Substituted 2,5'-bi-1H-benzimidazoles: Topoisomerase I inhibition and cytotoxicity

Article abstract of DOI:10.1021/jm950412w

Several 2'-aryl-5-substituted-2,5'-bi-1H-benzimidazole derivatives were synthesized and evaluated as topoisomerase I poisons and for their cytotoxicity toward the human lymphoblast cell line RPMI 8402. This study focused on 18 2,5'-bi-1H-benzimidazole der

Full text of DOI:10.1021/jm950412w

992
J . Med. Chem. 1996, 39, 992-998
Su bstitu ted 2,5′-Bi-1H-ben zim id a zoles: Top oisom er a se I In h ibition a n d
Cytotoxicity
J ung Sun Kim,† Barbara Gatto,‡ Chiang Yu,‡ Angela Liu,‡ Leroy F. Liu,‡ and Edmond J . LaVoie*,†
Department of Pharmaceutical Chemistry, Rutgers, The State University of New J ersey, Piscataway, New J ersey 08855, and
Department of Pharmacology, The University of Medicine and Dentistry of New J ersey, Robert Wood J ohnson Medical School,
Piscataway, New J ersey 08855
Received J une 7, 1995X
Several 2′-aryl-5-substituted-2,5′-bi-1H-benzimidazole derivatives were synthesized and evalu-
ated as topoisomerase I poisons and for their cytotoxicity toward the human lymphoblast cell
line RPMI 8402. This study focused on 18 2,5′-bi-1H-benzimidazole derivatives which contained
either a 5-cyano, a 5-(aminocarbonyl), or a 5-(4-methylpiperazinyl) group. Among these
bibenzimidazoles, the pharmacological activity of 2′-phenyl derivatives and the influence of
the different positional isomers of either a 2′-tolyl group or a 2′-naphthyl moiety on cytotoxicity
and topoisomerase I inhibitory activity were determined.
In tr od u ction
not been determined. In the present study, several
5-cyano-2′-aryl-2,5′-bi-1H-benzimidazole derivatives were
synthesized. Topoisomerase I poisoning and cytotoxicity
of the 2′-phenyl derivatives as well as all positional
isomers of either a 2′-tolyl group or a 2′-naphthyl group
were investigated. By the facile conversion of these
5-cyano analogs to their 5-(aminocarbonyl) derivatives,
the influence of a functional group capable of hydrogen
bonding at the 5-position of these various 2′-aryl-bi-1H-
benzimidazoles was also assessed. The pharmacological
activity of these analogs was compared with similar 2′-
aryl-2,5′-bi-1H-benzimidazole derivatives containing a
5-(4-methylpiperazinyl) moiety, which at physiological
pH is extensively protonated.
The chemotherapeutic action of several anticancer
agents has been linked to their ability to inhibit nuclear
DNA topoisomerases. Topoisomerases are involved in
producing the necessary topological and conformational
changes in DNA which are critical to many cellular
processes such as replication and transcription.1-3
There are two types of topoisomerases which have been
isolated from mammalian cells. These enzymes can be
distinguished by whether they function by producing a
transient protein-bridged DNA break on one (mam-
malian topoisomerase I) or both (mammalian topo-
isomerase II) DNA strands. There have been several
reviews on topoisomerases as targets for the develop-
ment of anticancer agents.4-6 Several of the anticancer
agents in clinical use have been shown to be potent
inhibitors of topoisomerase II. Etoposide (VP-16), ten-
iposide (VM-26), mitoxantrone, m-AMSA, adriamycin
(doxorubicin), and daunomycin have all been demon-
strated to possess significant activity as inhibitors of
topoisomerase II.4-6 In comparison to topoisomerase II
poisons, however, there are fewer known topoisomerase
I inhibitors. Camptothecin is the most extensively
studied mammalian topoisomerase I inhibitor. The
broad spectrum of potent antineoplastic activity ob-
served for camptothecin7,8 has resulted in efforts to
identify other agents which can poison mammalian
topoisomerase I.
Ch em istr y
The 5-cyano derivatives of several 2′-aryl-2,5′-bi-1H-
benzimidazoles (2a -f) were synthesized by coupling of
4-cyano-1,2-phenylenediamine (1) with the appropri-
ately substituted 5-formyl-2-arylbenzimidazoles (Scheme
1). The yields of these various 2′-aryl-5-cyano-2,5′-bi-
1H-benzimidazoles ranged from 63% to 94%. Com-
pounds 2a -f served as convenient intermediates for the
synthesis of the 2′-aryl-5-(aminocarbonyl)-2,5′-bi-1H-
benzimidazoles 3a -f. Treatment of 2a -f with H2O2 in
the presence of tetrabutylammonium hydrogen sulfate
and 5 N NaOH provided the 2′-aryl-5-(aminocarbonyl)-
2,5′-bi-1H-benzimidazoles 3a -f.
A series of 2-aryl-5-cyano-1H-benzimidazoles, 4a -f,
was prepared as outlined in Scheme 2 by the coupling
of 1 with the appropriately substituted benzaldehyde
derivative or naphthylaldehyde. These various benz-
imidazoles were formed in yields ranging from 51% to
71%. Reduction of 4a -f with Ni-Al in the presence of
formic acid followed by hydrolysis provided an effective
method for preparation of the 2-aryl-5-formyl-1H-benz-
imidazole intermediates 5a -f required for the formation
of the various 2,5′-bi-1H-benzimidazole derivatives in
this study.
The preparation of the 2′-aryl-5-(4-methylpipera-
zinyl)-2,5′-bi-1H-benzimidazoles 8a -f was accomplished
by coupling of 4-(4-methylpiperazinyl)-1,2-phenylene-
diamine, 7, with the appropriate 2-aryl-5-formyl-1H-
benzimidazole 5a -f as outlined in Scheme 3. Synthesis
of 2-nitro-5-(4-methylpiperazinyl)aniline, 6, was ac-
Hoechst 33258, 2′-(4-hydroxyphenyl)-5-(4-methyl-
piperazinyl)-2,5′-bi-1H-benzimidazole (NSC-32291, pi-
benzimol), and Hoechst 33342, 2′-(4-ethoxyphenyl)-5-
(4-methylpiperazinyl)-2,5′-bi-1H-benzimidazole, are in-
hibitors of topoisomerase I.9-11 These agents are known
to bind to the minor groove of DNA and have been
shown to bind with A + T specificity. While Hoechst
33258 is cytotoxic, the more lipophilic derivative,
Hoechst 33342, exhibits greater cytotoxicity. While
studies have been performed to assess the effects of
structural variation at the 5-position of 2,5′-bi-1H-
benzimidazoles on topoisomerase I poisoning,12 the
influence of structural variations at the 2′-position has
† Rutgers, The State University of New J ersey.
‡ The University of Medicine and Dentistry of New J ersey.
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0022-2623/96/1839-0992$12.00/0 © 1996 American Chemical Society

Substituted 2,5′-Bi-1H-benzimidazoles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 993
Sch em e 1
Sch em e 2
derivative 8f, the other derivatives within this series
of compounds were more than 1 order of magnitude less
cytotoxic than Hoechst 33342.
It has been recently demonstrated that within a series
of terbenzimidazole derivatives, similar activity as
topoisomerase I poisons and cytotoxicity to Hoechst
33342 can be achieved with compounds which do not
have a basic tertiary amine attached to the 5-position.13
Earlier studies have demonstrated that 5-cyano-2′-(p-
methoxyphenyl)-2,5′-bi-1H-benzimidazole has ca. one-
third the potency of Hoechst 33342 as an inhibitor of
topoisomerase I.13 In this series, the only 5-cyano
analog evaluated that had similar potency to Hoechst
33342 as a topoisomerase I poison was the 2′-(4-tolyl)
derivative 2d . 2′-Phenyl-5-cyano-2,5′-bi-1H-benzimid-
azole, 2a , as well as the other isomeric tolyl derivatives
2b,c, was significantly less potent. Both isomers of
5-cyano-2′-naphthyl-2,5′-bi-1H-benzimidazole, 2e,f, had
ca. one-tenth the potency of Hoechst 33342 as topo-
isomerase I poisons. The absence of a clear correlation
between cytotoxicity and potency as topoisomerase I
inhibitors was most evident within this series of 5-cy-
ano-2′-substituted-2,5′-bi-1H-benzimidazoles. While dif-
ferences in cellular absorption may be responsible for
the absence of such a correlation, it cannot be assumed
from these data that the mechanism associated with the
cytotoxicity of these various analogs is exclusively
mediated through their potential as topoisomerase I
poisons.
complished by reaction of 2-nitro-5-chloroaniline with
1-methylpiperazine. Catalytic hydrogenation of 6 pro-
vided 7 in 95% yield.
Resu lts a n d Discu ssion
The relative potency of these variously substituted
bibenzimidazoles as topoisomerase I poisons was deter-
mined by assessing their ability to induce DNA cleavage
in the presence of enzyme. The results of these assays
are summarized in Table 1. Within the series of
compounds 8a -f which possess a 5-(4-methylpiperazin-
yl) moiety, 8a ,c,d exhibited similar potency to Hoechst
33342 as topoisomerase I poisons. In comparison to 8a
which has a 2′-phenyl substituent, the presence of either
an o-tolyl group (8b) or a naphthyl moiety (8e,f) at the
2′-position was associated with a reduction in their
relative potency as topoisomerase poisons by ca. 1 order
of magnitude. This study does indicate that within this
series of 5-(4-methylpiperazinyl)-substituted-2,5′-bi-1H-
benzimidazoles, substituents with varied steric effects
can be accommodated at the 2′-position with retention
of topoisomerase I activity. In general, individual
compounds within this series, compounds 8a -f, were
among the more cytotoxic derivatives against the hu-
man lymphoblastoma cell line RPMI 8402. These
results are consistent with those previously observed
for other 2,5′-bi-1H-benzimidazoles where the presence
of a 5-(4-methylpiperazinyl) or 5-oxy(4-methylpiperi-
dinyl) substituent was associated with enhanced cyto-
toxicity.12 With the exception of the 2′-(2-naphthyl)
The 5-(aminocarbonyl)-2′-substituted-2,5′-bi-1H-benz-
imidazole derivatives 3a -f were all less active than
their corresponding 5-cyano-2,5′-bi-1H-benzimidazoles,
with the exception of 2a . Compounds 2a and 3a were
among the weaker topoisomerase I poisons and among
the least cytotoxic of the derivatives evaluated. None
of the 5-(aminocarbonyl)-2′-substituted-2,5′-bi-1H-benz-
imidazole derivatives 3a -f exhibited greater potency
than their corresponding 5-(4-methylpiperazinyl)-2,5′-
bi-1H-benzimidazole derivatives as either topoisomerase
I inhibitors or cytotoxic agents.
Evaluation of the data associated with each series of
compounds (the 5-cyano, 5-(aminocarbonyl), and 5-pip-
erazinyl series) did reveal similarities. It was observed,
for example, that within all three series of 5-substituted-
bi-1H-benzimidazoles, the 2′-(2-naphthyl) and 2′-(1-
naphthyl) derivatives are among the more cytotoxic
analogs. The increased lipophilicity which would be
expected with these naphthyl derivatives appears to
correlate with their greater cytotoxic activity. Similar
comparisons of the relative activity of these agents as

994 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Kim et al.
Sch em e 3
Ta ble 1. Cytotoxicity and Topoisomerase I-Mediated Cleavage of DNA Induced by 2,5′-Bi-1H-benzimidazoles
topo I-mediated
cytotoxicity
compound
X
Y
cleavagea
IC50 (µM)b
Hoechst 33342
4-ethoxyphenyl
phenyl
2-tolyl
3-tolyl
4-tolyl
1-naphthyl
2-naphthyl
phenyl
2-tolyl
3-tolyl
4-methylpiperazinyl
-CN
-CN
-CN
-CN
-CN
-CN
-CONH2
-CONH2
-CONH2
-CONH2
-CONH2
-CONH2
4-methylpiperazinyl
4-methylpiperazinyl
4-methylpiperazinyl
4-methylpiperazinyl
4-methylpiperazinyl
4-methylpiperazinyl
1.0
>100
.100
100
1
10
10
10
100
100
10
20
100
1
0.03
>25
1.2
0.86
0.57
0.65
0.13
>25
8.2
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
8a
8b
8c
8d
8e
8f
8.2
4-tolyl
>25
1-naphthyl
2-naphthyl
phenyl
2-tolyl
3-tolyl
4-tolyl
1-naphthyl
2-naphthyl
3.7
0.74
0.49
0.59
0.36
0.83
0.33
0.11
10
1
1
20
10
a
Topoisomerase I cleavage values are reported as REC, relative effective concentration, i.e., concentrations relative to that of Hoechst
33342, whose value is arbitrarily assumed as 1, that are able to produce the same cleavage on the plasmid DNA in the presence of calf
b
thymus topoisomerase I. Cleavage is calculated from the intensity of the strongest Hoechst specific band. IC50 has been calculated
after 4 days of continuous drug exposure. No indication of cytotoxicity was considered indicative for IC50 values substantially greater
than the highest dose assayed, 25 µM.
are reported in cm-1. Proton (1H NMR) and carbon (13C NMR)
topoisomerase I poisons indicate that those analogs with
nuclear magnetic resonance were recorded on a Varian Gemini-
a 2′-(4-tolyl) group are among the potent inhibitors of
topoisomerase I. These data suggest that the presence
of a lipophilic substituent at the para position within
this various series of 2,5′-bi-1H-benzimidazoles may
200 Fourier transform spectrometer. NMR spectra (200 MHz
1H and 50 MHz 13C) were recorded in CDCl3 (unless otherwise
noted) with chemical shifts reported in δ units downfield from
tetramethylsilane (TMS). Coupling constants are reported in
enhance topoisomerase I inhibitory activity. There is,
however, no similar consistency observed for these tolyl
derivatives with regard to the results obtained on their
relative cytotoxicity. Further studies are needed to
more fully assess those structural features of 2,5′-bi-
1H-benzimidazoles which are associated with optimal
activity as topoisomerase I poisons and cytotoxic activ-
ity.
hertz. Mass spectra were obtained from Midwest Center for
Mass Spectrometry within the Department of Chemistry at
the University of Nebraska-Lincoln. Combustion analyses
were performed by Atlantic Microlabs, Inc., Norcross, GA, and
are within (0.4% of the theoretical value.
3,4-Dia m in oben zon itr ile (1). 4-Amino-3-nitrobenzonitrile
(2.02 g, 12.4 mmol) in 100 mL of EtOAc was reduced by
hydrogenation using 45 psi of H2 and 10% Pd-C (500 mg) for
1.5 h.14,15 After passing through a bed of Celite, solvent was
removed in vacuo and 1.56 g (94.5%) of white solid was
obtained: mp 143-145 °C (lit.15 mp 146 °C); H NMR δ 3.39
Exp er im en ta l Section
1
(brs, 2H), 3.82 (brs, 2H), 6.68 (d, 1H, J ) 8.0), 6.95 (d, 1H, J
) 1.8), 7.05 (dd, 1H, J ) 8.0, 1.8); 13C NMR δ 99.8, 116.2, 118.7,
124.1, 125.3, 137.7, 143.2. The crude diamine obtained from
hydrogenation was typically used directly without further
purification.
Melting points were determined with a Thomas-Hoover
Unimelt capillary melting point apparatus. Column chroma-
tography refers to flash chromatography conducted on SiliTech
32-63 µm (ICN Biomedicals, Eschwegge, Germany) using the
solvent systems indicated. Chromatotron 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
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Cya n o-
2,5′-bi-1H-ben zim id a zoles: 2′-P h en yl-5-cya n o-2,5′-bi-1H-
ben zim id a zole (2a ). A stirred solution of 5a (218 mg, 0.98

Substituted 2,5′-Bi-1H-benzimidazoles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 995
mmol) and 1 (131 mg, 0.98 mmol) in 3 mL of nitrobenzene
was heated at 145 °C under N2 overnight. The cooled reaction
mixture was then purified directly by column chromatography.
Elution with 10% EtOAc/hexanes removed the nitrobenzene.
Product was obtained by employing a gradient from 100%
EtOAc to 5% MeOH/EtOAc. Concentration in vacuo provided
249 mg (76%) of a white solid: mp 240 °C dec; IR (KBr) 3149,
2222, 1442, 1291; 1H NMR (DMSO-d6 + 3 drops of CF3COOH)
δ 7.75-7.85 (m, 4H), 7.97 (d, 1H, J ) 8.7), 8.13 (d, 1H, J )
8.8), 8.25-8.30 (m, 2H), 8.36-8.41 (m, 2H), 8.66 (s, 1H); 13C
NMR (DMSO-d6 + 3 drops of CF3COOH) δ 106.6, 112.3, 114.2,
115.6, 115.7, 119.2, 120.1, 122.1, 125.3, 128.1, 128.4, 129.9,
132.1, 133.4, 133.7, 135.6, 137.9, 152.3, 152.8.
2′-o-Tolyl-5-cya n o-2,5′-bi-1H-ben zim id a zole (2b). A so-
lution of 5b (262 mg, 1.11 mmol) and 1 (148 mg, 1.11 mmol)
was treated as described for 2a . Column chromatography
using 40-100% EtOAc/hexanes provided 295 mg (76%) of
crude product. A portion of the crude product (57 mg) was
further purified by chromatotron chromatography using 40%
EtOAc/hexane to provide 49.3 mg of a white solid: mp >280
°C; IR (KBr) 3199, 2223, 1441, 1294; 1H NMR (CD3OD) δ 2.51
(s, 3H), 7.26-7.44 (m, 4H), 7.57-7.69 (m, 3H), 7.83 (s, 1H),
7.94 (dd, 1H, J ) 8.5, 1.7), 8.27 (s, 1H); 13C NMR (CD3OD) δ
20.9, 106.7, 121.0, 127.5, 131.2, 131.7, 132.6, 139.0.
vacuo. Column chromatography using a gradient from 100%
EtOAc to 25% MeOH/EtOAc afforded 126 mg (70.4%) of a
white solid: mp 242 °C dec; IR (KBr) 3364, 3169, 1651, 1621,
1441, 1390, 1287; 1H NMR (CD3OD) δ 7.49-7.52 (m, 3H), 7.59
(d, 1H, J ) 8.4), 7.68 (d, 1H, J ) 8.4), 7.78 (dd, 1H, J ) 8.4,
1.8), 7.97 (dd, 1H, J ) 8.5, 1.6), 8.05-8.09 (m, 2H), 8.16 (d,
1H, J ) 1.3), 8.30 (d, 1H, J ) 1.2); 13C NMR (CD3OD) δ 115.1,
115.2, 115.5, 116.3, 116.8, 123.3, 123.7, 125.4, 128.3, 129.7,
130.5, 130.8, 132.0, 155.7, 156.5, 173.2; HRMS (FAB) calcd
for C21H16N5O (M+ + 1) 354.1356, found 354.1361.
2′-o-T o ly l-5-(a m in o c a r b o n y l)-2,5′-b i-1H -b e n zim id -
a zole (3b). A biphasic reaction mixture consisting of a
solution of 2b (238 mg, 0.68 mmol) in 30% MeOH/CH2Cl2 in
the presence of 1.0 mL of 30% H2O2, 50 mg of tetrabutyl-
ammonium hydrogen sulfate, and 3 mL of 5 N NaOH was
performed as outlined for 3a . Column chromatography using
a gradient from 100% EtOAc to 5% MeOH/EtOAc afforded 39
mg (16%) of a white solid: mp >290 °C; IR (KBr) 3389, 3180,
1648, 1387, 1287; 1H NMR (CD3OD) δ 2.53 (s, 3H), 7.28-7.46
(m, 3H), 7.63 (d, 2H, J ) 7.7), 7.74 (d, 1H, J ) 8.4), 7.81 (dd,
1H, J ) 8.4, 1.6), 8.05 (dd, 1H, J ) 8.5, 1.4), 8.19 (s, 1H), 8.38
(s, 1H); 13C NMR (DMSO-d6) δ 21.3, 118.4, 119.5, 126.3, 129.8,
129.9, 131.6, 137.5, 168.7; HRMS (FAB) calcd for C22H18N5O
(M+ + 1) 368.1512, found 368.1502.
2′-m -Tolyl-5-cya n o-2,5′-bi-1H-ben zim id a zole (2c). A so-
lution of 5c (253 mg, 1.07 mmol) and 1 (142 mg, 1.07 mmol)
was treated as described for 2a . Column chromatography
using 0-100% EtOAc/hexanes and then 0-50% MeOH/EtOAc
provided 236 mg (63%) of a white solid: mp 245 °C dec; IR
(KBr) 3142, 2220, 1437; 1H NMR (CD3OD) δ 2.40 (s, 3H), 7.24-
7.43 (m, 3H), 7.59 (d, 2H, J ) 8.1), 7.78-7.88 (m, 4H), 8.14 (s,
1H); 13C NMR (CD3OD) δ 21.8, 106.4, 110.1, 121.0, 123.2,
123.2, 124.6, 125.4, 127.3, 128.7, 130.3, 130.5, 132.7, 140.4.
2′-p-Tolyl-5-cya n o-2,5′-bi-1H-ben zim id a zole (2d ). A so-
lution of 5d (235 mg, 0.99 mmol) and 1 (132 mg, 0.99 mmol)
was treated as described for 2a . Column chromatography
using 50-100% EtOAc and then 0-10% MeOH/EtOAc pro-
vided 258 mg (78%) of a white solid: mp 229 °C dec; IR (KBr)
3164, 2222, 1439, 1296; 1H NMR (DMSO-d6 + 3 drops of
CF3COOH) δ 2.47 (s, 3H), 7.56 (d, 2H, J ) 8.1), 7.70 (dd, 1H,
J ) 8.4, 1.4), 7.85 (d, 1H, J ) 8.4), 8.00 (d, 1H, J ) 8.5), 8.16
(d, 2H, J ) 8.1), 8.24 (s, 1H), 8.35 (dd, 1H, J ) 8.5, 1.4), 8.57
(s, 1H); 13C NMR (DMSO-d6 + 3 drops of CF3COOH) δ 21.5,
105.1, 113.3, 115.3, 115.9, 120.0, 120.6, 122.1, 124.4, 124.9,
126.7, 128.1, 130.4, 134.5, 136.0, 138.6, 140.7, 143.7, 152.0,
153.9.
2′-m -Tolyl-5-(a m in oca r b on yl)-2,5′-b i-1H -b e n zim id -
a zole (3c). A biphasic reaction mixture consisting of a
solution of 2c (183 mg, 0.53 mmol) in 30% MeOH/CH2Cl2 in
the presence of 1.0 mL of 30% H2O2, 38 mg of tetrabutyl-
ammonium hydrogen sulfate, and 3 mL of 5 N NaOH was
performed as outlined for 3a . Column chromatography using
a gradient from 0% to 10% MeOH/EtOAc afforded 22 mg (12%)
of a white solid: mp 250 °C dec; IR (KBr) 3393, 3186, 1652,
1
1600, 1438, 1386; H NMR (DMSO-d6) δ 2.46 (s, 3H), 7.36-
7.85 (m, 4H), 8.02-8.27 (m, 4H), 8.37 (s, 1H), 8.50 (d, 1H, J )
3.24); 13C NMR (DMSO-d6) δ 21.3, 124.1, 127.5, 129.2, 130.0,
131.2, 131.7, 138.6, 154.0, 168.7; HRMS (FAB) calcd for
C22H18N5O (M+ + 1) 368.1512, found 368.1517.
2′-p -T o lyl-5-(a m in oc a r b on yl)-2,5′-b i-1H -b e n zim id -
a zole (3d ). A biphasic reaction mixture consisting of a
solution of 2d (200 mg, 0.57 mmol) in 30% MeOH/CH2Cl2 in
the presence of 1.1 mL of 30% H2O2, 42 mg of tetrabutyl-
ammonium hydrogen sulfate, and 3 mL of 5 N NaOH was
performed as outlined for 3a . Column chromatography using
a gradient from 0% to 10% MeOH/EtOAc afforded 109 mg
(52%) of a white solid: mp 245 °C dec; IR (KBr) 3374, 3180,
1659, 1609, 1393, 1296; 1H NMR (CD3OD) δ 2.43 (s, 3H), 7.38
(d, 2H, J ) 8.0), 7.65 (d, 1H, J ) 8.7), 7.74 (d, 1H, J ) 8.4),
7.83 (dd, 1H, J ) 8.2, 1.7), 8.02 (d, 2H, J ) 8.2), 8.03 (dd, 1H,
J ) 7.0, 1.7), 8.19 (s, 1H), 8.35 (s, 1H); 13C NMR (CD3OD) δ
21.8, 123.3, 123.7, 125.4, 128.0, 128.3, 129.7, 131.1, 131.2,
2′-(Na p h th -1-yl)-5-cya n o-2,5′-bi-1H-ben zim id a zole (2e).
A solution of 5e (96 mg, 0.35 mmol) and 1 (47 mg, 0.35 mmol)
was treated as described for 2a . Column chromatography
using 20-50% EtOAc/hexanes provided 127 mg (94%) of a
white solid: mp >280 °C; IR (KBr) 3060, 2222, 1548, 1439,
142.8, 156.0, 173.2; HRMS (FAB) calcd for C22H18N5O (M+
1) 368.1512, found 368.1504.
+
1
1138; H NMR (DMSO-d6) δ 7.59-7.78 (m, 6H), 8.1-8.2 (m,
5H), 8.6 (s, 1H), 9.09 (dd, 1H, J ) 8.4, 2.2); 13C NMR (DMSO-
d6) δ 104.0, 120.4, 125.6, 125.9, 126.4, 126.7, 127.4, 127.5,
128.5, 128.7, 130.7, 130.9, 133.9.
2′-(Na p h t h -1-yl)-5-(a m in oca r b on yl)-2,5′-b i-1H -b e n z-
im id a zole (3e). A biphasic reaction mixture consisting of a
solution of 2e (78 mg, 0.20 mmol) in 30% MeOH/CH2Cl2 in
the presence of 0.3 mL of 30% H2O2, 14 mg of tetrabutyl-
ammonium hydrogen sulfate, and 3 mL of 5 N NaOH was
performed as outlined for 3a . Column chromatography using
a gradient from 0% to 10% MeOH/EtOAc afforded 63 mg (77%)
of a white solid: mp >288 °C; IR (KBr) 3378, 3190, 1653, 1387;
1H NMR (CD3OD) δ 7.56-7.66 (m, 4H), 7.78-7.90 (m, 3H),
7.95-8.11 (m, 3H), 8.19 (d, 1H, J ) 1.1), 8.43 (s, 1H), 8.49-
8.53 (m, 1H); 13C NMR (CD3OD) δ 115.4, 115.5, 115.6, 116.4,
116.9, 123.4, 123.7, 125.6, 126.5, 126.9, 127.9, 128.7, 128.9,
129.7, 129.8, 129.9, 132.3, 132.7, 135.7, 155.7, 156.6, 173.2;
HRMS (FAB) calcd for C25H18N5O (M+ + 1) 404.1512, found
404.1523.
2′-(Na p h th -2-yl)-5-cya n o-2,5′-bi-1H-ben zim id a zole (2f).
A solution of 5f (149 mg, 0.55 mmol) and 1 (73 mg, 0.55 mmol)
was treated as described for 2a . Column chromatography
using 50-100% EtOAc/hexanes provide 167 mg (80%) of a
white solid: mp 254 °C dec; IR (KBr) 3119, 2220, 1436, 1293;
1H NMR (CD3Cl3 + 3 drops of CD3OD) δ 7.29-7.34 (m, 3H),
7.49 (d, 1H, J ) 9.0), 7.56-7.80 (m, 6H), 7.85 (d, 1H, J ) 9.1),
8.08 (s, 1H), 8.24 (s, 1H); 13C NMR (CDCl3 + 3 drops of CD3OD)
δ 105.0, 115.7, 120.3, 122.2, 123.4, 123.6, 126.2, 126.4, 127.0,
127.1, 127.7, 128.0, 128.8, 129.0, 133.3, 134.4, 154.3, 156.1.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 2-(Ben z-
im idazol-5-yl)-5-(am in ocar bon yl)ben zim idazoles: 2′-P h en -
yl-5-(a m in oca r bon yl)-2,5′-bi-1H-ben zim id a zole (3a ).
A
2′-(Na p h t h -2-yl)-5-(a m in oca r b on yl)-2,5′-b i-1H -b e n z-
im id a zole (3f). A biphasic reaction mixture consisting of a
solution of 2f (122 mg, 0.32 mmol) in 30% MeOH/CH2Cl2 in
the presence of 0.3 mL of 30% H2O2, 22 mg of tetrabutyl-
ammonium hydrogen sulfate, and 3 mL of 5 N NaOH was
performed as outlined for 3a . Column chromatography using
a gradient from 0% to 10% MeOH/EtOAc afforded 85 mg (66%)
of a white solid: mp 270 °C dec; IR (KBr) 3410, 3180, 1643,
1543, 1429, 1392, 1287; 1H NMR (CD3OD) δ 7.37-7.42 (m, 2H),
solution of 2a (170 mg, 0.51 mmol) in 3 mL of 30% MeOH/
CH2Cl2 was cooled in an ice bath. To this solution were added
1.0 mL of 30% H2O2, 50 mg of tetrabutylammonium hydrogen
sulfate, and 3 mL of 5 N NaOH. The reaction mixture was
allowed to stir overnight at room temperature. The CH2Cl2
layer was removed, and the H2O layer was extracted with
EtOAc (3 × 3 mL). The organic layers were combined, washed
with brine and water, dried (Na2SO4), and concentrated in

996 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Kim et al.
7.48 (d, 1H, J ) 8.7), 7.57 (d, 1H, J ) 8.5), 7.69 (dd, 2H, J )
9.1, 1.5), 7.77-7.85 (m, 3H), 7.99 (dd, 1H, J ) 8.6, 1.7), 8.06
(s, 1H), 8.13 (s, 1H), 8.34 (s, 1H); 13C NMR (CD3OD) δ 114.9,
115.4, 116.3, 123.2, 123.6, 124.8, 125.2, 127.7, 128.0, 128.1,
128.6, 129.0, 129.5, 129.9, 130.1, 134.6, 135.8, 155.4, 156.2,
173.2; HRMS (FAB) calcd for C25H18N5O (M+ + 1) 404.1512,
found 404.1501.
126.9, 127.1, 127.4, 127.9, 128.1, 128.9, 129.0, 133.0, 134.1.
Anal. (C18H11N3) C, H, N.
Gen er a l Meth od for th e Con ver sion of 5-Cya n oben z-
im id a zoles t o 5-F or m ylb en zim id a zoles: 2-P h en yl-5-
for m ylben zim id a zole (5a ). Using a similar procedure to
that described previously,17 Ni-Al (3.93 g) was added to a
solution of 4a (820 mg, 3.74 mmol) in formic acid (59.6 mL)
and H2O (19.7 mL). The reaction mixture was heated at 95
°C for 6 h. The hot mixture was filtered through a bed of
Celite, and the reaction flask and Celite bed were rinsed with
water. The aqueous solution was concentrated to dryness.
After addition of H2O to this residue, a white precipitate
formed. The pH of this suspension was adjusted to 9 by the
dropwise addition of 2 N NaOH. The product was obtained
by extraction with EtOAc. The EtOAc extract was dried
(Na2SO4) and concentrated in vacuo. The residue was purified
by column chromatography using a gradient of 0-50% EtOAc/
hexanes to give 499 mg (60%) of a gum. Recrystallization with
hexanes provided a white solid: mp 173-174 °C; IR (KBr)
Gen er a l P r oced u r e for th e P r ep a r a tion of 5-Cya n o-
ben zim id a zoles: 2-P h en yl-5-cya n oben zim id a zole (4a ). A
solution of 1 (1.32 g, 9.9 mmol) and benzaldehyde (1.05 g, 9.9
mmol) in 20 mL of nitrobenzene was heated at 145 °C
overnight under N2 as described for 2a . Column chromatog-
raphy using 100% hexane to remove nitrobenzene and a
gradient elution from 10% to 40% EtOAc/hexanes provided
1.17 g (54%) of a pale yellow solid. Recrystallization from
EtOAc and hexanes provided white crystals (mp 210-212 °C)
as the hydrochloride: mp 270-271 °C (lit.16 mp 232-235 °C);
1
IR (KBr) 3259, 3046, 2230, 1615, 1538, 1459, 1305; H NMR
(CDCl3 + 2 drops of CD3OD) δ 7.39-7.47 (m, 4H), 7.56 (d, 1H,
J ) 9.0), 7.85 (s, 1H), 8.01-8.06 (m, 2H); 13C NMR (CDCl3 +
2 drops of CD3OD) δ 105.7, 120.0, 126.6, 127.4, 129.3, 129.6,
131.5. Due to hydration with H2O (C14H9N3‚1/3H2O), this
compound failed to give satisfactory microanalysis for C14H9N3.
Anal. (C14H9N3‚1/3H2O) C, H, N.
1
3259, 3057, 2831, 1662, 1608, 1460, 1294; H NMR (CDCl3 +
3 drops of CD3OD) 7.36-7.90 (m, 3H), 7.60 (d, 1H, J ) 9.0),
7.71 (dd, 1H, J ) 8.4, 1.5), 8.01-8.06 (m, 3H), 9.93 (s, 1H);
13C NMR (CDCl3 + 3 drops of CD3OD) δ 124.8, 127.4, 129.4,
129.5, 131.3, 132.0, 158.0, 193.1. Anal. (C14H10N2O) C, H, N.
2-o-Tolyl-5-for m ylben zim id a zole (5b). A reaction mix-
ture of Ni-Al (3.58 g) and 4b (896 mg, 3.84 mmol) in formic
acid (54.4 mL) and H2O (17.9 mL) was treated as 5a . Purifica-
tion by column chromatography using a gradient of 30-40%
EtOAc/hexanes gave 647 mg (71%) of a gum. Recrystallization
with hexanes provided a white solid: mp 139-140 °C; IR (KBr)
3045, 2926, 2737, 1692, 1609, 1437, 1283, 1146; 1H NMR δ
2.59 (s, 3H), 7.23-7.45 (m, 4H), 7.64 (d, 1H, J ) 7.7), 7.81
(dd, 1H, J ) 8.34, 1.34), 7.97 (s, 1H), 9.99 (s, 1H); 13C NMR δ
21.3, 124.4, 124.5, 126.7, 129.8, 130.2, 130.9, 132.0, 132.3,
138.0, 171.8, 192.6. Anal. (C15H12N2O) C, H, N.
2-o-Tolyl-5-cya n oben zim id a zole (4b). A solution of 1
(1.34 g, 10.1 mmol) and o-tolylaldehyde (1.21 g, 10.1 mmol) in
20 mL of nitrobenzene was treated as described for 4a and
provided 1.43 g (61%) of a pale yellow solid. Recrystallization
from hexanes and EtOAc gave white needles: mp 185-187
°C; IR (KBr) 3067, 2790, 2667, 2216, 1453, 1282; 1H NMR
(CDCl3 + 3 drops of CD3OD) δ 2.50 (s, 3H), 7.20-7.43 (m, 5H),
7.55 (d, 1H, J ) 7.5), 7.62 (s, 1H); 13C NMR (CDCl3 + 3 drops
of CD3OD) δ 21.0, 105.6, 120.4, 126.5, 126.7, 129.6, 130.2,
131.0, 131.9, 138.0, 156.0. Anal. (C15H11N3) C, H, N.
2-m -Tolyl-5-cya n oben zim id a zole (4c). A solution of 1
(1.29 g, 9.7 mmol) and m-tolylaldehyde (1.16 g, 9.7 mmol) in
20 mL of nitrobenzene was treated as described for 4a and
provided 1.54 g (69%) of a pale yellow solid. Recrystallization
from hexanes and EtOAc gave pale orange flakes: mp 180-
181 °C; IR (KBr) 3272, 3016, 2216, 1451, 1287; 1H NMR (CDCl3
+ 5 drops of CD3OD) δ 2.33 (s, 3H), 7.18-7.32 (m, 2H), 7.37
(dd, 1H, J ) 8.4, 1.5), 7.54 (d, 1H, J ) 8.4), 7.75-7.81 (m,
3H); 13C NMR (CDCl3 + 5 drops of CD3OD) δ 21.7, 105.3, 120.6,
124.5, 126.2, 128.1, 129.1, 129.4, 132.2, 139.8, 157.9. Anal.
(C15H11N3) C, H, N.
2-p-Tolyl-5-cya n oben zim id a zole (4d ). A solution of 1
(1.27 g, 9.5 mmol) and p-tolylaldehyde (1.14 g, 9.5 mmol) in
20 mL of nitrobenzene was treated as described for 4a and
provided 1.43 g (64%) of a pale yellow solid. Recrystallization
from hexanes and EtOAc gave white flakes: mp 243-244 °C;
IR (KBr) 3259, 3026, 2227, 1619, 1551, 1449, 1374, 1304, 1234,
1188; 1H NMR (CDCl3 + 3 drops of CD3OD) δ 2.29 (s, 3H),
7.18 (d, 2H, J ) 8.1), 7.35 (dd, 1H, J ) 8.3, 1.5), 7.52 (d, 1H,
J ) 8.3), 7.79 (s, 1H), 7.83 (d, 2H, J ) 8.1); 13C NMR (CDCl3
+ 3 drops of CD3OD) δ 21.7, 105.2, 120.4, 126.4, 127.3, 130.2,
141.8. Anal. (C15H11N3) C, H, N.
2-m -Tolyl-5-for m ylben zim id a zole (5c). A reaction mix-
ture of Ni-Al (4.62 g) and 4c (1.03 g, 4.41 mmol) in formic
acid (70.4 mL) and H2O (23.2 mL) was treated as 5a . Purifica-
tion by column chromatography using 40% EtOAc/hexanes
gave 535 mg (51%) of a gum. Recrystallization with hexanes
provided a white solid: mp 163-165 °C; IR (KBr) 3045, 2926,
1686, 1615, 1419, 1276; 1H NMR δ 2.20 (s, 3H), 7.20-7.32 (m,
2H), 7.65 (d, 1H, J ) 8.3), 7.79 (d, 1H, J ) 8.4), 7.96 (d, 1H, J
) 9.8), 7.99 (s, 1H), 8.01 (s, 1H), 9.98 (s, 1H); 13C NMR δ 21.7,
124.7, 125.1, 128.4, 129.4, 129.6, 132.2, 132.3, 139.6, 156.3,
193.0. Anal. (C15H12N2O) C, H, N.
2-p-Tolyl-5-for m ylben zim id a zole (5d ). A reaction mix-
ture of Ni-Al (3.70 g) and 4d (821 mg, 3.52 mmol) in formic
acid (56.1 mL) and H2O (18.5 mL) was treated as 5a . Purifica-
tion by column chromatography using a gradient of 30-40%
EtOAc/hexanes gave 535 mg (64%) of a gum. Recrystallization
with hexanes provided a white solid: mp 184-185 °C; IR (KBr)
3045, 2960, 1688, 1610, 1559, 1436, 1369, 1282, 1113; 1H NMR
δ 2.20 (s, 3H), 7.21 (d, 2H, J ) 8.1), 7.64 (d, 1H, J ) 8.2), 7.77
(dd, 1H, J ) 8.4, 1.2), 8.06 (d, 2H, J ) 8.1), 8.11 (s, 1H), 9.99
(s, 1H); 13C NMR δ 21.9, 126.7, 127.5, 130.5, 132.1, 142.0,
193.0. Anal. (C15H12N2O) C, H, N.
2-(Na p h th -1-yl)-5-cya n oben zim id a zole (4e). A solution
of 1 (1.12 g, 8.4 mmol) and 1-naphthylaldehyde (1.31 g, 8.4
mmol) in 20 mL of nitrobenzene was treated as described for
4a and provided 1.46 g (64%) of a pale yellow solid. Recrys-
tallization from hexanes and EtOAc gave pale orange crys-
tals: mp 217-218 °C; IR (KBr) 3046, 2780, 1677, 2215, 1405,
2-(Na p h th -1-yl)-5-for m ylben zim id a zole (5e). A mixture
of Ni-Al (1.60 g) and 4f (454 mg, 1.69 mmol) in formic acid
(25 mL) and H2O (8 mL) was treated as 5a . Purification by
column chromatography using a gradient of 20-40% EtOAc/
hexanes gave 293 mg (64%) of a gum. Recrystallization with
hexanes provided a white solid: mp 198 °C; IR (KBr) 3049,
1
1287; H NMR (CDCl3 + 3 drops of CD3OD) δ 7.41-7.52 (m,
1
5H), 7.73 (dd, 1H, J ) 7.2, 1.2), 7.75-7.88 (m, 2H), 7.91 (d,
1H, J ) 8.3), 8.52 (s, 1H); 13C NMR (CDCl3 + 3 drops of CD3-
OD) δ 105.6, 120.5, 125.4, 125.8, 126.5, 127.0, 127.3, 127.9,
128.7, 129.0, 131.3, 131.6, 134.3. Anal. (C18H11N3) C, H, N.
2-(Na p h th -2-yl)-5-cya n oben zim id a zole (4f). A solution
of 1 (1.18 g, 8.9 mmol) and 2-naphthylaldehyde (1.39 g, 8.9
mmol) in 20 mL of nitrobenzene was treated as described for
4a and provided 1.44 g (60%) of a pale yellow solid. Recrys-
tallization from hexanes and EtOAc gave pale yellow needles:
2955, 2720, 1678, 1614, 1531, 1367, 1273; H NMR δ 7.20 (d,
1H, J ) 7.5), 7.26-7.45 (m, 3H), 7.57 (d, 1H, J ) 7.3), 7.64 (s,
1H), 7.66 (d, 1H, J ) 8.4), 7.74-7.80 (m, 2H), 8.40 (d, 1H, J )
8.4), 9.79 (s, 1H); 13C NMR δ 124.1, 125.3, 125.8, 127.0, 127.5,
127.8, 128.7, 128.9, 131.4, 131.5, 132.1, 134.2, 155.4, 192.6.
Anal. (C18H12N2O) C, H, N.
2-(Na p h th -2-yl)-5-for m ylben zim id a zole (5f). A mixture
of Ni-Al (2.56 g) and 4e (731 mg, 2.71 mmol) in formic acid
(38.4 mL) and H2O (12.8 mL) was treated as 5a . Purification
by column chromatography using 40% EtOAc/hexanes gave
443 mg (60%) of a gum. Recrystallization with hexanes
provided a white solid: mp 190-191 °C; IR (KBr) 3294, 1668,
1
mp 237-238 °C; IR (KBr) 3261, 2232, 1535, 1444, 1286; H
NMR (DMSO-d6) δ 7.62-7.66 (m, 4H), 7.80 (d, 1H, J ) 9.0),
8.0-8.1 (m, 2H), 8.12 (d, 1H, J ) 9.1), 8.34 (dd, 1H, J ) 8.6,
1.6), 8.82 (s, 1H); 13C NMR (DMSO-d6) δ 104.3, 120.3, 124.2,
1
1609, 1383, 1300, 1128; H NMR δ 7.40-7.54 (m, 2H), 7.69-

Substituted 2,5′-Bi-1H-benzimidazoles
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 997
7.86 (m, 5H), 8.12 (s, 1H), 8.18 (dd, 1H, J ) 8.6, 1.8), 8.57 (s,
1H), 9.93 (s, 1H); 13C NMR δ 124.0, 125.4, 126.6, 127.5, 127.6,
128.2, 128.3, 129.1, 129.6, 132.4, 133.5, 134.8, 155.6, 192.8.
Anal. (C18H12N2O) C, H, N.
gradient from 0% to 30% MeOH/EtOAc gave the crude product
which was further purified by chromatotron chromatography
using 0-30% MeOH/EtOAc to provide 287 mg (85%) of a
1
greenish white solid: mp 240 °C dec (lit.18 mp >200 °C); H
NMR (CD3OD) δ 2.36 (s, 3H), 2.40 (s, 3H), 2.65 (t, 4H, J )
4.7), 3.20 (t, 4H, J ) 4.8), 7.03 (dd, 1H, J ) 8.8, 2.2), 7.12 (s,
1H), 7.34 (d, 2H, J ) 8.2), 7.50 (d, 1H, J ) 8.7), 7.68 (d, 1H, J
) 8.4), 7.92 (d, 1H, J ) 1.7), 7.98 (d, 2H, J ) 8.3), 8.25 (s, 1H);
13C NMR (CD3OD) δ 21.7, 46.4, 52.1, 56.4, 116.7, 122.9, 126.2,
128.2, 131.1, 142.6, 149.9.
5-(1-Meth yl-4-p ip er a zin yl)-2-n itr oa n ilin e (6). 5-Chloro-
2-nitroaniline (2.01 g, 11.59 mmol), 1-methylpiperazine (2.58
mL, 23.18 mmol), and K2CO3 (1.6 g, mmol) in 20 mL of DMF
was heated at 120 °C for 18 h. Purification on a silica gel
column with 20% MeOH/EtOAc as eluant gave 2.14 g (78%)
1
of a bright yellow solid: mp 153-4 °C (lit.18 mp 155 °C); H
2′-(Na p h t h -1-yl)-5-(4-m e t h ylp ip e r a zin yl)-2,5′-b i-1H -
ben zim id a zole (8e). A solution of 7 (109 mg, 0.5 mmol) and
5e (144 mg, 0.5 mmol) in 3 mL of nitrobenzene was heated
under N2 at 145 °C overnight. Column chromatography using
a gradient from 0% to 20% MeOH/EtOAc gave the crude
product which was further purified by chromatotron chroma-
tography using 0-20% MeOH/EtOAc to provide 133.6 mg
(55%) of an orange solid: mp 251-252 °C; IR (KBr) 3415, 3043,
2926, 2809, 1438, 1386, 1282; 1H NMR (CD3OD) δ 2.34 (s, 3H),
2.63 (t, 4H, J ) 4.7), 3.19 (t, 4H, J ) 4.3), 7.02 (dd, 1H, J )
8.8), 7.12 (s, 1H), 7.48-7.64 (m, 4H), 7.78 (d, 1H, J ) 8.5),
7.88 (d, 1H, J ) 7.2), 7.95-8.06 (m, 3H), 8.36 (s, 1H), 8.53 (m,
1H); 13C NMR (CD3OD) δ 46.4, 52.0, 56.4, 116.7, 123.1, 126.4,
126.5, 126.9, 127.9, 128.6, 129.0, 129.7, 129.9, 132.2, 132.7,
135.7, 149.9, 153.9, 155.4; HRMS (FAB) calcd for C29H27N6 (M+
+ 1) 459.2298, found 459.2282.
2′-(Na p h t h -2-yl)-5-(4-m e t h ylp ip e r a zin yl)-2,5′-b i-1H -
ben zim id a zole (8f). A solution of 7 (87 mg, 0.4 mmol) and
5f (115 mg, 0.4 mmol) in 3 mL of nitrobenzene was heated
under N2 at 145 °C overnight. Column chromatography using
50% MeOH/EtOAc gave the crude product which was further
purified by chromatotron chromatography using 0-10% MeOH/
EtOAc to provide 36 mg (18%) of an orange solid: mp 245-
246 °C (lit.19 mp 245 °C); 1H NMR (CD3OD) δ 2.7 (s, 3H), 3.14
(t, 4H, J ) 4.68), 3.17 (t, 4H, J ) 4.04), 6.91 (dd, 1H, J ) 8.8,
2.2), 6.98 (s, 1H), 7.38-7.46 (m, 3H), 7.58 (d, 1H, J ) 8.4),
7.79-7.86 (m, 4H), 8.04 (dd, 1H, J ) 8.0, 1.7), 8.12 (s, 1H),
8.39 (s, 1H); 13C NMR (CD3OD) δ 45.7, 51.4, 56.1, 102.6, 116.6,
116.7, 116.8, 122.9, 124.9, 125.9, 127.9, 128.0, 128.1, 128.7,
129.1, 129.9, 130.1, 134.7, 135.8, 140.3, 149.4, 153.8.
Top oisom er a se I-Med ia ted DNA Clea va ge Assa ys.
DNA topoisomerase I was purified from calf thymus gland as
reported previously.20,21 Plasmid YEpG was also purified by
the alkali lysis method followed by phenol deproteination and
CsCl/ethidium isopycnic centrifugation as described.22 The
end-labeling of the plasmid was accomplished as previously
described.23 The cleavage assays were performed as previously
reported.9 The drug and DNA in the presence of topoisomerase
I were incubated for 30 min at 37 °C. After development of
the gels, 24 h exposure was typically used to obtain auto-
radiographs outlining the extent of DNA fragmentation.
Cytotoxicity Assa y. The cytotoxicity as listed for several
of the compounds which were prepared as part of this study
was determined using human lymphoblast RPMI 8402 cells
and the MTT microtiter plate tetrazolium cytotoxicity assay
(MTA).24-26 The cytotoxicity assay was performed using 2000
cells/well, in 200 µL of growth medium, which were grown in
suspension at 37 °C in 5% CO2 and maintained by regular
passage in RPMI medium supplemented with 10% heat-
inactivated fetal bovine serum, L-glutamine (2 mM), penicillin
(100 U/mL), and streptomycin (0.1 mg/mL). The cells were
exposed continuously for 4 days to different drug concentra-
tions and assayed at the end of the fourth day. Each assay
was performed with a control which did not contain any drug.
All assays were performed at least twice in six replica wells.
NMR δ 2.33 (s, 3H), 2.51 (t, 4H, J ) 5.3), 3.37 (t, 4H, J ) 5.1),
5.95 (d, 1H, J ) 2.7), 6.16 (brs, 2H), 6.27 (dd, 1H, J ) 9.7,
2.6), 8.00 (d, 1H, J ) 9.7); 13C NMR δ 46.8, 47.3, 55.1, 98.8,
106.2, 125.2, 128.7, 147.6, 156.0.
5-(1-Met h yl-4-p ip er a zin yl)-1,2-d ia m in ob en zen e (7).
Compound 6 (464 mg, 1.96 mmol) was shaken with 45 psi of
H2 on 10% Pd-C (107 mg) in EtOAc for 6 h. Passing the
reaction mixture through a bed of Celite and concentrating
the solvent in vacuo gave 383 mg (95%) of an orange fluffy
solid: mp 85 °C; 1H NMR δ 2.30 (s, 3H), 2.52 (t, 4H, J ) 5.3),
3.02 (t, 4H, J ) 5.2), 3.29 (brs, 4H), 6.29 (dd, 1H, J ) 8.1, 2.3),
6.31 (d, 1H, J ) 2.2), 6.58 (d, 1H, J ) 8.4); 13C NMR δ 46.6,
51.0, 55.8, 106.5, 108.9, 118.6, 128.1, 136.8, 146.7. This
phenylenediamine18 was typically used directly after hydro-
genation without purification.
P r ep a r a tion of 5-(1-Meth yl-4-p ip er a zin yl)-2,5′-bi-1H-
ben zim id a zoles. These bibenzimidazole derivatives were
prepared as previously outlined for compounds 2a and 4a .
2′-P h e n yl-5-(4-m e t h ylp ip e r a zin yl)-2,5′-b i-1H -b e n z-
im id a zole (8a ). A solution of 7 (130 mg, 0.63 mmol) and 5a
(140 mg, 0.63 mmol) in 3 mL of nitrobenzene was heated under
N2 at 145 °C overnight. Column chromatography using a
gradient from 0% to 50% MeOH/EtOAc gave the crude product
which was further purified by chromatotron chromatography
using 0-30% MeOH/EtOAc; 80 mg (31%) was obtained: mp
224 °C (lit.18 mp 190 °C); 1H NMR (CD3OD) δ 2.73 (s, 3H),
2.66 (t, 4H, J ) 4.7), 3.18 (t, 4H, J ) 4.5), 6.90 (dd, 1H, J )
8.4, 1.7), 7.40 (d, 1H, J ) 2.0), 7.45-7.53 (m, 4H), 7.68 (d, 1H,
J ) 8.7), 7.93 (dd, 1H, J ) 8.5, 1.7), 8.06-8.11 (m, 2H), 8.24
(s, 1H); 13C NMR (CD3OD) δ 46.2, 51.9, 56.3, 102.5, 116.7,
116.9, 123.0, 126.2, 128.2, 130.5, 130.9, 132.0, 149.8, 153.9,
155.4; HRMS (EI) calcd for C25H24N6 408.2062, found 408.2062.
2′-o-Tolyl-5-(4-m e t h ylp ip e r a zin yl)-2,5′-b i-1H -b e n z-
im id a zole (8b). A solution of 7 (149 mg, 0.72 mmol) and 5b
(170 mg, 0.72 mmol) in 3 mL of nitrobenzene was heated under
N2 at 145 °C overnight. Column chromatography using a
gradient from 0% to 40% MeOH/EtOAc gave the crude product
which was further purified by chromatotron chromatography
using 0-30% MeOH/EtOAc to provide 172 mg (57%) of a pale
yellow solid: mp 248 °C; IR (KBr) 3067, 2933, 2800, 1437,
1
1280; H NMR (CD3OD) δ 2.38 (s, 3H), 2.55 (s, 3H), 2.68 (t,
4H, J ) 4.9), 3.23 (t, 4H, J ) 4.9), 7.06 (dd, 1H, J ) 8.8, 2.2),
7.15 (s, 1H), 7.34-7.46 (m, 3H), 7.52 (d, 1H, J ) 9.0), 7.66 (d,
1H, J ) 8.0), 7.76 (d, 1H, J ) 8.6), 8.02 (dd, 1H, J ) 8.0, 1.1),
8.33 (s, 1H); 13C NMR (CD3OD) δ 20.9, 46.4, 52.1, 56.5, 116.7,
122.4, 126.2, 127.4, 131.2, 131.4, 131.5, 132.5, 138.9, 149.9;
HRMS (FAB) calcd for C26H27N6 (M+ + 1) 423.2298, found
423.2296.
2′-m -Tolyl-5-(4-m e t h ylp ip e r a zin yl)-2,5′-b i-1H -b e n z-
im id a zole (8c). A solution of 7 (209 mg, 1.02 mmol) and 5c
(240 mg, 1.02 mmol) in 3 mL of nitrobenzene was heated under
N2 at 145 °C overnight. Column chromatography using a
gradient from 0% to 20% MeOH/EtOAc gave the crude product
which was further purified by chromatotron chromatography
using 0-20% MeOH/EtOAc. An orange solid, 370 mg (86%),
was obtained: mp 235 °C dec (lit.18 mp 236 °C); 1H NMR
(CD3OD) δ 2.37 (s, 3H), 2.46 (s, 3H), 2.66 (t, 4H, J ) 3.9), 3.20
(t, 4H, J ) 4.0), 7.03 (dd, 1H, J ) 8.8, 2.1), 7.12 (s, 1H), 7.35-
7.52 (m, 3H), 7.70 (dd, 1H, J ) 7.4, 2.3), 7.88-7.98 (m, 3H),
8.27 (s, 1H); 13C NMR (CD3OD) δ 21.8, 46.4, 52.1, 56.4, 116.7,
122.9, 123.0, 123.1, 125.4, 126.3, 128.8, 130.4, 130.8, 132.7,
140.5, 149.9.
Ack n ow led gm en t. We are grateful to the Midwest
Center for Mass Spectrometry at the University of
Nebraska-Lincoln for providing mass spectral data and
for the partial support of this facility by the National
Science Foundation, Biology Division (Grant No.
DIR9017262). This study was supported by Grant CA
39662 from the National Cancer Institute (L.F.L.) and
a fellowship grant from the J ohnson & J ohnson Dis-
covery Research Fund (E.J .L.).
2′-p -Tolyl-5-(4-m e t h ylp ip e r a zin yl)-2,5′-b i-1H -b e n z-
im id a zole (8d ). A solution of 7 (166 mg, 0.8 mmol) and 5d
(190 mg, 0.8 mmol) in 3 mL of nitrobenzene was heated under
N2 at 145 °C overnight. Column chromatography using a

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