First synthesis of an aziridinyl fused pyrrolo[1,2-a]benzimidazole and toxicity evaluation towards normal and breast cancer cell lines

Article abstract of DOI:10.1039/c1ob05694h

Anionic aromatic ipso-substitution has allowed an aziridine ring to be fused onto pyrrolo[1,2-a]benzimidazole. This diazole analogue of aziridinomitosene, and N-[(aziridinyl)methyl]-1H-benzimidazole are shown to be significantly more cytotoxic towards the

Full text of DOI:10.1039/c1ob05694h

View Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 6700
www.rsc.org/obc
PAPER
First synthesis of an aziridinyl fused pyrrolo[1,2-a]benzimidazole and toxicity
evaluation towards normal and breast cancer cell lines†
Sarah Bonham,a Liz O’Donovan,a Michael P. Cartyb and Fawaz Aldabbagh*a
Received 2nd May 2011, Accepted 22nd June 2011
DOI: 10.1039/c1ob05694h
Anionic aromatic ipso-substitution has allowed an aziridine ring to be fused onto
pyrrolo[1,2-a]benzimidazole. This diazole analogue of aziridinomitosene, and
N-[(aziridinyl)methyl]-1H-benzimidazole are shown to be significantly more cytotoxic towards the
human breast cancer cell lines MCF-7 and HCC1937 than towards a human normal fibroblast cell line
(GM00637). The aziridinyl fused pyrrolo[1,2-a]benzimidazole is less cytotoxic than the non-ring fused
aziridinyl analogue towards all three cell lines. The BRCA1-deficient HCC1937 cells are more sensitive
to mitomycin C (MMC) compared to GM00637 and MCF-7 cells. The evidence provided indicates that
different pathways may mediate cellular response to benzimidazole-containing aziridine compounds
compared to MMC.
Introduction
The mitomycin natural products are a family of pyrrolo[1,2-
a]indolequinones containing a fused aziridine.1 The most impor-
tant is mitomycin C (MMC, Fig. 1) used to treat solid tumours
(including breast cancer), despite its side-effects and sometimes
poor clinical efficacy.2 It is the archetypal bioreductive drug
incorporating a quinone for reductive activation and subsequent
electrophilic sites for alkylation of DNA.3 MMC induces apopto-
sis, and is reported to be more cytotoxic than other drugs, such as
docetaxel and cisplatin, towards human breast cancer cells (MCF-
7).4 Other breast cancer cell lines such as HCC1937, derived from
a primary breast cancer carcinoma from a patient with germ-line
mutation in the breast cancer susceptibility gene BRCA1,5 have
been reported to be hypersensitive to MMC and cisplatin.6 The
BRCA1 protein plays a key role in repair of DNA-strand breaks
arising as a result of intrastrand and interstrand DNA-crosslinks
induced by chemotherapeutic agents.
There have been many reported syntheses of the mit-
omycin skeleton or that of its bioactivated derivative
aziridinomitosene.7 Many related heterocyclic quinones have
been prepared, and evaluated as anti-tumour agents,8 in-
cluding alicyclic ring fused benzimidazolequinones,9–12 and
imidazobenzimidazolequinones.13,14 Some synthesized benzimi-
dazolequinones contain an aziridine ring substituent.9,12,15,16 For
diazoles the aziridinyl substituent and not the quinone is required
for hypersensitive killing of Fanconi anaemia (FA) cells deficient
Fig. 1 Aziridinyl-fused natural products and synthetic targets.
in FANCD2, with the effect on FA cell viability being similar
to that observed with MMC.12,16 The quinone functionality
decreases specificity towards cancer cells by increased toxicity
towards human normal fibroblast cells (GM00637) for aziridinyl
substituted diazoles precluding its requirement in the present
work.12
In this article we report heterocyclic system 1, a diazole analogue
of aziridinomitosene—it represents the first time an aziridine
is fused onto pyrrolo[1,2-a]benzimidazole. The synthesis uses a
novel protocol for fusion of aziridine involving an intramolecular
anionic aromatic ipso-substitution by the aziridinyl functionality
onto the benzimidazol-2-yl position. The toxicity of compound 1
is evaluated towards human normal (GM00637) and two breast
cancer cell lines; MCF-7 and HCC1937, and compared with N-
[(1-tritylaziridin-(2S)-yl)methyl]-1H-benzimidazole 2 (Fig. 1) in
order to assess the impact of ring fusion on toxicity.
aSchool of Chemistry, National University of Ireland Galway, University
Road, Galway, Ireland. E-mail: fawaz.aldabbagh@nuigalway.ie
bBiochemistry, School of Natural Sciences, National University of Ireland
Galway, University Road, Galway, Ireland
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra and HPLC chromatograms. See DOI: 10.1039/c1ob05694h
6700 | Org. Biomol. Chem., 2011, 9, 6700–6706
This journal is
The Royal Society of Chemistry 2011
©

View Online
the aziridinomitosene skeleton, and lithiation of the a-position on
pyrrole,19 and indole.21
Results and discussion
Synthesis
Selective lithiation at the benzimidazol-2-yl position was then
investigated by treatment of 9 with n-BuLi in THF at -78 ◦C with
the generated anion quenched with MeOD (Scheme 2). Selective
deuterium incorporation at the benzimidazol-2-yl position yielded
11 in 88% yield and replacing MeOD with diphenyl disulfide gave
10 in 60% yield.
The required aziridine fragment was prepared from N-trityl-
O-(tert-butyldimethylsilyl)-R-serinol 3,17 which was converted to
aldehyde 4 (Scheme 1) using a 2,2,6,6-tetramethyl-1-piperidiny-
loxy radical (TEMPO)/sodium hypochlorite oxidation.18
The less bulky organolithium, MeLi in THF at -78 ◦C was used
to initiate the cyclization from 10 by generating the aziridin-3-yl
anion (Scheme 3). This adds onto the electrophilic benzimidazol-
2-yl position followed by elimination of SPh. The latter ipso-
substitution gave tetracyclic target 1 in 75% yield with sulfide
8b (enantiomer of 8a) also isolated in 18% yield. Isolation of by-
product 8b confirmed the in situ generation of the aziridin-3-yl
anion, which was quenched by adventitious hydration. Repeated
attempts at this reaction did not improve the yield of target 1.
Organosulfur leaving groups have been previously displaced in
radical ipso-substitutions,22,23 however in this ionic case, SPh allows
deactivation of the benzimidazol-2-yl position (otherwise acidic)
during aziridine lithiation, as well as acting as an effective leaving
group for the subsequent cyclization.
Scheme 1 (i) TEMPO, NaClO, NaHCO3, NaBr, Tol, EtOAc, H2O, 0 ◦C,
92% (ii) n-Bu3SnLi, THF, -78 ◦C, diisopropyl azodicarboxylate (DIAD),
PPh3, Tol, 0 ◦C, 56% (iii) n-Bu4N+F-, THF, rt, 96%, (iv) 4-NO2C6H4SO2Cl,
Et3N, CH2Cl2, 0 ◦C, 75%.
Cytotoxicity evaluation
The formation of the new aziridine 5 used Bu3SnLi followed by
a Mitsunobu-type ring closure according to a modification of a
literature procedure.19 Our approach to aziridine 6 via TBDMS-
protected 5 circumvents the requirement for protection using
(tert-butyldimethylsilyloxy)methyl chloride, which requires prior
synthesis.20 TBDMS-protected aziridine 5 was converted to the
nosylate 6,19 and used rather than the analogous mesylate to
give reasonable yields (41%) of 1-{[(2S,3R)-3-(tributylstannyl)-1-
tritylaziridin-2-yl]methyl}-1H-benzimidazole 9 (Scheme 2). Steric
hindrance between the bulky tributylstannyl (Bu3Sn) and phenyl-
sulfanyl (PhS) groups prevented the successful coupling of 2-
phenylsulfanyl-1H-benzimidazole with 6 to directly give cycliza-
tion precursor 10, as indicated by reacting mesylate 7 lacking
the Bu3Sn group with 2-phenylsulfanyl-1H-benzimidazole to give
8a in good yield (72%). Target aziridine 2 was prepared in good
yield (83%) by coupling benzimidazole with mesylate 7. Vedejs
and co-workers have reported a Michael addition approach to
The toxicity of compounds was evaluated towards human normal
skin fibroblast cells (GM00637) and two human breast cancer
cell lines (MCF-7) and (HCC1937). Cells were treated in parallel
with MMC, which acts as a positive control in the MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) col-
orimetric assay.24 Aziridinyl-fused pyrrolo[1,2-a]benzimidazole 1
was found to be largely non-toxic at concentrations of £1 mM with
non-fused aziridinyl benzimidazole 2, and MMC showing greater
toxicity towards human normal fibroblast cells (GM00637, Fig. 2).
Aziridinyl fused compounds 1 and MMC show similar toxicity
towards the breast cancer cell line (MCF-7), while cell response
towards benzimidazole 2 is significantly greater (Fig. 3). In
contrast, the BRCA1-deficient breast cancer cell line HCC1937
showed a significantly greater response towards MMC, with an
effect on cell viability evident at doses as low as 10 nanomolar
(0.01 mM, Fig. 4). Non-ring fused aziridine 2 was 4 times more
Scheme 2 Synthesis of cyclization precursor.
The Royal Society of Chemistry 2011 Org. Biomol. Chem., 2011, 9, 6700–6706 | 6701
This journal is
©

View Online
Scheme 3 Anionic aromatic ipso-substitution onto benzimidazole.
Fig. 3 Viability of human breast cancer cell line (MCF-7) determined
Fig. 2 Viability of human normal skin fibroblast cell line (GM00637)
determined using the MTT assay following treatment with compounds 1
using the MTT assay following treatment with compounds 1 ( ), 2 (ꢀ)
᭹
and MMC (♦) under aerobic conditions for 24 h at 37 ◦C. Each data point
(
), 2 (ꢀ) and MMC (♦) under aerobic conditions for 24 h at 37 ◦C. Each
᭹
is the mean of at least three independent experiments.
data point is the mean of at least three independent experiments.
toxic than fused system 1 towards HCC1937 and 2–4 times
more toxic towards the other two cell lines evaluated (Table
1). The reduction in cytotoxicity for compound 1 compared to
analogue 2 is similar to a previously observed general increase
in cytotoxicity for 2-aromatic ring substituted benzimidazole-
4,7-diones compared to analogous alicyclic ring fused systems,
towards human normal and cancer cell lines.11 Table 1 shows the
aziridine-containing benzimidazoles 1 and 2 to be approximately
4–5 and 6–8 times, respectively, more toxic towards the breast
cancer cell lines than towards the normal cell line, which may
be of therapeutic advantage. In contrast the clinically used drug
MMC shows similar cytotoxicity towards the normal and the
breast cancer cell line, MCF-7.
Fig. 4 Viability of human breast cancer cell line (HCC1937) determined
using the MTT assay following treatment with compounds 1 ( ), 2 (ꢀ)
᭹
and MMC (♦) under aerobic conditions for 24 h at 37 ◦C. Each data point
is the mean of at least three independent experiments.
For MMC it is reported that the formation of crosslinks upon
DNA alkylation are primarily responsible for cell death.3,25 The
hypersensitivity of BRCA1-deficient cells (such as HCC1937) to-
wards MMC implicates BRCA1 in cellular response to crosslinks.6
The comparatively smaller cytotoxicity of benzimidazole contain-
ing aziridines 1 and 2 towards HCC1937 supports an alternative
mechanism underlying cellular response to these agents. Further-
more the chemical structures of 1 and 2 do not allow for the
formation of crosslinks, because there is only one position for
DNA-alkylation (at the aziridine) and the absence of a quinone
moiety indicates a lack of bioreductive activation. The latter is
however in line with reports of DNA-alkylation and reactions of
aziridinomitosene with nucleophiles in the absence of reduction.26
Table 1 Effect of aziridines on the viability of human normal skin
fibroblast cell line (GM00637) and human breast cancer cell lines (MCF-7)
and (HCC1937)
a
a
a
IC50 GM00637
IC50 MCF-7
IC50 HCC1937
Compound
[mM]
[mM]
[mM]
MMC (♦)
0.77 0.18
3.11 0.44
1.26 0.13
0.93 0.11
0.84 0.14
0.22 0.04
0.03 0.01
0.67 0.01
0.16 0.03
1 (
)
᭹
2 (ꢀ)
a IC50 represents the compound concentration required for the reduction
of the mean cell viability to 50% of the control after incubation for 24 h at
37 ◦C, as determined using the MTT assay.
6702 | Org. Biomol. Chem., 2011, 9, 6700–6706
This journal is
The Royal Society of Chemistry 2011
©

View Online
1
Future studies will examine detailed biochemical analysis of cell
response towards compounds 1 and 2, preparation of analogues,
as well as extending their toxicity evaluation to further cancer cell
lines.
by DEPT and H–13C NMR 2D spectra. Coupling constants (J)
are expressed in Hertz (Hz). High resolution mass spectra (HRMS)
for all compounds were carried out using electrospray ionization
(ESI) on a Waters LCT Premier XE spectrometer by manual peak
matching. The precision of all accurate mass measurements is
better than 5 ppm. Optical rotations were recorded on a UniPol
L1000 polarimeter. HPLC analysis was carried out using an
Agilent Technologies 1200 series instrument with a UV detector at
the specified wavelength. Absorbance was measured in the MTT
assay using a Wallac Victor 2 1420 multi-label Counter.
Conclusions
A new protocol for fusion of aziridine using anionic aromatic
ipso-substitution is described. This gave the first diazole ana-
logue of aziridinomitosene. The latter along with non-fused,
N-[(1-tritylaziridin-(2S)-yl)methyl]-1H-benzimidazole are shown
to possess 4–8 times greater toxicity towards human breast
cancer cell lines (MCF-7 and HCC1937) than towards a human
normal fibroblast (GM00637) cell line. Aziridine-ring fusion onto
diazole was found to reduce overall toxicity. The mitomycin
mechanism for cytotoxicity involving bioreductive activation with
subsequent formation of DNA-crosslinks is ruled out for the
new benzimidazole compounds because of differences in their
chemical structure. Bioreduction cannot take place since there
is no aminoquinone motif as with MMC, and the formation of
crosslinks is not possible since there is only one position for DNA-
alkylation. The inability to form DNA crosslinks is supported by
the comparatively smaller cell response of the BRCA1-mutated
cell line, HCC1937 towards the new aziridine compounds.
Synthesis of N-[(1-tritylaziridin-(2S)-yl)methyl]-1H-benzimi-
dazole (2). Mesylate 7 (0.450 g, 1.14 mmol) was added to
benzimidazole (96 mg, 0.81 mmol) and NaH (20 mg, 0.83 mmol)
in THF (5 mL), and heated at 30 ◦C for 24 h. The cooled mixture
was evaporated to dryness, and purified by dry column vacuum
chromatography with gradient elution of EtOAc and hexane.
Evaporation of the fractions containing the second component
gave the aziridine 2 (0.281 g 83%) as a clear oil; Rf 0.31 (1 : 9
ethyl acetate/hexane); [a]2D0 -58.9 (c 0.18 in CHCl3); nmax (neat,
cm-1) 2962, 1594, 1490, 1446, 1382, 1261, 1232, 1085, 1029; dH
(400 MHz, CDCl3) 1.18 (1H, d, J 6.0, aziridinyl–CHH), 1.67–1.75
(2H, m), 4.25 (1H, dd, J2 14.8, J3 5.6, NCHH), 4.59 (1H, dd, J2
14.8, J3 4.2, NCHH), 7.18–7.31 (12H, m), 7.44–7.46 (6H, m), 7.76–
7.79 (1H, m, BnIm-4-H), 7.94 (1H, s, BnIm-2-H); dC (100 MHz,
CDCl3) 26.6 (CH2), 31.9 (CH), 47.4 (NCH2), 74.2 (CPh3), 109.8
(BnIm-7-CH), 120.5 (BnIm-4-CH) 122.4, 123.0 (BnIm-5,6-CH),
127.0, 127.7, 129.4 (Ph–CH), 134.2 (C), 143.1 (BnIm-2-CH), 143.8
(C), 144.1 (C); HRMS (ESI): found MH+, 416.2109. C29H26N3
requires 416.2127.
Experimental
General
Materials. All materials were obtained from Sigma–Aldrich,
except (S)-serine methyl ester hydrochloride, which was obtained
fromTCI FineChemicals. Solvents werepurifiedanddriedprior to
use according to conventional methods. All reactions were carried
out under a nitrogen atmosphere. NaH was obtained as 60%
dispersion in oil and used without further purification. n-BuLi and
MeLi solutions were obtained as 1.6 M in hexanes and 1.4 M in
diethyl ether respectively, and titrated against diphenylacetic acid
before use. Monitoring of reactions by thin layer chromatography
(TLC) was carried out on aluminium-backed plates coated with
silica gel (Merck Kieselgel 60 F254). Column chromatography
was carried out using Merck Kieselgel silica gel 60 (particle
size 0.040–0.063 mm), and dry column vacuum chromatography
was carried out using Merck Kieselgel silica gel 60 (particle
size 0.015–0.040 mm).14,27 The two steps of the synthesis of N-
trityl-O-(tert-butyldimethylsiloxy)-R-serinol 3 from commercial
(S)-serine methyl ester hydrochloride were carried out accord-
ing to literature procedures.17,28 [(2S)-1-Tritylaziridin-2-yl]methyl
methanesulfonate 7 and 2-phenylsulfanyl-1H-benzimidazole were
prepared according to our previously reported methods.16,23 Cell
culture reagents were obtained from Sigma–Aldrich and sterile
plasticware was obtained from Sarstedt AG (Numbrecht, Ger-
many).
2-(Phenylsulfanyl)-1-{[(2S)-1-tritylaziridin-2-yl]methyl}-1H -
benzimidazole (8a). was prepared using the same procedure
as compound 2 (above), but replacing benzimidazole with 2-
phenylsulfanyl-1H-benzimidazole. Spectroscopic data were found
to be identical to enantiomer 8b (see below) with [a]2D0 -8.7.
Synthesis of N-trityl-O-(tert-butyldimethylsiloxy)-S-serinal (4).
An aqueous solution of NaClO (10.8 mL of 12% w/v,
17.48 mmol), NaHCO3 (2.030 g, 24.17 mmol) was added dropwise
to a mixture of alcohol 3 (1.370 g, 3.06 mmol), NaBr (0.33 g,
3.20 mmol), TEMPO (57 mg, 0.37 mmol), EtOAc (20 mL), toluene
(20 mL) and water (4 mL) at 0 ◦C, and stirred at room temperature
for 96 h. The aqueous layer was extracted with Et2O (2 ¥ 30 mL)
and the combined organic layers washed with KI in 10% KHSO4 (2
¥ 50 mL), 10% Na2S2O3 (2 ¥ 50 mL), brine (2 ¥ 50 mL), and dried
(MgSO4). The solution was evaporated and the residue purified by
column chromatography using silica gel as absorbent with gradient
elution of EtOAc and hexane to give the aldehyde 4 (1.250 g, 92%)
as a colourless oil; Rf 0.72 (1 : 9 ethyl acetate/hexane); [a]2D0 +32.1
(c 0.50 in CHCl3); nmax (neat, cm-1) 2950, 2929, 2856, 1732 (C O),
1573, 1522, 1491, 1447, 1462, 1377, 1330, 1253, 1205, 1176, 1109,
1031, 1005; dH (400 MHz, CDCl3) 0.14 (3H, s, CH3Si), 0.15 (3H, s,
CH3Si), 0.97 (9H, s, tBu), 3.25 (1H, dd, J2 9.6, J3 5.6, CHH), 3.51–
3.54 (1H, m, CH), 3.82 (1H, dd, J2 9.6, J3 4.0, CHH), 7.30–7.35
(9H, m), 7.59–7.61 (6H, m), 9.31 (1H, s, CHO); dC (100 MHz,
CDCl3) -5.5 ((CH3)2Si), 18.2 (C(CH3)3), 25.6 (C(CH3)3), 63.0
(CH), 64.2 (CH2), 70.8 (CPh3), 126.9, 128.0, 128.8 (Ph–CH), 146.2
(Ph-ipso-C), 205.1 (CHO). Aldehyde 4 was found to be unstable
Measurements. Melting points were determined on a Stuart
Scientific melting point apparatus SMP3. IR spectra were obtained
using a Perkin–Elmer Spectrum 1000 FT-IR spectrophotometer
with ATR accessory. NMR spectra were recorded using a JEOL
GXFT 400 MHz instrument equipped with a DEC AXP 300
computer workstation. Chemical shifts are reported relative to
Me4Si as internal standard and NMR assignments were supported
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6700–6706 | 6703
©

View Online
(could not be stored), and was used immediately in the next
step.
(BnIm-7-CH), 120.5 (BnIm-4-CH), 122.2, 122.9 (BnIm-5,6-CH),
126.8, 127.5, 129.3 (Ph–CH), 134.1 (C), 142.3 (BnIm-2-CH), 143.9
(C), 148.7 (C); HRMS (ESI): found MH+, 706.3204. C41H52N3Sn120
Synthesis of (2S,3R)-2-({[tert-butyl(dimethyl)silyl]oxy}methyl)-
3-(tributylstannyl)-1-tritylaziridine (5). n-BuLi (2.87 mL,
4.59 mmol) was added to N,N-diisopropylamine (0.43 mL,
requires 706.3183; m/z 706 (M + H+, 100%), 705 (C41H52N3Sn119
,
56%), 703 (C41H52N3Sn117, 39%), 702 (C41H52N3Sn116, 42%).
◦
3.05 mmol) in THF (3 mL) at -20 C. After 20 min, n-Bu3SnH
General procedure for the synthesis of 2-substituted benzimi-
dazoles 10 and 11. n-BuLi (0.10 mL, 0.16 mmol) was added
dropwise to benzimidazole 9 (0.100 g, 0.14 mmol) in THF (4 mL)
at -78 ◦C. The solution turned deep red, and after 15 min MeOD
or diphenyl disulfide (0.28 mmol) was added in THF (1 mL). The
solution was stirred at room temperature for 30 min, evaporated
to dryness, and the residue purified by column chromatography
with gradient elution of EtOAc and hexane. Evaporation of
fractions containing the second component gave the 2-substituted
benzimidazoles.
(0.82 mL, 3.05 mmol) was added and the yellow solution stirred
for 1 h. The solution was cooled to -78 ◦C and aldehyde 4
(0.680 g, 1.53 mmol) in THF (2 mL) added dropwise. The
resultant blue–black solution was stirred at -78 ◦C for 1 h.
Saturated NH4Cl (30 mL) was added, and the solution extracted
with Et2O (3 ¥ 40 mL), dried (Na2SO4), and evaporated to
dryness to give the amino alcohol intermediate as a yellow oil;
HRMS (ESI): found MH+, 738.3705. C40H64NO2SiSn120 requires
738.3728. This was unstable to purification and used directly
in the following Mitsunobu ring closure: DIAD (0.60 mL,
3.05 mmol) was added to the latter amino alcohol and Ph3P
(0.800 g, 3.05 mmol) in toluene (35 mL) at 0 ◦C. The solution
was stirred for 48 h at room temperature, water (30 mL) added,
extracted with CH2Cl2 (3 ¥ 30 mL), and dried (Na2SO4). The
solution was evaporated to dryness to give an orange residue,
which was purified by column chromatography using silica gel
as absorbent with gradient elution of CH2Cl2 and hexane to
give the aziridine 5 (0.620 g, 56%) as a colourless oil; Rf 0.60
(1 : 4 dichloromethane/hexane); [a]2D0 -6.1 (c 0.30 in CHCl3);
nmax (neat, cm-1) 2954, 2926, 2854, 1489, 1462, 1254, 1074, 907;
dH (400 MHz, CDCl3) 0.04 (3H, s, CH3Si), 0.06 (3H, s, CH3Si),
0.88–0.91 (18H, m, CH2CH3, and tBu), 0.93–1.03 (6H, m, CH2),
1.26–1.40 (7H, m, CH2 and aziridinyl–CHN), 1.43–1.51 (7H, m,
CH2 and aziridinyl–CHN), 3.65 (1H, dd, J2 10.8, J3 5.0, CHH),
3.82 (1H, dd, J2 10.8, J3 6.4, CHH), 7.19–7.23 (3H, m), 7.26–7.30
(6H, m), 7.53 (6H, d, J 7.2); dC (100 MHz, CDCl3) -5.2 (CH3Si),
-5.1 (CH3Si), 10.2 (CH2), 13.8 (CH3), 18.4 (C(CH3)3), 24.1 (CH),
26.0 (C(CH3)3), 27.5 (CH2), 29.4 (CH2), 38.2 (CH), 67.5 (OCH2),
75.2 (CPh3), 126.5, 127.4, 129.8 (Ph–CH), 144.6 (Ph-ipso-C);
HRMS (ESI): found MH+, 720.3625. C40H62NOSiSn120 requires
2-Deutero-1-{[(2S,3R)-3-(tributylstannyl)-1-tritylaziridin-2-
yl]methyl}-1H-benzimidazole (11). (87 mg, 88%) as a clear oil;
Rf 0.21 (1 : 4 EtOAc/hexane); nmax (neat, cm-1) 2929, 1489, 1466,
1439, 1216, 1100; dH (400 MHz, CDCl3) 0.86 (9H, t, J 7.2, CH3),
0.95–1.08 (6H, m, CH2), 1.09 (1H, d, J 6.8, aziridinyl–H), 1.23–
1.33 (6H, m, CH2), 1.42–1.50 (6H, m, CH2), 1.76 (1H, ddd, J 6.8,
6.8, 4.8, aziridinyl–H), 4.15 (1H, dd, J2 14.0, J3 4.8, NCHH), 4.26
(1H, dd, J2 14.0, J3 6.8, NCHH), 7.14–7.16 (9H, m), 7.26–7.27
(3H, m, BnIm-5,6,7-H), 7.35–7.37 (6H, m), 7.74–7.77 (1H, m,
BnIm-4-H); dC (100 MHz, CDCl3) 10.3 (CH2), 13.8 (CH3), 25.8
(CH), 27.5 (CH2), 29.3(CH2), 35.5(CH), 49.4(NCH2), 75.7 (CPh3)
109.6 (BnIm-7-CH), 120.5 (BnIm-4-CH), 122.1, 122.9 (BnIm-5,6-
CH), 126.8, 127.8, 129.3 (Ph–CH), 134.1, 143.9 (C); HRMS (ESI):
found MH+, 707.3204. C41H51DN3Sn120 requires 707.3205.
2-(Phenylsulfanyl)-1-{[(2S,3R)-3-(tributylstannyl)-1-tritylazi-
ridin-2-yl]methyl}-1H-benzimidazole (10). (68 mg, 60%) as a
clear oil; Rf 0.56 (1 : 4 EtOAc/hexane); [a]2D0 -30.0 (c 0.10 in
CHCl3); nmax (neat, cm-1) 2929, 1596, 1490, 1447, 1328, 1251,
1080, 1033, 907; dH (400 MHz, CDCl3) 0.87 (9H, t, J 7.4, CH3),
0.97–1.13 (7H, m, CH2 and aziridinyl–H), 1.25–1.35 (6H, m,
CH2), 1.45–1.53 (6H, m, CH2), 2.05 (1H, ddd, J 8.4, 8.4, 2.8,
aziridinyl–H), 4.15 (1H, dd, J2 14.4, J3 8.4, NCHH), 4.25 (1H, dd,
J2 14.4, J3 2.8, NCHH), 7.00–7.04 (9H, m), 7.06–7.10 (5H, m),
7.21–7.30 (9H, m), 7.71–7.73 (1H, m, BnIm-4-H); dC (100 MHz,
CDCl3) 10.3 (CH2), 13.8 (CH3), 25.5 (CH), 27.5 (CH2), 29.3 (CH2),
35.5 (CH), 49.8 (NCH2), 75.8 (CPh3), 110.2 (BnIm-7-CH), 120.1
(BnIm-4-CH), 122.3, 123.1 (BnIm-5,6-CH), 126.6, 127.2, 127.5,
129.4, 129.5, 129.9 (Ph–CH), 132.3, 136.3, 143.6, 143.9, 147.0 (all
C); HRMS (ESI): found MH+, 814.3229. C47H56N3SSn120 requires
814.3217; m/z 814 (M + H+, 100%), 813 (C47H56N3SSn119, 54%),
812 (C47H56N3SSn118, 66%).
720.3623; m/z 720 (M + H+, 100%), 719 (C40H62NOSiSn119
,
60%), 718 (C40H62NOSiSn118, 80%). Aziridine 5 was converted
into
[(2S,3R)-3-(tributylstannyl)-1-tritylaziridin-2-yl]methyl
4-nitrobenzenesulfonate 6 using the methods of Vedejs et al.19
Synthesis of 1-{[(2S,3R)-3-(tributylstannyl)-1-tritylaziridin-
2-yl]methyl}-1H-benzimidazole (9). Nosylate
6
(0.400 g,
0.51 mmol) was added to benzimidazole (55 mg, 0.47 mmol) and
NaH (13 mg, 0.54 mmol) in THF (5 mL), and heated at 50 ◦C for
24 h. The cooled mixture was evaporated to dryness, and purified
by dry column vacuum chromatography with gradient elution
of EtOAc and hexane. Evaporation of fractions containing the
second component gave the aziridine 9 (0.134 g, 41%) as a yellow
oil; Rf 0.28 (1 : 4 EtOAc/hexane); [a]2D0 -8.6 (c 0.50 in CHCl3); nmax
(neat, cm-1) 2927, 1597, 1492, 1447, 1216, 1032; dH (400 MHz,
CDCl3) 0.87 (9H, t, J 7.2, CH3), 0.94–1.07 (6H, m, CH2), 1.09 (1H,
d, J 6.8, aziridinyl–H), 1.24–1.33 (6H, m, CH2), 1.43–1.51 (6H, m,
CH2), 1.77 (1H, ddd, J 6.8, 6.8, 4.8, aziridinyl–H), 4.16 (1H, dd,
J2 14.0, J3 4.8, NCHH), 4.27 (1H, dd, J2 14.0, J3 6.8, NCHH),
7.14–7.16 (9H, m), 7.23–7.29 (3H, m, BnIm-5,6,7-H), 7.35–7.38
(6H, m), 7.75–7.77 (1H, m, BnIm-4-H), 7.79 (1H, s, BnIm-2-H);
dC (100 MHz, CDCl3) 10.3 (CH2), 13.8 (CH3), 25.8 (CH), 27.5
(CH2), 29.3 (CH2), 35.5 (CH), 49.4 (NCH2), 75.8 (CPh3) 109.6
Synthesis of (1aS,8aS)-1-trityl-1,1a,8,8a-tetrahydroazireno-
[2¢,3¢:3,4]pyrrolo[1,2-a]benzimidazole (1). MeLi (0.19 mL,
0.26 mmol) was added dropwise to benzimidazole 10 (60 mg,
0.074 mmol) in THF (2 mL) at -78 ◦C. The solution turned
deep red, and was brought to room temperature over 30 min.
The mixture was evaporated to dryness, and the residue purified
by column chromatography with gradient elution of EtOAc
and hexane. The following compounds were isolated in order of
elution: 2-(Phenylsulfanyl)-1-{[(2R)-1-tritylaziridin-2-yl]methyl}-
1H-benzimidazole 8b); (7 mg, 18%) as a yellow oil; Rf 0.65
(1 : 3 EtOAc/hexane); [a]2D0 +8.7 (c 0.52 in CHCl3); nmax (neat,
6704 | Org. Biomol. Chem., 2011, 9, 6700–6706
This journal is
The Royal Society of Chemistry 2011
©

View Online
cm-1) 1581, 1480, 1441, 1442, 1353, 1326, 1281, 1246, 1202,
1154, 1083, 1024; dH (400 MHz, CDCl3) 1.05 (1H, d, J 6.0,
aziridinyl–CHH), 1.60–1.65 (1H, m, aziridinyl–CH), 1.81 (1H, d,
J 2.8, aziridinyl–CHH), 4.23 (1H, dd, J2 14.8, J3 7.6, NCHH),
4.81 (1H, dd, J2 14.8, J3 4.0, NCHH), 7.20–7.26 (17H, m),
7.40–7.42 (6H, m), 7.74–7.76 (1H, m, BnIm-4-H); dC (100 MHz,
CDCl3) 28.1 (aziridinyl–CH2), 31.6 (CHN), 47.7 (NCH2), 74.2
(CPh3), 110.0 (BnIm-7-CH), 120.1 (BnIm-4-CH), 122.6, 123.5
(BnIm-5,6-CH), 126.9, 127.7, 129.4, 129.5, 130.3 (Ph–CH), 132.3,
136.1, 143.4, 144.1, 147.3 (all C); HRMS (ESI): found MH+,
524.2162. C35H30N3S requires 524.2160, and the target compound
(1) (23 mg, 75%) as a pale yellow oil; Rf 0.50 (1 : 3 EtOAc/hexane);
[a]D20 -9.3 (c 0.15 in CHCl3); nmax (neat, cm-1) 2929, 1623, 1545,
1490, 1448, 1354, 1265, 1216, 1151, 1051, 909; dH (400 MHz,
CDCl3) 2.97 (1H, d, J 4.8, 1a-H), 3.08–3.10 (1H, m, 8a-H), 4.06
(1H, dd, J2 11.2, J3 4.0, 8-HH), 4.42 (1H, d, J 11.2, 8-HH),
7.23–7.32 (12H, m), 7.47–7.49 (6H, m, Ph-H), 7.76–7.79 (1H, m,
3-H); dC (100 MHz, CDCl3) 33.3 (1a-CH), 40.2 (8a-CH), 45.2
(CH2), 73.4 (CPh3), 108.6 (6-CH), 119.5 (3-CH), 120.8, 121.6
(4,5-CH), 126.2, 127.0, 128.3 (Ph–CH), 132.0, 142.9, 146.9 (C),
157.6 (1b-C); HRMS (ESI): found MH+, 414.1971. C29H24N3
requires 414.1970.
by using GraphPad Prism software, v.5.02 (GraphPad Inc., San
Diego, CA, USA).
Acknowledgements
The
authors
thank
Science
Foundation
Ireland
(07/RFP/CHEF227) for financial support and Drs. Adrienne
Gorman and Paul Mullan (NUI Galway and QUB respectively)
for the breast cancer cell lines. We are grateful to Prof. Ian
D. Jenkins (Griffith University, Brisbane, Australia) on advice
regarding the Mitsunobu reaction.
References
1 W. A. Remers, in Anticancer Agents from Natural Products, ed., G. M.
Cragg, D. G. I. Kingston and D. J. Newman, Taylor & Francis, Boca
Raton, FL, USA, 2005, p. 475.
2 W. T. Bradner, Cancer Treat. Rev., 2001, 27, 35.
3 H. A. Seow, P. G. Penketh, R. P. Baumann and A. C. Sartorelli, Methods
Enzymol., 2004, 382, 221.
4 F. Pirnia, E. Schneider, D. C. Betticher and M. M. Borner, Cell Death
Differ., 2002, 9, 905.
5 G. E. Tomlinson, T. T.-L. Chen, V. A. Stastny, A. K. Virmani, M. A.
Spillman, V. Tonk, J. L. Blum, N. R. Schneider, I. I. Wistuba, J. W.
Shay, J. D. Minna and A. F. Gazdar, Cancer Res., 1998, 58, 3237.
6 (a) P. Tassone, P. Tagliaferri, A. Perricelli, S. Blotta, B. Quaresima, M.
L. Martelli, A. Goel, V. Barbieri, F. Costanzo, C. R. Boland and S.
Venuta, Br. J. Cancer, 2003, 22, 1285; (b) J. Yun, Q. Zhong, J.-Y. Kwak
and W.-H. Lee, Oncogene, 2005, 24, 4009; (c) M. Santarosa, L. D. Col,
E. Tonin, A. Caragnano, A. Viel and R. Maestro, Mol. Cancer Ther.,
2009, 8, 844.
7 J.-C. Andrez, Beilstein J. Org. Chem., 2009, 5No. 33.
8 (a) Y. Chen and L. Hu, Med. Res. Rev., 2009, 29, 29; (b) M. A. Colucci,
C. J. Moody and G. D. Couch, Org. Biomol. Chem., 2008, 6, 637.
9 (a) E. B. Skibo and W. G. Schulz, J. Med. Chem., 1993, 36, 3050; (b) R.
Zhou and E. B. Skibo, J. Med. Chem., 1996, 39, 4321; (c) E. B. Skibo
and C. Xing, J. Med. Chem., 2001, 44, 3545; (d) E. B. Skibo, A. Jamil,
B. Austin, D. Hansen and A. Ghodousi, Org. Biomol. Chem., 2010, 8,
1577.
Cell culture. A SV40-transformed normal human skin fibrob-
last cell line (repository number GM00637) was obtained from
the National Institute for General Medical Sciences (NIGMS)
Human Genetic Cell Repository (Coriell Institute for Medical
Research, New Jersey, USA). The MCF-7 cell line, a human
breast cancer cell line was obtained from Dr Adrienne Gorman,
Biochemistry, School of Natural Sciences, National University of
Ireland Galway. The HCC1937 breast cancer cell line was obtained
from Dr Paul Mullan, Centre for Cancer Research and Cell Bi-
ology, Queen’s University Belfast. The SV40-transformed normal
human skin fibroblast cell line (GM00637) was grown in minimum
essential media Eagle–Earle BSS (MEM) supplemented with 15%
non heat-inactivated fetal bovine serum (FBS), 1% penicillin-
streptomycin, 2mM L-glutamine, 2X essential and non-essential
amino acids and 2X vitamins. MCF-7 cells and HCC1937 cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
containing high glucose (4.5g mL-1) and supplemented with 10%
heat-inactivated FBS and 1% penicillin-streptomycin.
10 (a) M. Lynch, S. Hehir, P. Kavanagh, D. Leech, J. O’Shaughnessy, M.
P. Carty and F. Aldabbagh, Chem.–Eur. J., 2007, 13, 3218; (b) S. Hehir,
L. O’ Donovan, M. P. Carty and F. Aldabbagh, Tetrahedron, 2008, 64,
4196.
11 E. Moriarty, M. Carr, S. Bonham, M. P. Carty and F. Aldabbagh, Eur.
J. Med. Chem., 2010, 45, 3762.
12 K. Fahey, L. O’Donovan, M. Carr, M. P. Carty and F. Aldabbagh, Eur.
J. Med. Chem., 2010, 45, 1873.
13 (a) W. G. Schulz and E. B. Skibo, J. Med. Chem., 2000, 43, 629; (b) A.
Suleman and E. B. Skibo, J. Med. Chem., 2002, 45, 1211.
14 V. Fagan, S. Bonham, M. P. Carty and F. Aldabbagh, Org. Biomol.
Chem., 2010, 8, 3149.
Cytotoxicity measurements. Growth inhibition (cell viability)
was determined using the MTT colorimetric assay. Cells were
plated into 96-well plates at a density of 10 000 cells per well
for GM00637 and HCC1937 (200 mL per well) and 1,000 cells
per well for MCF-7 (200 mL per well), and allowed to adhere
over a period of 48 h. Compound 1 evaluation solutions were
applied in ethanol, MMC and compound 2 were applied in DMSO
(1% v/v final concentration in well). All cells were incubated at
37 ◦C under a humidified atmosphere containing 5% CO2 for
24 h. Control cells were exposed to an equivalent concentration
of ethanol and DMSO alone. MTT (20 mL, 5 mg ml-1 solution)
was added and the cells were incubated for another 3 h. The
supernatant was then removed carefully by pipetting. The resultant
MTT formazan crystals were dissolved in 100 mL of DMSO and
absorbance was determined on a plate reader at 550 nm with a
reference at 690 nm. Cell viability is expressed as a percentage of
the control solution value. Dose–response curves were analysed
by nonlinear regression analysis and IC50 values were estimated
15 C. M. Ahn, S. K. Kim and J. L. Han, Arch. Pharmacal Res., 1998, 21,
599.
16 L. O-Donovan, M. P. Carty and F. Aldabbagh, Chem. Commun., 2008,
5592.
17 E. Vedejs, A. Klapars, D. L. Warner and A. H. Weiss, J. Org. Chem.,
2001, 66, 7542.
18 J. Jurczak, D. Gryko, E. Kobrzycka, H. Gruza and P. Prokopowicz,
Tetrahedron, 1998, 54, 6051.
19 E. Vedejs, J. D. Little and L. M. Seaney, J. Org. Chem., 2004, 69,
1788.
20 L.-L. Gundersen, T. Benneche and K. Undheim, Acta Chem. Scand.,
1989, 43, 706.
21 (a) E. Vedejs and J. Little, J. Am. Chem. Soc., 2002, 124, 748; (b) E.
Vedejs and J. D. Little, J. Org. Chem., 2004, 69, 1794; (c) M. Kim and
E. Vedejs, J. Org. Chem., 2004, 69, 7262.
22 (a) W. B. Motherwell and A. M. K. Pennell, J. Chem. Soc., Chem.
Commun., 1991, 877; (b) S. Caddick, K. Aboutayab, K. Jenkins and R.
I. West, J. Chem. Soc., Perkin Trans. 1, 1996, 675; (c) S. Caddick, C.
L. Shering and S. N. Wadman, Tetrahedron, 2000, 56, 465; (d) W. B.
Motherwell and S. Va´zquez, Tetrahedron Lett., 2000, 41, 9667; (e) S.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 6700–6706 | 6705
©

View Online
M. Allin, W. R. Bowman, R. Karim and S. S. Rahman, Tetrahedron,
2006, 62, 4306.
23 F. Aldabbagh and W. R. Bowman, Tetrahedron, 1999, 55, 4109.
24 T. Mosmann, J. Immunol. Methods, 1983, 65, 55.
25 (a) V. N. Iyer and W. Szybalski, Science, 1964, 145, 55; (b) M. Tomasz,
R. Lipman, D. Chowdary, J. Pawlak, G. L. Verdine and K. Nakanishi,
Science, 1987, 235, 1204.
26 (a) V.-S. Li, D. Choi, M.-S. Tang and H. Kohn, J. Am. Chem. Soc.,
1996, 118, 3765; (b) E. Vedejs, B. N. Naidu, A. Klapars, D. L. Warner,
V.-S. Li, Y. Na and H. Kohn, J. Am. Chem. Soc., 2003, 125, 15796.
27 (a) L. M. Harwood, Aldrichim. Acta, 1985, 18, 25; (b) D. S. Pedersen
and C. Rosenbohm, Synthesis, 2001, 2431.
28 J.-M. Lee, H.-S. Lim and S.-K. Chung, Tetrahedron: Asymmetry, 2002,
13, 343.
6706 | Org. Biomol. Chem., 2011, 9, 6700–6706
This journal is
The Royal Society of Chemistry 2011
©