Synthesis and antitumor activity of novel enediyne-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids

Article abstract of DOI:10.1016/j.bmc.2008.12.036

A series of novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) hybrids linked with enediyne is described. These compounds were prepared by linking C-8 of DC-81 (1) with an enediyne (10-16) through carbon chain linkers to afford PBD hybrid agents 17-23 in good

Full text of DOI:10.1016/j.bmc.2008.12.036

Bioorganic & Medicinal Chemistry 17 (2009) 1172–1180
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antitumor activity of novel enediyne-linked
pyrrolo[2,1-c][1,4]benzodiazepine hybrids
Wan-Ping Hu a, Jium-Jia Liang b, Chai-Lin Kao b, You-Chiang Chen a, Chung-Yu Chen b, Feng-Yuan Tsai c,
Ming-Jung Wu d,f, Long-Sen Chang e,f, Yeh-Long Chen b, Jeh-Jeng Wang b,f,
*
a Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan
b Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan
c Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Miaoli, Taiwan
d Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan
e Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan
f National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) hybrids linked with enediyne is described. These
compounds were prepared by linking C-8 of DC-81 (1) with an enediyne (10–16) through carbon chain linkers
to afford PBD hybrid agents 17–23 in good yields. Most of the hybrids on human cancer cell lines exhibited
higher cytotoxicity,and anincrease inthesub-G1populationthan1. Inapreviousarticle, wehavedemonstrated
that DC-81-indole conjugate agents (3–6) arepotentinducers of cell apoptosis in melanoma.In thepresent arti-
cle, we investigatedwhetherDC-81-enediyneagentspossessmorecytotoxicity than6onhuman293Tcells. Our
data revealed that treatment of 293T cells with DC-81-enediyne resulted in a significant increase of annexin V
binding, caspase-3 degradation, and p53 arrest to identify apoptotic cells than 6. These results suggest that the
DC-81-enediyne agents are more efficient in inducing apoptosis than DC-81-indole in 293T cells.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 14 November 2008
Revised 12 December 2008
Accepted 13 December 2008
Available online 24 December 2008
Keywords:
Pyrrolo[2,1-c][1,4]benzodiazepine
DC-81
Enediyne
Apoptosis
1. Introduction
In the present study, we report the design, synthesis, and biolog-
ical evaluation of a homologous series of PBD-enediyne conjugates.
DNA is a target for many antitumor drugs currently used in the
clinic. However, there are only a few DNA-interactive agents that
bind to DNA with high sequence selectivity. The development of
low molecular weight molecules with the highly sequence-selective
DNA-interactive properties is a present study of interest. DC-81 (1),
an antitumor antibiotic produced by Streptomyces species, belongs
to the pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) which are potent
inhibitors of nucleic acid synthesis. Although the naturally occurring
PBDshave potentanticancer activity, they havebeenprecludedfrom
clinical application due to side effects.1 Therefore, conjugate agents
with active moieties of known antitumor and antiviral agents are
being designed and synthesized to provide highly sequence-selec-
tive DNA-interactive properties and antitumor activity (Fig. 1).2,3
An enediyne (7–9, Fig. 2)4–6 contains either DNA intercalating
groups7 or DNA minor groove binding functions8,9 and these are po-
tent DNA-damaging agent due to their ability to generate benzenoid
diradicals.10 These results encouraged us to design and synthesize a
diversity of novel PBD conjugate agents.
Meikrantz and Schlegel reported the control of cell death is linked to
the cell cycle.11 Cells with a defective cell cycle are more vulnerable
to some anticancer agents according to numerous preclinical stud-
ies. P53, the tumor-suppressor protein, is commonly associated with
anticancer agent-induced cell cycle arrest and apoptosis.12–15 Addi-
tionally, a major part of the phenomenon could be mediated by
deregulation in cell cycle progression governed by the families of
caspases, especially the caspase-3, which was the straight promoter
of apoptosis.16,17 In thisregard, we haveinitiatedexperimentsaimed
at characterizing the above molecules in the process of apoptosis
after 293T cells were treated with PBD conjugate agents.
The aim of this study was to investigate whether DC-81-ened-
iyne conjugates possessed more cytotoxicity than DC-81-indole
and verify whether hybrid agents induced antiproliferation, lead-
ing to cell growth cycle perturbation, a decrease in caspase-3 and
p53 expression, and subsequent apoptotic cell death.
2. Results and discussion
2.1. Syntheses
* Corresponding author at present address: Department of Medicinal and Applied
Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan. Tel.: +886 7 312
1101x2275; fax: +886 7 312 5339.
We prepared the enediyne moieties as shown in Scheme 1. (2-
Phenylethynyl)phenyl-alkyn-1-ols (7) were treated with carbon
E-mail address: jjwang@kmu.edu.tw (J.-J. Wang).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.036

W.-P. Hu et al. / Bioorg. Med. Chem. 17 (2009) 1172–1180
1173
tetrabromide in the presence of triphenyl phosphine to give corre-
2.3. Cellular sub-G1 accumulation
sponding bromides 10–12 in moderate to good yields (Eq. 1,
Scheme 1). While trans-4-alkyoxy-1-bromo-2-butenes (13 and
14) were prepared by the reaction of alcohols 7 with trans-1,4-dib-
rom-2-butene in basic condition with potassium iodide (Eq. 2). Un-
der similar reaction conditions, cis-4-alkyoxy-1-chloro-2-butenes
(15 and 16) were obtained in moderate yields (Eq. 3). We have pre-
viously reported the efficient synthesis of 1 in excellent yield.18
Condensation of 1 with bromide or chloride compounds 10–16 in
the presence of KI at 45 °C afforded the desired conjugate agents
17–23 in 32–83% yields (Scheme 2).
Agent action resulted in cells having a hypodiploid DNA content
(sub-G1 material) that is characteristic of apoptosis and reflects
fragmented DNA as shown in Figure 4. Treatment of 293T cells
with 4 lM agents for 24 h induced apoptosis effects in 6.3%
(control), 10.0% (1), 6.3% (17), 14.5% (18), 13.2% (19), 13.9% (20),
13.6% (21), 15.6% (22), and 13.3% (23) of sub-G1 DNA peak. Most
of the conjugate agents 17–23 were more efficient in inducing cel-
lular sub-G1 accumulation than 1 in 293T cells.
2.4. Apoptosis detection
2.2. In vitro cytotoxic effects
The changes in cellular morphology after treatment with com-
We evaluated the cytotoxicity of the DC-81-enediyne agents 17–
23 in various human cell lines by the MTT assay. As shown in Table 1,
most of the conjugate agents 17–23 on human cancer cell lines
exhibited higher cytotoxicity relative to 1 (DC-81). Because the
agents 17–23 exhibited a higher inhibitory activity on 293T cells
pounds (6 and 18) at a concentration of 4 lM or treated with
graded concentrations of 18 for 24 h is shown in Figure 5A. The
apoptotic morphology was dependent on drug concentration and
18 exhibited a higher apoptotic effect compared to that of 6 on
293T. To further characterize whether compound 18-induced cell
death involved apoptosis or not, we performed a biparametric
cytofluorimetric analysis using annexin V and PI double staining.
Fluorescein isothiocyanate (FITC)-conjugated annexin V has been
utilized to detect the externalization of phosphatidylserine that oc-
curs at an early stage of apoptosis. Propidium iodide (PI) is used as
a marker of necrosis due to cell membrane destruction.19 The dis-
tribution of stained cells is shown in Figure 5B. At a concentration
compared to other cell lines at a concentration of 4 lM, the 293T
cells were selected as a model for further studies. The compound
18 was selected by the US National Cancer Institute for evaluation
in the in vitro preclinical antitumor screening program against sixty
human tumor cell lines derived from nine cancer cell types. The se-
lected biological evaluation result for this compound is presented in
Table 2. The GI50 value of 18 was less than 1 lM, indicating that this
conjugate agent has the potency of the growth inhibition activity
against cancer cell lines. Moreover, to confirm whether conjugate
agent is more effective as an antiproliferative agent than 1, 11 (ened-
iyne) or a combination of 1 and 11, we used conjugate agent 18 for
this issue. Compound 18 exhibited a higher inhibitory activity com-
pared to that of other agents on 293T cells (Fig. 3).
of 1 l
M, compound 18 exhibited an apoptotic effect (annexin V+/
PIÀ) on 293T cells, and higher concentrations induced cell death
with increased cell permeability. In addition, to elucidate whether
18 induces more apoptotic cells than 6, treatment of 293T cells
with 4 lM agents for 24 h induced apoptosis effects in 2.0% (Con-
trol), 25.4% (18), and 19.4% (6) of annexin V-FITC cells (Fig. 5C).
N
N
HO
O
H
H
N
O
MeO
N
N
MeO
H
O
O
2
1
H
N
N
O
n
H
N
O
H
N
MeO
n = 3-6; 3-6
O
Figure 1. DC-81-indole conjugate agents.
O
O
Ar
O
R
O
ArCO
2
H N
R'
2
SugarO
7
8
9
Figure 2. Enediyne analogues.

1174
W.-P. Hu et al. / Bioorg. Med. Chem. 17 (2009) 1172–1180
CBr PPh
4/
3
eq 1
CH Cl
2
2
OH
n
Br
n
n = 1; 10 (83%)
n = 3; 11 (55%)
n = 4; 12 (65%)
Br
Br
eq 2
NaH/KI/CH Cl
2
2
O
n
OH
n
Br
n = 1; 13 (57%)
n = 2; 14 (40%)
Cl
Cl
eq 3
NaH/KI/CH Cl
2
2
Cl
O
n
OH
n
n = 1; 15 (62%)
n = 2; 16 (32%)
Scheme 1. Synthesis of enediyne analogues 10–16.
2.5. Apoptosis-related protein expression
than DC-81 (1), which may be associated with cellular apoptosis.
In addition, most DC-81-enediynes induced a markedly increased
accumulation of sub-G1 phase cells compared to that of 1 on
293T. Furthermore, compound 18 triggered more apoptotic cells
than 6 as revealed by the externalization of annexin V-targeted
PS residues at the periphery of the cells. It has been hypothesized
that the sensitivity of tumor cells to compound 18 might be asso-
ciated with the DNA repair process that involves p53. Data from
Western blot analysis showed that cells treated by 18 resulted in
Caspase-3 has been shown to be one of the most important cell
executioners for apoptosis.20,21 The p53 gene is considered a tu-
mor-suppressor gene,22 which might be associated with the DNA
repair process.23 The expression of apoptosis-related protein was
determined using immunoblotting assay. The results revealed that
the expression of caspase-3 was dependent on drug concentration
when 293T cells were exposed to compound 18. An increase in p53
protein expression was observed in the groups treated with 18 at
low concentrations, while DNA repair deficiency was observed at
concentration higher than 2 lM. Additionally, our results also
showed that 18-treated cells exhibited significantly decreased in
caspase-3 and p53 expression compared to those of 6 at a concen-
tration of 4 lM (Fig. 6A and B).
a decrease in p53 expression than 6 at a concentration of 4 lM.
Our result suggests that cells treated by 18 resulted in a decrease
in p53 expression, DNA repair deficiency, DNA synthesis block, cell
cycle arrest ultimately leading to 293T cell apoptosis. Moreover,
we also observed that 18 exposure resulted in a degradation in cas-
pase-3 than 6.
The data gathered in the present study demonstrated that DC-
81-enediyne agents are more efficient in inducing apoptosis than
DC-81-indole in 293T cells. In this article, we highlight a novel
DC-81-enediyne antitumor proliferation mechanism; this new re-
search will be helpful in further elucidation of undiscovered bio-
logical properties of these novel antitumor DC-81-enediynes.
3. Conclusions
We have previously combined DC-81 and an indole 2-carbonyl
moiety to synthesize DC-81-indole conjugate agents designed to
have much higher sequence selectivity in DNA interactivity. We
used human melanoma A2058 cells as a model as these provide
the hybridizing agents that are potent inducers of cell apoptosis.3
This novel result raised our interest and prompted us to synthesize
DC-81-enediyne designed to have much more biological potency
than DC-81-indole conjugate agents. Firstly, we used an MTT assay
to evaluate the cytotoxicity of tested compounds in five human cell
lines, A2058, A375, 293T, H1355, and MCF-7. Our results indicated
that most agents are more effective as an antiproliferative agent
4. Experimental
4.1. Syntheses
1H NMR and 13C NMR spectra were recorded at 400 and
100 MHz, respectively, using CDCl3 as a solvent. 1H NMR chemical
shifts are referenced to TMS or CDCl3 (7.26 ppm). 13C NMR was ref-

W.-P. Hu et al. / Bioorg. Med. Chem. 17 (2009) 1172–1180
1175
n = 1; 17 (57%)
n = 3; 18 (32%)
n = 4; 19 (38%)
10-12
N
O
n
H
N
MeO
O
N
HO
H
n = 1; 20 (25%)
n = 2; 21 (39%)
K2CO3 13, 14
KI/MEK
N
MeO
O
O
n
N
O
H
1
N
MeO
O
n = 1; 22 (20%)
n = 2; 23 (41%)
15, 16
N
O
O
n
H
N
O
MeO
Scheme 2. Synthesis of PBD-enediyne conjugate agents 17–23.
Table 1
Compounds against human-derived cancer cell lines in vitro
Survival (% control)
Compound (4 lm)
1
17
18
19
20
21
22
23
A2058 (melanoma)
A375 (melanoma)
293T (kidney)
H1355 (lung)
MCF-7 (breast)
63.4 4.4
50.2 3.2
68.9 1.8
73.9 3.6
72.3 3.2
46.1 4.8
13.1 6.6
48.9 1.6
70.9 1.6
48.9 1.6
47.2 1.3
22.3 6.8
28.7 0.8
87.7 0.8
49.7 0.8
49.5 2.9
28.3 4.8
19.2 3.5
75.2 3.5
85.2 3.5
43.0 4.1
42.9 1.6
48.4 0.7
67.4 0.7
70.4 0.7
46.2 4.9
33.4 2.3
35.2 1.4
85.2 1.4
59.2 1.4
39.1 4.9
33.9 1.6
44.0 2.4
44.0 2.4
44.0 2.4
84.5 5.6
85.7 0.7
60.7 0.2
71.0 5.6
83.8 5.6
Cells were cultured with agents at a concentration of 4 lM for 24 h before growth and viability were assessed using the MTT assay.
erenced to CDCl3 (77.0 ppm). Multiplicities were determined by
the DEPT sequence as s, d, t, q. Mass spectra and high resolution
mass spectra (HRMS) were measured using the electron-impact
(EI, 70 eV) technique by Kaohsiung Regional Instrument Center of
NSC at NSYSU. Flash chromatography was carried out on Silica
Gel 60 (E. Merck, 230–400 mesh).
dried over MgSO4, and concentrated to give crude product. The
crude mixture was subjected to chromatography (silica gel: 40–
63 l
M, hexane) to give desired product 10 (83%). 1H NMR
(400 MHz, CDCl3) d 7.62–7.58 (m, 2H), 7.54–7.52 (m, 1H), 7.48–
7.45 (m, 1H), 7.39–7.25 (m, 5H), 4.25 (s, 2H); 13C NMR
(100 MHz) d 132.1, 131.9, 131.8, 128.58, 128.51, 128.3, 127.9,
126,2, 124.6, 123.1, 93.7, 88.1, 87.8, 85.4, 15,1. HRMS (EI m/z) for
C17H11Br, calcd 294.0044, found 294.0046.
4.1.1. 1-(3-Brom-prop-1-ynyl)-2-phenylethynyl-benzene (10)
To the solution of 3-(2-phenylethynyl-phenyl)-prop-2-yn-1-ol
(500 mg, 1.7 mmol) in anhydrous CH2Cl2 (30 mL) was added CBr4
(1.68 g, 5.1 mmol) at 0 °C. After 10 min of being stirred at the same
conditions, PPh3 (888 mg, 3.4 mmol) was added to the reaction
then, the ice bath was removed and the reaction was stirred at
the ambient temperature for additional 6 h. The reaction was
quenched with ethanol (3 mL) then extracted with ethyl acetate
three times. The combined organic layer was washed with brine,
4.1.2. 1-(5-Brom-pent-1-ynyl)-2-phenylethynyl-benzene (11)
5-(2-Phenyl ethynyl-phenyl)-pent-4-yn-1-ol (500 mg, 1.9 mmol)
was subjected to the same procedure which was used to produce
10 afford desired product 11 with 55% yield. 1H NMR (400 MHz,
CDCl3) d 7.55–7.49 (m, 3H), 7.43–7.41 (m, 1H), 7.36–7.32 (m,
3H), 7.26–7.21 (m, 2H), 3.58 (t, J = 6 Hz, 2H), 2.68 (t, J = 6 Hz, 2H),
2.13 (quintet, J = 6 Hz, 2H). 13C NMR (100 MHz) d 131.9, 131.8,

1176
W.-P. Hu et al. / Bioorg. Med. Chem. 17 (2009) 1172–1180
Table 2
0.26 mmol). After 10 min of stirring at ambient temperature, the
resulting solution was heated at reflux temperature for 6 h. The
reaction was quenched with iced water and extracted with ethyl
acetate (30 mL). The organic layer was washed with brine, dried
over MgSO4, and concentrated to give crude product. The crude
Cytotoxic activity of 18 from the NCI’s in vitro selected human cancer cell lines
Panels/cancer cell lines
GI50 (lm)
Leukemia
CCRF-CEM
MOLT-4
0.422
0.648
0.285
mixture was purified over chromatography (silica gel: 40–63 lM,
CH2Cl2/hexane = 1:10) to give desired product (57%). 1H NMR
(400 MHz, CDCl3) d 7.56–7.28 (m, 9H), 5.94–5.75 (m, 2H), 4.47 (s,
2H), 4.18 (d, J = 6.8 Hz, 2H), 3.89 (d, J = 6.8 Hz, 2H); 13C NMR
(100 MHz) d 131.9, 131.8, 131.7, 130.9, 129.2, 128.5, 128.4, 128.2,
127.9, 125.9, 125.0, 123.1, 93.3, 88.9, 88.0, 85.4, 68.7, 58.2, 31.8.
PRMI-8226
Colon cancer
SW-620
0.478
0.924
Melanoma
SK-MEL-2
Ovarian cancer
IGROVI
OVCAR-3
OVCAR-8
0.549
0.520
0.394
4.1.5. 1-[4-((E)-4-Bromo-but-2-enyloxy)-but-1-ynyl]-2-phen-
ylethynyl-benzene (14)
4-(2-Phenylethynyl-phenyl)-but-3-yn-1-ol (200 mg, 0.81 mmol)
and trans-1,4-dibromo-but-2-ene (521 llL, 2.44 mmol) were sub-
jected to the same procedure which was used to produce 13 afford
desired product 11 with 40% yield. 1H NMR(400 MHz, CDCl3) d
7.56–7.46 (m, 4H), 7.38–7.32 (m, 3H), 7.27–7,22 (m, 2H), 5.96–
5.88 (m, 1H), 5.83–5.57 (m, 1H), 4.01 (dd, J = 1, 6 Hz, 2H), 3.91 (d,
J = 7 Hz, 2H), 3.67 (t, J = 7.2 Hz, 2H), 2.79 (t, J = 7.2 Hz, 2H); 13C
NMR (100 MHz) d 131.8, 131.7, 131.6, 130.4, 130.0, 128.6, 128.3,
128,3, 127,9, 126.0, 125.7, 123.3, 92.9, 90.9, 88.4, 80.4, 70.2, 68.2,
31.9, 21.0.
Renal cancer
CAKI-1
RXF393
0.485
0.686
Breast cancer
MCF7
MDA-MB-435
BT-549
0.305
0.468
0.641
0.779
T-47D
4.1.6. 1-[3-((Z)-4-Chloro-but-2-enyloxy)-prop-1-ynyl]-2-phen-
ylethynyl-benzene (15)
4 µM
125
100
75
3-(2-Phenylethynyl-phenyl)-prop-2-yn-1-ol (200 mg, 0.86 mmol)
and cis-1,4-dichloro-but-2-ene (322 llL, 2.59 mmol) were sub-
jected to the same procedure which was used to produce 13 afford
desired produce 15 with 62% yield.1H NMR(400 MHz, CDCl3) d
7.56–7.53 (m, 3H), 7.50–7.48 (m, 1H), 7.38–7.27 (m, 5H), 5.81–
5.69 (m, 2H), 4.48 (s, 2H), 4.28 (d, J = 6 Hz, 2H), 4.06 (d, J = 7 Hz,
2H); 13C NMR (100 MHz) d 132.0, 131.8, 131.7, 129.8, 129.2,
128.5, 128.4, 128.3, 128.0, 125.9, 125.0, 123.1, 93.4, 88.8, 88.1,
85.4, 64.4, 58.2, 39.1. HRMS (ESI m/z) for C21H17ClONa, calcd
343.0866, found 343.0863.
p < 0.01
50
25
0
Control
11
1
1 plus 11
18
4.1.7. 1-[4-((Z)-4-Chloro-but-2-enyloxy)-but-1-ynyl]-2-phen-
ylethynyl-benzene (16)
Figure 3. Cell viability of compounds tested against 293T cells (same experimental
conditions as in Table 1).
4-(2-Phenylethynyl-phenyl)-but-3-yn-1-ol (100 mg, 0.41 mmol)
and cis-1,4-dichloro-but-2-ene (152 llL, 1.22 mmol) were sub-
jected to the same procedure which was used to produce 13 afford
desired product 16 with 32% yield.1H NMR (400 MHz, CDCl3) d
7.57–7.49 (m, 3H), 7.44–7.42 (m, 1H), 7.38–7,33 (m, 3H), 7.27–
7,22 (m, 2H), 5.79–5.68 (m, 2H),4.10 (q, 4H), 3.67 (t, J = 7 Hz, 2H),
2.79 (t, J = 7 Hz, 2H); 13C NMR (100 MHz) d 131.8, 131.64, 131.64,
130.6, 128.4,128.3, 128.31, 127,9, 127.5, 126.0, 125.7, 123.3, 92.9,
90.9, 88.4, 80.3, 68.8, 66.0, 39.1, 21.0.
131.6, 128.4, 128.3, 127.9, 127.5, 126,0, 125.6, 123.2, 92.9, 92.3,
88.3, 80.4, 32.4, 31.5, 18.3. HRMS (ESI m/z) for C19H15Br, calcd
322.0357, found 322.0358.
4.1.3. 1-(6-Brom-hex-1-ynyl)-2-phenylethynyl-benzene (12)
6-(2-Phenylethynyl-phenyl)-hexa-5-yn-1-ol (500 mg, 1.92 mmol)
was subjected to the same procedure which was used to produce
10 afford desired product 12 with 65% yield. 1H NMR
(400 MHz,CDCl3) d 7.56–7.48 (m, 3H), 7.43–7.33 (4H, m), 7.27–
7.22 (m, 2H), 3.34 (t, J = 7 Hz, 2H), 2.54 (t, J = 7 Hz, 2H), 2.05 (quin-
tet, J = 7 Hz, 2H), 1.78 (quintet, J = 7 Hz, 2H); 13C NMR (100 MHz) d
131.8, 131.8, 131.6, 128.4, 128.3, 128.0, 127.4, 126,2, 125.6, 123.2,
93.7, 92.8, 88.4, 80.1, 33.4, 31.6, 27.1, 18.8. HRMS (EI m/z) for
C20H17Br, calcd 336.0514, found 336.0512.
4.1.8. 7-Methoxy-8-[3-(2-phenylethynyl-phenyl)-prop-2-ynyl-
oxy]-1,2,3,11a-tetrahydro-benzo[e]pyrrolo[1,2-a][1,4]diazepin-
5-one (17)
To the solution of 10 (120 mg, 0.406 mmol) in anhydrous MEK
(5 mL) was added KI (40 mg, 0.24 mmol) and irritated in sonicator
for 5 min. To the resulting mixture was added the solution of 1
(100 mg, 0.41 mmol) and K2CO3 (168 mg, 1.21 mmol) in anhydrous
butanone (25 mL). After the addition, the resulting solution was
heated to 45 °C for 24 h. The reaction quenched by iced water.
The resulting mixture was extracted with ethyl acetate. The organ-
ic layer was washed with brine, dried over MgSO4 to give a crude
product which was further purified over chromatography (silica
4.1.4. 1-[3-((E)-4-Bromo-but-2-enyloxy)-prop-1-ynyl]-2-phe-
nylethynyl-benzene (13)
To the solution of 3-(2-phenylethynyl-phenyl)-prop-2-yn-1-ol
(200 mg, 0.86 mmol) in anhydrous CH2Cl2 (30 mL) was added
NaH (60%, 34 mg, 1.44 mmol) at 0 °C. After 10 min of stirring, the
ice bath was removed. To the resulting solution was added trans-
1,4-dibromo-but-2-ene (553 llL, 2.59 mmol) and KI (43 mg,
gel, 40–63 lM, CH2Cl2/MeOH = 150:1) to give desired product
(17) with 57% yield. 1H NMR (400 MHz, CDCl3) d 7.60–7.30 (11H,
m), 7.10 (s, 1H), 5.13 (dd, J = 7, 16 Hz, 2H), 3.95 (s, 3H), 3.82–3.76
(m, 1H), 3.60–3.52 (m, 2H), 2.28–2.23 (m, 2H), 2.06–2.00 (m,

W.-P. Hu et al. / Bioorg. Med. Chem. 17 (2009) 1172–1180
1177
Figure 4. Effect of compound tested on the cellular sub-G1 content. 293T cells were treated with treated with 4
l
M agents for 24 h and stained with PI. Approximately 10,000
cells from each group were analyzed with the FACScan flow cytometer. Data represent the percentage of cell counts and display sub-G1.
2H); 13C NMR (100 MHz) d 164.5, 162,4, 149.4, 147.8, 140.5, 132.2,
131.8, 131.2 131.8, 131.7, 128.4, 128.3, 128.2, 127.8, 125.9, 124.6,
123.1, 121.0 111.7, 111.5, 93.5, 87.8, 87.0, 86.6, 57.5, 56.1, 53.5,
46.6, 29.6, 24.1. HRMS (ESI m/z) for C30H25N2O3, calcd 461.1865,
found 461.1868.
94.1, 92.8, 88.5, 80.1, 68.3, 56.1, 53.7, 46.6, 29.6, 28.1, 25.2, 24.1,
16.4. HRMS (EI m/z) for C33H31N2O3, calcd 503.2335, found 503.2332.
4.1.11. 7-Methoxy-8-[6-(2-phenylethynyl-phenyl)-hex-5-ynyl-
oxy]-1,2,3,11a-tetrahydro-benzo[e]pyrrolo[1,2-a][1,4]diazepin-
5-one (20)
4.1.9. 7-Methoxy-8-[4-(2-phenylethynyl-phenyl)-but-3-ynyl-
oxy]-1,2,3,11a-tetrahydro-benzo[e]pyrrolo[1,2-a][1,4]diazepin-
5-one (18)
Compound 1 (100 mg, 0.41 mmol) and 13 (137 mg, 0.41 mmol)
were subjected to the same procedure which was used to produce
17 afford desired product 20 with 25% yield. 1H NMR (400 MHz,
CDCl3) d 7.66 (d, J = 4 Hz, 1H), 7.56–7.46 (m, 5H), 7.36–7.25 (m,
5H), 6.78 (s, 1H), 5.99–5.86 (m, 2H), 4.64–4.54 (m, 2H), 4.47–
4.54 (m, 2H), 4.21 (d, J = 5 Hz, 2H), 3.94 (s, 3H), 3.87–3.79 (m,
1H), 3.73–3.69 (m, 1H), 3.61–3.54 (m, 1H), 2.32–2.28 (m, 2H),
2.08–2.00 (m, 2H); 13C NMR (100 MHz) d 164.6, 162.4, 150.2,
147.8, 140.5, 131.9, 131.7, 131.7, 130.3, 128.4, 128.3, 128.1,
127.9, 127.3, 125.9, 125.1, 123.1, 120.5, 111.6, 110.8, 93.3, 89.1,
88.1, 85.0, 69.1, 68.6, 58.1, 56.1, 53.7, 46.6, 29.6, 24.2. HRMS (EI
m/z) for C34H31N2O2, calcd 531.2284, found 531.2284.
Compound 1 (100 mg, 0.41 mmol) and 11 (125 mg, 0.41 mmol)
were subjected to the same procedure which was used to produce
17 afford desired product 18 with 32% yield. 1H NMR (400 MHz,
CDCl3) d 7.64 (d, J = 4 Hz, 1H), 7.52–7.49 (m, 4H), 7.44–7.41(m,
1H), 7.31–7.23 (m, 5H), 6.80 (s, 1H), 4.26–4.20 (m, 2H), 3.93 (s,
3H), 3.84–3.78 (m, 1H), 3.68–3.48 (m, 2H), 2.74 (t, J = 7 Hz, 2H),
2.31–2.27 (m, 2H), 2.23–2.16 (m, 2H), 2.08–2.01(m, 2H); 13C
NMR (100 MHz) d 162.3, 150.7, 147.8, 140.6, 131.9, 131.7, 131.6,
128.3, 128.2, 127.9, 127.4, 126.2, 125.6, 123.3, 120.2, 111.6,
110.6, 93.2, 93.2, 88.4, 80.1, 67.5, 56.1, 53.7, 46.6, 29.6, 28.7,
24.2, 16.4. HRMS (ESI m/z) for C32H29N2O3, calcd 489.2178, found
489.2180.
4.1.12. 7-Methoxy-8-{(E)-4-[3-(2-phenylethynyl-phenyl)-prop-
2-ynyloxy]-but-2-enyloxy}-1,2,3,11a-tetrahydro-benzo[e]pyr-
rolo[1,2-a][1,4]diazepin-5-one (21)
4.1.10. 7-Methoxy-8-[5-(2-phenylethynyl-phenyl)-pent-4-ynyl-
oxy]-1,2,3,11a-tetrahydro-benzo[e]pyrrolo[1,2-a][1,4]diazepin-
5-one (19)
Compound 1 (100 mg, 0.41 mmol) and 14 (147 mg, 0.41 mmol)
were subjected to the same procedure which was used to produce
17 afford desired product 21 with 39% yield. 1H NMR (400 MHz,
CDCl3) d 7.65 (d, J = 4.4 Hz, 1H), 7.56–7.46 (m, 5H), 7.36–7.25 (m,
5H), 6.78 (s, 1H), 5.99–5.89 (m, 2H), 4.66–4.57 (m, 2H), 4.05 (d,
J = 4.8Hz, 2H), 3.94 (s, 3H), 3.87–3.79 (m, 1H), 3.73–3.69 (m, 3H),
3.61–3.54 (m, 1H), 2.89 (t, J = 7 Hz, 2H), 2.31–2.27 (m, 2H), 2.08–
2.02 (m, 2H); 13C NMR (100 MHz) d 164.6, 162.4, 150.2, 147.8,
140.5, 131.9, 131.8, 131.7, 130.3, 128.4, 128.3, 127.9, 127.4,
126.9, 126.1, 125,7, 125,7, 123.4, 120.4, 111.6, 110.8, 93.0, 91.0,
88.4, 80.4, 70.6, 68.7, 56.1, 53.7, 46.6, 29.6, 24.2, 21.1. HRMS (ESI
m/z) for C35H33N2O4, calcd 545.2440, found 545.2443.
Compound 1 (100 mg, 0.41 mmol) and 12 (131 mg, 0.41 mmol)
were subjected to the same procedure which was used to produce
17 afford desired product 19 with 38% yield.1H NMR (400MHz, CDCl3)
d 7.64 (d, J = 4 Hz, 1H), 7.52–7.49 (m. 4H), 7.43–7.40 (m, 1H), 7.32–
7.23 (m, 5H), 6.75 (s, 1H), 4.04–4.20 (m, 2H), 3.93 (s, 3H), 3.84–3.78
(m, 1H), 3.72–3.68 (m, 1H), 3.61–3.54 (m, 1H), 2.59 (t, J = 7 Hz, 2H),
2.34–2.28 (m, 2H), 2.10–2.00 (m, 5H), 1.86–1.79 (m, 2H); 13C NMR
(100 MHz) d 164.6, 162,3, 150.7, 147.8, 140.6, 131.9, 131.7, 131.6,
128.3, 128.2, 127.9, 127.4, 126.2, 125,6, 123.3, 120.2, 111.6, 110.6,

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W.-P. Hu et al. / Bioorg. Med. Chem. 17 (2009) 1172–1180
Figure 5. Compound 18 induces 293T cell apoptosis. (A) Morphological changes of 293T cells treated with compound (6, 18) at a concentration of 4
lM or treated with
graded concentrations of 18 for 24 h. (B) Dot plots for 293T cells treated with graded concentrations of 18 for 24 h and then stained with PI and an annexin V-FITC conjugate
specifically detecting the exposure of PS residues at the cell surface. (C) To elucidate whether 18 induces more apoptotic cells than 6, cells were cultured with 4 lM agents for
24 h, then stained with annexin V-FITC and approximately 10,000 cells from each group were analyzed by FACS.

W.-P. Hu et al. / Bioorg. Med. Chem. 17 (2009) 1172–1180
1179
4.2. Cell culture
18
6
0
1
2
4
4 (µM)
Five human cell lines, A2058 (melanoma), A375 (melanoma),
293T (kidney), H1355 (lung) and MCF-7 (breast) purchased from
American Type Culture Collection (Manassas, VA), were main-
tained in DMEM (A2058, A375, 293T) or RPMI1640 (H1355, MCF-
7) medium supplemented with 10% FCS and 100 U/mL penicillin
32 kDa
Caspase-3
P53
G and 100 lg/mL streptomycin sulfate (Gibco, BRL) at 37 °C in a
humidified atmosphere containing 5% CO2.
Actin
4.3. Cell viability
Cell viability was assessed by the MTT assay, a mitochondrial
function assay based on the ability of viable cells to reduce the re-
dox indicator MTT to insoluble formazan crystals by mitochondrial
dehydrogenase. Briefly, cells were seeded in a 96-well plate at the
cell density of 2500 cells/well. After an overnight incubation, the
caspase-
3
p53
cells were treated with compounds at 4
24 h. The medium was then discarded and replaced with 10
of MTT dye. Plates were incubated at 37 °C for 2 h. The resulting
formazan crystals were solubilized in 100 L DMSO, and the opti-
lM and incubated for
lL
l
cal density was read at 540 nm with a microplate reader (MRX-II,
Dynex technology, Chantilly, VA).
0
1
2
4
4 (µM)
18
6
4.4. Sub-G1 region analysis
Figure 6. Immunoblot analysis showed the effect of compound on the degra-
dation of apoptotic molecules. (A) After exposure to different concentration of
293T cells were treated with compounds at 4 lM for 24 h.
18 or incubated with
4 lM 6 for 24 h, cell lysates were collected and
Cells were harvested by trypsinization and centrifugation. Cell
pellets were resuspended in 50% cold ethanol and fixed at
À20 °C. After fixation, cells were washed once with cold PBS
immunoblotted with specific antibodies as indicated. For the internal control,
the same amounts of protein extract were also probed with antibody against
actin. (B) The pattern of the caspase-3 and p53 by 293T cells treated with
compound (6, 18).
and incubated in 0.5 mL of PBS containing 100 lg/mL RNase
A for 20 min at 37 °C. Cells were harvested by centrifugation
at 400g for 5 min, and 250 L of PBS containing 50 g/mL pro-
l
l
pidium iodide (PI) was added to the pellet. Thirty minutes la-
ter, the DNA contents of 10,000 events were measured by
FACSscan flow cytometer (Elite ESP, Beckman Coulter, Brea,
CA). Histograms were analyzed using Windows Multiple Docu-
ment Interface software (WinMDI). Cells with DNA content less
than that in untreated cells in G0/G1 were considered
apoptotic.
4.1.13. 7-Methoxy-8-{(Z)-4-[3-(2-phenylethynyl-phenyl)-prop-
2-ynyloxy]-but-2-enyloxy}-1,2,3,11a-tetrahydro-benzo[e]py-
rrolo[1,2-a][1,4]diazepin-5-one (22)
Compound 1 (100 mg, 0.41 mmol) and 15 (130 mg, 0.41 mmol)
were subjected to the same procedure which was used to produce
17 afford desired product 22 with 20% yield. 1H NMR (400 MHz,
CDCl3) d 7.62 (d, J = 4 Hz, 1H), 7.57–7.44 (m, 5H), 7.34—7.22 (m,
5H), 6.77 (s, 1H), 5.92–5.76 (m, 2H), 4.70 (d, J = 6 Hz, 2H), 4.50–
4.46 (m, 2H), 4.31 (d, J = 6 Hz, 2H), 3.92 (s, 3H), 3.87–3.77 (m,
1H), 3.67–3.53 (m, 2H), 2.32–2.25 (m, 2H), 2.08–2.00 (m, 2H);
13C NMR (100 MHz) d 164.6, 162,2, 150.1, 147.8, 140.6, 131.9,
131.7, 131.6, 129.4, 128.5, 128.3, 128.2, 128.1, 127.9, 125,9,
125,0, 123.1, 120.4, 111.5, 110.8, 93.4, 88.9, 88.0, 85.3, 65.3, 65.0,
58.2, 56.1, 53.6, 46.6, 29.6, 24.1. HRMS (EI m/z) for C34H31N2O4,
calcd 531.2284, found 531.2283.
4.5. Morphology observation
239T (5 Â 105 cells/well) seeded on 6 well plate. After grow one
day, treated with compound 6 or 18 for 24 h. Take photos by using
microscope at 200Â phase.
4.6. Annexin V and PI binding assay
To assess the simultaneous observation of early phase of apop-
totic and necrotic features, 293T cells were treated with different
agents at 4 lM for 24 h. Cells were harvested by trypsinization
and centrifugation and measured by cytometry by adding annexin
V-FITC to 106 cells per sample according to the manufacturer’s
specifications (Bender MedSystems, Vienna, Austria). Simulta-
neously, the cells were stained with PI. Flow cytometry data were
analyzed by the WinMDI software.
4.1.14. 7-Methoxy-8-{(E)-4-[4-(2-phenylethynyl-phenyl)-but-3-
ynyloxy]-but-2-enyloxy}-1,2,3,11a-tetrahydro-benzo[e]p- yrrolo-
[1,2-a][1,4]diazepin-5-one (23)
Compound 1 (100 mg, 0.41 mmol) and 16 (135 mg, 0.41 mmol)
were subjected to the same procedure which was used to produce
17 afford desired product 23 with 41% yield. 1H NMR (400 MHz,
CDCl3) d 7.64 (d, J = 4.4 Hz, 1H), 7.55–7.48 (m, 4H), 7.43–7.41 (m,
1H), 7.36—7.31 (m, 3H), 7.25–7.23 (m, 2H), 6.80 (s, 1H), 5.89–
5.77 (m, 2H), 4.78–4.71 (m, 2H), 4.16(d, J = 5.6 Hz, 2H), 3.93 (s,
3H), 3.87–3.78 (m, 1H), 3.71–3.65 (m, 3H), 3.60–3.53 (m, 1H),
2.80 (t, J = 7.2 Hz, 2H), 2.30–2.25 (m, 2H), 2.07–2.00 (m, 2H); 13C
4.7. Protein extraction and western blot analysis
Total cell extracts from cultured 293T cells were obtained by
lysing the cells in ice-cold RIPA buffer (1 Â PBS, 1% NP-40, 0.5% so-
NMR (100 MHz)
d 164.5, 162.4, 150.1, 147.7, 140.5, 131.8,
dium deoxycholate, 0.1% SDS) containing 100 lg/mL PMSF, 2 lg/
131.61, 131.61, 130.1, 128.3, 128.2, 127.9, 127.5, 127.4, 126,0,
125.6, 123.3, 120.4, 111.5, 110.7, 92.9, 91.0, 88.3, 80.3, 68.8, 66.8,
65.0, 56.1, 53.6, 46.6, 29.5, 24.1, 21.0. HRMS (ESI m/z) for
C35H33N2O4, calcd 545.2440, found 545.2438.
mL aprotinin, 2 lg/mL leupeptin and 100 lg/mL NaF. After centri-
fugation at 14,000g for 30 min, protein in the supernatants was
quantified by Bradford method (Bio-Rad). Forty micrograms of pro-

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W.-P. Hu et al. / Bioorg. Med. Chem. 17 (2009) 1172–1180
tein per lane was applied in 10% SDS-poly-acrylamide gel. After
electrophoresis, protein was transferred from the gel to polyvinyl-
idene difluoride (PVDF) membrane (Millipore, Bedford, MA). The
membranes were blocked at room temperature for 1 h in
PBS + 0.1% Tween 20 (PBS-T) containing 5% skim milk. After being
briefly rinsed with PBS-T, the membrane was incubated with pri-
mary antibody at room temperature for 2 h or at 4 °C overnight.
Rabbit polyclonal antibodies against CPP32 (H-277) and mouse
monoclonal antibody P53 (DO-1) were purchased from Santa Cruz
Biotechnology. Mouse monoclonal antibody against actin was pur-
chased from Chemicon Int. Inc. (Temecula, CA). The membrane was
incubated with the corresponding horseradish peroxidase-labeled
secondary antibody (Santa Cruz Biotechnology) at room tempera-
ture for 1 h. Membranes were washed with PBS-T four times for
15 min and the protein blots were visualized with the Western
Lightning Chemiluminescence Reagent Plus (Perkin Elmer Life Sci-
ences, Boston, MA). The relative amounts of specific proteins were
quantified by densitometry scanning of X-ray films, and analyzed
by the Eagle Eye Image System (Stratagene, La Jolla, CA).
tion (M098004), and National Sun Yat-Sen University-Kaohsiung
Medical University Joint Research Center for financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.12.036.
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4.8. Statistical analysis
All results were expressed as means values standard deviation
(SD) and analyzed by using the statistical analysis system (SPSS,
SPSS Inc., Chicago, IL). Differences among groups were analyzed
by Student’s t-test. P values <0.05 were considered as significant
for all statistical tests.
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We would like to thank the National Science Council of the
Republic of China, Kaohsiung Medical University Research Founda-