Synthesis and biological evaluation of heterocyclic privileged medicinal structures containing (benz)imidazole unit

Article abstract of DOI:10.1007/s00706-016-1733-7

Abstract: New heterocyclic privileged medicinal scaffolds involving 4H-pyran, 1,4-dihydropyridine, quinazoline, and 2-aminochromene bearing a (benz)imidazole unit were prepared in good to excellent yields, and evaluated for their in vitro hepatotoxicity on HepG2 cells and antioxidant activity using DPPH radical-scavenging assay. From these results, 2-amino-4-(1-methyl-1H-imidazol-2-yl)-4H-benzo[h]chromene-3-carbonitrile has been identified as a racemic, readily available imidazole derivative, significantly less hepatotoxic than tacrine at 100?μM concentration. One compound was found to be a potent antioxidant agent. The catalytic effect of the 1-methylimidazole unit, in the synthesis of some heterocycle, was also demonstrated. Crystal X-ray structures are reported for six compounds. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-016-1733-7

Monatsh Chem
DOI 10.1007/s00706-016-1733-7
ORIGINAL PAPER
Synthesis and biological evaluation of heterocyclic privileged
medicinal structures containing (benz)imidazole unit
1
1
2
1
•
Houssem Boulebd
3
•
Sana Zama
Hocine Merazig
•
Bataiche Insaf
3
•
Abdelmalek Bouraiou
4
5
•
Sofiane Bouacida
Jos e´ Marco-Contelles
•
•
Alejandro Romero
•
Mourad Chioua
5
1
•
Ali Belfaitah
Received: 22 January 2016 / Accepted: 7 March 2016
Ó Springer-Verlag Wien 2016
Abstract New heterocyclic privileged medicinal scaffolds
involving 4H-pyran, 1,4-dihydropyridine, quinazoline, and
synthesis of some heterocycle, was also demonstrated.
Crystal X-ray structures are reported for six compounds.
Graphical abstract
2
-aminochromene bearing a (benz)imidazole unit were
prepared in good to excellent yields, and evaluated for their
in vitro hepatotoxicity on HepG2 cells and antioxidant
activity using DPPH radical-scavenging assay. From these
results, 2-amino-4-(1-methyl-1H-imidazol-2-yl)-4H-ben-
zo[h]chromene-3-carbonitrile has been identified as a
racemic, readily available imidazole derivative, significantly
less hepatotoxic than tacrine at 100 lM concentration. One
compound was found to be a potent antioxidant agent. The
catalytic effect of the 1-methylimidazole unit, in the
R
Ph
N
N
H2N
NC
N
O
H2N
O
NC
R
O
NC
NH2
O
N
N
CHO
O
Me
N
N
Me
H2N
CN
O
(Benz)imidazol-2-carbaldehyde
EtO2C
HO
HN
CO2Et
O
O
NC
O
H2N
N
H
Keywords (Benz)imidazole Á Polyheterocycle Á
Hepatotoxicity Á DPPH Á Crystal structure
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-016-1733-7) contains supplementary
material, which is available to authorized users.
&
Ali Belfaitah
abelbelfaitah@yahoo.fr
Introduction
1
Laboratoire des Produits Naturels d’Origine V e´ g e´ tale et de
Synth e` se Organique. Facult e´ des Sciences Exactes, Campus
de Chaabat Ersas, Universit e´ des fr e` res Mentouri-
Constantine, 25000 Constantine, Algeria
Heterocyclic compounds, and particularly those bearing
nitrogen and oxygen atoms, such as 4H-pyran, quinazoline,
1,4-dihydropyridine (DHP), and chromene, continue to
attract an increasing interest due to their wide range of
pharmacological activities [1–9]. The imidazole and ben-
zimidazole are common heterocyclic motifs present in
some well-known components of human organisms, such
as histidine, vitamin B12, purines, histamine, and biotin.
These cores were also found in many natural products [10,
2
3
Laboratoire de Mycologie, de Biotechnologie et de l’Activite´
Microbienne, Facult e´ des Sciences de la Nature et de vie,
Universit e´ des fr e` res Mentouri-Constantine, 25000
Constantine, Algeria
Unit e´ de Recherche de Chimie de l’Environnement et
Mol e´ culaire Structurale, Campus de Chaabat Ersas,
Universit e´ des fr e` res Mentouri-Constantine, 25000
Constantine, Algeria
11], as lepidiline A and B [12] or synthetic molecules such
as dacarbazine [13], zoledronic acid [14], tipifarnib [15],
and azathioprine [16, 17], showing significant cytotoxicity
against various types of human cancer cell lines. To
improve the pharmacological profile associated with
(benz)imidazoles, a number of investigations have been
4
5
Department of Toxicology and Pharmacology, School of
Veterinary, Complutense University of Madrid, 28040
Madrid, Spain
Laboratory of Medicinal Chemistry (IQOG, CSIC), C/Juan
de la Cierva 3, 28006 Madrid, Spain
123

H. Boulebd et al.
carried out by coupling them to other heterocyclic systems
(benz)imidazole-2-carbaldehyde to the corresponding a-
cyanoacrylonitriles 3 and 4 proceeded cleanly with 94 and
67 % yield, respectively, by reaction with malononitrile
(1–1.1 equiv), in EtOH at room temperature (in the case of
compound 4, a few drops of piperidine were added).
First of all, and based on our previous work [32–34], we
explored the synthesis of 4H-pyran derivatives. Polyfunc-
tionalized 4H-pyrans are common structural units in a
number of natural products [38]. The 4H-pyran ring can be
transformed into pyridine systems related to pharmaco-
logically important DHP type of calcium antagonists [39].
The presence of a 4H-pyran moiety in organic molecules
imparts them an extensive range of biological and phar-
macological properties, such as spasmolytic, diuretic, anti-
coagulant, anti-cancer, and anti-anaphylactic activities
[
18–22], or using them as bio-isostere [23–28].
In connection with our research on the preparation of
new heterocyclic compounds with biological interest pos-
sessing an imidazole unit [29–31] and the synthesis and
biological evaluation of tacrine analogs [32–34], we
describe here the synthesis of new highly functionalized
2
-substituted (benz)imidazole derivatives, and the evalua-
tion of cytotoxicity and antioxidant properties.
Furthermore, the in vitro hepatotoxicity test on HepG2 was
carried out in the context of a project targeted to identify
new suitable heterocyclic b-enaminonitrile precursors for
the synthesis of non-toxic tacrine analogs for the potential
treatment of Alzheimer’s disease (AD).
[
40–44]. Highly substituted pyran/chromenes tethered with
Results and discussion
Chemistry
–NH and –CN functionalities possess diverse pharmaco-
2
logical properties [45–47]; they can also be used as
precursors for designing new tacrine analogs for the
potential treatment of Alzheimer’s disease [32–34].
1
-Methyl-1H-(benz)imidazole derivatives 1–4 have been
2-Amino-3-cyano-4H-pyrans 5a–5f bearing an imida-
zole or benzimidazole moiety was prepared in a ‘‘one-pot’’
reaction. The treatment of (benz)imidazole 2-carbaldehy-
des 1 (or 2) with malononitrile gave (benz)imidazole-a-
cyanoacrylonitrile derivatives, which were immediately
reacted in situ with acetylacetone, ethyl acetoacetate, or
dimedone to afford the 2-(4H-pyran)-(benz)imidazole
derivatives 5a–5f (Table 1; Scheme 2). For the synthesis of
compounds 5a, 5c, and 5d, the reaction was generally over
in 2 h, in good yields, and no catalyst was required. The
molecular structures of compounds 5c, 5d, and 5f (Fig. 1
used as starting materials to prepare the diverse heterocy-
cle-(benz)imidazoles reported here. The synthetic
pathways adopted for their preparations are outlined in
Scheme 1. Known 1-methyl-1H-imidazole-2-carbaldehyde
(1) was prepared from 1-methyl-1H-imidazole via the
carbanion generated at C-2 with n-BuLi in the presence of
DMF, according to a modified procedure [35]. 1-Methyl-
1H-benzo[d]imidazole-2-carbaldehyde (2) was obtained
via an addition–cyclization/methylation sequence [36, 37],
followed by oxidation with SeO . The conversion of the
2
Scheme 1
1
- Glycolic acid,
NaOH , H2O/EtOH,
HCl 4N, 90 °C
NH2
NH2
N
OH
OH
(CH ) SO , 0-5 °C
3 2
4
N
N
2- NH4OH 10%
24 h, 88%
0.5 h, 72%
N
H
Me
1
2
3
-n-BuLi, THF, -15 °C
- DMF, THF, -15 °C
- H2O, rt
N
N
72%
NC
CH2(CN)2, EtOH, rt
(piperidine for 4)
Me
N
N
N
CN
CHO
For 3: 1 h, 94%
For 4: 2.5 h, 67%
N
SeO2, 80-90 °C,
toluene
Me
Me
1
, 2
3, 4
N
N
2
h, 79%
Me
OH
1
23

Synthesis and biological evaluation of heterocyclic privileged medicinal structures…
Table 1 Synthesis of
benz)imidazole-4H-pyran, -2-
aminochromene, and -1,4-
dihydropyridines
R = imidazole
Comp. Yield/%
R = benzimidazole
(
Structure
a
a
Comp.
Yield/%
R
EtO
2
C
CN
NH
5a
5c
5e
92
5b
85
Me
O
O
R
2
CN
90
69
5d
5f
73
66
O
R
NH
2
MeOC
Me
CN
O
R
NH
2
CN
NH
6
a
75
6b
95
O
2
R
O
CN
6
8
c
72
73
6d
8b
90
77
NH
2
R
EtO
2
C
CO
2
Et
a
Me
O
N
H
Me
R
O
8
c
80
N
H
a
Yield of pure product
Scheme 2
O
N
N
N
N
Me
Me
X
HO
NC
O
X=CH, N
EtOH, reflux
CN
NH2
3
CN
N
N
CN
EtOH, rt
4
7
2-95%
66-92%
O
2 O
a-5f
NH2
Me
3, 4
5
6
a-6d
O
OH
40%
OH
Ca(OH)2,
MeOH, rt.
N
N
Me
NC
OH
O
H N
2
O
7
123

H. Boulebd et al.
2-naphthol) to give the corresponding 2-aminochromene
derivatives 6a–6d (Table 1; Scheme 2).
Similarly, the reaction of 1-methyl-1H-imidazole-a-
cyanoacrylonitrile (3) with 2,4-dihydroxyacetophenone at rt
in the presence of Ca(OH) , according to the procedure
2
reported by Kolla and Lee [51], gives the corresponding 4H-
pyran 7 (Scheme 2) in moderate yield (40 %). Themolecular
structure of 7 was confirmed by single crystal X-ray
diffraction analysis (Fig. S4 in Supplementary Material).
Next, we expanded this work to the preparation of
azoles such as DHPs coupled to the (benz)imidazole unit.
4-Aryl-DHPs are a class of calcium channel blockers and,
to improve their pharmacological profile, a number of
investigations have been carried out by replacing the 4-aryl
substituent by various heterocycles [52–56]. Consequently,
we considered the synthesis of compounds containing a
DHP entity linked to (benz)imidazole unit.
Fig. 1 ORTEP plot of the X-ray crystal structure of compound 5c.
Displacement ellipsoids are drawn at the 50 % probability level
and Fig. S1, S2 in Supplementary Material) were confirmed
by crystal X-ray diffraction analysis.
Symmetrical DHP analogs 8a–8c (Table 1, Scheme 3)
were prepared by the classical Hantzsch reaction of the
(benz)imidazole-2-carbaldehyde 1 (or 2) with ethyl acetoac-
etate or dimedone in ethanol, and in the presence of a large
excess of ammonia. Single crystals of 8a were grown by slow
2
-Aminochromenes are important compounds in many
naturally occurring products used as cosmetics, pigments,
and potential biodegradable agrochemicals [48, 49]. These
compounds are usually synthesized by reaction of benzyli-
denemalononitriles and 1-naphthol in organic solvents in the
presence of organic bases such as piperidine or 4-methyl-
morpholine, which are frequently used in stoichiometric
amounts [50]. In this context, 1-methyl(benz)imidazole-a-
cyanoacrylonitrile 3 (or 4) was treated with 1-naphthol (or
evaporation of a CHCl solution and X-ray crystallographic
3
analysis confirmed the structural assignment (Fig. 2).
In a parallel approach, we explored the synthesis of
heterocycle containing two nitrogen atoms, such as
quinazoline possessing an imidazole subunit. It is well
known that modified quinazoline derivatives have shown
Scheme 3
O
1
- Dimedone, p-
N
N
anisidine, EtOH,
reflux, 24 h
N
N
Me
Me
O
O
1
, 2
CN 2-Et3N (cat.), reflux 1 h
NH4OH, EtOH, reflux
N
N
CHO
7
7%
73-80%
N
NH2
N
H
Me
8
a-8c
OMe
2
-Aminobenzophenone
3
h, 53%
AcONH4, I2, EtOH, 40 °C
9
N
N
N
Me
N
1
0
1
23

Synthesis and biological evaluation of heterocyclic privileged medicinal structures…
In vitro hepatotoxicity evaluation
Hepatotoxicity of drug candidates represents one of the
crucial factors for their possible clinical development, and
the in vitro screening is a valuable tool for preselection of
the best drug candidates in preclinical research reducing
the high costs in drug development [67]. This is particu-
larly of interest in projects targeted to design new tacrine
analogs for AD, due to the fact that tacrine was withdrawn
from the clinics for its secondary toxic effects on the liver
and hepatotoxicity. The in vitro screening of the hepato-
toxic activity of the newly synthesized compounds was
performed on human hepatoma cell line HepG2 [68], the
best-characterized and most frequently used cell line with
respect to hepatotoxic end points to test the metabolism and
liver toxicity of several drugs. For comparative purposes,
tacrine was used as a standard control. The results of the
cytotoxicity screening of the prepared compounds are
summarized in Table 2.
Fig. 2 ORTEP plot of the X-ray crystal structure of compound 8a.
Displacement ellipsoids are drawn at the 50 % probability level
The cytotoxicity of (benz)imidazole-4H-pyrans 5a–5f,
6
a–6d, 7, DHPs 8a–8c, 9, and quinazoline 10 was deter-
mined at micromolar concentrations ranging from 1, 3, 10,
0, 100, to 300 lM, using the MTT assay [69]. These
3
compounds were well tolerated after a 24 h incubation
period in human HepG2 cells compared with tacrine.
As shown in Table 2, and not surprisingly, tacrine sig-
nificantly decreased the number of living cells, being more
hepatotoxic gradually at higher doses, particularly at 100
and 300 lM concentrations. In contrast, all evaluated
compounds showed a reduced hepatotoxicity compared
with tacrine, especially at the highest concentrations used,
Fig. 3 ORTEP plot of the X-ray crystal structure of compound 10.
Displacement ellipsoids are drawn at the 50 % probability level
100 lM being the concentration that we are going to use
remarkable anti-cancer, anti-tubercular, and antibacterial
activities [57–62]. In this context and in accordance
with literature [63], 2-(1-methyl-1H-imidazol-2-yl)-4-
phenylquinazoline (10) was prepared in a ‘‘one-pot’’ three-
component reaction of 1-methyl-1H-imidazole-2-carb-
aldehyde (1), o-aminobenzophenone, ammonium acetate,
and iodine as catalyst (Scheme 3). It is important to note
that no purification was needed and compound 10 was
isolated in pure form in 53 % of yield by simple filtration
and washing with ethanol. The molecular structure of 10
was confirmed by single crystal X-ray diffraction analyses
next to discuss the observed structure–activity relationships
(SAR) on this series of compounds.
At the 100 lM concentration, and according to the
percentage of cell viability test, the four less toxic com-
pounds were, in decreasing order: 6a (85.7 %), 5c
(83.3 %), 5f (82.6 %), and 5e (82.2 %), with tacrine
showing a significant lower (64.3 %) value at the same
concentration. The least hepatotoxic compound, 6a, was
1.3-fold less toxic than tacrine. This means that the pyrans,
regardless of the type of the substituents at C2 and C3 in
the central heterocyclic core, are less toxic than DHPs.
Among the analyzed pyrans, 1-methyl(benz)imidazole-
4H-pyrans 5a–5f (Table 2, entries 1–6) are clearly less
hepatotoxic than tacrine at the different concentrations
tested. Compounds 5c (entry 3) and 5f (entry 6) have the
best non-hepatotoxic profile at 100 lM concentration with
83.3 and 82.6 % cell viability, respectively. For the same
type of substituent at C2 and C3, pyrans bearing the imi-
(
Fig. 3).
Note that the preparation of compound 3 and 1-methyl-
H-imidazol-2-yl derivatives 5a, 5c, 5e, 6a, 6c, 8a, and 8c
1
can be achieved well in a short time reaction, without
catalyst, the 2-substituted-1-methyl-1H-imidazole playing
itself this role [64–66]. In contrast, the synthesis of hete-
rocyclic compounds bearing a 1-methyl-1H-benzimidazole
unit required piperidine as catalyst and a longer time. A
plausible reaction mechanism has been detailed in Sup-
plementary Material (Scheme S1).
dazole ring are less (compare pyrans bearing C3 = CO Et;
2
C2 = Me: 5a (76.8 %) and 5b (71.7 %); C3 = COMe;
C2 = Me: 5c (83.3 %) and 5d (72.2 %)) or similarly
123

H. Boulebd et al.
Table 2 In vitro toxicity of 1-methyl-1H-(benz)imidazole-heterocycles 5–10 and tacrine in HepG2 cells
a
Entry
Compound
Viability (%) HepG2 cells
1
lM
3 lM
10 lM
30 lM
100 lM
300 lM
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
1
2
3
4
5
6
7
8
9
5a
5b
5c
99.9 ± 1.47
97.1 ± 0.79
95.4 ± 1.51
97.3 ± 1.58
99.3 ± 1.77
98.9 ± 0.22
97.1 ± 0.70
99.2 ± 1.33
98.6 ± 0.97
100 ± 0.73
96.6 ± 1.33
95.1 ± 2.89
94.1 ± 1.28
97.5 ± 0.88
95.4 ± 0.45
95.6 ± 1.19
93.4 ± 4.69
92.5 ± 0.95**
91.2 ± 0.4*
81.8 ± 1.40***
84.9 ± 2.50***
85.0 ± 0.62***
89.0 ± 2.39**
89.8 ± 0.54*
76.4 ± 1.32***
73.4 ± 1.73***
83.7 ± 0.49***
85.8 ± 1.83***
87.0 ± 1.23**
84.3 ± 0.57***
86.6 ± 0.32*
76.8 ± 1.75***
71.7 ± 1.81***
83.3 – 0.72***
72.2 ± 0.58***
82.2 ± 0.91***
82.6 – 1.79***
85.7 – 1.86***
81.6 ± 1.64***
77 ± 3.36***
72.6 ± 0.33***
72.3 ± 2.92***
80.8 ± 1.41***
74.9 ± 2.19***
71.7 ± 3.71***
77.6 ± 1.38***
78.0 ± 0.62***
78.2 ± 1.42***
70.5 ± 2.97***
80.8 ± 1.83***
67.4 ± 1.42***
73.0 ± 0.51***
63.4 ± 0.34***
78.2 ± 1.23***
71.4 ± 3.54***
61.5 ± 2.11***
40 ± 2.20***
91.5 ± 2.00*
90.9 ± 1.44*
ns
5d
5e
94.3 ± 1.06
5f
89.4 ± 0.95***
ns
95 ± 0.83
85.1 ± 1.00***
ns
93.2 ± 0.54
6a
6b
6c
ns
ns
96.2 ± 1.55
97 ± 1.08
83.4 ± 1.81*
ns
ns
93.5 ± 1.47
91.7 ± 2.08
78.8 ± 3.66*
ns
ns
10
11
12
13
14
15
16
17
6d
7
97.8 ± 1.04
96.6 ± 1.95
81.4 ± 0.85*
81.5 ± 0.39***
71.1 ± 1.64***
76.1 ± 1.55***
69.1 ± 4.15***
80.7 ± 0.68***
75.2 ± 2.91***
77.1 ± 1.47***
64.3 – 4.54***
ns
ns
88 ± 1.55
84.8 ± 1.08
82.3 ± 1.81*
8a
8b
8c
87.7 ± 0.99**
ns
85.8 ± 4.38
81.5 ± 1.90***
77.5 ± 3.69**
87.7 ± 1.34***
90.1 ± 2.03*
82.1 ± 2.12***
75.6 ± 4.05***
81.4 ± 1.22***
84.0 ± 1.69***
85.2 ± 1.46***
81.6 ± 4.88*
91.0 ± 2.30**
ns
94.2 ± 0.46
9
10
Tacrine
92.8 ± 1.40*
ns
90 ± 2.95
89.3 ± 1.38***
ns
88.7 ± 3.42
*** p \ 0.001, ** p \ 0.01, * p \ 0.05 and ns not significant, with respect to the control group. Comparisons between drugs and control group
were per-formed by one-way ANOVA followed by the Newman-Keuls post hoc test
a
Cell viability was measured as MTT reduction and data were normalized as % control. Data are expressed as the mean ± SEM of triplicate of
at least four different cultures. All compounds were assayed at increasing concentrations (1–300 lM)
(
compare pyrans bearing C2/C3: 3,3-dimethylcyclohexan-
-one: 5e (82.2 %) and 5f (82.6 %)) toxic than those
in pyrans of type 5, for the same arrangement
(C3 = CO Et; C2 = Me), the pyran bearing the imidazole
1
2
bearing the benzimidazole nucleus. In this group, we
observed also that for the pyrans bearing an imidazole ring
at C4, 5c bearing a C3 = COMe; C2 = Me substitution
was less toxic, and that for the pyrans bearing a benzimi-
ring at C4 (8a 76.1 %) was less hepatotoxic than the one
bearing a benzimidazole ring at C4 (8b 69.1 %).
Compound 10 (Table 2, entry 16) is the least efficient of
all compounds tested at higher concentration. Nevertheless,
it is much less toxic than tacrine with 77.1 vs. 64.3 % cell
viability at 100 lM.
dazole ring at C4, 5f bearing
dimethylcyclohexan-1-one substitution was less toxic.
Among the analyzed pyrans, 1-methyl(benz)imidazole-
a
C2/C3; 3,3-
4
H-pyrans 6a–6d (Table 2, entries 7–10) are clearly less
DPPH radical scavenging
hepatotoxic than tacrine at 100 lM concentration. Com-
pound 6a (entry 7) has the best non-hepatotoxic profile,
with 85.7 % cell viability. For the same type of naph-
thalene-fused ring, pyrans bearing the imidazole ring are
The in vitro antioxidant activity of all prepared compounds
was carried out by the DPPH assay [70] which measures
the hydrogen-donating ability of antioxidants to convert
stable DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical
to 2,2-diphenyl-1-picrylhydrazine. Ascorbic acid (Asc) was
used as a standard antioxidant. The results are expressed as
mean ± standard deviation (SD) and summarized in
Table 3.
0
0
less (compare pyrans bearing a C1 , C2 -naphthalene; 6a
85.7 %) and 6b (81.6 %)) or more [compare pyrans
(
0
0
bearing a C2 , C1 -naphthalene; 6c (77 %) and 6d
81.5 %)] toxic than those bearing the benzimidazole
(
nucleus. Finally, note that among the pyran derivatives,
compound 7 (Table 2, entry 11) was the most toxic.
Among the analyzed 1-methyl(benz)imidazole-1,4-di-
hydropyridines 8a–8c, 9 (Table 2, entries 12–15), DHP 8c
bearing a double 3,3-dimethylcyclohexan-1-one substitu-
tion at C2/C3 and C5/C6 showed lower hepatotoxicity than
tacrine (80.7 % at 100 lM). It is also interesting to high-
light that, in good agreement with what we have observed
As shown in Table 3, all the tested compounds show a
relatively poor radical-scavenging capability at t = 0
(*20 %), and no significant change was observed over
time (0–24 h) for most of them. The replacement of the
imidazole ring with benzimidazole in analogs does not
have an influence on the DPPH activity. However, a
noticeable increase in the antioxidant activity was detected
1
23

Synthesis and biological evaluation of heterocyclic privileged medicinal structures…
Table 3 DPPH radical-scavenging activities of 1-methyl-1H-(benz)imidazole-heterocycles 5–10
a
Entry
Compound
Percentage inhibition of DPPH free radical (I %)
0
min
30 min
3 h
24 h
1
2
3
4
5
6
7
8
9
5a
5b
5c
5d
5e
5f
19.65 ± 0.94
22.84 ± 2.46
21.66 ± 0.55
21.97 ± 2.93
20.19 ± 0.95
18.16 ± 0.20
20.09 ± 2.91
19.91 ± 3.34
21.35 ± 3.01
18.99 ± 0.56
23.63 ± 1.34
21.19 ± 3.56
19.56 ± 0.78
20.56 ± 0.79
26.13 ± 3.02
22.14 ± 0.61
86.22 ± 1.62
21.07 ± 3.02
19.44 ± 1.60
21.57 ± 1.41
21.99 ± 1.95
23.27 ± 1.05
18.14 ± 1.34
20.27 ± 1.00
18.42 ± 2.93
21.35 ± 1.30
17.88 ± 1.10
19.50 ± 0.88
22.08 ± 3.05
19.39 ± 0.34
18.89 ± 2.16
31.56 ± 2.33
19.30 ± 1.62
84.11 ± 1.55
24.51 ± 0.79
23.60 ± 2.30
23.91 ± 4.45
23.85 ± 1.68
25.12 ± 1.03
25.14 ± 0.23
21.88 ± 4.30
18.57 ± 3.78
21.74 ± 0.17
21.60 ± 0.31
23.96 ± 2.09
23.51 ± 1.33
22.53 ± 0.97
21.80 ± 3.04
46.10 ± 0.34
24.35 ± 1.26
83.83 ± 1.38
21.29 ± 3.43
19.16 ± 0.94
24.59 ± 1.71
22.39 ± 2.47
42.84 ± 0.50
36.06 ± 5.17
18.15 ± 1.88
23.71 ± 4.29
19.88 ± 3.33
27.39 ± 0.46
21.50 ± 0.83
22.13 ± 1.09
25.28 ± 4.25
18.34 ± 3.31
67.82 ± 0.70
20.86 ± 1.91
78.13 ± 1.19
6a
6b
6c
6d
7
10
11
12
13
14
15
16
8a
8b
8c
9
10
Asc
Standard
a
Data are an average for triplicate determinations
after 24 h incubation for 4H-pyran compounds 5e (Table 3,
entry 5) and 5f (Table 3, entry 6) tethered with –NH and –
motifs. Our synthetic approach allowed us to prepare 16 new
compounds of biological importance in good to excellent
yields, without the need of chromatographic separation. The
free radical-scavenging activity for all prepared compounds
was measured by DPPH. The data reported indicates that
compound 9 exhibited significantantioxidantactivity ([67 %)
after 24 h of incubation. The results obtained in the in vitro
hepatotoxicity test on human hepatoma cells HepG2 demon-
strated that all prepared compounds are significantly less toxic
than tacrine in the micromolar range, especially at higher
concentration (100–300 lM), and racemic 2-amino-4-(1-
methyl-1H-imidazol-2-yl)-4H-benzo[h]chromene-3-carboni-
trile (6a) is a readily available imidazole derivative. This result
is important because it paves the way to select and develop
further the2-amino-4H-benzo[h]chromene-3-carbonitrile core
as the appropriate heterocyclic core to design new tacrine
analogs for the potential treatment of Alzheimer’s disease, and
also confirms the suitability of the coupled imidazole ring for
the same purpose. All these endeavors are now in progress in
our laboratory and will be reported in due course.
2
CN functionalities bearing an acetyl group at C3, a methyl
at C2, and an imidazole ring or benzimidazole at C4
(
42.84 ± 0.50 vs. 20.19 ± 0.95 % for 5e, and
6.06 ± 5.17 vs. 18.16 ± 0.20 % for 5f). Among the tes-
ted compounds tethered with –NH2 and –CN
3
functionalities (5a–5f, 6a–6d, 7, and 9), compound 9
(
Table 3, entry 15) which contains a DHP entity, bearing at
C2/C3: 3,3-dimethylcyclohexan-1-one, a 4-methoxyphenyl
group on the nitrogen atom and a benzimidazole unit at C4,
shows higher DPPH radical-scavenging activity
(
67.82 ± 0.70 %). This result is in good agreement with
the values observed for ascorbic acid (78.13 ± 1.19 %) at
00 lM for 24 h. This can be explained by the presence of
1
high electron-releasing methoxy group, which facilitates
free radical inhibition after long reaction times [71]. The
remaining compounds (Table 3, entries 12–14, 16) exhib-
ited non-significant antioxidant activity compared to the
standard reference (\25 %).
Conclusion
Experimental
In conclusion, we have prepared and characterized by analyt-
ical spectroscopic data, in some cases, by X-ray diffraction
analyses, some novel heterocyclic compounds bearing a 4H-
pyran, quinazoline, DHP, and 2-aminochromene as central
heterocyclic core and possessing additional (benz)imidazole
IR spectra were recorded on a Shimadzu FT IR-8201 PC
spectrophotometer and only significant absorption band
1
13
frequencies are cited. H NMR and C NMR spectra were
recorded on Bruker Avance DPX250 or VARIAN Mercury-
300 spectrometers. Chemical shifts (d) are given in ppm and
123

H. Boulebd et al.
J values in hertz (Hz). Elemental analyses were recorded on a
Carlo-Erba CHNS apparatus/O. EA 1108. The measure-
ments of the diffracted intensities were recorded on an APEX
II diffractometer equipped with a two-dimensional detector
After complete reaction (TLC), the reaction mixture was
concentrated, and the solid residue was isolated by filtra-
tion, washed with cold EtOH, and dried on air.
Ethyl 6-amino-5-cyano-2-methyl-4-(1-methyl-1H-imidazol-
˚
kappa CCD (k Ka = 0.71073 A). Melting points were
2-yl)-4H-pyran-3-carboxylate (5a, C H N O )
14 16 4 3
determined on an Electrothermal Digital Melting Points
Apparatus IA 9200 or on a Kofler melting point apparatus.
Thin layer chromatography (TLC) was carried out on pre-
coated Merck silica gel aluminum sheets 60 F254 and
detection by UV light at 254 nm. Column chromatography
was performed on silica gel 60 (230 mesh). Solvents were
freshly distilled before use: MeOH from magnesium in the
presence of I , DMF was kept for few hours over CaCl and
Yield 92 %; m.p.: 230–232 °C; IR (KBr): vꢀ = 3390 (NH ),
2
-1 1
318 (CN), 1689 (CO) cm ; H NMR (300 MHz, DMSO-
2
d ): d = 6.96–6.94 (m, 3H, NH , H-imid.), 6.69 (s, 1H,
6
2
H-imid.), 4.59 (s, 1H, H-pyran), 3.99–3.87 (m, 2H,
OCH CH ), 3.65 (s, 3H, NCH ), 2.30 (s, 3H, CH ), 0.97
3
2
3
3
1
t, J = 7.1 Hz, 3H, OCH CH ) ppm; C NMR (75 MHz,
3
(
2 3
DMSO-d ): d = 165.2, 158.7, 157.6, 149.6, 126.6, 120.7,
6
2
2
119.8, 105.0, 60.0, 54.5, 32.1, 30.1, 18.2, 13.8 ppm.
distilled from CaO, and THF from Na/benzophenone.
Commercial-grade reagents were used as supplied.
Ethyl 6-amino-5-cyano-2-methyl-4-(1-methyl-1H-
benzo[d]imidazol-2-yl)-4H-pyran-3-carboxylate
(5b, C H N O )
General procedure for the synthesis of 1-methyl-1H-
(
1
8 18 4 3
benz)imidazole-a-cyanoacrylonitrile (3) and (4)
Yield 85 %; m.p.: 240–241 °C; IR (KBr): vꢀ = 3355 (NH ),
2
-1 1
198 (CN), 1720 (CO) cm ; H NMR (300 MHz, DMSO-
2
To a solution of 1-methyl-1H-imidazole-2-carbaldehyde
1) or 1-methyl-1H-benzo[d]imidazole-2-carbaldehyde (2)
d6): d = 7.54 (d, J = 7.5 Hz, 2H, H-benzimid.), 7.26–7.14
(m, 2H, H-benzimid.), 7.08 (br s, 2H, NH ), 4.92 (s, 1H,
(
2
3
in ethanol (e.g., 1.0 mmol in 5 cm ), 1–1.1 equivalent of
malononitrile and for compound 4 few drops of piperidine
were added, and the mixture was stirred at room temper-
ature for 1 h. The resultant residue was filtered off, washed
with cold ethanol, and dried on air to afford the pure
product.
H-pyran), 3.92–3.88 (m, 5H, OCH CH , NCH ), 2.39 (s,
3
2
3
1
3
3H, CH ), 0.88 (t, J = 7.1 Hz, 3H, OCH CH ) ppm;
3
C
2
3
NMR (75 MHz, DMSO-d ): d = 165.5, 159.3, 158.6,
6
157.4, 142.5, 135.7, 122.2, 121.9, 120.0, 119.0, 110.5,
104.9, 60.5, 54.2, 31.2, 30.0, 18.8, 14.0 ppm.
5-Acetyl-2-amino-6-methyl-4-(1-methyl-1H-imidazol-2-yl)-
2
-[(1-Methyl-1H-imidazol-2-yl)methylene]malononitrile
4H-pyran-3-carbonitrile (5c, C H N O )
1
3 14 4 2
(
3, C H N )
4
Yield 90 %; m.p.: 220–222 °C; IR (KBr): vꢀ = 3394 (NH ),
8
6
2
-1 1
2218 (CN), 1616 (CO) cm ; H NMR (300 MHz, DMSO-
Yield 94 %; m.p.: 200–202 °C; IR (KBr): vꢀ = 2210 (CN)
cm ; H NMR (250 MHz, DMSO-d ): d = 8.15 (s, 1H,
-
1 1
d ): d = 7.00–6.96 (s, 3H, NH , H-imid.), 6.71 (s, 1H,
6
6
2
H-imid.), 7.58 (s, 1H, H-imid.), 7.40 (s, 1H,
H-imid.), 4.76 (s, 1H, H-pyran), 3.66 (s, 3H, NCH ), 2.32
3
1
CH = C(CN) ), 3.79 (s, 3H, NCH ) ppm; C NMR
3
13
(s, 3H, OCH ), 2.01 (s, 3H, CH ) ppm; C NMR (75 MHz,
2
3
3
3
(
62.9 MHz, CD CN): d = 142.7, 141.1, 134.0, 129.2,
DMSO-d ): d = 197.5, 159.0, 155.9, 148.8, 126.7, 121.6,
3
6
1
15.8, 114.1, 79.3, 34.1 ppm.
119.9, 113.2, 54.2, 32.3, 30.9, 29.6, 18.8 ppm.
2
-[(1-Methyl-1H-benzo[d]imidazol-2-yl)methylene]-
5-Acetyl-2-amino-6-methyl-4-(1-methyl-1H-benzo[d]imi-
malononitrile (4, C H N )
1
dazol-2-yl)-4H-pyran-3-carbonitrile (5d, C H N O )
17 16 4 2
2
8
4
Yield 67 %; m.p.: [260 °C; IR (KBr): vꢀ = 2152 (CN)
Yield 73 %; m.p.: 230 °C; IR (KBr): vꢀ = 3301 (NH ), 2187
2
-
1
1
-1 1
(CN), 1661 (CO) cm ; H NMR (300 MHz, DMSO-d ):
cm ; H NMR (250 MHz, CDCl ): d = 7.99–7.95 (m,
3
6
1
H, H-benzimid.), 7.76 (s, 1H, CH = C(CN) ), 7.55–7.41
2
d = 7.54 (d, J = 7.0 Hz, 2H, H-benzimid.), 7.26–7.14 (m,
2H, H-benzimid.), 7.10 (br s, 2H, NH ), 5.02 (s, 1H,
1
3
(
m, 3H, benzimidazole), 3.98 (s, 3H, NCH ) ppm;
3
C
2
NMR (62.9 MHz, CDCl ): d = 144.8, 144.3, 143.0, 136.3,
H-pyran), 3.91 (s, 3H, NCH ), 2.36 (s, 3H, OCH ), 2.13 (s,
3
3
3
1
3
1
26.2, 124.5, 120.8, 114.5, 112.9, 111.8, 83.8, 30.3 ppm.
3H, CH ) ppm; C NMR (75 MHz, DMSO-d ): d = 197.5,
3
6
159.6, 157.6, 156.8, 142.4, 136.0, 122.3, 122.0, 120.1, 119.1,
General procedure for the synthesis of 4H-pyran
derivatives 5
113.8, 110.6, 54.1, 31.6, 30.5, 30.2, 19.6 ppm.
2
2
-Amino-7,7-dimethyl-4-(1-methyl-1H-benzo[d]imidazol-
-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carboni-
To
a
solution of the 2-[(1-methyl-1H-imidazol-2-
trile (5e, C H N O )
1
6 18 4 2
yl)methylene]malononitrile (3) or 2-[(1-methyl-1H-
benzo[d]imidazol-2-yl)methylene]malononitrile (4) in
3
ethanol (1 mmol/10 cm ), 1.3-dicarbonylic compound
Yield 69 %; m.p.: 192–194 °C; IR (KBr): vꢀ = 3367 (NH ),
2
-1 1
183 (CN), 1689 (CO) cm ; H NMR (300 MHz, DMSO-
2
d ): d = 7.01 (s, 2H, NH ), 6.92 (s, 1H, H-imid.), 6.66 (s,
6
2
(
1.1–1.2 equiv) and some drops of piperidine were added.
1
23

Synthesis and biological evaluation of heterocyclic privileged medicinal structures…
1
2
2
H, H-imid.), 4.44 (s, 1H, H-pyran), 3.70 (s, 3H, NCH3),
147.7, 142.6, 136.4, 131.3, 131.0. 130.5, 129.2, 128.1,
125.7, 123.6, 122.7, 122.4, 121.0, 119.4, 117.5, 113.5,
111.0, 53.7, 32.1, 30.7 ppm.
.94–2.84 (m, 2H, CH ), 2.69 and 2.47 (ABq, J = 16.0 Hz,
2
1
3
H, CH ), 1.03 (s, 3H, CH ), 0.99 (s, 3H, CH ) ppm;
3
C
2
3
NMR (75 MHz, DMSO-d ): d = 196.0, 162.6, 158.9,
6
3
-Amino-1-(1-methyl-1H-imidazol-2-yl)-4H-benzo[f]chro-
1
49.3, 126.5, 120.6, 119.9, 111.3, 55.5, 49.8, 32.3, 32.1,
mene-2-carbonitrile (6c, C H N O)
1
8 14 4
2
5.5, 26.9, 26.5 ppm.
Yield 72 %; m.p.: [260 °C; IR (KBr): vꢀ = 3440 (NH ),
2183 (CN) cm
2
-
1
1
2
2
-Amino-7,7-dimethyl-4-(1-methyl-1H-benzo[d]imidazol-
-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carboni-
;
H NMR (250 MHz, DMSO-d6):
d = 7.96–7.92 (m, 2H, H-naph.), 7.78–7.75 (m, 1H,
H-naph.), 7.51–7.43 (m, 2H, H-naph.), 7.30 (d,
trile (5f, C H N O )
2
0 20 4 2
Yield 66 %; m.p.: 250–251 °C; IR (KBr): vꢀ = 3429 (NH ),
J = 8.9 Hz, 1H, H-naph.), 7.15 (br s, 2H, NH ), 6.99 (d,
2
2
-
1 1
2
360 (CN), 1724 (CO) cm ; H NMR (250 MHz, DMSO-
d6): d = 7.56–7.48 (m, 2H, H-benzimid.), 7.26–7.11 (m,
H, H-benzimid.), 6.98 (br s, 2H, NH ), 4.79 (s, H,
J = 0.9 Hz, 1H, H-imid.), 6.67 (d, J = 0.9 Hz, 1H,
H-imid.), 5.67 (s, 1H, H-pyran), 3.66 (s, 3H, NCH )
3
1
3
2
ppm; C NMR (62.9 MHz, DMSO-d ): d = 160.4, 148.9,
2
6
H-pyran), 3.94 (s, 3H, NCH ), 2.64–2.46 (m, 2H, CH ),
3
146.8, 130.7, 130.4, 129.7, 128.5, 127.3, 126.6, 125.0,
2
2
.29 and 2.10 (ABq, J = 16.0 Hz, 2H, CH ), 1.07 (s, 3H,
2
123.2, 121.9, 120.5, 116.8, 113.1, 53.7, 32.4, 31.4 ppm.
1
CH ), 1.06 (s, 3H, CH ) ppm; C NMR (62.9 MHz,
3
3
3
3-Amino-1-(1-methyl-1H-benzo[d]imidazol-2-yl)-4H-ben-
C D N): d = 196.8, 163.9, 161.0, 158.1, 143.9, 136.7,
5
5
zo[f]chromene-2-carbonitrile (6d, C H N O)
2
2 16 4
1
22.7, 122.4, 121.2, 120.0, 112.6, 110.7, 56.8, 50.8, 40.8,
Yield 90 %; m.p.: [260 °C; IR (KBr): vꢀ = 3474 (NH ),
2
3
2.7, 30.5, 29.3, 29.0. 27.6 ppm.
-
1
1
2180 (CN) cm
;
H NMR (300 MHz, DMSO-d6):
d = 7.96 (d, J = 9.0 Hz, 1H, H-Ar), 7.92–7.90 (m, 1H,
H-Ar), 7.52–7.50 (m, 1H, H-Ar), 7.43–7.41 (m, 1H, H-Ar),
General procedure for the synthesis
of 2-aminochromene derivatives 6
7.43–7.33 (m, 4H, H-Ar), 7.23 (br s, 2H, NH ), 7.20–7.09
2
(
s, 2H, H-Ar), 5.96 (s, 1H, H-pyran), 3.97 (s, 3H, NCH )
3
To
2-[(1-methyl-1H-(benz)imidazol-2yl)methylene]-
3
malononitrile (3) (or 4) (1.0 mmol) dissolved in 10 cm
1
3
ppm; C NMR (75 MHz, DMSO-d ): d = 161.3, 156.8,
6
1
1
1
47.7, 142.6, 136.4, 131.3, 131.0. 130.5, 129.2, 128.1,
25.7, 123.6, 122.7, 122.4, 121.0, 119.4, 117.5, 113.5,
11.0, 53.7, 32.1, 30.7 ppm.
ethanol, 1 mmol of 1- or 2-naphtol and two to three drops
of piperidine were added under stirring, and then the
reaction mixture was heated at reflux overnight. After
completion (TLC), the reaction mixture was cooled, con-
centrated in a vacuum, and the resultant precipitate was
filtered off and washed with cold ethanol.
6-Acetyl-2-amino-5-hydroxy-4-(1-methyl-1H-imidazol-2-
yl)-4H-chromene-3-carbonitrile (7, C H N O )
16 14 4 3
To 158 mg 2-[(1-methyl-1H-imidazol-2-yl)methylene]-
3
malononitrile (3, 80 mmol, 1.0 equiv) dissolved in 5 cm
2
-Amino-4-(1-methyl-1H-imidazol-2-yl)-4H-benzo[h]chro-
methanol, 152 mg 2,4-dihydroxyacetophenone (1.0 equiv.)
and 75 mg Ca(OH)2 (1.0 equiv.) were added under
magnetic stirring. The reaction mixture was kept under
stirring at rt until the disappearance of the starting product
mene-3-carbonitrile (6a, C H N O)
1
8 14 4
Yield 75 %; m.p.:[260 °C; IR (KBr): vꢀ = 3433 (NH ), 2376
2
-1 1
(
CN) cm ; H NMR (250 MHz, CD CN): d = 7.95–7.90
3
(
m, 2H, H-naph.), 7.78–7.75 (m, 1H, H-naph.), 7.53–7.43 (m,
(
TLC). The residue was filtered and washed with ethyl
2
2
1
H, H-naph.), 7.30 (d, J = 8.9 Hz, 1H, H-naph), 7.15 (br s,
3
acetate (2 9 5 cm ) to remove residual 3. The solid was
H, NH ), 7.69 (s, 1H, H-imid.), 6.67 (s, 1H, H-imid.), 5.56 (s,
2
1
H, H-pyran), 3.65(s, 3H, NCH ) ppm; C NMR (62.9 MHz,
3
then dissolved in 15 cm THF and the mixture were filtered
3
3
off to remove the remaining Ca(OH) . The organic layer
2
CD CN): d = 160.4, 148.9, 146.8, 130.7, 130.6, 129.8, 128.5,
3
was dried over anhydrous MgSO , filtered, and the solvent
4
1
27.3, 126.6, 125.1, 123.2, 121.9, 120.5, 116.8, 113.1, 53.7,
removed under reduced pressure. The resultant residue was
purified by crystallization in dioxane/MeOH solution to
3
2.4, 31.5 ppm.
2
-Amino-4-(1-methyl-1H-benzo[d]imidazol-2-yl)-4H-ben-
give pure compound 7 as a white solid. Yield 40 %; m.p.:
1
zo[h]chromene-3-carbonitrile (6b, C H N O)
2
[260 °C; H NMR (300 MHz, DMSO-d ): d = 7.88 (d,
2
16
4
6
Yield 95 %; m.p.: [260 °C; IR (KBr): vꢀ = 3471 (NH ),
2
J = 8.9 Hz, H-Ar), 7.13 (s, 2H, NH ), 6.99 (s, 1H,
2
2
-
1
1
179 (CN) cm
;
H NMR (300 MHz, DMSO-d6):
H-imid.), 6.69–6.66 (m, 2H, H-imid., H-Ar), 4.98 (s, 1H,
d = 7.96 (d, J = 9.0 Hz, 1H, H-Ar), 7.92–7.90 (m, 1H,
H-Ar), 7.52–7.50 (m, 1H, H-Ar), 7.43–7.41 (m, 1H, H-Ar),
H-pyran), 3.76 (s, 3H, NCH ), 3.50 (br s, 1H, OH) 2.58 (s,
3
1
3
3H, CH3) ppm;
C NMR (75 MHz, DMSO-d6):
7
.43–7.33 (m, 4H, H-Ar), 7.23 (br s, 2H, NH ), 7.20–7.08
2
d = 204.6, 159.9, 159.8, 154.1, 149.4, 131.9, 126.6,
120.7, 120.2, 112.7, 110.1, 107.5, 54.3, 32.5, 27.6,
26.6 ppm.
(
m, 2H, H-Ar), 6.00 (s, 1H, H-pyran), 3.97 (s, 3H, NCH )
3
1
3
ppm; C NMR (75 MHz, DMSO-d ): d = 161.3, 156.8,
6
123

H. Boulebd et al.
General procedure for the synthesis of 1,4-
dihydropyridine-1-methyl-1H-(benz)imidazoles 8
yl)methylene]malononitrile (4, 1.0 equiv) and two to three
drops of triethylamine were added. The mixture was
refluxed again for 1 h. After cooling, the solid was filtered
off, washed with cold ethanol, and dried on air to give
compound 9 (375 mg) in a pure form as a yellow solid.
To 1.0 mmol of 1-methyl-1H-imidazole-2-carbaldehyde
(1) or 1-methyl-1H-benzimidazole-2-carbaldehyde (2)
dissolved in a minimum of EtOH, the activated methylene
Yield 77 %; m.p.: 214–216 °C; IR (KBr): vꢀ = 3452 (NH ),
2
-
1 1
2175 (CN), 1647 (CO) cm ; H NMR (300 MHz, DMSO-
compound (2.2 equiv) and NH OH (4.0 equiv) were added;
4
then the reaction mixture was refluxed until disappearance
of the starting product (TLC). After cooling to rt, the
residue was poured into cold-ice water, filtered off, and the
solid was washed with cold ethanol.
d6): d = 7.57–7.52 (m, 2H, H-Ar), 7.43–7.41 (m, 2H,
H-Ar), 7.23–7.13 (m, 4H, H-Ar), 5.51 (br s, 2H, NH ), 4.95
2
(s, 1H, H-pyrid.), 3.97 (s, 3H, OCH ), 3.84 (s, 3H, NCH ),
3
3
2.19 (d, J = 16.0 Hz, 2H, CH ), 2.00 and 1.85 (ABq,
2
J = 16.0 Hz, 2H, CH ), 0.97 (s, 3H, CH ), 0.86 (s, 3H,
2
3
Diethyl 2,6-dimethyl-4-(1-methyl-1H-imidazol-2-yl)-1,4-
1
3
CH ) ppm; C NMR (75 MHz, DMSO-d ): d = 195.5,
3
6
dihydropyridine-3,5-dicarboxylate (8a, C H N O )
1
7 23 3 4
1
1
5
60.0, 158.4, 152.9, 152.5, 142.8, 135.8, 135.8, 129.4,
21.9, 121.8, 121.6, 119.0, 115.6, 110.5, 110.4, 57.1, 56.4,
5.8, 49.6, 32.7, 30.1, 29.3, 29.1, 27.0, 18.9 ppm.
Yield 73 %; m.p.: 230–231 °C; IR (KBr): vꢀ = 1670 (CO)
-
1 1
cm ; H NMR (300 MHz, DMSO-d ): d = 9.19 (br s, 1H,
6
NH), 6.84 (s, 1H, H-imid.), 6.64 (s, 1H, H-imid.), 4.87 (s,
1
H, H-pyrid.), 4.00 (q, J = 7.1 Hz, 4H, OCH CH ), 3.72
2
2-(1-Methyl-1H-imidazol-2-yl)-4-phenylquinazoline
3
(
s, 3H, NCH ), 2.18 (s, 6H, CH ), 1.12 (t, J = 7.1 Hz, 6H,
3
(10, C H N )
18 14 4
3
1
3
OCH CH ) ppm;
2
C
NMR (75 MHz, DMSO-d6):
1-Methyl-1H-imidazol-2-carbaldehyde
3
(1,
2.0 mmol) dissolved in 5 cm EtOH was placed in a
315 mg,
3
d = 166.9, 152.6, 146.2, 125.9, 120.0, 99.6, 59.1, 32.5,
0.9, 18.3, 14.3 ppm.
3
50 cm round-bottomed flask, and under stirring 702 mg
3
2
-aminobenzophenone (1.0 equiv.), 193 mg ammonium
Diethyl 2,6-dimethyl-4-(1-methyl-1H-benzo[d]imidazol-2-
yl)-1,4-dihydropyridine-3,5-dicarboxylate
acetate (2.5 equiv.), and 13 mg iodine (5 % mmol) were
added successively. The reaction mixture was heated at
(
8b, C H N O )
21 25 3 4
4
0 °C for 3 h, and the reaction was then allowed to stand at
Yield 77 %; m.p.: [260 °C; IR (KBr): vꢀ = 3448 (NH),
rt overnight. The resultant solid was filtered off, washed
3
with H O (3 9 10 cm ) followed by ethanol (3 9 10 cm ),
-
1
1
1
631 (CO) cm
;
H NMR (300 MHz, DMSO-d6):
3
2
d = 9.00 (br s, 1H, NH), 7.48–7.43 (m, 2H, H-benzimid.),
and dried under reduced pressure to afford the compound
7
4
6
.16–7.07 (m, 2H, H-benzimid.), 5.11 (s, 1H, H-pyrid.),
10 (303 mg) as a pale green solid, which was crystallized
in an isopropanol/MeOH solution. Yield 53 %; m.p.:
.01–3.95 (m, 4H, OCH CH ), 3.90 (s, 3H, NCH ), 2.24 (s,
2
3
3
1
3
H, CH ), 1.13–1.10 (m, 6H, OCH CH ) ppm; C NMR
3
2
3
1
1
9
4
12 °C; H NMR (250 MHz, CDCl ): d = 8.18–7.57 (m,
3
(
75 MHz, DMSO-d ): d = 193.9, 167.5, 160.4, 147.2,
6
H, H-Ar), 7.27 (s, 1H, H-imid.), 7.06 (s, 1H, H-imid.),
1
.24 (s, 3H, NCH ) ppm; C NMR (62.9 MHz, CDCl3):
1
35.7, 122.2, 121.9, 119.0, 110.7, 100.0, 60.0, 31.2, 19.4,
3
3
1
4.9 ppm.
d = 153.8, 151.6, 144.2, 137.2, 133.9, 130.2, 130.0, 129.3,
128.5, 127.6, 127.0, 125.2, 121.5, 37.0 ppm.
3
3
,3,6,6-Tetramethyl-9-(1-methyl-1H-imidazol-2-yl)-
,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione
(
8c, C H N O )
2
Cell culture and treatment
1 27 3 2
Yield 80 %; m.p.: 228–230 °C; IR (KBr): vꢀ = 1654 (CO)
-
1
1
cm
;
H NMR (250 MHz, C D N): d = 7.30 (s, 1H,
The human hepatoma cell line HepG2 was cultured in
Eagl e´ s minimum essential medium (EMEM) supplemented
with 15 non-essential amino acids, 1 mM sodium pyruvate,
10 % heat-inactivated fetal bovine serum (FBS), 100 units/
6
5
H-imid.), 6.85 (s, 1H, H-imid.), 5.10 (br s, 1H, NH ), 4.94
2
(
s, 1H, H-pyrid.), 4.17 (s, 3H, NCH ), 2.40 (s, 4H, CH ),
3 2
2
.22 and 2.08 (ABq, J = 20 Hz, 4H, CH ), 0.94 (s, 12H,
2
1
3
3
3
CH ) ppm; C NMR (62.9 MHz, C D N): d = 197.4,
cm penicillin, and 100 lg/cm streptomycin (reagents
from Invitrogen, Madrid, Spain). Cultures were seeded into
flasks containing supplemented medium and maintained at
3
6 5
1
63.6, 127.8, 120.7, 114.3, 50.9, 40.9, 33.4, 32.6, 29.2,
2
7.3, 24.9 ppm.
3
7 °C in a humidified atmosphere of 5 % CO and 95 %
2
2
1
-Amino-1-(4-methoxyphenyl)-7,7-dimethyl-4-(1-methyl-
H-benzo[d]imidazol-2-yl)-5-oxo-1,4,5,6,7,8-hexahydro-
air. Culture media were changed every 2 days. Cells were
sub-cultured after partial digestion with 0.25 % trypsin–
quinoline-3-carbonitrile (9, C H N O )
27 27 5 2
EDTA. For assays, HepG2 cells were subcultured in
5
6-well plates at a seeding density of 1 9 10 cells per
Dimedone (150 mg, 1.07 mmol, 1.0 equiv) and 131 mg p-
3
anisidine (1.07 mmol, 1.0 equiv) were refluxed in 15 cm
9
well. When the HepG2 cells reached 80 % confluence, the
medium was replaced with fresh medium containing
ethanol for 24 h. The reaction mixture was cooled to rt, and
2
22 mg
2-[(1-methyl-1H-benzimidazol-2-
1
23

Synthesis and biological evaluation of heterocyclic privileged medicinal structures…
1–300 lM compounds or 0.1 % DMSO as a vehicle
control.
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