Discovery of Dihydrobenzoxazepinone (GS-6615) Late Sodium Current Inhibitor (Late INai), a Phase II Agent with Demonstrated Preclinical Anti-Ischemic and Antiarrhythmic Properties

Article abstract of DOI:10.1021/acs.jmedchem.6b00939

Late sodium current (late I_Na) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Na_v 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late I_Na, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia-ventricular fibrillation (VT-VF). We will describe structure-activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late I_Na inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S-T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC₅₀ values of 190 and 8000 nM, respectively. Compound 4 represents a new class of potent late I_Na inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

Full text of DOI:10.1021/acs.jmedchem.6b00939

Article
pubs.acs.org/jmc
Discovery of Dihydrobenzoxazepinone (GS-6615) Late Sodium
Current Inhibitor (Late INai), a Phase II Agent with Demonstrated
Preclinical Anti-Ischemic and Antiarrhythmic Properties
Jeff A. Zablocki,*,† Elfatih Elzein,† Xiaofen Li,† Dmitry O. Koltun,† Eric Q. Parkhill,† Tetsuya Kobayashi,†
Ruben Martinez,† Britton Corkey,† Haibo Jiang,† Thao Perry,† Rao Kalla,† Gregory T. Notte,†
Oliver Saunders,† Michael Graupe,† Yafan Lu,† Chandru Venkataramani,† Juan Guerrero,† Jason Perry,⊥
Mark Osier,§ Robert Strickley,∥ Gongxin Liu,# Wei-Qun Wang,# Lufei Hu,# Xiao-Jun Li,#
Nesrine El-Bizri,# Ryoko Hirakawa,# Kris Kahlig,# Cheng Xie,# Cindy Hong Li,# Arvinder K. Dhalla,#
Sridharan Rajamani,# Nevena Mollova,‡ Daniel Soohoo,‡ Eve-Irene Lepist,‡ Bernard Murray,‡
Gerry Rhodes,‡ Luiz Belardinelli,# and Manoj C. Desai†
‡
§
∥
⊥
†Medicinal Chemistry, Drug Metabolism, Drug Safety Evaluation, Formulation and Process Development, and Structural
Chemistry, Gilead Sciences Inc., 333 Lakeside Drive, Foster City, California 94404, United States
#Biology, Gilead Sciences Inc., 7601 Dumbarton Circle, Fremont, California 94555, United States
S
* Supporting Information
ABSTRACT: Late sodium current (late INa) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Nav
1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of
late INa, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy
(HCM), and ventricular tachycardia−ventricular fibrillation (VT−VF). We will describe structure−activity relationship (SAR)
leading to the discovery of 4 that is vastly improved from the first generation late INa inhibitor 1 (ranolazine). Compound 4 was
42 times more potent than 1 in reducing ischemic burden in vivo (S−T segment elevation, 15 min left anteriorior descending,
LAD, occlusion in rabbits) with EC50 values of 190 and 8000 nM, respectively. Compound 4 represents a new class of potent late
INa inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.
INa current (Figure 2).4 Late INa current leads up to a 50%
INTRODUCTION
■
increase in cytoplasmic sodium that subsequently leads to Ca2+
The high prevalence (7%) of ischemic heart disease (IHD) in
the United States (US) is a result of a western diet coupled with
overload through activity of the reverse mode Na−Ca2+
exchanger.5 Compound 1 inhibits late INa current, thereby
lack of exercise, resulting in atherosclerotic obstruction of
coronary vessels.1 Angina, a condition that afflicts nine million
lowering the ischemic burden (calcium imbalance and
sequelae).5 At its therapeutic concentration (2−8 μM in
people in the US, is a symptomatic manifestation of IHD that
causes chest pain.2 Ranolazine 1 (Figure 1) was approved in the
plasma), 1 inhibits a number of cardiac ion currents (e.g.,
US in 2006 for the treatment of chronic angina.3 Although the
mechanism of 1‘s antianginal effect has not been determined, at
therapeutic concentrations, 1 does inhibit cardiac late sodium
current. Reactive oxygen species (ROS) generated during
ischemia can modify the cardiac sodium channel resulting in
incomplete inactivation of the channel allowing repetitive
opening of the inactivation gate resulting in a persistent sodium
current that occurs late in the action potential, thus termed late
̀
human Ether-a-go-go-related gene, hERG) and has β-blocker
activity as well.6 In preclinical models of ischemia, 1 has been
shown to inhibit S−T segment elevation, a finding analogous to
its ability to inhibit S−T segment changes in IHD patients with
angina undergoing exercise treadmill testing (Monotherapy
Received: June 23, 2016
© XXXX American Chemical Society
A
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anemone toxin II (ATX-II) or tefluthrin that bind to an
external binding site that does not overlap with the lidocaine
binding site (Figure 2).9 Compound 1 (Figure 1) has an IC50 of
6.9 μM for ATX-II induced late INa inhibition (single cell
manual patch).6 Peak INa current (peak cardiac sodium current)
is responsible for the propagation of the action potential from
cell to cell starting from the pacemaker sinoatrial (SA) node
through the atria and ventricles. To avoid pro-arrhythmic
effects, it is critical to have an adequate separation of late INa
inhibition from peak INa current. Compound 1 has a separation
of >50-fold for inhibition of late INa current relative to peak INa
current, and it is important to maintain this separation.6
Herein, we describe our efforts to discover a potent late INa
current blocker selective against peak INa current and
demonstrate the antiarrhythmic and anti-ischemic effects of
our clinical candidate molecule 4 (Figure 1) in isolated heart, in
vivo S−T ischemia model, and ischemia induced ventricular
arrhythmia model. To avoid hERG and β-blockade, we
screened a small diverse library of in-house heterocyclic
compounds (approximately 800 compounds) without a basic
group for their late INa inhibitory activity using an automatic
patch clamp system (h Nav1.5 α-subunit human embryonic
kidney cell, HEK-293, Q-Patch system). The ensuing hit-to-
lead effort resulted in discovery of triazolopyridine 2 (Figure
1).10,11 The potent late INa current inhibitor 2 (GS-458967,10,11
IC50 of 333 nM) was found to have a good separation of late INa
current inhibition from peak sodium current but did not have a
favorable central nervous system (CNS) window due to high
brain penetration and high activity at brain sodium isoforms
(Nav1.1, 1.2, 1.3).10,11 We subsequently discovered 3 (GS-
462808,12,13 Figure 1), with an increased polar surface area
from 50 to 84 Å by adding a carbonyl to the core that helped
lower the brain penetration and serendipitously decreased
activity at the brain sodium isoforms resulting in a more
favorable CNS window (20-fold).12,13 Unfortunately, 3 was
found to have a metabolic liability, leading to liver lesions in 7
day rat toxicology studies that may be due to oxadiazole
cleavage or glutathione addition to the core.12,13 We will
describe the SAR leading to the discovery of 3,4-dihydrobenz-
[f ][1,4]oxazepin-5(2H)-one 4 (GS-6615,14,15 Figure 1) and its
improved properties relative to 1.14,15
Figure 1. Gilead late sodium channel current (late INa) inhibitors
progressed from 1 to a modified screening hit 2 with a narrow CNS
margin, then to 3 with a wider CNS margin but a metabolic liability,
followed by the development candidate 4 that meets all criteria.
Figure 2. Late sodium current (Late INa) in a control experiment (a)
or enhanced with aid of 1 nM (b) or 3 nM (c) ATX-II.
Assessment of Ranolazine In Stable Angina, MARISA Study).7
We describe the discovery of a second-generation late INa
inhibitor with improved properties relative to 1 with less hERG
and β-blockade.
In site directed mutagenesis studies where the key phenyl-
alanine or tyrosine of the lidocaine site have been
independently mutated to alanine, 1 loses its inhibitory effect
on late INa current, thus suggesting 1 binds at this site.8 In vitro
late INa current can be generated by the addition of toxins sea
RESULTS
■
Chemistry. The general route to all of the late INa inhibitors
in this article utilized an alkylation step of the amide nitrogen
a
Scheme 1. Preparation of Benzotriazinone 15
a
Reagents and conditions: (a) NaN3, 2N aq HCl; (b) 4-CF3OC6H4B(OH)2, Pd(dppf)Cl2·CH2Cl2, K2CO3, toluene/i-PrOH/H2O; (c) 3-
(chloromethyl)-5-cyclopropyl-1,3,4-oxadiazole, DMF, K2CO3, Et3N.
B
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and a palladium mediated Suzuki−Miyaura coupling, as
illustrated in Scheme 1 for compound 15. The benzotriazinone
synthesis commenced with commercially available compound 6
(Scheme 1).16 Diazotization and in situ ring closure to the
benzotriazinone core was invoked by treatment with sodium
nitrite and hydrochloric acid. The 5-bromoquinazolin-4(3H)-
one and 5-bromophthalazin-1(2H)-one cores were prepared
from commercial starting materials (Scheme 2). The lactam
unsaturated ring system and switch to a partially saturated 6,6
lactam system exemplified by 18 and 19 (Table 2) and that did
indeed result in improved water solubility to 28 and 60 μM,
respectively, and retained inhibition of late INa current in cells.
Both compounds had good metabolic stability, but neither
compound was very active in reversing the effects of ATX-II
induced late INa on monophasic action potential duration90
(MAPD90) in isolated hearts (IH assay described in detail
below). The addition of an oxygen and expanding to a seven-
membered 3,4-dihydrobenzo[1,4]oxazepinone ring as in
compounds 20 and 4 resulted in retaining good solubility
and inhibition of late INa current both in cells and isolated heart
preparations (62% reversal at 1 μM). It appears that the late INa
inhibitor pharmacophore prefers a H-bond acceptor at this
position consistent with series like the 3,4-dihydrobenzo[1,4]-
oxazepinone, providing good activity in isolated heart and
downstream anti-ischemic assays (rabbit S−T). Both 20 and 4
had a 10-fold separation of inhibition of late INa current in cells
at the cardiac Nav1.5 channel from the inhibition of high
frequency 25 Hz (Hz) brain isoform Nav1.1. Compound 4 with
the 4-trifluoromethoxyphenyl had better human microsomal
metabolic stability than the 4-trifluoromethylphenyl analogue
20, and it became the development compound based on this
stability and subsequent activities in downstream assays (e.g.,
rabbit ΔS−T).
We next explored ring C SAR to determine whether the 4-
trifluoromethoxyphenyl substituent of 4 was optimal. The 3-
trifluoromethoxyphenyl analogue 21 has less late INa inhibition
(2×), is less metabolically stable in human microsomes, and is
less active in reversing the effects of ATX-II induced late INa
MAPD90 in isolated hearts (Table 3). The 2- trifluoromethox-
yphenyl analogue 22 is much less active in late INa inhibition
relative to the 4-trifluoromethoxyphenyl of 4. Similarly, the 3-
trifluoromethylphenyl analogue 23 has less late INa inhibition
(3×) than the 4-trifluoromethylphenyl 20. The 4-chlorophenyl
derivative 24 has less late INa inhibition (2×) than 4, and it was
less metabolically stable in human microsomes. The importance
of the electronic and lipophilic effect of the 4-trifluoromethox-
yphenyl of 4 can be noted when comparing late INa inhibitory
activity for the 4-methoxyphenyl analogue 25 and the
unsubstituted derivative 26 that were found to be much less
active (clogD of 4 is 3.56 vs 3.01 for 25). The CF3O− group
has been referred to as a pseudohalogen with a Pauling
electronegativity of 3.7 that lies between F −4.0 and Cl −3.0,
and it is quite lipophilic with a π of 1.04.17 The CH3O− is
much less electron withdrawing, having a Pauling electro-
negativity of 2.7 (closer to Br −2.8) with a π of −0.02.17 In
addition, the conformation of the two groups are different
based on analysis of Cambridge Crystallographic Database data,
CH3O− carbon typically lies within the plane of the phenyl, but
the CF3O− carbon lies closer to 90° out of plane.18 This was
confirmed by single crystal X-ray for 4, where the carbon of the
Scheme 2. Preparation of Quinazolin-4(3H)-one and
a
Phthalazin-1(2H)-one Derivatives
a
(a) R1PhB(OH)2, Pd(dppf)Cl2·CH2Cl2, NaHCO3, DMF; (b) R2-X,
X = Cl or Br, NaH, DMF, 80 °C, 24 h.
core was prepared by a Beckmann rearrangement initiated by
treatment with sodium azide and methanesulfonic acid
(Scheme 3).16 3,4-Dihydrobenzo[f ][1,4]oxazepin-5(2H)-one
core commenced with commercially available 6-bromochro-
man-4-one that underwent ring expansion by Beckmann
rearrangement. The order of Suzuki−Miyaura coupling and
N-alkylation is interchangeable and depended on the focus of
the SAR effort (routes 1 and 2, Scheme 4).
Initially, we stayed close to the [1,2,4]triazolo[4,3-a]pyridin-
3(2H)-one core of 3 by adding another nitrogen to make the
6,6-benzo[d][1,2,3]triazin-4(3H)-one core of 15, but we
replaced the 3-methyl-1,2,4-oxadiazol-5-yl)methyl D-ring of 3
with a less metabolic labile 5-cyclopropyl-1,3,4-oxadiazol-2-
yl)methyl D-ring (Scheme 1). Compound 15 retained the high
polar surface area of 84 Å, had a lower brain to plasma ratio
than 3 (0.84 versus 1.5), and demonstrated anti-ischemic
activity in vivo with an EC50 of 970 nM in the rabbit S−T
model (Table 1).16 The benzotriazinone series had a number of
highly active compounds in the rabbit S−T model and the
lowest brain to plasma ratios as a class. However, we were not
comfortable developing a compound from the benzotriazinone
class due to the lack of precedence in pharmaceutical
development and the inherent low solubility (1−4 μM). We
tried removing a nitrogen to the more generally accepted
quinazolin-4(3H)-one 16 and phthalazin-1(2H)-one 17
(Scheme 2) that retained inhibition of late INa current, but
unfortunately this resulted in much higher brain to plasma
ratios 15.0 and 4.4, respectively.
To enhance water solubility for potential use as an
intravenous agent, we tried to break the planarity of the
a
Scheme 3. Preparation of Lactam Core Derivatives
a
Reagents and conditions: (a) NaN3, MeSO3H, 0 °C; (b) R1PhB(OH)2, Pd(dppf)Cl2·CH2Cl2, NaHCO3, DMF; (c) R2-X, X = Cl or Br, NaH, DMF,
80 °C, 24 h.
C
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a
Scheme 4. Preparation of 3,4-Dihydrobenzo[f ][1,4]oxazepin-5(2H)-one Derivatives
a
Reagents and conditions: (a) NaN3, MeSO3H, 0 °C; (b) R1PhB(OH)2, Pd(dppf)Cl2·CH2Cl2, NaHCO3, DMF; (c) R2-X, X = Cl or Br, NaH, DMF,
80 °C, 24 h.
Table 1. Fused Rings Comparison: Benzo[d][1,2,3]triazin-4(3H)-one, Quinazolin-4(3H)-one, and Phthalazin-1(2H)-one
15
51
16
17
late INa, % block at 1 μM
ΔS-T EC50, nM
B:P, fold
48
39
970
0.86
84
15
80
4.4
80
PSA, Å2
Table 2. Sodium Channel Blocking Properties of Lactam versus 3,4-Dihydrobenzo[1,4]oxazepinone Compounds
microsomal t1/2, min
R1
late INa block, IC50, μM solubility, μM peak Nav1.1, % block at 10 μM
h
r
d
isolated heart (1 μM), ΔAPD90, %
18
19
20
4
4-CF3
2.07
2.31
1.08
0.88
28
60
87
24
15
23
60
53
304
395
348
395
354
150
211
200
320
157
371
354
−25
−6
−62
−62
4-CF3O
4-CF3
4-CF3O
CF3O− group lies on average 83° out of plane of the phenyl
ring (Figure 3). The CF3− group has a comparable Pauling
electronegativity of 3.5 to the CF3O− group, and it is quite
lipophilic with a π of 0.88, so it is consistent that 20 would have
similar late INa inhibitory to 4 (cLogD of 20 is 3.37).17 The 4-
CHF2O− group of 27 imparts a decreased late INa inhibition
that may be imparted due to the decreased lipophilicity (cLogD
3.2) and the conformational effects where the CHF2O− θ
typically lies between 0° and 50°.18 Also, the 4-CHF2− group
of 28 imparts a decreased late INa inhibition that may be
imparted due to the decreased lipophilicity (28 cLogD
2.95).18,19 Highly polar groups such as 4-MeSO2− led to loss
of late INa inhibition for 29 consistent with the phenyl
substituent binding in a hydrophobic pocket. The 4-Cl,3-F-
phenyl derivative 30 was 2× less active than 4 in cells but
retained good late INa inhibition in the isolated heart assay.
Adding an ortho-F group (31) to 4 led to comparable late INa
inhibition but resulted in lower human microsomal stability.
Adding a meta-Cl (32) to 4 led to good activity, retention of
metabolic stability, but decreased activity in ex vivo assay (IH).
As expected, the addition of a 2-acetamido group to 4 affording
33 resulted in a considerable loss of late INa inhibition. The 4-
trifluoromethoxyphenyl group of 4 provided the best overall
properties, so this was fixed and we explored the D ring SAR
(Table 4). Prior to changing the D ring, we checked the linker
length. We found that longer linkers led to a loss in metabolic
stability as exemplified by the ethyl linker 34 (propyl was also
less stable, not shown). The pyridyl D ring analogue 35 (a one
nitrogen change from 4), resulted in retaining late INa
inhibition, a slight increase in peak sodium inhibition, and a
decrease in metabolic stability. Addition of a 5-trifluoromethyl
to 35 afforded 36 with improved metabolic stability, but the
solubility decreased below 1 μM. Replacing the 5-trifluor-
omethyl group with 5-fluoro 37 resulted in a very potent late
INa inhibitor, but the inhibition of peak Nav1.1 was too high and
the metabolic stability decreased substantially. The addition of a
D
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Table 3. Sodium Channel Blocking Properties of 3,4-Dihydrobenzo[f ][1,4]oxazepin-5(2H)-one Compounds
microsomal t1/2, min
late INa block, IC50 (or peak Nav1.5 block, % (at peak Nav1.1, % block at
isolated heart (1 μM)
ΔAPD90, %
R1
% at 1 μM)
conc, μM), 3 Hz
10 μM, 25 Hz
h
r
d
b
4
4-CF3O
4-CF3
0.88
1.08
24 (3)
27 (3)
53
60
395
348
200
211
354
371
−62
−62
a,b
20
b
b
b
b
b
b
b
b
b
b
b
b
b
21
22
23
24
25
26
27
28
29
30
31
32
33
3-CF3O
2-CF3O
3-CF3
4-Cl
2.03
(26)
3.03
1.62
(13)
(18)
(13)
(17)
(3)
3 (3)
18
31
297
194
294
96
43
63
−29
−68
17 (3)
4-MeO
H
4-CHF2O
4-CHF2
4-MeSO2
4-Cl,3-F
1.61
1.28
0.77
(4)
8 (1)
37 (3)
15 (3)
23
35
33
330
241
393
98
308
338
115
156
−58
−39
−23
4-CF3O,2-F
4-CF3O,3-Cl
4-CF3O,2-MeCONH
c
NT
a
b
c
Prepared by route 1, Scheme 4. Prepared by route 2, Scheme 4. NT = not tested.
opening, directing the residues I1485, F1486, and M1487 into
the hydrophobic pore.22 Various compounds described here
were docked into the three states of the channel, and a
consensus binding mode emerged for compounds docked to
the open and inactivated states. This binding mode, as
illustrated in Figure 4 for compound 4, suggests compounds
rest in the lidocaine pocket, with the biphenyl motif forming
stacking interactions with F1760 and Y1767 on the S6 helix of
domain IV. In support of this binding pose, 4 lost late INa
inhibition with the mutants F1760A or Y1767A in a similar
manner to 1. The trifluoromethoxy group of compound 4 is
positioned toward the hydrophobic opening of the channel,
interacting primarily with I1466 on the S6 helix of domain III
and I1768 on the S6 helix of domain IV. An interaction with
F1486 of the inactivation gate may also be possible in the
inactivated state. The more polar end of the molecule is
directed toward the selectivity filter, defined by the residues
D372, E898, K1419, and A1711.23
Next, we evaluated the antiarrhythmic and anti-ischemic
effects of 4 versus 1 in isolated heart, in vivo ischemia model
(rabbit ΔS−T), and ischemia induced ventricular arrhythmia
model. Late INa was induced in ex vivo isolated rabbit hearts
through the addition of toxin ATX-II that prolongs the
MAPD90 by approximately 100 ms (ms, Figure 5). Late INa
inhibitors 4 or 1 reverse the prolongation of MAPD90 in the
isolated heart but to a different degree and potency. Compound
4 reverses the ATX-II effect by 100% with an IC50 of 700 nM,
while 1 provides only a partial reversal by 60% with an IC50 of
16 μM. Compound 4 is 22 times more potent in reversal of late
INa effects than 1. It is noteworthy that neither 4 or 1 have L-
type calcium channel activity at the concentrations tested as this
would also shorten the MAPD90. The prolongation of MAPD90
by late INa translates into a longer QT interval in vivo. In these
isolated heart ex vivo studies, the effect of 4 is much more
pronounced on the QT interval with complete reversal of ATX-
Figure 3. Single crystal X-ray of 4.
3-methoxy 38 or 3-amino 39 to the D ring led to an increase in
the inhibition of peak Nav1.1. A highly polar group with H
bond donors was not well tolerated on the D ring (40). The
compounds of Table 2 and 3 begin to illustrate how challenging
it is to optimize all the properties of late INa inhibitors. The
original 4-trifluoromethoxyphenyl C ring and unsubstituted
pyrimidine D ring of 4 turned out to be optimal.
A homology model of Nav1.5 was built based on the crystal
structure of a bacterial voltage-gated sodium channel, NavAb
(3RVY, see Supporting Information).20 The model was
manipulated into several states, including the closed, open,
and inactivated states. The closed state differs from the open
and inactivated states by a translational motion of the S4 helices
in response to voltage changes. The shift in the S4 helices alters
the position of the S4−S5 linkers, which constrain the extent of
the pore aperture opening formed by the S6 helices.21 The
open and inactivated states differ by a conformational change in
the inactivation gate connecting domains III and IV. In the
inactivated state, the inactivation gate closes over the channel
E
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a
Table 4. Sodium Channel Blocking Properties of 3,4-Dihydrobenzo[f ][1,4]oxazepin-5(2H)-one Compounds
a
b
All compounds in this table were prepared by route 1, Scheme 4. NT = not tested.
II induced prolongation as compared to a partial reversal
observed with 1 (Figure 5). Patients with long QT-3 have a
persistent current late in the action potential duration due to
mutations in their sodium channel that lead to improper
inactivation of the channel. Both 1 and 4 have been shown to
shorten the long QT interval in LQT-3 patients that is
consistent with late INa inhibition.24,25 The ATX-II isolated
heart model is an ex vivo LQT3 model. Compound 1 has been
shown in chronic angina patients to increase their treadmill
exercise times before angina pain develops that correlates with
changes in their S−T segment during exercise.7 Preclinically, 1
inhibits the S−T segment elevation with an EC50 of 8 μM in a
rabbit model of ischemia where the LAD artery of a rabbit heart
is occluded for 15 min and 1 was infused prior to ischemia
(Figure 6). Compound 4 inhibits S−T segment elevation with
an EC50 of 190 nM, and it nearly abolishes S−T segment
elevation at 500 nM (93% inhibition). Compound 1 only
inhibits 60% of S−T segment elevation at 12 μM. Compound
20 inhibits S−T elevation with an EC50 of 300 nM, slightly
inferior to 4. The favorable activity of 4 in the rabbit S−T assay
is even more than expected based on cellular late INa inhibition
(IC50 = 600 nM, −120 mV, manual patch HEK-293).
Compound 4 has increased late INa potency, as the resting
membrane voltage decreases to −80 mV with an improvement
in IC50 to 100 nM. This enhanced voltage dependent block
(VDB) of 4 for late INa current is consistent with ischemic
states where resting membrane potentials are as low as −75
mV. Previously, Pierre Fabre researchers found their lead late
INa inhibitors that performed well in advanced ischemia models
had a high degree of VDB.26,27 While the VDB IC50 is more in
line with the rabbit S−T EC50, we did check all off target
cardiac ion channels that would affect efficacy and a full
selectivity panel of receptors where 4 was completely clean of
effects at 1 μM (see Supporting Information, Table 1). The
improved efficacy (93%) and potency (42 times) of 4 relative
to 1 in the rabbit S−T model prompted our decision to
advance 4 to development. We wanted to evaluate additional
indications for 4 and turned to an ischemia induced ventricular
arrhythmia model (Figure 7). In the VT−VF model, we
constrict the left circumflex artery in the rabbit for 30 min to
F
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consistent with advancement as a second-generation late INa
inhibitor, so we further characterized its ion channel and PK
properties.
Our initial triazolopyridine lead 2 has an IC50 of 400 nM
against the brain sodium isoforms Nav1.1, 1.2, 1.3 with a
stimulating frequency of 10 Hz, while 4 has an IC50 of 96, 60,
and 16 μM, respectively (Table 5).11 This enhanced selectivity
is thought to be important to avoid off-target CNS effects
observed with 2 that resulted in a narrow therapeutic index. It is
also important to avoid hitting the muscular isoforms Nav1.4
and 1.6 to avoid tremors in the peripheral nervous system
where 4 has low activity as well. The Nav1.7 and 1.8 are targets
for pain indications in the periphery where the high frequency
(25 Hz) inhibition by 4 may actually prove beneficial in
diabetic neuropathy, a common co-morbidity of ischemic
patients. It is noteworthy that 1 has demonstrated efficacy in
preclinical models of pain.29 Compound 4 has use dependent
block with increased activity for late INa inhibition with an IC50
less than 100 nM for 3 Hz stimulation (180 beats per minute).
The off-target activity for 4 does increase at the high frequency
of 25 Hz, but it is still above 3 μM for the brain isoforms.
Ultimately, 4 was found to be well tolerated in vivo as far as a
CNS effects with single ascending dose studies in rat and dog
supporting adverse events occurring above 10 μM. Compound
4 was found to be approximately 10 times more potent than 1
for late INa inhibition in cells (Table 6) while maintaining a
good separation from peak sodium inhibition. Compound 1 has
only a 2 fold separation of late INa inhibition from hERG
inhibition, but 4 has a 10-fold separation. The late INa inhibiting
effects of 1 on MAPD90 in isolated hearts are likely
compromised by its hERG inhibition that works against it.
This may be the reason for the partial efficacy observed with 1
in the isolated heart assay. Both 4 and 1 have low activity at the
brain isoform Nav1.1 at 10 μM with 10 Hz stimulation.
We evaluated the pharmacokinetics of 4 across species with a
good correlation between in vitro and in vivo clearance in rat
and dog but a slightly lower clearance (3×) observed in vivo in
the cynomolgus monkey (Table 7). The volume steady state
(Vss) across species was in an acceptable range where allometric
scaling projected a human volume of 1.5 L/kg. The half-life of 4
varied from 5 h in the cyno to 27.9 h in dog. Compound 4 had
Figure 4. Homology model of compound 4 bound to the inactivated
state of Nav1.5. Protein surface is colored by residue type: green is
hydrophobic, cyan is polar, blue is positively charged. The compound
is suggested to bind with its biphenyl motif sandwiched between
F1760 and Y1767, which define the lidocaine pocket. Its hydrophobic
end (trifluoromethoxy) is directed toward the inactivation gate, while
its polar end (pyrimidine) is directed toward the selectivity filter. Inset
shows the full length model. Domain I is blue, domain II is green,
domain III is yellow, and domain IV is pink. The inactivation gate
linking domains III and IV is orange. The C-terminus is red.
Compound 4 is seen to bind in the channel pore.
induce the control animals to have episodes of VT and VF.
Both the class Ic peak sodium current inhibitor flecainide and
class 1b mexiletine increase ventricular tachycardia/fibrillation
(VT−VF) and subsequent death due to their pro-arrhythmic
properties (Figure 7 and Supporting Information, Figure 2).28
Compound 4 (class Ib) completely inhibits VT−VF and death
at 2.5 μM with an EC50 of approximately 1.7 μM. Previously,
we found that 1 was only partially protective in a chemically
induced Torsades des Pointes (TdP) assay with 40% of the
animals having Tdp at 14 μM.13 Compound 4 was also
evaluated in the chemically induced TdP assay, and it was again
completely protective at 2.5 μM (see supplement). Overall,
compound 4 had marked improvement in potency and efficacy
relative to 1 in animal models of ischemia and arrhythmia
Figure 5. LQT-3 model 4 (orange) vs 1 (red), ex vivo isolated heart IC50 (rabbit).
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Article
Figure 6. Anti-ischemic effect of 1 versus 4 versus 20 in left anterior descending (LAD) coronary occlusion model (15 min, rabbit).
Table 5. Sodium Channel Blocking Properties of 3,4-
Dihydrobenzo[f][1,4]oxazepin-5(2H)-one 4 (Manual Patch
Clamp)
IC50 (μM) stimulating frequency (Hz)
Na+ channel isoform
10 Hz
25 Hz
Nav1.1
Nav1.2
Nav1.3
Nav1.4
Nav1.6
Nav1.7
Nav1.8
∼96
∼60
∼16
∼13
∼28
5
3.4
10
3.5
3.3
4
Figure 7. 4 suppresses ischemia induced VT−VF arrhythmias
recorded during 30 min of LCX ligation in anesthetized rabbits.
Flecainide (class 1c antiarrhythmic) and mexiletine (class 1b
antiarrhythmic, Supporting Information, Figure 2) induce VT−VF
and mortality at clinical exposures.
1
∼18.8
1.5
Table 6. Sodium Channel Blocking Properties of 3,4-
Dihydrobenzo[f][1,4]oxazepin-5(2H)-one 4 versus 1
(Manual Patch Clamp)
good oral bioavailability across species consistent with excellent
permeability (heterogeneous human epithelial colorectal
adenocarcinoma, CACO-2 A → B/B → A 34.6/29 × 10−6
cm/s) and low first-pass metabolism. The low clearance in
pooled human hepatocytes using tritiated 4 was consistent with
once a day dosing. The subsequent maintenance doses of 4
used in phase II trials were 3 and 6 mg per day.25 We do not
anticipate drug−drug interactions with 4 as there were no
significant inhibitory activity observed against the major
cytochrome P450s (>10 μM, Cyp 3A4, 2D6, 2C8, 1A, 2C9,
2C19, 2B6). We did measure the levels of 4 in the brain in the
rat after oral dosing, and the total brain to plasma ratios were
approximately 3:1. Fortunately, 4 was found to be well
4
1
late INa IC50 (μM) ATX II
0.62 (10-fold)
51 (82-fold)
6.0 (10-fold)
no effect
6.9
peak INa IC50 (μM) (peak/late)
hERG IC50 (μM) (hERG/late)
peak Nav1.1 @ 10 μM (10 Hz)
428 (62-fold)
12 (2-fold)
no effect
tolerated in vivo in the rat as far as a CNS effects due to the
decreased activity at the brain isoforms as mentioned above.
H
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HPLC system includes two sets of Gilson 332 pumps, a Gilson 156
UV/vis detector, and a Gilson 215 injector and fraction collector, with
Trilution LC software. A Phenomenex Luna 150 mm × 21.2 mm
column was used. The mobile phase was a gradient of 0.1% TFA water
(A) and 0.1% TFA acetonitrile (B). High-resolution mass spectra were
recorded on a Xevo G2 Q−T of mass spectrometer with an ESI
source.
Table 7. Preclinical PK Properties across Species for 4
beagle
dog
cyno
parameter
SD rat
0.16
monkey
human
<0.015
hepatocytes
Clint
(L/h/kg)
0.06
0.36
3H-4
7-(4-(Trifluoromethoxy)phenyl)-3,4-dihydrobenzo[f ][1,4]-
oxazepin-5(2H)-one (4). Commercially available 6-bromochroman-
4-one (1.0 g, 3 mmol) was dissolved in 10 mL of methanesulfonic acid.
The solution was cooled using an ice bath, and sodium azide (0.30 g,
4.5 mmol) was added over a period of 45 min. The mixture was stirred
at RT for 16 h. The mixture was neutralized using conc HCl. The
resulting solid was filtered and washed with water to afford 11 as
analytically pure sample.
IV
Cl (L/h/kg)
Vss (L/kg)
T1/2 (h)
0.14
2.5
0.042
1.6
0.11
1.5
13
27.9
5.0
PO
%F
91
100
39
CONCLUSION
■
Route 2: To a solution of 11 (20.0 g, 0.083 mol, 1 equiv) and 2-
(chloromethyl)pyrimidine (25.0 g, 0.15 mol, 1.8 equiv) in DMF (150
mL), NaOH solution (20 mL, 10M, 5 equiv) was slowly added at
room temperature (slightly exothermic) and stirred at rt for 10 min,
followed by heating at 95 °C for 2 h. After cooling the reaction
mixture, ethyl acetate (200 mL) was added and the organic layer was
separated. The organic layer was washed with water (20 mL), brine,
dried over sodium sulfate, and concentrated. The residue was dissolved
in 1,4-dioxane (50 mL) and to this 4 N HCl in dioxane (50 mL) and
conc HCl (2 mL) was added and stirred at room temperature for 4 h.
The resulting precipitate was filtered, washed with ethyl acetate, and
dried. Compound 13 obtained (30 g, 98.0%) was obtained as a light-
yellow solid. To the bromide 13 (15 g, 0.04 mol, 1 equiv), 4-
trifluoromethoxyphenylboronic acid (12.5 g, 0.06 mol, 1.5 equiv) and
potassium carbonate (22.0 g, 0.16 mol, 4 equiv) in a round-bottom
flask, solvent (150 mL, toluene/2-propanol/water, 2/1/1) was added
and stirred under nitrogen for 10 min. To the above solution, the
palladium catalyst Pd(dppf)Cl2·CH2Cl2 (1.0 g, 0.012 mol, 0.02 equiv)
was added and heated at 85 °C for 2 h. The reaction mixture was
diluted with ethyl acetate (300 mL), and the organic layer was
separated, filtered through a plug of Celite and silica gel, and
concentrated in vacuo. Flash column purification on silica gel using
ethyl acetate/hexane as eluent provided 4 (13.0 g 78%). This reaction
was repeated to afford an additional 13.0 g of 4. To a solution of 4 (26
g) in 1,4-dioxane (25 mL), 4 N HCl/dioxane (25 mL) was added
followed by conc. HCl (2 mL) and stirred at room temperature for 4
h. Solvent was distilled off, and dichloromethane was added and
distilled off. To the residue, ethyl acetate (150 mL) was added and
stirred at room temperature overnight. The precipitate was filtered,
washed with ethyl acetate and hexane, and dried under vacuum.
Compound 4 HCl obtained (24.8 g) was obtained as a white solid.
We identified 4 as an appropriate second-generation late INa
inhibitor with improved potency and efficacy relative to 1 in
cells, ex vivo isolated hearts, and in animal models of ischemia
and arrhythmia. Late sodium current is enhanced during
ischemia, leading to calcium overload that increases energy
demand during ischemia and increases the probability of early
after depolarizations and delayed after depolarizations, both
triggers for arrhythmias.4 The first-generation late INa inhibitor
1 within its therapeutic range (2−8 μM) does not completely
inhibit late INa (estimated at 15−30% inhibition based on rabbit
S−T), has only a 2-fold selectivity over hERG, has significant β-
blocker activity at therapeutic concentrations, requires twice a
day dosing at approved doses up to 1000 mg, and has dizziness
as a side effect due to CNS effects (6% at 1000 mg).
Compound 4 is estimated to have a 100% coverage of late INa
inhibition at 1 μM (based on rabbit S−T), has a 10-fold
selectivity over hERG, does not inhibit β-receptors at 10 μM, is
QD at 3 and 6 mg, and was well tolerated in phase I studies.
Compound 4 is 10 times more potent late INa inhibitor than 1
in cells and 22 times more potent in isolated heart ex vivo assay.
Compound 4 was 42 times more potent than 1 in reducing
ischemic burden in vivo (rabbit S−T segment) with 93%
inhibition at 500 nM, while 1 provided only 60% at 12 μM. In a
drug-induced model of TdP ventricular arrhythmia model
(supplement), 4 was completely protective at 2.4 μM, while 1
provides only 60% reduction at 14 μM. Compound 4 does not
potentiate ischemia-induced arrhythmias (VT−VF) or mortal-
ity as was observed with other class 1 antiarrhythmics in the
LCX model. Compound 4 is currently in clinical development
for treatment of LQT-3 syndrome, HCM, and VT−VF.
Compound 4 represents a new class of potent late INa
inhibitors that will be useful in further delineating the role of
inhibitors of this current in the treatment of patients with
cardiovascular disease.
1
Anal. HPLC 100% (6.78 min). H NMR (CDCl3) δ 8.72 (d, 2H, J =
5.2 Hz), 8.17 (d, 1H, J = 2.4 Hz), 7.59−7.63 (m, 3H), 7.26 (d, 2H, J =
3.2 Hz), 7.22 (t, 1H, J = 4.8 Hz), 7.10 (d, 1H, J = 8.4 Hz), 5.10 (s,
2H), 4.56 (t, 2H, J = 5.0 Hz), 3.77 (t, 2H, J = 5.0 Hz). LCMS m/z
416.1 (M + H). HRMS-ESI+: [M + H]+ calcd for C21H16F3N3O3,
416.1217; found, 416.1215.
6-Bromobenzo[d][1,2,3]triazin-4(3H)-one (7). To a mixture of
commercially available compound 6 (9.250 g, 43.03 mmol) and
sodium nitrite (8.909 g, 129.11 mmol) in 1,4-dioxane (40 mL) was
added dropwise 2N aqeuous HCl (80 mL, 160.00 mmol) with
vigorous stir over a period of 30 min, during which H2O was added
twice (40 mL each at 10th and 20th min). After completion of
addition, the reaction mixture was stirred overnight then diluted with
H2O (200 mL), sonicated, filtered, washed with H2O (500 mL), and
dried to afford the desired product as 7. LCMS m/z 226.0 (M + H),
228.0 (M + H + 2). Anal. HPLC > 98%. 1H NMR (400 MHz; DMSO-
d6) δ 8.30 (d, J = 2.3 Hz, 1H), 8.22 (dd, J = 8.6, 2.0 Hz, 1H), 8.10 (d, J
= 9.0 Hz, 1H).
EXPERIMENTAL SECTION
■
Nuclear magnetic resonance (NMR) spectra were recorded on Varian
Mercury Plus 400 MHz and a Varian Unity Plus 500 MHz
spectrometers at room temperature, with tetramethylsilane as an
internal standard. Chemical shifts (δ) are reported in parts per million
(ppm), and signals are reported as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), or br s (broad singlet). Purities of the final
compounds were determined by HPLC and were greater than 95%.
HPLC conditions to assess purity were as follows: Agilent HPLC,
Phenomenex Kinetix C18, 4.6 mm × 100 mm (2.6 μm); gradient of
0.1% TFA water (A) and 0.1% TFA acetonitrile (B) start at 98:2 A:B
with 8.5 min gradient to 0:100; flow rate, 1.5 mL/min; wavelength,
UV 254 nM. The LC-MS spectra were obtained on an Agilent system
with the following HPLC conditions: column, Phenomenex Luna C18,
4.6 mm × 75 mm (5 μm), gradient of 0.05% TFA water (A) and
0.04% TFA acetonitrile (B), flow rate, 1.0 mL/min. The preparative
6-(4-(Trifluoromethoxy)phenyl)benzo[d][1,2,3]triazin-4(3H)-
one (8). To a solution of 7 (1.130 g, 5.0 mmol) and 4-
trifluoromethoxyphenylboronic acid (1.544 g, 7.5 mmol) in DMF
(30 mL) was added K2CO3 (2.073 g, 15.0 mmol) and H2O (3 mL).
The reaction mixture was stirred for 5 min under an atmosphere of dry
N2. Pd(dppf)Cl2·CH2Cl2 (146 mg, 0.20 mmol) was added, and the
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resulting mixture was heated at 98 °C until 7 was consumed (LCMS).
The reaction mixture was cooled, diluted with EtOAc (70 mL), filtered
through a layer of Celite, washed with 20% DMF in EtOAc (100 mL),
transferred to a separation funnel, organic phase was washed with 0.5
M K2CO3 (50 mL, 25.0 mmol), 30% aqeuous NH4Cl (100 mL)and
brine (100 mL), dried, and concentrated. To the crude product was
added 10% EtOAc in n-hexane (10 mL), sonicated, filtered, and
washed with 10% EtOAc in n-hexane (20 mL) to afford the desired
1H). HRMS-ESI+: [M + H]+ calcd for C19H13F3N4O3, 403.1013;
found, 403.1011.
2-(Pyrimidin-2-ylmethyl)-7-(4-(trifluoromethyl)phenyl)-3,4-
dihydroisoquinolin-1(2H)-one (18). Compound 18 was prepared
using the procedures disclosed in the above scheme using the
appropriate starting materials. Anal. HPLC 99.3% (6.84 min). 1H
NMR (CD3OD) δ 8.72 (d, 2H, J = 4.8 Hz), 8.22 (d, 1H, J = 2.0 Hz),
7.77−7.81 (m, 3H), 7.72 (d, 2H, J = 8.0 Hz), 7.39 (d, 1H, J = 8.0 Hz),
7.34 (t, 1H, J = 5.0 Hz), 5.02 (s, 2H), 3.81 (t, 2H, J = 6.4 Hz), 3.14 (t,
2H, J = 6.4 Hz). MS m/z 384.1 (M + H). HRMS-ESI+: [M + H]+
calcd for C21H16F3N3O, 384.1318; found, 384.1316.
1
product as 8, MS m/z 308.0 (M + H), HPLC purity >97%. H NMR
matched the desired product. The combined filtrate was concentrated,
subjected to Gilson’s reverse-phase preparative HPLC with a gradient
0.1% TFA containing CH3CN/H2O (10% to 90%) to afford additional
desired product as 8. LCMS m/z 308.0 (M + H). Anal. HPLC > 99%.
The overall combined yield is 71%. 1H NMR (400 MHz; DMSO-d6) δ
8.42 (m, 1H), 8.39 (d, J = 2.3 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.00
(m, 2H); 7.52 (d, J = 8.2 Hz, 2H). 19F NMR (376 MHz; DMSO-d6) δ
−57.2 (s, 3F).
2-(Pyrimidin-2-ylmethyl)-7-(4-(trifluoromethoxy)phenyl)-
3,4-dihydroisoquinolin-1(2H)-one (19). Compound 19 was
prepared using the procedures disclosed in the above scheme using
1
the appropriate starting materials. Anal. HPLC 100% (6.96 min). H
NMR (DMSO-d6) δ 8.75 (d, 2H, J = 5.2 Hz), 8.17 (d, 1H, J = 2.4 Hz),
7.78 (m,3H) 7.35−7.45 (m,4H), 4.93 (s, 1H), 3.74−3.78 (t,
2H),3.07−3.10 (t, 2H). LCMS m/z 400.1 (M + H).
3-((5-Cyclopropyl-1,3,4-oxadiazol-2-yl)methyl)-6-(4-
(trifluoromethyl)phenyl)benzo[d][1,2,3]triazin-4(3H)-one (15).
To a solution of 8 (2.446 g, 7.96 mmol) and 3-(chloromethyl)-5-
cyclopropyl-1,3,4-oxadiazole (1.660 g, 10.47 mmol) in DMF (15 mL)
in a Biotage microwave tube (20 mL capacity) was added potassium
carbonate (1.881 g, 13.61 mmol) and triethyl amine (2 mL) with
stirring. The reaction mixture was stirred at room temperature for 5
min and then subjected to microwave heating at 120 °C until 8 was
consumed in LCMS. The mixture was cooled, diluted with 20% DMF
in EtOAc (50 mL), filtered, and washed with 20% DMF in EtOAc
(100 mL). The combined filtrate was concentrated in vacuo, dissolved
mostly in dichloromethane (20 mL), and filtered, and the filtrate was
subjected to Yamazen chromatography over Universal column, eluted
with a gradient EtOAc in n-hexane to afford, after drying, 15. Anal.
HPLC 97% (7.19 min). 15 was recrystallized from EtOAc/n-hexane
7-(4-(Trifluoromethyl)phenyl)-3,4-dihydrobenzo[f ][1,4]-
oxazepin-5(2H)-one (20). Route 1: 7-Bromo-3,4-dihydrobenzo[f ]-
[1,4]oxazepin-5(2H)-one (2.0 g), 4-trifluoromethoxyphenylboronic
acid (2.2 g), and potassium carbonate (2.0 g) were combined in a
mixture of toluene (20 mL), 2-propanol (10 mL), and water (10 mL),
and the resulting suspension was degassed with nitrogen. Palladium
catalyst Pd(dppf)Cl2·CH2Cl2 was added (0.42 g), and the reaction was
heated overnight at 85 °C. After cooling, the aqueous layer was
discarded and the organic layer was diluted 2-fold with ethyl acetate,
dried over MgSO4, and concentrated. Recrystallization was conducted
by dissolving in a minimum necessary amount of dichloromethane and
triturating with excess hexane, resulting in 7-(4-(trifluoromethyl)-
phenyl)-3,4-dihydrobenzo[f ][1,4]oxazepin-5(2H)-one as a gray solid
1
(1.43 g). H NMR: 8.42 (t, 1H), 8.12 (d, 1H), 7.86−7.76 (m, 5H),
1
7.13 (d, 1H), 4.38 (t, 2H), 3.37 (quartet, 2H). 20 was made via
alkylation with 2-(chloromethyl)pyrimidine using the conditions
described above for making 13. Anal. HPLC 100% (6.78 min).
Route 2: To the bromide 13 (28.0 g, 0.083 mol, 1 equiv), 4-
trifluoromethylphenylboronic acid (24.0 g, 0.125 mol, 1.5 equiv), and
potassium carbonate (45.0 g, 0.332 mol, 4 equiv) in a round-bottom
flask, solvent (200 mL, toluene/2-propanol/water, 2/1/1) was added
and stirred under nitrogen for 10 min. To the above solution, the
palladium catalyst Pd(dppf)Cl2·CH2Cl2 (1.4 g, 0.00166 mol, 0.02
equiv) was added and heated at 85 °C for 3 h. The reaction mixture
was diluted with ethyl acetate (200 mL), separated the organic layer,
washed with water, and brine. The organic layer was filtered through a
plug of Celite and concentrated. Column purification on silica gel
using ethyl acetate/hexane (70/30) as eluent provided the product 20
(28.9 g; 87%). 1H NMR (CDCl3) δ 8.80 (d, 2H, J = 5.2 Hz), 8.19 (d,
1H, J = 2.8 Hz), 7.66−7.71 (m, 5H), 7.33 (t, 1H, J = 5.0 Hz), 7.13 (d,
1H, J = 8.4 Hz), 5.14 (s, 2H), 4.59 (t, 2H, J = 4.8 Hz), 3.81 (t, 2H, J =
5.0 Hz). LCMS m/z 400.1 (M + H). HRMS-ESI+: [M + H]+ calcd for
C21H16F3N3O2, 400.1267; found, 400.1267.
and dried to give 15. H NMR (400 MHz, DMSO-d6) δ 8.57−8.44
(m, 2H), 8.36 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 8.1 Hz, 2H), 7.88 (d, J
= 8.2 Hz, 2H), 5.82 (s, 2H), 2.20 (ddd, J = 8.4, 4.9, 3.5 Hz, 1H), 1.15−
1.02 (m, 2H), 1.02−0.86 (m, 2H). HRMS-ESI+: [M + H]+ calcd for
C20H14F3N5O2, 414.1172; found, 414.1167.
3-((3-Methyl-1,2,4-oxadiazol-5-yl)methyl)-6-(4-
(trifluoromethoxy)phenyl)quinazolin-4(3H)-one (16). 16 was
prepared using the procedures disclosed herein above with the
appropriate starting materials. LC/MS (m/z): 403.0 (M + H+). Anal.
1
HPLC 100% (7.21 min). H NMR (chloroform-d) δ 8.50 (d, J = 2.2
Hz, 1H), 8.24 (s, 1H), 8.01 (dd, J = 8.5, 2.2 Hz, 1H), 7.86 (d, J = 8.5
Hz, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.33 (dd, J = 8.9, 1.0 Hz, 1H), 5.42
(s, 2H), 2.38 (s, 3H). HRMS-ESI+: [M + H]+ calcd for
C19H13F3N4O3, 403.1013; found, 403.1010.
2-((3-Methyl-1,2,4-oxadiazol-5-yl)methyl)-7-(4-
(trifluoromethoxy)phenyl)phthalazin-1(2H)-one (17). A mixture
of 7-bromophthalazinone (1.09g, 4.84 mmol), 4-trifluoromethox-
yphenyl boronic acid (1.20 g, 5.81 mmol), Pd(dppf)Cl2·CH2Cl2 (177
mg, 0.242 mmol), and potassium carbonate (1.34 g, 9.68 mmol) in
degassed toluene (4 mL), degassed water (2 mL), and degassed 2-
propanol (2 mL) was heated at 90 °C for 12 h. The layers were
separated, the organic layer was concentrated, and the residue was
purified by trituration with hexanes/ethyl acetate to provide 7-(4-
(trifluoromethoxy)phenyl)phthalazin-1(2H)-one as a white powder.
LCMS m/z C15H9F3N2O2. 307.2 (M + 1).
To a mixture of 7-(4-(trifluoromethoxy)phenyl)phthalazin-1(2H)-
one (50 mg, 0.163 mmol), 5-(chloromethyl)-3-methyl-1,2,4-oxadiazole
(47.5 mg, 0.359 mmol), and potassium carbonate (68 mg, 9.68 mmol)
was added DMF (1 mL), and the reaction was heated to 80 °C
overnight. The reaction was diluted with EtOAC and water, the layers
were separated, and the organic layer was concentrated to an oil. The
residue was purified by flash chromatography (Rf = 0.32, 1:1 hexanes/
ethyl acetate) to afford 2-((3-methyl-1,2,4-oxadiazol-5-yl)methyl)-7-
(4-(trifluoromethoxy)phenyl)phthalazin-1(2H)-one 17 as a white
solid. C19H13F3N4O3. LCMS m/z 403.1 (M + 1). Anal. HPLC
99.6% (7.18 min). 1H NMR (DMSO) δ 8.58 (s, 1H), 8.48 (d, J = 2.0
Hz, 1H), 8.34 (dd, J = 2.0, 8.4 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H),
7.98−8.02 (m, 2H), 7.54 (d, J = 8.0 Hz, 1H), 5.67 (s, 2H), 2.31 (s,
7-(3-(Trifluoromethoxy)phenyl)-3,4-dihydrobenzo[f ][1,4]-
oxazepin-5(2H)-one (21). Compound 21 was prepared according to
4 disclosed herein using the appropriate starting materials. Anal.
HPLC 100% (6.86 min). 1H NMR (400 MHz, DMSO-d6) δ 8.77 (d, J
= 4.8 Hz, 2H), 8.01 (d, J = 2.5 Hz, 1H), 7.81 (dd, J = 8.8, 2.4 Hz,
1H)7.77−7.65 (m, 1H), 7.66−7.53 (m, 2H), 7.43 (t, J = 4.9 Hz, 1H),
7.36 (d, J = 8.2 Hz, 1H), 7.17 (d, J = 8.5 Hz, 1H), 5.00 (s, 2H), 4.55 (t,
J = 4.8 Hz, 2H), 3.79 (t, J = 4.8 Hz, 2H). HRMS-ESI+: [M + H]+
calcd for C21H16F3N3O3, 416.1216; found, 416.1213.
Automated Ion Channel Assays. The late sodium current (late
INa) and peak sodium current (peak INa) assays were performed at
room temperature on the QPatch automated electrophysiology
platform (Sophion Bioscience, Copenhagen, Denmark), which used
the whole cell patch clamp technique to measure currents through the
cell membrane. Series resistance compensation was set to 100%, and
series resistance and whole-cell compensation were performed
automatically and currents were digitized at 25 kHz.
Cardiac hNaV1.5 Assays. HEK293 cells stably expressing the
human Nav1.5voltage-gated sodium channel (Millipore, Billerica, MA)
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were used to measure the block of INa at resting membrane potential of
−120 mV. For the late INa assay, a voltage step from the resting
membrane potential to −20 mV for 220 ms was performed at a
stimulating frequency of 0.1 Hz. To improve assay signal-to-noise, late
INa was enhanced using continuous application of tefluthrin (TEF, 10
μM) in the extracellular and intracellular solutions. Late INa was
measured as the magnitude of INa during the end phase of the voltage
step (210−220 ms). The extracellular solution was (in mM): 140
NaCl, 4 KCl, 1.8 CaCl2, 0.75 MgCl2, and 5 HEPES with pH adjusted
to 7.4 using NaOH. The intracellular solution was (in mM): 120 CsF,
20 CsCl, 5 EGTA, 5 HEPES, and pH adjusted to 7.4 with CsOH. For
the peak INa assay, a voltage step from the resting membrane potential
to −20 mV for 20 ms was performed at a stimulating frequency of 0.1
Hz. This protocol was performed in control solution and after the
superfusion of compound containing solution. The extracellular
solution was (in mM): 40 NaCl, 100 choline Cl, 4 KCl, 1.8 CaCl2,
0.75 MgCl2 and 5 HEPES with pH adjusted to 7.4 using NaOH. The
intracellular solution was (in mM): 120 CsF, 20 CsCl, 5 EGTA, 5
HEPES, and pH adjusted to 7.4 with CsOH.
of heart rhythm. Pressure-contact Ag-AgCl MAP electrodes were
placed on the epicardial ventricular free wall below the level of the
atrial−ventricular valve at the base of the left ventricle. Electrical
signals were amplified, filtered, and digitized in real time using a
BIOPAC Systems MP 150 signal processor and displayed on a
computer screen by AcqKnowledge 4.2 for MP150 software (BIOPAC
Systems Inc.). The duration of the monophasic action potential at 90%
repolarization (MAPD90) was used to monitor left ventricular
repolarization. MAPD90 was first prolonged by the perfusion of the
heart with ATX-II to activate late INa. The degree of MAPD90
shortening back to baseline was measured at steady-state following
compound perfusion.
Ischemia-Induced ECG S−T Segment Elevation by LAD
Ligation in Rabbits. Female New Zealand rabbits (3.0−4.0 kg) were
purchased from Western Oregon Rabbitry. Animals were housed on a
12 h light and dark cycle and received standard laboratory chow and
water. All experiments were performed in accordance with the Guide
for the Care and Use of Laboratory Animals published by The
National Research Council and with the experimental protocol
approved by the Institutional Animal Care Committee of Gilead
Sciences, Inc.
hERG Assay. CHO−hERG DUO cells stably expressing the human
ERG (Ether-a-go-go gene) KV11.1 voltage-gated potassium channel
̀
Rabbits were anesthetized with ketamine (35 mg/kg) and xylazine
(5 mg/kg) intramuscular injection (IM). A tracheotomy was
performed, and the trachea was intubated with an endotracheal tube.
The animal was ventilated with room air supplemented with oxygen
using a pressure control animal ventilator (Kent Scientific Corp.,
Torrington, CT) at a respiratory rate of 40 strokes/min and peak
inspiration pressure of 10 mmH2O, which was adjusted to keep blood
gases and pH within the physiological range (iSTAT clinic analyzer,
Heska Corp.; Waukesha, WI). The left femoral artery was cannulated
for the measurement of blood pressure (BP). Blood samples were also
withdrawn from femoral artery. The right external jugular vein was
cannulated for drug/vehicle administration. Needle electrodes were
inserted subcutaneously into the limbs for recording of the surface
electrocardiogram (ECG). The heart was exposed via an incision in
the fourth intercostal space (fourth and/or fifth ribs were cut for a
clear surgical vision). The chest was opened and a pericardial cradle
was formed using four retractors. A coronary artery occluder,
comprised of a snare made of 5 cm PE-10 tubing with a 6−0 Prolene
polypropylene suture in it, was placed loosely around the left anterior
descending artery (LAD) at its origin. Two unipolar electrodes, made
with Teflon coated silver wire attached to a small patch of filter paper,
were attached on the surface of the ischemic and normal regions of the
left ventricle to record epicardial electrocardiogram. Reference
electrodes were placed in the open incision of the neck. The body
temperature of the animal was monitored via a rectal thermometer and
maintained at 37−40 °C by adjusting the surface temperature of the
surgical table. Regional ischemia (15 min) was induced by ligating the
LAD, followed by 15 min of reperfusion caused by releasing the
ligation. BP and ECG were monitored and recorded continuously
using a computer data acquisition system (PowerLab, AD Instruments;
Mountain View, CA). S−T segment height in both epicardial and
surface ECG, and PR, QRS, and QT interval in surface ECG was
analyzed using an ECG analysis module (AD Instruments, Mountain
View, CA).
were obtained from Sophion. Inhibition of peak tail current was
measured from a holding potential of −80 mV. The voltage protocol
consisted of two contiguous steps: first a step to +20 mV for 4.8 s
followed by step to −40 mV for 5 s with a sweep frequency of 0.0667
Hz. Peak tail current IK was measured at the start of the step to −40
mV. This protocol was performed in control solution and after the
superfusion of compound containing solution. Then 1 μM Cisapride
solution was added near the end to define the baseline. The external
recording solution was (in mM): 140 NaCl, 4 KCl, 1.8 CaCl2, 1
MaCl2, 10 HEPES, and 10 glucose. The internal recording solution
was (in mM): 120 KCl, 31.25/10 KOH/EGTA, 10 HEPES, 5.374
CaCl2, 1.75 MgCl2, 4 Na2-ATP.
Manual Ion Channel Assays for Non-Cardiac NaV1.X
Channels. Assays were performed at room temperature using a
manual patch clamp in the whole-cell configuration using a
MultiClamp 700B amplifier, Digidata 1400 digitizer, and pClamp 10
software (Molecular Devices, Sunnyvale, CA, USA). Series resistance
was compensated 90% to ensure that the command potential was
reached within microseconds with a voltage error <2 mV. Leak
currents were subtracted by using an online P/4 procedure. Currents
were low-pass Bessel filtered at 4 kHz and digitized at 50 kHz. The
bath solution contained (mM): 140 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2,
10 dextrose, and 10 HEPES, with a pH of 7.35. The pipet solution
consisted of (mM) 10 NaF, 110 CsF, 20 CsCl, 2 EGTA, and 10
HEPES, with a pH of 7.35. Inhibition was assessed using a train of 50
square voltage steps (P1 to P50) from a holding potential of −120 mV
to a test potential of 0 mV (20 ms) at a frequency of either 10 Hz. The
ratio P50/P1 obtained in the presence of drug was normalized to the
ratio obtained in the absence of drug (Control) to calculate the degree
of inhibition.
Isolated Rabbit Heart Assay. Animal use was approved by the
Institutional Animal Care and Use Committee of Gilead Sciences
(Fremont, CA). New Zealand White female rabbits, weighing 2.5−3.5
kg, were sedated then anesthetized using IM and IV injections,
respectively, of xylazine and ketamine. The heart was excised and
placed in a modified Krebs−Heinsleit (K−H) solution (pH 7.4, gassed
with 95% O2 and 5% CO2). The K−H solution contained (in mM):
118 NaCl, 2.8 KCl, 1.2 KH2PO4, 2.5 CaCl2, 0.5 MgSO4, 2.0 sodium
pyruvate, 5.5 glucose, 0.57 Na2EDTA, and 25 NaHCO3. The aorta was
cannulated and the heart was perfused by the method of Langendorff
with K−H solution warmed to 36.5 °C. To ensure capture during
pacing, the right atrial wall was partially removed and complete
atrioventricular block was induced by thermoablation of the
atrioventricular nodal area. A bipolar Teflon-coated electrode was
placed on the right ventricular septum to pace the heart. Electrical
stimuli, 2 ms in width and 3-fold threshold amplitude, were delivered
to the pacing electrode at a frequency of 1 Hz using EP-4 stimulator
(EP MedSystems, ST. JUDE MEDICAL). After initiation of
ventricular pacing, a 30−50 min period was allowed for equilibration
The heart was excised at the end of the experiment, and the LAD
was religated. The ischemic area was visualized by perfusing the heart
with 1% Evans blue in saline and calculated as a percentage of total
ventricular weight. Rabbits with ischemic area less than 10% or larger
than 25% were excluded from the analysis.
Experimental Protocol. Animals were randomly assigned to vehicle,
GS-compound groups. After 30 min of dosing, the heart was subjected
to 15 min of ischemia followed by 15 min of reperfusion.
Blood samples were collected at 1, 5, 10, 30, and 60 min after drug
administration for determination of plasma concentrations of the
compound. Plasma concentrations of GS compound were determined
using a high performance liquid chromatograph−tandem mass
spectrometric (LC/MS/MS).
The 20 dose-dependently prevented the ischemia-induced S−T
elevation. The area under curve (AUC) for the S−T segment height
K
DOI: 10.1021/acs.jmedchem.6b00939
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
was reduced (vs control) by 38% and 88% at 0.28 and 0.52 μM plasma
concentration of 20. At the plasma concentration levels studied, 20
had no significant effect on blood pressure (BP), heart rate (HR), and
ECG intervals prior to the ischemia.
Similarly, 4 dose-dependently prevented the ischemia-induced ST
elevation. The area under curve (AUC) for the S−T segment height
was reduced (vs control) by 55% and 93% at 0.25 and 0.5 μM,
respectively, and plasma concentration of compound of 4. At the
plasma concentration levels studied, compound 4 had no significant
effect on blood pressure (BP), heart rate (HR), and ECG intervals
prior to the ischemia.
selectivity panel results, methoxyamine−clofilium in-
duced VT−VF model with 4, mexilitine data in a LCX
ligation model of VT−VF, details of homology model
including sequence analysis of sodium channel isoforms
(PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: 650-522-6360. E-mail: Jeff.Zablocki@Gilead.com.
■
Ischemia-Induced Arrhythmia by LCX Ligation in Rabbits.
Female New Zealand rabbits (3.0−4.0 kg) were purchased from
Western Oregon Rabbit Company. Animals were housed on a 12 h
light and dark cycle and received standard laboratory chow and water
ad libitum. All experiments were performed in accordance with the
Guide for the Care and Use of Laboratory Animals published by
National Research Council and with the experimental protocol
approved by the Institutional Animal Care Committee of Gilead
Sciences, Inc. Rabbits were anesthetized with sodium pentobarbital
given as an intravenous injection of 30 mg/kg followed by IV infusion
at 15 mg/kg/h. A tracheotomy was performed, and the trachea was
intubated with an endotracheal tube. The animal was ventilated with
room air supplemented with oxygen using a pressure control animal
ventilator (Kent Scientific Corp., Torrington, CT) at a respiratory rate
of 40 strokes/min and peak inspiration pressure of 10 mmH2O, which
was adjusted to keep blood gases and pH within the physiological
range (iSTAT clinic analyzer; Heska Corp., Waukesha, WI). The left
femoral artery was cannulated for the measurement of blood pressure
(BP). Blood samples were also withdrawn from femoral artery. The
right external jugular vein was cannulated for drug/vehicle
administration. Needle electrodes were inserted subcutaneously into
the limbs for recording of the surface electrocardiogram (ECG). The
heart was exposed via an incision at the fourth intercostal space (fourth
and/or fifth ribs were cut for a clear surgical vision). The chest was
opened, and a pericardial cradle was formed using four retractors. A
coronary artery occluder, comprised of a snare made of 5 cm PE-10
tubing with a 6−0 prolene polypropylene suture in it, was placed
loosely around the LCX 2−3 mm from its origin. The body
temperature of the rabbit was monitored via a rectal thermometer
and maintained between 37 and 40 °C by adjusting the surface
temperature of the surgical table. Regional ischemia (30 min) was
induced by ligating the LCX. BP and ECG were monitored and
recorded continuously using a computerized data acquisition system
(PowerLab; AD Instruments, Mountain View, CA). Incidences of
ventricular tachycardia (VT) and ventricular fibrillation (VF) were
examined during the ischemia period.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank John Shryock and Lin Wu for helpful discussions. All
authors were fully supported by Gilead Sciences Inc. with no
external funding of the research described.
ABBREVIATIONS USED
■
Å, angstrom; br s, broad singlet; CACO-2, heterogeneous
human epithelial colorectal adenocarcinoma; CNS, central
nervous system; Cyp, cytochrome P450; d, doublet; hERG,
̀
human Ether-a-go-go-related gene; HCM, hypertrophic cardio-
myopathy; HEK-293, human embryonic kidney cell; (Hz),
hertz; IHD, ischemic heart disease; IC50, inhibitory concen-
tration50; IH assay, isolated heart assay; late INa, late sodium
current; LAD, left anteriorior descending; LQT-3 syndrome,
long QT-3 syndrome; MAPD90, monophasic action potential
duration90; MARISA study, Monotherapy Assessment of
Ranolazine in Stable Angina Study; ms, milliseconds; m,
multiplet; NMR, nuclear magnetic resonance; peak INa current,
peak cardiac sodium current; ppm, parts per million; q, quartet;
ROS, reactive oxygen species; ATX-II, sea anemone toxin II;
SAR, structure−activity relationship; rabbit ΔS−T, rabbit
change in S−T segment elevation during ischemia; s, singlet;
SA, sinoatrial; TdP, Torsades des Pointes; t, triplet; US, United
States; VT−VF, ventricular tachycardia−ventricular fibrillation;
Vss, volume steady state
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* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00939.
Experimental for compounds 22−40, additional pharma-
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