One-pot double intramolecular homolytic aromatic substitution routes to dialicyclic ring fused imidazobenzimidazolequinones and preliminary analysis of anticancer activity

Article abstract of DOI:10.1039/c003511d

Bu₃SnH/1,1′-azobis(cyclohexanecarbonitrile) (ACN)-mediated five, six, and seven-membered double alkyl radical cyclizations onto imidazo[5,4-f]benzimidazole and imidazo[4,5-f]benzimidazole are described. The quinone derivatives evaluated show selective toxicity towards human cervical (HeLa) and prostate (DU145) cancer cell lines (with negligible toxicity towards a normal human cell line, GM00637). Only the Fremy oxidation of the 6-aminoimidazo[5,4-f]benzimidazole gave iminoquinone, which showed high specificity towards the prostate cancer cell line (DU145). The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c003511d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
One-pot double intramolecular homolytic aromatic substitution routes to
dialicyclic ring fused imidazobenzimidazolequinones and preliminary analysis
of anticancer activity†
Vincent Fagan,a Sarah Bonham,a Michael P. Cartyb and Fawaz Aldabbagh*a
Received 24th February 2010, Accepted 20th April 2010
First published as an Advance Article on the web 18th May 2010
DOI: 10.1039/c003511d
Bu3SnH/1,1¢-azobis(cyclohexanecarbonitrile) (ACN)-mediated five, six, and seven-membered double
alkyl radical cyclizations onto imidazo[5,4-f ]benzimidazole and imidazo[4,5-f ]benzimidazole are
described. The quinone derivatives evaluated show selective toxicity towards human cervical (HeLa)
and prostate (DU145) cancer cell lines (with negligible toxicity towards a normal human cell line,
GM00637). Only the Fremy oxidation of the 6-aminoimidazo[5,4-f ]benzimidazole gave iminoquinone,
which showed high specificity towards the prostate cancer cell line (DU145).
Introduction
Bu3SnH/azo-initiator mediated intramolecular homolytic aro-
matic substitution or radical cyclization onto (hetero)aromatics
is now a valuable protocol in organic synthesis.1–11 The pro-
tocol has attracted mechanistic interest, since the intermediate
p-cyclohexadienyl radical (s-complex) generated upon radical
addition formally loses a hydrogen atom to give the aromatic
substitution product, so is described as an oxidation in the presence
of the “reductant” Bu3SnH.1,9 Most recent examples achieve
optimized (often high) reaction yields in the presence of excess
amounts of appropriate azo-initiator.2–4,9–13 The latter is thought
to fulfil a dual role in the non-chain reaction, providing continual
initiation, as well as taking part in the oxidative rearomatiza-
tion. We reported the use of the protocol in the synthesis of
highly potent alicyclic [1,2-a] ring fused benzimidazolequinones
via five to seven-membered radical cyclizations of nucleophilic
N-alkyl radicals onto the benzimidazole-2-position activated by
quaternizing the pyridine-like 3-N of imidazole with camphor-
sulfonic acid (CSA).10 The pyrido[1,2-a]benzimidazolequinone 1
Fig. 1 Mitomycin C and synthetic benzimidazolequinone and imida-
zobenzimidazolequinone anticancer agents.
(Fig. 1) was the most potent compound prepared being more
than 300 times more cytotoxic than the clinically used drug,
mitomycin C (MMC) towards hypoxic (low pO2) human skin
fibroblast cells (GM00637).10 Moreover, compound 1 was more
to be excellent substrates for the quinone reductase enzyme,
cytotoxic than other benzimidazolequinones containing “DNA-
NAD(P)H:quinone oxidoreductase (NQO1,18 also known as
DT-diaphorase). This article contains the first cytotoxicity eval-
uation of 4 and 5, which we have carried out using normal cells
damaging functionality”, such as aziridine 214 or cyclopropane
310,15
This has led us to the present synthesis of the novel an-
ticancer agent dipyridoimidazo[5,4-f ]benzimidazolequinone 4
and dipyridoimidazo[4,5-f ]benzimidazolequinone 5. Dialicyclic
(GM00637), and two human cancer cell lines known to express
high levels of NQO1: cervical (HeLa, CCL-2)19 and prostate
(DU145)20 cancer.
ring fused imidazo[4,5-f ]benzimidazolequinones (including 5)
have been reported by Skibo and co-workers,16,17 and shown
The required double annulations of imidazobenzimidazole
have been achieved by the first one-pot double intramolecular
radical substitution onto heteroarenes. We are aware of only one
example of this protocol with arenes, which was reported by
Harrowven and co-workers7 involving the Bu3SnH/azoinitiator
mediated six-membered cyclization of aryl radicals to give
[5 and 7]helicenes. As part of our study, we now report
Bu3SnH/1,1¢-azobis(cyclohexanecarbonitrile) (ACN)-mediated
five, six and seven-membered alkyl radical cyclizations at the
aSchool of Chemistry, National University of Ireland, Galway, Ireland. E-
mail: fawaz.aldabbagh@nuigalway.ie
bBiochemistry, School of Natural Sciences, National University of Ireland,
Galway, Ireland
† Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra, HPLC chromatograms and cytotoxicity evaluations (dose
response curves). See DOI: 10.1039/c003511d
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Table 1 One-pot double intramolecular homolytic aromatic substitutions onto imidazo[5,4-f ]benzimidazolea
Entry
Precursors
Method
Products (%)b
1
2
3
4
5
6
7
7a
7a
7a
7b
7c
7c
7c
1
2
3
1
1
2
3
9a
(32)
10a
(31)
11a
(10)
c
c
c
—
—
—
9a
9b
9c
9c
9c
(47)
(90)
(29)
(54)
(34)
10a
10b
10c
10c
10c
(30)
(0)
(32)
(13)
(31)
11a
11b
11c
11c
11c
(0)
(0)
(8)
(0)
(0)
a i. Method 1: Bu3SnH (5 equiv, 0.06 M), ACN (5 equiv.), toluene, syringe pump addition (2.2 mL h-1), reflux, air; Method 2: Method 1 & CSA (2.1 equiv.);
Method 3: Method 1 & Ac2O (2.1 equiv.); ii. Sunlight (Ireland), 15–20 ◦C or 350 nm, Rayonet reactor, rt. b Isolated yields; c Intractable mixture.
Scheme 1 Synthesis of radical precursors.
imidazobenzimidazole 2- and 6-positions. This represents the first
protocol for annulation onto imidazobenzimidazole.
equivalents of Bu3SnH are required. However optimized yields
were obtained using five equivalents of Bu3SnH and ACN (or
2.5 equivalents per phenylselenide moiety). This gave dipyrido
ring fused imidazo[5,4-f ]benzimidazole 9b in excellent yield of
90% (Table 1, entry 4, method 1).
Results and discussion
It is noteworthy, that for all cyclizations (in Tables 1 and 2), the
homolytic aromatic substitutions were more efficient if carried out
in the absence of an inert atmosphere. Attempts to exclude air or
increase the number of ACN equivalents led to the formation of
complex mixtures of non-aromatic trapped p-radical adducts.4,13
The rearomatization of the inseparable mixture is aided by the
presence of air (or O2),3,24 with additional prolonged heating of the
reaction mixture or exposure to low-energy light (incl. sunlight)
required for full conversion to aromatic products. The requirement
for excess amounts of initiator indicates a non-chain mechanism,
however some direct hydrogen atom abstraction from intermediate
p-radicals by O2 resulting in propagating cycles cannot be ruled
out.25
The more difficult five and seven-membered radical cyclizations
onto imidazo[5,4-f ]benzimidazole gave low yields of diannulated
products 9a (32%) and 9c (29%) with similar respective yields
of single cyclization products 10a (31%) and 10c (32%) iso-
lated (Table 1, entries 1 and 5). It was apparent that radical
reduction was competing effectively with cyclization, with non-
cyclized compounds 11a (10%) and 11c (8%) also obtained. Thus
The two targeted isomeric heterocyclic systems were simulta-
neously accessed from imidazo[5,4-f (4,5-f )]benzimidazole 6a–6b
(benzo-bis(imidazole)).21,22 Tautomers 6a–6b exist in a 1 : 1
ratio observable in the 1H NMR spectrum as an “appar-
ent triplet” at 7.49–7.78 ppm. Reaction with 2.1 equivalents
of NaH and w-chloroalkyl phenyl selenides gave 1,5- and
1,7-dialkylphenylselenide radical precursors 7a–7c and 8a–8c in
excellent combined yields (Scheme 1). The obtained approximate
1 : 1 mixture of dialkylated isomers 7a–7c and 8a–8c was readily
separable by chromatography. Phenylselenides have previously
been shown to be effective diazole radical precursors8,10,11,23
because of their efficient radical, but poor SN2 leaving group,
ability. Unlike previous radical precursor preparations, alkylation
of the basic heterocycle was achieved without prior conver-
sion of the w-chloroalkyl phenyl selenide to the corresponding
iodide.
The conditions for the one-pot double homolytic aromatic
substitution protocol were optimized using the six-membered
cyclizations of dibutylphenylselenide 7b. Since two cyclizing
radicals are generated per molecule of substrate, at least two
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Table 2 One-pot double intramolecular homolytic aromatic substitutions onto imidazo[4,5-f ]benzimidazole
Entry
Precursors
Methoda
Products (%)b
1
2
3
8a
8b
8c
3
1
2
12a
12b
12c
(48)
(81)
(48)
13a
13b
13c
(27)
(0)
(16)
14a
14b
14c
(0)
(0)
(0)
a Reaction conditions as in Table 1. b Isolated yields.
activation10,26,27 of imidazo[5,4-f ]benzimidazole by protonation of
the 3,7-N basic sites (making the heterocycle more electrophilic)
was deemed necessary, and CSA (2.1 equivalents) was added
(method 2). Although the acid improved the double seven-
membered nucleophilic alkyl radical cyclization to give 9c in 54%
yield (and 10c (13%) entry 6), it led to an intractable mixture for the
attempted five-membered analogue (entry 2). Replacing CSA with
acetic anhydride (2.1 equivalents, method 3) facilitated the five
membered cyclization to give improved, but modest yields of 9a
(47%) and 10a (30%) (entry 3). Nevertheless, the present one-pot
double annulation yields compare favorably with literature single
alkyl radical cyclizations onto diazoles,8,10,11,23 since a 54% and
47% yield for the double five and seven-membered cyclizations (to
give 9a and 9c) correspond to a cumulative yield for two separate
cyclizations each proceeding in about 70% yield.
The optimized reaction conditions in Table 1 were applied
to the cyclizations onto imidazo[4,5-f ]benzimidazole (Table 2).
The double six-membered cyclization proceeded in excellent yield
without activation giving 12b in 81% yield (entry 2). Acetic
anhydride and CSA quaternization of the 3,5-N basic sites of
imidazo[4,5-f ]benzimidazole allowed respective five and seven
membered radical cyclizations to occur, with reduction minimized
(entries 1 and 3).
Scheme 2 Functionalization to give the target quinone.
Table 3 Cytotoxicity evaluation: IC50 values (mM)a
The new heterocyclic quinone 4 was obtained in high over-
all yield by nitration, reduction and oxidation, as depicted in
Scheme 2. Unexpectedly, iminoquinone 17 was obtained from the
Fremy oxidation of the intermediate amine 16 carried out at pH 4
in the presence of KH2PO4, with 17 easily hydrolyzed to 4 with
hydrochloric acid. The oxidation conditions were analogous to
those used by Suleman and Skibo,17 which we repeated to give
imidazo[4,5-f ]benzimidazolequinone 5 in high yield (69% from
12b). In agreement with the latter report no iminoquinone was
observed as part of the synthesis of quinone 5.
Cell lines
Compound
GM00637
HeLa CCL-2
DU145
MMC
0.46 0.09
> 5
> 5
0.27 0.16
1.67 0.05
3.33 0.47
1.55 0.35
0.17 0.02
1.99 0.39
1.73 0.23
0.30 0.01
4
5
17
3.63 0.52
a IC50 represents the compound concentration required for the reduction
of the mean cell viability to 50% of the control value after incubation for
72 h at 37 ◦C, as determined using the MTT assay.
The cytotoxicity of 4, 5 and 17 was evaluated against two
cancer cell lines known to contain high NQO1 activity: cervical
(HeLa, CCL-2)19 and prostate (DU145)20 cancer. For comparison
purposes cytotoxicity against a normal human fibroblast cell
line (GM00637)10,14 was also carried out. Each cell line was
treated with solutions of up to 5 mM of 4, 5, 17 and MMC
in parallel for 72 h. Cell viability was determined using the
well-known MTT colorimetric assay.28 Both 4 and 5 showed
negligible toxicity towards the normal cell line in this dose range
(Table 3). This may be of therapeutic advantage, as it represents
an improvement on earlier cytotoxicity evaluation of pyrido[1,2-
a]benzimidazolequinone 1,10 that deemed the compound to be
too toxic towards normal cells for further investigation in cancer
cell lines. Moreover, quinone 4 is found to be selectively toxic (in
comparison to normal cells) towards the two cancer cell lines, with
5 more toxic towards prostate than cervical cancer. Iminoquinone
17 showed a high specificity towards prostate cancer, being up
to twelve times more toxic towards this cell line than to normal
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cells. The elevated toxicity of 17 may indicate different cytotoxicity
pathways in comparison to quinones 4 and 5. In contrast, sub-
micromolar concentrations of MMC were found to be toxic
towards all three cell lines, with marginal selectivity towards the
two cancer cell lines.
give an approximate 1 : 1 mixture of the two tautomers of the title
compo◦und 6a–6b (0.542◦g, 98%) isolated as a pale brown solid; mp
>350 C (lit.,21 > 300 C); dH (400 MHz; DMSO-d6) 7.49 (1H,
bs, 8-H, 6b), 7.65 (2H, bs, 4,8-H, 6a), 7.78 (1H, bs, 4-H, 6b), 8.14
(4H, s, 2,6-H), 12.17 (4H, bs, N-H).
General procedure for the synthesis of radical precursors
Conclusions
1,5(7)-Di(x-(phenylselano)alkyl)imidazo[5,4-f (4,5-f )]benzimi-
dazoles. Imidazo[5,4-f (4,5-f )]benzimidazole 6a–6b (0.790 g,
5.0 mmol) and sodium hydride (0.252 g, 10.5 mmol) in
DMF (50 mL) were heated at 120 ◦C for 10 min. w-
Chloroalkyl phenyl selenide (10.5 mmol) was added, and the
mixture stirred for 1 h. The cooled mixture was evaporated,
dichloromethane added, and the insoluble solid removed by
filtration. The filtrate was evaporated, and the residue purified
by dry column vacuum chromatography with gradient elu-
tion of ethyl acetate and methanol yielding the separated 1,5-
di(w-(phenylselano)alkyl)imidazo[5,4-f ]benzimidazole 7a–7c and
1,7-di(w-(phenylselano)alkyl)imidazo[4,5-f ]benzimidazole 8a–8c
isomers.
The first one-pot double intramolecular homolytic aromatic sub-
stitution onto heterocycles is reported, as represented by the annu-
lation of imidazobenzimidazoles. Excellent yields are obtained for
the Bu3SnH/azo-initiator mediated double six-membered radical
cyclizations onto imidazo[5,4-f ]benzimidazole and imidazo[4,5-
f ]benzimidazole, while quaternization of the basic nitrogen atoms
is required for five and seven-membered analogous alkyl radical
cyclizations to proceed in reasonable yields. The derived annulated
imidazobenzimidazolequinones show specificity towards cancer
cell lines known to express elevated levels of NQO1, with the
evaluated iminoquinone intermediate showing very high toxicity
and specificity towards the prostate cancer cell line DU145. More
detailed anticancer activity screening is currently being carried
out, and the biochemical mechanisms for cytotoxicity investigated.
1,5-Di(3-(phenylselano)propyl)imidazo[5,4-f ]benzimidazole (7a).
(1.271 g, 46%); white solid; Rf 0.49 (EtOAc–MeOH 9 : 1); mp
144–146 ◦C; vmax (neat/cm-1) 3080, 2928, 1576, 1518, 1495, 1476,
1437, 1381, 1357, 1313, 1218, 1162, 1127, 1070, 1043, 1020; dH
(400 MHz, CDCl3) 2.17–2.24 (4H, m, CH2), 2.76 (4H, t, J 6.7,
CH2SePh), 4.29 (4H, t, J 6.6, NCH2), 7.18–7.21 (6H, m, Ph-H),
7.41–7.44 (4H, m, Ph-H), 7.68 (2H, s, Ar-4,8-H), 7.85 (2H, s,
Ar-2,6-H); dC (100 MHz, CDCl3) 24.0 (CH2SePh), 28.9 (CH2),
44.0 (NCH2), 98.9 (Ar-4,8-CH), 127.1 (Ph-CH), 128.8 (C), 129.0
(Ph-CH), 131.1 (C), 132.8 (Ph-CH), 141.4 (Ar-3a,7a-C), 144.1
(Ar-2,6-CH); m/z (ESI) 555.0557 (M + H+, C26H27N480Se80Se
requires 555.0566).
Experimental
General
Materials. All commercially available reagents were pur-
chased from Sigma Aldrich and were used throughout. Dimethyl-
formamide (DMF) and toluene were freshly distilled over
CaH. Thin layer chromatography (TLC) was carried out on
aluminium-backed plates coated with silica gel (Merck Kieselgel
60 F254). Dry column chromatography29 was carried out using
Merck Kieselgel silica gel 60 (particle size 0.015–0.040 mm) using
the specified eluents.
1,7-Di(3-(phenylselano)propyl)imidazo[4,5-f ]benzimidazole (8a).
(1.326 g, 48%); white solid; Rf 0.39 (EtOAc–MeOH 9 : 1); mp
89–91 ◦C; vmax (neat, cm-1) 3359, 3093, 2921, 2851, 1639, 1576,
1524, 1493, 1476, 1435, 1364, 1292, 1232, 1213, 1159, 1113, 1070,
1020, 1000; dH (400 MHz, CDCl3) 2.14–2.22 (4H, m, CH2), 2.77
(4H, t, J 6.9, CH2SePh), 4.25 (4H, t, J 6.6, NCH2), 7.16–7.21 (7H,
m, Ph-H & Ar-8-H), 7.38–7.43 (4H, m, Ph-H), 7.81 (2H, s, Ar-2,6-
H), 8.17 (1H, s, Ar-4-H); dC (100 MHz, CDCl3) 24.3 (CH2SePh),
29.3 (CH2), 44.2 (NCH2), 88.5 (Ar-8-CH), 110.5 (Ar-4-CH), 127.4
(Ph-CH), 129.1 (C), 129.4 (Ph-CH), 132.0 (C), 133.0 (Ph-CH),
141.2 (Ar-3a,4a-C), 143.8 (Ar-2,6-CH); m/z (ESI) 555.0576 (M +
H+, C26H27N480Se80Se requires 555.0566).
Measurements. Melting points were measured on a Stuart
Scientific melting point apparatus SMP3. Infrared spectra were
recorded using a Perkin-Elmer Spec 1 with ATR attached. 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 by DEPT and 1H-13C NMR 2D
spectra. High resolution mass spectra for compounds 7b, 7c, 8b,
8c, 9b, 9c and 12b were carried out at University College London,
Mass Spectroscopy Facility using a Thermo Finnigan MAT900xp
operating in chemical ionization (CI) mode with methane as
carrier gas, and accurate masses were calculated against reference
‘heptacosa’. High resolution mass spectra for all other 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.
1,5-Di(4-(phenylselano)butyl)imidazo[5,4-f ]benzimidazole (7b).
(1.509 g, 52%); white solid; Rf 0.50 (EtOAc–MeOH 9 : 1); mp
156–158 ◦C; vmax (neat, cm-1) 3074, 2930, 1797, 1579, 1521, 1477,
1446, 1356, 1236, 1212, 1124, 1072, 1022; dH (400 MHz, CDCl3)
1.67–1.74 (4H, m, CH2), 2.00-2.01 (4H, m, CH2), 2.86 (4H, t, J 7.2,
CH2SePh), 4.18 (4H, t, J 7.1, NCH2), 7.19–7.24 (6H, m, Ph-H),
7.39–7.43 (4H, m, Ph-H), 7.70 (2H, s, Ar-4,8-H), 7.86 (2H, s, Ar-
2,6-H); dC (100 MHz, CDCl3) 27.2 (CH2SePh), 27.3 (CH2), 29.4
(CH2), 44.9 (NCH2), 99.2 (Ar-4,8-CH), 127.1 (Ph-CH), 129.2 (Ph-
CH), 129.8 (C), 131.5 (C), 133.0 (Ph-CH), 141.7 (Ar-3a,7a-C),
144.2 (Ar-2,6-CH); m/z (CI) 583.0853 (M + H+, C28H31N480Se80Se
requires 583.0879).
Synthesis of imidazo[5,4(4,5)-f ]benzimidazole (6a–6b). 1,2,4,5-
Benzenetetraamine tetrahydrochloride (1.00 g, 3.5 mmol), con-
centrated formic acid (50 mL) and concentrated hydrochloric acid
(10 mL) were heated at reflux for 16 h. The cooled solution was
evaporated to dryness and sodium carbonate solution (5%, 50 mL)
was added. The precipitated product was collected via vacuum
filtration, washed with water and dried under vacuum (CaCl2) to
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1,7-Di(4-(phenylselano)butyl)imidazo[4,5-f ]benzimidazole (8b).
(1.277 g, 44%); white solid; Rf 0.40 (EtOAc–MeOH 9 : 1); mp 125-
127 ◦C; vmax (neat, cm-1) 3345, 2922, 1579, 1524, 1500, 1364, 1283,
1212, 1111, 1071, 1022; dH (400 MHz, CDCl3) 1.68–1.76 (4H, m,
CH2), 2.01–2.08 (4H, m, CH2), 2.87 (4H, t, J 7.0, CH2SePh), 4.15
(4H, t, J 7.1, NCH2), 7.14–7.19 (7H, m, Ph-H & Ar-8-H), 7.36–
7.39 (4H, m, Ph-H), 7.81 (2H, s, Ar-2,6-H), 8.18 (1H, s, Ar-4-H); dC
(100 MHz, CDCl3) 27.2 (CH2SePh), 27.3 (CH2), 29.2 (CH2), 44.7
(NCH2), 88.4 (Ar-8-CH), 110.6 (Ar-4-CH), 127.2 (Ph-CH), 129.2
(Ph-CH), 129.7 (C), 132.0 (C), 133.0 (Ph-CH), 141.3 (Ar-3a,4a-C),
143.6 (Ar-2,6-CH); m/z (CI) 583.0857 (M + H+, C28H31N480Se80Se
requires 583.0879).
[5,4-f (4,5-f )]benzimidazole (0.6 mmol) and acetic anhydride
(0.123 mL, 1.3 mmol) in toluene (50 mL) at reflux, and open
to the air, was added a solution of ACN (0.733 g, 3.0 mmol) and
tributyltin hydride (0.873 g, 3.0 mmol) in toluene (50 mL) using a
syringe pump at a rate of 2.2 mL h-1.
General work up and purification for methods 1–3
The cooled mixture was left in sunlight for 3 h and extracted
with acetic acid (80%, 5 ¥ 20 mL). The combined acid extracts
were washed with petroleum ether (3 ¥ 20 mL), and evaporated.
Sodium carbonate solution (5%, 50 mL) was added to the acid
residue, and extracted with dichloromethane (5 ¥ 50 mL). The
combined organic extracts were dried (Na2SO4), and evaporated
to dryness. The residue was purified by dry column vacuum
chromatography with a gradient elution of ethyl acetate and
methanol.
1,5-Di(5-(phenylselano)pentyl)imidazo[5,4-f ]benzimidazole (7c).
(1.308 g, 43%); white solid; Rf 0.50 (EtOAc–MeOH 9 : 1); mp 115–
117 ◦C; vmax (neat, cm-1) 3033, 2934, 2853, 1702, 1574, 1517, 1492,
1475, 1446, 1356, 1331, 1269, 1224, 1203, 1165, 1125, 1072, 1021;
dH (400 MHz, CDCl3) 1.41–1.49 (4H, m, CH2), 1.69–1.77 (4H,
m, CH2), 1.89–1.96 (4H, m, CH2), 2.86 (4H, t, J 7.3, CH2SePh),
4.20 (4H, t, J 6.9, NCH2), 7.22-7.27 (6H, m, Ph-H), 7.44–7.47
(4H, m, Ph-H), 7.71 (2H, s, Ar-4,8-H), 7.91 (2H, s, Ar-2,6-H);
dC (100 MHz, CDCl3) 26.9 (CH2), 27.4 (CH2SePh), 28.9 (CH2),
29.6 (CH2), 45.1 (NCH2), 99.1 (Ar-4,8-CH), 126.9 (Ph-CH), 129.0
(Ph-CH), 130.1 (C), 131.4 (C), 132.6 (Ph-CH), 141.6 (Ar-3a,7a-C),
144.1 (Ar-2,6-CH); m/z (CI) 611.1177 (M+H+, C30H35N480Se80Se
requires 611.1192).
One-pot double five-membered radical cyclizations onto
imidazo[5,4-f ]benzimidazole. Following method 1, using 7a and
after purification by chromatography; 2,3,8,9-Tetrahydro-1H, 7H-
pyrrolo[1,2-a]pyrrolo[1¢,2¢:1,2]imidazo[5,4-f ]benzimidazole (9a).
(46 mg, 32%), white solid; Rf 0.24 (CH2Cl2–Et2O–MeOH 7 : 2 : 1);
mp 295–305 ◦C decomp; vmax (neat, cm-1) 3009, 2942, 2895, 1546,
1489, 1434, 1411, 1347, 1324, 1267, 1161, 1100; dH (400 MHz,
CDCl3) 2.77–2.84 (4H, m, 2,8-CH2), 3.19 (4H, t, J 7.6, 3,9-CH2),
4.22 (4H, t, J 7.1, 1,7-CH2), 7.59 (2H, s, 5,11-H); dC (100 MHz,
CDCl3) 23.9 (3,9-CH2); 26.0 (2,8-CH2), 43.0 (1,7-CH2), 98.5 (5,11-
CH), 129.6 (5a,11a-C), 145.5 (4a,10a-C), 161.7 (3a,9a-C); m/z
(ESI) 239.1307 (M + H+, C14H15N4 requires 239.1297),
1-Propyl-7,8-dihydro-6H-imidazo[5,4-f ]pyrrolo[1,2-a]benzimi-
dazole (10a). (45 mg, 31%), white solid; Rf 0.32 (CH2Cl2–
Et2O–MeOH 7 : 2 : 1); mp 210–213 ◦C; vmax (neat, cm-1) 3019,
2935, 1538, 1493, 1444, 1418, 1357, 1291, 1222, 1189, 1163,
1110, 1056; dH (400 MHz, CDCl3) 0.97 (3H, t, J 7.3, CH3);
2.01–1.92 (2H, m, CH2CH3), 2.71–2.79 (2H, m, 7-CH2), 3.11 (2H,
t, J 7.6, 8-CH2), 4.15–4.21 (4H, m, NCH2), 7.63 (2H, s, Ar-4,10-
H), 7.92 (1H, s, Ar-2-H); dC (100 MHz, CDCl3) 11.4 (CH3), 22.8
(CH2), 23.8 (8-CH2), 25.9 (CH2), 42.9 (NCH2), 46.9 (NCH2), 98.4
(Ar-CH), 98.9 (Ar-CH), 130.0 (C), 131.0 (C), 140.6 (C), 143.5
(Ar-2-CH), 146.6 (C), 162.4 (Ar-8a-C); m/z (ESI) 241.1452 (M +
H+, C14H17N4 requires 241.1453), and
1,7-Di(5-(phenylselano)pentyl)imidazo[4,5-f ]benzimidazole (8c).
(1.248 g, 41%); white solid; Rf 0.40 (EtOAc–MeOH 9 : 1); mp
78–81 ◦C; vmax (neat, cm-1) 3089, 2936, 1577, 1522, 1477, 1446,
1361, 1288, 1261, 1211, 1142, 1107, 1070, 1021; dH (400 MHz,
CDCl3) 1.43–1.51 (4H, m, CH2), 1.70–1.77 (4H, m, CH2), 1.87–
1.95 (4H, m, CH2), 2.86 (4H, t, J 7.2, CH2SePh), 4.17 (4H, t, J
7.1, NCH2), 7.17–7.28 (7H, m, Ph-H & Ar-8-H), 7.42–7.46 (4H, m,
Ph-H), 7.88 (2H, s, Ar-2,6-H), 8.20 (1H, s, Ar-4-H); dC (100 MHz,
CDCl3) 27.0 (CH2), 27.5 (CH2SePh), 29.0 (CH2), 29.7 (CH2), 45.1
(NCH2), 88.5 (Ar-8-CH), 110.2 (Ar-4-CH), 126.9 (Ph-CH), 129.2
(Ph-CH), 130.2 (C), 132.0 (C), 132.6 (Ph-CH), 140.9 (Ar-3a,4a-C),
143.7 (Ar-2,6-CH); m/z (CI) 611.1185 (M + H+, C30H35N480Se80Se
requires 611.1192).
One-pot double homolytic aromatic substitution methods
1,5-Dipropylimidazo[5,4-f ]benzimidazole (11a). (14 mg, 10%),
white solid; Rf 0.39 (CH2Cl2-Et2O–MeOH 7 : 2 : 1); mp 205–
208 ◦C; vmax (neat, cm-1) 3091, 3025, 2960, 2930, 2874, 1741, 1518,
1491, 1450, 1382, 1361, 1312, 1227, 1208, 1167, 1113, 1084; dH
(400 MHz, CDCl3) 0.94 (6H, t, J 7.3, CH3), 1.90–1.99 (4H, m,
CH2), 4.18 (4H, t, J 7.1, NCH2), 7.72 (2H, s, Ar-4,8-H); 7.93 (2H, s,
Ar-2,6-H); dC (100 MHz, CDCl3) 11.3 (CH3), 22.7 (CH2), 46.9
(NCH2), 99.0 (Ar-4,8-CH), 131.4 (Ar-4a,8a-C), 141.6 (Ar-3a,7a-
C), 144.2 (Ar-2,6-CH); m/z (ESI) 243.1618 (M + H+, C14H19N4
calcd 243.1610).
Method 1: radical cyclizations without activation. To
a
solution of 1,5(7)-di(w-(phenylselano)alkyl)imidazo[5,4-f (4,5-
f )]benzimidazole (0.6 mmol) in toluene (50 mL) at re-
flux, and open to the air, was added a solution of 1,1¢-
azobis(cyclohexanecarbonitrile) (ACN) (0.733 g, 3.0 mmol) and
tributyltin hydride (0.873 g, 3.0 mmol) in toluene (50 mL) using a
syringe pump at a rate of 2.2 mL h-1.
Method 2: radical cyclizations with activation using camphor-
sulfonic acid. To a solution of 1,5(7)-di(w-(phenylselano)alkyl)-
imidazo[5,4-f (4,5-f )]benzimidazole (0.6 mmol) and camphorsul-
fonic acid (0.302 g, 1.3 mmol) in toluene (50 mL) at reflux, and
open to the air, was added a solution of ACN (0.733 g, 3.0 mmol)
and tributyltin hydride (0.873 g, 3.0 mmol) in toluene (50 mL)
using a syringe pump at a rate of 2.2 mL h-1.
Following method 2, using 7a an intractable mixture was
obtained. Following method 3, using 7a and after purification
by chromatography, 9a (67 mg, 47%) and 10a (43 mg, 30%) were
isolated only.
One-pot double six-membered radical cyclizations onto
imidazo[5,4-f ]benzimidazole. Following method 1, using
7b and after purification by chromatography; 1,2,3,4,8,9,10,
Method 3: radical cyclizations with activation using acetic anhy-
dride. To a solution of 1,5(7)-di(w-(phenylselano)alkyl)imidazo-
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11-Octahydropyrido[1,2-a]pyrido[1¢,2¢:1,2]imidazo[5,4-f ]benzimi-
dazole (9b). (0.144 g, 90%) white solid; Rf 0.23 (CH2Cl2–Et2O–
(4H, t, J 7.7, 3,7-CH2), 4.06 (4H, t, J 7.1, 1,9-CH2), 7.04 (1H, s,
11-H), 7.95 (1H, s, 5-H); dC (100 MHz, CDCl3) 23.8 (3,7-CH2),
26.1 (2,8-CH2), 42.8 (1,9-CH2), 88.7 (11-CH), 108.9 (5-CH), 129.5
(10a,11a-C), 145.6 (4a,5a-C), 161.2 (3a,6a-C); m/z (ESI) 239.1300
(M + H+, C14H15N4 requires 239.1297), and
1-Propyl-7,8-dihydro-6H-imidazo[4,5-f ]pyrrolo[1,2-a]benzimi-
dazole (13a). (40 mg, 27%) white solid; Rf 0.30 (CH2Cl2–Et2O–
MeOH 7 : 2 : 1); mp 168–171 ◦C; vmax (neat, cm-1) 3351, 3079, 2927,
1641, 1544, 1513, 1492, 1453, 1423, 1356, 1322, 1298, 1253, 1215,
1115; dH (400 MHz, CDCl3) 0.98 (3H, t, J 7.6, CH3), 1.91-2.00
(2H, m, CH2CH3), 2.70–2.78 (2H, m, 7-CH2), 3.09 (2H, t, J 7.8,
6-CH2), 4.12–4.17 (4H, m, NCH2), 7.16 (1H, s, Ar-10-H), 7.89
(1H, s, Ar-4-H), 8.08 (1H, s, Ar-2-H); dC (100 MHz, CDCl3) 11.5
(CH3), 22.9 (CH2), 23.8 (6-CH2), 26.1 (CH2), 42.9 (NCH2), 47.0
(NCH2), 88.5 (Ar-10-CH), 109.7 (Ar-4-CH), 130.6, 131.1, 140.7
(all C), 143.2 (Ar-2-CH), 146.2 (C), 161.9 (Ar-5a-C); m/z (ESI)
241.1457 (M + H+, C14H17N4 requires 241.1453).
◦
MeOH 7 : 2 : 1); mp 285–295 C decomp (lit.,30 >300 ◦C); vmax
(neat, cm-1) 2942, 1534, 1488, 1445, 1417, 1362, 1316, 1279, 1259,
1190, 1165, 1130, 1092; dH (400 MHz, CDCl3) 2.02–2.08 (4H, m,
CH2), 2.15–2.21 (4H, m, CH2), 3.16 (4H, t, J 6.4, 4,11-CH2), 4.14
(4H, t, J 6.0, 1,8-CH2), 7.55 (2H, s, 6,13-H); dC (100 MHz, CDCl3)
20.5 (CH2), 22.6 (CH2), 25.4 (4,11-CH2), 42.8 (1,8-CH2), 96.9
(6,13-CH), 132.1 (6a,13a-C), 138.1 (5a,12a-C), 152.4 (4a,11a-C);
m/z (CI) 267.1605 (M + H+, C16H19N4 requires 267.1610).
One-pot double seven-membered radical cyclizations onto
imidazo[5,4-f ]benzimidazole. Following method 1, using 7c
and after purification by chromatography; 2,3,4,5,10,11,12,13-
Octahydro-1H,9H -azepino[1,2-a]azepino[1¢,2¢:1,2]imidazo[5,4-
f ]benzimidazole (9c). (51 mg, 29%) white solid; Rf 0.38 (CH2Cl2–
Et2O–MeOH 7 : 2 : 1); mp 275–285 ◦C decomp; vmax (neat, cm-1)
2931, 2850, 1534, 1478, 1440, 1414, 1365, 1311, 1256, 1178, 1157,
1116, 1085; dH (400 MHz, CDCl3) 1.78–1.91 (8H, m, CH2), 1.92–
1.98 (4H, m, CH2), 3.11 (4H, t, J 5.6, 5,13-CH2), 4.19 (4H, t,
J 5.0, 1,9-CH2), 7.46 (2H, s, 7,15-H); dC (100 MHz, CDCl3) 25.7
(CH2), 28.8 (CH2), 30.5 (5,13-CH2), 31.0 (CH2), 44.7 (1,9-CH2),
97.1 (7,15-CH), 133.2 (7a,15a-C), 139.5 (6a,14a-C), 158.2 (5a,13a-
C); m/z (CI) 295.1916 (M + H+, C18H23N4 requires 295.1923),
1-Pentyl-7,8,9,10-tetrahydro-6H-azepino[1,2-a]imidazo[5,4-f ]-
benzimidazole (10c). (57 mg, 32%) white solid; Rf 0.43 (CH2Cl2–
Et2O–MeOH 7 : 2 : 1); mp 198–200 ◦C; vmax (neat, cm-1) 2924,
2855, 1528, 1476, 1447, 1358, 1313, 1260, 1205, 1188, 1118; dH
(400 MHz, CDCl3) 0.87 (3H, t, J 6.8, CH3), 1.27–1.38 (4H,
m, CH2), 1.80–2.00 (8H, m, CH2), 3.13 (2H, t, J 5.7, 10-CH2),
4.19–4.23 (4H, m, NCH2), 7.61 (2H, s, Ar-4,12-H), 7.91 (1H, s,
Ar-2-H); dC (100 MHz, CDCl3) 14.0 (CH3), 22.3, 25.7, 28.8, 29.0,
29.2 (all CH2), 30.5 (10-CH2), 31.0 (CH2), 44.7 (NCH2), 45.4
(NCH2), 98.0 (Ar-CH), 98.2 (Ar-CH), 131.1, 133.7, 140.3, 141.0
(all C), 143.6 (Ar-2-CH), 158.9 (Ar-10a-C); m/z (ESI) 297.2091
(M + H+, C18H25N4 requires 297.2079), and
1,5-Dipentylimidazo[5,4-f ]benzimidazole (11c). (15 mg, 8%)
white solid, Rf 0.49 (CH2Cl2–Et2O–MeOH 7 : 2 : 1); mp 161–
162 ◦C; vmax (neat, cm-1) 2923, 2855, 1715, 1517, 1494, 1452, 1375,
1357, 1318, 1222, 1194, 1164, 1118, 1061, 1029; dH (400 MHz,
CDCl3) 0.77 (6H, t, J 6.9, CH3), 1.17–1.28 (8H, m, CH2), 1.77–
1.85 (4H, m, CH2), 4.10 (4H, t, J 7.1, NCH2), 7.64 (2H, s,
Ar-4,8-H), 7.85 (2H, s, Ar-2,6-H); dC (100 MHz, CDCl3) 13.9
(CH3), 22.2, 29.0, 29.2 (all CH2), 45.3 (NCH2), 99.1 (Ar-4,8-CH),
131.5 (Ar-4a,8a-C), 141.6 (Ar-3a,7a-C), 144.2 (Ar-2,6-CH); m/z
(ESI) 299.2238 (M + H+, C18H27N4 requires 299.2236).
Following method 2, using 7c and after purification by chro-
matography 9c (95 mg, 54%) and 10c (23 mg, 13%) were isolated
only. Following method 3, using 7c and after purification by
chromatography, 9c (60 mg, 34%) and 10c (55 mg, 31%) were
isolated only.
One-pot double six-membered radical cyclizations onto
imidazo[4,5-f ]benzimidazole. Following method 1, using 8b
and after purification by chromatography; 1,2,3,4,8,9,10,11-
Octahydropyrido[1,2-a]pyrido[1¢,2¢:1,2]imidazo[4,5-f ]benzimida -
zole (12b). (0.129 g, 81%) white solid; Rf 0.20 (CH2Cl2–Et2O–
MeOH 7 : 2 : 1); mp 266–270 ◦C decomp (lit.,17 104–108 ◦C); vmax
(neat, cm-1) 3372, 2950, 2891, 1659, 1635, 1526, 1487, 1438, 1417,
1365, 1313, 1255, 1238, 1151, 1134; dH (400 MHz, CDCl3) 1.99–
2.05 (4H, m, CH2), 2.12–2.18 (4H, m, CH2), 3.09 (4H, t, J 6.3,
4,8-CH2), 4.07 (4H, t, J 6.0, 1,11-CH2), 7.02 (1H, s, 13-H), 7.95
(1H, s, 6-H); dC (100 MHz, CDCl3) 20.9 (CH2), 22.9 (CH2), 25.8
(4,8-CH2), 42.6 (1,11-CH2), 87.1 (13-CH), 107.4 (6-CH), 132.0
(12a,13a-C), 139.9 (5a,6a-C), 151.8 (4a,7a-C); m/z (CI) 267.1595
(M + H+, C16H19N4 requires 267.1610).
One-pot double seven-membered radical cyclizations onto
imidazo[4,5-f ]benzimidazole. Following method 2, using 8c
and after purification by chromatography; 2,3,4,5,10,11,12,13-
Octahydro-1H,9H -azepino[1,2-a]azepino[1¢,2¢:1,2]imidazo[4,5-
f ]benzimidazole (12c). (85 mg, 48%) white solid; Rf 0.32 (CH2Cl2–
Et2O–MeOH 7 : 2 : 1); mp 214–217 ◦C decomp; vmax (neat, cm-1)
3377, 2928, 2852, 1638, 1527, 1470, 1447, 1422, 1367, 1345, 1323,
1230, 1200, 1140; dH (400 MHz, CDCl3) 1.79–1.97 (12H, m, CH2),
3.11 (4H, t, J 5.6, 5,9-CH2), 4.18 (4H, t, J 5.1, 1,13-CH2), 7.00
(1H, s, 15-H), 7.95 (1H, s, 7-H); dC (100 MHz, CDCl3) 25.7
(CH2), 28.9 (CH2), 30.4 (5,9-CH2), 31.0 (CH2), 44.7 (1,13-CH2),
87.0 (15-CH), 108.0 (7-CH), 133.3 (14a,15a-C), 139.1 (6a,7a-C),
157.6 (5a,8a-C); m/z (ESI) 295.1911 (M + H+, C18H23N4 requires
295.1923).
1-Pentyl-7,8,9,10-tetrahydro-6H-azepino[1,2-a]imidazo[4,5-f ]-
benzimidazole (13c). (29 mg, 16%); colourless oil; Rf 0.41
(CH2Cl2–Et2O–MeOH 7 : 2 : 1); vmax (neat, cm-1) 2926, 2855, 1638,
1535, 1510, 1489, 1448, 1365, 1286, 1254, 1220, 1193, 1126; dH
(400 MHz, CDCl3) 0.89 (3H, t, J 6.9, CH3), 1.31–1.39 (4H, m,
CH2), 1.82-1.96 (8H, m, CH2), 3.11 (2H, t, J 5.6, 6-CH2), 4.16-4.21
(4H, m, NCH2), 7.10 (1H, s, Ar-12-H), 7.88 (1H, s, Ar-4-H), 8.07
(1H, s, Ar-2-H); dC (100 MHz, CDCl3) 14.0 (CH3), 22.3, 25.7,
28.9, 29.1, 29.2 (all CH2), 30.5 (6-CH2), 31.0 (CH2), 44.8 (NCH2),
45.3 (NCH2), 87.6 (Ar-12-CH), 109.2 (Ar-4-CH), 131.3, 134.1,
139.7, 140.7 (all C), 143.2 (Ar-2-CH), 158.2 (Ar-5a-C); m/z (ESI)
297.2089 (M + H+, C18H25N4 requires 297.2079).
One-pot double five-membered radical cyclizations onto
imidazo[4,5-f ]benzimidazole. Following method 3, using 8a and
after purification by chromatography, 2,3,8,9-Tetrahydro-1H,
7H -pyrrolo[1,2-a]pyrrolo[1¢,2¢:1,2]imidazo[4,5-f ]benzimidazole
(12a). (69 mg, 48%) w◦hite solid; Rf 0.22 (CH2Cl2–Et2O–MeOH
7 : 2 : 1); mp 266–268 C decomp (lit.16 240 ◦C decomp); vmax
(neat, cm-1) 3229, 2956, 1644, 1543, 1490, 1431, 1352, 1300, 1228,
1117; dH (400 MHz, CDCl3) 2.64–2.71 (4H, m, 2,8-CH2), 3.02
3154 | Org. Biomol. Chem., 2010, 8, 3149–3156
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Preparation of imidazobenzimidazolequinones
yield the title compound 4 (0.229 g, 9◦6%) as a yellow solid; Rf 0.32
(EtOAc–MeOH 9 : 1); mp 265–275 C decomp; vmax (neat, cm-1)
6-Nitro-1,2,3,4,8,9,10,11-octahydropyrido[1,2-a]pyrido[1¢,2¢:1,
=
2948, 1655 (C O), 1493, 1377, 1336, 1305, 1218, 1076, 1042; dH
2]imidazo[5,4-f ]benzimidazole
(15). Concentrated
HNO3
(400 MHz, CDCl3) 1.98–2.10 (8H, m, CH2), 2.99 (4H, t, J 6.2,
4,11-CH2), 4.35 (4H, t, J 5.9, 1,8-CH2); dC (100 MHz, CDCl3)
19.8 (CH2), 22.2 (CH2), 25.1 (4,11-CH2), 45.6 (1,8-CH2), 130.2
(10 mL) was added to a solution of 9b (0.240 g, 0.9 mmol) in
concentrated H2SO4 (10 mL) at 0 ◦C and heated to 80 ◦C for 3 h.
The cooled solution was neutralised with solid sodium carbonate
and extracted with dichloromethane (6 ¥ 50 mL). The combined
organic extracts were dried (MgSO4) and evaporated. The residue
was purified by dry column vacuum chromatography with a
gradient elution of ethyl acetate and methanol to yield the title
compound 15 (0.263 g, 94%); yellow solid; Rf 0.48 (EtOAc–MeOH
9 : 1); mp 230–236 ◦C decomp; vmax (neat, cm-1) 2952, 1735, 1542,
1514 (NO2), 1478, 1431, 1415, 1360 (NO2), 1335, 1310, 1272, 1255,
1205, 1170, 1145, 1076, 1043; dH (400 MHz, CDCl3) 2.01–2.29
(8H, m, CH2), 3.13–3.22 (4H, m, 4,11-CH2), 4.12–4.21 (4H, m,
1,8-CH2), 7.69 (1H, s, 13-H); dC (100 MHz, CDCl3) 20.1, 20.5,
22.5, 23.2 (all CH2), 26.0, 26.4 (4,11-CH2), 43.0, 45.7, (1,8-CH2),
102.8 (13-CH), 124.5, 124.6, 132.5, 134.1, 141.0 (all C), 154.4,
155.4, (4a,11a-C); m/z (ESI) 312.1460 (M + H+, C16H18N5O2
requires 312.1461).
=
(6a,13a-C), 141.8 (5a,12a-C), 151.4 (4a,11a-C), 171.6 (C O);
m/z (ESI) 297.1365 (M + H+, C16H17N4O2 requires 297.1352).
1,2,3,4,8,9,10,11-Octahydropyrido[1,2-a]pyrido[1¢,2¢:1,2]imida-
zo[4,5-f ]benzimidazol-6,13-dione (5). Following
a literature
procedure17 pyrido[1,2-a]pyrido[1¢,2¢:1,2]imidazo[4,5-f ]benzimi-
dazole 12b (0.158 g, 0.6 mmol) yielded the title compound 5
(0.121 g, 69%) as a yellow solid; Rf 0.30 (EtOAc–MeOH 9 : 1);
◦
◦
mp 221–223 C decomp (lit.,17 >260 C); vmax (neat, cm-1) 2941,
=
1675, 1652 (C O), 1493, 1442, 1421, 1331, 1295, 1163, 1073, 1039;
dH (400 MHz, CDCl3) 1.94-2.09 (8H, m, CH2), 2.98 (4H, t, J 6.4,
4,8-CH2), 4.32 (4H, t, J 6.0, 1,11-CH2); dC (100 MHz, CDCl3)
19.8 (CH2), 22.4 (CH2), 25.0 (4,8-CH2), 45.5 (1,11-CH2), 130.3
=
(12a,13a-C), 142.5 (5a,6a-C), 151.4 (4a,7a-C), 169.8 (13-C O),
+
=
175.5 (6-C O); m/z (ESI) 297.1352 (M + H , C16H17N4O2 requires
297.1355).
6-Imino-1,2,3,4,8,9,10,11-octahydropyrido[1,2-a]pyrido[1¢,2¢:1,
2]imidazo[5,4-f ]benzimidazol-13-one (17).
A mixture of 15
Cell culture and cytotoxicity evaluation
(0.275 g, 0.9 mmol) and Pd–C (10%, 25 mg) in methanol (100 mL)
was stirred under 40 psi H2 at room temperature for 16 h.
The catalyst was removed by filtration and the filtrate evap-
orated to give 6-amino-1,2,3,4,8,9,10,11-octahydropyrido[1,2-
a]pyrido[1¢,2¢:1,2]imidazo[5,4-f]benzimidazole (16) as a brown
residue (not purified further); dH (400 MHz, CDCl3) 1.97–2.08
(4H, m, CH2), 2.10–2.20 (4H, m, CH2), 3.10–3.06 (4H, m,
4,11-CH2), 4.07 (2H, t, J 6.1, NCH2), 4.36 (2H, bs, NH2), 4.62
(2H, t, J 6.1, NCH2), 6.97 (1H, s, 13-H). Monobasic potassium
phosphate solution (50 mL, 0.2 M) was added to the residue of 16,
and a separate solution containing potassium nitrosodisulfonate
(Fremy’s salt) (0.725 g, 2.7 mmol) in monobasic potassium
phosphate solution (50 mL, 0.2 M) was added. The solution
was stirred for 1 h at room temperature and extracted with
dichloromethane (6 ¥ 50 mL). The combined organic extracts
were dried (MgSO4) and evaporated. The residue was purified
by dry column vacuum chromatography with gradient elution of
ethyl acetate and methanol to yield the title compound 17 (0.238 g,
91%) as a yellow solid; Rf 0.30 (EtOAc–MeOH 9 : 1); mp 230–
Cell lines. An 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 HeLa cervical cancer cell line
(repository number CCL-2) was obtained from the American
Type Culture Collection (ATCC). The DU145 prostate cancer cell
line (ATCC repository number HTB-81) was obtained from Prof.
R.W.G Watson, School of Medicine & Medical Science, University
College Dublin, Ireland.
Cell culture. The SV40-transformed normal human skin fi-
broblast cell line (GM00637) was grown in minimum essential
media (MEM) Eagle-Earle BSS supplemented with 15% heat-
inactivated fetal bovine serum (FBS), penicillin-streptomycin,
2 mM L-glutamine, 2¥ essential and non-essential amino acids, and
vitamins. HeLa-CCL-2 cervical cancer cells were grown in Dul-
becco’s modified Eagle’s medium (DMEM) supplemented with
10% heat-inactivated fetal bovine serum, penicillin-streptomycin,
2 mM L-glutamine, and MEM non-essential amino acids. DU145
prostate cancer cells were grown in RPMI-1640 medium supple-
mented with 10% heat-inactivated fetal bovine serum, penicillin-
streptomycin and 2 mM L-glutamine.
240 ◦C decomp; vmax (neat, cm ) 2948, 2867, 1656, (C O), 1515,
-1
=
1494, 1442, 1425, 1405, 1377, 1336, 1306, 1272, 1219, 1167, 1075,
1042; dH (400 MHz, CDCl3) 1.93–2.08 (8H, m, CH2), 2.92–3.00
(4H, m, 4,11-CH2), 4.37–4.43 (4H, m, 1,8-CH2), 10.55 (1H, s,
NH); dC (100 MHz, CDCl3) 19.9, 20.0, 22.3, 22.7 (all CH2), 25.0,
25.3 (4,11-CH2), 45.4, 46.5, (1,8-CH2), 126.0 (C), 131.5 (C), 139.7
Cytotoxicity measurement. Cell viability was determined using
the MTT colorimetric assay.28 Normal cells were plated into 96-
well plates at a density of 10000 cells per well (200 mL per well) and
allowed to adhere over a period of 24 h. HeLa cells were plated
into 96-well plates at a density of 1000 cells per well (100 mL per
well) and allowed to adhere over a period of 24 h. DU145 cells
were plated into 96-well plates at a density of 2000 cells per well
(200 mL per well) and allowed to adhere over a period of 24 h.
Compound solutions were applied in ethanol–H2O (for 4)
or ethanol (for 5 and 17) (1% final concentration in well),
and the plates were incubated at 37 ◦C under a humidified
atmosphere containing 5% CO2 for 72 h. Control cells were
=
=
(2 ¥ C), 150.2 (2 ¥ C), 155.5 (C N), 172.0 (C O); m/z (ESI)
296.1502 (M + H+, C16H18N5O requires 296.1511).
1,2,3,4,8,9,10,11-Octahydropyrido[1,2-a]pyrido[1¢,2¢:1,2]imida-
zo[5,4-f ]benzimidazol-6,13-dione
(4). 6-Iminoquinone
17
(0.238 g, 0.8 mmol) was dissolved in hydrochloric acid (50 mL, 2
M) and stirred for 15 min. The pH of the solution was adjusted
to pH 6 using solid sodium carbonate and the product extracted
using dichloromethane (6 ¥ 50 mL). The combined organic
extracts were dried (MgSO4) and evaporated to dryness. The
residue was purified by dry column vacuum chromatography to
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exposed to an equivalent concentration of ethanol or ethanol–
H2O alone. MMC (Sigma) solutions were applied in DMSO
(1% final concentration in well) and the plates were incubated
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◦
at 37 C under a humidified atmosphere containing 5% CO2 for
72 h. Control cells were exposed to an equivalent concentration of
DMSO alone. MTT (20 mL, 5 mg ml-1 solution) was added, and the
cells were incubated for another 3 h. The supernatant was 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 EtOH–H2O, EtOH or
DMSO-only treated value. Dose–response curves were analysed by
nonlinear regression analysis and IC50 values were estimated using
GraphPad Prism software, v. 5.02 (GraphPad Inc., San Diego,
CA, USA).
Acknowledgements
This publication has emanated from research conducted
with the financial support of Science Foundation Ireland
(07/RFP/CHEF227). The authors thank the Irish Research
Council for Science Engineering and Technology: funded by the
National Development Plan for an Embark Scholar Award for
Vincent Fagan. We are grateful to Prof. William Watson (School of
Medicine & Medical Science, University College Dublin, Ireland)
for the DU145 cell line, and Dr Lisa Harris (Mass Spectrometry
Facility, Department of Chemistry, University College London,
UK) for mass spectra.
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3156 | Org. Biomol. Chem., 2010, 8, 3149–3156
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