INTRODUCTION:

Heterocyclic ring systems containing one oxygen and one nitrogen adjacently referred as isoxazole systems are well known for their medicinal importance¹. The synthesis of novel heterocyclic compounds continues to attract wide attention among synthetic chemists. Nitrogen-containing heterocyclics are of great importance to medical chemists². Isoxazole chemistry has obtained significant consideration over the past few decades due to their versatility in chemical synthesis as well as their usefulness in several fields like agriculture, industry and medicine.

Isoxazoles are involved in a wide spectrum of biological effects such as antitumor³, anthelmintic⁴, anti-inflammatory⁵ and anticonvulsant⁶. The marketed isoxazole drugs like Acetylsulfisoxazole, Cycloserine, Drazoxol and Zonisamide show tuberculostic⁷, anticonvulsant⁸, neurotoxic⁹ and antiepileptic^10-11activities. On the otherhand, benzimidazole moiety has been proved to be an important pharmaceutical agent.

Hence, it is worthwhile to exploit these simple molecules with different functionalities. Owing to their versatile chemotherapeutic importance, it appeared of interest to synthesize novel benzimidazolylisoxazole derivatives, to find their antioxidant property and to characterize structure of the most active compound by Density Functional Theory (DFT).

METHODS AND MATERIALS:

All the reagents used were of analytical grade purchased from E. Merck and were purified prior to use. Melting points were determined on Digital Program Rate melting point apparatus and were uncorrected. Proton NMR measurements were performed on a Bruker Advance III, 400MHz NMR spectrometer and chemical shifts were reported in δ(ppm). Mass spectral studies were performed on Waters UPLC - TQD mass spectrometer (ESI – MS).

Experimental Procedure:

Chalcone synthesis by Claisen-Schimidt Condensation:

Sodium hydroxide (22g) was dissolved in water (200ml) and rectified spirit (122.5 ml) and stirred well. The reaction mixture was immersed in a bath of crushed ice. 0.43 mol of 2-acetyl benzimidazole is added and start the stirrer. Then 0.43 mol of benzaldehyde derivative was added dropwise. Stirring was continued for 5 hours maintaining the temperature at 25ᵒC. The stirrer is removed and the reaction mixture was refrigerated overnight. The precipitate obtained was filtered and washed with cold water until the washings were neutral to litmus. The crude chalcone was dried and recrystallised from rectified spirit.

Isoxazole synthesis from chalcone:

Equimolar quantities of chalcone and hydroxylamine hydrochloride were taken, refluxed for 6 hours in the presence of sodium acetate in ethanol (25 ml). The mixture was then poured into ice water. The product obtained was filtered, dried and recrystallized from rectified spirit.

SCHEME:

5-phenyl-3-(benzimidazol-2-yl)-isoxazole C1:

¹H NMR (CDCl₃): 7.52-7.73(m, 4H, ArH), 7.26-7.51(m, 5H, ArH), 11.45(s, 1H, NH), 5.48(s, 1H, CH); Mass: m/z 262 (M⁺)

5-(4-methoxyphenyl)-3-(benzimidazol-2-yl)-isoxazole C2:

¹H NMR (CDCl₃): 7.51-7.73(m, 4H, ArH), 7.17-7.49(m, 4H, ArH), 11.77(s, 1H, NH), 5.40(s, 1H, CH), 3.59(s, 3H, OCH₃); Mass: m/z 292 (M⁺)

5-(4-chlorophenyl)-3-(benzimidazol-2-yl)-isoxazole C3:

¹H NMR (CDCl₃): 7.52-7.73(m, 4H, ArH), 7.16-7.52(m, 4H, ArH), 11.7(s, 1H, NH), 5.51(s, 1H, CH); Mass: m/z 295 (M⁺)

5-(4-methylphenyl)-3-(benzimidazol-2-yl)-isoxazole C4:

¹H NMR (CDCl₃): 7.42-7.73(m, 4H, ArH), 6.76-7.39(m, 4H, ArH), 11.83(s, 1H, NH), 5.60(s, 1H, CH), 2.34(s, 3H, CH₃); Mass: m/z 276 (M⁺)

DPPH scavenging assay:

The samples were made up with methanol to different concentrations (50, 100, 250, 500 and 750 μM). 2ml of each sample was allowed to react with 2ml of stable free radical, 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH) for half an hour at 37º C. The deep purple colour of the DPPH solution turns yellow in the presence of antioxidants. The concentration of DPPH was kept as 10^-5 mol. After incubation, the absorbance of all the solutions was measured at 517nm. tert-butyl-4-hydroxyanisole (BHA) was used as standard. Percent anti-oxidant activity of the samples was determined in comparison with a methanol treated control group by using the following formula:

OD test compound-OD Control

% Antioxidant activity=--------------------------------× 100

OD control

Where, OD is the optical density.

Two replicates of the analysis were undertaken and results averaged. A linear regression plot with abscissa and ordinate as concentration of samples and % antioxidant activity respectively was plotted to determine the IC₅₀ (effective concentration for scavenging 50% of the initial DPPH) values.

DFT calculation:

All the Density Functional Theory calculations were done using Gaussian 09 program package at Becke'sthree parameter hybrids function combined with the Lee-YangParr correlation functional (B3LYP) level using the 6-31G (d) basis set for the ground state optimization^12-13. The base of DFT is that the fundamental state of a polyelactronic system is determined through electronic density instead of wave function. The optimized geometry of the molecule was found by optimizing all geometrical variables. The electronic properties like frontier molecular orbital energy, ionisation potential, hardness, electronegativity were found.

RESULTS AND DISCISSON:

In our study, a new series of compounds C1-C4 were synthesized. The spectral data of all the compounds are in accordance with assigned structures. The multiplet obtained at δ 7.42-7.73 ppm shows the benzimidazole hydrogens. Compound C2, the 4-methoxy analog shows the presence of the methoxy group at δ 3.5ppm. The presence of the methyl group in C4 is indicated by the singlet at δ 2.34 ppm. The m/z peaks obtained in the mass spectra further confirmed the assigned structures.

Antioxidant activity:

The synthesized compounds were evaluated for their DPPH free radical scavenging activity. The results of the study shows that all the compounds showed significant antioxidant activity when compared with the standard antioxidant BHA and the scavenging rates were in the range of 34-72%. It is found from the IC50 data that compound C4 with lowest anti-radical capacity was having IC50 value 405 μM followed by C1 with IC50 386 μM and compound C3 was found to be more active (145 μM ) than C2 (188 μM) while BHA showed IC50 value 624 μM.

The synthesized compounds were considerably hydrophobic with estimated mean logP values of 3.25. Hence it appears that the compounds are less soluble in the hydrophilic fluids in the oral route.

Theoretical studies:

According to the frontier molecular orbital theory, the frontier molecular orbitals provide useful information about the biological activity. We selected the compound C3 which showed the highest antioxidant activity, for the DFT study.

Fig1: Energy minimized structure of C3

The HOMO of the compound is located on the phenyl ring of benzimidazole while, the LUMO of the title compound is located on the pyrazole ring. From Figure 2, the electron transition ocurrs from benzimidazole to pyrazole ring. The energy gap between the HOMO and LUMO is 0.2220 au. The higher energy gap shows the higher hardness and stability of the compound.

Table 1: Calculated electronic parameters of C3

Parameters (a.u)	Values
E_HOMO	-0.2498
E_LUMO	-0.0278
∆E	0.222
Electronegativity (χ)	0.1388
Ionisation Potential (I)	0.2498
Hardness (ղ)	0.111

The HOMO and LUMO enegies relate the structure of the compound to its reactivity. The chemical potential properties of C3 calculated by DFT is given in Table 1. Ionisation potential is the energy required to remove an electron from a molecule. The value obtained reveals that the molecule does not loss electrons easily^14-15. Electronegativity is thne tendency to attract electrons while, hardness is the measure of resistance to charge transfer. The results of the above properties show that C3 is having good antioxidant activity^16-17due to similarity with the chemical potential properties of (+)catechin and (-)epicatechin¹⁸.

CONCLUSION:

Some interesting benzimidazolylpyrazole derivatives were designed and synthesized. Their structures were confirmed using NMR and mass spectral analysis. The antioxidant study of the synthesized derivatives showed that among the four compounds, 5-(4-chlorophenyl)-3-(benzimidazol-2-yl)-isoxazole(C3) showed best anti-oxidant activity. It was selected for the DFT study. The frontier molecular orbitals were characterized.The reactivity descriptors of C3 such as HOMO-LUMO energy gap, ionisation potential, electronegativity and hardness describe the reactive sites of interaction. It is seen from these properties that the molecule donates electros easily which is essential for efficient antioxidant activity.

ACKNOWLEDGEMENT:

We are thankful to University Grants Commission (UGC), New Delhi for the financial support for carrying out the research and CDRI, Lucknow for spectral analysis.

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Received on 09.10.2017 Modified on 13.11.2017

Asian J. Research Chem. 2018; 11(1):65-68.

DOI:10.5958/0974-4150.2018.00014.7