Available online at www.pelagiaresearchlibrary.com Der Pharmacia Sinica, 2015, 6(1):11-15 ISS: 0976-8688 CDE (USA): PSIBD Glucose mediated solvent free synthesis of some -(2-oxoindolin-3-ylidene)- benzohydrazide derivatives and their antimicrobial activity G. Pavani Kumari*, M. ageshwara Rao, P. Gopal Krishna Padhy, P. Uday Shankar and P.. Kishore Babu Department of Pharmacy, Andhra University, Maharajah s College of Pharmacy, Vizianagaram (A.P.), India ABSTRACT A simple, efficient and green methodology has been developed for the synthesis of -(2-oxoindolin-3- ylidene)benzohydrazide derivatives using D-glucose as catalyst. All the compounds were synthesized under solid phase synthesis by simple physical grinding of reactants. All the synthesized compounds were evaluated for their antimicrobial activity, where compounds 3(d-f) shown moderate activity and others with none. Key words: Isatin, benzohydrazides, physical grinding, D-glucose and antimicrobial activity. ITRDUCTI Recent literature reveals the importance of nitrogen containing heterocycles as most prominent and dominant group existed in drug history. Several -containing heterocycles found its prominent position in deriving new compounds, which exhibit wide range of biological activities [1-4]. Among the -containing heterocycles isatin derivatives a 150 year old compound with its polyfunctional derivatives found to exhibit diverse therapeutic applications. Several isatin containing molecules found to exhibit excellent antimicrobial activities. Several of these derivatives were reported to possess good antimicrobial activity [5-8]. Recent reports reveal the use of glucose as catalyst in organic reactions, as it acts as active proton acceptor initiating the transformations. It was believed that glucose acts as crown ethers where it affects the molecule in cleaving the proton and by initiating the reaction. Present work deals with the synthesis of some -(2-oxoindolin-3-ylidene)-benzohydrazide derivatives catalysed by D-glucose and its preliminary antimicrobial study. MATERIALS AD METDS Melting points are uncorrected and were determined in open capillary tubes in sulphuric acid bath. TLC was performed on silica gel-g and spotting was done using iodine or UV light. IR spectra were recorded using Perkin- Elmer 1000 instrument in KBr phase, mass spectra on Agilent-LC-MS instrument giving only M +. +1 or M +. -1 values. 11
3 C R 1 R 2 S (a) (b) R C 3 (c) (d) Fig. 1: Compounds representing with good Antimicrobial Activity Synthesis General procedure for the synthesis of 3 under solid phase conditions: A mixture of substituted isatins 1 (10 mmol), D-glucose (10 mol%, 0.18 g) and Substituted benzohydrazides 2 (10 mmol) was taken in a mortar and grounded with pestle. The completion of reaction was monitored by using TLC analysis. After the completion of reaction the mixture was transferred into a beaker consisting of 30 g of crushed ice with stirring. Separated solid was filtered and dried to obtain crude 3(a-i). General procedure for the synthesis of 3 in solution phase: To a stirred solution of substituted isatins 1 (10 mmol) and D-glucose (10 mol%, 0.18g) in 20 ml of ethanol, substituted benzohydrazides 2 (10 mmol) was added at RT. Completion of reaction was monitored by TLC. After completion, the mixture was transferred into beaker consisting of crushed ice. Separated solid was filtered and dried to obtain crude. Recrystallization was performed using ethanol as solvent. Antimicrobial Activity The antimicrobial activity of the synthesized compounds was determined using well diffusion method (Amsterdam et. al., 1996) against different pathogenic reference strains, which were procured from the Microbial Type Culture Collection and Gene Bank (), CSIR-Institute of Microbial Technology, Chandigarh, India. The pathogenic reference strains were seeded on the surface of the media Petri plates, containing Muller-inton agar with 0.1 ml of previously prepared microbial suspensions, which individually containing 1.5 10 8 cfu ml -1 (equal to 0.5 McFarland). Wells of 6.0 mm diameter were prepared in the media plates using a cork borer and the synthesized compounds dissolved in 10% DMS at a dose range of 250-0.97 µg were added in each well, under sterile conditions in a laminar air flow chamber. Standard antibiotic solutions of ciprofloxacin (bacterial strains) and miconazole (Candida albicans) at a dose range of 250-0.97 µg well and the well containing methanol served as 12
positive and negative controls, respectively. The plates were incubated for 24 h at 30 C and the well containing the least concentration showing the inhibition zone is considered as the minimum inhibitory concentration. All experiments were carried out in duplicates and mean values are represented. RESULTS AD DISCUSSI 1-Indole-2,3-dione (1) was condensed with simple benzohydrazide (2), in the presence of D-glucose as catalyst under solvent free conditions by simple physical grinding of reactants using mortar and pestle for 2-3 mins at RT yielded (2-oxoindolin-3-ylidene)benzohydrazide (3) in 95% yield. The same above reaction was also done under solution phase conditions using ethanol as solvent and glucose as catalyst, yielded the product 3 in 75 % at 15 mins. When the above reaction of 1 with 2 attempted without the use of catalyst either in solution phase or solid phase led to the recovery of starting materials. Fig. 2: Solvent free synthesis of 3 from 1 and 2 using D-glucose as catalyst The effect of solvent on the reaction was checked by using other green solvents. Solvents like water, acetic acid, glycerol and PEG-600 was used. Among the solvents used, ethanol gave the better yields compared to other solvents. Yields and time were summarized in table I. Table I: Reaction of 2 with 1 in various green solvents at RT Entry Solvent Time (Mins) Yield (%) 1 Water 40 55 2 Ethanol 4 88 3 Acetic acid 5 75 4 Glycerol 35 60 5 PEG-600 35 70 By the results obtained in performing the reaction of 1 and 2 in solvent phase as well as in solid phase (i.e. Physical grinding), solid phase synthesis proved to be best comparing the yields and time of the reaction. The D-glucose used as catalyst has acted to act as proton acceptor, as it is believed that glucose acts as crown ethers and good proton acceptor. All the derivatives used were performed in solid phase synthesis using glucose as catalyst (Table-II). The minimum concentration of D-glucose as catalyst was also studied by altering the concentrations of glucose from 5, 10, 15, 20 and 25 mole percentages. Where maximum yields were obtained when 10 mol percentage of catalyst used yielding 3. Yields obtained were summarized in table III. 13
Table-II: Synthesis of Isatin derivatives using D-Glucose (10 mol %) as catalyst Product 3(a-i) 3a 3b 3c Structure 2 C 3 M.P ( 0 C) Solution Phase(Et) Time Yield a (min) (%) Time (min) Solid Phase Yield a (%) 212 4 90 2 95 230 11 80 7 89 198 14 85 5 90 3d >250 15 75 8 85 3e >250 120 70 10 90 3f 180 20 60 6 90 3g >250 15 80 15 92 3h >250 25 80 15 85 3i 167 20 80 10 90 a Isolated yields of final products. Table-III: Mole percentage calculated data for D-Glucose Solution Phase in ethanol Solid Phase Entry Catalyst (Mol %) Time (min) Yield (%) Time (min) Yield (%) 1 D-Glucose (5%) 8 72 10 83 2 D-Glucose (10%) 4 90 2 95 3 D-Glucose (15%) 10 80 3 93 4 D-Glucose (20%) 10 85 3 93 5 D-Glucose (30%) 10 87 4 92 6 D-Glucose (40%) 10 85 5 90 Antimicrobial Activity The derived compounds were evaluated for their preliminary antimicrobial activity towards Gram positive and negative bacteria and fungus. The bacterial strains of both Gram positive and negative species were used. The assessments of bacterial activity were measured in minimum inhibitory concentration (MIC) with standard broth dilution assay, where compounds 3(d-f) shown moderate activity and others with none. 14
Entry M. luteus 2470 S. aureus 96 Table IV: Antimicrobial activity of synthesized test compounds S. aureus MLS-16 2940 Minimum inhibitory concentration (µg/ml) B. subtilis 121 E. coli 739 P. aeruginosa 2453 K. planticola 530 C. albicans 3017 3a >250 >250 >250 >250 >250 >250 >250 >250 3b >250 >250 >250 >250 >250 >250 >250 >250 3c >250 >250 31.2 >250 >250 >250 >250 >250 3d 34.8 6.6 5.2 3.9 >250 >250 >250 >250 3e 7.8 18.9 9.1 15.6 >250 >250 >250 >250 3f 11.4 25.6 34.2 3.9 >250 >250 >250 >250 3g >250 >250 31.2 >250 >250 >250 >250 >250 3h >250 >250 >250 15.6 >250 >250 >250 >250 3i >250 >250 >250 >250 >250 >250 >250 >250 Ciprofloxacin Standard 0.9 - Miconazole Standard CCLUSI - 7.8 D-Glucose has been extensively used as a green catalyst in synthesizing the title compounds. The solvent free conditions provided in better yields and in lowering the times of the reaction conditions. Acknowledgments The authors are thankful for the authorities of Maharajah s College of Pharmacy, Vizianagaram, for providing laboratory facilities. REFERECES [1] Ansari KF, Lal C, J. Chem Sci., 2009, 121, 1017. [2] Arrowsmith JE, Cambell SF, Cross PE, Stobes JK, Burges RA and Blackburn KJ, J. Med. Chem., 1986, 29, 1698. [3] Janis RA, Triggle DJ, J. Med. Chem., 1983, 26, 775. [4] Sagyam RR, Padi PR, Ghanta MR and Virmudi, J. eterocycl. Chem., 2007, 44, 923. [5] Bari SB, Agarwal A, Patil UK, J. Sci., 2008, 19(3), 217-221. [6] Jarrahpour AA, Khalili D, Clerqu ED, Molecules, 2007, 12, 1720-1730. [7] Bari S, Talele G, Patel G, Sarangapani M, Iranian J. Pharm. Res., 2006, 4, 249-254. [8] Peter B, ovak T, Ludanyi K, Pete B, Toke L, Keglevich G, Tetrahedron: Assymetry, 1999,10(12), 2373. [9] Peter B, ovak T, Ludanyi K, Pete B, Toke L, Keglevich G, Phosphorus, Sulfur and Silicon Rel. Elements, 2008, 182(10), 2449. [10] Mahesh Kumar D and Dubey PK, Der Pharma Chemica, 2010, 2(4), 224. 15