Synthesis and Evaluation of Voltage-Gated Sodium Channel Blocking Pyrroline Derivatives Endowed with Both Antiarrhythmic and Antioxidant Activities

Article abstract of DOI:10.1002/cmdc.202000692

Under the hypothesis that cardioprotective agents might benefit from synergism between antiarrhythmic activity and antioxidant properties, a small series of mexiletine analogues were coupled with the 2,2,5,5-tetramethylpyrroline moiety, known for its antioxidant effect, in order to obtain dual-acting drugs potentially useful in the protection of the heart against post-ischemic reperfusion injury. The pyrroline derivatives reported herein were found to be more potent as antiarrhythmic agents than mexiletine and displayed antioxidant activity. The most interesting tetramethylpyrroline congener, a tert-butyl-substituted analogue, was at least 100 times more active as an antiarrhythmic than mexiletine.

Full text of DOI:10.1002/cmdc.202000692

Accepted Article
Title: Synthesis and evaluation of voltage-gated sodium channel
blocking pyrroline derivatives endowed with both antiarrhythmic
and antioxidant activities
Authors: Alessia Carocci, Roberta Budriesi, Matteo Micucci, Maria
Maddalena Cavalluzzi, Gualtiero Milani, Alessia Catalano,
Annalisa De Palma, Filomena Corbo, Carlo Franchini,
Solomon Habtemariam, and Lentini Giovanni
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000692
Link to VoR: https://doi.org/10.1002/cmdc.202000692
01/2020

10.1002/cmdc.202000692
ChemMedChem
Synthesis and evaluation of voltage-gated sodium channel blocking pyrroline derivatives
endowed with both antiarrhythmic and antioxidant activities
Alessia Carocci,*[a] Mariagrazia Roselli,[a] Roberta Budriesi,[b] Matteo Micucci,[b] Jean-François
Desaphy,[c] Concetta Altamura,[c] Maria Maddalena Cavalluzzi,[a] Maddalena Toma,[a] Giovanna Ilaria
Passeri,[a] Gualtiero Milani,[a] Angelo Lovece,[a] Alessia Catalano,[a] Claudio Bruno,[a] Annalisa De
Palma,[d] Filomena Corbo,[a] Carlo Franchini,[a] Solomon Habtemariam,[e] Giovanni Lentini.[a]
[a]Department of Pharmacy – Pharmaceutical Sciences, University of Bari Aldo Moro, via E. Orabona
n. 4, 70126 Bari, Italy.
[b]Department of Pharmacy and Biotechnologies, Alma Mater Studiorum-University of Bologna, Via
Belmeloro 6, Bologna, 40126, Italy.
[c]
Department of Biomedical Sciences and Human Oncology, University of Bari Aldo Moro,
Policlinico, piazza Giulio Cesare, 70126 Bari, Italy
[d]Department of Biosciences, Biotechnologies, and Biopharmaceutics, University of Bari Aldo Moro,
via E. Orabona 4, 70126 Bari, Italy.
[e]Pharmacognosy Research Laboratories & Herbal Analysis services UK, University of Greenwich,
Chatham-Maritime, Kent ME4 4TB, UK.
Abstract: Under the hypothesis that cardioprotecting agents might benefit from a synergism between
antiarrhythmic activity and antioxidant properties, a small series of mexiletine analogues were
coupled with the 2,2,5,5-tetramethylpyrroline moiety, known for its antioxidant effect, in order to
obtain dual acting drugs potentially useful in the protection of the heart against post ischemic
reperfusion injury. The pyrroline derivatives reported herein (2a–e, Fig. 1) were more potent as
antiarrhythmic agents than mexiletine and displayed antioxidant activity. The most interesting
tetramethylpyrroline congener, the tert-butyl-substituted analogue 2b, was at least 100 times more
active as an antiarrhythmic than mexiletine.
*Corresponding author: Prof. Alessia Carocci,
Department of Pharmacy – Pharmaceutical Sciences, University of Bari Aldo Moro, via E. Orabona
n. 4, 70126 Bari, Italy, phone number: (+39) 080-5442731; fax number: (+39) 080-5442724, E-mail
address: alessia.carocci@uniba.it
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Table of contents
Synthesis and evaluation of voltage-gated sodium channel blocking pyrroline derivatives
endowed with both antiarrhythmic and antioxidant activities
R
O
R
O
O
N
H
NH2
N
H
mexiletine (1a): R = CH3, EC50 = 11.6 µM
2b: R = tBu, EC50 = 0.054 µM
The investigation on the antiarrhythmic and antioxidant potential of a series of pyrroline derivatives
allowed to identify a potent antiarrhythmic agent (2b), more active than the parent compound
mexiletine.
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1. Introduction
Due to the presumed role of oxidative stress in a range of human diseases, such as cardiovascular
diseases (e.g. atherosclerosis[1,2] and ischemia-reperfusion injury[3]), neurodegenerative disorders[4]
(e.g. Alzheimer’s[5,6] and Parkinson’s diseases[7]), diabetes mellitus,[8] muscular dystrophy,[9] and
sepsis,[10] numerous efforts have been focused on the development of new effective antioxidant drugs.
Our interest in antioxidants stems from the consideration that mexiletine (1a, Figure 1), a well-known
voltage-gated sodium channel blocker used in the treatment of myocardial ischemia-
related arrhythmias, can also act as an antioxidant by inhibiting hydroxyl radical-mediated lipid
peroxidation in brain membranes, probably as a result of a direct interaction of the drug with the
biological membranes.[11] Indeed, mexiletine displayed in vivo neuroprotecting properties against
oxidative damage associated with diabetes[12] and in a model of focal, neocortal ischemia.[13]
Moreover, it has been demonstrated that an increase in the cardioprotective activity of mexiletine is
obtained with the introduction of a 2,2,5,5-tetramethylpyrroline structural moiety on the amino group,
possibly through the anti-oxidant effect of this moiety.[14,15] In the past decade we have developed
several mexiletine analogues with superior potency in blocking skeletal muscle voltage-gated sodium
channels than the parent compound.[16,17] Some of these compounds also showed better antiarrhythmic
activity along with similar or less cardiovascular effects than mexiletine, thus presenting a higher
selectivity of action and reduced side effects.[18] Recently, two tetramethylpyrroline derivatives of
both mexiletine and its potent use-dependent isopropyl analogue were tested for their ability to block
native NaV1.4 and to exert cytoprotective effects against oxidative-stress injury in myoblasts. Both
compounds showed an improved pharmacological profile as sodium channel blockers, with the
isopropyl analog being the strongest use-dependent mexiletine-like compound so far, and remarkable
cytoprotection at concentrations effective for use-dependent block.[19] Herein, we report the synthesis
of the above two pyrroline derivatives (2a,d) and of three additional analogues (2b,c,e, Figure 1)
which were studied for their antiarrhythmic and antioxidant properties.
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2. Results and discussion
2.1. Chemistry
Pyrroline derivatives (2a–e) were prepared as reported in Scheme 1, following a literature
procedure.[20–22] 2,2,6,6-Tetramethylpiperidin-4-one was brominated to obtain the 3,5-dibromo-
2,2,6,6-tetramethylpiperidin-4-one hydrobromide (4), which was coupled to the appropriate
aryloxyalkylamines (1a–e)[18] affording the corresponding pyrroline derivatives 2a–e. Amides 3a–e
were prepared coupling 4 with the appropriate alkylamines.
2.2. Biological results
2.2.1 Antiarrhythmic activity
All the synthesized compounds (2a–e, 3a–e) were tested in vitro for their antiarrhythmic activity on
guinea pig isolated left atria driven at 1 Hz. Results were reported in Table 1 along with data for
mexiletine (1a) and previously reported mexiletine analogues, amines 1b–e.[18] Pyrroline derivatives
2a–e increased the threshold of ac-arrhythmia more than mexiletine did, with the tert-butyl derivative
2b being the most active of the series, with an EC50 value in the nanomolar range. Compound 2b was
found to be 215-fold more active than mexiletine, 1a. Of particular interest, compound 2e shows an
activity of 0.41 µM, which was 10- and 28-fold higher than that of the parent amine 1e and
mexiletine, respectively. It should be noted that, except for compound 2c, all derivatives were more
active than the corresponding parent amines (cfr 2a–e with 1a–e, respectively). This result agrees with
those previously observed on myoblasts[19] and may be related to the 2a–e increased lipophilicity (cf.
Table 2). While pKa values were substantially unchanged throughout the three compounds series, Log
D7.4 values spanned a three order of magnitude range thus reflecting corresponding variations in Log
P values. Despite possible overestimation,[19] the observed positive relationship between lipophilicity
and antiarrhythmic potency agreed with previous observations.[18,19] The elongation of the
intermediate chain connecting the aryloxy moieties to the basic functional groups may also be
beneficial.[19,23,24] Furthermore, the tetramethylpyrroline basic nitrogen is inserted in a lipophilic
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structural environment and, when protonated, distributes the cationic charge in the form of partial
charges delocalized on the surrounding carbon atoms.[19] In agreement with previously reported
results,[16,19] the cationic charge delocalization is beneficial in mexiletine-like sodium channel
blocking agents. Finally, the possible additional contribution of hydrogen bond interactions at the
carboxamido group should not be discarded. Being this hypothesis true, the size of the substituents at
the stereogenic center might hinder efficacious hydrogen bond formation. The observed scale of
potencies does not support the hypothesis since the antiarrhythmic effect was positively related to the
size of the substituent onto the asymmetric center. On the other hand, the partially impaired rotation
around the amide bond might be conditioned by steric and electronic effects played by the same
substituents. This, in turn, might alter the conformer population distribution with the s-cis/s-trans
equilibrium more or less pending in favor of the latter. Being a proper orientation of the NHCO group
required for efficacious binding, s-cis/s-trans equilibrium variations should reflect into corresponding
variations in the observed EC50 value output. To explore the above hypothesis, a systematic
conformational search was performed on 2a−e through hybrid density functional theory (DFT)
B3LYP calculations with the 6-31G* basis set. Following previously developed procedures,[16 25] we
generated several conformers in a window of 5 kcal/mol from the global minimum conformation
(HF/6-31G*//HF/6-31G*) for each congener in the series 2a–e. Each conformer of the so-obtained
conformer populations underwent geometry optimization at the DFT B3LYP/6-31G* level.[26] The so-
obtained conformations were true conformers (no IR imaginary frequencies). Interestingly (Table 3),
the most active congeners 2b and 2e displayed the higher percentages of s-cis forms (14% and 4%,
respectively). This observation would suggest that the carboxamide geometry might contribute to the
pharmacological output.
Among amides 3a–e, lacking the 2,6-dimethylphenoxy moiety, only the benzyl derivative 3e showed
an anti-arrhythmic activity comparable to that of mexiletine, though it was less active than the
corresponding derivative 2e. This result suggests the importance of the presence of the 2,6-
dimethylphenoxy moiety for the antiarrhythmic activity. Conceivably, the activity of compound 2e is
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to be attributed to the presence of the benzyl substituent that takes the place of the missing 2,6-
dimethylphenoxy moiety.
In general, amides 3a–e resulted considerably less potent than the corresponding 2a–e analogues so
that their EC50 values have not been determined. The only exception is the benzyl derivative 3e
showing an anti-arrhythmic activity comparable to that of mexiletine (1a). Conceivably, the benzyl
moiety of 3e replaces the 2,6-dimethylphenoxy moiety of 1a during the binding with the target, thus
making the two molecules capable of establishing the same binding interactions. However, we
speculate that the tert-butyl amide 3b is an interesting compound since it displayed an ac-arrhythmia
threshold increase of about 50% at a concentration (0.5 M) lower than those of all other analogues.
Therefore, although less potent than derivatives 2a–e, amides 3a–e displayed the same trend of
activity, with the tert-butyl and benzyl analogues (2b,e and 3b,e) being the more potent of the
corresponding series. Conversely, compounds 1a–e, devoid of the tetramethylpyrroline moiety,
showed a completely different binding behavior, being the phenyl analogue 1c the most potent. This
finding suggests that the presence of the tetramethylpyrroline moiety could force the two series 2a–e
and 3a–e to establish the same binding poses within the target receptor. On the other hand, the
differences in potency hierarchy found between the two series (2b > 2e > 2c  2d  2a vs. 1c > 1b 
1e > 1a  1d) suggest that the amides 2a–e and 3a−e would share some binding sites with the
precursor 1a–e series but some differences in binding may be taken into account, with the
carboxamide group playing a major role (see above). To further explore the contribution of each
constituting moiety, a group efficiency (GE = ΔΔG/Δnumber of non-hydrogen atoms =
Δ1.37pEC50/ΔHA)[27,28] analysis on 2e was performed (Figure 2). Three mains putative
pharmacophoric elements were captured ― the xylyloxy substituent, the tetramethylpyrroline ring,
and the substituent at the asymmetric carbon atom ― and were color coded in red, green, and blue,
respectively. The GE analysis indicated that the substituent on the stereogenic center gave the highest
contribution (GE = 0.26) while the tetramethylpyrroline ring displayed the lowest GE value (0.14).
This could mean that the non-hydrogen atoms (HA) constituting the latter moiety contribute less
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efficiently to the binding of 2e with respect to the xylyloxy and the phenyl substituents (GE = 0.26
and 0.22, respectively). In Figure 3, the global minimum conformer electrostatic potential maps (DFT
B3LYP/6-31G*//DFT B3LYP/6-31G*) of compounds 2a−e are captured in a cartoon representing all
pharmacophoric elements discussed so far interacting with corresponding hypothetical binding sites.
In particular, we suggest that the substituent at the stereogenic center would be recognized by a
roughly spherical pocket that would be fully occupied by the tert-butyl group (2b) while resulting
relatively large (2a, 2c, and 2d) or narrow (2e) for the other substituents. The cartoon might also
explain why 2c is the only tetramethylpyrroline derivative less active than the parent compound (1c):
it may be hypothesised that the contemporary presence of three contiguous, flat and rigid moieties
(viz., phenyl substituent, amide group, and tetramethylpyroline ring) impedes the correct fit for
compound 2c.
To better define the cardiac profile of the pyrroline derivatives, their influence on additional cardiac
parameters was compared with that elicited by the reference compound, mexiletine (1a) (Table 4).
Pyrroline derivatives 2a–e decreased the developed tension on driven left atria less than mexiletine
did, except for the isopropyl derivative 2d whose effect was comparable with that of mexiletine. On
the other hand, the corresponding amides 3a–e showed a strong activity with EC50 values within the
range of 0.0019 and 0.11 µM. The most potent compound was 3b showing a negative inotropism 24-
fold higher than that of mexiletine. Regarding the negative chronotropic activity on spontaneously
beating right atrium, only compounds 2b and 2c showed a weak activity, which was 55-fold and 130-
fold lower than that of mexiletine. Furthermore, almost all compounds showed negative inotropism on
spontaneously beating right atrium even though with different degree of potency, ranging from 0.049
M for 3c to 1.48 M for 2a. It is noteworthy that this effect was not recorded for mexiletine and
compounds 2b and 2c due to their insignificant inotropism resulting from chronotropic effect.
Moreover, all the new pyrroline derivatives were tested on K+-depolarized (80 mM KCl) guinea pig
aortic strips to assess their vasorelaxant activity. Data are shown in Table 5 with mexiletine as the
reference drug. For all compounds, the intrinsic vasorelaxant activity percentage on aorta was lower
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than 30% and thus unremarkable. In order to rule out a possible inhibitory effect on nonvascular
smooth muscle, such as that displayed by Ca2+ channel antagonists, further investigation on relaxant
activity using K+-depolarized (80 mM KCl) guinea pig ileum longitudinal smooth muscle (GPILSM)
was pursued (Table 5). With regard to the pyrroline derivatives 2a–e, the observed profile of intrinsic
activity and potency for 2a and 2d was substantially similar to that displayed by mexiletine, while
compounds 2b, 2c and 2e showed a relaxant potency slightly higher than that of mexiletine, but in the
micromolar range. Overall, all pyrroline derivatives 2a–e appears to display a higher intrinsic activity
in nonvascular than vascular smooth muscle cells. The effect of these compounds on guinea pig ileum
longitudinal smooth muscle suggests a conceivable involvement of L-type calcium channels.
Notably, among the newly synthesized compounds, 2b and 2e were the most interesting mexiletine
derivatives since they displayed both the highest antiarrhythmic activity and the best cardiovascular
profile, showing both negative inotropism and cronotropism lower than the parent compound and a
negligible vasorelaxant activity on guinea-pig aortic strips.
2.2.2 Voltage-gated sodium channel blocking activity
Thus, they were tested in vitro on voltage-gated sodium currents recorded in HEK293 cells transiently
transfected with the human cardiac sodium channel, hNav1.5, using the whole-cell patch-clamp
method, in order to investigate the contribution of the block of this channel to the observed
antiarrhythmic activity. Both the compounds produced a concentration- and use-dependent inhibition
of sodium currents (Figure 3). Compared to the reference compound, mexiletine, tonic block
measured at 0.1 Hz frequency was increased about two-fold for 2e and four-fold for 2b (Table 6).
However, 2e was more use-dependent than mexiletine and 2b were; the IC50 ratios were similar for
mexiletine and 2b, whereas those of 2e were greater by 4.5-fold and about 7-fold at 2 and 10 Hz,
respectively, than those of 2b. Conceivably, increased lipophilia of the tested compounds, compared
to that of mexiletine (Table 2), may contribute to the increased tonic block exerted by the two
compounds. Such a behavior was observed with other compounds containing two aromatic rings, such
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as diphenylhydramine and orphenadrine.[29,30] These results are in line with what was found in our
previous studies.[31,32] The increased use-dependence of 2e suggests a stronger interaction with the
sodium channel in the inactivated state. Since the hierarchy of potency on NaV1.5 (2e > 2b) appeared
as opposite to that observed in the antiarrhythmic activity assay (2b > 2e), the sodium channel
blocking activity of our mexiletine derivatives, while possibly contributing to the observed cardiac
effects, is not sufficient to completely interpret the pharmacological profile of 2a−e.
2.2.3 Antioxidant activity
The in vitro antioxidant activity of the compounds under study (2a–e, 3a–e) was evaluated by means
of the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) cellular-based assay by measuring the
reducing effect of the test compounds against oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH)
to the fluorescent probe 2′,7′-dichlorofluorescein (DCF). Mexiletine was chosen as reference
compound. Results are reported in Table 7. Tests were performed on human hepatocellular liver
carcinoma (HepG2) cells because they have an enhanced oxidative metabolism that causes cellular
oxidative stress and/or generates reactive metabolites. Thus, it may be assumed that HepG2 cells are
suited to study protection against oxidative and cytotoxic effects, if any. The results of the DCFH-DA
assay showed that all pyrroline derivatives 2a–e significantly reduce the H2O2-induced oxidation,
being slightly more potent than mexiletine. Conversely, none of the amides 3a–e did show any
appreciable antioxidant activity. Based on these results it is possible to assume that the 2,6-
dimethylphenoxy moiety is crucial for the antioxidant activity as well as for the antiarrhythmic one.
Finally, the ability of mexiletine and some synthesized compounds (2a–d, 3a) to react against
hydroxyl radical using the antioxidant-sensitive non fluorescent probe benzoic acid in the presence of
hydroxyl radicals was measured. The results revealed that all tested compounds scavenge hydroxyl
radicals (IC50 about 70 µM), being slightly more active than mexiletine (IC50 93 ± 9 µM). The same
test run on 3a (IC50 198 ± 4µM) underlines once again the importance of the 2,6-dimethylphenoxy
moiety in the biological profile of such compounds.
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3. Conclusion
A small series of tetramethylpyrroline derivatives have been synthesized and 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. They all performed as potent
antiarrhythmic agents, even if with different degrees of potency. The observed activities were
probably related to the block of cardiac sodium and calcium channels. The most interesting
tetramethylpyrroline congener was the tert-butyl-substituted analog 2b, which was at least 100 times
more active as an antiarrhythmic than our lead compound mexiletine (1a). 2b also displayed the best
cardiovascular profile, showing both negative inotropism and cronotropism lower than the parent
compound and a negligible vasorelaxant activity on guinea-pig aortic strips. Interestingly, 2b presents
the highest fraction of sp3 hybridised carbon atoms (Fsp3) in the congeneric series 2a−e. A four-point
interaction with voltage-gated sodium and calcium channels was supposed to account for the observed
antiarrhythmic activity, with the latter probably playing a major role. This implies that chirality may
offer a way to further improve the pharmacological profile of the stated compounds. Both high Fsp3
and the presence of stereogenic centers have been related to clinical developability. The group
efficiency (GE) analysis indicated the aryloxy moiety as a pharmacophoric group and we have
previously reported that the introduction of a hydroxyl group onto the 3-position of the aromatic ring
of mexiletine improves potency on voltage-gated sodium channels while reducing the feared blocking
activity on hERG. Interestingly, the 3-hydroxy analog of mexiletine displayed stereoselectivity of
action as an antiarrhythmic (R > S). Since 2a−e displayed antioxidant activity, this series of mexiletine
derivatives may be considered as a starting point toward the development of dual-acting drugs
designed to obtain a combined activity on arrhythmias in the ischemic heart.
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4. Experimental protocols
4.1. Chemistry
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. Melting
points were determined on a Gallenkamp apparatus in open glass capillary tubes and are uncorrected.
1H NMR and 13C NMR spectra were recorded on either a Varian VX Mercury spectrometer operating
at 300 and 75 MHz for 1H and 13C, respectively, or an AGILENT 500 MHz operating at 500 and 125
1
MHz for H and 13C, respectively, using CDCl3 and CD3OD as solvents. Chemical shifts (δ) are
reported in ppm relative to the residual non-deuterated solvent resonance: CDCl3, δ = 7.26 (1H NMR)
and  = 77.3 (13C NMR); CD3OD, δ = 3.30 (1H NMR) and  = 47.8 (13C 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.[37] TLC analyses were performed on precoated silica
gel on aluminium sheets (Kieselgel 60 F254, Merck).
3,5-Dibromo-2,2,6,6-tetramethylpiperidin-4-one (4)
The title compound was prepared according to the literature procedure.[20] To a solution of 2,2,6,6-
tetramethylpiperidin-4-one (2.0 g) in 10 ml of glacial acetic acid a solution of Br2 in 8 ml of glacial
acetic acid was added dropwise. The reaction mixture was heated at 60 °C overnight. Then the
reaction mixture was filtered and the solid was washed with acetic acid, H2O and finally with Et2O.
After dried at room temperature for 15 days, a white solid (3.712 g) was obtained: yield 73%; mp: >
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250 °C ; 1H NMR (300 MHz, CD3OD): δ 1.45 (s, 6H, CH3), 1.83 (s, 6H, CH3), 5.47 (s, 2H, CH); 13C
NMR (75 MHz, CD3OD): δ 22.9 (2C), 28.0 (2C), 59.1 (2C), 65.85 (2C), 189.1 (1C). Other
spectroscopic data were in agreement with the literature.[20]
2,2,5,5-Tetramethyl-N-[1-(2,6-dimethylphenoxy)propan-2-yl]-2,5-dihydro-1H-pyrrole-3-
carboxamide (2a) To a solution of 1a.HCl (0.300 g, 1.4 mmol) in 10 mL of water, cooled in an ice
bath, trimethylamine (0.495 g, 4.9 mmol) was added. After removing the ice bath, 3,5-dibromo-
2,2,6,6-tetramethylpiperidin-4-one (4) (0.700 g, 1.7 mmol) was added in small portions over a period
of 6 hours. The reaction mixture was stirred at 50 °C for 24 h, then it was made alkaline with 6N
NaOH and extracted with EtOAc. Removal of the solvent under vacuo gave 0.500 g (61%) of 2a as a
yellow solid which was recrystallized from EtOAc/hexane. White crystals: yield: 61%; mp: 131–132
1
°C (EtOAc/hexane). GC-MS (70 eV) m/z (%) 315 (M+ –15, 45), 110 (100). H NMR (300 MHz,
CDCl3): δ 1.44–1.50 (m, 9H, 2CH3C + CH3CH), 1.62 (d, J = 3.9 Hz, 6H, 2CH3C), 2.26 (s, 6H,
CH3Ar), 2,95 (br s, 1H, NH), 3.74 (dd, J = 3.0, 9.1 Hz, 1H, CHH), 3.86 (dd, J = 3.9, 9.1 Hz, 1H,
CHH), 4.36–4.41 (m, 1H, CHCH2), 6.07 (s, 1H, CH), 6.26 (br d, J = 8.3 Hz, 1H, NHCO), 6.93 (t, J =
7.3 Hz, 1H, CHAr), 7.01 (d, J = 7.2 Hz, 2H, CHAr); 13C NMR (75 MHz, CDCl3): δ 16.4 (2C), 18.3
(1C), 30.3 (4C), 45.4 (1C), 63.9 (1C), 67.5 (1C), 74.0 (1C), 124.4 (1C), 129.3 (2C), 130.9 (2C), 140.2
.
(1C), 143.5 (1C), 155.0 (1C), 164.6 (1C). Anal. (C20H30N2O2 0.5H2O) C, H, N.
2,2,5,5-Tetramethyl-N-[3,3-dimethyl-1-(2,6-dimethylphenoxy)butan-2-yl)-2,5-dihydro-1H-
pyrrole-3-carboxamide (2b)
White crystals: mp 87–88 °C (EtOAc/hexane); GC-MS (70 eV) m/z (%) 357 (M+, –15, 100); 1H NMR
(300 MHz, CDCl3): δ 1.08 (s, 9H, CH3C), 1.30 (d, J = 6.3 Hz, 6H, CH3CNH), 1.42 (s, 6H, CH3CNH),
1.49 (s, 6H, CH3CNH), 1.78 (br s, 1H, NH), 2.15 (s, 6H, CH3Ar), 3.75 (dd, J = 9.6, 3.9 Hz, 1H,
CHH), 4.02 (dd, J = 9.9, 3.8 Hz, 1H, CHH), 4.06–4.97 (m, 1H, CHCH2), 6.11 (s, 1H, CH), 6.33 (br d,
13
J = 8.9 Hz, 1H, NHCO), 6.90–6.99 (m, 3H, CHAr); C NMR (75 MHz, CDCl3): δ 16.9 (2C), 27.6
12
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(3C), 30.1 (2C), 30.3 (2C), 34.9 (1C), 56.5 (1C), 64.3 (1C), 67.9 (1C), 71.3 (1C), 124.4 (1C), 129.3
.
(2C), 130.8 (2C), 139.2 (1C), 143.8 (1C), 155.5 (1C), 164.9 (1C). Anal. (C23H36N2O2 0.5H2O) C, H,
N.
2,2,5,5-Tetramethyl-N-[2-(2,6-dimethylphenoxy)-1-phenylethyl]-2,5-dihydro-1H-pyrrole-3-
carboxamide (2c)
Yellow crystals: mp 133–134 °C (EtOAc/petroleum ether); GC-MS (70 eV) m/z (%) 377 (M+ –15,
1
52), 110 (100). H NMR (300 MHz, CDCl3): δ 1.33 (s, 6H, CH3C), 1.48 (d, J = 3.9 Hz, 6H, CH3C),
2.11 (s, 7H, CH3Ar + NH), 4.03 (dd, J = 9.6, 4.1 Hz, 1H, CHH), 4.12 (dd, J = 9.4, 4.4 Hz, 1H, CHH),
5.34–5.44 (m, 1H, CHCH2), 6.19 (s, 1H, CH), 6.78 (br d, J = 8.0 Hz, 1H, NHCO), 6.90–6.99 (m, 3H,
13
CHAr), 7.31–7.47 (m, 5H, CHAr); C NMR (75 MHz, CDCl3): δ 16.4 (2C), 30.3 (2C), 30.4 (2C),
53.4 (1C), 63.9 (1C), 67.4 (1C), 74.1 (1C), 124.5 (1C), 127.1 (2C), 128.0 (2C), 128.9 (1C), 129.3
(2C), 130.9 (2C), 139.7 (1C), 140.8 (1C), 143.4 (1C), 155.0 (1C), 164.7 (1C). Anal.
.
(C25H32N2O2 05H2O) C, H, N.
2,2,5,5-Tetramethyl-N-[1-(2,6-dimethylphenoxy)-3-methylbutan-2-yl]-2,5-dihydro-1H-pyrrole-
3-carboxamide (2d)
White crystals: mp 96–97 °C (EtOAc/hexane); GC-MS (70 eV) m/z (%) 343 (M+ –15, 60), 110 (100).
1H NMR (500 MHz, CDCl3): δ 1.05 (d, J = 4.1 Hz, 3H, CH3CHCH3), 1.11 (d, J = 3.8 Hz, 3H,
CH3CHCH3), 1.32 (d, J = 4.7 Hz, 6H, CH3C), 1.47 (s, 3H, CH3C), 1.50 (s, 3H, CH3C), 2.18–2.23 (m,
1H, CHCH3), 3.78 (dd, J = 9.3, 3.9 Hz, 1H, CHH), 3.99 (dd, J = 9.3, 2.9 Hz, 1H, CHH), 4.02–4.05
(m, 1H, CHCH2), 6.09 (s, 1H, CH), 6.25 (br d, J = 9.3 Hz, 1H, NHCO), 6.93 (t, J = 7.6 Hz, 1H,
13
CHAr), 7.00 (d, J = 7.3 Hz, 2H, CHAr); C NMR (125 MHz, CDCl3): δ 16.4 (2C), 19.3 (1C), 20.0
(1C), 29.3 (2C), 30.1 (2C), 30.2 (1C), 54.5 (1C), 63.7 (1C), 67.3 (1C), 71.2 (1C), 124.2 (1C), 129.1
13
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.
(2C), 130.6 (2C), 139.5 (1C), 143.6 (1C), 155.0 (1C), 164.7 (1C). Anal. (C22H34N2O2 0.5H2O) C, H,
N.
2,2,5,5-Tetramethyl-N-[1-(2,6-dimethylphenoxy)-3-phenylpropan-2-yl)-2,5-dihydro-1H-pyrrole-
3-carboxamide (2e)
White crystals: mp 159–160°C (EtOAc/hexane); GC-MS (70 eV) m/z (%) 391 (M+ –15, 32), 110
1
(100). H NMR (500 MHz, CDCl3): δ 1.32 (s, 6H, CH3C), 1.41 (s, 3H, CH3C), 1.44 (s, 3H, CH3C),
2.26 (s, 6H, CH3Ar), 3.16 ( d, J = 7.3 Hz, 2H, CH2Ar), 3.80–3.88 (m, 2H, CH2CH), 4.54–4.62 (m, 1H,
CH), 5.97 (s, 1H, NH), 6.23 (br d, J = 8.3 Hz, 1H, NHCO), 6.93 (t, J = 6.6 Hz, 1H, CHAr), 7.00 (d, J
13
= 7.8 Hz, 2H, CHAr), 7.21–7.27 (m, 2H, CHAr), 7.28–7.34 (m, 3H, CHAr); C NMR (125 MHz,
CDCl3): δ 16.4 (2C), 29.8 (2C), 29.9 (2C), 37.7 (1C), 50.5 (3C), 71.7 (1C), 124.2 (1C), 126.7 (1C),
128.6 (2C), 129.1 (2C), 129.2 (2C), 130.6 (2C), 137.7 (1C), 139.6 (2C), 143.2 (2C), 154.9 (1C), 164.5
.
(1C). Anal. (C26H34N2O2 0.5H2O) C, H, N.
2,2,5,5-Tetramethyl-N-(propan-2-yl)-2,5-dihydro-1H-pyrrole-3-carboxamide (3a)
White crystals: mp 147–148 °C (EtOAc/hexane); GC-MS (70 eV) m/z (%) 195 (M+ –15, 4), 110
(100); 1H NMR (300 MHz, CD3OD): δ 1.18 (d, J = 6.6 Hz, 6H, CH3CH), 1.62 (s, 6H, CH3C), 1.71 (s,
6H, CH3C), 3.95–4.12 (m, 1H, CHCH3), 6.38 (s, 1H, CH); 13C NMR (75 MHz, CD3OD): δ 21.2 (2C),
26.0 (2C), 26.2 (1C), 41.5 (1C), 68.5 (1C), 71.9 (1C), 135.7 (1C), 138.7 (1C), 162.4 (1C). Anal.
(C12H22N2O) C, H, N.
2,2,5,5-Tetramethyl N-(3,3-dimethylbutan-2-yl)-2,5-dihydro-1H-pyrrole-3-carboxamide (3b)
White crystals: mp 96–98 °C (EtOAc/hexane); GC-MS (70 eV) m/z (%) 286 (M+ –15, 3), 110 (100);
1H NMR (500 MHz, CDCl3): δ 0.91 (s, 9H, CH3C), 1.08 (d, J = 6.8 Hz, 3H, CH3CH), 1.29 (s, 6H,
CH3CN), 1.43 (d, J = 4.9 Hz, 6H, 2CH3CH), 1.85 (br s, 1H, NH), 3.90–3.97 (m, 1H, CHCH3), 5.52
14
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(br d, J 9.3 Hz, 1H, NHCO), 5.99 (s, 1H, CH); 13C NMR (125 MHz, CDCl3): δ 16.2 (1C), 26.2 (3C),
30.1 (2C), 30.2 (2C), 34.3 (1C), 52.3 (1C), 63.5 (1C), 67.0 (1C), 139.0 (1C), 144.1 (1C), 164.6 (1C).
Anal. (C15H28N2O.0.33H2O) C, H, N.
2,2,5,5-Tetramethyl-N-(1-phenylethyl)-2,5-dihydro-1H-pyrrole-3-carboxamide (3c)
White crystals: mp 145–147°C (EtOAc/hexane); GC-MS (70 eV) m/z (%) 257 (M+ –15, 3), 110 (100);
1H NMR (300 MHz, CDCl3): δ 1,26 (d, J = 4.1 Hz, 6H, CH3C), 1,42 (d, J = 6.1 Hz, 6H, CH3C), 1,52
(d, J = 6.9 Hz, 3H, CH3CH), 1.76 (br s, 1H, NH), 5.08–5.22 (m, 1H, CHCH3), 5.98 (br d, J 7.4 Hz,
1H, NHCO), 6.03 (s, 1H, CH), 7.22–7.40 (m, 5H, CHAr); 13C NMR (75 MHz, CDCl3): δ 22.0 (1C),
30.4 (2C), 30.5 (2C), 48.8 (1C), 63.7 (1C), 67.3 (1C), 126.4 (1C), 127.6 (2C), 129.0 (2C), 140.1 (1C),
143.4 (1C), 143.7 (1C), 164.5 (1C). Anal. (C17H24N2O.0.33H2O) C, H, N.
2,2,5,5-Tetramethyl-N-(3-methylbutan-2-yl)-2,5-dihydro-1H-pyrrole-3-carboxamide (3d)
1
White crystals: mp 126–127 °C (EtOAc); GC-MS (70 eV) m/z (%) 238 (M+ –15, 25), 110 (100); H
NMR (300 MHz, CDCl3): δ 0.90 (dd, J = 6,8, 1.8 Hz, 6H, CH3CH), 1.09 (d, J = 6.9 Hz, 3H,
CH3CHN), 1.27 (s, 6H, CH3C), 1.42 (d, 6H, J = 1.9 Hz, CH3C), 1.78 (br s, 1H, NH), 3.80–3.98 (m,
1H, CHCH3), 5.52 (br d, J 8.3 Hz, 1H, NHCO), 5.99 (s, 1H, CH); 13C NMR (75 MHz, CDCl3): δ 17.6
(1C), 18.7 (1C), 18.8 (1C), 30.3 (1C), 30.4 (1C), 30.5 (2C), 33.3 (1C), 49.9 (1C), 63.7 (1C), 67.2
(1C), 139.4 (1C), 144.2 (1C), 164.9 (1C). Anal. (C14H26N2O) C, H, N.
2,2,5,5-Tetramethyl-N-(1-phenylpropan-2-yl)-2,5-dihydro-1H-pyrrole-3-carboxamide (3e)
White crystals: mp 149–150°C (EtOAc/hexane); GC-MS (70 eV) m/z (%) 271 (M+ –15, 79), 110
1
(100); H NMR (500 MHz, CDCl3): δ 1.15 (d, J = 6,4 Hz, 3H, CH3CH), 1.27 (d, J = 2.9 Hz, 6H,
CH3C), 1.40 (s, 6H, CH3C), 1.93 (br s, 1H, NH), 2.77 (dd, J = 13.7, 7.3 Hz, 1H, CHH), 2.86 (dd, J =
15
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13.5, 5.7 Hz, 1H, CHH), 4.27–4.33 (m, 1H, CHCH3), 5.53 (br d, J = 7.8 Hz, 1H, NHCO), 5.92 (s, 1H,
CHCN), 7.18–7.26 (m, 3H, CHAr), 7.28–7.31 (m, 2H, CHAr); 13C NMR (125 MHz, CDCl3): δ 20.0
(1C), 30.0 (2C), 30.1 (2C), 42.3 (1C), 45.9 (1C), 63.5 (1C), 67.0 (1C), 126.5 (1C), 128.4 (1C), 129.5
(1C), 137.9 (2C), 139.5 (2C), 143.6 (1C), 164.5 (1C). Anal. (C18H26N2O.0.25H2O) C, H, N.
4.2. Pharmacology
4.2.1. Details for functional studies
The pharmacological profile of all compounds was derived on guinea-pig isolated left and right atria
to evaluate their inotropic and/or chronotropic effects, respectively, and on K+-depolarized (80 mM)
guinea-pig vascular (aortic strips) and non-vascular [ileum longitudinal smooth muscle (GPILSM)] to
assess the calcium antagonist activity. The antiarrhythmic activity of all compounds was tested on
isolated guinea pig left atria driven at 1 Hz. Compounds were checked at increasing doses to evaluate:
i) antiarrhythmic activity, inducing arrhythmias by application of sinusoidal alternating current (50
Hz) of increasing strength to the isolated left atria driven at 1 Hz and assessing the “threshold of ac-
arrhythmia” (the current strength at which extra beats occurs) before and following the compound was
added to the tissue bath[38], ii) the percent decrease of developed tension on isolated left atrium driven
at 1 Hz and on spontaneously beating right atrium (negative inotropic activity), iii) the percent
decrease in atrial rate on spontaneously beating right atrium (negative chronotropic activity), and iv)
the percent inhibition of calcium-induced contraction on K+-depolarized aortic strips and GPILSM
(vascular and non-vascular relaxant activity respectively). Details have been reported on supporting
information. Data were analyzed using Student’s t-test and are presented as mean ± S.E.M.[39] Since
the analyzed compounds 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[39] or GraphPad Prism® software.[40,41]
16
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4.2.2. Details for patch clamp experiments.
Effects of compounds on human cardiac sodium channels were investigated as previously
described.[29,42] Transient transfection of the HEK293 cells (human embryonic kidney cell line) was
achieved using the calcium-phosphate co-precipitation method. The cells were incubated for 24 hours
with 0.1 µg/ml of the full-length SCN5A cDNA, encoding the α-subunit of the human cardiac sodium
channel subtype hNav1.5, and 0.05 µg/ml of a plasmid expressing the CD8 receptor and the auxiliary
human voltage-gated sodium channel β1 subunit (pCD8-IRES-hβ1). Cells marked with anti-CD8
antibody-coated microbeads (Dynal-Invitrogen, Milan, Italy) were used for patch clamp experiments
48−72 h after transfection. Whole-cell sodium currents were recorded at room temperature (20−22
°C) using Axopatch-1D amplifier (Axon Instruments Inc., Union City, CA, USA) and pClamp suite
software (Axon Instruments). The composition of the bath solution was (mM): 150 NaCl, 4 KCl, 2
CaCl2, 1 MgCl2, 5 HEPES and 5 glucose (pH 7.4). The pipette solution contained (mM): 120 CsF, 10
CsCl, 10 NaCl, 5 EGTA, and 5 HEPES (pH 7.2). With these solutions, pipette resistance was 2-4 mV.
After stabilization of the whole-cell configuration, sodium currents were elicited by a 25 ms-long test
pulse to -30 mV from a holding potential of −120 mV applied at 0.1, 2, and 10 Hz frequencies. The
patched cell was exposed to a continuous stream of control or drug-supplemented bath solution. To
limit bias due to rundown and shifts of channel voltage dependence, only two drug concentrations
were tested on each cell. The IDRUG/ICTRL ratio (mean ± S.E.M. from 3-8 cells) was reported as a
function of drug concentration. The relationships were fitted to a first-order binding function, Idrug
/
Ictrl = 1/{1 + ([drug]/IC50)nH}, allowing the calculation of half-maximum inhibitory concentration
(IC50) and slope factor (nH). The fit parameters are given with the standard error of the fit.
4.2.3. Details for antioxidant activity studies
4.2.3.1. Culture cells
17
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Human hepatocellular liver carcinoma (HepG2) cells (Sigma–Aldrich, St. Louis, MO) 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, Pasching, Austria), L-glutamine
(2 mM) (Sigma–Aldrich, St. Louis, MO), penicillin (100 g/ml) and streptomycin (100 g/ml)
(Sigma–Aldrich, St. Louis, MO) and incubated at 37 °C in a humid atmosphere of 5% CO2. For cell
assays, cells were trypsinized using Trypsin-EDTA 1X in PBS (Aurogene) and plated in 96-well
plates at a density of 10 000 cells per well in 125 μL of cell culture medium.
4.2.3.2. Detection of ROS generation
Generation of ROS was monitored using an oxidation-sensitive fluorescent probe, 2′,7′-
dichlorodihydrofluorescein diacetate (DCFH-DA, D6665; Sigma–Aldrich, St. Louis, MO) by slightly
modifying the procedure reported by Wang and James.[43] Briefly, viable cells (104/well) were seeded
in a black 96-well cell culture plate (PerkinElmer USA) and after 24 h were incubated with different
concentrations (10–100 µg/ml) of the tested compounds for 2 h at 37 °C in 5% CO2. DCFH-DA (50
μM final concentration) in medium without serum was added directly to each well, and the plate was
incubated at 37 °C for 30 min at 37 °C in 5% CO2. After washing using PBS, 100 μM H2O2 in PBS
was added to each well and the cells were incubated for an additional 30 min. The formation of
fluorescent dichlorofluorescein (DCF) due to oxidation of DCFH in the presence of ROS, was read at
480 nm using a multilabel plate counter Victor3 V (PerkinElmer) and DMSO medium was used for
control cells. At least three independent experiments with sex replicates were carried out, and the
results were averaged. Data were analyzed using Student’s t-test and are presented as mean ± S.E.M.
The mean difference between the control and experimental values at each concentration were tested
for a P value < 0.05.
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4.2.3.3. Measurement of hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity was measured according to the method of Toth et al.,[15]
with some modifications. Briefly, 3 mL of a solution containing 40 mM sodium phosphate buffer (pH
3), 0.1 mM benzoic acid, 0.02 mM hydrogen peroxide, 0.04 mM Fe(II)-EDTA, and tested compounds
(dissolved in DMSO) in different concentrations was incubated for 30 min at 37 °C. The fluorescence
was then monitored at the excitation wavelength of 305 nm and the emission wavelength of 407 nm
by a LS 55 Luminescence spectrometer (Perkin Elmer). All the tests were performed in triplicates and
reported as mean values.
4.3. Quantum mechanical calculations
The previously developed procedures were followed.[16,19,25,36] Briefly, the models of protonated
compounds 2a−e were generated from the atomic fragments incorporated into Spartan’16
(Wavefunction Inc., Irvine, CA) inner fragment library and assuming the suggested default starting
geometries. The generated geometries were optimized by the molecular mechanics MMFF routine
offered by the software[44] and then submitted to a systematic conformational distribution analysis
using the default step sizes. All conformers in a window of 10 Kcal/mol above the global minimum
conformer were retained. When two conformers differed by dihedral values lower than 10°, the less
stable conformer was left out. Conformers were then classified according to their ab initio gas phase
energy content calculated at the RHF/3-21G*level. All conformers falling within a window of 5
kcal/mol above global minimum were retained and submitted to RHF/3-21G* geometry optimization.
After removal of redundant conformers (i.e., each conformer differing from a more stable one by less
than 5° in their corresponding dihedral values), the single point energy content for all the remaining
conformers were calculated at the RHF/6-31G** level. The so-obtained set of conformers underwent
geometry optimization by density functional theory (DFT) implemented in Spartan’16 with B3LYP
functional[45] and the 6-31G* basis set[46] in the gas phase. The optimized structures were confirmed
19
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as real minima by IR frequency calculation (DFT B3LYP/6-31G*// DFT B3LYP/6-31G*). All
simulations were performed in vacuum.
R
O
R
O
O
N
H
NH2
N
H
1a-e
2a-e
a: R = CH3
b: R = tBu
c: R = Ph
d: R = iPr
e: R = Bn
R
O
N
H
N
H
3a-e
Figure 1. Structures of mexiletine analogues (1a–e), their pyrroline derivatives (2a–e) and amides
(3a–e).
20
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Figure 2. Group efficiency (GE) analysis on 2e: GE = ΔΔG/Δnumber of non-hydrogen atoms =
Δ1.37pEC50/ΔHA.[45]
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Figure 3. Putative pharmacophoric elements and interacting binding sites for compounds 2a-e
represented as the corresponding most stable conformers wrapped in their electrostatic potential maps
(DFT B3LYP/6-31G*//DFT B3LYP/6-31G*); for the sake of simplicity, the homochiral R series only
was considered; HB, hydrogen bond forming site.
22
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Figure 4:
Effects of mexiletine, 2b, and 2e on hNav1.5 channels: left column shows representative sodium
current traces elicited from a holding potential of −120 mV to a test potential of −30 mV in the
absence of drug (ctrl) and in the presence of 10 μM drug at 0.1, 2 or 10 Hz frequency stimulation.
right column reports the corresponding concentration−response relationships fitted with a first–order
binding equation described in material and methods. Each point is the mean ± SEM from 3-8 cells.
23
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ChemMedChem
O
O
R
O
Br
Br
O
i
ii
N
H
N
H
N
H
N
H
4
2a-e
iii
a: R = CH3
b: R = tBu
c: R = Ph
d: R = iPr
e: R = Bn
R
O
N
H
N
H
3a-e
Scheme 1. Reagents and conditions: (i) Br2, AcOH, 60 °C; (ii) 1a–e, TEA, H2O, 50 °C; (iii)
NH2CH(CH3)R, TEA, H2O, 50 °C
24
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Table 1. Anti-arrhythmic activity of compounds 2a–e, 3a–e, mexiletine 1a and its analogues 1b–e
b
Compd
Max % increasea
(mean ± SEM)
EC50
95% confidence limit
(µM)
(x 10–6)
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
90 ± 2.9c
46 ± 2.9c
144 ± 4.3d
48 ± 1.3e
281 ± 9.1
16 ± 0.7
5.73
0.054
4.04
4.57–7.19
0.024–0.12
3.05–5.83
1.01–1.74
76 ± 1.4c
25 ± 1.6
1.33
74 ± 1.9f
142 ± 6.5
0.41
8.07
11.61
2.11
0.43
18.4
4.36
0.37–0.83
6.38–8.94
8.71–13.47
1.38–3.20
0.34–0.54
14.9–22.7
1.47–6.38
mexiletine, 1a
1b
64 ± 1.4g
71 ± 2.4c
1c
1d
1e
135 ± 2.2f
145 ± 4g
169 ± 4c
aIncrease of threshold of ac-arrhythmia: increase in the current strength of 50 Hz alternating current required to produce
arrhythmia in guinea pig left atria driven at 1 Hz in the presence of each tested compounds at 5x10–5 M. For all data P <
0.05. bCalculated from log concentration-response curves (Probit analysis according to Litchfield and Wilcoxon[39] with
c
n = 6–8). When the maximum effect was < 50%, the EC50 values were not calculated. At 10–5 M. dAt 10–6 M. eAt 5
x10–7 M. fAt 5 x 10–6 M. gAt 10–4 M.
25
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Table 2. Physicochemical descriptors of compounds
under study
a
a
Compd
pKa
Log Pa
Log D7.4
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
9.0 ± 0.7
9.0 ± 0.7
9.0 ± 0.7
9.0 ± 0.7
8.9 ± 0.7
9.0 ± 0.7
9.0 ± 0.7
9.0 ± 0.7
9.0 ± 0.7
9.0 ± 0.7
5.0 ± 0.5
2.7 ± 0.4
6.2 ± 0.5
3.0 ± 0.4
6.3 ± 0.6
4.0 ± 0.5
5.8 ± 0.5
2.7 ± 0.4
6.6 ± 0.6
4.2 ± 0.5
3.4
1.1
4.6
1.4
4.8
2.5
4.3
1.1
5.1
2.6
mexiletine, 1a
8.6 ± 0.1
8.8 ± 0.2
8.5 ± 0.3
8.7 ± 0.1
7.8 ± 0.1
2.2 ± 0.2
3.4 ± 0.2
3.3 ± 0.3
3.0 ± 0.2
3.9 ± 0.2
1.0
2.0
2.2
1.7
3.3
1b
1c
1d
1e
aACDlabs 7.09, Toronto, Canada.
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Table 3. Conformational analysis on 2a−e in vacuum.
Compd
no. of conformersa
Boltzmann weights
s-trans
1.00
s-cis
0.00
0.14
0.00
0.02
0.04
2a
2b
2c
2d
2e
4
6
0.86
6
1.00
17
0.98
18
0.96
aTrue conformers found in a 5 Kcal/mol window above the global minimum conformer energy
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Table 4. Influence of tested compounds on cardiovascular parameters.
Left atrium
Right atrium
negative inotropy
negative inotropy
negative chronotropy
95%
conf lim
(x10-6)
95%
conf lim
(x10-6)
95%
b
b
b
Activitya
EC50
Activityc
EC50
Activityd
EC50
(µM)
Compd
conf lim
(x10-6)
(M ± S.E.M.)
(µM)
(M ± S.E.M.)
(µM)
(M ± S.E.M.)
90 ± 1
55 ± 2e
92 ± 2
68 ± 2e
97 ± 1f
78 ± 3e
95 ± 1g
80 ± 2
95 ± 1h
87 ± 4
90 ± 1
0.034
0.0073
0.18
0.022–0.058
0.0045–0.011
0.13–0.21
79 ± 3i
80 ± 2
1.48
0.93–2.07
31 ± 2i
5.0 ± 0.1
54 ± 2i
2a
0.075
0.051–0.10
3a
1.54
3.64
1.11–2.13
3.04–4.35
2b
0.0019
0.12
0.0012–0.029
0.079–0.17
0.022–0.036
0.0074–0.022
0.024–0.062
0.98–1.59
76 ± 2
0.080
0.056–0.11
5.0 ± 0.2 j
62 ± 2h
3b
2c
0.028
0.012
0.038
1.25
72 ± 4h
75 ± 2g
62 ± 2j
89 ± 2g
88 ± 2
0.049
0.22
0.040–0.060
0.16–0.30
10 ± 1
3c
6.0 ± 0.3g
8.0 ± 0.5 j
44 ± 1g
2d
0.063
0.057
0.65
0.049–0.082
0.041–0.082
0.40–0.98
3d
2e
0.11
0.079–0.14
0.035–0.058
19 ± 1
3e
0.045
85 ± 3k
0.028
0.023–0.035
mexiletine
aDecrease in developed tension on isolated guinea-pig left atrium at 10–5 M, expressed as percent changes from the control
(n = 5-6). The left atria were driven at 1 Hz. The 10–5 M concentration gave the maximum effect for most compounds.
bCalculated from log concentration-response curves (Probit analysis by Litchfield and Wilcoxon[39] with n = 6-7). When
c
the maximum effect was < 50%, the EC50 inotropic and EC50 chronotropic values were not calculated. Decrease in
developed tension on guinea-pig spontaneously beating isolated right atrium at 5x10–5 M, expressed as percent changes
d
from the control (n = 7-8). The 5x10–5 concentration gave the maximum effect for most compounds. Decrease in atrial
rate on guinea-pig spontaneously beating isolated right atrium at 5x10-5 M, expressed as percent changes from the control
(n = 7-8). The 5x10–5 M concentration gave the maximum effect for most compounds. Pretreatment heart rate ranged from
e
g
j
165 to 190 beats/min. At the 5x10–7 M. fAt the 5x10–5 M. At the 10–6 M. hAt the 5x10–6 M. iAt 10–5 M. At 10–4 M. kAt
the 10–7
28
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10.1002/cmdc.202000692
ChemMedChem
Table 5. Relaxant activity of compounds 2a–e, 3a–e, mexiletine on K+-depolarized guinea pig
vascular and non-vascular smooth muscle.
Cmpd
Aorta
Activitya
(M ± S.E.M.)
10 ± 1
Ileum
IC50
(μM)
b
Activitya
(M ± S.E.M.)
97 ± 1
95% conf lim
(x10-6)
2a
12.22
9.23–15.32
3a
0.4 ± 0.1
19 ± 1c
3.0 ± 0.2c
27 ± 1
2b
74 ± 1d
3.93
4.16
9.67
2.22
8.52
3.02–5.04
3.27–4.98
2.48–15.07
1.03–3.48
6.54–11.09
3b
41 ± 2
2c
29 ± 2
96 ± 1d
39 ± 2
3c
2.0 ± 0.1
18 ± 1
2d
96 ± 2
3d
2e
0.20 ± 0.01
30 ± 3c
28 ± 2
60 ± 2d
3e
9 ± 1
33 ± 2
mexiletine
5.0 ± 0.3
81 ± 2
aPercent inhibition of calcium-induced contraction on K+-depolarized (80 mM) guinea pig aortic strips and
longitudinal smooth muscle at 10–4 M. The 10–4 M concentration gave the maximum effect for most compounds.
bCalculated from log concentration-response curves (Probit analysis by Litchfield and Wilcoxon[39] with n = 6–7).
When the maximum effect was < 50%, the IC50 values were not calculated. cAt the 5x10–5 M. dAt the 10–5 M.
Table 6. Half-maximal concentrations and slope factors for tonic block (0.1 Hz) and use-dependent
block (2 or 10 Hz) of hNav1.5 sodium currents by mexiletine, 2b, and 2e compounds. Fit parameter
values are given with standard error of the fit.
Cmpd
2b
frequency (Hz)
nH
1.2 ± 0.1
1.2 ± 0.1
1.1 ± 0.2
1.1 ± 0.2
0.9 ± 0.1
IC50 (µM)
28 ± 2
13 ± 1
6.6 ± 1.1
47 ± 7
4.8 ± 0.5
IC50 ratio
0.1
2
10
0.1
2
2.2
4.2
2e
9.8
10
0.8 ± 0.1
1.6 ± 0.1
29.4
mexiletine
0.1
2
1.0 ± 0.1
1.0 ± 0.1
93 ± 14
40 ± 3
2.3
5.5
10
0.9 ± 0.1
17 ± 2
29
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