DEVELOPMENT DEVELOPMENT OF EFFECTIVE OF EFFECTIVE

ANTIVIRAL ANTIVIRAL AGENTSAGENTS

OF A NEW TYPE OF A NEW TYPEProfessor

Oleg ShadyroBelarusian State University

Department of Chemistry, Minsk, Belarusshadyro@open.by

The main goal of the study The main goal of the study was the development of was the development of

antivital agents based on the antivital agents based on the substances capable of substances capable of

regulating various types of free regulating various types of free radical reactions.radical reactions.

Some time ago, we have developed an antiviral product Some time ago, we have developed an antiviral product ButaminophenButaminophen that has proven to be effective against that has proven to be effective against

herpetic injuries of various types.herpetic injuries of various types.

OH

NH

Advantages of the product / technologyAdvantages of the product / technology

The ProductThe Product is an effective anti-herpetic agent,

particularly against strains resistant to acyclovir,it possesses also wound-healing, anti-inflammatory and antipyrotic action.

The TechnologyThe 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 A general scheme depicting synthetic pathways to obtain some sterically hindered pathways to obtain some sterically hindered

aminophenol derivativesaminophenol derivatives

OH

OH RNH2 / Et3N

NHR

OH

R = Ph (5), C6H4CH3 (6), C6H4OCH3 (7)

NH2

OHNH

OH

C CH3

O

1

Ac2O

R' = C2H5 (3), C3H7 (4)R'COCl/Et3N

NHR

OMe

9

(MeO)2 SO2 / K2CO3

(MeO)2 SO2 / K2CO3

R = Ph (9)

NH

OH

C R'

O

NH C CH3

OOMe

2 8

Membrane structureMembrane structure

Phospholipids

O

O

P

O

OX

O C

O

C O

O

Lipid peroxidation processLipid peroxidation process

Free-radical fragmentation of Free-radical fragmentation of cardiolipincardiolipin

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 Free-radical fragmentation of cerebrosidescerebrosides

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 A new approach to the regulation of free-radical processes in biosystems has free-radical processes in biosystems has

been proposedbeen proposed

OH

OH

O

O

O

OH

O

OH

.

.

ROO.

ROOH

R1

R1H

R2

R2H

. .

OH

X

O

X

.

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 Percent inhibition produced by aminophenols in reactions involving various radicalsinvolving various radicals

Test compounds Structure >CHOO >CH >CHOH

N-1 28 81 92

N-2 48 44 45

N-3 48 44 34

N-4 49 34 41

N-5 33 77 78

N-6 17 81 76

N-7 58 83 80

N-8 1,2 4,6 0,8

N-9 9,1 8,3 2,3

NH2

OH

NH

OH

NH CH3

OH

NH OCH3

OH

NHCOCH3

OH

NHCOC2H5

OH

NHCOC3H7

OH

NH

O CH3

NHCOCH3

O CH3

Effective concentrations of aminophenols Effective concentrations of aminophenols inhibiting the zymosan-stimulated production inhibiting the zymosan-stimulated production

of ROS by macrophagesof ROS by macrophagesTest

compoundsStructure Concentratio

n range, M EC50, M EC90, M

N-1 0.001-10 0.06 0.65

N-2 0.001-10 No inhibition

N-3 0.001-10No inhibition

N-4 0.001-10No inhibition

N-5 0.001-10 9.8 > 10

N-6 0.001-10No inhibition

N-7 0.001-10No inhibition

N-9 0.001-10No inhibition

NH2

OH

NH

OH

NH CH3

OH

NH OCH3

OH

NHCOCH3

OH

NHCOC2H5

OH

NHCOC3H7

OH

NH

O CH3

Antiviral properties of the test compounds in a cell culture Antiviral properties of the test compounds in a cell culture infected with HSVinfected with HSV

Test compounds

Structure MNTC, M EC50 (I95*), M EC90 (I95

*), M

N-1 113.2 87.3

(214.935.3) 288.2

(709.9117.2)

N-2 379.7 8.5

(10.56.9) 14.8

(18.212.1)

N-3 720.9 38.2

(41.335.3) 64.5

(69.659.5)

N-4 686.2 8.6

(10.37.2) 14.1

(17.211.7)

N-5 336.7 23.0

(56.49.4) 169.4

(316.290.9)

N-6 643.1 30.9

(37.025.7) 83.0

(99.469.1)

N-7 611.6 18.0

(22.414.5) 41.9

(52.333.6)

N-8 1444.0 798.0

(1053.8604.3)

1960.5(2588.81484.8

)

N-9 722.0 255.2

(569.2114.4) 623.8

(1373.9283.3)

NH2

OH

NH

OH

NH CH3

OH

NH OCH3

OH

NHCOCH3

OH

NHCOC2H5

OH

NHCOC3H7

OH

NH

O CH3

*I95 — is confidence interval at 95 % probability.

NHCOCH3

O CH3

Antiviral properties of the test compounds in Antiviral properties of the test compounds in mice infected with skin herpesmice infected with skin herpes

Normal ear Erythema Erythema and vesicles

Wounds in places of vesicle formation

0

0,2

0,4

0,6

0,8

1

1,2

1,4

Rel

ativ

e in

tens

ity o

f inf

ectio

n si

gns

1 2 3 4 5 6 7 8 9 10 11

Time after infection (days)

Control

N-5 ointment 1%

N-2 ointment 1%

Acyclovir 2.5%

Chemico-pharmacological advantages Chemico-pharmacological advantages of compound N-2 as compared to of compound N-2 as compared to

ButaminophenButaminophen®®

Lower toxicityLower toxicity Higher antiviral activity against Higher antiviral activity against

herpes virusesherpes viruses Higher chemical stabilityHigher chemical stability

Antiviral activity of compound N-2 against Antiviral activity of compound N-2 against influenza A/FPV/Rostok (H7N1) virus in influenza A/FPV/Rostok (H7N1) virus in

chicken embryo cell culturechicken embryo cell culture

CompounCompoundd

codecode

ConcentratiConcentration, on, MM

Titer of Titer of virus, virus,

lg PFU/mllg PFU/ml

ECEC5050, , MM MNTC/MNTC/ ECEC5050

N-2N-2 759759

380380

190190

4848

2424

1212

00

4.24.2

4.54.5

4.64.6

4.74.7

5.45.4

5.55.5

5.55.5

43.643.6 17.417.4

Antiviral activity of compound N-12 against Antiviral activity of compound N-12 against HIV-1 in a cell cultureHIV-1 in a cell culture

CompounCompound coded code

ConcentratiConcentration, on, MM

Percent of Percent of infected infected

cellscells

ECEC5050, , MM MNTC/ MNTC/ ECEC5050

N-12N-12 876876

221221

5555

1414

00

1212

2727

3232

4141

9696

7.97.9 111111

The obtained data indicate that The obtained data indicate that sterically hindered aminophenol sterically hindered aminophenol

derivatives possess antiviral properties derivatives possess antiviral properties and hence may be regarded as a novel and hence may be regarded as a novel

class of antiviral agents.class of antiviral agents.

Among the compounds tested, the Among the compounds tested, the most pronounced antiviral properties most pronounced antiviral properties

were found for N-acyl and N-aryl were found for N-acyl and N-aryl dertivatives of sterically hindered o-dertivatives of sterically hindered o-

aminophenol which were able to aminophenol which were able to interact with various organic radicals interact with various organic radicals while displaying low reactivity towards while displaying low reactivity towards

reactive oxygen species. reactive oxygen species.

Conclusions:Conclusions: