Biological study on novel coumarinyl 1,3,4-oxadiazoles

Article abstract of DOI:10.3906/kim-1703-2

Coumarinyl 1,3,4-oxadiazoles were synthesized from Schiff bases and acetic anhydride. All compounds were characterized by melting points and their structures confirmed by mass and ¹ H and ¹³ C NMR spectrometry. These novel coumarinyl

Full text of DOI:10.3906/kim-1703-2

Turk J Chem
Turkish Journal of Chemistry
(2018) 42: 146 – 157
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1703-2
Research Article
Biological study on novel coumarinyl 1,3,4-oxadiazoles
2
3
Maja MOLNAR1,∗, Ana AMIC , Valentina PAVIC , Tihomir KOVAC ,
´
ˇ4
´
ˇ1
3
¨
Marija KOVAC , Elizabeta HAS-SCHON
1Department of Applied Chemistry and Ecology, Faculty of Food Technology, J.J. Strossmayer University of Osijek,
Osijek, Croatia
2Department of Chemistry, J. J. Strossmayer University of Osijek, Osijek, Croatia
3Department of Biology, J. J. Strossmayer University of Osijek, Osijek, Croatia
4Inspecto Ltd., -DJ akovo, Croatia
Received: 01.03.2017
•
Accepted/Published Online: 02.10.2017
•
Final Version: 08.02.2018
Abstract: Coumarinyl 1,3,4-oxadiazoles were synthesized from Schiff bases and acetic anhydride. All compounds
were characterized by melting points and their structures confirmed by mass and 1 H and 13 C NMR spectrometry.
These novel coumarinyl derivatives were subjected to antibacterial, antifungal, antaflatoxigenic, and antioxidant activity.
Their activity varied depending on their structure, where 2-(3-acetyl-5-(((4-methyl-2-oxo-2H -chromen-7-yl)oxy)methyl)-
2,3-dihydro-1,3,4-oxadiazol-2-yl)-1,4-phenylene diacetate showed significant antioxidant and antibacterial activity on B.
subtilis. 4-(3-Acetyl-5-(((4-methyl-2-oxo-2H -chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-oxadiazol-2-yl)-1,2-phenylene
diacetate was found to possess excellent antifungal and antiaflatoxigenic activity.
Key words: Coumarin, 1,3,4-oxadiazole, antibacterial, antifungal, antimycotoxigenic activity
1. Introduction
Oxadiazoles are heterocyclic compounds showing a wide range of biological activities and they have been
explored by many researchers for decades. They can exist as four isomers, namely 1,2,4-oxadiazoles, 1,3,4-
oxadiazoles, 1,2,3-oxadiazoles, and 1,2,5-oxadiazoles.1,2 Due to their structural diversity their biological activi-
ties include antibacterial,3,4 antitubercular, antifungal, cytotoxic, anticancer,5,6 and many others. So far, our
work has been based on the synthesis of 1,3,4-oxadiazoles, which are present in some well-known drugs like
Furamizole (antibiotic), Raltegravir (antiretroviral), Zibotentan (anticancer), and tiodazosin and nesapidil (an-
tihypertensive). Considering the synthesis of 1,3,4-oxadiazole derivatives, there are some common procedures for
oxadiazole ring closure, depending on the starting compound. Dolman et al.7 prepared 1,3,4-oxadiazoles by to-
syl chloride/pyridine-mediated cyclization of thiosemicarbazides, Barbucenu et al.8 reacted thiosemicarbazides
with mercuric oxide in ethanol, while Narwade et al.9 used I2 /KI in sodium hydroxide solution. Dobrota et
al.10 prepared heterocycles of this type by oxidative cyclization of hydrazines with an excess of Dess–Martin
periodinane under mild conditions. Acetohydrazides were reacted with CS2 in alkaline medium followed by
acidic treatment.11 1,3,4-Oxadiazoles were also prepared by a reaction of different acylhydrazydes, aromatic
acids, and phosphoryl chloride by Amir and Kumar12 as well as by Khan and Akhtar.13 Khan and Akhtar13
performed a synthesis of oxadiazoles from acylhydrazide using cyanogen bromide, while Sharba et al.14 reacted
∗Correspondence: maja.molnar@ptfos.hr
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MOLNAR et al./Turk J Chem
N -formyl acid hydrazide with phosphorous pentasulfide to obtain a 1,3,4-oxadiazole ring. A well-known and of-
ten employed procedure for their preparation is the cyclization of Schiff bases in excess of acetic anhydride15−19
in the presence of chloramine T13,20,21 or ceric ammonium nitrate (CAN).22 Aside from conventional methods
in their preparation, ultrasound irradiation23 or one-pot reaction of aromatic hydrazides with aryl aldehydes
in the presence of catalytic amount of molecular iodine can be employed by grinding technique.24
Nowadays, many bacteria have genetic ability to acquire resistance to conventional antibiotic drugs.25
Side effects of overuse and misuse of antibiotics are also noticed, all harmful to vital organs and the immune
system in general. There is a constant pursuit for new antibiotics in order to eliminate the infections caused by
drug-resistant microbes.26
Our intention was to synthesize novel 1,3,4-oxadiazoles from coumarinyl Schiff bases and to investigate
them in terms of their antibacterial, antifungal, and antiaflatoxigenic activity. The obtained data will give us a
better insight into the structural activity relationship of these or similar compounds.
2. Results and discussion
Coumarinyl 1,3,4-oxadiazoles were synthesized from corresponding Schiff bases (Figure 1) and their structures
were confirmed by 1 H NMR, 13 C NMR, and mass spectrometry.
Figure 1. Synthetic route for coumarin derivatives.
The 1 H NMR spectra show characteristic peaks for acetyl groups on the 1,3,4-oxadiazole ring (2.13–
2.44 ppm), as well as typical shifts for aromatic protons (6.89–8.83 ppm) (see supplementary material for
NMR spectra). Moreover, the coumarin core was characterized by the coumarinyl–CH3 group in position
C-4 (2.40 ppm), one proton in position C-3 (6.18–6.22 ppm), and aromatic protons (6–8 ppm). All 1,3,4-
oxadiazole derivatives were synthesized from Schiff bases in excess of acetic anhydride, utilizing a very common
147

MOLNAR et al./Turk J Chem
procedure.16 A proposed mechanism, according to Desai et al.17 is shown in Figure 2. All hydroxyl groups that
were present in starting compounds were also acetylated. This was proven by 1 H NMR shifts for protons of
the –COCH3 group (1.21–2.30 ppm), as well as mass spectra where peaks of molecular ions were in accordance
with acetylated compounds. Our attempt was to synthesize 1,3,4-oxadiazoles from Schiff bases employing other
protocols too, like grinding Schiff bases with molecular iodine,24 or cyclization with chloramine T21 or ceric
ammonium nitrate.27 However, although some authors describe them as very effective procedures, they were
not suitable for our type of compounds. Low reactivity of Schiff bases towards these agents could be the reason
for the low yields we obtained in our protocol. Nevertheless, since our goal was to investigate these compounds
in terms of antimicrobial and antiaflatoxigenic activity, optimization of this synthetic route will be performed
in our future work.
Figure 2. Proposed mechanism for conversion of 2 to 3.
Antioxidant activity of synthesized compounds was performed with three different methods, each one
of them showing antioxidant activity gained by different mechanisms. Antioxidant activity of 1,3,4-oxadiazole
derivatives on DPPH• (2,2-diphenyl-1-picrylhydrazyl) (Table 1) was very low, since all hydroxyl groups, which
are usually responsible for high DPPH scavenging activity, were acetylated. Iron chelating activity was also
low and not comparable to standard compound EDTA. When antioxidant activity was performed with the
phosphomolybdenum method, where a different mechanism is employed, compounds like 3g and 3h showed
significant antioxidant activity. These compounds also showed good antibacterial activity (Table 2) on B.
subtilis, and 3h excellent activity on antifungal (Table 3) and antiaflatoxigenic activity (at 125 µg/mL) (Table
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MOLNAR et al./Turk J Chem
4). Compounds 3d, 3e, and 3m also showed very good antibacterial activity on B. subtilis and compound
3j revealed good antifungal activity. Although the antifungal activity of our compounds was low, their
antiaflatoxigenic activity was much higher. Compounds 3a, 3d, 3h, and 3j were excellent antiaflatoxigenic
agents, depending on their concentration. All of these data indicate that the combination of 1,3,4-oxadiazole
ring with coumarin core is a suitable system for achieving antimicrobial and antiaflatoxigenic activity, while
new and more efficient methods could be investigated in order to obtain them more easily and in greater yields.
Table 1. Antioxidant activity of oxadiazoles determined by DPPH, iron chelating, and phosphomolybdenum method
(Am – activity relative to ascorbic acid (AA) on a molar basis).
Compound
DPPH scavenging (%) Am
Iron chelatin activity (%) R2
Ascorbic acid 90.0
1
-
-
EDTA
3a
3b
3c
-
-
97
0
0.2
0
0.86
2-OCOCH3
3-OCOCH3
4-OCOCH3
3-OCH3
0.25 5.1
0.87 2.0
0
3d
3e
0
1.03
0.95
0
-
0.3
25
1.3
0
4-OCH3
3f
0.44 7.4
1.73 7.2
1.77 1.2
2,3-(OCOCH3)2
2,5-(OCOCH3)2
3,4-(OCOCH3)2
2-Cl
3g
3h
3i
3.2
1.3
2.9
0
0.76
0.66
0.71
0.87
0.81
0
-
3j
2-Br
3k
3l
-
4-Br
0
0
2-F
3m
0.4
3-F
3. Experimental
3.1. General
All chemicals were of p.a. quality and purchased from commercial suppliers. Melting points were determined
on a capillary melting point apparatus (Electrothermal, Rochford, Great Britain). NMR spectra were recorded
on a Bruker Avance 300 MHz NMR Spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) at 293 K
in DMSO-d6. The MS spectra were recorded on LCMS/MS API 2000 (Applied Biosystems/MDS SCIEX, CA,
USA) using an electrospray ionization (ESI) source. The elemental analysis for C, H, and N was done on a
PerkinElmer Analyzer 2400 Series II (PerkinElmer, Boston, MA, USA). The absorbance was measured on a
UV visible spectrophotometer Helios γ , (ThermoSpectronic, Cambridge, UK). Mycelia growth was performed
on a rotary shaker (KS 260 basic, IKA, Germany). Chromatographic analyses were performed in an Acquity
UPLC H-Class system (Waters, Milford, MA, USA) using an Acquity BEH C18 column (2.1 × 100 mm, 1.7
µm) (Waters). MS/MS detection of aflatoxins was performed using a Xevo TQD tandem quadrupole mass
spectrometer (Waters).
28
ˇ
Synthesis of hydrazide (1) was performed according to Sarkanj et al. and synthesis of Schiff bases (2)
was performed according to Molnar et al.29 (Figure 1).
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Table 2. Antibacterial activity of novel oxadiazoles on selected bacterial strains.
MIC (mg mL−1
G–
)
G+
Escherichia Pseudomonas Bacillus
coli aeruginosa subtilis
0.001953125 0.001953125
Staphylococcus
aureus
Compound
AMKC
3a
0.001953125 0.001953125
0.03125
0.03125
0.03125
0.015625
0.03125
0.03125
0.03125
0.03125
0.03125
0.03125
0.03125
0.03125
0.015625
0.015625
0.015625
0.015625
0.015625
0.015625
0.015625
0.015625
0.015625
0.015625
0.015625
0.015625
0.03125
0.03125
0.25
0.015625
0.03125
0.015625
0.015625
0.03125
0.03125
0.015625
0.015625
0.03125
0.03125
0.015625
0.03125
3b
3c
3d
3e
0.0078125
0.0078125
0.015625
0.0078125
0.03125
0.015625
0.03125
0.03125
0.0078125
3g
3h
3i
3j
3k
3l
3m
3.2. Synthesis of 1,3,4-oxadiazoles
A mixture of Schiff base and excess of acetic anhydride (10 mL) was refluxed for 1–4 h, and the reaction was
monitored by TLC (benzene:acetone:acetic acid). The excess acetic anhydride was distilled off and residue was
poured into ice-cold water. Upon formation of gummy/oily precipitate, the product was recrystallized from
ethanol and filtered. Structures of all oxadiazole derivatives were confirmed by mass spectrometry, 1 H and 13 C
NMR, and elemental analysis.
3.2.1. 2-(3-Acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-oxadiazol-
2-yl)phenyl acetate (3a)
Yield (3%), mp 186–187 ◦ C, Rf = 0.7: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, DMSO-d6):
2.13 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.39 (s, 3H, CH3), 5.06 (s, 2H, CH2), 6.21 (s, H, CH), 6.91 (1H, s, oxa),
6.93 (1H, d, coum-C6), 7.03 (1H, s, coum-C8), 7.22-7.55 (4H, m, arom), 7.62-7.67 (1H, d, coum-C5); 13 C NMR
(75 MHz, ppm, (CD3)2 SO): 11.3, 18.6, 21.2, 65.3, 65.7, 89.7, 102.0, 111.8, 112.8, 113.9, 124.4, 126.8, 128.1,
129.9, 131,7, 149.3, 153.8, 154.9, 157.3, 160., 161.5, 163.4, 169.3; MS m/z: 437.30 [M + H]+ , (M = 436.41);
Anal. Calcd. For C23 H20 N2 O7 : C, 63.30; H, 4.62; N, 6.42; O, 25.66%; Found: C, 63.39; H, 4.29; N, 6.24%.
3.2.2. 3-(3-Acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-oxadiazol-
2-yl)phenyl acetate (3b)
Yield (5%), mp 200–202 ◦ C, Rf = 0.53: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, DMSO-d6):
2.27 (s, 3H, CH3), 2.37 (s, 3H, CH3), 2.38 (s, 3H, 2CH3), 5.06 (s, 2H, CH2), 6.22 (s, H, CH), 6.89 (1H, s, oxa),
6.98–7.01 (1H, d, coum-C6), 7.06 (1H, s, coum-C8), 7.26–7.65 (4H, m, arom), 7.90–7.96 (1H, d, coum-C5); 13 C
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NMR (75 MHz, ppm, (CD3)2 SO): 18.6, 26.3, 65.8, 68.7, 102.1, 111.8, 113.0, 114.4, 114.7, 119.6, 125.6, 126.8,
131.7, 135.7, 153.8, 155.1, 160.6, 161.5, 164.4, 164.9, 168.7, 171.3; MS m/z: 437.20 [M + H]+ , (M = 436.41);
Anal. Calcd. For C23 H20 N2 O7 : C, 63.30; H, 4.62; N, 6.42; O, 25.66%; Found: C, 62.99; H, 4.29; N, 6.24%.
3.2.3. 4-(3-Acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-oxadiazol-
2-yl)phenyl acetate (3c)
Yield (13%), mp 193–194 ◦ C, Rf = 0.89: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, DMSO-d6):
1.88–2.27 (s, 3H, CH3), 2.36–2.47 (s, 6H, CH3), 5.39 (s, 2H, CH2), 6.12 (s, H, CH), 6.93 (1H, s, oxa), 6.98–7.01
(1H, d, coum-C6), 7.05 (1H, s, coum-C8), 7.26–7.65 (4H, m, arom), 7.90 (1H, d, coum-C5); 13 C NMR (75 MHz,
ppm, (CD3)2 SO): 18.6, 21.4, 26.2, 65.3, 68.8, 102.1, 111.8, 113.0, 113.9, 116.3, 123.1, 126.8, 130.0, 130.8, 153.8,
155.1, 160.6, 161.5, 166.5, 168.4, 169.4, 171.2; MS m/z: 437.30 [M + H]+ , (M = 436.41); Anal. Calcd. For
C23 H20 N2 O7 : C, 63.30; H, 4.62; N, 6.42; O, 25.66%; Found: C, 63.39; H, 4.51; N, 6.34%.
3.2.4. 7-((4-Acetyl-5-(3-methoxyphenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (3d)
Yield (38%), mp 190–191 ◦ C, Rf = 0.64: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, (CD3)2 SO):
2.39–2.42(s, 6H, CH3), 3.80 (s, 3H, OCH3), 5.31 (s, 2H, CH2), 6.22 (s, H, CH), 6.97 (1H, s, oxa), 7.01–7.04
(1H, d, coum-C6), 7.08 (1H, s, coum-C8), 7.15–7.46 (4H, m, arom), 7.65–7.74 (1H, d, coum-C5); 13 C NMR
(75 MHz, ppm, DMSO-d6): 18.6, 26.2, 55.7, 65.7, 68.8, 102.03, 111.8, 112.9, 113.1, 113.9, 118.7, 120.1, 121.8,
126.8, 126.9, 130.4, 134.6, 144.3, 153.8, 155.1, 160.0, 160.6, 161.5, 167.2, 168.4, 171.3; MS m/z: 409.10 [M +
H]+ , (M = 408.41); Anal. Calcd. For C22 H20 N2 O6 : C, 64.70; H, 4.94; N, 6.86; O, 23.50%; Found: C, 64.39;
H, 4.99; N, 6.74%.
3.2.5. 7-((4-Acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (3e)
Yield (7%), mp 184–185 ◦ C (lit.30 170 ◦ C), Rf = 0.82: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz,
ppm, DMSO-d6): 2.41(s, 6H, CH3), 3.86 (s, 3H, OCH3), 5.31 (s, 2H, CH2), 6.19 (s, H, CH), 6.89 (1H, s, oxa),
7.00–7.01 (1H, d, coum-C6), 7.05 (1H, s, coum-C8), 7.26–7.75 (4H, m, arom), 7.90–7.93 (1H, d, coum-C5); 13 C
NMR (75 MHz, ppm, (CD3)2 SO): 18.5, 25.9, 55.9, 68.9, 102.2, 111.8, 112.9, 114.1, 115.0, 125.8, 126.7, 130.8,
153.8, 155.1, 160.5, 161.6, 163.1, 167.5, 168.4, 171.2; MS m/z: 409.20 [M + H]+ , (M = 408.41); Anal. Calcd.
For C22 H20 N2 O6 : C, 64.70; H, 4.94; N, 6.86; O, 23.50%; Found: C, 64.59; H, 4.69; N, 6.74%.
3.2.6. 3-(3-Acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-oxadiazol-
2-yl)-1,2-phenylene diacetate (3f)
Yield (2%), mp 109–110 ◦ C, Rf = 0.55: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, (CD3)2 SO):
2.08–2.13 (s, 3H, CH3), 2.26–2.30 (s, 3H, CH3), 2.36–2.41 (s, 6H, CH3), 5.05 (s, 2H, CH2), 6.21 (s, H, CH),
6.91 (1H, s, oxa), 6.93 (1H, d, coum-C6), 7.05 (1H, s, coum-C8), 7.33–7.46 (3H, m, arom), 7.62–7.65 (1H, d,
coum-C5); 13 C NMR (75 MHz, ppm, DMSO-d6): 11.3, 18.6, 20.9, 65.6, 88.2, 89.4, 101.9, 111.8, 112.8, 113.1,
113.9,126.3, 126.8, 127.1, 130.1, 141.2, 143.3, 153.8, 154.9, 157.4, 160.6, 161.5, 163.6, 168.2, 168.5; MS m/z:
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MOLNAR et al./Turk J Chem
495.30 [M + H]+ , (M = 494.46); Anal. Calcd. For C25 H22 N2 O9 : C, 60.73; H, 4.48; N, 5.67; O, 29.12%;
Found: C, 60.40; H, 4.28; N, 5.24%.
3.2.7. 2-(3-Acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-oxadiazol-
2-yl)-1,4-phenylene diacetate (3g)
Yield (12%), mp 169–170 ◦ C, Rf = 0.53: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, DMSO-d6):
2.13 (s, 3H, CH3), 2.28–2.39 (s, 9H, 3CH3), 5.07 (s, 2H, CH2), 6.21 (s, H, CH), 6.91 (1H, s, oxa), 6.93 (1H,
d, coum-C6), 7.02 (1H, s, coum-C8), 7.28–7.32 (3H, m, arom), 7.62–7.68 (1H, d, coum-C5), 7.90–7.96 (2H, m,
arom); 13 C NMR (75 MHz, ppm, (CD3)2 SO): 11.3, 18.6, 21.2, 65.7, 88.9, 102.0, 111.8, 112.8, 113.9, 113.9,
122.8, 125.18, 125.4, 126.8, 129.1, 146.6, 148.3, 153.8, 154.9, 157.4, 160.6, 161.5, 163.7, 169.3, 169.6; MS m/z:
495.30 [M + H]+ , (M = 494.46); Anal. Calcd. For C25 H22 N2 O9 : C, 60.73; H, 4.48; N, 5.67; O, 29.12%;
Found: C, 60.79; H, 4.28; N, 5.24%.
3.2.8. 4-(3-Acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-oxadiazol-
2-yl)-1,2-phenylene diacetate (3h)
Yield (31%), mp 136–137 ◦ C, Rf = 0.53: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, DMSO-d6):
2.13 (s, 3H, CH3), 2.28–2.39 (s, 9H, 3CH3), 5.32 (s, 2H, CH2), 6.21 (s, H, CH), 6.92–6.94 (1H, s, oxa), 7.01
(1H, d, coum-C6), 7.29 (1H, s, coum-C8), 7.32-7.44 (3H, m, arom), 7.62–7.68 (1H, d, coum-C5); 13 C NMR (75
MHz, ppm, (CD3)2 SO): 11.3, 18.6, 21.2, 65.7, 68.8, 88.9, 102.0, 111.8,113.9, 120.7,122.8, 125.1, 126.8, 129.1,
146.6, 148.3, 153.8, 154.9, 157.4, 160.6, 161.5, 163.7, 169.3, 169.7, 171.13; MS m/z: 495.30 [M + H]+ , (M =
494.46); Anal. Calcd. For C25 H22 N2 O9 : C, 60.73; H, 4.48; N, 5.67; O, 29.12%; Found: C, 60.69; H, 4.39; N,
5.44%.
3.2.9. 7-((4-Acetyl-5-(2-chlorophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (3i)
Yield (17%), mp 190–192 ◦ C, Rf = 0.53: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, DMSO-
d6): 2.13 (s, 3H, CH3), 2.38 (s, 3H, CH3), 5.23 (s, 2H, CH2), 6.21 (s, H, CH), 6.95 (1H, s, oxa), 6.96 (1H, d,
coum-C6), 7.18 (1H, s, coum-C8), 7.42–7.57 (4H, m, arom), 7.63–7.67 (1H, d, coum-C5); 13 C NMR (75 MHz,
ppm, (CD3)2 SO): 11.4, 18.6, 65.8, 89.9, 102.0, 111.8, 112.9, 113.9, 126.8, 128.3, 129.6, 130.6, 132.3, 132.7,
132.9, 153.8, 154.9, 157.1, 160.6, 161.5, 164.0; MS m/z: 413.1 [M + H]+ , (M = 412.8); Anal. Calcd. For
C21 H17 ClN2 O5 : C, 61.10; H, 4.15; Cl, 8.59; N, 6.79; O, 19.38%; Found: C, 62.19; H, 4.29; N, 6.64%.
3.2.10. 7-((4-Acetyl-5-(2-bromophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (3j)
Yield (6%), mp 195–196 ◦ C, Rf = 0.64: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, DMSO-d6):
2.05–2.17 (s, 3H, CH3), 2.39 (s, 3H, CH3), 5.25 (s, 2H, CH2), 6.22 (s, H, CH), 6.96 (1H, s, oxa), 6.98 (1H, d,
coum-C6), 7.13 (1H, s, coum-C8), 7.33–7.48 (4H, m, arom), 7.64–7.73 (1H, d, coum-C5); 13 C NMR (75 MHz,
ppm, (CD3)2 SO): 11.4, 18.6, 65.3, 65.8, 91.7, 102.0, 111.8, 112.8, 112.9, 113.9, 122.4, 126.8, 126.9, 128.9, 129.5,
132.5, 133.8, 134.4, 153.8, 154.9, 157.0, 160.6, 161.5, 164.1; MS m/z: 459.1 [M + H]+ , (M = 457.3); Anal.
154

MOLNAR et al./Turk J Chem
Calcd. For C21 H17 BrN2 O5 : C, 55.16; H, 3.75; Br, 17.47; N, 6.13; O, 17.49%; Found: C, 55.11; H, 3.49; N,
6.25%.
3.2.11. 7-((4-Acetyl-5-(4-bromophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (3k)
Yield (10%), mp 190 ◦ C, Rf = 0.64: CHCl3 /MeOH (10:1). 1 H NMR (300 MHz, ppm, DMSO-d6): 2.16 (s,
3H, CH3), 2.44 (s, 3H, CH3), 5.32 (s, 2H, CH2), 6.18 (s, H, CH), 6.89 (1H, s, oxa), 6.93–6.95 (1H, d, coum-
C6), 7.00 (1H, s, coum-C8), 7.42–7.73 (2H, m, arom), 7.81–7.82 (1H, d, coum-C5); 13 C NMR (75 MHz, ppm,
(CD3)2 SO): 17.9.6, 25.4, 68.5, 101.9, 111.4, 112.3, 113.7, 125.6, 126.2, 128.7, 130.1, 132.2, 153.0, 159.9, 161.1,
164.3, 168.2, 170.8; MS m/z: 458.20 [M + H]+ , (M = 457.3); Anal. Calcd. For C21 H17 BrN2 O5 : C, 55.16;
H, 3.75; Br, 17.47; N, 6.13; O, 17.49%; Found: C, 55.20; H, 3.69; N, 6.19%.
3.2.12. 7-((4-Acetyl-5-(2-fluorophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (3l)
Yield (92%), mp 180–183 ◦ C, Rf = 0.86: C6 H6 /acetone/AcOH (8:1:1). 1 H NMR (300 MHz, ppm, DMSO-d6):
2.39 (s, 3H, OCH3), 2.43 (s, 3H, CH3), 5.33 (s, 2H, CH2), 6.21 (s, H, CH), 6.89 (1H, s, oxa), 6.93–6.95 (1H, d,
coum-C6), 7.00 (1H, s, coum-C8), 7.42–7.73 (4H, m, arom), 7.81–7.82 (1H, d, coum-C5); 13 C NMR (75 MHz,
ppm, (CD3)2 SO): 18.6, 26.2, 68.8, 102.0, 111.7, 112.9, 113.9, 116.7, 120.8, 125.7, 126.8, 128.0, 134.9, 153.8,
155.1, 159.8, 160.6, 161.5, 163.7, 168.5, 171.3; MS m/z: 397.10 [M + H]+ , (M = 396.38); Anal. Calcd. For
C21 H17 FN2 O5 : C, 63.63; H, 4.32; F, 4.79; N, 7.07; O, 20.18%; Found: C, 63.44; H, 4.49; N, 7.20%.
3.2.13. 7-((4-Acetyl-5-(3-fluorophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (3m)
Yield (46%), mp 150–151 ◦ C, Rf = 0.65: CHCl3 /MeOH (10:1). 1 H NMR (300 MHz, ppm, DMSO-d6): 2.40
(s, 3H, OCH3), 2.44 (s, 3H, CH3), 5.36 (s, 2H, CH2), 6.22 (s, H, CH), 6.96 (1H, s, oxa), 7.01–7.05 (1H, d,
coum-C6), 7.09 (1H, s, coum-C8), 7.40–7.71 (4H, m, arom), 7.74–7.76 (1H, d, coum-C5); 13 C NMR (75 MHz,
ppm, (CD3)2 SO): 18.6, 26.3, 68.7, 102.0, 111.7, 112.9, 113.9, 114.7, 119.3, 125.5, 126.8, 131.6,135.8, 153.8,
155.1, 160.6, 161.5, 164.4, 164.9, 168.6, 171.3; MS m/z: 397.10 [M + H]+ , (M = 396.38); Anal. Calcd. For
C21 H17 FN2 O5 : C, 63.63; H, 4.32; F, 4.79; N, 7.07; O, 20.18%; Found: C, 63.56; H, 4.15; N, 7.24%.
3.3. DPPH scavenging activity assay
DMSO solution of the corresponding oxadiazole derivative (0.2 mM) was added to DMSO solution of DPPH
radical (0.2 mM). The mixture was shaken and allowed to stand at room temperature. After 30 min the
absorbance at 517 nm was determined and the scavenging activity was calculated. Ascorbic acid (AA) was used
as a reference compound. All measurements were performed in triplicate.
3.4. Evaluation of antioxidant activity by phosphomolybdenum method
The antioxidant activity of tested coumarin derivatives was evaluated by the phosphomolybdenum method,
according to the procedure described by Prieto et al.31 The antioxidant activity was expressed relative to the
antioxidant activity of the same concentration of AA.
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MOLNAR et al./Turk J Chem
3.5. Iron chelating activity
32
ˇ
Iron chelating activity of the novel compounds was performed according to Caˇci´c et al. Briefly, solution of
FeCl2 (2 mM, 25 µL) was added to 2 mM solution of the desired compound followed by addition of ferrozine.
After incubation at room temperature the absorbance was measured at 562 nm. EDTA was used as standard
compound.
3.6. Antifungal and antiaflatoxigenic assay
Antifungal investigation was performed by submersed growing of Aspergillus flavus NRRL 3251 in aflatoxin-
inducing YES media,33 with the addition of coumarinyl 1,3,4-oxadiazoles to obtain final concentrations of 0,
15.625, 31.25, 62.5, 125, 250, and 500 mg mL−1 . Conidia suspension was prepared according to Sarkanj et
ˇ
al.28 After incubation at 29 ◦ C for 72 h, wet mycelia were separated and dry mycelia weight was determined.
Separated culture filtrates were used for antiaflatoxigenic activity determination by the “dilute and shoot”
method.34 Chromatographic analyses were performed with a gradient elution consisting of eluent A (water
with 0.1% formic acid) and eluent B (acetonitrile with 0.1% formic acid). Eluent A was held at 98% for the
first 0.5 min, followed by a decrease to 10% in 4.0 min, held for 0.5 min at 10%, followed by an increase to 98%
to 4.6 min, and equilibration for another 1.6 min, to give a total run time of 6 min. The flow rate was 0.5 mL
min−1 and the column temperature was 40 ◦ C. The capillary voltage was 3.5 kV, the source temperature was
150 ◦ C, and the desolvation gas temperature was 400 ◦ C with a flow of 650 L h−1 . Instrument control, data
acquiring, and processing were done by MassLynx and TargetLynx software (v. 4.1., Waters). Recovery was
92% for all aflatoxins. The limit of detection (LOD) was 0.15 ng mL−1 , and the limit of quantification (LOQ)
was 0.5 ng mL−1 for all aflatoxins.
3.7. Antibacterial assay
The antibacterial activities of coumarin derivatives were evaluated against four test bacteria strains as described
in our previous work.35 Briefly, two gram-positive Bacillus subtilis and Staphylococcus aureus and two gram-
negative Escherichia coli and Pseudomonas aeruginosa were investigated. The antibacterial activity was
assessed by a modified broth microdilution method in terms of minimum inhibitory concentrations (MICs),
defined as the lowest concentrations of a compound at which there was no visual turbidity due to microbial
growth.36 All assays were performed in duplicate.
4. Conclusions
We synthesized new coumarinyl derivatives bearing a 1,3,4-oxadiazole ring in their structure. This combination
was proven to be an excellent tool for gaining some bioactive compounds, considering antibacterial, antifungal,
and antiaflatoxigenic activity. Since some of the investigated compounds possess potent antibacterial and
antiaflatoxigenic activity, structures of this type are a good base for the design and synthesis of some new and
more efficient drugs. The results highlight these new coumarinyl derivatives as potential structures for further
investigation on new antibacterial and antiaflatoxigenic drug candidates.
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157

Supplementary material
2-(3-acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-
oxadiazol-2-yl)phenyl acetate (3a)
1

3-(3-acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-
oxadiazol-2-yl)phenyl acetate (3b)
2

4-(3-acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-
oxadiazol-2-yl)phenyl acetate(3c)
3

7-((4-acetyl-5-(3-methoxyphenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-
methyl-2H-chromen-2-one (3d)
4

7-((4-acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-
methyl-2H-chromen-2-one (3e)
5

3-(3-acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-
oxadiazol-2-yl)-1,2-phenylene diacetate (3f)
6

2-(3-acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-
oxadiazol-2-yl)-1,4-phenylene diacetate (3g)
7

4-(3-acetyl-5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-2,3-dihydro-1,3,4-
oxadiazol-2-yl)-1,2-phenylene diacetate (3h)
8

7-((4-acetyl-5-(2-chlorophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-
2H-chromen-2-one (3i)
9

7-((4-acetyl-5-(2-bromophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-
2H-chromen-2-one (3j)
10

7-((4-acetyl-5-(4-bromophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-
2H-chromen-2-one (3k)
11

7-((4-acetyl-5-(2-fluorophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-
2H-chromen-2-one (3l)
12

7-((4-acetyl-5-(3-fluorophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-
2H-chromen-2-one (3m)
13