Discovery and evaluation of Cav3.1-selective T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2017.09.063

We identified and characterized a series of pyrazole amides as potent, selective Ca_v3.1-blockers. This series culminated with the identification of pyrazole amides 5a and 12d, with excellent potencies and/or selectivities toward the Ca_v3.2- and Ca_v3.3-channels. This compound displays poor DMPK properties, making its use difficult for in vivo applications. Nevertheless, this compound as well as analogous ones are well-suited for in vitro studies.

Full text of DOI:10.1016/j.bmcl.2017.09.063

Accepted Manuscript
Discovery and evaluation of Cav3.1-selective T-type calcium channel blockers
Olivier Bezençon, Luboš Remeň, Sylvia Richard, Catherine Roch, Melanie
Kessler, Richard Moon, Jacques Mawet, Eric A. Ertel, Thomas Pfeifer, Bruno
Capeleto
PII:
S0960-894X(17)30971-X
https://doi.org/10.1016/j.bmcl.2017.09.063
BMCL 25327
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 August 2017
27 September 2017
29 September 2017
Please cite this article as: Bezençon, O., Remeň, L., Richard, S., Roch, C., Kessler, M., Moon, R., Mawet, J., Ertel,
E.A., Pfeifer, T., Capeleto, B., Discovery and evaluation of Cav3.1-selective T-type calcium channel blockers,
Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.09.063
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Discovery and evaluation of Cav3.1-selective
T-type calcium channel blockers
Leave this area blank for abstract info.
Olivier Bezençon∗, Luboš Remeň, Sylvia Richard, Catherine Roch, Melanie Kessler, Richard Moon, Jacques
Mawet, Eric A. Ertel, Thomas Pfeifer, and Bruno Capeleto

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery and evaluation of Cav3.1-selective T-type calcium channel blockers
Olivier Bezençon∗, Luboš Remeň, Sylvia Richard, Catherine Roch, Melanie Kessler, Richard Moon,
Jacques Mawet, Eric A. Ertel, Thomas Pfeifer, and Bruno Capeleto
Drug Discovery Chemistry, Biology and Pharmacology, Idorsia Pharmaceuticals Ltd., Hegenheimermattweg 91, CH-4123 Allschwil
ARTICLE INFO
ABSTRACT
Article history:
Received
We identified and characterized a series of pyrazole amides as potent, selective Cav3.1-blockers.
This series culminated with the identification of pyrazole amides 5a and 12d, with excellent
potencies and/or selectivities toward the Cav3.2- and Cav3.3-channels. This compound displays
poor DMPK properties, making its use difficult for in vivo applications. Nevertheless, this
compound as well as analogous ones are well-suited for in vitro studies.
Revised
Accepted
Available online
2009 Elsevier Ltd. All rights reserved
.
Keywords:
T-type calcium channel
Selective Cav3.1-blockers
Pyrazoles
———
∗ Corresponding author. Tel.: +41 61 565 65 77; fax: +41 61 565 65 00; e-mail: olivier.bezencon@idorsia.com

A first generation of T-channel blockers was developed in the
1990s, and culminated with the discovery of mibefradil. This
drug, developed at Roche for angina pectoris, and meanwhile
withdrawn from the market due to unacceptable drug-drug
interactions, is a rather potent, but unselective T-type calcium
channel blocker.1 A second generation of T-type calcium channel
blocker arose during the last years, with two compounds, MK-
89982 and Z-944,3 entering clinical trials. These compounds are
selective T-type calcium channel blockers, meaning that they
block the Cav3.1, Cav3.2, and Cav3.3 channels with similar
potencies, while they do not block other channels.
H
H
N
N
N
N
N
R
N
O
O
O
2a 2c
-
1
Fig 1. First pyrazole-based Cav3.1-blockers
Table 1. Potencies of a few T-channel blockers (FLIPR®)
We recently disclosed a series of dihydropyrazole derivatives
as selective T-type calcium channel antagonists4, and
Compound
R-
substituent
Cav3.1
Cav3.2
Cav3.3
Ratio
demonstrated their in vivo efficacy in two animal models of
epilepsy, the WAG/Rij rat model for absence seizures.
Simultaneously, we observed in spontaneously hypertensive rats
that these compounds induced a prolongation of the PR interval
on ECG. Due to the excellent PK/PD correlation and a strong
parallelism with the desired antiepileptic effect, we hypothesized
that this PR prolongation was directly linked to the blockade of
all T-type calcium channel subtypes in the rodent heart. We
conjectured that we might be able to dissociate both effects (i.e.
desired antiepileptic effect without PR prolongation) by applying
subtype selective T-type calcium channel blockers, i.e.
compounds that would block one of the three T-type calcium
channel only. A certain subtype selectivity was possible to
achieve. In this paper, we describe the discovery and the study of
Cav3.1-subtype selective blockers, whereas Cav3.2-subtype
selective blockers were described in the previous paper.
Furthermore, we described recently a compound with a moderate
selectivity for the Cav3.3 channel.5
IC50
(nM)a
IC50
(nM) a
IC50
(nM) a
Cav3.2/
Cav3.1
1
-
2100
200
130
59
5800
510
9000
-
2a
2b
2c
-H
>10000
>10000
>10000
2.6
15
17
-F
1900
1000
CF3CH2O-
aGeometric mean of at least two measurements
In a next step, we explored at first the position 4 of the
pyrazole ring. From commercial starting material, a bromination
led to compound 3 (Scheme 1). Saponification and amide
coupling led to a first derivative 4a. Formylation, reduction, and
fluorination led to compounds 4b, 4c, and 4d, respectively.
Vinylation of compound 4a led to derivative 4e, which was
transformed into compounds 4f and 4g via hydroboration and
oxidation. Eventually, reductive aminations of compounds 4g led
to secondary and tertiary amines 5a – 5c.
In the previous paper, we described a series of pyrrole-based
Cav3.2-subtype selective blockers. In an effort to increase the
polarity of such compounds, we investigated pyrazole analogues
from this series. Indeed, compound 16 (Fig. 1) had appeared as a
weak hit in an HTS campaign (Table 1). Two other hits,
Table 2. Potencies of first Cav3.1-subtype selective blockers
(FLIPR®)
Compound
R1R2N-
substituent
Cav3.1
Cav3.2
Cav3.3
Ratio
IC50
(nM)a
IC50
(nM) a
IC50
(nM) a
Cav3.2/
Cav3.1
compounds 2a and 2b, prepared via an amide coupling from
commercially available starting material, proved to be
4a
4c
4d
4e
4f
-
20
55
49
71
46
52
200
350
410
820
850
310
280
590
>10000
1370
17
moderately potent Cav3.1- and Cav3.2-blockers, with no
measurable potency against Cav3.3, this in in the FLIPR® assay
we had been using previously4,5 (Table 1). In particular, an ortho-
substituent seemed to confer a good subtype selectivity for the
Cav3.1 channel. In a first SAR study at the amide position, we
introduced an ortho-trifluoromethoxy substituent that had been
successful in the development of Cav3.2-blockers, as described in
the previous manuscript. Compound 2c proved to be a slightly
more potent and subtype selective Cav3.1-blocker. Compound 2c
was confirmed using patch-clamp (>99%, 83%, and 39% block
of Cav3.1, Cav3.2, and Cav3.3 at 10000 nM, respectively).
-
7.4
17
-
>10000
>10000
650
-
12
-
6.8
5.3
2.9
5a
5b
EtNH-
1900
Cyclopropyl-
NH
900
5c
Et2N-
240
2150
4370
9.0
aGeometric mean of at least two measurements

Scheme 1: Preparation of Cav3.1-subtype selective blockers 4a-4g and 5a-5c. (a) NBS, AcOH, microwave, 150 °C, 10 min, 87%; (b)
(i) 2.5M aq. NaOH, EtOH, reflux, 1 h; (ii) ortho-trifluoroethoxybenzylamine, EDC.HCl, HOBt, DMAP, DIPEA, CH2Cl2, rt, overnight,
50% over two steps; (c) 3M MeMgCl in THF, 1.6M tBuLi in pentanes, DMF, THF, -78°C to rt, 2 h, 46%; (d) NaBH4, MeOH, rt, 1 h,
73%; (e) DAST, CH2Cl2, rt, 2 weeks, 39%; (f) vinylboronic anhydride pyridine complex, Pd(tBu3P)2, K2CO3, H2O, dioxane, 80 °C,
overnight, 85%; (g) 0.5M 9-BBN in THF, 70 °C, overnight, 6M aq. NaOH, 30% H2O2, 50 °C, 1 h, 93%; (h) Dess-Martin periodinane,
CH2Cl2, 0 °C to rt, 3 h, 64%; (j) amine, NaBH(OAc)3, CH2Cl2, rt, 6 h, 71-94%.
The bromine substituent (compound 4a) increased slightly the
potency on the Cav3.1- and Cav3.2-channel compared to
compound 2c, while no potency was measurable on the Cav3.3-
channel. Overall, subtype selectivity toward the Cav3.2-channel,
the most difficult one to achieve, was maintained. From
compounds 4c – 4f, we concluded that polarity at this position is
not an important factor for the absolute potency on the Cav3.1-
channel. On the other hand, a polar primary hydroxyl substituent
tended to decrease selectivity toward the Cav3.3 channel. In a
further effort to improve polarity, we introduced primary and
secondary amines (compounds 5a – 5c). Here again, a polar ethyl
amine (5a) retained the potency toward Cav3.1 with a low
selectivity toward Cav3.2. The other amines, (5b and 5c) led to a
loss in potency.
Saponification and amide couplings delivered amides 10a-10f,
which were subsequently formylated (11a-11f) and reductively
aminated to compounds 12a-12f. Unfortunately, we were not
able to develop a chemistry that would allow the introduction of
diversity at position 1 at a later stage of the synthesis.
Considering the SAR, we focused our efforts by fixing the
substituent at position 4 to a bromine or to a difluoromethyl,
since the highest level of selectivity was achieved with them.
Introduction of a benzyl group at position 1 led to a loss of
potency (Table 3, compound 10a) and the presence of the small
methyl group led to compound 10e, with very similar potencies
as was measured with the benzyl group. On the other hand,
introducing a somewhat larger trifluoroethyl group (compound
10f) led to an increase in potency and selectivity. Overall, most
potent and selective compounds were obtained from an ortho-
fluorophenyl substituent (compound 10b). Other substitution
pattern led to a loss in potency. Generally, the difluoromethyl
sub-series (compounds 12a-12f) were less potent than their
bromo analogues (compounds 10a-10f). Finally, the excellent
selectivity ratio of compound 12d should be noted.
With these results in hand, we investigated the SAR at
position 1 of the pyrazole ring. For this, we prepared numerous
derivatives as depicted in Scheme 27. Cyclization between ethyl
oxaloacetate and the corresponding hydrazine led to compounds
6a-6f. Triflation (7a-7f), Suzuki couplings (8a-8f) and
bromination led to fully substituted pyrazoles 9a-9f.

Scheme 2: Preparation of compounds 10a-10f and 12a-12f. (a) RNHNH2, AcOH, H2O, toluene, reflux, overnight, 58-92%; (b) Tf2NPh,
Et3N, THF, 0 °C to rt, 1 h, 58-82%; (c) PhB(OH)2, Pd(PPh3)4, 2M aq. Na2CO3, toluene, 95 °C, overnight, 54-85%; (d) NBS, AcOH,
microwave, 150 °C, 10 min, 59-86%; (e) (i) 2.5M aq. NaOH, EtOH, reflux, 1 h, (ii) ortho-trifluoroethoxybenzyyl amine, EDC.HCl,
HOBt, DMAP, DIPEA, CH2Cl2, rt, overnight, 30-50%; (f) 3M MeMgCl in THF, 1.6 M tBuLi in pentanes, DMF, THF, -78°C to rt, 2 h,
35-58%; (g) DAST, CH2Cl2, rt, 2 weeks, 25-56%.
12c
12d
12e
12f
m-F-Ph-
p-F-Ph-
Me-
360
160
250
410
1500
5000
370
-
4.2
>10000 31
890
1.5
1.5
CF3CH2-
600
1400
aGeometric mean of at least two measurements
Table 3. Pyrazole derivatives 10a-f and 12a-f: SAR (FLIPR®)
Compound
R-
substituent
Cav3.1
Cav3.2
Cav3.3
Ratio
Modifications at position 5 were possible applying Suzuki
couplings in a similar manner as described in Scheme 2.
Unfortunately, such modifications let only to limited
improvement in terms of potency. The introduction of a
cyclopropyl group (compound 13a, Table 4), or of an ethyl group
(compound 13b) led to moderately potent derivatives with almost
no subtype-selectivity.
IC50
(nM)a
IC50
(nM) a
IC50
(nM)a
Cav3.2/
Cav3.1
10a
10b
10c
10d
10e
10f
Bn-
335
28
1450
300
580
410
220
890
170
1100
>10000 4.3
>10000 11
>10000 2.6
>10000 4.7
o-F-Ph-
m-F-Ph-
p-F-Ph-
Me-
220
88
Having optimized the substituents at the pyrazole ring, we
reinvestigated the amide substituent, focusing on more polar
heteroaryl systems (compounds 13c – 13e in Table 4). Again,
with this approach we were unable to improve potency and
selectivity with regard to the former compounds.
280
88
2850
1500
0.8
10
CF3CH2-
Bn-
12a
12b
250
190
>10000 0.7
8600 5.8
o-F-Ph-
Table 4. Last pyrazole derivatives (FLIPR®)

Compound
Cav3.1
IC50 (nM)a
100
Cav3.2
IC50 (nM) a
290
Cav3.3
IC50 (nM) a
110
Ratio
Cav3.1/Cav3.2
13a
13b
13c
13d
13e
2.8
3.4
12
110
380
1700
32
400
1800
45
380
>10000
>10000
8.5
10
58
600
a Geometric mean of at least two measurements.
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With apolar substituents only being tolerated for potency, the
compounds were bound with poor physicochemical and ADME
properties. Most compounds were quasi-insoluble in aqueous
medium at pH 7 (< 1mg/L). Compounds 5a – 5c, bearing an
aliphatic amine, formed an exception. In particular, compound 5a
displayed a solubility of 429 mg/L in an aqueous medium at pH 7
and a rat liver microsomal intrinsic clearance (RLM) of 377
µL/min.mg protein. We decided to administer this compound in
vivo to Wistar rats at 100 mg/kg po, and measured plasma and
brain concentrations after 1 h. Under these conditions, we
reached a plasma concentration of 810 nM (Cu,plasma = 4.9 nM),
and a total brain concentration of 80 17 nM. Under these
conditions the unbound plasmatic concentration remained below
the IC50-values, and the brain exposure was marginal, precluding
any in vivo pharmacological experiment. Compound 5a also
suffered from selectivity issues toward other channels. IC50-value
for the Cav1.2-channel was measured at 860 nM, and this
compound was strongly blocking sodium channels in rat cortical
neurons at 10 µM (72% and >99% blockade at the first and last
peak respectively). Under the same conditions, the slow- and fast
potassium channels were blocked 49% and 44% respectively.
In conclusion, we designed potent Cav3.1-blockers with
selectivity ratio up to 13 toward the Cav3.2 channel, and even
better toward the Cav3.3 channel. These compounds also block
partially sodium and potassium channels in organotypic slices. In
vivo exposition of compound 5a remains modest, nevertheless
this class of compounds can be used for in vitro studies.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We would like to thank Martin Faes, Sophie Moujon, Sven
Glutz, Claire Hinder, Jacques Mawet, Héléne Roellinger, Hélène
Massinet, Eileen Hubert, Alexandre Hasler, Isabelle Reymond,
and Johannes Mosbacher for their contribution to the results
presented here.
Abbreviations
AcOH: acetic acid; 9-BBN: 9-borabicyclo(3.3.1)nonane; DAST:
diethylaminosulfur trifluoride; DIPEA: N,N-
diisopropylethylamine; DMAP: 4-(dimethylamino)pyridine;
DMF: N,N-dimethylformamide; EDC.HCl: 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; HOBt:
Hydroxybenzotriazole; NBS: N-bromosuccinimide; tBu: tert-
butyl; Tf: trifluorosulfonyl; THF: tetrahydrofuran.
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