In vitro cytotoxicity on human ovarian cancer cells by T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2013.10.049

The growth inhibition of human cancer cells via T-type Ca²⁺ channel blockade has been well known. Herein, a series of new 3,4-dihydroquinazoline derivatives were synthesized via a brief SAR study on KYS05090 template and evaluated for both T-type Ca²⁺ channel (Ca _v3.1) blockade and cytotoxicity on three human ovarian cancer cells (SK-OV-3, A2780 and A2780-T). Most of compounds except 6i generally exhibited more potent cytotoxicity on SK-OV-3 than mibefradil as a positive control regardless of the degree of T-type channel blockade. In particular, eight compounds (KYS05090, 6a and 6c-6h) showing strong channel blockade exhibited almost equal and more potent cytotoxicity on A2780 when compared to mibefradil. On A2780-T paclitaxel-resistant human ovarian carcinoma, two compounds (KYS05090 and 6d) were 20-fold more active than mibefradil. With respect to cell cycle arrest effect on A2780 and A2780-T cells, KYS05090 induced large proportion of sub-G₁ phase in the cell cycle progression of A2780 and A2780-T, meaning the induction of cancer cell death instead of cell cycle arrest via blocking T-type Ca²⁺ channel. Among new analogues, compounds 6g and 6h induced cell cycle arrest at G₁ phase of A2780 and A2780-T cells in dose-dependent manner and exhibited strong anti-proliferation effects of ovarian cancer cells by blocking T-type Ca²⁺ channel. Furthermore, 6g and 6h possessing strong cytotoxic effects could induce apoptosis of A2780 cells, which was detected by confocal micrographs using DAPI staining.

Full text of DOI:10.1016/j.bmcl.2013.10.049

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6656–6662
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro cytotoxicity on human ovarian cancer cells by T-type calcium
channel blockers
Sun Jeong Jang a, , Heung Woo Choi a, , Doo Li Choi a, Sehyeon Cho a, Hong-Kun Rim b, Hye-Eun Choi b,
Ki-Sun Kim c, Minghua Huang d,e, Hyewhon Rhim d,e, Kyung-Tae Lee b, , Jae Yeol Lee a,
⇑
⇑
a Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
b Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
c Division of Human Health Sciences, Sehan University, Young Am, Chonnam 526-702, Republic of Korea
d Center for Neuroscience, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea
e Department of Neuroscience, University of Science and Technology, Daejeon 305-350, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
The growth inhibition of human cancer cells via T-type Ca2+ channel blockade has been well known.
Herein, a series of new 3,4-dihydroquinazoline derivatives were synthesized via a brief SAR study on
KYS05090 template and evaluated for both T-type Ca2+ channel (Cav3.1) blockade and cytotoxicity on
three human ovarian cancer cells (SK-OV-3, A2780 and A2780-T). Most of compounds except 6i generally
exhibited more potent cytotoxicity on SK-OV-3 than mibefradil as a positive control regardless of the
degree of T-type channel blockade. In particular, eight compounds (KYS05090, 6a and 6c–6h) showing
strong channel blockade exhibited almost equal and more potent cytotoxicity on A2780 when compared
to mibefradil. On A2780-T paclitaxel-resistant human ovarian carcinoma, two compounds (KYS05090
and 6d) were 20-fold more active than mibefradil. With respect to cell cycle arrest effect on A2780
and A2780-T cells, KYS05090 induced large proportion of sub-G1 phase in the cell cycle progression of
A2780 and A2780-T, meaning the induction of cancer cell death instead of cell cycle arrest via blocking
T-type Ca2+ channel. Among new analogues, compounds 6g and 6h induced cell cycle arrest at G1 phase of
A2780 and A2780-T cells in dose-dependent manner and exhibited strong anti-proliferation effects of
ovarian cancer cells by blocking T-type Ca2+ channel. Furthermore, 6g and 6h possessing strong cytotoxic
effects could induce apoptosis of A2780 cells, which was detected by confocal micrographs using DAPI
staining.
Article history:
Received 6 March 2013
Revised 11 October 2013
Accepted 23 October 2013
Available online 31 October 2013
Keywords:
T-type Ca2+ channel
Ovarian cancer
Cytotoxicity
Cell cycle arrest
Apoptosis
Ó 2013 Elsevier Ltd. All rights reserved.
Most ovarian cancers are either ovarian epithelial carcinomas
(cancer that begins in the cells on the surface of the ovary) or
malignant germ cell tumors (cancer that begins in egg cells).1
Estimated new 22,280 cases and 15,500 deaths from ovarian
cancer have been reported in the United States in 2012.2 Unfortu-
nately, at the present time there is no effective treatment for
ovarian cancer because the mechanism by which ovarian cancer
develops remains largely unknown.
Calcium plays a key role in intracellular signaling and controls
many different cell processes such as proliferation, differentiation,
growth, cell death and apoptosis.3,4 For example, it has been shown
that calcium signaling is required for cell cycle progression from
G1/S phase through mitosis.5–8 It has also been demonstrated that
depletion of intracellular calcium arrests the cell cycle in the G0/G1
and S interphases.9 Regulation of the changes in intracellular
calcium has been known to be via T-type calcium channels,10 of
which three isoforms have been identified so far: Cav3.1, Cav3.2,
and Cav3.3.11 The Cav3.1 and Cav3.2 are broadly expressed in vari-
ous tissues,12,13 while Cav3.3 expression is more restricted to the
central nervous system, thyroid, and adrenal gland tissue.14 The
regulation of Ca2+ homeostasis allows T-type Ca2+ channels to play
an important role in controlling cell proliferation and differentia-
tion.15–17,14,18 T-type Ca2+ channels are expressed in numerous
types of tumor cells as well as in normal tissues throughout the
body.19 Compared with the normal human cells, overexpression
of T-type Ca2+ channels has been demonstrated in the counterpart
tumor cells.20–22 In the case of ovarian cancer, in particular, Zhang’s
group reported that T-type Ca2+ channel expression in human
ovarian cancer tissues (A2780 and H08910 cells) is greatly in-
creased compared to normal ovarian tissues and also blockade of
T-type Ca2+ channel with NNC 55-0396 or mibefradil suppressed
the proliferation of two ovarian cancer cells and increased G0/G1
phase distribution in the cell cycle.23 Recently, our group also re-
ported that T-type Ca2+ channel blockers suppress the growth of
⇑
Corresponding authors. Tel.: +82 2 961 0726; fax: +82 2 966 3701.
E-mail addresses: ktlee@khu.ac.kr (K.-T. Lee), ljy@khu.ac.kr (J.Y. Lee).
The contributions of the authors are equal.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.049

S. J. Jang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6656–6662
6657
Figure 1. SAR study of KYS05090 for new synthetic analog (6).
O
O
O
O
OCH3
a
c
b
d
OCH3
OCH3
OCH3
NH
N
C N
NO2
NH2
O
N
X
H
X
1
2
3 (X = CH or N)
4 (X = CH or N)
O
O
CH3O
X
R2
N
H
X
e
N
N
N
R1
N
R1
5
6
Scheme 1. Reagents and conditions: (a) Zn, NH4Cl, MeOH, reflux; (b) biphenyl-4-carboxylic acid or 4-(2-pyridyl)benzoic acid, DPPA, Et3N, toluene, rt to 100 °C; (c) Ph3PꢀBr2,
Et3N, CH2Cl2, 0 °C; (d) R1H {compound 11 in Scheme 2 or N-[2-(dimethylamino)ethyl]piperazine}, toluene, rt; (e) R2-CH2NH2, TBD, rt.
tBoc
tBoc
H3C
CH3
a
b
( )n
7 (n = 2-4)
( )n
( )n
H2N
NH2
N
H
N
H
N
H
N
H
8
9
a
b
H3C
CH3
H3C
CH3
( )n
( )n
N
H
N
N
H
N
CH3
tBoc
11
10
Scheme 2. Reagents and conditions: (a) (tBoc)2O, HCl, MeOH, 0 °C to rt; (b) LiAlH4, dry THF, 0 °C to reflux.
some of human cancer cells including ovarian cancer.24–27 Thus, T-
(DPPA) in the presence of TEA, produced methyl trans-2-(3-biary-
lureido)cinnamate (3). The dehydration of 3 with Ph3PꢀBr2 and
Et3N provided methyl trans-2-(3-biarylcarbodiimido)cinnamate
(4) as a key intermediate of this reaction. Subsequently, the cou-
pling of 4 with CH3NH(CH2)nN(CH3)2 (11 in Scheme 2: where, n =
2–4) or N-[2-(dimethylamino)ethyl]-piperazine in toluene affor-
ded the corresponding 3,4-dihydroquinazoline compound (5) via
the tandem intramolecular conjugate addition. The treatment of
5 with ArCH2NH2 and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)
as a catalyst under solvent-free condition afforded directly the cor-
responding 3,4-dihydroquinazoline amide (6) as a final compound.
New 3,4-dihydroquinazoline analogs (6) and KYS05090 were
evaluated for their channel blocking effects against T-type channel
type Ca2+ channels represent promising targets for developing
novel and effective human ovarian cancer therapies. As a continu-
ous our research work, we report herein that a series of new 3,4-
dihydroquinazoline derivative (6) via brief SAR study on
KYS05090 template exhibited both significant T-type Ca2+ channel
blockade and cytotoxicity on three human ovarian cancer cells
(Fig. 1).
SAR study on the 3,4-dihydroquinazoline (KYS05090) was
mainly focused on the modification of its three positions (P-1, P-
2 and P-3) as illustrated in Figure 1. We used a short synthetic
route (see Schemes 1 and 2) that would allow the rapid modifica-
tion of the three variable positions (P-1, P-2 and P-3) to produce
new synthetic 3,4-dihydroquinazoline derivatives (6).26 The reduc-
tion of methyl trans-2-nitrocinnamate (1) with activated Zn and
NH4Cl in MeOH afforded methyl trans-2-aminocinnamate (2),
followed by an addition of a corresponding isocyanate, which
was in situ prepared from the reaction of biphenyl-4-carboxylic
acid or 4-(2-pyridyl)benzoic acid with diphenyl phosphorazidate
Cav3.1 (a1G) expressed on HEK293 cell at 1 lM concentration by
whole cell patch-clamp methods: T-type Ca2+ currents were
evoked every 15 s by a 50 ms depolarizing voltage step from
ꢁ100 to ꢁ30 mV and % inhibition was calculated as 100[(C ꢁ D)/
C], where D is the peak current in the presence of compound,
and C is the peak current before the application of compound.13

6658
S. J. Jang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6656–6662
Figure 2. Inhibition of T-type Ca2+ channel current by synthetic compounds as assessed by manual electrophysiology. Voltage protocol is that T-type Cav3.1 currents were
evoked every 15 s by a 50 ms depolarizing voltage step from ꢁ100 mV to ꢁ30 mV. (A) and (B) Representative current traces illustrating the inhibition of T-type Ca2+ channel
by 1 lM KYS05090 (most active) and 1 lM 6b (least active), respectively. (C) Dose–response curve for KYS05090, 6g, 6h and mibefradil yielded an IC50 values, respectively.
For only compounds showing more than 50% inhibition value, the
molar concentrations (IC50) of compounds required to produce 50%
inhibition of a1G T-type currents were determined from fitting raw
4-(2-dimethylaminoethyl)piperazine ring: Derivative 6e inhibited
moderately T-type calcium channel (46.8% @ 1 M) but was less
active than KYS05090. Insertion of p-methyl group to the benzyl
l
data into dose–response curves (Fig. 2).
amide ring (R2) showed the slightly recovered potency (6f, IC50
0.46 0.13 M), however the p-fluorobenzyl amide (6g, IC50
0.32 0.03
= 0.31 0.07
pound KYS05090 (IC50 = 0.26 0.01
=
=
All compounds were again evaluated for their cytotoxicities on
three human ovarian cancer cells [SK-OV-3, A2780 and A2780-T
paclitaxel-resistant human ovarian carcinoma] using MTT assay.28
Both assay results for the inhibition of calcium influx and the cyto-
toxicity on human ovarian cancer cells were summarized in Table 1
together with those of mibefradil as a positive control.
l
l
M) and p-methoxybenzyl amide derivative (6h, IC50
M) were nearly equipotent with the parent com-
M) against T-type channel.
l
l
It is worth noting that this simple SAR result provides valuable
information for our on-going diverse construction of 3,4-dihydro-
quinazoline library due to the synthetic difficulty of diamine side
change (R1). Like compound 6b, the insertion of N atom at biphenyl
With respect to the channel blockade of synthetic analogues,
the insertion of N atom at biphenyl ring led to a large decrease
(6b, 30.9% @ 1
ent compound (KYS05090, 76.6% @ 1
M), indicating the importance of hydrophobicity in these re-
gions for potent T-type activity. Next, compound 6c and 6d demon-
strated that an appropriate number of methylene spacer between
the diamine at R1 region is four and five unit (6c, IC50 = 0.31
l
M) in T-type channel potency compared to the par-
ring (6i, 32.3% @ 1
and 6k, 43.9 and 29.1% @ 1
crease in T-type channel potency compared to compounds
KYS05090 and 6e, indicating the importance of hydrophobicity
in these two regions for potent T-type activity.
With respect to in vitro cytotoxicity on three human ovarian
cancer cells, most of compounds were generally more cytotoxic
than mibefradil as a positive control, regardless of the degree of
T-type channel blockade. In addition, they showed the similar
relationship between T-type channel blocking effect and in vitro
cytotoxicity except compound 6b and 6e against SK-OV-3 cells,
even though it is non-linear. For example, compound 6k showing
weakest blocking effect against T-type channel produced weakest
l
M) or the pyridinomethyl amide analogs (6j
lM) and mibefradil (55.1% @
lM, respectively) led to a large de-
1
l
0.05
crease (6d, IC50 = 0.55 0.13
to compound KYS05090 (IC50 = 0.26 0.01
l
M), but the longer length (i.e., six unit) led to a twofold de-
M) in channel potency compared
M). The attempt to fill
l
l
the more hydrophobic region by attaching p-methoxy group to the
benzyl amide (R2) substituent showed a modest decreased potency
(6a, IC50 = 0.39 0.05
lM). Compounds 6e–6h were prepared to
explore changes to R1 region by replacing diamine chain with

S. J. Jang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6656–6662
6659
Table 1
T-type calcium channel blocking effect and cytotoxicity effect against human ovarian cancer cells of new 3,4-dihydroquinazoline derivatives (6)
O
R2
N
H
X
N
N
R1
R1
X
R2
Calcium channel blockade [T-type: Cav3.1 (
a
1G)]a
Cytotoxicity: IC50 (lM)
b
Entry
% inhibition (1
76.6 0.7
68.2 1.3
30.9 0.9
81.7 1.3
66.7 2.3
lM)
(IC50
:
l
M)c
SK-OV-3d
A2780e
1.06
1.38
—
A2780-Tf
( )3
( )3
( )3
( )2
( )4
N
N
N
N
N
N
KYS-05090
CH
CH
N
0.26 0.01
0.39 0.05
NDg
2.47
4.46
2.41
9.78
5.78
1.60
N
N
N
N
6a
6b
6c
6d
O
2.52
>5
CH
CH
0.31 0.05
0.55 0.13
1.65
1.19
3.97
1.74
N
N
N
N
N
N
N
6e
6f
CH
CH
CH
CH
N
46.8 1.4
57.8 2.8
81.0 2.5
75.9 2.2
32.3 2.8
43.9 1.7
29.1 0.8
ND
1.97
2.33
3.09
2.47
—
1.39
1.60
1.60
1.44
>5
2.39
3.52
4.00
3.82
>5
N
N
N
N
N
N
N
N
N
N
N
N
N
N
0.46 0.13
0.32 0.03
0.31 0.07
ND
F
6gh
6hh
6i
O
6j
CH
CH
ND
5.08
11.73
4.86
>5
>5
N
N
6k
ND
>5
O
H
O
N
O
Mibefradil
55.1 1.1
0.56 0.11
20.54
24.23
35.26
N
N
F
a
b
c
T-type Ca2+ channel (
MTT assay.
a1G) expressed on HEK293 cell.
Value was determined from dose–response curve and obtained from three independent experiments.
SK-OV-3 is the human ovarian carcinoma.
A2780 is the human ovarian carcinoma which was established from tumor tissue from an untreated patient.
A2780-T is the paclitaxel-resistant human ovarian carcinoma of A2780.
d
e
f
g
h
ND: not determined when less than 50% inhibition at 1
lM.
The cell viability of 6g and 6h against T-type Ca2+ channel non-transfected HEK293 cell are 10 and 9.28
lM, respectively.
cytotoxicity. KYS05090 showing the strongest potency against T-
type channel exhibited the most potent cytotoxicity on three
ovarian cancer cells. Interestingly, compound 6e showed stronger
cytotoxic effect on all three ovarian cancer cells in spite of rela-
tively lower T-type channel blockade. This interesting phenome-
non shows the possibility that compound 6e maybe have dual
purpose activities that involve some of other ion channels. In the
view of individual ovarian cancer cell, compounds showing >45%
such as physical property will be needed. On A2780 cells, once
again, all compounds showing strong channel blockade (>45% @
1
l
M) produced a short spectrum of cytotoxic effect (IC50 values
of 1.06 into 1.65 M) compared to that of SK-OV-3 cells and also
were from 14.7 to 22.8 times more active than mibefradil. In par-
ticular, KYS05090 showed an IC50 of 1.06 M as compared to
24.23 M for mibefradil. On A2780-T paclitaxel-resistant cells,
most compounds showing strong channel blockade (>45% @
M) exhibited a broader spectrum of cytotoxic effect (IC50 values
of 1.60 into 4.00 M) than those against A2780 cells, but still more
active than mibefradil (IC50 value of 35.26 M). As KYS05090
showed both strongest T-type channel blockade and cytotoxicity
(IC50 value of 1.06 M) against human ovarian A2780 and
A2780-T cells, in the meanwhile, we further examined the precise
l
l
l
channel inhibition at 1
effect (IC50 values of 1.97 into 9.78
l
M exhibited a broad spectrum of cytotoxic
M) against SK-OV-3 cells.
1 l
l
l
Interestingly, compounds 6b, 6c and 6e showed an inverse correla-
tion between T-type channel blockade and cytotoxic activity as
show in Table 1. At this preliminary stage, we cannot explain this
phenomenon properly and thus further studies on other factors
l
l

6660
S. J. Jang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6656–6662
Figure 3. Effect of KYS05090 on the cell cycle progression of A2780 and A2780-T cells. Each cell was treated with different concentrations (0, 2, 4 and 8 lM) of KYS05090 for
24 h. Cell cycle phase was determined by using flow cytometry (FACS). Data are presented as the means SD of three independent experiments. ⁄⁄⁄P <0.001 versus the control
group. Statistical significances were determined using ANOVA and Dunnett’s post-hoc test.
Figure 4. Cell cycle arrest effects of 6g and 6h in A2780 and A2780-T paclitaxel-resistant cells. Cells were treated with different concentrations (0, 2, 4, or 6 lM) of 6g and 6h
for 24 h. Cell cycle phase was determined by using flow cytometry (FACS). Data are presented as the means SD of three independent experiments. ⁄P <0.05 versus the control
group. Statistical significances were determined using ANOVA and Dunnett’s post-hoc test.
Table 2
phase (71.4% @ 8 lM) was evident depending on its concentration
The effect of synthetic compounds on cell-cycle progression of A2780 cell
compared to untreated control cells (3.1%). In the case of paclit-
axel-resistant A2780-T cells, a small proportion of sub-G1 phase
(14.7%) was induced compared to A2780 cells (61.4%) at low con-
Compounds @ 6
l
M
24 h after treatment
Sub-G1
G0/G1
S
G2/M
centration (2 or 4
again within 95.4% at 8
of cell death at high concentration (Fig. 3). Based on this result,
KYS05090 blocked T-type Ca2+ channel (
1G) significantly, de-
lM) but the proportion of sub-G1 phase was
Control
6g
6h
2.1
18.1
21.6
63.3
59.9
66.7
63.8
25.6
13.1
7.1
7.3
24.9
8.1
7.3
lM concentration, meaning the induction
KYS05090
5.8
5.3
a
creased intracellular Ca2+ concentrations and finally produced
strong cytotoxic effect on A2780 and A2780-T cells. On the other
hand, new analogues 6g and 6h were also selected for further eval-
uation on their effects of A2780 and A2780-T cell cycle distribution
based on T-type channel blocking effects (IC50 value), 2-week re-
peat-dose non-toxicity [40 mg/kg (po route): 0% mortality],30 and
effect of KYS05090 on the cell cycle progression of A2780 and
A2780-T cells using flow cytometry (FACS).29 Each cell were trea-
ted with different concentrations (0, 2, 4 and 8
for 24 h. With respect to A2780 cells, a large proportion of sub-G1
lM) of KYS05090

S. J. Jang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6656–6662
6661
Figure 5. Confocal micrographs: the nuclear morphological changes of active compounds 6g/6h-treated A2780 cells (stained with DAPI) showing an induced apoptosis in
A2780 cells.
the synthetic feasibilities compared to KYS05090. Two compounds
In summary, a series of new 3,4-dihydroquinazoline derivatives
induced cell cycle arrest in a concentration dependent manner (0,
were synthesized via a brief SAR study on KYS05090 template and
2, 4 or 6
lM) as shown in Figure 4. That is, the G1 phase propor-
evaluated for both T-type Ca2+ channel (
a1G) blockade and in vitro
tions of A2780 and A2780-T cells were increased compared to
untreated control cells after 24 h treatment of 6g and 6h, respec-
cytotoxicity on three human ovarian cancer cells. Evaluation of the
brief SAR indicated the importance of two hydrophobic regions
(the amide region at P-1 position and the biphenyl region at P-2
position) for optimal T-type Ca2+ channel inhibition and their
related cytotoxicity on human ovarian cancer cells. More impor-
tantly, the diamine side change (R2) of at P-1 position of
KYS05090 could be replaced with commercially available 4-(2-
dimethylaminoethyl)piperazine ring without losing the original
biological activity of KYS05090. Most of compounds showing sig-
nificant channel blockade exhibited more potent cytotoxicity on
these ovarian cancer cells than mibefradil as a positive control. In
addition, KYS05090 was found to induce increased sub-G1 phase
distribution (that is, cell death) in the cell cycle progression of
A2780 and A2789-T strongly by blocking T-type Ca2+ channel.
Among new analogues, compounds 6g and 6h induced cell cycle
arrest at G1 phase of A2780 and A2789-T in a dose-dependent
manner and also showed the strong cytotoxicity against ovarian
cancer cells. Two compounds possessing strong cytotoxic effect
could induce apoptosis of A2780 cells, which was detected by con-
focal micrographs using DAPI staining. Considered on T-type chan-
nel blocking effect and the related cytotoxic effect as well as the
potential synthetic feasibility, new analogues (6g and 6h)35 would
be also regarded as back-up compounds for KYS05090. Therefore,
our findings suggest that T-type Ca2+ channel blocker has potential
chemotherapeutic value for the treatment of human ovarian can-
cer as well as lung cancer via various modes of action.
tively. In the case of A2780 cell, 6
compounds induced the cell death as shown in Table 2. Thus, the
effect of 6 M compound 6g and 6h on G1 phase cell cycle arrest
was less than those of 4 M treatments. Thus, the real effect of
M 6g and 6h on cell cycle arrest at G1 phase of A2780 cell could
lM concentration of both
l
l
6
l
not be identified, which result is consistent with their higher cyto-
toxic effect on A2780 over A2780-T as shown in Table 1. Despite
the high structural similarity between three compounds
(KYS05090, 6g and 6h), on the other hand, the different effects
on the cell cycle among three compounds may stem from the
inherent stronger cytotoxicity of KYS05090 than 6g and 6h.
These overall results were perfectly consistent with our re-
cently reported work on human lung adenocarcinoma (A549) using
KYS05047: This compound, one of the same 3,4-dihydroquinazo-
line family, was found to have an anti-proliferative effect (IC50
1.87 0.05 M) on A549 cells by inducing G0/G1 phase cell cycle
arrest (>80% at 7 M concentration for 24 h) associated with a de-
crease in intracellular Ca2+ concentrations via T-type Ca2+ channel
blockade (IC50 = 0.17 0.13
M).27
=
l
l
l
Finally, the cell viability of active compounds 6g and 6h against
T-type Ca2+ channel non-transfected HEK293 cell viability was per-
formed in order to confirm the relationship between the blockade
of T-type calcium channel and cytotoxic effect on cancer cells. As
expected, these compounds showed less cytotoxicity (IC50 values
of 10 and 9.28
lM, respectively) against non-transfected HEK293
cell compared to those (IC50 value range of 1.44 into 4.00
l
M)
Acknowledgments
against the ovarian cancers above. In addition, we performed fur-
ther tests to elucidate whether their proliferation inhibition of
ovarian cancer cells was related to apoptosis. After A2780 cells
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (2011-
0027401).
were treated with 6g or 6h at concentrations of 6 lM for 24 h,
apoptotic characteristics were observed by DAPI staining.31 The
morphological changes of 6g (or 6h)-induced cells, such as cell
shrinkage, membrane blebbing, chromatin condensation and apop-
totic body formation, were canonical apoptotic markers (Fig. 5).
This result suggested that 6g (or 6h)-induced cytotoxicity of
A2780 cells arises from the induction of apoptosis.
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analyzed using the fluorescence-activated cell sorting cater-plus FACS (Becton
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times with PBS and stained with 0.1 l
g/mL DAPI (40,6-diamidine-20-
phenylindole: Roche) for 5 min. Cells were again washed three times with
PBS and mounted on glass slides in PBS containing 10% glycerol. DAPI stained
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35. The spectral data of 6g: 1H NMR (400 MHz, CDCl3) d 7.55–7.52 (2H, m, Ph), 7.48–
7.45 (2H, m, Ph), 7.44–7.39 (2H, m, Ph), 7.34–7.29 (1H, m, Ph), 7.24–7.16 (5H,
m, Ph), 7.11 (1H, d, Ph), 6.32 (1H, t, J = 5.6 Hz, –CO–NH–CH2-), 5.25 (1H, dd,
J = 9.6 and 5.6 Hz, –CO–CH2–CH–N–), 4.47–4.36 (2H, m, –NH–CH2–Ph–), 3.33
(4H, br s, –N–(CH2–CH2)2–N–CH2–), 2.58 (1H, dd, J = 14.4 and 9.6 Hz, –CO–
CH2–), 2.43 (1H, dd, J = 14.4 and 5.6 Hz, –CO–CH2–), 2.38–2.30 (4H, m, –N–
CH2–CH2–N–Me2), 2.22 (6H, s, –NMe2), 2.18 (4H, br s, –N–(CH2–CH2)2–N–CH2–
); 13C NMR (100 MHz, CDCl3) d 170.0, 153.0, 144.8, 143.4, 140.3, 137.0, 134.0,
130.0, 129.8, 128.8, 128.4, 127.8, 127.2, 126.8, 126.9, 125.0, 122.8, 122.7, 122.7,
115.6, 115.4, 60.9, 56.8, 56.6, 53.0, 45.9, 45.7, 43.0, 41.9; HRMS (FAB+) : calcd
for C37H42N6O1F1 (M+H) 605.3404, found: 605.3406.
28. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.; Tosini, S.; Skehan,
P.; Scudiero, D. A.; Monks, A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113. The
effect of synthetic compound on cell viability was assessed by using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in
3
The spectral data of 6h: 1H NMR (400 MHz, CDCl3) d 7.55–7.52 (2H, m, Ph),
7.48–7.45 (2H, m, Ph), 7.44–7.40 (2H, m, Ph), 7.34–7.29 (1H, m, Ph), 7.22–7.13
(6H, m, Ph), 6.99–6.96 (1H, m, Ph), 6.94–6.89 (1H, m, Ph), 6.81–6.78 (2H, m,
Ph), 5.83 (1H, t, J = 5.6 Hz, –CO–NH–CH2–), 5.48 (1H, dd, J = 9.6 and 5.6 Hz, –
CO–CH2–CH–N–), 4.45–4.30 (2H, m, –NH–CH2–Ph–), 3.75 (3H, s, –OCH3), 3.48
(4H, br s, –N–(CH2–CH2)2–N–CH2–), 2.57 (1H, dd, J = 14.4 and 9.6 Hz, –CO–
CH2–), 2.43 (1H, dd, J = 14.4 and 5.6 Hz, –CO–CH2–), 2.36 (4H, s, –N–CH2–CH2–
N–Me2), 2.22 (6H, s, –NMe2), 2.20 (4H, br s, –N–(CH2–CH2)2–N–CH2–); 13C NMR
(100 MHz, CDCl3) d 170.1, 160.9, 153.3, 144.8, 143.0, 140.3, 137.1, 134.5, 130.0,
129.9, 128.8, 128.4, 127.8, 127.2, 126.8, 126.6, 125.1, 122.9, 122.8, 122.2, 115.5,
115.3, 60.7, 56.7, 56.4, 52.8, 45.8, 42.9, 41.6; HRMS (FAB+): calcd for
replicates. Each cancer cells were seeded and incubated in 96-well, flat-
bottomed plates for 24 h and were exposed to various concentrations of test
agents dissolved in DMSO (final concentration, 0.1%) for 48 h. Controls received
DMSO vehicle at a concentration equal to that in drug-treated cells. The
medium was removed, replaced by 200 lL of 0.5 mg/mL MTT in 10% fetal
bovine serum containing RPMI, and cells were incubated in the carbon dioxide
incubator at 37 °C for 2 h. Supernatants were removed from the wells and the
reduced MTT dye was solubilized in 100
was determined on a plate reader..
lL/well DMSO. Absorbance at 540 nm
29. Lee, K. W.; Kim, H. J.; Lee, Y. S.; Park, H. J.; Choi, J. W.; Ha, J.; Lee, K.-T.
Carcinogenesis 1928, 2007, 28. A2780 cells were treated with various
C38H45N6O2 (M+H) 617.3604, found: 617.3602.