New quinoline-arylamidine hybrids: Synthesis, DNA/RNA binding and antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2017.05.054

Four series of new hybrid molecules with 7-chloroquinoline and arylamidine moieties joined through the rigid -O- (groups I (2a-g) and II (5a-g)) or flexible -NH-CH₂-CH₂-O- (groups III (8a-g) and IV (10a-g)) linker were synthesized, and their DNA/RNA binding properties and cytotoxic activity were tested, against several human cancer lines. The compounds and their interaction with DNA and RNA were studied by UV–Vis and CD spectroscopy. The obtained results showed that the binding affinity of the investigated compounds increases proportionally with the increase of the length and number of groups able to form hydrogen bonds with ds-polynucleotides. Improvement of binding was additionally achieved by reduction of the structural rigidity of the investigated compounds, new hybrid compounds preferentially bind to ctDNA. For most of them the DNA/RNA grooves are dominant binding sites, except for the compounds from group II for which intercalation in polyA-polyU was the dominant binding mode. The antiproliferative effects were tested by the MTT test on normal (MDCK1), carcinoma (HeLa and CaCo2) and leukemia cell lines (Raji and K462). The GI₅₀ values for all investigated compounds ranged from 5 to more than 100 × 10^?6 mol dm^?3. Carcinoma cells were more resistant to the investigated compounds than leukemia cells. The most effective compounds against leukemia cell lines were from group IV (10a-g), with GI₅₀ values ranging from of 5 and 35 × 10^?6 mol dm^?3. The cell cycle arrest was investigated by flow cytometry and the obtained results indicate that the selected compounds, 2d, 2e, 8a, 10d, 10e, and 10f, induce changes in the cell cycle of treated cells, but the cycle phase distribution varies between them. A significant decrease in the number of cells in S phase (p < 0.001) was observed in all treated cells, but only 10d and 10f induce cell cycle arrest at G0/G1 phase, dominantly.

Full text of DOI:10.1016/j.ejmech.2017.05.054

Accepted Manuscript
New quinoline-arylamidine hybrids: Synthesis, DNA/RNA binding and antitumor
activity
Luka Krstulović, Ivana Stolić, Marijana Jukić, Teuta Opačak-Bernardi, Kristina
Starčević, Miroslav Bajić, Ljubica Glavaš-Obrovac
PII:
S0223-5234(17)30423-3
10.1016/j.ejmech.2017.05.054
EJMECH 9484
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 April 2017
Revised Date: 23 May 2017
Accepted Date: 24 May 2017
Please cite this article as: L. Krstulović, I. Stolić, M. Jukić, T. Opačak-Bernardi, K. Starčević, M. Bajić,
L. Glavaš-Obrovac, New quinoline-arylamidine hybrids: Synthesis, DNA/RNA binding and antitumor
activity, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.05.054.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New Quinoline-Arylamidine Hybrids: Synthesis, DNA/RNA Binding and Antitumor Activity
Luka Krstulovića,1, Ivana Stolića,1, Marijana Jukićb, Teuta Opačak-Bernardib, Kristina
Starčevićc, Miroslav Bajića,*, Ljubica Glavaš-Obrovacb,*
a Department of Chemistry and Biochemistry, Faculty of Veterinary Medicine, University of
Zagreb, Heinzelova 55, HR-10000 Zagreb, Croatia
b Department of Medicinal Chemistry, Biochemistry and Clinical Chemistry, Faculty of
Medicine, J. J. Strossmayer University of Osijek, J. Huttlera 4, HR-31000 Osijek, Croatia
c Department of Animal Husbandry, Faculty of Veterinary Medicine, University of Zagreb,
Heinzelova 55, HR-10000 Zagreb, Croatia
*Corresponding authors.
E-mail addresses: mbajic@vef.hr (M. Bajić), lgobrovac@mefos.hr (Lj. Glavaš-Obrovac).
1 These authors contributed equally to this work.
Abstract
Four series of new hybrid molecules with 7-chloroquinoline and arylamidine moieties joined
through the rigid -O- (groups I (2a-g) and II (5a-g)) or flexible -NH-CH2-CH2-O- (groups III
(8a-g) and IV (10a-g)) linker were synthesized, and their DNA/RNA binding properties and
cytotoxic activity were tested, against several human cancer lines. The compounds and their
interaction with DNA and RNA were studied by UV-Vis and CD spectroscopy. The obtained
results showed that the binding affinity of the investigated compounds increases
proportionally with the increase of the length and number of groups able to form hydrogen
bonds with ds-polynucleotides. Improvement of binding was additionally achieved by
reduction of the structural rigidity of the investigated compounds, new hybrid compounds
preferentially bind to ctDNA. For most of them the DNA/RNA grooves are dominant binding
sites, except for the compounds from group II for which intercalation in polyA-polyU was the
dominant binding mode. The antiproliferative effects were tested by the MTT test on normal
(MDCK1), carcinoma (HeLa and CaCo2) and leukemia cell lines (Raji and K462). The GI50
values for all investigated compounds ranged from 5 to more than 100 × 10-6 mol dm-3.
Carcinoma cells were more resistant to the investigated compounds than leukemia cells. The
most effective compounds against leukemia cell lines were from group IV (10a-g), with GI50
values ranging from of 5 and 35 × 10-6 mol dm-3. The cell cycle arrest was investigated by
flow cytometry and the obtained results indicate that the selected compounds, 2d, 2e, 8a, 10d,
10e, and 10f, induce changes in the cell cycle of treated cells, but the cycle phase distribution
varies between them. A significant decrease in the number of cells in S phase (p < 0.001) was

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observed in all treated cells, but only 10d and 10f induce cell cycle arrest at G0/G1 phase,
dominantly.
Key words: Hybrid molecule, 7-Chloroquinoline, Aromatic amidine, DNA/RNA binding,
Antitumor activity
1. Introduction
The rising burden of cancer has a huge cost, enormous human potential is lost and the
treatment of cancer has an escalating economic impact. Although cancer therapies have
improved significantly in recent years, the clinical use of chemotherapeutics is often limited
due to chemo-resistance and/or the undesirable toxic effects caused by non-specific cell
killing at an effective dose of drugs [1, 2].
To overcome these problems, new drug discovery and development strategies are
focusing on molecules that act simultaneously on multiple targets, either by a combination of
two (or more) drugs or by combining two (or more) active pharmacophores covalently
connected in a single-hybrid molecule with dual activity [3, 4]. The clinical development of
an optimal combinational therapy is costly, and drug-drug interaction may result in additive
toxic side effects. On the other hand, dual-drugs are designed to interact with multiple targets,
induce synergistic effects and improve the efficacy and safety of drugs in the treatment of
complex diseases [5, 6]. Due to the complex chain of events that causes cancer, it is important
to address combinational modalities in order to provide a better response to cancer therapy.
An additional advantage of this method may arise from the fact that chemotherapy
usually causes immunosuppression in patients, and thus makes them susceptible to various
infectious diseases. As a result, the development of a single drug for treatment of more than
one disease would make an important contribution to the patient's therapy.
This approach has stimulated us to design, synthesize and evaluate a new class of
hybrid molecules, by joining two pharmacophores which are present in a number of natural
and synthetic agents: 7-chloroquinoline and phenyl- or phenylbenzimidazolyl-amidine
(arylamidine).
Chloroquine (CQ), 7-chloro-4-aminoquinoline, has been the mainstay of malaria
chemotherapy for much of the past seven decades. The drug has several advantages, including
limited host toxicity, ease of use, low cost, and effective synthesis. More recently, CQ and its
analogs have been the subject of intense interest in the development of safe and effective
anticancer drugs, due to the fact that parasites and cancer cells share basic characteristics
related to the metabolic requirements associated with their high proliferation rate [7]. Several

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4-aminoquinoline based derivatives have been reported for their significant activity against
different cancer cell lines [8-11]. Among the 7-chloro-4-amino(oxo)quinoline derivatives,
derivatives with N-heterocycles in the side chain have an important place. These pyrimidine
[12], triazine [13] or pyrazoline [14] based hybrids showed a wide range of activity. The
mechanism of action for their anticancer activity is not clearly understood, but data from
physiochemical studies suggest that CQ forms a complex with DNA, resulting in defects in
DNA synthesis and repair. It was been concluded that the ability to form a H-bond between
the N-diethylamino lateral side chain of CQ and a DNA base may contribute to the DNA
intercalative activity of the quinoline nucleus [15, 16].
On the other hand, aromatic amidines, DNA-affinic and highly water soluble moieties,
have exhibited a wide range of outstanding therapeutic applications, for instance as ACIS
inhibitors [17], botulinum neurotoxin A inhibitors [18], and anticancer [19], antiparasitic [20],
and antibacterial [21,22] agents. These compounds bind to the DNA minor groove by a
combination of ionic, hydrophobic and hydrogen bonding interactions [23]. Although the
mechanism of action of aromatic amidines is not fully elucidated, it has been proven that their
bioactivity is the result of DNA binding and the subsequent inhibition of DNA-dependent
enzymes (topoisomerases, polymerases, and nucleases), or possibly by direct inhibition of
transcription [24]. The generally accepted view is that the (un)symmetrical diamidines, with
two cationic terminal groups bind more tightly to the DNA and show better biological
activity, but recently published studies have shown that non-symmetrical mono-arylamidines
may be a new lead in the treatment of cancer and infectious diseases [25,26].
Although 7-chloroquinoline and arylamidines are present in many compounds with
broad pharmacological activities, their combination in a single hybrid molecule has only been
described in a few papers [27,28]. Aromatic amidine, as a pharmacophore, was chosen
because numerous benzimidazolyl- and phenyl-amidines previously synthesized and
evaluated in our group showed potent anticancer [29-31], antibacterial [22] and antiparasitic
activities [32]. Parallel studies showed a correlation between the biological activity and DNA
binding affinity of the investigated molecules. Compounds with stronger binding to DNA
showed more potent activity. These studies have indicated that the structure of the terminal
cationic groups of the tested compounds has a significant contribution to the strength of
binding to DNA, and biological activity. Therefore, we synthesized and evaluated the
antitumor activity of a series of compounds with a wide variety of amidine moieties:
unsubstituted amidine, alkyl substituted amidines and cyclic amidines.

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Since amidines show stronger interaction with DNA than 7-chloroquinoline, we
decided to keep 7-CQ as the unchanged standard in all the newly synthesized hybrid
molecules, and modify the second pharmacophore as well, as a link between them to study the
effect of these modifications on the compound’s DNA/RNA affinity and selectivity, and
biological activity. The results discussed in the present paper reflect our efforts to discover
new, potentially anticancer chemotherapeutic agents.
2. Results and discussion
2.1. Chemistry
R
Cl
O
Cl
N
· X HCl
HO
R
i
Cl
N
1a-g
2a-g
Cl
N
R
N
N
H
H2N
H2N
O
N
ii
Cl
O
HO
HO
· X HCl
+
i
N
H
H
R
R
3a-g
4a-g
Cl
N
5a-g
Compounds
1-5
a
b
c
d
e
f
g
NH
HN
NH
HN
NH
HN
N
NH
N
R
CN
N
H
NH2
HN
Scheme 1: Reagents and conditions: (i) (a) K2CO3, DMF, reflux; (b) EtOH/HCl(g); (ii) (a)
benzoquinone, MeOH; (b) EtOH/HCl(g).
O
R
O
CN
HN
HN
iii
v
· X HCl
Cl
N
Cl
N
OH
8b-g
Cl
N
Br
8a
HN
N
HN
N
i
ii
Cl
O
H
H2
N
N
N
Cl
Cl
O
O
7
6
4,7-dichloroquinoline
HN
N
HN
N
HN
H2
R
R
3a-g
iv
· X HCl
vi
Cl
Cl
9
10a-g

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Compounds
3,8,10
a
b
c
d
e
f
g
NH
HN
NH
HN
NH
HN
N
NH
N
R
CN
N
H
NH2
HN
Scheme 2: Reagents and conditions: (i) 1-ethanolamine, 130 °C, 5h, 90%; (ii) HBr, H2SO4,
170 °C, 3.5h 58%; (iii) 4-hydroxybenzonitrile, DMF, 70 °C, 66%; (iv) 4-
hydroxybenzaldehyde, DMF, 70 °C, 62%; (v) (a)HCl(g), MeOH; (b) appropriate amine,
MeOH, reflux or ambient temperature; (c) EtOH/HCl(g); (vi) (a)NaHSO3, EtOH, reflux; (b)
EtOH/HCl(g).
Schemes 1 and 2 outline our approach to the synthesis of the target hybrid molecules
containing two pharmacophores, 7-chloroquinoline and aromatic amidine, joined through a
short, rigid (-O-) or a long, more flexible (-NH-CH2-CH2-O-) linker. The phenol- and phenol-
benzimidazole amidines attached with a short linker to 7-CQ were synthesized according to
the procedure outlined in Scheme 1. 4,7-Dichloroquinoline was allowed to react with
substituted phenol (1a-g) or 2-(4-hydroxyphenyl)-1H-benzimidazole derivatives (4a-g) in the
presence of potassium carbonate in dry dimethylformamide (DMF) to give the expected
products as a free base. Stirring amidine derivatives with ethanolic HCl produced the
corresponding water soluble di(tri)hydrochloride salts 2b-g and 5b-g. This reaction path
proved to be better than the alternative route that would imply synthesis of nitrile
intermediates 2a or 5a and their conversion to amidines. Phenol amidines 1b-g, widespread
building blocks, were synthesized according to the Pinner method [31] from 4-
hydroxybenzonitrile (1a), dry methanol saturated with HCl(g) and the appropriate amine. The
synthesis and physical properties of precursors 4a,b,f and g from the phenol-benzimidazole
series have been described previously [33]. The remaining compounds from this series
(4c,d,e) were newly synthesized and a description of the synthesis, as well as the physical
properties of these compounds, is given in the present paper. All spectroscopic and analytical
data of earlier synthesized compounds correspond with information previously given, and will
not be shown in this paper.
Scheme 2 illustrates the synthetic route for the synthesis of compounds with an
aminoethoxy linker. The required intermediates 8a and 9 were prepared by the typical
aromatic substitution reaction using the method reported by Guantai et al. [34]. 4,7-
Dichloroquinoline was heated with excess ethanolamine to yield 2-(7-chloroquinolin-4-
ylamino)-ethanol (6). The latter compound was converted to (2-bromoethyl)-(7-
chloroquinolin-4-yl)-amine (7) by gentle reflux in a mixture of hydrobromic and sulfuric acid

ACCEPTED MANUSCRIPT
[35]. Bromide (7) reacted with 4-hydroxybenzonitrile (1a) in the presence of potassium
carbonate in dry dimethylformamide (DMF) to give 4-(2-(7-chloroquinolin-4-
ylamino)ethoxy)benzonitrile (8a) in moderate yield. This nitrile was then converted to
targeted amidine hydrochloride salts (8b-g) applying the Pinner reaction.
Synthesis of phenoxy-benzimidazol amidine trihydrochlorides 10b-g was performed
in two steps. The bromide 7 was first allowed to react with commercially available 4-
hydroxybenzaldehyde in the presence of potassium carbonate, to afford benzaldehyde
intermediate (9) [36]. The benzimidazoles 10a-g were prepared in a good yield by employing
a condensation reaction between aldehyde 9 and 3,4-diaminobenzonitrile (3a) or 3,4-
diaminobenzamidine derivatives 3b-g, by boiling in the presence of NaHSO3.
2.2. Spectroscopy
2.2.1. Spectroscopic characterization of compounds
Based on the structure, the compounds were divided into four groups: group I (2a-g,
Scheme 1), group II (5a-g, Scheme 1), group III (8a-g, Scheme 2), group IV (10a-g, Scheme
2). All compounds, except the nitrile derivatives, were dissolved in water, c = 1 × 10−3 mol
dm−3. Nitriles were dissolved in DMSO (c = 1 × 10−3 mol dm−3). The aqueous solutions of the
studied compounds were stable for several weeks. The absorbance of all solutions, measured
in the range of 220-900 nm, was proportional to their concentrations up to 3.5 × 10−5 mol
dm−3, indicating that there was no significant intermolecular stacking that could give rise to
hypochromicity effects. The UV-Vis spectra of compounds revealed negligible temperature
dependent changes (25–90 °C) and excellent reproducibility upon cooling to 25 °C.
Absorption maxima and the corresponding molar extinction coefficients (ε) are given in Table
S1.
2.2.2. Spectrophotometric titrations of compounds with ds-polynucleotides in aqueous
medium
UV-Vis titrations were performed in a buffered medium (sodium cacodylate buffer, I
= 0.05 mol dm−3, pH 7.0) at room temperature, Table 1. Positions of absorption maxima in
UV-Vis spectra of compounds 2a-g (<300 nm) inhibit use of UV-Vis titrations for
determination of the affinity and binding constants of investigated compounds toward
polynucleotides (ctDNA and polyA-polyU) due to the overlapping of the signals of
compounds and ds-polynucleotides. UV-Vis titrations with ds-polynucleotides were carried
out for compounds 5a-g by adding increasing aliquots of ds-polynucleotide solutions to a

ACCEPTED MANUSCRIPT
fixed concentration of each compound. The decrease in the absorption maxima (25-80%)
accompanied by a bathochromic shift (λ = 4-7 nm) indicates the formation of a new DNA-
ligand species, except for nitrile 5a. The absorption spectra were recorded until saturation was
observed. The binding constants for compounds 5b-g were determined in the range of 5 × 105
– 7 × 107 mol dm−3. To assess the sequence selectivity of these compounds, the experiment
was repeated with polyA-polyU. A bathochromic shift (λ = 2-6 nm) was observed along with
the decrease in new bands (7-70%), indicating the formation of new compound-polyA-polyU
complexes. The UV-Vis titration spectra revealed that alkylation of amidine 5b and its
replacement with cyclic amidine moieties increased affinity toward ctDNA and polyA-polyU.
Among amidines 5b-g, the isopropyl derivative 5c showed the highest affinity toward ctDNA,
while 5g showed the highest affinity toward polyA-polyU.
The change of linker between quinoline and aryl amidine, by substitution of oxygen
with 2-aminoethanol produced the next set of compounds, 8a-g and 10a-g. The UV-Vis
spectra of 8a-g showed minimal changes during titration, with ds-polynucleotides indicating
that those compounds interact only though a very weak electrostatic external mode, and as a
result, no further studies were conducted with them. Introduction of the benzimidazole group
between the phenyl and amidine moieties in the previous series resulted in a new group of
compounds, 10a-g. Titration of compounds from this series with ctDNA (58 % AT base pairs)
resulted in pronounced hypochromism accompanied by a bathochromic shift (∆λ = 6–11 nm),
showing the formation of a new DNA ligand species. Addition of ctDNA to the solution of
compound 10f, until r = 0.15, yielded a decrease in absorption maxima accompanied by
bathochromic shifts (∆λ = 7 nm). Further addition of the mentioned polynucleotides (r ≤ 0.15)
increased the position of maxima which produced sufficient data for accurate processing
using the Scatchard equation. To assess the sequence selectivity of the investigated
compounds, the experiment was repeated with ds-RNA polynucleotides (polyA-polyU).
Changes in the UV-Vis spectra of 10a-10c and 10g after the addition of polyA-polyU were
minor, indicating an external, electrostatic binding mode. The addition of polyA-polyU to the
solutions of compounds 10d-f resulted in hypochromic (24-50 %) and bathochromic (3 -7 nm)
changes as a result of the formation of complexes. The density of the binding sites (Table 1,
ratio n) and the absence of isosbestic points in the UV-Vis spectra obtained by titration with
both polynucleotides indicates several simultaneous binding modes. In general, high n values
suggest DNA/RNA groove binding as the dominant binding mode, possibly accompanied by
agglomeration along the DNA/RNA double helix, but this cannot exclude intercalation
accompanied by agglomeration or groove binding. The binding constants Ks and ratios n

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obtained by processing UV-Vis titration data with the Scatchard equation [37] are
summarized in Table 1.
Table 1. The hypochromic effects (H/%)a, binding constants (logKs) and ratios n
([compound]/[polynucleotide phosphate]) calculated from the UV/Vis titrations of
compounds with ds-polynucleotides (sodium cacodilate buffer, I = 0.05 M, pH = 7).
ctDNA
pApU
H/%
log Ks
n
H/%
log Ks
6.43
n
79.8
6.83
7.87
5.88
6.23
5.76
6.96
0.69
0.42
0.47
0.56
0.34
0.39
0.58b
0.58
0.45
0.35
0.48
6.9
0.44
0.36
5b
60.0
26.8
17.9
22.8
69.7b
15.5
5.36
6.05
6.31
5c
60.2
0.37 fix
0.45 fix
> 1
5d
36.7
5e
e
68.5
-
5f
25.5
26.2b
7.29
0.33
5g
e
c
c
c
-
-
-
-
8a
c
c
c
17.7
6.72
6.63
6.51
6.29
-
-
-
10b
10c
10d
10e
10f
10g
c
c
c
23.7
-
-
-
32.2
26.0
49.7
37.7
7.02
6.81
6.71
0.46
0.75
0.57
24.4
26.0, 21.6b
d
d
-
-
c
c
31.0
6.52
0.56 fix -c
-
-
a Titration data were processed using the Scatchard equation [37]; accuracy of the result
obtained n ± 10 − 30%, consequently log Ks values vary in the same order of magnitude.
b determine from experimental data
c too small changes
d mixed binding modes
e cannot be determined
2.2.3. Thermal melting studies
Thermal melting enables the rapid qualitative evaluation of the relative binding
affinities of compounds to ds-polynucleotides. Changes in the melting point (Tm) of ctDNA
and polyA-polyU upon the addition of compounds were measured in the range of r
[compound] / [polynucleotide] = 0.1–0.5 (Table 2). Compounds from group I (2b-g) showed
moderate stabilization of ctDNA. Toward polyA-polyU only the cyclic derivatives 2f and 2g
produced stabilization (r = 0.3 (2f) and r = 0.1 (2g)). In general, the data for compounds 5b-g

ACCEPTED MANUSCRIPT
show that as the aromatic stacking surface was extended the ctDNA binding affinity
increased. On examining the data for ctDNA it can be seen that the compounds from group II
have greater affinity than compounds from groups I, Table 2. The ∆Tm was also found to
increase as a function of the length and the flexibility of the compounds. Compounds from
group IV (10a-g) showed significantly higher stabilization of ctDNA in comparison with
compounds from other groups.
Table 2. ∆Tm values (°C)a of studied ds-polynucleotides upon addition of compounds at a
different ratio rb (pH = 7, sodium cacodilate buffer, I = 0.05 M).
ctDNA
Compd 0.1
PolyA-polyU
0.3
0.5
0.1
0.3
0.5
1.76
2.01
1.91
1.35
2.65
2.08
2.07
1.07
1.07
1.41
1.60
3.30
3.96
4.84
5.83
4.31
2.17
2.28
1.97
1.43
2.67
1.86
2.84
1.75
1.41
2,91
3.20
3.31
2.58
8.40
9.05
8.15
2.07
2.28
1.85
1.51
2.73
1.96
2.90
2.24
2.08
3,07
4.08
3,89
2.60
8.40
9.83
8.67
0
-0.16
-0.36
-0.80
0.65
2.18
1.22
0.72
1.32
0.78
4.06
0.78
1.98
-
-0.26
2b
2c
d
0.36
-0.71
0.64
1.18
1.62
2.98
-0.44
1.64
0.18
-0.16
2.84
-
-
2d
2e
-0.44
0.28
1.49
1.32
0.21
0.43
0.25
6.82
1.12
1.82
-
2f
2g
5b
5c
5d
5e
5f
5g
10a
10b
10c
10d
-
-
-
-
-
-
d
1.24
0.75
-
4.67/
35.35
8.86/
33.48
7.80
7.80
10e
3.71
3.02
1.14
d
d
-
-
10f
5.75
6.33
4.62
-
5.16
-
10.77
10g
9.76
-
a Error in ∆Tm: ±0.5 °C
b r = [compound]/[polynucleotide]
c - = not recorded
d cannot be determine

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Addition of extended compounds strongly stabilized the double helix of ctDNA,
where the obtained ∆Tm values suggested saturation of binding sites at about r = 0.3–0.5.
Biphasic curves observed for the interactions of compounds 10e and 10f with polyA-polyU at
higher ratios r may indicate agglomeration of compounds along the polynucleotide [38].
Furthermore, the stronger thermal stabilization of DNA in comparison to the RNA analogue
(poly A–poly U) may be attributed to the much narrower and hydrophobic minor groove of
the ds-DNA with respect to ds-RNA [39].
2.2.4. Circular dichroism (CD) experiments
In order to gain an insight into the changes to polynucleotide properties induced by
small molecule binding, we chose CD spectroscopy as a method, highly sensitive toward
conformational changes in the secondary structure of polynucleotides. The investigated
compounds do not possess intrinsic CD spectra, so induced CD spectra (ICD) upon the
binding of compounds to polynucleotides, will provide us useful information about binding
modes.
Interpretation of the CD spectra in the region of DNA absorbance is difficult due to
contributions arising from both changes in DNA conformation due to accommodating the
ligand, and changes in transitions of the ligand due to the new environment. For this reason,
we focused on the region of the spectrum >300 nm.
ctDNA
6
0.1
0.3
0.5
0.7
polyA-polyU
1.5
1.0
5
4
0.1
0.3
0.5
0.7
5f
3
0.5
2
0.0
1
-0.5
-1.0
-1.5
-2.0
0
-1
-2
250
300
350
400
250
300
350
400
λ
/ nm
λ
/ nm

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5
4
8
polyA-polyU
ctDNA
0.1
0.3
0.5
0.7
6
4
0.1
0.3
0.5
0.7
10e
3
2
2
1
0
0
-1
-2
-3
-2
-4
250
300
350
400
240
280
320
/ nm
360
400
λ
λ
/ nm
7
6
4
ctDNA
0.1
0.3
0.5
0.7
polyA-polyU
3
2
0.1
0.3
0.5
0.7
10f
5
4
3
1
2
0
1
-1
-2
-3
0
-1
-2
-3
240
280
320
360
400
250
300
350
400
λ
/ nm
λ
/ nm
Figure 1. CD titrations of ct-DNA (c = 2.0 ×10−5 mol dm−3) and polyA-polyU (c = 2.0 ×10−5
mol dm−3) with 5f, 10e and 10f (ratio, r = [compound]/[polynucleotide] = 0.1, 0.3, 0.5 and
0.7).
Depending on the compound’s relative orientation and distance towards the helical
axis, different changes in the CD spectra are obtained. The concentration dependent CD
signature of compounds from group II was determined first. As could be seen from the ICD
spectra, the changes to the ds-polynucleotides CD signals are directly proportional to the
concentration (Supporting Information, Figure S1). After the addition of compounds to the
solution of ctDNA a positive ICD band arises >300 nm, and increases again with increasing
amount of compound until the point of saturation. Meanwhile, the CD signal of
polynucleotide (around 280 nm) decreases without dramatic changes in shape, indicating its
binding into grooves but without any major disruption of the DNA helix. The addition of 10a-
g of group IV to the ctDNA solution resulted in a decrease in CD signal 280 nm and
appearance of a new positive maximum in the range of 300-400 nm (Fig. 1). In contrast to the
ICD spectra of compounds from the group II, addition of larger compounds induced drastic
changes to the CD signal of ctDNA, indicating significant disruption of the DNA helix. In
addition, the absence of isoelliptic points pointed toward the formation of several types of

ACCEPTED MANUSCRIPT
compound/DNA complexes, depending on the concentration ratio. This is in agreement with
the results obtained from UV-Vis titrations. The strongly pronounced non-linear dependence
of changes in the CD spectra on the ratio r indicates saturation of dominant binding sites at
about r = 0.3-0.5 (Fig. 2). These r values are again in good agreement with the experimental
and calculated data obtained from UV-Vis titrations (Table 1) and thermal melting
experiments.
b)
a)
5
4
3
2
1
0
0.4
0.2
5b
5c
5d
5e
5f
λ
λ
λ
λ
= 336 nm
= 333 nm
= 330 nm
= 335 nm
= 336 nm
= 333 nm
0.0
5b
5c
5d
5e
5f
λ
λ
λ
λ
λ
λ
= 318 nm
= 307 nm
= 307 nm
= 307 nm
= 320 nm
= 307 nm
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
-1.4
λ
5g
λ
10a
10b
10c
10d
10e
10f
λ
λ
λ
λ
λ
= 337 nm
= 337 nm
= 337 nm
= 337 nm
= 337 nm
= 343 nm
= 337 nm
5g
10d
10e
10f
λ
λ
λ
= 307 nm
= 307 nm
= 312 nm
λ
10g
λ
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
c(compound) / c (polyA-polyU)
c (compund) / c (ctDNA)
Figure 2. Dependence of CD signal on ratio r. (r = [compound]/[polynucleotide] = 0.1, 0.3,
0.5 and 0.7).
With regard to polyA-polyU, the appearance of a negative signal (> 300 nm) in the
ICD spectra of compounds 5b-g showed intercalation as the dominant binding mode. The
changes in the ICD spectra showed no saturation of binding sites at n ≈ 0.25 which may be
assumed according to the dominate binding mode. This result, together with the lack of
isoelliptic points in the ICD spectra, points to the possibility of several simultaneous binding
modes. Addition of the compounds 10d-f to ds-RNA (poly A–poly U) resulted in bisignate
ICD bands (> 300 nm) with a positive and negative part localized on either side of the
ligand´s absorption maximum. Such bisignate ICD bands could be attributed to the dimer
formation [31], most likely within the ds-RNA major groove, since the shallow and very wide
RNA minor groove is not convenient for the binding of small molecules. Due to the large size
of the binding site, dimeric aggregates are easily accommodated, and therefore the secondary
structure of the double helix is not significantly disturbed (Figure 1). In the ICD spectra of
compound 10f the increasing concentration of compound (r ≥ 0.3) generates an ICD signal of
the opposite sign, suggesting the presence of several different forms of compound aggregates
along the ds-RNA [40].

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2.3. Biological activity
2.3.1. Antiproliferative effects of the investigated compounds
Numbers of recent studies conducted on tumor cell lines have shown that chloroquinoline
derivatives and aromatic amidines individually are cytotoxic against tumor cells [41-43].
Since the results of the current study obtained from hybrid molecules in cell-free testing
suggest their various modes of interaction with nucleic acids (Tables 1 and 2; Figures 1, 2 and
S1), it was interesting to examine whether there are also differences in their activity in vitro
on the growth of different tumor cell lines.
The newly synthesized compounds were tested for their effects on the growth of
normal cells (MDCKI) and human tumor cell lines of different histological origin, CaCo2,
HeLa, K562 and Raji. The obtained results, presented as the concentration achieving 50% of
cell growth inhibition (GI50 value), show that the investigated compounds differentially
influenced tumor cell growth, depending on the cell line, as well as on the dose applied. As
shown in Table 3, GI50 values for all investigated compounds ranged from 5 to more than 100
× 10-6 mol dm-3. Carcinoma cell lines were more resistant to the investigated compounds than
leukemia cells. Most of the tested compounds with a short linker, group I (2a-g, Scheme 1)
and group II (5a-g, Scheme 1), exhibited strong inhibitory potential, but without significant
distinction between the normal and investigated tumor cell lines (GI50 values ranging from 30
to >
100 × 10-6 mol dm-3). Compounds 2d and 2e with GI50 ranging from 16 to 29 ×10-6 mol
dm-3 displayed the best effects on the leukemia cells. Unsubstituted amidine 5b possesses
poor inhibitory activity compared to other compounds from group II. That is in agreement
with its lower activity toward ctDNA. Alkyl substituted amidines (5c-e) and cyclic amidines
(5f,g) showed improved binding affinity as well as inhibitory effects on the treated cells’
growth (Table 2 and Table 3).
Furthermore, the phenol- and phenol-benzimidazole amidines attached with a short
linker to 7-CQ had less effect on the growth of Raji and K562 cells compared to the
compounds with a 2-aminoethanol linker (compounds 8a-g and 10a-g). The obtained results
prove that antiproliferative effects can be improved with the extension of the molecules’
central linker. This extension enhances the flexibility and adaptability of the molecular
conformation, causing variations in the DNA binding mode [44]. Unlike other compounds
from group III that had weak cytotoxicity on treated cell lines (GI50 values ranging 50 to >100
× 10-6 mol dm-3), compound 8a showed significant inhibitory potential against leukemia cells,
with GI50 values between 6 and 7 × 10-6 mol dm-3. Although the tested phenoxy-benzimidazol

ACCEPTED MANUSCRIPT
amidines (10a-g) showed significantly higher stabilization of ctDNA in comparison with
compounds from other groups (Fig. 1, Table 2), these compounds (10b-g) showed very weak
antiproliferative capacity on the normal cells and carcinoma cell lines. Unlike other
investigated groups of compounds, all compounds from group IV (10a-g) significantly
influenced the growth of leukemia cells with GI50 values between 5 and 27 × 10-6 mol dm-3.
Table 3. Sensitivity of human tumor and normal cells to investigated compounds.
GI50 (x10-6 mol dm-3)
Cell lines
MDCK1
HeLa
CaCo-2
K562
Raji
Group I
2a
88.5 ± 1.0
52.8 ± 4.7
57.7 ± 6.6
30.3 ± 4.8
35.0 ± 7.9
56.6 ± 3.3
>100
59.1 ± 9.4
56.6 ± 11.2 47.7 ± 6.0
52.3 ± 15.1 71.9 ± 17.6
48.6 ± 16.4 13.5 ± 8.0
57.7 ± 14.0 64.0 ± 15.6
>100
57.6 ± 3.1
86.7 ± 1.8
62.5 ± 15.9
82.9 ± 4.3
16.9 ± 3.1
18.8 ± 0.9
48.7 ± 12.3
62.0 ± 4.0
91.4 ± 10.8
53.0 ± 4.4
51.8 ± 6.2
25.5 ± 11.1
19.7 ± 10.6
40.8 ± 6.8
50.8 ± 5.2
2b
2c
2d
2e
2f
2g
37.5 ±1 0.2 57.0 ± 13.1 53.3 ± 11.9
Group II
5a
93.1 ±17.1 >100
>100
>100
>100
5b
5c
5d
5e
>100
>100
51.3 ± 0.6
82.9 ± 15.9
53.3 ± 5.6
>100
111.4 ± 33.1
48.2 ± 17.9
46.7 ± 7.2
41.5 ± 17.3
40.6 ± 8.6
50.5 ± 6.7
61.8 ± 3.1
53.5 ± 4.8
55.5 ± 5.5
77.1 ± 18.2 >100
67.1 ± 13.5 64.4 ± 5.6
69.0 ± 19.1
59.7 ± 7.4
54.6 ± 13.2
27.6 ± 8.9
21.6 ± 11.0
57.0 ± 18.8 43.2 ± 2.8
61.9 ± 11.1 47.4 ± 3.7
5f
5g
36.8 ± 18.3
34.5 ± 4.2
Group III
8a
8b
>100
74.4 ± 4.7
>100
84.4 ± 20.3 95.3 ± 35.0
7.2 ± 1.5
49.5 ± 4.6
>100
6.2 ± 0.9
58.9 ± 5.4
>100
74.0 ± 8.0
>100
68.6 ± 6.2
>100
8c
8d
8e
8f
8g
>100
>100
>100
>100
92.6 ± 28.5
84.5 ± 15.2
50.7 ± 5.9
>100
77.3 ± 15.2
76.9 ± 8.6
51.9 ± 3.7
>100
86.2 ± 26.0
96.9 ± 14.4
51.5 ± 7.2
>100
78.4 ± 5.6
>100
60.8 ± 5.3
>100
Group IV
10a
10b
10c
10d
10e
10f
22.1 ± 8.1
71.6 ± 2.7
>100
56.6 ± 22.1 28.3 ± 11.3
7.8 ± 3.3
5.2 ± 1.1
>100
>100
>100
>100
>100
>100
34.7 ± 4.5
17.2 ± 0.4
15.1 ± 0.8
11.4 ± 2.8
29.3 ± 8.3
19.6 ± 9.8
28.5 ± 1.2
17.1 ± 13.6
27.5 ± 1.4
94.1 ± 19.5 >100
99.2 ± 28.3 >100
>100
100.5
18.0
>100
117.4 ± 29.1 8.6 ± 1.9
10g
±
>100
>100 13.3 ± 1.6
19.0 ± 2.3
GI50 – Drug concentration that inhibited cell growth by 50%. Data represent mean GI50 (x10-6
mol dm-3) values ± standard deviation (SD) of the three independent experiments.

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Exponentially growing cells were treated with compounds during a 72-hr period. Growth
inhibition was analyzed using MTT survival assay
2.3.2. Cell cycle distribution
The cell cycle involves a complex series of molecular and biochemical signaling
.
pathways. Cell proliferation is governed by the cell cycle, which is a complex and stepwise
process, and uncontrolled cell proliferation is a hallmark of tumor cells [45]. Suppression of
cancer cell growth by many anticancer therapeutics correlates with perturbations in the cell
cycle progression [46]. To gain insights into the mechanism of suppression of cancer cell
proliferation, we examined the influence of selected compounds on the cell cycle distribution.
Six compounds (2d, 2e, 8a, 10d, 10e, and 10f), were selected for cell cycle analysis on K562
cells, based on their cytotoxicity results. Representative flow histograms, depicting cell cycle
distribution in K562 cells, following 24 hour exposure to the tested compounds are shown in
Figure 3. Tested compounds induce changes in the cell cycle of treated cells but the cycle
phase distribution varies between them. Cells treated with compounds from group I (2d and
2e) and the group III (8a) have a similar pattern of effect. Compared to the control, untreated
cells, the treated cells showed an enrichment of the G0-G1 fraction (Fig. 3). This was
accompanied by a decrease in both S phase and G2-M phase cells. DNA damage during the
G1 phase and antiproliferative signals induced a G1 phase arrest prior to entry into the S
phase. A SubG1 peak is indicative of DNA fragmentation, and that cells are dying, most
probably through apoptosis [47,48].
Although all the tested compounds showed a significant decrease in the number of
cells in the S phase (p < 0.001) only cells treated by two compounds (10d and 10f) from
group IV, had a significantly larger cell fraction in the subG0 phase than the control cells,
which means that these cells went into senescence. Compound 10d is the most effective,
affecting significantly both S and G2/M phases. Compounds 10e and 10f showed significant
changes in the S phase as well. Increased levels of p53 in normal cells and cells with a
functional p53 gene are responsible for the cell cycle arrests in the G1 and G2 phases in vitro,
and for signaling apoptosis in response to excessive or persistent damage [47]. In the case of
K562, which is a p53 null cell line, the drug may cause its effect through other alternate
pathways, which need to be investigated.

ACCEPTED MANUSCRIPT
Figure 3. Cell cycle distribution.
The K562 cells were treated with 10-5 mol dm-3 of tested compounds for 24 hrs, stained with PI and
analyzed by flow cytometry. PI stain histograms show a difference between the control (a), 2e (b), 8a
(c) and 10f (d). The chart (e) shows the numerical values for phase distribution. An asterisk (*)
denotes values statistically significantly different when compared to the control (p < 0.001)
4. Conclusion
This study reports the synthesis of four series of new molecular hybrids with two
moieties, 7-chloroquinoline and arylamidine, using various synthetic methods. All the novel
molecular hybrids contain a quinoline moiety, whereas the linkers between quinoline and
arylamidine, aryl core, and terminal amidine moiety were modified.

ACCEPTED MANUSCRIPT
The potential inhibitory capacity of the newly synthesized compounds on the human
tumor cell lines of different histological origin, as well as their interaction with DNA and
RNA was determined. It was shown that the binding affinity of the investigated compounds
increases proportionally to the increase in the length and number of groups able to form
hydrogen bonds with ds-polynucleotides. Improvement of binding was additionally achieved
by reduction of the structural rigidity of the investigated compounds
All the compounds bound preferentially to ctDNA, precisely in the DNA grooves.
Compounds from group IV showed the highest affinity to ds-polynucleotides. According to
their pronounced positive ICD bands, as well as moderate affinity and thermal stabilization
effects, compounds from groups II and IV preferentially bound into the minor groove of
ctDNA. The ICD spectra of compounds from group II with polyA-polyU showed
intercalation as the dominant binding mode, while compounds from group IV preferentially
bound into polyA-polyU grooves.
Antitumor selectivity was clearly seen in the effects on carcinoma and leukemia cells,
with the higher efficacy of the investigated compounds against leukemia cell lines. Unlike the
other groups of tested compounds, compounds from group IV significantly influenced the
growth of leukemia cells, with GI50 values between 5 and 27 × 10-6 mol dm-3. The highest
selectivity towards leukemia cells, and low cytotoxicity against normal cells, was obtained for
the compound 8a.
Based on these results regarding the antiproliferative capacity of tested compounds,
further research may be carried out to investigate the mechanisms of action of the compounds
from the group IV in different types of leukaemia cells in more detail. Compounds from this
series will serve as a template for the future design of new analogues with improved antitumor
activities.
4. Experimental section
4.1. Chemistry
The compounds 1a-g, 3a-g [29] and 4a,b,f,g [33] are already known. Procedures for the
preparation of all final products are presented below. All solvents and reagents were used
without purification from commercial sources. The progress of the chemical reaction was
monitored by TLC on silica-gel 60 F254 DC-plastikfolienC16H and detected under UV light.
Melting points were determined on a Büchi 510 melting point apparatus and were
uncorrected. IR spectra (νmax/cm–1) were obtained on a Bruker Vertex 70 spectrophotometer.

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The 1H and 13C NMR spectra were recorded on a Bruker Avance 300 MHz spectrometer and
chemical shifts (δ/ppm) were referred to TMS as internal standard. Theelectrospray ionization
(ESI) Q-ToF was used for the high-resolution mass spectra measurements. Elemental analyses
were performed by the Applied Laboratory Research department at INA d.d., Research and
Development Sector, Central Analytical Laboratory.
4.1.1. General method for the synthesis the compounds from group I (2a-g)
A solution of 4,7-dichloroquinoline (1.2 mmol), compounds 1a-g (1 mmol) and K2CO3 (0.03
mol) in dry DMF (50 mL) was refluxed with stirring under nitrogen for 8 hrs. The reaction
mixture was cooled at room temperature, diethyl ether was added, and the resulting solid was
filtered off. The products were dissolved in EtOH/HCl(g) and precipitated with mixture
acetone:diethyl ether (4:1), filtered off, and dried. It was repeated a few times until the
product were analytically pure.
4.1.1.1 4-(7-Chloroquinolin-4-yloxy)benzonitrile (2a).
Yield 0.1502 g (86.2 %). mp 152–154 °C; IR (νmax/cm–1): 3428, 2991, 2232, 1701, 1607,
1431, 1232, 1108, 785, 713, 462; 1H NMR (DMSO-d6) δ/ppm: 8.81 (d, 1H, J = 5.06 Hz, CH),
8.25 (d, 1H, J = 9.48 Hz, CH), 8.14 (d, 1H, J = 2.05 Hz, CH), 8.01 (d, 2H, J = 8.55 Hz, CH2),
7.71 (dd, 1H, J1 = 8.77 Hz, J2 = 1.79 Hz, CH), 7.50 (d, 2H, J = 8.93 Hz, CH2), 6.90 (d, 1H, J =
13
4.46 Hz, CH). C NMR (DMSO-d6) δ/ppm: 159.9, 158.4, 153.5, 150.3, 135.6, 135.5, 128.1,
127.9, 124.2, 121.8, 120.0, 118.8, 108.6, 107.4. HRMS calcd. for C16H10ClN2O (M + H)+:
281.0482; found: 281.0482. Anal. calcd. for C16H9ClN2O × 0.25H2O (Mr = 285.22): C 67.38,
H 3.36, N 9.82; found: C 67.55, H 3.36, N 9.51.
4.1.1.2. 4-(7-Chloroquinolin-4-yloxy)benzimidamide dihydrochloride (2b).
Yield 75.5%; mp 251–252 °C; IR (νmax/cm–1): 3085, 2387, 1683, 1629, 1587, 1483, 1421,
1
1288, 1200, 1080, 984, 804, 680, 600, 522, 459; H NMR (DMSO-d6) δ/ppm: 9.48 (s, 2H,
NH2), 9.26 (s, 2H, NH2), 8.96 (d, 1H, J = 5.21 Hz, CH), 8.39 (d, 1H, J = 9.22 Hz, CH), 8.28
(d, 1H, J = 1.96 Hz, CH), 8.05 (d, 2H, J = 8.97 Hz, CH2), 8.35 (dd, 1H, J1 = 9.29 Hz, J2 =
2.52 Hz, CH), 7.61 (d, 2H, J = 8.90 Hz, CH2), 6.92 (d, 1H, J = 5.49 Hz, CH). 13C NMR
(DMSO-d6) δ/ppm: 164.7, 163.7, 157.2, 150.0, 144.1, 137.8, 131.1, 128.9, 125.9, 124.7,
123.0, 121.2, 119.4, 106.0. HRMS calcd. for C16H13ClN3O (M + H)+: 298.0747; found:
298.0749. Anal. calcd. for C16H12ClN3O × 2HCl (Mr = 370.66): C 51.85, H 3.81, N 11.34;
found: C 51.71, H 3.92, N 11.12.
4.1.1.3. 4-(7-Chloroquinolin-4-yloxy)-N-isopropylbenzimidamide trihydrochloride (2c).

ACCEPTED MANUSCRIPT
Yield 45.2%; mp 198–199 °C; IR (νmax/cm–1): 3439, 2977, 2746, 1704, 1648, 1578, 1508,
1
1427, 1369, 1312, 1207, 1182, 1128, 1086, 858, 810, 623, 518, 458; H NMR (DMSO-d6)
δ/ppm: 9.69 (d, 2H, J = 8.59, NH2), 9.55 (s, 1H, NH), 9.22 (S, 1H, CH), 9.00 (d, 1H, J = 5.66
Hz, CH), 8.43 (d, 1H, J = 8.98 Hz, CH), 8.32 (d, 1H, J = 1.86 Hz, CH2), 7.95 (d, 2H, J = 8.72
Hz, CH), 7.86 (dd, 1H, J1 = 9.15 Hz, J2 = 2.01 Hz, CH), 7.61 (d, 2H, J = 8.90 Hz, CH), 6.84
(d, 1H, J = 5.67 Hz, CH), 4.10 (m, 1H, CH), 1.29 (d, 6H, J = 6.36 Hz, CH3) 13C NMR
(DMSO-d6) δ/ppm: 160.9, 156.7, 150.3, 137.5, 131.3, 128.8, 127.2, 124.5, 121.0, 119.4,
105.8, 45.2, 21.2. HRMS calcd. for C19H19ClN3O (M + H)+: 340.1212; found: 340.1218.
Anal. calcd. for C19H18ClN3O × 3HCl × 0.75H2O (Mr = 462.72): C 49.32, H 4.90, N 9.08;
found: C 49.33, H 5.15, N 8.95.
4.1.1.4. 4-(7-Chloroquinolin-4-yloxy)-N-isobutylbenzimidamide trihydrochloride (2d).
Yield 54.2%; mp 196–197 °C; IR (νmax/cm–1): 3057, 2965, 2710, 1670, 1590, 1474, 1439,
1
1364, 1301, 1207, 1081, 1011, 850, 700, 468; H NMR (DMSO-d6) δ/ppm: 10.01 (s, 1H,
NH), 9.64 (s, 1H, NH), 9.29 (s, 1H, NH), 9.05 (d, 1H, J = 5.84 Hz, CH), 8.47 (d, 1H, J = 8.99
Hz, CH), 8.38 (d, 1H, J = 1.66 Hz, CH), 8.00 (d, 2H, J = 8.65 Hz, CH), 7.90 (dd, 1H, J1 =
8.99 Hz, J2 = 1.75 Hz, CH), 7.63 (d, 2H, J = 8.62 Hz, CH), 6.89 (d, 1H, J = 5.85 Hz, CH),
3.30 (m, 2H, CH), 2.04 (m, 1H, CH2), 0.98 (d, 6H, J = 6.61 Hz, CH2). 13C NMR (DMSO-d6)
δ/ppm: 164.2, 162.5, 157.1, 150.6, 138.2, 131.7, 129.4, 127.5, 125.1, 123.6, 121.6, 119.9,
106.2, 50.1, 27.5, 20.4. HRMS calcd. for C20H21ClN3O (M + H)+: 354.1373; found: 354.1372.
Anal. calcd. for C20H20ClN3O × 3HCl × 1.5H2O (Mr = 490.26): C 49.00, H 5.35, N 8.57;
found: C 49.18, H 5.34, N 8.53.
4.1.1.5. 4-(7-Chloroquinolin-4-yloxy)-N-cyclopentylbenzimidamide trihydrochloride (2e).
Yield 45.2%; mp 198–199 °C; IR (νmax/cm–1): 2942, 2688, 1670, 1616, 1570, 1411, 1293,
1204, 1175, 1062, 842, 731, 446; 1H NMR (DMSO-d6) δ/ppm: 9.77 (d, 1H, J = 7.00 Hz, NH),
9.56 (s, 1H, NH), 9.19 (s, 1H, NH), 8.99 (d, 1H, J = 5.58 Hz, ArH), 8.41 8d, 1H, J = 8.91 Hz,
ArH), 8.31 (d, 1H, J = 1.57 Hz, ArH), 7.94 (d, 2H, J = 8.71 Hz, ArH), 7.84 (dd, 1H, J1 = 8,95
Hz, J2 = 1.96 Hz, ArH), 7.59 (d, 2H, J = 8.73 Hz, ArH), 6.84 (d, 1H, J = 5.63 Hz,ArH), 4.20
13
(m, 1H, CH), 2.07 (m, 2H, CH2), 1.74 (m, 4H, CH2), 1.59 (m, 2H, CH2); C NMR (DMSO-
d6) δ/ppm: 164.0, 161.5, 156.5, 149.9, 137.8, 131.5, 128.9, 127.2, 124.7, 122.9, 121.0, 119.4,
105.6, 54.4, 31.3, 23.7. HRMS calcd. for C21H21ClN3O (M + H)+: 366.1373; found: 366.1377.
Anal. calcd. for C21H20ClN3O × 3HCl (Mr = 475.24): C 53.07, H 4.88, N 8.84; found: C
53.18, H 4.98, N 8.51.

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4.1.1.6. 7-Chloro-4-(4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy)quinoline dihydrochloride
(2f).
Yield 74.4%; mp 201–202 °C; IR (νmax/cm–1): 2965, 1579, 1417, 1297, 1214, 1181, 1080,
1
856, 830, 806, 631, 515; H NMR (DMSO-d6) δ/ppm: 10.86 (s, 2H, NH), 8.96 (d, 1H, J =
5.53 Hz, CH), 8.37 (d, 1H, J = 8.22 Hz, CH), 8.28 (d, 1H, J = 2.09 Hz, CH), 8.24 (d, 2H, J =
8.79 Hz, CH), 7.83 (dd, 1H, J1 = 7.21 Hz, J2 = 2.00 Hz, CH), 7.65 (d, 2H, J = 8.76 Hz, CH),
7.00 (d, 1H, J = 5.54 Hz, CH), 4.02 (s, 4H, CH2); 13C NMR (DMSO-d6) δ/ppm: 163.6, 163.1,
157.8, 150.3, 137.6, 131.8, 128.8, 124.5, 123.4, 121.3, 120.0, 119.5, 106.5, 44.3; HRMS
calcd. for C18H15ClN3O (M + H)+: 324.0904; found: 324.0907. Anal. calcd. for C18H14ClN3O
× 2HCl × 1H2O (Mr = 414.72): C 52.13, H 4.37, N 10.13; found: C 52.21, H 4.76, N 9.99.
4.1.1.7. 7-Chloro-4-(4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenoxy)quinoline dihydrochloride
(2g).
Yield 68.9%; mp 198–199 °C; IR (νmax/cm–1): 2329, 1639, 1588, 1417, 1297, 1209, 1084,
1
856, 825, 737, 696, 594, 543, 511; H NMR (DMSO-d6) δ/ppm: 10.09 (s, 2H, NH), 8.91 (d,
1H, J = 1.06 Hz, CH), 8.35 (d, 1H, J = 8.95 Hz, CH), 8.23 (d, 1H, J = 1.68 Hz, CH), 8.01 (d,
1H, J = 8.76 Hz, CH), 7.92 (d, 2H, J = 8.76 Hz, CH), 7.78 (dd, 1H, J1 = 8.92 Hz, J2 = 1.99
Hz, CH), 7.57 (d, 2H, J = 8.74 Hz, CH), 6.86 (d, 1H, J = 5.46 Hz, CH), 3.50 (s, 4H, CH2) 1.98
13
(m, 2H, CH2); C NMR (DMSO-d6) δ/ppm: 163.3, 161.9, 158.1, 156.8, 150.4, 137.5, 131.3,
130.6, 128.7, 126.7, 126.3, 124.5, 123.6, 121.1, 121.0 119.4, 105.9, 38.8, 17.6; HRMS calcd.
for C19H17ClN3O (M + H)+: 338.1060; found: 338.1064. Anal. calcd. for C19H16ClN3O ×
2HCl × 1.5H2O (Mr = 437.75): C 52.13, H 4.84, N 9.60; found: C 52.06, H 4.75, N 9.60.
4.1.2. General method for the synthesis of hydrochloride derivatives of 2-(4-hydroxyphenyl)-
1H-benz[d]imidazole-6-carboximidamide (4c,d,e).
A solution of 4-hydroxybenzaldehyde (1.2 mmol), 3,4-diaminobenzamidine (3c,d,e) (1.0
mmol) and benzoquinone (1.2 mmol mol) in dry methanole (40 mL) was refluxed with
stirring under nitrogen for 8 hrs. The reaction mixture was cooled to room temperature,
diethyl ether was added, and the resulting solid was filtered off. The products were dissolved
in EtOH/HCl(g) and perticipitated with mixture acetone:diethyl ether (5:1), filtered off, and
dried. It was repeated a few times until the product were analytically pure.
4.1.2.1. 2-(4-Hydroxyphenyl)-N-isopropyl-1H-benzo[d]imidazole-6-carboximidamide
dihydrochloride (4c).
Yield 49.1%; mp > 290 °C; IR (νmax/cm–1): 3081, 2652, 1676, 1601, 1500, 1385, 1283, 1186,
1
843, 816, 700, 520, 566; H NMR (DMSO-d6) δ/ppm: 10.50 (brs, 1H, OH), 9.64 (d, 1H, J =

ACCEPTED MANUSCRIPT
8.93 Hz, NH), 9.49 (s, 1H, NH), 9.06 (s, 1H, NH), 8.25 (d, 2H, J = 8.72 Hz, CH), 8.05 (d, 1H,
J = 1.55 Hz, CH), 7.85 (d, 1H, J = 8.12 Hz, CH), 7.67 (dd, 1H, J1 = 8.91 Hz, J2 = 1.79 Hz,
13
CH), 7.05 (d, 2H, J = 8.77 Hz, CH), 4.09 (m, 1H, CH), 1.31 (d, 6H, J = 6.38 Hz, CH3). C
NMR (DMSO-d6) δ/ppm: 162.67, 162.21, 152,70, 130.84, 125,51, 125.01, 124.99, 116.84,
115.08, 114.37, 45.65, 21.73. HRMS calcd. for C17H19N4O (M + H)+: 295.1559; found:
295.1562. Anal. calcd. for C17H18N4O × 2HCl × 1.5H2O (Mr = 394.30): C 51.78, H 5.88, N
14,21; found: C 51.89, H 5.67, N 14.34.
4.1.2.2. 2-(4-Hydroxyphenyl)-N-isobutyl-1H-benzo[d]imidazole-6-carboximidamide
dihydrochloride (4d).
Yield 72.1%; mp > 290 °C; IR (νmax/cm–1): 3069, 2664, 1676, 1607, 1505, 1454, 1389, 1352,
1292, 1218, 1181, 862, 821, 705, 649, 566, 538, 515; 1H NMR (DMSO-d6) δ/ppm: 10.70 (brs,
1H, OH), 9.91 (s, 1H, NH), 9.58 (s, 1H, NH), 9.13 (s, 1H, NH), 8.30 (d, 2H, J = 8.72 Hz,
CH), 8.10 (s, 1H, CH), 7.89 (d, 1H, J = 8.49 Hz, CH), 7.73 (dd, 1H, J1 = 8.53 Hz, J2 = 1.35
Hz, CH), 7.06 (d, 2H, J = 8.77 Hz, CH), 3.29 (t, 2H, J1 = 6.97 Hz, J2 = 7.87 Hz, CH2), 2.05
(m, 1H, CH), 1.00 (d, 6H, J = 6.63 Hz, CH3). 13C NMR (DMSO-d6) δ/ppm: 163.0, 162.4,
152.1, 130.5, 125.0, 124.5, 116.4, 114.4, 114.0, 49.8, 27.0, 19.9. HRMS calcd. for C18H21N4O
(M + H)+: 309.1712; found: 309.1720. Anal. calcd. for C18H20N4O × 2HCl × 2.5 H2O (Mr =
426.34): C 50.71, H 6.38, N 13.14; found: C 50.43, H 6.59, N 13.23.
4.1.2.3. 2-(4-Hydroxyphenyl)-N-cyclopentyl-1H-benzo[d]imidazole-6-carboximidamide
dihydrochloride (4e).
Yield 73.6%; mp > 290 °C; IR (νmax/cm–1): 3081, 2664, 2364, 1670, 1601, 1497, 1289, 1197,
1
850, 700, 538; H NMR (DMSO-d6) δ/ppm: 10.53 (brs, 1H, OH), 9.72 (d, 1H, J = 7.35 Hz,
NH), 9.49 (s, 1H, NH), 8.99 (s, 1H, NH), 8.23 (d, 2H, J = 8.70 Hz, ArH), 8.04 (s, 1H, ArH),
7.83 (d, 1H, J = 8.49 Hz, ArH), 7.65 (dd, 1H, J1 = 8.51 Hz, J2 = 1.25 Hz, ArH), 7.04 (d, 2H, J
= 8.77 Hz, ArH), 4.17 (m, 1H, CH), 2.07 (m, 2H, CH2), 1.66 (m, 6H, CH3); 13C NMR
(DMSO-d6) δ/ppm: 162.9, 162.8 152.4, 136.5, 133.2, 131.1, 125.6, 125.4, 116.9, 115.1, 114.7,
114.3, 54.9, 31.9, 24.2. HRMS calcd. for C19H21N4O (M + H)+: 321.1715; found: 321.1717.
Anal. calcd. for C19H20N4O × 2HCl × 3 H2O (Mr = 447.36): C 51.01, H 6.31, N 12.52; found:
C 51.34, H 6.13, N 12.70.
4.1.3. General method for the synthesis the compounds from group II (5a-g)
A solution of 4,7-dichloroquinoline (1.2 mmol), compounds 4a-g (1 mmol) and K2CO3 (0.03
mol) in dry DMF (50 mL) was refluxed with stirring under nitrogen for 2d. The reaction

ACCEPTED MANUSCRIPT
mixture was cooled at room temperature, diethyl ether was added, and the resulting solid was
filtered off. The products were dissolved in EtOH/HCl(g) and precipitated with mixture
acetone:diethyl ether (4:1), solid was filtered off, and dried. It was repeated few times until
the product were analytically pure.
4.1.3.1. 2-[4-(7-Chloroquinolin-4-yloxy)phenyl]-1H-benzo[d]imidazole-6-carbonitrile (5a).
Yield 57.6 %; mp 178–179 °C; IR (νmax/cm–1): 3423, 2225, 1616, 1569, 1496, 1421, 1215,
1095, 858, 819; 1H NMR (DMSO-d6) δ/ppm: 8.75 (d, 1H, J = 5.09 Hz, CH), 8.34 (d, 2H, J =
7.72 Hz, CH), 8.31 (d, 1H, J = 8.55 Hz, CH), 8.11 (d, 1H, J = 2.29 Hz, CH), 7.71 (d, 1H, J =
2.60 Hz, CH), 7.69 (d, 1H, J = 1.86 Hz, CH), 7.90 (d, 1H, J = 1.49 Hz, CH2), 7.54 (d, 1H, J =
1.67 Hz, CH), 7.50 (d, 2H, J = 9.11 Hz, CH), 6.85 (d, 1H, J = 5.02 Hz, CH2); 13C NMR
(DMSO-d6) δ/ppm: 160.9, 155.7, 153.5, 152.6, 148.8, 135.5, 129.3, 127.4, 126.8, 126.7,
125.8, 123.9, 121.3, 119.9, 119.5, 105.9, 104.1; HRMS calcd. for C23H14ClN4O (M + H)+:
397.0856; found: 397.0849.
4.1.3.2. 2-[4-(7-Chloroquinolin-4-yloxy)phenyl]-1H-benzo[d]imidazole-6-carboximidamide
trihydrochloride (5b).
Yield 49.1%; mp > 250 °C; IR (νmax/cm–1): 3262, 3095, 2645, 1690, 1633, 1585, 1436, 1287,
1
1181, 1089, 859; H NMR (DMSO-d6) δ/ppm: 9.39 (s, 2H, NH), 9.04 (s, 2H, NH), 8.99 (d,
1H, J = 5.82 Hz, CH), 8.51 (d, 2H, J = 8.70 Hz, CH), 8.50 (d, 1H, J = 9.15 Hz, CH), 8.32 (d,
1H, J = 1.81 Hz, CH), 8.21 (s, 1H, CH), 7.90 (dd, 1H, J1 = 9.04 Hz, J2 = 1.96 Hz, CH), 7.86
(d, 1H, J = 8.98 Hz CH), 7.74 (dd, 1H, J1 = 8.59 Hz, J2 = 1.45 Hz, CH), 7.65 (d, 2H, J = 8.77
13
Hz, CH), 7.04 (d, 1H, J = 5.83 Hz, CH); C NMR (DMSO-d6) δ/ppm: 166.1, 155.5, 153.2,
151.8, 136.2, 129.6, 127.9, 127.0, 124.2, 124.0, 122.8, 121.6, 121.4, 119.5, 117.6, 116.1,
108.8, 105.9; HRMS calcd. for C23H17ClN5O (M + H)+: 414.1122; found: 414.1128. Anal.
calcd. for C23H16ClN5O × 3HCl × 3H2O (Mr = 577.29): C 47.85, H 4.36, N 12.13; found: C
48.16, H 4.34, N 12.12.
4.1.3.3. 2-[4-(7-Chloroquinolin-4-yloxy)phenyl]-N-isopropyl-1H-benzo[d]imidazole-6-
carboximidamide trihydrochloride (5c).
Yield 40.0%; mp > 250 °C; IR (νmax/cm–1): 3062, 2592, 2353, 1583, 1416, 1295, 1211, 1170,
1
1079, 859; H NMR (DMSO-d6) δ/ppm: 9.60 (d, 1H, J = 9.07 Hz, NH), 9.47 (s, 1H, NH),
9.06 (s, 1H, NH), 8.97 (d, 1H, J = 7.06 Hz, CH), 8.56 (d, 2H, J = 10.67 Hz, CH), 8.49 (d, 1H,
J = 9.70 Hz, CH), 8.30 (s, 1H, CH), 8.09 (s, 1H, CH), 7.88 (dd, 1H, J1 = 8.94 Hz, J2 = 2.54

ACCEPTED MANUSCRIPT
Hz, CH), 7.85 (d, 1H, J = 9.09 Hz, CH), 7.64 (d, 2H, J = 9.73 Hz, CH), 7.60 (s, 1H, CH), 7.01
(d, 1H, J = 6.26 Hz, CH), 4.11 (m, 1H, CH), 1.32 (d, 6H, J = 6.41 Hz, CH3); 13C NMR
(DMSO-d6) δ/ppm: 165.4, 161.8, 155.6, 151.4, 149.0, 142.1, 138.7, 138.2, 130.7, 129.5,
125.1, 124.9, 124.7, 124.3, 122.0, 121.44, 121.40, 119,3, 115.7, 114.6, 105.8, 45.2, 21.3.
HRMS calcd. for C26H23ClN5O (M + H)+: 456.1591; found: 456.1595. Anal. calcd. for
C26H22ClN5O × 3HCl × 3H2O (Mr = 619.37): C 50.42, H 5.04, N 11.31; found: C 50.23, H
4.65, N 11.04.
4.1.3.4. 2-[4-(7-Chloroquinolin-4-yloxy)phenyl]-N-isobutyl-1H-benzo[d]imidazole-6-
carboximidamide trihydrochloride (5d).
Yield 39.1%; mp 223–224 °C; IR (νmax/cm–1): 3062, 2645, 1681, 1609, 1425, 1296, 1192,
1109, 859, 821; 1H NMR (DMSO-d6) δ/ppm: 9.87 (t, 1H, J = 5.47 Hz, NH), 9.54 (s, 1H, NH),
9.11 (s, 1H, NH), 9.03 (d, 1H, J = 6.02 Hz, CH), 8.61 (d, 2H, J = 8.81 Hz, CH), 8.54 (d, 1H, J
= 9.00 Hz, CH), 8.40 (d, 1H, J = 1.99, CH), 8.14 (d, 1H, J = 1.27, CH), 7.93 (dd, 1H, J1 =
8.85 Hz, J2 = 1.98 Hz, CH), 7.89 (d, 1H, J = 8.66 Hz, CH), 7.70 (dd, 2H, J1 = 8.66 Hz, J2 =
1.89 Hz, CH), 7.69 (d, 2H, J = 8.85 Hz, CH), 7.08 (d, 1H, J = 6.00 Hz, CH), 3.30 (t, 2H, J =
6.65 Hz, CH), 2.06 (m, 1H, CH), 1.00 (d, 6H, J = 6.65 Hz, CH3); 13C NMR (DMSO-d6)
δ/ppm: 163.9, 163.5, 155.9, 152.9, 150.7, 144.9, 138.1, 130.5, 129.3, 126.6, 125.1, 124.1,
123.8, 123.6, 122.2, 119.9, 116.9, 106.3, 50.2, 27.5, 20.4. HRMS calcd. for C27H25ClN5O (M
+ H)+: 470.1748; found: 470.1753. Anal. calcd. for C27H24ClN5O × 3HCl × 2H2O (Mr =
615.39): C 52.70, H 5.08, N 11.38; found: C 52.51, H 5.08, N 11.06.
4.1.3.5. 2-[4-(7-Chloroquinolin-4-yloxy)phenyl]-N-cyclopentyl-1H-benzo[d]imidazole-6-
carboximidamide dihydrochloride (5e).
Yield 23.8%; mp 228–229 °C; IR (νmax/cm–1): 3069, 2954, 1607, 1571, 1420, 1291, 1205,
1169, 1083, 861, 818; 1H NMR (DMSO-d6) δ/ppm: 9.63 (d, 1H, NH), 9.41 (s, 1H, NH), 8.94
(s, 1H, NH), 8.88 (d, 1H, J = 5.41 Hz, CH), 8.44 (d, 2H, J = 8.66 Hz, CH), 8.41 (d, 1H, J =
8.91 Hz, CH), 8.21 (d, 1H, J = 1.82 Hz, CH), 8.06 (s, 1H, CH), 7.82 (d, 1H, J = 8.81 Hz, CH),
7.79 (dd, 1H, J1 = 9.04 Hz, J2 = 1.86 Hz, CH), 7.59 (dd, 1H, J1 = 8.77 Hz, J2 = 1.85 Hz, CH),
7.58 (d, 2H, J = 8.70 Hz, CH), 6.93 (d, 1H, J = 5.43 Hz, CH), 4.17 (m, 1H, CH), 2.07 (m, 2H,
CH2), 1.75 (m, 4H, CH2), 1.69 (m, 2H, CH2); 13C NMR (DMSO-d6) δ/ppm: 164.7, 162.7,
155.5, 151.8, 149.5, 138.2, 130.7, 130.4, 129.1, 126, 125.3, 124.8, 124.2, 123.8, 122.3, 121.8,
119.3, 116.5, 105.8, 54.3, 31.4, 23.7. HRMS calcd. for C28H25ClN5O (M + H)+: 482.1748;

ACCEPTED MANUSCRIPT
found: 482.1750. Anal. calcd. for C28H24ClN5O × 2HCl × 1.5H2O (Mr = 581.93): C 57.79, H
5.02, N 12.03; found: C 57.59, H 5.20, N 11.72.
4.1.3.6. 2-[4-(7-Chloroquinolin-4-yloxy)phenyl]-6-(4,5-dihydro-1H-imidazol-2-yl)-1H-
benzo[d]imidazole dihydrochloride (5f).
Yield 57.6 %; mp 227–228 °C; IR (νmax/cm–1): 3035, 2953, 2353, 1583, 1494, 1417, 1297,
1218, 1177, 1084, 858, 821; 1H NMR (DMSO-d6) δ/ppm: 10.58 (s, 2H, NH), 8.92 (d, 1H, J =
5.71 Hz, CH), 8.46 (d, 1H, J = 8.67 Hz, CH), 8.44 (d, 1H, J = 9.16 Hz, CH), 8.37 (s, 1H, CH),
8.25 (d, 1H, J = 2.29 Hz, CH), 7.85 (s, 1H, CH), 7.82 (d, 2H, J = 2.21 Hz, CH), 7.58 (d, 2H, J
13
= 8.89 Hz, CH), 6.97 (d, 1H, J = 6.16 Hz, CH2), 4.01 (s, 4H, CH2); C NMR (DMSO-d6)
δ/ppm: 165.7, 163.7, 155.8, 154.0, 151.3, 146.0, 137.5, 130.3, 128.9, 127.3, 124.9, 124.6,
123.4, 122.0, 119.9, 116.7, 116.3, 106.2, 44. HRMS calcd. for C25H19ClN5O (M + H)+:
440.1278; found: 440.1279. Anal. calcd. for C25H18ClN5O × 2HCl × 2.5H2O (Mr = 557.86): C
53.83, H 4.52, N 12.55; found: C 54.17, H 4.54, N 12.21.
4.1.3.7. 2-[4-(7-Chloroquinolin-4-yloxy)phenyl]-6-(1,4,5,6-tetrahydropyrimidin-2-yl)-1H-
benzo[d]imidazole dihydrochloride (5g).
Yield 74.4%; mp 137–138 °C; IR (νmax/cm–1): 3044, 2655, 1767, 1672, 1610, 1570, 1494,
1
1385, 1297, 1037, 814; H NMR (DMSO-d6) δ/ppm: 9.99 (s, 2H, NH), 8.95 (d, 1H, J = 5.54
Hz, CH), 8.49 (d, 2H, J = 9.28 Hz, CH), 8.45 (s, 1H, CH), 8.28 (d, 1H, J = 2.97 Hz, CH), 8.10
(d, 1H, J = 1.54 Hz, CH), 7.85 (d, 2H, J = 8.07 Hz, CH), 7.63 (d, 2H, J = 9.63 Hz, CH), 7.62
(d, 1H, J = 8.85 Hz, CH), 3.53 (t, 4H, J1 = 6.85 Hz, J2 = 5.71 Hz, CH2), 2.02 (m, 2H, CH2);
13C NMR (DMSO-d6) δ/ppm: 164.6, 159.3, 155.4, 151.9, 149.6, 138.1, 130.4, 129.1, 124.8,
123.6, 123.0, 122.3, 121.8, 119.3, 105.8, 38.8, 17.8. HRMS calcd. for C26H21ClN5O (M + H)+:
454.1435; found: 454.1438. Anal. calcd. for C26H20ClN5O × 2HCl × 3.2H2O (Mr = 584.50): C
53.43, H 4.90, N 11.98; found: C 53.31, H 4.67, N 12.02.
4.1.4.1. 4-[2-(7-Chloroquinolin-4-ylamino)ethoxy]benzonitrile (8a).
A suspension of (2-bromoethyl)-(7-chloroquinolin-4-yl)-amine (7) (1.52 g, 5.35 mmol), 4-
hydroxybenzonitrile (0.764 g, 6.41 mmol) and potassium carbonate (2.21 g, 16 mmol) in
dimethylformamide (40 mL) was heated at 60 °C for 24 h under nitrogen. After cooling to
room temperature, the reaction was diluted with 150 mL of dichloromethane and washed with
water. The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent
was removed under reduced pressure. The oil obtained, was diluted with methanol (5 mL),

ACCEPTED MANUSCRIPT
and the product precipitates with addition of water as white powder (1.15 g, 66%); mp 175
°C. IR (νmax/cm–1): 3434, 3273, 2223, 1608, 1578, 1510, 1257, 1172, 1146, 1052, 890, 844,
797, 549. 1H NMR (DMSO-d6) δ/ppm: 8.43 (d, J = 5.4 Hz, 1H, ArH), 8.27 (d, J = 9.1 Hz, 1H,
ArH), 7.80 (d, J = 2.2 Hz, 1H, ArH), 7.76 (d, J = 8.8 Hz, 2H, ArH), 7.46 (dd, J = 8.9, 2.2 Hz,
2H, NH + ArH), 7.13 (d, J = 8.8 Hz, 2H, ArH), 6.61 (d, J = 5.4 Hz, 1H, ArH), 4.35 (t, J = 5.4
13
Hz, 2H, CH2), 3.71 (q, J = 5.3 Hz, 2H, CH2). C NMR (DMSO-d6) δ/ppm: 161.89, 151.91,
149.95, 149.05, 134.15, 133.41, 127.54, 124.16, 124.01, 119.05, 117.40, 115.57, 102.92,
98.93, 66.11, 41.71. HRMS calcd. for C18H15ClN3O (M + H)+: 324.0903; found: 324.0901.
Anal. calcd. for C18H14ClN3O × 0.5H2O (Mr = 332.79): C 64.97, H 4.54, N 12.63; found: C
64.89, H 4.42, N 12.51.
4.1.5. General method for the synthesis the compounds from group III (8b-g)
A solution of nitrile 8a (600 mg, 1.85 mmol) in anhydrous methanol (50 mL) was cooled in
an ice bath and saturated with dry HCl gas. The reaction mixture was then sealed, and stirred
at room temperature until the starting nitrile was no longer detectable with IR. The reaction
mixture was evaporated to dryness and dried over KOH. The crude imidate ester
hydrochloride was suspended in anhydrous MeOH (30 mL) and the ammonia or appropriate
amine was added. The desired product was isolated with column cromatography
(CH2Cl2/MeOH 5:1; MeOH/NH4OH 3:1) and converted to hydrochloride salt using
anhydrous ethanol saturated with HCl(g).
4.1.5.1. 4-[2-(7-Chloroquinolin-4-ylamino)ethoxy]benzimidamide trihydrochloride (8b).
The crude imidate ester hydrochloride was dissolved in dry MeOH and saturated with dry
ammonia gas. A solution was stirred at room temperature and the progress of the reaction was
monitored by TLC. After three days the desired product was isolated using column
cromatography as white solid (207 mg, 23%); mp 143 °C. IR (νmax/cm–1): 1610, 1583, 186,
1457, 1269, 1211, 1187, 1171, 1087, 1045, 841, 814. 1H NMR (DMSO-d6) δ/ppm: 9.99 (t, J =
5.2 Hz, 1H, NH), 9.11 (s, 2H, NH), 8.68 (s, 2H, NH), 8.46 (d, J = 5.6 Hz, 1H, ArH), 8.32 (d, J
= 9.0 Hz, 1H, ArH), 7.82 (s, 1H, ArH), 7.81 (d, J = 8.9 Hz, 2H, ArH), 7.52 (dd, J = 9.0, 2.2
Hz, 1H, ArH), 7.18 (d, J = 8.9 Hz, 2H, ArH), 6.68 (d, J = 5.7 Hz, 1H, ArH), 4.38 (t, J = 5.3
13
Hz, 2H, CH2), 3.78 (q, J = 5.2 Hz, 2H, CH2). C NMR (DMSO-d6) δ/ppm: 164.27, 163.37,
162.72, 130.20, 129.35, 124.57, 119.68, 119.55, 114.85, 113.97, 110.58, 98.96, 66.18, 41.92,
40.20. HRMS calcd. for C18H18ClN4O (M + H)+: 341.1169; found: 341.1168. Anal. calcd. for

ACCEPTED MANUSCRIPT
C18H17ClN4O × 3HCl × 2H2O (Mr = 486.22): C 44.46, H 4.98, N 11.52; found: C 44.64, H
4.71, N 11.15.
4.1.5.2. 4-[2-(7-Chloroquinolin-4-ylamino)ethoxy]-N-isopropylbenzimidamide
trihydrochloride (8c).
A solution of crude imidate and isopropyl amine (438 mg, 7.41 mmol) in dry MeOH was
stirred at room temperature. The progress of the reaction was monitored by TLC. After three
days the desired product was isolated using column cromatography as white solid (274 mg,
29%); mp 78 °C. IR (νmax/cm–1): 3240, 3093, 2973, 1612, 1580, 1515, 1457, 1264, 1124,
1
1083, 1050, 838, 808, 743. H NMR (DMSO-d6) δ/ppm: 9.54 (s, 1H, NH), 9.38 (d, J = 8.1
Hz, 1H, NH), 9.26 (s, 1H, NH), 8.88 (s, 1H, NH), 8.67 (d, J = 9.2 Hz, 1H, ArH), 8.59 (d, J =
6.9 Hz, 1H, ArH), 8.05 (d, J = 1.9 Hz, 1H, ArH), 7.76 (dd, J = 9.1, 1.9 Hz, 1H, ArH), 7.70 (d,
J = 8.8 Hz, 2H, ArH), 7.15 (d, J = 8.8 Hz, 2H, ArH), 7.01 (d, J = 7.0 Hz, 1H, ArH), 4.42 (t, J
13
= 4.9 Hz, 1H, CH2), 4.1 – 3.9 (m, 3H, CH +CH2), 1.26 (d, J = 6.3Hz, 6H, CH3 ). C NMR
(DMSO-d6) δ/ppm: 161.88, 161.77 151.94, 150.13, 149.07, 133.46, 130.00 , 127.49, 124.26,
124.19 121.82, 117.48, 114.59, 98.90, 66.10, 41.75, 29.02, 21.53. HRMS calcd. for
C21H24ClN4O (M + H)+: 383.1638; found: 383.1641. Anal. calcd. for C21H23ClN4O × 3HCl ×
1H2O (Mr = 510.29): C 49.43, H 5.53, N 10.98; found: C 49.09, H 5.46, N 10.67.
4.1.5.3. 4-[2-(7-Chloroquinolin-4-ylamino)ethoxy]-N-isobutylbenzimidamide
trihydrochloride (8d).
A solution of crude imidate and isobutylamine (542 mg, 7.41 mmol) in dry MeOH was stirred
at room temperature. The progress of the reaction was monitored by TLC. After three days the
desired product was isolated using column cromatography as white solid (233 mg, 24%); mp
80 °C. IR (νmax/cm–1): 3441, 1688, 1612, 1579, 1512, 1456, 1384, 1261, 1190, 1143, 1049,
1
844, 813, 657. H NMR (DMSO-d6) δ/ppm: 9.72 (t, J = 4.8 Hz, 1H, NH), 9.38 (s, 1H, NH),
9.26 (s, 1H, NH), 9.03 (s, 1H, NH), 8.70 (d, J = 9.2 Hz, 1H, ArH), 8.52 (d, J = 6.5 Hz, 1H,
ArH), 8.06 (d, J = 2.1 Hz, 1H, ArH), 7.77 (d, J = 8.9 Hz, 2H, ArH), 7.64 (dd, J = 9.1, 2.0 Hz,
1H, ArH), 7.15 (d, J = 8.8 Hz, 2H, ArH), 6.88 (d, J = 6.6 Hz, 1H, ArH), 4.42 (t, J = 5.3 Hz,
2H, CH2), 3.91 (q, J = 4.7 Hz, 2H, CH2), 3.29 – 3.20 (m, 2H), 2.00 (m, 1H, CH), 0.94 (d, J =
6.6 Hz, 6H, CH3). 13C NMR (DMSO-d6) δ/ppm: 162.39, 162.13, 154.99, 144.31, 137.38,
130.13, 126.57, 125.52, 121.04, 120.41, 115.74, 114.76, 99.08, 66.14, 49.34, 42.48, 26.96,
19.87. HRMS calcd. for C22H26ClN4O (M + H)+: 397.1795; found: 397.1803. Anal. calcd. for

ACCEPTED MANUSCRIPT
C22H25ClN4O × 3HCl × 1H2O (Mr = 524.31): C 50.40, H 5.77, N 10.69; found: C 50.49, H
5.65, N 10.58.
4.1.5.4. 4-[2-(7-Chloroquinolin-4-ylamino)ethoxy]-N-cyclopentylbenzimidamide
dihydrochloride (8e).
A solution of crude imidate and cyclopentylamine (630 mg, 7.41 mmol) in dry MeOH was
stirred at room temperature. The progress of the reaction was monitored by TLC. After three
days the desired product was isolated using column cromatography as white solid (246 mg,
26%); mp 141–142 °C. IR(νmax/cm–1): 3353, 1607, 1589, 1513, 1462, 1412, 1368, 1292,
1
1260, 1166, 1141, 1044. H NMR (DMSO-d6) δ/ppm: 14.43 (s, 1H, NH), 9.79 (s, 1H, NH),
9.48 (d, J = 7.2 Hz, 1H, NH), 9.29 (s, 1H, NH), 8.87 (s, 1H, NH), 8.73 (d, J = 9.2 Hz, 1H,
ArH), 8.60 (d, J = 7.1 Hz, 1H, ArH), 8.10 (d, J = 2.0 Hz, 1H, ArH), 7.78 (dd, J = 9.1, 2.1 Hz,
1H, ArH), 7.71 (d, J = 8.9 Hz, 2H, ArH), 7.14 (d, J = 8.9 Hz, 2H, ArH), 7.04 (d, J = 7.1 Hz,
1H, ArH), 4.43 (t, J = 5.2 Hz, 2H, CH2), 4.18 – 4.05 (m, 1H, CH), 4.00 (q, J = 5.2 Hz, 2H,
CH2), 2.04 (dd, J = 14.0, 8.7 Hz, 2H, CH2), 1.78 – 1.48 (m, 6H, CH2). 13C NMR (DMSO-d6)
δ/ppm: 161.97, 161.71, 155.75, 142.91, 138.75, 137.84, 130.36, 126.76, 125.96, 121.05,
119.17, 115.49, 114.56, 99.02, 66.17, 54.09, 42.46, 31.32, 23.64. HRMS calcd. for
C23H26ClN4O (M + H)+: 409.1795; found: 409.1801. Anal. calcd. for C23H25ClN4O × 2HCl ×
1.75H2O (Mr = 513.37): C 53.81, H 5.99, N 10.91; found: C 53.86, H 5.83, N 10.73.
4.1.5.5. 7-Chloro-N-{2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]ethyl}quinolin-4-amine
dihydrochloride (8f).
A solution of crude imidate and ethane-1,2-diamine (277 mg, 4.63 mmol) in dry MeOH was
refluxed. The progress of the reaction was monitored by TLC. After 24 hours the desired
product was isolated using column chromatography as the white solid (265 mg, 29%); mp
144–145 °C. IR (νmax/cm–1): 3407, 3122, 3094, 1618, 1510, 1403, 1267, 1191, 1140, 1092,
1042, 842, 740. 1H NMR (DMSO-d6) δ/ppm: 14.55 (s, 1H, NH), 10.61 (s, 2H, NH), 9.82 (t, J
= 5.2 Hz, 1H, NH), 8.75 (d, J = 9.2 Hz, 1H, ArH), 8.59 (d, J = 7.1 Hz, 1H, ArH), 8.12 (d, J =
2.1 Hz, 1H, ArH), 8.04 (d, J = 8.9 Hz, 2H, ArH), 7.77 (dd, J = 9.1, 2.1 Hz, 1H, ArH), 7.21 (d,
J = 8.9 Hz, 2H, ArH), 7.03 (d, J = 7.2 Hz, 1H, ArH), 4.47 (t, J = 5.0 Hz, 2H, CH2), 4.01 (q, J
= 5.1 Hz, 2H, CH2), 3.95 (s, 4H, CH2). 13C NMR (DMSO-d6) δ/ppm: 163.96, 162.90, 155.85,
142.88, 138.63, 137.94, 130.88, 66.26, 44.09, 42.49. HRMS calcd. for C20H20ClN4O (M +
H)+: 367.1326; found: 367.1315. Anal. calcd. for C20H19ClN4O × 2HCl × 3H2O (Mr =
493.81): C 48.65, H 5.51, N 11.35; found: C 48.84, H 5.26, N 11.10.

ACCEPTED MANUSCRIPT
4.1.5.6. 7-Chloro-N-{2-[4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenoxy]ethyl}quinolin-4-amine
dihydrochloride (8g).
A solution of crude imidate and propane-1,2-diamine (343 mg, 4.63 mmol) in dry MeOH was
refluxed. The progress of the reaction was monitored by TLC. After 24 hours the desired
product was isolated using column cromatography as white solid (229 mg, 22%); mp 144–145
°C. IR (νmax/cm–1): 3407, 3204, 3008, 2932, 1634, 1609, 1582, 1452, 1374, 1316, 1260, 1196,
1
1092, 1142, 1091, 1050, 814, 704. H NMR (DMSO-d6) δ/ppm: 10.01 (s, 2H, NH), 8.50 (m,
3H, NH + ArH), 7.94 (d, J = 2.0 Hz, 1H, ArH), 7.78 (d, J = 8.8 Hz, 2H), 7.56 (dd, J = 9.0, 2.0
Hz, 1H, ArH), 7.16 (d, J = 8.8 Hz, 2H, ArH), 6.77 (d, J = 6.1 Hz, 1H, ArH), , 4.40 (t, J = 5.2
Hz, 2H, CH2), 3.83 (q, J = 5.1 Hz, 2H, CH2), 3.46 (s, 4H, CH2), 2.05 – 1.87 (m, 2H, CH2). 13C
NMR (DMSO-d6) δ/ppm: 162.49, 158.60, 153.31, 147.98, 144.56, 136.00, 129.97, 125.86,
125.65, 123.93, , 120.83, 117.01, 115.26, 99.44, 66.64, 42.51, 18.31. HRMS calcd. for
C21H22ClN4O (M + H)+: 381.1482; found: 381.1477. Anal. calcd. for C21H21ClN4O × 2HCl ×
1.75H2O (Mr = 566.24): C 44.55, H 4.90, N 9.89; found: C 44.64, H 4.75, N 9.58.
4.1.6.1. 2-{4-[2-(7-Chloroquinolin-4-ylamino)ethoxy]phenyl}-1H-benzo[d]imidazole-6-
carbonitrile (10a).
The solution of compound 9 (300 mg, 0.92 mmol), nitrile 3a (122 mg, 0.92 mmol) and water
solution of NaHSO3 (40%, 5 mL) in ethanol (50 mL) was refluxed for 6h. The hot solution
was filtered, filtrate was evaporated to dryness, and the residue was suspended in 30 mL of
water and stirred overnight. The solid was collected and dissolved in methanol, with addition
of water product precipitates as a brown powder (150 mg, 41%); mp 274 °C. IR (νmax/cm–1):
3421, 2920, 2850, 2368, 2217, 1610, 1581, 1490, 1450, 1425, 1249, 1180, 1141, 1053, 806.
1H NMR (DMSO-d6) δ/ppm 13.24 (s, 1H, NH), 8.47 (d, J = 5.6 Hz, 1H, ArH), 8.33 (d, J = 9.0
Hz, 1H, ArH), 8.14 (d, J = 6.8 Hz, 2H, ArH), 7.97 (brs, 1H), 7.82 (d, J = 2.1 Hz, 1H, ArH),
7.75 (s, 1H, ArH), 7.66 (s, 1H, ArH), 7.56 (d, J = 7.0 Hz, 1H, ArH), 7.51 (dd, J = 9.0, 2.1 Hz,
1H, ArH), 7.17 (d, J = 8.8 Hz, 2H, ArH), 6.70 (d, J = 5.7 Hz, 1H, ArH), 4.38 (t, J = 5.4 Hz,
2H, CH2), 3.79 (d, J = 5.0 Hz, 2H, CH2). 13C NMR (DMSO-d6) δ/ppm 160.42, 151.00,
150.51, 134.19, 128.57, 126.19, 124.61, 124.24, 121.84, 120.02, 117.13, 115.08, 103.64,
98.99, 65.92, 42.04. HRMS calcd. for C25H19ClN5O (M + H)+: 440.1278; found: 440.1291.
4.1.7. General method for the synthesis of the amidines from group IV (10b-g).
The solution of compound 9 (300 mg, 0.92 mmol) appropriate 3,4-diaminobenzamidine 3b-g
and water solution of NaHSO3 (40%, 5 mL) in ethanol (50 mL) was refluxed for 6h. The hot