Discovery of triazolopyridinone GS-462808, a late sodium current inhibitor (Late INai) of the cardiac Nav1.5 channel with improved efficacy and potency relative to ranolazine

Article abstract of DOI:10.1016/j.bmcl.2016.03.096

Previously we disclosed the discovery of potent Late I_Na current inhibitor 2 (GS-458967, IC₅₀ of 333 nM) that has a good separation of late versus peak Na_v1.5 current, but did not have a favorable CNS safety window due to high brain penetration (3-fold higher partitioning into brain vs plasma) coupled with potent inhibition of brain sodium channel isoforms (Na_v1.1, 1.2, 1.3). We increased the polar surface area from 50 to 84 ?² by adding a carbonyl to the core and an oxadiazole ring resulting in 3 GS-462808 that had lower brain penetration and serendipitously lower activity at the brain isoforms. Compound 3 has an improved CNS window (>20 rat and dog) relative to 2, and improved anti-ischemic potency relative to ranolazine. The development of 3 was not pursued due to liver lesions in 7 day rat toxicology studies.

Full text of DOI:10.1016/j.bmcl.2016.03.096

Accepted Manuscript
Discovery of Triazolopyridinone GS-462808, a Late Sodium Current Inhibitor
(Late INai) of the Cardiac NaV1.5 Channel with Improved Efficacy and Potency
Relative to Ranolazine
Dmitry O. Koltun, Eric Q. Parkhill, Elfatih Elzein, Tetsuya Kobayashi, Robert
H. Jiang, Xiaofen Li, Thao D. Perry, Belem Avila, Wei-Qun Wang, Ryoko
Hirakawa, Catherine Smith-Maxwell, Lin Wu, Arvinder K. Dhalla, Sridharan
Rajamani, Nevena Mollova, Brian Stafford, Jennifer Tang, Luiz Belardinelli,
Jeff A. Zablocki
PII:
S0960-894X(16)30328-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.096
BMCL 23739
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 February 2016
24 March 2016
25 March 2016
Please cite this article as: Koltun, D.O., Parkhill, E.Q., Elzein, E., Kobayashi, T., Jiang, R.H., Li, X., Perry, T.D.,
Avila, B., Wang, W-Q., Hirakawa, R., Smith-Maxwell, C., Wu, L., Dhalla, A.K., Rajamani, S., Mollova, N.,
Stafford, B., Tang, J., Belardinelli, L., Zablocki, J.A., Discovery of Triazolopyridinone GS-462808, a Late Sodium
Current Inhibitor (Late INai) of the Cardiac NaV1.5 Channel with Improved Efficacy and Potency Relative to
Ranolazine, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.03.096
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Bioorganic & Medicinal Chemistry Letters
Discovery of Triazolopyridinone GS-462808, a Late Sodium Current Inhibitor
(Late INai ) of the Cardiac NaV1.5 Channel with Improved Efficacy and Potency
Relative to Ranolazine
Dmitry O. Koltun*a, Eric Q. Parkhilla, Elfatih Elzeina, Tetsuya Kobayashia, Robert H. Jianga, Xiaofen Lia,
Thao D. Perrya, Belem Avilaa, Wei-Qun Wang,b Ryoko Hirakawa,b Catherine Smith-Maxwell,b Lin Wu,b
Arvinder K Dhalla,b Sridharan Rajamani,b Nevena Mollova,c Brian Stafford,c Jennifer Tang,c Luiz
Belardinelli,b Jeff A. Zablocki.*a
aDepartment of Medicinal Chemistry Gilead Sciences Inc., 333 Lakeside Drive, Foster City, CA, 94404, USA
bDepartment of Biological Sciences, Gilead Sciences Inc., 333 , Fremont, CA, 94, USA
cDepartment of Drug Metabolism, Gilead Sciences Inc., 333 Lakeside Drive, Foster City, CA, 94404, USA
ARTICLE INFO
ABSTRACT
Previously we disclosed the discovery of potent Late INa current inhibitor 2 (GS-458967, IC50 of 333
nM) that has a good separation of late vs. peak Nav1.5 current, but did not have a favorable CNS
safety window due to high brain penetration (3-fold higher partitioning into brain vs. plasma) coupled
with potent inhibition of brain sodium channel isoforms (Nav1.1, 1.2, 1.3). We increased the polar
surface area from 50 to 84 Å2 by adding a carbonyl to the core and an oxadiazole ring resulting in 3
GS-462808 that had lower brain penetration and serendipitously lower activity at the brain isoforms.
Compound 3 has an improved CNS window (>20 rat and dog) relative to 2, and improved anti-
ischemic potency relative to ranolazine. The development of 3 was not pursued due to liver lesions in
7 day rat toxicology studies.
Article history:
Received
Revised
Accepted
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords: Late INa current inhibitor; GS-
462808; anti-ischemic; ranolazine; angina.
Atherosclerotic narrowing of coronary vessels leads
(e.g. IKr ).6 Ranolazine provides its antianginal benefit without
causing bradycardia (slowing of heart rate) and/or lowering
systemic blood pressure.6 Ranolazine has been shown to
inhibit S-T segment elevation in pre-clinical models of
ischemia that correlates well with it’s ability to inhibit S-T
segment elevation in humans with angina undergoing exercise
treadmill testing (MARISA study).7 We describe our efforts
to identify a 2nd generation Late INa inhibitor with improved
properties relative to ranolazine with less hERG and -
blocking activity.
to ischemic heart disease (IHD) that has a high prevalence
(7%) within the United States (US).1 The initial stage of IHD
is silent ischemia that often progresses to more severe
ischemia that causes chest pain (angina), a condition that
afflicts 9 million people in the US.2 Ranolazine 1 was
approved for the treatment of chronic angina in the US in
2006.3 At therapeutic concentrations, ranolazine does inhibit
cardiac late sodium current, although the mechanism of
ranolazine’s antianginal effect has not been determined. In an
ischemic state, reactive oxygen species (ROS) can modify the
cardiac sodium channel resulting in incomplete inactivation
with repetitive opening of the inactivation gate resulting in a
persistent sodium current that occurs late in the action
potential, thus termed Late INa current (Figure 1).4 Late INa
current leads to sodium overload in the cytoplasm and
subsequent Ca+2 overload through activity of the reverse mode
Na-Ca+2 exchanger, and ranolazine inhibits this process
thereby lowering the ischemic burden (calcium imbalance and
sequelae).5 At its therapeutic concentration (2 – 8 M in
plasma), ranolazine inhibits a number of cardiac ion currents
Evidence provided through site directed mutagenesis studies
suggest that ranolazine binds to the lidocaine binding site in the
mouth of the cardiac sodium channel pore.8 Late INa current can
be generated in vitro by the addition of toxins ATX-II or
tefluthrin that bind to an external binding site that does not
overlap with the lidocaine binding site (Figure 1).9 Ranolazine
(1, Figure 2) has an IC50 6.9 M for ATX-II induced Late INa
inhibition (single cell manual patch).6
Peak INa current is responsible for the propagation of the action
potential from the pacemaker sinoatrial (SA) node through the

atria and ventricles, and it is critical to have a good separation of
Late INa inhibition from peak INa current. Ranolazine does not
inhibit peak INa current at therapeutic levels with a separation of
>50 fold.6
triazolone core using base (potassium carbonate) and alkyl
halides in an inert solvent (N,N-dimethylacetamide) by heating to
110 C for 2 h. 6-Bromo-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-
one
core was prepared from the corresponding 2-
hydrazinopyridine by reacting with carbonyldiimidazole (1.1
equivalents) in acetonitrile at reflux temperatures for 2 hours.
Figure 3. Preparation of 3.
In optimization of our compounds to widen the CNS
margin, we added the brain Nav 1.1 to our testing paradigm with
stimulation at 10 Hz frequency. Lead compounds were tested at
Nav 1.1, 1.2, and 1.3, but we found that activity for Nav 1.1
inhibition correlated well with activity at Nav 1.2 and 1.3, so we
screened against Nav 1.1. Our testing paradigm starts with
screening of our synthetic analogs for Late INa inhibition at 10
M concentration. With a few exceptions, compounds exhibiting
70% or greater inhibition at 10 M were subsequently tested to
determine an IC50. Selected compounds were then counter-
screened against Peak INa inhibition at concentration close to their
50% block of Late INa – typically 1 M. The screening for Peak
INa inhibition was performed at a pacing frequency 3 Hz,
corresponding to 180 beats per minute.
Figure 1. Late sodium channel current (Late INa) in a control
experiment (a), or enhanced with aid of 1 nM (b) or 3 nM (c)
ATX-II.
Herein, we describe our efforts to discover a potent Late
INa current blocker selective against peak current, and
demonstrate the anti-arrhythmic effects of our lead molecule 3 in
isolated heart and in vivo ventricular arrhythmia models,
respectively. Previously, we screened a number of in-house
heterocyclic compounds without a basic group, to decrease the
likelihood of hERG and -blockade, for their Late INa inhibitory
activity using automatic patch clamp system (hNav1.5 -subunit
HEK-293) that led to the discovery of triazolopyridine 2 (Figure
2).10 The potent Late INa current inhibitor 2 (IC50 of 333 nM)
has a good separation of Late INa current inhibition from peak
sodium current, but does not have a favorable CNS window due
to high brain penetration (brain to plasma 3:1 partitioning) and
high activity at brain sodium isoforms (Nav1.1, 1.2, 1.3). We
designed compound 3 with an increased polar surface area from
50 to 84 Å2 by adding a carbonyl to the core and an oxadiazole
ring with the hope of lowering brain penetration. We will
describe the SAR leading to the discovery of [1,2,4]triazolo[4,3-
a]pyridin-3(2H)-one 3 and it’s improved properties relative to
2.11
Table 1. Sodium channel blocking properties of 4-
trifluoromethoxyphenyl-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one
Late Na IC50
Peak Nav1.5 block, % (M)
R
H
Mic. T1/2
h:r:d
308:198:236
3 Hz
Nav1.5
10(10)
10 Hz
Nav1.1
8(10)
M
1.9
3
1.9
3.3
390:89:190
395:363:NT
24 (10)
10(10)
7
8
27(10)
28(10)
2.5
144:41:160
9
>10
4.0
10
11
3.5
12
>10
2.5
13
14
288:24:228
39:39:108
44(10)
Figure 2. Ranolazine (1), potent Late INa inhibitor (GS-458967 ,
2), and selective Late INa inhibitor (GS-462808, 3).
15
3.0
In general, the compounds were prepared via Suzuki coupling
using
a
palladium
catalyst,
for
example
[1,1'-
Initially, we fixed the 4-trifluoromethoxy phenyl C ring
bis(diphenylphosphino)ferrocene]palladium(II) dichloride,
a
and varied the D ring in our SAR studies on Late INa current
inhibition versus peak sodium current and Nav 1.1. The results
of our study are shown in Table 1. Early on in our SAR studies,
we prepared the 2-((3-methyl-1,2,4-oxadiazol-5-yl)methyl) D-
ring 3 that afforded acceptable Late INa inhibition (IC50 = 1.9
bromo core molecule, and an appropriately substituted boronic
acid derivative of formula R-Ar-B(OH)2 in the presence of a base
(potassium carbonate) using an inert solvent (degassed 2:1:1
toluene:isopropanol:water) at a temperature of 95 C, typically
for 3 days (3, Figure 3).11 Subsequently we alkylated the

longer QT interval. Long QT-3 patients have mutations in their
sodium channel that leads to improper inactivation of the channel
where it repetitively opens and closes affording a persistent
current late in the action potential duration.
M), a good separation from Peak INa inhibition, favorable
metabolic stability, and most importantly lower activity at the
brain isoform Nav 1.1. The unsubstituted triazolone core 7
retained the Late INa inhibition and metabolic stability, but picked
up more Peak INa inhibition. The 2-((5-methyl-1,2,4-oxadiazol-3-
yl)methyl) analog 8 was slightly less active for Late INa
inhibition, and exhibited more Peak INa inhibition than 3.
Removing a nitrogen from the D ring of 3 as in oxazole 9 and
isoxazole 7, resulted in less Late INa inhibition. Also, replacing
the 5-methyl of 3 with the larger 5-ethyl 11 and 2,6-
dichlorophenyl 9, led to less Late INa inhibition. The 2-((1-
methyl-1H-1,2,4-triazol-3-yl)methyl) 13 shows the importance of
D-ring electronics as it was completely inactive, in spite of
having a similar steric shape as 3. We tried to improve on
oxazole 9 by either moving the methyl to the 5-position as in 14
or replacing with a cyclopropyl group 15, but both compounds
were inferior to 3 (Peak INa inhibition and metabolic stability,
respectively).
Ranolazine shortens the long QT interval in LQT3
patients that is consistent with Late INa inhibition. The ATX-II
isolated heart model is an ex vivo model of LQT3 patients. Then,
we determined the anti-arrhythmic effect of 3 in an in vivo model
of chemically induced ventricular arrhythmia (Figure 5). In
anesthitized rabbits we slowed the heart rate through the addition
of an -blocker (methoxyamine) followed by the addition of a
hERG blocker (clofilium) that induced the control animals to
have torsade des pointes (TdP).
Table 2. Sodium channel blocking properties of 4-phenoxyphenyl-
[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one
Next, we decided to explore the SAR of replacing the
trifluoromethoxy group with either a 4-fluorophenoxy or 4-
chlorophenoxy group using some of the favorable D-rings as
shown in Table 2. The direct analogs of 3, 4-fluorophenoxy 16
and 4-chlorophenoxy 21, had improved Late INa inhibition by 10
fold, but both compounds had too much Peak INa inhibition and
were less metabolically stable. The enhanced lipophilicity
(measured Log D from 3.0 to 3.4) in this region really had a
pronounced effect on Late INa inhibition, but 16 and 21 are not
coming off the ion channel fast enough resulting in Peak INa
inhibition. Compound 18 and 21 are direct analogs of 8, and
they were much more potent for Late INa inhibition, but both have
an unacceptable increase in Peak INa inhibition. Adding a polar
hydroxyl group as in 19 (measured Log D 2.8) lessened the Peak
INa inhibition substantially, but it was not metabolically stable.
Changing to 2-((5-cyclopropyl-1,3,4-oxadiazol-2-yl)methyl) D-
ring 17 enhanced metabolic stability, but it had too much Peak INa
inhibition.
Late Na IC50 nM
Peak Nav1.5 block, % (M)
or %
Inhib
nM
R
H
Mic. T1/2
h:r:d
127:85:217
3 Hz
10 Hz
Nav1.5
Nav1.1
215
33(0.3) NT
16
R1=F
1903
541
395:43:273
28 (1)
46(1)
40(10)
17
R1=F
207:145:167
65(10)
18
R1=F
981
740
134
81:32:181
395:395:287
45:34:94
21(10)
69(1)
47(1)
56(10)
56(10)
42(10)
19
R1=F
20
R1=F
21
R1=Cl
Compound 3 had the best overall properties with
respect to low Peak INa and Nav 1.1, so we continued to profile it.
In manual patch assays, 3 had 10 fold lower activity than 2 at the
brain isoforms when tested at 10 Hz (Table 3). 12,13 Subsequently,
this translated to a larger CNS margin in both rat and dog (>20x).
In addition, the increase in PSA for 3 relative to 2 may have
accounted for diminished brain levels (brain vs. plasma 1.5:1 vs
3:1 for 2, rat). The hERG IC50 was very similar for the two
compounds.
60%
@300
nM
395:390:354
40(1)
79(10)
22
R1=Cl
Table 3. Sodium channel blocking properties of triazolopyridine 2
versus triazolonepyridine 3 (Manual patch clamp).
We evaluated PK across the preclinical species, and we
found a good in vitro-in vivo correlation for clearance (Table 4).
The half-life was moderate in the monkey and rabbit, short in the
rat, and relatively long in the dog that displayed a larger volume
of distribution. Allometric scaling projected an 8 hour half-life
in humans for compound 3.
The oral bioavailability for
3 was good in rat and dog. The free fraction for 3 was similar
across the species in the 4-5% range.
Assay
LatLate Nav1.5 IC50 µM
Peak Nav 1.5 IC50 µM
@ 1 Hz / 3 Hz
2
0.25
3
1.33
>10 / >3
>10 / 0.4
>10 / >10
>10 / 4.8
Next, we looked at the anti-arrhythmic effects of our
lead molecule 3 compared to ranolazine in isolated heart and in
vivo Torsades des pointes model. In isolated rabbit hearts, we
add the toxin ATX-II to induce Late INa that prolongs the mean
action potential duration (MAPD90) by approximately 100 msec.
The addition of Late INa inhibitors ranolazine or 3 reverse the
ATX-II effects in the isolated heart, albeit to a different degree
and potency. Compound 3 reverses the ATX-II effect by 90%
with an IC50 of 1.2 M, but ranolazine provides only a partial
reversal by 60% with an IC50 of 16 M. Late INa prolongs the
MAPD90 in the isolated heart that in vivo would translate into a
Nav 1.1 IC50 µM
@ 1 Hz / 10 Hz
Nav 1.2 IC50 µM
@ 1 Hz / 10 Hz
>10 / 0.4
7.9
>10 / 4.2
8.1
hERG IC50 µM
Compound 3 completely protected against Tdp at 3 M.
Ranolazine was only partially protective at much higher
concentrations (14 uM) with 40% of the animals having Tdp.

and we still observed the liver lesion in the 7 day rat toxicology
studies. We found that glutathione added to the
[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one core of both 3 and it’s
oxazole analog; therefore, ending further exploration of this
series.
Table 4. In vitro ADME and in vivo pharmacokinetic properties of compound
3..
Clearance (L/h/kg)
Rat:
Dog:
C. Monkey:
Human:
In vitro (mic.)
0.52
In vivo
0.719
0.479
0.478
0.26
0.40
0.21
We designed 3 with an increased polar surface area (from 50
to 84) by adding a carbonyl to the core and an oxadiazole ring
with the hope of lowering brain penetration. We found that 3
had decreased brain penetration than the previous lead 2, and
serendipitously had lower activity at the brain isoforms.
Although we had established a wider CNS margin with 3, the
[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one core had liabilities in rat
toxicology studies. We will report our efforts to replace the core
in due course.
Rabbit:
0.422
Terminal Half Life (h)
Rat:
Dog:
Monkey:
Rabbit:
2.10
17.3
4.78
4.81
Vol. of dist. (L/kg)
Rat:
Dog:
C. Monkey:
Rabbit:
1.95
4.23
2.29
2.24
Acknowledgments
Bioavailability (F)
Rat:
Dog:
44.3%a
72.8%b
We would like to thank Manoj Desai and Gerry Rhodes for
their suggestions on executing the Late INa program.
Protein binding (%)
Rat:
Dog:
95.4
94.3
95.0
95.7
References and Notes
Human:
Female Rabbit:
1
Fang, J.; Shaw, K. M.; Keenan, N. L. Morbidity and mortality weekly
a (15/10/75: NMP/Solutol HS 15/water)
b (5%EtOH, 40%PEG, 55% Water)
report. 2011, 40, 1377.
2 Kochanek, K. D.;Xu, J.; Murphy, S. L.; Minin, A.O.; Kung, H. C.
National Vital Statistics Report 2012, 60 (3), 1.
3Banon D; Filion, K.B.; Budlovsky, T.; Franck, C.; Eisenberg, M. J. , Am J
Cardiol. 2014, 113, 1075.
4Zaza, A.; Belardinelli, L.; Shryock, J. C. Pharmacol. Ther. 2008, 119, 326.
5Le Grand, B.; Vie, B; Talmant, J.; Coraboeuf, E.; John, G. W. Am. J.
Physiol. 1995, 269, H533.
Figure 4. Reversal of ATX-II increase in MAPD90 by compound 3 (1.2 uM
IC50, orange) and ranolazine (16 uM IC50, red) in rabbit isolated heart.
6Antzelevitch, C.; Belardinelli, L. Zygmunt, A. C.; Burashnikov A.; Di
Diego, J. M.; Fish, J. M., Cordeiro, J. M.; Thomas, G. Circulation 2004, 110,
904.
0
-20
7 Chaitman, B. R.; Skettino, S. L.; Parker, J. O.;Hanley, P.; Meluzin, J.; Kuch,
J; Pepine, C. J., Wang, W.; Nelson, J. J.; Hebert, D. A.; Wolff, A. A. J Am
Coll Cardiol. 2004, 43, 1375.
-40
8 Fredj, S; Sampson, K. J.; Liu, H; Kass, R. S. Br J Pharmacol. 2006, 148,
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9 Catteral, W. A.; Goldin, A. L.; Waxman, S. G. Pharmacol Rev 2005, 57,
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10 Abelman, M.; Elzein, E.; Jiang, R.; Kalla, R.; Kobayashi, T.; Li, X. ;
Perry, T. WO Patent 056527, 2010.
0
10
100 1,000 10,000 100,000
11 Kobayashi, T.; Koltun, D.; Notte, G.; Parkhill, E.; Zablocki, J.; WO
003392, 2012.
Drug Concentration (nM)
12 Belardinelli, L.; Liu, G.; Smith-Maxwell, C.; Wang, W.Q.; El-Bizri, N.;
Hirakawa, R.; Karpinski, S.; Li, C. H.; Hu, L.; Li, X.J.; Crumb, W.; Wu, L.;
Koltun, D.; Zablocki, J.; Yao, L.; Dhalla, A. K.; Rajamani, S.; Shryock, J. C.
J. Pharmacol. Exp. Ther. 2013, 344, 23.
Figure 5. Protection from hERG induced ventricular arrhythmias by 3
13 1H NMR of 3 (δ, d6-DMSO, 400 MHz): 7.95 (dd, 1H) 7.54 (d, 2H), 7.40
(dd, 1H), 7.32 (d, 2H), 7.20 (dd, 1H) 5.43 (s, 2H), 2.40 (s, 3H). For
experimental details see supplement.
(GS-462808) in anesthetized rabbits.
We went on to study 3 in 7 day rat and dog toxicology studies.
We were gratified to find a much wider CNS margin (>20x)
based on projected human Cmax for 3 in both rat and dog, a
dramatic improvement from the CNS margin for the initial
compound 2 (5x rat, 3x dog). These results were consistent with
both lower brain penetration and lower activity at the brain
isoforms. Unfortunately, 3 led to liver lesions in the rat that were
initially thought to be due to cleavage of the oxadiazole ring.
Subsequently, we replaced the oxadiazole ring with an oxazole,

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Discovery of Triazolopyridinone GS-462808,
a Late Sodium Current Inhibitor (Late INai )
of the Cardiac NaV1.5 Channel with
Improved Efficacy and Potency Relative to
Ranolazine
Leave this area blank for abstract info.
Dmitry O. Koltun*a, Eric Q. Parkhilla, Elfatih Elzeina, Tetsuya Kobayashia, Robert H. Jianga, Xiaofen Lia, Thao D Perrya, Belem Avilaa, Wei-Qun Wang,b
Ryoko Hirakawa,b Catherine Smith-Maxwell,b Lin Wu,b Arvinder K Dhalla,b Sridharan Rajamani,b Nevena Mollova,c Brian Stafford,c Jennifer Tang,c
Luiz Belardinelli,b Jeff A. Zablocki.*a