Synthesis and biological activity of novel inhibitors of topoisomerase I: 2-Aryl-substituted 2-bis-1H-benzimidazoles

Article abstract of DOI:10.1016/j.ejmech.2010.11.046

Inhibitors of topoisomerase I constitute a novel family of antitumor agents. The class of benzimidazole derivatives contains compounds possessing affinity to DNA. For example, fluorescent stains Hoechst 33342 and Hoechst 33258 interact with DNA as ligand

Full text of DOI:10.1016/j.ejmech.2010.11.046

European Journal of Medicinal Chemistry 46 (2011) 659e669
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of novel inhibitors of topoisomerase I:
2-Aryl-substituted 2-bis-1H-benzimidazoles
,
,
Manish Singh a b, Vibha Tandon a
*
a Department of Chemistry, University of Delhi, Delhi 110007, India
b Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of topoisomerase I constitute a novel family of antitumor agents. The class of benzimidazole
derivatives contains compounds possessing affinity to DNA. For example, fluorescent stains Hoechst
33342 and Hoechst 33258 interact with DNA as ligand and produce nonspecific inhibition of the catalytic
activity of many enzymes involved in DNA synthesis, including DNA topoisomerase and DNA helicase.
Several 2-aryl-5-substituted-2,5-bisbenzimidazole derivatives were synthesized and ability of these
derivatives to induce DNA cleavage in the presence of topoisomerase I was evaluated in vitro. These
analogs were also assayed for their cytotoxicity against U87, MCF7 and HeLa human tumor cells. All the
Received 26 March 2010
Received in revised form
25 November 2010
Accepted 30 November 2010
Available online 8 December 2010
Keywords:
Bisbenzimidazole
MTT
four compounds showed a potent growth inhibitory effect on all the cell lines, with IC50 in the mM range.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Hoechst 33342
Antitumor activity
Topoisomerase I
Calf Thymus DNA
1. Introduction
interactions with DNA and to prevent excessive supercoiling. There
are two fundamental types of topoisomerases, which differ in both
mechanism and cellular function [10]. Type I DNA topoisomerases
are classified into two subfamilies: type IA and type IB. The
enzymes of type IA subfamily, including bacterial DNA top-
oisomerase I and II, eukaryotic DNA topoisomerase III and reverse
gyrase [14,15] form a tyrosyl linkage with a 50-phosphate group of
the DNA strands generated due to the enzyme action [11], whereas
the enzymes of type IB subfamily, including eukaryotic and vaccinia
virus DNA topoisomerases I [16] and topoisomerase V, establish the
tyrosyl bond with the 30-phosphate group [11]. Type IA top-
oisomerases relax only negative supercoiled DNA with Mg2þ
requirement, whereas type IB topoisomerases relax both negative
and positive supercoiled DNA even in the absence of metallic
cofactors, although Mg2þ and Ca2þ stimulate the relaxation activity
[17,18].
Hoechst 33258, also known as Pibenzimol, which has two
benzimidazole groups linked in a head-to-tail manner [19]. This
compound was reported to have moderate in vitro activity on L1210
leukemia on repeated dose schedule but was not active against
P388 using the single-dose protocol [20] which was followed by
several phase I trials in humans [21]. The first trial was limited by
pancreatic toxicity; although in a second trial some responses were
seen in pancreatic cancer, a subsequent phase II trial did not show
any objective responses [22] and no further trial of this agent have
Studies on the noncovalent interactions of small molecules with
the minor groove of DNA continue to be a fruitful area for the
discovery of potential new therapeutic agents. The main repre-
sentatives of this class, which reached the clinic, are the antitumor
agents derived from CC-1065, that is, adozelesin, carzelesin and
bizelesin, and the distamycin A derivative tallimustine. These
‘classical’ minor groove binding agents (MGBs) have been shown to
be highly DNA sequence-specific [1,2] and to exert their cytotoxic
effect through the ability to directly alkylate DNA mainly at the N(3)
position of adenines exposed in (AT) rich sequences in the DNA
minor groove [3e7]. While certain DNA minor groove binding
agents, berenil, netropsin and various symmetric and asymmetric
bisbenzimidazoles can block DNA topoisomerase and helicase
activity by binding to duplex at specific base sequences [8,9].
DNA topoisomerases are ubiquitous enzymes that catalyze
essential enzymes to solve the topological problems accompanying
key nuclear processes such as DNA replication, transcription, repair
and chromatin assembly by introducing temporary single or double
strand breaks in the DNA [10e13]. In addition, these enzymes fine-
tune the steady-state level of DNA supercoiling to facilitate protein
* Corresponding author.
E-mail addresses: vibhadelhi@hotmail.com, vtandon@acbr.du.ac.in (V. Tandon).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.046

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M. Singh, V. Tandon / European Journal of Medicinal Chemistry 46 (2011) 659e669
been reported. Analogs of Hoechst 33258, bearing alkylation of the
terminal phenoxy moiety enhanced cytotoxic potency with respect
to parent compound Hoechst 33258. About the phenolic ether of
Hoechst 33258, such as Hoechst 33342 (R ¼ OC2H5) or Hoechst
33377 (R ¼ OC6H5), it has been suggested that these agents exhibit
greater cytotoxicity due to their superior ability to traverse the cell
membrane and reach its DNA target [23]. In fact, due to the ability
of these compounds in traversing both the cytoplasmic and nuclear
membranes and accumulating in the nucleus, they are capable of
inhibiting the binding of regulatory proteins. X-ray and NMR
studies of Hoechst 33258 bound to DNA as a 1:1 complex [24e26]
indicate how molecule accomplishes recognition of DNA: similar to
netropsin and distamycin, specific H-bonds are formed between
the benzimidazole NHs and adjacent adenine N(3)- and thymine O
(2)-atom on the floor of the minor groove. The binding preference
for AT base pairs is a result of the closer contact of the aromatic
H-atoms of Hoechst molecule and floor of the minor groove, thus
precluding the binding of the drug at GC base pairs due to the
presence of exocyclic N(2)-amino group of guanine. In addition, the
binding of Hoechst is stabilized by electrostatic interaction and
extensive van der Waals contacts with the walls of the minor
groove. Earlier, several analogs of this bisbenzimidazole have been
synthesized to further investigate the structureeactivity relation-
ships associated with their potency as topoisomerase I inhibitors
and the related cytotoxicity [27] (Fig. 1).
Most derivatives of Hoechst 33258 having the monosubstitu-
tion at para or meta position of phenyl ring, which kept whole
molecules to planar structures. To our knowledge, no literature
related to di-substituted groups, having halogen atoms as substit-
uents, at 2-position of benzimidazole as the derivatives of Hoechst
were reported so far. Moreover affinity increase in order for the
three DNA: p-OH < m-OCH3, p-OH < m-OH, p-OCH3 < bis-m-OH
Hoechst derivatives having various substitution (OH, m-OH,
p-OCH3, m-OCH3, bis-m-OH) on phenyl ring to poly[d(A-T)] [28].
Our interest in biologically active compounds as potential
antitumor agents focused our studies on the synthesis of several di
and tri substituted bisbenzimidazole, which have electron donating
(OCH3) and electron withdrawing groups (F, Cl) on the phenyl ring
and evaluate their potential to induce DNA cleavage in the presence
of mammalian topoisomerase I. The results associated with the
cytotoxicity observed for these analogs in various tumor cell lines is
also being presented in this manuscript.
mineral acids [32] has improved both the yield and purity of the
reaction [33]. On the other hand, the synthesis of benzimidazoles
via the condensation of 1,2-diaminobenzenes with aldehydes
requires an oxidative reagent to generate the benzimidazole
nucleus. Various oxidative reagents, such as nitrobenzene, benzo-
quinone, mercuric oxides, lead tetra acetate, iodine, copper(II)
acetate and Fe(III) mediated oxidation have been employed for
this purpose [34]. However, we determined during our early
attempts that such reported procedure for preparation of benz-
imidazole derivatives was not quite versatile nor compatible with
different substituted starting materials and also posed practical
difficulties in the form of laborious reaction workup protocols.
The dehydrogenating capacity of bisulfite has been demonstrated
in the condensation of 1,8 diaminonaphthalene and aldehyde to
2-substituted-1H-pyrimidines [35,36]. Hence, we are utilizing
bisulfite as an oxidizing reagent and condensation was carried out
in ethanol. Therefore, bisbenzimidazole derivatives 6aed were
easily prepared by using this procedure. A series of 2-aryl-5-cya-
no-1H-benzimidazole 2aed were synthesized by coupling of
4-cyano-1,2-phenylenendiamine with the appropriate substituted
benzaldehydes (Scheme 1). All benzimidazoles 2aed were
synthesized in yields ranging from 65% to 85%. Reduction of 2a-
d with Ni-Al alloy in the presence of formic acid followed by
hydrolysis provides an effective method for preparation of the 2-
aryl-5-formyl-1H-benzimidazole intermediates 3a-d (Scheme 2)
required for the formation of final compounds i.e. 2,5-bi-1H-
benzimidazole used in this study. The treatment of 5-chloro-2-
nitroaniline with 4-methylpiperazine in the presence of anhydrous
potassium carbonate in DMF afforded the required 5-(4-methyl-1-
piperazinyl)-2-nitroaniline 4 in nearly 85% yield. The required
diamine 5 was obtained by catalytic hydrogenation of compound 4,
using palladized carbon in a Parr reactor at ambient temperature.
The preparation of all four 20-aryl-5-(4-methylpiperazinyl)-2,50-bi-
1H-benzimidazoles 6aed was accomplished by coupling of 5-(4-
methylpiperazinyl)-1,2-phenylenediamine with the appropriate
2-aryl-5-formyl-1H-benzimidazole (Scheme 2).
3. Results and discussion
3.1. DNA binding
Tounderstand thephysico-chemicalproperties of thecompounds,
absorbance measurements were done in 20 mM sodium cacodylate
buffer, 100 mM NaCl, pH 7.2. Hoechst 33342 (hereafter Hoechst), 6c
and 6d showed absorption maxima at 340 nm whereas 6a and 6b
showed absorption maxima at 334 nm (Table 1). In the present paper
we studied the interaction of above four compounds with CT-DNA
using absorbance spectroscopy, where changes in absorbance and
absorption maxima indicated binding. The absorption maxima for
Hoechst, 6c and 6d showed a bathochromic shift of 10, 9 and 7 nm
respectively at r ¼ 4, DNA/drug ratio. Compounds 6a and 6b showed
a bathochromic shift of 12 and 6 nm respectively. Hoechst, 6c and 6d
showed38%, 28.1%and 27.1%hypochromicityatr ¼ 4, DNA/drugratio,
whereas in the case 6a, 20% hyperchromicity was observed only
(Fig. 2). In the case of 6c we did not observe any significant changes in
absorbance. A significant amountofhyperchromicityaswellas12 nm
bathochromic shiftobserved in case of 6a after binding to CT-DNA can
be attributed to the fluorine substitution at phenyl ring resulting in
a strong binding between compound and DNA. As our results suggest
that 6c, 6d and 6c after binding to CT-DNA observed hypochromic
shift whereas only 6a had shown hyperchromicity. The size of fluo-
rine atom is smallest as it has a small radii, because of which 6a may
not only be behaving as a minor groove binder rather it is able to slip
between DNA base pairs and bind to them as an intercalator causing
the unwinding of DNA duplex and resulting into the hyperchromicity
2. Chemistry
Traditionally, benzimidazoles have most commonly been
prepared from the reaction of 1,2-diaminobenzenes with carboxylic
acids under harsh dehydrating reaction conditions, utilizing the
strong acid such as polyphosphoric acid, hydrochloric acid, boric
acid or p-toluenesulfonic acid [29]. However, the use of milder
reagents, particularly Lewis acids, [30] inorganic clays [31] or
Fig. 1. Chemical structure of bisbenzimidazoles for Hoechst 33258, R1 ¼ R2 ¼ R4 ¼ H,
R3 ¼ OH; Hoechst 33342, R1 ¼ R2 ¼ R4 ¼ H, R3 ¼ OC2H5; Hoechst 33377,
R1 ¼ R2 ¼ R4 ¼ H, R3 ¼ OC6H5; 6a, R2 ¼ R4 ¼ H, R1 ¼ R3 ¼ F; 6b, R2 ¼ R4 ¼ H, R1 ¼ R3 ¼ Cl;
6c, R1 ¼ R4 ¼ H R2 ¼ R3 ¼ OCH3; and for 6d, R1 ¼ H, R2 ¼ R3 ¼ R4 ¼ OCH3.

M. Singh, V. Tandon / European Journal of Medicinal Chemistry 46 (2011) 659e669
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Scheme 1. Reagents, condition and yield: a) H2, 10% Pd/C, EtOH, rt, 30 min, 95%. b) Na2S2O5, MeOH/H2O, reflux 5 h, 65e85%; c) NieAl alloy, formic acid, 95 ꢂC, 5 h, 55e60%.
shift in UV absorption. Hence on the basis of these results we can
suggest that 6b, 6c and 6d are binding with CT-DNA in a non coop-
erative manner similar to Hoechst whereas 6a has a cooperative
binding with CT-DNA.
binding affinity as compared to methoxy substituted bisbenzimi-
dazoles (6b and 6d) as well as Hoechst (Fig. 3b).
3.2. Cytotoxicity
Upon binding to CT-DNA, fluorophore (6aed, Hoechst) exhibits
a significant fluorescence enhancement, due to the loss of solvent
exposure upon snugly binding (van der Waals and H-bonding
forces) within the DNA groove (Fig. 3a). Ligands 6aed and Hoechst
showed single emission maxima in presence of CT-DNA. Ligands
(6a, 6c, 6d) showed a blue shift of 13e31 nm, whereas 44 nm blue
shift was observed in case of Hoechst. The low dissociation
constant for 6a and 6b (1.86 ꢀ 10ꢁ6 and 1.98 ꢀ 10ꢁ6 respectively)
as compared to reference ligand Hoechst (4.50 ꢀ 10ꢁ6) suggested
that halogenated bisbenzimidazoles (6a and 6b) showed greater
Chemotherapy is a major therapeutic approach for the both
localized and metastasized cancers. The newly synthesized bisben-
zimidazole derivatives 6a, 6b, 6c and 6d were evaluated for their
cytotoxicity against human tumor cell lines, which are cervix carci-
noma cell line (HeLa), breast carcinoma cell line (MCF7) and brain
glioma cell line (U87) in comparison to Hoechst and Camptothecin
as reference compound (Fig. 4aec). The MTT assay was widely
applied to examine in vitro cytotoxicity. Compounds 6aed had
inhibitory effects on the growth of tumor cells (U87, MCF7 and HeLa)
Scheme 2. Reagents, condition and yield: a) Methylpiperazine, anhydrous K2CO3, dry DMF, 110 ꢂC, 24 h 85%. b) H2, 10% Pd/C, EtOH, rt, 2 h, 95%. c) Na2S2O5, EtOH/H2O, reflux 5 h,
62e68%.

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M. Singh, V. Tandon / European Journal of Medicinal Chemistry 46 (2011) 659e669
Table 1
and 6d for U87 cell line (72 h). But in case of MCF7, the IC50 was
observed at 5.3 and 16.8 M for 6c and 6d respectively. The IC50
UVevisible spectral changes of the bisbenzimidazoles derivatives upon titration
with CT-DNA at DNA/drug ratio r ¼ 4.
m
determined in the case of HeLa was 3.4 and 29
respectively.
mM for 6c and 6d
Compound
lex/lem (nm)
Red shift
Hypochromicity
log P
Hoechst 33342
340/496/
334/501
334/a
340/478
340/514
10
12
6
9
7
38
ꢁ20
28
1
27
3.95
4.05
4.85
3.48
3.36
Compound 6a was approximately 14e18 fold more cytotoxic
than 6c and 6d against U87 cell lines and 6b was even 3.6-fold more
cytotoxic than 6a and 2.8-fold more potent than Hoechst, but nearly
3-fold less cytotoxic than CPT. This analog (6b) was also active
against MCF7 and HeLa cells, but it was almost 2.6-fold and 2-fold
less potent with respect to the Hoechst. In case of Hela cells 6b was
observed 3-fold more cytotoxic thanCPT. TheIC50 values of 6a and 6c
6a
6b
6c
6d
A negative hypochromicity means a corresponding hyperchromicity.
a
Single emission value was not observed for 6b.
were 5.5 and 5.3
was 2.2-fold (IC50; 1.5
m
M against MCF7 whereas in HeLa, compound 6a
in dosage and time-dependent manners except compounds 6c and
6d in case of U87 (Fig. 4c). Meanwhile 6a, 6b showed higher anti-
proliferative activity than 6c and 6d against all screened cell lines,
demonstrating that our strategy of modifying these molecules was
successful (Table 2). Earlier it was observed in our laboratory that
dimethoxy analog of bisbenzimidazole (6c) is non cytotoxic in
comparison to Hoechst in BMG-1 (human glioma) cell lines [37]. The
non cytotoxicity may be due to low cellular uptake of these
compounds as their log P is 3.48 which is less than the Hoechst i.e.,
3.95. This trend was further confirmed by the synthesis of trime-
thoxyanalog 6d having log P value 3.36. The cellular uptake of 6c and
6d is reduced due to less lypophilicity of these compounds. The IC50
m
M) more potent than 6c (IC50; 3.4 M) at
m
72 h. Compound 6d was less potent in case of all the three above
mentioned cell lines. On the basis of above results it can be suggested
that 6b is more toxic in comparison to 6a, 6c, 6d and its toxicity is
comparable to Camptothecin in HeLa cells and little less in U87 and
MCF7 also. Certainly the analog 6b may serve as important lead in
the synthesis of more potent and selective anticancer agents.
3.3. Topoisomerase I inhibition
To investigate the inhibitory activity of these molecules against
value could not be determined even at 100 mM concentrations of 6c
purified human topo I, the enzyme was purified following the
Fig. 2. UV absorption spectra of Hoechst 33342 (A), 6a (B), 6b (C), 6c (D) and 6d (E) alone and in the presence of CT-DNA. The compound concentration was 5
r ¼ 2, r ¼ 3, r ¼ 4.
mM:free, r ¼ 0, r ¼ 1,

M. Singh, V. Tandon / European Journal of Medicinal Chemistry 46 (2011) 659e669
663
Fig. 3. a. A change in the fluorescence emission of ligand (6aed and Hoechst 33342) was observed on addition of CT-DNA at concentrations of 0.1e40
m
M. All measurements were
made after preincubating ligand with or without DNA at 25 ꢂC for 5 min. b. Plot of DF
/DFmax versus increasing concentration of CT-DNA.
procedure given in literature. A relaxation assay was performed by
simultaneous incubation of supercoiled plasmid, pBS (SKþ) DNA,
enzyme and compounds. The inhibitory activity of the compounds
was compared to that of standard topo I poison Camptothecin
(Fig. 5). Camptothecin have shown topoisomerase I inhibition
affinity of benzimidazole to CT-DNA but in addition to it, fluoro and
chloro (6a and 6b) showed significant cytotoxicity to human tumor
cell lines in comparison to methoxy substituted analogs of bis-
benzimidazole (6c and 6d). These compounds can be further
pruned to develop a chemotherapeutic drug against cancer.
below 10 mM concentration. which is evident from reduced top-
oisomer bands and w80% recovery of the supercoiled DNA band.
The di and trimethoxyphenyl bearing compounds 6c and 6d
showed less enzyme inhibition. The w90% topoisomerase activity
5. Experimental protocols
5.1. Chemistry
could be inhibited at w50
6d. Dihalogenated phenyl bearing derivatives 6a and 6b, however,
showed effective inhibition of the enzyme activity even at w25
concentration. This was evident from the recovery of the super-
coiled DNA band with increasing concentration of above
compounds. Compounds 6a and 6b were found to be more potent
topo I inhibitors, as compared to reference compound Hoechst.
mM and w75 mM concentration of 6c and
All the starting materials and reagents were purchased from E.
Merck (Germany), S.D. Fine (India), Spectrochem (India), Aldrich
and Fluka were used without further purification. Solvents were
dried and redistilled prior to use using standard method. Melting
points were recorded on Buchi 540 apparatus. The purity of the
compounds was confirmed by thin layer chromatography (TLC)
using Merck silica gel 60 F254 coated alumina plates and detected
under UV-light or I2 vapor. Column chromatography was performed
using silica gel mesh size 60e120. Infrared (IR) spectra were recor-
ded on a Perkin Elmer FT-IR 410 spectrometer in KBr.1H and 13C NMR
spectra were recorded using Bruker Avance-300 (300 MHz) NMR
mM
4. Conclusion
In view of above results we can conclude that introduction of
halogen groups at the phenyl ring not only increases the binding

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M. Singh, V. Tandon / European Journal of Medicinal Chemistry 46 (2011) 659e669
Fig. 4. a. Antiproliferative effects of the bisbenzimidazole derivatives 6aed, Hoechst 33342 on HeLa cell line. b. Antiproliferative effects of the bisbenzimidazole derivatives 6aed,
Hoechst 33342 on MCF7 cell line. c. Antiproliferative effects of the bisbenzimidazole derivatives 6aed, Hoechst 33342 on U87 cell line.
spectrometer. Mass spectra were recorded on ESI-TOF-MS-MS mass
spectrometer (Applied Biosystems). Elemental analysis was done on
GmbH VarioeEL elemental analyser.
7.61 (d, 1H, AreH, J ¼ 8.4), 7.77 (d, 1H, AreH, J ¼ 7.8), 8.154 (br s, 1H,
AreH), 8.28 (m, 1H); ESI-MS: m/z found 254.0976 (M ꢁ H); exact
mass 255.06. Anal. Calcd. for C14H7F2N3: C, 65.88; H, 2.76; N, 16.46.
Found: C, 65.82; H, 2.78; N, 16.52.
5.1.1. 4-Cyano-1,2-phenylenendiamine (1)
A solution of 4-amino-3-nitrobenzonitrile (0.52 g, 3.22 mmol)
in ethanol was treated with 10% Pd/C (140 mg), and mixture was
hydrogenated at rt at 42 psi pressure of hydrogen using Parr
hydrogenator (Parr Instrument Company, Illinois, USA). The reac-
tion was followed by TLC. After 30 min, the expected volume of
hydrogen had been taken up (22 psi) and reaction mixture was
colorless. The reaction mixture was filtered through Celite bed and
filtrate was used for next step without further purification;
Rf ¼ 0.52 in 100% EtOAc.
5.1.2.2. 5-Cyano-2-(2,4-dichlorophenyl)benzimidazole (2b). To a etha-
nolic solution of the hydrogenated product 1 (w0.40 g, w3.06
mmol), equivalent amount of 2,4 dichlorobenzaldehyde (0.53 g,
3.06 mol) and half equivalent of aqueous solution of Na2S2O5 (0.29 g,
1.53 mmol in 3 mL H2O) was treated as described for 2a and the
residue obtained was purified by column chromatography (eluent
pet. ether/EtOAc ¼ 5:5). The pure product was isolated as solid
(0.65 g, 2.29 mmol 75% yield); Rf ¼ 0.65 in 100% EtOAc;
mp ¼ 233.6e234.8 ꢂC; FTIR (KBr, cmꢁ1): 3294.28, 2230.73, 1590.20,
1413.56, 808.45, 625.50; 1H NMR (300 MHz, DMSO)
d
ppm ¼ 7.62
5.1.2. General procedure for preparation of 2-aryl-5-
cyanobenzimidazole
(s, 1H, AreH), 7.65 (s, 1H, AreH), 7.79 (d, 1H, AreH, J ¼ 8.4), 7.87
(s, 1H, AreH), 7.94 (d, 1H, AreH, J ¼ 8.4), 8.20 (s, 1H, AreH), 13.07
(br s, exch,1H, NH); ESI-MS: m/z found 285.7627 (M ꢁ H); exact mass
287.00. Anal. Calcd. for C14H7Cl2N3: C, 58.36; H, 2.45; N,14.58. Found:
C, 58.40; H, 2.38; N, 14.64.
5.1.2.1. 5-Cyano-2-(2,4-difluorophenyl)benzimidazole(2a). To a meth-
anolic solution of the hydrogenated product 1 (w0.40 g, w3.06
mmol), equivalent amount of 2,4-difluorobenzaldehyde (0.44 mL,
3.06 mmol) and half equivalent of aqueous solution of Na2S2O5
(0.29 g, 1.53 mmol in 3 mL H2O) were added. The resulting solution
was stirred at reflux for 5 h, and filtered through a bed of Celite. The
filtrate was concentrated under reduced pressure and residue was
purified by column chromatography (eluent pet. ether/EtOAc, 7:3).
The pure product was isolated as a solid (0.46 g, 1.83 mmol 65%
yield); Rf ¼ 0.73 in 100% EtOAc; mp ¼ 256.5e258.3 ꢂC; FTIR (KBr,
5.1.2.3. 5-Cyano-2-(3,4-dimethoxyphenyl)benzimidazole (2c). To a
ethanolic solution of the hydrogenated product
1 (w0.40 g,
w3.06 mmol), equivalent amount of 3,4 dimethoxybenzaldehyde
(0.50 g, 3.06 mmol) and half equivalent of solution of Na2S2O5
(0.29 g, 1.53 mmol in 3 mL H2O) were added. The resulting solution
was treated as described for 2a and the obtained residue was puri-
fied by column chromatography (fluent pet ether/EtOAc ¼ 2:8). The
pure product was isolated as light solid (0.68 g, 2.44 mmol 80%
1
cmꢁ1): 3328.68, 2229.86, 1623.70, 1480.75, 808.92, 664.58; H NMR
(300 MHz, DMSO)
d
ppm ¼ 7.32 (m, 1H, AreH), 7.54 (m, 1H, AreH),

M. Singh, V. Tandon / European Journal of Medicinal Chemistry 46 (2011) 659e669
665
Fig. 4. (continued).
yield); Rf ¼ 0.48 in 100% EtOAc; mp ¼ 223.1e224.8 ꢂC; FTIR (KBr,
95 ꢂC for 5 h under N2. The hot mixture was filtered through a bed
of Celite and the reaction flask and the Celite bed were rinsed with
water. The aqueous solution was concentrated to dryness. After
addition of H2O to this residue, a white precipitate formed. The pH
of this suspension was adjusted to 9 by the dropwise addition of 2 N
NaOH and the product was then extracted through EtOAc. The
organic layer was dried over anhydrous Na2SO4, then the solvent
was removed and column chromatography using silica gel was
done. The pure product was eluted out in 30e40% EtOAc as solid
(0.166 g, 0.64 mmol, 55% yield); Rf ¼ 0.60 in 100% EtOAc;
mp ¼ 214.5e216 ꢂC; FTIR (KBr, cmꢁ1): 3223.86, 1682.48, 1626.35,
1482.26, 1289.41, 1148.91, 806.85, 639.16; 1H NMR (300 MHz,
cmꢁ1): 3437.66, 2220.87, 1502.97, 1267.03, 1022.70, 813.60; 1H NMR
(300 MHz, DMSO)
d
ppm ¼ 3.83 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 7.13
(d, 1H, AreH, J ¼ 8.4), 7.54 (d, 1H, AreH, J ¼ 8.4), 7.69 (d, 1H, AreH,
J ¼ 7.8), 7.77 (s, 2H, AreH), 8.07 (s, 1H, AreH), 13.29 (br s, exch, 1H,
NH); ESI-MS: m/z found 279.882 (M þ H); exact mass 279. Anal.
Calcd. for C16H13N3O2: C, 68.81; H, 4.69; N,15.05. Found: C, 68.78; H,
4.65; N, 15.14.
5.1.2.4. 5-Cyano-2-(3,4,5-trimethoxyphenyl)benzimidazole (2d). To
a ethanolic solution of the hydrogenated product 1(w0.40 g,
w3.06 mmol), equivalent amount of 3,4,5 trimethoxybenzaldehyde
(0.60 g, 3.06 mmol) and half equivalent of aqueous solution of
Na2S2O5 (0.29 g, 1.53 mmol in 3 mL H2O) were added. The resulting
solutionwas treated as described for 2a andtheobtained residuewas
purified by column chromatography (eluent pet ether/EtOAc ¼ 3:7).
The pure product was isolated as solid (0.80 g, 2.60 mmol 85% yield);
Rf ¼ 0.52 in 100% EtOAc; mp ¼ 229.3e230.9 ꢂC; FTIR (KBr, cmꢁ1):
3435.99, 2222.98, 1590, 1465.25, 1239.69, 1127.10; 1H NMR
DMSO)
d
ppm ¼ 7.32 (m,1H, AreH), 7.56 (m,1H, AreH), 7.78 (m, 2H,
AreH), 8.14 (s, 1H, AreH), 8.28 (m, 1H, AreH), 10.07 (s, 1H, CHO),
13.07 (s, 1H, NH); ESI-MS: m/z found 259.3453 (M þ H); exact mass
258.06. Anal. Calcd. for C14H8F2N2O: C, 65.12; H, 3.12; N, 10.85.
Found: C, 65.06; H, 3.14; N, 10.89.
5.1.3.2. 5-Formyl-2-(2,4-dichlorophenyl)benzimidazole (3b). A reac-
tion mixture of NieAl alloy (1.25 g) and 2b (0.30 g, 1.04 mmol) in
formic acid (18 mL) and H2O (6.5 mL) was treated as 3a. Purification
by column chromatography using gradient of 50e60% EtOAc gave
solid (0.17 g, 0.58 mmol, 56% yield); Rf ¼ 0.56 in 100% EtOAc;
mp ¼ 208e210.5 ꢂC; FTIR (KBr, cmꢁ1): 3360.34, 1685.59, 1615.03,
1410.36, 1313.21, 1152.34, 1152.34, 812.71, 788.04, 630.42; 1H NMR
(300 MHz, DMSO)
d
ppm ¼ 3.73 (s, 3H, OCH3), 3.89 (s, 6H, OCH3), 7.56
(m, 3H, AreH), 7.74 (br s, AreH), 8.14 (br s, 1H, AreH), 13.39 (br s,
exch, 1H, NH); ESI-MS: m/z found 310.0189 (M þ H); exact mass
309.11. Anal. Calcd. for C17H15N3O3: C, 66.01; H, 4.89; N,13.58. Found:
C, 66.05; H, 4.92; N, 13.54.
5.1.3. General method of the conversion of 5-cyanobenzimidazole to
5-formylbenzimidazoles
5.1.3.1. 5-Formyl-2-(2,4-difluorophenyl)benzimidazole (3a). Using
the similar procedure to that described previously [38] NieAl alloy
(1.25 g) was added to a solution of 2a (0.30 g, 1.17 mmol) in formic
acid (18 mL) and H2O (6.5 mL). The reaction mixture was heated at
(300 MHz, DMSO)
d
ppm ¼ 7.65 (d, 1H, AreH, J ¼ 8.3), 7.82 (s, 2H,
AreH), 7.88 (s, 1H, AreH), 7.97 (d, 2H, AreH, J ¼ 8.3), 8.25 (s, 1H,
AreH), 10.08 (s, 1H, CHO), 13.24 (br s, exch, 1H, NH); ESI-MS: m/z
found 291.0148 (M þ H); exact mass 290. Anal. Calcd. for
C14H8Cl2N2O: C, 57.76; H, 2.77; N, 9.62. Found: C, 57.70; H, 2.82;
N, 9.56.

666
M. Singh, V. Tandon / European Journal of Medicinal Chemistry 46 (2011) 659e669
Fig. 4. (continued).
5.1.3.3. 5-Formyl-2-(3,4-dimethoxyphenyl)benzimidazole (3c). A reac-
tion mixture of NieAl alloy (2.50 g) and 2c (0.58 g, 2.08 mmol) in
formic acid (36 mL) and H2O (14 mL) was treated as 3a. Purification
by column chromatography using gradient of EtOAc to 4% MeOH/
EtOAc gave solid (0.35 g, 1.25 mmol, 60% yield); Rf ¼ 0.40 in 100%
EtOAc; mp ¼ 232.6e235.4 ꢂC; FTIR (KBr, cmꢁ1): 3429.26, 1686.19,
1619.11, 1505.75, 1265.35 1139.52, 1020.75, 810.45; 1H NMR
EtOAc and the product was obtained as solid (0.389 g,1.24 mmol, 60%
yield); Rf ¼ 0.47 in 100% EtOAc; mp ¼ 214.2e215.4 ꢂC; FTIR (KBr,
cmꢁ1): 3410.47, 1688.95, 1589.74, 1463.65, 1426.56, 1127.85, 1005.82,
787.58; 1H NMR (300 MHz, DMSO)
d
ppm ¼ 3.75 (s, 3H, OCH3), 3.92
(s, 6H, OCH3), 7.56 (s, 2H, AreH), 7.77 (s, 2H, AreH), 8.17 (br s, 1H,
AreH), 10.06 (s, 1H, CHO), 13.33 (br s, exch, 1H, NH); ESI-MS: m/z
found 313.1179 (M þ H); exact mass 312.12. Anal. Calcd. for
C17H16N2O4: C, 65.38; H, 5.16; N, 8.97. Found: C, 65.32; H, 5.20; N, 8.91.
(300 MHz, DMSO)
d
ppm ¼ 3.85 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 7.15
(d, 1H, AreH, J ¼ 8.3), 7.54 (d, 1H, AreH, J ¼ 8.4), 7.69 (d, 1H, AreH,
J ¼ 7.8), 7.77 (s, 2H, AreH), 8.07 (s,1H, AreH),10.05 (s,1H, CHO),13.29
(br s, exch, 1H, NH); ESI-MS: m/z found 282.9404 (M þ H); exact mass
282.10. Anal. Calcd. for C16H14N2O3: C, 68.07; H, 5.00; N, 9.92. Found:
C, 68.01; H, 4.91; N, 9.88.
5.1.3.5. 5-(4-Methyl-1-piperazinyl)-2-nitroaniline (4). 5-Chloro-2-
nitroaniline (2 g, 11.6 mmol), 4-methylpiperazine (1.53 mL,
13.8 mmol) and K2CO3 (2.5 g, 18 mmol) in 5 mL DMF was heated at
110 ꢂC for 26e30 h till TLC showed the disappearance of 5-chloro-
2-nitroaniline. The crude compound was suspended in water and
the product was extracted with ethyl acetate. The organic layer was
washed twice with water, dried over anhydrous Na2SO4 and
concentrated. The product was purified by column chromatography
using a gradient of 20% MeOH/EtOAc. The pure product was isolated
as a bright yellow solid (2.32 g, 9.86 mmol 85% yield); Rf ¼ 0.27 in
methanol/EtOAc, 4:6; mp ¼ 155.8e157.8 ꢂC; FTIR (KBr, cmꢁ1):
3442.03, 3283.01, 1619.30, 1619.30, 1247.39, 1000.30, 809.79,
5.1.3.4. 5-Formyl-2-(3,4,5-trimethoxyphenyl)benzimidazole (3d). A
reaction mixture of NieAl alloy (2.50 g) and 2d (0.642 g, 2.08 mmol)
in formic acid(36 mL) and H2O (14 mL) was treatedas 3a. Purification
by column chromatography using gradient of EtOAc to 2% MeOH/
Table 2
IC50 of the synthesized compounds against U87, MCF7 and HeLa cells in vitro after
72 h of continuous exposure of compound (IC50 is given in mM).
670.72; 1H NMR (300 MHz, DMSO)
d
ppm ¼ 2.18 (s, 3H, N-CH3),
2.38 (s, 4H, CH2), 3.29 (s, 4H, CH2), 6.24 (s, 1H, AreH), 6.32 (d, 1H,
AreH, J ¼ 9.4), 6.97 (br s, exch, 2H, NH2), 7.79 (d, 1H, AreH, J ¼ 9.0);
Compound
U87
MCF7
HeLa
13C NMR (75 MHz, DMSO)
d ppm: 45.50, 46.11, 54.08, 97.65, 105.34,
Hoechst 33342
6a
4.2
5.5
0.5
5.5
0.3
1.5
123.04,127.18,148.37, 154.97; ESI-MS: m/z found 237.1202 (M þ H);
exact mass 236.13. Anal. Calcd. for C11H16N4O2: C, 55.92; H, 6.83; N,
23.71. Found: C, 55.82; H, 6.87; N, 23.7.
6b
1.5
1.3
0.6
6c
6d
>100
>100
0.5
5.3
16.8
0.65
3.4
29.3
1.5
Camptothecin
5.1.3.6. 5-(4-Methyl-1-piperazinyl)-1,2-phenylenediamine (5). A solu-
All values are in
mM.
Values are mean of triplicates of three independent experiments.
tion of 5-(4-methyl-1-piperazinyl)-2-nitroaniline (0.2 g, 0.84 mmol)

M. Singh, V. Tandon / European Journal of Medicinal Chemistry 46 (2011) 659e669
667
of the hydrogenated product 5 (w0.15 g, w0.77 mmol), equivalent
amount of 2-(2,4-difluorophenyl)-5-formylbenzimidazole (0.20 g,
0.77 mmol) and half equivalent of solution of Na2S2O5 (0.072 g,
0.38 mmol in 1 ml H2O) were added. The resulting solution was
stirred at reflux for 6 h, then cooled to room temperature and
filtered through a bed of Celite. The filtrate was concentrated under
reduced pressure and the obtained residue was purified by column
chromatography using a gradient of 60e80% MeOH/EtOAc. The
pure product was isolated as an orange solid (0.215 g, 0.48 mmol
63% yield); Rf ¼ 0.29 in 100% MeOH; mp ¼ 234.1e235.8 ꢂC; FTIR
(KBr, cmꢁ1): 3421.67, 1628,57, 1447.68, 1267.23, 1142.34, 1010.00,
973.64, 817.86; 1H NMR (300 MHz, DMSO)
d
ppm ¼ 2.43 (s, 3H,
CH3), 2.77 (s, 4H, CH2), 3.21 (s, 4H, CH2), 6.95 (d, 1H, AreH, J ¼ 8.2),
7.05 (s, 1H, AreH), 7.33 (m, 1H, AreH), 7.47 (d, 1H, AreH, J ¼ 8.2),
7.53 (m, 1H, AreH), 7.75 (br s, 1H, AreH), 8.07 (br s, 1H, AreH), 8.31
(d, 1H, AreH, J ¼ 6.9), 8.36 (d, 1H, AreH, J ¼ 6.0), 12.88 (br s, exch,
1H, NH); 13C NMR (75 MHz, DMSO)
d
ppm ¼ 44.59, 49.13, 54.13,
105.06, 109.74, 112.6, 113.79, 114.76,116.43, 119.06, 120.8, 124.9,
131.8, 147.1, 151.4, 158.0, 161.4, 164.8; ESI-MS: m/z found 445.1476
(M þ H); exact mass 444.19. Anal. Calcd. for C25H22F2N6: C, 67.55; H,
4.99; N, 18.91. Found: C, 67.61; H, 4.95; N, 18.87.
5.1.4.2. 2-(2,4-Dichlorophenyl)-5-[5-(4-methylpiperazin-1-yl)-1H-
benzimidazol-2-yl]-1H-benzimidazole (6b). The ethanolic solution
of the hydrogenated product 5 (w0.21 g, w1.03 mmol), equivalent
amount of 2-(2,4-dichlorophenyl)-5-formylbenzimidazole (0.30 g,
1.03 mmol) and half equivalent of solution of Na2S2O5 (0.097 g,
0.515 mmol in 1.5 mL H2O) were treated as described for 6a and the
obtained residue was purified by column chromatography using
a gradient of 70e90% MeOH/EtOAc. The pure product was isolated
as a yellow solid (0.314 g, 0.66 mmol 65% yield); Rf ¼ 0.24 in 100%
MeOH; mp ¼ 189.8e194.2 ꢂC; FTIR (KBr, cmꢁ1): 3434.62, 1629.81,
1587.09, 1422.04, 1286.88, 1143.47, 1102.72, 1047.72, 820.09; 1H
NMR (300 MHz, DMSO)
d
ppm ¼ 2.24 (s, 3H, CH3), 2.50 (s, 4H, CH2),
3.12 (s, 4H, CH2), 6.94 (d, 1H, AreH, J ¼ 8.2), 7.02 (s, 1H, AreH), 7.44
(s, 1H, AreH), 7.64 (d, 1H, AreH, J ¼ 7.8), 7.78 (s, 1H, AreH), 7.87
(s, 1H, AreH), 7.98 (d, 1H, AreH, J ¼ 8.16), 8.09 (d, 1H, AreH, J ¼ 6.6),
8.40 (s, 1H, AreH), 12.65 (br s, exch, 1H, NH); 13C NMR (75 MHz,
DMSO)
d
ppm ¼ 45.7, 49.9, 54.8, 113.7, 116.3, 118.6, 121.08, 127.7,
128.6, 129.9, 132.7, 133.3, 135.2, 147.7, 149.5, 151.2; ESI-MS: m/z
found 476.9579 (M þ H); exact mass 476.13. Anal. Calcd. for
C25H22Cl2N6: C, 62.90; H, 4.64; N, 17.60. Found: C, 62.94; H, 4.60; N,
17.56.
5.1.4.3. 2-(3,4-Dimethoxyphenyl)-5-[5-(4-methylpiperazin-1-yl)-1H-
benzimidazol-2-yl]-1H-benzimidazole (6c). The ethanolic solution
of the hydrogenated product 5(w0.36 g, w1.77 mmol), equivalent
amount of 2-(3,4-dimethoxyphenyl)-5-formylbenzimidazole (0.50 g,
1.77 mmol) and half equivalent of solution of Na2S2O5 (0.168 g,
0.885 mmol in 3 mL H2O) were treated as described for 6a and the
obtained residue was purified by column chromatography using
a gradient of 100% MeOH. The pure product was isolated as a brown
orange solid (0.51 g, 1.09 mmol 62% yield); Rf ¼ 0.15 in 100% MeOH;
mp ¼ 253.7e254.8 ꢂC; FTIR (KBr, cmꢁ1): 3390.72, 1635.70, 1500.18,
1437.35, 163.87, 1226.25, 1146.13, 1021.17, 981.73, 811.22, 627.63; 1H
Fig. 5. Effects of DNA binding compounds on human topoisomerase I. Purified human
topoisomerase I was incubated with pBS (SKþ) plasmid 500 ng in the presence and
absence of compounds Hoechst 33342, 6a, 6b, 6c, 6d and Camptothecin at 37 ꢂC. Lane
1, pBS (SKþ) plasmid; lane 2, plasmid þ human topoisomerase I, two units; lanes 3e9,
incubation of plasmid with 1, 5, 10, 25, 50, 75 and 100 mM of compound (Campto-
thecin or Hoechst 33342 or 6aed) for 30 min at 37 ꢂC with simultaneous addition of
two units of human topoisomerase I; lane 10, incubation of plasmid with 100
compound without addition of topoisomerase I.
mM of
in ethanol was treated with 10% Pd/C (130 mg) and mixture was
hydrogenated at rt under 42 psi H2 pressure. The reaction was fol-
lowed by TLC. After 2 h, the expected volume of hydrogen had been
taken up (14 psi) and reaction mixture was colorless. Reaction
mixture was filtered through Celite and filtrate was used for next step
without further purification; Rf ¼ 0.14 in 40% methanol in EtOAc.
NMR (300 MHz, DMSO)
d
ppm ¼ 1.77 (s, 3H, CH3), 2.21 (s, 4H, CH2),
2.49 (s, 4H, CH2), 3.83 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.91 (d, 1H,
AreH, J ¼ 8.7), 6.92 (s, 1H, AreH), 7.12 (d, 1H, AreH, J ¼ 8.1), 7.43
(d, 1H, AreH, J ¼ 8.7), 7.67 (d, 1H, AreH, J ¼ 8.4), 7.82 (m, 2H, AreH),
8.00 (d, 1H, AreH, J ¼ 8.4), 8.31 (s, 1H, AreH), 12.91 (br s, exch, 1H,
NH); 13C NMR (75 MHz, DMSO)
d
ppm ¼ 45.8, 49.9, 54.9, 55.6, 109.9,
5.1.4. General synthesis of 2-aryl-5-[5-(4-methylpiperazin-1-yl)-
1H-benzimidazol-2-yl]-1H-benzimidazole
5.1.4.1. 2-(2,4-Difluorophenyl)-5-[5-(4-methylpiperazin-1-yl)-1H
benzimidazol-2-yl]-1H-benzimidazole (6a). To a ethanolic solution
111.7, 119.6, 120.6, 122.61, 124.4, 147.6, 148.9, 150.4, 151.6, 153.06; ESI-
MS: m/z found 469.25 (M þ H); exact mass 468.23. Anal. Calcd. for
C27H28N6O2: C, 69.21; H, 6.02; N, 17.94. Found: C, 69.25; H, 6.06;
N, 17.89.

668
M. Singh, V. Tandon / European Journal of Medicinal Chemistry 46 (2011) 659e669
5.1.4.4. 2-(3,4,5-Trimethoxyphenyl)-5-[5-(4-methylpiperazin-1-yl)-
1H-benzimidazol-2-yl]-1H-benzimidazole (6d). The ethanolic solu-
tion of the hydrogenated product 5 (w0.26 g, w1.28 mmol), equiva-
lent amount of 2-(3,4,5-trimethoxyphenyl)-5-formylbenzimidazole
(0.40 g, 1.28 mmol) and half equivalent of solution of Na2S2O5
(0.121 g, 0.64 mmol in 2 ml H2O) were treated as described for 6a and
the obtained residue was purified by column chromatography using
a gradient of 80e95% MeOH/EtOAc. The pure product was isolated as
solid (0.433 g, 0.87 mmol 68% yield); Rf ¼ 0.21 in 100% MeOH,
mp ¼ 260.2e262.5 ꢂC; FTIR (KBr, cmꢁ1): 3400.50, 1591.16, 1464.32,
1426.86,1238.37,1127.55, 997.76, 727.09; 1H NMR (300 MHz, DMSO)
5.2.4. Cell culture
Cells were routinely maintained in DMEM (D-7777) medium
supplemented with 10% heat-inactivated fetal calf serum (FCS),
5 U mLꢁ1 insulin, 100 U mLꢁ1 streptomycin, 100 g mLꢁ1 penicillin
m
and 45 m
g mLꢁ1 gentamicin. The cells were grown at 37 ꢂC, 5% CO2,
95% air and 84% relative humidity for 24 h prior to addition of
experimental compounds. The final concentration of methanol was
1% in the culture medium (v/v) (Methanol at this concentration did
not affect the viability of the cells).
5.2.5. MTT assay for cell viability
d
ppm ¼ 1.18 (s, 3H, CH3), 2.26 (s, 4H, CH2), 2.53 (s, 4H, CH2), 3.75
Various human tumor cells (U87, HeLa and MCF7) were main-
tained as monolayer at 37 ꢂC in 5% CO2 using DMEM medium.
Approximately 3000e8000 cells/well were seeded in 96-well
(s, 3H, OCH3), 3.93 (s, 6H, OCH3), 6.95 (d, 1H, AreH, J ¼ 6), 7.03 (s, 1H,
AreH), 7.46 (d,1H, AreH, J ¼ 8.2), 7.60 (s,1H,AreH), 7.72 (d,1H,AreH,
J ¼ 4.5), 8.04 (d, 1H, AreH, J ¼ 5.4), 8.34 (s, 2H, AreH); 13C NMR
plates containing 200
culture medium was replaced by fresh medium containing 1, 5, 10
and 20 M concentration of 6a, 6b, Hoechst, Camptothecin and 1,
10, 50, 100 M of 6c and 6d and incubated for 24, 48 and 72 h. The
mL of medium and incubated for 24 h. The
(75 MHz, DMSO)
d
ppm ¼ 45.5, 49.8, 54.7, 56.1, 60.1, 100.3, 104.1,
112.4, 113.6, 115.4, 120.9, 124.6, 125.2, 139.1, 147.6, 151.4, 152.7, 153.2,
164.5; ESI-MS: m/z found 499.1372 (M þ H); exact mass 498.24. Anal.
Calcd. for C28H30N6O3: C, 67.45; H, 6.06; N, 16.86. Found: C, 67.49; H,
5.98; N, 16.91.
m
m
cell viability was determined by the MTT assay following the
procedure described by Price and McMillan [39]. The light absor-
bance was measured at 570 nm wavelength using a microplate
reader (Infinite M200; Tecan Group Ltd., Männedorf, Switzerland).
5.2. Biological investigation
5.2.6. Plasmid relaxation assay
5.2.1. Materials
The type IB DNA topoisomerase was assayed by decreasing
mobility of the relaxed isomers of supercoiled pBS(SK þ ) DNA in an
All cell culture reagents were obtained from HIMEDIA (India).
DMEM, Hoechst 33342, Camptothecin (CPT) and 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were
purchased from Sigma Chemicals (St Louis, MO). The human
cancerous cell lines U87, HeLa and MCF7 were procured from
National Center for Cell Science, Pune, India. An extinction coeffi-
cient of 6420 Mꢁ1 cmꢁ1 was used to measure the nucleotide
concentration of CT-DNA (Calf thymus DNA, Pharmacia) solution.
The stock solution of bisbenzimidazoles was made at 10 mM
concentration in methanol. The molar extinction coefficients were
found to be 3334 ¼ 22,600 for 6a, 3334 ¼ 23,800 for 6b, 3340 ¼ 26,200
for 6c, 3340 ¼ 24,400 for 6d and 3340 ¼ 42,000 Mꢁ1 cmꢁ1 for Hoechst
in methanol at 25 ꢂC. Working stocks were prepared fresh before
each experiment from these by dilution with buffer (20 mM
sodium cacodylate buffer, 100 mM NaCl, pH 7.2).
agarose gel. The reaction mixture (25 mL) contained 25 mM Tris HCl,
pH 7.5, 5% glycerol, 50 mM KCl, 0.5 mM DTT, 10 mM MgCl2,
30
g mLꢁ1 BSA, 0.5
g {pBSK II(þ)} two units of enzyme (one unit
is defined as the amount of enzyme required to convert 50% of
m
m
0.5 mg supercoiled DNA substrate to the relaxed form under stan-
dard assay conditions). Reactions were carried out at 37 ꢂC for
30 min with an increasing concentration of 6aed and Hoechst (0, 1,
5, 10, 25, 50, 75, 100
EDTA, 0.5% SDS, 0.25
m
m
M) and then terminated by adding 10 mM
g mLꢁ1 bromophenol blue and 15% glycerol.
The samples were electrophoresed in a horizontal 1% agarose gel in
trisborate/EDTA buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8) at
1.5 V/cm for 14e16 h at rt. DMSO or Methanol concentrations in
each reaction were maintained at 1% by the addition of serially
diluted drug stocks so as not to produce solvent mediated inhibi-
tion of topoisomerase I. The gels were stained with ethidium
bromide (5 m
g mLꢁ1), destained in water and photographed under
UV illumination at alpha imager 2200.
5.2.2. Absorption titration
DrugeDNA interaction studies were done using spectroscopic
technique and their binding pattern was determined by the
changes in their absorption spectra. Increasing concentration of CT-
Acknowledgements
DNA (5, 10, 15, 20
mM) was titrated into a fixed concentration of
compounds (6aed and Hoechst) i.e., 5
m
M in 20 mM sodium
Funding: This work was funded by the Swedish International
Development Agency (Sweden) and Department of Biotechnology
cacodylate buffer, 100 mM NaCl, pH 7.2. These measurements were
performed at DNA/drug ratio, r ¼ 1e4. Absorption spectra were
recorded by UVevis Spectrophotometer (Cary Varian 300).
DrugeDNA solution was equilibrated for 15 min and spectral
measurements were recorded using 1 cm path length quartz cell at
25 ꢂC.
(India). MS received
a Junior Research Fellowship from the
Department of Science and Technology and Senior Research
Fellowship from Council of Scientific and Industrial Research
(New Delhi, India).
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Equilibrium binding experiments were conducted using a on
a Fluoromax-4 (JobinYvon) instrument at temperature 25 ꢂC. A
solution of ligand 6aed and Hoechst (100 nm concentrations) was
excited at its respective lmax (slits of 5 nm), and resulting emission
curves (from 350 to 650 nm) were recorded after serial additions
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