Design, synthesis, and evaluation of a novel 4-aminomethyl-4- fluoropiperidine as a T-type Ca2+ channel antagonist

Article abstract of DOI:10.1021/jm800419w

The novel T-type antagonist (S)-5 has been prepared and evaluated in in vitro and in vivo assays for T-type calcium ion channel activity. Structural modification of the piperidine leads 1 and 2 afforded the fluorinated piperidine (S)-5, a potent and selec

Full text of DOI:10.1021/jm800419w

3
692
J. Med. Chem. 2008, 51, 3692–3695
Design, Synthesis, and Evaluation of a Novel
-Aminomethyl-4-fluoropiperidine as a
4
2
+
T-Type Ca Channel Antagonist
,
†
†
†
William D. Shipe,* James C. Barrow, Zhi-Qiang Yang,
†
,b
†
†
Craig W. Lindsley, F. Vivien Yang,
†
†
Kelly-Ann S. Schlegel, Youheng Shu, Kenneth E. Rittle,
†
,∞
†
‡
Mark G. Bock, George D. Hartman, Cuyue Tang,
‡
‡
‡
Jeanine E. Ballard, Yuhsin Kuo, Emily D. Adarayan,
‡
‡
Thomayant Prueksaritanont, Matthew M. Zrada,
Victor N. Uebele, Cindy E. Nuss, Thomas M. Connolly,
§
§
§,⊥
§
§
§
Scott M. Doran, Steven V. Fox, Richard L. Kraus,
|
,×
|
Michael J. Marino, Valerie Kuzmick Graufelds,
#
,[
#
Hugo M. Vargas, Patricia B. Bunting,
#
#
Martha Hasbun-Manning, Rose M. Evans,
]
§
Kenneth S. Koblan, and John J. Renger
Departments of Medicinal Chemistry, Depression and Circadian
Disorders, Parkinson’s Disease, Drug Metabolism, and Safety and
Exploratory Pharmacology, Merck Research Laboratories, WP14-1,
P.O. Box 4, Sumneytown Pike, West Point, PennsylVania 19486
Figure 1. Profiles of 4-aminomethyl piperidine leads 1-3.
important role in the function of the thalamocortical loop, a
neural network structure implicated in absence epilepsy, arousal
state, and movement disorders such as essential tremor.
ReceiVed April 11, 2008
4
Abstract: The novel T-type antagonist (S)-5 has been prepared and
evaluated in in vitro and in vivo assays for T-type calcium ion channel
activity. Structural modification of the piperidine leads 1 and 2 afforded
the fluorinated piperidine (S)-5, a potent and selective antagonist that
displayed in vivo CNS efficacy without adverse cardiovascular effects.
Mibefradil, often described as the first selective T-type an-
tagonist, demonstrated efficacy as an antihypertensive. Shortly
5
after its introduction, mibefradil was withdrawn from the U.S.
6
market in 1998 because of drug-drug interactions, but in lieu
of other compounds, it remains the prototype T-type calcium
channel blocker. Additionally, ethosuximide has T-type antago-
nist properties (among others) and is used to suppress absence
Calcium is an important physiological signaling molecule,
and control of intracellular calcium concentration is a tightly
2
+
7
regulated process. Calcium ions (Ca ) can be released from
intracellular stores or enter the cell via ligand-gated or voltage-
gated calcium channels, initiating a wide range of physiological
seizures. Potent and selective agents are needed to better
elucidate the true functional role of T-type calcium channels in
these and other disease states. With increasing knowledge of
the localization, function, and properties of T-type calcium
channels, there has been heightened activity for the discovery
1
responses. Historically, voltage-gated calcium channels have
2
+
been divided by functional descriptors of the Ca currents (L-
type, N-type, T-type, etc.), but more recently molecular cloning
of the critical pore forming R subunit has resulted in three main
classes denoted Cav1.x (L-type), Cav2.x (N, P/Q, R-type), and
8
of novel inhibitors in recent years. Here, we detail the discovery
of a novel, orally bioavailable piperidine T-type calcium channel
antagonist that evolved via optimization of piperidines 1 and 2
and its in vivo evaluation in cardiovascular and CNS models.
2
Cav3.x (T-type). The Cav3.x family has three members (Cav3.1,
2+
Cav3.2, and Cav3.3), and these channels activate at more
negative membrane potential than members of the Cav1.x and
Cav2.x families and produce smaller and more transient currents.
They are widely expressed in CNS and peripheral tissues and
may have potential for the treatment of various indications such
as hypertension, epilepsy, pain, regulation of arousal states,
Screening efforts identified piperidines 1 and 2 as T-type Ca
channel antagonists (Figure 1). Their ability to block the Cav3.3
(R1I) channel was assessed using a high-throughput cell-based
a
calcium flux assay (FLIPR ) at a membrane potential of -20
9
mV (depolarized) or -60 mV (hyperpolarized). High-voltage
2+
activated Ca channel antagonists can have preferential affinity
for one state over another, and these properties can profoundly
3
tinnitus, and cancer. In the brain, T-type channels are found
1
0
in the thalamic and cortical regions where they have an
affect the efficacy and safety profiles of the compounds.
Although it is unknown whether these properties would impact
the physiological effect of T-type antagonists, the state-
dependent properties of all described compounds were also
characterized by standard voltage-clamp electrophysiological
assays. The compounds were counterscreened for activity on
*
To whom correspondence should be addressed. Telephone: 215-652-
6
527. Fax: 215-652-6345. E-mail: william_shipe@merck.com.
†
Department of Medicinal Chemistry.
Current address: Vanderbilt University.
Current address: Novartis Institutes for BioMedical Research, Inc.
Department of Drug Metabolism.
Department of Depression and Circadian Disorders.
Current address: Wyeth Pharmaceuticals.
Department of Parkinson’s Disease.
Current address: Cephalon, Inc.
b
∞
‡
§
a
Abbreviations: CSF, cerebrospinal fluid; DMF, dimethylformamide;
⊥
ECG, electrocardiography; EEG, electroencephalography; FLIPR, fluoro-
metric imaging plate reader; hERG, human ether-a-go-go-related gene;
HOBt, N-hydroxybenzotriazole; HR, heart rate; MAP, mean arterial blood
|
×
#
Department of Safety and Exploratory Pharmacology.
Current address: Amgen, Inc.
Basic Research Administration, Merck Research Laboratories, Rahway,
pressure; MP-BH(OAc) , macroporous polystyrene-bound triacetoxyboro-
3
[
hydride; MP-carbonate, macroporous polystyrene-bound carbonate; PS-
carbodiimide, polystyrene-bound carbodiimide; WAG/Rij, Wistar albino
Glaxo/Rijswijk.
]
NJ.
1
0.1021/jm800419w CCC: $40.75

2008 American Chemical Society
Published on Web 06/10/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3693
Figure 2. Profiles of piperidines 4 and 5.
Figure 3. Profiles of piperidines (R)-5 and (S)-5.
a
Scheme 1
16
quence was employed to convert 7 to primary amine 8. Amide
1 was obtained by carbodiimide-mediated coupling of 8 to 3,5-
1
dichlorobenzoic acid and subsequent removal of the tert-
butyloxycarbonyl protective group. Reductive amination with
1
7
tetrahydropyranyl aldehyde 12 provided the racemic tertiary
amine 5.
Racemic (()-5 was resolved by HPLC with a ChiralPak AD
column so that the biological activities of the enantiomers could
be individually evaluated. Little distinction could be made
1
8
between (R)-5 and (S)-5 on the basis of ion channel data
Figure 3), but (S)-5 was consistently superior in a WAG/Rij
(
rat model of absence epilepsy (vide infra) and was selected for
full characterization.
In agreement with the profile observed in the calcium flux
assays, the in vitro potencies of (S)-5 determined in a standard
voltage-clamp electrophysiological assay at membrane holding
potentials of -80 and -100 mV (115 and 84 nM, respectively)
confirmed that compounds in this piperidine series are state-
independent antagonists. Similar electrophysiology experiments
with Cav3.1 (R1G, 93 nM at -100 mV) and Cav3.2 (R1H, 196
nM at -100 mV) indicated that the compound potently inhibits
all T-type family members.
a
Conditions: (a) 70% HF ·pyridine, -10 °C f room temp; (b) p-TsCl,
pyridine, 0 °C f room temp; (c) potassium phthalimide, DMF, 150 °C;
d) ethanolamine, 60 °C; (e) HOBt, 3,5-dichlorobenzoic acid, i-Pr2EtN, PS-
carbodiimide, then MP-carbonate; (f) 4 N HCl/dioxane, CH2Cl2; (g) 12,
MP-BH(OAc)3, CH2Cl2.
(
the hERG K and L-type Ca2+ channels using binding
assays.
+
1
1,12
While 1 and 2 showed good T-type potency, a
concern was the lack of selectivity observed with respect to the
hERG and L-type ion channels. Blocking hERG or L-type ion
channels could have unwanted cardiac effects for a CNS
therapeutic agent; therefore, an early goal of the program was
to improve this selectivity margin.
Initial SAR showed the neohexyl group of 1 and the 3,5-
dichlorobenzoyl group of 2 to be preferred structural features
from a potency viewpoint. Combining those structural elements
of these leads about a 4-aminomethylpiperidine scaffold pro-
duced 3, which showed improved T-type potency and selectivity
with respect to the hERG and L-type ion channels. However,
submission of the compound to a broad external screen revealed
eight off-target activities with greater than 50% activity at 10
µM, reflecting its moderate selectivity versus other ion channels
and GPCRs.
Fluorinated piperidine analogues were then prepared. The
potential benefit of this strategy was 2-fold: (1) by reduction of
the basicity of the ring nitrogen atom, the ancillary pharmaco-
logical profile might be improved; (2) the fluorinated piperidine
ring might exhibit enhanced metabolic stability. Compound
, bearing a fluorine atom at the 4-position, possessed a potency
and selectivity profile similar to those of 3 (Figure 2), but
improved selectivity was observed by maintaining this fluorine
and varying the N-substituent. After extensive investigation, the
tetrahydropyran (()-5 displayed the best improvement on the
ion channel selectivity over analogues 3 and 4.
The pharmacokinetic parameters for (S)-5 following intra-
venous and oral doses in preclinical species are shown in Table
1
. The compound exhibited significant species differences in
clearance and volume of distribution but relatively short half-
life (∼2-5 h) across species. The oral bioavailability of (S)-5
was reasonably good (>35%) in all species.
P-Glycoprotein (P-gp) mediated transport of (S)-5 was found
to be minimal (B-A/A-B e 2), and the compound showed good
-
6
passive cellular permeability (Papp ) 34 × 10
cm/s).
Consistent with the in vitro data, (S)-5 showed good brain
penetration in rats (brain/plasma concentration ratio of 6.0 and
CSF/unbound plasma ratio of 2.8 at 9 h after dosing), supporting
its potential therapeutic use as a CNS agent.
To investigate the in vivo CNS activity of these compounds,
we employed a genetic model of absence epilepsy using Wistar
albino Glaxo rats bred in Rijswijk, The Netherlands (WAG/
Rij). The WAG/Rij rats display cortical EEG patterns and
physical behaviors characteristic of an epileptic condition,
1
9
including frequent seizures. Since T-type calcium channels
are involved in the regulation of thalamocortical rhythms,
measurement of EEG in these animals serves as a relevant
pharmacodynamic readout of brain penetration and T-type
channel activity. (S)-5 exhibited a dose- and exposure-dependent
decrease in the total seizure time during the 4 h period following
oral dosing (Figure 4). Notably, (S)-5 demonstrated robust CNS
efficacy in this model at a dose of 10 mg/kg with a plasma
level of 1 µM.
1
3
4
The synthesis of (()-5 proceeded as outlined in Scheme 1.
14
Regioselective epoxide opening of 6 with HF ·pyridine yielded
Another pathophysiological condition that may result from
1
5
20
the fluorinated alcohol 7. A straightforward three-step se-
thalamocortical dysfunction is essential tremor; therefore, (S)-5

3
694 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Letters
Table 1. Pharmacokinetic Profile of (S)-5
iv (in DMSO)
po (in 1% methylcellulose)
plasma protein
dose
CL
t1/2
Vdss
Cmax
Tmax
AUC0-24h
F
species
rat
binding (% bound)
(mg/kg)
(mL/min/kg)
(h)
(L/kg)
(µM)
(h)
(µM·h)
(%)
1
75 ( 21
1.8 ( 0.1
10 ( 2
94.3
99.0
97.5
1
0
0
1
0
2
0.48 ( 0.12
2.18 ( 1.97
0.58
0.7 ( 0.3
3.1 ( 4.3
1.5
1.94 ( 0.43
15.8 ( 10.9
3.2
36 ( 8
.5
.5
2.3
4.7
1.0
dog
87 ( 64
13.6
3.3
3.8
monkey
44
2
1
was also evaluated in the rat harmaline model of essential
tremor. Harmaline-induced tremor activity in the 6-12 Hz range
was monitored and quantified to assess tremor suppression. (S)-5
was found to significantly reduce tremor activity in a dose-
dependent manner (Figure 5), suggesting a possible role for
T-type antagonists in the treatment of movement disorders such
as essential tremor.
channel binding was also evident in the external screen (IC50
) 2.9 µM), so we examined the effect of compound on
25
cardiovascular function in a conscious dog model. Three dogs
were infused intravenously with (S)-5 first at a rate of 1.29 (mg/
kg)/60 min and then 4.30 (mg/kg)/60 min, resulting in mean
plasma levels of 1.5 ( 0.3 µM at the end of the first infusion
and 4.6 ( 1.7 µM at the end of the second. No behavioral effects
were noted during the study, and there were no significant
changes in mean arterial blood pressure (Figure 6), heart rate
(Figure 7), or ECG intervals (PR, QRS, QT, and QTc).
To gain further insight into potential ancillary pharmacology,
(
S)-5 was submitted to a large external screen, which revealed
only four off-target activities with greater than 50% activity at
0 µM. Most prominent was σ receptor binding (<100 nM),
1
Since T-type calcium channels are present in renal vascular
22
2
6
but follow-up in a vas deferens functional assay revealed only
weak antagonist activity (30% at 30 µM). Also noted was
sodium channel site 2 binding (IC50 ) 1.9 µM), but voltage
clamp studies with the Nav1.5 sodium channel showed an IC50
of 84 µM. (S)-5 also showed little inhibition of Cav2.2 in a
counterscreen (18% inhibition at 10 µM). hERG potassium
and tubular tissues, (S)-5 was evaluated for side effects on
renal function. Four dogs were administered a single oral dose
2
7
(
20 mg/kg) of (S)-5 and observed for 3 h after treatment. No
effects were observed on glomerular filtration rate, effective
2
3
2
4
Figure 6. Effect of (S)-5 on mean arterial blood pressure (MAP) in
Figure 4. Dose-response of (S)-5 in WAG/Rij rat model of absence
epilepsy, n g 6 rats per dose with PK taken from satellite animals, n
conscious dogs.
)
3 per group except 3 mg/kg group where n ) 1.
Figure 5. Dose-response of (S)-5 in harmaline-induced rat model of
essential tremor.
Figure 7. Effect of (S)-5 on heart rate (HR) in conscious dogs.

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3695
renal plasma flow, urine flow, urinary excretion of sodium and
potassium, or plasma electrolytes. While blood pressure and
ECG intervals were unaffected in this assay, increased heart
rate and minor behavioral changes (panting) were observed.
Peak plasma concentrations of (S)-5 were 34.5 ( 11.0 µM
during the experiment.
In conclusion, we identified (S)-5, a potent and selective
T-type calcium channel antagonist, starting from 1,4-disubsti-
tuted piperidine HTS leads. The introduction of a fluorine atom
at the 4-position served to improve the ancillary pharmacological
profile and to enhance metabolic stability. Robust efficacy was
seen in WAG/Rij epilepsy and harmaline-induced tremor models
at plasma levels well below the no effect level seen in
cardiovascular dog experiments. This suggests a good margin
between CNS and peripheral effects of selective T-type calcium
channel antagonists. Compounds such as (S)-5 hold promise
for the treatment of a diverse set of neurological indications
without adversely affecting cardiovascular function.
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(
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Cheng, H.; Yang, N.; Barrett, P. Q.; Perez-Reyes, E. Validation of
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v
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DeV. Technol. 2007, 5, 191–203.
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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 Susan Garson for technical
support of this work.
Supporting Information Available: Spectral and analytical data
for new compounds, a scheme detailing the preparation of 5, FLIPR
assay protocol, Na 1.5 electrophysiology measurements, dog renal
v
function assay protocol, and expanded references. This material is
available free of charge via the Internet at http://pubs.acs.org.
5
-HT1D receptor ligands with improved pharmacokinetic profiles.
J. Med. Chem. 1999, 42, 2087–2104.
(
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JM800419W