Molecules 2013, 18, 2683-2711; doi:10.3390/molecules18032683 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Synthesis, Antibacterial and Antifungal Activity of Some New Pyrazoline and Pyrazole Derivatives Seham Y. Hassan Department of Chemistry, Faculty of Science, University of Alexandria, PO Box 426, Ibrahimia 21321, Alexandria, Egypt; E-Mail: [email protected]; Tel.: +20-01-286-525-997; Fax: +20-3-5932-488 Received: 31 October 2012; in revised form: 7 January 2013 / Accepted: 9 January 2013 / Published: 28 February 2013 Abstract: A series of 2-pyrazolines 5–9 have been synthesized from α,β-unsaturated ketones 2–4. New 2-pyrazoline derivatives 13–15 bearing benzenesulfonamide moieties were then synthesized by condensing the appropriate chalcones 2–4 with 4-hydrazinyl benzenesulfonamide hydrochloride. Ethyl [1,2,4] triazolo[3,4-c][1,2,4]triazino[5,6-b]-5H- indole-5-ethanoate (26) and 1-(5H-[1,2,4]triazino[5,6-b] indol-3-yl)-3-methyl-1H-pyrazol- 5(4H)-one (32) were synthesized from 3-hydrazinyl-5H-[1,2,4]triazino[5,6-b]indole (24). On the other hand ethyl[1,2,4]triazolo[3,4-c][1,2,4]triazino[5,6-b]-5,10-dihydroquinoxaline- 5-ethanoate (27) and 1-(5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-methyl-1H- pyrazol-5(4H)-one (33) were synthesized from 3-hydrazinyl-5,10-dihydro-[1,2,4]triazino[5,6- b]quinoxaline (25) by reaction with diethyl malonate or ethyl acetoacetate, respectively. Condensation of 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde (1') with compound 24 or 25 afforded the corresponding Schiff's bases 36 and 37, respectively. Reaction of the Schiff's base 37 with benzoyl hydrazine or acetic anhydride afforded benzohydrazide derivative 39 and the cyclized compound 40, respectively. Furthermore, the pyrazole derivatives 42–44 were synthesized by cyclization of hydrazine derivative 25 with the prepared chalcones 2–4. All the newly synthesized compounds have been characterized on the basis of IR and 1H-NMR spectral data as well as physical data. Antimicrobial activity against the organisms E. coli ATCC8739 and P. aeruginosa ATCC 9027 as examples of Gram-negative bacteria, S. aureus ATCC 6583P as an example of Gram-positive bacteria and C. albicans ATCC 2091 as an example of a yeast-like fungus have been studied using the Nutrient Agar (NA) and Sabouraud Dextrose Agar (SDA) diffusion methods. The best performance was found for the compounds 16, 17, 19 and 20. Molecules 2013, 18 2684 Keywords: chalcones; pyrazoline; pyrazole; carbothioamide; benzenesulfonamide; thiazolidine; indole; quinoxaline 1. Introduction Chalcones have been recently the subject of great interest due to their interesting pharmacological activities, including antioxidant [1,2], antibacterial [3], antileishmanial [4], anticancer [5], antiangiogenic [6], anti-infective, anti-inflammatory [7], antifungal [8], anti-malarial [9], anti-tumor [10], anti-protozoal [11] and cytotoxic properties [12]. Many pyrazole derivatives are reported to have a broad spectrum of biological activities, such as anti-inflammatory [13], antifungal [14], antiviral [15], cytotoxic [12], A3 adenosine receptor antagonists [16], antioxidant [13], antihypertensive [17], tranquilizing, muscle relaxant, psychoanaleptic, hypnotic, ulcerogenic, antidepressant, antibacterial and analgesic effects [18]. Pharmacologically-interesting heterocyclic systems like pyrazolines have been widely studied owing to their pharmacological activities, which include anti-tumor [19,20], anti-inflammatory [21–32], anti-parasitary [33], anticonvulsant [34], antimicrobial [35–39], antinociceptives [40], antimalarial [41], nitric oxide synthase inhibitory, associated with diseases such as Alzheimer, Huntington, and inflammatory arthritis [42], antidepressant [43,44], anticancer [45–47], antibacterial [48], antitubercular, analgesic [49], antiviral [46], antioxidant [50], antiamoebic [51–53], cytotoxic [53], antidiabetic [20], antifungal [54,55], antinociceptive [56], antimycobacterial [57], antihepatotoxic [58] and pesticidal properties [59]. Substituted 2-pyrazolines have been synthesized from α,β-unsaturated ketones and hydrazine hydrate with acetic/formic acid in ethanol/dimethyl sulfoxide (DMSO) [60], hydrazine in dimethyl formamide (DMF) or acetic acid [46], nicotinic acid hydrazide in n-butanol [41], phenyl hydrazine hydrochloride in the presence of sodium acetate [39], hydrazine hydrate in ethanol and DMF [25], and phenyl hydrazine in the presence of hot pyridine [27]. Some new substituted 2-pyrazoline derivatives bearing benzenesulfonamide moieties [21–23,26] were synthesized by condensing appropriate chalcones with 4-hydrazinobenzenesulfonamide hydrochloride. In view of these observations and in continuation of our research programme on the synthesis of five-membered heterocyclic compounds [61–66], we report herein the synthesis of some new pyrazoline and pyrazole derivatives bearing an indoline and quinoxaline moiety, which have been found to possess an interesting profile of antimicrobial activity. 2. Results and Discussion 2.1. Chemistry 2.1.1. Preparation of the Chalcones 2–4 The chalcones 2–4 were prepared as starting material to obtain the desired pyrazoline and pyrazole derivatives. The sequence leading to the title compounds is outlined in Scheme 1. The desired compounds were prepared by the reaction of 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2- carbaldehyde (1') [67] with different acetophenones (p-bromo-, p-chloro-, or p-methoxyacetophenones) in aqueous ethanolic KOH in good yield (Scheme 1). Their 1H-NMR spectra showed the -CH=CH- Molecules 2013, 18 2685 protons as a multiplet in the 7.52–7.63 ppm range for compound 3, and two doublet peaks at 7.54, 7.60 and 7.48, 7.60 ppm with coupling constants of 15.3 Hz for compounds 2 and 4, respectively. The 13C-NMR spectrum of prototypical compound 2 showed the two carbonyl carbons at 187.8 and 192.7 ppm. 2.1.2. Synthesis of Pyrazoline Derivatives 5–9 and Isoxazoline Derivatives 10–12 The compounds 2–4 were converted into the corresponding 3-(aryl)-5-(6,6-dimethyl-4-oxo-4,5,6,7- tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamides 5–7 by treatment with thiosemicarbazide (Scheme 1). Scheme 1. Synthesis of chalcones 2–4, pyrazoline derivatives 5–9 and isoxazoline derivatives 10–12. Their 1H-NMR spectra showed multiplets within the 3.33–4.82 range corresponding to H , H of 4 4' the pyrazoline ring, where a multiplet at 6.92–6.98 ppm is observed for compound 7 corresponding to H . A doublet of doublets at 6.73–6.88 corresponding to H of the pyrazoline ring was observed for 5 5 compounds 5 and 6, respectively. In addition to a broad signal corresponding to the exchangeable NH 2 protons was observed in the 7.25–8.01 ppm range. The 13C-NMR spectrum of compound 6 chosen as a prototype showed C=S and C=O peaks at 180.0 and 204.6 ppm, respectively. Reaction of compounds 2 and 3 with hydrazinum chloride gave rise to 2-(3-(aryl)-4,5-dihydro-1H-pyrazol-5-yl)-6,6-dimethyl- 6,7-dihydro-1H-indol-4(5H)-ones 8 and 9 (Scheme 1). In their 1H-NMR spectra, the appearance of signals in the ranges 3.33–3.75 and 5.49–6.70 ppm corresponding to (H , H ) and H of the pyrazoline 4 4' 5 ring respectively was observed. The product of compound 4 with hydrazinium chloride could not be separated in a pure form. The 2-(3-(aryl)-4,5-dihydroisoxazol-5-yl)-6,6-dimethyl-6,7-dihydro-1H- indol-4(5H)-ones 10–12 were synthesized by cyclization of 2–4 in presence of hydroxylamine hydrochloride. Their 1H-NMR spectra showed three signals within the ranges 3.34–4.22 and 5.55–6.52 ppm corresponding to the (H , H ) and H of the pyrazoline ring, respectively. The 13C-NMR spectrum 4 4' 5 of compound 11 selected as a prototype showed the carbonyl carbon at 193.7 ppm. Molecules 2013, 18 2686 2.1.3. Synthesis of Benzenesulfonamide Derivatives 13–21 Reaction of the prepared chalcones 2–4 with 4-hydrazinyl benzenesulfonamide hydrochloride afforded 4-(3-(aryl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-4,5-dihydro-1H-pyrazol- 1-yl)benzenesulfonamides 13–15 (Scheme 2). Scheme 2. Synthesis of benzenesulfonamide derivatives 13–21. Their 1H-NMR spectra showed three signals at 3.15–3.40, 3.36–4.00, and 4.80–5.56 ppm corresponding to the H , H , and H of the pyrazoline ring. In addition a broad singlet was observed in 4 4' 5 the 6.63–7.35 ppm range corresponding to the NH protons. The 13C-NMR spectrum of compound 15 2 as a prototype showed C=O at 193.8 ppm. On the other hand, the reaction of pyrazolines 13–15 with bromine in acetic acid [68] at room temperature in order to obtain the pyrazoles, afforded the corresponding substituted 4-(4-bromo-3-(4-bromophenyl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro- 1H-indol-2-yl)-1H-pyrazol-1-yl)benzenesulfonamide derivatives 16–18, respectively in 79–84% yield. Furthermore, the prepared compound 17 was treated with phenyl isothiocyanate to furnish 4-(4- bromo-3-aryl-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)-1H-pyrazol-1-yl)-N-(phenyl- carbamothioyl)benzene sulfonamide 19 in 76.8% yield (Scheme 2). The proton NMR spectrum showed three broad singlets at 8.62, 9.76, and 11.11 ppm corresponding to three NH protons. The prepared substituted benzenesulfonamides 13 and 14 were allowed to react with phenyl isothiocyanate to correspondingly furnish 4-(3-(aryl)-5-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol- 2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N-(phenylcarbamothioyl)benzenesulfonamides 20 and 21 (Scheme 2). Molecules 2013, 18 2687 Their 1H-NMR spectra showed D O exchangeable signals at the ranges 9.00–9.15, 10.54–10.60, and 2 11.03–11.08 ppm, corresponding to three NH protons. 2.1.4. Synthesis of [1,2,4]Triazolo[3,4-c][1,2,4]triazino[5,6-b]-5-N-(phenylcarbamothioyl) Ethanoic Acid Hydrazide Derivatives 30, 31 and 3-Methyl-4-(propan-2-ylidene)-1H-pyrazol-5(4H)-one Derivatives 34, 35 Reaction of indoline-2,3-dione (1'') [69,70] with thiosemicarbazide gave rise to 5H-[1,2,4] triazino[5,6-b]indole-3-thiol (22) [70] (Scheme 3). The 1H-NMR spectrum showed two broad singlets exchangeable with D O at 12.43 and 14.54 ppm, corresponding to the two NH protons, which confirm 2 the structure of 22. The 13C-NMR also confirmed the structure of 22 with a peak at 179.5 corresponding to the C=S group. Treatment of the thiol derivative 22 with hydrazine hydrate afforded 3-hydrazinyl-5H-[1,2,4]triazino[5,6-b]indole (24) [70] in 92.6% yield (Scheme 3). The proton NMR spectrum showed two broad singlets at 4.31 and 8.54 ppm corresponding to NH and NH protons of 2 hydrazine chain, in addition to a broad singlet at 11.82 ppm corresponding to the indole ring NH. The reaction of 24 with diethyl malonate gave rise to the corresponding ester 26. The proton NMR spectrum showed a triplet signal at 1.16 ppm corresponding to the CH protons, and a quartet signal at 3 4.31 ppm corresponding to CH of the ester moiety, and a singlet signal at 4.12 corresponding to CH 2 2 protons. Reaction of the ester 26 with hydrazine hydrate afforded the corresponding 5-ethanoic hydrazide 28 (Scheme 3). From the proton NMR spectrum the disappearance of CH and CH protons 3 2 of the ester chain can be observed. Treatment of the prepared hydrazide 28 with phenyl isothiocyanate afforded the corresponding 5-N-(phenylcarbamothioyl)ethanoic acid hydrazide 30 in 80.1% yield (Scheme 3). Its structure was confirmed by 1H-NMR, 13C-NMR spectra, and elemental analysis. The 13C-NMR spectrum showed C=S and C=O carbons at 168.7 and 189.4 ppm respectively (see Experimental part). On the other hand, treatment of the prepared hydrazine derivative 24 with ethyl acetoacetate in acetic acid afforded 1-(5H-[1,2,4]triazino[5,6-b]indol-3-yl)-3-methyl-1H-pyrazol-5(4H)-one (32) (Scheme 3). The proton NMR spectrum of this compound showed the CH protons as a singlet at 1.80 3 ppm, and the CH protons as a singlet at 2.43 ppm. 1-(5H-[1,2,4]Triazino[5,6-b]indol-3-yl)-3-methyl- 2 4-(propan-2-ylidene)-1H-pyrazol-5(4H)-one (34) was prepared from the previous pyrazoline-5-one derivative 32 by its reaction with acetone (Scheme 3). The proton NMR spectrum showed two methyl protons as a singlet at 1.87 ppm. Cyclization of quinoxaline-2,3(1H,4H)-dione (1''') with thiosemicarbazide afforded 5,10-dihydro- [1,2,4]triazino[5,6-b]quinoxaline-3-thiol (23) in good yield (Scheme 3). Its proton NMR spectrum showed two broad singlets, exchangeable with D O, at 11.88 and 14.50 ppm, corresponding to the 2 three NH protons, which confirm the structure of 23. Treatment of this thiol derivative 23 with hydrazine hydrate gave 3-hydrazinyl-5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxaline (25, Scheme 3). Its proton NMR spectrum showed NH protons as a broad singlet signal at 4.55 ppm, in addition to 2 three NH protons, see Experimental part. Treatment of the prepared hydrazine derivative 25 with diethyl malonate gave rise to the corresponding ester 27 (Scheme 3). The proton NMR spectrum showed the ester protons (CH , CH ) as a triplet and a quartet signals at 1.09 and 4.09 ppm, 3 2 respectively, in addition to CH protons at 4.66 ppm as a singlet signal. 2 Molecules 2013, 18 2688 Scheme 3. Synthesis of [1,2,4]triazolo[3,4-c][1,2,4]triazino[5,6-b]-5-N-(phenylcarbamothioyl) ethanoic acid hydrazide derivatives 30, 31 and 3-methyl-4-(propan-2-ylidene)-1H-pyrazol- 5(4H)-one derivatives 34, 35. Reaction of the ester 27 with hydrazine hydrate leads to the corresponding 5-ethanoic hydrazide 29. The proton NMR spectrum showed the disappearance of CH and CH protons of the ester chain, and 3 2 NH and NH protons at 8.90 and 9.61 ppm were observed as a two broad singlets. Its 13C-NMR 2 spectrum showed the carbonyl carbon at 168.4 ppm. Treatment of the prepared hydrazide 29 with phenyl isothiocyanate leads to corresponding 5-N-(phenylcarbamothioyl) ethanoic acid hydrazide 31 (Scheme 3). Its structure was also confirmed by 1H-NMR, and elemental analysis. Reaction of the hydrazine derivative 25 with ethyl acetoacetate in acetic acid afforded 1-(5,10- dihyro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-methyl-1H-pyrazol-5(4H)-one (33, Scheme 3). The proton NMR spectrum showed the CH protons at position 3 of the pyrazoline ring as a singlet signal 3 at 2.29 ppm, and the CH protons (H ) of the pyrazoline ring as a singlet signal at 2.92 ppm, with the 2 4 disappearance of the peak corresponding to NH protons. 13C-NMR spectrum showed the carbonyl 2 carbon at 170.0 ppm. Reaction of the prepared pyrazoline-5-one 33 with acetone gave rise to 1-(5,10- dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-3-methyl-4-(propan-2-ylidene)-1H-pyrazol-5(4H)-one (35, Scheme 3). Its proton NMR spectrum showed three methyl groups at 2.30, 2.42, and 2.93 ppm. Molecules 2013, 18 2689 2.1.5. Synthesis of Schiff’s Bases 36, 37, 4-Oxo-4,5,6,7-tetrahydro-1H-indol-2-yl)thiazolidin-4-one (38), Benzohydrazide Derivative 39, 1,2,4]Triazolo[3,4-c]-5,10-dihydro [1,2,4]triazino[5,6-b] quinoxaline (40), and Pyrazole Derivatives 41–44 Condensation of hydrazine derivative 24 with 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2- carbaldehyde (1') afforded the corresponding Schiff's base 36 (Scheme 4). Its proton NMR spectrum showed the disappearance of the NH signal, and a singlet signal corresponding to a CH=N proton at 2 8.03 ppm was observed. The 13C-NMR spectrum showed the carbonyl carbon at 192.6 ppm. Treatment of the prepared compound 36 with thioglycolic acid in dry benzene gave rise to corresponding 3-(5H- [1,2,4]triazino[5,6-b]indol-3-yl)-2-(6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-2-yl) thiazolidin- 4-one (38, Scheme 4). The proton NMR spectrum showed the CH (H , H ) protons of the thiazolidine 2 5 5' ring at 3.35–3.49 ppm as a multiplet and the CH proton of thiazolidine ring (H ) as a singlet signal at 2 7.93 ppm. Scheme 4. Synthesis of Schiff’s bases 36, 37, 4-oxo-4,5,6,7-tetrahydro-1H-indol-2- yl)thiazolidin-4-one (38), benzohydrazide derivative 39, 1,2,4]triazolo[3,4-c]-5,10-dihydro [1,2,4]triazino[5,6-b] quinoxaline (40), and pyrazole derivatives 41–44. Condensation of the hydrazine derivative 25 with 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole- 2-carbaldehyde (1') furnished to the corresponding Schiff's base 37 in 86.6% yield (Scheme 4). Its proton NMR spectrum showed the CH=N proton as a singlet at 9.45 ppm. Condensation of 37 with benzoyl hydrazine afforded the corresponding N'-2-((2-(5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxalin- 3-yl)hydrazono)methyl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-ylidene)benzohydrazide (39). Its Molecules 2013, 18 2690 13C-NMR spectrum showed the C=O group at 162.7 ppm. On the other hand, oxidative cyclization of Schiff's base 37 with acetic anhydride afforded the cyclized compound 40 (Scheme 4). The proton NMR spectrum showed the disappearance of CH=N proton. We expected to obtain the acetylated product, but the 1H-NMR spectrum confirmed the structure of the cyclized compound 40 as shown, with an exchangeable peak corresponding to three NH protons being observed at 12.23 ppm, and no peak observed corresponding to the acetyl methyl group. Oxidative cyclization of 3-hydrazinyl-5H- [1,2,4]triazino[5,6-b]indole (24) with 2-(3-(4-bromophenyl)-3-oxoprop-1-enyl)-6,6-dimethyl-6,7-dihydro- 1H-indol-4(5H)-one (2) afforded 2-(1-(5H-[1,2,4]triazino[5,6-b]indol-3-yl)-3-(4-bromo-phenyl)-1H- pyrazol-5-yl)-(6,6-dimethyl-6,7-dihydro-1H-indol-4 (5H)-one (41) in 80.8% yield. The proton NMR spectrum showed the CH proton of pyrazole ring as a singlet signal at 6.12 ppm. The 13C-NMR spectrum showed C=O carbon 190.8 at ppm. Scheme 5. Charge distribution on nitrogen atoms N , N of compound 37. 1 3 On the other hand, the hydrazine derivative 25 was allowed to react with the prepared chalcones 2–4 to correspondingly furnish 2-(3-(aryl)-1-(5,10-dihydro-[1,2,4]triazino[5,6-b]quinoxalin-3-yl)-1H- pyrazol-5-yl)-6,6-dimethyl-6,7-dihydro-1H-indol-4(5H)-ones 42–44, respectively (Scheme 4). Their proton NMR spectra showed the CH protons of the pyrazole ring (H ) and indole ring (H ) at 6.80 and 4 3 6.91, 6.90 and 7.27 and 6.78 and 6.85, ppm respectively. The 13C-NMR spectra of compounds 42–44 showed the C=O of the indole ring at 192.8–193.3 ppm. According to the charge distribution determined using ChemDraw Ultra, the N nitrogen atom has a better nucleophile character compared 1 to the N nitrogen atom, which is in accordance with the proposed structure of compound 40 (Scheme 5). 3 2.2. Pharmacological Screening Four test organisms representing different groups of microorganisms were used to evaluate the bioactivity of the designed products. The utilized test organisms were: Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC 9027 as Gram-negative bacteria, Staphylococcus aureus ATCC 6538P as an example of Gram-positive bacteria, and Candida albicans ATCC 2091 as yeast-like fungi. The inhibition zone (IZ) and minimal inhibitory zone (MIC) results are given in Table 1. Molecules 2013, 18 2691 Table 1. In vitro antimicrobial activity of the test compounds and evaluation of the inhibition zone (IZ) and the minim. inhibitory concentration (MIC). Escherichia Staphylococcus Candida Pseudomonas Microorganism coli aureus albicans aeruginosa IZ MIC IZ MIC IZ MIC IZ MIC ampicillin 10.0 µg/disc 18 25 22 12.5 ----- ------ ----- ----- ciprofloxacin 5.0 µg/disc 28 12.5 30 25 ----- ------ 38 25 clotrimazole 100.0 µg/disc ---- ----- ----- ------ 40 12.5 ----- ------ imipenam 10.0 µg/disc 26 ----- 30 ------ ----- ---- 30 ------ 2 18 200 17 200 21 200 16 200 3 19 200 17 200 24 200 18 200 4 19 200 15 200 23 200 18 200 5 19 200 15 200 23 200 19 200 6 19 200 16 200 25 200 20 200 7 18 200 13 200 21 200 16 200 8 19 200 17 200 22 200 18 200 9 18 200 16 200 24 200 18 200 10 19 200 16 200 24 200 20 200 11 19 200 18 100 23 200 19 200 12 19 200 15 200 23 200 18 200 13 18 200 19 200 23 200 18 200 14 18 200 15 200 23 200 18 200 15 18 200 16 100 22 200 19 200 16 19 200 26 25 27 50 20 200 17 19 200 >50 100 >40 50 21 100 18 19 200 20 100 23 200 19 200 19 19 200 16 100 25 12.5 19 200 20 18 200 16 200 26 12.5 18 200 21 18 200 16 200 28 50 20 200 22 19 200 16 200 25 50 18 200 23 18 200 8 200 24 200 20 200 24 18 200 16 200 23 200 20 200 25 19 200 15 200 22 200 18 200 26 19 200 13 200 23 200 19 200 27 19 200 17 200 25 200 17 200 28 19 200 17 200 23 200 18 200 29 18 200 15 200 21 200 18 200 30 19 200 17 200 26 200 16 200 31 19 200 16 200 34 25 20 200 32 19 200 16 200 23 200 20 200 33 19 200 17 200 24 200 18 200 34 19 200 17 200 24 200 17 200 35 19 200 16 100 23 200 19 200 36 19 200 17 200 24 200 18 200 37 17 200 15 200 22 200 18 200 38 19 200 17 200 21 200 18 200 Molecules 2013, 18 2692 Table 1. Cont. Escherichia Staphylococcus Candida Pseudomonas Microorganism coli aureus albicans aeruginosa IZ MIC IZ MIC IZ MIC IZ MIC 39 19 200 17 200 22 200 16 200 40 19 200 17 200 21 200 18 200 41 18 200 13 200 21 200 16 200 42 18 200 16 200 23 200 18 200 43 18 200 17 100 24 200 19 200 44 20 200 17 100 23 200 19 200 DMF 18 13 21 16 The compounds under investigation 2–44 did not show any activity against the test organisms Escherichia coli and Pseudomonas aeruginosa. The inhibition Zone (IZ) listed in Table 1 showed that compound 16 has good antimicrobial activity against Staphylococcus aureus, comparable to that of ampicillin, while compound 17 has remarkable antimicrobial activity against Staphylococcus aureus exceeding that of ampicillin, ciprofloxacin and imipenam.. The minimal inhibitory concentration (MIC) value showed that compound 16 has good antimicrobial activity against Staphylococcus aureus, comparable to that of ciprofloxacin, while its activity is about 50% of that of ampicillin. In addition, compound 17 has an IZ against Candida albicans comparable to that of clotrimazole. The minimal inhibitory concentration (MIC) of compound 17 against Candida albicans is about 25% of that clotrimazole. On the other hand, the minimal inhibitory concentration (MIC) of compounds 19 and 20 against Candida albicans was good, and comparable to that of clotrimazole, while compound 31 has 50 % activity compared to that of clotrimazole. 3. Experimental 3.1. General Methods Fresh solvents were used without purification. Melting points were obtained in open capillary tubes by using a MEL-Temp II melting point apparatus and are uncorrected. Infrared spectra (IR) were recorded on a Perkin-Elmer 1600 series Fourier Transform instrument with the samples as KBr pellets. 1H-NMR and 13C-NMR spectra were recorded on a JEOL 500 MHz spectrometer at ambient temperature using tetramethylsilane as an internal reference. Elemental analyses were carried out by the University of Cairo Microanalytical Laboratories. The antimicrobial tests were carried out at the Pharmaceutical Chemistry Department, Faculty of Pharmacy, Alexandria University. ChemDraw- Ultra-11.0 has been used for the nomenclature of the prepared compounds. 3.2. General Procedure for the Preparation of Compounds 2–4 An equimolar mixture of 6,6-dimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde (1', 1.91 g, 0.01 mol) [67] and the substituted acetophenone (0.01 mol) in 2% ethanolic KOH (20 mL) was stirred at room temperature (R.T.) for 5 h. The solid product was cooled, collected by filtration, washed with water, dried and recrystallized from chloroform/ethanol.