Synthesis of new 2,6-prolylxylidide analogues of tocainide as stereoselective blockers of voltage-gated Na+ channels with increased potency and improved use-dependent activity

Article abstract of DOI:10.1021/jm000931l

A series of tocainide chiral analogues were designed, synthesized, and evaluated in vitro, in pure enantiomeric form, as use-dependent blockers of skeletal muscle sodium channels to better understand the structural requirements responsible for the antimyotonic activity. The voltage clamp recordings showed a remarkable increase of both potency and use-dependent behavior with the analogue N-(2,6-dimethylphenyl)-2-pyrrolidinecarboxamide (1a). In fact (R)-1a was 5-fold more potent than (R)-tocainide in producing the tonic block, i.e., the reduction of peak sodium current in resting conditions after application of the compound, but it was 21-fold more potent in condition of high frequency of stimulation (phasic block). Furthermore, as opposite to tocainide, this compound was also stereoselective, (S)-1a being 2-3-fold less potent than (R)-1a. The introduction in 1a of a methyl group in place of the hydrogen bonded to either the aminic nitrogen atom [N-(2,6-dimethylphenyl)-1-methyl-2-pyrrolidinecarboxamide (2a)] or the amidic nitrogen atom [N-(2,6-dimethylphenyl)-N-methyl-2-pyrrolidinecarboxamide (3a)] led unexpectedly to an inversion of stereoselectivity, the (S)-enantiomers being 3-fold more potent than the (R)-ones. The comparison between eutomers showed that (S)-2a and (S)-3a are almost equieffective to (R)-1a in producing a tonic block, the half-maximal concentrations being about 100 μM; however, the use-dependent behavior was remarkably decreased by the presence of the methyl group: i.e., the gain of potency observed at high frequency of stimulation amounted to 3 and 1.6 times for 2a and 3a, respectively. The replacement of both hydrogens bonded to the aminic and amidic nitrogen atoms resulted in N-(2,6-dimethylphenyl)-N,1-dimethyl-2-pyrrolidinecarboxamide (4a) in which the (S)-isomer was still twice as potent as the (R)-one, but the absolute potency and mostly the use-dependent behavior were strongly reduced, showing therefore no clear advantages with respect to tocainide. The use-dependent behavior, which plays a pivotal role for antimyotonic activity, is strongly reduced by the presence of methyl groups on the nitrogen atoms, likely for modification of pK(a) and/or for constraint of molecular conformation.

Full text of DOI:10.1021/jm000931l

3792
J . Med. Chem. 2000, 43, 3792-3798
Brief Articles
Syn th esis of New 2,6-P r olylxylid id e An a logu es of Toca in id e a s Ster eoselective
Block er s of Volta ge-Ga ted Na + Ch a n n els w ith In cr ea sed P oten cy a n d Im p r oved
Use-Dep en d en t Activity
Carlo Franchini,*,† Filomena Corbo,† Giovanni Lentini,† Gemma Bruno,† Antonio Scilimati,† Vincenzo Tortorella,†
Diana Conte Camerino,‡ and Annamaria De Luca‡
Dipartimento Farmaco-Chimico and Farmaco-Biologico, Universita` di Bari, Via Orabona n.4, 70125 Bari, Italy
Received J une 6, 2000
A series of tocainide chiral analogues were designed, synthesized, and evaluated in vitro, in
pure enantiomeric form, as use-dependent blockers of skeletal muscle sodium channels to better
understand the structural requirements responsible for the antimyotonic activity. The voltage
clamp recordings showed a remarkable increase of both potency and use-dependent behavior
with the analogue N-(2,6-dimethylphenyl)-2-pyrrolidinecarboxamide (1a ). In fact (R)-1a was
5-fold more potent than (R)-tocainide in producing the tonic block, i.e., the reduction of peak
sodium current in resting conditions after application of the compound, but it was 21-fold more
potent in condition of high frequency of stimulation (phasic block). Furthermore, as opposite
to tocainide, this compound was also stereoselective, (S)-1a being 2-3-fold less potent than
(R)-1a . The introduction in 1a of a methyl group in place of the hydrogen bonded to either the
aminic nitrogen atom [N-(2,6-dimethylphenyl)-1-methyl-2-pyrrolidinecarboxamide (2a )] or the
amidic nitrogen atom [N-(2,6-dimethylphenyl)-N-methyl-2-pyrrolidinecarboxamide (3a )] led
unexpectedly to an inversion of stereoselectivity, the (S)-enantiomers being 3-fold more potent
than the (R)-ones. The comparison between eutomers showed that (S)-2a and (S)-3a are almost
equieffective to (R)-1a in producing a tonic block, the half-maximal concentrations being about
100 µM; however, the use-dependent behavior was remarkably decreased by the presence of
the methyl group: i.e., the gain of potency observed at high frequency of stimulation amounted
to 3 and 1.6 times for 2a and 3a , respectively. The replacement of both hydrogens bonded to
the aminic and amidic nitrogen atoms resulted in N-(2,6-dimethylphenyl)-N,1-dimethyl-2-
pyrrolidinecarboxamide (4a ) in which the (S)-isomer was still twice as potent as the (R)-one,
but the absolute potency and mostly the use-dependent behavior were strongly reduced, showing
therefore no clear advantages with respect to tocainide. The use-dependent behavior, which
plays a pivotal role for antimyotonic activity, is strongly reduced by the presence of methyl
groups on the nitrogen atoms, likely for modification of pKa and/or for constraint of molecular
conformation.
In tr od u ction
Finally, by virtue of its ability to block sodium channels
in a use-dependent manner, i.e., with an increased
potency in condition of high-frequency discharges of
action potentials, tocainide has been proposed as a clin-
ically useful antimyotonic drug. In fact, myotonic syn-
dromes are hereditary disorders of skeletal muscle due
to genetic defects in sodium or chloride channels whose
main result is an abnormal membrane hyperexcitability
that triggers muscle stiffness. Due to the wide spectrum
of pharmacological activity, the use of tocainide as
antimyotonic is hindered by unwanted side effects.
The antiarrhythmic action of the type I antiarrhyth-
mic drugs is thought to be mediated via blockade of the
fast voltage-gated sodium channel.1 Models based on
electrophysiological data have been developed to explain
the interaction between antiarrhythmic drugs and the
cardiac sodium channel.2,3 According to these models,
type I antiarrhythmic drugs bind to specific site(s) or
receptor(s) on the functional R-subunit of sodium chan-
nels.4
Tocainide hydrochloride is a class Ib antiarrhythmic
drug once used in the treatment of symptomatic life-
threatening ventricular arrhythmias. It has also a
marked analgesic effect in trigeminal neuralgia in
humans5-7 and antinociceptive effect in rats as well.8
Tocainide is a chiral R-amino acid derivative clinically
used as racemate, although most of its biological activi-
ties reside mainly in the (R)-enantiomer. In fact, the
(-)-(R)-stereoisomer represents the eutomer in binding
cardiac sodium channels9 and in blocking cardiac-like
embryonic sodium channels expressed in cultured muscle
cells10 but fails to show any significant stereoselectivity
on sodium channel of adult skeletal muscle.11,12 The (-)-
* To whom correspondence should be addressed. Tel: +39 080
5442743. Fax: +39 080 5442231. E-mail: cfranc@farmchim.uniba.it.
† Dipartimento Farmaco-Chimico.
‡ Dipartimento Farmaco-Biologico.
10.1021/jm000931l CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/19/2000

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3793
Sch em e 1a
a
Reagents and conditions: (a) Boc-ON, dioxane/H2O; (b) 2,6-
dimethylaniline, EEDQ/Et3N, CHCl3; (c) HCl(g).
F igu r e 1. Str u ctu r es of N-2-(2,6-d im eth ylp h en yl)-2-p yr -
r olid in eca r boxa m id e (1) a n d d er iva tives. F r ee ba ses of
com p ou n d s 1-4 w ill be in d ica ted a s 1a -4a .
Sch em e 2a
(R)-tocainide is the eutomer also responsible for exerting
analgesia. Pharmacological tests have shown that the
antinociceptive effect of the (-)-(R)-enantiomer, like the
analgesia induced by lidocaine, procaine, and mexilet-
ine, is due to a central presynaptic cholinergic mecha-
nism of action.9
These observations suggest that stereoselectivity
plays an important role for the pharmacological activi-
ties of tocainide.9 The identification of the chemical
features specifically responsible for the various biologi-
cal activities of tocainide can help to identify new
compounds with a more selective pharmacological spec-
trum and therefore with less side effects.
In our previous work,13 we have synthesized both
enantiomers of a rigid analogue of tocainide [e.g., N-(2,6-
dimethylphenyl)-2-pyrrolidinecarboxamide, 1a ] and
started to evaluate their pharmacological activity as
well. Such an anilide was still endowed with analgesic
activity, but it exerted antinociception without any
stereoselectivity.13 Moreover, it is worthy of note that
all structural modifications previously described13 shifted
the antinociceptive activity from central cholinergic to
a different, not well characterized mechanism of anal-
gesia. In addition, N-methyltocainide (methyl group
bonded to the amidic nitrogen) has no analgesic activity,
which makes the pharmacological dissociation of tocain-
ide activities conceivable, still being more active than
tocainide on the cardiac sodium channel.14 Concomi-
tantly, voltage clamp experiments on sodium currents
of adult muscle fibers have shown that chiral tocainide
derivatives with substitutions on the asymmetric carbon
atom, i.e., with an isopropyl group in place of the methyl
one, are more potent and stereoselective, corroborating
the importance of this part of the molecule for the
antimyotonic activity as well.12
These findings prompted us to synthesize further
anilide derivatives, structurally related to tocainide,
sterically hindered at the level of the chiral carbon atom,
and therefore conformationally restricted, in which only
one nitrogen (aminic or amidic) or both bear a methyl
group (Figure 1) to evaluate their pharmacological
activity and to gain a better insight into the molecular
determinants specifically related to the antimyotonic
activity. In this paper the results of the above-mentioned
studies are reported, and the rationale of the pharma-
cological data will be discussed.
a
Reagents and conditions: (a) CH3I, Ba(OH)2, DMF/H2O; (b)
CH3COOH/HBr; (c) HCHO/HCOOH; (d) 48% aq HBr; (e) HCl; (f)
i. NaOH, ii. 5% Pd/C, H2 (10 atm), HCHO, iii. HCl (g), Et2O.
1: R-proline was converted into its N-Boc derivative 6
by reaction with 2-tert-butoxycarbonylimino-2-pheny-
lacetonitrile (Boc-ON)/triethylamine (Et3N) in dioxane/
water. Then, N-Boc-R-proline (6) was condensed with
2,6-dimethylaniline in the presence of EEDQ affording
the desired 1-(tert-butoxycarbonyl)-N-(2,6-dimethylphe-
nyl)-2-pyrrolidinecarboxamide (7) that in turn was
converted into N-(2,6-dimethylphenyl)-2-pyrrolidinecar-
boxamide (1a ) (free amines will be identified with the
same number of the corresponding hydrochloride or
hydrobromide followed by a letter, such as 1a, 2a, etc.;
see also Figure 1) and its hydrochloride 1.
1-(tert-Butoxycarbonyl)-N-(2,6-dimethylphenyl)-N-
methyl-2-pyrrolidinecarboxamide (8) was prepared by
reacting 7 with CH3I in DMF/H2O in the presence of
Ba(OH)2 (Scheme 2). N-(2,6-Dimethylphenyl)-N-methyl-
2-pyrrolidinecarboxamide hydrobromide (3) was then
obtained treating 8 with acetic acid and 48% aqueous
hydrobromic acid. N-(2,6-Dimethylphenyl)-N,1-dimethyl-
2-pyrrolidinecarboxamide (4a ) was obtained from 3,
through the classical route of reductive formylation
HCHO/HCOOH. The corresponding hydrobromide 4
was prepared by treating 4a with 48% aqueous hydro-
bromic acid.
N-(2,6-Dimethylphenyl)-1-methyl-2-pyrrolidinecar-
boxamide (2) was obtained from 1 (previously converted
into its free base 1a by treatment with 2 M NaOH) by
reaction with HCHO followed by reduction (H2/5% Pd-
C). 2a was converted into its hydrochloride by gaseous
HCl.
Ch em istr y a n d Biologica l Resu lts
The structural modifications of tocainide were de-
signed (Figure 1) to evaluate in vitro potential antimyo-
tonic activity. We evaluated the capability of the new
compounds to exert both tonic and phasic block of Na+
current (INa) of skeletal muscle fibers, by using the pulse
protocols described in the Experimental Section. Single
Assuming tocainide as a “lead compound”, we syn-
thesized new analogues. The proline derivatives (object
of this study) are reported in Figure 1 and were
synthesized as described in Schemes 1 and 2.
The synthesis of N-(2,6-dimethylphenyl)-2-pyrrolidi-
necarboxamide was carried out as indicated in Scheme

3794 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Brief Articles
Ta ble 1. Concentrations for Half-Maximal Block of Sodium
Currents of Skeletal Muscle Fibers by Tocainide and Its Chiral
Proline-like Derivatives
IC50 (µM)a
tonic/
absolute
config
tonic
block
phasic
block
phasic antimyotonic
compd
block
activityb
tocainide
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
580 ( 11 270 ( 5
2.1
2.4
8.5
5.2
3
3.4
1.6
1.5
1.1
1.4
1
1
21
5
2.7
8
1
3
0.6
1.1
520 ( 8
110 ( 12
230 ( 5
220 ( 5
13 ( 1
44 ( 1
1
2
3
4
300 ( 35 100 ( 15
106 ( 10
28 ( 3.5
340 ( 3.6 217 ( 18
104 ( 5.3
>500
286 ( 11 198 ( 2.5
67 ( 1.6
440 ( 2 0
a
The half-maximal concentrations (IC50) of each compound for
producing a tonic block (block of sodium channel at resting
conditions evaluated during infrequent depolarizing pulses) and
a phasic block (cumulative sodium current reduction by the drug
at 10-Hz stimulation frequency) have been obtained by concentra-
b
tion-response curves (see the Experimental Section). Antimyo-
F igu r e 2. Concentration-response curves for the tonic block
exerted by (R)-tocainide and the tonic and phasic block at 10
Hz for its derivative (R)-1 obtained by introducing the chiral
carbon atom in a rigid proline-like cycle. Each point is the
mean ( SEM from 3-7 fibers. The curves have been obtained
by fitting the experimental point to the logistic equation
described in the Experimental Section.
tonic activity: potency in phasic block in relation to tocainide (see
the Experimental Section); tocainide ) 1.
enantiomers were tested with the aim of evaluating the
presence of stereoselectivity in blocking INa
.
The tonic block shown by tested compounds is due to
their ability to interact with Na+ channels in the resting
state; thus, tonic block reflects block in tissues at normal
firing frequency. However, the use-dependent blockers
showed a selectivity of action in favor of tissues char-
acterized by higher frequency of firing and higher
depolarized membrane potential than normal ones.
Thus, at high frequency of stimulation, an additional
cumulative block over the tonic block occurs; this
cumulative INa reduction reflects the highest affinity of
the drug for open and/or inactivated channels. For their
preferential action on hyperexcitable membranes, use-
dependent blockers are the first choice drugs in the
therapy of a number of muscolar disorders, like myo-
tonic syndromes. All tested compounds are able to
produce both tonic and phasic block, although with
different potency. From data reported in Table 1, some
considerations can be derived.
F igu r e 3. Each bar shows the ratio between the half-maximal
concentration (IC50) of the distomer and that of the eutomer,
for tonic and phasic block calculated at a 10-Hz frequency.
Note that for compound 1 stereoselectivity increases during
high stimulation frequency, demonstrating that this analogue
has a high-use-dependent behavior.
The introduction of a rigid proline-like cycle, as in
N-(2,6-dimethylphenyl)-2-pyrrolidinecarboxyamide (1a ),
remarkably increased the potency in producing both
tonic and phasic block, with respect to tocainide. In fact,
the calculated IC50 value for tonic block by (R)-1a is
5-fold lower than that of the parent compound (R)-
tocainide, the IC50 being 110 µM vs 580 µM. At a 10-Hz
frequency, the protocol used for evaluating phasic block,
the IC50 value of (R)-1a was 21 times lower than that
calculated for (R)-tocainide, demonstrating that this
analogue has a notable use-dependent behavior (Table
1, Figure 2). In support of this view, at high frequency
of stimulation the IC50 value of tocainide was twice
lower than that for tonic block, while the IC50 of (R)-1a
was 8.5 times lower when passing from tonic to phasic
block. As previously shown12 and also in the experimen-
tal conditions reported here, no significant difference
between tocainide enantiomers in producing both tonic
and use-dependent block was found, the two enanti-
omers being almost equieffective. In contrast, 1a pro-
duced a stereoselective block of INa current, with the
(R)-antipode being the eutomer. In particular, the
eudismic ratio (IC50 distomer/IC50 eutomer) was about
2 and 4 for tonic and phasic block, respectively (Figure
3). Thus, stereoselectivity was enhanced during phasic
block.
The introduction of a methyl moiety on the ionizable
amino group as in compound 2a , as well as on the
amidic nitrogen atom as in 3a , led to an unexpected
reversal stereoselectivity, with the (S)-isomers being the
eutomers, about 3-fold more potent than the opposite
(R)-enantiomer for tonic block. A comparison between
eutomers showed that for tonic block both (S)-2a and
(S)-3a are equieffective to (R)-1a , being therefore still
more active than tocainide. However, the presence of
the methyl group of the nitrogen atoms markedly
decreased the use-dependent behavior, with respect to
1a . In fact, the potency at high frequency of stimulation
was increased by a factor of about 3 and 1.5 for 2a and
3a , respectively, which was considerably less than that
observed with 1a (Table 1). In this respect, compound
3a was even less use-dependent than tocainide; indeed
the distomer showed an IC50 for phasic block similar to
that of tocainide (Table 1).
On the other hand, the introduction of a further

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3795
methyl group on the amidic nitrogen atom led to 4a , of
which the (S)-isomer was the eutomer being about twice
as potent as (S)-tocainide for tonic block but almost
equieffective for phasic block, while the (R)-4a was much
less potent than tocainide.
All the tested compounds behaved as inactivated
channel blockers, being able to shift the steady-state
inactivation curves toward more negative potentials in
a manner correlated to their potency in blocking INa
(data not shown).
by virtue of the voltage gradient across the mem-
brane.20,21 Physicochemical aspects such as basicity and
lipophilicity can account for the different use-dependent
behavior of each compound; however, other factors
should be taken into account in order to justify the
reversal of stereoselectivity of action observed when
passing from 1a to its methylated derivatives. As may
be expected, the introduction of conformational con-
straints increases the eudismic ratio [ER ) (S) - IC50/
(R) - IC50], and a higher stereoselectivity of action was
found in all tocainide cyclic analogues. Less predictable
was the inversion of the stereoselectivity pattern which
assigned the role of eutomer to 2a , 3a , and 4a (S)-
isomers in opposition to that observed with 1a . The
reason of this finding is far from obvious, even if some
hypotheses may be envisaged considering some recent
results coming from molecular pharmacology stud-
ies.22,23 The aromatic ring of local anesthetic-like drugs
has been proposed to bind to a tyrosine residue at
position 1586 in the rat skeletal muscle sodium channel,
while the terminal amine group of the drugs may
associate with a neighboring phenylalanine residue
through π electron interaction (position 1579). Recently,
site-specific mutagenesis experiments have added other
evidence to the importance of hydrophobicity near or
at the local anaesthetic-like drug receptor for drug
binding affinity.24,25 This was corroborated by our stud-
ies with a series of mexiletine derivatives, showing that
the drug potency for blocking sodium channels is highly
correlated to the hydrophobicity of the groups linked to
the chiral carbon atom,26 suggesting the occurrence of
a strong π-π interaction. Very recently an asparagine
has also been claimed to be involved in the binding of
local anaesthetic-like drugs.27 These findings led to a
hypothesis of a rough receptor model presenting three
possible interaction sites, two out of three being repre-
sented by aromatic residues. The driving force in
binding should be given by groups relatively far from
the stereogenic center (xylidide moiety). This model
might explain the low ERs observed even in the more
potent analogues and the different orientations and/or
“three-point” interactions possibly assumed by each
ligand.
Discu ssion
Previous results, confirmed by the present study, have
underlined that tocainide, able to exert various phar-
macological actions in a stereoselective manner, is
poorly stereoselective in blocking sodium channels of
adult skeletal muscle fibers.12 Nonetheless, the chiral
carbon atom of tocainide is an important pharmacoph-
oric part also for the interaction with voltage-gated
sodium channel and therefore for antimyotonic activity.
In fact, we previously demonstrated that an increase of
the steric hindrance at this level increases the potency
and stereoselectivity.12 In the present study we synthe-
sized a series of new tocainide analogues in which the
stereogenic center is constrained in a rigid proline-like
cycle. All the compounds were effective sodium channel
blockers, and in particular 1a , 2a , and 3a were more
potent than tocainide and mostly differ for stereoselec-
tivity and use-dependent behavior. The most interesting
congener was compound 1a , which showed a great
increase of stereoselectivity with respect to tocainide,
the (R)-isomer being 2-3 times more potent than (S)-
1a in both tonic and phasic block. Also 1a was markedly
more potent and use-dependent than tocainide; in fact,
(R)-1a was 5-fold more potent than (R)-tocainide for
tonic block of sodium currents and up to 20-fold more
potent than the parent compound at high stimulation
frequency: i.e., the IC50 of (R)-1a decreased by 8-fold
passing from tonic to phasic blockade. The use-depend-
ent behavior, i.e., the increase of drug potency observed
in condition of high frequency of stimulation, plays an
important role for the therapeutic activity of sodium
channel blockers, use-dependent compounds being very
active on pathological tissues discharging at high fre-
quency, such as muscle fibers affected by myotonic
syndromes. In fact we previously demonstrated that new
derivatives of mexiletine characterized by a remarkable
use-dependent behavior were more potent than the
parent compound in suppressing in vitro the hyperex-
citability of genetically myotonic mouse.15 For absolute
potency and high-use-dependent behavior, the tocainide
derivative 1a seems a compound particularly promising
as antimyotonic. The remarkable use-dependent activity
of 1a may be explained assuming that this molecule is
provided with more basicity and lipophilicity, both
properties being favorable for a ready access to the
receptor site supposed to be located in the channel pore.
Since the drug reaches its receptor from the intracel-
lular side of the channel16-18 and we applied the
compounds outside the cell, a difference in apparent
affinity may come from difference in drug lipophilicity
or pKa, the former facilitating the diffusion through the
membrane and the latter through the hydrophilic
pathway of the open channel from the cytoplasm19 and
Con clu sion
Given the lack of central analgesic action in both 1a
and N-methyl(amidic)tocainide, the introduction of con-
formational constraints was assumed to be favorable to
the dissociation of the main tocainide activities (central
analgesic and antimyotonic actions).
1a -4a were prepared as depicted in Schemes 1 and
2 in good yields and tested in vitro as possible antimyo-
tonic agents by evaluating the ability to block in a use-
dependent manner (phasic block) the sodium current
of skeletal muscle fibers recorded by means of voltage
clamp experiments. Compounds 1a and 2a were mark-
edly more potent than tocainide in blocking sodium
currents; however, a reduction of the antimyotonic
activity resulted in the order 1a -4a (Table 1). Notably,
(R)-1a was 21 times more active than tocainide in
exerting a phasic block, and the IC50 value relative to
phasic block was at least 8 times lower when compared
with the corresponding value derived from tonic block
experiments. This highly use-dependent behavior pro-

3796 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Brief Articles
CDCl3 or DMSO-d6 on a Varian EM 390 or Bruker 300 MHz
spectrometer (ASPECT 3000 model) and chemical shifts are
reported in parts per million (δ) from internal Me4Si. Absolute
values of the coupling constant are reported. IR spectra were
recorded on a Perkin-Elmer 681 spectrometer. Optical rota-
tions were measured on a Perkin-Elmer digital polarimeter,
model 241 MC. Thin-layer chromatography (TLC) was per-
formed on silica gel sheets with fluorescent indicator (Sta-
tocrom SIF, Carlo Erba), and the spots on the TLC were
observed under ultraviolet light or were visualized with I2
vapor. Chromatography was conducted by using silica gel with
an average particle size of 60 µm, a particle size distribution
of 40-63 µm and 230-400 ASTM. GC-MS analyses were
performed on an HP 5995C model and microanalyses on a
elemental analyzer 1106, Carlo Erba instrument. (-)-(S)-2-
(4-Chlorophenoxy)-2-phenylacetic acid (CPPA)29 was used as
chiral solvating agent for determining the enantiomeric ratio
poses (R)-1a as a candidate for more complex studies
aimed at evaluating its actual usefulness in the treat-
ment of myotonias.
Further studies are in progress by SAR investigation
to find a correlation between potency, stereoselectivity,
and conformational characteristics of the compounds
presented in this paper.
Exp er im en ta l Section
P h a r m a cology. 1. P r ep a r a tion a n d Solu tion s. Record-
ings of Na+ current were performed on a segment of undam-
aged single muscle fibers (1-2 cm in length), obtained by
microsurgery from the ventral branch of semitendinosus
muscle, removed from Rana Esculenta. The cut end fiber was
superfused with an internal solution, having the following
composition (mM): CsF 105; MOPS 5; EGTA 5; MgSO4 2; Na2-
ATP 0.55 (pH at 7.2 with standard NaOH concentrated
solution); then it was transferred in the recording chamber
(filled with the same solution) that consisted of three partitions
delineating four pools; the width of the gap of the central pool
A was set to 70-100 µm. With the fiber in this position, three
strips of grease were applied and then carefully sealed to
reduce leakage. Four KCl/Agar bridge electrodes, one for each
pool, connected the recording chamber and the voltage clamp
amplifier, based on methods described by Hille and Campbell28
and detailed elsewhere.11 The solution level was adjusted to
have the four pools physically and electrically independent
from each other. The solution in the central pool A was
replaced with an external solution, of the following composition
(mM): NaCl 77; choline Cl 38; CaCl2 1.8; KCl 2.5; Na2HPO4
2.15; NaH2PO4 0.85. All the tested compounds were solubilized
in the external solution; stock solutions of drug were prepared
daily and diluted to obtain the required concentration.
2. Recor d in g of Na+ Cu r r en t a n d P u lse P r otocols. After
an equilibration time of 10 min Na+ currents recordings were
performed at 10 °C. The holding potential used was -100 mV.
The voltage clamp amplifier was connected via a 12-bit AD/
DA Digidata 1200 interface (Axon Inc.) to a 486 DX2/66 PC.
The stimulation protocols and data acquisition were driven
by the Clampex Program (pClamp 6 software packge, Axon
Inc.). The Na+ currents flowing in response to depolarizing
stimuli were low-pass filtered at 10 kHz, visualized on a
oscilloscope, sampled at 20 kHz and stored on the hard disk;
when necessary, leak and capacity currents were subtracted
using the P/4 method. Data analysis were conducted using the
Clampfit program. The voltage dependence of the Na + current
(I/V curve) was evaluated by using a single test pulse of 10-
ms duration, from the h.p. to -20 mV, able to elicit a nearly
maximal Na+ current (INamax). Interpulse duration was long
enough to allow complete recovery of Na+ channels from
inactivation. Tonic block was calculated as percentage reduc-
tion of the peak Na+ current exerted by each compound. 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 by normalizing the residual
current at the end of the stimulation protocol with respect to
the current in the absence of drug.
1
of 3a by recording H NMR spectra of a mixture of (R)-3a [or
(S)-3a ] (10 mg, 0.043 mmol) and CPPA (25 mg, 0.083 mmol)
in C6D6. The relative intensities of the singlets, due to the two
methyl groups bonded to the phenyl ring, gave the ratio of (R)-
3a ‚(S)-CPPA:(S)-3a ‚(S)-CPPA [95:5 for 3a prepared starting
from (R)-proline and vice versa for 3a derived from (S)-proline].
Ch em ica ls a n d Rea gen ts. BOC-ON reagent [2-(tert-bu-
toxycarbonyloxyimino)-2-phenylacetonitrile], (R)- and (S)-2-
pyrrolidinecarboxylic acid, 2,6-dimethylaniline and EEDQ (2-
ethoxycarbonyl-1,2-dihydroquinoline) were purchased from
Aldrich. Other chemicals were of the highest quality com-
mercially available. Procedure to prepare the compounds
shown in Schemes 1 and 2 is as follows.
1-(ter t-Bu toxyca r bon yl)-2-p yr r olid in eca r boxylic Acid
(6). A mixture of (R)[or (S)]-2-pyrrolidinecarboxylic acid (1
mmol), 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile30
(1.1 mmol) and triethylamine (1.5 mmol) in 50 mL of dioxane/
water (1:1) was stirred at room temperature for 24 h. The
reaction mixture was protected from the light for the entire
reaction time. Then, the dioxane layer was evaporated and the
aqueous residue extracted twice with ethyl ether. The aqueous
phase, acidified with 10% HCl solution (pH ) 2), was then
extracted six times with ethyl acetate; the organic phases were
combined, washed with water and dried over MgSO4, and the
solvent evaporated under reduced pressure to give a crude oil,
which was purified by crystallization. The product was fully
characterized by routine spectroscopic analysis and, all the
analytical data were in agreement with those ones reported
for the autentical sample.13
1-(ter t-Bu toxyca r bon yl)-N-(2,6-d im eth ylp h en yl)-2-p yr -
r olid in eca r boxa m id e (7). A mixture of 1-(tert-butoxycarbo-
nyl)-2-pyrrolidinecarboxylic acid (6) (1 mmol), 2,6-dimethyla-
niline (1.1 mmol), freshly recrystallized EEDQ (1.2 mmol), and
triethylamine (1.5 mmol) in 50 mL of CHCl3 was refluxed for
6 h. The solvent was evaporated and the residue, dissolved in
ethyl acetate, was extracted with a 10% HCl solution. The
organic layer was then washed with 0.1 M NaOH and H2O,
dried over MgSO4, and then evaporated under reduced pres-
sure to give a crude product, which was purified by recrystal-
lization (86-98% yield): mp 166-167 °C (EtOAc); [R]20
)
D
-126 (c ) 1.0, CHCl3) for (S)-enantiomer, [R]20 ) +124 (c )
D
3. Sta tistica l An a lysis. The data obtained were expressed
as mean ( standard error of the mean (SEM). The molar
concentrations of each drug producing a 50% block of INamax
(IC50) were determined by using a nonlinear least-squares fit
of the concentration-response curves to the following logistic
equation:
0.9, CHCl3) for (R)-enantiomer; GC-MS (70 eV) m/z (rel inten.)
318 (M+, 1), 70 (100), 114 (69), 57 (47).
Full characterization was performed on the deprotected
product [N-(2,6-dimethylphenyl)-2-pyrrolidinecarboxamide] and
its hydrochloride 1 [N-(2,6-dimethylphenyl)-2-pyrrolidinecar-
boxamide hydrochloride] as well (see below).
N-(2,6-Dim eth ylph en yl)-2-pyr r olidin ecar boxam ide Hy-
d r och lor id e (1). A solution of 1-(tert-butoxycarbonyl)-N-(2,6-
dimethylphenyl)-2-pyrrolidinecarboxamide (7) (1 mmol) in 3
mL anhydrous ethyl ether was saturated with gaseous HCl
and stirred at room temperature for 15 min. After removing
the solvent under reduced pressure, the residue was purified
effect ) -100/1 + (K/[drug])n
where effect ) percentage reduction of INa, -100 ) maximal
percent block of INa, K ) IC50 of tested drug, n ) logistic slope
factor, and [drug] ) molar concentration of the tested drug.
The h∞ curves were fitted with a single Boltzmann distribution
as described in detail elsewere.12
by recrystallization from EtOH/Et2O (70% yield): [R]20 ) -2
D
(c ) 1.0, MeOH) and mp ) 240 °C dec (EtOH/Et2O) for (S)-
Ch em istr y. Melting points taken on Electrothermal ap-
enantiomer, [R]20 ) +1 (c ) 2, MeOH) and mp ) 240 °C dec
D
1
paratus were uncorrected. H NMR spectra were recorded in
(EtOH/Et2O) for (R)-enantiomer; 1H NMR (DMSO-d6, δ)

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3797
10.24-10.05 (bs, 1H, NHCO: exchange with D2O); 9.50-8.20
(bs, 2 H, +NH2-ring: exchange with D2O); 7.09 (s, 3 H, aromatic
protons); 4.50-4.45 (t, J ) 7.4 Hz, 1 H, CHCO); 3.27-3.18
(m, 2 H, +NH2CH2); 2.58-2.37 (m, 1 H, COCHCH2; this
multiplet overlaps to the DMSO signals (used as solvent to
record the spectrum); by addition of D2O the two signals
appeared at 2.5 ppm for DMSO (as expected) and 2.48-2.30
ppm for COCHCH2, respectively); 2.15 (s, 6 H, CH3); 2.10-
1.87 (m, 3 H, 1H of COCHCH2 and 2 H of NCHCH2CH2). Anal.
(C13H18N2O‚HCl‚0.5H2O) C, H, N.
N-(2,6-Dim eth ylph en yl)-2-pyr r olidin ecar boxam ide (1a).
N-(2,6-Dimethylphenyl)-2-pyrrolidinecarboxamide hydrochlo-
ride (1) (5 mmol) was treated with 10 mL AcOEt and 2 M
NaOH (5.5 mmol). The aqueous phase was extracted three
times with EtOAc. The organic extracts were combined and
dried over Na2SO4. The solvent was evaporated under reduced
pressure giving the product: 1H NMR (CDCl3, δ) 9.30-9.00
(bs, 1 H, NHCO: exchange with D2O); 7.09 (s, 3H, aromatic
protons); 3.93-3.89 (dd, J ) 5.0 Hz and 9.1 Hz, 1 H, CHCO);
3.15-2.97 (m, 2 H, NHCH2); 2.32-2.13 (m, 1 H, COCHCH2);
2.19 (s, 6 H, CH3); 2.12-2.02 (m, 1 H, COCHCH2); 2.01-1.90
(bs, 1 H, NH-ring: exchange with D2O); 1.90-1.70 (m, 2 H,
NCHCH2CH2); GC-MS (70 eV) m/z (rel inten.) 218 (M+, 2),
70 (100), 41 (7).
1-Meth yl-N-(2,6-d im eth ylp h en yl)-2-p yr r olid in eca r box-
a m id e (2a ). N-(2,6-Dimethylphenyl)-2-pyrrolidinecarboxamide
hydrochloride (1) (5 mmol) was treated with 10 mL AcOEt
and 2 M NaOH (5.5 mmol). The aqueous phase was extracted
three times with EtOAc. The organic extracts were combined
and dried over Na2SO4. The solvent was evaporated under
reduced pressure giving the N-(2,6-dimethylphenyl)-2-pyrro-
lidinecarboxamide, used without any further purification. A
40% aqueous formaldehyde was added to an equimolar etha-
nolic solution of N-(2,6-dimethylphenyl)-2-pyrrolidinecarboxa-
mide. This solution was transferred into the autoclave and
treated, in the presence of 5% Pd/C as catalyst, with H2 at 10
atm for 24 h. The solution was filtered on Celite and the
solvent removed under reduced pressure. The residue was
purified by chromatography on silica gel (eluent: petroleum
ether/ethyl acetate ) 8/2). The product obtained was converted
into its hydrochloride as previous described: 1H NMR (CDCl3,
δ) 9.10-8.55 (bs, 1 H, NHCO: exchange with D2O); 7.15-6.90
(m, 3H, aromatic protons); 3.22-3.13 (m, 1 H, NCH2); 3.12-
3.02 (dd, J ) 4.9 and 10.4 Hz, 1 H, CHCO); 2.52 (s, 3 H, NCH3);
2.50-2.38 (dd, J ) 8.8 and 17.0 Hz, 1 H, NCH2); 2.37-2.24
(m, 1 H, COCHCH2); 2.21 (s, 6 H, CH3); 2.07-1.93 (m, 1 H,
COCHCH2); 1.92-1.80 (m, 2 H, NCHCH2CH2); GC-MS (70
eV) m/z (rel inten.) 232 (M+, 0.5), 85 (14), 84 (100), 42 (45).
(2,6-dimethylphenyl)-N-methyl-2-pyrrolidinecarboxamide (8)
(5 mmol) was solubilized in 40 mL of a mixture (1/1) AcOH/
48% aqueous HBr. The reaction mixture was stirred at room
temperature for 15 min. Then, the excess of AcOH/aqueous
HBr mixture was evaporated under reduced pressure and the
crude crystalized by EtOH/Et2O (75% yield): mp 166-167 °C
(EtOH/Et2O); [R]20D ) +6.0 (c) 1.1, MeOH) for (R)-enantiomer,
[R]20 ) -7.6 (c ) 1.3, MeOH) for (S)-enantiomer; 1H NMR
D
(DMSO-d6, δ) 9.80-8.20 (bs, 2H, +NH2-ring: exchange with
D2O); 7.32-7.18 (m, 3H, aromatic protons); 3.67 (t, 1 H, J )
8.5 Hz, CHCO); 3.26-3.15 (m, 1 H, NCH2); 3.14-3.02 (m, 1
H, NCH2); 3.11 (s, 3 H, NCH3); 2.19 (s, 3 H, CH3); 2.18 (s, 3 H,
CH3); 1.95-1.68 (m, 1 H, NCHCH2); 1.67-1.55 (m, 3 H,
NCHCH2 (1 H) and NCHCH2CH2 (2 H)). Anal. (C14H20N2O‚
HBr) C, H, N.
N-(2,6-Dim et h ylp h en yl)-N-m et h yl-2-p yr r olid in eca r -
boxa m id e (3a ). N-(2,6-Dimethylphenyl)-N-methyl-2-pyrro-
lidinecarboxamide hydrobromide (3) dissolved in 2 M NaOH
(20 mL) was extracted with ethyl acetate (4 × 25 mL). The
organic layer was dried over Na2SO4 and then evaporated
under reduced pressure to give the product: 1H NMR (CDCl3,
δ) 7.18-7.00 (m, 3 H, aromatic protons); 3.21 (t, J ) 8.0 Hz,
1H, COCH); 3.14 (s, 3 H, NCH3); 3.17-3.05 (m, partially
overlapped to the previous singlet, 1 H); 2.70-2.57 (m, 1 H);
2.20 (s, 6 H, CH3); 1.80-1.44 (m, 4 H, CHCH2 and NCH2CH2).
1-Met h yl-N-(2,6-d im et h ylp h en yl)-N-m et h yl-2-p yr r o-
lid in eca r boxa m id e (4a ). N-(2,6-Dimethylphenyl)-N-methyl-
2-pyrrolidinecarboxamide hydrobromide (3) (7.3 mmol) was
dissolved in 1 mL of 40% aqueous formaldehyde to which 1.3
mL of 70% aqueous formic acid was added. The reaction
mixture was stirred at 80 °C for 8 h, then it was concentrated
under reduced pressure and the residue, dissolved in 2 M
NaOH (20 mL) was extracted with ethyl acetate (4 × 25 mL).
The organic layer was dried over Na2SO4 and then evaporated
under reduced pressure to give a crude oil which was purified
as hydrobromide, prepared as previous described: 1H NMR
(CDCl3, δ) 7.20-7.02 (m, 3 H, aromatic protons); 3.12 (s, 3 H,
CONCH3); 3.11-3.02 (t, J ) 8.6 Hz, 1 H, COCH; the upper
field signal of the triplet appeared split by a long-range
coupling constant, J ) 2.2 Hz); 2.47-2.35 (t, J ) 7.8 Hz, 1 H,
NCH2; the central signal of the triplet appeared splitted by a
long-range coupling constant, J ) 3.1 Hz); 2.27 (s, 3 H, NCH3);
2.20 (s, 3 H, CH3); 2.17 (s, 3 H, CH3); 2.04-1.91 (m, 1 H,
NCHCH2); 1.90-1.70 (m, 2 H, 1 H of NCHCH2 and 1 H of
NCH2); 1.64-1.63 (m, 2 H, NCH2CH2); GC-MS (70 eV) m/z
(rel inten.) 246 (M+, 0.3), 84(100), 42 (25).
1-Met h yl-N-(2,6-d im et h ylp h en yl)-N-m et h yl-2-p yr r o-
lid in eca r boxa m id e h yd r och lor id e (4): mp 197 °C dec
(EtOH/Et2O); 30% yield; [R]20 ) +30 (c ) 1.15, MeOH) for
D
1-Meth yl-N-(2,6-d im eth ylp h en yl)-2-p yr r olid in eca r box-
a m id e h yd r och lor id e (2): mp 215-217 °C (EtOH/Et2O);
(R)-enantiomer, [R]20 ) -33.5 (c ) 2.1, MeOH) for (S)-
D
enantiomer; 1H NMR (DMSO-d6, δ) 10.40-8.30 (bs, 1H,
+NHCH3-ring: exchange with D2O); 7.37-7.18 (m, 3 H,
aromatic protons); 3.56 (t, 1 H, J ) 8.5 Hz, CHCO); 3.49-
3.37 (m, 1 H, NCH2); 3.11 (s, 3 H, CONCH3); 3.00-2.84 (m, 1
H, NCHCH2); 2.70 (s, 3 H, NCH3-ring); 2.18 (s, 3 H, CH3); 2.17
(s, 3 H, CH3); 1.88-1.55 (m, 4 H, NCHCH2 (1 H), NCH2 (1 H)
and NCH2CH2 (2 H)). Anal. (C15H22N2O‚HBr‚2/3H2O) C, H, N.
63-98% yield; [R]20 ) +14.6 (c ) 1.5, MeOH) for (R)-
D
enantiomer, [R]20D ) -16 (c ) 1.5, MeOH) for (S)-enantiomer;
1H NMR (DMSO-d6, δ) 10.50-10.15 (bs, 1H, NHCO: exchange
with D2O); 8.25-7.80 (bs, 1H, +NHCH3: exchange with D2O);
7.15-7.05 (m, 3 H, aromatic protons); 4.32-4.08 (m, 1 H,
CHCO); 3.60-3.45 (m, 1 H, NCH2); 3.20-3.00 (m, 1 H, NCH2);
2.80 (s, 3 H, NCH3); 2.70-2.53 (m, 1 H, COCHCH2); 2.15 (s, 6
H, CH3); 2.13-1.83 (m, 3 H, COCHCH2 (1 H) and NCHCH2CH2
(2 H)). Anal. (C14H20N2O‚HCl) C, H, N.
Ack n ow led gm en t. Financial support by the Min-
istero dell′Universita` e della Ricerca Scientifica e Tec-
nologica (Rome) and Telethon-Italy (No. 1208) is ac-
knowledged.
1-(t er t -Bu t oxyca r b on yl)-N -(2,6-d im e t h ylp h e n yl)-N -
m eth yl-2-p yr r olid in eca r boxa m id e (8). To a mixture of
1-(tert-butoxycarbonyl)-N-(2,6-dimethylphenyl)-2-pyrrolidine-
carboxamide (7) (2.2 mmol) and Ba(OH)2 (13.2 mmol) in DMF/
H2O (30 mL/10 mL) was added CH3I (79.2 mmol). This
suspension was stirred at room temperature for 12 h; after
this time the yellow reaction mixture was filtered to remove
the excess of Ba(OH)2 and then extracted three times with
petroleum ether. The organic layer was dried over Na2SO4.
The solvent was then removed under reduced pressure afford-
ing the reaction crude which was purified and fully character-
ized as hydrobromide (94% yield): GC-MS (70 eV) m/z (rel
inten.) 232 (M+, 3), 114 (87), 70 (100), 57 (64).
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