Synthesis and toxicopharmacological evaluation of m-Hydroxymexiletine, the first metabolite of mexiletine more potent than the parent compound on voltage-gated sodium channels

Article abstract of DOI:10.1021/jm201197z

The first synthesis of m-hydroxymexiletine (MHM) has been accomplished. MHM displayed hNav1.5 sodium channel blocking activity, and tests indicate it to be ～2-fold more potent than the parent mexiletine and to have more favorable toxicological properties than mexiletine. Thus, MHM and possible related prodrugs might be studied as agents for the treatment of arrhythmias, neuropathic pain, and myotonias in substitution of mexiletine (metabolite switch), which has turned out to be tainted with common toxicity.

Full text of DOI:10.1021/jm201197z

Brief Article
pubs.acs.org/jmc
Synthesis and Toxicopharmacological Evaluation of
m-Hydroxymexiletine, the First Metabolite of Mexiletine More Potent
Than the Parent Compound on Voltage-Gated Sodium Channels
Alessia Catalano,† Jean-Francois Desaphy,‡ Giovanni Lentini,*,† Alessia Carocci,† Antonia Di Mola,†
̧
Claudio Bruno,† Roberta Carbonara,‡ Annalisa De Palma,§ Roberta Budriesi,∥ Carla Ghelardini,⊥
Maria Grazia Perrone,† Nicola Antonio Colabufo,† Diana Conte Camerino,‡,# and Carlo Franchini†,#
‡
§
†Dipartimento Farmaco-Chimico, Sezione di Farmacologia, Dipartimento Farmaco-Biologico, and Sezione di Biochimica,
Dipartimento Farmaco-Biologico, Universita degli Studi di Bari “Aldo Moro”, Via E. Orabona, 4, 70126 Bari, Italy
di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
̀
∥Dipartimento di Scienze Farmaceutiche, Universita
⊥Dipartimento di Farmacologia Preclinica e Clinica, Universita
̀
di Firenze, Viale Pieraccini 6, 50139 Firenze, Italy
̀
S
* Supporting Information
ABSTRACT: The first synthesis of m-hydroxymexiletine (MHM) has been accomplished. MHM displayed hNav1.5 sodium
channel blocking activity, and tests indicate it to be ∼2-fold more potent than the parent mexiletine and to have more favorable
toxicological properties than mexiletine. Thus, MHM and possible related prodrugs might be studied as agents for the treatment
of arrhythmias, neuropathic pain, and myotonias in substitution of mexiletine (metabolite switch), which has turned out to be
tainted with common toxicity.
INTRODUCTION
■
The past decade has witnessed a dramatic reduction in the
number of new drugs (new molecular entities) approved for
therapy.1 Correspondingly, research costs for a new drug2 have
compelled researchers to look for new strategies to cope with the
current, relative shortage of new lead compounds. A relatively
less explored approach is the so-called metabolite switch, i.e., the
selection of an active metabolite as the substitute for the parent
compound, provided that the former has more favorable
properties when compared with the latter.3 In the past, this
strategy has scored only a few anecdotic successes, such as the
well-known history of paracetamol, an active metabolite of
phenacetine and safer analgesic than the retired parent com-
pound,4 or the replacement of terfenadine, a nonsedative H1
antagonist possibly cardiotoxic when coadministered with
several xenobiotics, by its active metabolite fexofenadine.3
Further examples of successful application of the metabolite
switch strategy have been recently reviewed.5 Mexiletine, 1-(2,6-
dimethylphenoxy)-2-propanamine (Figure 1), is a class IB
antiarrhythmic drug.6,7 Although it is primarily used in
treating ventricular arrhythmias, its recent uses include
chronic pain8−11 and myotonia.12−15 Mexiletine is adminis-
tered in doses of 150−200 mg two to three times a day and is
generally well tolerated. At high doses, mexiletine causes
drowsiness, confusion, nausea, hypotension, sinus bradycar-
dia, paresthesia, seizures, bundle branch block, atrioven-
tricular heart block, ventricular arrhythmias, asystole,
cardiovascular collapse, and coma.16 In Europe, the use
and commerce of mexiletine hydrochloride (Mexitil,
Boehringer Ingelheim) were discontinued in 2008 but it
has been reintroduced in some European countries owing to
specific national pharmaceutical productions. The treatment
Figure 1. Structures of mexiletine and its hydroxylated metabolites
(PHM, HMM, MHM).
of high-dose mexiletine intoxication, for example, in the case
of suicide attempt, includes decontamination and supportive
therapy and recently hemodialysis.16 Mexiletine is eliminated
slowly in humans with a half-life of 10−12 h, although the
drug undergoes extensive metabolism.17 Indeed, mexiletine
is metabolized in the liver by oxidation, deamination,
reduction, and conjugation.18,19 Unchanged mexiletine in
urine accounts for only 10% of the administered dose.20
Eleven metabolites of mexiletine, most of which are
eliminated as glucuronide conjugates, were identified, but
none of them were found to possess any pharmacological
activity.17,21,22 Indeed, we recently demonstrated that two
metabolites, hydroxymethylmexiletine (HMM, Figure 1) and
p-hydroxymexiletine (PHM), conserve only residual block-
ing activity on sodium currents recorded in skeletal muscle
fibers, when compared to mexiletine.23−25 In this paper we
Received: September 9, 2011
Published: December 21, 2011
© 2011 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Synthesis of m-Hydroxymexiletine MHM
a
Reagents and conditions: (i) phthalic anhydride, Et3N, toluene, reflux; (ii) 4-chloro-2,6-dimethylphenol, PPh3, DIAD, anhyd THF, room temp.;
(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.
report the synthesis and biological activity of a minor metabolite of
mexiletine, m-hydroxymexiletine (MHM), which in humans
accounts for approximately 2% of an administered oral dose of
mexiletine. Since mexiletine is clinically used as a racemic mixture,
MHM and the parent compound were studied in this form.
Table 1. Concentrations for Half-Maximal Tonic (0.1 Hz)
and Use-Dependent (10 Hz) Block of Sodium Currents
(IC50) and Slope Factor (nH) of Mexiletine and MHM on
Heterologously Expressed hNav1.5 Channels
0.1 Hz
IC50 (μM)
420 70
174
10 Hz
IC50 (μM)
58 13
30
compd
nH
nH
RESULTS AND DISCUSSION
■
mexiletine
MHM
1.0 0.2
1.0 0.3
1.0 0.2
0.8 0.1
Chemistry. The synthetic sequence to MHM (Scheme 1) is
similar to the one previously reported for PHM.23 The amino
propanol 1 was protected with phthalic anhydride in quantitative
yield to give the phthalimido alcohol 2 which was reacted with 4-
chloro-2,6-dimethylphenol under Mitsunobu conditions.26,27
Friedel−Crafts acylation, run on alkyl aryl ether 3, gave the
acetyl derivative 4.28 Then 4 was dehalogenated to give 529
which underwent Bayer−Villiger oxidation28 to give the desired
acetoxy derivative 6. Hydrazinolysis on 6 allowed the removal
of the acetyl and phthaloyl groups, furnishing MHM.30 All
compounds were characterized by routine spectrometric and
6
3
1
spectroscopic analyses. The H NMR spectrum of MHM was
fully assigned and compared to that of the literature.31 The
correct assignment of aromatic protons was obtained on the
basis of NOESY experiments, partially disproving what was
reported in the literature.31
Biological Results. Drugs were tested in vitro on voltage-
gated sodium currents recorded in HEK293 cells transiently
transfected with the human cardiac sodium channel, hNav1.5,
using the whole-cell patch-clamp method. Sodium currents
were elicited by 25 ms long depolarizing test pulses at −30 mV
from the holding potential of −120 mV at two stimulation
frequencies, 0.1 Hz for determination of tonic block and 10 Hz
for use-dependent block determination. The IC50 values were
calculated by fitting the concentration/effect relationships with
a first-order binding function and are reported with the SE of
the fit (Table 1, Figure 2).32
Surprisingly, MHM was about 2-fold more potent than
mexiletine in tonic and phasic block. These positive results led us
to evaluate MHM activity on isolated cardiac tissue. Mexiletine
and MHM were tested for antiarrhythmic activity on guinea pig
isolated left atria driven at 1 Hz. MHM increased the threshold
of ac arrhythmia more than mexiletine did. The efficacy of MHM
was not significantly different from that of mexiletine (Table 2).
Figure 2. Effects of mexiletine and MHM on hNav1.5 channels:
representative sodium current traces elicited from a holding potential
of −120 mV to a test potential of −30 mV in the absence of drug
(CTRL) and in the presence of 100 μM drug at 0.1 or 10 Hz
frequency stimulation. Concentration−response relationships were
constructed at 0.1 and 10 Hz stimulation and fitted with binding
equation described in Supporting Information. Each point is the mean
SEM from at least three cells.
To better define the cardiovascular profile of mexiletine
metabolite, its inotropic effect was measured in guinea pig left
atrium stimulated at 1 Hz, its chronotropism was evaluated in
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Journal of Medicinal Chemistry
Brief Article
MHM may display a more favorable pharmacological/
toxicological profile than mexiletine. In addition we verified if
these ligands may display different ability to cross the blood−
brain barrier (BBB) by defining their P-glycoprotein (P-gp)
interacting activity. For this purpose, the apparent permeability
(Papp) of both compounds across epithelial colorectal
adenocarcinoma (Caco-2) cell monolayer overexpressing
human P-gp was evaluated in the basolateral−apical (BA)
and apical−basolateral (AB) fluxes. The Caco-2 cells over-
expressing P-gp grown on permeable filters are commonly
employed to characterize P-gp substrates. In this assay, ligands
that behave as P-gp substrates are effluxed and therefore unable
to cross the BBB. Generally, ligands displaying BA/AB > 2 are
defined as P-gp substrates, whereas nonsubstrates or P-gp
inhibitors display BA/AB < 2.34 The results shown in Table 5
suggest that mexiletine and MHM may not be substrates for
P-gp, since the BA/AB values were approximately 2.
Table 2. Antiarrhythmic Activity of Compounds
a
b
max % increase
EC50
95% confidence
compd
(mean SEM)
(μM)
limit (×10−6)
c
amiodarone
lidocaine
10 0.5
34 2.6
11 0.4
69 0.4
procainamide
quinidine
mexiletine
MHM
10.26
11.61
10.18
8.44−12.46
8.71−13.47
8.19−12.67
c
64 1.4
c
72 2.7
a
Max % increase of threshold of ac arrhythmia after pretreatment with
compounds. Increase of threshold of ac arrhythmia is the increase in
the intensity of 50 Hz alternating current required to produce arrhythmia
in guinea pig left atria driven at 1 Hz in the presence of each tested
b
compounds at 5 × 10−5 M. For all data P < 0.05. Calculated from log
concentration−response curves (Probit analysis according to Litchfield
and Wilcoxon33 with n = 6−8). When the maximum effect was <50%,
c
the EC50 values were not calculated. At 10−4 M.
spontaneously beating right atrium, and its vasorelaxant activity
was assessed in K+-depolarized (80 mM) guinea pig aortic
strips. Data are collected in Table 3 with those of mexiletine
and other well-known antiarrhythmic drugs.
CONCLUSION
■
In this paper, we report the first synthesis and complete
characterization of MHM, a minor metabolite of mexiletine,
and its biological evaluation. The synthetic route was designed
on the basis of that of PHM.23 It was easily performed and
brought to the final compound in moderate overall yield. MHM
antiarrhythmic activity was evaluated and compared to that of
mexiletine. Interestingly, in vitro assays revealed that MHM
does possess about twice the blocking activity of mexiletine on
cardiac sodium channels, which may account for its increases of
antiarrhythmic and vasorelaxant activities on isolated tissues. In
addition, MHM at the highest tested doses, did not impair
motor coordination, in contrast to mexiletine, and showed
no cytotoxicity. Our preliminary in vitro studies on a BBB
permeation model indicate that none of the two compounds
are effluxed by transporters present in Caco-2 cells over-
expressing P-gp. This suggests that differences in CNS toxicity
may not be a result of selective BBB permeability. Nevertheless,
keeping in mind the complexity of the BBB that expresses a
large number of transporters, we cannot definitely exclude a
role for the BBB in the observed different toxicological profiles.
Since new drugs for the treatment of arrhythmias are needed,
MHM may be viewed as a promising starting point toward
clinical candidates for the treatment of these diseases. The
metabolite switch3 from mexiletine to MHM might be possible,
MHM exhibited a similar negative inotropism, an increased
vasorelaxant activity, and a very limited negative chronotrop-
ism. Since mexiletine clinical use is often associated with CNS
toxicity, a preliminary toxicological evaluation of MHM was
performed by assessing the motor coordination of treated
animals using the rotarod test. MHM, even injected at a dose of
50 mg/kg, did not increase the number of falls in comparison
with control animals treated with saline. By contrast, mexiletine
at the same dosage caused an increase of falls from rotating rod
in comparison with saline treated animals (Table 4).
Since mexiletine undergoes extensive, first-pass oxidative
metabolism and since oxidative metabolism may in turn cause
cellular oxidative stress and/or generate reactive metabolites,
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay on human hepatocellular liver carcinoma
cells (HepG2) was performed. Mexiletine and MHM displayed
low toxicity, presenting EC50 of 2.0
0.1 and >2 mM,
respectively (Figure S1, Supporting Information). Considering
that mexiletine plasma concentrations range between 4 and
11 μM in clinical use,20 both compounds are safe drugs as far as
cytotoxicity is concerned. Altogether these results suggest that
Table 3. Influence of Tested Compounds on Cardiovascular Parameters
EC50 of inotropic negative
EC30 of chronotropic
negative activity
% decrease (mean SEM)
negative inotropic negative chronotropic
activity
c
c
dm
,
EC50
95% conf lim
EC30
95% conf lim
vasorelaxant activity
(mean SEM)
am bm
, ,
compd
activity
activity
(μM)
(×10−6)
(μM)
(×10−6)
e
amiodarone
lidocaine
procaine
quinidine
mexiletine
MHM
30 2.6*
88 3.0*
92 1.4*
71 3.6*
90 1.3*
96 1.9*
72 4.5*
5.57
4.93−6.02
3
0.1
14 0.9*
0.2
30 1.6*
0.3
19 0.7*
f
jk
29 0.9*, ,
0.017
0.014
3.38
0.012−0.024
0.011−0.017
2.69−4.25
0.035−0.058
0.066−0.14
g
k
9
0.6
3
h
l
86 0.5*
85 2.6*
39 2.2*
3.99
3.81−4.06
0.009−0.023
i
0.045
0.098
0.014
5
a
Activity: decrease in developed tension on isolated guinea pig left atrium at 5 × 10−5 M, expressed as percentage change from the control (n = 4−6).
b
The left atria were driven at 1 Hz. Activity: decrease in atrial rate on guinea pig spontaneously beating isolated right atria at 10−4 M, expressed as
c
the percentage change from the control (n = 6−8). The pretreatment heart rate ranged from 170 to 195 beats/min. Calculated from log
concentration−response curve (Probit analysis according to Litchfield and Wilcoxon33 with n = 6−8). When the maximum effect was <50%,
d
the EC50 inotropic, EC30 chronotropic, and IC50 vasorelaxant values were not calculated. Activity: percent inhibition of calcium-induced
e
contraction on K+-depolarized guinea pig aortic strip at 10−4 M. The 10−4 M concentration gave the maximum effect for most compounds. At
f
g
h
i
j
k
l
m
10−4 M. At 10−6 M. At 5 × 10−7 M. At 5 × 10−5 M. At 10−5 M. At 5 × 10−6 M. Positive chronotropic effect. At 10−7 M. An asterisk indicates
P < 0.05.
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Journal of Medicinal Chemistry
Brief Article
a
Table 4. Effect of Mexiletine and MHM on Motor Coordination Expressed as Number of Falls from Rotarod
falls from the rota-rod
after treatment
treatment
dose, mg kg−1po
before treatment
15 min
30 min
45 min
saline
2.5 0.4
2.8 0.5
2.4 0.3
3.1 0.5
3.0 0.5
1.9 0.3
2.1 0.4
4.6 0.2*
1.8 0.3
2.0 0.4
1.2 0.3
1.3 0.4
3.8 0.3*
1.0 0.2
1.2 0.3
0.9 0.2
0.8 0.3
3.5 0.2*
0.7 0.2
0.9 0.3
mexiletine
mexiletine
MHM
25
50
25
50
MHM
a
Each value represents the mean of six to eight mice. ∗, P < 0.01 in comparison with saline controls.
residue was filtered off. After evaporation of the filtrate, the residue
was taken up with EtOAc and extracted with 2 N HCl (3 × 10 mL).
Then the aqueous phase was brought to 9 < pH < 11 with 2 N NaOH
(20 mL) and 2 N Na2CO3 (20 mL) and extracted twice with EtOAc.
The combined organic layers were dried (Na2SO4) and concentrated
under vacuum. The final product was a reddish solid (44 mg, 64%)
which was recrystallized from Et2O: mp 109−110 °C; IR (KBr) 3346,
Table 5. Apparent Permeability of Mexiletine and MHM
across Caco-2 Cell Monolayers Overexpressing Human P-gp
from the Basolateral to Apical (BA) Sides and from the
Apical to Basolateral (AB) Compartments
a
Papp
BA
,
nm/s
AB
a
compd
P
app(BA)/Papp(AB)
λ (nm)
ε
1
3279 (NH2, OH). H NMR (CD3OD, δ 3.30) chemical shifts were
mexiletine
MHM
2818
3006
1214
1425
2.3
2.1
210
220
9530
7670
attributed on the basis of NOESY experiment: δ 1.18 (d, J = 6.3 Hz,
3H, CH3CH), 2.10 (s, 3H, ArO CH3C-2), 2.15 (s, 3H, ArO CH3C-6),
3.23−3.34 (m, 1H, CH), 3.46−3.65 (m, 2H, CH2), 6.45 (d, J = 8.1 Hz,
1H, Ar HC-4), 6.77 (d, J = 8.1 Hz, 1H, Ar HC-5); 13C NMR
(CD3OD, δ 47.8) δ 8.32 (1C), 14.9 (1C), 18.0 (1C), 47.0 (1C), 77.3
(1C), 110.4 (1C), 117.5 (1C), 121.1 (1C), 127.7 (1C), 154.5 (1C),
156.1 (1C); MS (70 eV) m/z (%) 195 (M+, 6), 58 (99), 44 (100).
Anal. (C11H17NO2·0.50H2O) C, H, N.
a
Data are the mean of three independent determinations (samples in
triplicate) each with SEM < 10%.
assuming that MHM presents a more favorable profile than
mexiletine. Indeed, the use of prodrugs of mexiletine and its
active metabolites has been recently suggested for the treatment
of neuropathic pain and arrhythmias.35 Finally, to verify possible
distinct biological activities for MHM enantiomers paralleling the
light stereoselectivity observed in mexiletine pharmacodynamics
and pharmacokinetics,36 the preparation of MHM enantiomers
has been undertaken in view of a possible switch from the use of
(RS)-MHM to that of one of the enantiomers (chiral switch).3
Additional Information. For details on patch clamp, functional,
motor coordination, cytotoxicity, and permeability experiments, see
Supporting Information sections S5, S6, S8, S9, and S10, respectively.
ASSOCIATED CONTENT
* Supporting Information
■
S
Elemental analysis results, procedures for the synthesis of
intermediates, procedure for biological studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
EXPERIMENTAL SECTION
■
Materials and Methods. Chemicals were purchased from Sigma-
Aldrich or Lancaster. Yields refer to purified products and were not
optimized. The structures of the compounds were confirmed by routine
spectrometric and spectroscopic analyses. Compounds 2, 3, and mexiletine
hydrochloride were prepared as previously described.26 For the synthesis
and characterization of 4, 5, and 6, see Supporting Information. Only
spectra for compounds not previously described are given. Melting
points were determined on a Gallenkamp apparatus in open glass
capillary tubes and are uncorrected. Infrared spectra were recorded on a
Perkin-Elmer (Norwalk, CT) Spectrum One FT spectrophotometer, and
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 080 5442744. Fax: +39 0805442724. E-mail:
glentini@farmchim.uniba.it.
■
Author Contributions
#These authors contributed equally as senior authors.
ACKNOWLEDGMENTS
This work was accomplished with financial support of the Italian
Ministero dell’Istruzione, dell’Universita
■
band positions are given in reciprocal centimeters (cm−1). 1H and 13
C
NMR spectra were recorded on a Varian VX Mercury spectrometer
̀
e della Ricerca (Grant
1
operating at 300 and 75 MHz for H and 13C, respectively, using
MIUR 2005033023-001 to C.F.), and the French “Association
Francaise contre les Myopathies” (Grant 15020 to D.C.C.).
CDCl3 as solvent unless otherwise indicated. Chemical shifts are reported
in parts per million (ppm) relative to the residual nondeuterated
solvent resonance: CDCl3, δ 7.26 (1H NMR) and δ 77.3 (13C NMR). J
values are given in Hz. EI mass spectra were recorded on a Hewlett-
Packard 6890-5973 MSD gas chromatograph/mass spectrometer at
low resolution. Elemental analyses (C, H, N) were used to confirm the
purity of all new compounds (>95%) and were performed on a
Eurovector Euro EA 3000 analyzer (results within 0.4% of the
theoretical values; see Supporting Information). Chromatographic
separations were performed on silica gel columns by flash
chromatography (Kieselgel 60, 0.040−0.063 mm, Merck, Darmstadt,
Germany) as described by Still et al.37 TLC analyses were performed
on precoated silica gel on aluminum sheets (Kieselgel 60 F254, Merck).
Synthesis of (RS)-3-(2-Aminopropoxy)-2,4-dimethylphenol
(MHM). To a stirred solution of (RS)-6 (0.13 g, 0.35 mmol) in MeOH
(5 mL), glacial AcOH (2.1 mmol) and aqueous hydrazine (2.1 mmol)
were added. The mixture was kept under reflux for 5 h. The solid
̧
ABBREVIATIONS USED
■
AB, apical−basolateral; BA, basolateral−apical; BBB, blood−
brain barrier; HMM, hydroxymethylmexiletine; MHM,
m-hydroxymexiletine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; Papp, apparent permeability; P-gp,
P-glycoprotein; PHM, p-hydroxymexiletine
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