New mustard-linked 2-aryl-bis-benzimidazoles with anti-proliferative activity

Article abstract of DOI:10.1039/b600567e

We describe new methodology for the synthesis of symmetric bis-benzimidazoles carrying 2-aryl moieties, including 2-[4-(3′- aminopropoxy)phenyl] and 2-[4-(3′-aminopropanamido)phenyl] substituents, together with the synthesis of novel hybrid molecules comprising bis-benzimidazoles in ester and amide combination with the N-mustard chlorambucil. The in vitro activities of these compounds against five cancer cell lines are also provided. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b600567e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
New mustard-linked 2-aryl-bis-benzimidazoles with anti-proliferative activity
Christine Le Sann,a Anne Baron,a John Mann,*a Hendrik van den Berg,b Mekala Gunaratnamc and
Stephen Neidle*c
Received 17th January 2006, Accepted 9th February 2006
First published as an Advance Article on the web 9th March 2006
DOI: 10.1039/b600567e
We describe new methodology for the synthesis of symmetric bis-benzimidazoles carrying 2-aryl
moieties, including 2-[4-(3ꢀ-aminopropoxy)phenyl] and 2-[4-(3ꢀ-aminopropanamido)phenyl]
substituents, together with the synthesis of novel hybrid molecules comprising bis-benzimidazoles
in ester and amide combination with the N-mustard chlorambucil. The in vitro activities of these
compounds against five cancer cell lines are also provided.
Introduction
We have recently described the synthesis and DNA-binding affini-
ties of the symmetrical bis-benzimidazole 11 and the dimeric bis-
benzimidazole 2.2 The former exhibited high sequence-selectivity
for four consecutive A/T base pairs in the dodecanucleotide
sequence d[CGCGAATTCGCG], which is a model for longer
oligonucleotide sequences in the A/T region of the minor groove.
It also exhibited sub-micromolar activity against nine cancer cell
lines in vitro, including IC50 values of 50 nM against MCF-7 (breast
cancer) and 80 nM against H838 (lung cancer) cells.3 In contrast,
compound 2 exhibits levels of activity at least ten times less than
exhibited by 1, but has high affinity for ten base pairs4 of the DNA
sequence d[A.T]4–[G.C]–[A.T]4. This site-size selectivity compares
well with the best selectivities reported by Dervan5 and Bruice.6
In this paper we report on bis-benzimidazole molecules with
covalent-binding functionality and similar site-size requirements
to compound 2, that further define the structural requirements for
potent anticancer activity in this series.
Results and discussion
We decided initially to investigate the coupling of the well-
employed extensive simulation methods to produce a set of
detailed structures. The initial, energy-minimised structure of
a DNA complex with compound 18 is shown in Fig. 1. The
ligand binding site-size is 10 base pairs, with one arm of each
alkylating group positioned to interact covalently with N3
sites on purines. Inter-strand cross-linking is also feasible. The
ester–(CH2)3-linkers are in stereochemically acceptable, extended
conformations, suggesting that this linker length is appropriate.
We also find that the replacement of the ester group in the linkage
by an amide group is similarly feasible, now with the potential for
a hydrogen bond between the amide nitrogen atom and a thymine
O2 atom (Fig. 2).
established alkylating agent chlorambucil to the bis-benzimidazole
motif. Chlorambucil, in common with the majority of DNA
alkylating compounds, has a preference for cross-linking via N7 of
guanine in the DNA major groove,7 but can also bind to phosphate
groups and to N3 in the minor groove, as has been found with
a chlorambucil–polyamide conjugate.8 We have used computer
modelling to provide a guide to the optimal length of linker groups
between bis-benzimidazole moieties and chlorambucil, assuming
alkylation at N3.
Molecular modelling has been used to develop
a
stereochemically-plausible structural model that would guide
the synthesis of possible targets, and we have not at this stage
Our general synthetic strategy is shown in Scheme 1 and
involved condensation of the commercially available 3,3ꢀ,4,4ꢀ-
tetraaminobiphenyl 3 with 4-(3ꢀ-bromopropoxy)benzaldehyde 4
(prepared by a Mitsunobu reaction9 between 3-bromopropanol
and 4-hydroxybenzaldehyde) in the presence of the oxidising agent
aSchool of Chemistry, Queen’s University Belfast, Stranmillis Road, Belfast,
UK BT9 5AG. E-mail: j.mann@qub.ac.uk; Fax: +44 (0)28 9097 4890;
Tel: +44 (0)28 9097 5525
bDepartment of Oncology, Queen’s University Belfast, City Hospital, Belfast,
UK BT9 7AR
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Oxoneꢀ.10 This reagent proved to be more convenient than the
cCRUK Biomolecular Structure Group, The School of Pharmacy, University
of London, 29–39 Brunswick Square, London, UK WC1N 1AX. E-mail:
stephen.neidle@pharmacy.ac.uk
nitrobenzene that we used in our earlier work.1 The resultant
bis-benzimidazole 5 was then reacted with the requisite amine
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The relatively high activities of compounds 6a–c prompted
an examination of similar compounds, e.g. 9 wherein an aniline
replaced the phenol, since such compounds would allow us to
probe the effect of an O to N replacement in terms of efficacy
of binding to the minor groove of DNA. These new compounds
were prepared from the N–BOC derivative 10 of 4-aminobenzyl
alcohol12 which, after oxidation with MnO2 was condensed with
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3,3ꢀ,4,4ꢀ-tetraaminobiphenyl in the presence of Oxoneꢀ to produce
the expected bis-benzimidazole 11. Upon treatment with TFA
this provided the key 2-(4ꢀ-aminophenyl)-bis-benzimidazole 12,
and this was reacted with chloropropionyl chloride and then
dimethylamine13 to yield the dimethylaminopropyl derivative 13,
and this chemistry is shown in Scheme 2. Unfortunately, we have
not yet been able to synthesise the reduced compound 9.
Alternative routes to the key 2-(4ꢀ-aminophenyl)-bis-benzimida-
zole 12 were explored as shown in Scheme 3. Thus condensation
of the tetraaminobiphenyl 3 with 4ꢀ-nitrobenzaldehyde in the pres-
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ence of Oxoneꢀ provided the 2-(4ꢀ-nitrophenyl)-bis-benzimidazole
14 (83% yield) and condensation of 3 with 4-fluorobenzaldehyde
provided the 2-(4ꢀ-fluorophenyl-bis-benzimidazole) 15 (84%).
Unfortunately, attempted reduction of 14 to produce 12 using a
number of reducing agents (H2/Pd, transfer hydrogenation with
ammonium formate and Pd, Zn/HOAc, and Fe/HCl) all failed.
In addition, attempted displacement of fluoride from compound
15 with 3-dimethylaminopropylamine was also non-productive.
Despite these failures, this new methodology for producing
2-aryl-bis-benzimidazoles has proved to be both high-yielding
and compatible with a variety of 4-substituted benzaldehydes.
As a first foray into the production of hybrid molecules that
combine a bis-benzimidazole (for binding to the DNA minor
groove) and a known anticancer agent, we have prepared two
novel hybrids that incorporate chlorambucil (Scheme 4). Thus
reaction of 2-(4ꢀ-hydroxyphenyl)-bis-benzimidazole 16 (already
prepared by us,1 but now prepared more efficiently from 3 and 4-
Fig. 1 Molecular model of compound 18 bound to a 12-mer DNA
sequence. The ligand is shown in space-filling mode.
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hydroxybenzaldehyde in conjunction with Oxoneꢀ) with the acid
chloride of chlorambucil 17 (oxalyl chloride in DMF) provided the
novel ester hybrid 18. Similarly, reaction of 2-(4ꢀ-aminophenyl)-
bis-benzimidazole 12 with the acid chloride of chlorambucil
provided the novel amide hybrid 19. Interestingly, this amide
hybrid 19 was around 12-fold more cytotoxic (IC50 of 0.47 lM)
than the ester hybrid 18 (IC50 6.2 lM), possibly due to greater
metabolic stability, though the possibility of more effective binding
to the DNA minor groove by means of hydrogen bonding as shown
in Fig. 2 cannot be excluded. A range of similar compounds with
different side-chains will be prepared to explore further the SARs
for these hybrids.
These IC50 values were obtained in MCF-7 breast carcinoma
cell lines, in which the non-covalently-binding analogues 6a–c have
also been evaluated (Fig. 3–5). These also have an IC50 of ca. 1 lM,
which suggests that the mustard group in compounds 18 and 19
is not contributing significantly to the cytotoxicity, probably on
account of the known preference of this group for binding to N7
of guanine in the major groove of DNA, rather than the minor
groove.
Fig. 2 View of the modelled structure of compound 19 bound to the
12-mer DNA sequence, showing a potential hydrogen bond between the
linker amide nitrogen atom and a thymine O2 atom.
(piperidine or N-methylpiperazine) to provide the required bis-
benzimidazoles 6a,b. For pyrrolidine, a higher yield was obtained
using a sequence involving Michael addition of the amine to
methylacrylate, followed by reduction (LiAlH4), chloride forma-
tion (SOCl2), and reaction of the 3-(N-pyrrolidinyl)propyl chloride
7 with 4-hydroxybenzaldehyde (Cs2CO3, NaI)11 to yield the 4-
(N-pyrrolidinyl)propoxybenzaldehyde 8, ready for condensation
These results suggest that it could be profitable to attach
alternative anticancer drugs with different modes of action (e.g.
anthracyclinones) to the side-chains of bis-benzimidazoles in order
to probe the efficacy of these molecules as drug delivery agents,
and this work is in progress.
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with 3,3ꢀ,4,4ꢀ-tetraaminobiphenyl in the presence of Oxoneꢀ to
yield bis-benzimidazole 6c. These compounds were tested in vitro
against four cancer cell lines and the inhibition curves are shown
in Fig. 3–5.
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Scheme 1 Preparation of analogues of bis-benzimidazoles 1 using OxoneꢀR .
Scheme 2 Preparation of a nitrogen isostere of 1.
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Fig. 3 The effect of piperidinyl analogue (compound 6a) on the prolifer-
ation of human cancer cells in vitro.
Scheme 3 An alternative method of preparing bis-benzimidazoles.
Fig. 4 The effect of N-methylpiperazinyl analogue (compound 6b) on the
proliferation of human cancer cells in vitro.
Fig. 5 The effect of pyrrolidyl analogue (compound 6c) on the prolif-
eration of human cancer cells in vitro. Human cells used were: MCF-7,
MDA468-Breast carcinoma; HT29, RKO-Colon carcinoma; MalMe3M,
SKMe128-Melanoma.
Scheme 4 Coupling of bis-benzimidazoles 12 and 16 with chlorambucil.
for electron impact (EI) and chemical ionisation (CI) techniques,
or a VG Quattro spectrometer for the electrospray method.
Infrared spectra were obtained on a Perkin Elmer Spectrum
RX I FTIR System instrument. Melting points were taken on
Experimental
General
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a Gallenkampꢀ apparatus and are uncorrected.
1H-NMR spectra were obtained using a Bruker 300 NMR Avance
DPX spectrometer or a Bruker DRX Avance spectrometer at
300 MHz and 500 MHz respectively. 13C-NMR spectra were
recorded on a Bruker Avance DPX or Avance DRX spectrometer
at a frequency of 75 MHz or 125 MHz respectively. 1H–13C-NMR
correlation experiments were carried out on a Bruker Avance DRX
spectrometer. Chemical shifts (d) are given in parts per million
(ppm). Mass spectra were recorded on a VG Autospec instrument
Methyl-3-pyrrolidin-1ꢀ-yl propanoate. A solution of methyl
acrylate (1.21 g, 1.26 mL, 14.06 mmol) in dichloromethane
(15 mL) at 0 ◦C was treated dropwise with pyrrolidine (1 g,
14.06 mmol). The mixture was left stirring and warming to
room temperature for 4 h, and was then evaporated to yield
the desired product as a pale yellow oil (2.12 g, 96%). mmax
(film)/cm−1 2953, 2790, 1740, 1653, 1436, 1205; dH (300 MHz,
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CDCl3, Me4Si) 1.80 (4H, m, CH2 × 2), 2.54 (4H, m, CH2N × 2),
2.54 (2H, t, J 7 Hz, CH2CO), 2.76 (2 H, t, J 7 Hz, CH2N),
3.68 (3H, s, CH3); dC (125 MHz, CDCl3, Me4Si) 23.8 (CH3),
4 hours. The reaction mixture was then evaporated to dryness to
give a thick yellow oil that was purified by column chromatography
using 10% ethyl acetate in petroleum ether on neutralised silica gel.
The title compound 4 was obtained as a pale yellow oil (536 mg,
61%). mmax (film)/cm−1 2940, 2880, 2828, 1688, 1600, 1468 and
1159; dH (300 MHz, CDCl3, Me4Si) 2.36 (2H, quintet, J 6 Hz,
2ꢀ-H2), 3.62 (2H, t, J 6 Hz, 3ꢀ-H2), 4.20 (2H, t, J 6 Hz, 1ꢀ-H2), 7.02
(2H, d, J 8.5 Hz, 3-H and 5-H), 7.69 (2H, d, J 8.5 Hz, 2-H and
6-H), 9.89 (1H, s, CHO); dC (125 MHz, CDCl3, Me4Si) 25.7 (C-
2ꢀ), 29.9 (C-3ꢀ), 66.0 (C-1ꢀ), 115.2 (C-3 and C-5), 130.6 (C-1), 132.4
(C-2 and C-6), 164.1 (C-1), 191.2 (CHO); m/z (EI) 241.9940 (M+,
C10H11O2Br requires 241.9942), 244 (40%), 242 (41), 138 (40), 122
(47), 121 (100), 77 (23), 65 (33).
34.3 (CH2 × 2), 51.8 (CH2CO), 51.9 (CH2N), 54.4 (CH2N), 173.3
+
=
(C O); m/z (CI) 158 (MH , 100%), 130 (47), 84 (30), 70 (15).
3-Pyrrolidin-1ꢀ-yl propan-1-ol. A suspension of lithium alu-
minium hydride (525 mg, 13.84 mmol) in dry diethyl ether
◦
(10 mL) at 0 C was treated dropwise with a solution of methyl-
3-pyrrolidin-1ꢀ-yl propanoate (2.12 g, 13.84 mmol) in diethyl
ether (10 mL). The reaction mixture was left to stir for 1 hour.
Water (525 lL) was then added slowly, followed by a solution of
sodium hydroxide (15%, 525 lL), and finally water (1.58 mL). A
white precipitate formed. Dichloromethane (15 mL) was added to
solubilise the alcohol, the suspension was filtered and the filtrate
was concentrated in vacuo to give the title product as a colourless
oil (1.52 g, 85%). mmax (film)/cm−1 3367, 2944, 2877, 2803, 1457,
1134, 1065; dH (300 MHz, CDCl3, Me4Si) 1.56–1.78 (6H, m, 2-H2
and CH2 × 2), 2.56 (4H, CH2N × 2), 2.73 (2 H, t, J 6 Hz, 3-H2),
3.81 (2H, t, J 6 Hz, 1-H2), 5.50 (1H, s, OH); dC (125 MHz, CDCl3,
Me4Si) 23.7 (CH2 × 2), 29.5 (C-2), 54.6 (CH2N × 2), 56.8 (C-3),
65.1 (C-1); m/z (CI) 130 (MH+, 100%), 84 (22), 58 (100).
2,2-Bis[4ꢀ-(3ꢀꢀ-pyrrolidinopropoxy)phenyl]-5,5-bi-1H-benzimida-
zole 6c. A solution of 3,3ꢀ-diaminobenzidine tetrahydrochloride
dehydrate (200 mg, 541 mmol), in N,N-dimethylformamide
(15 mL) was treated with sodium hydroxide (80 mg) in water
(1 mL), followed by a solution of aldehyde 8 (252 mg, 1.082 mmol)
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in DMF (14 mL), and finally Oxoneꢀ (432 mg, 0.703 mmol).
The reaction mixture was left stirring for 18 hours. A solution of
potassium carbonate in water (1 M, 2 mL) was added, followed
by water (30 mL). The solid that precipitated was then filtered
under vacuum, and washed with water. It was then left to dry in
the air. The title compound was obtained as a tan-coloured solid
4-(3ꢀ-Pyrrolidin-1ꢀꢀ-ylpropoxy)benzaldehyde 8. A solution of
3-pyrrolidin-1ꢀ-yl propan-1-ol (250 mg, 1.93 mmol) in
dichloromethane was cooled to 0 ◦C, then treated dropwise with
thionyl chloride (3 mL, 41 mmol). The reaction mixture was stirred
in the cold for 2.5 h. The solvent and excess reagent were then
removed in vacuo and the solid residue was then dried under high
vacuum to give the desired chloride 7 as a white solid that was
used fresh and without further purification in the next step.
A solution of 4-hydroxybenzaldehyde (207 mg, 1.70 mmol) in
dry DMF (15 mL) was treated with caesium carbonate (1.4 g,
4.3 mmol), followed after 5 minutes by sodium iodide (220 mg,
1.46 mmol) and the chloride 7 (270 mg, 1.46 mmol). The reaction
mixture was stirred vigorously and heated to 100 ◦C, in the dark,
for 10 hours. After cooling, the mixture was treated with a solution
of sodium hydroxide (2 M, 50 mL). Sodium chloride was added
to help the separation of the layers, and the aqueous mixture
was extracted with ethyl acetate (4 × 50 mL). The combined
organic layer was washed with brine (100 mL), and a solution
of sodium hydroxide (2 M, 100 mL). It was then dried over
magnesium sulfate, concentrated, and dried under high vacuum to
give the desired product as an orange oil. (253 mg, 74%). mmax (KBr
disc)/cm−1 2957, 2796, 1690, 1601, 1259 and 1159; dH (300 MHz,
CDCl3, Me4Si) 1.82 (4H, m, CH2 × 2), 2.04 (2H, quintet, J 6.5 Hz,
2-H2), 2.52 (4H, m, CH2N × 2), 2.63 (2H, t, J 7 Hz, 3-H2), 4.12 (2H,
t, J 6.5 Hz, 1-H2), 7.04 (2H, d, J 7 Hz, 2ꢀ-H and 6ꢀ-H), 7.82 (2H, d,
J 7 Hz, 3ꢀ-H and 5ꢀ-H), 9.87 (1H, s, CHO); dC (125 MHz, CDCl3,
Me4Si) 23.8 (C-3ꢀꢀ and C-4ꢀꢀ), 29.1 (C-2ꢀ), 53.3 (C-3ꢀ), 54.6 (C-2ꢀꢀ and
C-5ꢀꢀ), 67.2 (C-1ꢀ), 115.2 (C-3 and C-5), 130.2 (C-1), 132.36 (C-2
◦
(301 mg, 87%). Mp 192–194 C (decomp.); mmax (KBr disc)/cm−1
3400, 2959, 1612, 1254 and 1179; dH (500 MHz, DMSO-d6) 1.66
(8H, m, 3ꢀꢀꢀ-H2 and 4ꢀꢀꢀ-H2), 1.89 (4H, quintet, J 7 Hz, 2ꢀꢀ-H2),
2.48 (8H, m, 2ꢀꢀꢀ-H2 and 5ꢀꢀꢀ-H2), 2.52 (4H, t, J 7 Hz, 3ꢀꢀ-H2), 4.08
(4H, t, J 6.5 Hz, 1ꢀꢀ-H2), 7.10 (4H, d, J 8.5 Hz, 3ꢀ-H2 and 5ꢀ-H2),
7.50 (2H, d, J 8.5 Hz, 7-H), 7.61 (2H, d, J 8.5 Hz, 6-H), 7.78
(2H, s, 4-H), 8.12 (4H, d, J 8.5 Hz, 2ꢀ-H and 6ꢀ-H); dC (125 MHz,
DMSO-d6) 24.0, 29.1, 53.1, 54.4, 67.0, 115.7, 122.4, 123.5, 128.9,
136.4, 152.9, 160.9; m/z (ES) 641.6 (M, 17), 321.6 (100).
General procedure for the preparation of 2,2-bis[4ꢀ-(3ꢀꢀ-
piperidinylpropoxy)phenyl]-5,5-bi-1H-benzimidazole 6a and
2,2-bis[4ꢀ-(3ꢀꢀ-N-methylpiperazinylpropoxy)phenyl]-5,5-bi-1H-
benzimidazole 6b
A solution of 3,3ꢀ-diaminobenzidine tetrahydrochloride dehydrate
(1 equivalent), in N,N-dimethylformamide (29 mL) was treated
with sodium hydroxide (3 equivalents) in water (1 mL), followed
by a solution of 4-bromopropoxybenzaldehyde (2 equivalents) in
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DMF, and Oxoneꢀ (1.3 equivalents). The reaction mixture was left
to stir for 18 hours, then excess methyl piperazine or piperidine
was added. The mixture was left to stir for an additional hour. A
solution of potassium carbonate in water (1 M, 1.5 mL/100 mg
starting tetraamine) was added, followed by water (30 mL/100 mg
starting tetraamine). The solid that precipitated was filtered under
vacuum, and washed with water. It was then left to dry in air.
+
+
=
and C-6), 164.5 (C-4), 191.2 (C O); m/z (CI ) 234.1489 (MH ,
Bis-benzimidazole derivative 6a. The reaction was carried
C14H20NO2 requires 234.1494), 234 (100%), 112 (12), 84 (43).
out following the procedure described above with tetraamine
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4-(3ꢀ-Bromo-1ꢀꢀ-propoxy)benzaldehyde 4. A solution of bro-
mopropanol (500 mg, 3.6 mmol), triphenylphosphine (1.575 g,
5.4 mmol) and 4-hydroxybenzaldehyde (440 mg, 3.6 mmol) in
dry tetrahydrofuran (15 mL) at 0 ◦C was treated with diethyl
azodicarboxylate (DEAD) (0.62 mL, 5.4 mmol), dropwise. The
reaction mixture was left to stir and warm to room temperature for
3 (394 mg), aldehyde 4 (480 mg), Oxoneꢀ (782 mg), sodium
hydroxide (163 mg), DMF (29 mL), water (1 mL), and piperidine
(2.5 mL). The title compound◦was obtained as a tan-coloured solid
(541 mg, 82%). Mp 164–166 C (decomp.); mmax (KBr disc)/cm−1
3400, 3152, 2854, 2818, 1611, 1492, 1180, 1038 and 803; dH
(500 MHz, DMSO-d6) 1.37 (4H, m, 4ꢀꢀ-H2), 1.49 (8H, m, 3ꢀꢀ-H2
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and 5ꢀꢀ-H2), 1.89 (4H, J 7 Hz, CH2CH2CH2), 2.32 (8H, m, 2ꢀꢀ-
H2 and 6ꢀꢀ-H2), 2.38 (4H, t, J 7 Hz, CH2N), 4.07 (4H, t, J 6.5 Hz,
CH2O), 7.12 (4H, d, J 8.5 Hz, 3ꢀ-H and 5ꢀ-H), 7.53–7.91 (6H, m, 4ꢀ-
H, 6ꢀ-H, 7ꢀ-H), 8.14 (4H, d, J 8.5 Hz, 2ꢀ-H and 6ꢀ-H); dC (75 MHz,
DMSO-d6) 24.5, 25.9, 26.6, 54.3, 55.4, 67.0, 115.1, 122.9, 128.4,
136.1, 145, 152, 160.4; m/z (ES) 669.5 (M+, 100%).
carbonate. The title compou◦nd was obtained as a brown solid
(179 mg, 84%). Mp 158–160 C (decomp.); mmax (KBr disc)/cm−1
3400, 3109, 1608, 1505, 1250 and 840; dH (300 MHz, DMSO-d6)
7.45 (4H, m, 3ꢀ-H and 5ꢀ-H), 7.62 (2H, d, J 8 Hz, 6-H), 7.71 (2H,
d, J 8 Hz, 7-H), 7.88 (2H, s, 4-H), 8.29 (4H, m, 2ꢀ-H and 6ꢀ-H); dC
(125 MHz, DMSO-d6) 115.8, 116.8, 117.2, 123.2, 126.92, 129.8,
129.9, 136.9, 137.7, 148.9, 151.6; 162; m/z (ES) 422.9 (M+, 90%),
318 (100), 172 (41).
Bis-benzimidazole derivative 6b. The reaction was carried
out following the procedure described above with tetraamine 3
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(448 mg), aldehyde 4 (540 mg), Oxoneꢀ (890 mg), sodium hydrox-
4ꢀ,4ꢀ-(5,5-Bi-1H-benzimidazol)-2,2-diylbis(phenol) 16. The re-
action was carried out as described above using 3,3ꢀ-
diaminobenzidine tetrahydrochloride dehydrate (400 mg), 4-
hydroxybenzaldehyde (246 mg), DMF (29 mL), water (1 mL),
ide (180 mg), DMF (29 mL), water (1 mL), and methylpiperazine
(3 mL). The title compound was obtained as a tan-coloured solid
◦
(701 mg, 90%). Mp 158–160 C (decomp.); mmax (KBr disc)/cm−1
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sodium hydroxide (160 mg) and Oxoneꢀ (800 mg). The product
3400, 2939, 2807, 1610, 1490, 1177, 1048, 837 and 808; dH
(300 MHz, DMSO-d6) 1.90 (4H, m, 2ꢀꢀ-H2), 2.19 (6H, s, CH3N),
2.23–2.41 (20H, m, 3ꢀꢀ-H2 and CH2N), 4.08 (4H, t, J 6 Hz, 1ꢀꢀ-
H2), 7.11 (4H, d, J 8.5 Hz, 3ꢀ-H2 and 5ꢀ-H2), 7.52 (2H, d, J 8 Hz,
6-H), 7.64 (2H, d, J 8 Hz, 7-H), 7.80 (2H, s, 4-H), 8.13 (4H, d,
J 8.5 Hz, 2ꢀH and 6ꢀ-H); dC (125 MHz, DMSO-d6) 26.5 (2C, 2ꢀꢀ-C),
31.1 (10C, 3ꢀꢀ-C and CH2N), 54.8 (2C, 1ꢀꢀ-C), 115.2 (4C, C-3ꢀ and
C-5ꢀ), 122.9 (2-C, C-6), 128.4 (2C, C-2ꢀ and C-6ꢀ), 135.0 (4C, C-3a
and C-7a), 152.3 (2C, C-2), 160.4 (2C, C-4ꢀ); m/z (ES) 699.4 (M+,
28%), 680 (100), 617 (52).
was precipitated in water without addition of the solution of
potassium carbonate. The bis-benzimidazole 16 was obtained as
a tan-coloured solid (238 mg, 57%). (Spectroscopic data identical
to that previously described.1)
4-[N-(tert-Butoxycarbonyl)amino]benzyl alcohol 10. (As syn-
thesised in reference 12.)
4-[N-(tert-Butoxycarbonyl)amino]benzylaldehyde. A solution
of alcohol 10 (200 mg, 0.95 mmol) in dry dichloromethane (5 mL)
was treated with manganese dioxide (165 mg, 1.9 mmol). The
reaction mixture was regularly monitored by TLC. Once no more
progress of the reaction was observed, the mixture was filtered over
General procedure for the preparation of bis-benzimidazoles 14, 15
and 16
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Celiteꢀ to remove the residues of oxidising agent and the resulting
A solution of 3,3ꢀ-diaminobenzidine tetrahydrochloride dehy-
drate (1 equivalent) in DMF (20 mL) was treated with sodium
filtrate was treated with an additional portion of manganese
dioxide. After 2 h, TLC revealed that the reaction had gone to
R
hydroxide (3–4 equivalents) in water (1 mL). Oxoneꢀ (1.3
R
completion. The reaction mixture was filtered through Celiteꢀ
equivalents) was then added to the resulting red solution,
followed by 4-nitrobenzaldehyde, 4-fluorobenzaldehyde or 4-
hydroxybenzaldehyde (2 equivalents) in solution in DMF (10 mL),
using a syringe pump, over 3 hours. The reaction mixture was
left to stir for 18 hours after the end of the addition. It was
then treated with a solution of potassium carbonate in water
(1 M, 1.5 mL/100 mg starting tetraamine), and poured in water
(60 mL/200 mg starting tetraamine). The resulting suspension was
filtered, washed with water and diethyl ether, and left to dry in air
on the filter paper. It was subsequently collected and dried in vacuo.
and the filtrate was concentrated in vacuo. The title compound
was obtained as a white crystalline solid (152 mg, 72%). Mp
◦
◦
130–132 C (from ethyl acetate–hexane) (lit.8 138 C); mmax (KBr
disc)/cm−1 3253, 1734, 1674, 1150 and 836; dH (300 MHz, CDCl3,
Me4Si) 1.57 (9H, s, (CH3)3), 7.56 (2H, d, J 8.5 Hz, 2-H and 6-H),
7.86 (2H, d, J 8.5 Hz, 3-H and 5-H), 9.92 (1H, s, CHO); m/z (CI)
222 (MH+, 62%), 166 (100), 122 (9).
2,2-Bis(4ꢀ -[N -(tert-butoxycarbonyl)amino]phenyl)-5,5-bi-1H-
benzimidazole 11. A solution of 3,3ꢀdiaminobenzidine tetrahy-
drochloride dehydrate (188 mg, 0.475 mmol) in DMF (15 mL)
was treated with a solution of sodium hydroxide (75 mg) in water
(1 mL), followed by a solution of BOC-protected aminobenzalde-
2,2-Bis(4ꢀ-nitrophenyl)-5,5-bi-1H-benzimidazole 14. The re-
action was carried out as described above using 3,3ꢀ-
diaminobenzidine tetrahydrochloride dehydrate (200 mg), 4-
nitrobenzaldehyde (152 mg), DMF (29 mL), water (1 mL), sodium
R
hyde (200 mg, 0.95 mmol) in DMF (14 mL), and Oxoneꢀ (376 mg,
R
hydroxide (80 mg) and Oxoneꢀ (400 mg). The product was
0.61 mmol). A solution of potassium carbonate in water (1 M,
2 mL) was added, followed by water (30 mL). The solid that
precipitated was filtered off under vacuum, and washed with water.
It was then left to dry in air. The title compound was obtained as
a yellowish solid (246 mg, 84%). Mp 252–254 ◦C (decomp.); mmax
(KBr disc)/cm−1 3421, 2977, 1697, 1614 and 1158; dH (500 MHz,
DMSO-d6) 1.52 (18H, s, (CH3)3), 7.52–7.96 (6H, m, 4-H, 6-H, 7-
H), 7.65 (4H, d, J 8.5 Hz, 3ꢀ-H and 5ꢀ-H), 8.10 (4H, d, J 8.5 Hz,
2ꢀ-H and 6ꢀ-H); dC (125 MHz, DMSO-d6) 28.9, 80.3, 112.1, 118.9,
127.9, 142.0, 153.4, 257.4; m/z (ES) 617.5 (M+, 100%), 309.4 (94).
precipitated in water after addition of a solution of potassium
carbonate. The bis-benzimidazole 14 was obtained as a brown
solid (200 mg, 83%). Mp >300 ◦C; mmax (KBr disc)/cm−1 3400,
1603, 1515, 1346 and 856; dH (500 MHz, DMSO-d6) 7.69 (2H, d,
J 8.5 Hz, 6-H), 7.81 (2H, d, J 8.5 Hz, 7-H), 7.98 (2H, s, 4-H), 8.49
(4H, d, J 8.5 Hz, 3ꢀ-H and 5ꢀ-H), 8.52 (4H, d, J 8.5 Hz, 2ꢀ-H and
6ꢀ-H); dC (125 MHz, DMSO-d6) 121.7, 123.3, 126.3, 135.2, 135.3,
146.6, 148.9; m/z (ES) 477.0 (M+, 25%), 223 (100).
2,2-Bis(4ꢀ-fluorophenyl)-5,5-bi-1H-benzimidazole 15. The re-
action was carried out as described above using 3,3ꢀ-
diaminobenzidine tetrahydrochloride dehydrate (200 mg), 4-
fluorobenzaldehyde (125 mg), DMF (29 mL), water (1 mL),
4ꢀ,4ꢀ-(5,5-Bi-1H-benzimidazol)-2,2-diylbis(aniline)
12. The
solid, 11, was then treated with trifluoroacetic acid (5 mL) and
left to stir for 20 minutes. The acid was evaporated at room
temperature and the resulting solid was triturated in a solution
of potassium carbonate (1 M, 10 mL). The resulting dark brown
R
sodium hydroxide (80 mg) and Oxoneꢀ (400 mg). The product
was precipitated in water after addition of a solution of potassium
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solid was filtered off and dried under high vacuum (180 mg, 91%).
Mp 242–244 ◦C (decomp.); mmax (KBr disc)/cm−1 3380, 1610, 1489
and 804; dH (500 MHz, DMSO-d6) 5.62 (4H, s, NH2), 6.67 (4H,
d, J 8.5 Hz, 3ꢀ-H2 and 5ꢀ-H2), 7.44 (2H, dd, J 8, 1.5 Hz, 6-H),
7.55 (2H, d, J 8 Hz, 7-H), 7.87 (4H, d, J 8.5 Hz, 2ꢀ-H and 6ꢀH);
dC (125 MHz, DMSO-d6) 114.5, 118.2, 121.9, 128.7, 136.1, 151.5,
154.1; m/z (ES) 417.04 (M+, 100%).
added. A precipitate formed and was filtered off under reduced
pressure. The desired bis-benzimidazole was obtained as a light
brown powder (138 mg, 82%). Mp > 250 ◦C mmax (KBr disc)/cm−1
3460, 1675, 1602, 841, 738; dH (300 MHz, DMSO-d6) 2.90 (4H,
t, J 7 Hz, 3ꢀꢀ-H2), 3.92 (4H, t, J 7 Hz, 2ꢀꢀ-H2), 7.54–7.86 (6H, m,
4-H, 6-H, 7-H), 7.81 (4H, d, J 8.5 Hz, 3ꢀ-H and 5ꢀ-H), 8.16 (4H,
d, J 8.5 Hz, 2ꢀ-H and 6ꢀ-H); dC (75 MHz, DMSO-d6) 57.2, 113.5,
119.5, 119.8, 124.3, 125.5, 127.5, 128.2, 132.1, 136.5, 145.7, 149.9,
163.8, 168.7; m/z (ES) 600.1/597.1 (M+, 5/19%), 563.4/562.1
(25/100), 527 (48).
Coupling of bis-benzimidazole 12 and bis-benzimidazole 16 with
chlorambucil. Oxalyl chloride (50 lL, 0.58 mmol) was added
dropwise to dry DMF (4 mL) at −20 ◦C under argon. A solution
of chlorambucil (73 mg, 0.24 mmol) in dry DMF 90.5 mL) was
added to the resulting slurry and the reaction mixture was left
to warm to room temperature. After two hours, the solution was
cooled to −20 ◦C and a solution of bis-benzimidazole 12 or 16
(50 mg, 0.12 mmol) in dry pyridine (0.5 mL) was added. The
reaction mixture was left to warm to room temperature, then to
stir for 48 h. The mixture was then poured into water (40 mL).
After a few seconds a dark brown solid precipitated. It was filtered
off and rinsed with water (3 × 10 mL). The dry solid was then
triturated in acetone (1 mL). The resulting suspension was filtered,
and the filtrate was concentrated under vacuum. Both adducts
were prepared on the same scale.
2,2-Bis(4ꢀ-[3ꢀꢀ-N,N-dimethylaminopropanamido]phenyl)-5,5-bi-
1H-benzimidazole 13. The reaction mixture containing bis-
benzimidazole prepared above was treated with excess
dimethylamine in solution in methanol (2 M, 2 mL) before
work up. The reaction mixture was left stirring overnight at
room temperature. Methanol was removed under vacuum, then a
solution of potassium carbonate (1 M, 10 mL) was added, followed
by water (60 mL). The precipitate that formed was filtered off and
dried under reduced pressure. The desired bis-benzimidazole was
◦
obtained as a dark brown powder (18 mg, 11%). Mp > 250 C,
mmax (KBr disc)/cm−1 3423, 1664, 1602, 1536; dH (500 MHz,
DMSO-d6) 2.15 (12H, s, N(CH3)2), 2.48 (4H, m, 3ꢀꢀ-H2), 2.55
(4H, t, J 7 Hz, 2ꢀꢀ-H2), 7.51 (2H, d, J 8.5 Hz, 6-H), 7.62 (2H,
d, J 8.5 Hz, 7-H), 7.75 (4H, d, J 8.5 Hz, 3ꢀ-H and 5ꢀ-H), 7.79
(2H, s, 4-H), 8.12 (4H, d, J 8.5 Hz, 2ꢀ-H and 6ꢀ-H); dC (75 MHz,
DMSO-d6) 35.7, 45.8, 55.9, 114.4, 119.9 (4 C), 122.5, 125.75,
127.9 (4 C), 136.5, 141.5, 152.8, 163.1, 171.4; m/z (ES) 615.3 (M+,
12%), 308.2 (100), 279.6 (30).
2,2-Bis{4ꢀ -[4ꢀꢀ -(p-N,N-(chloroethyl)aminophenyl)butanamido]-
phenyl}-5,5-bi-1H-benzimidazole 19. The bis-benzimidazole 19
was obtained as a light brown solid (53 mg, 41%). Mp 108–112 ◦C;
mmax (KBr disc)/cm−1 3400, 2957, 1666, 1613, 1518 and 804; dH
(500 MHz, DMSO-d6) 1.71 (4H, quintet, J 7 Hz, 3ꢀꢀ-H2), 2.17
(4H, t, J 7.5 Hz, 2ꢀꢀ-H2 or 4ꢀꢀ-H2), 2.45 (4H, J 7.5 Hz, 2ꢀꢀ-H2 or
4ꢀꢀ-H2), 3.66 (8H, m, NCH2CH2Cl), 5.58 (2H, s, NH), 6.64 (4H,
d, J 8.5 Hz, Ar CHCN and CHCC), 6.66 (4H, d, J 8.5 Hz, Ar
CHCC), 7.01 (4H, d, J 8.5 Hz, 3ꢀ-H and 5ꢀ-H), 7.43 (2H, dd,
J 8.5, 1.5 Hz, 6-H), 7.53 (2H, d, J 8.5 Hz, 7-H), 7.70 (2H, s, 4-H),
7.85 (4H, d, J 8.5 Hz, 2ꢀ-H and 6ꢀ-H); dC (125 MHz, DMSO-d6)
27.5, 33.9, 34.2, 42.1, 53.1, 112.8, 114.4, 118.2, 121.9, 128.7, 130.2,
Molecular modelling
The crystal structure1 of the 12-mer duplex DNA, of sequence
d(CGCGAATTCGCG), with the bis-benzimidazole compound
1 bound (PDB id number 1FTD), was used as a starting-point
for modelling. The chlorambucil groups and linkers were added
using the HYPERCHEM program,14 and conformations manually
adjusted in order to bring the mustard groups into close proximity
to base edges. The conformation of the resulting ligands and DNA
were minimised using the AMBER force-field as implemented in
HYPERCHEM.
130.6, 136.1, 145.4, 151.5, 153.9, 175.2; m/z (ES) 703.4 (M+
C14H16NOCl2, 13%), 417 (100).
−
2,2-Bis{4ꢀ-[4ꢀꢀ-(p-N,N-(chloroethyl)aminophenyl)butanoyl(oxy)]-
phenyl}-5,5-bi-1H-benzimidazole 18. The bis-benzimidazole 18
was obtained as a golden solid (48 mg, 40%). Mp 111–114 ◦C; mmax
(KBr disc)/cm−1 3401, 2929, 1612, 1179 and 803; dH (500 MHz,
MeOD) 1.89 (4H, quintet, J 7 Hz, 3ꢀꢀ-H2), 2.27 (4H, t, J 7.5 Hz,
2ꢀꢀ-H2 or 4ꢀꢀ-H2), 2.55 (4H, J 7.5 Hz, 2ꢀꢀ-H2 or 4ꢀꢀ-H2), 3.71 (8H,
m, NCH2CH2Cl), 6.69 (4H, d, J 8.5 Hz, Ar CHCN), 6.97 (4H, d,
J 8.5 Hz, Ar CHCC), 7.06 (4H, d, J 8.5 Hz, 3ꢀ-H and 5ꢀ-H), 7.58
(2H, dd, J 8.5, 1.5 Hz, 6-H), 7.65 (2H, d, J 8.5 Hz, 7-H), 7.84
(2H, s, 4-H), 7.98 (4H, d, J 8.5 Hz, 2ꢀ-H and 6ꢀ-H); dC (75 MHz,
DMSO-d6) 26.8, 33.3, 33.6, 41.5, 52.6, 112.3, 121.9, 122.9, 128.0,
129.8, 130.1, 134.9, 135.9, 151.5, 152.0, 159.4, 171.9; m/z (ES)
998.4 (21%), 990.6 (M+, 54).
Sulforhodamine B (SRB) short term cytotoxicity assay
Short term growth inhibition was measured using the SRB assay as
described previously.1,3 Briefly, cells were seeded (4000 cells/wells)
into the wells of a 96 well-plate in Dulbecco modified Eagle
medium (DMEM) and incubated overnight as before to allow the
cells to attach. Subsequently cells were exposed to freshly made
compounds at increasing concentrations of 0, 0.1, 1, 5 and 25 lM
in quadruplicate and incubated for a further 96 h. Following this
the cells were fixed with ice-cold trichloroacetic acid (TCA) (10%
w/v) for 30 min and stained with 0.4% SRB dissolved in 1%
acetic acid for 15 min. All incubations were carried out at room
temperature. The IC50 values, concentrations required to inhibit
cell growth by 50%, were determined from the mean absorbances
at 540 nm for each drug concentration expressed as a percentage
of the control untreated well absorbance.
2,2-Bis(4ꢀ -[3ꢀꢀ -chloropropanamido]phenyl)-5,5-bi-1H-benzimi-
dazole. A solution of chloropropionyl chloride (0.3 mL, 400 mg,
3.14 mmol) in dry DMF (3 mL) at −10 ◦C was treated dropwise
with a solution of bis-benzimidazole 12 (118 mg, 0.28 mmol) in
dry pyridine (2 mL). The reaction mixture was left stirring for
30 min. A solution of potassium carbonate (1 M, 5 mL) was then
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6 A. L. Satz and T. C. Bruice, Acc. Chem. Res., 2002, 35, 86.
Acknowledgements
7 J. A. Hartley, in Molecular Aspects of Anticancer Drug-DNA Interac-
tions, ed. S. Neidle and M. J. Waring, Macmillan Press, London, 1993,
vol 1, p. 1.
C. Le Sann acknowledges support from the EU Framework 6
Programme: Molecules and Cancer.
8 Y.-D. Wang, J. Dziegielewski, N. R. Wurtz, B. Dziegielewska, P. B.
Dervan and T. A. Beerman, Nucleic Acids Res., 2003, 31, 1208–1215.
9 Y. Ueda, J. M. Chuang, L. B. Crast and R. A. Partyka, J. Antibiot.,
1989, 42, 1379.
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