Synthesis and biological activity evaluation of 1H-benzimidazoles via mammalian DNA topoisomerase I and cytostaticity assays

Article abstract of DOI:10.1016/j.ejmech.2008.06.018

Benzimidazoles are important compounds because of their antibacterial, antifungal, antimicrobial, antiprotozoal and antihelmintic activities. Some benzimidazole derivatives also interfere with the reactions of DNA topoisomerases, enzymes functioning at al

Full text of DOI:10.1016/j.ejmech.2008.06.018

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 2280e2285
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological activity evaluation of 1H-benzimidazoles via
mammalian DNA topoisomerase I and cytostaticity assays
c
c
Gunes Coban a,1, Sevil Zencir b,1,2, Istvan Zupko , Borbala Rethy ,
´
´
´
´
H. Semih Gunes a, Zeki Topcu d,
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey
b Department of Medical Biology, School of Medicine, Pamukkale University, Denizli 20020, Turkey
c Department of Pharmacodynamics and Biopharmacy, University of Szeged, Szeged H-6720, Hungary
d Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Ege University, Izmir 35100, Turkey
Received 15 April 2008; received in revised form 18 June 2008; accepted 20 June 2008
Available online 26 June 2008
Abstract
Benzimidazoles are important compounds because of their antibacterial, antifungal, antimicrobial, antiprotozoal and antihelmintic activities.
Some benzimidazole derivatives also interfere with the reactions of DNA topoisomerases, enzymes functioning at almost all stages of the cell
cycle. In this study, nine 1H-benzimidazole derivatives with substituents at positions 2 and 5 were synthesized and the structure of the com-
pounds was elucidated by instrumental methods. The characterized compounds were screened to identify if they interfered with mammalian
type I DNA topoisomerase activity via in vitro supercoil relaxation assays. Selected compounds were subjected to cytostatic assays using
HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells. Our results showed that 5-
chloro-2-(2-hydroxyphenyl)-1H-benzimidazole exerted the most profound topoisomerase I inhibition and cytotoxicity.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: 1H-Benzimidazole derivatives; Synthesis; Type I DNA topoisomerase; MTT assay; Plasmid Supercoil relaxation assays
1. Introduction
recombination and transposition [8,9]. These enzymes are
classified as type I (topo I) and type II (topo II) topoiso-
merases according to the reaction mechanisms; type I topoi-
somerases catalyze topological changes in duplex -DNA by
reversibly nicking one strand, whereas type II enzymes cata-
lyze the transient breakage of both strands simultaneously [9].
DNA topoisomerases were shown to be important targets for sev-
eral chemotherapeutic agents [7,10e14]. In this study, nine 1H-
benzimidazole derivatives with substituents at positions 2 and
5 were synthesized, namely: 2-(1H-benzimidazol-2-yl) phenol
(I), 2-[2-(2-morpholin-4-ylethoxy)phenyl]-1H-benzimidazole
(II), 2-[2-(2-piperidin-1-ylethoxy)phenyl]-1H-benzimidazole
(III), 2-[2-(2-pyrrolidin-1-ylethoxy)phenyl]-1H-benzimidazole
Benzimidazoles are important agents found in many bioac-
tive compounds from natural and synthetic sources. These
compounds have been intensively studied over last years be-
cause of their antibacterial [1], antifungal [2], antimicrobial
[3], antiprotozoal [4] and antihelmintic [5] activities. Some
benzimidazole derivatives are also active as type I DNA top-
oisomerase inhibitors [6,7]. DNA topoisomerases are essential
enzymes that regulate conformational changes in DNA topology
by transient breakage of nucleic acid backbone during many
genetic processes, including DNA replication, transcription,
* Corresponding author. Tel.: þ90 232 388 4000x1931; fax: þ90 232 388
(IV),
5-chloro-2-[2-(2-morpholin-4-ylethoxy)phenyl]-1H-
5258.
benzimidazole (V), 5-chloro-2-[2-(2-piperidin-1-ylethoxy)-
phenyl]-1H-benzimidazole (VI), 5-chloro-2-[2-(2-pyrrolidin-
1-ylethoxy)phenyl]-1H-benzimidazole (VII), 2-[2-(5-chloro-
1H-benzimidazol-2-yl)phenoxy]-N,N-diethylethanamine (VIII)
E-mail address: zeki.topcu@ege.edu.tr (Z. Topcu).
These authors had equal contribution.
1
2
Current address: Department of Biochemistry, Faculty of Science, Ege
University, Izmir 35100, Turkey.
0223-5234/$ - see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.06.018

G. Coban et al. / European Journal of Medicinal Chemistry 44 (2009) 2280e2285
2281
O
O
H
C
A
CHO
OH
H
O
- Na+
S
+
NaHSO3
O
OH
O
O
H
C
B
NH2
NH2
R
H
N
O
- Na+
+
S
O
N
H
R
OH
HO
R
R
N
N
R1
+
Cl
N
H
N
H
O
HO
R1
R1
-
R
C
I
H
II
H
-N
O
O
III
IV
V
H
H
-N
-N
-N
-N
-N
Cl
Cl
Cl
VI
VII
VIII
IX
Cl
Cl
-N(C2H5)2
-
Fig. 1. The synthesis pathway and the structures of the compounds.
and 2-(5-chloro-1H-benzimidazol-2-yl)phenol (IX) (Figs. 1 and
2). Following characterization of a number of parameters includ-
ing melting points, UV, IR, 1H NMR, 13C NMR and mass spec-
tral data, the compounds were evaluated for their interference
with topo I reactions. Selected derivatives were subjected to
the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide]) assay in vitro against three human tumoral cell lines:
HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma)
and A431 (skin epidermoid carcinoma).
10 min at room temperature. The mixture was filtered and so-
dium hydroxy (2-hydroxyphenyl)methane sulfonate salt
(86%) was obtained from the crude extract (Fig. 1, panel
A). The crude salt (0.01 mol) and o-phenylenediamine
(0.01 mol) in 30 mL of dimethyl formamide (DMF) were re-
fluxed for 2 h at 150 ꢀC in an oil bath and the mixture was
poured in an ice bath. 2-(2-Hydroxyphenyl)-1H-benzimid-
azole (53.5%) was filtered and recrystallized from methanol/
water (Fig. 1, panel B) [15]. 5-Chloro-2-(2-hydroxyphenyl)-
1H-benzimidazole (43.73%) was synthesized with 4-chloro-
o-phenylenediamine as described and recrystallized from
methanol/water [16]. After reacting 2-(2-hydroxyphenyl)-
1H-benzimidazole (0.005 mol) and 4-(2-chloroethly) morpho-
line (0.01 mol) with sodium hydroxide (0.015 mol) in 10 mL
ethanol, the mixture was refluxed at 95 ꢀC for 2 h in an oil
bath. Ethanol was evaporated and the residue was extracted
with ether. The compounds II (7.7%) and V (8.14%) were ob-
tained by column chromatography and crystallization with
2. Experimental
2.1. Synthesis of the benzimidazole derivatives
The preparation of the substituted benzimidazoles is out-
lined in Fig. 1 [15,16]. Briefly, 0.04 mol of 2-hydroxybenzal-
dehyde and 0.04 mol of sodium bisulfide, dissolved in 20 mL
of ethanol and water, respectively, were mixed and stirred for

2282
G. Coban et al. / European Journal of Medicinal Chemistry 44 (2009) 2280e2285
2.3. Plasmid supercoil relaxation assays
A
1
2
3
4
5
6
7
8
9
10
11
Plasmid supercoil relaxation assays were carried out as de-
scribed [17]. Briefly, 20 mL of reaction mixture contained
1 unit of calf thymus topoisomerase I, 0.5 mg of supercoiled
(sc) pBR322 (Takara, Otsu-Shiga, Japan), in the presence or ab-
sence of the test compounds in 35 mM TriseHCl (pH 8.0),
72 mM KCl, 5 mM MgCl2, 5 mM DTT, 5 mM spermidine,
and 0.1% bovine serum albumin. A stock solution of 10 mg/
mL CPTin DMSO was serially diluted for comparison purposes.
The relaxation products were analyzed on 1% agarose gels in
TBE buffer (45 mM Tris Borate and 1 mM EDTA, pH 8.0) in
a horizontal electrophoresis apparatus (5 V/cm) (Thermo
EC250) and photographed under UV light after staining in ethid-
ium bromide (EtdBr) solution (0.5 mg/mL). The relationship be-
tween the binding of EtdBr and the amount of fluorescencegiven
by sc and relaxed DNA (rlx DNA) under UV light was carried
out as described [11]. DNA bands were quantified from gel
photo images using BioRad Multianalyst (version 1.1). One
unit of the enzyme activity was defined as the activity removing
the supercoils from 500 ng of sc plasmid substrate at 37 ꢀC.
B 100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
2.4. Cytostatic assays
cpd I
cpd IV
cpd IX
Antiproliferative effects of the test compounds were mea-
sured in vitro on three human tumoral cell lines: HeLa (cervix
adenocarcinoma), MCF7 (breast adenocarcinoma) and A431
(skin epidermoid carcinoma), by using the MTT ([3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay
[18]. Briefly, cancer cells (5000/well) were seeded onto
a 96-well microplate and attached to the bottom of the well
overnight. On the second day, 200 mL of new medium contain-
ing the test substances was added. After incubation for 72 h,
the living cells were assayed by the addition of 20 mL of
5 mg/mL MTT solution. MTT was converted by intact mito-
chondrial reductase and precipitated as blue crystals during
a 4 h contact period. The medium was then removed, and
the precipitated crystals were dissolved in 100 mL of dimethyl
sulfoxide (DMSO) during a 60 min period of shaking. Finally,
the reduced MTT was assayed at 545 nm, using a microplate
reader; wells with untreated cells were utilized as controls.
All in vitro experiments were carried out on two microplates
with at least five parallel wells. Doxorubicin was used as pos-
itive control. Stock solutions of the tested substances (30 mM)
were prepared with DMSO. The highest DMSO concentration
(0.3%) of the medium did not have any significant effect on
the cell proliferation. Their antiproliferative effects were
determined in the concentration range 0.1e30 mM, the
doseeresponse curves were fitted by means of the computer
program GraphPad Prism 4 (GraphPad Software, San Diego,
CA, USA) and the IC50 values were calculated.
Fig. 2. (A) A representative assay using 1 mg/mL concentration of the test com-
pounds in topo I reactions, lane 1, pBR322 DNA without enzyme; lane 2, su-
percoil relaxation with 1 unit of DNA topoisomerase I; lanes 3e11, same as
lane 2 in the presence of the test compounds from I to IX, respectively. (B)
Quantitative assessment of the inhibition obtained with the compounds (see
Section 2 for the details).
methanol/water. 4-(2-Chloroethly) morpholine was replaced
with 1-(2-chloroethly) piperidine for the synthesis of com-
pounds III (10.29%) and VI (7.45%), 1-(2-chloroethly)pyrro-
lidine for the synthesis of compounds IV (6.84%) and VII
(9.70%), and 2-chloro-N,N-diethlyethanamine for the synthe-
sis of compound VIII (11.18%) (Fig. 1, panel C).
2.2. Characterization of the benzimidazole derivatives
Melting points were determined with a capillary melting
point apparatus (Buchi 510, BUCHI, Flawil, Switzerland).
The IR spectra of compounds were monitored as potassium
bromide pellets (FT/IR-430, JASCO, Tokyo, Japan). The
NMR spectra (400 MHz for 1H and 100 MHz for 13C) were re-
corded in CD3OD and CDCl3 (AS 400 Mercury Plus NMR
Varian, Palo Alto, USA). Chemical shifts were measured as
parts per million (d). The J values, given in Hz. Mass spectra
(CI MS) were measured in methanol solution (HP 6890 Series
GC System Mass spectrometer and HP 6890 Mass Selective
Detector, Hewlett Packard, Palo Alto, USA). UV spectra
were taken on a spectrometer in methanol solution (UV-160,
Shimadzu, Kyoto, Japan). Analytical thin-layer chromatogra-
phy (TLC) was run on Merck silica gel plates (Kieselgel
60F254) with detection by UV light. All starting materials
and reagents were high-grade commercial products.
3. Results and discussion
The results of chemical analyses of the synthesized 1H-
benzimidazole derivatives are summarized in Tables 1 and 2.
Table 1 contains the data for the melting point, UV spectra,

G. Coban et al. / European Journal of Medicinal Chemistry 44 (2009) 2280e2285
2283
Table 1
Results of the chemical analyses of the compounds IeIX
Compound
m.p. (ꢀC)
UV lmax (nm)
IR nmax (cmꢁ1
)
CI MS [M þ 1]þ
I
237
157
130
115
180
144
141
126
277
329, 318, 292, 213
311, 293, 211
301, 211
3325, 2924, 1602, 1490, 1452, 961
211
325
323
309
359
357
343
345
245
II
3302, 2961, 2817, 1604, 1584, 1470, 1450, 955
3291, 2933, 2852, 1583, 1462, 1443, 942
3065, 2965, 2820, 1603, 1581, 1459, 1440, 960
3314, 2963, 2823, 1603, 1579, 1461, 1422, 948
3276, 2938, 2824, 1602, 1582, 1459, 1422, 955
3220, 2963, 2815, 1602, 1581, 1459, 1420, 965
3071, 2964, 2822, 1602, 1582, 1460, 1420, 951
3325, 2923, 1600, 1584, 1489, 1420, 964
III
IV
V
302, 211
315, 215
VI
VII
VIII
IX
301, 212
308, 212
305, 212
332, 321, 216
1
IR spectra and mass spectroscopy. Our data in Table 1 show
that the ethylenic bands at 205e210 nm and benzenoid bands
at 250e350 nm are in total agreement with the literature data
[19e21]. The IR spectra obtained in solid phase at 2400e
3200 cmꢁ1 are characteristic for benzimidazole derivatives
(Table 1) [22]. H NMR results are given in Table 2. The ar-
omatic proton signals of o-hydroxy phenyl substituent at posi-
tion 2 were observed within prospective chemical shift values
and divisions while the hydrogen atoms at positions 4 and 7
were not detected at the prospective divisions [23]. The
Table 2
1H and 13C NMR results for the compounds IeIX
Compound
NMR
I
1H NMR 7.00 (1H, td, J ¼ 1.2, 7.4 Hz, H-50), 7.02 (1H, d, J ¼ 8.4 Hz, H-30), 7.27 (2H, m, H-5, H-6), 7.37 (1H, td, J ¼ 1.6, 7.8 Hz,
H-40), 7.59 (1H, d, J ¼ 7.6 Hz, H-7), 7.70 (1H, d, J ¼ 7.6 Hz, H-4), 8.04 (1H, dd, J ¼ 1.6, 8 Hz, H-60), 13.15e13.12 (1H, bs, NeH)
13C NMR 113.3 (C-10), 117.9 (C-50), 119.8 (C-30), 123.5 (C-5, C-6), 126.9 (C-60), 132.4 (C-40), 152.4 (C-2), 158.7 (C-20)
1H NMR 2.66 (4H, t, J ¼ 4.8 Hz, H-3000, H-5000), 2.94 (2H, t, J ¼ 5.6 Hz, H-200), 3.84 (4H, q, J ¼ 4.8 Hz, H-2000, H-6000), 4.34 (2H, t,
J ¼ 5.6 Hz, H-100), 7.02 (1H, dd, J ¼ 0.8, 7.6 Hz, H-30), 7.16 (1H, td, J ¼ 1.2, 7.6 Hz, H-50), 7.26 (1H, t, J ¼ 5.6 Hz, H-6*), 7.28
(1H, t, J ¼ 6.4 Hz, H-5*), 7.41 (1H, td, J ¼ 2, 7.8 Hz, H-40), 7.67 (2H, bs, H-4, H-7), 8.59 (1H, dd, J ¼ 2, 7.8 Hz, H-60)
13C NMR 53.5 (C-200), 57.6 (C-3000, C-5000), 64.1(C-100), 67.0 (C-2000, C-6000), 112.9 (C-4, C-7, C-30), 119.0 (C-10), 122.3 (C-50), 122.7
(C-5, C-6), 130.7 (C-60), 131.1 (C-3a, C-7a, C-40), 150.1 (C-2), 156.3 (C-20)
II
III
IV
V
1H NMR 1.55 (2H, m, H-4000), 1.72 (4H, m, H-3000, H-5000), 2.60 (4H, bs, H-2000, H-6000), 2.87 (2H, t, J ¼ 5.6 Hz, H-200), 4.33 (2H, t,
J ¼ 5.6 Hz, H-100), 7.02 (1H, d, J ¼ 8.4 Hz, H-30), 7.15 (1H, t, J ¼ 7.6 Hz, H-50), 7.25 (1H, t, J ¼ 6.6 Hz, H-6*), 7.26 (1H, t,
J ¼ 6.4 Hz, H-5*), 7.40 (1H, td, J ¼ 2, 7.8 Hz, H-40), 7.66 (1H, bs, H-7), 7.68 (1H, bs, H-4), 8.54 (1H, dd, J ¼ 1.6, 7.8 Hz, H-60)
13C NMR 11.2 (C-4000), 46.8 (C-3000, C-5000), 54.4 (C-2000, C-6000), 57.7 (C-200), 64.7 (C-100), 113.1 (C-4, C-7, C-30), 119.7 (C-10), 122.2
(C-50), 122.4 (C-5, C-6), 130.7 (C-60), 131.1 (C-40, C-3a, C-7a), 150.3 (C-2), 156.4 (C-20)
1H NMR 1.93 (4H, m, H-2000, H-5000), 2.78 (4H, m, H-3000, H-4000), 3.04 (2H, t, J ¼ 5.4 Hz, H-200), 4.33 (2H, t, J ¼ 5.2 Hz, H-100), 7.02
(1H, d, J ¼ 8.4 Hz, H-30), 7.15 (1H, td, J ¼ 1.2, 7.5 Hz, H-50), 7.23 (1H, t, J ¼ 6.6 Hz, H-6*), 7.24 (1H, t, J ¼ 7.2 Hz, H-5*), 7.39
(1H, td, J ¼ 2, 7.7 Hz, H-40), 7.60 (2H, bs, H-4, H-7), 8.50 (1H, dd, J ¼ 1.6, 8 Hz, H-60)
13C NMR 24.0 (C-3000, C-4000), 53.6 (C-2000, C-5000), 55.3 (C-200), 67.3 (C-100), 113.9 (C-4, C-7, C-30), 120.2 (C-10), 122.3 (C-5, C-6, C-
50), 130.7 (C-60), 130.9 (C-3a, C-7a, C-40), 150.4 (C-2), 156.7 (C-20)
1H NMR 2.67 (4H, t, J ¼ 4.7 Hz, H-3000, H-5000), 2.96 (2H, t, J ¼ 5.4 Hz, H-200), 3.83 (4H, t, J ¼ 4.7 Hz, H-2000, H-6000), 4.35 (2H, t,
J ¼ 5.5 Hz, H-100), 7.03 (1H, d, J ¼ 8 Hz, H-30), 7.18 (1H, dd, J ¼ 0.8, 7.6 Hz, H-50), 7.23 (1H, t, J ¼ 1.6, 8.8 Hz, H-6), 7.42 (1H, td,
J ¼ 1.6, 7.8 Hz, H-40), 7.52 (1H, bs, H-4), 7.58 (1H, bs, H-7), 8.53 (1H, dd, J ¼ 1.6, 8 Hz, H-60)
13C NMR 53.5 (C-200), 57.5 (C-3000, C-5000), 64.0 (C-100), 66.9 (C-2000, C-6000), 112.9 (C-4, C-7, C-30), 118.8 (C-10), 122.4 (C-50), 123.2
(C-6), 128.2 (C-5), 130.7 (C-60), 131.6 (C-3a, C-7a, C-40), 151.2 (C-2), 156.2 (C-20)
VI
VII
VIII
IX
1H NMR 1.56 (2H, m, H-4000), 1.70 (4H, m, H-3000, H-5000), 2.59 (4H, bs, H-2000 H-6000), 2.86 (2H, t, J ¼ 5.2 Hz, H-200), 4.32 (2H, t,
J ¼ 5.2 Hz, H-100), 6.99 (1H, d, J ¼ 8 Hz, H-30), 7.14 (1H, t, J ¼ 7.8 Hz, H-50), 7.19 (1H, dd, J ¼ 1.6, 8.4 Hz, H-6), 7.38 (1H, td,
J ¼ 1.6, 7.8 Hz, H-40), 7.70 (1H, bs, H-7), 7.75 (1H, bs, H-4), 8.49 (1H, dd, J ¼ 1.6, 7.8 Hz, H-60), 12.03 (1H, bs, NH)
13C NMR 24.5 (C-4000), 25.9 (C-3000, C-5000), 54.4 (C-200), 57.7 (C-2000, C-6000), 64.7 (C-100), 113.0 (C-4, C-7, C-30), 119.4 (C-10), 122.2
(C-6), 123.0 (C-50), 130.8 (C-60), 131.3 (C-3a, C-7a, C-40), 151.5 (C-2), 156.4 (C-20)
1H NMR 1.91 (4H, m, H-3000, H-4000), 2.78 (4H, bs, H-2000, H-5000), 3.04 (2H, t, J ¼ 5.2 Hz, H-200), 4.33 (2H, t, J ¼ 5.2 Hz, H-100), 7.02
(1H, d, J ¼ 8.8 Hz, H-30), 7.15 (1H, t, J ¼ 7.4 Hz, H-50), 7.19 (1H, dd, J ¼ 1.2, 8.6 Hz, H-6), 7.40 (1H, td, J ¼ 2, 7.8 Hz, H-40),
7.80e7.56 (2H, 2ꢂ bs, H-4, H-7), 8.55 (1H, dd, J ¼ 1.6, 7.8 Hz, H-60)
13C NMR 24.0 (C-3000, C-4000), 53.6 (C-2000, C-5000), 55.3 (C-200), 67.3 (C-100), 114.0 (C-4, C-7, C-30), 119.9 (C-10), 122.4 (C-50), 122.8
(C-6), 130.7 (C-60), 131.3 (C-3a, C-7a, C-40), 151.5 (C-2), 156.7 (C-20)
1H NMR 1.15 (6H, t, J ¼ 7.6 Hz, H-2000), 2.81 (4H, q, J ¼ 7.6 Hz, H-1000), 2.96 (2H, t, J ¼ 5.2 Hz, H-200), 4.31 (2H, t, J ¼ 5.2 Hz, H-
100), 7.05 (1H, d, J ¼ 8 Hz, H-30), 7.15 (1H, t, J ¼ 7.6 Hz, H-50), 7.20 (1H, dd, J ¼ 1.6, 7.6 Hz, H-6), 7.40 (1H, td, J ¼ 2, 7.6 Hz, H-
40), 7.70 (1H, bs, H-4), 7.78 (1H, bs, H-7), 8.50 (1H, dd, J ¼ 1.6, 7.6 Hz, H-60), 12.85 (1H, bs, NeH)
13C NMR 11.1 (C-2000), 46.7 (C-1000), 51.6 (C-200), 65.8 (C-100), 114.0 (C-4, C-7, C-30), 119.8 (C-10), 122.5 (C-50), 122.9 (C-6), 130.6
(C-60), 131.3 (C-3a, C-7a, C-40), 151.2 (C-2), 156.5 (C-20)
1H NMR 7.01 (1H, td, J ¼ 1.2, 7.8 Hz, H-30), 7.02 (1H, dd, J ¼ 1.2, 7.4 Hz, H-50), 7.28 (1H, d, J ¼ 6.8 Hz, H-6), 7.38 (1H, td,
J ¼ 1.2, 7.8 Hz, H-40), 7.78e7.61 (2H, 2ꢂ bs, H-4, H-7), 8.04 (1H, dd, J ¼ 1.6, 7.8 Hz, H-60), 13.25e12.71 (1H, br s, NeH)
13C NMR 113.3 (C-10), 117.9 (C-50), 120 (C-30), 123.7 (C-6), 127.3 (C-60), 132.8 (C-40), 153.5 (C-2), 158.5 (C-20)

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G. Coban et al. / European Journal of Medicinal Chemistry 44 (2009) 2280e2285
compounds substituted with the terminal of ethoxy chain at
position 2 of phenyl group gave rise to similar chemical shift
values and divisions at the ring of the benzimidazole nucleus
as I and IX (Table 2). The lack of the signal belonging to
3a, 4, 7 and 7a carbon in 13C NMR is noteworthy in that it
may suggest a proton exchange due to 1,3-tautomerization
(Table 2) [23].
A431 cells
A 100
Cpd I
Cpd IV
Cpd IX
75
50
25
0
We employed plasmid supercoil relaxation assays to iden-
tify if the synthesized derivatives interfere with topo I reac-
tions. Our assay uses sc plasmid DNA and relies on the
ability of topoisomerase I to rlx DNA, which can be separated
as discrete bands using gel electrophoresis. An inhibition of
the relaxation activity due to the presence of a particular inter-
fering agent is monitored in the form of an accumulated faster-
migrating sc DNA (form I). A representative gel analysis of
supercoil relaxation activity of DNA topoisomerase I in the
presence of 1 mg/mL of the compounds IeIX is given in
Fig. 2. As seen in Fig. 2A, the sc DNA (Fig. 2A, lane 1)
was fully relaxed by the enzyme (Fig. 2A, lane 2). Relaxation
of sc plasmid substrate, pBR322, was inhibited upon incuba-
tion with the compounds I, IV and IX (Fig. 2A, lanes 3, 6
and 11, respectively) while the other compounds, II, III, V,
VI, VII and VIII (Fig. 2A, lanes 4, 5,and 7e9, respectively)
did not interfere with the enzyme’s ability to relax the super-
coils. We previously reported that the enzyme’s relaxation ac-
tivity was not affected by DMSO [7]. We quantified the
percent values of the sc vs. rlx DNA bands using the gel photo
images for these three compounds (Fig. 2B). The average
quantitative values showed that the strongest inhibition was
obtained with I. The percent inhibition for I, IV and IX was
found to be 95.4%, 90.2% and 99%, respectively (Fig. 2B).
When we made a serial comparison of topo I reactions using
decreased concentrations of the test compounds, the inhibition
diminished gradually, which shows that the effects were con-
centration-dependent (data not shown).
0.0
0.5
1.0
Log concentration (µM)
1.5
2.0
MCF7 cells
B
100
75
50
25
0
Cpd I
Cpd IV
Cpd IX
0.0
0.5
1.0
Log concentration (µM)
1.5
2.0
Fig. 3. The graphical representation of the cytostaticity obtained by the com-
pounds I, IV and IX. (A) The plots are given as the inhibitory percent vs. the
concentration for the test compounds against A431 cell line using the calcu-
lated exact concentrations at which the cell proliferation is inhibited by
50%. (B) The same parameter plotted for the results obtained with MCF7
cell line.
Many reports in recent years indicated that interference
with normal DNA topoisomerase functions coincide with cyto-
toxic properties for several naturally occurring and synthetic
compounds [10,14]. We therefore subjected I, IV and IX to
MTT assay in HeLa (cervix adenocarcinoma), MCF7 (breast
adenocarcinoma) and A431 (skin epidermoid carcinoma) cells
to identify a possible correlation, if any. The results of cyto-
static analyses are summarized in Table 3. We also plotted
the percent effect for A431 and MCF7 cell lines by calculating
the exact concentrations at which the cell proliferation is in-
hibited by 50% (Fig. 3). The most effective compounds in
this set of compounds were IX as it exhibited relatively low
calculated IC50 values on all three cell lines (Table 3 and
Fig. 3). The compound I was a strong inhibitor in topo I relax-
ation but it gave rise to a high IC50 (Fig. 3). A slight change in
the inhibitory activity was manifested upon substitution with
chlorine at the 5-position of benzimidazole nucleus (data not
shown). On the other hand, IV, which also gave a high degree
of interference in topo I reactions, was not active as a cytostatic
agent (Table 3 and Fig. 3). This differs from II, III, V and VI
by the presence of either a 5C ring within the amine structure
at the terminal of ethoxy chain or chlorine atom (Fig. 1). Be-
cause of the solubility concern, VIII was not differentiated
with the same parameters.
The compounds interfering with DNA topoisomerases, in
general, are not structurally related to each other. This is
most probably due to the presence of multiple mechanisms
in the course of topoisomerase inhibition. Our results showed
that the topo-inhibition obtained by the three compounds in
topo I reactions was not correlated to their cytostatic effects.
Therefore, the interpretation of the effects of I, IV and IX in
both topo reactions and cytotoxicity requires further kinetic
analyses.
Table 3
The results of the cytostaticity assays for the compounds I, IV and IX
Calculated IC50 values (mM)
Compound
A431
HeLa
MCF7
I
IV
>30
>30
26.45
19.82
6.04
16.03
>30
6.94
IX
Doxorubicin
6.16
0.19
0.16
0.31

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