Synthesis and cytotoxicity evaluation of some benzimidazole-4,7-diones as bioreductive anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2007.11.020

New benzimidazole-4,7-diones substituted at 2-position were synthesized via a microwave-assisted reaction using 2-chloromethyl-1,5,6-trimethyl-1H-benzimidazole-4,7-dione 5b as a key intermediate compound. Their cytotoxicity has been evaluated on colon, br

Full text of DOI:10.1016/j.ejmech.2007.11.020

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1858e1864
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxicity evaluation of some benzimidazole-4,7-diones
as bioreductive anticancer agents
a
Armand Gellis , Herve Kovacic , Narimene Boufatah , Patrice Vanelle
b
a
a,
*
´
`
a Laboratoire de Chimie Organique Pharmaceutique, UMR CNRS 6517, Universite´ de la Me´diterrane´e, Faculte´ de Pharmacie,
27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
b Laboratoire de Biophysique, FRE CNRS 2737, Universite´ de la Me´diterrane´e, Faculte´ de Pharmacie, 27 Boulevard Jean Moulin,
13385 Marseille Cedex 5, France
Received 26 September 2007; received in revised form 12 November 2007; accepted 22 November 2007
Available online 15 December 2007
´
Dedicated to Professor Jose Maldonado for his contribution to the Science of Pharmaceutical Organic Chemistry.
Abstract
New benzimidazole-4,7-diones substituted at 2-position were synthesized via a microwave-assisted reaction using 2-chloromethyl-1,5,6-tri-
methyl-1H-benzimidazole-4,7-dione 5b as a key intermediate compound. Their cytotoxicity has been evaluated on colon, breast and lung cancer
cell lines. The dimer 17 was shown to possess excellent cytotoxicity comparable to that of mitomycin C.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: Benzimidazole-4,7-dione; Mitomycin C; Antitumor; Microwave
1. Introduction
two-electron reduction by different cellular enzyme complexes
such as cytochrome P450 oxidoreductase, cytochrome b5 ox-
idoreductase and DT-diaphorase. The DT-diaphorase is an
NADPH-dependent enzyme that reduces quinones and other
substrates into the corresponding two-electron reduction prod-
ucts [5]. These reductive alkylating quinone methide species,
which are formed upon reduction of leaving groups, are highly
reactive and produce cytotoxic effects by reacting with DNA
of tumor cells [6]. Among these agents, the pyrrolo[1,2-
a]benzimidazoles (PBIs) were found to possess cytotoxicity
with minimal antitumor activity [7]. The indoloquinone EO9
was considered to be a promising antitumor agent, but phase
I clinical trials revealed short plasma half-lives as well as tox-
icity [8].
Biological properties also have been observed in some ben-
zimidazolediones [9], and 5-amino-6-bromobenzimidazole-4,7-
dione showed good antitumor activity in vivo on P388 leukemia
cell line [10]. In most cases the activity is related to the ability
of quinone to accept one or two electrons, forming reactive cy-
totoxic species [11]. The substituents on the quinone ring play
a significant role in determining the biological properties. The
Mitomycin C (Fig. 1) is an antitumor drug discovered in the
1950s by Japanese microbiologists. Several close structural
variants of mitomycin C have since been isolated, collectively
called as mitomycins [1]. Mitomycin C has a variety of spe-
cific biological effects in mammalian cells, including selective
inhibition of DNA synthesis, mutagenesis, and increase of
genetic recombination, chromosome breakage and sister chro-
matid exchange [2]. Many solid tumors possess viable hypoxic
cells that are resistant to radiation therapy and some anticancer
chemotherapy protocols. Strategies designed to overcome this
radioresistance include the use of bioreductive drugs, which
exhibit selective toxicity towards hypoxic cells, both in vitro
and in vivo [3]. Mitomycins and the corresponding mitosenes
are well-known examples of reductive alkylating quinones [4].
Bioreduction of such quinones is mediated through one- or
* Corresponding author.
E-mail address: patrice.vanelle@pharmacie.univ-mrs.fr (P. Vanelle).
0223-5234/$ - see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2007.11.020

A. Gellis et al. / European Journal of Medicinal Chemistry 43 (2008) 1858e1864
1859
O
50%), which had been previously developed in our laboratory
[26], for the preparation of 2-(chloromethyl)-4,7-dimethoxy-1-
methyl-1H-benzimidazole derivatives.
The 2-chloromethyl-1,5,6-trimethyl-1H-benzimidazole-4,7-
dione 5b was obtained after an oxidative demethylation of
4b by treatment with cerium ammonium nitrate (CAN), as
shown in Scheme 2.
NH2
O
O
O
O
O
OH
N
H2N
H3C
OCH3
OH
N
N
NH
CH3
Mitomycin C
EO-9
The dihydroxy compound 6 was obtained by demethylation
of 4a, using BBr3/CH2Cl2 at ꢀ78 ꢁC. Oxidation of 6 using po-
tassium dichromate in water at room temperature gave 5a in
59% overall yield (Scheme 3).
The bromo derivative 8 was obtained in 58% yield from 4a
via refluxing in HBr (48%) followed by oxidation with
K2Cr2O7 (Scheme 4).
O
O
H2N
Br
N
N
N
N
R
R1
N
H3C
CH3
Benzimidazole-4,7-diones
O
O
PBI
The sulfonyl derivatives 9 and 10 were prepared under micro-
wave irradiation (H2O, 4 min), bytreatment of 5bwith the sodium
salts of the corresponding sulfinic acid in aqueous solution,
according to a previously described method (Scheme 5) [26]. Re-
action of 5b with an excess of nitronate anions, under microwave
irradiation, afforded the corresponding benzimidazole-4,7-diones
11e13, in 73e85% yield via an SRN1 mechanism (Scheme 5).
The malonate anions gave, under microwave irradiation, the cor-
responding diesters 14 and 15, in 67 and 72% yield, respectively.
Treatment of 11 with TBAOH resulted in the elimination of
HNO2 with the formation of the alkene 16 in 78% yield
(Scheme 6).
According to a study recently carried out at our laboratory
on bis-alkylating derivatives [27], we sought thereafter to pre-
pare a new bioreductive bis-alkylating agent in quinone series,
aiming at comparing the cytotoxic activity of the quinone
derivatives comprising various substituents in 2-position with
that of a dimer presenting two reducible chloromethyl groups.
Thus, oxidation of para-dimethoxybenzene derivatives by
CAN generally leads to the corresponding quinones. There
are, however, some unsubstituted para-dimethoxybenzene deriv-
atives, for which an oxidative coupling is observed, leading to
a dimer [28]. Indeed, the oxidative demethylation of 4a, by treat-
ment with CAN, gave the dimer 17 in 38% yield (Scheme 7).
The exact structure of this compound was determined accord-
ing to HMBC experiment.
Fig. 1. Structures of azamitosene, benzimidazole-4,7-dione, and indoloquinone
derivatives and of mitomycin C.
presence of electron-withdrawing or electron-donating groups,
by varying the quinone redox properties, influences its capacity
to interfere with DNA synthesis [12]. Moreover, studies on aryl-
sulfone containing antitumor agents showed that substituents of
the sulfone group play an important role in the biological activ-
ity [13e16].
Concurrently, the beneficial effects of microwave irradiation
are finding an increased role in process chemistry, especially
when conventional methods require forcing conditions or pro-
longed reaction times [17e19]. The possibilities offered by
this technology are particularly attractive for multi-step synthesis
[20e22] and drug discovery process, where high yielding proto-
cols and avoidance or ease of purification are highly desirable.
On the basis of the above considerations, we have synthe-
sized a series of variously substituted benzimidazole-4,7-dio-
nes and tested them for their antitumor activity. Substituents
were selected with different electronic and solubility charac-
teristics, while others were chosen for their alkylating proper-
ties, with the aim of investigating the substituent effects at the
2-, 5- and 6-position.
2. Chemistry
We applied Day’s method [23] to prepare (4,7-dimethoxy-
5,6-dimethyl-1H-benzimidazol-2-yl)-methanol 2b with signif-
icant modifications (Scheme 1) [24,25]. We present herein
a synthetic method based on a six-step synthesis (overall yield
3. Biological studies
Evaluation of cytotoxicity of the synthesized compounds
was performed on different cancer cell lines using MTT assay.
OH
OCH3
OCH3
OCH3
OCH3
R
R
R
R
NH2
NH2
R
R
R
R
R
R
OH
OH
N
N
Cl
N
N
N
N
a, b, c
d
e
f
H
CH3
CH3
OH
OCH3
OCH3
OCH3
OCH3
1a, R = H (40%)
1b, R = CH3 (84%)
2a, R = H (83%)
2b, R = CH3 (92%)
3a, R = H (83%)
3b, R = CH3 (85%)
4a, R = H (92%)
4b, R = CH3 (80%)
Scheme 1. Reagents and conditions: (a) KOH, (CH3)2SO4, CH3OH, reflux, 1 h; (b) R ¼ H: HNO3 62%, 1 h at 0 ꢁC, 1 h at rt, 1 h at 100 ꢁC or R ¼ CH3:
(CH3CO)2O, HNO3 fuming, 90 ꢁC, 1 h; (c) Sn, HCl; (d) HOCH2CO2H, 4 N HCl, microwave, 800 W, 1 h 30 min; (e) LiN(TMS)2, THF, 20 min, then CH3I,
7 h; (f) SOCl2, CHCl3, reflux, 4 h.

1860
A. Gellis et al. / European Journal of Medicinal Chemistry 43 (2008) 1858e1864
O
compared to mitomycin C. Compound 17 exhibits a similar dose
OCH3
OCH3
effect to mitomycin C (Fig. 2). Inhibition of DT-diaphorase by
2 mM of dicoumarol significantly increases the IC50 value of
mitomycin C from 0.9 ꢂ 0.1 to 2.1 ꢂ 0.2. In contrast, IC50 of
compound 17 is unchanged by addition of dicoumarol
2.8 ꢂ 0.2 versus 2.7 ꢂ 0.2.
H3C
H3C
Cl
N
N
Cl
H3C
N
N
a
H3C
4b
CH3
CH3
O
5b
85%
For derivative 17, the results show that the activity of the
compound is insensitive to DT-diaphorase activity modulation,
which would tend to show that the mechanism of action of the
dimer 17 passes primarily by a mechanism of monoelectronic
bioreduction. These results suggest the possibility to obtain
new bioreductive alkylating agents with limited resistance
compared to mitomycin C.
Scheme 2. Reagents and conditions: (a) Ce(NH4)2(NO3)6, CH3CN, rt, 3 h.
A first screening on breast (T47D), lung (A549) and colon
(HT29) cancer cell lines using 10ꢀ5 and 10ꢀ4 M of each com-
pound was done. Compounds which do not exhibit inhibition
of cell viability higher than 50% at 10ꢀ4 M are not further char-
acterized and their IC50 value are reported as >10ꢀ4 M. Com-
pounds exhibiting inhibition of cell viability higher than 50%
at 10ꢀ4 M have been further evaluated using the Chou and
Talalay method [29] to determine the IC50 values (see Section 5).
Summary of the results obtained is presented in Table 1.
Compound 17 is far more effective in all cell lines tested
with an efficient IC50 close to 3 mM. Other compounds exhibit
variable results depending on the cell line tested. The other
compounds are arranged in an order of decreasing activity:
17 [ 9 ¼ 10 > 11 > 5b for the mammary T47D cell line;
however, 17 [ 11 > 9 > 5b for the lung A549 cell line and
17 [ 5b ¼ 11 for the colon HT29 cell line.
4. Conclusion
The microwave-assisted reaction gave new benzimidazole-
4,7-diones from the intermediate 2-chloromethyl-1,5,6-tri-
methyl-1H-benzimidazole-4,7-dione 5b, which was prepared
in a high overall yields (50%) in seven steps. The compounds
thus obtained were tested against different cancerous mamma-
lian cell lines to evaluate their doseeresponse relationships.
Among all the tested compounds, the dimer 17 proved to be
most effective on breast (T47D), lung (A549) and colon (HT29)
cancer cells. This efficiency is comparable to that of the refer-
ence prototype bioreductive anticancer drug mitomycin C. It is
well-known that this drug requires metabolism conversion to
produce reactive intermediates which irreversibly binds to
DNA. Mitomycin C reduction, through two-electron addition
mechanism, is catalyzed by various enzyme complexes and
in particular by DT-diaphorase. The limitation of some biore-
ductions is a factor of resistance to mitomycin C. Based on
our in vitro results, compound 17, insensitive to DT-diaphorase
bioreduction, may participate in the development of mitomycin
C-like agents, presenting lower tumoral cell resistance.
Apart from the diquinone 17, the cytotoxic activity of other
tested quinones are shown by those bearing a methyl group in
positions 5 and 6, and a chloromethyl or a substituted sulfonyl
group in position 2 (compounds 5b, 9, 10, and 17). The qui-
nones having no substituents at positions 5 and 6 (compound
5a and 8) showed lower activity on the different cellular lines
tested.
Besides the changes in structure, which explain the differen-
tial effects of these compounds, variable effects observed de-
pending on the different cell lines that might result from
a different metabolic background. Indeed these benzimidazole-
4,7-dione derivatives, as the prototypic molecule mitomycin C,
are known to be bioactivated through one- or two-electron reduc-
tion [30]. One important mechanism for two-electron reduction
occurs through DT-diaphorase (NADPH: quinone oxidoreduc-
tase 1; NQO1), which is significantly active in HT29 cell [31].
Compound 17 exhibits the lowest IC50 concentration whatever
the cell line considered compared to other compounds. This
derivative 17 with two chloride nucleofuges, corresponding to
a bioreductive bis-alkylating agent susceptible to undergo two
monoelectronic transfers [32], exhibits a cytotoxic activity higher
than the ones of the derivatives presenting one chloride nucleo-
fuge. The previous biological data suggest that compound 17
would present a different profile of DT-diaphorase bioactivation
5. Experimental protocols
5.1. Chemistry
Melting points were determined on Buchi B-540 and are un-
¨
corrected. Elemental analyses were performed by the Micro-
analyses center of the University of Aix-Marseille 3 and of
1
the INP-ENSCT (Toulouse, France). Both H and 13C NMR
spectra were determined on a Bruker ARX 200 spectrometer.
The 1H chemical shifts are reported as ppm downfield from tet-
ramethylsilane (Me4Si), and the 13C chemical shifts were refer-
enced to the solvent peak: CDCl3 (76.9 ppm). Solvents were
O
OCH3
OH
Cl
Cl
Cl
N
N
N
N
N
N
a
b
75%
78%
6
4a
5a
CH3
CH3
CH3
O
OCH3
OH
Scheme 3. Reagents and conditions: (a) BBr3, CH2Cl2, ꢀ78 ꢁC, 3 h; (b) K2Cr2O7, H2O, rt, 2 h.

A. Gellis et al. / European Journal of Medicinal Chemistry 43 (2008) 1858e1864
1861
OCH3
O
OH
.HBr
Br
Cl
Br
N
N
N
N
N
N
a
b
58%
83%
CH3
4a
7
8
CH3
CH3
O
OH
OCH3
Scheme 4. Reagents and conditions: (a) HBr (48%), reflux, 3 h; (b) K2Cr2O7, H2O, rt, 2 h.
dried by conventional methods. The following adsorbent was
used for column chromatography: silica gel 60 (Merck, particle
size 0.063e0.200 mm, 70e230 mesh ASTM). TLC was per-
formed on 5 cm ꢃ 10 cm aluminum plates coated with silica
gel 60F-254 (Merck) in an appropriate solvent.
Multimode reactor: ETHOS Synth Lab station (Ethos start,
Milestone Inc.). The multimode microwave has a twin magne-
tron (2 ꢃ 800 W, 2.45 GHz) with a maximum delivered power
of 1000 W in 10 W increments (pulse irradiation). Built-in
magnetic stirring (Teflon-coated stirring bar) was used in all
operations. During experiments, time, temperature and power
were measured with the ‘‘easy WAVE’’ software package. The
temperature was measured throughout the reaction and evalu-
ated by an infrared detector, which indicated the surface tem-
perature. Analysis indicated by the symbols of the elements,
were within ꢂ0.4% of the theoretical values (see supplemen-
tary data).
with chloroformeethanol (9/1) and recrystallization from
propan-2-ol gave 5a (78%) as a yellow solid; mp 164e
166 ꢁC; 1H NMR (200 MHz, DMSO-d6) d 3.95 (3H, s,
NCH3), 5.03 (2H, s, CH2), 6.74 (2H, d, J ¼ 8 Hz, CH); 13C
NMR (50 MHz, DMSO-d6) d 32.36 (CH2), 35.93 (NCH3),
131.79 (C), 136.57 (2CH), 140.05 (C), 149.97 (C), 178.54
(C), 180.83 (C); Anal. C9H7ClN2O2 (C, H, N).
5.1.2. 2-(Bromomethyl)-1-methyl-1H-benzimidazole-4,
7-dione 8
The above-mentioned procedure reported for 5a was fol-
lowed using 2-(bromomethyl)-1-methyl-1H-benzimidazole-
4,7-diol hydrobromide 7 to give 8 (83%) as a white solid; mp
1
154e156 ꢁC [34]; H NMR (200 MHz, CDCl3) d 4.03 (3H,
s, NCH3), 4.57 (2H, s, CH2), 6.66 (2H, d, J ¼ 8 Hz, CH); 13C
NMR (50 MHz, CDCl3) d 20.04 (CH2), 32.53 (NCH3),
131.54 (C), 136.27 (CH), 136.55 (CH), 140.82 (C), 149.79
(C), 178.37 (C), 180.41 (C); Anal. C9H7BrN2O2 (C, H, N).
The derivatives 1be5b, 9, 10 [26], 1ae4a, 6 and 7 [33]
were prepared as previously described.
5.1.3. 1,5,6-Trimethyl-2-(2-methyl-2-nitropropyl)-1H-
benzimidazole-4,7-dione 11
5.1.1. 2-Chloromethyl-1-methyl-1H-benzimidazole-4,
7-dione 5a
To a solution of 2-nitropropane lithium salt (0.35 g,
3.7 mmol) in H2O (3 mL), 2-chloromethyl-1,5,6-trimethyl-1H-
benzimidazole-4,7-dione 5b (0.3 g, 1.26 mmol) was added drop-
wise. The reaction mixture was irradiated in a microwave oven
(Ethos start) for 10 min at 100 ꢁC, with a power of 800 W. The
crude product was dissolved in dichloromethane (20 mL). The
organic layer was then dried over anhydrous magnesium sulfate,
and evaporated under reduced pressure. Purification by column
To a solution of potassium dichromate (13.8 g, 46.9 mmol)
in water (50 mL), 2-(chloromethyl)-1-methyl-1H-benzimid-
azole-4,7-diol 6 (2 g, 9.4 mmol) was added portionwise. The
reaction mixture was stirred for 2 h and extracted with chloro-
form (3 ꢃ 50 mL). The combined organic layer was dried over
magnesium sulfate and evaporated under reduced pressure.
Purification of the crude product on silica gel by eluting
O
O
O
R3
R2
H3C
H3C
SO2R1
H3C
H3C
H3C
H3C
Cl
N
N
N
N
N
N
a
b
NO2
CH3
CH3
CH3
O
O
O
5b
9, R1 = C6H5 (87%)
10, R1 = C4H9 (88%)
11, R2 = R3 = CH3
12, R2 + R3 = -(CH2)4- (78%)
(85%)
c
13, R2 + R3 = -(CH2)5- (73%)
O
O
R4
CO2CH2CH3
CO2CH2CH3
H3C
H3C
N
N
14, R4 = CH3 (67%)
15, R4 = C6H5 (72%)
CH3
Scheme 5. Reagents and conditions: (a) R-SOꢀ2 Naþ, H2O, microwave, 800 W, 80 ꢁC, 4 min; (b) N(Bu)þ4 ꢀCR1R2NO2, H2O, microwave, 800 W, 10 min, 100 ꢁC;
(c) N(Bu)þ4 ꢀC(CO2CH2CH3)2, H2O, microwave, 800 W, 10 min, 100 ꢁC.

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A. Gellis et al. / European Journal of Medicinal Chemistry 43 (2008) 1858e1864
Table 1
Cytotoxicity IC50 values in mM for the tested compounds against three cancer
cell lines
O
O
O
H3C
H3C
CH3
NO2
H3C
H3C
CH3
78%
H3C
H3C
N
N
N
N
a
Compound Formula
T47Da A549a HT29a
(breast) (lung) (colon)
CH3
CH3
O
O
11
16
5a
>100 >100 >100
Cl
N
N
Scheme 6. Reagents and conditions: (a) CH2Cl2, TBAOH 40%, 3 h, rt.
CH
O
3
chromatography on silica gel using chloroformeethyl acetate (7/
3) as eluent and recrystallization from propan-2-ol gave 11
(85%) as a yellow solid; mp 183e184 ꢁC; 1H NMR
(200 MHz, CDCl3) d 1.78 (6H, s, 2CH3), 2.06 (3H, s, CH3),
2.09 (3H, s, CH3), 3.39 (2H, s, CH2), 3.92 (3H, s, NCH3); 13C
NMR (50 MHz, CDCl3) d 12.08 (CH3), 12.41 (CH3), 26.37
(2CH3), 32.29 (NCH3), 36.31 (CH2), 87.11 (C), 130.77 (C),
139.95 (C), 140.69 (C), 141.08 (C), 149.19 (C), 178.76 (C),
180.96 (C); Anal. C14H17N3O4 (C, H, N).
O
O
H C
3
Cl
N
5b
100 ꢂ 8 40
5
15 4
N
H C
3
CH
3
O
O
Br
N
N
8
>100
>100
>100
CH
3
5.1.4. 1,5,6-Trimethyl-1H-benzimidazole-4,7-dione
derivatives 12e15
O
O
H C
3
S
O
To a solution of 40% tetrabutylammonium hydroxide in
water (3.7 mmol) and corresponding nitroalkane or malonate
derivative (3.7 mmol), 2-chloromethyl-1,5,6-trimethyl-1H-
benzimidazole-4,7-dione 5b (0.3 g, 1.26 mmol) was added.
The reaction mixture was irradiated in a microwave oven
(Ethos start) for 10 min at 100 ꢁC, with a power of 800 W.
The crude product was dissolved in dichloromethane
(20 mL). The organic layer was then dried over anhydrous
magnesium sulfate, and evaporated under reduced pressure.
Purification by column chromatography on silica gel using
chloroformeethyl acetate (7/3) as eluent and recrystallization
from propan-2-ol gave corresponding products 12 and 13 (78
and 73%) or 14 and 15 (67 and 72%).
N
9
10
10
3
8
2
>100
>100
>100
>100
>100
>100
N
CH
H C
3
O
O
3
C H
4
9
O
H C
3
S
N
N
O
10
11
12
13
14
4
10 3
H C
3
CH
O
O
3
H C
3
CH
H C
3
3
N
N
NO
>100
>100
>100
>100
>100
>100
>100
>100
2
H C
3
CH
O
O
3
3
H C
3
N
N
NO
2
H C
3
5.1.4.1.
1,5,6-Trimethyl-2-[(1-nitrocyclopentyl)methyl]-1H-
CH
O
O
benzimidazole-4,7-dione 12. Yellow solid; mp 162e163 ꢁC;
1H NMR (200 MHz, CDCl3) d 1.78e1.84 (4H, m, 2CH2),
2.04 (3H, s, CH3), 2.08 (3H, s, CH3), 2.19e2.63 (4H, m,
2CH2), 3.46 (2H, s, CH2), 3.90 (3H, s, NCH3); 13C NMR
(50 MHz, CDCl3) d 12.06 (CH3), 12.39 (CH3), 24.25
(2CH2), 32.16 (NCH3), 34.86 (CH2), 37.73 (2CH2), 97.73
(C), 130.75 (C), 139.91 (C), 140.66 (C), 141.11 (C), 149.70
(C), 178.78 (C), 180.99 (C); Anal. C16H19N3O4 (C, H, N).
H C
3
N
N
NO
2
H C
3
CH
O
O
3
H C
3
CO CH CH
3
CO CH CH
H C
3
2
2
N
N
2
2
3
H C
3
CH
O
O
3
5.1.4.2.
1,5,6-Trimethyl-2-[(1-nitrocyclohexyl)methyl]-1H-
benzimidazole-4,7-dione 13. Yellow solid; mp 183e184 ꢁC;
1H NMR (200 MHz, CDCl3) d 1.78e1.84 (6H, m, 3CH2),
CO CH CH
3
H C
3
2
2
N
N
15
16
>100
>100
>100
>100
>100
>100
CO CH CH
2
2
3
H C
3
CH
O
O
O
3
H3C
OCH3
OCH3
N
N
O
Cl
N
N
H C
3
a
H C
3
CH
N
N
3
Cl
N
N
Cl
H C
3
O
CH
CH3
O
3
CH3
O
38%
4a
17
(continued on next page)
Scheme 7. Reagents and conditions: (a) Ce(NH4)2(NO3)6, CH3CN, rt, 2 h.

A. Gellis et al. / European Journal of Medicinal Chemistry 43 (2008) 1858e1864
1863
Table 1 (continued)
(CH3), 12.33 (CH3), 13.98 (2CH3), 31.22 (NCH3), 32.84
(CH2), 60.89 (2CH2), 126.85 (3CH), 127.43 (2CH), 140.11
(C), 140.12 (C), 141.21 (C), 141.35 (C), 148.14 (C), 169.32
(C), 169.53 (C), 178.87 (2C), 180.50 (2C); Anal.
C24H26N2O6 (C, H, N).
Compound Formula
T47Da
(breast) (lung) (colon)
A549a HT29a
O
O
17
3
1
3
1
3 1
H C
3
N
O
O
Cl
N
N
N
Cl
CH
5.1.5. 1,5,6-Trimethyl-2-(2-methylprop-1-enyl)-1H-
benzimidazole-4,7-dione 16
3
O
To
a solution of 1,5,6-trimethyl-2-(2-methyl-2-nitro-
NH
2
O
O
O
propyl)-1H-benzimidazole-4,7-dione 11 (0.2 g, 0.68 mmol)
in dichloromethane (30 mL) was added dropwise an aqueous
solution of 40% tetrabutylammonium hydroxide (1.3 g,
0.68 mmol). The reaction mixture was stirred at room temper-
ature for 3 h. The organic layer was then dried over anhydrous
magnesium sulfate, and evaporated under reduced pressure.
Purification by column chromatography on silica gel using
chloroformeethyl acetate (7/3) as eluent and recrystallization
from propan-2-ol gave 16 (78%) as a red solid; mp 153e
H N
2
OCH
MC
e
e
0.9 0.1
3
N
H C
3
NH
a
Results express the mean IC50 ꢂ SEM obtained from three independent
experiments.
2.04 (3H, s, CH3), 2.06 (3H, s, CH3), 2.16e2.25 (4H, m,
2CH2), 3.46 (2H, s, CH2), 3.90 (3H, s, NCH3); 13C NMR
(50 MHz, CDCl3) d 12.04 (CH3), 12.40 (CH3), 24.20
(2CH2), 25.60 (CH2), 32.33 (NCH3), 34.79 (CH2), 37.73
(2CH2), 97.65 (C), 130.68 (C), 139.85 (C), 140.60 (C),
141.01 (C), 149.60 (C), 178.88 (C), 180.90 (C); Anal.
C17H21N3O4 (C, H, N).
1
154 ꢁC; H NMR (200 MHz, CDCl3) d 2.00 (3H, s, CH3),
2.05 (3H, s, CH3), 2.09 (3H, s, CH3), 2.22 (3H, s, CH3),
3.89 (3H, s, NCH3), 6.01 (1H, s, CH); 13C NMR (50 MHz,
CDCl3) d 12.11 (CH3), 12.44 (CH3), 20.98 (CH3), 27.42
(CH3), 31.84 (NCH3), 109.53 (CH), 130.65 (C), 139.72 (C),
140.53 (C), 141.00 (C), 149.10 (C), 178.64 (C), 180.86 (C);
Anal. C14H16N2O2 (C, H, N).
5.1.4.3. Diethyl 2-methyl-2-[(1,5,6-trimethyl-4,7-dioxo-4,7-di-
hydro-1H-benzimidazol-2-yl)methyl]malonate 14. Yellow oil;
1H NMR (200 MHz, CDCl3) d 1.24 (6H, t, J ¼ 7.2 Hz,
2CH3), 1.70 (3H, s, CH3), 2.06 (3H, s, CH3), 2.09 (3H, s,
CH3), 3.48 (2H, s, CH2), 3.84 (3H, s, NCH3), 4.20 (4H, q,
J ¼ 7.2 Hz, 2CH2); 13C NMR (50 MHz, CDCl3) d 12.36
(CH3), 12.40 (CH3), 13.96 (2CH3), 20.56 (CH3), 32.50
(NCH3), 32.76 (CH2), 61.91 (2CH2), 140.06 (C), 140.50 (C),
141.02 (C), 148.10 (C), 169.27 (C), 169.48 (C), 178.81
(2C), 180.59 (2C); Anal. C19H24N2O6 (C, H, N).
5.1.6. 2,20-Bis(chloromethyl)-1,10-dimethyl-5,50-bi
(1H-benzimidazole)-4,40,7,70-tetraone 17
To a solution of 2-(chloromethyl)-4,7-dimethoxy-1-methyl-
1H-benzimidazole 4a (5 g, 20.7 mmol) in acetonitrile (100 mL)
was added dropwise a mixture of CAN (cerium ammonium ni-
trate) (32.6 g, 59.5 mmol) in water (40 mL). The reaction mix-
ture was stirred for 3 h and the solvent was evaporated under
vacuum. The residue was dissolved in chloroform and washed
with water (3 ꢃ 30 mL). The organic layer was dried over mag-
nesium sulfate and the solvent was removed under vacuum. Pu-
rification of the crude product on silica gel eluting with
chloroformeethyl acetate (9/1) and recrystallization from
propan-2-ol gave 17 (38%) as a yellow solid; mp 221e222 ꢁC;
1H NMR (200 MHz, CDCl3) d 4.01 (3H, s, NCH3), 4.59 (2H,
s, CH2), 6.50 (1H, s, CH); 13C NMR (50 MHz, CDCl3) d 32.2
5.1.4.4. Diethyl 2-phenyl-2-[(1,5,6-trimethyl-4,7-dioxo-4,7-di-
hydro-1H-benzimidazol-2-yl)methyl]malonate 15. Yellow oil;
1H NMR (200 MHz, CDCl3) d 1.15 (6H, t, J ¼ 7.0 Hz,
2CH3), 2.05 (3H, s, CH3), 2.08 (3H, s, CH3), 3.60 (2H, s,
CH2), 3.85 (3H, s, NCH3), 4.45 (4H, q, J ¼ 7.0 Hz, 2CH2),
7.35 (5H, m, H ar); 13C NMR (50 MHz, CDCl3) d 12.30
1.5
1.0
0.5
1.5
1.0
0.5
Compound 17
Compound 17 + Dicoumarol 2µM
0.0
Mitomycin C
Mitomycin C + Dicoumarol 2µM
0.0
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Log doses
Log Doses
-0.5
-0.5
Fig. 2. Effect of DT-diaphorase inhibition on compound 17 efficiency compared to mitomycin C.

1864
A. Gellis et al. / European Journal of Medicinal Chemistry 43 (2008) 1858e1864
(NCH3), 35.2 (CH2), 131.6 (C), 135.7 (CH), 140.3 (C), 150.2 (C),
177.7 (C), 180.4 (C); Anal. C18H12Cl2N4O4 (C, H, N).
Appendix. Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.ejmech.2007.11.020.
5.2. Evaluation of cell cytotoxicity
5.2.1. Cell culture
References
Human lung (A549), breast (T47D) and colon (HT29) can-
cer cell lines were used to evaluate the impact on cell viability
of each compound. A549 and T47D cells were cultured in
RPMI 1640 medium and HT29 cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM), all the me-
diums were supplemented with 10% fetal bovine serum (FBS)
and 2 mM l-glutamine at 37 ꢁC under a 5% CO2 atmosphere.
For each cell line, 70% confluent cell culture flask was trypsi-
nized and cells were seeded in 96 well plates at a density of
3000 cells by well in the appropriate complete media; 24 h
after seeding, the cells were treated with the different com-
pounds or control vehicle solution (DMSO 0.1% in phosphate
saline buffer); 48 h after treatment, viability was accessed by
MTT assay.
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Acknowledgment
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This work was supported by the Centre National de la
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