Influence of phenyl ring disubstitution on bisbenzimidazole and terbenzimidazole cytotoxicity: Synthesis and biological evaluation as radioprotectors

Article abstract of DOI:10.1021/jm030114w

DNA minor groove binders, Hoechst 33258 and Hoechst 33342, have been reported to protect against radiation-induced DNA-strand breakage, but their mutagenicity and cytotoxicity limit their use as protectors of normal tissue during radiotherapy and as biological radioprotectors during accidental radiation exposure. On the basis of these observations, two new nontoxic disubstituted benzimidazoles were synthesized, one having two methoxy groups (5-(4-methylpiperazin-1-yl)-2-[2′-(3,4-dimethoxyphenyl) -5′-benzimidazolyl]benzimidazole, 5) and another having a methoxy and a hydroxyl group (5-(4-methylpiperazin-1-yl)-2-[2′{2″-(4-hydroxy-3-methoxyphenyl) -5″-benzimidazolyl}-5′-benzimidazolyl]benzimidazole, 6) ortho to each other on the phenyl ring. The radiomodifying effects of these nontoxic ligands were investigated with a human glioma cell line exposed to low linear energy transfer radiation by determining cell survival and cell proliferation compared with effects of the parent compound, Hoechst 33342. Cytotoxicity assayed by analyzing clonogenicity, cell growth, and metabolic Viability showed that both 5 and 6 were nontoxic at 100 μM after 72 h of exposure, whereas Hoechst 33342 resulted in lysis of 77% of these cells in 24 h. Macrocolony assay (clonogenicity) showed that 73%, 92%, and 10% of the cells survived when treated with 100 μM 5, 6, and Hoechst 33342, respectively. Both 5 and 6 did not affect the growth of BMG-1 cells. At 10 μM, 5 and 6 showed 82% and 37% protection against radiation-induced cell death (macrocolony assay) while 100% protection was observed against growth inhibition. Disubstitution of the phenyl ring has not only reduced cytotoxicity but also enhanced DNA-ligand stability, conferring high degree of radioprotection.

Full text of DOI:10.1021/jm030114w

J . Med. Chem. 2003, 46, 3785-3792
3785
Articles
In flu en ce of P h en yl Rin g Disu bstitu tion on Bisben zim id a zole a n d
Ter ben zim id a zole Cytotoxicity: Syn th esis a n d Biologica l Eva lu a tion a s
Ra d iop r otector s
Urmila Tawar,† Akash K. J ain,† B. S. Dwarakanath,‡ Ramesh Chandra,† Yogendra Singh,†,§ N. K. Chaudhury,‡
Divya Khaitan,‡ and Vibha Tandon*,†
Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India, Institute of Nuclear Medicine
and Allied Sciences, Brig. S. K. Majumdar Road, Delhi 110054, India, and Institute of Genomics and Integrative Biology,
Mall Road, Delhi 110007, India
Received March 10, 2003
DNA minor groove binders, Hoechst 33258 and Hoechst 33342, have been reported to protect
against radiation-induced DNA-strand breakage, but their mutagenicity and cytotoxicity limit
their use as protectors of normal tissue during radiotherapy and as biological radioprotectors
during accidental radiation exposure. On the basis of these observations, two new nontoxic
disubstituted benzimidazoles were synthesized, one having two methoxy groups (5-(4-
methylpiperazin-1-yl)-2-[2′-(3,4-dimethoxyphenyl)-5′-benzimidazolyl]benzimidazole, 5) and an-
other having a methoxy and a hydroxyl group (5-(4-methylpiperazin-1-yl)-2-[2′{2′′-(4-hydroxy-
3-methoxyphenyl)-5′′-benzimidazolyl}-5′-benzimidazolyl]benzimidazole, 6) ortho to each other
on the phenyl ring. The radiomodifying effects of these nontoxic ligands were investigated with
a human glioma cell line exposed to low linear energy transfer radiation by determining cell
survival and cell proliferation compared with effects of the parent compound, Hoechst 33342.
Cytotoxicity assayed by analyzing clonogenicity, cell growth, and metabolic viability showed
that both 5 and 6 were nontoxic at 100 µM after 72 h of exposure, whereas Hoechst 33342
resulted in lysis of 77% of these cells in 24 h. Macrocolony assay (clonogenicity) showed that
73%, 92%, and 10% of the cells survived when treated with 100 µM 5, 6, and Hoechst 33342,
respectively. Both 5 and 6 did not affect the growth of BMG-1 cells. At 10 µM, 5 and 6 showed
82% and 37% protection against radiation-induced cell death (macrocolony assay) while 100%
protection was observed against growth inhibition. Disubstitution of the phenyl ring has not
only reduced cytotoxicity but also enhanced DNA-ligand stability, conferring high degree of
radioprotection.
In tr od u ction
interactions,18 and these interactions are suggested to
contribute to overall bonding affinity.19 The hydrophobic
interaction with DNA bonding sites is more likely to
represent the main driving force for complex forma-
tion.20 Synthesis of a number of bisbenzimidazoles
equipped with various side chains have been re-
ported.21-26 The central part of the parent bisbenzimi-
dazole has also been modified with the aim of designing
compounds capable of recognizing GC containing se-
quences.27,28 Recently several bisbenzimidazoles and
terbenzimidazoles have been identified as topoisomerase
I inhibitors.29-39
DNA ligands such as bisbenzimidazoles Hoechst
33258 and Hoechst 33342 form strong and noncovalent
bonds with the adenine- and thymine-rich regions in the
minor groove of DNA, significantly altering chromatin
structure.1,2 Dyes Hoechst 33258 and Hoechst 33342 are
frequently used in cytometry to stain chromosomal
materials in situ because these two compounds become
highly fluorescent upon bonding to DNA.3 It has been
known for a long time that Hoechst 33258 bonds
specifically to AT-rich sequences in DNA.4-8 NMR and
X-ray crystal structures of Hoechst 33258 bound to
different oligonucleotide duplexes reveal that the drug
fits into the minor groove of the double helix for a
sequence of four contiguous AT base pairs.9-17 The
Hoechst 33258-DNA interaction appears to be stabi-
lized by several H-bonding and van der Waals bonding
Hoechst 33258 and also Hoechst 33342 have been
shown to protect nonbonded as well as genomic DNA
against radiation-induced damage.40 Administration of
these compounds prior to irradiation affords protection
against the formation of primary strand breaks in
aqueous solutions of DNA as well as in the intact cell
nucleus. These DNA-ligand interactions have also been
observed to reduce radiation-induced cytogenetic dam-
age and cell death in cell cultures and to protect whole
body irradiated animals.41-43 However, postirradiation
* To whom correspondence should be addressed. Telephone: 91-11-
27666272, extension 248. Fax: 91-11-27666248. E-mail address:
vibhadelhi@hotmail.com.
† University of Delhi.
‡ Institute of Nuclear Medicine and Allied Sciences.
§ Institute of Genomics and Integrative Biology.
10.1021/jm030114w CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/26/2003

3786 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Tawar et al.
Sch em e 1
treatment of cells with these ligands has been observed
to enhance cell death in vitro.44 Free radical scavenging
and quenching of DNA radicals appear to be the
mechanisms responsible for the protection of DNA
against radiation-induced damage by Hoechst com-
pounds,44-47 while mechanisms involved in the enhance-
ment of radiation-induced cell death, when administered
after irradiation, are not clearly understood.
The mutagenic, clastogenic, and cytotoxic effects of
these ligands arising from DNA lesions caused by
topoisomerase inhibition, resulting in altered gene
expression and DNA repair inhibition, limit their use
as radioprotectors in humans. Therefore, the develop-
ment of DNA bonding ligands (minor groove binding
ligands particularly) that afford a radioprotective effect
without causing mutagenic and cytotoxic effects can
play a significant role in biological radioprotection.
In the present study, detailed synthesis of two benz-
imidazoles 5-(4-methylpiperazin-1-yl)-2-[2′-(3,4-dimeth-
oxyphenyl)-5′-benzimidazolyl]benzimidazole 5 and 5-(4-
methylpiperazin-1-yl)-2-[2′{2′′-(4-hydroxy-3-methoxyphe-
nyl)-5′′benzimidazolyl}-5′-benzimidazolyl]benzimida-
zole 6 and the relative structure-activity of 3,4-di-
methoxybisbenzimidazole and 4-hydroxy-3-methoxyphe-
nylterbenzimidazole as radioprotective agents were
compared to those of Hoechst 33342 using a human
glioma cell line (BMG-1). Addition of an extra methoxy
group to the phenyl ring was expected to significantly
reduce cytotoxic effects in vitro.33,37 The reduced cyto-
toxicity of these ligands makes them promising candi-
dates as radioprotective agents. Effects on cell survival,
macrocolony formation, and growth inhibition have been
investigated as parameters relevant to protection against
radiation-induced damage. Results indicate that these
novel ligands not only demonstrate radioprotective
effects but also are not toxic under in vitro conditions.
dazol-2′-yl]-2-nitroaniline, which was hydrogenated over
5% Pd/C to give 1 in 100% yield. 4 was prepared by
condensing 3,4-diaminobenzonitrile with 3-methoxy-4-
hydroxybenzaldehyde (vanillin), a methodology de-
scribed by La Voie et al.34-37 The nitrile group was
reduced to a formyl group using a Ni-Al alloy to obtain
4 (Schemes 1 and 2).
Two chemical modifications were introduced into
bisbenzimidazole and terbenzimidazole. For bisbenz-
imidazole, two methoxy groups were introduced at the
phenyl ring, which are electron donors, whereas in the
terbenzimidazole, one of the methoxy groups was re-
placed by a hydroxyl group. These modifications pro-
duced increased lipophilicity but reduced cytotoxicity.
At the same time, these ligands were found to provide
better protection against radiation-induced cytotoxicity
than monosubstituted Hoechst analogues.
Resu lts a n d Discu ssion
Hoechst 33342 has been shown to be cytotoxic to
various types of mammalian cells.53 The increased
cytotoxicity of Hoechst 33342 has been suggested to be
due to lipophilicity of the molecule, which increases cell
penetration of the drug.29 Earlier studies have also
shown that substitution of the phenyl ring at the 5′-
position of terbenzimidazole increased its cytotoxicity
toward mammalian cells.33 Compounds 5 and 6 were
different from analogues synthesized by La Voie et
al.34-37 and Leupin et al.54 because they have a meth-
ylpiperazine ring at the 5-position and methoxy and
hydroxyl groups at the 3′- and 4′-positions on the phenyl
ring. It was suggested that substitution with a methoxy
group would increase the lipophilicity, leading to a
higher uptake by mammalian cells and resulting in
enhanced cytotoxicity. Besides a lipophilicity-linked
uptake, the intracellular concentration of the drug that
determines its toxicity is also regulated by the P-
glycoprotein (Pgp) mediated drug efflux.55 Therefore, our
results suggest that both 5 and 6 are handled by the
Pgp pump similar to Hoechst 33342. The lipophilicity
of 5 and 6 determined by measuring the uptake of these
compounds by lymphocytes isolated from Wistar rat
spleen was compared to that of Hoechst 33342 (Figure
1). The uptake of Hoechst 33342, 5, and 6 as indicated
by the fluorescence intensity of live lymphocytes sug-
gests that all three ligands enter cells and bond to
nuclear DNA. The difference in fluorescence intensity
after 1 h suggests that Hoechst 33342 and 5 are secreted
from cells in a similar fashion whereas 6 seems to
remain inside the cells for a longer time. It is notable
here that the inherent fluorescence of 5 and 6 is less
Ch em istr y
In general, the synthesis of 2-arylbenzimidazoles have
been effected by condensation of o-arylenediamine and
arylcarboxylic acids and their derivatives ester, amide,
imidate esters, chlorides, and anhydrides in the pres-
ence of a strong acid such as a mineral acid or poly-
phosphoric acid. 2-Arylbenzimidazole can also be ob-
tained from o-arylenediamines and aldehydes in the
presence of an oxidant.
The synthesis of 5 was carried out basically as
described by Kelly et al.48 using 2-amino-4- [5′-(4′′-
methylpiperazin-1′′-yl)benzimidazol-2′-yl]aniline (1) and
3,4-dimethoxybenzaldehyde in 27% overall yield. This
methodology is different from the method described by
Lown et al.49-51 and Douglas et al.52 where they have
condensed 2-arylbenzimidazole with o-arylenediamine.
6 was prepared using a methodology described by La
Voie et al.33 using an equimolar mixture of 1 and
5-formyl-[3-methoxy-4-hydroxybenzimidazole] (4) in ni-
trobenzene that was heated at 140-150 °C for 36 h. An
overall yield of 25% was obtained for 6. In this reaction,
the initially formed Schiff base undergoes oxidative
cyclization in nitrobenzene to give 6. 1 was synthesized
by condensing ethyl-4-amino-3-nitrobenzenecarboximi-
date hydrochloride48 and 5-(4′-methylpiperazin-1′yl)-2-
nitroaniline in dry ethanol/glacial acetic acid refluxed
for 1 h to give 4-[5′-(4′′-methylpiperazin-1′′-yl)benzimi-

Influence of Phenyl Ring Disubstitution
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3787
Sch em e 2
than that of Hoechst 33342, but upon bonding to DNA,
these two ligands show higher fluorescence in compari-
son to Hoechst 33342, Rf ) 16 and Rf ) 100, respec-
tively.61 It was observed that addition of a methoxy
group increases cellular uptake of these compounds in
comparison to Hoechst 33342. The cytotoxicity of these
ligands was investigated using a human glioma cell line
(BMG-1) by studying their effects on cell survival,
metabolic viability, and cell proliferation. For survival
and proliferation studies, cells were incubated with 5,
6, and Hoechst 33342 for 1 h and washed to remove the
drug, and growth was monitored as colony formation.
Cell survival was determined following the addition of
0.1, 1, 10, or 100 µM drug. At low concentrations, <1
µM, Hoechst 33342 had no effect on cell survival,
whereas at 10 or 100 µM, it killed 55% or 90% of these
cells, respectively. On the other hand, 6, even at 100
µM, did not show any adverse effect on cell survival
(Figure 2). Similarly, 5 was also not cytotoxic at
concentrations up to 10 µM, while 27% cells were killed
with the addition of 100 µM. Effects of 5 and 6 on the
proliferation of exponentially growing cells at 100 µM
were also studied. In these experiments, cells were
incubated with 5, 6, or Hoechst 33342 for 1 h, washed
to remove the drug, and incubated further for 24, 48,
and 72 h to observe growth. Kinetics of cell growth
treated with 5 or 6 did not differ significantly from the
kinetics of the nontreated cells, and doubling time was
comparable to that of the nontreated cells (Figure 3).
However, 100 µM Hoechst 33342 induced complete lysis
of these cells (Figure 3). Taken together, these results
suggest that 5 and 6 are significantly less toxic than
Hoechst 33342.
Cytotoxicity was further investigated by oxidation of
MTT as described earlier49,51,56 by incubating cells with
drugs continuously. It was observed that when 10 µM
Hoechst 33342 was incubated with BMG-1 cells for 72
h, 35% of the cells were killed whereas 100 µM 5 and 6
showed no cytotoxicity. Incubation of cells with 100 µM
Hoechst 33342 for 72 h showed complete lysis of cells.
Interestingly only a marginal increase in the cytotoxicity
F igu r e 2. Effect of 5 and 6 on the clonogenicity of BMG-1
cells exposed to various concentrations at 24, 48, and 72 h.
Cells were plated and allowed to form colonies in the presence
of various concentrations of drugs. Results are shown as the
percent of the control. 5 and 6 were found to have no effect on
clonogenicity of BMG-1 cells.
F igu r e 1. Cellular uptake of 10 µM Hoechst 33342, 5, and 6
by live lymphocytes isolated from Wistar rat spleen. Cells were
incubated with ligands for 15 min and 1 h and dropped on
clean chilled glass slides. Results are shown as the mean of
the fluorescence intensity for 100 cells in each group. Hoechst
33342 and 5 appear to be secreted by the Pgp mediated pump,
whereas 6 seemed to be retained inside these cells.
F igu r e 3. Effect of 100 µM 5 and 6 on growth kinetics of
BMG-1 cells. Cells were grown in the presence of these drugs,
and the number of proliferating cells were counted using a
hemocytometer at different time intervals. Results are shown
in terms of the number of cells (in millions per milliliter). 5
and 6 do not show any effect on the proliferation kinetics of
BMG-1 cells.

3788 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Tawar et al.
F igu r e 4. Cytotoxicity of 5 and 6 compared to Hoechst 33342 using the MTT assay of human brain malignant glioma cells,
BMG-1. Cells were grown in the presence of drugs at various concentrations up to 72 h, and the metabolic activity of these cells
was measured using MTT. Results are shown as the percent survival. 5 and 6 were found to be noncytotoxic even at 100 µM up
to 72 h.
Ta ble 1. DNA Binding Properties of the Ligands with CTDNA
was observed with 100 µM 5 and 6 (Figure 4). The
in 2 mM Sodium Cacodylate Buffer (pH 6.9)
cytotoxicity of Hoechst 33342 is primarily due to the
λex
λem
(nm)
Tm ∆Tm
stabilization of cleavable complexes, the protein-linked
DNA strand breaks, an intermediate process of DNA
topoisomerase action.57 Cleavable complexes have been
shown to induce interphase death via apoptosis58 as well
as mitotic death linked to the cytogenetic damage in the
form of chromosomal aberrations and micronuclei for-
mation.59 The absence of cell death with 5 and 6
observed here implies that either the intracellular
concentration of these ligands is significantly lower than
the cellular concentration of Hoechst 33342 or they do
not stabilize the cleavable complexes, unlike Hoechst
33342. However, significant differences in the uptake
of these ligands and Hoechst 33342 were not observed.
Therefore, it appears that the inability of these ligands
to stabilize cleavable complexes is mainly responsible
for reduced toxicity.
study system
CTDNA (100 µM)
(nm)
(°C) (°C)
70
CTDNA+ Hoechst 33342 (r ) 0.1a) 354 484
76
6
CTDNA + 5 (r ) 0.1a)
CTDNA + 6 (r ) 0.1a)
Hoechst 33342 (10 µM)
5 (10 µM)
354 484
356 391, 418, 441 78
340 500
340 500
350 500
76.4 6.4
8
6 (10 µM)
a
r is the ratio of drug to DNA (drug/DNA).
ability is not the only factor responsible for increased
cytotoxicity of Hoechst 33342 compared to Hoechst
33258.
The limitation of the parent Hoechst 33342 compound
being mutagenic and, as a result, cytotoxic prevents its
use as a radioprotective agent. Since 5 and 6 did not
possess these limitations, their potential as radiopro-
tective agents was explored and compared with that of
Hoechst 33342. Macrocolony assay was performed fol-
lowing irradiation with one of three radiation doses (2,
5, or 10 Gy) and incubation with medium containing
10 µM ligand (Figure 5). With a radiation dose of 2 Gy,
Hoechst 33342 produced 10% protection whereas 5 and
6 produced 24% protection, and with a radiation dose
of 5 Gy, Hoechst 33342 produced 15% cell survival, 5
produced 82% cell survival, whereas 6 produced 37%
cell survival. Strikingly, with 10 Gy, both 5 and 6
produced 100% protection with respect to irradiated
control cells, whereas Hoechst 33342 produced 15%
survival. Similar results were obtained for radiation-
induced growth inhibition, where 84% protection was
observed at 24 h with 5 whereas 6 produced 100%
The DNA bonding property of 5, 6, and Hoechst 33342
was determined with calf thymus DNA (CTDNA) by
thermal denaturation, absorption, and emission spec-
troscopy. Incubation of 10 µM drug with 100 µM CTDNA
was performed, and thermal denaturation spectra were
recorded. Results suggest that 5 stabilizes the duplex,
similar to Hoechst 33342, but 6 conferred higher duplex
stability (Table 1). In their DNA bonding activity,
Hoechst 33342 and 5, both being bisbenzimidazoles,
have similar properties whereas 6, a terbenzimidazole,
binds DNA in a different manner as evident from UV
and fluorescence spectrophotometry and thermal dena-
turation studies. The disubstituted 5 and 6 are more
lipophilic and have higher cell permeability compared
to Hoechst 33342, which again suggests that cell perme-

Influence of Phenyl Ring Disubstitution
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3789
to the stability and selectivity of these complexes.6,19
6
has a longer structure and is able to recognize more base
pairs that are evident through the relative fluorescence
enhancement and Tm studies. The higher bonding
affinity but noncytotoxicity of 6 is suggested to be due
to the cavities that remain after the bonding of 6 to DNA
that allow the religation as well as rotation of single-
stranded DNA to facilitate DNA replication. Since metal
chelation has been convincingly demonstrated to reduce
the radiation damage in a number of systems,60 the
possibilities of minor groove binding ligands to facilitate
this process is worth investigating.
F igu r e 5. Radioprotection afforded by 5 and 6 compared to
Hoechst 33342 studied by macrocolony assay. BMG-1 cells
were seeded in Petri dishes and exposed to radiation doses of
2, 5, and 10 Gy at room temperature. These irradiated cells
were plated and allowed to form colonies. Results are shown
as the percent of control cells irradiated at the respective doses.
5 and 6 are shown to be better radioprotectors compared to
Hoechst 33342.
Con clu sion
5 and 6 have electron-donating groups on the phenyl
ring compared to Hoechst 33342; thus, the complex
formed by these ligands with DNA have enhanced
stability and also selectivity, which may prevent the
formation of free radical species on these bases. These
ligands also scavenge the free radicals being generated
in the vicinity and thereby afford better protection
against radiation-induced DNA damage. Hence, it can
be concluded that disubstitution of electron-donating
groups on the phenyl ring of 5 and 6 enhances cellular
uptake but reduces cytotoxicity and affords better
radioprotection.
Exp er im en ta l Section
Ch em ica ls. Chemicals and reagents used in the present
studies were analytical reagent grade or equivalent and were
procured from Sigma Chemical Co., E. Merck (Germany), Fine
Chemicals (India), and Fluka Chemicals. Organic Solvents
were freshly distilled before use. The purity and homogeneity
of all organic compounds were confirmed using NMR and
reverse-phase HPLC. IR spectra were recorded with an FTIR-
8300 Shimadzu spectrophotometer. NMR spectral character-
ization of all synthetic compounds were performed with a
Bruker 300 MHz NMR instrument. The FAB mass spectra
were recorded on a J EOL SX 102/DA-6000 mass spectrometer
using m-nitrobenzyl alcohol (NBA) as the matrix. Mass spectra
of compound 6 were obtained with a MALDI Kratos Analytical
Kompact SEQ IV, U.K., mass spectrometer using R-cyano-4-
hydroxycinnamic acid as a matrix.
F igu r e 6. Radioprotective effect of 5 and 6 determined as
growth kinetics of BMG-1 cells after exposure to a 5 Gy
radiation dose. Cells were irradiated after treatment with a
ligand and then counted using a hemocytometer at 24, 48, and
72 h. Results are shown as cell number (in millions per
milliliter). BMG-1 cells showed similar proliferation kinetics
in the presence of 5 and 6 as control, whereas Hoechst 33342
caused cell killing after irradiation.
Gen er a l P r oced u r e. The synthesis of 4-[5′-(4′′-methylpi-
perazin-1′′-yl)benzimidazol-2′-yl]-2-nitroaniline and ethyl-4-
amino-3-nitrobenzenecarboximidate hydrochloride were per-
formed according to the procedures described by Kelly et al.48
and Lown et al.26-28
2-Am in o-4-[5′-(4′′-m eth ylp ip er a zin -1′′-yl)ben zim id a zol-
2′-yl]a n ilin e (1). A solution of 4-[5′-(4′′-methylpiperazin-1′′-
yl)benzimidazol-2′-yl]-2-nitroaniline (1.04 g, 30 mmol) in ethyl
acetate/methanol (80 mL of 2:1 mixture) was reduced with 5%
palladium on carbon (250 mg) at room temperature and
atmospheric pressure. When hydrogen uptake ceased, the
solution was filtered through a Celite bed and the filtrate was
concentrated without delay to afford the orange-brown diamine
1 in 100% yield.
3,4-Dia m in oben zon itr ile (2). A solution of 4-aminoben-
zonitrile (1.5 g, 9.4 mmol) in ethyl acetate/methanol (80:20)
was reduced over 10% Pd/C at room temperature and atmos-
pheric pressure. After the hydrogen uptake ceased, the solution
was filtered with the aid of Celite and the filtrate was
concentrated to dryness to obtain the desired diamine 2 in
100% yield.
5-Cya n o-2-[3-m eth oxy-4-h yd r oxyben zim id a zole]-5′-yl
(3). A solution of 1.224 g (9.2 mmol) of the diamine 2 and 1.4
g (9.2 mmol) of 3-methoxy-4-hydroxybenzaldehyde in nitroben-
zene was allowed to react in a three-necked round-bottom flask
under nitrogen at 140 °C. The reaction mixture was heated
for 18 h with stirring and nitrobenzene was then removed
protection (Figure 6). Bonding of these ligands to the
DNA is characterized by a blue shift in emission and
an increase in fluorescence intensity that is suggested
to be due to the displacement of water molecules from
the spine of hydration in DNA.12,13 Displacement of
water molecules minimizes free radical generation upon
radiolysis of water due to ionizing radiation, which is a
primary cause of radiation-induced damage. Both 5 and
6 produced high relative fluorescence enhancement
upon bonding to DNA (Rf ) 16 and Rf ) 100, respec-
tively),61 displacing the spine of hydration from the
DNA, and thus showed enhanced protection against
radiation compared to Hoechst 33342. The majority of
strand breaks in irradiated DNA may be produced by
radical species formed near the nucleic acid bases, which
subsequently abstract H atoms from deoxyribosyl nu-
cleic acid bases. Delocalization of the radical center on
these bases could involve the O2 of pyrimidines, and it
is clear that H atom donation is possible from these
ligands. The proximity of H atom donors (viz. benzimi-
dazole NHs) on the ligand and H-bond acceptors (viz.
thymine O2s and adenine N3s) on the DNA contributes

3790 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Tawar et al.
under reduced pressure to obtain the brown crude product 3.
The final product was then obtained using silica gel (60-120
mesh size) column chromatography with EtOAc/MeOH as
eluent: mp 226-230 °C; IR 3425.3 (O-H), 3263.3 (-NH),
2221.8 (-CN), 1278.7 (C-O-C) cm-1; 1H NMR δ (in ppm) 13.5
(b, 1H, -NH), 9.52 (s, 1H, -OH), 7.85 (d, 1H, C7), 7.82 (d,
1H, C6), 7.72 (s, 1H, C3′), 7.63 (d, 1H,C5′), 6.93 (d, 1H, C6′),
3.90 (s, 3H, -OCH3).
performed with asynchronously growing cells in the exponen-
tial growth phase (24 h after plating).
Cytotoxicity Assa y. Cytotoxicity was determined using the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide] assay49,51,56 using a 96-well microtiter plate. Three-
thousand cells per well were plated in 200 µL of the complete
medium, and treatment with these ligands was performed 24
h after plating. For IC50 determination, cells were exposed
continuously with varying concentrations of drug and MTT
assays were performed at the end of the fourth day. At the
end of the treatment, control and treated cells were incubated
with MTT at a final concentration of 0.5 mg/mL for 2 h at 37
°C and then the medium was removed. The cells were lysed
and the formazan crystals were dissolved using 150 µL of
DMSO. The absorbance was read at 570 nm using 630 nm as
the reference wavelength using an enzyme linked immunosor-
bent assay (ELISA) reader.
Ma cr ocolon y Assa y. BMG-1 cells were washed with
HBSS, plated, and grown for 4 days before harvesting using
0.05% trypsin. Depending on the treatments, 200-1200 cells
were plated in 90 mm Petri dishes and incubated at 37 °C in
a 5% CO2 humidified atmosphere for 8-10 days. Colonies were
fixed with methanol and stained with 1% crystal violet.
Colonies containing more than 50 cells were counted.
P r olifer a tion Kin etics. BMG-1 cells were seeded at 7000-
8000 cells/cm2, and their proliferation kinetics was studied at
24 h intervals following trypsinization and counting total cells
per flask using a hemocytometer.
Ir r a d ia tion P r oced u r es. Exponentially growing cells, 24
h after plating, were irradiated at room temperature in growth
medium with a Co-60 source (γ cell, AECL, Canada) at a dose
rate of 0.5-1.2 Gy/min. A concentration of 10 µM drug was
added 1 h before irradiation. Following this procedure, cells
were grown for various time intervals to study growth kinetics
and plated for macrocolony assay.
5-F or m yl-[3-m eth oxy-4-h yd r oxyben zim id a zole] (4). To
a solution of 1 g (3.77 mmol) (3) in 60 mL formic acid and 20
mL of water, Ni-Al alloy (3.96 g) was added in several
portions. The reaction mixture was heated at 95 °C for 4 h.
The hot mixture was filtered with the aid of Celite and washed
thrice with water. The solution was concentrated to dryness
and water added to the residue to obtain a white precipitate.
The pH of this suspension was adjusted to 9 by the dropwise
addition of 2 N NaOH. The product was obtained by extraction
with ethyl acetate. The ethyl acetate extract was dried over
Na2SO4 and concentrated in vacuo to give the yellow colored
compound in 50% yield: mp 260-264 °C; IR 3435.6 (-OH),
3194.55 (-NH), 1675.6 (-CHO), 1594, 1506, 1441.6, 1280.7
-1
cm
;
1H NMR δ (in ppm) 13.7 (b, 1H, -NH), 10.2 (s, 1H,
-CHO), 9.5 (s, 1H, -OH), 8.25 (d,1H, C6), 7.78 (s,1H, C4),
7.75 (d,1H, C7), 7.7 (s, 1H, C3′), 7.65 (d,1H, C5′), 6.92 (d,1H,
C6′), 3.92 (s,3H, -OCH3).
5-(4-Meth ylpiper azin -1-yl)-2-[2′-(3,4-dim eth oxyph en yl)-
5′-ben zim id a zolyl]ben zim id a zole (5). A solution of freshly
prepared diamine 1 (1.18 g, 3.67 mmol) and 3,4-dimethoxy-
benzaldehyde (0.61 g, 3.67 mmol) in 110 mL of nitrobenzene
is heated at 140-150 °C for 24 h. The solvent is then removed
under reduced pressure to give the final crude product as a
brown solid. The product is purified by column chromatogra-
phy on a Buchi 688 liquid (MPLC) pump using silica gel (70-
230 mesh size) and EtOAc/MeOH as eluent to give a yellow
compound that was characterized by spectroscopic tech-
niques: mp 220 °C; yield 60%; IR 3556, 2922, 1629, 1508, 1417,
Dr u g-DNA Bin d in g Stu d ies. CTDNA was purchased
from E. Merck, Germany, and used without further purifica-
tion. Absorbance of 5 (ꢀ340 ) 20 300) and 6 (ꢀ354 ) 16 600) was
compared with that of Hoechst 33342 in sodium cacodylate
buffer (2 mM, pH 7.2), and the absorbance spectra were
1
1371, 1296, 1022, 810 cm-1; H NMR (DMSO-d6) δ (in ppm)
2.24 (s, 3H, -NCH3), 2.46 (t, 4H,CH2, J ) 4 Hz), 3.5 (t, 4H,
CH2, J ) 4 Hz), 3.86 (s, 3H,OCH3), 6.77 (d, 1H, Ar-H, J ) 8
Hz), 7.13 (d, 2H, Ar-H, J ) 9 Hz), 7.64 (d, 1H, Ar-H, J ) 8.5
Hz), 7.78 (d, 1H, Ar-H, J ) 8 Hz), 8.03 (m, 1H, Ar-H), 8.17
(d, 2H, Ar-H, J ) 9 Hz), 8.3 (s, 1H, Ar-H), 13.0 (bs, 2H, NH).
D2O exchange resulted in the disappearance of the peak at δ
13.0. EIMS 467 (M+), 425, 411, 261, 235, 220, 194, 118, 91,
55, 44. C27H28N6O2: C ) 69.23 (calcd), 69.25 (obsd); H ) 5.98
(calcd), 5.97 (obsd); N ) 17.95 (calcd), 17.93 (obsd).
recorded with
a UV-visible GBC-916 spectrophotometer
(Australia). Drug-DNA solutions (drug/DNA ratio r ) 0.1)
were prepared in sodium cacodylate buffer (2 mM, pH 7.2).
Solutions of drug and DNA were incubated for 25 min for
equilibration. The spectral measurements were recorded using
a 1 cm path length quartz cell in the range 200-500 nm.
Fluorescence measurements were made with
a custom-
5-(4-Meth ylp ip er a zin -1-yl)-2-[2′{2′′-(4-h yd r oxy-3-m eth -
oxyp h en yl)-5′′-ben zim id a zolyl}-5′-ben zim id a zolyl]ben z-
im id a zole (6). A mixture of freshly prepared diamine 1 (218
mg, 0.82 mmol) and 4 (219 mg, 0.82 mmol) was dissolved in
30 mL of nitrobenzene and heated at 140 °C for 36 h. The
addition of 4 was performed as three additions to the reaction
mixture to obtain a better yield. Nitrobenzene was removed
under reduced pressure and the resulting solid was purified
by column chromatography (EtOAc/MeOH) on a Buchi 688
liquid (MPLC) pump using silica gel (70-230 mesh size) to
obtain the final product in 54% yield as a brown solid: mp
designed integrated steady-state and time-resolved spectro-
fluorimeter model FS900/FL900CDT Edinburgh Analytical
Instruments, U.K. The instrument parameters were as fol-
lows: λ ex ) 340 nm (for drugs) and λ ex ) 350 nm (for complex),
slit width ) 10 mm, EHT ) 6.7, gap ) 0.8 mm, frequency )
40 kHz. Data acquisition was based a on time-correlated
single-photon counting (TCSPC) technique. Melting temper-
ature (Tm) measurements were performed with a Cary UV-
vis spectrophotometer using 1 cm path length quartz cells
equipped with a thermoprogrammer. Absorbance was moni-
tored at 260 nm, while the temperature was raised from 25 to
100 °C at the rate of 0.5 °C/min. The transition melting
temperature, Tm, was determined as the midpoint of the
normalized curves.
Cellu la r Up ta k e by Lym p h ocytes. Lymphocytes were
isolated from Wistar rat spleen as single cell suspensions by
passing the isolate through Nylon wool. Isolated lymphocytes
were resuspended in HBSS and divided into three groups. The
viability of these cells was determined by staining them with
Trypan blue. A sample of 10 µM Hoechst 33342, 5, or 6 was
added to different groups, and cells were incubated for 15 min
and 1 h. Treated cells were dropped on clean, chilled glass
slides and mounted. The fluorescence intensity of 100 cells was
measured using a Nikon fluorescence microscope and the
software ImagePro Plus. The mean values of the intensity were
calculated and plotted as intensity vs ligand concentration for
15 min and for 1 h.
1
>290 °C; IR 3435, 3195, 1632, 1560, 1413, 1281; H NMR δ
(in ppm) 13.4-13.55 (b, 3H), 9.5 (s, 1H), 8.45 (s, 1H), 8.39 (s,
1H), 8.03-8.08 (m, 4H), 7.9 (d, 1H), 7.68 (d, 1H), 7.38 (d, 1H),
6.91-6.98 (m, 3H), 3.92 (s, 3H), 3.23 (t, 4H), 2.68 (t, 4H), 2.60
(s, 3H). The mass to charge ratio of 6 was observed with
MALDI: 570.3. C33H30N8O2: C ) 69.35 (calcd), 69.34 (obsd);
H ) 5.429 (calcd), 5.43 (obsd); N ) 19.62 (calcd), 19.64 (obsd).
Cell Cu ltu r e. The human glioma cell line BMG-1 (DNA
index of 0.96; wild-type TP 53) was established from a human
mixed glioma.62 Cells were maintained as monolayers at 37
°C in 25 cm2 tissue culture flasks (Tarsons, India) using
Dulbecco’s modified Eagle’s medium (Sigma) supplemented
with 5% fetal calf serum (Biologicals, Israel). Cells were
passaged routinely in the exponential growth phase twice a
week using 0.05% trypsin-EDTA solution in phosphate-
buffered saline (PBS) for trypsinization. All experiments were

Influence of Phenyl Ring Disubstitution
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18 3791
(19) Haq, I.; Ladbury, J . E.; Chowdhury, B. Z.; J enkins, T. C.;
Chaires, J . B. Specific Binding of Hoechst 33258 to the d(CG-
CAAATTTGCG)2 duplex: Calorimetric and spectroscopic studies.
J . Mol. Biol. 1997, 271, 244-257.
(20) Czarny, A.; Boykin, D. W.; Wood, A. A.; Nunn, C. M.; Neidle, S.;
Zhao, M.; Wilson, W. D. Analysis of van der Waals and
electrostatic contributions in the interactions of minor groove
binding benzimidazoles with DNA. J . Am. Chem. Soc. 1995, 117,
4716-4717.
(21) Wood, A. A.; Nunn, C. M.; Czarny, A.; Boykin, D. W.; Neidle, S.
Variability in DNA minor groove width recognized by ligand
binding: the crystal structure of a bisbenzimidazole compound
to the DNA duplex d(CGCGAATTCGCG)2. Nucleic Acids Res.
1995, 23, 3678-3684.
Ap p en d ix
Abbr evia tion s. DMA 5, 5-(4-methylpiperazin-1-yl)-
2-[2′-(3,4-dimethoxyphenyl)-5′-benzimidazolyl]benzimi-
dazole; TBZ 6, 5-(4-methylpiperazin-1-yl)-2-[2′{2′′-(4-
h ydr oxy-3-m et h oxyph en yl)-5′′-ben zim ida zolyl}-5′-
benzimidazolyl]benzimidazole; CTDNA, calf thymus
DNA; Tm, melting temperature; Gy, gray; Rf, relative
fluorescence enhancement; LET, linear energy transfer.
Refer en ces
(22) Clark, G. R.; Boykin, D. W.; Czarny, A.; Neidle, S. Structure of
a bis-amidinium derivative of Hoechst 33258 complexed to the
dodecanucleotide d(CGCGAATTCGC)2: the role of hydrogen
bonding in the minor groove drug-DNA interaction. Nucleic
Acids Res. 1997, 25, 1510-1515.
(23) Bostock-Smith, C. E.; Embrey, K. J .; Searle, M. S. DNA minor
groove recognition by a tetrahydropyrimidinium analogue of
Hoechst 33258: NMR and molecular dynamics studies of the
complex with d(GGTAATTACC)2. Nucleic Acids Res. 1998, 26,
1660-1667.
(24) Ebrahimi, S. E. S.; Parkinson, J . A.; Fox, K. R.; McKie, J . H.;
Barber, J .; Douglas, K. T. Studies of the interaction of a meta-
hydroxy analogue of Hoechst 33258 with DNA by melting
temperature, footprinting and high-resolution 1H NMR spec-
troscopy. J . Chem. Soc., Chem. Commun. 1992, 1398-1399.
(25) Clark, G. R.; Gray, E. J .; Neidle, S.; J i, Y.-H.; Leupin, W.
Isohelicity and phasing in drug-DNA sequence recognition:
Crystal structure of trisbenzimidazole-oligonucleotide complex.
Biochemistry 1996, 35, 13745-13752.
(26) Bathini, Y.; Rao, K. E.; Shea, R. G.; Lown, J . W. Molecular
recognition between ligands and nucleic acids: Novel pyridine-
and benzoxazole-containing agents related to Hoechst 33258 that
exhibit altered DNA sequence specificity deduced from foot-
printing analysis and spectroscopic studies. Chem. Res. Toxicol.
1990, 3, 268-280.
(1) Hilwig, I.; Gropp, A. Decondensation of consecutive heterochro-
matin in L cell chromosomes by a bisbenzimidazole compound
(Hoechst 33258). Exp. Cell Res. 1973, 81, 474-477.
(2) Loontiens, F. G.; Regenfuss, P.; Zechel, A.; Durmortier, L.; Clegg,
R. M. Binding characteristics of Hoechst 33258 with calf thymus
DNA, poly[d(A-T)] and [d(CCGGAATTCCGG)]: Multiple stoi-
chiometries and determination of tight binding with a wide
spectrum of site affinities. Biochemistry 1990, 29, 9029-9039.
(3) Stokke, T.; Steen, H. B. Multiple Binding Modes for Hoechst
33258 to DNA. J . Histochem. Cytochem. 1985, 33, 333-338.
(4) Portugal, J .; Waring, M. J . Assignment of DNA binding sites
for 4′,6-diamidino-2-phenylindole and bisbenzimide (Hoechst
33258). A comparative footprinting study. Biochim. Biophys.
Acta 1987, 949, 158-168.
(5) Pjura, P. E.; Grzeskoniak, K.; Dickerson, R. E. Binding of
Hoechst 33258 to the minor groove of B-DNA. J . Mol. Biol. 1987,
197, 257-271.
(6) Loontiens, F. G.; McLaughlin, L. W.; Diekmann, S.; Clegg, R.
M. Binding of Hoechst 33258 and 4′,6-diamidino-2-phenylindole
to self-complementary decadeoxynucleotides with modified exo-
cyclic base substituents. Biochemistry 1991, 30, 182-189.
(7) Quintana, J . R.; Lipanov, A. A.; Dickerson, R. E. Low-temper-
ature crystallographic analyses of the binding of Hoechst 33258
to the double-helical DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-
G. Biochemistry 1991, 30, 10294-10306.
(8) Sriram, M.; van der Marel, G. A.; Roelen, H. L. P. F.; van Boom,
J . H.; Wang, A. H.-J . Conformation of B-DNA containing O6-
ethyl-GC base pairs stabilized by minor groove binding drugs:
molecular structure of d(CGC[e6G]AATTCGCG) complexed with
Hoechst 33258 or Hoechst 33342. EMBO J . 1992, 11, 225-232.
(9) de C. T. Carronodo, M. A. A. F.; Coll, M.; Aymami, J .; Wang, A.
H.-J .; van der Marel, G. A.; van Boom, J . H.; Rich, A. Binding of
Hoechst dye to d(CGCGATATCGCG) and its influence on the
conformation of the DNA fragment. Biochemistry 1989, 28,
7849-7859.
(10) Searle, M. S.; Embrey, K. J . Sequence-specific interaction of
Hoechst 33258 with the minor groove of an adenine-tract DNA
duplex studied in solution by 1H NMR spectroscopy. Nucleic
Acids Res. 1990, 18, 3753-3762.
(11) Embrey, K. J .; Searle, M. S.; Craik, D. J . Probing the interaction
of Hoechst 33258 with an A-T rich oligonucleotide using 1H NMR
spectroscopy. J . Chem. Soc., Chem. Commun. 1991, 1770-1771.
(12) Embrey, K. J .; Searle, M. S.; Craik, D. J . Interaction of Hoechst
33258 with the minor groove of the A+T rich DNA duplex
d(GGTAATTACC)2 studied in solution by NMR spectroscopy.
FEBS Lett. 1993, 211, 437-447.
(13) Fede, A.; Labhardt, A.; Bannwarth, W.; Leupin, W. Dynamics
and binding mode of Hoechst 33258 to d(CGCGAATTCCAC)2
in the 1:1 solution complex as determined by two-dimensional
1H NMR. Biochemistry 1991, 30, 11377-11388.
(14) Parkinson, J . A.; Barber, J .; Douglas, K. T.; Rosamond, J .;
Sharples, D. Minor-groove recognition of the self-complementary
duplex (CGCGAATTCGCG)2 by Hoechst 33258: a high field
NMR study. Biochemistry 1990, 29, 10181-10190.
(15) Parkinson, J . A.; Barber, J .; Douglas, K. T.; Rosamond, J .;
Sharples, D. Molecular design of DNA-directed ligands with
specific interactions: Solution NMR studies of the interaction
of a meta-hydroxy analogue of Hoechst 33258 with d(CGC-
GAATTCGCG)2. Biochemistry 1994, 33, 8442-8452.
(16) Vega, M. C.; Garcia Saez, I.; Aymami, J .; Eritja, R.; Van der
Marel, G. A.; Van Boom, J . H.; Rich, A.; Coll, M. Three-
dimensional crystal structure of the A-tract DNA dodecamer
d(CGCAAATTTGCG) complexed with the minor-groove-binding
drug Hoechst 33258. Eur. J . Biochem 1994, 222, 721-726.
(17) Gavathiotis, E.; Sharman, G. J .; Searle, M. S. Sequence depend-
ent variation in DNA minor groove width dictates orientational
preference of Hoechst 33258 in A tract recognition, solution NMR
structure of the 2:1 complex with d(CTTTTGCAAAAG)2. Nucleic
Acids Res. 2000, 28, 728-735.
(18) Fede, A.; Billeter, M.; Leupin, W.; Wuthrich, K. Determination
of the NMR solution structure of the Hoechst 33258-d(GTG-
GAATTCCAC)2 complex and comparison with the X-ray crystal
structure. Structure 1993, 1, 177-186.
(27) Kumar, S.; J oseph, T.; Singh, M. P.; Bathini, Y.; Lown, J . W.
Sequence specific molecular recognition and binding by a GC
recognizing Hoechst 33258 analogue to the decadeoxynucleotide
d-[CATGGCCATG]2; structural and dynamic aspects deduced
from high field 1H NMR studies. J . Biomol. Struct. Dyn. 1990,
8, 331-357.
(28) Singh, M. P.; J oseph, T.; Kumar, S.; Bathini, Y.; Lown, J . W.
Synthesis and sequence-specific DNA binding of a topoisomerase
inhibitory analog of Hoechst 33258 designed for altered base and
sequence recognition. Chem. Res. Toxicol. 1992, 5, 597-607.
(29) Chen, A. Y.; Yu, C.; Bodley, A. L.; Peng, L. F.; Liu, L. A new
mammalian DNA topoisomerase I poison Hoechst 33342: Cy-
totoxicity and drug resistance in human cell cultures. Cancer
Res. 1993, 53, 1332-1337.
(30) Chen, A. Y.; Yu, C.; Gatto, B.; Liu, L. F. DNA minor groove-
binding ligands: A different class of mammalian DNA topo-
isomerase I inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
8131-8135.
(31) Beerman, T. A.; McHugh, M. M.; Sigmund, R.; Lown, J . W.; Rao,
K. E.; Bathini Y. Effects of analogs of the DNA minor groove
binder Hoechst 33258 on topoisomerase II and I mediated
activities. Biochim. Biophys. Acta 1992, 1131, 53-61.
(32) Sun, Q.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J .
Structure activity of topoisomerase I poisons related to Hoechst
33342. Bioorg. Med. Chem. Lett. 1994, 4, 2871-2876.
(33) Sun, Q.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J .
Synthesis and evaluation of terbenzimidazoles as topoisomerase
I inhibitors. J . Med. Chem. 1995, 38, 3638-3644.
(34) Kim, J . S.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J .
Substituted 2,5′-Bi-1H-benzimidazoles: Topoisomerase I inhibi-
tion and cytotoxicity. J . Med. Chem. 1996, 39, 992-998.
(35) Kim, J . S.; Sun, Q.; Yu, C.; Liu, L.; Liu, L. F.; LaVoie, E. J .
Quantitative structure-activity relationships on 5-substitued
terbenzimidazoles as topoisomerase I poisons and antitumor
agents. Bioorg. Med. Chem. 1998, 6, 163-172.
(36) Xu, Z.; Li, T.-K.; Kim, J . S.; LaVoie, E. J .; Breslauer, K. J .; Liu,
L. F.; Rich, D. J . DNA minor groove binding-directed poisoning
of human DNA topoisomerase I by terbenzimidazoles. Biochem-
istry 1998, 37, 3558-3566.
(37) Kim, J . S.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J . Terbenzimi-
dazoles: Influence of 2′′-, 4-, and 5-substituents on cytotoxicity
and relative potency as topoisomerase I poisons. J . Med. Chem.
1997, 40, 2818-2824.
(38) Mekapati, S. B.; Hansch, C. Comparative QSAR studies on
bibenzimidazoles and terbenzimidazoles inhibiting topoisomerae
I. Bioorg. Med. Chem. 2001, 9, 2885-2893.

3792 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 18
Tawar et al.
(39) Pilch, D. S.; Xu, Z.; Sun, Q.; LaVoie, E. J .; Liu, L. F.; Breslauer,
K. J . A terbenzimidazole that preferentially binds and confor-
mationally alters structurally distinct DNA duplex domains: A
potential mechanism for topoisomerase I poisoning. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 13565-13570.
(40) Denison, L.; Haigh, A.; D’Cunha, G. M.; Martin, R. F. DNA
ligands as radioprotectors: molecular studies with Hoechst
33342 and Hoechst 33258. Int. J . Radiat. Biol. 1992, 61, 69-
81.
(41) Lyubimova, N. V.; Coultas, P. G.; Yuen, K.; Martin, R. F. In vivo
radioprotection of mouse brain endothelial cells by Hoechst
33342. Br. J . Radiol. 2001, 74, 77-82.
(42) Singh, S. P.; J ayanth, V. R.; Chandna, S.; Dwarakanath, B. S.;
Adhikari, J . S.; J ain, V. Radioprotective effects of DNA ligands
Hoechst 33342 and 33258 in whole body irradiated mice. Indian
J . Exp. Biol. 1998, 36, 375-384.
(43) Young, S. D. Hill radiation sensitivity of tumour cells stained
in vitro and in vivo with the bisbenzimide fluorochrome Hoechst
33342. Br. J . Cancer 1989, 60, 715-721.
(44) Martin, R. F.; Denison, L. DNA ligands as radiomodifiers:
Studies with minor-groove binding bisbenzimidazoles. Int. J .
Radiat. Oncol. Biol. Phys. 1992, 23, 579-586.
(45) Adhikary, A.; Bothe, E.; J ain, V.; von Sonntag, C. Inhibition of
radiation-induced DNA strand breaks by Hoechst 33258: OH
radical scavenging and DNA radical quenching. Radioprotection
1997, 32, 89.
(46) Adhikary, A.; Bothe, E.; J ain, V.; von Sonntag, C. Pulse radiolysis
of the DNA-binding bisbenzimidazole derivatives Hoechst 33258
and 33342 in aqueous solutions. Int. J . Radiat. Biol. 2000, 76,
1157-1166.
(47) Martin, R. F.; Anderson, R. F. Pulse radiolysis studies indicate
that electron transfer is involved in radioprotection by Hoechst
33342 and methylprolamine. Int. J . Radiat. Oncol. Biol. Phys.
1998, 42, 827-831.
(48) Kelly, D. P.; Bateman, S. A.; Martin, R. F.; Reum, M. E.; Rose,
M.; Whittaker, A. R. D. DNA binding compounds V Synthesis
and characterization of boron-containing bibenzimidazoles re-
lated to the DNA minor groove binder, Hoechst 33258. Aust. J .
Chem. 1994, 47, 247-262.
(49) Wang, H.; Gupta, R.; Lown, J . W. Synthesis, DNA binding,
sequence preference and biological evaluation of minor groove
selective N1-alkoxyalkyl-bis-benzimidazoles. Anti-Cancer Drug
Des. 1994, 145-157.
(50) Yadagiri, B.; Lown, J . W. Convenient routes to substituted
benzimidazoles and imidazolo[4,5-b]pyridines using nitrobenzene
as oxidant. Synth. Commun. 1990, 20, 955-963.
(51) Singh, A. K.; Lown J . W. Design, synthesis and antitumor
cytotoxicity of novel bis-benzimidazoles. Anti-Cancer Drug Des.
2000, 15, 265-275.
(52) Ebrahimi, S. E. S.; Wilton, A. N.; Douglas, K. T. Unique binding
site for bis-benzimidazoles on transfer RNA. Chem. Commun.
1991, 385-386.
(53) Turner, P. R.; Denny, W. A. The mutagenic properties of DNA
minor-groove binding ligands. Mutat. Res. 1996, 355, 141-169.
(54) J i, Y.-H.; Bur, D.; Hasler, W.; Schmitt, V. R.; Dorn, A.; Bailly,
C.; Waring, M. J .; Hochstrasser, R.; Leupin, W. Tris-benzimi-
dazole derivatives: Design, synthesis and DNA sequence recog-
nition. Bioorg. Med. Chem. 2001, 9, 2905-2919.
(55) Wang, E.-j.; Casciano, C. N.; Clement, R. P.; J ohnson W. W. Two
transport binding sites of P-glycoprotein are unequal yet con-
tingent: initial rate kinetics analysis by ATP hydrolysis dem-
onstrates intersite dependence. Biochim. Biophys. Acta 2000,
1481, 63-74.
(56) Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J .
Immunol. Methods 1983, 65, 55-63.
(57) Capranico, G.; Binaschi, M.; Borgnetto, M. E.; Zunino, F.;
Palumbo, M. A protein-mediated mechanism for the DNA
sequence specific action of topoisomerase II poisons. Trends
Pharmacol. Sci. 1991, 18, 323-329.
(58) Ganapathi, R.; Vaziri, S. A. J .; Tabata, M.; Takigawa, N.;
Grabowski, D. R.; Bukowski, R. M.; Ganapathi, M. K. Inhibition
of NF-κâ and proteasome activity in tumors: Can we improve
the therapeutic potential of topoisomerase I and topoisomerase
II poisons. Curr. Pharm. Des. 2002, 8, 1945-1958.
(59) Kallio, M.; Lahdetie, J . Analysis of micronuclei induced in mouse
early spermatids by mitomycin C, vinblastine sulphate or
etoposide using fluorescence in situ hybridisation. Mutagenesis
1993, 8, 561-567.
(60) Sorenson, J . R. J . Cu, Fe, Mn, and Zn chelates offer a medicinal
chemistry approach to overcoming radiation injury. Curr. Med.
Chem. 2002, 9, 639-662.
(61) Tawar, U.; J ain, A. K.; Chandra, R.; Singh, Y.; Chaudhury, N.
K.; Dwarakanath, B. S.; Tandon, V. Submitted for publication
2002.
(62) Dwarakanath, B. S.; J ain, V. Energy linked modification of
radiation response in a human cerebral glioma cell line. Int. J .
Radiat. Oncol. Biol. Phys. 1986, 17, 1033-1040.
J M030114W