benzo[d] isoxazole: In vitro Antimicrobial and Antioxidant activity

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Supplementary Information
Synthesis and characterization of new thiourea and urea derivatives
of 6-fluoro-3-(piperidin-4-yl)benzo[d] isoxazole: In vitro
Antimicrobial and Antioxidant activity
HASTI SUDHAMANIa, SK THASLIM BASHAa, NAGAM VENKATESWARLUb, TARTTE
VIJAYAb and CHAMARTHI NAGA RAJUa,*
a
Department of Chemistry, S. V. University, Tirupati-517 502, Andhra Pradesh, India
b
Department of Botany, S. V. University, Tirupati-517 502, Andhra Pradesh, India
e-mail: rajuchamarthi10@gmail.com.
S.No
Content
Table/Figure
1
Antibacterial activity
2
In vitro bacterial activity of the title compounds (8a-g/9a-d)
3
Antifungal activity
4
In vitro evaluation of antifungal activity of the synthesized
Page No
2
Table S1
3
4
Table S2
5
compounds (8a-g/9a-d).
5
H2O2 Scavenging Assay
6
6
Antioxidant activity of the synthesized compounds
Table S3
7
byH2O2method
7
DPPH radical scavenging activity
8
8
Antioxidant activity of the synthesized compounds by DPPH
method.
1
Table S4
9
9
Title compounds
Figure S1
9
10
Proton and Carbon NMR of compound 8c
Figure S2&3
10
11
Proton and Carbon NMR of compound 9d
Figure S4&5
11
BIOLOGICAL ACTIVITY
Antibacterial activity
All the newly synthesized compounds (8a-g and 9a-d) have been assayed for their in vitro
antibacterial activity by disc diffusion method23-24 in comparison with the reference compound
Tetracycline against pathogenic representative Gram-positive Bacillus subtilis (MTCC-441) and
Staphylococcus aureus (MTCC-737)and Gram-negative Escherichia coli (MTCC-443)and
Pseudomonas aeruginosa (MTCC-741). The bacterial strains selected for this study are most
common and easily available. Tetracycline was used as a standard drug, where as sterile DMSO
poured disc was used as negative control and solvent for dilution of the compounds showed no
effect on test organisms. The bacterial strains were maintained on liquid LB (Luria Bertani) agar
medium for 24 hrs, adjusted at a concentration of 106 cells/mL and spread (100 μL) on petri
dishes containing Nutrient agar mediumhomogeneously. Each dish was placedequidistantly, four
disks of sterile filter paper Whatman no. 4 (6 mm) inoculated with 10μL (1mg/mL
concentration)of extracts. As negative controls, paper disks were inoculated with autoclaved
distilled water as positive control, Tetracycline (50 μg/mL in DMSO) was used. The dishes were
incubated at 37 °C in B.O.D for 24 hrs. After an overnight incubation, the bacterial growth
around each disc is observed. The antibacterial activity was evaluated by measuring the diameter
of clear Zone of inhibition (ZI) in mm of each zone. The inhibition zones of the test compounds
2
were measured and compared with standard. In all determinations, tests were performed in
triplicate and the results were reported as mean of three determinations presented in Table S1.
Table S1. In vitro antibacterial activity of the title compounds (8a-g/9a-d).*
Zone of Inhibition (mm)
Compd.
Bacillus subtilis
Escherichiacoli
Pseudomonas
aeruginosa
Staphylococcus
aureus
8a
12.0 ± 0.08
12.2 ± 0.26
12.2 ± 0.16
11.8 ± 0.14
8b
12.2 ± 0.14
12.6 ± 0.18
12.5 ± 0.14
12.0 ± 0.15
8c
12.0 ± 0.12
12.5 ± 0.15
11.8 ± 0.22
12.5 ± 0.20
8d
10.6 ± 0.16
10.6 ± 0.14
10.5 ± 0.21
10.8 ± 0.16
8e
9.8 ± 0.18
10.2 ± 0.18
10.0 ± 0.22
9.8 ± 0.22
8f
10.8 ± 0.22
11.2 ± 0.22
10.6 ± 0.08
11.2 ± 0.18
8g
12.8 ± 0.26
12.6 ± 0.16
12.5 ± 0.16
12.6 ± 0.26
9a
11.4 ± 0.14
11.8 ± 0.14
11.4 ± 0.16
11.7 ± 0.14
9b
11.5 ± 0.16
11.2 ± 0.15
11.0 ± 0.15
10.3 ± 0.18
9c
12.5 ± 0.14
12.7 ± 0.17
12.7 ± 0.15
12.5 ± 0.16
9d
11.1 ± 0.16
11.5 ± 0.10
11.4 ± 0.14
11.5 ± 0.12
Std
15.5 ± 0.15
15.2 ± 0.12
15.4 ± 0.15
15.5 ± 0.08
Std: Tetracycline
Values were mean of the triplicates ± SD.
* Antibacterial activity was carried out at 100 µg/mL.
3
Antifungal activity
Antifungal activity was screened against two fungal strains, Candida albicans and Candida non
albicans using Ampotericin -B as a standard drug. Stock solutions of the test compounds and
antibiotics were dissolved in DMSO (Merck) was used as solvent control. Antifungal activity
was determined using the disc diffusion method25 according to the National Committee for
Clinical Laboratory Standards (NCCLS), 2003. Fungal stains Candida albicans, Candida non
albicans grown on pre-warmed Mueller-Hinton agar (MHA), is first evenly seeded throughout
the plate with the isolate of interest that has been diluted at a standard concentration. Test
compound dissolved in DMSO (1 mg/mL) were pipetted (10 µL) onto sterile paper discs (6 mm
diameter) and placed onto the surface of inoculated agar plates. Plates were inverted and
incubated for 37ºC in BOD incubator for 72 hrs. The zone of inhibition was measured for
pathogenecity of test compound. The experiment was repeated thrice. Triplicates were
maintained for each test compound at every 24 hours interval. After the incubation period, the
diameter of inhibition zone was measured and documented as an indicator for the activity of the
compounds. The data of the antifungal activity are listed in the Table S2.
4
Table S2. In vitro evaluation of antifungal activity of the synthesized compounds (8a-g/9a-d).*
Zone of Inhibition (mm)
Compd.
Candia albicans
Candida non
albicans
8a
11.2 ± 0.14
11.8 ± 0.22
8b
11.8 ± 0.15
12.4 ± 0.12
8c
10.5 ± 0.25
9.8 ± 0.16
8d
11.8 ± 0.16
12.2 ± 0.14
8e
12.2 ± 0.20
13.4 ± 0.15
8f
10.6 ± 0.12
11.3 ± 0.18
8g
11.5 ± 0.20
11.8 ± 0.12
9a
10.4 ± 0.16
12.8 ± 0.20
9b
10.6 ± 0.18
12.4 ± 0.14
9c
11.8 ± 0.16
12.0 ± 0.18
9d
10.6 ± 0.10
11.2 ± 0.16
Std
15.2 ± 0.18
15.8 ± 0.20
Std: Ampotercin-B
Values were mean of the triplicates ± SD.
*Antifungal activity was carried out at 100 µg/mL.
5
Antioxidant activity
The radical scavenging activities of the newly synthesized compounds (8a-g, and 9a-d) were
screened for their free radical scavenging activity. The free radical scavenging activity of the
synthesized compounds was determined by hydrogen peroxide(H2O2) and 1,1-diphenyl-2-picrylhydrazil (DPPH) method.
H2O2 Scavenging Activity
The antioxidant activity of the test compounds and standard (Ascorbic acid) were assessed on the
basis of the radical scavenging effect of the stable hydrogen peroxide free radical.26-27 A solution
of hydrogen peroxide (40 mM) was prepared in phosphate buffer at pH 7.4. 25, 50, 75 and 100
µg/mL concentrations of the test compounds in 3.4 mL of phosphate buffer were added to H2O2
solution (0.6 mL, 40 mM). The test tubes were incubated for 30 min in dark with occasional
shaking. The absorbance of the test samples and phosphate buffer without hydrogen peroxide
were measured at 230 nm using UV spectrophotometer. Ascorbic acid was used as the standard.
The percentage of hydrogen peroxide radical scavenging activity was calculated using the given
formula:
Where, Abscontrol was the absorbance of the standard H2O2; Abssample was the absorbance
in the presence of the sample and standard H2O2. The experiment was performed in triplicates
and standard deviation values were reported in TableS3.
6
Table S3. Antioxidant activity of the synthesized compounds byH2O2method.
Percentage of radical scavenging activity by H2O2 method
Compd.
25µg/mL
50 µg/mL
75 µg/mL
100 µg/mL
8a
43.87±0.18
48.34±0.22
62.14±0.24
70.62±0.28
8b
44.21±0.20
50.14±0.18
66.43±0.30
72.86±0.24
8c
48.16±0.36
59.68±0.28
70.32±0.25
80.12±0.32
8d
44.28±0.14
56.15±0.22
65.74±0.35
75.18±0.24
8e
46.14±0.32
58.42±0.12
69.29±0.14
78.14±0.20
8f
47.48±0.18
56.89±0.24
67.14±0.24
72.18±0.14
8g
51.46±0.24
62.16±0.18
72.42±0.32
84.82±0.14
9a
46.52±0.30
54.21±0.25
63.12±0.26
72.86±0.32
9b
45.86±0.32
53.14±0.24
61.14±0.26
70.24±0.34
9c
51.68±0.18
59.63±0.32
66.46±0.12
75.02±0.25
9d
46.16±0.25
58.78±0.28
68.52±0.15
74.16±0.16
Ascorbic acid
74.84
81.68
88.14
92.42
Blank
----
----
----
----
Blank- Methanol. (----) indicates no scavenging activity. Values were the means of three replicates ± SD
7
DPPH free radical scavenging assay
The antioxidant activity of the extracts was determined by the DPPH (1,1-diphenyl-2picrylhydrazyl) assay. DPPH radicals are considered as a representative method for the
preliminary screening of compounds able to scavenge activated oxygen species, since they are
more stable and easier to handle than oxygen free radicals and DPPH assay has been largely
used. The antioxidant activity of the compounds (8a-g and 9a-d) and the standard were assessed
on the basis of radical scavenging effect of DPPH free radical.28-29 2 mg of the test compounds
were dissolved in 10 mL of methanol and further diluted to various concentrations (25, 50, 75,
100 µg/mL). DPPH solution was prepared for the assay by mixing of 4 mg of DPPH in 100 mL
of methanol. The mixture was shaken vigorously and allowed to stand at room temperature for
30 min. After this, the absorbance at 517 nm was determined by using UV-Vis
spectrophotometer Beckman DU-40 and the solution of ascorbic acid was used as a standard for
this study was prepared in distilled H2O. The percentage of scavenging activity was calculated
by using the formula shown below. The experiment was repeated in triplicates and the values
mentioned in Table S4.
% inhibition of DPPH
8
Table S4. Antioxidant activity of the synthesized compounds by DPPH method.
Percentage of radical scavenging activity by DPPH method
Compd.
25 µg/mL
50 µg/mL
75 µg/mL
100 µg/mL
8a
35.70 ± 0.18
40.34 ± 0.12
57.50 ± 0.24
66.15 ± 0.28
8b
36.18 ± 0.24
44.14 ± 0.12
58.43 ± 0.30
64.25 ± 0.24
8c
40.80 ± 0.42
51.48 ± 0.16
56.26 ± 0.32
77.42 ± 0.28
8d
41.56 ± 0.22
49.64 ± 0.26
63.38 ± 0.12
73.80 ± 0.40
8e
42.36 ± 0.40
55.82 ± 0.24
60.40 ± 0.24
76.56 ± 0.24
8f
41.65 ± 0.20
49.20 ± 0.14
62.84 ± 0.14
67.20 ± 0.18
8g
48.55 ± 0.32
58.76 ± 0.18
67.54 ± 0.22
79.60 ± 0.32
9a
42.50 ± 0.25
51.86 ± 0.15
58.12 ± 0.26
66.48 ± 0.20
9b
38.40 ± 0.12
45.52 ± 0.15
57.36 ± 0.16
65.84 ± 0.40
9c
49.20 ± 0.28
56.64 ± 0.28
66.46 ± 0.12
76.44 ± 0.18
9d
43.54 ± 0.22
52.20 ± 0.14
64.25 ± 0.15
72.46 ± 0.22
Ascorbic acid
74.65
82.50
90.15
94.50
Blank
----
----
----
----
Blank- Methanol. (----) indicates no scavenging activity. Values were the means of three replicates ± SD
9'
10'
X
18 17R
12
16
N 11
N 13
15
10 H
14
3
2 1 8
4
N 9
O
F 5 6 7
Figure S1. Numbering of the title compound
9
Figure S2.1H NMR spectrum of compound 8c.
Figure S3.13C NMR spectrum of compound8c.
10
FigureS4.1H NMR Spectrum of compound 9d.
Figure S5.13C NMR spectrum of compound9d.
11
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