Topoisomerase I inhibition and cytotoxicity of 5-bromo- and 5-phenylterbenzimidazoles

Article abstract of DOI:10.1016/S0968-0896(00)00188-7

Topoisomerase I is an enzyme that is essential for maintaining the three-dimensional structure of DNA during the processes of transcription, translation and mitosis. With the introduction of new clinical agents that are effective in poisoning topoisomeras

Full text of DOI:10.1016/S0968-0896(00)00188-7

Bioorganic & Medicinal Chemistry 8 (2000) 2591±2600
Topoisomerase I Inhibition and Cytotoxicity of 5-Bromo- and
5-Phenylterbenzimidazoles
Meera Rangarajan,a Jung Sun Kim,a Sai-Peng Sim,b Angela Liu,b
Leroy F. Liub,c and Edmond J. LaVoiea,c,*
aDepartment of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA
bDepartment of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School,
Piscataway, NJ 08854, USA
cThe Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
Received 20 December 1999; accepted 6 July 2000
AbstractÐTopoisomerase I is an enzyme that is essential for maintaining the three-dimensional structure of DNA during the pro-
cesses of transcription, translation and mitosis. With the introduction of new clinical agents that are e€ective in poisoning topo-
isomerase I, this enzyme has proved to be an attractive molecular target in the development of anticancer drugs. Several
terbenzimidazoles have been identi®ed as potent topoisomerase I poisons. Structure±activity data on various terbenzimidazoles
have revealed that the presence of lipophilic substituents at the 5-position of various terbenzimidazoles correlates with enhanced
cytotoxicity. While the e€ect of having substituents at both the 5- and 6-positions had not been evaluated, previous studies did
indicate that the presence of a fused benzo-ring at the 5,6-position results in a signi®cant decrease in topoisomerase I poisoning
activity and cytotoxicity. In the present study we investigated whether substituents at both the 5- and 6-positions of varied terben-
zimidazoles would allow for retention of topo I poisoning activity. The 6-bromo, 6-methoxy, or 6-phenyl derivatives of both
5-bromo- and 5-phenylterbenzimidazole were synthesized and evaluated for topo I poisoning activity, as well as their cytotoxicity
toward human lymphoblastoma cells. The data indicate that such derivatives do retain similar topo I poisoning activity and possess
cytotoxicity equivalent to either 5-bromo- or 5-phenylterbenzimidazole. Signi®cant enhancement in the topoisomerase I poisoning
activity and cytotoxicity of 5-phenylterbenzimidazole is observed when the 200-position is substituted with either a chloro or tri-
¯uoromethyl substituent. The in¯uence of such substituents on the biological activity of 5,6-dibromoterbenzimidazole (6a) was also
explored. In the case of either 200-chloro-5,6-dibromoterbenzimidazole (6b) or 200-tri¯uoromethyl-5,6-dibromoterbenzimidazole (6c),
topoisomerase I poisoning was not enhanced relative to 6a. While cytotoxicity toward RPMI 8402 was also not signi®cantly
a€ected, comparative studies performed against several solid human tumor cell lines did reveal a signi®cant increase in cytotoxicity
observed for 6c as compared to 6a. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Several bibenzimidazoles and terbenzimidazoles have
been synthesized and identi®ed as topo I poisons.5 10
Extensive studies have been performed on the structure±
activity of terbenzimidazoles. Chart 1 lists several terben-
zimidazoles that have potent activity as topo I poisons
when evaluated in a subcellular assay. Structure±activity
data on these various terbenzimidazoles revealed that
the presence of lipophilic substituents at the 5-position
of various terbenzimidazoles did correlate with enhanced
cytotoxicity.8 10 Consequently, 1 and 2 were signi®cantly
less cytotoxic than 3a±c and 4a in the human lympho-
blastoma cell line, RPMI 8402. These di€erences in
cytotoxic activities have been attributed to di€erences in
cellular penetration/uptake. Comparison of the relative
cytotoxic activities of 4-phenylterbenzimidazole and
5-phenylterbenzimidazole also demonstrated that the
DNA topoisomerases I and II are nuclear enzymes
necessary for maintaining the three-dimensional struc-
ture of DNA, especially during the processes of tran-
scription, translation and mitosis.1 4 Topoisomerase I
(topo I) causes transient single-strand breaks, relieving
torsional tension along the fork of replicating DNA.
Topoisomerase poisons produce their antitumor e€ects
by stabilizing the cleavable ternary complex consisting
of topo I enzyme, DNA, and drug.
*Corresponding author. Tel: +1-732-445-2674; fax: +1-732-445-
6312; e-mail: elavoie@rci.rutgers.edu
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00188-7

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M. Rangarajan et al. / Bioorg. Med. Chem. 8 (2000) 2591±2600
Table 1. 5-Phenylterbenzimidazoles and 5-bromoterbenzimidazoles
evaluated for topoisomerase I poisoning activity and cytotoxicity
Compound
R1
R2
R3
3a
3b
3c
4a
4b
4c
5
6a
6b
6c
7
Phenyl
Phenyl
Phenyl
Br
Br
Br
p-Cl-Phenyl
Br
Br
H
H
H
H
H
H
H
Br
Br
Br
Phenyl
OCH3
OCH3
H
Cl
CF3
H
OH
CH2CH2CH3
Chart 1.
CF3
H
Cl
CF3
H
H
Br
5-substituted analogue was signi®cantly more potent.9
While the e€ect of having substituents at both the 5-
and 6-position had not been evaluated, previous studies
did indicate that the presence of a fused benzo-ring at the
5,6-position was associated with a signi®cant decrease in
topoisomerase I poisoning activity and cytotoxicity.9
Phenyl
Br
Phenyl
8
9
H
presence of H2 in case of 12b and Raney Nickel in etha-
nol in case of 12a and 12c to provide the diamines 13a±c.
Compound 13a was reacted with 14 and 15 to give pro-
ducts 4b and 4c, respectively. Compound 13b was con-
densed with 16 to give 5. Reaction of 13c with 17
provided 6b. Aldehydes 14±16 were prepared from their
corresponding nitriles by HCOOH/Ni/Al reduction.
The requisite nitrile intermediates were obtained by
reacting 4-cyano-1,2-phenylenediamine with urea,15
propionaldehyde16 or tri¯uoroacetic acid.16 Aldehyde
17 was prepared as previously described.11
The objective of the present study was primarily focused
on determining whether substituents at both the 5- and
6-position of varied terbenzimidazoles would allow for
retention of their activity as topo I poisons. Speci®cally,
the in¯uences of 6-bromo, 6-methoxy, or 6-phenyl sub-
stituents attached to either 5-phenyl- or 5-bromo-
terbenzimidazole on topo I poisoning activity, as well as
their cytotoxicity toward human lymphoblastoma cells,
were examined. Several 200-substituted derivatives of 3a
were recently synthesized and evaluated as topo I poi-
sons and for cytotoxic activity.11 Enhanced cytotoxicity
and topo I poisoning activity were observed when a
chloro or tri¯uoromethyl substituent was at the 200-
position, as in 3b and 3c. In the present study, we also
examined whether, in the case of 5,6-dibromoterbenzi-
midazole, the presence of a chloro or tri¯uoromethyl
substituent at the 200-position would enhance its topo-
isomerase I poisoning activity and cytotoxicity.
The methods used for the synthesis of compounds 6a, 6c
and 7±9 are outlined in Scheme 2. The phenylenedi-
amines, 18±20, were prepared by reduction of their spe-
ci®c nitroaniline precursors. The nitroaniline precursor
for 18 was prepared by Suzuki coupling of 3,4-dibromo-
6-nitroaniline using phenylboronic acid, wherein both
of the bromine atoms were replaced by phenyl groups
using Pdꢀ as the catalyst.17 3-Bromo-4-methoxy-6-
nitroaniline was commercially available and was
reduced with Raney Nickel to give 19. Suzuki coupling
of 3-bromo-4-methoxy-6-nitroaniline provided 4-meth-
oxy-2-nitro-5-phenylaniline, which was converted to 20
by hydrogenation using Pd/C as catalyst. The o-
phenylenediamines 10c, 18, 19 and 20 were condensed
with 21 at 145 ꢀC to yield terbenzimidazoles 6a, 7, 8 and
9, respectively. Reaction of 10c with 22 provided 6c.
Chemistry
Comparative studies were performed on several 5-
phenyl- and 5-bromoterbenzimidazole derivatives. Table
1 summarizes the various terbenzimidazoles that were
evaluated in this study. Methods for the preparation of
3a±c and 4a have been previously described.8 11 Com-
pounds 4b, 4c, 5 and 6b were prepared as shown in
Scheme 1. Intermediates 10a±c were prepared by reduc-
tion of the requisite nitroaniline. A literature procedure
was employed for the preparation of 10c.12 The o-phe-
nylenediamines 10a±c were condensed with the 3,4-
dinitrobenzaldehyde, 11, to give 12a±c.13 3,4-Dinitro-
benzaldehyde was prepared from 3,4-dinitrobenzoic
acid by reduction to the alcohol with borane/THF and
subsequent oxidation of the alcohol with pyridinium
chlorochromate to the aldehyde.14 Reduction of the
dinitro intermediates was carried out using Pd/C in the
Aldehyde 21 was prepared as described in the litera-
ture.8 Compound 22 was synthesized as outlined in
Scheme 3. The 4-cyano-o-phenylenediamine (23) was
re¯uxed in tri¯uoroacetic acid for 6 h to give the 5-
cyano-2-tri¯uoromethylbenzimidazole.11,18 The result-
ing nitrile was reduced to the formyl derivative, 24, with
Ni/Al and formic acid in 41% yield. Compound 24 was
condensed with 23 and reduced to give 22 in 40%
yield.11

M. Rangarajan et al. / Bioorg. Med. Chem. 8 (2000) 2591±2600
2593
Scheme 1. (a) PhNO2, 145 ꢀC; (b) Pd/C; H2 or NiAl (Ra-Ni); EtOH.
Scheme 2. (a) PhNO2, 145 ꢀC.
Results and Discussion
activity. In a comparison of the relative topo I poison-
ing activity of various 5-bromoterbenzimidazoles, how-
ever, the presence of either a 200-hydroxyl group (4b) or a
200-propyl group (4c) did not in¯uence topo I poisoning
activity relative to the 200-unsubstituted analogue, 4a.
Compound 5, wherein the phenyl moiety of 5-phenyl-
terbenzimidazole is replaced with a p-chlorophenyl group,
had similar topo I poisoning activity and cytotoxicity to
Topoisomerase I poisoning activity
Major di€erences were not observed in the topo I poi-
soning activities of 3a±c, 4a±c, 5, 6a±c, and 7±9 (Table
2). Previous studies indicated that variation of the 200-
substituents within a series of 5-phenylterbenzimidazole
derivatives did signi®cantly in¯uence topo I poisoning

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M. Rangarajan et al. / Bioorg. Med. Chem. 8 (2000) 2591±2600
Scheme 3. (a) CF3COOH, 90 ꢀC; (b) HCOOH, NiAl (R); (c) PhNO2, 145 ꢀC; (d) HCOOH, NiAl (Ra-Ni).
Table 2. Relative topoiosmerase I poisoning activities of cytotoxi-
cities of 5-phenylterbenzimidazoles and 5-bromoterbenzimidazoles
retain activity as topo I poisons. The data listed in Table
2 clearly indicate that 5,6-dibromoterbenzimidazole (6a)
and 5,6-diphenylterbenzimidazole (7) exhibit similar
activity to 5-phenylterbenzimidazole (3a). Among the
other 5,6-disubstituted terbenzimidazoles that were
evaluated, 5-bromo-6-methoxyterbenzimidazole (8) was
only slightly less potent than 6a as a topo I poison. 5-
Phenyl-6-methoxyterbenzimidazole (9), however, did
appear somewhat more potent than 3a as a topo I poi-
son. These data demonstrate that 5,6-disubstituted ter-
benzimidazoles can have activity as topo I poisons.
While there was some minor variation in activity, for
the derivatives evaluated in this study it would appear
that such substituents did not exert a major impact on
topo 1 poisoning activity.
Cytotoxicityb
Compound
Relative topo
I-mediated
DNA cleavagea
RPMI-8402
IC50 (mM)
CPT-K5
IC50 (mM)
3a
3b
3c
4a
4b
4c
5
6a
6b
6c
7
1.0
0.1
0.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
2.0
0.5
0.09
0.07
0.04
1.6
0.39
6.0
>10
2.0
>10
>10
1.4
6.5
0.21
0.33
0.26
0.11
0.25
1.6
2.0
0.09
8.8
>10
>10
>10
The presence of electron-withdrawing substituents at
the 200-position in the case of 5-phenylterbenzimidazoles
was observed to increase their relative activity as topo I
poisons. We investigated whether a similar e€ect could
be seen in the case of derivatives of 5,6-dibromoter-
benzimidazole (6a). As shown in Table 2, the presence
of either a 200-chloro (6b) or a 200-tri¯uoromethyl group
(6c) did not signi®cantly in¯uence topo 1 poisoning activ-
ity within this subset of terbenzimidazole derivatives.
8
9
1.6
0.35
aTopoisomerase I cleavage values are reported as REC, relative e€ective
concentration, i.e. concentrations relative to compound 3a, whose value
is arbitrarily assumed as 1, that are able to produce the same cleavage on
the plasmid DNA in the presence of human topoisomerase I.
bIC50 values were calculated after 4 days of continuous drug exposure.
The methods employed for these assays are detailed in Experimental.
3a. While studies did indicate that steric bulk could be
tolerated at the 5-position, the 5,6-benzofused analogue
of terbenzimidazole, 2-[20-(benzimidazol-500-yl)benzimi-
dazol-5-yl]-1H-naphth[2,3-d]imidazole, did not exhibit
signi®cant cytotoxic activity.9 The weak minor groove
DNA binding anity of this naphthimidazole derivative
relative to terbenzimidazole and 5-phenylterbenzimida-
zole has been correlated with its lack of activity as a
topo I poison.19 The strong potential for this naphthi-
midazole derivative to self associate, rather than steric
properties incompatible with retention of topo I poi-
soning activity, o€ers an alternative explanation for the
lack of activity observed with this terbenzimidazole
derivative.
Cytotoxicity
The relative cytotoxic activities of 3a±c, 4a±c, 5, 6a±c,
and 7±9 against the human lymphoblastoma cell line,
RPMI 8402, and its camptothecin-resistant variant,
CPT-K5, are listed in Table 2. Previous studies on ter-
benzimidazoles revealed that presence of a lipophilic
substituent at either the 5- or 6-position was an impor-
tant factor associated with cytotoxic activity.8 10
The e€ects of a 200-substituent on cytotoxicity was also
examined for several 5-phenylterbenzimidazole deriva-
tives.11 No similar studies were performed with derivatives
of 5-bromoterbenzimidazole, 4a. The presence of a 200-
hydroxyl group as in 4b resulted in a decrease in cytotoxi-
city. This was especially apparent in the camptothecin-
We were uncertain prior to the present study whether
5,6-disubstituted terbenzimidazole derivatives would

M. Rangarajan et al. / Bioorg. Med. Chem. 8 (2000) 2591±2600
2595
resistant cell line, CPT-K5. As observed with 5-phenyl-
terbezimidazoles, the presence of a 200-n-propyl group as
in 4c enhanced cytotoxicity relative to 4a in RPMI 8402
cells. As previously observed with 5-phenylterbenzimi-
dazole,11 the presence of the 200-propyl group, however,
resulted in cross-resistance against CPT-K5 cells that
was not apparent in the 200-unsubstituted analogue, 4a.
Compound 5, which di€ers from 5-phenylterbenzimida-
zole with respect to the presence of a p-chloro substituent
on the phenyl moiety and the 200-tri¯uoromethyl group,
exhibited only slightly less cytotoxicity relative to 3a in
both of these cell lines. When compared with compound
3c, where the only di€erence was the p-chloro group on
the 5-phenyl moiety, slightly lower toxicity was
observed for 5 in RPMI 8402 cells. Both 3c and 5
exhibited cross-resistance to CPT-K5 cells.
Table 3. Cytotoxicity of 3c, 6a and 6c in various solid human tumor
cell lines
IC50 values (mM)a
Compound MDA-MB-435 OVCAR-3 SK-OV-3 PC-3 DU-145
3c
6a
6c
0.08
0.80
0.15
0.26
>30
>5.0
0.55
>30
1.4
0.06
>30
0.23
0.59
>30
2.0
aIC50 values were calculated after 4 days of continuous drug exposure.
The methods employed for these assays are similar to those detailed in
Experimental. These cytotoxicity assays were performed under con-
tract by Southern Research Institute, Birmingham, AL, USA.
terbenzimidazole and 5-phenylterbenzimidazole and the
6-phenyl derivative of 5-phenylterbenzimidazole. 5,6-
Diphenylterbenzimidazole (7) and 5-phenyl-6-methoxy-
terbenzimidazole (9) were less cytotoxic than 3a. In
addition, both of these substituted 5-phenylterbenzimi-
dazoles were cross-resistant to CPT-K5. While 5-
bromo-6-methoxyterbenzimidazole (8) had similar
cytotoxic activity to 6a in RPMI 8402 cells, it was also
cross-resistant to CPT-K5 cells.
The 5,6-benzofused analogue of terbenzimidazole, 2-
[(20-(benzimidazol-500-yl)benzimidazol-5-yl]-1H-naphtho
[2,3-d]imidazole, in earlier studies did not exhibit sig-
ni®cant cytotoxic activity, despite the fact that previous
studies did indicate that substantial steric bulk could be
tolerated at the 5-position.9 While these data are con-
sistent with the lack of topo I poisoning activity
observed with this derivative, the strong potential
observed for this naphthimidazole derivative to self
associate could explain the lack of cytotoxicity observed
with this terbenzimidazole derivative. In either event, it
remained uncertain as to the relative cytotoxic activity
that one would observe with 5,6-disubstituted terbenzi-
midazoles. The 5,6-disubstituted terbenzimidazoles that
we targeted for synthesis incorporated within their
structure at least one lipophilic moiety, either a phenyl
or bromo group at the 5-position. All three 5,6-dibromo
compounds, 6a±c, exhibited cytotoxicity comparable to
5-phenylterbenzimidazole in RPMI 8402 cells. As was
the case with 5-phenylterbenzimidazole derivative, no
cross-resistance to CPT-K5 cells was observed with 5,6-
dibromoterbenzimidazole, 6a, or 200-chloro derivative,
6b. As is the case with 3c, however, the presence of a
tri¯uoromethyl substituent at the 200-position (6c) was
associated with signi®cant cross-resistance.
There are two structural modi®cations that appear to
have a pronounced e€ect on the cytotoxicity of terben-
zimidazole derivatives. The presence of lipophilic sub-
stituents at either 5- and/or 6-position consistently
appears to favor cytotoxicity. While a 5,6-benzo-fused
ring does cause a major loss in cytotoxic activity, the
presence of functionality at both the 5- and 6-position
does not necessarily result in a reduction in cytotoxic
activity. The presence of a chloro or 2-tri¯uoromethyl
substituent at the 200-position does not necessarily alter
activity against RPMI 8402 cells. Analogues with a 200-
tri¯uoromethyl group, however, do tend to exhibit sig-
ni®cant cross-resistance to its camptothecin-resistant
variant, CPT-K5. In comparative studies in other solid
tumor cell lines, however, the 200-tri¯uoromethyl deri-
vative of 5,6-dibromoterbenzimidazole (6c) proved to be
signi®cantly more cytotoxic than the 200-unsubstituted
analogue (6a). While strong electron-withdrawing sub-
stituents on the 200-position can signi®cantly alter the
pKa of hydrogen attached to N100, the in¯uence of this
speci®c modi®cation of the physicochemical properties
of terbenzimidazoles on biological activity has not been
determined. Further studies are needed to elucidate the
impact of such modi®cations on cell penetration/uptake
as well as on the interaction with the components asso-
ciated with the pharmacological target, the cleaved
complex comprised of topo I and DNA.
It was anticipated that electron-withdrawing sub-
stituents at the 200-position of 5,6-dibromoterbenzimi-
dazole would exhibit signi®cantly increased cytotoxicity.
Neither the 200-chloro nor the 200-tri¯uoromethyl deriva-
tive, 6b and 6c, were more cytotoxic than 6a toward
RPMI 8402 cells. In an extended evaluation of cyto-
toxicity, which included several di€erent human tumor
lines, however, signi®cantly enhanced cytotoxic activity
was observed for 6c relative to 6a (Table 3). In the
highly di€erentiated mammary tumor cell line, MDA-
MB-435, and the ovarian tumor cell line OVCAR-3, 6c
was ®ve to six times more cytotoxic than 6a. Much
greater di€erences in cytotoxicity were seen with the ovar-
ian tumor cell line, SK-OV-3, and the prostate tumor cell
lines PC-3 and DU-145. However, in these human tumor
cell lines, 6c was consistently less cytotoxic than 3c, the 200-
tri¯uoromethyl derivative of 5-phenylterbenzimidazole.
Experimental
Melting points were determined with a Thomas-Hoover
Unimelt capillary melting point apparatus. Column
chromatography refers to ¯ash chromatography con-
ducted on SiliTech 32±63 mm (ICN Biomedicals,
Eschwegge, Germany) using the solvent systems indi-
cated. Radial chromatography refers to the use of a
Model 8924 chromatotron (Harrison Research, CA).
Infrared spectral data (IR) were obtained on a Perkin±
Elmer 1600 Fourier transform spectrophotometer and
Other 5,6-disubstituted terbenzimidazoles evaluated in
this study were the 6-methoxy derivatives of 5-bromo-

2596
M. Rangarajan et al. / Bioorg. Med. Chem. 8 (2000) 2591±2600
are reported in cm 1. Proton (1H NMR) and carbon
(13C NMR) nuclear magnetic resonance spectra were
recorded on a Varian Gemini-200 Fourier Transform
was removed with a Kugelrohr and the compound pur-
i®ed by ¯ash column chromatography. Elution with 5±
15% methanol/ethyl acetate gave 41% (47.5 mg,
0.1 mmol) of pure yellow compound: mp>260 ꢀC; IR
(KBr): 3246, 2965, 2873, 1627, 1548, 1438, 1284; UV
(MeOH) 330, 235 nm (log e=4.75, 4.73); 1H NMR
(DMSO-d6+3 drops CF3COOH) d 0.95 (t, 3H), 1.88±
1.97 (m, 2H), 3.18 (t, 2H), 7.7 (dd, 1H, J=1.7, 8.8), 7.83
(d, 1H, J=8.6), 8.07±8.18 (m, 3H), 8.22±8.26 (m, 1H),
8.42 (d, 1H, J=8.1), 8.65 (d, 2H, J=3.5); 13C NMR
(DMSO-d6+3 drops CF3COOH) d 13.4, 20.3, 28.4,
113.7, 115.2, 115.6, 116.1, 116.9, 119.0, 123.7, 124.2, 125.2,
128.9, 131.7, 131.9, 133.6, 134.0, 136.8, 139.2, 150.8,
153.1, 157.3; HRMS (FAB) calculated for C24H20BrN6
(MH+) 471.0933, found 471.0923.
1
spectrometer. NMR spectra (200 MHz H and 50 MHz
13C) were recorded in the deuterated solvent indicated
with chemical shifts reported in d units down®eld from
tetramethylsilane (TMS). Coupling constants are repor-
ted in hertz (Hz). A few drops of CF3COOH improved
13C NMR spectra by allowing for increased solubility
and formation of the protonated form of the terbenzi-
midazoles, thereby eliminating tautomeric di€erences
among carbon atoms. Mass spectra were obtained from
Washington University Resource for Biomedical and
Bio-organic Mass Spectrometry within the Department
of Chemistry at Washington University, St. Louis, MO.
The purity of all compounds for which HRMS data are
provided was determined by analytical reverse-phase
HPLC. Compounds were analyzed using both of the
following conditions: (method A) a Vydac C-18 column
(The Separations Group) using methanol:H2O (87:13)
with a ¯ow rate of 1 mL/min; (method B) a Microsorb
C-8 column (Rainin Instrument Co., Inc.) using metha-
nol:0.1 M potassium phosphate bu€er (pH 7.0) (70:30)
with a ¯ow rate of 1 mL/min. HPLC analyses were per-
formed with a Hewlett±Packard 1090 liquid chromato-
graph equipped with a diode array UV detection
monitoring at 254 and 335 nm. The % purity of these
compounds was calculated from the peak area assuming
that the extinction coecient of the compound of inter-
est and the impurity are the same. On the basis of these
analyses, all the compounds were found to be 98.0±99%
pure in these systems. Combustion analyses were per-
formed by Atlantic Microlabs, Inc., Norcross, GA, and
were within Æ 0.4% of the theoretical value. The
syntheses of 3a±c and 4a have been detailed in the lit-
erature.8 11
5-(p-Chlorophenyl)-2-[20-(200-tri¯uoromethylbenzimidazol-
500-yl)benzimidazol-50-yl]benzimidazole (5). 5-(p-Chloro-
phenyl)-2-(3,4-diaminophenyl)benzimidazole
(65 mg,
0.19 mmol) and 5-formyl-2-tri¯uoromethylbenzimid-
azole11 were heated together in nitrobenzene (4 mL) at
150 ꢀC. The nitrobenzene was removed with a Kugel-
rohr, and the crude mixture was loaded on a column.
Elution with 90:10 ethyl acetate:hexanes gave the
product (32 mg, 0.06 mmol) in 30% yield: mp>260 ꢀC;
IR (KBr) 3153, 2965, 1546, 1444, 1162, 810; UV
(MeOH) 335, 240 nm (log e=4.45, 4.40); 1H NMR
(DMSO-d6+3 drops CF3COOH) d 7.56±7.64 (m, 2H),
7.73±8.06 (m, 6H), 8.17±8.25 (m, 2H), 8.33 (dd, 1H,
J=1.5, 8.8), 8.67 (d, 1H, J=1.4); 13C NMR (DMSO-
d6+3 drops CF3COOH) d 114.8, 115.5, 116.0, 117.3,
117.5, 123.3, 123.9, 124.1, 125.1, 125.2, 127.45, 127.49,
129.3, 129.4, 129.4, 132.5, 133.1, 133.5, 136.9, 137.0,
138.2, 138.6, 140.6, 150.6, 150.8, 154.4; HRMS (FAB)
calculated for C28H17ClF3N6 (MH+) 529.1155, found
529.1156.
5-Bromo-2-[20-(200-hydroxybenzimidazol-500-yl)benzimida-
zol-50-yl]benzimidazole (4b). 5-Bromo-2-(3,4-diamino-
phenyl)benzimidazole (33.3 mg, 0.11 mmol) and 5-
formyl-2-hydroxybenzimidazole11 (17.7 mg, 0.11 mmol)
were heated at 145 ꢀC in nitrobenzene (3 mL) overnight.
Nitrobenzene was removed with a Kugelrohr and the
compound was puri®ed by ¯ash column chromato-
graphy. Elution with 5±15% methanol/ethyl acetate
gave 23.5 mg (0.05 mmol) of yellow colored compound
in 48% yield: mp>260 ꢀC; IR (KBr) 3409, 3211, 1698,
1558, 1482, 1384, 1279; UV (MeOH) 335, 240 nm (log
e=4.73, 4.69); 1H NMR (DMSO-d6+3 drops CF3
COOH) d 7.30 (d, 1H, J=8.3), 7.69 (dd, 1H, J=1.8,
8.7), 7.82 (s, 1H), 7.86±7.99 (m, 2H), 8.09±8.13 (m, 2H),
8.32 (dd, 1H, J=1.4, 8.7), 8.61 (s, 1H), 11.38 (s, 1H);
13C NMR (DMSO-d6+3 drops CF3COOH) d 108.0,
109.6, 113.9, 115.2, 115.3, 116.3, 117.2, 117.9, 121.1, 122.4,
125.2, 128.7, 130.8, 132.7, 133.2, 134.9, 134.9, 135.6, 150.4,
153.1, 155.7; HRMS (FAB) calculated for C21H14BrN6O
(MH+) 445.0412, found 445.0408.
5,6-Dibromo-2-[20-(benzimidazol-500-yl)benzimidazol-50-yl]
benzimidazole (6a). 4,5-Dibromo-1,2-phenylenediamine
(128 mg, 0.48 mmol) and 5-formyl-2-(benzimidazol-50-
yl)benzimidazole8 (126 mg, 0.48 mmol) were heated in
nitrobenzene (6 mL) at 145 ꢀC overnight. The nitro-
benzene was removed with a Kugelrohr and the mixture
loaded on a column for puri®cation. Elution with 1±
10% methanol/ethyl acetate gave 0.1 g (41%) of pure
compound: mp>260 ꢀC; IR (KBr) 3405, 3198, 1626,
1
1544, 1385, 1292; H (DMSO-d6+3 drops CF3COOH)
d 8.03±8.13 (m, 2H), 8.17 (s, 2H), 8.25 (d,1H, J=9.2),
8.42 (d, 1H, J=8.6), 8.59 (s, 1H), 8.74 (s, 1H), 9.75 (s, 1H);
13C (DMSO-d6+3 drops CF3COOH) d 114.7, 114.9,
115.8, 116.0, 118.9, 119.6, 120.7, 123.3, 124.5, 125.6,
131.5, 133.5, 134.8, 135.8, 138.0, 151.9, 152.3; HRMS
(FAB) calculated for C21H13Br2N6 (MH+) 506.9568,
found 506.9574.
5,6-Dibromo-2-[20-(200-chlorobenzimidazol-500-yl)benzimi-
dazol-50-yl]benzimidazole (6b). 5,6-Dibromo-2-(3,4-di-
aminophenyl)benzimidazole (95 mg, 0.25 mmol) and 5-
formyl-2-chlorobenzimidazole11 (45 mg, 2.5 mmol) were
re¯uxed in nitrobenzene (2 mL) overnight. Nitrobenzene
was removed with a Kugelrohr and the compound pur-
i®ed by ¯ash column chromatography. Elution with 1±
20% methanol/ethyl acetate gave 51.9% (70 mg,
5-Bromo-2-[20-(200-n-propylbenzimidazol-500-yl)benzimida-
zol-50-yl]benzimidazole (4c). 5-Bromo-2-(3,4-diamino-
phenyl)benzimidazole (75 mg, 0.25 mmol) and 5-formyl-
2-n-propylbenzimidazole11 (46.5 mg, 0.25 mmol) were
re¯uxed in nitrobenzene (2 mL) overnight. Nitrobenzene

M. Rangarajan et al. / Bioorg. Med. Chem. 8 (2000) 2591±2600
2597
0.13 mmol) pure yellow compound: mp>270 ꢀC; IR
(KBr) 3259, 2961, 1708, 1629, 1542, 1440, 1289; 1H
NMR (DMSO-d6+3 drops CF3COOH) d 8.01±8.02 (m,
2H), 8.08±8.09 (m, 2H), 8.14-8.19 (m, 1H), 8.28±8.32
(m, 1H), 8.50 (s, 1H), 8.55 (s, 1H); 13C NMR (DMSO-
d6+3 drops CF3COOH) d 112.7, 113.1, 115.2,117.2,
118.5, 119.5, 122.6, 124.0, 129.7, 134.9, 139.1, 152.6,
153.3; HRMS (FAB) calculated for C21N6Br2H12Cl
(MH+) 540.9178, found 540.9180.
(s, 1H), 9.69 (s, 1H); 13C NMR (DMSO-d6+3 drops
CF3COOH) d 56.2, 96.4, 114.1, 115.0, 115.6, 115.8, 115.9,
116.4, 118.0, 118.4, 123.1, 125.3, 125.6, 126.3, 127.4, 131.9,
133.1, 133.1, 138.4, 140.8, 149.0, 153.4; HRMS (FAB)
calculated for C22H16BrN6O (MH+) 459.0569, found
459.0573.
5-Phenyl-6-methoxy-2-[20-(benzimidazol-500-yl)benzimid-
azol-50-yl]benzimidazole (9). 4-Methoxy-5-phenyl-1,2-
phenylenediamine (139 mg, 0.65 mmol) and 5-formyl-2-
(benzimidazol-50-yl)benzimidazole8 (170 mg, 0.65 mmol)
were heated together in nitrobenzene (5 mL) overnight
at 145 ꢀC. Nitrobenzene was removed with a Kugelrohr
and the compound was loaded onto a column. (1±10%)
Methanol/ethyl acetate gave 120 mg (41%) of pure pro-
duct: mp>260 ꢀC; IR (KBr) 3298, 3050, 2987, 1630,
1541, 1438, 1283; UV (MeOH) 335, 265 (log e=4.4,
4.36); 1H NMR (DMSO-d6+3 drops CF3COOH) d
3.92 (s, 3H), 7.39±7.57 (m, 6H), 7.72 (s, 1H), 8.11±8.15
(m, 1H), 8.19±8.27 (m, 2H), 8.47 (dd, 1H, J=1.46,
8.07), 8.64 (s, 1H), 8.76 (s, 1H), 9.75 (s, 1H); 13C NMR
(DMSO-d6+3 drops CF3COOH) d 56.4, 95.7, 114.6,
115.2, 116.0, 116.2, 119.0, 119.1, 125.6, 126.3, 127.6, 127.8,
128.3, 129.8, 130.7, 131.5, 132.4, 133.4, 137.6, 148.8, 152.8,
156.8; HRMS (FAB) calculated for C28H20N6O (MH+)
457.1777, found 457.1770.
5,6-Dibromo-2-[20-(200-tri¯uoromethylbenzimidazol-500-yl)
benzimidazol-50-yl]benzimidazole (6c). 4,5-Dibromo-1,2-
phenylenediamine (60 mg, 0.22 mmol) and 73 mg
(0.22 mmol) of 5-formyl-2-(20-tri¯uoromethylbenzimid-
azol-50-yl)benzimidazole11 were heated in nitrobenzene
(10 mL) overnight at 150 ꢀC. The nitrobenzene was
removed with a Kugelrohr and the mixture loaded on a
column. Elution with 90:10 ethyl acetate:n-hexanes yiel-
ded 25 mg (20% yield, 0.05 mmol) of compound:
mp>260 ꢀC; IR (KBr) 3047, 2931, 1720, 1544, 1441,
1290, 1155, 981; UV (MeOH) 335, 265 nm (log e=4.45,
1
4.48); H NMR (DMSO+3 drops CF3 COOH) d 8.02±
8.09 (m, 2H), 8.14 (s, 2H), 8.56±8.29 (m, 2H), 8.57 (s,
1H), 8.69 (s, 1H); 13C NMR (DMSO+3 drops CF3
COOH) d 113.6, 115.4, 118.2, 119.4, 120.8, 124.0, 124.1,
124.4, 135.0, 136.6, 137.8, 152.6, 152.9; HRMS (FAB)
calcd. for C22H12BrF3N6 (MH+) 574.9441, found
574.9431.
4-Bromo-1,2-phenylenediamine (10a). 4-Bromo-2-nitro-
aniline (600mg, 2.76mmol) was dissolved in 25mL abso-
lute ethanol and 2.72g (14 mmol) SnCl2 was added. The
mixture was re¯uxed overnight. Ethanol was removed in
vacuo and the mixture basi®ed with 2N NaOH to pH 11.
Ether extraction, drying the ether layer over anhydrous
Na2SO4 and concentration in vacuo a€orded 486 mg
(2.6 mmol, 94% yield) of the crude diamine which was
used for the next step without characterization.
5,6-Diphenyl-2-[20-(benzimidazol-500-yl)benzimidazol-50-yl]
benzimidazole (7). 4,5-Diphenyl-1,2-phenylenediamine
(151 mg, 0.58 mmol) and 5-formyl-2-(benzimidazol-50-
yl)benzimidazole8 (152 mg, 0.58 mmol) were heated in
nitrobenzene (4 mL) overnight at 145 ꢀC under nitrogen.
The nitrobenzene was removed with a Kugelrohr.
Compound was puri®ed by ¯ash column chromato-
graphy. Elution with 2±10% methanol/ethyl acetate
gave 108 mg (0.22 mmol) of pure yellow compound in
37% yield: mp>260 ꢀC; IR (KBr) 3399, 3059, 1629,
1551, 1441, 1292; UV (MeOH) 345, 245 (log e 4.50,
4.43); 1H NMR (DMSO-d6+3 drops CF3COOH) d
7.17±7.33 (m, 10H), 7.84 (s, 2H), 8.06±8.25 (m, 3H),
8.49 (dd, 1H, J=1.2, 8.9), 8.66 (s, 1H), 8.75 (s, 1H), 9.69
(s, 1H); 13C NMR (DMSO-d6+3 drops CF3COOH) d
113.9, 115.2, 115.8, 116.2, 116.3, 117.9, 123.3, 126.3,
126.7, 127.9, 128.4, 128.4, 128.5, 130.1, 131.8, 131.9,
133.0, 138.9, 139.0, 140.6, 141.2, 153.7, 161.0; HRMS
(FAB) calculated for C33H23N6 (MH+) 503.1984, found
503.1989.
4-(p-Chlorophenyl)-1,2-phenylenediamine (10b). 4-(p-
Chlorophenyl)-2-nitroaniline (0.79 g, 3.2 mmol) in a
mixture of methanol/ethyl acetate in equal parts
(40 mL) was reduced using 115 mg of 10% Pd/C.
Hydrogenation was carried out at 40 psi pressure for 10
h. The mixture was ®ltered through Celite and the bed
washed with methanol. The methanol layer was con-
centrated in vacuo to yield 0.71 g (3.2 mmol) of the
crude diamine. The yield was quantitative. The crude
diamine was used as such without puri®cation: 1H
NMR (DMSO-d6+3 drops CF3COOH) d 7.09 (d, 1H,
J=8.3), 7.24 (dd, 1H, J=8.3, 2.12), 7.32 (d, 1H,
J=2.1), 7.47±7.60 (m, 4H); 13C NMR (DMSO-d6+3
drops CF3COOH) d 118.9, 120.9, 121.9, 126.3, 127.4,
127.9, 129.2, 132.1, 133.3, 138.5, 139.7.
5-Bromo-6-methoxy-2-[20-(benzimidazol-500-yl)benzimida-
zol-50-yl]benzimidazole (8). 4-Bromo-5-methoxy-1,2-
phenylenediamine (123 mg, 0.57 mmol) and 5-formyl-2-
(benzimidazol-50-yl)benzimidazole8 (150 mg, 0.57 mmol)
in nitrobenzene (4 mL) were heated at 145 ꢀC overnight
under nitrogen. Nitrobenzene was removed with a
Kugelrohr. Chromatographic separation with 1±10%
methanol/ethyl acetate a€orded 104 mg (40%) of pure
compound: mp>260 ꢀC; IR (KBr) 3456, 1631, 1385,
1195, 1159, 810; UV (MeOH) 335, 225 (log e=4.5,
4.63); 1H NMR (DMSO-d6+3 drops CF3COOH) d
3.91 (s, 3H), 7.17±7.29 (m, 1H), 7.78 (d, 1H, J=8.8),
8.00±8.13 (m, 3H), 8.49±8.53 (m, 1H), 8.66 (s, 1H), 8.79
4-(p-Chlorophenyl)-2-nitroaniline. 4-Bromo-2-nitroani-
line (1 g, 4.6 mmol) was dissolved in DME (13 mL). Tet-
rakis(triphenylphosphine)palladium (266mg, 0.23mmol),
p-chlorophenylboronic acid (1.14 g, 6.9 mmol), and 2 M
Na2CO3 (5 mL) were added to the reaction mixture and
re¯uxed at 90 ꢀC overnight. The reaction mixture was
concentrated in vacuo and loaded onto a column. Flash
column chromatography using 1±3% ethyl acetate/n-
hexanes a€orded 0.79 g (0.32 mmol) of compound in
69% yield: mp 172±173 ꢀC; IR (KBr) 3479, 3358, 1639,

2598
M. Rangarajan et al. / Bioorg. Med. Chem. 8 (2000) 2591±2600
1
1487, 1414, 1344, 1240, 1093, 822; H NMR (CDCl3) d
6.14 (brs, 2H), 6.90 (d, 1H, J=8.7), 7.37±7.51 (m, 4H),
7.61 (dd, 1H, J=2.2, 8.7), 8.35 (d, 1H, J=2.1); 13C
NMR (CDCl3) 119.9, 124.4, 128.0, 128.1, 129.6, 132.9,
133.9, 134.7, 134.7, 134.8, 137.7, 144.4; HRMS (EI)
calculated for C12H9ClN2O2 m/z 248.0352, found
248.0353.
nitrobenzene was removed with a Kugelrohr. Column
puri®cation (5±20% ethyl acetate/hexanes) a€orded
230 mg (0.51 mmol, 54.7% yield) of the pure product:
mp 95±97 ꢀC; IR (KBr) 3313, 3054, 1604, 1551, 1437,
1
1362, 1238; H NMR (DMSO-d6+3 drops CF3COOH)
d 7.48 (dd, 1H, J=2.0, 8.6), 7.71 (d, 1H, J=8.6), 7.97
(d, 1H, J=1.6), 8.25 (s, 1H), 8.43 (s, 1H); 13C NMR
(DMSO-d6+3 drops CF3COOH) d 116.7, 117.9, 118.3,
123.6, 126.8, 131.1, 134.5, 137.9, 140.2, 142.4, 142.9,
148.5.
4,5-Dibromo-1,2-phenylenediamine (10c). 3,4-Dibromo-
6-nitroaniline (189 mg, 0.64 mmol) was dissolved in
anhydrous ethanol (8 mL) to which was added Raney
Nickel catalyst (400 mg).12 The hydrogenation appara-
tus was at a hydrogen pressure of 50 psi. After 45 min
the deep yellow color originally present was completely
discharged, indicating complete reduction of the nitro
into the amino groups. The reaction mixture was ®ltered
through Celite and the bed was washed with methanol.
The methanol was concentrated in vacuo to give 135 mg
of the crude diamine in 82% yield. The crude diamine
was used as such without puri®cation: 1H NMR
(CDCl3) d 3.37 (br, 4H), 6.93 (s, 2H); 13C NMR
(CDCl3) d 114.0, 121.0, 135.8.
5-Bromo-2-(3,4-diaminophenyl)benzimidazole (13a). 5-
Bromo-2-(3,4-dinitrophenyl)-benzimidazole
(140 mg,
0.38 mmol) was dissolved in absolute ethanol (8 mL).
SnCl2 (0.8 g, 4.2 mmol) was added and the mixture
re¯uxed overnight. Ethanol was removed in vacuo and
the mixture basi®ed with 2N NaOH to pH 11. Repeated
extraction with ether, drying the ether layer over anhy-
drous Na2SO4 and concentration in vacuo yielded 0.11 g
(0.37 mmol) of the crude diamine in 98% yield. The
diamine was used without further puri®cation: 1H
NMR (DMSO) d 6.63 (d, 1H, J=8.1), 7.26 (m, 2H),
7.39 (m, 2H), 6.65 (d, 1H, J=1.8).
5 - Bromo - 2 - (3,4 - dinitrophenyl)benzimidazole (12a). 4-
Bromo-1,2-phenylenediamine (275 mg, 1.5 mmol) and
3,4-dinitrobenzaldehyde (300 mg, 1.5 mmol) in 2 mL
nitrobenzene were heated at 145 ꢀC overnight. The
nitrobenzene was removed with a Kugelrohr. Column
puri®cation (1±10% ethyl acetate/hexanes) a€orded
209 mg (0.57 mmol, 39% yield) of the pure product: mp
79±80 ꢀC; IR (KBr) 3133, 1613, 1544, 1436, 1359, 1086,
832; 1H NMR (DMSO+3 drops CF3COOH) d 7.48
(dd, 1H, J=1.8, 8.8), 7.70 (d, 1H, J=8.8), 7.96 (d, 1H,
J=1.46), 8.45 (d, 1H, J=8.5), 8.68 (dd, 1H, J=1.8,
8.5), 8.93 (d, 1H, J=1.8); 13C NMR (DMSO+3 drops
CF3COOH) d 116.4, 117.5, 118.7, 123.7, 127.1, 132.1,
134.5, 137.6, 140.2, 142.4, 142.8, 148.5; HRMS (EI)
calculated for C13H7BrN4O4 m/z 361.9650, found m/z
361.9648.
2-(3,4-Diaminophenyl)-5-(p-chlorophenyl)benzimidazole
(13b). 2-(3,4-Dinitrophenyl)-5-(p-chlorophenyl)benzimid-
azole (125 mg, 0.32 mmol) was dissolved in ethyl acetate
(25 mL) and reduced by hydrogenation using 10% Pd/C
(25 mg) as the catalyst overnight. The catalyst was
removed by Celite ®ltration. The ethyl acetate was con-
centrated in vacuo and the residue was dried to yield
0.11 g (0.33 mmol) of crude solid. The yield was quanti-
tative. This crude solid was further used without pur-
1
i®cation: H NMR (DMSO-d6+3 drops CF3COOH) d
6.92 (d, 1H, J=8.42), 7.57±7.62 (m, 4H), 7.65±7.81 (m,
4H), 7.92 (s, 1H); 1H NMR (DMSO-d6+3 drops
CF3COOH) d 110.2, 111.3, 114.1, 115.3, 117.7, 123.8,
124.7, 129.18, 129.22, 129.33, 129.34, 131.9, 132.9,
133.0, 136.7, 138.7, 143.7, 150.8.
2-(3,4-Dinitrophenyl)-5-(p-chlorophenyl)benzimidazole
(12b). A stirred solution of 4-(p-chlorophenyl)-1,2-diami-
nobenzene (0.7g, 3.2 mmol) and 3,4-dinitrobenzaldehyde
(0.63 g, 3.2 mmol) in nitrobenzene (13 mL) was heated at
150 ꢀC under N2 overnight. Nitrobenzene was removed
with a Kugelrohr and the crude mixture was puri®ed by
¯ash column chromatography. Elution with 10% ethyl
acetate/hexanes provided 0.55 g (1.39 mmol) of a bright
yellow±red compound in 44% yield: mp 75±76 ꢀC; IR
5,6-Dibromo-2-(3,4-diaminophenyl)benzimidazole (13c).
5,6-Dibromo-2-(3,4-dinitrophenyl)benzimidazole (0.2 g,
0.45 mmol) was dissolved in 20 mL anhydrous ethanol
to which was added about 0.4 g Raney Nickel catalyst.
Hydrogenation was carried out at a hydrogen pressure
of 50 psi. After 2 h, the deep yellow color originally
present was completely discharged, indicating complete
reduction of the nitro into the amine group. The reac-
tion mixture was ®ltered through Celite and the bed was
washed with ethanol. The ethanol layer was con-
centrated in vacuo to give 155 mg (0.41 mmol) of the
crude diamine in 91.5% yield. The crude diamine was
1
(KBr) 3392, 2927, 1542, 1359, 1093, 845, 809; H NMR
(DMSO-d6+3 drops CF3COOH) d 7.55 (d, 2H, J=8.3),
7.65 (d, 1H, J=8.4), 7.77±7.84 (m, 3H), 7.96 (s, 1H),
8.47 (d, 1H, J=8.8), 8.68±8.73 (m, 1H), 8.95 (s, 1H); 13C
NMR (DMSO-d6+3 drops CF3COOH) d 113.7, 116.6,
123.4, 123.4, 123.5, 127.2, 127.3, 129.0, 129.2, 131.8, 132.4,
135.1, 135.2, 139.0, 139.6, 139.7, 142.1, 142.9, 148.3;
HRMS (EI) calculated for C19H11ClN4O4 m/z 394.0463,
found 394.0463.
1
used as such without puri®cation: H NMR (DMSO-
d6+3 drops CF3COOH) d 3.7 (brs, 4H), 7.45 (dd, 1H,
J=8.4), 7.75 (d, 1H, J=8.4), 7.95 (d, 1H, J=1.6), 8.18
(s, 1H), 8.51 (s, 1H).
5 - Formyl - 2 - propylbenzimidazole (15). 5-Cyano-2-pro-
pylbenzimidazole (0.33 g, 1.83mmol) was dissolved in
HCOOH (26 mL). Water (2 mL) and Ni/Al catalyst (1.8 g)
were added and the mixture was re¯uxed at 110 ꢀC for
6 h. The hot reaction mixture was ®ltered through a bed
of Celite and the ®ltrate concentrated in vacuo. pH was
5,6-Dibromo-2-(3,4-dinitropheny)benzimidazole
(12c).
4,5-Dibromo-1,2-phenylenediamine (250mg, 0.94mmol)
and 3,4-dinitrobenzaldehyde (188mg, 0.94mmol) in 3 mL
nitrobenzene were heated at 145 ꢀC overnight. The

M. Rangarajan et al. / Bioorg. Med. Chem. 8 (2000) 2591±2600
2599
adjusted to 9 with 2N NaOH and the mixture extracted
with ethyl acetate. Puri®cation was done with ¯ash col-
umn chromatography. Elution with 30:70 ethyl acetate:
n-hexanes gave 0.32 g (1.7 mmol, 95% yield) of pure
product: mp 79±80 ꢀC; IR (KBr) 2935, 2850, 1690, 1420,
NMR (CDCl3) d 3.20 (br, 2H), 3.39 (br, 2H), 3.8 (s,
3H), 8.36 (s, 1H), 6.89 (s, 1H); 13C NMR (CDCl3) d
57.4, 100.4, 102.3, 122.5, 128.7, 136.7, 150.9.
4-Methoxy-5-phenyl-1,2 phenylenediamine (20). 4-Meth-
oxy-2-nitro-5-phenylaniline (165 mg, 0.68 mmol) in ethyl
acetate (20 mL) was reduced in the presence of H2 at
atmospheric pressure using 20 mg of 10% Pd/C as the
catalyst, overnight. The mixture was ®ltered through
Celite bed and the bed was washed with methanol. All
the washings were concentrated in vacuo to yield 139 mg
(96% yield) of the crude diamine, which was used with-
1
1283; H NMR (DMSO+3 drops CF3 COOH) d 0.99
(t, 3H), 1.91 (sextet, 2H), 3.13 (t, 2H), 8.03 (m, 2H), 8.36
(s, 1H), 10.17 (s, 1H); 13C NMR (DMSO+3 drops CF3
COOH) d 13.5, 20.2, 28.4, 114.9, 116.7, 125.9, 131.5,
133.8, 135.1, 157.6, 192.5; HRMS (EI) calculated for
C11H12N2O m/z 189.1031, found 189.1029.
1
5-Cyano-2-propylbenzimidazole. 3,4-Diaminobenzonitrile
(0.5 g, 3.8 mmol) and butyraldehyde (0.4 mL) were
heated in 5 mL nitrobenzene overnight in a sealed tube.
Nitrobenzene was removed with a Kugelrohr and the
mixture loaded on a column for chromatographic pur-
i®cation. Elution with 30:70 ethyl acetate:n-hexanes
provided 0.34 g (1.8 mmol, 49% yield) of the pure com-
pound: mp 160±161 ꢀC; IR (KBr) 2965, 2874, 2223,
1625, 1543, 1412, 1295, 825; 1H NMR (DMSO+3
drops CF3 COOH) d 0.98 (t, 3H), 1.86 (sextet, 2H), 3.12
(t, 2H), 7.93±7.96 (m, 2H), 8.42 (s, 1H); 13C NMR
(DMSO+3 drops CF3 COOH) d 13.3, 20.1, 28.3, 106.7,
115.5, 118.6, 119.3, 129.0, 131.1, 134.1; HRMS (EI)
calculated for C11H11N3 m/z 187.1109, found 187.1104.
out further puri®cation: H NMR (CDCl3) d 3.49 (brs,
4H), 3.71 (s, 3H), 6.42 (s, 1H), 6.74 (s, 1H), 7.26±7.52 (m,
5H); 13C NMR (CDCl3) d 56.8, 101.8, 120.9, 127.5, 128.4,
128.5, 128.5, 129.8, 130.0, 136.6, 139.2, 142.4, 153.7.
4-Methoxy-2-nitro-5-phenylaniline. 3-Bromo-4-methoxy-
6-nitroaniline (400mg, 1.62mmol) was dissolved in
DME (20 mL). Tetrakispalladium triphenyl phosphine
(94 mg, 0.08 mmol) served as the catalyst. To this mix-
ture were added phenyl boronic acid (300 mg,
1.01 mmol) and 2 M Na2CO3 (1.8 mL) and the mixture
re¯uxed at 90 ꢀC overnight. The reaction mixture was
concentrated in vacuo and puri®ed by ¯ash column
chromatography. 1±10% Ethyl acetate/n-hexanes gave
280 mg (1.14 mmol) of pure product in 71% yield: mp
105±106 ꢀC; IR (KBr) 3435, 3328, 2933, 1570, 1480,
4,5-Diphenyl-1,2-phenylenediamine (18). 4,5-Diphenyl-2-
nitroaniline (200 mg, 0.69 mmol) in ethanol (50 mL) was
reduced using 40 mg 10% Pd/C. Hydrogenation was
carried out at 40 psi pressure for 10 h. The mixture was
®ltered through Celite and the bed washed with metha-
nol. The methanol layer was concentrated in vacuo to
yield 151 mg of the crude diamine in 84% yield. The
crude diamine was used as such without puri®cation: 1H
NMR (CD3OD) d 6.78 (s, 2H), 7.01±7.16 (m, 10H); 13C
(CD3OD) d 118.1, 121.2, 127.2, 127.7, 130.4, 131.8, 138.3.
1
1227, 1029, 692; H NMR (CDCl3) d 3.80 (s, 3H), 5.94
(br, 2H), 6.79 (s, 1H), 7.39±7.64 (m, 5H), 7.64 (s, 1H);
13C NMR (CDCl3) d 56.6, 106.8, 121.3, 128.7, 128.7,
128.8, 128.8, 128.9, 129.7, 136.7, 140.2, 141.4, 148.7;
HRMS (EI) calculated for C13H12N2O3 m/z 244.0848,
found m/z 244.0847.
Topoisomerase I-mediated DNA cleavage assays.
Human topo I was expressed in Escherichia coli and
isolated as a recombinant protein using a T7 expression
system as previously described.20 DNA topoisomerase I
was puri®ed from calf thymus gland as reported pre-
viously.21 Plasmid YepG was puri®ed using the QIA-
GEN Plasmid MAXI kit. DNA end labeling was
performed by digesting YEpG with BamH I, followed
by end-®lling using Klenow polymerase. The topo I-
mediated cleavage assay was performed by incubating
topo I with labeled DNA in the presence of di€erent
concentrations of drugs or solvent. Reaction was carried
out at 23 ꢀC for 15 min and subsequently stopped by
addition of SDS±proteinase K, followed by 37 ꢀC incu-
bation for 1 h. Samples were added with loading dye
containing 200 mM NaOH to denature the double
stranded DNA. DNA fragments were separated on an
agarose gel followed by autoradiography. Topo I-medi-
ated DNA cleavage values are reported as REC, Relative
E€ective Concentration, i.e. concentrations relative to
3a, whose value is arbitrarily assumed as 1.0. Compound
3a has approximately 50% of the potency of Hoechst
33342 as a topo I poison. Cleavage was calculated on the
intensity of the strongest speci®c band. The DNA clea-
vage pattern for camptothecin is distinctly di€erent
from terbenzimidazoles, making comparisons of relative
potency dicult. An estimation of the relative potency
of camptothecin was made by comparing the doses of
4,5-Diphenyl-2-nitroaniline. 3,4-Dibromo-6-nitroaniline
(332mg, 1.12mmol) was dissolved in DME (20 mL). Tet-
rakis (triphenylphosphine) palladium (65 mg, 0.06mmol),
phenyl boronic acid (200 mg, 1.64 mmol), and 2 M
Na2CO3 (10 mL) were added to the reaction mixture
and re¯uxed at 90 ꢀC overnight. The reaction mixture
was concentrated in vacuo and loaded onto a column.
1±3% Ethyl acetate/n-hexanes a€orded 259 mg of pure
yellow compound in 80% yield: mp 139±141 ꢀC; IR
(KBr) 3476, 3363, 2924, 1621, 1476, 1263, 1089; 1H
NMR (CDCl3) d 6.11 (brs, 2H), 6.86 (s, 1H), 7.04±7.09
(m, 5H), 7.15±7.26 (m, 5H), 8.21 (s, 1H); 13C NMR
(CDCl3) d 120.6, 127.2, 127.7, 128.2, 128.3, 128.5, 128.5,
129.3, 129.4, 129.5, 129.8, 130.1, 130.9, 131.8, 139.9, 139.9,
144.1, 149.0; HRMS (EI) calculated for C18H14N2O2 m/z
290.1059, found 290.1055.
4-Bromo-5-methoxy-1,2-phenylenediamine (19). 3-Bromo-
4-methoxy-6-nitroaniline (150 mg, 0.61 mmol) was dis-
solved in ethanol (10 mL) and hydrogenation was car-
ried out using 350 mg Raney Nickel for 30 min. The
reaction mixture was ®ltered through Celite bed and
washed with methanol. The methanolic layer was dried
in vacuo to give 127 mg of the crude diamine in 97%
1
yield, which was used as such without puri®cation: H

2600
M. Rangarajan et al. / Bioorg. Med. Chem. 8 (2000) 2591±2600
compound 3a and camptothecin that induced fragmen-
tation of approximately 10% of the labeled DNA.
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MTT-microtiter plate tetrazolinium cytotoxicity assay.
The cytotoxicity was determined by the MTT
assay.22 24 The human lymphoblast RPMI 8402 and its
camptothecin-resistant variant cell line, CPT-K5, were
provided by Dr. Toshiwo Andoh (Aichi Cancer Center
Research Institute, Nagoya, Japan). Cells were main-
tained in suspension at 37 ꢀC with 5% CO2 in RPMI
1640 medium supplemented with 10% heat inactivated
fetal bovine serum, l-glutamine (2 mM), penicillin
(100 U/mL), and streptomycin (0.1 mg/mL). The cyto-
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Acknowledgements
Mass spectrometry was provided by the Washington
University Mass Spectrometry Resource, an NIH
Research Resource (Grant No. P41RR0954). We
acknowledge the contribution of Mr. Song Jin in the
preparation of 5,6-dibromo-200-chloroterbenzimidazole.
This study was supported by Grant CA 39662 from the
National Cancer Institute (L.F.L.) and a fellowship
grant from the Johnson & Johnson Discovery Research
Fund (E.J.L.).
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References and Notes
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