Pharmacological and insect antifeedant activities of some 3-(2,4-dichloro-5-fluorophenyl)-5-(substituted phenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamides

A series containing twelve titled compounds were synthesized by solvent-free method and their purities were examined by literature procedure. These compounds were subjected to study the pharmacological effects such as antibacterial, antifungal and antioxidant activities and the insect antifeedant activities using Bauer-Kirby disc diffusion method with their bacterial and fungal strains, DPPH radical scavenging and leaf disc bio-assay method with 4 th instar larvae Achoea janata L.

In these derivatives, the formation of pyrazoline ring was confirmed by infrared and NMR spectra. From infrared spectra the cyclic C=N and N-N are the characteristics stretches (ν, cm -1 ) [29]. The protons of the pyrazoline rings are existed in the trans-positions gave doublet of doublets and this is confirmed by 1 H NMR spectra. The Hammett spectral correlation study was reported in the literature [30].
The infrared stretches, 1H and 13C chemical shifts data of the pyrazolines were correlated with Hammett equation using substituent constants, F and R parameters applying single and multiple correlation equations [31]. From the results of statistical analysis, the effects of substituents on the spectral frequencies were studied. Ranganathan et al have studied the synthesis, spectral correlations and antimicrobial activities of some 3-(5chlorothiophen-2-yl)-4,5-dihydro-5-(substituted phenyl)-1Hpyrazoline derivatives [28].
Thirunarayanan and Sekar have studied the solvent-free synthesis and spectral correlations of some 1-acetyl pyrazolines [32]. Within the view there is no report available for the study of pharmacological and insect antifeedant activities of some pyrazoline carbothioamides in past in the literature. Therefore the author have taken efforts for studying the pharmacological effects such as antibacterial, antifungal and antioxidant activities and the insect antifeedant activities using Bauer-Kirby [33] disc diffusion method with their bacterial and fungal strains, DPPH [34] radical scavenging and leaf disc bio-assay method [35] with 4 th instar larvae Achoea janata L.

1. Synthesis of pyrazoline thiocarbamides
The titled pyrazoline thiocarbamides were synthesized and their purities were examines by literature method [36]. The general structure of the pyrazoline thiocarbamides was shown in Fig. 1.
The disc diffusion technique followed the Bauer-Kirby [33] method, at a concentration of 250 µg/mL with ampicillin and streptomycin used as the standard drugs. For the study of antifungal activities of all pyrazoline thiocarboamides with Candida albicans the disc diffusion technique was followed, while the two other strains (Penicillium sp. and Aspergillus niger), the dilution method [33] was used. The drug dilution was 50 µg/mL. Griseofulvin was used as the standard drug.

1. Measurement of antibacterial sensitivity
The antibacterial sensitivity assay was performed using the Bauer-Kirby [33] disc diffusion technique. In each Petri plate about 0.5 mL of the bacterial test sample was spread uniformly over solidified Mueller-Hinton agar using a sterile glass spreader.
Then 5 mm discs made from Whatman No. 1 filter paper were saturated with the potential inhibitor solution and placed on the medium using sterile forceps. The plates were incubated for 24 h at 37 °C upside down to prevent the collection of water droplets over the medium.
After 24 h, the plates were examined and the diameter values of the zone of inhibition were measured. Triplicate results were recorded.

Measurement of antifungal sensitivity
Antifungal sensitivity was determined by using the Bauer-Kirby [33] disc diffusion technique. The PDA medium was prepared and sterilized as above and added to the Petri plate containing 1 mL of the fungal species. The plate was rotated clockwise and counter clockwise for uniform spreading. The discs were impregnated with the test solution, prepared by dissolving 15 mg of the 3-(2,4-dichloro-5-fluorophenyl)-5-(substituted phenyl)-4,5dihydro-1H-pyrazole-1-carbothioamides in 1 mL of DMSO solvent.
The medium was allowed to solidify and incubate for 24 h. The plates were examined and the diameter of the zone of inhibition was measured. Triplicate results were recorded.
After 30 min at room temperature the absorbance of each solution was measured by UV spectrophotometry at 517 nm. A mixture of buffer solution and ethanol was used as the reference for the spectrophotometer. A graph was plotted with the weight of the 3-(2,4dichloro-5-fluorophenyl)-5-(substituted phenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamides versus absorption and IC 50 values were determined. The antioxidant activity was expressed in terms of IC 50 (µg/mL, concentration required to inhibit DPPH radical formation by 50 %). α-Tocopherol was used as a positive control. The radical scavenging activity was calculated as DPPH radical scavenging activity (% of inhibition) = 100 absorbance Control absorbance Sample -absorbance Control ×
This test was performed with a 4 th instar larva Achoea janata L against castor semilooper, were reared as described on the leaves of castor, Ricinus communis in the laboratory at the temperature range of 26 °C ±1 °C and a relative humidity of 75-85 %. The leaf -disc bioassay method was used against the 4 th instar larvae to measure the antifeedant activity [36]. The 4 th instar larvae were selected for testing because the larvae at this stage feed very voraciously.
Castor leaf discs of a diameter of 1.85 cm were punched and intact with the petioles. The synthesized aryl 3-(2,4-dichloro-5-fluorophenyl)-5-(substituted phenyl)-4,5-dihydro-1Hpyrazole-1-carbothioamides were dissolved in acetone at a concentration of 200 ppm dipped for 5 minutes. The leaf discs were air-dried and placed in one litre beaker containing little water in order to facilitate translocation of water.

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These latter compounds are moderately active, with 13-19 mm zones of inhibition. Carbothioamides 7, 8, 11 and 12 were active with an 8-12 mm of zone of inhibition.
The compounds 1 and 4 were inactive. The pyrazoline thiocarbamides 2, 3 and 6 were found to be effective against S. aureus strain with 20-24 mm of zones of inhibition. Compounds 5 and 10 are moderately active with 13-19 mm of zones of inhibition. The thioamides 4, 11 and 12 were moderately active with an 8-12 mm zone of inhibition .  Compounds 1, 7, 8 and 9 were inactive against S. aureus.
The pyrazoline thiocarboamide derivatives 3 and 5 were shown to be more active against Pseudomonas, with greater than a 20 mm zone of inhibition, while the other derivatives showed zones of inhibition between 12-19 mm. Compound 4 and 6 are inactive against the Pseudomonas aeruginosa strain. Pyrazoline thiocarbamides 2, 3, 6 and 9 were more effective against the Klebsiella pneumoniae strain with 20-24 mm zones of inhibition, while the thioamides 5 and 10 showed moderate activity with a 13-19 mm zone of inhibition. The parent compound 1, 11 and 12 were active with an 8-12 mm zone of inhibition. Compounds 4, 7 and 8 were inactive against the K. pneumoniae species.
The zone of inhibition of pyrazoline thiocarboamides 3, 6 and 10 were most effective against Aspergillus niger relative to compounds 1, 2, 4, 8 and 9. The compounds 4, 11 and 12 shows two fungal colonies and compound 7 showed little to no effectiveness with any fungal strain. The presence of a chloro, methoxy and nitro substituents appear to be responsible for the antimicrobial activities of pyrazoline thiocarboamides.
The chloro-, methoxy-and nitro-substituents of the pyrazoline thiocarboamides have good antimicrobial activities. The antioxidant activities of the thiocarboamides derivatives were measured by a DPPH radical scavenging method; the compounds containing hydroxy and methoxy substituents showed antioxidant activity. Compound 3 shows good insect antifeedant activity against the 4 th instar larvae Achoea Janata L with castor leaf disc bioassay method.