Antioxidative and antiproliferative activities of novel pyrido[1,2-a]benzimidazoles

Article abstract of DOI:10.1007/s11030-016-9702-y

A series of pyrido[1,2-a]benzimidazoles has been designed, and novel examples are synthesized and evaluated for their potential antiproliferative activity against four human tumour cell lines—cervical (HeLa), colorectal (SW620), breast (MCF-7) and hepatocellular carcinoma (HepG2). In addition, their antioxidative potency has been evaluated by in vitro spectrophotometric assays. Preliminary structure–activity relationships among the synthesized compounds are discussed. Evaluation of their antioxidative capacity has shown that two compounds (25 and 26) possess promising reducing characteristics and free radical scavenging activity. Selective antiproliferative effect in the single-digit micromolar range was observed for compound 25 on MCF-7 (IC50=6μM) and HeLa (IC50=8μM) cell lines, comparable to the standards 5-fluorouracil and cisplatin. The combination of the radical scavenging activity and antiproliferative activity of compound 25 positions this compound as a potential lead candidate for further optimization.

Full text of DOI:10.1007/s11030-016-9702-y

Mol Divers
DOI 10.1007/s11030-016-9702-y
ORIGINAL ARTICLE
Antioxidative and antiproliferative activities of novel
pyrido[1,2-a]benzimidazoles
Martina Tireli1 · Kristina Starcˇevic´2 · Tamara Martinovic´3
·
Sandra Kraljevic´ Pavelic´3 · Grace Karminski-Zamola4 · Marijana Hranjec4
Received: 22 February 2016 / Accepted: 12 September 2016
© Springer International Publishing Switzerland 2016
Abstract A series of pyrido[1,2-a]benzimidazoles has been
designed, and novel examples are synthesized and eval-
uated for their potential antiproliferative activity against
four human tumour cell lines—cervical (HeLa), colorec-
tal (SW620), breast (MCF-7) and hepatocellular carcinoma
(HepG2). In addition, their antioxidative potency has been
evaluated by in vitro spectrophotometric assays. Prelimi-
nary structure–activity relationships among the synthesized
compounds are discussed. Evaluation of their antioxida-
tive capacity has shown that two compounds (25 and 26)
possess promising reducing characteristics and free radical
scavenging activity. Selective antiproliferative effect in the
single-digit micromolar range was observed for compound
25 on MCF-7 (IC50 = 6 μM) and HeLa (IC50 = 8 μM)
cell lines, comparable to the standards 5-fluorouracil and cis-
platin. Thecombinationoftheradicalscavengingactivityand
antiproliferative activity of compound 25 positions this com-
pound as a potential lead candidate for further optimization.
Keywords Pyrido[1,2-a]benzimidazoles · Amides ·
Antiproliferative activity · Antioxidative activity ·
Cyclization
Introduction
Azine azoles, including pyrido-, pyrimido-, and triazino-
benzimidazole heterocycles, have attracted considerable
attention in recent years because of their promising biolog-
ical activities and their use as important building blocks in
natural and synthetic bioactive compounds [1–3]. The benz-
imidazole nucleus itself has been widely incorporated as one
of the most important heterocyclic pharmacophores in vari-
ous biomedical compounds, thus possessing a wide spectrum
of biological activities. Furthermore, benzimidazole deriv-
atives as a structural isostere of nucleotides could easily
interact with the biomacromolecules of a living system and
significantly impact their biological activities and functions.
In addition to their well-known anticancer [4], antibacterial
[5], antiviral [6] and others activities, in the last decade, the
antioxidative potency of versatile benzimidazole derivatives
has also been studied [7]. The implication of free radicals in
the cell signalling pathway of many chronic diseases, such
as cancer, inflammation, neurodegenerative syndrome or dia-
betes is very important. In general, the rate of reactive oxygen
species (ROS) production is increased in most diseases and
could be regulated by a proper application of antioxidants [8].
Antioxidants are considered as compounds that can prevent
oxidation of easily oxidizable substrates and prevent excess
of ROS. Therefore, the antioxidative evaluation of natural
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-016-9702-y) contains supplementary
material, which is available to authorized users.
Marijana Hranjec
mhranjec@fkit.hr
B
1
Laboratory for synthesis and processes of self-assembling of
organic molecules, Division of Physical Chemistry, Rud¯er
Boškovic´ Institute, Bijenicˇka cesta 54, 10000 Zagreb, Croatia
2
Department of Animal Husbandry, Faculty of Veterinary
Medicine, University of Zagreb, Heinzelova 55, 10000
Zagreb, Croatia
3
Department of Biotechnology, Centre for high-throughput
technologies, University of Rijeka, Radmile Matejcˇic´ 2,
51000 Rijeka, Croatia
4
Department of Organic Chemistry, Faculty of Chemical
Engineering and Technology, University of Zagreb,
Marulic´ev trg 20, P. O. Box 177, 10000 Zagreb, Croatia
123

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and synthetic compounds is extremely important in terms of
prevention of diseases mentioned above.
substituted 2-cyanomethylbenzimidazoles 5–8, were pre-
pared through a cyclocondensation reaction between 4-
substituted-1,2-phenylenediamines 1–4 and 2-cyanoaceta-
mide at high temperature.
Pyrido[1,2-a]benzimidazoles are attractive compounds
due to their wide range of significant biological activ-
ities including anticancer, antineoplastic [9,10], antiviral
[11], anxiolytic [12,13], antimalarial [14,15] and antimi-
crobial activities [16] and potential pharmaceutical appli-
cations. Pyrido[1,2-a]benzimidazoles with substitution on
the N5-nitrogen have been found to possess high affin-
ity for the benzodiazepine (BZD) site on the GABA-A
receptor [17,18]. The most promising of these compounds
was an ethoxymethyl analogue which has activity in the
nanomolar concentration range for the BZD site on the
GABA-A receptor and has been advanced to human clin-
ical trials [15]. Moreover, Badaway et al. have published
the synthesis and antineoplastic activity of several substi-
tuted pyrido[1,2-a]benzimidazole derivatives where some
analogues exhibited moderate in vitro antineoplastic activ-
ity against most of the leukaemia and Artina salina larvae
cell lines [19,20]. From previous studies, it was concluded
that the biological activity is strongly dependent on the
pyrido[1,2-a]benzimidazole tricyclic core as well as the posi-
tion and type of the side chain substituents.
Recently, our attention was focused on fused, benzan-
nulated benzimidazole derivatives with a highly conjugated
planar tetracyclic chromophore bearing different side chain
substituents that could be important for additional interac-
tions with possible biological targets or could improve their
spectroscopic properties, namely fluorescence intensity, due
to their applications in fluorescent microscopy as potential
fluorescent probes for the detection of biomacromolecules in
living systems [21,22]. Additional biological activity stud-
ies of the above-mentioned class of compounds including
spectroscopic characterization of interactions with DNA,
topoisomerase I-mediated DNA relaxation, DNase I foot-
printing and fluorescent microscopy experiments confirmed
their promising anticancer potential [23].
The synthesis of cyano-substituted pyrido[1,2-a]benzi-
midazoleswasaccomplishedbyheatinganethanolicsolution
of 2-cyanomethylbenzimidazoles 5–8 with perchloric acid
at reflux producing cyclic derivatives 9–12 in good yields.
Within the acidic hydrolysis, the corresponding carboxylic
acids 13–16 were obtained as main precursors for the syn-
thesis of amido-substituted pyrido[1,2-a]benzimidazoles.
Activation of 13–16 with 1,1ꢀ-carbodiimidazole in DMF
followed by the addition of an amine afforded desired amido-
substituted derivatives. In light of the poor solubility of these
compounds, these compounds were dissolved in ethanol and
converted to their hydrochloride salts 17–24 by bubbling
excess HCl(g) through the ethanolic solution.
8-Amino-substituted pyrido[1,2-a]benzimidazole 25 was
synthesized by reducing the 8-nitro precursor 10 which
was isolated from a regioisomer mixture via column chro-
matography. All substituted pyrido[1,2-a]benzimidazoles
17–24 were prepared as a mixture of two regioisomers. The
structures of all pyrido[1,2-a]benzimidazoles 17–24 were
confirmed by 1H and 13C NMR spectroscopy, and mass spec-
trometry.
NMR analysis was based on the values of H-H coupling
constantsandchemicalshiftsinthe1Hand13C NMRspectra.
Antiproliferative activity
Antiproliferative activities of the pyrido[1,2-a]benzimida-
zole derivatives 17–26 were assessed against four human
tumour cell lines: cervical carcinoma (HeLa), colorec-
tal metastatic adenocarcinoma (SW620), breast metastatic
epithelial adenocarcinoma (MCF-7), hepatocellular carci-
noma (HepG2) cell lines, and against normal diploid human
fibroblasts (BJ). Cisplatin and 5-fluorouracil were used as
positive controls and the results for the most active com-
pounds 19, 23–26 are summarized in Table 1.
Compounds 19, 25 and 26 exhibited antiproliferative
activity against three cancer cell lines (MCF7, SW620 and
HeLa), with IC50 values of 6–43 μM and no activity towards
HepG2 cells. The pyrido[1,2-a]benzimidazole 23, bearing
a nitro group at position 3 and an N,N-dimethylamino-
1-propyl carboxamide moiety at position 7(8), exhibited
moderate cytotoxicity towards two cell lines: SW620 and
HeLa (IC50 = 28 and 36 μM, respectively). The most potent
and selective growth inhibitory activity observed in the series
was with compound 25 against MCF-7 and HeLa cancer cell
lines (IC50 = 6 μM), with a comparable inhibitory value to
5-fluorouracil and cisplatin.
Based on previous knowledge, and considering the pro-
mising anticancer potential of fused benzimidazole deriva-
tives, herein we present the design and synthesis of novel
amino and amido-substituted pyrido[1,2-a]benzimidazoles
via a key catalytic amidation reaction and their antiprolifer-
ative and antioxidative biological profiles.
Results and discussion
Chemistry
All novel pyrido[1,2-a]benzimidazoles 9–26 were prepared
according to the experimental procedure shown in Scheme 1
startingfrom4-substituted-1,2-phenylenediamines. Themain
precursors for the synthesis of pyrido[1,2-a]benzimidazoles,
All other analogues 17, 18, 20–22 and 24 did not show
any effect on tested tumour cell lines (IC50 > 100 μM).
123

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Scheme 1 Synthesis of
pyrido[1,2-a]benzimidazoles
NH2
NH2
CN
N
180 °C
CNCH2CONH2
N
H
R
R
1 R=H
5
6
7
8
R=H
2 R=NO2
3 R=Cl
R=NO2
R=Cl
4 R=CN
R=CN
acetyl-acetone
HClO4,
EtOHabs.
reflux
COOH
CH3
CN
N
N
H2SO4/H2O
reflux
R
R
N
N
CH3
H3C
H3C
13 R=H (93%)
9 R=H (59%)
10 R=NO2 (80%)
11 R=Cl (50%)
12 R=CN (56%)
14 R=NO2 (72%)
15 R=Cl (59%)
.
SnCl2 2H2O,
MeOH
16 R=COOH (14%)
1. DMF, 1,1'-CDI
NH2R1
2. EtOHabs.; HClgas
CN
N
H2N
N
CH3
O
H3C
C NHR1
N
25 (96%)
.
HCl
R
N
CH3
EtOHabs.; HClgas
H3C
CN
N
-Cl+H3N
17 R=H, R1=CH3 (30%)
18 R=H, R1=(CH2)3CH3 (71%)
19 R=H, R1=(CH2)3N(CH3)2 (93%)
20 R=NO2, R1=CH3 (54%)
N
CH3
H3C
21 R=NO2, R1=(CH2)3CH3 (25%)
,
22 R=NO2, R1=(CH2)3N(CH3)2 (50%)
23 R=Cl, R1=(CH2)3N(CH3)2 (51%)
26 (73%)
24 R=CONH(CH2)3N(CH3)2, R1=(CH2)3N(CH3)2 (67%)
Table 1 In vitro anticancer activity (aIC50) of selected pyrido[1,2-
a]benzimidazole derivatives
rable to the two standards used in this study. By exploring
the variation in selectivity of the compounds towards the
four tumour cell lines used in our work, it was revealed
that variations of substituents at positions 3 and 7(8) of
the pyrido[1,2-a]benzimidazole derivatives led to different
cytotoxic activities. Structure–activity relationships (SAR)
demonstrated that compounds with hydrogen or nitro sub-
stituent at position 7(8), and a methyl or butyl carboxamide
group at position 3 of the pyrido[1,2-a]benzimidazole skele-
ton did not exhibit any activity against the tumour cell lines.
Placing a N,N-dimethylamino-1-propyl carboxamide group
at position 3 of unsubstituted or 7(8)-chloro-substituted
derivative exerted moderate activity towards three tumour
cell lines (MCF-7, SW620 and HeLa). However, the pres-
ence of a nitro group at the 7(8)-position was detrimental
for antiproliferative activity. The best observed antiprolif-
erative activity was for compound 25 with an amino and
Comp.
ICa50 (μM)
MCF-7
HepG2
SW620
HeLa
BJ
19
43
>100
>100
>100
>100
2.41
35
34
26
23
>100
6
28
36
22
25
27
8
35
26
41
24
34
26
Cisplatin
12.61
42.0
4.01
3.4
0.35
10.3
0.22
18.5
5-FU
>100
a
IC50 values are the concentrations that cause 50% inhibition of cancer
cell growth (μM)
We also investigated the effect of these compounds on nor-
mal fibroblasts (BJ) proliferation and found they exhibited
IC50 values in the range of 21–100 μM which are compa-
123

Mol Divers
cyano group at positions 7(8) and 3, respectively. Interest-
ingly, the hydrochloride salt of 25 (26) showed a decrease
in antiproliferative activity and selectivity compared to the
salt-free precursor 25. This could be probably addressed to
the pKa of the aniline NH2 group which is known to be 9.2.
Therefore, we could assume that at physiological pH of the
biological assay medium, the aniline NH2 group of com-
pound 26 is still protonated. In addition, the presence of two
electron-donating methyl groups and one electron-accepting
cyano group probably does not have a significant influence
on the pKa value of the aniline NH2 group. The presence
of a positive charge in compound 26 is unfavourable to enter
the cell membrane which results in a weaker antiproliferative
activity.
Table 2 cLogP values of compounds 17–26
Comp
clogPa
17
18
19
20
21
22
23
24
R=H, R1=CONHCH3 × HCl
2.88
4.47
3.57
2.74
4.33
3.43
4.28
3.47
R=H, R1=CONH(CH2)3CH3 × HCl
R=H, R1=CONH(CH2)3N(CH3)2 × HCl
R=NO2, R1=CONHCH3 × HCl
R=NO2, R1=CONH(CH2)3CH3 × HCl
R=NO2, R1=CONH(CH2)3N(CH3)2 × HCl
R=Cl, R1=CONH(CH2)3N(CH3)2 × HCl
R=CONH(CH2)3N(CH3)2,
Nevertheless, the observed SAR among the evaluated
compounds suggests that an increase of antiproliferative
activity is due to the placement of an electron-withdrawing
group at the 3-position and an electron-donating substituent
at 7(8)-position of the pyrido[1,2-a]benzimidazole scaffold.
Hence, the targeted compounds could efficiently enter the
cell and act as radical scavengers. Overall, the most potent
and selective growth inhibitory activity was observed in the
micromolar range against the MCF-7 and HeLa cancer cell
lines, with compound 25 being most potent (IC50 = 6 μM),
comparable to the standards, 5-fluorouracil and cisplatin.
It is accepted that adequate lipophilicity is necessary for
compounds to penetrate and cross the cell membrane. LogP is
defined as 1-octanol/water partition coefficient and is indica-
tive for lipophilicity or hydrophilicity of novel compounds.
The calculated LogP values, also known as clogP values,
were obtained using ChemDraw Ultra 10.0 [24]. The clogP
values for the neutral form of the prepared compounds are
presented in Table 2. The cLogP values of compounds 17–26
increased with the length of the alkyl groups on position R1
resulting in reduced or no antiproliferative activity for com-
pounds 17, 18, 20 and 21. Increasing further the length of
the alkyl chain by N-dimethylamino group led to a decrease
in cLogP values and enhancement of antiproliferative activ-
ity. From the results, it is clear that despite of increasing the
clogP of the compounds, the enhancement of their antimi-
crobial activities is not consistent indicating there is a poor
correlation between the clogP values and antiproliferative
activity of these compounds.
R1=CONH(CH2)3N(CH3)2
25
26
R=NH2, R1=CN
3.07
3.07
R=NH2x HCl, R1=CN
a Calculated as neutral species
The radical scavenging capacity of final compounds was
measured against DPPH and ABTS stable radicals as well
as ferric reducing ability/antioxidant power (FRAP) abil-
ity to reduce ferric to ferrous ion. The radical scavenging
method using DPPH stable radical indicates the ability of
tested species to donate proton/electron. In particular, this is
a measure of reducing ability in iron-free medium [26,27].
The DPPH final concentration of the test compounds was
100 μM and results were read after 30 min (Table 3). The
results obtained in this study were expressed as IC50 val-
ues with the exception of 19, 20, 21, 23 and 25 which did
not react under the experimental condition. Compounds 17,
18 and 22 showed poor reducing ability expressed as a very
high concentration of IC50 (410 and 450 μM, respectively).
The best activity was observed for compound 24 (100 μM)
in comparison to BHT (IC50 25
4.2 μM). Regarding the
substitution pattern of pyrido[1,2-a]benzimidazole scaffold,
the best activity was observed for compound bearing N,N-
dimethylamino-1-propyl carboxamide group at positions 3
and 7(8). The different alkyl substitutions of carboxamide
at 3-position of heterocycle scaffold exhibited poor reducing
ability (high IC50). The expansion of alkyl chain from 1 to
4 carbon atoms of carboxamide at 3-position in combination
with EWG substituent at 7(8)-position showed no activity at
all.
Compound 25 bearing a nitrile group at the 3-position
and an amino group at 7(8)-position of the pyrido[1,2-
a]benzimidazole scaffold did not show any reduction in
activity under the experimental condition, while its hydro-
chloride salt 26 exhibited limited activity (IC50 = 262 μM),
about 10× higher than BHT. The radical scavenging activ-
ity of all other compounds was very week in comparison to
BHT.
Thus, this suggests that the observed antiproliferative
activity could be explained by the fact that protonated species
were unfavourable for being delivered through the cell mem-
brane.
Antioxidative activity
Antioxidative activity could be estimated by different in vitro
and in vivo biological assays. In vitro assays rely on spec-
trophotometric measurements [25].
123

Mol Divers
Table 3 Antioxidant activity of
compounds 13–21 and 27 by
DPPH, FRAP and ABTS
methods
Comp
DPPH IC50 μM
FRAP mmolFe2+/mmolC
ABTS mmolTEAC/mmolC
17
>300
>300
No
8.54 0.34
8.64 0.95
41.36 4.75
55.01 15.54
52.60 2.73
19.52 3.62
23.02 2.99
92.97 1.44
177.59 1.47
176.40 5.82
754.09 0.71
680.88 1.69
679.15 37.48
18
19
48.15 1.43
78.48 6.91
57.79 14.28
5.45 0.06
20
No
21
No
22
>300
No
23
21.64 1.17
23.69 1.25
4407.21 93.46
4838.34 105.81
2089.34 55.98
24
100 40
No
25
26
261 30
25 4.2
BHT
no no activity under the experimental condition
The reducing power of tested compounds refers to
their antioxidant activity. The ferric reducing ability of
plasma (FRAP) assay [28,29] was used to determine the
reducing power of novel compounds and the results are
presented in Table 3. The FRAP values showed that com-
25 as an attractive lead compound for further optimization
and development against cancer.
Materials and methods
Synthesis
pounds 25 and 26 (4407.21
93.46 and 4838.34
105.81 mmolFe2+/mmolC, respectively) have notable rad-
ical scavenging activity, while the rest of the compounds
showed very low activity (8–78 mmolFe2+/mmolC). It
should to be emphasized that compounds 25 and 26 showed
the highest level of scavenging in comparison to BHT.
General methods
All chemicals and solvents were purchased from commercial
suppliers Aldrich and Acros. Chemicals were used with-
out further purification, while solvents were distilled from
appropriate drying agents. Melting points were determined
by using SMP11 Bibby and Büchi 535 apparatus and were
Conclusions
1
uncorrected. The H and 13C NMR spectra were recorded
In this manuscript, we present the synthesis, antiprolifera-
tive and antioxidative activities of novel amido-substituted
pyrido[1,2-a]benzimidazoleswithdifferenttypesandlengths
of amide side chains placed on the tricyclic skeleton. The
most pronounced antiproliferative effect in the micromo-
lar range among all newly synthesized derivatives was
observed for compound 25 on MCF-7 (IC50 = 6μM)
and HeLa (IC50 = 8μM) cell lines. Additionally, N,N-
dimethylamino-1-propyl carboxamide group in the unsubsti-
tuted or 7(8)-chloro-substituted derivative slightly enhanced
the antiproliferative activity unlike the nitro group that
decreased antiproliferative activity. On the other hand, proto-
nated amino derivative 26 showed decrease in the antiprolif-
erative activity in comparison to 25 probably due to reduced
cell membrane penetration.
on a Varian Gemini 300 or Varian Gemini 600 at 300, 600
and 150 and 75 MHz, respectively, in DMSO-d6 solutions.
Chemical shifts in ppm ( ) are in reference to TMS (s =
δ
singlet, d = doublet, t = triplet, q = quartet and m = mul-
tiplet). TLC was carried out on Merck silica gel 60F-254
glass plates. Elemental analysis obtained on a Perkin-Elmer
2400 elemental analyser and the obtained analytical results
are within 0.4% of the theoretical value.
General method for preparation of amido-substituted
pyrido[1,2-a]benzimidazoles hydrochloride salts 17–24
A suspension containing a pyrido[1,2-a]benzimidazolecar-
boxylic acid (13–16) in anhydrous DMF (2–7 mL) was
stirred for several minutes at room temperature and the 1,1ꢀ-
carbonyldiimidazole was added. After stirring for 1.5 h at
room temperature, an amine was added and the reaction mix-
ture was stirred at room temperature overnight. The reaction
mixture was evaporated to reach a volume of 1–2 mL and the
precipitate was filtered off and washed with small amount
A biological evaluation concerning antioxidant activity of
novel pyrido[1,2-a]benzimidazoles indicates that the com-
pounds 25 and 26 have significant reducing power and
promisingradicalscavengingactivityintheFRAPandABTS
assays with comparable activity to the reference compound
BHT. All these results position pyrido[1,2-a]benzimidazole
123

Mol Divers
of water (2 mL). The product was dried and suspended in
absolute ethanol (5 mL), saturated with the gaseous HCl and
stirred for 24 h at room temperature. After the filtration, the
product was washed with small amount of diethylether (10
mL) to obtain hydrochloride salt.
afforded desired hydrochloride salt (0.275 g, 93%) as grey
powdered; mp 275–279 ◦C; 1H NMR (DMSO-d6, 600 MHz)
10.20 (bs, 1H), 9.16 (t, 1H, J = 5.8 Hz), 8.50 (d, 1H,
δ
J = 8.4 Hz), 7.95 (d, 1H, J = 8.3 Hz), 7.77 (t, 1H,
J = 8.8 Hz), 7.61 (t, 1H, J = 8.85 Hz), 7.35 (s, 1H), 3.17
(q, 2H, J = 6.1 Hz), 3.05–2.98 (m, 4H), 2.78 (s, 6H), 2.22
(s, 3H), 1.98 (s, 3H); 13C NMR (DMSO-d , 150 MHz)
N-methyl-1,3-dimethylpyrido[1,2-a]benzimidazole-4-car-
δ
6
boxamide hydrochloride 17
163.1, 147.8, 143.0, 142.6, 141.9, 128.8, 128.2, 124.1, 119.5,
118.4, 117.7, 114.8, 54.9, 42.2 (2C), 37.2, 24.3, 21.1, 20.2;
From 13 (0.40 g, 1.60 mmol), 1,1ꢀ-carbonyldiimidazole (0.28
g, 1.70 mmol) and a 30% solution of methylamine in water
(0.17 mL, 1.60 mmol) were added in DMF (4 mL) to obtain
0.061 g (15%) of crude amide (grey powder) which in turn
afforded desired hydrochloride salt (0.19 g, 30%) as grey
powdered;mp232–235 ◦C;1H NMR (DMSO-d6, 600 MHz)
UV (EtOH) λmax: 350, 250, 245; IR (diamond) ( /cm−1):
ν
3567, 3356, 2700, 1670, 1639; Anal. (C19H25ClN4O) Calc.
C 63.24, H 6.98, N 15.53%; Found C 62.98, H 6.80, N
15.38%.
N-methyl-1,3-dimethyl-7(8)-nitropyrido[1,2-a]benzimida-
zole-4-carboxamide hydrochloride 20
8.98 (d, 1H, J = 8.8 Hz), 8.70 (bs, 1H), 8.53 (t, 2H,
δ
J = 8.4 Hz), 7.95 (d, 1H, J = 8.0 Hz), 7.85 (t, 2H,
J = 8.4 Hz), 7.70 (d, 1H, J = 8.0 Hz), 7.41 (s, 1H),
3.18 (s, 3H, CH3), 2.93 (d, 3H, J = 4.6 Hz, CH3), 2.57
From 14 (0.40 g,1.40 mmol), 1,1ꢀ-carbonyldiimidazole (0.28
g,1.70 mmol) and a 30% solution of methylamine in water
(0.17 mL,1.60 mmol) were added in DMF (2.5 mL) to obtain
0.29 g (69%) of crude amide (grey powder) which in turn
afforded desired hydrochloride salt (0.155 g, 54%) as light
yellow powdered; mp 265–267 ◦C; 1H NMR (DMSO-d6,
(s, 3H, CH3); 13C NMR (DMSO-d6, 150 MHz) 162.4,
δ
149.6, 148.7, 146.4, 135.1, 128.7, 122.3, 121.8, 118.7, 117.9,
117.5, 96.1, 23.1, 21.9; UV (EtOH) λmax: 339, 250, 243; IR
(diamond) ( /cm−1): 3394, 3206, 3039, 2573, 1713, 1662,
ν
1646, 1540; Anal. (C15H16ClN3O) Calc. C 62.18, H 5.57, N
600 MHz) 90.20 (bs, 1H), 9.16 (bs, 1H), 9.05 (bs, 1H), 8.92
δ
14.50%; Found C 62.04, H 5.69, N 14.73%.
(bs, 1H), 8.85 (s, 1H), 8.76 (s, 1H), 8.46 (d, 1H, J = 8.4 Hz),
8.38 (d, 1H, J = 8.5 Hz), 8.14 (d, 1H, J = 8.5 Hz), 7.94 (d,
1H, J = 8.7 Hz), 6.88 (s, 1H), 6.87 (s, 1H), 3.19 (s, 3H), 2.99
(d, 3H, J = 4.6 Hz), 2.54 (s, 3H); 13C NMR (DMSO-d6,
N-butyl-1,3-dimethylpyrido[1,2-a]benzimidazole-4-car-
boxamide hydrochloride 18
150 MHz) 164.5, 164.4, 159.7, 159.6, 150.9, 150.1, 149.4,
δ
From 13 (0.40 g, 1.60 mmol), 1,1ꢀ-carbonyldiimidazole
(0.28 g (1.70 mmol) and a 30% solution of n-buthylamine
in water (0.16 mL, 1.60 mmol) were added in DMF (4
mL) to obtain 0.035 g (7%) of crude amide (grey pow-
der) which in turn afforded desired hydrochloride salt (0.024
g, 71%) as grey powdered; mp 239–243 ◦C; 1H NMR
(DMSO-d , 600 MHz) 8.97(t, 1H, J = 4.8 Hz), 8.80 (bs,
149.1, 144.2, 144.0, 140.9, 140.6, 140.3, 139.5, 132.2, 128.9,
121.1, 120.8, 118.9, 117.1, 116.8, 115.3, 114.5, 113.00, 44.5,
44.1, 31.8, 30.9, 21.1, 20.2; UV (EtOH) λmax: 385, 293, 276,
237; IR (diamond) ( /cm−1): 3220, 3054, 2918, 1662, 1504;
ν
Anal. (C15H15ClN4O3) Calc. C 63.24, H 6.98, N 15.53%;
Found C 62.98, H 6.80, N 15.38%.
δ
6
1H), 8.51 (d, 1H, J = 8.5 Hz), 7.93 (d, 1H, J = 8.4 Hz),
7.78 (t, 1H, J = 7.8 Hz), 7.61 (t, 1H, J = 8.0 Hz), 7.37 (s,
1H), 3.42 (q, 2H), 3.15 (s, 3H), 2.57 (s, 3H), 1.57 (m, 2H),
1.39 (m, 2H), 0.94 (s, 3H); 13C NMR (DMSO-d6, 150 MHz)
N-butyl-1,3-dimethyl-7(8)-nitropyrido[1,2-a]benzimida-
zole-4-carboxamide hydrochloride 21
From 14 (0.40 g,1.40 mmol), 1,1ꢀ-carbonyldiimidazole (0.28
g, 1.70 mmol) and a 30% solution of n-buthylamine in water
(0.17 mL, 1.60 mmol) were added in DMF (2.5 mL) to obtain
0.153 g (32%) of crude amide (grey powder) which in turn
afforded desired hydrochloride salt (0.045 g, 25%) as light
yellow powdered; mp 214–217 ◦C; 1H NMR (DMSO-d6,
164.8, 147.6, 143.9, 140.8, 139.7, 129.3, 125.2, 120.5,
δ
119.0, 118.5, 115.4, 114.8, 56.3, 44.6, 36.8, 264, 20.4, 20.1;
UV (EtOH) λmax: 343, 250, 244; IR (diamond) ( /cm−1):
3492, 3197, 2941, 2606, 1526; Anal. (C18H22ClN3O) Calc.
C 65.15, H 6.68, N 12.66%; Found C 65.02, H 6.80, N
12.87%.
ν
600 MHz) 14a: 9.30 (bs, 1H), 9.24 (bs, 1H), 9.03 (t, 1H,
δ
J = 2.3 Hz), 9.02 (t, 1H, J = 2.4 Hz), 8.89 (bs, 1H), 8.58
(d, 1H, J = 2.2 Hz), 8.44 (d, 1H, J = 9.2 Hz), 8.35 (dd,
1H, J1 = 9.1 Hz, J2 = 2.1 Hz), 8.11 (dd, 1H, J1 = 9.1 Hz,
J2 = 2.3 Hz), 7.88 (d, 1H, J = 9.1 Hz), 6.93 (s, 1H), 6.83
(s, 1H), 3.41–3.34 (m, 2H), 3.33–3.30 (m, 2H), 3.10 (s, 3H),
3.04 (s, 3H), 2.63 (s, 3H), 2.54 (s, 3H), 1.63–1.56 (m, 2H),
1.53–1.48 (m, 2H), 1.49–1.42 (m, 2H), 1.41–1.32 (m, 2H),
0.97(t, 3H, J = 7.3 Hz), 0.96(t, 3H, J = 7.3 Hz);13C NMR
N-(3-N,N-dimethylamino-1-propyl)-1,3-dimethylpyrido[1,
2-a]benzimidazole-4-carboxamide hydrochloride 19
From 13 (0.50 g, 2.10 mmol), 1,1ꢀ-carbonyldiimidazole (0.37
g, 2.25 mmol) and 3-N,N-dimethylamino-1-propylamine
(0.29 mL, 2.30 mmol) were added in DMF (7 mL) to obtain
0.262 g (40%) of crude amide (grey powder) which in turn
123

Mol Divers
(DMSO-d , 150 MHz) 164.3, 164.0, 159.9, 159.4, 151.4,
142.1, 141.9, 133.2, 132.9, 125.0, 124.6, 119.6, 119.3, 118.1,
117.7, 114.3, 113.4, 41.7 (2C, q), 38.0, 37.7, 33.5, 33.1,
29.1, 28.7, 27.6, 27.2, 24.1, 23.7 (q); UV (EtOH) λmax: 352,
δ
6
151.1, 149.8, 149.6, 145.0, 144.8, 141.2, 140.9, 140.7, 139.9,
131.0, 129.9, 122.0, 121.7, 118.6, 118.1, 117.0, 116.5, 114.6,
113.1, 36.4, 35.7, 32.7, 31.9, 31.6, 30.8, 28.5, 28.2, 24.4, 24.2,
21.4, 20.8; UV (EtOH) λmax: 390, 296, 238; IR (diamond)
303, 281, 273, 254, 247, 219; IR (diamond) ( /cm−1): 3214,
ν
2989, 1656, 1649; Anal. (C19H24Cl2N4O) Calc. C 57.73, H
6.12, N 14.17%; Found C 57.90, H 5.98, N 14.05%.
( /cm−1): 3197, 2964, 1653, 1616; Anal. (C18H21ClN4O3)
ν
Calc. C 57.37, H 5.62, N 14.87%; Found C 57.55, H 5.80,
N 15.05%.
4,7(8)-N,N-di(3-N,N-dimethylamino-1-propyl)-1,3-dime-
thylpyrido[1,2-a]benzimidazole-4-carboxamide dihydro-
chloride 24
N-(3-N,N-dimethylamino-1-propyl)-1,3-dimethyl-7(8)–
nitropyrido[1,2-a]benzimidazole-4-carboxamide
chloride 22
hydro-
From 16 (0.05 g, 0.17 mmol), 1,1ꢀ-carbonyldiimidazole (0.07
g, 0.37 mmol) and 3-N,N-dimethylamino-1-propylamine
(0.15 mL, 0.74 mmol) to obtain 0.09 g of yellow oil which
in turn afforded desired hydrochloride salt (0.07 g, 67%) as
yellow oil; 1H NMR (DMSO-d , 600 MHz) 9.85 (bs, 2H),
From 14 (0.80 g, 0.70 mmol), 1,1ꢀ-carbonyldiimidazole (0.15
g, 0.77 mmol) and 3-N,N-dimethylamino-1-propylamine
(0.10 ml, 0.77 mmol) were added in DMF (3.6 mL) to obtain
0.020 g (20%) of crude amide (yellow powder) which in turn
afforded desired hydrochloride salt (0.011 g, 50%) as yellow
powdered; mp 209–211 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ
6
9.80 (bs, 2H), 9.31 (bs, 2H), 9.10 (d, 1H, J = 9.0 Hz), 9.00
(d, 1H, J = 8.9 Hz), 8.16 (bs, 4H), 7.80 (s, 1H), 7.78 (s, 1H),
7.68 (d, 1H, J = 8.8 Hz), 7.62 (d, 1H, J = 8.8 Hz), 3.42 (bs,
24H), 3.19 (s, 3H), 3.15 (s, 3H), 2.90 (q, 8H, J = 6.4 Hz),
2.79–2.74 (m, 8H), 2.74 (s, 3H), 2.70 (s, 3H), 2.16–1.97 (m,
8H);UV(EtOH) λmax:345, 258, 219, 216, 210;IR(diamond)
9.46 (bs, 1H), 9.40 (bs, 1H), 9.14 (t, 1H, J = 2.2 Hz),
δ
9.08 (t, 1H, J = 2.3 Hz), 8.40 (dd, 1H, J1 = 9.1 Hz,
J2 = 2.2 Hz), 8.34 (dd, 1H, J1 = 9.1 Hz, J2 = 2.2 Hz),
7.91 (d, 1H, J = 9.0 Hz), 7.83 (d, 1H, J = 9.0 Hz), 7.62
(s, 1H), 7.57 (s, 1H), 6.99 (s, 1H), 6.93 (s, 1H), 3.39 (s,
6H), 3.34 (s, 6H), 3.30 (s, 3H), 3.22 (s, 3H), 2.92 (q, 2H,
J = 6.1 Hz), 2.88 (q, 2H, J = 6.0 Hz), 2.73–2.66 (m, 4H),
2.66 (s, 3H), 2.60 (s, 3H), 1.24–1.19 (m, 4H); 13C NMR
( /cm−1): 3202, 2995, 1666, 1660; Anal. (C25H38Cl2N6O2)
ν
Calc. C 57.14, H 7.29, N 15.99%; Found C 56.98, H 7.44,
N 15.70%.
8-Amino-1,3-dimethylpyrido[1,2-a]benzimidazole-4-car-
bonitrile 25
(DMSO-d , 150 MHz) 166.2, 166.1, 162.4, 162.0, 153.3,
δ
6
153.0, 150.7, 150.4, 147.3, 146.8, 143.0, 142.6, 141.6, 140.6,
133.0, 132.4, 125.2, 124.7, 119.8, 119.1, 118.3, 117.8, 114.9,
113.9, 41.8 (2C), 37.6, 36.7, 33.8, 33.2, 28.9, 28.3, 27.9, 27.5,
24.2, 23.8 (q); UV (EtOH) λmax: 389, 292, 237; IR (diamond)
From 10 (0.20 g, 0.75 mmol) and solution of SnCl2 × 2H2O
(1.40 g, 0.00585 mmol) in MeOH (2.2 mL) and concentrated
HCl (2.2 mL) after stirring at refluxed for 0.5 h. After cooling,
the reaction mixture was evaporated under vacuum and dis-
solved in water (25 mL). The resulting solution was treated
with 20% NaOH to pH = 14. Resulting product was filtered
off and washed with water (10 mL) to obtain 0.17g (96%) of
( /cm−1): 3209, 2988, 1664, 1646; Anal. (C19H24ClN5O3)
ν
Calc. C 56.22, H 5.96, N 17.25%; Found C 56.50, H 5.82,
N 17.09%.
1
crude product (yellow powder); mp 285–286 ◦C; H NMR
N-(3-N,N-dimethylamino-1-propyl)-7(8)-chloro-1,3-dime-
thylpyrido[1,2-a]benzimidazole-4-carboxamide hydrochlo-
ride 23
(DMSO-d , 600 MHz) 7.48 (d, 1H, J = 1.9 Hz), 7.42 (d,
δ
6
1H, J = 1.8 Hz), 7.00 (dd, 1H, J1 = 8.7 Hz, J2 = 1.9 Hz),
6.92 (dd, 1H, J1 = 8.7 Hz, J2 = 1.9 Hz), 6.70 (s, 1H),
6.66 (s, 1H), 5.25 (s, 2H), 5.20 (s, 2H), 2.95 (s, 3H), 2.90 (s,
3H), 2.55 (s, 3H) 2.53 (s, 3H); 13C NMR (DMSO-d6, 150
From 15 (0.40 g, 0.72 mmol), 1,1ꢀ-carbonyldiimidazole (0.14
g, 0.72 mmol) and 3-N,N-dimethylamino-1-propylamine
(0.16 mL, 0.98 mmol) were added in DMF (7.5 mL) to obtain
0.20 g (80%) of yellow oil which in turn afforded desired
MHz) 146.9, 146.93, 144.9, 144.7, 144.4, 144.3, 143.6,
δ
143.4, 136.5, 136.3, 130.7, 130.5, 123.4, 123.1, 119.6, 119.2,
115.7, 115.6, 115.4, 115.2, 111.9, 111.7, 98.0, 97.8, 20.6,
20.4, 20.3, 20.0 (q); UV (EtOH) λmax: 420, 312, 271, 219;
Anal. (C14H12N4) Calc. C 71.17, H 5.12, N 23.71%; Found
C 71.40, H 5.02, N 23.90%.
hydrochloride salt (0.11 g, 51%) as yellow powdered; mp
1
239–241 ◦C; H NMR (DMSO-d6, 600 MHz) 9.91 (bs,
δ
2H), 9.25 (t, 1H, J = 2.4 Hz), 9.12 (t, 1H, J = 2.3 Hz),
8.54 (s, 1H), 8.47 (s, 1H), 8.38 (d, 1H, J = 8.3 Hz), 8.30
(d, 1H, J = 8.4 Hz), 7.90 (d, 1H, J = 8.4 Hz), 7.71 (d, 1H,
J = 8.4 Hz), 7.10 (s, 1H),7.06 (bs, 1H), 3.68 (s, 6H), 3.64 (s,
6H), 2.64 (s, 6H), 3.50–3.43 (t, 4H, J = 6.9 Hz), 3.40–3.22
(m, 4H), 2.83(s, 3H), 2.81(s, 3H), 2.54(s, 6H), 2.03–1.97(m,
4H); 13C NMR (DMSO-d , 150 MHz) 165.6, 165.1, 162.1,
8-Amino-1,3-dimethylpyrido[1,2-a]benzimidazole-4-car-
bonitrile hydrochloride 26
A suspension of 25 (0.05 g, 0.0021 mmol) in absolute
δ
6
161.5, 153.8, 153.3, 150.5, 150.0, 147.1, 146.5, 143.4, 143.0,
ethanol (15 mL) was saturated with HCl(g). After 24 hours
123

Mol Divers
of stirring, small amount of diethylether (5 mL) was added,
and the resulting product was filtered off and washed with
diethylether (10 mL) to obtain 0.06 g (73%) of crude prod-
tion[25,26]. Briefly, tosolutionofDPPH(finalconcentration
100 μM) in absolute ethanol was added equal volume of
tested compounds of various concentrations dissolved in
DMSO. The assay was carried out in a 96-well microtitre
plate. Ethanol and DMSO were used as control solution.
Sample blank was also performed. After 30 min in dark at
room temperature, the absorbance was recorded at 517 nm on
microplate reader μQuant (Biotec Inc.). All measures were
done in triplicate and the results presented in Table 3 were
averaged. The percentage scavenging (PS) of test samples at
each concentration was calculated using the following for-
mula:
1
uct (yellow powder); mp > 250 ◦C; H NMR (DMSO-d6,
600 MHz) 10.42 (bs, 3H), 10.40 (bs, 3H), 8.47 (d, 1H,
δ
J = 1.7 Hz), 8.45 (d, 1H, J = 1.6 Hz), 8.04 (d, 1H, J =
8.8 Hz), 8.00 (d, 1H, J=8.7 Hz), 7.59 (dd, 1H, J1 = 8.7 Hz,
J2 = 1.9 Hz), 7.57 (dd, 1H, J1 = 8.7 Hz, J2 = 1.9 Hz),
7.03 (s, 1H), 7.00 (s, 1H), 3.07 (s, 3H), 3.05 (s, 3H), 2.66 (s,
3H), 2.64 (s, 3H); 13C NMR (DMSO-d , 150 MHz) 150.7,
δ
6
150.4, 148.0, 147.6, 144.6, 144.5, 142.1, 141.9, 140.1, 139.9,
129.0, 128.7, 121.5, 121.1, 119.3, 119.0, 114.7, 114.6, 114.0,
113.7, 110.9, 110.6, 96.4, 96.2, 21.0, 20.6, 20.5, 20.4 (q); UV
(EtOH) λmax: 407, 271, 218; Anal. (C14H13ClN4) Calc. C
61.65, H 4.80, N 20.54%; Found C 61.75, H 4.64, N 20.70%.
[(Acontrol − Acompound)/Acontrol] × 100
The IC50 values for each compound were calculated from
dose–response curves using linear regression analysis by
using GraphPad Prism 6 program.
Antiproliferative activity
Cell culturing
Determinationofferricreducing/antioxidantpower(FRAP
assay)
The experiments were carried out on four human cell lines
HeLa (cervical carcinoma), SW620 (colorectal adenocar-
cinoma, metastatic), MCF-7 (breast epithelial adenocarci-
noma, metastatic), HepG2 (hepatocellular carcinoma); and
BJ (normal diploid human fibroblasts) which were cultured
as monolayers. The cell lines were maintained in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10%
foetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL
penicillin and 100 μg/mL streptomycin in a humidified
atmosphere with 5 % CO2 at 37 ◦C.
The FRAP method was conducted according to the previ-
ously reported procedure [28] with minor modifications for
assay on a 96-well microplate. The antioxidant capacity of
the tested compounds was estimated as their power to reduce
the TPTZ-Fe(III) complex to TPTZ-Fe(II) complex, which
is simple, fast and reproducible. A solution of 10 mM TPTZ
and 20 mM ferric chloride was diluted in 300 mM sodium
acetate buffer (pH 3.6) at a ratio of 1:1:10. The tested com-
pound solution (20 μL) was added to the 96-well microplate
followed by working FRAP solution (280 μL). The mix-
ture was shaken and incubated 30 min at 37 ◦C in the dark.
Finalconcentrationoftestedcompoundswas(0.01mM). The
absorbance at 593 nm was recorded using microplate reader
μQuant (Biotec Inc.). For FRAP assay, ferrous sulphate
(FeSO4 × 7H2O) was used to develop a 20–2000 μmol/L
standard curve.
Proliferation assays
The panel cell lines were inoculated onto a series of stan-
dard 96-well microtiter plates on day 0, at 5000 cells
per well according to the doubling times of specific cell
line. Test compounds and control compounds (cisplatin and
5-fluorouracile, 5_FU) were then added in five 10-fold dilu-
tions (0.01–100 μM) followed by a 72-h incubation. The
experiments were implemented according to the standard
previously published experimental procedure [30].
All results were then expressed as Fe2+ equivalents
(Fe2+ μmol). All tests were done in triplicate and the results
were averaged and presented in Table 2.
Antioxidative activity
Free radical scavenging ability by the use of a stable
ABTS radical cation (2,20 azinobis-(3-ethylbenzthiazoline-
6-sulphonic acid)
Each in vitro experiment was performed at least in tripli-
cate, the results were averaged and the standard deviation of
absorbance was less than 10% of the mean, and the results
are presented in Table 3.
The total antioxidant activity (TEAC) method [29–31] was
modifiedandadjustedformicrotiterplatereader. Forthestan-
dard TEAC assay, ABTS·+ was prepared by mixing an ABTS
stock solution (7 mM in water) with 2.45 mM potassium
persulfate. This mixture was allowed to stand for 12–16 h at
room temperature in the dark until reaching a stable oxidative
state. Onthedayofanalysis, theABTS·+ solutionwasdiluted
Determination of the reducing activity of the stable radical
1,1-diphenyl-picrylhydrazyl (DPPH)
The determination of reducing activity was measured accord-
ing to the previously reported procedure with some modifica-
123

Mol Divers
with PBS (pH 7.4) to an absorbance of 0.700
0.01 at 734
10. Refaat HM (2012) Synthesis of potential anticancer derivatives
of pyrido[1,2 a]benzimidazoles. Med Chem Res 21:1253–1260.
doi:10.1007/s00044-011-9636-y
nm. The radical was stable in this form for more than two
days when stored in the dark at room temperature. Standards
and solutions of tested compounds (10 μL) were mixed with
working ABTS·+ radical cation solution (200 μL) to each
well of the microplate, shake and incubate at room temper-
ature for 5 min. The decrease of absorbance at 734 nm was
recorded by μQuant (Biotec Inc.). Aqueous phosphate buffer
solution (without ABTS·+ solution) and Trolox (0.2–1.25
mmol/L) were used as a control and main calibrating stan-
dard, respectively. Results were expressed as average of three
independent measures as Trolox equivalents (mmol TEAC/
mmolC) and presented in Table 2.
11. Badawey E, Kappe T (1995) Benzimidazole condensed ring
system. IX. Potential antineoplastics. New synthesis of some
pyrido[1,2-a]benzimidazoles and related derivative. Eur J Med
Chem 30:327–332. doi:10.1016/0223-5234(96)88241-9
12. Maryanoff BE, Nortey SO, McNall JJ, Sanfilippo PJ, McCom-
sey DF, Dubinsky B, Shank RP, Reitz AB (1999) Poten-
tial anxiolytic agents. 3. Novel A-ring modified pyrido[1,2-
a]benzimidazoles. Bioorg Med Chem Lett 9:1547–1552. doi:10.
1016/S0960-894X(99)00240-1
13. Kotovskaya SK, Baskakova ZM, Charushin VN, Chupakhin
ON, Belanov EF, Bormotov NI, Balakhnin SM, Serova OA
(2005) Synthesis and antiviral activity of fluorinated pyrido[1,2-
a]benzimidazoles. Pharm Chem J 39:574–578. doi:10.1007/
s11094-006-0023-9
14. Reitz AB, Gauthier DA, Ho W, Maryanoff BE (2000) Tau-
tomerism and physical properties of pyrido[1,2-a]benzimidazole
(PBI) GABA-A receptor ligands. Tetrahedron 56:8809–8812.
doi:10.1016/S0040-4020(00)00874-7
15. Jordan AD, Vaidya AH, Rosenthal DI, Dubinsky B, Kordik
CP, Sanfilippo PJ, Wu WN, Reitza AB (2002) Potential
anxiolytic agents. Part 4: novel orally-active N5-substituted
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Acknowledgments We greatly appreciate the financial support of
the Croatian Ministry of Science Education and Sports (Project 125-
0982464-1356), the Croatian Science Foundation under the project
5596 (Synthesis and cytostatic evaluations of novel nitrogen hetero-
cycles library) and University of Rijeka research Grant 13.11.1.1.11.
16. Ndakala AJ, Gessner RK, Gitari PW et al (2011) Antimalarial
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