New series of quinazolinone derived Schiff’s bases: synthesis, spectroscopic properties and evaluation of their antioxidant and cytotoxic activity

Article abstract of DOI:10.1007/s11696-017-0345-y

A series of novel 2,3-di-substituted-2,3-dihydro-quinazolin-4(1H)-one derived Schiff’s bases (1–7) have been designed, synthesized and characterized on the basis of their physical and spectral data. The presented microwave-assisted, phosphomolybdic acid (

Full text of DOI:10.1007/s11696-017-0345-y

Chemical Papers
https://doi.org/10.1007/s11696-017-0345-y
ORIGINAL PAPER
New series of quinazolinone derived Schif’s bases: synthesis,
spectroscopic properties and evaluation of their antioxidant
and cytotoxic activity
Zuzana Hricovíniová1 · Michal Hricovíni2 · Katarína Kozics3
Received: 7 August 2017 / Accepted: 18 November 2017
© Institute of Chemistry, Slovak Academy of Sciences 2017
Abstract
A series of novel 2,3-di-substituted-2,3-dihydro-quinazolin-4(1H)-one derived Schif’s bases (1–7) have been designed,
synthesized and characterized on the basis of their physical and spectral data. The presented microwave-assisted, phos-
phomolybdic acid (PMoA) catalysed, protocol provides an efcient and convenient route for the synthesis of structurally
diverse and potentially biologically active compounds. The molecular structures of these Schif’s bases related to quina-
zolinones were confrmed by various spectroscopic methods (NMR, FTIR, UV–Vis) and their antioxidant activities were
evaluated by UV–Vis and EPR spectroscopy employing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
assay. Derivatives 1–7 were examined for their cytotoxicity in vitro against human hepatocellular carcinoma cells (HepG2)
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphentltetrazolium bromide (MTT) assay. The structure–activity relationships study
revealed that the position and nature of functional groups attached to the quinazolinone moiety alter their physico-chemical
and biological properties. Derivatives 5, 6 and 7 bearing multiple electron-donating groups were found to be the most active
members of this series.
Keywords Quinazolinones · Heterogeneous catalysis · NMR · EPR spectroscopy · Antioxidant · Cytotoxic efect
Introduction
a lone electron pair in the sp2 hybridized orbital of the
nitrogen atom. The mechanism of action likely proceeds
through the hydrogen bond formation between the azome-
thine groups at the active centres of cellular entities (Zoubi
2013). An azomethine group (C=N) has been observed
in several natural, natural product-derived, and synthetic
compounds (Guo et al. 2007; Ernst et al. 2014; Zhang et al.
2016). Heterocyclic compounds of this group, depending
on their molecular structure, have many possibilities for
chemical permutation and can exhibit antimicrobial, anti-
fungal, antitubercular, antiviral, anticancer, anticonvulsant
and many other properties (Przybylski et al. 2009; Da Silva
et al. 2011; Zoubi 2013). Moreover, Schif’s bases belong
to an important class of nitrogen donor ligands and their
metal complexes are used in pharmaceuticals for cancer
targeting (Cozz 2004). Compounds with a quinazolinone
skeleton isolated from plants and microorganisms (e.g.
peganine, febrifugine, isofebrifugine, luotonin, boucha-
rdatine) have been the subject of considerable interest
due to their important therapeutic efects (Michael 2005;
Mhaske and Argade 2006; Rakesh et al. 2015). Conse-
quently, considerable efort has been directed towards the
Schif’s bases related to quinazolinones have been the sub-
ject of ongoing interest in medicinal feld. Schif’s bases,
also known as azomethines, possess potent biological
activities and have been explored for their remarkable ver-
satility (Schif 1864; Sztanke et al. 2013). Their chemical
and biological signifcance arises from the presence of
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-017-0345-y) contains
supplementary material, which is available to authorized users.
* Zuzana Hricovíniová
Zuzana.Hricoviniova@savba.sk
1
Institute of Chemistry, Slovak Academy of Sciences,
845 38 Bratislava, Slovakia
2
Faculty of Chemical and Food Technology, Institute
of Physical Chemistry and Chemical Physics, Slovak
University of Technology, 812 37 Bratislava, Slovakia
3
Cancer Research Institute, Biomedical Research Center,
Slovak Academy of Sciences, 845 05 Bratislava, Slovakia
Vol.:(0123456789)
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Chemical Papers
study of new quinazolinone derivatives due to their diverse
pharmacological properties. A number of new derivatives
and their complexes have been synthesised and their prop-
erties extensively investigated, including their chemistry,
molecular structure, biological activities, and applications
in various felds (Sawant et al. 2009; Khan et al. 2010;
Chawla and Batra 2013; Rakesh et al. 2015). Recently,
quinazolinone chemistry has undergone rapid development
because of its extensive potential in medicinal chemistry
and its relevance to cancer chemotherapy (Zahedifard et al.
2015). Derivatives which displayed considerable antioxi-
dant properties were screened for their DNA protective
activity (Kuntikana et al. 2016). The properties of quina-
zolinone derivatives vary considerably with even minor
modifcations in their molecular structure. Introduction of
substituents together with their specifc positions in the
aromatic ring determine whether they behave as antioxi-
dants, cytotoxic or mutagenic agents (Gawad et al. 2010;
Kumar et al. 2011).
potential of new 2,3-di-substituted-2,3-dihydro-quinazolin-
4(1H)-ones aiming at potentially useful anticancer agents.
Experimental
Materials and methods
All chemicals were of reagent grade and used without further
purifcation. High-resolution NMR spectra were recorded
in a 5 mm cryoprobe at 25°C on a Bruker Avance III HD
spectrometer, at 14 T in DMSO-d6. The proton and carbon
chemical shifts were referenced to TMS. One-dimensional
1
600 MHz H and 150 MHz 13C NMR spectra as well as
two-dimensional COSY, HSQC and HMBC were used for
determination of 1H and 13C chemical shifts. Chemical shifts
(δ) are quoted in ppm and spin–spin coupling constants (J)
in Hertz. FT-IR spectra were measured on a Nicolet 6700
spectrometer (Thermo Fisher Scientifc) with DTGS detec-
tor and OMNIC 8.0 software using 128 scans at the resolu-
tion of 4 cm−1 with diamond ATR technique. Melting points
were determined on a Kofer hot stage and are uncorrected.
HRMS were acquired on an Orbitrap Velos PRO (Thermo
Scientifc), with electrospray ionization method, operated
in positive mode. Microwave reactions were performed in a
multimode microwave reactor CEM Discover consisting of a
continuously focused microwave power delivery system with
an operator-selectable power of 0–300 W and a microwave
frequency source of 2.45 GHz. The progress of the reactions
was monitored by thin layer chromatography (TLC) on alu-
minium sheets pre-coated with Silica Gel 60 F254 (Merck).
Solvent A (ethyl acetate/n-hexane/methanol 7:1:2 v/v/v);
solvent B (ethyl acetate/chloroform/methanol 1:1:0.5 v/v/v).
In the current context of sustainability, there is a grow-
ing demand for development of more efcient synthetic
strategies, which can involve improvement in selectivity,
elimination of hazardous chemicals, recovery, and reuse
of reagents. Exploration of alternative green technologies
in the synthesis of quinazolinone-derived Schif’s bases
(the use of metallic or non-metallic catalysts, mechano-
chemical, ultrasonic or microwave activation) led to the
development of novel efcient synthetic protocols (Mar-
tins et al. 2009; Wang et al. 2011; Zhang et al. 2016;
Kuntikana et al. 2016). However, there is still a need to
develop economical and environmentally friendly routes
to obtain the desired compounds. Recently, solid heter-
opoly acids (HPA) have received a signifcant attention as
they exhibit high catalytic activity and selectivity. HPA
possess high Brønsted acidity and show higher catalytic
activity than mineral acids but, at the same time, they are
environmentally friendly, low in toxicity, non-corrosive,
and economically cost efective (Kozhevnikov 1998; Fir-
ouzabadi and Jafari 2005). Applications of HPA in combi-
nation with unconventional energy sources have markedly
improved the traditional methods of organic synthesis.
Microwave irradiation (MW), as a clean energy source, has
gained acceptance as an efcient tool and environmentally
friendly technique (Loupy 2006).
Determination of antioxidant activity
UV/Vis measurements were performed by means of a UV/
Vis spectrometer (Shimadzu UV-3600) at 25 °C. The solu-
tion of semi-persistent free radical cation ABTS•+ was pre-
pared by dissolving of 2,2′-azino-bis(3-ethylbenzothiazo-
line-6-sulfonate diammonium salt), (ABTS, Sigma-Aldrich)
and potassium persulfate in distilled water and storing in
the dark for 16 h before use (Walker and Everette 2009).
The stock solution of ABTS•+ was prepared by dilution
of the stored solution (1 ml) with 60 ml of distilled water
to an overall concentration of 75 µM (Re et al. 1999). The
ABTS•+ solution was mixed with 100 µl of the sample solu-
tions (DMSO, 1 mM) to a total volume of 3 ml (30:1 v/v)
before the measurement. The antioxidant capacity of the
studied compounds 1–7 was monitored by EPR spectroscopy
on EPR spectrometer Bruker EMX Plus, using the ABTS•+
free radical. A water-soluble analogue of vitamin E (Trolox,
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid,
Taking into consideration the wide range of applica-
tions of quinazolinone-based compounds in the feld of
pharmaceutical chemistry, we decided to synthesize a new
series of quinazolinone-derived Schif’s bases. We wish to
demonstrate that phosphomolybdic acid, supported on sil-
ica gel (PMoA/SiO2), could act as an efcient catalyst for
their preparation. Herein we report the microwave-assisted
and PMoA-catalysed synthesis, structural analysis, radical
scavenging capacity and evaluation of the antiproliferative
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Chemical Papers
Sigma-Aldrich) was used as an antioxidant standard. The
solution of ABTS•+ (67 µM) with a total volume of 1000 µl
was added to one syringe. The samples (1 mM) dissolved
in DMSO were transferred to the second syringe and meas-
ured under identical conditions as during the UV/Vis experi-
ments. After simultaneous injection of both solutions into
the cell (WG-814-Q, Wilmad LabGlass), the radical con-
centration was monitored for 5 min. The EPR spectra were
recorded and evaluated using WinEPR (Bruker) software.
Calculations and plots were processed using OriginPro 2016
(OriginLab).
50 μl of 1 mM MTT solution for 4 h. After incubation, the
MTT solution was removed, the formazan crystals in each
well dissolved in DMSO (100 μl) and placed on an orbital
shaker for 30 min. The absorbance intensity was measured
using an xMark™ Microplate Spectrophotometer (Bio-Rad
Lab) at 540 nm with a reference wavelength of 690 nm. All
experiments were performed in triplicate, and the relative
cell cytotoxicity was expressed as a percentage relative to
the untreated control cells.
Synthesis and spectral data
Cytotoxicity screening assay
General procedure for the synthesis
of 2,3‑disubstituted‑2,3‑dihydro‑quinazolin‑4‑one derived
Schif’s bases under conventional conditions
The in vitro cytotoxicity screening was assessed using a
malignant human hepatocellular cell line (HepG2) obtained
from Prof. A. R. Collins (University of Oslo). The cells were
cultured in RPMI 1640 medium supplemented with 10%
fetal calf serum and antibiotics (penicillin 200 U/ml, strep-
tomycin 100 μg/ml, kanamycin 100 μg/ml) on Petri dishes at
37 °C in humidifed atmosphere of 5% CO2. The cytotoxicity
of compounds 1–7 was monitored by MTT cell proliferation
assay (Mosmann 1983; Berridge et al. 2005). In this assay
the yellow tetrazolium salt MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) is reduced by meta-
bolically active cells to purple formazan that can be spectro-
photometrically quantifed. HepG2 cells were seeded into a
set of 96 well plates at a density of 2 × 104/well, and cultured
in the RPMI 1640 medium. Exponentially growing cells
were then pre-incubated in the presence of diferent con-
centrations of the tested compounds (1, 2, 5, 10, 20, 50, 120,
240, 480 µg/ml) for 24 h. Stock solutions of the tested sam-
ples 1–7 (96 mg/ml) in DMSO, and serial dilutions thereof,
were prepared before the particular experiment. Cells treated
with the medium only served as a negative control. After
the treatment with 1–7, the properly treated HepG2 cells
were incubated in 100 μl of complete RPMI medium and
A solution of 2-aminobenzhydrazide (ABH, 1 mmol) and the
corresponding aromatic aldehyde (2 mmol) in dry ethanol
(30 ml), in the presence of a catalytic amount of PMoA/
SiO2 (0.06 mmol, based on PMoA) (Hricovíniová 2016)
was refuxed for 3−6 h. The progress of the reaction was
checked by TLC and the composition of the reaction mix-
ture was determined by 1H NMR spectroscopy. The catalyst
was fltered, washed with a small amount of ethanol, and
dried. The reaction mixture was concentrated to syrup and
the crude product thus obtained was purifed by recrystal-
lization (Scheme 1).
General procedure for the synthesis of 2,3‑disubsti‑
tuted‑2,3‑dihydro‑quinazolin‑4‑one derived Schif’s bases
under microwave‑assisted conditions
A solution of ABH (1 mmol) and the corresponding aro-
matic aldehyde (2 mmol) in dry ethanol (30 ml), in the pres-
ence of a catalytic amount of PMoA/SiO2 (0.06 mmol, based
on PMoA) (Hricovíniová 2016) was mixed in a Pyrex glass
tube and sealed with a Tefon septum. The reaction mixture
5
O
O
NH2
EtOH, PMoA/SiO2
MW 10-20 min
N
O
H
N
H
N
+
R
C
NH2
N
H
5
1-7
NO2
OH
OCH3
NO2
OH
OH
OH
OH
NO2
Cl
OH
5 ꢀ
OCH3
OCH3
OCH3
NO2
Scheme 1 Microwave-assisted, PMoA-catalysed synthesis of Schif’s bases 1–7
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Chemical Papers
was exposed to microwave irradiation (200 W) for an appro-
priate time (5–20 min). Samples (0.5 ml) were measured at
selected intervals and the composition of the reaction mix-
ture was determined by 1H NMR spectroscopy. The PMoA/
SiO2 catalyst from the reaction mixture was fltered of. The
desired products 1–7 were obtained using the same work-up
procedure as listed for the conventional approach.
J = 8.2 Hz, 1H, H-8), 6.811 (dd, J = 8.2 Hz , J = 7.4 Hz, 1H,
H-6), 6.675 (d, J = 3.2 Hz, 1H, H-2); 13C NMR (DMSO-d6,
150 MHz) δ: 160.53 (C=O), 147.83 (C-3″), 147.76 (=CH),
147.38 (C-3′), 145.65 (C-8a), 140.81 (C-1′), 135.07 (C-1″),
134.68 (C-7), 132.29 (C-5″), 132.01 (C-5′), 131.82 (C-6″),
131.39 (C-6′), 128.32 (C-5), 126.31 (C-4″), 125.14 (C-4′),
123.90 (C-2′), 123.76 (C-2″), 118.62 (C-6), 115.17 (C-8),
114.35 (C-4a), 70.73 (C-2).
3‑((4‑nitrobenzylidene)
amino)‑2‑(4‑nitrophenyl)‑2,3‑dihydroquinazo‑
lin‑4(1H)‑one (1)
3‑((2‑hydroxy‑5‑nitrobenzylidene)amino)‑2‑(2
‑hydroxy‑5‑nitrophenyl)‑2,3‑dihydroquinazo‑
lin‑4(1H)‑one (3)
Yellow solid; yield 94.8%; m.p. 209–211 °C; Ref (Hricov-
íni and Hricovíni 2017) m.p. 207–208 °C; Ref (Fülöp et al.
1992) m.p. 215–217 °C; Rf 0.78 (solvent A); FT-IR (ATR,
diamond): νmax: 3380 (N–H), 3110 (C–H)Ar, 1648 (C=O),
Yellow solid; yield 96.9%; m.p. 247–249 °C; Ref
(Zahedifard et al. 2015) m.p. 250–252 °C); Rf 0.61 (sol-
vent A); UV–Vis (DMSO): λmax 298, 427 nm; HRMS for
C21H15N5O7Na: calcd [M + Na]+ 472.3629; found 472.3631;
analytical data (1H NMR, 13C NMR, FT-IR) were in agree-
ment with those recently published (Zahedifard et al. 2015).
Our 1H NMR and 13C NMR data are shown for better com-
1608 (HC=N), 1335, 1333 (NO2), 1290 (N–N) cm−1
;
UV–Vis (DMSO): λmax 344 nm; HRMS for C21H15N5O5Na:
calcd [M + Na]+ 440.3641; found 440.3638; 1H NMR and
13C NMR were in agreement with those recently published
(Hricovíni and Hricovíni 2017) and our data are shown for
better comparison with the other quinazolinone derivatives.
1H NMR (DMSO-d6, 600 MHz) δ: 9.092 (s, 1H, N=C–H),
8.296 (d, J = 8.8 Hz, 2H, H-3″, H-5″), 8.230 (d, J = 8.9 Hz,
2H, H−3′, H-5′), 8.154 (d, J = 3.4 Hz, 1H, N–H), 7.968
(d, J = 8.8 Hz, 2H, H-2″, H-6″), 7.748 (d, J = 7.5 Hz, 1H,
H-5), 7.637 (d, J = 8.9 Hz, 2H, H-2′, H-6′), 7.350 (dd,
J = 8.6 Hz, J = 6.8 Hz, 1H, H-7), 6.853 (d, J = 8.2 Hz, 1H,
H-8), 6.787 (dd, J = 7.5 Hz, J = 6.8 Hz, 1H, H-6), 6.763 (d,
J = 3.4 Hz, 1H, H-2); 13C NMR (DMSO-d6, 150 MHz) δ:
160.61 (C=O), 148.12 (C-4″), 147.91 (=CH), 147.48 (C-4′),
146.95 (C-1′), 145.81 (C-8a), 140.71 (C-1″), 134.62 (C-7),
128.32 (C-5), 128.22 (C-6″), 128.22 (C-2″), 127.63 (C-6′),
127.63 (C-2′), 124.06 (C-5″), 124.06 (C-3″), 123.79 (C-5′),
123.79 (C-3′), 118.45 (C-6), 115.14 (C-8), 114.41 (C-4a),
71.03 (C-2).
1
parison with other quinazolinone derivatives. H NMR
(DMSO-d6, 600 MHz) δ: 8.825 (s, 1H, N=C–H)), 8.442 (d,
J = 2.9 Hz, 1H, H-6″), 8.159 (dd, J = 9.2 Hz, J = 2.9 Hz,
1H, H-4″), 8.107 (d, J = 9.2 Hz, J = 2.9 Hz, 1H, H-4′),
7.879 (d, J = 2.9 Hz, 1H, H-6′), 7.824 (d, J = 7.9 Hz, 1H,
H-5), 7.697 (d, J = 2.5 Hz, 1H, N–H), 7.336 (dd, J = 8.3 Hz,
J = 6.7 Hz, 1H, H-7), 7.101 (d, J = 9.2 Hz, 1H, H-3″), 7.086
(d, J = 9.2 Hz, 1H, H-3′), 6.849 (mult., 1H, H-8), 6.841
(mult., 1H, H-2), 6.796 (dd, J = 7.9 Hz, J = 6.7 Hz, 1H,
H-6); 13C NMR (DMSO-d6, 150 MHz) δ: 162.73 (C-6″),
161.67 (C-6′), 160.40 (C=O), 146.64 (=CH), 145.96 (C-8a),
139.82 (C-1″), 139.14 (C-1′), 134.60 (C-7), 128.18 (C-5),
126.86 (C-4″), 126.38 (C-4′), 125.10 (C-3′), 124.73 (C-2″),
122.21 (C-2′), 119.45 (C-3″), 118.11 (C-6), 117.36 (C-5″),
116.38 (C-5′), 114.98 (C-8), 113.14 (C-4a), 66.41 (C-2).
3‑((4‑hydroxy‑3‑methoxy‑5‑nitroben‑
zylidene)amino)‑2‑(4‑hydroxy‑3‑meth‑
oxy‑5‑nitrophenyl)‑2,3‑dihydro‑
quinazolin‑4(1H)‑one (4)
3‑((4‑chloro‑3‑nitrobenzylidene)amino)‑2‑(4‑chloro‑
3‑nitrophenyl)‑2,3‑dihydroquinazolin‑4(1H)‑one (2)
Light yellow solid; yield 90.4%; m.p. 211–213 °C; Rf 0.74
(solvent A); FT-IR (ATR, diamond): νmax: 3377 (N–H), 3103
(C–H)Ar, 1650 (C=O), 1529 (HC=N), 1353 (NO2), 1299
(N–N), 1149 (C–N); 808 (C–Cl) cm−1; UV–Vis (DMSO):
λmax 304, 364 nm; HRMS for C21H13Cl2N5O5Na: calcd
[M + Na]+ 509.2542; found 509.2546; 1H NMR (DMSO-d6,
600 MHz) δ: 9.068 (s, 1H, N=C–H), 8.353 (d, J = 1.9 Hz,
1H, H-2″), 8.128 (d, J = 2.2 Hz, 1H, H-2′), 8.081 (d,
J = 3.2 Hz, 1H, N–H), 8.018 (d, J = 8.5 Hz, J = 1.9 Hz, 1H,
H-6″), 7.866 (d, J = 8.5 Hz, 1H, H-5″), 7.772 (d, J = 8.6 Hz,
1H, H-5′), 7.751 (dd, J = 7.7 Hz, J = 1.2 Hz, 1H, H-5),
7.598 (d, J = 8.6 Hz, J = 2.2 Hz 1H, H-6′), 7.370 (ddd,
J = 8.2 Hz, J = 7.4 Hz, J = 1.2 Hz, 1H, H-7), 6.855 (d,
Orange solid; yield 77.4%; m.p. 225–227 °C; Rf 0.74
(solvent A); FT-IR (ATR, diamond): νmax: 3497 (O–H),
3281 (C–H)Ar, 3083 (N–H), 1662 (C=O), 1543 (HC=N),
1262 (N–N), 1143 (C–N), 1101 (C–O), 1383 (NO2), 1059
(O–CH3) cm−1; UV–Vis (DMSO): λmax 365, 455 nm; HRMS
for C23H19N5O9Na: calcd [M + Na]+ 532.4148; found
1
532.4135; H NMR (DMSO-d6, 600 MHz) δ: 10.959 (s,
1H, OH”), 10.572 (s, 1H, OH’), 8.877 (s, 1H, N=C–H),
7.873 (d, J = 2.3 Hz, 1H, N–H), 7.806 (d, J = 1.3 Hz, 1H,
H-6″), 7.741 (d, J = 7.8 Hz, 1H, H-5), 7.559 (d, J = 1.3 Hz,
1H, H-2″), 7.401 (m, 2H, H-6′, H-2′), 7.346 (dd, J = 8.2 Hz,
J = 7.8 Hz, 1H, H-7), 6.839 (d, J = 8.2 Hz, 1H, H-8), 6.788
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Chemical Papers
(t, J = 7.8 Hz, 1H, H-6), 6.462 (d, J = 2.3 Hz, 1H, H-2),
3.904 (s, 3H, OMe′), 3.821 (s, 3H, OMe″); 13C NMR
(DMSO-d6, 150 MHz) δ: 160.66 (C=O), 150.67 (=CH),
149.78 (C-3″), 149.54 (C-3′), 146.10 (C-8a), 144.49 (C-4″),
142.74 (C-4′), 137.10 (C-5″), 136.35 (C-5′), 134.24 (C-7),
130.25 (C-1′), 128.13 (C-5), 125.02 (C-1″), 118.23 (C-6),
116.26 (C-6″), 114.87 (C-8), 114.57 (C-6′, C-2′), 113.84
(C-4a), 112.54 (C-2″), 71.56 (C-2), 56.59 (OMe′), 56.52
(OMe″).
130.93 (C-1′), 127.88 (C-5), 125.81 (C-1″), 122.11 (C-2″),
118.98 (C-6), 117.57 (C-2′), 115.40 (C-3″) 114.88 (C-3′),
114.61 (C-8), 111.07 C-6′), 109.67, C-6″), 72.11 (C-2),
55.56 (OCH3″), 55.52 (OCH3′).
3‑((3,4‑dihydroxybenzylidene)amino)‑2‑(3,4‑dihydr
oxyphenyl)‑2,3‑dihydroquinazolin‑4(1H)‑one (7)
Of white solid; yield 65.8%; m.p. 201–203 °C; Rf 0.57 (sol-
vent B); FT-IR (ATR, diamond): νmax: 3421 (O–H), 3291
(N–H), 3067 (C–H)Ar, 1608 (C=O), 1514 (HC=N), 1281
(N–N), 1143 (C–N), 1115 (C–O) cm−1; UV–Vis (DMSO):
λmax 302, 338 nm; HRMS for C21H17N3O5Na: calcd
[M + Na]+ 414.3666; found 414.3662; 1H NMR (DMSO-
d6, 600 MHz) δ: 9.280 (s, 2H, O–H(4)’, O–H(4)”), 8.890 (s,
2H, O–H(3)’, O–H(3)”), 8.530 (s, 1H, N=C–H), 7.686 (m,
1H, H-5), 7.682 (m, 1H, H-2′) 7.602 (s, N–H), 7.262 (dd,
J = 8.0 Hz, J = 7.3 Hz, 1H, H-7), 7.192 (s, 1H, H-2″), 6.926
(d, J = 8.1 Hz, 1H, H-6″), 6.763 (m, H, H-8), 6.760 (m, 1H,
H-5′’), 6.707 (t, 1H, J = 8.0 Hz, J = 7.1 Hz, H-6), 6.640 (m,
2H, H-5′, H-6′), 6.214 (s, 1H, H-2); 13C NMR (DMSO-d6,
150 MHz) δ: 160.37 (C=O), 152.34 (=CH), 148.24 (C-3″),
146.06 (C-8a), 145.59 (C-3′), 145.30 (C-4′), 144.97 (C-4″),
133.56 (C-7), 131.49 (C-2′), 131.15 (C-1′), 127.89 (C-5),
125.84 (C-1″), 121.03 (C-6″), 117.44 (C-6), 117.35 (C-5′),
115.33 (C-8), 114.84 (C-4a), 113.77 (C-5″), 112.86 (C-2″),
71.51 (C-2).
3‑((4‑hydroxy‑3,5‑dimethoxybenzylidene)amino)
‑2‑(4‑hydroxy‑3,5‑dimethoxyphenyl)‑2,3‑dihydro‑
quinazolin‑4(1H)‑one (5)
Light yellow solid; yield 66.9%; m.p. 175–176 °C; Rf 0.69
(solvent B); FT-IR (ATR, diamond): νmax: 3507 (O–H), 3380
(N–H), 3117 (C–H)Ar, 1641 (C=O), 1609 (HC=N), 1250
(N–N), 1151 (C–N), 1111 (C–O), 1042 (O–CH3) cm−1
;
UV–Vis (DMSO): λmax 335 nm; HRMS for C25H251N3O7Na:
calcd [M + Na]+ 502.4717; found 502.4726; H NMR
(DMSO-d6, 600 MHz) δ: 8.714 (s, 1H, N=C−H), 7.968 (dd,
J = 8.2 Hz, J = 7.1 Hz, 1H, H-7), 7.712 (d, J = 7.8 Hz, 1H,
H-5), 7.659 (s, 1H, N–H), 6.923 (s, 2H, H-2″ H-6″), 6.816
(d, J = 8.2 Hz, 1H, H-8), 6.745 (dd, J = 7.8 Hz, J = 7.1 Hz,
1H, H-6), 6.704 (s, 2H, H-2′, H-6′), 6.287 (s, 1H, H-2);
13C NMR (DMSO-d6, 150 MHz) δ: 160.64 (C=O), 152.76
(=CH), 148.02 (C-3″, C-5″), 147.66 (C-3′, C-5′), 146.45
(C-8a), 138.23 (C-4″), 135.72 (C-4′), 133.69 (C-7), 130.05
(C-1′), 127.86 (C-5), 124.61 (C-1″), 117.67 (C-6), 114.89
(C-4a), 114.61 (C-8), 104.90 (C-2″, C-6″), 104.61 (C-2′,
C-6′), 72.64 (C-2).
Results and discussion
Chemistry
3‑((4‑hydroxy‑3‑methoxybenzylidene)amino)‑2‑(4
‑hydroxy‑3‑methoxyphenyl)‑2,3‑dihydroquinazo‑
lin‑4(1H)‑one (6)
The frst part of our work was aimed at the synthesis of
Schif’s bases derived from the quinazolinone heterocyclic
skeleton. We have focused on the development of a method
that is simple, fast, and offers high yields of products.
Schif’s bases are formed by condensation of the primary
amines and structurally diverse aldehydes. The direct con-
densation reaction occurs at high temperatures and this ther-
mal approach is very practical in many cases (Fülöp et al.
1992; Gawad et al. 2010). We decided to build on our pre-
vious experience in this feld by exploring the efect of the
reusable heterogeneous PMoA/SiO2 catalyst and microwaves
(Hricovíniová 2016) on the synthesis of a homologous series
of quinazolin-4-one derived Schif’s bases.
Of white solid; yield 74.9%; m.p. 190–192 °C; Rf 0.75
(solvent B); FT-IR (ATR, diamond): νmax: 3497 (O–H),
3281 (C–H)Ar, 3083 (N–H), 1662 (C=O), 1543 (HC=N),
1262 (N–N), 1143 (C–N), 1101 (C–O), 1383 (NO2), 1059
(O–CH3) cm−1; UV–Vis (DMSO): λmax 332 nm; HRMS
for C23H21N3O5Na: calcd [M + Na]+ 442.4197; found
442.4192; 1H NMR (DMSO-d6, 600 MHz) δ: 9.552 (s, 1H,
O–H″), 9.033 (s, 1H, O–H′), 8.683 (s, 1H, N=C–H), 7.698
(d, J = 7.7 Hz, 1H, H-5), 7.652 (d, J = 2.2 Hz, 1H, N–H),
7.284 (dd, J = 8.1 Hz, J = 7.3 Hz, 1H, H-7), 7.265 (s, 1H,
H-6″), 7.105 (d, J = 8.1 Hz, 1H, H-2″), 7.056 (s, 1H, H-6′),
6.818 (d, J = 8.1 Hz, 1H, H-3″), 6.788 (d, J = 8.1 Hz, 1H,
H-8), 6.735 (m, 2H, H-2′, H-6), 6.672 (d, J = 8.2 Hz, 1H,
H-3′), 6.287 (d, J = 2.2 Hz 1H, H-2), 3.783 (s, 3H, OCH3″),
3.701 (s, 3H, OCH3′); 13C NMR (DMSO-d6, 150 MHz) δ:
160.57 (C=O), 153.12 (=CH), 149.25 (C-5″), 147.87 (C-4″),
147.42 (C-4′), 146.59 (C-5′), 146.31 (C-8a), 133.65 (C-7),
Adjacent amino and hydrazino functional groups in het-
erocyclic compounds provide opportunities for construction
of an additional ring system. Primary amines and aldehydes
react under acid catalysis to form imine derivatives as the
condensation products. The reaction is reversible and the
formation of imine requires the presence of acid, which is
necessary for subsequent elimination of water. Our attention
1 3

Chemical Papers
Table 1 Data of the microwave-assisted, PMoA-catalysed cyclocon-
densation reaction of ABH with variably substituted aromatic alde-
hydes
was aimed at the modifcation of the aryl moiety at positions
2 and 3 of the quinazolin-4-one scafold to yield structural
analogues with appreciable biological activity.
Schif’s base
Microwave feld
Oil-bath heating
Acid hydrazides are useful intermediates for the syn-
thesis of N-heterocyclic compounds. The procedure for
building up such a heterocyclic system is based on the
cyclization reaction in dry solvent. As shown in Scheme 1,
acid-catalysed cyclocondensation of aromatic hydrazide
with various aromatic aldehydes provided the correspond-
ing 2,3-disubstituted-2,3-dihydro-quinazolin-4-ones. In
a typical experiment, a mixture of 2-aminobenzhydrazide
(ABH, 1.0 equiv.) and an aromatic aldehyde (2.0 equiv.)
was dissolved in absolute ethanol, and, in the presence of
acid catalyst, was refuxed in an oil-bath at 85 °C. After
several hours, the crude product thus obtained was puri-
fed by recrystallization from an appropriate solvent. In this
study, a new catalytic system based on phosphomolybdic
acid supported on silica gel (PMoA/SiO2) has been tested.
We screened the efect of diferent acid promoters by test-
ing several acid catalysts. Experiments using glacial acetic
acid, sulfuric acid, p-toluenesulfonic acid and PMoA/SiO2
gave interesting results. We also examined whether MW
irradiation could facilitate the cyclo condensation reaction
to form the desired Schif’s bases. The efect of MW irradia-
tion on conversion was explored using a multimode micro-
wave reactor consisting of a continuous focus MW power
delivery system with operator-selectable power. Prelimi-
nary results showed considerable improvements in Schif’s
base formation when using MW irradiation compared to the
conventional method. Both yields and the composition of
the reaction mixtures depended on the length of exposure,
as well as on the structure of the aromatic aldehyde. The
thermal efects of irradiation arise from the dissipation of
energy into heat as a result of intermolecular friction when
the dipoles change their mutual orientation on the alterna-
tion of the electric feld at a high frequency. Thus, MW
irradiation caused a remarkable acceleration of the conden-
sation reaction due to efcient energy transfer and, conse-
quently, both the reaction kinetics and the yields markedly
increased. The Schif’s base syntheses were carried out in
a microwave reactor at a power of 200 W, and took around
10–20 min. The three-component reaction employing ABH,
aromatic aldehyde and acid catalyst in ethanol proceeded
smoothly, and all catalysts tested exhibited good activity.
However, the cyclo condensation reaction proceeded most
efectively in the presence of PMoA/SiO2. At the end of
the reaction, the catalyst was recovered by simple fltration,
washed with ethanol and dried at 160 °C for 2 h. The cata-
lyst could be reused several times in the subsequent reaction
without notable loss of activity. This approach yielded the
required 2,3-disubstituted-quinazolinones in higher yields
in considerably shorter times (Table 1). It should be noted
that applying power exceeding 200 W did not improve the
Time (min)
Yield %
Time (min)
Yield %
1
2
3
4
5
6
7
10
15
15
15
20
15
20
95
90
97
77
67
75
66
180
180
240
300
300
360
360
88
80
89
65
56
66
48
Conversions are also shown for conventional oil-bath heating to
demonstrate the diferences between microwave and conventional
approaches
product yield. Experimental results clearly showed that MW
irradiation increased the reaction rate (10–20 min) and the
yield (66–97%) compared to conventional heating (3–6 h,
yield 48–89%). Our experimental results are in agreement
with recent developments in quinazolinone synthesis, where
microwave irradiation has provided a major advantage over
conventional thermal methods (Besson and Chosson 2007).
The worth of microwave-assisted synthesis has been proven
in spectacular acceleration of reaction times (reduced from
hours to minutes), increased yields, decreased side-products,
and simple reaction setup also in other organic reactions
(Hricovíniová 2006, 2010, 2016).
NMR spectral analysis
The structures of the new compounds were established
by 1D and 2D NMR spectroscopic studies. The structural
assignments based on the NMR, IR, and HRMS data are
listed in the experimental part. High-resolution NMR spec-
tral data confrmed the proposed structures of the synthe-
sized compounds 1–7. The chemical shifts and three-bond
proton–proton coupling constants (3JH–H) for H-5–H-8 were
not much afected by substitution at the aromatic rings in
the various compounds. Chemical shifts for these four pro-
tons varied by about 0.1 ppm for 1–7 in DMSO. The most
deshielded one was H-5 (having chemical shifts close to
7.7 ppm in all seven derivatives) whereas H-6 resonated in
the interval of 6.7–6.8 ppm. Slightly bigger variations in
chemical shifts values were shown by azomethine and the
N–H protons—both varied by about 0.4 ppm for 1–7. The
azomethine proton was most deshielded in 1 (9.09 ppm)
and 2 (9.07 ppm) which contain nitro group (para- or meta-
substituted) but have no substituent at the ortho- position
in the ring. On the other hand, the presence of OH groups
caused shielding of this proton resulting in lower values of
1 3

Chemical Papers
the chemical shift in 3 (8.83 ppm) and 7 (8.53 ppm). Pro-
tons in the NH or OH (in 3–7) groups showed much higher
variability in chemical shift values. The NH protons were
most deshielded in 1 and 2 (δ > 8 ppm), while protons in
all the other derivatives showed chemical shifts in the range
FT‑IR spectra
The FT-IR spectra also provided valuable structural informa-
tion on the synthesised compounds. Formation of the new
quinazolin-4-one derived Schif’s bases 1–7 was inferred
from the appearance of sharp absorption bands in the IR
spectra due to the characteristic azomethine (CH=N) stretch
at 1514–1609 cm−1. Moreover, the broad absorption bands
observed in the spectra near 3300–3400 cm−1 could be
attributed to the (N–H) stretching vibrations of the quina-
zoline ring. The NO2 group stretch in 1, 2, 3, and 4 was
confrmed by the presence of strong bands in the range of
1333–1383 cm−1. The C–Cl (halogen) absorption frequency
band appeared at 808 cm−1(2). Bands characteristic for the
phenolic group (O–H) were observed at 3366–3507 cm−1
and the bands at approximately 1650 cm−1 were assigned to
the carbonyl groups (C=O).
1
of 7.6–7.9 ppm. As expected, the biggest variations in H
chemical shifts were detected for OH groups. Intramolecular
hydrogen bonds caused a strong increase of chemical shift
values in 3 (12.341 and 11.936 ppm) whereas the shift val-
ues of the OH protons fanked by the two methoxy groups
were considerably smaller in 5 (8.942 and 8.447 ppm). The
presence of these methoxy groups at each of the aromatic
rings thus strongly afected the shielding of these OH pro-
tons. Furthermore, the temperature dependence of the OH
groups showed that the intramolecular H-bonds were com-
peting with intermolecular hydrogen bonds with the solvent
(Hricovíni and Hricovíni 2017). In 3 the H–bond was formed
between the OH group and the nitrogen in the azomethine
group.
UV–Vis absorption spectra
The chemical shifts of protons at the two aromatic
rings (one linked to C-2 and the second to the azome-
thine group) varied considerably upon substitution. The
effects of the electron-withdrawing groups, such as the
nitro group, were seen clearly in 2, where the protons
H-2′ (δ = 8.13) and H-2″ (δ = 8.35 ppm) were con-
siderably deshielded. This group also showed similar
deshielding effects in 1 (for protons H-3′, H-3″, H-5′
and H-5″) and 4 (H-6″). On the other hand, the effect
of introducing methoxy groups was lower chemical
shifts values for the ring protons in 4 (H-2′ and H-2″,
δ = 7.40 ppm and δ = 7.56 ppm), in 5 (H-2′, H-2″,
H-6′ and H-6″, all having δ = 6.70–6.99 ppm) and in
6 (H-6′ and H-6″, δ = 7.06 ppm and δ = 7.27 ppm).
Obviously, the chemical shift values of the aromatic
protons were also affected by the presence of other sub-
stituents (OH and Cl). Apart from the methoxy protons,
the most shielded signal in 1–7 was that of the proton
in the –N–C2(H) group which resonated between 6.21
and 6.85 ppm. Similarly, to proton chemical shifts, the
carbon δ values also varied for 1–7. As expected, the
carbonyl signal was most deshielded (approximately
160 ppm), and, apart from the methoxy groups present
in 4–6 (δ ~ 56 ppm), the most shielded carbon was C-2
linked to two nitrogen atoms. The chemical shifts of the
latter carbon varied between 70.73 and 72.11 ppm for
all derivatives, except for 3. The considerably lower δ
value detected for C-2 in 3 (66.41 ppm) is due to the
effect of the OH group in the ortho- position in the
two aromatic rings linked to C-2 and to the azomethine
We also used UV–Vis spectroscopy to study the electronic
absorption spectra of the Schif’s bases 1–7. DMSO solu-
tions of the compounds were prepared at concentration of
0.1 mM and measured immediately after preparation. The
individual absorption maxima (λmax), and extinction coef-
fcients (εmax) of 1–7 are given in Table 2. The diferences
between the derivatives, bearing various functional groups,
are clearly visible from the individual maxima and the cal-
culated values of the extinction coefcients. Compounds 2–4
and 7 showed two separated absorption maxima, with the
frst in the range of 300‒370 nm and the second between
350 and 500 nm.
The excitations around 300 nm point to the presence
of a C=N bond conjugated with the aromatic ring system.
The absorption bands in the range from 330‒350 nm can
be attributed to π–π* transitions in systems where the phe-
nyl rings are conjugated with the non-bonding pairs and the
C=C double bonds. Such conjugation is typical for systems
with substituents containing groups with non-bonding pairs,
such as OH and NH groups. In the case of compound 1,
bearing the NO2 groups, the broad maximum is visible at
344 nm, belonging to the π‒π* transitions of the phenyl sys-
tems. The non-bonding pairs contribute to the conjugation
and are responsible for the relatively high absorbance. The
same is seen for derivative 7, which possesses only OH sub-
stituents, with λmax at 338 nm (π‒π* transitions) and 302 nm
(azomethine conjugation with aromatic system). The difer-
ences among these compounds are caused by the diferent
excited states arising from the varying type and position of
the substituent (Blevins and Blanchard 2004). This results in
a diferent distribution of electrons over the system, afect-
ing the excitation energies (Crompton and Lewis 2004). The
strong conjugation of the non-bonding electron pairs in the
1
group. The H and 13C NMR spectra of derivative 4,
as representative of these compounds, is presented in
Fig. 1.
1 3

Chemical Papers
Fig. 1 1H and 13C NMR spectra of compound 4
1 3

Chemical Papers
Table 2 The absorption maxima
and the extinction coefcients
of Schif’s bases 1–7 in DMSO
Schif’s base
λmax/nm
1
2
3
4
5
6
7
344
304
298
365
335
332
302
364
15380
7320
427
19700
29700
455
15560
3970
338
29210
26700
εmax/dm3 mol−1 cm−1
17860
17040
17330
OH groups, along with the positional efect of the substitu-
ents, results in high absorbance for compound 7 as seen from
the high extinction coefcient values. On the other hand, 5
and 6 have similar spectra and the maxima are close to each
other (λmax 335 and 332 nm, respectively) due to their very
similar structure, difering only in that 5 has two para-meth-
oxy groups and 6 only one. Methoxy groups are less conju-
gated and have a slightly less intense substituent efect than
the OH groups (Crompton and Lewis 2004), which results
in lower absorbance of compounds. Spectrum of compound
4 is slightly shifted (λmax 365 nm) due to the solvation efect
of DMSO, associated with intramolecular hydrogen bonds
causing a bathochromic shift of the maximum to a longer
wavelength (Díaz et al. 2009). The absorption maxima in the
range 350‒500 nm correspond to the n‒π* transitions typi-
cal for chromophores (N=N) and found in similar systems
(Nielsen et al. 2006). The very strong absorption band of 3
and 4 (λmax 427 nm for 3 and 455 nm for 4, respectively) is
caused by the auxochromic efect of NO2 coupled with the
efect of the OH groups. Moreover, the solvation efect of
DMSO molecules, as well as possible intramolecular hydro-
gen bonds, increases the bathochromic shift of the maximum
to longer wavelengths, as seen previously for similar systems
(Kaur et al. 2017). In the case of 2, the efect of the NO2
group is diminished by the chlorine substituent, which has a
very strong mesomeric efect and causes a redistribution of
electrons in the aromatic rings. Consequently, the absorption
maximum is there decreased to 364 nm and 304 nm (azome-
thine conjugation with aromatic system).
semi-persistent radicals with the investigated molecules. The
ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid)] salt was used as a source of the semi-stable radical
cation (ABTS•+). The antioxidant activity is determined by
the decolourisation of the ABTS•+ solution, i.e. the reduc-
tion of the radical cation is measured by its concomitant
decrease in absorbance. The UV–Vis measurements for
all investigated compounds were performed with aliquots
of ABTS•+ diluted to an absorbance of ~ 1.0 at 735 nm.
The kinetics of the reaction of ABTS•+ with the studied
compounds was monitored at a fxed wavelength (735 nm)
for a 15 min period, which was found to be sufcient time
to allow the system to reach a steady-state level. A signif-
cant drop in absorbance was observed in all samples (1–7)
immediately after starting the measurements. The absorb-
ance curves recorded during the reaction of ABTS•+ with
the selected Schif’s bases are shown in Fig. 2.
The results thus obtained indicated signifcant antioxi-
dant activity for all compounds. The number and position
of the aromatic hydroxyl groups is of crucial importance
in modulating the antioxidant capacity. Electron-donating
groups, especially alkyl and methoxy groups had previ-
ously been reported to increase the electron density of
phenoxyl radicals, leading to enhancement of the radi-
cal scavenging and antioxidant activity (Kajiyama and
Ohkatsu 2001; Kuntikana et al. 2016). The high activity of
α-tocopherol (vitamin E) was attributed to the para-alkoxy
Determination of antioxidant activity
Radical scavenging properties investigated by UV/Vis
spectroscopy
Antioxidant activity is directly related to the ability of com-
pounds to release hydrogen atoms. The mechanism lead-
ing to elimination of free radicals is based on the donation
of hydrogen to free radicals, reducing them to unreactive
species (Halliwell et al. 1999). The characterisation of
antioxidant properties by Trolox equivalent antioxidant
capacity (TEAC) is a suitable approach for investigations
of the radical-scavenging activity of compounds containing
condensed aromatic ring systems (Re et al. 1999; Tian and
Schaich 2013). A TEAC assay is based on the reaction of
Fig. 2 Time-dependent absorbance changes of ABTS•+ at 735 nm, in
the presence of Schif’s bases 1–7 in DMSO, compared to the refer-
ence solution (DMSO)
1 3

Chemical Papers
group and the methyl groups on the aromatic ring (Brees
et al. 2000). The data clearly showed that compound 7,
bearing multiple hydroxyl groups, exhibited the highest
quenching activity—the absorbance is a fat zero, indicat-
ing a quenching reaction so fast, it was over before record-
ing could begin. Compound 5, bearing four methoxy and
two hydroxyl groups on the aromatic rings, showed very
similar activity to that of 7. The NH group present in the
quinazolinone moiety also participates in the antioxidant
activity, but its efect is minor compared to the radical
scavenging activity of the OH or OCH3 groups. Both
derivatives thus manifested very high antioxidant activ-
ity, showing a massive initial absorbance drop with no
further reaction thereafter. The reaction between ABTS•+
and compound 6 showed a small decrease in antioxidant
activity compared to 5 and 7. The large initial drop in
absorbance was followed by an ongoing slow decrease of
the kinetic curve. This behaviour is typical for many small
aromatic compounds containing multiple OH groups (or
combined with an OCH3 group) along with an aliphatic
system, e.g. gallic acid and coniferyl alcohol (Walker and
Everette 2009).
The decrease of ABTS•+ concentration caused by the
antioxidant action of the tested compounds can be calculated
from the TEAC value, assuming that two ABTS•+ molecules
are eliminated by one Trolox molecule (Tian and Scha-
ich 2013). The TEAC values of the studied Schif’s bases
1–7 are summarized in Table 3. The absorbance changes
Table 3 The TEAC values of Schif’s bases 1–7 calculated from
kinetic and EPR measurements
Schif’s Structure
TEACABTS UV/Vis
,
TEACABTS,EPR
0.91 0.02
base
1
2
3
0.87 0.03
0.51 0.01
0.98 0.03
0.46 0.01
1.04 0.03
An interesting feature of this series of compounds is
the efect of halogenation, methoxylation and nitration of
aromatic rings. Replacement of a methoxy group with the
electron-withdrawing nitro group (4) led to diminution of
the antioxidant activity. The drop in absorbance showed a
similar but more gradual trend. Removal of the methoxy
group and variation of the position of the hydroxyl group
(3) resulted in another reduction of the free radical quench-
ing ability, caused by diferent electron distribution in the
aromatic ring system. In this case, a smaller intense decrease
at the start of recording was observed, followed by a slow
decrease of the absorbance, but not as signifcant as seen
for 4. The complete absence of OH groups in compound
(1) meant that the hydrogen of the NH group was the only
one involved in the radical-scavenging reaction. In this case,
there was a sharp initial drop at the start of recording, but
very little change afterwards. Completely diferent behav-
iour was observed when a chlorine atom was present on
the phenyl ring along with the nitro group (2). This curve
looked rather diferent to others, with a far more leisurely
initial absorbance drop, followed by gradual, steady decrease
spanning over the whole measurement period.
4
5
1.09 0.03
1.17 0.03
1.19 0.04
1.30 0.04
6
7
1.17 0.04
1.21 0.04
1.27 0.03
1.36 0.04
The antioxidant activity of a given compound is charac-
terized by the TEAC value (Trolox equivalent antioxidant
capacity):
(Af − A0)sample
TEAC =
,
(Af − A0)trolox
where Af is the fnal absorbance and A0 is the initial ABTS•+
absorbance value of the sample or Trolox standard.
1 3

Chemical Papers
observed in our data suggest that the molecular structure and
steric accessibility to the hindered radical site in ABTS•+
are the dominant factors controlling the reaction rates of our
compounds with ABTS•+.
are in good agreement with the kinetic experiments. The
highest TEAC values were found for 5, 6 and 7, refecting
their high antioxidant activity, as seen in the individual spec-
tra of 6 in Fig. 3. The spectra of compounds 3, 4 and 6 were
recorded with 10 × diluted samples with a concentration of
1.10−4 M, to avoid signal extinction before the experiment
could begin. It should also be noted that the signal intensi-
ties of 5 and 7 diminished quite rapidly during the record-
ing period (even at 1.10−4 M concentration) due to the fast
extinction of the radicals. The EPR results thus confrmed
the strong antioxidant properties of the investigated Schif’s
bases antioxidants in the presence of radical ABTS•+. Minor
diferences (without any efect on the antioxidant activi-
ties of 1–7) between the TEAC values obtained from the
UV–Vis and EPR measurements were possibly caused by
the diferent settings of the experiments. A minor delay
arose between the mixing of the solutions and the start of
measurement, due to transfer of the solution mixture from
syringes to the cell in EPR spectrometer. Nevertheless, both
experimental methods, UV–Vis and EPR, indicated all the
investigated Schif’s bases to possess very good antioxidant
properties, making them worthy of further study regarding
their biological activity.
Radical scavenging properties investigated by EPR spec‑
troscopy
The antioxidant activity of the investigated Schif’s bases
was also monitored via EPR spectroscopy. For this purpose,
a set of experiments was performed with all compounds 1–7
using the ABTS•+ radical. All the experiments were initiated
by mixing a sample of compound dissolved in DMSO with a
solution of ABTS•+ in distilled water. Figure 3 presents the
characteristic changes over time of the EPR spectra of fve
selected compounds.
The plots of the selected spectra over time show the
decrease of the ABTS•+ signal. No changes in the intensity
of the EPR spectra were observed in the sample-free refer-
ence (Fig. 3).
When performed with the Schif’s bases, however, all of
them showed clear changes in the ABTS•+ concentrations
over the period of the experiment (including 5 and 7, not
shown here). The double integral intensities of ABTS•+ were
evaluated for the time evolutions of all the experimental EPR
spectra. The ABTS•+ concentrations of individual samples
and the reference were calculated and used for determination
of the TEAC value. All data are summarized in Table 3 and
Determination of cytotoxic activity
MTT assay is commonly used for the evaluation of cell
proliferation and hence is a good indicator of cell death or
inhibition of growth. The synthesized compounds 1–7 were
subjected to the colorimetric MTT assay to be evaluated
for their in vitro cytotoxicity against the malignant human
hepatocellular HepG2 cell line. HepG2 was chosen because
these cells are used as a model system for studies of liver
metabolism and toxicity of xenobiotics, the detection of
environmental and dietary cytotoxic and genotoxic agents.
IC50 values (median inhibitory concentrations that cause
approximately 50% cell death) were: 2–417; 5–300; 6–310;
7–279 µg/ml (Fig. 4).
100
80
60
40
20
0
1
2
3
4
5
6
7
0
1
2
5
10 20 50 120 240 480
Concentraꢀon (µg/mL)
Fig. 3 Evolution over time of the EPR signals recorded after mixing a
solution of a Schif’s bases (1–4 and 6), or reference (DMSO), with a
Fig. 4 The in vitro cytotoxicity results toward the HepG2 cell line at
diferent concentrations of the Schif’s bases (1–7)
solution of the radical oxidant (ABTS•+
)
1 3

Chemical Papers
Acknowledgements The authors would like to acknowledge fnancial
support from the Slovak Grant Agency, VEGA Grants No. 2/0100/14,
1/0041/15, 2/0027/16, 2/0022/18 and SP Grant 2003SP200280203 sup-
ported by the Research & Development Operational Program funded
by the ERDF.
The experimental results revealed that the nature and
position of substituents on the aromatic rings afected
the magnitude of cytotoxic activity. Compound 1 bear-
ing an electron-withdrawing nitro group and 4 with mixed
electron-withdrawing and electron-donating substituents
did not show any cytotoxic activity. The presence of nitro
and hydroxy groups made 3 slightly active. Exchanging
hydroxy groups for chlorine (2) rendered this compound
more active, markedly enhancing the cytotoxic activ-
ity toward the HepG2 cells. The compounds 5, 6 and 7,
bearing multiple electron-donating methoxy and hydroxy
groups, afected the cell viability in a dose-dependent
manner, proving themselves the most active members of
this series. Overall, compounds 2, 3, 5–7 caused disrup-
tions in the cell cycle of the HepG2 cells. The studied
compounds exhibited only mild levels of antiproliferative
activity against HepG2 cell line. These preliminary results
merit additional investigations. Thus, these compounds
will be further studied with a more efficient disease-
oriented screening strategy employing various microbial
strains, and both normal and tumour human cell lines.
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