Amino substituted benzimidazo[1,2-a]quinolines: Antiproliferative potency, 3D QSAR study and DNA binding properties

Article abstract of DOI:10.1016/j.ejmech.2016.07.007

We describe the synthesis, 3D-derived quantitative structure-activity relationship (QSAR), antiproliferative activity and DNA binding properties of a series of 2-amino, 5-amino and 2,5-diamino substituted benzimidazo[1,2-a]quinolines prepared by environme

Full text of DOI:10.1016/j.ejmech.2016.07.007

Accepted Manuscript
Amino substituted benzimidazo[1,2-a]quinolines: Antiproliferative potency, 3D QSAR
study and DNA binding properties
Nataša Perin, Raja Nhili, Maja Cindrić, Branimir Bertoša, Darko Vušak, Irena Martin-
Kleiner, William Laine, Grace Karminski-Zamola, Marijeta Kralj, Marie-Hélène David-
Cordonnier, Marijana Hranjec
PII:
S0223-5234(16)30554-2
10.1016/j.ejmech.2016.07.007
EJMECH 8725
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 December 2015
Revised Date: 27 June 2016
Accepted Date: 5 July 2016
Please cite this article as: N. Perin, R. Nhili, M. Cindrić, B. Bertoša, D. Vušak, I. Martin-Kleiner, W. Laine,
G. Karminski-Zamola, M. Kralj, M.-H. David-Cordonnier, M. Hranjec, Amino substituted benzimidazo[1,2-
a]quinolines: Antiproliferative potency, 3D QSAR study and DNA binding properties, European Journal
of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.007.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Amino substituted benzimidazo[1,2-a]quinolines: Antiproliferative potency,
D QSAR study and DNA binding properties
3
a
b
a
c
c
Nataša Perin , Raja Nhili , Maja Cindrić , Branimir Bertoša , Darko Vušak , Irena Martin-
d
b
a
d
Kleiner , William Laine , Grace Karminski-Zamola , Marijeta Kralj ,
b
a*
Marie-Hélène David-Cordonnier and Marijana Hranjec
R1
ac tv i iyt er du ec dby
s i de ch a ni el n gh t
CH3
R
2
N
N
NH
CH
N
N CH3
ac tv i iyt ni c er as e dby
s i de ch an i el n gh t
N
ht e m os
3
t ac vt ie co mp o un ds
NC
R
R2 =N HR ,N R2
,
1
I C =0 3. - 0 .8
M
50
µ

ACCEPTED MANUSCRIPT
Amino substituted benzimidazo[1,2-a]quinolines: Antiproliferative potency,
3
D QSAR study and DNA binding properties
a
b
a
c
c
Nataša Perin , Raja Nhili , Maja Cindrić , Branimir Bertoša , Darko Vušak , Irena
d
b
a
d
Martin-Kleiner , William Laine , Grace Karminski-Zamola , Marijeta Kralj ,
b
a*
Marie-Hélène David-Cordonnier and Marijana Hranjec
a
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulićev trg 20, P. O. Box 177, HR-10000 Zagreb, Croatia;
b
INSERM UMR-S1172, Jean-Pierre Aubert Research Centre (JPARC), Université de Lille,
Institut pour la Recherche sur le Cancer de Lille, Place de Verdun, F-59045 Lille cedex,
France;
c
Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-
1
0000 Zagreb, Croatia;
d
Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička cesta 54, P. O. Box
80, HR-10000 Zagreb, Croatia;
1
*
Corresponding author: Dr. Marijana Hranjec, Assoc. Prof., Department of Organic
Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb,
Marulićev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia, Phone No. +38514597245; Fax
No. +38514597250; e-mail: mhranjec@fkit.hr

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Abstract
We describe the synthesis, 3D-derived quantitative structure-activity relationship (QSAR),
antiproliferative activity and DNA binding properties of a series of 2-amino, 5-amino and 2,5-
diamino substituted benzimidazo[1,2-a]quinolines prepared by environmentally friendly
uncatalyzed microwave assisted amination. The antiproliferative activities were assessed in
vitro against colon, lung and breast carcinoma cell lines; activities ranged from
submicromolar to micromolar. The strongest antiproliferative activity was demonstrated by 2-
amino-substituted analogues, whereas 5-amino and or 2,5-diamino substituted derivatives
resulted in much less activity. Derivatives bearing 4-methyl- or 3,5-dimethyl-1-piperazinyl
substitutents emerged as the most active. DNA binding properties and the mode of interaction
of chosen substituted benzimidazo[1,2-a]quinolines prepared herein were studied using
melting temperature studies, a series of spectroscopic studies (UV/Visible, fluorescence, and
circular dichroism), and biochemical experiments (topoisomerase I-mediated DNA relaxation
and DNase I footprinting experiments). Both compound 36 and its bis-quaternary iodide salt
3
7 intercalate between adjacent base pairs of the DNA helix while compound 33 presented a
very weak topoisomerase I poisoning activity. A 3D-QSAR analysis was performed to
identify hydrogen bonding properties, hydrophobicity, molecular flexibility and distribution
of hydrophobic regions as these molecular properties had the highest impact on the
antiproliferative activity against the three cell lines.
Key words: benzimidazoles, benzimidazo[1,2-a]quinolines, 3D-QSAR, antiproliferative
activity, DNA binding properties

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1
. Introduction
The benzimidazole moiety is one of the most important organic structural motifs found
in a variety of bioactive natural products as well as numerous synthetic medical and
biochemical agents, which possess different chemical and pharmacological features [1–5].
Importantly, due to the structural similarity with naturally occurring nucleotides,
benzimidazole derivatives can easily interact with biomolecules like DNA, RNA or proteins
in living systems, thus playing a crucial role in their function. The DNA molecule is still one
of the principal targets in anticancer drug development strategies since it has a central role in
many life processes [6]. Understanding the molecular basis for cytotoxicity by anticancer
agents is critical for the rational development of novel, more selective and efficient agents
with greater specificity of action [7]. The most used classes of chemotherapeutic agents are
comprised of molecules that interact with DNA such as intercalators, alkylating agents or
groove binders [8]. Organic intercalators are a class of polycyclic aromatic compounds that
usually consist of planar and fused aromatic or heteroaromatic rings that can insert or
intercalate between two adjacent base pairs of duplex DNA and inhibit nucleic acid synthesis
[
9]. The combination of the benzimidazole moiety with other heteroaromatic groups can lead
to highly conjugated, planar benzannulated benzimidazoles with improved biological
properties. Their high fluorescence intensity and possibility of interaction with
biomacromolecules suggest the potential use of benzannulated benzimidazoles as fluorescent
probes for detection of biologically important molecules such as DNA or proteins in
biomedicinal diagnostics [10, 11]. Moreover, the quinoline ring system, which widely exists
in a large number of alkaloids and synthetic analogues with various biological actions, is also
of great importance in medicinal and natural product chemistry [12, 13].
Quinoline-fused benzimidazoles, recently prepared and published in our research
group, have been shown to be a very promising class of tetracyclic intercalators; studies of
their biological activity comprising cytostatic evaluation, DNA/RNA interaction, inhibition of
topoisomerase I and II, and proteomic profiling have confirmed the anticancer potential of
this class of compounds [14, 15]. Positively charged amidino-substituted benzimidazo[1,2-
a]quinolines intercalate into double-stranded DNA or RNA with the pronounced selectivity
towards colon carcinoma cells [14].
In addition, we have reported on the biological action of 2-aminobenzimidazo[1,2-
a]quinoline-6-carbonitriles as antiproliferative agents [15]. The obtained biological activity
demonstrated that the structural features and length of the amino side chains influence the

ACCEPTED MANUSCRIPT
antiproliferative potency of these compounds. Some of the examined amino side chains
yielded analogues with activities in the sub-micromolar range. In a more recent paper, we
reported the synthesis, antiproliferative activity and DNA binding properties of a series of 5-
aminobenzimidazo[1,2-a]quinoline-6-carbonitriles and showed that the displacement of the
amino side chain to position 5 of the quinoline-fused benzimidazole moiety leads to
compounds with significantly decreased potency [16]. Based on previous knowledge and in
consideration of the promising anticancer potential of the benzimidazo[1,2-a]quinoline
scaffold, herein we described the design and synthesis of novel 2-amino, 5-amino and 2,5-
diamino substituted analogues, aimed at improving antiproliferative activity and selectivity.
The newly synthesized compounds were tested in vitro for their antitumor properties using
three human tumor cell lines, and for each cell line 3D-QSAR models were obtained.
Moreover, we present the DNA binding ability results of selected compounds.
2
. Methods
2
.1 Chemistry
The new target compounds 5–9 and 18–39 were synthesized according to the
procedures shown in Schemes 1 and 2, respectively, by employment of conventional methods
for construction of structurally related fused benzimidazole derivatives. Thus, 4-
fluorobenzaldehyde 1 (Scheme 1) or benzoyl chlorides (Scheme 2) were condensed with 2-
(
1H-benzimidazol-2-yl)acetonitrile 2 in the presence of piperidine in absolute ethanol to
provide the corresponding benzimidazol substituted nitriles 3, 12 and 15 in suitable yields
75 %, 44 %, and 49 %, respectively) according to previously published procedures [15, 16].
(
As shown in Scheme 1, compound 3 underwent a thermal cyclization in sulfolane to afford 2-
fluorobenzimidazo[1,2-a]quinoline-6-carbonitrile 4 (72 %), which was the common precursor
for the preparation of the desired compounds 5–9. On the other hand, the acyclic
intermediates 12 and 13 were subjected to thermal cyclization reaction in DMF under basic
conditions (potassium tert-butoxide) to give the corresponding 5-oxo-5,7-
dihydrobenzimidazo[1,2-a]quinoline-6-carbonitriles 14 and 15 in 57 % and 96 % yields,
respectively (Scheme 2). Subsequent treatment of these intermediates with POCl and POCl5
3
gave the respective 5-chloro substituted benzimidazo[1,2-a]quinoline-6-carbonitriles 16
(70 % yield) and 17 (96 % yield), which were the precursors for the preparation of the final
products 18–39.

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H
Cl NC
N
H
F
Cl
N
N
CN
EtOHabs
+
CH2
N
piperidine
F
CHO
3
1
2
sulfolane or
EtOH, h
ν
F
R
amine
N
CH CN
N
3
MW
N
N
CN
CN
-9
4
5
8
R=
R=
N
N CH
3
5
6
7
R=NHCH(CH3)2
R=NHCH CH CH(CH )
3 2
2
2
CH3
NH
R=NHCH CH CH CH CH CH
3
2
2
2
2
2
9
N
CH3
Scheme 1. Synthesis of 2-amino substituted derivatives 5–9
The halogen compounds 4, 16 and 17 were converted to the targeted amino and
diamino analogues 5–9 and 18–39, respectively in 15–74 % yields upon reaction with five- to
seven-fold excess of the appropriate amine in acetonitrile under microwave irradiation. After
determining the optimal yields in the shortest reaction time, this microwave assisted
amination was conducted by using 800 W power, 170 °C and 40 bar pressure. The bis-
quaternary iodide salt 37 was obtained in 47 % yield by treating piperazine analogue 36 with
an excess of methyl iodide in the presence of anhydrous potassium carbonate in acetonitrile.
1
The structures of all prepared compounds were determined by NMR spectroscopy ( H
1
3
1
and C NMR), mass spectrometry, and elemental analysis. H NMR spectra of the cyclization
reaction products showed a downfield shift of the aromatic protons and absence of the
1
benzimidazol NH proton in comparison with the respective acyclic intermediates. These H
NMR spectroscopic differences confirmed the formation of the benzimidazo[1,2-a]quinoline
heterocycle.

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H
Cl NC
N
X
Cl
H
N
piridine
CH2
N
H
Cl
R1
C
N
CN
O
1
0 R =H
2
12 R =H
1
O
1
DMF
t-KOBu
1
1 R =F
13 R =F
1
1
R1
R1
R1
N
N
3
POCl /PCl5
amine
N
CH CN
MW
3
N
N
H
R2
O
N
Cl
CN
CN
CN
1
8-42
R =H; R =NHCH(CH )
3 2
16 R
1
=H
1
14 R =H
15 R =F
1
1
1
7 R =F
1
1
2
8
9
0
1
2
3
3
3 R =R =
N
N
R =H; R =NHCH CH CH(CH )
R =H; R =NHCH CH CH CH CH CH
3
1
2
1
2
2
2
3 2
1
2
2
2
2
2
2
4 R =R =
1
2
2
1
R =H; R =
N
N CH3
1
2
CH3
NH
35 R =R =
N
N
N
O
1
2
2
2
R =H; R =
N
1
2
3
6 R =R =
NH
1
2
CH3I
CH3
CH
3
CH CN
3
2
2
2
2
2
2
3
4
5
6
7
8
R =R =NHCH
37 R =R =
1 2
N
1
2
3
R =R =NHCH CH CH CH
CH3
1
2
2
2
2
3
I
R =R =NHCH(CH )
1 2 3 2
38 R
=R
=
N
N
CH3
R =R =NHCH CH(CH )
3 2
1
2
1
2
2
R =R =NHCH CH CH(CH )
1
2
2
2
3 2
CH3
NH
R =R =NHCH CH CH CH CH CH
3
1
2
2
2
2
2
2
29
30
31
32
R =R =N(CH )
39 R =R =
N
1
2
3 2
1
2
R =R =N(CH CH )
1
2
2
3 2
CH3
R =R =N(CH CH CH )
1
2
2
2
3 2
R =R =N(CH CH CH CH CH )
3 2
1
2
2
2
2
2
Scheme 2. Synthesis of 5-amino (18–22) and 2,5-diamino (23–39) substituted derivatives
2
.2 3D-QSAR analysis
3
D-QSAR models were derived using antiproliferative activity data against H460,
HCT 116, MCF-7 of the compounds presented in this paper and similar compounds whose
antiproliferative activities have been measured in the same laboratory and published
previously [15, 16]. Altogether, 51 compounds were used (Table S1 in the Supplement).
The 3D structure of each compound was generated from the SMILES code using VolSurf+
3
D structure generator. Molecular descriptors for each compound were generated based on its
D structure using the VolSurf+ program [17]. A series of 128 descriptors that refer to
3
molecular size and shape, to hydrophilic and hydrophobic regions and to the balance between
them, to the "charge state" descriptors, to lipophilicity, to molecular diffusion (logP, logD) to
the presence/distribution of pharmacophoric descriptors, to molecular flexibility, to H-bond

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interaction, and to descriptors of some other relevant ADME properties were considered. The
definition of all 128 VolSurf+ descriptors is given in the VolSurf+ manual [17, 18].
Using Partial Least Square (PLS) analysis, the relationship between the 3D structure-
based molecular descriptors and biological activities was studied. Autoscaling pretreatment,
by which every variable is the mean centered and scaled to give unit variance, was applied.
For each cell line, different 3D-QSAR models were generated (models labeled 1, 2, and 3, for
the cell lines H460, HCT 116, and MCF-7, respectively). The number of significant latent
variables (nLV) and quality of the models were determined using the leave-one-out (LOO)
cross-validation procedure. Standard deviation of error of calculation (SDEC) and standard
deviation of error of prediction (SDEP) were calculated for each model. The PLS coefficients
of the obtained models were analyzed in order to investigate influence of each descriptor on
compounds’ antiproliferative activity.
3
D-QSAR analysis was used to explore the physical and chemical properties having
the highest influence on the antiproliferative activities of investigated benzimidazo[1,2-
a]quinoline-6-carbonitriles. 3D-QSAR models 1, 2, and 3 (Table 1, Figure 1) were derived
using the antiproliferative activities of the herein presented compounds together with the
antiproliferative activities of similar compounds [15, 16] against H460, HCT 116 and MCF-7
cell lines, respectively.
Table 1. Statistical properties of 3D-QSAR models.
a
b
2
2 c
d
e
Cell line
Model nO
LV
R
Q
SDEC SDEP
H460
HCT 116
MCF-7
1
2
3
49
50
50
5
5
5
0.817
0.790
0.489
0.427
0.614
0.345
0.370
0.266
0.577
0.611
0.437
0.857
a
b
c
2
Number of objects used to build the model. Number of latent variables. Q is the cross-validated predictive
n
2
(
yexp(i) −ypred (i)
)
∑
n=1
n
2
performance and is given by Q = 1−
; where ypred(i) corresponds to the predicted and yexp(i) to
2
(
yexp(i) − yexp
)
∑
n=1
d
the experimentally determined inhibition, pIC for the compound i, respectively. SDEC is standard deviation of
error of calculation. SDEP is the standard deviation in cross-validated prediction and is given by
50
e
n
2
(
yexp(i)−ypred(i)
)
∑
i=1
SDEP =
.
n

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Figure 1. Predicted vs experimental antiproliferative activity (expressed as pIC – negative
5
0
logarithmic value of concentration that causes 50 % growth inhibition of the tumor cell lines
of: A) model 1 (H460), B) model 2 (HCT 116) and C) model 3 (MCF-7).
3
3
3
. Results and Discussion
.1 Antiproliferative activity
.1.1. Antiproliferative activity in human cell lines
The antiproliferative activities of new benzimidazo[1,2-a]quinoline-6-carbonitriles
bearing different amino side chains were tested on human colon, breast and lung tumor cell
lines (Table 2) [15, 16, 18]. The most active and/or structurally interesting compounds were
also tested on immortalized human mammary epithelial cells to test their potential selectivity

ACCEPTED MANUSCRIPT
towards tumor cells. The obtained results were compared with known antitumor agents
doxorubicin and etoposide.
a
Table 2. IC values (in ꢀM)
5
0
Cell lines
Compounds HCT 116
MCF-7
H460
HMLE
5
6
0.8±0.2
3±0.3
5±0.3
0.6±0.3
0.3±0.08
13±0.4
5±1
0.7±0.4
2±0.09
5±0.6
2±1
2±0.8
4±0.3
5±2
1±0.1
3±0.4
c
7
N.T.
8
9
1±0.8
0.5±0.04
5±2
0.5±0.4
0.2±0.03
N.T.
0.6±0.2
20±3
1
8
9
0
1
2
3
4
1
2
2
2
2
2
4±0.5
3±0.6
3±0.3
6±2
2±0.5
2±0.3
4±0.6
4±0.3
≥100
N.T.
4±1
N.T.
3±0.2
3±0.2
7±3
N.T.
3±0.4
N.T.
2±1
7±1
5±0.8
7±1
4±1
N.T.
b
25
≥10
21±2
N.T.
2
2
2
3
3
3
3
3
3
3
3
3
3
6
7
8
0
1
2
3
4
5
6
7
8
9
2±0.2
6±0.3
>100
2±0.6
2±0.05
>100
3±0.2
5±0.2
>100
N.T.
6±0.8
N.T.
16±4
22±3
25±2
N.T.
14±5
21±6
29±0.2
>100
N.T.
>100
>100
N.T.
2±0.5
11±3
2±0.6
10±3
12±1
N.T.
15±2
N.T.
11±5
9±1
18±8
N.T.
1.5±0.3
>100
1±0.001
>100
4±0.6
>100
2±0.8
N.T.
2±0.5
0.4±0.09
4±1
3±0.5
0.8±0.3
0.03±0.01
0.1±0.04
N.T.
1±0.2
0.02±0.01
1±0.7
0.2±0.01
0.01±0.002
0.4±0.09
doxorubicin 0.07±0.02
etoposide
5±2
a
b
c
IC ; the concentration that causes 50 % growth inhibition
The highest tested concentration was 10 µM.
N.T.; not tested
50

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Among the 2-amino substituted derivatives (5–9), three compounds strongly inhibited
the growth of all cell lines. The most cytotoxic (IC < 1 µM) were the 4-methyl-1-piperazinyl
5
0
and 3,5-dimethyl-1-piperazinyl substituted compounds 8 and 9.
The comparison of antiproliferative activity with different amino side chains lengths
(5–7) displayed that the side chain length decreased the activity, probably due to the increase
of the amphiphilic moment (A) and the unbalanced distribution of hydrophobic regions in the
molecule (see Section 3.1.2). On the other hand, the results obtained for compounds 18, 19
and 20 revealed the opposite effect of the side chain length on the activities; for these
compounds, the amino side chain is displaced to the 5- position and since the aliphatic chain
does not decrease the unbalanced distribution of hydrophobic regions in the molecule, an
increase in antiproliferative activity results. Therefore, the activities of compounds 19 and 20
are comparable to 6 and 7 bearing the 2-methylbutyl and 1-hexyl amino side chains. The
activity of compound 5, bearing the 2-methylethyl side chain, is more pronounced in
comparison to the activity of compound 18. Among the most abundant group of 2,5-diamino
substituted derivatives, 1-piperazinyl substituted compounds 36, 38 and 39 displayed the most
pronounced antiproliferative activity, whereby 3,5-dimethyl-1-piperazinyl substituted
derivative 39 was the most active one. The high antiproliferative activity of these compounds
can be explained by the presence of an additional N heteroatom, which contributes to
additional interactions with potential biological targets. Interestingly, the substituted
quaternary iodide salt 37 was completely inactive, which is comparable to the previously
obtained antiproliferative activity of a similar derivative with the substituent placed at the
position 5 [16], and is captured by QSAR analysis of the results regarding the percentage of
unionized species (see Section 3.1.2). 1-Piperidinyl and 4-morpholinyl substituted analogues
3
4 and 35 showed moderate activity (IC50 10–20 µM), while 1-pyrrolidinyl substituted
analogue 33 showed pronounced activity on HCT 116 and MCF-7 cell lines. 2,5-diamino
substituted derivatives with secondary amino side chains 23–27 showed moderate
antiproliferative activity in comparison to the less active derivatives 30–32 with tertiary
amino side chains.
The lower antiproliferative activity displayed by derivatives with tertiary, rather than
secondary, amino side chains confirmed that the secondary amine side chain is generally
slightly preferred for antiproliferative activity, which is in agreement with previously
published results [15, 16]. Moreover, disubstituted derivatives 28 and 32 bearing the longest
amino side chains did not show any antiproliferative effect towards the tested cell lines. In
general, there was no significant difference in the sensitivity towards the tested compounds

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between the cell lines, which may indicate common mechanisms of action of the new
compounds. The exception was compound 23, with less pronounced activity toward H460 cell
line. Furthermore, although the selected compounds similarly affected the proliferation of
healthy (non-tumor) and tumor cells, it is evident that the control anti-tumor compounds -
doxorubicin and etoposide were more cytotoxic towards the non-tumor cells.
Within this scientific study we have additionally confirmed that the antiproliferative
activity is highly dependent both on the type and length as well as the position of the amino
substituent on the tetracyclic skeleton (Figure 2). The obtained results indicated that the most
promising antiproliferative agents are those bearing the amino substituent at the position 2,
and displacement of the amino side chain from position 2 to position 5 significantly decreased
the activity. Introduction of two amino substituents at the positions 2 and 5, in comparison to
the 2-substituted derivatives, slightly decreased the antiproliferative activity.
2
the most pronounced
R1
antiproliferative activity
with submicromolar IC50
2
,5
decrease of antiproliferative
activity by introduction of
two amino substituents
N
N
R2
5
CN
significant decrease of antiproliferative activity by
displacement of the amino side chain from position 2 to position 5
Figure 3. SAR of amino substituted benzimidazo[1,2-a]quinoline-6-carbonitriles
Since previously published results clearly showed that the most active compounds are
those bearing the 2-methylpropylamino and 1-piperazinyl side chains at position 2 [15], we
designed and prepared the derivatives with the isoalkylamine and substituted 1-piperazinyl
side chains. 2-methylethyl and 2-methylbutylamino side chains were used to evaluate the
influence of the isoalkylamine side chain length on the antiproliferative activity. The obtained
results confirmed that the 2-methylpropylamino substituted derivative displayed the strongest
activity, in sub-micromolar range. Furthermore, by choosing the 4-methyl-1-piperazinyl and
3
,5-dimethyl-1-piperazinyl side chains, our intention was to confirm the assumption that the
presence of another N heteroatom contributes to the interactions with biological targets
probably due to hydrogen bonding. The obtained results indicated that the antiproliferative

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activity decreased in comparison to the 1-piperazinyl substituted derivatives bearing the side
chain at 2-, 5- and 2,5-positions. The lowest activity among the 1-piperazinyl substituted
derivatives was observed for compounds bearing 4-methyl-1-piperazinyl side chains, which
do not posses a N atom accessible for interactions with biological targets. Derivatives bearing
the 3,5-dimethyl-1-piperazinyl side chains showed decreased activity due, presumably, to the
steric hindrance of methyl groups which reduce the interactions of N heteroatom with
potential biological targets. Benzimidazo[1,2-a]quinoline-6-carbonitriles, especially those
bearing amino side chains at position 2 of the tetracyclic skeleton, should be further optimized
as promising compounds for anticancer therapy. Of particular interest would be the synthesis
of derivatives with different types and lengths of amino side chains at different positions by
combining the amino side chains that showed the greatest impact on the antiproliferative
activity herein.
3
.1.2 QSAR analysis of antiproliferative activities
QSAR analysis of the obtained models identified the molecular properties with the
highest influence on the antiproliferative activities against the studied cell lines (Figure 3A).
Analysis of model 1 (H460 cell line) revealed that molecular flexibility (FLEX_RB),
descriptors related to H-bond properties (WN5, WN6, WO1-4, DRDRDO, DRACAC,
DRACDO, ACACDO, ACACAC), protein binding (PB) and hydrophobicity (D3-7, ID2-4,
CD3-6) have the largest positive influence on the antiproliferative activity. Also, the
percentage of unionized species at different pH values (%FU4, %FU5), amphiphilic moment
(A; the vector that connects the center of hydrophobic and center of hydrophilic regions) and
ratio of molecular volume to molecular surface (R) were identified as the descriptors with the
greatest negative influence on antiproliferative activity against H460 cell line.
Similar descriptors are found to be important for the activity against cell line HCT 116
(Figure 3B). The descriptors having the highest positive influence on antiproliferative activity
are those related to H-bonding properties (WN6, WO1-WO5, DRDRDO, DRACAC,
DRACDO, ACACDO, ACACAC), molecular flexibility (FLEX_RB), hydrophobic regions
(D3-D8, ID3, ID4, CD3, CD4, DD1-DD4) and protein binding (PB). The percentage of
unionized species at different pH values (%FU4, %FU5), amphiphilic moment (A) and integy
moment (ID1; the vector that connects the center of a molecule and the center of hydrophobic
regions) had the largest negative influence on antiproliferative activity on the HCT 116 cell
line. In the case of the MCF-7 cell line (Figure 3C), the same descriptors as for cell line HCT
1
16 had the greatest positive influence on antiproliferative activity.

ACCEPTED MANUSCRIPT
Figure 3. PLS coefficients of 3D-derived QSAR model: A) model 1 (H460), B) model 2
(
HCT 116) and C) model 3 (MCF-7). Descriptors with the highest impact on the activity are
labeled; list and description of all 128 VolSurf+ descriptors is given in the VolSurf+ manual
17].
[

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Furthermore, the descriptors related to the unbalance between the centre of mass of the
molecule and the barycentre of its hydrophobic regions (amphiphilic moment, A, and integy
moment, ID1), the partition coefficient of cyclohexane/water (LOGP c-Hex) and the ratio of
molecular volume and molecular surface (R) showed the highest negative influence on the
antiproliferative activity.
Therefore, an increase of H-bonding properties, hydrophobicity and flexibility, as well as
decrease of the uneven distribution of hydrophobic and hydrophilic regions in the molecule
should lead to an increase of antiproliferative activity against all three studied cell lines.
3
.2. DNA Binding Properties
Considering the simplicity and mutual similarity of the studied structures, fast
screening by thermal denaturation experiments were carried out. The temperature at which
half of the double stranded DNA is degenerated to single stand DNA (T ) may increase upon
m
binding of the tested compound to DNA (positive ∆T ), thus suggesting the stabilization of
m
the DNA helix; rarely a decrease is observed upon DNA binding, evidencing DNA
denaturation by the bound compound (negative ∆T ). Evaluation of the DNA degeneration
m
temperature values obtained after binding of the chosen compounds 9, 23, 24, 26 and 30 to 37
to circulating tumor DNA (ct-DNA) revealed compounds 36 and 37 as strong DNA binders
(Table 3).
Table 3. DNA melting temperature studies. The measurement were performed at the
indicated drug/DNA ratios (R); nd = not determined.
Compounds
∆Tm (°C)
R=0.25
R=0.5
R=0.1
9
0
1.2
0
nd
nd
nd
nd
nd
nd
nd
nd
5.1
23
31
33
34
35
36
nd
0.6
0.7
0.5
20.1
nd
nd
nd
15.9
R=0.25
R=0.1
20.7
R=0.05
2.1
37
nd

ACCEPTED MANUSCRIPT
Compounds bearing amino substituents on position 2 which showed significant
antiproliferative activity were not chosen since the DNA binding of similar compounds was
already studied [16]. Exception was compound 9 with 3,5-dimethylpiperazin-1-yl substituent.
Interestingly, only compounds 36 and 37 presented potent DNA helix stabilization whereas
none of the other tested compounds changed the intrinsic degeneration temperature of ct-
DNA. Compound 36 revealed ∆T values 15.9 °C and 20.1 °C at 1:2 and 1:4 compound/DNA
m
ratios, respectively. ∆T values using higher compound/DNA ratios could not be determined
m
since less than half of the DNA denaturation could be achieved using temperature as high as
1
00 °C. This observation suggested very strong and significant DNA stabilization due to the
binding of 37 to DNA.
Based on the aforementioned considerations, compounds 36 and 37 were chosen for
further evaluation by using spectrophotometric analysis. The DNA binding properties of 36
and 37 were underlined firstly by using UV/Visible spectroscopy. UV/Visible measurements
were performed using a fixed concentration of compounds incubated with increasing
concentrations of ct-DNA. Obtained results showed the absorption spectra modification for
both studied compounds 36 and 37 (Figure 4A); hypsochromic and bathochromic shifts were
observed, which are frequently associated with DNA intercalation. However, spectral changes
did not reveal any isosbestic point (typically observed for a single binding mode), suggesting
that the mode of binding of the compounds to the DNA helix is rather a complex. In addition,
the intrinsic fluorescence properties of compounds 36 and 37 were studied by using
fluorescence spectroscopy in the presence of increasing ct-DNA/compound ratios (Figure 4B)
and evidenced an increase of the fluorescence intensity of compounds 36 or 37 upon binding
to ct-DNA.

ACCEPTED MANUSCRIPT
A)
B)
Figure 4. A) UV/Vis titration of compound 36 with ct-DNA, B) Fluorimetric titration of
compound 36 with ct-DNA
To gain insight into the binding mode of compounds 36 and 37, circular dichroism
measurements and topoisomerase I-induced DNA relaxation were performed. By using
circular dichroism, the orientations of the molecules relative to the DNA helix (intercalation,
groove binding) were investigated. The absence of intrinsic CD of 36 and 37 was validated
using the highest evaluated dose (60 µM, Figure 5, dashed lines).

ACCEPTED MANUSCRIPT
40
30
20
10
0
40
36
30
20
37
10
0
-10
-20
-30
-40
-10
-20
-30
-40
250
300
350
400
450
500
250
300
350
400
450
500
Wavalength (nm)
Wavelength (nm)
Figure 5. Circular dichroism spectra. ct-DNA (50 µM of base pairs) was incubated alone
dashed bold lines) with increasing concentrations of 36 or 37 from 1 to 60 (full bold lines)
µM (top to bottom). Pointed bold lines correspond to the CD spectra of the compound alone
60 µM).
(
(
Binding of either compound 36 or 37 on ct-DNA decreased the intensity of the positive
intrinsic CD of ct-DNA at 275 nm, thus suggesting modifications in the base stacking and/or
the ellipticity of the DNA helix. Furthermore, binding of the compounds to DNA induced the
appearance of a new negative induced circular dichroic (ICD) signal from 290 to 440 nm
corresponding to the absorption bands of either compounds 36 or 37, suggesting the
intercalation of the planar compound/chromophore between adjacent base pairs of the DNA
and creation of a new dichroic signal. Both changes were dependent on the compounds
concentration.
In order to compare the DNA intercalation profile of 36 and 37 with other derivatives,
the DNA topoisomerase I enzyme was used to dissipate the constraints of a negatively
supercoiled circular DNA and to generate positively supercoiled circular DNA upon
intercalation between adjacent base pairs [18]. Intercalation between adjacent base pairs
lengthens the DNA helix, diminishes the angle of rotation between each base pair and thus
induces major constraints to the DNA structure. The topoisomerase I enzyme resolves the
constraints that are generated upon negative supercoiling of the plasmid DNA when produced
in bacteria (Sc) (Figure 6) and thus generates different DNA topoisomers (Topo)
corresponding to different number of released supercoils in the circular plasmid and up to
fully relaxed circular DNA (Rel). Blockage of the topoisomerase I re-ligation step by poison
drugs such as camptothecin (CPT) results in the generation of a nicked plasmid (Nck) that is

ACCEPTED MANUSCRIPT
only opened on one strand whereas DNA intercalation is associated with the formation of
progressive positive supercoiled plasmid with each topoisomers being more difficult to assign
individually due to binding of the compounds that reduce DNA migration within the agarose
gel, as seen with 36 and 37 in Figure 5. Within this experiment we confirmed the DNA
intercalation propensity of 36 and 37 but not the other evaluated compounds that were not
found to bind to DNA.
Observed DNA intercalation is not associated with the topoisomerase I poisoning
effect of 36 and 37 as demonstrated by the lack of increase in the intensity of the nicked form
(Nck) of the DNA. Such an intercalation process is not associated with sequence selective
binding as evaluated using DNase I footprinting experiments with increasing concentrations
of either 36 or 37 (data shown in Fig. S35).
Topo I
CPT
34
33
35
23
36
31
0
20 50 5 20 50 5 20 50 5 20 50 5 20 50 5 20 50 5 20 50 0
Rel/Nck
Topo
Sc
Topo I
CPT
31
37
0
10 20 50 0 0.5 1 2 5 10 20 50 0 0.5 1 2 5 10 20 50 0
Rel/Nck
Topo
Sc
Figure 6. Topoisomerase I-induced DNA relaxation. The pUC19 supercoiled plasmid
“DNA”) was incubated with topoisomerase I (“0”) alone or in the presence of the indicated
(
concentrations (µM) of compounds as specified on the top of the lanes. Rel, relaxed DNA;
Nck, nicked DNA; Topo, topoisomers; Sc, supercoiled DNA.

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Compounds 33 and 23 displayed a small increase of the band corresponding to the
relaxed/nicked form. However, only compound 33 presented a very weak topoisomerase I
poisoning activity as confirmed upon migration in ethidium bromide-containing agarose gels
(see the weak increase in the nicked form using 100 µM as the highest drug concentration)
whereas no poisoning activity was evidenced using compound 23 (Figure 7).
Figure 7. Topoisomerase I poisoning effects of compounds 33 and 23. Native supercoiled
pUC19 plasmid DNA (lanes “DNA”) was incubated with topoisomerase I in the absence
(lanes “Topo I”) or presence of increasing concentrations of compounds 33 and 23 and the
reference drugs camptothecin (CPT). DNA samples were separated under electrophoresis on
an ethidium bromide containing 1 % agarose gel. The gels were photographed under UV
light. Sc, supercoiled; Rel, relaxed; Nck; nicked; Lin, linear.
4
. Conclusions
Herein we present the synthesis of amino substituted benzimidazo[1,2-a]quinoline-6-
carbonitriles with different structural features and length of amino side chains incorporated at
either one or two positions on the tetracyclic skeleton. The new compounds have been
designed and synthesized by uncatalyzed microwave-assisted amination and explored with
3
D-QSAR analysis, and their antiproliferative activities and DNA binding abilities were
evaluated. The antiproliferative activities have been evindenced on human colon, breast and
lung tumor cell lines. 2-amino substituted analogues 5–9 demonstrated a more pronounced
antiproliferative activity when compared to 5- (18–22) and 2,5-diamino substituted
derivatives 23–39.

ACCEPTED MANUSCRIPT
The results obtained from antiproliferative activity examination were in good
agreement with previously published results. The antiproliferative activity results obtained for
the synthesized compounds, in particular those with the most pronounced activity of the 2-
substituted derivatives, were in good agreement with our previously published results [15].
Among the 2-substituted derivatives, 4-methyl-1-piperazinyl 8 or 3,5-dimethyl-1-piperazinyl
9
substituted analogues were the most active. Moreover, derivatives with long and/or
branched side chains were less active (mono-substituted analogues) or completely inactive
di-substituted analogues).
In general, 3D-QSAR analysis revealed the same molecular properties having the
(
largest positive and the largest negative influence on antiproliferative activity on all three
studied cell lines. The increase of antiproliferative activity against all three studied cell lines
could be explained by increased H-bonding properties, hydrophobicity and flexibility, as well
as the decrease of unbalance in distribution of hydrophobic and hydrophilic regions in the
molecule.
Compounds 36 and 37 were to be the most potent DNA binders of the compounds
studied and was associated with strong DNA helix stabilization evidenced by using melting
temperature studies and intercalative mode of DNA interaction (Figures 5 and 6). Additional
experiments with topoisomerase I-induced DNA relaxation confirmed that DNA binding is
not associated with sequence selective binding. The DNA intercalation process of the studied
compounds does not correlate with the cytotoxic effects evaluated on colon (HCT 116), breast
(
MCF-7) and lung (H460) carcinoma cells.
-Piperazinyl substituted compound 36 displayed a potent cytotoxic effect in all cell
lines (1.5, 1 and 4 µM, respectively), while the quaternary iodide salt 37 was totally inactive
IC > 100 µM). The new series of derivatives, bearing the amino substituent at position 2,
1
(
5
0
demonstrated a high potential for expansion and optimization for their antiproliferative
activity, in particular when either 2-methylpropyl or 1-piperazinyl amino substituents were
used.
5
5
. Experimental part
.1 General methods
All chemicals and solvents were purchased from commercial suppliers Aldrich and
Acros. Melting points were recorded on SMP11 Bibby and Büchi 535 apparatus. All NMR
1
spectra were measured in DMSO-d solutions using TMS as an internal standard. The H and
6

ACCEPTED MANUSCRIPT
1
3
C NMR spectra were recorded on a Varian Gemini 300 (at 300 and 75 MHz) or Varian
Gemini 600 (at 600 and 150 MHz) respectively. Chemical shifts are reported in ppm (δ)
relative to TMS. All compounds were routinely checked by TLC with Merck silica gel 60F-
2
54 glass plates. Microwave-assisted synthesis was performed in a Milestone start S
microwave oven using quartz cuvettes under the pressure of 40 bar. Mass spectra were
recorded on an Agilent 1200 series LC/6410 QQQ instrument. The electronic absorption
spectra were recorded on Varian Cary 50 spectrometer using quartz cuvette (1 cm). Elemental
analysis for carbon, hydrogen and nitrogen were performed on a Perkin-Elmer 2400 elemental
analyzer. Analyses are indicated by the symbols of the elements, and the results obtained were
within ±0,4 % of the theoretical values.
5
.2 Synthesis of 2-amino substituted benzimidazo[1,2-a]quinolines
.2.1 General method for preparation of compounds 5–9
5
Compounds 5–9 were prepared by reacting 4 with excess of the appropriate amine in
acetonitrile (10 mL) under microwave irradiation (800 W power) at 170 °C and 40 bar
pressure for the required reaction time. After cooling, the reaction mixture was filtered off and
and the crude solid was purified by column chromatography on SiO2 using
dichloromethane/methanol 100:1 as eluent.
5
.2.1.1 2-[(1-Methylethyl)amino]benzimidazo[1,2-a]quinoline-6-carbonitrile 5. Following the
above general method, compound 4 (100 mg, 0.38 mmol) reacted with isopropylamine (0.15
ml, 1.76 mmol) after 6 h of irradiation to yield 46 mg (43 %) of 5 as yellow crystals; m.p.
1
2
30–233 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.51 (d, 1H, J=8.10 Hz, Harom.), 8.46
6
(
s, 1H, Harom.), 7.94 (dd, 1H, J =1.28 Hz, J =7.50 Hz, Harom.), 7.81 (d, 1H, J=8.85 Hz, Harom.),
1 2
7
.77 (s, 1H, Harom.), 7.58 (dt, 1H, J =1.28 Hz, J =7.30 Hz, H
), 7.52 (dt, 1H, J =1.28 Hz,
arom. 1
1
2
J2=7.30 Hz, Harom.), 7.27 (d, 1H, J=7.56 Hz, NH), 6.91 (dd, 1H, J =1.75 Hz, J =8.85 Hz,
1
2
13
Harom.), 3.98–3.87 (m, 1H, CH), 1.28 (d, 6H, J=6.59 Hz, CH ); C NMR (150 MHz, DMSO-
3
d6): δ/ppm= 152.97 (s), 145.90 (s), 144.18 (d), 140.12 (d), 138.11 (s), 132.93 (d), 130.40 (s),
1
24.70 (d), 122.40 (d, 2C), 119.56 (d), 116.76 (s), 114.33 (d, 2C), 111.04 (s), 91.92 (s), 43.36
(
d), 22.24 (q, 2C); Found: C, 75.73; H, 5,52; N, 18,75. Calc. for C H N : C, 75.98; H, 5.37;
1
9
16
4
+
N, 18.65 %; MS (ESI): m/z= 301.2 ([M+1] ).
5
.2.1.2 2-[(3-Methylbutyl)amino]benzimidazo[1,2-a]quinoline-6-carbonitrile 6. Following
the above general method, compound 4 (80 mg, 0.30 mmol) reacted with isopentylamine

ACCEPTED MANUSCRIPT
(0.25 ml, 2.15 mmol) after 5 h of irradiation to yield 29 mg (29 %) of 6 as yellow crystals;
1
m.p. 200–205 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.50 (d, 1H, J=8.36 Hz, Harom.),
6
8
.46 (s, 1H, Harom.), 7.94 (dd, 1H, J =1.40 Hz, J =7.77 Hz, Harom.), 7.80 (d, 1H, J=8.93,
1 2
Harom.), 7.74 (s, 1H, Harom.), 7.57 (dt, 1H, J =1.00 Hz, J =7.39 Hz, Harom.), 7.51 (dt, 1H,
1
2
J =1.28 Hz, J =7.52 Hz, H
), 7.31 (t, 1H, J=5.17 Hz, NH), 6.90 (dd, 1H, J =1.70 Hz,
1
1
2
arom.
J2=8.85 Hz, Harom.), 3.32 (q, 2H, J=7.08 Hz, CH ), 1.80–1.75 (m, 1H, CH), 1.57 (q, 2H,
2
13
J=6.85 Hz, CH ), 0.98 (d, 6H, J=6.54 Hz, CH ); C NMR (150 MHz, DMSO-d ): δ/ppm=
2
3
6
1
1
53.84 (s), 145.92 (s), 144.21 (s), 140.10 (d), 138.11 (s), 132.81 (d), 130.40 (s), 124.34 (d),
22.34 (d), 119.56 (d), 116.73 (s), 114.32 (d), 111.16 (s), 92.00 (s), 40.60 (t), 37.28 (t), 25.23
(
d), 22.37 (q, 2C); Found: C, 76.93; H, 5.99; N, 17.08. Calc. for C H N : C, 76.80; H, 6.14;
21 20 4
+
N, 17.06 %; MS (ESI): m/z= 329.3 ([M+1] ).
5
.2.1.3 2-(Hexylamino)benzimidazo[1,2-a]quinoline-6-carbonitrile 7. Following the above
general method, compound 4 (90 mg, 0.34 mmol) reacted with hexylamine (0.24 ml, 1.20
mmol) after 3 h of irradiation to yield 60 mg (54 %) of 7 as yellow crystals; m.p. 97–104 °C.
1
H NMR (600 MHz, DMSO-d ): δ/ppm= 8.48 (d, 1H, J=8.28 Hz, Harom.), 8.43 (s, 1H, Harom.),
6
7
.92 (d, 1H, J=7.62 Hz, Harom.), 7.78 (d, 1H, J=8.76 Hz, Harom.), 7.70 (s, 1H, Harom.), 7.56 (t,
H, J=7.62 Hz, Harom.), 7.50 (dt, 1H, J =1.06 Hz, J =7.72 Hz, Harom.), 7.31 (t, 1H, J=5.10 Hz,
1
1
2
NH), 6.88 (dd, 1H, J =1.74 Hz, J =8.76 Hz, H
), 3.35-3.28 (m, 2H, CH ), 1.66 (p, 2H,
2
1
2
arom.
J=7.26 Hz, CH ), 1.44 (p, 2H, J=7.36 Hz, CH ), 1.36–1.31 (m, 4H, CH ), 0.88 (t, 3H, J=7.08
2
2
2
13
Hz, CH ); C NMR (150 MHz, DMSO-d ): δ/ppm= 153.87 (s), 145.85 (s), 144.00 (s), 140.14
3
6
(d), 130.34 (s), 124.73 (d), 122.38 (d, 2C), 119.45 (d), 116.72 (s), 114.34 (d, 2C), 111.14 (s),
9
6
1.61 (s), 42.40 (t), 31.06 (t), 28.36 (t), 26.27 (t), 22.68 (t), 13.90 (q); Found: C, 76.98; H,
.28; N, 16.74. Calc. for C H N : C, 77.16; H, 6.48; N, 16.36 %; MS (ESI): m/z= 343.2
2
2
22
4
+
(
[M+1] ).
5
.2.1.4 2-(4-Methyl-1-piperazinyl)benzimidazo[1,2-a]quinoline-6-carbonitrile 8. Following
the above general method, compound 4 (90 mg, 0.34 mmol) reacted with 1-methylpiperazine
(0.10 ml, 0.90 mmol) after 3 h of irradiation to yield 65 mg (56 %) of 8 as yellow crystals;
1
m.p. 267–273 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.56 (s, 1H, Harom.), 8.55 (d, 1H,
6
J=7.68 Hz, Harom.), 7.94 (dd, 1H, J =1.36 Hz, J =7.48 Hz, Harom.), 7.91 (d, 1H, J=9.15 Hz,
1
2
Harom.), 7.80 (d, 1H, J=1.05 Hz, Harom.), 7.59 (t, 1H, J=6.78 Hz, Harom.), 7.54 (dt, 1H, J =1.42
1
Hz, J =7.34 Hz, H
), 7.33 (dd, 1H, J =1.72 Hz, J =9.25 Hz, Harom.), 3.61 (t, 4H, J=4.80 Hz,
1 2
2
arom.
1
3
CH ), 2.54 (t, 4H, J=5.09 Hz, CH ), 2.27 (s, 3H, CH ); C NMR (75 MHz, DMSO-d ):
2
2
3
6

ACCEPTED MANUSCRIPT
δ/ppm= 154.61 (s), 146.15 (s), 144.57 (s), 140.34 (d), 138.60 (s), 132.83 (d), 130.78 (s),
1
25.38 (d), 123.46 (d), 120.17 (d), 116.87 (s), 115.35 (d), 113.02 (d), 112.84 (s), 98.13 (d),
9
4.88 (s), 54.79 (t, 2C), 47.14 (t, 2C), 46.16 (t, 2C); Found: C, 73.98; H, 5.71; N, 20.31. Calc.
+
for C H N : C, 73.88; H, 5.61; N, 20.51 %; MS (ESI): m/z= 342.2 ([M+1] ).
2
1
19
5
5
.2.1.5 2-(3,5-Dimethyl-1-piperazinyl)benzimidazo[1,2-a]quinoline-6-carbonitrile 9.
Following the above general method, compound 4 (90 mg, 0.34 mmol) reacted with 2,6-
dimethylpiperazine (0.150 g, 1.30 mmol) after 6 h of irradiation to yield 39 mg (33 %) of 9 as
1
yellow powder; m.p. 263–268 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.47 (s, 1H,
6
Harom.), 8.43 (dd, 1H, J =1.19Hz, J =7.13 Hz, H
), 7.94 (dd, 1H, J =1.41 Hz, J =7.56 Hz,
1 2
1
2
arom.
Harom.), 7.84 (d, 1H, J=9.12 Hz, Harom.), 7.68 (s, 1H, Harom.), 7.58 (dt, 1H, J =1.21, J =6.59 Hz,
1
2
Harom.), 7.53 (dt, 1H, J =1.28 Hz, J =6.69 Hz, H
), 7.27 (dd, 1H, J =1.66 Hz , J =9.13 Hz,
1 2
1
2
arom.
Harom.), 4.01 (d, 2H, J=10.53 Hz, CH ), 2.95–2,90 (m, 2H, CH), 2.55 (d, 2H, J=11.70 Hz,
2
1
3
CH ), 1.13 (d, 6H, J=6.21 Hz, CH ); C NMR (75 MHz, DMSO-d ): δ/ppm= 154.27 (s),
2
3
6
1
1
5
7
46.15 (s), 144.56 (s), 140.18 (d), 138.57 (s), 132.74 (d), 130.72 (s), 125.29 (d), 123.38 (d),
20.13 (d), 116.92 (s), 115.14 (d), 112.93 (d), 112.53 (s), 97.85 (d), 94.51 (s), 53.67 (t, 2C),
0.61 (d, 2C), 19.56 (q, 2C); Found: C, 74.40; H, 6.08; N, 19.52. Calc. for C H N : C,
2
2
21
5
+
4.34; H, 5.96; N, 19.70 %; MS (ESI): m/z= 356.3 ([M+1] ).
5
.3 Synthesis of 5-amino substituted benzimidazo[1,2-a]quinolines
.3.1 General method for preparation of compounds 18–22
5
Compounds 18–22 were prepared by reacting 16 with excess of the appropriate amine in
acetonitrile (10 mL) under microwave irradiation (800 W power) at 170 °C and 40 bar
pressure for the required reaction time. After cooling, the reaction mixture was filtered off and
and the crude solid was purified by column chromatography on SiO2 using
dichloromethane/methanol 100:1 as eluent.
5
.3.3.1 5-[(1-Methylethyl)amino]benzimidazo[1,2-a]quinoline-6-carbonitrile 18. Following
the above general method, compound 16 (100 mg, 0.38 mmol) reacted with isopropylamine
(0.10 ml, 1.12 mmol) after 2 h of irradiation to yield 42 mg (39 %) of 18 as white powder;
1
m.p. 260–263 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.66 (d, 1H, J=8.08 Hz, Harom.),
6
8
.55 (dd, 1H, J =0.92 Hz, J =7.76 Hz, Harom.), 8.43 (d, 1H, J=8.08 Hz, Harom.), 7.93 (dt, 1H,
1
2
J =0.92 Hz, J =8.12 Hz, H
), 7.75 (dd, 1H, J =0.60 Hz, J =7.86 Hz, Harom.), 7.59 (t, 1H,
1 2
1
2
arom.
J=7.60 Hz, Harom.), 7.55 (t, 1H, J=6.81 Hz, NH), 7.42 (t, 1H, J=7.50 Hz, Harom.), 7.33 (dt, 1H,

ACCEPTED MANUSCRIPT
1
3
J =1.17 Hz, J =7.40 Hz, H
), 4.90–4.79 (m, 1H, CH), 5.99 (d, 6H, J=6.68 Hz, CH ); C
3
1
2
arom.
NMR (150 MHz, DMSO-d ): δ/ppm= 149.15 (s), 148.99 (s), 144.67 (s), 135.05 (s), 132.96
6
(d), 130.79 (s), 124.78 (d), 124.13 (d), 123.88 (d), 121.28 (d), 118.36 (d), 117.53 (s), 116.54
(s), 116.14 (d), 113.62 (d), 71.84 (s), 45.87 (d), 23.00 (q, 2C); Found: C, 75.77; H, 5.69; N,
+
1
8.54. Calc. for C H N : C, 75.98; H, 5.37; N, 18.65 %; MS (ESI): m/z= 301.2 ([M+1] ).
1
9
16
4
5
.3.3.2 5-[(3-Methylbutyl)amino]benzimidazo[1,2-a]quinoline-6-carbonitrile 19. Following
the above general method, compound 16 (50 mg, 0.18 mmol) reacted with i-pentylamine
(0.11 ml, 0.90 mmol) after 2 h of irradiation to yield 9 mg (15 %) of 19 as white powder; m.p.
1
1
05–108 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.67 (d, 1H, J=8.65 Hz, Harom.), 8.49
6
(d, 1H, J=8.35 Hz, Harom.), 8.43 (d, 1H, J=8.35 Hz, Harom.), 8.11 (t, 1H, J=5.68 Hz, NH), 7.93
(t, 1H, J=7.46 Hz, Harom.), 7.74 (d, 1H, J=7.76 Hz, Harom.), 7.59 (t, 1H, J=7.76 Hz, Harom.), 7.41
(
t, 1H, J=7.46 Hz, Harom.), 7.33 (t, 1H, J=7.61 Hz, Harom.), 3.91 (q, 2H, J=6.86 Hz, CH ), 1.67–
2
13
1
.58 (m, 1H, CH), 1.43 (q, 2H, J=7.16 Hz, CH ), 0.88 (q, 6H, J=6.27 Hz, CH ); C NMR
2 3
(
150 MHz, DMSO-d ): δ/ppm= 149.95 (s), 149.03 (s), 144.67 (s), 135.02 (s), 132.95 (d),
6
1
1
7
3
30.81 (s), 124.40 (d), 124.28 (d), 123.88 (d), 121.27 (d), 118.34 (d), 117.60 (s), 116.57 (s),
16.25 (d), 113.62 (d), 71.46 (s), 42.59 (t), 37.21 (t), 22.39 (d), 22.09 (q, 2C); Found: C,
7.05; H, 6.02; N, 16.93. Calc. for C H N : C, 76.80; H, 6.14; N, 17.06 %; MS (ESI): m/z=
21
20
4
+
29.3 ([M+1] ).
.3.3.3 5-(Hexylamino)benzimidazo[1,2-a]quinoline-6-carbonitrile 20. Following the above
5
general method, compound 16 (100 mg, 0.36 mmol) reacted with hexylamine (0.20 ml, 1.52
mmol) after 3 h of irradiation to yield 56 mg (48 %) of 20 as yellow powder; m.p. 121–125
1
°
C. H NMR (600 MHz, DMSO-d ): δ/ppm= 8.65 (d, 1H, J=8.34 Hz, Harom.), 8.44 (dd, 1H,
6
J =0.84 Hz, J =8.37 Hz, Harom.), 8.41 (d, 1H, J=8.27 Hz, Harom.), 8.07 (t, 1H, J=5.58 Hz, NH),
1
2
7
.90 (dt, 1H, J =0.87 Hz, J =7.73 Hz, Harom.), 7.73 (d, 1H, J=7.80 Hz, Harom.), 7.57 (t, 1H,
1 2
J=7.59 Hz, Harom.), 7.40 (t, 1H, J=7.47 Hz, Harom.), 7.32 (dt, 1H, J =0.93 Hz, J =7.73 Hz,
1
2
Harom.), 3.86 (q, 2H, J=6.58 Hz, CH ), 1.78 (p, 2H, J=7.19 Hz, CH ), 1.42 (p, 2H, J=7.28 Hz,
2
2
13
CH ), 1.31 (m, 4H, CH ), 0.86 (t, 3H, J=7.08 Hz, CH ); C NMR (150 MHz, DMSO-d ):
2
2
3
6
δ/ppm= 149.88 (s), 149.00 (s), 144.65 (s), 135.00 (s), 132.11 (d), 130.79 (s), 124.31 (d),
1
7
6
24.25(d), 123.86 (d), 121.25 (d), 118.32 (d), 117.54 (s), 116.51 (s), 116.22 (d), 113.59 (d),
1.42(s), 44.05 (t), 30.92 (t), 29.37 (t), 25.56 (t), 21.96 (t), 13.60 (q); Found: C, 77.36; H,
.18; N, 16.46. Calc. for C H N : C, 77.16; H, 6.48; N, 16.36 %; MS (ESI): m/z= 343.2
2
2
22
4
+
(
[M+1] ).

ACCEPTED MANUSCRIPT
5
.3.3.4 5-(4-Methyl-1-piperazinyl)benzimidazo[1,2-a]quinoline-6-carbonitrile 21. Following
the above general method, compound 16 (100 mg, 0.36 mmol) reacted with 1-
methylpiperazine (0.10 ml, 0.90 mmol) after 3 h of irradiation to yield 53 mg (43 %) of 21 as
1
yellow powder; m.p. 253–257 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.77 (d, 1H,
6
J=8.46 Hz, Harom.), 8.59 (d, 1H, J=7.86 Hz, Harom.), 8.17 (dd, 1H, J =0.78 Hz, J =8.04 Hz,
1
2
Harom.), 7.95 (dt, 1H, J =0.95 Hz, J =7.38 Hz, H
), 7.89 (dd, 1H, J =1.08 Hz, J =7.44 Hz,
1 2
1
2
arom.
Harom.), 7.63 (t, 1H, J=7.70 Hz, Harom.), 7.52 (t, 1H, J=7.20 Hz, Harom.), 7.46 (dt, 1H, J =1.06
1
Hz, J =7.69 Hz, H
), 3.63 (t, 4H, J=4.58 Hz, CH ), 2.66 (t, 4H, J=4.51 Hz, CH ), 2.33 (s,
2 2
2
arom.
1
3
3
H, CH ); C NMR (150 MHz, DMSO-d ): δ/ppm= 157.28 (s), 146.94 (s), 144.31 (s), 136.10
3 6
(
s), 133.18 (d), 130.63 (d), 127.53 (d), 124.65 (d), 124.60 (d), 122.58 (d), 119.46 (s), 119.41
d), 116.33 (d), 115.91 (s), 114.26 (d), 89.09 (s), 54.88 (t, 2C), 52.10 (t, 2C), 45.77 (q);
(
Found: C, 73.76; H, 5.81; N, 20.40. Calc. for C H N : C, 73.88; H, 5.61; N, 20.51 %; MS
2
1
19
5
+
(
ESI): m/z= 342.2 ([M+1] ).
5
.3.3.5 5-(3,5-Dimethyl-1-piperazinyl)benzimidazo[1,2-a]quinoline-6-carbonitrile 22.
Following the above general method, compound 16 (100 mg, 0.36 mmol) reacted with 2,6-
dimethylpiperazine (0.100 g, 0.87 mmol) after 3 h of irradiation to yield 60 mg (47 %) of 22
1
as yellow powder; m.p. 247–250 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.73 (d, 1H,
6
J=8.46 Hz, Harom.), 8.56 (d, 1H, J=7.86 Hz, Harom.), 8.14 (d, 1H, J=8.19 Hz, Harom.), 7.93 (t,
1
1
H, J=7.88 Hz, Harom.), 7.87 (d, 1H, J=7.86 Hz, Harom.), 7.61 (t, 1H, J=7.53 Hz, Harom.), 7.51 (t,
H, J=7.45 Hz, Harom.), 7.44 (t, 1H, J=7.63 Hz, Harom.), 3.60 (d, 2H, J=11.16 Hz, CH ), 3.10
2
13
(
m, 2H, CH), 3.04 (d, 2H, J=11.11 Hz, CH ), 1.05 (d, 6H, J=5.97 Hz, CH ); C NMR (150
2
3
MHz, DMSO-d ): δ/ppm= 157.06 (s), 147.13 (s), 144.36 (s), 136.14 (s), 133.08 (d), 130.54
6
(
s), 127.53 (d), 127.68 (d), 124.60 (d), 124.45 (d), 122.47 (d), 119.49 (s), 119.41 (s), 119.33
d), 116.30 (d), 115.99 (s), 114.19 (d), 100.41 (s), 59.01 (t, 2C), 50.85 (d, 2C), 39.24 (q, 2C);
(
Found: C, 74.18; H, 6.16; N, 19.60. Calc. for C H N : C, 74.34; H, 5.96; N, 19.70 %; MS
2
2
21
5
+
(
ESI): m/z= 356.3 ([M+1] ).
5
.4 Synthesis of 2,5-diamino substituted benzimidazo[1,2-a]quinolines
5
.4.1 -(1H-benzimidazol-(3H)-idene)-2-chloro-4-fluoro- -oxobenzenepropanenitrile 13. A
α
γ
solution of (1H-benzimidazol-2-yl)acetonitrile 2 (1.63 g, 10.40 mmol) and 2-chloro-4-
fluorobenzoyl chloride 11 (2.00 g, 10.40 mmol) in pyridine (13 ml) was refluxed for 1.5 h.
After being cooled to room temperature, the reaction mixture was poured into water (100 ml)
and the precipitated solid was filtered off and recrystallized from ethanol to obtain 13 as a

ACCEPTED MANUSCRIPT
1
brown powder (1.36 g, 49 %); m.p. >300 °C. H NMR (300 MHz, DMSO-d ): δ/ppm=13.10
6
13
(
bs, 2H, NH), 7.59–7.51 (m, 4H, Harom.), 7.36–7,30 (m, 3H, Harom.); C NMR (75 MHz,
DMSO-d ): δ/ppm= 184.75 (s), 162.40 (s, J =247.17 Hz), 150.84 (s), 137.56 (s, J =3.50
6
CF
CF
Hz), 131.24 (s, J =10.75 Hz), 130.86 (s), 130.65 (d, J =9.17 Hz), 124.14 (d, 2C), 120.30
CF
CF
(
s), 117.46 (d, J =25.01 Hz), 114.83 (d, J =21.13 Hz), 112.74 (d, 2C), 67.77 (s).
CF CF
5
.4.2 2-Fluoro-5-oxo-5,7-dyhidrobenzimidazo[1,2-a]quinoline-6-carbonitrile 15. A solution
of compound 13 (1.00 g, 3.37 mmol) and t-BuOK (0.88 g, 7.84 mmol) in DMF (12 ml) was
refluxed for 2 h. After cooling, the reaction mixture was evaporated under vacuum, and the
residue was triturated with water (50 ml). The resulting solid was filtered off and
1
recrystallized from ethanol to obtain 15 as a white powder (0.85 g, 96 %); m.p. >300 °C. H
NMR (300 MHz, DMSO-d ): δ/ppm= 8.29 (dd, 1H, J =6.99 Hz, J =8.79 Hz, Harom.), 8.19 (d,
6
1
2
1
H, J=8.10 Hz, Harom.), 8.13 (dd, 1H, J =2.30 Hz, J =11.06 Hz, H
), 7.48 (dd, 1H, J =0.84
1
2
arom.
1
Hz, J =7.86 Hz, H
), 7.27 (dd, 1H, J =0.95 Hz, J =6.20 Hz, H
), 7.23 (dt, 1H, J =2.19
2
arom.
1
2
arom.
1
1
3
Hz, J =8.53 Hz, H
), 7.11(dt, 1H, J =1.10 Hz, J =7.87 Hz, H
); C NMR (150 MHz,
2
arom.
1
2
arom.
DMSO-d ): δ/ppm= 171.64 (s), 163.59 (s, J =245.44 Hz), 154.08 (s), 137.13 (s, J =5.85
6
CF
CF
Hz), 130.90 (s), 128.02 (d, J =10.45 Hz), 122.99 (d), 121.30 (s), 120.27 (s), 119.06 (d),
CF
1
16.35 (d), 112.31 (d), 110.33 (d, J =21.68 Hz), 101.83 (d, J =27.06 Hz), 73.59 (s).
CF CF
5
.4.3 5-chloro-2-fluorobenzimidazo
[
1,2-a
]
quinoline-6-carbonitrile 17.
A solution of 0.60 g (2.15 mmol) 2-fluoro-5-ketobenzimidazo[1,2-a]quinoline-6-carbonitrile
and 0.24 g (1.15 mmol) PCl in POCl (12 ml) was refluxed for 1.5 h. After cooling, the
5
3
reaction mixture was evaporated under vacuum and the residue was triturated with water (10
mL). The resulting solid was filtered off and washed with water to obtain 17 as a yellow
1
powder (0.62 g, 96 %); m.p. 250–257 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.71 (d,
6
1
H, J=8.49 Hz, Harom.), 8.58 (dd, 1H, J =2.27 Hz, J =10.37 Hz, H
), 8.41 (dd, 1H, J =6.06
arom. 1
1
2
Hz, J =9.12 Hz, H
), 8.00 (dd, 1H, J =1.32 Hz, J =7.67 Hz, Harom.), 7.67–7.60 (m, 2H,
1 2
2
arom.
13
Harom.), 7.57 (dt, 1H, J =1.34 Hz, J =7.61 Hz, Harom.); C NMR (150 MHz, DMSO-d6):
1
2
δ/ppm= 165.29 (s, J =253.77 Hz), 144.34 (s), 143.82 (s), 142.18 (s), 136.82 (s, J =12.18
CF
CF
Hz), 130.91 (d, J =10.95 Hz), 130.21 (s), 125.66 (d), 124.13 (d), 120.27 (d), 116.32 (s,
CF
J =1.61 Hz), 114.99 (d), 114.01 (d, J =23.40 Hz), 113.32 (s), 103.43 (d, J =27.53 Hz),
CF
CF
CF
1
01.64 (s).
.4.4 General method for preparation of compounds 23–39
5

ACCEPTED MANUSCRIPT
Compounds 23–39 were prepared by reacting 17 with excess of the appropriate amine in
acetonitrile (10 mL) under microwave irradiation (800 W power) at 170 °C and 40 bar
pressure for the required reaction time. After cooling, the reaction mixture was filtered off and
and the crude solid was purified by column chromatography on SiO2 using
dichloromethane/methanol 100:1 as eluent.
5
.4.4.1
2,5-Bis(methylamino)benzimidazo[1,2-a]quinoline-6-carbonitrile
23.
Following the above general method, compound 17 (50 mg, 0.50 mmol) reacted with 33 %
solution of methylamine in ethanol (0.26 ml, 1.90 mmol) after 12 h of irradiation to yield 35
1
mg (57 %) of 23 as light brown powder; m.p. >280 °C. H NMR (300 MHz, DMSO-d ):
6
δ/ppm= 8.29 (d, 1H, J=8.07 Hz, Harom.), 8.08 (d, 1H, J=9.21 Hz, Harom.), 7.89 (d, 1H, J=5.16
Hz, NH), 7.69 (dd, 1H, J =0.78 Hz, J =7.89 Hz, Harom.), 7.55 (d, 1H, J=1.62 Hz, Harom.), 7.41
1
2
(
t, 1H, J=7.52 Hz, Harom.), 7.33 (dt, 1H, J =1.03 Hz, J =7.73 Hz, Harom.), 7.06 (d, 1H, J=4.62
1
2
Hz, NH), 6.78 (dd, 1H, J =1.86 Hz, J =9.18 Hz, H
), 3.39 (d, 3H, J=4.98 Hz, CH ), 2.93
3
1
2
arom.
1
3
(
d, 3H, J=4.59 Hz, CH ); C NMR (75 MHz, DMSO-d ): δ/ppm= 153.99 (s, 2C), 152.41 (s),
3
6
1
37.26 (s), 131.06 (s), 126.07 (d), 124.61 (d), 121.41 (d), 118.93 (s), 117.96 (d), 113.94 (d),
09.72 (d), 104.98 (s), 96.56 (d), 94.79 (s), 82.64 (s), 32.41 (q), 29.74 (q); Found: C, 71.60;
1
H, 5.09; N, 23.31. Calc. for C H N : C, 71.74; H, 5.02; N, 23.24 %; MS (ESI): m/z= 302.4
1
8
15
5
+
(
[M+1] ).
5
.4.4.2 2,5-Bis(butylamino)benzimidazo[1,2-a]quinoline-6-carbonitrile 24. Following the
above general method, compound 17 (100 mg, 0.34 mmol) reacted with n-butylamine (0.18
ml, 1.80 mmol) after 4 h of irradiation to yield 75 mg (57 %) of 24 as light yellow powder;
1
m.p. 150–153 °C. H NMR (600 MHz, DMSO-d ): δ/ppm= 8.26 (d, 1H, J=8.22 Hz, Harom.),
6
8
.16 (d, 1H, J=9.18 Hz, Harom.), 7.67 (d, 1H, J=7.86 Hz, Harom.), 7.64 (t, 1H, J=6.09 Hz, NH),
.61 (s, 1H, Harom.), 7.37 (t, 1H, J=7.67 Hz, Harom.), 7.28 (dt, 1H, J =1.08 Hz, J =7.76 Hz,
7
1
2
Harom.), 6.94 (t, 1H, J=5.22 Hz, NH), 6.77 (dd, 1H, J =1.98 Hz, J =9.18 Hz, Harom.), 3.81 (q,
1
2
2
1
H, J=6.88 Hz, CH ), 3.25 (q, 2H, J=6.36 Hz, CH ), 1.74 (m, 1H, CH ), 1.63 (m, 2H, CH ),
2 2 2 2
.46 (m, 2H, CH ), 1.42 (m, 2H, CH ), 0.96 (t, 3H, J=7.38 Hz, CH ), 0.94 (t, 3H, J=7.38 Hz,
2
2
3
13
CH ); C NMR (75 MHz, DMSO-d ): δ/ppm= 153.19 (s), 151.18 (s), 150.61 (s), 145.43 (s),
3
6
1
1
1
7
37.37 (s), 131.28 (s), 126.40 (d), 124.08 (d), 120.98 (d), 119.06 (s), 118.42 (d), 113.74 (d),
09.01 (d), 105.08 (s), 97.17 (d), 68.35 (s), 44.09 (t), 42.51 (t), 32.27 (t), 31.09 (t), 20.25 (t),
9.72 (t), 14.24 (q, 2C); Found: C, 74.75; H, 7.00; N, 18.25. Calc. for C H N : C, 74.72; H,
2
4
27
5
+
.06; N, 18.17 %; MS (ESI): m/z= 386.5 ([M+1] ).

ACCEPTED MANUSCRIPT
5
.4.4.3 2,5-Bis[(1-methylethyl)amino]benzimidazo[1,2-a]quinoline-6-carbonitrile 25.
Following the above general method, compound 17 (100 mg, 0.34 mmol) reacted with
isopropylamine (0.40 ml, 2.35 mmol) after 7 h of irradiation to yield 31 mg (26 %) of 25 as
1
white powder; m.p. 240–245 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.27 (d, 1H,
6
J=8.19 Hz, Harom.), 8.21 (d, 1H, J=9.24 Hz, Harom.), 7.68 (d, 1H, J=7.80 Hz, Harom.), 7.62 (d,
1
H, J=1.30 Hz, Harom.), 7.38 (t, 1H, J=7.56 Hz, Harom.), 7.29 (t, 1H, J=7.40 Hz, Harom.), 7.11 (d,
H, J=8.55 Hz, NH), 6.90 (d, 1H, J=7.68 Hz, NH), 6.78 (dd, 1H, J =1.29 Hz, J =9.03 Hz,
1
1
2
Harom.), 4.83–4.72 (m, 1H, CH), 3.91–3.80 (m, 1H, CH), 1.38 (t, 6H, J=6.30 Hz, CH ), 1.25 (t,
3
1
3
6
1
1
H, J=6.27 Hz, CH ); C NMR (75 MHz, DMSO-d ): δ/ppm= 152.37 (s, 2C), 152.33 (s),
3
6
50.53 (s), 145.44 (s), 137.49 (s), 131.28 (s), 126.93 (d), 124.10 (d), 121.02 (d), 118.93 (s),
18.45 (d), 113.73 (d), 109.14 (d), 105.06 (s), 97.56 (d), 68.90 (s), 46.12 (d), 43.66 (d), 23.67
(
q, 2C), 22.78 (q, 2C); Found: C, 73.84; H, 6.53; N, 19.63. Calc. for C H N : C, 73.92; H,
22 23 5
+
6
.49; N, 19.59 %; MS (ESI): m/z= 358.2 ([M+1] ).
5
.4.4.4 2,5-Bis[(2-methylpropyl)amino]benzimidazo[1,2-a]quinoline-6-carbonitrile
26.
Following the above general method, compound 17 (100 mg, 0.34 mmol) reacted with
isobutylamine (0.50 ml, 5.04 mmol) after 6 h of irradiation to yield 25 mg (19 %) of 26 as
1
yellow crystals; m.p. 128–133 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.29 (d, 1H,
6
J=8.10 Hz, Harom.), 8.19 (d, 1H, J=9.24 Hz, Harom.), 7.76 (t, 1H, J=6.16 Hz, NH), 7.68 (d, 1H,
J=7.86 Hz, Harom.), 7.67 (s, 1H, Harom.), 7.37 (t, 1H, J=7.37 Hz, Harom.), 7.30 (dt, 1H, J =1.05
1
Hz, J =7.67 Hz, H
), 7.04 (t, 1H, J=5.48 Hz, NH), 6.81 (dd, 1H, J =1.64 Hz, J =9.17 Hz,
1 2
2
arom.
Harom.), 3.62 (t, 2H, J=6.63 Hz, CH ), 3.10 (t, 2H, J=6.11 Hz, CH ), 2.20–2.11 (m, 1H, CH),
2
2
13
1
.99–1.91 (m, 1H, CH), 1.03 (d, 6H, J=6.66 Hz, CH ), 0.98 (d, 6H, J=6.63 Hz, CH ); C
3 3
NMR (75 MHz, DMSO-d ): δ/ppm= 153.39 (s), 151.25 (s), 150.60 (s), 145.44 (s), 137.39 (s),
6
1
1
7
3
31.29 (s), 126.41 (d), 124.08 (d), 120.97 (d), 118.97 (s), 118.43 (d, 2C), 113.73 (d, 2C),
05.11 (s), 51.38 (t), 50.60 (t), 28.72 (d), 28.13 (d), 20.83 (q, 2C), 20.04 (q, 2C); Found: C,
4.25; H, 7.10; N, 18.56. Calc. for C H N : C, 74.77; H, 7.06; N, 18.17 %; MS (ESI): m/z=
24
27
5
+
86.3 ([M+1] ).
.4.4.5 2,5-Bis[(3-methylbutyl)amino]benzimidazo[1,2-a]quinoline-6-carbonitrile
5
27.
Following the above general method, compound 17 (200 mg, 0.72 mmol) reacted with
isopentylamine (0.17 ml, 1.50 mmol) after 6 h of irradiation to yield 34 mg (28 %) of 27 as
1
white powder; m.p. 111–113 °C. H NMR (300 MHz, DMSO-d ): δ/ppm= 8.27 (d, 1H,
6
J=8.19 Hz, Harom.), 8.13 (d, 1H, J=9.26 Hz, Harom.), 7.68 (d, 1H, J=7.48 Hz, Harom.), 7.62 (s,