Synthesis, structure and cytotoxic activity of mono- and dialkoxy derivatives of 5,8-quinolinedione

Article abstract of DOI:10.3390/molecules21020156

A series of 5,8-quinolinedione derivatives containing one or two alkoxy groups was synthesized and characterized by ¹H- and ¹³C-NMR, IR and MS spectra. X-ray diffraction was used to investigate the crystal structures of 6-chloro-7-(2

Full text of DOI:10.3390/molecules21020156

molecules
Article
Synthesis, Structure and Cytotoxic Activity of Mono- and
Dialkoxy Derivatives of 5,8-Quinolinedione
Monika Kadela 1,*, Maria Jastrze˛bska 2,3, Ewa Be˛benek 1, Elwira Chrobak 1,
4
5
Małgorzata Latocha , Joachim Kusz , Maria Ksia˛zek 2 and Stanisław Boryczka 1
˙
1
Department of Organic Chemistry, School of Pharmacy with the Division of Laboratory Medicine in
Sosnowiec, Medical University of Silesia in Katowice, 4 Jagiellon´ska Str., 41-200 Sosnowiec, Poland;
ebebenek@sum.edu.pl (E.B.); echrobak@sum.edu.pl (E.C.); boryczka@sum.edu.pl (S.B.)
Department of Solid State Physics, Institute of Physics, University of Silesia, 4 Uniwersytecka Str.,
2
40-007 Katowice, Poland; maria.jastrzebska@us.edu.pl (M.J.); maria.ksiazek@us.edu.pl (M.K.)
Silesian Center for Education and Interdisciplinary Research, University of Silesia, 75 Pułku Piechoty 1,
3
41-500 Chorzów, Poland
4
Department of Cell Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec,
Medical University of Silesia in Katowice, 8 Jednos´ci Str., 41-200 Sosnowiec, Poland; mlatocha@sum.edu.pl
Department of Physics of Crystals, Institute of Physics, University of Silesia, 4 Uniwersytecka Str.,
5
40-007 Katowice, Poland; joachim.kusz@us.edu.pl
*
Correspondence: mkadela@sum.edu.pl; Tel.: +48-32-364-1666
Academic Editor: Annie Mayence
Received: 20 November 2015 ; Accepted: 11 December 2015 ; Published: 27 January 2016
Abstract: A series of 5,8-quinolinedione derivatives containing one or two alkoxy groups was
synthesized and characterized by 1H- and 13C-NMR, IR and MS spectra. X-ray diffraction
was used to investigate the crystal structures of 6-chloro-7-(2-cyjanoethoxy)-5,8-quinolinedione
and 6,7-di(2,2,2-trifloroethoxy)-5,8-quinolinedione. All studied compounds were tested in vitro
for their antiproliferative activity against three human cancer cell lines and human normal
fibroblasts. Most of the compounds showed higher cytotoxicity than the starting compound,
6,7-dichloro-5,8-quinolinedione, and cisplatin, which was used as a reference agent.
Keywords: alkoxy-5,8-quinolinedione; crystal structure; cytotoxic activity
1. Introduction
Cancer diseases are nowadays one of the most important health problems in the world. According
to a World Health Organization report published in 2012, more than 14.1 million new cancer cases
were diagnosed and about 8.2 million cancer-related deaths were recorded. The increase in cancer
incidents is most likely associated with aging of population and unhealthy lifestyle including physical
inactivity, smoking and poor diet.
Antibiotics containing the 5,8-quinolinedione moiety, like Streptonigrin, Lavendamycin and
Steptonigrone, are known for their high anticancer, antibacterial, and antiviral activities
(Figure 1) [2–4].
In the 1970s, the anticancer activity of the Streptonigrin against leukemia cell lines was recognized
to be one of great interest to researchers. However, because of its toxic side effects, a clinical trial
was ended in 1977 [5,6] The structure–activity relationship of 5,8-quinolinedione antibiotics have
been studied over the years. The research has shown that the most important part of the molecule
is the 5,8-quinolinedione moiety, which is responsible for inhibiting DNA and RNA synthesis, and
interfering with topoisomerase II action.
Moreover, 5,8-quinolinedione can be reduced by nicotinamide adenine dinucleotide
phosphate(NAD(P)H) as a cofactor to form semiquinone or hydroquinone intermediates [7–10].
Molecules 2016, 21, 156; doi:10.3390/molecules21020156
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These compounds can react with oxygen yielding a regenerated 5,8-quinolinedione fragment and
creating the hydroxyl radicals that are ultimately responsible for DNA strand cleavage. Furthermore,
it was also found that the introduction of amine, hydroxyl or thiol substituents at position 6 or 7 of the
5,8-quinolinedione moiety results in an enhanced biological activity [3,4,11–14].
(a)
(b)
(c)
Figure 1. Structures of 5,8-quinolinedione antibiotics: (
(c) Steptonigrone.
a
) Streptonigrin; (b) Lavendamycin; and
There are many reports on the synthesis and biological activity of the amine derivatives of
5,8-quinolinedione, whereas studies on the alkoxy analogues are rather scarce in the literature [9,15].
In the present work, we report the synthesis of mono- and disubstituted alkoxy derivatives
of 5,8-quinolinedione and the structural characterization of some alkoxy compounds. All newly
synthesized compounds were tested for cytotoxic activity against cancer cell lines such as human
melanoma (C-32), glioblastoma (SNB-19), and breast cancer (MDA-MB-231) as well as normal
fibroblasts (HFF-1) in order to obtain more information about the influence of one or two alkoxy
groups on the anticancer activity of the compounds under study.
2. Results and Discussion
2.1. Chemistry
The starting compound, 6,7-dichloro-5,8-quinolinedione
The chemical structure of
1, was obtained from 8-hydroxyquinoline.
1
was determined using the proton nuclear magnetic resonance (1H-NMR),
the carbon nuclear magnetic resonance (13C-NMR) and the infrared spectroscopy (IR). The band
assignment of the spectra was done using literature data [16].
As was previously described by Jastrze˛bska et al. [17], reaction of compound
1 with an amine
group carried out in tetrahydrofuran (THF) and in the presence of potassium carbonate, gives
two isomeric products. The major product of this is a compound containing an amino substituent at
the 7 position [17]. In this study, the alkoxy derivatives of 5,8-quinolinedione were prepared using
the same reaction conditions. The reactions of 6,7-dichloro-5,8-quinolinedione
performed in tetrahydrofuran in the presence of potassium carbonate giving products
1
with alcohols were
(Scheme 1).
After purification by column chromatography, derivatives 2–9 were obtained with yields of 53%–88%.
2–9
Structures of all new compounds were confirmed based on 1H-, 13C-NMR, IR, electron ionization mass
spectrometry (EI-MS) and high-resolution mass spectrometry (HR MS) methods.
In order to confirm the regioselectivity of the substitution reaction, 13C-NMR spectroscopy and
X-ray diffraction analyses were performed. According to literature data [16], the 13C-NMR spectra of
aliphatic amine derivatives show low intensity signals of C7, C6 and C8 atoms for the 6-substituted
isomer, while those of C5, C6 and C7 atoms are of low intensity for the 7-substituted derivative.
Therefore, the signal intensity of C5 and C8 atoms should be different depending on the position of the
substituent. For the described products 2–9, the signal of the C5 atom is of lower intensity than the

Molecules 2016, 21, 156
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signal of C8 atom. Therefore, it has been found that the compounds
2
–9
contain the alkoxy group at
the 7 position. Moreover, the X-ray diffraction analysis confirmed that 7 is a 7-substituted derivative.
2
3
4
5
6
7
R = CH3
R = CH2CH3
R = CH2CH2CH3
R = CH2CH CH2
R = CH2CH2Cl
R = CH2CH2CN
O
O
Cl
Cl
Cl
K2CO3, THF
t. pok., 3-24 h
+
ROH
N
N
OR
O
R =
8
9
O
1
2-9
R =
Scheme 1. Synthesis of the 7-substituted derivatives of 5,8-quinolinedione, 2–9.
In the literature [18], the nucleophilic substitution occurring at the C6 or C7 position was assumed
to follow the SN2 mechanism. It was found that the solvent plays an important role in the reaction of
6,7-dichloro-5,8-quinolinedione
1 with amines and arylthiols. The 7-substituted derivative was the
major product of the reaction when an aprotic solvent, like THF or dimethylformamide (DMF), was
used. The reaction carried out in protic solvent such as water, led to 6-substituted derivatives with
higher yields [17
a solvent [19].
,18], whereas 6,7-dithioarylo compounds were synthesized when ethanol was used as
In our work, dimethyl sulfoxide (DMSO) was used as a solvent in order to obtain 6,7-dialkoxy
derivatives. Treatment of 6,7-dichloro-5,8-quinolinedione with alcohols in DMSO in the presence of
potassium carbonate gave 6,7-disubstituted products 10–18 with the good yields (Scheme 2).
1
10
11
12
13
14
15
R = CH3
R = CH2CH3
R = CH2CH2CH3
R = CH2CH CH2
R = CH2CH2Cl
R = CH2CF3
O
O
Cl
Cl
OR
K2CO3, DMSO
t. pok., 3-24 h
+
ROH
N
N
OR 16
R = CH2CH2CN
O
O
17 R =
1
10-18
18
R =
Scheme 2. Synthesis of the 6,7-disubstituted derivatives of 5,8-quinolinedione, 10–18.
Structures of all new compounds 10–
18 were confirmed by 1H-, 13C-NMR, IR and MS spectra.
In addition, the crystal structure of the derivative 15 was determined by the X-ray diffraction analysis.
Formation of the disubstituted products in this reaction can be explained by the influence of the
solvent on the alcohol nucleophilicity. In the presence of the dimethyl sulfoxide, the DMSO-alcohol
complex can be formed via the hydrogen bond (Scheme 3) [20,21].
CH3
CH3
O
+
R OH
R OH O
S
H3C
CH3
Scheme 3. Forming the hydrogen bond between alcohol molecule and DMSO.
Formation of the H-bond leads to an increase in the nucleophilic character of the alcohol molecule,
enabling the substitution of two chlorine atoms into the 5,8-quinolinedione moiety. In tetrahydrofuran,

Molecules 2016, 21, 156
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the interaction between solvent and alcohol molecule does not occur, giving the monoalkoxy derivative
as the product of the reaction.
2.2. Crystal Structure
X-ray diffraction analysis was performed for monocrystals of the following compounds:
6-chloro-7-cyanoethoxy-5,8-quinolinedione
7 and 6,7-di(2,2,2-trifluoroethoxy)-5,8-quinolinedione 15.
Both crystals were grown from dichloromethane. Figure 2 presents the molecular structure and atom
numbering of the crystals. The derivatives
7 and 15 were crystallized in different monoclinic space
groups, C 2/c and P21/n, respectively.
Figure 2. Molecular structures with the atom numbering of (a) 6-chloro-7-(2-cyanoethoxy)-5,8-
quinolinedione and ( ) 6,7-di(2,2,2-trifluoroethoxy)-5,8-quinolinedione 15. Displacement ellipsoids
are shown at the 50% probability level. H atoms are shown as small spheres of arbitrary radii.
7
b
The unit cell of
7 contains eight molecules (Table S1). The angle between mean planes of
rings and the 2-cyanoethoxy chain O3-C9-C10 is equal to 33.36˝, while a similar angle in the
crystal structures of 6-(7-chloro-6-(4-methoxyphenyl)-5,8-quinolinedione [15], 6-chloro-7-propylamine
˝
˝
-5,8-quinolinedione, and 7-chloro-6-propylamine-5,8-quinolinedione [17] were equal to 82.03 , 89.77 ,
and 81.79˝, respectively. This significant difference results from the various molecular packing that
occurs in these crystals. As shown in Figure 3, molecules of
7 are connected by weak hydrogen bonds,
which form the “ring” pattern. The 5,8-quinolinedione moiety is located inside the ring, whereas the
substituent chain is arranged outside of the ring. All parameters of the hydrogen bonds are shown in
Table S3.
Figure 3. Molecular arrangement in 6-chloro-7-(2-cyanoethoxy)-5,8-quinolinedione
7 showing a “ring”
pattern. View down the “b” axis.
Moreover, the neighboring rings are linked by the following hydrogen bonds: C9-H9B¨ ¨ ¨ N2 and
C10-H10B¨ ¨ ¨ N1 (Figure S1, Table S3). Both interactions have a highly bent geometry, which does not

Molecules 2016, 21, 156
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really correspond to a significant hydrogen bond interaction. According to the literature data [17
the hydrogen bonds character decreases with a decreasing <DHA angle.
,22],
The unit cell of 15 contains four molecules (Table S1). The molecules form an infinite spiral
along the “b” axis, as shown in Figure 4. The angles between the mean planes of rings and the
2,2,2-trifluoroethoxy chain at positions C-7 (O31C91C101) and C-6 (O32C92C102) are equal to 14.37˝
˝
and 56.16 , respectively.
The crystal structure of 6,7-di(2,2,2-trifluoro)-5,8-quinolinedione 15 is stabilized by the intra- and
inter-unit hydrogen bonds, which are formed between the C-H groups as H-bond donors (D) and N, O
or fluorine atoms as H-bond acceptors (A) (Table S3). The C-H¨ ¨ ¨ O, C-H¨ ¨ ¨ N and C-H¨ ¨ ¨ F hydrogen
bonds are moderately strong, as their H¨ ¨ ¨ A distances cover the range of 2.35–2.64 Å (Figure S2) [22].
Figure 4. Molecular arrangement in the 6,7-(2,2,2-trifloroethoxy)-5,8-quinolinedione 15 crystal showing
a “spiral” pattern. View down the “b” axis.
2.3. Cytotoxic Activity
The antiproliferative activities in vitro of the new compounds
2–18 and for
6,7-dichloro-5,8-quinolinedione were tested against the three human cancer lines: melanoma (C-32),
1
glioblastoma (SNB-19), and breast cancer (MDA-MB-231), as well as normal fibroblasts cell lines
(HFF-1) using the WST-1 test. The IC50 parameter (µM), defined as the compound concentration that
inhibits the proliferation of 50% of tumor cells as compared to the control untreated cells, was used
to characterize all cytotoxic activities under study. As a reference, the cytotoxic agent cisplatin was
applied. The results of the cytotoxicity analysis are collected in Table 1.
As shown in Table 1, the introduction of the alkoxy group at the C7 and/or C6 position
leads to changes in the antiproliferative activity against the tested cancer lines compared
to 6,7-dichloro-5,8-quinolinedione
for compound , exhibits a high activity against melanoma (C-32) cancer cell lines. Moreover,
compounds and show higher activity than cisplatin. Compound exhibits a selective
effect for the C-32 lines with the IC50 value being twenty-five and twenty times higher than those
for 6,7-dichloro-5,8-quinolinedione and cisplatin, respectively. In the series of monosubstituted
derivatives , 6-chloro-7-methoxy-5,8-quinolinedione shows the highest cytotoxic activity against
the human breast cancer (MDA-MB-231) cell line and its selectivity index is equal to 18.4. Compound
is nearly seventeen times more active than both compound and cisplatin. The introduction of the
cyanoethoxy or chloroethoxy substituents to the 5,8-quinolinedione moiety in compounds and
1. The series of the 7-subtsituted derivatives 2–9, except
7
4–
5
8–
9
9
1
2–
9
2
2
1
5
6
leads to a decrease in the cytotoxic activity. The presence of a double bond in the substituent causes an
increase in the activity against melanoma cells (C-32). The structure–activity relationship indicates
that the rank order of the cytotoxic activity against the glioblastoma (SNB-19) cell line observed in the
7-substituted derivatives
2–5 is as follows: propoxy > ethoxy > methoxy; i.e., it is influenced by the
length of the chain substituent.
Comparing the activity of the mono- and disubtituted derivatives, one can notice that the
introduction of the two alkoxy groups at the C6 and C7 positions leads to changes in the anticancer

Molecules 2016, 21, 156
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activity against tested human cancer lines. For the derivatives 10
–
18, only compounds 11 and 14
show a higher activity than the monosubstituted derivatives
2–9 with respect to the C-32 cancer line.
Derivatives and 17 exhibit very similar cytotoxic effects against both the melanoma (C-32) and
8
glioblastoma (SNB-19) cell lines. Thus, the replacement of the chlorine atom in the cyclohexyloxy
group did not change the activity of the compound. Moreover, compound 15 exhibits a selectivity
effect against breast cancer (MDA–MB-231) cell line, for which the IC50 value is equal to 2.7
The selectivity index (SI = 4.6) is twenty times higher than that of cisplatin. For derivatives 11
rank order of the anticancer activity against the SNB-19 cell lines is as follows: 13 12 11 10
15 16. Similar to the monosubstituted derivatives, the substituent chain length and the double bond
˘
0.1
16, the
14
µ
M.
–
>
>
>
=
>
>
lead to an increase in the cytotoxic activity. On the other hand, the halogen atoms and cyano group
causes a reduction in the cytotoxicity.
Table 1.
Cytotoxic activity of 6,7-dichloro-5,8-quinolinedione 1, alkoxy derivatives of
5,8-quinolinedione 2–18 and cisplatin as a reference compound. The incubation time is 72 h.
Human Cell Line/IC50 ˘ SD [µM]
Compound
R
C-32
SNB-19
MDA-MB-231
HFF-1
1
2
3
4
5
6
7
-
22.8 ˘ 0.7
19.2 ˘ 2.1
30.7 ˘ 1.1
0.20 ˘ 0.01
0.28 ˘ 0.09
22.8 ˘ 2.5
119.4 ˘ 4.3
26.2 ˘ 0.8
42.2 ˘ 0.1
10.3 ˘ 2.4
0.36 ˘ 0.03
0.30 ˘ 0.01
18.3 ˘ 1.4
58.3 ˘ 2.8
25.1 ˘ 3.1
1.5 ˘ 0.2
15.7 ˘ 0.5
27.6 ˘ 2.1
1.3 ˘ 0.5
Neg
CH3
CH2CH3
CH2CH2CH3
CH2CH =CH2
CH2CH2Cl
CH2CH2CN
24.0 ˘ 2.8
22.4 ˘ 2.7
17.2 ˘ 2.8
17.0 ˘ 3.0
164.5 ˘ 3.9
Neg
15.8 ˘ 1.2
2.5 ˘ 0.4
8
9
1.4 ˘ 0.1
1.8 ˘ 0.1
3.5 ˘ 0.3
13.9 ˘ 3.0
0.81 ˘ 0.01
21.5 ˘ 2.0
17.6 ˘ 1.6
1.1 ˘ 0.1
2.3 ˘ 0.5
3.5 ˘ 0.7
271.3 ˘ 9.8
3.1 ˘ 0.1
2.0 ˘ 0.3
3.1 ˘ 0.8
Neg
Ph
10
11
12
13
14
15
16
CH3
CH2CH3
CH2CH2CH3
CH2CH =CH2
CH2CH2Cl
CH2CH3
2.1 ˘ 0.1
18.6 ˘ 2.0
3.0 ˘ 0.3
0.7 ˘ 0.02
0.2 ˘ 0.01
3.5 ˘ 0.3
Neg
Neg
1.5 ˘ 0.01
12.1 ˘ 0.2
10.9 ˘ 1.3
134.4 ˘ 10.7
12.6 ˘ 2.7
10.9 ˘ 1.4
2.7 ˘ 0.1
1.9 ˘ 0.2
12.4 ˘ 1.2
18.2 ˘ 2.6
14.9 ˘ 0.7
203.7 ˘ 14.3
CH2CH2CN
110.7 ˘ 3.7
17
18
1.4 ˘ 0.1
1.7 ˘ 0.3
13.8 ˘ 0.1
Neg
18.7 ˘ 0.9
137.4 ˘ 0.7
107.9 ˘ 10.5
25.5 ˘ 0.2
17.2 ˘ 1.7
Ph
cisplatin
16.4 ˘ 1.2
18.9 ˘ 0.4
9.1 ˘ 1.8
Neg—Negative in the concentration used.
3. Materials and Methods
3.1. General Techniques
Melting points were measured on an Electrothermal IA 9300 melting point apparatus. The 1H- and
13C-NMR spectra were determined using a Bruker Avance 600 spectrometer (Bruker, Billerica, MA,
USA) in CDCl3; chemical shifts (δ) are reported in ppm and J values in Hz. The peak multiplicity is
designated as singlet (s), doublet (d), triplet (t), doublet of doublets (dd), doublet of triplets (dt) and
multiplet (m). Mass spectra were recorded under EI conditions on a Finnigan MAT 95 instrument
(Thermo Fisher Scientific, Waltham, MA, USA). High-resolution mass spectral analysis was performed
on a Bruker Impact II instrument (Bruker). Infrared spectra were recorded on a IRAffinity-1 Shimadzu
spectrophotometer (Shimadzu Corporation, Kioto, Japan). Thin layer chromatography (TLC) was
carried out on silica gel 60 254F plates (Merck, Darmstadt, Germany) using a mixture of chloroform
and ethanol (40:1 or 15:1, v/v) as an eluent. The spots were visualized by UV light (254 nm) and iodine.
All new compounds were purified by column chromatography. As a solid phase silica gel 60 was used
and the eluent was a mixture of chloroform and ethanol (40:1, v/v).
The starting compound, 6,7-dichloro-5,8-quinolinedione
described previously [16,17]. M.p. 220–221 C (lit. m.p. 219–221 C [16]; 220–221 C [17]).
1
, ˝was obtained according to methods
˝
˝

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3.2. General Procedure for the Synthesis of 6-Chloro-7-alkoxy-5,8-quinolinedione
The mixture of 6,7-dichloro-5,8-quinolinedione
1 (0.100 g, 0.441 mmol) and potassium carbonate
(0.061 g, 0.441 mmol) in dry tetrahydrofuran (1 mL) was added to a solution of alcohol (1.2 eqv.,
0.529 mmol) in dry tetrahydrofuran (0.5 mL). Stirring at room temperature was continued for 3–24 h.
Subsequently, the reaction mixture was concentrated under reduced pressure. The crude product was
purified by column chromatography (chloroform/ethanol, 40:1, v/v) to give pure product 2–9.
6-Chloro-7-methoxy-5,8-quinolinedione (
4.42 (s, 3H, OCH3), 7.72 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz, 1H, H-3), 8.50 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H,
H-4), 9.06 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2). 13C-NMR (CDCl3, 150 MHz)
62.2 (OCH3), 127.6
(C-6), 128.0 (C-3), 128.1 (C-4a), 134.9 (C-4), 146.6 (C-8a), 154.7 (C-2), 157.4 (C-7), 177.7 (C-8), 177.9 (C-5).
2 δ
). Yield: 53%, m.p. 161–163 ˝C. 1H-NMR (CDCl3, 600 MHz)
δ
´1
EI MS (70 eV) m/z: 223 [M+] (100), 193 (31), 152 (9), 137 (36), 124 (21), 89 (2), 77 (8). IR (KBr, cm
)
ν
:
max
3078–2857, 1696, 1662, 1594–1560, 1096. HR-MS (APCI) m/z: C10H6ClNO3 [M + H]+, Calcd. 224.0114;
Found 224.0101.
˝
6-Chloro-7-ethoxy-5,8-quinolinedione (3 δ 1.49
). Yield: 58%, m.p. 112–114 C. 1H-NMR (CDCl3, 600 MHz)
(t, J = 7.8 Hz, 3H, CH3), 4.73 (q, J = 6.6 Hz, J = 7.8 Hz, 2H, OCH2), 7.69 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz,
1H, H-3), 8.47 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 9.03 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2).
13C-NMR (CDCl3, 150 MHz)
δ
16.0 (CH2
134.8 (C-4), 146.6 (C-8a), 154.7 (C-2), 157.3 (C-7), 177.8 (C-8), 178.0 (C-5). EI MS (70 eV) m/z: 237 [M+]
(100), 222 (12), 193 (67), 181 (65), 137 (98), 90 (10), 77 (10). IR (KBr, cm´1
max: 3080–2850, 1691, 1669,
1591–1560, 1094. HR-MS (APCI) m/z: C11H8ClNO3 [M + H]+, Calcd. 238.0271; Found 238.0261.
CH3), 71.2 (OCH2), 127.9 (C-6), 128.2 (C-3), 128.5 (C-4a),
)
ν
6-Chloro-7-propoxy-5,8-quinolinedione (
). Yield: 87%, m.p. 121–122 ˝C. 1H-NMR (CDCl3, 600 MHz)
1.07 (t, J = 7.2 Hz, 3H, CH3), 1.85–1.87 (m, 2H, CH2CH3), 4.63 (t, J = 6.6 Hz, OCH2), 7.70 (dd, J23 = 4.8 Hz,
34 =7.8 Hz, 1H, H-3), 8.48 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 9.05 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz,
1H, H-2). 13C-NMR (CDCl3, 150 MHz)
10.2 (CH3), 23.8 (CH2CH3), 75.8 (OCH2), 127.9 (C-6), 128.1
(C-3), 128.1 (C-4a), 134.8 (C-4), 146.6 (C-8a), 154.6 (C-2), 157.4 (C-7), 177.7 (C-8), 178.0 (C-5). EI MS
4
δ
J
δ
(70 eV) m/z: 251 [M+] (46), 209 (50), 181 (100), 137 (75), 125 (15), 90 (8), 70 (11). IR (KBr, cm´1
)
ν
:
max
3042–2898, 1694, 1671, 1596–1570, 1097. HR-MS (APCI) m/z: C12H10ClNO3 [M + H]+, Calcd. 252.0427;
Found 252.0417.
˝
5
). Yield: 88%, m.p. 89–90 C. 1H-NMR (CDCl3, 600 MHz)
6-Chloro-7-(2-propenoxy)-5,8-quinolinedione (
5.19 (dt, J = 1.2 Hz, J = 6.0 Hz, 2H, OCH2), 5.33 (dt, J = 1.2 Hz, J = 10.2 Hz, 1H, CH=CH2), 5.48 (dt,
δ
J = 1.2 Hz, J = 16.2 Hz, 1H, CH=CH2), 6.04–6.10 (m, 1H, CH=CH2), 7.69 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz,
1H, H-3), 8.47 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 9.03 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2).
13C-NMR (CDCl3, 150 MHz)
132.4 (
m/z: 249 [M+] (10), 221 (59), 193 (6), 176 (53), 124 (33), 90 (6), 77 (23). IR (KBr, cm´1
1671, 1595–1569, 1097. HR-MS (APCI) m/z: C12H8ClNO3 [M + H]+, Calcd. 250.0271; Found 250.0265.
δ
75.0 (OCH2), 120.0 (CH=
H=CH2), 134.9 (C-4), 146.6 (C-8a), 154.7 (C-2), 156.8 (C-7), 177.7 (C-8), 178.0 (C-5). EI MS (70 eV)
max: 3038, 1694,
CH2), 127.9 (C-6), 128.1 (C-3), 128.2 (C-4a),
C
)
ν
˝
1
6-Chloro-7-(2-chloroethoxy)-5,8-quinolinedione (
600 MHz) 3.86 (t, J = 6.0 Hz, 2H, CH2Cl), 4.89 (t, J = 0.6 Hz, 2H OCH2), 7.72 (dd, J23 = 4.8 Hz,
J34 = 7.8 Hz, 1H, H-3), 8.49 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 9.05 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz,
1H, H-2). 13C-NMR (CDCl3, 150 MHz)
42.9 (CH2Cl), 74.0 (OCH2), 127.7 (C-6), 128.1 (C-3), 129.2
(C-4a), 134.9 (C-4), 146.5 (C-8a), 154.8 (C-2), 156.7 (C-7), 177.6 (C-8), 177.8 (C-5). EI MS (70 eV) m/z: 273
[M+] (25), 236 (100), 209 (31), 181 (66), 137 (89), 90 (15), 77 (33). IR (KBr, cm´1
max: 3078–2958, 1685,
1668, 1593–1564, 1096. HR-MS (APCI) m/z: C11H7Cl2NO3 [M + H]+, Calcd. 271.9881; Found 271.9872.
6). Yield: 78%, m.p. 114–116 C. H-NMR (CDCl3,
δ
δ
)
ν
˝
1
6-Chloro-7-(2-cyanoethoxy)-5,8-quinolinedione (
600 MHz) 3.02 (t, J = 5.4 Hz, 2H, CH2CN), 4.71 (t, J = 6.0 Hz, 2H, OCH2), 7.85 (dd, J23 = 4.8 Hz,
J34 = 7.8 Hz, 1H, H-3), 8.40 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 9.02 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz,
1H, H-2). 13C-NMR (CDCl3, 150 MHz)
19.0 ( H2CN), 68.7 (OCH2), 118.4 (CH2 N), 127.8 (C-6), 128.0
7): Yield: 68%, m.p. 177–179 C. H-NMR (CDCl3,
δ
δ
C
C

Molecules 2016, 21, 156
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(C-3, C-4a), 134.1 (C-4), 146.9 (C-8a), 154.2 (C-2), 156.9 (C-7), 177.1 (C-8), 177.9 (C-5). EI MS (70 eV) m/z:
262 [M+] (38), 227 (20), 222 (56), 193 (19), 137 (100), 90 (17), 77 (34). IR (KBr, cm´1
) νmax: 3079, 2965,
2917, 2257, 1691, 1666, 1596–1568, 1094. HR-MS (APCI) m/z: C12H7ClN2O3 [M + H]+, Calcd. 263.0223;
Found 263.0219.
˝
8
): Yield: 79%, m.p. 123–124 C. 1H-NMR (CDCl3, 600 MHz)
6-Chloro-7-cyclohexyloxy-5,8-quinolinedione (
1.37–1.40 (m, 4H, 2 CH2), 1.69–1.72 (m, 2H, CH2), 1.82–1.84 (m, 2H, CH2), 1.97–1.99 (m, 2H, CH2),
5.17–5.20 (m, 1H, OCH), 7.69 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz, 1H, H-3), 8.48 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz,
1H, H-4), 9.02 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2). 13C-NMR (CDCl3, 150 MHz)
23.3 (CH2), 25.2
δ
ˆ
δ
(2 ˆ CH2), 32.8 (2 ˆ CH2), 83.2 (OCH2), 127.8 (C-3), 128.2 (C-6), 129.9 (C-4a), 134.8 (C-4), 146.7 (C-8a),
154.6 (C-2), 157.2 (C-7), 177.8 (C-8), 178.2 (C-5). EI MS (70 eV) m/z: 291 [M+] (8), 211 (92), 181 (65), 137
´1
) νmax: 3048, 2948–2854, 1682, 1665, 1594–1565, 1096. HR-MS
(42), 90 (8), 77 (15), 55 (100). IR (KBr, cm
(APCI) m/z: C15H14ClNO3 [M + H]+, Calcd. 292.0740; Found 292.0730.
6-Chloro-7-(1-methylbenzyloxy)-5,8-quinolinedione (
): Yield: 65%, m.p. 215–217 ˝C. 1H-NMR (CDCl3,
600 MHz) 1.77 (d, J = 6.6 Hz, 3H, CH3), 6.49 (q, J = 3.6 Hz, J = 6.6 Hz, 1H, CHCH3), 7.25–7.26 (m, 1H,
9
δ
Ph), 7.30–7.31 (m, 2H, Ph), 7.42–7.43 (m, 2H, Ph), 7.64 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz, 1H, H-3), 8.39 (dd,
J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 8.97 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2). 13C-NMR (CDCl3,
150 MHz)
δ 23.9 (CH3), 62.6 (CH), 126.5 (Ph), 127.8 (C-3), 128.0 (Ph), 128.3 (C-6), 128.4 (Ph), 128.6 (Ph),
128.7 (Ph), 130.2 (C-4a), 134.8 (C-4), 141.1 (Ph), 146.5 (C-8a), 154.6 (C-2), 156.7 (C-7), 177.6 (C-8), 178.2
(C-5). EI MS (70 eV) m/z: 313 [M+] (9), 278 (21), 211 (20), 181 (15), 137 (10), 105 (100), 77 (30). IR (KBr,
cm´1 max: 3064-2854, 1690, 1671, 1592–1559, 1094. HR-MS (APCI) m/z: C17H12ClNO3 [M + H]+,
) ν
Calcd. 314.0584; Found 314.0572.
3.3. General Procedure for the Synthesis of 6,7-Dialkoxy-5,8-quinolinedione
The mixture of 6,7-dichloro-5,8-quinolinedione
1 (0.100 g, 0.441 mmol) and potassium carbonate
(0.138 g, 0.882 mmol) in dry dimethyl sulfoxide (1 mL) was added to a solution of alcohol (2.2 eqv.,
0.970 mmol) in dry dimethyl sulfoxide (0.5 mL). Stirring at room temperature was continued for 3–24 h.
Subsequently, the reaction mixture was concentrated under reduced pressure. The crude product was
purified by column chromatography (chloroform/ethanol, 40:1, v/v) to give pure product 10–18.
˝
6,7-Dimethoxy-5,8-quinolinedione (10): Yield: 49%, m.p. 132–133 C. 1H-NMR (CDCl3, 600 MHz)
(s, 3H, CH3), 4.19 (s, 3H, CH3), 7.67 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz, 1H, H-3), 8.43 (dd, J24 = 1.8 Hz,
J34 = 7.8 Hz, 1H, H-4), 9.02 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2). 13C-NMR (CDCl3, 150 MHz)
δ 4.17
δ
61.6 (OCH3), 61.7 (OCH3), 127.5 (C-3), 127.7 (C-4a), 134.3 (C-4), 146.7 (C-8a), 147.2 (C-7), 148.4 (C-6),
154.5 (C-2), 180.2 (C-8), 180.9 (C-5). EI MS (70 eV) m/z: 221 [M+] (9), 204 (100), 189 (69), 174 (66), 148
(37), 105 (71), 77 (63). IR (KBr, cm´1
) νmax: 3024–2845, 1690, 1672, 1607–1570. HR-MS (APCI) m/z:
C11H9NO4 [M + H]+, Calcd. 220.0609; Found 220.0600.
6,7-Diethoxy-5,8-quinolinedione (11): Yield: 65%, m.p. 69–70 ˝C. 1H-NMR (CDCl3, 600 MHz)
δ 1.46 (t,
J = 13.8 Hz, 6H, 2
(dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 9.01 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2). 13C-NMR (CDCl3,
150 MHz) 15.7 (2 CH3), 70.0 (OCH2), 127.4 (C-3), 127.7 (C-4a), 134.2 (C-4), 146.8 (C-7), 147.1 (C-6),
148.2 (C-8a), 154.4 (C-2), 180.5 (C-8), 181.2 (C-5). EI MS (70 eV) m/z: 247 [M+] (13), 232 (65), 218 (100),
203 (54), 192 (54), 106 (26), 79 (72). IR (KBr, cm´1
max: 3078–2904, 1684, 1669, 1602–1560. HR-MS
(APCI) m/z: C13H13NO4 [M + H]+, Calcd. 248.0922; Found 248.0912.
ˆ
CH3), 4.41 (m, 4H, 2
ˆ
OCH2), 7.65 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz, 1H, H-3), 8.42
δ
ˆ
)
ν
˝
1
6,7-Dipropoxy-5,8-quinolinedione (12): Yield: 62%, m.p. 133–135 C. H-NMR (CDCl3, 600 MHz)
1.03–1.05 (m, 6H, 2
CH3), 1.81–1.86 (m, 4H, 2 ˆ CH2CH3), 4.30 (t, J = 6.6 Hz, 2H, OCH2), 4.33 (t,
J = 6.6 Hz, 2H, OCH2), 7.63 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz, 1H, H-3), 8.38 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz,
1H, H-4), 8.98 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2). 13C-NMR (CDCl3, 150 MHz)
10.2 (2 CH3),
δ
ˆ
δ
ˆ
23.5 (2 ˆ CH2CH3), 75.8 (OCH2), 75.9 (OCH2), 127.3 (C-3), 127.7 (C-4a), 134.1 (C-4), 146.9 (C-7), 147.4
(C-6), 148.8 (C-8a), 154.3 (C-2), 180.4 (C-8), 181.1 (C-5). EI MS (70 eV) m/z: 275 [M+] (9), 246 (27),

Molecules 2016, 21, 156
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233 (33), 191 (93), 163 (100), 135 (50), 79 (40). IR (KBr, cm´1
)
ν
max: 2968–2879, 1671, 1604, 1579–1570.
HR-MS (APCI) m/z: C15H17NO4 [M + H]+, Calcd. 276.1235; Found 276.1225.
˝
6,7-di(2-Propenyloxy)-5,8-quinolinedione (13): Yield: 71%, m.p. 135–136 C. 1H-NMR (CDCl3, 600 MHz)
4.90 (dt, J = 1.2 Hz, J = 6.0 Hz, 2H, OCH2), 4.93 (dt, J = 1.2 Hz, J = 6.0 Hz, 2H, OCH2), 5.30 (dt, J = 1.2 Hz,
J = 9.0 Hz, 2H, 2 CH=CH2), 5.43 (dt, J = 1.2 Hz, J = 16.2 Hz, 2H, 2 CH=CH2), 6.04–6.10 (m, 2H,
ˆ CH=CH2), 7.64 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz, 1H, H-3), 8.40 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H,
H-4), 8.99 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2). 13C-NMR (CDCl3, 150 MHz)
74.5 (2 OCH2),
δ
ˆ
ˆ
2
δ
ˆ
119.4 (CH=CH2), 119.6 (CH=CH2), 127.5 (C-3), 127.7 (C-4a), 132.8 (2 ˆ CH=CH2), 134.3 (C-4), 146.8
(C-6), 147.1 (C-7), 148.3 (C-8a), 154.5 (C-2), 180.3 (C-8), 181.0 (C-5). EI MS (70 eV) m/z: 271 [M+] (1), 230
´1
) νmax: 3075–2929, 1696, 1676, 1648–1517.
(59), 213 (19), 174 (22), 146 (17), 105 (19), 77 (31). IR (KBr, cm
HR-MS (APCI) m/z: C15H13NO4 [M + H]+, Calcd. 272.0922; Found 272.0910.
˝
6,7-di(2-Chloroethoxy)-5,8-quinolinedione (14): Yield: 65%, m.p. 189–191 C. 1H-NMR (CDCl3, 600 MHz)
3.85–3.88 (m, 4H, 2 CH2Cl), 4.67–4.69 (m, 4H, 2 OCH2), 7.66 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz,
1H, H-3), 8.40 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 9.00 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2).
13C-NMR (CDCl3, 150 MHz)
42.7 (CH2Cl), 42.8 (CH2Cl), 73.5 (OCH2), 73.5 (OCH2), 127.6 (C-3), 127.6
(C-4a), 134.3 (C-4), 146.6 (C-7), 147.7 (C-6), 147.8 (C-8a), 154.6 (C-2), 179.9 (C-8), 180.5 (C-5). EI MS
δ
ˆ
ˆ
δ
(70 eV) m/z: 317 [M+] (11), 280 (76), 218 (100), 192 (45), 164 (25), 90 (14), 77 (57). IR (KBr, cm´1
)
ν
:
max
3457, 1804, 1776, 1667–1610, 1077. HR-MS (APCI) m/z: C13H11Cl2NO4 [M + H]+, Calcd. 316.0143;
Found 316.0133.
˝
1
6,7-di(2,2,2-Trifluoroethoxy)-5,8-quinolinedione (15): Yield: 82%, m.p. 110–111 C. H-NMR (CDCl3,
600 MHz) 4.79 (q, J = 8.4 Hz, J = 7.8 Hz, 2H, OCH2), 4.85 (q, J = 8.4 Hz, J = 7.8 Hz, 2H, OCH2),
7.71 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz, 1H, H-3), 8.42 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 9.05 (dd,
24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2). 13C-NMR (CDCl3, 150 MHz)
69.1–69.6 (CH2), 127.4 (CF), 128.0
(C-3), 129.7 (C-4a), 134.5 (C-4), 145.7 (C-7), 146.2 (C-6), 146.7 (C-8a), 155.0 (C-2), 179.1 (C-8), 179.7 (C-5).
δ
J
δ
´1
EI MS (70 eV) m/z: 355 [M+] (33), 336 (18), 286 (61), 272 (100), 174 (47), 105 (75), 77 (50). IR (KBr, cm
ν
)
max: 2959–2855, 1685, 1667, 1596–1560, 1101-1066. HR-MS (APCI) m/z: C13H7F6NO4 [M + H]+, Calcd.
356.0357; Found 356.0341.
6,7-di(2-Cyanoethoxy)-5,8-quinolinedione (16): Yield: 59%, m.p. 45–47 ˝C. 1H-NMR (CDCl3, 600 MHz)
δ
2.95–2.98 (m, 4H, 2
1H, H-3), 8.43 (dd, J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 9.05 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2).
13C-NMR (CDCl3, 150 MHz)
18.6 (2 CH2), 67.3 (2 OCH2), 116.1 (CN), 116.2 (CN), 126.5 (C-3),
126.9 (C-4a), 133.4 (C-4), 145.4 (C-7), 145.6 (C-6), 146.6 (C-8a), 153.8 (C-2), 178.8 (C-8), 179.3 (C-5). EI
MS (70 eV) m/z: 297 [M+] (19), 257 (41), 243 (50), 215 (89), 192 (100), 105 (42), 78 (62). IR (KBr, cm´1
max: 2966–2850, 2253, 1669, 1611–1581. HR-MS (APCI) m/z: C15H11N3O4 [M + H]+, Calcd. 298.0827;
Found 298.0817.
ˆ
CH2CN), 4.66–4.68 (m, 4H, 2
ˆ
OCH2), 7.71 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz,
δ
ˆ
ˆ
)
ν
1
6,7-Dicyclohexloxy-5,8-quinolinedione (17): Yield: 51%, oil. H-NMR (CDCl3, 600 MHz)
δ
1.26–1.36 (m,
CH2),
8H, 4 CH2), 1.54–1.58 (m, 4H, 2 CH2), 1.71–1.74 (m, 4H, 2 CH2), 1.98–2.04 (m, 4H, 2
ˆ
ˆ
ˆ
ˆ
4.69–4.70 (m, 1H, OCH), 4.77–4.78 (m, 1H, OCH), 7.61 (dd, J23 = 4.8 Hz, J34 = 7.8 Hz, 1H, H-3), 8.37 (dd,
J24 = 1.8 Hz, J34 = 7.8 Hz, 1H, H-4), 8.97 (dd, J24 = 1.8 Hz, J23 = 4.8 Hz, 1H, H-2). 13C-NMR (CDCl3,
150 MHz)
34.5 (2 CH2), 80.6 (2
(C-8a), 153.2 (C-2), 179.8 (C-8), 180.5 (C-5). EI MS (70 eV) m/z: 355 [M+] (1), 273 (12), 192 (100), 163
(34), 135 (11), 83 (18), 77 (4). IR (KBr, cm´1
max: 2935, 2858, 1669, 1599-1559. HR-MS (APCI) m/z:
C21H25NO4 [M + H]+, Calcd. 356.1862; Found 356.1853.
δ
22.7 (CH2), 22.8 (CH2), 23.1 (2
ˆ
CH2), 24.4 (CH2), 24.5 (CH2), 31.6 (CH2), 31.7 (CH2),
ˆ
ˆ
OCH2), 126.3 (C-3), 126.8 (C-4a), 133.2 (C-4), 146.0 (C-7), 146.9 (C-6), 148.3
)
ν
1
6,7-di(1-Methylbenzyloxy)-5,8-quinolinedione (18): Yield: 57%, oil. H-NMR (CDCl3, 600 MHz)
(m, 6H, 2 CH3), 1.73–1.75 (m, 6H, 2 CH3), 5.93–6.10 (m, 4H, 4 CH), 7.27–7.30 (m, 4H, Ph),
7.37–7.43 (m, 16H, Ph), 7.54–7.60 (m, 2H, 2 H-3), 8.25–8.30 (m, 2H, 2 H-4), 8.89–8.92 (m, 2H,
H-4). 13C-NMR (CDCl3, 150 MHz)
22.5 (2 CH3) 22.6 (2 CH3), 69.0 (2 CH), 69.3 (2 CH),
δ 1.69–1.71
ˆ
ˆ
ˆ
ˆ
ˆ
2
ˆ
δ
ˆ
ˆ
ˆ
ˆ

Molecules 2016, 21, 156
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125.3 (2
ˆ
Ph), 125.5 (4
ˆ
Ph), 126.4 (2
ˆ
Ph), 127.2 (2
ˆ
Ph), 127.3 (2
ˆ
C-3), 127.4 (4
ˆ
Ph), 127.5
(4 Ph), 132.1 (2
ˆ
ˆ
C-4a), 133.0 (2
ˆ
C-4), 144.9 (2
ˆ
Ph), 145.5 (C-7), 145.6 (C-7), 146.6 (C-6), 146.7
(C-6), 148.0 (C-8a), 148.1 (C-8a), 153.1 (2
MS (70 eV) m/z: 399 [M+] (0.16), 295 (4), 279 (0.3), 191 (32), 163 (3), 105 (100), 77 (10). IR (KBr, cm´1
max: 3087–2899, 1684, 1680, 1669, 1663, 1601–1560. HR-MS (APCI) m/z: C25H21NO4 [M + H]+, Calcd.
ˆ
C-2), 179.3 (C-8), 179.4 (C-8), 180.0 (C-5), 180.1 (C-5). EI
)
ν
400.1549; Found 400.1559.
3.4. Crystal Structure Determination
X-ray diffraction measurement was performed at 100
˘
1 K Before measurement, the single
crystals of a good quality were preselected under a polarization microscope Oxford Cryosystem
Equipment, Oxfordshire, UK). For compounds and 15, brown and green crystals with the dimensions
of 0.34 0.29 0.08
0.19 mm3 and 0.44 0.03 mm3 were selected, respectively. The crystals were
7
ˆ
ˆ
ˆ
ˆ
mounted on a glass capillary and cooled down by a cold, dry nitrogen gas stream (Oxford Cryosystem
Equipment, Oxfordshire, UK). The data were collected using a SuperNova kappa diffractometer with
a Sapphire3 CCD detector (Oxford Diffraction Ltd., Yarnton, UK). For the integration of the collected
data, CrysAllis RED program (versions 1.171.32.29, Agilent Technologies, Santa Clara, CA, USA)
was used.
Refinement
The solving and refining procedures were similar for both compounds. The structures were
solved using direct methods with SHELXS97 software and then the solutions were refined using the
SHELXL97 program [23]. The aromatic hydrogen atoms were treated as “riding” on their parent
carbon atoms with d(C
´
H) = 0.95 Å and assigned isotropic atomic displacement parameters equal
to 1.2 times the value of the equivalent atomic displacement parameters of the parent carbon atom
(Uiso(H) = 1.2Ueq(C)) was used. The methylene H atoms were constrained to an ideal geometry with
d(C
´
H) = 0.99 Å or d(C – H) = 0.95 Å (for terminal methylene group) and Uiso(H) = 1.2Ueq(C). Methyl
H atoms were constrained as riding atoms, fixed to the parent atoms with distance of 0.98 Å, only
torsion angles were refined and Uiso(H) = 1.5Ueq(C). Hydrogen atoms involved in hydrogen bonding
were refined freely with Uiso(H) = 1.2Ueq of their parent atom.
CCDC-1436478 and CCDC-1436479 contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
3.5. Biological Study
3.5.1. Cell Culture
The synthesized compounds were tested against tumor cells SNB-19 (human glioblastoma,
DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany),
MDA-MB-231 (human adenocarcinoma, breast cancer; ATCC), C-32 (human melanoma; ATCC) and
nontumor cells HFF-1 (normal human fibroblasts derived from foreskin, ATCC). The cultured cells
˝
were kept at 37 C and in 5% CO2. The cells were seeded (103 cells/well/100 mL DMEM supplemented
with FBS (Fetal Bovine Serum, Lonza) to a final concentration of 10% and streptomycin 10 mg/mL and
penicillin 1000 IU/mL (Sigma), using 96-well plates (Corning).
3.5.2. Analysis of the Biological Activity
The antiproliferative effect of the synthesized compounds was determined using the Cell
Proliferation Reagent WST-1 assay (Roche Diagnostics, Mannheim, Germany). This colorimetric
assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in
viable cells, leading to formazan formation. After an exposure to the tested compounds at concentration
varying in the range 0.4–400 nM/mL, the cell viability was quantified by a cell proliferation assay.

Molecules 2016, 21, 156
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The amount of WST-1-formazan was measured at 450 nm and appropriate calculations were performed
as described previously. The cytotoxic activity of the tested compound was compared to the cisplatin
(positive control). The experiments were repeated in triplicate for each concentration of the tested
compound. The initial concentration of the tested compounds was 1 mg/mL DMSO. Solvent control
(DMSO) was included to check that the DMSO had no effect at the concentration used. Calculations
of the IC50 values were performed using the GraphPad Prism 6 (GraphPad Software, San Diego,
CA, USA).
4. Conclusions
The mono- and disubstituted derivatives
2–18 were synthesized with good yields
using 6,7-dichloro-5,8-quinolinedione as a starting material. The reactions of compound
1
1
with alcohols were performed in THF solution in the presence of potassium carbonate,
leading to formation of 7-substituted derivatives. Using DMSO solvent instead of THF, we
were able to obtain the 6,7-disubstituted derivatives. The molecular structures of the title
1
compounds were confirmed by H-, 13C-NMR, IR and MS spectra. Furthermore, structures of
6-chloro-7-(2-cyanoethoxy)-5,8-quinolinedione
7 and 6,7-di(2,2,2-trifluoroethoxy)-5,8-quinolinedione
15 were also tested using X-ray diffraction analysis. The evaluation of the in vitro cytotoxic activity
was carried out against three human cancer lines: C-23, SNB-19 and MDA-MB-231. Most of the
synthesized compounds showed high cytotoxic activity, depending on the type of substituents and
applied tumor cells.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
2/156/s1.
Acknowledgments: This work was supported by the Medical University of Silesia in Katowice, Poland (grant
No. KNW-2-003/N/5/N).
Author Contributions: M.K. and S.B. designed and contributed to analyzing spectra. E.B. and E.C. contributed to
the synthesis and purification all new compounds. M.L. performed biological activity experiments. M.J. and J.K.,
M.K. performed the X-ray analysis. M.K. wrote the paper. All authors have given approval to the final version of
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. World Health Organization. Global Health Estimates Summary. Available online: http://www.who.int
(accessed on 1 June 2013).
2.
Rao, K.V.; Cullen, W.P. Streptonigrin, an antitumor substance. I. Isolation and characterization. Antibiot. Annu.
1960, 7, 950–953.
3. Boger, D.L.; Yasuda, M.; Mitscher, L.A.; Drake, S.D.; Kitos, P.A.; Thompson, S.C. Streptonigrin and
lavendamycin partial structures. Probes for the minimum, potent pharmacophore of streptonigrin,
lavendamycin, and synthetic quinoline-5,8-diones. J. Med. Chem. 1987, 30, 1913–1928. [CrossRef]
4. Bringmann, G.; Reichert, Y.; Kane, V.V. The total synthesis of streptonigrin and related antitumor antibiotic
natural products. Tetrahedron 2004, 60, 3539–3574. [CrossRef]
5.
Bringmann, G.; Reichert, M.; Hemnberger, J. The absolute configuration of streptonigrin. Tetrahedron 2008, 64,
515–528. [CrossRef]
6. Donohoe, T.J.; Jones, C.R.; Kornahrens, A.F.; Barbosa, L.C.; Walport, L.J.; Tatton, M.T.; O’Hagan, M.;
Rathi, A.H.; Baker, B.D. Total synthesis of the antitumor antibiotic ( )-streptonigrin: First- and
˘
second-generation routes for de novo pyridine formation using ring-closing metathesis. J. Org. Chem.
2013, 78, 12338–12350. [CrossRef] [PubMed]
7. Fryatt, T.; Goroski, D.T.; Nilson, Z.D.; Moody, C.J.; Beall, H. Novel quinolinequinone antitumor agents:
Structure-metabolism studies with NAD(P)H:quinone oxidoreductase (NQO1). Bioorg. Med. Chem. Lett.
1999, 9, 2195–2198. [CrossRef]

Molecules 2016, 21, 156
12 of 12
8. Ryu, C.-K.; Jeong, H.J.; Lee, S.K.; You, H.-J.; Chio, K.U.; Shim, J.-Y.; Heo, Y.H.; Lee, C.-O. Effects of
6-arylamine-5,8-quinolinediones and 6-chloro 7-arylo-5,8-isoquinolinediones on NAD(P)H: Quinone
oxidoreductase (NQO1) activity and their cytotoxic potential. Arch. Pharm. Res. 2001, 24, 390–396. [CrossRef]
[PubMed]
9. Rhee, H.K.; Park, H.J.; Lee, S.K.; Lee, C.-O.; Choo, H.-Y. Synthesis, cytotoxicity, and DNA topoisomerase
II inhibitory activity of benzofuroquinolinediones. Bioorg. Med. Chem. 2007, 15, 1651–1658. [CrossRef]
[PubMed]
10. Leteurtre, F.; Kohlhagen, G.; Pommier, Y. Streptonigrin induced topoisomerase II sites exhibit base preferences
in the middle of the enzym stragger. Biochem. Biophys. Pres. Commun. 1994, 203, 1259–1267. [CrossRef]
[PubMed]
11. Kim, Y.-S.; Park, S.-Y.; Lee, H.-J.; Suh, M.-E.; Schollmeyer, D.; Lee, C.-O. Synthesis and cytotoxicity of
6,11-dihydro-pyrido- and 6,11-dihydro-benzo[2,3-b]phenazine-6,11-dione derivatives. Bioorg. Med. Chem.
2003, 11, 1709–1714. [CrossRef]
12. Shaikh, I.A.; Johnson, F.; Grollman, A.P. Streptonigrin. 1. Structure-activity relationships among simple
bicyclic analogs. Rate dependence of DNA degradation on quinone reduction potential. J. Med. Chem. 1986
29, 1329–1340. [CrossRef] [PubMed]
,
13. Cai, W.; Hassani, M.; Karki, R.; Walter, E.D.; Koelsch, K.K.; Seradj, H.; Lineswala, J.P.; Mirzaei, H.; York, Y.S.;
Olang, F.; et al. Synthesis, metabolism and in vitro cytotoxicity studies on novel lavendamycin antitumor
agents. Bioorg. Med. Chem. 2010, 18, 1899–1909. [CrossRef] [PubMed]
14. Hassani, M.; Cai, W.; Holley, D.C.; Lineswala, J.P.; Maharjan, B.R.; Ebrahimian, G.R.; Seradj, H.;
Stocksdale, M.G.; Mohammadi, F.; Marvin, C.C; et al. Novel lavendamycin analogues as antitumor agents:
Aynthesis, in vitro cytotoxicity, structure-metabolism, and computational molecular modeling studies with
NAD(P)H: Quinone oxidoreductase 1. J. Med. Chem. 2005, 48, 7733–7749. [CrossRef] [PubMed]
15. Lee, D.M.; Ko, J.H.; Lee, K.I. Cesium carbonate-mediated reaction of dichloronaphthoquinone derivatives
with O-nucleophiles. Monatsh. Chem. 2007, 138, 741–746. [CrossRef]
16. Mulchin, B.J.; Newton, C.G.; Baty, J.W.; Grasso, C.H.; Martin, W.J.; Walton, M.C.; Dangerfield, E.M.;
Plunkett, C.H.; Berridge, M.V.; Harper, J.L; et al. The anti-cancer, anti-inflammatory and tuberculostatic
activities of a series of 6,7-substituted-5,8-quinolinequinones. Bioorg. Med. Chem. 2010, 18, 3238–3251.
[CrossRef] [PubMed]
17. Jastrzebska, M.; Boryczka, S.; Kadela, M.; Wrzalik, R.; Kusz, J.; Nowak, M. Synthesis, crystal structure
and infrared spectra of new 6- and 7-propylamine-5,8-quinolinediones. J. Mol. Struct. 2014, 1067, 160–168.
[CrossRef]
18. Yoon, E.Y.; Choi, H.Y.; Shin, K.J.; Yoo, K.H.; Chi, D.Y.; Kim, D.J. The regioselectivity in the reaction of
6,7-dihaloquinoline-5,8-diones with amine nucleophiles in various solvents. Tetrahedron Lett. 2000, 41,
7475–7480. [CrossRef]
19. Ryu, C.-K.; Sun, Y.-J.; Shim, J.-Y.; You, H.-J.; Choi, K.U.; Lee, H. Synthesis and antyfungal activity of
6,7-bis-[S-(aryl)tio]-5,8-quinolinediones. Arch. Pharm. Res. 2002, 25, 795–800.
20. Bhuiyan, M.M.H.; Ferdaush, J.; Uddin, M.H. Densities and viscosities of binary mixtures of
{dimethylsulfoxide + aliphatic lower alkanols (C1–C3)} at temperatures from T = 303.15 K to T = 323.15 K.
J. Chem. Thermodyn. 2007, 39, 675–683. [CrossRef]
21. Kiefer, J.; Noack, K.; Kirchner, B. Hydrogen bond in mixtures of dimethyl sulfoxide and cosolvents. Curr. Phys.
Chem. 2011, 1, 340–351. [CrossRef]
22. Steiner, T. The hydrogen bond in the solid state. Angew. Chem. 2002, 41, 48–79. [CrossRef]
23. Sheldrick, G.M. A short history of SHELX. Acta Cryst. A 2008, 64, 112–122. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).