DEVELOPMENT OF EFFECTIVE ANTIVIRAL AGENTS OF A NEW TYPE Professor Oleg Shadyro Belarusian State University Department of Chemistry, Minsk, Belarus shadyro@open.by

The main goal of the study was the development of antivital agents based on the substances capable of regulating various types of free radical reactions.

Advantages of the product / technology Some time ago, we have developed an antiviral product Butaminophen that has proven to be effective against herpetic injuries of various types. Advantages of the product / technology The Product is an effective anti-herpetic agent, particularly against strains resistant to acyclovir, it possesses also wound-healing, anti-inflammatory and antipyrotic action. The Technology is simple and easy to put into practice, is a low-cost manufacturing process, the starting raw material is readily available.

A general scheme depicting synthetic pathways to obtain some sterically hindered aminophenol derivatives

Membrane structure Phospholipids O P X C

Lipid peroxidation process

Free-radical fragmentation of cardiolipin Shadyro O.I., Yurkova I.L., Kisel M.A., Brede O., Arnhold J. Radiation-induced fragmentation of cardiolipin in a model membrane. International Journal of Radiation Biology, 2004, 80, 239-245. Shadyro O.I., Yurkova I.L., Kisel M.A., Brede O., Arnhold J. Radiation-induced free-radical transformations of Phospholipids: MALDI-TOF MS study. Chemistry and Physics of Lipids, 2004, 132, 235-246.

Free-radical fragmentation of cerebrosides Shadyro O.I., Yurkova I.L., Kisel M.A., Brede O., Arnhold J. Formation of phosphatidic acid, ceramide and diglyceride on radiolysis of lipids: identification by MALDI-TOF mass spectrometry. Free Radical Biology & Medicine, 2004, 36, 1612-1624. Shadyro O.I., Yurkova I.L., Kisel M.A., Arnhold J. Free-radical fragmentation of galactocerebrosides: a MALDI-TOF mass spectrometry study. Chemistry and Physics of Lipids, 2005, 134, 41-49. Shadyro O.I., Yurkova I.L., Kisel M.A., Arnhold J. Iron-mediated free-radical formation of signaling lipids in a model system. Chemistry and Physics of Lipids, 2005, 137, 29-37.

A new approach to the regulation of free-radical processes in biosystems has been proposed Diphenol and aminophenol derivatives were found to be capable of regulating free-radical transformations occurring in bioorganic compounds with participation of both oxygen-centered (oxidation) and carbon-centered (fragmentation) radicals. Shadyro O.I. et al. Quinones as free-radical fragmentation inhibitors in biologically important molecules. Free Rad. Res., 2002, 36, 859-867. Shadyro O.I., Murase H., Kagiya T. et al. Effects of phenolic compounds on reactions involving various organic radicals. Free Rad. Res., 2003, 37, 1087-1097. Shadyro O.I. et al. Reactions of arylamine and aminophenol derivatives, and riboflavin with organic radicals. Free Rad. Res., 2004, 38, 1183-1190.

Percent inhibition produced by aminophenols in reactions involving various radicals Test compounds Structure >CHOO >CH >CHOH N-1 28 81 92 N-2 48 44 45 N-3 34 N-4 49 41 N-5 33 77 78 N-6 17 76 N-7 58 83 80 N-8 1,2 4,6 0,8 N-9 9,1 8,3 2,3

Concentration range, M Effective concentrations of aminophenols inhibiting the zymosan-stimulated production of ROS by macrophages Test compounds Structure Concentration range, M EC50, M EC90, M N-1 0.001-10 0.06 0.65 N-2 No inhibition N-3 N-4 N-5 9.8 > 10 N-6 N-7 N-9

Antiviral properties of the test compounds in a cell culture infected with HSV Structure MNTC, M EC50 (I95*), M EC90 (I95*), M N-1 113.2 87.3 (214.935.3) 288.2 (709.9117.2) N-2 379.7 8.5 (10.56.9) 14.8 (18.212.1) N-3 720.9 38.2 (41.335.3) 64.5 (69.659.5) N-4 686.2 8.6 (10.37.2) 14.1 (17.211.7) N-5 336.7 23.0 (56.49.4) 169.4 (316.290.9) N-6 643.1 30.9 (37.025.7) 83.0 (99.469.1) N-7 611.6 18.0 (22.414.5) 41.9 (52.333.6) N-8 1444.0 798.0 (1053.8604.3) 1960.5 (2588.81484.8) N-9 722.0 255.2 (569.2114.4) 623.8 (1373.9283.3) *I95 — is confidence interval at 95 % probability.

Wounds in places of vesicle formation Antiviral properties of the test compounds in mice infected with skin herpes Normal ear Erythema Erythema and vesicles Wounds in places of vesicle formation

Higher antiviral activity against herpes viruses Chemico-pharmacological advantages of compound N-2 as compared to Butaminophen® Lower toxicity Higher antiviral activity against herpes viruses Higher chemical stability

Antiviral activity of compound N-2 against influenza A/FPV/Rostok (H7N1) virus in chicken embryo cell culture Compound code Concentration, M Titer of virus, lg PFU/ml EC50, M MNTC/ EC50 N-2 759 380 190 48 24 12 4.2 4.5 4.6 4.7 5.4 5.5 43.6 17.4

Antiviral activity of compound N-12 against HIV-1 in a cell culture Compound code Concentration, M Percent of infected cells EC50, M MNTC/ EC50 N-12 876 221 55 14 12 27 32 41 96 7.9 111

Conclusions: The obtained data indicate that sterically hindered aminophenol derivatives possess antiviral properties and hence may be regarded as a novel class of antiviral agents. Among the compounds tested, the most pronounced antiviral properties were found for N-acyl and N-aryl dertivatives of sterically hindered o-aminophenol which were able to interact with various organic radicals while displaying low reactivity towards reactive oxygen species.