All chemicals were purchased from Fisher Scientific, Suwanee, GA and used without further purification. All NMR spectra were obtained on a 400 MHz varian NMR spectrometer. Chemical shifts are given in ppm with TMS as internal reference. All spectra also recorded at room temperature. Mass data was recorded on a Varian LC-MS with ESI.

4-amino-3,5-dimethyl-1,2,4-triazole was synthesized from acetonitrile and hydrated hydrazine in methanol following literature procedure [17] . A mixture of 3.0 ml hydrated hydrazine, 1.5 ml acetonitrile and 1 ml methanol was heated in a 15 mL Teflon- lined autoclave at 120˚C for 3 days (Scheme 1). The reaction mixture was cooled to room temperature slowly and finally cooled in ice bath. The white crystal product was filtered off. Yield 83%; ¹HNMR (δ, DMSO-d₆): 2.3 (s, 6H, CH₃), 5.77 (s, 2H, NH₂); ¹³CNMR (δ, DMSO-d₆): 10.18, 151.78

1,10-phenanthroline-2,9-dicarboxaldehyde (9) was synthesized form 2,9-dimethyl-1,10- phenanthroline hemihydrate (8) following previously reported procedure [18] [19] . Neocuproine hemihydraes (3.0 g, 0.0144 mol) was dissolved in 200 ml 1,4-dioxane containing 4% water and SeO₂ (6.0 g, 0.054 mol) was added and the reaction mixture was refluxed for 6 h (Scheme 2). Then the reaction mixture was filtered through thick celite pad. Light yellow product was precipitated on slow cooling to room temperature. The product was isolated and dried under vacuum. The crude product was purified by recrystallization in chloroform to get pure compound (Y: 70%). ¹HNMR (δ, DMSO-d₆):

Scheme 1. Synthesis of 4-amino-3,5-dimethy-1,2,4-triazole.

Scheme 2. Synthesis of Schiff base 1 from 1,10-phenanthroline-2,9-dicarboxaldehyde.

10.36 (s, 2H, CHO), 8.9 (d, J_ortho= 7.8Hz, 2H, Ar-H), 8.36 (m, 4H, Ar-H); ¹³CNMR (δ, DMSO-d₆): 194.13, 152.68, 145.77, 138.86, 131.95, 129.76, 120.67.

[2,2’-Bipyridine]-4,4’-dicarboxaldehyde (11) was synthesized from 4,4’-dimethyl-2,2’- bipyridine (10) through direct oxidation by SeO₂ under nitrogen condition following literature procedure [20] . 4,4’-dimethyl-2,2’-bipyridine (2.0 g, 0.012 mol) was dissolved in 100 mL 1,4-dioxane containing 4% water and SeO₂ (4.8 g, 0.048 mol) was added and the reaction mixture was refluxed for 48 h (Scheme 3). The reaction was carried out under N₂ environment. Then the hot reaction mixture was filtered through a thick Celite pad. The filtrate was kept overnight in dark place to isolate dialdehyde as solid precipitate. The product was isolated and dried under vacuum. The product was further purified by recrystallization in chloroform(Y: 43%). ¹HNMR (δ, DMSO-d₆): 10.18 (s, 2H, CHO), 9.22 (d, J_meta= 2.32Hz, 2H, Ar-H), 8.65 (d, J_ortho= 8.6Hz, 2H, Ar-H), 8.45 (dd, J_ortho= 8.6Hz, J_meta= 2.32Hz, 2H, Ar-H); ¹³CNMR (δ, DMSO-d₆): 192.96, 158.62, 151.95, 138.36, 132.19, 122.55.

[2,2’-Bipyridine]-5,5’-dicarboxaldehyde (13) was synthesized from 5,5’-dimethyl-2,2’- bipyridine (12) through direct oxidation by SeO₂. [2,2’-Bipyridine]-5,5’-dicarboxaldehyde was afforded from the treatment of 5.56 mmol 5,5’-dimethyl-2,2’-bipyridine with 20 mmol SeO₂ in 40 ml 1,4-dioxane by reflux for 48 h under N₂ environment (Scheme 4).

Scheme 3. Synthesis of Schiff base 2 from 2,2’-bipyridyl-4,4-dialdehyde.

Then the hot reaction mixture was filtered through a thick Celite pad. The filtrate was kept over night in dark place to isolate dialdehyde as solid precipitate. The dialdehyde was isolated and dried under vaccum. The pure compound was obtained by washing with hot chloroform (Y: 47%). ¹HNMR (δ, DMSO-d₆): 10.22 (s, 2H, CHO), 9.05 (d, J_ortho= 7.88Hz, 2H, Ar-H), 8.8 (s, 2H, Ar-H), 7.95 (d, J_ortho= 7.88Hz, 2H, Ar-H); ¹³CNMR (δ, DMSO-d₆): 193.81, 156.33, 151.56, 143.31, 123.21, 119.88.

[2,2’-Bipyridine]-6,6’-dicarboxaldehyde (15) was synthesized from 6,6’-dimethyl-2,2’- bipyridine (14) through direct oxidation by SeO₂ following literature procedure [21] [22] . 6,6’-dimethyl-2,2’-bipyridine (2.0 g, 0.012 mmol) was dissolved in 160 mL glacial acetic acid and SeO₂ (12.0 g, 0.12 mol) was added and the reaction mixture was refluxed for 48 h (Scheme 5). Then the hot reaction mixture was filtered through a thick Celite pad. The filtrate was kept overnight in dark place to isolate dialdehyde as solid precipitate. The product was isolated and dried under vacuum. The crude product was dissolved in hot chloroform and then cooled to room temperature slowly. The impurities precipitated leaving the impurities in solution. The impurities were filtered off and the filtrate was evaporated under vacuum to get pure product (Y: 44%). ¹HNMR (δ, DMSO-d₆): 10.12 (s, 2H, CHO), 9.77 (d, J_ortho= 7.92Hz, 2H, Ar-H), 8.3 (t, J= 7.88, 2H, Ar-H), 8.05 (d, J_ortho= 7.64Hz, 2H, Ar-H); ¹³CNMR (δ, DMSO-d₆): 193.85, 155.26, 152.55, 139.65, 125.6, 122.9.

0.5 mmol aldehyde was dissolved in 20 ml hot ethanol or methanol. 1.1 mmol amine 1 was added to the hot solution followed by reflux for 2 h. The reaction mixture was cooled to room temperature and solvent was evaporated under vacuum. Product that obtained was washed with hot acetonitrile to get pure products. Addition of 2 - 3 drops of H₂SO₄ increased the yield by at least 5%.

All the cell lines were purchased from American Type Culture Collection (ATCC), Manassas, VA. Dulbecco’s Modified Eagle’s Medium (DMEM), bovine calf serum (BCS) and penicillin-streptomycin were purchased from GIBCO, New York, USA. MTT assay kit, Glutathione assay kit and reduced glutathione were purchased from Calbiochem, California, USA.

All four different cell lines were cultured under 5% CO₂ atmosphere at 37˚C in DMEM supplemented with 10% (v/v) BCS and 1% (v/v) penicillin-streptomycin following literature procedure [23] . Media was changed in every two days. At 80% confluence in T-75 flasks, cells were trypsinized with 3 ml of Trypsin-EDTA solution and seeded in either 6 or 96 well plates.

Cell viability was determined as previously reported [23] [24] . Briefly, 1 × 10⁴ cells/well were seeded in 96-well plates and treated with 100 µL different doses (10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 200 µM) of the synthesized Schiff bases dissolved in DMSO. The plates were then incubated in an incubator (Thermo Scientific, Marietta, OH) at 37˚C, 5% CO₂ for 24 h. following the incubation 0.5 mg/ml MTT (3-4,5-dimethylthiazol-2-yl-2, 3-diphenyl tetrazolium bromide) were added to the wells and incubated at 37˚C for 4 hours. Supernatants were aspirated and the formazan crystals were dissolved in 100 µL DMSO and the absorbance read at 570 nm in a synergic microplate reader (BioTek, Winooski, VT). The percentage of cell viability was calculated using previously reported formula [20] . The experiments were repeated 3× with each treatment at 4 replicates.

2 × 10⁶ cells/well for each cell lines were seeded in 6-well plates and cells were treated with 25, 50, 75 and 100 µM for each of synthesized Schiff bases and incubated for 24 h at 37˚C under 5% CO₂. Supernatants were aspirated and 500 µL of Trypsin-EDTA was added and incubated for 5 - 10 minutes at 37˚C to detach cells from wells. 1.50 ml DMEM was added and the cell solutions (2 ml) were transferred to a 2 ml Eppendorf tubes. Cells were pelleted at 3000 x g for 10 minutes at 4˚C in a refrigerated centrifuge (Thermo Scientific, Marietta, OH). Supernatants were discarded and cells were resuspended in 200 µL of 5% metaphosphoric acid (MPA). Reduced GSH was analyzed according to previously reported method [25] . The cells were sonicated under ice for 30 seconds using a cell dismembrator (Fisher Scientific, Suwanee, GA) at a setting of 3. Samples were then centrifuged at 4˚C at 4000 × g for 8 minutes and the supernatant was used for the GSH analysis as described by the GSH Assay Kit (Cat# 354102). A standard curve was prepared to calculate GSH levels expressed as nmol GSH/10⁶ cells [25] [26] . The experiments were repeated 2× with each treatment at 4 replicates.

Antibacterial screening test was done using the disc diffusion method [10] [27] [28] . Previously reported procedure [10] was followed to prepare media plate and culture bacteria. Stock solution (10 mg/ml DMSO) was prepared for both synthesized compounds and antibacterial drug tetracycline. Stock solution soaked sterile 6 mm whatman filter paper was placed in the middle of bacteria spread media plate and incubated for 24 h at 37˚C. Zone of inhibition was measured after 24 h incubation.

The starting amine 7 was synthesized following previously reported procedure (Scheme 1) [17] . 2,9-phenanthroline-1,10-dialdehyde (9) was also synthesized using literature procedure [18] [19] . The dialdehyde was then reacted with 1 to produce Schiff base 1 (Scheme 2). All product structures were determined by spectral methods. The dialdehyde 11 and 15 have been prepared using previously published procedure [20] [21] [22] following SeO₂ oxidation. Dialdehyde 13 has been synthesized previously via multiple step reactions such as bromination of 5,5’-dimethyl-2,2’-bipyridine followed by hydrolysis in acetic acid [29] . In this paper we are reporting an efficient SeO₂ oxidation reaction to synthesize dialdehyde 13. Procedure involves the use of 1:4 equivalents of the precursor to SeO₂ ratio producing the best yield (47%). Bipyridylaldehydes (11, 13 and 15) were then subjected to react with amine 7 to produce corresponding Schiff bases 2, 3 and 4 (Schemes 3-5). The synthesis of Schiff bases were carried out in both methanol as well as in ethanol. Higher yields of all four Schiff bases were obtained in ethanol. No reaction occurred in tetrahydrofuran and in chloroform. Solvent Polarity and reflux temperature may have had tremendous effect on Schiff base formation. Addition of 2 - 3 drops of sulfuric acid to the reaction mixture further increased the yield of Schiff bases due to the enhanced nucleophilic attack by amine group of 4-amino-3,5-dimethyl-1,2,4-triazole to carbonyl carbon upon protonation of the carbonyl group. Percent yields of Schiff bases for different reaction conditions are listed in Table 1.

The synthesized Schiff bases were tested to determine their effects on cell viability in A549, BT549, PC3 and 3T3-L1 cell lines using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Di- phenyltetrazolium Bromide (MTT) assay [23] [24] (Table 2). Briefly cells were treated with the respective compounds dissolved in DMSO. Controls containing 2% DMSO was used as the negative control. The anticancer drug tamoxifen was used as positive control. IC₅₀ for tamoxifen for the A549, BT549, PC3 and 3T3-L1 cell lines were 55, 55, 60 and 35 µM respectively. Compound 4 decreased cell viability in A549 and BT549 cell lines with IC₅₀ values of 70 µM and 50 µM respectively.

However, cell viability did not significantly decrease for compound 4 following exposure to the PC3 and 3T3-L1 cells respectively. On the other hand, tamoxifen was found to have more cytotoxicity against 3T3-L1 cells compared to the A549, BT549 and 3T3- L1 cell lines. In addition compound 4 was found to have a better anticancer activity against BT549 with an IC₅₀ of 50 µM compared to anticancer drug tamoxifen with an IC₅₀ of 55 µM. Compound 1, 2 and 3 did not show any significant effects on cell viability in any of the four cell lines tested. These findings suggest that apart from compound 4 which showed some cytotoxic effects to cell lines A549 and BT549, all the others did not affect cell lines tested as well as their anti-proliferative activities.

Oxidative stress causes cancer [30] , Alzheimer [31] , Parkinson [32] , infection [33] and heart failure [34] . Glutathione (GSH) plays an important role in the antioxidant status of cells. We measured GSH levels in A549, BT549, PC3 and 3T3-L1 after 24 h exposure to our synthesized compounds. Table 3 shows the levels of GSH in controls and treated samples for all the four cell lines. Compound 1 increased GSH level slightly for all the doses tested for four cell lines. The increases in GSH for compound 1 from 10 µM to 100 µM were 1.17, 0.98, 1.51 and 1.89 nMole/106 cells for A549, BT549, PC3 and 3T3- L1 cell lines respectively. Compounds 2 and 3 did not have any significant effect on GSH levels for the doses tested. Furthermore, Compound 4 did not have significant effect on GSH in PC3 and 3T3-L1 cells for the doses tested from 0 - 75 µM. However, a slight drop in GSH level was noticed for both the PC3 and 3T3-L1 cells following exposure to 100 µM. GSH levels also dropped significantly for A549 and BT549 for the said compound for all the doses tested. The observed decreases in GSH for the cells for 4 may be due to its cytotoxic effects. Thus, most of the tested Schiff bases did not cause the loss of GSH as shown in Table 3. The above findings may indicate the abilities of these compounds in maintaining and preserving any oxidative damage to the cells.

Antibacterial screening test of all four Schiff bases were analyzed using both gram positive and negative bacteria. None of the tested Schiff bases was active against E. coli MC4100, E. coli DH5α, Listeria monocytogenes, Staphylococcus aureus and Bacillus subtilus. Zone of inhibitions were zero for all Schiff bases tested in all 5 bacteria strains.