4-Methyl-7-hydroxycoumarin antifungal and antioxidant activity enhancement by substitution with thiosemicarbazide and thiazolidinone moieties

Article abstract of DOI:10.1016/j.foodchem.2013.01.027

According to literature data, thiosemicarbazide and thiazolidinone moieties should enhance biological properties of coumarin. Antioxidant, metal-chelating and antifungal activities of all compounds were investigated and compared to the activity of the sta

Full text of DOI:10.1016/j.foodchem.2013.01.027

Food Chemistry 139 (2013) 488–495
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
4-Methyl-7-hydroxycoumarin antifungal and antioxidant activity enhancement
by substitution with thiosemicarbazide and thiazolidinone moieties
b
ˇ ´ a
Bojan Šarkanj a, Maja Molnar a, , Milan Cacic , Lars Gille
⇑
ˇ
a
ˇ
Department of Applied Chemistry and Ecology, Faculty of Food Technology, J.J. Strossmayer University, Franje Kuhaca 20, 31 000 Osijek, Croatia
b Institute of Pharmacology and Toxicology, Department of Biomedical Sciences, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2012
Received in revised form 6 December 2012
Accepted 10 January 2013
Available online 23 January 2013
According to literature data, thiosemicarbazide and thiazolidinone moieties should enhance biological
properties of coumarin. Antioxidant, metal-chelating and antifungal activities of all compounds were
investigated and compared to the activity of the starting material, 7-hydroxy-4-methylcoumarin, and
were proven to possess potent antioxidant and antifungal activity. In general, thiosemicarbazides showed
higher scavenging activity towards DPPH and galvinoxyl radicals than did 4-thiazolidinones and some of
them had the same or even better activity than had ascorbic acid itself, depending on the free radical
used. In antifungal activity tests towards four foodborne mycotoxigenic fungi, Aspergillus flavus, Aspergil-
lus ochraceus. Fusarium graminearum and Fusarium verticillioides, coumarin derivatives were proven to
possess a very high activity in terms of growth inhibition, depending on the fungi investigated. In general,
4-thiazolidinones showed better antifungal activity than did thiosemicarbazides. F. graminearum was the
most susceptible to the compounds investigated and F. verticillioides was proven to be the most resistant.
Two compounds, both coumarinyl thiosemicarbazides, were found to possess both antifungal and anti-
oxidant activity which could be useful for applications in medicine, food industry and agriculture.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Coumarin
Antioxidant activity
Antifungal activity
Thiosemicarbazide
4-Thiazolidinone
1. Introduction
Substituted coumarins have important effects in plant biochem-
istry and physiology, acting as antioxidants (Kostova, 2005), anti-
´
One of the main goals in the food industry today is maintaining
the quality of food during handling, processing and storage. Many
factors can cause loss of nutritive and organoleptic properties of
food, such as molds and free radical-induced oxidation. Apart from
causing changes in appearance, some molds can cause numerous
health problems in both humans and animals.
fungals (Basile et al., 2009; Radulovic et al., 2006), enzyme
inhibitors and precursors of toxic substances (Kostova, 2005) and
the coumarinic ring is present in many pesticides and pharmaceu-
tical drugs (Lopes et al., 1995). Coumarins, both natural and syn-
thetic are compounds with many diverse biological activities
(Farshori, Banday, Ahmad, Khan, & Rauf, 2011; Manojkumar, Ravi,
& Subbuchettiar, 2009; Mohareb, El-Arab, & El-Sharkawy, 2009;
Sardari, Nishibe, & Daneshtalab, 2000). An antioxidant, auraptene
(7-geranyloxycoumarin), isolated from the peel of citrus fruit (Cit-
rus natsudaidai Hayata) has been reported to have chemopreven-
tive effects on chemically induced carcinogenesis (Kostova,
2005). Natural coumarin derivatives affect the formation and scav-
enging of ROS, and influence free radical-mediated oxidative dam-
age (Fylaktakidou, Hadjipavlou-Litina, Litinas, & Nikolaides, 2004)
and coumarins with a 1,3-thiazolidine-4-one moiety possess po-
According to many researchers there is a rapid emergence in
pathogen resistance to conventional antimicrobial agents (Radul-
´
ovic et al., 2006; Soltani, Dianat, & Sardari, 2009); thus, discovery
of new potent antifungal compounds is necessary.
New drugs with antioxidant and antifungal properties would be
of interest due to finding that the application of antioxidants af-
fects mycotoxin biosynthesis. It has been shown that high levels
of reactive oxygen species (ROS) can increase mycotoxin biosyn-
thesis (Narasaiah, Sashidhar, & Subramanyam, 2006; Reverberi,
Ricelli, Zjalic, Fabbri, & Fanelli, 2010). In fact, the results of Mab-
rouk and EI-Shayeb (1992) have confirmed that some coumarin
compounds possess antiaflatoxigenic properties on Aspergillus fla-
vus, especially on aflatoxin B1 biosynthesis.
ˇ
ˇ ´
tent antioxidant activity (Cacic, Molnar, Šarkanj, Has-Schön, &
Rajkovic, 2010). 7-Hydroxy-4-methylcoumarin derivatives possess
´
diverse biological properties, such as neuroleptic, antibacterial,
antitubercular, antifungal, antineoplastic, anti HIV, and antihel-
mintic (Pankaj, Sanjib, Namita, Ashish, & Baghel, 2010) and couma-
rins substituted with a thiosemicarbazide moiety also possess
antibacterial and antifungal properties (Arshad, Osman, Chan,
Goh, & Fun, 2010).
⇑
Corresponding author. Tel.: +385 31 224 378; fax: +385 31207 115.
E-mail addresses: bsarkanj@ptfos.hr (B. Šarkanj), maja.molnar@ptfos.hr (M.
ˇ
ˇ ´
Molnar), mcacic@ptfos.hr (M. Cacic), Lars.Gille@vetmeduni.ac.at (L. Gille).
0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.01.027

B. Šarkanj et al. / Food Chemistry 139 (2013) 488–495
489
Thiosemicarbazones and 4-thiazolidinones are two groups of
compounds with interesting biological activities (Haraguchi et al.,
2011; Ottana et al., 2007; Rawal, Tripathi, Katti, Pannecouquec, &
De Clercq, 2007; Vigorita et al., 2003). Thiosemicarbazides possess
many bioactivities, such as antibacterial, antifungal (Pelosi, 2010;
Sasse, Scheinpflug, & Grews, 1972; Yamaguchi et al., 2009), tran-
quillising, muscle relaxing, psychoanaleptic, hypnotic, ulcerogenic,
antidepressant, analgesic and anti-inflammatory (Khan, Kumar,
Joshi, Iqbal, & Saleem, 2008), as well as antitumor and antiviral
(Pelosi, 2010). Zhong, Zhong, Xing, Li, and Mo (2010) have shown
that the active –NH– and C@S groups in the chitosan thiosemicar-
bazone derivatives strongly increase the ferrous ion-chelating ef-
fect and free radical-scavenging activity. According to Singh,
Barwa, and Tyagi (2006) and Bagihallia, Patila, and Badami
(2009), N and S atoms in various molecules play an important role
in the coordination of metals at the active sites of numerous
metallobiomolecules.
4-Thiazolidinone derivatives are known to possess antibacte-
rial, antifungal, antiviral and antituberculosis properties (Küçükgü-
zel, Kocatepe, De Clercq, Sßahin, & Güllüce, 2006).
The substitution of 4-methyl-7-hydroxycoumarin at position 7
enhances its biological activity (Mouri, Yano, Kochi, Ando, & Hori,
2005). The aim of the present work was to synthesize derivatives
of 4-methyl-7-hydroxycoumarin, substituted in position 7 with
thiosemicarbazide and 4-thiazolidinone moieties, to elucidate
their potential role as antioxidants, antifungal agents and iron che-
lators with enhanced activity.
was used as a solvent (Taskova, Mitova, Mikhova, & Duddeck,
2003), due to the low solubility of synthesized compounds in eth-
anol and methanol.
Seven hundred and 50 ll of DMSO solution of the correspond-
ing synthesized compound (0.2 mM) was added to a DMSO solu-
tion of DPPH radical (0.2 mM), so that the final concentration of
DPPH radical and the synthesized compound in a solution was
0.1 mM. The mixture was shaken and allowed to stand at room
temperature. After 30 min the absorbance at 517 nm was deter-
mined and the scavenging activity was calculated according to
the formula below. Ascorbic acid (AA) was used as a reference
compound.
Ab þ As ꢀ Am
% DPPH scanenging ¼
ꢁ 100
ð1Þ
Ab
where Ab – absorbance of 0.1 mM DMSO solution of DPPH radical at
517 nm; As – absorbance of 0.1 mM DMSO solution of test com-
pound at 517 nm; Am – absorbance of DMSO mixture of test com-
pound and DPPH radical at 517 nm.
2.2.1.2. EPR determination of DPPHÅ (1,1-diphenyl-2-picryhydrazyl
radical)-scavenging activity. The DPPH radical-scavenging activity
of coumarin derivatives was also determined with an EPR spec-
trometry method. All reaction mixtures contained 25
ll of
0.2 mM DPPHÅ and 25
l
l of 0.2 mM DMSO solutions of coumarin
compounds and the control solution contained no antioxidant.
Ascorbic acid was used as a reference compound.
Finding of new compounds possessing both antifungal and anti-
oxidant properties could be of a great importance for applications
in medicine, agriculture or the food industry.
EPR signals were recorded 30 min after incubation at room tem-
perature. Spectrometer conditions were: microwave frequency,
9.72 GHz; microwave power, 20 mW; modulation amplitude,
0.95 G; sweep 100 G; gain 2 ꢁ 105 and sweep time, 41.9 s.
The DPPHÅ-scavenging activity of test compounds was calcu-
lated using the following equation:
2. Materials and methods
h0 ꢀ hx
2.1. General
% DPPH scanenging ¼
ꢁ 100
ð2Þ
h0
Melting points were determined on a capillary melting point
apparatus (Electrotermal, Rochford, Great Britain) and are uncor-
rected. Thin-layer chromatography was performed with fluores-
cent silica gel plates HF254 (Merck, Darmstadt, Germany), which
were checked under UV (254 and 365 nm) light, using ben-
zene:acetone:acetic acid (8:1:1) as a solvent. The elemental analy-
sis for C, H and N was done on a Analyzer 2440 (Perkin–Elmer,
where hx and h0 are the EPR signal intensities of samples in the
presence and absence of test compounds.
2.2.1.3. EPR determination of galvinoxyl radical-scavenging activ-
ity. Twenty five
ll of the tested compound (0.2 mM DMSO solu-
tion) was mixed with a 0.2 mM solution of galvinoxyl radical.
Ascorbic acid was added as positive control. EPR signals were re-
corded 20 min after incubation at room temperature. Spectrometer
conditions were: microwave frequency, 9.72 GHz; microwave
power, 20 mW; modulation amplitude, 0.48 G; sweep 40 G; gain
2 ꢁ 105 and sweep time, 41.9 s.
Boston, MA, USA). Infrared spectra (m
max-cmꢀ1) were recorded on
a FT-IR 3303 spectrometer (Beckman Instruments, Inc., USA), using
KBr disks. 1H NMR spectra were recorded on a Bruker Avance
600 MHz NMR Spectrometer (Bruker Biospin GmbH, Rheinstetten,
Germany) at 293 K in DMSO-d6. The MS spectra were recorded on
LC/MS/MS API 2000 (Applied Biosystems/MDS SCIEX, CA, USA). The
absorbance was measured on a UV visible spectrophotometer He-
The galvinoxyl-scavenging activity of test compounds was cal-
culated using the following equation:
h0 ꢀ hx
lios c, (Thermo Spectronic, Cambridge, UK). Microplates were read
% galvaninoxyl scanenging ¼
ꢁ 100
ð3Þ
h0
on a Sunrise absorbance reader (Tecan Group Ltd., Männedorf,
Switzerland). Incubation was carried in Aqualytic AL 500-8 incuba-
tor (Aqualytic, Dortmund, Germany).
Determination of correlation coefficients (Pearson method) and
student t-test were performed in Statistica 8.0 (StatSoft Inc., Tulsa,
OK, USA).
where hx and h0 are the EPR signal intensities of samples in the
presence and absence of test compounds.
2.3. Evaluation of antioxidant activity by phosphomolybdenum
method
2.2. Antioxidant activity
The antioxidant activity of tested coumarin derivatives was
evaluated by the phosphomolybdenum method, according to the
procedure of Prieto, Pineda, and Aguilar (1999). An aliquot of
2.2.1. DPPHÅ-scavenging activity
2.2.1.1. Spectrophotometric determination of DPPH (1,1-diphenyl-2-
picryhydrazyl radical)-scavenging activity. Determination of antiox-
idant activity was performed according to the procedure described
in the literature (Manojkumar et al., 2009; Wu, Huang, Lin, Ju, &
Ching, 2007) with some modifications. DMSO (dimethyl sulfoxide)
100 ll of sample solution (2 mM in DMSO) was mixed with 1 ml
of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phos-
phate and 4 mM ammonium molybdate). The samples were incu-
bated in a water bath at 95 °C for 90 min. Samples were cooled
to room temperature and the absorbance was measured at

490
B. Šarkanj et al. / Food Chemistry 139 (2013) 488–495
695 nm. The antioxidant activity was expressed relative to the
antioxidant activity of the same concentration of ascorbic acid.
tre plates (96 U-shaped wells; Brand, Wertheim, Germany) were
used in the microdilution test. Rows 2–11 contained the series of
drug dilutions in 100
pension. Row 1 contained 200
ium and served as the sterility control, while row 12 contained
100 l of drug-free medium and 100 l of inocula and served as
l
l volumes and 100
l
l of the 2ꢁ spore sus-
2.4. Iron chelating activity
ll of uninoculated, drug-free med-
The chelating activity of coumarin derivatives for ferrous ions
Fe2+ was measured according to the literature method (Zhao,
Xiang, Ye, Yuan, & Guo, 2006) with some modifications. Twenty
l
l
growth control (the final volume in each well was 200 ll).
five
DMSO solution (4:1) of the compound investigated. After 30 s,
50 l of ferrozine (5 mM) were added. Samples were incubated
at room temperature for 10 min and the absorbance of the complex
formed between Fe2+ and ferrozine was measured at 562 nm. Me-
tal-chelating efficiency of samples was compared to the chelating
activity of EDTA disodium salt. The chelating activity of the extract
for Fe2+ was calculated as:
ll of FeCl2 (2 mM) were added to 1 ml of 2 mM methanol/
2.5.6. Incubation and MIC determination
Following inoculation, all plates were incubated at 35 °C in an
atmospheric incubator. After 48 h of incubation, plates were read
on a microplate reader at 450 nm. Minimal inhibitory concentra-
tion for 100% cell death (MIC100) was defined as the lowest concen-
tration reducing the optical density by 100% at 450 nm compared
with growth control (Clement, Tremblay, Lange, Thibodeau, & Bel-
humeur, 2008; Santos et al., 2010).
l
A0 ꢀ A1
% Chelating rate ¼
ꢁ 100
ð4Þ
A0
2.6. Synthesis of the target compounds
where A0 – absorbance of the control (blank, without samples) at
562 nm; A1 – absorbance in the presence of the methanol/DMSO
sample solution at 562 nm.
2.6.1. General
The synthesis of the target compounds was carried out as out-
lined in Fig. 1.
2.5. Antifungal activity
2.6.2. Preparation of thiosemicarbazide (2a–e)
2.5.1. General
A mixture of hydrazide (10 mmol) and an appropriate isothio-
cyanate (10 mmol) in 50 ml of absolute ethanol was refluxed for
3 h. The crude product thus obtained was filtered and recrystal-
lized from ethanol.
Broth microdilution assays were performed in accordance with
the guidelines detailed in CLSI document M38-A.
2.5.2. Tested fungi
Fungi which were used in this experiment are major producers
of mycotoxins and food contaminants (Hussein & Brasel, 2001): A.
flavus (NRRL 3251); Aspergillus ochraceus (CBS 589.68), Fusarium
graminearum (CBS 110.250) and Fusarium verticillioides (CBS
119.825).
2.6.3. 4-Methyl-1-(2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)acetyl)thiosemicarbazide (2a)
M.p. 218 °C, Rf = 0.25, Y = 54%. FT-IR (KBr) m
max (cmꢀ1): 3136.36
(N–H), 3061.13 (C–H, Ar–H), 2933.83, 1693.50 (C@O), 1440.87,
1267.27(C@S), 1136.11; 1H NMR d (ppm): 2.40 (s, 3H, CH3, C-4),
3.53 (s, 3H, CH3), 4.73 (s, 2H, OCH2), 6.23 (s, 1H, H-3), 6.96 (d,
1H, H-6), 7.07 (d, 1H, H-8), 7.70 (d, 1H, H-5), 8.05 (s, 1H, NH),
9.31 (s, 1H, NH), 9.43 (s, 1H, NH); MS m/z: 320.0 [MꢀH+],
(M = 321.35); Anal. Calcd. for C14H15N3O4S (321.35): C, 52.33; H,
4.70; N, 13.08; O, 19.92; S, 9.98; Found: C, 52.27; H, 4.75; N,
13.12; O, 19.95; S, 9.92%.
2.5.3. Preparation of inoculum
The inocula were obtained from cultures that were grown on
potato dextrose agar (PDA) slants. Aspergillus strains were grown
at 35 °C for 7 days, and Fusarium strains were grown at 35 °C for
2 days, followed by 7 days at 25 °C. Fungal spores were harvested
by pouring 5 ml of sterile saline onto the culture slants and scrap-
ing the surface with an inoculation loop. The number of spores in
the stock suspension was adjusted to 106 CFU/ml, using a Bür-
ker–Türk counting chamber (Haemocytometer). Turbidity of each
stock suspension was checked with a 0.5 McFarland standard read
at 530 nm. Working 2ꢁ spore suspension was prepared by diluting
2.6.4. 4-Ethyl-1-(2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)acetyl)thiosemicarbazide (2b)
M.p. 216 °C, Rf = 0.26, Y = 56%. FT-IR (KBr) m
max (cmꢀ1): 3173.01
(N–H), 3068.85 (C–H, Ar–H), 2958.90, 2034.97, 1720.56 (C@O),
1438.94, 1267.27 (C@S), 1138.04; 1H NMR d (ppm): 1.09 (t, 3H,
CH3), 2.40 (s, 3H, CH3, C-4), 3.49 (q, 2H, CH2), 4.74 (s, 2H, OCH2),
6.22 (s, 1H, H-3), 6.96 (d, 1H, H-6), 7.01 (d, 1H, H-8), 7.96 (d, 1H,
H-5), 9.24 (s, 1H, NH), 9.43 (s, 1H, NH), 9.54 (s, 1H, NH); MS m/z:
333.9 [MꢀH+], (M = 335.38); Anal. Calcd. for C15H17N3O4S
(335.38): C, 53.72; H, 5.11; N, 12.53; O, 19.08; S, 9.56; Found: C,
53.69; H, 5.15; N, 12.50; O, 19.11; S, 9.56%.
200 ll of stock suspension into 10 ml of RPMI 1640 medium (Na-
tional Committee for Clinical Laboratory Standards, 2002).
2.5.4. Medium
Antifungal susceptibility testing was performed, using RPMI
1640 medium buffered with 0.164 M MOPS (34.53 g/l) and ad-
justed to pH 7.0 with (1 M) NaOH, as recommended by the Na-
tional Committee for Clinical Laboratory Standards. Medium was
sterilized by filtration using a 0.45
l
m filter.
2.6.5. 1-(2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-4-
phenylthiosemicarbazide (2c)
2.5.5. Drug dilutions
M.p. 189 °C, Rf = 0.28, Y = 62%. FT-IR (KBr) m
max (cmꢀ1): 3215.44
The drug dilutions were prepared following the additive two
fold drug dilution scheme described in NCCLS M28-A method for
water-insoluble compounds (National Committee for Clinical Lab-
oratory Standards, 2002). Briefly, stock drug solutions were first di-
luted by 20ꢁ of the final concentrations in 100% dimethyl sulfoxide
(DMSO), followed by further dilutions to the final drug concentra-
(N–H), 3095.85 (C–H, Ar–H), 2960.83, 2033.04, 1708.99 (C@O),
1446.66, 1261.49 (C@S), 1138.04; 1H NMR d (ppm): 2.42 (s, 3H,
CH3, C-4), 4.81 (s, 2H, OCH2), 6.25 (s, 1H, H-3), 6.96 (d, 1H, H-6),
7.07 (s, 1H, H-8), 7.01–7.46 (d, 5H, arom.), 7.73 (d, 1H, H-5), 9.70
(s, 1H, NH), 10.26 (s, 1H, NH), 10.35 (s, 1H, NH); MS m/z: 382.0
[MꢀH+], (M = 383.42); Anal. Calcd. for C19H17N3O4S (383.42): C,
59.52; H, 4.47; N, 10.96; O, 16.69; S, 8.36; Found: C, 59.48; H,
4.51; N, 10.90; O, 16.73; S, 8.38%.
tions, 10, 1, 0.1 and 0.01
by filtration, using a 0.45
l
l
g/ml. All drug dilutions were sterilized
m filter. Sterile polypropylene microti-

B. Šarkanj et al. / Food Chemistry 139 (2013) 488–495
491
Fig. 1. Synthesis of thiosemicarbazide and 4-thiazolidinone derivatives from 7-hydroxy-4-methyl coumarin (R/Ar: a – methyl; b – ethyl; c – phenyl; d – p-tolyl; e – p-
methoxyphenyl).
2.6.6. 1-(2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-4-p-
tolylthiosemicarbazide (2d)
(C@O), 1612.54 (C@N), 1440.87, 1147.68, 675.11 (C–S–C); 1H NMR
d (ppm): 2.40 (s, 3H, CH3, C-4), 3.66 (s, 3H, CH3), 4.11 (s, 2H, CH2),
5.45 (s, 2H, OCH2), 6.23 (s, 1H, H-3), 6.96 (d, 1H, H-6), 6.97 (d, 1H,
H-8), 7.70 (d, 1H, H-5), 9.54 (s, 1H, NH); MS m/z: 360.2 [MꢀH+],
(M = 361.37); Anal. Calcd. for C16H15N3O5S (361.37): C, 53.18; H,
4.18; N, 11.63; O, 22.14; S, 8.87; Found: C, 53.22; H, 4.15; N,
11.68; O, 22.09; S, 8.88%.
M.p. 122–124 °C, Rf = 0.38, Y = 58%. FT-IR (KBr) mmax (cmꢀ1):
3171.08(N–H), 3103.57 (C–H, Ar–H), 2960.83, 2011.82, 1718.63
(C@O), 1425.44, 1230.63 (C@S), 1126.47; 1H NMR d (ppm): 2.34
(d, 3H, CH3, C0-4, arom.), 2.40 (s, 3H, CH3, C-4), 4.83 (s, 2H,
OCH2), 6.23 (s, 1H, H-3), 6.94 (d, 1H, H-6), 7.02 (d, 1H, H-8),
6.98–7.35 (d, 4H, arom.), 7.70 (d, 1H, H-5), 9.68 (s, 1H, NH),
10.20 (s, 1H, NH), 10.52 (s, 1H, NH); MS m/z: 396.1 [MꢀH+],
(M = 397.45); Anal. Calcd. for C20H19N3O4S (397.45): C, 59.52; H,
4.47; N, 10.96; O, 16.69; S, 8.36; Found: C, 59.48; H, 4.51; N,
10.90; O, 16.73; S, 8.38%.
2.6.10. (Z)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N-(2-
(ethylimino)-4-oxothiazolidin-3-yl)acetamide(3b)
M.p. 188 °C, Rf = 0.38, Y = 81%. FT-IR (KBr) m
max (cmꢀ1): 3433.41
(N–H), 3088.14 (C–H, Ar–H), 2978.19 (C–H, CH2), 2960.83, 1695.49
(C@O), 1614.47 (C@N), 1431.23, 1147.68, 675.11 (C–S–C); 1H NMR
d (ppm): 1.33 (t, 2H, CH3), 1.76 (q, 2H, CH2), 2.42 (s, 3H, CH3, C-4),
4.17 (s, 2H, CH2), 5.46 (s, 2H, OCH2), 6.24 (s, 1H, H-3), 6.99 (d, 1H,
H-6), 7.04 (s, 1H, H-8), 7.73 (d, 1H, H-5), 9.46 (s, 1H, NH); MS m/z:
374.1 [MꢀH+], (M = 375.40); Anal. Calcd. for C17H17N3O5S (375.40):
C, 54.39; H, 4.56; N, 11.19; O, 21.31; S, 8.54; Found: C, 54.33; H,
4.60; N, 11.16; O, 21.38; S, 8.52%.
2.6.7. 4-(4-Methoxyphenyl)-1-(2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)acetyl)thiosemicarbazide (2e)
M.p. 180 °C, Rf = 0.27, Y = 60%. FT-IR (KBr) m
max (cmꢀ1): 3203.87
(N–H), 3068.85 (C–H, Ar–H), 2966.62, 2034.97, 1693.56 (C@O),
1438.94, 1253.77 (C@S), 1139.97; 1H NMR d (ppm): 2.42 (s, 3H,
CH3, C-4), 3.77 (s, 3H, OCH3, C0-4, arom.), 4.80 (s, 2H, OCH2), 6.24
(s, 1H, H-3), 6.93–7.31 (d, 4H, arom.), 6.94 (d, 1H, H-6), 7.06 (d,
1H, H-8), 7.73 (d, 1H, H-5), 9.62 (s, 1H, NH), 10.12 (s, 1H, NH),
10.31 (s, 1H, NH); MS m/z: 412.2 [MꢀH+], (M = 413.45); Anal.
Calcd. for C20H19N3O5S (413.45): C, 58.10; H, 4.63; N, 10.16; O,
19.35; S, 7.76; Found: C, 58.15; H, 4.60; N, 10.20; O, 19.29; S, 7.76%.
2.6.11. (Z)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-
(phenylimino)thiazolidin-3-yl)acetamide (3c)
M.p. 175–178 °C, Rf = 0.42, Y = 77%. FT-IR (KBr) mmax (cmꢀ1):
3435.34 (N–H), 2993.62 (C–H, Ar–H), 2972.40, 1699.34 (C@O),
1612.54 (C@N), 1427.37, 1155.40, 692.47 (C–S–C); 1H NMR d
(ppm): 2.38 (s, 3H, CH3, C-4), 4.14 (s, 2H, CH2), 5.26 (s, 2H,
OCH2), 6.22 (s, 1H, H-3), 6.99 (d, 1H, H-6), 7.00 (d, 1H, H-8),
6.88–7.63 (d, 5H, arom.), 7.65 (d, 1H, H-5), 11.20 (s, 1H, NH); MS
m/z: 424.3 [M+H+], (M = 423.44); Anal. Calcd. for C21H17N3O5S
(423.44): C, 59.57; H, 4.05; N, 9.92; O, 18.89; S, 7.57; Found:C,
59.62; H, 3.99; N, 9.98; O, 18.84; S, 7.57%.
2.6.8. Preparation of thiazolidinone(3a-e)
Thiosemicarbazide 2a–e (10 mmol) and of ethyl bromoacetate
(11 mmol) were refluxed in 30 ml of absolute EtOH in the presence
of anhydrous NaOAc (40 mmol) for 9 h. The reaction mixture was
cooled, diluted with water and allowed to stand overnight. The so-
lid precipitated was washed with water, dried and recrystallized
from ethanol.
2.6.12. (Z)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N-(4-oxo-2-(p-
tolylimino)thiazolidin-3-yl)acetamide (3d)
2.6.9. (Z)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N-(2-
(methylimino)-4-oxothiazolidin-3-yl)acetamide (3a)
M.p. 205 °C, Rf = 0.33, Y = 92%. FT-IR (KBr)
(N–H), 3061.13 (C–H, Ar–H), 2980.12 (C–H, CH2), 2962.76, 1693.56
M.p. 213–217 °C, Rf = 0.52, Y = 84%. FT-IR (KBr) mmax (cmꢀ1):
3433.41 (N–H), 3064.99 (C–H, Ar–H), 2929.27, 1697.41 (C@O),
1616.40 (C@N), 1429.30, 1149.61, 702.11 (C–S–C); 1H NMR d
m
max (cmꢀ1): 3435.34

492
B. Šarkanj et al. / Food Chemistry 139 (2013) 488–495
(ppm): 1.18 (s, 3H, CH3, C0-4, arom.), 2.37 (s, 3H, CH3, C-4), 4.11 (s,
2H, CH2), 5.23 (s, 2H, OCH2), 6.21 (s, 1H, H-3), 6.89 (d, 1H, H-6),
6.99 (d, 1H, H-8), 7.30–7.71 (d, 4H, arom.), 7.39 (d, 1H, H-5),
11.30 (s, 1H, NH); MS m/z: 436.4 [MꢀH+], (M = 437.47); Anal.
Calcd. for C22H19N3O5S (437.47): C, 60.40; H, 4.38; N, 9.61; O,
18.29; S, 7.33; Found: C, 60.35; H, 4.44; N, 9.68; O, 18.33; S, 7.32%.
elimination of stable free radicals (Polovka, 2006). In contrast to
other methods, EPR spectroscopy directly determines the free
DPPHÅ concentration and is, therefore, less prone to the interfer-
ence of coloured antioxidants or their coloured reaction products
(Becker, Nissen, & Skibsted, 2004).
3.2.3. Galvinoxyl radical-scavenging activity (EPR)
Galvinoxyl radical is a commercially available O-centred stable
free radical with the absorbance maximum in the visible region
(432 nm). Galvinoxyl radicals are more reactive toward phenolics
than is DPPHÅ (Tirzitis & Bartosz, 2010) and the ability of some
compounds to scavenge galvinoxyl radicals is much higher than
that for the DPPH radical (Zhao & Liu, 2010). This is probably be-
cause the free electron of DPPH is sterically more protected than
that of the galvinoxyl radical (Koprivnjak et al., 2008).
2.6.13. (Z)-N-(2-(4-methoxyphenylimino)-4-oxothiazolidin-3-yl)-2-
(4-methyl-2-oxo-2H-chromen-7-yloxy)acetamide (3e)
M.p. 200 °C, Rf = 0.65, Y = 86%. FT-IR (KBr) m
max (cmꢀ1): 3433.41
(N–H), 3072.71 (C–H, Ar–H), 2931.90, 1697.41 (C@O), 1618.33
(C@N),1448.59, 1155.40, 623.03 (C–S–C); 1H NMR d (ppm): 2.36
(s, 3H, CH3, C-4), 3.81 (s, 3H, OCH3, C0-4, arom.), 4.10 (s, 2H, CH2),
5.23 (s, 2H, OCH2), 6.20 (s, 1H, H-3), 6.90 (d, 1H, H-6), 6.99 (d,
1H, H-8), 7.11–7.44 (d, 4H, arylidene), 7.61 (d, 1H, H-5), 11.18 (s,
1H, NH); MS m/z: 452.8 [MꢀH+], (M = 453.47); Anal. Calcd. for C22-
H19N3O6S (453.47): C, 58.27; H, 4.22; N, 9.27; O, 21.17; S, 7.07;
Found: C, 58.21; H, 4.25; N, 9.32; O, 21.19; S, 7.03%.
Both DPPHÅ and galvinoxyl are stable free radicals which accept
either an electron plus a proton (sequential proton loss electron
transfer, SPLET) or a hydrogen atom (hydrogen atom transfer,
HAT) to become diamagnetic molecules. They are often used as
substrates to evaluate the antioxidant capacity and various com-
pounds usually exhibit the same scavenging pattern for DPPHÅ
and galvinoxyl radicals (Rodrıguez et al., 2007).
3. Results and discussion
3.1. General
According to the data in Table 1. the best DPPHÅ-scavengers are
thiosemicarbazides 2c and 2e, substituted with a phenyl and a 4-
methoxyphenyl group in position 4 of the thiosemicarbazide chain.
With the exception of 2d, thiosemicarbazides with an aryl (phenyl)
group show higher DPPHÅ-scavenging activity than those with alkyl
substituents (methyl and ethyl). Also, the substituents on a phenyl
ring show a great influence on DPPHÅ-scavenging activity of thiose-
micarbazides in a way that p-methoxy substitution on the phenyl
ring increases and p-methyl substitution decreases DPPHÅ-scaveng-
ing activity of thiosemicarbazide with a phenyl ring. Substitution
on the phenyl ring in 4-thiazolidinones did not show any signifi-
cant influence on antioxidant activity. Antioxidant activity of thi-
osemicarbazides is assigned to the presence of sulphur in the
C@S group (Nicolaides, Fylaktakidou, Litinas, & Hadjipavlou-Litina,
1998), although it is obvious that the presence of a phenyl ring and
its substitution has a great impact on the activity, as well.
The best galvinoxyl radical-scavengers are thiosemicarbazides
3c, 3e, 3b and 3a, showing activities higher than 80%. Since the
same conditions, in both galvinoxyl and DPPHÅ assays, are applied,
it is likely that the mechanisms of DPPHÅ- and galvinoxyl radical-
scavenging in our experiments are similar. This assumption is sup-
ported by the observation that the sequence of activity of the syn-
thesized coumarin derivatives is highly correlated for both radicals
(r = 0.67, p = 0.0118). Although the mechanism is probably similar,
galvinoxyl radicals are faster reduced by the tested compounds
than are DPPH radicals in the polar solvent, DMSO. A correspond-
ing effect was also described for other antioxidants (Koprivnjak
et al., 2008; Zhao & Liu, 2010).
In our attempt to obtain new coumarin derivatives with poten-
tial antioxidant and antifungal activity, we performed a synthesis
of thiosemicarbazides and 4-thiazolidinones from 4-methyl-7-
hydroxycoumarin (Fig. 1). Newly synthesized compounds were
purified and the structures of the products were elucidated and
confirmed from their analytical and spectral data. The 1H NMR
spectrum of thiosemicarbazides exhibited two signals due to
NH–CS–NH, which are absent in 1H NMR spectra of 4-thiazolidi-
nones. IR spectra of thiosemicarbazides showed absorption signals
at 1230–1268 cmꢀ1 (C@S) which are absent in IR spectra of 4-thia-
zolidinones which is characterised with 623–692 cmꢀ1 (C–S) and
1612–1618 cmꢀ1 (C@N) signals.
3.2. Antioxidant activity
3.2.1. General
In the DPPHÅ, galvinoxyl and phosphomolybdenum assay meth-
ods hydrogen and electron transfer from antioxidants to DPPHÅ,
galvinoxyl radical and Mo(VI) complex occurs. These transfers de-
pend upon the structure of the antioxidants. The reason ascorbic
acid was used as a standard in all antioxidant investigations is that
ascorbic acid has the ability to donate hydrogen and electrons and
can be detected by both assay models (Marwah et al., 2007).
3.2.2. Scavenging of 1,1-diphenyl-2-picrylhydrazyl radical
3.2.2.1. Spectrophotometric determination of DPPH radical-scavenging
activity. The DPPH free radical exhibits a strong absorption maxi-
mum at 517 nm, resulting in a purple colour. Upon reduction of
DPPHÅ by antioxidants to DPPHÅ-H, the colour turns from purple
to yellow due to an absorption maximum of DPPHÅ-H in the UV
range. The method used in this work determines the antioxidant
capacity by measuring the remaining amount of DPPHÅ after
30 min of incubation with the respective compounds (endpoint).
This DPPHÅ-scavenging activity was compared to ascorbic acid as
a standard compound.
In all cases thiosemicarbazides showed better DPPHÅ- and gal-
vinoxyl-scavenging activity than 4-thiazolidinones with the same
substituent. The assumption is that thiosemicarbazides, when
donating an electron to a free radical, can form a structure stabi-
lized by delocalization of electrons throughout the molecule.
Synthetic modification significantly increased (student t-test,
p < 0.05) galvinoxyl-scavenging activity of the new derivatives in
comparison to the starting compounds.
3.2.4. Evaluation of antioxidant activity by phosphomolybdenum
method
3.2.2.2. EPR determination of DPPHÅ-scavenging activity. EPR is an
analytical method that directly measures free radicals, relying on
the absorption of microwave energy when the samples are placed
This method is based on the reduction of Mo(VI) to Mo(V) by
the tested compounds, followed by formation of a green phos-
phate/Mo(V) complex at acid pH (Prieto et al., 1999).
Substitution of 4-methyl-7-hydroxycoumarin in position 7 in-
creases the antioxidant activity of the starting compound, which
ˇ
ˇ
´
in a variable magnetic field (Djilas, Canadanovic-Brunet, & Cetk-
´
ovic, 2002). This method includes scavenging of reactive free radi-
cals and antioxidant activity can be monitored through the

B. Šarkanj et al. / Food Chemistry 139 (2013) 488–495
493
Table 1
DPPHÅ- and galvinoxyl radical-scavenging activity of coumarinyl thiosemicarbazides and 4-thiazolidinones (0.1 mM).c
compound
DPPHÅ-scavenging activity (%) (spp)a
DPPHÅ-scavenging activity (%) (EPR)b
Galvinoxyl radical-scavenging activity (%)
AAd
85.2 6.1
2.4 0.82
14.4 4.1
14.4 5.3
2.5 0.4
23.4 5.7
3.5 0.6
54.1 5.6
1.2 1.2
9.9 0.9
2.3 0.6
45.4 2.5
1.8 1.4
88.6 0.9
4.9 4.6
10.2 0.6
9.6 4.2
9.5 0.9
24.6 5.8
9.7 1.8
58.1 6.8
4.1 2.7
1.0 0.4
4.8 4.0
57.5 1.7
0.1 2.1
82.6 1.4
4.2 3.2
Ke
1
32.9 2.5
84.2 0.7
40.9 2.3
96.3 0.5
36.1 2.2
98.4 0.2
53 4.5
40.6 1.6
62 1.4
97.2 0.3
30.5 1.0
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
a
b
c
spectrophotometrically determined.
EPR determined.
data are means standard deviation of three replicates.
ascorbic acid was used as standard.
7-hydroxy-4-methylcoumarin.
d
e
has a very low antioxidant activity compared to ascorbic acid (only
20%) (Table 2). Hydrazide itself has the highest activity in this ser-
ies and the introduction of the thiosemicarbazide and 4-thiazolid-
inone moiety decreases its activity. Thiosemicarbazides with alkyl
substituents show better antioxidant activity than do those with
aryl substituents. When comparing the activity of thiosemicarba-
zide and 4-thiazolidinone with the same substituent pattern from
2b and 3b, it is evident that thiosemicarbazide, 2b, shows better
activity than does thiazolidinone 3b. Other thiosemicarbazides
and thiazolidinones could not be compared because during the as-
say thiazolidinones precipitated, thereby interfering with spectro-
photometric measurements.
When comparing other antioxidant assays performed in this
work (DPPHÅ, galvinoxyl radicals) with the phosphomolybdenum
method, there is no correlation. This is probably because the
phosphomolybdenum method operates via different mechanisms.
Similar discrepancies were also observed by other authors (Jayap-
rakasha, Girennavar, & Patil, 2008; Matkowski & Piotrowska, 2006).
result that the red colour of the complex is decreased. Therefore,
the chelating activity of the coexisting chelator is estimated by
measurement of colour reduction at 562 nm.
In this assay, only compounds 3a, 3b and 2d showed iron che-
lating activities, 62.2%, 46.6% and 4.54%, compared to that of EDTA
disodium salt (97%). In this case, 4-thiazolidinones are much better
iron chelators than are thiosemicarbazides, especially those with
alkyl substituents on the 4-thiazolidinone ring.
3.3. Antifungal activity
Antifungal activity was determined against four common food-
borne mycotoxigenic fungi: A. flavus, A. ochraceus, F. graminearum
and F. verticillioides.
According to Table 3, compounds 2c, 3c and 3e possess the best
antifungal activity against A. flavus and all of them are character-
ised by the presence of a phenyl group in their structure, which
was previously proven to enhance antifungal activity (Sasse
et al., 1972). One of these compounds is thiosemicarbazide and
two of them are 4-thiazolidinones. These compounds showed bet-
ter antifungal activity towards A. flavus than the starting com-
pound, 7-hydroxy-4-methylcoumarin. Both thiosemicarbazides
and 4-thiazolidinones with alkyl substituents did not show signif-
icant antifungal activity on this mold compared to other coumarin
derivatives. The most prominent antifungal activity towards A. och-
raceus is observed with compounds 3a, 3d, 2e, 3e; all of them, ex-
cept 3a, exhibit a phenyl ring in their structure. In this case, 4-
3.2.5. Iron chelating activity
This investigation is based upon the complex formation be-
tween ferrozine and Fe2+, which is red in colour. In the presence
of chelating agents, the complex formation is disrupted, with the
Table 2
Antioxidant activities of coumarin derivatives 2a–e and 3a–e
(2 mM) relative to ascorbic acid (Am – activity relative to ascorbic
acid (AA) on a molar basis).a
compound
Am
Table 3
AAb
1.00 0.01
0.20 0.01
0.90 0.01
0.59 0.05
–
0.70 0.07
0.22 0.01
0.55 0.02
–
0.52 0.10
–
0.55 0.00
–
Antifungal activity of tested coumarinyl thiosemicarbazides and thiazolidinones.
Kc
Compound
MIC100
(l
g/ml)
A. ochraceus
1
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
A. flavus
F. graminearum
F. verticillioides
Ka
1
1–10
0.1–1
>10
>10
>10
0.1–1
0.1–1
>10
<0.1
>10
>10
>10
>10
0.1–1
<0.1
<0.1
<0.1
<0.1
<0.1
0.1–1
<0.1
0.1–1
0.1–1
1–10
<0.1
<0.1
<0.1
<0.1
<0.1
>10
>10
>10
0.1–1
>10
>10
>10
<0.1
>10
>10
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
>10
0.1–1
0.1–1
1–10
<0.1
>10
–, indicated compounds precipitated during assay (not possible
to determine activity under these conditions).
0.1–1
0.1–1
a
data are means standard deviation of three replicates.
ascorbic acid was used as standard.
7-hydroxy-4-methylcoumarin.
<0.1
b
a
c
7-Hydroxy-4-methylcoumarin.

494
B. Šarkanj et al. / Food Chemistry 139 (2013) 488–495
Haraguchi, S. K., Silva, A. A., Vidotti, G. J., dos Santos, P. V., Garcia, F. P., Pedroso, R. B.,
et al. (2011). Antitrypanasomal activity of novel benzaldehyde-thiosemicarbazone-
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thiazolidinones showed better antifungal activity than did 4-thi-
osemicarbazides, which emphasises the importance of both phenyl
and 4-thiazolidinone rings for achieving antifungal activity. For
this mold, the presence of a substituted phenyl ring is important,
since the substitution with the unsubstituted phenyl ring caused
lower activity. The antifungal activity of 7-hydroxy-4-methyl-
coumarin is increased by substitution in position 7 with these spe-
cific thiosemicarbazide and 4-thiazolidinone moieties, bearing the
substituted phenyl ring mentioned above. Among fungi tested, F.
graminearum is the most susceptible towards the compounds
tested, as most of them show an excellent antifungal activity. On
the other hand, F. verticillioides is the least susceptible species to-
wards the tested compounds. The best antifungals for this species
are compounds 2c, 3c, 2e and 3e, all of them containing substituted
phenyl ring in their structure. Introduction of alkyl substituents
and an unsubstituted phenyl ring did not show significant increase
in antifungal activity towards this mold. Once more, the substitu-
tion of a starting compound in position 7 by thiosemicarbazide
and 4-thiazolidinone moieties increased the antifungal activity to-
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thiazolidinones, expressed as DPPHÅ- and galvinoxyl-scavenging
activity, but lower iron chelating activity. 4-Thiazolidinones
showed better antifungal activity on all four examined fungal spe-
cies than did thiosemicarbazides. The mould most susceptible to-
wards tested compounds was F. graminearum, while F.
verticillioides was the least susceptible.
In both antioxidant and antifungal assays, substitution of 7-hy-
droxy-4-methylcoumarin in position 7 was proven to be important
for increasing of the activity investigated.
Compounds 2c and 2e showed an excellent antioxidant and
antifungal activity and could be further investigated for potential
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Acknowledgement
This work was supported by the Croatian Ministry of Science,
Education and Sports (Grant No. 113-1130473-0334).
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