Inhibition of cellular proliferation and induction of apoptosis in human lung adenocarcinoma A549 cells by T-type calcium channel antagonist

Article abstract of DOI:10.1016/j.bmcl.2014.01.071

The anti-proliferative and apoptotic activities of new T-type calcium channel antagonist, 6e (BK10040) on human lung adenocarcinoma A549 cells were investigated. The MTT assay results indicated that BK10040 was cytotoxic against human lung adenocarcinoma (A549) and pancreatic cancer (MiaPaCa2) cells in a dose-dependent manner with IC₅₀ of 2.25 and 0.93 μM, respectively, which is ca. 2-fold more potent than lead compound KYS05090 despite of its decreased T-type calcium channel blockade. As a mode of action for cytotoxic effect of BK10040 on lung cancer (A549) cells, this cancer cell death was found to have the typical features of apoptosis, as evidenced by the accumulation of positive cells for annexin V. In addition, BK10040 triggered the activations of caspases 3 and 9, and the cleavages of poly (ADP-ribose) polymerase (PARP). Moreover, the treatment with z-VAD-fmk (a broad spectrum caspase inhibitor) significantly prevented BK10040-induced apoptosis. Based on these results, BK10040 may be used as a potential therapeutic agent for human lung cancer via the potent apoptotic activity.

Full text of DOI:10.1016/j.bmcl.2014.01.071

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1565–1570
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of cellular proliferation and induction of apoptosis
in human lung adenocarcinoma A549 cells by T-type calcium channel
antagonist
Doo Li Choi a, Sun Jeong Jang a, Sehyeon Cho a, Hye-Eun Choi b, Hong-Kun Rim b, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
The anti-proliferative and apoptotic activities of new T-type calcium channel antagonist, 6e (BK10040)
on human lung adenocarcinoma A549 cells were investigated. The MTT assay results indicated that
BK10040 was cytotoxic against human lung adenocarcinoma (A549) and pancreatic cancer (MiaPaCa2)
Received 11 November 2013
Revised 15 January 2014
Accepted 24 January 2014
Available online 3 February 2014
cells in a dose-dependent manner with IC50 of 2.25 and 0.93 lM, respectively, which is ca. 2-fold more
potent than lead compound KYS05090 despite of its decreased T-type calcium channel blockade. As a
mode of action for cytotoxic effect of BK10040 on lung cancer (A549) cells, this cancer cell death was
found to have the typical features of apoptosis, as evidenced by the accumulation of positive cells for
annexin V. In addition, BK10040 triggered the activations of caspases 3 and 9, and the cleavages of poly
(ADP-ribose) polymerase (PARP). Moreover, the treatment with z-VAD-fmk (a broad spectrum caspase
inhibitor) significantly prevented BK10040-induced apoptosis. Based on these results, BK10040 may
be used as a potential therapeutic agent for human lung cancer via the potent apoptotic activity.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
T-type calcium channel
Antagonist
Human lung adenocarcinoma (A549) cells
Pancreatic cancer (MiaPaCa2) cells
Apoptosis
Cell cycle arrest
Caspases
Calcium plays a key role in intracellular signaling and controls
many different cell processes such as proliferation, differentiation,
growth, cell death and apoptosis.1,2 Thus, alterations in calcium
signaling can cause defects in cell growth and are associated with
certain types of cancer.3,4 A number of research groups have sug-
gested a potential role for voltage-activated Ca2+ channels, in par-
ticular T-type (Cav3), in the regulation of tumor growth and
progression.5–9 This T-type Ca2+ channel family (Cav3) contains
calcium channel blockade.17–20 In an effort to improve anti-prolif-
erative activity of lead compound KYS050590 as 3,4-dihydroqui-
nazoline derivative, we incorporated bioisosteres into our further
optimization process because this strategy has been frequently
used for such a purpose in conventional medicinal chemistry.21
Accordingly, the modification of three parts (P-1, P-2, and P-3 part)
in KYS05090 led to new eight 3,4-dihydroquinazoline derivatives
as shown in Figure 1 and Table 1. Among these compounds, 6e
(BK10040) was found to be more potent than KYS05090 against
human lung adenocarcinoma (A549) and pancreatic cancer
(MiaPaCa2) cells. Compound 6e (BK10040) was obtained by the
replacement of N,N0,N0-trimethyl-1,5-pentanediamine chain at
P-1 part and biphenyl ring at P-2 part with 4-[2-(dimethyl-
amino)ethyl]piperazine ring and p-cyclohexylphenyl ring, respec-
tively, as shown in Figure 1. Herein we focus on how BK10040
can contribute to anti-proliferative activity in human lung adeno-
carcinoma (A549) cells via T-type calcium channel blockade.
We have synthesized new 3,4-dihydroquinazoline derivatives
through the modification of P-1, P-2, and P-3 parts by the same
procedure as described previously by our group as outlined in
Schemes 1 and 2.17–20 The reduction of trans-2-nitrocinnamate 1
with activated Zn and NH4Cl in MeOH afforded trans-2-aminocin-
namate 2, followed by an addition of commercially available or
three members: Cav3.1 (a1G), Cav3.2 (a1H) and Cav3.3 (a
1I).10 There
is a growing body of evidence that some of T-type Ca2+ channels
are abnormally expressed in cancerous cells compared to normal
cells and thus the blockade of these channels may reduce cell pro-
liferation in addition to inducing apoptosis in cancer cells.11–15
Lung cancer is the most common malignant tumor in the world.
Non-small cell lung cancer (NSCLC) is the majority of lung cancer,
approximately 80% of total malignancies, with a 5-year survival of
only 15%. The other 20% of total lung cancer is small cell lung can-
cer (SCLC).16 In our previous papers, our group have reported that
3,4-dihydroquinazoline derivatives exhibited in vitro and in vivo
anti-proliferative effects against lung cancer (A549) cells via T-type
⇑
Corresponding author. Tel.: +82 2 961 0726; fax: +82 2 966 3701.
E-mail address: ljy@khu.ac.kr (J.Y. Lee).
http://dx.doi.org/10.1016/j.bmcl.2014.01.071
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1566
D. L. Choi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1565–1570
Figure 1. Discovery of new 3,4-dihydroquinazoline derivative 6e (BK10040) via a modification of substituents of lead compound KYS05090.
Table 1
T-type Ca2+ channel blocking effect and anti-proliferative effect against lung (A549) and pancreatic (MiaPaCa2) cancer cells of T-type Ca2+ channel antagonists
O
X
R2
R3
N
N
R1
Entry
R1
R2
R3
X
Calcium channel blockade [T-type: Cav3.1 (
a
1G)]a
Cytotoxicity: IC50 (l
M)c
% Inhibition (@ 1
76.6 0.7
l
M)b
(IC50
:
l
M)b
A549
MiaPaCa2
( )3
N
N
KYS05090
5b
H
H
NH
O
0.26 0.01
1.81 0.21
4.07
1.94
N
46.4 4.7
26.4 2.8
55.2 2.3
4.29
5.44
6.65
2.70
2.54
3.99
N
N
N
( )4
N
N
6a
6b
H
NH
NH
NDd
( )4
N
N
OMe
F
0.73 0.07
O
6c
NH
53.5 1.9
0.60 0.06
14.95
7.43
N
H
N
N
N
N
N
6d
NO2
NH
NH
NH
NH
38.1 1.5
53.2 1.5
39.2 1.6
64.3 3.5
NDd
4.69
2.25
2.18
0.93
N
N
N
N
N
N
N
N
6e (BK10040)
H
1.04 0.10
NDd
6f
8
H
7.57
3.02
N(SO2Me)2
0.52 0.08
20.70
16.11
O
H
N
O
O
Mibefrail
55.1 1.1
0.56 0.11
24.8
18.4
N
N
F
a
b
c
T-type calcium channel (a1G) expressed on HEK293 cell.
Value was determined from dose–response curve and obtained from three independent experiments.
MTT assay.
d
ND: not determined when less than 40% inhibition at 1 lM.
in situ generated isocyanate from the reaction of the corresponding
carboxylic acid and diphenyl phosphorazidate (DPPA) in the pres-
ence of TEA produced urea compound 3. The amine chains for R2
substituent were synthesized as outlined in Scheme 2: N,N0,N0-
trimethyl-1,6-hexanediamine (13) was prepared according to the
previously reported method.19 For 3-(N,N-dimethylureidopropyl)-
1-methylamine 17, selective mono-protection of secondary amine
in 14 with di-tert-butyl dicarbonate and subsequent N-acylation
with dimethylcarbamoyl chloride afforded compound 16, which
was treated with 3 N HCl to give the target compound 17. The
dehydration of 3 with Ph3PꢀBr2 and Et3N provided carbodiimide
4, which was coupled with commercially available piperazine ring
or the above amine chain (13 or 17) to afford the 3,4-dihydroqui-
nazoline ester derivative 5a–b, respectively. The treatment of
methyl ester 5a (R = Me) with p-R3-benzylamine and 1,5,7-
triazabicyclo[4.4.0]dec-5-ene (TBD) as a catalyst under solvent-free

D. L. Choi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1565–1570
1567
O
O
O
O
OR
a
b
c
OR
R2
OR
OR
NH
N
C N
NO2
NH2
O
N
H
R2
1a: R = Me
2a: R = Me
2b
3a: R = Me
3b
4a: R = Me
1b
4b
: R = Bn
: R = Bn
: R = Bn
: R = Bn
d
O
O
RO
N
H
R2
R1
R2
e or f
R3
N
N
(for 5a)
N
N
R1
6d: R3 = NO2
5a
5b
: R = Me
: R = Bn
g
:
R3 = NH2
R3 = N(SO2Me)2
7
h
8
:
Scheme 1. Reagents and conditions:(a) Zn, NH4Cl, MeOH, reflux, 12 h, 95–96%; (b) R2-CO2H, DPPA, Et3N /or R2-NCO, toluene, rt to 100 °C, 12 h, 55–79%; (c) Ph3PꢀBr2, Et3N,
CH2Cl2, 0 °C, 12 h, 64–81%; (d) compound 13 or 17 in Scheme 2 or 4-[2-(dimethylamino)ethyl]piperazine, toluene, rt, 3 h, 44–97%; (e) p-R3-BnNH2, TBD, rt, 3 h, 61–89%; (f) (i)
LiOHꢀH2O, THF–H2O (1:1), 70 °C, 12 h, quantitative, (ii) EDC, HOBt, p-NO2-BnNH2, THF–CH2Cl2 (1:1), 0 °C to rt, 54% (for 6d); (g) H2, 5% Pd/C, MeOH, rt, 48 h, 74%; (h) Me2SO2Cl
(1 equiv), TEA, CH2Cl2, rt, 1 h, 67%.
condition afforded directly the corresponding 3,4-dihydroquinazoline
amide derivative 6, respectively. In the case of only p-nitrobenzyl
amide 6d, the methyl ester 5a was hydrolyzed with LiOH to
provide the free carboxylic compound in quantitative yield. This
acid compound was coupled with p-nitrobenzylamine by using
EDC and HOBt to give the p-nitrobenzylamide 6d, followed by
hydrogenation with 5% Pd/C afforded p-aminobenzyl amide 7.
The coupling of 7 with 1 equiv of methansulfonyl chloride in the
presence of Et3N afforded unexpectedly bis(methanesulfonyl)amino
compound 8. Finally, a series of 6a–f were prepared as HCl salt by
treatment with concd HCl in EtOAc solution.
human lung adenocarcinoma (A549) and pancreatic cancer
(MiaPaCa2) cells using MTT assay.23 Both assay results for the inhi-
bition of calcium influx and the cytotoxicity on human cancer cells
were summarized in Table 1 together with those of mibefradil and
KYS05090 as positive controls.
With respect to T-type channel blocking effect, all trials of
replacement of P-1 and/or P-2 and/or P-3 part in KYS05090 with
various substituents led to a decrease in potency (both % inhibi-
tions at 1 lM and IC50 values) and four analogs (5, 6a, 6d, and
6f) was particularly far less potent than KYS05090, indicating the
importance of the appropriate methylene unit (n = 3) of diamine
chain at P-1 part, the biphenyl ring at P-2 part, and the amide bond
at P-3 part for potent T-type channel blockade. This assay result
was practically consistent with our previously extensive SAR stud-
ies on a variety of 3,4-dihydroquinazoline derivatives for T-type
Ca2+ channel blockade.17–20
With respect to cytotoxic effect on both cancer cells, first of all,
all new analogs disclosed a broad spectrum of cytotoxicity with
IC50 values of 0.93 into 20.78 lM irrespective of a degree of T-type
The channel blocking activity for new 3,4-dihydroquinazoline
derivatives was evaluated against T-type channel Cav3.1 (a1G
)
expressed on HEK293 cell at 1 lM concentration by whole cell
patch-clamp methods.22 For only compounds showing more than
40% inhibition value, the molar concentrations (IC50) of compounds
required to produce 50% inhibition of a1G T-type currents were
determined from fitting raw data into dose–response curves. All
derivatives were evaluated for anti-proliferative activity against
a
b
c
Boc
Boc
( )4
N
H
N
Boc
( )4
( )4
( )4
N
H
N
H
N
H
H2N
NH2
N
H
9
10
11
12
b
( )4
N
H
N
13
O
O
c
d
e
N
H
NH2
N
Boc
NH2
N
Boc
N
N
N
N
N
H
H
H
14
15
16
17
Scheme 2. Reagents and conditions: (a) (Boc)2O, MeOH, rt, 1 h, 91%; (b) LiAlH4, THF, reflux, 12 h, 96% for 11 and 50% for 13; (c) (Boc)2O, MeOH, concd HCl, 0 °C to rt, 3 h, 33%
for 12 and 23% for 15; (d) dimethylcarbamoyl chloride, CH2Cl2, ꢁ10 °C, 4 h, 75%; (e) 3 N HCl, MeOH, rt, quantitative.

1568
D. L. Choi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1565–1570
Figure 2. Inhibition of T-type Ca2+ channel current by KYS05090 and 6e (BK10040) 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) Representative current traces illustrating the inhibition of T-type Ca2+ channel by
18 lM KYS05090. (B) Dose–response curve for BK10040 yielded an IC50 value.
Table 2
The cell cycle phase distribution of human lung adenocarcinoma (A549) cells by T-type Ca2+ channel antagonists
Compound
Cell cycle phase (%) in A549a
For 24 h
For 48 h
Sub-G1
G0/G1
S
G2/M
Sub-G1
G0/G1
S
G2/M
KYS05090
6e (BK10040)
Mibefradilb
Controlc
55.3
15.8
9.6
33.6
55.4
66.1
53.1
8.8
2.2
16.0
13.4
21.6
98.1
77.8
10.0
4.9
1.2
15.7
66.8
57.8
0.6
4.6
12.0
12.0
0.1
1.9
11.2
25.4
12.8
10.9
17.6
7.8
a
b
c
Effect of each compound @ 5
l
M on the cell cycle progression of A549 cells.
Effect of mibefradil @ 10
Untreated A549 cells.
lM on the cell cycle progression of A549 cells.
Figure 3. The effect of T-type Ca2+ channel antagonist on the cell cycle arrest in A549 lung cancer cells. A549 cells were treated with 5
lM concentration of each antagonist
for 24 or 48 h, respectively. Cell cycle phases were detected by FACS.
channel blockade, as shown in Table 1. Interestingly, all derivatives
as well as KYS05090 were about 2-fold more potent against pan-
creatic (MiaPaCa2) cancer cells than against lung (A549) cancer
cells. Among them, new derivative 6a containing 1,6-diamine at
R1/a naphthalene ring at R2, 6d containing a piperazine ring at
R1/a nitro group at R3, and 6f containing a piperazine ring at R1/a
p-isopropylphenyl ring at R2 exhibited appropriate activities with
be noticed that 6e (BK10040) possessing a piperazine ring at R1
and a p-cyclohexylphenyl ring at R2 was ca. 2-fold and 4-fold less
potent (IC50 = 1.04 0.10 lM) than mibefradil and KYS05090,
respectively, for T-type Ca2+ channel (Cav3.1) blockade as shown
in Figure 2. Despite of this relatively less degree of T-type channel
blockade, however, 6e (BK10040) showed ca. 2-fold more potency
(IC50 = 2.25 and 0.93 lM) against both human cancer cells than
IC50 values of 5.44, 4.69 and 7.57
channel blocking effects (>40% inhibitions at 1
l
M, respectively, despite of lower
KYS05090.
l
M). On the con-
In order to investigate this discordance between T-type Ca2+
channel blocking effect and anti-proliferative effect, therefore,
the effect of 6e (BK10040) on cell cycle progression of A549 cells
was performed using flow cytometry (FACS)24 together with those
of mibefradil and KYS05090 as positive controls. Flow cytometry-
generated cell cycle phase distribution revealed that A549 cells
trary, new derivative 6c containing
x
-ureido group at R1/ fluoro
atom at R3, and 8 containing a piperazine ring at R1/p-bis(methan-
sulfone)amido group at R3 showed less activities with IC50 values
of 14.95 and 20.70
lM, respectively, despite of potent channel
blocking effects comparable to that of KYS05090. Finally, it should

D. L. Choi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1565–1570
1569
Figure 4. Compound 6e (BK10040) induces apoptosis in A549 lung cancer cells. A549 cells were stained with Annexin V (FITC) and propidium iodide (PI) after treatment with
6e (BK10040). Fluorescence-activated cell sorting analysis of A549 cells at 24 h following treatment with 0, 1, 3, and 5 M 6e (BK10040) (C, D, E, F, respectively). Percentage
l
of each 2 and 4 area represents Annexin V-positive/PI-negative (early apoptotic) and Annexin V-positive/PI-positive cells (apoptotic), respectively.
Figure 5. (A) 6e (BK10040) alters pro-caspase-3, -9 and PAPR levels in A549 lung cancer cells. Western blot analysis of cells treated without or with 6e (BK10040) (1, 3 or
5 lM). (B) Cell death treated with 5 lM 6e (BK10040) in the absence or presence of 5 lM z-VAD-fmk.
treated with each compound for 24 or 48 h harvest exhibited a dis-
tribution of cell cycle phase as shown in Table 2 and Figure 3. The
very high percentage of sub-G1 phase for KYS05090-treatment
indicated the A549 cell death for 24 or 48 h harvest due to its
inherent strong cytotoxicity as well as T-type channel blockade.
In the case of 6e (BK10040)-treatment for 24 h, A549 cells showed
a little increment in the sub-G1 phase, and a little reduction in S
and G2/M phase of the cell cycle compared to those of untreated
control. After 48 h, the percentage of cells in the sub-G1 phase
was significantly increased and the G0/G1 phase was significantly
decreased in response to 6e (BK10040)-treatment comparable to
that of KYS05090-treated cells. Therefore, T-type Ca2+ channel
blockade-induced sub-G1 phase (cell death) increment may be
one of possible mechanisms of anti-proliferative activity for 6e
(BK10040), which is consistent with the reported literatures.25–28
Generally, the sub-G1 phase (cell death) may be related with the
induction of apoptosis or necrosis in A549 cells via T-type Ca2+
channel blockade. Thus, fluorescence-activated cell sorting analy-
sis was performed to identify the possibility of induction of apop-
tosis in A549 cell when treated with 6e (BK10040). FACS analysis
of Annexin V-FITC and PI stained A549 cells was used to determine
the percentage of viable and apoptotic cells following treatment
with 6e (BK10040) for 24 h.29 As shown in Figure 4, a dose-dependent
effect of 6e (BK10040) in inducing apoptosis in A549 cells was
observed. The percentage sum of early apoptotic cells (Annexin
V-positive/PI-negative) and late apoptotic cells (Annexin V-positive/
PI-positive) was 6.1% for control, 5.7% for 1
lM, 32.3% for 3 lM
and 45.4% for 5 M of 6e (BK10040), respectively.
l
The induction of apoptosis essentially needs the activation of
caspases as a group of intracellular proteases, which are responsi-
ble for planning the cell into apoptotic bodies during apoptosis.30–
33
Caspases are present as inactive proenzymes that are activated
by proteolytic cleavage.34 In particular, caspases-3, -8, and -9 are
situated at pivotal junctions in apoptosis pathways.35–37 Therefore,
the effect of 6e (BK10040) on the expression of proteins related to
apoptosis was tested. As shown in Figure 5(A), treatment of A549
cells with 5 lM 6e (BK10040) for 24 h significantly reduced the
expression of pro-caspase-3 and -9, indicating the potential activa-
tion of both of caspase-3 and -9 via proteolytic cleavage. In addi-
tion, increased cleavage of PARP protein was seen after a 24 h
exposure to 5 lM 6e (BK10040). To further confirm the involve-
ment of caspases in 6e (BK10040)-induced apoptosis, z-VAD-fmk
(a broad spectrum caspase inhibitor) was used at concentrations
that completely blocked the activations of caspases.38 As shown

1570
D. L. Choi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1565–1570
19. Kang, H. B.; Rim, H. K.; Park, J. Y.; Choi, H. W.; Choi, D. L.; Seo, J. H.; Chung, K. S.;
Huh, G.; Kim, J.; Choo, D. J.; Lee, K.-T.; Lee, J. Y. Bioorg. Med. Chem. Lett. 2012, 22,
1198.
20. Rim, H. K.; Lee, H. W.; Choi, I. S.; Park, J. Y.; Choi, H. W.; Choi, J. H.; Cho, Y. W.;
Lee, J. Y.; Lee, K.-T. Bioorg. Med. Chem. Lett. 2012, 22, 7123.
21. Shu, Y.-Z.; Johnson, B. M.; Yang, T. J. AAPS J. 2008, 10, 178. and references
therein.
22. Monteil, A.; Chemin, J.; Bourinet, E.; Mennessier, G.; Lory, P.; Nargeot, J. J. Biol.
Chem. 2000, 275, 6090.
23. 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-
in Figure 5(B), z-VAD-fmk significantly suppressed 6e (BK10040)-
induced apoptosis from 50% to 39%, indicating that 6e (BK10040)
induced apoptosis is partly dependent on the activation of caspas-
es. Taken together, these biological results demonstrate that 6e
(BK10040) displayed an induction of apoptosis in A549 cells via
caspase-3 and caspase-9-dependent pathway triggered by T-type
Ca2+ channel blockade.
In conclusion, new synthetic 3,4-dihydroquinazoline derivative
6e (BK10040) was found to be more potent than lead compound
KYS05090 against human lung adenocarcinoma (A549) and pan-
creatic cancer (MiaPaCa2) cells, whereas it showed the decreased
effect of T-type Ca2+ channel blockade compared to that of
KYS05090. As a study on mode of action for cytotoxic effect on
A549 cells, this compound was found to induce the death of human
lung cancer (A549) cell via T-type Ca2+ channel blockade based on
the increased cell cycle phase at sub-G1 phase 48 h after treatment.
The induction of apoptosis as a cell death pathway is also found to
be mediated through the activation of both of caspase-3 and -9.
Thus, 6e (BK10040)39 was decided to further evaluate in vivo effi-
cacy using xenograft animal model in order to identify a potential
therapeutic agent for human lung cancer. In vivo experimental as
well as pharmacokinetics profiles are in progress and will be an-
nounced in the future.
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in
3
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
24. Lee, K. W.; Kim, H. J.; Lee, Y. S.; Park, H. J.; Choi, J. W.; Ha, J.; Lee, K.-T.
Carcinogenesis, 2007, 28, 1928: A549 cells were treated with 5 lM
concentrations of each compound, washed with 1 mL phosphate buffered
saline (PBS), fixed in 70% ice-cold ethanol, and kept in a freezer overnight. The
fixed cells were centrifuged, washed twice with PBS and re-suspended in PBS
containing 50 mg/mL PI and 100 lg/mL DNase-free RNase A. The cell
suspension, which was hidden from light, was incubated for 30 min and
analyzed using the fluorescence-activated cell sorting cater-plus FACS (Becton
Dickinson Co, Heidelberg, Germany). Raw data for the distribution of DNA
content were expressed as the percentage of cells in the sub-G1, G0/G1, S, and
G2/M populations.
Acknowledgment
25. Hwang, J. A.; Hwang, M. K.; Jang, Y.; Lee, E. J.; Kim, J. E.; Oh, M. H.; Shin, D. J.;
Lim, S.; Ji, G. O.; Oh, U.; Bode, A. M.; Dong, Z.; Lee, K. W.; Lee, H. J. J. Nutr.
Biochem. 2013, 24, 1096.
26. Kondratskyi, A.; Yassine, M.; Kondratska, K.; Skryma, R.; Slomianny, C.;
Prevarskaya, N. Front Physiol. 2013, 4, 272.
This work was supported by a Grant from the Kyung Hee Uni-
versity in 2012 (KHU-20120770).
27. Miri, R.; Motamedi, R.; Rezaei, M. R.; Firuzi, O.; Javidnia, A.; Shafiee, A. Arch.
Pharm. 2011, 344, 111.
Supplementary data
28. Giordanetto, F.; Knerr, L.; Selmi, N.; Llinàs, A.; Lindqvist, A.; Wang, Q.-D.;
Ståhlberg, P.; Thorstensson, F.; Ullah, V.; Nilsson, K.; O’Mahony, G.; Högberg,
Å.; Lindhardt, E.; Åstrand, A.; Duker, G. Bioorg. Med. Chem. Lett. 2011, 21, 5557.
29. Ki, Y. S.; Park, E.Y.; Lee, H. W.; Oh, M. S.; Cho, Y. W.; Kwon, Y. K.; Moon, J. H.; Lee
K.-T. Biol. Pharm. Bull. 2010, 33, 1054: For PI and Annexin V double staining,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.
071.
A549 cells were suspended with 100
NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and stained with 5
conjugated Annexin and of PI (50 g/mL) for 15 min at room
temperature in dark place and then added 400 binding buffer, and
analyzed by the fluorescence-activated cell sorting (FACS) cater-plus flow
cytometry (Becton Dickinson Co, Heidelberg, Germany).
l
L of binding buffer (10 mM HEPES/
l
L of FITC-
References and notes
V
5
l
L
l
lL
1. Berridge, M. J.; Lipp, P.; Bootman, M. D. Nat. Rev. Mol. Cell Biol. 2000, 1, 11.
2. Berridge, M. J.; Bootman, M. D.; Roderick, H. L. Nat. Rev. Mol. Cell Biol. 2003, 4,
517.
3. Santoni, G.; Santoni, M.; Nabissi, M. Br. J. Pharmacol. 2012, 166, 1244.
4. Zhang, Y.; Zhang, J.; Jiang, D.; Zhang, D.; Qian, Z.; Liu, C.; Tao, J. Br. J. Pharmacol.
2012, 166, 1247.
5. Gray, L. S.; Perez-Reyes, E.; Gamorra, J. C.; Haverstick, D. M.; Shattock, M.;
McLatchie, L.; Harper, J.; Brooks, G.; Heady, T.; Macdonald, T. L. Cell Calcium
2004, 36, 489.
6. Lory, P.; Bidaud, I.; Chemin, J. Cell Calcium 2006, 40, 135.
7. (a) Berridge, M. J. Neuron 1988, 21, 13; (b) Yunker, A. M.; McEnery, M. W. J.
Bioenerg. Biomembr. 2003, 35, 533.
8. Monteil, A.; Chemin, J.; Leuranguer, V.; Altier, C.; Mennessier, G.; Bourinet, E.;
Lory, P.; Nargeot, J. J. Biol. Chem. 2000, 275, 16530.
9. Chemin, J.; Monteil, A.; Lory, P. J. Soc. Biol. 2003, 197, 235.
10. Perez-Reyes, E.; Lory, P. CNS Neurol. Disord.: Drug Targets 2006, 5, 605.
11. Perez-Reyes, E. Physiol. Rev. 2003, 83, 117.
12. Li, M.; Xiong, Z.-G. Int. J. Physiol. Pathophysiol. Pharmacol. 2011, 3, 156.
13. Latour, I.; Louw, D. F.; Beedle, A. M.; Hamid, J.; Sutherland, G. R.; Zamponi, G.
W. Glia 2004, 48, 112.
14. Taylor, J. T.; Zeng, X.-B.; Pottle, J. E.; Lee, K.; Wang, A. R.; Yi, S. G.; Scruggs, J. A.;
Sikka, S. S.; Li, M. World J. Gastroenterol. 2008, 14, 4984.
15. Li, W.; Zhang, S.-L.; Wang, N.; Zhang, B.-B.; Li, M. Cancer Invest. 2011, 29, 339.
16. Smith, R. A.; Brooks, D.; Cokkinides, V.; Saslow, D.; Brawley, O. W. CA Cancer J.
Clin. 2013, 63, 88.
30. Thornberry, N. A.; Lazebnik, Y. Science 1998, 281, 1312.
31. Nagata, S. Cell 1997, 88, 355.
32. Nicholson, D. W. Cell Death Differ. 1999, 6, 1028.
33. Earnshaw, W. C.; Martins, L. M.; Kaufmann, S. H. Annu. Rev. Biochem. 1999, 68,
383.
34. Kaufmann, S. H.; Earnshaw, W. C. Exp. Cell Res. 2000, 256, 42.
35. Ashkenazi, A.; Dixit, V. M. Science 1998, 281, 1305.
36. Green, D. R.; Reed, J. C. Science 1998, 281, 1309.
37. Budihardjo, I.; Oliver, H.; Lutter, M.; Luo, X.; Wang, X. Annu. Rev. Cell Dev. Biol.
1999, 15, 269.
38. Mohamed, A. E.; Zhu, Q.; Wang, Q.; Gulzar, W.; Altaf, A. W. Int. J. Cancer 2005,
117, 409.
39. The spectral data for 6e (BK10040: free form): mp 228–240 °C (salt); 1H NMR
(400 MHz, CDCl3) d 7.29–7.23 (3H, m, Ph), 7.22–7.16 (3H, m, Ph), 7.13–7.11
(1H, m, Ph), 7.06–6.99 (4H, m, Ph), 6.96–6.87 (2H, m, Ph), 5.81 (1H, t, J = 5.6 Hz,
–CO–NH–CH2–), 5.17 (1H, dd, J = 9.6 and 5.6 Hz, –CO–CH2–CH–N–), 4.47–4.38
(2H, m, –NH–CH2–Ph–), 3.32 (4H, m, –N–(CH2–CH2)2–NCH2–), 2.58 (1H, dd,
J = 14.0 and 9.6 Hz, –CO–CH2–), 2.42 (1H, dd, J = 14.0 and 9.6 Hz, –CO–CH2–),
2.45–2.40 (1H, m, Ph-cyclohexyl), 2.34 (4H, s, –N–CH2–CH2–NMe2), 2.22 (6H, s,
–NMe2), 2.17 (4H, m, –N–(CH2–CH2)2–N–CH2–), 1.83–1.81 (4H, m, cyclohexyl),
1.75–1.71 (1H, m, cyclohexyl), 1.39–1.32 (4H, m, cyclohexyl), 1.29–1.21 (1H,
m, cyclohexyl); 13C NMR (100 MHz, CDCl3) d 170.0, 153.4, 144.1, 143.6, 143.3,
138.0, 128.7, 128.2, 128.1, 127.6, 127.4, 126.3, 125.0, 122.6, 122.5, 122.5, 56.8,
56.6, 52.9, 45.9, 45.7, 43.9, 43.8, 42.2, 34.5, 34.4, 26.9, 26.1; HRMS (FAB+): m/z
estimated for C37H49N6O [M+H]+ 593.3968, found: 593.3969.
17. Heo, J. H.; Seo, H. N.; Choe, Y. J.; Kim, S.; Oh, C. R.; Kim, Y. D.; Rhim, H.; Choo, D.
J.; Kim, J.; Lee, J. Y. Bioorg. Med. Chem. Lett. 2008, 18, 3899.
18. Jung, S. Y.; Lee, S. H.; Kang, H. B.; Park, H. A.; Chang, S. K.; Kim, J.; Choo, D. J.; Oh,
C. R.; Kim, Y. D.; Seo, J. H.; Lee, K.-T.; Lee, J. Y. Bioorg. Med. Chem. Lett. 2010, 20,
6633.