Synthesis, characterization and cytotoxicity of some novel 1,3-disubstituted-2,3-dihydro-2-iminobenzimidazoles

Article abstract of DOI:10.1016/j.ejmech.2011.04.061

Some new 1,3-disubstituted-2,3-dihydro-2-iminobenzimidazoles were synthesized using 1-(un)substituted-2-aminobenzimidazoles as precursors in order to determine their cytotoxicity. The structures of the compounds were confirmed by IR, ¹H NMR, ¹³C NMR and elemental analysis. Compounds 4, 7-11 and 13-14 were evaluated for their cytotoxical effect on two cancer cell lines: human colorectal cancer cell line HT-29, breast cancer cells MDA-MB-231 and as well as normal spleen cells. The distinctly marked antiproliferative activity of 1,3-bis(3-phenylpropyl-1)-1,3-dihydro-2H-benzimidazol-2-imine hydro bromide 7, N-(aminopropyl)-2-(3-{2-[(aminopropyl)-amino]-2-oxoethyl}-2-imino-2, 3-dihydro-1H-benzimidazol-1-yl)acetamide 9 and 1,3-bis[2-(4-methylpiperazin-1- yl)-2-oxoethyl]-2,3-dihydro-2H-benzimidazol-2-imine 11 against human colorectal cancer cell line HT-29 was ascertained and the calculated IC₅₀ were 9.26, 0.56 and 0.013 nM respectively. Compounds 4, 9, 10 and 13 exhibited relative high cytotoxic activity against MDA-MB-231 cells. The calculated IC₅₀ values were in the range 0.123-1.65 nM. All tested compounds excluding compound 1,3-bis[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2,3-dihydro- 2H-benzimidazol-2-imine (11) revealed proliferative activities to normal spleen cells. The computed EC₅₀ values varied from 0.05 to 16.91 nM.

Full text of DOI:10.1016/j.ejmech.2011.04.061

European Journal of Medicinal Chemistry 46 (2011) 3362e3367
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, characterization and cytotoxicity of some novel 1,3-disubstituted-2,
3-dihydro-2-iminobenzimidazoles
,
b
c
c
Anelia Ts. Mavrova a , Diana Wesselinova , Nikolay Vassilev , Jordan A. Tsenov
*
a Department of Organic Synthesis, University of Chemical Technology and Metallurgy, 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-disubstituted-2,3-dihydro-2-iminobenzimidazoles were synthesized using 1-(un)
substituted-2-aminobenzimidazoles as precursors in order to determine their cytotoxicity. The struc-
tures of the compounds were confirmed by IR, 1H NMR, 13C NMR and elemental analysis.
Compounds 4, 7e11 and 13e14 were evaluated for their cytotoxical effect on two cancer cell lines: human
colorectal cancer cell line HT-29, breast cancer cells MDA-MB-231 and as well as normal spleen cells. The
distinctlymarkedantiproliferativeactivityof1,3-bis(3-phenylpropyl-1)-1,3-dihydro-2H-benzimidazol-2-imine
hydro bromide 7, N-(aminopropyl)-2-(3-{2-[(aminopropyl)-amino]-2-oxoethyl}-2-imino-2,3-dihydro-1H-
benzimidazol-1-yl)acetamide 9 and 1,3-bis[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2,3-dihydro-2H-benzi-
midazol-2-imine 11 against human colorectal cancer cell line HT-29 was ascertained and the calculated IC50
were 9.26, 0.56 and 0.013 nM respectively. Compounds 4, 9,10 and 13 exhibited relative high cytotoxic activity
against MDA-MB-231 cells. The calculated IC50 values were in the range 0.123e1.65 nM. All tested compounds
excluding compound 1,3-bis[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2,3-dihydro-2H-benzimidazol-2-imine
(11) revealed proliferative activities to normal spleen cells. The computed EC50 values varied from 0.05 to
16.91 nM.
Received 6 February 2011
Received in revised form
25 April 2011
Accepted 27 April 2011
Available online 6 May 2011
Keywords:
2-Aminobenzimidazoles
2,3-Dihydro-2-iminobenzimidazoles
Solideliquid phase transfer catalysis
Cytotoxicity
Anticancer activity
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
compounds for the development of new anticancer drugs [10],
while mebendazole was identified as a potent, melanoma-specific
2-Aminobenzimidazole is one of the well known biologically
accepted pharmacophores. The polyfunctionality resulting from
the cyclic guanidine residue has made it a building block for the
synthesis of a wide diversity of benzimidazole derivatives of
pharmacological interest. The demonstrated potent antihelminthic
activities of different substituted 2-aminobenzimidazoles as well as
the development of various drugs support further the importance
of this heterocycle in generating better chemotherapeutical agents
against parasitic diseases [1e3]. On the other hand a number of
2-aminobenzimidazoles have exhibited antiproliferative properties
[4e7]. Albendazole, a benzimidazole carbamate with extensive
clinical use as an antihelminthic drug, can also inhibit hepatocel-
lular carcinoma cell proliferation under both in vitro and in vivo
experimental conditions [8]. It was reported that carbendazim
(FB642) is an anticancer agent that induces apoptosis of cancer cells
[9] and together with benomyl show interesting and diverse
cytotoxic mechanisms of action and seem suitable as leading
cytotoxic agent [11]. The 2-aminoderivatives revealed cytotoxicity
against human colorectal cancer cell line (HT-29) and a human
prostate cancer cell line (PC-3) [12]. There are data in the literature
that the antihelminthic flubendazole inhibits microtubule function
through a mechanism distinct from Vinca alkaloids and displays
preclinical activity in leukemia and myeloma [13]. Series of Schiff
bases of 2-aminobenzimidazole and substituted aromatic alde-
hydes exhibited cytotoxic activity against the cells of human cancer
cell lines as SW707 (rectal), HCV29T (bladder), A549 (lung) and
T47D (breast cancer) [14]. Despite advances in the field of cancer
treatment the finding of cytotoxic agents that have specificity for
cancer and tumor cells is indispensable.
On the basis of all above observations and following our aim to
synthesize new benzimidazole derivatives we undertook investiga-
tion over the interaction of 2-aminobenzimidazole with different
alkylation agents both under solideliquid phase transfer catalysis
conditions and in the presence of DBU. In this paper we report the
synthesis of new 1,3-disubstitutet-2,3-dihydro-2-iminobenzimida-
zoles and their cytotoxicity against human colorectal cancer cell line
HT-29, breast cancer cells MDA-MB-231 and normal spleen cells.
* Corresponding author. Tel.: þ359 28163207; fax: þ359 2685488.
E-mail address: anmav@abv.bg (A.Ts. Mavrova).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.061

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 46 (2011) 3362e3367
3363
Because of the structural similarity of benzimidazole nucleus to
naturally occurring compounds as purines it could be expected the
benzimidazole derivatives easily to interact with biological targets
in the living systems. As 2-aminobenzimidazole compounds are
usually associated with antiparasitic, anticancer, antiviral and some
other pharmacological activities, it may be anticipated 1,3-dis-
ubstitutes-2-iminobenzimidazoles to display potential biological
activity not only against parasites but also against tumor cells.
(2-ethoxy-2-oxoethyl)-2-imino-2,3-dihydro-1H-benzimidazol-1-yl]
acetate hydrogen bromide 4 with hydrazine hydrate afforded the
hydrazide 12, which was reacted with the corresponding arylalde-
hydes yielding hydrazones 13e14.
The reaction of ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-2,3-
dihydro-1H-benzimidazol-1-yl]acetate 4 with 1,3-diaminopropane,
benzyl amine and 1-methylpiperazine led to the formation of
2-imino-2,3-dihydrobenzimidazol-1,3-diacetamides 10e12.
The chemical structures of the compounds were established by
elemental analyses, IR-, 1H NMR and 13C NMR spectra and the
results are presented in the Experimental part. The elemental
analyses indicated by the symbols of the elements were within
ꢀ0.4% of theoretical values.
2. Chemistry
The synthesis of 1,3-disubstituted-2,3-dihydro-2-iminobenzi-
midazoles is illustrated and outlined in Fig. 1.
The starting 1-(un)substituted-2-aminobenzimidazoles 1e3 were
synthesized according to the method described by us early in [15].
The nucleophilic substitution between the 2-aminobenzimidazoles
and the appropriated halogen derivatives under solideliquid phase
transfer catalysis conditions in dry acetonitrile as well as in the
presence of DBU resulted in the formation of 1,3-disubstituted-2,3-
dihydro-2-iminobenzimidazoles 4e8.The condensation of ethyl [3-
3. Pharmacology
3.1. Cytotoxicity
Compounds 4, 7e8 and 10e13 were evaluated for their cyto-
toxicity to human colorectal cancer cell line HT-29, breast cancer
O
N
N
a (or b)
N
N
N
a (or b)
N
NH2
NH
N
R
O
1-3
O
1. R = H
8.
2. R = CH3
3. R = C3H7
7.
a (or b)
O
O
O
NH2
NH
O
N
NH2
NH2
N
e
N
c
NH
NH
NH
NH
N
N
R
N
NH
NH2
12
4 - 6
4. R = CH2COOC2H5
5. R = CH3
O
O
6. R = C3H7
9.
f
d
d
F
N
N
NH
O
N
O
NH
NH
NH
N
N
N
N
O
NH
O
N
NH
O
N
O
NH
N
N
F
10.
N
11.
13-14
Fig. 1. Synthesis of 1,3-disubstituted-2,3-dihydro-2-iminobenzimidazoles. Regents and conditions: a) DBU, acetonitrile, halogen derivative, 20 ꢁC; acetonitrile, TBAB, dry K2CO3,
halogen derivative, 25 ꢁC; c) ethanol, 1,3-diaminopropane, 25 ꢁC; d) ethanol, benzyl amine or 1-methylpiperazine, refluxing; e) hydrazine hydrate, ethanol, refluxing; f) ethanol,
4- or 3-floro-benzaldehyde, refluxing.

3364
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 46 (2011) 3362e3367
cells MDA-MB-231 and normal spleen cells by using the MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrasolium inner salt) e test [16].
400
350
300
250
200
150
100
50
concentration
0.5
4. Results and discussion
0.05
We have already reported the synthesis of 1-(un)substituted-2-
aminobenzimidazoles [15]. In order to synthesize new derivatives
of 2-aminobenzimidazole and study their cytotoxicity we under-
took investigation on the reaction of 1-(un)substituted-2-amino-
benzimidazoles with halogen containing reagents in the presence
of DBU in dry acetonitrile. It was established that the reaction led to
the formation of 1,3-disubstituted-2,3-dihydro-2-iminobenzimi-
dazoles in good yields, 66e75%. The same interaction was studied
under solid-liquid phase transfer catalysis using anhydrous potas-
sium carbonate and tetrabutyl ammonium bromide as catalyst. It
was established that the reaction runs best at mol ratio of 2-ami-
nobenzimidazoles 4e6, halogen compounds and potassium
carbonate 1:2:2 and has led to a range of new 1,3-disubstituted
derivatives being prepared easily and in good yields.
The synthesis of 1,3-disubstituted-2,3-dihydro-2-iminobenzi-
midazoles was reported earlier but by using the reduction of
2-halogen-nitrobenzenes and condensation with BrCN [17e19].
The reaction performed with ethyl 2-bromopropanoate resulted
in formation of fused imidazo [1,2-a]benzimidazole ring, possess-
ing on third position the ethyl propionate residue. The IR-spectral
0.005
0.0005
0
0.005
0.5
4
7
8
9
10
11
13
14
substance Nr.
Fig. 2. Relative HT-29 cells viability (%).
at which the compound excited cytotoxic effect on HT-29 cells.
That fact is an indicator for the selectivity effect of the compound
9. It could be underlined, that excluding compound 11, all
compounds possessing cytotoxicity against HT-29 and MDA-MB-
231 revealed proliferative activities to normal spleen cells.
Stimulating effect on the three types of cells showed ethyl 2-(3-
methyl-2-oxo-2,3-dihydro-9H-imidazo[1,2-a]benzimidazol-9-yl)
propanoate 8. Statistical significant differences in the level of
cells in both control and experimental groups were determined
(p ꢃ 0.05).
To make a conclusion about the mechanism of the action of the
studied compounds, it is necessary to perform additional investi-
gations, but it can be pointed out that the cell proliferation MTS-
assay is based on the fact that the MTS tetrazolium compound is
bioreduced by cells into a colored formazan product that is soluble
in the tissue culture medium. This conversion is presumably
accomplished by NADPH or NADH, produced by dehydrogenase in
metabolically active cells [20]. The bigger release amount of for-
mazan indicates to a higher vitality of the cells (proliferation). The
low vitality demonstrates a cytotoxic influence of the experimental
compounds.
data characteristics of both carbonyl groups are e
y
C]O ꢂ1737,
53 cmꢂ1 and
y
C]O ꢂ1695, 32 cmꢂ1
.
The test for estimating the in vitro cytotoxicity against human
colorectal cancer cell line HT-29, breast cancer cells MDA-MB-231
and normal spleen cells was performed with compounds 4, 7e10,
as well as 13e14 according to MTS method, as described in [16]. 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 samples MTS colorimetric assay of cell survival
was performed. The wells were treated with MTS solution and
incubated for 2 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). Relative cell
viability, expressed as a percentage of the untreated control (100%
viability), was calculated for each concentration. All data points
represent an average of three independent assays and are given in
Figs. 2e4. The obtained results were plotted and IC50 and EC50 were
calculated. The data are given in Table 1 and Table 2.
5. Conclusion
Improved conditions have been developed and optimized for
the synthesis of new 1,3-substituted-2,3-dihydro-2-iminobenzi-
midazoles using 1-(un)substituted-2-aminobenzimidazoles as
precursors and different halogen derivatives under solideliquid
PTC as well as by means of DBU. This methodology, combined with
a “catch and release” purification strategy, has led to a range of new
derivatives being prepared easily and in excellent yield.
Among all tested compounds only compounds 7, 9 and 11
revealed cytotoxicity against HT-29 cells. The substituted with
4-methylpiperazine-1,3-diacetamide 11 showed highest cyto-
The initial biological screening in vitro showed that the studied
compounds 7, 9, 11 possessed relative high cytotoxicity against
toxic effect on HT-29 cells, IC50
¼
0.013 nM, followed by
the compound 9, IC50 ¼ 0.56 nM. Compounds 4, 9, 10 and 13
exhibited relative high cytotoxic activity against MDA-MB-231
cells. Most toxic were N-(aminopropyl)-2-(3-{2-[(aminopropyl)
amino]-2-oxoethyl}-2-imino-2,3-dihydro-1H-benzimidazol-1-yl)
acetamide 9 and the hydrazone 13 with IC50 e 0.123 nM and
IC50 e 0.78 nM respectively.
250
200
concentration
150
0.5
0.05
0.005
0.0005
As it can be seen from the results, given in Table 2, all tested
compounds excluding compound 11 revealed proliferative effects
to the normal spleen cells. The EC50 values varied in the range
from 0.05 nM for compound 9 to 16.91 nM for compound 13. If
the results, obtained for compound 9 are taken in consideration it
should be noted that N-(aminopropyl)-2-(3-{2-[(aminopropyl)
amino]-2-oxoethyl}-2-imino-2,3-dihydro-1H-benzimidazol-1-yl)
acetamid revealed proliferative activity against normal spleen
cells at lower concentration in comparison to the concentration
100
50
0
0.005
0.5
4
7
8
9
substance Nr.
10
11
13
14
Fig. 3. Relative MDA-MB-231 cells viability (%).

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 46 (2011) 3362e3367
3365
Table 2
Proliferative effects in vitro.
2500
Comp.
EC50 ꢀ SE (nM)
2000
1500
1000
500
0
HT-29
MDA-MB-231
Normal
concentration
spleen cells
0.5
0.05
0.005
0.0005
4
7
8
9
10
11
13
14
2.37 ꢀ 0.65
e
e
14.32 ꢀ 0.02
14.13 ꢀ 0.59
0.542 ꢀ 0.56
0.05 ꢀ 0.29
7.74 ꢀ 0.26
e
1.388 ꢀ 0.22
5.68 ꢀ 0.17
e
2.019 ꢀ 0.13
e
0.205 ꢀ 0.36
e
e
1.59 ꢀ 0.12
e
0.005
0.5
4.30 ꢀ 0.14
3.51 ꢀ 0.45
16.91 ꢀ 0.21
11. 02 ꢀ 0.50
4
7
8
9
0.002 ꢀ 0.20
10
11
13
14
substance Nr.
Fig. 4. Relative normal spleen cells viability (%).
(DBU) in 20 ml dry acetonitrile 0.024 mol of the corresponding
halogen derivative was dropped by cooling. The solution was stir-
red for 2e4 h at ambient temperature and the obtained precipitate
was filtered. Additional quantity of the compounds was received
through refluxing the filtrate for 1 h, removing the solvent and
crystallizing the obtained oil product with suitable solvent.
HT-29 cells. IC50 values were in the range 0.013e9.26 nM. In respect
to MDA-MB-231 cells most cytotoxic was compound 9 with IC50
e 0.123 nM. All investigated compounds excluding compound 11
revealed proliferative effects on normal spleen cells, the EC50 values
were in the range 0.05e16.91 nM.
The above results confirmed also the hypothesis that the
introduction of different substituents at 1 and 3-th position in the
structure of 2-aminobenzimidazole are auspicious to the interac-
tion of these molecules with the biological targets.
6.1.2. Method B
To
a solution of 1-(un)substituted-2-aminobenzimidazole
(0.01 mol) in dry acetonitrile (50 ml) was added anhydrous
potassium carbonate (2.7 g, 0.02 mol) and tetrabutyl ammonium
bromide (TBAB) (0.9 g, 0.003 mol) and 0.02 mol of the halogen
organic reagent was dropped by cooling. The mixture was stirred
for 2e5 h vigorously at 25 ꢁC and monitored by TLC over the
reaction period. After completion of the reaction, the mixture was
filtered to separate the solid K2CO3, and the organic solvent was
evaporated. The residue was then crystallized from appropriate
solvent to give products 4e8.
6. Experimental part
Melting points (mp) were determined on an Electrothermal AZ
9000 3MK4 apparatus and were uncorrected. The thin layer chro-
matography (TLC, Rf values) was performed on silica gel plates F254
(Merck, 0.2 mm thick) and visualization was effected with ultra-
violet light. IR spectra were recorded on a Bruker Equinox 55
spectrophotometer as potassium bromide discs. 1H and 13C NMR
spectra were recorded on a Bruker Avance II þ 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 accom-
plished by measurement of 2D homonuclear correlation (COSY),
DEPT-135 and 2D inverse detected heteronuclear (CeH) correla-
tions (HMQC and HMBC). The microanalyses for C, H, N and S were
performed on PerkineElmer elemental analyzer.
6.1.3. Ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-2,3-dihydro-1H-
benzimidazol-1-yl]acetate hydro bromide (4)
After removing of acetonitrile the compound was crystallized
with ethanol. Yield e 66.48% (method A); 82.5% (method B); Mp e
245e246 ꢁC; Rf ¼ 0.68, mobile phase: benzene/ethanol e 1:1; 1H
NMR (DMSO-d6)
d
(ppm): 1.245 (t, J ¼ 7.1 Hz, 6H, CH3), 4.201 (q,
J ¼ 7.1 Hz, 4H, OCH2), 5.250 (s, 4H, NCH2), 7.34 (AA’ part of AA’XX’
system, 2H, 5-H and 6-H) and 7.63 (XX’ part of AA’XX’, 2H, 4-H and 7-
H), 9.200 (bs, 2H, NH.HBr).13C NMR(DMSO-d6)
d(ppm): 14.02 (CH3),
44.24 (NCH2), 61.84 (OCH2), 110.61 (4-C and 7-C),124.03 (5-C and 6-
C), 129.56 (3a-C and 7a-C), 150.71 (C]N), 166.49 (C]O). Analysis:
Calc. forC15H20 BrN3O4;C, 46.64; H, 5.22; Br, 20.69;N,10.88; O,16.57;
Found: C, 46.61; H, 5.25; Br, 20.72; N, 10.86; O, 16.54.
2-Amino-1H-benzimidazoles 1e3 were synthesized by heating
of 1-(un)substituted-1H-benzimidazol-2-sulphonic acid and 25%
ammonium hydroxide as described in [15].
6.1. General procedures for preparation of compounds 4e8
6.1.4. Ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-2,3-dihydro-
1H-benzimidazol-1-yl]acetate (4.1)
6.1.1. Method A
Some quantity of compound 4 was washed with water to afford
To
a solution of 1-(un)substituted-2-aminobenzimidazole
4.1. Mp e 161e162 ꢁC; Rf ¼ 0.66, mobile phase: benzene/ethanol
(0.004 mol) and 0.016 mol of 1,8-diazabicyclo[5.4.0]undec-7-ene
e 1:1; 1H NMR (DMSO-d6)
d d (ppm):
(ppm): 1H NMR (DMSO-d6)
1.208 (t, J ¼ 7.1 Hz, 6H, CH3), 4.138 (q, J ¼ 7.1 Hz, 4H, OCH2), 4.758 (s,
4H, NCH2), 6.91e6.94 (AA’ part of AA’BB’ system, 2H, 5-H and 6-H)
and 6.96e6.99 (BB’ part of AA’BB’, 2H, 4-H and 7-H); 13C NMR
Table 1
In vitro cytotoxicity against HT-29, MDA-MB-231 and normal spleen cells.
Comp
IC50 ꢀ SE (nM)
(DMSO-d6) d (ppm): 14.04 (CH3), 42.44 (NCH2), 60.91 (OCH2),106.79
HT-29
MDA-MB-231
Normal
spleen cells
(4-C and 7-C), 120.56 (5-C and 6-C), 131.31 (3a-C and 7a-C), 152.41
(C]N), 168.16 (C]O); Analysis: Calc. for C15H19N3O4; C, 59.01; H,
6.27; N, 13.76; O, 20.96; Found: C, 59.04; H, 6.23; N, 13.78; O, 20.91.
4
7
8
e
1.15 ꢀ 0.12
e
e
e
9.26 ꢀ 0.11
e
e
e
6.1.5. Ethyl (2-imino-3-methyl-2,3-dihydro-1H-benzimidazol-1-yl)
acetate hydro bromide (5)
Yield e 75% (method A), 88% (method B); re-crystallized with
ethanol; Mp. 242e244 ꢁC; Rf ¼ 0.60, mobile phase: benzene/
ethanol e 1:1.
9
0.56 ꢀ 0. 28
0.123 ꢀ 0.43
1.65 ꢀ 0.23
e
10
11
12
13
e
0.013 ꢀ 0.81
1.38 ꢀ 0.04
e
e
0.78 ꢀ 0.29
e
e
e

3366
A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 46 (2011) 3362e3367
1H NMR (DMSO-d6)
d
(ppm): 1H NMR (DMSO-d6)
d
(ppm): 1.246
1.5 ml (0.018 mol) 1,3-diaminopropane was added and the solution
was stirred at ambient temperature for 6 h. After completion of the
reaction the solvent was removed under reduced pressure. The oily
residue was crystallized with 5 ml chloroform and re-crystallized
with ethyl acetate.
(t, J ¼ 7.1 Hz, 6H, CH3), 3.704 (s, 3H, NCH3), 4.200 (q, J ¼ 7.1 Hz, 4H,
OCH2), 5.204 (s, 2H, NCH2), 7.323 (dt, J ¼ 1.1, 8.0 Hz,1H) and 7.364 (dt,
J ¼ 1.1, 8.0 Hz, 1H, 5-H and 6-H), 7.59e7.61 (m, 2H, 4-H and 7-H),
8.986 (s, 2H, NH). 13C NMR (DMSO-d6)
d (ppm): 13.99 (CH3), 29.67
(NCH3), 44.05 (NCH2), 61.78 (OCH2),110.38 (4-C and 7-C),123.62 and
123.80 (5-C and 6-C),129.62 and 130.00 (3a-C and 7a-C),150.37 (C]
N),166.66 (C]O); Analysis: Calc. for C12H15N3O2; C, 61.79; H, 6.48; N,
18.01; O, 13.72; Found: C, 61.82; H, 6.45; N, 18.04; O, 13.69.
Yield e 68%; Mp e 146e148 ꢁC; Rf ¼ 0.32, mobile phase:
benzene/methanol e 3:1; 1H NMR (D2O)
d (ppm): 1.710 (sextet,
J ¼ 7.1 Hz, 4H, CH2), 2.794 (t, J ¼ 7.1 Hz, 4H, NCH2), 3.204 (q,
J ¼ 7.1 Hz, 4H, NCH2), 4.564 (s, 4H, NCH2), 6.93e6.97 (AA’ part of
AA’BB’ system, 2H, 5-H and 6-H) and 7.04e7.07 (BB’ part of AA’BB’,
6.1.6. Ethyl (2-imino-3-propyl-2,3-dihydro-1H-benzimidazol-1-yl)
acetate hydro bromide (6)
2H, 4-H and 7-H); 13C NMR (D2O)
d (ppm): 27.41 (CH2), 36.30
(NCH2), 37.26 (NCH2), 44.21 (NCH2), 107.58 (4-C and 7-C), 122.28
The compound was crystallized with ethanol; Yield: 73 %
(method A), 85.0% (method B), the compound was crystallized with
ethanol; Mp. 213e215 ꢁC; Rf ¼ 0.46, mobile phase: benzene/
(5-C and 6-C), 130.67 (3a-C and 7a-C), 155.07 (C]N), 161.23 (C]O).
6.2.1. Analysis
ethanol e 2:1. 1H NMR (DMSO-d6)
d
(ppm): 0.907 (t, J ¼ 7.1 Hz, 3H,
Calc. for C17H27N7O2; C, 56.49; H, 7.53; N, 27.13; O, 8.85; Found:
C, 56.46; H, 7.51; N, 27.17; O, 8.87.
CH3), 1.238 (t, J ¼ 7.1 Hz, 3H, CH3), 1.726 (sextet, J ¼ 7.1 Hz, 2H, CH2),
4.185 (t, J ¼ 7.1 Hz, 2H, NCH2), 4.194 (q, J ¼ 7.1 Hz, 2H, OCH2), 5.196
(s, 2H, NCH2), 7.324 (dt, J ¼ 1.1, 7.6 Hz, 1H) and 7.357 (dt, J ¼ 1.1,
7.5 Hz, 1H, 5-H and 6-H), 7.616 (dd, J ¼ 1.1, 7.5 Hz, 1H) and 7.381 (dt,
J ¼ 1.1, 7.6 Hz, 1H, 4-H and 7-H), 8.977 (s, 2H, NH). 13C NMR (DMSO-
6.3. General procedure for preparation of compounds 10 and 11
To solution of 0.5 g (0.0013 mol) ethyl [3-(2-ethoxy-2-oxoethyl)-
2-imino-2,3-dihydro-1H-benzimidazol-1-yl]acetate 4 in 10 ml
d6)
d
(ppm): 10.47 (CH3), 13.96 (CH3), 20.79 (CH2), 43.84 (NCH2),
44.02 (NCH2), 61.79 (OCH2), 110.48 and 110.59 (4-C and 7-C), 123.66
and 123.88 (5-C and 6-C),129.35 and 129.60 (3a-C and 7a-C),149.92
(C]N), 166.63 (C]O). Analysis: Calc. for C14H19N3O2; C, 64.35; H,
7.33; N, 16.08; O, 12.25; Found: C, 64.32; H, 7.35; N, 16.04; O, 12.27.
ethanol 0.0052 mol of the corresponding amine was added. The
mixture was refluxed for 2e6 h. After cooling the obtained
precipitate was filtered and re-crystallized with ethanol.
6.3.1. N-Benzyl-2-{3-[2-(benzylamino)-2-oxoethyl]-2-imino-2,3-di
hydro-1H-benzimidazol-1-yl}acetamide (10)
6.1.7. 1,3-Bis(3-phenylpropyl-1)-1,3-dihydro-2H-benzimidazol-2-
imine hydro bromide (7)
Yield e 94%; Mp e 178e181 ꢁC, re-crystallized with ethanol;
Rf ¼ 0.45, mobile phase: benzene/ethanol e 2:1; 1H NMR (DMSO-
The compound crystallized after addition of ethyl acetate. Yield
e 84% (method B); Mp e 222e224 ꢁC; Rf ¼ 0.67, mobile phase:
d6),
d (ppm): 4.395 (s, 4H, 2CH2); 4.565 (s, 4H, 2CH2); 7.211e7.248
benzene/ethanol e 2:1; 1H NMR (DMSO-d6)
d
(ppm): 1.990 (m, 4H,
(m, 10H, 2Ph); 7. 391e7.448 (dd, J ¼ 7.57 Hz, 2H, 2CH); 8.145 (d,
J ¼ 7.65, 2H, 2CH); Analysis: Calc. for C25H25N5O2: C, 70.24; H, 5.89;
N, 16.38; O, 7.49; Found: C, 70.22; H, 5.87; N, 16.41; O, 7.46.
2-CH2), 2.686 (m, 4H, 3-CH2), 4.224 (t, J ¼ 7.4 Hz, 4H, 1-CH2), 7.154
(t, J ¼ 7.2 Hz, 2H, p-Ph), 7.178 (d, J ¼ 7.4 Hz, 4H, o-Ph), 7.246 (t,
J ¼ 7.6 Hz, 4H, m-Ph), 7.32 (AA’ part of AA’XX’ system, 2H, 5-H and
6-H) and 7.56 (XX’ part of AA’XX’, 2H, 4-H and 7-H), 8.875 (bs, 2H,
6.3.2. 1,3-Bis[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2,3-dihydro-
2H-benzimidazol-2-imine (11)
NH.HBr). 13C NMR (DMSO-d6)
d (ppm): 29.19 (2-CH2), 31.84 (3-
CH2), 42.52 (1-CH2), 110.37 (4-C and 7-C), 123.47 (5-C and 6-C),
125.96 (p-Ph), 128.07 (o-Ph), 128.34 (m-Ar), 129.56 (3a-C and 7a-C),
140.93 (i-Ph), 149.03 (C]N); Analysis: Calc. for C25H27N3; C, 81.26;
H, 7.37; N, 11.37; Found: C, 81.29; H, 7.39; N, 11.35.
Yield e 65%; Mp e 265e268 ꢁC (decomp.); Rf ¼ 0.50, mobile
phase: benzene/ethanol e 1:1; 1H NMR (DMSO-d6)
d (ppm): 2.174
(s, 6H, CH3), 2.37 (m, 8H, NCH2), 2.89 (m, 8H, NCH2), 4.54 (s, 4H,
CH2), 6.84e6.94 (AA’ part of AA’BB’ system, 2H, 5-H and 6-H) and
7.02e7.12 (BB’ part of AA’BB’, 2H, 4-H and 7-H). Analysis: Calc. for:
C21H31N7O2; C, 61.00; H, 7.56; N, 23.71; O, 7.74; Found: C, 61.04; H,
7.53; N, 23.74; O, 7.70.
6.1.8. Ethyl 2-(3-methyl-2-oxo-2,3-dihydro-9H-imidazo[1,2-a]benz
imidazol-9-yl)propanoate (8)
The compound crystallized after addition of water. Yield: 86%
(Method B); Mp. 128e130 ꢁC; Rf ¼ 0.39, mobile phase: benzene/
ethanol e 5:1.
6.4. Procedure for preparation of 2-[3-(2-hydrazino-2-oxoethyl)-2-
imino-2,3-dihydro-1H-benzimidazol-1-yl]acetohydrazide (12)
1H NMR (DMSO-d6)
d
(ppm): 1.114 (t, J ¼ 7.0 Hz, 3H, CH3), 1.637
(d, J ¼ 7.1 Hz, 3H, CH3), 1.744 (d, J ¼ 7.4 Hz, 3H, CH3), 4.118 (q,
J ¼ 7.0 Hz, 2H, OCH2), 5.090 (q, J ¼ 7.1 Hz, 1H, CH), 5.105 (q,
J ¼ 7.4 Hz,1H, CH), 7.14 (t, J ¼ 7.1 Hz,1H) and 7.16 (t, J ¼ 7.2 Hz,1H, 6-
H and 7-H), 7.50 (d, J ¼ 7.0 Hz, 1H) and 7.54 (d, J ¼ 6.8 Hz, 1H, 5-H
To a solution of 1 g (0.002 mol) ethyl [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 98% hydrazine hydrate and the
mixture was refluxed for 2 h. After cooling the obtained precipitation
was filtered and re-crystallized ethanol. Yield e 95%; Mp e 254e256 ꢁC
and 8-H); 13C NMR (DMSO-d6)
d (ppm): 13.93 (CH3), 14.11 (CH3),
15.73 (CH3), 49.06 (CH), 54.98 (CH), 109.27 and 118.43 (5-C and 8-
C), 121.45 and 121.50 (6-C and 7-C), 131.07 and 144.40 (4a-C and 8a-
C), 152.45 (C]N), 169.19 (OC]O), 175.06 (NC]O). Analysis: Calc.
for C15H17N3O3; C, 62.71; H, 5.96; N,14.63; O,16.71; Found: C, 62.74;
H, 5.91; N, 14.65; O, 16.69.
(decomp.); Rf
¼
0.31, mobile phase: benzene/diethyl ether/
ethanol ¼ 8:8:1; 1H NMR (DMSO-d6),
d (ppm): 4.43 (ds, 2H, CH2); 4.52
(s, 2H CH2); 6.93e7.10 (m, 4H, Ar); 9.28 (bs,1H, NH); 10.21 (s, 2H, 2NH);
13C NMR (DMSO-d6): 40.10 (2CH2); 107.8 (CH); 131.35 (CH); 143.68
(C]N); 152.62 (C); 157.45 (C); 165.84 (C); Analysis: Calc. for
C11H15N7O2; C, 47.65; H, 5.45; N, 35.36; O, 11.54; Found: C, 47.68; H,
5.43; N, 35.38; O, 11.51.
6.2. Procedure for preparation of N-(aminopropyl)-2-(3-{2-[(amino
propyl)amino]-2-oxoethyl}-2-imino-2,3-dihydro-1H-benzimidazol-
1-yl)acetamide (9)
6.5. General procedure for preparation of compounds 13e14
To 1 g (0.003 mol) ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-2,3-
dihydro-1H-benzimidazol-1-yl]acetate 4 diluted in 10 ml ethanol
0.3 g (0.001 mol) acetohydrazide (12) and 0.003 mol of the
relevant aldehyde were refluxed in 10 ml absolute ethanol for

A.Ts. Mavrova et al. / European Journal of Medicinal Chemistry 46 (2011) 3362e3367
3367
4e5 h. After completion of the reaction the solution was cooled, the
obtained precipitate was filtered and re-crystallized with ethanol.
[CellTiter 96 Non-Radioactive Cell proliferation assay Technical
Bulletin #TB112, Promega Corporation]. This consisted of 1e3 h
incubation with MTS solution at 37 ꢁC under 5% carbon dioxide and
95% air. The absorbance of each well at 490 nm was read by an
automatic microplate reader (Absorbance Reader “Tecan”, Austria).
Relative cell viability, expressed as a percentage of the untreated
control (100% viability), was calculated for each concentration. All
data points represent an average of three independent assays.
6.5.1. 2-(3-{2-[2-(4-Fluoro)-benzylidenehydrazino]-2-oxoethyl}-2-
imino-2,3-dihydro-1H-benzimidazol-1-yl)-N0-[(4-fluoro)-benzylide
ne]acetohydrazide (13)
1H NMR (DMSO-d6)
d (ppm): 5.543 (s, 4H, NCH2), 7.29e7.32 (AA’
part of AA’XX’ system, 2H, 5-H and 6-H) and 7.60e7.62 (XX’ part of
AA’XX’, 2H, 4-H and 7-H), 7.340 (t, J ¼ 8.8 Hz, 4H, 3-Ar and 5-Ar),
7.879 (dd, J ¼ 5.6, 8.8 Hz, 4H, 2-Ar and 6-Ar), 8.096 (s, 2H, CH), 8.96
(bs, 1H, NH), 11.96 (bs, 2H, NNH); 13C NMR (DMSO-d6)
d (ppm):
References
44.84 (NCH2), 110.68 (4-C and 7-C), 116.10 (2JCF ¼ 22.0 Hz, 3-Ar and
5-Ar), 123.86 (5-C and 6-C), 129.49 (3JCF ¼ 8.6 Hz, 2-Ar and 6-Ar),
130.34 (3a-C and 7a-C), 130.69 (4JCF ¼ 2.7 Hz, 1-Ar), 143.454 (CH),
151.48 (C]N), 163.32 (1JCF ¼ 248.0 Hz, 4-Ar), 166.67 (C]O); 19F
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d
(ppm): ꢂ110.4; Analysis: Calc. for C25H25N5O2: C,
70.24; H, 5.89; N, 16.38; O, 7.49; Found: C, 70.21; H, 5.91; N, 16.41;
O, 7.47.
6.5.2. 2-(3-{2-[2-(3-Fluoro)-benzylidenehydrazino]-2-oxoethyl}-2-
imino-2,3-dihydro-1H-benzimidazol-1-yl)-N0-[(3-fluoro)-benzylide
ne]acetohydrazide (14)
1H NMR (DMSO-d6)
d (ppm): 5.565 (s, 4H, NCH2), 7.301 (dd,
J ¼ 6.1, 8.8 Hz, 1H, 4-Ar), 7.29e7.31 (AA’ part of AA’XX’ system, 2H,
5-H and 6-H) and 7.59e7.61 (XX’ part of AA’XX’, 2H, 4-H and 7-H),
7.619 (d, J ¼ 7.7 Hz, 1H, 6-Ar), 7.702 (ddd, J ¼ 1.4, 2.3, 9.2 Hz, 1H, 2-
Ar), 7.530 (dt, J ¼ 6.1, 7.9 Hz,1H, 5-Ar), 8.100 (s, 2H, CH), 9.00 (bs,1H,
NH), 12.05 (bs, 2H, NNH); 13C NMR (DMSO-d6),
d (ppm): 44.76
(NCH2), 110.51 (4-C and 7-C), 112.90 (2JCF ¼ 22.6 Hz, 2-Ar), 117.00
(2JCF ¼ 21.5 Hz, 4-Ar), 123.87 (5-C and 6-C), 124.88 (4JCF ¼ 2.5 Hz,
6-Ar), 130.31 (3a-C and 7a-C), 131.02 (3JCF ¼ 8.2 Hz, 5-Ar), 136.59
(3JCF ¼ 7.9 Hz, 1-Ar), 142.92 (4JCF ¼ 2.8 Hz, CH), 151.46 (C]N), 162.51
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ꢂ112.8; Analysis: Calc. for C25H25N5O2: C, 70.24; H, 5.89; N, 16.38;
ꢀ
A. Nasulewicz, M. Pe1czynska, A. Opolski, II Farmaco 59 (2) (2004) 83e91.
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6.6. MTS test
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The cells were seeded in 96-well flat-bottomed microplates
(Orange scientific) at a concentration of 2 ꢄ 104 cells/well. At the
24th h cells from monolayers were washed and covered with media
modified with different concentrations of the compound tested.
Samples of cells grown in non-modified medium served as
a control. After 2 h of incubation MTS colorimetric assay of cell
survival was performed as described by the protocol of “Promega”
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