In vitro antifungal screening effects of the investigated compound were tested against specific fungal species
(Aspergillus niger and Candida albicans). We found that compound 5 exhibits antifungal activity against Aspergillus niger
and Candida albicans (Fig. 1b).
Antioxidant Activity. Most antioxidant compounds in a typical diet are derived from plant sources and belong to
classes of compounds with a wide variety of physical and chemical properties. Some compounds, such as gallates, have strong
antioxidant activity, while others, such as monophenols, are only weak antioxidants [22]. The role of antioxidants is to remove
free radicals. One important mechanism by which this is achieved is the donation of hydrogen to free radicals to reduce them
to nonreactive species. Addition of hydrogen removes the unpaired electron that is responsible for radical reactivity. Free
radicals have been a subject of significant interest among scientists for the past decade. Compound 5 has good antioxidant
activity (Fig. 2) compared with the reference compound ascorbic acid.
EXPERIMENTAL
All chemicals used were of reagent grade (supplied by either Merck or Fluka) and used as supplied. The FTIR spectra
were recorded as KBr discs on an FTIR 8300 Shimadzu spectrophotometer. The UV-visible spectra were measured using a
Shimadzu UV-Vis 160 A spectrophotometer. Proton NMR spectra were recorded on a Bruker DPX 300 MHz spectrometer.
Elemental microanalysis was performed using the CHN elemental analyzer (model 5500) Carlo Erba instrument.
Synthesis of Ethyl 2-(2-Oxo-2H-chromen-7-yloxy)acetate (2). A suspension of umbelliferone (1) (6.17 mmol) in
acetone (30 mL) was refluxed with ethyl bromoacetate (9.15 mmol) and K CO (4.69 g, 33.91 mmol) for 12 h. After cooling,
2
3
the mixture was evaporated to dryness, and the residue was partitioned between CHCl (50 mL) and water (50 mL). The
3
organic phase was dried (Na SO ), filtered, and evaporated to dryness. The residue was recrystallized from acetone [19].
2
4
Colorless needles (yield 76%); mp 112.5ꢂC.
Synthesis of 2-(2-Oxo-2H-chromen-7-yloxy)acetic acid (3). A solution of compound 2 (2.7 mmol) and sodium
hydroxide 5% (2.16 mL) in ethanol (15 mL) was stirred under reflux for 2 h. After removal of the solvent, the residue was
dissolved in water and acidified with 6 M HCl. The white solid collected by filtration was washed with cold water, dried, and
crystallized from ethanol [19]. White powder (yield 93%); mp 210.5ꢂC.
Synthesis of 7-((5-Amino-1,3,4-thiadiazol-2-yl) methoxy)-2H-chromen-2-one (4). Phosphorus oxychloride (20 mL)
was added to compound 3 (0.05 mol), and the mixture was stirred for 1 h at room temperature. Thiosemicarbazide (4.56 g,
0.05 mol) was added, and the mixture was heated under reflux for 5 h. Upon cooling, the mixture was poured onto ice. After
4 h, the mixture was stirred for 15 min to decompose the excess phosphorus oxychloride, heated under reflux for 30 min,
cooled, and neutralized with 5% potassium hydroxide. The precipitate was filtered, washed with water, dried, and recrystallized
–1
1
from ethanol, yield 51%, mp 105ꢂC. C H N O S. IR (ꢁ, cm ): 3302 and 3343 (N-H, amine), 1747 (C=O, lactone). H NMR
12
9 3 3
13
(CDCl , ꢀ, ppm): 4.94 (1H, s, NH ), 5.760, 5.891 (each 1H, s, H-3, 4), 7.510–7.283 (m, C-H aromatic ring). C NMR
3
2
(DMSO-d , ꢀ, ppm): 101.9, 111.0, 113.1, 114.1, 128.1, 149.5, 156.3, 160.8, 161.2, 163.5, 168.3, 176.4.
6
Synthesis of 5-((2-Oxo-2H-chromen-7-yloxy)methyl)-1,3,4-thiadiazol-2(3H)-one (5).A10% aqueous sodium nitrite
solution (10 mL) was added dropwise with continuous stirring over a period of 20 min to a cooled (ice-bath) suspension of
compound 4 (0.01 mol) and hydrochloric acid (5 mL) in cold water (20 mL). The mixture was allowed to rise to room
temperature, heated to boiling for 10 min, cooled, and allowed to stand overnight. The separated crude product was filtered,
–1
washed with water, dried, and recrystallised from ethanol, yield 47%, mp 133ꢂC. C H N O S. IR (ꢁ, cm ): 3397 (N-H,
12
8 2 4
1
amine), 1713 (C=O, lactone). H NMR (CDCl , ꢀ, ppm, J/Hz): 7.81 (1H, d, J = 2.2, H-8), 7.51 (1H, d, J = 8.7, H-5), 7.29 (1H,
3
dd, J = 8.5, 2.2, H-6), 6.43 (1H, s, H-4), 5.81 (1H, s, H-3), 5.31 (1H, br.s, H-4ꢃ), 5.21 (1H, d, J = 7.5, H-1ꢃ), 4.61 (1H, d, J = 2.1,
13
H-1ꢃ). C NMR (ꢀ, ppm, J/Hz): 161.1 (d, J = 256, C-2), 115.1 (d, J = 3, C-3), 155.9 (s, C-4), 123.2 (s, C-5), 117.5 (s, C-6),
166.2 (s, C-7), 103.2 (C-8), 117.9 (s, C-1ꢃ), 155.3 (d, J = 21, C-2ꢃ), 171.2 (s, C-5ꢃ).
Evaluation ofAntibacterialActivities. The in vitro antibacterial effects of the synthesised compound 5 were evaluated
against one species of Gram-positive bacteria (Staphylococcus aureus) and four Gram-negative bacterial species (Escherichia
coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Proteus vulgaris) using the disc diffusion method [23, 24] on
nutrient agar medium. The bacteria were subcultured in the agar medium and incubated for 24 h at 37ꢂC. The 5 mm discs were
soaked in test solutions (sterile filter paper discs, Whatman No. 1.0) with the appropriate amount of compound 5 dissolved in
–1
sterile dimethylsulfoxide (DMSO) at concentrations of 0.1–1.0 mg disc , placed on an appropriate medium previously seeded
with organisms in Petri dishes, and stored in an incubator for the above-mentioned period of time. The inhibition zone around
952