Pyridyl amides as potent inhibitors of T-type calcium channels

Article abstract of DOI:10.1016/j.bmcl.2011.01.089

A novel series of amide T-type calcium channel antagonists were prepared and evaluated using in vitro and in vivo assays. Optimization of the screening hit 3 led to identification of the potent and selective T-type antagonist 37 that displayed in vivo eff

Full text of DOI:10.1016/j.bmcl.2011.01.089

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1692–1696
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyridyl amides as potent inhibitors of T-type calcium channels
Thomas S. Reger a, , Zhi-Qiang Yang a, Kelly-Ann S. Schlegel a, Youheng Shu a, Christa Mattern a,
⇑
Rowena Cube a, Kenneth E. Rittle a, Georgia B. McGaughey a, George D. Hartman a, Cuyue Tang a,
Jeanine Ballard b, Yuhsin Kuo b, Thomayant Prueksaritanont b, Cindy E. Nuss a, Scott M. Doran c,
Steven V. Fox c, Susan L. Garson c, Yuxing Li c, Richard L. Kraus c, Victor N. Uebele c, John J. Renger c,
James C. Barrow a
a Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, United States
b Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, West Point, PA 19486, United States
c Department of Depression and Circadian Disorders, Merck Research Laboratories, West Point, PA 19486, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of amide T-type calcium channel antagonists were prepared and evaluated using in vitro
and in vivo assays. Optimization of the screening hit 3 led to identification of the potent and selective T-
type antagonist 37 that displayed in vivo efficacy in rodent models of epilepsy and sleep.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 30 November 2010
Revised 19 January 2011
Accepted 20 January 2011
Available online 26 January 2011
Keywords:
T-type calcium channel
Structure–activity relationships
Pregnane X receptor
Electrocorticogram
Epilepsy
Voltage-gated calcium channels (VGCCs) are membrane-span-
ning proteins that allow controlled entry of Ca2+ ions into cells. Cal-
cium is the most common signal transduction element in cells and
its influx initiates a wide range of physiological processes.1 VGCCs
are divided into three distinct classes based on sequence homology
Despite their potential role in diverse physiological and patho-
physiological processes, assessment of the functional role of T-type
calcium channels has been hampered by the lack of selective
antagonists.10 The anti-hypertensive agent mibefradil was initially
described as a selective T-type calcium channel inhibitor.11 How-
ever, it was later shown that its effects on cardiovascular function
likely resulted from L-type channel blockade,12 and it was ulti-
mately withdrawn from the market due to concerns related to
of the critical pore-forming
a1 subunit, voltage gating, current
kinetics and pharmacology: Cav1.x (L-type); Cav2.x (N, P/Q,
R-type); and Cav3.x (T-type).2 The T-type calcium channel family
has three members (
a
1G/Cav3.1,
a1H/Cav3.2, and
a1I/Cav3.3) that
drug–drug interactions.13 The recent development of
a1G knock-
are differentiated from the L, N, P/Q, and R-type families by their
activation at lower membrane potentials, smaller conductance,
out mice14 as well as specific anti-sense knockdown of each T-type
sub-type15 further underscores the channel’s potential and interest
as a therapeutic target. Several laboratories have utilized pharma-
cophore models for the rational design of selective T-type small
molecule antagonists, but these reports are limited to in vitro stud-
ies.16 We have recently detailed efforts to discover potent, selec-
tive, and brain penetrant T-type antagonists.17,18 Piperidine 117a
and quinazolinone 218 both demonstrated in vivo efficacy in rat
models of epilepsy and tremor, while 2 was also shown to suppress
active wake in a rat sleep experiment. An additional lead series
exemplified by amide 3 was identified in the same high-through-
put screen that uncovered the precursors to 1 and 2 (Fig. 1). Com-
pound 3 was an attractive starting point due to its good potency,
low molecular weight, and modular structure. Herein, we describe
the optimization of this novel series as we sought to identify
and faster inactivation. The
a1H subtype is broadly expressed in
both peripheral and central tissues while the
a
1G and 1I subtypes
a
are primarily expressed in CNS neurons.3 As such, T-type calcium
channels are implicated in many physiological processes including
smooth muscle contraction, hormone secretion, pain processing,
and thalamocortical signaling.4 Given these diverse functions,
modulators of T-type channels have significant potential for the
treatment of hypertension and CNS disorders such as absence epi-
lepsy,5 essential tremor,6 insomnia,7 schizophrenia,8 and neuro-
pathic pain.9
⇑
Corresponding author. Tel.: +1 215 652 2438; fax: +1 215 652 3971.
E-mail address: thomas_reger@merck.com (T.S. Reger).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.089

T. S. Reger et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1692–1696
1693
Table 1
Early SAR around HTS lead 3.
another distinct class of potent and selective T-type calcium chan-
nel inhibitors for evaluation in CNS models.
R3
N
The potency of compounds on the T-type calcium channel was
assessed against the inactivated, or depolarized, state of the
a1I
channel subtype utilizing a FLIPR assay as we have previously re-
ported.19 Lead compound 3 is a single enantiomer and, as illus-
trated in Table 1, displayed good potency in the FLIPR assay with
an IP of 235 nM. Moving to either the S-enantiomer (4) or the
gem-dimethyl (5) analogs resulted in a significant loss of potency.
Likewise, N-methylation of 3 to produce 6 led to an inactive
compound.
In order to improve T-type potency, we next examined modifi-
cations to the benzylamine group and the results are shown in
Table 2. ortho-, meta- and para-Substitution was tolerated with
compounds 7–10 essentially equipotent to 3. Electronic factors
did not significantly influence activity as a 4-chloro and 4-methoxy
substituent resulted in similar potency. In an effort to reduce the
overall hydrophobicity of the molecules, a nitrogen atom was
incorporated at each position of the ring to produce pyridines
11–13. The 2-pyridyl analog maintained modest T-type potency
with a FLIPR IP of 427 nM whereas the 3- and 4-pyridyl isomers
had reduced activity. The combination of the 4-chloro or 4-meth-
oxy groups with the 2-pyridyl template to generate 14 and 15,
respectively, was synergistic as a dramatic potency enhancement
was observed.
R1 R2
O
Compd
R1
R2
R3
a1I FLIPR, IPa (nM)
3
4
5
6
Me
H
Me
Me
H
H
H
H
Me
235
4678
5475
Me
Me
H
>10,000
a
Values are the mean of two or more experiments.
Table 2
Modifications to the benzylamine group
H
N
Ar
Me
O
In addition to its activity in the functional FLIPR assay, com-
pound 15 (TTA-A1)20 also had high affinity in an
a1I membrane
Compd
Ar
a1I FLIPR, IPa (nM)
7b
8
9
10
11b
12b
13b
14
15
2-Chlorophenyl
4-Chlorophenyl
3-Methoxyphenyl
4-Methoxyphenyl
2-Pyridyl
3-Pyridyl
4-Pyridyl
5-Chloropyridin-2-yl
5-Methoxypyridin-2-yl
332
123
146
146
427
binding assay with a Ki of 5 nM (Fig. 2).19,21 Counterscreening in
binding assays against the hERG potassium channel and L-type cal-
cium channel revealed excellent selectivity as 15 was inactive at
the highest tested concentration of 10
lM. Compound 15 was
not substrate for the rat P-glycoprotein transporter (B:A/
a
5383
>10,000
30
A:B = 1.2)22 and exhibited a brain/plasma concentration ratio of
0.27 at 1 h after oral dosing. In vivo CNS activity of 15 was evalu-
ated in a genetic model of absence epilepsy using Wistar albino
Glaxo rats bred in Rijswijk, The Netherlands (WAG/Rij).23 These
rats display abnormal thalamocortical oscillatory activities and a
characteristic EEG pattern of frequent seizures. Because T-type cal-
cium channels are involved in the regulation of thalamocortical
rhythms that underlie these seizures, measurement of EEG in
WAG/Rij rats serves as a relevant pharmacodynamic readout for
brain penetration and T-type channel efficacy of test compounds.
Despite its similar in vitro potency to piperidine 1 and quinazoli-
none 2, amide 15 demonstrated inferior WAG/Rij efficacy with a
modest 49% inhibition of total seizure duration over the initial
4 h period after a 10 mg/kg oral dose.17,18 This is likely due to a
poor pharmacokinetic profile in rat characterized by high clearance
(CL = 52 mL/min/kg) and low bioavailability (F% = 6).
22
a
Values are the mean of two or more experiments.
Tested as a racemic mixture.
b
SEAP assay.24 This activity can lead to induction of the CYP3A4 en-
zyme which is a potential risk for clinical drug–drug interactions.25
We sought to reduce this liability as well as to improve oral phar-
macokinetics in subsequent analogs.
Molecular modeling suggested that replacement of the tert-bu-
tyl substituent in 15 with smaller, more polar groups may reduce
the propensity for PXR activation.24 As illustrated in Table 3, how-
ever, changes to the tert-butyl group generally resulted in reduced
T-type potency. Indeed, para-substitution with a fluoro (17), chloro
(18), methylsulfone (22), cyano (23), or methoxy (24) group pro-
duced less active compounds. Interestingly, a 4-carbomethoxy or
Compound 15 was also a potent activator of the Pregnane X
Receptor (PXR, 87% of positive control rifampicin) in an in vitro
Cl
H
N
Cl
NC
F
O
H
N
N
H
Cl
N
CF3
O
O
N
O
H
1 (TTA-P2)
2 (TTA-Q6)
3
Figure 1. Structurally diverse T-type calcium channel antagonists.

1694
T. S. Reger et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1692–1696
Table 4
OMe
Modifications to the alkoxy group
H
N
OR2
N
H
O
Me
15 (TTA-A1)
a1I FLIPR IP
N
N
O
Me
R1
22 nM
5 nM
a1I binding Ki
hERG binding IC50
L-type binding IC50
Compd
R1
R2
a1I FLIPR, IPa (nM)
PXRb
>10 uM
>10 uM
30
31
32
33
34
35
36
37
iPr
iPr
iPr
iPr
cyPr
cyPr
cyPr
cyPr
H
920
16
10
NT
Benzyl
Propyl
57%
42%
30%
NT
38%
12%
14%
PGP (B:A/A:B)
WAG/Rij (%inh@4h)
PXR (%Rif@10uM)
1.2
49% (10 mg/kg)
87%
Trifluoroethyl
H
5
1722
21
11
Benzyl
Propyl
Figure 2. In vitro/in vivo profile of 15.
Trifluoroethyl
6
NT = not tested.
a
Values are the mean of two or more experiments.
% response of PXR activation relative to Rifampicin at 10 lM (see Ref. 24).
Table 3
b
Modifications to the phenylacetic acid group
OMe
H
N
O
CF3
N
R
H
N
N
O
Me
Compd
R
a1I FLIPR, IPa (nM)
PXRb
O
Me
15
16
17
18
19
20
21
22
23
24
25
26
4-tBu
H
4-F
4-Cl
3-Cl
22
>10,000
>10,000
1331
2778
>5000
252
>10,000
>5000
1742
439
1452
87%
NT
NT
NT
NT
NT
35%
NT
NT
NT
37 (TTA-A2)
a1I FLIPR IP
a1I binding Ki
hERG binding IC50
L-type binding IC50
6 nM
1.2 nM
>10 uM
>10 uM
2-Cl
3,4-Dichloro
4-SO2Me
4-CN
4-OMe
4-OPh
4-CO2Me
HO
PGP (B:A/A:B)
WAG/Rij (%inh@4h)
0.7
87% (10 mg/kg)
Figure 3. In vitro/in vivo profile of 37.
29%
À2.7%
27
4-
1133
À7.1%
Me Me
Vh = -100mV
28
29
4-Isopropyl
4-Cyclopropyl
45
70
26%
22%
100
75
50
25
0
NT = not tested
a
Values are the mean of two or more experiments.
% response of PXR activation relative to Rifampicin at 10
b
l
M (see Ref. 24).
Vh = -80mV
tertiary alcohol substitution gave 26 and 27 that, while less active
against the T-type calcium channel, did not activate PXR. It was
ultimately discovered that just slightly reducing the steric bulk of
the tert-butyl substituent had a significant impact on PXR activa-
tion. Compounds 28 and 29 containing an isopropyl and cyclopro-
pyl group, respectively, exhibited reduced PXR activation and were
only slightly less potent than 15.
In an effort to regain some potency, we prepared a series of ana-
logs in which the methoxy substituent of either 28 or 29 was mod-
ified (Table 4). Removal of the methyl group gave the
hydroxypyridines 30 and 34 that had reduced activity. Incorpora-
tion of a benzyloxy, propyloxy, or trifluoroethoxy group on either
the isopropyl or cyclopropyl scaffold, however, generated very po-
tent compounds. The trifluorethoxy analogs in particular were ex-
-10
-9
-8
-7
-6
-5
Compound 37 [M]
Figure 4. Inhibition of the T-type calcium channel
determined by standard voltage clamp. Data points reflect means SEs of three
determinations. IC50 values of 98 nM and 3.7
a1I subtype by 37 (TTA-A2) as
l
M were determined at À80 and
À100 mV, respectively. In some cases, error bars are hidden behind the symbols.
Like compound 15, it was not a substrate for the rat P-glycoprotein
transporter (B:A/A:B = 0.7). When tested in the in vivo WAG/Rij
epilepsy model, compound 37 displayed robust efficacy with an
87% reduction in total seizure time over the initial 4 h period after
a 10 mg/kg oral dose.
The functional potency of 37 (TTA-A2) was confirmed in a stan-
dard voltage-clamp electrophysiology assay.26 Similar to the previ-
ously described quinazolinone 2,18 amide 37 exhibited state-
tremely potent in the a1I FLIPR assay. The cyclopropyl-containing
analogs 35–37 showed reduced PXR activation compared to the
corresponding isopropyl analogs 31–33. Compound 37 (TTA-A2),
therefore, displayed a favorable overall profile as a potent T-type
calcium channel antagonist with reduced PXR activation.
Compound 37 (TTA-A2) also exhibited high affinity in the
a1I
dependent inhibition of a1I with potencies of 98 nM and 3.7 lM
binding assay with a Ki of 1.2 nM and had excellent selectivity over
at membrane holding potentials of À80 and À100 mV, respectively
the hERG potassium channel and L-type calcium channel (Fig. 3).19
(Fig. 4).27 As an additional screen for selectivity towards T-type

T. S. Reger et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1692–1696
1695
Table 5
OH
a
O
CF3
O
CF3
b, c
Pharmacokinetic parameters of 37 (TTA-A2)
HO
N
N
N
Species
CLp (mL/min/kg)
T1/2 (h)
Vdss (L/kg)
F (%)
38
39
40
Rata
6
0.9
17
1.5
11
0.9
0.8
0.7
0.9
68
43
7
Dogb
d, e
Rhesusc
O
CF3
O
CF3
a
b
c
2 mg/kg iv, DMSO, n = 2; 10 mg/kg p.o., 1% methylcellulose, n = 3.
0.5 mg/kg iv, DMSO, n = 2; 1 mg/kg p.o., 1% methylcellulose, n = 2.
0.5 mg/kg iv, DMSO, n = 2; 3 mg/kg p.o., 1% methylcellulose, n = 2.
H
f
N
N
S
N
S
N
O
O
42
41
g
channels, compound 37 was submitted to an external panel of 170
O
CF3
O
CF3
H
binding and functional assays. No activities with an IC50 of less
Cl
H3N
N
than 10 l
M were identified.28
h
N
N
O
A summary of pharmacokinetic (PK) parameters is shown in
Table 5. Compound 37 exhibited a favorable PK profile in rat and
dog with low clearance, low volume of distribution, and good bio-
availability. These data, coupled with excellent in vitro potency
and selectivity and in vivo efficacy in the Wag/Rij epilepsy model,
prompted us to evaluate the effects of 37 on sleep and wake in ro-
dents. Regulation of arousal includes a significant thalamocortical
component and T-type channels play a role in maintaining normal
signaling activities.29 In the experiment, telemetry implanted rats
were dosed with compound or vehicle 30 min prior to the inactive
phase in a seven-day crossover design. Electrocorticogram (ECoG)
and electromyogram (EMG) recordings were collected and scored
for the amount of time spent awake or in each state of arousal
43
37
Scheme 1. Synthesis of T-type calcium channel inhibitor 37 (TTA-A2). Reagents and
Conditions: (a) CF3CH2OTf, Cs2CO3, DMF; (b) mCPBA, CHCl3; (c) Ac2O, 100 °C, then
K2CO3, MeOH; (d) NaOCl, TEMPO, KBr, NaHCO3, CH2Cl2, H2O; (e) (R)-2-methyl-2-
propanesulfinamide, CuSO4, CH2Cl2; (f) MeMgBr, CH2Cl2, À78 °C; (g) HCl, MeOH; (h)
4-cyclopropylphenylacetic acid, EDC, HOAt, iPr2NEt, CH2Cl2.
(light sleep, delta sleep, REM) and binned into 30 min intervals.
As shown in Figure 5, a 3 mg/kg dose of 37 produced significant
changes in sleep architecture in rats.30 A reduction in active wake
soon after dosing was observed with a concurrent increase in delta
sleep and decrease in REM sleep. These effects persisted for up to
4 h post-dose. This pattern is consistent with the effects observed
with piperidine 1 and quinazolinone 2, suggesting the common
involvement of T-type calcium channels.31 A parallel PK study per-
formed in non-implanted satellite animals revealed that 37
reached a Cmax of ꢀ4
lM in plasma at 1.2 h post-dose where the
changes in vigilance state were the most pronounced.
The synthesis of compound 37 (TTA-A2) is described in Scheme
1. Commercially-available 5-hydroxy-2-methylpyridine (38) was
O-alkylated with trifluoroethyl triflate to give 39. Treatment with
mCPBA produced the pyridine N-oxide which underwent rear-
rangement in acetic anhydride to generate an acetoxymethyl pyr-
idine intermediate. Hydrolysis of the acetate with potassium
carbonate in methanol gave the hydroxymethyl compound 40. A
TEMPO-mediated oxidation to the corresponding aldehyde was
followed by condensation with (R)-2-methyl-2-propanesulfina-
mide to provide 41. In the key step, a highly diastereoselective
addition of MeMgBr to 41 afforded 42 as the major isomer (14:1;
R,R:R,S) that was readily separated from the minor isomer.32
Deprotection of the tert-butyl sulfinyl group with HCl gave amine
4333 that was coupled to 4-cyclopropylphenylacetic acid34 under
standard conditions to furnish amide 37.
In summary, we have described the evolution of the promising
HTS lead compound 3 into the potent and selective T-type calcium
channel antagonist 37 (TTA-A2). By varying the substitution of the
two aromatic rings flanking the central amide bond, compounds
with improved T-type potency and pharmacokinetics were identi-
fied. The optimized amide 37 exhibited robust efficacy in rodent
models of epilepsy as well as sleep, and it represents a useful
preclinical tool for further investigation of the physiological role
of T-type calcium channels.35,36
Figure 5. Effects of a 3 mg/kg dose of 37 (TTA-A2) on sleep architecture in rats. The
duration of time spent in Active Wake, Light Sleep, Delta sleep, and REM sleep in
vehicle (d) vs. drug (s) treated animals following a 3 mg/kg p.o. dose of 37 in 0.5%
methylcellulose administered at 15:00. Values are mean SEM. The time bar below
the x-axis denotes periods of lights on (h, inactive phase) and lights off (j, active
phase). Vertical gray lines represent statistically significant differences from vehicle
treatment as determined using mixed-model ANOVAs at each time point. Signif-
icance levels are indicated by length of tic marks (short <0.5, medium <0.01, long
<0.001).
Acknowledgements
The authors thank Ken Anderson, Anne Taylor, and Debra
McLoughlin for PK analysis; Nicole Pudvah for P-gp measurements;
Charles Ross and Joan Murphy for high-resolution mass spectral
analysis; and Richard Ball, Arlene McKeown, and Nancy Tsou for
X-ray crystallography.

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T. S. Reger et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1692–1696
Adarayan, E. D.; Prueksaritanont, T.; Zrada, M. M.; Marino, M. J.; Graufelds, V.
Supplementary data
K.; DiLella, A. G.; Reynolds, I. J.; Vargas, H. M.; Bunting, P. B.; Woltmann, R. F.;
Magee, M. M.; Koblan, K. S.; Renger, J. J. J. Med. Chem. 2008, 51, 6471.
18. Barrow, J. C.; Rittle, K. E.; Reger, T. S.; Yang, Z. Q.; Bondiskey, P.; McGaughey, G.
B.; Bock, M. G.; Hartman, G. D.; Tang, C.; Ballard, J.; Kuo, Y.; Prueksaritanont, T.;
Nuss, C. E.; Doran, S. M.; Fox, S. V.; Garson, S. L.; Kraus, R. L.; Li, Y.; Marino, M. J.;
Graufelds, V. K.; Uebele, V. N.; Renger, J. J. ACS Med. Chem. Lett. 2010, 1, 75.
19. Uebele, V. N.; Nuss, C. E.; Fox, S. V.; Garson, S. L.; Cristescu, R.; Doran, S. M.;
Kraus, R. L.; Santarelli, V. P.; Li, Y.; Barrow, J. C.; Yang, Z. Q.; Schlegel, K. S.;
Rittle, K. E.; Reger, T. S.; Bednar, R. A.; Lemaire, W.; Mullen, F. A.; Ballard, J. E.;
Tang, C.; Dai, G.; McManus, O. B.; Koblan, K. S.; Renger, J. J. Cell Biochem.
Biophys. 2009, 55, 81.
20. The TTA-Ax nomenclature is provided for consistency across publications, such
as Refs. 18 and 19.
21. Due to its potency, favorable physical properties, and ease of preparation
compound 15 was labeled with tritium and demonstrated to be an effective
radioligand for binding assays. See Ref. 19 for more details.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.089.
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Compound 37 exhibited excellent selectivity against the Cav1.2 (L-type), Cav2.1
(P/Q-type), Cav2.2 (N-type), and Cav2.3 (R-type) channels which all had IC50
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31. Compound 37 (TTA-A2) and quinazolinone
2 (TTA-Q6) are both state-
dependent T-type channel blockers while piperidine 1 (TTA-P2) is a state-
independent blocker. The consistent effect of all three compounds on sleep and
wake in rodents suggests that binding to the inactivated state of the channel is
sufficient for pharmacological effects.
32. For a discussion on the diastereoselectivity of Grignard additions to chiral 2-
pyridyl tert-butyl (Ellman) sulfinimines, see: Kuduk, S. D.; DiPardo, R. M.;
Chang, R. K.; Ng, C.; Bock, M. G. Tetrahedron Lett. 2004, 45, 6641–6643.
33. The absolute stereochemistry of a closely related analog of 43 was firmly
established by X-ray crystallographic analysis. See Supplementary data for
details.
34. The synthesis of 4-cyclopropylphenylacetic acid is described in the
Supplementary data. For
a
leading reference on the use of
cyclopropylboronic acid in Suzuki cross-coupling reactions, see: Wallace, D.
J.; Chen, C. Y. Tetrahedron Lett. 2002, 43, 6987–6990.
35. We have recently reported that 37 inhibits high-fat diet-induced weight gain
in mice. See Ref. 28 for details.
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K. S.; Shu, Y.; Rittle, K. E.; Bock, M. G.; Hartman, G. D.; Tang, C.; Ballard, J. E.;
Kuo, Y.; Adarayan, E. D.; Prueksaritanont, T.; Zrada, M. M.; Uebele, V. N.; Nuss,
C. E.; Connolly, T. M.; Doran, S. M.; Fox, S. V.; Kraus, R. L.; Marino, M. J.;
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