First synthesis of N-[(aziridin-2-yl)methyl]benzimidazolequinone and analysis of toxicity towards normal and Fanconi anemia cells

Article abstract of DOI:10.1039/b814706j

A diazole is N-substituted with 1-trityl-2-methylaziridine and demethylated and oxidised with NBS under acidic conditions to give a benzimidazolequinone; this novel anti-tumour agent is marginally more cytotoxic than mitomycin C (MMC) towards the normal h

Full text of DOI:10.1039/b814706j

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First synthesis of N-[(aziridin-2-yl)methyl]benzimidazolequinone and
analysis of toxicity towards normal and Fanconi anemia cellsw
Liz O’Donovan,a Michael P. Carty*b and Fawaz Aldabbagh*a
Received (in Cambridge, UK) 22nd August 2008, Accepted 3rd September 2008
First published as an Advance Article on the web 29th September 2008
DOI: 10.1039/b814706j
A diazole is N-substituted with 1-trityl-2-methylaziridine and de-
methylated and oxidised with NBS under acidic conditions to give a
benzimidazolequinone; this novel anti-tumour agent is marginally
more cytotoxic than mitomycin C (MMC) towards the normal
human fibroblast cell line GM00637, while the MMC-hypersensi-
tive human Fanconi anaemia (FA) cell line, PD20i, lacking the
FANCD2 protein, is also hypersensitive to the benzimidazole-
quinone, with expression of FANCD2 protein decreasing sensitivity
to both MMC and the benzimidazolequinone.
lines were found to be more sensitive than mouse cancer cell
lines to benzimidazolequinones with aziridine substituted
directly onto the quinone.6 Other cytotoxic benzimidazolequi-
nones without aziridine exist,7–11 including [1,2-a] alicyclic ring
fused benzimidazolequinones containing an additional fused
cyclopropane ring 1.9–11 It is speculated that, upon single
electron reductive activation, ring-opening of the cyclo-
propane produces a highly reactive cyclopropyl radical capable
of hydrogen abstraction from DNA leading to strand clea-
vage.2,10,12 However, benzimidazoles and the corresponding
benzimidazolequinones N-substituted with 1-trityl-2-methyl-
aziridines (in order to impart DNA-alkylating ability) are an
unknown class of compounds. We now report the synthesis of
N-[(1-tritylaziridin-(2S)-yl)methyl]-1H-benzimidazole-4,7-
dione 2 and compare its cytotoxicity with the clinically used
drug MMC. Cells from Fanconi anemia (FA) patients are
known to be hypersensitive to killing by DNA-crosslinking
agents such as MMC.13 We therefore compared the cytotoxic
effects of MMC and 2 towards FA cell line, PD20i, lacking
FANCD2 protein,14 as well investigating the effect of
FANCD2 protein expression on the cellular sensitivity
towards MMC and 2.
Many bioreductive antitumour agents utilise a quinone for
reductive activation and a strained aziridine ring as a DNA-
alkylating center.1,2 This includes mitomycin C (MMC, Fig. 1)
the naturally occurring prototype bioreductive antitumour
agent.1–3 Cytotoxicity is known to be initiated by enzymatic
reduction giving rise to electrophilic sites at C1 and C10 due to
respective aziridine ring-opening and carbamate elimination.
The C1 DNA-alkylation always precedes reaction at C10,
resulting in inter- and intrastrand crosslinks that prevent
DNA replication.4
Skibo and co-workers introduced pyrrolo[1,2-a]benzimida-
zoles (PBI) as bioreductive antitumour agents, and an alter-
native hydrolytic strand cleavage mechanism was reported via
nucleophilic attack by the DNA phosphate to open the
aziridine of PBI.5 In contrast to MMC, human cancer cell
The aziridine fragment originated from commercial
(S)-serine methyl ester hydrochloride, which was used to
prepare (2S)-1-tritylaziridine-2-methanol 3 using three litera-
ture synthetic steps15–18 in an overall B65% yield (Scheme 1).
The reaction of the alcohol 3 with triethylamine and methane-
sulfonyl chloride gave the novel mesylate 4 in 77% yield.
Treatment of 4,7-dimethoxybenzimidazole19 with sodium
hydride followed by 4 in DMF gave 4,7-dimethoxy-N-[(1-trity-
laziridin-(2S)-yl)methyl]-1H-benzimidazole 5 in 80% yield
(Scheme 2). We believe that this is the first time that N-diazole
nucleophiles have been used to substitute at the methyl carbon
of 2-methyl aziridines.
As perhaps expected, conversion of the 4,7-dimethoxy substi-
tuents of 5 into the target quinone 2, while maintaining the
integrity of the fragile aziridine moiety proved challenging
Fig. 1
a School of Chemistry, National University of Ireland, Galway,
Ireland. E-mail: Fawaz.Aldabbagh@nuigalway.ie;
Tel: +353-91-493120
b Department of Biochemistry, School of Natural Sciences, National
University of Ireland, Galway, Ireland.
. E-mail: Michael.Carty@nuigalway.ie
w Electronic supplementary information (ESI) available. Synthetic
and biological procedures, and NMR spectra for compounds 2, and
4–10. See DOI: 10.1039/b814706j
Scheme 1 (i) TrCl, Et3N, CH2Cl2, 0 1C,15 (ii) SO2Cl2, Et3N, Tol,
À50 1C,16 (iii) LiAlH4, THF, À20 1C,17 or DIBAL, Tol, À78 1C.18
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Scheme 2
initially. Hydrobromic acid induced demethylation of 5 followed
by room temperature oxidation with ferric chloride of the in situ
formed hydroquinone, as successfully utilised in our [1,2-a]
alicyclic ring fused benzimidazolequinone syntheses (including
in the synthesis of 1)9,10 resulted in the opening of the aziridine.
The aziridine of 5 equally did not survive oxidative-demethylation
attempts using cerium(IV) ammonium nitrate (CAN)7 or hyper-
valent iodine(III) reagents.20 This led us to the procedure reported
by Chi and co-workers21 for the facile room temperature conver-
sion of fused 1,4-dimethoxybenzenes and 5,8-dimethoxy-2-
methylquinoline to the respective quinones. To our delight, this
procedure of N-bromosuccinimide (NBS, 1.1 equiv.) and a
catalytic amount of H2SO4 in aqueous THF resulted in the
isolation of 2 in 56% yield with minor amounts of 5-bromo
and 6-bromo-N-[(1-tritylaziridin-(2S)-yl)methyl]-1H-benzimida-
zole-4,7-diones (6 and 7, respectively)22 separated after column
chromatography (Scheme 2). Less than full equivalents of
NBS per OMe of 5 are required for oxidative-demethylation,21
as the electrophilic bromide supplied by NBS is required
to substitute at only one OMe position in order to facilitate
hydrolytic-demethylation.
Fig. 2 Viability of normal human skin fibroblast cells (GM00637)
determined using the MTT assay following treatment with MMC (’)
and benzimidazolequinone 2 (&) for 24 h at 371C (logarithmic scale).
Each data point is the mean of at least three independent experiments.
IC50, the drug concentration required to reduce viability to 50%, was
calculated by drawing lines of best fit on linear scale plots.
the 5-H of 9 at 7.10 ppm. The 5-H of 9 would be expected to be
relatively downfield due to it being in closer proximity to the
more electronegative pyridine-like N-3 of imidazole compared
to the 6-H of 8.11,23
Purified 5-bromo isomer 8 was then readily oxidised to benzi-
midazolequinone 6 using NBS under acidic conditions (Scheme 3).
This indicated that quinone formation occurred prior to
bromination in Scheme 2, seemingly due to the benzimidazo-
lequinone 2 and 4,7-dimethoxy precursor 5 being predomi-
nantly brominated at different positions by NBS.
The cytotoxicity of synthetic target 2 was first measured using
the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
In order to unambiguously confirm the location of the
bromine substituent in benzimidazolequinones 6 and 7 (due
to very similar spectroscopic data of these isomers22), we
treated 5 with an excess of NBS (5 equiv.) in aqueous THF
in the absence of H2SO4 in order to favour the bromination
reaction.21 This gave 5-bromo-4,7-dimethoxy-N-[(1-trityl-
aziridin-(2S)-yl)methyl]-1H-benzimidazole 8 in 60% yield, with
trace amounts of the 6-bromo isomer 9 and 5,6-dibrominated
benzimidazole 10 isolated in 30% yield (Scheme 3). Isomers 8
and 9 were principally distinguished by 1H NMR spectroscopy
using the upfield shift of the 6-H of 8 at 6.72 ppm relative to
Fig. 3 Viability of PD20i (K, J) and PD20:RV (m, n) cells
determined using the MTT assay to measure the effect of FANCD2
expression on cell viability following treatment with MMC (closed
symbols) and benzimidazolequinone 2 (open symbols) for 24 h at
371C. For comparison, the viability of human skin fibroblast
(GM00637) (’, & from Fig. 2) is also shown. Each data point is
the mean of at least three independent experiments.
Scheme 3
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bromide) assay10,11,24 following treatment of the normal human
skin fibroblast cell line, GM00637, with 2 and MMC in parallel.
MMC acts as a positive control for cytotoxicity in the MTT
assay. Benzimidazolequinone 2 was found to be marginally more
cytotoxic than MMC, with IC50 = 0.5 mM for 2 and 0.8 mM for
MMC (Fig. 2).
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FA is a rare human genetic disease, characterised by an
increased incidence of cancer in early adulthood. FA cells are
characterised by a hypersensitivity to agents that induce cross-
links in DNA, in particular MMC.13 FA cells are mutant in
one of a number of genes encoding proteins in the FANC
multiprotein complex, which plays a key role in processing
MMC-induced DNA damage.13 The human FA fibroblast cell
line (PD20i) that lacks the FANCD2 protein and, as a control,
an isogenic cell line (PD20:RV) expressing wild-type
FANCD2 protein from an inserted transgene14 were treated
with benzimidazolequinone 2 and MMC in parallel (Fig. 3).
MMC and 2 exhibited cytotoxicity in the nanomolar range
(10À9 M) towards the FA (PD20i) cell line, which lacks the
FANCD2 protein. PD20:RV cells, which express FANCD2,
were found to be less sensitive to both MMC and 2 (Fig. 3).
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cells are also more sensitive to 2, and that expression of wild-
type FANCD2 protein partially corrects the cellular sensitivity
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The authors thank the Irish Research Council for Science,
Engineering and Technology: funded by the National Develop-
ment Plan for an Embark Scholar Award for Liz O’Donovan.
This publication emanated from research conducted with
financial support from Science Foundation Ireland (07/RFP/
CHEF227) and the Health Research Board.
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22 Isomers may be tentatively assigned using NMR spectroscopy:
5 - bromo-N-[(1-tritylaziridin-(2S)-yl)methyl]-1H-benzimidazole- 4,
7-dione 6: 1H NMR: d 7.10, Ar-6-H; 13C NMR: d 137.1, Ar-6-CH;
6-bromo-N-[(1-tritylaziridin-(2S)-yl)methyl]-1H-benzimidazole-4,7-
dione 7: 1H NMR: d 7.16–7.26 (within), Ar-5-H; 13C NMR: d 137.7,
Ar-5-CH.
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5594 | Chem. Commun., 2008, 5592–5594