Synthesis, antiarrhythmic activity, and toxicological evaluation of mexiletine analogues

Article abstract of DOI:10.1016/j.ejmech.2016.05.046

Four mexiletine analogues have been tested for their antiarrhythmic, inotropic, and chronotropic effects on isolated Guinea pig heart tissues and to assess calcium antagonist activity, in comparison with the parent compound mexiletine. All analogues showed from moderate to high antiarrhythmic activity. In particular, three of them (1b,c,e) were more active and potent than the reference drug, while exhibiting only modest or no negative inotropic and chronotropic effects and vasorelaxant activity, thus showing high selectivity of action. All compounds showed no cytotoxicity and 1b,c,d did not impair motor coordination. All in, these new analogues exhibit an interesting cardiovascular profile and deserve further investigation.

Full text of DOI:10.1016/j.ejmech.2016.05.046

Accepted Manuscript
Synthesis, antiarrhythmic activity, and toxicological evaluation of mexiletine
analogues
Mariagrazia Roselli, Alessia Carocci, Roberta Budriesi, Matteo Micucci, Maddalena
Toma, Lorenzo Di Cesare Mannelli, Angelo Lovece, Alessia Catalano, Maria
Maddalena Cavalluzzi, Claudio Bruno, Annalisa De Palma, Marialessandra Contino,
Maria Grazia Perrone, Nicola Antonio Colabufo, Alberto Chiarini, Carlo Franchini,
Carla Ghelardini, Solomon Habtemariam, Giovanni Lentini
PII:
S0223-5234(16)30444-5
10.1016/j.ejmech.2016.05.046
EJMECH 8638
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 March 2016
Revised Date: 20 May 2016
Accepted Date: 21 May 2016
Please cite this article as: M. Roselli, A. Carocci, R. Budriesi, M. Micucci, M. Toma, L. Di Cesare
Mannelli, A. Lovece, A. Catalano, M.M. Cavalluzzi, C. Bruno, A. De Palma, M. Contino, M.G. Perrone,
N.A. Colabufo, A. Chiarini, C. Franchini, C. Ghelardini, S. Habtemariam, G. Lentini, Synthesis,
antiarrhythmic activity, and toxicological evaluation of mexiletine analogues, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.046.
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ACCEPTED MANUSCRIPT
Table of Contents graphic
Synthesis, Antiarrhythmic Activity, and Toxicological
Evaluation of a Series of Mexiletine Analogues.
R
O
mexiletine: R = Me, EC50 = 11.6 µM
b: R = tBu, EC50 = 2.1 µM
NH2
1

ACCEPTED MANUSCRIPT
Synthesis, Antiarrhythmic Activity, and Toxicological Evaluation of Mexiletine Analogues.
†
*,†
¥
¥
†
†
Mariagrazia Roselli, Alessia Carocci, Roberta Budriesi, Matteo Micucci, Maddalena Toma,
ˆ
†
†
Lorenzo Di Cesare Mannelli, Angelo Lovece, Alessia Catalano, Maria Maddalena Cavalluzzi,
†
§
†
†
Claudio Bruno, Annalisa De Palma, Marialessandra Contino, Maria Grazia Perrone, Nicola
†
¥
†
ˆ
¶
Antonio Colabufo, Alberto Chiarini, Carlo Franchini, Carla Ghelardini, Solomon Habtemariam,
†
Giovanni Lentini.
†
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, via E.
¥
Orabona n. 4, 70126 Bari, Italy. Dipartimento di Farmacia e Biotecnologie, Università di
ˆ
Bologna, Via Belmeloro 6, 40126 Bologna, Italy. Dipartimento di Farmacologia Preclinica e
§
Clinica, Università di Firenze, Viale Pieraccini 6, 50139 Firenze, Italy. Dipartimento di
Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari Aldo Moro, Via
¶
Orabona 4, 70126 Bari, Italy. Pharmacognosy Research Laboratories & Herbal Analysis services,
Chatham-Maritime, Kent ME4 4TB, UK.
*Corresponding author: Dott.ssa Alessia Carocci,
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari A. Moro, via
Orabona 4, 70126 Bari, Italy, phone number: (+39) 080-5442745; fax number: (+39) 080-5442724,
E-mail address: alessia.carocci@uniba.it

ACCEPTED MANUSCRIPT
Abstract
Four mexiletine analogues have been tested for their antiarrhythmic, inotropic, and chronotropic
effects on isolated guinea pig heart tissues and to assess calcium antagonist activity, in comparison
1
with the parent compound mexiletine. All analogues showed from moderate to high antiarrhythmic
activity. In particular, three of them (1b,c,e) were more active and potent than the reference drug,
while exhibiting only modest or no negative inotropic and chronotropic effects and vasorelaxant
activity, thus showing high selectivity of action. All compounds showed no cytotoxicity and 1b,c,d
did not impair motor coordination. All in, these new analogues exhibit an interesting cardiovascular
profile and deserve further investigation.
Keywords: Mexiletine; Antiarrhythmics; P-glycoprotein; Inotropism; MW-assisted synthesis
1
Abbreviations used: CNS, central nervous system; DMEM, Dulbecco’s modified eagle’s medium;
GPILSM, guinea pig ileum longitudinal smooth muscle; HepG2, human hepatocellular liver
carcinoma cells; MDCK, Madin-Darby canine kidney; MDCK-MDR1, MDCK overexpressing P-
gp; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MW, microwave; P-gp, P-
glycoprotein ; PSS, physiological salt solution.

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1. Introduction
One of the major causes of mortality is represented by cardiovascular diseases and about 25% of
cardiovascular-related adverse events are caused by arrhythmias. The term arrhythmia defines an
abnormality in the sequence and/or shape of electrical impulses during the cardiac cycle. A critical
role in the regulation of electrical activity of cardiac cells is played by ion channels such as sodium,
calcium and potassium channels. Abnormalities in the expression and/or function of ion channels
are responsible of many arrhythmias [1,2], thus, they represent one of the targets for antiarrhythmic
therapy. Indeed, most of the existing antiarrhythmic agents, such as lidocaine, tocainide, and
mexiletine [3,4] are ion channel blockers. Mexiletine (1a, Fig. 1) is an antiarrhythmic drug
belonging to class IB. It is a voltage-gated sodium channel blocker whose mechanism of action is
related to its preferential binding to the open and/or inactivated states of the channels. The selective
action of mexiletine on pathologically hyperactive tissues seems to be related to the use-dependent
blockade of sodium channels [5]. Mexiletine undergoes extensive metabolism; eleven phase I
metabolites of mexiletine have been identified [6,7]. Mexiletine is predominantly used in the
treatment of ventricular arrhythmias, however, recent studies have demonstrated its therapeutic
value for treating many other disorders associated with voltage-gated sodium channel dysfunction
including chronic painful diabetic neuropathy [8], neuropathic pain [9], myotonic syndromes [10],
and Timothy syndrome [11]. Recently, mexiletine has been successfully used in the treatment of
sporadic amyotrophic lateral sclerosis [12–14]. Despite all these reported therapeutic values, the use
of mexiletine is characterized by several drawbacks including epigastric discomfort, atrioventricular
heart block, and CNS disturbances [15]. Hence, it has now been discontinued in many countries
including the US and the UK [16]. Consequently, numerous attempts have been made in recent
years to develop an alternative to mexiletine, including the design of new analogues that offer the
same pharmacological effect but without the unwanted side effects. In the past decade, our own
laboratories have been focusing on the development of such analogues through which the structural
determinants for blocking voltage-gated sodium channels of skeletal muscles have been established

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[
17–20]. As these identified compounds were more potent channel blockers than the parent
compound, they have been proposed as mexiletine alternatives for the treatment of myotonias. We
also established that the activity of mexiletine-like sodium channel blockers is strongly linked to the
part of the molecule nearby the asymmetric carbon atom. In order to evaluate the potential of these
compounds for treating arrhythmias, we selected the most interesting compounds from the
previously reported series (1c–e, Fig. 1) that bear a bulky group at the stereogenic center. These
compounds, along with a new additional compound, 1b, bearing a tert-butyl group as substituent
were prepared by alternative and efficient synthetic routes. The biological activity of all compounds
was assessed with due comparison with mexiletine.
2. Results and discussion
2.1 Chemistry
Mexiletine (1a) and its analogues (1b,c) were prepared in two steps according to the synthetic route
depicted in Scheme 1, carried out in both traditional conditions and microwave-assisted synthesis.
In the first step, 2,6-dimethylphenol underwent Williamson reaction [21] with the suitable halo
ketone (2a–c) to obtain aryloxyalkyl ketones (3a–c) which were converted into the corresponding
amines 1a–c by reductive amination under both traditional and microwave conditions [22]. The
corresponding hydrochloride salts (1a–c·HCl) were obtained by treating 1a–c with gaseous HCl.
Mexiletine analogues 1d,e were prepared as depicted in Scheme 2. Compounds 5d,e were obtained
by protecting the corresponding commercial valinol and phenylalaninol, respectively, with di-tert-
butyl dicarbonate [23,24]. Compounds 5d,e were subjected to Mitsunobu reaction with 2,6-
dimethylphenol as previously described [25]. The so obtained aryl alkyl ethers 6d,e were treated
with gaseous HCl to give the corresponding amines as hydrochloride salts (1d,e·HCl).
2.2 Biological results

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Mexiletine and its analogues (1a–e) were tested in vitro for their antiarrhythmic activity on guinea
pig isolated left atria driven at 1 Hz. Compound 1b,c,e increased the threshold of ac-arrhythmia
more than mexiletine did, while 1d showed an anti-arrhythmic activity comparable to that of the
parent compound, mexiletine (Table 1). In particular, compound 1c was found to be 27-fold more
active than mexiletine. To better define the cardiac profile of mexiletine analogues, their influence
on additional cardiac parameters were compared with that elicited by the reference compound,
mexiletine (Table 2). All the tested compounds strongly decreased the developed tension on driven
left atria with EC values within the range of 0.012 and 0.042 mM. The most potent analogue was
5
0
1e showing a negative inotropism 4-fold higher than that of mexiletine (Tab. 2), while its
antiarrhythmic potency was 2.7-fold higher than that of mexiletine (Table 1). The effect of 1b was
comparable to that of mexiletine. It is noteworthy that the introduction of an aromatic moiety, in
lieu of the methyl one, on the stereogenic center as in 1c and 1e, strongly increased antiarrhythmic
and negative inotropic potency. On the other hand, the introduction of an isopropyl group, as in 1d,
while slightly affecting the antiarrhythmic activity, enhanced two times the negative inotropic
effect. With regard to the negative chronotropic activity on spontaneously beating right atrium,
only compound 1d showed a weak activity, about 900-fold lower than that of mexiletine.
Furthermore, 1b,c,e showed negative inotropism on spontaneously beating isolated right atrium,
too. It is noteworthy that this effect was not recorded for mexiletine and 1d due to their insignificant
inotropism resulting from chronotropic effect. Moreover, all the new mexiletine analogues were
+
tested on K -depolarized (80 mM KCl) guinea pig aortic strips to assess their vasorelaxant activity.
Data are shown in Table 3 with mexiletine as the reference drug. For all compounds, the intrinsic
vasorelaxant activity percentage on aorta was lower than 40% and thus unremarkable. In order to
2
+
rule out a possible inhibitory effect on nonvascular smooth muscle, such as that displayed by Ca
+
channel antagonists, further investigation on relaxant activity using K -depolarized (80 mM KCl)
guinea pig ileum longitudinal smooth muscle (GPILSM) was pursued (Table 3). The observed
profile of intrinsic activity and potency for 1b,c,d was, however, substantially similar to that

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displayed by mexiletine. Only compound 1e showed a relaxant potency slightly higher than that of
the parent compound, but in the micromolar range. Over all, all the compounds appears to display a
higher intrinsic activity in nonvascular than vascular smooth muscle cells. Since mexiletine clinical
use is often associated to CNS toxicity, a preliminary toxicological evaluation of mexiletine
analogues was performed by assessing the motor coordination of treated animals using the rota-rod
test (Table 4). All compounds at the dose of 25 mg/kg ip did not provoke any change in normal
behavior of mice as demonstrated in rota-rod experiments. At the dose of 50 mg/kg ip, only
compound 1e showed a significant impairment of mouse motor coordination, with a slight increase
in the number of falls being displayed. Since mexiletine is known to undergo extensive first-pass
oxidative metabolism [16,26,27] that is linked to the generation of reactive species metabolites
and/or cellular oxidative stress, the cytotoxicity of the four analogues was assessed using the MTT
cell viability assay [28]. When the human hepatocellular liver carcinoma cells (HepG2) were
challenged with the four mexiletine analogues at two concentrations (the first close to their
corresponding EC values, and the second 10-fold higher than the former), no cytotoxicity was
5
0
observed (data not shown). This finding was in agreement with the previous report for mexiletine
where cell viability was not affected at similar concentration range [16]. In addition, we tried to
verify if these analogues may display differential ability to interact with P-glycoprotein (P-gp), an
ATPase protein highly expressed in biological membranes, that generally affects pharmacokinetics,
efficacy, safety, and tissue levels of drugs. For this, drug-protein interaction was assessed for each
compound by using the Madin-Darby canine kidney (MDCK) cells overexpressing the ATPase
protein P-gp (MDCK-MDR1). As shown in Table 5, only compounds that bear a phenyl ring near
the basic center (1c,e) was able to bind P-gp. This data was in good agreement with a recent study
on a series of structurally related rigid analogues [29].
3. Conclusions

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Four mexiletine analogues differing for the substituent on the stereogenic center have been tested
for their antiarrhythmic, inotropic, and chronotropic effects on isolated guinea pig heart tissues and
for their calcium antagonist activity on GPILSM, in comparison with mexiletine. Even if with
different degrees of potency, antiarrhythmic activity has been observed in all the studied
compounds, conceivably suggesting the hypothesis that the substitution of the methyl group of
mexiletine with a bulky one, strengthens the interaction with the biological structures involved in
the regulation of heart activity. In particular, compounds 1b,c,e showed high antiarrhythmic
activity, exhibiting EC values in the range between 0.43 and 4.36 µM, thus comparing favorably
50
with the reference drug mexiletine (EC = 11.6 µM). These compounds exhibit an interesting
5
0
cardiovascular profile, being endowed with high antiarrhythmic activity and potency with only
modest or no negative inotropic and chronotropic effects and vasorelaxant activity. When
–
1
administered intraperitoneally (25 mgkg ), all compounds did not impair motor coordination as
demonstrated in rota-rod experiments and showed no in vitro cytotoxicity at concentration 10 times
higher than the corresponding antiarrhythmic EC values. On the basis that the observed favorable
5
0
profile should not be affected by interaction with P-gp, compounds 1b and 1d could be considered
as the preferred choices. Taken all the data together, this work afforded compounds endowed with
better antiarrhythmic activity along with similar or less cardiovascular effects than mexiletine, thus
presenting a higher selectivity of action and reduced side effects. In this respect, the t-butyl
substituted mexiletine analogue 1b seems to be the most interesting compound. Further
investigation would allow more complete knowledge about its pharmacological potential and
mechanism of action, which in turn might allow the design and development of even more active
and potent antiarrhythmic agents.
4. Experimental Protocols
4.1 Chemistry.

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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. Only spectra for compounds not previously described are given. The
reaction under microwaves irradiation were carried out at constant temperature in a CEM Discover
BenchMate microwave reactor, with continuous stirring. The temperature was measured and
controlled by a built-in infrared detector. 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 band positions are given in
–1
1
13
reciprocal centimeters (cm ). H NMR and C NMR spectra were recorded on either a Varian VX
1
13
Mercury spectrometer operating at 300 and 75 MHz for H and C, respectively, or an AGILENT
1
13
5
00 MHz operating at 500 and 125 MHz for H and C, respectively, using CDCl and CD OD as
3 3
solvents. Chemical shifts (δ) are reported in ppm relative to the residual non-deuterated solvent
1
13
1
resonance: CDCl , δ = 7.26 ( H NMR) and δ = 77.3 ( C NMR); CD OD, δ = 3.30 ( H NMR) and δ
3
3
1
3
=
47.8 ( C NMR) as internal references. Coupling constants (J) are given in Hz. Gas
chromatography (GC)/mass spectroscopy (MS) was performed on a Hewlett-Packard 6890–5973
MSD at low resolution. Liquid chromatography (LC)/mass spectroscopy (MS) was performed on a
spectrometer Agilent 1100 series LC-MSD Trap System VL. Elemental analyses were performed
on a Eurovector Euro EA 3000 analyzer and the data for C, H, N were within ± 0.4 of theoretical
values. Chromatographic separations were performed on silica gel columns by column
chromatography on silica gel (Kieselgel 60, 0.040–0.063 mm, Merck, Darmstadt, Germany) as
described by Still et al [30]. TLC analyses were performed on precoated silica gel on aluminum
sheets (Kieselgel 60 F , Merck).
2
54
4.1.1. 1-(2,6-Dimethylphenoxy)propan-2-amine (1a). The preparation of 1a was performed under
both traditional (method A) and microwaves conditions (method B) [31].
Method A

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Ammonium acetate (23.8 g, 310 mmol) and sodium cyanoborohydride (1.4 g, 21.7 mmol) were
added to a solution of 1-(2,6-dimethylphenoxy)propan-2-one (3a) (5.5 g, 31 mmol) in 40 mL of
MeOH. The reaction mixture was stirred for four days at room temperature. 6 M HCl was added
dropwise to the mixture reaction to destroy the excess hydride. The organic solvent was removed
under vacuum and the residue aqueous phase was made alkaline with NaOH pellets. Then, the
aqueous phase was extracted with AcOEt and the combined organic phases were dried (Na SO ).
2
4
The solvent was evaporated under reduced pressure to give the desired amine (1a) as a yellow oil
4.5 g, 81%). Spectroscopic data were in agreement with the literature [21]. Method B. In a
(
microwave tube, ammonium acetate (7.35 g, 95.5 mmol) and sodium cyanoborohydride (0.42 g, 6.7
mmol) were added to a solution of 1-(2,6-dimethylphenoxy)propan-2-one (3a) (1.7 g, 9.55 mmol)
in 15 mL of MeOH. The reaction mixture was stirred for 11 min at 110 °C in a microwave reactor
at 95 watt as power max. The processing of reaction mixture was carried out as reported for method
A, obtaining 1a in 72% yield. Spectrometric and spectroscopic data were in agreement with the
literature [21].
.
4
.1.2. 1-(2,6-Dimethylphenoxy)propan-2-amine hydrochloride (1a HCl). 1a was treated with
.
gaseous HCl to obtain the corresponding hydrochloride salt (1a HCl) as a white solid which was
1
recrystallized from EtOH/Et O to afford 2.9 g (40%) of white crystals: mp 215–216 °C; H NMR
2
(
500 MHz, d6-DMSO): δ 1.35 (d, J = 6.8 Hz, 2H, CH CH), 2.23 (s, 6H, CH Ar), 3.50–3.58 (m, 1H,
3 3
CH), 3.82 (d, J = 5.4 Hz, 2H, CH ), 6.91 (t, J = 6.8 Hz, 1H, Ar), 6.93 (t, J = 7.3 Hz, 1H, Ar), 7.00
2
+
3
13
(
d, J = 7.8 Hz, 2H, Ar), 8.50 (br s, 3H, NH ) ; C NMR (125 MHz, d6-DMSO): δ 15.5 (2C), 16.4
(1C), 47.3 (1C), 72.2 (1C), 124.7 (1C), 128.9 (2C), 129.3 (2C), 130.9 (2); 154.8 (1C); Anal.
(
C H NOCl) C, H, N.
1
1
18
4.1.3. 1-(2,6-Dimethylphenoxy)-3,3-dimethylbutan-2-amine (1b). Prepared as reported for 1a
1
(
Method B) starting from 3b. The synthetic pathway provided 1b as a yellow oil in 34% yield. H
NMR (500 MHz, CDCl ): δ 0.97 (s, 9H, (CH ) C), 1.99 (br s, 2H, NH ), 2.30 (s, 6H, CH Ar), 3.02
3
3 3
2
3
(dd, J = 9.3, 2.9 Hz, 1H, CHH), 3.63 (t, J = 9.3 Hz, 1H, CH), 3.84 (dd, J = 9.3, 2.9 Hz, 1H, CHH),

ACCEPTED MANUSCRIPT
1
3
6
1
.93 (t, J = 7.3 Hz, 1H, Ar) 7.02 (d, J = 7.3 Hz, 2H, Ar); C NMR (125 MHz, CDCl ): δ 15.3 (1C),
3
6.4 (1C), 26.5 (3C), 33.0 (1C), 60.5 (1C), 65.9 (1C), 123.9 (1C), 128.9 (2C), 130.9 (2C), 155.8
+
(
1C); GC-MS (70 eV) m/z (%) 221 (M , 1), 86 (100).
.
4
.1.4. 1-(2,6-Dimethylphenoxy)-3,3-dimethylbutan-2-amine hydrochloride (1b HCl). Obtained as
.
reported above for 1a HCl: yield: 71%; white crystals; mp: 215–216 °C (abs EtOH/Et O). Anal.
2
(
C H NOCl) C, H, N.
14 24
4.1.5. 2-(2,6-Dimethylphenoxy)-1-phenylethanamine (1c). Prepared as reported for 1a (Method B)
1
starting from 3c. The synthetic pathway provided 1c as a yellow oil in 26% yield. H NMR (500
MHz, CDCl ): δ 2.17 (s, 6H, CH Ar), 3.88–4.08 (m, 2H, CH ), 4.50–4.57 (m, 1H, CH), 6.88–7.03
3
3
2
1
3
(
m, 3H, Ar), 7.13–7.22 (m, 3H, Ar), 7.24–7.38 (m, 2H, Ar), 7.50 (d, J = 6.8 Hz, 2H, CH ); C
2
NMR (125 MHz, CDCl ): δ 15.3 (1C), 16.3 (1C), 21.5 (1C), 65.9 (1C), 124.2 (1C), 125.3 (1C),
3
1
1
4
26.8 (1C), 127.4 (1C), 128.2 (1C), 128.6 (1C), 128.7 (1C), 128.9 (1C), 129.0 (2C), 130.8 (1C),
+
37.9 (1C); GC-MS (70 eV) m/z (%) 241 (M , <1), 106 (100).
.
.1.6 2-(2,6-dimethylphenoxy)-1-phenylethanamine hydrochloride (1c HCl). Obtained as reported
.
above for 1a HCl: yield: 80%; white crystals; mp: 259–261 °C (abs EtOH/Et O); Anal.
2
(
C H NOCl) C, H, N.
16 20
.
4
.1.7. 1-(2,6-Dimethylphenoxy)-3-methylbutan-2-amine hydrochloride (1d HCl). A solution of 5d
(
2.5 g, 8.1 mmol) in anhydrous Et O (15 mL) was saturated with gaseous HCl and stirred at room
2
temperature for 15 min. Removal of the solvent under reduced pressure gave a solid (1.5 g, 75%)
which was recrystallized from EtOH/Et O: white crystals: mp 171–172 °C (abs EtOH/Et O); Anal.
2
2
.
(
C H NOCl 0.17H O) C, H, N.
13 22 2
4.1.8. 1-(2,6-Dimethylphenoxy)-3-methylbutan-2-amine (1d). Obtained as a yellow oil by extraction
1
from the corresponding hydrochloride salt. H NMR (500 MHz, CDCl ): δ 0.98 (d, J = 7.3 Hz, 3H,
3
CH CHCH ), 0.99 (d, J = 6.8 Hz, 3H, CH CHCH ), 1.73–1.82 (m, 1H, CHCH ), 1.88 (br s, 2H,
3
3
3
3
3
NH ), 2.30 (s, 6H, CH Ar), 2.98–3.08 (m, 1H, CHCH ), 3.64 (t, J = 8.8 Hz, 1H, CHHCH ), 3.77
2
3
2
2

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13
(
dd, J = 3.8, 8.8 Hz, 1H, CHHCH ), 6.92 (t, J = 7.8 Hz, 1H, Ar), 7.01 (d, J = 7.8 Hz, 2H, Ar); C
2
(
125 MHz, CDCl ): δ 15.3 (1C), 16.4 (1C), 18.2 (1C), 19.4 (1C), 30.8 (1C), 57.1 (1C), 65.9 (1C),
3
+
1
23.8 (1C), 128.9 (2C). 129.0 (1C), 130.1 (1C), 155.7 (1C); MS (70 eV) m/z (%) 207 (M , 3), 72
(100).
.
4
.1.9. 1-(2,6-Dimethylphenoxy)-3-phenylpropan-2-amine hydrochloride (1e HCl). Prepared as
.
.
reported for 1d HCl starting from 5e. The synthetic pathway provided 1e HCl as a white solid in
9
2
3
2% yield. The solid was recrystallized from abs EtOH/Et O to give white crystals (40%); mp:
2
1
18–219 °C. H NMR (300 MHz, CD OD): δ 2.21 (s, 6H, CH Ar), 3.02–3.24 (m, 2H, CH Ar),
3
3
2
1
3
.72–4.0 (m, 3H, CH CH), 6.95–7.05 (m, 3H, Ar), 7.25–7.45 (m, 5H, Ar); C NMR (100 MHz,
2
CD OD): δ 15.3 (2C), 35.4 (1C), 53.5 (1C), 70.0 (1C), 124.6 (1C), 127.4 (1C), 129.0 (4C), 129.1
3
(
2C), 130.5 (2C). 135.5 (1C), 154.6 (1C). Anal. (C H NOCl) C, H, N.
17 22
4.2. Pharmacology
4
4
.2.1. Details for functional studies.
.2.1.1. Guinea-Pig Atrial Preparations.
Female guinea pigs (300−400 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 CaCl , 1.0 MgCl , 0.4
2
2
NaH PO xH O, 11.9 NaHCO and 5.5 glucose. The physiological salt solution (PSS) was buffered
2
4
2
3
at pH 7.4 by saturation with 95% O -5% CO gas, and the temperature was maintained at 35 °C.
2
2
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, and hung vertically in a 15 mL organ bath containing the PSS
continuously bubbled with 95% O -5% CO gas at 35 °C, pH 7.4. The contractile activity was
2
2
recorded isometrically by means of a force transducer (FT 0.3, Grass Instruments Corporation,

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®
Quincy, MA, USA) using Power Lab software (AD-Instruments Pty Ltd, Castle Hill, Australia).
The left atria were stimulated by rectangular pulses of 0.6–0.8 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
length-tension curve was determined, and the muscle length was maintained which elicited 90% of
maximum contractile force observed at the optimal length. A stabilization period of 45–60 min was
allowed before the atria were used to test 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 activity of the
compounds (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 µM) dissolved in PSS. During the
construction of cumulative dose-response curves, the next higher concentration of the compounds
was added only after the preparation reached a steady state.
4.2.1.2. Alternating Current Induced Arrhythmia in Guinea Pig Left Atrial Preparations [32,33].
Left atria from guinea pigs were fixed to platinum wire electrodes and placed between two parallel
platinum field electrodes. The preparations were mounted vertically in a 15 mL organ bath
containing a modified Krebs-Henselait solution of the following composition (mM): 118 NaCl, 4.7
KCl, 2.5 CaCl , 1.2 MgSO , 1.2 NaH PO xH O, 24.9 NaHCO , and 10.1 glucose. The solution was
2
4
2
4
2
3
buffered at pH 7.4 by saturation with 95 % O -5 % CO gas, and the temperature was maintained at
2
2
35 °C. The preparations were connected to a force transducer. Isometric force contraction was
®
recorded using Power Lab software. Arrhythmias were induced by application of sinusoidal
alternating current (50 Hz) of increasing strength to the isolated heart preparations, 2 and 4 mA/
2
(
cm • s) to induce arrhythmia corresponding to an increase in field intensity of 0.3 V/(cm • s). The
2
current intensity (mA/cm ) at which extra beats occurred was called “threshold of ac-arrhythmia”
[
34]. The preparations were allowed to stabilize for 60 min. Then concentration-response curves for

ACCEPTED MANUSCRIPT
threshold of ac-arrhythmia were recorded by cumulative application of compounds to the bathing
solution every 30 min.
4
.2.1.3. Guinea-Pig Aortic Strips. The thoracic aorta was removed and placed in Tyrode solution
of the following composition (mM): 118 NaCl, 4.75 KCl, 2.54 CaCl , 1.20 MgSO , 1.19 KH PO ,
2
4
2
4
2
5 NaHCO and 11 glucose equilibrated with 95 % O -5 % CO gas at pH 7.4. The vessel was
3
2
2
cleaned of extraneous connective tissue. Two helicoidal strips (10 mm x 1 mm) were cut from each
aorta beginning from the end most proximal to the heart. Vascular strips were then tied with
surgical thread (6-0) and suspended in a jacketed tissue bath (15 mL) containing aerated PSS at 35
°
C. Aortic strips were subjected to a resting force of 1 g. Strips were secured at one end to a force
displacement transducer (FT 0.3, Grass Instruments Corporation) to monitor 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 containing 80 mM KCl (equimolar substitution
+
+
of K for Na ). After the contraction reached a plateau (about 45 min) the compounds (0.1, 0.5, 1, 5,
10, 50, and 100 µM) were added cumulatively to the bath allowing for any relaxation to obtain an
+
equilibrated level of force. Addition of the drug vehicle had no appreciable effect on K -induced
contraction (PSS for all compounds).
4.2.1.4. Statistical analysis. Data were analyzed using Student’s t-test and are presented as mean ±
S.E.M. Since the drugs were added in cumulative manner, the difference between the control and
the experimental values at each concentration were tested for a P value < 0.05. The potency of
drugs defined as EC50 and IC50 was evaluated from log concentration-response curves (Probit
analysis using Litchfield and Wilcoxon) [35] in the appropriate pharmacological preparations.
4.2.2. Details for motor coordination studies.

ACCEPTED MANUSCRIPT
4.2.2.1 Rota-rod test. The apparatus consisted of a base platform and a rotating rod of 3 cm
diameter with a non-slippery surface. The rod was placed at a height of 15 cm from the base. The
rod, 30 cm in length, was divided into five equal sections by six disks. Thus, up to five mice were
tested simultaneously on the apparatus, with a rod-rotating speed of 16 rpm. The integrity of motor
coordination was assessed on the basis of the number of falls from the rod in 30 s according to
Vaught et al. [36]
4
4
.2.3. Details for cytotoxicity studies.
.2.3.1. Cell cultures
HepG2 (Sigma-Aldrich, St. Louis, MO) cells were cultured in DMEM-Dulbecco’s modified
Eagle’s medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% (v/v) inactivated fetal
bovine serum (PAA Laboratories GmbH, Austria), 2 mM L-glutamine (Sigma-Aldrich, St. Louis,
MO), 100 µg/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO) at 37°C
in 5% CO . For experiments, the cells were grown to 70% confluence and then were treated with
2
test compounds in serum free media for the indicated times.
4.2.3.2. Cell viability assay.
HepG2 cell viability was assessed using a conventional MTT reduction assay [37]. This method is
based on the ability of viable cells to metabolize MTT, a water-soluble salt (yellow color), by
cellular oxidoreductase into a water-insoluble blue formazan product. Therefore, the amount of
4
formazan produced is proportional to the viable cells. Briefly, viable cells (10 /well) were plated
into sterile 96-Well Cell Culture Cluster (Corning, NY 14831) and incubated with different
concentrations (0.05–2 mM) of tested compounds for 2 h at 37 °C in 5% CO . At the end of
2
incubation, the culture medium was replaced by a solution of MTT 0.5 mg/mL (Sigma-Aldrich,
Milan, Italy) in PBS (Sigma-Aldrich, Milan, Italy). After 2 h incubation at 37 °C in 5% CO this
2

ACCEPTED MANUSCRIPT
solution was removed and 200 µl of DMSO was added to each well to dissolve the formazan
3
product. Absorbance values at 570 nm were measured using a plate reader Victor V (Perkin-Elmer)
and DMSO medium was used as blank solution. Results are expressed as the percentage of MTT
reduction respect to control cells. All experiments were carried out in sextuplicate and were
repeated twice.
4
4
.2.4. Details for P-gp interaction studies.
.2.4.1. Cell cultures
MDCK-MDR1 cells are a gift of Prof. P. Borst, NKI-AVL Institute, Amsterdam, Nederland.
MDCK cells were grown in DMEM high glucose supplemented with 10% fetal bovine serum, 2
mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, in a humidified incubator at 37 °C
with a 5 % CO atmosphere.
2
4.2.4.2. Calcein-AM experiment
These experiments were carried out as described by Feng et al. with minor modifications [38].
Each cell line (50,000 cells per well) was seeded into black CulturePlate 96/wells plate with 100 ꢀl
medium and allowed to become confluent overnight. 100 ꢀl of test compounds were solubilized in
culture medium and added to monolayers. 96/Wells plate was incubated at 37 °C for 30 min.
Calcein-AM was added in 100 ꢀl of Phosphate Buffered Saline (PBS) to yield a final concentration
of 2.5 ꢀM and plate was incubated for 30 min. Each well was washed 3 times with ice cold PBS.
Saline buffer was added to each well and the plate was read to Victor3 (PerkinElmer) at excitation
and emission wavelengths of 485 nm and 535 nm, respectively. In these experimental conditions
Calcein cell accumulation in the absence and in the presence of tested compounds was evaluated
and fluorescence basal level was estimated by untreated cells. In treated wells the increase of

ACCEPTED MANUSCRIPT
fluorescence with respect to basal level was measured. EC values were determined by fitting the
5
0
fluorescence increase percentage versus log [dose].
Acknowledgment. This work was accomplished thanks to the financial support of the Italian
Ministero dell’Istruzione, dell’Università e della Ricerca.
Supporting Information Available: Elemental analyses. Procedures for the synthesis of
intermediates.
References
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ACCEPTED MANUSCRIPT
R
a: R = Me
b: R = tBu
O
NH
2
: R = Ph
c
d: R = iPr
e: R = Bn
1
a-e
Figure 1. Structures of mexiletine (1a) and its analogues (1b–e).

ACCEPTED MANUSCRIPT
O
R
a: R = CH , X = Cl
3
O
O
O
R
ii, iii
NH .HCl
2
X
i
b: R = tBu, X = Br
c: R = Ph, X = Br
R
2a−c
3a−c
1a−c
Scheme 1. Reagents and conditions: (i) 2,6-xylenol, K CO , anhyd DMF, rt, 24h (for 2a); 2,6-
2
3
xylenol, K CO , MeOH, MW, 100 °C, 20 min (for 2b); 2,6-xylenol, NaOH, DMSO, MW, 60 °C,
2
3
1
5 min (for 2c); (ii) NaBH CN, AcONH , MeOH, rt (or MW, 110 °C, 11 min); (iii) anhyd Et O,
3
4
2
gaseous HCl.
R
R
R
R
O
O
i
ii
NH-tBoc
iii
NH .HCl
HO
2
HO
NH-tBoc
NH2
4d,e
5d,e
6d,e
1d,e
d: R = iPr
e: R = Bn
Scheme 2. Reagents and conditions: (i) Boc O, 1M NaOH, THF, rt; (ii) 2,6-xylenol, DIAD, PPh ,
2
3
anhyd THF, rt; (iii) anhyd Et O, gaseous HCl.
2

ACCEPTED MANUSCRIPT
Table 1. Antiarrhythmic Activity of Compounds 1a–e.
a
b
Compd
Max % increase
EC50
95% confidence
–
6
(
mean ± SEM)
(µM)
limit (x 10 )
8.71–13.5
1.38–3.20
0.34–0.54
14.9–22.7
1.47–6.38
1
1
a
64 ± 1.4
1
2
0
1
4
1.6
.11
.43
8.4
.36
c
b
71 ± 2.4
d
1
c
135 ± 2.2
1
d
145 ± 4
c
1
e
169 ± 4
a
Increase of threshold of ac-arrhythmia: increase in the intensity of 50 Hz alternating current required to
–4
produce arrhythmia in guinea pig left atria driven at 1 Hz in the presence of each tested compounds at 10 M.
For all data P < 0.05. Calculated from log concentration-response curves (Probit analysis according to
b
Litchfield and Wilcoxon [35] with n = 6−8). When the maximum effect was < 50 %, the EC values were not
50
c
−5
d
−6
calculated. At 10 M. At 5x10 M.
Table 2. Influences of Tested Compounds on Cardiovascular Parameters.
Left atrium
Right atrium
negative inotropy
negative inotropy
negative chronotropy
9
5%
95%
95%
a
b
c
b
d
b
Activity
EC50
Activity
EC50
Activity
EC50
Compd
confidence lim
confidence lim
confidence lim
(% ± SEM)
(µM)
(% ± SEM)
(µM)
(% ± SEM)
(µM)
-
6
-6
-6
(
x10 )
(x10 )
(x10 )
*
*g
1
1
a
90 ± 1
0.045
0.042
0.029
0.021
0.012
0.035–0.058
0.027–0.060
0.017–0.052
0.012–0.037
0.010–0.015
85 ± 3
0.014
0.009–0.023
*
i
b
92 ± 1
77 ± 1
1.02
8.2
0.85–3.05
4.6–10.4
7.0 ± 0.3
*
e
h
i
1
c
94 ± 2
84 ± 2
54 ± 2
11 ± 1
*f
*f
1
d
55 ± 2
13.1
10.1–15.0
*
g
*
1
e
79 ± 2
57 ± 3
0.066
0.048–0.091
24 ± 2
a
–5
Decrease in developed tension on isolated guinea-pig left atrium at 10 M, expressed as percentage change from the
–5
control (n = 5–6). The left atria were driven at 1 Hz. The 10 M concentration gave the maximum effect for most
compounds. Calculated from log concentration-response curves (Probit analysis by Litchfield and Wilcoxon [35]
b
with n = 6–7). When the maximum effect was < 50%, the EC50 inotropic, EC50 chronotropic and IC50 vasorelaxant
c
values were not calculated. Decrease in developed tension on guinea-pig spontaneously beating isolated right atrium
-
6
–6
at 5x10 M, expressed as percent changes from the control (n = 7–8). The 5x10 M concentration gave the maximum
d
–5
effect for most compounds. Decrease in atrial rate on guinea-pig spontaneously beating isolated right atria at 10 M,
expressed as the percentage change from the control (n = 7–8). The 10 M concentration gave the maximum effect
for most compounds. The pretreatment heart rate ranged from 170 to 195 beats/min. At 5x10 M. At 10 M. At 10
M. At 10 M. P > 0.05. An asterisk indicates P < 0.05.
–5
e
–6
f
–4
g
–
7
h
–5
i

ACCEPTED MANUSCRIPT
+
Table 3. Relaxant Activity of Compounds on K -Depolarized Guinea Pig Vascular and non-
vascular Smooth Muscle.
Aorta
Ileum
a
a
b
Activity
Activity
IC50
95% confidence lim
compd
-
6
(
% ± S.E.M.)
5.0 ± 0.3
38 ± 2
(% ± S.E.M.)
(µM)
8.5
(x10 )
6.5–11.1
9.0–12.8
10.8–17.5
12.0–25.0
2.8–4.6
1
a
81 ± 2
c
1
b
73 ± 2
10.7
13.8
19.6
3.6
d
1
c
31 ± 2
75 ± 3
1
d
11 ± 1
88 ± 1
d
1
e
33 ± 1
73 ± 2
a
+
Percent inhibition of calcium-induced contraction on K -depolarized (80 mM) guinea pig aortic strips and
–
4
–4
longitudinal smooth muscle at 10 M. The 10 M concentration gave the maximum effect for most
compounds. Calculated from log concentration-response curves (Probit analysis by Litchfield and Wilcoxon
35] with n = 6–7). When the maximum effect was < 50%, the IC50 values were not calculated. At 5x10 M.
At 10 M.
b
c
–5
[
d
–5
Table 4. Effect of mexiletine and its analogues on motor coordination expressed as number of falls
a
from Rota-rod.
Falls from the rota-rod
–
1
Treatment
Dose, mg kg ip
Before treatment
After treatment
3
0 min
45 min
60 min
Saline
4.4 ± 1.1
2.8 ± 0.5
2.4 ± 0.3
4.6 ± 0.5
4.7 ± 0.4
4.3 ± 0.5
4.5 ± 0.6
4.8 ± 0.7
4.5 ± 0.6
4.5 ± 0.9
2.5 ± 0.7
1.3 ± 0.4
3.8 ± 0.3
2.7 ± 0.4
3.1 ± 0.3
2.4 ± 0.3
3.2 ± 1.6
2.6 ± 0.8
3.0 ± 1.4
2.9 ± 0.8
1.2 ± 0.5
0.8 ± 0.3
3.5 ± 0.2
1.7 ± 0.4
1.8 ± 0.5
1.8 ± 0.4
2.8 ± 0.7
2.2 ± 0.6
2.1 ± 1.3
1.9 ± 0.5
0.9 ± 0.3
-
b
1
1
1
1
1
1
1
1
1
a
a
b
b
c
25
50
25
50
25
50
25
50
25
50
b
-
0.8 ± 0.2
1.2 ± 0.6
1.3 ± 0.3
1.0 ± 0.2
1.1 ± 0.3
1.0 ± 1.2
1.2 ± 0.5
1.3 ± 1.1
c
d
d
e
*
*
1
e
4.2 ± 0.8
4.4 ± 1.1
3.5 ± 0.9
a
b
Each value represents the mean of 8 mice. Ref.[16] *P < 0.05 in comparison with saline-treated mice.

ACCEPTED MANUSCRIPT
Table 5. P-gp interaction of mexiletine (1a) and its analogues (1b–e).
a
Compd
EC50 ± SEM, µM
P-gp
b,c
1
1
a
NA
b
b
NA
1
c
27 ± 5
b
1
d
NA
1
e
8.4 ± 1.5
a
The value is the mean of three different
b
experiments, samples in duplicate; NA =
No activity at 100µM; Ref. [31].
c

ACCEPTED MANUSCRIPT
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•
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Search for new antiarrhythmic agents.
Antiarrhythmic effects evaluation of mexiletine analogues.
Identification of potent and selective antiarrhythmic agents.