Short-acting T-type calcium channel antagonists significantly modify sleep architecture in rodents

Article abstract of DOI:10.1021/ml100170e

A novel phenyl acetamide series of short-acting T-type calcium channel antagonists has been identified and evaluated using in vitro and in vivo assays. Heterocycle substitutions of the 4-position of the phenyl acetamides afforded potent and selective antagonists that exhibited desired short plasma half-lives across preclinical species. Lead compound TTA-A8 emerged as a compound with excellent in vivo efficacy as indicated by its significant modulation of rat sleep architecture in an EEG telemetry model, favorable pharmacokinetic properties, and excellent preclinical safety. TTA-A8 recently progressed into human clinical trials, and in line with our predictions, preliminary studies (n = 12) with a 20 mg oral dose afforded a high C_max of 1.82 ± 0.274 μM with an apparent terminal half-life of 3.0 ± 1.1 h.

Full text of DOI:10.1021/ml100170e

pubs.acs.org/acsmedchemlett
Short-Acting T-Type Calcium Channel Antagonists
Significantly Modify Sleep Architecture in Rodents
Zhi-Qiang Yang,*,† Kelly-Ann S. Schlegel,† Youheng Shu,† Thomas S. Reger,† Rowena Cube,†
Christa Mattern,† Paul J. Coleman,† Jim Small,† George D. Hartman,† Jeanine Ballard,‡
Cuyue Tang,‡ Yuhsin Kuo,‡ Thomayant Prueksaritanont,‡ Cindy E. Nuss,§ Scott Doran,§
Steve V. Fox,§ Susan L. Garson,§ Yuxing Li,§ Richard L. Kraus,§ Victor N. Uebele,§
Adekemi B. Taylor,‡ Wei Zeng,‡ Wei Fang,‡ Cynthia Chavez-Eng,‡ Matthew D. Troyer,
Julie Ann Luk, Tine Laethem, William O. Cook,^ John J. Renger,§ and James C. Barrow†
Departments of †Medicinal Chemistry, ‡Drug Metabolism and Pharmacokinetics, §Depression and Circadian Disorders,
Clinical Pharmacology, and ^Toxicology Sciences, Merck Research Laboratories, WP14-3, P.O. Box 4, 770 Sumneytown Pike,
West Point, Pennsylvania 19486
ABSTRACT A novel phenyl acetamide series of short-acting T-type calcium
channel antagonists has been identified and evaluated using in vitro and in vivo
assays. Heterocycle substitutions of the 4-position of the phenyl acetamides
afforded potent and selective antagonists that exhibited desired short plasma
half-lives across preclinical species. Lead compound TTA-A8 emerged as a com-
pound with excellent in vivo efficacy as indicated by its significant modulation of
rat sleep architecture in an EEG telemetry model, favorable pharmacokinetic
properties, and excellent preclinical safety. TTA-A8 recently progressed into human
clinical trials, and in line with our predictions, preliminary studies (n=12) with a
20 mg oral dose afforded a high Cmax of 1.82 ( 0.274 μM with an apparent terminal
half-life of 3.0 ( 1.1 h.
KEYWORDS Calcium channel antagonists, sleep, T-type calcium channels, electro-
corticogram, pharmacokinetics
oltage-gated calcium channels regulate the entry of
Ca2þ into cells in response to membrane depolariza-
tion. The consequences of Ca2þ influx include further
with hyperpolarization and slow-wave sleep (SWS).6,7 Thus,
we sought to identify selective and brain-penetrant T-type
channel blockers to evaluate how modulation of these
channels could impact the thalamocortical network and
vigilance state.
V
depolarization of the cell membrane, muscle contraction,
neurotransmitter release, and many other events.1 On the
basis of cloning of the main pore-forming R-subunit, they
can be divided into three main families: Cav1.x (L-type),
Cav2.x (N-, P/Q-, R-type), and Cav3.x (T-type).2 Recently, the
T-type calcium channel has attracted a lot of interest for
treatment of both peripheral and central nervous system
(CNS) disorders.3 Of the three T-type calcium channels, the
Cav3.1 and Cav3.3 subtypes are primarily expressed in the
brain, while Cav3.2 has a broader central and peripheral
expression.4 In the CNS, T-type calcium channels are highly
expressed in the thalamus and cortex regions and play imp-
ortant roles in thalamocortical signaling.5 The corticothala-
mic loop consists of cortical pyramidal, thalamic relay, and
reticular thalamic neurons whose resting membrane poten-
tial is modified by inputs from multiple neurotransmitter
systems such as histamine, noradrenaline, and serotonin, re-
sulting in control of the arousal state of the network. The
resting membrane potential and the state of the T-type cal-
cium channel determine one of two distinct network acti-
vities: tonic activity, which is associated with depolarization
and the wake state, or bursting activity, which is associated
Our laboratories have previously disclosed two structu-
rally distinct classes of T-type calcium channel antagonists:
piperidines 1 and 2 and quinazolinone 3 (Figure 1).8-10 Both
series showed good in vivo efficacy in epilepsy and tremor
models while having minimal effects on cardiovascular para-
meters.8,9 Moreover, with both series, we observed signifi-
cant effects on sleep architecture in rodent and rhesus
electrocorticogram (ECoG) models.10,11 More recently, we
disclosed another series of T-type antagonists exemplified
by phenyl acetamide 4.11,12,15 Similar to structurally distinct
piperidines 1 and 2 and quinazolinone 3, phenyl acetamide 4
exhibited robust effects on sleep architecture in rats, suggest-
ing the common involvement of T-type calcium channels.
These promising results led us to focus on identifying
compounds with shorter half-lives and good bioavailability,
Received Date: July 14, 2010
Accepted Date: August 18, 2010
Published on Web Date: August 24, 2010
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Figure 1. T-type calcium channel antagonists.
Scheme 1. General Synthetic Route to Heterocycle Amides 9
ideal for clinical evaluation as novel sleep agents. Here,
we wish to report compounds from the phenyl aceta-
mide class exhibiting desirable pharmacokinetic properties
(Figure 1).
Potency on the T-type calcium channel was determined by
a binding assay and two FLIPR assays designed to measure
potency on the inactivated state of the channel (depolarized
assay) and the resting state of the channel (hyperpolarized
assay) as previously described.15 All compounds were also
counter-screened against other ion channels, such as the
human ether-a-go-go potassium channel (hERG)16,17 and
L-type calcium channels.14 A group of representative exam-
ples containing diverse heterocycles are shown in Table 1,
including saturated and aromatic heterocyclic rings. Com-
paring the two FLIPR assays, we noted a greater than 80-fold
difference in potency depending on the resting membrane
potential of the test cell line. Previously, we have shown that
both state-independent piperidines 1 and 2 and state-
dependent quinazolinones such as 3 were efficacious in our
in vivo models, suggesting that binding to the inactivated state
of the channel is sufficient for pharmacological effects. As a
general trend, the phenyl acetamide class of compounds
displayed excellent potency on T-type calcium channels and
weak activity on hERG and L-type channels (IC50 values >
10 μM). As shown in Table 1, compound 10 bearing a 3-fluoro
pyrolidine is extremely potent with an IC50 value of 4 nM in
the depolarized FLIPR assay and a Ki value of 0.3 nM in the
binding affinity assay.18 The 4-methyl oxazole-containing
compound 11 and 3-substituted pyrazolo[1,5-b]pyridazine
During the discovery of compound 4,11 it was noted that
the trifluoroethoxypyridyl group is optimal for both potency
and metabolic stability, whereas heterocycles are well toler-
ated as a replacement of the cyclopropyl group. In addition,
heterocycle modifications generally provided compounds
with improved physical properties and minimal or no preg-
nane X receptor (PXR) activity. Activation of PXR could lead
to the induction of CYP3A enzymes and thus poses a poten-
tial risk of drug-drug interactions; therefore, minimization
of this activity is desirable.13 To this end, we developed an
efficient strategy to allow extensive screening of a wide array
of heterocycles. The general synthetic route was illustrated
in Scheme 1. Chiral amine 6 was synthesized from commer-
cially available 3-hydroxyl picoline 5 in 31% over six steps
with excellent stereoselectivity.12 Amine 6 was coupled to
either a 4-halo phenylacetic acid or 4-boronic ester phenyla-
cetic acid to give intermediate 7. Subsequent cross-coupling to
install the heterocycles afforded compounds 9. Alternatively,
para-heterocycle-substitutedphenylaceticacid(generalstruc-
ture 8) was constructed first and then coupled to amine 6 to
afford the final amides 9.
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Table 1. In Vitro and in Vivo Data for T-Type Calcium Channel Antagonists
aData presented as the mean ( standard deviation as described in reference 15. b Data presented as the mean ( standard deviation as described in
reference 15 or the average of n = 2 measurements. c Data are the average of n = 2 measurements. The assayis described in reference14. d Data are the
average of n = 2 measurements. The assay is described in reference 17. e Inhibition of seizure duration was calculated 4 h after 3 mpk oral dosing
relative to vehicle dosing on the previous day as an average of n = 2 rats. f Basolateral to apical/apical to basolateral transport ratio in human MDR1
transfected cells (see reference 23). g Percent response of PXR activation relative to Rifampicin at 10 μM (see reference 27). h Dog intravein cassettes
in beagles, 0.125 mpk dose in DMSO (n = 2).
Table 2. Pharmacokinetic Parameters of 11
analogue 12 also exhibited excellent binding affinity and
functional potency. A six-membered ring such as 2-methyl
pyrazine derivative 13 showed good potency with an IC50
value of 31 nM.
CLp
(mL/min/kg)
T1/2
(h)
Vss
(L/kg)
plasma
species
F (%)
PB (% bound)a
rat
35
0.4
1.1
1.1
0.3
100
50
5.2
1.6
To be effective in inhibiting central T-type channels in vivo,
compounds subjected to the efflux mediated by P-glycopro-
tein transporter (P-gp) should be avoided as this transporter
can often limit brain penetration.22 Despite introducing addi-
tional hydrogen bond acceptors into general structure 9,
which might limit brain penetration,23 compounds 10-13
showed low directional transport ratios (BA/AB < 2) in
human P-gp expressing cell line and maintaining excellent
cell permeability.24 To rapidly evaluate compounds' ability
to block central T-type channels, we employed a rat genetic
model of absence epilepsy using Wistar Albino Glaxo rats
bred in Rijswijk, The Netherlands (WAG/Rij), as reported pre-
viously.15 These rats display cortical EEG patterns and physi-
cal behaviors characteristic of absence epilepsy, including
frequent seizures.25 Because T-type calcium channels are
involved in the regulation of thalamocortical rhythms that
underlie these seizures, measurement of EEG in these ani-
mals serves as a relevant pharmacodynamic readout of brain
penetration and T-type channel blockade. Consistent with
our previous findings with compounds 1-4, compounds
11-13 all displayed robust inhibition of time in seizure, with
greater than 70% reduction over the first 4 h after oral dosing
3 mg/kg (Table 1).
dog
3.6
aDetermined by ultracentrifugation with 14C-labeled 11 at 5 μM.
the volume of distribution at steady state (Vss), T1/2 = 0.693
Vss/Cl, a shorter half-life could be achieved via enhancing Cl
or lowering Vss. However, a high clearance compound is
not desired due to the high propensity for drug-drug inter-
actions.26 Thus, another strategy is to strive for low to mode-
rate clearance compounds with low Vss. In the structural scaf-
folds that we have explored, the phenylacetamides generally
showed these characteristics. To screen compounds for
potential short half-lives in humans, we employed cassette
intravenous administration to dogs as our primary pharma-
cokinetics screening tool to get a quick readout on clearance
and Vss. Among the compounds evaluated, compounds 11-13
appeared to possess the desired profile as their short half-lives
were mainly attributed to their low Vss (Table 1). On the basis
of its potency and promising results from the initial pharma-
cokinetic screening, compound 11 was chosen for further
single-dose pharmacokinetic profiling in preclinical species.
As shown in Table 2, consistent with the finding from the dog
intravenous cassette study, compound 11 displayed a T1/2
=
Optimally, a compound designed to treat sleep disorders
would be rapidly absorbed, readily crossing the blood-brain
barrier and engaging the target at efficacious levels for the
∼8 h duration of the sleep period. We preferred a compound
with a restricted (predicted) human half-life. As the value
of half-life is determined by systemic clearance (Cl) and
1.1 h in this species with a good bioavailability of 50%. In
rats, compound 11 displayed a T1/2 = 24 min and was 100%
bioavailable after dosing at 10 mg/kg.
However, further profiling of 11 revealed a high potential
for bioactivation. Incubation of 11 in liver microsomes gave
rise to a significant amount glutathione (GSH) adduct, which
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was confirmed by NMR spectroscopy.27 The formation of a
covalent adduct was confirmed with a covalent labeling
experiment.28 Incubation of [14C]-labeled 11 (10 μM) with
human liver microsomes (1 mg/mL) for 60 min in the
presence of NADPH yielded high levels of irreversible binding
to microsomal proteins (1558 pmol equivalent/mg protein)
(Table 3). Bioactivation was quickly addressed by replacing
the oxazole ring with different heterocycles. For instance,
fused bicyclic compound 12 showed a low amount of GSH
adduct formation, which translated to a moderate level of
covalently labeled human microsomal proteins (244 pmol
equivalent/mg protein). Gratifyingly, no GSH adduct was
detected with methylpyrazine derivative 13. In agreement,
incubation of [14C]-labeled 13 with human liver microsome
(1 mg/mL) yielded a minimal level of irreversible binding
to microsomal proteins (<10 pmol equivalent/mg protein),
indicating a low potential of bioactivation. As a result, 13 was
further characterized as a key compound of interest.
The potency and state-dependent profile observed in the
FLIPR assays were confirmed with whole cell patch clamp
recording at two holding potentials, -100 and -80 mV.
Compound 13 showed potencies of 12 μM and 264 nM,
respectively (Figure 2). To examine ion channel selectivity,
compound 13 was assayed against important cardiac sodium
and potassium channels using planar patch clamp technol-
ogy. At the highest tested concentration of 30 μM, compound
13 inhibited hERG current (IKr) by 41 ( 4% and had no effect
on IKs. Likewise, at the highest tested concentration of 30 μM,
compound 13 had little effect on INa (20% inhibition at 3 Hz)
and L-type ICa (12% at 30 μM). In addition, the off-target
activity of compound 13 was evaluated in a panel of 170
receptor binding or enzyme assays (MDS Pharma Services)
and revealed no activities with IC50 values < 10 μM.
To aid in the prediction of human pharmacokinetics, the
pharmacokinetic profile of compound 13 was evaluated in
three preclinical species, and data are shown in Table 4. It
exhibits moderate to high plasma clearance (CLp), low vo-
lume of distribution (Vss), and a short half-life (T1/2) in three
preclinical species. In light of the greater extent of plasma
protein binding of 13 in human than preclinical species
(Ta bl e 4), the volume of distribution of 13 could be lower
than the values observed in animals. The T1/2 of 13 was
predicted to be less than 5 h in humans.29 As the distribu-
tion of 13 into human brain is not expected to be hampered
by P-gp-mediated efflux, its free brain concentration may be
reasonably reflected by its free plasma concentration. In
preclinical settings, drug concentrations in the cerebrospinal
fluid (CSF) have been widely used as the surrogate for free
brain concentration. It was found that 30 min after oral
dosing in rats, the concentration of 13 in the CSF and brain
accounted for 4.3 and 38% of that in plasma, giving a free
fraction in the brain of 11%, consistent with its free fraction
in the plasma.
Table 3. Covalent Protein Binding of 11-13 in Human Microsome
human in vitro
GSH
covalent protein binding
compound
adducta
(pmol/mg protein)b
11
12
13
high
low
no
1558 ( 109
244 ( 28
8 ( 1
aAmount was semiquantified using LC-MS. b Labeled protein mea-
sured after incubation of [14C]-labeled 11-13 (10 μM) with human liver
microsomes (1 mg/mL) for 60 min in the presence of NADPH.
To evaluate the effects of T-type antagonists on sleep and
wake, telemetric recordings of ECoG and electromyogram
(EMG) signals were measured in rats. In a 7 day cross-over
design, vehicle or compound 13 was dosed orally every day,
30 min before the inactive phase (lights on for rats). The
ECoG and EMG signals were collected and scored for the
amount of time awake or each phase of sleep (light sleep,
delta sleep, and REM).
Remarkably, despite having a half-life of 14 min, a 5 mg/kg
dose of 13 to rats prior to their inactive period produced a
significant suppression of active wake for 1.5 h after dosing
(Figure 3). In addition, a dose-dependent increase of delta
Figure 2. Inhibition of the T-type calcium channel Cav3.3 subtype
by 13 as determined by standard voltage clamp. Data points reflect
means ( SEs of three determinations. The IC50 values of 264 nM
and 12 μM were determined at -80 and -100 mV, respectively.
Table 4. Pharmacokinetic Parameters of 13
CL (predicted)a
(mL/min/kg)
CLp (observed)
(mL/min/kg)
plasma PB
species
T
1/2 (h)
V
ss (L/kg)
F (%)
(% unbound)b
rat
8
13
30.3
11.8
19.3
0.24
0.81
0.53
0.59
0.36
0.74
71
23
5
12.3
15.5
14.6
3.2
dog
rhesus
human
22
∼1c
<5c
<0.4c
aCL was predicted based on in vitro metabolic Clint with the well-stirred model. b Determined by equilibrium dialysis with 14C -labeled 13 at 5 μM.
cPredicted human values.
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Figure 3. Effect of 13 on N = 7 male SD rat active sleep for 23 h after dosing 1, 5, and 10 mg/kg po. Data are average minutes of active wake,
Delta sleep, and REM sleep in each 30 min bin ((SEM) starting at dose time (abscissa 15:00). The time bar below the x-axis denotes periods
of lights on (0, inactive phase) and lights off (9, active phase). The significance at each time point is reported as tick marks (short p < 0.05,
medium p < 0.01, and long p < 0.001) and gray vertical lines through significantly different comparisons based on a linear mixed effects
ANOVA at each time point.
sleep was observed for 0.5-2.0 h after dose. Maximal
plasma concentrations, as estimated from dosing nonim-
planted satellite animals, were between 0.12 and 2.7 μM for
doses ranging from 1 to 10 mg/kg at 0.1-0.7 h postdose.
On the basis of a favorable preclinical safety profile,
desired PK properties, and good in vivo efficacy, compound
13 was advanced to human safety and tolerability studies. In
line with our predictions, preliminary studies in human
subjects (n = 12) with a 20 mg oral dose afforded a high
Cmax of 1.82 ( 0.274 μM with an apparent terminal half-life
of 3.0 ( 1.1 h.
In conclusion, replacement of the cyclopropane substituent
of 4 with various heterocycles maintained potency and selec-
tivity for the T-type calcium channel while modulating phar-
macokinetics and resulted in the identification of the 2-methyl-
pyrazine phenyl acetamide 13. Compound 13 displayed
excellent potency and selectivity for the T-type channel. It
showed significant suppression of active wake and increased
delta sleep in rats. Compound 13 progressed into human clinical
trials and displayed a short half-life and good exposure as
predicted. Phase I studies with a structurally related compound
suggest that the qEEG effects may be species-dependent. The
results will be disclosed in a separate account in due course.
ACKNOWLEDGMENT We thank Nicole Pudvah for P-gp mea-
surements; Ken Anderson, Leslie Geer, and Debra McLoughlin for
PK analysis; Charles Ross and Joan Murphy for high-resolution mass
spectral analysis; Elena S. Trepakova for ion channel profiling; and
Yuming Zhao, Joseph Simeone, and Eric Soli for 14C-labeled com-
pound synthesis.
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Corresponding Author: *To whom correspondence should
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2010 American Chemical Society
509
DOI: 10.1021/ml100170e ACS Med. Chem. Lett. 2010, 1, 504–509
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