Res.J.Chem.Sci.______________________________________________Research Journal of Chemical Sciences Vol. 1(3) June (2011) ISSN 2231-606X New Biologically Active Compounds from 1, 3-Diketones Mulongo George, Mbabazi Jolocam2*, Odongkara B., Twinomuhwezi H. and Mpango G.B.Department of Chemistry, Gulu University, P.O. Box 166, Gulu, UGANDA Department of Chemistry, Makerere University, P.O. Box 7062, Kampala, UGANDA Department of Paediatrics, Faculty of Medicine, Gulu University, P.O. Box 166, Gulu, UGANDA Available online at: www.isca.in (Received 30th April 2011, revised 28th May 2011, accepted 07th June 2011) Abstract The ready availability of cyclohexanones and the enhanced reactivity at their -positions render them starting materials of choice in the present study. The synthesis of new compounds of antimicrobial activity was undertaken by the coupling of aromatic amines with 5,5-dimethyl cyclohexan-1,3-dione (dimedone). The products were refluxed with N–benzyl-N-phenylhydrazine in acetic acid. The structures of the products were elucidated using micro- and IR-spectral analyses. They were confirmed using H NMR at 60MHz and TMS as internal standard. The diketone derivatives were tested for their biological activity against gram-positive Cocci and Bacilli, and gram-negative Bacilli. The study showed that the derivatives gave a wide range of activity from inactive to highly active, which proves it to be of fresh pharmaceutical interest. Key words: Dimedone, Antimicrobial activity, Gram-positive Cocci and Bacilli, Gram-negative Bacilli Introduction Cyclohexanedione derivatives far exceed any other alicyclic system in number. In nature the preponderance of cyclohexanedione derivatives over those of other alicyclic systems is overwhelming. The introduction of the second carbonyl group into the cyclohexane ring has a profound effect on the enolisation of the first carbonyl1,2. Cyclohexan-1,3-diones are also completely enolised and possess an acidity comparable to that of carboxylic acids. Cyclohexan-1,3-diones are synthesised by several methods, among which are by reduction of benzenoid compounds, application of the Dieckmann, and by Michael condensation5-7. Cyclohexan-1,3-diones react with amino compounds in various ways, including reactions between simple amines to give enaminones, and with aminothiophenol to give phenothiazinones. They also undergo coupling reactions with diazonium salts to give the 2-arylazo-cyclohexan-1,3-dione. The object of the present study was to synthesise new compounds of 1,3-diketones with diazonium salts and -benzyl--phenyl hydrazine with a view to investigating their biological activity towards various micro-organisms. Material and MethodsMaterials and apparatus: All chemicals and solvents were of reagent grade (Merck, Fluka and Sigma – Aldrich) and used without further purification. All melting points () were determined by the open tube capillary method and quoted uncorrected. The purity of the compounds was controlled by thin layer chromatography (TLC). IR spectra were recorded in KBr pellets on Mattson 5000 FTIR spectrophotometer (USA). The C, H, N, Cl data was estimated by using Perkin–Elmer Instrument (200B, USA). The H NMR spectra of the compounds were measured in CDCl and DMSO-d solution on a DRX – 300MHz spectrometer (Bruker, UK) using TMS as internal standard. Res.J.Chem.Sci.______________________________________________Research Journal of Chemical Sciences Vol. 1(3) June (2011) ISSN 2231-606X Synthesis of 2-arylazo-1, 3-diketones): The appropriate aromatic amines (seeScheme, 15 m mol) in sodium acetate solution (1.23g, 15 m mol, 10 ml) and hydrochloric acid (15 m mol) (warmed where necessary) were diazotized with sodium nitrite solution (1.04g, 15 m mol) at 0–5. This solution was added to 5,5-dimethylcyclohexan-1,3-dione (, 2.1g, 15 m mol) dissolved in ethanol (20 ml) in the cold (0-5C) and left overnight at ambient temperature, filtered and left to dry in a desiccator. The obtained crystals were re-crystallised from ethanol and the melting points together with percentage yields determined. Synthesis of 2-arylazo-1, 3-diketones derivatives (3): 2-arylazocyclohexan-1,3-dione (, 0.002 mol) were dissolved in a mixture of acetic acid (25 ml) and -benzyl--phenyl hydrazine (0.396g, 0.4 ml, 2 m mol), refluxed for 3 – 4 hours, left to cool, filtered and the product re-crystallised from suitable solvents. The coupling process was carried out at temperatures of 0–5 and the products isolated and re-crystallised from methanol and water (50–80% yields). Biological activity tests8-9: Requisite quantities of the liquid agar media were poured into sterile Petri dishes to a depth of 3-4 mm. After solidifying, the liquid media test organism was spread over the solidified agar media and incubated in the Petri dish at 37C for 24 hours to allow the micro-organisms to grow. With the help of a sterile rod, a hole was made on the medium and poured on the known (10 mL of 100 or 1000 µg/mL concentrations) test solution in that hole. The biological activity of the derivatives was evaluated by determining the average diameter of the inhibition zone (figure-1). Results and Discussion Compound (2a) was obtained as yellow crystals in 69% yield, m.p. 143C; IR, Vmax in cm-1: 3069, 1636, 1498 (CH-aromatic), 2951, 2873, 1387 (CH), 1686 (�C=O). Compound (2b) was obtained as yellow crystals in 54.5% yield, m.p 157C, 1415 (RMM, 273.28). Compound (2c) was obtained as light brown crystals in 68%, m.p. 217C, 1415ClO (RMM, 278.74). Compound (2d) was obtained as red crystals in 80% yield, m.p. 178C, 1414 (RMM, 272.27). Compound (2e) was obtained as yellow crystals in 50% yield, m.p. 183C, C1415 (RMM, 272.27). Compound (2f) was obtained as yellow crystals in 59% yield, m.p. 224, C1415 (RMM, 273.28). Compound (2g) was obtained as red crystals in 79% yield, m.p. 224 C1415 (RMM, 273.28); IR, Vmax in cm: 3110 (aromatic), 2956, 2875, 1434, 1332 (-CH), 1697, (�C=O), 747, 774 (=CH). Compound (2h) was obtained as golden yellow crystals in 59% yield, 1410 (RMM, 272.3). Derivatives of 2-arylazo-1, 3-diketones were afforded in good yield. The products (3a – 3h) were isolated, re-crystallised (from mixtures of methanol and water) and obtained in 40 – 60% yields. Compound (3a) was obtained as red crystals in 54% yield, m.p. 81. Anal. calcd for C2226ClO2 (RMM, 465.41): C, 65.72; H, 5.31; N, 11.36; Cl, 14.37; Found: C, 65.35; H, 5.70; N, 11.21; Cl, 14.37%. Compound (3b) was obtained as dark brown crystals in 49% yield, m.p. 100 ; IR, Vmax in cm-1: 3058, 3027, (CH, aromatic), 2953, 2877, 1455, 1358, (CH), 695, 739, (=CH), 792, (C-Cl), 1609, 1492, (CH, aromatic), 738, 693, (=CH). Anal. calcd for 22273 (RMM, 469.53): C, 69.06; H, 5.80; N, 14.92; Found: C, 69.39; H, 5.50; N, 14.81%. Compound (3c) was obtained as orange crystals in 52% yield, m.p 115; IR, Vmax in cm-1: 3539 – 3208, (NH), 3058, 1649, 1598 (CH, aromatic), 2957, 2857, 1456, 1347, (CH), 696 (=CH), 823 (C-Cl). H NMR spectra at 60MHz ( units ppm) showed; 1.033(q, 3H, CH), 0.933(t, 2H, CH), 7.653(s, 5H, CHar), 7.416(s, 1H, CH), 7.369(s, 1H, CHar), 6.234(d, 1H,=NH, -OH), 7.351( s, 5H, CH), 7.351(s, 5H, CH), and 7.149(s,5H, CH). Anal. calcd for C2727ClO (RMM, 458.98): C, 70.65; H, 5.93; N, 12.21; Cl, 7.73; Found: C, 70.55; H, 5.75; N, 12.21; Cl, 7.69%. Compound (3d) was obtained as dark brown crystals in 58% yield, m.p. 98C; H NMR spectra at 60 MHz ( unit in ppm); 0.98(q, 3H, CH), 4.09(t, 2H, CH), 7.589(s, 5H, CH), 7.277(s, 5H, CH), 7.233(s, Res.J.Chem.Sci.______________________________________________Research Journal of Chemical Sciences Vol. 1(3) June (2011) ISSN 2231-606X 5H, CH), 8.019(s, 5H, CH), 7.333(d, 1H, CH), 7.52(s, 5H, CH), 7.24(s, 5H, CH), and 6.712(s, 5H, CH). Anal. calcd for C2826ClO (RMM, 503.98): C, 64.34; H, 5.20; N, 13.90; Cl, 7.04; Found C, 64.61; H, 5.35; N, 13.65; Cl, 7.15%. Compound (3e) was obtained as brown crystals in 40% yield, m.p. 180C; IR, Vmax in cm-1: 3086, 3059, 1609, 1490 (CH, aromatic) 2957, 2929, 1339, (CH), 739, 697, (=CH), 892 (C-Cl). Anal. calcd for 2726ClO (RMM, 503.98): C, 64.34; H, 5.20; N, 13.90; Cl, 7.04; Found C, 64.66; H, 5.29; N, 13.59; Cl, 7.10%. Compound (3f) was obtained as red crystals in 60% yield, m.p. 79C; IR, Vmax in cm; 3555 – 3253 (NH, OH), 3084 – 3003, 1585, 1497, (CH, aromatic) 2957, 2925, 1452, 1323, (CH), 737, 697 (C=H). Anal. calcd for C2727 (RMM, 469.53): C, 69.06; H, 5.80; N, 14.92; Found: C, 69.39; H, 5.75; N, 14.69%. Compound (3g) was obtained as dark brown crystals in 49.2% yield, m.p. 118C; IR, Vmax in cm-1: 3511 – 3404 (NH), 2956, 2929, 1453, 1348, (CH), 1659, 1529, (CH, aromatic), 735, 694 (=CH). Anal. calcd for C2726 (RMM, 469.53): C, 69.21; H, 5.59; N, 14.95; Found C, 69.41; H, 5.65; N, 14.75%. Compound (3h) was obtained as red crystals in 51.2% yield m.p. 112C, RMM, 468.53. Anal.calcd for C2828: C, 71.77; H, 6.02; N, 11.96; Found: C, 71.91; H, 6.11; N, 12.01%. Biological screening: The 2-arylazo-1,3-diketone derivatives () were examined in vitro against bacterial species which included gram-positive Cocci, gram-positive Bacilli and gram-negative Bacilli. The photographs provided (figure 1) represent the situations that prevailed. Tables 1 and 2 show the spectral data of antimicrobial activity of the synthesised compounds (3a – 3h) at 100 and 1000 µg/mL concentration levels, respectively, against the micro-organisms. The test results presented in table 1 suggested that compounds (3b 3f, and 3g) showed high antimicrobial activity (i.e. at 100µg/ml) against all the tested micro-organisms. This is attributed to the presence of the nitro (-NO) group. The reactivity might be strongly dependent on the electronic richness of the nitrogen atoms and on the steric hindrance of the substituent10,11. Compounds (3a and 3c) with a chlorine atom onthe arylazo- group showed high antimicrobial activity against the grampositive Cocci and gram-positive Bacilli. On the contrary, both compounds (3a and 3c) were inactive against all the gram-negative Bacilli. Compounds (3h) with a carboxyl group on the arylazo moiety showed relative biological activity on all the micro-organisms tested. Compound (3h) was highly active against all the gram-positive Cocci and gram-positive Bacilli. The test results presented on gram-negative Bacilli by compound (3h) ranged from inactive to moderate activity against the tested micro-organisms. It showed moderate activity on Aerobacterium klebsiella, Bacillus Arizona, Bacillus Proteus, Bacillus Pseudomonas, Escherichia Coliand Salmonella Paratyphi A. Compound (3h) was inactive against Salmonella Paratyphi B, Salmonella Paratyphi C, Shigella flexneri and Shigella sonnei. Compound (3d) was highly active against both gram-positive Cocci and gram-positive Bacilli. Compound (3f) was biologically inactive against Aerobacterium klebsiella and Bacillus Arizona, and moderately active against the rest of the gram-negative Bacilli. Compound (3e) was moderately active against all the tested gram-negative Bacilli,highly active on gram-positive Bacilli. The results show the effect of the nitro and chlorine substituents on the biological activity. When the concentration was increased to 1000 µg/ml, there was a slight change in the antimicrobial activity of most of the products (3a – 3h) (table 2). It should be noted that with exception of product (3a) which was highly active against Bacillus subtilis, product (3e), became highly active against Sarcina lutea Bacillus permal and subtilis. Products (3e and 3h) were inactive against Aerobacterium klebsiella, Bacillus Arizona pseudomonas and proteus, Salmonella and proteus Salmonella paratyphi A, B and C, Shigella flexneri and sonnei, and Escherichia coli (table 2). Res.J.Chem.Sci.______________________________________________Research Journal of Chemical Sciences Vol. 1(3) June (2011) ISSN 2231-606X Conclusion This study shows that 5,5-dimethylcyclohexan-1,3-dione represents an adaptable starting material for the preparation of new biologically active compounds that might prove to be of pharmaceutical interest. Some of the products however exhibited total inactivity towards the tested micro-organisms. Acknowledgements Our sincere thanks go to Dr. A. Metwally (formerly at Makerere University) of Faculty of Science, Mansoura University, Egypt for providing elemental, IR and NMR analyses. We also wish to express our gratitude to Chemistry Department, Makerere University (Uganda) for laboratory facilities. References 1. French H.S. and Holden M.E.T., Absorption Spectra of Certain -Unsaturated Ketones, including Benzal Compounds, J. Amer. Chem. Soc., 67, 1239 (1945) 2. Schwarzenbach G. and Wittwer Ch., Über das Keto-Enol-Gleichgewicht bei cyclischen Diketonen, Helv. Chim. Acta 30, 663 (1947) 3. Conroy H., Picrotoxin. II., The Skeleton of Picrotoxinin. The Total Synthesis of dl Picrotoxadiene, J. Amer. Chem. Soc., 74, 3046 1952) 4. Meek E. G., Turnbull J. H. and Wilson W., Alicyclic compounds. Part II. The preparation of cyclohexane-1:3-diones and their enol ethers, J. Chem. Soc. 811 (1953) 5. Shriner R.L. and Todd H.R., 1,3-Cyclohexadione-5,5-dimethyl, Org. Synth., II, 200 (1943)6. Frank R.L. and Hall H.K., Monocyclic Terpenes from Cyclic 1,3-Diketones, J. Chem. Soc. 72, 1645 (1950)7. Pal B.C., Dehydration of Phenylethylcyclohexanol-3, J. Amer. Chem. Soc., 11, 3397 (1955)8. Chitra M., Shyamala D.C.S. and Sukumar E., Antibacterial Activity of Embelin, Filotropia, 74, 401 (2003)9. Manjudar S.H., Chakra G.S. and Kulkarni K.S., Medicinal Potential of Semecarpus anacardium Nut., J. Herb. Med. Toxicol.,, 9 (2008) 10. Cousinité S., Gressier M., Alphonse P. and Menu M.J., Silica-Based Nanohybrids containing Dipyridine, Urethan, or Urea Derivatives, Chem. Matters, 19, 6492 (2007) 11.Bares J., Richard P., Meunier P., Pirio N., Padelkova Z., Cernoisck Z., Cysarova I. and Ruzicka, A., Reactions of C,N-chelated Tin(II) and Lead(II) Compounds with Zirconocene Dichloride Derivatives, Organometallics, 28, 3105 (2009) Res.J.Chem.Sci.______________________________________________Research Journal of Chemical Sciences Vol. 1(3) June (2011) ISSN 2231-606X SCHEME R1 R2 R3 R4 R5 R1 R2 R3 R4 R5 +N2 N=N O O H3C H3C O O H3C H3C R1 R2 R3 R4 R5 N=N O OH H3C H3C R1 R2 R3 R4 R5 N=N OH OH H3C H3C N O O H3C H3C H R1 R2 R3 R4 R5 N N O H3C H3C N R1 R2 R3 R4 R5 N(CH)C C6H5 1 2 3 An illustrated reaction pathway for the synthesis of antimicrobial products using dimedone (), aromatic amines and -benzyl-phenyl hydrazine 3 i) R=R=R=H, R=R=Cl (2a) (3a) ii) R=R=R=R=H, R=NO (2b) (3b) iii) R1==R=R=H, R=Cl (2c) (3c) iv) R=R=R=H, R=NO, R=Cl (2d) (3d) v) R=R=R=H, R=Cl, R=NO (2e) (3e) vi) R=R=R=R=H, R=NO (2f) (3f) vii) R=R=R=R=H, R=NO (2g) (3g) viii) R=R=R=R=H, R=COOH (2h) (3h)   Res.J.Chem.Sci.______________________________________________Research Journal of Chemical Sciences Vol. 1(3) June (2011) ISSN 2231-606X Figure 1 Table-1: Collective data showing the spectra of antimicrobial activity of the compounds (3a – 3h) at 100µg/ml concentration level against micro-organisms used Test strains of micro-organisms Test Compounds 3a 3b 3c 3d 3e 3f 3g 3h A)Gram-Positive Cocci1.Staphylococcus aureus 2.Staphylococcus epidermis 3.Sarcina lutea B)Gram- Positive Bacilli1.Bacillus permal 2.Bacillus subtilis C)Gram-Negative Bacilli1.Aerobacterium klebsiella 2.Bacillus Arizona 3.Bacillus proteus 4.Bacillus pseudomonas 5.Escherichia coli 6.Salmonella paratyphi A 7.Salmonella paratyphi B 8.Salmonella paratyphi C 9.Shigella flexneri 10.Shigella sonnei + + + + + - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + - - - - - - - - - - + + + + + - - ± ± ± ± ± ± ± ± ± ± + + + ± ± ± ± ± ± ± ± ± ± + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ± ± ± ± ± ± - - - - N.B. (+) High growth (inactive), (±) Moderate growth (moderate activity), (-) No growth (Highly active)   Res.J.Chem.Sci.______________________________________________Research Journal of Chemical Sciences Vol. 1(3) June (2011) ISSN 2231-606X Table-2: Collective data showing the spectra of antimicrobial activity of the compounds (3a – 3h) at 1000µg/ml concentration level against micro-organisms used Test strains of micro-organisms Test Compounds 3a 3b 3c 3d 3e 3f 3g 3h A)Gram-Positive Cocci1.Staphylococcus aureus 2.Staphylococcus epidermis 3.Sarcina lutea B)Gram- Positive Bacilli1.Bacillus permal 2.Bacillus subtilis C)Gram-Negative Bacilli1.Aerobacterium klebsiella 2.Bacillus Arizona 3.Bacillus proteus 4.Bacillus pseudomonas 5.Escherichia coli 6.Salmonella paratyphi A 7.Salmonella paratyphi B 8.Salmonella paratyphi C 9.Shigella flexneri 10.Shigella sonnei ± ± ± + + - - - - - - - - - - ± ± ± ± ± + + + + + + + + + + + + + ± + - - - - - - - - - - + ± + ± + - - - - - - ± ± - - ± ± - - - - - - - - - - - - - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ± ± ± + + - - - - - - - - - - N.B. (+) High growth (inactive), (±) Moderate growth (moderate activity), (-) No growth (Highly active) \n