DNA binding compounds. VII synthesis, characterization and DNA binding capacity of 1,2-dicarba-closo-dodecaborane bibenzimidazoles related to the DNA minor groove binder Hoechst 33258

Article abstract of DOI:10.1071/C98148

A series of bibenzimidazole derivatives based on the known DNA minor groove binder Hoechst 33258 have been prepared to include a 1,2-dicarba-closo-dodecaborane cage for potential use in boron neutron capture therapy (BNCT). The carborane derivatives (5)-(7) were chosen to reduce the steric inhibition of minor groove DNA binding displayed by the previously prepared carborane ligand (4). The synthesis and preliminary DNA binding studies of these bibenzimidazole derivatives are presented herein.

Full text of DOI:10.1071/C98148

C S I R O P U B L I S H I N G
Australian Journal
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Volume 52, 1999
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Aust. J. Chem., 1999, 52, 291–301
.
DNA Binding Compounds. VII†
Synthesis, Characterization and DNA Binding Capacity of
1,2-Dicarba-closo-dodecaborane Bibenzimidazoles Related
to the DNA Minor Groove Binder Hoechst 33258
Stuart A. Bateman,A David P. Kelly,A Roger F. MartinB and Jonathan M. WhiteA,C
A
School of Chemistry, The University of Melbourne, Parkville, Vic. 3052.
Molecular Sciences Group, Peter MacCallum Cancer Institute,
B
St Andrews Place, East Melbourne, Vic. 3001.
C
To whom correspondence should be addressed.
A series of bibenzimidazole derivatives based on the known DNA minor groove binder Hoechst 33258
have been prepared to include a 1,2-dicarba-closo-dodecaborane cage for potential use in boron neutron
capture therapy (BNCT). The carborane derivatives (5)–(7) were chosen to reduce the steric inhibition
of minor groove DNA binding displayed by the previously prepared carborane ligand (4). The synthesis
and preliminary DNA binding studies of these bibenzimidazole derivatives are presented herein.
Introduction
The 2 3 MeV shared by the Li and He atoms is
dissipated over a relatively short range, of the order of
a cell diameter, so the intense radiochemical damage
is con ned to the cell containing the 10B atom, and
will be targeted to tumour cells if they are preferen-
tially labelled with a 10B-containing pharmaceutical.
However, the distribution of the boron within the cell
is also important. Since nuclear DNA is the critical
radiobiological target for the cytotoxic e ect of ionizing
radiation, the high LET (linear energy transfer) tracks
of the ssion particles must pass through the cell nuclei
to be e ective. The probability of nuclear traverse
depends on the location of the nuclear capture event
in the cell, and calculations assuming idealized cellular
geometries indicate that the e ciency of a capture
event in the cell membrane is much lower than one
in the nucleus. For example, compared to when all
the 10B is in the cell membrane, moving just 10%
into the cytoplasm and 5% into the nucleus increases
the radiation dose to the nucleus by >70%.18 Aside
from this advantage of directing the neutron capture
event to the nucleus, the use of 10B–DNA ligands also
exploits the large reservoir of binding sites in DNA;
cell kill is directly proportional to the concentration
of 10B atoms.
Hoechst 33258 (1) is a crescent-shaped bibenzimida-
zole that has been shown to bind in the minor groove of
duplex DNA by a range of physiochemical techniques.1
4
X-Ray crystallography5
and n.m.r.9
studies of
8
11
Hoechst 33258 d(CGCGAATTCGCG)2 complexes indi-
cate that the benzimidazole NH groups are in close
proximity to hydrogen-bond acceptors on N 3 of adenines
and O 2 of thymines at appropriate locations. A num-
ber of studies using DNA sequencing gel analysis have
indicated that these ligands prefer to bind to sites
comprised of 3–5 consecutive A–T base pairs.12
14
The rst of these12 used 125I-labelled Hoechst 33258
(2), which, as a consequence of 125I-decay, induces
strand breaks at the ligand binding sites. The more
usual approach of ‘footprinting’ revealed a similar
sequence speci city,13 as did later experiments with
125I-iodoHoechst 3325814 in both puri ed DNA and
intact human cells.
Boron neutron capture therapy is an experimental
form of cancer treatment, which relies on the high
cross-section of the 10B isotope of boron for thermal
neutron capture compared with other elements found
18
in tissue.15
Upon irradiation an unstable entity 11
B
is formed that readily decays producing helium and
lithium atoms,
(equation (1)).
Experiments completed by Corder and coworkers19
showed that an increased e ciency of cell kill resulted
from incubating cells with boronic acid derivatized
Hoechst 33258 (3) upon irradiation with thermal neu-
trons. We have already reported the preparation of a
radiation, and 2 79 MeV of energy
† Part VI, Aust. J. Chem., 1994, 47, 1751.
Manuscript received 25 September 1998
᭧ CSIRO 1999
0004-9425/99/040291$ 05.00
10.1071/CH98148

292
S. A. Bateman et al.
bibenzimidazole directly substituted with a 1,2-dicarba-
closo-dodecaborane (carborane) cage (4) in an attempt
to further optimize the e ciency of neutron capture.20
However, preliminary biological testing of the latter
derivative indicated that the molecule had a reduced
DNA binding capacity which may be attributed to
steric hindrance between the carborane substituent
and the minor groove; the diameter of the minor
groove of 10B–DNA is approximately 0 57 nm,21 and
the diameter of the carborane cage22 0 86 nm. In
an attempt to increase binding we now report the
preparation and DNA binding capacity of three new
carborane bibenzimidazole derivatives (5)–(7), whereby
the carborane cage is linked to the bibenzimidazole by
a short tether.
of aldehydes.28,29 Thus, (9) was reduced under mild
conditions with sodium borohydride to give the benzyl
alcohol (10) which was then treated with acetyl chloride
and pyridine to provide the acetate ester (11).28,30
Reaction of (11) with bis(acetonitrile)–decaborane
complex (12) was carried out by using the lower
temperatures and longer reaction times described by
Miura30 and was found to be more e cient than the
procedure previously adopted to prepare bibenzimida-
zole (4);20 this is possibly a result of decreased steric
hindrance to the formation of the carborane cage in
(13) compared with that in the preparation of (4). In
our hands, also, we found that a greater overall yield
was obtained by rst isolating the borane complex
before reaction with alkyne (11) as per the method of
Schae er.31 The ethynyl group of (11) is less sterically
hindered compared with the corresponding precursor
used to prepare (4) where it is attached directly to the
phenyl ring; this may also contribute to the increased
yield for the reaction.
The i.r. spectrum of (13) showed the absence of
1
the ethyne signal at 2120 cm and the appearance of
a distinct BH stretch at 2590 cm 1. The f.a.b. mass
spectrum showed the typical isotopic cluster for these
compounds centred about the pseudo molecular ion
M+H at m/z 323 with a second cluster centred at about
263 arising from the loss of the acetate group. The
10B-enriched carborane (13), which was prepared in an
analogous fashion by using 10B-enriched decaborane,
showed no isotopic cluster in the f.a.b. mass spectrum
due to the presence of only one B isotope (not the 8 : 2
ratio of 11B to 10B of natural abundance B) and a
pseudo molecular ion (M+H) at m/z 315, eight mass
units less than natural abundance (13) as expected.
Removal of the acetate protecting group from (13)
was achieved by acid rather than base hydrolysis to
avoid degradation of the carborane cage15,25,26 and
was con rmed by 13C n.m.r. spectroscopy (loss of the
Results and Discussion
Synthesis
The carboranyl derivatives (5) and (6) incorporate
the less basic piperidine unit as in (4) rather than
the N -methylpiperazinyl substituent which is present
in the parent compound Hoechst 33258 (1).23 This
is due to the failure of our earlier attempts to pre-
pare carboranyl-substituted bibenzimidazoles related
to Hoechst 33258,24 a result which we believed was
caused by the incompatibility of the base-sensitive
carborane cage15,25,26 and the basic piperazine sub-
stituent. Like analogue (4),19,20 derivatives (5) and (6)
were prepared in a convergent procedure but a milder
coupling method was employed; this involved use of
an aromatic diamine and aldehyde in the presence
of sodium metabisul te27 to promote cyclization and
oxidation in the nal step.
carbonyl signal at
170 7). Intermediate (8) was
obtained by the mild oxidation of alcohol (14) with
pyridinium chlorochromate and showed a sharp singlet
at 9 97 in the 1H n.m.r. spectrum due to the aldehyde
proton, and the loss of the benzyl protons (4 40).
Di erences which were observed in the aliphatic
region of the proton n.m.r. spectra of the natural
and enriched forms of the carborane compounds might
be attributed to the 10B quadrupole moment being
approximately twice that of the 11B nucleus.32 In the
natural abundance compounds the BH signals appear
as four distinct broad but overlapping peaks in the
region 1 2 and 3 4 whilst in the 10B-enriched form
these signals appear as a at hump.
With the aldehyde (8) in hand, metabisulfate-
mediated coupling with the benzimidazole diamine
(15) was then attempted. Thus, the diamine (15) was
added to a solution of (8) and sodium metabisul te
(Scheme 1). This method of addition ensured that
the bisul te adduct is always present in excess and
Synthesis of Bibenzimidazoles (5) and (6)
Carborane (5) was prepared according to Scheme
1. The propargyl ether (9) was prepared from meta-
hydroxybenzaldehyde by treatment with propargyl
bromide in the presence of potassium bicarbonate.
Conversion of the aldehyde group of (9) into the ester
in derivative (11) was necessary prior to treatment
with bis(acetonitrile)–decaborane (12) owing to the
known instability of such complexes in the presence

DNA Binding Compounds. VII
293
Scheme 1. Reagents and conditions: (i) K2CO3, ethanol; (ii) NaBH4, ethanol; (iii) AcCl, pyridine, tetrahydrofuran; (iv) toluene,
10H12(CH3CN)2 (12); (v) HCl, methanol, H2O; (vi) pyridinium chlorochromate, CH2Cl2; (vii) NaHSO3, H2O, methanol.
B
thus decreases the amount of diamine decomposition
during the reaction, the products of which often have
similar chromatographic properties to the required
bibenzimidazole.20 On completion of the reaction and
evaporation of the reaction solvent a highly insoluble
residue was obtained, a fact presumably indicating that
the sulfate salt of the bibenzimidazole had formed.
Neutral alumina chromatography using a very polar
solvent mixture of methanol and ethyl acetate produced
a residue upon evaporation which was soluble in dilute
acetic acid and methanol.
similar couplings to H 4 and H 6. Protons 2 and 6
resonate as a multiplet between 7 82 and 7 91 which
has cross-peaks to H 4 and H 5, and also to H 700
(8 05) and H 400 (8 25), connectivities thus identifying
H 600 from the second benzimidazole ring also in this
multiplet. Protons 700 and 400, on the second benzim-
idazole ring, appear respectively as an ortho-coupled
doublet and meta-coupled doublet due to interactions
with H 600. Protons on the rst benzimidazole ring
(Bz 1) generally resonate at higher frequency due
to the deshielding e ects of the two adjacent aro-
matic ring systems, and were identi ed from their
coupling patterns and the required cross-peaks in the
two-dimensional spectrum.
The proton n.m.r. spectrum of (5), in natural
abundance form, showed the carborane cage as the
usual broad multiplet between
piperidine protons were present as three multiplets
at
1 85–1 95 (H 4000), 2 04–2 19 (H 3000,5000), and
1 2 and 3 4. The
The 13C n.m.r. spectra of bibenzimidazoles are often
di cult to obtain due to their low solubility in n.m.r.
solvents.33 The carbon resonances in the 13C n.m.r.
spectrum of (5), where possible, were assigned by
analogy with other previously reported compounds.33
These compounds have been fully assigned by HET-
COR, and long-range heteronuclear correlation (COLOC
or HMBC, inverse detection) experiments. Kelly and
coworkers20,33 have shown that changing the nature and
position of the substituents in the terminal aromatic ring
has relatively little e ect on the bibenzimidazole carbon
resonances. Similarly, the resonances attributed to
the N -methylpiperazine or piperidine ring also remain
relatively constant. The f.a.b. mass spectrum of (5)
gave the correct pseudo molecular ion for both the
natural abundance and 10B-enriched forms of (M+H)
at m/z 566 and 558 respectively.
3 75–3 78 (H 2000,6000), the relative deshielding due to
their relative proximity to the bibenzimidazole ring
systems. The carborane CH was a broad singlet at
4 77 and the methylene group at 4 71 appeared
as a sharp two-proton singlet. The aromatic n.m.r.
region had numerous overlapping signals which were
assigned by two-dimensional n.m.r. experiments as
well as by analogy with other previously prepared
compounds.20,33
The terminal phenyl protons were assigned from
H 4, which appears as a dd at lowest frequency (
7 40) due to shielding e ects of the alkoxy group,
and connectivity to H 5 (7 69) and H 6 (7 82–7 91)
in the two-dimensional spectrum. Proton 5 appears
as an apparent triplet due to two non-equivalent but

294
S. A. Bateman et al.
Synthesis of the novel bibenzimidazole (6) was
doublet at 8 20 for H 2 and H 6. 13C n.m.r. spectroscopy
gave all of the required 18 aromatic resonances and an
accurate f.a.b. mass spectrum gave the correct pseudo
molecular ion cluster centred at m/z 566 (M+H).
Owing to di culties associated with the isola-
tion of compounds (5) and (6) by means of the
sodium metabisul te mediated coupling methodology,
the preparations were repeated with nitrobenzene as
the reaction solvent and oxidizing reagent.35,36 The
aldehyde and diamine were heated in nitrobenzene at
140 for extended reaction periods. Following evapora-
tion of the nitrobenzene solvent, the residue obtained
was found to be very soluble in a range of organic
solvents. However, it was found to be contaminated
by a signi cant proportion of decomposition products
that were di cult to remove. Degradation presumably
results from the high temperatures employed in the
reaction, leading to decomposition of the diamine and
aldehyde precursors in competition with the desired
coupling reaction.
attempted rst in a manner similar to the production
of the meta derivative (5), a method thus requiring the
preparation of aldehyde (16). However, di culties with
the premature removal of the acetate protecting group
and reduced yields lead to the utilization of thioacetal
protecting groups as reported by Threadgill.29 By using
this methodology, (17) was protected as its thioacetal
(not shown) and treatment with (12) furnished the
carborane (18) (Scheme 2), the f.a.b. mass spectrum
of which showed the required pseudo molecular ion
cluster centred at m/z 354 (M+H), with a fragment
due to the loss of the thioacetal protecting group
centred at 263. The proton n.m.r. spectrum showed
the characteristic broadened BH protons at 1 2–3 4
and the broadened carborane CH at 4 08.
Synthesis of the Bibenzimidazole (7)
Unfortunately, compounds (5) and (6), incorporat-
ing a tethered carborane cage (as with (4); see later),
4
showed little shift in
in the u.v. spectrum1 upon
max
interaction with calf thymus DNA, a classical indicator
of bibenzimidazole minor groove binding. Accordingly,
it was proposed that replacement of the piperidine
moiety with N -methylpiperazine might improve the
binding of these carborane derivatives since the proto-
nated N -methylpiperazine nitrogen has been reported
to interact with negatively charged phosphate groups
in the minor groove of DNA.4
Initially the formation of bibenzimidazole (7) was
attempted by using a metabisul te coupling strategy.
Aldehyde (8) was converted into its metabisul te adduct
and then treated with diamine (19). On workup of
the reaction mixture, however, a complex mixture of
products was obtained, possibly due to reaction of
the basic N -methylpiperazine moiety of (19) with the
carborane cage. It was envisaged that bibenzimidazole
(7) might successfully be prepared by adopting an
imidate/diamine strategy as used for the preparation
of (4), in which the coupling procedure is completed
in an acidic medium which would ensure protonation
of the piperazine ring.20
Scheme 2. Reagents and conditions: (i) (CH2SH)2, BF3.OEt2,
CH2Cl2; (ii) (12), toluene; (iii) Hg(ClO4)2.3H2O, tetrahydro-
furan; (iv) (15), NaHSO3, MeOH, H2O.
The thioacetal protecting group of (18) was readily
cleaved by treatment with mercury perchlorate trihy-
drate for 5 min to yield (16).34 The integrity of the
carborane cage was demonstrated by the observation
of the typical isotopic cluster centred about the pseudo
molecular ion (M+H) at m/z 279 in the f.a.b. mass
spectrum. The characteristic broadened BH protons
( 1 2–3 4) and the aldehyde functionality (9 91) were
also clearly evident in the proton n.m.r. spectrum.
Conversion of aldehyde (16) into its corresponding
metabisul te adduct (as with the synthesis of (5)) and
treatment with diamine (15) gave a residue suitable
for puri cation. Puri cation involved trituration of
the highly insoluble residue in boiling diethyl ether, to
remove unreacted aldehyde (16) and soluble degradation
products, and then a combination of neutral alumina
chromatography and gel permeation chromatography
to give (6) in low yield (20%). This yield does not so
much re ect the ine ciency of the coupling reaction,
but rather the di culty in puri cation.
This approach required the imidate (22) which was
prepared according to Scheme 3. 3-Hydroxybenzonitrile
was reacted with propargyl bromide to give the ethyne
(21), in 86% yield after puri cation. The infrared
spectrum indicated both ethyne and nitrile stretches
1
at 2115 and 2232 cm
respectively. As with other
The proton n.m.r. spectrum of bibenzimidazole (6)
showed three aromatic spin systems, which were not
overlapping as in the meta isomer (5). The AA0XX0
spin system of the 1,4-disubstituted terminal phenyl
ring was easily identi ed as a pair of pseudo coupled
doublets resonating at 7 31 for H 3 and H 5 due to
shielding a ects from the methyleneoxy bridge and a
ethynes, there was a four-bond coupling between
the ethyne CH and methylene CH2 in the proton
n.m.r. spectrum. Precursor (21) was reacted with
bis(acetonitrile)–decaborane complex (12) to give the
carborane intermediate (20), the X-ray structure of
which has been reported elsewhere.37

DNA Binding Compounds. VII
295
abundance and the 10B-enriched compounds showed
the correct pseudo molecular ions (M+H) centred at
m/z 581 and 573 respectively.
The N -methylpiperazine-substituted bibenzimida-
zole (7) has proton n.m.r. resonances due to the
second benzimidazole ring at much lower frequency
compared with the piperidine-substituted derivatives.
This may be due to increased electron donation into
this ring system by through-bond interactions between
the piperazine nitrogens. This type of electron delo-
calization also accounts for the greater nucleophilicity
of piperazine compared with piperidine.38,39
DNA Binding
The DNA binding was assessed rst by using ultra-
4
violet (u.v.) absorption spectroscopy1
and secondly
by the use of circular dichroism (c.d.)1,2,4 spectroscopy.
The qualitative results from these studies were com-
pared to those obtained with Hoechst 33258 (1) in
order to verify minor groove binding.
It has been suggested that Hoechst 33258 (1) can
bind to DNA by modes other than the high-a nity
minor groove binding mode apparent from the X-ray
4
di raction data.1
It has been reported that these
secondary modes include charge-, dye-, and structure-
mediated interactions4 and increase as the drug to
base pair ratio becomes greater than 0 1.1 DNA
binding experiments on compounds (1) and (4)–(7)
were undertaken in bu ers containing 0 2 M sodium
chloride and the ligand to base pair ratio was kept
below 0 1, since these conditions have been reported
to reduce the amount of secondary binding.1,4,40 The
bu er also contained 20% methanol due to the low
aqueous solubility of the ligands, and this addition
also favours minor groove binding, but the absolute
K value is reduced considerably.4
Scheme 3. Reagents and conditions: (i) (12), toluene; (ii)
HCl(g), EtOH; (iii) AcOH/EtOH (2 : 1).
Carborane (20) was also prepared in 10B-enriched
form, and, as with the trend shown by other carborane
derivatives, the BH protons in the n.m.r. spectrum of
10B-enriched (20) is devoid of ne structure compared
with natural abundance (20). Following treatment
with anhydrous HCl gas in anhydrous ethanol the
imidate hydrochloride salt (22) was obtained in 86%
yield. F.a.b. mass spectrometric analysis showed the
typical cluster centred at m/z 322 corresponding to
the pseudo molecular ion minus hydrogen chloride
[(M+H) HCl]. The 10B-enriched sample did not have
the isotopic cluster associated with it, as expected,
and appeared at m/z 314, eight mass units less.
The target bibenzimidazole (7) was successfully pre-
pared from the reaction of the imidate hydrochloride
(22), with diamine (19) as outlined in Scheme 3 under
milder conditions than those generally employed for
coupling reactions.23 The target compound (7) pre-
cipitated from the reaction mixture (very likely as
the hydrochloride salt) and, following washing with
diethyl ether, was found to be pure. Both natural
abundance and 10B-enriched derivatives were prepared
analogously. The proton n.m.r. spectrum of (7) was
assigned by two-dimensional n.m.r. experiments as well
as by analogy with bibenzimidazole (5). All but one
of the required carbon resonances appeared in the
13C n.m.r. spectrum with the missing signal possibly
overlapping with the signal at 115 0 since its inten-
sity was uncharacteristically large. Both the natural
U.V. Absorption Spectroscopy
DNA binding ligands such as Hoechst 33258 (1)
show changes in their u.v. absorption spectrum upon
interaction with DNA,1,2 generally characterized by
a bathochromic shift in the u.v. absorption band at
approximately 340 nm.40
Table 1 shows the change in max absorption on addi-
tion of ct (calf thymus) DNA to the bibenzimidazoles
(1) and (4)–(7). Hoechst 33258 (1) gave the greatest
shift (5 nm) in
absorption of the compounds
max
examined. It should be noted that the shift in
max
for the bibenzimidazoles analysed (including Hoechst
33258) was less than that previously reported for
Hoechst 33258 (18 nm)1 and this might be attributed
to the 20% methanol concentration required to solubi-
lize the analogues which presumably also reduces the
ligand–DNA binding constant.3
The bibenzimidazole derivatives showed both hyp-
sochromic [(4) and (7)] and bathochromic [(5) and (6)]
shifts in max; however, these are very small. The
u.v. absorption data alone are insu cient to delineate

296
S. A. Bateman et al.
the mode of binding of the carborane derivatives and
it was therefore necessary to assess the propensity of
the bibenzimidazole derivatives for DNA minor groove
binding by other spectroscopic means.
DNA (as evident from the induced ellipticity across
their absorption region) the carborane derivatives (4)–
(7) show smaller levels of ellipticity compared with
Hoechst 33258 (1), presumably owing to weaker minor
groove binding interactions that may result from the
carborane cage sterically interfering with binding. The
N -methylpiperazine carborane derivative (7) produces
a marginally greater amount of induced ellipticity com-
pared with the piperidine (5), a result which suggests
that inclusion of the N -methylpiperazine moiety of
(7) aides in the minor groove binding of carborane-
derivatized bibenzimidazoles.
Table 1. Change in ligand
(nm) on binding to ct DNA
max
in the u.v. spectrum
DNA
ligand
Ligand
(
)
max
max
with DNA
max
(1)
(2)
(5)
(6)
(7)
339 3
341 0
340 2
340 7
347 5
344 2
339 5
341 3
342 3
345 8
4 9
1 5
1 1
1 6
1 8
Table 2. Induced ellipticity of bibenzimidazole derivatives
at
max
Com-
pound
Ellipticity
(deg)
Com-
pound
Ellipticity
(deg)
Circular Dichroism (C.D.) Spectroscopy
Circular dichroism (c.d.) is a property exhibited by
optically active substances and provides information
on the con guration of small molecules or the confor-
mation of a protein or biopolymer.41 The magnitude of
the c.d. e ect increases as the di erence in absorption
between the left- and right-handed circularly polarized
light increases, and reaches a maximum (or minimum)
corresponding to the u.v. absorption maxima.
Hoechst 33258 (1) and the derivatives (4)–(7) pre-
pared are not optically active2,40 since they do not
contain chiral centres or axes; however, DNA is chiral
and produces a c.d. spectrum. In order to form a DNA
complex the ligand must adopt a conformation which is
complementary to the minor groove and this induces a
twist between the two benzimidazole rings, giving rise
to a chiral axis in the molecule. The bibenzimidazole
is held in that conformation by strong minor groove
binding forces such as hydrogen bonding and van der
Waals interactions as discussed previously. Thus, c.d.
has been used to provide information on the binding
of Hoechst 33258 (1),1,4,40,42 and on the binding of
bibenzimidazole derivatives.40 The c.d. spectrum that
results has a component due to DNA at wavelengths less
than approximately 300 nm, and a second component
due to the bibenzimidazole centred at approximately
350 nm.
The amount of induced ellipticity can be related
to the concentration of the optically active species
by the Beer–Lambert law, since ellipticity is propor-
tional to the di erence in absorption of the left- and
right-handed components of plane polarized light.41
Thus, if it is assumed that, on binding to DNA, the
bibenzimidazole derivatives (1) and (4)–(7) have simi-
lar extinction coe cients, then the relative amount of
induced ellipticity they produce (hence the proportion
of ligand bound in the minor groove) may be compared
with the induced ellipticity produced from the known
DNA minor groove binder Hoechst 33258 (1).36
The amount of induced ellipticity for the biben-
zimidazole derivatives observed at their absorption
maximum is given in Table 2. These results show that
while all of the compounds form complexes with ct
(1)
(4)
(5)
6 4
0 5
0 4
(6)
(7)
0 5
1 8
Conclusion
The tethered 1,2-dicarba-closo-dodecaborane cage
substituted Hoechst derivatives (5)–(7) were success-
fully prepared; however, they appear to bind only
weakly to DNA, a fact limiting their use as BNCT
agents. The most likely reason for this weak binding
is steric interference between the boron cage and the
minor groove; therefore, further studies will be aimed at
preparing analogues containing longer tethers between
the bibenzimidazole and the boron cage.
Experimental
General
Melting points were determined on a Gallenkamp capillary
melting point apparatus and are uncorrected. 1H and 13C
n.m.r. spectra were recorded as solutions in the stated solvent
on a JEOL GX400, FX-90Q or Varian Unity 300 spectrometer
operating at 399 5, 89 5 and 299 9 MHz respectively for 1H
and 100, 22 5 and 72 5 MHz for 13C n.m.r. 11B n.m.r.
spectra were recorded on a JEOL FX-100 spectrometer oper-
ating at 31 9 MHz. Proton n.m.r. spectra were referenced to
tetramethylsilane (0 00 ppm) or to residual CHCl3 (7 26) if
run in CDCl3, residual CD2HOD (3 30) in (D4)methanol,
or residual (D5)dimethyl sulfoxide (2 49) in (D6)dimethyl
sulfoxide. The addition of a few drops of an acid such as
tri uoroacetic acid to CD3OD solutions of the bibenzimida-
zoles was found to reduce peak broadening and enhance the
de nition of multiplets in the aromatic region. Such cases
are designated (solvent+acid). For the acquisition of carbon
spectra in CD3OD, the addition of a few drops of an acid such
as methanesulfonic acid was used to increase the solubility
of the otherwise sparingly soluble material. Such cases are
designated (CD3OD+acid). 13C n.m.r. spectra were referenced
to the following solvent peaks: 77 0 (CDCl3), 49 0 (CD3OD)
or 39 5 (CD3)2SO as appropriate. Boron chemical shifts are
quoted relative to internal boron tri uoride diethyl etherate.
Positive-ion electron-impact mass spectra were recorded on
a Micromass VG7070F instrument at an ionization potential
of 70 eV, unless otherwise stated. F.a.b. mass spectra were
recorded on a JEOL AX-505H spectrometer, with xenon gas
and a glycerol or thioglycerol matrix. Infrared spectra were

DNA Binding Compounds. VII
297
recorded as either thin lms on sodium chloride plates (oils) or
as potassium bromide plates (solids) on a Perkin–Elmer 938G
spectrometer.
closo-dodecaborane hydrochloride20 (65 8 mg, 0 2 mmol) in
glacial acetic acid (3 ml) and the mixture heated at re ux
under nitrogen for 6 h. Workup as before20 gave (4), identical
with a previous sample.
U.v.–visible spectra were recorded on either a Perkin–Elmer
UV/Vis Lambda 2 or Hitachi 150–20 UV/Vis spectrometer
by using matched quartz cells of 1 cm path length and either
spectroscopic-grade solvents or water puri ed by the ‘Milli Q’
method. Bu ers for each sample run were freshly prepared.
The instrument scan speed was set at 120 nm/s with a threshold
of 0 01. Circular dichroism (c.d.) spectra were recorded on
a Varian spectrometer with AVIV 60DS V4.1t software with
a 1 0 nm bandwidth, a scan step of 0 2 nm and by using
a 0 5 mm path length. The spectrum was corrected for the
interference of unbound ligand, and polynomially smoothed.
High-performance liquid chromatography (h.p.l.c.) was per-
formed on a Waters instrument by using a reverse-phase
Phenomonex Bondclone PRP18 (5 by 200 mm) column (A) or a
PLFP-S, 8 m–100 A (75 by 300 mm) column (B) at 1 ml/min
with the solvents speci ed in the text for analytical runs.
Elution was monitored simultaneously at 214 and 366 nm. Size
exclusion chromatography was completed on either Sephadex
LH20 (700 by 25 mm column) by using methanol/2% acetic
acid or on G25 Sephadex (350 by 12 5 mm column) by using
a bu ered eluent (100 mmol saline, 50 mmol bicarbonate).
Analytical t.l.c. was performed on silica (Alugram, SIL G) or
on neutral alumina plates (Merck #5551) as indicated, and
the plates were analysed under 254 or 366 nm light.
Short-path distillation is quoted as (bath temperature, pres-
sure). 1,2-Dimethoxyethane, tetrahydrofuran, and diethyl ether
were distilled from sodium benzophenone ketal under an atmo-
sphere of nitrogen. Toluene, acetonitrile and dichloromethane
were distilled from barium oxide at reduced pressure and stored
over 4 A molecular sieves. Other chemicals and solvents were
puri ed by literature procedures.43 M13RF DNA was obtained
from Boehringer–Mannheim. Bovine serum albumin (BSA) was
obtained from the Sigma Chemical Company. 10B-enriched
decaborane was obtained from Boron Biologicals Inc. (U.S.A.)
at a 96 9% enrichment level. The 10B-enriched derivatives,
unless stated, were prepared in an analogous fashion to that
discussed for the natural abundance compound but used 96 9%
10B-enriched decaborane. Many of the starting materials,
reagents and solvents were purchased from the Aldrich Chemi-
cal Company. Where possible these were obtained as analytical
grade.
3-(Propargyloxy)benzaldehyde (9)
Potassium carbonate (8 24 g, 59 6 mmol) was added to
a solution of 3-hydroxybenzaldehyde (4 88 g, 40 0 mmol) in
ethanol (40 ml). Propargyl bromide (80% solution in toluene,
7 13 g, 48 0 mmol) was added dropwise over 30 min at room
temperature and the mixture heated at re ux for 4 h under
nitrogen. After cooling the mixture to room temperature,
the solvent and excess of propargyl bromide were evaporated
at reduced pressure and the residue was suspended in water
(150 ml). The organic components were extracted with ethyl
acetate (3 50 ml) and the combined extracts washed with
saturated brine, water and dried (MgSO4). Filtration through
Celite and evaporation gave a low-melting solid that was
puri ed by short-path distillation and gave a clear oil (9) that
soon crystallized (5 7 g, 89%) (b.p. 90 bath temperature,
0 2 mmHg) (lit.44 82–86 /0 03 mmHg), RF 0 43 (SiO2; 20%
EtOAc/petrol). 1H n.m.r. (100 MHz, CDCl3)
2 56, t, J
2 8 Hz, 1H, ethyne CH; 4 76, d, J 2 8 Hz, 2H, CH2; 7 31–
7 52, m, 4H, H 2,4,5,6; 9 98, s, 1H, CHO. 13C n.m.r. (25 MHz,
CDCl3) 56 8, CH2; 76 1, CH; 78 0, ethyne C; 114 2, C 2;
122 5, C 4; 124 6, C 6; 130 9, C 5; 138 6, C 1; 158 5, C 3;
192 5, CHO.
1320, 1286, 1166, 1147, 994, 891, 732 cm
(NaCl) 3287, 2121, 1689, 1580, 1590, 1384,
max
1
.
3-(Propargyloxy)benzyl Alcohol (10) 30
Sodium borohydride (1 0 g, 26 mmol) was added to a solu-
tion of the benzaldehyde (9) (1 7 g, 11 mmol) in ethanol (20 ml)
over 20 min whilst the temperature was maintained below 20 .
The solution was stirred overnight under nitrogen at room
temperature and then water (100 ml) was cautiously added.
The resulting suspension was extracted with diethyl ether
(3 50 ml), and the combined organic extracts were washed
with a 5% sodium metabisul te solution (2 50 ml), water and
then dried (MgSO4). Filtration through Celite and evaporation
gave (10) as a pale oil (1 52 g, 90%) (b.p. 100 bath temper-
ature, 0 5 mmHg), RF 0 76 (SiO2; 50% EtOAc/petrol). 1H
n.m.r. (90 MHz, CDCl3) 2 51, t, J 2 5 Hz, ethyne CH; 4 67,
s, 2H, ArCH2; 4 76, d, J 2 5 Hz, 2H, ArOCH2; 6 8–7 3, m,
4H, H 2,4,5,6. 13C n.m.r. (22 5 MHz, CDCl3) 54 4, ArOCH2;
63 5, ArCH2; 74 2, ethyne CH; 78 5, ethyne C; 111 8, 112 6,
Synthetic Procedures
4-[5 0-(Piperidin-1 00-yl)benzimidazol-2 0-yl]benzene-1,2-
diamine (15)
C 2,6; 118 6, C 4; 128 2, C 5; 141 1, C 3; 153 3, C 1.
max
(NaCl) 3250br, 3286, 2121, 1584, 1453, 1374, 1262, 1033, 930,
1
879, 756 cm
.
A solution of 2-nitro-4-[50-(piperidin-100-yl)benzimidazol-20-
yl]aniline (100 mg, 0 3 mmol) in ethyl acetate/methanol (2 : 1,
60 ml) was treated with 5% palladium on carbon (150 mg)
and stirred under an atmosphere of hydrogen gas at room
temperature. When no more hydrogen was consumed (4 h)
the solution was ltered under nitrogen through Celite, and
concentrated without delay to a ord the title diamine (15) as
an orange-brown solid (90 mg, 96%), m.p. 240 (dec.) (lit.14
244 ). 1H n.m.r. (400 MHz, CD3OD+methanesulfonic acid)
1 80–1 90, m, 2H, H 400; 2 10–2 20, m, 4H, H 300,500; 3 76–
3 78, m, 4H, H 200,600; 7 05, d, J 8 8 Hz, 1H, H 6; 7 90–7 94,
m, 2H, H 5,60; 7 98, d, J 8 8 Hz, 1H, H 70; 8 10, d, J 2 2 Hz,
1H, H 40; 8 27, d, J 2 0 Hz, 1H, H 3. 13C n.m.r. (100 MHz,
3-(Propargyloxy)benzyl Acetate (11)
The alcohol (10) (1 4 g, 8 7 mmol) was dissolved in dry
tetrahydrofuran (30 ml) and the solution cooled to 0 before
pyridine (4 ml, 52 mmol) and an acetyl chloride solution (1 92 g,
24 0 mmol, in 10 ml of tetrahydrofuran) were added. After
the resulting solution had warmed to room temperature, it
was heated at re ux for 1 5 h and then kept overnight. Next,
water (150 ml) was added and the mixture extracted with
diethyl ether (3 50 ml). The combined organic extracts were
sequentially washed with saturated brine and water, dried
(MgSO4) and then evaporated to give (11) as a near colourless
oil (1 67 g, 93%) (b.p. 120 bath temperature, 0 8 mmHg)
(lit.30 123 bath temperature, 5 mmHg), RF 0 8 (SiO2; 40%
EtOAc/petrol). 1H n.m.r. (100 MHz, CDCl3) 2 10, s, 3H,
CH3; 2 53, t, J 2 4 Hz, 1H, ethyne CH; 4 69, d, J 2 4 Hz,
2H, ArOCH2; 5 08, s, 2H, ArCH2; 6 92–6 98, m, 3H, H 2,4,6;
7 28, apparent t, J 8 0 Hz, 1H, H 5. 13C n.m.r. (25 MHz,
CD3OD+methanesulfonic acid)
21 9, C400; 24 8, C300,500;
59 0, C200,600; 108 9, 110 4, 116 7, 118 1, 118 5, 120 6, 126 0,
130 6, 133 1, 133 4, 141 3, 148 4, 152 8.
4-(1,2-Dicarba-closo-dodecaboranyl)-1-{5 0-[5 00-(piperidin-
1 000-yl)benzimidazol-2 00-yl]benzimidazol-2 0-yl}benzene (4) 20
CDCl3)
ethyne CH; 78 3, ethyne C; 114 4, 114 6, C 2,4; 121 1, C 6;
129 6, C 5; 137 5, C 1; 157 6, C 3; 170 7, CO2. (NaCl)
20 9, CH3; 55 7, ArOCH2; 65 9, ArCH2; 75 6,
Freshly prepared diamine (15) (50 mg, 0 16 mmol) was
added to a solution of [4-[imino(ethoxy)methyl]phenyl]dicarba-
max

298
S. A. Bateman et al.
3284, 3036, 2955, 2120, 1735, 1602, 1586, 1487, 1453, 1378,
3-[(1,2-Dicarba-closo-dodecaboranyl)methoxy]-
benzaldehyde (8)
1
1244, 1150, 940, 783 cm
.
Aldehyde (8) was prepared from alcohol (14) by the reported
method.30
Bis(acetonitrile)–Decaborane (12) 31
3-[(1,2-Dicarba-closo-dodecaboranyl)methoxy]-1-{5 0-[5 00
(piperidin-1 000-yl)benzimidazol-2 00-yl]benzimidazol-2 0-
yl}benzene (5)
-
Decaborane (1 5 g, 12 3 mmol) was dissolved in dry acetoni-
trile (12 ml) and the solution heated at re ux under nitrogen
for 2 h. Upon cooling overnight, colourless prisms separated
from the mixture; these were collected by ltration and dried
under reduced pressure to a ord the title borane (12) (two
crops, 2 1 g, 87%), m.p. >300 .
Aldehyde (8) (200 mg, 0 72 mmol) was dissolved in methanol
(10 ml) and the stirred solution treated with a solution of sodium
metabisul te (152 mg, 0 8 mmol) in aqueous methanol (1 : 1,
0 5 ml) and then the freshly prepared diamine (15) (218 mg,
0 71 mmol). The solution was heated at 90 for 4 h under
nitrogen and, after cooling (1 5 h), the mixture was evapo-
rated to dryness and the highly insoluble residue dissolved
in dimethylformamide (20 ml) and pre-adsorbed onto alumina.
Chromatography on neutral alumina (5% MeOH/EtOAc), and
collection of the second uorescent band, gave a yellow solid upon
evaporation. This was dissolved in 2% acetic acid/methanol
and after 5 min the title bibenzimidazole (5) precipitated from
solution. The yellow solid was collected at the pump and dried
under vacuum (183 mg, 45%), m.p. >190 (dec.), RF 0 48
(Al2O3; 2% MeOH/EtOAc) (Found: (M+H)+ , 566 39091.
3-[(1,2-Dicarba-closo-dodecaboranyl)methoxy]benzyl Acetate
(13) 30
The alkyne (11) (100 mg, 0 49 mmol) was added to a solu-
tion of bis(acetonitrile)–decaborane (12) (198 mg, 0 98 mmol)
in dry toluene (20 ml) and the mixture heated at 80 for 60 h
under nitrogen. After ltration through Celite and evaporation
of the solvent, the residue was dissolved in ethanol (15 ml) and
stirred overnight. Concentration of the solvent gave the crude
acetate (13), which was puri ed by radial chromatography
(20–50% Et2O/hexane gradient). Collection of the fastest
moving band revealed (13) as a white solid on evaporation
(136 mg, 86%), m.p. 46–48 , RF 0 57 (SiO2; Et2O/hexane,
C
28H35B10N5O requires (M+H)+ , 566 39233). 1H n.m.r.
(400 MHz, CD3OD+tri uoroacetic acid) 1 2–3 4, br m,
1 : 1). 1H n.m.r. (400 MHz, CDCl3)
1 2–3 4, br m, 10H,
10H, BH; 1 85–1 95, br m, 2H, H 4000; 2 04–2 19, br m, 4H,
H 3000,5000; 3 75–3 78, m, 4H, H 2000,6000; 4 71, s, 2H, CH2;
4 77, br s, 1H, carborane CH; 7 40, dd, J 8 5, 2 2 Hz, 1H,
H 4; 7 69, apparent t, J 8 1 Hz, 1H, H 5; 7 82–7 91, m, 3H,
H 2,6,600; 8 05, d, J 9 0 Hz, 1H, H 700; 8 14, d, J 8 7 Hz,
1H, H 70; 8 25, d, J 2 0 Hz, 1H, H 400; 8 35, dd, J 8 5,
1 7 Hz, 1H, H 60; 8 73, d, J 1 5 Hz, 1H, H 40. 13C n.m.r.
(100 MHz, CD3OD/(CD3)2SO (5 drops), acetic acid) 25 1,
C 4000; 27 2, C 3000,5000; 54 0, C 2000,6000; 62 2, carborane CH;
70 4, CH2; 74 1, carborane C; 101 7, C 400; 113 8, 113 9,
116 3, 116 4, 117 7, 121 2, 122 3, 125 2, 131 2, 131 7, 134 9,
139 4, 140 2, 141 2, 150 0, 152 4, 153 6, 158 7 (one aro-
matic resonance overlapping). m/z (f.a.b. thioglycerol) 566
BH; 2 11, s, 3H, CH3; 4 10, br s, 1H, carborane CH; 4 41,
s, 2H, ArOCH2; 5 06, s, 2H, ArCH2; 6 80, dm, J 8 3 Hz,
1H, H 4; 6 84–6 85, m, 1H, H 2; 7 02, dm, J 8 3 Hz, 1H, H 6;
7 30, apparent t, J 8 1 Hz, 1H, H 5. 13C n.m.r. (100 MHz,
CDCl3) 20 9, CH3; 57 4, carborane CH; 65 7, ArCH2; 69 0,
ArOCH2; 73 1, carborane C; 114 2, 114 4, C 2,4; 122 2, C 6;
130 0, C 5; 138 0, C 1; 157 1, C 3; 170 7, CO2. m/z (f.a.b.,
thioglycerol) 323 (M+H), 263.
1731, 1647, 1596, 1394, 1303, 1255, 1205, 1178, 1125, 951,
(KBr) 3083br, 2931, 2590,
max
1
891 cm
.
1H n.m.r. of 10B-enriched compound (300 MHz,
CDCl3) 1 4–3 0, br m, 10H, BH; 2 11, s, 3H, CH3; 4 10,
br s, 1H, carborane CH; 4 42, s, 2H, ArOCH2; 5 07, s, 2H,
ArCH2; 6 80, dd, J 8 3, 2 7 Hz, 1H, H 4; 6 84–6 86, m, 1H,
H 2; 7 03, d, J 7 6 Hz, 1H, H 6; 7 30, apparent t, 1H, J
7 8 Hz, H 5. m/z (f.a.b., thioglycerol) 315 (M+H), 255.
(M+H).
1382, 1056, 872 cm
(400 MHz, CD3OD+tri uoroacetic acid) 1 4–3 0, br m, 10H,
BH; 1 80–1 90, m, 2H, H 4000; 2 05–2 15, m, 4H, H 3000,5000
(KBr) 3426, 2921, 2521, 2589, 2314, 1629, 1449,
max
1
.
1H n.m.r. of 10B enriched compound
;
3 74–3 78, m, 4H, H 2000,6000; 4 72, s, 2H, CH2; 4 83, br s,
1H, carborane CH; 7 41, dd, J 8 3, 2 2 Hz, 1H, H 4; 7 69,
apparent t, J 8 0 Hz, H 5; 7 82–7 88, m, 3H, H 2,6,600; 8 01,
d, J 9 0 Hz, 1H, H 700; 8 11, d, J 8 8 Hz, 1H, H 70; 8 17, d, J
2 2 Hz, 1H, H 400; 8 34, dd, J 8 8, 1 7 Hz, 1H, H 60; 8 67–8 69,
m, 1H, H 40. m/z (f.a.b., thioglycerol) 558 (M+H). H.p.l.c. RT
8 70 min (A) (55% MeCN/0 1% tri uoroacetic acid/H2O).
3-[(1,2-Dicarba-closo-dodecaboranyl)methoxy]benzyl Alcohol
(14) 30
Carborane (13) (136 mg, 0 42 mmol) was dissolved in a
solution of methanol and concentrated HCl (80 : 1, 10 ml) and
the resulting solution stirred at 60 overnight. After this period,
t.l.c. indicated the removal of the protecting groups (SiO2;
Et2O/petrol, 1 : 1, RF 0 38) and the solution was concentrated
under vacuum. Dichloromethane was added to the residue
and the solution washed with water, dried with magnesium
sulfate and then evaporated. The title alcohol (14) was isolated
by ash chromatography (SiO2; CH2Cl2) to give a clear oil
which slowly crystallized (99 mg, 84%), m.p. c. 25 . 1H n.m.r.
(400 MHz, CDCl3) 1 2–3 4, br m, 10H, BH; 4 08, br s, 1H,
carborane CH; 4 40, s, 2H, ArOCH2; 4 65, s, 2H, ArCH2;
6 75, dd, J 8 3, 2 7 Hz, 1H, H 4; 6 86–6 88, m, 1H, H 2;
6 98, dm, J 7 8 Hz, 1H, H 6; 7 27, apparent t, J 7 8 Hz,
4-(Propargyloxy)benzaldehyde (17)
4-Hydroxybenzaldehyde (4 88 g, 40 0 mmol) and potassium
carbonate (8 24 g, 59 2 mmol) in dry ethanol (40 ml) were
treated with propargyl bromide (80% solution in toluene, 7 13 g,
48 0 mmol) dropwise over 30 min, and the resulting mixture
was heated at re ux for 4 h. After being cooled, the solvent
and excess of propargyl bromide were distilled o at reduced
pressure, and the residue was suspended in water (250 ml).
The mixture was extracted with ethyl acetate (3 60 ml) and
the combined extracts were washed with brine, water and dried
(MgSO4). Following ltration and evaporation at reduced
pressure, the o -white solid that remained was recrystallized
from petrol to give (17) as a white solid (5 1 g, 80%), m.p.
79–80 (lit.45 79–80 ), RF 0 68 (SiO2; EtOAc/petrol, 2 : 8).
1H n.m.r. (90 MHz, CDCl3) 2 58, t, J 2 8 Hz, 1H, ethyne
CH; 4 85, d, J 2 8 Hz, 2H, CH2; 7 09, d, J 8 8 Hz, 2H,
H 3,5; 7 86, d, J 8 8 Hz, 2H, H 2,6; 9 90, s, 1H, CHO. 13C
1H, H 5. 13C n.m.r. (100 MHz, CDCl3)
57 4, carborane
CH; 64 7, ArCH2; 69 0, ArOCH2; 71 4, carborane C; 112 9,
113 8, C 2,4; 120 8, C 6; 129 9, C 5; 143 0, C 1; 157 2, C 3.
m/z (f.a.b., thioglycerol) 280 (M), 264, 250.
3398br, 2600, 1584, 1484, 1451, 1257, 1154, 1032, 736 cm
1H n.m.r. of 10B-enriched compound (300 MHz, CDCl3)
(NaCl)
1
max
.
1 4–3 0, br m, 10H, BH; 4 09, s, 1H, carborane CH; 4 42, s,
2H, ArOCH2; 4 67, s, 2H, ArCH2; 6 76, dd, J 8 0, 2 4 Hz,
1H, H 4; 6 88–6 90, m, 1H, H 2; 7 00, br d, 1H, H 6; 7 28,
apparent t, J 7 8 Hz, 1H, H 5. m/z (f.a.b., thioglycerol) 272
(M).
n.m.r. (22 5 MHz, CDCl3)
56 6, CH2; 76 0, ethyne CH;
78 0, ethyne C; 115 8, C 3,5; 132 0, C 1; 132 5, C 2,6; 162 0,

DNA Binding Compounds. VII
299
C 4; 191 3, CHO.
1214, 1021, 828, 765 cm
(KBr) 3204, 1679, 1601, 1425, 1250,
1
2745, 2592, 1682, 1600, 1578, 1507, 1251, 1215, 1162, 830, 810,
max
1
.
620 cm
.
4-[(1,2-Dicarba-closo-dodecaboranyl)methoxy]-1-{5 0-[5 00
(piperidin-1 000-yl)benzimidazol-2 00-yl]benzimidazol-2 0-
yl}benzene (6)
-
2-[4 0-(Propargyloxy)phenyl]-1,3-dithiolan29
4-(Propargyloxy)benzaldehyde (17) (1 0 g, 6 3 mmol) was
dissolved in dry dichloromethane (20 ml) and ethane-1,2-dithiol
(880 mg, 9 4 mmol) and a solution of boron tri uoride diethyl
etherate (156 mg, 1 6 mmol in 2 ml of CH2Cl2) added to the
solution. After stirring the mixture overnight at room tempera-
ture, a 5% aqueous sodium hydroxide solution (50 ml) was added
and the mixture extracted with dichloromethane (3 20 ml).
The combined organic extracts were washed sequentially with
brine, water and then dried (MgSO4). Upon evaporation, the
title compound was isolated as a near colourless oil (1 2 g,
80%), which could be further puri ed by distillation (b.p. 130
bath temperature, 0 2 mmHg), or by chromatography, RF 0 83
(SiO2; Et2O/petrol, 1 : 1). 1H n.m.r. (300 MHz, CDCl3) 2 44,
t, J 2 5 Hz, 1H, ethyne CH; 3 33–3 39, m, 2H, thioacetal
protons; 3 48–3 52, m, 2H, thioacetal protons; 4 60, d, J
2 5 Hz, 2H, CH2; 5 60, s, 1H, H 2; 6 91, d, J 8 5 Hz, 2H,
H 30,50; 7 46, d, J 8 5 Hz, 2H, H 20,60. 13C n.m.r. (72 5 MHz,
The benzaldehyde (16) (60 mg, 0 22 mmol) was dissolved
in methanol (5 ml) and treated with a solution of sodium
metabisul te (45 mg, 0 24 mmol) in warm aqueous methanol
(1 : 1, 1 ml). A solution of freshly prepared diamine (15) (61 mg,
0 2 mmol) in methanol (5 ml) was then added and the resulting
solution heated under nitrogen at 90 for 6 h. After the solution
was cooled, the solvent was removed under vacuum and the
mud-yellow residue triturated with diethyl ether. The highly
insoluble solid that remained was preadsorbed onto a neutral
alumina chromatography column by using dimethylformamide,
and eluted with methanol/ethyl acetate (5–15% MeOH gradi-
ent). Collection of the uorescent band gave a bright yellow
solid after evaporation; this was subjected to Sephadex LH20
size exclusion chromatography (2% acetic acid/MeOH). The
solid obtained on evaporation of the second uorescent band
was further puri ed by neutral alumina chromatography (2%
MeOH/EtOAc) to give the bibenzimidazole (6) (25 mg, 20%),
m.p. 180 (dec.), RF 0 6 (Al2O3; 2% MeOH/EtOAc) (Found:
CDCl3)
CH; 78 4, ethyne C; 114 8, C 30,50; 129 1, C 20,60; 132 9, C 10;
157 3, C 40.
(NaCl) 3281, 2919, 2334, 2119, 1603, 1451,
1301, 1263, 1110, 1024, 923, 834 cm
40 1, C 4,5; 55 8, 55 9, CH2, C 2; 75 6, ethyne
max
(M+H)+ , 566 39158. C28H35B10N5O requires (M+H)+
566 39233). 1H n.m.r. (300 MHz, CD3OD+tri uoroacetic
acid) 1 2–3 4, br m, 10H, BH; 1 80–1 90, br m, 2H,
,
1
.
2-[4 0-{(1,2-Dicarba-closo-dodecaboranyl)methoxy}phenyl]-
H 4000; 2 05–2 15, br m, 4H, H 3000,5000; 3 73–3 77, br m, 4H,
H 2000,6000; 4 73, s, 2H, CH2; 4 80, br s, 1H, carborane CH;
7 31, d, J 8 8 Hz, 2H, H 3,5; 7 84, dd, J 9 0, 2 2 Hz, 1H,
H 600; 8 01, d, J 9 0 Hz, 1H, H 700; 8 07, d, J 8 8 Hz, 1H,
H 70; 8 10, d, J 2 0 Hz, 1H, H 400; 8 20 d, J 8 8 Hz, 2H, H 2,6;
8 33, dd, J 8 8, 1 7 Hz, 1H, H 60; 8 66, d, J 1 4 Hz, 1H, H 40.
13C n.m.r. (72 5 MHz, CD3OD+(CD3)2SO (5 drops)+acetic
1,3-dithiolan (18) 29
Bis(acetonitrile)–decaborane (12) (563 mg, 2 8 mmol) sus-
pended in dry toluene (10 ml) was treated with 2-[40-
(propargyloxy)phenyl]-1,3-dithiolan (540 mg, 2 3 mmol) and
the mixture heated at 80 under nitrogen for 60 h. After being
cooled, the solution was ltered through Celite and the ltrate
evaporated to dryness. The solid residue was dissolved in
ethanol, whereupon hydrogen was released, and the resulting
solution stirred overnight. The residue obtained on evaporation
was subjected to radial chromatography (30% Et2O/petrol).
and collection of the rst band gave (18) as a white, low-melting
solid (490 mg, 61%), RF 0 55 (40% Et2O/petrol). 1H n.m.r.
(300 MHz, CDCl3) 1 2–3 4, br m, 10H, BH; 3 33–3 37, m,
2H, thioacetal protons; 3 47–3 52, m, 2H, thioacetal protons;
4 08, br s, 1H, carborane CH; 4 39, s, 2H, CH2; 5 60, s,
1H, H 2; 6 78, d, J 8 8 Hz, 2H, H 30,50; 7 46, d, J 8 8 Hz,
acid (5 drops)) 25 1, C 4000; 27 2, C 3000,5000; 54 0, C 2000,6000
62 2, carborane CH; 70 4, CH2; 74 1, carborane C; 102 6,
C 400; 114 3, 116 5, 116 6, 117 3, 122 6, 124 5, 124 7, 129 8,
134 8, 139 5, 140 6, 142 0, 150 3, 151 5, 153 0, 154 8, 160 6.
;
m/z (f.a.b., thioglycerol) 566 (M+H), 410, 325.
(KBr)
max
3410, 2939, 2590, 1664, 1605, 1555, 1464, 1301, 1256, 1030,
1
949, 835, 720, 685 cm
.
4-[5 0-(4 00-Methylpiperazin-1 00-yl)benzimidazol-2 0-
yl]benzene-1,2-diamine (19)
2H, H 20,60. 13C n.m.r. (72 5 MHz, CDCl3)
55 6, C 2; 57 7, carborane CH; 69 2, CH2; 71 3, carborane
C; 114 6, C 30,50; 129 5, C 20,60; 134 3, C 10; 156 7, C 40. m/z
40 3, C 4,5;
Hydrogenation of 4-[50-(400-methylpiperazin-100-yl)benzimi-
dazol-20-yl]-2-nitroaniline (750 mg, 2 2 mmol) as for the prepa-
ration of (15) gave diamine (19) as an orange-brown solid
(560 mg, 82%), m.p. 259–261 (lit.14 268 ). 1H n.m.r. (400 MHz,
CD3OD+methanesulfonic acid) 3 03, s, 3H, CH3; 3 38–3 48,
m, 4H, piperazine protons; 3 71–3 77, m, 2H, piperazine
protons; 3 90–3 95, m, 2H, piperazine protons; 7 05, d, J
8 8 Hz, 1H, H 6; 7 37, dd, J 2 2, 9 0 Hz, 1H, H 60; 7 43, d,
J 2 2 Hz, 1H, H 40; 7 64, d, J 9 0 Hz, 1H, H 70; 7 76, dd, J
9 0, 2 2 Hz, 1H, H 5; 7 96, d, J 2 2 Hz, 1H, H 3. 13C n.m.r.
(100 MHz, CD3OD+methanesulfonic acid) 43 7, CH3; 49 3,
C 200,600; 53 9, C 300,500; 102 4, 111 5, 115 5, 118 7, 118 8,
119 0, 125 1, 128 4, 129 7, 133 5, 146 9, 147 8, 149 9.
(f.a.b., thioglycerol) 354 (M+H), 263.
(NaCl) 3435, 3075,
max
2592, 1683, 1600, 1578, 1428, 1302, 1251, 1163, 1053, 870, 824,
1
723 cm
.
4-[(1,2-Dicarba-closo-dodecaboranyl)methoxy]benzaldehyde
(16) 29
Carborane (18) (250 mg, 0 71 mmol) was dissolved in dry
tetrahydrofuran (15 ml) and the solution treated with mercury
perchlorate trihydrate (704 mg, 1 55 mmol) in tetrahydrofuran
(10 ml). After stirring the mixture at room temperature for
5 min, the white precipitate was removed by ltration through
Celite, and the ltrate evaporated to dryness. The yellow oil
thus obtained was dissolved in dichloromethane (10 ml) and
washed sequentially with brine, water, and then dried over
anhydrous magnesium sulfate to yield the title aldehyde (16)
(165 mg, 84%), m.p. 146–148 . 1H n.m.r. (300 MHz, CDCl3)
1 2–3 4, br m, 10H, BH; 4 07, br s, 1H, carborane CH; 4 51,
s, 2H, CH2; 6 97, d, J 8 8 Hz, 2H, H 3,5; 7 86, d, J 8 8 Hz,
2H, H 2,6; 9 91, s, 1H, CHO. 13C n.m.r. (72 5 MHz, CDCl3)
57 9, carborane CH; 69 1, CH2; 70 8, carborane C; 114 9,
C 3,5; 131 4, C 1; 132 0, C 2,6; 161 4, C 4; 190 4, CHO. m/z
3-(Propargyloxy)benzonitrile (21)
3-Hydroxybenzonitrile (1 19 g, 10 mmol) was dissolved in
dry ethanol (30 ml) and treated with potassium carbonate
(1 8 g, 10 mmol). Propargyl bromide (1 6 ml, 11 mmol, 80%
solution in toluene) was added dropwise and the mixture heated
at re ux for 6 h under nitrogen. After the mixture was cooled,
the solvent and excess of propargyl bromide were evaporated
under vacuum and water (200 ml) was added to the residue.
The solution was extracted with dichloromethane (3 50 ml)
and the combined organic extracts were washed with water and
brine, dried (MgSO4), and evaporated to give an o -white solid
(f.a.b., thioglycerol) 279 (M+H).
(KBr) 3441, 3070, 2830,
max

300
S. A. Bateman et al.
(1 50 g, 94%). Recrystallization from petrol gave the ether
(21) as white plates (1 35 g, 86%), m.p. 55–57 , RF 0 8 (SiO2;
CH2Cl2/hexane, 1 : 1) (Found: M+ , 157 0445. C10H7NO
requires M+ , 157 0449). 1H n.m.r. (300 MHz, CDCl3) 2 59,
t, J 2 5 Hz, 1H, CH; 4 75, d, J 2 5 Hz, 2H, CH2; 7 22–7 45,
m, 4H, H 2,4,5,6. 13C n.m.r. (72 5 MHz, CDCl3) 56 0, CH2;
76 5, acetylene CH; 77 4, acetylene C; 113 2, C 1; 118 0,
118 5, C 4, CN; 120 1, C 2; 125 3, C 6; 130 3, C 5; 157 5, C 3.
in glacial acetic acid/ethanol (1 : 2, 30 ml) and the resulting
solution heated at re ux under nitrogen for 4 5 h. After
the mixture was cooled in the fridge for 3 h, the resultant
precipitate was collected by ltration, washed sequentially with
cold glacial acetic acid/ethanol (1 : 2) and nally diethyl ether.
The yellow solid thus obtained was dried under vacuum to
give 130 mg (51%) of the bibenzimidazole (7), m.p. 220 (dec.),
RF 0 2 (Al2O3; EtOAc/MeOH, 20 : 1) (Found: (M+H)+
,
m/z (positive e.i., 70 eV) 157 (M+).
2232, 2115, 1601, 1577, 1478, 1229, 1037, 877 cm
(KBr) 3252, 3071,
1
581 4015. C28H36B10N6O requires (M+H)+ , 581 4032). 1H
n.m.r. (300 MHz, CD3OD+tri uoroacetic acid) 1 2–3 4, br
m, 10H, BH; 3 01, s, 3H, CH3; 3 67, br s, 2H, piperazine
protons; 3 69, br s, 2H, piperazine protons; 3 97, br s, 2H,
piperazine protons; 4 01, br s, 2H, piperazine protons; 4 75,
s, 2H, CH2; 4 84, br s, 1H, carborane CH; 7 36–7 46, m, 2H,
H 4,400; 7 45, dd, J 9 3, 2 2 Hz, 1H, H 600; 7 70, apparent t,
J 8 8 Hz, 1H, H 5; 7 78, d, J 9 0 Hz, 1H, H 700; 7 82–7 85,
m, 2H, H 2,6; 8 13, d, J 8 8 Hz, 1H, H 70; 8 28, dd, J 8 8,
1 5 Hz, 1H, H 60; 8 64, d, J 1 4 Hz, 1H, H 40. 13C n.m.r.
(72 5 MHz, CD3OD+tri uoroacetic acid) 43 6, CH3; 48 7,
C 2000,6000; 53 8, C 3000,5000; 62 1, carborane CH; 70 6, CH2;
73 7, carborane C; 102 3, C 4000; 115 0, 116 2, 117 1, 119 7,
121 5, 122 7, 123 0, 124 1, 126 7, 128 4, 132 7, 133 1, 133 8,
135 6, 148 3, 148 8, 152 7, 159 5, one signal missing (possible
overlapping with signal at 115 0). m/z (f.a.b., thioglycerol)
max
.
3-[(1,2-Dicarba-closo-dodecaboranyl)methoxy]benzonitrile
(20)
3-(Propargyloxy)benzonitrile (21) (500 mg, 3 2 mmol) and
bis(acetonitrile)–decaborane complex (12) (770 mg, 3 89 mmol)
were stirred in dry toluene (30 ml) at 80 under nitrogen. The
decaborane complex slowly dissolved and after 24 h the solu-
tion was allowed to cool to room temperature. Following
ltration through Celite, the ltrate was evaporated under
vacuum. Ethanol was added to the oily residue and the
solution stirred for a further 24 h at room temperature, after
which the colour of the solution had lightened. Removal of the
solvent gave a residue which was subjected to chromatography
(SiO2; CH2Cl2/hexane, 1 : 1, RF 0 6) and evaporation of the
rst band collected gave crystalline (20) (470 mg, 52%), m.p.
581 (M+H), 510, 483.
(KBr) 3412, 2957, 2587, 796, 1667,
max
1637, 1587, 1452, 1201, 1086, 1053 cm 1. H.p.l.c. RT 7 10 min
(55% MeCN/0 1% tri uoroacetic acid/H2O). 1H n.m.r. of 10B-
enriched compound (300 MHz, CD3OD+tri uoroacetic acid)
1 4–3 0, br m, 10H, BH; 3 01, s, 3H, CH3; 3 67, br s, 2H,
piperazine CH2; 3 71, br s, 2H, piperazine CH2; 3 96, br s,
2H, piperazine CH2; 4 02, br s, 2H, piperazine CH2; 4 73, s,
2H, CH2; 4 82, br s, 1H, carborane CH; 7 37, d, J 2 2 Hz, 1H,
H 400; 7 43–7 48, m, 2H, H 4,600; 7 71, apparent t, J 7 8 Hz,
1H, H 5; 7 78, d, J 9 0 Hz, 1H, H 700; 7 86–7 88, m, 2H, H 2,6;
8 15, d, J 8 8 Hz, 1H, H 70; 8 30, dd, J 1 7, 8 8 Hz, 1H, H 60;
8 65, br s, 1H, H 40. m/z (f.a.b., thioglycerol) 573 (M+H).
110–111 . 1H n.m.r. (300 MHz, CDCl3)
1 2–3 4, br m,
10H, BH; 4 03, br s, 1H, carborane CH; 4 44, s, 2H, CH2;
7 08–7 13, m, 2H, H 2,4; 7 31, dm, J 7 5 Hz, 1H, H 6; 7 43,
apparent td, J 7 3, 1 7 Hz, 1H, H 5. 13C n.m.r. (72 5 MHz,
CDCl3)
57 8, carborane CH; 69 4, CH2; 70 6, carborane
C; 113 7, C 1; 117 8, 117 9, C 4, CN; 119 6, C 2; 126 4, C 6;
130 8, C 5; 156 9, C 3. m/z (positive e.i., 70 eV) 275 (M+).
(KBr) 3199, 3059, 2529, 2228, 1595, 1489, 1478, 1394,
1257, 1160, 1052, 935, 790 cm
max
1
.
1H n.m.r. of 10B-enriched
compound (300 MHz, CDCl3) 1 4–3 0, br m, 10H, BH; 4 03,
br s, 1H, carborane CH; 4 44, s, 2H, CH2; 7 09–7 12, m, 2H,
H 2,4; 7 36, dm, J 7 6 Hz, 1H, H 6; 7 44, apparent td, J 7 3,
1 7 Hz, H 1,5. m/z (f.a.b., thioglycerol) 268 (M+H).
DNA Binding Experiments
Ethyl 3-[(1,2-Dicarba-closo-dodecaboranyl)methoxy]-
benzenecarboximidate Hydrochloride (22)
Bu er solutions were freshly prepared for each series of
experiments and aliquots taken by using Socorex microlitre
pipettes. Calf thymus (ct) DNA was stored at 20 and fresh
samples were aliquoted each day. The method of sample prepa-
ration for ultraviolet and circular dichroism experiments was
identical and outlined in Table 3. Experiments were carried out
in 20% methanol, which increased the solubility of the highly
insoluble DNA binding ligands. It was found that the order
of addition of each of the components was also important in
preventing precipitation, as was the need for constant vortexing
of the solution during additions. Samples prepared in this
Nitrile (20) (190 mg, 0 69 mmol) was suspended in dry
ethanol (20 ml) and dry HCl gas bubbled through the solution
for 45 min. The mixture was protected with a CaCl2 drying
tube, stirred overnight and then evaporated to give a colourless
oil which soon crystallized. Trituration of the solid residue in
dry diethyl ether, and ltration under nitrogen, gave (22) as a
white, moisture-sensitive solid (210 mg, 86%), m.p. 125–127 .
1H n.m.r. (300 MHz, (CD3)2SO) 1 47, t, J 7 0 Hz, 3H, CH3;
1 2–3 4, br m, 10H, BH; 4 60, q, J 7 0 Hz, 2H, imidate CH2;
4 77, s, 2H, ArOCH2; 5 47, br s, 1H, carborane CH; 7 43, dd,
J 8 0, 2 2 Hz, 1H, H 4; 7 58, apparent t, J 8 0 Hz, 1H, H 5;
7 70, br d, J 8 0 Hz, 1H, H 6; 7 80–7 81, m, 1H, H 2. 13C
n.m.r. (72 5 MHz, (CD3)2SO) 13 5, CH3; 62 2, carborane
CH; 68 9, 69 9, imidate CH2, ArOCH2; 73 4, carborane C;
115 1; 122 1; 122 6; 127 4; 130 7; 157 2; 170 4, imine C.
m/z (f.a.b., thioglycerol) 322 (M+H HCl). 1H n.m.r. of 10B-
enriched compound (300 MHz, (CD3)2SO) 1 47, t, J 6 8 Hz,
3H, CH3; 1 4–3 0, br m, 10H, BH; 4 59, q, J 6 9 Hz, 2H,
imidate CH2; 4 76, s, 2H, ArOCH2; 5 54, br s, 1H, carborane
CH; 7 42, dd, J 8 0, 2 2 Hz, 1H, H 4; 7 57, apparent t, J
7 8 Hz, 1H, H 5; 7 70, br d, J 7 8 Hz, 1H, H 6; 7 77, br s,
1H, H 2. m/z (f.a.b., thioglycerol) 314 (M+H HCl).
5
5
way contained between 3 5 10
and 4 0 10
mmol of the
ligand, and, on addition of ct DNA, a ligand to base pair ratio
of between 0 08 and 0 09. Details of the instrument type and
conditions employed to record the spectra are reported in the
General Experimental.
Table 3. Method of sample preparation for DNA binding
experiments
Reagents
used
Amounts used ( l)
Control
DNA+salt
Tris (2 0 M, pH 7 5)
EDTA (0 5 M, pH 7 5)
H2O
1 70 mM bp ct DNA
MeOH
DNA ligands, 6 6–8 0 mM,
in 1 : 1 MeOH/H2O
NaCl (2 M)
50
50
3900
—
904
96
50
50
3400
250
904
96
3-[(1,2-Dicarba-closo-dodecaboranyl)methoxy]-1-{5 0-[5 00
-
(4 000-methylpiperazin-1 000-yl)benzimidazol-2 00
-
yl]benzimidazol-2 0-yl}benzene (7)
Freshly prepared diamine (19) (149 mg, 0 44 mmol) was
added to a solution of imidate (22) (180 mg, 0 50 mmol)
—
250

DNA Binding Compounds. VII
301
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1927.
Hook, R. J., unpublished data.
Acknowledgments
20
Financial support from the Australian Research
Council is gratefully acknowledged. S.A.B. thanks the
Australian Research Council for an Australian Post-
graduate Research Award. We thank Dr Michael Rose
for completing a literature search for the preparation
of (8) and (16), and Mr Marshal Pardee for help in
obtaining h.p.l.c. data.
21
22
23
24
25
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M., Meriaty, H., Allen, B. J., and Martin, R. F., Abstract,