Synthesis, characterization and anticancer activity of novel ferrocene containing quinolinones: 1-Allyl-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-ones and 1-allyl-2-ferrocenylquinolin-4(1H)-ones

Article abstract of DOI:10.1016/j.jorganchem.2018.08.004

A two new series of ferrocene containing quinolinones – 1-allyl-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-ones and 1-allyl-2-ferrocenylquinolin-4(1H)-ones – were prepared and characterized by standard spectroscopic techniques and cyclic voltammetry. The in vitro antitumor activity of all synthesized compounds was investigated against Human Cervix Carcinoma (HeLa), Chronic Myelogenous Leukemia (K562) and normal endothelial (HUVEC) cell lines using the MTT method. Quinolone derivative 5c exhibited the highest cytotoxic activity in the cell growth inhibition of HeLa cell line while 5f was the most toxic against K562 cells.

Full text of DOI:10.1016/j.jorganchem.2018.08.004

Accepted Manuscript
Synthesis, characterization and anticancer activity of novel ferrocene containing
quinolinones: 1-Allyl-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-ones and 1-allyl-2-
ferrocenylquinolin-4(1H)-ones
Anka Pejović, Józef Drabowicz, Marcin Cieslak, Julia Kazmierczak-Baranska,
Karolina Królewska-Golińska
PII:
S0022-328X(18)30696-X
10.1016/j.jorganchem.2018.08.004
JOM 20531
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 20 April 2018
Revised Date: 2 August 2018
Accepted Date: 7 August 2018
Please cite this article as: A. Pejović, Jó. Drabowicz, M. Cieslak, J. Kazmierczak-Baranska, K.
Królewska-Golińska, Synthesis, characterization and anticancer activity of novel ferrocene containing
quinolinones: 1-Allyl-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-ones and 1-allyl-2-ferrocenylquinolin-4(1H)-
ones, Journal of Organometallic Chemistry (2018), doi: 10.1016/j.jorganchem.2018.08.004.
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ACCEPTED MANUSCRIPT
Synthesis, characterization and anticancer activity of novel ferrocene
containing quinolinones: 1-allyl-2-ferrocenyl-2,3-dihydroquinolin-
4
(1H)-ones and 1-allyl-2-ferrocenylquinolin-4(1H)-ones
∗
a
b,c
∗ ∗ d
d
Anka Pejović, Józef Drabowicz,
Marcin Cieslak,
Julia Kazmierczak-Baranska,
d
Karolina Królewska-Golińska
a
Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovića 12,
3
4000 Kragujevac, Serbia.
b
The Institute of Chemistry, Environmental Protection and Biotechnology, Faculty of
Mathematics and Natural Sciences, The Jan Dlugosz University in Czestochowa, Armii
Krajowej 13/15, 42-218, Czestochowa.
c
Department of Heteroorganic Chemistry, Centre of Molecular and Macromolecular Studies,
Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.
d
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies,
Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland.
E-mail address: apejovic@kg.ac.rs (A. Pejović) mjcieslak@gmail.com (M. Cieslak)
Abstract: A two new series of ferrocene containing quinolinones – 1-allyl-2-ferrocenyl-2,3-
dihydroquinolin-4(1H)-ones and 1-allyl-2-ferrocenylquinolin-4(1H)-ones – were prepared
and characterized by standard spectroscopic techniques and cyclic voltammetry. The in vitro
antitumor activity of all synthesized compounds was investigated against Human Cervix
Carcinoma (HeLa), Chronic Myelogenous Leukemia (K562) and normal endothelial
(HUVEC) cell lines using the MTT method. Quinolone derivative 5c exhibited the highest
cytotoxic activity in the cell growth inhibition of HeLa cell line while 5f was the most toxic
against K562 cells.
Key words: Ferrocene, Quinolinones, 2,3-Dihydroquinolin-4(1H)-ones, Quinolin-4(1H)-
ones, Electrochemistry, Anticancer activity
∗
∗
Corresponding author. e-mail: apejovic@kg.ac.rs
∗ Corresponding author. e-mail: mjcieslak@gmail.com

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1
. Introduction
Since 1950s rapid progress has been made in the area of cancer cell biology. Most of the
cancer treatments are still multimodal, involving chemotherapy, radiotherapy, and extensive
surgery. However, chemotherapy remains the most significant pharmacological approach to
,
the cancer treatment [1]. Due to the limitations associated with the existing agents – like side
effects and acquired tumor resistance, there is a permanent need for the new anticancer agents
which will have better therapeutic profile [1]. Among the wide range of compounds tested as
potential anticancer agents, ferrocene derivatives have attracted significant attention over the
last few decades. This great interest in ferrocene–containing compounds as anticancer agents
is caused by the discovery of ferrocifens, analogues of hydroxytamoxifen, the active
tamoxifen metabolite in inhibition of cancer cell proliferation by competitive binding to the
estrogen receptors in endocrine therapy [2]. Ferrocifen and its analogues specially ones which
bears an additional hydroxyl group are the most important examples for the application of
organometallic molecules in medicinal and pharmaceutical chemistry. The above mentioned
molecules represent new and very promising drug candidates for treatment of hormone-
dependent breast cancer [3, 4] and melanoma cells [5]. The huge success of ferrocenyl-
tamoxifen derivatives as anticancer agents has triggered the interest on novel hybrids'
molecules (ferrocene and different pharmacophores) with greater efficiencies and minor side
effects than the drugs used nowadays in the treatment of cancer. Novel compounds of this
kind have been synthesized and biologically evaluated against certain diseases, and it turned
out that many ferrocenyl heterocycles, especially ferrocenyl quinolines, display interesting
cytotoxic activities (Figure 1) [6–8]. Quinoline ring is pharmacologically valuable scaffold
which occurs in the numerous biologically active compounds – synthetic and natural [9].
th
Throughout the 20 century, the research in this field covered the intensive studies of
quinolines which pointed out the wide range of their interesting bioactivities such as,
antibacterial, antifungal, anti-inflammatory, and antimalarial [10–14]. Additionaly, the thieno
[
3,2-c]quinolin-4-one was found to be a potential Hh signaling inhibitor [15]. Based on this
compound, the inhibitory effects of furo-, pyrrolo- and piperazine-derivatives on Hh
signaling were evaluated using Gli-luc reporter assay. Moreover, in vivo studies in mouse
model showed that these compounds completely suppressed medulloblastoma tumor growth
[
16]. Quinoline derivatives have also shown remarkable anticancer activity against other
lines, such as breast, prostate, gastrointestinal tract, colon and liver cancer [17–20].
Interestingly, although cytotoxic activity of 4-quinolone derivatives led to design new

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anticancer agents, to the best of our knowledge there are no reports on the anticancer activity
of ferrocene derivatives of 4-quinolone (neither 2-ferrocenyl-2,3-dihydroquinolin-4(1H)-ones
nor 2-ferrocenyl-quinolin-4(1H)-ones.
Having in mind the above considerations, we decided to combine the ferrocene scaffold
with quinolon-4-one pharmacophore in hope that resulting novel heterocyces would be
biologically active. Knowing from the literature studies devoted to the development of new
antimicrobial and anticancer drugs that the presence of an allyl group on the heterocyclic
nitrogen improves biological effectiveness of the corresponding N allyl derivatives [21–23].
so we also chose in our syntheses the allyl group as a substituent on the nitrogen of the
quinolone sceleton. Therefore, in the present paper we report the synthesis, spectral and
electrochemical characterization of 1-allyl-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-ones and
1
-allyl-2-ferrocenylquinolin-4(1H)-ones, as well as evaluation of their antiproliferative
activity against different cancer cell lines.
Figure 1. Structures of some bioactive ferrocene-containing quinolines
2
. Results and Discussion
2
.1. Synthesis
The preparation of target dihydroquinolinones and quinolinones is schematically described
in Scheme 1. The synthesis of desired 1-allyl-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-ones
a–f started with commercially available of o-aminoacetophenones 1a–c which were
4
allylated with corresponding allyl bromides according to known literature procedure [24].
The compounds 2 were then subjected to aldol condensation with ferrocenecarboxaldehyde in
the presence of sodium hydroxide using ethanol as a solvent yielding expected ferrocene
analogues of chalcones 3a–f. After purification on column chromatography, the chalcones
3
a–f were treated with the mixture of acetic and orthophosphoric acids in ultrasound bath
affording desired dihydroquinolin-4(1H)-ones 4a–f in good to excellent yield. These
compounds, upon treatment with an excess of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) in the dichloromethane, gave their oxidized analogues 5a–i (Scheme 1). The overall

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yields for the synthesis of 4a–i and 5a–i are given in Table 1. As can be seen a variety of
substituents such as Cl, Br, H, at the quinolone ring, and allyl on the nitrogen atom, are
compatible with the applied reaction conditions.
Table 1. The synthesis of the newly prepared 1-allyl-2-ferrocenyl-2,3-dihydroquinolin-
4
(1H)-ones (4a–f) and 1-allyl-2-ferrocenylquinolin-4(1H)-ones (5a–f) as well as the yield of
the reactions
R
R
NH2
NH
O
Fe
O
HN
Br
R
X
Fe
X
X
K CO , DMF
EtOH, NaOH
2
3
O
O
1a-c
2a-f
3a-f
CH COOH
3
H PO
3
4
R
R
N
N
DDQ
X
X
Fe
a-f
Fe
CH Cl2
2
O
O
5
4a-f
a)
a)
Entry
X
R
H
Product Yield
Entry
X
R
Product Yield
1
2
3
4
5
6
H
H
4a
4b
4c
4d
4e
4f
80%
94%
70%
90%
96%
68%
7
8
H
H
H
CH3
H
5a
5b
5c
5d
5e
5f
73%
CH3
H
77%
74%
61%
76%
65%
Cl
Cl
Br
Br
9
Cl
Cl
Br
Br
CH3
H
10
11
12
CH3
H
CH3
CH3
a) After purification by means of column chromatography on SiO2
.
2
.2. Spectral characterization
1
13
The structures of quinolones 4a–f and 5a–f were deduced based on their IR, H- and C-
NMR spectral data.
The IR spectra of quinolones 4a–f contain two strong bands at 3079–3085 cm and 1664–
-1
-1
1
672 cm which are attributed to the alkenyl C–H stretching and carbonyl C=O stretching,

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1
respectively. On the other hand, in the H NMR spectra of all six compounds signals of the
cyclopentadienyl protons appear at the similar positions (δ = 4.15 ppm for the unsubstituted,
and 3.57–4.30 ppm for substituted rings, respectively). The protons of the –N-CH unit in 4a–
2
f are also located in same region as the substituted cyclopentadienyl rings protons (3.57 to
1
4
.30 ppm). In H NMR spectra of 4a–f, three signals appear at about 4.50, 3.24 and 3.14
ppm. They correspond to the methine and methylene protons of the quinolinone moiety,
respectively. Signals of CH=C protons exhibit a doublet of doublets of triplets at 5.75 ppm
while signals of C=CH protons appear as a doublet of quartets (4a, 4c, 4e) and singlet or
2
doublet (4b, 4d, 4e) at 4.91–5.24 ppm.
The NMR and IR spectral data of the synthesized quinolin-4-ones also clearly confirm
their structures. Thus, the IR spectra of these ferrocene derivatives (5a–f) contain the
-1
-1
intensive band at 3056–3100 cm and 1616–1622 cm , which is attributed to the alkenyl and
1
carbonyl group, respectively. The H NMR spectra of 5a–f contain characteristic signals for –
1
N-CH , aromatic and ferrocenyl protons. They are located in the expected regions of the H
2
NMR spectra. Signals of the quinolone proton appear as a singlet at about 6.96–7.01 ppm. In
1
addition, in the H NMR spectra of 5a–f, three signals appear at about 5.95, 5.32 and 5.01
ppm. They correspond to the CH=C and C=CH protons, respectively.
2
2
.3. Electrochemistry
The electrochemical properties of the synthesized compounds 4a–f and 5a–f were
investigated by cyclic voltammetry in dichloromethane containing 0.1 mol/L
tetrabutylammonium perchlorate as a supporting electrolyte. All measurements were carried
out using ferrocene as an internal standard.
2.3.1. Electrochemical properties of the 1-allyl-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-ones 4a–f
The voltammogram of compounds 4b is presented here (Figure 2), as representative
example, whereas the data of the other compounds are listed in Tables 2. As it can be seen
from Figure 2, curve a, the ferrocene containing quinolin-4-one 4b exhibited at the first scan
two well defined oxidation waves on the forward potential sweep (O1, at 0.668 and O2, at
1
.443 V, respectively) and three reduction waves on the back potential sweep (R1, at 1.114,
R2, at 1.102 and R3, at 0.528 V). The reduction peak R3 appeared also when the potential
was reversed after O1, and, therefore, O1 and R3 should correspond to interdependent

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Table 2.
The electrochemical data for compounds 4a–f
Compound
E (V)
E (V)
E (V)
E (V)
E (V)
E (V)
∆E (V)
0.155
0.140
0.223
0.153
0.144
0.152
O1
O2
R1
R2
R3
1/2
4
a
0.674
0.668
0.717
0.693
0.684
0.692
1.453
1.443
1.651
1.593
1.575
1.579
1.074
1.114
1.019
1.410
1.086
1.073
1.007
1.119
0.974
1.080
0.864
1.078
0.519
0.528
0.494
0.540
0.540
0.540
0.596
0.598
0.605
0.616
0.612
0.616
4
b
4
c
4
d
4
e
4f
Figure 2. Cyclic voltammograms of
methylallyl)quinolin-4(1H)-one (4b) at the glassy carbon electrode by a 0.1 V s scan rate in
.1 M dichloromethane solution of Bu NClO : (a) the first scan, (b) the second scan.
1
mM solution of 2-ferrocenyl-1-(2-
-1
0
4
4
electrochemical events. Since the difference between the values of these two potentials is
close to the theoretical one, O1 and R3 apparently belong to a reversible redox couple,
appearing due to the presence of the ferrocene nucleus. The second oxidation wave (O2) is
occurring due to the quasi reversible electrochemical event. We believe that an electron
transfer from the nitrogen atom to anode is liable for it appearance. This assumption is based
on the certain degree of similarity of this class of compounds with cyclic voltammetry of N-
alkyl anilines, as we reported earlier [25].

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2
.3.2. Electrochemical properties of 1-allyl-2-ferrocenylquinolin-4(1H)-ones 5a–f
All quinolones 5a–f exhibit a reversible one-electron redox couple at the similar potential
–
oxidation waves appear at 0.647–0.894 V and the reduction ones at 0.552–696 V, Table 3.
We attributed them to the oxidation of ferrocene unit by the forward potential sweep, and the
reduction of the obtained ferricinium ion at the back potential sweep. As the representative
example we give the voltammograms of compound 5b (Figure 3). The difference between
anodic and cathodic peak potentials (Table 3) is close to the theoretical value and
independent of the scan rate v. Both, anodic and cathodic peak currents are proportional to
the square root of the scan rate, and their ratio is independent of the scan rate, indicating a
diffusion-controlled process.
Table 3.
The electrochemical data for compounds 5a–f
Compound
E (V)
O3
E (V)
R4
E (V)
1/2
∆E (V)
5
a
0.784
0.806
0.894
0.821
0.674
0.833
0.696
0.690
0.653
0.690
0.552
0.690
0.740
0.748
0.773
0.755
0.613
0.761
0.088
0.116
0.241
0.131
0.122
0.143
5
b
5
c
5
d
5
5
e
f
Figure 3. Cyclic voltammograms of
methylallyl)quinolin-4(1H)-one (5b) at the glassy carbon electrode by a 0.1 V s scan rate in
.1 M dichloromethane solution of Bu NClO : (c) the first scan, (d) the second scan.
1
mM solution of 2-ferrocenyl-1-(2-
-1
0
4
4

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2
.4. Biology
We have screened the cytotoxic properties of 12 ferrocene-quinolinone derivatives in 2 model
human cancer cell lines (HeLa cervix carcinoma and K562 chronic myelogenous leukemia)
and in normal endothelial cells (HUVEC). As the control, cells treated with 1 % DMSO were
used. Initially, we measured cell viability after 48 h incubation with test compounds at the
concentration of 100 µM. Subsequently, for compounds that reduced the cell survival below
5
0% of the control, the IC50 values were calculated based on concentration dependent cell
viability experiments. To compare the cytotoxic activity of ferrocene-quinolinone derivatives,
IC values were calculated after 48 h incubation of both cancer and normal cells with test
5
0
compounds. Data presented in Table 4 indicate that the cytotoxic activity of test compounds
is rather moderate. This is evidenced by IC50 values in the range of 19 to 88 µM.
Interestingly, comparison of IC values suggest that K562 leukemia cells are more sensitive
5
0
to treatment with ferrocene-quinolinone derivatives than HeLa cells. Interestingly, our results
indicate that normal endothelial cells are the least sensitive to test compounds because only
compounds 4b and 5f displayed IC50 below 100 µM (80 µM and 72 µM, respectively).
Compound 5c showed the highest toxicity against HeLa cells (IC 28 µM), while 5f was the
5
0
most toxic against K562 cells (IC50 19 µM). As reported previously, ferrocene is rather
nontoxic in animals and cellular models [26]. In agreement with this report, we also haven’t
observed toxicity of ferrocene toward HeLa, K562 and normal human endothelial cells. In
cell lines used, IC of ferrocene was above 100 µM (Table 4). In general, data presented in
5
0
Table 4 demonstrate that ferrocene-quinolinone conjugates are more toxic toward cancer cells
than ferrocene alone. This is evidenced by lower IC50 values of compounds 4a, 4b, 5a, 5b,
5
c, 5d, 5e, 5f compared to ferrocene.
Table 4. Cytotoxicity of test compounds against HeLa, K562 and HUVEC cells
expressed as IC values [µM] after 48 h incubation. Mean values ± SD are shown
5
0
Compound
HeLa
K562
HUVEC
4
a
90±8.3
38±4.2
>100
33±6.8
22±5.6
>100
>100
80±4.8
>100
>100
>100
>100
>100
>100
4
b
4
c
4
d
>100
>100
4
e
>100
85±5.9
80±8.0
32±3.2
25±0.9
4
f
>100
5
a
88±7.2
70±7.4
5
b

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5
c
28±3.3
45±5.8
135±8.4
75±2.9
>100
56±4.8
25±4.1
42±3.4
19±0.5
>100
>100
>100
5
d
5
e
>100
5
f
72±8.3
>100
Ferrocene
Ferrocene and ferrocenium compounds contain iron atoms that exist at +2 or +3 oxidation
state, respectively. Several reports suggest that cytotoxicity of ferrocene/ferrocenium
derivatives results from reactive oxygen species formation (ROS), mainly superoxide anion.
ROS cause damage of DNA, proteins and cell membranes leading to cell death [27, 28]. To
explain the observed cytotoxicity of ferrocene-quinolinone derivatives, we measured the
relative amount of ROS generated in cells in the presence of the most toxic compounds. ROS
were quantified using DCFDA reporter dye in K562 cells incubated 4 hours with test
compounds at the concentration of 20, 50 or 100 µM. Data shown in Table 5 clearly indicate
that H O (positive control) used at 50 and 100 µM strongly induced formation of ROS 4-fold
2
2
and 9.5-fold, respectively. In the presence of ferrocene, we observed a concentration
dependent ROS induction, reaching 1.8-fold at 100 µM. Surprisingly, we didn’t observe any
induction of ROS in the presence of ferrocene-quinolinone derivatives. Moreover, some of
these compounds i.e. 4a, 4d, 4e, 5a, 5b inhibit the formation of ROS in K562 cells. This
inhibition is up to 40 % compared to control (K562 cells + 1% DMSO). Therefore, we
conclude that oxidative stress is not responsible for the observed cytotoxicity of ferrocene-
quinolinone derivatives toward K562 or HeLa cells. It has been reported that quinolone
derivatives are strong inhibitors of ROS formation in endothelial cells exposed to NADH [29,
3
0]. This observation can explain the lack of ROS induction or even inhibition of ROS
formation in the presence of ferrocene-quinolinone derivatives.
Table 5. ROS generation in K562 cells incubated with ferrocene-quinolinone derivatives (20,
5
0 or 100 µM). Mean fold change of DCF fluorescence intensity ± SD is shown.
Fluorescence intensity in control sample (DMSO) was taken as 1.
Compound/
DCF Fluorescence Intensity
concentration
2
0 µM
50 µM
100 µM
a
DMSO
H O
1 (±0.0)
Nd
b
c
4.0(±0.15)
9.5(±0.19)
2
2
4
a
0.8 (±0.04)
0.7 (±0.04)
0.8 (±0.02)
0.7 (±0.04)
0.8 (±0.02)
0.8 (±0.05)
0.7 (±0.09)
0.8 (±0.08)
0.7 (±0.06)
4b
4
e

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4
f
0.9 (±0.02)
0.7 (±0.05)
0.6 (±0.01)
0.9 (±0.01)
0.9 (±0.02)
0.9 (±0.02)
0.9 (±0.01)
0.9 (±0.02)
0.8 (±0.01)
0.9 (±0.02)
0.6 (±0.04)
0.8 (±0.03)
1.1 (±0.03)
1.0 (±0.02)
0.8 (±0.02)
1.0 (±0.04)
1.8 (±0.06)
5
a
0.6 (±0.03)
0.7 (±0.01)
1.0 (±0.04)
1.0 (±0.01)
0.8 (±0.05)
0.8 (±0.03)
1.2 (±0.02)
5b
5
c
5d
5
e
5
f
Ferrocene
a)
b)
c)
d)
Control (K562 + 1% DMSO)
Positive control (K562 + H O 50 µM)
2
2
Positive control (K562 + H O 100 µM)
2
2
nd – not determined
2
.5. Conclusion
In summary, the novel 1-allyl-ferrocenyl-2,3-dihydroquinolin-4(1H)-ones and 1-allyl-
-ferrocenylquinolin-4(1H)-ones were prepared. The structures of all new compounds were
2
determined by suitable spectroscopic methods. Furthermore, electrochemical properties of
synthesized compounds were examined by cyclic voltammetry.
The prepared ferrocene derivatives were tested in 2 model human cancer cell lines
(HeLa cervix carcinoma and K562 chronic myelogenous leukemia) and in normal endothelial
cells (HUVEC). These compounds showed better cytotoxic activity on the K562 cell line than
on the HeLa cell line for all concentrations and incubation times. The quinolinone derivative
5
c has showed the highest toxicity against HeLa cells (IC 28 µM) while 5f was the most
50
toxic against K562 cells (IC 19 µM). The normal endothelial cells were the least sensitive
5
0
to test compounds. In addition, our cytotoxicity studies (Table 4) indicate that ferrocene-
quinolinone derivatives have better anticancer activity than ferrocene.
3
. Experimental
3
.1. General information
All chemicals were commercially available and used as received, except that the solvents
were purified by distillation. Chromatographic separations were carried out using silica gel
0 (Merck, 230–400 mesh ASTM), whereas silica gel 60 on Al plates, layer thickness 0.2
6
mm (Merck) was used for TLC. Electrochemical measurements were performed at room
temperature in a three-electrode cell using an Autolab potentiostat (PGSTAT 302N). A

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standard three-electrode cell (5 mL) equipped with a platinum wire and a silver wire
immersed in 0.1 M Bu NClO solution in CH Cl as the counter and reference electrode,
4
4
2
2
respectively. A glassy carbon (d = 2 mm) was used as the working electrode. Melting points
uncorrected) were determined on a Mel-Temp capillary melting points apparatus, model
(
1
13
IA9100. The H and C NMR spectra of the samples in CDCl were recorded on a Varian
3
Gemini (200 MHz) NMR spectrometer. Chemical shifts are expressed in δ (ppm), relative to
1
13
residual solvent protons as an internal standard (CDCl : 7.26 ppm for H and 77 ppm for C).
3
The IR measurements were carried out with a Nexus Nicolet FTIR spectrophotometer.
Ultrasonic cleaner Sonic-3, 2 x 160W was used for the ultrasonically supported syntheses.
3
.2. Cells and cytotoxicity assay
The HeLa (human cervix carcinoma) and K562 (leukemia), cells were cultured in RPMI
1
640 (HeLa) medium supplemented with antibiotics and 10 % fetal calf serum (HeLa, K562)
3
in a 5 % CO - 95 % air atmosphere. 7x10 cells were seeded on each well on 96-well plate
2
(Nunc). 24h later cells were exposed to the test compounds for additional 48 hours. Stock
solutions of test compounds were freshly prepared in DMSO. The final concentrations of the
-1
-1
-2
-2
compounds tested in the cell cultures were: 2x10 mM, 1x10 mM, 5x10 mM, 1x10 mM,
-3
-4
1
x10 mM, 1x10 mM. The concentration of DMSO in the cell culture medium was 1%.
Human umbilical vein endothelial cells (HUVEC) were from Life Technologies and
cultured in Medium 200 with low serum growth supplement (Life Technologies) according to
4
manufacturer’s instructions. 10 cells were seeded on each well on 96-well plate (Nunc).
The values of IC (the concentration of test compound required to reduce the cell survival
5
0
fraction to 50 % of the control) were calculated from dose-response curves and used as a
measure of cellular sensitivity to a test compound.
The cytotoxicity of compounds was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-
2
,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO] assay as described [31]. Briefly,
after 48 h of incubation with drugs, the cells were treated with the MTT reagent and
incubation was continued for 2 h. MTT-formazan crystals were dissolved in a buffer
containing 20 % SDS and 50 % DMF (pH 4.7) and absorbance was read at 570 and 650 nm
on an ELISA-PLATE READER (FLUOstar Omega). As a control (100 % viability), we used
cells grown in the presence of 1% DMSO (vehicle).

ACCEPTED MANUSCRIPT
3
.3. Reactive oxygen species (ROS) measurement
Intracellular ROS levels were assessed with a 2,7-dichlorofluorescein diacetate (DCFDA,
Sigma-Aldrich) assay. DCFDA diffuses to the cells and in the presence of ROS can be
oxidized to a highly fluorescent 2',7'–dichlorofluorescein (DCF). DCF can be easily detected
by fluorescence spectroscopy with excitation and emission wavelengths of 485 nm and 520
nm, respectively.
K562 cells were cultured in RPMI 1640 medium supplemented with antibiotics and 10%
fetal calf serum in a 5% CO2 - 95% air atmosphere. 105 cells were stained with 20 µM
DCFDA in the culture media and incubated for 30 min at 37 °C. After staining cells were
centrifuged (700 rpm, 6 min) and suspended in a complete medium without phenol red. 105
cells per well were seeded on a 96-well plate (Perkin-Elmer). Test compounds (end
concentration in the cell culture 20 µM, 50 µM and 100 µM) were dissolved in DMSO and
added to cells and incubated for 4 h. The concentration of DMSO in the cell culture medium
was 1%. Cells treated with 50 µM and 100 µM H O served as the positive control. After
2
2
incubation, fluorescence in each well was measured (exc 485 nm, em 520 nm) with
FLUOStar Omega microplate reader (BMG-Labtech, Germany). DCF fluorescence intensity
in the control cells (exposed to 1 % DMSO) was taken as 1.0.
3
.4. Ferrocenecarboxaldehyde and 1-(2-(allylamino)phenyl)ethanones (2a–f)
Ferrocenecarboxaldehyde and 2a is known and their spectral data are already described
[
24, 31]. Spectra of the unknown acetophenones (2b–f) are listed below.
1
-(2-(2-Methylallylamino)phenyl)ethanone (2b). Yield (54 %); IR (KBr): ν = 3317, 2917,
-1 1
1
636, 1570, 1518, 1417, 746 cm ; H NMR (200 MHz, CDCl , ppm): δ = 9.11 (s, 1H), 7.73
3
(
4
dd, J = 8.0, 1.4 Hz, 1H), 7.35 – 7.23 (m, 1H), 6.65 – 6.52 (m, 2H), 4.93 (d, J = 1.3 Hz, 1H),
.87 (d, J = 1.3 Hz, 1H), 3.76 (d, J = 5.8 Hz, 2H), 2.57 (s, 3H), 1.77 (s, 3H); C NMR (50
1
3
MHz, CDCl , ppm): δ = 199.6, 149.8, 140.2, 133.6, 131.4, 116.2, 112.8, 110.8, 109.7, 47.2,
3
2
6.7, 19.1.
1
3
8
5
-(2-(Allylamino)-5-chlorophenyl)ethanone (2c). Yield (58 %), m.p. 42 °C; IR (KBr): ν =
-1 1
317, 3072, 2926, 1640, 1565, 1511, 1417, 812 cm ; H NMR (200 MHz, CDCl , ppm): δ =
3
.96 (s, 1H), 7.66 (d, J = 2.5 Hz, 1H), 7.24 (dd, J = 9.1, 2.5 Hz, 1H), 6.60 (d, J = 9.1 Hz, 1H),
.88 (ddt, J = 17.2, 10.0, 4.9 Hz, 1H), 5.23 (dq, = 17.2, 1.6 Hz, 1H), 5.16 (dq, J = 10.0, 1.6
1
3
Hz, 1H), 3.84 (dt, J = 4.9, 1.6 Hz, 2H), 2.54 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ =
3
1
99.3, 148.1, 133.5, 132.6, 130.4, 117.3, 116.9, 115.0, 112.4, 43.7, 26.7.

ACCEPTED MANUSCRIPT
1
3
9
8
1
1
-(5-Chloro-2-(2-methylallylamino)phenyl)ethanone (2d). Yield (51 %); IR (KBr): ν =
-1 1
441, 3087, 2916, 1652, 1599, 1512, 1411, 821 cm ; H NMR (200 MHz, CDCl , ppm): δ =
3
.32 (t, J = 5.4 Hz, 1H), 7.66 (d, J = 2.4 Hz, 1H), 7.19 (dd, J = 8.9, 2.4 Hz, 1H), 6.63 (d, J =
.9 Hz, 1H), 5.07 – 4.99 (m, 1H), 4.96 – 4.89 (m, 1H), 3.79 (d, J = 5.4 Hz, 2H), 2.67 (s, 3H),
13
.83 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ 156.7, 129.3, 129.1, 129.0, 127.8, 121.5,
3
20.1, 111.8, 109.5, 48.0, 119.5, 117.7.
1
3
8
5
1
1
-(2-(Allylamino)-5-bromophenyl)ethanone (2e). Yield (56 %), m.p. 62 °C; IR (KBr): ν =
-1 1
281, 3082, 2912, 1636, 1564, 1507, 1418, 805 cm ; H NMR (200 MHz, CDCl , ppm): δ
3
.97 (s, 1H), 7.79 (d, J = 2.4 Hz, 1H), 7.35 (dd, J = 9.1, 2.4 Hz, 1H), 6.54 (d, J = 9.1 Hz, 1H),
.88 (ddt, J = 17.2, 10.0, 4.9 Hz, 1H), 5.23 (dq, = 17.2, 1.6 Hz, 1H), 5.16 (dq, = 10.0, 1.6 Hz,
1
3
H), 3.83 (dt, J = 4.9, 1.6 Hz, 2H), 2.54 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ = 198.6,
3
48.4, 136.1, 133.4, 132.5, 117.6, 115.0, 112.8, 104.0, 43.7, 26.7.
1
-(5-Bromo-2-(2-methylallylamino)phenyl)ethanone (2f). Yield (50 %), m.p. 37 °C; IR
-1 1
(KBr): ν = 3310, 3079, 2914, 1636, 1559, 1511, 1418, 812 cm ; H NMR (200 MHz, CDCl ,
3
ppm): δ = 9.07 (s, 1H), 7.79 (d, J = 2.3 Hz, 1H), 7.34 (dd, J = 9.1, 2.3 Hz, 1H), 6.51 (d, J =
.1 Hz, 1H), 4.87 (d, J = 1.3 Hz, 1H), 4.86 (d, J = 1.3 Hz, 1H), 3.73 (d, J = 5.8 Hz, 2H), 2.54
9
1
3
(s, 3H), 1.74 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ = 198.6, 148.6, 139.7, 136.1,
3
1
33.3, 117.5, 112.9, 109.9, 103.9, 47.2, 26.7, 19.0.
3
.5. 1-(2-(Allylamino)phenyl)-3-ferrocenylprop-2-en-1-ones (3a–f)
1
-(2-(Allylamino)phenyl)-3-ferrocenylprop-2-en-1-ones
(3a–f)
were
synthesized
according to previously described procedure [32]. The all synthesized chalcones are unknown
and their spectral data are described below.
1
3
-(2-(Allylamino)phenyl)-3-ferrocenylprop-2-en-1-one (3a). Yield (94%); IR (KBr): ν =
437, 3002, 2922, 1636, 1607, 1570, 1514, 1204, 748 cm ; H NMR (200 MHz, CDCl ,
3
-1
1
ppm): δ = 9.20 (t, J = 4.7 Hz, 1H), 7.85 (dd, J = 8.1, 1.3 Hz, 1H), 7.65 (d, J = 15.2 Hz, 1H),
7
6
1
4
1
4
.35 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 7.25 (d, J = 15.2 Hz, 1H), 6.70 (dd, J = 8.5, 1.0 Hz, 1H),
.63 (ddd, J = 8.1, 7.2, 1.0 Hz, 1H), 5.97 (ddt, J = 17.2, 10.1, 5.0 Hz, 1H), 5.32 (dq, J = 17.2,
.6 Hz, 1H), 5.19 (dq, J = 10.3, 1.6 Hz, 1H), 4.56 (t, J = 1.8 Hz, 2H), 4.43 (t, J = 1.8 Hz, 2H),
1
3
.16 (s, 5H), 3.89 (dt, J = 5.0, 1.6 Hz, 2H); C NMR (50 MHz, CDCl , ppm): δ = 189.8,
3
50.3, 142.8, 133.3, 133.1, 130.0, 118.8, 117.2, 114.9, 112.9, 110.8, 78.5, 69.7, 68.4, 67.5,
3.9.
3
-Ferrocenyl-1-(2-(2-methylallylamino)phenyl)prop-2-en-1-one (3b). Yield (84%), m.p.
-1 1
8
8 °C; IR (KBr): ν = 3277, 3080, 2911, 1653, 1605, 1569, 1517, 1201, 746 cm ; H NMR
(
200 MHz, CDCl , ppm): δ = 9.31 (t, J = 5.7 Hz, 1H), 7.85 (dd, J = 8.1, 1.5 Hz, 1H), 7.65 (d,
3
J = 15.2 Hz, 1H), 7.33 (ddd, J = 8.5, 7.2, 1.5 Hz, 1H), 7.26 (d, J = 15.2 Hz, 1H), 6.67 (dd, J =
8
1
.5, 1.0 Hz, 1H), 6.62 (ddd, J = 8.1, 7.2, 1.0 Hz, 1H), 4.99 (d, J = 1.4 Hz, 1H), 4.90 (d, J =
.4 Hz, 1H), 4.57 (t, J = 1.8 Hz, 2H), 4.43 (t, J = 1.8 Hz, 2H), 4.16 (s, 5H), 3.79 (d, J = 5.7

ACCEPTED MANUSCRIPT
1
3
Hz, 2H), 1.80 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ = 189.8, 150.6, 142.7, 140.3,
3
1
33.2, 130.0, 118.8, 117.1, 112.9, 110.9, 109.8, 78.5, 69.7, 68.4, 67.5, 47.5, 19.2.
1
-(2-(Allylamino)-5-chlorophenyl)-3-ferrocenylprop-2-en-1-one (3c). Yield (74%); IR
-1 1
(KBr): ν = 3477, 2924, 2854, 1640, 1465, 1363, 1249, 1080, 821 cm ; H NMR (200 MHz,
CDCl , ppm): δ = 9.14 (t, J = 4.8 Hz, 1H), 7.77 (d, J = 2.5 Hz, 1H), 7.67 (d, J = 15.1 Hz, 1H),
3
7
=
.27 (dd, J = 9.1, 2.5 Hz, 1H), 7.14 (d, J = 15.1 Hz, 1H), 6.63 (d, J = 9.1 Hz, 1H), 5.93 (ddt, J
17.3, 10.2, 5.0 Hz, 1H), 5.28 (dq, J = 17.3, 1.6 Hz, 1H), 5.19 (dq, J = 10.4, 1.6 Hz, 1H),
4
.58 (t, J = 1.8 Hz, 2H), 4.46 (t, J = 1.8 Hz, 2H), 4.17 (s, 5H), 3.87 (dt, J = 5.0, 1.6 Hz, 2H);
1
3
C NMR (50 MHz, CDCl , ppm): δ = 188.8, 148.8, 144.1, 133.0, 132.7, 129.1, 117.9, 117.4,
3
1
15.1, 112.7, 112.3, 78.2, 70.0, 68.5, 67.7, 44.0.
1
-(5-Chloro-2-(2-methylallylamino)phenyl)-3-ferrocenylprop-2-en-1-one (3d). Yield
-1 1
(67%); IR (KBr): ν = 3437, 3088, 2917, 1638, 1568, 1511, 1190, 810 cm ; H NMR (200
MHz, CDCl , ppm): δ 9.25 (t, J = 5.6 Hz, 1H), 7.78 – 7.62 (m, 2H), 7.30 – 7.08 (m, 2H), 6.66
3
–
4
6.51 (m, 1H), 4.95 (d, J = 1.2 Hz, 1H), 4.89 (d, J = 1.2 Hz, 1H), 4.57 (t, J = 1.8 Hz, 2H),
.46 (t, J = 1.8 Hz, 2H), 4.16 (s, 5H), 3.76 (d, J = 5.6 Hz, 2H), 1.78 (s, 3H); C NMR (50
1
3
MHz, CDCl , ppm): δ = 189.1, 149.3, 144.3, 133.2, 129.9, 129.3, 127.8, 118.2, 117.6, 112.7,
3
1
10.2, 78.4, 70.2, 68.7, 67.9, 47.8, 19.4.
1
-(2-(Allylamino)-5-bromophenyl)-3-ferrocenylprop-2-en-1-one (3e). Yield (85%); m.p.
-1 1
9
1 °C; IR (KBr): ν = 3281, 3083, 2840, 1634, 1598, 1562, 1505, 1188, 803 cm . H NMR
(
200 MHz, CDCl , ppm): δ = 9.15 (t, J = 4.8 Hz, 1H), 7.90 (d, J = 2.2 Hz, 1H), 7.66 (d, J =
3
1
1
1
1
1
5.1 Hz, 1H), 7.38 (dd, J = 9.1, 2.2 Hz, 1H), 7.12 (d, J = 15.1 Hz, 1H), 6.58 (d, J = 9.1 Hz,
H), 5.93 (ddt, J = 17.2, 10.1, 5.0 Hz, 1H), 5.28 (dq, J = 17.2, 1.5 Hz, 1H), 5.18 (dq, J = 10.3,
.5 Hz, 1H), 4.58 (t, J = 1.8 Hz, 2H), 4.46 (t, J = 1.8 Hz, 2H), 4.17 (s, 5H), 3.86 (dt, J = 5.0,
1
3
.5 Hz, 2H); C NMR (50 MHz, CDCl , ppm): δ = 188.8, 149.1, 144.2, 135.6, 132.6, 132.0,
3
29.6, 117.9, 115.1, 112.7, 104.2, 78.2, 70.0, 68.5, 67.7, 43.9.
1
-(5-Bromo-2-(2-methylallylamino)phenyl)-3-ferrocenylprop-2-en-1-one (3f). Yield
-
(
94%), m.p. 121 °C; IR (KBr): ν = 3280, 3082, 2912, 1636, 1598, 1563, 1507, 1188, 805 cm
1
1
;
H NMR (200 MHz, CDCl , ppm): δ = 9.26 (t, J = 5.8 Hz, 1H), 7.90 (d, J = 2.3 Hz, 1H),
3
7
.66 (d, J = 15.1 Hz, 1H), 7.36 (dd, J = 9.1, 2.3 Hz, 1H), 7.13 (d, J = 15.1 Hz, 1H), 6.55 (d, J
=
9.1 Hz, 1H), 4.95 (d, J = 1.6 Hz, 1H), 4.90 (d, J = 1.6 Hz, 1H), 4.58 (t, J = 1.7 Hz, 2H),
1
3
4
.46 (t, J = 1.7 Hz, 2H), 4.17 (s, 5H), 3.76 (d, J = 5.8 Hz, 2H), 1.78 (s, 3H); C NMR (50
MHz, CDCl , ppm): δ = 188.8, 149.3, 144.1, 139.9, 135.6, 132.0, 117.9, 112.9, 110.1, 110.0,
3
1
04.2, 78.2, 70.0, 68.5, 67.7, 47.5, 19.1.
3
4
.6. General procedure for the preparation of 1-allyl-2-ferrocenyl-2,3-dihydroquinolin-
(1H)-ones (4a–f) and 1-allyl-2-ferrocenylquinolin-4(1H)-ones (5a–f)
The solution of ferrocenyl chalcones 3a–f (1 mmol) in glacial acetic (3 mL) acid and
orthophosphoric acid (3 mL) was irradiated for 50 minutes in ultrasonic cleaner. The reaction
mixture was poured into ice–water mixture, extracted with ethyl acetate (three 20 mL

ACCEPTED MANUSCRIPT
portions), the obtained solution washed with NaHCO , and dried overnight (anhydrous
3
Na SO ). The solvent was evaporated, and the residue chromatographed over a short pad of
2
4
SiO (hexane/ethyl acetate 9:1, v/v). After the evaporation of the solvent, the obtained
2
products (dihydroquinolones 4a–f) were dissolved in anhydrous dichloromethane (20 mL) to
which was added DDQ (1.2 mmol). The solution first stirred 30 minutes at room temperature
and then was brought to reflux over the night. After cooling, the solution was added to ice
water (50 mL), and 20% Na S O solution (10 mL) was added. The resulting yellow brown
2
2
5
precipitate was extracted with dichloromethane (three 20 mL portions), the dichloromethane
layer dried (anhydrous Na SO ), and the solvent removed under reduced pressure to give the
2
4
crude product (5a–f). The residue was purified by column chromatography (SiO , ethyl
2
acetate/methanol 9:1, v/v).
1
-Allyl-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-one (4a). Yield (80%), m.p. 108 °C; IR
-1 1
(KBr): ν = 3081, 2964, 1664, 1643, 1600, 1488, 1224, 813 cm ; H NMR (200 MHz, CDCl ,
3
ppm) δ = 7.88 (dd, J = 7.8, 1.7 Hz, 1H), 7.31 – 7.22 (m, 1H), 6.69 – 6.58 (m, 1H), 6.58 – 6.46
m, 1H), 5.80 (ddt, J = 17.2, 9.7, 4.8 Hz, 1H), 5.24 (dq, J = 17.2, 1.5 Hz, 1H), 5.17 (dq, J =
(
9
=
.7, 1.5 Hz, 1H), 4.50 (dd, J = 5.7, 4.7 Hz, 1H), 4.30 – 3.64 (m, 6H), 4.15 (s, 5H), 3.25 (dd, J
16.3, 5.7 Hz, 1H), 3.14 (dd, J = 16.3, 4.7 Hz, 1H); C NMR (50 MHz, CDCl , ppm): δ =
3
1
3
1
6
92.1, 148.0, 134.3, 132.4, 126.3, 118.2, 115.3, 115.0, 112.3, 85.6, 67.6, 67.4, 67.3, 66.3,
5.4, 56.8, 50.5, 42.6.
2
-Ferrocenyl-1-(2-methylallyl)-2,3-dihydroquinolin-4(1H)-one (4b). Yield (94%), m.p. 98
-1
1
°
C; IR (KBr): ν = 3079, 2887, 1672, 1600, 1484, 1222, 817 cm ; H NMR (200 MHz,
CDCl , ppm): δ = 7.88 (dd, J = 7.8, 1.4 Hz, 1H), 7.25 (ddd, J = 8.6, 7.4, 1.4 Hz, 1H), 6.62
3
(dd, J = 7.8, 7.4 Hz, 1H), 6.46 (d, J = 8.6 Hz, 1H), 4.91 (s, 2H), 4.49 (dd, J = 5.7, 4.5 Hz,
1
H), 4.19 – 3.64 (m, 6H), 4.15(s, 5H), 3.24 (dd, J = 16.2, 5.7 Hz, 1H), 3.14 (dd, J = 16.2, 4.5
13
Hz, 1H), 1.75 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ = 192.1, 148.2, 139.0, 134.3,
3
1
26.2, 118.0, 114.9, 112.2, 110.3, 85.4, 68.2, 67.6, 67.3, 66.3, 65.4, 56.8, 53.8, 42.5, 18.8.
1
-Allyl-6-chloro-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-one (4c). Yield (70%), m.p. 140
1
°
C; IR (KBr): ν = 3085, 2922, 1668, 1599, 1485, 1210, 817; H NMR (400 MHz, CDCl ,
3
ppm): δ = 7.83 (d, J = 2.7 Hz, 1H), 7.18 (dd, J = 9.1, 2.7 Hz, 1H), 6.47 (d, J = 9.1 Hz, 1H),
.76 (ddt, J = 17.4, 9.7, 4.7 Hz, 1H), 5.23 (dq, J = 17.4, 1.4 Hz, 1H), 5.16 (dq, J = 9.7, 1.4
Hz, 1H), 4.50 (dd, J = 5.5, 4.9 Hz, 1H), 4.15 – 3.75 (m, 6H), 4.14 (s, 5H), 3.24 (dd, J = 16.4,
5
1
3
5
1
4
.5 Hz, 1H), 3.13 (dd, J = 16.4, 4.9 Hz, 1H); C NMR (50 MHz, CDCl , ppm): δ = 191.0,
3
46.4, 134.0, 131.9, 125.5, 120.6, 118.8, 115.6, 114.1, 85.1, 67.6, 67.4, 66.5, 65.3, 56.9, 50.7,
2.5.
6
-Chloro-2-ferrocenyl-1-(2-methylallyl)-2,3-dihydroquinolin-4(1H)-one
(4d).
Yield
-
1 1
(90%), m.p. 150 °C; IR (KBr): ν = 3080, 2839, 1670, 1599, 1490, 1209, 817 cm ; H NMR
(
200 MHz, CDCl , ppm): δ = 7.82 (d, J = 2.7 Hz, 1H), 7.17 (dd, J = 9.1, 2.7 Hz, 1H), 6.40 (d,
3
J = 9.1 Hz, 1H), 4.91 (d, J = 1.2 Hz, 1H), 4.88 (d, J = 1.2 Hz, 1H), 4.49 (dd, J = 5.5, 4.8 Hz,
H), 4.15 – 3.57 (m, 6H), 4.14 (s, 5H), 3.23 (dd, J = 16.3, 5.5 Hz, 1H), 3.14 (dd, J = 16.3, 4.8
1
1
3
Hz, 1H), 1.73 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ = 191.0, 146.6, 138.6, 134.0,
3
1
25.4, 120.5, 118.7, 114.0, 110.5, 85.0, 67.6, 67.4, 67.2, 66.5, 65.3, 57.0, 54.0, 42.3, 18.8.

ACCEPTED MANUSCRIPT
1
-Allyl-6-bromo-2-ferrocenyl-2,3-dihydroquinolin-4(1H)-one (4e). Yield (96%), m.p. 122
-1
1
°
C; IR (KBr): ν = 3084, 2834, 1669, 1594, 1422, 1210, 819 cm ; H NMR (200 MHz,
CDCl , ppm): δ = 7.97 (d, J = 2.5 Hz, 1H), 7.31 (dd, J = 9.1, 2.5 Hz, 1H), 6.42 (d, J = 9.1 Hz,
3
1
1
1
1
6
H), 5.76 (ddt, J = 14.4, 9.4, 4.6 Hz, 1H), 5.22 (dq, J = 14.4, 1.5 Hz, 1H), 5.16 (dq, J = 9.4,
.5 Hz, 1H), 4.50 (dd, J = 5.5, 4.9 Hz, 1H), 4.15 – 3.79 (m, 6H), 4.15 (s, 5H), 3.27 (d, J =
1
3
6.4, 5.5 Hz, 1H), 3.13 (dd, J = 16.4, 4.9 Hz, 1H); C NMR (50 MHz, CDCl , ppm): δ =
3
90.9, 187.8, 146.7, 136.6, 131.8, 128.6, 119.3, 115.6, 114.5, 107.6, 85.1, 67.6, 67.4, 67.4,
6.5, 65.3, 56.9, 50.6, 42.4.
6
-Bromo-2-ferrocenyl-1-(2-methylallyl)-2,3-dihydroquinolin-4(1H)-one
(4f).
Yield
-
1 1
(68%), m.p. 158 °C; IR (KBr): ν = 3079, 2837, 1672, 1594, 1421, 1210, 816 cm ; H NMR
(
200 MHz, CDCl , ppm): δ = 7.96 (d, J = 2.5 Hz, 1H), 7.29 (dd, J = 9.1, 2.5 Hz, 1H), 6.35 (d,
3
J = 9.1 Hz, 1H), 4.91 (d, J = 1.4 Hz, 1H), 4.87 (dd, d, J = 1.4 Hz, 1H), 4.48 (dd, J = 5.6, 4.7
Hz, 1H), 4.16 – 4.00 (m, 4H), 4.14 (s, 5H), 3.88 – 3.63 (m, 2H), 3.23 (d, J = 16.4, 5.6 Hz,
1
3
2
1
6
H), 3.12 (d, J = 16.4, 4.7 Hz, 2H), 1.73 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ =
3
90.8, 146.9, 138.5, 136.6, 128.5, 119.2, 114.4, 110.5, 107.5, 85.0, 67.6, 67.4, 67.2, 66.5,
5.3, 56.9, 54.0, 42.2, 18.8.
1
3
8
-Allyl-2-ferrocenylquinolin-4(1H)-ones (5a). Yield (73%), m.p. 141 °C; IR (KBr): ν =
-1 1
059, 3010, 1620, 1597, 1566, 1472, 1270, 818 cm ; H NMR (200 MHz, CDCl , ppm): δ =
3
.44 (dd, J = 7.9, 1.0 Hz, 1H), 7.57 (ddd, J = 8.2, 7.2, 1.0 Hz, 1H), 7.42 – 7.27 (m, 2H), 6.99
(s, 1H), 5.97 (ddt, J = 17.3, 10.5, 3.5 Hz, 1H), 5.31 (d, J = 10.5 Hz, 1H), 5.01 (d, J = 17.3 Hz,
1
H), 4.76 (d, J = 3.5 Hz, 2H), 4.61 (t, J = 1.5 Hz, 2H), 4.37 (t, J = 1.5 Hz, 2H), 4.24 (s, 5H);
1
3
C NMR (50 MHz, CDCl , ppm): δ = 176.1, 152.3, 140.2, 131.9, 130.7, 125.6, 125.2, 122.2,
3
1
16.2, 115.7, 112.7, 81.9, 81.2, 69.5, 69.2, 67.9, 49.7.
2
-Ferrocenyl-1-(2-methylallyl)quinolin-4(1H)-one (5b). Yield (77%), m.p. 87 °C; IR
-1 1
(KBr): ν = 3078, 2966, 1617, 1596, 1541, 1488, 1270, 819 cm ; H NMR (200 MHz, CDCl ,
3
ppm): δ = 8.43 (dd, J = 7.9, 1.0 Hz, 1H), 7.56 (ddd, J = 8.3, 7.2, 1.0 Hz, 1H), 7.37 – 7.22 (m,
2
1
1
H), 7.00 (s, 1H), 5.02 (s, 1H), 4.77 –4.42 (m, 5H), 4.37 (t, J = 1.6 Hz, 2H), 4.22 (s, 5H),
1
3
.77 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ = 176.1, 152.6, 140.3, 138.9, 130.6, 125.4,
3
25.0, 122.3, 115.8, 112.4, 111.1, 80.7, 69.2, 68.7, 68.0, 53.1, 18.8.
1
-Allyl-6-chloro-2-ferrocenylquinolin-4(1H)-one (5c). Yield (74%), m.p. 188 °C; IR
-1 1
(KBr): ν = 3100, 2924, 1710, 1622, 1589, 1545, 1478, 1294, 812 cm ; H NMR (200 MHz,
CDCl , ppm): δ = 8.38 (d, J = 2.5 Hz, 1H), 7.48 (dd, J = 9.2, 2.5 Hz, 1H), 7.32 (d, J = 9.2 Hz,
3
1
H), 6.97 (s, 1H), 5.95 (ddt, J = 17.7, 10.7, 3.7 Hz, 1H), 5.32 (dt, J = 10.7, 1.8 Hz, 1H), 4.99
(
(
dt, J = 17.7, 2.0 Hz, 1H), 4.74 (ddd, J = 3.7, 2.0, 1.8 Hz, 2H), 4.59 (t, J = 1.8 Hz, 2H), 4.38
13
t, J = 1.8 Hz, 2H), 4.23 (s, 5H); C NMR (50 MHz, CDCl , ppm): δ = 174.8, 152.6, 138.6,
3
1
31.6, 130.8, 128.5, 126.7, 124.4, 117.6, 116.4, 112.9, 80.8, 69.5, 69.2, 68.0, 49.9.
6
=
=
-Chloro-2-ferrocenyl-1-(2-methylallyl)quinolin-4(1H)-one (5d). Yield (61%), IR (KBr): ν
-1 1
3045, 2922, 1618, 1587, 1564, 1445, 1282, 817 cm ; H NMR (200 MHz, CDCl , ppm): δ
3
8.66 – 8.19 (m, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.27 – 6.87 (m, 1H), 6.96 (s, 1H), 5.10 – 4.98
1
3
(m, 1H), 4.91 – 4.44 (m, 5H), 4.39 (m, 2H), 4.22 (s, 5H), 1.77 (s, 3H); C NMR (50 MHz,
CDCl , ppm): δ = 175.6, 152.9, 138.8, 132.4, 130.8, 129.2, 128.6, 126.0, 124.3, 117.8, 111.3,
3
8
0.4, 71.0, 69.9, 69.3, 68.2, 67.5, 53.2, 18.8.
1
-Allyl-6-bromo-2-ferrocenylquinolin-4(1H)-one (5e). Yield (76%), m.p. 168 °C; IR
-1; 1
(KBr): ν = 3056, 2921, 1617, 1589, 1566, 1541, 1466, 1293, 818 cm . H NMR (200 MHz,
CDCl , ppm): δ = 8.55 (d, J = 1.4 Hz, 1H), 7.62 (dd, J = 8.8, 1.4 Hz, 1H), 7.26 (d, J = 8.8 Hz,
3
1
H), 6.98 (s, 1H), 5.95 (ddt, J = 17.3, 10.6, 3.2 Hz, 1H), 5.33 (d, J = 10.6 Hz, 1H), 4.99 (d, J

ACCEPTED MANUSCRIPT
=
17.3 Hz, 1H), 4.74 (d, J = 3.2 Hz, 2H), 4.60 (t, J = 1.4 Hz, 2H), 4.39 (t, J = 1.4 Hz, 2H),
1
3
4
1
.24 (s, 5H); C NMR (50 MHz, CDCl , ppm): δ = 174.8, 152.6, 141.3, 139.0, 133.5, 131.5,
27.7, 117.8, 116.4, 116.2, 113.0, 80.8, 69.5, 69.2, 68.0, 49.8.
3
6
-Bromo-2-ferroceny1-(2-methylallyl)quinolin-4(1H)-one (5f). Yield (65%), m.p. 189 °C;
-1 1
IR (KBr): ν = 3074, 2934, 1616, 1587, 1538, 1461, 1293, 830 cm ; H NMR (200 MHz,
CDCl , ppm): δ = 8.55 (d, J = 2.3 Hz, 1H), 7.61 (dd, J = 9.1, 2.3 Hz, 1H), 7.13 (d, J = 9.1
3
yHz, 1H), 7.01 (s, 1H), 5.03 (s, 1H), 4.72 – 4.44 (m, 5H), 4.39 (t, J = 1.7 Hz, 2H), 4.22 (s,
1
3
5
1
H), 1.76 (s, 3H); C NMR (50 MHz, CDCl , ppm): δ = 174.7, 153.0, 139.1, 138.6, 133.5,
3
27.6, 126.8, 117.9, 116.2, 112.7, 111.3, 80.4, 69.4, 69.2, 68.2, 53.2, 18.7.
Acknowledgements
This work was supported by the Ministry of Education and Science of the Republic of
Serbia (grant 172034). Studies carried out in the Lodz laboratory was financially supported
by the National Science Center, Poland fund awarded based on the decision UMO-
2
012/06/A/ST5/00227 (MAESTRO to JD).
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Figure captions
Figure 1. Some bioactive ferrocene-containing quinolines
Figure 2. Cyclic voltammograms of
methylallyl)quinolin-4(1H)-one (4b) at the glassy carbon electrode by a 0.1 V s scan rate in
.1 M dichloromethane solution of Bu NClO : (a) the first scan, (b) the second scan.
1
mM solution of 2-ferrocenyl-1-(2-
-1
0
4
4
Figure 3. Cyclic voltammograms of
methylallyl)quinolin-4(1H)-one (5b) at the glassy carbon electrode by a 0.1 V s scan rate in
.1 M dichloromethane solution of Bu NClO : (c) the first scan, (d) the second scan.
1
mM solution of 2-ferrocenyl-1-(2-
-1
0
4
4

ACCEPTED MANUSCRIPT
Highlights
-
-
-
Two novel series of ferrocene containing quinolinones were synthesized and characterized
Their electrochemical behavior was examined using cyclic voltammetry
Cytotoxicity of all synthesized quinolinones was measured against HeLa, K562 and HUVEC
cell lines
All tested ferrocene compounds displayed the better activity against K562 cell lines
-