Current Organic Chemistry, 2012, 16, - 1385-2728/12 $58.00+.00 © 2012 Bentham Science Publishers 5-Aminouracil as Effective Inhibitor of Peroxyl Radicals. Experimental and Theoreti- cal Studies Stanislav A. Grabovskiy,* Irina G. Konkina, Yuri I. Murinov and Natalia N. Kabal’nova Institution of the Russian Academy of Science Institute of Organic Chemistry, Ufa Research Centre of the Russian Academy of Sciences, 71 prosp. Oktyabrya, 450054 Ufa, Russia Abstract: The reaction of 5-aminouracil with peroxyl radicals generated by the thermal decomposition of 2,2-azo-bis(2- methylpropionitrile) (AIBN) and 2,2-azo-bis(2-amidinopropane) dihydrochloride was studied at 50° in ethanol and water (pH 7.0) solu- tion respectively. The oxidation product of 5-aminouracil formed by peroxyl radicals was dihydro-5,5,6-trihydroxypyrimidine-2,4-dione. The relative rate constant of 5-aminouracil vs. quercetin and 2,6-di-tert-butyl-4-methylphenol by peroxyl radicals generated from AIBN was measured in ethanol and found to be 0.19 (50°C) and 3.6 (70°C) respectively. Theoretical data of the redox potential and the bound dissociation energy oppose against single electron/proton transfer mechanism and provide support for a hydrogen atom abstraction mechanism. Transition structures and activation barriers of the hydrogen abstraction from 5-aminouracil, 5-hydroxy-6-methyluracil and 2,6-di-tert-butyl-4-methylphenol by methyl peroxyl radical were determined with the BB1K/6-31+G(d,p) level of theory. The relative theoretical reactivity was found to be in a good agreement with the experimental results and also supported the hydrogen abstraction mechanism. Keywords: DFT, uracils, oxidation, peroxyl radical, relative rate constant. INTRODUCTION Properties of differently substituted derivatives of uracil are im- portant in view of their medical application. Some derivatives of uracil exhibit a significant pharmacological activity and have been used as antitumor, antibacterial and antiviral drugs. So 6- aminouracil is a competitive inhibitor of thymidine phosphorylase (TP) activity. The TP inhibitors include 6-aminouracil derivatives such as 6-aminothymine, 6-amino-5-bromouracil, 6-amino-5- chlorouracil, and 6-(2-aminoethyl)-amino-5-chlorouracil [1]. The reactions of pyrimidines with reactive oxygen species are important for understanding the mechanisms of damage and protec- tion of DNA. One of the main factors leading to DNA damage is the radical oxidation of nucleic bases by the hydroxyl ( • ) and peroxyl (ROO • ) radicals [2, 3]. Data about the products and reac- tions mechanism of pyrimidines with peroxyl radicals are few and far between [3-5]. The structure/activity relationship of 5,6- diaminouracils against free radicals has already been described. In order to study the scavenging mechanism of diaminouracils against lipid radicals, they were also tested against the azo-initiated peroxi- dation of ether methyl linoleate in organic solvents or in liposomal suspension of dilinoleolylphosphatidylcholine. Diaminouracils were quickly consumed (like trolox in the two phase systems or - tocopherol in monophasic) and stoichiometric coefficients in or- ganic solvent were 1 mol of lipid radical per 1 mole of uracil de- rivative. Monoalkylation was possible on each nitrogen atom with- out any loss in reducing activity, the amino group in 5-position of uracil ring was necessary for high reactivity while 5-nitroso and 6- amino uracil were totally inactive [6]. In this work, we studied the reaction of 5-aminouracil with the peroxyl radicals generated by the thermal decomposition of 2,2- azo-bis(2-methylpropionitrile) (AIBN) in ethanol and 2,2-azo- *Address correspondence of this author at the Institution of the Russian Academy of Science Institute of Organic Chemistry, Ufa Research Centre of the Russian Academy of Sciences, 71 prosp. Oktyabrya, 450054 Ufa, Russia; Tel: +79173499696; Fax: +73472356066; E-mail: stas_g@anrb.ru bis(2-amidinopropane)dihydrochloride (AAPH) in water solution at pH 7.0 (0.1 M phosphate buffer) in the presence of oxygen. The products were identified by the HPLC-MS and 13 C NMR. The rela- tive rate constants of 5-aminouracil towards quercetin and 2,6-di- tert-butyl-4-methylphenol in reaction with peroxyl radicals were measured in competition experiments. Also calculations of redox potentials, bond dissociation energies and activation parameters for hydrogen abstraction from 5-aminouracil, 5-hydroxy-6-methyl- uracil and 2,6-di-tert-butyl-4-methylphenol were carried out in addition to the experiments. METHODS Water was purified by passing through a Millipore Milli-Q sys- tem (Bedford, MA) and ethanol was HPLC grade. 5-Aminouracil (1) (98.0%), quercetin (Q) (>99%), 2,6-di-tert-butyl-4-methyl- phenol (BHT) (99.0%), 2,2-azo-bis(2-amidinopropane)dihydro- chloride (AAPH) (97%), 2,2-azobis(2-methylpropionitrile) (AIBN) (98.0%) and dimethyl sulfoxide-d 6 were purchased from Sigma- Aldrich. All chemicals were used without further purification. The reaction product was studied by 1 H, 13  NMR, HPLC and MS. The NMR spectra were recorded on a Bruker AM-300 spec- trometer (DMSO-d 6 as solvent, Me 4 Si as internal standard). The chromatographic analyses of 5-aminouracil and oxidation products were carried out on a Shimadzu LC-20. The chromatographic sepa- rations were performed on Luna C18 stationary phase Luna C18 phase (Column Phenomenex 5 μm, 2504.6 mm, USA). A water- acetonitrile (95:5) eluent with a flow rate of 1 mL min –1 was used as the mobile phase. Detection was carried out at a wavelength of 215 nm. MS spectra were recorded on Esquire 3000 Plus Bruker instrument equipped with electrospray ionization source by direct sample insertion. The consumption of 5-aminouracil, quercetin and BHT in the reactions with the peroxyl radicals generated from AIBN or AAPH was monitored by HPLC and spectrophotometry on a Specord M40 instrument (Carl Zeiss Jena) on absorption maxima of 1 at 287 nm