Synthesis of 4-aminoantipyrine Schiff bases and their antimicrobial activities            
Olatunde S. Oladeji, Monisola I. Ikhile*, Carine M. D. Fotsing, Messai Mamo, Patrick G. Ndungu, Derek T. Ndinteh*
Department of Applied Chemistry, Faculty of Science, University of Johannesburg, P.O Box 2028, South Africa 
 
 
Abstract: Various compounds of 4-aminoantipyrine Schiff bases (M1M12) were synthesized via a condensation reaction of 4-aminoantipyrine with different benzaldehydes through a conventional method of refluxing the mixture for 34 h. The synthesizedSchiff bases were characterized by using elemental analyses, FT-IR, UV-Vis, Mass, 1H and 13C NMR spectroscopy. The antimicrobial activity of the synthesized Schiff bases was investigated against 12 bacterial strains(Mycobacterium smegmatis, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus epidermidis, Klebsiella pneumonia, Escherichia coli, Enterobacter cloacae,Klebsiella oxytoca, Proteus vulgaris, Enterobacter aerogenes, and Pseudomonas aeruginosa), and antifungal activities were tested against seven fungal strains (Aspergillus flavus, Aspergillus carbonarious, Aspergillus parasiticus, Aspergillus fumigatus,Aspergillus niger, Fusarium verticillioides and Fusarium proliferatum). The antimicrobial activities of the synthesized compounds were compared with standard streptomycin and nalidixicacid. The results obtained from antibacterial assay indicated that M1M12 inhibited potential growth of Proteus vulgaris with minimum inhibitory concentrations (MICs) ranging from 15.6–250 µg/mL compared with the standard nalidixic acid with an MIC of 500 µg/mL. Moreover, we could conclude that most of the tested compounds experienced mild to low activities at 15.6 µg/mL. Their activities could be attributed to their low concentrations.The antifungal analysis showed that the tested fungi were not sensitive to the prepared Schiff bases at the prepared concentration of 500 µg/mL. Therefore, we recommended further analysis on both cytotoxicity and minimum bactericidal concentration (MBC) to ascertain their potential effects against human cells.  
Keywords: 4-Aminoantipyrine; Antibacterial; Schiff bases; Antifungal; Synthesis; Inhibition   
CLC number: R916                Document code: A                 Article ID: 10031057(2018)1175314
 
 
1. Introduction
The compound, 4-aminoantipyrine (4-AAP) is well-known for many decades. It has several applications, such as their use for the calorimetric identification of various derivatives of phenol group and propoxur in water samples and pesticide formulation[1,2]. They have also application in several biological systems as a result of the presence of an amino group and in the area of bioinorganic chemistry for the provision of the metallic model in metalloenzymes[35]. Despite its good applications in pharmaceutical industry, 4-AAP usually produces agranulocytosis as a side effect, and because of this effect, 4-AAP is scarcely administered as energetic agent in these days[6]. Therefore, this limitation has led many researchers to explore the use of Schiff bases, especially in the pharmaceutical industry. Generally, Schiff bases are important in both the pharmaceutical industry and medicinal field for the treatment of cancer and infections caused by both bacteria and fungi[3]. Schiff bases derived from 4-AAP have wide applicationin pharmaceutical industries as an active component used in the treatment of several diseases, such as antipyretic,analgesic, antiproliferative antioxidant, fungicidal, bactericidal, antiviral, anti-inflammatory,antitumor[7], antiallergic and antiphlogistic agents[815]. The presence of active components (electron donors N and O) from various derivatives of 4-AAP Schiff bases makes their activities outstanding with respect to their biological performance[16]. Biological activities of some 4-AAP Schiff base derivatives have been established[17]. In addition, the biochemical reaction of some 4-AAP Schiff base derivatives, as a reagent that produces peroxides and phenol, has been recently reported[18,19]. However, various intra- or intermolecular hydrogen bonds can be obtained from 4-AAP Schiff bases substituted aromatic aldehyde, and these bonds are often used to evaluate the chemical and physicochemicalproperties of the compounds[20,21]. The unique performanceof 4-AAP Schiff base derivatives has been attributed to the presence of imine groups derived from heteroaromaticcontaining nitrogen atoms, which show efficient biological activities[22]. Furthermore, in order to perform structural elucidation of various Schiff base derivatives prior to their antimicrobial activities, different spectroscopic techniques and theoretical calculations have been employed and stated in some of the listed articles[23,24].
In the past decades, herbal medicine that consists of herbs, herbal materials, herbal preparations and finished herbal products have wide applications in the treatment of many diseases. These medicines are found in plants or plant materials as active products, and they have been used for the prevention and treatment of various ailments worldwide, especially in Africa[26]. As of date, substantial active components of modern drugs are from plants, such as morphine from papaversomniferumand cocaine from colchicumautumnale for the treatment of body pain and pericardial diseases[25]. They have also been used for the treatment of other diseases, like tuberculosis, pneumonia, cholera and others[25,26]. In the course of regular use of herbal medicine for prevention and treatment of various diseases, some of these herbal medicines have side effects, while many of them have been resisted by an infection, which is usually referred to as methicillin-resistant Staphylococcus aureus (MRSA)[27,28]. Their activities pose a serious challenge all over the world. For this reason, researchers have shifted their attention to organic synthesis of various compounds to augment the activities of natural products against microbial (bacterial and fungal) activities[29]. Due to the limitations of herbal products to resist the activities of microorganism in the human body, it is therefore of paramount importance to synthesize new organic compounds that will combat this limitation. Therefore, the condensation of 4-AAP with aromatic aldehyde will limit the cytotoxic effect exhibited by 4-AAP. This is because that the presence of imine and amino groups as earlier reported in 4-AAPSchiff bases enhances their activities for various applications, especially in drug manufacture against various microbial activities[30,31]
In the present study, we reported the synthesis, characterization and antimicrobial activities of 12 Schiff base derivatives consisting of a 4-AAP core with different anchoring electron donors against 12 bacterial strains and seven MRSA strains.
2. Materials and methods
All solvents were of analytical grade and used without further purification. 4-AAP and aromatic aldehydes were purchased from Merck (Pty) Ltd., South Africa. RPMI-1640 and 96-well plates were purchased from Sigma Aldrich, while bacterial strains (Bacillus cereus ATCC10876, Bacillus subtilis ATCC19659,Pseudomonas aeruginosa ATCC27853, Klebsiella pneumonia ATCC13882, Enterococcus faecalis ATCC13047, Mycobacterium smegmatis MC2155, Staphylococcus epidermidis ATCC14990, Escherichia coli ATCC25922, Enterobacter cloacae ATCC13047, Klebsiella oxytoca ATCC8724, Enterobacter aerogenes,Proteus vulgaris ATCC6380) and fungi strains (Aspergilluscarbonarious, Aspergillus flavus, Aspergillus fumigatus, Aspergillus parasiticus, Aspergillus niger and Fusariumproliferatum and Fusarium verticillioides) were obtained from Department of Mycology, National Collection of Fungi and Bacteria, Agricultural ResearchCouncil, South Africa. FT-IR analyses of the spectra weredetermined with Perkin-Elmer 783 spectrophotometer using KBr pellets within the range of 4500–400 cm1. Agilent technologies Cary 60 UV-Vis absorption was used to record the absorption spectra. The NMR spectral analyses were reported on a 500 MHz Bruker advance using CDCl3 solvent. Melting point values of the compounds were determined using Agilent melting point, while the elemental analyses were conducted on 2400 series llCHNS/O PerkinElmer analyzer. The mass spectrometry analysis was determined with Agilent Technologies 7890B, Pegasus GC-HR TOFMS.
3. Experimental details
3.1. General synthesis of 4-AAP Schiff base derivatives (M1M12)
The synthesis of 4-AAP Schiff base derivatives was synthesized via condensation of 4-AAP and aromatic aldehyde according to reported procedures[6,29]. The stirring of the reaction mixtures was performed at 5000 r/min and heated at 120 ºC under reflux in dry ethanol condition for 3–4 h.
3.2.1. Synthesis of 4-[(2-methoxy-benzylidene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M1)
4-AAP (0.24 g, 0.0012 mol) and 2-methoxybenzaldehyde (0.16 g, 0.0012 mol). The product was collected as chartreuse color powder: 0.2825 g, yield 76%, mp. 230.4–232.3 ºC, UV-Vis (nm): 319, 418; IR (KBr cm1): 673, 1134, 1377, 1456, 1570, 1650, 2852, 2957, 3069, 3104; 1H NMR (500 MHz, CDCl3) δ: 2.46 (s, 3H, CH3); 3.11 (s, 3H, N-CH3); 3.82 (s, 3H, OCH3); 6.88 (d, 2H, J 8.40 Hz, C6H6); 6.97 (t, 1H, J 9.50 Hz, C6H6); 7.27 (m, 2H, C6H6); 7.32 (m, 1H, C6H6); 7.42 (m, 2H, C6H6); 8.10 (d, 1H, J 9.5 Hz, C6H6); 10.08 (s, 1H, N=CH); 13C NMR (125 MHz, CDCl3) δ: 10.13, 35.98, 55.50, 120.46, 126.51, 129.07, 131.38, 135.02, 152.71, 160.96; m/z (ESI): 321.15, (M+, 100%); Anal. Calc. for C19H19N3O2 (%): H, 5.79, C, 90.62., N, 13.19; Found (%): H, 5.96, C, 91.01, N, 13.15.
3.2.2. Synthesis of 4-[(4-hydroxy-2-methoxy-benzylidene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M2)
4-AAP (0.20 g, 0.001 mol) and 2-methoxy-4-hydroxybenzaldehyde (0.15 g, 0.001 mol). The product was collected as permesean color powder: 0.3618 g, yield 94%, mp. 277–279 ºC, UV-Vis (nm): 273, 309, 408; IR (KBr cm1): 878, 1065, 1306, 1465, 1573, 16541, 2845, 2931, 3220, 3410, 3554, 3708; 1H NMR (500 MHz, CDCl3) δ: 2.39 (s, 3H, CH3); 3.09 (s, 3H, N-CH3); 3.76 (s, 3H, OCH3); 6.45 (d, 1H, J 10.0 Hz, C6H6); 7.32 (m, 3H, C6H6,); 7.50 (t, 2H, J 9.50 Hz, C6H6); 7.85 (d, 2H, J 9.5 Hz, C6H6); 9.75 (s, 1H, N=CH); 9.99 (s, 1H, -OH); 13C NMR (125 MHz, CDCl3) δ: 10.13, 35.98, 55.40, 108.45, 122.17, 124.23, 126.62, 129.03, 130.77, 152.11, 159.01, 188.19; m/z (ESI): 337.37, (M+, 100%); Anal. Calc. for C19H19N3O3 (%): H, 5.61, C, 68.27, N, 12.58; Found (%) H, 5.68, C, 67.64, N, 12.46.
3.2.3. Synthesis of 3-[(5-bromo-2-hydroxy-benzylidene)-amino]-4,5-dimethyl-1-phenyl-1,5-dihydro-pyrrol-2-one (M3)
4-AAP (0.20 g, 0.001 mol) and 2-hydroxy-4-bromobenzaldehyde (0.20 g, 0.001 mol). The product was collected as lime color powder: 0.3628 g, yield 94%, mp. 208.6–210.5 ºC , UV-Vis (nm): 327, 458;IR (KBr cm1): 875, 1075, 1304, 1481, 1589, 1652, 2943, 3057, 3247, 3413, 3475, 3520; 1H NMR (500 MHz, CDCl3) δ: 2.35 (s, 3H, CH3); 3.14 (s, 3H, N-CH3); 6.86 (d, 1H, J 8.5 Hz, C6H6); 7.17 (m, 3H, C6H6); 7.34 (m, 3H, C6H6); 7.47 (t, 2H, J 8.0 Hz, C6H6); 9.72 (s, 1H, N=CH); 13.31(s, 1H, -OH); 13C NMR (125 MHz, CDCl3) δ: 9.97, 35.20, 115.39, 130.52, 131.17, 133.95, 149.61, 158.40, 159.73; m/z (ESI): 385.25, (M+, 100%); Anal. Calc. for C19H17BrN2O2 (%): H, 4.12, C, 58.58, N, 6.08; Found (%) H, 4.45, C, 59.23, N, 6.27.
3.2.4. Synthesis of 4-[(2,4-dihydroxy-benzylidene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M4)
4-AAP (0.20 g, 0.001 mol) and 2,4-dihydroxy-benzaldehyde (0.13 g, 0.001 mol). The product was collected as canary color powder: 0.3003 g, yield 93%, mp. 208.8–310.5 ºC, UV-Vis (nm): 349, 413; IR (KBr cm1): 844, 1334, 1592, 1654, 2901, 3086, 3612; 1H NMR (500 MHz, CDCl3) δ: 2.33 (s, 3H, CH3,); 3.13 (s, 3H, N-CH3); 5.70 (s, 1H, C6H6); 6.25 (d, 1H, J 2.0 Hz, C6H6); 6.33 (d, 1H, J 6.50 Hz, C6H6); 7.35 (m, 3H, C6H6); 7.50 (m, 3H, C6H6); 9.54 (s, 1H, N=CH); 13.33 (s, 1H, -OH); 13C NMR (125 MHz, CDCl3) δ: 9.80, 35.38, 102.49, 112.59, 124.59, 126.90, 132.95, 134.38, 149.53, 158.34, 159.42, 161.21; m/z (ESI): 323.35, (M+, 100%); Anal. Calc. for C18H17N3O3 (%): H, 5.43, C, 64.87, N, 12.57; Found (%) H, 5.30, C, 64.86, N, 13.00.
3.2.5. Synthesis of 4-[(4-chloro-2-hydroxy-benzylidene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M5)
4-AAP (0.20 g, 0.001 mol) and 4-chloro-2-hydroxybenzaldehyde (0.24 g, 0.001 mol). The product was collected as lemon color powder: 0.3205 g, yield 92.9%, mp. 230.5–232.5 ºC, UV-Vis (nm): 357, 369; IR (KBr cm1): 816, 1035, 1334, 1591, 1659, 2929, 3083, 3363, 3503; 1H NMR (500 MHz, CDCl3) δ: 2.35 (s, 3H, CH3); 3.11 (s, 3H, N-CH3); 6.79 (d, 1H, J 8.0 Hz, C6H6); 7.29 (m, 3H, C6H6); 7.32 (m, 3H, C6H6); 7.45 (d, 1H, J 1.0 Hz, C6H6); 9.69 (s, 1H, N=CH); 13.33 (s, 1H, -OH); 13C NMR (125 MHz, CDCl3) δ: 10.11, 35.35, 110.44, 121.68, 129.26, 133.65, 134.05, 149.71, 158.55, 159.34, 159.87, 161.62; m/z (ESI): 341.79, (M+, 100%); Anal. Calc. for C18H16ClN3O2 (%): H, 4.68, C, 62.91, N, 12.26; Found (%) H, 4.72, C, 63.25, N, 12.29.
3.2.6. Synthesis of 4-[(4-diethylamino-2-hydroxy-benzylidene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M6)
4-AAP (0.20 g, 0.001 mol) and 2-hydroxy-4-diethylaminobenzaldehyde (0.24 g, 0.001 mol). The product was collected as orange color powder: 0.3532 g, yield 93%, mp. 245.6–248.5 ºC, UV-Vis (nm): 357, 369, 446; IR (KBr cm1): 843, 1001, 1330, 1502, 1590, 1693, 2922, 3092, 3345, 3460; 1H NMR (500 MHz, CDCl3) δ: 1.02 (t, 6H, J 9.0 Hz, C2H6); 2.20 (s, 3H, CH3); 2.93 (s, 3H, N-CH3); 3.24 (m, 4H, N-CH2); 6.06 (m, 2H, C6H6); 6.18 (d, 1H, J 2.5 Hz, C6H6); 6.99 (d, 1H, J 2.5 Hz, C6H6); 7.12 (m, 1H ,C6H6); 7.38 (m, 3H, C6H6); 9.48 (s, 1H, N=CH); 13.61 (s, 1H, -OH); 13C NMR(125 MHz, CDCl3) δ: 10.23, 12.69, 36.17, 44.49, 97.61, 103.43, 109.63, 117.52, 124.01, 126.63, 129.04, 133.36, 134.72, 148.69, 150.95, 160.87, 162.85; m/z (ESI): 378.21, (M+, 100%); Anal. Calc. forC22H26N4O2 (%): H, 6.71, C, 70.31, N, 14.93; Found (%) H, 6.92, C, 69.82, N, 14.80.
3.2.7. Synthesis of 4-[(2-nitro-benzylidene)-amino]-1,5-Dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M7)
4-AAP (0.20 g, 0.001 mol) and 2-nitrobenzaldehyde (0.18 g, 0.001 mol). The product was collected as goldencolor powder: 0.3208 g, yield 95.5%, mp. 234.3–236.3 ºC; IR (KBr cm1): 844, 1034, 1330, 1503, 1590, 1643, 2922, 3083, 3480; 1H NMR (500 MHz, CDCl3) δ: 2.45 (s, 3H, CH3); 3.24 (s, 3H, N-CH3); 7.29 (t, 2H, J 1.5 Hz, C6H6); 7.37 (m, 3H, C6H6); 7.45 (d, 1H, C6H6); 7.59 (m, 1H, C6H6); 7.82 (d, 1H, J 1.0 Hz, C6H6); 8.12 (d, 1H, J 1.2 Hz, C6H6); 9.99 (s, 1H, N=CH); 13C NMR (125 MHz, CDCl3) δ: 10.02, 35.54, 118.34, 124.67, 127.17, 129.77, 132.27, 134.57, 149.19, 151.51, 152.53, 160.26; m/z (ESI): 336.12, (M+, 100%); Anal. Calc. for C18H16N4O3 (%): H, 4.82, C, 64.30, N, 17.21; Found (%) H, 4.79, C, 64.28, N, 16.66.
3.2.8. Synthesis of 4-[(4-ethylamino-benzylidene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M8)
4-AAP (0.20 g, 0.001 mol) and 4-ethylamino-benzaldehyde (0.17 g, 0.001 mol). The product was collected as butter color powder: 0.3250 g, yield 97%, mp. 141.1–143.2 ºC, UV-Vis (nm): 339, 454; IR (KBr cm1): 905, 1054, 1332, 1533, 1543, 1675, 2937, 3063, 3384; 1H NMR (500 MHz, CDCl3) δ: 2.43 (s, 3H, CH3); 2.99 (s, 6H, N-C2H6); 3.05 (s, 3H, N-CH3); 6.69 (d, 1H, J 8.5 Hz, C6H6); 7.25 (m, 3H, C6H6); 7.42 (m, 4H, C6H6); 7.72 (d, 1H, J 8.5 Hz, C6H6); 9.63 (s, 1H, N=CH); 13C NMR (125 MHz, CDCl3) δ: 10.13, 36.23, 40.02, 111.69, 124.00, 126.28, 129.30, 135.20, 151.12, 157.93, 161.35; m/z (ESI): 334.18, (M+, 100%); Anal. Calc. for C20H22N4O (%): H, 6.45, C, 72.06, N, 16.81; Found (%) H, 6.78, C, 71.83, N, 16.75.
3.2.9. Synthesis of 4-[(2,4-dinitro-benzylidene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M9)
4-AAP (0.20 g, 0.001 mol) and 2,4-dinitrobenzaldehyde (0.23 g, 0.001 mol). The product was collected as candycolor powder: 0.3797 g, yield 99.7%, mp. 265.3–267.5 ºC, UV-Vis (nm): 424; IR (KBr cm1): 828, 1059, 1321, 1458, 1513, 1567, 1651, 2975, 3063, 3410; 1H NMR (500 MHz, CDCl3) δ: 2.49 (s, 3H, CH3); 3.26 (s, 3H, N-CH3); 7.35 (t, 2H, J 7.0 Hz, C6H6); 7.48 (t, 2H, C6H6); 8.40 (m, 3H, C6H6); 8.68 (s, 1H, C6H6); 10.04 (s, 1H, N=CH); 13C NMR (125 MHz, CDCl3) δ: 9.99, 35.13, 117.82, 125.26, 127.81, 129.45, 130.02, 134.07, 147.93, 152.56, 159.60; m/z (ESI): 381.34, (M+, 100%); Anal. Calc. for C18H15N5O5 (%): H, 4.15, C, 56.97, N, 18.43; Found (%) H, 3.96, C, 56.69, N, 18.37.
3.2.10. Synthesis of 4-[(2,3-dimethoxy-benzylidene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M10)
4-AAP (0.20 g, 0.001 mol) and 2,3-dimethoxy-benzaldehyde (0.19 g, 0.001 mol). The product was collected as permesean color powder: 0.0587 g, yield 46%, mp. 187.9–189.8 ºC, UV-Vis (nm): 330, 410; IR (KBr cm1): 680, 1054, 1337, 1514, 1567, 1652, 1692, 2937, 3023, 3472; 1H NMR (500 MHz, CDCl3) δ: 2.48 (s, 3H, CH3); 3.13 (d, 3H, N-CH3); 3.85 (s, 3H, OCH3); 3.97 (s, 3H, OCH3); 6.92 (t, 2H, J 2.0 Hz, C6H6); 7.12 (m, 2H, C6H6 ); 7.38 (m, 3H, C6H6); 7.44 (d, 1H, J 2.0 Hz,C6H6); 10.00 (s, 1H, N=CH); 13C NMR (125 MHz, CDCl3)δ: 10.25, 35.84, 55.90, 118.09, 123.86, 126.94, 129.23, 134.90, 150.06, 151.88, 153.04, 160.86; m/z (ESI): 351.40, (M+, 100%); Anal. Calc. for C20H21N3O3 (%): H, 5.95 C, 68.86, N, 12.03; Found (%) H, 6.02, C, 68.36, N, 11.96.
3.2.11. Synthesis of 4-{[2-(3,4,5,6-tetrahydroxy-tetrahydro-pyran-2-yloxy)-benzylidene]-amino}1,5-Dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M11)
4-AAP (0.20 g, 0.001 mol) and 2-(3,4,5,6-Tetrahydroxy-tetrahydro-pyran-2-yloxy)-benzaldehyde (0.34 g, 0.001 mol). The product was collected as banana color powder: 0.4523 g, yield 96%, mp. 258.7–260.7 ºC, UV-Vis (nm): 348, 435; IR (KBr cm1): 720, 1028, 1348, 1590, 1680, 3020, 3323; 1H NMR (500 MHz, CDCl3) δ: 2.47 (s, 3H, CH3); 3.13 (s, 3H, N-CH3); 3.69 (s, 6H, C6H12O6); 3.94 (s, 6H, C6H12O6); 7.12 (s, 1H, C6H6 ); 7.38 (d, 1H, J 1.0 Hz, C6H6); 7.46 (m, 3H, C6H6); 8.11 (d, 1H, J 8.0 Hz, C6H6); 9.60 (s, 1H, N=CH); 13C NMR (125 MHz, CDCl3) δ: 9.97, 25.27, 27.05, 30.82, 35.10, 65.72, 70.51, 112.31, 118.90, 121.23, 129.42, 132.42, 150.20, 158.12, 160.7, 161.61; m/z (ESI): 470.16, (M+, 100%); Anal. Calc. for C23H25N3O7 (%): H, 5.75, C, 60.43, N, 8.55; Found (%) H, 5.53, C, 60.65, N, 9.23.
3.2.12. Synthesis of 4-[(6-methoxy-benzo[1,3]dioxol-4-ylmethylene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (M12)
4-AAP (0.20 g, 0.001 mol) and 6-Methoxy-benzo[1,3]dioxole-4-carbaldehyde (0.21 g, 0.001 mol). The product was collected as permesean color powder: 0.1355 g, yield 37%, mp. 187.6–188.5 ºC, UV-Vis (nm): 301, 338; IR (KBr cm1): 720, 1028, 1348, 1590, 1660, 3020, 3323; 1H NMR (500 MHz, CDCl3) δ: 2.18 (s, 3H, CH3); 3.17 (s, 3H, N-CH3); 3.96 (s, 3H, OCH3); 4.98 (s, 2H, O-CH2O); 6.19 (s, 1H, C6H6); 7.16 (s, 1H, C6H6); 7.34 (t, 2H, J 8.0 Hz, C6H6); 7.46 (m, 3H, C6H6); 9.65 (s, IH, N=CH); 13C NMR (125 MHz, CDCl3) δ: 10.20, 35.92, 56.92, 62.55, 101.84, 108.84, 124.35, 126.88, 129.17, 134.90, 143.77, 149.17, 156.69, 160.93; m/z (ESI): 365.38, (M+, 100%); Anal. Calc. for C20H19N3O4 (%): H, 5.24 C, 65.12, N, 11.26; Found (%) H, 5.20, C, 65.69, N, 11.49.
3.3. Antibacterial investigation of the minimum inhibitory concentration (MIC) of prepared Schiff bases against 12 bacterial strains
The antimicrobial activities of synthesized 4-APP Schiff base derivatives were evaluated against the Gram-positive and Gram-negative pathogenic strains. These strains included: Bacillus cereus ATCC10876, Bacillus subtilis ATCC19659, Proteus vulgaris ATCC6380, Enterococcus faecalis ATCC13047, Staphylococcus epidermidis ATCC14990, Escherichia coliATCC25922, Klebsiella oxytoca ATCC8724, Klebsiella pneumonia ATCC13882, Mycobacterium smegmatis MC2155, Pseudomonas aeruginosa ATCC27853, Enterobacter aerogenes,and Enterobacter cloacae ATCC13047. A stock solution (1 mg/mL) of each compound was prepared in DMSO, and a serial dilution (500, 250, 125, 62.5, 31.2, 15.6 µg/mL) was performed in 100 µL and seeded in 96-well plates. Thereafter, an overnight fresh culture (50 µL) containing 1.5×106 cfu/mL of each bacterial strain in Muller-Hilton broth was seeded in 96-well plates containing 50 µL different concentrationsin duplicate to make a final volume of 100 µL and allowed bacteria to grow overnight with 5% CO2 flow at 37 ºC. Streptomycin (STM) and nalidixic acid (NLD) were used as standard controls. Incubation and the MIC were determined for each compound after 5 h, and resazurin dye was used to determine the cell viability.
3.4. Antifungal investigation
The antifungal activities of 12 synthesized compounds of 4-AAP derivatives against seven fungal strains were determined. The fungal strains included Aspergillus niger, Aspergillus carbonarious, Aspergillus fumigatus, Fusarium verticillioides, Aspergillus parasiticus, Fusariumproliferatum and Aspergillus flavus. Antifungal investigation was performed using disc diffusion assay by impregnating 15 µL (1 mg/mL) of each compound which was dissolved in DMSO (approximately 15 µg) on 6-mm Ø sterile blank discs. These discs containing dissolved compounds were then deposited on Petri dishes in triplicate containing 20 mL of solidified potato dextrose agar (PDA), or conditioned with 200 µL Ringer solution containing 1×104 spores of a 10-day fungal cultures. Throughout the experiment, Amphotericin B and Nystatin were used as positive controls.  
3.5. Determination of minimum fungicidal concentration
The killing power (minimum fungicidal concentration) of the synthesized compounds was tested against the same strains mentioned above. The inoculation was obtained as described by CLSI, (2008) standard M38 A2[32]. Suspensions were diluted in RPM1-1640 to a concentration of 1×105 spores corresponding to 0.5 Mac Farland standard. Micro-dilution technique was used to seed 100 µL working solutions of 500, 250, 125, 62.5, 31.2 and 15.6 µg/mL of drugs into 96-well plates. Subsequently, 100 µL of inoculation was then added to each well in duplicate and incubated at 37ºC for 3 d. Cell viability was verified colorimetrical using resazurin dye, which turns pink enzymatically inthe presence of living cells and remains blue in the non-living cells (dead cells). After 72 h, each well was added with 10 µL (0.02%) dye and allowed to stand for more than 30 min in the incubator, and minimum fungicidal concentration was recorded. 
 
 
                      1                                              2 (m1m12)                                                                             3 (M1M12)
Scheme 1. Synthesis of 4-APP Schiff bases. Rreaction condition: (i) EtOH, Reflux 3–4 h.  
4. Results and discussion
4.1. Physical properties of synthesised Schiff bases
Table 1 summarizes the physical properties of 4-AAP Schiff bases M1M12. Thin layer chromatography (TLC) was used to monitor the formation of the products together with FT-IR.  
 
Table 1. Percentage yield and molecular weight of M1M12for 4-AAP Schiff bases. 
 
Yd: yield; M.P: melting point; Pdt: product and MW: molecular weight. 
 
4.2. Photophysical characterization
The UV-Vis electronic absorption spectra of M112 in CH2Cl2 (Fig. 1) displayed three main absorption bands in the region of (273–290 nm), (300–400 nm) and (400–457 nm). The bands (273–290 nm) regions were assigned to the π-π* transition of the aromatic group. The second absorption bands in the high-energy region of (300–400 nm) were attributed to the n-π*region of carbonyl and imine group (C=O and C=N). The absorptions of (400–450 nm) corresponded to n-π* intramolecular charge transfer interaction, ICT. CompoundM2 experienced a blue-shift of up to 10 nm in comparison with M1. The reason for this behavior could be assigned to the presence of electron donating effect of a hydroxyl group (OH), which was in accordance with the results obtained by Animesh et al.[33] that APSAL hass only hydroxyl group substituent at the o-position on the aromatic compound. Compound M9 with the absorption of 424 nm also experienced a red-shift by up to 30 nm compared with 454 nm absorption of M6, and this shift was attributed to electron withdrawing effect of a methylgroup (N-CH3)2 at the para position. Compound M6 gave the highest absorption of 475 nm, which was attributed to the presence of OH interaction with p-diethylaminobenzaldehyde. No absorption peak was revealed for M7 in DCM, which was quite surprising, and this was attributed probably to the presence of NO2group at the ortho-aromatic compound, while compound M5 experienced a blue-shift compared with compound M4.Furthermore, compound M11 experienced a blue-shift absorbance as compared with M10 and M12 compounds. This was attributed to the presence of multiple hydroxylgroups of sugar attached to the compound at the o-position. In addition, compounds M1M12 had different absorptions, some had one absorption, some had two absorptions, and others had three absorptions. Various substituents attached to aromatic compound at different positions, which caused different behaviours in DCM, could be responsible for the absorption differences.
 
 
 
Figure 1. UV-Vis spectra for M1M12 Schiff bases. 
 
 
Table 2. UV-Vis spectroscopy bands for M1M12 Schiff bases. 
 
CMD: Compounds. 
  
4.3. Infrared spectra
FT-IR analyses of all the compounds were conducted to confirm their functional groups. The summaries of analyses for some selected bands are discussed as follows. The presence of asymmetric and symmetric stretching bands at 3555–3317 cm1 was assignedtoν(N-H) of the antipyrine (APP) ring. The peak vibration around 3247–3017 cm1 was ascribed to symmetric and asymmetric stretching of ν(C-H) bands on the aromatic compound. The presence of 1597–1533 cm1was assigned to ν(C=N) bands on antipyrine (APP) Schiff bases. The bands at 1650–1621 cm1 were also ascribed to intense functional groups of ν(C=O) on antipyrine ring[34,35]. Bands at 14851406 cm1 were assigned to (N-CH3) groups with lower wave numbers as a result of intramolecular hydrogen bond[22]. The vibrational mode of the aliphatic functional group at 29842716 cm1 was ascribed to (C-H) stretching band, and their deformation vibrations of γ(C-H) ring gave lower bands at 891–724 cm1. The bands at 3520, 3473, 3554 and 3402 cm1 for M3M5 and M11 corresponded to (O-H) broad functional groups, and the bands at 3708 and 3694 cm1 for M2 and M6 wereascribed to (O-H) sharp hydroxyl groups. The peaks at 2852–2830 cm1 were attributed to the methoxy group (C-OCH3) on M1, M2, M10,andM11. In addition, the peak at 650 cm1 was assigned to Br, while the presence of a peak at 770 cm1 was ascribed to Cl functional group. Compound M7 and M9 also displayed peaks at 1463 and 1375 cm1, which were ascribed to the presence of (N=O) bands. All compounds showed γ(CH=N) deformation at around 1147–1057 cm1.
4.4. Antimicrobial studies of Schiff bases against 12 bacterial and seven fungal strains
Antimicrobial evaluation of 12 synthesized compounds against 12 bacterial strains and seven fungal strains was carried out, and the results are tabulated in Table 3. MIC was evaluated in accordance with the standard recommendation of the clinical and laboratory institute[36]. The potential susceptibility of microbes was observedagainst the test compounds as an inhibition zone, which occurred around the disc. Thereafter, the antimicrobial activity of each ligand was evaluated by measuring the inhibition zone against the test microorganism, and the values were compared with obtainable values of the standard. This exercise was performed for all the synthesized compounds (M1M12). MIC under a standard condition was performed. All analyses were performed thrice, and the results obtained were shown in Table 3, showing that each tested compound had different activities against the test microorganisms at the concentration of 15 µg/mL. Antibacterial activities of all the tested compounds M1M12 against Proteus vulgaris were outstanding (MIC ranging from 15.6–250 µg/mL). Their MIC levels were lower compared with NLD (MIC of 500 µg/mL), which made them more potent than the recommended NLD against the afore-mentionedstrains. Their good activities were attributed to the presence of imine group in conjunction with other functional groups like (O, N, C; electron donors) that caused transformation reaction in the biological system[31]. M2 compound showed good activities against Staphylococcus epidermidis (MIC of 62.5 µg/mL) and against Proteus mirabilis (MIC of 125 µg/mL). These activities were better than NLD against the mentioned microorganisms (MIC of 64 and 128 µg/mL). A similar trend was observed with M3 against Enterococcus faecalis (MIC of 125 µg/mL) and against Pseudomonas aeruginosa (MIC of 15.6 µg/mL) as compared with the streptomycin activity (MIC of 128 µg/mL) and NLD (MIC of 500 µg/mL). The presence of OH group that allowed the protonation of compound M4 displayed good antibiotic activities[37] against Enterobacter cloacae (MIC of 15.6 µg/mL), which was lower than the value of streptomycin standard (MIC of 16 µg/mL). It also displayed good activities against Pseudomonas aeruginosa and Enterobacter aerogenes (MIC of 62.5 and 15.6/mL). The mild activityof M4 for the above microorganisms was attributed to the reaction of imine functional group with two hydroxyl groups at ortho and para substituted on the aromatic compound, which are capable of donating electrons to the ring. The results of compound M5 revealed good activities at the concentration of 15 µg/mL for Enterococcus faecalis, Staphylococcus aureus and Pseudomonas aeruginosa, showing good activities (MIC of 15.5, 125 and 125 µg/mL) but low activities against other tested microbes. However, compounds M6and M7 experienced good activities against Enterococcus faecalis (MIC of 15.6/mL). Their activities against Enterococcus faecalis were far better compared with streptomycin (MIC of 128 µg/mL). M7 also showed good activities than NLD against Proteus mirabilis and Staphylococcus epidermidis (MIC 31.2 and 15.6 µg/mL). The tested compounds M9 and M10 against Staphylococcus epidermidis experienced good activities (MIC of 15.6 and 31.2 µg/mL), and they were found to also show good activities against Staphylococcus aureus and Enterococcus faecalis (MIC of 15.6 µg/mL) when compared with streptomycin standard with an MIC value of 256 and 64 µg/mL, respectively. Most of the tested strains of bacteria against M9 and M10 showed bad activities at 15 µg/mL. In other words, compounds M7, M8, M9and M10 experienced relative good activities (MIC value of 125 µg/mL) against Pseudomonas aeruginosa. Furthermore, compounds M1 and M3 showed no activities against Pseudomonas aeruginosa at a concentration of 15 µg/mL. Their no antibacterial activities were attributed to the presence of electron donating effects of themethoxy group (O-CH3) and the concentration of the test compound. Compound M3  experienced very low activities against other tested microorganisms except for Enterococcus faecalis and Pseudomonas aeruginosa at the mentioned concentration. The presence of OH group (electrons donor) and brominesubstituted atom (electron withdrawer) could be responsible for their activities. The different performance was observed with compound M4 against Bacillus cereus and Bacillus subtilis (MIC value of 62.5 and 125 µg/mL), which displayed low activities compared with standards (MIC of 16 µg/mL). At the same concentration of the sample, compound M8 showed low activities against the tested strains, but it displayed good antibacterial activities only with Escherichia coli (MIC of 31.2 µg/mL) as compared with streptomycin (MIC of 54 µg/mL). Compound M8 activity was attributed to para-substituted ethyl amino on benzene ring that donates electrons. The tested compounds M10M12 revealed no activity against Klebsiella pneumonia. Generally, the antimicrobial activities of the test compounds toward microorganisms at a concentrationof 15 µg/mL showed better activities with Gram-positivebacteria than Gram-negative bacteria, except for compoundsM5 and M10 (Table 3). Compound M11 showed very low activities against all tested bacteria strains. The low activities experienced by compound M11 against all the tested bacteria strains could be attributed to the presence of sugar molecules at the o-position through a breakdown cleavage of a glycosidic bond (C1-O-C4)[38]. Gram-negative strains, like Proteus vulgaris and Escherichia coli,were sensitive at a lower concentration of M1, M2, M5 and M8 (MIC: 15.6 µg/mL, 62.5 µg/mL, 62.5 µg/mL and 31.2 µg/mL). In contrast, Gram-positive strains, such as Enterococcus faecalis and Mycobacterium smegmatis, inhibited the bacterial growth at the minimum concentration of M1, M2, M5and M7 (MIC: 15.6 µg/mL). 
 
Table 3. Potential inhibitory activities of the synthesized compound against 12 bacterial strains. 
 
STM: streptomycin; NLD: nalidixic acid, M.org: microorganism, G.P: gram-positive, G.N; gram-negative. 
 
The antifungal results showed that the tested fungi were not sensitive to the prepared Schiff bases at the prepared concentration of 500 µg/mL. This simply means that the cells were viable. This was attributed to the use of a smaller amount of the prepared compounds as well as the presence of free ligands that allowed the multiplicity of the microbes, by leaving their active sites exposed[31].
5. Conclusions
A total of 12 Schiff bases of 4-AAP derivatives were synthesized via condensation of 4-aminoantipyrine and aromatic aldehydes. The targeted imines were achieved with relatively high yield, and the elemental analyses result agreed with calculated values. The compounds were successfully characterized with UV-Vis spectroscopy,FT-IR, EA, 1H NMR and 13C NMR spectroscopy. The melting points were successfully assessed, and all analysis obtained were in order with the proposed structures. The UV-Vis spectroscopy data showed three major absorptions between 273–457 nm. Theantibacterial activities of all the tested compounds M1M12 against Proteus vulgaris showed good activitieswith MIC ranging from 15.6–250 µg/mL in comparison with the standard NLD with an MIC of 500 µg/mL. We could conclude that most of the tested compounds experienced mild to low activities against major bacterial strains at 15 µg/mL. Their activities were attributed to the low concentration of the compounds. The fungi activities of the tested samples revealed no activities at the concentration of 500 µg/mL. Therefore, further analysis will be carried out on minimum bactericidal concentration (MBC). Moreover, cytotoxicity of the tested compounds will be determined in order to establish the potential effects of these compounds against human cells.
Acknowledgements
This project was supported by the Faculty of Science, Department of Applied Chemistry, the University of Johannesburg for providing enabling environment to perform this work and the National Research Foundation (NRF) for the provision of running cost of this work.
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Received: 2018-04-27, Revised: 2018-06-18, Accepted: 2018-09-13.
Foundation items: This project was supported by the Faculty of Science, Department of Applied Chemistry, the University of Johannesburg for providing enabling environment to perform this work and the National Research Foundation (NRF) for the provision of running cost of this work.
*Corresponding author. Tel.: +27-115596160, E-mail: miikhile@gmail.com; dndinteh@uj.ac.za       
 
 

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