Synthesis and toxicity towards normal and cancer cell lines of benzimidazolequinones containing fused aromatic rings and 2-aromatic ring substituents

Article abstract of DOI:10.1016/j.ejmech.2010.05.025

A facile 6-exo-trig cyclization of σ-aromatic radicals has allowed the synthesis of various aromatic ring fused benzimidazoles and benzimidazolequinones. The most highly conjugated naphthyl fused benzimidazolequinone, (5-methyl-5,6-dihydrobenzimidazo[2,1-a]benzo[f] isoquinoline-8,11-dione) showed the highest specificity towards human cervical (HeLa) and prostate (DU145) cancer cell lines with little toxicity towards a human normal (GM00637) cell line at doses of <1 μM. In contrast, 2-aromatic ring substituted (benzimidazole-4,7-diones) analogues, benzimidazolequinone with a pyridine ring and mitomycin C were more toxic than the highly conjugated naphthyl fused benzimidazolequinone towards the normal cell line. The naphthyl fused benzimidazolequinone showed the highest specificity towards human cervical (HeLa) and prostate (DU145) cancer cell lines, with little toxicity towards a human normal (GM00637) cell line at doses of <1 μM.

Full text of DOI:10.1016/j.ejmech.2010.05.025

European Journal of Medicinal Chemistry 45 (2010) 3762e3769
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and toxicity towards normal and cancer cell lines of
benzimidazolequinones containing fused aromatic rings and 2-aromatic
ring substituents
,
Eoin Moriartya, Miriam Carr a, Sarah Bonham a, Michael P. Carty b, Fawaz Aldabbagh a
*
a School of Chemistry, National University of Ireland, Galway, Ireland
b Biochemistry, School of Natural Sciences, National University of Ireland, Galway, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
A facile 6-exo-trig cyclization of s-aromatic radicals has allowed the synthesis of various aromatic ring
Received 15 April 2010
Received in revised form
7 May 2010
Accepted 10 May 2010
Available online 19 May 2010
fused benzimidazoles and benzimidazolequinones. The most highly conjugated naphthyl fused benzi-
midazolequinone, (5-methyl-5,6-dihydrobenzimidazo[2,1-a]benzo[f]isoquinoline-8,11-dione) showed
the highest specificity towards human cervical (HeLa) and prostate (DU145) cancer cell lines with little
toxicity towards a human normal (GM00637) cell line at doses of <1 mM. In contrast, 2-aromatic ring
substituted (benzimidazole-4,7-diones) analogues, benzimidazolequinone with a pyridine ring and
mitomycin C were more toxic than the highly conjugated naphthyl fused benzimidazolequinone towards
the normal cell line.
Keywords:
Bioreductive
Heterocyclic compounds
NQO1
Ó 2010 Elsevier Masson SAS. All rights reserved.
Quinones
1. Introduction
We now report the first biological evaluation of benzimidazole-
quinones containingfusedaromatic rings(aryl, pyridinyl ornaphthyl
Many antitumor agents require metabolic activation to exert
their cytotoxic or cytostatic effects [1,2]. The clinically used natural
product, mitomycin C (MMC, Fig. 1) is a prodrug that requires bio-
reductive activation in ordertoexert antitumoractivitybydamaging
DNA through monofunctional and bifunctional alkylation [3]. One or
two electron reductive activation occurs via the quinone moiety
leading to reactive sites at C-1 (after aziridinyl ring-opening) and C-
10 (after carbamate elimination) for DNA alkylation. Benzimidazo-
lequinone alternatives containing aziridinyl [4e8] and other labile
groups [9e11] have been developed; however there are a number of
reported benzimidazolequinones that have anticancer activity
despite lacking reactive sites that upon reductive activation can
undergo DNA alkylation [12e16]. This includes the series of benz-
imidazole-4,7-diones containing pyridinyl [12,14] and thiazolyl [13]
groups at the 2-position, reported by Garuti and co-workers. The
nature of the quinone substituent, and the structure of the hetero-
cyclic group attached at the benzimidazole-2-position were shown
to influence cytotoxicity against selected human tumor cell lines.
Others have prepared benzimidazole-4,7-dione with a 2-phenyl
substituent reporting little toxicity towards human tumor cell lines
(incl. DU145 [17]), and 6-arylamino-5-chloro-2-(2-pyridinyl)benz-
imidazole-4,7-diones exhibiting antifungal activity [18].
rings: compounds 1e3, Fig. 1), and cytotoxicity of 2-aromatic ring
substituted (benzimidazole-4,7-diones) analogues (compounds
4e6). The premise is that the highlyconjugated structure will lead to
an increased tendency for bioreduction due to the subsequent
stability of the reduced intermediates formed. This is expected to be
manifested in selective toxicity towards cancer cell lines having
elevated levels of reductase enzymes. There is evidence for several
benzimidazolequinones [4,11,19] acting as substrates for the obligate
two electron quinone reductase enzyme, NAD(P)H:quinone oxido-
reductase (NQO1, also known as DT-diaphorase [20,21]). This article
evaluates the cytotoxicity of compounds using two human cancer
cell lines known to have high NQO1 activity: cervical (HeLa [22]) and
prostate (DU145 [23]) cancer cell lines. Cytotoxicity towards these
cancer cell lines is compared to a normal human skin fibroblast cell
line (GM00637 [7,8,16]).
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of benzimidazole-4,7-diones possessing 2-aromatic
ring substituents
2-Phenyl and 2-pyridinyl benzimidazoles 7 and 8 (Scheme 1)
were obtained using literature procedures [14,18] from the
* Corresponding author. Tel.: þ353 91 493120; fax: þ353 91 495700.
E-mail address: fawaz.aldabbagh@nuigalway.ie (F. Aldabbagh).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.025

E. Moriarty et al. / European Journal of Medicinal Chemistry 45 (2010) 3762e3769
3763
O
O
10
O
O
OCONH2
13
5
14
N
N
N
N
H2N
Me
OMe
10
9
1
3
1
2
X
NH
N
4
6
O
O
1: X = CH
2: X = N
3
MMC
O
O
O
O
N
N
N
N
X
4: X = CH
5: X = N
6
Fig. 1. Mitomycin C and aromatic ring fused and 2-substituted benzimidazolequinones.
condensation of 3,6-dimethoxybenzene-1,2-diamine with the
appropriate aromatic aldehyde. Analogous condensation using
2-naphthaldehyde gave novel 4,7-dimethoxy-2-(2-naphthyl)-1H-
benzimidazole 11 in 79% yield (Scheme 2). Reaction with ethyl
iodide in the presence of sodium hydride gave 1-ethyl-4,7-dime-
thoxy-2-aromatic ring substituted benzimidazoles in yields of
54e79%. Formation of target quinones 4e6 via hydrobromic acid
induced demethylation of the 4,7-dimethoxy substituents of 9, 10
and 12, and oxidation of the reactive hydroquinones occurred in
61e77% yield. It should be noted that the latter intermediates are
difficult to isolate and characterize prior to room temperature
oxidation using ferric chloride, and thus are not evaluated in this
article.
from an ammonium chloride-mediated condensation using nic-
otinaldehyde, according to a modification of a literature method for
making 2-aryl substituted benzimidazoles [26]. Radical precursors
14a and 14b and 19a and 19b were obtained in yields of 86e95% by
reaction with allyl bromide in the presence of sodium hydride. The
radical cyclizations proceeded to give 16a and 16b and 20a and 20b
in about 70% yield. The presence of 4,7-methoxy substituents in 14b
and 19b was found to have no discernible effect on the cyclization.
Target quinones 2 and 3 were obtained in over 75% yield from
dimethoxy cyclized adducts 16b and 20b respectively using the
well-established hydrobromic acid/ferric chloride procedure [16].
2.2. Cytotoxicity evaluation
2.1.2. Synthesis of aromatic ring fused benzimidazolequinones
The formation of the new 5,6-dihydrobenzimidazo[2,1-f]-1,
6-naphthyridine ring system was achieved via a 6-exo-trig radical
The cytotoxicity of aromatic ring fused benzimidazolequinones
1e3 and non-cyclized analogues with an aromatic ring at the
2-position 4e6 was evaluated against cancer cell lines reported to
contain high DT-diaphorase activity: cervical (HeLa [22]) and
prostate (DU145 [23]) cancer. For comparison purposes cytotoxicity
against a normal human skin fibroblast cell line (GM00637) was
also determined. Each cell line was treated with solutions of
cyclization of reactive
s-pyridinyl radical 15a closing to form the
more stable methylene radical 15b under chain reaction conditions
(Scheme 3). The efficient Bu3SnH-mediated six-membered cycli-
zation of
communication [24]. We now report the synthesis of 5-methyl-5,6-
dihydrobenzimidazo[2,1-a]benzo[f]isoquinoline-8,11-dione in
s-aryl and s-naphthyl radicals was the subject of a recent
0.001e10 mM of compound, and with MMC in parallel. Aryl ring
3
fused compounds 1 and 3 were about 10e60 times less toxic
towards the three cell lines than 2-phenyl and 2-naphthyl
substituted benzimidazolequinone analogues 4 and 6 (Table 1).
Aryl ring fused compounds were more toxic towards the cancer cell
lines than the normal GM00637 cell line, with the highest toxicity
towards the prostate cancer cell line. The naphthyl fused compound
3 showed the highest specificity towards the cancer cell lines in this
full (Scheme 4). The condensation of (3,6-dimethoxy)benzene-1,2-
diamine with 1-bromo-2-naphthoic acid 17 in the presence of
polyphosphoric acid (PPA) was effective in giving 2-(1-bromo-2-
naphthyl)-1H-benzimidazoles 18a and 18b, but attempts to
condense 2-bromonicotinic acid did not give high yields of 2-(2-
bromopyridin-3-yl)-1H-benzimidazoles 13a and 13b [24,25]. The
latter compounds could be efficiently prepared in 85e98% yield
dose range (up to 5 mM, Fig. 2). The negligible toxicity of 3 towards
OMe
OMe
OMe
(ii), (iii)
X
N
N
N
(i)
4 (68%), 5 (61%)
N
H
X
OMe
7: X = CH
8: X = N
9: X = CH (79%)
10: X = N (54%)
Scheme 1. Reagents and conditions; (i) NaH, Et-I, THF, reflux, 2 h; (ii) 48% HBr (aq), reflux, 3 h; (iii) FeCl3 (aq), rt, 18 h.

3764
E. Moriarty et al. / European Journal of Medicinal Chemistry 45 (2010) 3762e3769
OMe
OMe
OMe
O
H
NH2
NH2
N
Tol, reflux, 5 h
+
N
H
OMe
11 (79%)
OMe
OMe
(ii), (iii)
N
(i)
6 (77%)
N
12 (60%)
Scheme 2. Reagents and conditions; (i) NaH, Et-I, THF, reflux, 2 h; (ii) 48% HBr (aq), reflux, 3 h; (iii) FeCl3 (aq), rt, 18 h.
the normal cell line at sub-micromolar concentrations may be of
therapeutic advantage. The higher specificity of compound 3
(compared to other benzimidazolequinones evaluated) towards
cancer cell lines reported to have elevated reductase activity, may
be related to the resonance stabilization of the chemically reduced
intermediates formed, since this is the most conjugated quinone
system prepared. In contrast benzimidazole-4,7-diones 4e6
without fused aromatic rings showed little specificity towards the
two cancer cell lines, with 1-ethyl-2-(2-naphthyl)benzimidazole-
4,7-dione 6 showing very high toxicity towards all three cell lines
evaluated. Perhaps the flexibility of the 2-aromatic ring substituent
in aryl substituted compounds 4 and 6 increases the overall toxicity,
and reduces the specificity of compounds towards the cancer cell
lines. Pyridinyl fused and substituted compounds 2 and 5 were
more toxic towards the prostate than the cervical cancer cell line,
however both compounds were significantly toxic towards the
normal cell line. The requirement for the quinone moiety in cyto-
toxicity is confirmed by evaluating benzimidazole 20a, the
analogue of the most promising benzimidazolequinone 3. This non-
quinone containing compound was found to be essentially non-
toxic towards the normal and cervical cancer cell lines evaluated.
Similarly to 1-ethyl-2-phenyl-1H-benzimidazole-4,7-dione 4, sub-
micromolar concentrations of MMC were cytotoxic towards all
three cell lines, although MMC showed marginal selectivity
towards the two cancer cell lines.
3. Conclusion
The synthesis of new aromatic ring fused benzimidazoles and
benzimidazolequinones has been achieved via a facile 6-exo-trig
R
R
(i)
NH2
NH2
O
H
N
NH4Cl, rt, 8 h
+
N
H
N
N
Br
Br
R
R
13a: R = H (98%)
13b: R = OMe (85%)
R
R
R
R
N
6-exo-trig
(ii)
Bu3Sn
N
N
N
N
N
N
N
N
Br
R
R
15a
15b
14a: R = H (95%)
14b: R = OMe (94%)
R
R
O
O
1
2
(iii), (iv)
N
N
Bu3SnH
N
N
10
3
9
R = OMe
N
N
5
6
16a: R = H (71%)
16b: R = OMe (71%)
2 (78%)
Scheme 3. Reagents and conditions; (i) NaH, CH2]CHCH2Br, THF, reflux, 4 h; (ii) Bu3SnH (1.4 equiv), ACN (0.2 equiv), 8 h, Tol, reflux; (iii) 48% HBr (aq), reflux, 3 h; (iv) FeCl3 (aq), rt,18 h.

E. Moriarty et al. / European Journal of Medicinal Chemistry 45 (2010) 3762e3769
3765
R
R
R
PPA, 150 °C, 5 h
O
(i)
N
NH2
NH2
+
N
H
HO
Br
Br
R
17
18a: R = H (61%)
18b: R = OMe (76%)
R
R
N
N
N
(ii)
(iii), (iv)
3 (76%)
N
R = OMe
Br
R
R
19a: R = H (86%)
19b: R = OMe (88%)
20a: R = H (70%)
20b: R = OMe (71%)
Scheme 4. Reagents and conditions; (i) NaH, CH2]CHCH2Br, THF, reflux, 4 h; (ii) Bu3SnH (1.4 equiv), ACN (0.2 equiv), 8 h, Tol, reflux; (iii) 48% HBr (aq), reflux, 3 h; (iv) FeCl3 (aq), rt,
18 h.
cyclization of
s
-aromatic radicals. The naphthyl fused benzimida-
7-dimethoxy-2-pyridin-3-yl-1H-benzimidazole 8 were prepared
zolequinone 3 showed the greatest specificity towards two cancer
cell lines (HeLa and DU145). This supports our premise of
increasing toxicity towards cancer cell lines that express higher
levels of reductases, with increased quinone conjugation. Incor-
porating a pyridine ring was found to increase toxicity towards the
normal (GM00637) cell line. 1-Ethyl-2-aromatic ring substituted
benzimidazole-4,7-diones showed higher toxicity than aromatic
ring fused analogues towards GM00637 cells, with little specificity
towards cancer cell lines. 1-Ethyl-2-(2-naphthyl)benzimidazole-
4,7-dione 6 (the non-fused analogue of 3) was exceptionally toxic
towards all three cell lines evaluated.
according to the literature from the condensation of 3,6-dime-
thoxybenzene-1,2-diamine
with
benzaldehyde
and
nic-
otinaldehyde respectively [14,18]. 1-Bromo-2-naphthoic acid 17
was prepared according to the literature oxidation of 1-bromo-2-
methylnaphthalene [28].
4.2. Instrumentation
Melting points were measured on a Stuart Scientific melting
point apparatus SMP3. Infrared spectra were recorded using a Per-
kin-Elmer Spec 1 with ATR attached. NMR spectra were recorded
using a Joel 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 1He13C NMR 2D spectra. High resolution mass spectra
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.
4. Experimental
4.1. Materials
Syntheses were carried out using commercially available
starting materials from SigmaeAldrich (apart from 2-bromonico-
tinaldehyde obtained from Frontier Scientific). 3,6-Dimethox-
ybenzene-1,2-diamine was obtained from the reduction of 1,
4-dimethoxy-2,3-dinitrobenzene [27]. All compounds described
below are reported for the first time, apart from the preparation of
5-methyl-5,6-dihydrobenzimidazo[2,1-a]isoquinoline-8,11-dione 1
and 5-methyl-5,6-dihydrobenzimidazo[2,1-a]benzo[f]isoquinoline
20a described in the preliminary synthetic communication on this
work [24]. 4,7-Dimethoxy-2-phenyl-1H-benzimidazole 7 and 4,
4.3. Preparation of 4,7-dimethoxy-2-(2-naphthyl)-1H-
benzimidazole (11)
3,6-Dimethoxybenzene-1,2-diamine (1.800 g, 11 mmol) and
2-naphthaldehyde (1.400 g, 9 mmol) in toluene (30 mL) were
100
80
60
40
20
0
Table 1
Effect of compounds on the viability of human normal skin fibroblast (GM00637),
and cervical (HeLa) and prostate (DU145) cancer cell lines.
a
a
a
Compound
IC50
(
m
mol/L)
IC50
HeLa
(
m
mol/L)
IC50
(mmol/L)
GM00637
DU145
MMC
1
2
3
4
5
6
20a
0.46 ꢀ 0.09
1.65 ꢀ 0.57
0.74 ꢀ 0.14
2.44 ꢀ 0.65
0.15 ꢀ 0.04
0.48 ꢀ 0.06
0.04 ꢀ 0.01
>10
0.27 ꢀ 0.16
1.49 ꢀ 0.48
1.51 ꢀ 0.41
1.17 ꢀ 0.31
0.18 ꢀ 0.08
1.50 ꢀ 0.08
0.07 ꢀ 0.01
>10
0.17 ꢀ 0.02
1.09 ꢀ 0.38
0.70 ꢀ 0.11
0.78 ꢀ 0.40
0.07 ꢀ 0.02
1.27 ꢀ 0.05
0.06 ꢀ 0.02
e
0.001
0.01
0.1
1
10
Concentration (µ M)
Fig. 2. Viability of a normal human skin fibroblast cell line (GM00637) (-) and human
cervical (HeLa) (C) and prostate (DU145) ( ) cancer cell lines determined using the
MTT assay following treatment with 5-methyl-5,6-dihydrobenzimidazo[2,1-a]benzo[f]
isoquinoline-8,11-dione 3 under aerobic conditions for 72 h at 37 ꢁC. Each data point is
the mean of at least three independent experiments.
6
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 MMT assay.

3766
E. Moriarty et al. / European Journal of Medicinal Chemistry 45 (2010) 3762e3769
refluxed for 5 h. The solution was cooled and evaporated to dryness
to yield a brown residue, which was purified by column chroma-
tography using silica gel as absorbent with chloroform as eluent to
give 11 (2.181 g, 79%) as a brown solid; Rf 0.45 (50% petrol:50%
EtOAc); mp 235e237 ꢁC. IR (neat) 1071, 1094, 1167, 1204, 1264,
129.7 (Naph-1-CH), 128.2 (CH), 128.1 (C), 127.9, 127.1, 127.0, 126.8,
126.7 (all CH), 126.3 (C), 103.3, 101.8 (BnIm-5,6-CH), 56.1, 56.0
(OCH3), 41.6 (CH2), 17.3 (CH3); HRMS (ESIþ): m/z calcd for
C21H21N2O2: 333.1603; found 333.1606 [M þ H]þ.
1338, 1401, 1418, 1444, 1499, 1519, 1541, 2933 cmꢂ1
;
1H NMR
8.83 (s, 1H, Naph-1-H), 8.35 (m, 1H, Naph-
4.5. General method for preparation of benzimidazolequinones
(400 MHz, DMSO-d6):
d
H), 8.01e7.95 (m, 3H, Naph-H), 7.58e7.52 (m, 2H, Naph-H), 6.64 (d
(ABq), J ¼ 8.3, 1H, BnIm-5(6)-H), 6.55 (d(ABq), J ¼ 8.3, 1H, Ar-(5)6-
H), 3.89 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), NH not observed; 13C NMR
4.5.1. 1-Ethyl-2-phenyl-1H-benzimidazole-4,7-dione (4)
Benzimidazole 9 (0.530 g, 1.9 mmol) in 48% hydrobromic acid
(13 mL) was refluxed for 3 h. The reaction was cooled and evapo-
rated to dryness. FeCl3 solution (14.4 mL, 0.7 M) was added, and
stirred at room temperature for 18 h. The solution was extracted
with CHCl3, the combined organic extracts dried and evaporated to
dryness to give an orange solid, which was purified by column
chromatography using silica gel as absorbent with gradient elution
of petrol, CH2Cl2 and EtOAc to give 4 (0.321 g, 68%) as an orange
solid; Rf 0.51 (50% CH2Cl2:50% EtOAc); mp 129e132 ꢁC. IR (neat)
1046, 1116, 1213, 1278, 1353, 1429, 1483, 1589, 1650 (C]O), 1675
(100 MHz, DMSO-d6):
d 150.4 (BnIm-2-C), 146.1, 141.0, 136.0, 133.8,
133.4 (all C), 129.0, 128.8, 128.3, 128.2, 127.5, 127.3, 127.1 (all CH),
126.5, 124.7 (C), 103.8, 103.0 (BnIm-5,6-CH), 56.3 (2 ꢃ OCH3); HRMS
(ESIꢂ): m/z calcd for C19H15N2O2: 303.1134; found 303.1148
[M ꢂ H]ꢂ.
4.4. General method for preparation of 1-ethyl-4,7-dimethoxy-2-
aromatic ring substituted benzimidazoles
(C]O) cmꢂ1 1H NMR (400 MHz, CDCl3):
; d 7.61e7.59 (m, 2H),
4.4.1. 1-Ethyl-4,7-dimethoxy-2-phenyl-1H-benzimidazole (9)
7.47e7.41 (m, 3H), 6.62 (d(ABq), J ¼ 10.4, 1H, BnIm-(6)5-H), 6.58 (d
Benzimidazole
7
(0.740 g, 2.9 mmol) and NaH (86 mg,
(ABq), J ¼ 10.4, 1H, BnIm-(5)6-H), 4.34 (q, J ¼ 7.1, 2H, CH2), 1.39 (t,
3.6 mmol) in THF (10 mL) were refluxed for 30 min. Iodoethane
(0.23 mL, 2.9 mmol) was added and refluxed for 1.5 h. The solution
was evaporated to dryness and the residue purified by column
chromatography using silica gel as absorbent with gradient elution
of petrol and EtOAc to give 9 (0.647 g, 79%) as a yellow oil; Rf 0.43
(50% petrol:50% EtOAc). IR (neat) 1059, 1102, 1145, 1204, 1280, 1380,
J ¼ 7.1, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d 181.2 (C]O), 178.2
(C]O), 153.7 (BnIm-2-C), 142.0 (C), 136.6, 136.1 (BnIm-5,6-CH),
130.8 (C),130.6,129.3,129.2,128.9 (all CH),128.4 (C), 41.7 (CH2),16.1
(CH3); HRMS (ESIþ): m/z calcd for C15H13N2O2: 253.0977; found
253.0970 [M þ H]þ.
1462, 1518, 2834 cmꢂ1; 1H NMR (400 MHz, CDCl3):
d
7.70e7.66 (m,
4.5.2. 1-Ethyl-2-pyridin-3-yl-1H-benzimidazole-4,7-dione (5)
Benzimidazole 10 (55 mg, 0.2 mmol) was treated with 48%
hydrobromic acid (1.5 mL) and FeCl3 solution (1.5 mL, 0.7 M), and
after purification by column chromatography gave 5 (30 mg, 61%)
as an orange solid; Rf 0.25 (EtOAc); mp 147e148 ꢁC. IR (neat) 1024,
1052, 1114, 1215, 1288, 1379, 1412, 1528, 1595, 1657 (C]O), 1676
2H), 7.45e7.42 (m, 3H), 6.56 (d(ABq), J ¼ 8.6, 1H, BnIm-(6)5-H), 6.52
(d(ABq), J ¼ 8.6, 1H, BnIm-(5)6-H), 4.37 (q, J ¼ 7.1, 2H, CH2), 3.94 (s,
3H, OCH3), 3.88 (s, 3H, OCH3), 1.35 (t, J ¼ 7.1, 3H, CH3); 13C NMR
(100 MHz, CDCl3):
d 152.8 (BnIm-2-C), 146.1, 141.6, 135.2, 130.7 (all
C), 129.7, 129.6, 128.5 (all CH), 126.2 (C), 103.2, 101.7 (BnIm-5,6-CH),
55.9 (2 ꢃ OCH3), 41.5 (CH2), 17.3 (CH3); HRMS (ESIþ): m/z calcd for
C17H19N2O2: 283.1447; found 283.1445 [M þ H]þ.
(C]O), 2853, 2927 cmꢂ1 1H NMR (400 MHz, CDCl3):
; d 8.93 (d,
J ¼ 1.4, 1H, Pyr-2-H), 8.78 (dd, J ¼ 5.0, J ¼ 1.4, 1H, Pyr-6-H), 8.08 (d,
J ¼ 8.3, 1H, Pyr-4-H), 7.50 (dd, J ¼ 8.3, J ¼ 5.0, 1H, Pyr-5-H), 6.75 (d
(ABq), J ¼ 10.1, 1H, Ar-5(6)-H), 6.69 (d(ABq), J ¼ 10.1, 1H, Ar-(5)6-H),
4.46 (q, J ¼ 7.1, 2H, CH2), 1.51 (t, J ¼ 7.1, 3H, CH3); 13C NMR (100 MHz,
4.4.2. 1-Ethyl-4,7-dimethoxy-2-pyridin-3-yl-1H-benzimidazole
(10)
Benzimidazole 8 (0.200 g, 0.8 mmol), NaH (24 mg, 1.0 mmol),
and iodoethane (0.06 mL, 0.8 mmol), after purification by column
chromatography gave 10 (0.120 g, 54%) as a yellow oil; Rf 0.33 (90%
EtOAc:10% MeOH). IR (neat) 1059, 1102, 1145, 1204, 1280, 1380,
CDCl3): d 181.0 (C]O), 178.2 (C]O), 151.5 (Pyr-6-CH), 150.8 (C),
149.4 (Pyr-2-CH), 142.2 (C), 137.1 (Pyr-4-CH), 136.6, 136.4 (BnIm-
5,6-CH),131.1 (C),125.0 (C),123.8 (Pyr-5-CH), 42.0 (CH2),16.3 (CH3);
HRMS (ESIþ): m/z calcd for C14H12N3O2: 254.0930; found 254.0924
[M þ H]þ.
1462, 1518, 2834, 2934 cmꢂ1; 1H NMR (400 MHz, CDCl3):
d 8.93 (d,
J ¼ 1.8, 1H, Pyr-2-H), 8.70 (dd, J ¼ 4.8, J ¼ 1.8, 1H, Pyr-6-H), 8.06 (d,
J ¼ 7.6,1H, Pyr-4-H), 7.43e7.40 (dd, J ¼ 7.6, J ¼ 4.8,1H, Pyr-5-H), 6.60
(d(ABq), J ¼ 8.5, 1H, BnIm-(6)5-H), 6.55 (d(ABq), J ¼ 8.5, 1H, BnIm-
(5)6-H), 4.41 (q, J ¼ 7.1, 2H, CH2), 3.96 (s, 3H, OCH3), 3.91 (s, 3H,
4.5.3. 1-Ethyl-2-(2-naphthyl)-1H-benzimidazole-4,7-dione (6)
Benzimidazole 12 (0.100 g, 0.3 mmol) was treated with 48%
hydrobromic acid (2.5 mL) and FeCl3 solution (2.5 mL, 0.7 M),
and after purification by column chromatography gave 6 (70 mg,
77%) as an orange solid; Rf 0.57 (50% petrol:50% EtOAc); mp
135e137 ꢁC. IR (neat) 1069, 1287, 1426, 1471, 1527, 1590, 1651
OCH3), 1.39 (t, J ¼ 7.1, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d 150.5
(Pyr-6-CH), 149.9 (Pyr-2-CH), 149.6, 146.1, 141.6 (all C), 137.4 (Pyr-4-
CH), 135.3, 127.0, 126.3 (all C), 123.5 (Pyr-5-CH), 103.6, 101.9 (BnIm-
5,6-CH), 56.0 (2 ꢃ OCH3), 41.7 (CH2), 17.4 (CH3); HRMS (ESIþ): m/z
calcd for C16H18N3O2: 284.1399; found 284.1413 [M þ H]þ.
(C]O), 1676 (C]O) cmꢂ1 1H NMR (400 MHz, CDCl3):
; d 8.21 (s,
1H, Naph-1-H), 7.98 (d, J ¼ 8.5, 1H), 7.93e7.91 (m, 2H), 7.76 (d,
J ¼ 8.7, 1H), 7.62e7.56 (m, 2H), 6.73 (d(ABq), J ¼ 10.4, 1H, Ar-5(6)-
H), 6.67 (d(ABq), J ¼ 10.4, 1H, Ar-(5)6-H), 4.50 (q, J ¼ 7.1, 2H, CH2),
4.4.3. 1-Ethyl-4,7-dimethoxy-2-(2-naphthyl)-1H-benzimidazole
(12)
1.51 (t, J ¼ 7.1, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d 181.3 (C]
Benzimidazole 11 (0.500 g, 1.6 mmol), NaH (48 mg, 2.0 mmol)
and iodoethane (0.12 mL, 1.6 mmol) after purification by column
chromatography gave 12 (0.319 g, 60%) as a brown oil; Rf 0.52 (50%
petrol:50% EtOAc). IR (neat) 1079, 1102, 1145, 1222, 1260, 1354,
O), 178.2 (C]O), 153.8 (BnIm-2-C), 142.2 (C), 136.6, 136.2 (BnIm-
5,6-CH), 133.0, 132.9, 130.9 (all C), 129.7 (Naph-1-CH), 128.8,
128.7, 127.9, 127.8, 127.1, 125.7 (all CH), 41.9 (CH2), 16.2 (CH3);
HRMS (ESIþ): m/z calcd for C19H15N2O2: 303.1134; found
303.1143 [M þ H]þ.
1389, 1462, 1520, 2836, 2934 cmꢂ1 1H NMR (400 MHz, CDCl3):
;
d
8.23 (s, 1H, Naph-1-H), 7.93e7.80 (m, 4H, Naph-H), 7.52e7.49 (m,
2H, Naph-H), 6.59 (d(ABq), J ¼ 8.6, 1H, Ar-5(6)-H), 6.55 (d(ABq),
J ¼ 8.6, 1H, Ar-(5)6-H), 4.46 (q, J ¼ 7.1, 2H, CH2), 3.98 (s, 3H, OCH3),
3.89 (s, 3H, OCH3), 1.39 (t, J ¼ 7.1, 3H, CH3); 13C NMR (100 MHz,
4.5.4. 5-Methyl-5,6-dihydrobenzimidazo[2,1-f]-1,6-naphthyridine-
8,11-dione (2)
Benzimidazole 16b (0.500 g, 1.7 mmol) was treated with 48%
hydrobromic acid (13 mL) and FeCl3 solution (0.7 M, 15 mL), and
CDCl3):
d 152.8 (BnIm-2-C), 146.2, 141.7, 135.5, 133.6, 133.0 (all C),

E. Moriarty et al. / European Journal of Medicinal Chemistry 45 (2010) 3762e3769
3767
after purification by column chromatography gave 2 (0.351 g, 78%)
4.7. General method for preparation of 2-(1-bromo-2-naphthyl)-
1H-benzimidazoles
as an orange solid; Rf 0.55 (100% EtOAc); mp 199e200 ꢁC. IR (neat)
1094, 1249, 1413, 1489, 1517, 1570, 1585, 1656 (C]O) cmꢂ1; 1H NMR
(400 MHz, CDCl3):
d
8.63 (dd, J ¼ 4.9, J ¼ 1.6, 1H, 3-H), 8.47 (dd,
4.7.1. 2-(1-Bromo-2-naphthyl)-1H-benzimidazole (18a)
J ¼ 7.9, J ¼ 1.6, 1H, 1-H), 7.34 (dd, J ¼ 7.9, J ¼ 4.9, 1H, 2-H), 6.72 (d
(ABq), J ¼ 10.4, 1H, (9)10-H), 6.66 (d(ABq), J ¼ 10.4, 1H, 9(10)-H),
4.73 (dd, J ¼ 13.9, J ¼ 5.7, 1H, 6-HH), 4.51 (dd, J ¼ 13.9, J ¼ 6.8, 1H, 6-
HH), 3.54e3.49 (m, 1H, 5-H), 1.44 (d, J ¼ 7.0, 3H, CH3); 13C NMR
A mixture of naphthoic acid 17 (3.000 g, 12 mmol), 1,2-phenyl-
enediamine (1.296 g, 12 mmol) and polyphosphoric acid (17.000 g)
was heated at 150 ꢁC for 6 h. The mixture was poured onto crushed
ice, and the precipitate filtered. Na2CO3 solution (0.5 M, 300 mL)
was added and the mixture stirred for 30 min. The benzimidazole
free base was filtered, and recrystallized from methanol to give 18a
(2.390 g, 61%) as a white solid; mp 267e268 ꢁC. IR (neat) 1014,1125,
(100 MHz, CDCl3):
d 181.0 (C]O), 178.5 (C]O), 158.0 (C), 151.6 (3-
CH), 148.2 (C), 142.7 (C), 136.7, 136.3 (9,10-CH), 133.2 (1-CH), 130.5
(C), 123.1 (2-CH), 120.4 (C), 48.2 (6-CH2), 35.0 (5-CH), 17.3 (CH3);
HRMS (ESIþ): m/z calcd for C15H12N3O2: 266.0930; found 266.0923
[M þ H]þ.
1226, 1276, 1337, 1408, 1441, 1499, 1534 cmꢂ1
DMSO-d6):
;
1H NMR (400 MHz,
8.34 (d, J ¼ 8.2, 1H, Naph-3(4)-H), 8.10e8.06 (m, 2H,
d
Naph-H), 7.80 (d, J ¼ 8.2, 1H, Naph-(3),4-H), 7.78e7.67 (m, 2H,
4.5.5. 5-Methyl-5,6-dihydrobenzimidazo[2,1-a]benzo[f]
isoquinoline-8,11-dione (3)
Naph-H), 7.65e7.62 (m, 2H, BnIm-H), 7.25e7.22 (m, 2H, BnIm-H),
NH not observed; 13C NMR (100 MHz, DMSO-d6):
d 151.7 (BnIm-2-
Benzimidazole 20b (0.100 g, 0.3 mmol) was treated with 48%
hydrobromic acid (2.5 mL) and FeCl3 solution (0.7 M, 5.0 mL), and
after purification by column chromatography gave 3 (70 mg, 76%)
as a red solid; Rf 0.48 (50% petrol:50% EtOAc); mp 145e147 ꢁC. IR
(neat) 1057, 1092, 1196, 1239, 1257, 1272, 1424, 1479, 1512, 1654 (C]
C), 134.7, 132.1, 131.6 (all C), 129.2, 129.1, 129.0 (ꢃ2), 127.9, 127.9 (all
Naph-CH), 122.8 (C-Br), 122.7, 122.6, 115.9, 115.8 (all BnIm-CH);
HRMS (ESIþ): m/z calcd for C17H1179BrN2: 322.0100; found 322.0108
[M]þ.
O) cmꢂ1; 1H NMR (400 MHz, CDCl3):
d
8.35 (d, J ¼ 8.7, 1H), 8.08 (d,
4.7.2. 2-(1-Bromo-2-naphthyl)-4,7-dimethoxy-1H-benzimidazole
(18b)
J ¼ 8.2, 1H), 7.92e7.87 (m, 2H), 7.64e7.56 (m, 2H), 6.72 (d(ABq),
J ¼ 10.4, 1H, (9)10-H), 6.66 (d(ABq), J ¼ 10.4, 1H, 9(10)-H), 5.08 (d,
J ¼ 13.7, 1H, 6-HH), 4.34 (dd, J ¼ 13.7, J ¼ 5.0, 1H, 6-HH), 4.14e4.05
(m, 1H, 5-H), 1.34 (d, J ¼ 7.3, 3H, CH3); 13C NMR (100 MHz, CDCl3):
Naphthoic acid 17 (2.000 g, 8.0 mmol), 3,6-dimethoxybenzene-
1,2-diamine (1.360 g, 8.1 mmol) and polyphosphoric acid (11.400 g)
after recrystallization from methanol gave 18b (2.329 g, 76%) as
a light brown solid; mp 189e191 ꢁC. IR (neat) 1105, 1263, 1452,
d
181.3 (C]O), 178.5 (C]O), 149.5 (C), 142.7 (C), 136.7, 136.3 (9,10-
CH), 136.4, 135.1, 130.7, 129.8 (all C), 129.5, 128.5, 127.5, 127.4, 123.4,
122.7 (all CH), 121.2 (C), 48.3 (CH2), 28.8 (5-CH), 19.9 (CH3); HRMS
(ESIþ): m/z calcd for C20H15N2O2: 315.1134; found 315.1131
[M þ H]þ.
1508, 1538, 2547, 3302 cmꢂ1; 1H NMR (400 MHz, DMSO-d6):
d 8.43
(d, J ¼ 8.4, 1H, Naph-H), 8.19e8.16 (m, 2H, Naph-H), 7.88e7.85 (m,
1H, Naph-H), 7.82e7.78 (m, 2H, Naph-H), 6.76 (s, 2H, BnIm-5,6-H),
4.02 (s, 6H, 2 ꢃ CH3), NH not observed; 13C NMR (100 MHz, DMSO-
d6): d 150.4 (BnIm-2-C), 134.7, 132.0, 131.6 (all C), 129.1, 129.0, 128.9,
4.6. General method for preparation of 2-(2-bromopyridin-3-yl)-
1H-benzimidazoles
128.3, 128.1, 127.8 (all Naph-CH), 123.3 (C-Br), 103.4 (2 ꢃ BnIm-5,6-
CH), 56.2 (2 ꢃ CH3); HRMS (EIþ): m/z calcd for C19H1579BrN2O2:
383.0395; found 383.0396 [M þ H]þ.
4.6.1. 2-(2-Bromopyridin-3-yl)-1H-benzimidazole (13a)
2-Bromonicotinaldehyde (0.930 g, 5.0 mmol), benzene-1,2-
diamine (0.540 g, 5.0 mmol) and NH4Cl (1.100 g, 20.5 mmol) in
CH3OH (25 mL) were stirred at room temperature for 8 h. The
solution was evaporated to dryness, CHCl3 (25 mL) added, filtered,
and recrystallized from toluene to give 13a (1.350 g, 98%) as
a brown solid; mp 206e208 ꢁC. IR (neat) 1052, 1097, 1261, 1399,
4.8. General method for preparation of radical precursors
4.8.1. 1-Allyl-2-(2-bromopyridin-3-yl)-1H-benzimidazole (14a)
Bromide 13a (1.000 g, 3.6 mmol) and NaH (81 mg, 3.4 mmol) in
THF (40 mL) were refluxed for 1 h. Allyl bromide (0.50 mL,
5.8 mmol) in THF (20 mL) was added over 30 min, and refluxed for
8 h. The mixture was filtered, evaporated to dryness and purified by
column chromatography using silica gel as absorbent with gradient
elution of petrol and EtOAc to give 14a (1.080 g, 95%) as an oil; Rf
1531, 2922 cmꢂ1
;
1H NMR (400 MHz, DMSO-d6):
d
8.52 (d, J ¼ 3.0,
1H, Pyr-6-H), 8.16 (d, J ¼ 6.9, 1H, Pyr-4-H), 7.69 (bs, 1H, BnIm-H),
7.63e7.56 (m, 2H, Pyr-5-H and BnIm-H), 7.23 (bs, 2H, BnIm-H), NH
not observed; 13C NMR (100 MHz, DMSO-d6):
d
151.0 (Pyr-6-CH),
0.38 (50% petrol:50% EtOAc). IR (neat) 1330, 1381, 1454, 1553 cmꢂ1
1H NMR (400 MHz, CDCl3):
;
148.7 (C), 143.3 (C), 140.8 (Pyr-4-CH), 140.6 (C), 134.6 (C), 130.0 (C),
123.4 (Pyr-5-CH), 123.0, 121.9, 119.3 (all BnIm-CH), 111.8 (BnIm-7-
CH); HRMS (ESIþ): m/z calcd for C12H1079BrN3: 273.9980; found
273.9991 [M þ H]þ.
d
8.45 (dd, J ¼ 4.8, J ¼ 2.0, 1H, Pyr-6-H),
7.85e7.82 (m, 1H, BnIm-4-H), 7.78 (dd, J ¼ 7.5, J ¼ 2.0, 1H, Pyr-4-H),
7.45e7.40 (m, 1H, BnIm-7-H), 7.36 (dd, J ¼ 7.5, J ¼ 4.8, 1H, Pyr-5-H),
7.33e7.28 (m, 2H, BnIm-5,6-H), 5.85e5.75 (m, 1H, 20-H), 5.10 (d,
J ¼ 10.3, 1H, 30-cis-H), 4.90 (d, J ¼ 17.2, 1H, 30-trans-H), 4.63 (d,
4.6.2. 2-(2-Bromopyridin-3-yl)-4,7-dimethoxy-1H-benzimidazole
(13b)
J ¼ 5.0, 2H, NCH2); 13C NMR (100 MHz, CDCl3):
d 151.4 (Pyr-6-CH),
149.9, 142.9, 142.8 (all C), 140.7 (Pyr-4-CH), 134.7 (C), 131.8 (20-CH),
129.7 (C), 123.6 (BnIm-CH), 122.8 (BnIm-CH), 122.7 (Pyr-5-CH),
120.2 (BnIm-4-CH), 118.0 (30-CH2), 110.8 (BnIm-7-CH), 47.1 (NCH2);
HRMS (ESIþ): m/z calcd for C15H1379BrN3: 314.0293; found 314.0295
[M þ H]þ.
2-Bromonicotinaldehyde (1.116 g, 6.0 mmol), 3,6-dimethox-
ybenzene-1,2-diamine (1.008 g, 6.0 mmol) and NH4Cl (1.320 g,
24.6 mmol) in CH3OH (30 mL), after recrystallization from toluene
gave 13b (1.710 g, 85%) as a brown solid; mp 160e161 ꢁC. IR (neat)
1057, 1226, 1279, 1315, 1378, 1553, 1578 cmꢂ1
CDCl3):
;
1H NMR (400 MHz,
8.81 (dd, J ¼ 7.8, J ¼ 2.1, 1H, Pyr-6-H), 8.40 (dd, J ¼ 4.6,
d
4.8.2. 1-Allyl-2-(2-bromopyridin-3-yl)-4,7-dimethoxy-1H-
benzimidazole (14b)
J ¼ 2.1, 1H, Pyr-4-H), 7.40 (dd, J ¼ 7.8, J ¼ 4.6, 1H, Pyr-5-H), 6.60 (s,
2H, BnIm-5,6-H), 3.97 (s, 6H, CH3), NH not observed; 13C NMR
Bromide 13b (2.590 g, 7.8 mmol), NaH (0.188 g, 7.8 mmol), allyl
bromide (0.680 mL, 7.8 mmol) in THF (80 mL) after purification by
column chromatography gave 14b (2.726 g, 94%) as a brown oil; Rf
0.42 (50% petrol:50% EtOAc); mp 119e120 ꢁC. IR (neat) 1102, 1259,
(100 MHz, CDCl3):
d 150.4 (Pyr-6-CH), 145.6 (C), 141.3 (Pyr-4-CH),
138.7 (C), 128.2 (C), 123.2 (Pyr-5-CH), 103.1 (BnIm-5,6-CH), 56.0
(2 ꢃ CH3); HRMS (ESIþ): m/z calcd for C14H1379BrN3O2: 334.0191;
found 334.0198 [M þ H]þ.
1378, 1446, 1519 cmꢂ1 1H NMR (400 MHz, CDCl3):
; d 8.34 (dd,

3768
E. Moriarty et al. / European Journal of Medicinal Chemistry 45 (2010) 3762e3769
J ¼ 4.8, J ¼ 1.8, 1H, Pyr-6-H), 7.67 (dd, J ¼ 7.6, J ¼ 1.8, 1H, Pyr-4-H),
7.24 (dd, J ¼ 7.6, J ¼ 4.8, 1H, Pyr-5-H), 6.50 (d(ABq), J ¼ 8.4, 1H,
BnIm-5(6)-H), 6.44 (d(ABq), J ¼ 8.4, 1H, BnIm-(5)6-H), 5.75e5.65
(m,1H, 20-H), 4.84 (d, J ¼ 10.3,1H, 30-cis-H), 4.76 (bs, 2H, NCH2), 4.49
(d, J ¼ 17.2, 1H, 30-trans-H), 3.83 (s, 3H, CH3), 3.74 (s, 3H, CH3); 13C
CH), 123.4 (CH), 122.9 (2 ꢃ CH), 121.9 (C), 119.9 (CH), 109.4 (11-CH),
46.6 (CH2), 35.4 (5-CH), 18.0 (CH3); HRMS (ESIþ): m/z calcd for
C15H14N3: 236.1188; found 236.1185 [M þ H]þ.
4.9.2. 8,11-Dimethoxy-5-methyl-5,6-dihydrobenzimidazo[2,1-f]-
1,6-naphthyridine (16b)
NMR (100 MHz, CDCl3): d 151.2 (Pyr-6-CH), 149.1, 146.0, 143.0, 141.6
(all C), 141.1 (Pyr-4-CH), 134.9 (C), 133.8 (20-CH), 129.9 (C), 125.5 (C),
122.4 (Pyr-5-CH), 116.4 (30-CH2), 104.1, 102.1 (BnIm-5,6-CH), 56.0
(CH3), 55.9 (CH3), 48.5 (NCH2); HRMS (ESIþ): m/z calcd for
C17H1779BrN3O2: 374.0504; found 374.0498 [M þ H]þ.
Bromide 14b (2.200 g, 5.9 mmol), Bu3SnH (2.390 g, 8.2 mmol)
and ACN (0.287 g, 1.2 mmol) gave after purification by column
chromatography 16b (1.237 g, 71%) as a white solid; Rf 0.28 (50%
petrol:50% EtOAc); mp 137e139 ꢁC. IR (neat) 1084, 1107, 1165, 1246,
1259, 1458, 1527 cmꢂ1; 1H NMR (400 MHz, CDCl3):
d 8.57e8.52 (m,
4.8.3. 1-Allyl-2-(1-bromo-2-naphthyl)-1H-benzimidazole (19a)
Bromide 18a (2.907 g, 9.0 mmol), NaH (0.216 g, 9.0 mmol) and
allyl bromide (0.78 mL, 9.0 mmol) in THF (80 mL) after purification
by column chromatography gave 19a (2.823 g, 86%) as a yellow oil;
Rf 0.48 (50% petrol:50% EtOAc). IR (neat) 1248, 1281, 1327, 1388,
2H, 1 and 3-H), 7.25 (dd, J ¼ 7.7, J ¼ 5.0, 1H, 2-H), 6.52 (d(ABq),
J ¼ 8.5, 1H, 10(9)-H), 6.47 (d(ABq), J ¼ 8.5, 1H, (10)9-H), 4.71 (dd,
J ¼ 13.1, J ¼ 5.5, 1H, 6-HH), 4.55 (dd, J ¼ 13.1, J ¼ 6.5, 1H, 6-HH), 3.94
(s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.45e3.43 (m, 1H, 5-H), 1.41 (d,
J ¼ 6.9, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d 158.5 (C), 150.3 (3-
1455, 1492 cmꢂ1
;
1H NMR (400 MHz, CDCl3):
d
8.34 (d, J ¼ 8.3, 1H,
CH),146.6, 146.1, 141.7, 136.0 (all C),133.0 (1-CH), 125.8 (C), 122.7 (2-
CH), 121.9 (C), 103.6, 101.7 (9,10-CH), 55.9 (2 ꢃ OCH3), 48.8 (CH2),
35.7 (5-CH), 17.6 (CH3); HRMS (ESIþ): m/z calcd for C17H18N3O2:
296.1399; found 296.1408 [M þ H]þ.
Naph-5(8)-H), 7.95e7.92 (m, 1H, BnIm-4-H), 7.85 (d, J ¼ 8.5, 1H,
Naph-3(4)-H), 7.82 (d, J ¼ 8.0, 1H, Naph-(5)8-H), 7.61e7.51 (m, 2H,
Naph-6,7-H), 7.48 (d, J ¼ 8.5, 1H, Naph-(3)4-H), 7.42e7.39 (m, 1H,
BnIm-7-H), 7.35e7.30 (m, 2H, BnIm-5,6-H), 5.78e5.71 (m, 1H, 20-
H), 5.06 (d, J ¼ 10.3, 1H, 30-cis-H), 4.91 (d, J ¼ 17.2, 1H, 30-trans-H),
4.9.3. 8,11-Dimethoxy-5-methyl-5,6-dihydrobenzimidazo[2,1-a]
benzo[f]isoquinoline (20b)
4.63e4.52 (bs, 2H, NCH2); 13C NMR (100 MHz, CDCl3):
d 152.7
(BnIm-2-C), 143.0 (BnIm-3a-C), 134.6 (2 ꢃ C), 131.9 (20-CH), 131.8
(C), 130.0 (C), 128.3, 128.2, 128.0, 127.9, 127.8, 127.7 (all Naph-CH),
124.9 (C-Br), 123.0, 122.4 (BnIm-5,6-CH), 120.2 (BnImþ-4-CH), 117.7
(30-CH2), 110.6 (BnIm-7-CH), 46.9 (NCH2); HRMS (ESI ): m/z calcd
for C20H1679BrN2: 363.0497; found 363.0498 [M þ H]þ.
Bromide 19b (1.000 g, 2.4 mmol), Bu3SnH (1.000 g, 3.4 mmol)
and ACN (0.117 g, 0.5 mmol) gave after purification by column
chromatography 20b (0.587 g, 71%) as a white solid; Rf 0.41 (50%
petrol:50% EtOAc); 1H NMR (400 MHz, CDCl3):
d
8.54 (d, J ¼ 8.7, 1H),
8.02 (d, J ¼ 8.2, 1H), 7.82 (d, J ¼ 8.7, 2H), 7.53e7.44 (m, 2H), 6.53 (d
(ABq), J ¼ 8.5, 1H, 10(9)-H), 6.50 (d(ABq), J ¼ 8.5, 1H, 9(10)-H), 5.04
(d, J ¼ 13.0, 1H, 6-HH), 4.36e4.32 (dd, J ¼ 13.0, J ¼ 4.6, 1H, 6-HH),
3.99 (s, 3H, OCH3), 3.96e3.84 (m, 1H, 5-H), 3.87 (s, 3H, OCH3), 1.32
4.8.4. 1-Allyl-2-(1-bromo-2-naphthyl)-4,7-dimethoxy-1H-
benzimidazole (19b)
Bromide 18b (2.000 g, 5.2 mmol), NaH (0.144 g, 6.0 mmol) and
allyl bromide (0.45 mL, 5.2 mmol) in THF (80 mL) gave after puri-
fication by column chromatography 19b (1.937 g, 88%) as a yellow
oil; Rf 0.46 (50% petrol:50% EtOAc). IR (neat) 1095, 1173, 1195, 1259,
(d, J ¼ 7.1, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d 148.2, 146.2, 141.7,
136.2, 136.0, 134.6, 130.2 (all C), 129.2, 127.7, 126.9, 126.6 (all CH),
126.2 (C), 123.4 (CH), 123.3 (CH), 122.9 (C), 103.4, 101.5 (9,10-CH),
56.0 (OCH3), 55.9 (OCH3), 48.8 (CH2), 29.3 (5-CH), 19.9 (CH3); HRMS
(ESIþ): m/z calcd for C22H21N2O2: 345.1603; found 345.1605
[M þ H]þ.
1384, 1448, 1518, 2932 cmꢂ1; 1H NMR (400 MHz, CDCl3):
d 8.36 (d,
J ¼ 7.8, 1H, Naph-5(8)-H), 7.88 (m, 2H, 2 ꢃ Naph-H), 7.66e7.57 (m,
2H, Naph-6,7-H), 7.51 (d, J ¼ 8.4, 1H, Naph-3(4)-H), 6.63 (d(ABq),
J ¼ 8.5, 1H, BnIm-5(6)-H), 6.58 (d(ABq), J ¼ 8.5, 1H, BnIm-(5)6-H),
5.84e5.76 (m, 1H, 20-H), 5.20e5.10 (bs, 1H, NCHH), 4.95 (d, J ¼ 10.3,
1H, 30-cis-H), 4.65 (d, J ¼ 17.2, 1H, 30-trans-H), 4.58e4.47 (bs, 1H,
NCHH), 3.98 (s, 3H, CH3), 3.89 (s, 3H, CH3); 13C NMR (100 MHz,
4.10. Cell culture and cytotoxicity evaluation
4.10.1. 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 DU145 prostate cancer cell line (ATCC repository
number HTB-81) was obtained from Prof. R.W.G Watson, School
of Medicine and Medical Science, University College Dublin,
Ireland.
CDCl3): d
152.0 (BnIm-2-C),146.3, 141.7, 135.1, 134.8 (all C), 134.1 (20-
CH), 132.0 (C), 130.4 (C), 128.3, 128.2, 127.9, 127.7, 127.6, 127.5 (all
Naph-CH), 125.5 (C), 125.2 (C), 116.4 (30-CH2), 103.8, 101.9 (BnIm-
5,6-CH), 56.1 (CH3), 56.0 (CH3), 48.5 (NCH2); HRMS (ESIþ): m/z calcd
for C22H20N2O2 79Br: 423.0708; found 423.0718 [M þ H]þ.
4.9. General method for radical cyclization
4.9.1. 5-Methyl-5,6-dihydrobenzimidazo[2,1-f]-1,6-naphthyridine
(16a)
Bu3SnH (1.250 g, 4.3 mmol) and ACN (0.150 g, 0.6 mmol) in
toluene (50 mL) were added over 8 h via a syringe pump to
bromide 14a (1.000 g, 3.2 mmol) in toluene (85 mL) at reflux. The
cooled solution was evaporated to dryness and purified by column
chromatography using silica as absorbent with gradient elution of
petrol, CH2Cl2 and EtOAc to give 16a (0.535 g, 71%) as a white solid;
Rf 0.32 (43% petrol:43% EtOAc:14% methanol); mp 119e120 ꢁC. IR
(neat) 1327, 1413, 1448, 1484, 1570, 2926, 2967, 3397 cmꢂ1; 1H NMR
4.10.2. Cell culture
The SV40-transformed normal human skin fibroblast cell line
(GM00637) was grown in minimum essential media (MEM) Eagle-
Earle BSS supplemented with 15% heat-inactivated fetal bovine
serum (FBS), penicillinestreptomycin, 2 mM
L-glutamine, 2ꢃ
essential and non-essential amino acids, and vitamins. HeLa-CCL-2
cervical cancer cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% heat-inactivated fetal
(400 MHz, CDCl3):
d
8.59 (dd, J ¼ 4.8, J ¼ 1.6, 1H, 3-H), 8.49 (dd,
bovine serum, penicillinestreptomycin, 2 mM L-glutamine, and
MEM non-essential amino acids. DU145 prostate cancer cells were
grown in RPMI-1640 medium supplemented with 10% heat-inac-
tivated fetal bovine serum, penicillinestreptomycin and 2 mM L-
glutamine.
J ¼ 7.8, J ¼ 1.6, 1H, 1-H), 7.80e7.78 (m, 1H), 7.37e7.26 (m, 4H), 4.43
(dd, J ¼ 12.5, J ¼ 5.6, 1H, 6-HH), 4.13 (dd, J ¼ 12.5, J ¼ 6.0, 1H, 6-HH),
3.58e3.54 (m,1H, 5-H),1.44 (d, J ¼ 7.1, 3H, CH3); 13C NMR (100 MHz,
CDCl3):
d
158.6 (C), 150.7 (3-CH), 147.6, 144.2, 134.9 (all C), 133.0 (1-

E. Moriarty et al. / European Journal of Medicinal Chemistry 45 (2010) 3762e3769
3769
4.10.3. Cytotoxicity measurement
References
Cell viability was determined using the MTT colorimetric
assay [29]. Normal cells were plated into 96-well plates at
[1] L. Garuti, M. Roberti, D. Pizzirani, Mini Rev. Med. Chem. 7 (2007) 481e489.
[2] Y. Chen, L. Hu, Med. Res. Rev. 29 (2009) 29e64.
a density of 10,000 cells per well (200
were plated into 96-well plates at a density of 1000 cells per
well (100 L per well). DU145 cells were plated into 96-well
plates at a density of 2000 cells per well (200 L per well). Cells
mL per well). HeLa cells
[3] H.A. Seow, P.G. Penketh, R.P. Baumann, A.C. Sartorelli, in: H. Sies, L. Packer
(Eds.), Quinones and Quinone Enzymes, Part B, Methods in Enzymology, vol.
382, Elsevier, 2004, pp. 221e233.
[4] E.B. Skibo, S. Gordon, L. Bess, R. Boruah, M.J. Heileman, J. Med. Chem. 40 (1997)
1327e1339.
m
m
were allowed to adhere for 24 h before exposure to benzimida-
zolequinone or MMC.
[5] E.B. Skibo, I. Islam, W.G. Schulz, R. Zhou, L. Bess, R. Boruah, Synlett (1996)
297e309.
[6] C.M. Ahn, S.K. Kim, J.L. Han, Arch. Pharm. Res. 21 (1998) 599e609.
[7] L. O’Donovan, M.P. Carty, F. Aldabbagh, Chem. Commun. (2008) 5592e5594.
[8] K. Fahey, L. O’Donovan, M. Carr, M.P. Carty, F. Aldabbagh, Eur. J. Med. Chem. 45
(2010) 1873e1879.
[9] A.K. Singh, J.W. Lown, Anticancer Drug Des. 15 (2000) 265e275.
[10] A. Gellis, H. Kovacic, N. Boufatah, P. Vanelle, Eur. J. Med. Chem. 43 (2008)
1858e1864.
Benzimidazolequinone and MMC (Sigma) were applied in
DMSO (1% final concentration in well) and the plates were incu-
bated 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 m
L, 5 mg mLꢂ1 solution) was added, and
[11] C. Flader, J. Liu, R.F. Borch, J. Med. Chem. 43 (2000) 3157e3167.
[12] L. Garuti, M. Roberti, M. Malagoli, T. Rossi, M. Castelli, Bioorg. Med. Chem. Lett.
10 (2000) 2193e2195.
[13] L. Garuti, M. Roberti, A. Pession, E. Leoncini, S. Hrelia, Bioorg. Med. Chem. Lett.
11 (2001) 3147e3149.
[14] L. Garuti, M. Roberti, D. Pizzirani, A. Pession, E. Leoncini, V. Cenci, S. Hrelia, IL
Farmaco 59 (2004) 663e668.
[15] K.-H. Chung, S.-Y. Hong, H.-J. You, R.-E. Park, C.-K. Ryu, Bioorg. Med. Chem. 14
(2006) 5795e5801.
[16] M. Lynch, S. Hehir, P. Kavanagh, D. Leech, J. O’Shaughnessy, M.P. Carty,
F. Aldabbagh, Chem. Eur. J. 13 (2007) 3218e3226.
[17] O. Lavergne, A.-C. Fernandes, L. Bréhu, A. Sidhu, M.-C. Brézak, G. Prévost,
B. Ducommun, M.-O. Contour-Galcera, Bioorg. Med. Chem. Lett. 16 (2006)
171e175.
the cells were incubated for another 3 h. The supernatant was
removed carefully by pipetting. The resultant MTT formazan crys-
tals were dissolved in 100 mL of DMSO and absorbance was deter-
mined on a plate reader at 550 nm with a reference at 690 nm. Cell
viability is expressed as a percentage of the DMSO-only treated
value. Each data point used is the mean of at least three indepen-
dent experiments. Doseeresponse curves were analysed by non-
linear regression analysis and IC50 values were estimated using
GraphPad Prism software, v. 5.02 (GraphPad Inc., San Diego, CA,
USA).
[18] C.-K. Ryu, E.-H. Song, J.-Y. Shim, H.-J. You, K.U. Choi, I.H. Choi, E.Y. Lee, M.
J. Chae, Bioorg. Med. Chem. Lett. 13 (2003) 17e20.
[19] J.J. Newsome, M.A. Colucci, M. Hassani, H.D. Beall, C.J. Moody, Org. Biomol.
Chem. 5 (2007) 3665e3673.
Acknowledgements
[20] M.A. Colucci, C.J. Moody, G.D. Couch, Org. Biomol. Chem. 6 (2008) 637e656.
[21] D. Ross, D. Siegel, in: H. Sies, L. Packer (Eds.), Quinones and Quinone Enzymes,
Part B, Methods in Enzymology, vol. 382, Elsevier, 2004, pp. 115e144.
[22] R.I. Bello, C. Gómez-Díaz, F. Navarro, F.J. Alcaín, J.M. Villalba, J. Biol. Chem. 276
(2001) 44379e44384.
This publication has emanated from research conducted with
financial support from Science Foundation Ireland (SFI) (07/RFP/
CHEF227). The PhD of EM was supported by the Environmental
Protection Agency (EPA) Doctoral Scheme (2006-PhD-ET-6). We
thank the SFI and the EPA, Department of Environment, Heritage
and Local Government funded under the National Development
Plan 2000e2006 for this financial support. We thank Prof.
William Watson of the Conway Institute, School of Medicine and
Medical Science, University College Dublin, Ireland for the
DU145 cell line.
[23] S.A. Fitzsimmons, P. Workman, M. Grever, K. Paull, R. Camalier, A.D. Lewis,
J. Natl. Cancer Inst. 88 (1996) 259e269.
[24] E. Moriarty, F. Aldabbagh, Tetrahedron Lett. 50 (2009) 5251e5253.
[25] K.R. Reddy, G.G. Krishna, Tetrahedron Lett. 46 (2005) 661e663.
[26] B.C. Raju, N.D. Theja, J.A. Kumar, Synth. Commun. 39 (2009) 175e188.
[27] I.A. Shaikh, F. Johnson, A.P. Grollman, J. Med. Chem. 29 (1986) 1329e1340.
[28] D. Hellwinkel, S. Bohnet, Chem. Ber. 120 (1987) 1151e1173.
[29] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.