Design, synthesis, and biological evaluation of pyrrolo[2,1-c][1,4] benzodiazepine and indole conjugates as anticancer agents

Article abstract of DOI:10.1021/jm050956q

A series of novel pyrrolo[2,1-c][1,4]benzodia/epine (PBD) hybrids linked with indole carboxylates is described. These compounds were prepared by linking C-8 of 3 (DC-81) with an indole 2-carbonyl moiety (9) through carbon chain linkers to afford PBD hybrid agents 17-21 in good yields. Preliminary in vivo tests show that these hybrid agents have potent antitumor activity. The cytotoxic studies of the hybrid agents on human melanoma A2058 cells indicate most of the hybrids induced higher cytotoxicity, better DNA-binding ability, an increase in the apoptotic sub-G1 population, and a significant reduction in ΔΨ_mt relative to compound 3. In addition, DNA flow cytometric analysis shows that hybrids actively induce a marked loss of cells from the G2/M phase of the cell cycle, which progresses to early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide (PI). Thus, we suggest that the hybrid agents are potent inducers of cell apoptosis in A2058 cells.

Full text of DOI:10.1021/jm050956q

1442
J. Med. Chem. 2006, 49, 1442-1449
Design, Synthesis, and Biological Evaluation of Pyrrolo[2,1-c][1,4]benzodiazepine and Indole
Conjugates as Anticancer Agents
Jeh-Jeng Wang,*,† Yu-Kai Shen,† Wan-Ping Hu,‡ Ming-Chu Hsieh,† Fu-Lung Lin,† Ming-Kuan Hsu,† and Mei-Hui Hsu†
Faculty of Medicinal and Applied Chemistry, and Faculty of Biotechnology, Kaohsiung Medical UniVersity, Kaohsiung City 807, Taiwan
ReceiVed September 26, 2005
A series of novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) hybrids linked with indole carboxylates is
described. These compounds were prepared by linking C-8 of 3 (DC-81) with an indole 2-carbonyl moiety
(9) through carbon chain linkers to afford PBD hybrid agents 17-21 in good yields. Preliminary in vivo
tests show that these hybrid agents have potent antitumor activity. The cytotoxic studies of the hybrid agents
on human melanoma A2058 cells indicate most of the hybrids induced higher cytotoxicity, better DNA-
binding ability, an increase in the apoptotic sub-G1 population, and a significant reduction in ∆Ψmt relative
to compound 3. In addition, DNA flow cytometric analysis shows that hybrids actively induce a marked
loss of cells from the G2/M phase of the cell cycle, which progresses to early apoptosis as detected by flow
cytometry after double-staining with annexin V and propidium iodide (PI). Thus, we suggest that the hybrid
agents are potent inducers of cell apoptosis in A2058 cells.
Introduction
Pyrrolo[2,1-c][1,4]benzodiazepines (1) (PBDs) are a group
of potent, naturally occurring antitumor antibiotics produced by
Streptomyces species.1 The cytotoxic and antitumor effects of
these compounds are believed to arise from modification of
DNA, which leads to inhibition of nucleic acid synthesis and
production of excision-dependent single- and double-strand
breaks in cellular DNA.2 These antibiotics have been proposed
to covalently bond to N2 of guanine to form a neutral minor
groove adduct.3,4 Molecular modeling, solution NMR, fluorim-
etry, and DNA footprinting experiments reveal that the PBD
Figure 1. PBD analogues.
monomers recognize three base pairs of DNA with an alkylating
preference for 5′-AGA sequences.1 1 (tomaymycin), 2 (anthra-
indole moiety incorporated into natural and synthetic anticancer
agents such as 5 (CC-1065),16 6 (bizelesin),17 7 (UTA-6026),18
and 8 (K-252a)19,20 (Figure 2) shows potent cytotoxicity. These
results encouraged us to design and synthesize a diversity of
novel PBD conjugate agents.
mycin), and 3 (DC-81); a PBD natural product from Strepto-
myces roseiscleroticus,5 are the best known examples of the
PBDs (Figure 1).6 Although the natural occurring PBDs have
potent anticancer activity they have been precluded from clinical
application due to side effects.7 Synthetic monoalalkylating
analogues devoid of cardiotoxicity manifest relatively poor in
vitro and in vivo potency. Recently, there has been increasing
interest in the design and synthesis of DNA interstrand cross-
linking as well as conjugate agents to enhance the sequence
selectivity and increase selectivity for tumor cells.8-13
A template-directed approach studied by solution NMR and
molecular modeling showed that two tomaymycin molecules
can be covalently bound to a 10 mer duplex DNA, where the
drug molecules are positioned on opposite strands six base pairs
apart.14 Further examination demonstrated that 4 (DSB-120,
Figure 2),8 a head-to-head linked tomaymycin dimer, generates
a guanine-guanine interstrand cross-link on the same 10 mer,
with drug-DNA adducts maintaining self-complementarity.
However, an unusual conformation at the 8I nucleotide indicates
that the five membered ring of 4 is more shallowly immersed
in the minor groove, perhaps due to strained cross-linking with
a very short linker unit.15 Thurston and co-workers reported that
DSB-120 and related synthetic analogues have promising in vitro
cytotoxicity and interstrand DNA cross-linking activity.8-10 An
In the present study, we report the design, synthesis, and
biological evaluation of a homologous series of PBD-indole
conjugates.21 The human melanoma cell line A2058 was selected
as a model; it is a highly metastasizable cell line resistant to
radio- and chemotherapy.22 Meikrantz et al. reported the control
of cell death is linked to the cell cycle.23 Cells with a defective
cell cycle are more vulnerable to some anticancer agents
according to numerous preclinical studies. Many reports have
indicated that mitochondria play a critical role in the commit-
ment of cells to apoptosis.24 Anticancer drugs may damage the
mitochondria by increasing the permeability of the outer
mitochondrial membrane, which is associated with collapse of
mitochondrial membrane potential (∆Ψmt). Disruption in ∆Ψmt
can be measured using rhodamine 123, a cationic lipophilic
fluorochrome;25 the extent of fluorescent dye uptake reflects
the redox potential across the mitochondrial membrane.26
The aim of this study was to investigate whether hybrids
possessed more cytotoxicity than 3, and verify whether hybrid
agents induced antiproliferation, leading to cell growth cycle
perturbation, a decrease in ∆Ψmt, and subsequent apoptotic cell
death.
* To whom correspondence should be addressed. Tel. (886)-7-312-1101
ext 2275, fax (886)-7-312-5339, e-mail: jjwang@kmu.edu.tw.
† Faculty of Medicinal and Applied Chemistry.
Results and Discussion
Chemistry. We prepared the indole-2-carbonyl moieties as
shown in Scheme 1. Indole-2-carboxylic acid (9) was treated
‡ Faculty of Biotechnology.
10.1021/jm050956q CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/26/2006

Anticancer Benzodiazepine-Indole Conjugates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1443
Figure 2. Antitimor agents.
Scheme 1. Synthesis of Indole-2-carbonyl Analogues 13-16
Scheme 2. Synthesis of PBD Conjugate Agents 17-21
after 24 h is shown in Figure 3. The inhibitory effect is
dependent on drug concentration. At concentrations larger than
3 µM, hybrid agents with the exception of 20 exhibited a higher
inhibitory activity than 3 on A2058 cells.
The compounds 17 and 18 were selected by the US National
Cancer Institute for evaluation in the in vitro preclinical
antitumor screening program against 60 human tumor cell lines
derived from nine cancer cell types. The selected biological
with commercially available 3-bromopropylamine (n ) 3) in
the presence of EDCI in dry THF and DMF at room temperature
to afford N-(3-bromopropyl)-1H-2-indolecarboxamide (13) in
82% yield. The homologous analogues (n ) 4 to 6) of indole-
2-carbonyl 14-16 were prepared by bromination of hydroxyl
compounds 10-12 with CBr4 and PPh3 in dry CH2Cl2. These
hydroxyl compounds were obtained from condensation of
indole-2-carboxylic acid with amine alcohols under standard
EDCI coupling conditions in high yields. We have previously
reported the efficient synthesis of 3 in excellent yield.6,27
Reaction of 3 with 9 generated conjugate compound 17 in 75%
yield (Scheme 2). Condensation of 3 with bromide compounds
13-16 in the presence of KI at room temperature afforded the
desired homologous conjugate agents 18-21 in 60-70% yields
(Scheme 2). This is the first example of the 3 molecule applied
in this chemical reaction.
In Vitro Cytotoxic Effects. Conjugate agents 17-21 were
assessed for their in vitro cytotoxicity on human melanoma cell
line A2058. The activity of mitochondrial dehydrogenase
enzymes, detectable by catalyzing MTS reagent, correlated with
cell viability.28 The cytotoxic effects of the six compounds 3,
and hybrid agents 17-21 on human melanoma cell line A2058
was examined using an MTS cell proliferation assay. The cell
viability of A2058 cells treated with agents at different dosages
Figure 3. Dose-response curves for compounds tested against A2058
cells. Cells were seeded in a 96-well plate at 2500 cells per well and
cultivated overnight until cell attachment. Compounds at the indicated
concentration were added into the culture media in triplicate and
incubated for 24 h before MTS being added. The conversion of MTS
to formazan was measured at 490 nm. The absorbance is directly
proportional to the number of living cells.

1444 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Wang et al.
Table 1. In Vitro Cytotoxicity of Compounds 17 and 18 in Selected
Table 2. Energies (kcal/mol) of Covalent Complexes between Hybrid
Compounds and d(CICGATCICG)2
Cancer Cell Linesa
GI50 (µM)
complex
energy (kcal/mol)
panels/cancer cell lines
17
18
DNA-17
DNA-18
DNA-19
DNA-20
DNA-21
-329.3
-333.5
-331.4
-326.2
-331.1
Non-Small Cell Lung Cancer
EKVX
NCI-H522
0.674
0.198
0.604
0.0141
Colon Cancer
0.196
COLO 205
HT29
0.118
0.274
0.185
CNS Cancer
0.669
SF-268
U251
0.027
0.0394
0.302
Melanoma
0.162
M14
UACC-62
0.173
0.203
0.290
Renal Cancer
0.155
RXF 393
SN12C
Meanb
0.025
0.41
0.182
0.21
0.38
a Data obtained from NCI’s in vitro disease-oriented tumor cells screen.
b Mean values over 60 cell lines tested.
Figure 4. Agarose electrophoresis gel showing incubation of BamHI
with complexes of pBR322, 3 and hybrid agents. Lane 1: control
pBR322; lane 2: complete digest of pBR322 by BamHI; lane 3-8:
agent-pBR322 complexes at 5 µM digested by BamHI were 3, 17, 18,
19, 20, and 21, respectively. (OC ) open-circular, SC ) supercoiled,
L ) linear).
evaluation results for the two compounds are presented in Table
1. The mean GI50 values for hybrid 17 and 18 are 0.38 and
0.182 µM, respectively, indicating that these agents have the
potential for use as a highly potent broad-spectrum anticancer
compound to inhibit the growth of a variety of cancer cell lines.21
In Vivo Cytotoxicity. Compounds 17 and 18 were further
examined in an in vivo hollow fiber assay conducted by the
National Cancer Institute, in which an intraperitonael (ip) sample
and a subcutaneous (sc) sample were tested. In this assay, if a
tested compound is observed to have a total of the ip plus sc
scores larger than 20, it will be considered to be active and to
have potential as an antitumor/anticancer drug candidate.29 The
preliminary in vivo testing results showed compounds 17 and
18 have total scores of 22 and 30, respectively. This indicates
that they have potent antitumor/anticancer activity.
Enzyme Inhibition. The restriction endonuclease digestion
assay is based on the ability of agent to inhibit the cleavage
activity of restriction endonuclease BamHI.30 The digestion
assay gel includes two controls: open-circular (OC) and
supercoiled (SC) pBR322 DNA in lane 1, and fully linearized
DNA in lane 2. The experimental results show that hybrid agents
(18, 19, 21) inhibit BamHI digestion (Figure 4). Our finding
suggested that hybrid agents bind to DNA more efficiently than
3.
Figure 5. Effect of compound tested on the cellular sub-G1 content.
A2058 cells were treated with 5 µM agents for 18 h and stained with
PI. Cells were analyzed with the FACScan flow cytometer. Data
represent the percentage of cell counts and display sub-G1.
two I (inosine). Lacking the exocyclic 2-amino group, inosine
is unreactive toward alkylation by 3. The results obtained
demonstrate that the hybrids 17-21 exhibit different DNA-
binding activity (Table 2). It was found that hybrid 18, 19, and
21 form more stable complexes with DNA as compared to the
other hybrids. The reason might be that the carbon chain linkers
in the above hybrids form a better isohelical fit, giving rise to
more favorable interaction within the minor groove than other
hybrids. In addition, Baraldi et al.32 mentioned that hydrogen
bonds, electrostatic forces, and van der Waals interactions are
responsible for the stability of the complex between these hybrid
compounds and DNA. The binding of hybrid agents to double
helix will be further studied.
Cell Cycle Effects. To investigate the effects of 3 and hybrids
on cell cycle progression of A2058 cells, the DNA content of
cell nuclei was measured by flow cytometric analysis. 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 5. Treatment of A2058
cells with 5 µM agents for 18 h induced apoptosis effects in
Molecular Modeling Studies. To achieve monospecific
binding GC,31 we modified the 10-mer d(CGCGATCGCG)2 in
which the two G (guanine) underlined were each replaced with

Anticancer Benzodiazepine-Indole Conjugates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1445
Figure 7. Effect of agent on the mitochondrial membrane potential
(∆Ψmt). Cells were cultured with different agents at a concentration of
4 µM for 24 h and then stained with rhodamine 123 and analyzed
immediately by flow cytometry as described under Materials and
Methods. The number in M1 indicates the percentage of cells with
reduced ∆Ψmt.
investigated whether ∆Ψmt disruption was involved in agent-
induced apoptosis. A2058 cells were treated with 3 and 17-21
at a concentration of 4 µM for 24 h and then analyzed by flow
cytometry after rhodamine 123 dye labeling. The dye binds to
the inner and outer membrane of mitochondria and undergoes
a red shift in fluorescence during membrane depolarization.33
The results are shown in Figure 7. Most hybrids exhibited a
marked reduction in cellular uptake of the fluorochrome
compared to 3. The decrease of fluorescence intensity reflects
the collapse of ∆Ψmt, which generally defines an early but
already inreversible stage of apoptosis.34 These results reveal
that exposure of melanoma A2058 cells to hybrids inducing
the drop of ∆Ψmt may be a possible cause for the apoptotic
process.
Apoptosis Detection. Fluorescein isothiocyanate (FITC)-
conjugated annexin V has been utilized to detect the external-
ization of phosphatidylserine that occurs at an early stage of
apoptosis propidium iodide (PI) is used as a marker of necrosis
due to cell membrane destruction.35 Although hybrids are highly
potent antitumor agents, the precise mechanism of action remain
unclear. To further characterize whether hybrid 21-induced cell
death involved apoptosis or not, we performed a biparametric
cytofluorimetric analysis using annexin V and PI double
staining. The distribution of stained cells is shown in Figure 8.
A 2 µM concentration exhibited an apoptotic effect on A2058
cells, and higher concentrations induced cell death with
increased cell permeability. The apoptotic effect was dependent
on drug concentration.
Figure 6. Flow cytometric analyses of cell cycle distribution of A2058
cells after exposure to hybrid 21 (0-4 µM) for 24 h before cell cycle
analysis. M1 ) apoptotic sub-G1 area; M2 ) G1 area; M3 ) S area;
M4 ) G2/M area.
45.0% (3), 38.5% (17), 46.8% (18), 46.8% (19), 2.9% (20), and
50.6% (21) of sub-G1 DNA peak. Our results show that hybrids
18, 19, and 21 were more efficient in inducing apoptosis than
3 in A2058 cells. Moreover, to verify whether cell damage might
be attributable to the cell cycle program or might have become
arrested at any cell cycle phases by hybrid-induced apoptosis
in A2058 cells, we used hybrid 21 at various concentrations
(Figure 6). The cells were treated with graded concentrations
of the drug for 24 h. The majority of control cells exposed to
DMSO of the cell cycle were in the G1 phase 54.0%, S phase
14.4%, and G2/M phase 30.5%. At a concentration of 2 µM,
hybrid 21 treatment resulted in cells progressing to S and G2/M
phases, while concomitantly the G1 population decreased.
Furthermore, there is a marked loss of cells from the G2/M
phase (from 47.7 to 12.5%) by hybrid 21 at a concentration of
4 µM. Our data indicate that hybrid 21 had cytotoxic effects
on human melanoma A2058 cells, and the cytotoxic effects may
be through apoptosis induction.
Conclusions
We have previously reported an efficient synthesis of 3, as a
starting point for the design novel dimeric and conjugate agents
that would be expected to be more biologically potent. We next
combined 3 and an indole 2-carbonyl moiety to synthesize
hybrids designed to have much higher sequence selectivity in
DNA interactivity. NCI screening results indicate that these
agents have the potential for use as highly potent broad-spectrum
antitumor/anticancer compounds to inhibit the growth of a
variety of cancer cell lines. Preliminary in vivo tests show that
these hybrid agents have potent antitumor/anticancer activity.
In further study, we used an MTS cell proliferation assay to
evaluate the cytotoxicity of tested compounds in human
Mitochondrial Membrane Potential (∆Ψmt) Disruption.
Previous studies have suggested that a decline of ∆Ψmt may
be an early event in the process of cell death. Therefore, we

1446 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Wang et al.
Figure 8. Hybrid 21 induces externalization of PS. Dot plots for A2058 cells treated with graded concentrations of 21 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. For each drug concentration tested,
the percentage of Annexin V+ cells is given.
to TMS or CDCl3 (7.26 ppm). 13C NMR was referenced 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
Taichung Regional Instrument Center of NSC at NCHU. Flash
chromatography was carried out on Silica Gel 60 (E. Merck, 230-
400 mesh).
The purity of the final compounds was analyzed with a
Waters1525 Binary HPLC system connected to a Waters 2487 UV
detector and following the peaks at λ) 221 nm. Method A: The
flow was 1 mL/min and the gradient was from 70% acetonitrile-
water, until 80% over a period of 10 min. The column for the
analysis was Symmetry C18 (5 µm, 150 × 4.6 mm). Method B:
The flow was 3 mL/min and the gradient was from 80% acetoni-
trile-water, until 90% over a period of 40 min. The column for
the analysis was C18 of Hypersil ODS (5 µm, 250 × 21.2 mm).
melanoma A2058 cells. Our results indicate that most of the
hybrid agents are more effective as an antiproliferative agent
than 3. One can speculate that this is because hybrids recognize
more DNA-binding sites and increase the stability of the drug/
DNA complex. Thus, these compounds with DNA binding
ability were further evaluated by restriction endonuclease BamHI
and molecular modeling studies. The experimental results show
that most of the hybrids inhibit BamHI digestion to a greater
degree than 3, and hybrids 18, 19, and 21 form more stable
complexes with DNA as compared to the other hybrids. To
identify whether the antiproliferative effect of hybrids were
associated with cell cycle progression, hybrid 21 was selected
against A2058 cells. The results obtained demonstrate that
hybrid 21 induces a marked loss of cells from the G2/M phase
and triggers apoptosis as revealed by the externalization of
annexin V-targeted PS residues at the periphery of the cells.
Owing to studies suggesting that a decline of ∆Ψmt may be an
early event in the process of cell death, this prompted us to
investigate whether ∆Ψmt disruption was involved in hybrid-
induced apoptosis. In this study, most of the hybrids exhibited
a marked reduction in ∆Ψmt. Taken together, we provide
evidence that hybrid agents are potent inducers of apoptosis in
A2058 cells. We expect our studies can provide an important
mechanistic insight into the action of hybrids.
General Procedure for the Syntheses of N2-(Alkanol)-1H-2-
indolecarboxamides (10-12). To a stirred solution of indole-2-
carboxylic acid (1 g, 6.2 mmol) and amino-1-alkanols (6.8 mmol)
in THF (15 mL) and DMF (3 mL) was added EDCI (1.3 g, 6.8
mmol) in one portion under nitrogen at 0 °C. The resulting solution
was stirred at room temperature for 24 h. The reaction mixture
was poured into ice-water (150 mL) and extracted four times with
ethyl acetate. The combined organic phases were washed with H2O
and brine and dried over MgSO4. After removal of solvent, the
residue was purified by flash chromatography (hexane/AcOEt )
5:1) to give the products.
N2-(4-Butanol)-1H-2-indolecarboxamide (10): white solid;
yield 1.10 g (82%); mp 141-143 °C. 1H NMR (CDCl3 + DMSO-
d6, 400 MHz) δ 10.68 (bs, 1H), 7.76 (bs, 1H), 7.61(d, J ) 7.6 Hz,
1H), 7.45 (dd, J ) 8, 0.8 Hz, 1H), 7.24 (dt, J ) 8, 0.8 Hz, 1H),
7.08 (t, J ) 7.6 Hz, 1H), 7.03 (d, J ) 1.2 Hz, 1H), 3.65 (t, J ) 6
Experimental Section
Synthetic Chemistry. Infrared spectra were recorded on a
1
Perkin-Elmer Series 2000 spectrophotometer. H NMR and 13C
NMR spectra were recorded at 400 and 100 MHz, respectively,
using CDCl3 as a solvent. 1H NMR chemical shifts are referenced

Anticancer Benzodiazepine-Indole Conjugates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4 1447
Hz, 2H), 3.67-3.46 (m, 2H), 2.90 (bs, 1H), 1.79-1.63 (m, 4H);
13C NMR (CDCl3 + DMSO-d6, 100 MHz) δ 161.3 (s), 136.0 (s),
131.2 (s), 127.0 (s), 123.2 (d), 121.1 (d), 119.5 (d), 111.6 (d), 102.4
(d), 61.3 (t), 38.9 (t), 29.5 (t), 25.8 (t); LRMS (EI, m/z) 232 (M+);
HRMS (EI, m/z) for C13H16N2O2, calcd 232.1213, found 232.1209.
N2-(5-Pentanol)-1H-2-indolecarboxamide (11): white solid;
yield 1.17 g (76%); mp 120-122 °C. 1H NMR (CDCl3 + DMSO-
d6, 400 MHz) δ 10.68 (bs, 1H), 7.60 (d, J ) 8 Hz, 1H), 7.52 (t, J
) 6 Hz, 1H) 7.44 (dd, J ) 6.0, 0.8 Hz, 1H), 7.22 (dt, J ) 8.0, 1.2
Hz, 1H), 7.07 (dt, J ) 8.0, 0.8 Hz, 1H), 7.04 (dd, J ) 6.0, 0.8 Hz,
1H), 3.59 (t, J ) 6.4 Hz, 2H), 3.45 (q, J ) 6.8 Hz, 2H), 2.97 (bs,
1H), 1.68-1.41 (m, 6H); 13C NMR (CDCl3 + DMSO-d6, 100 MHz)
δ 161.6 (s), 136.3 (s), 131.1 (s), 127.1 (s), 123.5 (d), 121.3 (d),
119.7 (d), 111.8 (d), 102.8 (d), 61.6 (t), 39.2 (t), 31.9 (t), 28.9 (t),
22.9 (t); LRMS (EI, m/z) 246 (M+); HRMS (EI, m/z) for
C14H18N2O2, calcd 246.1369, found 246.1372.
N2-(6-Hexanol)-1H-2-indolecarboxamide (12): white solid;
yield 1.15 g (75%); mp 120-122 °C. 1H NMR (CDCl3 + DMSO-
d6, 400 MHz) δ 10.72 (bs, 1H), 7.60 (d, J ) 8 Hz, 1H), 7.49 (t, J
) 5.6 Hz, 1H), 7.44 (d, J ) 8 Hz, 1H), 7.22 (t, J ) 8 Hz, 1H),
7.07 (t, J ) 8 Hz, 1H), 7.04 (d, J ) 2.4 Hz, 1H), 3.56 (t, J ) 5.6
Hz, 1H), 3.44 (q, J ) 6.8 Hz, 1H), 2.98 (bs, 1H), 1.62 (t, J ) 6.8
Hz, 2H), 1.53 (t, J ) 6.4 Hz, 2H), 1.41-1.36 (m, 4H); 13C NMR
(CDCl3 + DMSO-d6, 100 MHz) δ 161.6 (s), 136.3 (s), 131.1 (s),
127.2 (s), 123.5 (d), 121.3 (d), 119.7 (d), 111.8 (d), 102.8 (d), 61.7
(t), 39.2 (t), 32.2 (t), 29.2 (t), 26.3 (t), 25.1 (t); LRMS (FAB, m/z)
261 (M + H); HRMS (ESI, m/z) for C15H21N2O2 [(M + H)+], calcd
261.1600, found 261.1603.
N2-(3-Bromopropyl)-1H-2-indolecarboxamide (13). To a stirred
solution of indole-2-carboxylic acid (100 mg, 0.62 mmol) and
3-bromoproppylamine (6.8 mmol) in THF (3 mL) and DMF (1 mL)
was added EDCI (133.6 mg, 0.68 mmol) in one portion under
nitrogen at 0 °C. The resulting solution was stirred at room
temperature for 24 h. The reaction mixture was poured into ice-
water (20 mL) and extracted four times with ethyl acetate. The
combined organic phases were washed with H2O and brine and
dried over MgSO4. After removal of solvent, the residue was
purified by flash chromatography (hexane/AcOEt ) 4:1) to give a
light yellow solid 13: yield 143.2 mg (82%); mp 101-103 °C. 1H
NMR (CDCl3 + DMSO-d6 400 MHz) δ 10.66 (s, 1H), 7.96 (s,
1H), 7.61 (d, 1H, J ) 7.8 Hz), 7.46 (d, 1H, J ) 7.4 Hz), 7.18-
7.27 (m, 1H), 7.04-7.11 (m, 2H), 3.53-3.70 (m, 4H), 2.04-2.24
(m, 2H); 13C NMR (CDCl3 + DMSO-d6, 100 MHz) δ 161.6, 135.1,
130.9, 127.0, 123.3, 121.2, 119.5, 111.6, 103.1, 37.4, 32.0, 30.9;
LRMS (EI, m/z) 280 (M+); HRMS (EI, m/z) for C12H13N2OBr,
calcd 280.0212, found 280.0215.
General Procedure for the Syntheses of N2-(Bromoalkyl)-
1H-2-indolecarboxamides (14-16). To a mixture of N2-(Alkanol)-
1H-2-indolecarboxamides (1.94 mmol) and carbon tetrabromide
(2.03 g, 5.82 mmol) in anhydrous dichloromethane (11 mL) was
added, at 0 °C triphenylphosphine (1.03 g, 3.88 mmol). The
resulting solution was stirred at room temperature for 3 h. After
removal of solvent, the residue was purified by flash chromatog-
raphy (hexane/AcOEt ) 5:1) to give the products.
+ DMSO-d6, 100 MHz) δ 161.7 (s), 136.3 (s), 131.1 (s), 127.3
(s), 123.7 (d), 121.5 (d), 120.0 (d), 111.8 (d), 102.7 (d), 39.1 (t),
33.5 (t), 32.0 (t), 28.6 (t), 25.2 (t); LRMS (EI, m/z) 388 (M +
HBr); HRMS (EI, m/z) for C14H17N2OBr+HBr, calcd 387.9786,
found 387.9783.
N2-(6-Bromohexyl)-1H-2-indolecarboxamide (16): white solid;
yield 445 mg (57%); mp 118-120 °C. 1H NMR (CDCl3 400 MHz)
δ 9.85 (bs, 1H), 7.63 (dd, J ) 8.0, 0.8 Hz, 1H), 7.45 (dd, J ) 8.0,
0.8 Hz, 1H) 7.28 (dt, J ) 8.0, 1.2 Hz, 1H), 7.13 (dt, J ) 8.0, 0.8
Hz, 1H), 6.84 (d, J ) 1.2 Hz, 1H), 6.29 (t, J ) 5.6 Hz, 1H), 3.51
(t, J ) 6.4 Hz, 2H), 3.39 (t, J ) 6.4 Hz, 2H), 1.89-1.82 (m, 2H),
1.77 (s, HBr), 1.70-1.63 (m, 2H), 1.53-1.38 (m, 4H); 13C NMR
(CDCl3, 100 MHz) δ 161.8 (s), 136.4 (s), 130.8 (s), 127.6 (s), 124.4
(d), 121.8 (d), 120.6 (d), 112.0 (d), 101.7 (d), 39.6 (t), 33.7 (t),
32.5 (t), 29.6 (t), 27.8 (t), 26.1 (t). LRMS (FAB, m/z) 323 [(M +
H)+]; HRMS (ESI, m/z) for C15H20N2OBr [(M + H)+] calcd
323.0759, found 323.0760.
(11aS)-8-(1H-2-Indolecarbonyloxy)-7-methoxy-1,2,3,11a-tet-
rahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (17). To a
stirred solution of compound 3 (100 mg, 0.41 mmol) in THF (5
mL) and water (1 mL) was added NaHCO3 (86 mg, 0.82 mmol) in
one portion at 0 °C for 30 min. 1H-2-Indolecarbonyl chloride (118
mg, 1.12 mmol) in THF (5 mL), generated freshly by thionyl
chloride, was added to the solution dropwise. The resulting solution
was stirred at room temperature for 24 h. The reaction mixture
was poured into ice water (30 mL) and extracted with ethyl acetate.
The combined organic phases were washed with H2O and brine
dried over MgSO4 and concentrated under vacuum. The crude
product was subjected to flash chromatography (CH2Cl2/MeOH )
70:1) to give a white solid 17: yield 119.6 mg (75%); mp 108-
1
110 °C. H NMR (CDCl3, 400 MHz) δ 9.37 (s, 1H), 7.75-7.71
(m, 2H), 7.67 (s, 1H), 7.49-7.47 (m, 1H), 7.38 (dd, J ) 6.8 and
1.2 Hz, 1H), 7.21-7.17 (m, 3H), 3.88-3.79 (m, 5H), 3.65-3.60
(m, 1H), 2.36-2.32 (m, 2H), 2.10-2.05 (m, 2H); 13C NMR (CDCl3,
100 MHz) δ 164.2 (s), 163.2 (d), 159.3 (s), 149.8 (s), 141.7 (s),
139.9 (s), 137.4 (s), 127.4 (s), 126.3 (s), 126.0 (d), 125.8 (s), 122.8
(d), 121.9 (d), 121.1 (d), 113.8 (d), 112.1 (d), 110.9 (d), 56.3 (q),
53.7(d), 46.8 (t), 29.6 (t), 24.1 (t); LRMS (FAB, m/z) 390 [(M +
H)+]; HRMS (FAB, m/z) for C22H20N3O4 [(M + H)+] calcd
390.1455, Found 390.1445.
General Procedure for the Syntheses of (11aS)-8-[3-(1H-2-
Indolycarboxamido)]alky-oxyl-7-methoxy-1,2,3,11a-tetrahydro-
5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (18-21). To a solu-
tion of 3 (100 mg, 0.41 mmol) in acetone (5 mL) was added K2CO3
(84 mg, 0.62 mmol) at 0 °C and stirred for 30 min. N2-
(bromoalkyl)-1H-2-indolecarboxamides (13-16) 0.70 mmol, gener-
ated freshly in acetone (5 mL) and KI (41 mg, 0.60 mmol), was
added to the solution dropwise. The resulting solution was stirred
at room temperature for 24 h. The reaction mixture was poured
into ice-water (30 mL) and extracted with ethyl acetate. The
combined organic phases were washed with H2O and brine dried
over MgSO4 and concentrated under vacuum. The residue was
subjected to flash chromatography (CH2Cl2/MeOH ) 40:1) to give
products.
N2-(4-Bromobutyl)-1H-2-indolecarboxamide (14): white solid;
yield 371 mg (65%); mp 135-137 °C. 1H NMR (CDCl3 + DMSO-
d6, 400 MHz) δ 10.69 (bs, 1H), 7.75 (t, J ) 5.6 Hz, 1H), 7.61 (dd,
J ) 8.4, 1.2 Hz, 1H,), 7.46 (dd, J ) 8.4, 0.8 Hz, 1H), 7.22 (dt, J
) 8.4, 1.2 Hz, 1H), 7.10-7.06 (m, 2H), 3.50-3.45 (m, 4H), 2.00-
1.93 (m, 2H), 1.82-1.75 (m, 2H); 13C NMR (CDCl3 + DMSO-d6,
100 MHz) δ 161.4 (s), 136.1 (s), 131.0 (s), 127.0 (s), 123.2 (d),
121.1 (d), 120.0 (d), 111.6 (d), 102.7 (d), 38.0 (t), 33.1 (t), 29.5
(t), 27.8 (t); LRMS (EI, m/z) 294 (M+); HRMS (EI, m/z) for
C13H15N2OBr, calcd 294.0369, found 294.0370.
N2-(5-Bromopentyl)-1H-2-indolecarboxamide hydrobromide
(15): light yellow solid; yield 527 mg (70%); mp 101-103 °C.
1H NMR (CDCl3 + DMSO-d6, 400 MHz) δ 10.36 (bs, 1H), 7.63
(d, J ) 7.6 Hz, 1H), 7.45 (dd, J ) 8.0, 0.8 Hz, 1H), 7.33 (t, J )
5.6 Hz, 1H), 7.24 (dt, J ) 8.0, 0.8 Hz, 1H), 7.10 (dt, J ) 8.0, 0.8
Hz, 1H), 7.01 (d, J ) 1.6 Hz, 1H), 3.49-3.40 (m, 4H), 1.94-1.87
(m, 2H), 1.70-1.63 (m, 2H), 1.57-1.50 (m, 2H); 13C NMR (CDCl3
(11aS)-8-[3-(1H-2-Indolycarboxamido)]propoxyl-7-methoxy-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
one (18): white solid; yield 123 mg (67%); mp 106-108 °C. H
1
NMR (CDCl3, 400 MHz) δ 9.59 (s, 1H), 7.67 (d, J ) 4.4 Hz, 1H),
7.64 (dd, J ) 0.8, 4.4 z, 1H), 7.57 (s,1H), 7.44 (dd, J ) 8 and 0.8
Hz, 1H), 7.30-7.26 (m, 1H), 7.14 (dt, J ) 7.2 and 1.2 Hz, 1H),
6.99 (dd, J ) 2 and 0.8 Hz, 1H), 6.85 (s, 1H), 4.32-4.18 (m, 2H),
3.97 (s, 3H), 3.87-3.55 (m, 5H), 2.33-2.01 (m, 6H). 13C NMR
(CDCl3, 100 MHz) δ 164.5 (s), 162.6 (d), 161.7 (s), 150.3 (s), 147.6
(s), 140.7 (s), 136.3 (s), 131.1 (s), 127.7 (s), 124.3 (d), 121.7 (d),
120.7 (d), 120.6 (s), 112.0 (d), 111.7 (d), 110.4 (d), 102.3 (d), 69.0
(t), 56.0 (q), 53.7 (d), 46.7 (t), 38.5 (t), 29.6(t), 28.6 (t), 24.2 (t);
LRMS (FAB, m/z) 447 [(M + H)+]; HRMS (FAB, m/z) for
C25H26N4O4 [(M + H)+] calcd 447.2034., Found 447.2028.
(11aS)-8-[3-(1H-2-Indolycarboxamido)]butoxyl-7-methoxy-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
1
one (19): white solid; yield 113 mg (60%); mp 114-116 °C. H

1448 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 4
Wang et al.
NMR (CDCl3, 400 MHz) δ 9.53 (bs, 1H), 7.67 (d, J ) 4.4 Hz,
1H), 7.62 (dd, J ) 8.0 and 0.8 Hz, 1H), 7.55 (s, 1H), 7.43 (dd, J
) 8.0 and 0.8 Hz, 1H), 7.27 (dt, J ) 8.0 and 1.2 Hz, 1H), 7.13 (dt,
J ) 8.0, 1.2 Hz, 1H), 7.02 (t, J ) 6.4 Hz, 1H), 6.89 (d, J ) 1.2
Hz, 1H), 6.83 (s,1H), 4.21-4.07 (m, 2H), 3.92 (s, 3H), 3.86-3.54
(m, 5H), 2.34-2.28 (m, 2H), 2.10-1.81 (m, 6H); 13C NMR (CDCl3,
100 MHz) δ164.6 (s), 162.6 (d), 161.7 (s), 150.4 (s), 147.6 (s),
140.7 (s), 136.2 (s), 131.0 (s), 127.7 (s), 124.3 (d), 121.8 (d), 120.5
(d), 120.3 (s), 111.9 (d), 111.5 (d), 110.2 (d), 101.9 (d), 68.7 (t),
56.1 (q), 53.7 (d), 46.7 (t), 38.7 (t), 29.6 (t), 26.6 (t), 25.6 (t), 24.2
(t); LRMS (FAB, m/z) 461 [(M + H)+]; HRMS (FAB, m/z) for
C26H29N4O4 [(M + H)+] calcd 461.2191, Found 461.2190.
(11aS)-8-[3-(1H-2-Indolycarboxamido)]pentoxyl-7-methoxy-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
temperature at 4 °C for 10 min. Then 20 µL (150 ng of plasmid
DNA) from each digestion was added to 4 µL of loading buffer
and loaded onto a 1.0% horizontal agarose gel which was run in
Tris-acetate EDTA buffer (TAE-40 mM Tris base, pH 8.0, 18 mM
glacial acetic acid, 1 mM EDTA) at 100 V for 2 h. The gels were
stained with ethidium bromide (2 µg/mL) for 30 min and then
destained in TAE buffer for 10 min. The DNA bands were
visualized by UV and photography.
Molecular Modeling Studies. All of the calculations described
below were performed using INSIGHT II suite of software (MSI,
Inc.) running on a Silicon Graphics O2 system.
1. Molecular Mechanics (MM) Calculations. The model of
d(CICGATCICG)2 and hybrid compounds were constructed using
the BIOPOLYMER and BUILDER module, respectively. Docking
of a hybrid compound into the minor groove of the DNA duplex
was carried out manually and formed a covalent bond between C11
of the drug and a single reactive guanine (G4) on one strand of
d(CICGATCICG)2. The potentials and the charges were fixed using
the AMBER force field.36 The complex was minimized using
steepest descent and conjugate gradient techniques until the energy
root-mean-square gradient was less than 0.1 kcal/molÅ.
2. Molecular Dynamics (MD) Simulations. The minimized
models were then subjected to 100 ps of molecular dynamics at
300 K with a gradual increase in temperature from 10 to 300 K
over the first 10 ps. During simulations, all of the intermittent
structures of the complexes formed at every successive 5 ps were
saved and later they were subjected to energy reminimization to
<0.1 kcal/molÅ.
Cell Cycle Analysis. A2058 cells were treated with compounds
at 5 µM for 18 h or treated with various concentrations (0-4 µM)
of hybrid 21 for 24 h. Cells were harvested by trypsinization and
centrifugation. After being washed with PBS, the cells were fixed
with ice-cold 70% ethanol for 30 min, washed with PBS, and then
treated with 1 mg/mL of RNase A solution (containing 0.112 mg/
mL of trisodium citrate) at 37 °C for 30 min. Cells were harvested
by centrifugation at 400g for 5 min and further stained with 250
µL of DNA staining solution (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 H2O) at room temperature for 30 min in
the dark. After loading 750 µL of PBS, 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 Document interface software (WinMDI).
1
one (20): white solid; yield 119 mg (61%); mp 101-103 °C. H
NMR (CDCl3, 400 MHz) δ 9.62 (bs, 1H, NH), 7.67 (d, J ) 3.6
Hz, 1H), 7.62 (d, J ) 8 Hz, 1H), 7.51 (s, 1H), 7.43 (dd, J ) 8.0
and 0.8 Hz, 1H), 7.26 (dt, J ) 8.0 and 0.8 Hz, 1H), 7.12 (dt, J )
7.6 and 0.8 Hz, 1H), 6.86 (d, J ) 2.8 Hz, 1H), 6.81 (s,1H), 6.57 (t,
J ) 6.0 Hz, NH), 4.12-4.00 (m, 2H), 3.88 (s, 3H), 3.85-3.50 (m,
5H), 2.34-1.54 (m, 10 H); 13C NMR (CDCl3, 100 MHz): δ 164.7
(s), 162.4 (d), 161.7 (s), 150.8 (s), 147.7 (s), 140.6 (s), 136.2 (s),
130.9 (s), 127.6 (s), 124.3 (d), 121.8 (d), 120.5 (d), 120.1 (s), 111.9
(d), 111.6 (d), 110.4 (d), 101.9 (d), 68.8 (t), 56.1 (q), 53.7 (d),
46.7 (t), 39.5 (t), 29.6 (t), 29.2 (t), 28.4 (t) 24.2 (t), 23.5 (t). LRMS
(FAB, m/z) 475 [(M + H)+]; HRMS (FAB, m/z) for C27H30N4O4
[(M + H)+] calcd 475.22347, Found 475.2353.
(11aS)-8-[3-(1H-2-Indolycarboxamido)]hexoxyl-7-methoxy-
1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-
1
one (21): white solid; yield 140 mg (70%); mp 104-106 °C. H
NMR (CDCl3, 400 MHz) δ 9.69 (bs, 1H), 7.67 (d, J ) 4.0 Hz,
1H), 7.63 (d, J ) 7.6 Hz, 1H), 7.52 (s, 1H), 7.42 (dd, J ) 8.0 and
0.8 Hz, 1H), 7.27 (dt, J ) 8.0 and 0.8 Hz, 1H), 7.12 (dt, J ) 7.6
and 0.8 Hz, 1H), 6.86 (d, J ) 1.6 Hz, 1H), 6.83 (s,1H), 6.46 (t, J
) 5.6 Hz, 1H), 4.14-4.00 (m, 2H), 3.92 (s, 3H), 3.85-3.45 (m,
5H), 2.34-2.28 (m, 2H), 2.09-1.96 (m, 2H), 1.90-1.42 (m, 8H);
13C NMR (CDCl3, 100 MHz) δ 164.7 (s), 162.4 (d), 161.7 (s), 150.8
(s), 147.8 (s), 140.6 (s), 136.3 (s), 130.9 (s), 127.6 (s), 124.3 (d),
121.8 (d), 120.5 (d), 120.0 (s), 112.0 (d), 111.5 (d), 110.4 (d), 101.9
(d), 68.8 (t), 56.1 (q), 53.7 (d), 46.7 (t), 39.5 (t), 29.7 (t), 29.6 (t),
28.6 (t), 26.5 (t), 25.5 (t), 24.2 (t); LRMS (FAB, m/z) 489 [(M +
H)+]; HRMS (FAB, m/z) for C28H32N4O4 [(M + H)+] calcd
489.2504, Found 489.2511.
Cell Culture. Human melanoma A2058 cells purchased from
American Type Culture Collection (Manassas, VA) were maintained
in Dulbecco’s minimal essential medium (DMEM) supplemented
with 10% FCS and 100 U/mL penicillin G and 100 µg/mL
streptomycin sulfate (Gibco, BRL). A2058 cells were passaged at
confluence after treatment with 5 mM EDTA (Gibco, BRL) and
incubated at 37 °C in a humidified atmosphere containing 5% CO2.
MTS Cell Proliferation Assay. A commercially available kit
(CellTiter96 Aqueous proliferation assay kit, Promega, Madison,
WI) was used to detect the proliferation according to the manu-
facturer’s instruction. Cells were seeded in a 96-well plate at the
cell density of 2500 cells/well. After an overnight incubation, the
cells were treated with 3 or hybrid 17-21 at 5 µM and incubated
for 24 h. The MTS reagent contains tetrazolium salt, [3-(4,5-
dimethylthiazol-2-y1)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-
nyl)-2H-tetrazolium], premixed with the electron coupling reagent
(phenazine ethosulfate) and was added into each well in 20 µL
portions. The plate was then incubated for 1-2 h at 37 °C. The
optical density value was detected by a microplate reader (MRX-
II, Dynex technology, Chantilly, VA), whose detecting and refer-
ence wavelengths were set at 490 and 690 nm, respectively.
Restriction Endonuclease Digestion Assay. Compound-DNA
complexes were prepared by incubating plasmid DNA (0.5 µg) with
compound at 5 µM in restriction buffer (pH 7.5) and bovine serum
albumin (BSA) (0.1 mg/mL) in a reaction volume of 19 µl at 37
°C for 1 h. An excess of BamHI (20 units in 1 µL) was added and
incubated at 37 °C for a further 1 h. Each digestion was stopped
by incubation at 65 °C for 20 min followed by lowering the
Assessment of Mitochondrial Membrane Potential (∆Ymt).
A2058 cells were cultured in six-well plates and allowed to reach
exponential growth for 24 h before treatment. The cells were
harvested 24 h after treatment with compound (3, hybrid 17-21)
at a concentration of 4 µM. The medium was removed, and the
adherent cells were trypsinized. The cells were pelleted by
centrifugation at 400g for 5 min and further resuspended in 1 mL
of rhodamine 123 (10 µg/mL) for 30 min at room temperature and
washed with PBS twice and resuspended in PBS. The samples were
analyzed for fluorescence (FL-1 detector, filter 530/30 nm band-
pass) by using the WinMDI software for flow cytometry.
Annexin V and PI Binding Assay. To assess the simultaneous
observation of early phase of apoptotic and necrotic features, A2058
cells were treated with graded concentration of hybrid 21 for 24 h,
and then cells were 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.
Acknowledgment. We would like to thank the National
Science Council of the Republic of China for financial support.
Supporting Information Available: Spectra and HPLC data
for target compounds 17-21. This material is available free of
charge via the Internet at http://pubs.acs.org.

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