Synthesis of 4-aminoantipyrine derived Schiff bases and their evaluation for antibacterial, cytotoxic and free radical scavenging activity
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 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.
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), antioxidant activities (Thorat et al., 2012), antiviral (Maity et al., 2012), antipyretic (Kalaivani et al., 2012), antifungal (Cinarli et al., 2011), insecticidal (Patil et al., 2012), antitumor (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. beta-lactams (Raman et al., 2009).
Schiff bases are reported for antimicrobial, antitumor and antioxidant potentials herein we report the synthesis and antibacterial, cytotoxic and antioxidant 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).
Scheme 1: Synthesis of Schiff bases (G1-G8)
Materials and Methods
Chemistry
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 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 (d) 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-aminoantipyrene (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), d: 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), d: 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), d: 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), d: 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), d: 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), d: 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), d: 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), d: 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 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 ug/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.
Antioxidant 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:
% of inhibition of DPPH activity = (A-B)/A x 100
A is absorption in blank and B is absorption of test sample.
Result 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) as shown in Table I.
Table I: Antibacterial activity of G1-G8
| Zone of inhibition | Zone of Inhibition | |||
|---|---|---|---|---|
| Compounds | 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 | |||
% Lethality = {(NSN Control - NSN Test)/ NSN Control} x 100
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 antitumor (Khafagi et al., 2004), enzyme inhibition and iron regulation activities (Venugopal et al., 2011).
Table II: Percent death and LC50 values for brine shrimp assay of synthesized compounds
| Compounds | Percent lethality at 24 hours | Total Exposed | LC50 | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Negative control | 1 ppm |
5 ppm |
10 ppm | 50 ppm | 100 ppm | 250 ppm | 500 ppm | 750 ppm | |||
| G1 | 0 | 0 | 30 | 30 | 50 | 60 | 80 | 90 | 100 | 10 | 50 ppm |
| G2 | 0 | 30 | 50 | 50 | 60 | 70 | 70 | 80 | 100 | 10 | 5 pm |
| G3 | 0 | 20 | 30 | 50 | 70 | 80 | 90 | 100 | 100 | 10 | 10 ppm |
| G4 | 0 | 30 | 50 | 50 | 70 | 80 | 90 | 100 | 100 | 10 | 5 ppm |
| G5 | 0 | 50 | 50 | 60 | 70 | 70 | 70 | 80 | 100 | 10 | 1 ppm |
| G6 | 0 | 30 | 30 | 50 | 70 | 80 | 80 | 80 | 100 | 10 | 10 ppm |
| G7 | 0 | 0 | 30 | 30 | 70 | 70 | 70 | 100 | 100 | 10 | <50 ppm |
| G8 | 0 | 20 | 30 | 50 | 70 | 80 | 80 | 80 | 100 | 10 | 10 ppm |
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 antioxidant capability was evaluated through IC50, the effective concentration at which 50% of the radicals were scavenged. A compound is potent antioxidant if its IC50 value is less than 10 µg/mL (Zhang et al., 2013).
Table III: DPPH scavenging activity of synthesized shiff bases of 4-aminoantipyrene
| Compounds | IC50 | Compounds | IC50 |
|---|---|---|---|
| G1 | 31.3a | G5 | >500 |
| G2 | 1.1a | G6 | 285 |
| G3 | 133.0 | G7 | 2.4a |
| G4 | >500 | G8 | 118 |
G1 benzylidene phenyl ring is unsubstituted, gives IC50 at 31.3 µg/mL and shows no significant antioxidant activity. Antioxidant 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 did not 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 antioxidant activity.
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 antitumor potentials and when substituted with electron donating group have positive effect on its antioxidant activity.
Acknowledgement
The authors are grateful to the University of Malakand, Chakdara, Lower Dir KPK, Pakistan for making the resources available to carry out the research work.
References
Alam MS, Lee DU. Synthesis, molecular structure and antioxidant activity of (E)-4[benzylideneamino]-1, 5-dimethyl-2-phenyl-1H-pyrazol-3 (2H)-one, a Schiff base ligand of 4-aminoantipyrine. J Chem Crystallogr. 2012; 42: 93-102.
Ali N, Ali Shah S, Ahmad B. Calcium channel blocking activity of fruits of Callistemon citrinus. 2011; 33: 245-48.
Awad L, Ibrahim E, Bdeewy OK. Synthesis of antipyrine derivatives derived from dimedone. China J Chem. 2007; 25: 570-73.
Burdulene D, Palaima A, Stumbryavichyute Z, Talaikite Z. Synthesis and anti-inflammatory activity of 4-aminoantipyrine derivatives of succinamides. Pharm Chem J. 1999; 33: 191-93.
Cinarli A, Gürbüz D, Tavman A, Birteksöz AS. Synthesis, spectral characterizations and antimicrobial activity of some Schiff bases of 4-chloro-2-aminophenol. Bull Chem Soc Ethiop. 2011; 25: 407-17.
Colwell WT, Lange JH, Henry DW. Chemotherapeutic nitroheterocycles. Nitropyrrole-2-carboxaldehyde derivatives. J Med Chem. 1968; 11: 282-85.
Evstropov A, Yavorovskaya V, Vorob'ev E, et al. Synthesis and antiviral activity of antipyrine derivatives. Pharm Chem J. 1992; 26: 426-30.
Harpstrite SE, Collins SD, Oksman A, Goldberg DE, Sharma V. Synthesis, characterization, and antimalarial activity of novel Schiff-base-phenol and naphthaleneamine ligands. Med Chem. 2008; 4: 392-95.
Ilahi I, Samar S, Khan I, Ahmad I. In vitro antioxidant activities of four medicinal plants on the basis of DPPH free radical scavenging. Pak J Pharm Sci. 2013; 26: 949-52.
Kabak M, Elmali A, Elerman Y, Durlu T. Conformational study and structure of bis-N,N′-p-bromo-salicylideneamine-1, 2-diaminobenzene. J Mol Struct. 2000; 553: 187-92.
Kalaivani S, Priya NP, Arunachalam S. Schiff bases: Facile synthesis, spectral characterization and biocidal studies. Int J App Bio Pharm Tech. 2012; 3: 219-23.
Karthikeyan MS, Prasad DJ, Poojary B, Subrahmanya Bhat K, Holla BS, Kumari NS. Synthesis and biological activity of Schiff and Mannich bases bearing 2, 4-dichloro-5-fluoro-phenyl moiety. Bioorg Med Chem. 2006; 14: 7482-89.
Khafagi I, Dewedar A, Farouk S. In vitro cytotoxicity and antimicrobial activities of some common essential oils. Egypt J Bio. 2004; 2: 20-27.
Kumar D, Chadda S, Sharma J, Surain P. Syntheses, spectral characterization, and antimicrobial studies on the coordination compounds of metal ions with Schiff base containing both aliphatic and aromatic hydrazide moieties. Bioinorg Chem App. 2013; 2013: 1-10.
Maity S, Khan SA, Ahmad S. Synthesis, characterization, antimicrobial and antioxidant activity of some novel Schiff bases derived from 8-hydroxy quinoline. IJBPS. 2012; 3: 90-98.
Malladi S, Isloor AM, Isloor S, Akhila D, Fun H-K. Synthesis, characterization and antibacterial activity of some new pyrazole based Schiff bases. Arabian J Chem. 2013; 6: 335-40.
Metzler CM, Cahill A, Metzler DE. Equilibriums and absorption spectra of Schiff bases. J Am Chem Soc. 1980; 102: 6075-82.
Patil S, Jhadav S, Patil U. Natural acid catalyzed synthesis of Schiff base under solvent-free condition: As a green approach. Arch Ap Sci Res. 2012; 4: 1074-78.
Raman N, Mitu L, Sakthivel A, Pandi M. Studies on DNA cleavage and antimicrobial screening of transition metal complexes of 4-aminoantipyrine derivatives of N2O2 type. J Iran Chem Soc. 2009; 6: 738-48.
Shivarama HB, Veerendra B, Shivananda M, Poojary B. Synthesis characterization and anticancer activity studies on some Mannich bases derived from 1, 2, 4-triazoles. Eu J Med Chem. 2003; 38: 759-67.
Thorat B, Mandewale M, Shelke S, et al. Synthesis of novel Schiff bases of 4-hydroxy-3-methoxy-5-nitrobenzaldehyde and development of HPLC chromatographic method for their analysis. J Chem Pharm Res. 2012; 4: 14-17.
Venkatesan K, Satyanarayana V, Sivakumar A. Microwave-assisted synthesis and evaluation of antibacterial activity of 2, 2'-(naph-thalene-2, 7-diylbis (oxy)) bis (N'-substituted acetohydrazide) derivatives. Bull Chem Soc Ethiop. 2012; 26: 257-65.
Venugopal T, Swathi D, Suchitha Y, et al. Mineral composition, cytotoxic and anticariogenic activity of Scleropyrum pentandrum (Dennst.) Mabb. Int J Drug Develop Res. 2011; 3: 344-50.
Zhang Y, Fang Y, Liang H. Synthesis and antioxidant activities of 2-oxo-quinoline-3-carbaldehyde Schiff-base derivatives. Bioorg Med Chem lett. 2013; 23: 107-11.
