Substituted Aryl )-1-benzofuranyl-2-propenones : Antimicrobial Properties of Some Chalcones-Type Compounds and their 2-Pyrazoline Derivatives

2-Acetylbenzofuran on condensation with furan-2-carboxaldehyde and pyrrole-2-carboxaldehyde in methanolic KOH solution yielded the corresponding benzofuran chalcones. These two compounds and nine benzofuran chalcones were synthesized before, were further reacted with hydrazine hydrate in ethanol which led to the formation of 2-pyrazoline derivatives. All the synthesized compounds were characterized by elemental analysis, melting point determination, infrared spectroscopy and nuclear magnetic resonance spectroscopy. Nine chalcone-type compounds and eleven 2-pyrazolines were evaluated for their biological activities against the six bacteria and the three yeast and it was seen that thirteen compounds showed activity. Four of them are chalcone-type compounds showed more or less activity


Experimental
All chemicals were purchased from Fluka and Aldrich.All melting points were measured using a differential scanning calorimeter (Shimadzu DSC-50) and are uncorrected.Elemental analyses were performed on a Leco CHNS-932 apparatus. 1H-and 13 C NMR spectra were recorded on a Bruker AC 300 (300 MHz) and a Varian Mercury-Plas NMR (400 MHz) in the specified deuterated solvents.Chemical shifts were expressed in ppm from internal tetramethysilane.Infrared (IR) spectra were recorded as KBr pellets on a Perkin-Elmer Spectrum One FTIR spectrometer.

General method for the synthesis of 3-(substituted aryl and heteroaryl)-1benzofuranyl-2-propenones (1j and 1k)
To a mixture of 2-acetylbenzofuran (2.187 g, 13.6 mmol) and the appropriate aldehyde (13.6 mmol) in methanol (25 mL) a solution of sodium hydroxide (1 M, 18 mL) was added.The reaction mixture was stirred for 3 h on a magnetic stirrer at room temperature and was allowed to stand in refrigerator for overnight.The product was precipitated by pouring to 250 mL of cold water, was filtered and dried and crystallized from ethanol.

General method for the synthesis of 3-(2-benzofuranyl)-5-aryl and heteroaryl-2pyrazolines (2a-k)
The pyrazolines were synthesized as follow; Compounds 2a-k: The required chalcone-type compound (example, 1a, 1.320 g, 5 mmol) was suspended in absolute ethanol (10 mL) and stirred at room temperature.hydrazine hydrate (0.375 g, 7.5 mmol) was added and the mixture stirred at reflux for 30 min.Upon cooling, the mixture was poured to cold water (500 mL) and the precipitate was filtered off and washed with water and dried.The product (2a) was crystallized from ethanol solution, yield: 1.029 g, 74%, mp: 211 o C.
The reflux time in the transformation of the chalcones to the pyrazolines varied from 10 to 60 min according to the kinds of the chalcones (10 min for 2j and 2e, 15 min for 2k, 30 min 2c and 2i, 40 min for 2a,2b,2d, 2f and 2 h and 60 min for 2g).

Evaluation of biological action
Six bacteria and three yeast were used as test organisms.These bacteria were Staphylococcus aureus COWAN  All of the synthesized compounds were screened against six bacteria and three yeasts using the method given in literature 22 .The bacteria strains were inoculated into nutrient broth and yeast strain inoculated into malt extract broth for 24 and 48 h, respectively.In the disc-diffusion method, sterile Mueller-Hinton agar for bacteria and Malt extract agar for yeasts were separately inoculated with the test bacteria (10 5 bacteria per mL) and yeasts (10 4 yeast per mL).In order to prepare the disc a part containing 1000 µg of compound from its solution in DMSO was added onto the discs.In addition, disk containing DMSO were placed (10 µL).Discs were applied on the solid agar medium by pressing slightly.Petri dishes were placed at 4 o C for 2 h.The bacteria samples were incubated at 35±0.1 o C for 24 h and yeast samples were incubated at 25±0.1 o C for 72 h.At the end of the period, inhibition zones were measured in millimeter.The inhibition zone of compound was calculated by subtracting that of pure DMSO from the zone of solution in DMSO of the compound.

Results and Discussion
This work is the second part of the studies on 3-(substituted aryl)-1-benzofuranl-2propenones and describes the synthesis and characterization of 2-pyrazoline derivatives (2a-k) from the chalcones (1a-i) given previously 21 and two new chalcones (1j and 1k) (Scheme 1) and the biological activities of the twenty products.The synthesized chalcones compounds were suspended in absolute ethanol, hydrazine hydrate was added and the mixture stirred at reflux for a period of 10-60 min as depending on the kind of chalcones.The cyclization of chalcones were carried out at a short period of time 13,14 .The cyclization of some chalcones with hydrazine hydrate was required to make a reflux of 7 h in ethanol 13 .2-Pyrazoline derivatives were formed in 74-90 % yields 13 .The yields in formation of 3-(benzofuran-2yl)-5-(3-nitrophenyl)-2-pyrazoline (2f) and bispyrazoline (2h) are 60% and 45%, respectively and relatively low.

Structural characterization (FTIR, 1 H-and 13 C-NMR)
The newly synthesized two benzofuran chalcones (1j and 1k) showed the characteristic absorption bands at 1659 cm -1 ( C=O stretching in 1j), at 1648 cm -1 (C=O stretching in 1k), at 3000-3150 cm -1 (=C-H stretching in furan ring) and at 3284 cm -1 (N-H stretching vibration in pyrrole ring) in FTIR spectra.In 1 H-NMR, the signals of olefinic β-H and α-H on double bond conjugated to the carbonyl group were seen at 7.68 and 7.46 ppm as the both doublet (J≈15.02Hz) for 1j and at 7.68 and 7.52 ppm as the both doublet (J≈15.60Hz) for 1k.
The disappearance of carbonyl absorptions between 1660-1640 cm -1 in FT-IR spectra of chalcones 21 and arising a new band between 3290-3360 cm -1 (NH stretching vibration) confirm the cyclization to 2-pyrazoline derivatives of the chalcones. 1H-NMR spectra of 2-pyrazolines derivatives show a signal due to NH proton between 8.09-6.92ppm as depending on substituted aromatic group bonded to pyrazoline ring at 5-position.
Vicinal coupling constants of NH proton with pyrazoline ring proton at 5-position were in the range of 1.60-2.54Hz. 13 C NMR spectra of two pyrazolines (2b and 2i) have also been taken and they show characteristic signals for 2-pyrazoline ring carbons at about 151 ppm (C=N carbon), at about 41 ppm (CH 2 ring carbon) and at about 60 ppm (CH ring carbon).

Biological activity
All the synthesized chalcones and pyrazolines, except 1j and 1k, were screened in vitro for antibacterial activity against Staphylococcus aureus COWAN 1, Pseudomonas aeruginosa DMS 50071 SCOTTA, Proteus vulgaris FMC 1, Escherichia coli ATCC 25922, Klebsiella pneumoniae FMC, Bacillus megaterium DMS 32 at absorbed 1000 µg per disc and for antifungal activity against Candida albicans FMC 17, Candida glabrata ATCC 66032 and Candida tropicalis ATCC 13803 at 1000 µg per disc by agar diffusion method 22 given in detail in experimental section .
Antibacterial and antifungal activities of the obtained compounds were measured as a function of diameter of zone of inhibition (mm).DMSO used as control showed activity at different ratios against the strains of all the bacteria and yeast.The results were compared with standard drugs amoksilin for antibacterial activitiy and nystatin for antifungal activity by measuring the zone of inhibition in mm at 1000 µg.Compound 2j was found most effective of the synthesized compounds against S. aureus with zone of inhibition 23 mm, compound 1a is the most effective against P. aeruginosa and P. vulgaris with zone of inhibition 16 mm, 2k is the most effective against K. pneumoniae and E. coli with zone of inhibition 18 mm and 17 mm, respectively, and compound 1a is the most effective among the synthesized compounds against B. megaterium with zone of inhibition 27 mm.The compound 2j exhibited the maximum diameter of zone of inhibition against C. albicans, C. glabrata and C. tropicalis (21 mm, 23 mm and 20 mm, respectively).Compounds 1d, 1f, 1g, 1h, 1i, 2d and 2h showed no activity against the strains of all the bacteria and the yeast used in the work.Although compounds 1a, 2f, 2g and 2i showed an activity against most bacteria, they showed no activity against the yeast.The results of the antibacterial and antifungal activity are given in Table 1.Another striking result is to show more or less activity against all the bacteria and the yeast of compounds 2a, 2b, 2c, 2e and 2k which are pyrazolines with hydroxyl group moiety on the phenyl ring of their first four and the last is a pyrazoline having pyrrole ring.Compound 1a possessing hydroxyl group on ortho position of phenyl ring has shown very good antibacterial activity against B. megaterium and the activity is comparable with that of the standard drug (amoksilin) (with 90% inhibition of that of the standard).Compound 1a against other bacteria and compounds 1b, 2a, 2b, 2c, 2i and 2k against all the bacteria showed moderate activity with 30-63% inhibition of that of the standard drug.While compound 2j and 2k showed high antifungal activity against Candida albicans, Candida glabrata and Candida tropicalis, with 56-78% inhibition of that of the standard drug (nystatin), compounds 1c, 1e, 2a, 2b, 2c and 2e showed moderate activity against all of the three yeast, with 28%-47% inhibition of that of the standard.The results obtained for antibacterial and antifungal activities of 2-pyrazoline derivatives used in this study are comparable with the values given in literature for some 2-pyrazolines 23 .

Conclusion
Syntheses and structural characterizations of two new chalcones and eleven pyrazolines were given in this study.The cyclization of the chalcones, given 2-pyrazoline was carried out at a shorter period of time than that of the some other chalcones given in literature using absolute ethanol in reflux.Antibacterial and antifungal activities of twenty compounds from this new class of benzofurane chalcones and pyrazoline derivatives against the six bacteria and three yeast were investigated.Seven compounds (1d, 1f, 1g, 1h, 1i, 2d and 2h) showed no activity against any bacteria and any yeast.Five compounds (2a, 2b, 2c, 2e and 2k) which are the all pyrazoline derivatives have exhibited an activity against all of the bacteria and yeast.Compound 1a possessing hydroxyl group on ortho position of phenyl ring has exhibited a comparable antibacterial activity against B. megaterium with that of the standard drug (amoksilin).While the compounds 1b, 1c, 1e and 2j are effective against most of bacteria and yeast used in this work, the compounds 2f, 2g and 2i are effective against most bacteria but no yeast.