Synthesis and Characterization of Some New Pyrazoline and Isoxazoline Derivatives as Antibacterial Agents

In this paper some chalcones (C1-C8) are prepared based on the reaction of one mole of substituted acetophenone with one mole of substituted benzaldehydes in the presence of (40%) sodium hydroxide as a base. Pyrazolines (P1–P8) are prepared from the reaction of chalcones (C1-C8) with hydrazine hydrate. Isoxazoline (I1-I8) is prepared from the reaction of chalcones (C1-C8) with hydroxyl amine hydrochloride in the presence of (10%) sodium hydroxide as a base. These compounds are characterized by using various physical and spectral methods. The compounds are screened for their in vitro antibacterial activity using grampositive bacteria and gram-negative bacteria. Several derivatives of pyrazolines and isoxazolines are produced well to moderate activities against number of bacteria.


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In view of these observations and continuation of the research work on bioactive heterocycles [25][26][27][28], it is intended to design and synthesize some new isoxazoline and pyrazoline derivatives and evaluate them for antimicrobial activities.

Materials and Methods:
Melting points are uncorrected and measured in open capillary tubes using a Gallenkamp electric melting point apparatus.IR spectra are recorded on FT-IR 100 Fisher company thermo scientific spectroph-otometers, using samples in KBr disks. 1 H-NMR spectra are taken on FT.NMR-Bruker, Shield, Model 2003 Ultra spectrometer (300 MHz) using DMSO-d 6 as solvent and TMS as the internal standard at Al-Albiat university in Jordan.

Synthesis of Substituted Pyrazoline and Isoxazoline Derivatives:
Step-1.Procedure for the Synthesis of Substituted Chalcone Derivatives: A solution of sodium hydroxide (40%) in water and rectified spirit is placed in a flask provided with a mechanical stirrer.The flask is immersed in a bath of crushed ice.Substituted acetophenones (A1) (0.006 M) are poured with constant stirring; and substituted benzaldehydes (B1) (0.006 M) are added to the solution.The temperature of the mixture is kept at about 25ºC and is stirred vigorously until the mixture becomes thick enough to retard the stirring (4-6 hr).The stirrer is removed and the reaction mixture is kept at 8ºC overnight.The products (C1-C8) are 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.

Step-2. Procedure for the Synthesis of Substituted Pyrazoline Derivatives:
A mixture of substituted chalcones (C1-C8) and hydrazine hydrate in ethanol is taken in a round bottom flask.The reaction mixture is refluxed for 4 hrs on a water bath followed with the addition of ice cold water at room temperature.The precipitated crude products are filtered, washed with distilled water and dried.The product is filtered and recrysta-llized from ethanol to get the final products (P1-P8).

Step-3. Procedure for the Synthesis of Substituted Isoxazoline Derivatives:
A mixture of substituted chalcones (C1-C8) and hydroxylamine hydrochloride in ethanol is taken in a round bottom flask.The reaction mixture is refluxed for 6 hrs on a water bath followed with the addition of ice cold water at room temperature.The mixture is kept overnight at 8ºC.The precipitates are filtered, washed with distilled water and dried.The product is recrystallized with ethanol to get the final products (I1-I8).

Cylinder Plate Method [29]:
A definite volume of the microbial suspension (inoculums) is poured into the sterilized nutrient agar media (cooled at 40ºC) and mixed thoroughly.About 20 ml of this suspension is poured aseptically in the petri plates and kept till the solidification.The surface of agar plates is pierced using a sterile cork borer.The prepared wells are filled with equal volume of a solution of synthesized compounds and standard drugs, separately.After a period of preincubation diffusion, the plates are incubated face up for a definite time under specified conditions.The zones of inhibition are measured as a parameter of antimicrobial properties of synthesized derivatives.

Results and Discussion:
New pyrazoline (P1-P8) and isoxazoline (I1-I8) derivatives were synthesized based on the cyclization of substituted chalcone derivatives in the presence of hydrazine hydrate or hydroxylamine hydrochloride successsively (Scheme 1).
Physiochemical properties of synthesized compounds were determined in terms of melting point, Color and percentage yield with the elemental analysis Table (1).The synthesized compounds are also characterized by using FT-IR and 1 H-NMR.The IR spectrum of the synthesized compounds reveales presence of C=O (1630-1689 cm -1 C=O str.), C=C(aromatic) stretching at 1645-1672 cm -1 , 850-640 cm -1 (aromatic C-H oop) [30], 1223-1244 cm -1 (C-O str.),3412-3363 cm -1 (O-H str.) for chalcones, and 3445-3416 cm -1 (N-H str.), 1645-1670 cm -1 (C=N str.), 1025-1106 cm -1 (C-N str.), 1040-1099 cm -1 (N-N str.), etc. for pyrazoline derivatives and the isoxazoline derivatives reveales presence of C-O stretching at 1070-1263 cm -1 , 1024-1071 cm -1 (N-O str.), etc.In 1 H-NMR spectra δ value of various synthesized compounds are found in the range of 3.06-3.8for methyl proton, 5.3-5.4 for hydroxyl proton and 8.3-6.5 for benzyl proton Table (2).Antibacterial activities are also performed as in-vitro antimicrobial screening against bacterial strain (Table 3).According to the preliminary antibacterial screening by paper disc method, some compounds are found to have comparable antibacterial activity against S. aureus and B. subtilis as Gram positive bacteria with E. coli and P.aeruuginosa as Gram negative bacteria compared to Norfloxacin as a standard drug.The antimicrobial screening reveales that the compound (P7) and compound (I7) exhibited potent antibacterial activity as compared to other derivatives.Compound P7 is found to exhibit potent in-vitro antibacterial activity against Escherichia coli and Pseudomonas aeruuginosa while compound I7 is found to exhibit potent in-vitro antibacterial activity against Staphylococcus aureus and Bacillsubtilis Table (3).

Conclusion:
The present research work involves synthesis of novel substituted pyrazoline and isoxazoline derivatives to explore their antibacterial activity.Compounds P7 exhibit the highest antibacterial activity for Gram negative bacteria and compound I7 exhibits potent antibacterial activity for Gram positive bacteria respectively.Hence, it is concluded that there is an ample scope for further study in developing these as good lead compounds for the treatment of bacterial strain as well as fungal strain.