was heated under reflux for
1 h. The reagent was distilled off and the residue, consisting
of the corresponding acyl chloride 1, was dissolved in
20 mL of anhydrous toluene. This solution was added drop-
wise to a solution of the suitable amine [5.0 mmol of
2-hydroxy-5-chloro-aniline (2a), 2-hydroxy-5-methyl-aniline
(2b), 2-methoxy-5-nitro-aniline (2c), 2-methyl-4-nitro-aniline
(2d), 4-nitro-aniline (2e), 2-fluoro-aniline (2f) or aniline (2g)]
and TEA (0.7 mL, 5.0 mmol) in 20 mL of anhydrous toluene
and the mixture was refluxed for 12 h.
Noteworthy, all these three compounds possess a phenolic
hydroxy group at the ortho-position of the benzene ring at
the acidic side of the amide linker, which seems to represent
a significant indication about the need of such a structural
feature as an useful requirement for the interaction with the
biological target (BK channel). This remark seems to be well
For the isolation of 3a and 3b, the toluene solution, after
cooling, separated a crystalline solid which was collected by
filtration and treated with H2O to dissolve the possible TEA

V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 792e799
797
hydrochloride. The insoluble material consisted of a first por-
tion of 3a or 3b. The toluene filtrate was evaporated in vacuo,
the residue was dissolved in CHCl3 and the new solution, after
washing with 10% HCl and 5% NaHCO3, was dried (MgSO4)
and evaporated to give a further amount of 3a or 3b. The
combined fractions were purified by crystallization (Table 1).
For the isolation of 3e, the toluene solution, after cooling,
separated a crystalline solid which was collected by filtration
and consisted of TEA hydrochloride. The filtrate, worked up
as above, provided 3e which was purified by crystallization
(Table 1).
toluene and the mixture was refluxed for 12e16 h. After cool-
ing, a crystalline precipitate of triethylamine hydrochloride
was formed which was separated by filtration. The filtrate
was evaporated in vacuo and the residue was dissolved in
CHCl3. The chloroform solution, after washing with 10%
HCl and 10% NaOH, was dried (MgSO4) and evaporated to
give the title compounds as a residue which was purified by
crystallization (Table 1).
5.1.5. N-(Substituted-benzyl)-2-hydroxy-phenylacetamides
(9aec)
For the isolation of 3c, 3d, 3f and 3g, the toluene solution
was evaporated and worked up in the usual manner (Table 1).
A solution of 2.0 mmol of the appropriate methoxy deriva-
tive 8a, 8b or 8c in 80e100 mL of anhydrous CH2Cl2 was
cooled at ꢁ20 ꢀC and, under stirring, a solution of BBr3
(2 mL, y20 mmol; 3 mL, y30 mmol for 9b) in 8e10 mL
of anhydrous CH2Cl2 was added drop by drop. After 1 h of
stirring at ꢁ20 ꢀC, the reaction mixture was left in an ice-
chamber (ꢁ20 ꢀC) overnight. The reagent was decomposed
by addition of MeOH (8 mL) followed by H2O (30e40 mL)
and the organic phase was separated from the aqueous one.
The organic phase (CH2Cl2), after washing with H2O, was ex-
tracted with 10% NaOH, then dried (MgSO4) and evaporated
to give no residue or little amounts of the starting material.
Acidification of the combined alkaline extracts provided the
title compounds which were collected by filtration and purified
by crystallization (Table 1).
5.1.2. N-(Substituted-phenyl)-2-hydroxy-phenylacetamides
(4ceg)
To a solution of 2.0 mmol of the suitable methoxy deriva-
tive 3cee, 3f or 3g in 100e120 mL of anhydrous CH2Cl2,
cooled at ꢁ30 ꢀC, a solution of BBr3 (z2 mL, z20 mmol;
z3 mL, z30 mmol for 3c) in 10 mL of anhydrous CH2Cl2
was added drop by drop, under stirring. After 1 h at this tem-
perature, the reaction mixture was left at ꢁ20 ꢀC overnight. The
excess of the reagent was decomposed by cautious addition of
MeOH (z10 mL) and H2O (z30 mL). The organic phase, af-
ter washing with H2O, was extracted with 10% NaOH. From
the alkaline solution, cooled in an ice-bath, by acidification
with 36% HCl, precipitated the product which was collected
by filtration and purified by crystallization (Table 1).
The CH2Cl2 solution, dried (MgSO4) and evaporated, left
traces of solid residue.
5.1.6. N-(Substituted-benzyl)-2-methoxy-benzamides
(11aec)
A solution of 2-methoxy-benzoic acid (0.761 g, 5.0 mmol) in
8 mL of SOCl2 was heated under reflux for 1 h. The reagent was
distilled off and the residue, consisting of the corresponding acyl
chloride 10, was dissolved in 20 mL of anhydrous toluene. This
solution was added dropwise to a solution of the suitable amine
[5.0 mmol of benzylamine (7a), 2-methoxy-benzylamine (7b)
or 2-nitro-benzylamine (7c)] and TEA (1 mL, 7.2 mmol) in
20 mL of anhydrous toluene and the mixture was refluxed for
15 h. After cooling a precipitate was formed which was col-
lected by filtration and treated with H2O. The insoluble material
consisted of a fraction of the title compounds. The filtrated
toluene solution was evaporated in vacuo and the residue was
dissolved in CHCl3. The chloroform solution, after washing
with 10% NaOH and 10% HCl, was dried (MgSO4) and evapo-
rated to give a further fraction of the title compounds. The
combined fractions of product were purified by crystallization
(Table 1).
5.1.3. 2-Trifluoromethyl-phenylacetanilides (6a and 6b)
A solution of 2-trifluoromethyl-phenylacetic acid (1.22 g,
6.0 mmol) in 10 mL of SOCl2 was heated under reflux for
1 h. The reagent was distilled off and the residue, consisting
of the corresponding acyl chloride 5, was dissolved in
30 mL of anhydrous toluene. This solution was added
dropwise to a solution of the suitable amine [6.0 mmol of
2-hydroxy-5-chloro-aniline (2a) or 2-fluoro-aniline (2f)] and
TEA (0.85 mL, 6.0 mmol) in 50 mL of anhydrous toluene
and the mixture was heated at 100 ꢀC overnight. After cooling
the solution was evaporated in vacuo and the residue was
dissolved in CHCl3. The chloroform solution, after washing
with 10% HCl and 5% NaHCO3, was dried (MgSO4) and
evaporated to give the title compounds as a solid residue
(Table 1).
5.1.4. N-(Substituted-benzyl)-2-methoxy-phenylacetamides
(8aec)
5.1.7. N-(Substituted-benzyl)-2-hydroxy-benzamides
(12aec)
A solution of 2-methoxy-phenylacetic acid (0.830 g,
5.0 mmol) in 10 mL of SOCl2 was heated under reflux for
1 h. The reagent was distilled off and the residue, consisting
of the corresponding acyl chloride 1, was dissolved in 20 mL
of anhydrous toluene. This solution was added dropwise to
a solution of the suitable amine [5.0 mmol of benzylamine
(7a), 2-methoxy-benzylamine (7b) or 2-nitro-benzylamine
(7c)] and TEA (0.7 mL, 5.0 mmol) in 20 mL of anhydrous
A solution of 2.0 mmol of the appropriate methoxy deriva-
tive 11a, 11b or 11c, in 100 mL of anhydrous CH2Cl2 was
cooled at ꢁ20 ꢀC and, under stirring, a solution of BBr3
(2 mL, y20 mmol; 3 mL, y30 mmol for 12b) in 8e10 mL
of anhydrous CH2Cl2 was added drop by drop. After 1 h of
stirring at ꢁ20 ꢀC, the reaction mixture was left in an ice-
chamber (ꢁ20 ꢀC) overnight. The reagent was decomposed
by addition of MeOH (8 mL) followed by H2O (40 mL) and

798
V. Calderone et al. / European Journal of Medicinal Chemistry 43 (2008) 792e799
the organic phase (CH2Cl2) after washing with H2O was ex-
tracted with 10% NaOH, then dried (MgSO4) and evaporated
to give no residue (or little amounts of the starting material).
Acidification of the combined alkaline extracts provided the ti-
tle compounds which were collected by filtration and purified
by crystallization (Table 1).
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 of 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 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 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 <50%. Com-
pounds exhibiting an efficacy level <20% were considered as
ineffective. The parameters of efficacy and potency were
expressed as mean ꢂ 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 by
a computer fitting procedure (software: GraphPad Prism 3.0).
5.2. Pharmacology
All the experimental procedures were carried out following
the guidelines of the European Community Council Directive
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 a light ether anaesthesia, the rats
were sacrificed by cervical dislocation and bleeding. The
aortae were immediately excised and freed of extraneous tis-
sues. The endothelial layer was removed by gently rubbing
the intima surface of the vessels with a hypodermic needle.
Five millimeter-wide aortic rings were suspended, under a pre-
load 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), thermostated at 37 ꢀC and continuously gassed with
a mixture of O2 (95%) and CO2 (5%). Changes in tension
were recorded by means of an isometric transducer (Grass
FTO3), connected with a unirecord microdynamometer
(Buxco Electronics).
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