Synthesis and Antioxidant Ability of 5-amino-1 , 3 , 4-oxadiazole Derivitives Containing 2 , 6-dimethoxyphenol

4-(((4-hydroxy-3,5-dimethoxybenzyl)oxy)methyl)benzoic acid was synthesized from multisteps and converted to their corresponding hydrazide. The corresponding hydrazide was cyclized to their corresponding 5-amino-1,3,4-oxadizole. Newly Schiff bases (7a-7e) were synthesized from reaction the 5-amino-1,3,4-oxadizole with several substituted of 4hydroxybenzylaldehyde. The resulting compounds were characterized based on their IR, 1HNMR, 13C-NMR, and HRMS data. 2,2-Diphenyl-1-picrylhydrazide (DPPH) and ferric reducing antioxidant power (FRAP) assays were used to test the antioxidant properties of the synthesized compounds. Compound 7d and 7e exhibited significant free-radical scavenging ability in both assays.


INTRODUCTION
The reactive oxygen species (ROS) and other related free radicals species are capable to react either directly or indirectly, to damage all biomolecules, such as proteins, lipids, DNA and carbohydrates resulting in cell damage and subsequently this damage can cause many diseases 1 .Many researchers reported the implication of these free radicals will cause several diseases such as inflammatory 2 , cancers 3 , degenerative 4 and chronic diseases 5 .Phenolic antioxidant compounds are one of the most important antioxidant species which can inhibit the Generally, antioxidants donate protons to become stable free radicals.This stability increases with the extent of delocalization and enhances antioxidant ability 8,9 .As such, many synthesized compounds containing long chain resonance exhibited high antioxidant activity.Furthermore, the compounds which can be classified as a strong antioxidant usually shared common structure features.They often own multiple phenolic hydroxyl groups like flavonoids 10 or which have full conjugation À system like carotenoids 11 .Moreover, exhibited substituted groups might influence on the scavenging ability.This indicates the existence of a close relationship between the chemical structure and the ability to scavenge free radicals.

Chemistry
The IR spectra were obtained with a Perkin Elmer 400 Fourier Transform Infrared (FTIR) Spectrometer. 1 H and 13 C-NMR spectra were recorded at Joel Lambda and ECA DELTA spectrometers at 400 MHz) UM, Malaysia).CDCl 3 and DMSO-d 6 were used as solvents with TMS as the internal standard.The mass spectra were recorded using an Agilent 5975 system for EI/MS and a Finnigan TSQ7000 for HREIMs (NUS, Singapore).For UV spectroscopy, a Power Wave X340 (BIO-TEK Instruments, Inc., Winooski, VT, USA) was used to record the FRAP and DPPH.Melting points were measured on a Gallenkamp melting point apparatus in open-end capillary tubes.Flash column chromatography on silica gel 60 (230-400 mesh, E. Merck) was employed.General grade solvents and reagents were purchased from commercial suppliers and used without further purification.

Synthesis of 4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl) benzohydrazide (5)
Thionylchloride (4 mL) was added in small portions of 4-(((4-hydroxy-3,5-dimethoxybenzyl) oxy)methyl)benzoic acid to (2 g,6.2 mmol).The mixture was refluxed for 3h, the excess of thionyl chloride was removed under reduced pressure.0.1 moles of acid chloride (without further purification) were dissolved in dry benzene(15mL), and it was transferred to an addition funnel hydrazine hydrate (98%) 5 mL in dried benzene (10 mL) was added into a two neck flask that equipped with a condenser.The addition funnel was then fixed onto the flask and secured firmly.The acid chloride was added dropwise at 0 C.After that, the mixture was allowed to stand for 1 h at an ambient temperature.It was then stirred and refluxed for 3h.The excess solvent was removed under reduced pressure and the crude solid was collected, wash with water and recrystallized from ethanol afforded white solid.Yield 84%, Mp 92-94 C , IR (KBr) v max 3418.
All synthesized compounds were characterized from their IR, 1 H-NMR, 13 C-NMR spectrum beside the EIMS and HRMIS.
The IR spectrum of compound 3, showed an interesting peak at 2248 cm -1 for Ca"N which is disappearing from compound 4 and a new peak of C=O was appeared at 1673 cm -1 , as well interesting peak of C=N of the oxadiazole ring for compound 6 was appearing at 1609 cm -1 .Moreover, the C=N of Schiff bases of compounds 7a-7e were appeared at 1614-1621 cm -1 beside the C=N of the oxadiazole ring.The 1 H-NMR spectrum of compound 2 showed the disappearing of the proton of aldehyde from 9.85 ppm and appearing of two protons of CH 2 beside the OH which represents the successful reduction of aldehyde group.Furthermore, the new two doublets for compound 3 at 7.41 and 7.49 ppm ( H-8, H-9 )with copling constant (J) 8.2 and 7.9 Hz respectively represented successfully of reaction compound 2 with 4-(bromo methyl)benzonitrile.As well 1 H-NMR of compound 4 showed disappearing of trimethyl lsilyl grop from 0.18 ppm and appears of the broad singlet of OH for the converting the nitrile group to carboxylic acid.The 1 H-NMR spectrum of compound 5 showed the disappearing of H of carboxylic and appeared of new two board singlet of NH 2 and NH at 4.74 and 8.83 ppm respectively, while compound 6 showed disappear these peaks and appeared new signal at 7.51 which is represented the NH 2 attached the oxadiazole ring.The 1 H-NMR of compounds 7a-7e showed appearance of new peaks represented the Schiff base (Imin proton) at 8.57-8.68ppm.The peaks of the substituted 4-hydroxylphenol appeared in their expected areas (see the experimental section).The 13 C-NMR spectrum of compound 2 showed the carbons of trimethylsilyl at -0.05 as well the carbon of CH 2 -OH.While the 13   As well as all the substituent group for 7b-7e were appeared in the expected range.The EIMS spectra showed the molecular ion M •+ for all compounds and the base peak (100%) as well Fig. 3: FRAP value of 7a-7e the HREIMs value was confirmed the accurate mass and the molecular formula as depicted in Table 1.

Antioxidant activity
The synthesized compounds (7a-7e) were exhibited high antioxidant ability in both assaysBased on the type of substitute group of phenol.Compound 7e, which I possess 2,6 di-tertbutylphenol group shows antioxidant ability slightly higher than ascorbic acid in both assays (DPPH and FRAP) as shown in Figure 1 While, compound 7d which is posses 2,6di methoxy phenol group exhibited antioxidant ability higher than BHT also, all compounds showed antioxidant activity more than 2,6-dimethoxy phenol itself.The antioxidant of the substituted phenol followed the following sequence: 2,6-di-tert-butyl>2,6-dimethoxy>2-methoxyH"2-ethoxy> non substituted phenol.These results are in agreement with many articles xxx that the more hindered phenol increase the antioxidant stability.Furthermore, we assume that the long chain resonance between the substituted phenol and the main group (CH=N) attached the oxadiazole ring after donating a proton could enhance the free radical stability, which can enhance the free-radical scavenging ability.As shown in Figure 2.
The FRAP value was higher than DPPH when comparing the results with the references and this difference could be attributed to the different mechanisms for FRAP and DPPH.FRAP involves a single electron transfer mechanism, whereas DPPH assay depends on the H-atom transfer mechanism 30 beside the hindrances around the phenol.Figure 3 displayed the FRAP value of the synthesized compound 7a-7e CONCLUSIONS A series of new Schiff base compounds from 5amino-1,3,4-oxadizole incorporating hindered phenol moieties were successfully synthesized and characterized.All of the new compounds were screened for antioxidant activity using the FRAP and DPPH assays.The antioxidant ability of these compounds increases with increasing the hindered phenol.

Table 1 : The substituent group and selected properties of synthesized compound 7a-7e
C-NMR spectrum of compound 3 displayed two carbons of CH 2 OCH 2 at 70.76 & 71.14 beside the Ca"N at 101.22 also the carbons of benzonitrile ring.Disappearing of carbon