Synthesis and Antioxidant Ability of Some New 6-amino-7 H-[ 1 , 2 , 4 ] triazolo [ 3 , 4-b ] [ 1 , 3 , 4 ] thiadiazin-3-yl ) Derivatives Bearing 2 , 6-Dimethoxy-4-( methoxymethyl ) Phenol Moiety

Compound 4-(((6-amino-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-3-yl)methoxy)methyl)2,6-dimethoxyphenol (6) was synthesized by multi steps. The corresponding acetonitrile thioalkyl (7) was cyclized by refluxing with acetic acid to afford 4-(((6-amino-7H-[1,2,4]triazolo[3,4b][1,3,4]thiadiazin-3-yl)methoxy)methyl)-2,6-dimethoxyphenol (8). Two new series of 4-(((6-(3(4-aryl)thioureido)-7H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazin-3-yl)methoxy)methyl)-2,6dimethoxyphenol (9a-c) and of 4-(((6-(substitutedbenzamido)7H-[1,2,4]triazolo[3,4b][1,3,4]thiadiazin-3-yl)methoxy)methyl)-2,6-dimethoxyphenol (10a-c) were synthesized as new derivatives for fused 1,2,4-trizaole-thiadiazine(8). The antioxidants of newly compounds were evaluated by DPPH and FRAP assays. Compound 9b showed significant antioxidant ability in both assays(higher than ascorbic acid) as well compound 6,8 and 10a-c showed antioxidant higher than BHT.


INTRODUCTION
The free radicals are one of the most important factors that cusses critical disadvantagetobiomolecules, for instance, proteins, carbohydrates,lipids, and DNA 1 .Such this disadvantage considered as source of many infirmity such as inflammatory 2 and cancers infirmity 3 , degenerative infirmity 4 and Chronic infirmity.For that, theantioxidants compounds considered one of thesignificant materials due to their capacity to terminate the free radicals ordiminish the oxidation effect.Furthermore, many anti-inflammatory and antinecrotic medications reported it possess antioxidant ability besides their therapeutic proper ties 5 .Commonly, the free radical scavengingcompounds endue protons and transform toextra stable free radicals.This stability rises with the presence of delocalization and rises the antioxidant ability 6,7 .Otherwise,the presence of multiple hydroxyl groups or presence full conjugation (π system) in structure and steric hindrance these factors has positive influence on the antioxidant ability [8][9][10] .

Chemistry
The melting point was detected by open capillary tube method utilizingOMEGA MPS10, apparatus and it is uncorrected.The purities of synthesized compounds were checked with a thin layer chromatography (Silica gel TLC) plates and the spots were visualized either with UV lights or iodine vapors.The FTIR spectra were obtained with Perkin Elmer 400 Fourier Transform Infrared (FTIR) Spectrometer.1D NMR spectra were recorded by Bruker AVN 400MHz.CDCl 3 and DMSO-d 6 wereutilized as a solvent with TMS as internal standard, measurements.The accurate mass spectra were recorded by utilizing a Finnigan TSQ7000 for HREIMs (NUS, Singapore).The FRAP Assay and DPPH assay (as antioxidant assay) were recording byusing ultravioletspectroscopy Power Wave X340,BIO-TEK instrument.

2-((4-hydroxy-3,5-dimethoxybenzyl)oxy)acetohydrazide 5
Thionylchloride (5ml) was added droop wise to 2-((4-hydroxy-3,5-dimethoxybenzyl)oxy)acetic acid.(4) (3.6 g, 14.86mmol).The stirring mixture was refluxed for 3h.The remains of thionyl chloride was evaporated under reduce pressure.The resulting acid chloride (without further purification) was transferred to an addition funnel with 10 ml dry benzene.5 ml of hydrazine hydrate (98 %) in 10 ml dried benzene was added into a two neck flask that equipped with a condenser.After that the addition funnel fixed onto the flask and the solution of acid chloride was added dropwise at 0 °C.Aftercompletion the addition, the mixture incubated for 1 h with stirring at an ambient temperature, then for further it was refluxed 3 h .The solvent was removed under reduce pressure.The crude product collected and washed with water then recrystallized from aqueous ethanol to afford white needle crystal.Yield 82%.Mp103

Antioxidant
The DPPH assay was carried out as reported by Gerhauser et al. 24 and the FRAP assay was carried out according to the Benzie and Strain 25 method as described in previous publications 26,27 .2,6-dimethoxyphenol (2,6-DMP), BHT and ascorbic acid were used as standard references.
Refluxing compound (7) in acetic acid afforded corresponding fused 1,24-triazolethiadiazine-6-amine ring (8).Compound 8 react with aryl isothiocyanate to afford the corresponding thioureaderivatives (9a-c), while it react with aryl acid chloride afford corresponding amide derivatives (10a-c).The para substituent and some physical properties were tabulated in Table 1.Thestructures of all synthesized compounds were confirmed from their FTIR, 1 H NMR, 13 C NMR besides to HREIMs spectra.The FTIR of compound 3 exhibitedband at 1725 cm -1 attributed to carbonyl group which also appeared in 13 C NMR spectrum at 171.04.The 1 H NMR spectrum of this compound displayed new two singlet peak for CH 2 OCH 2 as well the ethoxy group of ester as triplet for CH 3 and quartet for CO 2 CH 2 .The FTIR of compounds4-7 were convenient with proposed structure as well the 1 H NMR and 13 C NMR. Furthermore, the HREIMs spectra of these compounds were compatible with calculated mass.The FTIR spectrum of compound 8 was exhibited disappearanceof the CN band at 2249 cm -1 .Moreover it shows the band of OH at 3542 cm -1 , while the band of NH 2 located at3343 and 3219 cm -1 .The band of C=N was appeared at 1618 cm -1 .

Scheme. 1. Synthetic route ofsynthesis compound 3-8 besides to 9a-c and 10a-c
The 1H NMR of this compound showed the two protons of H-9 at 4.41ppm and the two protons of NH 2 at 7.34 as broad singlet peak besides to all expected protons were appeared at their expected regions .The 13 C NMR also showed disappearance carbon signal of CN group as well it exhibited new peak at37.57ppm attributed to C-9.three carbons signals at were located at 154.48, 157.16 and 157.70 ppm respectively attributed to three C=N for fused heterocyclic.The FTIR spectra of compounds 9a-c displayed new band at 3230-3235 cm -1 attributed to NH of thiourea part as well the band of C=S was located at 1247-1257 cm -1 besides to the popular band in their structure such as OH, CH aromatic & aliphatic and C=N.The 1HNMR spectra showed the new two doublet peaks each on with integration for two protons belonging toarylthiourea part, moreover the 1HNMR displayed peak at 2.21ppm for three protons of para CH 3 of compound 9b and peak at 3.84 ppm for nine protons for three set of OCH 3 for compound 9c.Furthermore, two broad singlets were located at 8.84-8.86 and 8.90-8.93ppm attributed to two NH of thiourea part.13C NMR spectra of these compounds were exhibited four new carbons attributed to arylthiourea part and interesting peak for C=S was located at 181.09-181.92ppm besides to the expected carbons were located in their expected region.The accurate mass spectra (HREIMs) were in agreement with the structure and the molecular formula for synthesized compound 9a-c (Table 1).
The FTIR spectra of compounds 10a-c showed interested two band, first one at 3320-3345 cm -1 of NH and the second one at 1668-1671 to C=O which are indicated to successfully formation of amide.The 1 H NMR spectra exhibited the peak of NH amide at 11, 21-11.57ppm and new peaks for aromatic protons of aryl partof the amide as two doublets for four protons, each one integral for two protons.Furthermore, new broad singlet peak was located with compound 10a attributed to para OH at 9.87 ppm.The para CH 3 of compound 10b was located at 2.29 ppm.The 13 C NMR spectra of these compounds showed characteristic peak for carbonyl of amide at 172.71-173.46ppm.Furthermore, the spectra exhibited all carbons of aryl amide group at their expected regions.The HREMs spectra were compatible with the calculated molecular mass.

Antioxidant Activity
The antioxidant ability of newly synthesized compounds 6-8, 9a-c and 10a-c were evaluated with DPPH assay which prefer atom transfer mechanism (HAT) 28 .Furthermore, the antioxidant ability of these compounds were evaluated utilizing FRAP assay which is undergoes single electron transfer mechanism(SET) 29 .The DPPHinhibition % and theIC 50 valuefor these compounds tabulated in Table 2. Compound 6 exhibited antioxidant ability higher than BHT and less than ascorbic acid.And their IC 50 value was less BHT.The antioxidant of compound 7 (alkyl derivative of compound 6) showed eminent deficiency in antioxidant ability.This deficiencycould be point to fade the thioamide group which werereported as free radical scavengers. 30hioamide isconsidered as a part of thiourea system.Compound 8 showed DPPH inhibition slightly less than BHT as well their IC 50 is higher than BHT.The antioxidant capacity of compounds 9a-c showed significant antioxidant capacity.Compound 9b exhibited DPPH inhibitions higher than ascorbic acid, although their IC50 was higher than ascorbic acid.Compound 9c exhibited antioxidant less than ascorbic acid and 9ashowed DPPH inhibition less than compound 9b and 9c.Increase the antioxidant ability of compounds 9a-c when compared to antioxidant ability of compound 8 could be attributed to thiourea system which is known as effective antioxidant 31 .Furthermore, the difference in DPPH results between 9a, 9b and 9capprove that the inductive effectof electrondonating groups (EDG) of substituent group at para position play vitalrole to enhance the antioxidant ability, while the as electron-withdrawing groups  All compounds were characterized successfully and screened their antioxidant ability.Compound 9a showed significant antioxidant ability, moreover 9ac exhibited antioxidant higher than 10a-c and that could be attributed to existent of thiourea part which enhances the antioxidant ability.

Table . 2: DPPH inhibition % and IC 50 for synthesized compounds 6-8,9a-cand 10a-c
EWG) demotethe antioxidant ability.Compounds 10a-c exhibited moderated antioxidant ability.Their antioxidant was higher than compound 8, but less than 9a-c.Compound 10a showed higher antioxidant than10b and 10c and that could due the existence of another phenolic hydroxyl group which can enhances the antioxidant properties.