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