Hydroxylated analogs of mexiletine as tools for structural-requirements investigation of the sodium channel blocking activity

Article abstract of DOI:10.1002/ardp.200900218

[2-(2-Aminopropoxy)-1,3-phenylene]dimethanol 1 and 4-(2-aminopropoxy)-3- (hydroxymethyl)-5-methylphenol 2, two dihydroxylated analogs of mexiletine - a well known class IB anti-arrhythmic drug - were synthesized and used as pharmacological tools to investigate the blockingactivity requirements of human skeletal muscle, voltage-gated sodium channel. The very low blocking activity shown by newly synthesized compounds corroborates the hypothesis that the presence of a phenolic group in the para-position to the aromatic moiety and/or benzylic hydroxyl groups on the aromatic moiety of local anesthetic-like drugs impairs either the transport to or the interaction with the binding site in the pore of Na⁺ channels.

Full text of DOI:10.1002/ardp.200900218

Arch. Pharm. Chem. Life Sci. 2010, 343, 325–332
A. Catalano et al.
325
Full Paper
Hydroxylated Analogs of Mexiletine as Tools for Structural-
Requirements Investigation of the Sodium Channel Blocking
Activity
Alessia Catalano1, Alessia Carocci1, Maria M. Cavalluzzi1, Antonia Di Mola1, Giovanni Lentini1,
Angelo Lovece1, Antonella Dipalma2, Teresa Costanza2, Jean-Franꢀois Desaphy2, Diana Conte
Camerino2, and Carlo Franchini1
1 Department of Medicinal Chemistry, University of Bari, Bari, Italy
2 Department of Pharmacology, University of Bari, Bari, Italy
[2-(2-Aminopropoxy)-1,3-phenylene]dimethanol 1 and 4-(2-aminopropoxy)-3-(hydroxymethyl)-5-
methylphenol 2, two dihydroxylated analogs of mexiletine – a well known class IB anti-arrhyth-
mic drug – were synthesized and used as pharmacological tools to investigate the blocking-
activity requirements of human skeletal muscle, voltage-gated sodium channel. The very low
blocking activity shown by newly synthesized compounds corroborates the hypothesis that the
presence of a phenolic group in the para-position to the aromatic moiety and/or benzylic
hydroxyl groups on the aromatic moiety of local anesthetic-like drugs impairs either the trans-
port to or the interaction with the binding site in the pore of Na+ channels.
Keywords: Hydroxylation / Ion channel / Mexiletine /
Received: September 9, 2009; Accepted: November 3, 2009
DOI 10.1002/ardp.200900218
muscle voltage-gated sodium channels. In contrast, the
b2-agonist salbutamol (Fig. 1) had no effect on the sodium
currents (INa). On the basis of these results it was hypothe-
sized that the presence of hydroxyl groups on the aro-
matic moiety may impede the hydrophobic interaction
between the aforementioned aromatic moiety and the
local anesthetic receptor [6]. In fact, it should be noted
that most of the b2-agonists present two hydroxyl groups
on their aromatic moieties, and that the atypical clenbu-
terol was the unique b2-agonist able to block sodium
channels [6]. Similar experiments with the chemically
similar b2-antagonists propranolol and nadolol (Fig. 1)
suggested the presence of two hydroxyl groups on the
aromatic moiety of the drugs as a molecular requisite for
impeding sodium channel block [6]. Thus, we suggest
that a similar modification on mexiletine (i. e., hydroxyla-
tion of the aromatic ring) might cause a reduction of the
sodium channel blocking activity. The hypotesis is sup-
ported by our previous experience that two of the main
monohydroxylated mexiletine metabolites, the so-called
p-hydroxymexiletine (PHM, Fig. 1) and hydroxymethyl-
mexiletine (HMM), are less active than mexiletine in
Introduction
Voltage-gated sodium channels [1] are the target of clini-
cally important drugs such as anti-arrhythmics [2], anti-
myotonics [3], anticonvulsants [4], and local anaesthetics
[5]. Mexiletine (Fig. 1) is an orally effective lidocaine ana-
log belonging to the IB anti-arrhythmic drug group. Its
clinical usefulness is due to its ability to block voltage-
gated sodium channels more potently in situations of
excessive burst of action potentials (use-dependent or
phasic block). This occurrs in diseased tissues, rather
than under conditions of physiological excitability (tonic
block). A few years ago [6], we reported that the b2-adren-
ergic agonist clenbuterol (Fig. 1) exhibited blocking
effects similar to those shown by mexiletine on skeletal
Correspondence: Catalano Alessia, Dr. Ph.D., Dipartimento Farmaco-
Chimico, Facoltꢀ di Farmacia, Universitꢀ di Bari, Via Orabona 4, 70126
Bari, Italy.
E-mail: a.catalano@farmchim.uniba.it
Fax: +39 080 544-2724
Abbreviations: hydroxymethylmexiletine (HMM); p-hydroxymexiletine
(PHM)
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Arch. Pharm. Chem. Life Sci. 2010, 343, 325–332
Figure 1. Structures of mexiletine, clenbuterol, salbutamol, and
hydroxylated analogs of mexiletine (PHM, HMM, 1 and 2).
reducing INa in both tonic and phasic block [7–9]. Herein,
we report the synthesis and complete characterization of
two new dihydroxylated analogs of mexiletine (1 and 2),
which present themselves as valuable pharmacological
tools for the study of sodium channel blocking activity.
Scheme 1. Synthesis of compound 1.
Results and discussion
Chemistry
isolated by column chromatography and fully character-
ized. The formation of the latter brought us to hypothe-
size the unknown side-product as 9 (Scheme 1).
Our strategy for the synthesis of compound 1 is shown in
Scheme 1. 2-[2-(2,6-Dimethylphenoxy)-1-methylethyl]-1H-
isoindole-1,3(2H)-dione 6 was prepared as previously
described [7, 9, 10], by protecting 2-amino-1-propanol 3
with phthalic anhydride 4 [11], and then by reacting the
phthalimido alcohol 5 with 2,6-dimethylphenol, under
Mitsunobu conditions [12]. Then, compound 6 was
reacted with N-bromosuccinimide and benzoyl peroxide
for 72 h to give the dibromoderivative 7 in 43% yield by
modifying a procedure reported in the literature [9, 13].
Compound 7 was refluxed for 19 h in a mixture of diox-
ane and water to give the corresponding diol 8 [14]. The
phthalimido diol 8 was deprotected by hydrazinolysis
[15] to give the amine 1 which was converted into its
hydrochloride salt (1 N HCl) by treatment with a few drops
of 2 N HCl and azeotropically removing water. It is note-
worthy that in our efforts to raise the yield in favor of 7,
N-bromosuccinimide and benzoyl peroxide were added
together, in four portions, to compound 6 at approx. 4 h-
intervals and stopping the reaction after three days. Sur-
prisingly, we noticed that, instead of rising, yields of
compound 7, were lowered (32%), because of the forma-
tion of a new unknown side-product. Hydrolysis [14] of
this mixture gave 8 along with aldehyde 10, which was
The synthesis of compound 2 is shown in Scheme 2.
The key step was 4-[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-
yl)propoxy]-3,5-dimethylphenyl acetate 14 which was
obtained following two alternative routes. The first was
performed following the previously described synthesis
[7], by submitting compound 6 to Friedel–Craft acylation
and then Baeyer–Villiger oxidation reactions [16]. By this
route, 14 was obtained in four steps, in 54% overall yield.
An alternative route starts from commercially available
2,6-dimethylphenol 11, which was converted into its
acetoxy derivative 12 by reaction with iodine and silver
acetate [17], which was then submitted to a Mitsunobu
reaction with 2-(2-hydroxy-1-methylethyl)-1H-isoindole-
1,3(2H)-dione 5 [12], to give the acetoxy intermediate 14.
By this route, compound 14 was obtained in three steps
of a convergent synthesis, in 70% overall yield; the latter
route is more convenient and efficient than the former
and obviates the use of large amounts of silver acetate, an
expensive reagent. Then, in order to obtain the bromo-
derivative 15, compound 14 was reacted with N-bromo-
succinimide and benzoyl peroxide [9, 13]. Unfortunately,
the yield of this reaction was very low (2%) because of the
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Arch. Pharm. Chem. Life Sci. 2010, 343, 325–332
Novel Hydroxylated Analogs of Mexiletine
327
between the amino group and the hydroxyl group on the
methyl in 2-position of the aromatic moiety (3,9-
dimethyl-2,3,4,5-tetrahydro-1,4-benzoxazepin-7-ol hydro-
bromide). It was assessed by spectroscopic analyses, as
already observed in the synthesis of HMM [9]. Subse-
quently, a satisfactory extraction of 2 from the hydrazi-
nolysis deprotection of 16 was obtained using a buffer
solution at pH 9 to 11 and with product finally obtained
as the acetate salt 2 N CH3COOH.
Pharmacology
The actions of the drugs were tested in vitro on INa cur-
rents recorded in HEK293 cells permanently transfected
with the human skeletal muscle sodium channel,
hNav1.4, using the whole-cell patch-clamp method.
Sodium currents were elicited by 25 ms-long depolariz-
ing test pulses at –30 mV from the holding potential of –
120 mV at two stimulation frequencies, 0.1 Hz for deter-
mination of the tonic block and 10 Hz for the phasic
block determination [6]. The IC50 values were calculated
by fitting the concentration/effect relationships with a
first-order binding function, and are reported in Table 1
with the S. E. of the fit. Formal introduction of hydroxyl
groups onto the aromatic ring of mexiletine (a phenolic
one in the para-position or a benzylic one) always caused
a dramatic fall of potency of blockade under both tonic
and phasic conditions. The effect was additive, each
hydroxyl group determining a lowering effect of about
one order of magnitude. Thus, 1 and 2 were at least one
hundred times less potent than mexiletine in their
sodium channel blocking activity. However, this residual
activity was dependent on the frequency of stimulation:
a clear-cut dependency of action was found in all the
tested compounds. The IC50 values at 10 Hz stimulation
frequency were from 2 to 10 times lower than those at
0.1 Hz. The lowering effect on the IC50 values of the mono-
hydroxyl derivatives, PHM and HMM, seemed more pro-
nounced for the compound with the benzylic carbinol
(HMM). However, this trend is reversed when activities of
the dihydroxyl derivatives 1 and 2 are compared: the ana-
log bearing both a para-phenolic and a benzyl carbinolic
group has shown the most evident lowering effect of the
series. Further studies are necessary in order to clarify
the reasons of the peculiar behavior of mono and dihy-
droxyl analogs of mexiletine in lowering the blocking
activity on Na+ channels.
Scheme 2. Synthesis of compound 2.
formation of a dibrominated derivative as a side-com-
pound, with a Rf value very close to the one of compound
15. Therefore, compound 14 was subjected to an alterna-
tive bromination reaction, by modifying a procedure
used in the literature for the bromination of xylenes [18]
to give 15 in a mixture with 14. This mixture, without
any further purification, was submitted to hydrolysis in
water and dioxane, and the dihydroxy derivative 16 was
isolated. The first attempt to obtain amine 2 was by sub-
mitting compound 16 to hydrazinolysis [15]. The work-up
of this reaction was quite difficult due to the fact that
amine 2 and the 2,3-dihydrophthalazine-1,4-dione – by-
product of the hydrazinolysis – are both soluble in the
aqueous layer. Compound 2 was obtained in an amount
just sufficient for its characterization by 1H-NMR analysis
and GC/MS, but not for its conversion into the corre-
sponding salt. Thus, unpurified amine 2 was directly sub-
mitted to protection with Boc2O giving the correspond-
ing carbamate 17, which was deprotected with gaseous
HCl, in an ice-bath, to give 2 N HCl as an oil that could not
be recrystallized. On the other hand, deprotection with
gaseous HBr afforded a mixture of 2 N HBr and a by-prod-
uct resulting from an intramolecular condensation
Conclusion
We have described the synthesis, full characterization,
and biological evaluation of two novel dihydroxylated
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A. Catalano et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 325–332
Table 1. Concentrations for half-maximal tonic and use-dependent block of sodium currents (IC50) and logD of mexiletine, PHM,
HMM, 1, 2, and several b2-adrenergic receptor ligands.
Compound
Tonic block
Use-dependent block
logDb
IC50 at 0.1 Hz (lM)a
IC50 at 10 Hz (lM)a
Mexiletine
260 € 30
23.9 € 1.7
0.53
PHM
1140 € 220
2320 € 400
390 € 80
–0.06
–0.59
HMM
800 € 140
1
12 100 € 1500
5100 € 700
–0.85
2
>30 000
76 € 5
18 400 € 7000
26 € 5
–1.28
0.255
clenbuterol
propranolol
nadolol
68.9 € 2.8
>30 000
>30 000
7.9 € 0.2
>30 000
>30 000
1.12
–1.55
–2.01
salbutamol
a
The half-maximum inhibitory concentrations (IC50) were calculated from the fit with a first-order binding function of the concen-
tration/sodium current block relationships measured at 0.1 Hz and 10 Hz stimulation frequencies. The IC50 values are expressed
with the S.E. of the fit.
logD values were determined using a Sirius pKa analyzer on the basis of experimentally determined pKa and logP values (see
Experimental).
b
analogs of mexiletine, [2-(2-aminopropoxy)-1,3-pheny- mined for Mex, 1, 2, and for the aforementioned b-adren-
lene]dimethanol 1 and 4-(2-aminopropoxy)-3-(hydroxy- ergic receptor ligands, in order to confirm our hypothe-
methyl)-5-methylphenol 2. Our present data demonstrate sis that the difference in activity is related to variations
that structural modifications on the aromatic moiety of in lipophilicity. Table 1 shows clearly that the lower the
mexiletine (i. e. introduction of two hydroxyl groups) lipophilicity, the lower is the activity of compounds on
reduce the activity towards voltage-gated sodium chan- sodium channels. In addition, we previously reported
nels (Table 1). These findings confirm our previous that PHM and HMM [7–9], two metabolites of mexiletine,
hypothesis that the introduction of two hydroxyl groups bearing one hydroxyl group (para-phenolic or a benzyl
onto the aromatic moiety of drugs acting on such chan- carbinolic, respectively), did not show the same blocking
nels (anti-arrhythmics and/or b-adrenergic receptor activity as mexiletine on voltage-gated sodium channels,
ligands) impairs either the transport to or the interaction but only a slight residual activity on such channels, deter-
with the binding site in the pore of the Na channels mined in voltage-clamp experiments, and these results
(Table 1) [6]. Experimental logD values were also deter- have been confirmed herein by means of patch-clamp
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Arch. Pharm. Chem. Life Sci. 2010, 343, 325–332
Novel Hydroxylated Analogs of Mexiletine
329
1698 (C=O) cm–1; 1H-NMR d: 1.57 (d, J = 7.1 Hz, 3H, CH3), 4.25 (dd, J
= 9.3, 5.2 Hz, 1H, CHHCH), 4.39 (d, J = 10.2 Hz, 2H, CHHBr), 4.57
(d, J = 9.9 Hz, 2H, CHHBr), 4.76 (apparent t, J = 9.6 Hz, 1H,
CHHCH), 4.90–5.10 (m, 1H, CH), 7.08 (t, J = 7.7 Hz, 1H, ArO), 7.31
(d, J = 7.7 Hz, 2H, ArO), 7.65–7.80 (m, 2H, Ar), 7.80–7.95 (m, 2H,
Ar); 13C-NMR d: 15.2 (1C), 27.9 (2C), 47.2 (1C), 73.7 (1C), 123.6 (2C),
125.5 (1C), 132.3 (4C), 132.5 (2C), 134.2 (2C), 155.1 (1C), 168.9
(2C); GC/MS (70 eV) m/z (%): 465 [M+] (a1), 188 (100). Anal. calcd.
for C19H17NO3Br2 (467.15): C, 48.85; H, 3.67; N, 3.00. Found: C,
48.51; H, 3.63; N, 3.06.
recordings (Table 1). A possible explanation for the lack
of activity of the dihydroxylated analogs is that the two
hydroxyl groups may prevent these compounds from
crossing the plasma membrane and/or they may hamper
the proposed relevant interaction of the xylyloxy moiety
of these compounds with a Tyr residue in the local anes-
thetic-like drug binding site receptor [19, 20].
2-{2-[2,6-Bis(hydroxymethyl)phenoxy]-1-methylethyl}-
1H-isoindole-1,3(2H)-dione 8
Experimental
Materials and methods
0.20 g (0.43 mmol) of 7 were dissolved in 4 mL of H2O/dioxane
(1:1). The reaction mixture was kept under reflux for 19 h. Diox-
ane was removed by evaporation under vacuum and the aqueous
phase was extracted several times with EtOAc. The combined
organic layers were dried (Na2SO4) and concentrated under vac-
uum to give 4.27 g of a yellow oil. Purification of the crude resi-
due by silica gel column chromatography (EtOAc/petroleum
ether, 1:1) gave 8 (0.14 g, 0.41 mmol, 95%) as a yellow oil: IR
(neat): 3458 (OH), 1773, 1708 (C=O) cm–1; 1H-NMR d: 1.50 (d, J = 7.1
Hz, 3H, CH3), 2.47 (br s, 2H, exch D2O, OH), 3.98 (dd, J = 9.5, 5.4 Hz,
1H, CHHCH), 4.48–4.65 (m overlapping s at 4.59, 1H, CHHCH),
4.59 (br s overlapping m at 4.48–4.65, 4H, CH2OH), 4.78–4.94 (m,
1H, CH), 7.05 (t, J = 7.6 Hz, 1H, ArO), 7.26 (d, J = 7.4 Hz, 2H, ArO),
7.66–7.76 (m, 2H, Ar), 7.78–7.88 (m, 2H, Ar); 13C-NMR d: 15.2 (1C),
47.4 (1C), 60.8 (2C), 74.4 (1C), 123.6 (2C), 125.0 (1C), 129.4 (2C),
132.0 (2C), 134.1 (2C), 134.4 (2C), 154.7 (1C), 169.0 (2C); GC/MS (70
eV) m/z (%): 341 [M+] (a1), 188 (100).
Chemicals were purchased from Sigma-Aldrich (Italy) or Lancas-
ter (Lanchester Chemical, USA) in the highest quality commer-
cially available. Yields refer to purified products and were not
optimized. The structures of the compounds were confirmed by
routine spectrometric and spectroscopic analyses. Compounds
5, 6, and 12–14 were prepared as described previously [7, 9, 10,
17]. Only spectra for compounds not previously described are
given. Melting points were determined on a Gallenkamp appara-
tus (Weiss-Gallenkamp, London, UK) in open glass capillary
tubes and are uncorrected. Infrared spectra were recorded on a
Perkin-Elmer (Norwalk, CT, USA) Spectrum One FT spectropho-
tometer and band positions are given in reciprocal centimeters
(cm–1). 1H-NMR and 13C-NMR spectra were recorded on a Varian
VX Mercury spectrometer (Varian Inc., Palo Alto, CA, USA) oper-
ating at 300 and 75 MHz for1H and 13C, respectively, using CDCl3
as solvent unless otherwise indicated. Chemical shifts are
reported in parts per million (ppm) relative to solvent reso-
nance: CDCl3, d: 7.26 (1H-NMR) and d: 77.3 (13C-NMR). J values are
given in Hz. EI-MS spectra were recorded on a Hewlett-Packard
6890-5973 MSD gas chromatograph/mass spectrometer (Hew-
lett-Packard, Palo Alto, CA, USA) at low resolution. ESI+/–/MS/MS
analyses were performed with an Agilent 1100 series LC-MSD
trap system VL Workstation (Agilent, Palo Alto, CA, USA). Ele-
mental analyses were performed on a Eurovector Euro EA 3000
analyzer. Electrospray ionization (ESI) time-of-flight (TOF) reflec-
tron experiments are performed on an Agilent ESI-TOF mass
spectrometer (Agilent). Samples are electrosprayed into the TOF
reflectron analyzer at a ESI voltage of 4000 V and a flow rate of
200 lL/min. 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. [21]. TLC analyses were performed on precoated silica gel on
aluminum sheets (Kieselgel 60 F254, Merck).
2-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)propoxy]-3-
(hydroxymethyl)benzaldehyde 10
2.1 g of a mixture of 7 (55%, 1.16 g, 2.49 mmol) and 9 (45%, 0.94
g, 1,72 mmol) was reacted as above described for the synthesis of
8. Purification of the crude residue (1.50 g) by silica gel column
chromatography (EtOAc/petroleum ether, 1:1) gave 0.9 g of 8
and 0.4 g of 10. Compound 10 was recrystallized from CHCl3/hex-
ane to give 0.30 g (0.88 mmol, 52%) of 10 as white crystals: m.p.:
133–1348C; IR (KBr): 3441 (OH), 1771, 1705 (C=O) cm–1; 1H-NMR d:
1.53 (d, J = 7.1 Hz, 3H, CH3), 2.26 (br s, 1H, exch D2O, OH), 4.12 (dd,
J = 9.5, 5.1 Hz, 1H, CHHCH), 4.66 (s overlapping apparent t at
4.71, 2H, CH2OH), 4.71 (apparent t overlapping s at 4.66, J = 9.6
Hz, 1H, CHHCH), 4.84–5.02 (m, 1H, CH), 7.21 (apparent t, J = 7.7
Hz, 1H, ArO), 7.62 (dd, 1H, J = 6.9, 1.1 Hz, ArO), 7.69–7.78 (m, 2H,
Ar), 7.82–7.91 (m, 2H, Ar + 1H, ArO), 10.3 (s, 1H, CHO); 13C-NMR d:
15.2 (1C), 47.2 (1C), 60.2 (1C), 76.9 (1C), 123.6 (3C), 125.0 (1C),
129.5 (1C), 132.0 (2C), 134.4 (2C), 135.2 (1C), 135.9 (1C), 159.4
(1C), 168.8 (2C), 189.6 (1C); GC/MS (70 eV) m/z (%): 339 [M+] (3), 188
(100).
Chemistry
2-{2-[2,6-Bis(bromomethyl)phenoxy]-1-methylethyl}-1H-
isoindole-1,3(2H)-dione 7
To a stirred solution of 6 (0.50 g, 1.62 mmol) in CCl4 (10 mL), N-
bromosuccinimide (0.57 g, 3.24 mmol) and benzoyl peroxide (54
mg, 0.23 mmol) were added. The reaction mixture was refluxed
for 48 h; then, additional N-bromosuccinimide (0.15 g, 0.81
mmol) and benzoyl peroxide (27 mg, 0.11 mmol) were added.
The mixture was refluxed for 24 h and then the solid residue
was filtered off. After evaporation of the solvent under vacuum,
the residue (1.30 g) was purified by silica gel column chromatog-
raphy (EtOAc/petroleum ether, 0.5:9.5) to give 7 (0.32 g, 0.69
mmol, 43%) as a white solid: m.p.: 117–1198C; IR (KBr): 1774,
2-[2-(4-Acetyl-2,6-dimethylphenoxy)-1-methylethyl]-1H-
isoindole-1,3(2H)-dione 13
Prepared as reported in the literature for R- and S-isomers in 78%
yield [7]. The solid obtained was recrystallized from EtOAc/petro-
leum ether to give 13 as white crystals (61% yield): m.p.: 93–948C
(EtOAc/petroleum ether); IR (KBr): 1773, 1708, 1676 (C=O) cm–1;
1H-NMR d: 1.55 (d, J = 7.1 Hz, 3H, CH3CH), 2.22 (s, 6H, CH3ArO),
2.51 (s, 3H, CH3CO), 3.93 (dd, J = 9.3, 5.6 Hz, 1H, CHHO), 4.43
(apparent t, J = 9.2 Hz, 1H, CHHO), 4.80–4.94 (m, 1H, CH), 7.57 (s,
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Arch. Pharm. Chem. Life Sci. 2010, 343, 325–332
2H, ArO), 7.70–7.78 (m, 2H, Ar), 7.82–7.90 (m, 2H, Ar); 13C-NMR d:
15.3 (1C), 16.7 (2C), 26.7 (1C), 47.3 (1C), 71.9 (1C), 123.4 (2C), 129.6
(2C), 131.2 (2C), 132.2 (2C), 133.2 (2C), 134.2 (1C), 159.7 (1C),
168.6 (2C), 197.8 (1C); GC/MS (70 eV) m/z (%): 351 [M+] (2), 188
(100). Anal. calcd. for C21H21NO4 (351.39): C, 71.78; H, 6.02; N,
3.99. Found: C, 72.22; H, 6.46; N, 4.04.
under vacuum to give 1.23 g of a yellow oil. Purification of the
crude residue by silica gel column chromatography (EtOAc/
petroleum ether, 1:1 fi EtOAc) gave 16 as a yellow oil (0.77 g,
2.26 mmol, >99%), which was crystallized from CHCl3/hexane to
give 0.68 g (1.99 mmol, 88%) of 16 as white crystals: m.p.: 153–
1548C (CHCl3/hexane); IR (KBr): 3396 (OH), 1771, 1705 (C=O) cm–1;
1H-NMR d: 1.48 (d, J = 7.1 Hz, 3H, CH3CH), 2.06 (s, 3H, CH3ArO),
3.33 (s, 2H, exch. with D2O, OH), 3.81 (dd, J = 9.5, 5.4 Hz, 1H,
CHHCH), 4.35 (apparent t, J = 9.5 Hz, 1H, CHHCH), 4.45 (d overlap-
ping d at 4.52, J = 12.7 Hz, 1H, AB system, CHHOH), 4.52 (d over-
lapping d at 4.45, J = 12.7 Hz, 1H, AB system, CHHOH), 4.72–4.88
(m, 1H, CH), 6.47 (d, J = 3.0 Hz, 1H, ArO), 6.58 (d, J = 3.0 Hz, 1H,
ArO), 7.64–7.72 (m, 2H, Ar), 7.78–7.86 (m, 2H, Ar);13C-NMR d: 15.2
(1C), 16.3 (1C), 47.4 (1C), 60.9 (1C), 73.2 (1C), 113.5 (1C), 117.5 (1C),
123.5 (3C), 132.0 (2C), 132.3 (1C), 134.3 (2C), 148.1 (1C), 152.4
(1C), 169.1 (2C); GC/MS (70 eV) m/z (%): 341 [M+] (4), 188 (100).
4-[2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)propoxy]-
3,5-dimethylphenyl acetate 14
Prepared as reported in the literature [7] for R- and S-isomers
obtaining 14 as a white solid (95% yield): m.p.: 75–778C; IR (KBr):
1766, 1706 (C=O) cm–1; 1H-NMR d: 1.54 (d, J = 6.9 Hz, 3H, CH3CH),
2.17 (s, 6H, CH3ArO), 2.23 (s, 3H, CH3CO), 3.88 (dd, J = 9.3, 5.8 Hz,
1H, CHHO), 4.35 (apparent t, 1H, J = 9.2 Hz, CHHO), 4.76–4.92 (m,
1H, CH), 6.66 (s, 2H, ArO), 7.68–7.76 (m, 2H, Ar), 7.80–7.88 (m, 2H,
Ar); 13C-NMR d: 15.4 (1C), 16.6 (2C), 21.3 (1C), 47.3 (1C), 71.9 (1C),
121.6 (2C), 123.4 (4C), 132.2 (2C), 134.2 (2C), 146.4 (1C), 153.1
(1C), 168.7 (2C), 170.1 (1C); GC/MS (70 eV) m/z (%): 367 [M+] (4), 188
(100).
tert-Butyl {2-[4-hydroxy-2-(hydroxymethyl)-6-
methylphenoxy]-1-methylethyl}carbamate 17
To a stirred solution of 16 (0.75 g, 2.2 mmol) in MeOH (5 mL), gla-
cial AcOH (0.25 mL, 4.38 mmol) and N2H4 . H2O (8.8 mmol) were
added and the mixture was kept under reflux for 3 h. After evap-
oration of the solvent under vacuum, the residue was dissolved
in 3 mL of 1 N NaOH and kept in an ice bath. A solution of Boc2O
(1.15 g, 5.26 mmol) in THF (12 mL) was then added dropwise and
the mixture was stirred at room temperature for 24 h. After stir-
ring, the solvent was removed under vacuum and the aqueous
phase was acidified with 2 N HCl and extracted twice with
EtOAc. The combined organic layers were dried (Na2SO4) and
concentrated under vacuum to give an oil consisting of 17 and
poli-Boc derivatives of 2. Purification of the residue by silica gel
column chromatography (EtOAc/petroleum ether, 2:8) gave 17
(68 mg, 0.22 mmol, 10%) as a yellow oil: IR (neat): 3356 (NH, OH),
1688 (C=O) cm–1; 1H-NMR d: 1.26 (d, J = 7.0 Hz, 3H, CH3CH), 1.44 (s,
9H, t-Bu), 2.12 (s, 3H, CH3Ar), 3.50–3.75 (m, 2H, CH2CH), 4.48 (d
overlapping d at 4.55, J = 12.8 Hz, 1H, AB system, CHHOH), 4.55 (d
overlapping d at 4.48, J = 12.8 Hz, 1H, AB system, CHHOH), 4.95–
5.20 (br m, 1H, CH), 6.52 (d, J = 2.9 Hz, 1H, Ar), 6.63 (d, J = 2.6 Hz,
1H, Ar), 7.62 (br s, 3H, exch. with D2O, OH + NH);13C NMR: d = 16.3
(1C), 17.9 (1C), 28.6 (3C), 46.9 (1C), 60.6 (1C), 75.9 (1C), 80.1 (1C),
113.6 (1C), 117.6 (1C), 132.2 (1C), 134.4 (1C), 148.0 (1C), 152.6
(1C), 156.1 (1C); ESI+/MS m/z: 334 [M + Na+]; ESI+/MS/MS m/z: 234
(100).
3-(Bromomethyl)-4-[2-(1,3-dioxo-1,3-dihydro-2H-
isoindol-2-yl)propoxy]-5-methylphenyl acetate 15
Method A
Prepared as above described for 7 starting from 14 (1.90 g, 5.18
mmol), N-bromosuccinimide (0.92 g, 5.18 mmol) and benzoyl
peroxide (0.10 g, 0.4 mmol) in CCl4 (77 mL). Purification by col-
umn chromatography (toluene/EtOAc, 10:0.5) gave only a small
amount of pure 15 (40 mg, 0.09 mmol, 2%): IR (neat): 1771, 1709
(C=O) cm–1; 1H-NMR d: 1.55 (d, J = 6.9 Hz, 3H, CH3CH), 2.20 (s, 3H,
CH3ArO), 2.24 (s, 3H, CH3CO), 4.03 (dd, J = 9.3, 5.6 Hz, 1H, CHHCH),
4.34 (d, J = 9.9 Hz, 1H, CHHBr), 4.548 (d overlapping t at 4.553, J =
10.2 Hz, 1H, CHHBr), 4.553 (apparent t overlapping d at 4.548, J =
9.3 Hz, 1H, CHHCH), 4.86–5.02 (m, 1H, CH), 6.82 (d, J = 3.0 Hz, 1H,
ArO), 6.91 (d, J = 3.0 Hz, 1H, ArO), 7.66–7.76 (m, 2H, Ar), 7.80–7.90
(m, 2H, Ar); 13C-NMR d: 15.3 (1C), 16.7 (1C), 21.3 (1C), 28.0 (1C),
47.2 (1C), 72.7 (1C), 121.7 (1C), 123.5 (2C), 125.0 (1C), 128.4 (1C),
129.3 (1C), 132.2 (2C), 134.2 (2C), 146.6 (1C), 152.9 (1C), 168.8
(2C), 169.6 (1C); GC/MS (70 eV) m/z (%): 445 [M+] (a1), 188 (100).
Method B
To a solution of 14 (0.50 g, 1.36 mmol) in EtOAc (3 mL), a solution
of NaBrO3 (0.61 g, 4.08 mmol) in water (2 mL) was added. Then, a
solution of Na2S2O5 (0.39 g, 2.04 mmol) in water (4 mL) was added
dropwise over a period of 15 min. Cyclohexane (3 mL) was then
added and the reaction mixture was stirred at room tempera-
ture for 4 h. The aqueous layer was extracted twice with ethyl
ether and the combined organic layers were dried (Na2SO4) and
concentrated under vacuum to give 0.60 g of a yellow oil. Purifi-
cation of the residue by silica gel column chromatography
(EtOAc/petroleum ether, 3:7) gave 15 in 71% yield (0.43 g, 0.97
mmol) along with 14 (0.14 g, 0.39 mmol, 29%).
[2-(2-Aminopropoxy)-1,3-phenylene]dimethanol 1
To a stirred solution of 8 (0.22 g, 0.65 mmol) in absolute EtOH
(2.6 mL), glacial AcOH (0.11 mL, 1.93 mmol) and N2H4 N H2O (1.93
mmol) were added and the mixture was kept under reflux for
2 h. The solid residue was filtered off. After evaporation of the
filtrate, the residue was taken up with EtOAc and extracted with
2 N HCl; then, the aqueous phase was treated with 2 N NaOH
and extracted several times with EtOAc. The combined organic
layers were dried (Na2SO4) and concentrated under vacuum to
give 1 (0.12 g, 0.57 mmol, 87%) as a slightly yellowish oil:1H-NMR
d: 1.19 (d, J = 6.6 Hz, 3H, CH3), 1.76 (br s, 4H, exch. with D2O, OH +
NH2), 3.35–3.50 (m, 1H, CH), 3.72 (dd, J = 9.2, 7.8 Hz, 1H, CHHCH),
4.06 (dd, J = 9.3, 3.0 Hz, 1H, CHHCH), 4.63 (d, J = 12.6 Hz, 2H,
CHHOH), 4.77 (d, J = 12.4 Hz, 2H, CHHOH), 7.09 (t, J = 7.6 Hz, 1H,
Ar), 7.20–7.30 (m, 2H, Ar).
2-{2-[4-Hydroxy-2-(hydroxymethyl)-6-methylphenoxy]-1-
methylethyl}-1H-isoindole-1,3(2H)-dione 16
1.43 g of a mixture of 15 (1.01 g, 2.26 mmol, 71%) and 14 (0.42 g,
1.14 mmol, 29%) were dissolved in a mixture of H2O (29 mL) and
dioxane (29 mL). The reaction mixture was kept under reflux for
20 h. Dioxane was removed by evaporation under vacuum and
the aqueous phase was extracted several times with EtOAc. The
combined organic layers were dried (Na2SO4) and concentrated
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Arch. Pharm. Chem. Life Sci. 2010, 343, 325–332
Novel Hydroxylated Analogs of Mexiletine
331
oil which was crystallized from abs. EtOH/iPr2O to give
2 N CH3COOH (123 mg, 0.45 mmol, 61%) as yellow crystals: m.p.:
169–1708C (abs. EtOH/i-Pr2O);1H-NMR (CD3OD, d: 3.08) d: 1.15 (d, J
= 6.6 Hz, 3H, CH3CH), 1.68 (s, 3H, CH3Ar), 2.01 (s, 3H, CH3COO),
3.36–3.48 (m, 1H, CH), 3.57 (dd, J = 10.1, 7.0 Hz, 1H, CHHCH), 3.67
(dd, J = 10.1, 3.7 Hz, 1H, CHHCH), 4.33 (s, 2H, CH2OH), 6.33 (d, J =
2.7 Hz, 1H, Ar), 6.44 (d, J = 2.7 Hz, 1H, Ar); 13C-NMR (CD3OD, d:
47.8) d: 14.5 (1C), 15.2 (1C), 22.8 (1C), 47.8 (1C), 59.6 (1C), 73.8
(1C), 113.6 (1C), 116.8 (1C), 131.7 (1C), 134.7 (1C), 147.3 (1C),
153.7 (1C), 178.8 (1C); ESI+/MS m/z: 234 [M + Na+]; ESI+/MS/MS m/z:
177 (100); Anal. calcd. for C11H17NO3 N CH3COOH (271.31): C,
57.55; H, 7.80; N, 5.16. Found: C, 57.33; H, 7.88; N, 5.12.
[2-(2-Aminopropoxy)-1,3-phenylene]dimethanol
Hydrochloride 1 N HCl
0.125 g (0.59 mmol) of 1 was treated with 1 mL of 2 N HCl and
the water was azeotropically removed (toluene/abs. EtOH) giving
a solid which was recrystallized from MeOH/Et2O to give 1 N HCl
(42 mg, 0.16 mmol, 27%) as white crystals: m.p.: 167–1688C
(MeOH/Et2O); IR (KBr): 3363 (OH, NH) cm–1; 1H-NMR (DMSO-d6; d:
2.48) d: 1.28 (d, J = 6.6 Hz, 3H, CH3), 3.25–3.70 (m, 3H, exch. with
D2O, NH3 + 1H, CH), 3.8–4.0 (m, 2H, CH2CH), 4.54 (s, 4H, CH2OH),
7.12 (t, J = 7.4 Hz, 1H, Ar), 7.32 (d, J = 7.7 Hz, 2H, Ar), 8.22 (br s, 2H,
exch. with D2O, OH); 1H-NMR (CD3OD; d: 3.31) d: 1.41 (d, J = 6.6 Hz,
3H, CH3), 3.65–3.80 (m, 1H, CH), 4.03 (dd, J = 10.2, 7.3 Hz, 1H,
CHHCH), 4.15 (dd, J = 10.2, 3.7 Hz, 1H, CHHCH), 4.66 (s, 4H,
CH2OH), 7.17 (t, J = 7.7 Hz, 1H, Ar), 7.37 (d, J = 7.7 Hz, 2H, Ar); 13C-
NMR (CD3OD, d: 47.8) d: 14.1 (2C), 47.8 (1C), 59.4 (1C), 74.9 (1C),
124.7 (1C), 129.6 (2C), 134.2 (2C), 154.6 (1C); ESI+/MS m/z: 234 [M +
Na+]; ESI+/MS/MS m/z: 176 (100). Anal. calcd. for
C11H17NO3 N HCl N 0.33 H2O (253.72): C, 52.07; H, 7.42; N, 5.52.
Found: C, 52.24; H, 7.13; N, 5.87.
Physicochemical data
Physicochemical data of the compounds shown in Table 1 were
obtained by a pH-metric technique using a GlpKa apparatus (Sir-
ius Analytical Instruments Ltd., Forrest Row, East Sussex, UK)
[22–25]. Because of the low solubility of the investigated com-
pounds in aqueous medium, methanol was used as a co-solvent
for pKa measurements. Three separate solutions of a concentra-
tion approximately 10–5 M, in 10–30% w/w (MeOH/H2O), were
prepared. They were acidified with 0.5 M HCl to pH 4. The solu-
tions were titrated with 0.5 M KOH to pH 12. Initial pKa values,
which are the apparent ionization constants relative to the mix-
ture of the solvents, were obtained by Bjerrum Plot i. e., the curve
obtained by the difference between the titration curve of the ion-
isable substance and that of the blank solution. These values
were optimized by a weighted nonlinear least-squares procedure
(Refinement Pro 1.0 software) to obtain pKa values in the absence
of the cosolvent by extrapolation using the Yasuda–Shedlovsky
equation [26]. To obtain logP data, at least three separate titra-
tions were performed for each compound. The concentration of
the analite was approximately 10–5 M, in mixtures of H2O (7.5
mL) and n-octanol, (0.1 to 10 mL). The biphasic solutions were
acidified to pH 4 with 0.5 M HCl and then titrated with 0.5 M
KOH until pH 12. The obtained data were optimized as described
above and the average of these data gave the logP value for each
compound [23, 24]. All titrations were carried out at 25 € 0.18C
under nitrogen atmosphere to exclude CO2.
4-(2-Aminopropoxy)-3-(hydroxymethyl)-5-methylphenol 2
To a stirred solution of 16 (0.34 g, 1.0 mmol) in absolute EtOH (10
mL), glacial AcOH (2.0 mmol) and aqueous hydrazine (4.0 mmol)
were added and the mixture was kept under reflux for 5 h. The
solid residue was filtered off. After evaporation of the filtrate,
the residue was taken up with EtOAc and extracted with 2 N HCl
(3610 mL); then, the aqueous phase was brought to 9 a pH a 11
with 2 N NaOH (20 mL) and 2 N Na2CO3 (20 mL) and extracted sev-
eral times with EtOAc. The combined organic layers were dried
(Na2SO4) and concentrated under vacuum. The final product was
a slightly yellowish oil (147 mg, 0.70 mmol, 70%): IR (neat): 3353
(OH, NH) cm–1; 1H-NMR (CD3OD, d: 3.09) d: 0.96 (d, 3H, J = 6.6 Hz,
CH3CH), 1.99 (s, 3H, CH3Ar), 3.0–3.15 (m overlapping CH3OH sig-
nal, 1H, CH), 3.35 (apparent t, J = 8.1 Hz, 1H, CHHCH), 3.44 (dd, J =
9.1, 4.4 Hz, 1H, CHHCH), 4.36 (s, 2H, CH2OH), 6.31 (d, J = 3.0 Hz,
1H, Ar), 6.46 (d, J = 3.0 Hz, 1H, Ar); 13C-NMR (CD3OD, d: 47.8) d:
15.3 (1C), 18.1 (1C), 47.0 (1C), 59.2 (1C), 78.6 (1C), 113.0 (1C), 116.5
(1C), 131.6 (1C), 134.9 (1C), 147.7 (1C), 153.4 (1C); GC/MS (70 eV)
m/z (%): 211 [M+] (2), 58 (100).
Pharmacology
4-(2-Aminopropoxy)-3-(hydroxymethyl)-5-methylphenol
In-vitro drug testing was performed as previously described [6].
Permanent transfection of HEK293 cells (human embryonic kid-
ney cell line) with the full-length hNav1.4 cDNA, encoding the
human skeletal muscle sodium channel subtype, was obtained
using the calcium-phosphate precipitation method followed by
clone selection with geneticin (Gibco-Invitrogen, Italy). More
than 95% of the cloned cells express robust whole-cell INa of 1–4
nA amplitude with voltage- and time-dependent properties typi-
cal of voltage-gated sodium channels. Whole-cell sodium cur-
rents were recorded at room temperature (20 to 228C) using an
Axopatch 1D amplifier (Axon Instruments, Union City, CA, USA).
Voltage-clamp protocols and data acquisition were performed
with pClamp 6.0 software (Axon Instruments, USA) through a
12-bit A-D/D-A Digidata 1200 interface (Axon Instruments). The
external solution contained (in mM): 150 NaCl, 4 KCl, 2 CaCl2, 1
mg Cl2, 5 Hepes and 5 glucose; the pH was set to 7.4 with NaOH.
The pipette solution contained (in mM): 120 CsF, 10 CsCl, 10
NaCl, 5 EGTA, and 5 Hepes; The pH was set to 7.2 with CsOH. Cur-
rents were low-pass filtered at 2 kHz (–3 dB) by the four-pole Bes-
sel filter of the amplifier and digitized at 10–20 kHz. After rup-
turing the patch membrane, sodium currents were elicited by a
Hydrochloride 2 N HCl
A solution of 17 (70 mg, 0.22 mmol) in Et2O (4 mL) in an ice-bath
was treated with gaseous HCl for a few seconds until the solution
turns into a suspension under formation of a white gum, which
was filtrated giving 2 N HCl (38 mg, 0.14 mmol, 73%) as a white
gum, when taken at –208C, that became an oil at room temper-
ature: 1H-NMR (CD3OD, d: 3.31) d: 1.41 (d, J = 6.9 Hz, 3H, CH3CH),
2.23 (s, 3H, CH3Ar), 3.65–3.75 (m, 1H, CH), 3.80–3.90 (m, 1H,
CHHCH), 3.93 (dd, J = 10.2, 3.8 Hz, 1H, CHHCH), 4.51 (s, 2H,
CH2OH), 6.56 (d, J = 3.0 Hz, 1H, Ar), 6.67 (d, J = 3.0 Hz, 1H, Ar); 13C-
NMR (CD3OD, d: 46.3) d: 12.6 (1C), 13.7 (1C), 46.5 (1C), 58.0 (1C),
71.7 (1C), 112.1 (1C), 115.3 (1C), 130.1 (1C), 133.1 (1C), 145.7 (1C),
152.2 (1C).
4-(2-Aminopropoxy)-3-(hydroxymethyl)-5-methylphenol
Acetate 2 N CH3COOH
To a solution of 2 (157 mg, 0.74 mmol) in MeOH (4 mL), a solution
of glacial AcOH (0.88 mmol) in MeOH (2 mL) was added. The sol-
vent was removed by evaporation under vacuum to give a brown
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www.archpharm.com

332
A. Catalano et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 325–332
25 ms-long test pulse to –30 mV from a holding potential of –120
mV applied at 0.1 Hz frequency, until stabilization of the current
amplitude was achieved (typically 5 min). Then, the drug
(directly dissolved in saline solution) was applied at the desired
concentration around the cell through a plastic capillary, and
the effects of the drug on INa was measured first at 0.1 Hz stimu-
lation frequency, then at 10 Hz. Little or no run-down was
observed during the experiments. Analysis was performed off-
line. The mean ratio IDRUG/ICTRL calculated from at least three cells
was reported as a function of drug concentration. At least three
drug concentrations were used to obtain each relationship,
which were fitted by a first-order binding function:
[8] M. De Bellis, A. De Luca, F. Rana, M. M. Cavalluzzi, et al., Br.
J. Pharmacol. 2006, 149, 300–310.
[9] M. M. Cavalluzzi, A. Catalano, C. Bruno, A. Lovece, et al.,
Tetrahedron Asymmetry 2007, 18, 2409–2417.
[10] A. Carocci, A. Catalano, F. Corbo, A. Duranti, et al., Tetrahe-
dron Asymmetry 2000, 11, 3619–3634.
[11] A. K. Bose, F. Greer, C. C. Price, J. Org. Chem. 1958, 23,
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[12] O. Mitsunobu, Synthesis 1981, 1.
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IDRUG/ICTRL = 1/(1 + ([DRUG]/IC50))
[14] H.-J. Buysch, E.-G. Hoffmann, U. Jansen, D. Ooms, B.-U.
Schenke, D.E. Patent 19520612, 1996; Chem. Abstr. 1997,
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allowing the calculation of half-maximum inhibitory concentra-
tion values. Because of the limited drug quantity available and
to avoid drug-solubility problems, the maximum drug concen-
tration used was 10 mM. Thus, the IC50 value calculated for mex-
iletine derivatives are to be considered merely qualitative.
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This work was accomplished thanks to financial support from the Min-
istero dell'Istruzione, dell'Universitꢀ e della Ricerca (MIUR, grant num-
ber 2005033023) to CF and Telethon-Italy (grant number GGP04140) to
DCC.
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The authors have declared no conflict of interest
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