Reaction of indole-3-carboxaldehydes 4 with hydrazine derivatives and different substituted acid hydrazides afforded the corresponding hydrazine derivatives 5a-c and acid hydrazide derivatives 7-11 respectively. Condensation of indole-3-carboxaldehydes 4 with phenacyl bromide and thiourea gives 1,3-thiazol-2-amine derivative 18. On the other hand, reaction 4 with 3-acetylchromene-2-one afforded chalcone derivative 19. Compound 4 undergoing Knoevenagel condensation with cyanoacetamide, ethyl cyanoacetate, benzimidazol-2-ylacetonitrile, rhodanine-3-acetic acid, 2,3-dihydropyrimidin-4-one derivative and 2,4-dihydropyrazol-3-one afforded the compounds 20a,b, 22, 23, 27 and 28 respectively. The structure of the newly synthesized compounds has been confirmed by elemental analysis and spectra data. The antimicrobial activities of the some newly synthesized compounds were measured and showed that most of them have high activities
In the recent past, bacterial infections have increased at an alarming rate causing deadly diseases and wide- spread epidemics in humans. All types of bacterial diseases have taken a high toll on humanity. The resistance of antibiotics to control emerging and pre-emerging bacterial pathogens focused the medicinal chemists to search potential new antimicrobial agents to cure microbial infections effectively [1] .
Heterocyclic compounds containing nitrogen have been described for their biological activity against various micro-organisms. The indole unit is the key building block for a variety of compounds which have crucial roles in the functions of biologically important molecules. Many indole alkaloids are recognized as one of the rapidly growing groups of marine invertebrate metabolites for their broad spectrum of biological properties [2] [3] . For example, five new indole alkaloids, meridianins A-E have been isolated from the tunicate Aplidium meridianum, which showed cytotoxicity toward murine tumor cell lines [4] .
Introduction of different groups to the modified indole structure can produce a series of compounds with multiple activities. Various 3-substituted indoles had been used as starting materials for the synthesis of a number of alkaloids, agrochemicals, pharmaceuticals and perfumes. Also 3-substituted indole derivatives possess various types of broad spectrum’s biological activities such as antimicrobial, antitumor, hypoglycemic, anti-inflamma- tory, analgesic and antipyretic activities [5] [6] . Moreover the substitution at the 3-position of the indole ring can take place by connecting an additional heterocyclic ring, such as imidazole (topsentins, nortopsentins) [7] [8] , dihydroimidazole (discodermindole) [9] , oxazole (pimprinols A-C, almazole C) [10] [11] , thiazole (bacillamide A) [12] , quinazoline (tremorgens) [13] , and pyrimidine [14] . Therefore, 3-substituted indoles still represent a significant synthetic challenge. In view of the important biological properties of the indole ring, we planned to synthesize a new series of 3-substituted indole derivatives bearing side chains with different structures; as such derivatives could possess interesting and useful antimicrobial activity.
2. Materials and Methods2.1. Experimental
Melting points were measured on a Gallenkamp apparatus and are uncorrected. IR spectra were recorded on Shimadzu FT-IR 8101 PC infrared spectrophotometer (υmax in cm−1). The 1H NMR and 13C NMR spectra were determined in DMSO-d6 at 300 MHz on a Varian Mercury VX 300 NMR spectrometer using TMS as an internal standard. Mass spectra were measured on a GCMS-QP1000 EX spectrometer at 70 Ev. Elemental analyses were carried out at the Microanalytical Center of Cairo University. Spectral data of the synthesized compounds were given in Table 1.
2.1.1. General Procedure for the Synthesis of 2-Substituted-Indole (3a-d)
Synthesis of 2-substituted-1H-indole 3a-d was carried out by the procedure of Fischer indole synthesis. Phenylhydrazone derivatives 2a-d were prepared by warming a mixture of compounds 1a-d (0.04 mol) and phenyl hydrazine (0.072 ml, 0.04 mol) with 60 ml of ethanol and few drops of glacial acetic acid. The resulting reaction mixture was allowed to stirring for about 2 h. The reaction mixture was then poured into ice water (50 ml) where upon the crude compound was precipitated. The residue obtained after filtration was washed with water and used in second step. A mixture of 2a-d (0.01 mol) and polyphosphoric acid (20 ml) was refluxed for 6 h. After the completion of the reaction, it was filtered and filtrate was poured into ice cooled water. The solid obtained was filtered and recrystallized from the ethanol to give 3a-d.
2-(4-Methylphenyl)-1H-indole 3a
Yellow crystals. Yield: (1.24 g, 60%); m.p.: 220-221˚C. Anal. calcd. for C15 H13 N(207.27): C, 86.92; H, 6.32; N, 6.76. Found: C, 86.62; H, 6.12; N, 6.56.
4-(1H-Indol-2-yl)aniline 3b
White crystals. Yield (1.37 g, 66%); m.p.: 250-252˚C. Anal. calcd. for C14 H12 N2 (208.25):C, 80.74; H, 5.81; N,13.45.Found: C, 80.54; H, 5.61; N, 13.35.
2-(4-Bromophenyl)-1H-indole 3c
Yellow crystals. Yield (1.63, 60%); m.p.: 220-222˚C. Anal. calcd. for C14 H10 Br N (272.14): C, 61.79; H, 3.70; Br, 29.36; N, 5.15. Found: C, 61.59; H, 3.50; Br, 29.16; N, 5.00.
3-(1H-Indol-2-yl)-2H-chromen-2-one 3d
Dark brown crystals. Yield (1. 57, 60%; m.p.: 240-242˚C (DMF). Anal. calcd. for C17 H11 NO2 (261.27): C, 78.15; H, 4.24; N, 5.36. Found: C, 78.00; H, 4.04; N, 5.06.
Phosphorous oxychloride (21.47 ml, 0.14 mol) was added drop wise to N,N/-dimethylformamide (DMF) (10.23 ml, 0.14 mol) under cooling with an ice bath and the reaction mixture was stirred for 2 h. to prepare the Vilsmeier reagent. Then compound 3c (19.59 g, 0.072 mol) in DMF (20 ml) was added drop wise into the Vilsmeier
Spectral data of the newly prepared compounds 3-31
reagent and continuous stirring and kept at room temperature for 2 h. The reaction mixture was allowed to stand overnight and was then refluxed for 2 h. under vigorous stirring. The mixture was then poured onto ice cold water and neutralized with dilute ammonia solution till the precipitation occurs. The formed precipitate was collected by filtration and recrystallized from ethanol to give 4 as yellow crystals. Yield (15.13 g, 70%, m.p.: 270-272˚C. Anal. calcd. for C15 H10 Br NO (300.15): C, 60.02; H, 3.36; Br, 26.62; N, 4.67. Found: C, 59.89; H, 3.16; Br, 26.42; N, 4.37.
2.1.3. General Procedure for the Synthesis of 5a-c
An equimolecular mixture of 4 (3 g, 0.01 mol) and the hydrazine derivatives (0.5 ml, 0.01 mol) were refluxed in absolute ethanol (20 ml) in the presence of 2 - 3 drops of glacial acetic acid for the appropriate time. The reaction mixture was cooled to room temperature and poured into ice-cold water. The separated product was filtered, washed with cold water, dried and recrystallized from the appropriate solvent to give 5a-c.
Compound 5b was prepared from phenyl hydrazine for 4 h. Pale brown powder. Yield (2.5 g, 64%); m.p.: 115-117˚C (hexane). Anal.calcd. for C21H16BrN3 (390.28): C, 64.63; H, 4.13; Br, 20.47; N, 10.77. Found: C, 64.43; H, 4.00; Br, 20.27; N, 10.57.
Compound 5c was prepared from 2-hydrazinyl-1,3-benzothiazole for 4 h. Pale yellow crystal. Yield (2.46 g, 55%); m.p.: 280-282˚C (ethanol/DMF). Anal. calcd. for C22H15BrN4S (447.35): C, 59.07; H, 3.38; Br, 17.86; N, 12.52; S, 7.17. Found: C, 58.98; H, 3.18; Br, 17.66; N, 12.40; S, 7.00.
2.1.4. General Procedure for the Synthesis of 7-10
An equimolecular mixture of 4 (3 g,0.01 mol) and the acid hydrazide derivatives 6a-d (0.01 mol) was refluxed for 2 h. in absolute ethanol (20 ml) in the presence of 2 - 3 drops of glacial acetic acid. The reaction mixture was cooled to room temperature and poured into ice-cold water. The separated product was filtered, washed with cold water, dried and recrystallized from the appropriate solvent.
An equimolecular mixture of 4 (3 g, 0.01 mol) and cyanoacetohydrazide (1.98 g, 0.02 mol) in absolute ethanol (30 ml) was heated under reflux for 2 h. The precipitate formed after cooling was filtered off, washed with cold ethanol, dried and recrystallized from DMF to give 11 as pale brown powder. Yield (3.24 g, 85%); m.p.: 290-292˚C. Anal. calcd for C18H13BrN4O (381.22): C, 56.71; H, 3.44; Br, 20.96; N, 14.70. Found: C, 56.41; H, 3.24; Br, 20.86; N, 14.50.
To a cold solution of 11 (3.81 g, 0.01 mol) in ethanol (20 ml) containing sodium acetate (3.0 g) was added with continuous stirring 4-methylbenzene diazonium salt (0.01 mol) [prepared by adding sodium nitrite (1.38 g, 0.02 mol) in water (8 ml) to a cold solution of p-toluidine (1.07 g, 0.01 mol) in the appropriate amount of hydrochloric acid]. The reaction mixture was stirred for 2 h. and the formed solid was collected by filtration and recrystallized from ethanol to give 12 as orange crystals. Yield (3.5 g, 70%); m.p.: 240-242˚C. Anal. calcd for C25H19 Br N6O (499.36): C, 60.13; H, 3.84; Br, 16.00; N, 16.83. Found: C, 60.00; H, 3.54; Br, 15.98; N, 16.75.
2.1.7 General Procedure for the Synthesis of 13a,b and 14
Equimolecular mixture of 11 (3.81 g, 0.01 mol) and the selected aldehydes such as 1,3-diphenyl-1H-pyrazole-4- carboxaldehyde, p-nitrobenzaldehyde and salicyaldehyde (0.01 mol) in 1,4-dioxane (20 ml) containing piperidine (0.5 ml) was heated under reflux for 3 h. The reaction mixture was left to cool then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration and recrystallized from the appropriate solvent.
Brown crystals. Yield (2.9 g, 60%); m.p.: 130-132˚C (hexane). Anal. calcd for C25H17BrN4O2 (485.33): C, 61.87; H, 3.53; Br, 16.46; N, 11.54. Found: C, 61.57; H, 3.33; Br, 16.26; N, 11.34.
2.1.8. General Procedure for the Synthesis of 15 and 16
To a solution of compound 11 (3.81 g, 0.01 mol) in absolute ethanol (50 ml) containing triethylamine (1 ml) either ethyl cyanoacetate (1.13 g, 0.01 mol) or phenylisothiocyanate (1.39 g, 0.01 mol) together with elemental sulfur (0.32 g, 0.01 mol) were added. Reaction mixture was heated under reflux for 8 h. then poured onto ice/water mixture and the formed solid product, in each case, was collected by filtration recrystallized from ethanol.
A solution of compound 16 (5.48 g, 0.01 mol) in a mixture of acetic acid (5 ml) and acetic anhydride (10 ml) was heated under reflux for 8 h. and then allowed to cool. The precipitate that formed was collected by filtration, dried and recrystallized from acetic acid to give compound 17 as yellow crystals; Yield (3.4 g, 60%); m.p.: 316-318˚C. Anal. Calcd. C27 H18 Br N5 O S2 (572.50): C, 56.64; H, 3.17; Br, 13.96; N, 12.23; S, 11.2. Found: C, 56.44; H, 3.07; Br, 13.66; N, 12.03; S, 11.00.
A mixture of compound 4 (3 g, 0.01 mol), phenacyl bromide (0.01 mol) and thiourea (0.78 g, 0.01 mol) in absolute ethanol (30 ml) containing acetic acid (1 ml) were heated under reflux for 8 h. Reaction mixture poured in an ice cold water, the solid obtained was filtered, dried and recrystallized from ethanol to give 18 as brown powder. Yield (3.21 g, 70%); m.p.: 230-232˚C. Anal. calcd. for C24H16Br N3S (458.37): C, 62.89; H, 3.52; Br, 17.43; N, 9.17; S, 7.00. Found: C, 62.59; H, 3.22; Br, 17.23; N, 9.00; S, 6.81.
A mixture of 4 (3 g, 0.01 mol), 3-acetyl-2H-chromen-2-one (1.88 g, 0.01 mol) in 20 ml absolute ethanol and 0.5 ml piperdine was refluxed for 30 min. The reaction mixture was left overnight at room temperature, the obtained solid was filtered off and recrystallized from ethanol to give 19 as yellow crystals. Yield (4.01 g, 64%); m.p.: 280-282˚C. Anal. calcd. for C26H16BrNO3 (470.31): C, 66.40; H, 3.43; Br, 16.99; N, 2.98. Found: C, 66.30; H, 3.23; Br, 16.79; N, 2.78.
2.1.12. General Procedure for the Synthesis of 20a,b and 22
To a solution of compound 4 (3 g, 0.01 mol) in 20 ml ethanol, the appropriate active methylene compounds such as cyanoacetamide, ethyl cyanoacetate and 1H-benzimidazol-2-ylacetonitrile (0.01 mol) and few drops of triethylamine was added. The reaction mixture was refluxed for 5 h. and then allowed to cool. The formed solid product was collected by filtration, washed with ethanol and recrystallized from the appropriate solvent.
A mixture of compound 20b (3.95 g, 0.01 mole) and hydrazine hydrate (0.75 ml, 0.015 mole) in ethanol (20 ml) was refluxed for 3 h, then poured into water. The resulting solid was collected and recrystallized from ethanol to give 21 as yellowish white crystals. Yield (1.91 g, 50%); m.p.: 198-200˚C. Anal. calcd for C18H13BrN4O (381.23): C, 56.71; H, 3.44; Br, 20.96; N, 14.70. Found: C, 56.51; H, 3.24; Br, 20.76; N, 14.50.
To a solution of rhodanine-3-acetic acid (1.91 g, 0.01 mol) and anhydrous sodium acetate (0.5 g) in glacial acetic acid was added the 1H-indole-3-carboxaldehyde 4 (3 g, 0.01 mol). The mixture was stirred under reflux for 6 h and then poured into ice-cold water. The precipitate was filtered, washed with water, dried and recrystallized from xylene to gives 23 as orange powder. Yield (3.08 g, 65%); m.p.: 223-225˚C. Anal. calcd. For C20H13BrN2O3S2 (473.36): C, 50.75; H, 2.77; Br, 16.88; N, 5.92; S, 13.55. Found: C, 50.45; H, 2.57; Br, 16.58; N, 5.62; S, 13.25.
A mixture of an equimolar amount of 4-phenyl-2-aminothiazole 24 (1.76 g, 0.01 mol) and diethylmalonate 1.6 g, 0.01 mol) was heated in an oil bath at 180˚C for 2 hours then left to cool. The product was collected and used in second step.
A mixture of ester 25 (2.9 g, 0.01 mol) and thiourea (0.76 g, 0.01 mol) in ethanol (30 ml) containing sodium ethoxide was heated under reflux for 6 h. The reaction mixture was poured into cold water and the formed solid product was collected by filtration, washed, dried and recrystallized from ethanol to gives 26 as yellow crystals. Yield (3.8 g, 60%); m.p.:200-202˚C. Anal. calcd. for C13 H10 N4 O S2 (302.37): C, 51.64; H, 3.33; N, 18.53; S, 21.21. Found: C, 51.44; H, 3.13; N, 18.33; S, 21.01.
A mixture of pyrimidine derivative 26 (3 g, 0.01 mol) and 1H- indole-3-carboxaldehyde 4 (3 g, 0.01 mol) in ethanol (30 ml) was heated under reflux for 6 h., then left to cool. The solid product was collected by filtration and recrystallized from xylene to give 27 as green powder. Yield (2.34 g, 40%. m.p.: 208-210˚C. Anal. calcd. for C28H18BrN5OS2 (584.50): C, 57.54; H, 3.10; Br, 13.67; N,11.98; S,10.97. Found: C, 57.34; H, 3.00; Br, 13.47; N, 11.68; S, 10.67.
A mixture of 1H-pyrazol-5(4H)-one (1.74 g, 0.01 mol) and 1H- indole-3-carboxaldehyde 4 (3 g, 0.01 mol) in acetic acid in the presence of anhydrous sodium acetate was refluxed for 5 h. The reaction mixture was cooled to room temperature and poured into ice cold water. The solid separated out was filtered washed with water and recrystallized from (ethanol/DMF) to give 28 as orange crystals. Yield (3.01g, 66%); m.p.: 190-192˚C. Anal. calcd. for C25H18BrN3O (456.33): C, 65.80; H, 3.98; Br,17.51; N, 9.21. Found: C, 65.56; H, 3.68; Br, 17.31; N, 9.00.
A mixture of compound 28 (4.56 g, 0.01 ml) and hydrazine hydrate (0.5 ml, 0.01 ml) in ethanol in present of few drops of acetic acid was refluxed for 7 h. Reaction mixture was cooled at room temperature and poured in ice cold water. The solid separated was filtered, washed with water and recrystallized from ethanol to give 29a as yellow crystals. Yield (2.81 g, 60%); m.p.: 180-182˚C. Anal.calcd. for C25H18BrN5 (468.34): C, 64.11; H, 3.87; Br, 17.06; N, 14.95. Found: C, 64.00; H, 3.57; Br, 16.89; N, 14.65.
A mixture of compound 28 (4.56 g, 0.01 ml), phenyl hydrazine (1.08 ml, 0.01 mol), anhydrous sodium acetate (0.5 g) and acetic acid (20 ml) was refluxed for 7 h. Reaction mixture was cooled to room temperature and poured in ice cold water. The solid separated out was filtered, washed with water and recrystallized from ethanol to give 29b as yellow crystals. Yield (3.54 g, 65%); m.p.: 105-106˚C. Anal. calcd. for C31H22BrN5 (544.44): C, 68.39; H, 4.07; Br, 14.68; N, 12.86. Found: C, 68.09; H, 4.00; Br, 14.48; N, 12.66.
A mixture of compound 28 (4.56 g, 0.01 mol) and N-(4-methylphenyl)thiosemicarbazide (1.81 g, 0.01 mol) was refluxed in ethanol in the presence of NaOH/H2O (10%, 5 ml) for 8 h. Reaction mixture was cooled to room temperature and poured in ice-cold water. The solid separated out was filtered, washed with water and recrystallized from ethanol to give 30 as yellow crystals, Yield (3.89 g, 63%); m.p.: 240-242˚C (ethanol). Anal. Calcd. for C33H25BrN6S (617.56): C, 64.18; H,4.08; Br,12.94; N, 13.61; S, 5.19. Found: C, 64.00; H, 4.00; Br, 12.70; N, 13.36; S, 5.09.
A mixture of compound 28 (4.56 g, 0.01 mol), N-(4-nitrophenyl)thiourea (1.97 g, 0.01 mol) and potassium hydroxide (0.5 g) in ethanol (20 ml) was refluxed with stirring for 4 h. The reaction mixture was left overnight and then concentrated under reduced pressure. The solid residue was collected, washed with water and recrystallized from ethanol to gives 31 as orange powder, Yield (3.48 g, 55%); m.p.: 150-152˚C. Anal. calcd. for C32H21BrN6O2S (633.52): C, 60.67; H, 3.34; Br, 12.61; N, 13.27; S, 5.06. Found: 60.47; H, 3.14; Br, 12.41; N, 13.07; S, 4.89.
2.2. Antimicrobial Assays
Synthesized compounds 5c, 7, 9, 11, 13a, 27, 30 and 31 were screened for their antimicrobial activitiesin vitro against two species of Gram-positive bacteria, namely Staphylococcus saureus RCMB 0100010 (SA), Bacillus subtilis RCMB 010067 (BS) and two negative bacteria, namely Pseudomonas aeuroginosa RCMB 010043 (PA), Escherichia coli CMB 010052 (EC). Two fungal strains Aspergillus fumigatus RCMB 02568 (AF) and Candida albicans RCMB 05036 (CA) are used for antifungal activity. The antibacterial and antifungal activities were determined by means of inhibition% ± standard deviation at a concentration of 100 μg/ml of tested samples [15] [16] . Optical densities of antimicrobial were measured after 24 hours at 37˚C to bacteria and measured after 48 hours at 28˚C to fungal using a multidetection microplate reader at the Regional Center for Mycology and Biotechnology (Sun Rise-Tecan, USA at 600 nm) Al-Azhar University. Ampicillin, gentamicin were used as bacterial standards and amphotericin B was used as fungal standards for references to evaluate the efficacy of the tested compounds under the same conditions. The MICs of the compounds assays were determined by using microbroth kinetic system [17] .
3. Results and Discussion3.1. Chemistry
The synthesis of the new compounds is outlined in Schemes 1-6. 2-Substituted-indole reported to be obtained via Fischer indole synthesis using phenyl hydrazine and acetophenone derivatives 1a-d in present of polyphosphoric acid as catalysis [18] . Synthesis of 1H-indole-3-carboxaldehyde derivative 4 from the 2-(4-bromo- phenyl)-1H-indole 3c via Vilsmeir Haack’s formylation using phosphorus oxychloride (POCl3) and N,N/-dime- thylformamide (DMF) [19] (Scheme 1). The IR spectrum of 4 revealed C=O stretching band of formyl group at 1672 cm−1. 1H NMR spectrum showed an D2O-exchangeable signal at 12.40 ppm assigned to the NH proton and a non exchangeable signal at δ 9.95 ppm corresponding to the formyl proton. The mass spectrum showed the molecular ion peak at m/z 300 corresponding to the molecular formula C15H10BrNO.
The hydrazine derivatives 5a-c were obtained by the reaction of 1H-indole-3-carboxaldehyde derivative 4 with different substituted hydrazines [20] namely, hydrazine hydrate, phenyl hydrazine and 2-hydrazinyl-1,3- benzothiazole (Scheme 1). The molecular structure of the synthesis compounds were established based on analytical and spectral data. For example, 1H NMR spectrum of compound 5c showed an D2O -exchangeable signal at 7.95 and 11.82 assigned to the two NH protons and a non exchangeable signal at 8.42 ppm corresponding =CH proton. On other hand mass spectrum of 5c showed a molecular ion peak m/z at 447 corresponding to the molecular formula C22H15BrN4S.
Reaction of 1H-indole-3-carboxaldehyde derivative 4 with different substituted acid hydrazides [21] such as 2-(quinolin-8-yloxy)acetohydrazide 6a, 2-(1H-indol-3-yl)acetohydrazide 6b, 4-(1H-indol-3-yl)butanehydrazide 6c and 1-benzofuran-2-carbohydrazide 6d in presence of catalytic amount of acetic acid in absolute ethanol
Scheme 1. Synthesis of compounds 3-5.
Scheme 2. Synthesis of compounds 7-10.
Scheme 3. Synthesis of compounds 11-17.
afforded the corresponding the acid hydrazide derivatives 7-10 respectively (Scheme 2). The assignment of the structure of the synthesis compounds were based on analytical and spectroscopic data. For example IR spectrum of 8 exhibit absorption band at 1604 cm−1 and 1654 cm−1 due to -C=N and CO groups. 1H NMR of 8 exhibits signal at δ 8.90 ppm for =CH proton and D2O-exchangeable signal at δ 4.31 and 12.03 ppm assigned to the 2NH protons. The mass spectrum of compound 8 showed the molecular ion peak at m/z 471 corresponding to the molecular formula C25H19BrN4O.
Reaction 1H-indole-3-carboxaldehyde 4 with cyanoacetohydrazide in absolute ethanol [22] to form the N'- [1H-indol-3-ylmethylene]-2-cyanoacetohydrazide derivative 11. The assignment of the structure of compound
Scheme 4. Synthesis of compounds 18-23.
11 was based on analytical and spectroscopic data. Thus, the 1H NMR showed a singlet at δ 4.33 for the CH2 group, a singlet at δ 8.33ppm for the =CH proton and D2O-exchangeable single at δ 11.33, 11.93 ppm for the two NH protons. 13C NMR spectrum of 11 displayed signals at δ 24.39, 116.19, 157.73 and 163.58 ppm for CH2, CN, C=N and CO respectively. Further structure elucidation of compound 11 was obtained through the study of its reactivity towards chemical reagents. Thus, the reaction of 11 with 4-methylbenzene diazonium chloride [23] gave the hydrazone derivatives 12 (Scheme 3). The structures of the compound 12 were determined from spectroscopic and elemental analytical data (see Experimental section).
Knoevengel condensation of the 2-cyanoacetohydrazide derivatives 11 with aromatic aldehydes namely 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde and p-nitrobenzaldehyde [24] afforded benzylidene derivatives 13a, b (Scheme 3). The IR spectrum of compound 13a, taken as a typical example of the series prepared, revealed absorption bands at 1686 cm−1, 2204 cm−1, 3335 cm−1 and 3126 cm−1 corresponding to carbonyl, nitrile and 2 NH groups, respectively. Where the 1H NMR spectra showed the absence of the active methylene proton and showed signals at δ 9.21 ppm for N=CH proton and D2O-exchangeable signal at δ 11.73 and 12.04 assigned to the 2NH protons. Its mass spectrum showed a molecular ion peak at m/z 611 corresponding to the molecular formula C34H23BrN6O.
Cyclocondensation of 2-cyanoacetohydrazide derivatives 11 with salicyaldehyde in dioxane in the presence of
Scheme 5. Synthesis of compounds 25-27.
Scheme 6. Synthesis of compounds 28-31.
a catalytic amount of piperidine afforded 2-imino-2H-chromene-3-carbohydrazide 14. The plausible mechanism for the formation of compound 14 may be attributed to the initial Knoevenagel condensation of the active methylene nitrile of 11 with carbonyl group of salicyaldehyde followed by an intramolecular 1,6-dipolar cyclization via the addition of the phenolic OH group to the cyano function to afford the target compounds [25] (Figure 1). 1H NMR spectrum of 14 showed three D2O-exchangeable signal at δ 11.44, δ 11.56 and δ 12.14 ppm due to three NH protons. Its mass spectrum showed a molecular ion peak at m/z 485 corresponding to the molecular formula C25H17BrN4O2.
The reaction of 11 with ethyl cyanoacetate and elemental sulfur in the presence of triethylamine gave the thiophene derivatives [26] 15. The structure of compound 15 was confirmed by its infrared spectrum which indicated the absence of CN absorption band and contain the characteristic absorption bands for NH and CO func-
tional groups. On the other hand the reaction of 11 with elemental sulfur and phenylisothiocyanate [27] gave the thiazole derivative 16. Compounds 15 and 16 were obtained according to the proposed following mechanism (Figure 2). The structure of compounds 15 and 16 were elucidated on the basis of elemental analysis and spec- tral data. The IR spectrum of thiazoline 16 revealed the absence of CN absorption band and the presence of new absorption bands at 3395, 3269 cm−1 assignable to NH2 group and band at 1237 cm−1 due to C=S group. The 13C NMR data showed signals at δ 183.91, δ 161.36, δ 153 and δ 147.39 ppm to CS, CO, C-NH2 and C=N. Cyclization of thiazoline 16 with acetic anhydride afforded 1,3-thiazolo[4,5-d]pyrimidin-7(6H)-one derivative 17 (Scheme 3).
Proposed mechanism of formation of compound 14
Proposed mechanism of formation of compounds 15 and 16
N-[1H-indol-3-ylmethylene]-1,3-thiazol-2-amine 18 was synthesized by the one-pot three compounds. Thus, condensation of phenacyl bromide, 1H-indole-3-carboxaldehyde 4 and thiourea [28] under conventional heating in absolute ethanol using catalytic amount of acetic acid. The 1H NMR spectra of compound 18 showed the absence of the aldehyde proton, moreover D2O-exchangeable signal at δ 12.43 ppm due to the NH proton and signal at δ 6.85 ppm for =CH proton. Condensation of 1H-indole-3-carboxaldehyde 4 with 3-acetyl-2H-chromen- 2-one [29] afforded 3-[3-(1H-indol-3-yl)prop-2-enoyl]-2H-chromen-2-one 19 (Scheme 4).
Condensation of 4 with cyanoacetamide, ethyl cyanoacetate and 1H-benzimidazol-2-yl-acetonitrile [30] afforded 3-(1H-indol-3-yl)-2-cyanoprop-2-enamide, ethyl 3-(1H-indol-3-yl)-2-cyanoprop-2-enoate 20a,b and 2- (1H-benzimidazol-2-yl)-3-(1H-indol-3-yl)crylonitrile 22 respectively (Scheme 4). The structure of the reaction product 20a,b and 22 were ascertained on the basis of its elemental analysis and spectral data. The IR spectrum of compound 20a exhibited characteristic absorption bands at 3466 cm−1, 3310 cm−1, 2203 cm−1 and 1688 cm−1 corresponding to NH2, CN and CO groups respectively. The 1H NMR spectrum of 20a indicated the presence of one singlet peak at δ 8.22 ppm of the =CH proton and the disappearance of a singlet at δ 9.95 ppm of CHO proton. Cyclization of 20b by hydrazine hydrate to 5-oxopyrazolidine-4-carbonitrile derivative 21 was achieved by refluxing in ethanol. The 1H NMR spectrum of compound 21 indicated the presence of D2O-ex- changeable singlet at δ 10.18, δ 11.41 and δ 12.22 ppm which correspond to three NH groups.
On the other hand, the reaction of 1H-indole-3-carboxaldehyde 4 with rhodanine-3-acetic acid [31] afforded [5-(1H-indol-3-ylmethylene)-1,3-thiazolidin-3-yl]acetic acid derivative 23. The 1H-NMR spectrum of compound 23 indicated the presence of singlet at δ 4.71 ppm of the CH2 and D2O-exchangeable singlet at δ 12.60 ppm of the OH proton. Mass spectrum of 23 showed a molecular ion peak m/z at 473 corresponding to the molecular formula C20H13BrN2O3S2 (Scheme 4).
The key substrate ester 25 was synthesized from the reaction of 4-phenyl-2-amino-thiazole 24 and diethyl malonate. Reaction of ester 25 with thiourea in ethanolic sodium ethoxide solution afforded 6-[1,3-thiazol- 2-ylamino]-2-thioxo-2,3-dihydropyrimidin-4(5H)-one derivative [32] 26. Treatment of pyrimidinone derivative 26 with 1H-indole-3-carboxaldehyde 4 afforded 5-[1H-indol-3-ylmethylene]-2-thioxo-2,5-dihydropyrimidin- 4(3H)-one 27 (Scheme 5). The structure of 27 was identified as the reaction product on the basis of its elemental analysis and spectroscopic data. The 1H NMR spectrum of compound 27 indicated the presence of singlet signal at δ 9.96 ppm of the =CH proton and D2O-exchangeable singlet at δ 12.18, δ 12.22 and δ 12.40 ppm corresponding to the three NH protons.13C NMR spectrum showed signal at δ 185.37 (C=S) and 168.01 (C=O).
Condensation of 1H-indole-3-carboxaldehyde 4 with 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one afforded 4-[1H-indol-3-ylmethylene]-1H-pyrazol-5(4H)one 28 (Scheme 6). The IR spectrum of 28 exhibited characteristic absorption bands at 3275 cm−1 and 1706 cm−1 corresponding to NH and CO groups, respectively.13C NMR spectrum showed signal at δ 12.58 (CH3), δ 153 (C=C) and δ 160 (C=O). Mass spectrum of 28 showed a molecular ion peak m/z at 456 corresponding to the molecular formula C25H18BrN3O.
Compound 28 was used as key intermediates in the synthesis of novel pyrazolo[3,4-c]pyrazolone and pyrazole[3.4-d] pyrimidine derivatives via their interaction with different reagents. Thus, the reaction of 28 with hydrazine hydrate, phenyl hydrazine and N-(4-methylphenyl)thiosemicarbazide by cyclocondensation reaction [33] afforded 3-(4-methyl-pyrazolo[3,4-c]pyrazol-3-yl)-1H-indole derivatives 29a,b and pyrazolo[3,4-c]-pyrazole- 2(6H)-carbothioamide 30 respectively (Scheme 6). The structure of the newly synthesis compounds were based on their correct elemental analysis and spectral data. 1H NMR spectrum of 30 exhibited a singlet signal at δ 2.28 ppm due to CH3 protons of tolyl moiety. Its mass spectrum, the compound displayed the molecular ion peak at m/z 617 corresponding to the molecular formula C33H25BrN6S. Alternatively, treatment of the compound 28 with N-(4-nitrophenyl)thiourea [34] afforded pyrazolo[3,4-d]pyrimidine-6-thione derivatives 31. The structures of the compound 31 were determined from spectroscopic and elemental analytical data (see Experimental section).
3.2. Antimicrobial Activity
The newly synthesized compounds 5c, 7, 9, 11, 13a, 27, 30 and 31 were evaluated for their in vitro antibacterial activity against Gram-positive namely Staphylococcus aureus RCMB 0100010 (SA) and Bacillus subtilis RCMB 010067 (BS) and Gram-negative Pseudomonas aeuroginosa RCMB 010043 (PA) and Escherichia coli RCMB 010052 (EC). They were also evaluated for their in vitro antifungal activity against Aspergillus fumigatus RCMB 02568 (AF) and Candida albicans RCMB 05036 (CA). Ampicillin was the standard used for the evaluation of antibacterial activity against gram positive bacteria and Gentamicin was used as a standard in assessing the activity of the tested compounds against gram negative bacteria, while Amphotericin B was taken as a reference for the antifungal effect. The inhibitory effects of the synthetic compounds against these organisms are given in Table 2, Figure 3 and Figure 4.
In general, most of the tested compounds revealed better activity against the Gram-positive rather than the Gram-negative bacteria. All test compounds were found to be inactive against Pseudomonas aeuroginosa RCMB 010043 (PA). It was shown (Figure 3) that the majority of the compounds studied possessed significant antibacterial activity towards Staphylococcus aureus RCMB 0100010 (SA), Bacillus subtilis RCMB 010067 (BS) and Escherichia coli RCMB 010052 (EC). The highest activities were observed for compounds 9 and 30, followed by 11, 13a and 31.Compounds 5c, 7 and 27 showed the least antibacterial activity.
It was shown (Figure 4) that the compounds 5c, 11, 31 strong antifunger activity against Aspergillus fumigates RCMB 02568 (AF) and Candida albicans RCMB 05036 (CA) comparable to Amphotericin B. The com-
Graphical representation of the antibacterial activity of tested compounds compared to Ampicillin and Gentamicin
Graphical representation of the antifungal activity of tested compounds compared to Amphotericin B
pounds 9, 13a and 30 showed moderate activities against Aspergillus fumigates RCMB 02568 (AF) and Candida albicans RCMB 05036 (CA) comparable to Amphotericin B. While the compounds 7 and 27 weak antifungal activity against Aspergillus fumigates RCMB 02568 (AF) and Candida albicans RCMB 05036 (CA) comparable to Amphotericin B.
The minimum inhibitory concentration (MIC) was considered to be the lowest concentration of the tested compound which inhibits growth of the microorganisms. The initial screening of the tested compounds showed promising activity of the compounds 5c, 9, 30 and 31 which encouraged the determination of their minimum inhibitory concentration (MIC) (Table 3).
The best results were demonstrated by compounds 9, 30 and 31 as antibacteria, it possessed double the activity of the standard, Ampicillin against Bacillus subtilis RCMB 010067 (BS) 1.95 and 3.9 μg/ml respectively. Moderate activity against Staphylococcus aureus RCMB 0100010 (SA) and Escherichia coli RCMB 010052 (EC) were also demonstrated by compounds 9 and 30. On other hand moderate activity against Aspergillus fumigatus RCMB 02568 (AF) and Candida albicans RCMB 05036 (CA) were also demonstrated by compounds 5c and 31.
Antimicrobial evaluation of the some synthesized compounds
: Staphylococcus aureus RCMB 0100010, (BS)
Comp. No.
Inhibition % ± standard deviation
Gram positive bacteria
Ggram negative e bacteria
Fungal
SA
BS
PA
ES
AF
CA
5c
56.85 ± 0.58
68.32 ± 1.2
NA
46.32 ± 0.58
89.25 ± 0.72
80.23 ± 1.2
7
21.25 ± 0.58
25.63 ± 0.28
NA
14.63 ± 0.2
11.22 ± 0.28
9.32 ± 0.72
9
82.63 ± 0.75
90.42 ± 0.28
NA
72.46 ± 0.28
64.35 ± 0.58
52.14 ± 0.63
11
76.8 ± 0.58
89.4 ± 0.63
NA
63.5 ± 0.72
90.3 ± 0.58
83.6 ± 0.28
13a
72.14 ± 0.58
81.32 ± 0.63
NA
64.21 ± 0.63
52.63 ± 0.58
42.18 ± 1.2
27
32.6 ± 0.63
40.4 ± 0.85
NA
23.6 ± 1.2
20.3 ± 1.2
15.4 ± 0.58
30
90.3 ± 1.2
92.4 ± 0.72
NA
71.6 ± 0.93
72.4 ± 0.58
62.5 ± 0.28
31
76.8 ± 0.58
89.4 ± 0.63
NA
63.5 ± 0.72
90.3 ± 0.58
83.6 ± 0.28
Ampicillin
96.52 ± 0.2
99.65 ± 0.3
Gentamicin
89.23 ± 0.1
82.14 ± 0.3
Amphotericin B
96.25 ± 0.1
91.29 ± 0.1
Minimum inhibitory concentration of compounds 5c, 9, 29 and 31
Comp. No.
Minimum inhibitory concentration (µg/ml)
Gram positive bacteria
Gram negative e bacteria
Fungi
SA
BS
PA
EC
AF
CA
5c
31.25
15.63
NA
62.5
3.9
3.9
9
3.9
1.95
NA
7.81
15.63
31.25
30
1.95
1.95
NA
7.81
7.81
15.63
31
7.81
1.95
NA
15.63
1.95
3.9
Ampicillin
0.98
3.9
Gentamicin
1.95
3.9
Amphotericin B
0.98
1.95
4. Conclusion
In the present work, we synthesized novel series of 3-substituted indole by reaction of indole-3-carboxaldehyde derivative with different reagents. Screening for some selected compounds was carried for their potential antibacterial, antifungal activity. Most of the tested compounds revealed better activity against the Gram-positive rather than the Gram negative bacteria. All test compounds were found to be inactive against Pseudomonas aeuroginosa. Compounds 9, 30 and 31 exhibited excellent activity against Staphylococcus aureus, Bacillus subtilis and Escherichia coli compared with the standards drugs, while compounds 5c, 11 and 31 have strong antifunger activity against Aspergillus fumigatus and Candida albicans comparable to Amphotericin B.
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