Synthesis and biological activity of novel substituted benzanilides as potassium channel activators. V

Article abstract of DOI:10.1016/j.ejmech.2004.02.006

As part of our program toward designing potassium channel openers, the synthesis of a novel series of substituted benzanilides and their vasodilating activity are presented. The facile synthetic pathway generally involves coupling between the appropriate

Full text of DOI:10.1016/j.ejmech.2004.02.006

European Journal of Medicinal Chemistry 39 (2004) 491–498
www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of novel substituted benzanilides
as potassium channel activators. V
Giuliana Biagi a, Irene Giorgi a, Oreste Livi a, Antonio Nardi a,*, Vincenzo Calderone b,
Alma Martelli b, Enrica Martinotti b, Oreste LeRoy Salerni c
a Dipartimento di Scienze Farmaceutiche, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, via Bonanno 6, 56126 Pisa, Italy
c Butler University College of Pharmacy and Health Sciences, 4600 Sunset, Indianapolis, IN 46208, USA
Received 29 September 2003; received in revised form 9 February 2004; accepted 11 February 2004
Available online 02 April 2004
Abstract
As part of our program toward designing potassium channel openers, the synthesis of a novel series of substituted benzanilides and their
vasodilating activity are presented. The facile synthetic pathway generally involves coupling between the appropriate benzoyl chloride and
commercial available anilines, followed by the selective or non-selective cleavage of methyl ether substituent(s), affording the corresponding
phenol or bisphenol derivatives. The pharmacological evaluation of these structurally novel potential BK-openers on vascular contractile
activity was studied in vitro, using isolated rat aortic rings pre-contracted with KCl 20 mM. Some derivatives were found to be potent smooth
muscle relaxants and the vasodilation effects of these compounds were inhibited by tetraethylammonium (TEA) and iberiotoxin (IbTX),
suggesting that the opening of BK channels is prevalent in the mechanism of action of these compounds. The best compound of the series was
N-(2-hydroxy-5-phenyl)-(2-methoxy-5-chloro)-benzamide (16b) showing a full vasorelaxant efficacy and almost nanomolar potency index.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Benzanilides; BK channels; BK-openers; Vasodilator activity
1. Introduction
psychoses, post-stroke neuroprotection, convulsions and
anxiety. As far as the cardiovascular system is concerned,
since the activation of relatively few BK channels can have a
strong influence on membrane potential, the physiological
function of these ion channels represents a fundamental
steady state mechanism, modulating vessel depolarisation,
vasoconstriction and increases of intravascular pressure [17].
It has been clearly documented that the role and/or the ex-
pression of vascular BK channels is enhanced during chronic
hypertension, acting as a protective mechanism triggered by
the pathological status itself [17]. An increased role of BK
channels in the vasodilator responses of several vascular beds
has also been observed in hypercolesterolemic conditions. A
decreased modulation by BK channels has been observed in
the coronary vessels of aged humans and rats, a possible
cause of senile coronary vasospasm [18].
Large-conductance calcium-activated potassium chan-
nels, also known as BK or Maxi-K channels, almost ubiqui-
tously distributed among tissues, are expressed in both excit-
able and non-excitable cells, where they are involved in the
regulation of cell excitability and function. Their physiologi-
cal role has been especially studied in the nervous system
[1–3], where they are key regulators of neuronal excitability
and of neurotransmitter release, and in smooth muscle [4–8],
where they are crucial in modulating the tone of vascular,
broncho-tracheal, urethral, uterine or gastro-intestinal mus-
culature. Given these implications, small agents with BK-
opener properties, named BK-openers or BK-activators,
could have a potentially powerful influence in the modulation
and control of numerous consequences of muscular and neu-
ronal hyperexcitability [1,9,10–16], such as asthma, urinary
incontinence and bladder spasm, gastroenteric hypermotility,
Diabetes causes an approximate fourfold increased risk of
coronary, cerebrovascular and further cardiovascular prob-
lems. In fact, diabetes represents a condition with impaired
production of reactive oxygen species, determining a de-
creased availability of endogenous NO and a higher produc-
* Corresponding author.
E-mail address: nardi@farm.unipi.it (A. Nardi).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.02.006

492
G. Biagi et al. / European Journal of Medicinal Chemistry 39 (2004) 491–498
opener properties. The other aromatic ring, if not substituted
H
N
Cl
Cl
F3C
by an o-hydroxyphenyl group, often displays electron with-
drawing groups. The spacer unit, when present, may vary. It
can be represented by a heterocyclic ring, that may or may
not be fused to one of the two phenyls, or by an acyclic
moiety. For the latter, chemically heterogeneous linkers of
various lengths seems to be tolerated: the phenyl systems
may be separated by one bond (no spacer), such as in the
compound 3 [21] or 4 (magnolol) [23], two bonds, such as in
the compounds 5 and 6 [21] that are, respectively, character-
ised by a methylene and a thioether spacer or even four, such
as in the compound 7 (NS 1608) [24], where an urea linker is
present (Fig. 1).
O
OH
N
OH
HO
Cl
OH
HO
Cl
X
1 (NS004), X = Cl
2 (NS1619), X = CF3
3
4
Cl
S
Cl
F3C
Cl
Cl
OH
CH2
OH
Cl
Cl
OH
NH
NH
In the course of our studies focused on structural modifi-
cation of the prototypical BK-opener NS 004, we elected to
synthesise and evaluate a number of new potential BK-
opener compounds [25–28]. On pursuing our researches, we
sought, in an effort to explore the scope of the linker, to insert
an amide moiety as a new three-bond-spacer unit between an
o-hydroxyphenyl and a multi-substituted phenyl ring or be-
tween two multi-substituted phenol nuclei. This spacer,
where the amidic NH could also represent one of the required
H-bond-donor sites, has recently demonstrated to be a valid
choice, as shown by the easy synthetic pathway and vasodi-
lation properties of some 2-hydroxy-N-phenyl-benzamide
derivatives (general formula A, R1: NO2, Fig. 2) [28] (in this
series compound 8 is the most representative). These com-
pounds represent key intermediates to the synthesis of some
potassium channel openers, the 5-substituted-1-(2-
hydroxybenzoyl)-benzotriazoles [28], which have been
evaluated for activity as potential potassium channel activa-
tors. Thus, to further investigate this new scaffold, we
planned a simple synthesis and pharmacological evaluation
of some new benzanilides, the 2-hydroxy-N-phenyl-
benzamides (general formula A, Fig. 2), bearing new sub-
stituents in the N-phenyl ring (compounds 12a–g, Fig. 4) and
new N-(2-hydroxyphenyl)-benzamides (general formula B,
Fig. 2), represented by compounds 16a–b and 17 (Fig. 6).
Here, we also report the pharmacological evaluation of two
related benzanilides (compounds 18a and 18b, Fig. 3), which
O
OH
Cl
OH
Cl
Cl
7
5
6
Fig. 1. Chemical structures of some BK-openers.
tion of peroxynitrite. Both these factors can play a major role
in the inactivation of vascular BK channels, reported in
several experimental models [17]. Given the above consider-
ations, development of selective activators of BK channels
appears to be a promising research field for the pharmaco-
therapy of vascular diseases, including hypertension, coro-
nary diseases and vascular complications associated with
diabetes or hypercolesterolemia.
The synthetic benzimidazolone derivatives NS 004 (1)
and NS 1619 (2) (Fig. 1) are pioneer BK-activators [19,20]
and represent the reference models, which led to the design
of several chemically heterogeneous BK-openers [9]. Many
of these compounds show a general pharmacophoric model,
involving the presence of two phenyl rings, that may or may
not be symmetrically, linked by a spacer unit, and at least two
H-bond-donor sites [21,22]. One of the two phenyl rings is
often represented by an o-hydroxyphenyl group; a halogen
substituent, if present, is preferred in the p-position to the
hydroxy function. The latter seems to be essential for BK-
R2
R3
R1
N
OH
H3C
NO2
H
H
H
N
OH R1
N
R2
R4
OH
R4
O
O
O
R3
8
A
B
General formulaA
General formula B
R : Cl, CH , H
R : OCH , OH, NO
2
1
3
1
3
R : OCH , OH, NO
R : OH, NO , H
2
3
2
2
2
R
Cl, H
R
Cl, NO , H
2
3:
3:
R : CH , H
R :CH , H
4
3
4
3
Fig. 2. Reference compound 8 and general formula A and B of the tested compounds.

G. Biagi et al. / European Journal of Medicinal Chemistry 39 (2004) 491–498
493
2. Chemistry
OH
OH
H
H
H3C
N
NO2
N
NO2
A series of new 2-methoxybenzanilides (11a–e) was pre-
pared in excellent yield, by the acylation reaction between
the 2-methoxybenzoylchloride (9) [29] and commercial
available anilines (10a–e) (see Table 1 for the experimental
conditions). The methoxy derivatives (11a–e), on treatment
with an excess of boron tribromide, underwent a demethyla-
tion reaction to give the corresponding salicylanilides
(12a–g) (Fig. 4). (Compounds 12d [30], 12f [31], 12g [32]
were previously reported in literature but obtained by differ-
ent procedures.) The demethylation reaction on benzanilides
11b–e, obtained from anilines containing a methoxy group
(10b–e), provided the expected dihydroxy compounds 12b,
12d, 12f, 12g. In the case of dimethoxy derivatives 11c and
11d, bearing nitro substituents and a methoxy group in the
o-position of the aniline ring, the treatment with boron tribro-
mide at a temperature not higher than –70 °C, did not cleave
the methoxy group in the N-aryl substituent and the
monomethoxy derivatives 12c and 12e were isolated in good
yields. Unequivocal proof of chemical structure of 12e was
demonstrated by its preparation by an alternate synthetic
pathway, which involves reaction of the acetylsalicylic acid
chloride (13) [33] with the 2-methoxy-5-nitroaniline (10d),
as presented in Fig. 5. It is assumed that ester hydrolysis
occurred upon evolution of HCl. Selective demethylation of
some aryl methyl ethers by use of the analogue Lewis acid
BCl3 has been reported [34]. Therefore, under controlled
experimental conditions, rapid and selective demethylation
of the methoxy function in o-position to the carbonyl group
takes place. This occurs even if in the presence of a large
excess of BBr3. Finally, in an effort to further explore this
new chemotype, we prepared two other different amido de-
rivatives, the N-(2-hydroxyphenyl)-2-methoxybenzamides
16a and 16b (Fig. 4), bearing a methoxy substituent on the
acid part of the benzanilide and an hydroxy function in the
o-position of the aniline ring. This was accomplished by
reaction of 2-methoxy-5-chloro-benzoylchloride (14) [35]
with 2-amino-4-chlorophenol (15a) or 2-amino-4-methyl-
phenol (15b), respectively. The cleavage of methyl ether 16b,
again by treatment with boron tribromide, led to compound
17 in good yield (Table 2).
O
O
Cl
18a
18b
Fig. 3. Compounds 18a–b, previously prepared and structurally correlated
to 16a–b, 17.
we previously prepared as intermediates to the synthesis of
some benzotriazinones [27] whose vasodilating properties
have not yet been investigated. The pharmacological study
was performed on in vitro assays (rat aortic rings) by the
pharmacological evaluation of their vasodilating activity and
of the possible involvement with BK channels in the mecha-
nism of action.
R2
R1
COCl
+
R3
NH2
OCH3
i
R4
10
9
R2
R3
R1
R2
R3
R1
ii
H
H
OCH3
N
N
OH
R
4O
R
4O
11
12
Cmpd
R1
R2
R3
R4
10 or11a
10 or11b
NO2
NO2
H
OCH3
NO2
H
H
H
CH3
H
10 or11c OCH3
10 or11d OCH3
10 or11e OCH3
H
H
NO2
Cl
H
H
H
H
12a
12b
12c
12d
12e
12f
12g
NO2
NO2
OCH3
OH
H
CH3
H
OH
NO2
NO2
H
H
H
H
H
H
OCH3
OH
NO2
NO 2
Cl
H
H
H
Structures of all the new compounds prepared were con-
firmed by analytical and spectroscopic methods. Table 3
reports the 1H-NMR data.
OH
H
H
Fig. 4. Preparation of benzanilides 11a–e and 12a–g. Reagents and condi-
tions: (i) toluene (100 °C); (ii) BBr3.
OCH3
+
NH2
OCH3
H
COCl
i
O2N
O2N
N
OH
OCOCH3
O
10d
13
12e
Fig. 5. Preparation of 12e by one pot synthesis. Reagents and conditions: (i) toluene (100 °C).

494
G. Biagi et al. / European Journal of Medicinal Chemistry 39 (2004) 491–498
OH
OH
Cl
COCl
OCH3
i
+
H
R
NH2
R
N
OCH3
Cl
O
R = CH3 = 15a
R = Cl = 15b
14
R = CH3 = 16a
R = Cl = 16b
OH
H
ii
Cl
N
OH
O
17
Cl
Fig. 6. Preparation of compounds 16a–b and 17. Reagents and conditions: (i) toluene (16a: 100 °C; 16b: r.t.); (ii) BBr3.
3. Pharmacology
vasodilating mechanism of action. In the presence of tetra-
ethylammonium (TEA), a blocker of different subtypes of
KCa channels, the dilating action of 16a and 16b was dramati-
cally reduced. A higher inhibition was observed in the pres-
ence of iberiotoxin (IbTX), a selective BK channel blocker
which significantly reduced the effects of these two com-
pounds, suggesting that BK channels play a major role in the
mechanism of action of 16a and 16b. The pharmacological
results allow us to make some initial observations concerning
structure–activity relationships. According to our previous
studies [28], the 2-nitrosalicylanilide structures (general for-
mula A, R1: NO2, Fig. 2) showed good vasodilating activity.
In particular, compound 12a (Fig. 4) was a more potent
vasodilator (pIC50: 6.60) than the reference compound NS
1619 (pIC50: 5.18) and the isomeric compound 8 (Fig. 2),
previously reported (pIC50: 5.60) [28]. Compound 12b
(pIC50: N.C.) (Fig. 4) showed that an introduction of an
hydroxy function in the m-position to the nitro group on the
anilino moiety of the salicylanilide, combined with the re-
moval of the methyl substituent, caused a clear decrease of
activity. On the contrary, the replacement of the nitro group
in the o-position of the aniline moiety with a methoxy sub-
stituent, provided compounds with good vasodilating activ-
ity (12c, pIC50: 6.72 and 12e, pIC50: 6.35, Fig. 4). The
demethylation of the methoxy substituent of compounds 12c
and 12e (Fig. 4) to give bisphenol derivatives 12d (pIC50:
5.84) and 12f (pIC50: 5.09) (Fig. 4) induced a remarkable
lowering of the efficacy parameter. In contrast, the presence
of a phenolic acid function in the 2 position of the aniline
moiety and a methoxy group in the 2 position of the salicylic
The vasodilating effect of the novel potential BK-openers
12a–g, 16a–b, 17, 18a–b on vascular contractile function
was studied in vitro, using isolated rat aortic rings pre-
contracted with KCl 20 mM (see later for the pharmacologi-
cal details).
4. Results and discussion
The experimental results are summarised in Table 4. All
the tested compounds showed a profile of full or nearly full
vasodilation action, except for the compounds 12b (Fig. 4),
18a and 18b (Fig. 3) which exhibited a modest vasodilating
effect (<50%). Compound 12f (Fig. 4), which is character-
ised by a value of efficacy of approximately 50%, and a value
of potency lower than all the other compounds tested, proved
to be comparable with that recorded for the reference com-
pound NS 1619. Compound 12d (Fig. 4) presented an index
of potency slightly higher than that of NS 1619, while 12a,
12c, 12e, 12g (Fig. 4) and 17 (Fig. 6) have shown a signifi-
cant improvement (>10-fold) of potency. This parameter
reached remarkable values for compounds 16a and 16b
(Fig. 6), respectively, approximately 200- and 2000-fold
higher than the value of potency of NS 1619. For this reason,
compounds 16a and 16b were investigated further to identify
the pharmacodynamic profile and a possible involvement of
the potassium channels, especially the BK channels, in the
Table 1
Experimental conditions for the preparation of the benzanilides 11a–e and 16a–b
Compounds
11a
11b
11c
11d
11e
16a
16b
Acyl chloride (mmol)
Amine (mmol)
12.88
Solvent (ml)
150
Temperature (°C)
Time (h)
11.7
105
85
20
20
24
20
16
16
3
5.86
5.86
150
11.73
5.86
11.73
150
100
85
5.86
150
3.55
3.55
50
85
10.71
10.71
10.71
150
20
10.71
150
100

G. Biagi et al. / European Journal of Medicinal Chemistry 39 (2004) 491–498
495
Table 2
Table 4
Chemical and physical properties of all synthesised compounds
Experimental results: potency (pIC50) and efficacy (Emax %) values of the
tested compounds
Compounds Yield
(%)
Crystallisation
solvent
Melting Analyses (C, H, N)
point
(°C)
Compounds
12a
Efficacy (Emax %)
100a
pIC50
6.60 0.052
N.C.
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
12f
12g
16a
16b
17
72
96
85
82
70
94
90
42
MeOH/H2O
EtOH
112–114
148–150
216–218
257–259
134–136
143–145
178–181
205–206
197–200
244–246
267–270
192–195
170–171
240–242
235–236
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
15H14N2O4
15H14N2O5
15H14N2O5
15H14N2O5
15H14NO3Cl
14H12N2O4
13H10N2O5
14H12N2O5
13H10N2O5
14H12N2O5
13H10N2O5
13H10NO3Cl
15H14NO3Cl
14H11NO3Cl2
13H9NO3Cl2
12b
12c
12d
12e
12f
12g
16a
+TEA
+IbTX
16b
+TEA
+IbTX
17
18a
18b
NS 1619
41.2 9.7
100a
6.72 0.23
5.84 0.039
6.35 0.17
5.09 0.095
6.87 0.37
7.56 0.10
N.C.
DMF/H2O
DMF/H2O
EtOH
100a
100a
54.6 10.6
90.2 6.4
86.4 6.6
27.7 16.0*
MeOH/H2O
EtOH
MeOH
61
a
MeOH/H2O
DMF/H2O
DMF/H2O
EtOH/H2O
EtOH/H2O
EtOH
3
4*
N.C.
100a
8.49 0.091
N.C.
18 b
34
15.8 4.2*
3
2*
N.C.
52
89.8 2.3
32.5 16.6
24.0 11.6
100a
6.43 0.11
N.C.
81
62
MeOH/H2O
N.C.
a Procedure A: 66%; procedure B: 84%.
b After recrystallisation (analytical sample).
5.18 0.055
The asterisk * indicates a significant difference from the control value. N.C.
indicates that the parameter could not be calculated because of low efficacy
(<50%). The symbol a indicates that a full vasorelaxing effect was reached in
all the experiments performed, hence the S.E. could not be expressed.
moiety (compounds 16a, pIC50: 7.56 and 16b, pIC50: 8.49,
Fig. 6), provided the most potent compounds of this series.
However, poorly active compounds were obtained when the
methoxy substituent was replaced with a nitro group (com-
pounds 18a and 18b, efficacy <50%, Fig. 3). Substitution of a
chloro for a nitro function, generally induced an increased
vasodilation (compounds 16a and 16b; and 12g: pIC50:
6.87). Finally, the concurrent presence of a hydroxy group on
both benzanilide rings caused a remarkable lowering of ac-
tivity (compounds 12b, 12d, 12f and 12g, Fig. 4, and 17,
pIC50: 6.43, Fig. 6). In this series, both the amido and phe-
nolic function groups show acid properties and the biological
data suggests that the introduction of another phenolic group,
a third weakly acidic function, would be detrimental to BK
channel activation.
Table 3
1H-NMR spectra in DMSO-d6 (d)
11a
2.38 (s, 3H, CH3); 3.97 (s, 3H, OCH3); 7.06–7.82 (m, 7H, Ar);
10.13 (s, 1H, NH)
11b
3.86 and 4.05(2s, 6H, 2OCH3); 7.13–8.48 (m, 7H, Ar); 11.44 (s,
1H, NH)
11c
11d
4.11 (s, 6H, 2OCH3); 7.14–7.82 (m, 7H, Ar); 11.02 (s, 1H, NH)
4.11 and 4.13 (2s, 6H, 2OCH3); 7.16–8.41 (m, 6H, Ar); 9.39 (s.1H,
Ar); 10.86 (s, 1H, NH)
5. Experimental protocols
5.1. Chemistry
11e
12a
12b
12c
12d
12e
12f
12g
16a
16b
17
3.97 and 4.08 (2s, 6H, 2OCH3); 7.12–8.54 (m, 7H, Ar); 10.74 (s,
1H, NH)
2.36 (s, 3H, CH3); 6.95–7.97 (m, 7H, Ar); 10.51 and 11.81 (2s, 2H,
NH and OH)
All 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 on aVarian Gemini 2000 spectrometer
in DMSO-d6 in d units, using TMS as an internal standard.
Elemental analyses (C, H, N) were within 0.4% of the
theoretical values and were performed in a Carlo Erba El-
emental Analyser Mod. 1106 apparatus.
6.93–8.13 (m, 7H, Ar); 11.31, 11.38 and 11.79 (3s, 3H, NH and
2OH)
6.97–8.06 (m, 6H, Ar); 8.74 (d, 1H, Ar); 11.31 and 11.91 (2s, 2H,
NH and OH)
6.97–8.03 (m, 6H, Ar); 8.68 (d, 1H, Ar);11.27, 11.29 and 11.85 (3s,
3H, NH and 2OH)
6.98–8.08 (m, 6H, Ar); 9.40 (d, 1H, Ar); 11.13 and 11.86 (2s, 2H,
NH and OH)
6.97–8.08 (m, 6H, Ar); 9.39 (d, 1H, Ar);11.09, 11.81 and 11.90 (3s,
3H, NH and 2OH)
5.1.1. General procedure for
the N-(substitutedphenyl)-2-methoxybenzamides (11a–e)
6.87–8.49 (m, 7H, Ar); 10.43, 10.91 and 11.74 (3s, 3H, NH and
2OH)
Equimolar amounts of 2-methoxybenzoylchloride (9) and
the appropriate substituted aniline (10a–e) (Fig. 4) in anhy-
drous toluene were reacted under the experimental condi-
tions reported in Table 1. After cooling, the solvent was
evaporated in vacuo and the residue was stirred with 10%
NaOH, 10% HCl and H2O. The residue obtained after every
2.23 (s, 3H, CH3); 4.04 (s, 3H, OCH3); 8.18–8.71 (m, 6H, Ar);
10.00 and 10.50 (2s, 2H, NH and OH)
4.05 (s, 3H, OCH3); 6.89–8.42 (m, 6H, Ar); 10.61 and 10.65 (2s,
2H, NH and OH)
6.88–8.45 (m, 6H, Ar); 10.50, 10.92 and 12.09 (3s, 3H, NH and
2OH)

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G. Biagi et al. / European Journal of Medicinal Chemistry 39 (2004) 491–498
decantation, consisting of the title compound, was collected
by filtration and purified by crystallisation (Table 2).
5.1.6. N-(2-methoxy-5-nitrophenyl)-salicylanilide (12e)
A) (Fig. 4) To a stirred solution of dimethoxybenzanilide
11d (0.272 g, 0.90 mmol) in 120 ml of anhydrous CH2Cl2,
cooled at –60 °C and under a nitrogen flow, a solution of
BBr3 (1.0 ml, 10.6 mmol) in 8 ml of anhydrous CH2Cl2 was
slowly added (≈1 h). The reaction mixture was kept at –20 °C
for 15–16 h, then it was worked up as described for 12a, to
give the title compound (Table 2).
5.1.2. N-(2-nitro-6-methylphenyl)-salicylanilide (12a)
To a stirred solution of monomethoxybenzanilide 11a
(0.400 g, 1.40 mmol) in 100 ml of anhydrous CH2Cl2, cooled
at –60 °C and under a nitrogen flow, a solution of BBr3
(1.0 ml, 10.6 mmol) in 8 ml of anhydrous CH2Cl2 was slowly
added. The reaction mixture was left at –20 °C for 15 h, then
again placed in an ice–salt bath, and finally the reagent was
destroyed by the addition of MeOH and H2O, drop by drop
and with stirring. The organic layer was separated, washed
with H2O, then extracted with 7% NaOH. The combined
alkaline extracts were acidified and again extracted with
CHCl3. The chloroform extracts, after washing with H2O,
were dried (MgSO4) and evaporated to give 12a as a solid
residue which was purified by crystallisation (Table 2).
B) (Fig. 5) A solution of acetylsalicylic acid chloride (13)
(1.70 g, 8.61 mmol) and 2-methoxy-5-nitroaniline (10d)
(1.45 g, 8.63 mmol) in 120 ml of anhydrous toluene was
heated at 105 °C for 24 h. The solvent was evaporated in
vacuo and the residue was stirred with 6% NaHCO3, 10%
HCl and H2O. The new residue obtained after every decanta-
tion, consisting of 12e, was collected by filtration and puri-
fied by crystallisation (Table 2).
5.1.7. N-(2-hydroxy-5-nitrophenyl)-salicylanilide (12f)
5.1.3. N-(4-hydroxy-2-nitrophenyl)-salicylanilide (12b)
To a stirred solution of dimethoxybenzanilide 11d
(0.500 g, 1.65 mmol) in 150 ml of anhydrous CH2Cl2, cooled
at –10 °C and under a nitrogen flow, a solution of BBr3
(2.0 ml, 21 mmol) in 7 ml of anhydrous CH2Cl2 was slowly
added. After stirring at r.t. for 24 h, the reaction mixture was
worked up as described for 12a. The title compound, precipi-
tated by acidification as a brown gel, was collected by paper
filtration. This gel had to be dissolved in hot MeOH and
reprecipitated by the addition of H2O as a crude brown solid,
which was purified by treatment with EtOH/H2O then recrys-
tallised (Table 2).
To a stirred solution of dimethoxybenzanilide 11b
(0.600 g, 1.98 mmol) in 50 ml of anhydrous CH2Cl2, cooled
at –78 °C and under a nitrogen flow, a solution of BBr3
(2.0 ml, 21 mmol) in 8 ml of anhydrous CH2Cl2 was slowly
added (≈40 min) and the temperature was raised to –60 °C.
The reaction mixture was left at –20 °C for 15 h, then it was
worked up as described for the preparation of 12a. The title
compound, precipitated by acidification as an orange solid,
was collected by filtration (Table 2).
5.1.4. N-(2-methoxy-4-nitrophenyl)-salicylanilide (12c)
To a stirred solution of dimethoxybenzanilide 11c
(0.400 g, 1.32 mmol) in 150 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 anhydrous CH2Cl2 was slowly
added. The cooling-bath was removed when the temperature
was raised to –50 °C and the reaction mixture was kept at
–20 °C for 15 h, then at room temperature (r.t.) for 6 h. The
reaction was worked up as described for the preparation of
12a and the title compound, precipitated by acidification as a
clear solid, was collected by filtration (Table 2).
5.1.8. N-(2-hydroxy-5-chlorophenyl)-salicylanilide (12g)
To a stirred solution of dimethoxybenzanilide 11e
(0.600 g, 2.06 mmol) in 30 ml of anhydrous CH2Cl2, cooled
at –5 °C and under a nitrogen flow, a solution of BBr3 (2.0 ml,
21 mmol) in 8 ml of anhydrous CH2Cl2 was slowly added.
The ice-bath was removed and the stirring continued at r.t. for
15–16 h. The reaction mixture was worked up as described
for the preparation of 12a, to give 12g as a solid residue
which was purified by crystallisation (Table 2).
5.1.9. N-(2-hydroxy-5-methylphenyl)-(2-methoxy-5-chloro)-
benzamide (16a) and N-(2-hydroxy-5-chlorophenyl)-(2-
methoxy-5-chloro)-benzamide (16b)
5.1.5. N-(2-hydroxy-4-nitrophenyl)-salicylanilide (12d)
To a stirred solution of dimethoxybenzanilide 11c
(0.400 g, 1.32 mmol) in 80 ml of anhydrous CH2Cl2, cooled
at –5 °C and under a nitrogen flow, a solution of BBr3 (1.0 ml,
10.6 mmol) in 8 ml of anhydrous CH2Cl2 was slowly added.
The reaction mixture was left at 4 °C for 15 h and at r.t. for
12 h, then it was worked up as described for the preparation
of 12a. The title compound, precipitated by acidification as a
solid and consisting of a mixture of the expected didemethy-
lated compound 12d and ≈5% of the monodemethylated 12c,
was purified by fractional crystallisation from MeOH/H2O,
taking advantage of the lower solubility of 12c (Table 2).
Equimolar amounts of 2-methoxy-5-chlorobenzoyl-
chloride (14) and 2-hydroxy-5-methylaniline (15a) or
2-hydroxy-5-chloro-aniline (15b) in anhydrous toluene were
reacted under the experimental conditions reported in
Table 1. For the isolation of 16a the solvent was evaporated
and the residue was washed with 10% HCl, 6% NaHCO3 and
H2O (Table 2). Compound 16b, crystallised by the cooling of
the reaction mixture, was collected by filtration (65% yield).
A further fraction was obtained by the evaporation of the

G. Biagi et al. / European Journal of Medicinal Chemistry 39 (2004) 491–498
497
filtrate and purification of the residue by the procedure de-
scribed for 16a (Table 2).
nel blocker tetraethylammonium chloride (TEA 10 mM) or
the BK-selective blocker iberiotoxin (IbTX, 100 nM) were
added, after the KCl (20 mM)-induced contraction, followed
by the administration of selected compounds. The reference
drug NS 1619 (Sigma) was dissolved (10 mM) in EtOH 95%
and further diluted in Tyrode solution. Acetylcholine chlo-
ride (Sigma) was dissolved (100 mM) in EtOH 95% and
further diluted in bidistilled water whereas KCl and TEA
were both dissolved in Tyrode solution. Most of the synthe-
sised derivatives (12a–g, 16a, 18a–b) were dissolved
(10 mM) in NaOH 0.1 N whereas two of them (16b and 17)
were dissolved (10 mM) in DMSO; they all were further
diluted in Tyrode solution. All the solutions were freshly
prepared immediately before the pharmacological experi-
mental procedures. Previous experiments showed a complete
ineffectiveness of the vehicles. The vasodilating efficacy was
evaluated as maximal vasodilating response, expressed as a
percentage (%) of the contractile tone induced by KCl
20 mM. When the limit concentration 0.1 mM (the highest
concentration, which could be administered) of the tested
compounds did not reach the maximal effect, the parameter
of efficacy represented the vasodilating response, expressed
as a percentage (%) of the contractile tone induced by KCl
20 mM, evoked by this limit concentration. The parameter of
potency was expressed as pIC50, calculated as negative Loga-
rithm of the molar concentration of the tested compounds,
evoking a half reduction of the contractile tone induced by
KCl 20 mM. The pIC50 could not be calculated for those
compounds showing an efficacy parameter lower than 50%.
The parameters of efficacy and potency were expressed as
mean standard error (S.E.), for 5–10 experiments. Stu-
dent’s t-test was selected as statistical analysis, P < 0.05 was
considered representative of a significant statistical differ-
ence. Experimental data were analysed by a computer fitting
procedure (software: GraphPad Prism 3.0).
5.1.10.N-(2-hydroxy-5-chlorophenyl)-(2-hydroxy-5-chloro)-
benzamide (17)
To a stirred solution of benzanilide 16b (0.500 g,
1.68 mmol) in 160 ml of anhydrous CH2Cl2, cooled at –70 °C
and under a nitrogen flow, a solution of BBr3 (1.5 ml, 15.75
mmol) in 8 ml of anhydrous CH2Cl2 was slowly added (≈1
h). The reaction mixture was allowed to reach r.t. and stirring
was maintained for a night, then again placed in an ice–salt
bath, to destroy the excess of the reagent by the addition of
MeOH and H2O, drop by drop and under stirring. This
mixture was extracted with 7% NaOH and the combined
alkaline extracts were acidified. The white solid precipitated
was collected by filtration and purified by crystallisation
(Table 2).
5.1.11. Pharmacology
All the experimental procedures were carried out follow-
ing the guidelines of the European Community Council Di-
rective 86-609. To determine a possible vasodilator mecha-
nism of action, the compounds were tested on isolated
thoracic aortic rings of male normotensive Wistar rats (250–
350 g). The rats were sacrificed by cervical dislocation under
light ether anaesthesia and bled. The aortae were immedi-
ately excised and freed of extraneous tissues. The endothelial
layer was removed by gently rubbing the intimal surface of
the vessels with a hypodermic needle. Five millimetres wide
aortic rings were suspended, under a preload of 2 g, in 20 ml
organ baths, containing Tyrode solution (composition of sa-
line in mM: NaCl 136.8; KCl 2.95; CaCl2 1.80;
MgSO4·7H2O 1.05; NaH2PO4 0.41; NaHCO3 11.9; Glucose
5.5), thermostated at 37 °C and continuously gassed with a
mixture of O2 (95%) and CO2 (5%). Changes in tension were
recorded by means of an isometric transducer (Grass FTO3),
connected with an unirecord microdynamometer (Buxco
Electronics). After an equilibration period of 60 min, the
endothelial removal was confirmed by the administration of
acetylcholine (ACh) (10 µM) to KCl (20 mM)-pre-
contracted vascular rings. A relaxation <10% of the KCl-
induced contraction was considered representative of an ac-
ceptable lack of the endothelial layer, while the organs,
showing a relaxation ≥10% (i.e. significant presence of the
endothelium), were discarded. From 30 to 40 min after the
confirmation of the endothelium removal, the aortic prepara-
tions were contracted by treatment with a single concentra-
tion of KCl (20 mM) and when the contraction reached a
stable plateau, threefold increasing concentrations of the
tested compounds or of the reference drug NS 1619 (a well-
known BK-activator) were added cumulatively. Preliminary
experiments showed that the KCl (20 mM)-induced contrac-
tions remained in a stable tonic state for at least 40 min. In
other sets of experiments, the non-selective potassium chan-
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