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.
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 [
Chelating agents containing nitrogen are a group of compounds of continuing interests for their ability to interact with biological systems. It is known that the nitrogen rich 1,2,4-Triazole derivatives are a class of compounds that show antitumor [
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 hetero- cyles have been reported to have cytotoxic [
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 [
bases on three cancer cells and one obese cell lines.
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 [
1,10-phenanthroline-2,9-dicarboxaldehyde (9) was synthesized form 2,9-dimethyl-1,10- phenanthroline hemihydrate (8) following previously reported procedure [
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, Jortho= 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 [
[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, Jortho= 7.88Hz, 2H, Ar-H), 8.8 (s, 2H, Ar-H), 7.95 (d, Jortho= 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 [
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%.
1HNMR (δ, DMSO-d6): 9.13 (s, 2H, Ar-H), 8.75 (d, Jortho= 8.4Hz, 2H, Ar-H), 8.55 (d, Jortho= 8.4Hz, 2H, Ar-H), 8.19 (s, 2H, CH), 2.55 (s, 12H,CH3); 13CNMR (δ, DMSO-d6): 160.28, 152.32, 148.55, 145.48, 138.39, 130.56, 128.85, 121.10, 11.01; ESI-MS m/z (% rel. abund.): 424 [M]+ (74), 447 [M+Na]+ (100).
Scheme 4. Synthesis of Schiff base 3 from 2,2’-bipyridyl-5,5-dialdehyde.
Scheme 5. Synthesis of Schiff base 4 from 2,2’-bipyridyl-6,6-dialdehyde.
1HNMR (δ, DMSO-d6): 9.25 (d, Jmeta= 2.52Hz, 2H, Ar-H), 9.05 (s, 2H, Ar-H), 8.66 (d, Jortho= 8.52Hz, 2H, Ar-H), 8.55 (dd, Jortho= 8.52Hz, Jmeta= 2.52Hz, 2H, Ar-H), 2.44 (s, 12H, CH3); 13CNMR (δ, DMSO-d6): 160.53, 157.44, 150.87, 148.09, 136.83, 129.69, 121.88, 11.1; EI-MS m/z (% rel. abund.): 423 [M + Na]+ (100), 401 [M]+ (25).
1H NMR (δ, CD3OD): 9.00 (m, 6H, Ar-H), 8.00 (s, 2H, CH), 2.60 (s, 12H, CH3); 13CNMR (δ, CD3OD): 156.15, 157.06, 152.68, 150.15, 139.53, 124.87, 123.69, 11.12; EI-MS m/z (% rel. abund.): 401 [M]+ (100), 423 [M + Na]+, (30).
1H NMR (δ, CDCl3): 8.70 (m, 4H, 2 Ar-H, 2 CH), 8.30 (d, Jortho= 7.92Hz, 2H, Ar-H), 8.07 (t, J= 7.52Hz, 2H, Ar-H), 2.8 (s, 12H,CH3); 13CNMR (δ, CDCl3): 160.26, 155.37, 150.99, 148.46, 138.18, 123.69, 121.84, 11.96; EI-MS m/z (% rel. abund.): 423 [M + Na]+ (100), 401 [M + H]+ (20).
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 [
Cell viability was determined as previously reported [
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 [
Antibacterial screening test was done using the disc diffusion method [
The starting amine 7 was synthesized following previously reported procedure (Scheme 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 [
Compound | % yield in | |||
---|---|---|---|---|
Methanol | Methanol + 2 - 3 drops H2SO4 | Ethanol | Ethanol + 2 - 3 drops H2SO4 | |
1 | 58 | 63 | 70 | 81 |
2 | 51 | 61 | 73 | 80 |
3 | 63 | 74 | 69 | 76 |
4 | 68 | 81 | 78 | 93 |
Compound | IC50 (µM) | |||
---|---|---|---|---|
A549 | BT549 | PC3 | 3T3-L1 | |
1 | >200 | >200 | >200 | >200 |
2 | >200 | >200 | >200 | >200 |
3 | >200 | >200 | >200 | >200 |
4 | 70 | 50 | >200 | >200 |
Tamoxifen | 55 | 55 | 60 | 35 |
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 [
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.
Compound | Concentration (µM) | GSH level (nmol/106 cells) in | ||||
---|---|---|---|---|---|---|
A459 | BT549 | PC3 | 3T3-L1 | |||
1 | 10 | 12.88 | 14.83 | 8.08 | 18.42 | |
25 | 13.04 | 14.99 | 8.34 | 18.79 | ||
50 | 13.37 | 15.18 | 8.71 | 19.24 | ||
75 | 13.88 | 15.43 | 9.11 | 19.88 | ||
100 | 14.05 | 15.81 | 9.59 | 20.31 | ||
2 | 10 | 11.66 | 14.69 | 9.01 | 19.13 | |
25 | 11.69 | 14.58 | 9.07 | 19.09 | ||
50 | 11.68 | 14.65 | 8.94 | 19.18 | ||
75 | 11.63 | 14.72 | 8.99 | 19.18 | ||
100 | 11.67 | 14.60 | 8.91 | 19.15 | ||
3 | 10 | 13.17 | 15.17 | 8.6 | 19.01 | |
25 | 13.14 | 15.03 | 8.62 | 18.97 | ||
50 | 13.16 | 15.18 | 8.71 | 18.91 | ||
75 | 13.21 | 15.24 | 8.64 | 18.99 | ||
100 | 13.18 | 15.21 | 8.64 | 19.01 | ||
4 | 10 | 12.53 | 14.39 | 8.22 | 19.21 | |
25 | 11.97 | 11.95 | 8.15 | 19.29 | ||
50 | 6.08 | 6.18 | 8.11 | 19.31 | ||
75 | 2.98 | 2.06 | 8.19 | 19.15 | ||
100 | 1.91 | 0.79 | 7.27 | 17.88 | ||
DMSO | 2% | 12.84 | 14.94 | 8.39 | 19.07 |
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. The decrease in GSH levels for compound 4 may be due to its cytotoxicity.
We would like to thank Department of Chemistry, Tennessee State University, USA for necessary support to pursue the research. We are also grateful to the Department of Education, USA, Title III funds for providing necessary instrumental support.
Islam, Md.S., Karim, M.R., Boadi, W., Falekun, S. and Mirza, A.H. (2016) Biological Evaluation of New Schiff Bases: Synthesized from 4-Amino-3,5- dimethyl-1,2,4-triazole, Phenathroline and Bipyridine Dicarboxaldehydes. Advances in Biological Chemistry, 6, 180-192. http://dx.doi.org/10.4236/abc.2016.66016