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Volume: 9, Issue: 9, September, 2019
DOI: 10.7324/JAPS.2019.90915



Research Article

Synthesis, in-vitro antioxidant activity and in-silico prediction of drug-likeness properties of a novel compound: 4-(3,5-Di-tert-butyl- 4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one

Madhavi Kuchana, Durga Rani Bethapudi, Ragini Katamarajugari Ediga, Yoshitha Sisapuram

  Author Affiliations


Abstract

A simple and eco-friendly method has been used for the synthesis of novel compound 4-(3,5-di-tert-butyl-4- hydroxybenzylidene)-3-methylisoxazol-5(4H)-one. The synthesized compound was characterized by its physical and spectral data. This compound was screened for in-vitro antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide free radical scavenging assays at 100 μM concentration. The DPPH scavenging ability of evaluated compound was superior to the synthetic antioxidant butylated hydroxytoluene (BHT) and comparable to the standard ascorbic acid. The nitric oxide scavenging ability of the compound was also found greater than the BHT. The in silico prediction of molecular properties and bioactivity scores of synthesized compound and BHT were carried out using Molinspiration Cheminformatics online software. The study revealed that the predicted compounds obeyed Lipinski’s rule of five and showed good intestinal absorption and membrane permeability indicating the drug-likeness properties of predicted compounds. Finally, the in silico study identified the title compound as a nuclear receptor ligand compared to BHT.

Keywords:

Butylated hydroxytoluene, 2, 6-Di-tert-butylphenol, 4-Substituted benzylidene-3- methylisoxazol-5(4H)-one, In-vitro antioxidant activity, In-silico prediction.



Citation: Kuchana M, Bethapudi DR, Ediga RK, Sisapuram Y. Synthesis, in-Vitro antioxidant aActivity and in-silico prediction of drug likeness properties of a novel compound: 4-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one. J Appl Pharm Sci, 2019; 9(09):105–110.


Copyright: The Author(s). This is an open access article distributed under the Creative Commons Attribution Non-Commercial License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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INTRODUCTION

Butylated hydroxytoluene (BHT), a synthetic antioxidant primarily used as a preservative in food, pharmaceuticals, and cosmetics. It can be used as an antioxidant in many industrial products and laboratory chemicals. It has several other names, which include 3,5-di-tert-butyl-4-hydroxytoluene, 2,6-di-tert-butyl-p-cresol, 2,6-di-tert-butyl-4-methylphenol, and Ionol. Generally, it can be prepared from p-cresol by dibutylation or from 2,6-di-tert-butylphenol by aminomethylation or hydroxymethylation followed by hydrogenolysis. Various synthetic derivatives containing BHT or 2,6-di-tert-butylphenol moiety such as chalcones (Cabral et al., 2017; Won et al., 2005), hydrazones (Cuadro et al., 1998; Duarte et al. 2007; Podyachev et al., 2012), styryl heterocycles (Flynn et al., 1991; Lazer et al., 1989; Kelarev et al., 2003; Madhavi et al., 2010; Mullican et al., 1993), benzylidene compounds (Clemens et al., 1991; Hori et al., 2002; Ikuta et al., 1987; Inagaki et al., 2000; Katsumi et al., 1986; Phillips et al., 1992; Unangst et al., 1994; Wong et al., 1992), and other heterocyclic systems (Gowdra et al., 2014; Isomura et al., 1983a; 1983b; 1984; Jeong et al., 2004; Unangst et al., 1992; Ziakas et al., 2006) were reported to have anti-inflammatory activity with dual inhibition of 5-lipoxygenase and cyclooxygenase enzymes as well as low ulcerogenic liability. Several of these compounds were also reported to have anti-obesity, anti-tumor, and antioxidant activities. The drugs containing BHT or 2,6-di-tert-butylphenol were available commercially, which include Tazofelone®, Darbufelone®, Prifelone®, and Tebufelone® used to treat inflammatory conditions and Eldacimibe® used for the treatment of hyperlipidemia.

Isoxazol-5(4H)-one belongs to the class of five-membered heterocyclic compound and known to possess a variety of biological and pharmacological activities (Ishioka et al., 2003; Kafle and Cho, 2012; Laughlin et al., 2005; Srikantamurthy et al., 2018). Especially, 4-substituted benzylidene-3-methylisoxazol-5(4H)-ones were utilized as versatile building blocks for the synthesis of fused heterocycles (Soliman et al., 2013). In the literature, the synthesis of 4-substituted benzylidene-3-methylisoxazol-5(4H)-ones was reported through a one-pot reaction of ethyl acetoacetate with hydroxylamine hydrochloride and substituted benzaldehyde using different reagents, catalysts, and/or reaction medium (Chavan et al., 2014; Vekariya and Patel, 2017). However, no research reports found regarding the preparation of 4-(3,5-di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one. Hence, the present study aimed to prepare the title compound and to evaluate preliminarily its in vitro antioxidant properties.

A number of computational techniques to identify drug-like molecules were developed in recent years (Kadam and Roy, 2007). Molinspiration Cheminformatics software, a free online service (https://www.molinspiration.com) used for the computational prediction of molecular properties (Molinspiration property engine v2018.10) such as log p, polar surface area, molecular weight, hydrogen bond donors, hydrogen bond acceptors and others, as well as bioactivity score (Molinspiration bioactivity score v2018.03) as GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor. Therefore, the present study also aimed to evaluate drug-likeness properties of the title compound using Molinspiration Cheminformatics software.


MATERIALS AND METHODS

Reagents and Instruments

3,5-Di-tert-butyl-4-hydroxybenzaldehyde was procured from Alfa Aesar, and all other chemicals were obtained from Sigma Aldrich and SD fine chemicals. The melting points were determined in open capillaries on tempo melting point apparatus and they were uncorrected. The purity of compounds was checked by thin-layer chromatography using the glass plates coated with silica gel-G and spots were detected by iodine vapor. The IR spectrum was recorded using KBr Pellet on a BRUKER Infrared spectrophotometer (cm−1). 1H NMR and 13C NMR spectra were recorded on BRUKER NMR spectrometers (400 MHz for 1 H NMR and 100 MHz for 13C NMR) and the chemical shifts expressed as δ values (ppm) downfield from tetramethylsilane as an internal standard. The mass spectrum was recorded on LC-MS, Agilent Technology 1200 infinity series, Apex chromatogram model.

Synthesis of 4-(3,5-di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one (V, Scheme—I)

To a solution of 0.139 gm (0.002 mol) of hydroxylamine hydrochloride (I) in 2 ml of absolute alcohol, added 0.164 gm (0.002 mol) of anhydrous sodium acetate and 0.25 ml (0.002 mol) of ethyl acetoacetate (II). The reaction contents were stirred for 1 hour to give an oxime initially, followed by its cyclization to form an intermediate 3-methylisoxazol-5(4H)-one (III). The formation of intermediate compound III was confirmed by TLC. To the above reaction mixture, added 0.468 gm (0.002 mol) of 3,5-di-tert-butyl-4-hydroxybenzaldehyde (IV) and the reaction contents were refluxed for 1 hour. The completion of the reaction was monitored by TLC. Then the solution was cooled and the crude product obtained was collected by filtration. Finally, recrystallization of the crude compound with aqueous alcohol resulted in a bright yellowish crystalline powder. Yield 72%; m.p 200°C–202ºC; IR (KBr, cm-1) ʋmax: 3615 (O-H stretch, free), 3446 (O-H stretch, bonded), 3116 (Ar C-H stretch), 2961, 2922 (Ali C-H stretch), 1742 (C=O stretch), 1616 (C=N stretch), 1569 (C=C stretch), 1218, 1118 (C-O stretch), 812 (C-H def); 1H NMR (DMSO-d6) δ ppm: 1.43 (s, 18H, -C(CH3)3), 2.27 (s, 3H, -CH3), 7.87 (s, 1H, -CH=), 8.41 (s, br, 1H, OH), 8.46 (s, 2H, Ar-H) ppm; 13C NMR (DMSO-d6) δ ppm: 168.9 (C=O), 162.3 (C=N), 160.7 (C-OH), 152.8 (CH= benzylidene), 138.4 (2C C-C(CH3)3 Aromatic), 132.8 (C-CH= Aromatic), 124.8 (C=CH isoxazole), 113.8 (2C Ar), 34.7 (2C -C(CH3)3), 30.0 ( 6C -C(CH3)3), 11.3 (CH3 isoxazole); MS m/z: 314 (100%, M-H¯).

Scheme I. Synthesis of 4-(3,5-di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one (V).



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In-vitro antioxidant activity

DPPH free radical scavenging assay

To the test compound in absolute alcohol (100 µM), freshly prepared DPPH solution in absolute alcohol (100 µM) was added and the solution kept at room temperature for 20 minutes and the absorbance measured at 517 nm (Blois, 1958). The control experiment carried out in a similar manner without the test compound. The standard compounds ascorbic acid and BHT were used as positive controls for comparing the results. The results were expressed as the average of triplicate measurements. The percentage of the DPPH radical scavenging was calculated using the following formula.

Percentage of DPP Hradical scavenging = ( Control Test ) Control × 100

Nitric oxide free radical scavenging assay

Sodium nitroprusside (5 mM) in phosphate-buffered saline was added to 100 µM concentration of test compound dissolved in alcohol and incubated at 25°C for 150 minutes. The control experiment was carried out in a similar manner without test compound but with an equal amount of solvent. The standard BHT was used as a positive control for comparison. After incubation, 2 ml of incubated solution was added to 2 ml of Griess reagent (1% Sulfanilamide, 2% H3PO4, and 0.1% N-(1-Naphthyl)ethylenediamine dihydrochloride). The absorbance of chromophore formed during the diazotization of nitrite with sulfanilamide and subsequent coupling with N-(1-Naphthyl) ethylenediamine was read at 546 nm (Sreejayan and Rao, 1997). The percentage of the nitric oxide free radical scavenging was calculated using the following formula.

Percentage of nitric oxide scavenging = ( Control Test ) Control × 100

Prediction of molecular properties and bioactivity score of 4-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one using Molinspiration Cheminformatics software

The Molinspiration Cheminformatics online molecular property calculation toolkit was used to know the drug-likeness of the synthesized compound and standard BHT based on Lipinski’s rule of five. The structures of the title compound and standard BHT were generated and their molecular properties such as log P value, molecular weight, Topological Polar Surface Area (TPSA), hydrogen bond acceptor/donor, rotatable bonds, and molecular volume were calculated for the prediction of absorption, distribution, metabolism, and elimination properties. Percentage of absorption of the compounds was estimated using the following equation: %ABS = 109 – (0.345 X TPSA) (da Silva et al., 2015). The bioactivity scores of the synthesized compound and BHT as GPCR ligands, ion channel modulators, kinase inhibitors, nuclear receptor ligands, protease inhibitors, and enzyme inhibitors were also predicted using the same Molinspiration Cheminformatics software.


RESULTS AND DISCUSSION

Chemistry

In the present research work, a novel compound 4-(3,5-di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one (V) was synthesized by condensation of active methylene group of 3-methylisoxazol-5(4H)-one (III) with 3,5-di-tert-butyl-4-hydroxybenzaldehyde (IV). The intermediate 3-methylisoxazol-5(4H)-one (III) was generated by stirring equivalent amounts of hydroxylamine hydrochloride (I) and ethyl acetoacetate (II) in alcohol (Cocivera et al., 1976; Wazalwar et al., 2017) containing equimolar quantity of sodium acetate and the resultant intermediate was utilized for the next step without isolation. The acetic acid generated during the preparation of 3-methylisoxazol-5(4H)-one (III) acts as a catalyst in the condensation reaction. Hence, this reaction may be considered as an eco-friendly and simple synthetic method. The yield of the synthesized compound was good and found to be 72%. Furthermore, it was characterized by physical and spectral analytical data.

The IR spectrum of the compound V showed absorption bands at 3615 and 3446 cm-1 due to free as well as bonded O-H stretching vibrations. The absorption bands due to aromatic C-H stretching vibrations appeared at 3116 cm−1 and the aliphatic stretching vibrations appeared at 2961 and 2922 cm−1. The absorption band due to α,β unsaturated carbonyl group appeared at 1742 cm-1. The spectrum also revealed the presence of absorption bands at 1616 and 1569 cm−1 due to C=N and C=C stretching, respectively. The 1H NMR spectrum of the compound V revealed the presence of more intense singlet peak at δ 1.43 due to 18 protons of the di-tert-butyl group. A singlet at δ 2.27 indicates the protons of the methyl group of isoxazole ring. The benzylidene proton and aromatic protons appeared as two different singlets at δ 7.87 and δ 8.46. The phenolic proton appeared as a broad singlet at δ 8.41. The 13C NMR spectrum of compound V showed assignable peaks at δ 168.9, δ 162.3, δ 160.7, δ 152.8, δ 138.4 (2C), δ 132.8, δ 124.8, δ 113.8 (2C), δ 34.7 (2C), δ 30.0 (6C), and δ 11.3 ppm. The mass spectrum of the compound V showed characteristic [M-H]̅ at 314. These spectral analytical data confirm the structure of the title compound.

In-vitro antioxidant activity

In the present study, the synthesized compound was evaluated preliminarily for in-vitro antioxidant property as it contains the synthetic antioxidant BHT in its structure. Two different models, DPPH and nitric oxide free radical scavenging assays, were selected for screening the antioxidant potential of compound V at 100 µM concentration. The activity data were presented in Table 1. The scavenging of DPPH free radical by compound V was 84.64%, near to the value found with the standard compound ascorbic acid (87.15%). Furthermore, the DPPH free radical scavenging ability of compound V appeared greater than the standard compound BHT (55.17%). The compound V also showed better activity in scavenging nitric oxide free radical (57.71%) when compared with the standard BHT (38.19%). The greater activity of compound V may be due to the cyclic α,β unsaturated carbonyl system formed by linkage of BHT moiety to 3-methylisoxazol-5(4H)-one at 4-position. This observation was in confirmation with our previous report, that the α-cyano-N-(4-hydroxyphenyl) cinnamamide with BHT moiety/3,5 di-tert-butyl-4-hydroxy substitution exhibited good radical scavenging properties (Madhavi et al., 2019).

Prediction of molecular properties and bioactivity score of 4-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one using Molinspiration Cheminformatics software

The molecular properties of the synthesized compound and the reference compound BHT were calculated using Molinspiration Cheminformatics software. The data were presented in Table 2. The predicted data revealed that both the compounds obeyed the Lipinski’s rule of five; thus, indicated their drug-likeness. However, the log P value of compound V was found 4.66, within the acceptable criteria (log p ≤ 5), whereas the log P value of standard compound BHT was 5.43. The log P measurement used to understand the substance solubility behavior and hence their oral absorption and bioavailability. The compound V possesses an adequate number of hydrogen bond acceptors (HBA = 4) and hydrogen bond donor (HBD = 1), ensuring efficient interaction with hydrogen bonding groups of the receptor. The number of rotatable bonds found with compound V was 3, indicating the conformational flexibility when compared with BHT. The computational TPSA value of compound V was found 63.33 Ǻ2 and %ABS was 87.15% indicative of good intestinal absorption and membrane permeability, thus encouraging its use as a new drug candidate.

Table 1. In vitro antioxidant activity of 4-(3,5-di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one (V).



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Prediction of bioactivity scores of synthesized compound and BHT was performed by Molinspiration Cheminformatics software and the data presented in Table 3. As a general rule, the larger the bioactivity score, higher the probability that the predicted compound would be active. Therefore, a molecule having bioactivity score more than 0.00 expected to possess considerable biological activities, while the values between −0.50 and 0.00 considered to be moderately active and the score less than −0.50 presumed to be inactive (Verma, 2012). The results of in-silico prediction indicated that the compound V was more active as nuclear receptor ligand compared to standard compound BHT. Furthermore, the bioactivity score of compound V as nuclear receptor ligand was found more than the previously synthesized and predicted compound containing 3,5-di-tert-butyl-4-hydroxy substitution on the phenyl ring of α-cyano-N-(2-hydroxyphenyl) cinnamamide (Madhavi and Renuka, 2018). The higher predictive bioactivity score of the compound V as nuclear receptor ligand may be attributed to the overall estimated TPSA value of the molecule and other molecular properties, mainly hydrogen bond characteristics, hydrophobicity, electronic distribution, molecule size, and flexibility. In addition, the presence of pharmacophoric groups and/or structural similarity with the training set molecules may also contribute to the predictive high bioactivity score as nuclear receptor ligand.

Table 2. Molecular properties prediction of 4-(3,5-di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one (V) using molinspiration cheminformatics software.



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Table 3. Bioactivity score prediction of 4-(3,5-di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one (V) using molinspiration cheminformatics software.



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CONCLUSION

4-(3,5-Di-tert-butyl-4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one, a novel isoxazol-5(4H)-one derivative containing sterically hindered phenolic moiety BHT, was successfully synthesized with good yield and purity by cyclocondensation of ethyl acetoacetate and hydroxylamine hydrochloride in the presence of sodium acetate followed by subsequent condensation of resultant 3-methylisoxazol-5(4H)-one with 3,5-di-tert-butyl-4-hydroxybenzaldehyde. The in-vitro antioxidant study revealed that the title compound possesses extremely good DPPH free radical scavenging ability comparable to the standard compound ascorbic acid and greater than the BHT. The title compound also exhibited good nitric oxide scavenging ability better than the BHT. The better in-vitro antioxidant properties of compound V indicates the structural importance of 3,5-di-tert-butyl-4-hydroxybenzylidene moiety conjugating to the carbonyl group of 3-methylisoxazol-5(4H)-one. Since the title compound exhibited better in-vitro antioxidant activity, it can be further investigated against the diseases associated with oxidative stress and inflammation.

The in-silico prediction of molecular properties of the synthesized compound and the standard BHT revealed that these compounds obeyed Lipinski’s rule of five and showed good intestinal absorption as well as membrane permeability indicating the drug-likeness properties. In addition, the bioactivity score prediction identified the title compound as the most promising nuclear receptor ligand compared to the standard BHT. Therefore, further studies needed to be performed to know the safety and therapeutic efficacy of the title compound.


ACKNOWLEDGMENTS

The Authors thank the DST-CURIE Centre, Sri Padmavati Mahila Visvavidyalayam (Women’s University) for providing IR spectrum and Laila Impex R & D Centre, Vijayawada, Andhra Pradesh, for providing 1H NMR, 13C NMR, and Mass spectra.


CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.


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