Growth inhibition of human cancer cells in vitro by T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2006.07.046

This paper describes the preliminary biological results that novel T-type calcium channel blockers inhibit the growth of human cancer cells by blocking calcium influx into the cell, based on unknown mechanism on the cell cycle responsible for cellular proliferation. Among the selected compounds from compound library, compound 9c (KYS05041) was identified to be nearly equipotent with Cisplatin against some human cancers in the micromolar range.

Full text of DOI:10.1016/j.bmcl.2006.07.046

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5014–5017
Growth inhibition of human cancer cells in vitro by T-type
calcium channel blockers
Jae Yeol Lee,a,* Seong Jun Park,b,c Sung Jun Park,a Min Joo Lee,a Hyewhon Rhim,c
Seon Hee Seoc and Ki-Sun Kimd
aResearch Institute for Basic Sciences and Department of Chemistry, College of Sciences,
Kyung Hee University, 1 Hoegi-Dong, Seoul 130-701, Republic of Korea
bDepartment of Chemistry, Korea University, 1-Anamdong, Seoul 136-701, Republic of Korea
cLife Sciences Division, Korea Institute of Science & Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
dDepartment of Chemical Engineering, Daebul University, Young Am, Chonnam 526-702, Republic of Korea
Received 12 March 2006; revised 3 June 2006; accepted 17 July 2006
Available online 28 July 2006
Abstract—This paper describes the preliminary biological results that novel T-type calcium channel blockers inhibit the growth of
human cancer cells by blocking calcium influx into the cell, based on unknown mechanism on the cell cycle responsible for cellular
proliferation. Among the selected compounds from compound library, compound 9c (KYS05041) was identified to be nearly equi-
potent with Cisplatin against some human cancers in the micromolar range.
Ó 2006 Elsevier Ltd. All rights reserved.
Calcium plays a critical role as an intracellular signal,
and controls many different cell processes,1 of which cal-
cium appears to play an important role in cell growth.2
For example, it has also been shown that calcium signal-
ing is required for cell cycle progression from G1/S
phase through mitosis.3–6 It has been demonstrated that
depletion of intracellular calcium arrests the cell cycle in
the G0/G1 and S interphases.7 Regulation of the chang-
es in intracellular calcium has been proposed to be via a
T-type calcium channel.8 Lined with this proposition, it
has recently been reported that T-type calcium channel
blockers (CCBs) inhibited cellular proliferation.9,10
Opposed to T-type CCBs, however, it remains to discus-
sion that some L-type CCB as anti-hypertensive agents
may be related to the risk of cancer in the elderly and
promote growth of pre-existing cancer cells in human
by inhibition of apoptosis.11 Therefore, selective T-type
CCBs could be another tool to treat cancer where the
cell cycle has become aberrant. Based on these findings,
we also decided to evaluate the inhibitory activity of 3,4-
dihydroquinazoline derivatives as novel T-type calcium
channel blockers against the growth of human cancer
cells in order to find another relationship between
T-type channel blocking and the growth inhibition of
cancer cells.
By using the synthetic methods according to Scheme 1,
we have recently prepared a library of 3,4-dihydroqui-
nazoline derivatives and evaluated their channel block-
ing effects against T-type channel (a1G) expressed on
HEX293 cell by whole cell patch-clamp methods: T-type
Ca2+ currents were evoked every 15 s by a 50 ms depo-
larizing voltage step from À100 to À30 mV and percent-
age inhibition was calculated as 100[(C À D)/C], where
D is the peak current in the presence of drug, and C is
the peak current before the application of drug.12 As a
result, we have disclosed a few of lead compounds
(KYS05064 and KYS05071) as novel T-type channel
blockers showing a good potency and channel selectivity
through intensive SAR study.13 From the compound li-
brary, we have selected eight compounds showing a
broad range of channel blocking effects (34–91% inhibi-
tion at 10 lM concentration) to find their relationship
between channel blocking effect and growth inhibition
of cancer cells, and the structures of selected compounds
are shown in Figure 1.
Keywords: Cancer cell; Growth inhibition; T-type calcium channel
blockers; Cisplatin.
The selected compounds were evaluated for their growth
inhibition against five cancer cell lines, including human
*
Corresponding author. Tel.: +82 2 9610726; fax: +82 2 9663701;
e-mail: ljy@khu.ac.kr
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.046

J. Y. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5014–5017
5015
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
R1
R2
d
e
f
g
N
N
R3
N
N
N
R2
N
5
6
7a-b
O
O
N
H
N
H
O
O
R1
R2
S
R1
N
h
H2N
N
N
H
N
R4
N
R2
8
9a-d
Scheme 1. Reagents and conditions: (a) SnCl2Æ2H2O, EtOAc, 70 °C; (b) R1NCO, benzene, rt; (c) Ph3PÆBr2, Et3N, CH2Cl2, 0 °C; (d) R2H, THF, rt; (e)
LiOHÆH2O, 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.
O
O
O
O
N
H
N
N
H
H
CO
3
H
N
CH
H
N
2
N
N
CH
CH
3
3
3
N
N
N
N
N
N
N
N
N
H
NH
2
CH
3
5 (KYS05044)
7a (KYS05075)
7b (KYS05074)
8 (KYS05040)
O
O
O
O
N
H
N
H
N
H
N
H
N
O
O
O
O
O
O
O
O
S
S
S
S
N
H
N
N
CH
CH
N
N
CH
3
CH
3
N
N
N
H
CH
3
3
3
H
H
N
N
N
N
N
N
N
CH
CH
3
3
F
H
C
H C
3
3
H C
3
9a (KYS05071)
9b (KYS05070)
9c (KYS05041)
9d (KYS05064)
Figure 1. 3,4-Dihydroquinazoline derivatives studied in SRB assay.
lung carcinoma (A-549), human colon cancer (HCT-15),
human epidermoid carcinoma (KB), human malignant
melanoma (SK-MEL-2) and human ovarian cancer
(SKOV3) using sulforhodamine B (SRB) assay:14 All
cell lines were grown in RPMI 1640 (Gibco BRL) sup-
plemented 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 cells are in the
exponential phase of growth at the time of drug addi-
tion. After incubation, 100 lL of formalin solution
was gently added to the wells. Microplates were left
for 30 min at room temperature, washed five times with
tap water. One hundred microliters of 0.4% SRB solu-
tion was added to each well and left at room tempera-
ture for 30 min. SRB was removed and the plates were
washed five times with 1% acetic acid before air-drying.
Bound SRB was solubilized with 100 lL 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 a microplate reader (Versa-
max, Molecular Devices) with a 520 nm wavelength and
the growth inhibition concentration was expressed as
GI50. The results of assay are summarized in Table 1
as well as the channel blocking effect (% inhibition val-
ues at 10 lM concentration). Cisplatin was used as a ref-
erence for anti-cancer activity and also tabulated with
data for comparison. In addition, blocking effects
against N-type channel are also inserted into Table 1
in order to investigate the N-type channel blocking effect
on growth inhibition of cancer cells.
Before discussing the results, we have already confirmed
that all of the selected compounds did not show any
cytotoxicity on HEX293 cell at 100 lM concentration
as reported previously.13 From these data, a brief rela-
tionship profile emerged as follows: as expected, Cisplat-
in barely blocked T-type Ca2+ currents and the mean
percentage of its inhibition is 3.9 2.1 (n = 3) at
10 lM concentration of Cisplatin, which acts by

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J. Y. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5014–5017
Table 1. Biological data for selected compounds
Compound
Channel blocking effect
(% inhibition)a,b
Growth inhibition of cancer cell: GI50 (lM)c
T-type (a1G
)
N-type (a1B
)
A-549d
HCT-15e
KBf
3.77 0.07
>200
SK-MEL-2g
SKOV3h
5 (KYS05044)
7a (KYS05075)
7b (KYS05074)
8 (KYS05040)
9a (KYS05071)
9b (KYS05070)
9c (KYS05041)
9d ((KYS05064)
Cisplatin
82.5 0.7
34.4 1.4
59.3 1.9
43.5 4.5
62.7 2.3
81.3 0.8
89.9 1.3
91.3 0.6
3.9 2.1
No blocking
27.6 0.5
9.3 0.5
3.39 0.11
>200
3.64 0.24
>200
3.32 0.20
50.19 2.45
42.48 0.00
30.73 5.97
31.36 1.53
24.21 5.08
2.70 0.21
3.52 0.18
4.40 1.40
3.24 0.31
>200
95.10 19.97
36.60 1.19
66.10 19.10
39.16 6.97
3.06 0.10
23.03 0.75
0.67 0.67
74.16 9.63
33.86 3.86
38.62 3.13
33.89 2.75
2.80 0.28
25.67 8.61
2.80 1.00
108.14 24.43
37.08 0.82
41.63 7.41
33.83 0.56
3.11 0.25
22.28 1.80
1.50 1.20
77.84 12.62
39.32 1.28
46.48 5.29
33.44 0.00
2.93 0.00
22.78 5.15
1.80 0.67
8.9 1.8
No blocking
8.9 1.8
14.1 2.1
No blocking
NDi
a % Inhibition on HEX293 cell at 10 lM.
b Value was determined from dose–response curve and obtained from three independent experiments.
c GI50 value was determined from dose–response curve and obtained from three independent experiments.
d Human lung carcinoma (A-549).
e Human colon cancer (HCT-15).
f Human epidermoid carcinoma (KB).
g Human malignant melanoma (SK-MEL-2).
h Human ovarian cancer (SKOV3).
i ND, not determined.
crosslinking DNA in several different ways, making it
impossible for rapidly dividing cells to duplicate their
DNA for mitosis. In general, all compounds showed
the similar correlation between cell growth inhibitory
activity and T-type channel blocking effect, even though
it is non-linear. That is, compound 7a showing weak
activity against T-type channel produced weak activity
against cancer cells. Compounds 5 and 9c showing
strong potency against T-type channel exhibited strong
activity against cancer cells. All compounds also exhib-
ited the similar growth inhibitory activities against five
cancer cells except for compounds 7a, 7b, and 9d. Inter-
estingly, compound 9d, which is the most active com-
pound (91.3 0.6%) against T-type channel, exhibited
less activity than compounds 5 and 9c except for
SKMEL2 cell line. This result could be explained by
comparing IC50 values of compounds 5, 9c, and 9d,
which are 0.56 0.10, 0.17 0.08, and 0.96 0.22 lM,
respectively (the data not inserted in Table 1). With re-
spect to IC50 value, therefore, the most active compound
9c exhibited the meaningful inhibition of cancer cell’s
growth compared to Cisplatin, which is our reference
compound.
preliminary data. This result is obviously found to
coincide with the previous works by other research
groups and also endows T-type calcium channel
blocker with the possibility as another anti-cancer
agent.15,16 However, more work is needed in order
to understand the calcium influx and cell proliferation
via T-type calcium channel. Encouraged by these
results, therefore, the synthesis and biological evalua-
tion of a variety of compounds are in progress for
providing the direct linkage and will be announced
in the future.
Acknowledgement
This study was supported by Vision 21 Program from
Korea Institute of Science and Technology.
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