Carboxylic acids and skeletal muscle chloride channel conductance: Effects on the biological activity induced by the introduction of methyl groups on the aromatic ring of chiral α-(4-chloro-phenoxy)alkanoic acids

Article abstract of DOI:10.1016/S0014-827X(01)01041-2

One or two methyl groups have been introduced on the aromatic ring of two chiral clofibric acid analogs, 2-(4-chloro-phenoxy)propanoic and 2-(4-chloro-phenoxy)butanoic acids. The biological activity of the derivatives obtained (3-6) has been evaluated on the skeletal muscle chloride conductance (gCl). The results confirm the hypothesis of two different sites modulating chloride channel function, an excitatory site that increases channel activity and an inhibitory site that produces a channel block. In fact, this chemical modification strongly reduces the blocking activity of the (R)-and (S)-enantiomers in comparison with the parent compounds, but does not markedly affect the ability of the (R)-enantiomers to increase chloride channel conductance.

Full text of DOI:10.1016/S0014-827X(01)01041-2

www.elsevier.nl/locate/farmac
Il Farmaco 56 (2001) 239–246
Short Communication
Carboxylic acids and skeletal muscle chloride channel conductance:
effects on the biological activity induced by the introduction of
methyl groups on the aromatic ring of chiral
a-(4-chloro-phenoxy)alkanoic acids
Savina Ferorelli a, Fulvio Loiodice a, Vincenzo Tortorella a,*, Diana Conte-Camerino b,
Anna Maria De Luca b
a Dipartimento Farmaco-Chimico, Uni6ersita` di Bari, 6ia Orabona 4, 70126 Bari, Italy
b Dipartimento Farmaco-Biologico, Uni6ersita` di Bari, 6ia Orabona 4, 70126 Bari, Italy
Received 4 October 2000; accepted 15 November 2000
Abstract
One or two methyl groups have been introduced on the aromatic ring of two chiral clofibric acid analogs, 2-(4-chloro-phen-
oxy)propanoic and 2-(4-chloro-phenoxy)butanoic acids. The biological activity of the derivatives obtained (3–6) has been
evaluated on the skeletal muscle chloride conductance (gCl). The results confirm the hypothesis of two different sites modulating
chloride channel function, an excitatory site that increases channel activity and an inhibitory site that produces a channel block.
In fact, this chemical modification strongly reduces the blocking activity of the (R)-and (S)-enantiomers in comparison with the
parent compounds, but does not markedly affect the ability of the (R)-enantiomers to increase chloride channel conductance.
© 2001 Elsevier Science S.A. All rights reserved.
Keywords: Enantioselective synthesis; Absolute configuration; Chiroptical properties; Chloride channel conductance; Myotonic-inducing effects
1. Introduction
particular, the two enantiomers of 2-(4-chloro-phen-
oxy)propanoic (1) and butanoic (2) acids (Fig. 1), show
different and in some cases opposite biological re-
sponses. In fact, the (R)-isomers show the same antilipi-
demic effect with respect to the (S)-isomers and
clofibric acid, but they have higher antiaggregatory
activity [8] and lower myotonic [9] and hepatocarcino-
genicity inducing effects [10–12]. Also, the two optical
antipodes have been demonstrated to have opposite
activity on chloride channel conductance, a parameter
which is directly related to the myotonic effects on
skeletal muscle: in fact the (S)-isomers reduce the chlo-
ride conductance whereas the (R)-enantiomers increase
it [13]. In order to obtain more information on the
receptor site of the skeletal muscle chloride channel and
improve the pharmacological profile of these lead com-
pounds, we prepared both racemic and enantiomeric
forms of the analogs 3–6 (Fig. 1) in which one or two
Clofibric
acid,
[2-(4-chloro-phenoxy)-2-methyl-
propanoic acid] represents the active metabolite of clofi-
brate, an antilipidemic drug which is clinically used for
the management of hyperlipidemia. As a result of the
adverse effects on skeletal muscle (myalgias and myoto-
nias) [1–5] and on liver (carcinogenicity) [6,7], therapy
with clofibrate, however, should be limited to substan-
tial hyperlipidemia with high risk of associated morbid-
ity and mortality.
In order to improve the pharmacological activity of
clofibrate, in the past, several chiral a-substituted-a-
aryloxyacetic acid analogs of clofibric acid have been
synthesized and resolved into their optical antipodes. In
* Corresponding author.
E-mail address: vtor@farmchim.uniba.it (V. Tortorella).
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01041-2

240
S. Ferorelli et al. / Il Farmaco 56 (2001) 239–246
tained compounds 4–6; so they were prepared by con-
densation of phenol or with ethyl
7
8
2-bromopropanoate (9a) or ethyl 2-bromobutanoate
(9b) to give the corresponding esters 10–13 which were
hydrolyzed by methanolic KOH (Scheme 1).
The optical resolution of racemic acids has been
achieved by crystallization of the diastereomeric salts
with optically active amines. Compound 3 has been
resolved by a little modification (see Section 4) of the
procedure reported in literature [14]. In addition we
prepared the optical isomers by enantioselective synthe-
sis which allows a ready assignment of the absolute
configuration. For this purpose, the same procedure
reported in literature for the synthesis of the enan-
tiomers of some O-arylated lactic acid derivatives has
been performed [15]. So we started from (S)-ethyl
lactate (14a) or (S)-methyl 2-hydroxy-butanoate (14b)
which were condensed with phenol 7 or 8 in Mitsunobu
conditions [16]. This reaction occurs with inversion of
configuration giving the corresponding esters (R)-15–
18 which were hydrolyzed to acids (R)-3–6, respectively
(Scheme 2).
Fig. 1.
methyl groups have been introduced on the aromatic
ring of 2-(4-chloro-phenoxy)propanoic (1) and butanoic
(2) acids. The activity on the chloride channel conduc-
tance of the (R)- and (S)-enantiomers is reported
herein.
In the same way the (S) optical isomers could be
obtained starting from (R)-methyl lactate or (R)-methyl
2-hydroxy-butanoate.
The chiroptical properties of compounds 3–6 have
been determined as well. The ORD curves are reported
in Fig. 2.
The CD analysis (Fig. 3) of compounds (R)-3–6
shows a high dichroic band in the carboxylic group
absorption region (around 230 nm). (R)-3,4 present,
also, a strong positive Cotton effect in the aromatic
2. Chemistry
Racemic acid 3 has been prepared as previously
reported [14]. Following the same procedure we ob-
Scheme 1.
Scheme 2.

S. Ferorelli et al. / Il Farmaco 56 (2001) 239–246
241
Fig. 2. ORD curves of acids (R)-3–6. Concentration range: 1.0–2.0 g/100 ml, MeOH.
Fig. 3. CD curves of acids (R)-3–6. Concentration range: 0.1–0.4 mg/ml, MeOH.
absorption region, whereas (R)-5,6 show a low dichroic
band which is not distinguishable from the instrument
noise as a consequence, probably, of the reduced conju-
gation effect of the phenolic system due to the steric
hindrance of the two ortho methyl groups.
This compound is almost equally as effective as its
analog having an ethyl instead of a methyl group on
the chiral carbon atom, 2-(4-chloro-phenoxy)butanoic
acid (2) [13]. As detailed elsewhere [17], the effect
observed with the (S)-enantiomers of this class of com-
pounds might be due to the specific drug interaction
with an inhibitory site able to decrease chloride channel
activity. As shown in Fig. 4A, the introduction of an
ortho methyl group on the aromatic ring of 1 (com-
pound 3) led to a marked decrease of drug potency; in
fact the 50% decrease of gCl was observed at about 100
mM and no much greater block was observed at con-
centrations as high as 300 mM. The introduction of an
ortho methyl group on the aromatic ring of 2 (com-
pound 4) also led to a similar decrease in the potency,
3. Results and discussion
3.1. Effects of the (S)-enantiomers
In agreement with previous results [17], the (S)-enan-
tiomer of 2-(4-chloro-phenoxy)propanoic acid (1) pro-
duced the typical concentration-dependent decrease of
gCl with a half maximal concentration of about 15 mM.

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S. Ferorelli et al. / Il Farmaco 56 (2001) 239–246
or, more particularly, two bulky groups ortho to the
phenolic oxygen hampers the coplanarity and the con-
sequent conjugation effect between this atom and the p
electronic cloud of the aromatic ring. In this way, the
electronic density in this part of the molecule would
change, perhaps thereby reducing the strength of the
interaction with complementary functional groups of
the aminoacids constituting the drug binding site in the
channel protein.
although the concentration of 300 mM produced a 70%
block of gCl. The propanoic acid derivative having two
ortho methyl groups on the aromatic ring (compound
5) was very poorly effective, 300 mM producing only a
34% decrease of gCl, while the dimethyl derivative 6
was even less potent (Fig. 4A). Thus the introduction of
methyl groups on the aromatic moiety strongly de-
creases the blocking ability of the (S)-enantiomers of
these chiral clofibric acid analogs. This effect could be
explained by suggesting the involvement of either steric
or electronic factors. In the former case, the increased
steric hindrance of these molecules would reduce their
ability to interact properly with the inhibitory site. In
the latter, it can be supposed that the presence of one
3.2. Effects of the (R)-enantiomers
As already shown, (R)-1 produced the typical bipha-
sic effect on gCl, increasing it up to 25% at low
Fig. 4. Effect of (S)- and (R)-enantiomers (A and B, respectively) on resting chloride conductance (gCl) of rat extensor digitorum longus muscle
fibers. For each compound the effect is shown as percent change of conductance9S.E. produced by each concentration with respect to the related
control value recorded in the absence of drug. In section A, concentration is reported in logarithmic scale. 1: 2-(4-chloro-phenoxy)propanoic acid;
3: 2-(4-chloro-2-methyl-phenoxy)propanoic acid; 4: 2-(4-chloro-2-methyl-phenoxy)butanoic acid; 5: 2-(4-chloro-2,6-dimethyl-phenoxy)propanoic
acid; 6: 2-(4-chloro-2,6-dimethyl-phenoxy)butanoic acid.

S. Ferorelli et al. / Il Farmaco 56 (2001) 239–246
243
4.1. General procedure for the preparation of the
racemic esters 10–13
concentrations (1–5 mM) and producing a decrease of
gCl at higher concentration even though never more
than 30% (Fig. 4B). This peculiar behavior is related to
the ability of this compound to act as a double agonist
on both an excitatory site, that increases channel activ-
ity, and the inhibitory site described above [13,17]. The
(R)-enantiomers of the two derivatives bearing one
methyl group on the aromatic ring (compounds 3 and
4) produced a singular effect. In fact, both compounds
produced an increase of gCl (12–17%) up to concentra-
tions as high as 100 mM (Fig. 4B). A 16% decrease of
gCl was observed with (R)-3 at 700 mM (data not
shown). These results suggest that the introduction of
the ortho methyl group does not markedly affect the
ability of these enantiomers to interact with the excita-
tory site, while the action on the inhibitory site is
strongly reduced. On the contrary, the (R)-5 isomer,
bearing two ortho methyl groups, produced biphasic
effects similar to those observed with the parent com-
pound 1, while the dimethyl derivative 6 was almost
devoid of significant effects, the only effect observed
being a slight decrease of gCl at high concentrations
(Fig. 4B).
They were prepared in the usual way [14] by refluxing
equivalent amounts of ethyl 2-bromopropanoate or 2-
bromobutanoate and the appropriate sodium phenate
in abs. EtOH for 4–12 h. After cooling, the mixture
was filtered and evaporated to dryness. The oily residue
was taken up with CHCl3 and washed with 2N NaOH
and brine; the organic layer was dried over Na2SO4 and
the solvent removed under reduced pressure affording a
red oil which was hydrolyzed without any further
purification.
4.2. General procedure for the hydrolysis of the
racemic esters 10–13
A mixture of the ester (1 mmol) and of 10% KOH (2
mmol) methanol solution was kept at reflux for 2–5 h.
After cooling, the mixture was evaporated to dryness
and the residue taken up with H2O and washed with
CHCl3; the aqueous phase was acidified with 2 N HCl
and extracted with CHCl3; the organic layer was dried
over Na2SO4 and the solvent removed under reduced
pressure affording a solid which was crystallized from
hexane or pet. ether.
These results corroborate the hypothesis of two dif-
ferent sites modulating chloride channel function. In
fact, the introduction of one or two methyl groups on
the aromatic ring of both enantiomers of compounds 1
and 2, strongly reduces the blocking activity, but leaves
practically unchanged the ability of the (R)-enan-
tiomers to increase chloride channel conductance. The
lack of effect of both (R)-6 and (S)-6 suggests that the
concomitant presence of the two methyl groups on the
aromatic ring and of the ethyl group on the chiral
carbon atom represents a limiting factor for the interac-
tion with both binding sites as a consequence, proba-
bly, of the lower molecular flexibility.
4.2.1. (R,S)-2-(4-Chloro-2-methyl-phenoxy)propanoic
acid ((R,S)-3)
White crystals; m.p. 92–94°C (hexane). Total yield:
93%. IR (KBr); 1700 cm−1 (CꢀO). NMR (CDCl3): l
1.65 (d, 3H, CH3CH); 2.23 (s, 3H, ArCH3); 4.72 (q,
1H, CH); 6.59–7.13 (m, 3H, aromatic); 10.40 (broad,
1H, COOH). MS, m/z (rel. abund.): 214 (M+, 50), 142
(100). Anal. (C10H11ClO3) C,H.
4.2.2. (R,S)-2-(4-Chloro-2-methyl-phenoxy)butanoic
acid ((R,S)-4)
4. Experimental
White crystals; m.p. 87–89°C (pet. ether). Total
yield: 46%. IR (KBr); 1720 cm−1 (CꢀO). NMR
(CDCl3): l 1.05 (t, 3H, CH3CH2); 1.80–2.20 (m, 2H,
CH2); 2.25 (s, 3H, ArCH3); 4.50 (t, 1H, CH); 6.45–7.25
(m, 3H, aromatic); 8.60 (broad, 1H, COOH). MS, m/z
(rel. abund.): 228 (M+, 31), 142 (100). Anal.
(C11H13ClO3) C,H.
Melting points were taken on a Gallenkamp appara-
tus and are uncorrected. Mass and IR spectra were
determined with a Kratos MS 80 spectrometer and a
1
Perkin–Elmer 283 spectrophotometer, respectively. H-
NMR spectra were recorded on a Varian EM390 spec-
trometer using Me4Si as internal standard (chemical
shifts (l) are expressed in ppm). Optical rotations at
different wavelengths were measured in MeOH with a
Perkin–Elmer 241 MC polarimeter: Concentrations are
expressed as g · 100 ml−1. CD curves were measured in
MeOH with a Cary 61 dichrograph using a 10 mm cell.
Microanalyses were carried out with a HP Model 185
CHN analyzer (the analytical results are within 0.4% of
the theoretical values).
4.2.3. (R,S)-2-(4-Chloro-2,6-dimethyl-phenoxy)-
propanoic acid ((R,S)-5)
White crystals; m.p. 97–99°C (hexane). Total yield:
72%. IR (KBr); 1740 cm−1 (CꢀO). NMR (CDCl3): l
1.50 (d, 3H, CH3CH); 2.25 (s, 6H, 2ArCH3); 4.55 (q,
1H, CH); 6.95 (s, 2H, aromatic); 9.55 (broad, 1H,
COOH). MS, m/z (rel. abund.): 228 (M+, 36), 155
(100). Anal. (C11H13ClO3) C,H.

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S. Ferorelli et al. / Il Farmaco 56 (2001) 239–246
4.2.4. (R,S)-2-(4-Chloro-2,6-dimethyl-phenoxy)butanoic
acid ((R,S)-6)
lamine. After several crystallizations from ethyl acetate,
the salt, with [h]2D0: +14.6 (c 1.5, MeOH) was treated
as reported above affording (+)-5 which was crystal-
lized from hexane; m.p.: 97–99°C; [h]2D0: +34.0 (c 1.0,
MeOH).
White crystals; m.p. 64–65°C (hexane). Total yield:
23%. IR (KBr); 1740 cm−1 (CꢀO). NMR (CDCl3): l
1.05 (t, 3H, CH3CH2); 1.75–2.15 (m, 2H, CH2); 2.25 (s,
6H, 2ArCH3); 4.45 (t, 1H, CH); 7.00 (s, 2H, aromatic);
9.90 (broad, 1H, COOH). MS, m/z (rel. abund.): 242
(M+, 29), 156 (100). Anal. (C12H15ClO3) C,H.
4.2.8. Resolution of (R,S)-6
(R,S)-6 (21.2 g, 0.088 mol) and (1R,2R)-2-amino-1-
(4-nitro-phenyl)-1,3-propanediol (19.2 g, 0.088 mol)
were mixed and crystallized several times from ethyl
acetate to constant rotation. The salt, with [h]2D0: +4.5
(c 1.0, EtOH), was treated with 6 N HCl and extracted
with CHCl3 affording (+)-6 as a colorless oil which
solidified on cooling; m.p.: 49–50°C; [h]2D0: +25.7 (c
1.0, MeOH). The mother liquors from the first crystal-
lization of (1R,2R)-2-amino-1-(4-nitro-phenyl)-1,3-
propanediol salt were evaporated to dryness and the
salt hydrolyzed to recover the acid which was mixed
with an equimolar amount of (1S,2S)-2-amino-1-(4-ni-
tro-phenyl)-1,3-propanediol. After several crystalliza-
tions from ethyl acetate, the salt with [h]2D0: −4.7 (c 1.0,
EtOH) was treated as reported above affording (−)-6
as a colorless oil which solidified on cooling; m.p.:
49–50°C; [h]2D0: −25.4 (c 1.0, MeOH).
4.2.5. Resolution of (R,S)-3
(R,S)-3 (27.3 g, 0.13 mol) and (S)-1-phenylethy-
lamine (15.7 g, 0.13 mol) were mixed and crystallized
several times from ethyl acetate to constant rotation.
The salt, with [h]2D0: −10.0° (c 0.6, EtOH), was treated
with 6 N HCl and extracted with CHCl3 affording
(−)-3 as a white solid which was crystallized from
hexane; m.p.: 96–97°C; [h]2D0: −18.5 (c 0.75, MeOH).
The mother liquors from the first crystallization of the
(S)-1-phenylethylamine salt were evaporated to dryness
and the salt hydrolyzed to recover the acid which was
mixed with an equimolar amount of (R)-1-phenylethy-
lamine. After several crystallizations from ethyl acetate,
the salt, with [h]2D0: +10.0 (c 1.95, EtOH), was treated
as reported above affording (+)-3 which was crystal-
lized from hexane; m.p.: 95–96°C; [h]2D0: +20.6 (c 0.5,
MeOH).
4.3. General procedure for the preparation of the esters
(R)-15–18
4.2.6. Resolution of (R,S)-4
(R,S)-4 (10 g, 0.043 mol) and (R)-1-phenylethylamine
(5.3 g, 0.043 mol) were mixed and crystallized several
times from ethyl acetate to constant rotation. The salt,
with [h]2D0: +24.0 (c 0.4, EtOH), was treated with 6 N
HCl and extracted with CHCl3 affording (+)-4 as a
white solid which was crystallized from pet. ether; m.p.:
74–76°C; [h]2D0: +30.0 (c 0.4, MeOH). The acid recov-
ered from the mother liquors of the first crystallization
of the (R)-1-phenylethylamine salt was mixed with an
equimolar amount of (S)-1-phenylethylamine and crys-
tallized several times from ethyl acetate. When the salt
had [h]2D0: −24.0 (c 0.4, EtOH) it was treated as
reported above affording (−)-4 as a white solid which
was crystallized from pet. ether; m.p.: 74–76°C; [h]2D0:
−30.0 (c 0.4, MeOH).
A solution of diethyl azodicarboxylate (55 mmol) in
anhyd. THF (35 ml) was added dropwise to a mixture
of (S)-ethyl lactate or (S)-methyl 2-hydroxy-butanoate
[18] (50 mmol), the appropriate phenol (50 mmol) and
triphenylphosphine (50 mmol) in anhyd. THF (75 ml).
The reaction mixture was stirred at r.t., under a nitro-
gen atmosphere, for 12 h. The solvent was removed
under reduced pressure and a mixture of diethyl ether–
hexane (1:1) was added in order to precipitate the
triphenylphosphine oxide formed which was then
filtered off. This procedure was repeated several times.
Finally, the solvent was removed under reduced pres-
sure affording an oil which was chromatographed on
silica gel (E. Merck) using a mixture of pet.ether–ethyl
acetate 9:1 as eluent.
4.2.7. Resolution of (R,S)-5
4.3.1. (R)-Ethyl
(R,S)-5 (27.4 g, 0.12 mol) and (R)-1-phenylethy-
lamine (14.6 g, 0.12 mol) were mixed and crystallized
several times from ethyl acetate to constant rotation.
The salt, with [h]2D0: −14.7 (c 1.0, MeOH), was treated
with 6 N HCl and extracted with CHCl3 affording
(−)-5 as a white solid which was crystallized from
hexane; m.p.: 97–98°C; [h]2D0: −34.1 (c 1.0, MeOH).
The mother liquors from the first crystallization of the
(R)-1-phenylethylamine salt were evaporated to dryness
and the salt hydrolyzed to recover the acid which was
mixed with an equimolar amount of (S)-1-phenylethy-
2-(4-chloro-2-methyl-phenoxy)propanoate ((R)-15)
Colorless oil: yield 77%. [h]2D0: +23.2 (c 2.0, MeOH).
NMR (CDCl3): l 1.25 (t, 3H, CH3CH2); 1.60 (d, 3H,
CH3CH); 2.30 (s, 3H, ArCH3); 4.25 (q, 2H, CH2); 5.00
(q, 1H, CH); 6.50–7.20 (m, 3H, aromatic). MS, m/z
(rel. abund.): 242 (M+, 46), 169 (100).
4.3.2. (R)-Methyl
2-(4-chloro-2-methyl-phenoxy)butanoate ((R)-16)
Colorless oil: yield 24%. [h]2D0: +40.0 (c 1.5, MeOH).
NMR (CDCl3): l 1.20 (t, 3H, CH3CH2); 1.95 (m, 2H,

S. Ferorelli et al. / Il Farmaco 56 (2001) 239–246
245
CH2); 2.25 (s, 3H, ArCH3); 3.65 (s, 3H, OCH3); 4.50 (t,
4.4.2. (R)-2-(4-Chloro-2-methyl-phenoxy)butanoic acid
1H, CH); 6.50–7.20 (m, 3H, aromatic). MS, m/z (rel.
((R)-4)
abund.): 242 (M+, 35), 142 (100).
White crystals; m.p.: 74–76°C. Yield: 53%. [h]2D0:
+31.0 (c 1.0, MeOH).
4.3.3. (R)-Ethyl
4.4.3. (R)-2-(4-Chloro-2,6-dimethyl-phenoxy)propanoic
acid ((R)-5)
2-(4-chloro-2,6-dimethyl-phenoxy)propanoate ((R)-17)
Colorless oil: yield 55%. [h]2D0: +44.3 (c 1.6, MeOH).
NMR (CDCl3): l 1.55 (t, 3H, CH3CH2); 1.95 (d, 3H,
CH3CH); 2.25 (s, 6H, 2ArCH3); 2.90 (q, 2H, CH2);
4.60 (q, 1H, CH); 7.00 (s, 2H, aromatic). MS, m/z (rel.
abund.): 256 (M+, 46), 156 (100).
White crystals; m.p.: 97–99°C. Yield: 36%. [h]2D0:
+35.0 (c 1.0, MeOH).
4.4.4. (R)-2-(4-Chloro-2,6-dimethyl-phenoxy)butanoic
acid ((R)-6)
Colorless oil; yield: 60%. [h]2D0: +24.6 (c 1.4,
MeOH).
4.3.4. (R)-Methyl
2-(4-chloro-2,6-dimethyl-phenoxy)butanoate ((R)-18)
Colorless oil: yield 52%. [h]2D0: +44.8 (c 1.8, MeOH).
NMR (CDCl3): l 0.98 (t, 3H, CH3CH2); 1.77–2.08 (m,
2H, CH2); 2.23 (s, 6H, 2ArCH3); 3.70 (s, 3H, OCH3);
4.32 (t, 1H, CH); 6.95 (s, 2H, aromatic). MS, m/z (rel.
abund.): 265 (M+, 19), 156 (100).
In the same way, the (S)-enantiomers of esters 15–18
were obtained starting from (R)-methyl lactate or (R)-
methyl 2-hydroxy-butanoate [18].
Following the same procedure, the (S)-enantiomers
of acids 3–6 were obtained by hydrolysis of the esters
(S)-15–18.
4.4.5. (S)-2-(4-Chloro-2-methyl-phenoxy)propanoic
acid ((S)-3)
White crystals; m.p.: 98–99°C. Yield: 47%. [h]2D0:
−17.5 (c 0.5, MeOH).
4.4.6. (S)-2-(4-Chloro-2-methyl-phenoxy)butanoic acid
((S)-4)
4.3.5. (S)-Ethyl
White crystals; m.p.: 73–75°C. Yield: 60%. [h]2D0:
−30.0 (c 1.0, MeOH).
2-(4-chloro-2-methyl-phenoxy)propanoate ((S)-15)
Colorless oil: yield 58%. [h]2D0: −23.0 (c 2.0, MeOH).
4.4.7. (S)-2-(4-Chloro-2,6-dimethyl-phenoxy)propanoic
acid ((S)-5)
4.3.6. (S)-Methyl
2-(4-chloro-2-methyl-phenoxy)butanoate ((S)-16)
White crystals; m.p.: 97–99°C. Yield: 41%. [h]2D0:
−34.0 (c 1.0, MeOH).
Colorless oil: yield 53%. [h]2D0: −39.4 (c 1.5, MeOH).
4.3.7. (S)-Ethyl
4.4.8. (S)-2-(4-Chloro-2,6-dimethyl-phenoxy)butanoic
acid ((S)-6)
2-(4-chloro-2,6-dimethyl-phenoxy)propanoate ((S)-17)
Colorless oil: yield 44%. [h]2D0: −44.2 (c 1.6, MeOH).
Colorless oil which solidified on cooling; m.p.: 49–
50°C. Yield: 35%. [h]2D0: −25.4 (c 1.4, MeOH).
4.3.8. (S)-Methyl
2-(4-chloro-2,6-dimethyl-phenoxy)butanoate ((S)-18)
4.5. Pharmacology
Colorless oil: yield 40%. [h]2D0: −44.0 (c 1.8, MeOH).
4.5.1. Methods
4.4. General procedure for the hydrolysis of the esters
(R)-15–18
The newly synthesized compounds have been tested
in vitro on membrane ionic conductance of extensor
digitorum longus (EDL) muscle of adult male Wistar
rats of 350–400 g. The muscle was removed under
urethane anesthesia and placed in a temperature con-
trolled muscle chamber at 30°C and bathed with a
physiological solution in the absence and presence of
the test compounds. The normal physiological solution
had the following composition (in mM): 148 NaCl, 4.5
KCl, 2.0 CaCl2, 1.0 MgCl2, 0.44 NaH2PO4, 12
NaHCO3, 5.5 glucose. The physiological solution was
continuously bubbled with 95% O2 and 5% CO2 (pH
7.2). The total resting ionic conductance of sarcolemma
(gm) of muscle fibers was calculated from the cable
parameters and in particular from the membrane resis-
To a solution of the ester (1 mmol) in THF (3 ml), 3
ml of 1 N NaOH were added and the mixture stirred at
r.t. for 5 h. After removing THF under reduced pres-
sure, the remaining aqueous phase was acidified with 2
N HCl and extracted with CHCl3. The organic layer,
dried with Na2SO4 and concentrated in vacuo, gave a
white solid which was crystallized from pet. ether.
4.4.1. (R)-2-(4-Chloro-2-methyl-phenoxy)propanoic
acid ((R)-3)
White crystals; m.p.: 99–100°C. Yield: 45%. [h]2D0:
+17.5 (c 0.5, MeOH).

246
S. Ferorelli et al. / Il Farmaco 56 (2001) 239–246
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tance (Rm) values, measured by standard cable analysis
with the two intracellular microelectrode technique. In
brief, a voltage sensitive microelectrode (3 M KCl) was
used to measure the membrane potential and the
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observed modifications of conductance were considered
as change of gCl, assuming no effect of the newly
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stock solutions of each compound were prepared daily
and the final concentrations used were obtained with
appropriate dilution with normal physiological solu-
tion. On each preparation no more than three concen-
trations were tested and each concentration had been
incubated for at least 20 min before recordings to allow
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Acknowledgements
This work was accomplished thanks to the financial
support of MURST.
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