T-type Ca2+ channel blockers suppress the growth of human cancer cells

Article abstract of DOI:10.1016/j.bmcl.2008.06.034

In order to further clarify the role of T-type Ca²⁺ channels in cell proliferation, we have measured the growth inhibition of human cancer cells by using our potent T-type Ca²⁺ channel blockers. As a result, KYS05090, a most potent T-type Ca²⁺ channel blocker, was found to be as potent as doxorubicin against some human cancer cells without acute toxicity. Therefore, this letter provides the biological results that T-type calcium channel is important in regulating the important cellular phenotype transition leading to cell proliferation, and thus novel T-type Ca²⁺ channel blocker presents new prospects for cancer treatment.

Full text of DOI:10.1016/j.bmcl.2008.06.034

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3899–3901
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
T-type Ca2+ channel blockers suppress the growth of human cancer cells
Jae Ho Heo a, Han Na Seo a, Yun Jeong Choe a, Sujin Kim a, Chun Rim Oh b, Young Deuk Kim b,
Hyewhon Rhim c, Dong Joon Choo a, Jungahn Kim a, Jae Yeol Lee a,
*
a Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung Hee University, 1 Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, Republic of Korea
b DongWoo Syntech Co., Ltd, Bondae-Ri Geumwang-Eup, Eumsung-Goon, Choongcheungbook-Do 369-901, Republic of Korea
c Life Sciences Division, Korea Institute of Science & Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
In order to further clarify the role of T-type Ca2+ channels in cell proliferation, we have measured the
growth inhibition of human cancer cells by using our potent T-type Ca2+ channel blockers. As a result,
KYS05090, a most potent T-type Ca2+ channel blocker, was found to be as potent as doxorubicin against
some human cancer cells without acute toxicity. Therefore, this letter provides the biological results that
T-type calcium channel is important in regulating the important cellular phenotype transition leading to
cell proliferation, and thus novel T-type Ca2+ channel blocker presents new prospects for cancer
treatment.
Article history:
Received 20 May 2008
Revised 6 June 2008
Accepted 11 June 2008
Available online 14 June 2008
Keywords:
T-type calcium channel blocker
3,4-Dihydroquinazoline
Doxorubicin
Ó 2008 Elsevier Ltd. All rights reserved.
Anti-cancer
Calcium plays a critical role as an intracellular signal and con-
trols many different cell processes,1 of which intracellular calcium
(Ca2+) regulates proliferation, differentiation, growth, cell death,
and apoptosis.2 Thus, alterations in Ca2+ signaling can be the cause
of defects in cell growth and are associated with some cancers. A
number of research groups have suggested a potential role of
T-type Ca2+ channels in controlling cell proliferation.3–8 If a prolif-
eration is a principal characteristic of cancer cells, therefore, a
modulator of T-type Ca2+ channels in cancer cells responsible for
proliferation is of potential clinical significance. For example, the
known inhibitors of T-type Ca2+ channels, for example, both mibe-
fradil and pimozide, have been demonstrated to be effective in
decreasing cell proliferation in normal cells as well as breast cancer
cells (Fig. 1).9–15 In addition, it is recently reported that inhibition
of T-type channels reduces cell proliferation via a p53-dependent
p21CIP1 pathway in certain esophageal carcinomas.16 In light of
these findings, we have recently demonstrated that through the as-
say of eight compounds showing a broad range of channel blocking
channel blocking activity of selected compounds was evaluated
against HEK293 cells which stably express both T-type calcium
channel Cav3.1 with a1G subunit and potassium channel Kir2.1.23
The selected compounds were again evaluated for their growth
inhibition against five human cancer cell lines [human lung carci-
noma (A549), human prostate cancer (DU 145), human colon can-
cer (HT-29), human malignant melanoma (SK-MEL-2), and human
ovarian cancer (SK-OV-3)] using sulforhodamine B (SRB) assay.24
Table 1 provides the results of assays used to measure the inhi-
bition of calcium influx and the growth inhibition of human cancer
cells by the compounds as illustrated in Figure 1. The entry of com-
pounds was arranged in order of the channel blocking activity for
the easy data comparison.
Doxorubicin was used as a reference for anti-cancer activity and
also tabulated with data for comparison. Before discussing the
results, we have already confirmed that most compounds showed
little cytotoxicity on HEX293 cell at 100 lM concentration except
KYS05080, which showed the serious cytotoxicity at that concen-
tration. From these data, a brief relationship profile emerged as fol-
lows: first, most compounds showed the linear correlation
between cell growth inhibitory activity and T-type channel block-
ing effect except KYS05055 and KYS05056, which showed no anti-
cancer activity against three cell lines (A549, DU 145, and SK-OV-3)
and the poor activity against another cell lines (HT-29 and SK-MEL-
2) although they show the good channel blocking effect. Second,
most compounds exhibited the similar growth inhibitory activities
against five cancer cells except for compounds KYS05055 and
KYS05056. Among compounds, KYS05090, the most potent chan-
nel blocker, showed the best activity (their GI50 values are less than
effects (34–91% inhibition at 10 lM concentration), there is a close
connection between channel blocking effect and growth inhibition
of human cancer cells.17
As a continuous study, we have set out to select and screen 13
compounds showing the good inhibitory activity (its IC50 is less
than 1.0 lM) against calcium influx from our library of compounds
as illustrated in Figure 2.18–22 The general synthetic method of 3,4-
dihydroquinazoline compounds is described in Scheme 1.20 The
* Corresponding author. Tel.: +82 2 961 0726; fax: +82 2 966 3701.
E-mail address: ljy@khu.ac.kr (J.Y. Lee).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.034

3900
J. H. Heo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3899–3901
O
O
O
OH
OH
OH
F
OCH3
H3C
CH3
O
H
OH
O
N
N
N
O
HN
CH3O
O
O
N
N
CH3
F
CH3
F
H2N OH
mibefradil
pimozide
doxorubicin
Figure 1. Structures of mibefradil, pimozide, and doxorubicin.
O
O
O
O
N
H
O
N
H
O
O
O
S
MeO
N
H
S
N
N
N
N
CH3
H
H
CH3
N
N
N
N
N
N
Boc
Boc
CH3
N
N
H
N
N
N
N
H
H3
C
F
H
H
KYS05065
KYS05046
KYS05057
KYS05085
NH2
O
O
O
O
N
H
N
H
N
H
MeO
H2
N
N
O2N
N
N
N
CH3
CH3
N
N
N
N
N
N
N
N
N
CH3
CH3
CH3
H
KYS05056
KYS05055
KYS05043
KYS05080
Boc
HN
O
O
O
O
N
H
O
O
H3CO
N
N
S
H
H
N
N
N
H
N
N
CH3
N
N
N
O
N
N
CH3
Boc
N
N
N
N
H
N
CH3
H
H
F
KYS05089
KYS05047
KYS05048
KYS05042
N
H
N
CH3
N
N
N
CH3
CH3
KYS05090
Figure 2. 3,4-Dihydroquinazoline derivatives studied in SRB assay.
O
O
O
O
a
b
c
OCH3
OCH3
OCH3
OCH3
R1
NO2
NH2
NH
NH
R1
N C N
O
1
2
3
4
O
O
O
CH3O
HO
N
H
R1
R2
R1
R2
R1
R2
d
e
f
g
N
N
R3
N
N
N
N
5
6
7
O
O
N
H
N
O
O
H
R1
R2
S
R1
N
h
H2N
N
N
H
N
R4
N
R2
8
9
Scheme 1. Reagents and conditions: (a) SnCl2Á2H2O, EtOAc, 70 °C; (b) R1NCO, benzene, rt; (c) Ph3PÁBr2, Et3N, CH2Cl2, 0 °C; (d) R2H, THF, rt; (e) LiOHÁH2O, THF–H2O (1:1), 60 °C;
(f) p-R3-BnNH2, HOBT, EDC, rt; (g) 10% Pd(C), MeOH, rt; (h) p-R4-PhSO2Cl, pyridine, 0 °C to rt.

J. H. Heo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3899–3901
3901
Table 1
Biological data for selected compounds
Compound
T-type calcium channel
Growth inhibition of cancer cell (GI50: l
M)b
blocking effect (IC50: l
M)a,b
A549c
DU 145d
HT-29e
SK-MEL-2f
SK-OV-3g
KYS05065
KYS05046
KYS05057
KYS05085
KYS05056
KYS05055
KYS05043
KYS05080
KYS05089
KYS05047
KYS05048
KYS05042
KYS05090
Doxorubicin
1.04 0.25
0.68 0.18
0.63 0.04
0.57 0.05
0.38 0.15
0.35 0.07
0.30 0.09
0.26 0.01
0.23 0.03
0.17 0.03
0.16 0.02
0.11 0.06
0.041 0.001
NDh
24.46 5.79
3.83 0.55
14.27 1.05
7.74 2.60
>100
17.21 1.39
3.83 1.08
6.52 1.00
4.26 0.56
>100
13.94 1.13
2.08 0.43
3.15 0.66
3.18 0.52
28.28 1.42
22.31 1.00
1.08 0.28
0.61 0.12
0.52 0.24
1.70 0.16
1.67 0.18
1.71 0.17
0.04 0.01
0.21 0.04
17.03 1.56
4.31 2.56
8.53 1.65
2.02 0.40
43.95 7.68
31.62 3.05
1.80 0.48
0.69 0.15
1.91 0.11
1.59 0.27
1.48 0.31
1.73 0.28
0.48 0.13
0.11 0.01
26.14 6.08
4.61 0.30
15.61 1.14
7.18 1.18
>100
>100
>100
>100
2.90 0.38
0.87 0.18
1.77 0.16
1.87 0.05
1.83 0.22
1.93 0.13
0.17 0.02
0.16 0.01
4.09 0.71
2.49 0.53
1.78 0.17
1.79 0.04
1.64 0.09
1.71 0.16
0.19 0.02
0.06 0.01
3.99 1.07
2.76 0.76
1.95 0.04
2.01 0.32
1.61 0.09
1.95 0.23
0.66 0.13
0.12 0.02
a
T-type calcium channel (a1G) expressed on HEX293 cell.
Value was determined from dose–response curve and obtained from three independent experiments.
Human lung carcinoma (A549).
Human prostate cancer (DU 145).
Human colon cancer (HT-29).
Human malignant melanoma (SK-MEL-2).
Human ovarian cancer (SK-OV-3).
b
c
d
e
f
g
h
ND, not determined.
10. Rodman, D. M.; Reese, K.; Harral, J.; Fouty, B.; Wu, S.; West, J.; Hoedt-Miller, M.;
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1 lM) across all the cell lines in the series as shown in Table 1. This
compound was found to be as potent as doxorubicin against hu-
man lung carcinoma (A549) and showed in particular 4-fold more
potency against human colon cancer (HT-29) compared to doxoru-
bicin. For the acute toxicity study, KYS050590 as a corn oil mixture
was orally single administered at dose of 1000 mg/kg to two CD-
1(ICR) mice, and no mortality was observed during 5 days (the data
not shown). Therefore, KYS050590 did not appear to have signifi-
cant toxicity. This result implies a possibility that KYS050590
works by stopping cancer cells from multiplying, and thus it stops
cancer from growing (cytostatic effect) rather than killing cancer
cells (cytotoxic effect).
In conclusion, our potent T-type channel blockers were eval-
uated for the growth inhibition of human cancer cells and
KYS050590 possess GI50 similar to that of Doxorubicin. This re-
sult provided a strong evidence for the correlation between inhi-
bition of calcium influx and anti-cancer activity. Therefore, this
study presents new prospects for cancer treatment. Now, the
evaluation of in vivo anti-tumor efficacy and chronic toxicity in
CD-1(ICR) mice is in progress and its data will be announced
in the future.
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23. Kim, T.; Choi, J.; Kim, S.; Kwon, O.; Nah, S. Y.; Han, Y. S.; Rhim, H. Biochem.
Biophys. Res. Commun. 2004, 324, 401. For the recordings of a1G T-type Ca2+
currents, the standard whole-cell patch–clamp method was utilized. Briefly,
borosilicate glass electrodes with a resistance of 3–4 M
X were pulled and
filled with the internal solution containing (in mM): 130 KCl, 11 EGTA, 5 Mg-
ATP, and 10 HEPES (pH 7.4). The external solution contained (in mM): 140
NaCl, 2 CaCl2, 10 HEPES, and 10 glucose (pH 7.4). a1G T-type Ca2+ currents
were evoked every 15 s by a 50 ms depolarizing voltage step from À100 to
À30 mV. The molar concentrations of test compounds required to produce
50% inhibition of peak currents (IC50) were determined from fitting raw data
into dose–response curves. The current recordings were obtained using an
EPC-9 amplifier and Pulse/Pulsefit software program (HEKA, Germany).
24. Skehan, P.; Streng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren,
J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107. All
cell lines were grown in RPMI 1640 (Gibco BRL) supplemented with 10% (V/V)
heat inactivated Fetal Bovine Serum (FBS), and maintained at 37 °C in a
humidified atmosphere with 5% CO2. The cells were seeded into 96-well plate.
Various concentrations of samples were added to each well in triplicate, and
then incubated at 37 °C with 5% CO2 for 72 h such that time cells are in the
exponential phase of growth at the time of drug addition. After incubation, the
Acknowledgments
This work was supported by the Korea Research Foundation
Grant funded by the Korean Government (MOEHRD) (KRF-2007-
313-C00475) and DongWoo Syntech Co., Ltd. (20071065).
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100
left for 30 min at room temperature, washed 5 times with tap water. The
100 of 0.4% SRB solution was added to each well and left at room
temperature for 30 min. SRB was removed and the plates were washed 5 times
with 1% acetic acid before air drying. Bound SRB was solubilized with 100 L of
10 mM unbuffered Tris-base solution (Sigma) and plates were left on a plate
shaker for at least 10 min. The optical density was measured using
microplate reader (Versamax, Molecular Devices) with a 520 nm wavelength
and the growth inhibition concentration was expressed as a GI50
lL of formalin solution was gently added to the wells. Microplates were
l
L
l
a
.