Novel DNA intercalators without basic side chains as efficient antitumor agents: Design, synthesis and evaluation of benzo-[c,d]-indol-malononitrile derivatives

Article abstract of DOI:10.1016/j.bmc.2010.03.017

Several 2-(substituted benzo[c,d]indol-2(1H)-ylidene)malononitriles have been designed and synthesized. Their DNA binding, antitumor and DNA damaging properties were evaluated. All the compounds exhibited efficient antitumor activities with preference to

Full text of DOI:10.1016/j.bmc.2010.03.017

Bioorganic & Medicinal Chemistry 18 (2010) 3279–3284
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel DNA intercalators without basic side chains as efficient antitumor
agents: Design, synthesis and evaluation of benzo-[c,d]-indol-malononitrile
derivatives
a
Xiaolian Li a, Qianqian Wang a, Yang Qing c, Yanjie Lin a, Yingli Zhang a, Xuhong Qian a,b, , Jingnan Cui
*
a State Key Laboratory of Fine Chemicals, Dalian University of Technology, PO Box 89, 158 Zhongshan Road, Dalian 116012, China
b Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, PO Box 544, 130 Meilong Road, Shanghai 200237, China
c Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Several 2-(substituted benzo[c,d]indol-2(1H)-ylidene)malononitriles have been designed and synthe-
sized. Their DNA binding, antitumor and DNA damaging properties were evaluated. All the compounds
exhibited efficient antitumor activities with preference to be against the tumor cell line 7721 rather than
the tumor cell line MCF-7. Compound 1f could intercalate into DNA entirely presumably by the good con-
jugation of carbonyl group with benzo[c,d]indol moiety. What’s more, 1f exhibited potent toxicity against
Received 27 December 2009
Revised 7 March 2010
Accepted 9 March 2010
Available online 21 March 2010
MCF-7 cells with IC50 at 0.003
l
M and against 7721 cells at 0.115
lM, respectively.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Benzo[c,d]indol-2(1H)-one
Antitumor agents
DNA intercalator
DNA cleavage
1. Introduction
(Fig. 1h). Many researches focus on optimizing its structure to
broaden its application. Appelt14 reported that naphthostyril
(Fig. 1e) derivatives showed good binding affinity for thymidylate
synthase (TS) as well as amazing inhibitory activity against them.
By substituting the hydrogen atom of lactam with different cylic
As cancer cells are highly proliferative tissues, the DNA becomes
one of the most promising biological targets of developing antitu-
mor agents to specifically constrain tumor cell growth.1 In recent
years, much attention has been paid to design and synthesize
new and efficient DNA-targeted antitumor agents.2,3 DNA-interca-
lating molecules are those which intercalate DNA base pairs via
electron-deficient chromophores with flexible side chains. These
specially designed groups confer DNA sequence selectivity and al-
low aromatic heterocycles to position at proper sites or interact
with topoisomerases so as to interfere with DNA replication and
transcription.4–6 Several reported bifunctional DNA binders, which
carry two such functional groups, have proven this mechanism by
the increase of DNA binding affinity and biological activity.7,8 DNA
intercalators have been investigated for more than 40 years,9 suc-
cessful examples including (a) amonafide,10 (b) DACA,11 (c)8-oxo-
8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile12 and (d) ametant-
rone13 (Fig. 1).
amines, López-Rodrlguez15 designed a series of 5-HT7R antagonists
´
(Fig. 1f), which exhibited satisfactory affinities and pharmacologi-
cal properties. Our group16 synthesized a novel DNA intercalator
tailing with straight amine side chain (Fig. 1g), which manifested
good DNA binding and anticancer activities.
In terms of the biological activities of benzo[c,d]indolone deriv-
atives, regular design concept is that the lactam group on the tricy-
clic rings can form hydrogen bond with its targets (protein or the
backbone of nucleic acids) and its planarity would facilitate its
embedding to DNA base pairs. Planarity is one of the most impor-
tant factors of intercalators.17 However, most researches focused
on restructuring of sites of 1 and 3 (Fig. 1h), but paid little attention
to the alteration of the carbonyl group (site 2). Although the
benzo[c,d]indolone derivatives were widely used as protein inhib-
itors, their interaction with DNA was rarely reported. Therefore,
our group focused on the modification of the carbonyl group (site
2) to design and synthesis of novel DNA-targeted benzo[c,d]indo-
lone derivatives. Unlike reported DNA intercalators with amine
side chains, compounds we report here only carry alkyl groups in
an attempt to expand the diversity of intercalators’ structures
and substituting groups.
Early in 1920s, benzo[c,d]indol-2(1H)-one derivatives were
originally developed as dyes, and then used as electronic typing
materials. Until recently, their prominent bioactivities have been
discovered. Benzo[c,d]indol-2(1H)-one has three active sites, 1–3
* Corresponding author. Tel.: +86 21 64253589; fax: +86 21 64252603.
E-mail address: xhqian@ecust.edu.cn (X. Qian).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.017

3280
X. Li et al. / Bioorg. Med. Chem. 18 (2010) 3279–3284
N
H
N
O
O
HN
HN
OH
OH
O
N
O
N
N
O
N
H
NH2
(a) Amonafide
N
H
(b) DACA
(d) ametantrone
O
CN
N
R1
HN
R2
O
(e)enzyme thymidylate synthase inhibitor
(c) 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile
site2
site I
O
HN
N
O
O
N
n
N
Y
N
n=1,3,4,5
Y=N or CH
site3
(h)Benzo[c,d]indol-2(1H)-one
(f) 5-HT7R Antagonism.
(g)1-(2-(dimethylamino)ethyl)benzo[cd]indol-2(1H)-one
Figure 1. Structures of some reported compounds.
Cyano group is known to be a good photophore for one-electron
DNA oxidation due to its forceful electron withdraw ability.18
Therefore, we introduced dicyano moiety to the site of oxygen
atom, not only enlarging the conjugated plane but also reducing
electron density so as to facilitate binding of the electron-rich
DNA. Although amine side chains have been widely used as essen-
tial toxic groups in DNA intercalators,19 we demonstrated here
electron-deficient chromophore with non-basic side chains (1a–
g) also behaved as efficient antitumor agents with intercalation
to DNA. Several unsaturated hydrocarbons were linked to nitrogen
atom (1a–c) to increase electrostatic interaction between com-
pounds and acidic DNA molecules through the similar mechanism
as amine side chains follows. Meanwhile, compounds 1d–e were
synthesized to behave as potential DNA alkylating agents and re-
sult in inhibition of tumor growth by cross-linking of basic
groups.20 Furthermore, the intercalator conjugated with acylamide
had been reported to have better antitumor activity,21–23 so com-
pound 1f–g were designed as new anticancer agents by changing
lactam to acylamide (Fig. 2).
2. Results and discussion
2.1. Synthesis and spectra data
Preparation of the compounds (1a–g) was shown in Figure 2.
Benzo[c,d]indol-2(1H)-one was reacted with propanedinitrile in
toluene at 100 °C in the presence of POCl3 for 4 h to give interme-
diate 2, which was a Knoevenagel Condensation. Then 2 under-
went three different routes to give all the compounds. (Route 1):
2 was reacted with the corresponding bromo-reactants in acetoni-
trile with K2CO3 as catalyst to afford compounds 1a–c; (route 2):
dihaloalkane was used as reactant as well as solvent to combine
with 2 to result in compounds 1d–e; (route 3): acyl chloride was
added to the mixture of 2 and acetic acid at 0–5 °C, then heating
to reflux for 5 h to give 1f–g. Structures of all these final products
were confirmed by 1H NMR, HRMS and IR.
The different substitutes were found to have slight effect on the
UV–vis spectra, with maximal absorptions around 440 nm with
different intensities (Table 1). One exception is that 1b exhibited
NC
R1
CN
CN
CN
1a R1=CH2C
1b R1=CH2C
CH
N
N
ii
1c R1=CH2CH=CH2
NC
NC
NC
O
R2
CN
HN
HN
iii
N
1d R2=CH2CH2Br
i
1e R2=CH2CH2CH2Cl
2
1
O
R3
iv
C CH3
1f R3=
1g R3=
N
O
C
Figure 2. Structures and synthesis of target compounds. Regents and conditions: (i) CNCH2CN, toluene, POCl3, 100 °C, 83% yield; (ii) correspond bromo-reactants, acetonitrile,
K2CO3, 80 °C; (iii) dihaloalkane, K2CO3, 100 °C; (iv) acyl chloride, acetic acid, reflux.

X. Li et al. / Bioorg. Med. Chem. 18 (2010) 3279–3284
3281
C1a/10-6M
Table 1
0.25
UV–vis spectra of the compounds
c=20
R=0.4
c=10
R=0.2
c=5
R=0.1
c=2
R=0.04
c=1
R=0.02
Compound
kmax (nm)
log n
n (104 L/mol cm)
0.20
1a
1b
1c
1d
1e
1f
446
377
445
452
446
440
447
4.21
3.94
4.26
4.24
4.03
4.30
4.30
1.62
0.87
1.82
1.74
1.07
1.99
0.15
0.10
1g
1.99
In absolute DMSO.
0.05
remarkable blue shift to 377 nm due to the strong electron-with-
drawing ability of cyano group, increasing the energy of electronic
transition.
0.00
300
350
400
450
500
550
600
650
wavelength
2.2. Studies on mechanism of interaction with DNA
Figure 4. Absorbance changes after interaction of 1a and calf thymus DNA
(CDNA = 50 M, R = Ccom/CDNA, R value varies at 0.02, 0.04, 0.1, 0.2, 0.4).
l
The affinities of the target compounds for CT DNA were deter-
mined by spectroscopic technique and viscosity measurement.
its absorption, this was hypochromicity; when R >0.1, hyper-
chromicity occurred. It could be concluded that different binding
mode existed during the increase of 1a’s concentration. At low
compound/DNA ratio, a complete intercalation mode dominated
the whole process, so the intensity was reduced; at high com-
pound/DNA ratio, part of the compounds might just stack along
DNA surface which induced hyperchromicity. We also used 1a to
calculate binding constant, reaching 1.65 Â 105 molÀ1 L, suggesting
good binding ability.
2.2.1. UV–vis spectra
It is widely accepted that if the compound can intercalate DNA,
the UV–vis curve of their complex will be induced bathochromic
shift and hypochromicity.24,25 The UV–vis spectra were measured
in two ways: (1) Fixed the concentration of compounds
(Ccom = 25
lM), increasing the concentration of CT DNA; (2) kept
DNA at 50
lM, changing compounds’ concentration.
In the first way, as the DNA concentration was increased, all the
compounds showed significant hypochromicities and slight batho-
cromic shifts (1–3 nm). The most attractive spectrum was 1f, 2-(1-
acetylbenzo[c,d]indol-2(1H)-ylidene)malononitrile (Fig. 3). When
DNA was added, 1f was forced to lose all of its own absorption
peaks. The reason could be that the acetyl group was conjugated
with the whole aromatic heterocycle, leading 1f to be a big planar
rigid system which could insert into the double helix entirely, so
that its spectrum could not be observed. However, the molecular
volume of 1g was bigger than that of 1f, so it did not have the same
behavior. In the second way, with titration of DNA at 50 lM, bath-
ochromic shift and hypochromicity could be found in all the com-
pounds. One typical curve was 1a (Fig. 4).
When binding with DNA, distinguishable changes happened to
1a with different relative concentration. When R = 0.1, the maxi-
mal isosbestic point was at 452 nm; when R <0.1, DNA decreased
The UV–vis spectra indicated that the binding mode for com-
pounds with DNA was of intercalation.
2.2.2. Fluorescence spectra
Ethidium bromide (EB) has no fluorescence itself, but when
incubated with DNA, it will intercalate exclusively into DNA and
its fluorescence intensity will be enhanced greatly. If the com-
pound can replace EB from DNA, its fluorescence will be quenched,
indicating that the drug has stronger intercalation ability than
EB.26 1a was employed to evaluate the potential affinity capabili-
ties (Fig. 5). It can be seen from the curve 1aD in Figure 5, 1a ap-
peared little change in fluorescence while interacting with DNA,
eliminating the effect of 1a on the change of fluorescence. Then,
1a was added to the complex of EB and DNA, distinctive decrease
happened to the intensity of EB, which gave another proof for
the deducement of intercalation model.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1f
15
R=5
E
R=2.5
R=1.25
R=0.8
R=0.6
R=0.5
R=0.4
ED
1a(3)
10
5
1aD(4)
1aED(5)
0
-5
-10
350
400
450
500
550
600
650
400
450
500
550
600
650
700
750
800
850
Wavelength(nm)
Wavelength(nm)
Figure 3. Absorbance changes after interaction of 1f and calf thymus DNA
(Ccom = 25 M, R = Ccom/CDNA, R value varies at 5, 2.5, 1.25, 0.8, 0.6, 0.5, 0.4).
Figure 5. Fluorescence spectrum of E, ED, 1a, 1aD and 1aED [E: EB only; ED:
EB+DNA; 1aD: 1a+DNA; 1aED: 1a+EB+DNA (E = 5 M, D = 10 M, 1a = 5 M)].
l
l
l
l

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X. Li et al. / Bioorg. Med. Chem. 18 (2010) 3279–3284
2.2.3. Viscosity measurement
In the absence of crystal figures, viscosity measurement is the
most effective method to explore the binding mode of drug and
DNA which is more persuasive than spectra.27 Generally, when
the molecule intercalates into DNA, the distance between adjacent
base pair will be largened, causing lengthening, unwinding and
stiffening of the helix and usually accompanied by an increases
in solution viscosity. When drug interacts with DNA in other ways
(electrostatic attraction or groove binding), the DNA length will re-
main and the viscosity of the complex solution has minor change.
Take 1a as an example to study its effect on CT DNA viscosity. In
Figure 6, the increase in viscosity of DNA solution was observed
versus the increase in concentration of 1a, implying intercalation
binding by further evidence.
Figure 7. The photocleavage of plasmid DNA pBR322 (25 ng/
1a–g, in 20 mM Tris–HCl, pH 7.5 under light irradiation for 3 h. Lane 1: DNA alone;
lane 2: DNA under light irradiation for 3 h; lane 3–9, DNA plus 50
under light irradiation for 3 h.
lL) by compounds,
lM compounds
2.3. Damage of plasmid DNA
DNA damaging activities of 1a–g were evaluated in 20 mM
Tris–HCl (pH 7.5) in the presence of supercoiled plasmid DNA
pBR322 under dark for 12 h as well as light irradiation for 3 h. Ex-
posed under 365 nm light, all the compounds could cleave the
closed supercoiled DNA into relaxed, open circular form (Form II)
and compounds 1a–c could cleave closed supercoiled plasmid
DNA into linear form (Form III) (Fig. 7). These results seemed to
be in accordance with our prediction that under photo-irradiation,
the electron-deficient compounds owing to the dicyano group
could facilitate the electron transferring from DNA to themselves,
causing oxidation of DNA.18 The compounds and DNA formed D–
A complex in this process, where compounds acted as electron
acceptor (A) while DNA as electron donor (D).28 Thus, it might be
concluded that the cleaving process was charge-transfer
mechanism.
More interestingly, the reaction mixtures containing closed
supercoiled plasmid DNA and compounds at 4°C for 12 h under
dark, more linear DNA (Form III) were observed (Fig. 8). This phe-
nomenon hinted that photo-irradiation might not the essential
condition for cleavage. It might be caused by hydrolysis mecha-
nism, which was similar to our previous report using naphthali-
mide derivatives as DNA cleavers.29
Figure 8. The cleavage of plasmid DNA pBR322 (25 ng/
the buffer of Tris–HCl (20 mM, pH 7.5) at 4 °C for 12 h under no photo-irradiation.
Lane 1: DNA alone no h , lane 2–8: DNA plus 50 M compounds at 4 °C for 12 h.
lL) by compounds, 1a–g, in
m
l
The IC50 values were listed in Table 2 and revealed that all the com-
pounds possessed desirable antitumor activities with higher effi-
ciency against 7721 cells than MCF-7 cells, except that 1f was
the strongest inhibitor against MCF-7 cells than 7721 cells with
IC50 of 0.003 lM and 0.115 lM, respectively. The IC50 of most of
the compounds could be as low as 10À7 M against 7721 cells, 10-
times lower than most reported compounds with IC50 of around
10À6 M.
The strong electron-withdrawing ability of the dicyano group
played an important role in anticancer effect. The moderate activ-
ities of 1d and 1e might be the result of alkylation which was con-
sistent with previous report,30 excellent leaving groups such as
chloride and bromide are required for cytotoxicity in an alkylation
mechanism. The acetyl group of 1f could form hydrogen bond with
DNA bases, so it could interact with DNA forcefully and showed the
most outstanding cytotoxic ability, indicating molecules that have
intense interaction with DNA often put up favorable antitumor
activities. The phenyl of 1g could conjugate with carbonyl to re-
duce its competence of forming hydrogen bond and its volume
was bigger than 1f, therefore, its antitumor activity was much low-
er than 1f. Moreover, the good potency of 1a–c conferred new
functions to unsaturated aliphatic side chains, though the exact
mechanism was still under study.
2.4. Cytotoxicity
The antitumor activity of the compounds, 1a–g, were evaluated
in vitro against MCF-7 cells (human mammary cancer cell) and
7721 cells (human liver cancer cell) by MTT tetrazolium assay.
The basic side chains in many anticancer agents such as naph-
thalimide, pyridocarbazoles and acridines, were well known strat-
egy to enhance intercalator’s antitumor activity. However, the
compounds without N-dialkyl group basic side chains, we
presented here, also exhibited good bioactivity, suggesting that
this series of compounds hold entirely different structure–activity
relationship (SAR) from traditional DNA intercalators.
Table 2
Antitumor activities of compounds against 7721 and MCF-7 cells
Compound
Cytotoxicity (IC50
,
lm)
7721
MCF-7
1a
1b
1c
1d
1e
1f
0.728 0.02
0.260 0.07
0.175 0.01
0.210 0.05
0.354 0.08
0.115 0.06
8.80 0.23
12.5 0.13
13.5 0.15
4.73 0.07
5.38 0.14
6.93 0.09
0.003 0.0002
15.3 0.27
Figure 6. Effect of increasing amounts of compounds 1a on the relative viscosity of
CT DNA at 25 °C. [DNA] = 100
lM in Tris–HCl (30 mM, pH 7.5). g is the viscosity of
1g
DNA in the presence of the compounds and g0 is the absence of the compounds.

X. Li et al. / Bioorg. Med. Chem. 18 (2010) 3279–3284
3283
In summary, comparing with existing compounds, our com-
pounds have two distinct novelty in anticancer effects: (1) the
introduction of dicyano group was the first report on modifying
the carbonyl group of benzo[c,d]indol-2(1H)-one to act as DNA poi-
sons. Its forceful electron-withdrawing ability played an important
role in anticancer effect. (2) These compounds contained no basic
side chains which had been widely used as functional groups in
many DNA intercalators, however, they also showed attractive bio-
logical activities, which might expand the diversity of intercalators’
structures and substituting groups.
Stirred and refluxed at 80 °C until the reaction was completed.
The mixture was cooled, dropped into ice-water, extracted with
CH2Cl2 and dried with MgSO4. The crude product was separated
on silica gel chromatography.
4.2.2.1. 2-(1-(Prop-2-ynyl)benzo[c,d]indol-2(1H)-ylidene)malon-
onitrile (1a). Yield 70%, mp: >300 °C. 1HNMR (DMSO-d6,
400 MHz): d (ppm): 2.50 (s, 1H), 5.23 (s, 2H), 7.24 (d, J = 7.2 Hz,
1H), 7.62 (dd, J1 = 8.0 Hz, J2 = 7.2 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H),
7.82 (dd, J1 = 7.6 Hz, J2 = 7.6 Hz, 1H), 8.14 (d, J = 8.0 Hz, 1H), 8.69
(d, J = 7.6 Hz, 1H. IR (KBr cmÀ1) 2214, 3415, 3290, 600, 675. HRMS
(ESI) m/z (M+H)+ calcd for C17H9N3 255.0796; found: 255.0796.
3. Conclusions
4.2.2.2. 2-(1-(Cyanomethyl)benzo[c,d]indol-2(1H)-ylidene)mal-
ononitrile (1b). Yield 68%, mp: >300 °C. 1HNMR(DMSO-d6,
400 MHz): d (ppm): 4.36 (s, 2H), 7.60 (dd, J1 = 6.8 Hz, J2 = 7.6 Hz,
1H), 7.68 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.2 Hz, 1H), 7.75 (dd,
J1 = 8.4 Hz, J2 = 7.2 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 8.01 (d,
J = 6.8 Hz, 1H). IR (KBr cmÀ1) 2250, 2209, 2196, 2178, 3435. HRMS
(ESI) m/z (M+H)+ calcd for C16H8N4 256.0749; found: 256.0749.
The strategy we report here was the first one on restructuring
the carbonyl group of Benzo[c,d]indol-2(1H)-one, which had al-
ready been used as DNA-targeted drugs. Though they did not have
traditional basic side chains, they put up well chemical and biolog-
ical activities. Their DNA binding properties were evaluated and
manifested that the compounds could efficiently intercalate CT
DNA, giving a presumption that the flat conjugated aromatic struc-
ture of compounds made the large part contribution to intercalate
DNA base pairs. DNA damaging assay using plasmid DNA pBR322
showed all the compounds were efficient to photocleave super-
coiled DNA, while 1a–f could photocleave supercoiled DNA into
linear forms. What’s more, they could cleave DNA in darkness.
The tumor cell growth inhibitory test using 7721 and MCF-7 cell
lines exhibited that these compounds exhibited excellent antitu-
mor activities and IC50 value of 1f against MCF-7 cells was as low
as 3 nM.
4.2.2.3. 2-(1-Allylbenzo[c,d]indol-2(1H)-ylidene)malononitrile
(1c). Yield 60%, mp: 223.5 °C. 1HNMR(CDCl3, 400 MHz): d (ppm):
5.07 (d, J = 4.4 Hz, 2H), 5.20 (d, J = 13.2 Hz, 1H), 5.36 (d, J = 10.0 Hz,
1H), 6.07 (m, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.58 (dd, J1 = 8.0 Hz,
J2 = 7.6 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.81 (dd, J1 = 7.6 Hz,
J2 = 8.0 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.69 (d, J = 7.6 Hz, 1H). IR
(KBr cmÀ1) 2214, 3400, 3000, 1670, 665. HRMS (ESI) m/z (M+H)+
calcd for C17H11N3 257.0953; found: 257.0953.
4.2.3. General procedure for synthesis of 1d–e
4. Experimental
2-(Benzo[c,d]indol-2(1H)-ylidene) (2, 0.217g, 1mmol) was dis-
solved in relevant 1-bromo side chain (1.2mmol), K2CO3 (0.331 g,
2.4 mmol) were added to the solution as catalyst. Stirred at
100 °C for 4h. The mixture was cooled, filtrated directly to get
the product. The crude product was separated on silica gel
chromatography.
4.1. Materials and methods
All the solvents were of analytic grade. The closed supercoiled
pBR322 DNA was a bought from Takara Biotech Co. Ltd (Dalian).
1H NMR was measured on a Bruker AV-400 spectrometer with
chemical shifts reported as ppm (in DMSO/CDCl3-d6, TMS as inter-
nal standard). Mass spectra were measured on a HP 1100 LC–MS
spectrometer. Melting points were determined with an X-6 mi-
cro-melting point apparatus and are uncorrected. IR spectra were
recorded on a Nicolet Nexus 770 spectrometer. Fluorescence spec-
tra were determined on a Hitachi F-4500. Absorption spectra were
determined on a PGENERAL TU-1901 UV–vis. spectrophotometer.
4.2.3.1. 2-(1-(2-Bromoethyl)benzo[c,d]indol-2(1H)-ylidene)mal-
ononitrile(1d). Yield 77%, mp: 232.3–234.0 °C. 1HNMR(CDCl3,
400 MHz): d (ppm): 4.86 (t, J1 = 6.8 Hz, J2 = 6.4 Hz, 2H), 3.83 (t,
J1 = 6.8 Hz, J2 = 6.4 Hz, 2H), 7.24 (d, J = 8.0 Hz, 1H), 7.61 (dd,
J1 = 8.0 Hz, J2 = 7.6 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.86 (dd,
J1 = 8.0 Hz, J2 = 8.4 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.71 (d,
J = 7.6 Hz, 1H). IR (KBr cmÀ1) 2220, 2197, 1640, 600, 2970. HRMS
(ESI) m/z (M+H)+ calcd for C16H10BrN3 323.0058; found: 323.0058.
4.2. Synthesis
4.2.3.2. 2-(1-(3-Chloropropyl)benzo[c,d]indol-2(1H)-ylidene)mal-
ononitrile(1e). Yield 71%, mp: 222.8–223.4 °C. 1HNMR(CDCl3,
400 MHz): d (ppm): 2.44 (m, 2H), 3.71 (t, J1 = 6.8 Hz, J2 = 6.8 Hz, 2H),
4.63 (t, J1 = 6.8 Hz, J2 = 6.8 Hz, 2H), 7.29 (d, J = 7.6 Hz, 1H), 7.81 (dd,
J1 = 8.4 Hz, J2 = 7.2 Hz, 1H), 8.69 (d, J = 7.2 Hz, 1H), 7.61 (dd,
J1 = 7.6 Hz, J2 = 7.6 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 8.13 (d, J = 8.4 Hz,
1H). IR (KBr cmÀ1)2218, 2192, 3423, 3343, 3240. HRMS(ESI) m/z
(M+H)+ calcd for C17H11ClN3 293.0720; found: 293.0720.
4.2.1. 2-(Benzo[c,d]indol-2(1H)-ylidene)malononitrile (2)
Benzo[c,d]indol-2(1H)-one (1.69 g, 0.01 mol) and malononitrile
(0.6 g, 0.01 mol) were dissolved in 15 mL toluene. POCl3 (1.1 mL)
was added dropwise while the mixture was stirring. Then tempera-
ture of the reaction mixture was raised to 100 °C. After 4 h, the mix-
ture was cooled and added with 10 mL methanol, filtered, and dried,
separated on silica gel chromatography (CH2Cl2) to afford the prod-
uct 2 (1.82 g, 83% yield). Mp: >300 °C. 1HNMR (DMSO-d6, 400 MHz):
d (ppm): 5.30 (s, 1H), 7.21 (d, J = 7.2 Hz, 1H), 7.58 (t, J1 = 7.6 Hz,
J2 = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.83 (t, J1 = 8.0 Hz,
J2 = 7.2 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H). IR
(KBr cmÀ1) 2216, 2237, 2197, 3427, 1642, 655. HRMS (ESI) m/z
(M+H)+ calcd for C14H7N3217.0640; found: 217.0640.
4.2.4. General procedure for synthesis of 1f–g
2-(Benzo[c,d]indol-2(1H)-ylidene) (2, 0.217 g, 1 mmol) was dis-
solved in 6 mL acetic acid, acyl chloride was added dropwise, and
refluxed for 3 h, then cooled. The solution was dropped into water,
and adjusted the pH to 7 using NaOH to get brown solid, filtrated
and dried over vacuum. The crude product was separated on silica
gel chromatography.
4.2.2. General procedure for synthesis of 1a–c
2-(Benzo[c,d]indol-2(1H)-ylidene) (2, 0.217 g, 1 mmol) was dis-
solved in 10 mL acetonitrile, K2CO3 (0.331 g, 2.4 mmol) and rele-
vant 1-bromo side chain (1.2 mmol) were added to the solution.
4.2.4.1. 2-(1-Acetylbenzo[c,d]indol-2(1H)-ylidene)malononitrile
(1f). Yield 71%, mp: >300 °C. 1HNMR(CDCl3, 400 MHz): d (ppm):

3284
X. Li et al. / Bioorg. Med. Chem. 18 (2010) 3279–3284
3.05 (s, 3H), 7.32 (d, J = 7.2 Hz, 1H), 7.52 (dd, J1 = 8.0 Hz, J2 = 7.2 Hz,
1H), 7.78 (d, J = 8.4 Hz, 1H), 7.97 (dd, J1 = 7.2 Hz, J2 = 8.4 Hz, 1H),
compounds dissolved in DMSO (the final volume of DMSO/medium
was less than 0.001, v/v). After treatment, MTT solution (5 mg/mL
in PBS) was added to each well. After 3 h incubation lysis buffer
(200 g/L SDS, 50% Formamide, pH 4.7) was added to each well to
dissolve formazan. The absorbance was measured at 570 nm with
a Microplate reader. All experiments were performed at least three
times and average of the percentage absorbance was plotted
against concentration. The results were expressed as percentage
relative to untreated control and IC50 value was calculated for each
compound.
8.28 (d, J = 8.0 Hz, 1H), 8.74 (d, J = 7.2 Hz, 1H). IR (KBr cmÀ1
)
2217, 2205, 1670, 1640, 2970. HRMS (ESI) m/z (M+H)+ calcd for
C16H9N3 O259.0746; found: 259.0746.
4.2.4.2. 2-(1-Benzoylbenzo[c,d]indol-2(1H)-ylidene)malononit-
rile (1g). Yield 68%, mp: >300 °C. 1HNMR(CDCl3, 400 MHz): d
(ppm): 6.39 (d, J = 7.6 Hz, 1H), 7.39 (dd, J1 = 8.0 Hz, J2 = 7.6 Hz,
1H), 7.64 (dd, J1 = 7.6 Hz, J2 = 7.2 Hz, 2H), 7.72 (dd, J1 = 6.0 Hz,
J2 = 7.6 Hz, 1H), 7.73 (d, J = 7.6 Hz, 2H), 7.84 (d, J = 8.4 Hz, 1H),
7.95 (dd, J1 = 7.2 Hz, J2 = 8.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.75
(d, J = 7.2 Hz, 1H). IR (KBr cmÀ1)2220, 3433, 1655, 693, 738. HRMS
(ESI) m/z (M+H)+ calcd for C21H11N3O 321.0902; found: 321.0902.
Acknowledgments
We are grateful to the National Natural Science Foundation of
China (20536010), Program for Changjiang Scholars and Innovative
Research Team in University (IRT07110), and interdiscipline pro-
ject of Dalian University of Technology.
4.3. Spectroscopic measurements
The compounds were dissolved in absolute DMSO to give 10
À5 M solutions. Following spectra testing were read with Shimadzu
UV for absorption spectra and with Perkin–Elmer LS 50 for fluores-
cence spectra.
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