Synthesis and toxicity of new ring-fused imidazo[5,4-f] benzimidazolequinones and mechanism using amine N-oxide cyclizations

Article abstract of DOI:10.1002/ejoc.201101687

A new synthetic route to ring-fused imidazo[5,4-f]benzimidazoles is reported that can be used to access symmetrical and unsymmetrical quinone anticancer agents. Oxone in formic acid allows cyclisation of o-tert-aminoacetanilides to give ring-fused benzimi

Full text of DOI:10.1002/ejoc.201101687

FULL PAPER
DOI: 10.1002/ejoc.201101687
Synthesis and Toxicity of New Ring-Fused Imidazo[5,4-f]benzimid-
azolequinones and Mechanism Using Amine N-Oxide Cyclizations
Vincent Fagan,[a] Sarah Bonham,[a] Patrick McArdle,[a] Michael P. Carty,[b] and
Fawaz Aldabbagh*[a]
Keywords: Synthetic methods / Medicinal chemistry / Nitrogen heterocycles / Reaction mechanisms
A new synthetic route to ring-fused imidazo[5,4-f]benzimid-
azoles is reported that can be used to access symmetrical and
unsymmetrical quinone anticancer agents. Oxone in formic
acid allows cyclisation of o-tert-aminoacetanilides to give
ring-fused benzimidazoles and imidazobenzimidazoles in su-
perior yields. A mechanism for these oxidative cyclisations is
proposed that proceeds through a hydrogen-bonded amine
N-oxide intermediate. The amine N-oxide is shown to act as
an oxidant in the aromatisation to the imidazole ring. X-ray
crystal structures of the dimorpholine N-oxide intermediate
and bis[1,4]oxazinoimidazo[5,4-f]benzimidazolequinone bis-
(trifluoroacetate) adduct are included. Two unsymmetrical
quinones are shown to have greater cytotoxicity than the pre-
viously reported imidazobenzimidazolequinones.
Introduction
Heterocyclic quinone compounds are well-known biore-
ductive antitumour agents.[1,2] One reductase enzyme shown
to be responsible for reductive activation is NAD(P)H:quin-
one oxidoreductase 1 (NQO1).[3] Dipyrroloimidazo[4,5-f]-
benzimidazolequinone (1a) was shown in cytotoxicity as-
says[4] to have high specificity towards human melanoma
cell lines, and dipyridoimidazo[4,5-f]benzimidazolequinone
(1b), which was modelled into the human NQO1 active site,
was reported to be a better substrate than 1a (Figure 1).[5]
More recently, isomeric dipyridoimidazo[5,4-f]benzimid-
azolequinone (2a) and iminoquinone derivative 2b were re-
ported; their cytotoxicity was evaluated using human nor-
mal fibroblast cells (GM00637), and two human cancer cell
lines that were reported to express high levels of NQO1:
cervical (HeLa, CCL-2) and prostate (DU-145) cancer. Qui-
nones 1b and 2a showed toxicity towards human cancer cell
lines, with negligible toxicity towards human normal cell
line, and iminoquinone 2b was about twelve times more
toxic towards the prostate cancer cell line than towards the
normal cell line.[6]
Figure 1. Reported and target imidazo-benzimidazole(imino)quin-
ones.
There are few reliable methods for forming alicyclic ring-
fused imidazobenzimidazoles. Our recent report used
Bu3SnH and azo-initiator-mediated alkyl radical cyclisa-
tions onto the 2- and 6-positions of imidazobenzimidazole,
giving five- to seven-membered alicyclic ring-fused imid-
azo[4,5-f]benzimidazoles, and -[5,4-f]benzimidazoles, in
yields of 47–90%.[6] It is, however, difficult to form an imid-
azobenzimidazole containing two different fused rings
using this radical methodology. Furthermore, the mecha-
nism of cyclisation involved a non-chain radical reaction,
which typically requires large amounts of initiator to pro-
ceed adequately.[7,8] To date, this radical route is the only
reported viable synthesis of ring-fused imidazo[5,4-f]benz-
[a] School of Chemistry, National University of Ireland Galway,
University Road, Galway, Ireland
Fax: +353-91-495576
E-mail: Fawaz.Aldabbagh@nuigalway.ie
[b] Centre of Chromosome Biology, Biochemistry, School of
Natural Sciences, National University of Ireland Galway,
University Road, Galway, Ireland
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101687.
Eur. J. Org. Chem. 2012, 1967–1975
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1967

F. Aldabbagh et al.
FULL PAPER
imidazoles. Older reports describe a dipyrido-ring fused im- dinitrobenzene was used in place of 5. Reduction and acet-
idazo[5,4-f]benzimidazole, but in very low yields.[9,10] Ac- ylation of the nitro groups of 6a–c gave the cyclisation pre-
cess to dipyrrolo- and dipyrido-fused imidazo[4,5-f]benz- cursors 7a–c in high yields (79–92%).
imidazoles has been achieved using cyclisations of in situ
generated pyrrolidine and piperidine amine N-oxides with
adjacent acetanilide groups, albeit in low yields (10–
31%).[4,5] Meth-Cohn and Suschitzky proposed a mecha-
nism for these acid-catalysed cyclisations involving initial
nucleophilic addition of the amine N-oxide onto the amide
carbonyl group.[11,12] We now propose an alternative mecha-
nism, as part of a new synthetic route to symmetrical and
unsymmetrical ring-fused imidazo[5,4-f]benzimidazoles.
New [1,4-oxazino]- and alicyclic ring-fused imidazo[5,4-f]-
benzimidazoles are converted into quinone targets 3a–c,
and their toxicity was assessed against two human cancer
cell lines (HeLa CCL-2 and DU-145), and one human nor-
mal fibroblast cell line (GM00637).
Results and Discussion
Synthesis and Mechanism
Scheme 2. Synthesis of N,NЈ-[2,5-bis(tert-amino)-1,4-phenylene]di-
1,4-Difluoro-2,5-dinitrobenzene (5) can be separated in
acetamides 7a–c: (i) NaHCO3, morpholine for 6b at room temp.,
low yield from a mixture of nitration products of 1,4-di-
piperidine for 6c at –30 °C, THF, 16 h; (ii) piperidine for 6b, pyr-
rolidine for 6c, THF, room temp., 1 h.
fluorobenzene.[13] Compound 5 is, however, a useful syn-
thetic building block, and we now report its preparation
from commercial 2,5-difluoroaniline, without the need for
chromatography (Scheme 1). Acetylation and nitration
gives N-(2,5-difluoro-4-nitrophenyl)acetamide 4 as the sole
product by using a modification of a procedure reported
for the dibromo analogue.[14] A one-pot deprotection of the
amine and oxidation to a nitro group was achieved using
hydrogen peroxide in methanesulfonic acid to give 5 in 71%
overall yield.
Oxidative cyclisations of cyclic amines with neighbouring
acetamides have been reported to give alicyclic ring-fused
benzimidazoles and imidazo[4,5-f]benzimidazoles using
peroxy acids (formic acid[4,5,11,12,15,16] or trifluoroacetic
acid[17,18] and H2O2). However, when formic acid and 30%
H2O2 was used, the yield of double cyclisation imidazo[5,4-f]-
benzimidazole products from 7a–c were low (Ͻ30%),
prompting us to explore alternative oxidising systems. Acet-
anilide 8, used for these optimisation studies, was obtained
without the need for chromatography from commercial 2,5-
dibromonitrobenzene in 83% yield. After surveying several
oxidising agents in various acids, Oxone (active form is
KHSO5, 3 equiv.) in 90% formic acid was found to be the
most efficient oxidising system, because it involved the easi-
est work up, allowing isolation of the cyclised products by
precipitation (Scheme 3). Acetanilide 8 gave 8-bromo[1,4]-
oxazino[4,3-a]benzimidazole (9) in 80% yield, and diacetan-
ilides 7a and 7b gave [1,4]oxazino-fused imidazo[5,4-f]benz-
imidazoles 10a and 10b in 58 and 47% yield, respectively
(Scheme 3). The unsymmetrical dialicyclic ring-fused imid-
azo[5,4-f]benzimidazole 10c was obtained in 52% yield. Al-
Scheme 1. Synthesis of 1,4-difluoro-2,5-dinitrobenzene 5.
1,4-Difluoro-2,5-dinitrobenzene (5) underwent double though these yields are modest, they are a significant im-
nucleophilic aromatic substitutions with the appropriate provement on reported yields for forming dialicyclic ring-
secondary cyclic amines to give 1,4-di-tert-amino-2,5-di- fused imidazo[4,5-f]benzimidazoles using formic acid and
nitrobenzenes 6a–c in 69–96% yield (Scheme 2). The syn- H2O2.[4,5]
thesis of the unsymmetrical compounds 6b and 6c was pos-
These oxidative cyclisations are reported to occur via an
sible because monosubstitution proceeded almost to com- amine N-oxide intermediate.[11,12] Thus, to investigate the
pletion before disubstitution occurred. Aryl fluorides mechanism for cyclisation, morpholine N-oxide 11 and di-
underwent nucleophilic aromatic substitution more ef- morpholine N-oxide 12, were prepared in 84 and 76% yield,
ficiently than other aryl halides, and it was found that the respectively, by treatment of acetanilide 8 and diacetanilide
second substitution did not occur when 1,4-dichloro-2,5- 7a, with m-chloroperoxybenzoic acid (mCPBA; Scheme 4).
1968
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 1967–1975

Ring-Fused Imidazo[5,4-f]benzimidazolequinones
Figure 2. Crystal structure of N,NЈ-[2,5-bis(4-oxidomorpholin-4-
yl)-1,4-phenylene]diacetamide (12) showing the orientation of
amide groups, the distance between H(2) of amide and O(2) of N-
oxide, and the angle between O(2) of N-oxide, H(2) and N(2) of
amide.[19]
Scheme 3. Oxidative cyclisations mediated by Oxone in formic acid.
The X-ray crystal structure of dimorpholine N-oxide 12
showed that the orientation of the amide was influenced by
hydrogen bonding of the amide NH to the N-oxide residues
(Figure 2), which may account for the absence of the amide
simultaneous formation of an iminium ion and an imidol
(species 13). Nucleophilic addition of the imidol onto the
iminium ion gives rise to an intermediate N-acetyldihydrob-
enzimidazole 14, which is oxidised by Oxone to an imid-
azolium formate 15. Hydrolysis and neutralisation precipi-
tates benzimidazole 9.
1
NH peaks in the H NMR spectra of the amine N-oxides.
NMR analysis of N-(2-benzoylaminophenyl)pyrrolidine N-
oxide led Meth-Cohn to assign an analogous strongly hy-
drogen-bonded NH residue,[11] which results in the carbonyl
group being orientated away from the N-oxide oxygen.
However, counter to this molecular orientation, the pro-
posed mechanism for cyclisation, giving pyrrolo[1,2-a]benz-
imidazole, involves initial nucleophilic addition of the
amine N-oxide onto the amide carbonyl.[11,12] A new
mechanism for the cyclisation, using the hydrogen-bonding
orientation of the amine N-oxide, is now proposed.
Scheme 5. New mechanism taking into account hydrogen bonding
of the N-oxide with the amide.
Scheme 4. Preparation of N-oxides 11 and 12.
Heating morpholine N-oxide 11 in 90% formic acid re-
The o-tert-aminoacetanilide 8 is oxidised to the amine N- sulted in the formation of a ca. 1:1 mixture of benzimid-
oxide 11 and protonated in the acidic medium, causing the azole 9 and acetanilide 8 in an overall yield of 94%
NCH2 of the morpholine N-oxide ring to become acidic (Scheme 6). We propose that an acid-catalysed cyclisation
(Scheme 5). A water molecule is expelled, resulting in the occurred to give the intermediate N-acetyldihydrobenzimid-
Eur. J. Org. Chem. 2012, 1967–1975
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1969

F. Aldabbagh et al.
FULL PAPER
azole 14, which, in the absence of an external oxidant, was
Acetanilide 8 remained unchanged when heated for pro-
oxidised by morpholine N-oxide 11, giving rise to the mix- longed periods in formic acid, indicating that formation of
ture of acetanilide 8 and benzimidazole 9. This result sup- the amine N-oxide is essential. Amine N-oxides did not re-
ports the requirement for two equivalents of oxidant in the act when heated to reflux temperature in acetonitrile for
proposed mechanism, and explains why the intermediate prolonged times, indicating that the acid is also essential.
acetyldihydrobenzimidazole 14 was not isolated.
Quinones 3a–c were obtained by nitration, reduction and
oxidation using Fremy’s salt [K2NO(SO3)2], as depicted in
Scheme 8. Nitration of bis[1,4]oxazinoimidazo[5,4-f]benz-
imidazole (10a), with a mixture of concentrated nitric and
sulfuric acid, gave only the 6-nitro derivative 18 in 79%
yield. Analogous nitration of 10b and 10c gave an insepa-
rable mixture of both possible nitro isomers, which were
hydrogenated and oxidised to give quinones 3b and 3c in 67
and 50% yield, respectively. It is interesting to note that
no iminoquinone (cf. 2b)[6] was observed from the Fremy
oxidation of 10a and 10b, whereas a mixture containing ca.
30% iminoquinone (hydrolyzed with dilute acid) was ob-
tained for 10c. Bis[1,4]oxazinoimidazo[5,4-f]benzimid-
azolequinone 3a was isolated in 58% yield under the same
reaction conditions starting from 18.
Scheme 6. Intermolecular oxidation by morpholine N-oxide.
Heating dimorpholine N-oxide 12 in 90% formic acid
gave [1,4]oxazino[4,3-a]benzimidazole 17 as the sole prod-
uct in 80% yield (Scheme 7). We propose that an acid-cata-
lysed cyclisation occurred, giving N-acetyldihydrobenzimid-
azole intermediate 16, which, in the absence of an external
oxidant, was oxidised through the internal conjugated sys-
tem by the second amine N-oxide, leading to benzimidazole
17.
Scheme 8. Conversion of imidazo[5,4-f]benzimidazoles into quin-
one derivatives: (i) HNO3, H2SO4, 80 °C, 3 h; (ii) H2 (6 bar), Pd-
C, AcOH, room temp., 16 h; (iii) Fremy’s salt, pH 4 buffer, room
temp., 1 h; (iv) HCl (2 m), room temp., 15 min.
Quinone 3a was found to be insoluble in organic sol-
vents, water and aqueous acids, and therefore was not suit-
able for cytotoxicity evaluation. Trifluoroacetic acid (TFA)
was used to solubilize 3a, and an X-ray crystal structure
was obtained of the formed salt, with trifluoroacetate coun-
terions coordinated at the basic nitrogen atoms (7,14-N) of
imidazo[5,4-f]benzimidazole (Figure 3).
Scheme 7. Proposed intramolecular oxidation in formic acid lead-
ing to benzimidazole 17.
1970
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 1967–1975

Ring-Fused Imidazo[5,4-f]benzimidazolequinones
evaluation of unsymmetrical ring-fused imidazo[5,4-f]benz-
imidazolequinones indicated that the oxygen atom of the
1,4-oxazino ring greatly increases toxicity, and replacing the
fused pyridine ring with a pyrrole ring also increases po-
tency. A report is in preparation containing more extensive
structure–activity evaluations, as well as examining the rela-
tionship between the toxicity of these compounds and
NQO1 activity.
Figure 3. Crystal structure of 3,4,10,11-tetrahydro-1H,8H-[1,4]-
oxazino[4,3-a][1,4]oxazino[4Ј,3Ј:1,2]imidazo[5,4-f]benzimidazolium-
6,13-dione bis(trifluoroacetate) [3a·2F3CCO2H].[19]
Experimental Section
Cytotoxicity Evaluation
General: Thin-layer chromatography (TLC) was carried out on alu-
minium-backed plates coated with silica gel (Merck Kieselgel 60
The cytotoxicity of imidazo[5,4-f]benzimidazolequinones
3b and 3c was evaluated against cervical (HeLa CCL-2) and
prostate (DU-145) cancer cells, and, for comparison,
against a human normal fibroblast cell line (GM00637).
Cell viability was determined using the well-established
MTT colorimetric assay (Table 1).[20]
F254). Flash chromatography was carried out using Merck Kieselgel
60H silica gel (particle size 0.040–0.060 mm) with the specified elu-
ent. Melting points were measured with a Stuart Scientific melting
point apparatus SMP3. Infrared spectra were recorded with a Per-
kin–Elmer Spec 1 with ATR attached. NMR spectra were recorded
with a Joel GXFT 400 MHz instrument equipped with a DEC
AXP 300 computer workstation. Chemical shifts for quinone 3a
are reported relative to DSS (4,4-dimethyl-4-silapentane-1-sulfonic
acid sodium salt) as an internal standard. All other compounds are
reported relative to Me4Si as internal standard. All NMR assign-
ments were supported by DEPT and 1H–13C NMR 2D spectra.
High-resolution mass spectroscopy (HRMS) was carried out using
electrospray ionization (ESI) with a Waters LCT Premier XE spec-
trometer by manual peak matching. The precision of all accurate
mass measurements were better than 5 ppm. Elemental analysis
was carried out with a Perkin–Elmer 2400 Series II analyser. HPLC
was carried out with an Agilent Technologies 1200 series instru-
ment fitted with a UV detector operating at 300 nm [hyperclone 5μ
ODS (C18) 120A, 250ϫ4.6 mm; MeOH/water (60:40) containing
1% Et3N; 1 mL min–1]. Hydrogenation reactions were carried out
with a Parr 5500 Series Compact reactor.
Table 1. IC50 values (μm)[a] obtained for 3b and 3c, as well as values
reported[6] for imidazobenzimidazolequinones evaluated under
identical conditions.
Entry Compd.
Cell lines
HeLa CCL-2
GM00637
DU-145
1
2
3
4
5
1b
2a
2b
3b
3c
Ͼ 5.0
Ͼ 5.0
3.63Ϯ0.52
0.60Ϯ0.03
1.27Ϯ0.05
3.33Ϯ0.47
1.67Ϯ0.05
1.55Ϯ0.35
0.58Ϯ0.03
0.95Ϯ0.10
1.73Ϯ0.23
1.99Ϯ0.39
0.30Ϯ0.01
0.66Ϯ0.02
0.79Ϯ0.05
[a] IC50 represents the compound concentration required to reduce
mean cell viability to 50% of the control value, after incubation for
72 h at 37 °C, as determined by using the MTT assay.
The oxygen atom of the 1,4-oxazino ring was found to
significantly increase cytotoxicity against all three cell lines,
as can be seen by comparing 3b with analogue 2a, which
differs in only this atom (Figure 1). Quinone 3c was also
found to be more toxic than the dipyrido analogue 2a, with
greater potency towards the two cancer cell lines than the
normal cell line.
1,4-Difluoro-2,5-dinitrobenzene (5): 2,5-Difluoroaniline (1.555 g,
12.0 mmol) and Ac2O (5.7 mL, 60.2 mmol) were heated at reflux
in MeOH (40 mL) for 30 min. The cooled reaction mixture was
evaporated, ice-water (200 mL) was added and the mixture was
stirred for 3 h. The precipitate was collected, washed with water
and dried to give N-(2,5-difluorophenyl)acetamide (2.018 g,
11.8 mmol). The latter was slowly added to a stirred solution of
HNO3 (70%, 15 mL) and H2SO4 (conc., 30 mL) at 0 °C. The solu-
tion was stirred for 1 h, poured onto crushed ice (400 g) and stirred
for 30 min. The precipitate was collected, washed with water, and
dried to give to give N-(2,5-difluoro-4-nitrophenyl)acetamide 4
(2.498 g, 11.6 mmol). H2O2 (30 wt.-%, 11.95 mL, 0.116 mol) was
added dropwise to the latter in MeSO3H (90%, 40 mL) and the
solution was heated at 55 °C for 2 h. The cooled solution was
poured onto ice (400 g) and the precipitate was collected, washed
with water and dried to give the 5 (1.746 g, 71% from 2,5-difluoro-
aniline); yellow solid; m.p. 103–104 °C (ref.[13] 110–112 °C). 1H
NMR (400 MHz, CDCl3): δ = 8.06 (t, J = 7.5 Hz, 2 H) ppm. 13C
NMR (100 MHz, CDCl3): δ = 150.7 (dd, J = 268.7, 5.5 Hz, CF),
Conclusions
The ring-fused imidazo[5,4-f]benzimidazole heterocyclic
system has been prepared using a new synthetic route in-
volving a key oxidative cyclisation of o-tert-aminoacetani-
lides. Oxone in 90% formic acid was found to give superior
yields of alicyclic and 1,4-oxazino ring-fused adducts. A
new mechanism has been proposed for the formation of
ring-fused benzimidazoles and imidazobenzimidazoles via
a hydrogen-bonded amine N-oxide intermediate. In acidic
solution, amine N-oxides have been shown to act as oxi-
dants in imidazole ring-forming reactions. The symmetrical
bis[1,4]oxazinoimidazo[4,5-f]benzimidazolequinone 3a was
found to have poor solubility, but an X-ray crystal structure
140.4–140.1 (m, CNO ), 117.0–116.5 (m, CH) ppm. IR (neat): ν =
˜
2
1033, 1059, 1108, 1199, 1292, 1343 (NO2), 1412, 1498, 1547 (NO2),
3075 cm–1. HRMS (ESI): m/z calcd. for C6H2N2O4F2-H– 202.9904
[M – H]–; found 202.9911.
4,4Ј-(2,5-Dinitro-1,4-phenylene)dimorpholine (6a): 1,4-Difluoro-2,5-
dinitrobenzene
5
(0.793 g, 3.9 mmol), NaHCO3 (1.638 g,
of its bis(trifluoroacetate) salt was obtained. Cytotoxicity 19.5 mmol) and morpholine (1.37 mL, 15.6 mmol) were heated in
Eur. J. Org. Chem. 2012, 1967–1975
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1971

F. Aldabbagh et al.
FULL PAPER
THF (30 mL) at 50 °C for 3 h. Water (100 mL) was added, and the
precipitate was collected, washed with water and dried to give 6a
(1.262 g, 96%); red solid; m.p. 236–237 °C. 1H NMR (400 MHz,
CDCl3): δ = 7.49 (s, 2 H), 3.82 (t, J = 4.6 Hz, 8 H, OCH2), 3.01 (t,
J = 4.6 Hz, 8 H, NCH2) ppm. 13C NMR (100 MHz, CDCl3): δ =
146.2 (C), 140.4 (C), 118.7 (CH), 66.7 (OCH2), 52.2 (NCH2) ppm.
N,NЈ-[2-(Morpholin-4-yl)-5-(piperidin-1-yl)-1,4-phenylene]diacetamide
(7b): Yield 1.758 g (92%); white solid; m.p. 230–240 °C (dec.). H
1
NMR (400 MHz, CDCl3): δ = 8.50 (br. s, disappears with D2O, 1
H, NH), 8.43 (br. s, disappears with D2O, 1 H, NH), 8.26 (s, 1 H),
8.23 (s, 1 H), 3.84 (t, J = 4.5 Hz, 4 H, OCH2), 2.85 (t, J = 4.5 Hz,
4 H, NCH2), 2.77 (t, J = 5.2 Hz, 4 H, NCH2), 2.18 (s, 6 H, CH3),
1.73–1.64 (m, 4 H, CH2), 1.61–1.53 (m, 2 H, CH2) ppm. 13C NMR
(100 MHz, CDCl3): δ = 168.1 (C=O), 168.0 (C=O), 139.7, 137.1,
129.7, 129.4 (all C), 112.2 (CH), 112.0 (CH), 67.7 (OCH2), 53.8
(NCH2), 52.7 (NCH2), 27.0 (CH2), 25.0 (CH3), 24.0 (CH2) ppm.
IR (neat): ν = 1055, 1064, 1109, 1210, 1230, 1249, 1283, 1300, 1324,
˜
1358 (NO2), 1378, 1409, 1501 (NO2), 1528 (NO2), 2838, 2859,
2974 cm–1. HRMS (ESI): m/z calcd. for C14H18N4O6 + H+
339.1305 [M + H]+; found 339.1290.
IR (neat): ν = 1017, 1035, 1053, 1071, 1115, 1197, 1245, 1298, 1376,
˜
4-(2,5-Dinitro-4-piperidin-1-ylphenyl)morpholine (6b): 1,4-Difluoro-
1414, 1451, 1528, 1661 (C=O), 2491, 2853, 3265 cm–1. HRMS
(ESI): m/z calcd. for C19H28N4O3-H– 359.2083 [M – H]–; found
359.2082.
2,5-dinitrobenzene
5 (0.600 g, 2.9 mmol), NaHCO3 (1.218 g,
14.5 mmol) and morpholine (0.28 mL, 3.2 mmol) were stirred in
THF (30 mL) at room temperature for 16 h. Piperidine (2.87 mL,
29.0 mmol) was added and the mixture was stirred for 1 h. Water
(100 mL) was added and the precipitate was collected and purified
by flash chromatography (petroleum ether/acetone, 19:1) to give 6b
N,NЈ-[2-(Piperidin-1-yl)-5-(pyrrolidin-1-yl)-1,4-phenylene]diacetamide
1
(7c): Yield 1.432 g (79%); white solid; m.p. 214–216 °C. H NMR
(400 MHz, CDCl3): δ = 8.53 (br. s, 1 H, disappears with D2O, NH),
(0.702 g, 71%); red solid; Rf = 0.12 (petroleum ether/acetone, 9:1);
m.p. 175–176 °C. H NMR (400 MHz, CDCl3): δ = 7.50 (s, 1 H), 1 H), 3.05–2.95 (m, 4 H, NCH2), 2.77 (t, J = 5.1 Hz, 4 H, NCH2),
8.24 (s, 1 H), 8.18 (br. s, 1 H, disappears with D2O, NH), 8.12 (s,
1
7.41 (s, 1 H), 3.80 (t, J = 4.5 Hz, 4 H, OCH2), 2.97 (t, J = 4.8 Hz,
8 H, NCH2), 1.73–1.65 (m, 4 H, CH2), 1.63–1.53 (m, 2 H,
CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 147.2, 144.7, 142.3,
138.6 (all C), 119.3 (CH), 117.9 (CH), 66.9 (OCH2), 53.0 (NCH2),
2.17 (s, 6 H, CH3), 1.96–1.89 (m, 4 H, CH2), 1.72–1.66 (m, 4 H,
CH2), 1.61–1.50 (m, 2 H, CH2) ppm. 13C NMR (100 MHz,
CDCl3): δ = 168.1, 168.0 (C=O), 138.4, 136.8, 129.8, 129.1 (all C),
112.9, 110.9 (CH), 53.8, 52.8 (NCH2), 27.1 (CH2), 25.0 (CH3), 24.5
52.5 (NCH ), 25.9 (CH ), 23.8 (CH ) ppm. IR (neat): ν = 1030,
(CH ), 24.1 (CH ) ppm. IR (neat): ν = 1014, 1033, 1108, 1216,
˜
2 2
˜
2
2
2
1049, 1066, 1113, 1210, 1226, 1253, 1323, 1351 (NO2), 1379, 1407,
1499 (NO2), 1516 (NO2), 1530 (NO2), 2855, 2936 cm–1. HRMS
(ESI): m/z calcd. for C15H20N4O5 + H+ 337.1512 [M + H]+; found
337.1503.
1239, 1295, 1366, 1411, 1475, 1526, 1663 (C=O), 2809, 2937,
3318 cm–1. HRMS (ESI): m/z calcd. for C19H28N4O2-H– 343.2134
[M – H]–; found 343.2126.
N-[5-Bromo-2-(morpholin-4-yl)phenyl]acetamide (8): 2,5-Dibro-
monitrobenzene (2.000 g, 7.1 mmol), NaHCO3 (2.982 g,
35.5 mmol) and morpholine (3.10 mL, 35.5 mmol) were stirred in
DMF (50 mL) at 80 °C for 1 h. The cooled mixture was evaporated,
water (100 mL) was added and the mixture was extracted with
EtOAc (5ϫ50 mL). The combined organic extracts were washed
with water (3ϫ50 mL), dried (Na2SO4), and the solvents evapo-
rated. SnCl2·2H2O (8.010 g, 35.5 mmol) in MeOH/THF (1:1,
50 mL) was added to the residue and the mixture was heated at
reflux for 1 h. The cooled mixture was evaporated, neutralised with
NaOH (5 m), and the precipitate was collected and dissolved in
MeOH (50 mL). Ac2O (6.7 mL, 71.0 mmol) was added and the
1-[2,5-Dinitro-4-(pyrrolidin-1-yl)phenyl]piperidine
(6c):
1,4-Di-
fluoro-2,5-dinitrobenzene 5 (0.750 g, 3.7 mmol), NaHCO3 (1.554 g,
18.5 mmol) and piperidine (0.41 mL, 4.1 mmol) were stirred in
THF (30 mL) at –30 °C for 16 h. Pyrrolidine (3.04 mL, 37.0 mmol)
was added and the mixture was stirred at room temperature for
1 h. Water (100 mL) was added and the precipitate was collected
and purified by flash chromatography (petroleum ether/acetone,
19:1) to give the 6c (0.818 g, 69%); red solid; Rf 0.22 (petroleum
ether/acetone, 9:1); m.p. 162–164 °C. 1H NMR (400 MHz, CDCl3):
δ = 7.54 (s, 1 H), 7.14 (s, 1 H), 3.18 (t, J = 6.5 Hz, 4 H, NCH2),
2.85 (t, J = 5.3 Hz, 4 H, NCH2), 2.02–1.96 (m, 4 H), 1.70–1.63 (m,
4 H, CH2), 1.56–1.49 (m, 2 H) ppm. 13C NMR (100 MHz, CDCl3): mixture was heated at reflux for 30 min. The cooled mixture was
δ = 148.8, 138.5, 138.1, 135.5 (all C), 120.7, 111.7 (CH), 54.4, 50.6 evaporated, ice-water was added (200 mL) and the mixture was
(NCH ), 26.3, 25.8, 23.9 (all CH ) ppm. IR (neat): ν = 1032, 1106, stirred for 3 h. The precipitate was collected, washed with water
˜
2
2
1211, 1221, 1248, 1264, 1306 (NO2), 1360, 1449, 1497, 1534 (NO2),
1630, 2856, 2946 cm–1. HRMS (ESI): m/z calcd. for
C15H20N4O4 + H+ 321.1563 [M + H]+; found 321.1578.
and dried to give 8; yield 1.766 g (83%); white solid; m.p. 139–
141 °C. 1H NMR (400 MHz, CDCl3): δ = 8.55 (d, J = 2.2 Hz, 1
H, 6-H), 8.42 (br. s, disappears with D2O, 1 H, NH), 7.16 (dd, J =
8.5, 2.2 Hz, 1 H, 4-H), 7.00 (d, J = 8.5 Hz, 1 H, 3-H), 3.84 (t, J =
4.6 Hz, 4 H, OCH2), 2.82 (t, J = 4.6 Hz, 4 H, NCH2), 2.19 (s, 3 H,
CH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 168.1 (C=O), 139.5
(C), 134.8 (C), 126.7 (4-CH), 122.4 (6-CH), 122.2 (3-CH), 119.2
General Procedure for the Reduction and Acetylation of Nitro-
benzenes: 2,5-Dinitrobenzene 6a–c (5.3 mmol), Pd-C (5%, 0.178 g)
and Ac2O (5.0 mL, 53.0 mmol) in glacial AcOH (50 mL) were
stirred under H2 (6 bar) at room temperature for 16 h. The mixture
was filtered, evaporated, Na2CO3 (5%, 50 mL) was added and the
mixture was stirred for 1 h. The precipitate was collected, washed
with water, and dried to give 7a–c.
(C), 67.6 (OCH ), 52.6 (NCH ), 25.1 (CH ) ppm. IR (neat): ν =
˜
2
2
3
1033, 1112, 1226, 1374, 1410, 1507, 1683 (C=O), 2951, 3332 cm–1.
HRMS (ESI): m/z calcd. for C12H15N2O279Br – H– 297.0239 [M –
H]–; found 297.0237.
N,NЈ-[2,5-Di(morpholin-4-yl)-1,4-phenylene]diacetamide (7a): Yield
1.606 g (84%); white solid; m.p. 266–268 °C. H NMR (400 MHz,
CDCl3): δ = 8.42 (br. s, disappears with D2O, 2 H, NH), 8.27 (s, 2
H), 3.84 (t, J = 4.5 Hz, 8 H, OCH2), 2.85 (t, J = 4.5 Hz, 8 H,
NCH2), 2.19 (s, 6 H, CH3) ppm. 13C NMR (100 MHz, CDCl3): δ
= 168.1 (C=O), 137.8 (C), 129.6 (C), 112.4 (CH), 67.7 (OCH2),
General Procedure for the Synthesis of Fused Benzimidazoles and
Imidazo[5,4-f]benzimidazoles: O-tert-Aminoacetanilides 8, 7a–c
(1.5 mmol) and Oxone (2.766 g, 4.5 mmol per cyclisation) were
heated in HCO2H (90%, 30 mL) at 40 °C for 6 h. The mixture was
evaporated, water (50 mL) was added and the solution was neutral-
ised with solid Na2CO3. The precipitate was collected, washed with
water and dried to give 9, 10a–c, respectively.
1
52.6 (NCH ), 25.0 (CH ) ppm. IR (neat): ν = 1007, 1057, 1071,
˜
2
3
1113, 1179, 1229, 1259, 1294, 1368, 1418, 1525, 1677 (C=O), 2843,
2969, 3341 cm–1. HRMS (ESI): m/z calcd. for C18H26N4O4+Na+ 8-Bromo-3,4-dihydro-1H-[1,4]oxazino[4,3-a]benzimidazole (9): Yield
385.1852 [M + Na]+; found 385.1847.
0.304 g (80%); white solid; m.p. 213–214 °C. H NMR (400 MHz,
1
1972
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 1967–1975

Ring-Fused Imidazo[5,4-f]benzimidazolequinones
CDCl3): δ = 7.84 (s, 1 H, 9-H), 7.36 (dd, J = 8.5, 1.4 Hz, 1 H, 7- NMR (400 MHz, CDCl3): δ = 9.13 (s, 2 H), 4.65 (t, J = 12.8 Hz,
H), 7.19 (d, J = 8.5 Hz, 1 H, 6-H), 5.01 (s, 2 H, 1-CH2), 4.22–4.11
(m, 4 H, 3,4-CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 149.1,
144.1, 133.1 (all C), 125.5 (7-CH), 122.5 (9-CH), 115.6 (C), 110.0
4 H), 3.95–3.87 (m, 8 H), 3.47 (d, J = 10.6 Hz, 4 H), 2.20 (s, 6 H,
CH3) ppm, NH not observed. 13C NMR (100 MHz, CDCl3): δ =
169.3 (C=O), 138.9 (C), 130.2 (C), 113.1 (CH), 66.3 (CH2), 61.6
(6-CH), 65.5 (1-CH ), 63.9 and 42.2 (3,4-CH ) ppm. IR (neat): ν =
(CH ), 25.7 (CH ) ppm. IR (neat): ν = 1019, 1033, 1054 (N–O),
˜
2 3
˜
2
2
1049, 1091, 1112, 1170, 1317, 1333, 1369, 1407, 1432, 1481, 1520,
2 9 3 7 , 2 9 7 6 , 3 0 3 7 c m– 1 . H R M S ( E S I ) : m/ z c a l c d . fo r
C10H9N2O81Br + H+ 254.9956 [M + H]+; found 254.9957.
1118, 1247, 1301, 1404, 1428, 1475, 1532, 1655 (C=O), 2940, 2973,
3223, 3512 cm–1. HRMS (ESI): m/z calcd. for C18H26N4O6 + H+
395.1931 [M + H]+; found 395.1912.
3,4,10,11-Tetrahydro-1H,8H-[1,4]oxazino[4,3-a][1,4]oxazino[4Ј,3Ј:
1,2]imidazo[5,4-f]benzimidazole (10a): Yield 0.235 g (58%); white
solid; m.p. Ͼ350 °C. 1H NMR (400 MHz, CDCl3): δ = 7.58 (s, 2
H), 5.06 (s, 4 H, 1,8-CH2), 4.27–4.21 (m, 8 H, CH2) ppm. 13C NMR
(100 MHz, CDCl3): δ = 148.9, 139.9, 132.1 (all C), 97.7 (CH), 65.7
N-(8-Morpholin-4-yl-3,4-dihydro-1H-[1,4]oxazino[4,3-a]benzimid-
azol-7-yl)acetamide (17): Dimorpholine N-oxide 12 (0.150 g,
0.4 mmol) was heated in HCO2H (90%, 20 mL) at 40 °C for 6 h.
The mixture was evaporated and Na2CO3 (5%, 50 mL) was added.
The precipitate was collected, washed with water and dried to give
17 (96 mg, 80 %); white solid; m.p. 251–252 °C. 1H NMR
(400 MHz, CDCl3): δ = 8.88 (br. s, disappears with D2O, 1 H, NH),
8.47 (s, 1 H), 7.52 (s, 1 H), 4.98 (s, 2 H, 1-CH2), 4.18–4.13 (m, 4
H, 3,4-CH2), 3.91–3.86 (m, 4 H, 2Ј,6Ј-CH2), 2.92–2.85 (m, 4 H,
3Ј,5Ј-CH2), 2.24 (s, 3 H, CH3) ppm. 13C NMR (100 MHz, CDCl3):
δ = 168.2 (C=O), 148.2, 138.5, 137.6, 131.6, 129.6 (all C), 111.9
(CH), 99.7 (CH), 67.9 (2Ј,6Ј-CH2), 65.6 (1-CH2), 64.1 (3-CH2), 53.5
(1,8-CH ), 64.2 (CH ), 42.2 (CH ) ppm. IR (neat): ν = 1099, 1106,
˜
2
2
2
1149, 1191, 1275, 1336, 1353, 1426, 1447, 1490, 1541, 3137 cm–1.
HRMS (ESI): m/z calcd. for C14H14N4O2 + H+ 271.1195 [M +
H]+; found 271.1199.
3,4,8,9,10,11-Hexahydro-1H-[1,4]oxazino[4,3-a]pyrido[1Ј,2Ј:1,2]imid-
azo[5,4-f]benzimidazole (10b): Yield 0.189 g (47%); white solid; m.p.
330–340 °C (dec.). 1H NMR (400 MHz, CDCl3): δ = 7.53 (s, 1 H),
7.52 (s, 1 H), 5.04 (s, 2 H, 1-CH2), 4.25–4.20 (m, 4 H, 3 & 4-CH2),
4.13 (t, J = 6.1 Hz, 2 H, 11-CH2), 3.11 (t, J = 6.3 Hz, 2 H, 8-CH2),
2.20–2.14 (m, 2 H, CH2), 2.07–2.01 (m, 2 H, CH2) ppm. 13C NMR
(100 MHz, CDCl3): δ = 151.4, 146.9, 138.8, 138.1, 131.4, 130.4 (all
C), 96.2 (CH), 95.5 (CH), 64.4 (1-CH2), 62.8 (3-CH2), 41.3 (11-
CH2), 40.8 (4-CH2), 24.4 (8-CH2), 21.4 (CH2), 19.5 (CH2) ppm. IR
(3Ј,5Ј-CH ), 42.3 (4-CH ), 25.2 (CH ) ppm. IR (neat): ν = 1033,
˜
2
2
3
1046, 1097, 1108, 1164, 1242, 1338, 1365, 1459, 1471, 1507, 1591,
1672 (C=O), 2846, 2957 cm–1. HRMS (ESI): m/z calcd. for
C16H20N4O3-H– 315.1457 [M – H]–; found 315.1456.
6-Nitro-3,4,10,11-tetrahydro-1H,8H-[1,4]oxazino[4,3-a][1,4]oxazino-
[4Ј,3Ј:1,2]imidazo[5,4-f]benzimidazole (18): Imidazo[5,4-f]benzimid-
azole 10a (0.321 g, 1.2 mmol) was heated in concd. HNO3 (5 mL)
and concd. H2SO4 (10 mL) at 80 °C for 3 h. The cooled mixture
was diluted with water (100 mL) and neutralised with solid
Na2CO3. The precipitate was collected, washed with water and
dried to give 18 (0.299 g, 79%); yellow solid; m.p. Ͼ350 °C. 1H
NMR (400 MHz, CDCl3): δ = 7.84 (s, 1 H), 5.13 (s, 2 H, 1,8-CH2),
5.09 (s, 2 H, 1,8-CH2), 4.33 (m, 2 H, CH2), 4.27 (s, 4 H, CH2), 4.18
(t, J = 5.4 Hz, 2 H, CH2) ppm. 13C NMR (100 MHz, CDCl3): δ =
152.1, 151.1, 141.3, 134.5, 132.3, 125.1, 125.0 (all C), 104.1 (CH),
65.8, 65.7 (1 & 8-CH2), 64.4, 63.8, 45.6, 42.5 (all CH2) ppm. IR
(neat): ν = 1089, 1147, 1187, 1280, 1360, 1417, 1445, 1486, 1537,
˜
2944 cm–1. HRMS (ESI): m/z calcd. for C15H16N4O + H+ 269.1402
[M + H]+; found 269.1391.
2,3,7,8,9,10-Hexahydro-1H-pyrido[1,2-a]pyrrolo[1Ј,2Ј:1,2]imidazo-
[5,4-f]benzimidazole (10c): Yield 0.197 g (52%); white solid; m.p.
1
280–290 °C (dec.). H NMR (400 MHz, CDCl3): δ = 7.51 (d, J =
0.7 Hz, 1 H, 5 or 12-H), 7.49 (d, J = 0.7 Hz, 1 H, 5 or 12-H), 4.17–
4.11 (m, 4 H, 1,7-CH2), 3.12–3.07 (m, 4 H, 3,10-CH2), 2.77–2.70
(m, 2 H, 2-CH2), 2.20–2.12 (m, 2 H, CH2), 2.02–2.00 (m, 2 H,
CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 161.7, 152.3, 145.7,
139.6, 132.0, 130.0 (all C), 97.7 (5,12-CH), 43.0, 42.6 (1,7-CH2),
(neat): ν = 1054, 1095, 1116, 1210, 1273, 1331 (NO ), 1356 (NO ),
˜
2
2
1435, 1501 (NO2), 2867, 3021 cm–1. HRMS (ESI): m/z calcd. for
26.0 (2-CH ), 25.8, 23.9, 22.8, 20.9 (all CH ) ppm. IR (neat): ν =
˜
2
2
C14H13N5O4 + H+ 316.1046 [M + H]+; found 316.1031.
1097, 1131, 1162, 1265, 1313, 1334, 1353, 1412, 1433, 1443, 1484,
1541, 2941 cm–1. HRMS (ESI): m/z calcd. for C14H16N4 + H+
253.1453 [M + H]+; found 253.1453.
3,4,10,11-Tetrahydro-1H,8H-[1,4]oxazino[4,3-a][1,4]oxazino[4Ј,3Ј:
1,2]imidazo[5,4-f]benzimidazol-6,13-dione (3a): 6-Nitroimidazo[5,4-
f]benzimidazole 18 (0.726 g, 2.3 mmol) and Pd-C (5%, 73 mg) were
stirred under H2 (6 bar) in glacial AcOH (50 mL) at room tempera-
ture for 16 h. The mixture was filtered, evaporated, and KH2PO4
(0.2 m, 100 mL) was added. A separate solution of Fremy’s salt
(1.852 g, 6.9 mmol) in KH2PO4 (0.2 m, 50 mL) was added and the
mixture was stirred at room temperature for 1 h. The resultant pre-
cipitate was collected, washed with water and dried to give 3a
(0.401 g, 58%); yellow solid; m.p. Ͼ350 °C. 1H NMR (400 MHz,
TFA): δ = 5.59 (br. s, 4 H), 5.00 (br. s, 4 H), 4.65 (br. s, 4 H, 4,11-
CH2) ppm. 13C NMR (100 MHz, TFA): δ = 167.0 (C=O), 152.1,
134.3, 132.0 (all C), 65.0 (CH2), 64.4 (CH2), 48.7 (4,11-CH2) ppm.
N-[5-Bromo-2-(4-oxidomorpholin-4-yl)phenyl]acetamide (11): N-[5-
Bromo-2-(morpholin-4-yl)phenyl]acetamide 8 (0.342 g, 1.1 mmol)
and mCPBA (70 %, 0.678 g, 2.75 mmol) were heated in CH2Cl2
(40 mL) at reflux for 1 h. The cooled mixture was washed with
Na2CO3 (5%, 3ϫ50 mL), dried (Na2SO4) and evaporated to give
11 (0.302 g, 84 %); white solid; m.p. 164–166 °C. 1H NMR
(400 MHz, CDCl3): δ = 8.78 (d, J = 2.2 Hz, 1 H, 6-H), 7.16 (d, J
= 8.9 Hz, 1 H, 3-H), 7.12 (dd, J = 8.9, 2.2 Hz, 1 H, 4-H), 4.64–
4.57 (m, 2 H), 3.85–3.72 (m, 4 H), 3.44 (d, J = 11.0 Hz, 2 H), 2.12
(s, 3 H, CH3) ppm, NH not observed. 13C NMR (100 MHz,
CDCl3): δ = 168.8 (C=O), 138.3 (C), 136.5 (C), 126.0 (6-CH), 125.4
(4-CH), 124.0 (C), 119.2 (3-CH), 65.9 (OCH2), 61.6 (NCH2), 25.6
IR (neat): ν = 1051, 1104, 1128, 1369, 1439, 1485, 1502, 1664
˜
(C=O), 2949 cm–1. HRMS (ESI): m/z calcd. for C14H12N4O4 + H+
(CH ) ppm. IR (neat): ν = 1020, 1106 (N–O), 1226, 1286, 1370,
˜
3
301.0937 [M + H]+; found 301.0937.
1409, 1443, 1514, 1590, 1655 (C=O), 2942, 2969 cm–1. HRMS
(ESI): m/z calcd. for C12H15N2O379Br-H– 313.0188 [M – H]–; found
313.0176.
3,4,8,9,10,11-Hexahydro-1H-[1,4]oxazino[4,3-a]pyrido[1Ј,2Ј:1,2]imid-
azo[5,4-f]benzimidazol-6,13-dione (3b): Imidazo[5,4-f]benzimidazole
10b (0.381 g, 1.4 mmol) was heated in concd. HNO3 (5 mL) and
concd. H2SO4 (10 mL) at 80 °C for 3 h. The cooled mixture was
diluted with water (100 mL), neutralised with solid Na2CO3, and
extracted with CH2Cl2 (5ϫ50 mL). The combined organic extracts
were dried (Na2SO4) and evaporated to give a mixture of nitro
N,NЈ-[2,5-Bis(4-oxidomorpholin-4-yl)-1,4-phenylene]diacetamide
(12): Diacetamide 7a (0.204 g, 0.6 mmol) and mCPBA (70 %,
0.740 g, 3.0 mmol) were heated in acetone at reflux for 1 h. The
resultant white precipitate was collected, washed with acetone and
1
dried to give 12 (0.168 g, 76%); white solid; m.p. 234–236 °C. H
Eur. J. Org. Chem. 2012, 1967–1975
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1973

F. Aldabbagh et al.
FULL PAPER
isomers (0.383 g, 1.2 mmol), which were stirred under H2 (6 bar) in
glacial AcOH (50 mL), with Pd-C (5%, 38 mg) at room tempera-
ture for 16 h. The mixture was filtered, evaporated, and KH2PO4
(0.2 m, 100 mL) was added. A separate solution of Fremy’s salt
(0.966 g, 3.6 mmol) in KH2PO4 (0.2 m, 50 mL) was added, and the
(MEM) Eagle-Earle BSS supplemented with 15% non-heat-inacti-
vated fetal bovine serum (FBS), penicillin-streptomycin, 2 mm l-
glutamine, 2ϫ essential and non-essential amino acids, and vita-
mins. HeLa-CCL-2 cervical cancer cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% non-
mixture was stirred at room temperature for 1 h. Extraction with heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin,
CH2Cl2 (5ϫ50 mL), drying (Na2SO4), evaporation and recrystalli-
sation from water gave 3b (0.284 g, 67 %); yellow solid; m.p.
Ͼ350 °C. H NMR (400 MHz, CDCl3): δ = 4.94 (s, 2 H, 1-CH2),
2 mm l-glutamine, and MEM nonessential amino acids. DU-145
prostate cancer cells were grown in RPMI-1640 medium sup-
plemented with 10% non-heat-inactivated fetal bovine serum, peni-
1
4.38 (t, J = 5.1 Hz, 2 H, 4-CH2), 4.32 (t, J = 5.9 Hz, 2 H, 11-CH2), cillin-streptomycin and 2 mm l-glutamine.
4.16 (t, J = 5.1 Hz, 2 H, 3-CH2), 2.98 (t, J = 6.2 Hz, 2 H, 8-CH2),
Cytotoxicity Measurement: Cell viability was determined using the
2.09–1.98 (m, 4 H, 9,10-CH2) ppm. 13C NMR (100 MHz, CDCl3):
δ = 171.4, 171.3 (C=O), 151.9, 147.7, 141.7, 141.6, 130.5, 130.3 (all-
C), 65.0 (1-CH2), 63.5 (3-CH2), 45.7 (11-CH2), 45.0 (4-CH2), 25.1
MTT colorimetric assay.[20] Normal cells were plated into 96-well
plates at a density of 10,000 cells per well (200 μL per well) and
allowed to adhere for 24 h. HeLa cells were plated into 96-well
plates at a density of 1000 cells per well (100 μL per well) and
allowed to adhere for 24 h. DU-145 cells were plated into 96-well
plates at a density of 2000 cells per well (200 μL per well) and
allowed to adhere for 24 h.
(8-CH ), 22.2, 19.7 (9,10-CH ) ppm. IR (neat): ν = 1045, 1071,
˜
2
2
1099, 1116, 1136, 1223, 1249, 1302, 1315, 1374, 1433, 1479, 1496,
1657 (C=O), 2869, 2953 cm–1. HRMS (ESI): m/z calcd. for
C15H14N4O3 + H+ 299.1144 [M + H]+; found 299.1148; elemental
analysis calcd. (%) for C15H14N4O3: C 60.40, H 4.73, N 18.78;
found C 60.00, H 4.64, N 18.80.
Compounds were applied in ethanol (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 ex-
posed to an equivalent concentration of ethanol alone. MTT
(20 μL, 5 mg/mL solution) was added, and the cells were incubated
for 3 h. The supernatant was removed by pipetting. The resultant
MTT formazan crystals were dissolved in DMSO (100 μL) and ab-
sorbance was measured on a plate reader at 550 nm with a refer-
ence at 690 nm. Cell viability is expressed as a percentage of the
EtOH-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).
2,3,7,8,9,10-Hexahydro-1H-pyrido[1,2-a]pyrrolo[1Ј,2Ј:1,2]imidazo-
[5,4-f]benzimidazol-5,12-dione (3c): Pyrido[1,2-a]pyrrolo[1Ј,2Ј:1,2]-
imidazo[5,4-f]benzimidazole 10c (0.100 g, 0.4 mmol) was heated in
concd. HNO3 (5 mL) and concd. H2SO4 (10 mL) at 80 °C for 3 h.
The cooled mixture was diluted with water (100 mL), neutralised
with solid Na2CO3, and extracted with CH2Cl2 (5ϫ50 mL). The
combined organic extracts were dried (Na2SO4) and evaporated to
give a mixture of nitro isomers (97 mg, 0.3 mmol), which were
stirred under H2 (6 bar) in glacial AcOH (50 mL) with Pd-C (5%,
38 mg) at room temperature for 16 h. The mixture was filtered,
evaporated, and KH2PO4 (0.2 m, 100 mL) was added. A separate
solution of Fremy’s salt (0.966 g, 3.6 mmol) in KH2PO4 (0.2 m,
50 mL) was added and the mixture was stirred at room temperature
for 1 h. Extraction with CH2Cl2 (5ϫ50 mL), drying (Na2SO4) and
evaporation gave a mixture of compounds containing ca. 30%
iminoquinone. HCl (2 m, 50 mL) was added to the residue and the
solution was stirred for 15 min. The acid solution was neutralised
with solid Na2CO3 and extracted with CH2Cl2 (5ϫ50 mL). The
combined organic extracts were dried (Na2SO4), evaporated and
recrystallised from water to give 3c (56 mg, 50%); yellow solid; m.p.
Supporting Information (see footnote on the first page of this arti-
1
cle): H NMR and 13C NMR spectra, HPLC chromatogram and
cell viability plots are available.
Acknowledgments
We thank the Irish Research Council for Science Engineering and
Technology funded by the National Development Plan for award-
ing an Embark Scholar Award to Vincent Fagan. We also gratefully
acknowledge for financial support the Science Foundation Ireland
for a Research Frontiers Programme Award (07/RFP/CHEF227).
We are grateful to Prof. William Watson (School of Medicine &
Medical Sciences, University College Dublin, Ireland) for the DU-
145 cell line.
1
Ͼ350 °C. H NMR (400 MHz, CDCl3): δ = 4.34 (t, J = 5.9 Hz, 2
H, 1 or 7-CH2), 4.28 (t, J = 7.2 Hz, 2 H, 1 or 7-CH2), 2.99–2.93
(m, 4 H, 3,10-CH2), 2.76–2.68 (m, 2 H, 2-CH2), 2.08–1.96 (m, 4
H, 8,9-CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 171.9, 171.2
(C=O), 160.3, 151.4, 146.3, 141.3, 130.8, 129.9 (all-C), 45.7, 45.4
(1,7-CH2), 26.5 (2-CH2), 25.1, 23.1 (3,10-CH2), 22.3, 19.8 (8,9-
CH ) ppm. IR (neat): ν = 1043, 1072, 1164, 1222, 1270, 1302, 1375,
˜
2
1404, 1440, 1482, 1654 (C=O), 2871, 2952 cm–1. HRMS (ESI): m/z
calcd. for C15H14N4O2 + H+ 283.1195 [M + H]+; found 283.1187.
Cell Culture and Cytotoxicity Evaluation
[1] Y. Chen, L. Hu, Med. Res. Rev. 2009, 29, 29–64.
[2] L. Garuti, M. Roberti, D. Pizzirani, Mini-Rev. Med. Chem.
2007, 7, 481–489.
Cell Lines: An SV40-transformed normal human skin fibroblast
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 DU-145 prostate cancer cell line (ATCC
repository number HTB-81) was obtained from Prof. R. W. G. Wat-
son, School of Medicine & Medical Science, University College
Dublin, Ireland.
[3] D. Ross, D. Siegel, Methods Enzymol. 2004, 382, 115–144.
[4] W. G. Schulz, E. B. Skibo, J. Med. Chem. 2000, 43, 629–638.
[5] A. Suleman, E. B. Skibo, J. Med. Chem. 2002, 45, 1211–1220.
[6] V. Fagan, S. Bonham, M. P. Carty, F. Aldabbagh, Org. Biomol.
Chem. 2010, 8, 3149–3156.
[7] W. R. Bowman, J. M. D. Storey, Chem. Soc. Rev. 2007, 36,
1803–1822.
[8] M. Lynch, S. Hehir, P. Kavanagh, D. Leech, J. O’Shaughnessy,
M. P. Carty, F. Aldabbagh, Chem. Eur. J. 2007, 13, 3218–3226.
[9] I. Baxter, D. W. Cameron, J. Chem. Soc. C 1968, 1747–1752.
[10] I. Baxter, D. W. Cameron, Chem. Ind. 1967, 1403.
[11] O. Meth-Cohn, J. Chem. Soc. C 1971, 1356–1357.
Cell Culture: The SV40-transformed normal human skin fibroblast
cell line (GM00637) was grown in minimum essential media
1974
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 1967–1975

Ring-Fused Imidazo[5,4-f]benzimidazolequinones
[12] O. Meth-Cohn, H. Suschitzky, J. Chem. Soc. 1963, 4666–4669.
[18]
[19]
M. D. Nair, R. Adams, J. Am. Chem. Soc. 1961, 83, 3518–3521.
[13] G. W. Bradley, D. G. Morris, J. Chem. Res. Synop. 1993, 220–
CCDC-816319 (for 12) and -816444 [for 3a·2F3CCO2H] con-
tain the supplementary crystallographic data for this paper.
These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
221.
[14] J. J. S. Lamba, J. M. Tour, J. Am. Chem. Soc. 1994, 116, 11723–
11736.
[15] K. Fahey, F. Aldabbagh, Tetrahedron Lett. 2008, 49, 5235–
5237.
[20] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.
Received: November 24, 2011
Published Online: February 13, 2012
[16] R. Zhou, E. B. Skibo, J. Med. Chem. 1996, 39, 4321–4331.
[17] A. R. Freedman, D. S. Payne, A. R. Day, J. Heterocycl. Chem.
1966, 3, 257–259.
Eur. J. Org. Chem. 2012, 1967–1975
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1975