Vol-3, Issue-3, July-2012 ISSN: Panda et al

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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES SYNTHESIS OF SOME ANTIBACTERIAL, ANALGESIC AND ANTI- INFLAMMATORY AGENTS CONTAINING ISATIN NUCLEUS Jnyanaranjan Panda Post Graduate Department of Pharmaceutical Chemistry, Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha , India. ABSTRACT Isatin on reaction with equimolar quantity of primary aromatic amines in presence of acetic acid yields the precursor Schiff bases. Using this precursors a series of N-Mannich bases were prepared by reacting with methyl phenyl amine in presence of formaldehyde. The chemical structures of the title compounds have been confirmed and elucidated by means of their physical and spectral data respectively. The N-Mannich bases were evaluated for their possible antibacterial, analgesic and anti-inflammatory activities by cup plate method, acetic acid induced writhing and carrageen induced paw edema method respectively. Among the tested compounds, the compound containing chloro group showed significant biological activity. Keywords: Isatin, Mannich bases, Antibacterial, Analgesic, Anti-inflammatory. INTRODUCTION Isatin (indole-2,3-dione) is an endogenous compound having wide range of biological activities. The well documented biological profiles of isatin derivatives have attracted much attention over the years. Schiff bases are some of the most widely used organic compounds. They are used as pigments and dyes, catalysts, intermediates in organic synthesis, as polymer stabilizers along with some biological activities. Schiff and Mannich bases of isatin represent one of the most important classes of organic compounds because of their broad spectrum of pharmacological activities such as antibacterial [1,2], antifungal [3], antiviral [4], anti-hiv [5], antidepressant [6], antiinflammatory [7], analgesic [8] and anticonvulsant [9]. In the present study, aromatic primary amines were subjected to react with isatin to form the Schiff bases. The corresponding N- Mannich bases were synthesized by reacting the Schiff bases with the secondary amine and formaldehyde. The chemical structures of the synthesized compounds were confirmed by means of their physical, IR, 1 H-NMR and mass data. The synthesized compounds were tested for their antibacterial, analgesic and anti-inflammatory activities by the standard methods. IC Value

2 MATERIALS AND METHODS Chemistry All the melting points were determined by open-ended capillary tube on Veego electrical melting point apparatus, expressed in ο C and are uncorrected. The IR spectra of the compounds were recorded on Shimadzu IR Affinity series-1 in KBr and the values were expressed in cm -1. The 1 H-NMR spectra of the compounds were recorded on a Bruker Advance II 400 MHz spectrophotometer and the values were expressed in δ ppm. The mass spectra of the compounds were recorded on Micromass Q-Tof Micro; in m/z. The purity of the compounds was checked by thin layer chromatography on silica gel G coated plates. Procedure for synthesis of Schiff bases of isatin Equimolar (0.01 mol) quantity of isatin and substituted anilines were dissolved in a sufficient amount of ethanol and refluxed for 3 hr in presence of glacial acetic acid. After 24 hr at room temperature, the products were separated by filtration, dried under vacuum and recrystallized from warm ethanol. Procedure for Synthesis of Mannich bases of isatin Equimolar quantity of methyl phenyl amine (0.004mol) in 10ml of ethanol was added to the solution containing appropriate isatin and formaldehyde solution (37% v/v). The reaction mixture was stirred for 2 hr at room temperature and kept under refrigeration for 24 hr. The products were separated by suction filtration, dried under vacuum and recrystallized from warm ethanol. The molecular formula, molecular weight, melting point, yield, R f and spectral data were presented in table 1 and 2. TLC was monitored by using solvent system benzene: chloroform (55:45) and the spots were identified by placing the dried plate in iodine chamber. O R-NH 2 NR HCHO NR N H O N H O HN CH 3 N O CH 3 N Scheme IC Value

3 Pharmacology Antibacterial study The synthesized compounds were screened for in-vitro antibacterial activity against Staphylococcus aureus (MTCC-87), Escherichia coli (MTCC-40), Staphylococcus epidermidis (MTCC-2639), Pseudomonas aeruginosa (MTCC-424) and Proteus vulgaris (MTCC 426) using cup plate method [10]. The compounds were tested at 500µg concentration in DMSO, using nutrient agar as the medium. After 24hr of incubation at 37 o C, the zone of inhibition formed were measured in mm against standard drug tetracycline and the data were presented in table 3. Analgesic activity The analgesic activity of the synthesized compounds was studied using acetic acid induced writhing response [11]. In this model (chemical method), the animals were divided into different groups (n=6). Group I served as control (1% Carboxy Methyl Cellulose as vehicle, 1ml/kg, p.o.), group II served as standard (Indomethacin, 10 mg/kg, p.o.) and other groups were served as test groups and received the test compounds each at the dose of 200 mg/kg/p.o. The vehicle, standard and test compounds were administered in the suspension form in Carboxy Methyl Cellulose to the respective groups, 30 min before the induction of pain by acetic acid. Healthy Swiss albino mice (20-30 gm) were placed into individual restraining cages. The animals were then allowed to adapt in the cages for 30 minutes before testing. Writhing was induced in mice by administration of 0.6% acetic acid (10 ml/kg body weight, i.p.). The number of wriths was calculated over the period of 20 min after acetic acid injection. A writh is indicated by an abdominal constriction followed by full extension of hind limb. The data represent the total numbers of wriths observed over the 20 min period. Anti-inflammatory study The anti-inflammatory activity was determined by carrageenan induced paw edema method [12] in Wistar rats by using digital plethysmometer (Panlab LE 7500). Wistar rats of either sex ( gm) were selected and housed under standard laboratory conditions, given standard rat pellet and tap water ad libitum and maintained under standard environmental conditions throughout the period of experimentation. The animals were housed in cages under standard laboratory condition. They had free access to IC Value

4 standard diet and water. The animals were divided into different groups of six animals each and fasted for 12hr before the experiment. Group I served as control and received vehicle, group II served as standard group and received indomethacin (10 mg/kg, p.o.) and other groups are served as test groups and received the test compounds (200 mg/kg/p.o.) one hour prior to carrageenan injection. The initial right hind paw volume of the rats were measured using a digital plethysmometer and then 0.1 ml of 1% w/v carrageen solution in normal saline was injected into the sub plantar region of the right hind paw. The volume of right hind paw was measured at 1, 2, 3, 4 and 5 hr after carrageenan injection by using digital plethysmometer. The data were expressed is paw volume (ml), compared with the initial hind paw volume of each rat. Procedures employed for evaluation of analgesic and anti-inflammatory activity were reviewed and approved by the Institutional Animal Ethical Committee. TABLE 1: PHYSICAL DATA OF THE SYNTHESIZED COMPOUNDS Compd R M.F. M.W. M.P. Yield ( o C) (%) R f 6A phenyl C 22 H 19 N 3 O B 2-nitrophenyl C 22 H 18 N 4 O C 3-nitrophenyl C 22 H 18 N 4 O D 4-nitrophenyl C 22 H 18 N 4 O E 3-chlorophenyl C 22 H 18 ClN 3 O F 4-chlorophenyl C 22 H 18 ClN 3 O G 4-fluorophenyl C 22 H 18 FN 3 O H 4-bromophenyl C 22 H 18 BrN 3 O I 2,4-dinitrophenyl C 22 H 17 N 5 O J 2,6-dichlorophenyl C 22 H 17 Cl 2 N 3 O K 3,4-dichlorophenyl C 22 H 17 Cl 2 N 3 O L 4-chloro-2- nitrophenyl C 22 H 17 ClN 4 O M 2-chloro-4- nitrophenyl C 22 H 17 ClN 4 O N 3-chloro-4- fluorophenyl C 22 H 17 ClFN 3 O IC Value

5 TABLE 2: IR, 1 H-NMR AND MASS SPECTRAL DATA OF THE SYNTHESIZED COMPOUNDS Compounds 6A 6B 6C 6D 6E 6F 6G 6H 6I 6J 6K 6L 6M 6N IR (KBr), 1 H NMR (400 MHz, DMSO-d6), Mass (m/z) IR (cm -1 ): 1709(C=O), 1614(C=N), 1465(C=C) 1 H NMR δ (ppm): (t, 3H, CH 3 ), 4.57 (s, 2H, CH 2 ), (m, 14H, Ar-H). m/z 341 IR (cm -1 ): 1719(C=O), 1614(C=N), 1342 & 1510(NO 2 ) 1 H NMR δ (ppm): 1.24 (t, 3H, CH 3 ), 4.23(s, 2H, CH 2 ), (m, 13H, ArH). m/z 386 IR (cm -1 ): 1716(C=O), 1616(C=N), 1354 & 1529(NO 2 ) 1 H NMR δ (ppm):1.25(t, 3H, CH 3 ), 4.22(s, 2H, CH 2 ), (m, Ar-H, 13H). m/z 386 IR (cm -1 ): 1716(C=O), 616(C=N), 1354 &1529(NO 2 ) 1 H NMR δ (ppm): 1.25(t, 3H, CH 3 ), 4.22(s, 2H, CH 2 ), (m, Ar-H, 13H). m/z 386 IR (cm -1 ): 1716(C=O), 1614(C=N), 1463(C=C), 721(C=Cl) (m, Ar-H, 12H) 1 H NMR δ (ppm): (3H, CH 3 ), 4.09(s, 2H, CH 2 ), (m, Ar-H, 12H). m/z 376 IR (cm -1 ): 1716(C=O), 1612(C=N), 1097(C-F), 1458(C=C) 1 H NMR δ (ppm): 1.34(t, 3H, CH 3 ), 4.41(s, 2H, CH 2 ), (m, Ar-H,13H). m/z (m, Ar-H, 12H) IR (cm -1 ): 1716(C=O), 616(C=N), 1354 & 1529(NO 2 ) 1 H NMR δ (ppm): 1.25(t, 3H, CH 3 ), 4.22(s, 2H, CH 2 ), (m, Ar-H, 13H) (m, Ar-H, 12H) (m, Ar-H, 12H) 1 H NMR δ (ppm): (t, 3H, CH 3 ), 4.09 (s, 2H, CH 2 ), (m, Ar-H, 12H). m/z (m, Ar-H, 12H) (m, Ar-H, 12H) IC Value

6 TABLE 3: IN-VITRO ANTIBACTERIAL ACTIVITY OF THE SYNTHESIZED COMPOUNDS Compounds Diameter of zone of inhibition (mm) P. vulgaris P. aeruginosa E. coli S. aureus S. epidermidis 6A ± ± ± B ± ± ± C ± ± ± ± ± D ± ± ± ± ± E ± ± ± ± F ± ± G ± ± ± ± H ± ± ± ± I ± ± ± ± ± J ± ± ± ± ± K ± ± ± N ± ± ± ± 0.57 Control Standard ± ± ± ± ± 1.52 Results were expressed as Mean ± S.D. (n = 3), - indicates no zone of inhibition IC Value

7 Figure 1 Effects of the synthesized compounds on acetic acid induced writhing in mice. Results were expressed as mean ± SEM (n=6). *p < 0.05, **p < 0.01, ***p < compared to control. Figure 2 Effects of the synthesized compounds on the rat paw edema-induced by sub plantar injection of carrageenan. Results were expressed as Mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < as compared to control. IC Value

8 Statistical analysis The results of statistical analysis for animal experiments were expressed as mean ± SEM. The number of animals in each group were six (n=6). The results were statistically analyzed by two way ANOVA followed by Bonferroni post tests in case of carrageen induced paw edema and in case of acetic acid induced writhing model where the data were analyzed by one way ANOVA followed by Dunnet s multiple comparison test by using GraphPad Prism software, v 5.0 (trial), (GraphPad Inc, USA). *p < 0.05, **p < 0.01, ***p < compared to control were considered to be statistically significant. RESULTS AND DISCUSSION The spectral and TLC data proved the structure and purity of the synthesized compounds. The synthesized compounds were evaluated for in vitro antibacterial activity by cup plate method. The results were summarized in table 3 including the activity of standard. The compound 6J showed most promising effect whereas compound 6D was found to have moderate effect and other compounds of this series were found to have mild effect against P. vulgaris. The compound 6J was found to have moderate effect against P. aeruginosa whereas compounds 6C, 6D, 6I as well as 6N were found to have mild effect against P. aeruginosa. The compound 6D and 6J were found to be most effective against E. coli whereas compound 6C was found to be moderately effective and compounds 6E, 6F, 6G, 6H and 6I were found to have mild effect against this species. The compound 6D and 6J were found to be moderately effective against S. aureus whereas other compounds of this series were found to have mild effect against S. aureus. The compound 6J also showed moderately inhibitory effect against S. epidermidis whereas other compounds of this series were found to have mild effect against this species except compound 6F. Among the tested compounds the compound 6J showed highest activity against all the test organisms but the activity was less than that of the standard drug Tetracycline in this test concentration. The analgesic activity of the synthesized compounds was evaluated using chemical method. Acetic acid induced writhing test used for detecting both central and peripheral analgesia. The compound 6F and 6J were found to be significant (p < 0.01) in reducing the number of wriths. The compounds 6C, 6E and 6K were also found to be significant (p < 0.05) in reducing the number of wriths, but the activity was comparatively less than IC Value

9 that of 6F and 6J. The standard drug Indomethacin was found to be more potent than the test compounds (Fig.1). Carrageenan induced paw edema is a multimediated phenomenon that liberates diversity of mediators. It is believed to be biphasic, the first phase (1hr) involves the release of serotonin and histamine while the second phase (over 1hr) is mediated by prostaglandins, the cyclooxygenase products and the continuity between the two phases is provided by kinins. The result of this study shows that some of the synthesized compounds have significant anti-inflammatory activity. Among these, the compounds 6E and 6F showed significant anti-inflammatory activity at fourth (p < 0.05) and fifth (p < 0.001) hour whereas they were found to be non-significant at first, second and third hour of the study. Similarly the compound 6J showed significant activity at fourth (p < 0.01) and fifth (p < 0.001) hour and was non-significant at first, second and third hour. Other compounds of this series were found to be non-significant in this test concentration (Fig. 2). ACKNOWLEDGEMENTS The author is very much thankful to the Principal and Management, Roland Institute of Pharmaceutical Sciences, Berhampur for providing necessary facilities and SAIF, Panjab University for characterization of the compounds. REFERENCES 1. Ramesh A, Sridhar SK and Saravanan M. Synthesis and antibacterial screening of hydrazones, Schiff and Mannich Bases of isatin derivatives. Eur J Med Chem. 2001; 36: Sriram D, Bal TR and Yogeeswari P. Synthesis, antiviral and antibacterial activities of isatin mannich bases. Med Chem Res. 2005; 14: Pandeya SN, Sriram D, Nath G and De Clercq E. Synthesis and antimicrobial activity of Schiff and Mannich bases of isatin and its derivatives with pyrimidine. Farmaco. 1999; 54: Aliasghar J, Dariush K, De Clercq E, Chanaz S and Jean MB. Synthesis, Antibacterial, Antifungal and Antiviral Activity. Evaluation of Some New bis- Schiff Bases of Isatin and Their Derivatives. Molecules. 2007; 12: IC Value

10 5. Bal TR, Anand B, Yogeeswari P and Sriram D. Synthesis of anti-hiv activity of isatin β-thiosemicarbazone derivatives. Bioorg Med Chem Lett. 2005; 15: Singh TJ and Gujral PK. Neuropharmacological Actions of Indoline-2,3-dione. Ind. J. Pharmac. 1971; 3(4): Ramesh A and Sridhar SK. Synthesis and Pharmacological activities of Schiff and Hydrazones of isatin derivatives. Indian Drugs. 2001; 38(4): Khan SA, Haque SW, Imran M and Siddiqui N. Synthesis and biological evaluation of some novel Mannich Bases of isatin. J. Pharm. Res. 2006; 5(2): Varma M, Pandeya S N, Singh K N and Stables J P. Anticonvulsant activity of Schiff bases of isatin derivatives. Acta Pharm. 2004; 54: Barry AL: The antimicrobial susceptibility test: principle and practices. Philadelphia, USA, Ghosh MN: Fundamentals of Experimental Pharmacology, Scientific Book Agency, Calcutta, India, Second Edition Vogel GH: Drug discovery and evalution-pharmacological Assay. New York, Springer, For Correspondence: Jnyanaranjan Panda jrpanda77@gmail.com IC Value