INTRODUCTION:

Free radicals are reactive species produced during normal metabolic and biochemical pathways as a part of normal immune reaction and for redox balance. Overproduction of free radicals results in cell damage as they can react with important cellular constituents like nucleic acids, lipids and proteins. Free radical involvement in pathophysiology of diseases and conditions like rheumatoid arthritis¹, Parkinson`s disease², diabetes³, cancer and inflammation⁴ is reported.

Flavonoids are polyphenolic substances occurring in wide range of plants. They are antioxidants as they can scavenge free radicals⁵, chelate metal ions and protect low density lipoproteins from oxidation⁶. In the present work, we report, for the first time in literature, the microwave assisted synthesis and antioxidant screening of novel 2-[3-(2,6-halophenyl)-4-methylisoxazol-5-yl]-substituted-4H-chromen-4-one derivatives.

MATERIAL AND METHODS:

All the chemicals were obtained from Sigma Chemical Co., Merck and SD Fine chemicals. Microwave synthesis was done on LG domestic microwave oven model MG-555F. TLC analyses were performed on a 3–10 cm aluminium sheet precoated with silica gel 60_254F (Merck). Silica, 200–400 mesh size (Merck), was used for column chromatography. Melting points were determined on open capillary tubes and are uncorrected.

Optical density was measured on Perkin Elmer Lambda 10 UV-visible spectrophotometer. IR spectra were recorded on Shimadzu FT-IR spectrum 100 spectrophotometer and the values are expressed in cm^-1. NMR spectra were recorded on a Brucker AV-III 400 NMR spectrometer using CDCl₃ as solvent.

General methods of synthesis:⁷

2-substituted-chromen-4-ones were prepared as per scheme 1. Substituted/unsubstituted phenols (1a-e) were acetylated with acetic anhydride to yield 2a-e; on refluxing 2a-e with anhydrous AlCl₃, acetylated phenols underwent Fries rearrangement⁸ to afford respective ortho-hydroxy acetophenones (3a-e) in good yield. Compounds 3a-e yielded acyl esters on treatment with aromatic acid chlorides, which further, in presence of base (pyridine/KOH), underwent Baker-Venkataraman rearrangement to 1,3-diketones⁹ (4a-j). These diketones afforded corresponding flavones (5a-j) by cyclization under strongly acidic condition¹⁰.

Synthesized flavones were characterized by disappearance of –OH stretch at 3240 cm^-1 and appearance of –C=O stretch at 1670-1650 cm^-1. A singlet peak at 2.8 δ corresponding to –CH₃ protons of methyl group of isoxazole ring in proton NMR confirmed the structure of the flavones. The physical characteristics of all the compounds are given in table 1.

Synthesis of acetylated phenols (2a-e):

Freshly distilled pyridine (10 ml) was added to solution of phenols 1a-e (0.05 mole) in acetic anhydride (0.10 mole). Reaction mixture was exposed to microwave (455 watt) for 6 min.

After confirming the completion of reaction by TLC, reaction mixture was cooled and product was extracted thrice with ethyl acetate-water (1:1). Ethyl acetate layer was separated, dried with anhydrous sodium sulphate and concentrated by vacuum distillation. Since the intermediate product obtained was semisolid, it was not possible to find out % yield in this step.

Synthesis of 2-hydroxyacetophenones by Fries rearrangement (3a-e):

Aluminum chloride (0.05 M) in carbon tetrachloride (15 ml) was added drop wise to acetylated phenol 2a-e (0.05 M). After complete addition of aluminum chloride reaction mixture was irradiated to microwave (245 watt) for 5 min. Intensity was increased to 455 watt and exposed for 3 minutes, so as to get ortho derivative as major product. After confirming the completion of reaction by TLC, solvent was removed by distillation. Ice cold 6N HCl was added to the residue. HCl gas liberated was allowed to escape. Product was extracted with ethyl acetate (3 x 15 ml), separated and dried over anhydrous sodium sulphate. Ethyl acetate was removed by distillation. The crude product obtained was used for next step as such.

Synthesis of substituted propane-1,3-dione derivatives (4a-j):

To the solution of 4-methyl-3-(2,6-substituted-phenyl)-isoxazole-5-carbonyl chloride (0.02 mole) in pyridine (10 ml), substituted salicylaldehyde (0.01 mole) and powdered potassium hydroxide (0.03 mole) was added by stirring and then exposed to microwave irradiation at 280 watt for 5 min. After cooling the reaction mixture, product was precipitated by pouring it to a container containing ice cold 6N HCl (50 ml). The solid collected by filtration was washed with ice cold water and recrystallized from aqueous ethanol.

Synthesis of chromen-4-one derivatives¹¹ (5a-j):

Intermediate 1,3-dione (0.01 mole) was dissolved in acetic acid (10 ml) and 0.1 ml of Sulphuric acid was added as condensing reagent. Reaction mixture was irradiated in microwave (490 watt) for 5 min and poured onto crushed ice after cooling, made basic by adding NaHCO₃. Precipitate formed was collected by filtration, washed with ice cold water and dried. The product was recrystallized from aqueous ethanol.

Table I – Physical data of compounds 5a-j.

Code	phenol	R₁	R₂	R₃ & R₄	Yield (%)	m.p. (°C)	Mol.formula	Mol. Wt.
5a	o-cresol	-H	-CH₃	-Cl	68	198-200	C₂₀H₁₃Cl₂NO₃	386.32
5b	p-cresol	-CH₃	-H	-Cl	70	113-114	C₂₀H₁₃Cl₂NO₃	386.32
5c	o-nitro phenol	-H	-NO₂	-Cl	76	132	C₁₉H₁₀Cl₂N₂O₅	417.09
5d	p-nitro phenol	-NO₂	-H	-Cl	79	160-161	C₁₉H₁₀Cl₂N₂O₅	417.09
5e	α-napthol	--	-C₆H₄-	-Cl	67	158	C₂₃H₁₃Cl₂NO₃	422.15
5f	o-cresol	-H	-CH₃	-F	59	135-136	C₂₀H₁₃F₂NO₃	353.32
5g	p-cresol	-CH₃	-H	-F	64	180-181	C₂₀H₁₃F₂NO₃	353.32
5h	o-nitro phenol	-H	-NO₂	-F	70	132-133	C₁₉H₁₀F₂N₂O₅	384.29
5i	p-nitro phenol	-NO₂	-H	-F	73	96-97	C₁₉H₁₀F₂N₂O₅	384.29
5j	α-napthol	--	- C₆H₄-	-F	71	149-150	C₂₃H₁₃F₂NO₃	389.35

2-[3-(2,6-dichlorophenyl)-4-methylisoxazol-5-yl]-8-methyl-4H-chromen-4-one (5a):

IR (KBr cm^-1): 3074.63 (=C-H), 1654.98 (-C=O), 748.21 cm^-1 (-C-Cl). ¹H NMR (400 MHz, CDCl₃) δ 7.48-7.06 (m, 7H, aromatic protons), 2.8447 (s, 3H, -CH₃ protons of isoxazole), 1.0442 (s, 3H, -CH₃ protons of chromene ring).

2-[3-(2,6-dichlorophenyl)-4-methylisoxazol-5-yl]-6-nitro-4H-chromen-4-one (5d):

IR (KBr cm^-1): 1658.84 (-C=O), 728.73 cm^-1 (-C-Cl). ¹H NMR (400 MHz, CDCl₃) δ 7.57-7.05 (m, 7H, aromatic protons), 2.85 (s, 3H, -CH₃ protons of isoxazole).

2-[3-(2,6-difluorophenyl)-4-methylisoxazol-5-yl]-8-nitro-4H-chromen-4-one (5h):

IR (KBr cm^-1): 1670.41 (-C=O), 828.41 cm ^-1(-C-F). ¹H NMR (400 MHz, CDCl₃) δ 7.58-6.91 (m, 7H, aromatic protons), 2.84 (s, 3H, -CH₃ protons of isoxazole).

2-[3-(2,6-difluorophenyl)-4-methylisoxazol-5-yl]-4H-benzo[h]chromen-4-one (5j):

IR (KBr cm^-1): 1670.41 (-C=O), 815.67 cm^-1 (-C-F). ¹H NMR (400 MHz, CDCl₃) δ 7.56-6.73 (m, 10H, aromatic protons), 2.77 (s, 3H, -CH₃ protons of isoxazole).

Antioxidant activity:

DPPH radical scavenging activity:

The DPPH free radical scavenging ability of the compound was assessed by the method described by Gülçin et al.¹² Various concentrations of sample solution (2 ml) were incubated at room temperature with DPPH (1ml, 0.1 mM) for 10 min and the absorbance was measured at 515 nm against blank. The free radical scavenging ability was calculated using following equation.

Scavenging effect (%) = (A_{C 515 nm}- A_{S
515 nm}/ A_{C 515 nm}) x 100

Where A_cis the initial absorbance of stable DPPH^. radical without test compound and A_s is the absorbance of DPPH^. radical in presence of sample.

Measurement of reducing power:

The reducing power of the samples was determined by the method described by Elmastas et al.¹³ with minor modifications. Reaction mixture containing different concentrations of sample in phosphate buffer (0.2 M, pH 6.6; 1ml), was incubated with potassium ferricyanide (0.5 ml, 1%W/V) at 50˚C for 20 min. After cooling, 1.5 ml of trichloroacetic acid solution (10% W/V) was added to terminate the reaction and the contents were centrifuged at

3000 rpm (10 min). To 1.5 ml of supernatant, equal amount of distilled water and 0.5 ml ferric chloride (0.1% W/V) was added. The absorbence was measured at 700 nm.

RESULTS:

Free radical scavenging activity:

The stable free radical DPPH has been extensively used to measure free radical scavenging activity. DPPH has unpaired electron in outermost orbit thereby showing a strong absorbance at 515 nm. The antioxidant capacity of the sample is measured by its ability to pair the odd electron of DPPH radical, thereby causing stoichiometrical decrease in absorbance. Decreased absorbance of the reaction mixture indicated radical scavenging activity of the sample. Ascorbic acid was used as reference compound. All the estimations were done in triplicates and free radical scavenging activity was expressed in terms of IC₅₀ ± SEM. Figure 1 shows IC₅₀ values of active compounds. Ascorbic acid had shown IC₅₀ value of 6.44 ± 0.076 µg/ml, where as compound 5a, 5b, 5c, 5e, 5h and 5j showed IC₅₀ value 88.03, 60.06, 126.46, 92.94, 169.5, 66.71 µg/ml respectively.

Figure 1: DPPH radical scavenging activity of the active compounds expressed in IC50 value.

Reducing power assay:

Figure 2 depicts the reducing power of the compounds. The reducing power of the compounds was concentration dependant. Reducing power shows the ability of molecule to break free-radical chain reaction by donating hydrogen atom. The compound 5a was found to have highest reducing power among all the synthesized chromen-4-ones.

DISCUSSION:

The antioxidant properties of several substituted 2-[3-(2,6-halophenyl)-5-methyl-isoxazolyl]-chromen-4-ones, synthesized by novel microwave irradiation method, seem to be dependent on the type of substituent’s and their position. In general, the presence of electron-donating groups (-Me, -aromatic ring) increased the reducing power as evidenced in compounds 5a, 5b, 5e, 5f and 5j. The order of activity decreased from Naphthyl>Methyl>Nitro group. Compounds with Fluorine atom in the ortho-position of isoxazole ring have less activity than their Chloro counterparts, possibly because of strong electron-withdrawing effect of fluorine as compared to Chlorine. Although compounds 5b, 5e and 5j showed free radical scavenging activity in DPPH assay, they did not exhibit significant reducing potential. This indicates that the antioxidant potential of these compounds is not mediated by inhibiting iron involvement in radical generation.

Figure 2: Reducing power of synthesized compounds. Each value is expressed as mean (n=3)

CONCLUSION:

Novel 2-[3-(2,6-substituted-phenyl)-4-methylisoxazol-5-yl]-substituted-4H-chromen-4-one derivatives were synthesized using microwave irradiation method and their antioxidant potential was investigated. Further studies to evaluate the mechanism of antioxidant action and substantiation of their antioxidant claims by synthetic strategies are under progress.

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Received on 16.09.2009 Modified on 14.11.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 106-109