Synthesis and DNA interactions of benzimidazole dications which have activity against opportunistic infections

Article abstract of DOI:10.1021/jm9507946

Considerable evidence now indicates that DNA is the receptor site for dicationic benzimidazole anti-opportunistic infections agents (Bell, C. A.; Dykstra, C. C.; Naiman, N. A. I.; Cory, M.; Fairley, T. A.; Tidwell, R. R. Antimicrob. Agents Chemother. 1993

Full text of DOI:10.1021/jm9507946

1452
J . Med. Chem. 1996, 39, 1452-1462
Syn th esis a n d DNA In ter a ction s of Ben zim id a zole Dica tion s Wh ich Ha ve
Activity a ga in st Op p or tu n istic In fection s
Richard L. Lombardy,† Farial A. Tanious,† Kishore Ramachandran,‡ Richard R. Tidwell,‡ and W. David Wilson*,†
Department of Chemistry, Georgia State University, Atlanta, Georgia 30303, and Department of Pathology,
University of North Carolina, Chapel Hill, North Carolina 27599
Received October 27, 1995X
Considerable evidence now indicates that DNA is the receptor site for dicationic benzimidazole
anti-opportunistic infections agents (Bell, C. A.; Dykstra, C. C.; Naiman, N. A. I.; Cory, M.;
Fairley, T. A.; Tidwell, R. R. Antimicrob. Agents Chemother. 1993, 37, 2668-2673. Tidwell, R.
R.; J ones, S. K.; Naiman, N. A.; Berger, I. C.; Brake, W. R.; Dykstra, C. C.; Hall, J . E. Antimicrob.
Agents Chemother. 1993, 37, 1713-1716). To obtain additional information on benzimidazole-
receptor complexes, the syntheses and DNA interactions of series of symmetric benzimidazole
cations, linked by alkyl or alkenyl groups, have been evaluated. Biophysical techniques, thermal
denaturation measurement (∆Tm), kinetics, and circular dichroism (CD) have been used in
conjunction with NMR and molecular modeling to evaluate the affinities, binding mode, and
structure of complexes formed between these compounds and DNA. All of the compounds bind
strongly to DNA samples with four or more consecutive AT base pairs, and they bind negligibly
to GC rich DNA or to RNA. Spectral and kinetics characteristics of the benzimidazole complexes
indicate that the compounds bind in the DNA minor groove at AT sequences. NMR and
molecular modeling of the complex formed between an ethylene-linked benzimidazole derivative,
5, and the self-complementary oligomer d(GCGAATTCGC) have been used to establish
structural details for the minor groove complex. These results have been used as a starting
point for molecular mechanics calculations to refine the model of the minor groove-
benzimidazole complex and to draw conclusions regarding the molecular basis for the effects
of substituent changes on benzimidazole-DNA affinities.
In tr od u ction
specific DNA binding interactions and microbial enzyme
inhibition selectivity.18-20 Alkyl-linked benzimidazoles
(Table 1) show improved anti-PCP activity and de-
creased toxicity relative to pentamidine.20 Initial stud-
ies indicate that the compounds interact with DNA more
strongly than pentamidine and inhibit the topo-
isomerase II enzyme of the microorganism to give the
observed biological activity.13,21 We report here a
comparative study of the binding strength, kinetics, and
binding mode of a variety of alkyl-linked benzimida-
zoles. These studies are essential for establishing the
biological mode of action of these compounds and they
form the basis of design experiments for the next
generation of synthetic anti-PCP agents. Compound 6
of Table 1 is in preliminary clinical trials against PCP.
Organic cations that bind noncovalently in the minor
groove of DNA generally fall into two classes: (i) alkyl-
linked amines (such as spermine and spermidine) that
bind tightly at low ionic strength but with limited
specificity and (ii) extended conjugated systems with
unfused aromatic rings that are directly bonded (such
as DAPI, Hoechst 33258, furamidine) or are connected
through conjugated systems (such as netropsin, dista-
mycin, berenil, and stilbamidine).1-10 Structures of
some of these compounds are compared in Chart 1.
The aromatic amidine, pentamidine (Chart 1), which
is being used clinically against Pneumocystis carinii
pneumonia (PCP), the most common and leading cause
of morbidity and mortality in AIDS patients,11,12 is a
different type structure with the aromatic groups linked
through an alkyl chain. The mechanism of action of
pentamidine and derivatives is believed to involve
formation of a complex with the DNA of the microor-
ganism, followed by selective inhibition of a DNA
interactive microbial enzyme. There is now consider-
able evidence that the enzyme inhibited by the penta-
midine-DNA complex is topoisomerase II of Pneu-
mocystis,13-15 and this inhibition rapidly leads to cell
death.
Ch em istr y
The starting reagents for the bisbenzimidazoles 8-10
are the appropriate diamine 13a or 13b, which can
readily be prepared according to reported procedures.17,18
Reaction of 13a or 13b with (14a ) under oxidative
conditions in refluxing ethanol22 provided the N-aceta-
midobisbenzimidazoles (15a or 15b). The reaction
proceeds readily and purification is straightforward.
Deacetylation in 2 N HCl followed by reduction of the
nitro group with catalytic hydrogenation provided the
diamine 17a and 17b. All the intermediates were fully
characterized by 1H NMR and microanalysis as de-
scribed in the Experimental Section. The final step in
the preparation of compounds 8-10 was first attempted
using the Pinner synthesis.23 Reactions of the imidates
prepared from succinonitrile or 1,4-dicyanobutane with
17a or 17b were not successful in achieving the desired
coupling reaction. The problem could be attributed to
Pentamidine binds relatively weakly in the DNA
minor groove in AT sequences16,17 and shows some side
effects that limit its drug use.14 We are involved in the
design, synthesis, and study of minor groove binding
cations with the goal of improving efficacy by enhancing
† Georgia State University.
‡ University of North Carolina.
X Abstract published in Advance ACS Abstracts, March 1, 1996.
0022-2623/96/1839-1452$12.00/0 © 1996 American Chemical Society

Synthesis and DNA Interactions of Benzimidazole Dications
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1453
Ch a r t 1
Ta ble 1. Nucleic Acid Binding
the insolubility of the diamines in appropriate organic
solvents including glacial acetic acid, which is the
standard solvent used for the coupling of imidates with
diamines to give the desired imidazoles. The reaction
of either 17a or 17b with succinic acid or adipic acid in
4 N HCl was carried out using a method described by
Phillips.24 The reaction, monitored by HPLC, required
1 week of reflux for gradual conversion of the diamine
to the desired product. The reaction appears to progress
through the intermediate o-aminoanilide.25,26 When the
reaction is complete, the aqueous solution is concen-
trated, and the residue purified by repeated recrystal-
lization from ethanol to provide the desired products.
Bin d in g Affin ity a n d Sp ecificity
Representative Tm curves for the sequence d(CGC-
GAATTCGCG)2 and its complexes with 1 and 5 are
shown in Figure 1. Increasing the ratio of compound
to duplex from 1 to 2 did not result in a significant
additional increase in the complex Tm. The ∆Tm values
(Tm complex - Tm DNA) cover a range of >20 °C for
1-10 with the weakest binding observed for 2 and 3,
intermediate binding for 6 and 7, and very strong
binding for the other compounds (Table 1). A larger
magnitude but similar order of ∆Tm values is observed
with the polymer poly(dA)‚poly(dT), and the Tm curves

1454 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Lombardy et al.
F igu r e 2. Spectrophotometric titrations of compound 1 (A),
and compound 5 (B) with d(CGCGAATTCGCG)2. Titrations
were conducted in 1 cm cell in MES buffer with 0.2 M NaCl
added at 25 °C. (A) The concentrations are as follows: 3 ×
10-5 M of compound 1, and d(CGCGAATTCGCG)2 concentra-
F igu r e 1. (A) The effect of 1 as a function of ratio on the
melting curve of d(CGCGAATTCGCG)2. Experiments were
conducted in MES buffer with 0.1 M NaCl added; the concen-
tration of d(CGCGAATTCGCG)2 was 3 × 10-6 in duplex and
the molar ratio of compound/duplex were 0 (b), 0.5 (2), 1.0
(4), 1.5 (O), and 2 (0). (B) Comparison of the effect of 1 (2),
and 5 (0) on the melting curve of d(CGCGAATTCGCG)2 (b).
Experiments were conducted in MES buffer with 0.1 M NaCl
added, the concentration of d(CGCGAATTCGCG)2 was 3 ×
10-6 in duplex, and the molar ratio of compound/duplex was
1.0.
tions in duplex of zero, 9.20 × 10-7, 1.83 × 10-6, 2.74 × 10-6
,
,
3.65 × 10-6, 4.55 × 10-6, 5.44 × 10-6, and 6.33 × 10-6
respectively, from the top to the bottom curves at 360 nm. (B)
The concentrations are as follows: 2 × 10-5 M of compound 5,
and d(CGCGAATTCGCG)2 concentrations in duplex of zero,
3.07 × 10-6, 6.08 × 10-6, 9.03 × 10-6, 1.19 × 10-5, 1.48 × 10-5
,
1.76 × 10-5, and 2.03 × 10-5, respectively, from the top to the
bottom curves at 275 nm.
2-6 (not shown) which do not have an extended
conjugated system as in 1. The spectrum of 5 signifi-
cantly overlaps that of the DNA bases, but Figure 2B
is a difference spectrum obtained by subtraction of DNA
absorbance. Compound 5 does not have the initial
aggregation-induced absorbance drop observed with 1
(Figure 2A), but shows a gradual shift to longer wave-
length and slight decrease in extinction coefficient on
titration with DNA. A clear isosbestic point is not
obtained in the titration, but this could be due to slight
errors in subtraction of the large DNA absorbances. The
low absorbance wavelengths of 2-7 and the duplex-
induced aggregation problems of 1 prevent direct cal-
culation of binding equilibrium constants from the
spectral results, and we have turned to other methods
to evaluate binding constants.
The results of an ethidium displacement assay with
complexes of 1-10 with polyd(A-T)2 are in general
agreement with the results from Tm studies (Figure 3).
The highest concentration required for 50% displace-
ment of ethidium is observed with 2, and this is followed
closely by 3 (Figure 3). Intermediate, but much lower,
concentrations of 6 and 7 are required for ethidium
displacement while 1, 4, 5, and 8-10 require the lowest
concentrations for displacement. Although a number
of assumptions are involved in calculation of binding
constants by displacement of the intercalator ethidium
by these compounds, it is clear that the binding con-
stants of 1 and 4-10 are predicted to be greater than
107 under these conditions while the binding constants
of 2 and 3 are close to 106. The conclusion from all of
these results is that 1 and 4-10 bind very strongly to
AT sequences and much more weakly to GC sequences
for complexes of 1, 4, 5, and 8-10 are shifted to greater
than 95 °C. Melting experiments were also conducted
with the corresponding sequence RNA polymer, poly-
(A)‚poly(U), but no significant Tm increase was observed
for the RNA sequence with any of the benzimidazoles.
A similar lack of Tm increase with 1-10 is observed with
the GC base pair duplex formed by sequence
d(GCGCGCGC)2. These results agree with those of Bell
et al.13 for melting of AT and GC polymer DNA samples
with compounds 1-7 at low salt concentrations. Re-
sults for the aromatic diamidines pentamidine and
DAPI, which have been crystallized in complex with the
oligomer d(CGCGAATTCGCG)2, are shown in Table 1
for comparison.
Spectral changes on addition of d(CGCGAATTCGCG)2
to 1 are shown in Figure 2A. In the initial stages of
the titration at high ratios of 1 to duplex, there is a large
decrease in absorbance with little shift in wavelength.
On continued addition of oligomer duplex there is a
gradual shift to longer wavelength, and a limiting
complex spectrum is reached near a ratio of one com-
pound per duplex. This spectral behavior along with
the Tm results suggests that, up to a ratio of one
compound per duplex, the compound is bound only at
the strong AATT binding site. At higher ratios weaker
binding of the compound, probably primarily by elec-
trostatic interactions, is observed. The observed spec-
tral changes and extended conjugated system of 1 both
supported this dual type of binding process.
A similar titration of 5 with the oligomer duplex is
shown in Figure 2B. The wavelength maximum of 5 is
shifted to lower wavelength as with all of the derivatives

Synthesis and DNA Interactions of Benzimidazole Dications
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1455
F igu r e 3. The decrease in ethidium fluorescence at 596 nm
(excitation at 546 nm) of a complex of ethidium and polyd(A-
T)2 on addition of various concentrations of compounds 1 (b),
2 (9), 3 (O), 4 ([), 5 (0), 6 (2), and 7 (4).
F igu r e 4. Stopped-flow kinetics traces for the SDS-driven
dissociation of compound 1 with polyd(A-T)2. The experiments
were conducted at 20 °C in MES buffer with [Na+] ) 0.135 M
at a ratio of 1:10 compound to polymer base pairs. The
concentration of compound 1 after mixing was 1.0 × 10-6 M.
The smooth lines in the panels are the two exponential fits to
the experimental data. Residual plots are shown under the
experimental plot.
in DNA or to any sequence in RNA. The order of
binding of 1-10 to AT sequences is 1 > 8-10 > 4, 5 >
6, 7 . 2, 3.
From the ∆Tm values for all bisbenzimidazoles com-
pounds complexed with d(CGCGAATTCGCG)2, binding
constants were calculated by method of Crothers27
according to the equation shown below, and the results
are shown in Table 1
1/Tm° - 1/Tm ) R/∆H° ln(1 + KaL) ) ∆Tm/Tm‚Tm°
where Tm° is the melting temperature of the free DNA,
∆H° is the enthalpy of melting for the free DNA, Tm is
the melting temperature of the complex, K is the binding
equilibrium constant, and aL is the free ligand concen-
tration.
Bin d in g Kin etics
Both UV-visible and fluorescence spectral changes
can be used to monitor the kinetics of binding. A plot
of fluorescence spectral changes as a function of time
for dissociation of a 1-polyd(A-T)2 complex as an
example is shown in Figure 4. Two exponential fits to
the data are also shown along with plots of residuals.
Single exponential fits give unsatisfactory residuals and
significantly higher RMS deviations with all the com-
plexes of these compounds with the polymer. Three
exponential fits do not significantly improve the residu-
als or RMS deviations.
F igu r e 5. Plots of log kapp vs -log [Na+] for dissociation from
polyd(A-T)2 of 1 (9), 4 (2), 5 (4), 7 (b), and DAPI (0).
Experiments were conducted in MES buffer at different ionic
strengths in the manner described in Figure 4.
fluorescence spectral signals for 2-7 are not satisfactory
for experiments at the low concentrations required to
measure association by stopped-flow techniques. The
association results are plotted in Figure 7 as a function
of salt concentration. Results for DAPI, the dicationic
intercalator propidium, and a naphthalenediimide that
binds by a threading intercalation mode are included
in Figure 7 for reference. As with the dicationic
diamidine DAPI, 1 binds to AT sequence in DNA with
a ka >107 M-1 s-1 that does not vary with the salt
concentration as extensively as with the intercalators.
The calculated equilibrium constant for 1 with a ka of
>107 M-1 s-1 and a kd of ∼0.1 s-1 at 0.1 M Na+ is >108
M-1, in agreement with the high values calculated from
the Tm studies (Table 1).
For the purposes of comparison, the dissociation
lifetime (τ) and apparent rate constant (kapp ) 1/τ) were
calculated from the computer-derived, best-fit values for
rate constants and amplitudes:
τ ) 1/(A1k1 + A2k2)
where A and k values refer to the amplitudes and rate
constants for the two exponential fits to the dissociation
results. Plots of the dissociation rate constants for
complexes of 1, 4, 5, and 7 determined as in Figure 4
are shown in Figure 5 as a function of log [Na+] at
constant temperature. The slopes of the plots are all
1.7 ( 0.1 in agreement with the slope obtained for the
DAPI-polyd(A-T)2 complex4,28 (Figure 5).
All of benzimidazoles have similar slopes for their
dissociation constants, and if they have similar associa-
tion rate constants, close to the diffusion-limit value,
the calculated equilibrium constants from the associa-
tion and dissociation kinetics results are 5-10 times
lower for 4 and 5 than for 1 while 6 and 2 have binding
The association of 1 with polyd(A-T)2 was also fol-
lowed by stopped-flow methods (Figure 6), but the

1456 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Lombardy et al.
F igu r e 6. Stopped-flow kinetics traces for the association of
compound 1 with polyd(A-T)2. The experiments were conducted
at 20 °C in MES buffer with [Na+] ) 0.319 M at a ratio of
1:10 compound to polymer base pairs. The concentration of
compound 1 was 2.5 × 10-8 M and the DNA was 2.5 × 10-7
M
in base pairs after mixing. The smooth lines in the panels are
the two exponential fits to the experimental data. Residual
plots are shown under the experimental plot.
F igu r e 7. Plots of log kapp vs -log [Na+] for association of 1
(2) with polyd(A-T)2, propidium with CTDNA (9), naphthalene
diimide with polyd(C-G)2 (4), and DAPI with polyd(A-T)2 (b).
Experiments were conducted in MES buffer at different ionic
strengths in the manner described in Figure 6.
constants that are 50-100 times lower than for 1 in
agreement with the calculated equilibrium constants in
(Table 1).
F igu r e 8. CD spectra of compounds 1 (A), 4 (B), 5 (C), and 7
(D) with polyd(A-T)2. Experiments were conducted in a 1 cm
cell in MES buffer with 0.1 M NaCl added at 25 °C. The ratio
of compound/polymer in base pairs is 0 (2), 0.05 (O), 0.10 (9),
0.15 (0), 0.20 ([), 0.25 (4), and 0.3 (b).
CD Sp ectr a
CD spectra were obtained for benzimidazole com-
plexes in both the wavelength region above 300 nm,
where DNA does not absorb and all bands are due to
induced CD in bound compounds, and the region below
300 nm, where both DNA and the compounds absorb
(Figure 8A-D). None of the benzimidazoles derivatives
has an intrinsic CD spectrum. Addition of compound 1
to polyd(A-T)2 in 0.1 M NaCl results in a small decrease
of the DNA signal at 260 nm, increase in the DNA signal
at 230 nm, and a strong positive CD signal at 375 and
395 nm (Figure 8A). Addition of compound 2 to poly-
d(A-T)2 results in a very weak CD signal at 295 and no
change in the DNA signal in 0.1 M NaCl (not shown).
Addition of compound 4 to polyd(A-T)2 results in a
decrease of the DNA signal at 260 nm, an increase in
the DNA signal at 230 nm, a strong positive CD signal
at 290 nm, and very clear isoeleptic points at 270 and
250 nm. The CD results for complex with 5 are very
similar to those with 4. The only significant difference
between the two results is that the increase in the DNA
signal at 230 nm is less with compound 5 (Figure 8B,C).
Addition of 7 to polyd(A-T)2 results in a decrease in the
DNA signal at 260 nm, a strong positive CD signal at
290 nm, and very clear isoeleptic points at 270 and 250
nm (Figure 8D). The induced signal at 290 nm is less

Synthesis and DNA Interactions of Benzimidazole Dications
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1457
F igu r e 9. The aromatic and thymine methyl regions of the spectra for the titration of compound 5 into DNA are shown. For the
two ratios of the complex the guanine H8 protons were exchanged with deuterium to simplify the spectrum.
for 7 than 5 at the same ratio in agreement with the
observed greater binding constant for 5.
NMR
To obtain additional structural information on the
benzimidazole-DNA complexes, NMR spectra of 5
complexed with d(GCGAATTCGC)2 were obtained. The
chemical shifts of the nonexchangeable protons were
assigned through a combination of 1D titration spectra,
as well as NOESY and COSY 2D experiments. For the
free DNA, the chemical shift assignments of the non-
exchangeable protons have been reported.29-31 In the
1D titrations at 35 °C (Figure 9) major downfield shifts
are seen for A5-H2 and the aromatic protons of 5, while
large upfield shifts are seen for the H6 and H1′ of T7.
The change in chemical shifts upon binding the ligand
for several protons along the duplex are plotted in
Figure 10, and it can be seen that the most dramatic
F igu r e 10. The change in chemical shift upon ligand binding
(complex-free) for the assigned protons of the DNA is shown.
H8, H6 (9); H5, H2, Me (b); H1′ ([); H2′ (2); H2′′ (1); H3′
(×).
changes occur in the central AT rich region. The
spectrum at a 2:1 ratio of DNA to ligand (Figure 9)
contains resonances from both the free DNA and the
DNA-ligand complex, indicating that the complex is in
slow exchange with free DNA on the NMR time scale.
Figure 11 contains plots of the aromatic region of the
NMR spectrum for the 1:1 complex at temperatures
ranging from 5 to 60 °C. At low temperature, substan-
tial line-broadening occurs. This can be attributed to
exchange between different species and is probably due
to the sliding motion of the drug along the DNA. Above
25 °C the signals are sharper and the bound species in
the complex are in fast exchange. The H8/H6 to H1′
region of the 2D NOESY spectrum for the 1:1 complex
is shown in Figure 12. This plot also shows one of the
strong cross peaks between the ligand (H4) and DNA
(A5-H2) while the other two strong cross peaks, L-H4
to A5-H1′ and L-H4 to T7-H1′, are shown in Figure 13.
data described above, a model of the decamer complexed
to 5 was built, and energy minimized as described in
the Methods Section (Figure 14). The compound binds
at the central AT region with the bound ligand spanning
five base pairs. The model indicates that a number of
hydrogen bonds between the ligand and DNA are
possible. These can occur between the imidazole NH
group and T17-O2 (2.7 Å), A4-N3 (2.9 Å), and A5-N3
(3.8 Å), and between the benzimidazole NH group and
T16-O2 (1.9 Å), T17-O2 (3.8 Å), T6-O2 (3.2 Å), and A5-
N3 (3.9 Å) at one end of the ligand. At the other end of
5 in the complex the imidazole NH can hydrogen bond
with T7-O2 (2.0 Å) and A14-N3 (3.1 Å) while the
benzimidazole NH can hydrogen bond with T7-O2 (1.9
Å) and A15-N3 (3.1 Å). The imidazole NH at this end
of the molecule is only 2.0 Å away from the O2 of C8
while the corresponding distance at the other end of the
molecule with C18-O2 is 3.9 Å. This difference and
Molecu la r Mod elin g
On the basis of the X-ray structure of pentamidine
bound to d(GCGAATTCGC)2 and the NMR structural

1458 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Lombardy et al.
F igu r e 13. The cross peaks between the H4 of compound 5
and DNA from the 350 ms NOESY are shown. The x-axis
range is from 8.4 to 7.8 ppm. The cross peak to A4-H2 is the
weakest.
d(CGCGAATTCGCG)2 as observed with a number of
minor groove binding agents such as DAPI and penta-
midine.16,32 The compounds bind very strongly to DNA
in extended sequences of AT base pairs but bind very
poorly to DNA GC rich sequences and to RNA. These
results are all characteristic of a minor groove binding
mode,3-5 and this binding mode for the benzimidazoles
is confirmed by results from CD, kinetics, and NMR
experiments.
F igu r e 11. The aromatic region of the spectra for the 1:1
complex of compound 5 with the decamer are shown at
temperatures ranging from 5 °C to 60 °C.
Minor groove binding, for example, is characterized
by relatively large association rate constants with much
lower dissociation rate constants. The association rate
constants have a very low dependence on salt concen-
tration, while the dissociation rate constants have high
dependence on salt concentration33 and this is observed
with the benzimidazoles (Figures 4-7). In addition, the
NMR NOESY intermolecular cross peaks between 5 and
the DNA oligomer duplex d(GCGAATTCGC)2 are all to
minor groove protons on AT base pairs (for example, to
AH2 and to H1′ protons of A and T). The NMR cross
peaks and X-ray structure of pentamidine were used as
guides to dock 5 into the AATT minor groove binding
site of the oligomer duplex, and the structure was
energy minimized with the NMR intermolecular con-
straints. A final model was then obtained by energy
minimization of the structure with all constraints
removed (Figure 14). The model generated for the
complex has the expected features of minor groove
interactions.19 The imidazole-benzimidazoles units
slide deeply into the groove and their NH groups at the
floor of the groove form hydrogen bonds to TO2 and AN3
acceptor groups. Specificity is generated by the tight
fit of the benzimidazoles at the edge of the AT base pairs
that would be completely disrupted by the 2-NH2 group
of G in a GC base pair.
The NMR spectra show a single set of NMR signals
at temperatures above 30 °C, but become broader and
more complex at lower temperatures. This result sug-
gests that the benzimidazoles may have two (or more)
binding sites that are in fast exchange above 30 °C but
move into intermediate exchange below 30 °C. The
broad peaks down to 0 °C preclude analysis of the
spectra in the 0-30 °C region, but the molecular
modeling results indicate that in the preferred binding
mode 5 is not symmetrically placed in the AATT binding
sites of the d(GCGAATTCGC)2 duplex. Each end of the
AATT site could provide an identical binding environ-
ment, and the broadening at low temperature may
represent slow exchange for sliding of the symmetrical
F igu r e 12. The aromatic to H1′ cross peak region of the 350
ms NOESY spectrum at 35 °C is shown. The cross peak
between the H6 and H1′ of thymine 6 was not resolved, and
its position is indicated by a box.
differences in the above hydrogen bond distances, point
out the unsymmetrical nature of the model. The ligand
can bind at two nearly symmetric sites spanning G3-
T7 and A4-C8, and exchange between these sites may
explain the broadened, more complicated NMR spectra
observed for the 1:1 complex at low temperature.
Discu ssion
The Tm studies conducted as a function of ratio
indicate that the benzimidazoles of Table 1 have a
single, strong binding site on the oligomer duplex

Synthesis and DNA Interactions of Benzimidazole Dications
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1459
F igu r e 14. Stereoview of compound 5 bound to the central (GAATTC)2 region of the decamer.
benzimidazole back and forth between the two sym-
metrical binding sites. Dissociation, rotation, and re-
association of benzimidazole would accomplish the same
transformation.
the two benzimidazoles that allows only one benzimi-
dazole amidine/imidazoline group at a time to fit into
the minor groove of DNA, and this results in a large
decrease in interaction energetics. On the other hand,
compounds with additional benzimidazole groups which
can fit into the minor groove, 8-10, have significantly
enhanced binding with equilibrium association con-
stants more than 20 times greater than those of corre-
sponding bisbenzimidazoles.
It is interesting that enhanced binding of compounds
such as 1 and 8-10 does not necessarily translate into
better drugs. 1 and 8-10 have good activity, but they
all display unacceptable toxicity with the PCP mouse
model.20 The very strong binding of these compounds
may lead to binding at secondary sites that can cause
toxicity. Compound 7 has a binding constant of 5 × 106
M-1 at AATT sites, and this is obviously enough to give
a strong cellular biological response. Future develop-
ment in this series is focused on benzimidazoles that
maintain this strong DNA binding, but which have
enhanced DNA-associated enzyme inhibition that is
responsible for the anti-PCP activity of this class of
compounds.
This model correctly predicts the dramatic difference
in interaction observed for the benzimidazoles deriva-
tives at AT and GC sites in DNA as well as the observed
difference for AT DNA and AU RNA binding sites. The
minor groove base interaction chemistry for DNA and
RNA is the same so that the benzimidazole could slide
deeply into the RNA minor groove and hydrogen bond
to AN3 and UO2 groups at the floor of the groove. The
problem with this model is that in the A-form helical
conformation of RNA the minor groove is not deep and
narrow, as in AT sites in the B-form DNA, but wide and
shallow.34 The shift from a B-form to an A-form
geometry thus destroys the structure and favorable
electrostatics of the minor groove required for strong
binding of compounds such as the benzimidazoles,
netropsin, pentamidine, and related minor groove bind-
ing drugs.1-10,16,17,28,32
Finally, the model in Figure 14 can be used to
interpret the binding constant differences observed as
function of structure for the benzimidazole derivatives
(Table 1). Compound 1 has a 5-10 times higher binding
constant than 4, and this is explained by the fit of the
benzimidazoles into the narrow minor groove of AT sites
in DNA. The more planar conjugated system of 1 fits
better into the groove and makes better contacts with
the walls of the groove than 4. Compounds 4 and 5 with
two methylene groups bind 5-10 times more strongly
than 6 and 7 which have four methylene groups. The
added methylenes of 6 and 7 do not form particularly
favorable contacts with the minor groove in AT se-
quences, and there is a significant entropy loss on
binding. This entropy loss, which results from the
reduction in the multiple rotational configurations of 6
and 7 in solution to the much small number of configu-
rations available for binding in the minor groove,
accounts for the equilibrium constant reduction for 6
and 7 relative to 4 and 5. It should be noted however,
that even with 4 and 5 the binding constants are greater
than 106 M-1 for AT sites under physiological relevant
conditions.
Exp er im en ta l Section
Melting points are uncorrected and were measured on a
Thomas Hoover capillary melting point apparatus or a Mel-
Temp II apparatus. IR spectra were recorded in Nujol mull
1
or KBr pellet on a Perkin-Elmer 1320 spectrophotometer. H
NMR spectra were recorded on a Bruker AC 300 MHz or a
Varian XL 400 MHz spectrometer. All spectra were recorded
in DMSO-d25 unless specified otherwise. Anhydrous ethanol
was distilled over Mg immediately prior to use. Reaction
products were dried over P2O5 at 77 or 110 °C at 0.2 mmHg.
Unless stated otherwise, reactions were monitored by TLC on
silica or by reverse phase HPLC. HPLC chromatograms were
recorded on a Hewlett-Packard 1090 chromatograph interfaced
with a Hewlett-Packard 3396 integrator utilizing UV detection
(230 nm). Chromatography was performed on a Dupont
Zorbax Rx C8 column (4.6 mm × 25 cm). Mobile phases
consisted of mixtures of acetonitrile (5-67.5% v/v) in water
containing 10 mM tetramethylammonium chloride, 10 mM
sodium heptanesulfonate, and 2.2 mM phosphoric acid. Elec-
tron impact mass spectra were recorded on a VG 70-SE, a VG
70-SEQ Hybrid, or a J MS 0-100 double focusing spectrometer.
FAB mass spectra were recorded on a VG 70-SEQ Hybrid
spectrometer (cesium ion gun, 30 kV). Microanalyses were
performed by Atlantic Microlab, Norcross, GA. The synthesis
and physical properties of bisbenzimidazoles 1-7 (Table 1) and
diamines 13a and 13b (Scheme 1) have been described in
previous communications.17,18 Preparation of 14a (Scheme 1)
was carried out according to a reported procedure.35
The reason for the dramatic drop in binding for the
single methylene compounds 2 and 3 is quite clear from
the model. The tetrahedral geometry for the methylene
group in 2 and 3 creates a significant angle between

1460 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Lombardy et al.
Sch em e 1a
a
Key: (a, b, c2); (d8); (e) 1,4-benzoquinone, ethanol, ∆, 4.5 h; (f) 2 N HCl, ∆, 2 h; (g) H2, 10% Pd/C, MeOH, water, 1 h; (h) HOOC(X)COOH
(where X ) (CH2)2 or (CH2)4), 4 N HCl, ∆, 1 week.
P r ep a r a tion of 15a a n d 15b. A mixture of the appropri-
ate diamine (13a or 13b, 5.0 mmol), 4-N-acetamido-3-ni-
trobenzaldehyde (14a , 5.0 mmol), and 1,4-benzoquinone (5.9
mmol) in ethanol (80.0 mL) was heated to reflux for 4.5 h. The
flask was cooled in ice bath, and the solid that separated was
filtered, washed with ethanol, and dried.
H), 6.92 (d, 1 H, J ) 9 Hz), 7.74-7.93 (m, 4 H), 8.35 (s, 1 H),
10.8 (s 2 H). Anal. for C16H16N6‚2HCl‚3H2O, C, H, N.
2-(3,4-Dia m in op h en yl)-5-a m id in oben zim id a zole (17b):
0.990 g (88%); mp 280-282 °C; H NMR (ppm) 6.9 (d, 1 H),
7.71-7.84 (m, 4 H), 8.12 (s, 1 H), 9.17 (s, 2 H), 9.44 (s, 2 H).
Anal. for C14H18N6‚2HCl‚2H2O, C, H, N.
1
P r ep a r a tion of 8-10. To a well-stirred solution of the
appropriate diamine (17a or 17b, 3.70 mmol) in 4 N HCl (140
mL) was added the appropriate diacid (1.85 mmol). The
solution was heated to reflux for 1 week and then concentrated
to provide a dark green solid. Repeated recrystallization from
hot ethanol provided the pure desired product.
2-(4-N-Acet a m id o-5-n it r op h en yl)-5-im id a zolin ob en z-
im id a zole (15a ): 1.8 g (90%); mp 240-242 °C; 1H NMR (ppm)
2.1 (s, 3 H), 4.02 (s, 4 H), 7.7-8.7 (m, 6 H), 10.65 (bs, 2 H),
14.11 (bs, 1 H). Anal. for C18H16N6O3‚HCl, C, H, N.
2-(4-N-Acet a m id o-5-n it r op h en yl)-5-a m id in ob en zim i-
1
d a zole (15b): 1.64 g (88%); mp 270-272 °C; H NMR (ppm)
1,2-Bis[5-(5′-(2-im id a zolin yl)-2′-ben zim id a zolyl)-2-ben -
zim id a zolyl]eth a n e (8): 1.44 g (17%); mp 340-342 °C; FAB-
MS m/z 631 (MH+ of free base); 1H NMR (DMSO-d6 + NaOD)
(ppm) 3.14 (s, 4 H), 3.62 (s, 8 H), 7.25-7.95 (m, 12 H), 8.2 (s,
2 H). Anal. for C36H30N12‚6HCl‚2.5H2O‚0.5C2H5OH, C, H, N.
1,2-Bis[5-(5′-(2-a m id in o)-2′-ben zim id a zolyl)-2-ben zim i-
d a zolyl]bu ta n e (9): 0.89 g (25%); mp >325 °C; FAB-MS 607
(MH+ of free base); 1H NMR (ppm) 2.08 (s, 4 H), 3.30 (s, 4 H),
7.74-7.87 (m, 4 H), 7.98-8.05 (m, 4 H), 8.22 (2s, 2 H), 8.52
(d, 2 H, J ) 8 Hz), 8.78 (bs, 2 H), 9.16 (s, 2 H), 9.46 (s, 2 H).
Anal. for C34H30N12‚6HCl‚H2O‚1.9C2H5OH, C, H, N.
1,2-Bis[5-(5′-(2-im id a zolin yl)-2′-ben zim id a zolyl)-2-ben -
zim id a zolyl]bu ta n e (10): 1.0 g (30%); mp 325 °C; FAB-MS
659 (MH+ for free base); 1H NMR (ppm) 2.09 (s, 4 H), 3.31 (s,
4 H), 4.03 (s, 8 H), 7.78-8.0 (m, 8 H), 8.40-8.50 (m, 4 H), 8.73
(s, 2 H), 10.71 (s, 4 H). Anal. for C38H34N12‚6HCl‚H2O‚0.5C2H5-
OH, C, H, N.
2.13 (s, 3 H), 7.73-7.89 (m, 3H, J ) 8, 10 Hz), 8.26 (s, 1 H),
8.55 (s, 1 H), 8.78 (s, 1 H), 9.03 (s, 2 H), 9.34-9.39 (2s, 2 H),
10.64 (s, 1 H). Anal. for C16H14N6O3‚HCl, C, H, N.
P r ep a r a tion of 16a a n d 16b. The appropriate N-aceta-
midobenzimidazole (15a or 15b, 14.25 mmol) in 2 N HCl (120
mL) was heated to reflux for 2 h. The solution was cooled in
ice bath, and the solid that separated was filtered, washed with
cold acetone, and dried to provide the appropriate nitro amine.
2-(4-Am in o-5-n it r op h en yl)-5-im id a zolin ob en zim id a -
zole (16a ): 4.9 g (96%); mp >325 °C; 1H NMR (ppm) 4.03 (s,
4 H), 7.25 (d, 1 H), 7.85 (d, 1 H), 7.96 (d, 1 H), 8.34-8.39 (m,
2 H), 9.01 (d, 1 H), 10.7 (s, 2 H). Anal. for C16H14N6O2‚
2HCl‚2.5H2O, C, H, N.
2-(4-Am in o-5-n it r op h en yl)-5-a m id in ob en zim id a zole
1
(16b): 5.0 g (87%); mp >325 °C; HNMR (ppm) 7.2 (d, J ) 9
Hz, 1 H), 7.77-7.87 (2d, 2 H, J ) 9 Hz), 8.18 (s, 1 H), 8.37-
8.40 (m, 1 H), 9.05 (s, 1 H), 9.19 (s, 2 H), 9.50 (s, 2 H). Anal.
for C14H16N6O2‚2HCl‚2H2O, C, H, N.
Bu ffer . MES buffer contained 0.01 M MES 1 × 10-3
M
EDTA. Sodium chloride was added to adjust the ionic strength,
and the pH was adjusted to 6.2 with NaOH.
P r ep a r a tion of 17a a n d 17b. To the appropriate nitro
amine (16a or 16b, 3.01 mmol) dissolved in water (140 mL)
was added 10% Pd/C (100 mg) and the solution hydrogenated
at room temperature at 50 psi. The reaction was stopped after
cessation of H2 uptake. The catalyst was filtered through a
bed of Celite, washed with water (4 × 15 mL), and concen-
trated to provide a dark green solid. Recrystallization from
hot ethanol furnished the pure desired product.
DNA a n d RNA. The polymers poly(dA)‚poly(dT), polyd(A-
T)2 (Pharmacia), and poly(A)‚poly(U) (Sigma) were prepared
as previously described.36 The oligomers d(GCGCGCGC),
d(GCGAATTCGC), and d(CGCGAATTCGCG) (Midland Certi-
fied Reagent Co.) were purified by HPLC. The concentrations
were determined optically using extinction coefficients per
mole of strand at 260 nm determined by the nearest neighbor
procedure.37 For NMR, 1 mmol of DNA duplex solution was
added to 0.5 mL of 7.5 mM sodium phosphate, 100 mM NaCl,
2-(3,4-Dia m in op h en yl)-5-im id a zolin ob en zim id a zole
1
(17a ): 0.89 g (90%); mp 248-250 °C; H NMR (ppm) 4.0 (s, 4

Synthesis and DNA Interactions of Benzimidazole Dications
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1461
and 100 mM EDTA, pH 7.0, in an NMR tube. The H2O was
removed under N2 and the sample rehydrated in 0.5 mL of
99.996% D2O (Isotec). This removal of solvent and rehydration
with D2O was repeated two times. The final solution was 2
mM in GCGAATTCGC duplex.
internal reference. The two-dimensional experiments were
obtained with a spectral width of 3000 Hz in both dimensions
with 1024 complex data points in the t2 dimension and 512
points in the t1 dimension (256 complex points in t1 for the
uncomplexed oligomer). Phase sensitive NOESY spectra were
obtained with a mixing time of 350 ms using the method of
States.45
Molecu la r Mech a n ics a n d Mod elin g. All models were
built and energy minimized using the software package Sybyl
(v6.03, Tripos). The Kollman all-atom forcefield46,47 was used
with minor modifications.48 To better approximate the tor-
sional barrier of biphenyl and related unfused aromatic
compounds, some modifications were made to the forcefield of
Veal and Wilson.48 The HA (aromatic protons) van der Waals
parameters were changed from r* ) 1.30 and e ) 0.0125 to r*
) 1.35 and e ) 0.025. Additionally, an explicit torsional term
was added for CA-CA-CA-CA with V2/2 ) 2.0 and V2/2 for
*-CA-CA-* was raised from 2.65 to 3.75 (where CA is an
aromatic carbon atom type). The combination of these three
changes retain the N,N-dimethyluracil stacking and benzene-
benzene interaction results of Veal and Wilson48 while also
giving a better description of the biphenyl torsional barrier.
DNA a n d RNA Th er m a l Meltin g. Thermal melting
experiments were conducted with Cary 3 or Cary 4 spectro-
photometers interfaced to microcomputers as previously de-
scribed.38 A thermistor fixed into a reference cuvette was used
to monitor the temperature. The DNA or RNA were added to
1 mL of buffer (MES with 0.1 M NaCl added) in 1 cm path
length reduced volume quartz cells, and the concentration was
determined by measuring the absorbance at 260 nm. Experi-
ments were generally conducted at a concentration of 5 × 10-5
M base pairs for the poly(dA)‚poly(dT) and poly(A)‚poly(U), and
3 × 10-6 M in duplex for d(G-C)4 and d(CGCGAATTCGCG)2.
For the complex Tm experiments a ratio of 0.6 compound per
base pair for poly(dA)‚poly(dT), poly(A)‚poly(U), and d(G-C)4
and a ratio of one compound per oligomer duplex for d(CGC-
GAATTCGCG)2 were used.
Absor p tion Sp ectr oscop y. UV-vis scans were obtained
as previously described36,39 with Cary 3 or Cary 4 spectropho-
tometers in MES with 0.1 M NaCl added. Difference spectra
were obtained by addition of the same amount of DNA to the
sample cell and reference cell to subtract the DNA absorbance.
Cir cu la r Dich r oism . CD spectra were obtained on a
J ASCO J -710 spectrometer interfaced to an IBM-PC computer.
The software supplied by J ASCO provided instrument control,
data acquisition, and manipulation. Solutions of the com-
pounds in MES buffer at 25 °C were scanned in 1 cm quartz
cuvettes. A solution of the DNA was scanned, and the
compound was then added and the sample rescanned at all
desired ratios.
Eth id iu m Disp la cem en t Exp er im en ts. Ethidium bro-
mide was added to 3 mL of MES buffer to give a concentration
of 1.3 × 10-6 M. DNA was added to the ethidium solution at
a concentration of 1.8 × 10-6 M in base pairs, and the
maximum fluorescence was measured with an SLM 8000
spectrofluorometer at an excitation wavelength of 546 nm and
an emission wavelength of 600 nm. Aliquots of a compound
stock solution were added to the DNA-ethidium solution, and
fluorescence was measured after each addition until greater
than a 50% reduction of fluorescence was obtained. The
apparent binding constant was calculated as previously
described40-43 by using the concentration of drug that gave a
50% reduction of the maximum fluorescence of the DNA-
ethidium complex.
Kin etics. Kinetics measurements were conducted on a Hi-
Tech SF-51 stopped-flow spectrometer interfaced to an HP 330
computer as previously described.4,44 The SF-51 is equipped
with a micromixing chamber and microcell that allow use of
less than 50 mL of sample solution in each mixing event.
Single wavelength kinetic records of absorbance or fluorescence
versus time were collected.
NMR Exp er im en ts. Compound 5 was dissolved in 99.96%
D2O (Isotec) to a concentration of 20 mM. For the 2:1 ratio of
DNA duplex to ligand 0.5 mmol of compound 5 solution was
added to the DNA duplex solution in the NMR tube. The
solvent was removed under N2 and 0.5 mL of 99.996% D2O
added. For the 1:1 ratio sample an additional 0.5 mmol of
compound 5 solution was added to the 2:1 sample, solvent was
removed, and the sample was rehydrated with 0.5 mL of
99.996% D2O. For the NMR of the uncomplexed compound 5,
1 mmol of solution was added to 0.5 mL of the phosphate buffer
solution, the solvent was removed, and the sample was
rehydrated with 0.5 mL of 99.996% D2O. The removal of
solvent and rehydration with D2O was repeated two times to
minimize the amount of HDO. All of the 1D and 2D 1H spectra
were collected on Varian spectrometers and the data trans-
ferred to a Silicon Graphics workstation for processing with
the software package Felix (v2.30, Biosym). A 1D temperature
study (data not shown) indicated that 35 °C is the optimal
temperature for minimal signal overlap. The one-dimensional
experiments were obtained with a spectral width of 4500 Hz,
16000 complex data points, a 1-s relaxation delay, and 100-
500 transients. The residual HDO peak was used as an
The X-ray coordinates of the pentamidine complex with
16
d(CGCGAATTCGCG)2 were from the Brookhaven Protein
Databank (PDB file 1D64) were used as a starting point for
the DNA structure. The terminal CG base pairs of this
dodecamer were removed and the ends capped with 5′- and
3′-OH groups. Compound 5 was built using the fragment
library of Sybyl and the geometry optimized in an extended
conformation using MNDO charges. This model was then
visually docked into the pentamidine binding site of the DNA
and the pentamidine was deleted. For the initial geometry
optimization of this complex, the DNA was treated as an
aggregate in Sybyl and the three NMR cross peaks between
the ligand and DNA were used as symmetric range constraints
(2-4 Å, 25 kcal/mol force constant). This complex was then
energy minimized to a rms gradient of 0.08 kcal/(mol Å) using
the Powell method. The aggregate on the DNA was removed
and the complex energy minimized to a rms gradient of 0.08
kcal/(mol Å). Finally, the constraints were removed and the
complex energy minimized to a rms gradient of 0.05 kcal/(mol
Å), followed by minimization to a rms gradient of 0.02 kcal/
(mol Å) using the BFGS method in SYBYL.
Ack n ow led gm en t. This research was supported by
NIH Grant AI-33363. The NMR, UV-visible, and CD
instruments were purchased through funds from NSF
and the Georgia Research Alliance.
Refer en ces
(1) Zimmer, C.; Wahnert, U. Nonintercalating DNA-binding
ligands: specificity of the interaction and their use at tools in
biophysical, biochemical and biological investigations of the
genetic material. Prog. Biophys. Mol. Biol. 1986, 47, 31-112.
(2) Pjura, P. E.; Grzeskowiak, K.; Dickerson, R. E. Binding of
Hoechst 33258 to the minor groove of B-DNA. J . Mol. Biol. 1987,
197, 257-271.
(3) Wilson, W. D. Reversible Interactions of Small Molecules with
Nucleic Acids. Nucleic Acids in Chemistry and Biology; Black-
burn, G. M., Gait, M. J ., Eds.; IRL Press: Oxford, 1993; Chapter
8, pp 295-336.
(4) Wilson, W. D.; Tanious, F. A.; Barton, H. J .; J ones, R. L.; Fox,
K.; Wydra, R. L.; Strekowski, L. DNA Sequence dependent
binding modes of 4′,6-diamidino-2-phenylindole (DAPI). Bio-
chemistry 1990, 29, 8452-8461.
(5) Wilson, W. D.; Tanious, F. A.; Buczak, H.; Ratmeyer, L.;
Venkatramanan, M. K.; Kumar, A.; Boykin, D. W.; Munson, R.
Molecular Factors that Control the Nucleic Acid Binding Mode
Selection by Unfused Aromatic Cations. Structure & Function:
Nucleic Acids; Sarma, R. H., Sarma, M. H., Eds.; Adenine
Press: Schenectady, NY, 1992; Vol. 1, pp 83-105.
(6) Coll, M.; Frederick, C. A.; Wang, A. H.-J .; Rich, A. A bifurcated
hydrogen-bonded conformation in the d(A.T) base pairs of the
DNA dodecamer d(CGCAATTGCG) and its complex with dista-
mycin. Proc. Natl. Acad. Sci. USA. 1987, 84, 8385-8389.
(7) Kopka, M. L.; Pjura, P.; Yoon, C.; Goodsell, D.; Dickerson, R. E.
Structure and Motion: Membranes, Nucleic Acids and Proteins;
Clementi E., Corongiu, G., Sarma, M. H., Sarma, R., Eds.;
Adenine Press: New York, 1985; pp 461ff.

1462 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Lombardy et al.
(8) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E.
The molecular origin of DNA-drug specificity in netropsin and
distamycin. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 1376-1380.
(9) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E.
Binding of an antitumor drug to DNA. Netropsin and d(CGC-
GAATTBrCGCG). J . Mol. Biol. 1985, 183, 553-563.
(10) Kopka, M. L.; Larsen, T. A. In Nucleic Acid Targeted Drug
Design; Propst, C. L., Perun, T. J ., Eds.; Marcel Dekker, Inc.:
New York, 1992; pp 303-374.
(29) Hare, D. R.; Wemmer, D. E.; Chou, S. H.; Drobny, G.; Reid, B.
R. J . Mol. Biol. 1983, 171, 319-336.
(30) Klevit, R. E.; Wemmer, D. E.; Reid, B. R.
1H NMR Studies on
the Interaction between Distamycin A and a Symmetrical DNA
Dodecamer. Biochemistry 1986, 25, 3296-3303.
(31) Lane, A. N.; J enkins, T. C.; Brown, T.; Neidle, S. Interaction of
berenil with EcoRI dodecamer d(CGCGAATTCGCG)2 in solution
studied by NMR. Biochemistry 1991, 30, 1372-1385.
(32) Larsen, T. A.; Goodsell, D. S.; Cascio, D.; Grzeskowiak, K.;
Dickerson, R. E. The structure of DAPI bound to DNA. J . Biomol.
Struct. Dynam. 1989, 7, 477-491.
(33) Wilson, W. D.; Tanious, F. A. Kinetic Analysis of Drug-Nucleic
Acid Binding Modes: Absolute Rates and Effects of Salt Con-
centration. Molecular Aspects of Anticancer Drug-DNA Interac-
tions. Topics in Molecular and Structural Biology; Neidle, S.,
Waring, M. J ., Eds.; The Macmillan Press Ltd.: London, 1994;
Vol. 2, pp 243-269.
(34) Saenger, W. Principles of Nucleic Acid Structure; Cator, C. R.,
Ed.; Springer-Verlag: New York, 1984; pp 220-282.
(35) Arnett, C. D.; Wright, J .; Zenker, N. Synthesis and Adrenergic
Activity of Benzimidazole Bioisoesters of Norepinephrine and
Isoproterenol. J . Med. Chem. 1978, 21, 72-78.
(36) Wilson, W. D.; Wang, Y.-H.; Kusuma, S.; Chandrasekaran, S.;
Yang, N. C.; Boykin, D. W. Binding Strength and Specificity in
DNA Interactions: The Design of A)T Specific Intercalators. J .
Am. Chem. Soc. 1985, 107, 4989.
(37) Fasman, G. D. Nucleic Acids. Handbook of Biochemistry and
Molecular Biology, 3rd ed.; CRC Press: Cleveland, OH, 1975;
Vol. 1, p 589.
(38) Kibler-Herzog, L.; Kell, B.; Zon, G.; Shinozuka, K.; Mizan, S.;
Wilson, W. D. Sequence Dependant Effects in Methylphospho-
nate Deoxyribonucleotide Double and Triple Helical Complexes.
Nucleic Acids Res. 1990, 18, 3545-3555.
(39) Wilson, W. D.; Lopp, I. G. Analysis of Cooperativity and Ion
Effects in the Interaction of Quinacrine with DNA. Biopolymers
1979, 18, 3025-3041.
(40) Morgan, A. R.; Lee, J . S.; Pulleyblank, D. F.; Murray, N. L.;
Evans, D. H. Ethidium fluorescence assays, Pari 1, Physio-
chemical studies. Nucleic Acids Res. 1979, 7, 547-569.
(41) Debart, F.; Perigaud, C.; Gosselin, G.; Mrani, D.; Rayner, B.;
LeBer, P.; Auclair, C.; Balzarini, J .; DeClercq, E.; Paoletti, C.;
Imbach., J .-L. Synthesis, DNA binding, and biological evalution
of synthetic precursors and novel analogues of netropsin. J . Med.
Chem. 1989, 32, 1074-1083.
(42) Lee, M.; Rhodes, A. L.; Wyatt, M. D.; Forrow, S.; Hartley, J . A.
GC Base Recognition by Olig(imidazolecarboxamide) and C-
Terminus-Modified Analogues of Distamycin Deduced from
Circular Dichroism, Proton Nuclear Magnetic Resonance, and
Methidiumpropylethylenediaminetetraacetate-Iron(II) Footprint-
ing Studies. Biochemistry 1993, 32, 4237-4245.
(43) Lee, M.; Rhodes, A. L.; Wyatt, M. D.; Forrow, S.; Hartley, J . A.
Design, synthesis, and biological evaluation of DNA sequence
and minor groove selective alkylating agents. Anti-Cancer Drug
Des. 1993, 8, 173-192.
(44) Tanious, F. A.; Yen, S.-F.; Wilson, W. D. Kinetic and equilibrium
analysis of a threading intercalation mode: DNA sequence and
ion effects. Biochemistry 1991, 30, 1813-1819.
(45) States, D. J .; Haberkorn, R. A.; Ruben, D. J . A Two-Dimensional
Nuclear Overhauser Experiment with Pure Absorption Phase
in Four Quadrants. J . Magn. Reson. 1982, 48, 286-292.
(46) Weiner, S. J .; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio,
C.; Alagona, G.; Profeta, S., J r.; Weiner, P. A new force field for
molecular simulation of nucleic acids and proteins. J . Am. Chem.
Soc. 1984, 106, 765-784.
(47) Weiner, S. J .; Kollman, P. A.; Nguyen, D. T.; Case, D. A. An all
atom force field for simulations of proteins and nucleic acids. J .
Comput. Chem. 1986, 7, 230-252.
(48) Veal, J . M.; Wilson, W. D. Modeling of Nucleic Acids Complexes
with Cationic Ligands: A Specialized Molecular Mechanics Force
Field an Its Application. J . Biomol. Struct. Dynam. 1991, 8,
1119-1145.
(11) Niedt, G. W.; Schinella, R. A. Arch. Pathol. Lab. Med. 1985, 109,
727-734.
(12) Moskowitz, L.; Hensley, G. T.; Chan, J . C.; Adams, K. Arch.
Pathol. Lab. Med. 1985, 109, 735-738.
(13) Bell, C. A.; Dykstra, C. C.; Aiman, N. A. I.; Cory, M.; Fairley, T.
A.; Tidwell, R. R. Structure-Activity Studies of Dicationically
Substituted Bis-Benzimidazoles against Giardia lamblia: Cor-
relation of Antigiardial Activity with DNA Binding Affinity and
Giardial Topoisomerase II Inhibition. Antimicrob. Agents
Chemother. 1993, 37, 2668-2673.
(14) Bell, C. A.; Cory, M.; Fairley, T. A.; Hall, J . E.; Tidwell, R. R.
Structure-Activity Relationships of Pentamidine Analogs against
Giardia Lamblia and Correlation of Antigiardial Activity with
DNA-Binding Affinity. Antimicrob. Agents Chemother. 1991, 35,
1099-1107.
(15) Dykstra, C. C.; Tidwell, R. R. Inhibition of topoisomerases from
Pneumocystis carinii by aromatic dicationic molecules. J . Pro-
tozool. 1991, 38, 78S-81S.
(16) Edwards, K. J .; J enkins, T. C.; Nedidle, S. Crystal Structure of
a Pentamidine Oligonucleotide Complex: Implications for DNA-
Binding Properties. Biochemistry 1992, 31, 7104-7109.
(17) Fairley, T. A.; Tidwell, R. R.; Donkor, I.; Naiman, N. A.;
Ohemeng, K. A.; Lombardy, R. J .; Bentley, J . A.; Cory, M.
Structure, DNA minor groove binding, and base pair specificity
of alkyl- and aryl-linked bis(amidinobenzimidazoles) and bis-
(amidinoindoles). J . Med. Chem. 1993, 36, 1746-1753.
(18) Tidwell, R. R.; Geratz, J . D.; Dann, O.; Volz, G.; Zeh, D.
Diarylamidine Derivatives with One or Both of the Aryl Moieties
Consisting of an Indole or Indole-like Ring. Inhibitiors of
Arginine-Specific Esteroproteases. J . Med. Chem. 1978, 21, 613-
623.
(19) Boykin, D. W.; Kumar, A.; Spychala, J .; Zhou, M.; Lombardy,
R. J .; Wilson, W. D.; Dykstra, C. C.; J ones, S. K.; Hall, J . E.;
Tidwell, R. R.; Laughton, C.; Nunn, C. M.; Neidle, S. Dicationic
Diarylfurans as Anti-Pnemocystis carinii Agents. J . Med. Chem.
1995, 38, 912-916.
(20) Tidwell, R. R.; J ones, S. K.; Naiman, N. A.; Berger, I. C.; Brake,
W. R.; Dykstra, C. C.; Hall, J . E. Activity of ctionically substi-
tuted bis-benzimidazoles against experimental Pneumocystis
carinii pneumonia. Antimicrob. Agents Chemother. 1993, 37,
1713-1716.
(21) Dykstra, C. C.; McClernon, D. R.; Elwell, L. P.; Tidwell, R. R.
SelectiveInhibition of Topoisomerases from Pneumocystis carinii
Compared with That of Topoisomerases from Mammalian Cells.
Antimicrob. Agents Chemother. 1994, 38, 1890-1898.
(22) Kumar, S.; Kansal, V. K.; Bhaduri, A. P. Possible Anthelmintic
Agents : Synthesis of Ethyl 5(6)-[5(6)-Substituted-2-benzimi-
dazolyl]benzimidazole-2-carbamates and Ethyl 4, 6-Dinitro-5-
substituted-amino-benzimidazole-2-carbamates. Indian J . Chem.
1981, 20B, 254-256.
(23) Pinner, A.; Klein, F. Umwandlung der nitrile in imide. (Conver-
sion of nitriles to imides.) Chem. Ber. 1887, 10, 1889-1897.
(24) Phillips, A. M. The Formation of 2-Substituted Benzimidazoles.
J . Chem. Soc. 1928, 2393-2399.
(25) Holan, G.; Evans, J . J .; Linton, M. Formation of Benzimidazoles
at High Pressure. J . Chem. Soc., Perkin Trans. 1 1977, 1200-
1203.
(26) Morgan, K. J .; Turner, A. M. Studies in Heterocyclic Chemistry.
III. Tetrahedron 1969, 25, 915-927.
(27) Crothers, D. M. Statistical thermodynamics of nucleic acid
melting transitions with coupled binding equilibria. Biopolymers
1971, 10, 2147-2160.
(28) Wilson, W. D.; Tannious, F. A.; Buczak, H.; Venkatramanan,
M. K.; Das, B. P.; Boykin, D. W. The effects of ligand structure
on binding mode and specificity in the interaction of unfused
aromatic cations with DNA. In Molecular Basis of Specificity in
Nucleic Acid-Drug Interactions: J erusalem Symposia on Quan-
tum Chemistry and Biochemistry; Pullman, B., J ortner, J ., Eds.;
Kluwer Academic Publishers: Printed in the Netherlands, 1990;
Vol. 23, pp 331-353.
J M9507946