Synthesis, cytotoxic activity, and fluorescence properties of a set of novel 3-hydroxyquinolin-4(1H)-ones

Article abstract of DOI:10.1016/j.tetlet.2014.04.121

The targeted solid-phase synthesis of 3-hydroxyquinolin-4(1H)-one derivatives is described. Primary and secondary amines, 3-amino-4- (methoxycarbonyl)benzoic acid and 2-bromo-1-(4-chloro-3-nitrophenyl)ethanone were used as starting materials. The structures of the final compounds were designed in accordance with previous information obtained from structure-activity relationship studies of similar cytotoxic derivatives. Representative prepared compounds were subjected to in vitro screening of cytotoxic activity against various cancer cell lines; the results obtained are discussed. Fluorescence properties of selected compounds were also studied to compare the data with those obtained in analogous derivatives.

Full text of DOI:10.1016/j.tetlet.2014.04.121

Accepted Manuscript
Synthesis, cytotoxic activity and fluorescence properties of a set of novel 3-
hydroxyquinolin-4(1H)-ones
Jasna Kadrić, Kamil Motyka, Petr Džubák, Marián Hajdúch, Miroslav Soural
PII:
S0040-4039(14)00758-8
DOI:
http://dx.doi.org/10.1016/j.tetlet.2014.04.121
TETL 44583
Reference:
Tetrahedron Letters
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Revised Date:
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28 January 2014
23 March 2014
30 April 2014
Please cite this article as: Kadrić, J., Motyka, K., Džubák, P., Hajdúch, M., Soural, M., Synthesis, cytotoxic activity
and fluorescence properties of a set of novel 3-hydroxyquinolin-4(1H)-ones, Tetrahedron Letters (2014), doi: http://
dx.doi.org/10.1016/j.tetlet.2014.04.121
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Graphical Abstract
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Synthesis, cytotoxic activity and fluorescence
properties of a set of novel 3-hydroxyquinolin-4(1H)-ones
Jasna Kadrić, Kamil Motyka, Petr Džubák, Marián Hajdúch, Miroslav Soural

1
Tetrahedron Letters
jo u rn al h ome p ag e : w ww .e lse vie r .c om
Synthesis, cytotoxic activity and fluorescence properties of a set of novel 3-
hydroxyquinolin-4(1H)-ones
Jasna Kadrić a, Kamil Motyka a, Petr Džubák b, Marián Hajdúch b, Miroslav Soural a,∗
a Department of Organic Chemistry, Institute of Molecular and Translational Medicine, Faculty of Science, University of Palacky, 17. listopadu 12, 771 46
Olomouc, Czech Republic
b Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Hněvotínská 5,
CZ-775 15 Olomouc, Czech Republic
ARTICLE INFO
ABSTRACT
Article history:
Received
The targeted solid-phase synthesis of 3-hydroxyquinolin-4(1H)-one derivatives is described.
Primary and secondary amines, 3-amino-4-(methoxycarbonyl)benzoic acid and 2-bromo-1-(4-
chloro-3-nitrophenyl)ethanone were used as starting materials. The structures of the final
compounds were designed in accordance with previous information obtained from structure-
activity relationship studies of similar cytotoxic derivatives. Representative prepared compounds
were subjected to in vitro screening of cytotoxic activity against various cancer cell lines; the
results obtained are discussed. Fluorescence properties of selected compounds were also studied
to compare the data with those obtained in analogous derivatives.
Received in revised form
Accepted
Available online
Keywords:
Solid-phase synthesis
Hydroxyquinolinones
Fluorescence
2011 Elsevier Ltd. All rights reserved.
Cytotoxicity
Compounds based on the 3-hydroxyquinolin-4(1H)-one
scaffold (3HQs) represent a new class of biologically promising
compounds.1 For this reason, synthetic approaches to such
derivatives have often been studied and simple methods for their
preparation have been developed.2 2-Phenyl-3-hydroxyquinolin-
4(1H)-ones were identified as strong anticancer agents with
significant in vitro cytotoxicity against various cancer cell lines.3-
6 Aside from this, some derivatives exhibit high antiprotozoal and
immunosupressive activity.6 With respect to their limited
solubility, current research in this field is focused particularly on
the improvement of the bioavailability of selected compounds.7,8
In addition to the biological properties, 3-hydroxyquinolin-
4(1H)-ones have been studied for their interesting fluorescence
effects. Dual emission spectra and promising quantum yields of
some derivatives have led to several studies on their possible use
as fluorescence labels.9-13 Alongside our recent focus on
pharmacokinetic studies of selected compounds, we have also
been interested in the development of novel structural analogues
to obtain substances with improved biological properties,
particularly higher activity, low toxicity and better selectivity. In
this article, we describe the preparation and study of derivatives
that have been designed as a combination of the structural motifs
found in the most active compounds. In the past, compounds
bearing a carboxamide group on the benzene ring of the
quinolinone scaffold were synthesized using solid-phase
synthesis.14 With the use of combinatorial chemistry and
3HQs bearing specific 3-nitro-4-amino substitution at the 2-
phenyl group were described as potent anticancer and
antiprotozoal agents.6 Some of the most active compounds are
displayed in Figure 1.
Figure 1: Some of the most active representatives of 3HQs
Based on previous information, target derivatives III were
designed as a combination of the most active compounds I and II
(Figure 2). For the preparation of the desired derivatives,
polystyrene-supported chemistry was chosen to enable the
structure-cytotoxicity relationship studies, substitution of the
———
5
carboxamide group was targeted to specific ligands. Similarly,
∗ Corresponding author. Tel.: +420-585634418; fax: +420-585634465; e-mail: soural@orgchem.upol.cz

2
Tetrahedron
preparation of
a
small chemical library of the target
After cleavage from the polymer support the final cyclization
of intermediates 3 was performed by heating in the presence of
an acid. Two different methods were tested: reflux in TFA or
AcOH. The latter gave significantly better results in terms of the
crude purity of the final compounds 4. Despite this, the
cyclization step afforded variable results and the crude purity of
the final products was limited in some cases (Table 1).
quinolinones. The detailed structure-activity relationships of
these targets could then be studied.
Table 1: Purities/yields of synthesized compounds after precipitation
aPurity calculated from LC-UV traces (220 nm), NI=not isolated
bOverall yields after the whole reaction sequence and purification
Product
R1
R2
Puritya
(%)
45
Yield b
(%)
NI
4a
4b
4c
4d
4e
4f
cyclopentyl
cyclopentyl
cyclopentyl
Bn
piperidin-1-yl
pyrrolidin-1-yl
N,N-dipropylamino
piperidin-1-yl
32
NI
60
NI
Figure 2: Design of target compounds and ligand selection
93
20
Bn
pyrrolidin-1-yl
N,N-dipropylamino
piperidin-1-yl
95
23
The key intermediates 1 were prepared according to the
14
known procedure (Scheme 1). The results achieved were the
same as those previously published, and compounds 1 were
obtained with crude purities of more than 95% (calculated from
LC-UV traces).
Bn
55
20
4g
4h
4i
pentyl
pentyl
pentyl
decyl
decyl
decyl
98
39
pyrrolidin-1-yl
N,N-dipropylamino
piperidin-1-yl
97
38
95
20
4j
98
21
4k
4l
pyrrolidin-1-yl
N,N-dipropylamino
92
20
85
10
Separation of side products 2a proved simple; after
evaporation of acetic acid, the crude material was suspended in
diethyl ether. The quinolinones 4 precipitated while side products
2a remained dissolved. With simple filtration it was possible to
separate some of the products in excellent purity. On the other
hand, some compounds 4 were accompanied by numerous side
products and purification by precipitation or crystallization was
not successful. For compounds synthesized from diethanolamine,
the cyclization was not applicable due to partial acetylation of
both hydroxyl groups. Subsequent attempts to hydrolyze the
acetates failed due to total decomposition of the molecule.
Scheme 1: Preparation of the target compounds
However, the subsequent reaction with amines did not lead to
the pure intermediates 2. We observed unexpected cleavage of
the ester bond and side products 2a were detected (Scheme 2).
The quantities of side products 2a were too high (40-60%,
calculated from LC-UV traces), to allow for continuation of this
route under these conditions. It was clear that the ester cleavage
had to occur during the reaction, but not during the substitution;
after analysis of the cleaved samples, we did not detect any other
compounds except derivatives 3 and their carboxylic analogues.
On the other hand, formation of 2a was surprising as aminolysis
of the ester should afford an amide instead of the free carboxylic
group. To suppress the unwanted hydrolysis, we used anhydrous
reagents (dry DMSO and amines). Under such conditions the side
product was still detected, but in limited quantities (5-10% for
piperidine and pyrrolidine, 15-20% for diethanolamine and
dipropylamine).
Compounds that were isolated in sufficient quantity and high
purity were subjected to MTT colorimetric assay and their
cytotoxicity against various cancer cell lines was evaluated. To
compare the results with the cytotoxicities of previously studied
compounds I and II, similar cell lines were selected: human
myeloid leukemia (K562), human myeloid leukemia resistant to
paclitaxel (K562-tax), T-lymphoblastic leukemia (CEM), T-
lymphoblastic leukemia resistant to doxorubicine (CEM-DNR-
bulk) and lung adenocarcinoma (A549). Results of the MTT
testing are summarized in Table 2.
Table 2: Results of biological screening of representative compounds
(IC50, µM)a
Product CEM CEM-DNR-bulk K562 K562-tax A549
4i
4g
4h
4d
4e
4k
182.9
3.0
150.2
9.3
152.6
2.7
150.7
11.1
4.7
168.8
5.2
3.0
9.9
4.1
2.4
9.1
41.0
41.7
9.8
39.5
11.4
7.2
62.6
67.6
9.4
10.9
8.7
6.7
2.9
2.9
aAverage IC50 values from 3-4 independent experiments with SDs ranging
from 10-25% of the average values.
Scheme 2: Formation of side products during the nucleophilic substitution
step

3
Some interesting structure-activity relationships were
observed in compounds 4g, 4h and 4i. It can be concluded that
the presence of the five-membered ring (pyrrolidine) or the six-
membered ring (piperidine) gives the same results, while the
aliphatic analogue (dipropylamine) does not exhibit any activity.
As well as the presence of R2 substituents effecting the activity,
varying the R1 ligand also caused a difference in the biological
properties. This is shown by the comparison of 4h and 4e in
which the locked pyrrolidine scaffold at position R2 gave 14
times higher activity for R1=pentylamine compared to
R1=benzylamine (K562 cell line). However, from the overall
results, it is evident that the combination of selected ligands does
not lead to an improvement in the anticancer activity. No single
compound from the analyzed set exhibited a lower IC50 value
than that reported for model compounds I and II.5,6
introduction of the carboxamide group increases the quantum
yield values. On the other hand, the quantum yield values of
analogous 3HQs II with different 2-phenyl substitution were
higher.10
400
300
200
100
0
The fluorescence properties of representative synthesized
compounds 4g-i were investigated to compare the results with
previously studied analogues of type I and II (Figure 3).9,13 The
excitation spectra of 4g-i showed mostly several relatively
narrow distinctive local maxima at wavelengths around 290, 322
and 360 nm (Figure 3, Table 3).
400
450
500
550
600
650
700
Wavelength (nm)
500
400
300
200
100
0
Figure 4: Emission spectra of 4i in various solvents. (– -) EtOAc; (—)
CHCl3; (–•–) THF; (---) DMSO; (•••) MeOH; (– –) toluene. (Excitation
wavelength for all solvents = 370 nm; 4i conc = 20 µmol.L-1).
Additionally, the influence of solvents and pH on the
fluorescence properties of compound 4i were studied. The effect
of solvents on the dual emission spectrum was significant (Figure
4, compare the emission spectrum in dimethyl sulfoxide and
chloroform), nevertheless, no obvious relationship between the
solvent polarity and fluorescence was observed.
Also the pH (4i was dissolved in a phosphate buffer at a conc.
of 100 mg.L-1) significantly affected the shape of the emission
spectrum (Figure 5). The fluorescence measured at a low
wavelength maximum (451 nm) reached the maximum intensity
at a pH of 3.26 and then gradually decreased. The fluorescence
intensity measured at 555 nm decreased nearly linearly (R2 =
0.9687) with increasing pH (Figure 5).
300
400
500
600
700
Wavelength (nm)
Figure 3: Excitation and emission spectra of compounds 4g-i in methanol.
(—) 4i; (– –) 4h; (•••) 4g. (for excitation and emission wavelengths see Table
3; 4i conc = 20 µmol.L-1).
140
120
100
80
Table 3: Spectroscopic properties of 4g, 4h and 4i in methanol.
b
c
Product
εa
λex
(nm)
λem,1
(nm)
λem,2d (nm)
φf
(%)
(mol-1
cm-1)
4i
4h
4g
9808
11294
6986
370
360
360
451
450e
450e
555
558
522
4.85
2.35
5.06
60
a ε, molar extinction coefficient for λex.
b λex, excitation wavelength.
40
c λem,1, the fluorescence emission maximum at lower wavelengths.
d λem,2, the fluorescence emission maximum at higher wavelengths.
e indistinguishable local maximum, see Figure 1.
20
0
f φ, fluorescence quantum yield (determined with quinine sulfate in 0.5 M
H2SO4 (φ = 0.57715) taken as a reference fluorescence standard).
400
450
500
550
600
650
700
Wavelength (nm)
Figure 5: Emission spectra of 4i depending on the pH (for better lucidity
emission spectra for selected pH values are depicted, for complete data see
Figure 6). (—) pH 2.86; (– –) pH 3.99; (---) pH 7.09; (– • –) pH 8.82; (•••) pH
10.23. (Excitation wavelength for all solvents = 370 nm; 4i conc = 20 µmol.L-
1).
The emission spectra had a dual behavior that is characteristic
for 3-hydroxyquinolones resulting from the formation of two
excited state tautomeric forms.9-13 Interestingly, only in the case
of 4i was the lower wavelength local maximum of the emission
spectrum sufficiently distinguished (Figure 3). Quantum yields
were detected within the range of 2 to 5 percent. Such values are
generally higher than in the case of 3HQs I9 which shows that
However, the dependence of I2/I1 on the pH was not linear,
which limits the possible applicability of 4i as a fluorescent pH

4
Tetrahedron
9. Motyka K.; Hlaváč J.; Soural M.; Hradil P.; Krejčí P.; Kvapil L.;
Weiss M. Tetrahedron Lett. 2011, 52, 715-717.
10. Motyka K.; Hlaváč J.; Soural M.; Funk P. Tetrahedron Lett. 2010,
51, 5060-5063.
indicator with the dual emission spectrum. The fluorescence of
dual fluorescence labels is not dependent on the concentration in
the case when the ratio of the two band intensities can be applied
as a signal.9,13
11. Yushchenko D. A.; Shvadchak V. V.; Klymchenko A. S.;
Duportail G.; Mely Y.; Pivovarenko V. G. New J. Chem. 2006,
30, 774-781.
Figure 6: Fluorescence intensities at local maxima of the emission spectra
and their ratio for 4i depending on pH. ■ I1 451 nm; ▲ I2 555 nm; ● I2/I1.
(excitation wavelength for all solvents = 370 nm; 4i conc = 20 mol.L-1).
12. Yushchenko D. A.; Bilokin´ M. D.; Pyvovarenko O. V.; Duportail
G.; Mely Y.; Pivovarenko V. G. Tetrahedron Lett. 2006, 47, 905-
908.
13. Motyka K.; Vaňková B.; Hlaváč J.; Soural M. J. Fluoresc. 2011,
21, 2207-2212.
14. Soural M.; Krchnak V. J. Comb. Chem. 2007, 9, 793-796.
In conclusion, we have identified several new promising 3HQ
derivatives. Although the studied compounds exhibited high
anticancer activity and interesting fluorescence, both their
emission properties and cytotoxicity were not significantly
improved in comparison to analogues studied previously. Despite
this fact, some surprising results have been obtained. Regardless
of the structural similarity, compound 4i showed quite different
biological/spectral properties and other derivatives exhibited
significantly different activities towards the same cell lines.
Further, the linear dependence of fluorescence intensity measured
at 555 nm and pH allows the possible application of compound 4i
as a pH indicator.
Acknowledgements
The authors are grateful to projects CZ.1.07/2.3.00/20.0009,
CZ.1.07/2.3.00/30.0060 and CZ.1.07/2.4.00/31.0130 from the
European Social Fund. The infrastructural part of this project
(Institute of Molecular and Translational Medicine) was
supported from the Operational Program Research and
Development for Innovations (project CZ.1.05/2.1.00/01.0030).
The authors would also like to acknowledge the project
TE01020028.
References and notes
1. Hradil P.; Hlaváč J.; Soural M.; Hajdúch M.; Kolář M.; Večeřová
R. Mini-Rev. Med. Chem. 2009, 9, 696-702.
2. Soural M.; Hradil P.; Křupková S.; Hlaváč J. Mini-Rev. Org.
Chem. 2012, 9, 426-432.
3. Hradil P.; Krejčí P.; Hlaváč J.; Wiedermannová I.; Lyčka A.;
Bertolasi, V. J. Heterocycl. Chem., 2004, 41, 375-379
4. Soural M.; Hlaváč J.; Hradil P.; Fryšová I.; Hajdúch M.; Bertolasi
V.; Maloň M. Eur. J. Med. Chem., 2006, 41, 467-474.
5. Soural M.; Hlaváč J.; Funk P.; Džubák P.; Hajdúch, M. ACS
Combi. Sci. 2011, 13, 39-44.
6. Krejčí P.; Hradil P.; Hlaváč J.; Hajdúch M. Preparation of 2-
phenyl-3-hydroxyquinolin-4(1H)-ones for treatment of immune
system and proliferative disorders, WO2008028427 A1.
7. Cagno M.; Stein P.C.; Stýskala J.; Hlaváč J.; Skalko-Basnet N.;
Brauer-Brandl A. Eur. J. Pharm. Biopharm. 2012, 80, 657-662.
8. di Cagno M.; Stýskala J.; Hlaváč J.; Brandl M.; Brauer-Brandl A.;
Skalko-Basnet, N. J. Liposome Res. 2011, 21, 272-278.

5
Graphical Abstract
Synthesis, cytotoxic activity and fluorescence
Leave this area blank for abstract info.
properties of a set of novel 3-hydroxyquinolin-4(1H)-ones
Jasna Kadrić, Kamil Motyka, Petr Džubák, Marián Hajdúch, Miroslav Soural