Synthesis and Microbial Activity of Novel 3-Methyl-2-pyrazolin-5-one Derivatives

2-Oxo-2H-chromene-3-carbohydrazide derivatives 2a,b react with 2-{[4-(substituted thiazol-2-yl)iminoethyl)phenyl]hydrazono}-3-oxo-butyric acid ethyl esters 4a–c to give 3-methyl-1-[(2-oxo-2H-chromen-3-yl) carbonyl]-4-{[4(substituted thiazol-2-yl)iminoethyl)-phenyl]hydrazono}]-2-pyrazolin-5-one derivatives 5a–f. A considerable increase in the reaction rate had been observed with better yield using microwave irradiation for the synthesis of compounds 2a, b, 3a–c, and 5a–f. The synthesized products were tested against B. subtilis, S. aureus, and E. coli as well as C. albicans compared with tetracycline and nystatin as reference drugs.


Experimental
Melting points were estimated using a Stuart apparatus in open capillaries.Purity of compounds was checked by TLC.IR spectra were recorded as KBr pellets on a Jasco FTIR 460 plus spectrophotometer. 1 H-NMR spectra were recorded using a Bruker AV 500 MHz spectrometer using DMSO-d 6 as solvent and TMS as an internal standard.The mass spectral data were obtained with a Micro Spectrometer model 7070 at 70 eV and a 90 ∘ C inlet temperature.Elemental analyses were performed on a Perkin-Elmer 240 microanalyser in the Faculty of Science, Cairo University.Microwave irradiations (MWIs) were carried out in a SANYO EM-700T domestic oven (700 W).

Method II.
A solution of 4-aminoacetophenone (0.01 mole) in methanol (30 mL) and 2-aminothiazoles (0.01 mole) was put in round-bottomed flask placed in a microwave oven and irradiated for 2.0-3.5 min, and then the solvent was removed by vacuum distillation.The solid product was filtered, dried, and recrystallized from ethanol to give 3a-c (Table 3).

Synthesis of 2-{[4-(Substituted thiazol-2yl)iminoethyl)-phenyl]Hydrazono}-3-oxo-Butyric Acid Ethyl Ester 4a-c
A solution of sodium nitrite (0.01 mole) in water (10 mL) was added to an ice-cooled mixture of 3a-c (0.01 mole) in concentrated HCl (10 mL) and water (10 mL).The diazotized compound was dropped while cooling with stirring over a cold mixture of ethyl acetoacetate (0.01 mole) and sodium acetate (2 g in 10 mL water) in ethanol (20 mL).The reaction mixture was stirred at room temperature for 8 h.The precipitated solid was collected by filtration.Then it was washed with water, dried, and recrystallized to afford 4a-c (Tables 1 and 2).
The organic layer was washed with water.Then, it was dried with MgSO 4 and filtered.After that, the solvent was removed under vacuum to give 5a-f (Tables 1 and 2).

Method II.
A mixture of 4a-c (0.01 mole for each), 2a,b (0.01 mole for each), and glacial acetic acid (2 mL) in an Erlenmeyer flask was exposed to pulsed microwave irradiation using microwave oven for 3 min.The reaction mixture was poured onto crushed ice; the separated solid was filtered, then washed with water, and dried to give the pyrazolin-5-ones 5a-f (Table 3).
It has been found that the 4-aminoacetophenone reacted with 2-amino-5-methylthiazole or 2-aminobenzothiazole in DMF to give the imino derivatives (3b,c), similar to compound (3a) [23].It was found that the higher yields of compounds 3a-c were obtained at 500 watt for 2-3 min of microwave irradiation (Figure 2).
IR spectra of 4a-c revealed no absorption band in amino group region; furthermore, it displayed absorption bands at 1760 and 1560 cm −1 for ester group and (N=N), respectively.

Antimicrobial Activity
The antimicrobial activity of new compounds was investigated against a variety of microorganisms, including the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus and the gram-negative bacteria Escherichia coli as well as the unicellular fungi Candida albicans.The agar plate disc-diffusion method [24] was used to assess the activity of the compounds.Sterilized filter paper discs (5 mm in diameter) were wetted with 10 L each of a solution of the tested compound (10 mg/mL of the compound in DMF).The discs were then allowed to dry and were placed on the surface of agar plates seeded with the test organism.Nutrient agar was used for bacterial plating and sabourauds dextrose agar for fungi.Each plate contained 15 mL of the agar medium, previously seeded with 0.2 mL of an 18 h-old broth culture of each organism.The inoculated plates were incubated at 37 ∘ C for 48 h with the test discs in place, and the inhibition zones were measured in mm.Controls including the use of the solvent DMF without test compounds that showed no antimicrobial activity for this solvent.The antibacterial reference tetracycline discs and the antifungal reference nystatin discs were tested concurrently as standards.

Conclusion
This paper describes the synthesis, spectral characterization, and screening of antimicrobial activity of some 3methylpyrazolin-5-one derivatives bearing side chains, imino-thiazole-phenylhydrazone derivatives, and coumarin moiety.Microwave-assisted synthesis was also used to improve the yield and reaction time.The synthesized compounds showed a wide range of potentially promising antimicrobial activities.

Table 1 :
Characterization data of synthesized compounds.

Table 2 :
Comparison of conventional and microwave synthesis.

Table 3 :
Spectral data of synthesized compounds.

Table 4 :
Antimicrobial activity of prepared compounds.