Differential antiproliferative activity of new benzimidazole-4,7-diones

Article abstract of DOI:10.1016/j.farmac.2004.04.001

Ten benzimidazole-4,7-diones were synthesized and tested in vitro on two tumor cell lines. Several compounds showed a significant antiproliferative activity on K562 cells, although to a different extent, whereas compound 1i showed a highly significant activity on SW620 cells, comparable to that of doxorubicin. Both the substituents in the quinone ring and the position of the nitrogen atom in the pyridine moiety play a crucial role for the biological activity.

Full text of DOI:10.1016/j.farmac.2004.04.001

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IL FARMACO 59 (2004) 663–668
www.elsevier.com/locate/farmac
Differential antiproliferative activity of new benzimidazole-4,7-diones
Laura Garuti a,*, Marinella Roberti a, Daniela Pizzirani a, Annalisa Pession b,
Emanuela Leoncini c, Valentina Cenci b, Silvana Hrelia c
a Department of Pharmaceutical Science, University of Bologna, via Belmeloro 6, Bologna 40126, Italy
b Department of Experimental Pathology, University of Bologna, via S. Giacomo, Bologna 40126, Italy
c Department of Biochemistry G. Moruzzi University of Bologna, via Irnerio 48, Bologna 40126, Italy
Received 12 March 2004; accepted 17 April 2004
Available online 25 May 2004
Abstract
Ten benzimidazole-4,7-diones were synthesized and tested in vitro on two tumor cell lines. Several compounds showed a significant
antiproliferative activity on K562 cells, although to a different extent, whereas compound 1i showed a highly significant activity on SW620
cells, comparable to that of doxorubicin. Both the substituents in the quinone ring and the position of the nitrogen atom in the pyridine moiety
play a crucial role for the biological activity.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Antiproliferative activity; Benzimidazoles; Quinones; Pyridine
1. Introduction
The aim of this work is to correlate the substituent effect with
the antiproliferative activity on two different tumor cell lines.
We report herein the synthesis of some benzimidazole-4,7-
diones related to general structure 1 and their effectiveness in
inhibiting DNA synthesis in tumor cells (Fig. 1).
A number of benzimidazolediones displays good antitu-
mor activity [1–8]. In most cases the activity is related to the
ability of quinone to accept one or two electrons to form
reactive species toxic to cells [9]. The substituents on the
quinone ring play a significant role in determining the bio-
logical properties. The presence of electron-attracting or
-donating groups, by varying the quinone redox properties,
influences its capacity to interfere with DNA synthesis [10].
Our recent studies on benzimidazole-4,7-diones [11,12] re-
vealed that the presence of an electron-donating group, such
as methoxy, increased the antiproliferative activity. These
results prompted us to prepare new quinones bearing the
methoxy or the methyl substituent at C-5(6). Studies on
various quinones bearing the 2-pyridyl ring [13–15] showed
that the substituents on this moiety influence the biological
activity. Thus, the presence of a methyl function at the
pyridine-6-position is evaluated. Moreover, it is known that
in heterocyclic quinones containing nitrogen atoms, the posi-
tion of nitrogen is an important feature for the cytotoxicity
[7].
2. Chemistry
Compounds 1a, namely 2-(2-pyridyl)benzimidazole-4,7-
dione and the 5(6)-methoxy derivative 1b are reported in the
literature [10].
Refluxing
2 [16] with 6-methyl-2-pyridinecarbox-
aldehyde in nitrobenzene gave the corresponding 4,7-
dimethoxybenzimidazole 3a (Scheme 1). 2-(3-Pyridyl)-4,7-
dimethoxybenzimidazole 3b and 2-(4-pyridyl)-4,7-
dimethoxybenzimidazole 3c were prepared according to a
known procedure [17]. Oxidation of 3(a–c) with cerium
ammonium nitrate (CAN) in aqueous acetic acid gave the
quinones 1(c–e). Reaction of 1(c–e) with ammonium hy-
droxide (NH4OH) in methanol at room temperature yielded
the 5(6)-methoxy derivative 1(f–h) (Scheme 2).
The quinones 1(i–n) were synthesized from commercially
available 4-methyl-2-nitroaniline (4).
This led us to examine the effect of the shifting of the
nitrogen in the pyridine ring from 2- to 3- and 4-positions.
Reduction of 4 with tin chloride (SnCl2) in boiling
methanol, afforded 5. Cyclization of the compound 5 with
appropriate arylaldehydes such as 2-, 3-, 4- pyridinecarbox-
* Corresponding author.
E-mail address: lauga@alma.unibo.it (L. Garuti).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.04.001

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L. Garuti et al. / IL FARMACO 59 (2004) 663–668
Fig. 1 . Studied quinones
Scheme 1. Reagents and conditions: (a) 6-methyl-2-pyridinecarboxaldehyde, nitrobenzene.
Scheme 2. Reagents and conditions: (a) CAN, AcOH/H2O; (b) NH4OH, MeOH.
aldehyde or 6-methyl-2-pyridinecarboxaldehyde in nitroben-
zene, gave the corresponding 2-aryl-5(6)-methyl-
benzimidazoles 6(a–d). The bromination of 6(a–d) to
provide 6-bromo derivatives 7(a–d) was carried out so as to
direct nitration to the 4-position in the next step. Nitration of
7(a–d) afforded 8(a–d), as a mixture of 4- and 7-nitro iso-
mers. One-pot reductive removal of the bromo- and nitro-
group reduction, utilizing Pd on carbon and H2, afforded
9(a–d) as a mixture of 4- and 7- amino isomers. Fremy’s
oxidation of these mixtures gave a single 5(6)-methyl
quinone 1(i–n) (Scheme 3).
they were treated with the different compounds and doxoru-
bicin, utilized a known antiproliferative drug in these cell
lines, dissolved in dimethyl sulfoxide (DMSO) and diluted in
medium containing 5% fetal-calf serum to give the final
concentrations ranging from 0.1 to 100 µM. The final DMSO
concentration was kept constant at 0.2% and was also added
to control wells. After 24 h of incubation, cell proliferation
analyses were performed. Daily observations were made by
phase contrast microscopy.
Evaluation of cell proliferation was performed by measur-
ing DNA synthesis by means of methyl-[3H]-thymidine incor-
poration as previously reported [18,19]. All data represent
the mean S.D. of quadruplicate assays. Cytotoxicity data
were expressed as IC50 values, i.e. the concentrations of the
test agent inducing 50% reduction in DNA synthesis com-
pared with the control cultures.
3. Biology
The cell lines used were derived from human tumors: the
K562 cell line (from ATCC, Rockville, MA) was from an
erythroleukemia and the SW620 cell line (from ATCC,
Rockville, MA) was from a colon carcinoma. All cell lines
were maintained in RPMI 1640 medium supplemented with
0.5% non-essential amino acids, 100 IU/ml penicillin,
0.1 mg/ml streptomycin, 0.1 mg/ml L-glutamine and 10%
fetal-calf serum. Cells were incubated at 37 °C in a humidi-
fied atmosphere of 5% CO2 95% air. Cells of each line were
seeded in six-well plates (5 × 105 cells/well), and after 24 h
4. Results and discussion
The antiproliferative activity of the synthesized com-
pounds were determined in two different cell lines after 24 h
drug exposure. Cytotoxicity of doxorubicin, a drug with high
antiproliferative action against these cell lines, was also de-
termined in the same conditions. The effect of the adminis-

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L. Garuti et al. / IL FARMACO 59 (2004) 663–668
665
Scheme 3. Reagents and conditions: (a) SnCl2 MeOH; (b) 2-, or 3-, or 4-pyridinecarboxaldehyde, or 6-methyl-2-pyridinecarboxaldehyde, nitrobenzene; (c)
bromine, AcOH; (d) HNO3, H2SO4; (e) H2, Pd on carbon; (f) Fremy’s salt, pH 3.
tration of the different compounds on DNA synthesis was
reported in Table 1 as the IC50 value. The antiproliferative
activity was variously manifested in the two cell lines. Nine
out of 12 compounds showed a significant activity on K562
cells, the IC50 values ranging between 1 and 10 µM. A fewer
number of compounds was active on SW620 cell line, only
four of them showed a IC50 value below 10 µM, and com-
pound 1i was the most active, with a IC50 value comparable
to that of doxorubicin. The presence of a substituent on the
quinone ring is crucial, all the compounds with a significant
antiproliferative activity (<10 µM) are 5(6) substituted (a part
from compound 1a whose IC50 value in SW620 cells is
9.92 µM, very close to 10 µM).
The two electron-donating groups at C-5(6) on the
quinone ring were able to influence to a different extent the
antiproliferative action: the methoxy group was a better sub-
stituent than the methyl group on K562 cell line (see 1b), on
the contrary, the presence of a methyl function on the
quinone ring, led to the best activity on SW620 (see 1i). The
introduction of a methyl substituent on the pyridine-6-
position caused an increase in the IC50 value in comparison
to the corresponding quinone-substituted compound (com-
pare 1b with 1f, 1i with 1n) independent of the cell line taken
into account. This effect is probably due to the increased
steric hindrance of the quinone–pyridine-substituted com-
pounds in comparison to the quinone-substituted ones.
The position of the nitrogen atom in the pyridine ring
showed an important role in modulating the activity: the
2-pyridyl-substituted quinones (1b and 1i) were the most
active in both the two cell lines. Shifting the nitrogen from 2-
to 3- and 4-positions progressively decreased the antiprolif-
erative action, independent of the presence or the absence of
substituents on the quinone ring (compare 1b with 1g and 1h;
1i with 1l and 1m). This indicates that not only the degree of
substitution of the pyridine ring but also the distance of the
pyridine nitrogen from the benzimidazole ring play a crucial
role in the interactions between the compounds and the
biological target(s) accountable for the antiproliferative ef-
fect. The different behavior of these quinones in the two
different cancer cell lines is difficult to explain. The total
efficacy of an antiproliferative molecule is affected not only
by the intrinsic activity of the drug molecule but also by its
physical properties, such as water solubility, surface hydro-
phobicity and drug release rate. Different chemical com-
pounds may evidence different ability to partition into the
cell membranes and to enter the cancer cell. The physico-
Table 1
Inhibition of human cancer K562 and SW620 cell proliferation by derivati-
ves 1(a–n)
Compound
IC50 (µM)
K562
SW620
Doxorubicin
0.66 0.11
18.38 3.26
0.56 0.14
5.05 1.03
23.12 1.95
28.06 2.03
9.18 1.38
1.99 0.54
3.21 0.75
1.18 0.22
4.06 0.81
7.64 1.06
2.52 0.33
0.72 0.10
9.92 2.57
18.96 2.84
23.32 3.18
17.22 2.98
21.12 2.48
21.78 2.19
22.47 1.73
27.18 2.36
0.98 0.08
2.58 0.14
5.02 0.26
14.31 1.78
1a
1b
1c
1d
1e
1f
1g
1h
1i
1l
1m
1n
IC50 represents the drug concentration that causes 50% inhibition of DNA
synthesis in comparison to control cells. IC50 values were evaluated by
measuring DNA synthesis in the presence of the different drugs as reported
in methods.Values are mean S.D. of at least four independent experiments.

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L. Garuti et al. / IL FARMACO 59 (2004) 663–668
chemical properties of the lipid bilayers of different cell lines
could in turn influence the ability of the drug to partition into
and cross through tumor cell membranes. It has been sug-
gested that unsaturated fatty acids (UFAs) may influence the
cytotoxic activity of cancer chemotherapeutic agents [20]
supporting the hypothesis that some UFAs can be considered
as modulators of tumor cell chemosensitivity. So, the previ-
ously demonstrated different fatty acid composition of phos-
pholipids of tumor cells [18,19] could in turn influence the
biological activity of the tested molecules. Due to the fact
that all the new derivatives possess the same benzimidazole-
4,7-dione moiety, the differences in behavior have to be
attributed to the different side chains. Although the impor-
tance of the methoxy group in the quinone ring was clearly
evidenced as far as the antiproliferative activity in K562 cell
line is concerned, different chemical modifications of the
structure moiety of the parent compound should be war-
ranted to improve the biological activity on the colon carci-
noma cell line. Data reported in this paper allow us to affirm
the crucial role carried out by the presence of suitable side
chains and in this framework structures reported here can
constitute an useful tool for the development of rationales
devoted to the synthesis of new drugs endowed with better
specificity of action towards solid tumor cell lines.
give 0.60 g (62.5%) of 3a as a solid, which was crystallized
from acetone–petroleum ether. M.p. 173 °C, 1H NMR
(DMSO-d6): d 2.60 (s, 3H), 3.90 (s, 6H), 6.65 (m, 2H), 7.34
(m, 1H), 7.85 (m, 1H), 8.07 (m, 1H); elemental analysis (%):
calc. for C15H15N3O2 (269.29): C 66.74, H 5.6, N 15.60;
found: C 66.79, H 5.70, N 15.52.
5.1.3. General procedure for the synthesis
of 2-arylbenzimidazole-4,7-diones 1(c–e)
A solution of the appropriate 4,7-dimethoxybenzimi-
dazole 3(a–c) (6.0 mmol) in acetic acid (20 ml) was added of
a solution of CAN (6.67 g, 12.7 mmol) in water (10 ml) and
stirred at room temperature for 3 h. The aqueous solution was
extracted with ethyl acetate (4 × 50 ml). The organic phase
was dried sodium sulfate (Na2SO4), evaporated in vacuo and
chromatographed on
a
silica gel column, using
chloroform/methanol, 95:5 as the eluent. The purified prod-
ucts were crystallized from EtOH.
1
1c: m.p. 230 °C decomposed; H NMR (DMSO-d6): d
2.60 (s, 3H), 6.77 (s, 1H), 7.40 (m, 1H), 7.86 (m, 1H), 8.02
(m, 1H); elemental analysis (%): calc. for C13H9N3O2
(239.22): C 65.26, H 3.79, N 17.56; found: C 65.48, H 3.57,
N 17.39.
1d: m.p. 235 °C, 1H NMR (DMSO-d6): d 6.80 (s, 2H), 7.4
(s, 1H), 7.85 (b, 1H), 8.80 (m, 2H), 9.40 (s, 1H); elemental
analysis (%): calc. for C12H7N3O2 (225.19): C 63.99, H 3.13,
N 18.65; found: C 63.71, H 3.28, N 18.89.
5. Experimental
1e: m.p. 198 °C, 1H NMR (DMSO-d6): d 6.86 (s, 1H), 7.01
(s, 1H), 8.38 (m, 2H), 8.91 (m, 2H); elemental analysis (%):
calc. for C12H7N3O2 (225.19): C 63.99, H 3.13, N 18.65;
found: C 64.03, H 3.21, N 18.46.
5.1. Chemistry
5.1.1. Materials and methods
Reaction courses and product mixtures were routinely
monitored by thin-layer chromatography (TLC) on silica gel
(precoated F254 Merck plates) and visualized with iodine or
5.1.4. General procedure for the synthesis of 2-aryl-5(6)-
methoxybenzimidazole-4,7-diones 1(f–h)
1
aqueous potassium permanganate. H NMR and 13C NMR
A solution of the appropriate benzimidazole-4,7-dione
1(c–e) (1.46 mmol) in methanol was added of aqueous
NH4OH (30% NH3 in water) (15 ml) and stirred at room
temperature for 1 h. The solution was then evaporated in
vacuo and the residue was chromatographed on a silica gel
column eluted with ethyl acetate–petroleum ether, 6:4. The
purified products were crystallized from acetone–petroleum
ether.
were determined in DMSO-d6 solutions with a Varian VXR
300 spectrometer, peaks positions are given in parts per
million (d) downfield from tetramethylsilane as internal stan-
dard. The EI-MS were recorded on a VG 7070E at 70 eV; the
positive ion is reported as m/z. Melting points (m.p.) were
determined on a Buchi–Tottoli instrument and are uncor-
rected. Chromatographies were performed with Merck 60–
200-mesh silica gel. All products reported 1H NMR spectra
in agreement with the assigned structures. Elemental analy-
ses were performed by the micro-analytical laboratory of
Dipartimento di Chimica, University of Ferrara, and agreed
within 0.4% of the theoretical values.
1f: m.p. 170 °C; 1H NMR (DMSO-d6): d 2.62 (s, 3H), 3.99
(s, 3H), 7.28 (s, 1H), 7.40 (m, 1H), 7.89 (m, 1H), 8.30 (m,
1H); elemental analysis (%): calc. for C14H11N3O3 (269.23):
C 62.45, H 4.11, N 15.60; found: C 62.54, H 4.19, N 15.52.
1
1g: m.p. >300 °C, H NMR (DMSO-d6): d 4.01 (s, 3H),
7.29 (s, 1H), 7.60 (m, 1H), 8.57 (m, 1H), 8.70 (m, 1H), 9.42
(m, 1H); elemental analysis (%): calc. for C13H9N3O3
(255.22): C 61.67, H 3.55, N 16.46; found: C 61.28, H 3.43,
N 16.52.
5.1.2. 2-(6-Methyl-2-pyridyl)-4,7-dimethoxybenzimidazole
(3a)
A solution of 2,3-diamino-1,4-dimethoxybenzene 2
(0.6 g, 3.56 mmol) in nitrobenzene (10 ml) was added of
6-methyl-2-pyridinecarboxaldehyde (0.431 g, 3.56 mmol)
and refluxed for 2 h. After cooling the solution was evapo-
rated in vacuo. The residue was chromatographed on a silica
gel column, eluted with ethyl acetate–petroleum ether, 8:2 to
1
1h: m.p. >300 °C, H NMR (DMSO-d6): d 3.92 (s, 3H),
6.81 (s, 1H), 8.11 (m, 2H), 8.77 (m, 2H); elemental analysis
(%): calc. for C13H9N3O3 (255.22): C 61.67, H 3.55, N
16.46; found: C 61.26, H 3.40, N 16.63.