Synthesis and characterization of 1,3,5-trisubstituted pyrazoline derivatives by ultrasonic irradiation method and evaluation of its antibacterial activity

A series of novel 1,3,5-trisubstituted pyrazoline derivatives (P 1 -P 10 ) have been synthesized by the reaction of substituted chalcones (C 1 -C 10 ) with 4-hydroxybenzhydrazide. The starting material, chalcones were prepared by claisen Schmidt condensation of 4-methylacetophenone with substituted aldehydes in the presence of sodium hydroxide in ethanol. 4-hydroxybenzhydrazide was synthesized by condensing methyl 4-hydroxybenzoate with hydrazine hydrate. The cycloaddition of chalcones with 4-hydroxybenzhydrazide gives 1, 3, 5-trisubstituted pyrazoline derivatives. The structures of synthesized derivatives were confirmed by IR, 1 HNMR and 13 C NMR spectrum. The synthesized compounds were screened for their antibacterial and antifungal activity.


INTRODUCTION
Nitrogen containing heterocycles are perhaps by far the most explored heterocyclic compounds because of their occurrence in a myriad of natural products and biologically active compounds. For this reason, synthetic chemists continue to be interested in the construction and functionalization of these heterocycles. The most common examples of naturally occurring N-heterocycles which otherwise too are of fundamental importance to life are haemoglobin and chlorophyll.
Pyrazoline derivatives have attracted attention of medicinal chemists for both with regard to heterocyclic chemistry and the pharmacological activities associated with them. Many pyrazole derivatives are reported to have a broad spectrum of biological activities, such as anti-inflammatory 1 , antifungal 2 , antiviral 3 , cytotoxic 4 , A3 adenosine receptor antagonists 5 , antioxidant 1 , antihypertensive 6 , tranquilizing, muscle relaxant, psychoanaleptic, hypnotic, ulcerogenic, antidepressant, antibacterial and analgesic effects 7 . Sonochemistry is attracting considerable research activity within the synthetic chemistry community, because it offers a new approach to the preparation of organic compounds. In the last two decades, sonochemical methods have become widely used in organic synthesis 8 . Now a days, the ultrasonic irradiation technique has been employed, not only to decrease reaction times, but also to improve yields in a large variety of polyfunctionalized heterocycles. Compared with traditional methods, this method is more convenient and easily controlled. A large number of organic reactions can be carried out in a higher yield shorter reaction time and milder conditions under ultrasound 9 . Pyrazoles and their derivatives are widely used as pharmaceutical [10][11][12] and agrochemical agents 13 and consequently a large number of synthetic routes to pyrazoles has been reported [14][15][16][17][18] In view of these observations and in continuation of our research programme on the synthesis of five-membered heterocyclic compounds, we report herein the synthesis of some pyrazoline derivatives from substituted chalcone using ultrasonic irradiation , which have been found to possess an interesting profile of antimicrobial activity.

ILCPA Volume 39
In the present work, (E)-3-(substitutedphenyl)-1-p-tolylprop-2-en-1-one (C 1 -C 10 ) was prepared by reaction of 4-methylacetophenone with substituted benzaldehyde in dilute ethanolic sodium hydroxide solution under ultrasonic irradiation in the water bath of an ultrasonic cleaner at room temperature in accordance with the method described in the literature. The application of ultrasound in synthetic organic chemistry became more and more interesting. "Sonochemistry" is a new trend in organic chemistry, offering a versatile and pathway for a large variety of syntheses. Therefore, a large number of organic reactions can be carried out under ultrasonic irradiation in high yields, short time and mild conditions. Cavitation is the formation, growth and collapse of bubbles in an irradiated liquid. This effect induces very high local pressure and temperatures inside the bubbles and enhances mass transfer and turbulent flow in the liquid. Ultrasound has been utilized to accelerate a number of synthetically useful reactions, especially in heterocyclic chemistry The synthetic route of compound C 1 -C 10 and P 1 -P 10 is outlined in Scheme 1. The physical data along with reaction time and yield of synthesized chalcone compounds were reported in Table 1.

RESULTS AND DISCUSSION
The IR spectra for synthesized chalcones (C 1 -C 10 ) observed the shifting of absorption band of carbonyl group for the two reactants 4-methylacetophenone and substituted benzaldehyde is higher to lower wave number, which is a strong evidence for the formation of conjugated enone of chalcone. Other strong bands appeared at 1589-1602 cm -1 , corresponding to C=C of the enone and aromatic rings, also the disappearance of aldehydic (CH) bands is a good evidence for the formation of chalcones.
The IR spectra of condensed product (P 1 -P 10 ) displayed disappearance of the characteristic absorption at 1651-1663 cm -1 for -C=O of conjugated carbonyl and presence of characteristic absorption band at 1589-1595 cm -1 due to C=N of pyrazoline in the IR spectrum 8 . Synthesized compounds (P 1 -P 10 ) showed weak C-H stretching bands near 3066-3076 cm -1 and absorption around 2914-2956 cm -1 for aliphatic nature of compounds /substituents. The 1 H NMR spectra of products showed characteristics ABX system due to geminalvicinal multiple coupling between 4-CH 2 and 5-CH protons 19,20 . The high field double doublet at δ 3.01-4.36 ppm and δ 3.10-4.74 ppm due to H A and H B respectively of C-4 protons and low field δ 4.19-5.77 ppm due to Hx at C-5 are characteristics signals due to vicinal coupling with the two magnetically nonequivalent protons of methylene group at position 4 of the pyrazolines ring. In all the compounds absorption as multiplet at δ 6.73-8.35 ppm was assigned to aromatic protons. In all the compounds absorption as singlet at δ 2.15-2.67 ppm was assigned to -CH 3 protons 13 C-NMR spectra of all compounds were recorded in DMSO and CDCl 3 , spectral signals which are in good agreement with the probable structures.
The C 4 and C 5 carbon of pyrazolines resonated at 42.37-56.97 and 56.53-69.19 ppm, respectively. The carbon of -CH 3 in all compounds resonates at 14.42-29.69 ppm, respectively. The carbon of (C=O) displayed signals at 160.49-169.45 ppm. All the compounds showed signal at 119.46-139.69 ppm were assigned to the aromatic carbon. All the compounds showed signals at 147.30-156.29 ppm assigned to (C=N).
International Letters of Chemistry, Physics and Astronomy Vol. 39

1. Antibacterial studies
The synthesized pyrazoline derivatives were screened for the antibacterial activity against three Gram-positive bacteria viz., Streptococcus pyogenes, Bacillus subtilis and Staphylococcus aureus and two Gram-negative bacteria viz., Escherichia coli and Pseudomonas aeruginosa by using the disc diffusion method 21 . Ciprofloxacin was used as reference standard for comparing the results. The antibacterial activity of the pyrazoline derivatives are shown in Fig. 1 for Plates 1-10, and the zone of inhibition values are given Table 2. The Clustered column Fig. 2 Showed that pyrazoline derivatives of (P 1 ) to (P 10 ) posses significant activity almost equipotent with the standard Ciprofloxacin against both Gram +ve and Gram -ve pathogenic organism. Thus the substituent's place a vital role in imparting enhanced antibacterial activity to the compounds. The screening results indicate that compounds (P 6 ), (P 8 ), (P 9 ) and (P 10 ) were found to be active against S. aureus. Compounds(P 1 ), (P 2 ) and (P 5 ) were found to moderately active be active against S. aureus, whereas compounds (P 3 ), (P 4 ) and (P 7 ) were found to be inactive be active against S. aureus. Compounds (P 4 ), (P 6 ), (P 7 ) and (P9) were found to be active against B. subtilis. Compounds (P 8 ) and (P 10 ) were found to be moderately active against B. subtilis. where as compound (P 1 ) and (P 2 ), (P 3 ) and (P 5 ) was found to be inactive against B. subtilis. Compound (P 9 ) and (P 10 ) was found to active against E. coli. Compounds (P 4 ), (P 5 ) and (P 8 ) were found to be moderately active against E. coli, all other compounds were found to be inactive against E. coli. Table 1. The antibacterial activities of pyrazoline derivatives by disc diffusion method (P 1 -P 10 ).

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ILCPA Volume 39 whereas compounds (P 1 ) and (P 2 ) were found to be less active be active against P. aeruginosa.

2. Experimental protocols
Melting points were determined by open capillary method and are uncorrected. The IR spectra (in KBr pellets) were recorded on a Shimadzu FT-IR 157 spectrophotometer. 1 H NMR spectra were recorded either on a Bruker 400 MHz NMR spectrometer using TMS as an internal standard. The purity of the compounds was checked by thin layer chromatography (TLC) on silica gel plate using petroleum ether and ethyl acetate.

2. 1. General Procedure for the preparation of (E)-1,3-dip-tolylprop-2-en-1-one (C 1 -C 10 )
4-methylacetophenone (2.5 mmol), Substituted benzaldehydes (2.5 mmol) 95 % Ethanol (20 ml) and 2 N NaOH (3 ml) were taken into a 100 ml conical flask. The mixture was irradiated by an ultrasonic generator in a water-bath at 30-35 °C for 3 min. The product was filtered with suction on a Buchner funnel, washed with cold water until the washings were neutral to litmus and then with ice cold ethanol. The crude product was recrystallized from ethanol.