Synthesis and evaluation of α,α′-disubstituted phenylacetate derivatives for T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2008.06.037

We have synthesized and evaluated α,α′-disubstituted phenylacetate derivatives that were designed as T-type calcium channel blockers. Among them, compound 10e (IC₅₀ = 8.17 ± 0.48 nM) showed the most potent T-type calcium current blocking activity and higher potency than Mibefradil (IC₅₀ = 1.34 ± 0.49 μM). The PK profile and subtype selectivity over L-type calcium channel were satisfied for further animal assay using disease model.

Full text of DOI:10.1016/j.bmcl.2008.06.037

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4424–4427
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a,
a0-disubstituted phenylacetate derivatives
for T-type calcium channel blockers
Hyung Kook Lee a,b, Yun Suk Lee a, Eun Joo Roh a, Hyewhon Rhim a, Jae Yeol Lee b, Kye Jung Shin a,
*
a Life Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongyang, Seoul 130-650, Republic of Korea
b Research Institute for Basic Sciences and Department of Chemistry, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
We have synthesized and evaluated a,
a0-disubstituted phenylacetate derivatives that were designed as
T-type calcium channel blockers. Among them, compound 10e (IC50 = 8.17 0.48 nM) showed the most
potent T-type calcium current blocking activity and higher potency than Mibefradil (IC50 = 1.34
Article history:
Received 2 April 2008
Revised 4 June 2008
Accepted 11 June 2008
Available online 14 June 2008
0.49
lM). The PK profile and subtype selectivity over L-type calcium channel were satisfied for further
animal assay using disease model.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
T-type calcium channel
Neuropathic pain
Patch-clamp
Voltage-dependent calcium channels have crucial roles in trans-
lating electrical signals into biochemical events such as enzyme
activity, neurotransmitter release, neuronal excitability, neurite
outgrowth, and gene transcription.1 They are subdivided into two
major classes, high-voltage activated (HVA or L-type) and low-
voltage activated (LVA or T-type) calcium channels based on their
biophysical and pharmacological properties. Mibefradil (PosicorÒ,
Hoffman-La Roche) which was launched in 1998 for the treatment
of hypertension and angina pectoris,2–4 blocks T-type calcium
channels at concentration lower than that needed to block L-type
calcium channels.5,6 Unfortunately, the drug was withdrawn from
the market due to drug–drug interaction with antihistamine such
as astemizole, but this side effect is not related to T-type calcium
channel blockade.7,8 T-type calcium channels are involved in car-
diac pacemaking,9 regulation of vascular tone, and secretion of var-
ious types of hormones.10–13 Thus, T-type calcium channel is now
considered to be a novel therapeutic target for the treatment of
cardiovascular, neuronal, and endocrine systems.14 Currently
several compounds including Mibefradil analogues15 have been
reported to inhibit T-type calcium channels; however, none of
them have shown high potency and selectivity to this channels.
In this study, we designed and synthesized compounds with po-
tency and selectivity to T-type calcium channel using the 3D ligand
based pharmacophore model, which was generated by hypothesis
approach (HipHop) implemented in CATALYST program16,17
(Fig. 1). As a result of in vitro inhibition assay, most of compounds
showed higher inhibition activities than Mibefradil. Especially,
compound 10e showed the most potent T-type calcium channel
blocking activity and good selectivity over L-type calcium channel.
10a–g and 11a–j were synthesized via a routine procedure
shown in Schemes 1 and 2. Aminobenzimidazole intermediates
5a–c were prepared from 4-methylaminobutyric acid 1.18 Protec-
tion of the secondary amine with benzyloxycarbonyl (Cbz) affor-
ded compound 2, which was then treated with isobutyl
chloroformate and o-phenylenediamines to provide compounds
3a–c in 59–94% yields. They were treated with p-toluenesulfonic
acid in toluene under reflux condition to give cyclized benzimidaz-
oles 4a–c in 33–77% yields. Deprotection of Cbz group from the
secondary amines with 10% Pd/C gave compounds 5a–c in
69–95% yields.
The methods for the preparation of the title compounds were
shown in Scheme 2. Starting from phenylacetic acids 6a–c, the car-
boxylic acids were esterified to make esters 7a–c in 90–95% yields,
which were alkylated with t-BuOK and isopropyl bromide to give
compounds 8a–c. Compounds 9a–c were obtained via treating
8a–c with LDA and dibromopropane in THF under anhydrous con-
dition. The coupling reaction of 9a–c with previously prepared
benzimidazole derivatives 5a–c and commercially available piper-
azine derivatives under basic condition (K2CO3, EtOH) produced
compounds 10a–g in 20–46% yields and compounds 11a–j in
47–69% yields, respectively.
In vitro calcium channel blocking activities of 10a–g and 11a–j
were tested with T-type calcium channels expressed in HEK293
cells (a1G). All the compounds exhibited promising activities on
a1G calcium channels expressed in HEK293 cells at 10
lM concen-
tration by the whole-cell patch-clamp method in a preliminary
* Corresponding author. Tel.: +82 2 958 5154; fax: +82 2 958 5189.
E-mail address: kjshin@kist.re.kr (K.J. Shin).
assay.19,20 Then the molar concentrations needed to produce 50%
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.037

H. K. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4424–4427
4425
O
O
O
R'
R
R
N
N
O
O
O
Y
O
N
N
N
N
N
H
HN
F
X
X
11a-j
10a-g
Mibefradil
R=Me, Et
R'=various benzyl or phenyl
X=H, Br, OCH3
Y=H, OCH3, di-Me
Figure 1. Designed structures for T-type calcium channel blocker and mapping result of suggested pharmacophore with 10a.
R1
R2
O
Cbz
N
O
Cbz
N
O
H
i
ii
N
OH
OH
N
H
NH2
2
1
3a-c
R1
R1
R2
R2
iii
iv
Cbz
N
N
N
H
N
N
N
H
H
4a-c
5a-c
R1, R2 = H
1 = H, R2 = OMe
R1, R2 = Me
R
Scheme 1. Reagents and condition: (i) benzyl chloroformate, NaOH, EtOH/H2O (1:1), 0 °C to rt, 63%; (ii) isobutyl chloroformate, o-phenylenediamines, Et3N, THF, ꢀ15 °C to rt;
(iii) p-TsOH, toluene, reflux; (iv) H2, 10% Pd/C, MeOH, rt.
OR4
OH
OR4
ii
i
O
O
R3
R3
O
R3
6a-c
7a-c
8a-c
R1
O
R2
N
OR4
iv
N
N
H
R3
O
OR4
10a-g
Br
iii
R3
R5
O
N
OR4
9a-c
R3 = H, Br, OMe
v
N
R3
R
4 = Me, Et
11a-j
Scheme 2. Reagents and condition: (i) H2SO4, MeOH, reflux; (ii) isopropyl bromide, t-BuOK, DMF, 0 °C to rt; (iii) LDA, 1,3-dibromopropane, THF, ꢀ78 °C to rt; (iv) compound
5, K2CO3, EtOH, reflux; (v) 1-substituted piperazine derivatives, Et3N, NaI, CH3CN, reflux.
inhibition of peak currents (IC50) were measured and summarized
in Tables 1 and 2 with Mibefradil as a positive control for compar-
ison. According to the assay results, most of derivatives exhibited
significant inhibitory activities on HEK293 cells and numbers of

4426
H. K. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4424–4427
Table 1
In vitro calcium channel blocking effects of benzimidazole derivatives
R1
O
R2
N
OR4
N
N
H
R3
10a-g
Compound
R1
R2
R3
R4
Patch-clamp HEK293 cell (T-type
a
1G, n = 3)21
IC50 M)
% Inhibition (10
l
M)
(l
10a
10b
10c
10d
10e
10f
10g
Mibefradil
H
Me
H
OMe
H
OMe
Me
H
Me
H
H
H
H
H
OMe
OMe
Br
Et
Et
98.3 0.9
91.4 0.4
93.5 3.2
91.7 1.9
96.9 0.5
94.5 1.2
0.25 0.005
0.10 0.01
0.19 0.001
0.88 0.07
8.17 0.48 (nM)
0.25 0.01
53.02 4.87 (nM)
1.34 0.49
Me
Me
Me
Me
Me
H
Me
Br
Br
94.2 0.4 (1
95.9 1.7
lM)
Table 2
In vitro calcium channel blocking effects of piperazine derivatives
R5
O
N
OR4
N
R3
11a-j
Compound
R3
R4
R5
Patch-clamp HEK293 cell (T-type
a
1G, n = 3)21
M)
% Inhibition (10 M) IC50 (l
l
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
Mibefradil
H
H
Et
Et
4-Methoxybenzyl
4-Fluorobenzyl
2-Fluorobenzyl
3-Fluorobenzyl
4-Fluorobenzyl
3-Trifluorobenzyl
2-Methoxyphenyl
4-Methoxybenzyl
96.3 1.7
94.0 2.5
90.4 3.2
89.3 1.6
96.5 1.7
93.0 1.5
94.1 1.5
96.0 1.7
95.1 1.9
94.9 1.5
95.9 1.7
0.34 0.02
0.26 0.03
0.74 0.02
0.98 0.11
1.11 0.05
0.28 0.02
95.04 14.78 (nM)
0.48 0.08
0.32 0.01
0.39 0.03
1.34 0.49
Br
Br
Br
Br
Br
Br
Br
Br
Me
Me
Me
Me
Me
Me
Me
Me
2,3,4-Trimethoxybenzyl
3-Methylbenzyl
compounds showed excellent blocking efficacy. Especially, com-
pounds 10e, 10g, and 11g had 14- to 160-fold higher potency than
Mibefradil.
Table 4
Plasma and brain pharmacokinetic parameters in mouse obtained via a single oral
dose
Among them, 10e22 exhibited the most potent T-type calcium
current blocking activity (8.17 0.48 nM). Thus, further evalua-
tions of 10e such as hERG inhibition, pharmacokinetic profile
assay, and selectivity test over L-type calcium channel were carried
out. According to the hERG inhibition assay, IC50 of 10e was
178 nM (Table 3).23 Although the absolute IC50 value of hERG inhi-
bition was low, the relative ratio over two channels was moder-
10e
Brain
Plasma
B/Pa
Doseb (mg/kg)
Tmax (h)
50
50
1.0
1615
3902
1.0
274
973
Cmax (ng/ml)
0.17
0.25
c
AUC0–t (h ng/ml)
a
B/P = AUCbrain/AUCplasma
.
b
ately high (hERG IC50/T-type a1G IC50 = 21.8). Moreover,
%
The vehicle of PK experiment: DMA/Cremophor EL/PBS (10/10/80 v/v %).
Area under curve (AUC) values were calculated by linear trapezoidal rule.
c
inhibition value against L-type calcium channel at 1 M of 10e
l
was 0%. It means that 10e could distinguish T-type and L-type
calcium channel effectively and block T-type calcium channel
selectively (T-type/L-type > 1000). In order to examine in vivo
dynamics, 10e was subjected to pharmacokinetics analysis in
mouse. 10e was administered as single oral dose (50 mg/kg) and
evaluated in the plasma and brain. Based on the pharmacokinetic
data, compound 10e exhibited competitive pharmacokinetic pro-
files in plasma. It is inferred that 10e is metabolically stable in liver
enzymes (T1/2 = 1 h) and has comparable absorption in gastrointes-
Table 3
Selectivity data of compound 10e against hERG and L-type calcium channel
Compound T-type IC50 (nM) hERG IC50
10e 8.17 0.178
(l
M) % Inhibition of L-type at 1
0
lM

H. K. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4424–4427
4427
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tinal system. Also, 10e could penetrate BBB and reach to brain in
25% ratio compared to plasma (Table 4).
In summary, 10a–g and 11a–j were designed and synthesized
based on pharmacophore mapping study and most of compounds
showed higher T-type calcium channel inhibition activities than
Mibefradil. Among them, compound 10e exhibited the most potent
T-type calcium current blocking activity and good pharmacokinetic
profiles. Furthermore, 10e showed excellent selectivity over L-type
calcium channel. Therefore, the series of title compounds were
suggested to elucidate the potential of new therapeutics for T-type
calcium channel associated disease such as hypertension, neuro-
pathic pain, and migraine based on the fundamental etiology.
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Acknowledgments
21. Experimental procedure for patch-clamp (electrophysiological recording).
For the recordings of a1G T-type Ca2+ currents, the standard whole-cell
patch-clamp method was utilized. Briefly, borosilicate glass electrodes
The authors express thanks to Prof. Seong-Woo Jeong and Dr. Ki
Ho Lee for their contributions to subtype selectivity and PK profil-
ing. This work was financially supported by a grant (2E 20430)
from KIST vision 21 program.
with
solution contained (in mM): 130 KCl, 11 EGTA,
Hepes (pH 7.4). The external solution contained (in mM): 140 NaCl,
CaCl2, 10 Hepes, and 10 glucose (pH 7.4). a1G T-type Ca2+ currents were
evoked every 15 s by
50 ms depolarizing voltage step from ꢀ100 mV
to ꢀ30 mV. The molar concentrations of test compounds required to
produce 50% inhibition of peak currents (IC50 were determined from
a
resistance of 3–4 M
X
were pulled and filled with the internal
5
Mg–ATP, and 10
2
a
References and notes
)
fitting raw data into dose–response curves. The current recordings were
obtained using an EPC-9 amplifier and Pulse/Pulsefit software program
(HEKA, Germany).
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22. Compound 10e: 1H NMR (400 MHz, CDCl3) d 7.45 (br s, 2H), 7.39 (d, J = 8.6 Hz,
2H), 7.18 (dd, J = 6.0, 3.1 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 3.69 (s, 3H), 3.04 (t,
J = 6.4 Hz, 2H), 2.48 (t, J = 5.8 Hz, 2H), 2.35–2.40 (m, 3H), 2.21 (s, 3H), 2.04–2.10
(m, 1H), 1.89–1.98 (m, 3H), 1.23–1.43 (m, 2H), 0.85 (d, J = 6.8 Hz, 3H), 0.74 (d,
J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 175.0, 155.4, 138.6, 130.7, 130.1,
121.7, 120.5, 59.0, 58.4, 58.2, 51.7, 41.5, 34.6, 33.9, 28.9, 24.2, 22.7, 18.9, 18.0.;
Anal. (C26H34BrN3O22HCl) Calcd: C, 54.46; H, 6.33; N, 7.33. Found: C, 54.16; H,
6.23; N, 7.61.
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