Bangladesh Journal of Pharmacology Research Article Synthesis of 4Synthesis of 4Synthesis of 4---aminoantipyrine de-aminoantipyrine de-aminoantipyrine de- rived Schiff bases and their evalua-rived Schiff bases and their evalua-rived Schiff bases and their evalua- tion for antibacterial, cytotoxic and tion for antibacterial, cytotoxic and tion for antibacterial, cytotoxic and free radical scavenging activityfree radical scavenging activityfree radical scavenging activity BJP O N N NH2 R1 R2 O + O N N N R1 R2 rt, neat, grinding Introduction Irrational use of medicine has resulted in resistance to available antimicrobial agents. The resistant microorganisms have resulted in high morbidity and mortality. Schiff bases, the easy synthesis, wide range of pharmacological activities and its azomethane functional group –CN– have tilted research in pharmaceutical and medicinal chemistry (Malladi et al., 2013). They are reported for various pharmacological activities such as bacteriostatic (Karthikeyan et al., 2006), bactericidal (Metzler et al., 1980), antimalarial (Harpstrite et al., 2008), anti-oxidant activities (Thorat et al., 2012), antiviral (Maity et al., 2012), anti-pyretic (Kalaivani et al., 2012), antifungal (Cinarli et al., 2011), insecticidal (Patil et al., 2012), anti-tumor (Venkatesan et al., 2012) and anti-inflammatory (Shivarama et al., 2003). Schiff bases synthesized from aromatic reactants have variety of applications in biological, inorganic and analytical chemistry (Kabak et al., 2000). 4-aminoantipyrine is a pyrazolone derivative, it has shown wide range of biological activities such as antimicrobial activity (Awad et al., 2007), analgesic (Burdulene et al., 1999), antiviral (Evstropov et al., 1992) and also used as precursors in the synthesis of bioactive compounds i.e. β-lactams (Raman et al., 2009). 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-aminoanti- pyrene 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). Materials and Methods Chemistry A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2015; 10: 332-336 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract 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 anti-tumor 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 anti-oxidant 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 anti-oxidant activity. Article Info Received: 7 March 2015 Accepted: 28 March 2015 Available Online: 17 April 2015 DOI: 10.3329/bjp.v10i2.22471 Cite this article: Shoaib M, Rahman G, Shah SWA, Umar MN. Synthesi s of 4 - aminoantipyrine derived Schiff bases and their evaluation for antibacterial, cytotoxic and free radical scavenging activity. Bangladesh J Pharmacol. 2015; 10: 332-36. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Synthesis of 4-aminoantipyrine derived Schiff bases and their evaluation for antibacterial, cytotoxic and free radical scavenging activity Mohammad Shoaib1, Ghawsur Rahman1, Syed Wadood Ali Shah1 and Mohammad Naveed Umar2 Department of 1Pharmacy and 2Chemistry, University of Malakand, Chakdara Lower Dir, Khyber Pakhtunkhwa, Pakistan. 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 (254/365 =ג nm). Bruker AV spectrometer was used for recording 1H-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 4- aminoanti-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. (Z)-4-(benzylideneamino)-2, 3-dimethyl-1-phenyl-1, 2- dihy-dropyrazol-5-one (G1) Cream color crystalline solid, Yield: 75%, mp 178ºC, Rf 0.70, 1H-NMR (300 MHz, CDCl3), δ: 2.51 (s, 3H, =C- CH3), 3.17 (S, 3H, -N-CH3),7.27-7.57 (m, 8H, ArH), 7.81- 7.93 (m, 2H, ArH), 9.79 (s, 1H, -N=CH) IR (KBr) 1646 (>C=O), 1594.2 (C=N) cm-1. (Z)-4-(4-hydroxy-3-methoxybenzylideneamino)-2, 3- dimethyl-1-phenyl-1, 2dihydropyrazol-5-one (G2) Cream color crystalline solid, Yield: 73%, mp 207ºC, Rf 0.61, 1H-NMR (300 MHz, CDCl3), δ: 2.48 (s, 3H, =C- CH3 ), 3.16 ( s, 3H, -N-CH3), 3.95 (s, 3H, OCH3), 6.10 (s, 1H, OH ), 6.94 (s,1H,ArH ),7.44 ( m, 7H, ArH), 9.68 ( s,1H, -N=C-H); IR (KBr) 1624 (>C=O), 1576 (C=N)cm-1. (Z)-4-(2-nitrobenzylideneamino)-2, 3-dimethyl-1- phenyl-1-2-dihydropyrazol-5-one (G3) Deep yellow color crystalline solid, Yield: 76%, mp 212ºC, Rf 0.5, 1H-NMR (300 MHz, CDCl3), δ: 2.49 (s, 3H, =C-CH3), 3.22 (s, 3H, -N-CH3), 6.96-8.44 (m, 9H, ArH), 10.04 (s, 1H, -N=CH); IR (KBr) 1646 (>C=O), 1567 (C=N) cm-1. (Z)-4-(2-hydroxybenzylideneamino)-2, 3-dimethyl-1- phenyl-1-2-dihydropyrazol-5-one (G4) Light yellow crystalline solid, Yield, 72%, mp 200ºC, Rf 0.14, 1H-NMR (300 MHz, CDCl3), δ: 2.43 (s, 3H, =C- CH3), 3.19 (s, 3H, -N-CH3), 6.85-7.02 (m, 5H, ArH), 7.46- 7.57 (m, 2H, ArH), 9.85 (s, 1H, -N=CH), 13.37 (s, 1H, ArOH); IR (KBr) 1652 (>C=O), 1589(C=N) cm-1. (Z)-4-(3-nitrobenzylideneamino)-2, 3-dimethyl-1- phenyl-1-2-dihydropyrazol-5-one (G5) Yellow color crystalline solid, Yield: 73%, mp 218ºC, Rf 0.75, 1H-NMR (300 MHz, CDCl3), δ: 2.54 (s, 3H, =C-H3), 3.23 (s, 3H, -N-CH3), 7.31-7.65 (m, 6H, ArH), 7.83-8.40 (m, 2H, ArH), 8.77 (s, 1H, -CH-NO2), 9.83 (s, 1H, - N=CH); IR (KBr) 1645 (>C=O), 1592 (C=N) cm-1. (Z)-4-(4-nitrobenzylideneamino)-2, 3-dimethyl-1- phenyl-1-2-dihydropyrazol-5-one (G6) Golden color crystalline solid, Yield: 71%, mp 254ºC, Rf 0.6, 1H-NMR (300 MHz, CDCl3), δ: 2.54 (s, 3H, =C-CH3), 3.25 (s, 3H, -N-CH3), 7.23-7.58 (m, 5H, ArH), 7.99 (d, J=8.3Hz, 2H, ArH), 8.27 (d, J=8.3Hz, 2H, ArH), 9.82 (s, 1H, -N=CH); IR (KBr) 1640 (>C=O), 1572 (C=N) cm-1. (Z)-4-(4-dimethylamino)benzylideneamino)-2, 3- dimethyl-1-phenyl-1-2-dihydropyrazol-5 one (G7) Yellow color crystalline solid, Yield: 75%, mp 219ºC, Rf 0.8, 1H-NMR (300 MHz, CDCl3), δ: 2.48 (s, 3H, =C-CH3), 3.04 (s, 6H, -N-(CH3)2), 3.11 (s, 3H, -N-CH3), 6.68-6.79 (m, 2H, ArH), 7.15-7.92 (m, 7H, ArH), 9.68 (s, 1H, - N=CH); IR (KBr) 1645 (>C=O), 1577 (C=N) cm-1; (Z)-4-(1-(2 hydroxy phenyl) ethylilideneamino)-2, 3- dimethyl-1-phenyl-1-2-dihydropyrazol-5-one (G8) Yellow color crystalline solid, Yield: 71%, mp 166, Rf 0.37, 1H-NMR (300 MHz, CDCl3), δ: 2.32 (s, 3H, N=C- CH3), 2.54 (s, 3H, =C-CH3), 3.14 (s, 3H, N(CH3)2), 6.82- 7.05 (m, 2H, ArH), 7.26-7.56 (m, 6H, ArH), 7.68 (dd, J=8.1, 1.6Hz, 1H, ArH), 1.5 (s, 1H, OH); IR (KBr) 1658 (>C=O), 1592 (C=N) cm-1. Pharmacological evaluation 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 O N N NH2 R1 R2 O + O N N N R1 R2 rt, neat, grinding Bangladesh J Pharmacol 2015; 10: 332-336 333 Scheme 1: Synthesis of Schiff bases (G1-G8) http://en.wiktionary.org/wiki/%D7%92 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. Cytotoxicity The cytotoxicity of G1, G2, G3, G4, G5, G6, G7 and G8 was carried out on active nauplii of brine shrimp (Artemia salina). Eggs were hatched in sterile artificial seawater having composition of sea salt 38.0 g/L and pH 8 then left in continuous aeration for 48 hours. Different concentrations (1, 5, 10, 50, 100, 250, 500 and 750 ppm) of compounds (G1, G2, G3, G4, G5, G6, G7 and G8) were prepared in dimethyl sulfoxide (DMSO). After evaporation of DMSO, 10 brine shrimp larvae were added to each concentration (Ali et al., 2011). The percent mortality was determined after 24 hours after counting survived and dead shrimps. The readings were taken in triplicate. LC50 value was calculated using graph pad prism software version V. 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-2- picrylhydrazyl (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 suscep- tible 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) as shown in Table I. NSN Control – NSN Test % Lethality = × 100 NSN Control NSN control - Number of surviving napulii in control; NSN Test - Number of surviving napulii in test 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). DPPH scavenging activity (Table III) of synthesized compounds G1, G2, G3, G4, G5, G6, G7 and G8 was carried out at concentration of 20, 40, 60, 80 and 100 ppm giving IC50 at 31.26a, 1.13a, 133, >500, >500, 285, 2.4a, 118 µg/mL respectively. The anti-oxidant capability was evaluated through IC50, the effective concentration at which 50% of the radicals were scavenged. A compound is potent anti-oxidant if its IC50 value is less than 10 µg/mL (Zhang et al., 2013). G1 benzylidene phenyl ring is unsubstituted, gives IC50 at 31.3 µg/mL and shows no significant anti-oxidant 334 Bangladesh J Pharmacol 2015; 10: 332-336 Table I Antibacterial activity of G1-G8 Zone of inhibition Zone of Inhibition Com- pounds Proteus mirabilis Staph aureus Escherichia coli Bacillus cereus G1 10 26 10 10 G2 30 20 15 25 G3 15 18 15 15 G4 32 20 20 25 G5 38 20 21 18 G6 25 25 15 5 G7 5 25 20 8 G8 20 22 20 7 Ceftriaxone 45 42 30 45 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. 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