Synthesis and study of antiviral and anti-radical properties of aminophenol derivatives

Article abstract of DOI:10.1016/j.bmcl.2008.02.055

A number of sterically-hindered o-aminophenol derivatives have been synthesized, and their antiviral activity in parallel with reactivity towards commonly encountered free-radical intermediates was investigated. Of the compounds tested, the highest activity in suppressing replication of Herpes simplex type l viruses was displayed by N-acyl and N-aryl derivatives of 4,6-di-tert-butyl-2-aminophenol, which were able to interact with organic free radicals and, at the same time, manifested low reactivity towards reactive oxygen species.

Full text of DOI:10.1016/j.bmcl.2008.02.055

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2420–2423
Synthesis and study of antiviral and anti-radical properties
of aminophenol derivatives
O. Shadyro,a,* G. Ksendzova,a G. Polozov,a V. Sorokin,a E. Boreko,b
O. Savinova,b B. Dubovikc and N. Bizunokc
aDepartment of Chemistry of the Belarusian State University, Nezavisimosty Avenue 4, 220030 Minsk, Belarus
bResearch Institute of Epidemiology and Microbiology, Belarus Public Health Ministry, Minsk, Belarus
cBelarusian Medicinal University, Minsk, Belarus
Received 24 January 2008; revised 19 February 2008; accepted 21 February 2008
Available online 26 February 2008
Abstract—A number of sterically-hindered o-aminophenol derivatives have been synthesized, and their antiviral activity in parallel
with reactivity towards commonly encountered free-radical intermediates was investigated. Of the compounds tested, the highest
activity in suppressing replication of Herpes simplex type l viruses was displayed by N-acyl and N-aryl derivatives of 4,6-di-tert-
butyl-2-aminophenol, which were able to interact with organic free radicals and, at the same time, manifested low reactivity towards
reactive oxygen species.
Ó 2008 Elsevier Ltd. All rights reserved.
Although studies in the area of free-radical virology
have started relatively recently, it has already been
shown that the course of a number of viral infections,
including rhino-, cytomegalo-, influenza, HIV, and var-
ious neuroviral infections, as well as chronic viral hepa-
titis,1 is accompanied by generation of reactive oxygen
species (ROS) and activation of lipid peroxidation
(LPO) processes. Therefore, investigation of the possi-
bility of using antioxidants for prevention and treatment
of viral diseases is a trend in pharmacological research
that is worthy of notice.1 Nevertheless, the information
available at present does not allow a relationship to be
found between the effects produced by chemical com-
pounds on free-radical processes and antiviral activity
of these compounds. The complexity of the problem
(i.e., finding out such relationships) is due to an ambig-
uous role of free-radical processes in the development of
viral pathology. The reason is that the reactions involv-
ing ROS generally take place in a non-selective manner,
so they can cause damage to both viruses and host cells.
Thus, the question as to which compounds should be
introduced into biosystems to modify the direction of
free-radical reactions so that a predominantly antiviral
effect is obtained, remains open. The search for such
compounds and investigation of their properties is the
main purpose of this study.
Some time ago, we have developed an antiviral product,
ButaminophenÒ, based on a sterically-hindered o-amino-
phenol derivative, effective against herpetic injuries of
various types.2,3 Many aminophenol derivatives are
known to be effective antioxidants4,5 and to be capable of
modifying the direction of various free-radical processes.6
We have synthesized a number of o-aminophenol deriv-
atives and carried out investigations of their antiviral
activity in parallel with their reactivity towards various
free radicals—for the purpose of finding out interrela-
tions between structures, antiviral and anti-radical prop-
erties of these compounds, which would be helpful for
the search for new antiviral agents.
A general scheme depicting the synthetic pathways is
shown in Figure 1.
Synthetic procedures used to prepare compounds (1–9)
and other aminophenol derivatives are described in
7–10
Refs.
Structures of all the compounds synthesized
have been confirmed by 1H NMR and mass spectrometry.
Keywords: Aminophenols; Radicals; Viruses; Inhibition.
*
Antiviral properties of the synthesized compounds were
studied in cell cultures infected with Herpes simplex
Corresponding author. Tel.: +375 17 206 6146; fax: +375 17 209
5464; e-mail: shadyro@open.by
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.055

O. Shadyro et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2420–2423
2421
OMe
OH
(MeO)2 SO2 / K2CO3
CH3
Ac2O
O
O
CH3
NH C
NH C
OH
2
8
NH2
OH
R'COCl/Et3N
RNH2 / Et3N
1
R' = C2H5 (3), C3H7 (4)
O
R'
NH C
OMe
NHR
OH
OH
OH
(MeO)2 SO2 / K2CO3
9
NHR
R = Ph (5), C6H4CH3 (6), C6H4OCH3 (7)
R = Ph (9)
Figure 1. A general scheme depicting synthetic pathways.
virus of type I (HSV-1). The investigation was per-
formed using the method based on the evaluation of
cytopathic effects (CPE) produced by the virus in the cell
culture of human rhabdomyosarcoma (RD) infected
with HSV-1.11 The compound tested was predissolved
in 10% ethanol and then twofold dilutions in mainte-
nance medium (medium 199, Sigma Chemical Co.) were
prepared. Virus titer reduction in the presence of the test
compounds as compared to control was taken as a crite-
rion of antiviral activity. Based on the data obtained,
concentrations of the test compounds that suppressed
virus replication by 50% and 90% were calculated
(EC50 and EC90, respectively) (Table 1). Maximum
non-toxic concentrations (MNTC) of the substances
were determined in non-infected cell cultures after 72-h
incubation. As seen from Table 1, the MNTC values
are much higher than EC50 and EC90.
rats by particles of opsonized zymosan.12 The oxidant
burst in phagocytes provoked an effective production
of ROS due to the activation of the NADPH-oxidase
mechanism that generates superoxide radical anions
ð O2ꢀÞ.13
Å
The intensity of ROS production was evaluated by the
luminol-derived chemoluminescence method in the
absence and in the presence of the test compounds.
Effective inhibitory concentrations (EC50 and EC90)
of the compounds under study were measured, that
is, the concentrations, at which the luminescence
intensity decreased by 50% and 90%, respectively (see
Table 2).
It follows from the obtained data that compound (1) re-
acts with ROS by two or three orders of magnitude
more actively as compared with other aminophenol
derivatives. Compounds (2–4, 6, and 7) and (9), taken
at concentrations within the range studied, produced
no effect on the intensity of chemoluminescence, while
compound (5) decreased it slightly, evidencing the ab-
sence or low reactivity of the named compounds to-
wards ROS. Similar results were obtained in our
experiments performed in order to assess reactivity of
aminophenol derivatives towards ROS generated using
fermentative systems.14
The obtained results show that the aminophenol deriva-
tives under study possess antiviral properties expressed
to a variable degree, depending on the respective struc-
tures. The named properties are the most pronounced
for those structures, in which one hydrogen atom in
the amino group is replaced by an acyl (2–4) or an aryl
(5–7) group. For structures, where free amino and hy-
droxyl groups are present (compound 1), or where H
atom in the hydroxyl group is replaced by a methyl
group (compounds 8 and 9), low antiviral activity is
observed.
The ability of the synthesized compounds to modify the
probability of free-radical reactions involving alkyl (RÅ),
peroxyalkyl (ROOÅ) and a-hydroxyalkyl (RÅCHOH) rad-
icals, was investigated using c-radiation as initiator of
various free-radical processes. Radical intermediates of
such types are known to arise in the course of ROS-in-
The reactivity of aminophenol derivatives towards ROS
was investigated in a cellular model named ‘the oxidant
burst’, which was induced in peritoneal macrophages of
Table 1. Antiviral properties and toxicity of the test compounds
a
a
Table 2. Effective concentrations of aminophenols inhibiting the
Compound MNTC EC50 ðI95
(lM)
Þ
(lM)
EC90 ðI95
Þ
(lM)
zymosan-stimulated production of ROS by macrophages
Test
compound
Concentration
range (lM)
EC50 (lM)
1
2
3
4
5
6
7
8
9
113.2 87.3 (214.9–35.3)
379.7 8.5 (10.5–6.9)
720.9 38.2 (41.3–35.3)
686.2 8.6 (10.3–7.2)
336.7 23.0 (56.4–9.4)
643.1 30.9 (37.0–25.7)
611.6 18.0 (22.4–14.5)
288.2 (709.9–117.2)
14.8 (18.2–12.1)
64.5 (69.6–59.5)
14.1 (17.2–11.7)
169.4 (316.2–90.9)
83.0 (99.4–69.1)
41.9 (52.3–33.6)
1
2
3
4
5
6
7
9
0.001–10
0.001–10
0.001–10
0.001–10
0.001–10
0.001–10
0.001–10
0.001–10
0.06
0.65
>10
No inhibition
No inhibition
No inhibition
9.8
1444.0 798.0 (1053.8–604.3) 1960.5 (2588.8–1484.8)
722.0 255.2 (569.2–114.4) 623.8 (1373.9–283.3)
No inhibition
No inhibition
No inhibition
a I95 is confidence interval at 95% probability.

2422
O. Shadyro et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2420–2423
duced processes of oxidation and fragmentation occur-
ring in cell membrane components.15 c-Irradiated hex-
ane and ethanol have been selected as model systems
because radiolysis of these substrates was extensively
studied and its features are well known. The methods
used for analysis of products formed in radiolysis of eth-
anol and hexane, as well as those employed for the
determination of the respective yields, are described in
detail in Ref. 16.
Reactivity of the additives towards a-hydroxyalkyl rad-
icals can be evaluated by studying the effects of additives
on radiolysis of ethanol. This appears to be important
because radicals of such kind are formed when ROS at-
tack hydroxyl-containing biologically relevant sub-
stances, such as carbohydrates, lipids and nucleosides,
and further transformations of these radicals result in
fragmentation of the starting material.18–23
Radiolysis of ethanol has been studied in detail,24 and its
main product was found to be 2,3-butanediol (2,3-BD),
formed by the reaction of a-hydroxyalkyl radicals:
The main products obtained on radiolysis of hexane in
the absence of oxygen are dodecanes of various struc-
tures formed by recombination of hexyl radicals on
Å
ÅC2 and C3 atoms.16
γ
.
CH3CHOH
CH3CH2OH
.
.
γ
.
C6H13
CH3CHOH
C6H13
C12H26
C6H14
CH3CH(OH)CH(OH)CH3
Hence, by measuring the overall yield of dodecanes in
the presence of additives, reactivity of the latter towards
alkyl radicals can be evaluated (Table 3).
Evaluation of yields of 2,3-butanediol in the presence
of various additives makes it possible to judge about
reactivity of the latter towards CH3 CHOH radicals.
The data obtained on this issue are presented in
Table 3.
Å
When c-irradiation of hexane is performed in the pres-
ence of O2, the formation of dodecanes does not occur
because oxygen is an effective acceptor of alkyl radicals,
transforming them into peroxyalkyl radicals (ROOÅ).
From the latter, after further transformations, the corre-
sponding alcohols and ketones are formed, hence, the
main products to be expected on radiolysis of hexane
in the presence of O2 are hexanols and hexanones.17
It follows from the data shown in Table 3 that com-
pounds (8) and (9) produce virtually no effect on the
probability of processes involving alkyl, peroxyalkyl
and a-hydroxyalkyl radicals. Compounds (1–7) display
the ability to inhibit, to a greater or lesser extent, the
processes involving various organic free radicals.
γ
While assessing the relationship between anti-radical
and antiviral activities of the test compounds we pro-
ceeded from the following experimental facts. Com-
pound (1) effectively interacted with both ROS (Table
2) and the organic radicals (Table 3), but manifested
low antiviral activity (Table 1). Compounds (2–7), as
well as compound (9), displayed low activity towards
ROS (Table 2), however, unlike (8) and (9), reacted with
organic radicals (Table 3) and manifested marked anti-
viral properties (Table 1). Hence, the obtained data indi-
cate that those aminophenol derivatives, which interact
with organic radicals while being indifferent towards
ROS, display the highest activity as inhibitors of HSV
replication.
.
.
O2
C6H14
+ H
C6H13
C6H13OO
hexanol-2, hexanol-3,
C6H13OOH
hexanone-2, hexanone-3
The overall yield of hexanols and hexanones (RG(ox)) in
the presence of aminophenols depends on the effective-
ness of interaction of the latter with C6H13OOÅ radicals
(Table 3).
Table 3. The effects of compounds (1–9) on yields (G, mol/J) of
dodecanes G(C12H26), hexane oxidation products RG(ox) and 2,3-
butanediol G(2,3-BD)
The question why just such compounds manifest antivi-
ral activity is open at the moment, and we shall try to
find out an answer to it in our further studies.
Test compound
(C = 10ꢀ3 M)
G(C12H26
)
RG(ox)
G(2,3-BD)
The facts set forth above indicate that sterically hin-
dered aminophenol derivatives possess antiviral proper-
ties and hence may be regarded as a novel class of
antiviral agents. Of the compounds tested, N-acyl and
N-aryl derivatives of 4,6-di-tert-butyl-2-aminophenol,
which are able to interact with organic free radicals
and, at the same time, display low reactivity towards
reactive oxygen species, possess the most marked activ-
ity in suppressing replication of Herpes simplex type l
viruses in cell cultures.
—
1
0.48 0.03
0.09 0.01
0.27 0.01
0.27 0.02
0.32 0.02
0.11 0.01
0.09 0.01
0.08 0.01
0.46 0.02
0.44 0.02
2.04 0.11
1.46 0.11
1.07 0.09
1.07 0.09
1.06 0.08
1.36 0.09
1.70 0.13
0.87 0.05
2.02 0.14
1.99 0.09
1.29 0.04
0.10 0.01
0.72 0.04
0.85 0.06
0.76 0.05
0.28 0.01
0.31 0.01
0.25 0.02
1.28 0.05
1.26 0.07
2
3
4
5
6
7
8
9

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2423
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Acknowledgment
This work was supported by the International Science
and Technology Centre (Grant No. B-1206).
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