Synthesis and Biological Evaluation of Some Novel Pyrazoline Derivatives Derived from Chalcones

 

K. Ishwar Bhat, Abhishek Kumar*

Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences, Nitte University, Paneer, Deralakatte-575018, Mangalore, Karnataka.

 

ABSTRACT:

A novel series pyrazoline derivatives have been developed as chemotherapeutic agents and have found wide clinical applications such as antibacterial, anticancer, antifungal and antitubercular agents. Chalcones are known to exhibit interesting pharmacological activities like anti-inflammatory, antileishmanial, antibacterial, antitumor, antimalarial and antitubercular activity. A series of pyrazoline derivatives [Substituted 1,5-diphenyl-3-(m-tolyl)-4,5-dihydro-1H-pyrazole]  were prepared from chalcones by the reported method, characterized by IR, ¹H NMR and mass spectra. The synthesized compounds were evaluated for antitubercular and cytotoxic activity studies. Most of the compounds have exhibited promising antitubercular and cytotoxic activities.

 

 

 

INTRODUCTION:

Heterocyclic compounds containing nitrogen like pyrazolines have attracted considerable attention in the recent years due to its versatile biological and pharmacological activities. Pyrazoline derivatives have been found to possess wide range of therapeutic activity such as antitubercular¹, anti-depressant², antitumor³, anti-inflammatory⁴, analgesic⁵, antibacterial⁶ and antifungal⁷ activities. The intermediate used are substituted chalcones from various aldehydes and ketones which are known for their antimalarial⁸, anticancer⁹, antioxidant¹⁰, analgesic¹¹ and anti-inflammatory¹² activities. Based on the observations it was decided to synthesize a novel series pyrazoline derivatives derived from chalcones to improve the biological spectrum of title compounds.

 

MATERIALS AND METHODS:

All the chemicals were of analytical grade: 4-Methyl acetophenone, substituted benzaldehyde, phenylhydrazine, sodium hydroxide, ethanol and glacial acetic acid.

 

Melting points were determined by open capillary method and are uncorrected. Purity of the intermediates and final compounds were monitored by thin layer chromatography (TLC) using silica gel G plates. The spots were visualized under UV light and by the exposure to iodine vapors. Chloroform: Ethylacetate (9:1) was used as solvent for running the TLC of these compounds. All IR spectra were recorded in Alpha Bruker using ATR method. ¹H NMR spectra were recorded on Bruker spectrophotometer (400 MHz) in DMSO solvent using tetra methyl silane (TMS) as an internal standard.. Mass spectra was recorded by using a Jeol-D 300 mass spectrometer (70 eV), Shimadzu (Japan) by FAB.

 

General Procedure:

Synthesis of Substituted 3-phenyl-1-(p-tolyl)prop-2-en-1-one¹³

A mixture of 4-methylcetophenone (0.01 mol) and substituted benzaldehyde (0.01 mol) in ethanol (20 ml)  were stirred for 24 hrs in presence of 20% NaOH (4 ml). The mixture was poured into crushed ice and acidified with 5% HCl. The product (chalcones) was filtered and recrystallised from alcohol.

 

Synthesis of Substituted 1,5-diphenyl-3-(p-tolyl)-4,5-dihydro-1H-pyrazole¹⁴ (PZ₁-PZ₆)

A mixture of substituted chalcones (0.01 mol) in 20 ml of glacial acetic acid and phenyl hydrazine (0.01 mol) were added and refluxed for 5-8 h. Then the reaction mixture was poured in 250 ml ice cold water. The solid separated was washed with cold water and recrystallised from ethanol. Chloroform: Ethylacetate (9:1) was used as solvent for running TLC and purity was confirmed.

 

Figure 1: Reaction Scheme for Pyrazoline derivatives

 

Spectral data:

1,5-Diphenyl-3-(p-tolyl)-4,5-dihydro-1H-pyrazole (PZ₁):

IR (cm^-1): 1645 (C=N str), 2925 (C-H aliphatic), 3020 (C-H str), 730 (C-Cl str), 1550 (C=C str).

 

¹H NMR (400 MHz, DMSO):

δ 7.1-7.2 (m, 13H, Ar-H), 2.2 (s, 3H of CH₃), 3.4-3.6 (dd, IH of H_a), 3.7-3.9 (dd, IH of H_b), 5.2-5.6 (dd, IH of H_c).

MS (M⁺): m/z 346.

 

1-Phenyl-3-(p-tolyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazole (PZ₂):

IR (cm^-1): 1642 (C=N str), 2931 (C-H aliphatic), 3024 (C-H aromatic), 1522 (C=C aromatic).

¹H NMR (400 MHz, DMSO): δ 7.1-7.5 (m, 13H, Ar-H), 2.1 (s, 3H of CH₃), 3.3-3.5 (dd, IH of H_a), 3.6-3.8 (dd, IH of H_b), 5.1-5.3 (dd, IH of H_c).

MS (M⁺): m/z 328.

 

5-(4-nitrophenyl)-1-phenyl-3-(p-tolyl)- 4,5-dihydro-1H-pyrazole (PZ₃):

IR (cm^-1): 1638 (C=N str), 2926 (C-H aliphatic), 3018 (C-H aromatic), 1549 (C=C aromatic).

¹H NMR (400 MHz, DMSO): δ 7.2-7.6 (m, 13H, Ar-H), 2.2 (s, 3H of CH₃), 3.1-3.3 (dd, IH of H_a), 3.4-3.7 (dd, IH of H_b), 5.2-5.6 (dd, IH of H_c).

MS (M⁺): m/z 359.

 

Antitubercular activity:

The antitubercular activity of synthesized compounds were assessed against Mycobacterium tuberculosis using microplate Alamar blue assay¹⁵. This methodology is non toxic, uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method. 200 µl of sterile deionized water was added to all outer perimeter wells of sterile 96 well plate to minimised evaporation of medium in the test wells during incubation. The 96 well plate received 100µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate. The final drug concentrations of the tested compounds were 0.01 to 20.0 µl/ml. The plates were covered and sealed with parafilm and incubated at 37^οC for 5 days. After this, 25  µl of freshly prepared 1:1 mixture of Alamar blue reagent and 10% tween 80 was added to the plate and incubated for 24 hours. A blue color in the well was interpreted as no bacterial growth and pink color was interpreted as growth. The minimum inhibitory concentration was defined as lowest drug concentration which prevented the color change from blue to pink. The compounds PZ₁, PZ₃, PZ₅ and PZ₆ induced significant effect on bacterial growth with an activity at a concentration of 6.25 µg/ml compared to the standard drug streptomycin and pyrazinamide. The results are tabulated in Table 2.

 

Cytotoxic Activity:

All the test compounds were screened for cytotoxic activity against Ehrlich Ascites Carcinoma cells (EAC). The tumor cells aspirated from peritoneal cavity of tumor bearing mice was washed thrice with normal saline and checked for viability using Trypan blue exclusion method¹⁶. The cell suspension (1 million cells in 0.1 ml) was added to tubes containing various concentrations of the test compounds and volume was made upto 1 ml using phosphate buffered saline. Control tubes contained only cell suspension. The assay mixtures were incubated for 3 h, at 37^οC and then percent of dead cells were evaluated by trypan blue exclusion method. Compounds PZ₁, PZ₃, PZ₅ and PZ₆ induced the greatest effect on EAC cells with an activity more than 60% at a concentration of 200 µg/ml. The results are summarized in Table 3.

 

RESULTS AND DISCUSSION:

Table 1: Physical data of Substituted Pyrazolines

 

Table 2: Antitubercular activity of compounds  (PZ₁-PZ₆) by Microplate Alamar blue assay.

 

Table 3: Cytotoxic activity of Compounds (PZ₁-PZ₆) by Trypan Blue exclusion method

 

CONCLUSION:

The study reports the successful synthesis of substituted pyrazoline derivatives with moderate yields and most of the synthesized compounds showed good potent antitubercular and cytotoxic activity.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Nitte University for providing the necessary facilities to carry out this research. The authors are grateful to Head, SAIF, Panjab University, Chandigarh for providing spectroscopic data and Amala Cancer Research Center, Thrissur.

 

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Accepted on 04.04.2017           © RJPT All right reserved