Human iPSC-derived cardiomyocytes and pyridyl-phenyl mexiletine analogs

Article abstract of DOI:10.1016/j.bmcl.2021.128162

In the United States, approximately one million individuals are hospitalized every year for arrhythmias, making arrhythmias one of the top causes of healthcare expenditures. Mexiletine is currently used as an antiarrhythmic drug but has limitations. The purpose of this work was to use normal and Long QT syndrome Type 3 (LQTS3) patient-derived human induced pluripotent stem cell (iPSC)-derived cardiomyocytes to identify an analog of mexiletine with superior drug-like properties. Compared to racemic mexiletine, medicinal chemistry optimization of substituted racemic pyridyl phenyl mexiletine analogs resulted in a more potent sodium channel inhibitor with greater selectivity for the sodium over the potassium channel and for late over peak sodium current.

Full text of DOI:10.1016/j.bmcl.2021.128162

Bioorg. Med. Chem. Lett. 46 (2021) 128162
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Human iPSC-derived cardiomyocytes and pyridyl-phenyl
mexiletine analogs
Mark Johnsona, Jorge Gomez-Galenoa, Daniel Ryana, Karl Okolotowicza,
,
*
Wesley L. McKeithanb, Kevin J. Sampsonc, Robert S. Kassc, Mark Mercolab, John R. Cashmana
a Human BioMolecular Research Institute, 6351 Nancy Ridge Dr. Suite B, San Diego, CA 92121, USA
b Cardiovascular Institute and Department of Medicine, Stanford University, Stanford, CA 94305, USA
c Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the United States, approximately one million individuals are hospitalized every year for arrhythmias, making
arrhythmias one of the top causes of healthcare expenditures. Mexiletine is currently used as an antiarrhythmic
drug but has limitations. The purpose of this work was to use normal and Long QT syndrome Type 3 (LQTS3)
patient-derived human induced pluripotent stem cell (iPSC)-derived cardiomyocytes to identify an analog of
mexiletine with superior drug-like properties. Compared to racemic mexiletine, medicinal chemistry optimiza-
tion of substituted racemic pyridyl phenyl mexiletine analogs resulted in a more potent sodium channel inhibitor
with greater selectivity for the sodium over the potassium channel and for late over peak sodium current.
Human induced pluripotent stem cell-derived
cardiomyocytes
Long QT syndrome Type 3
Action potential duration
Early after depolarizations
Sodium channel
Potassium channel
Mexiletine analogs
Introduction
arrhythmogenic response to mexiletine to treat LQTS3 because the drug
has off-target effects on other important ion channels including the
Human induced pluripotent stem cell (hiPSC)-derived car-
diomyocytes offer a cell-based method to test drug candidates in a
comprehensive way to foretell pro-arrhythmia effects. Patient-derived
hiPSC cardiomyocytes can be used alongside normal hiPSC-derived
cardiomyocytes to help develop safe and effective drugs to treat ar-
rhythmias. In this work, a human genetic heart disorder characterized
by long delay (i.e., Long Q-T interval syndrome Type 3, LQTS3) between
hERG channel.3 hERG conducts the rapidly activating component of the
delayed rectifier potassium current (IKr) that is responsible for the bulk
of ventricular repolarization in cardiomyocytes. Mexiletine also has
minor but detectable adverse drug properties including hepatotoxicity
and blood dyscrasias.2 In addition, mexiletine suffers from a relatively
short half-life that necessitates frequent doses. Further doses of mex-
iletine can elicit CNS toxicity. Other drugs have been withdrawn from
the market because they induced QT prolongation and potentially fatal
ventricular tachycardia (VT), or Torsade de Pointes (TdP).4
heart beats caused by mutations in a Na+ channel
α subunit was used to
identify drug candidates related to mexiletine and pyridyl phenyl mex-
iletine analogs. Sodium channel inhibition potency is considered
responsible for mexiletine’s pro-arrhythmic effect1 but potassium (i.e.,
Previously, we reported on a kinetic image cytometry (KIC) primary
assay using normal and LQTS3 hiPSC cardiomyocytes.5,6 In normal
cardiomyocytes, late Na+ current (i.e., INaL) is only a small part of the
action potential (AP). In cardiomyocytes from LQTS3 patients with
mutations in genes encoding the cardiac sodium channel SCN5A, the
amplitude of INaL can be much greater. In individuals with LQTS3, a
small percentage of sodium channels do not inactivate and cause a INaL
that can protract the ventricular action potential (AP). This is manifested
by a lengthening of the QT interval of the ECG (Fig. 1).
K+ human ether-a-go-go (hERG)) channel inhibition is a potentially
`
detrimental off-target effect. Compounds that are efficacious against the
“leaky” Na+-channel yet minimize off-target effects against the hERG
channel could have significant impact on LQTS3 and other cardiovas-
cular diseases.
Mexiletine is an important class of antiarrhythmic agent used to treat
human genetic heart disorders. LQTS3 is characterized by abnormal
heart beats caused by mutations in the Na+ channel
α
subunit.2 Certain
LQTS3 is caused by mutations in the Nav1.5 sodium channel (enco-
ded by SCN5A) that weakens channel inactivation and accelerates
patients are genetically predisposed to
a
potentially fatal
* Corresponding author.
E-mail address: JCashman@HBRI.org (J.R. Cashman).
https://doi.org/10.1016/j.bmcl.2021.128162
Received 12 February 2021; Received in revised form 16 April 2021; Accepted 26 May 2021
Available online 29 May 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

M. Johnson et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128162
hERG channel.1 From approximately 30 substituted pyridyl mexiletine
and pyridyl phenyl mexiletine analogs synthesized and tested, potent
and selective compounds were identified.
This report describes the development of a new drug candidate that
uses patient hiPSC-derived cardiomyocytes to develop a safer and more
potent drug candidate to treat a serious human genetic heart health
disorder characterized by long Q-T interval between heart beats caused
by mutations in the Na+ channel
α subunit.
Effect of mexiletine analogs on human cardiomyocytes
Previously, mexiletine analogs were synthesized and tested.10,11 In
this report, new racemic pyridine analogs of mexiletine and phenyl
mexiletine were examined for their effect on normal and patient-derived
(pathogenic) human LQTS3 iPSC-derived cardiomyocytes using KIC
assays.5 The design was to identify analogs with increased on-target
potency (i.e., sodium channel inhibition) and decreased off-target (i.e.,
potassium channel inhibition) effects in human cardiomyocytes. We
sought compounds that would decrease the APD of LQTS3 car-
diomyocytes and not have any detectable toxic effect on normal car-
diomyocytes. Patient-derived LQTS3 cardiomyocytes harbored
mutations that caused prolonged APD. Normal cardiomyocytes
employed did not have any sodium or potassium channel mutations and
did not have prolonged APs. Thus, the effects of pyridine analogs of
mexiletine and phenyl mexiletine on cardiomyocytes with LQTS3 were
compared to their effects on normal cardiomyocytes. The pharmaco-
logical effect of each compound was tested in a dose–response study (i.
Fig. 1. A. Representative trace of a normal ECG shows the Q, R, S and T waves
for a normal individual. B. the lower trace shows a representative ECG trace for
a LQTS3 patient with a delayed action potential duration and an early after
depolarization (EAD). In response to mexiletine, the T wave is shortened for
LQTS3 patients and reverts to a normal action potential duration.
recovery from the inactivated state.7–9 In the presence of pathological
cardiomyocytes from LQTS3 patients, administration of mexiletine can
shorten the QT interval. There is a correspondence between the QT in-
terval on the ECG and the action potential duration in cardiomyocytes;
thus, LQTS3 cardiomyocytes can be used to identify compounds that
cause shortening of the AP duration (APD). Normal non-pathological
cardiomyocytes without sodium channel mutations can be used for
general toxicity assessment (i.e., compound-induced appearance of early
after depolarizations, (EADs)) in the drug discovery process.
e., 0–200 μM, nine concentrations, in triplicate) in normal and LQTS3
human cardiomyocytes to determine a concentration of cessation of cell
beating, an EC50 for shortening the APD, a fold-shortening of the APD
and a concentration that caused shortening of the AP. Toxicity of com-
pounds was assessed by quantifying a concentration that caused cessa-
tion of cell beating and/or a concentration that caused pro-arrhythmic
induction of EADs in normal and LQTS3 cardiomyocytes.
In addition to measuring desired APD shortening in LQTS3 car-
diomyocytes and undesired APD prolongation and EADs in normal
cardiomyocytes, on-target potency for inward INaL, selectivity for INaL
versus INaP and selectivity against potassium channels (i.e., hERG) was
obtained with selected compounds in whole cell patch clamp
studies.13,14 On the basis of IC50 values, selectivity for INaL versus INaP
provided a measure of on-target effects INaL versus INaP off-target effects
and afforded a measure of selectivity.
LQTS3 cardiomyocytes are sensitive to drug-induced sodium tran-
sient anomalies and AP prolongation typical of hERG blockade.6 The KIC
assay is sensitive, relatively high throughput and can be diagnostic for
identifying pro-arrhythmia effects in a drug development campaign.5 In
addition, to distinguish whether compounds diminished hERG blockade
directly, we developed secondary patch clamp assays that employed
HEK cells that overexpressed hERG and another cell line that overex-
pressed the mutated sodium channel.1
Patient-derived iPSC cardiomyocytes harboring a de novo SCN5A
LQTS3 mutation (i.e., F1473C) associated with drug-induced LQTS3 was
used in the study herein. Both normal hiPSC cardiomyocytes and
patient-derived LQTS3 cardiomyocytes were previously fully charac-
terized for ion channels, receptors and effects of standard drugs on
function.6 Normal hiPSC-cardiomyocytes and LQTS3 hiPSC car-
diomyocyte primary assays were used to test for pro-arrhythmia effects
of mexiletine and synthetic analogs using KIC readouts (i.e., Na+ tran-
sients, action potential voltage kinetics) to identify compounds for
retention or persistence of INaL channel current inhibition and removal
of patient specific drug-induced arrythmogenicity.6 These results were
compared to peak Na+ channel current (i.e., INaP) inhibition to deter-
mine selectivity ratios. In addition to channel inhibition, toxicity of
compounds to cardiomyocytes was determined by quantifying
arrhythmia, including EADs, and a concentration whereby a compound
caused cessation of cardiomyocyte beating. To obtain primary assay
data, LQTS3 hiPSC-cardiomyocytes and normal cardiomyocytes were
treated through dose escalation of mexiletine and synthetic test com-
pounds. Shortening of the APD in LQTS3 cardiomyocytes was a key
criteria of potency of synthetic compounds. Prolongation of the APD was
a key parameter to assess hERG blockade.5 In a secondary assay, channel
inhibition was confirmed with patch clamp physiology measurements in
HEK cells transfected with F1473C Na+ channel mutation and wild type
2-Pyridine analogs of mexiletine
Structure-activity relationship (SAR) studies were designed to
improve the antiarrhythmic properties of racemic mexiletine. In addi-
tion, because previously it was observed racemic phenyl mexiletine
possessed pharmacological advantages over mexiletine, most of the
compounds examined herein were racemic pyridyl phenyl mexiletine
analogs.
Initially, a small group of 2-pyridyl analogs of mexiletine was syn-
thesized and tested (Table 1). The results of these studies showed that
replacement of the aryloxy group with a 2-pyridine moiety did not
afford markedly greater fold-shortening of the APD. For example,
compared to mexiletine or phenyl mexiletine (i.e., 1 and 2, respec-
tively), 3 did not shorten the APD in LQTS3 cardiomyocytes nor have
much effect on normal cardiomyocytes (Table 1).
Compound 4 showed fold-shortening of the APD in LQTS3 car-
diomyocytes (Table 1) but also caused cessation of beating at a relatively
low dose (i.e., 7.4 µM). This indicated some potential toxicity for 4.
Compound 4 did not have any apparent effect on normal car-
diomyocytes (Table 1). Compound 4 did not cause EADs. Overall, the
results suggested improvement in the antiarrhythmic properties could
be possible with substitution of the aryloxy moiety for a pyridyl analog
of mexiletine. However, because alpha phenyl-substituted mexilitene
2

M. Johnson et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128162
Table 1
Effect of mexiletine, phenyl mexiletine and 2-pyridyl mexiletine analogs on cardiovascular properties in human normal and LQTS3 iPSC-derived cardiomyocytes.
Compound
Number
Structure
Normal CMsa EC50 (µM)
Normal CMs Cessation
LQTS3 CMs EC50 (µM)
LQTS3Fold-
LQTS3-Shortening
Prolongation
Dose (µM)b
Shorteningc
Shorteningc
Dose (µM)d
Mexiletine, 1
20.4
–
1.8
1.3
1.2
–
22.0
Phenyl
4.0
133
–
< 0.8
2.5
Mexiletine, 2
3
4
Does Not Prolong AP
Does Not Prolong AP
–
–
Does Not Shorten
7.4
–
1.5
a
CMs, cardiomyocytes. Kinetic imaging cytometer assay results for racemic mexiletine, racemic phenyl mexiletine and racemic 2-pyridyl mexiletine derivatives.
Data is representative of 4 different measurements. The test compound concentration range was from 100 nM to 100 µM as described in the Supporting Information.
The EC50 value is the concentration causing one half the maximum stimulation of APD prolongation in normal cardiomyocytes.
b
The symbol “-” denotes that cessation of cardiomyocyte beating was not observed up to 200 µM of the test compound examined.
c
The symbol “-” denotes that shortening of cardiomyocyte action potential duration (APD) was not observed up to 200 µM of the test compound examined in LQTS3
cells. The EC50 value is the concentration causing one half the maximum stimulation of APD shortening in LQTS3 cardiomyocytes.
d
LQTS3 shortening dose is the lowest concentration where shortening of cardiomyocyte APD was observed up to 200 µM of the test compound examined in LQTS3
cells.
analogs have shown greater promise compared to mexiletine,10,11 and
because some toxicity was noted in the 2-pyridyl mexiletine analogs, we
elected to examine racemic pyridine analogs of phenyl mexiletine.
3-Pyridine phenyl mexiletine analogs
The effect of chemical structure of pyridine phenyl mexiletines on
cardiovascular properties in hiPSC-derived cardiomyocytes was
extended to racemic 3-pyridine phenyl mexiletine analogs. Mexiletine
did not cause cessation of cell beating in either LQTS3 or normal car-
diomyocytes but did induce EADs at 200 µM in normal cells (Table 1).
Compound 12 also induced EADs at 133 µM in LQTS3 cardiomyocytes
and actually prolonged the APD in normal and LQTS3 cardiomyocytes.
In contrast, 6-methyl-, 6-CF3-, 4-methyl-, 2,6-dimethyl-, 2,4-dimethyl-,
and 4,6-dimethyl-3-pyridyl phenyl mexiletines (i.e., 14, 15, 18, 20,
21, 22, and 23, respectively) did not cause cessation of beating and
compounds 13–27 did not cause EADs in normal cardiomyocytes. As a
representative example, data for compound 14 is shown (Fig. 2).
Fig. 2A shows the shortening of the APD of LQTS3 hiPSC-derived
cardiomyocytes in the presence of compound 14 (66 µM). Fig. 2B
shows the dose response relationships for AP shortening in the presence
of compound 14 that afforded an EC50 of 8.8 µM.
2-Pyridine analogs of phenyl mexiletine
In the presence of LQTS3 or normal cardiomyocytes, 2-pyridyl
phenyl mexiletine analogs (i.e., 5–10) did not cause cessation of
beating. Compound 11 was an exception but only caused cessation of
beating at elevated concentrations (i.e., 133 µM) (Table 2). However,
compounds 6, 8 and 9 caused EADs but only in normal and not LQTS3
cardiomyocytes. These compounds were therefore judged to be toxic.
Compared to compound 5, compounds 6–9 caused shortening of the AP
in LQTS3 cardiomyocytes. In normal cardiomyocytes, 5, 7–11 did not
prolong the AP. Compounds 6 and 8 prolonged the AP but only at
elevated concentrations (Table 2).
In contrast to unsubstituted 2-pyridine-substituted phenyl mexilitene
5, 2-methyl, 2-CF3, 3-methyl, and 3-CF3-substituted pyridyl phenyl
mexiletine derivatives (i.e., 6–9, respectively) caused fold-shortening of
the AP in LQTS3 but not normal cells. This suggested some potency and
selectivity in this series. However, fold-shortening for the 2-pyridyl
phenyl mexiletine compounds was not significantly greater than
phenyl mexiletine itself. To examine a different chemotype, an oxime
was synthesized to replace the primary amine of 2-pyridyl phenyl
mexiletines (Table 2). The unsubstituted 2-pyridyl phenyl mexiletine
oxime (i.e., 10) did not affect fold-shortening of the AP in either LQTS3
or normal cardiomyocytes. In contrast, the 2-methyl pyridyl-substituted
oxime, compound 11, was more potent than phenyl mexiletine at
shortening the APD in LQTS3 cells. However, in normal cardiomyocytes,
11 caused cessation of beating at greater concentrations (i.e., 133 µM).
The results suggested N-substituted 2-pyridine analogs of phenyl mex-
iletine could afford detectable shortening of the APD and this was
further explored. However, oxime derivatives were not further
examined.
Of the other substituted 3-pyridyl phenyl mexiletine analogs tested,
(i.e., 13, 16, 17, 19, 24–27) cessation of beating in normal car-
diomyocytes was observed (i.e., 66–200 µM range). The results sug-
gested that for certain substituted 3-pyridyl phenyl mexiletine analogs
(Table 3), cardiomyocyte toxicity was apparent. However, compound 14
showed promise and was not toxic and was examined further in patch
clamp electrophysiology studies (see below)
For substituted 3-pyridyl phenyl mexiletine analogs, generally, the
potency for shortening the APD (i.e., EC50 for shortening) did not exactly
correspond to the potency of the concentration causing shortening of the
APD in LQTS3 cardiomyocytes (Table 3) although compound 26 was
similarly potent at both measures. In some cases (i.e., 13, 17, 25, and
26), significant fold-shortening of the APD was observed in LQTS3 cells.
To further explore the effect of structure on potency, additional N-
substituted 3-pyridyl phenyl mexiletine analogs were synthesized and
tested. Notable potency for compounds (i.e., 23, 25 and 26) were
observed for N-substituted 3-pyridyl phenyl mexiletine analogs. Based
on the potency of fold-shortening by substituted 3-pyridyl phenyl
3

M. Johnson et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128162
Table 2
Effect of substituted 2-pyridyl phenyl mexiletine derivatives on cardiovascular properties in human normal and LQTS3 iPSC-derived cardiomyocytes.
Compound
Number
Structure
Normal CMsa EC50 (µM)
Normal CMs Cessation
LQTS3-EC50 (µM)
LQTS3Fold-
Shortening
LQTS3-Shortening
Prolongation
Dose (µM)b
Shorteningc
Dose (µM)d
5
Does Not Prolong AP
–
Does Not Shorten
Does Not Shorten
Does Not Shorten
6
7
18.48
–
–
12.82
20.7
1.29
1.36
66
Does Not Prolong AP
133
8
9
133
–
–
–
–
1.22
1.3
22
22
Does Not Prolong AP
10
11
Does Not Prolong AP
Does Not Prolong AP
–
Does Not Shorten
1.38
Does Not Shorten
1.41
Does Not Shorten
66
133
a
CMs, cardiomyocytes. Kinetic imaging cytometer assay results for racemic 2-pyridyl phenyl mexiletine derivatives. Data is representative of 4 different mea-
surements. The test compound concentration range was from 100 nM to 100 µM as described in the Supporting Information. The EC50 value is the concentration
causing one half the maximum stimulation of APD prolongation in normal cardiomyocytes.
b
The symbol “-” denotes that cessation of cardiomyocyte beating was not observed up to 200 µM of the test compound examined.
c
The symbol “-” denotes that shortening of cardiomyocyte action potential APD was not observed up to 200 µM of the test compound examined in LQTS3 cells. The
EC50 value is the concentration causing one half the maximum stimulation of APD shortening in LQTS3 cardiomyocytes.
d
LQTS3 shortening dose is the lowest concentration where shortening of cardiomyocyte APD was observed up to 200 µM of the test compound examined in LQTS3
cells.
Fig. 2. Effect of compound 14 on action
potential of LQT3 hiPSC-cardiomyocytes.
Plots of representative optically recorded
AP traces of LQTS3 hiPSC-derived car-
diomyocytes treated with compound 14 (66
µM, in red) versus DMSO vehicle control (in
black) (A). Three biological replicates are
shown. Dose response curve for APD75
shortening in LQT3 hiPSC-derived car-
diomyocytes treated with compound 14 (B).
Error bars = s.e.m. The data represents the
average of three biological replicates. The
EC50 for AP shortening in this experiment
was 8.8 µM. APD75 refers to the width of the
action potential at 75% repolarization. (For
interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
mexiletines, additional 4-pyridyl phenyl mexiletine derivatives were
synthesized and tested in further structure function studies.
Pyridyl-phenyl mexiletines (i.e., 28, 29 and 30) caused 1.2–1.3-fold
shortening in LQTS3 cells. Compounds 29 and 31 caused prolongation
of the APD in normal cardiomyocytes (Table 4). The results suggested 4-
pyridyl phenyl mexiletine analogs were similar to mexiletine itself but
less potent.
4-Pyridyl phenyl mexiletine analogs
Of the 4-pyridyl phenyl mexiletine analogs examined, none caused
cessation of beating at any of the concentrations examined (Table 4) and
additional compounds in this series was not pursued.
Automated patch clamp studies in HEK cells.
Compound 29 caused EADs but only in normal and not LQTS3 car-
diomyocytes and was therefore judged to possess some toxicity. 4-
Mexiletine, phenyl mexiletine and compound 14 were tested in
automated patch clamp experiments to determine IC50s for INaP and INaL
4

M. Johnson et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128162
Table 3
Effect of substituted 3-pyridyl phenyl mexiletine derivatives on cardiovascular properties in human normal and LQTS3 iPSC-derived cardiomyocytes.
Compound
Number
Structure
Normal CMsa EC50 (µM)
Normal CMs Cessation
LQTS3-EC50 (µM)
LQTS3 Fold-
Shortening
LQTS3-Shortening
Prolongation
Doseb (µM)
Shorteningc
Dose (µM)d
12
33.8
–
62.6 (Prolongation and
EADs)
1.5 (Prolongation)
133
13
Does Not Prolong AP
133
8.57
8.8
–
1.43
Does Not Shorten
14
15
18.5
25.7
–
–
1.3
66
Does Not Shorten
Does Not Shorten
16
17
7.24
200
200
–
Does Not Shorten
1.42
Does Not Shorten
22
Does Not Prolong AP
(no EC50 – but
shortens)
18
19
Does Not Prolong AP
Does Not Prolong AP
–
67.5
1.23
22
66
–
Does Not Shorten
Does Not Shorten
20
21
22
23
8.22
–
–
–
–
–
Does Not Shorten
1.12
Does Not Shorten
Does Not Prolong AP
(no EC50 – but
22
shortens)
7.33
–
Does Not Shorten
1.14
Does Not Shorten
22
25.42
2.58
24
25
Does Not Prolong AP
Does Not Prolong AP
200
200
46.57
1.19
1.4
66
66
(no EC50 – but
shortens)
26
Does Not Prolong AP
Does Not Prolong AP
66
66
(no EC50 – but
1.6
7.4
shortens)
27
–
Does Not Shorten
Does Not Shorten
a
CMs, cardiomyocytes. Kinetic imaging cytometer assay results for racemic 3-pyridyl phenyl mexiletine derivatives. Data is representative of 3–4 different mea-
surements. The test compound concentration range was from 100 nM to 100 µM as described in the Supporting Information. The EC50 value is the concentration
causing one half the maximum stimulation of APD prolongation in normal cardiomyocytes.
b
The symbol “-”denotes that cessation of cardiomyocyte beating was not observed up to 200 µM of the test compound examined.
c
The symbol “-” denotes that shortening of cardiomyocyte APD was not observed up to 200 µM of the test compound examined in LQTS3 cells. The EC50 value is the
concentration causing one half the maximum stimulation of APD shortening in LQTS3 cardiomyocytes. EADs are early after depolarizations. The term “no EC50 - but
shortens” indicates shortening was observed but the dose response did not afford an EC50 value.
d
LQTS3 shortening dose is the lowest concentration where shortening of cardiomyocyte APD was observed up to 200 µM of the test compound examined in LQTS3
cells.
5

M. Johnson et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128162
Table 4
Structures and KIC assay results for 4-pyridyl phenyl mexiletine derivatives.
Compound
Number
Structure
Normal CMsa EC50 (µM)
Normal CMs Cessation
LQTS3-EC50 (µM)
LQTS3Fold-
Shortening
LQTS3-Shortening
Prolongation
Dose (µM)b
Shorteningc
Dose (µM)d
28
Does Not Prolong AP
No cessation of beating
3.82
1.3
7.4
29
133
No cessation of beating
–
1.2
133
30
Does Not Prolong AP
97.68
No cessation of beating
No cessation of beating
(no EC50 – but
1.23
22
shortens)
31
–
Does Not Shorten
Does Not Shorten
a
CMs, cardiomyocytes. Kinetic imaging cytometer assay results for racemic 4-pyridyl phenyl mexiletine derivatives. Data is representative of 4 different mea-
surements. The test compound concentration range was from 100 nM to 100 µM as described in the Supporting Information. The EC50 value is the concentration
causing one half the maximum stimulation of APD prolongation in normal cardiomyocytes.
b
Cessation of cardiomyocyte beating was not observed up to 200 µM of the test compound examined.
c
The symbol “-“ denotes that shortening of cardiomyocyte APD was not observed up to 200 µM of the test compound examined in LQTS3 cells. The EC50 value is the
concentration causing one half the maximum stimulation of APD shortening in LQTS3 cardiomyocytes. The term “no EC50 - but shortens” indicates shortening was
observed but the dose response did not afford an EC50 value.
d
LQTS3 shortening dose is the lowest concentration where shortening of cardiomyocyte APD was observed up to 200 µM of the test compound examined in LQTS3
cells.
current inhibition. In separate studies, IKr inhibition was examined.
Compound 14 was chosen as a representative example of a pyridyl
phenyl mexiletine analog because 14 was potent at shortening the APD,
did not produce cessation of beating and did not cause EADs.
In HEK cells transfected with mutant cardiac sodium channel SCN5A,
the effect of mexiletine showed an IC50 of 22.5 ± 4.4 µM and 182.8 ±
41.8 µM for INaL and INaP current, respectively. In CHO cells transfected
with a normal potassium channel, mexiletine showed an IC50 value of
53.7 ± 8.0 µM for IKr (Table 5).
mexiletine (i.e., IC50 INaP/IC50 INaL ratio of 8.1), 14 possessed significant
selectivity for INaL (i.e., IC50 INaP/IC50 INaL ratio of 90). Compared to
mexiletine (i.e., IC50 IKr/IC50 INaL ratio of 2.4), 14 showed significant
IKr/INaL selectivity (i.e., IC50 IKr/IC50 INaL ratio of 11.2, meaning inhi-
bition of the IKr was 11.2-fold less potent than for INaL).
Compared to phenyl mexiletine, compound 14 showed significant
channel selectivity. Phenyl mexiletine, compound 2 had IC50s of 11.7 ±
0.8 µM and 20.6 ± 2.1 µM for INaL and INaP current, respectively, and 8.0
± 1.3 µM for IKr (Table 5). Compared to phenyl mexiletine (i.e., IC50
INaP/IC50 INaL ratio of 1.8), 14 possessed significant selectivity for INaL (i.
The values listed in Table 5 for mexiletine were in the range of values
previously reported for these ion currents.12,13 Compared to mexiletine,
introduction of an ortho-methyl 3-pyridyl group to phenyl mexiletine (i.
e., 14) showed significant changes in channel selectivity. Compound 14
had IC50s of 1.0 ± 0.2 µM and 90.9 ± 8.0 µM for INaL and INaP current,
respectively, and 11.2 ± 1.6 µM for IKr (Table 5). Compared to
e., IC50
I
/IC50 INaL ratio of 90). Compared to phenyl mexiletine (i.e.,
NaP
IC50 IKr/IC50 INaL ratio of 0.7), 14 showed significant IKr/INaL selectivity
(i.e., IC50 IKr/IC50 INaL ratio of 11.2, meaning inhibition of the IKr was
16.7-fold less potent than for INaL).
Selective inhibition of the sodium late current showed 14 was se-
lective over mexiletine or phenyl mexiletine for both peak INaL and IKr.
Results showed that compound 14 was 21.6- and 11.2-fold more potent
than mexiletine or phenyl mexiletine, respectively, at inhibiting the INaL
current. Selectivity for the INaL versus the IKr (i.e., IKr/IC50 INaL) was
almost 5-fold and 16-fold for 14 compared to mexiletine and phenyl
mexiletine, respectively. The results showed that at least in one case, a 3-
pyridine analog of phenyl mexiletine can afford a relatively potent and
selective sodium late channel inhibitor.
Table 5
Summary of autopatch dose–response data for mexiletine, phenyl mexiletine
and compound 14.
Compd
Name
IC50
INaL,
µMa
Nb
IC50 INaP,
N
IC50 IKr,
N
Selectivityc
µMa
µMb
Mexiletine,
22.5 ±
4
5
4
182.8 ±
41.8
4
53.7 ±
8.0
7
6
9
2.4
0.7
4.4d
Normal and LQTS3 patient-derived hiPSCs are a useful source to
create human cardiomyocytes to evaluate synthetic or other drug can-
didates in a drug development campaign. In the study described herein,
dynamic medicinal chemistry was done on a practical time scale using a
384-well multi-well format assay to test drug candidates for selectivity
and potency to inhibit cardiac sodium and potassium channels. The use
of LQTS3 patient-derived cardiomyocytes allowed a disease-in-a-dish
approach to address an important human disease, namely QT prolon-
gation and potentially fatal VT and TdP.12 Cardiomyocytes encoding
mutant sodium channels afforded a genetic congenital model of LQTS3
that predisposes a patient to VT and sudden death.14 A goal was to
provide a way to identify compounds to reverse disease phenotype.
1
2
11.7 ±
0.8
20.6 ±
2.1
6
8.0 ±
1.3
14
1.04 ±
0.2
90.9 ±
8.0
12
11.2 ±
1.6
11.2
a
Late sodium current (INaL), Peak sodium current (INaP), Potassium current
(IKr).
b
N is the number of independent replicate determinations conducted.
Selectivity is IC50 IKr/IC50 INaL
c
d
Data is presented as mean ± standard error.
6

M. Johnson et al.
Bioorganic & Medicinal Chemistry Letters 46 (2021) 128162
Studies conducted in parallel with normal human cardiomyocytes were
also useful to identify compounds that could potentially be toxic to
cardiomyocytes. Identification of drug candidates that are toxic to car-
diomyocytes could provide insight to structural aspects of drug candi-
dates that causes toxicity or understand why established drugs possess
toxicity.
Acknowledgments
We are grateful for the financial support of the California Institute for
Regenerative Medicine (CIRM) (Grant Number TR4-06857 to JRC and
MM). The contents of this publication are solely the responsibility of the
authors and do not necessarily represent the official views of CIRM or
any other agency of the State of California. Flow cytometry and high
throughput screening services were supported by NIH P30CA030199 at
the Sanford-Burnham-Prebys Medical Discovery Institute.
Another goal was to re-engineer mexiletine to new compounds with
superior pharmacological or pharmaceutical properties because mex-
iletine possesses adverse properties. Development of compounds with
increased on target (i.e., INaL) versus off-target (i.e., IKr) selectivity was
another goal. hERG inhibition is known to be associated with clinically
relevant arrhythmias and EADs. Herein, chemical-induced production of
EADs or ectopic beats reminiscent of PVCs, or measurement of cessation
of normal cardiomyocyte beating was used as a measure of car-
diomyocyte toxicity.13
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128162.
Previously, we reported on use of molecules known to cause hERG
inhibition and afforded clinically relevant arrhythmia to validate dose-
dependent drug induction of toxicity in the cardiomyocytes used here-
in.6 We used molecules known to cause EADs and observed dose-
dependent ectopic beats to further validate the cellular test system.6
For the compounds examined, cell toxicity was dependent on the pyridyl
substitution pattern of the compounds tested. For example, EADs were
observed for 2-pyridyl phenyl mexiletine (i.e., 6, 8 and 9) and 4-pyridyl
phenyl mexiletine (i.e., 29) (Tables 2 and 4). However, with the
exception of 12, among 3-pyridyl phenyl mexiletines tested (i.e.,
13–27), most did not produce EADs in normal cardiomyocytes. Because
mexiletine can cause seizures in humans at elevated doses15,16 exami-
nation of potential cell toxicity in human cardiomyocytes in a drug
development campaign may be an efficient means of identifying un-
tenable drug candidates.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
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