Constrained analogues of tocainide as potent skeletal muscle sodium channel blockers towards the development of antimyotonic agents

Article abstract of DOI:10.1016/j.ejmech.2008.01.023

1-Benzyl-N-(2,6-dimethylphenyl)piperidine-3-carboxamide and 4-benzyl-N-(2,6-dimethylphenyl)piperazine-2-carboxamide, two conformationally restricted analogues of tocainide, were designed and synthesized as voltage-gated skeletal muscle sodium channel blockers. They showed, with respect to tocainide, a marked increase in both potency and use-dependent block.

Full text of DOI:10.1016/j.ejmech.2008.01.023

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2535e2540
http://www.elsevier.com/locate/ejmech
Original article
Constrained analogues of tocainide as potent skeletal muscle sodium
channel blockers towards the development of antimyotonic agents
Alessia Catalano a, Alessia Carocci a, Filomena Corbo a, Carlo Franchini a,
*
,
Marilena Muraglia a, Antonio Scilimati a, Michela De Bellis b, Annamaria De Luca b,
Diana Conte Camerino b, Maria Stefania Sinicropi c, Vincenzo Tortorella a
a Department of Medicinal Chemistry, University of Bari, via E. Orabona n. 4, 70125 Bari, Italy
b Department of Pharmacology, University of Bari, via E. Orabona n. 4, 70125 Bari, Italy
c Department of Pharmaceutic Sciences, University of Calabria, Arcavacata di Rende (CS) 87036, Italy
Received 11 December 2007; received in revised form 15 January 2008; accepted 16 January 2008
Available online 31 January 2008
Abstract
1-Benzyl-N-(2,6-dimethylphenyl)piperidine-3-carboxamide and 4-benzyl-N-(2,6-dimethylphenyl)piperazine-2-carboxamide, two conforma-
tionally restricted analogues of tocainide, were designed and synthesized as voltage-gated skeletal muscle sodium channel blockers. They
showed, with respect to tocainide, a marked increase in both potency and use-dependent block.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: Sodium channel blockers; Myotonia; Benzylation; Tocainide
1. Introduction
human skeletal muscle sodium channel Nav1.4 that prevent
the normal fast inactivation of sodium channel [7e9]. Cur-
rently, tocainide is among the few drugs clinically used for
the symptomatic treatment of muscle hyperexcitability in
myotonic syndromes. However, its use as antimyotonic is hin-
dered by unwanted adverse side-effects [10]. Thus, there is
a need to develop new safer use-dependent sodium channel
blockers with an improved pharmacological profile. A few
years ago, a comprehensive model of the sodium channel
was reported, showing that the increase in lipophilicity and
molecular size of antiarrhythmic drugs can reinforce hydro-
phobic interactions with the binding site during use-dependent
block [11,12]. As a part of our program aimed at developing
new antimyotonic drugs using tocainide as a ‘‘lead com-
pound’’, a series of tocainide analogues were designed with
the purpose of identifying novel potent voltage- and use-de-
pendent skeletal muscle sodium channel blockers with very
high affinity constants for the inactivated channels [13,14].
In particular, potency and use-dependent behaviour were
found to be strongly increased by constraining the amino ter-
minal group of 1a in both a rigid alpha- and beta-proline cycle
Sodium channels are involved in many cellular functions
and are altered in many pathological conditions, as in the
channelopathies. Therefore, drugs targeting sodium channels
constitute important therapeutic interventions for a number
of diseases. Among these, tocainide (1a, Fig. 1), a well-known
sodium channel blocker, is a class Ib antiarrhythmic drug once
used in the treatment of symptomatic life-threatening ventric-
ular arrhythmias [1,2]. It has also a marked analgesic effect in
trigeminal neuralgia in humans [3,4] and antinociceptive effect
in rats [5]. Furthermore, tocainide has been proposed as a clin-
ically useful antimyotonic drug being able to block sodium
channels in a use-dependent manner, i.e., with an increased
potency in condition of high-frequency discharges of action
potentials [6]. Myotonic syndromes are hereditary disorders
of the skeletal muscle caused by missense mutations in the
* Corresponding author. Tel.: þ39 080 5442743; fax: þ39 080 5442724.
E-mail address: cfranc@farmchim.uniba.it (C. Franchini).
0223-5234/$ - see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.01.023

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A. Catalano et al. / European Journal of Medicinal Chemistry 43 (2008) 2535e2540
H
N
NHR
O
1a (R = H, tocainide)
1b (R = Bn)
X
H
N
H
N
H
N
N
N
N
R
R
R
O
O
O
2a (R = H)
2b (R = Me)
2c (R = Bn)
3a (R = H)
4a (R = H, X = CH2)
4b (R = Bn, X = CH2)
5a (R = H, X = NH)
5b (R = Bn, X = NH)
3b (R = Bn)
Fig. 1. Structures of tocainide analogues.
(2a and 3a, Fig. 1). A further improvement was still achieved
by introducing a benzyl on the amino group of proline-derived
compounds (2c and 3b) [15,16]. To confirm the importance of
both molecular rigidity and presence of the benzyl group, we
designed and synthesized two compounds, 4 and 5, having
a chemical structure that combines alpha- and beta-proline
features (2 and 3) in a six-membered ring.
Thus, the two tocainide derived compounds were designed
with the aim to merge the characteristics of 2c and 3b, and
with the consideration that several local anaesthetics currently
used in therapy, such as mepivacaine, bupivacaine and ropiva-
caine, are substituted 2-piperidine carboxanilides, structurally
related to our compounds [18] and that 2-piperazine carboxa-
nilides were already investigated as anaesthetic and antiar-
rhythmic drugs [19,20].
The introduction of an N-benzyl versus the more classic N-
alkyl was designed as an attempt to investigate the effect of
the presence of a second aromatic moiety in the molecule,
which could establish specific additional hydrophobic interac-
tions with the binding site in the protein. As we have previ-
ously reported [16], lipophilicity seems to affect the tonic
block; in fact, all the N-alkyl derivatives showed higher tonic
block values than their corresponding unsubstituted precur-
sors. This, in turn, is related to the increase of the block
potency of the channel. Recently, docking studies [17] on to-
cainide (1a) and N-benzyltocainide (1b) showed that the ab-
sence of an alkyl linked to the amino group of the tocainide
led to the reduction of the van der Waals interactions with
the side chain of Phe1579. In fact, the benzyl group in 1b
adopts an off-centered parallel orientation relative to the aro-
matic ring of the side chain of Phe1579. The additional energy
of interaction explains the higher binding affinity of 1b than
tocainide (1a). We found that N-benzyltocainide analogues
2c and 3b showed a remarkable increase of potency, 3b being
the most active, also with respect to the corresponding N-
methyl derivative [15]. Thus, to better clarify the role of the
amino group as a pharmacophore in the drugesodium channel
interaction and confirm our previous findings, we designed
two anilide derivatives, 4b and 5b that simultaneously resem-
ble tocainide and alpha- and beta-proline. This has been ac-
complished by including the pharmacophore amino group
into N-benzyl substituted piperidine or piperazine rings.
2. Chemistry
Compounds 4a,b and 5a,b were synthesized as depicted in
Scheme 1. N-t-Boc derivatives 8 and 9 were prepared by react-
ing 6 (or 7) and 2-tert-butoxycarbonylimino-2-phenylacetoni-
trile (Boc-ON) in a mixture of dioxane/water in the presence
of Et3N. Then, 8 (or 9) was reacted with 2,6-dimethylaniline
in the presence of IIDQ (2-isobutoxy-1-isobutoxycarbonyl-
1,2-dihydroquinoline) to afford the corresponding carboxa-
mides 4c and 5c, which were, in turn, deprotected by treatment
with 3 N HCl or 48% HBr to give 4a and 5a, respectively. The
free amines were converted into their N-benzyl derivatives 4b
and 5b by reacting 4a and 5a with benzyl bromide. We attrib-
uted the preferential benzylation of the nitrogen atom in the
beta position of 5a to several factors: the sterical hindrance
of the xylididic moiety; the electron-withdrawing effect of
the same moiety that determines a reduction of basicity of
the alpha nitrogen atom and, finally, the possibility of the
same nitrogen atom to be involved in a five-membered
pseudo-cycle with the carbonyl oxygen.
3. Pharmacology
The effects of the newly synthesized N-benzyl analogues of
tocainide in which the asymmetric carbon atom is constrained

A. Catalano et al. / European Journal of Medicinal Chemistry 43 (2008) 2535e2540
2537
X
X
X
a
b
H
N
HO
N
HO
N
N
H
t-Boc
t-B o c
O
O
O
4c (X = CH2)
6 (X = CH2)
7 (X = NH)
8 (X = CH2)
5c (X = Nt-Boc)
c
9 (X = Nt-Boc)
X
X
H
H
N
N
H
N
N
d
Bn
O
O
4b (X = CH2)
5b (X = NH)
4a (X = CH2)
5a (X = NH)
Scheme 1. Reagents and conditions: (a) Boc-ON, Et3N, dioxane/water, rt; (b) 2,6-dimethylaniline, IIDQ, Et3N, CHCl3, reflux; (c) 48% HBr or 3 N HCl, EtOAc, rt;
(d) BnBr, dioxane/water, reflux.
in a piperidine or piperazine ring were then evaluated on Naþ
currents (INa) of native frog skeletal muscle fibers. Naþ current
(INa) measurements executed in the presence of 4a or 5a were
not performed because the corresponding compounds in the
set of alpha- and beta-proline (2a and 3a, respectively) were
less active than the corresponding N-benzyl derivatives (2c
and 3b) in use-dependent block.
synthesis of constrained analogues of tocainide has been pro-
posed. The synthetic routes herein described, though not in-
novative and with not very high overall yields, gave access
to 4b and 5b which present themselves as valuable pharma-
cological tools, both being more potent and use-dependent
than tocainide. In particular, 5b showed a marked increase
in both potency and use-dependence of action; compared to
tocainide, it was 18- and 114-fold more potent in tonic and
phasic block experiments, respectively. In conclusion, the
candidates 4b and 5b will be subjected to a complete pharma-
cological characterization as antimyotonic agents possibly
overcoming tocainide’s adverse side-effects. Further in vivo
studies on the compounds 4b and 5b will be performed.
Moreover, given that a stereoselective site for sodium channel
blockers on adult skeletal muscle fibers has been evidenced
[14], we have recently started the synthesis of the optical iso-
mers of 4b and 5b; further studies are in progress to find
a correlation between potency, stereoselectivity and confor-
mational characteristics of the compounds presented in this
paper.
4. Results and discussion
The research herein described is based upon our previous
works dealing with tocainide derivatives with enhanced po-
tency and selectivity for use-dependent inhibition of the skel-
etal muscle Nav1.4 channel. In particular, additional tonic
potency was attained by introduction of the hydrophobic ben-
zyl group, whereas ring constraint (proline ring) affected both
potency and use-dependence of channel block. The present
work shows that similar potency and use-dependence inhibi-
tion values are still possible by expanding the proline, from
five- to a six-membered ring, also incorporating an additional
nitrogen atom. In fact, by constraining the stereogenic center
of tocainide in a rigid piperidine and piperazine cycle, as in
4b and 5b, respectively, a marked increase of the potency
for producing both tonic and use-dependent blocks of INa
was obtained. Tonic block IC50 values of the two anilide de-
rivatives 4b and 5b were similar being about 15- and 18-fold
more potent than tocainide at ꢀ100 mV, respectively. During
the 10 Hz stimulation, the potency of both compounds in-
creased. Indeed, the IC50 values for use-dependent block of
4b and 5b were about 61- and 114-fold lower, respectively
(Table 1), than tocainide. In particular, a very high increase
in potency for use-dependent block was observed with the
5b analogue, with a ratio (IC50 tonic block/IC50 use-depen-
dent block at 10 Hz ¼ 31.6:2.4) of 13. In summary, the
Table 1
Concentrations for half-maximal tonic and phasic (use-dependent) blocks
(IC50, mM) of sodium currents, potency ratio (IC50 tocainide/IC50 derivatives),
clog P and pKa of tocainide (1a) and its analogues (4b,5b)
Compound
Tonic blocka (IC50
)
Phasic blockb (IC50
)
clog Pc
1a (Tocainide)
580.7 ꢁ 37.9
38.5 ꢁ 1.4
31.6 ꢁ 0.8
273.3 ꢁ 23.3
4.5 ꢁ 0.3
0.76 ꢁ 0.48
3.53 ꢁ 0.34
2.81 ꢁ 0.46
4b
5b
a
2.4 ꢁ 0.4
Tonic block: block of sodium channel at resting conditions, evaluated dur-
ing infrequent depolarizing pulses.
b
Phasic block: cumulative sodium current reduction by the drug at 10 Hz
stimulation frequency, obtained by concentrationeresponse curves.
c
Calculated using Advanced Chemistry Development (ACD) Software So-
laris V4.76.

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A. Catalano et al. / European Journal of Medicinal Chemistry 43 (2008) 2535e2540
5. Experimental protocols
5.1. Chemistry
EtOAc/petroleum ether 2:8) of the residue gave 0.39 g (17%
yield) of 4c as a white solid: mp: 59e61 ꢂC; FT-IR (KBr):
3421, 2988, 2944, 2860, 1675, 1488, 1467, 1368, 1300,
1270, 1149, 957, 989, 858 cmꢀ1
;
1H NMR: d ¼ 1.45 (s,
Yields refer to purified products and were not optimized. All
chemicals were purchased from SigmaeAldrich or Lancaster
in the highest quality commercially available. The structures
of the compounds were confirmed by routine spectrometric
analyses. For compounds not previously described, complete
spectroscopic characterization is given; for known compounds
only spectra not described in the literature are given. Melting
points were determined on a Gallenkamp melting point appara-
tus in open glass capillary tubes and are uncorrected. 1H NMR
and 13C NMR spectra were recorded on a Varian VX Mercury
9H, partially overlapped to multiplet at 1.42e1.60 ppm, t-
Bu), 1.42e1.60 (m, 1H, partially overlapped to singlet at
1.45 ppm), 1.64e1.75 (m, 1H), 1.84e1.90 (m, 1H), 2.18 (s,
6H, CH3eAr), 2.40e2.60 (m, 1H), 2.95e3.15 (m, 1H),
3.25e3.40 (m, 1H), 3.65e3.85 (m, 1H), 3.95e4.10 (m,
2H), 6.95e7.10 (m, 3H, Ar), 7.35 ppm (br s, 1H: exchange
with D2O, NH ); 13C NMR: d ¼ 18.6 (2C), 24.4 (1C), 28.0
(2C), 28.6 (3C), 43.2 (1C), 44.7 (1C), 46.3 (1C), 80.3 (1C),
127.5 (1C), 128.4 (3C), 135.6 (1C), 155.2 (1C), 171.8 ppm
(1C);
GCeMS
(70 eV)
m/z
(rel.
int.):
332
spectrometer, operating at 300 and 75 MHz for H and 13C,
[Mþ, 1], 121 (100).
1
respectively, using CDCl3 as solvent. Chemical shifts are
reported in parts per million (ppm) relative to solvent reso-
nance: d 7.26 (1H NMR) and d 77.3 (13C NMR). Absolute
values of J are given in hertz. EIMS spectra were recorded
on a HewlettePackard 6890-5973 MSD gas chromatograph/
mass spectrometer at low resolution. Elemental analyses
were performed with a Eurovector Euro EA 3000 analyzer.
Flash chromatography was performed on silica gel (Kieselgel
60, 0.040e0.063 mm, Merck, Darmstadt, Germany) packed
columns as described by Still et al. [21]. TLC analyses were
performed on precoated silica gel on aluminum sheets (Kiesel-
gel 60 F254, Merck). PLC analyses were performed on pre-
coated silica gel on glass plates (Kieselgel 60 F254, Merck).
5.1.4. Di-tert-butyl 2-{[(2,6-dimethylphenyl)amino]-
carbonyl}piperazine-1,4-dicarboxylate (5c)
Prepared as reported above for the synthesis of 4c, but start-
ing from 9. Yield 51%; white crystals, mp: 189e190 ꢂC
(EtOAc/petroleum ether); FT-IR (KBr): 3273, 2979, 2885,
1675, 1478, 1403, 1366, 1250, 1169, 1111, 1041, 974, 868,
1
764 cmꢀ1; H NMR: d ¼ 1.44 (s, 9H, t-Bu), 1.50 (s, 9H, t-
Bu), 2.19 (s, 6H, CH3eAr), 2.95e3.40 (m, 3H), 3.80e4.10
(m, 2H), 4.50e4.85 (m, 2H), 6.95e7.15 (m, 3H, Ar),
7.39 ppm (br s, 1H, NH ); 13C NMR: d ¼ 18.7 (2C), 28.5
(6C), 43.3 (2C), 60.6 (2C), 80.6 (1C), 81.9 (1C), 127.7 (2C),
128.5 (3C), 135.5 (1C), 154.8 (2C), 168.0 ppm (1C); GCe
MS (70 eV) m/z (rel. int.): 333 [Mþ ꢀ 100, 1], 85 (100).
5.1.1. 1-(tert-Butoxycarbonyl)piperidine-3-
carboxylic acid (8)
5.1.5. N-(2,6-Dimethylphenyl)piperidine-
3-carboxamide (4a)
Yield 75%; white solid, mp: 160e162 ꢂC, lit [22] 149e
151 ꢂC (abs EtOH/H2O); GCeMS (70 eV): m/z (rel. int.):
229 [Mþ, 10], 57 (100); 13C NMR: d ¼ 24.3 (1C), 27.4 (1C),
28.6 (3C), 41.3 (1C), 44.1 (1C), 45.7 (1C), 80.2 (1C), 155.0
(1C), 179.1 ppm (1C). Other spectroscopic data were in agree-
ment with the literature [22].
To a solution of 4c (0.40 g, 1.2 mmol) in EtOAc (9.5 mL),
3 N HCl (3.1 mL) was added. The reaction mixture was stirred
at room temperature for 3 h. The solvent was removed under
reduced pressure to give a white solid (0.41 g) which was re-
crystallized from EtOH/Et2O to afford 0.21 g (65% yield) of
the desired amine hydrochloride (4a$HCl): mp: 199e201 ꢂC
(abs EtOH/Et2O), lit [18] mp: 196e210 ꢂC (abs EtOH/
Et2O). Calcd for C14H20N2O$HCl$0.17H2O % C 61.87, H
7.91, N 10.31; found C 62.14, H 7.96, N 10.34. Compound
4a as free amine was recovered by extraction of the corre-
sponding hydrochloride: FT-IR (CHCl3): 3421, 2988, 2944,
2860, 1675, 1488, 1467, 1368, 1300, 1270, 1149, 957, 989,
5.1.2. 1,4-Bis(tert-butoxycarbonyl)piperazine-2-
carboxylic acid (9)
Yield 75%; white solid, mp: 148e150 ꢂC, lit [23] 143.0e
144.5 ꢂC for the (S )-enantiomer; 13C NMR: d ¼ 28.4 (6C),
40.3 (1C), 41.6 (1C), 53.6 (1C), 54.9 (1C), 80.9 (1C), 81.3
(1C), 154.8 (1C), 155.4 (1C), 174.3 ppm (1C). Other spectro-
scopic data were in agreement with those reported for the (S )-
enantiomer [23].
858 cmꢀ1
;
1H NMR: d ¼ 1.51e1.62 (m, 1H, NH ), 1.72e
2.04 (m, 3H), 2.19 (s, 6H, CH3eAr), 2.46e3.04 (m, 5H),
3.10e3.24 (m, 1H), 7.03 (s, 3H, Ar), 9.53 ppm (br s, 1H,
NHeCO); 13C NMR: d ¼ 18.8 (2C), 23.4 (1C), 27.5 (1C),
42.5 (1C), 46.6 (1C), 48.4 (1C), 127.4 (1C), 127.8 (2C),
128.6 (2C), 135.2 (1C), 173.7 ppm (1C); GCeMS (70 eV)
m/z (rel. int.): 232 [Mþ, 20], 84 (100).
5.1.3. tert-Butyl 3-{[(2,6-dimethylphenyl)amino]carbonyl}-
piperidine-1-carboxylate (4c)
IIDQ (2.46 mL, 8.3 mmol), 2,6-dimethylaniline (0.93 mL,
7.6 mmol) and Et3N (1.4 mL, 10.4 mmol) were successively
added to a stirring solution of 8 (1.60 g, 6.9 mmol) in
CHCl3 (220 mL). The reaction mixture was heated under re-
flux for 6 h. The solvent was removed under reduced pres-
sure and the residue, taken up with EtOAc, was washed
three times with 2 N HCl, twice with 2 N NaOH, and then
dried over anhydrous Na2SO4. Flash chromatography (eluent
5.1.6. N-(2,6-Dimethylphenyl)piperazine-2-
carboxamide (5a)
To a solution of 5c (0.90 g, 2.1 mmol) in EtOAc (12 mL),
48% HBr (48 mL) was added. The reaction mixture was
stirred at room temperature for 3 h. The solvent was

A. Catalano et al. / European Journal of Medicinal Chemistry 43 (2008) 2535e2540
2539
evaporated under reduced pressure and the aqueous phase was
5.1.8. 4-Benzyl-N-(2,6-dimethylphenyl)piperazine-
2-carboxamide (5b)
washed with EtOAc, made alkaline with 2 N NaOH and
extracted with EtOAc. The organic phase was dried over an-
hydrous Na2SO4. The solvent was removed under pressure to
give 5a (0.22 g, 45% yield) as a white solid: mp: 172e
174 ꢂC, lit [19] 171e172 ꢂC (CHCl3/CCl4); FT-IR (KBr):
3251, 3200, 3021, 2932, 2850, 2805, 1657, 1594, 1531,
It was obtained as reported above for the preparation of 4b,
but starting from 5a. Compound 5b was obtained as a yellow
oil containing also the dibenzylderivative of 5a, which were
separated by preparative chromatography (PLC) using silica
gel on glass plates (eluent EtOAc/petroleum ether 1:1) to
give 5b in 21% yield as a slightly yellowish oil: FT-IR
(neat): 3356, 3201, 3066, 3031, 2983, 2942, 2822, 1676,
1601, 1497, 1445, 1373, 1303, 1248, 1145, 1047, 1028, 846,
1
1477, 1224, 1118, 897, 769, 759 cmꢀ1; H NMR: d ¼ 1.95
(s, 2H: exchange with D2O, NH ), 2.21 (s, 6H, CH3eAr),
2.74e3.14 (m, 5H), 3.28 (dd, J ¼ 3.1 and 12.1 Hz, 1H),
3.55 (dd, J ¼ 3.1 and 8.0 Hz, 1H), 7.07 (s, 3H, Ar),
8.50 ppm (br s, 1H: exchange with D2O, NHeCO); 13C
NMR: d ¼ 18.9 (2C), 45.5 (1C), 46.5 (1C), 49.5 (1C), 59.8
(1C), 127.3 (3C), 128.4 (2C), 135.2 (1C), 170.8 ppm (1C);
GCeMS (70 eV) m/z (rel. int.): 233 [Mþ, 1], 85 (100). Com-
pound 5a was purified by recrystallization of the correspond-
ing hydrochloride salt, obtained by adding to the free amine
a few drops of aq. HCl and then, removing the water azeo-
tropically (toluene/abs EtOH): mp >250 ꢂC (abs EtOH/
Et2O). Calcd for C13H19N3O$2HCl$1.5H2O% C 46.85, H
7.26, N 12.61; found C 46.84, H 7.24, N 12.68.
1
792 cmꢀ1; H NMR: d ¼ 1.93 (br s, 1H: exchange with D2O,
NH ), 2.21 (s, 6H, CH3eAr), 2.31e2.44 (m, 1H), 2.50e2.66
(m, 2H), 2.88e3.02 (m, 2H), 3.04e3.16 (m, 1H), 3.54 (2d,
J ¼ 13.0 Hz, 2H, benzylic protons), 3.65 (dd, J ¼ 7.1 and
3.6 Hz, 1H), 7.02e7.10 (m, 3H, Ar), 7.22e7.34 (m, 5H, Ar),
8.55e8.64 ppm (br s, 1H: exchange with D2O, NHeCO);
13C NMR: d ¼ 18.9 (2C), 44.2 (1C), 53.6 (1C), 56.2 (1C),
58.9 (1C), 63.5 (1C), 127.3 (2C), 127.5 (2C), 128.4 (2C),
128.6 (2C), 129.4 (2C), 135.3 (1C), 137.7 (1C), 170.8 ppm
(1C); GCeMS (70 eV) m/z (rel. int.): 223 [Mþ ꢀ 100, 1], 91
(100). Compound 5b$HCl was obtained as reported above
for 4b$HCl in 17% yield: mp: 241e243 ꢂC (abs EtOH/
Et2O). Calcd for C20H25N3O$HCl$1.67H2O % C 61.61, H
7.58, N 10.78; found C 61.60, H 7.55, N 10.79.
5.1.7. 1-Benzyl-N-(2,6-dimethylphenyl)piperidine-
3-carboxamide (4b)
To a stirring solution of 4a (0.15 g, 0.65 mmol) in dioxane
(8 mL), a solution of K2CO3 (0.26 g, 1.88 mmol) in H2O
(8 mL) was added. The reaction mixture was heated to
70 ꢂC, and then, benzyl bromide (0.09 mL, 0.76 mmol) was
added dropwise. The heating was continued for 45 min.
Then, the dioxane was removed under reduced pressure and
the aqueous residue was taken up with EtOAc and extracted
with 2 N HCl. The aqueous phase was made alkaline with
2 N NaOH and extracted twice with EtOAc. The combined or-
ganic layers were dried over anhydrous Na2SO4 and concen-
trated under vacuum to give 90 mg (43% yield) of 4b as
a white solid: mp: 133e135 ꢂC; FT-IR (KBr): 3246, 3029,
2935, 2920, 2848, 2804, 1649, 1516, 1469, 1364, 1223,
5.2. Pharmacology
5.2.1. Recording of Naþ current and pulse protocols
The actions of the two anilide derivatives of tocainide 4b
and 5b were tested in vitro on sodium currents (INa) of single
fibers of frog semitendinosus muscle by vaseline-gap voltage
clamp method, as described in detail elsewhere [24]. The tonic
block (TB) exerted by each compound was calculated as per-
centage reduction of the maximal peak sodium transient
(INa max) elicited by infrequent depolarizing steps to ꢀ20 mV
from the holding potential (h.p.) of ꢀ100 mV at a frequency
of 0.3 Hz. The use-dependent block exerted by each drug
was evaluated by using trains of 10 ms test pulses, from the
h.p. to ꢀ20 mV at 10 Hz frequency for 30 s and then normal-
izing the residual current at the end of the stimulation protocol
with respect to the current in the absence of drug.
1
1071, 1010, 768, 739, 699 cmꢀ1; H NMR: d ¼ 1.55e1.80
(m, 2H), 1.90e2.25 (m, 2H, partially overlapped to a singlet
at 2.15 ppm), 2.15 (s, 6H, overlapped to a multiplet at 1.90e
2.25 ppm, CH3eAr), 2.30e2.55 (m, 2H), 2.65e2.80 (m,
1H), 2.82e3.00 (m, 1H), 3.05e3.25 (m, 1H), 3.55 (s, 2H, ben-
zylic protons), 7.08 (s, 3H, Ar), 7.16e7.32 (m, 5H, Ar), 9.50e
9.80 ppm (br s, 1H: exchange with D2O, NH ); 13C NMR:
d ¼ 18.9 (2C), 22.9 (1C), 26.9 (1C), 42.1 (1C), 54.0 (1C),
55.3 (1C), 63.8 (1C), 127.0 (2C), 127.8 (2C), 128.4 (2C),
128.7 (2C), 129.6 (2C), 135.3 (1C), 137.3 (1C), 173.7 ppm
(1C); GCeMS (70 eV) m/z (rel. int.): 322 [Mþ, 23], 91
(100). Compound 4b was transformed into its hydrochloride
salt by treatment with a few drops of 2 N HCl and then remov-
ing the water azeotropically (toluene/abs EtOH). The white
solid so obtained (4b$HCl) was recrystallized from EtOH/
Et2O to afford 65 mg (67% yield) of white crystals: mp:
235e237 ꢂC (abs EtOH/Et2O). Calcd for C21H26N2O$HCl$
0.25H2O % C 69.41, H 7.63, N 7.71; found C 69.42,
H 7.68, N 7.71.
5.2.2. Statistical analysis
The data obtained were expressed as mean ꢁ standard error
of the mean (SEM). The molar concentrations of each drug
producing a 50% block of INa max (IC50) were determined by
using a non-linear least-squares fit of the concentrationere-
sponse curves to the following logistic equation:
n
Effect ¼ ꢀ100=1 þ ðK=½drugꢃÞ
where effect ¼ percentage change of INa, ꢀ100 ¼ maximal
percentage block of INa, K ¼ IC50 of tested compound,
n ¼ logistic slope factor, and [drug] ¼ molar concentration of
the tested compound [24].

2540
A. Catalano et al. / European Journal of Medicinal Chemistry 43 (2008) 2535e2540
[11] V. Yarov-Yarovoy, J. Brown, E.M. Sharp, J.J. Clare, T. Scheuer,
W.A. Catterall, J. Biol. Chem. 276 (2001) 20e27.
Acknowledgment
[12] V. Yarov-Yarovoy, J.C. McPhee, D. Idsvoog, C. Pate, T. Scheuer,
W.A. Catterall, J. Biol. Chem. 277 (2002) 35393e35401.
[13] C. Franchini, F. Corbo, G. Lentini, G. Bruno, A. Scilimati, V. Tortorella,
D. Conte Camerino, A. De Luca, J. Med. Chem. 43 (2000) 3792e3798.
[14] S. Talon, A. De Luca, M. De Bellis, J.-F. Desaphy, G. Lentini,
A. Scilimati, F. Corbo, C. Franchini, P. Tortorella, H. Jockusch,
D. Conte Camerino, Br. J. Pharmacol. 134 (2001) 1523e1531.
[15] M. Muraglia, C. Franchini, F. Corbo, A. Scilimati, M.S. Sinicropi, A. De
Luca, M. De Bellis, D. Conte Camerino, V. Tortorella, J. Heterocycl.
Chem. 44 (2007) 1099e1103.
This work was carried out under the framework of the Na-
tional Projects ‘‘Progettazione, Sintesi e Valutazione Biologica
di Nuovi Farmaci Cardiovascolari’’ supported by the Ministero
`
dell’Universita e della Ricerca (MiUR, Rome) shared also
with the University of Bari.
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