Discovery of 1,4-substituted piperidines as potent and selective inhibitors of T-type calcium channels

Article abstract of DOI:10.1021/jm800830n

The discovery of a novel series of potent and selective T-type calcium channel antagonists is reported. Initial optimization of high-throughput screening leads afforded a 1,4-substituted piperidine amide 6 with good potency and limited selectivity over hE

Full text of DOI:10.1021/jm800830n

J. Med. Chem. 2008, 51, 6471–6477
6471
Discovery of 1,4-Substituted Piperidines as Potent and Selective Inhibitors of T-Type Calcium
Channels
,
†
†
†
†
†
†
Zhi-Qiang Yang,* James C. Barrow, William D. Shipe, Kelly-Ann S. Schlegel, Youheng Shu, F. Vivien Yang,
‡
†
,|
†
†,⊥
†
‡
Craig W. Lindsley, Kenneth E. Rittle, Mark G. Bock, George D. Hartman, Victor N. Uebele, Cindy E. Nuss,
Steve V. Fox, Richard L. Kraus, Scott M. Doran, Thomas M. Connolly, Cuyue Tang, Jeanine E. Ballard, Yuhsin Kuo,
Emily D. Adarayan, Thomayant Prueksaritanont, Matthew M. Zrada, Michael J. Marino, Valerie Kuzmick Graufelds,
Anthony G. DiLella, Ian J. Reynolds, Hugo M. Vargas, Patricia B. Bunting, Richard F. Woltmann, Michael M. Magee,
‡
‡
‡
‡,]
§
§
§
§
§
§
¶,∞
¶
¶
¶
#,×
#
#
#
‡
‡
Kenneth S. Koblan, and John J. Renger
Departments of Medicinal Chemistry, Depression and Circadian Disorders, Drug Metabolism, Parkinson’s Disease, and Safety and
Exploratory Pharmacology, Merck Research Laboratories, West Point, PennsylVania 19486
ReceiVed July 8, 2008
The discovery of a novel series of potent and selective T-type calcium channel antagonists is reported.
Initial optimization of high-throughput screening leads afforded a 1,4-substituted piperidine amide 6 with
good potency and limited selectivity over hERG and L-type channels and other off-target activities. Further
SAR on reducing the basicity of the piperidine and introducing polarity led to the discovery of 3-axial
fluoropiperidine 30 with a significantly improved selectivity profile. Compound 30 showed good oral
bioavailability and brain penetration across species. In a rat genetic model of absence epilepsy, compound
3
0 demonstrated a robust reduction in the number and duration of seizures at 33 nM plasma concentration,
with no cardiovascular effects at up to 5.6 µM. Compound 30 also showed good efficacy in rodent models
of essential tremor and Parkinson’s disease. Compound 30 thus demonstrates a wide margin between CNS
and peripheral effects and is a useful tool for probing the effects of T-type calcium channel inhibition.
Introduction
Voltage-gated calcium channels regulate the entry of Ca +
2
into cells in response to membrane depolarization. The conse-
2+
quences of Ca influx include further depolarization of the cell
membrane, muscle contraction, neurotransmitter release, and
many others. Early electrophysiology studies classified calcium
channels as either high- or low-voltage-activated. The former
class includes L-, N-, P/Q-, and R-types. The last is designated
as T-type owing to their fast inactivation (transient) and small
conductance. Molecular cloning studies of all high and low
voltage-gated calcium channels identified 10 genes encoding
Figure 1
is due to its T-type activity. However, a more recent study using
conditional L-type knockout mice indicates that the antihyper-
tensive effects of 2 depend on its L-type antagonist properties.
In this study, mean arterial blood pressure in control mice was
reduced significantly by a relevant dose of 2 but not changed
in conditional L-type KO mice. 2 also blocks voltage-gated
sodium and potassium channels, so conclusions based on in
vivo observations with 2 should be viewed with caution.
The R1G and R1I subtypes are primarily expressed in CNS
neurons. Recent evidence has linked T-type Ca channels to
6
1
2+
the main pore-forming R1 subunit. As a result, the T-type Ca
channel family has three members: Cav3.1 (R1G), Cav3.2 (R1H),
and Cav3.3 (R1I). The R1H subtype is found in many tissues,
2
including brain, liver, kidney, heart, and smooth muscle, and
7
on the basis of this distribution, the T-type channel has been
proposed to play a role in cardiac pace-making and blood
pressure regulation. Researchers at Roche identified the dual
T-/L-type calcium channel antagonist mibefradil (2, Figure 1),
8
2+
9
,10
3
many potential therapeutical indications, including epilepsy,
which was briefly marketed as an antihypertensive agent.
1
1-15
16
17
pain,
movement disorders, tinnitus/hearing loss, arousal
Studies have shown that 2 is a potent T-type blocker with 10-
18
19
20
4
,5
states, overactive bladder, and cancer. In the brain, T-type
channels are found throughout the thalamus and cortex regions
and play an important role in the integration of neuronal firing.
The proper function of thalamocortical signaling is critical for
to 30-fold selectivity for T- over L-type channels. Therefore,
it has been widely used as a pharmacological tool for studying
T-type channels. Some have suggested its antihypertensive effect
2
1
*
To whom correspondence should be addressed. Phone: 215-652-2171.
generating a normal rhythmic pattern of oscillatory activities.
Fax: 215-652-3971. E-mail: zhiqiang_yang@merck.com.
Alternations in the normal functioning of the thalamocortical
signaling have been attributed to many neurological disorders,
including aforementioned epilepsy, pain, and movement disor-
†
Department of Medicinal Chemistry.
Current affiliation: Vanderbilt University.
Current affiliation: Novartis Institute for BioMedical Research, Inc.
Department of Depression and Circadian Disorders.
Current affiliation: Wyeth Pharmaceuticals.
Department of Drug Metabolism.
Department of Parkinson’s Disease.
Current affiliation: Cephalon, Inc.
|
⊥
2
2
‡
ders. In fact, ethosuximide (1, Figure 1), a marketed drug for
]
epilepsy has been extensively investigated and found to block
T-type calcium channels at therapeutic concentrations.
Despite their huge potential, the physiological roles of T-type
channels have remained elusive because of the lack of truly
selective blockers. Efforts in several laboratories have focused
§
2
3,24
¶
∞
#
Department of Safety and Exploratory Pharmacology.
Current affiliation: Amgen, Inc.
×
25
1
0.1021/jm800830n CCC: $40.75

2008 American Chemical Society
Published on Web 09/26/2008

6
472 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Yang et al.
Scheme 1
Figure 2. T-Type calcium channel antagonist leads.
Table 1. Piperidine Leads
T-type, FLIPR IP
hERG, IP
(nM)
L-type binding, IC50
a
b
c
compd
(nM)
(nM)
1
2
4
5
6
>5 000 000
126 ( 52
281 ( 80
245 ( 38
61 ( 16
806
135
88
7
566
206
1934
1191
a
All values are the mean ( standard deviation of at least n ) 3
b
c
measurements. All values are the average of n ) 2. Inhibition of diltiazem
binding to L-type calcium channels in rabbit muscle cells. All values are
the average of n ) 2.
ity by comparison of FLIPR functional T-type potency versus
binding assays for hERG and the L-type channel is not optimal,
this method provided robust SAR in each assay that would be
later confirmed in vivo. Combining two leads together provided
a hybrid lead 6, which is not only more potent but also more
selective against hERG and L-type channel screening. Com-
pound 6 displayed an IP value of 61 nM, which is approximately
a 5-fold improvement over 4. Its hERG binding liability was
reduced by more than 10-fold (IC50 )1.9 µM) compared to 4.
Compound 6 also displayed a 2-fold improvement on L-type
binding (IC50 ) 1.2 µM). Thus, a new potent lead was identified,
and it is significantly more selective over hERG and L-type
channels.
on the identification of new classes of selective agents, but these
2
6
reports are restricted to in vitro studies. Recently we reported
the in vitro and in vivo evaluation of a novel class of selective
and potent T-type channel antagonists based on a piperidine
2
7
scaffold exemplified by 3. Compound 3 has good potency,
selectivity, brain penetration, and pharmacokinetic profile and
demonstrated excellent separation between CNS target-engage-
ment and adverse CV effects. Herein, we expand on the SAR
and in vitro and in vivo properties of this promising series of
T-type calcium channel antagonists.
Results and Discussion
An HTS campaign was initiated, and screening of Merck
sample collection led to identification of two structurally similar
leads 4 and 5 (Figure 2). The former is a 1,4-substituted
piperidine amide, while the latter is a 1,4,4-substuituted pip-
eridine urea. Their activity in blocking the R1i subtype of the
T-type channel was evaluated using a high-throughput cell-based
Accordingly, a more detailed profiling of 6 was initiated. We
employed a rat genetic model of absence epilepsy to evaluate
the pharmacodynamics of T-type channel blockers. These rats
belong to a strain of Wistar albino Glaxo rats from Rijswijk
34
(
WAG/Rij). They display abnormal thalamocortical oscillatory
activities, which show a characteristic EEG pattern of excessive
seizures. Because of the role of T-type channels in the regulation
of thalamocortical rhythms, the WAG/Rij model provided a
quick readout of compounds’ efficacy on T-type channels. In
this model, compound 6 showed a robust effect on reducing rat
seizure count and time (data reported as 68% inhibition of
seizure duration over a 4 h period after dosing). A pharmaco-
kinetic analysis of the compound in rat revealed that compound
a
28,29
calcium flux assay (FLIPR ).
potency with a FLIPR IP value of 281 nM (Table 1). Compound
is slightly more active, with an IP value of 245 nM.
Compound 4 showed good
5
Counterscreening of these compounds, however, showed that
they provided no selectivity over human ether-a-go-go related
2+
gene (hERG) and L-type Ca channels. Blocking either hERG
+
2+
K or L-type Ca channels could raise serious cardiovascular
issues. The hERG channel is responsible for the IKr current, a
critical component in ventricular repolarization. Its blockade can
lead to increased QTc interval and potentially to ventricular
6
was highly bioavailable (100% F). Compound 6 was evaluated
in a P-glycoprotein (P-gp) assay and proved not to be a P-gp
substrate but did have good cell permeability, properties
consistent with good brain penetration. A broad based screen
of 170 enzymes, receptors, and ion channels (MDS Pharma
Services, Bothell, WA) identified eight off-target activities of
3
0
arrhythmias. Blockers of L-type channel, like nifedipine, can
3
1,32
cause hypotension. In hERG
(
1
and L-type calcium channel
diltiazem ligand) binding assays, 4 showed IC50 values of
35 and 566 nM, respectively, while 5 gave IC50 of 88 and 206
3
3
<
10 µM, suggesting further improvement on selectivity was
needed.
Because of the positive in vivo profile of 6, we set out to
nM against these two channels. While measurement of selectiv-
a
Abbreviations: CSF, cerebrospinal fluid; DAST, diethylaminosulfur
optimize this structural series to improve further on potency
and selectivity. We first screened the N-alkyl group of the
piperidine. Some representative examples are shown in Scheme
1. Their FLIPR data as well as hERG and L-type binding
activities were determined and listed in Table 2. One notable
trend of the SAR on FLIPR was that lipophilicity at the terminal
end is crucial for potency. Introduction of a nonsubstituted
trifluoride; DIEA, diisopropylethylamine; DMF, dimethylformamide; ECG,
electrocardiography; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride; EEG, electroencephalography; FLIPR, fluorometric imaging
plate reader; hERG, human ether-a-go-go-related gene; HOAt, 1-hydroxy-
7
-azabenzotriazole; HR, heart rate; MAP, mean arterial blood pressure; MP-
BH(OAc)3, macroporous polystyrene-bound triacetoxyborohydride; PBSF,
perfluoro-1-butanesulfonyl fluoride; THF, tetrahydrofuran; WAG/Rij, Wistar
albino Glaxo/Rijswijk.

1
,4-Substituted Piperidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6473
Scheme 3
Table 2. SAR on the N-Alkyl Group
T-type, FLIPR IP
hERG, IP
(nM)
L-type binding, IC50
a
b
c
compd
(nM)
(nM)
8
9
1
1
1
1
1
93 ( 9
7613
483
ND
279
ND
d
d
0
1
2
3
4
552 ( 213
771 ( 253
109 ( 30
38 ( 4
655
1556
d
d
ND
ND
2208
883
438
254
d
d
3480
ND
ND
a
All values are the mean ( standard deviation of at least n ) 3
b
measurements except for 9 and 14, where n ) 1. All values are the average
c
of n ) 2. Inhibition of diltiazem binding to L-type calcium channels in
d
rabbit muscle cells. All values are the average of n ) 2. ND ) not
determined.
Scheme 2
Table 4. Introducing Polarity and Reducing pKa
T-type, FLIPR hERG, IP L-type binding,
a
b
c
d
e
compd
IP (nM)
(nM)
IC50 (nM)
pKa calcd pKa
19
20
21
23
25
30
32
6
3
97 ( 40
252 ( 166
169 ( 64
22 ( 8
42 ( 13
32 ( 10
411 ( 112
61 ( 16
82 ( 27
2047
4810
4731
5097
3891
1526
1416
1567
2134
1049
1191
6330
8.0
9.2
7.3
8.8
8.1
7.4
8.6
7.5
7.5
9.5
7.5
>10000
7.6
1260
4114
3034
1934
3790
7.9
6.7
8.7
7.9
a
All values are the mean ( standard deviation of at least n ) 3
b
c
measurements. All values are the average of n ) 2. Inhibition of diltiazem
binding to L-type calcium channels in rabbit muscle cells. All values are
the average of n ) 2. Measured pKa by potentiometric titrations.
Calculated pKa using ACD/pKa database.
d
e
Table 3. SAR on the Benzoic Acid Moiety
potency (17f,g). However, none of these modifications improved
selectivity on hERG and L-type channels.
T-type, FLIPR IP
hERG, IP
(nM)
L-type binding, IC50
a
b
c
compd
(nM)
(nM)
Previously it has been proposed that basic amine-containing
hERG blockers are involved in a π-cation interaction with
1
1
1
1
1
1
1
a
7a
7b
7c
7d
7e
7f
41 ( 4
45 ( 7
1799
1047
4823
739
616
3
5,36
Y652
and thus suggested a need to reduce the basicity of
37,38
351 ( 98
2552 ( 1110
1683 ( 591
99 ( 34
2946
piperidine N.
Several electron-withdrawing groups, includ-
d
d
ND
ND
ND
ing hydroxyl, fluorine, and ether, were placed onto the N-alkyl
chain at ꢀ-position, while a keto or amide group was incorpo-
rated at the R-position. Chemistry for preparation of these
molecules was straightforward (Scheme 3). Alkylation of
piperidine precursor 7 with R-bromoketone 18 under basic
conditions gave the keto derivative 19. Reduction of 15 with
NaBH4 provided the corresponding alcohol 20. Conversion of
the hydroxyl group to fluorine proved to be nontrivial. Common
fluorinating reagents, such as DAST or bis(2-methyoxyethyl)-
aminosulfur trifluoride, failed to give any product, presumably
because of the presence of the basic amine. Under a one-pot
d
d
ND
3674
1021
984
944
7g
27 ( 3
All values are the mean ( standard deviation of at least n ) 3
b
c
measurements. All values are the average of n ) 2. Inhibition of diltiazem
binding to L-type calcium channels in rabbit muscle cells. All values are
the average of n ) 2. ND ) not determined.
d
pyridine (9) or terminal hydroxyl group (14) resulted in >50-
fold loss of potency vs 6. Even replacement of tert-butyl with
a cyclopropyl group led to greater than 10-fold reduction of
potency (11 vs 6). In addition, none of these analogues showed
improvement on selectivity as judged by hERG and L-type
binding values.
3
9
procedure developed by the Merck process group, we were
able to convert 20 to its fluorinated counterpart 21 by treatment
with Et N, Et N(HF) , and perfluoro-1-butanesulfonyl fluoride
3
3
3
Next, we shifted our focus on optimizing the benzoic acid
moiety. Some representative examples are shown in Scheme 2,
and their FLIPR, hERG, and L-type binding data are listed in
Table 3. We found that both 3,5- and 3,4-disubstituted benzoic
amides helped potency (17a,b), while mono-Cl analogue at
(PBSF) in THF. Treatment of 7 with R-chloroacetamide 22
yielded the bis-amide 23. Finally, the ether derivative 25 was
prepared from 7 under reductive amination of aldehyde 24. The
pKa of these compounds was either measured or calculated using
ACD/pKa DB. In comparison to lead 6, all of these analogues
proved to be less basic (Table 4). However, only the bis-amide
23 displayed a significant improvement on selectivity over
hERG. It is a potent T-type blocker with IP value of 17 nM in
3-position resulted in a 5-fold decrease of FLIPR potency (17c).
Heterocyclic replacements were not tolerated (17d-e). Interest-
ingly, substitution with 4-substituted phenylacetyl group retained

6
474 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Yang et al.
Scheme 4
Table 5. Ion-Channel Selectivity of 30
a
a
R1I VC
R1G VC
a
-
100 mV, -80 mV, -100 mV, -80 mV, L-type VC,
hERG EP,
nM
nM
nM
nM
nM
-100 mV, nM
4
3
25
62
44
14900
2400
a
Obtained from whole-cell patch-clamp recordings on HEK-293 cells
expressing human Cav 3.1 and 3.3 T-type or Cav 1.2 L-type calcium
channels.
a
Table 6. Pharmacokinetic Parameters of 30
plasma
protein
iv dose Clp ((mL/ T1/2 po dose AUC Cmax
F
binding
species (mg/kg) min)/kg) (h) (mg/kg) (µM h) (nM) (%) (% bound)
rat
2
41
4.9 10
3.1
0.19
432 30
33
93
1
0
.3 ∼0.01
dog
rhesus
a
1
1
9.1
9.6
10.8
4.6
3
3
9.5 1175 67
97
97
2.4
184 18
Plasma levels from PK experiments carried out as described in
Supporting Information.
the depolarized FLIPR assay, while its hERG binding IC50 is
greater than 10 µM. Unfortunately, this compound is a P-gp
substrate and still possessed significant L-type channel binding
activity.
We then shifted our focus on incorporating fluorine into the
piperidine ring. Previously, we have had success with 4-F
2
7
derivative such as compound 3. Here, we focused our efforts
on introducing 3-F at the piperidine. Both cis (single enantiomer)
and trans isomers (racemic mixture) were prepared using
standard transformations from intermediates 26 or 28 previously
40
published by Merck (Scheme 4). As expected, the trans isomer
2 is a much weaker base than the cis counterpart 30. The pKa
value of 32 is 2 log units lower than that of 6, while 30 (like its
-fluoro counterpart 3) is 1 log unit lower. In depolarized FLIPR
assay, compound 30 is 2-fold more potent than nonfluorinated
Figure 3. Brain and plasma level of 30 following an oral dose (10
mpk) to rats.
3
4
activity (IC50 ≈ 15 µM), which was not predicted by our high-
throughput L-type binding assay (IC50 ) 2.1 µM). This is not
uncommon because of multiple binding sites for L-type channels
6, while 32 is 8-fold weaker than 6. To our surprise, cis isomer
30 (pKa ) 7.9) is more selective than the less basic trans isomer
32 (pKa ) 6.7) on hERG and L-type channels. Overall,
4
2
or voltage-dependence of the inhibitors and underscores the
need to check high-throughput binding results with functional
assays.
compound 30 is 4-fold more selective on T-type than hERG
and L-type channels than 6. In addition, 30 showed only four
off-target activities below 10 µM in a broad based screen of
The pharmacokinetic parameters for compound 30 following
intravenous and oral administration in preclinical species are
shown in Table 6. The compound exhibited moderate clearance
and moderate to good bioavailability across all three species.
P-Glycoprotein (P-gp) mediated transport of 30 was minimal
(B-A/A-B < 2 in both human and rat), and the compound
1
70 enzymes, receptors, and ion channels (MDS Pharma
Services, Bothell, WA), representing another significant im-
provement over 6. Among those four off-target activities, the
most prominent were σ1 and σ2 receptor binding (IC50 < 20
4
1
-6
nM), but follow-up in a tissue functional assay revealed only
weak antagonism (30% inhibition at 30 µM)). The others
included IC50 ) 1.4 µM on 5HT4, IC50 ) 8 µM on muscarinic
M2, and IC50 ) 6 µM on histamine H1). Thus, a highly potent
and selective T-type channel antagonist, 30, was identified.
Overall Profile and Cardiovascular Effect of 30. The
FLIPR potency of 30 on T-type calcium channels was confirmed
using standard voltage clamp electrophysiology assays (see
Supporting Information for assay protocol). It had similar
potency on the R1I subtype at two different holding potentials
showed good passive cellular permeability (Papp ) 37 × 10
cm/s). Consistent with the in vitro data, 30 showed excellent
brain penetration in rats (brain/plasma concentration ratio of 9
and CSF/plasma ratio of 0.14 at 9 h after oral dosing) (Figure
3), supporting its therapeutic potential as a CNS agent.
The in vivo efficacy of compound 30 was first evaluated in
3
3
the WAG/Rij rat absence epilepsy model. The WAG/Rij rats
display cortical EEG patterns and physical behaviors charac-
teristic of an epileptic condition, including frequent seizures.
Since T-type calcium channels are involved in the regulation
of thalamocortical rhythms, this model served as a relevant
pharmacodynamic readout. The data are reported as total seizure
time reduction (in seconds) over the 4 h period after dosing
orally compared with vehicle control. As shown in Figure 4a,
T-type channel blocker 30 exhibited a nice dose-dependent
reduction of seizure time, evident as a brain-penetrant, centrally
(
-100 and -80 mV), suggesting a state-independent profile. It
also had similar potency on another T-type submember, R1G,
indicating that it is not subtype selective. In functional patch
clamp, compound 30 showed similar hERG activity as predi-
cated by hERG binding screening assay, with an IC50 of 2.4
µM. However, in an L-type voltage clamp assay, 30 had reduced

1
,4-Substituted Piperidines as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6475
Figure 5. (a) Dose response of 30 in harmaline-induced rat model of
essential tremor after po dosing in 90% PEG200. (b) Effect of 30 on
haloperidol induced catalepsy after po dosing.
Figure 4. Dose response (po in 90% PEG400) in WAG/Rij rat model
of absence epilepsy of 30 (a) and ethosuximide (b), n g 5 rats per
dose. Values represent decrease in total seconds spent in seizure during
the 4 h period after dosing compared to vehicle.
effects using a CV dog model. The three dogs used are
anesthetized, ventilated, and vagotomized. In this assay, systolic/
diastolic and mean arterial pressures, heart rate, and blood flow
were measured, as well as PR, QRS, QT/QTc intervals. The
compound was dosed in 30 min consecutive iv infusions (i.e.,
three rising doses). T-Type blocker 30 did not alter the intervals
of QTc and QRS up to 33 µM plasma levels, despite having an
IC50 of 2.4 µM in hERG functional clamp assay. This could be
partially due to that fact that compound 30 is highly protein-
bound in dogs (97%). Compound 30 had no effect on PR
interval and displayed modest bradycardia (5-15% up to 33
µM) and hypotension (8-13% at 1-3 mg/kg; 32% at 33 µM).
In fact, the hypotension effect of 30 became significant only at
plasma exposures greater than 5.6 µM, possibly because of its
activity on the L-type channel. In comparison, the mixed T-
and L-type blocker 2 has significant effects on PR interval
(+30%), heart rate (-22%), and blood pressure (-27%) at
lower plasma exposures (6.4 µM) in this assay (see Supporting
Information for detailed data).
active blocker of T-type channels. The exposures at each dose
were determined and listed in Table 6. Robust seizure suppres-
sion was observed at 1 mpk dose (AUC ) 0.19 µM·h) with
Cmax ≈ IC50. In comparison, ethosuximide suppressed seizure
to a similar degree at a much higher dose (100 mpk, Figure
4
b), consistent with its reduced potency on the T-type channel.
Like absence epilepsy, essential tremor has also been cor-
4
3
related to thalamocortical dysfunction; therefore, 30 was
44
evaluated in rat harmaline model of essential tremor. Harma-
line-induced tremor activity in the 6-12 Hz range was
monitored and quantified to assess tremor suppression. 30 was
found to significantly reduce tremor activity in a dose-dependent
manner (Figure 5a), highlighting the potential role of T-type
antagonists as a novel treatment for movement disorders such
as essential tremor.
Given the success of 30 in the essential tremor model, it was
examined in a rat model of Parkinson’s disease, another potential
indication of T-type calcium blockers. In this mode, a cataleptic
state is induced by treatment of rats with the dopamine
Since T-type channels are expressed in renal vascular and
tubular tissues, compound 30 was evaluated for potential side
effects on renal function. Four dogs were administered a single
oral dose (10 mpk) of 30 and observed for 3 h after dosage. No
effects were observed on glomerular filtration rate, effective
renal plasma flow, urine flow, urinary excretion of sodium and
potassium, or plasma electrolytes. While blood pressure and QTc
and QRS intervals were unaffected in this assay, increased heart
rate and minor behavioral changes (lethargy) were observed,
consistent with data collected from anesthetized dogs. Peak
plasma concentration of 30 was 2 µM during the experiment.
45
antagonist haloperidol as previously described, and therapeutic
agents are evaluated for their ability to reverse this state.
Compound 30 was dosed orally in 90% PEG400 to rats 1 h
after haloperidol administration; after an additional 30 min,
catalepsy was measured by placing rats on a vertical grid and
measuring latency to forepaw movement. As shown in Figure
5b, in the vehicle control group, the level of haloperidol induced
catalepsy is reflected by a latency to forepaw movement of
approximately 110 s. Compound 30 when dosed at 10 mpk
significantly reduced haloperidol induced catalepsy, which is
encouraging for potential palliative treatment of the motor
deficits of Parkinson’s disease. Notably, counterscreening
showed that the compound does not bind to dopamine receptors
or the dopamine transporter (D1, D2, D3, D4, DAT < 50%
inhibition at 10 µM). Thus, T-type blockers present a potential
novel mechanism for treatment of Parkinson’s disease.
In summary, the T-type selective blocker 30 had no effect
on QTc, QRS, and PR intervals and showed only modest
decreases in heart rate and blood pressure at plasma concentra-
tions greater than 5.6 µM, exposure where its L-type activity
might begin to operate. In comparison, we observed robust CNS
effects at 33 nM. These results clearly demonstrated that such
Bearing in mind that compound 30 still has micromolar hERG
and L-type activities, we evaluated potential cardiovascular
2
+
a selective T-type Ca channel blocker has great therapeutical

6
476 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Yang et al.
potential with significant safety margin for undesired cardio-
vascular effects.
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2
+
(
(
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12) Altiers, C.; Zamponi, G. W. Targeting Ca channels to treat pain:
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Conclusion
Truly selective T-type calcium channel antagonists are po-
tential therapeutic agents for treatment of a variety of neurologi-
cal disorders. We identified a potent and highly selective T-type
blocker (30) starting from HTS 1,4-substituted piperidine leads.
We found that introducing 3-S F onto the piperidine ring
increased potency on T-type channels, as well as decreasing
activity on L-type and hERG channels. Compound 30 displays
good pharmacokinetics in three preclinical species. Robust
efficacy was seen in WAG/Rij rat absence epilepsy and
harmaline-induced tremor models at plasma exposure well below
the no effect level in CV dog experiments. In addition,
compound 30 demonstrates a significant reversal of haloperidol
induced catalepsy in a preclinical model of Parkinson’s disease.
These data suggest that 30 has a good margin between on- and
off-target effects. Therefore, selective T-type calcium channel
antagonists such as 30 hold promise for the treatment of a variety
of neurological disorders without adverse cardiovascular side
effects.
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the treatment of pain. Drug DiscoVery Today 2006, 11, 245–253.
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channels in peripheral and central pain processing. CNS Neurol.
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(
(
(
(
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association between T-type Ca current and outward current in
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Acknowledgment. We thank Carl F. Homnick, David D.
Wisnoski, Wei Lemaire, Scott D. Mosser, Rodney A. Bednar,
Charles W. Ross, III, Joan S. Murphy, Kevin B. Albertson,
William H. Leister, Ray T. McClain, Anna Dudkina, Emily
Wang, Debra McLoughlin, and Dorothy Levorse for technical
support of this work.
Shattock, M.; McLatchie, L.; Harper, J.; Brooks, G.; Heady, T.;
2
+
Macdonald, T. L. The role of voltage gated T-type Ca channel
2
+
isoforms in mediating “capacitative” Ca entry in cancer cells. Cell
Calcium 2004, 36, 489–497.
(
21) Contreras, D. The role of T-channels in the generation of thalamo-
cortical rhythms. CNS Neurol. Disord.: Drug Targets 2006, 5, 571–
5
85.
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22) McCormick, D. A. Are thalamocortical rhythms the rosetta stone of
a subset of neurological disorders? Nat. Med. 1999, 5, 1349–1351.
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of cloned human T-type calcium channels by succinimide antiepileptic
drugs. Mol. Pharmacol. 2001, 60, 1121–1132.
Supporting Information Available: Experimental procedures
and analytical data for the synthesis of compounds 6, 8-14, 17,
(
1
9-21, 23, 25, 30, 32; FLIPR assay protocol; assay protocol for
voltage-clamp assay; pharmacokinetic experiment procedure; dog
renal function assay protocol; and anesthetized CV dog data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(
24) Broicher, T.; Seidenbecher, T.; Meuth, P.; Munsch, T.; Meuth, S. G.;
Kanyshkova, T.; Pape, H.-C.; Budde, T. T-Current related effects of
2
+
antiepileptic drugs and a Ca channel antagonist on thalamic relay
and local circuit interneurons in a rat model of absence epilepsy.
Neuropharmacology 2007, 53, 431–446.
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25) Lory, P.; Chemin, J. Towards the discovery of novel T-type calcium
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