Design, synthesis and antiproliferative properties of some new 5-substituted-2-iminobenzimidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.03.010

Some new 1,3,5-substituted-2,3-dihydro-2-imino-benzimidazoles were synthesized under solid-liquid phase transfer catalysis conditions using 5-substituted-2-aminobenzimidazoles as precursors in order to assess their cytotoxicity respectively proliferative activity. The structures of the compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis. Compounds 9-10, 12 and 16-17 were evaluated for their cytotoxical effect on four cancer cell lines: HT-29, breast cancer cells MDA-MB-231, HeLa, HepG2 and as well as human diploid cell line Lep-3. Significant cytotoxicity of hydrazone 16 against MDA-MB-231 was established by biologically study, the IC₅₀ was 6.2 nM while the EC₅₀ value to Lep 3 is 0.21 nM. Relative high antiproliferative effects of the acetate 12 and compound 16 against HT-29 were ascertained and the calculated IC₅₀ values were IC₅₀ - 0.85 nM and IC₅₀ - 2.83 nM respectively. Cytotoxic activity against HeLa and HepG2 cells was demonstrated by hydrazone 17, IC₅₀ was 7.2 nM and 117 nM respectively. All tested compounds revealed proliferative activities to human diploid cell line Lep-3. The EC₅₀ values were in the range from 0.05 to 16.91 nM. The obtained results prove the selective cytotoxicity of the tested compounds and are promising for further evaluation of the investigated compounds in vivo experiments using experimentally induced tumors in laboratory animals.

Full text of DOI:10.1016/j.ejmech.2013.03.010

European Journal of Medicinal Chemistry 63 (2013) 696e701
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and antiproliferative properties of some new
5-substituted-2-iminobenzimidazole derivatives
,
b
c
c
Anelia Ts. Mavrova a , Diana Wesselinova , Nikolay Vassilev , Jordan A. Tsenov
*
a University of Chemical Technology and Metallurgy, Department of Organic Synthesis, 8 Kliment Ohridski Blvd., 1756 Sofia, Bulgaria
b Institute of Parasitology and Experimental Pathology, Bulgarian Academy of Science, 1113 Sofia, Bulgaria
c Institute of Organic Chemistry, Bulgarian Academy of Science, 1113 Sofia, Bulgaria
a r t i c l e i n f o
a b s t r a c t
Article history:
Some new 1,3,5-substituted-2,3-dihydro-2-imino-benzimidazoles were synthesized under solideliquid
phase transfer catalysis conditions using 5-substituted-2-aminobenzimidazoles as precursors in order to
assess their cytotoxicity respectively proliferative activity. The structures of the compounds were
confirmed by IR, 1H NMR, 13C NMR and elemental analysis.
Received 30 October 2012
Received in revised form
3 March 2013
Accepted 5 March 2013
Available online 18 March 2013
Compounds 9e10, 12 and 16e17 were evaluated for their cytotoxical effect on four cancer cell lines:
HT-29, breast cancer cells MDA-MB-231, HeLa, HepG2 and as well as human diploid cell line Lep-3.
Significant cytotoxicity of hydrazone 16 against MDA-MB-231 was established by biologically study,
the IC50 was 6.2 nM while the EC50 value to Lep 3 is 0.21 nM. Relative high antiproliferative effects
of the acetate 12 and compound 16 against HT-29 were ascertained and the calculated IC50 values were
IC50 e 0.85 nM and IC50 e 2.83 nM respectively. Cytotoxic activity against HeLa and HepG2 cells was
demonstrated by hydrazone 17, IC50 was 7.2 nM and 117 nM respectively. All tested compounds revealed
proliferative activities to human diploid cell line Lep-3. The EC50 values were in the range from 0.05 to
16.91 nM. The obtained results prove the selective cytotoxicity of the tested compounds and are
promising for further evaluation of the investigated compounds in vivo experiments using experimen-
tally induced tumors in laboratory animals.
Keywords:
2-Aminobenzimidazoles
5-Substituted-2,3-dihydro-2-
iminobenzimidazoles
Solideliquid phase transfer catalysis
Cytotoxicity
Anticancer activity
Proliferation
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
times more cytotoxic than the parent drug albendazole [7]. Flu-
bendazole induced cell death in leukemia and myeloma cell lines
The 2-aminobenzimidazole occurs as pharmacophore in the
structure of many clinically useful chemotherapeutic agents and is of
crucial importance as precursor to generating new derivatives with a
wide spectrum of biological activity as antiviral, diuretic, as H3
agonist and selective NOD1-inhibitors [1e5]. It is known, that
2-aminobenzimidazole anthelmintics block microtubule function in
cells, so that research efforts are aimed at testing their activity
against cancer cells and elucidation of the mechanism of action.
Albendazole, a benzimidazole carbamate (methyl 5-propylthio-1H-
benzimidazol-2-yl carbamate) with extensive clinical use as an
anthelmintic drug, can also inhibit hepatocellular carcinoma cell
proliferation under both in vitro and in vivo experimental conditions
[6]. The cytotoxicity of albendazole derivatives was evaluated
against a human colorectal cancer cell line (HT-29) and a human
prostate cancer cell line (PC-3) and some of them were up to ten
and primary patient samples at nanomolar concentrations and
inhibited tubulin polymerization by binding tubulin at a site distinct
from vinblastine [8]. Cytotoxicity assay on HEp-2 (Human larynx
cancer cell line) was performed with 2-aminobenzimidazole de-
rivatives. Some of the tested compounds showed cytotoxicity, which
is comparable to that of the standard drug 5-Fluorouracil [9]. Anti-
parasitic mebendazole showed survival benefit in 2 preclinical
models of glioblastoma multiforme and it was also reported that
mebendazole induces apoptosis via Bcl-2 inactivation in chemo-
resistant melanoma cells [10,11]. Many other 2-aminobenzimidazole
derivatives were synthesized and studied for antitumor activity
[12,13]. On the other hand several new nitrobenzimidazoles were
created as substrates for DT-diaphorase and reported to possess
cytotoxic activity [14,15].
On the base of the above mentioned facts, the present work is an
extension of our ongoing efforts toward a development of new
potent anticancer agents using 5-substituted-2-aminobenzimida
zoles as precursors. Our previous study on the 1,3-substituted-2,3-
dihydro-2-imino-benzimidazoles showed that some of the studied
* Corresponding author. Tel.: þ359 28163207; fax: þ359 2685488.
E-mail addresses: anmav@abv.bg, mavrova_anelia@abv.bg (A.Ts. Mavrova).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.010

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 63 (2013) 696e701
697
compounds exhibited remarkable activity in vitro against HT-29 and
MDA-cancer cell lines and in the same time the tested compound
revealed proliferative effect against human spleen cells [16]. Stim-
ulated by these encouraging and promising results we undertook
investigation on the synthesis of some novel 5-nitro- and 5-benzoyl-
1,3-disubstituted-2,3-dihydro-2-imino-benzimidazoles and phar-
macological evaluation of the compounds against four human cancer
cell lines and human diploid cell line.
benzimidazol-2-sulfonic acids. The yields of thiols were in the range
of 84e98%, while these of the sufonic acids varied from 54% to 69%.
The lower yields of the sulfonic acids can be explained with the fact
that the reaction took place in heterogenous medium. The nucleo-
philic substitution between the 2-aminobenzimidazoles and the
appropriated halogen derivatives under solideliquid phase transfer
catalysis conditions in dry acetonitrile resulted in the formation of
1,3,5-substituted-1,3-dihydro-2-imino-benzimidazoles 9e13. The
condensation of ethyl [3-(2-ethoxy-2-oxoethyl)-5-nitro-2-imino-5-
nitro-2,3-dihydro-1H-benzimidazol-1-yl]acetate respectively ethyl
[3-(2-ethoxy-2-oxoethyl)-2-imino-5-benzoyl-2,3-dihydro-1H-ben
zimidazol-1-yl]acetate with hydrazine hydrate in molar ratio
afforded the hydrazides 14e15, which were reacted with the cor-
responding arylaldehydes yielding hydrazones 16e17.
2. Chemistry
The synthesis of 1,3-disubstituted-2,3-dihydro-2-iminobenzimi
dazoles is illustrated and outlined in Scheme 1.
The starting 5-substituted-2-aminobenzimidazoles were syn-
thesized in three steps starting with the obtaining of the corre-
sponding benzimidazol-2-thiols 3e4, subsequent oxidation of the
thiols to the corresponding sulfonic acids 5e6 and substitution of
sulfonic-group by interaction with ammonium hydroxide according
to the method described by us early in Ref. [17]. The refluxing of the
solution of 4-substituted-1,2-diaminobenzen, carbon disulfide and
sodium hydroxide in ethanol medium afforded the corresponding
thiols. The oxidation of the thiols with potassium permanganate in
water solution of sodium hydroxide led to the obtaining of
The chemical structures of the compounds were established by
elemental analyzes, IR-, 1H NMR and 13C NMR spectra and the re-
sults are presented in the Experimental part. The elemental ana-
lyzes indicated by the symbols of the elements were within ꢀ0.4%
of the theoretical values.
In the 1H NMR spectra, particularly meaningful are character-
istic NCH2CO signals (singlets) of compounds 11e17, shifted
downfield due to deshielding effect of both N-atom and the CO
group. The chemical shift values varied in the range from 4.3 to
H
R
R
O
S
O
R
R
NH2
NH2
N
N
N
b
a
SH
OH
S
N
H
N
H
N
H
1- 2
3 - 4
5 - 6
1 R = COC6H5
2 R = NO2
c
CN
R
R
N
N
N
d
R
d
N
NH
NH2
NH
N
N
H
7- 8
d
CN
9. R = NO2
d
10. R = COC6H5
OCH3
O
O
NH2
O
NH
O
NH
NH
NH
R
R
N
N
N
e
N
O
R
N
N
NH2
NH
O
O
O
14 - 15
12 - 13
12. R = COC6H5
13. R = NO2
f
OCH3
11. R = COC6H5
O
N
CH Ar
CH Ar
O
R
NH
NH
N
16 R = COC6H5, Ar =
O
N
NH
N
17 R = NO2, Ar = 4-F-C6H5
O
16 -17
Scheme 1. Synthesis of 1,3,5-substituted-2,3-dihydro-2-iminobenzimidazoles., Regents and conditions: a) sodium hydroxide, ethanolewater solution, carbon disulphide, refluxing;
b) KMnO4, 25% NaOH, reflux; c) 25% NH4OH, 145 ꢁC, in welded ampoule; d) acetonitrile, TBAB, dry K2CO3, halogen derivative, 25 ꢁC; e) hydrazine hydrate, ethanol, refluxing; f)
ethanol, substituted benzaldehydes, refluxing.

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5.3 ppm depending of the substituents. The aromatic protons in the
1H NMR spectra show typical pattern for mono-, di- and three-
substituted phenyls. The labile NH protons are not characteristic
and their chemical shifts depend on the water quantity in the
solvent. In the 13C NMR spectra, the characteristic signals are NCH2
(43e45 ppm), C]N (w158 ppm), NeC]O (w166 ppm) and C]O
(w195 ppm). The 19F NMR spectra of fluorine-containing com-
pounds were useful to check the purity. Some 1H NMR and 13C NMR
spectra are given in the Supplementary material.
3. Pharmacology
3.1. Cytotoxicity
Compounds9e10, 12 and 16e17 were evaluated for their cyto-
toxicity to human colorectal cancer cell line HT-29, breast
cancer cells MDA-MB-231, cervical cancer cells HeLa, human liver
carcinoma cell line HepG2 and human normal diploid cell line
Lep3 by using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrasolium inner salt) e
test [18].
Fig. 1. Relative viability of HT-29 and Lep 3 cells (%) after treatment with, compounds
12 and 16.
compounds 12 and 16 with IC50 e 0.64 nM and IC50 e 2.83 nM
respectively. As regards to the effects of the compounds on HeLa
and Hep G2-cell lines it may be pointed out, that only compound 17
revealed cytotoxic activity and the IC50 values were 117 nM and
7.27 nM respectively.
4. Results and discussion
All studied compounds manifested proliferative effects to the
human diploid cell line Lep-3. The EC50 values varied in the range
from 0.21 nM for compound 16 to 11.40 nM for compound 9.
Substances 9 and 10 exhibited proliferative activity against all
others cell lines (Table 1; Supplementary material e Figs. 12e16).
If the results, obtained for compound 16 are taken in to
consideration it should be noted that N0-[1,3-benzodioxol-5-
ylmethylene]-2-(3-{2-[2-(1,3-benzodioxol-5-ylmethylene)]-2-
oxoethyl}-5-benzoyl-2-imino-2,3-dihydro-1H-benzimidazol-1-yl)
acetohydrazide revealed proliferative activity against human
diploid cell line Lep-3 at lower concentration in comparison to the
concentration at which the compound excited cytotoxic effect on
HT-29 cells. That fact is an indicator for the selectivity effect of the
compound 16. Similar results were observed also for compounds 17,
which showed cytotoxicity effect at higher concentration on HeLa
cells and proliferative effect on Lep-3 at lower concentration. The
relation between cell viability and the concentrations of com-
pounds 12, 16 and 17 was plotted to obtain the survival curve of
MDA-MB 231, HT-29, HeLa and Hep G2-cell lines as well as that of
Lep-3, Figs. 1e3. As it can be seen from figures, the viability of the
treated with the above mentioned compounds cancer cells was
many times lower than the vitality of Lep-3 cells.
In order to synthesized new 5-substituted-2,3-dihydro-2-
aminobenzimidazole derivatives and to compare their effects on
human cancer cell lines to these of the no-substituted in 5-position
derivatives we improved the method for unsubstituted benzimid-
azoles, described by us earlier [16,17]. The required 2-amino
benzimidazoles were obtained by the reaction of the relevant
5-substituted-1,2-diaminobenzene, carbon disulfide, sodium hy-
droxide in ethanol, followed by the oxidation of the synthesized
thiols to the corresponding sulfonic acids. The reaction of
5-substituted-2-aminobenzimidazoles with halogen containing re-
agents under solideliquid phase transfer catalysis using anhydrous
potassium carbonate and tetrabutyl ammonium bromide as catal-
ysis led to the obtaining of 1,3-disubstituted-2,3-dihydro-2-imino-
benzimidazoles. It was established that the reaction runs best at mol
ratio of 2-aminobenzimidazoles 7e8, halogen compounds and po-
tassium carbonate 1:2:2 and has led to obtaining of a range of new
1,3-disubstituted derivatives being prepared easily and in good
yields e 75e87%.
The cytotoxicity respectively proliferative effect was assessed by
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrasolium inner salt (MTS) assay, which is based
on the reduction of yellow tetrazolium salt by metabolically active
viable cells to a formazan product that can be measured spectro-
photometrically. Hence, the intensity of the color in the solution is
directly proportional to cell viability [19,20]. In order to evaluate
the effects of some of the newly synthesized compounds on cell
proliferation, four cancer cell lines: human colorectal cancer cell
line HT-29, breast cancer cells MDA-MB-231, cervical cancer cells
HeLa, human liver carcinoma cell line HepG2 and human diploid
cell line Lep-3 were used. The test was performed with compounds
9, 10, 12 as well as 16e17 according to MTS method, as described in
Refs. [18,19]. Relative cell viability, expressed as a percentage of the
untreated control (100% viability), was calculated for each con-
centration. All data points represent an average of three indepen-
dent assays and are given in Figs. 1e3. The obtained results were
plotted and IC50 and EC50 were calculated. Statistical significant
differences in the level of cells in both control and experimental
groups were determined (p ꢂ 0.05).
The biological data of the synthesized compounds highlighted
that the nature and position of the substituent on the benzene ring
of benzimidazolone moiety as well as these at 1,3-position greatly
influence both the cytotoxicity to the cancer cells and the prolif-
eration properties of Lep3 of the tested compounds. The intro-
duction of the benzoyl group in position 5 and the disubstitution in
1 and 3 positions by means of butyl or cyanopentyl groups of the
benzimidazole ring generally led to a proliferation activity as
Among all tested compounds only compound 16 revealed
cytotoxicity against MDA-MB-231 cells. The calculated IC50 value
was 6.2 nM. The highest cytotoxicity against HT29 cell lines showed
Fig. 2. Relative viability of MDA-MB-231 and Lep 3 cells (%) after treatment with,
compound 16.

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 63 (2013) 696e701
699
examined substances is indicating that their biological role can be
examined further in in vivo experiments using experimentally
induced tumors in laboratory animals. In case these results become
similar, we could suppose that exactly that derivatives could be
proceed further as potential oncotherapeutics.
6. Experimental part
The reactions were monitored by thin layer chromatography,
which was performed on Merck pre-coated plates (silica gel. 60
F254, 0.25 mm) and was visualized by fluorescence quenching
under UV light (254 nm).
Fig. 3. Relative viability of HepG2, Hela and Lep 3 cells (%) after treatment, with
compound 17.
Melting points (mp) were determined on an Electrothermal AZ
9000 3MK4 apparatus and were uncorrected. IR spectra were
recorded on a Bruker spectrophotometer as potassium bromide
discs. 1H and 13C NMR spectra were recorded on a Bruker Avance
IIþ 250 MHz and a Bruker Avance IIþ 600 MHz NMR instrument.
The spectra are referred to the solvent signal. Chemical shifts are
expressed in ppm and coupling constants in Hz. The precise
assignment of the 1H and 13C NMR spectra was accomplished by
measurement of 2D homonuclear correlation (COSY), DEPT-135 and
2D inverse detected heteronuclear (CeH) correlations (HMQC and
HMBC) and as well as 19F NMR for the compounds containing F
atom. The microanalyses for C, H, N and S were performed on
PerkineElmer elemental analyzer.
regards to all tested cells. In particular, the presence of a benzoyl
group at the 5 position and ethoxycarbonylmethyl groups at the 1
and 3 position of the 2-imino-benzimidazole system provoked the
appearance of cytotoxicity and provided the most active compound
12 against HT-29 (IC50 e 0.85 nM), while the unsubstituted analog
showed EC 50 to HT-29 cells [16]. The hydrazone 17 containing a
nitro group in the 5 place revealed cytotoxicity to HeLa and Hep and
in comparison to the unsubstituted analog showed proliferative
properties to HT-29 [16]. To display more precise the relationship
between structure and activity of the synthesized compounds it is
necessary to be carried out further research on the rest of the
synthesized compounds.
The starting 4-substituted-1,2-diaminobenzenes 1e2 were
commercially available.
Moreover, we have planned further structural modifications
such as the conversion of the imino group of the benzimidazole
nucleus into the isosteric thiocarbonyl group and as well as the
introduction of different substituents at the 1, 3 and 5-th position.
6.1. General procedure for compounds 3e4
4-Substituted-1,2-diaminobenzene (0.019 mol) and water (3 ml)
were added to a solution of sodium hydroxide (0.022 mol) in
ethanol (20 ml) and carbon disulphide (0.022 mol). The mixture
was heated under reflux for 3 h. Charcoal was added cautiously and
removed by filtration after the mixture has been refluxed for 10 min
more. The filtrate was heated to 60e70 ꢁC and quenched with
warm water (70ꢁ, 20 ml), and then 50% acetic acid (9 ml) was added
by good stirring. After cooling the solution in refrigerator for 3 h the
crystallization was completed and the relevant thiol was separated.
5. Conclusion
Optimized reaction conditions for the synthesis of new 1,3-
substituted-2,3-dihydro-2-iminobenzimidazoles were developed
using 5-substituted-2-aminobenzimidazoles and different halogen
derivatives as precursors under solideliquid PTC and a range of
new derivatives have being prepared easily and in good yields.
The initial biological screening in vitro showed that the studied
compound 16 possessed relative high cytotoxicity against
MDA-MB-231 as well as against HT-29 cell lines, the IC50 values
were 6.63 nM and 2.83 nM, but the compound 12 was more toxic to
HT-29 cells than compound 16 with a IC50 value of 0.64 nM. Only
compound 17 revealed cytotoxic activity to HeLa and Hep G2-cell
lines and the IC50 values were 117 nM and 7.27 nM respectively.
All investigated compounds revealed proliferative effects on hu-
man diploid cells, the EC50 values were in the range 0.2e114 nM.
The above results confirmed also the hypothesis that the pres-
ence of different substituents at 5-th position in the structure of
1,3-disubstituted-2,3-dihydro-2-imino-benzimidazoles not only
contribute to the expression of cytotoxicity, but also to the mani-
festation of proliferative activity to human diploid cells. That fact
could be regarded as a sign of selectivity. The in vitro effect of the
6.1.1. (2-Mercapto-1H-benzimidazol-5-yl)(phenyl)methanone 3
Yield e 96,8%; Mp e 248e250 ꢁC; Rf ¼ 0.68, mobile phase: 1H
NMR (DMSO-d6)
d
(ppm): 1.877 (s, 1H, SH), 7.251 (d, J ¼ 8.2 Hz, 1H,
7-H), 7.462 (d, J ¼ 1.6 Hz, 1H, 4-H), 7.545 (dd, J ¼ 8.2 Hz, J ¼ 1.6 Hz,
1H, 6-H), 7.558 (t, J ¼ 7.7 Hz, 2H, m-Ph), 7.658 (dt, J ¼ 7.4 Hz,
J ¼ 1.4 Hz,1H, p-Ph), 7.700 (dd, J ¼ 8.1 Hz, J ¼ 1.4 Hz, 2H, o-Ph),12.75
(br s, 1H, NH);
13C NMR (DMSO-d6)
d (ppm): 109.30 (7-C), 110.81 (4-C), 125.12
(6-C), 128.48 (m-Ph), 129.35 (o-Ph), 130.51 (3a-C), 132.12
(p-Ph), 132.92 (7a-C), 137.14 (5-C), 137.99 (i-Ph), 170.78 (C]N),
195.08 (C]O).
Analysis: Calc. for C14H10N2OS; C, 66.12; H, 3.96; N, 11.02; O,
6.29; S, 12.61; Found: C, 66.09; H, 3.91; N, 11.12; O, 6.32; S, 12.64;
6.1.2. 5(6)-Nitro-1H-benzimidazole-2-thiol 4
Yield e 84%; Mp e 272 ꢁC (decomp); Rf ¼ 0.41, mobile phase:
CHCl3/ethyl acetate ¼ 6:1; 1H NMR (DMSO-d6): 7.94 (dd, J ¼ 9.1 Hz,
1H, CH), 8.13 (m, 1H, CH), 8.34 (d, 1H), 12.32 (s, 1H, SH). Analysis:
Calc for C7H5N3O2S: C, 43.07; H, 2.58; N, 21.53; O, 16.39; S, 16.43;
Found: C, 43.17; H, 2.53; N, 21.57; O, 16.41; S, 16.42;
Table 1
The proliferative activity (EC50) of the studied compounds.
No EC50 ꢀ SE (nM)
MDA-MB231 HT-29
HeLa
158 ꢀ 0.05
HepG2
Lep3
9.
86 ꢀ 0.23
177 ꢀ 0.196
142 ꢀ 0.073 114 ꢀ 0.012
6.2. General procedure for compounds 5e6
10. 119 ꢀ 0.06
46.7 ꢀ 0.22 0.015 ꢀ 0.016 94.1 ꢀ 0.04
68 ꢀ 0.013
73 ꢀ 0.077
12. 0.28 ꢀ 0.023
e
0.002 ꢀ 0.001 70.1 ꢀ 0.068
16.
17.
e
e
0.25 ꢀ 0.02 10.2 ꢀ 0.056 0.21 ꢀ 0.102
To a boiling solution of 4-substituted-1H-benzimidazol-2-yl-tiol
(0.04 mol) in water (25 ml) and 50% sodium hydroxide (15 ml) a
17 ꢀ 0.023 24.5 ꢀ 0.08
e
e
12.2 ꢀ 0.0170

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A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 63 (2013) 696e701
solution of potassium permanganate (0.08 mol) in 200 ml was
added in small portions by stirring. After the complete addition of
potassium permanganate the reaction solution was refluxed for
30 min more. The formed manganese dioxide was filtered. The
filtrate was treated with hydrochloric acid to pH ¼ 1 by cooling. The
obtained precipitate of 1-(un)substituted-1H-benzimidazol-2-yl-
sulphonic acid was filtered and washed with water.
phase: dichloromethane/methanol ¼ 8:1; 1H NMR (DMSO-d6)
d
(ppm): 0.919 (t, J ¼ 7.4 Hz, 6H, CH3), 1.292 (sextet, J ¼ 7.4 Hz, 4H,
CH2), 1.544 (pentet, J ¼ 7.4 Hz, 4H, CH2), 3.138 (m, 4H, CH2), 6.315 (s,
1H, NH), 7.012 (d, J ¼ 8.6 Hz, 1H, 7-Ar), 7.733 (dd, J ¼ 2.2, 8.6 Hz, 1H,
6-Ar), 7.82 (d, J ¼ 2.2 Hz, 1H, 4-Ar); 13C NMR (DMSO-d6)
d (ppm):
13.60 (CH3), 19.30 (CH2), 23.14 (CH2), 57.59 (CH2), 106.11 (4-Ar),
110.63 (7-Ar), 115.57 (6-Ar), 128.22 (3a-Ar), 128.50 (7a-Ar), 137.99
(5-Ar), 164.19 (C]N). Analysis: Calc. for C25H27BrN4O2; C, 60.61; H,
5.49; Br, 16.13; N, 11.31; O, 6.46; Found: C, 60.55; H, 5.52; Br, 16.15;
N, 11.29; O, 6.49;
6.2.1. 5-Benzoyl-1H-benzimidazole-2-sulfonic acid 5
Yield e 69.4%; Mp e 275e277 ꢁC; Rf ¼ 0.53, mobile phase:
dichloromethane/methanol ¼ 5: 1; 1H NMR (DMSO-d6)
d (ppm):
7.59 (m, J ¼ 7.2 Hz, 2H, H-18, H-20), 7.67 (m, J ¼ 1.9 Hz,1H, H-9), 7.88
(m, J ¼ 7.4 Hz, 2H, H-17, H-21), 7.98 (dd, 1H, H-7), 8.04 (s, 1H, H-9),
8.14 (dd, J ¼ 8.6 Hz, 1H, H-6), 12.36 (br s, 1H, OH). Analysis: Calc. for
6.4.2. 5-[3-(4-Cyanobutyl)-5-benzoyl-2-imino-2,3-dihydro-1H-ben
zimidazol-1-yl]pentanenitrile 10
Yield ꢃ75%; Mp e 180 ꢁC; Rf ¼ 0,48, mobile phase: dichloro-
C
14H10N2O4S; C, 55.62; H, 3.33; N, 9.27; O, 21.17; S, 10.61; Found: C,
methane/methanol ¼ 8:1; NMR (DMSO-d6)
d (ppm): 1.792 (m, 8H,
55.68; H, 3.31; N, 9.30; O, 21.14; S, 10.68;
4CH2, J ¼ 7.35 Hz); 2.62 (t, 4H CH2CN, J ¼ 6.41); 4.15 (t. 4H, CH2eN,
J ¼ 6.4 Hz); 7.52 (dd, 2H, CH, J ¼ 7.21 Hz) 7.82 (dd, 2H, 2CH,
J ¼ 7.63 Hz); 8.61 (br s, 1H, NH exchangeable with D2O);
6.2.2. 5-Nitro-1H-benzimidazole-2-sulfonic acid 6
Analysis: Yield e 52.4%; Mp ꢃ310 ꢁC (decomp.); Rf ¼ 0.58,
mobile phase: dichloromethane/methanol ¼ 8:1; 1H NMR (DMSO-
6.4.3. 1,3-Bis(4-methoxyphenyl)-2-oxoethyl)-5-benzoyl-2,3-dihydro-
1H-benzimidazol-2-imine 11
d6)
d
(ppm): 8.07 (dd, 1H, H-7), 8.36 (d, J ¼ 9.1 Hz, 1H, H-6), 8.51 (d,
J ¼ 2.3, 1H, H-9). Analysis: Calc. for C7H5N3O5S; C, 34.57; H, 2.07; N,
17.28; O, 32.89; S, 13.18; Found: C, 34.61; H, 2.010; N, 17.24; O,
32.85; S, 13.14;
After removing of acetonitrile the compound was crystallized
with dichloromethane. Yield e 75%; Mp e 206e208 ꢁC, recrystal-
lized with dichloromethane; Rf ¼ 0.46 phase: dichloromethane/
methanol ¼ 8:1; NMR (DMSO-d6)
d (ppm): 3.95 (s, 3H, 2OeCH3);
6.3. General procedure for compounds 7e8
6.25 (s, 4H, 2CH2); 7.18 (dd, 4H, 4CH, J ¼ 8.56); 7.65 (m, 2H, CH,
J ¼ 7.15); 7.82 (m, 6H, 6CH, J ¼ 7.28); 8.18 (m, 4H, CH, J ¼ 8.57).
Analysis: Calc. for C22H27N3O5; C, 72.03; H, 5.10; N, 7.88; O, 14.99;
Found: C, 72.07; H, 5.13; N, 7.85; O, 14.97;
4-Substituted-1H-benzimidazol-2-sulphonic acid (0.002 mol)
and 25% ammonium hydroxide (1 ml) were heated in welded
ampoule for 5 h at 145e150 ꢁC. After cooling the formed crystals of
2-aminobenzimidazole were filtered and washed with ammonium
hydroxide (0.3 ml) and water (4 ml) and re-crystallized with ethanol.
6.4.4. Ethyl[3-(2-ethoxy-2-oxoethyl)-2-imino-5-benzoyl-2,3-dihydro-
1H-benzimidazol-1-yl]acetate 12
Yield - 86.4%; Mp e 138e140 ꢁC; Rf ¼ 0.66, mobile phase:
6.3.1. (2-Amino-1H-benzimidazol-5-yl)(phenyl)methanone 7
Yield e 64.5%; Mp e 172e174 ꢁC (destruction); Rf ¼ 0.44, mobile
phase: dichloromethane/methanol ¼ 8:1; 1H NMR (DMSO-d6)
dichloromethane/methanol ¼ 8:1; 1H NMR (DMSO-d6)
d (ppm):
1.191 (t, J ¼ 7.1 Hz, 6H, CH3), 4.152 (q, J ¼ 7.1 Hz, 4H, OCH2), 4.262 (s,
4H, NCH2), 7.107 (d, J ¼ 8.1 Hz, 1H, 7-Ar), 7.346 (d, J ¼ 8.1 Hz, 1H,
6-Ar), 7.446 (s, 1H, 4-H), 7.536 (t, J ¼ 7.7 Hz, 2H, m-Ph), 7.640 (t,
J ¼ 7.4 Hz, 1H, p-Ph), 7.678 (dd, J ¼ 8.2, 1.2 Hz, 2H, o-Ph); 13C NMR
d
(ppm): 6.91 (bs, 1H, NH), 7.18 (dd, J ¼ 9.1 Hz, 1H, H-6), 7.65 (m, 1H,
H-7), 7.70 (dd, J ¼ 2.1 Hz, 1H, H-9); Analysis. Calc. for C14H11N3O; C,
70.87; H, 4.67; N,17.71; O, 6.74; Found: C, 70.80; H, 4.71; N,17.74; O,
6.73;
(DMSO-d6) d (ppm): 13.97 (CH3), 47.48 (NCH2), 60.14 (OCH2), 105.77
(7-C), 107.92 (4-Ar), 109.58 (6-Ar), 128.37 (m-Ph), 129.10 (7a-C),
129.30 (o-Ph), 131.95 (p-Ph), 132.25 (3a-C), 138.06 (i-Ph), 152.22
(C]N), 167.77 (OeC]O), 171.52 (C]O). Analysis: Calc. for
C22H23N3O5; C, 64.54; H, 5.66; N,10.26; O,19.54; Found: C, 64.59; H,
5.61; N, 10.28; O, 19.49;
6.3.2. 5-Nitro-1H-benzimidazol-2-amine 8
Analysis: Yield e 77.4%; Mp e 137e139 ꢁC; Rf ¼ 0.64, mobile
phase: dichloromethane/methanol ¼ 8:1; 1H NMR (DMSO-d6)
d
(ppm): 7.26 (bs,1H, NH), 7.98 (dd, J ¼ 9.1 Hz,1H, H-6), 8.05 (dd,1H,
H-7), 7.70 (dd, J ¼ 2.2 Hz, 1H, H-9). Analysis: Calc. for C7H6N4O2; C,
47.19; H, 3.39; N, 31.45; O, 17.96; Found: C, 47.23; H, 3.42; N, 31.41;
O, 17.92.
6.4.5. Ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-5-nitro-2,3-dihydro-
1H-benzimidazol-1-yl]acetate hydro bromide 13
Yield e 78%; Mp e 240e242 ꢁC; Rf ¼ 0.48, mobile phase:
dichloromethane/methanol ¼ 8:1; 1H NMR (DMSO-d6)
d (ppm):
6.4. General procedure for compounds 9e13
1.225 (t, J ¼ 6.9 Hz, 6H, CH3), 4.168 (q, J ¼ 7.1 Hz, 4H, CH2), 5.072
(s, 4H, CH2), 7.045 (s, 1H, NH), 7.207 (d, J ¼ 8.8 Hz, 1H, 4-Ar), 7.953
(dd, J ¼ 2.3, 8.8 Hz, 1H, 5-Ar), 8.131 (d, J ¼ 2.3 Hz, 1H, 7-Ar); 13C
0.004 mol of 2-aminobenzimidazole was diluted in 20 ml dry
acetonitrile and TBAB (tetrabutylammonium bromide) (0.0012 mol)
was added to the solution. The halogen derivative (0.008 mol) was
added (in the case of the halogen esters drop by drop) by intensive
stirring and after that dry K2CO3 (0.008 mol) was placed. The reac-
tion mixture was allowed to be stirred for 3e6 h at room tempera-
ture. After completing of the reaction, which was determined
through TLC, K2CO3 was filtrated and the solvent was removed under
reduced pressure. The obtained residue was crystallized through the
addition of suitable solvent to obtained compounds 9e13.
NMR (DMSO-d6)
d (ppm): 14.12 (CH3), 43.59 (NCH2), 61.40
(OCH2), 104.11 (7-Ar), 113.66 (4-Ar), 118.33 (5-Ar), 134.36 (3a-Ar),
142.23 (7a-Ar), 149.72 (6-Ar), 159.50 (C]N). 167.88 (C]O).
Analysis: Calc. for C15H19BrN4O6; C, 41.78; H, 4.44; Br, 18.53; N,
12.99; O, 22.26; Found: C, 41.73; H, 4.40; Br, 18.60; N, 12.90; O,
22.22;
6.5. General procedure for preparation of acetohydrazides 14e15
To
a solution of 0.002 mol of the corresponding ethyl
6.4.1. 1,3-Dibutyl-5-nitro-1,3-dihydro-2H-benzimidazol-2-imine 9
After removing of acetonitrile the compound was crystallized
with ethanol. Yield e 81%; Mp e 245e247 ꢁC; Rf ¼ 0.62, mobile
[3-(2-ethoxy-2-oxoethyl)-2-imino-2,3-dihydro-1H-benzimidazol-
1-yl]acetate hydro bromide (4) in 20 ml ethanol were added 0.6 ml
(0.012) mol 98% hydrazine hydrate and the mixture was refluxed

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 63 (2013) 696e701
701
for 2 h. After cooling the obtained precipitation was filtered and re-
crystallized with ethanol.
4.700 and 4.729 (s, 4H, NCH2), 7.205 (d, J ¼ 8.7 Hz, 1H, 4-Ar), 7.943
(d, J ¼ 8.7 Hz,1H, 5-Ar), 7.316 (t, J ¼ 8.8 Hz, 4H, 3-Ar and 5-Ar), 7.865
(dd, J ¼ 2.2, 8.8 Hz, 4H, 2-Ar and 6-Ar), 7.952 (s, 1H, 7-C), 8.05 (s, 2H,
CH), 8.76 (br s, 1H, NH), 9.44 (br s, 2H, NNH); 13C NMR (DMSO-d6)
6.5.1. 2-[3-(2-Hydrazino-2-oxoethyl)-5-benzoyl-2-imino-2,3-dihydro-
1H-benzimidazol-1-yl] acetohydrazide 14
d
(ppm): 43.49 (NCH2), 103.72 (7-C), 109.53 (4-C), 115.98
Yield - 81%; Mp 254e255 ꢁC, re-crystallized with ethanol;
Rf ¼ 0.41, mobile phase: dichloromethane/methanol ¼ 8:1; 1H NMR
(2JCF ¼ 21.4 Hz, 3-Ar and 5-Ar), 118.27 (5-C), 129.32 (3JCF ¼ 8.3 Hz,
2-Ar and 6-Ar), 130.77 (4JCF ¼ 2.7 Hz, 1-Ar), 134.40 (3a-Ar), 140.20
(7a-Ar), 142.78 (5JCF ¼ 2.8 Hz, CH), 149.83 (6-C), 159.80 (C]N),
163.15 (1JCF ¼ 247.7 Hz, 4-Ar), 165.72 (C]O). 19F NMR (DMSO-d6)
(DMSO-d6) d (ppm): 4.3 (bs, 4H, NH2), 4.451 (s, 2H) and 4.531 (s, 2H,
NCH2), 7.108 (d, J ¼ 8.2 Hz, 1H, 7-Ar), 7.426 (dd, J ¼ 8.2, 1.6 Hz, 1H, 6-
Ar), 7.462 (d, J ¼ 1.6 Hz, 1H, 4-H), 7.552 (t, J ¼ 7.7 Hz, 2H, m-Ph),
7.652 (tt, J ¼ 7.5, 1.3 Hz, 1H, p-Ph), 7.695 (dd, J ¼ 8.3, 1.3 Hz, 2H, o-
Ph), 9.351 (s, 1H, C]NH), 10.351 (s, 2H, HNC]O); 13C NMR (DMSO-
d
(ppm): ꢃ111.2; Analysis: Calc. for C23H20F2N8O4; C, 54.12; H, 3.95;
F, 7.44; N, 21.95; O, 12.54; Found: C, 54.06; H, 3.89; F, 7.47; N, 21.90;
O, 12.57.
d6)
d (ppm): 42.76 and 43.95(NCH2), 106.12 (7-C), 107.69 (4-Ar),
126.48 (6-Ar), 128.56 (m-Ph), 128.76 (7a-C), 129.42 (o-Ph), 137.65
(5-C), 138.13 (p-Ph), 131.09 (3a-C), 138.13 (i-Ph), 157.78 (C]N),
165.68 (NeC]O), 194.72(C]O). Analysis: Calc. for C18H19N7O3; C,
56.69; H, 5.02; N, 25.71; O,12.59; Found: C, 56.62; H, 5.06; N, 25.75;
O, 12.53;
6.7. MTS test
The compounds were dissolved in DMSO at the concentration of
0.5 mg/ml. The investigation was carried out by dilution of the stock
solution in ratio 1:10, 1:100, 1:1000 and 1:10 000. Samples of cells,
grown in non-modified medium served as a control. After 24 h of
incubation of the cells with the compounds MTS colorimetric assay
of cell survival was performed. The wells were treated with MTS
solution and incubated for 4 h at 37 ꢁC under 5% carbon dioxide and
95% air atmosphere. The absorbance of each well at 490 nm was
read by an automatic microplate reader (“Tecan”, Austria).
6.5.2. 2-[3-(2-Hydrazino-2-oxoethyl)-2-imino-5-nitro-2,3-dihydro-
1H-benzimidazol-1-yl]acetohydrazide 15
Yield e 88,2%; Mp 284 ꢁC (decomp), re-crystallized with
ethanol; Rf ¼ 0.41, mobile phase: dichloromethane/methanol ¼ 8:1;
1H NMR (DMSO-d6)
d (ppm): 4.700 (s, 2H) and 4.730 (s, 2H, NCH2),
6.974 (bs, 2H) and 7.244 (br s, 2H, NH2), 7.18 (d, J ¼ 8.7 Hz, 1H, 7-Ar),
7.865 (dd, J ¼ 8.7, 2.0 Hz, 1H, 6-Ar), 7.922 (d, J ¼ 2.0 Hz, 1H, 4-H),
8.76 (s, 1H, C]NH), 9.44 (s, 2H, HNC]O); 13C NMR (DMSO-d6)
Appendix A. Supplementary data
d
(ppm): 43.49 and 43.68 (NCH2), 103.60 (7-C), 107.34 (4-Ar), 109.52
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.03.010.
(6-Ar), 114.90 (5-C), 134.40 (7a-C), 138.93 (3a-C), 158.25 and 159.79
(C]N), 165.63 and 165.72 (NeC]O). Analysis: Calc. for
C
11H14N8O4; C, 40.99; H, 4.38; N, 34.77; O,19.86; Found: C, 40.91; H,
References
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6.6.1. N0-[1,3-Benzodioxol-5-ylmethylene]-2-(3-{2-[2-(1,3-benzodio
xol-5-ylmethylene)]-2-oxoethyl}-5-benzoyl-2-imino-2,3-dihydro-1H-
benzimidazol-1-yl)acetohydrazide 16
Yield e 76.7%; Mp 263e265 ꢁC; Rf ¼ 0.73, mobile phase:
dichloromethane/methanol ¼ 8:1; NMR (DMSO-d6)
d
(ppm): 1H
d (ppm): 4.586 (bs, 2H) and 5.056 (bs, 2H, NCH2),
NMR (DMSO-d6)
6.079 (s, 8H, OCH2), 6.979 (d, J ¼ 8.0 Hz, 2H, 5-Ar), 7.153 (dd, J ¼ 1.5,
8.0 Hz, 2H, 6-Ar), 7.207 (d, J ¼ 8.3 Hz, 1H, 7-Ar), 7.363 (d, J ¼ 1.5 Hz,
2H, 2-Ar), 7.428 (dd, J ¼ 1.4, 8.2, 1H, 6-Ar), 7.482 (d, J ¼ 1.6 Hz, 1H, 4-
Ar), 7.553 (t, J ¼ 7.7 Hz, 2H, m-Ph), 7.653 (dt, J ¼ 1.3, 7.5 Hz, 1H, p-
Ph), 7.709 (dd, J ¼ 1.3, 7.7 Hz, 2H, o-Ph), 7.940 (s, 2H, CH), 9.35 (s,1H,
C]NH), 10.342 (s, 2H, HNC]O); 13C NMR (DMSO-d6)
d (ppm):
ꢀ
ꢁ
ꢁ
ꢀ
ꢁ
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107.72 (4-Ar), 108.52 (5-Ar), 123.37 (6-Ar), 126.57 (6-Ar), 128.44
(7a-C), 128.54 (m-Ph), 129.30 (i-Ar). 129.41 (o-Ph), 130.86 (3a-C),
132.07 (p-Ph), 134.29 (5-Ar), 138.00 (3-Ar), 138.17 (i-Ph), 143.81 (4-
Ar), 144.04 (CH). 157.64 (C]N). 167.62 (NC]O), 194.70 (C]O).
Analysis: Calc for C34H27N7O7; C, 63.25; H, 4.22; N, 15.19; O, 17.35;
Found: C, 63.30; H, 4.27; N, 15.15; O, 17.31;
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6.6.2. N0-[4-Fluorophenyl)methylene]-2-(3-{2-[2-(4-fluorophenyl)
methylenehydrazino]-2-oxoethyl}-2-imino-5-nitro-2,3-dihydro-1H-
benzimidazol-1-yl)acetohydrazide 17
Yield 72%; Mp e 273e275 ꢁC; Rf ¼ 0.57, mobile phase:
dichloromethane/methanol ¼ 8:1; 1H NMR (DMSO-d6)
d (ppm):