Pyrazolines as Potent Antioxidant Agents

 

K. Ishwar Bhat¹, Abhishek Kumar^2*

¹Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences, Nitte (Deemed to be University), Paneer, Deralakatte-575018, Mangalore, Karnataka.

²Assistant Professor, (Department of Pharmaceutical Chemistry) NGSM Institute of Pharmaceutical Sciences Paneer, Deralakatte, Mangalore 575018, Karnataka, India.

 

ABSTRACT:

A series of novel pyrazoline derivatives have been synthesized known for their 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 antioxidant activity studies. Most of the compounds have exhibited promising antioxidant activities.

 

 

 

INTRODUCTION:

Pyrazolines are well known and important nitrogen containing 5-membered heterocyclic compounds and various methods have been worked out for their synthesis. 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, phenyl hydrazine,  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.¹ H 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.

 

Evaluation of Antioxidant Activity:

DPPH radical scavenging activity:

The stable radical DPPH was used as the reagent in this spectrophotometric assay¹⁵. 3 ml of 0.1 mM solution of DPPH and 1 ml of the compounds containing various concentrations (10-200 μg/ml) was mixed and preserved in dark for 30 min. Then it was incubated at 30 min at room temperature. Then the absorbance was measured against blank at 517 nm.

 

Nitric oxide free radical scavenging method:

By using this reagent sodium nitroprusside, nitric oxide radicals (NO) were generated. To this different concentrations (10-200 μg/ml) of the test compounds, 1 ml of sodium nitroprusside (10 mM) and 1.5 ml of phosphate buffer saline (0.2 M, pH 7.4) were added. The resulting mixtures were incubated for 150 min at 25⁰C and 1 ml of the reaction mixture was treated with 1 ml of Griess reagent. The absorbance was measured at 546 nm. The results obtained were expressed for both antioxidant methods as percentage of inhibition, which was calculated according to the following equation¹⁶.

 

% Inhibition = Control absorbance - Test absorbance

                                     Control absorbance

 

RESULTS AND DISCUSSION:

Table 1: Physical data of substituted Pyrazolines

Comp. code	R	Mol. wt	M.P oC	Physical state	% Yield
PZ1	4-Cl	346	140-142	White crystals	81
PZ2	4- CH3	326	89-91	White crystals	76
PZ3	4-NO2	357	172-174	Yellow crystals	74
PZ4	4-OH	328	120-122	Brown crystals	83
PZ5	4-Br	390	180-182	Orange crystals	78
PZ6	4-F	330	157-159	White crystals	77

 

Table 2: Antioxidant activity of substituted Pyrazolines (PZ₁-PZ₆) by DPPH radical scavenging method.

Compound	R	% Inhibition at different concentration (μg/ml)
		10	50	100	200
PZ1	4-Cl	49	58	69	79
PZ2	4- CH3	40	49	54	60
PZ3	4-NO2	51	64	73	86
PZ4	4-OH	44	56	60	63
PZ5	4-Br	48	59	68	80
PZ6	4-F	49	61	75	84
Standard	Ascorbic acid	65	83	88	90

 

Table 3: Antioxidant activity of substituted Pyrazolines (PZ₁-PZ₆) by Nitric oxide free radical scavenging method.

Compound	R	% Inhibition at different concentration (μg/ml)
		10	50	100	200
PZ1	4-Cl	46	57	68	78
PZ2	4- CH3	39	46	55	61
PZ3	4-NO2	53	64	74	85
PZ4	4-OH	40	51	56	62
PZ5	4-Br	50	58	64	81
PZ6	4-F	51	59	67	86
Standard	Ascorbic acid	65	83	88	90

 

Antioxidant studies:

The test compounds were subjected to in vitro antioxidant activity by using both DPPH radical scavenging and nitric oxide free radical scavenging method by using ascorbic acid as standard drug. Compounds PZ₁, PZ₃, PZ₅ and PZ₆ showed very good activity compared to standard drug ascorbic acid. Radical scavenging activity in DPPH and nitric oxide methods increases with concentration and % inhibition at highest concentration 200 μg/is shown in the Table 2 and Table 3.

 

CONCLUSION:

The study reveals the successful synthesis of various substituted pyrazoline derivatives with good yields and most of the compounds showed potent antioxidant 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 the spectroscopic data.

 

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Accepted on 23.02.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(5):1978-1980.