Novel antitumor agent family of 1H-benzo[c,d]indol-2-one with flexible basic side chains: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2006.11.016

A series of mono-1H-benzo[c,d]indol-2-one with different amine side chains and bis-1H-benzo[c,d]indol-2-one as novel family of DNA intercalators were designed and synthesized, the contributions of aromatic chromophores and amine side chains for DNA binding properties, for example, intercalation and electrostatic binding, respectively, were evaluated. Among them, A3 tailed with N,N-dimethylamino-ethyl-ethane-1,2-diamine showed selective anti-tumor activities against cell lines A549 and P388 with IC₅₀ 0.428 μm and 1.69 μm.

Full text of DOI:10.1016/j.bmc.2006.11.016

Bioorganic & Medicinal Chemistry 15 (2007) 1356–1362
Novel antitumor agent family of 1H-benzo[c,d]indol-2-one with
flexible basic side chains: Synthesis and biological evaluation
Hong Yin, Yufang Xu and Xuhong Qian*
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China, University of Science and Technology,
Shanghai 200237, China
Received 8 October 2006; revised 4 November 2006; accepted 6 November 2006
Available online 11 November 2006
Abstract—A series of mono-1H-benzo[c,d]indol-2-one with different amine side chains and bis-1H-benzo[c,d]indol-2-one as novel
family of DNA intercalators were designed and synthesized, the contributions of aromatic chromophores and amine side chains
for DNA binding properties, for example, intercalation and electrostatic binding, respectively, were evaluated. Among them, A3
tailed with N,N-dimethylamino-ethyl-ethane-1,2-diamine showed selective anti-tumor activities against cell lines A549 and P388
with IC50 0.428 lm and 1.69 lm.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
to contribute to the binding. On the basis of the similar-
ity between naphthostyril or benzo[c,d]indol-2-one and
naphthalimide as famous DNA intercalative antitumor,
we wondered if the lactam group could interact with
DNA as well through the hydrogen bond between the
lactam oxygen and the backbone of nucleic acids and
also acted as intercalating chromophore moiety, which
was required for useful antitumor activity (Fig. 1).
The discovery of new compounds with antitumor activ-
ity became one of the most important goals in medicinal
chemistry. DNA-targeted chemotherapeutic agents were
the mainstay for cancer therapy.1,2 Most of DNA’s
interacting binders were characterized by the presence
of a planar chromophore, generally a tri- or tetracyclic
ring system with flexible basic side chains. The rationale
was mostly based on that the planar aromatic rings such
as naphthalimides, pyridocarbazole, acridine, anthracy-
clines, benzimidazo[1,2-c]quinazoline, and so on provid-
ed DNA binding affinity,3,4 the basic side chains with
multitude of cationic site charges supported the DNA
sequence selectivity and allowed the molecule to adjust
its structure to follow the groove, additionally contrib-
uting to interference with topoisomerases and perhaps
facilitating the cellular penetration of the molecule.5
Some meaningful examples were amonafide (a), elinafide
(b),6 SN 16713 (c), and DACA (d) (Fig. 1).7
Meanwhile, it is well known, N,N-dimethyl ethyldiami-
no group and its analogues commonly applied in
anticancer agents11 conjugated into the intercalating
N
O
O
H
H
N
N
N
O
N
O
N
O
O
(b) Elinafide
NH2
(a) Amonafide
N
Naphthostyril (e) derivatives played an important role
not only in good binding affinity for Thymidylate Syn-
thase (TS) but also favorable inhibitor against them
especially human TS.8,9 The region of the lactam group
on the tricyclic rings could form hydrogen bond9,10 with
the active site of TS, and their interaction was thought
NMe2
N
O
NH+
H
O
H3CO2SHN
+HN
N
(d) DACA
N
H
H
R1
OCH3
R2
HN
(c
) SN
1671
3
O
(e)enzyme thymidylate synthase inhibitor
Figure 1. Structures of some reported compounds.
*
Corresponding author. Tel.: +86 021 64253589; fax: +86 021
64252603; e-mail: xhqian@ecust.edu.cn
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.016

H. Yin et al. / Bioorg. Med. Chem. 15 (2007) 1356–1362
1357
O
gest intensity with 4.08 (log e), and the maximal emis-
sions were at 496 nm because of the two
chromophores in the molecule. All of the compounds
had low quantum efficiency.
A1 R = CH2CH2N(CH3)2
NR
A2 R = CH2CH2CH2NH(CH3)2
A3 R = CH2CH2NHCH2CH2N(CH3)2
O
Cl
O
O
NH
N
N
N
2.2. DNA interaction property
The binding properties between the compounds and
ctDNA were evaluated. With titration of ctDNA, the
mixed solutions were incubated at 25 ꢁC for 6 h, then
the UV–vis absorption and the fluorescent spectra were
measured, and the representative curves for compounds
A1 and A3 are illustrated in Figures 4 and 5,
respectively.
NO
2
A5
A4
Figure 2. Structures of the novel potential antitumor compounds.
moiety as the basic side chain. Therefore, we designed
two novel compounds A1 and A2 as potential anticancer
agents. Moreover, in recent years, much attention had
been paid to polyamines, which were positively charged
and present in prokaryotic and eukaryotic cells.12 The
polyamine cations had ‘high’ DNA affinity but were
‘loosely’ bound and could ‘read’ DNA.13 At molecular
level, it could modulate DNA conformation.14,15 Fur-
thermore, the intercalator conjugated with polyamine
cations had been reported to have better antitumor
activity and selectivity,16,11d so compound A3 was de-
signed as new kind of potential antitumor agents. Addi-
tionally, we were interested in bis-intercalators by
linking two planar rings with a linkage such as Elinafide,
and the compound A5 was therefore designed. In order
to find a novel hybrid compound, we also synthesized 1-
(2-chloro-ethyl)-5-nitro-1H-benzo[c,d]indol-2-one (A4),
which would be monocrosslinked with DNA as alkylat-
ing agents and resulted in inhibition of tumor growth.2
Their spectra, anti-tumor properties, and DNA-binding
affinities are also studied herein (Fig. 2).
In Figure 4, representative UV–vis spectra for com-
pounds A1 and A3 are shown in (a) and (b), in which
spectra 1 and 2 corresponded to their UV–vis spectra be-
fore and after the addition of ctDNA at the DNA/A3
molar ratio of 2.0, respectively. It was observed that
the addition of ctDNA to the solution of A1 induced
bathochromic shifts (1–7 nm) and hypochromicities
(4.8–10%), the bathochromic shifts (1–9 nm) and strong
hypochromicities (7.1–19.4%) were observed in the spec-
tra (b) of Figure 4, which showed A3 binding ctDNA
with higher affinity in the UV–vis absorption spectra.17
On the other hand, for A1 there was only one isosbes
(308 nm) in the UV–vis absorption, however for A3
three obvious isosbestic points at around 318, 409, and
545 nm were observed, which indicated that there were
different equilibriums for A1 and A3 between binding
and free drug. The reasons were probably due to the fact
that the different cationic side chains of A1 and A3
resulted in different charge-charge attraction with the
phosphate backbone of the DNA.
2. Results and discussion
From the analyses of the relationship between the fluo-
rescence intensities and the DNA concentrations by
nonlinear curve fitting methods, these significant chang-
es make the association steady constants of A1 and A3
with DNA available with 3.0 · 104 Mꢀ1 and
5.7 · 104 Mꢀ1, respectively,18 which indicated that A3
was more stable than A1 in binding to ctDNA.
2.1. Synthesis and spectral data
The synthetic route is shown in Figure 3. 1H-Benzo[c,-
d]indol-2-one was reacted at refluxing for 5 h with 1,2-
dibromoethane and 1,3-dichloropropane, respectively,
in acetonitrile in presence of K2CO3 as catalyst to give
intermediates (1) and (2), then (1) was reacted with dim-
ethylamine solution and N,N-dimethylethylenediamine
to give the target compounds A1 and A3. (2) was reacted
with dimethylamine solution to afford compound A2.
1H-Benzo[c,d]indol-2-one was stirred in concentrated
sulfuric acid at 0–5 ꢁC with HNO3 in concentrated sulfu-
ric acid added drop-wise and then stirred at room tem-
perature to give 3, then the solid was refluxed in 1,2-
dichloroethane to produce A4. Diethanolamine in chlo-
roform was added to SOCl2 in chloroform drop-wise at
0–5 ꢁC, then stirred at room temperature to afford
dichloroethylamine. The amine reacted with 1H-ben-
zo[c,d]indol-2-one to give A5. Structures of all these final
From (a) in Figure 5, it is found that the fluorescence of
A1 was quenched as most intercalators did with the
addition of increasing ctDNA concentration, and A3
exhibited enhanced intensities as (b) in Figure 5. It
was also very interesting that, for A3 when it was incu-
bated with ctDNA from 2 h to 6 h as shown in Figure
5(c), the fluorescence was quenched first, then began to
enhance. In order to provide further insight into the spe-
cial interaction, the fluorescence spectra of the mixture
were evaluated along with the increasing time as shown
in Figure 6. From (b) in Figure 6, it was found that the
fluorescence of A3 + DNA1 (10:1) was quenched after
incubated for 1 h, 2 h, then kept almost constant, and
the fluorescence began to enhance 4 h later. The sample
A3 + DNA2 (1:10) had almost the same trend as shown
in Figure 6(c), which indicated that the interaction was
possibly associated with thermodynamic equilibrium
process. Taken the structure into account, the reasons
were probably due to the fact that the tri-cyclic ring of
1
products were identified by H NMR, HRMS, and IR.
The aminoalkyl side chains were found to have slight ef-
fect on the UV–vis and fluorescent spectra of A1–A3
(Table 1). Their maximal absorptions were around
336 nm with different intensities. A5 showed the stron-

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H. Yin et al. / Bioorg. Med. Chem. 15 (2007) 1356–1362
Figure 3. Synthesis of target compounds. Reagents and conditions: (a) BrCH2CH2Br, acetonitrile, K2CO3, 81% yield; (b) dimethylamine solution,
reflux, 54% yield; (c) N,N-dimethylethylenediamine, acetonitrile, 68% yield; (d) ClCH2CH2CH2Cl, acetonitrile, 53% yield; (e) dimethylamine
solution, reflux, 44% yield; (f) HNO3, H2SO4, ice bath, 85% yield; (g) ClCH2CH2Cl, acetonitrile, K2CO3, 65%; (h) SOCl2, CHCl3, 82% yield; (i)
K2CO3, DMF, reflux, 10 h, 49% yield.
Table 1. Spectra dataa,b of A1–A5
Compound
UV kmax/nm (lge)
FL kmax/nm (U)
Excitation wavelength k/nm
A1
A2
A3
A4
A5
336.3 (3.60)
336.3 (3.94)
336.3 (3.56)
336.5 (2.57)
337.2 (4.08)
490.7 (0.0016)
495.6 (0.0015)
492.5 (0.0011)
493.7 (0.0003)
496.1 (0.0014)
336.3
336.3
336.3
336.5
337.2
a In absolute ethanol.
b With quinine sulfate in sulfuric acid as quantum yield standard (U = 0.55).
A3 was intercalated into base pair at first, then the long
and flexible polyamine side chain continued to modulate
itself to interact with ctDNA in an apropos conforma-
tion. Because the polyamine interacted readily with the
DNA phosphate anionic backbone, causing condensa-
tion by charge neutralization, this effect was also a key
first step in minimizing the size of foreign DNA for gene
therapy.19 On the other hand, Gershon et al.20 demon-
strated that at high and low charge ratios, the fluores-
cence intensity was at its extreme values. However, at
the key intermediate charge ratios during the DNA con-
densation process, fluorescence was time dependent.
Our results also proved that with moderate polyamine
length, the fluorescence of the compounds with ctDNA
was time dependent.
2.3. Damage of plasmid DNA property
In an effort to find further biological properties, we ex-
plored the damage of plasmid DNA property for A1–
A5, on the basis that small molecules characterized with
a DNA intercalator tailed with dimethylamino alkyldii-
mide could cleave supercoiled plasmid DNA.21 Herein,
Agarose gel electrophoresis was carried out at 70 ꢁC
for 4 h as shown in Figure 7.
Figure 7 shows that all of the compounds could cleave
the closed supercoiled DNA but with low efficiency.
A3 was found to have better ability than others, which
indicated that the polyamine side chain of A3 probably
contributed favorable biological activities.

H. Yin et al. / Bioorg. Med. Chem. 15 (2007) 1356–1362
1359
0.1
a
0.1
b
1
2
1
2
1
1
0.0
2
2
0.0
300
400
500
600
300
400
500
600
wavelength(nm)
wavelength(nm)
Figure 4. UV–vis absorption spectra of A1 (a), A3 (b) (10 lM, 25 ꢁC, 6 h), in 20 mM Tris–HCl buffer (pH 7.5) before (1) and after (2) the addition of
ctDNA. The concentration ratio of DNA/drug was 2:1. The arrows indicate the isosbes.
Figure 5. Fluorescence spectra of (a) A1 (6 h) and (b) A3 (6 h); (c) A3 (2 h) (10 lM) with ctDNA. The concentrations of ctDNA were increased from
0 to 400 lM in 20 mM Tris–HCl (pH 7.5) at 25 ꢁC. The insets indicated the relationship between the fluorescent intensity and the concentration of
ctDNA (lM).
2.4. Antitumor evaluation
cancer cell) and P388 (murine leukemia cell), respective-
ly, listed in Table 2. The IC50 represents the drug con-
centration (lM) required to inhibit the cell growth by
50%.
The antitumor activities of compounds A1–A5 were
evaluated in vitro against cell lines A549 (human lung

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H. Yin et al. / Bioorg. Med. Chem. 15 (2007) 1356–1362
35
30
25
20
15
10
5
a
A3
A3+CD1
A3+CD2
0
450
500
550
600
650
wavelength(nm)
c
23.0
22.5
22.0
21.5
21.0
20.5
20.0
19.5
b
32
A3+DNA2 (1:10)
A3+DNA1 (10:1)
31
30
29
28
27
0
2
4
6
8
10
0
2
4
6
8
10
Time (hr)
Time (hr)
Figure 6. Fluorescence spectra of (a) A3 before and after the addition of ctDNA: A3 + DNA1 (1:10), A3, A3 + DNA2 (10:1) incubated at 25 ꢁC for
2 h. (b) A3 + DNA1 incubated for 1–10 h in 20 mM Tris–HCl (pH 7.5) at 25 ꢁC. (c) A3 + DNA2 incubated for 1–10 h in 20 mM Tris–HCl (pH 7.5) at
25 ꢁC.
Table 2. Cytotoxicity of compounds A1–A5 against cell lines A549a
and P388b
Compound
Cytotoxicity (IC50, lm)
A549
300
22.9
0.489
P388
A1
A2
A3
A4
A5
79.4
7.76
1.69
17.8
13.8
52.5
29.5
a Cytotoxicity (CTX) against human lung cancer cell (A549) was
measured by sulforhodamine B dye-staining method.16b
b CTX against murine leukemia cells (P388) was measured by micro-
culture tetrazolium–formazan method.11d
Figure 7. 1% Agarose gel electrophoresis assay of plasmid PBR
322DNA cleaved by compounds A1–A5. Lane 1, DNA marker; lane 2,
200 ng pBR322 DNA (incubated at 70 ꢁC for 4 h); lanes 3–7, 200 ng
pBR322 DNA and A1, A2, A3, A4, A5 (100 lM), respectively,
incubated at 70 ꢁC for 4 h in 20 mM Tris–HCl (pH 7.5).
reflecting the excellent selectivity for a special human
lung cell type. The different antitumor activities
showed that the modification of such agents for potent
antitumor drugs seemed to be dependent on structures
of the amine side chains. Specially, the high antitumor
activity of compound A3, which was incorporated in
additional ethyleneamine groups in the side chain,
was probably associated with the polyamine-induced
DNA condensation in a suitable conformation, so
the selectivity and biological activity of the compounds
were improved.20
It is shown in Table 2 that A1 had low activity, A2,
A4, and A5 (bis-1H-benzo[c,d]indol-2-one) displayed
increased potency. They were found to be more cyto-
toxic against P388 than A549. On the other hand,
A3 was the strongest growth inhibitor against A549
and P388 with IC50 of 0.489 and 1.69 lM, respectively.
Interestingly, it was also found that the cytotoxicity of
this compound against A549 was higher than P388,

H. Yin et al. / Bioorg. Med. Chem. 15 (2007) 1356–1362
1361
3. Conclusion
the dimethylamine solution (30mL) and kept refluxing
for 10 h, extracted with methylene chloride, the oil layer
was gathered and evaporated in vacuum, separated on
silica gel column chromatograph using eluent ethyl ace-
tate/petroleum ether (1:1, v/v) to give the yellow solid
product A1 0.54 g, 54% yield. Mp 134–135 ꢁC. 1H
NMR (CDCl3) d (ppm): 2.35 (s, 6H, CH3N), 2.69 (t,
J = 8.02 Hz 2H, CH2N (CH3)2), 4.05 (t, J = 7.06 Hz,
2H, CH2NCO), 6.95 (d, J = 6.93 Hz, 1H, 6-H), 7.46 (t,
J = 7.21 Hz, 1H, 7-H), 7.52 (d, J = 8.43 Hz, 1H, 8-H),
7.69 (t, J = 7.13 Hz, 1H, 2-H), 7.99 (d, J = 8.10 Hz,
1H, 1-H), 8.05 (d, J = 6.96 Hz, 1H, 3-H). IR (KBr):
2957, 2890, 1684, 1625, 1470, 770 cmꢀ1; HRMS:
C15H16N2O calculated: 240.1263; found: 240.1263.
In summary, we synthesized a novel class of compounds
(A1–A5) featuring tricyclic ring with different side
chains. Their antitumor activities together with DNA
binding affinities were evaluated, and interaction prop-
erty with ctDNA (represented as A1 and A3) had been
further studied. Also, their damage of supercoiled
DNA property was explored. Among the compounds,
A3 exhibited not only the high antitumor activity and
the good selectivity, but also distinctive interaction with
ctDNA. This work also provided the influence of poly-
cation side chains on their biological activities, which re-
vealed that could be advantageous modulation for
antitumor agents.
4.3.2. 1-[2-(2-Dimethylamino-ethylamino)-ethyl]-1H-ben-
zo[c,d]indol-2-one (A3). N,N-Dimethylethylenediamine
(0.31 g, 3.6 mmol) and K2CO3 (0.1 g) was added to
4. Experimental
1(bromo-ethyl-1H-benzo[c,d]indol-2-one)
(0.82 g,
4.1. Materials and methods
3 mmol) in acetonitrile 30 ml, the mixture was stirred
and refluxed for 5 h, then filtered and evaporated, sepa-
rated on silica gel with eluent ethyl acetate/CHCl3 (5:1,
v/v), yellow solid product A3 was obtained 0.58 g, yield
All the solvents were of analytical grade. The closed
supercoiled pBR322 DNA was purchased from Takara
Biotech Co. Ltd (Shanghai). H NMR was measured
1
1
68%. Mp 96–97 ꢁC. H NMR (CDCl3) d (ppm): 2.19 (s,
on a Bruker AV-500 spectrometer with chemical shifts
reported as parts per million (in acetone-d6/DMSO-d6/
CDCl3, TMS as an internal standard). Mass spectra
were measured on a HP 1100 LC–MS spectrometer.
Melting points were determined by an X-6 micro-melt-
ing point apparatus and are uncorrected. Absorption
spectra were determined on PGENERAL TU-1901
UV–VIS Spectrophotometer.
6H, CH3N), 2.41 (t, J = 8.4 Hz, 2H, CH2N CH3), 2.76
(t, J = 8.0 Hz, 2H, CH2NH), 3.03 (t, J = 8.8 Hz, 2H,
CH2CH2NCO), 4.05 (t, J = 9.2 Hz, 2H, CH2NCO),
6.97 (d, J = 9.2 Hz, 1H, 6-H), 7.45 (d, J = 9.2 Hz, 1H,
8-H), 7.51 (t, J = 11.2 Hz, 1H, 7-H), 7.69 (t,
J = 9.6 Hz, 1H, 2-H), 8.00 (d, J = 8.4 Hz, 1H, 1-H),
8.05 (d, J = 9.6 Hz, 1H, 3-H). IR (KBr): 3310, 2925,
2920, 1689, 1601, 756 cmꢀ1; HRMS: C17H21N3O calcu-
lated: 283.1685; found: 283.1683.
4.2. Spectrophotometric titration experiments
4.3.3. 1-(3-Dimethylamino-propyl)-1H-benzo[c,d]indol-2-
one (A2). 1H-Benzo[c,d]indol-2-one (1.69 g, 0.01 mol),
1,3-dichloropropane (2 g, 0.012 mol) and K2CO3
(0.05 g) were refluxed in acetonitrile (50 ml) for 5 h, then
filtered and evaporated in vacuum, separated on silica
gel column chromatograph with eluent ethyl acetate/pe-
troleum (3:1, v/v) to give 2(chloro-propyl-1H-benzo[c,-
d]indol-2-one) 1.3 g, yield 81%. 2 (0.82 g, 3 mmol) was
then added to the dimethylamine solution (2 mmol)
and kept refluxing for 10 h, extracted with methylene
chloride, the oil layer was gathered and evaporated in
vacuum, separated on silica gel column chromatograph
with ethyl acetate/petroleum ether = 1:1, v/v) to give
yellow solid product A2 0.22 g, yield 44%, Mp 122–
123 ꢁC; 1H NMR (CDCl3) d (ppm): 2.26 (t, J =
6.91 Hz, 2H, 2-H, CH2CH2N), 2.64 (s, 6H, CH3N),
3.03 (s, 2H, CH2NCH3), 4.09 (t, J = 6.92 Hz, 2H,
CH2NCO), 7.32 (d, J = 7.05 Hz, 1H, 6-H), 7.55 (t,
J = 7.16 Hz, 1H, 7-H), 7.62 (d, J = 8.43 Hz, 1H, 8-H),
7.81 (t, J = 7.16 Hz, 1H, 2-H), 8.02 (d, J = 6.92 Hz,
1H, 1-H), 8.15 (d, J = 8.11 Hz, 1H, 3-H); IR (KBr):
2949, 2855, 1690, 1470, 1330, 780 cmꢀ1; HRMS:
C16H18N2O calculated: 254.1419; found: 254.1419.
The concentrations of compounds and calf thymus
DNA were 10 lM and 0–400 lM, respectively, in
20 mM Tris–HCl buffer, pH 7.5, and DMSO (1% by vol-
ume), the solution in a final volume of 10 ml, which was
used for fluorescence-quenching experiments. An equi-
librium period of 6h for constant stirring at 25 ꢁC in
the dark of the mixed solution was allowed before
recording each spectrum. The association constants
(Ka’s) were derived according to the equation
I = I0 + {(I1 ꢀ I0/2[Q]0)} · {([DNA]0 + [Q]0 + 1/Ka)2 ꢀ
4[DNA]0[Q]0}1/2}, wherein I0, I, and I represent the
1
fluorescence intensities of compounds alone, the sample,
and DNA totally bound, respectively. [DNA]0 and [Q]0
were the initial analytical concentrations of DNA and
the agents, respectively.
4.3. Synthesis
4.3.1. 1-(2-Dimethylamino-ethyl)-1H-benzo[c,d]indol-2-
one (A1). 1H-Benzo[c,d]indol-2-one 0.35 g (2 mmol),
1,2-dibromo ethane 2.5 ml (0.03 mol), and potassium
carbonate anhydrous 0.05 g were added to acetonitrile
20 ml, the mixture was stirred and refluxed for 5 h, then
filtered and evaporated in vacuum, separated on silica
gel column chromatograph with eluent ethyl acetate/pe-
troleum ether (3:1, v/v) to give the intermediate 1(bro-
mo-ethyl-1H-benzo[c,d]indol-2-one) (0.45 g, yield 81%).
Then the intermediate (0.82 g, 3 mmol) was added to
4.3.4. 2-(2-Chloro-ethyl)-6-nitro-2H-acenaphthylen-1-one
(A4). 1H-Benzo[c,d]indol-2-one (4.0 g, 0.025 mol) was
stirred in concentrated sulfuric acid (20 ml, 98%) at
0–5 ꢁC. The mixed acids (HNO3 1.73 ml 0.025 mol,
d = 1.4, 6.5%) in concentrated sulfuric acid (5 ml) was

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H. Yin et al. / Bioorg. Med. Chem. 15 (2007) 1356–1362
added drop-wise, keeping the temperature below 20 ꢁC,
then stirred at room temperature for 1.5 h. The reactant
was then poured into water (400 ml), filtered, and
washed with water to get 3 (4-nitro-1H-benzo[c,d]in-
dol-2-one) 4.5 g, 85% yield; 3 (0.85 g, 4 mmol) was
refluxed with 1,2-dichloroethane 5 ml (0.06 mol) and
K2CO3 (0.1 g) in acetonitrile (30 ml) for 2 h, cooled,
removed the solvent, and separated on silica gel chro-
matograph with ethyl acetate/petroleum ether (1:1, v/v)
to afford the yellow needle product A4 0.7 g, yield
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1
65%. Mp 217–218 ꢁC. H NMR (CDCl3) d (ppm): 4.03
(t, J = 6.11 Hz, 2H, CH2NCO), 4.4 (t, J = 6.12 Hz, 2H,
CH2Cl), 7.44 (d, J = 8.09 Hz, 1H, 5-H), 8.10 (td,
J1 = 7.13 Hz, J2 = 1.31 Hz, 1H, 6-H), 8.22 (d,
J = 6.99 Hz, 1H, 7-H), 8.69 (d, J = 8.09 Hz, 1H, 2-H),
8.97 (d, J = 8.50 Hz, 1H, 1-H), IR (KBr): 3093, 2910,
1731, 1513, 1326, 749 cmꢀ1; HRMS: C13H9N2O3 35Cl
calculated: 276.0302; found: 276.0294; C13H9N2O3 37Cl
calculated: 278.0272; found: 278.0284.
4.3.5. Di-1H-benzo[c,d]indol-2-one-ethyl-amine (A5).
Diethanolamine (15.75 g, 0.14 mol) in CHCl3 20 ml
was added to SOCl2 in CHCl3 30 ml drop-wise at
0–5 ꢁC, then stirred at room temperature for 3 h,
the excessive SOCl2 was evaporated in vacuum to
give the white solid N,N-dichloroethylamine, 17.5 g,
82%
yield.
1H-Benzo
[c,d]indol-2-one
(2.0 g,
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0.01 mol), N,N-dichloroethylamine (0.6 g, 0.004 mol),
and K2CO3 (0.1 g) were stirred and refluxed in
DMF 30 ml for 10 h, the mixture was then poured
into ice-water to precipitate yellow solid, filtered,
and separated on silica gel column chromatograph
with
eluent
ethyl
acetate/ethanol/chloroform
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(1:0.4:0.4, v/v/v) to give pale yellow product A5
0.8 g, yield 50%. Mp 144–145 ꢁC. 1H NMR (CDCl3)
d (ppm): 3.05 (t, J = 6.36 Hz, 2H, CH2NH), 3.99 (t,
J = 6.33 Hz, 2H, CH2NCO) 7.09 (d, J = 7.09 Hz,
1H, 6-H), 7.43 (t, J = 8.39 Hz, 1H, 7-H), 7.54 (d,
J = 8.44 Hz, 1H, 8-H), 7.77 (t, J = 7.76 Hz, 1H, 2-
H), 7.96 (d, J = 6.91 Hz, 1H, 1-H), 8.10 (d,
J = 8.09 Hz, 1H, 3-H), IR (KBr): 3307, 2925, 2851,
1696, 1625, 773 cmꢀ1; HRMS: C26H21N3O2 calculat-
ed: 408.1712 (M+1), 409.1746 (M+2), 430.1531
(M+Na); found: 408.1703 (M+1), 409.1774 (M+2),
430.1558 (M+Na).
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4.4. Cytotoxicity in vitro evaluation
The cytotoxicities of the synthesized compounds were
evaluated by The National Center for Drug Screening.
Acknowledgments
The National Basic Research Program of China
(2003CB114400), National Natural Science Foundation
of China, and The Science and Technology Foundation
of Shanghai supported this study.
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