Synthesis and potential cytotoxic activity of new phenanthrylphenol-pyrrolobenzodiazepines

Article abstract of DOI:10.1016/j.ejmech.2010.01.054

New phenanthrylphenol-pyrrolobenzodiazepine (PP-PBD) conjugates have been synthesized and evaluated for their biological activity. One of the compounds 4a has been evaluated for its antiproliferative activity on 57 human tumour cell lines. The growth inhi

Full text of DOI:10.1016/j.ejmech.2010.01.054

European Journal of Medicinal Chemistry 45 (2010) 2173–2181
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and potential cytotoxic activity of new
phenanthrylphenol-pyrrolobenzodiazepines
,
a
a
b
a
Ahmed Kamal a , Kokkonda Sreekanth , P. Praveen Kumar , Nagula Shankaraiah , G. Balakishan ,
*
M. Janaki Ramaiah a, S.N.C.V.L. Pushpavalli a, Paramita Raya, Manika Pal Bhadra a
,
*
a Chemical Biology Laboratory, Division of Organic Chemistry-I, Indian Institute of Chemical Technology, Hyderabad 500 607, India
b National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
a r t i c l e i n f o
a b s t r a c t
Article history:
New phenanthrylphenol-pyrrolobenzodiazepine (PP-PBD) conjugates have been synthesized and eval-
uated for their biological activity. One of the compounds 4a has been evaluated for its antiproliferative
activity on 57 human tumour cell lines. The growth inhibition of 4a–c has been determined by MTT
viability assay on MCF-7 cell line. Among them, 4c showed most potent growth inhibition. Based on this,
an attempt was made to rationalize their mechanism of action through cell cycle analysis and DNA
interaction studies. The effect of the lead compound 4c on MCF-7 cell growth associated with cell cycle
arrest in G1 phase, followed by apoptosis. Our findings suggested the phenanthrylphenol-PBD conjugate
4c, which is a cyclin D1 inhibitor could be considered as a promising lead compound against breast
cancer for further investigation.
Received 10 December 2009
Received in revised form
19 January 2010
Accepted 21 January 2010
Available online 2 February 2010
Keywords:
Pyrrolobenzodiazepines
Phenanthrylphenol
DNA binding affinity
Anticancer activity
G1 cell cycle arrest
Apoptosis
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
activity against breast cancer [11]. Similarly, the pyrrolo [9,10]
phenanthrene molecules that effectively intercalate with DNA
In spite of great progress in chemotherapy, there has been
significant development in recent years in the number of new
anticancer agents, with the emphasis on creating new DNA inter-
active drugs [1–3]. Presently, most of the anticancer drugs interact
with DNA mainly by two binding modes, which are DNA minor
groove binding through a combination of hydrophobic, electro-
static, hydrogen-bonding interactions and intercalative binding in
which a planar aromatic moiety slides between the DNA base pairs
[4–6]. A number of polycyclic aromatic hydrocarbons (PAHs) and
their derivatives in view of their planar ring system are known to
intercalate with DNA resulting in the anticancer activity [7,8]. PAHs
have the potential to disrupt the normal function of cellular DNA
and could lead to mutagenesis, carcinogenesis and even cell death
[9,10]. A variety of phenanthrene ring systems intercalate with
DNA, giving rise to many drugs that possess chemotherapeutic
activity. As shown in Fig. 1 (1 and 2), substituted phenanthrenes
with basic amino side chains have shown significant anticancer
results in synergetic enhancement of DNA immobilization/cross
linking [12]. Recently, several phenanthrene based tylophorine
derivatives as potential anticancer agents have been reported [13].
These results together with other related studies indicate that
a linear tricyclic chromophore is the minimum requirement for
efficient intercalative binding [14].
On the other hand, it has been demonstrated that small mole-
cules may recognize an increasing range of DNA sequences, which
have DNA binding properties. These include pyrrolo
[2,1-c][1,4]benzodiazepines (PBDs) and lexitropsins [15]. PBDs are
well known naturally occurring DNA interactive antitumour anti-
biotics like anthramycin, chicamycin and DC-81 (3), which are
produced from various Streptomyces bacteria [16]. They exert their
biological activity through covalent binding via their N10–C11
imine/carbinolamine moiety to the C2-amino position of a guanine
residue within the minor groove of duplex DNA. PBD monomers
span three DNA base pairs with a preference for 50-Pu-G-Pu,
particularly 50-AG or 50-GA sequences [17]. The cytotoxicity of these
agents is thought to be due to their ability to inhibit cellular
processes such as replication and transcription.
* Corresponding authors. Tel.: þ91 40 27193157; fax: þ91 40 27193189.
E-mail addresses: ahmedkamal@iict.res.in (A. Kamal), manika@iict.res.in (M.P.
Bhadra).
Previously, in our laboratory a few hybrid molecules have been
synthesized wherein chrysene and pyrene are linked to PBD moiety
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.054

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A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 2173–2181
by cross coupling of commercially available 9-bromophenanthrene
and 4-methoxyphenylboronic acid (5) using Pd(PPh3)4 and K3PO4
in 1,4-dioxane/H2O. Then, demethylation step is carried out with
borontribromide (BBr3) in anhydrous CH2Cl2 to give the required
phenathrylphenol intermediate 7.
The key precursor 8 has been prepared from commercially available
vanillin through several straightforward functional group trans-
formations as reported in our earlier studies [20]. Then the debenzy-
lation of intermediate 8 with BF3.OEt2/EtSH gives (2S)-N-((4-hydroxy)-
MeO
MeO
OMe
Cl
N
N
H
R1 = Me, Et
Cl
R2 = (CH2)4, (CH2)5
1
O
2
NR1R2
5-methoxy-2-nitrobenzoyl)pyrrolidine-2-carboxaldehyde
diethyl
N
( )nO
10
N
B11a
5
O
H
9
11
thioacetal (9). Etherification of this hydroxyl compound 9 with various
dibromoalkanes in the presence of K2CO3 produces the (2S)-N-
{4-[3-bromoalkoxy-5-methoxy-2-nitrobenzoyl}pyrrolidine-2-ca-
rboxaldehyde diethyl thioacetal intermediates (10a–c). Later, the
HO
H
8
1
A
N
MeO
C
N
MeO
7
2
O
6
4
3
O
4a; n = 3
4b; n = 4
4c; n = 5
phenanthrylphenol precursor
7 is coupled to each of the
3
intermediate 10 using K2CO3 in acetone to provide the nitro-
thioacetal intermediates 11a–c. Finally, these upon reduction
with SnCl2.2H2O in methanol followed by deprotective-
cyclization using HgCl2–CaCO3 affords the desired target PBD
conjugates 4a–c in good yields ranging from 58 to 60% as
depicted in Scheme 2.
Fig. 1. Representative chemical structures of phenanthrenes (1 and 2), DC-81 (3), and
phenanthrylphenol-PBD conjugates (4a–c).
at C8 position [18,19]. We observed that, such hybrids of PBD that
are linked to polycyclic aromatic hydrocarbons (PAH) enhance the
anticancer activity compared to DC-81. Using similar design
concepts, with the aim of obtaining new anticancer compounds we
have synthesized new phenanthrylphenol linked PBD (PP-PBD)
conjugates and evaluated the effect of these compounds on MCF-7
cell proliferation. Cell cycle arrest and apoptotic properties have
also been evaluated. The reason for choosing MCF-7 cell line for our
experimentation is that we have been interested in finding out the
relationship between substituted phenanthrenes and breast anti-
cancer activities as some of the phenanthrene derivatives are
known to exhibit antiproliferative effect against breast cancer cells
[11]. Moreover, the expression of cyclin D1 has shown to increase
the proliferation rate of MCF-7 cells. We carried out our studies
2.2. Evaluation of biological activity
2.2.1. Anticancer activity
Representative compound 4a has been evaluated by the
National Cancer Institute to determine its growth inhibitory prop-
erties against 57 human cancer cell lines producing a growth
inhibition (GI50) ranging from 0.04 to 1.76 mM as shown in Table 1.
Further, we examined the antiproliferative activity of these new PP-
PBDs (4a–c) on human breast cancer cell line MCF-7 using MTT
assay by treating cells at 0.5, 1, 2 and 4 mM of DC-81, compounds
4a–c for 24 h.
The new PP-PBD conjugates exhibit enhanced potency over the
parent compound DC-81 (Fig. 2). The results reveal that increasing
the length of alkyl chain results in excellent cytotoxicity. Accord-
ingly, the compound 4c exhibited its activity to cause significant
using this cell line since it has shown a GI50 value of 0.17 mM
obtained from the National Cancer Institute (NCI) anticancer
screening on the compound 4a.
The results suggest that the synthetic PP-PBD is indeed
a candidate for anticancer agent causing sub G1 arrest and
apoptosis following the intrinsic mitochondrial death pathway as
the mechanism of action. This proposed mechanism of intrinsic
mitochondrial path is also consistent with the release of cyto-
chrome c, activation of caspase-9 followed by PARP cleavage and
decrease in cyclin D1 levels.
cytotoxicity (up to 50%) at 1
show significant cytotoxicity (up to 50%) at 2
control cells have been taken as 100% in each of the compound
treatment. All the compounds (4a–c) are equally effective at 4 M.
Thus, it can be concluded that compound 4c is the most effective
cytotoxic agent.
m
M concentration while 4a and 4b
mM. Herein; the
m
2.2.2. RED100-restriction endonuclease digestion assay
Several reports in the literature have employed restriction
endonuclease inhibition study to confirm the relative binding
affinity of DNA interactive small molecule ligands [21–23]. A
quantitative restriction enzyme digestion (RED100) assay has been
developed in which the inhibition of DNA cleavage by BamHI is
used to probe the DNA binding capability of PBD monomers [24].
We have earlier investigated this assay for preferences of base pair
selectivity of imine–amide PBD dimers [25]. The drug when pre-
incubated by PP-PBD compounds suggest that these molecules
selectively interact with G-rich sequence of DNA. Therefore, PBD
compounds with DNA were subjected to BamHI digestion, and the
protection of cleavage of the GGATCC sites is due to affinity of PBDs
in covalent interaction with the free amino group attached to the N2
of guanine in DNA.
2. Results and discussion
2.1. Chemistry
Accordingly, Scheme 1 shows the synthesis of the required
phenanthrylphenol (7) moiety for the preparation of DNA inter-
active PP-PBD conjugates. This synthetic strategy involves the use
of Pd-catalyzed Suzuki cross coupling reaction for the arylation
followed by demethylation. Starting material 6 has been obtained
OMe
OH
OMe
b
a
In order to investigate the binding capability of these
compounds to DNA at G-rich regions and inhibit restriction
digestion by restriction endonuclease BamHI, we have carried out
B(OH)2
5
6
7
the assay at various concentrations (2, 4, 8, 16 and 32 mM) of these
compounds 4a–c. Interestingly, the concentration dependent
differential inhibitory activity by PBDs is observed for 100%
Scheme 1. Reagents and conditions: (a) 9-bromophenanthrene, Pd(PPh3)4, K3PO4,
dioxane–H2O, 80 ꢀC, 16 h, 70%; and (b) BBr3, dry CH2Cl2, ꢁ78 ꢀC, 5 h, 80%.

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 2173–2181
2175
CH(SEt)2
NO2
BnO
NO2 CH(SEt)2
N
HO
a
N
H3CO
H3CO
O
O
9
8
b
NO2
NO2
CH(SEt)2
CH(SEt)2
O
Br
O
O
( )n
( )n
c
N
N
MeO
MeO
O
O
11a−c; n = 3−5
10a−c; n = 3−5
d
NH2
O
CH(SEt)2
O
( )n
4a; n = 3
4b; n = 4
4c; n = 5
e
N
MeO
12a−c; n = 3−5
O
Scheme 2. Reagents and conditions: (a) BF3$OEt2/EtSH, dry CH2Cl2, r.t., 8 h, 88%; (b) dibromoalkanes, K2CO3, dry DMF, r.t., 24 h, 88–93%; (c) 7, K2CO3, dry acetone, reflux, 48 h, 85–
90%; (d) SnCl2.2H2O, MeOH, reflux, 4 h, 75–80%; and (e) HgCl2, CaCO3, CH3CNeH2O (4:1), r.t., 12 h, 58–60%.
inhibition at 16 mM in all the PP-PBD compounds tested (Fig. 3).
Based on the results of DNA binding studies and cytotoxicity, it has
been considered of interest to understand the mechanism of
tumour cell proliferation inhibition or cell death for these
conjugates.
Table 1
GI50 values of compound 4aa on 57 human cancer cell lines.
Cancer panel/cell line
GI50
(m
M)
Cancer panel/cell line
GI50 (mM)
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
0.13
0.17
0.27
0.18
0.19
0.22
1.02
1.59
0.57
0.04
1.26
1.06
1.36
0.63
2.2.3. Cell cycle perturbations and apoptosis induction
Cell cycle analysis has been performed to explore the basis for
antiproliferative properties of these PP-PBD conjugates (4a–c) at
2 mM and 4 mM concentrations in MCF-7 cells. G1 cell cycle arrest is
observed in all the three compounds tested, as indicated by
concentration dependent accumulation of cells in G0 phase. The
control cells treated with DMSO (the solvent in which the
compound is dissolved) showed 63.35% of G1 phase. Compound 4a
showed 73.46% and 83.68%, compound 4b showed 81.39% and
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Ovarian cancer
IGROV1
0.72
1.05
1.00
0.36
1.17
1.41
1.59
1.04
0.12
0.12
0.25
0.58
1.20
0.42
1.56
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
In vitro-cytotoxicity
120%
100%
Prostate cancer
DU-145
0.40
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
Renal cancer
786-0
A498
80%
1.19
1.21
0.25
1.18
0.89
0.78
0.51
0.70
0.69
1.26
0.40
0.24
0.51
0.60
0.16
DC-81
4a
4b
4c
60%
ACHN
CAKI-1
RXF 393
SN12C
TK-10
40%
20%
0%
UO-31
CNS cancer
SF-268
SF-539
SNB-19
SNB-75
U251
Breast cancer
MCF-7
NCI/ADR-RES
MDA-MB-231/ATCC
HS-578T
MDA-MB-435
BT-549
T-47D
0.5
1
2
4
Con
C+D
0.86
1.76
1.16
0.21
0.91
0.17
1.67
0.29
1.02
0.24
0.27
0.08
Compound Treatments
Fig. 2. Effect of PP-PBD compounds (4a–c) on cell viability (invitro cytotoxicity). MCF-
7 cells were treated with 0.5, 1, 2 and 4 M concentrations of PP-PBD compounds as
m
indicated for 24 h in 96-well plates seeded with 10,000 cells/well. O.D. readings were
taken at 570 nm wavelength to measure the percentage of cell viability after treatment
with the respective compound. DC-81 was used as the positive control. Con: control
cells (untreated cells), C þ D: control cells treated with DMSO. Numbers 0.5, 1, 2 and 4
represent concentrations of the compounds in micromolar.
a
Data obtained from the NCI’s in vitro disease-oriented human cancer cell line
screening.

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A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 2173–2181
observed that the decrease in cyclin D1 is significant in case of
compound 4c thus indicating that the cells are effectively arrested
at G1 phase of the cell cycle (Fig. 6).
2.2.6. PP-PBDs induced release of cytochrome c into the cytoplasm
Caspases are the family of cysteine proteases that are known to
play a key role in the process of apoptosis [29]. Moreover, apoptosis
occurs through two different pathways, that is the intrinsic and
extrinsic pathways. Intrinsic pathway involves mitochondria that
play an important role in apoptosis, whereas extrinsic pathway is
mediated by cell death receptors such as Fas, TNF
a after receiving
the death signal. Cytochrome c levels in the cytoplasm have been
determined by western blot analysis after treatment of MCF-7 cells
with these conjugates (4a–c) at 2
mM as well as 4 mM, and
compared to DC-81 (4 M concentrations for 24 h). It is observed
m
that there is an increase in cytosolic cytochrome c levels for all the
compounds including for DC-81. These results clearly show that PP-
PBD conjugates induce apoptosis through the intrinsic pathway
involving mitochondria (Fig. 7).
2.2.7. Activation of caspase-8 and 9 by PP-PBD conjugates
As induction of apoptotic cell death is mediated by caspases, the
involvement of various caspases and their role in the process of
apoptosis has been investigated. MCF-7 cells lack endogenous
caspase-3 protein that has been reported to play an important role
in drug-induced apoptosis [29]. MCF-7 cells were treated with 4a–c
Fig. 3. Inhibitory activity of compounds 4a–c on cleavage of PBR322 by restriction
endonuclease BamHI (10 U/m
l) for 2 h at 37 ꢀC. The cut (C) and the uncut (UC) were
at 2 and 4 mM concentration and DC-81 at 4 mM. It is observed that
used as controls. 2, 4, 8, 16 And 32 represents concentrations in micromolar used for
RED assay. Products were separated by 0.8% agarose gel electrophoresis and visualized
by ethidium bromide staining under UV illumination.
caspase-8 activity is measured by Calorimetry method, increases
when the MCF-7 cells were treated with PP-PBD conjugates (4a–c)
in comparison to the untreated control cells and DC-81 (Fig. 8a).
Further it is determined that the caspase-9 activity increases
91.16% and compound 4c showed 88.92% and 91.78% G1 phase at
significantly when MCF-7 cells have been treated with 4c at 2
mM
2
mM and 4
m
M concentrations, respectively. Whereas, DC-81 that
and 4 M concentrations using a fluorimetry based assays. This
m
has been taken as a positive control showed 69.17% and 83.15% of
G1 phase. Therefore, compound 4c could be considered as the most
effective one to cause cell cycle arrest. Moreover, the increase of G0
and G1 phase and decrease of G2/M phase of cell cycle clearly
indicates that these compounds follow an apoptotic pathway
(Fig. 4a, b).
activity reduced to the levels below that of control when caspase-9
inhibitor (LEHD-CHO) is added. A similar increase of caspase-9
activity (by twofolds) in comparison to untreated controls has
been observed when cells were treated with compounds 4a and 4b
at 4 mM concentrations (Fig. 8b).
2.2.8. Induced cleavage of PARP by PP-PBD conjugates
2.2.4. DNA fragmentation assay
Activation of caspases during apoptosis results in the cleavage of
The key feature of apoptotic response is the DNA ladder
formation mediated by enzymes called caspase-3, 8 and 9, that are
cysteine proteases [26]. DNA fragmentation assay has been carried
out to investigate apoptosis by treating the MCF-7 cells with the PP-
PBD conjugates. It is observed that DNA laddering is significant in
PARP (Poly ADP-ribose Polymerase) a 116 kDa protein [30].
Cleavage of PARP inactivates the enzyme and hence cannot bind to
DNA strand breaks during drug-induced apoptosis in many
different cell lines [31]. Since these compounds have shown an
increase in the caspase-9 activity and cytochrome c release, it is
considered of interest to understand their effect on cleavage of
PARP by western blot analysis using antiPARP antibody (89 kDa) on
MCF-7 cells. It is observed that there is an increase of cleaved PARP
expression in all the compounds tested as shown in Fig. 7.
compounds 4b and 4c at 4 mM concentration. The effectiveness of
the compounds in causing DNA fragmentation is in the order of
4b > 4c > 4a (Fig. 5). DNA laddering caused by the compounds
depends on the p53 protein expression level. Earlier studies report
that the threshold expression of p53 is important for DNA frag-
mentation [27]. It has been observed that p53 protein expression
level is more in 4b compared to 4c (data has not shown). However,
apart from p53, there could be some other proteins that may
regulate this event.
3. Conclusion
We have synthesized a set of phenanthrylphenol-PBD conju-
gates and evaluated them for the anticancer properties by a battery
of assays. RED100 assay has revealed that these compounds show
effective DNA binding activity. MTT assay, which indicates cyto-
toxicity, has revealed that PP-PBD derivative 4c to be highly effec-
tive. Flow cytometric data of these compounds showed increased
sub G1 peak, which is suggestive of apoptosis and G1 cell cycle
arrest. It is known that over expression of cyclin D1 increases the
phosphorylation status of retinoblastoma protein and transition
from G1 to S phase. Furthermore, MCF-7 cells have been found to be
dependent on cyclin D1/CDK4 pathway for their continued prolif-
eration. Interestingly, these compounds are effective as cyclin D1
2.2.5. Effect of PP-PBD conjugates on cyclin D1 levels
The cell cycle regulatory protein that control the G1 to S phase
transition is cyclin D1 that is accumulated during late G1 phase of
cell cycle [28]. In the present investigation the FACS data clearly
indicates that cell cycle arrest occurs at G1 phase and possibly has
an effect on cell cycle regulatory proteins particularly cyclin D1. The
levels of cyclin D1 have been determined by western blot analysis
for these PP-PBD conjugates (4a–c) in MCF-7 cells at 2
mM as well as
4
m
M concentrations and compared to DC-81 (4 M for 24 h). It is
m

Fig. 4. (a) DNA histograms obtained by flowcytometry. The percentages of cells in sub G0, G1, S, and G2/M cell cycle phase, after the treatment of MCF-7 cells with 4a–c at 2
M for 24 h. (b). FACS analysis of cell cycle distribution of MCF-7 cells after treatment with PP-PBD conjugates (4a–c) for 24 h. DC-81 was used as the positive control. C þ D:
control cells treated with DMSO. Numbers 2 and 4 in parenthesis represent concentrations of the treated compounds in micromolar.
mM and
4
m

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A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 2173–2181
Fig. 6. Effect of PP-PBD compounds on cyclin D1 levels. MCF-7 cells were treated with
PP-PBDs 4a–c at 2 M, 4 M and DC-81 at 4 M concentrations for 24 h. The cell lysates
were collected and expression levels of cyclin D1 were determined by western blot
analysis.
-Tubulin was used as loading control. C þ D: control þ DMSO.
m
m
m
b
hydride). 1H NMR spectra were recorded on Varian Gemini
200 MHz and Avance 300 MHz spectrometer using tetramethyl
silane (TMS) as an internal standard. Chemical shifts are reported in
parts per million (ppm) down field from tetramethyl silane. Spin
multiplicities are described as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet). Coupling constants are reported in Hertz
(Hz). Optical rotations are measured on Horiba, high sensitive
polarimeter, SEPA-300.
4.1. Synthesis of phenanthrylphenol-PBD conjugates
4.1.1. 9-(4-Methoxyphenyl) phenanthrene (6)
To
a stirred solution of 9-bromophenanthrene (257 mg,
1.0 mmol) in dioxane/H2O (10 mL/2 mL) in a sealable tube, was
added 4-methoxyphenylboronic acid (5) (304 mg, 2.0 mmol) and
K3PO4 (636 mg, 3.0 mmol). The mixture was bubbled with N2 over
5 min, and then Pd(PPh3)4 (110 mg, 0.1 mmol) was added and
heated to 80 ꢀC. The reaction mixture was then poured into water
(10 mL) and extracted with ethyl acetate (4 ꢂ15 mL). The combined
organic phases were washed with H2O, brine and dried over
Na2SO4. After removal of solvent, the residue was purified by
column chromatography. (198 mg, 70%): Rf ¼ 0.85 (EtOAc–hexane
1:9); White solid, mp: 142–144 ꢀC; 1H NMR (CDCl3, 200 MHz)
d:
3.90 (s, 3H, eOCH3), 7.0 (d, J ¼ 7.8 Hz, 2H, ArH), 7.4 (d, J ¼ 7.8 Hz, 2H,
ArH), 7.48–7.73 (m, 5H, ArH), 7.79–7.99 (m, 2H, ArH), 8.70 (m, 2H,
ArH).
Fig. 5. Agarose gel electrophoresis of DNA extracted from MCF-7 cells. The figure
represents MCF-7 cells treated with PP-PBD compounds for 24 h. DNA from the cells
was extracted and electrophoresed in 1% agarose gel and visualized by ethidium
bromide staining under UV illumination. Lane 1: 100 bp marker, Lane 2: control cells
(without treatment), Lane 3: control cells þ DMSO, Lane 4: treated by PP-PBD (4a-
4.1.2. 4-(9-Phenanthryl)phenol (7)
Compound 6 (284 mg, 1.0 mmol) was dissolved in dry CH2Cl2
(5 mL) to which borontribromide (35 mmol) was slowly added. This
solution was stirred at ꢁ78 ꢀC for 1 h under nitrogen atmosphere.
After this procedure, the reaction was slowly brought to ambient
temperature. TLC was indicated that the reaction reached by
completion usually within 5 h. Ice was added slowly to this mixture
and was neutralized with aqueous ammonium chloride and then
4
m
M). Lane 5: treated with PP-PBD (4b-4
mM). Lane 6: treated with PP-PBD (4c-4 mM).
Lanes 5 and 6 show a clear DNA fragmentation.
inhibitors and have shown a pronounced increase in caspase-9
activity, cytosolic cytochrome c and cleaved PARP. Moreover,
a correlation has been obtained with the earlier studies that have
demonstrated that cyclin D1 regulates the size and function of
mitochondria. Therefore, these results support the inhibition of
cyclin D1/CDK4 complex and development of such inhibitors as
potential anticancer agents.
4. Experimental protocols
Reaction progress was monitored by thin-layer chromatography
(TLC) using GF254 silica gel with fluorescent indicator on glass
plates. Visualization was achieved with UV light and iodine vapor
unless otherwise stated. Chromatography was performed using
Acme silica gel (100–200 and 60–120 mesh). The majority of
reaction solvents were purified by distillation under nitrogen from
the indicated drying agent and used fresh: dichloromethane
(calcium hydride), tetrahydrofuran (sodium benzophenone ketyl),
methanol (magnesium methoxide), and acetonitrile (calcium
Fig. 7. Effect of PP-PBD compounds on cytochrome c release and levels of cleaved
PARP. MCF-7 cells were treated with compounds 4a–c at 2
mM, 4 mM and DC-81 at
4
mM concentrations for 24 h. The cell lysates were collected and expression levels of
cytochrome c and cleaved PARP were determined by western blot analysis. b-Tubulin
was used as loading control. C þ D: control þ DMSO.

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 2173–2181
2179
1 mmol). The reaction mixture was heated to reflux for 48 h. After
completion of the reaction as indicated by TLC, potassium carbonate
was removed by suction filtration and the solvent was removed
under vacuum. The crude product thus obtained was purified by
column chromatography to afford pure compound 11a (639 mg,
90%); Rf ¼ 0.61 (EtOAc–hexane 3:7); Light yellow solid, mp: 64–
Caspase-8 Activity
a
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
ꢀ
23
66 ꢀC; [
a]
¼ ꢁ58 (c ¼ 0.9 in CHCl3); 1H NMR (CDCl3, 200 MHz)
d:
D
1.16–1.45 (m, 6H, e(CH3)2), 1.51–1.91 (m, 2H, eCH2e) 2.26–2.35 (m,
2H, eCH2CH2e), 2.50–2.62 (m, 2H, eCH2e), 2.63–2.90 (m, 4H,
2ꢂeSCH2e), 3.16–3.32 (m, 2H, eNCH2e), 3.99 (s, 3H, eOCH3), 4.27
(t, J ¼ 5.7 Hz, 2H, eOCH2-), 4.36 (t, J ¼ 5.7 Hz, 2H, eOCH2e), 4.65–
4.72 (m, 1H, eNCHe), 4.8 (d, J ¼ 3.0 Hz, 1H, eCHS2e), 6.82 (s, 1H,
ArH), 7.06 (d, J ¼ 8.6 Hz, 2H, ArH), 7.45 (d, J ¼ 8.6 Hz, 2H, ArH), 7.50–
7.72 (m, 5H, ArH), 7.76 (s, 1H, ArH), 7.82–8.07 (m, 2H, ArH), 8.63–8.82
(m, 2H, ArH). MS (ESI): m/z 711 [M þ H]þ.
C+D
DC-
81(4)
4a(2) 4a(4) 4b (2) 4b (4) 4c(2) 4c(4)
Compound Treatments
Caspase-9 Assay
4.1.4. (2S)-N-{4-(4-[4-(9-Phenanthryl)phenoxy]butyl)oxy-5-
methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehyde diethyl
thioacetal (11b)
2.5
b
The compound 11b was prepared according to the method
described for the compound 11a by employing compounds 7 and
10b (535 mg, 1 mmol) to afford the crude compound, which was
further purified by column chromatography affords compound 11b
(638 mg, 88%); Light yellow solid, Rf ¼ 0.62 (EtOAc–hexane 3:7);
2
1.5
1
ꢀ
¼ ꢁ66 (c ¼ 0.86 in CHCl3); 1H NMR (CDCl3,
23
mp: 60–62 ꢀC; [
200 MHz)
a]
D
d
:
1.16–1.42 (m, 6H, e(CH3)2), 1.75–1.93 (m, 4H,
2ꢂeCH2e), 2.05–2.32 (m, 4H, 2ꢂeCH2e), 2.67–2.89 (m, 4H,
2ꢂeSCH2e), 3.15–3.30 (m, 2H, eNCH2e), 3.89 (s, 3H, eOCH3),
4.03–4.31 (m, 4H, eOCH2e), 4.63–4.77 (m, 1H, eNCHe), 4.87 (d,
J ¼ 3.6 Hz, 1H, eCHS2e), 6.82 (s, 1H, ArH), 7.03 (d, J ¼ 8.6 Hz, 2H,
ArH), 7.46 (d, J ¼ 7.9 Hz, 2H, ArH), 7.51–7.74 (m, 6H, ArH), 7.83–8.05
0.5
0
(m, 2H, ArH), 8.66–8.81 (m, 2H, ArH); 13C NMR (CDCl3, 75 MHz)
d:
166.5, 154.4, 138.3, 137.2, 133.1, 131.4, 131.1, 130.6, 129.7, 128.5, 128.1,
127.3, 126.7, 126.3, 122.8, 122.4, 114.2, 109.2, 108.1, 69.2, 67.3, 61.0,
56.4, 52.8, 50.1, 27.2, 26.6, 26.2, 26.0, 25.7, 24.6, 15.1, 14.9 ppm; MS
(ESI): m/z 725 [M þ H]þ; Anal. calcd for C41H44N2O6S2: C 67.93, H
6.12, N 3.86, S 8.85, found: C 67.72, H 6.48, N 3.58, S 8.44.
I
I
I
)
)
)
)
)
)
)
4
D
+
4
2
(
4
2
4
2
+
+
+
)
(
(
a
(
(
(
(
c
)
)
c
1
a
4
C
4
b
b
4
4
4
(
4
4
8
(
(
b
4
4
-
a
c
C
4
4
4
D
Compound Treatments
Fig. 8. (a) Effect of PP-PBD conjugates on caspase-8 activity in MCF-7 cells. The
increased enzymatic activity of caspase-8, in apoptosis after the treatment of PP-PBDs
(4a–c) was determined by photometry. The cleavage of peptide by caspase-8 releases
the pNA, which can be quantified at wavelength of 405 nm using multimode varioskan
4.1.5. (2S)-N-{4-(5-[4-(9-Phenanthryl)phenoxy]pentyl)oxy-5-
methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehydediethyl
thioacetal (11c)
Flash. The concentrations of the compounds in micromolar (mM) are represented in
brackets. Here DC-81 was used as positive control. Numbers 2 and 4 in brackets
represent concentrations of the treated compounds in micromolar. (b). Effect of PP-
PBD conjugates on caspase-9 activity in MCF-7 cells. The increased enzymatic
activity of caspase-9, in apoptosis after the treatment of PP-PBDs (4a–c) was deter-
mined by flourimetry. The cleavage of peptide by caspase-9 releases the fluorophore
AMC that was quantified at excitation wavelength of 400 nm and emission wavelength
The compound 11c was prepared according to the method
described for the compound 11a by employing compounds 7 and 10c
(457 mg,1 mmol) to afford the crude compound, which was purified
by column chromatography, affords compound 11c. (628 mg, 85%);
Rf ¼ 0.63 (EtOAc–hexane 3:7); Light yellow solid, mp: 58–60 ꢀC;
ꢀ
of 505 nm. The concentrations of the compounds in micromolar (mM) are represented
23
[a]
¼ ꢁ54 (c ¼ 1 in CHCl3); 1H NMR (CDCl3, 200 MHz)
d: 1.19–1.42
D
in brackets. ‘I’ represents the inhibitor used. DC-81 was used as the positive control.
C þ D: control cells treated with DMSO. Numbers 2 and 4 in brackets represent
concentrations of the treated compounds in micromolar.
(m, 8H, e(CH3)2, 2ꢂeCH2e),1.44–1.62 (m, 2H, eCH2e),1.68–1.87 (m,
2H, eCH2e), 1.90–2.05 (m, 4H, eCH2e), 2.62–2.88 (m, 4H,
2ꢂeSCH2e), 3.14–3.33 (m, 2H, eCH2e), 3.89 (s, 3H, eOCH3), 4.06–
4.20 (m, 4H, 2ꢂeOCH2e), 4.63–4.78 (m, 1H, eCHS2e), 4.86 (d,
J ¼ 3.7 Hz, 1H, eNCHe), 6.80 (s, 1H, ArH), 7.01 (d, J ¼ 9.0 Hz, 2H), 7.44
(d, J ¼ 8.3 Hz, 2H, ArH), 7.47–7.71 (m, 6H, ArH), 7.83–7.95 (m, 2H,
organic layer was washed with water (3 ꢂ 10 mL). After drying by
Na2SO4, the product was concentrated under reduced pressure. The
target product was isolated using column chromatography.
(216 mg, 80%): Rf ¼ 0.79 (EtOAc–hexane 1:9); White solid, mp:
ArH), 8.66–8.79 (m, 2H, ArH); 13C NMR (CDCl3, 75 MHz)
d: 166.6,
150–152 ꢀC; 1H NMR (CDCl3, 200 MHz)
d
: 6.93 (d, J ¼ 8.59 Hz, 2H,
158.4, 154.4, 148.4, 138.3, 137.2, 133.0, 131.5, 131.0, 130.6, 129.7, 128.5,
128.0, 127.3, 126.7, 126.3, 122.8, 122.4, 114.2, 109.2, 108.1, 69.4, 67.6,
61.0, 56.4, 52.8, 50.1, 28.9, 28.6, 27.1, 26.6, 26.2, 24.6, 22.6, 15.1,
14.9 ppm; MS (ESI): m/z 739 [M þ H]þ; Anal. calcd. for C42H46N2O6S2:
C 68.27, H 6.27, N 3.79, S 8.68, found: C 67.99, H 6.22, N 3.46, S 8.42.
ArH), 7.4 (d, J ¼ 8.59 Hz, 2H, ArH), 7.46–7.71 (m, 5H, ArH), 7.80–7.97
(m, 2H, ArH), 8.71 (m, 2H, ArH).
4.1.3. (2S)-N-{4-(3-[4-(9-Phenanthryl)phenoxy]propyl)oxy-5-
methoxy-2-nitrobenzoyl}-pyrrolidine-2-carboxaldehyde diethyl
thioacetal (11a)
4.1.6. 7-Methoxy-8-[N-(4-(9-phenanthryl)phenoxy)propyl]oxy-
(11aS)-1,2,3,11a-tetrahy-dro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-
5-one (4a)
A solution of amino thioacetal 12a (681 mg, 1 mmol), HgCl2
(596 mg, 2.2 mmol), and CaCO3 (240 mg, 2.4 mmol) in CH3CN–H2O
To
a solution of (2S)-N-[4-(3-bromopropoxy)-5-methoxy-2-
nitrobenzoyl]pyrrolidine-2-carboxaldehyde diethyl thioacetal (10a)
(521 mg, 1 mmol) in acetone (10 mL) was added anhydrous K2CO3
(552 mg, 4 mmol) and 4-(9-phenanthryl)phenol (7) (270 mg,

2180
A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 2173–2181
(16:4) was stirred slowly at room temperature until TLC (EtOAc)
indicates complete loss of starting material. The reaction mixture
was diluted with ethyl acetate (30 mL) and filtered through celite.
The clear yellow organic supernatant was extracted with ethyl
acetate (2 ꢂ 20 mL). The organic layer was washed with saturated
aqueous NaHCO3 (2 ꢂ 20 mL), brine (2 ꢂ 20 mL), and then
combined organic phase was dried (Na2SO4). The organic layer was
evaporated under vacuum and the crude product was purified by
column chromatography to afford the compound 4a as a white
solid; (334 mg, 60%); Rf ¼ 0.70 (MeOH–CHCl3, 1:9); mp 112–114 ꢀC.
medium (DMEM) (Invitrogen), supplemented with 10% fetal calf
serum and 100 U/mL Pencillin and 100 mg/mL streptomycin sulfate
(Sigma). The cells were passaged and maintained at 37 ꢀC in
a humidified atmosphere containing 5% CO2.
4.3. Cell viability (MTT assay)
Cell viability was assessed by the MTT assay using Vybrant MTT
cell proliferation assay kit (Invitrogen), which is based on the ability
of viable cells to reduce the MTT to insoluble formazan crystals by
mitochondrial dehydrogenase. Briefly, MCF-7 cells were seeded in
a 96-well plate (TPP) at a cell density of 10,000 cells/well. After
overnight incubation, the cells were treated with compounds 4a–c
and DC-81, and incubated for 24 h. The medium was then discarded
ꢀ
23
[
a]
¼ þ448 (c ¼ 1 in CHCl3). 1H NMR (CDCl3, 300 MHz)
d: 2.02–
D
2.13 (m, 2H, eCH2e), 2.27–2.38 (m, 2H, eCH2e), 2.39–2.51 (p, 2H,
eCH2e), 3.54–3.66 (m, 1H, eNCH2e), 3.69–3.78 (m, 1H, eNCH2e),
3.79–3.91 (m, 1H, eNCHe), 3.98 (s, 3H, eOCH3), 4.28–4.38 (m, 4H,
2ꢂeOCH2e), 6.92 (s, 1H, ArH), 7.08 (d, J ¼ 9.0 Hz, 2H, ArH), 7.47 (d,
J ¼ 9.0 Hz, 2H, ArH), 7.56 (s, 1H, ArH), 7.58–7.72 (m, 6H, 5ꢂArH,
imine-H), 7.88–7.99 (m, 2H, ArH), 8.71–8.83 (m, 2H, ArH); 13C NMR
and replaced with fresh 100
of MTT dye. Plates were incubated at 37 ꢀC for 2 h. The resulting
formazan crystals were solubulized in 100 L of extraction buffer
mL media followed by addition of 10 mL
m
(CDCl3, 75 MHz)
d
: 161.6, 136.3, 131.1, 127.1, 123.5, 121.3, 119.7, 111.8,
(SDS). The optical density (O.D.) was read at 570 nm using Multi-
mode Varioskan FLASH (Thermoscientifics).
102.8, 61.6, 39.2, 31.9, 28.9, 22.9 ppm; MS (ESI): m/z 557 [M þ H]þ;
HRMS-EI: m/z [M þ Na]þ calcd for C36H32N2O4 579.2259, found
579.2233; Anal. calcd for C36H32N2O4: C 77.68, H 5.79, N 5.03 found:
C 77.26, H 5.44, N 4.87.
4.4. Cell cycle analysis
5 ꢂ105 Cells of MCF-7 were seeded in 60 mm dish and were
allowed to grow for 24 h. Compounds 4a–c and DC-81 at 2 and
4 mM were added to the culture media and the cells were incubated
for an additional 24 h. Harvesting of cells was done with Trypsin-
EDTA, fixed with ice-cold 70% ethanol at 4 ꢀC for 30 min, washed
with PBS and incubated with 1 mg/mL RNaseA solution (Sigma) at
37 ꢀC for 30 min. Cells were collected by centrifugation at 2000 rpm
4.1.7. 7-Methoxy-8-[N-(4-(9-phenanthryl)phenoxy)butyl]oxy-
(11aS)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
one (4b)
The compound 4b has been prepared by following the method
described in the compound 4a employing 12b (695 mg, 1 mmol) to
afford the compound 4b as a white solid (342 mg, 60%); Rf ¼ 0.72
23
(MeOH–CHCl3, 1:9); mp 102–104 ꢀC; [
a
]
¼ þ250ꢀ (c ¼ 0.9 in
for 5 min and further stained with 250 ml of DNA staining solution
D
CHCl3). 1H NMR (CDCl3, 400 MHz)
d
: 1.95–2.23 (m, 6H, 3ꢂeCH2e),
[10 mg of Propidium Iodide (PI), 0.1 mg of trisodium citrate, and
0.03 mL of Triton X-100 were dissolved in 100 mL of sterile MilliQ
water at room temperature for 30 min in the dark]. The DNA
contents of 20,000 events were measured by flowcytometer (DAKO
CYTOMATION, Beckman Coulter, Brea, CA). Histograms were
analyzed using Summit Software.
2.25–2.40 (m, 2H, eCH2e), 3.52–3.67 (m, 1H, eNCH2e), 3.64–3.77
(m, 1H, eNCH2e), 3.78–3.90 (m, 1H, eNCHe) 3.96 (s, 3H, eOCH3),
4.10–4.26 (m, 4H, 2ꢂeOCH2e), 6.85 (s, 1H, ArH), 7.05 (d, J ¼ 9.0 Hz,
2H, ArH), 7.46 (d, J ¼ 8.3 Hz, 2H, ArH), 7.53 (s, 1H, ArH), 7.55–7.70
(m, 6H, 5ꢂArH, imine-H), 7.86–7.98 (m, 2H, ArH), 8.70–8.80 (m, 2H,
ArH); 13C NMR (CDCl3, 100 MHz)
d: 162.2, 158.2, 150.6, 147.6, 140.4,
138.2, 132.8, 131.3, 130.9, 130.4, 129.6, 128.4, 127.2, 126.7, 126.2,
122.5, 120.0, 114.1, 111.3, 110.2, 68.5, 67.3, 56.1, 53.6, 46.7, 29.6, 25.9,
24.2 ppm; MS (ESI): m/z 571 [M þ H]þ; Anal. calcd for C37H34N2O4:
C 77.87, H 6.01, N 4.91 found: C 77.59, H 6.00, N 4.37.
4.5. RED100 assay
The restriction endonuclease digestion is based on the activity of
a compound to inhibit the cleavage activity of restriction endonu-
clease. Compounds 4a–c with the final concentrations of 2, 4, 8, 16
4.1.8. 7-Methoxy-8-[N-(4-(9-phenanthryl)phenoxy)pentyl]oxy-(11aS)-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (4c)
The compound 4c has been prepared following the method
described in the compound 4a employing 12c (709 mg, 1 mmol) to
and 32 mM were added to the PBR322 vector DNA and incubated for
16 h at 37 ꢀC and restriction enzyme (BamHI) was added to check
whether the compound binds to DNA and inhibits the enzymatic
digestion. The digestion assay gel includes two controls uncut (UC)
PBR322 DNA and C indicates cut (without the addition of the
afford the compound 4c as a white solid (339 mg, 58%); Rf ¼ 0.75
23
(MeOH–CHCl3, 1:9); mp 96–98 ꢀC; [
a]
D
¼ þ384ꢀ (c ¼ 0.89 in CHCl3);
compound). The plasmid (1
above compound in a final volume of 17
l) and 1 l of 10 U/m
m
g) was incubated with each of the
l for 16 h at 37 ꢀC. Next
l of BamHI enzyme was
1H NMR (CDCl3, 300 MHz)
d
: 1.62–1.83 (m, 2H, eCH2e),1.85–2.16 (m,
m
4H, eCH2e), 2.23–2.45 (m, 2H, eCH2e), 3.38 (t, J ¼ 6.7, 7.5 Hz, 2H,
eCH2e), 3.51–3.64 (m, 1H, eNCH2e), 3.67–3.76 (m, 1H, eNCH2e),
3.78–3.89 (m, 1H, eNCHe), 3.96 (s, 3H, eOCH3), 3.99–4.27 (m, 4H,
2ꢂeOCH2e), 6.85 (s, 1H, ArH), 7.05 (d, J ¼ 7.8 Hz, 2H, ArH), 7.47 (d,
J ¼ 8.5 Hz, 2H, ArH), 7.54 (s, 1H, ArH), 7.63–7.75 (m, 6H, 5ꢂArH,
imine-H), 7.85–8.01 (m, 2H, ArH), 8.68–8.83 (m, 2H, ArH); 13C NMR
10ꢂ BamHI buffer (2
m
m
added and incubated for 2 h at 37 ꢀC. The contents were loaded on
to 1% agarose gel. The electrophoresis was carried out in 0.5 ꢂ Tris–
acetate EDTA buffer at 80 V for 1 h and the gel was stained with
ethidium bromide and photographed.
(CDCl3, 100 MHz)
d: 164.4, 162.2, 158.2, 150.6, 140.6, 140.4, 138.3,
4.6. DNA fragmentation assay
132.7, 131.5, 131.1, 130.9, 130.5, 129.6, 128.4, 127.3, 126.7, 126.2, 122.7
122.4, 120.0, 114.2, 111.4, 110.5, 68.8, 67.6, 56.2, 53.7, 46.6, 29.6, 29.1,
28.7, 24.2, 22.7 ppm; MS (ESI): m/z 585 [M þ H]þ; Anal. calcd for
C38H36N2O4: C 78.06, H 6.21, N 4.79, found: C 77.84, H 6.13, N 4.27.
The 1.5 ꢂ106 MCF-7 cells were grown in 60 mm dish and treated
with compounds 4a–c at a concentration of at 4 mM, the effective
concentration which was obtained from FACS analysis. The cells
were washed twice in 1ꢂ TBS and lysed in 10 mM Tris–Hcl pH 7.5;
10 mM EDTA and 0.5% Triton X-100. The lysate was then centri-
4.2. Cell lines
fuged at 13 K for 10 min and the supernatant was treated with 1
of RNaseA and 40
g of proteinase K for 2 h at 37 ꢀC, respectively.
After phenol–chloroform extraction, the DNA was precipitated with
mg
MCF-7 (human breast cancer cells) was obtained from ATCC,
USA. MCF-7 cells were maintained in Dulbecco’s modified Eagle’s
m

A. Kamal et al. / European Journal of Medicinal Chemistry 45 (2010) 2173–2181
2181
1/10th volume of 3 M sodium acetate and 2 volumes of ethanol and
stored at ꢁ70 ꢀC for 2 h and centrifuged at 12 K for 30 min. After
70% ethanol wash the DNA samples were analyzed by agarose gel
protein blots were visualized with chemiluminescence reagent
(Thermo Fischer Scientifics Ltd.). The X-ray films were developed
with developer and fixed with fixer solution.
electrophoresis in 1.2% agarose gel containing 1
ethidium bromide.
ml of 10 mg/mL
Acknowledgements
4.7. Caspase-8 assay
We are thankful to the National Cancer Institute (NCI), USA for
providing the data in the 57-cell panel of human cancer cell lines.
The authors K.S, P.P.K and G.B.K are also grateful to CSIR, New Delhi,
for the award of Research Fellowships.
Caspase-8 assay was carried out using the Apoalert caspase-8
calorimetry assay kit (Clonetech, CA) according to manufacturer’s
recommendations. MCF-7 cells were treated with compounds 4a–c
and DC-81 and the substrate used in this assay was IETD-pNA. The
caspase-8 substrate and cell lysate was incubated at 37 ꢀC for 1 h
and readings were taken at 405 nm.
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We have used Apoalert caspase-9/6 fluorescent assay kit (Clo-
netech, CA) according to the manufacturer’s recommendations.
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4.9. Protein extraction and western blot analysis
Total cell lysates from cultured MCF-7 cells after compound
treatments as mentioned earlier were obtained by lysing the cells
in ice-cold RIPA buffer (1ꢂ PBS, 1% NP-40, 0.5% sodium deoxy-
cholate and 0.1% SDS) and containing 100
mg/mL PMSF, 5
m
m
g/mL
g/mL
Aprotinin, 5 mg/mL Leupeptin, 5 mg/mL Pepstatin and 100
NaF. After centrifugation at 12,000 rpm for 10 min, the protein in
supernatant was quantified by Bradford method (BIO-RAD) using
Multimode varioscan instrument (Thermo Fischer Scientifics).
Thirty micrograms of protein per lane was applied in 12% SDS-
polyacrylamide gel. After electrophoresis, the protein was trans-
ferred to polyvinylidine difluoride (PVDF) membrane (Amersham
Biosciences). The membrane was blocked at room temperature for
2 h in TBS þ 0.1% Tween20 (TBST) containing 5% blocking powder
(Santacruz). The membrane was washed with TBST for 5 min, and
primary antibody was added and incubated at 4 ꢀC overnight (O/N).
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4 (2002)
Rabbit polyclonal
b-Tubulin (1:100), mouse monoclonal cyto-
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chrome c (1:300), rabbit polyclonal cyclin D1 (1:200) and mouse
monoclonal PARP (1:200) antibodies were purchased from Imge-
nex, USA. After three TBST washes, the membrane was incubated
with corresponding horseradish peroxidase-labeled secondary
antibody (1:2000) (Santa Cruz) at room temperature for 1 h.
Membranes were washed with TBST three times for 15 min and the