Synthesis of 4-aminoantipyrine derived Schiff bases and their evaluation for antibacterial , cytotoxic and free radical scavenging activity

The work was aimed to synthesize 4-Aminoantipyrine derived Schiff bases in economical way and to screen it for study the effect of nitro group on its antibacterial potential, conduct antitumor preliminary study, the effects of group presence in benzylidene phenyl ring on the cytotoxic potentials and study the effects of electron withdrawing and donating group on antioxidant potential. We used green method with 75% reduction in general synthesis time of Schiff bases. Synthesized compound possess antibacterial potentials and nitro group presence enhances this potential. G2, G3, G4, G5, G6, G7 and G8 have significant cytotoxic and no significant antioxidant activity.

Schiff bases are reported for antimicrobial, anti-tumor and anti-oxidant potentials herein we report the synthesis and antibacterial, cytotoxic and anti-oxidant screening of Schiff bases derived from 4-aminoantipyrene and aromatic carbonyl compounds.The Schiff bases were synthesized by grinding equimolar quantities of 4-aminoantipyrene and carbonyl compounds at room temperature in mortar using pestle (Scheme 1).
All the solvents and chemicals were Sigma-Aldrich brand.On Barnstead electrothermal melting point apparatus melting points (mp) were determined in open capillaries and are uncorrected.Thin-layer chromatography (TLC) on Merck silica gel 60 F254 aluminum sheets (Merck; Darmstadt, Germany) was used to monitor the reaction progress, using various developing system in various ratios and observed under UV light ‫=ג(‬ 254/365 nm).Bruker AV spectrometer was used for recording 1 H-NMR spectra at 300 MHz and tetramethyl silane (TMS) was used as internal standard.Chemical shift values (δ) were mentioned in ppm.FTS 3000 MX, Bio-Rad Merlin Fourier Transform Infra Red spectrophotometer was used to record IR spectra.KBr pellets were used for recording their spectra.

General procedure for synthesis of compounds G1-G8
Solid carbonyl compounds (2 mmol) were first grinded in a mortar with a pestle to fine powder then 4aminoanti-pyrene (2 mmol, 0.4 g) was added and grinded up to 30 min at room temperature without the use of any solvent and catalyst.The progress of the reaction was monitored by thin layer chromatography (TLC).Upon completion of reaction the product was crystallized in absolute ethanol.

Antibacterial activity
G1-G8 were evaluated for antimicrobial activity by well diffusion method (Kumar et al., 2013) against two Gram negative bacteria (Escherichia coli 739, Proteus mirabilis rt, neat, grinding 13315) and two Gram positive (Staph aureus 29213 and Bacillus cereus locally collected).Each petri plate was filled with 20 mL of agar medium then swabbed with 10 μg/mL test microorganism and kept for 15 min for adsorption.Wells were bored into the seeded agar plates through sterile cork borer of 5 mm diameter and loaded with a 10 mg/mL of test compounds.Ceftriaxone was used as positive controls for bacteria.All the plates were incubated at 37°C for 24 hours.The antimicrobial activity of G1-G8 was evaluated by measuring the zone of inhibition against the test microorganisms with scale.

Anti-oxidant activity
DPPH free radical scavenging assay of the test sample and standard was accessed as described in protocols with a slight modification (Ilahi et al., 2013).Briefly, 6 mg of each G1, G2, G3, G4, G5, G6, G7 and G8 was dissolved in 600 µL of methanol to prepare stock solutions (10,000 ppm).The stock solutions were then serially diluted to get a concentration of 20, 40, 60, 80 and 100 ppm.Ascorbic acid, a standard, was also prepared in same concentrations.DPPH (0.002%) was dissolved in methanol.1 mL stable 2, 2-diphenyl-2picrylhydrazyl (DPPH) of was added to each concentration.The solution mixture was then incubated for 30 min in dark area of the laboratory at room temperature.Methanol containing DPPH was used as blank.Ascorbic acid was also accessed as that of test sample.After required time the absorption of the compounds were measured at 520 nm on spectrophotometer (Shimadzu UV-1700).
The % absorption was then calculated by using the following formula:

A-B % of inhibition of DPPH activity = x 100 A
A is absorption in blank and B is absorption of test sample.

Results and Discussion
Proteus mirabilis 13315 was found susceptible to G2, G4, G5, G6 and G8, intermediate to G3 and resistant to G1 and G7.Staphylococcus aureus 29213 was found susceptible to all test compounds G1-G8.Escherichia coli was found susceptible to G4, G5, G7 and G8, intermediate to G2, G3 and G6 and resistant to G1. Bacillus cereus (locally collected) was found susceptible to G2, G4 and G5, intermediate to G3 and resistant to G1, G6, G7 and G8 (Colwell et al., 1968)  G2 to G8 all showed significant cytotoxicity because their LC50 values were below 20 µg/mL (Table II).We can speculate that test compounds show better activity when a group is attached to benzylidene phenyl ring.These results not only tell us about toxicity of test compound but it also gives us preliminary data about anti-tumor (Khafagi et al., 2004), enzyme inhibition and iron regulation activities (Venugopal et al., 2011).
G1 benzylidene phenyl ring is unsubstituted, gives IC50 at 31.3 µg/mL and shows no significant anti-oxidant

Inoculums control
Growth in all concentrations activity.Anti-oxidant data of electron donating groups (OH, OMe) at various positions in benzylidene phenyl ring has been reported (Alam and Lee, 2012).We introduced strong electron withdrawing group (NO2) at various positions and didn't find any significant activity.The comparative study shows that introducetion of electron donating groups to benzylidene phenyl ring of the Schiff base analogues of 4-aminoantipyrine are important for anti-oxidant activity.
In conclusion, 4-aminoantipyrine derived Schiff bases have antibacterial potentials and cytotoxic potentials which can be enhanced in presence of nitro group and when a group is attached to benzylidene phenyl ring respectively.4-Aminoantipyrine derived Schiff bases have anti-tumor potentials and when substituted with electron donating group have positive effect on its antioxidant activity.
as shown in TableI.