Synthesis, anticancer activity and UPLC analysis of the stability of some New Benzimidazole-4,7-dione derivatives

Article abstract of DOI:10.3390/molecules19010400

In this work, a sensitive analytical method to study the stability of two new series of synthesized heterocyclic compounds, the benzimidazole-4,7-diones 5 and N-oxide benzimidazole-4,7-dione derivatives 6 was established and validated. These derivatives w

Full text of DOI:10.3390/molecules19010400

Molecules 2014, 19, 400-413; doi:10.3390/molecules19010400
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Anticancer Activity and UPLC Analysis of the
Stability of Some New Benzimidazole-4,7-dione Derivatives
1
,
2
2
Katarzyna Błaszczak-Świątkiewicz *, Diogo Correia Almeida , Maria De Jesus Perry and
1
Elżbieta Mikiciuk-Olasik
1
Department of Pharmaceutical Chemistry and Drug Analysis, Medical University, Muszyńskiego 1,
Łódź 90-151, Poland; E-Mail: elzbieta.mikiciuk-olasik@umed.lodz.pl
2
Department of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy, University of
Lisbon, Av. Prof. Gama Pinto, Lisboa 1649-003, Portugal;
E-Mails: diogo.correia.almeida@gmail.com (D.C.A.); mjprocha@ff.ul.pt (M.D.J.P.)
*
Author to whom correspondence should be addressed;
E-Mail: katarzyna.blaszczak-swiatkiewicz@umed.lodz.pl.
Received: 21 October 2013; in revised form: 9 December 2013 / Accepted: 16 December 2013 /
Published: 31 December 2013
Abstract: In this work, a sensitive analytical method to study the stability of two new
series of synthesized heterocyclic compounds, the benzimidazole-4,7-diones 5 and
N-oxide benzimidazole-4,7-dione derivatives 6 was established and validated. These
derivatives were developed as potential anticancer substances to be activated under
hypoxic conditions. At this point we were concerned with establishing their stability in
some specific environments for further biological studies. For that, we developed and
validated an RP-UPLC method. Next, selected compounds were tested in vitro for possible
anticancer activity. Their effect on A549 tumour cell lines under normoxia and hypoxia
conditions was determined by a WST-1 test. Four of the examined compounds (compounds
5
a–c and 6c) showed very good antiproliferative effects and three of them (compounds 6a,
b and 6d) were specific for hypoxia conditions. The hypoxia/normoxia cytotoxic coefficient
6
of compound 6b is close to that of tirapazamine—a reference compound in our
experiments—and this parameter locates it between mitomycin C and 2-nitroimidazole
(
misonidazole).
Keywords:
anticancer
prodrug;
hypoxia;
benzimidazole-4,7-dione; N-oxide
benzimidazole-4,7-dione; UPLC

Molecules 2014, 19
. Introduction
Cancer is still one of the leading causes of death in the World, particularly in developing countries.
401
1
Hypoxia is present in most of those tumours and its characteristic feature is the variable incidence and
severity within a given patient population, which happens because solid tumour tissue is poorly
perfused. There is evidence that tumours exhibiting extensive hypoxia are more aggressive and,
therefore, they have a poorer prognosis, since hypoxia is implicated in radiotherapeutic and
chemotherapeutic resistance. Currently there is a wide range of drugs, mainly hypoxic-activated
bioreductive prodrugs, that attempt to address this issue, tirapazamine being the most representative
molecule of such prodrugs. A prodrug is defined as an inactive compound that is converted in the
body, either spontaneously or through metabolism by some endogenous enzymes, into the desired,
pharmacologically active species [1]. Bioreductive prodrugs are converted in hypoxic areas into
cytotoxic species by enzymatic reduction [2,3]. Nitroaromatic groups, quinones, aromatic N-oxides
and aliphatic N-oxides are some of the chemical groups which have the potential to be metabolised by
enzymatic reduction under hypoxic conditions, and thus provide the basis for the design of
bioreductive prodrugs [2]. The most common mechanism by which bioreductive prodrugs are selective
for hypoxic cells involves one-electron reduction which generates a prodrug radical that can be
re-oxidized by oxygen (reaction 1 in Figure 1) in normal cells (forming superoxide), but generates an
active drug in hypoxic cells, either by fragmentation of the prodrug radical (reaction 2 in Figure 1) or
by its further reduction, usually by disproportionation (reaction 3 in Figure 1) and subsequent
reduction of the two electron reduction of product X, (reactions 4 and 5 in Figure 1). This is
characteristic of nitro and aromatic N-oxide compounds and results in hypoxia-selective cell killing
provided that the prodrug radical (or its downstream products) are more cytotoxic than superoxide or
the unreduced prodrug [2,3].
Figure 1. One-electron versus two-electron reduction of bioreductive prodrug [2].
The second mechanism, typical of quinones, includes bioreductive activation with two sequential
one-electron reductions [2]. Nitroaromatic compounds, such as CB1954, are hypoxia-activated
prodrugs. The one-electron adduct (the nitro radical anion) can be scavenged by molecular oxygen,
restricting activation in hypoxic cells [3–5]. The aromatic N-oxide class is best represented by
tirapazamine (TPZ), which is the clinically most useful hypoxia-activated prodrug discovered to date.
TPZ shows a high hypoxic selectivity (100 to 200 fold) in cell suspension cultures, but its diffusion

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402
through tissue is limited by its metabolism into the non-diffusible, radical species. Nevertheless, this
drug is selectively toxic to the hypoxic fraction of cells in several animal tumours in vivo. This specific
hypoxic cytotoxicity results from the fact that the TPZ radical is much more cytotoxic than the
superoxide radical [3,6,7]. It has been suggested that TPZ has a dual mechanism of action, both
generating DNA radicals and, also oxidizing these radicals to form DNA breaks [8]. It was shown that
TPZ enhances the antitumor efficacy of some other antitumor drugs, namely cisplatin [9–14].
Derivatives containing benzimidazole rings are active as human DNA topoisomerase I inhibitors [15–
1
7] and are expected to possess anticancer properties [18] and selective affinity for cells under hypoxic
conditions [15]. In this context, benzimidazole-4,7-dione derivatives play an important role, as
quinones are known to have a noticeable antitumor activity [19–23].
These compounds are being worked on intensively as they might have new potential anticancer
properties [24–28]. This was the reason for initiating our experiments with a group of new
benzimidazole-4,7-dione derivatives 5 and their analogues 6 with N-oxide bonds (Figure 2).
Figure 2. Structural formula of benzimidazole-4,7-dione derivatives. a: p-chlorophenyl, b:
o-nitrophenyl, c: piperonyl, d: naphthyl.
The main aim of this study was to describe the method of synthesis to obtain derivatives 5 and 6,
evaluate their stability in different environments by UPLC analysis, and also quantify their biological
activities under normoxic and hypoxic conditions.
2
. Results and Discussion
2
.1. Chemistry
The syntheses of the benzimidazole-4,7-dione derivatives 5 compounds and the N-oxide
benzimidazole-4,7-dione derivatives 6 comprises a series of known reactions (Scheme 1). Reactions A
aromatic nitration) and B (reduction of nitro compounds) are common to all products. Reaction C was
(
adapted from the literature and comprises the reaction between diamine 3 and different aldehydes to
produce benzimidazole-4,7-dione derivatives 4 [15]. They were obtained by the direct cyclocondensation
of a suitable diamine with a suitable aldehyde in an anhydrous solvent at its boiling point [15].

Molecules 2014, 19
403
Reaction D is an oxidation of product 4, and it was accomplished using oxidizing agents such as nitric
acid, which works well for highly substituted 1,4-dimethoxybenzene derivatives and requires strongly
acidic media [29]. Reaction E was performed to produce the N-oxide benzimidazole-4,7-dione
derivatives, which were obtained by the direct reaction of 30% solution of hydrogen peroxide in
glacial acetic acid with the previously obtained benzimidazole-4,7-dione derivatives [15]. The
benzimidazole ring structure of the main product of cyclocondensation of a diamine with an aldehyde
was established by X-ray crystal structure analysis [15].
Scheme 1. General synthesis reaction.
2
.2. UPLC Analysis
2
.2.1. Optimisation
Using an isocratic system consisting of a mixture of acetonitrile and ammonium formate buffer, the
retention times for compounds 5b and 6b were 4.3 and 3.9 min, respectively (Table 1). This valuable
difference may be due to the protonation of the Schiff base without the N-oxide bond. This does not
occur with the N-oxide derivative. This theory is supported by the chromatogram (Supplementary
Material Figure S1) of product 5b, which presents its minimum and the maximum absorbance for the
protonated and non-protonated forms, respectively. The chromatogram of 6b only shows the maximum
absorbance for the non-protonated form. The use of this mobile phase at a flow rate of 0.5 mL/min was
found to be optimum and provided adequate peak separation and resulted in the best resolution among
all the other combinations tested.

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404
Table 1. The obtained values of Rt, k, n for the isocratic system acetonitrile/ammonium
formate buffer (30:70 v/v, pH = 2.4).
−1
Concetration 10 mg/mL Rtmean (min)
k
mean
n
mean
5
6
b
b
4.3
3.9
1.24e^1 7.74e^3
1.20e^1 6.31e^3
Rt, retention time; k, retention coefficient; n, number of theoretical plates.
2.2.2. Validation
Precision: To determine the compound content in the sample, a calibration curve was generated for
each compound as described earlier. The following parameters: arithmetic mean ( ), standard
deviation (s), mean ± SD (µ), and relative standard deviation [RSD (%)] were used to define the
precision of the method for each compound. The results are presented in Supplementary Material
Table S1).
Accuracy: To determine the compound content in the sample, a calibration curve was generated for
each compound as described earlier. A degree of recovery was defined for each substance content
expressed in mg/mL and the parameters: , s, µ, RSD (%) were used to determine the accuracy of the
method for each compound. The results are presented in Supplementary Material Table S2).
Linearity: A linear relationship between the particular sample concentration and peak area was
drawn. Equations (1) and (2) represent the calibration curves for compounds 5b and 6b, respectively.
Correlation coefficients were also determined:
6
3
2
y = 6.54 × 10 x − 3.39 × 10
R = 0.9999
(1)
(2)
6
4
2
y = 9.10 × 10 x − 1.41 × 10
R = 0.9998
LOD: This parameter was calculated as an arithmetical mean from three individual measurements. The
limit of detection, at a signal-to-noise ratio of 3, were 0.304 µg/mL and 0.849 µg/mL for compounds
5
b and 6b, respectively.
LOQ: This parameter was calculated as an arithmetical mean from three individual measurements. The
limit of detection, at a signal-to-noise ratio of 10, were 1.01 µg/mL and 2.83 µg/mL for compounds 5b
and 6b, respectively.
The accuracy and precision of the method were within the acceptable limits of ±5%.
2
.2.3. Stability of Compounds in Different Environments
The purpose of this analysis was to assess the media in which compounds 5b and 6b are more
stable, so that it can be used in further biological studies. For this purpose, it is necessary for the
compounds to be as stable as possible in these media, i.e., possessing a high t1/2. The percentage of
sample recovery in each environment is presented in Table 2. The results of the degradation process
for compounds 5b and 6b (t and degradation rate) are summarized in Table 3.
1
/2
Both compounds seem to be rather stable in all solvents and within a time of up to 72 h do not
undergo relevant decomposition. Apparently, compound 5b did not suffer any degradation in water
and methanol. This fact is proved by the high mean recovery in these solvents (Table 2). In theory, all

Molecules 2014, 19
405
of these environments can be used in further biological studies, and the choice falls on the most
suitable one.
Table 2. Sample recovery (arithmetic mean) after 72 h in the specific environment.
Compound 5b
Solvent
Recovery (%)
77.0
0
.9% NaCl
H
2
O
100.0
0
.2% DMSO
MeOH
83.0
98.2
Compound 6b
.9% NaCl
0
78.2
77.9
90.4
76.1
2
H O
0
.2% DMSO
MeOH
DMSO, dimethylsulfoxide; MeOH, methanol.
Table 3. Degradation kinetic parameters in tested solvents.
Compound 5b
−
1
2
Solvent
k (h )
t
1/2 (h)
R
0
.9% NaCl
0.0004
155 0.98
a
a
a
2
H O
0
.2% DMSO
MeOH
0.0002
215 0.93
a
a
a
Compound 6b
0.0004
0
.9% NaCl
142 0.97
146 0.86
413 0.99
161 0.99
H
2
O
0.0004
0
.2% DMSO
MeOH
0.00009
0.0002
a
No degradation.
2
.3. Biological Activities under Normoxia and Hypoxia Conditions
The anticancer activity of newly synthesized benzimidazole derivatives 5 and N-oxide benzimidazole
derivatives 6 was investigated in vitro on human lung adenocarcinoma A549 cells. The results were
expressed as a fraction of viable cells. Changes in cell morphology induced by the compounds were
visualized with a phase-contrast microscope. Tirapazamine was used as a reference drug.
2
.3.1. Cytotoxicity at Normoxia
The concentration-response analysis was performed to determine the compound concentrations
required to inhibit the growth of cancer cells by 50% (IC ) after an incubation of 48 h. The
5
0
synthesized compounds were tested in a wide range of concentrations, from 1 to 500 μM. Four out of
eight tested compounds (compounds 5b and 5d, 6b and 6d) showed IC50 values less than 100 μM in
cells cultured under normoxic conditions (Table 4). In comparison, under the same experimental

Molecules 2014, 19
406
conditions 166 μM tirapazamine was required to inhibit the growth of A549 cells by 50%. Compounds
5
a, 5c and 6c were found to have a higher anticancer activity, with an IC50 value of 30.2 ± 1.2 μM,
0.9 ± 1.9 μM and 79.5 ± 1.9, respectively. The culture of A459 cells in the presence of compound 5a
8
at 10 µM caused approximately 33% inhibition of the cell growth compared to control cells. However,
the treatment of cells with compound 6a at the same concentration decreased cell viability by 20%.
These results demonstrated a much higher effectiveness of compound 5a compared to compound 6a at
low concentration (10 µM). A smaller effect on the cell growth was observed after the treatment with
benzimidazole-4,7-dione derivatives 5b and 6a (IC values of 100.0 ± 1.8 μM and 115.7 ± 1.9 μM,
5
0
respectively). Other tested benzimidazole-4,7-diones (compounds 5b–d and 6b–d) were less effective
and showed a much higher value of IC , over the range 252–500 μM (Table 4).
5
0
Table 4. In vitro growth inhibition of selected tumor cell lines by new benzimidazole-4,
7-dione derivatives.
IC50 [μM] A549
Compound
Differential cytotoxicity O/H
Normoxia (O)
Hypoxia (H)
36.1 ± 1.2
47.4 ± 1.1
51.2±1.5
5
a
30.2 ± 1.2
100.0 ± 1.8
80.9 ± 1.9
479.5 ± 3.6
115.7 ± 1.9
500.6 ± 1.2
79.5 ± 1.9
252 ± 2.5
0.83
2.13
1.58
2.06
3.28
4.31
1.80
2.62
4.61
5
b
5
c
5d
6a
6b
232.4 ± 1.4
35.0 ± 1.6
116.0 ± 0.8
44.0 ± 2.5
96.8 ± 1.9
30.0 ± 1.5
6
c
6
d
Tirapazamine
166.2 ± 1.6
WST-1 assay was used to determine the inhibition of the cell growth after 48 h incubation with
the tested compounds. IC50 values (concentration of the tested compounds causing 50%
inhibition of the cell growth compared to control cells) were calculated and expressed as mean
±
SD, n = 3.
Our conclusions from the results are that benzimidazole derivatives which have a chlorophenyl
(
compounds 5a, 6a) or nitrophenyl substituent (compounds 5b, 6b) inhibited the growth of A549 cells
more potently than the analogous benzimidazole-4,7-dione derivatives with piperonyl (compounds 5c,
6
c) or naphthyl (compounds 5d, 6d) groups.
Comparing these results with our earlier study with other benzimidazoles, we found that both
groups—benzimidazole derivatives and benzimidazole-4,7-dione derivatives—possess a very similar
cytotoxic activity against the selected tumor cell lines [15,30,31].
2.3.2. Cytotoxicity at Hypoxia
The tumour microenvironment characterized by hypoxia is the target of novel potential anticancer
substances which have a bioreductive mechanism of action. For these reasons we also evaluated the
effects of our compounds on hypoxic cancer cells. A549 cells were exposed to hypoxia (1% O ) for
4 h before treatment and were maintained under hypoxic conditions during culture in the presence of
the compounds. As shown in Table 4, the most active agents among series 5 and 6 under hypoxic
2
2

Molecules 2014, 19
407
conditions were compounds 5a and 6a (IC50 values of 36.1 ± 1.2 μM and IC50 value 35.0 ± 1.6 μM,
respectively, Supplementary Material Figure S2). Under hypoxic conditions, the cell viability was
significantly decreased when cells were cultured in the presence of compounds 5b and 6b (IC50 values
of 47.4 ± 1.1 μM and IC value 116.0 ± 0.8 μM, respectively). Moreover, compound 6b was more
5
0
effective in hypoxic cells compared to normoxic cells with IC50 values of 116.0 ± 0.8 μM and
00.6 ± 1.2 μM, respectively. The same activity is characteristic for compound 6a (Supplementary
Material Figure S2).
5
3
. Experimental
3.1. Materials and Instrumentation
1
IR spectra (KBr discs) were recorded using a Mattson Infinity Series FT-IR spectrophotometer. H
1
3
and C spectra were recorded on a 300 MHz Varian Mercury spectrometer (Darmstadt, Germany) in
DMSO-d or CDCl -d as solvents and tetramethylsilane (TMS) as internal reference. MS spectra
FAB method, M+1, M-1, matrix-glycerin) were recorded on a Finnigan Mat 95 spectrometer,
6
3
6
(
Bremen, Germany). Carbon, hydrogen and nitrogen elemental analyses were performed using a
Perkin Elmer 2400 series II CHNS/O (Madison, WI, USA), and agreed with the proposed structures
within ± 0.3% of theoretical values.
Chromatographic purification was performed on silica gel plates (Merck F254, Darmstadt, Germany)
with the indicated eluents. Chemicals and solvents were obtained from commercial sources. The UPLC
system consisted of an Acquity Ultra Performance LC Waters H-class system (Waters, Corporation,
Milford, MA, USA). Chromatographic separation was achieved on an Acquity UPLC HSS T3 column
(
1.8 µm × 2.1 × 50 mm). The chromatographic peaks were identified with the Waters PDA eλ Acquity
UPLC Detector. Compound 5b was monitored at a wavelength of 261 nm and compound 6b at
89 nm. All chromatographic experiments were performed in an isocratic mode. The mobile phase
2
consisted of a mixture of solvents of chromatographic purity, acetonitrile/buffer (30:70 v/v). Buffer
was a 0.01 M mixture of ammonium formate and formic acid pH = 2.4. The flow rate was set to
0
.5 mL/min and the oven temperature to 25 °C. Compound samples were weighed on analytical scales
with an accuracy of 0.01 mg. All the samples were prepared in 5, 10 and 25 mL volumetric flasks.
Solution volume injections of 1.5 µL were performed with the use of an autosampler. Each sample was
analysed three times and the run time analysis was five minutes. All solutions were filtered through a
0
.45 µm membrane filter. The final result was presented as an arithmetical mean. The control of the
®
UPLC system and detection was achieved by the Empower 3 Waters software. Solvents for
UPLC analysis were of UPLC grade and obtained from J. T. Baker (Center Valley, PA, USA). The
method was validated in terms of precision, accuracy, specificity, detectability, and stability for
compounds 5b and 6b.
The human lung adenocarcinoma A549 cell line, purchased from Health Protection Agency Culture
Collections (ECACC, Salisbury, UK), was cultured in F12K medium (HyClone, Loughborough, UK)
supplemented with 10% heat-inactivated foetal bovine serum (FBS), penicillin (10 U/mL) and
2
streptomycin (10 µg/mL) in air with 5% CO at 37 °C. Hypoxic cells were obtained by culturing in a
hypoxic incubator in 1% O and 5% CO at 37 °C for 24 h before treatment.
2
2

Molecules 2014, 19
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In order to determine the anticancer activity of the analysed compounds we evaluated the cell viability
using THE WST-1 assay (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.
The absorbance was determined at 420 nm using a microplate reader (Synergy H1, Bio-Tek,
Winooski, VT, USA) after 3 h of incubation at 37 °C. IC50 values (concentration of the tested
compounds required to reduce cell density to 50%) were calculated by the concentration-response
curve fitting using a Microsoft Excel-based analytic method.
3
.2. Methods
3.2.1. Chemistry
The synthesis of compounds 2 and 3 are well known reactions [32]. The chemical reaction to
produce derivatives 4 was described by the authors in another paper [15].
Synthesis of 1H-benzimidazole-4,7-dione Derivatives 5 [31]. The 4,7-dimethoxybenzimidazole
derivative 4 (2.89 g; 10 mmol) was suspended in HCl 30% (15 mL). The suspension was heated under
reflux at 80–90 °C, with constant stirring. Next, HNO 90% (5 mL), was added dropwise over one
3
hour. The mixture was stirred at room temperature for 24 h and the crude precipitate was filtered off.
The final product was recrystallized from ethanol. This synthesis was repeated in the same way,
individually, to give products 5a–d.
−
1
2
-(4-Chlorophenyl)-1H-benzimidazol-4,7-dione (5a). Yield 65%, IR (KBr) υ/cm : 3615 (NH), 1690
1
(
C=O), 1485 (C=N); H-NMR (DMSO-d
6
) δ: 14.5 (s,1H,NH), 8.2 (d, 2H, CH, J = 2.0 Hz), 7.6 (d, 2H,
13
CH, J = 2.0 Hz), 7.5 (d, 2H, CH, J = 7.9); C-NMR (DMSO-d
36.8, 135.7, 129.9, 129.3, 128.7, 127.9; MS m/z [M+1, M−1]: 259, 257; calculated for
ClN : C 60.36, H 2.73, N 10.83; found: C 60.43, H 2.80, N 10.60. R (chloroform/
methanol—6.25% v/v) = 0.49.
6
) δ: 180.0, 179.1, 150.2, 143.4, 142.8,
1
C
13
H
7
2
O
2
f
−
1
2
-(2-Nitrophenyl)-1H-benzimidazol-4,7-dione (5b). Yield 45%, IR (KBr) υ/cm : 3391 (NH), 1690
1
(C=O), 1526 (NO
2
asym), 1347 (NO
2
sym), 1481 (C=N); H-NMR (DMSO-d
6
) δ: 9.2 (s, 1H, NH), 8.1
1
3
(
m, 4H, CH), 7.8 (d, 2H, CH, J = 7.9 Hz); C-NMR (DMSO-d6) δ: 181.2, 179.1, 148.7, 147.2, 146.7,
46.1, 142.9, 141.9, 133.1, 131.9, 128.1, 125.5, 124.6: MS m/z [M+1, M−1]: 270, 268; calculated
: C 58.00, H 2.62, N 15.61; found: C 58.12, H 2.64, N 15.67. R (chloroform/
methanol—6.25% v/v) = 0.51.
1
for C13
H
7
N
3
O
4
f
−
1
2
-Benzo[1,3]dioxol-1H-benzimidazol-4,7-dione (5c). Yield 65%, IR (KBr) υ/cm : 3324 (NH), 2960
1
(CH
2
), 1701 (C=O), 1503 (C=N), 1264 (C-O-Csym), 1036 (C-O-Casym); H-NMR (DMSO-d
6
) δ: 4.0 (s,
1
2
1
3
H, NH), 7.9 (d, 2H, CH, J = 8.0 Hz), 7.2 (d, 1H, CH, J = 7.9 Hz), 7.0 (d, 2H, CH, J = 1.98 Hz), 6.2 (s,
1
3
H, CH
29.1, 120.3, 115,1, 113.2, 91; MS m/z [M+1, M−1]: 269, 267; calculated for C14
.01, N 10.44; found: C 62.55,H 2.99, N 10.50. R (chloroform/methanol—6.25% v/v) = 0.50.
2
); C-NMR (DMSO-d
6
) δ: 179.5, 179.0, 149.1, 148.6, 147.6, 142.1, 141.5, 141.3, 141.1,
8 2 4
H N O
:C 62.69, H
f
−
1
2
-Naphthyl-1H-benzimidazol-4,7-dione (5d). Yield 55%, IR (KBr) υ/cm : 3424 (NH), 3033 (ArH),
1
1
677 (C=O ); 1452 (C=N); H-NMR (DMSO-d
6
) δ: 13.0 (s,1H,NH), 8.7 (s,1H,CH) 8.3 (d, 2H, CH,

Molecules 2014, 19
409
J = 1.8 Hz), 8.1 (d, 2H, CH, J = 1.8 Hz), 7.9 (d, 2H, CH, J = 1.8 Hz), 7.6 (d, 2H, CH, J = 8.1 Hz);
1
3
C-NMR (DMSO-d
33.6, 128.5, 126.5, 124.3; MS m/z [M+1, M−1]: 275, 273; calculated for C17
.67,N 10.21; found: C 74.30, H 3.62, N 10.47. R (chloroform/methanol—6.25% v/v) = 0.50.
6
) δ: 179.9, 179.1, 150.2, 148.6, 148.5, 147.7, 147.3, 142.2, 141.2, 134.1, 133.8,
1
3
10 2 2
H N O
: C 74.44, H
f
Synthesis of N-oxide 1H-benzimidazole-4,7-dione derivatives 6 [15]. The benzimidazole-4,7-dione
derivative 5 (2.59 g; 10 mmol) was mixed with anhydrous acetic acid (15 mL). Next, one first portion
of hydrogen peroxide (5 mmol) was added. The mixture was heated under reflux at 50–60 °C. After six
hours, the second equal portion of hydrogen peroxide was added. After 24 h heating the mixture was
concentrated in vacuum to a small volume, diluted with methylene chloride and washed with sodium
carbonate solution. The organic layer was dried, concentrated in vacuum and diluted with diethyl ether.
The solid precipitate was filtered off and recrystallized from isopropanol. This synthesis was made in
the same way, individually, to compounds 5a–d to give products 6a–d, respectively. The
chromatographic purity of the obtained compounds was confirmed by TLC as above.
−
1
2
-(4-Chlorophenyl)-1H-benzimidazol-4,7-dione N-oxide (6a). Yield 50%, IR (KBr) υ/cm : 3445
1
(NH), 1685 (C=O), 1661 (C=O), 1484 (C=N), 1282 (N-O); H-NMR (DMSO-d
6
) δ: 14.5 (s, 1H, NH),
8
.1 (d, 2H, CH, J = 2.0 Hz), 7.6 (d, 2H, CH, J = 2.0 Hz), 7.5 (d, 2H, CH, J = 8.0 Hz);
1
3
C-NMR (DMSO-d
6
) δ: 180.2, 179.3, 157.2, 143.1, 142.6, 136.5, 135.2, 129.8, 129.1, 128.6, 128.0;
ClN : C 56.85, H 2.57, N 10.20; found: C
f
6.53, H 2.70, N 10.45. R (chloroform/methanol—6.25% v/v) = 0.50.
MS m/z [M+1, M−1]: 275, 273; calculated for C13
H
7
2 3
O
5
−
1
2
1
1
1
-(2-Nitrophenyl)-1H-benzimidazol-4,7-dione N-oxide (6b). Yield 75%, IR (KBr) υ/cm : 3480 (NH),
1
682 (C=O), 1541 (NO2asym), 1347 (NO2sym) 1434 (C=N), 1251 (N-O); H-NMR (DMSO-d
6
) δ: 10 (s,
) δ: 180.2, 179.3, 157.7,
47.6, 146.2, 146.0, 143.0, 142.4, 133.5, 131.5, 128.4, 125.3, 124.1: MS m/z [M+1, M−1]: 286, 284;
: C 54.74, H 2.47, N 14.73; found: C 54.92, H 2.22, N 15.02. R
chloroform/methanol—6.25% v/v) = 0.50.
1
3
H, NH), 8.2 (m, 4H, CH), 7.9 (d, 2H, CH, J = 7.9 Hz); C-NMR (DMSO-d
6
calculated for C13
H
7
N
3
O
5
f
(
−
1
2
-Benzo[1,3]dioxol-1H-benzimidazol-4,7-dione N-oxide (6c). Yield 35%, IR (KBr) υ/cm : 3346
1
(NH), 2961 (CH
2
), 1680 (C=O), 1470 (C=N), 1255 (C-O-Csym), 1045 (C-O-Casym), 1249 (N-O); H-
NMR (DMSO-d ) δ: 10.0 (s, 1H, NH), 7.8 (d, 2H, CH, J = 8.0 Hz), 7.7 (d, 1H, CH, J = 8.0 Hz), 7.6 (d,
6
1
3
2
H, CH, J = 2.0 Hz), 6.0 (s, 2H, CH
42.0, 141.7, 141.5, 141.0, 129.3, 120.1, 115.5, 113.3, 91.2; MS m/z [M+1, M−1]: 285, 283; calculated
: C 59.16, H 2.84, N 9.86; found: C 59.11, H 2.89, N 9.80. R (chloroform/methanol—
.25% v/v) = 0.50
2 6
); C-NMR (DMSO-d ) δ: 18025, 179.8, 159.1, 148.2, 147.1,
1
for C14
H N O
8 2 5
f
6
−
1
2
-Naphthyl-1H-benzimidazol-4,7-dione N-oxide (6d). Yield 70%, IR (KBr) υ/cm : 3300 (NH), 3031
1
(
ArH), 1670 (C=O), 1465 (C=N), 1261 (N-O); H-NMR (DMSO-d ) δ: 13.1 (s, 1H, NH), 8.8 (s, 1H,
6
CH), 8.3 (d, 2H, CH, J = 2.0 Hz), 8.0 (d, 2H, CH, J = 2.0 Hz), 7.6 (d, 2H, CH, J = 2.0 Hz), 7.4 (d, 2H,
13
CH, J = 7.9 Hz); C-NMR (DMSO-d
6
) δ: 179.5, 179.2, 160.1, 147.7, 147.5, 142.6, 142.1, 141.0,
1
40.2, 134.3, 133.7, 133.5, 128.1, 126.2, 124.7; MS m/z [M+1, M−1]: 291, 289; calculated for

Molecules 2014, 19
: C 70.34, H 3.47, N 9.65; found: C 70.30, H 3.62, N 10.17. R
410
C
17
H
10
N
2
O
3
f
(chloroform/methanol—
6
.25% v/v) = 0.50
3.2.2. UPLC Analysis
3
.2.2.1. Optimisation
The optimisation of the analytical system allowed determining the separation parameters for
compounds 5b and 6b. The feasibility of various combinations of solvents such as acetonitrile and
methanol in an isocratic system with altered flow rates was investigated for the complete
chromatographic resolution of the compounds with the best sensitivity, efficiency, and peak shape.
3
.2.2.2. Validation and Evaluation Procedures of the Compounds Stability in Different Media
Precision: Solutions were prepared containing from 25% to 100% (v/v) of the studied compound.
Accuracy: Solutions were prepared containing from 25% to 100% (v/v) of the studied compound.
Linearity: Solutions of compound 5b at concentrations of 0.104, 0.052, 0.0104 mg/mL, 0.0052 and
.00104 mg/mL and solutions of compound 6b at concentrations of 0.104, 0.052, 0.0104 and
.0052 mg/mL were prepared. The calibration curve was obtained by plotting the peak area against the
0
0
amount of the analysed substance at those concentrations, and studied by fitting the results by linear
least-squares regression.
Limit of Detection (LOD): Solutions of each compound with a concentration of 0.1 µg/mL
were prepared.
Limit of Quantification (LOQ): Solutions of each compound with a concentration of 0.1 µg/mL
were prepared.
Stability: Each medium (1 mL, 0.2% DMSO, water, 0.9% NaCl, and HCl pH = 1.5) was added to
1
0 mL volumetric flasks containing products 5b and 6b (1 mg). The flasks were filled up with the
mobile phase. Each solution was analysed at t and after 6, 24, 48, and 72 h. The obtained calibration
0
curve was used for the determination of degradation kinetics of the compounds in the solvents. The
degradation kinetic parameters: rate constant (k) and t1/2 (half-life) were calculated with the use of
Equation (3), where c
0
is the concentration at time zero, c is the remaining concentration, k is the rate
−1
constant (h ) and t is the time (h) [30]:
(
3)
3.2.3. Cytotoxicity
In order to determine the anticancer activity of the analysed compounds we evaluated the cell
viability using the WST-1 assay, which is based on the conversion of the tetrazolium salt WST-1 to
formazan by cellular mitochondrial dehydrogenases. Therefore, the amount of formazan dye formed
directly correlates with the number of live cells in the culture.

Molecules 2014, 19
411
A549 cells were seeded in 96-well plates at a density of 5,000 cells/well and cultured under
normoxic conditions. To investigate the effect of compounds on hypoxic cancer cells, A549 cells were
exposed to hypoxia (1% O
prepared in DMSO and diluted in complete medium to give final concentrations in the range from 1 to
00 µM. Normoxic and hypoxic cells were treated with different concentrations of the tested
2
) for 24 h before treatment. Stock solutions of the tested compounds was
5
compounds or vehicle (0.2% DMSO) for control cells. The cell viability was assessed after a 48 h
incubation under normoxic or hypoxic conditions with the compounds. Briefly, WST-1 reagent was
added to the cells and the percentage (%) of cell viability related to control cells was calculated by the
expression [A] test/[A] control × 100 where [A] test is the absorbance of the cells treated with
compounds and [A] control is the absorbance of control cells. IC values are presented in Table 4.
5
0
4
. Conclusions
The work developed in this analytical investigation allowed us to reach some conclusions about
some new heterocyclic compounds as new chemotherapeutics, namely bioreductive prodrugs,
which are activated under hypoxic conditions. To study the stability of some benzimidazole-4,7-dione
and N-oxide benzimidazole-4,7-dione derivatives in a few solvent environments, UPLC analyses
were executed.
The established analytical method allowed us to study the stability of compounds in some specific
environments. The compounds were stable in all the solvents, as shown by the high half-lifetime. This
information was very useful for our further biological studies with cancer cell lines, in which potential
bioreductive prodrugs (such as the ones developed in this research work) were tested.
The biological study was conducted in 0.2% DMSO and it proved that compound 5a was the most
effective in inhibiting the growth of normoxic as well as hypoxic A549 cells. Compounds 6b and 6a
were more potent to specifically inhibit the cell viability of hypoxic cancer cells while they were less
effective in normoxic cells. Moreover, hypoxic/aerobic cytotoxicity coefficient of compound 6b was
4
.31 while for tirapazamine it was 4.61. This parameter classifies compound 6b as a new bioreductive
agents between mitomycin C (cytotoxic coefficient from 1 to 5) and 2-nitroimidazole (misonidazole–
toxicity coefficient from 5 to 15) [15,29,30].
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/1/400/s1.
Acknowledgments
The study was supported by the Medical University of Lodz, Poland (503-3015-1).
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2014, 19
412
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Sample Availability: Samples of the compounds 5a–d and 6a–d are available from the authors.
©
2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(