Vol-3, Issue-4, Suppl-2, Nov 2012 ISSN: Joshi et al PHARMA SCIENCE MONITOR

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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME NOVEL SCHIFF S BASES OF PYRAZOLE ALDEHYDE Aashka Joshi*, Arun Parikh, Hansa Parikh and Vijayalakshmi Gudaparthi Department of Pharmaceutical Chemistry, L. J. Institute of Pharmacy, S.G.Highway, Ahmedabad ABSTRACT Efficient syntheses of some novel potent compounds are described to overcome problem of multi-drug resistant (MDR) bacteria and fungi resulting from the widespread use and misuse of classical anti-microbial agents. The synthetic procedure involves the condensation of pyrazole aldehyde bearing 2,4-dichlorosubstituent with different aromatic primary amines furnish schiff s bases namely N-aryl((3-(2,4-dichlorophenyl)-1- phenyl-1h-pyrazol-4-yl)methylene)amines (Ia-Ij). The structures of all the synthesised compounds are assigned on the basis of IR, 1 H NMR, Mass spectral data. All the synthesised compounds are evaluated for antibacterial and antifungal activities against S.aureus, E.coli, Candida albicans.the result revealed that all the synthesised compounds showed significant activity. Keywords: Schiff s bases, Pyrazole aldehyde, Aromatic amines, Antibacterial, Antifungal. INTRODUCTION The chemistry of heterocyclic compounds is as logical as that of aliphatic or aromatic compounds. Heterocycles containing sulphur and nitrogen atoms in the core structure, it shows number of pharmacologically and biologically active compounds. Heteroaromatic compounds have attracted considerable attention in the design of biologically active molecules and advanced organic materials. Heterocyclic chemistry is of great importance to the medicinal chemists because of their drug utility. Hence, a practical method for the preparation of such compounds is of great interest in synthetic organic chemistry. Structurally, a schiff s base (also known as imine or azomethine) is a nitrogen analogue of an aldehyde or ketone in which the carbonyl group has been replaced by an imine or azomethine group, many methods for the synthesis of schiff s bases have been developed and the simplest method appears is to condense by boiling them into alcohols. Aldehydes and ketones react with primary amine (R-NH 2 ) and with other ammonia derivatives (Z- NH 2 ) to form schiff s base (imine).schiff s bases of pyrazole aldehydes and aromatic IC Value

2 amines exhibit wide range of biological activities as antifungal [1], antibacterial [2] and antitubercular [3] etc. The biological significance of this class of compounds impelled us to continue working on the synthesis of new schiff s bases of pyrazole derivatives. Accordingly the various schiff s bases of pyrazole aldehyde have been synthesized. Antimicrobial acivity: Vora J et al. [4] have reported some novel schiff s base derivatives of 4-methylpyridin-2- amine (1) as antimicrobial agents. (1) R 1 =H, Cl ; R 2 =H; R 3 = H, Cl Malladi S et al. [5] reported the synthesis of some novel [3-(4-aryl)-1H-pyrazol-4- yl]methylidene}amino)-4h-1,2,4-triazole-3-thiol (2) as antimicrobial agents. (2) R' =OCH 3, F, Cl During the last past decades, human population affected with life treating infectious diseases caused multi-drug resistant bacteria increase an alarming level around the world. In recent years, much attention has been focused on addressing the problem of multi-drug resistant (MDR) bacteria and fungi resulting from the widespread use and misuse of IC Value

3 classical anti-microbial agents. Due to this reason, new classes of antibacterial agent with novel mechanisms are crucial need to combat with the multidrug resistant infections. So there is need to synthesize new heterocycles to overcome this problem. From review of literature, various schiff s bases show potent antimicrobial and antitubercular and activity. So the aim is to schiff s bases of pyrazole aldehyde with different amines of therapeutic importance. Schiff s bases of pyrazole aldehyde are to be synthesized by using different aromatic amines like 2-aminobenzthiazole, 2- aminopyridine, p-aminosalicylic acid, 4-aminoantipyrine, 5-aminotetrazole, isoniazide and 4-methoxy-2-nitroaniline,p-arsanalic acid,p-chloroaniline and p-anisidine. MATERIALS AND METHODS Materials: The chemicals and reagents used in the project work were of AR and LR grade, procured from Astron chemicals, Ahmedabad and they are used as they obtained. Analytical Techniques: Melting points were determined in open capillary tubes and are uncorrected. Compounds were checked for their purity by TLC on silica gel G plates and spots were located by iodine vapors. In some cases TLC GF-254 was used and spot were visualized under UV light. Insruments: IR spectra of all synthesized compounds were recorded in on FT-IR 84 Shimadzu spectrophotometer. Mass spectra were recorded on 2010 EV LCMS Shimadzu instrument at 70eV. 1 H NMR spectra were obtained in CDCl 3, DMSOd 6 on BRUKER Avance-II 400MHz instrument and chemical shift were measured as parts per million (ppm) downfield from tetramethylsilane (TMS) as internal standard. Method: General Procedure for the synthesis of N-aryl((3-(2,4-dichlorophenyl)-1-phenyl- 1H-Pyrazol-4-yl)methylene)amines (Ia-Ij) Step 1: Synthesis of 1-(1-(2,4-dichlororophenyl)ethylidene)-2-phenylhydrazine The mixture of 2,4-dichloroacetophenone (1.89g, 0.01 mol) and phenylhydrazine(1.08g, 0.98mL,0.01 mol) in methanol in presence of 5 drops of glacial acetic acid was refluxed IC Value

4 on water bath for 2 hrs at C.Product obtained after cooling was recrystallized from methanol. Step 2 : Synthesis of 3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 2-(1-(2,4-dichlorophenyl)ethylidene)-1-phenylhydrazine (2.79g, 0.01 mol) was added in Vilsmeier-Haack reagent(prepared by dropwise addition of 3 ml POCl 3 in ice cooled 25 ml DMF) and refluxed for 6 hrs at C. The reaction mixture was poured onto crushed ice followed by neutralisation with sodium bicarbonate. Crude Product was isolated and recrystallised from methanol. Step 3 : Synthesis of N-aryl((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)amine (Ia-Ij) A mixture of 3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (3.17g, 0.01 mol) and aromatic amine (0.01 mol) was refluxed in absolute ethanol for 10 hrs at C using glacial acetic acid as catalyst. The contents were cooled and product isolated was recrystallised from ethanol. TABLE 1: PHYSICAL CONSTANTS DATA OF SCHIFF S BASES Compound R Molecular Formula I a Benzthiazol -2-yl Mol. wt. C 23 H 14 Cl 2 N 4 S M.P. C I b 2-Pyridyl C 21 H 14 Cl 2 N I c I d I e I f 3-Carboxyl- 2-hydroxyl phenyl Antipyrin- 4-yl Tetrazol-5- yl ɤ-Pyridoyl amine C 23 H 15 Cl 2 N 3 O C 27 H 21 Cl 2 N 5 O C 17 H 11 Cl 2 N C 22 H 15 Cl 2 N 5 O Yield % Rf Value Solvent System n-hexane: Toluene: (8:2) n-hexane: n-hexane: Toluene: (8:2) n-hexane: IC Value

5 I g I h 4-Methoxy 2- nitrophenyl Arseno phenyl C 23 H 16 Cl 2 N 4 O C 22 H 16 AsCl 2 N 3 O I i p-anisyl C 23 H 17 Cl 2 N 3 O I j p- Chlorophen yl C 22 H 14 Cl 3 N Toluene: n-hexane: n-hexane: n-hexane: Scheme for the Synthesis of N-aryl-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4- yl)methylene)amines IC Value

6 Spectral data of schiff s bases (Ia) N-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) benzo[d]thiazol-2-amine IR(v,cm-1): 783(C-Cl), 1650 (C=N), 1224 (C-N), 626 (C-S); MASS(m/z): 449.1(M + ), 451.1(M+2); 1 HNMR(δ,ppm): 8.78(s,1H,CH), 8.40(s,1H,CH), (d,1H,ArH), (dd,2H,ArH), (dd,2H,ArH), (d,1H,ArH),7.4(s,1H,ArH), (m,5H,ArH). (Ib)N-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) pyridin- 2-amine IR(v,cm-1): 800(C-Cl), 1600 (C=N), 1200 (C-N), 1595(C=N:pyridine); MASS(m/z): 392.0(M + ),394.0(M+2); 1 HNMR(δ,ppm): 8.79(s,1H,CH), 8.44(s,1H,CH), (d,1H, ArH), (d,1H,ArH), (d,1H,ArH), 7.40(d,1H,ArH), 7.66(d,1H,ArH), (d,1H, ArH),7.43(s,1H,ArH), (m,5H,ArH). (Ic) 4-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene amino)-3- hydroxybenzoic acid IR(v,cm-1): 850(C-Cl), 1590 (C=N), 1225 (C-N), 2810(C-H), 3120(O-H:carboxyl), 3400(O-H); MASS(m/z): 453.4(M + ),455.4(M+2); 1 HNMR(δ,ppm): 8.81(s,1H,CH), 8.42(s,1H,CH), (d,1H,ArH), (d,1H,ArH), (d,1h,arh), 7.73(s,1H,ArH), 11.00(s,1H, OH),5.43(s,1H,OH), (m,5H,ArH). (Id) 4-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene amino)-1,2- dihydro-2,3-dimethyl-1-phenylpyrazol-5-one IR(v,cm-1): 826(C-Cl), 1598 (C=N), 1218 (C-N), 1725(C=O), 1698(C=O)MASS(m/z): 501.2(M + ), 503.2(M+2); 1 HNMR(δ,ppm): 8.40(s,1H,CH), 7.50(s,1H,CH), (d,1H ArH), (d,1H,ArH), (d,1h,arh), (m,4H,ArH), 2.45(s,1H, CH 3 ),3.22(s,1H,CH 3 ), (m,5H,ArH). (Ie) N-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-2Htetrazol-5-amine IR(v,cm-1): 812(C-Cl), 1605 (C=N), 1220 (C-N), 3210 (N-H); MASS(m/z): 383.1(M + ) 385.2(M+2). (If) N-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene) isonicotinohydrazide IR(v,cm-1): 827(C-Cl), 1616 (C=N), 1228 (C-N), 1668 (C=O) 3385 (N-H); MASS(m/z): 435.9(M + ),437.2(M+2); 1 HNMR(δ,ppm): 8.87(s,1H,CH), 8.18(s,1H,CH), (d,1H, ArH), (d,1H,ArH), (d,1H,ArH), (d,1H,ArH), (d,1H, ArH), (d,1h,arh), 7.62(s,1H,ArH), (m,5H,ArH), 7.00(s,1H,NH), (m,5H,ArH). IC Value

7 (Ig) N-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4- methoxy-2-nitrobenzenamine IR(v,cm-1): 831(C-Cl), 1595 (C=N), 1228 (C-N), 1296 (C-O),1672 (N-O); MASS(m/z): 466.0(M + ),468.0(M+2); 1 HNMR(δ,ppm): 8.90(s,1H,CH), 8.40(s,1H,CH), (d,1H, ArH), 7.8(s,1H,ArH), (d,1H,ArH), 7.46(d,1H,ArH),7.44(d,1H, ArH), (m,5h,arh). (Ih) 4-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methyleneamino)phenyl arsonic acid IR(v,cm-1): 836(C-Cl), 1589 (C=N), 1200 (C-N), 3221 (O-H); MASS(m/z): 516.2(M + ), 518.2(M+2). (Ii) N-((3-(2,4-dichlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)4-methoxy benzenamine IR(v,cm-1): 846(C-Cl), 1522(C=N), 1212 (C-N), 1227(C-O); MASS(m/z): 421.0(M + ), 423.0(M+2). (Ij) N-((3-(2,4-dichlorophenyl)-1-phenyl-1H- pyrazol-4-yl)methylene)4-chloro benzenamine IR(v,cm-1): 828(C-Cl), 1590(C=N), 1210(C-N); MASS(m/z): 425.0(M + ), 427.0(M+2). RESULT AND DISCUSSION Antimicrobial Screening All synthesized compounds were screened for antimicrobial activity using Broth Dilution Method. It is one of the non-automated in vitro bacterial susceptibility tests. This classic method yields a quantitative result for the amount of antimicrobial agents that is needed to inhibit growth of specific microorganisms. 1. All synthesized compounds were tested for antibacterial activity. 2. All necessary controls maintained are : Drug control Vehicle control Agar control Organism control Known antibacterial drugs control IC Value

8 Mueller Hinton Broth was used as nutrient medium to grow and dilute the drug suspension for the test bacteria. Inoculum size for test strain was adjust to 10 8 Cfu [Colony Forming Unit] per milliliter by comparing the turbidity. Following common strains were used for screening of antibacterial activity. E.coli S.aureus Candidia albicans MTCC MTCC MTCC DMSO was used as diluents/vehicle to get desired concentration of drugs to test upon standard bacterial strains. Procedure Serial dilutions were prepared in primary and secondary screening. Primary screen: In primary screening 1000 ug/ml, 500 ug/ml, and 250 ug/ml concentrations of the test compounnds were taken. The active test compound found in this primary screening were further tested in a second set of dilution against all microorganisms. Secondary screen: The drugs found active in primary screening were similarly diluted to obtain 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, and µg/ml, concentrations. The control group containing no antibiotic is immediately sub cultured [before inoculation] by spreading a loopful evenly over a quarter of plate of medium suitable for the growth of the test organism and put for incubation at 37 0 C overnight. The tubes are then incubated overnight. The MIC of the control organism is read to check the accuracy of the drug concentrations. The lowest concentration inhibiting growth of the organism is recorded as the MIC. The amount of growth from the control tube before incubation [which represents the original inoculums] is compared. IC Value

9 The highest dilution showing at least 99 % inhibition zone is taken as MIC. The result of this is much affected by the size of the inoculum. The test mixture should contain 10 8 organism/ml. TABLE 2: EVALUATION OF ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF SYNTHESIZED COMPOUNDS Minimum Inhibitory Concentration(μg/mL) Comp. Code S.aureus E.Coli Candida Albicans Ia Ib Ic Id Ie If Ig Ih Ii Ij Ampicillin Chloramphenicol Griseofulvin Nystatin Figure 1 Comparison of MIC of Synthesized Compounds against S.aureus and E.Coli with Standard drugs IC Value

10 Figure 2 Comparison of MIC of Synthesized Compounds against Candida albicans with Standard drugs All the compounds were screened for their antibacterial activity by Broth Dilution Method using different strain like E.Coli and S.aureus at 1000, 500, , 100, 50, 25 and 12.5 µg/ml. Ampicillin and Chloramphenicol were taken as the standard drugs for antibacterial activity. Nystatin and Griseofulvin were taken as the standard drugs for antifungal activity. The test compounds Ia (R = benzthiazol-2-yl), Id (R = antipyrin-4-yl), Ie (R = tetrazol-5- yl), Ig (R=4-methoxy-2-nitrophenyl), Ih (R=arsenophenyl), Ii (R=p-anisyl), Ij (R=pchlorophenyl) showed lower MIC than standard Ampicillin against S.aureus. The test compound Ia (R = benzthiazol-2-yl) exhibited euipotent activity with standard drug Chloramphenicol against S.aureus and E.Coli. The test compound Ig (R=4-methoxy-2- nitrophenyl) and Ii (R=p-anisyl) exhibited euipotent activity with standard drug Chloramphenicol against S.aureus. The test compounds Ia (R = benzthiazol-2-yl), Ib (R =2-pyridyl), Ie (R=4-methoxy-2- nitrophenyl) and If (R = ɤ-pyridoylamine), Ig (R=4-methoxy-2-nitrophenyl), Ih (R=arsenophenyl), Ii (R=p-anisyl), Ij (R=p-chlorophenyl) showed lower MIC than standard Griseofulvin against Candida albicans.the test compounds Ia (R = benzthiazol- IC Value

11 2-yl) and Ie (R = tetrazol-5-yl) showed lower MIC than standard Nystatin against Candida albicans.compounds Ig (R=4-methoxy-2-nitrophenyl), Ih (R=arsenophenyl) and Ij (R=p-chlorophenyl) exhibited equipotent activity with the standard drug Nystatin against Candida albicans. CONCLUSION All the compounds were found to be mild to moderately active against gm(+ve) and gm(-ve) bacterial strains and fungal strain. REFERENCES 1. Akhaja T: 1,3-dihydro-2H-indol-2-ones derivatives: Design, Synthesis, in vitro antibacterial, antifungal and antitubercular study. Eur. J. Med. Chem. 2011; Pandey V, Chawla V, Saraf S: Comparative study of conventional and microwave-assisted synthesis of some schiff bases and their potential as antimicrobial agents. Med. Chem. Res. 2011; Masunari A: A new class of nifuroxazide analogues : synthesis of 5-nitrophene derivatives with antimicrobial activity against multidrug-resistant Staphylococcus aureus. Bioorg. Med. Chem. 2007; Vora J, Parmar K, Chauhan C: Synthesis, Spectral and Microbial Studies of Some Novel Schiff Base Derivatives of 4-Methylpyridin-2-amine. E.Journal of Chemistry 2009;6: Malladi S: Synthesis characterization and antibacterial activity of some new pyrazole based schiffbases. Arabian Journal of Chemistry 2011; For Correspondence: Aashka Joshi aashka_161@yahoo.com IC Value