Discovery of Tirasemtiv, the First Direct Fast Skeletal Muscle Troponin Activator

Article abstract of DOI:10.1021/acsmedchemlett.7b00546

The identification and optimization of the first activators of fast skeletal muscle are reported. Compound 1 was identified from high-throughput screening (HTS) and subsequently found to improve muscle function via interaction with the troponin complex. Optimization of 1 for potency, metabolic stability, and physical properties led to the discovery of tirasemtiv (25), which has been extensively characterized in clinical trials for the treatment of amyotrophic lateral sclerosis.

Full text of DOI:10.1021/acsmedchemlett.7b00546

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Letter
Discovery of Tirasemtiv, the First Direct
Fast Skeletal Muscle Troponin Activator
Scott Collibee, Gustave Bergnes, Alexander R. Muci, William F Browne, Aaron C Hinken,
Alan J Russell, Ion Suehiro, James Hartman, Raja Kawas, Pu-Ping Lu, Kenneth H. Lee,
David Marquez, Matthew Tomlinson, Donghong Xu, Adam Kennedy, Darren Hwee, Julia
Schaletzky, Kwan H. Leung, Fady I. Malik, David J. Morgans, and Bradley P. Morgan
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00546 • Publication Date (Web): 13 Feb 2018
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Discovery of Tirasemtiv, the First Direct Fast Skeletal Muscle Troponin
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Activator.
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Scott Collibee,* Gustave Bergnes, Alexander Muci, William F. Browne IV,↑ Aaron C. Hinken,○ Alan J.
Russell,‡ Ion Suehiro, James Hartman, Raja Kawas, Pu-Ping Lu, Kenneth H. Lee, David Marquez,
Matthew Tomlinson,δ Donghong Xu, Adam Kennedy, Darren Hwee, Julia Schaletzky, Kwan Leung,
Fady I. Malik, David J. Morgans, Jr.,# and Bradley P. Morgan
Cytokinetics, Inc., 280 East Grand Avenue, South San Francisco, California 94080
KEYWORDS amyotrophic lateral sclerosis (ALS), skeletal muscle activator, troponin, Tirasemtiv, imidazo[4,5-b]pyrazin-2-one
This paper is dedicated to the memory of Alex R. Muci, a significant contributor to this research effort.
ABSTRACT: The identification and optimization of the first activators of fast skeletal muscle are reported. Compound 1 was
identified from high-throughput screening (HTS) and subsequently found to improve muscle function via interaction with
the troponin complex. Optimization of 1 for potency, metabolic stability and physical properties led to the discovery of
tirasemtiv (25), which has been extensively characterized in clinical trials for the treatment of ALS.
Direct activation of skeletal muscle has the potential to im-
prove physical performance in patients with compromised mus-
cle function, such as those suffering from amyotrophic lateral
sclerosis (ALS),1 spinal muscular atrophy2, and Charcot-Marie-
Tooth disease.3 Muscle impairment in these disease states oc-
curs from diminished neuromuscular input and can lead to disa-
bility and increased mortality.4 Current treatment options are
limited or nonexistent and represent a significant unmet medical
need.5-7 Precedent for activation of muscle function exists with
omecamtiv mecarbil, a direct cardiac muscle activator that im-
proves contractility and is undergoing clinical trials for the
treatment of heart failure.8-9 This report describes the discovery
and optimization of the first direct activators of fast skeletal
muscle, culminating in the discovery of tirasemtiv (25), a com-
pound that was found to selectively sensitize the sarcomere of
fast skeletal muscle to calcium and increase its force production
via interaction with the regulatory troponin complex.10
Skeletal muscle is classified into two main forms, fast skeletal
muscle (Type II fibers) and slow skeletal muscle (Type I), which
differ in usage, metabolism and sarcomere composition. Fast
skeletal muscle fibers have unique myosin and troponin compo-
nents compared to cardiac and slow skeletal muscle, while slow
skeletal muscle fibers have the same myosin and troponin C
(TnC, a subunit of the regulatory complex) as cardiac muscle.
Myosin is the molecular motor responsible for contractility
where chemical energy from adenosine triphosphate (ATP)
hydrolysis is coupled to force generation. The troponin complex
plays a regulatory role by acting as a calcium sensor and resides
in the thin filament along with actin and tropomyosin. The se-
quence homology between fast and slow muscle myosin and
troponins C, T and I is about 50%, providing a good opportunity
to find selective skeletal muscle troponin activators.11 Because
skeletal muscle is mainly responsible for voluntary movement
and has unique myosin and troponin components compared to
other muscle types, fast skeletal muscle represents a compelling
target for small molecule intervention.
Table 1. Activity and selectivity of HTS hit and close-in ana-
logs.
FSK
AC40
(µM)
7
>49
>49
CV
SSK
AC40
(µM)
>39
-
#
R
AC40
(µM)
>49
-
1
2
3
S-Me
R-Me
H
-
-
FSK = Fast Skeletal Muscle; CV = Cardiac Muscle; SSK = Slow
Skeletal Muscle
Compounds with the potential to modulate human muscle
function were identified using a high-throughput screen (HTS)
that measures the rate of ATP hydrolysis in rabbit fast skeletal
muscle myofibrils as a surrogate for force generation in skeletal
muscle.12 Biochemical activity (AC40) is defined as the com-
pound concentration that produces a 40% increase in myofibril
ATPase activity at 25% of the calcium concentration needed for
a maximal response in control preparations. Among the hits was
imidazopyrazinone 1 (CK-1303249). Compound 1 displayed
modest biochemical potency (AC40 = 7.0 µM), ligand and lipo-
philic efficiencies (LE and LipE) of 0.32 and 5.01, respectively,13-
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14
and specificity for fast skeletal muscle over slow skeletal,
pounds, DF30 is defined as the compound concentration that
1
2
3
4
5
6
7
8
cardiac, and smooth muscle (Table 1, data not shown for smooth
muscle). Additionally, 1 showed a eudismic ratio (> 7) that is
suggestive of a specific interaction between the molecule and a
biological target (1 vs. 2 in Table 1). To determine whether
activation occurs via the myosin or regulatory complex, mixed
sarcomeres were created using myosin and thin filament prepa-
rations from fast, slow, and cardiac muscle sources, and each
was then assayed in the presence of 1. Because activation was
observed only in combinations containing fast skeletal thin fila-
ments, 1 was determined to be a direct troponin complex activa-
tor.10
produces a 30% increase in tension at 25% of the calcium con-
centration needed for a maximal response in control prepara-
tions.
The initial optimization goals were based loosely on our
previous experience with the optimization of cardiac muscle
activators.9 The biochemical potency needed for compounds
to show physiologically significant cardiac muscle activation
was between 0.1 - 5 µM. The desired pharmacokinetic profile
would ensure that efficacious concentrations could be sus-
tained by dosing orally once or twice per day. Therefore, the
9
target compound profile included biochemical potency (AC40
)
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24
25
26
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less than 1 µM, minimal in vitro clearance and good in vivo
exposure with oral dosing.
DMSO 1%
Synthesis of the imidazo[4,5-b]pyrazin-2-one analogs of 1
was achieved using Scheme 1. Addition of primary amines to 2-
amino-3,5-halopyrazine (31) gave diaminopyrazines 32 in good
yields, followed by ring closure using carbonyl diimidazole (CDI)
to afford imidazo[4,5-b]pyrazin-2-ones 33. The targeted ana-
logs were produced in one or two steps from 34.16
CK1303249 100uM
A
CK1303249 30uM
CK1303249 10uM
120
100
80
60
40
20
0
8
7
6
5
4
pCa
DMSO 1%
B
CK1303249 100uM
120
100
80
60
40
20
0
Scheme 1. Synthesis of imidazopyrazinone derivatives: a)
NHR1. b) CDI, THF. c) (R2=furyl or phenyl) R2B(OH)3,
Cl2Pd(dppf), Na2CO3, dioxane/water. d) (R2=SMe) NaSMe, NMP.
e) (R2=OMe) NaOMe, NMP. f) (R2=CN) Zn(CN)2, (Ph3P)4Pd, DMF.
g) (R2=Ac) tributyl(1-ethoxyvinyl)tin, Cl2Pd(dppf), dioxane. h)
(R2=H) H2, Pd/C, EtOH. i) (R2=Me): trimethylboroxine,
Cl2Pd(dppf), Na2CO3, dioxane/water. j) (R2 = H3CC≡C) H3CC≡CH,
Cl2Pd(dppf), CuI, TEA, dioxane/water. k) (R2 = HC≡C) l) 1.
TMSC≡CH, Cl2Pd(dppf), CuI, TEA, dioxane/water; 2. KF,
8
7
6
5
4
MeOH/THF/water. m) (R2
=
vinyl) trimethylboroxine,
pCa
Cl2Pd(dppf), Na2CO3, dioxane/water. n) (R2 = Et) from R2 = vinyl;
H2, Pd/C, EtOH.
Figure 1. The calcium-force relationship of 1 on skinned
skeletal fibers from (A) rabbit psoas muscle (fast) and (B) rabbit
semimembranosus muscle (slow) plotted as % of maximal ten-
sion vs. (-)log of calcium concentration (pCa)
Having established calcium sensitization in isolated muscle
fibers, we were interested in demonstrating improved functional
activity in an animal model. We initially focused on replacing for
the furan in 1 to improve the metabolic stability and physical
properties while exploring the structure activity relationship
(SAR) at that position. Overall, the tolerance for substitution at
R2 was found to be somewhat limited (Table 2). The observed
potency improvement with vinyl (5) but not phenyl (4) illustrat-
ed the limited space in that region and hinted at the potential for
more potent, lower molecular weight structures. The combina-
tion of reduced potency with ethyl 6 and methoxy 7, but im-
proved potency with SMe 8 and bromo 9, suggested a preference
for polarizable electron rich groups. In contrast, analogs with
greater polarity (10-12) were much less active, pointing to in-
tolerance for certain polar interactions at this position.
The observed increase in ATP hydrolysis in the presence of 1
was subsequently shown using the skinned fiber assay to trans-
late to improved force generation in a skinned, fast muscle fiber
(Figure 1).15 This assay is performed by mounting a muscle fiber
(pretreated with detergent to remove the membranes and cyto-
plasm) to a force transducer rig to enable force measurement
across the muscle. Treatment of primarily fast rabbit psoas
muscle fibers with 1 increased the calcium sensitivity of the
muscle fibers without increasing the maximum force or the
shape of the curve (Figure 1A). A related control experiment
using skinned, primarily slow, rabbit semimembranosus muscle
fibers failed to show increased calcium sensitivity in the pres-
ence of 1 (Figure 1B). For potency comparison across com-
The SAR around the N1-position (R1) was examined in the
context of a bromine substituent at the 6-position (Table 3). The
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focus was on defining a minimum pharmacophore at this posi-
generally preferred in this position, as exemplified by the poten-
cy loss observed when bromine (14) or chloro (21) substituent
was replaced with a trifluoromethyl (22) or cyano (27) group.
Vinyl-analog 24 was among the more potent compounds, but the
strongest preference was for an ethynyl group (25, CK-
2017357), which was 3-fold more potent than bromo 14. Nota-
bly, propynyl analog 26 was much less active, again indicating a
steric limitation in that part of the binding pocket. The micro-
somal stability of 14, 21, and 25 were roughly equivalent, with
25 displaying the most promising combination of potency and
metabolic stability.
1
2
3
4
5
6
7
8
tion and reducing oxidative potential for the series (as measured
from microsome clearance experiments).17 Replacement of the
phenyl group from the methylbenzyl substituent of 9 with an
ethyl group (13) maintained potency and displayed improved
stability in rat and human microsomes. A 3-pentyl group (14)
provided a further 3-fold potency improvement in biochemical
potency, excellent functional activity in the skinned fiber assay,
eliminated stereochemistry from the molecule, and provided
similar microsomal stability to 13. Isopropyl compound 15 lost
significant potency compared to 14. The t-butyl substituent of
16 recovered much of the biochemical potency and presented a
favorable microsomal profile, but the activity in skinned fibers
was higher than expected based on its biochemical potency.
Interestingly, cyclic R1 substituents such as cyclopropyl 17 and
cyclohexyl 18 had significantly lower potencies, indicating a
strong conformational preference. These results pointed to the
3-pentyl group as the optimal substituent at this position based
on its potency profile and stability in human liver microsomes.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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Table 4. Assessment of R2, N-1 Methylbenzyl Analogs
AC40
(µM)
14.0
2.0
0.5
0.3
11.3
2.2
1.1
0.1
>49
36.3
DF30
(µM)
NT
4.4
0.6
0.4
NT
Re
(r)
NT
Re
(h)
NT
#
R2
19
20
21
14
22
23
24
25
26
27
H
Me
Cl
Br
Table 2. Assessment of R2, N-1 Methylbenzyl Analogs
0.7
0.4
0.4
0.2
>0.7
>0.7
0.3
NT
<0.2
<0.2
<0.2
<0.2
<0.2
0.5
<0.2
NT
<0.2
CF3
OMe
CH=CH2
C≡CH
C≡CCH3
CN
NT
1.1
0.3
NT
AC40
(µM)
DF30
(µM)
#
R2
1
4
5
6
7
8
9
10
11
12
2-Furyl
Ph
CH=CH2
CH2CH3
OMe
SMe
Br
SO2Me
CN
COMe
7.0
>49
3.5
21.8
8.9
2.6
30.6
NT
4.6
NT
NT
3.8
3.0
NT
NT
NT
NT
<0.2
The importance of the hydrogen bond donor/acceptor re-
gion of 25 to biochemical potency is highlighted in Figure 2.
Compound 25 may exist as a mixture of tautomers in solution
with 25a.18 Removal of the H-bond donor by either isosteric
replacement of nitrogen with oxygen (29) or methylation at
nitrogen (28) or oxygen (30) led to a dramatic reduction in
potency.
1.6
>49
>49
>49
Table 3. Assessment of N-1 position (R1)
AC40
(µM)
DF30
(µM)
3.0
5.1
0.4a
2.2
4.6
NT
NT
Re
Re
(h)
<0.3
<0.2
<0.2
NT
0.2
NT
NT
#
R1
Figure 2. SAR assessment of the donor/acceptor region of 25.
(r)
0.5
0.3
0.4
NT
0.3
NT
NT
9
(S)-Me-benzyl 1.6
The rat pharmacokinetic profiles for compounds with opti-
mal potency and microsomal stability were assessed to de-
termine their suitability for in situ live fiber experiments (Ta-
ble 5). In rats, analogs 14, 21, and 25 all showed low to mod-
erate clearance values and good oral bioavailability when
dosed as crystalline suspensions. The free fraction (Fu) for 25
in both human and rat plasma was greater than 14 and 21.
Based on superior activity and oral free exposure, 25 was
selected for advanced in vivo testing.
13
14
15
16
17
18
(S)-2-butyl
3-pentyl
i-Pr
t-butyl
cyclopropyl
cyclohexyl
1.0
0.3
6.0
0.8
>49
10
Re = Ratio of extraction in microsomes (r =rat, h = human) a14 was
tested multiple times in the skinned fiber assay (n=10, standard devia-
tion = 0.2)
The SAR exploration at the R2 position in the context of a 3-
pentyl R1 group revealed specific steric and electronic prefer-
ences (Table 4). Compact, electron rich lipophilic groups were
Table 5. Summary Rat Pharmacokinetic Results for 14, 21
and 25
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Compound 25 exhibited moderate inhibition and time-
1
2
3
4
5
6
7
8
dependent inhibition of CYP1A2 (IC50 = 20 µM, IC50 shift of >3
following a 30-minute preincubation).21 In clinical studies
involving ALS patients taking both 25 and Riluzole, a drug-
drug interaction (DDI) occurred that resulted in an approxi-
mate doubling of Riluzole concentrations.22 Riluzole is a
commonly prescribed drug for ALS that is cleared mainly by
CYP1A2 and CYP1A1 mediated metabolism.23 This DDI was
managed in the clinic by reducing the dose of Riluzole from
100 mg/day b.i.d. to 50 mg/day q.d.
CL
Vss
F
Fu (r, h)
(%)
1.3, -0.5
1.1, 0.8
6.9, 1.9
#.
R2
(mL/min/kg) (L/Kg) (%)
14 Br
21 Cl
25 C≡CH
19.0
7.7
2.6
6.5
3.4
2.6
62
100
98
Fu(r) or Fu(h): fraction unbound in rat (r) or human (h) plasma.
9
The effect of compound 25 (tirasemtiv) on muscle perfor-
mance has been evaluated in preclinical and clinical models.
In a mouse model of ALS with functional deficits (B6SJL-
SOD1G93A), a single dose of tirasemtiv significantly increased
submaximal isometric force, forelimb grip strength, grid hang
time, and rotarod performance.24 Tirasemtiv also increased
grip strength in rats treated with the anti-nAchRα antibody in
the PT-EAMG (passive transfer experimental autoimmune
myasthenia gravis) rat model.10 In a clinical study performed
in healthy humans, tirasemtiv increased force produced by the
tibialis anterior in a dose, concentration, and frequency de-
pendent manner with the largest increases produced at sub-
tetanic nerve stimulation frequencies.25 This effect was simi-
lar to what was observed in rat EDL studies.
The sensitization of fast skeletal muscle to calcium in the
presence of 25 was demonstrated using an in situ preparation
of the extensor digitorum longus (EDL) muscle in a rat model
where the nerve and the blood supply are intact.19 Systemic
infusion of 25 resulted in rapid, dose-dependent increases in
muscle force at sub-maximal stimulation rates (Fig. 2) with-
out increasing the maximum tension at tetanic stimulation
rates. This result was consistent with the effects of 25 on the
force-calcium relationship of skinned rabbit psoas muscle
fibers and the increase in ATPase rate (a surrogate for force
production) in the biochemical assay.
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25
26
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In conclusion, HTS hit imidazopyrazinone 1 was identified
using a fast skeletal ATPase assay and demonstrated im-
proved calcium sensitivity and force production in muscle
fibers. To enable in vivo pharmacological testing, 1 was opti-
mized for biochemical potency and oral exposure, leading to
compound 25 (tirasemtiv). Upon intravenous administration
in preclinical models, tirasemtiv showed a concentration de-
pendent improvement in muscle force production in situ. In a
Phase 3 clinical trial (VITALITY-ALS, NCT02496767) in pa-
tients with ALS, tirasemtiv did not meet its primary endpoint,
change from baseline in slow vital capacity (SVC) at 24 weeks,
or any of the secondary endpoints. Though not reaching sta-
tistical significance, the decline in SVC was smaller in patients
who received any dose of tirasemtiv compared to patients
receiving placebo. The largest differences from placebo were
observed in patients randomized to the mid- and high-dose
groups of tirasemtiv who could tolerate and remain on their
target dose, suggestive of pharmacologic activity for the
mechanism of action in ALS. More patients discontinued dou-
ble-blind treatment on tirasemtiv than on placebo primarily
due to non-serious adverse events related to tolerability, po-
tentially confounding the assessment of its efficacy. The limi-
tations of tirasemtiv may be addressed with CK-2127107, the
next-generation fast skeletal muscle troponin activator cur-
rently being assessed in several mid-stage clinical trials.26
Figure 2. Rat in situ EDL muscle plasma concentration-
force response curve with 25 (CK-20171357).
To support pharmacology and safety studies as well as human
PK and dose projections, a pharmacokinetic assessment of 25
was performed (Table 6). In a Caco-2 cell monolayer system, 25
demonstrated high permeability (permeability coefficient of >50
x 10-6 cm/sec) and no apparent efflux. It showed low to moder-
ate liver microsomal stability and relatively low in vivo clearance
across species with values less than 15% of hepatic blood flow.
Following oral administration, 25 was well absorbed with oral
bioavailability ranging from 43% in mice to greater than 90% in
rats and dogs. The low clearance and high bioavailability of 25
observed in nonclinical species are consistent with the high
exposure following oral administration of the clinical dose in
humans.20
SUPPORTING INFORMATION AVAILABLE
Experimental procedures and analytical data. This material is
available free of charge via the Internet at http://pubs.acs.org.
Table 6. In vitro and in vivo pharmacokinetics of 25
CL
Vss
F
(%)
-
93
98
43
AUTHOR INFORMATION
Species
Re
(mL/min/kg)) (L/kg)
Corresponding Author: * To whom correspondence should
be addressed. Tel: 650-624-3229. Fax: 650-624-3010. E-mail:
scollibee@cytokinetics.com.
Human
Dog
Rat
<0.21
<0.21
0.26
-
-
4.3
2.6
13.9
1.7
0.8
1.0
Mouse
0.24
Present Addresses
Re = Ratio of extraction in microsomes, PK dose = 1 mg/kg
○ For ACH: GlaxoSmithKline, 709 Swedeland Road, King of
Prussia, PA 19406
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‡For AJR: Edgewise Therapeutics, 3415 Colorado Ave, Boul-
der, CO 80303
# For DJM: 700 Saginaw Drive, Suite 150, Redwood City, CA
94063
↑ For WFB IV: Cornell University Medical Center, 525 E 68th St
# STAR8, New York, NY, 10065
1
2
3
4
5
6
Δ For MT: BMS, 521 Cottonwood Dr, Milpitas, CA 95035
7
8
Notes
9
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
We thank all members of the much larger Skeletal Muscle
Program Team at Cytokinetics for their contributions.
ABBREVIATIONS
HTS, high-throughput screening; ALS, amyotrophic lateral
sclerosis; TnC, troponin C; ATP, adenosine triphosphate;
ATPase, enzymatic hydrolysis of adenosine triphosphate; FSK,
fast skeletal muscle; CV cardiovascular muscle; SSK, slow
skeletal muscle; LE, ligand efficiency; LipE, lipophilic efficien-
cy; SAR, structure-activity relationship; CYP, cytochrome P450
enzyme; EDL, extensor digitorum longus muscle; AC40, con-
centration that increases native ATPase function by 40%;
DF30, concentration that increases muscle fiber tension by
30%; Re(r) and Re(h), microsomal extraction ratio in rat (r) or
human (h) microsomes; Fb, fraction bound in plasma, (r, rat);
PK, pharmacokinetics; DDI, drug-drug interaction; SVC, slow
vital capacity.
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