Searching for new antiarrhythmic agents: Evaluation of meta- hydroxymexiletine enantiomers

Article abstract of DOI:10.1016/j.ejmech.2013.05.008

Mexiletine is a very well-known class IB antiarrhythmic drug, whose enantiomers differ in both pharmacodynamic and pharmacokinetic properties, the (R)-isomer being the eutomer on experimental arrhythmias and in binding studies on cardiac voltage-gated sodium channels. meta-Hydroxymexiletine (MHM) is a minor metabolite of mexiletine, which has demonstrated to be more potent than the parent compound. Herein we report the synthesis and biological evaluation of MHM enantiomers for their potential antiarrhythmic activity. The same stereoselectivity pattern observed for mexiletine was found for MHM: the (R)-enantiomer of MHM was the eutomer on ac-arrhythmia also showing a negative inotropism higher than the one displayed by mexiletine and, at the same time, a decreased vasorelaxant activity on guinea-pig left atrium and guinea-pig ileum longitudinal smooth muscle.

Full text of DOI:10.1016/j.ejmech.2013.05.008

European Journal of Medicinal Chemistry 65 (2013) 511e516
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Searching for new antiarrhythmic agents: Evaluation
of meta-hydroxymexiletine enantiomers
Alessia Catalano a, Roberta Budriesi b, Claudio Bruno a, Antonia Di Mola a, Ivana Defrenza a,
,
a
Maria Maddalena Cavalluzzi a, Matteo Micucci b, Alessia Carocci a , Carlo Franchini ,
*
Giovanni Lentini a
a Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, Via E. Orabona n. 4, 70126 Bari, Italy
b Dipartimento di Farmacia e Biotecnologie, Università di Bologna, Via Belmeloro n. 6, 40126 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Mexiletine is a very well-known class IB antiarrhythmic drug, whose enantiomers differ in both phar-
macodynamic and pharmacokinetic properties, the (R)-isomer being the eutomer on experimental ar-
rhythmias and in binding studies on cardiac voltage-gated sodium channels. meta-Hydroxymexiletine
(MHM) is a minor metabolite of mexiletine, which has demonstrated to be more potent than the parent
compound. Herein we report the synthesis and biological evaluation of MHM enantiomers for their
potential antiarrhythmic activity. The same stereoselectivity pattern observed for mexiletine was found
for MHM: the (R)-enantiomer of MHM was the eutomer on ac-arrhythmia also showing a negative
inotropism higher than the one displayed by mexiletine and, at the same time, a decreased vasorelaxant
activity on guinea-pig left atrium and guinea-pig ileum longitudinal smooth muscle.
Received 14 March 2013
Received in revised form
2 May 2013
Accepted 10 May 2013
Available online 18 May 2013
Keywords:
Antiarrhythmic drugs
Sodium channel blockers
Mexiletine
Ó 2013 Elsevier Masson SAS. All rights reserved.
meta-Hydroxymexiletine
1. Introduction
study of mexiletine metabolites in their racemic and homochiral
form [17e21]. Our major interest was focused on meta-hydrox-
Several drugs are now marketed or being developed as single
enantiomers in place of a previous racemic mixture, a process
known as “chiral switching” [1]. The potential advantages of chiral
switching include an improved therapeutic index through
increased potency and selectivity and decreased side-effects.
Mexiletine, 1-(2,6-dimethylphenoxy)-2-propanamine (Fig. 1), is a
very well-known class IB antiarrhythmic drug [2,3]. Its primary use
has been the treatment of ventricular arrhythmias, but it can also be
used in chronic pain [4e7] and myotonia [8e10]. High doses of
mexiletine cause drowsiness, confusion, nausea, hypotension, sinus
bradycardia, paresthesia, seizures, bundle branch block, atrioven-
tricular heart block, ventricular arrhythmias, asystole, cardiovas-
cular collapse, and coma [11,12]. Mexiletine is eliminated slowly in
humans with a half-life of 10e12 h, although the drug undergoes
extensive metabolism [13]. Indeed, mexiletine is metabolized in the
liver by oxidation, deamination, reduction, and conjugation [14,15].
Unchanged mexiletine in urine accounts for only 10% of the
administered dose [16]. Since 2004, we have been involved in the
ymexiletine (MHM, Fig. 1), that is a minor metabolite of mexiletine,
which in humans accounts for approximately 2% of an administered
oral dose of mexiletine. We found that MHM is equipotent to
mexiletine on voltage-gated skeletal muscle sodium channels and
even 2-fold more potent than the parent compound on the cardiac
ones and it has more favorable toxicological properties than mex-
iletine since it did not impair motor coordination in contrast to
mexiletine and showed no cytotoxicity [22e24]. Therefore, we
proposed MHM for a metabolite switch. MHM and mexiletine are
chiral compounds. Differentiation between stereoisomers is a
fundamental phenomenon as chiral compounds interact in a ster-
eoselective way with each other, as recently reviewed by Leach
et al. [25]. Therefore, being our interest devoted to the study of
chiral molecules, we have developed stereospecific syntheses for
the preparation of chiral compounds in their homochiral forms
[26]. The two enantiomers of mexiletine show differences in both
pharmacodynamic effects and pharmacokinetic profiles. Both pre-
vention of ventricular tachycardia and tonic block of the skeletal
muscle channels effects exerted by R-mexiletine were higher than
those displayed by its enantiomer [27e29]. Thus, given the higher
activity of MHM, we hypothesized that similar differences could be
found for MHM enantiomers. Therefore, we reported a synthetic
* Corresponding author. Tel.: þ39 080 5442745; fax: þ39 080 5442724.
E-mail address: alessia.carocci@uniba.it (A. Carocci).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.008

512
A. Catalano et al. / European Journal of Medicinal Chemistry 65 (2013) 511e516
observed at a 100-fold lower concentration. To better define the
O
cardiovascular profile of mexiletine metabolite enantiomers, their
corresponding inotropic effects were measured in guinea-pig left
atrium stimulated at 1 Hz, their chronotropism was evaluated in
spontaneously beating right atrium, and their vasorelaxant activ-
ities were assessed in Kþ-depolarized (80 mM KCl) guinea-pig
aortic strips. Data are collected in Table 2 together with those of
mexiletine and racemic MHM previously reported [22]. (R)-MHM
performed as the eutomer, exhibiting a negative inotropism higher
than the one displayed by mexiletine, being twice as active as the
latter, while the negative inotropism was very low for (S)-MHM. All
the studied compounds displayed no activity on spontaneously
beating right atrium. Negative inotropism was lower than 40% for
all the tested compounds. Moreover, both the enantiomers of MHM
showed a decreased vasorelaxant activity. Therefore, in order to
verify a potential inhibitory effect on nonvascular smooth muscle,
such as that displayed by Ca2þ channel antagonists, further in-
vestigations on the relaxant activity on Kþ-depolarized (80 mM
KCl) guinea-pig ileum longitudinal smooth muscle (GPILSM) were
investigated (Table 3). When passing from aorta strips to nonvas-
cular smooth muscle, (R)-MHM was still inactive, whereas a slightly
increase in activity was registered for (S)-MHM.
NH2
O
NH2
OH
mexiletine
MHM
Fig. 1. Structures of mexiletine and MHM.
route to (R)- and (S)-MHM [30] and proposed a chiral switch from
MHM to one of its enantiomers, provided that the two enantiomers
differ in pharmacodynamics and/or pharmacokinetics. Herein, we
report the synthesis of MHM enantiomers and the evaluation of
their corresponding antiarrhythmic activities.
2. Results and discussion
2.1. Chemistry
The synthetic sequence to (R)- and (S)-MHM (Scheme 1) ex-
ploits the one previously reported for the racemate [22], by using
homochiral aminopropanols
1 as starting materials. All in-
termediates and final compounds were characterized by routine
spectrometric and spectroscopic analyses. Ee values of MHM en-
antiomers were evaluated by chiral 1H NMR analysis [31], using
(ꢀ)-(R)-2-phenyl-2-(quinolin-2-yloxy)acetic acid as a chiral sol-
vating agent, and were 98% for both enantiomers.
3. Conclusion
In this paper we report the synthesis of MHM enantiomers, a
minor metabolite of mexiletine, which is more potent than the
parent compound on cardiac voltage-dependent skeletal muscle
sodium channels [22]. Actually, it is known that mexiletine enan-
tiomers differ in both pharmacodynamic and pharmacokinetic
properties, the (R)-isomer being the eutomer on experimental ar-
rhythmias [32], in binding studies on cardiac sodium channels [27],
and in blocking skeletal muscle sodium channels [28,29]. MHM
enantiomers were tested for antiarrhythmic activity and the same
pattern of stereoselectivity of mexiletine was found: (R)-MHM was
the eutomer on ac-arrhythmia also showing a negative inotropism
higher than mexiletine and, at the same time, it showed a
decreased vasorelaxant activity on guinea-pig left atrium and on
GPILSM. Thus, the use of (R)-MHM in lieu of MHM should be
envisaged in view of further studies.
2.2. Biological results
MHM enantiomers were tested for antiarrhythmic activity on
guinea-pig isolated left atria driven at 1 Hz (Table 1). MHM showed
a stereoselective behavior in the increase of threshold of alternating
current (ac)-arrhythmia showing the same stereoselectivity pattern
of mexiletine. This was conceivable since the spatial disposition
around the chiral center of MHM is the same of mexiletine. (R)-
MHM was 20-fold more active than (RS)-MHM, while (S)-MHM
increased the threshold of ac-arrhythmia much less than (RS)-
MHM did. It is noteworthy that, despite the intrinsic activity of (R)-
MHM did not differ too much from that of (RS)-MHM, it was
O
O
O
O
O
OH
iii
OH
N
N
ii
i
N
H2N
Cl
O
Cl
O
O
O
(R)- and (S)-1
(R)- and (S)-2
(R)- and (S)-3
(R)- and (S)-4
iv
O
N
O
O
O
O
N
NH2
vi
v
O
O
O
OH
O
O
(R)- and (S)-MHM
(R)- and (S)-6
(R)- and (S)-5
Scheme 1. Reagents and conditions: (i) phthalic anhydride, Et3N, toluene, reflux; (ii) 4-chloro-2,6-dimethylphenol, PPh3, DIAD, anhyd THF, rt.; (iii) CH3COCl, AlCl3, anhyd CH2Cl2,
reflux; (iv) HCOONa, Pd(OAc), 2,2-(di-tert-butylphosphino)biphenyl, CH3OH, reflux; (v) MCPBA (77%), anhyd CH2Cl2, reflux; (vi) aq N2H4, AcOH, MeOH, reflux.

A. Catalano et al. / European Journal of Medicinal Chemistry 65 (2013) 511e516
513
Table 1
Table 3
Antiarrhythmic activity of compounds.
Relaxant activity of tested compounds on Kþ-depolarized guinea-pig ileum longi-
tudinal smooth muscle.
Max % increasea
(M ꢂ SEM)
EC50 (mM)
95% Conf.
b
Compd.
lim. (ꢃ10ꢀ6
)
Comp
Activitya (M ꢂ SEM)
IC50
(
m
M)
95% Conf lim. (ꢃ10ꢀ6
)
b
Mexiletinec
MHMc
(R)-MHM
(S)-MHM
64 ꢂ 1.4
72 ꢂ 2.7
66 ꢂ 1.4d
13 ꢂ 0.7
11.61
10.18
0.56
8.71e13.47
8.19e12.67
0.37e0.84
Mexiletine
MHM
(R)-MHM
(S)-MHM
81 ꢂ 1.9
51 ꢂ 1.6
47 ꢂ 1.3
75 ꢂ 3.1
8.52
25.83
6.54e11.09
19.45e28.43
13.58
9.71e19.01
a
a
Max % increase of threshold of arrhythmia after pretreatment with compounds.
Increase of threshold of ac-arrhythmia in the increase in the intensity of 50 Hz
alternating current required to produce arrhythmia on guinea-pig left atria driven at
1 Hz in the presence of each tested compounds at 10ꢀ4 M. For all data P < 0.05.
Percent inhibition of calcium-induced contraction on Kþ-depolarized (80 mM
KCl) guinea-pig longitudinal smooth muscle at 10ꢀ4 M. The 10ꢀ4 M concentration
gave the maximum effect for most compounds.
b
Calculated from log concentrationeresponse curves (Probit analysis according
b
Calculated from log concentrationeresponse curves (Probit analysis according
to Litchfield and Wilcoxon [40] with n ¼ 6e8). When the maximum effect was
to Litchfield and Wilcoxon [40] with n ¼ 6e8). When the maximum effect was
<50%, the IC50 values were not calculated.
<50%, the EC50 values were not calculated.
c
Ref. [22].
d
At 10ꢀ6 M.
HewlettePackard 6890-5973 MSD gas chromatograph/mass spec-
trometer at low resolution. Elemental analyses were performed
4. Experimental protocols
on
a Eurovector Euro EA 3000 analyzer. Optical rotations
were measured on a Perkin Elmer (Norwalk, CT) Mod 341 spec-
tropolarimeter; concentrations are expressed in g/100 mL, and
4.1. Chemistry
20
the cell length was 1 dm, thus [
a
]
values are given in units
D
of 10ꢀ1 deg cm2 gꢀ1. Chromatographic separations were performed
on silica gel columns by flash chromatography (Kieselgel 60,
0.040e0.063 mm, Merck, Darmstadt, Germany) using the tech-
nique as described elsewhere [34e37]. TLC analyses were per-
formed on precoated silica gel on aluminum sheets (Kieselgel 60
Chemicals were purchased from SigmaeAldrich or Lancaster.
(ꢀ)-(R)-2-phenyl-2-(quinolin-2-yloxy)acetic acid, used as chiral
solvating agent (CSA), was homemade [33]. Solvents were RP grade
unless otherwise indicated. Both enantiomers of 2 and 3 and
racemic MHM and mexiletine hydrochloride were prepared as
previously described [22]. Yields refer to purified products and
were not optimized. Compound structures were confirmed by
routine spectrometric and spectroscopic analyses. Only spectra for
compounds not previously described are given. Melting points
were determined on a Gallenkamp melting point apparatus in open
glass capillary tubes and are uncorrected. The infrared spectra were
recorded on a PerkineElmer (Norwalk, CT) Spectrum One FT
spectrophotometer and band positions are given in reciprocal
centimeters (cmꢀ1). 1H NMR and 13C NMR spectra were recorded on
a Varian Mercury-VX spectrometer operating at 300 and 75 MHz
for 1H and 13C, respectively using CDCl3 or CD3OD (where indicated)
as solvents. Chemical shifts are reported in parts per million (ppm)
F254, Merck).
4.1.1. (ꢀ)-(R)-2-[2-(3-acetyl-4-chloro-2,6-dimethylphenoxy)-1-
methylethyl]-1H-isoindole-1,3(2H)-dione [(ꢀ)-(R)-4]
To a suspension of aluminum chloride (3.34 g, 25.0 mmol) in dry
dichloromethane (15 mL) at room temperature, under nitrogen at-
mosphere, acetyl chloride (1.47 g, 18.7 mmol) and successively a
solution of (ꢀ)-(R)-3 (2.15 g, 6.25 mmol) in dry CH2Cl2 (15 mL) were
added dropwise. The reaction was brought to 50 ꢁC and taken at this
temperature for 5 h and then it was stirred at room temperature
overnight. The reaction was quenched by addition of ice and 2 N HCl.
Dichloromethane was then removed under vacuum and the residue
was taken up with ethyl ether. It was then washed three times with
water and dried over anhydrous Na2SO4. Purification of the residue
by flash chromatography (EtOAc/petroleum ether 1:9) gave a
slightly yellowish foam (1.54 g, 64%), which was crystallized from
relative to solvent resonance: CDCl3,
NMR); CD3OD,
d
7.26 (1H NMR) and
d C
77.3 (13
d
3.30 (1H NMR) and
d
47.8 (13C NMR). J values are
given in Hz. When determining ee values, free amines were dis-
solved with 2.0 equiv. of CSA in CDCl3. Spectra were registered at
25 ꢁC and the splitting of the
a-methyl groups doublet was
EtOAc/petroleum ether to give 1.30 g of (ꢀ)-(R)-4 as white crystals:
20
mp 100e101 ꢁC (EtOAc/petroleum ether); [
a]
¼ ꢀ43.1 (c 2,
observed (Dd ¼ 0.30 ppm). EIMS spectra were recorded on a
D
Table 2
Influence of tested compounds on cardiovascular parameters.
Compd.
% Decrease (M ꢂ SEM)
Negative inotropic activitya
EC50 of inotropic negative activity
Vasorelaxant activitye
(M ꢂ SEM)
Negative chronotropic activityb
EC50
(
m
M)
95% Conf lim. (ꢃ10ꢀ6
)
c
Mexiletined
MHMd
(R)-MHM
(S)-MHM
90 ꢂ 1.3f
96 ꢂ 1.9
89 ꢂ 2.4g
93 ꢂ 1.1
85 ꢂ 2.6h
39 ꢂ 2.2i
34 ꢂ 1.6
16 ꢂ 0.7
0.045
0.098
0.025
0.12
0.035e0.058
0.066e0.14
0.016e0.036
0.089e0.16
5 ꢂ 0.3
19 ꢂ 0.7
1 ꢂ 0.09l
4 ꢂ 0.2
a
Activity: decrease in developed tension on isolated guinea-pig left atrium at 5 ꢃ 10ꢀ5 M, expressed as percentage change from the control (n ¼ 4e6). For all data P < 0.05.
The left atria were driven at 1 Hz.
b
Activity: decrease in atrial rate in guinea-pig spontaneously beating isolated right atria at 10ꢀ5 M, expressed as the percentage change from the control (n ¼ 6e8). The
pretreatment heart rate ranged from 170 to 195 beats/min.
c
Calculated from log concentrationeresponse curve (Probit analysis according to Litchfield and Wilcoxon [40] with n ¼ 6e8).
d
Ref. [22].
e
Activity: percent inhibition of calcium-induced contraction on Kþ-depolarized guinea-pig aortic strip at 10ꢀ4 M. The 10ꢀ4 M concentration gave the maximum effect for
most compounds.
f
At 10ꢀ5 M.
g
At 10ꢀ6 M.
h
At 10ꢀ7 M.
i
At 10ꢀ4 M.
l
At 5 ꢃ 10ꢀ5 M.

514
A. Catalano et al. / European Journal of Medicinal Chemistry 65 (2013) 511e516
CHCl3); IR (KBr): 1772, 1757, 1708 (C]O); 1H NMR:
d
1.53 (d,
becomes pink, and then dried over anhydrous Na2SO4. After
J ¼ 7.2 Hz, 3H, CH3CH), 2.06 (s, 3H, ArO CH3C-6), 2.17 (s, 3H, ArO
CH3C-2), 2.46 (s, 3H, CH3CO), 3.84 (dd, J ¼ 9.4, 5.5 Hz, 1H, CHH), 4.35
(apparent t, J ¼ 9.4 Hz, 1H, CHH), 4.77e4.92 (m, 1H, CH), 6.98 (s, 1H,
ArO), 7.68e7.76 (m, 2H, Ar HC-5,6), 7.80e7.88 (m, 2H, Ar HC-4,7);
GCeMS (70 eV) m/z (%) 385 (Mþ, 3), 188 (100). 13C NMR spectrum
was in agreement with that reported for the racemate [22]. Anal.
Calcd for (C21H20ClNO4$0.25H2O): C, 64.62; H, 5.29; N, 3.59. Found:
C, 64.89; H, 5.27; N, 3.68.
evaporation of the solvent under vacuum, the residue was purified
by flash chromatography (EtOAc/petroleum ether 2:8) to give
20
(ꢀ)-(R)-6 as a colorless oil (95 mg, 62%): [
a
]
¼ ꢀ61.2 (c 2, CHCl3);
D
IR (neat): 1776, 1759, 1709 (C]O); 1H NMR:
d
1.54 (d, J ¼ 7.2 Hz, 3H,
CH3CH), 2.00 (s, 3H, ArO CH3C-2), 2.16 (s, 3H, ArO CH3C-6), 2.27 (s,
3H, CH3CO), 3.88 (dd, J ¼ 9.4, 5.5 Hz, 1H, CHH), 4.37 (apparent t,
J ¼ 9.3 Hz, 1H, CHH), 4.79e4.93 (m, 1H, CH), 6.67 (d, J ¼ 8.2 Hz, 1H,
ArO HC-4), 6.95 (d, J ¼ 8.2 Hz, 1H, ArO HC-5), 7.66e7.75 (m, 2H, Ar
HC-5,6), 7.79e7.89 (m, 2H, Ar HC-4,7); GCeMS (70 eV) m/z (%) 367
(Mþ, 6), 188 (100). 13C NMR spectra were in agreement with those
reported for the racemate [22].
4.1.2. (þ)-(S)-2-[2-(3-acetyl-4-chloro-2,6-dimethylphenoxy)-1-
methylethyl]-1H-isoindole-1,3(2H)-dione [(þ)-(S)-4]
Prepared via the above reaction starting from (þ)-(S)-3. Yield:
72% (66% after crystallization); mp 101e102 ꢁC (EtOAc/petroleum
4.1.6. (þ)-(S)-3-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
propoxy]-2,4-dimethylphenyl acetate [(þ)-(S)-6]
20
ether); [
a
]
¼ þ42.9 (c 2, CHCl3). Spectroscopic and spectrometric
D
data were in agreement with those found for the (R)-isomer and
the racemate [22]. Calcd for C21H20ClNO4$0.33H2O % C 64.37, H
5.32, N 3.57; found C 64.04, H 5.11, N 3.72.
Prepared via the above reaction starting from (þ)-(S)-5. Yield:
20
33%. Colorless oil: [
a
]
¼ þ61.0 (c 2, CHCl3). GCeMS, IR and 1H
D
NMR spectra were in agreement with the one reported for the (R)-
isomer; 13C NMR spectrum was in agreement with the one reported
for the racemate [22].
4.1.3. (ꢀ)-(R)-2-[2-(3-acetyl-2,6-dimethylphenoxy)-1-methylet-
hyl]-1H-isoindole-1,3(2H)-dione [(ꢀ)-(R)-5]
A stirring solution of Pd(OAc)2 (60 mg, 0.27 mmol) and (2-
biphenyl)di-tert-butylphosphine (0.12 g, 0.40 mmol) in meth-
anol (10 mL) was taken under nitrogen atmosphere. After 5 min,
(ꢀ)-(R)-4 (1.58 g, 4.09 mmol) and methanol (10 mL) were added.
Then, sodium formate (0.90 g, 13.2 mmol) was added to the
black solution. The reaction was taken under reflux for 4 h. The
catalyst was removed by filtration through celite, and the sol-
vent was evaporated in vacuo to give a crude residue that was
taken up with EtOAc, washed with brine (three times) and water
(three times) to give 1.50 g of a brown oil. Purification of the
residue by column chromatography (EtOAc/petroleum ether,
3:7) gave 0.63 g (44%) of (ꢀ)-(R)-5 as a colorless oil which was
crystallized from EtOAc/petroleum ether to give 0.53 g of white
4.1.7. (ꢀ)-(R)-3-(2-aminopropoxy)-2,4-dimethylphenol [(ꢀ)-(R)-
MHM]
Prepared in 92% yield as reported in the literature for the race-
mate [22] starting from (ꢀ)-(R)-6. Slightly yellowish solid: mp 98e
20
99 ꢁC; [
a
]
¼ ꢀ1.4 (c 1.1, CH3OH); 98% ee. Spectroscopic data were
D
in agreement with the literature [30]. Calcd for C11H17NO2$0.25H2O
% C 66.14, H 8.83, N 7.01; found C 66.48, H 8.57, N 7.01.
4.1.8. (þ)-(S)-3-(2-aminopropoxy)-2,4-dimethylphenol [(þ)-(S)-
MHM]
Prepared in 70% yield as reported in the literature [22] for
the racemate starting from (þ)-(S)-6. Slightly yellowish oil.
20
[
a
]
¼ þ1.6 (c 1.1, CH3OH); 98% ee; GCeMS (70 eV) m/z (%) 195
D
crystals:
mp
111e112
ꢁC
(EtOAc/petroleum
ether);
(Mþ, 9), 58 (100), 44 (98). Chemical purity of this compound was
assured by converting it into its hydrochloride salt by treating the
free amine with few drops of 2 N HCl and azeotropically removing
water (absolute EtOH/toluene). The solid obtained was recrystal-
lized from MeOH/Et2O/i-Pr2O to give slightly yellowish crystals:
20
[a]
¼ ꢀ66.1 (c 2.1, CHCl3); IR (KBr): 1774, 1709, 1676 (C]O);
D
1H NMR:
d
1.54 (d, J ¼ 7.2 Hz, 3H, CH3CH), 2.21 (s, 3H, ArO CH3C-
6), 2.32 (s, 3H, ArO CH3C-2), 2.50 (s, 3H, CH3CO), 3.87 (dd,
J ¼ 9.4, 5.5 Hz, 1H, CHH), 4.34 (apparent t, J ¼ 9.2 Hz, 1H, CHH),
4.80e4.94 (m, 1H, CH), 7.00 (d, J ¼ 8.0 Hz, 1H, ArO HC-5), 7.30 (d,
J ¼ 7.7 Hz, 1H, ArO HC-4), 7.68e7.75 (m, 2H, Ar HC-5,6), 7.80e
7.88 (m, 2H, Ar HC-4,7); GCeMS (70 eV) m/z (%) 351 (Mþ, 3), 188
(100). 13C NMR spectrum was in agreement with the one re-
ported for the racemate [22]. Calcd for C21H21NO4 % C 71.78, H
6.02, N 3.99; found C 71.89, H 6.10, N 4.21.
mp 199e200 ꢁC; 1H NMR (CD3OD):
d
1.43 (d, J ¼ 6.6 Hz, 3H,
CH3CH), 2.13 (s, 3H, ArO CH3C-2), 2.19 (s, 3H, ArO CH3C-6), 3.65e
3.90 (m, 3H, CH þ CH2), 6.50 (d, J ¼ 8.2 Hz, 1H, Ar HC-4), 6.82 (d,
J ¼ 8.0 Hz, 1H, Ar HC-5); 13C NMR (CD3OD):
d 8.2 (CH3), 14.2 (CH3),
14.7 (CH3), 47.8 (CH), 71.9 (CH2), 111.0 (CH), 117.4 (C), 120.9 (C),
127.9 (CH), 154.7 (C), 155.1 (C). Calcd for C11H17NO2$HCl$0.25H2O
% C 55.93, H 7.89, N 5.93, found C 56.16, H 7.90, N 5.77. For bio-
logical studies and the determination of ee value of this com-
pound, (S)-MHM as free amine was recovered by extraction of a
sample of the corresponding hydrochloride salt. Spectroscopic
data were in agreement with the literature [30].
4.1.4. (þ)-(S)-2-[2-(3-acetyl-2,6-dimethylphenoxy)-1-methylet-
hyl]-1H-isoindole-1,3(2H)-dione [(þ)-(S)-(5)
Prepared via the above reaction starting from (þ)-(S)-4. Yield:
66%. White crystals: mp 111e112 ꢁC (EtOAc/petroleum ether);
20
[
a]
¼ þ65.1 (c 2.1, CHCl3). GCeMS, IR and 1H NMR spectra were
D
in agreement with the one reported for the (R)-isomer; 13C NMR
spectrum was in agreement with the one reported for the racemate
[22]. Calcd for C21H21NO4$0.50H2O % C 69.98, H 6.15, N 3.89; found
C 70.03, H 5.99, N 3.98.
4.2. Pharmacology
4.2.1. Pharmacology. Guinea-pig atrial preparations
Female guinea pigs (300e400 g) were sacrificed by cervical
dislocation. After thoracotomy the heart was immediately
removed and washed by perfusion through the aorta with
oxygenated Tyrode solution of the following composition (mM):
136.9 NaCl, 5.4 KCl, 2.5 CaCl2, 1.0 MgCl2, 0.4 NaH2PO4 ꢃ H2O, 11.9
NaHCO3, and 5.5 glucose. The physiological salt solution (PSS) was
buffered at pH 7.4 by saturation with 95% O2/5% CO2 gas, and the
temperature was maintained at 35 ꢁC. Isolated guinea-pig heart
preparations were used, spontaneously beating right atria and left
atria driven at 1 Hz. For each preparation, the entire left and right
atria were dissected from the ventricles, cleaned of excess tissue,
4.1.5. (ꢀ)-(R)-3-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
propoxy]-2,4-dimethylphenyl acetate [(ꢀ)-(R)-6]
A solution of (ꢀ)-(R)-5 (0.15 g, 0.43 mmol) and 77% m-chlor-
operbenzoic acid (MCPBA; 0.20 g, 1.16 mmol) in dichloromethane
(4 mL) was vigorously refluxed for 6 h, during which time a solid
precipitated from the solution. Dichloromethane was removed by
evaporation under vacuum and the residue was dissolved in ethyl
acetate, washed with a saturated solution of sodium hydrogen
carbonate, water and 0.5 N NaOH until the aqueous NaOH solution

A. Catalano et al. / European Journal of Medicinal Chemistry 65 (2013) 511e516
515
and hung vertically in a 15 mL organ bath containing the PSS
continuously bubbled with 95% O2/5% CO2 gas at 35 ꢁC, pH 7.4. The
contractile activity was recorded isometrically by means of a force
transducer (FT 0.3, Grass Instruments Corp., Quincy, MA) using
Power Lab software (AD-Instruments Pty Ltd., Castle Hill,
Australia). The left atria were stimulated by rectangular pulses of
0.6e0.8 ms duration and about 50% threshold voltage through two
platinum contact electrodes in the lower holding clamp (Grass S88
stimulator). The right atrium was in spontaneous activity. After the
tissue was beating for several minutes, a lengthetension curve was
determined, and the muscle length was maintained, which elicited
90% of the maximum contractile force observed at the optimal
length. A stabilization period of 45e60 min was allowed before the
atria were used to test the compounds. During the equilibration
period, the bathing solution was changed every 15 min and the
threshold voltage was ascertained for the left atria. Atrial muscle
preparations were used to examine the inotropic and chronotropic
activities of the compounds (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, and
for monitoring changes in isometric contraction and washed
every 20 min with fresh PSS for 1 h. After the equilibration period,
guinea-pig aortic strips were contracted by washing in PSS con-
taining 80 mM KCl (equimolar substitution of Kþ for Naþ). When
the contraction reached a plateau (about 45 min) various con-
centrations of the compounds (0.01, 0.05, 0.1, 0.5, 1, 5, 10 50
and 100
mM) dissolved in PSS. All data are presented as
mean ꢂ S.E.M. The IC50 were calculated from log concentratione
response curves [40].
Acknowledgments
This work was accomplished thanks to the financial support of
the Ministero dell’Istruzione, dell’Università e della Ricerca (Grant
MIUR 2005033023-001 to C.F.).
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