Antiarrhythmic Hit to Lead Refinement in a Dish Using Patient-Derived iPSC Cardiomyocytes

pubs.acs.org/jmc

Article

Antiarrhythmic Hit to Lead Reﬁnement in a Dish Using Patient-

Derived iPSC Cardiomyocytes

John R. Cashman,* Daniel Ryan, Wesley L. McKeithan, Karl Okolotowicz, Jorge Gomez-Galeno,

Mark Johnson, Kevin J. Sampson, Robert S. Kass, Arash Pezhouman, Hrayr S. Karagueuzian,

and Mark Mercola

Cite This: J. Med. Chem. 2021, 64, 5384 −5403

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: Ventricular cardiac arrhythmia (VA) arises in acquired or congenital heart disease. Long QT syndrome type-3

(LQT3) is a congenital form of VA caused by cardiac sodium channel (I_NaL) SCN5A mutations that prolongs cardiac action

potential (AP) and enhances I_NaLcurrent. Mexiletine inhibits I_NaLand shortens the QT interval in LQT3 patients. Above therapeutic

doses, mexiletine prolongs the cardiac AP. We explored structure−activity relationships (SAR) for AP shortening and prolongation

using dynamic medicinal chemistry and AP kinetics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Using patient-derived LQT3 and healthy hiPSC-CMs, we resolved distinct SAR for AP shortening and prolongation eﬀects in

mexiletine analogues and synthesized new analogues with enhanced potency and selectivity for I_NaL. This resulted in compounds

with decreased AP prolongation eﬀects, increased metabolic stability, increased I_NaLselectivity, and decreased avidity for the

potassium channel. This study highlights using hiPSC-CMs to guide medicinal chemistry and “drug development in a dish”.

INTRODUCTION

■

Approximately one million individuals are hospitalized every

year for arrhythmias in the United States, making arrhythmias

one of the top causes of healthcare expenditures with a direct

cost of almost $50 billion annually for diagnosis, treatment,

and rehabilitation.¹Another 300,000 individuals die of sudden

arrhythmic death syndrome every year.²Arrhythmias are very

common in older adults, but unfortunately, drugs used to treat

arrhythmias have liabilities. In addition, numerous drugs have

been withdrawn from the market because they induce QT

prolongation (Figure 1) and a potentially fatal ventricular

tachycardia (VT), torsade de pointes (TdP) (Figure 2).³

Cardiac sodium current (I_NaP) occurs in less than a

millisecond and underlies the rapid upstroke or phase 0 of

the AP (action potential) (Figure 1). Thereafter, an activation

gate and an inactivation gate on the sodium channel are both

open. After several milliseconds, the current begins to decay,

Figure 1. Schematic representation of cardiac cell membrane action

contributing to a notch in the AP called phase 1. At this point,

potential (AP) and its relation with surface electrocardiogram.

some of the channels are inactivated (shown as “I” with the

inactivation gate closed). There is no commonly accepted

name for this phase of I_Na, but here, it is labeled “early I_Na”

(Figure 1). After several milliseconds, I_Nanormally decays to

Received: September 3, 2020

Published: May 4, 2021

<1% of I_NaP, but a residual current ﬂows as late I_NaLand this

depolarizing current along with calcium currents support phase

2 or the plateau of the AP. The mechanisms for I_NaLat the

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

5384

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 2. Eﬀect of slow membrane depolarization on the QT interval on a surface electrocardiogram.

sodium current level are multiple but can generally be thought

of as incomplete inactivation. Eventually, activating potassium

currents repolarize the membrane (phase 3 of the AP), and

when the voltage decreases below the sodium channel

threshold, the activation gate closes.

Given that these latter properties are potentially proar-

rhythmic, it is important to determine if they are mediated by

distinct determinants in the chemical structure of mexiletine as

those responsible for the desirable inhibition of I_NaLand

whether structure modiﬁcations might decrease the AP

prolonging eﬀects, increase metabolic stability (and decrease

the required dose), and increase selectivity for the sodium

channel, thus decreasing avidity for the potassium channel and

decrease toxicity.

Herein, we report the application of human-induced

pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)

to comprehensively evaluate proarrhythmic eﬀects of mex-

iletine analogues and deﬁne chemical determinants responsible

for antiarrhythmia. We used patient-derived hiPSC-CMs

carrying a previously described LQT3 mutation in SCN5A

(F1473C)¹⁷to quantify the extent of AP shortening of

mexiletine and analogues. LQT3 human CMs were used to

determine the mexiletine analogue’s beneﬁcial eﬀects,

quantiﬁed as the EC₅₀for shortening the APD, a fold

shortening of the APD, and a concentration that caused

shortening of the action potential. These results were

compared to the concentration of a test compound required

for cessation of cell beating, an EC₅₀for shortening the APD,

and fold shortening of the APD in normal cardiomyocytes.

Both normal and patient-derived LQT-3 hiPSC-CMs were

previously characterized for the eﬀects of benchmark reference

drugs on AP kinetics and arrhythmia.¹³

Mexiletine is an orally available inhibitor of muscles and

neuronal sodium channels and is a class 1B antiarrhythmic

drug in the Vaughan−Williams classiﬁcation scheme.⁴

Mexiletine has been used to treat life-threatening ventricular

arrhythmia and also long QT syndrome type 3 (LQT3).^5,6

Cardiac AP is initiated by the opening of the cardiac sodium

channel Na_v1.5 (encoded by SCN5A) conducting the large

peak sodium current (I_NaP) responsible for the AP upstroke. In

healthy individuals, the sodium channels inactivate with

depolarization. However, LQT3 causal mutations in Na_v1.5

impair channel inactivation and accelerate recovery from the

inactivated state.⁷⁻⁹Increased I_NaLopposes repolarization and

prolongs the AP, thus prolonging the QT interval on the

surface electrocardiogram.^7,10Mexiletine inhibits I_NaLand

consequently shortens the QT interval in LQT3 patients¹¹

and decreases their risk of developing ventricular tachycardia

and ventricular ﬁbrillation.¹²However, at concentrations

slightly greater than its therapeutic plasma concentration and

IC₅₀for inhibition of I_NaL, mexiletine also prolongs the cardiac

AP and promotes potentially proarrhythmic events (including

early after depolarizations (EADs)) in vitro.^13,14Observations

of proarrhythmia have restricted mexiletine use although

cardiologists generally predetermine a safe and eﬃcacious

dose. Mexiletine has liabilities. The FDA-approved label states

that severe liver injury and blood dyscrasias (i.e., leukopenia or

agranulocytosis) and other adverse reactions including

reversible gastrointestinal and nervous system problems have

been reported after mexiletine treatment. Mexiletine also has a

relatively short half-life (i.e., t_1/2α-phase 3−12 min and β-

phase 6−12 h¹⁵) that necessitates multiple doses per day.

Greater doses of mexiletine produce side eﬀects in the central

nervous system.¹⁶Accordingly, mexiletine analogues were

designed to replace metabolically labile groups with metabol-

ically stable moieties to increase bioavailability, decrease the

dose, and improve the therapeutic-to-toxicity ratio.

To quantify the eﬀect of chemical structural modiﬁcation on

the CM response, each test compound was tested in a dose−

response study (i.e., 0−200 μM, nine concentrations, in

triplicate) to identify the concentration of cessation of cell

beating, determine the concentration at which EADs occurred,

and quantify the EC₅₀for prolonging the APD.

Optical recordings of AP kinetics¹³were used as the primary

assay, and results were conﬁrmed by automated patch clamp

recording¹⁷of exogenously expressed SCN5A variants as a

secondary assay. Based on results for AP kinetics, inhibition of

I_NaLand I_NaP, and blockade of the potassium current I_Kr(i.e.,

hERG) that mediates the repolarizing potassium current, a

SAR arose for the mexiletine analogues synthesized and tested.

5385

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

The results led to identiﬁcation of signiﬁcantly more potent

and selective I_NaLinhibitors with decreased proarrhythmic

eﬀects and much improved physicochemical properties.

Compounds 30, 33, 39, and 62 were shown to be eﬀective

antiarrhythmic compounds in hiPSC-CM models of LQT3-

associated arrhythmia.

oximes and their subsequent reduction with borane to give the

desired amines (2−42).

Secondary racemic amines were directly prepared by a one-

step reductive amination procedure to give N-substituted

products 43−50 and 55−63 as reported.²²N-Acylated

analogues 51−54 were generated from the corresponding

primary amines by treatment with either acetyl chloride or

benzoyl chloride in the presence of triethylamine. Chiral

analogues were generated by the method illustrated in Scheme

3 for (R)-30 and (R)-59. Chiral N-acylated 4-phenyl 1,2,3-

oxathiazolidine-2,2-dioxide derivatives were combined with

substituted phenols under basic conditions.²³To convert to

the primary amines, the N-acyl group used was a t-Boc

substituent that was removed under acidic conditions. For the

methoxyethyl-substituted amine 59, an N-methoxy acetyl

protecting group was reduced with lithium aluminum hydride

and aﬀorded the secondary amine (Scheme 3).

Scheme 1. Regions of Mexiletine Modiﬁed to Lead to

Greater Potency, Selectivity, and Less Toxicity. Region I

(red) Is Alpha to the Amine, Region II (Green) Is the

Phenoxy Moiety, and Region III (Blue) Contained N-

Substituents

The hydrochloride salts of the amine products were used for

biological testing and judged to be greater than 95% pure by

¹H NMR and HPLC.

BIOLOGICAL RESULTS

RESULTS

■

Eﬀect of Mexiletine Analogues on Human Cardio-

myocytes. Mexiletine, 1 was re-engineered by chemical

synthesis to aﬀord analogues with increased on-target potency

(i.e., sodium channel) and decreased oﬀ-target (i.e., potassium

channel) eﬀects. The structure−activity relationship (SAR) for

the eﬀect of mexiletine analogues on normal and patient-

derived hiPSC-CMs was systematically evaluated in CMs

derived from an LQT3 patient (i.e., carrying the SCN5A

F1473 mutation) and from an unrelated healthy donor. Thus,

the eﬀects of mexiletine analogues on CMs with channelo-

pathies (i.e., disease in a dish) were compared to the eﬀects of

compounds on normal nonpathogenic CMs. To quantify the

eﬀect of modiﬁcation of 1, the molecule was conceptually

divided into three exploratory regions (Scheme 1): (I) the

region alpha to the primary amine moiety (red), (II) the

phenoxy region (green), and (III) the N-substituted region

(blue). Each compound was tested in a dose−response study

(i.e., 0−200 μM, nine concentrations, in triplicate) in LQT3

hiPSC-CMs to determine a concentration of cessation of cell

beating, an EC₅₀for shortening the APD, a fold shortening of

the APD, and a concentration that caused shortening of the

AP. In normal cardiomyocytes, the concentration that caused

cessation of cell beating, the concentration that caused

proarrhythmic induction of EADs, and the EC₅₀for shortening

the APD were determined. These latter measures were indices

of cell toxicity.

Chemistry. Mexiletine and analogues were prepared using

the synthetic routes described in Scheme 2. Some synthetic

Scheme 2. General Synthesis of Mexiletine Analogues and

N-Substituted Mexiletine Analogues

a

(a) PhCH₂ONH₂-HCl, pyridine, ethanol, 21 °C. (b) BH₃-THF, 65

°C, followed by treatment with HCl-ether. (c) R′-NH₂, NaBH₃CN,

ethanol, 21 °C, followed by treatment with HCl-ether.

procedures were adapted from literature reports.^18,19Mex-

iletine was divided into three hypothetical regions for

theoretical molecular dissection as follows: Region I included

substituents α to the amine, Region II included substituents

associated with the phenoxy moiety, and Region III included

N-substituents. In a typical synthesis, substituted phenols were

combined with α-bromoketones in the presence of potassium

carbonate to aﬀord phenoxy alkyl ketones.²⁰α-Aryloxy ketones

with variable substituents²¹were the precursors for one of the

two synthetic routes. To prepare primary amines, a two-step

protocol was employed that involved preparation of benzyl

Initially, a small group of approximately 20 compounds

directed at structural modiﬁcations of the portion of 1 alpha to

the primary amine (i.e., Region I) were synthesized and tested

a

Scheme 3. Syntheses of Chiral Analogues (R)-30 and (R)-59

a

(a) 2-Triﬂuoromethylphenol (for (R)-30, R = tert-BuO−) or 2,3-dimethylphenol (for (R)-59, R = CH₂OCH₃), NaH, DMF, 21 °C, 16 h. (b) For

(R)-30: HCl, methanol, 21 °C, 16 h. For (R)-59: LiAlH₄, THF, 75 °C, 16 h.

5386

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 1. Eﬀect of Alpha Substituents of Mexiletine on Cardiovascular Properties in Human iPSC-Derived Cardiomyocytes

a

b

c

Kinetic imaging cytometer assay results for alpha-substituted mexiletine derivatives. Lowest dose that cessation of beating was observed. EC₅₀

d

e

f

for shortening the AP. Ratio of AP untreated/AP treated. Maximal dose that shortens the AP. Dose that early after depolarization was observed.

g

EC₅₀for prolongation of the AP. The symbol “-” denotes that the indicated eﬀect was not observed. EAD dose indicates the concentration at

which the compound induced early after depolarizations.

(Tables 1 and 2). The results of these studies showed that an

alpha aryl moiety showed greater fold shortening than aliphatic

substituents. However, partial aryl characters (i.e., cyclopropyl

compounds) (Table 2) were less eﬀective at fold shortening

than aryl compounds. Modiﬁcations of the phenoxy moiety

(Tables 3 and 4) (i.e., Region II) resulted in compounds with

potent APD shortening. N-Modiﬁcation of selected com-

pounds (Tables 5 and 6, Region III) yielded additional

derivatives with potent fold shortening and in some cases less

toxicity to normal CMs.

LQT3 patient-derived hiPSC-CMs were resistant to the

adverse eﬀects of mexiletine, suggestive of the previously

reported patient that tolerated a high dose.¹⁷Therefore, the

detrimental eﬀects on AP prolongation and EADs were most

apparent in healthy normal donor hiPSC-CMs. Thus, the

results from studies with normal CMs served as a control and

an indicator of toxicity of compounds tested. In addition to

measuring desired APD shortening in LQT3 cardiomyocytes

and undesired APD prolongation and EADs in normal

cardiomyocytes, on-target potency for inward sodium late

currents (I_NaL), selectivity for I_NaLversus I_NaP, and selectivity

against potassium currents (I_Kr) were obtained with whole cell

automated planar patch clamp studies in which the SCN5A

and hERG channels were expressed in CHO cells.

Region I. Analogues of Mexiletine. In LQT3 hiPSC-

CMs, compared to mexiletine, 1, tert-butyl, cyclopropyl, and

cyclohexyl analogues (i.e., 2, 3, and 4) were about 12−15% less

potent at shortening the APD (Table 1). In contrast, aryl-

substituted derivatives, 4-methyl phenyl, 4-CF₃phenyl, and 4-

methoxy phenyl (i.e., 6, 7, and 8) were about 7−19% more

potent than mexiletine at shortening the APD. The naphthyl

derivative, compound 9, was about as potent as mexiletine at

shortening the APD. In contrast, in normal hiPSC-CMs,

compounds 1−9 did not cause detectable shortening of the

APD.

Mexiletine did not cause cessation of cell beating in either

LQT3 or normal hiPSC-CMs but induced EADs at 200 μM in

normal cells in primary screening (Table 1). Cessation of

beating is an indication of cardiotoxicity, and EADs are

indicative of proarrhythmic activity for a compound.

Compounds 2 and 3 induced EADs at 7.4 and 66 μM,

respectively, in normal CMs. Of the alpha amine-substituted

compounds examined, cessation of beating was observed (i.e.,

66−133 μM range) but the cyclopropyl derivative (i.e., 3) was

less potent and less toxic.

5387

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 2. Eﬀect of Phenoxy Substituents of Alpha Cyclopropane Mexiletine Analogues on Cardiovascular Properties in Human

a

iPSC-Derived Cardiomyocytes

a

b

c

Kinetic imaging cytometer assay results for alpha cyclopropane mexiletine derivatives. Lowest dose that cessation of beating was observed. EC₅₀

d

e

f

for shortening the AP. Ratio of AP untreated/AP treated. Maximal dose that shortens the AP. Dose that early after depolarization was observed.

g

EC₅₀for prolongation of the AP. The symbol “-” denotes that the indicated eﬀect was not observed.

Generally, the potency for shortening the APD (i.e., EC₅₀for

shortening) did not exactly correspond to the potency of the

concentration causing fold shortening of the APD in LQT3

cardiomyocytes (Table 1) although compounds 2 (isopropyl),

4 (cyclohexyl), and 5 (phenyl) were similarly potent at both

measures based on the lack of potency of cessation of beating.

Based on the potency of shortening by cyclopropyl compound

3, additional cyclopropyl derivatives were synthesized and

tested (Table 2).

Like mexiletine or its individual enantiomers, the cyclo-

propyl-substituted compounds in this class (i.e., compounds

12, 13, 15, and 16) only caused cessation of cell beating at 200

μM or not at all in either LQT3 or normal CMs. Compound

14 caused cessation of cell beating at 66 μM and was not

extensively examined further. However, compounds 12, 13, 15,

and 16 did induce EADs at 66 μM in normal CMs and were

deemed to possess some potential for toxicity (Table 2).

In contrast to mexiletine or its stereoisomers, for this class of

cyclopropyl mexiletine analogues, the potency for fold

shortening the APD (i.e., EC₅₀for shortening) corresponded

to the potency of the concentration causing shortening of the

APD in LQT3 CMs (Table 2).

However, based on the potency that these cyclopropyl

analogues caused toxic EADs at relatively low concentrations

(i.e., 66 μM), additional cyclopropyl derivatives were not

synthesized and tested. Rather, exploration of the phenoxy

portion of the molecule was investigated to determine

substituents that would aﬀord less toxicity (i.e., cessation of

beating, APD prolongation, and EADs).

Region II. Eﬀect of Phenoxy Substituents on

Mexiletine and Phenyl Mexiletine Analogues. Table 3

shows the results of modifying the phenoxy portion of

mexiletine to aﬀord analogues. In LQT3 CMs, compared to

mexiletine, 1, ortho monosubstituted methyl-, ethyl-, or propyl-

5388

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 3. Eﬀect of Phenoxy-Substituted Mexiletine Analogues on Cardiovascular Properties in Human iPSC-Derived

a

Cardiomyocytes

a

b

c

Kinetic imaging cytometer assay results for phenoxy-substituted mexiletine derivatives. Lowest dose that cessation of beating was observed. EC₅₀

d

e

f

for shortening the AP. Ratio of AP untreated/AP treated. Maximal dose that shortens the AP. Dose that early after depolarization was observed.

g

EC₅₀for prolongation of the AP. The symbol “-” denotes that the indicated eﬀect was not observed.

substituted compounds (i.e., 17, 18, and 19) were less potent

at shortening the APD based on fold shortening. In contrast,

phenoxy-substituted derivatives, 2-CF₃, 3-CH₃, and 3-CF₃(i.e.,

20, 22, and 23, respectively) were about 4−24% more potent

than mexiletine at shortening the APD. The 3,5-disubstituted

derivatives, compounds 24 and 25, were slightly less potent

than mexiletine at shortening the APD. In contrast, in normal

CMs, compounds 17−26 did not cause detectable shortening

of the APD and in some cases (i.e., compounds 19 and 24)

caused prolongation of the APD at relatively low concen-

trations.

Except compound 25 (i.e., at 66 μM) and compounds 19,

23, and 24 (at elevated doses), compounds in this class did not

cause cessation of cell beating in either LQT3 or normal CMs.

Compound 22 induced EADs at 66 μM. However, it required

high concentrations (133−200 μM) to induce EADs in normal

CMs for compound 17, 21, or 26 (Table 3).

However, compound 19 was potent at both measures. Based

on the increase in potency of shortening the APD by certain

monosubstituted phenoxy derivatives of mexiletine, additional

analogues combining beneﬁcial properties of the phenoxy ring

and aryl substituent alpha to the amine were synthesized and

tested.

Eﬀect of Phenoxy Substituents on Alpha Phenyl

Mexiletine Analogues. Because certain phenoxy-substituted

mexiletine compounds and certain alpha amino aryl mexiletine

analogues showed potency at shortening the APD, synthesis

and testing of compounds that combined those two moieties

were undertaken. Compared to the alpha amino phenyl

analogue of mexiletine, certain phenoxy-substituted alpha

amino phenyl derivatives potently increased fold shortening

of the APD (Table 4). In LQT3 CMs, compared to phenyl

mexiletine, 5, ortho monosubstituted methyl-, ethyl-, or propyl-

substituted compounds (i.e., 27, 28, and 29) were more potent

at shortening the APD by 37, 69, and 13%, respectively.

Compared to phenyl mexiletine, 2-CF₃, 2-OCH₃, and 2-O-

methoxy-CF₃, 2-O-CF₃(i.e., 30, 31, and 32 and 33,

respectively) were 52, 26, 29, and 21% more potent at

As observed for chemotypes described above, generally, the

potency for shortening the APD (i.e., EC₅₀for shortening) did

not exactly correspond to the potency of the concentration

causing fold shortening of the APD in LQT3 CMs (Table 3).

5389

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 4. Eﬀect of Phenoxy Substituents of Alpha Phenyl Mexiletine Analogues on Cardiovascular Properties in Human iPSC-

a

Derived Cardiomyocytes

a

b

c

Kinetic imaging cytometer assay results for phenyl mexiletine derivatives. Lowest dose that cessation of beating was observed. EC₅₀for

d

e

f

shortening the AP. Ratio of AP untreated/AP treated. Maximal dose that shortens the AP. Dose that early after depolarization was observed.

g

EC₅₀for prolongation of the AP. The symbol “-” denotes that the indicated eﬀect was not observed.

5390

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 5. Eﬀect of N-Substituted Mexiletine Analogues on Cardiovascular Properties in Human iPSC-Derived Cardiomyocytes

LQT3 cells

Normal cells

Cessation dose EC₅₀shortening

Fold

shortening

Maximal shortening

Cessation dose

EAD dose EC₅₀prolongation

f g

b

c

d

e

b

Number

R =

(μM)

dose (μM)

(μM)

1

H

Methyl

Ethyl

1-Propyl

1-Butyl

2-Methoxyethyl

Phenyl

Benzyl

2-Phenylethyl

Acetyl

Benzoyl

-

1.8

1.5

-

<0.8

2.3

-

1.335

1.112

-

1.199

1.180

1.267

-

1.264

1.706

-

22

-

2.5

7.4

-

22

2.5

-

66

-

200

133

-

200

22

133

66

-

22

-

20.4

7.8

21.5

10.4

7.8

32.0

-

5.7

2.5

-

43

44

45

46

47

48

49

50

51

52

133

-

200

66

-

133

7.4

-

200

133

-

133

57.82

1.45

66

-

a

b

c

Kinetic imaging cytometer assay results for N-modiﬁed mexiletine derivatives. Lowest dose that cessation of beating was observed. EC₅₀for

d

e

f

shortening the AP. Ratio of AP untreated/AP treated. Maximal dose that shortens the AP. Dose that early after depolarization was observed.

g

EC₅₀for prolongation of the AP. The symbol “-” denotes that the indicated eﬀect was not observed.

shortening the APD. The 3-substituted derivatives (i.e.,

compounds 34−38) were slightly more potent than phenyl

mexiletine at shortening the APD (i.e., 3−31%). Compared to

phenyl mexiletine, compounds containing 4-substituted groups

(i.e., 39 and 40) increased shortening of the APD 2 and 17%,

respectively. 1- and 2-Naphthyl derivatives of phenyl

mexiletine increased shortening 12 and 25%, respectively,

compared to phenyl mexiletine. In contrast, in normal CMs,

compounds 5−42 did not cause detectable shortening of the

APD and in some cases (i.e., compounds 5 and 31) caused

prolongation of the APD at relatively low concentrations.

Except compounds 41 and 42, phenoxy-substituted

derivatives of phenyl mexiletine (i.e., 27−40) caused cessation

of cell beating in both LQT3 and normal CMs (albeit at

elevated concentrations). Compound 31 induced EADs at a

relatively low concentration (i.e., 7.4 μM) in normal CMs

(Table 4).

As a chemotype, generally, the phenoxy-substituted phenyl

mexiletine analogues examined possessed great potency for

shortening the APD (i.e., EC₅₀for shortening) and most were

in the 0.8 μM or lower range in LQT3 CMs (Table 4). Based

on the increase in potency of shortening the APD by certain

monosubstituted phenoxy derivatives of phenyl mexiletine,

additional derivatives combining beneﬁcial properties of the

phenoxy ring substituents and the amine nitrogen were

synthesized and tested.

Region III. N-Substituted Analogues of Mexiletine.

The eﬀect of N-substitution on the pharmacological properties

of mexiletine was investigated. Compared to mexiletine, most

N-substituted mexiletine derivatives examined decreased the

shortening dose of the APD (Table 5). In LQT3 CMs,

compared to mexiletine, 1, N-methyl-, N-propyl-, N-butyl-, N-

ethyl methoxy-, and N-benzyl-substituted compounds (i.e., 43,

44, 45, 46, and 47) were less potent at fold shortening the

APD by 17, 10, 10, 5, and 5%, respectively. However, more

lipophilic N-ethyl phenyl mexiletine (i.e., 50) was 28% more

potent at shortening the APD than mexiletine. Compared to

mexiletine, the more polar N-acetyl phenyl-substituted

compound 52 increased shortening of the APD 9%. In

contrast, in normal CMs, compounds 45−52 did not cause

detectable shortening of the APD and in some cases (i.e.,

compounds 43, 45, 46, 49, and 50) caused prolongation of the

APD at relatively low doses (Table 5).

Except compound 52, of the N-substituted derivatives of

mexiletine tested (i.e., 45−51), cessation of cell beating in

normal CMs was observed only at elevated doses (i.e., 133−

200 μM). However, in LQT3 CMs, lipophilic compounds 46

and 50 caused cessation of cell beating at relatively low doses

(i.e., 66 and 7.4 μM, respectively) (Table 5). Except

compounds 43 and 50, in normal CMs, compounds 44−49

either did not cause EADs or only caused EADs at elevated

doses (i.e., 66 μM).

Generally, N-substituted mexiletines examined possessed

signiﬁcant potency for shortening the APD (i.e., EC₅₀for

shortening), and aside from compound 52, most compounds

that provided EC₅₀values were below 2 μM in LQT3 CMs

(Table 5). Based on the potency of shortening the APD by N-

substituted derivatives of mexiletine, additional derivatives

combining beneﬁcial properties of the phenoxy ring and

substituents on the amine nitrogen of phenyl mexiletine were

synthesized and tested.

Region III. N-Substituted Analogues of Phenoxy-

Substituted Phenyl Mexiletine. The eﬀect of N-substitu-

tion on the pharmacological properties of phenyl mexiletine

and analogues was investigated. Compared to phenyl

mexiletine, most N-substituted-2,3-dimethyl phenoxy-substi-

tuted phenyl mexiletine compounds examined decreased fold

shortening of the APD (Table 6). In LQT3 CMs, compared to

phenyl mexiletine, (i.e., compound 5), N-methyl, N-butyl, and

N-ethyl methoxy-2,3-dimethyl phenoxy-substituted com-

pounds (i.e., 55, 58, and 59) were more potent at shortening

the APD by 9, 18, and 22%, respectively. The more lipophilic

N-ethyl methoxy 3,5-ditriﬂuoromethyl phenoxy-substituted

phenyl mexiletine (i.e., 61) was 28% more potent at shortening

5391

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 6. Eﬀect of N-Substituted Phenoxy-Substituted Phenyl Mexiletine Analogues on Cardiovascular Properties in Human

a

iPSC-Derived Cardiomyocytes

a

b

Kinetic imaging cytometer assay results for N-substituted phenyl mexiletine derivatives. Lowest dose that cessation of beating was observed.

c

d

e

f

EC₅₀for shortening the AP. Ratio of AP untreated/AP treated. Maximal dose that shortens the AP. Dose that early after depolarization was

g

observed. EC₅₀for prolongation of the AP. The symbol “-” denotes that the indicated eﬀect was not observed.

the APD than phenyl mexiletine. Compared to phenyl

mexiletine, the somewhat more polar N-ethyl hydroxyl 3,5-

dimethyl phenoxy-substituted phenyl mexiletine, compound

62, increased shortening of the APD by 28%. In contrast, in

normal CMs, compounds 53−63 did not cause detectable

shortening of the APD nor did these compounds cause

prolongation of the APD (Table 6).

Except compounds 62 and 63 of the N-substituted

derivatives of phenyl mexiletine examined (i.e., 53−63),

cessation of cell beating in normal cardiomyocytes was

observed only at elevated concentrations (i.e., 133−200

μM). However, in LQT3 CMs, somewhat polar compounds

62 and 63 and compound 56 caused cessation of cell beating

at a relatively low concentration (i.e., 22 μM, respectively)

(Table 6). In normal CMs, compounds 53−63 did not cause

EADs.

in LQT3 CMs (Table 6). The potency of shortening the APD

by N-substituted derivatives of phenyl mexiletine was

signiﬁcant and showed that N-substituents on phenyl

mexiletine were tolerated. In general, results indicated that

N-alkylation decreased prolongation eﬀects and did not induce

EADs. Compared to phenyl mexiletine, the N-methoxyethyl

derivative of a number of phenyl mexiletines displayed similar

shortening to the parent compound and decreased prolonga-

tion eﬀects (Table 6). Certain N-substituted phenyl mexiletine

analogues examined possessed signiﬁcant potency for short-

ening the APD (i.e., EC₅₀for shortening). For example,

compounds 53 and 54 aﬀorded EC₅₀values of 0.8 μM in

LQT3 CMs (Table 6). Compounds 58, 59, and 61 were less

potent at shortening the APD (i.e., EC₅₀for shortening: 20,

5.7, and 6.6 μM, respectively).

Automated Patch Clamp Studies of Mexiletine

Analogues. Automated patch clamp experiments were done

to determine IC₅₀s for peak (I_NaP) and late sodium current

(I_NaL) inhibition and potassium current (I_Kr) inhibition for

selected compounds. Mexiletine and analogues were applied

Generally, N-substituted phenyl mexiletine analogues

examined possessed signiﬁcant potency for shortening the

APD (i.e., EC₅₀for shortening), and aside from compound 58,

most compounds that provided EC₅₀values were below 7 μM

5392

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 3. Traces of automated patch clamp studies. Panel (A) shows low-gain Na⁺channel signals where peak block was measured for a given

concentration and high-gain (inset) traces for late current. Panel (B) shows dose−response relationships for the peak and late Na⁺current. Panels

(C) and (D) show the analogous information for I_Krblock with current traces in panel (C) and dose−response in panel (D). Recordings were

analyzed at low (I_NaP) and high (I_NaL) gains for each compound concentration.

Table 7. Eﬀect of Mexiletine and Analogues on Automated Patch Clamp Dose−Response Data for Sodium and Potassium

Channel-Transfected Cells

a

Compound

IC₅₀I_NaL(μM)

n

IC₅₀I_NaP(μM)

n

IC₅₀I_Kr(μM)

n

Selectivity

b

1

2

3

22.5 4.4

4

5

4

7

6

5

7

4

5

4

7

6

10

6

5

6

4

182.8 41.8

91.4 12.7

130.4 12.0

128.6 12.8

128.2 10.0

161.6 21.0

76.8 9.0

153.7 8.3

20.6 2.1

34.2 6.7

41.1 8.4

20.1 4.1

20.0 4.1

10.9 0.7

124.1 5.9

25.8 6.6

38.0 7.1

45.1 9.2

4

5

8

6

5

4

7

5

6

9

7

9

13

10

5

7

53.7 8.0

27.6 12.5

30.1 5.2

84.0 7.5

54.3 11.1

98.0 4.8

45.8 11.8

49.3 8.4

8.0 1.3

22.9 4.7

37.5 14.0

6.2 0.8

7.16 1.2

9.2 1.3

7

5

7

5

6

9

6

10

4

12

5

9

7

9

6

2.4

2.42

4.1

4.42

1.81

1.9

4.6

2.84

0.7

35.6

36.1

31

19.4

8.9

1.2

34.5

6.8

11.4 3.7

7.4 0.4

19.0 4.6

29.9 5.4

51.8 5.9

10.0 4.9

17.3 10.5

11.7 0.8

0.643 0.047

1.04 0.34

0.20 0.054

1.03 0.06

1.03 0.1

10

11

15

16

24

26

27

30

36

37

38

43

59

60

63

36.0 3.2

44.1 2.2

27.6 4.2

5.1 0.85

16.8 2.4

0.747 0.063

0.753 0.094

1.02 0.02

10

16.5

a

b

Selectivity is deﬁned as IC₅₀for the lesser of the ratios of I_NaP/I_NaLor I_Kr/I_NaL. Data presented as mean standard error, n = number of replicate

determinations. Sodium channel SCN5A (F1473C mutant) was transfected into HEK cells and potassium channel was transfected into CHO cells.

over a concentration range from 0.1 to 100 μM, and inhibition

relative to vehicle control was determined at a steady state (∼3

min) for each concentration. For I_NaL, data were recorded and

peak channel currents were measured in response to 100 ms

voltage steps to −10 mV imposed from a holding potential of

−100 mV at a rate of 0.1 Hz (Figure 3, panel A). Compounds

were applied to the extracellular compartment in increasing

concentrations from 0 (i.e., DMSO vehicle control) to 100

μM. The ratio of IC₅₀values for inhibition of Na⁺channels was

determined by normalizing measured current to control

recordings for both I_NaPand I_NaLin the presence of a constant

vehicle (DMSO, 0.1%). Tetradotoxin (TTX), 50 μM, was

applied at the end of the experiments, so TTX currents could

be subtracted from the rest of the traces to identify only

sodium channels.

For I_Krexperiments, cells were held at −80 mV and

activating pulses (500 ms) were applied at +40 mV followed by

a return potential of −40 mV (0.1 Hz) whereupon deactivating

tail currents were measured. Currents were recorded again over

a concentration range of 0.1 to 100 μM and were normalized

5393

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

to control recordings (including 0.1% vehicle (DMSO)). The

resulting peak tail current was then plotted versus test

compound concentration to determine concentration−re-

sponse relationships for test compounds and I_Krinhibition

(Figure 3, panels C and D). The selective I_Krinhibitor E-4031,

5 μM, was used as a control inhibitor.

phosphate buﬀer, 37 °C) (data not shown) as judged by

HPLC analysis. At pH 3, compounds 27 and 30 had T_1/2of

>15 days and 33 and 59 had T_1/2of >30 days. (R)- and (S)-

Enantiomers of 27, 30, and 36 were prepared and tested for

stereoselective hepatic metabolism. Time-course studies (i.e.,

up to 1 h incubations at 37 °C in the presence of 0.5 mM

NADPH) were conducted. In the presence of human liver S-9,

the (R)-enantiomers of 27 and 36 appeared to be more

metabolically labile than their corresponding (S)-enantiomers.

Compared to (R)- and (S)-mexiletine and (R)- and (S)-59 and

-30 and (S)-27 and (S)-36 that did not have detectable

metabolism in the presence of S-9, compounds (R)-27 and

(R)-36 had T_1/2values of 408 and 1015 min, respectively. In

the presence of human liver microsomes, the (R)-enantiomers

of 30 and 36 appeared to be more metabolically labile than

their corresponding (S)-enantiomers. Both (R)- and (S)-

mexiletine, -27 and -59, did not have detectable metabolism in

the presence of human liver microsomes. Highly puriﬁed

human CYP3A4, 3A5, and 2D6 metabolized racemic

mexiletine (46.2, 48.8, and 6.0 pmol/min/μg of protein,

respectively). Highly puriﬁed CYP3A4, 3A5, and 2D6

metabolized racemic compound 30 (15.4, 42.6, and 43.6

pmol/min/μg of protein, respectively). Racemic compound 36

was detectably metabolized by highly puriﬁed CYP2D6 (6.9

pmol/min/μg of protein) but not CYP3A4 and CYP3A5.

Metabolism of racemic 27 and 59 in the presence of highly

puriﬁed CYP was not conducted.

In Vivo Studies of Mexiletine and Analogues. Based on

in vitro metabolism data, pharmacokinetics (PK) studies of

mexiletine, racemic 30, and its enantiomers were examined

(Table 9). Animals were administered by intravenous (5 mg/

kg) or oral (25 mg/kg) routes to obtain bioavailability (F)

data. Compared to PK parameters of mexiletine, racemic 30,

(R)-30, and (S)-30 aﬀorded superior oral administration PK

properties. This included acceptable half-lives, greater peak

onset (C_max), and greater bioavailability (F). Compared to

mexiletine (F = 37%), (R)-30 (100%) possessed superior oral

PK properties. Bioavailability values for (S)-30 and rac-30 were

42 and 38%, respectively.

Table 7 shows automated patch clamp IC₅₀values for I_NaL

_NaP, and I_Krobtained for each of the 18 compounds examined

,

I

with an n ≥ 4 for computed IC₅₀values. Also included is a

calculation for overall selectivity for inhibiting peak sodium

current over late sodium current (i.e., I_NaP/I_NaL) or I_Krover late

sodium current (i.e., I_Kr/I_NaL). Essentially, all compounds

examined were selective for late sodium current over peak

sodium current and hERG. Compared to mexiletine, there

were compounds that were more selective and more potent

late sodium current inhibitors. Consistent with a prior report,²⁴

mexiletine was measured to have IC₅₀values of 22.5 4.4 and

182.8 41.8 μM for late and peak Na currents, respectively,

and 53.7

8.0 μM for I_Kr. Compared to mexiletine,

replacement of the alpha methyl group with tert-butyl (i.e.,

2) or cylopropyl (i.e., 3) had only modest changes in channel

selectivity. Modiﬁcation of the phenoxy ring of mexiletine (i.e.,

15, 16, or 24) also had modest changes on channel ratios. In

contrast, alpha phenyl group modiﬁcation (i.e., 30, 33, 39, 40,

and 41) aﬀorded compounds with, in some cases, relatively

great selectivity for I_NaP(i.e., I_Kr/I_NaPratios of 31−36-fold).

The selectivity of N-methyl substituted mexiletine (i.e., 43)

was no diﬀerent from that of mexiletine itself. However, N-

substitution of alpha phenyl mexiletine analogues (i.e., 59, 60,

and 63) in some cases showed a signiﬁcant I_Kr/I_NaPselectivity

(i.e., 17−35-fold). Accordingly, based on the kinetic image

cytometry results and the automated patch clamp IC₅₀proﬁles

of channel inhibition, certain compounds were advanced to

further study. For selective late current block, we examined

compounds in greater detail that were maximally selective over

both I_NaPand I_Krin electrophysiology experiments.

Pharmaceutical Properties of Mexiletine and Ana-

logues. The chemical and metabolic stabilities of certain

mexiletine analogues were examined in advance of in vivo or ex

vivo studies. Because one of the objectives was to develop a

compound that possessed lower metabolic clearance than

mexiletine, the eﬀect of varying substituents of select

compounds on metabolic stability was investigated (Table

8). Racemic compounds 27, 30, 36, and 59 possessed

metabolic stability (in human liver microsomes + NADPH of

Behavioral Studies of Mexiletine and Analogues in

Mice. Previously, it was reported that mexiletine has an LD₅₀

of 115 mg/kg in mice.²⁵In our hands, signiﬁcant behavioral

eﬀects (i.e., seizures and deaths) were observed for mexiletine

at 100 and 200 mg/kg (i.p.). In contrast, we observed that

synthetic phenyl mexiletine analogues examined possessed

LD₅₀> 200 mg/kg (i.p.) in mice. Administration of 200 mg/kg

(i.p.) of (R)- or (S)-enantiomers of 27, 30, 36, or 59 did not

show any lethality. Thus, the LD₅₀was >200 mg/kg for the

phenyl mexiletine compounds examined.

T_1/2> 60 min) and chemical stability (T_1/2> 30 days, pH 7.4,

Table 8. Metabolic Stability of Mexiletine and Analogues

with Human Liver Preparations

Among the adverse reactions in rodents²⁶and human

patients²⁷administered (R)-mexiletine are nausea and seizures.

We examined the behavioral properties of enantiomerically

pure 27, 30, 36, or 59 and compared them to (R)- or (S)-

mexiletine at 100 mg/kg (Table 10). The phenyl mexiletine

enantiomers (i.e., enantiomers of 27, 30, 36, or 59) were

relatively well-tolerated in vivo (100 mg/kg, i.p.). For (R)-

mexiletine, administration of 100 mg/kg produced immobili-

zation, seizures, and death. (S)-Mexiletine (100 mg/kg)

produced lethargy and immobilization, but severe seizures

were not observed. In contrast, mice treated with enantiomer

(R)-30, 36, or 59 (100 mg/kg) showed no apparent behavioral

eﬀects although (R)-27 showed some immobilization eﬀects.

Lethargy and other central nervous system eﬀects were

T_1/2(min)

Compound number

Human liver S-9

Human liver microsomes

(R)-mexiletine

(S)-mexiletine

(R)-27

>95% after 1 h

408 min

>95% after 1 h

365 min

(S)-27

(R)-30

(S)-30

(R)-36

>95% after 1 h

1015 min

136 min

>95% after 1 h

406 min

(S)-36

(R)-59

(S)-59

>95% after 1 h

1040 min

>95% after 1 h

5394

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 9. Eﬀect of Mexiletine and Analogues on Pharmacokinetic Parameters in Rats

Compound

Mexiletine

Route of administration

T_max(h)

C_max(mg/mL)

AUC (h × ng/mL)

T_1/2(h)

i.v.

oral

0.17

1.0

289 39

167 41

447 47

839 456

10.6

11

F = 37%

i.v.

oral

F = 38%

i.v.

oral

F = 100%

i.v.

oral

(Rac)-30

(R)-30

(S)-30

0.17

1.5

250 16

226 15

544 27

1008 227

3.2

6.5

0.18

1.5

249

258

2

280 59

1556 19

1.2

5.3

0.17

1.5

219 15

226 15

407 34

1008 227

3.8

6.5

F = 42%

a

Two-four male rats for each route of administration.

potent than the eﬀect of ranolazine (10 μM) in the same ex

vivo model²⁸or GS-967 or roscovitine in related ex vivo

models.²⁸The conclusion is that (R)-30 is more eﬃcacious

than the currently used standard of care.

Table 10. Eﬀect of Mexiletine and Analogues on Behavior in

Mice

Behavioral eﬀect

((R)-enantiomer)

Behavioral eﬀect

a

Compound

((S)-enantiomer)

Mexiletine 1 seizure, 1 death, 2

immobilized

4/4 immobilized

CONCLUSIONS

■

27

30

36

59

4/4 immobilized

3/4 immobilized

2/4 slightly lethargic

2/4 lethargic

Patient-derived iPSC CMs combined with dynamic medicinal

chemistry were used to re-engineer mexiletine. Replacement of

metabolically labile mexiletine substituents with more meta-

bolically resilient groups (e.g., −CF₃for −CH₃) improved the

pharmaceutical and pharmacological properties of the

molecule. The combination of drug design in a dish and

disease in a dish approaches was shown to be useful to identify

more potent, selective, less toxic analogues of mexiletine that

may hold clinical relevance. In addition, compounds such as

relatively long-lived (R)-30 possessing later T_maxand greater

oral C_maxand AUC with good ex vivo eﬃcacy showed that

major shortcomings of mexiletine (the need for multiple and

repeated administration and toxicity) have potentially been

overcome.

No detectable eﬀect

2/4 shaking

a

Four mice were administered 100 mg/kg compound, i.p. Behavioral

eﬀects were monitored for 30 min. Lethality was measured after 24 h.

observed for (S)-27, 30, 36, or 59 at 100 mg/kg. Thus,

behavioral eﬀects for phenyl mexiletines 30, 36, or 59 were

stereoselective (i.e., behavioral toxicity was (S)- > (R)-

enantiomer). In conclusion, compared to mexiletine, it was

apparent that (R)-enantiomers of compounds 30, 36, and 59

examined showed considerably less toxicity (i.e., seizures and

death) than that observed for mexiletine enantiomers.

Ex Vivo Studies of (R)-30 with Rat Heart Preparations.

As an adjunct to LQT3 in vitro studies, we conducted ex vivo

studies of (R)-30 in aged rats. In this model, conduction

velocity (CV), action potential duration (APD), and

responsiveness to drugs were measured in perfused ex-vivo

rat heart preparations (Figure 4a). Ex vivo heart preparations

can be used to study the eﬀect of compounds to decrease

arrhythmias because CV and APD are preserved in buﬀer-

perfused rat hearts for up to 2 days. We chose a rat heart

model to test the eﬀects of (R)-30 ex vivo because (R)-30 is

potent and stereoselective, not extensively metabolized, has

good bioavailability and solubility (8−10 mg/mL water), and

no apparent toxicity up to 200 mg/kg (i.p.). In this perfused

ex-vivo rat heart model, the continuous presence (perfusion) of

H₂O₂(0.1 mM) produced EADs and ectopic ventricular beats

6 min after exposure that degenerated to ventricular

tachycardia (VT) and ventricular ﬁbrillation (VF) after 12

min (Figure 4b). Left untreated, these perfused hearts

generally die within 45 min. Using this preparation, complete

resolution of all forms of arrhythmias to normal sinus rhythm

was observed 30−40 min after administration of compound

(R)-30 (10 μM) in the continuous presence of H₂O₂(0.1

mM) (Figure 4c). This shows that administration of (R)-30 to

a heart suﬀering from severe arrhythmias potently reverses VT

and VF and corrects EADs in a very short time. This may be

clinically relevant. The eﬀect of (R)-30 is similar to or more

METHODS

■

General. Reagents, starting materials, and solvents were purchased

in the highest purity available from commercial suppliers and used as

received. Mexiletine, (R)-mexiletine, and (S)-mexiletine were

purchased from Toronto Research (Toronto, CA). Hydrochloride

salts were prepared by dissolution of the appropriate compound in a

minimum amount of dichloromethane and addition of excess 2 M

HCl in ether. The solvent was evaporated, and hydrochlorides were

used directly for evaluation. Phosphate-buﬀered saline (PBS) and

SYBR Green used in cell proliferation assays were purchased from

Life Technologies (Carlsbad, CA). Fluorescence was determined

using a Tecan SPECTRAFluor Plus plate reader (Tecan, San Jose,

CA). Luminescence was recorded on a Wallac Victor plate reader

(PerkinElmer Inc., Waltham, MA).

Chemistry Experimental Section. Experiments were carried out

under an inert atmosphere when oxygen- or moisture-sensitive

reagents or intermediates were employed. Commercial solvents and

reagents were used without further puriﬁcation. Microwave reactions

were conducted using a Biotage Initiator microwave synthesizer

(Biotage, Uppsala, Sweden). Reaction products were puriﬁed, when

necessary, using an Isco Combiﬂash R_fﬂash chromatography system

(Teledyne-Isco, Lincoln, NE) with the solvent systems indicated.

Nuclear magnetic resonance (NMR) data were recorded on a Varian

Mercury 300 MHz spectrometer (Agilent, Santa Clara, CA) unless

speciﬁed or in a Bruker 500 MHz instrument at NuMega Resonance

Laboratories (San Diego, CA) in the indicated solvents. Chemical

shifts for nuclear magnetic resonance (NMR) data were expressed in

5395

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

compounds were assessed for purity by HPLC via the following

conditions: column: Waters Atlantis dC18 4.6 × 50, 5 um; mobile

phase A: 0.05% TFA in water (v/v); mobile phase B: 0.05% TFA in

acetonitrile (v/v); gradient: 95.0% water/5.0% acetonitrile linear to

5% water/95% acetonitrile in 4.0 min, hold at 5% water/95%

acetonitrile to 5.0 min; ﬂow: 2 mL/min. Samples analyzed for ESI-

TOF high-accuracy mass spectroscopy were sent for analysis at the

mass spectroscopy service at The Scripps Research Institute, San

Diego, CA.

Chiral compounds were analyzed using a Hitachi-7100 HPLC

system equipped with a Phenomenex Lux 5 μm Cellulose-1 150 × 4.6

mm column. Detection was achieved with a Hitachi-7400 UV

detector with monitoring at 220 nm. Compounds were eluted with a

mixture of 70:20:5 hexanes/isopropanol/acetonitrile containing

0.02% perchloric acid with a ﬂow rate of 0.5 mL/min.

Syntheses of Amines via Reduction of O-Benzyloximes. 1-

(2,6-Dimethylphenoxy)-3,3-dimethylbutan-2-amine (2). Step 1: 1-

(2,6-Dimethylphenoxy)-3,3-dimethylbutan-2-one. To a mixture of

1-bromo-3,3-dimethylbutan-2-one (0.75 mL, 5.6 mmol) and 2,6-

dimethylphenol (1.02 g, 8.3 mmol) in DMF (5 mL) was added 1.1 g

(8.3 mmol) of potassium carbonate. After stirring at 21 °C for 16 h

water was added and extracted with diethyl ether. The organic phase

was washed (water, 2 M sodium hydroxide, brine), dried over

magnesium sulfate, and concentrated under reduced pressure. The

residue was puriﬁed by ﬂash chromatography on silica gel using an

ethyl acetate/hexane gradient. The target ketone was obtained (0.49

1

g) in 40% yield. H NMR (300 MHz, CDCl₃): 7.00−7.02 (m, 2H),

6.93−6.96 (m, 1H), 4.64 (s, 2H), 2.26 (s, 6H), 1.22 (s, 9H). MS:

+

221.0 (MH ).

Step 2: (E/Z)-1-(2,6-Dimethylphenoxy)-3,3-dimethylbutan-2-one

O-Benzyl Oxime. To a ﬂask containing 400 mg (1.8 mmol) of 1-(2,6-

dimethylphenoxy)-3,3-dimethylbutan-2-one in 8 mL of ethanol were

added O-benzylhydroxylamine hydrochloride (879 mg, 5.5 mmol)

and pyridine (0.882 mL, 10.9 mmol) and stirred at 21 °C for 16 h,

diluted with water, and extracted with diethyl ether. The organic

phase was washed (water, brine), dried over sodium sulfate, and

concentrated. Chromatography on silica gel using a gradient of ethyl

acetate in hexane was conducted to yield 365 mg (62%) of the

1

product as a colorless oil. H NMR (CDCl₃): 1.31 (s, 9H), 2.26 (s,

6H), 4.59 (s, 2H), 5.08 (s, 2H), 6.87−6.92 (m, 1H), 6.96−6.98 (m,

2H), 7.28−7.31 (m, 5H). MS: 326.0 (MH⁺).

Step 3: 1-(2,6-Dimethylphenoxy)-3,3-dimethylbutan-2-amine

(2). To a ﬂask containing 100 mg (0.31 mmol) of the above O-

benzyl oxime in 1 mL of THF was added 1.2 mL (1.2 mmol) of a 1 M

solution of borane in THF and heated at 50 °C for 16 h. The mixture

was allowed to cool to room temperature and carefully stopped by

addition of 6 M aqueous HCl. After about 10 min, the mixture was

taken to pH > 10 by addition of an excess of 6 M aqueous sodium

hydroxide and extracted with ethyl acetate. The organic phase was

washed (water, brine), dried (sodium sulfate), and concentrated.

Chromatographic puriﬁcation using 5% methanol in dichloromethane

aﬀorded the target product. ¹H NMR (CDCl₃): 1.03 (s, 9H), 2.31 (s,

6H), 3.12−3.16 (m, 1H), 3.75−3.87 (m, 2H), 6.89−6.94 (m, 1H),

6.96−7.02 (m, 2H) MS: 222.0 (MH⁺).

Figure 4. (A) Initiation of early after depolarizations-mediated

ectopic ventricular beats and ventricular ﬁbrillation in an isolated

perfused aged rat heart exposed to hydrogen peroxide. (B)

Representative example of administration of compound (R)-30 (10

μM) after 20 min to an aged rat heart perfused with H₂O₂(0.1 mM).

Note that compound (R)-30 suppressed ventricular ﬁbrillation (VF)

by conversion to monomorphic ventricular tachycardia (VT) and

then to sinus rhythm. (C) In the aged rat heart (treated as in Figure

4A) with continued perfusion of H₂O₂(0.1 mM) and treatment with

compound (R)-30 (10 μM) after 40 min, the heart continued to show

sustained sinus rhythm. Complete resolution of all forms of

arrhythmias to normal sinus rhythm 30−40 min after perfusion of

compound (R)-30 (10 μM) was observed in an isolated perfused aged

heart in the presence of hydrogen peroxide.

The following compounds were prepared using similar sequences

with the appropriate starting materials:

1-Cyclopropyl-2-(2,6-dimethylphenoxy)ethanamine (3). ¹H

NMR (CDCl₃): 7.03−6.90 (m, 3H), 3.90−3.77 (AB of ABX, J_AB

=

9.0 Hz, 2H), 2.41 (m, 1H, overlapping with neighboring peak), 2.32

(s, 6H), 0.96 (m, 1H), 0.57 (m, 2H), 0.32 (m, 2H) ppm. MS: 206.2

(MH⁺). R_f= 0.17 (10% methanol in dichloromethane, silica gel).

1-Cyclohexyl-2-(2,6-dimethylphenoxy)ethan-1-amine (4). ¹H

NMR (300 MHz, methanol-d₄) δ 1.15−1.39 (m, 5H), 1.63−2.05

(m, 6H), 2.30 (s, 6H), 3.41 (td, J = 3.7, 7.2 Hz, 1H), 3.66 (s, 1H),

3.86−4.05 (m, 1H), 6.87−7.07 (m, 2H). MS: 248.2 (MH⁺).

2-(2,6-Dimethylphenoxy)-1-phenylethanamine (5). ¹H NMR

(CDCl₃): 7.43−7.46 (m, 2H), 7.27−7.37 (m, 3H), 6.97−6.99 (m,

2H), 6.87−6.92 (m, 1H), 4.46 (dd, J = 4.7 and 8.0 Hz, 1H), 3.79−

3.89 (m, 2H), 2.25 (s, 6H). MS: 242.2 (MH⁺).

parts per million (ppm, δ) referenced to residual peaks from

deuterated solvents. Mass spectrometry (MS) data were reported

from liquid chromatography−mass spectrometry (LCMS) instrumen-

tation. Electrospray ionization (ESI) mass spectral data were obtained

using an Agilent 1100 LC/MS (Agilent, Santa Clara, CA). Final test

compounds were pure based on LCMS analysis using UV−vis

detection at 275 and 220 nM. Mexiletine and analogue compound

purities were determined by high-performance liquid chromatography

(HPLC), and all ﬁnal test compounds were of >95% purity. Final

5396

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2-(2,6-Dimethylphenoxy)-1-p-tolylethanamine (6). ¹H NMR

(CDCl₃): 2.29 (s, 6H), 2.36 (s, 3H), 3.78−3.87 (m, 2H), 4.43 (dd,

J = 4.1 and 8.0 Hz, 1H), 6.88−6.93 (m, 1H), 6.98−7.01 (m, 2H),

7.17 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H). MS: 256.2 (MH⁺).

2-(2,6-Dimethylphenoxy)-1-(4-(triﬂuoromethyl)phenyl)-

MS: 183.1 (MH⁺). R_f= 0.29 (15% methanol in dichloromethane on

silica gel).

1-(m-Tolyloxy)propan-2-amine (22). ¹H NMR (500 MHz,

CD₃OD) 7.11−7.23 (m, 1H), 6.69−6.90 (m, 3H), 4.09−4.23 (m,

1H), 3.91−4.04 (m, 1H), 3.66−3.76 (m, 1H), 2.32 (s, 3H), 1.41 (d, =

7.1 Hz, 3H). MS: 166.1 (MH⁺).

1

ethanamine (7). H NMR (CDCl₃): 2.23 (s, 6H), 3.78−3.88 (m,

2H), 4.51 (dd, J = 4.4 and 4.4 Hz, 1H), 6.88−6.93 (m, 1H), 6.97−

7.00 (m, 2H), 7.56−7.72 (m, 4H). MS: 309.2 (MH⁺).

2-(2,6-Dimethylphenoxy)-l-(4-methoxyphenyl)ethanamine (8).

¹H NMR (CDCl₃): 7.36 (s, J = 8.8 Hz, 2H), 6.97−6.99 (m, 2H),

6.85−6.92 (m, 3H), 4.39−4.43 (m, 1H), 3.80 (s, 3H), 3.79−3.82 (m,

2H), 2.26 (s, 6H). MS: 272.1 (MH⁺).

1-(3-(Triﬂuoromethyl)phenoxy)propan-2-amine (23). ¹H NMR

(500 MHz, CD₃OD) 6.90−6.95 (m, 1H), 7.15−7.30 (m, 3H), 4.31

(d, J = 3.3 Hz, 1H), 4.23 (d, J = 6.6 Hz, 1H), 2.77−2.98 (m, 1H),

2.28 (s, 6H), 1.12−1.27 (m, 1H), 0.80−0.93 (m, 1H), 0.71−0.81 (m,

1H), 0.56−0.70 (m, 1H), 0.43−0.52 (m, 1H). MS: 220.1 (MH⁺).

1-(3,5-Dimethylphenoxy)propan-2-amine (24). ¹H NMR (500

MHz, CD₃OD) 6.67 (none, 1H), 6.65 (s, 2H), 4.31 (d, J = 3.29 Hz,

1H), 4.23 (d, J = 6.57 Hz, 1H), 2.77−2.98 (m, 1H), 2.28 (s, 6H),

1.12−1.27 (m, 1H), 0.80−0.93 (m, 1H), 0.71−0.81 (m, 1H), 0.56−

0.70 (m, 1H), 0.43−0.52 (m, 1H). MS: 180.0 (MH⁺).

2-(2,6-Dimethylphenoxy)-1-(naphthalen-2-yl)ethan-1-amine (9).

¹H NMR (300 MHz, DMSO-d₆) δ 8.95 (s, 3H), 8.15 (s, 1H), 7.97

(m, 3H), 7.77 (d, J = 8.5 Hz, 1H), 7.57 (dd, J = 5.7, 3.8 Hz, 2H),

7.01−6.87 (m, 3H), 4.90 (s, 1H), 4.38−4.00 (m, 2H), 2.10 (s, 6H).

MS: 292.1 (MH⁺). R_f= 0.52 (5% methanol in dichloromethane on

silica).

1-(3,5-Bis(triﬂuoromethyl)phenoxy)propan-2-amine (25). ¹H

NMR (500 MHz, CD₃OD) 7.63−7.44 (m, 1H), 7.04−7.26 (m,

1H), 6.72−6.92 (m, 1H), 4.65−4.80 (m, 1H), 4.32−4.41 (m, 1H),

4.25−4.33 (m, 1H), 2.32 (s, 3H). MS: 288.1 (MH⁺).

1-Cyclopropyl-2-(2,6-dimethylphenoxy)ethan-1-amine (12). ¹H

NMR (CDCl₃): 7.03−6.90 (m, 3H,), 3.90−3.77 (AB of ABX, J_AB

=

2-(2,4-Dimethylphenoxy)-1-phenylethan-1-amine (26). ¹H NMR

(500 MHz, CD₃OD) 7.41−7.69 (m, 5H), 6.90−7.01 (m, 2H), 6.77−

6.84 (m, 1H), 4.75−4.82 (m, 1H), 4.26−4.34 (m, 2H), 2.23 (s, 3H),

2.22 (s, 2H). MS: 242.1 (MH⁺).

9.0 Hz, 2H), 2.41 (m, 1H, overlapping with neighboring peak), 2.32

(s, 6H), 0.96 (m, 1H), 0.57 (m, 2H), 0.32 (m, 2H). MS: 206.2

(MH⁺).

1-Cyclopropyl-2-(2,4-dimethylphenoxy)ethan-1-amine (13). ¹H

NMR (300 MHz, CD₃OD) 6.90−7.00 (m, 2H), 6.77−6.86 (m, 1H),

4.06−4.28 (m, 2H), 2.84 (ddd, J = 3.4, 6.3, 10.0 Hz, 1H), 2.24 (broad

s, 6H), 1.11 (m, 1H), 0.78 (ddd, J = 2.0, 3.8, 8.0 Hz, 2H), 0.45−0.63

(m, 2H). MS: 206.2 (MH⁺).

1-Phenyl-2-(o-tolyloxy)ethanamine (27). ¹H NMR (300 MHz,

CDCl₃): 7.44−7.47 (m, 1H), 7.29−7.37 (m, 4H), 7.08−7.13 (m,

2H), 6.83−6.88 (m, 1H), 6.75−6.78 (m, 1H), 4.45−4.49 (m, 1H),

4.13 (dd, J = 4.1 and 9.1 Hz, 1H), 3.98−4.05 (m, 1H), 2.88 (bs, 2H),

2.24 (s, 3H). MS: 228.0 (MH⁺). ¹³C NMR (125 MHz, CD₃OD):

157.14, 135.27, 130.99, 130.55, 128.88, 128.23, 123.04, 115.62, 71.35,

55.81, 28.86. Calcd m/z for [C₁₅H₁₇NO + H]⁺: 228.1383. Observed:

228.1387. MS: 228.0 (MH⁺).

1-Cyclopropyl-2-(3,4-dimethylphenoxy)ethan-1-amine (14). ¹H

NMR (300 MHz, CD₃OD) 7.03 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 2.8

Hz, 1H), 6.72 (dd, J = 2.7, 8.2 Hz, 1H), 4.23 (dd, J = 3.5, 10.3 Hz,

1H), 4.09 (dd, J = 7.0, 10.3 Hz, 1H), 2.79 (ddd, J = 3.5, 7.0, 10.4 Hz,

1H), 2.2.4 (s, 3H), 2.20 (s, 3H), 1.07−1.24 (m, 1H), 0.70−0.82 (m,

2H), 0.54 (qd, J = 4.9, 10.2 Hz, 2H). MS: 206.2 (MH⁺).

1-Cyclopropyl-2-(3,5-dimethylphenoxy)-N-(2-methoxyethyl)-

1

2-(2-Ethylphenoxy)-1-phenylethan-1-amine (28). H NMR (300

MHz, CDCl₃): 7.44−7.47 (m, 1H), 7.29−7.38 (m, 4H), 7.08−7.15

(m, 2H), 6.86−6.91 (m, 1H), 6.77−6.80 (m, 1H), 4.45−4.49 (m,

1H), 4.12 (dd, J = 4.1 and 9.1 Hz, 1H), 3.99−4.05 (m, 1H), 2.65 (q, J

= 7.4 Hz, 2H), 2.52 (bs, 2H), 1.18 (t, J = 7.4 Hz, 3H). MS: 242.0

(MH⁺).

1

ethan-1-amine (15). H NMR (300 MHz, CD₃OD) 6.58 (s, 1H),

6.54 (s, 2H), 4.05−4.16 (m, 1H), 4.05−4.16 (m, 1H), 3.44−3.63 (m,

2H), 3.37 (s, 3H), 2.95−3.16 (m, 1H), 2.73−2.95 (m, 1H), 2.10−

2.22 (m, 1H), 0.45−0.61 (m, 1H), 0.32−0.47 (m, 2H), 0.14−0.32

(m, 2H). MS: 264.2 (MH⁺).

1-Phenyl-2-(2-propylphenoxy)ethanamine (29). ¹H NMR (300

MHz, CDCl₃): 7.44−7.47 (m, 1H), 7.29−7.38 (m, 4H), 7.08−7.14

(m, 2H), 6.84−6.90 (m, 1H), 6.77−6.80 (m, 1H), 4.44−4.48 (m,

1H), 4.11 (dd, J = 4.4 and 9.1 Hz, 1H), 3.98−4.04 (m, 1H), 2.56−

2.61 (m, 2H), 2.36 (bs, 2H), 1.57 (sextet, J = 7.4 Hz, 2H), 0.95 (t, J =

7.4 Hz, 3H). MS: 256.0 (MH⁺).

1-Cyclopropyl-2-(3,5-dimethylphenoxy)ethan-1-amine (16). ¹H

NMR (300 MHz, CDCl₃): 8.67 (br. s., 3H), 6.68 (s, 2H), 6.58 (s,

1H), 3.85−4.39 (m, 2H), 2.26 (s, 6H), 2.16−2.39 (m, 1H), 1.82−

2.10 (m, 1H), 0.93−1.30 (m, 2H), 0.52 (m, 2H). MS: 206.2 (MH⁺).

1-Phenyl-2-(2-(triﬂuoromethyl)phenoxy)ethanamine (30). ¹H

NMR (300 MHz, CDCl₃): 7.54−7.58 (m, 1H), 7.44−7.49 (m,

2H), 7.29−7.42 (m, 4H), 6.98−7.03 (m, 1H), 6.91−6.94 (m, 1H),

4.48−4.58 (m, 1H), 4.19−4.27 (m, 1H), 4.02−4.11 (m, 1H), 2.38

(bs, 2H). MS: 282.0 (MH⁺). ¹³C NMR (125 MHz, CD₃OD): 157.50,

135.56, 132.08, 130.88, 130.54, 128.75, 128.25, 122.97, 113.09, 70.40,

56.11. Calcd m/z for [C₁₅H₁₄F₃NO + H]⁺: 282.1100. Observed:

282.1089.

1

1-(2-Methylphenoxy)propan-2-amine (17). H NMR (300 MHz,

CDCl₃): 7.16 (m, 2H), 6.87 (td, J = 7.5, 1.1 Hz, 1H), 6.82 (m, 1H),

3.93−3.70 (AB of ABX, J_AB= 8.8 Hz, 2H), 3.41 (m, 1H), 2.27 (s,

3H), 1.23 (d, J = 6.6 Hz, 1H). MS: 166.1 (MH⁺). R_f= 0.20 (15%

methanol in dichloromethane on silica gel).

1-(2-Ethylphenoxy)propan-2-amine (18). ¹H NMR (300 MHz,

CDCl₃): 7.16 (m, 2H), 6.92 (td, J = 7.4, 1.1 Hz, 1H), 6.82 (dd, J =

8.8, 0.9 Hz, 1H), 3.92−3.71 (AB of ABX, J_AB= 9.0 Hz, 2H), 3.41 (m,

1H,), 2.69 (q, J = 7.5 Hz, 2H), 1.24 (t, J = 7.5 Hz, 3H), 1.23 (d, J =

6.6 Hz, 1H). MS: 180.1 (MH⁺). R_f= 0.22 (15% methanol in

dichloromethane on silica gel).

2-(2-Methoxyphenoxy)-1-phenylethanamine (31). ¹H NMR

(300 MHz, CDCl₃): 7.47−7.50 (m, 1H), 7.30−7.39 (m, 4H),

6.86−6.98 (m, 4H), 4.48−4.52 (m, 1H), 4.15−4.20 (m, 1H), 4.05−

4.11 (m, 1H), 3.79 (m, 3H), 2.68 (bs, 2H).). MS: 244.0 (MH⁺).

1-Phenyl-2-(2-(triﬂuoromethoxy)phenoxy)ethan-1-amine (32).

¹H NMR (300 MHz, CDCl₃): 7.44−7.58 (m, 2H), 7.31−7.43 (m,

3H), 7.14−7.30 (m, 2H), 6.84−7.03 (m, 2H), 4.51 (dd, J = 3.58, 8.53

Hz, 1H), 4.15 (dd, J = 3.58, 8.80 Hz, 1H), 3.91−4.07 (m, 1H) for the

1

1-(2-Propylphenoxy)propan-2-amine (19). H NMR (300 MHz,

CDCl₃): 7.15 (m, 2H), 6.89 (td, J = 7.4, 1.4 Hz, 1H), 6.82 (m, 1H),

3.92−3.70 (AB of ABX, J_AB= 8.8 Hz, 2H), 3.41 (m, 1H), 2.64 (t, J =

7.5 Hz, 2H), 1.64 (m, 2H), 1.23 (d, J = 6.6 Hz, 1H), 0.98 (t, J = 7.1

Hz, 3H). MS: 194.1 (MH⁺). R_f= 0.24 (15% methanol in

dichloromethane on silica gel).

1

free base amine. H NMR (300 MHz, CD₃OD) d 7.41−7.63 (m,

5H), 7.26−7.39 (m, 2H), 7.17−7.25 (m, 1H), 7.00−7.13 (m, 1H),

4.80 (dd, J = 4.95, 7.15 Hz, 1H), 4.34−4.53 (m, 2H) for the

hydrochloride salt. MS: 298.0 (MH⁺). R_f= 0.5 (5% methanol in

dichloromethane on silica gel).

1-(2-(Triﬂuoromethyl)phenoxy)propan-2-amine (20). ¹H NMR

(300 MHz, CDCl₃): 7.57 (dd, J = 7.7, 1.3 Hz, 1H), 7.48 (m, 1H),

7.00 (m, 2H), 4.03−3.76 (AB of ABX, J_AB= 8.5 Hz, 2H), 3.44 (m,

1H), 1.23 (d, J = 6.6 Hz, 1H) ppm. MS: 220.0 (MH⁺). R_f= 0.16 (15%

methanol in dichloromethane on silica gel).

1-Phenyl-2-(2-(2,2,2-triﬂuoroethoxy)phenoxy)ethan-1-amine

1

(33). H NMR (CDCl₃): 7.25−7.59 (m, 5H), 6.81−7.13 (m, 4H),

1-(2-Methoxyphenoxy)propan-2-amine (21). 1H NMR (300

4.48 (d, J = 4.40 Hz, 1H), 4.29 (qd, J = 2.06, 8.39 Hz, 2H), 4.20 (td, J

= 3.44, 4.75 Hz, 1H), 3.99−4.14 (m, 1H) for the amine free base. ¹H

NMR (300 MHz, DMSO-d₆) δ 8.57 (br. s., 3H), 7.63−7.53 (m, 2H),

MHz, CDCl₃): 6.96−6.88 (m, 4H), 3.98−3.70 (AB of ABX, J_AB

=

9.4 Hz, 2H), 3.88 (s, 3H), 3.41 (m, 1H), 1.20 (d, J = 6.6 Hz, 1H).

5397

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

7.39−7.52 (m, 2H), 7.06−7.24 (m, 3H), 6.91−7.06 (m, 2H), 4.76

(m, 1H), 4.53−4.70 (m, 2H), 4.23−4.36 (m, 2H) for the

hydrochloride. MS: 311.0 (MH⁺). R_ffor the free base = 0.5 (5%

methanol in dichloromethane on silica gel).

dichloromethane to provide the title compound (22.6 mg, 38% yield)

as a pale yellow oil that solidiﬁed over time. H NMR (CDCl₃):

7.02−6.93 (m, 3H), 6.43 (broad s, 1H), 3.93 (m, 2H), 3.58 (m, 1H),

2.86 (s, 3H), 2.30 (s, 6H), 1.52 (d, J = 6.6 Hz, 3H) ppm. R_f= 0.23

(10% methanol in dichloromethane on silica gel).

1

1-Phenyl-2-(m-tolyloxy)ethan-1-amine (34). ¹H NMR (500

MHz, CD₃OD): δ 7.63−7.44 (m, 1H), 7.04−7.26 (m, 1H), 6.72−

6.92 (m, 1H), 4.65−4.80 (m, 1H), 4.32−4.41 (m, 1H), 4.25−4.33

(m, 1H), 2.32 (s, 3H). MS: 228.0 (MH⁺).

The following compounds were synthesized by the same sequence

as 43:

1-(2,6-Dimethylphenoxy)-N-ethylpropan-2-amine (44). ¹H NMR

(300 MHz, CDCl₃): 7.02−6.92 (m, 3H), 6.23 (broad s, 1H), 4.03−

3.96 (AB of ABX, J_AB= 10.1 Hz, 2H), 3.61 (m, 1H), 3.24 (m, 2H),

2.30 (s, 6H), 1.51 (d, J = 6.9 Hz, 3H), 1.45 (t, J = 6.4 Hz, 3H). MS:

208.2 (MH⁺). R_f= 0.19 (10% methanol in dichloromethane on silica

gel).

1

1-Phenyl-2-(3-(triﬂuoromethyl)phenoxy)ethan-1-amine (35). H

NMR (CDCl₃): 7.27−7.49 (m, 8H), 4.48 (dd, J = 3.8, 8.6 Hz, 1H),

4.15 (dd, J = 3.8, 8.7 Hz, 1H), 4.02 (t, J = 8.7 Hz, 1H). MS: 282.0

(MH⁺).

2-(3,5-Dimethylphenoxy)-1-phenylethanamine Hydrochloride

1

(36). H NMR (300 MHz, CD₃OD): δ 7.56−7.46 (m, 5H), 6.64

1-(2,6-Dimethylphenoxy)-N-propylpropan-2-amine (45). ¹H

NMR (300 MHz, CDCl₃): 7.03−6.90 (m, 3H), 3.70 (m, 2H), 3.17

(m, 1H), 2.82−2.62 (m, 2H), 2.50 (broad s, 1H), 2.31 (s, 6H), 1.62

(m, 2H), 1.28 (d, J = 6.6 Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H). MS: 222.3

(MH⁺). R_f= 0.24 (10% methanol in dichloromethane on silica gel).

1-(2,6-Dimethylphenoxy)-N-butylpropan-2-amine (46). ¹H

NMR (300 MHz, CDCl₃): 7.02−6.91 (m, 3H), 4.21 (broad s, 1H),

3.83−3.73 (AB of ABX, J_AB= 9.6 Hz, 2H), 3.27 (m, 1H), 2.95−2.76

(m, 2H), 2.30 (s, 6H), 1.65 (m, 2H), 1.43 (m, 2H,), 1.28 (d, J = 6.6

Hz, 3H), 0.98 (t, J = 7.2 Hz, 3H). MS: 236.2 (MH⁺). R_f= 0.31 (10%

methanol in dichloromethane on silica gel).

(s, 3H), 4.74 (dd, J = 8.3, 4.4 Hz, 1H), 4.35−4.24 (AB of ABX, J_AB

=

10.5 Hz, 2H), 2.27 (s, 6H). MS: 242.0 (MH⁺). ¹³C NMR (125 MHz,

CD₃OD): 159.47, 140.71, 135.41, 130.86, 130.54, 128.75, 124.65,

113.61, 69.94, 56.07, 21.07. MS: 242.0 (MH⁺). Calcd m/z for

[C₁₆H₁₉NO + H]⁺: 242.1540. Observed: 242.1542.

2-(3,5-Bis(triﬂuoromethyl)phenoxy)-1-phenylethan-1-amine

(37). ¹H NMR (300 MHz, CDCl₃): 7.27−7.49 (m, 8H), 4.48 (dd, J =

3.8, 8.6 Hz, 1H), 4.15 (dd, J = 3.8, 8.7 Hz, 1H), 4.02 (t, J = 8.7 Hz,

1H). MS: 350.0 (MH⁺).

2-(2,3-Dimethylphenoxy)-1-phenylethanamine (38). ¹H NMR

(300 MHz, CDCl₃): 7.50−7.47 (m, 2H), 7.42−7.28 (m, 3H), 7.03 (t,

J = 7.7 Hz, 1H), 6.79 (d, J = 7.5 Hz, 1H), 6.68 (d, J = 8.0 Hz, 1H),

4.47 (dd, J = 7.7, 3.8 Hz, 1H), 4.13−3.95 (AB of ABX, J_AB= 9.1 Hz,

2H), 2.39 (broad s, 2H), 2.30. MS: 242.1 (MH⁺).

1-(2,6-Dimethylphenoxy)-N-butylpropan-2-amine (47). ¹H

NMR (300 MHz, CDCl₃): δ 7.02−6.89 (m, 3H), 3.76−3.67 (m,

2H), 3.59 (app t, J = 4.9 Hz, 2H), 3.40 (s, 3H), 3.22−3.12 (m, 2H),

3.01−2.85 (m, 2H), 2.54 (broad s, 1H), 2.31 (s, 6H,), 1.22 (d, J = 6.6

Hz, 3H). MS: 238.2 (MH⁺). R_f= 0.36 (10% methanol in

dichloromethane on silica gel).

2-(2,4-Dimethylphenoxy)-1-phenylethan-1-amine (39). ¹H NMR

(300 MHz, CD₃OD): 7.42−7.60 (m, 5H), 6.94 (m, 2H), 6.80 (d, J =

8.1 Hz, 1H), 4.79 (dd, J = 4.9, 7.1 Hz, 1H), 4.22−4.36 (m, 2H), 2.23

(s, 3H), 2.24 (s, 3H). MS: 242.3 (MH⁺).

1-(2,6-Dimethylphenoxy)-N-phenylpropan-2-amine (48). ¹H

NMR (300 MHz, CDCl₃): 7.20 (t, J = 7.7 Hz, 2H), 7.00 (m, 2H),

6.92 (m, 1H), 6.75−6.69 (m, 3H), 4.13 (broad s, 1H), 3.89 (m, 1H),

3.85−3.80 (m, 2H), 2.27 (s, 6H), 1.47 (d, J = 6.6 Hz, 3H). MS: 256.2

(MH⁺). R_f= 0.34 (20% ethyl acetate in hexane on silica gel).

1-(2,6-Dimethylphenoxy)-N-benzylpropan-2-amine (49). ¹H

NMR (300 MHz, CDCl₃): 7.40−7.3 (m, 4H), 7.27 (m, 1H,

overlapping with the solvent signal), 7.01 (m, 2H), 6.93 (m, 1H),

4.00−3.87 (AB quartet, J = 13.1 Hz, 2H), 3.77−3.69 (m, 2H), 3.20

(m, 1H), 2.29 (s, 6H), 1.22 (d, J = 6.0 Hz, 3H). MS: 270.2 (MH⁺). R_f

= 0.24 (5% methanol in dichloromethane on silica gel).

2-(3,4-Dimethylphenoxy)-1-phenylethan-1-amine (40). ¹H NMR

(300 MHz, CD₃OD): 7.42−7.58 (m, 5H), 7.03 (d, J = 8.2 Hz, 1H),

6.82 (d, J = 2.7 Hz, 1H), 6.73 (dd, J = 2.8, 8.2 Hz, 1H), 4.73 (dd, J =

4.2, 8.4 Hz, 1H), 4.18−4.37 (m, 2H), 2.24 (s, 3H), 2.19 (s, 3H). MS:

242.1 (MH⁺).

2-(Naphthalen-2-yloxy)-1-phenylethan-1-amine (41). ¹H NMR

(300 MHz, CD₃OD): 8.81−8.35 (m, 3H), 7.82 (dd, J = 16.8, 10.6

Hz, 3H), 7.63 (d, J = 6.5 Hz, 4H), 7.56−7.09 (m, 5H), 4.81 (s, 1H),

4.40 (s, 2H). MS: 264.1 (MH⁺). R_f= 0.45 (5% methanol in

dichloromethane on silica gel).

1-(2,6-Dimethylphenoxy)-N-(2-phenylethyl)propan-2-amine

1

2-(Naphthalen-1-yloxy)-1-phenylethan-1-amine (42). ¹H NMR

(300 MHz, DMSO-d₆): 8.8 (s, 3H), 8.44 (d, J = 9.6 Hz, 1H), 7.96−

7.76 (m, 1H), 7.49 (m, 9H), 6.99 (d, J = 7.5 Hz, 1H), 4.9 (m, 1H),

4.46 (d, J = 5.7 Hz, 2H). MS: 264.1 (MH⁺). R_f= 0.50 (5% methanol

in dichloromethane on silica gel).

(50). H NMR (300 MHz, CDCl₃): 7.31 (m, 2H), 7.26 (m, 2H,

overlapping with the solvent signal), 7.22 (t, J = 7.1 Hz, 1H), 7.01 (m,

2H), 6.92 (m, 1H), 3.67 (m, 2H), 3.15 (m, 1H), 3.06 (m, 1H), 2.96−

2.83 (m, 2H), 2.25 (s, 6H), 1.18 (d, J = 6.1 Hz, 3H). MS: 284.1

(MH⁺). R_f= 0.25 (5% methanol in dichloromethane on silica gel).

2-(2,3-Dimethylphenoxy)-N-methyl-1-phenylethanamine (55).

¹H NMR (300 MHz, CDCl₃): 7.48−7.46 (m, 2H), 7.42−7.3 (m,

3H), 7.01 (t, J = 7.7 Hz, 1H), 6.78 (d, J = 7.4 Hz, 1H), 6.68 (d, J = 8.3

Hz, 1H), 4.02 (m, 3H), 2.42 (s, 3H), 2.29 (s, 3H), 2.18 (s, 3H). MS:

256.2 (MH⁺). R_f= 0.33 (5% methanol in dichloromethane on silica

gel).

Synthesis of N-Alkylated Analogues via Reductive Amina-

tion. 1-(2,6-Dimethylphenoxy)-N-methylpropan-2-amine (43).

Step 1: 1-(2,6-Dimethylphenoxy)propan-2-one. To a solution of

α-bromoacetone (3.17 g, 16.66 mmol) in 30 mL of DMF were added

3.10 g (25.4 mmol) of 2,6-dimethylphenol and 3.40 g (24.6 mmol) of

potassium carbonate. The mixture was stirred at 21 °C for 20 h and

then at 55 °C for an additional 2 h. The reaction mixture was poured

on water and extracted with ether. The organic phase was washed (2

M sodium hydroxide, brine), dried over sodium sulfate, and

concentrated under reduced pressure. Chromatography on silica gel

using a gradient of ethyl acetate in hexanes aﬀorded 1.01 g (34%

1

2-(2,3-Dimethylphenoxy)-N-ethyl-1-phenylethanamine (56). H

NMR (300 MHz, CDCl₃): 7.49−7.46 (m, 2H), 7.41−7.29 (m, 3H),

7.01 (t, J = 8.0 Hz, 1H), 6.78 (d, J = 7.4 Hz, 1H), 6.68 (d, J = 8.3 Hz,

1H), 4.20 (app dd, J = 8.0, 4.7 Hz, 1H), 4.12−4.03 (m, 2H), 2.64 (q,

J = 7.2 Hz, 2H), 2.29 (s, 3H), 2.17 (s, 3H), 1.17 (t, J = 7.1 Hz, 3H).

MS: 270.3 (MH⁺). R_f= 0.18 (4% methanol in dichloromethane on

silica gel).

1

yield) of the target ketone. H NMR (CDCl₃): 7.04−6.94 (m, 3H),

4.37 (s, 2H), 2.38 (s, 3H,), 2.30 (s, 6H). R_f= 0.31 (10% diethyl ether

in hexanes on silica gel).

2-(2,3-Dimethylphenoxy)-N-propyl-1-phenylethanamine (57).

¹H NMR (300 MHz, CDCl₃): 7.50−7.46 (m, 2H), 7.42−7.29 (m,

3H), 7.03 (t, J = 7.7 Hz, 1H), 6.79 (d, J = 7.1 Hz, 1H), 6.68 (d, J = 8.0

Hz, 1H), 4.18 (app dd, J = 8.5, 4.1 Hz, 1H), 4.10−3.99 (m, 2H), 2.56

(t, J = 7.1 Hz, 2H), 2.30 (s, 3H), 2.19 (s, 3H), 1.58 (m, 2H), 0.96 (t, J

= 7.7 Hz, 3H). MS: 284.2 (MH⁺). R_f= 0.23 (2% methanol in

dichloromethane on silica gel).

Step 2. 1-(2,6-Dimethylphenoxy)-N-methylpropan-2-amine (43).

Sodium cyanoborohydride (38 mg, 0.602 mmol) was added to a

solution of methylamine hydrochloride (76.3 mg, 1.13 mmol) and 1-

(2,6-dimethylphenoxy)propan-2-one (54.5 mg, 0.31 mmol) in

ethanol (2.7 mL) at 21 °C. The reaction was heated at 90 °C in a

microwave reactor for 1 h and stopped by dropwise addition of 10%

(w/w) Na₂CO_3(aq)until pH 9; then Celite was added, and the mixture

was concentrated to dryness. Product-infused Celite was loaded onto

a silica gel column and eluted using a gradient of methanol in

1

2-(2,3-Dimethylphenoxy)-N-butyl-1-phenylethanamine (58). H

NMR (300 MHz, CDCl₃): 7.49−7.46 (m, 2H), 7.41−7.29 (m, 3H),

7.02 (t, J = 8.0 Hz, 1H), 6.79 (d, J = 7.4 Hz, 1H), 6.67 (d, J = 8.0 Hz,

5398

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1H), 4.17 (app dd, J = 8.2, 3.9 Hz, 1H), 4.10−3.99 (m, 2H), 2.59 (t, J

= 6.9 Hz, 2H), 2.30 (s, 3H), 2.19 (s, 3H), 1.54 (m, 2H), 1.40 (m,

2H), 0.94 (t, J = 7.4 Hz, 3H). MS: 298.2 (MH⁺). R_f= 0.25 (2%

methanol in dichloromethane on silica gel).

temperature to aﬀord 350 mg (72%) of (R)-30. ¹H NMR (300 MHz,

DMSO-d₆) δ 8.58 (br. s., 3H), 7.51−8.05 (m, 4H), 7.37−7.51 (m,

2H), 7.32 (d, J = 8.53 Hz, 1H), 7.14 (t, J = 7.56 Hz, 1H), 4.72 (t, J =

20

6.05 Hz, 1H), 4.32−4.59 (m, 2H). MS: 282.0 (MH⁺). [α]

=

589

2-(2,3-Dimethylphenoxy)-N-methoxy-ethyl-1-phenylethan-

−19.9° (c = 0.23; methanol). Chiral HPLC retention time: 10.22 min.

R_f= 0.3 (5% methanol in dichloromethane on silica gel, weak UV

activity, stains with ninhydrin) for the free base.

1

amine (59). H NMR (300 MHz, CDCl₃): 7.25 (s, 1H), 6.96−7.05

(m, 2H), 6.92 (dd, J = 6.2, 8.4 Hz, 1H), 4.14−4.31 (m, 1H), 3.58−

3.79 (m, 2H), 2.29 (s, 6H), 1.27 (d, J = 6.5 Hz, 7H). MS: 300.1

(MH⁺). ¹³C NMR (125 MHz, CD₃OD): 157.22, 139.42, 133.80,

131.31, 130.69, 129.87, 124.84, 111.27, 69.93, 68.14, 63.04, 59.41,

49.32, 20.23. MS: 300.1 (MH⁺). Calcd m/z for [C₁₉H₂₅NO₂+ H⁺]:

300.1958. Observed: 300.1952.

The following compounds were synthesized using the same

sequence as (R)-30 with appropriate modiﬁcations:

(S)-1-Phenyl-2-(2-(triﬂuoromethyl)phenoxy)ethanamine (S)-30.

¹H NMR (300 MHz, DMSO-d₆) δ 8.60 (br. s., 3H), 7.59 (dd, J =

7.84, 15.54 Hz, 4H), 7.36−7.51 (m, 3H), 7.32 (d, J = 8.53 Hz, 1H),

7.14 (t, J = 7.84 Hz, 1H), 4.73 (t, J = 6.05 Hz, 1H), 4.34−4.59 (m,

2-(3,5-Dimethylphenoxy)-N-(2-methoxyethyl)-1-phenylethan-1-

1

20

2H). MS: 282.0 (MH⁺). [α] +20.2° (c = 0.24; methanol). Chiral

amine (60). H NMR (300 MHz, CD₃OD) δ 7.22−7.47 (m, 5H),

589

6.49−6.61 (m, 3H), 3.96−4.13 (m, 2H), 3.33−3.56 (m, 2H), 3.35 (s,

3H), 2.53−2.76 (m, 1H), 2.24 (d, J = 0.7 Hz, 6H). MS: 300.1 (MH⁺).

2-(3,5-Bis(triﬂuoromethyl)phenoxy)-N-(2-methoxyethyl)-1-phe-

HPLC retention time: 10.96 min. R_f= 0.3 (5% methanol in

dichloromethane on silica gel, for the free base).

(R)-1-Phenyl-2-(2-methylphenoxy)ethanamine (R)-27. H NMR

1

nylethan-1-amine (61). H NMR (300 MHz, CDCl₃): 7.32−7.50

(500 MHz, CD₃OD) 7.55−7.63 (m, 2H), 7.43−7.55 (m, 3H), 7.09−

(m, 6H), 7.27 (d, J = 10.2 Hz, 2H), 4.08−4.21 (m, 4H), 3.50 (m,

1H), 3.38 (s, 3H), 2.62−2.85 (m, 2H). MS: 407.2 (MH⁺).

2-((2-(3,5-Dimethylphenoxy)-1-phenylethyl)amino)ethan-1-ol

(62). ¹H NMR (300 MHz, CDCl₃): 2.28 (s, 6H), 2.61−2.87 (m, 2H),

3.52−3.74 (m, 2H), 3.94−4.10 (m, 2H), 4.10−4.19 (m, 1H), 6.50−

6.64 (m, 2H), 7.22−7.46 (m, 6H). MS: 286.2 (MH⁺).

7.20 (m, 2H), 6.84−6.99 (m, 2H), 4.30−4.40 (m, 2H), 2.26 (s, 3H).

20

MS: 228.0 (MH⁺). [α] −25.4° (c = 0.22; methanol) Chiral HPLC

589

retention time: 7.42 min.

(S)-1-Phenyl-2-(2-methylphenoxy)ethanamine (S)-27. H NMR

1

(500 MHz, CD₃OD) 7.54−7.61 (m, 2H), 7.42−7.54 (m, 3H), 7.06−

7.19 (m, 2H), 6.82−6.99 (m, 2H), 4.28−4.43 (m, 2H), 2.26 (s, 3H).

2-((2-(3,5-Bis(triﬂuoromethyl)phenoxy)-1-phenylethyl)amino)-

20

MS: 228.0 (MH⁺). [α] +26.0° (c = 0.19; methanol). Chiral HPLC

1

589

ethan-1-ol (63). H NMR (300 MHz, CDCl₃): 7.37−7.61 (m, 6H),

retention time: 8.34 min.

7.33 (d, J = 10.2 Hz, 2H), 4.08−4.21 (m, 4H), 3.50 (m, 1H), 2.62−

(R)-2-(3,5-Dimethylphenoxy)-1-phenylethanamine (R)-36. ¹H

NMR (500 MHz, CD₃OD) 7.66−7.41 (m, 5H), 6.65 (s, 3H), 4.74

2.85 (m, 2H). MS: 394.0 (MH⁺).

Synthesis of Amide Analogues. N-(1-(2,6-Dimethylphenoxy)-

propan-2-yl)acetamide (51). To 15 mg (0.06 mmol) of mexiletine

hydrochloride and triethylamine (0.020 mL, 0.14 mmol) in

dichloromethane (1 mL), acetyl chloride (0.006 mL, 0.08 mmol)

was added. The mixture was stirred at 21 °C for 15 h, concentrated to

dryness, and puriﬁed by ﬂash chromatography (silica gel, ethyl

(dd, J = 8.4, 4.1 Hz, 1H), 4.35−4.25 (m, 2H), 2.27 (s, 6H). MS:

20

242.0 (MH⁺). [α] −26.7° (c = 0.19, CD₃OD). Chiral HPLC

589

retention time: 12.66 min. R_f= 0.42 (1:20 methanol/dichloro-

methane on silica gel).

(S)-2-(3,5-Dimethylphenoxy)-1-phenylethanamine (S)-36. ¹H

NMR (500 MHz, CD₃OD) 7.93−7.15 (m, 5H), 6.65 (s, 3H), 4.74

1

acetate/hexane gradient) to aﬀord 10.2 mg (65%) of 51. H NMR

(dd, J = 8.4, 4.1 Hz, 1H), 4.36−4.24 (m, 2H), 2.27 (s, 6H). MS:

(300 MHz, CDCl₃): 6.86−7.08 (m, 3H), 5.93 (s, 1H), 4.28−4.44 (m,

1H), 3.66−3.86 (m, 2H), 2.26 (s, 6H), 2.04 (s, 3H), 1.41 (d, J = 6.8

Hz, 3H). MS: 222.2 (MH⁺).

20

242.0 (MH⁺). [α] +28.2° (c = 0.1875, methanol). Chiral HPLC

589

retention time: 9.71 min. R_f= 0.42 (1:20 methanol/dichloro-

methane).

N-(1-(2,6-Dimethylphenoxy)propan-2-yl)benzamide (52). ¹H

NMR (300 MHz, CDCl₃): 7.73−7.92 (m, 2H), 7.38−7.57 (m,

3H), 6.87−7.05 (m, 3H), 6.64 (d, J = 8.4 Hz, 1H), 4.57 (m, 1H), 3.95

(dd, J = 3.8, 9.1 Hz, 1H), 3.90 (s, 2H), 3.78−4.00 (m, 2H), 2.28 (s,

6H), 1.54 (d, J = 6.8 Hz, 3H). MS: 284.2 (MH⁺).

Synthesis of (R)-59. Step 1: (R)-N-(2-Hydroxy-1-phenylethyl)-2-

methoxyacetamide. Methoxyacetic acid (1.595 mL, 20.8 mmol) and

EDCI (4.12 g 21.5 mmol) were added to a solution of (R)-

phenylglycinol (3.17 g, 23.1 mmol) in dichloromethane (150 mL).

The mixture was vigorously shaken to break the aggregates formed

and then stirred at 21 °C for 5 h. After cooling to −20 °C and ﬁltering

through a Celite pad, the ﬁltrate was concentrated under reduced

pressure and puriﬁed by chromatography on silica gel using a

methanol/dichloromethane gradient to give 750 mg of the N-(2-

hydroxy-1-phenylethyl)-2-methoxyacetamide as an oﬀ-white solid that

was used without characterization.

Step 2: (4R)-1,2,3-Oxathiazolidine-3-methoxyacetamido-4-phe-

nyl-2,2-dioxide. A solution of imidazole (1.75 g, 25.6 mmol) and

triethylamine (2.14 mL, 15.4 mmol) in dichloromethane (30 mL) was

cooled to −60 °C. Thionyl chloride (0.56 mL, 7.69 mmol) was added

dropwise. Temperature was kept at −60 to −50 °C. (R)-N-(2-

Hydroxy-1-phenylethyl)-2-methoxyacetamide (1.34 g, 6.4 mmol) in

dichloromethane (100 mL) was added dropwise over a period of 1 h.

The mixture was stirred at −60 °C for an additional 3 h and then

allowed to reach ambient temperature. After 20 min at 21 °C, water

was added and the organic phase was separated. The organic layer was

washed with brine, dried over sodium sulfate, and concentrated under

reduced pressure. The residue was dissolved in 3:1 acetonitrile/water

(100 mL) and cooled in an ice−water bath (4−5 °C). Sodium

periodate (2.92 g, 13.6 mmol) was added followed by RuCl₃(H₂O)₃

(5 mg, 0.02 mmol). The mixture was stirred for 30 min and allowed

to reach ambient temperature. More water was added to bring the

acetonitrile/water ratio to 1:1. After stirring for an additional hour,

the mixture was extracted with ether. The organic phase was washed

with brine, dried over sodium sulfate, and concentrated under

reduced pressure. The residue was puriﬁed by chromatography on

silica gel using an ethyl acetate/hexane gradient to aﬀord (4R)-1,2,3-

N-(2-(2,6-Dimethylphenoxy)-1-phenylethyl)acetamide (53). ¹H

NMR (300 MHz, CDCl₃): 7.29−7.43 (m, 4H), 6.87−6.98 (m, 3H),

6.39−6.43 (m, 1H), 5.33−5.39 (m, 1H), 3.98−4.10 (m, 2H), 2.11 (s,

6H). MS: 284.1 (MH⁺).

N-(2-(2,6-Dimethylphenoxy)-1-phenylethyl)benzamide (54). ¹H

NMR (300 MHz, CDCl₃): 7.84−7.88 (1H), 7.44−7.53 (m, 4H),

7.30−7.41 (m, 4H), 7.15−7.18 (m, 1H), 6.88−6.98 (m, 4H), 6.39−

6.43 (m, 1H), 5.52−5.58 (m, 1H), 4.21 (dd, J = 4.4 and 9.4 Hz, 1H),

4.11 (dd, J = 3.9 and 9.4 Hz, 1H), 2.13 (s, 6H). MS: 346.0 (MH⁺).

(R)-1-Phenyl-2-(2-(triﬂuoromethyl)phenoxy)ethanamine (R)-30.

Step 1: (R)-tert-Butyl 1-Phenyl-2-(2-(triﬂuoromethyl)phenoxy)-

ethylcarbamate. To sodium hydride (78 mg of 60% suspension in

oil, 0.76 mmol) in DMF (5 mL) was added 299 mg of 2-

triﬂuoromethylphenol (1.8 mmol). After ca. 5 min, 499 mg (1.7

mmol) (4R)-1,2,3-oxathiazolidine-3-carboxylic acid, 4-phenyl 1,1-

dimethylethyl ester-2,2-dioxide²³in DMF (1 mL) was added. The

mixture was stirred at 21 °C for 16 h and diluted with water. The

product was extracted with ethyl acetate. The organic phase was

washed (1 M aqueous sodium hydroxide, water, and brine), dried

(magnesium sulfate), and concentrated under reduced pressure.

Puriﬁcation of the residue aﬀorded 587 mg (92% yield) of a colorless

oil that was used without further characterization. R_f= 0.7 (30% ethyl

acetate in hexanes on silica gel).

Step 2. To a solution of the precursor from Step 1 above (587 mg,

1.5 mmol) in methanol (10 mL) at 21 °C was added a solution of

hydrogen chloride in ether (2 M, 2 mL, 4.0 mmol). After stirring for

16 h, the solvent and excess hydrogen chloride were removed under a

stream of nitrogen and the solid obtained was vacuum dried at room

5399

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

oxathiazolidine-3-methoxyacetamido-4-phenyl-2,2-dioxide as a white

solid. ¹H NMR (500 MHz, CDCl₃) δ 7.48−7.33 (m, 5H), 5.61 (dd, J

= 7.1, 6.0 Hz, 1H), 5.02−4.86 (m, 1H), 4.53 (dd, J = 9.4, 5.9 Hz, 1H),

4.32 (dd, J = 33.6, 15.7 Hz, 2H), 3.42 (s, 3H). MS: 272.1 (MH⁺). R_f=

0.43 (40% hexanes/EtOAc on silica gel).

At day 25, cells were dissociated and plated onto Matrigel coated

384-well tissue culture plates (Greiner Bio-One) at a density of

20,000 cells/well. Experiments were conducted day 28 post-

diﬀerentiation.

Preparation of VF2.1.Cl Loading Solution and Automated

Image Acquisition. A VF2.1.Cl dye used was synthesized as

described previously (Fluovolt, ThermoFisher).³¹One microliter of 2

mM VF2.1.Cl in DMSO was mixed with 1 μL of 10% Pluronic F127

(diluted in 1.7 mL water) by agitating and centrifuging three times.

Separately, Hoechst 33258 was diluted into Tyrode’s solution (136

mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose,

10 mM HEPES, pH 7.4) to a concentration of 4 μg/mL. One

milliliter of Hoechst/Tyrode’s solution was added to a 1.7 mL

VF2.1.Cl/Pluronic F127 mixture and mixed for 10 s. Each test

compound was diluted in Tyrode’s solution to a 2× concentrated

stock and added to a well and incubated at 37 °C and 5% CO₂for 5

min before image acquisition. Time series images were acquired

automatically using a IC200 KIC instrument (Vala Sciences, San

Diego, CA) at an acquisition frequency of 100 Hz for a duration of 6.5

s or 33 Hz for 20 s, with an excitation wavelength of 485/20 nm and

emission ﬁlter of 525/30 nm using a 0.75 NA 20× Nikon Apo VC

objective. A single image of the Hoechst/Tyrode solution was

acquired after the time series. Optimized dye loading and imaging

conditions were replicated using a diﬀerent high content imager, the

ImageXpress Micro XLS platform (Molecular Devices), and hiPSC-

CMs produced by a novel diﬀerentiation protocol.³⁰

Image Analysis, Physiological Parameter Calculations, and

Data Analysis. Image analysis and physiological parameter

calculations were conducted using Cyteseer (Vala Sciences) as

previously described.^32,33Brieﬂy, an image output by a Cyteseer

scanner was loaded into Cyteseer and a cardiac time series algorithm

was executed on image ﬁles. Data were analyzed using Microsoft Excel

2013, and dose−response curves were calculated using GraphPad

Prism 7 software (Prism, San Diego, CA).

Meta-Analysis of Experimental Data. An R-script (R version

3.3.1) was developed in-house that read CSV ﬁles generated by

Cyteseer and amalgamated all the physiological parameters of interest

into a single ﬁle. Using this dataset, meta-analysis of experimental data

was conducted and showed intra- and intervariability in studies

examining thawing cells in diﬀerent ways (i.e., APD₇₅) and the

relationship between APD₇₅and beat rate. Because of the nonlinear

relationship of APD₇₅and beat rate, statistical signiﬁcance was

determined using a sliding window (center value 3) to mirror

experimental ranges. A Mann−Whitney test was applied to the data

(using the Bonferroni criterion) for multiple testing.

Automated Patch Clamp Studies. Whole cell planar patch

clamp recordings were used to study the eﬀects of test compounds in

transfected cells using ion channel currents including: I_NaP, I_NaL, and

I_Krchannels.³⁴For each compound studied, at least six increasing

concentrations were included to obtain a dose−response relationship.

For Na⁺channel experiments, a stable HEK cell line expressing a long

QT mutant, F1473C,³⁵was used. For I_Kr, a commercially available

CHO stable cell line, hERG Duo (Bsys, Basel, Switzerland), was used.

Cells were grown to ∼75% conﬂuence, dissociated with trypsin−

EDTA (Gibco, Waltham, MA), and resuspended in extracellular

recording solutions at a density of ∼1 million cells/mL. Cells in

solution were added to the Patchliner system (Nanion, Munich, DE)

for voltage clamp recordings. The following solutions were used:

internal solution for K⁺channel experiments: 50 mM KCl, 10 mM

NaCl, 60 mM K-ﬂuoride, 20 mM EGTA, and 10 mM HEPES/KOH,

pH 7.2; internal solution for Na⁺channel experiments: 50 mM CsCl,

10 mM NaCl, 60 mM Cs-ﬂuoride, 20 mM EGTA, 10 mM HEPES/

CsOH, pH 7.2; external solutions for K⁺and Na⁺channel

experiments; 140 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 5 mM D-

glucose monohydrate, and 10 mM HEPES/NaOH H 7.4. Test

compounds (and controls) were prepared in extracellular solution

with 0.01% DMSO. All reagents, unless otherwise noted, were

obtained from Sigma-Aldrich (St. Louis, MO). Speciﬁc channel

blockers tetrodotoxin (Calbiochem, San Diego, CA) and E-4031 were

prepared in extracellular solution.

Step 3: (R)-N-(2-(2,3-Dimethylphenoxy)-1-phenylethyl)-2-me-

thoxyacetamide. 2,3-Dimethyl phenol (396 mg, 3.2 mmol) in

DMF (10 mL) was treated with NaH, 60% dispersion in mineral oil

(141 mg, 3.5 mmol), and stirred for 20 min. (R)-Cyclic sulfamidate

(obtained in Step 2 above) (800 mg, 2.9 mmol) was added. The

mixture was stirred for 4 h and partitioned between brine and ethyl

acetate. The organic phase was washed with additional brine and

dried over sodium sulfate. The solvent was removed in vacuo, and the

crude product was puriﬁed on silica gel using an ethyl acetate/hexane

gradient to aﬀord 479 mg of (R)-N-(2-(2,3-dimethylphenoxy)-1-

phenylethyl)-2-methoxyacetamide as a clear oil. ¹H NMR (300 MHz,

CDCl₃) δ 7.22−7.49 (m, 5H), 7.04 (t, J = 7.98 Hz, 1H), 6.80 (d, J =

7.70 Hz, 1H), 6.68 (d, J = 8.25 Hz, 1H), 5.39−5.59 (m, 1H), 4.18−

4.39 (m, 2H), 3.97 (s, 2H), 3.39−3.51 (m, 3H), 2.28 (s, 3H), 2.10−

2.18 (m, 3H). MS: 314.2 (MH⁺). R_f= 0.7 (19:1 dichloromethane/

methanol on silica gel).

Step 4: (R)-2-(2,3-Dimethylphenoxy)-N-(2-methoxyethyl)-1-phe-

nylethanamine (R)-59. To 479 mg (1.5 mmol) of (R)-N-(2-(2,3-

dimethylphenoxy)-1-phenylethyl)-2-methoxyacetamide in 15 mL of

anhydrous THF was added 1.0 M LiAlH₄in THF (6.0 mL). The

mixture was heated at 80 ° C for 24 h, cooled in an ice/water bath,

and diluted with anhydrous THF. The excess reagent was neutralized

by dropwise addition of H₂O (240 μL), dropwise addition of 3.5 M

NaOH (240 μL), and further addition of H₂O (720 μL). After stirring

for 10 min, magnesium sulfate was added; the mixture was allowed to

reach ambient temperature and ﬁltered through Celite. The ﬁltrate

was concentrated in vacuo and puriﬁed by chromatography on silica

gel using a methanol/dichloromethane gradient. The amine was

transferred to a vial using an appropriate amount of 0.5 M HCl in

methanol. After thoroughly drying the product in vacuo, the desired

HCl salt was obtained as a white powder. ¹H NMR (500 MHz,

CD₃OD) δ 7.61 (dd, J = 7.7, 1.9 Hz, 2H), 7.58−7.45 (m, 3H), 7.04

(t, J = 7.9 Hz, 1H), 6.82 (t, J = 7.9 Hz, 2H), 4.88−4.83 (m, 1H), 4.48

(dd, J = 10.6, 7.1 Hz, 1H), 4.43−4.33 (m, 1H), 3.40 (s, 3H), 2.33−

20

2.18 (m, 3H), 2.17 (s, 3H). MS: 300.1 (MH⁺). [α] −25.1° (c =

589

0.278, methanol). Chiral HPLC retention time: 13.37 min. R_f= 0.45

(5% dichloromethane in methanol on silica gel).

(S)-59 was prepared by the same route as (R)-59 except that (S)-

phenylglycinol was used as the starting material.

(S)-2-(2,3-Dimethylphenoxy)-N-(2-methoxyethyl)-1-phenyle-

thanamine (S)-59. ¹H NMR (500 MHz, CD₃OD) δ 7.61 (dd, J = 7.7,

1.8 Hz, 2H), 7.57−7.48 (m, 3H), 7.04 (t, J = 7.9 Hz, 1H), 6.82 (t, J =

7.3 Hz, 2H), 4.49 (dd, J = 10.6, 7.1 Hz, 1H), 4.39 (dd, J = 10.6, 4.7

Hz, 1H), 3.77−3.57 (m, 1H), 3.40 (s, 3H), 2.26 (s, 3H), 2.15 (s, 3H).

20

MS: 300.1 (MH⁺). [α] +24.4° (c = 0.281, methanol). Chiral HPLC

589

retention time: 14.17 min. R_f= 0.45 (5% dichloromethane: methanol

on silica gel).

BIOLOGY EXPERIMENTAL SECTION

■

Cell Culture and Cell Diﬀerentiation to hiPSC-CMs. MyCell

(LQTS3) and iCell (healthy individual) cardiomyocytes (Cellular

Dynamics International, Madison, Wisconsin) were prepared as

described previously.³⁰Media were exchanged every other day by

removing 50 μL of media and adding 50 μL of fresh iCCMM for 14

days prior to imaging. Human iPSCs were dissociated using 0.5 mM

EDTA (ThermoFisher Scientiﬁc) in PBS without CaCl₂or MgCl₂

(Corning) for 7 min at room temperature. Dissociated hiPSCs were

plated at a density of 3 × 10⁵cells per well in a Matrigel coated 12-

well plate in mTeSR1 media (StemCell Technologies) supplemented

with 2 μM thiazovivin (Selleck Chemicals). After 24 h, the media

were replaced with mTESR1 without thiazovivin and were

replenished daily for 3−5 days until the cells reached ≥90%

conﬂuence to begin diﬀerentiation. Cardiomyocytes were diﬀer-

entiated by methods previously described.^29,30

5400

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Data Analysis. Normalized blocking ratio data, for each of I_Kr, I_NaP

,

in accordance with the current IACUC protocol and all applicable

local, state, and federal regulations and guidelines.

and I_NaL, were ﬁtted by a four parameter logistic ﬁt. Patch clamp data

were acquired with PatchMaster (HEKA, Lambrecht, DE) and

PatchLiner Data acquisition (Nanion, Munich, DE) and analyzed

with Origin 7.0 (OriginLab, Northampton, MA, USA), IgorPro

(WaveMetrics, Portland, OR, USA), and MATLAB (Mathworks,

Natick, MA, USA). Data were shown as mean SEM. Statistical data

analyses were assessed with Student’s t-test; diﬀerences at P < 0.05

were considered as statistically signiﬁcant.

Data Analysis. Oﬄine data analysis, curve ﬁtting, and statistical

evaluations were conducted with OriginPro 2016 (OriginLab,

Northampton, MA) software. Dose−response curves for test

compound inhibition were determined by measuring “peak” and

“late” sodium current components on the same current trace. “Peak”

values for the each response were deﬁned automatically by the

“negative peak searching” algorithm in OriginPro, whereas “late”

component values were deﬁned as an average of 20 ms of the current

response after the current amplitude reached the steady-state level.

Values for each test compound concentration were normalized to

control currents recorded during the control solution applications.

Dose−response data for each sodium current response component

were ﬁtted using a Marquardt−Levenberg iteration algorithm with a

standard Hill equation. Data were presented as mean s.d.

Chemical Stability of Mexiletine Analogues at Various

Temperatures and pHs. A typical chemical stability incubation

contained 100 μM of the test compound prepared in 50 mM PBS

buﬀer (pH 3.0 or 7.4,) with 1% ethanol. Test compounds were

incubated at 37 °C. An aliquot from each incubation was taken at

various times and injected onto an RP-HPLC. Samples were run on a

Hitachi D-7000 HPLC system (Hitachi High Tech) using an L-7100

analytical pump, L-7400 UV−vis variable wavelength detector, and L-

7600 automatic sample injector. A Gemini C18 column (250 × 4.6

mm, 5 μm particle size; Phenomenex) with a C18 guard column

(Phenomenex) was used for chromatographic separation of mexiletine

analogues. The mobile phase used was an isocratic system using 75%

water (0.05% TFA) and 25% acetonitrile (0.05% TFA) with a ﬂow

rate of 1.25 mL/min and monitored at 275 nM. The disappearance of

the analyte was monitored over time. A plot of the area under the

curve for the normalized analyte versus time aﬀorded the half-life

Adult male Balb/C mice or Sprague Dawley rats (from Charles

River, San Diego, CA) were housed in groups of four and maintained

in a temperature-controlled environment on a 12 h:12 h light cycle

(0600H on−1800H oﬀ) upon arrival to the laboratory. Animals were

given free access to food and water during a 1 week habituation

period to the laboratory. For PK studies, mexiletine and test

compounds (in 0.9% saline) were administered to individually

housed, starved jugular-cannulated Sprague Dawley rats (5 mg/kg

i.v. or 25 mg/kg oral). At 10 time points over 25 h, blood was

withdrawn, cooled, and centrifuged. Serum was combined with an

internal standard (compound 44), extracted with aqueous NaCl,

Na₂CO₃, and ethyl acetate, separated, evaporated to dryness, taken up

in methanol, and injected into the Agilent LCMS described above.

Peak areas were quantiﬁed, and pharmacokinetic parameters were

determined with a WinNonlin-Pro program (Pharsight, Inc., Prince-

ton, NJ). For behavioral studies, test compounds or mexiletine were

dissolved in DMSO (7%), PEG400 (57%) isotonic saline (40%), and

administered i.p. to separate groups in a volume of 0.1 mL/mouse.

Ex Vivo Studies. For ex-vivo studies, the eﬀect of (R)-30 on

isolated heart tissue isolated from aged Sprague Dawley rats was

examined. Vulnerability to EAD formation was investigated by

examining the arrhythmogenic eﬀects of hydrogen peroxide (H₂O₂)

perfusion in isolated Langendorﬀ rat hearts. Oxidative stress was

shown to readily induce EADs at the isolated myocyte level in aged

hearts from 24−26 month old rats. H₂O₂exposure consistently

promoted EADs and triggered activity in >90% of the aged ﬁbrotic

hearts that lead to VT/VF.

AUTHORSHIP CONTRIBUTIONS

■

J.R.C., K.O., D.R., R.S.K., H.S.K., and M.M. participated in the

research design. J.R.C., D.R., J.G.-G., K.O., M.J., W.L.M., A.P.,

and K.S. conducted experiments. D.R., J.G.-G., K.O., and M.J.

contributed new reagents or analytical tools. J.R.C., W.M.,

K.J.S., R.S.K., H.S.K., and M.M. performed data analysis.

J.R.C., J.G.-G., R.S.K., W.L.M., and M.M. wrote or contributed

to the writing of the manuscript.

values and k_app

.

Metabolic Stability Studies in the Presence of Human Liver

Microsomes and S-9. Human liver S-9, human liver microsomes,

highly puriﬁed cytochrome P-450 (CYP) 3A4, 3A5, and 2D6, and

NADPH-generating system were obtained from BD Biosciences

(Waltham, MA). A typical incubation contained human liver

microsomes or S-9 (0.4−0.5 and 1 mg of protein, respectively), 50

mM potassium phosphate buﬀer (pH 7.4), 50 μM test compound, an

NADPH-generating system consisting of 0.5 mM NADP⁺, 0.5 mM

glucose-6-phosphate, 5 U/mL glucose-6-phosphate dehydrogenase, 1

mg/mL diethylenetriaminepentaacetic acid (DETAPAC), and 3 mM

MgCl₂in a ﬁnal incubation volume of 0.25 mL. Incubations were run

for 0, 7, 15, 30, and 60 min with constant shaking at 37 °C in a water

bath and were terminated by addition of 0.75 mL of cold acetonitrile.

After centrifugation at 3000 rpm for 5 min, the organic fraction was

collected and solvent was removed with a stream of argon. The

residue was reconstituted in 125 μL of MeOH and 125 μL of H₂O,

mixed thoroughly, centrifuged at 13,000 rpm for 5 min, and analyzed

by high-performance liquid chromatography. Samples were run on a

Hitachi D-7000 HPLC system (Hitachi High Tech) as described

above with a Gemini C18 column (250 × 4.6 mm, 5 μm particle size;

Phenomenex) with a C18 guard column (Phenomenex). The mobile

phase used was an isocratic system using 75% water (0.05% TFA) and

25% acetonitrile (0.05% TFA) with a ﬂow rate of 1.25 mL/min and

monitored at 275 nM. The disappearance of the analyte was

monitored over time. A plot of the area under the curve for

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01545.

Molecular formula strings (CSV)

HPLC Methods and traces (PDF)

AUTHOR INFORMATION

■

Corresponding Author

John R. Cashman − Human BioMolecular Research Institute,

San Diego, California 92121, United States; orcid.org/

0000-0002-8628-9129; Phone: (858) 458-9305;

Email: JCashman@HBRI.org; Fax: (858) 458-9311

Authors

Daniel Ryan − Human BioMolecular Research Institute, San

Diego, California 92121, United States

Wesley L. McKeithan − Cardiovascular Institute and

Department of Medicine, Stanford University, Stanford,

California 94305, United States; Graduate School of

Biomedical Sciences, Sanford Burnham Prebys Medical

Discovery Institute, San Diego, California 92037, United

States

normalized analyte versus time aﬀorded the half-life values and k_app

.

In Vivo Eﬀects of Mexiletine and Analogues. Animal work

followed the Guide for Care and Use of Animals as adopted by NIH.

Formal approval was obtained from the IACUC of HBRI and UCLA.

Animals used in the research studies were handled, housed, and killed

Karl Okolotowicz − Human BioMolecular Research Institute,

San Diego, California 92121, United States

5401

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(3) Koplan, B. A.; Stevenson, W. G. Ventricular tachycardia and

sudden cardiac death. Mayo Clin. Proc. 2009, 84, 289−297.

(4) Singh, S.; Kerndt, C.; Zeltser, R. Mexiletine; StatPearls: Treasure

Island (FL), 2020.

Jorge Gomez-Galeno − Human BioMolecular Research

Institute, San Diego, California 92121, United States

Mark Johnson − Human BioMolecular Research Institute, San

Diego, California 92121, United States

(5) Al-Khatib, S. M.; Stevenson, W. G.; Ackerman, M. J.; Bryant, W.

J.; Callans, D. J.; Curtis, A. B.; Deal, B. J.; Dickfeld, T.; Field, M. E.;

Fonarow, G. C.; Gillis, A. M.; Granger, C. B.; Hammill, S. C.; Hlatky,

M. A.; Joglar, J. A.; Kay, G. N.; Matlock, D. D.; Myerburg, R. J.; Page,

R. L. 2017 AHA/ACC/HRS guideline for management of patients

with ventricular arrhythmias and the prevention of sudden cardiac

death: A report of the American college of cardiology/American heart

association task force on clinical practice guidelines and the heart

rhythm society. Heart Rhythm. 2017, 15, e190−e252.

(6) Priori, S. G.; Blomstrom-Lundqvist, C.; Mazzanti, A.; Blom, N.;

Borggrefe, M.; Camm, J.; Elliott, P. M.; Fitzsimons, D.; Hatala, R.;

Hindricks, G.; Kirchhof, P.; Kjeldsen, K.; Kuck, K. H.; Hernandez-

Madrid, A.; Nikolaou, N.; Norekval, T. M.; Spaulding, C.; Van

Veldhuisen, D. J.; ESC Scientific Document Group. 2015 ESC

guidelines for the management of patients with ventricular

arrhythmias and the prevention of sudden cardiac death: The task

force for the management of patients with ventricular arrhythmias and

the prevention of sudden cardiac death of the European society of

cardiology (ESC). Endorsed by: Association for European paediatric

and congenital cardiology (AEPC). Eur Heart J. 2015, 36, 2793−

2867.

Kevin J. Sampson − Department of Pharmacology, College of

Physicians and Surgeons, Columbia University, New York

10032, United States

Robert S. Kass − Department of Pharmacology, College of

Physicians and Surgeons, Columbia University, New York

10032, United States

Arash Pezhouman − Division of Cardiology, Department of

Medicine, David Geﬀen School of Medicine, University of

California Los Angeles, Los Angeles, California 90095,

United States

Hrayr S. Karagueuzian − Division of Cardiology, Department

of Medicine, David Geﬀen School of Medicine, University of

California Los Angeles, Los Angeles, California 90095,

United States

Mark Mercola − Cardiovascular Institute and Department of

Medicine, Stanford University, Stanford, California 94305,

United States; Graduate School of Biomedical Sciences,

Sanford Burnham Prebys Medical Discovery Institute, San

Diego, California 92037, United States

(7) Bennett, P. B.; Yazawa, K.; Makita, N.; George, A. L., Jr.

Molecular mechanism for an inherited cardiac arrhythmia. Nature

1995, 376, 683−685.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01545

(8) Wang, Q.; Shen, J.; Splawski, I.; Atkinson, D.; Li, Z.; Robinson, J.

L.; Moss, A. J.; Towbin, J. A.; Keating, M. T. SCN5A mutations

associated with an inherited cardiac arrhythmia, long QT syndrome.

Cell 1995, 80, 805−811.

(9) Wang, Q.; Shen, J.; Li, Z.; Timothy, K.; Vincent, G. M.; Priori, S.

G.; Schwartz, P. J.; Keating, M. T. Cardiac sodium channel mutations

in patients with long QT syndrome, an inherited cardiac arrhythmia.

Hum. Mol. Genet. 1995, 4, 1603−1607.

(10) Moss, A. J.; Kass, R. S. Long QT syndrome: from channels to

cardiac arrhythmias. J. Clin. Invest. 2005, 115, 2018−2024.

(11) Schwartz, P. J.; Priori, S. G.; Locati, E. H.; Napolitano, C.;

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We are grateful for the ﬁnancial support of the California

Institute for Regenerative Medicine (CIRM) (Grant number

TR4-06857 to J.R.C. and M.M.). 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. We are grateful to Dr. Jim Weiss

(UCLA) for help with the data of Figure 4. Some HPLC

traces were done at AxisPharm, San Diego, CA, 92121.

̀

Cantu, F.; Towbin, J. A.; Keating, M. T.; Hammoude, H.; Brown, A.

M.; Chen, L.-S. K.; Colatsky, T. J. Long QT syndrome patients with

mutations of the SCN5A and HERG genes have differential responses

to Na⁺channel blockade and to increases in heart rate: Implications

for gene-specific therapy. Circulation 1995, 92, 3381−3386.

(12) Mazzanti, A.; Maragna, R.; Faragli, A.; Monteforte, N.; Bloise,

R.; Memmi, M.; Novelli, V.; Baiardi, P.; Bagnardi, V.; Etheridge, S. P.;

Napolitano, C.; Priori, S. G. Gene-specific therapy with mexiletine

reduces arrhythmic events in patients with long QT syndrome type 3.

J. Am. Coll. Cardiol. 2016, 67, 1053−1058.

ABBREVIATIONS

■

APD, action potential duration; AUC, area under the curve; F,

bioavailability; CF, conduction velocity; C_max, concentration

(13) McKeithan, W. L.; Savchenko, A.; Yu, M. S.; Cerignoli, F.;

Bruyneel, A. A. N.; Price, J. H.; Colas, A. R.; Miller, E. W.; Cashman,

J. R.; Mercola, M. An automated platform for assessment of

congenital and drug-induced arrhythmia with hiPSC-derived

cardiomyocytes. Front. Physiol. 2017, 8, 766.

(14) Gualdani, R.; Tadini-Buoninsegni, F.; Roselli, M.; Defrenza, I.;

Contino, M.; Colabufo, N. A.; Lentini, G. Inhibition of hERG

potassium channel by the antiarrhythmic agent mexiletine and its

metabolite m -hydroxymexiletine. Pharmacol. Res. Perspect. 2015, 3,

No. e00160.

̀

max; EADs, early after depolarizations; hERG, human ether-a-

go-go; iPSC-CMs, induced pluripotent stem cell-derived

cardiomyocytes; I_Kr, potassium current; ERG, inward potas-

sium channel; KIC, kinetic image cytometry; I_NaL, late inward

sodium current; LQTS3, long QT syndrome Type 3; I_NaP, peak

sodium current; PK, pharmacokinetics; SAR, structure−

activity relationship; TTX, tetradotoxin; VT, ventricular

tachycardia; VF, ventricular ﬁbrillation

(15) Campbell, N. P.; Kelly, J. G.; Adgey, A. A.; Shanks, R. G. The

clinical pharmacology of mexiletine. Br. J. Clin. Pharmacol. 1978, 6,

103−108.

REFERENCES

■

(1) Wu, E. Q.; Birnbaum, H. G.; Mareva, M.; Tuttle, E.; Castor, A.

R.; Jackman, W.; Ruskin, J. Economic burden and co-morbidities of

atrial fibrillation in a privately insured population. Curr. Med. Res.

Opin. 2005, 21, 1693−1699.

(2) Zheng, Z. J.; Croft, J. B.; Giles, W. H.; Mensah, G. A. Sudden

cardiac death in the United States, 1989 to 1998. Circulation 2001,

104, 2158−2163.

(16) Rudel, R.; Lehamann, H. F.; Ricker, K. The nondystrophic

myotonias. In Myology, Chapt 49; Engel, A. G.; Franzini-Armstrong,

C., eds; pp 1291−1302, McGraw-Hill: New York 1994.

(17) Terrenoire, C.; Wang, K.; Tung, K. W. C.; Chung, W. K.; Pass,

R. H.; Lu, J. T.; Jean, J.-C.; Omari, A.; Sampson, K. J.; Kotton, D. N.;

Keller, G.; Kass, R. S. Induced pluripotent stem cells used to reveal

5402

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

drug actions in a long QT syndrome family with complex genetics. J.

Gen. Physiol. 2013, 141, 61−72.

(18) De Bellis, M.; De Luca, A.; Desaphy, J. F.; Carbonara, R.;

Heiny, J. A.; Kennedy, A.; Carocci, A.; Cavalluzzi, M. M.; Lentini, G.;

Franchini, C.; Camerino, D. C. Combined modifications of mexiletine

pharmacophores for new lead blockers of Na(v)1.4 channels. Biophys.

J. 2013, 104, 344−354.

(35) Bankston, J. R.; Yue, M.; Chung, W.; Spyres, M.; Pass, R. H.;

Silver, E.; Sampson, K. J.; Kass, R. S. A novel and lethal de novo LQT-

3 mutation in a newborn with distinct molecular pharmacology and

therapeutic response. PLoS One 2007, 2, No. e1258.

(19) De Luca, A.; Natuzzi, F.; Desaphy, J. F.; Loni, G.; Lentini, G.;

Franchini, C.; Tortorella, V.; Camerino, D. C. Molecular determinants

of mexiletine structure for potent and use-dependent block of skeletal

muscle sodium channels. Mol. Pharmacol. 2000, 57, 268−277.

(20) Huang, P.-Q.; Zheng, X.; Wei, H. Synthesis of (S )-vasicol and

(S )-3-hydroxy-2-pyrrolidinone. Heterocycles 2003, 60, 1833−1841.

́

(21) Huang, K.; Ortiz-Marciales, M.; Stepanenko, V.; De Jesus, M.;

Correa, W. A Practical and efficient route for the highly

enantioselective synthesis of mexiletine analogues and novel beta-

thiophenoxy and pyridyl ethers. J. Org. Chem. 2008, 73, 6928−6931.

̀

(22) Giardina, D.; Crucianelli, M.; Angeli, P.; Buccioni, M.; Gulini,

U.; Marucci, G.; Sagratini, G.; Melchiorre, C. Structure-activity

relationships among novel phenoxybenzamine-related beta-chloroe-

thylamines. Bioorg. Med. Chem. 2002, 10, 1291−1303.

(23) Kang, S.; Han, J.; Lee, E. S.; Choi, E. B.; Lee, H. K.

Enantioselective synthesis of cyclic sulfamidates by using chiral

rhodium-catalyzed asymmetric transfer hydrogenation. Org. Lett.

2010, 12, 4184−4187.

(24) Wang, D. W.; Kiyosue, T.; Sato, T.; Arita, M. Comparison of

the effects of class I anti-arrhythmic drugs, cibenzoline, mexiletine and

flecainide, on the delayed rectifier K⁺current of guinea-pig ventricular

myocytes. J. Mol. Cell. Cardiol. 1996, 28, 893−903.

(25) Fenster, P. E.; Comess, K. A. Pharmacology and clinical use of

mexiletine. Pharmacotherapy 1986, 6, 1−7.

(26) Alexander, G. J.; Kopeloff, L. M.; Alexander, R. B.; Chatterjie,

N. Mexiletine: biphasic action on convulsive seizures in rodents.

Neurobehav. Toxicol. Teratol. 1986, 8, 231−235.

(27) Akıncı, E.; Yuzbasi̧ oglu, Y.; Cosķ un, F. Hemodialysis as an

̈

alternative treatment of mexiletine intoxication. Am. J. Emerg. Med.

2011, 29, 1235−1235.e6.

(28) Karagueuzian, H. S.; Pezhouman, A.; Angelini, M.; Olcese, R.

Enhanced late Na and Ca currents as effective antiarrhythmic drug

targets. Front. Pharmacol. 2017, 8, 36.

(29) Burridge, P. W.; Matsa, E.; Shukla, P.; Lin, Z. C.; Churko, J. M.;

Ebert, A. D.; Lan, F.; Diecke, S.; Huber, B.; Mordwinkin, N. M.;

Plews, J. R.; Abilez, O. J.; Cui, B.; Gold, J. D.; Wu, J. C. Chemically

defined generation of human cardiomyocytes. Nat. Methods 2014, 11,

855−860.

(30) Cunningham, T. J.; Yu, M. S.; McKeithan, W. L.; Spiering, S.;

Carrette, F.; Huang, C. T.; Bushway, P. J.; Tierney, M.; Albini, S.;

Giacca, M.; Mano, M.; Puri, P. L.; Sacco, A.; Ruiz-Lozano, P.; Riou, J.

F.; Umbhauer, M.; Duester, G.; Mercola, M.; Colas, A. R. Id genes are

essential for early heart formation. Genes Dev. 2017, 31, 1325−1338.

(31) Miller, E. W. Small molecule fluorescent voltage indicators for

studying membrane potential. Curr. Opin. Chem. Biol. 2016, 33, 74−

80.

(32) Cerignoli, F.; Charlot, D.; Whittaker, R.; Ingermanson, R.;

Gehalot, P.; Savchenko, A.; Gallacher, D. J.; Towart, R.; Price, J. H.;

McDonough, P. M.; Mercola, M. High throughput measurement of

Ca²⁺dynamics for drug risk assessment in human stem cell-derived

cardiomyocytes by kinetic image cytometry. J. Pharmacol. Toxicol.

Methods 2012, 66, 246−256.

(33) Lu, H. R.; Whittaker, R.; Price, J. H.; Vega, R.; Pfeiffer, E. R.;

Cerignoli, F.; Towart, R.; Gallacher, D. J. High throughput

measurement of Ca⁺⁺dynamics in human stem cell-derived

cardiomyocytes by kinetic image cytometery: A cardiac risk

assessment characterization using a large panel of cardioactive and

inactive compounds. Toxicol. Sci. 2015, 148, 503−516.

(34) Farre, C.; Fertig, N. HTS techniques for patch clamp-based ion

channel screening - advances and economy. Expert. Opin. Drug.

Discovery 2012, 7, 515−524.

5403

https://doi.org/10.1021/acs.jmedchem.0c01545

J. Med. Chem. 2021, 64, 5384−5403

Article Doi

Antiarrhythmic Hit to Lead Refinement in a Dish Using Patient-Derived iPSC Cardiomyocytes

DOI: 10.1021/acs.jmedchem.0c01545

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.0c01545

Full text of DOI:10.1021/acs.jmedchem.0c01545

Products guided by the article

R&D Labs maybe for 1036762-56-7

Relevant to this article

Hot Product