Biological Evaluation of New Schiff Bases : Synthesized from 4-Amino-3 , 5-dimethyl-1 , 2 , 4-triazole , Phenathroline and Bipyridine Dicarboxaldehydes

Four new Schiff bases with promising anticancer activity have been synthesized from 4-amino-3,5-dimethyl-1,2,4-triazole and di-pyridyl-aldehydes. Structures have been established by various spectroscopic methods. The compounds were tested in vitro to study their cytotoxicity and anti-oxidative activity in human lung carcinoma (A549), breast carcinoma (BT549), prostate adenocarcinoma (PC3) and mouse preadipocytes (3T3-L1) cells. Compound 1 was found to increase Glutathione (GSH) level slightly in all four cell lines. Compound 4 showed better selectivity and cytotoxicity against both BT549 and A549 cells compared to the anticancer drug tamoxifen. With the exception of compound 4 which reduced GSH levels in A549 and BT549, all other compounds maintained GSH levels in comparison to their respective controls.


Introduction
Cancer is the major cause of death in the present world.Approximately 14 million cancer cases was diagnosed and 8.2 million people died because of cancer in 2012 [1] [2].
These numbers are more likely to increase over the following decades [1].Accordingly, developing new anticancer drugs has become an urgent need.Currently a number of reactive oxygen species (ROS) [3] [4], acting as antioxidants thereby reducing the incidence of certain cancers.Schiff bases form an important class of organic compounds with a wide variety of biological properties.
Schiff bases and their metal complexes are usually considered one of the most biologically active chemical compounds.The imine bonds or Schiff bases are also found in different enzymes such as transaminases, transketolases and tryptophan synthase to mention a few.Schiff bases synthesized from aldehydes or amines containing heterocyles have been reported to have cytotoxic [7] [8], antiproliferative [9], antibacterial acitivity [8] [9] [10] [11], antiviral [8] [11], antifungal and anticancer activities [11].For example, Schiff bases and Copper(II) complexes of Schiff bases synthesized from 4amino-3,5-dimethyl-1,2,4-triazole have been reported to inhibit endocytosis [12] and protein tyrosine phosphatases [13] respectively.Triazole containing Schiff bases has also been reported to have antitumor activity [14].Previous workers in our laboratory had synthesized new Schiff bases containing less flexible, structurally rigid 1,10-phenanthroline backbone, with O,N,S and F-containing amines and studied their antibacterial activity [10].New Schiff bases have also been synthesized in our laboratory and their antibacterial properties are studied.Since 4-amino-3,5-dimethyl-1,2,4-triazole derivatives have been found to exhibit variety of inhibition properties, we have decided to synthesize various Schiff bases using this 4-amino-3,5-dimethyl-1,2,4-triazole with phenanthroline and bi-pyridyl aldehydes and study their anticancer and antioxidant activities.Particularly, we wanted to test the efficacy of our synthesized Schiff bases in maintaining the antioxidant potential of several cell lines by measuring the effects of the said compounds on levels of glutathione, a natural antioxidant, in cells.Also we have used tamoxifen (Figure 1), a breast cancer drug [15], as our internal standard in comparison to our synthesized Schiff bases for cell viability tests.The structure of tamoxifen is as follows.
Glutathione (GSH) is a tripeptide whose reducing and nucleophilic properties have a vital role in metabolic pathways.GSH has been reported to be the main line of defense for the maintenance of the appropriate mitochondrial redox environment to avoid or repair oxidative modifications leading to mitochondrial dysfunction and cell death [16].Thiol group of cysteine is oxidized when target molecules are reduced by GSH [16].GSH also plays an important role in metabolism of xenobiotics, thiol disulfide exchange reactions, cellular signaling such as apoptosis, proliferation and cell-cycle regulation [16].In this paper, we report the cell viability and oxidative properties of four Schiff bases on three cancer cells and one obese cell lines.

Chemicals and Instrumentation
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.
The product was isolated and dried under vacuum.The crude product was purified by recrystallization in chloroform to get pure compound (Y: 70%).

General Procedure for the Synthesis of Schiff Bases
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 H 2 SO 4 increased the yield by at least 5%.

Biological Materials
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.

Cell Culture
All four different cell lines were cultured under 5% CO 2 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.

Glutathione (GSH) Assay
2 × 10 6 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% CO 2 .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/10 6 cells [25] [26].The experiments were repeated 2× with each treatment at 4 replicates.
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.

Cell Viability Activities in Vitro
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-Diphenyltetrazolium 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.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 IC 50 of 50 µM compared to anticancer drug tamoxifen with an IC 50 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.

Effects on Glutathione (GSH) Level
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 Studies
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.The decrease in GSH levels for compound 4 may be due to its cytotoxicity.

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 IC 50 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 IC 50 values of 70 µM and 50 µM respectively.

Four
Schiff bases have been synthesized successfully and reaction condition for Schiff base preparation has been optimized to better yield.Addition of 2 -3 drops of sulfuric acid increases the yield by 5% -15%.Compound 1 containing rigid phenanthroline backbone may act as an antioxidant since it can increase and maintain GSH levels of cells.Compound 4 exhibited promising anticancer activity with excellent selectivity towards A549 and BT549 over PC3 and 3T3-L1 as opposed to the anticancer drug tamoxifen which killed the cells randomly.To use 4 as an anticancer drug and in clinical studies further studies are anticipated.In conclusion, exposure of several cell lines to different doses of Schiff bases with the exception of one (i.e.compound 4) did not cause significant losses in intracellular levels of GSH compared to their respective controls.