One-pot microwave assisted synthesis under green chemistry conditions, antioxidant screening, and cytotoxicity assessments of benzimidazole Schiff bases and pyrimido[1,2-a]benzimidazol-3(4H)-ones

Article abstract of DOI:10.1016/j.ejmech.2010.11.018

The synthesis of a number of benzimidazole Schiff bases 3 and 3-oxo-pyrimido[1,2-a]benzimidazoles 4 in excellent yields by a one-step sequence from the reaction of 2-aminobenzimidazole under green chemistry conditions is described. Structural assignments

Full text of DOI:10.1016/j.ejmech.2010.11.018

European Journal of Medicinal Chemistry 46 (2011) 297e306
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
One-pot microwave assisted synthesis under green chemistry conditions,
antioxidant screening, and cytotoxicity assessments of benzimidazole
Schiff bases and pyrimido[1,2-a]benzimidazol-3(4H)-ones
,
Constantinos G. Neochoritis a, Tryfon Zarganes-Tzitzikas a, Constantinos A. Tsoleridis a
*
,
Julia Stephanidou-Stephanatou a , Christos A. Kontogiorgis , Dimitra J. Hadjipavlou-Litina ,
,
b
b
*
Theodora Choli-Papadopoulou c
a Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, University Campus, Thessaloniki 54124, Macedonia, Greece
b Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Macedonia, Greece
c Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Macedonia, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2010
Received in revised form
29 October 2010
Accepted 11 November 2010
Available online 19 November 2010
The synthesis of a number of benzimidazole Schiff bases 3 and 3-oxo-pyrimido[1,2-a]benzimidazoles 4 in
excellent yields by a one-step sequence from the reaction of 2-aminobenzimidazole under green chem-
istry conditions is described. Structural assignments of the new compounds as well as complete assign-
ment of 1H and 13C NMR signals have been unambiguously achieved based on the analysis of their 1H and
13C NMR (1D and 2D), IR, MS and elemental analysis data. To the synthesized Schiff bases the E-config-
uration was assigned on the basis of comparison of experimental and calculated (DFT) 13C NMR chemical
shifts. Compounds 3 and 4 were evaluated as inhibitors of lipoxygenase (LOX) and of lipid peroxidation
(LPO). All the tested derivatives showed inhibition of lipid peroxidation, whereas most of them were found
to have higher activation than the reference compound trolox; The Schiff bases 3e, 3h, and 3i, and the
pyrimidobenzimidazoles 4a, 4e and 4f were found to be the most potent. The most potent LOX inhibitor
within the subset of Schiff bases was found compound 3i, followed by 3f, whereas compounds 4a and 4g
were found the most potent of the 3-oxo-pyrimido[1,2-a]benzimidazole group. Moreover, some
cytotoxicity assessments were undertaken, whereupon it was found that Schiff base 3i and
pyrimidobenzimidazoles 4e and 4f did not exhibit cytotoxicity at similar concentrations resembling thus
the inhibitory activity of lipid peroxidation. The most cytotoxic Schiff base and pyrimidobenzimidazole
were found to be 3d and 4c, respectively.
Keywords:
2-Aminobenzimidazole
Acetophenones
Cytotoxicity
E/Z configuration of imines
Green chemistry
Lipid peroxidation inhibition
Lipoxygenase inhibition
Microwave irradiation
Pyrimido[1,2-a]benzimidazoles
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
effective scavengers of 2,2-diphenyl-1-picrylhydrazyl (DPPH)
stable free radical [5]. On the other hand, it has been reported that
2-Aminobenzimidazole has attracted considerable attention
since its derivatives such as astemizole, mebendazole, enviroxime,
carbendazime, and benomyl have been widely used [1,2]. Con-
cerning benzimidazoles it is well known that they exhibit antioxi-
dant activities causing inhibition of NADPH-dependent lipid
peroxidation (LPO) in the rat liver microsomes [3,4]. It seems that
they have variable tissue dependent in vitro effects on LPO due to
their distinct effects on CYP activities, but not on NADPH-cyto-
chrome P450 reductase activity in rats [4]. They also have strong
inhibitory effects on superoxide anion formation and act as
Schiff bases are known to display good level of anti-inflammatory
activity as well as inhibition of lipoxygenase (LOX) [6e8]. Finally,
dihydropyrimidine derivatives represent a family of heterocycles
with significant therapeutic and medicinal properties [9e11].
Several marine alkaloids having the dihydropyrimidine core-unit
were found to show interesting biological activities such as anti-
viral, antibacterial, antioxidant and anti-inflammatory [12,13].
Today, there is an increased interest in the combination of two
pharmacophores on the same scaffold leading to hybrid molecules
or conjugates. These hybrid drugs combine two drugs in a single
molecule with the goal of creating a chemical entity more medically
effective than its individual components [14e19]. As a result, pyr-
imido[1,2-a]benzimidazoles, which combine two biologically active
heterocycliccores, havebeenfoundtobe ofpharmacological interest
* Corresponding authors. Tel.: þ30 2310 997865; fax: þ30 2310 997679.
E-mail addresses: tsolerid@chem.auth.gr (C.A. Tsoleridis), ioulia@chem.auth.gr
(J. Stephanidou-Stephanatou).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.018

298
C.G. Neochoritis et al. / European Journal of Medicinal Chemistry 46 (2011) 297e306
R2
Table 1
4
N
N
Reaction conditions and products.
a
N3
N1
R1
R2
4
'
NH2
+
R1
COMe
R1
R2
Time (min)
Temp (ꢀC)
Prod
Yield (%)
N
Ketone
7
1'
3
'
H
1
2a-i
R2
H
H
2a
2b
2c
2d
2e
2f
H
Me
H
OMe
H
Cl
H
H
Me
H
OMe
H
5
4
4
10
4
5
160
160
160
160
160
160
160
160
250
250
250
250
250
250
200
200
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4e
4f
92
91
90
89
90
93
85
89
92
81
72
86
70
92
93
88
Me
H
3a-i
R1
H
Me
H
R2
R1
9
b
N10
a
b
c
d
e
H
f
Cl
Br
H
7
H
Me
H
g
h
i
N1
5
N
R2
Br
(-CH=)4
4
3
'
4''
OMe
H
3
2g
2h
2i
2a
2b
2c
2e
2f
Br
H
H
Br
4
4
10
5
7
10
10
5
5
5
R1
'
4
MeO
OMe
R1
3''
R2
1-Naphthyl
H
Me
H
H
Cl
Br
H
4a-c, 4e-h
H
H
Me
OMe
H
Scheme 1. Reagents and conditions: (a) MW irradiation, 160 ꢀC, 4e10 min; (b) MW
irradiation, 250 ꢀC, 5e10 min including catalytic amount of acetic acid in both cases
(molar ratio of benzimidazole:ketone:acetic acid ¼ 1:2.1:0.2).
2g
2h
H
Br
4g
4h
for their diverse biological activities [20e22]. Therefore, recently
considerable attention has been devoted towards their synthesis by
a variety of synthetic procedures [23e26], the two predominating
being either cyclocondensation reactions of 2-aminobenzimidazole
with a variety of a,b-unsaturated carbonyl compounds [27e29] or
multicomponent condensation reactions [30e33] involving 2-ami-
nobenzimidazole. The synthesis of 3-oxo-pyrimido[1,2-a]benz-
2. Chemistry
All reactions were carried out by environmentally friendly
conditions (‘green chemistry’) by application of microwave irradia-
tion without solvent. The syntheses were carried out simply by
mixing the 2-aminobenzimidazole (1 mmol) with the acetophe-
none 2 (2.1 mmol) in the presence of 0.2 mmol of acetic acid and
irradiating in the microwave oven, whereupon the benzimidazole
derivatives 3 or 4 were obtained in very good yields and were
purified by column chromatography. The desired reaction condi-
tions (temperature and time) were set as reported in Table 1.
Analytically, initial studies were conducted with acetophenone 2a
whereupon at 160 ꢀC only the Schiff base 3awas isolated (92% yield).
At higher temperatures (180e230 ꢀC), mixtures of the two products
3a and 4a, their ratio depending upon the reaction temperature,
were formed, whereas finally at 250 ꢀC only the pyr-
imidobenzimidazole 4a was isolated (81% yield). After the reaction
conditions were optimized, the generality of the reaction was
investigated, and the results (Table 1) show that the reaction has
a broad applicability. In all studied reactions at 160 ꢀC the Schiff
bases 3aeh were formed with the exception of the 1-naphthyl-
derivative 3i for the formation of which a higher temperature
(250 ꢀC) was required. Generally, at 250 ꢀC pyrimidobenzimidazoles
4aec and 4eef were the isolated reaction products, with exception
of the bromo derivatives 4g and 4h for which the optimal reaction
imidazoles by the reaction of propiolic esters and a,b-unsaturated
esters with 2-aminobenzimidazole has also been investigated since
they were found to show immunotropic properties [34,35]. To the
contrary, concerning the 3-oxo-pyrimido[1,2-a]benzimidazole
derivatives to our knowledge only one reference appeared in the
literature for the 4-methyl-2,4-diphenyl-derivative prepared by
a two step reaction sequence [36]. Finally, although available data
confirm that benzimidazole Schiff bases exhibit antineoplastic
activity [37], the corresponding acetophenone-benzimidazole Schiff
bases are not even reported in the literature.
Against this literature background, concerning the biological
activity of benzimidazole and pyrimidobenzimidazole derivatives,
in the present work we wish to describe a one-pot synthetic
methodology for the preparation of a series of novel benzimidazole
Schiff bases 3 and 3-oxo-pyrimido[1,2-a]benzimidazoles 4 in
excellent yields occurring upon microwave irradiation in a simple
and straight-forward manner (Scheme 1) along with a preliminary
study of compounds 3 and 4 as inhibitors of lipoxygenase (LOX) and
of lipid peroxidation (LPO). In addition, their potential of medical
applications as inhibitors in certain cell metabolic pathways as lipid
peroxidation or in the catalysis of dioxygenation of poly-
unsaturated fatty acids in lipids, is articulated and supported by cell
toxicity assessments on human lung fibroblasts.
R1
R1
R2
R1
R2
R2
N
N
N
+H
2
+
N
N
NH2
O
-H2O
N
N
H
N
OH
Me
Me
Me
Me
H
3
1
2
R1
5A
R2
R1
R2
N
N
N
N
-H2O
R1
[O]
R1
N
N
N
R2
R1
R2
R1
N
N
H
R1
CH2
OH
Me
Me
Me
O
R2
R2
4
6
5B
R2
Scheme 2. Plausible mechanism for the formation of compounds 3 and 4.

H
H
H
H
H
H
H
H
H
H
H
H
N
N
H
H
H
H
N
N
H
H
N
H
N
H
H
H
H
H
H
H
H
N
H
H
H
N
H
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N
H
H
H
H
N
H
N
H
H
H
H
H
N
H
H
N
H
H
N
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
N
H
H
H
H
H
N
N
H
N
N
H
H
H
H
N
H
H
Fig. 1. Chemical structure and selected numbering for the four conformers investigated in the gas phase by DFT method (B3LYP/6-311G(df,p)). ΔE is the energy difference relative to
3a-E A.
temperature was found 200 ꢀC, and the lack of formation of the 4-
methoxy-substituted compound 4d and the naphthlyl derivative 4i.
Concerning the reaction mechanism, for the formation of the
pyrimidobenzimidazoles 4, the benzimidazole amino-group
attacks the acetophenone carbonyl group, whereas the NH moiety
attacks a second acetophenone molecule to form the imine inter-
mediate 5A, which undergoes further tautomerization to the
enamine 5B. Cyclization under water abstraction and formation of
the 6-membered ring gives rise to the colourless compound 6
(Scheme 2). Quite unexpectedly the 3-methylene group of the
pyrimidine ring is air oxidized to give finally the yellow or orange
3-oxo-pyrimido[1,2-a]benzimidazole 4. An analogous methylene
group air oxidation has been previously observed in some benzo-
diazepine derivatives [38]. Concerning the lack of formation of the
4-methoxy derivative 4d the reduced nucleophilicity of the NH
moiety (compared to that of NH2), renders attack to the compara-
tively poor electrophilic 4-methoxy-acetophenone carbonyl carbon
prohibited, therefore only the Schiff base 3d is always formed. To
the contrary, the enhanced electrophilicity of the 4-bromo-aceto-
phenone carbonyl carbon allows the reaction to proceed success-
fully at lower temperature (200 ꢀC). Finally, the behavior of the
1-acetylnaphthalene (2i) could be attributed to stereochemical
reasons.
Concerning the Z- or E-configuration of Schiff bases, there is
generally an ambiguity on account of the many different experi-
mental and theoretical studies [39e41], with exception cases
where a particular configuration is favoured [40] because of
formation of an intramolecular hydrogen bond between

300
C.G. Neochoritis et al. / European Journal of Medicinal Chemistry 46 (2011) 297e306
substituents and heteroatoms. It has also been reported that imines
having E- or Z-configuration in the solid state, afford in solution at
ambient temperature equilibrium mixtures, even in non polar
solvents (cyclohexane) [42]. Concerning the geometry of the iso-
lated Schiff bases 3aei a theoretical approach was undertaken,
since NOE measurements were not applicable.
In Fig. 1 for both isomers 3a-E and 3a-Z, where the imino group
is coplanar with the benzimidazole ring, two conformers A and B
are presented, with the NH group anti or syn to the imino double
bond, respectively.
H
H
N
CH3
H
H
N
N
H
H
H
H
H
H
3a
H
H
N
Initially, all structures were optimized at the AM1 level [43, 44],
whereupon conformer 3a-E B was converted to conformer 3a-E A,
probably due to stereochemical interactions of 9-CH3 with 1-H. For
the two conformers of 3a-Z, after optimization, a small difference in
heat of formation (DDHf ¼ 1.85 kcal/mol) is calculated to favour the
3a-Z A. Finally, 3a-E A is calculated to be more stable from 3a-Z A by
about 1.4 kcal/mol. Since from the above results no definite
conclusion could be drawn, quantum chemical calculations were
performed using DFT at the B3LYP/6-311G(df,p) level of theory in
order to find out which structure is preferred, using as input the
geometries produced from the AM1 treatment. After optimization
each geometry was checked by frequency calculation to assure that
it does not represent a transition state (no imaginary frequencies).
The optimized geometry for 3a-Z B is calculated to be similar to that
of 3a-Z A, with the substituents on the imino-carbon in symmetric
position to the N1eC2eN8 plane (Fig. 1). The final geometry for 3a-
E A is calculated to be favoured over 3a-Z A (or B) by 5.7 kcal/mol.
Since this energy difference is small to lead to an unambiguous
conclusion the experimental 13C NMR chemical shifts were
compared with the calculated ones (DFT, GIAO) [45e47]. The
comparison is limited only to the nuclei around the imino group
(Table 2).
Comparing the calculated NMR chemical shifts with the experi-
mental ones a much betterapproximation is observed for the methyl
carbon of 3a-E A, wherethe methyl group is closer tothe C2]N3 and
most probably shielded from the magnetic anisotropic effect of this
double bond. Furthermore, from the better approximation for C9 it
can be concluded that in solution (CDCl3) the prepared Schiff bases
most probably exist in the E-configuration and consequently the
same configuration is adopted in the solid state.
The assigned molecular structures of all new compounds 3 and 4
are based on rigorous spectroscopic analysis including IR, NMR (1H,
13C, COSY, NOESY, HETCOR or HMQC and COLOC or HMBC), MS and
elemental analysis data.
H
H
N
N
H
H
Me
CH3
O
H
H
H
H
CH3
4b
2
3
Fig. 2. COLOC correlations between protons and carbons (via JCH and JCH
compound 4b. Only critical COLOC correlations in 3a are depicted.
) in
23.8 ppm). Long-range CeH correlations (COLOC) spectra were
optimized for J ¼ 8 Hz, thus aromatic protons show COLOC corre-
lations mainly via 3JCH, whereas protons on saturated carbons show
COLOC via JCH and JCH couplings. Consequently, the 4-methyl
group protons gave COLOC correlations with the quaternary
carbons at 188.7 (CO), 133.6 (C-100), and 69.9 (C-4) ppm. With the C-
4 carbon (69.9 ppm) correlated also the two o-protons (
the one p-substituted phenyl ring, whereas the o-protons (
of the second p-substituted phenyl ring correlated with the
quaternary carbon at 158.5 ppm (C-2). In Fig. 2 all the observed
COLOC correlations in compound 4b and some critical correlations
in compound 3a are depicted.
2
3
d
¼ 6.93) of
d
¼ 8.01)
3. Pharmacology
3.1. Antioxidant activity
In the present investigation compounds 3 and 4 are tested in
vitro, with regard to their antioxidant ability as well as their ability
to inhibit soybean LOX. All results are shown in Table 3 and are
discussed in terms of structural characteristics.
There is a growing body of evidence on the possible role of free
radicals in the inflammatory process [49], in the etiology of
atherosclerosis, nephritis and carcinogenesis [50]. It is also known
that lipid peroxidation is a free radical chain reaction [51], which
causes the degeneration of cell membranes. Most products of lipid
peroxidation are known to have mutagenic and/or carcinogenic
properties. Antioxidants are defined as substances that even at low
concentration significantly delay or prevent oxidation of easy
oxidizable substrates. There is an increased interest of using anti-
oxidants for medical purposes in the recent years. Thus, drugs
possessing antioxidant and free radical scavenging properties are
considered for preventing and/or treatment of diseases, which are
directly related to the lack of the antioxidant capacity of the
organisms. Many non-steroidal anti-inflammatory drugs have been
reported to act either as inhibitors of free radical production or as
radical scavengers [52].
Concerning the structure of the isolated 3-oxo-pyrimido[1,2-a]
benzimidazoles 4 the assignment of 4b is described. In the IR
spectrum the presence of a carbonyl (1710 cmꢁ1) was observed. In
the 1H NMR spectrum, in addition to the two p-substituted phenyl
rings, the presence of the four benzimidazole aromatic protons
resonating as a double doublet of doublets at
0.5 Hz, 9-H) [48], a double doublet of doublets at
7.2, 0.95 Hz, 8-H), a double doublet of doublets at
d
¼ 7.90 (J ¼ 8.2, 1.0,
d
d
¼ 7.32 (J ¼ 8.2,
¼ 7.20 (J ¼ 8.2,
7.2, 1.0 Hz, 7-H) and as a double doublet of doublets at
d
¼ 7.06
(J ¼ 8.2, 0.95, 0.5 Hz, 6-H), with their carbons resonating at 121.8,
123.4, 124.9, and 112.2 ppm, respectively, was identified. Moreover,
the 4-position methyl group appears as a singlet at
d
¼ 2.33 (C at
Table 2
Comparison of experimental and calculated 13C NMR chemical shifts for critical
carbon atoms for the E- and Z-isomers of imine 3a.
Factors such as solubility or steric hindrance, which may be of
overriding importance in one environment but not in another, can
be varied and the antioxidant ability of a compound in a variety of
milieus may be evaluated. For these reasons several assays should
be used in order to assess in vitro antioxidant activity [53]. Thus, we
have used two different types of assays to measure the antioxidant
Carbon
Expl
3a-E A
3a-Z A
2
9
10
155.0
174.3
139.5
19.8
158.7
177.0
146.4
20.5
156.6
183.2
143.2
35.3
9-Me

C.G. Neochoritis et al. / European Journal of Medicinal Chemistry 46 (2011) 297e306
301
Table 3
Interaction % with DPPH; In vitro % inhibition of lipid peroxidation (AAPH); In vitro % inhibition of soybean lipoxygenase (LOX % inh).
N
R1
H
Me
H
OMe
H
R2
H
H
Me
H
R1
Cl
Br
H
R2
H
H
N
a
b
c
d
e
f
N
R1
R2
R1
N
g
h
i
N
R1
Br
N
H
Me
R2
O
(-CH=)4
Me
R2
3a-i
OMe
4a-c, 4e-h
Compds
C log P
DPPH % 0.05 mM
20 min
AAPH % inhibition
0.01 mM
LOX % inhibition
0.01 mM
60 min
0.1 mM
0.1 mM
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
4e
4f
4g
4h
NDGA
BHT
Trolox
3.51
4.01
4.01
3.88
3.59
4.60
4.37
4.37
5.06
5.03
6.03
6.03
4.87
6.45
6.75
6.56
3.92
5.43
3.09
0.3
No
No
4
No
3
No
No
5
2
No
4
3
2
5
8
No
3
No
4
4
6
7
1
No
5
2
2
2
3
83
60
18
47
63
12
69
nt
22
65
26
11
8
67
84
77
68
100
26
50
97
97
100
80
84
100
97
78
88
Noa
No
No
22
No
62
No
No
18
No
No
8
65
42
85
18
No
98
100
40
49
No
No
No
No
51
35
3
33
9
19
No
No
61
12
84
3
No
81
31
51
No
40
63
a
No activity detectable under the reported experimental conditions. Each in vitro experiment was performed at least in triplicate and the standard deviation of absorbance
was less than 10% of the mean.
activity of compounds 3 and 4: a) Interaction with 1,1-diphenyl-2-
picrylhydrazyl stable free radical (DPPH) and b) interaction with
the water soluble azo compound 2,20-azobis(2-amidinopropane)
dihydrochloride (AAPH) and the results are compared to well
known antioxidant agents e.g. nordihydroguairetic acid (NDGA),
butylated hydroxyl toluene (BHT) and 6-hydroxy-2,5,7,8-tetrame-
thylchroman-2-carboxylic acid (trolox).
Both protocols require a spectrophotometric measurement and
a certain reaction time in order to obtain reproducible results [54].
DPPH has been used in a radical scavenging measuring method. In
its oxidized form, the DPPH radical has an absorbance maximum
centred at about 517 nm [55]. The DPPH method is described as
a simple, rapid and convenient method independent of sample
polarity for screening many samples for radical scavenging activity
[56]. Azo compounds generating free radicals through spontaneous
thermal decomposition are useful for free radical production
studies in vitro [57]. The water soluble azo compound AAPH has
been extensively used as a clean and controllable source of ther-
mally produced alkylperoxyl free radicals. We have used AAPH as
a free radical initiator to follow oxidative changes of linoleic acid to
conjugated diene hydroperoxide.
The interaction of the examined compounds with the stable free
radical DPPH indicates their radical scavenging ability and electron
donating activity in an iron free system. All compounds presented
very low, if any, interaction to DPPH at 0.05 mM. The low interac-
tion values of the derivatives, compared to BHT, should be mainly
attributed to the absence of easily oxidized functionalities like the
ones present in BHT. The absence of hydrogen atoms in phenolic
hydroxyl groups, which could be donated to stabilize the DPPH
radical is obvious within the given results.
dependent. Comparing the anti-lipid peroxidation activities of the
tested Schiff bases, compound 3e is the most potent followed by 3h
and 3i, whereas the unsubstituted Schiff base 3a presents moderate
activity. Although high inhibition values are observed for Schiff bases
(3e, 3h, 3i) with high lipophilicities (C log P 3.59e5.06), there are
some exceptions, such as compound 3f presenting high C log P 4.60
and low anti-LPO activity. Perusal of the % inhibition values at 0.1 mM
(Table 3) did not show significant differences among the pyrimido
[1,2-a]benzimidazoles 4 (78e100%), 4a and 4e (100%) being the most
potent followed by 4f (97%). Compound 4e with the lower C log P
value (4.87) within the 4 series is very potent. The standard inhibitor
trolox obviously exerts its inhibitory effect on lipid peroxidation
mainly through the ability of its 6-hydroxy-5,7,8-trimethylchromane
moiety to break the radical chain. Although no phenol moieties are
present within these compounds, they could break the radical chain
through the initial abstraction of a hydrogen. The thus created new
radicals are efficiently stabilized through resonance.
The role of molar lipophilicity, as theoretically calculated C log P
[58] in the series of pyrimido[1,2-a]benzimidazoles 4, is contra-
dictory since the three most active analogs 4a, 4e and 4f present big
differences in terms of calculated values of lipophilicity, e.g. C log P
values 5.03, 4.87 and 6.45. It is worth to mention that between the
two isomers 3b and 3c as well as 4b and 4c, not big differences are
observed. On the contrary, significant differences are noticed
between isomers 3g and 3h.
Lipoxygenases (LOXs) play a role in membrane lipid peroxidation
by forming hydroperoxides in the lipid bilayer [59,60]. Leukotrienes
play an important role as mediators of a variety of inflammatory and
allergic reactions and are derived from the biotransformation of
arachidonic acid catalyzed by LOXs. Inhibitors of LOX have attracted
attention initially as potential agents for the treatment of inflamma-
tory and allergic diseases but their therapeutic potential has now
been expanded to certain types of cancer and cardiovascular diseases
The majority of the studied compounds 3 and 4 effectively inhibit
AAPH induced lipid peroxidation (LPO), showing higher activity than
the reference compound trolox. This activity is concentration

302
C.G. Neochoritis et al. / European Journal of Medicinal Chemistry 46 (2011) 297e306
[61,62]. Thus, we decided to further evaluate the synthesized mole-
cules for their ability to inhibit soybean LOX by the UV absorbance
based enzyme assay [63]. Most of the LOX inhibitors are antioxidants
or free radical scavengers [64]. Other studies suggest a relationship
betweenLOXinhibition and theabilityof theinhibitors toreduceFe3þ
at the active site to the catalytically inactive Fe2þ. Several LOX
inhibitorsareexcellentligandsforFe3þ andthisinhibitionisrelatedto
their ability to reduce the iron species in the active site to the cata-
lytically inactive ferrous form [64]. It seems that the inhibition
increaseswiththeconcentration’sincrease. Perusalofthe%inhibition
values (Table 3) shows that among the Schiff bases the most potent
inhibitor is the naphthyl substituted compound 3i followed by 3f and
3d, whereas 4a and 4g are the most active among the pyrimido[1,2-a]
benzimidazoles. Notably, the parent compound 3a seemed essen-
tially inactive under our experimental conditions, while the parent 4a
showed the higher % LOX inhibition. It is worth to mention that
between the two isomers 4g and 4h most potent is the p-analogue.
Although lipophilicity is referred to as an important physicochemical
property for LOX inhibitors [65], herein the most potent compounds
3i and 4a with 98% and 100% inhibition at 0.1 mM present C log P
values 5.06 and 5.03 and do not follow this concept. Overall lip-
ophilicity, as C log P values does not influence biological activity.
Compared to the standard inhibitor NDGA and taking into
consideration the mode of action of LOX, the most active analogs of
the present set of compounds do not contain a catechol moiety,
which efficiently scavenges free radicals and might also chelate
Fe3þ and reduce it to the catalytically inactive Fe2þ. Our experi-
mental findings suggest that the tested compounds may bind to the
active site of the enzyme or simultaneously act as antioxidants.
The present study indicates that LOX or LPO inhibitory activity is
not always accompanied by DPPH radical scavenging activity. Thus,
compounds 3f, 3i, 4a and 4g are potent LOX inhibitors possessing
very low DPPH radical scavenging activity. This is in accordance
with the finding of Curini et al. [66] and Hadjipavlou-Litina et al.
[67], who have studied antioxidant and LOX inhibitory activity and
showed that the most efficient LOX inhibitors are not the most
active DPPH radical scavenger. However, a better correlation exists
between LOX and LPO inhibitory activity.
Fig. 4. Cytotoxic activity of pyrimidobenzimidazoles 4c, 4e and 4f assessed by MTT
assay on human lung fibroblasts over an incubation period of 24 h. Values shown are
the standard deviation of at least three measurements.
exceed 1%) and after 24 h of cell adherence/culture, under the
described conditions in the experimental part of this work, the
compounds were added at different concentrations from 1
mg/mL to
50 g/mL to each vial. The results obtained after 24 h by performing
m
the MTTassay are indicated below in Figs. 3 and 4 for Schiff bases 3d,
3g, 3i and pyrimidobenzimidazoles 4c, 4e, 4f, respectively. In
particular, Schiff bases 3i and 3g exhibited a non toxic uptake by the
fibroblasts at 0.003e0.017 mM and 0.003e0.015 mM, respectively.
In higher concentrations 0.17 mM and 0.16 mM cell viability was
decreased, but however remained higher than 50% (62.5% and 73.6%,
respectively). These assessments are in accordance with those
obtained for LOX or AAPH inhibition and corroborate a possible
medical use of these compounds. On the contrary, cell viability
studies of 3d indicated that at 0.003e0.037 mM cell viability was
over 50% (96e56%, respectively), while for higher concentrations
(>0.037 mMe0.18 mM) the viability was ranked between 33% and
29%, respectively. It has to be mentioned that this compound
exhibited a promising activity profile for concentrations up to
0.1 mM for LOX or AAPH inhibition.
Concerning the pyrimidobenzimidazoles, compounds 4f and 4e
(Fig. 4) exhibited a non toxic uptake by fibroblasts up to 0.02 mM
and 0.04 mM, respectively. In higher concentrations cell viability
was reduced to 37% and 63%, respectively. Additionally, 4e seems to
be a good candidate for medical use, taken into consideration its
AAPH inhibitory activity at 0.1 mM being 100%. The compound 4c
showed a similar cell viability profile as 4f at lower however
concentrations.
3.2. Cytotoxicity in human lung fibroblasts
The cytotoxicity of Schiff bases 3d, 3g and 3i, as well as of pyr-
imidobenzimidazoles 4c, 4e and 4f was assessed by means of
a colorimetric microculture assay (MTT assay) in human lung
fibroblast passage 19. The investigated compounds were dissolved
in DMSO (its end concentration in the culture medium did not
4. Conclusion
In conclusion, we have developed a direct method for the
synthesis of a series of benzimidazole Schiff bases 3 and 3-oxo-
pyrimido[1,2-a]benzimidazoles 4 in excellent yields by a one-pot
synthetic procedure from the reaction of 2-aminobenzimidazole
with acetophenones by applying environmentally friendly condi-
tions (‘green chemistry’) namely, microwave irradiation without
solvent. In addition, a highly unusual air oxidation of a methylene
group to carbonyl was observed. Full assignment of all 1H and 13C
NMR chemical shifts has been unambiguously achieved. To the
Schiff bases 3 the E-configuration was assigned comparing the
experimental and calculated (by DFT) 13C NMR chemical shifts in
the proximity of the imino bond. In addition, compounds 3 and 4
were evaluated as inhibitors of lipoxygenase (LOX) and of lipid
peroxidation (LPO). All the tested derivatives showed inhibition of
lipid peroxidation, whereas most of them were found to have
higher activation than the reference compound trolox. The Schiff
Fig. 3. Cytotoxic activity of Schiff bases 3d, 3g and 3i, assessed by MTT assay on human
lung fibroblasts over an incubation period of 24 h. Values shown are the standard
deviation of at least three measurements.

C.G. Neochoritis et al. / European Journal of Medicinal Chemistry 46 (2011) 297e306
303
bases 3e, 3h, and 3i as well as the pyrimidobenzimidazoles 4a, 4e
and 4f were found to be the most potent. The most potent LOX
inhibitor within the subset of Schiff bases was found compound 3i,
followed by 3f, whereas compounds 4a and 4g are the most potent
of the 3-oxo-pyrimido[1,2-a]benzimidazole group. Compounds 3i
and 4a can be used as lead molecules for the design of agents with
excellent LOX and LPO inhibitory activities. The role of lipophilicity
is contradictory.
Moreover, based on cell viability investigations it seems that 3i
and 3g could be possible candidates for medical use concerning
their inhibitory activity for LOX or AAPH. On the contrary, 3d seems
to be toxic for the cells and therefore its inhibitory activity cannot
be recommended for medical applications, at least at the moment.
On the other hand, the uptake of pyrimidobenzimidazole 4e from
fibroblasts points out a promising compound for medical applica-
tions. The pyrimidobenzimidazoles 4c and 4f could be used as
inhibitors at lower concentrations than 4e.
gel using petroleum ethereethyl acetate (10:1) of slowly increased
polarity to afford in elution order compounds 3 or 4.
5.2.2. N-[(1E)-1-phenylethylidene]-1H-benzimidazol-2-amine 3a
Yield: 0.216 g (92%); white crystals; mp 185e187 ꢀC (ethanol).
1H NMR (CDCl3):
d
¼ 2.85 (s, 3H, N]CeCH3), 7.15e7.25 (m, 2H, 5,6-
H), 7.32 (d, J ¼ 6.4 Hz, 1H, 7-H), 7.42e7.52 (m, 2H, 30,50-H), 7.51 (t,
J ¼ 7.5 Hz, 1H, 40-H), 7.73 (d, J ¼ 6.5 Hz, 1H, 4-H), 8.08 (d, J ¼ 7.5 Hz,
2H, 20,60-H), 9.52 (br s, 1H, NH). 13C NMR (CDCl3):
d
¼ 19.8 (N]
CeCH3), 110.1 (C-7), 119.5 (C-4), 122.2 (C-5),* 122.5 (C-6),* 127.9 (C-
20,60), 128.6 (C-30,50), 131.6 (C-40), 133.1 (C-7a), 139.5 (C-10), 143.4 (C-
3a), 155.0 (C-2), 174.3 (2-N]C). LCeMS (ESI, 1.65 eV): m/z (%) ¼ 268
(Mþ þ H þ MeOH, 8), 258 (Mþ þ Na, 100), 236 (Mþ þ H, 100). Anal.
Calcd for C15H13N3 (235.28): C, 76.57; H, 5.57; N, 17.86%. Found: C,
76.65; H, 5.70; N, 17.68%. (*The assignments may be interchanged).
5.2.3. N-[(1E)-1-(4-methylphenyl)ethylidene]-1H-benzimidazol-2-
amine 3b
Yield: 0.227 g (91%); yellow crystals; mp 175e176 ꢀC (ethanol).
5. Experimental
1H NMR (CDCl3):
d
¼ 2.41 (s, 3H, 40-CH3), 2.79 (s, 3H, N]CeCH3),
7.15e7.23 (m, 2H, 5,6-H), 7.24 (d, J ¼ 7.3 Hz, 2H, 30,50-H), 7.50e7.55
5.1. General
(m, 2H, 4,7-H), 7.92 (d, J ¼ 7.3 Hz, 2H, 20,60-H), 9.40 (br s,1H, NH). 13C
NMR (CDCl3):
d
¼ 19.7 (N]CeCH3), 21.5 (40-CH3), 114.6 (br, C-4,7),
DPPH, trolox, BHT and NDGA were purchased from Aldrich
Chemical Co. (Milwaukee, WI, USA). Soybean LOX and linoleic acid
sodium salt were obtained from Sigma Chemical Co. (St. Louis, MO,
USA). DMEM was purchased from Invitrogen, Carlsbad, CA. FBS was
purchased from Intergen, NY, and penicillin and streptomycin were
purchased from GIBCO. For the in vitro tests a Lambda 20 (Per-
kineElmer) UVeVis double beam spectrophotometer was used.
Melting points were measured on a Kofler hot-stage and are
uncorrected. Column chromatography was carried out using Merck
silica gel. TLC was performed using precoated silica gel glass plates
0.25 mm containing fluorescent indicator UV254 purchased from
MachereyeNagel using a 3:1 mixture of petroleum ethereethyl
acetate. Petroleum ether refers to the fraction boiling between 60
and 80 ꢀC. NMR spectra were recorded at room temperature on
a Bruker AM 300 or AVANCE 300 spectrometer at 300 MHz for 1H
and 75 MHz for 13C, respectively, using CDCl3 as solvent. Chemical
122.3 (C-5,6), 127.9 (C-30,50), 129.3 (C-20,60), 136.6 (C-10), 138.0 (br,
C-3a,7a), 142.3 (C-40), 155.0 (C-2), 174.4 (2-N]C). LCeMS (ESI,
1.65 eV): m/z (%) ¼ 272 (Mþ þ Na, 35), 250 (Mþ þ H, 100). Anal.
Calcd for C16H15N3 (249.31): C, 77.08; H, 6.06; N, 16.85%. Found: C,
77.15; H, 5.96; N, 16.90%.
5.2.4. N-[(1E)-1-(3-methylphenyl)ethylidene]-1H-benzimidazol-2-
amine 3c
Yield: 0.224 g (90%); yellow crystals; mp 155e157 ꢀC (ethanol).
1H NMR (CDCl3):
d
¼ 2.40 (s, 3H, 30-CH3), 2.79 (s, 3H, N]CeCH3),
7.17e7.23 (m, 2H, 5,6-H), 7.29e7.37 (m, 2H, 40,50-H), 7.48e7.56 (m,
2H, 4,7-H), 7.77e7.83 (m, 1H, 60-H,). 7.84 (br s, 1H, 20-H), 9.33 (br s,
1H, NH). 13C NMR (CDCl3):
d
¼ 19.7 (N]CeCH3), 21.4 (30-CH3), 114.6
(br, C-4,7), 122.2 (C-5,6), 125.1 (C-60), 128.3 (C-20), 128.4 (C-50), 132.4
(C-40), 138.0 (br, C-3a,7a), 138.2 (C-30), 139.1 (C-10), 154.9 (C-2), 174.7
(2-N]C). LCeMS (ESI, 1.65 eV): m/z (%) ¼ 250 (Mþ þ H, 100). Anal.
Calcd for C16H15N3 (249.31): C, 77.08; H, 6.06; N, 16.85%. Found: C,
77.19; H, 6.11; N, 16.71%.
shifts are expressed in
d values (ppm) relative to TMS as internal
standard for 1H and relative to TMS (0.00 ppm) or to CDCl3
(77.05 ppm) for 13C NMR spectra. Coupling constants nJ are reported
in Hz. Second order 1H NMR spectra were analyzed by simulation
[48]. IR spectra were recorded on a PerkineElmer 1600 series FTIR
spectrometer and are reported in wave numbers (cmꢁ1). Low-
resolution LCeMS (ESI, 1.65 eV) mass spectra were recorded on
LCMS-2010 EV system (Shimadzu). Elemental analyses performed
with a PerkineElmer 2400-II CHN analyzer.
5.2.5. N-[(1E)-1-(4-methoxyphenyl)ethylidene]-1H-benzimidazol-
2-amine 3d
Yield: 0.236 g (89%); yellow crystals; mp 210e212 ꢀC (ethanol).
1H NMR (CDCl3):
d
¼ 2.74 (s, 3H, N]CeCH3), 3.86 (s, 3H, OeCH3),
6.93 (dt, J ¼ 8.8, 2.0 Hz, 2H, 30,50-H), 7.17e7.22 (m, 2H, 5,6-H),
7.48e7.53 (m, 2H, 4,7-H), 7.99 (dt, J ¼ 8.8, 1.9 Hz, 2H, 20,60-H), 12.67
(br s,1H, NH). 13C NMR (CDCl3):
d
¼ 19.5 (N]CeCH3), 55.5 (OeCH3),
5.2. Chemistry
113.8 (C-30,50), 114.9 (C-7), 122.0 (C-4,5,6), 129.7 (C-20,60), 131.5
(C-10), 132.1 (C-7a), 144.2 (C-3a), 155.3 (C-2), 162.6 (C-40), 173.4
(2-N]C). LCeMS (ESI, 1.65 eV): m/z (%) ¼ 320 (Mþ þ Na þ MeOH,
40), 288 (Mþ þ Na, 35), 266 (Mþ þ H, 100). Anal. Calcd for
C16H15N3O (265.31): C, 72.43; H, 5.70; N, 15.84%. Found: C, 72.55; H,
5.62; N, 15.77%.
5.2.1. General procedure for the microwave synthesis of Schiff bases
3 and of pyrimido[1,2-a]benzimidazoles 4
The syntheses were carried out simply by mixing thoroughly the
2-aminobenzimidazole 1 (1 mmol) with the acetophenone deriv-
ative 2 (2.1 mmol) in the presence of catalytic amount of acetic acid
(one drop, ∼0.2 mmol) and irradiating in a Biotage Initiator 2.0
microwave oven at a power and time indicated in Table 1, where-
upon the benzimidazole derivatives were obtained in very good
yields. At the temperature of 160 ꢀC only the Schiff bases 3 were
isolated, with exception of the 1-naphthyl derivative 3i, which was
formed at 250 ꢀC. Increasing the temperature to 250 ꢀC led to the
synthesis of only derivatives 4, with exception of the bromo-
substituted ones, 4g and 4h, which were formed at 200 ꢀC. The
crude products were purified by column chromatography on silica
5.2.6. N-[(1E)-1-(3-methoxyphenyl)ethylidene]-1H-benzimidazol-
2-amine 3e
Yield: 0.239 g (90%); brownish solid; mp 181e183 ꢀC (ethanol).
1H NMR (CDCl3):
d
¼ 2.78 (s, 3H, N]CeCH3), 3.83 (s, 3H, OeCH3),
7.05 (dd, J ¼ 6.4, 2.3 Hz, 1H, 40-H), 7.18e7.25 (m, 2H, 5,6-H), 7.34
(t, J ¼ 8.1 Hz, 1H, 50-H), 7.52e7.62 (m, 5H, 4,7,20,60-H,NH). 13C NMR
(CDCl3):
d
¼ 20.0 (N]CeCH3), 55.4 (OeCH3), 112.5 (C-20), 114.6 (br,
C-4,7), 117.8 (C-40), 120.5 (C-60), 122.3 (C-5,6), 129.6 (C-50), 133.4 (br,
C-7a), 138.0 (br, C-3a), 140.4 (C-10), 154.8 (C-2), 159.7 (C-30), 174.3

304
C.G. Neochoritis et al. / European Journal of Medicinal Chemistry 46 (2011) 297e306
(2-N]C). LCeMS (ESI, 1.65 eV): m/z (%) ¼ 298 (Mþ þ H þ MeOH, 8),
266 (Mþ þ H, 100). Anal Calcd for C16H15N3O (265.31): C, 72.43; H,
5.70; N, 15.84%. Found: C, 72.59; H, 5.78; N, 15.70%.
7.42 (tt, J ¼ 7.2, 1.5 Hz, 2H, 30,50-H), 7.50 (tt, J ¼ 7.2, 1.5 Hz, 1H, 40-H),
7.94 (dd, J ¼ 8.2, 1.0 Hz,1H, 9-H), 8.08 (dt, J ¼ 7.2, 1.5 Hz, 2H, 20,60-H).
13C NMR (CDCl3):
d
¼ 23.9 (4-Me), 70.1 (C-4), 112.1 (C-6), 122.0 (C-
90)0, 123.5 (C-8),0125.1 (C-7), 126.0 (C-200,600), 128.04 (C-300,500), 129.1 (C-
4 ), 129.5 (C-3 ,50),* 129.9 (C-20,60),* 132.3 (C-4 ), 132.7 (C-10), 134.0
(C-5a), 136.5 (C-100), 144.2 (C-9a), 147.9 (C-10a), 158.5 (C-2), 188.4
(C-3). LCeMS (ESI, 1.65 eV): m/z (%) ¼ 370 (Mþ þ H þ H2O, 30). 352
(Mþ þ H, 5), 270 (100) Anal. Calcd for C23H17N3O (351.40): C, 78.61;
H, 4.88; N, 11.96%. Found: C, 78.75; H, 4.73; N, 11.87%. (*The
assignments may be interchanged).
5.2.7. N-[(1E)-1-(4-chlorophenyl)ethylidene]-1H-benzimidazol-2-
amine 3f
Yield: 0.251 g (93%); yellow crystals; mp 218e220 ꢀC (ethanol).
1H NMR (CDCl3):
d
¼ 2.80 (s, 3H, N]CeCH3), 7.15e7.25 (br m, 2H,
5,6-H), 7.40 (d, J ¼ 8.2 Hz, 2H, 30,50-H), 7.54 (br m, 2H, 4,7-H), 7.95
(d, J ¼ 8.2 Hz, 2H, 20,60-H), 9.05 (br s, 1H, NH). 13C NMR (CDCl3):
d
¼ 19.6 (N]CeCH3), 112.5 (br, C-4,7), 122.5 (C-5,6), 128.8 (C-20,60),*
128.9 (C-30,50),* 137.8 (C-10), 138.0 (C-40), 142.1 (br, C-3a,7a), 154.6
(C-2), 172.7 (2-N]C). LCeMS (ESI, 1.65 eV): m/z (%) ¼ 292/294
(Mþ þ Na, 10), 270/272 (Mþ þ H, 100). Anal Calcd for C15H12ClN3
(269.73): C, 66.79; H, 4.48; N, 15.58%. Found: C, 66.58; H, 4.37; N,
15.39%. (*The assignments may be interchanged).
5.2.12. 4-Methyl-2,4-bis(4-methylphenyl)pyrimido[1,2-a]
benzimidazole-3(4H)-one 4b
Yield: 0.273 g (72%); yellow crystals; mp 237e239 ꢀC (ethanol).
IR (KBr): 1710 (C]O) cmꢁ1. 1H NMR (CDCl3):
d
¼ 2.28 (s, 3H, 400-Me),
2.33 (s, 3H, 4-Me), 2.39 (s, 3H, 40-Me), 6.93 (d, J ¼ 8.4 Hz, 2H, 200,600-
H), 7.06 (ddd, J ¼ 8.2, 0.95, 0.5 Hz, 1H, 6-H), 7.11 (d, J ¼ 8.4 Hz, 2H,
300,500-H), 7.20 (ddd, J ¼ 8.2, 7.2, 1.0 Hz, 1H, 7-H), 7.22 (d, J ¼ 8.4 Hz,
2H, 30,50-H), 7.32 (ddd, J ¼ 8.2, 7.2, 0.95 Hz, 1H, 8-H), 7.90 (ddd,
J ¼ 8.2, 1.0, 0.5 Hz, 1H, 9-H), 8.01 (d, J ¼ 8.4 Hz, 2H, 20,60-H). 13C NMR
5.2.8. N-[(1E)-1-(4-bromophenyl)ethylidene]-1H-benzimidazol-2-
amine 3g
Yield: 0.267 g (85%); green crystals; mp 220e221 ꢀC (ethanol).
1H NMR (CDCl3):
d
¼ 2.79 (s, 3H, N]CeCH3), 7.19e7.28 (m, 2H, 5,6-
(CDCl3):
d
¼ 21.0 (400-Me), 21.7 (40-Me), 23.8 (4-Me), 69.9 (C-4),112.1
H), 7.51e7.62 (m, 4H, 4,7,30,50-H), 7.87 (d, J ¼ 7.7 Hz, 2H, 20,60-H),
(C-6), 121.8 (C-9), 123.4 (C-8), 124.9 (C-7), 126.0 (C-200,600), 129.3 (C-
30,50), 129.9 (C-20,60), 130.1 (C-10), 130.3 (C-300,500), 133.6 (C-100), 134.1
(C-5a), 139.1 (C-400), 143.3 (C-40), 144.2 (C-9a), 148.2 (C-10a), 158.5
(C-2), 188.7 (C-3). LCeMS (ESI, 1.65 eV): m/z (%) ¼ 434
(Mþ þ Na þ MeOH, 100), 402 (Mþ þ Na, 65), 380 (Mþ þ H, 70). Anal.
Calcd for C25H21N3O (379.45): C, 79.13; H, 5.58; N, 11.07%. Found: C,
79.05; H, 5.53; N, 11.22%.
9.15 (br s, 1H, NH). 13C NMR (CDCl3):
d
¼ 19.7 (N]CeCH3), 114.8 (br,
C-4,7), 122.5 (C-5,6), 126.6 (C-40), 129.3 (C-20,60), 131.8 (C-30,50),
137.9 (br, C-3a,7a), 138.0 (C-10), 154.4 (C-2), 173.1 (2-N]C). LCeMS
(ESI, 1.65 eV): m/z (%) ¼ 336/338 (Mþ þ Na, 15), 314/316 (Mþ þ H,
100). Anal Calcd for C15H12BrN3 (314.18): C, 57.34; H, 3.85; N,
13.37%. Found: C, 57.45; H, 3.77; N, 13.41%.
5.2.9. N-[(1E)-1-(3-bromophenyl)ethylidene]-1H-benzimidazol-2-
amine 3h
5.2.13. 4-Methyl-2,4-bis(3-methylphenyl)pyrimido[1,2-a]
benzimidazole-3(4H)-one 4c
Yield: 0.280 g (89%); yellow crystals; mp 145e146 ꢀC (ethanol).
Yield: 0.326 g (86%); yellow crystals; mp 160e162 ꢀC (ethanol).
1H NMR (CDCl3):
d
¼ 2.73 (s, 3H, N]CeCH3), 7.15e7.23 (m, 2H, 5,6-
IR (KBr): 1706 (C]O) cmꢁ1. 1H NMR (CDCl3):
d
¼ 2.27 (s, 3H, 300-Me),
H), 7.25 (t, J ¼ 7.9 Hz, 1H, 50-H), 7.48e7.57 (m, 2H, 4,7-H), 7.59 (d,
2.35 (s, 3H, 4-Me), 2.39 (s, 3H, 30-Me), 6.84 (dd, J ¼ 7.7, 1.2 Hz, 1H,
600-H), 6.89 (d, J ¼ 1.2 Hz,1H, 200-H), 7.05 (dd, J ¼ 8.2,1.0 Hz,1H, 6-H),
7.13 (dd, J ¼ 7.6,1.2 Hz,1H, 400-H), 7.20 (ddd, J ¼ 8.2, 7.2, 1.0 Hz,1H, 7-
H), 7.21 (t, J ¼ 7.6 Hz, 1H, 500-H), 7.25e7.36 (m, 3H, 8,40,50-H),
7.83e7.88 (m, 1H, 60-H), 7.93 (dd, J ¼ 8.4, 1.0 Hz, 1H, 9-H), 7.96 (d,
J ¼ 7.9 Hz, 1H, 40-H), 7.83 (dd, J ¼ 7.9, 1.6 Hz, 1H, 60-H), 8.13 (d,
J ¼ 1.6 Hz, 1H, 20-H), 9.05 (br s, 1H, NH). 13C NMR (CDCl3):
¼ 19.7
d
(N]CeCH3), 114.7 (br, C-4,7), 122.5 (C-5,6), 122.8 (C-30), 126.4 (C-
60), 130.0 (C-50), 130.7 (C-20), 134.4 (C-40), 138.0 (br, C-3a,7a), 140.9
(C-10), 154.5 (C-2), 172.4 (2-N]C). LCeMS (ESI, 1.65 eV): m/z (%) ¼
336/338 (Mþ þ Na, 10), 314/316 (Mþ þ H, 100). Anal Calcd for
C15H12BrN3 (314.18): C, 57.34; H, 3.85; N, 13.37%. Found: C, 57.47; H,
3.70; N, 13.48%.
J ¼ 1.2 Hz, 1H, 20-H). 13C NMR (CDCl3):
d
¼ 21.4 (300-Me), 21.6 (30-
Me), 23.9 (4-Me), 70.0 (C-4), 112.1 (C-6), 121.9 (C-9), 123.1 (C-600),
123.4 (C-8), 125.0 (C-7), 126.6 (C-60), 127.3 (C-200), 128.3 (C-20), 129.4
(C-400), 129.9 (C-50), 130.3 (C-500), 132.7 (C-10), 133.2 (C-40), 134.1
(C-5a), 136.6 (C-100), 138.2 (C-30), 139.5 (C-300), 144.2 (C-9a), 148.0
(C-10a), 158.7 (C-2), 188.6 (C-3). LCeMS (ESI, 1.65 eV): m/z (%) ¼ 412
(Mþ þ H þ MeOH, 50), 402 (Mþ þ Na, 10), 380 (Mþ þ H, 100). Anal
Calcd for C25H21N3O (379.45): C, 79.13; H, 5.58; N, 11.07%. Found: C,
79.21; H, 5.50; N, 10.97%.
5.2.10. N-[(1E)-1-(1-naphthyl)ethylidene]-1H-benzimidazol-2-
amine 3i
Yield: 0.262 g (92%); yellow crystals; mp 134e136 ꢀC (ethanol).1H
NMR (CDCl3):
d
¼ 3.02 (s, 3H, 4-Me), 6.78 (d, J ¼ 7.8 Hz, 1H, 7-H), 7.10
(dd, J ¼ 8.0, 7.3 Hz, 1H, 5-H), 7.19 (dd, J ¼ 7.8, 7.3 Hz, 1H, 6-H),
7.48e7.58 (m, 3H, 30,60,70-H), 7.69 (dd, J ¼ 7.1, 0.9 Hz, 1H, 50-H), 7.75 (d,
J ¼ 8.0 Hz,1H, 4-H), 7.91 (dd, J ¼ 6.2, 3.5 Hz,1H, 40-H), 7.95 (dd, J ¼ 8.0,
0.9 Hz, 1H, 80-H), 8.36 (dd, J ¼ 6.2, 3.5 Hz, 1H, 20-H), 10.28 (br s, 1H,
5.2.14. 4-Methyl-2,4-bis(3-methoxyphenyl)pyrimido[1,2-a]
benzimidazole-3(4H)-one 4e
Yield: 0.288 g (70%); orange crystals; mp 58e60 ꢀC (ethanol). IR
NH). 13C NMR (CDCl3):
d
¼ 25.2 (4-Me), 110.30(C-7), 119.6 (C-4), 122.1
(KBr): 1707 (C]O) cmꢁ1
.
1H NMR (CDCl3):
d
¼ 2.36 (s, 3H, 4-Me),
(C-6),* 122.4 (C-5),* 125.0 (C-30),125.5 (C-20,8 ),126.3 (C-60),127.0 (C-
70), 128.7 (C-50), 130.2 (C-8a0), 130.3 (C-40), 134.2 (C-4a0), 139.1 (C-10),
143.3 (C-3a), 147.5 (C-10a), 154.2 (C-2), 178.7 (2-N]C). LCeMS (ESI,
1.65 eV): m/z (%) ¼ 308 (Mþ þ Na, 20), 286 (Mþ þ H,100). Anal. Calcd
for C19H15N3 (285.34): C, 79.98; H, 5.30; N,14.73%. Found: C, 80.15; H,
5.43; N, 14.77%. (*The assignments may be interchanged).
3.71 (s, 3H, 300-OMe), 3.87 (s, 3H, 30-OMe), 6.61 (dd, J ¼ 7.6, 1.3 Hz,
1H, 600-H), 6.62 (dd, J ¼ 2.1,1.3 Hz,1H, 200-H), 6.86 (dd, J ¼ 7.6, 2.1 Hz,,
1H, 400-H), 7.08 (dd, J ¼ 7.6, 2.1 Hz, 1H, 40-H), 7.11 (dd, J ¼ 8.1, 1.0 Hz,
1H, 6-H), 7.22 (t, J ¼ 7.6 Hz, 1H, 7-H), 7.24 (ddd, J ¼ 8.1, 7.0, 1.0 Hz,
1H, 500-H), 7.33 (t, J ¼ 7.6 Hz, 1H, 50-H), 7.34 (ddd, J ¼ 8.1, 7.0, 1.0 Hz,
1H, 8-H), 7.64 (m, 1H, 60-H), 7.74 (dd, J ¼ 2.1, 1.3 Hz, 1H, 20-H), 7.94
(d, J ¼ 8.1 Hz,01H, 9-H). 13C NMR (CDCl3):
d
¼ 23.8 (4-Me),0055.3 (300-
5.2.11. 4-Methyl-2,4-diphenylpyrimido[1,2-a]benzimidazole-3(4H)-
one 4a
Me), 55.5 (3 -Me), 70.0 (C-4), 112.1 (C-6),*00 112.3 (C-2 ),* 113.4
(C-4000), 114.0 (C-20), 118.2 (C-40),* 119.6 (C-6 ),* 122.0 (C-9), 123.0
(C-6 ),* 123.5 (C-8),* 125.2 (C-7), 129.4 (C-500), 130.6 (C-50), 134.0 (C-
10),* 134.1 (C-050 a),* 138.1 (C-0100), 144.2 (C-9a), 147.9 (C-10a), 158.3 (C-
2), 159.7 (C-3 ),* 160.4 (C-3 ),* 188.2 (C-3). LCeMS (ESI, 1.65 eV): m/
z (%) ¼ 430 (Mþ þ H þ H2O, 100), 412 (Mþ þ H, 20). Anal Calcd for
Yield: 0.285 g (81%); orange crystals; mp 115e117 ꢀC (ethanol).
IR (KBr): 1703 (C]O) cmꢁ1. 1H NMR (CDCl3):
d
¼ 2.36 (s, 3H, 4-Me),
7.05 (dd, J ¼ 8.2, 1.0 Hz, 1H, 6-H), 7.05e7.10 (m, 2H, 200,600-H), 7.20
(ddd, J ¼ 8.2, 7.2, 1.1 Hz, 1H, 7-H), 7.28e7.37 (m, 4H, 8,300,400,500-H),

C.G. Neochoritis et al. / European Journal of Medicinal Chemistry 46 (2011) 297e306
305
C25H21N3O3 (411.45): C, 72.98; H, 5.14; N, 10.21%. Found: C, 73.11; H,
5.22; N, 10.07%. (*The assignments may be interchanged).
values of compounds 3 and 4 as C log P values in n-octanol/buffer
by C log P Programme of Biobyte Corp. [43].
5.2.15. 4-Methyl-2,4-bis(4-chlorophenyl)pyrimido[1,2-a]
benzimidazole-3(4H)-one 4f
5.4. Antioxidant activity
Yield: 0.387 g (92%); orange crystals; mp 215e217 ꢀC (ethanol). IR
Each in vitro experiment was performed at least in triplicate, the
results were averaged and the standard deviation of absorbance was
less than 10% of the mean and the results are presented in Table 2.
(KBr): 1698 (C]O) cmꢁ1
.
1H NMR (CDCl3):
d
¼ 2.34 (s, 3H, 4-Me),
6.99 (dd, J ¼ 8.1, 1.1 Hz, 1H, 6-H), 7.01 (d, J ¼ 8.9 Hz, 2H, 200,600-H), 7.24
(ddd, J ¼ 8.1, 7.0, 1.1 Hz, 1H, 7-H), 7.32 (d, J ¼ 8.9 Hz, 2H, 300,500-H), 7.35
(ddd, J ¼ 8.1, 7.0, 1.1 Hz, 1H, 8-H), 7.42 (d, J ¼ 8.9 Hz, 2H, 30,50-H), 7.95
(dd, J ¼ 8.1, 1.1 Hz, 1H, 9-H), 8.09 (d, J ¼ 8.9 Hz, 2H, 20,60-H). 13C NMR
5.4.1. Determination of the reducing activity of the stable radical
DPPH [49]
(CDCl3):
d
¼ 24.3 (4-Me), 69.7 (C-4),112.0 (C-6), 122.3 (C-9),123.8 (C-
To an ethanolic solution of DPPH (0.05 mM) in absolute ethanol
an equal volume of the compounds (final concentration 0.05 mM)
dissolved in DMSO was added. The mixture was shaken vigorously
and allowed to stand for 20 min or 60 min; absorbance at 517 nm
was determined spectrophotometrically and the percentage of
activity was calculated. All tests were undertaken on three repli-
cates and the results were averaged (Table 2).
8), 125.6 (C-7), 127.6 (C-200,600), 128.9 (C-300,500), 129.8 (C-30,50), 131.1
(C-10),131.3 (C-20,60),133.9 (C-5a),135.2 (C-100),135.6 (C-400), 139.1 (C-
40), 144.4 (C-9a), 147.5 (C-10a), 156.8 (C-2), 188.1 (C-3). LCeMS (ESI,
1.65 eV): m/z (%) ¼ 474/476/478 (Mþ þ Na þ MeOH, 40), 452/454/
456 (Mþ þ H þ MeOH, 50), 442/444/446 (Mþ þ Na, 35), 420/422/424
(Mþ þ H, 100). Anal. Calcd for C23H15Cl2N3O (420.29): C, 65.73; H,
3.60; N, 10.00%. Found: C, 65.65; H, 3.53; N, 10.17%.
5.4.2. Inhibition of linoleic acid lipid peroxidation [42]
5.2.16. 4-Methyl-2,4-bis(4-bromophenyl)pyrimido[1,2-a]
benzimidazole-3(4H)-one 4g
The water soluble azo compound AAPH is used as a free radical
initiator for in vitro studies of free radical production. Production of
conjugated diene hydroperoxide by oxidation of linoleic acid
sodium salt in an aqueous solution is monitored at 234 nm. This
assay can be used to follow oxidative changes and to understand
the contribution of each tested compound.
Yield: 0.474 g (93%); orange crystals; mp 210e212 ꢀC (ethanol).
IR (KBr): 1705 (C]O) cmꢁ1. 1H NMR (CDCl3):
d
¼ 2.34 (s, 3H, 4-Me),
6.94 (d, J ¼ 8.4 Hz, 2H, 200,600-H), 6.99 (dd, J ¼ 8.1,1.1 Hz,1H, 6-H), 7.24
(dd, J ¼ 8.1, 7.1,1.1 Hz,1H, 7-H), 7.35 (ddd, J ¼ 8.1, 7.1,1.1 Hz, 1H, 8-H),
7.48 (d, J ¼ 8.3 Hz, 2H, 300,500-H), 7.58 (d, J ¼ 8.5 Hz, 2H, 30,50-H), 7.95
(dd, J ¼ 8.1, 1.1 Hz, 1H, 9-H), 8.01 (d, J ¼ 8.5 Hz, 2H, 20,60-H),. 13C NMR
10
mL of the 16 mM linoleic acid sodium salt solution was added
to the UV cuvette containing 0.93 mL of 0.05 M phosphate buffer,
(CDCl3):
d
¼ 24.2 (4-Me), 69.8 (C-4),112.0 (C-6),122.3 (C-9),123.8 (C-
pH 7.4 prethermostated at 37 ꢀC. The oxidation reaction was initi-
400), 123.9 (C-8), 125.7 (C-7), 127.9 (C-200,600), 128.0 (C-40), 131.4 (C-
300,500), 131.5 (C-10), 131.9 (C-20,60), 132.8 (C-30,50), 133.9 (C-5a), 135.7
(C-100), 144.4 (C-9a), 147.5 (C-10a), 156.9 (C-2), 187.9 (C-3). LCeMS
(ESI, 1.65 eV): m/z (%) ¼ 562/564/566 (Mþ þ Na þ MeOH, 30), 540/
542/544 (Mþ þ H þ MeOH, 50), 530/532/534 (Mþ þ Na, 20), 508/
510/512 (Mþ þ H, 100). Anal. Calcd for C23H15Br2N3O (509.19): C,
54.25; H, 2.97; N, 8.25%. Found: C, 54.36; H, 3.03; N, 8.37%.
ated at 37 ꢀC under air by the addition of 50
m
L of 40 mM AAPH
solution. Oxidation was carried out in the presence of aliquots
(10 L) in the assay without antioxidant, lipid oxidation was
m
measured in the presence of the same level of DMSO. The rate of
oxidation at 37 ꢀC was monitored by recording the increase in
absorption at 234 nm caused by conjugated diene hydroperoxides.
5.4.3. Soybean LOX inhibition study in vitro [49]
5.2.17. 4-Methyl-2,4-bis(3-bromophenyl)pyrimido[1,2-a]
benzimidazole-3(4H)-one 4h
The tested compounds dissolved in DMSO were incubated at RT
with sodium linoleate (0.1 mL) and 0.2 mL of enzyme solution
(1/9 ꢂ 10ꢁ4 w/v in saline). The conversion of sodium linoleate to 13-
hydroperoxylinoleic acid at 234 nm was recorded and compared
with the appropriate standard inhibitor.
Yield: 0.448 g (88%); orange crystals; mp 62e64 ꢀC (ethanol). IR
(KBr): 1705 (C]O) cmꢁ1. 1H NMR (CDCl3):
d
¼ 2.32 (s, 3H, 4-Me), 6.91
(ddd, J ¼ 7.9, 1.7, 1.0 Hz, 1H, 600-H), 6.97 (dd, J ¼ 8.1 Hz, 1H, 6-H), 7.18 (t,
J ¼ 7.9 Hz, 1H, 500-H), 7.23 (dd, J ¼ 8.1, 7.2 Hz,1H, 7-H), 7.30 (t, J ¼ 7.9 Hz,
1H, 50-H), 7.33 (dd, J ¼ 8.1, 7.2 Hz, 1H, 8-H), 7.37 (t, J ¼ 1.7 Hz, 1H, 200-H),
7.48 (ddd, J ¼ 7.9, 1.7, 1.0 Hz, 1H, 400-H), 7.64 (ddd, J ¼ 7.9, 1.7, 1.0 Hz, 1H,
40-H), 7.94 (dd, J ¼ 8.1 Hz,1H, 9-H), 8.07 (ddd, J ¼ 7.9, 1.7, 1.1 Hz, 1H, 60-
5.4.4. Cytotoxicity in human lung fibroblast cell line
5.4.4.1. Cell culture. Human lung fibroblasts p19 were cultured in
DMEM supplemented with 10% fetal bovine serum (FBS) and
antibiotics penicillin and streptomycin. Cell line was maintained in
continuous culture at 37 ꢀC, in 5% CO2, in a fully humidified incu-
bator, using standard aseptic techniques and cell growth was
monitored by determining the cell number/mL with the use of
a Coulter counter model ZBI.
H), 8.33 (t, J ¼ 1.7 Hz, 1H, 20-H). 13C NMR (CDCl3):
¼ 24.4 (4-Me), 69.7
d
(C-4),112.0 (C-6), 122.5 (C-9), 122.7 (C-300), 123.93 (C-30), 123.090 6 (C-8),
124.8 (C-600),125.800 (C-7),128.6 (C-60),129.4 (C-200),* 130.0 (C-5 ),* 131.0
(C-50), 132.6 (C-4 ),* 132.7 (C-20),* 133.9 (C-5a), 134.6 (C-10), 135.2 (C-
40), 139.0 (C-100), 144.5 (C-9a), 147.3 (C-10a), 156.2 (C-2), 187.8 (C-3).
LCeMS (ESI, 1.65 eV, pos. polarity): m/z (%) ¼ 562/564/566
(Mþ þ Na þ MeOH, 1), 540/542/544 (Mþ þ H þ MeOH, 3), 508/510/
512 (Mþ þ H, 5), 153 (100). LCeMS (ESI, 1.65 eV, neg. polarity): m/z
(%) ¼ 538/540/542 (Mþ e H þ MeOH, 100), 524/526/528 (30), 508/
510/512 (Mþ þ H, 30), 314/316 (60), 205 (98). Anal. Calcd for
C23H15Br2N3O (509.19): C, 54.25; H, 2.97; N, 8.25%. Found: C, 54.12; H,
3.12; N, 8.15%. (*The assignments may be interchanged).
5.4.4.2. MTT assays. Cell proliferation was assessed by monitoring
the conversion of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) to formazan. The reduction of MTT is
catalyzed by mitochondrial dehydrogenase enzyme and is there-
fore a measure for cell viability [68]. The MTT assay was used as
a cytotoxicity assay for lung fibroblastic cells grown in the presence
of the studied compounds. Briefly, cells (3 ꢂ104 cells mLꢁl) were
seeded in 48-well plates and after 24 h were treated with varying
5.3. Physicochemical studies
concentrations of the studied compounds (1e50 mg/mL) for 24 h.
5.3.1. Determination of lipophilicity as C log P values
Briefly, 400 mL of freshly prepared MTT solution (5 mg/mL in PBS)
were added to each well e after removal of culture medium and
washing with PBS. Following incubation at 37 ꢀC for 4e6 h in a fully
humidified atmosphere at 5% CO2, MTT was taken up by active cells
Since lipophilicity is a significant physicochemical property
determining distribution, bioavailability, metabolic activity and
elimination, we tried to calculate theoretically the lipophilicity

306
C.G. Neochoritis et al. / European Journal of Medicinal Chemistry 46 (2011) 297e306
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71 (2006) 7165e7179.
and reduced in the mitochondria to insoluble formazan granules.
Subsequently, the medium was discarded and the precipitated
formazan was dissolved in isopropanol (400 mL/well). The Organon
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Teknika @ Enterprise Ireland plate was used to measure the
absorbance of the plates at 570 nm. Values shown are the standard
deviation of at least three measurements.
[44] MOPAC 2000 ver. 1.11, Fujitsu.
Acknowledgement
[45] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb,
J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant,
J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi,
G. Scalmani, N. Rega, G.A. Peterson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota,
R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai,
M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi,
C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador,
J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain,
O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz,
Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko,
P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, Y.C. Peng,
A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen,
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ingford, CT, 2004.
We thank Dr C. Hansch and Biobyte Corp. 201 West 4th Str.,
Suite 204, Claremont CA California, 91711, USA for free access to the
C-QSAR program.
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Abbreviations
AAPH: 2,20-azobis(2-amidinopropane) dihydrochloride
BHT: butylated hydroxy toluene
C log P: logarithm of partition coeficient log(Coctanol/Cwater
)
COLOC: correlation via long-range coupling 2JCH or JCH
3
n
COSY: 2D HeH correlation spectroscopy via JHH
CYP: cytochrome P450
DMEM: Dulbecco’s/Vogt modified eagle minimal essential medium
DPPH: 2,20-diphenyl-1-picrylhydrazyl
FBS: fetal bovine serum
GIAO: gauge independent atomic orbital
HETCOR: 2D heteronuclear correlation via JCH
HMQC: heteronuclear multiple quantum coherence, 2-D inverse H,C correlation
HMBC: heteronuclear multiple bond coherence, 2-D inverse H,C correlation
LOX: lipoxygenase peroxydation
LPO: lipid peroxydation
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MW: microwave irradiation
1
NADPH: nicotinamide adenine dinucleotide phosphate
NDGA: nordihydroguairetic acid
Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid