untitled European Journal of Chemistry 2 (2) (2011) 243‐250 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2011 EURJCHEM DOI:10.5155/eurjchem.2.2.243‐250.257 European Journal of Chemistry Journal homepage: www.eurjchem.com Synthesis and characterization of a novel series of benzenesulfonylurea and thiourea derivatives of 2H‐pyran and 2H‐pyridine‐2‐ones as antibacterial, antimycobacterial and antifungal agents Hassan Mostafa Faidallah*, Khalid Ali Khan and Abdullah Mohammad Asiri Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah‐21589, Saudi Arabia *Corresponding author at: Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah‐21589, Saudi Arabia. Tel.: +966‐567743180; fax: +966‐2‐6952293. E‐mail address: hfaidallahm@hotmail.com (H.M. Faidallah). ARTICLE INFORMATION ABSTRACT Received: 19 August 2010 Received in revised form: 28 October 2010 Accepted: 04 November 2010 Online: 30 June 2011 KEYWORDS Arylhydrazines reacted with dehydroacetic acid (1) to give the corresponding 2H‐pyran‐2‐one hydrazones (2), which on treatment with hydrazine hydrate afforded the corresponding 1‐amino‐2H‐pyridin‐2‐ones (3). Reaction of 3 with nitrous acid, aromatic aldehyde and substituted benzenesulfonyl chlorides yielded the corresponding 2H‐pyridine‐2‐one derivatives. A series of urea and thiourea derivatives were also prepared. Some of these compounds have shown significant antibacterial and mild to moderate antimycobacterial and antifungal activities. 2H‐pyran‐2‐one hydrazones 1‐Amino‐2H‐pyridin‐2‐ones Benzenesulfonylurea derivatives Thiourea derivatives Antimicrobial activity Antimycobacterial activity 1. Introduction In recent years, the number of life threatening infections caused by multi‐drug resistant Gram positive and Gram negative pathogenic bacteria has reached an alarming level in many countries around the world; consequently the need for the synthesis of novel antibiotics is a reality. However, investigations in the chemistry and biology of 2‐pyrones have become that they constitute an essential pharmacophore in many naturally occurring and biologically active agents [1]. Literature survey revealed that a simple change in the substitution pattern on the 2‐pyrone ring often leads to diverse biological activities. For example, some 2‐pyrone derivatives are yeast lipase (CRLI), cholesterol esterase (CRL3) inhibitors [3], anti‐ inflammatory [4,5] and immune suppressive [6] agents. Particular attention has been focused on the distinctive chemotherapeutic activity of 2‐pyrones as cytotoxic agents against some human cancer cell lines [7,8], anti‐HIV [9,10] and potential antimicrobial agents [11,12]. On the other hand, a broad spectrum of pharmacological properties has been ascribed to the structurally‐relevant 2‐pyridone derivatives. In addition to their pronounced effects on the cardiovascular system as cardiotonic [13,14], calcium channel blocking [15] and tissue factor VII inhibitory agents [16], some pyridones were reported to exhibit potential antimicrobial [17‐20], antitubercular [21,22], antiamoebic [23], antiparasitic [24], antimalarial [25], antifungal and antiviral [26‐28] activities. N‐Substituted pyrazolyl‐benzensulfonamides are known to show COX‐2 selective inhibition [29], thioureide analogs of 2‐phenetilbenzoic acid and pyridazine derivatives carrying urea, thiourea, and sulfonamide moieties were reported to exhibit potential antimicrobial and antifungal activities [30‐34]. Hence urea, thiourea and sulfonamides are considered, one of the active areas of medicinal research where efforts are focused in order to have new and better therapeutic agents. On the basis of these findings, the aim of this study was to synthesize the hybrid molecule through a combination of pyridone and hydrazide pharmacophores in one structure with the hope of obtaining better antibacterial and/or antifungal agents. Therefore, we have synthesized a series of new 2‐ pyrone and their 2‐pyridone analogs (Scheme 1 and 2), carrying urea, thiourea and sulfonamide groups in order to investigate their antibacterial and antifungal activities. 2. Experimental 2.1. Synthesis Melting points were determined in open glass capillaries on a Gallenkamp melting point apparatus and were uncorrected. The infrared (IR) spectra were recorded on Perkin‐Elmer 297 infrared spectrophotometer using the plate technique. The 1H NMR and 13C NMR spectra were recorded on Bruker DPX‐400‐ FT spectrometer using CDCl3 and DMSO‐d6 as a solvent and tetramethylsilane as the internal standard. Elemental analyses were performed at the Microanalytical Unit, Faculty of Science, Cairo University, Cairo, Egypt. Follow up of the reactions and checking the homogeneity of the compounds were made by Thin layer chromatography (TLC) on silica gel‐protected aluminum sheets (Type 60 F254, Merck) and the spots were detected by exposure to UV lamp at λ = 254 nm. Biological testing was performed in the Faculty of Science, University of Alexandria, Egypt. Dehydroacetic acid was purchased from 244 Faidallah et al. / European Journal of Chemistry 2 (2) (2011) 243‐250 Scheme 1 Scheme 2 Aldrich Chemical Co., Milwaukee, USA and was used without further purification. The characterization data of the synthesized compounds (2‐6) (Scheme 1 and 2) are given in Table 1‐3. 2.1.1. 4‐Hydroxy‐6‐methyl‐3‐[1‐(substituted‐hydrazono) ethyl]pyran‐2‐ones (2a‐e) To a solution of 1 (10 mmol) in benzene (20 mL) was added the appropriate aryl hydrazine (10 mmol). The mixture was refluxed for 15 min and allowed to stand at room temperature for 2 h. After the mixture was cooled, the hydrazone was collected and recrystallized from ethanol. 2.1.2. 1‐Amino‐4‐hydroxy‐6‐methyl‐3‐[1‐(substituted‐ hydrazono)ethyl]‐1H‐pyridin‐2‐ones (3a‐d) A solution of the appropriate hydrazone 2 (10 mmol) in ethanol (20 mL) was refluxed with hydrazine hydrate 98% (1.1 mL, 22 mmol) for 2 h. The reaction mixture was concentrated to half its volume and allowed to cool. The solid product separated was filtered, washed with cold ethanol and recrystallized from ethanol. Faidallah et al. / European Journal of Chemistry 2 (2) (2011) 243‐250 245 Table 1. Characterization data of compounds (2‐6). Comp. R R1 or R2 Yield (%) M.p. (oC) Mol. Formula Calculated % Found % C H N S C H N S 2a C6H5 82 212a C14H14N2O3 65.11 5.46 10.85 56.21 5.54 10.94 2b p‐H2NSO2C₆H₄ 85 255 C14H15N3O5S 49.84 4.48 12.46 9.51 49.72 4.51 12.47 9.62 2c p‐ClC₆H₄ 78 218 C14H13ClN2O3 57.44 4.48 9.57 57.45 4.53 9.62 2d p‐FC₆H₄ 74 212 C14H13FN2O3 60.87 4.74 14.01 60.81 4.76 13.98 2e p‐NO2C₆H₄ 78 216 C14H13N3O5 55.45 4.32 13.86 55.48 4.42 13.93 3a C6H5 69 233 C14H16N4O2 61.75 5.92 20.58 61.84 6.02 20.67 3b p‐H2NSO2C₆H₄ 70 192 C14H17N5O4S 47.85 4.88 19.93 9.13 47.92 4.92 20.11 9.23 3c p‐ClC₆H₄ 63 250 C14H15ClN4O2 54.82 4.93 18.26 54.97 5.04 18.18 3d p‐FC₆H₄ 60 216 C14H15FN4O2 57.92 5.21 19.31 57.84 5.12 19.42 4a C6H5 204 C14H15N3O2 65.35 5.88 16.33 65.45 5.97 16.45 4b p‐ClC₆H₄ 231 C14H14ClN3O2 57.64 4.84 14.41 57.81 4.93 14.38 4c p‐FC₆H₄ 223 C14H14FN3O2 61.08 5.13 15.26 61.12 5.16 15.37 5a C6H5 Cyclohexyl 68 169 C21H27N5O3 63.46 6.85 17.62 63.58 6.92 17.74 5b C6H5 C6H5 70 205 C21H21N5O3 64.44 5.41 17.89 64.54 5.38 17.92 5c C6H5 p‐ClC₆H₄ 68 214 C21H20ClN5O3 59.23 4.73 16.44 59.34 4.82 16.54 5d p‐ClC₆H₄ Cyclohexyl 72 264 C21H26ClN5O3 58.41 6.07 16.21 58.33 6.18 16.38 5e p‐ClC₆H₄ C6H5 74 249 C21H20ClN5O3 59.23 4.73 16.44 59.28 4.84 16.36 5f p‐ClC₆H₄ p‐ClC₆H₄ 70 259 C21H19Cl2N5O3 54.79 4.16 15.21 54.82 4.27 15.22 5g p‐FC₆H₄ C6H5 66 218 C21H20FN5O3 61.61 4.92 17.11 61.73 5.11 17.31 5h p‐FC₆H₄ p‐ClC₆H₄ 68 248 C21H19ClFN5O3 56.83 4.31 15.78 56.71 4.23 15.87 6a C6H5 CH3 69 260 C16H19N5O2S 55.63 5.54 20.27 9.28 55.54 5.48 20.32 9.31 6b C6H5 CH3(CH3)2CH2 66 249 C19H25N5O2S 58.89 6.51 18.07 8.27 58.97 6.58 17.18 8.38 6c C6H5 C6H5 72 254 C21H21N5O2S 61.91 5.19 17.19 7.87 61.87 5.23 16.76 7.94 6d C6H5 p‐CH3C₆H₄ 70 262 C22H23N5O2S 62.69 5.51 16.61 7.61 62.79 5.62 16.58 7.62 6e C6H5 p‐FC₆H₄ 69 216 C21H20FN5O2S 59.28 4.74 16.46 7.54 59.41 4.78 16.48 7.53 6f p‐ClC₆H₄ C6H5 72 266 C21H20ClN5O2S 57.07 4.56 15.85 7.26 57.14 4.66 15.92 7.34 6g p‐ClC₆H₄ p‐CH3C₆H₄ 74 241 C22H22ClN5O2S 57.95 4.86 15.36 7.03 58.12 4.89 15.44 7.11 6h p‐ClC₆H₄ p‐FC₆H₄ 68 257 C21H19ClFN5O2S 54.84 4.16 15.23 6.97 54.86 4.23 15.41 7.04 6i p‐FC₆H₄ C6H5 67 252 C21H20FN5O2S 59.28 4.74 16.46 7.54 59.37 4.86 16.55 7.63 7a C6H5 C6H5 72 238 C21H20N4O2 69.98 5.59 15.55 70.11 5.62 15.64 7b C6H5 p‐CH3C₆H₄ 74 226 C22H22N4O2 70.57 5.92 14.96 70.48 6.02 15.04 7c C6H5 p‐CH3OC₆H₄ 68 230 C22H22N4O3 67.68 5.68 14.35 67.79 5.74 14.42 7d C6H5 p‐ClC₆H₄ 72 242 C21H19ClN4O2 63.88 4.85 14.19 63.92 4.94 14.25 7e C6H5 2‐Thienyl 76 235 C19H18N4O2S 62.28 4.95 15.29 62.31 5.11 15.41 7f p‐ClC₆H₄ C6H5 78 199 C21H19ClN4O2 63.88 4.85 14.19 63.77 4.76 14.28 7g p‐ClC₆H₄ p‐ClC₆H₄ 76 216 C21H18Cl2N4O2 58.75 4.23 13.05 58.84 4.24 13.16 7h p‐ClC₆H₄ 2‐Thienyl 72 222 C19H17ClN4O2S 56.93 4.27 13.98 57.12 4.39 14.01 7i p‐FC₆H₄ C6H5 69 220 C21H19FN4O2 66.66 5.06 14.81 66.75 5.08 14.92 7j p‐FC₆H₄ p‐ClC₆H₄ 66 218 C21H18ClFN4O2 61.09 4.39 13.57 61.12 4.42 13.64 7k p‐FC₆H₄ 2‐Thienyl 68 209 C19H17FN4O2S 59.36 4.46 14.57 59.43 4.61 15.46 8a C6H5 C6H5 76 178 C20H20N4O4S 58.24 4.89 13.58 7.77 58.13 4.91 13.62 7.86 8b C6H5 p‐CH3C₆H₄ 78 162 C21H22N4O4S 59.14 5.21 13.14 7.52 59.24 5.32 13.24 7.45 8c p‐ClC₆H₄ C6H5 77 136 C20H19ClN4O4S 53.75 4.29 12.54 7.17 53.88 4.41 12.63 7.21 8d p‐FC₆H₄ C6H5 76 138 C20H19FN4O4S 55.81 4.45 13.02 7.45 55.92 4.61 13.12 7.56 8e p‐FC₆H₄ p‐CH3C₆H₄ 78 164 C21H21FN4O4S 56.75 4.76 12.61 7.21 56.82 4.79 12.75 7.34 9a Cyclohexyl 74 172 C21H26N4O6S 54.53 5.67 12.11 6.93 54.64 5.73 12.21 7.11 9b C6H5 72 192 C21H20N4O6S 55.26 4.42 12.27 7.02 55.37 4.43 12.36 7.15 9c p‐ClC₆H₄ 78 163 C21H19ClN4O6S 51.38 3.91 11.41 6.53 51.49 4.02 11.56 6.48 10a CH3 70 189 C16H18N4O5S2 46.82 4.42 13.65 15.62 46.88 4.34 13.78 15.74 10b Cyclohexyl 72 199 C21H26N4O5S2 52.71 5.48 11.71 13.41 52.82 5.52 11.82 13.48 10c CH2C6H5 70 168 C22H22N4O5S2 54.31 4.56 11.51 13.18 54.42 4.64 15.68 13.02 10d C6H5 70 152 C21H20N4O5S2 53.38 4.27 11.86 13.57 53.51 4.38 11.99 13.43 10e p‐CH3C₆H₄ 77 148 C22H22N4O5S2 54.31 4.56 11.51 13.18 54.44 4.62 11.54 13.08 10f p‐FC₆H₄ 72 151 C21H19FN4O5S2 51.42 3.91 11.42 13.07 51.56 4.14 11.58 13.15 11a Cyclohexyl 72 215 C28H39N7O6S 55.89 6.53 16.29 5.33 55.92 6.58 16.41 5.38 11b C6H5 70 243 C28H27N7O6S 57.04 4.62 16.63 5.44 57.13 4.73 16.74 5.42 11c p‐ClC₆H₄ 72 236 C28H25Cl2N7O6S 51.07 3.83 14.89 4.87 51.12 3.94 14.78 5.96 12a Cyclohexyl 73 229 C28H39N7O4S3 53.06 6.21 15.47 15.18 53.18 6.12 15.52 15.09 12b CH3 70 237 C18H23N7O4S3 43.45 4.66 19.71 19.33 43.61 4.75 19.83 19.32 12c CH3(CH2)2CH2 68 244 C24H35N7O4S3 49.55 6.06 16.85 16.53 49.64 6.17 16.92 16.64 12d C6H5 70 242 C28H27N7O4S3 54.09 4.38 15.77 15.47 54.12 4.42 15.91 15.52 12e p‐CH3C₆H₄ 69 250 C30H31N7O4S3 55.45 4.81 15.09 14.81 55.54 4.96 15.13 14.71 12f p‐FC₆H₄ 67 232 C28H25F2N7O4S3 51.13 3.83 14.91 14.63 51.24 3.74 15.04 14.68 aLit. [7] M.p.: 209 oC. 2.1.3. 4‐Hydroxy‐6‐methyl‐3‐[1‐(substituted‐hydrazono) ethyl]‐1H‐pyridon‐2‐ones (4a‐c) A solution of the appropriate 1‐amino‐2‐pyridone derivative 3 (2 mmol) in glacial acetic acid (10 mL) was treated (drop wise) with an aqueous solution of sodium nitrite (0.5 g) with stirring for 2 h. The reaction mixture was then poured onto ice cold water and the separated solid was recrystallized from ethanol. 2.1.4. 1‐{4‐Hydroxy‐6‐methyl‐2‐oxo‐3‐[1‐(substituted‐ hydrazono)ethyl]‐2H‐pyridin‐1‐yl}‐3‐substituted ureas (5a‐ h) To the solution to the appropriate aminopyridone 3 (2 mmol) in pyridine (10 mL) was added the appropriate isocyanate (2.1 mmol), and the reaction mixture was heated under reflux for 8 h. After cooling to room temperature, the reaction mixture was poured on crushed ice and the separated solid product was filtered, washed thoroughly with water, dried and crystallized from ethanol. 246 Faidallah et al. / European Journal of Chemistry 2 (2) (2011) 243‐250 Table 2. 1H NMR spectral data (/ppm)a of compounds (2‐10). Comp. R R1 or R2 CH3 H‐5 Ar‐H NH OH Others (s, 3H) (s, 1H) (m) (s, 1H) (s, 1H) 2a C6H5 1.03, 1.52 5.99 6.62‐7.01 7.85 14.28 2b p‐NH2SO2C₆H₄ 1.17, 1.62 6.22 6.85‐7.72 8.02 14.46 2c p‐ClC₆H₄ 0.95, 1.41 6.36 6.45‐6.98 7.62 14.82 2d p‐FC₆H₄ 1.05, 1.38 6.24 6.48‐6.76 7.71 14.26 2e p‐NO2C₆H₄ 1.08, 1.47 6.21 6.25‐7.01 8.96 15.02 3a C6H5 1.13, 1.43 5.02 6.62‐7.12 7.58 14.52 3b p‐NH2SO2C₆H₄ 1.12, 1.41 5.34 6.47‐7.68 7.94 13.84 4a C6H5 1.03, 1.54 5.52 6.62‐7.68 7.72, 8.12 14.92 4b p‐ClC₆H₄ 1.12, 1.42 5.34 6.71‐7.03 7.64, 8.06 15.03 4c p‐FC₆H₄ 0.98, 1.38 5.28 6.74‐7.26 7.52, 8.03 14.74 5a C6H5 Cyclohexyl 1.10, 1.55 5.46 6.81‐7.71 6.62, 6.75, 8.57 13.98 1.32‐2.63 (m, 11H, cyclohexyl) 5b C6H5 C6H5 1.04, 1.52 5.29 6.93‐7.68 6.74, 6.84, 8.61 14.05 5e p‐ClC₆H₄ C6H5 1.18, 1.49 5.34 7.01‐7.79 6.88, 6.94, 8.43 13.24 5f p‐ClC₆H₄ p‐ClC₆H₄ 1.05, 1.54 5.22 6.74‐7.84 7.26, 8.64, 8.94 14.46 5g p‐FC₆H₄ C6H5 0.98, 1.37 5.34 6.62‐7.68 7.85, 8.32, 8.75 14.04 6a C6H5 CH3 0.95, 1.41 5.29 6.63‐7.02 7.24, 7.56, 8.66 15.13 3.45 (d, 3H, NCH3) 6c C6H5 C6H5 1.01, 1.46 5.38 6.72‐7.41 7.64, 7.83, 9.12 14.28 6d C6H5 p‐CH3C₆H₄ 1.24, 1.53 5.42 6.83‐7.78 7.83, 8.72 12.98 3.13 (s, 3H, CH3) 6g p‐ClC₆H₄ p‐CH3C₆H₄ 1.18, 1.49 5.39 6.64‐7.76 7.94, 8.82 13.56 3.08 (s, 3H, CH3) 6i p‐FC₆H₄ C6H5 1.07, 1.33 5.26 6.56‐7.64 7.74, 7.92, 8.45 14.03 7a C6H5 C6H5 1.12. 1.46 5.28 6.78‐7.75 7.95 12.98 8.21 (s, 1H, CH=) 7b C6H5 p‐CH3C₆H₄ 1.23, 1.50 5.36 6.67‐7.58 8.38 13.56 3.14 (s, 3H, CH3), 8.16 (s, 1H, CH=) 7c C6H5 p‐CH3OC₆H₄ 1.25, 1.46 5.42 6.89‐7.79 8.61 13.42 3.85 (s, 3H, CH3O), 8.12 (s, 1H, CH=) 7f p‐ClC₆H₄ C6H5 1.18, 1.38 5.39 6.77‐7.68 8.56 14.05 8.24 (s, 1H, CH=) 8a C6H5 C6H5 1.20, 1.36 5.25 6.80‐7.72 8.05, 8.42 14.36 8e p‐FC₆H₄ p‐CH3C₆H₄ 1.22, 1.48 5.34 6.74‐7.81 8.16, 8.89 13.88 3.12 (s, 3H, CH3) 9a Cyclohexyl 0.98, 1.50 6.22 6.68‐7.42 7.82, 8.05, 8.56 12.98 1.35‐2.84 (m, 11H, cyclohexyl) 9b C6H5 1.15, 1.54 6.34 6.72‐7.64 8.12, 8.34, 8.74 14.45 10a CH3 1.12, 1.49 6.28 6.84‐7.36 8.06, 8.42, 8.68 13.92 3.38 (d, 3H, CH3) 10c CH2C6H5 1.20, 1.56 6.42 6.94‐7.65 7.98, 8.14, 8.78 12.78 4.57 (d, 2H, CH2) 10d C6H5 1.17, 1.65 6.27 6.68‐7.62 7.86, 8.03, 8.66 14.15 11a Cyclohexyl 1.14, 1.68 5.28 6.72‐7.34 7.94, 8.05, 8.56, 8.84 14.25 1.22‐3.05 (m, 22H, 2cyclohexyl) 11b C6H5 1.22, 1.56 5.32 6.64‐7.58 7.82, 8.14, 8.35, 8.82 13.98 12a Cyclohexyl 1.02, 1.59 5.41 6.76‐7.49 8.01, 8.25, 8.67, 9.11 13.67 1.18‐3.14 (m, 22H, 2cyclohexyl) 12d C6H5 1.06, 1.63 5.29 6.65‐7.78 8.14, 8.42, 8.90, 9.24 14.18 12e p‐CH3C₆H₄ 1.17, 1.65 5.34 6.89‐7.82 7.98, 8.06, 8.67, 8.98 14.28 3.12 (s, 3H, CH3), 3.18 (s, 3H, CH3) a Solution in a mixture of CDCl3 and DMSO‐d6. 2.1.5. N1‐{4‐Hydroxy‐6‐methyl‐2‐oxo‐3‐[1‐(substituted‐ hydrazono)ethyl]‐2H‐pyridin‐1‐yl}‐N3‐substituted thioureas (6a‐i) To a solution of the appropriate 3 derivative (2 mmol) in pyridine (10 mL) was added the appropriate isothiocyanate (2.1 mmol), and the reaction mixture was refluxed for 5 h and worked up as above. 2.1.6. 1‐(Arylideneamino)‐4‐hydroxy‐6‐methyl‐3‐[1‐ (substituted‐hydrozono)ethyl]‐1H‐pyridin‐2‐ones (7a‐k) A mixture of the appropriate 3 derivate (1 mmol) and the appropriate aldehyde (1 mmol) in benzene (10 mL) was refluxed for 5 h. Excess solvent was removed under reduced pressure, and the remaining residue was treated with methanol, filtered and recrystallized from ethanol. 2.1.7. 1‐(N‐substituted benzenesulfonylamino)‐4‐hydroxy‐6‐ methyl‐3‐[1‐substituted‐hydrazono)ethyl]‐1H‐pyridin‐2‐ ones (8a‐e) To a solution of the appropriate 3 derivative (1 mmol) in pyridine (10 mL) was added the appropriate benzenesulfonyl chloride derivative (1.1 mmol), and the mixture was heated under reflux for 4 h. After cooling to room temperature, the reaction mixture was poured on crushed ice and the separated solid product was filtered, washed thoroughly with water, dried and crystallized from a mixture of ethanol and benzene. 2.1.8. General procedure for the preparation of N1‐ substituted N3‐{4‐[(4hydroxy‐3‐methyl‐2‐oxopyran‐3‐yl) ethylidenehydrazino]benzenesulfonyl]}ureas and thioureas (9a‐c, 10a‐f) To a solution of 2b (0.34 g, 1 mmol) in pyridine (10 mL) was added the appropriate isocyanate or isothiocyanate (1.1 mmol), and the reaction mixture was heated under reflux for 5‐ 8 h. After cooling to room temperature, the reaction mixture was poured on crushed ice and the separated solid product was filtered, washed thoroughly with water, dried and crystallized from ethanol. 2.1.9. General procedure for the preparation of 2‐pyridone diureas and dithioureas derivatives (11a‐c, 12a‐f) To a solution of 3b (0.35g, 2 mmol) in pyridine (10 mL) was added the appropriate isocyanate or isothiocyanate (4.2 mmol), and the reaction mixture was heated under reflux for 6‐ 10 h. After cooling to room temperature, the reaction mixture was poured on crushed ice and the separated solid product was filtered, washed thoroughly with water, dried and crystallized from the dimethylformamide containing few drops of water. 2.2. Antimicrobial screening 2.2.1. Inhibition zone (IZ) measurement Standard sterilized filter paper discs (5 mm diameter) impregnated with a solution of the test compound in dimethyl sulfoxide (DMSO) (1 mg/mL) was placed on an agar plate seeded with the appropriate test organism in triplicates. The utilized test organisms were: Staphylococcus aureus (ATCC 25923) and Bacillus subtilis ( ATCC 6051) as examples of Gram positive bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) as examples of Gram negative bacteria, Candida albicans (ATCC 10231) and Aspergillus niger (recultured) as representatives of fungi. Ampicillin trihydrate and clotrimazole were used as standard antibacterial and antifungal agents, respectively. DMSO alone Faidallah et al. / European Journal of Chemistry 2 (2) (2011) 243‐250 247 Table 3. 13C NMR spectral data (/ppm)a of compounds (2‐10). Comp. R R1 or R2 CH3 Pyrone or Pyridone C ArC C=N Others 2a C6H5 13.5, 22.8 92.0, 102.8, 144.1, 115.1, 118.5, 129.3, 140.5 155.6 161.0, 175.2 2b p‐NH2SO2C₆H₄ 12.7, 20.9 94.3, 105.2, 145.4, 117.2, 126.3, 129.4, 137.4 152.4 160.7, 174.1 2c p‐FC₆H₄ 11.8, 21.4 95.2, 103.7, 143.1, 116.3, 120.2, 142.3, 152.1 156.3 160.9, 172.3 3a C6H5 12.0, 20.3 94.4, 103.7, 133.7, 120.2, 126.3, 129.4, 142.3 154.2 160.2, 171.2 3b p‐NH2SO2C₆H₄ 96.6, 108.2, 147.3, 118.6, 125.9, 129.7, 148.3 155.4 162.8, 173.4 4a C6H5 94.9, 102.6, 138.4, 120.7, 126.3, 129.8, 143.7 157.1 163.3, 169.9 5b C6H5 Cyclohexyl 11.98, 21.2 92.8, 106.2, 135.8, 121.3, 126.8, 129.5, 138.8 23.1, 29.2, 34.6, 49.5 161.7, 170.4 163.2 (cyclohexyl) (CO) 6a C6H5 CH3 12.6, 20.7, 32.3 94.6, 109.4, 136.8, 118.9, 126.3, 129.2, 140.4 185.6 (CS) 160.4, 168.6 6d C6H5 p‐CH3C₆H₄ 13.6, 18.6, 93.2, 104.6, 135.9, 115.4, 120.3, 122.2, 127.8 155.8 181.5 (CS) 22.3 161.3, 171.8 128.1, 130.7, 139.2, 140.1 7a C6H5 C6H5 11.6, 20.2 94.6, 110.4, 133.8, 120.2, 121.7, 127.5, 129.8 152.7 126.2 (CH=) 163.6, 168.4 131.8, 136.7, 137.9, 141.2 7b C6H5 p‐CH3C₆H₄ 12.9, 21.4, 92.6, 108.4, 142.1, 117.3, 121.2, 124.5, 126.4 156.6 125.4 (CH=) 23.2 160.5, 171.6 128.2, 130.6, 138.7, 143.5 8a C6H5 C6H5 13.7, 20.9 94.6, 105.2, 145.3, 118.8, 121.2, 122.6, 128.4 155.4 22.9, 28.1, 34.2, 48.4 161.9, 170.4 129.3, 132.6, 138.8, 142.1 163.8 (cyclohexyl) (CO) 9a C6H5 Cyclohexyl 12.7, 21.3 93.6, 106.4, 142.7, 118.8, 126.9, 129.4, 142.3 153.4 186.4 (CS) 160.8, 173.4 10a CH3 13.2, 20.8, 98.9, 110.3, 146.2, 120.3, 127.9, 137.2, 142.9 33.2 161.9, 172.9 a Solution in a mixture of CDCl3 and DMSO‐d6. was used as control at the same above‐mentioned concentration. The plates were incubated at 37 oC for 24 h for bacteria and 72 h for fungi. The results were recorded for each tested compound as the average diameter of inhibition zones of bacterial growth around the discs in mm (Table 4). 2.2.2. Minimal inhibitory concentration (MIC) measurement MICs were measured for compounds that showed significant growth inhibition zones (≥ 12 mm) using the two‐ fold serial dilution technique [35]. The microdilution susceptibility test in Muller‐Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid) was used for the determination of antibacterial and antifungal activity, respectively. Stock solutions of the tested compounds, ampicillin trihydrate and clotrimazole were prepared in DMSO at concentration of 1600 µg/mL followed by two‐fold dilution at concentrations of 800, 400, 200, 100, 50, 25, 12.5 and 6.25 µg/mL). The microorganism suspensions at 106 CFU/mL (Colony Forming Unit/mL) concentration were inoculated to the corresponding wells. Plates were incubated at 36 oC for 24 h to 48 h and the minimal inhibitory concentrations (MIC) were determined. Control experiments were also done. 2.2.3. Antimycobacterial screening The available Mycobacterium tuberculosis strain was cultured in tubes with 10 µL of a suspension of the strain in physiological saline. The strain was collected from the slant after four weeks incubation [36]. The inoculum was prepared with 3‐5 weeks old M. tuberculosis colonies from Loewenstein‐ Jensen slants, emulsified in dilution fluid containing 2% fatty acid free albumin and 0.02% Tween 80, pH = 6.9. Suspensions were then diluted in saline to a turbidity of McFarland no.1 standard and then diluted to obtain inocula of 3x105 cells per well. The MICs measurements were determined by agar dilution technique. Agar supplemented with 10% OADC (oleic acid‐albumin‐dextrose‐catalase) enrichment, was used to prepare quadrant plates with serial DMSO two‐fold dilutions of the test compounds. The following concentrations were used: 1000, 500, 250, 125, 62, 32, 16, 8, 4 µg/mL. A 100 µL sample of the mycobacterial suspension was inoculated onto each compound‐containing quadrant. Control quadrant consisted of agar alone, culture medium with DMSO and culture medium with reference antimycobacterial drugs; rifampicin and 1HN was performed. All plates were then incubated at 37 oC in a CO2 (5% CO2 / 95% humidified air) incubator, for 3‐4 weeks. The MIC was defined as the lowest chemical dilution associated with at least a 99% reduction in the number of visible colonies. 248 Faidallah et al. / European Journal of Chemistry 2 (2) (2011) 243‐250 Table 4. In vitro antimicrobial activity of the target compounds 2‐12. Comp. S. aureus (ATCC 25923) B. subtilis (ATCC 6051) E. coli (ATCC 25922) P. aeruginosa (ATCC 27853) C. albicans (ATCC 10231) IZa MICb IZa MICb IZa MICb IZa MICb IZa MICb 2a 12 >200 10 ...c 7 ... NAd ... 7 ... 2b 20 100 17 100 14 200 10 ... 14 100 2c 14 100 12 200 8 ... NA ... 9 ... 3a 18 100 15 200 9 ... NA ... 9 ... 3b 22 100 17 100 14 200 10 ... 16 100 3c 24 50 20 50 15 200 11 ... 23 50 4a 19 50 18 100 6 ... NA ... 8 ... 5a 28 12.5 22 50 17 50 14 200 29 25 5b 12 >200 11 …. 8 ... 6 ... 18 50 5c 23 50 18 100 16 200 11 ... 18 50 5d 20 100 17 100 12 200 10 ... 14 100 6a 3 ... 4 ... NA ... NA ... NA ... 6b 5 ... 6 ... NA ... NA ... NA ... 7a 12 >200 10 ... 8 ... NA ... 10 ... 7b 6 ... 7 ... NA ... NA ... NA ... 7c 4 ... 3 ... 6 ... NA ... 9 ... 8a 18 100 16 200 10 ... NA ... 8 ... 8b 20 50 18 50 14 200 10 ... 16 100 9a 13 >200 10 ... 8 ... 6 ... 14 100 9b 18 100 12 200 10 ... NA ... 9 ... 9c 12 >200 9 ... 8 ... NA ... 6 ... 10a 3 ... 4 ... NA ... NA ... NA ... 10b 5 ... 6 ... NA ... NA ... NA ... 11a 14 100 15 200 12 200 10 ... 22 50 11b 16 100 17 50 14 100 13 ... 18 100 11c 24 50 20 50 10 100 12 ... 16 100 12a 3 …. 4 … NA ... NA ... 2 ... A* 36 12.5 30 25 32 25 27 50 ... ... C** … … ... ... ... ... ... ... 42 12.5 a Inhibition zone (mm). b Minimal inhibitory concentration (g/mL). c … : not tested. d NA: not active. e A*: Ampicillin trihydrate; C**: Clotrimazole. 3. Results and discussion 3.1. Chemistry Dehydroacetic acid has been reported to generate a number of heterocyclic compounds through ring opening and recyclization upon treatment with a variety of binucleophiles [37‐41]. In 1991 Bendaas et al. reported the synthesis of 4‐ acylacetyl‐5‐hydroxy‐1‐phenylpyrazoles (I) [42] (Scheme 3). In more recent work Djerrari et al. [43] isolated 3‐hydroxy‐5‐ methyl‐4‐[3‐methyl‐1‐phenylpyrazol‐5‐yl]‐1‐phenylpyrazole (II) from the reaction of I with phenyl hydrazine. However, as early as 1884 Perkin [44] reported that phenylhydrazine reacts readily with dehydroacetic acid in ethanol to yield 3‐(1‐ phenylhydrazonoethyl)dehydroacetic acid (III). Scheme 3 On the basis of the results we investigated the action of aryl hydrazines on 1 following the literature method. We found that the hydrazinolysis reaction of 1 afforded the corresponding hydrazones (2a‐e). The IR spectra of these hydrazones revealed carbonyl absorption at 1720‐1738 cm‐1 as well as an OH absorption in the region 3520‐3600 cm‐1. Their 1H NMR spectra in agreement with the suggested structures which showed besides the methyl and aromatic protons two singlets at  5.99‐6.36 and 14.26‐15.02 for H‐5 and OH, respectively (Table 2). The structures were further confirmed by 13C NMR spectral data (Table 3). Reaction of the hydrazone derivatives 2 with hydrazine hydrate afforded the corresponding 1‐amino‐2‐ pyridones 3 which in their turn, were allowed to react with nitrous acid to give the pyridine derivatives 4. The IR spectra of compounds 3 and 4 exhibited an absorption band at 1655‐ 1668 cm‐1 due to the carbonyl group and a broad band at 3300‐ 3370 cm‐1 for the NH2 or NH absorptions. Their structures were further supported by 1H and 13C NMR data (Tables 2 and 3). Condensation of 2‐pyridones 3 with the appropriate isocyanate and isothiocyanate derivatives in pyridine as alkaline medium afforded the corresponding ureas 5 and thioureas 6, respectively. The IR spectra of these compounds exhibited a urea carbonyl band at 1647‐1668 cm‐1 in case of compounds 5 and a thiourea carbonyl absorption in the region 1148‐1166 cm‐1 in case of the thiourea derivatives 6. The 1H NMR spectra of 5 and 6, exhibited besides the aromatic and methyl protons, a singlet at 5.22‐5.46 for H‐5 and exchangeable signals in the regions  6.62‐9.12 and 12.98‐15.13 for the NH and OH groups, respectively (Table 2). Their 13C NMR spectra (Tables 3) showed characteristic signals at  163.2 and 181.5‐185.6 corresponding to the CO and CS, respectively. On the other hand, condensing the 2‐pyridones 3 with the appropriate aldehyde gave rise to the corresponding 1‐ arylideneamino derivatives 7. Their 1H NMR spectra are characterized by the presence of singlets at  8.12 to 8.24 due to the CH=N proton (Table 2). Moreover, reacting 1‐amino‐2‐ pyridones 3 with benzenesulfonyl chloride and p‐toluene‐ sulfonyl chloride in the presence of pyridine led to the formation of the N‐substituted benzenesulfonyl derivative 8. Their IR spectra showed two absorption bands at 1173‐1185 cm‐1 and 1338‐1345 cm‐1 for the SO2N groups. Their structures were further supported by 1H and 13C NMR data (Tables 2 and 3). Furthermore condensation of the 2‐pyrone hydrazone 2b with different isocyanates and isothiocyanates yield the corresponding substituted sulfonylureido and sulfonyl‐ thioureido derivatives 9 and 10, respectively (Scheme 2). The IR spectra of derivatives 9 showed the sulfonylureido carbonyl absorption at 1658‐1662 cm‐1, whereas those of derivatives 10 were characterized by the presence of the C=S absorption at 1120‐1150 cm‐1. The 1H NMR spectra of urea and thiourea derivatives 9 and 10, revealed the new NH protons at their expected ranges in addition to other signals assigned for the Faidallah et al. / European Journal of Chemistry 2 (2) (2011) 243‐250 249 ureido and thioureido nitrogens and the respective substituents (Table 2). Their 13C NMR spectra are recorded in Table 4 which showed a urea carbonyl carbon at 163.8 and a thiocarbonyl carbon at 186.4 for compounds 9a and 10a, respectively. Finally, reacting the 2‐pyridone 3b with 2 moles of the appropriate isocyanate or isothiocyanate in pyridine, afforded the corresponding substituted sulfonylureido and thioureido analogs 11 and 12, respectively. Their IR and 1H NMR spectral data of some representatives were concordant with the proposed structures showed the functional groups and compounds protons at their expected chemical shifts. 3.2. Antimicrobial screening Compounds, 2a‐c, 3a‐c, 4a, 5a‐d, 6a,b, 7a‐c, 8a,b, 9a‐c, 10a,b, 11a‐c, 12a, were evaluated for their in vitro antimicrobial activity against Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6051) as examples of Gram positive bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) as examples of Gram negative bacteria, Candida albicans (ATCC 10231) and Aspergillus niger (recultured) as representatives of fungi. Agar diffusion method was used for the determination of the preliminary antibacterial and antifungal activity. Ampicillin trihydrate and clotrimazole were used as reference drug. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial growth around the discs in mm. The minimum inhibitory concentration (MIC) measurement was determined for compounds that showed significant growth inhibition zones (≥ 12 mm) using the two‐ fold serial dilution method [39]. The IZ (mm) and MIC (µg/mL) values are recorded in Table 4. The results revealed that most of the tested compounds displayed greater inhibitory effect on the growth of the tested Gram positive strain compared to Gram negative ones. Most of the compounds showed weak or no antibacterial activity against the Gram negative, P. aeruginosa. Moreover, few compounds were able to exert potential antifungal activity against C. albicans, while, all the tested compounds lacked antifungal activity against the Aspergillus niger fungus. A close examination of the structures of the active compounds revealed that the antimicrobial profile of the pyridine‐2‐one compounds seemed to be more interesting than their corresponding pyran‐ 2‐one isosteres, as evidenced by their IZ diameters and MIC values recorded in Table 4. Among the pyran‐2‐one series, compound 2a showed weak antimicrobial activity against all the tested microbial strains (IZ≤ 12 mm). Introduction of chlorine atom in the phenyl group of the hydrazone moiety as in 2c resulted in a slight improvement in the activity against the Gram positive S. aureus and B. subtilis (MIC values 100 and 200 µg/mL, respectively). Whereas, replacement of the chlorine atom in 2c with sulfamyl group resulted in a more potent compound 2b, with an appreciable broad spectrum of antibacterial activity against the tested Gram positive, Gram negative bacteria (MIC values 100‐ 200 µg/mL), and moderate antifungal activity towards C. albicans (MIC 100 µg/mL). On the other hand, bioisosteric shift from the pyran‐2‐one to the pyridine‐2‐one structure resulted in an obvious improvement in the antimicrobial spectrum of the target compounds (Table 4). In this view, the prototype 4a displayed an appreciable activity against the Gram positive S. aureus and B. subtilis (MIC 50 and 100 µg/mL, respectively) when compared with the bioisosteric pyran‐2‐one 2a. Introduction of an amino group at position‐1 in the pyridine ring as in compound 3c resulted in significant change of the overall antimicrobial spectrum. It showed two‐fold improvement in the potency against B. subtilis when compared with 4a (MIC 50 νs 100 µg/mL, respectively), while it revealed moderate activity against E. coli (MIC 200 µg/mL). It also showed antifungal activity towards C. albicans (MIC 50 µg/mL). Furthermore, replacement of the 1‐amino group in 3a with a urea moiety (compound 5a produced the most potent antimicrobial activity in the current series of compounds. Compound 5a is as potent as ampicillin (MIC 12.5 µg/mL) against S. aureus, whereas its activity against B. subtilis and E. Coli was 50% lower than that of ampicillin (MIC 50 νs 25 µg/mL, respectively). Moreover, it displayed a remarkable antifungal activity towards C. albicans, which was about 50% of that of Clotrimazole (MIC 25 νs 12.5 µg/mL, respectively). However, it is worthy to mention that, structure modification of the urea derivatives to thiourea ones led to almost complete abolishment of the antimicrobial activity (IZ≤ 10 mm). 3.3. Antimycobacterial screening Determination of in νitro antimycobacterial activity of the target compounds 2a‐c, 3a‐c, 4a, 5a‐d, 6a,b, 7a‐c, 8a,b, 9a‐c, 10a, b, 11a‐c, 12a; was performed by employing the two‐fold agar dilution method slightly modified from that described by Cantos et al. [40]. A strain of Mycobacterium tuberculosis (locally isolated, Alexandria, Egypt) was utilized in this assay. Rifampicin and isonicotinic acid hydrazide (INH) were used as reference antimycobacterial drugs. The MIC was defined as the lowest concentration of the tested compound that yielded no visible growth on the plate. Among the compounds tested, only compound 7a was able to exert weak growth inhibitory effect (MIC 250 µg/mL) against the Mycobacterium tuberculosis used in this screening, while the rest of the synthesized compounds were totally inactive. Acknowledgements The authors are very grateful to the Department of Biology, Faculty of Science, Alexandria University for carrying out the microbiological screening. References [1]. Dickinson, J. M. Nat. Prod. Rep. 1993, 10(1), 71‐98. [2]. Douglas, C. J.; Sklenika, H. M.; Shen, H. C.; Mathias, D. S.; Degen, S. J.; Golding, G. M.; Morgan, C. D.; Shin, R. A.; Mueller, K. L.; Scurer, L. M.; Johnson, E. W.; Hsung, R. P. Tetrahedron 1999, 55, 13683‐13696. [3]. Hatch, M. S.; Brown, W. M.; Deck, J. A.; Hunsaker, L. A.; Deck, L. M.; Vander Jagt, D. L. B. B. A. ‐Protein Struct. M. 2002, 1596, 381‐391. [4]. Tuchinda, P.; Reutrakul, V.; Claeson, P.; Pongprayoon, U.; Sematong, T.; Santisuk, T.; Taylor, W. C. Phytochemistry 2002, 59, 169‐173. [5]. Rao, P. N. P.; Uddin, J.; Knaus, E. E. J. Med. Chem. 2004, 47, 3972‐3990. [6]. Fujimoto, H.; Okamoto, Y.; Sone, E.; Maeda, S.; Akiyama, K.; Ishibashi, M. Chem. Pharm. Bull. 2005, 53, 923‐929. [7]. Marrison, L. R.; Dickinson, J. M.; Fairlamb, I. J. S. Bioorg. Med. Chem. Lett. 2002, 12, 3509‐3515. [8]. Marrison, L. R.; Dickinson, J. M.; Fairlamb, I. J. S. Bioorg. Med. Chem. Lett. 2003, 13, 2667‐2671. [9]. De Clercq, E. J. Med. Chem. 1995, 38, 2491‐2517. [10]. De Clercq, E. B. B. A. ‐ Mol. Basis Dis. 2002, 1587, 258‐275. [11]. Fairlamb, I. J. S.; Marrison, L. R.; Dickinson, J. M.; Lu, F. J.; Schmidt, J. P. Bioorg. Med. Chem. 2004, 12, 4285‐4299. [12]. McGlacken, G. P.; Fairlamb, I. J. S. Nat. Prod. Rep. 2005, 22, 369‐385. [13]. Fossa, P.; Menozzi, G.; Dorigo, P.; Floreani, M.; Mosti, L. Bioorg. Med. Chem. 2003, 11, 4749‐4759. [14]. Krauze, A.; Vitolina, R.; Garaliene, V.; Sile, L.; Kluša, V.; Duburs, G. Eur. J. Med. Chem. 2005, 40, 1163‐1167. [15]. Ochoa, E.; Suarez, M.; Verdecia, Y.; Pita, B.; Martin, N.; Quinteiro, M.; Seoane, C.; Soto, J. L.; Duque, J.; Pomes, R. Tetrahedron 1998, 54, 12409‐12420. [16]. Parlow, J. J.; South, M. S. Tetrahedron 2003, 59, 7695‐7701. [17]. Abdel‐Aziz, A. A.; El‐Subbagh, H. I.; Kunieda, T. Bioorg. Med. Chem. 2005, 13, 4929‐4935. [18]. Srivastava, B. K.; Solanki, M.; Mishra, B.; Soni, R.; Jayadev, S.; Valani, D.; Jain, M.; Patel, P. R. Bioorg. Med. Chem. 2007, 15, 1924‐1929. [19]. Aanandhi, M. V.; George, S.; Vaidhyalingam, V. Arkivoc 2008, 11, 187‐ 194. 250 Faidallah et al. / European Journal of Chemistry 2 (2) (2011) 243‐250 [20]. Narayana, B. L.; Rao, A. R. R.; Rao, P. S. Eur. J. Med. Chem. 2009, 44, 1369‐1376. [21]. Ranft, D.; Seyfarth, T.; Schaper, K. J.; Lehwark‐Yvetot, G.; Bruhn, C.; Buege, A. Arch. Pharm. Pharm. Med. Chem. 1999, 332, 427‐430. [22]. Khoshneviszadeh, M.; Edraki, N.; Javidnia, K.; Alborzi, A.; Pourabbas, B.; Mardaneh, J.; Miri, R. Bioorg. Med. Chem. 2009, 17, 1579‐1586. [23]. Abid, M.; Kakul Husain, K.; Azam, A. Bioorg. Med. Chem. Lett. 2005, 15, 4375‐ 4379. [24]. Goebel, T.; Ulmer, D.; Projahn, H.; Kloeckner, J.; Heller, E.; Glaser, M.; Ponte‐Sucre, A.; Specht, S.; Sarite, S. R.; Hoerauf, A.; Kaiser, A.; Hauber, I.; Hauber, J.; Holzgrabe, U. J. Med. Chem. 2008, 51, 238‐250. [25]. Rodrigues, T.; Guedes, R. C.; dos Santos, D. J. V. A.; Carrasco, M.; Gut, J.; Rosenthal, P. J.; Moreira, R.; Lopes, F. Bioorg. Med. Chem. Lett. 2009, 19, 3476‐3480. [26]. Tiwari, A. K.; Mishra, A. K.; Bajpai, A.; Mishra, P.; Sharma, R. K.; Pandey, V. K.; Singh, V. K. Bioorg. Med. Chem. Lett. 2006, 16, 4581‐4585. [27]. Gudmundsson, K. S.; Johns, B. A.; Wang, Z.; Turner, E. M.; Allen, S. H.; Freeman, G. A.; Boyd, F. L. Jr.; Sexton, C. J.; Selleseth, D. W.; Moniri, K. R.; Creech, K. L. Bioorg. Med. Chem. 2005, 13, 5346‐5361. [28]. Allen, S. H.; Johns, B. A.; Gudmundsson, K. S.; Freeman, G. A.; Boyd, F. L. Jr.; Sexton, C. H.; Selleseth, D. W.; Creech, K. L.; Moniri, K. R. Bioorg. Med. Chem. 2006, 14, 944‐954. [29]. Croitoru, M.; Pintilie, L.; Tanase, C.; Caproiu, M. T.; Draghici, C. Rev. Chem. ‐Bucharest 2004, 55(12), 993‐997. [30]. Limban, C.; Misisir, A.; Chirita, I.; Ilie, C.; Caproiu, M. T. Rev. Chem. ‐ Bucharest 2008, 59(10), 1136‐1139. [31]. Limban, C.; Misisir, A.; Chirita, I.; Niculescu, G. M.; Ilie, C.; Caproiu, M. T. Rev. Chem. ‐Bucharest 2008, 59(11), 1245‐1249. [32]. Limban, C.; Misisir, A.; Chirita, I.; Niculescu, G. M.; Ilie, C.; Caproiu, M. T. Rev. Chem. ‐Bucharest 2009, 60(7), 657‐661. [33]. Morusceag, L.; Misisir, A.; Ilie, C.; Guta, R.; Andreescu, D. N.; Caproiu, M. T. Rev. Chem. ‐Bucharest 2009, 60(8), 805‐809. [34]. Dogruer, D. S.; Urlu, S.; Onkol, T.; Ozcelik, B.; Sahin, M. F. Turk. J. Chem. 2010, 34, 57‐65. [35]. Conte, J. E.; Barriere, S. L. Manual of Antibiotics and Infectious Disease. 1st ed., Lea and Febiger, USA, 1988, 135‐38. [36]. Mamolo, M. G.; Vio, L. Il Farmaco 1992, 47, 1055‐1066. [37]. Ait‐Baziz, N.; Rachedi, Y.; Silva, A. M. S. Arkivoc 2010, 10, 86‐97. [38]. Akhrem, A. A.; Moiseenkov, A. M.; Lakhvich, F.A. Smul’Skii, S. P. Izy. Akad. N. SSR Ser.1971, 5, 1098‐1100. [39]. Habart, M. H.; Pene, C.; Royer, R. Chim. Ther. 1973, 8, 314‐318. [40]. Cantos, A.; De March, P.; Manas, M. M.; Pla, A.; Ferrando, F. S.; Vergili, A. Bull. Chem. Soc. Japan. 1987, 60, 4425‐4431. [41]. Susnik, I.; Furak, J. V.; Durakovic, S.; Kopuvanoc, S.; Lasniger, J. Monatsch. Chem. 1992, 123, 817‐822. [42]. Bendaas, A.; Hamdi, M.; Sellier, N. J. Heterocylic Chem. 1999, 36, 1291‐ 1294. [43]. Djerrari, B.; Essasi, E.; Fifani, J. Bull. Soc. Chim. France 1991, 128, 521‐ 524. [44]. Perkin, Jr., W. H.; Bernhart, C. Ber. 1884, 17, 1522‐1527.