Synthesis and antiviral activity of 1-hydroxy-2-(2-hydroxyphenyl)imidazoles against vaccinia virus

Article abstract of DOI:10.1007/s11172-019-2467-6

2-(2-Hydroxyplienyl)imidazole derivatives were synthesized and tested for antiviral activity against vaccinia virus in Vero cell culture. 1-Methylimidazole 3-oxides, 1-methoxyimidazoles, and 1H-imidazoles showed no activity, whereas some 1-hydroxyimidazole derivatives hold promise, exhibiting antiviral activity and weak cytotoxicity.

Full text of DOI:10.1007/s11172-019-2467-6

Russian Chemical Bulletin, International Edition, Vol. 68, No. 3, pp. 634—637, March, 2019
634
Synthesis and antiviral activity
of 1-hydroxy-2-(2-hydroxyphenyl)imidazoles against vaccinia virus
P. A. Nikitina,a,b N. I. Bormotov,c L. N. Shishkina,c A. Ya. Tikhonov,d and V. P. Perevalova
aD. I. Mendeleev University of Chemical Technology of Russia,
9 pl. Miusskaya, 125047 Moscow, Russian Federation
bA. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
28 ul. Vavilova, 119991 Moscow, Russian Federation.
E-mail: polinandrevna@yandex.ru
cState Research Center of Virology and Biotechnology VECTOR,
Koltsovo, Novosibirsk Region, 630559, Russian Federation
dN. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry,
Siberian Branch of the Russian Academy of Sciences,
9 prosp. Akad. Lavrentieva, 630090 Novosibirsk, Russian Federation
2-(2-Hydroxyphenyl)imidazole derivatives were synthesized and tested for antiviral activ-
ity against vaccinia virus in Vero cell culture. 1-Methylimidazole 3-oxides, 1-methoxyimidazoles,
and 1H-imidazoles showed no activity, whereas some 1-hydroxyimidazole derivatives hold
promise, exhibiting antiviral activity and weak cytotoxicity.
Key words: 1-hydroxyimidazoles, vaccinia virus, antiviral activity, cytotoxicity.
Scheme 1
In 1966—1980, the World Health Organization (WHO)
undertook the Smallpox Eradication Programme, and this
disease was officially declared eradicated in 1980. Never-
theless, smallpox virus is considered a potential threat as
a biological weapon.1,2 Hence, there is still a need for new
antiviral agents, including small molecule representatives,3
with activity against smallpox virus and other orthopox-
viruses pathogenic for humans.
1-Hydroxyimidazole derivatives were found to exhibit
different biological activities. This class of compounds
includes potential pesticides,4 herbicides,5 molecules hav-
ing hypotensive,6 antiprotozoal,7 antibacterial,8 and anti-
viral9 activities, potential selective inhibitors of kinases
with different functions,10 and xanthine oxidase inhibi-
tors.11,12 The goal of the present work is to synthesize
1-hydroxy-2-(2-hydroxyphenyl)imidazoles, evaluate their
antiviral activity against vaccinia virus, and compare their
activity with that of other imidazole derivatives.
1-Hydroxyimidazoles 1a—h were prepared by the
condensation of salicylaldehyde derivatives 2a—d with
oximes 3a—d and ammonium acetate in glacial acetic acid
(Scheme 1).
The reaction was performed at room temperature
(synthesis of compounds 1a—c,e,f) or under reflux (when
using oxime 3d in the synthesis of derivatives 1d,g,h).
To compare antiviral activity of 1-hydroxy-2-(2-hy-
droxyphenyl)imidazoles with that of 2-(2-hydroxyphenyl)
imidazole derivatives, we synthesized 1-methylimidazole
1
a
b
c
d
e
f
R1
H
H
H
H
R2
Me
Me
R3
Me
OEt
CH2C(Me)2CH2
Ph
Me
Me
Ph
NHPh
Me
OEt
NHPh
NHPh
5-Cl
4-OCH2Ph
5-Cl
5-Br
g
h
Ph
3-oxides 4a—c and 1-methoxyimidazoles 5a—c (Scheme 2)
according to procedures described previously.13
1H-Imidazole 6a unsubstituted at nitrogen atoms was
formed as a by-product in the synthesis of 1-methoxy
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 0634—0637, March, 2019.
1066-5285/19/6803-0634 © 2019 Springer Science+Business Media, Inc.

1-Hydroxy-2-(2-hydroxyphenyl)imidazoles
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 3, March, 2019
635
Scheme 2
4, 5: R2 = R3 = Me (a); R2 = Me, R3 = OEt (b); R2 + R3 = CH2C(Me)2CH2 (c)
derivative 5a.13 Imidazoles 6b,c were prepared by the re-
duction of 1-hydroxyimidazoles 1b,c with triphenylphos-
phine in glacial acetic acid under reflux (Scheme 3).
1-Hydroxyimidazoles 1a—h were tested for activity
against vaccinia virus in Vero cell culture. The results are
given in Table 1.
Scheme 3
1-Hydroxy-2-(2-hydroxyphenyl)imidazole derivatives
1a—c, which were synthesized starting from unsubstituted
salicylaldehyde 2a and which contain an acetyl (1a), ethoxy-
carbonyl (1b), or fixed carbonyl group (1c) in position 5 of
the heterocycle, exhibited potential antiviral activity and low
cytotoxicity. Structure 1c showed most promise. The
characteristics of antiviral activity of compounds 1b,c were
reliably different from those of compound 1a (see Table 1).
The presence of substituents (Hal, OCH2Ph) in the
2-hydroxyphenyl moiety of 1-hydroxyimidazoles 1e,f,g,h
led to an increase in cytotoxicity. 1-Hydroxyimidazoles
1d,g,h containing the N-phenylcarbamoyl substituent in
6: R2 = Me, R3 = OEt (b); R2 + R3 = CH2C(Me)2CH2 (c)
position 5 appeared to be cytotoxic and showed no in-
hibitory activity.
We also tested imidazole 3-oxides 4a—c, 1-methoxy-
imidazoles 5a—c, and 1H-imidazoles 6a—c containing
a set of substituents in positions 2, 4, and 5 of the hetero-
cycle, which are similar to those in promising 1-hydroxy-
imidazoles 1a—c (Table 2).
Table 1. Antiviral activity of 1-hydroxyimidazoles 1a—h against vaccinia virus (Copenhagen strain) in Vero cell culture
Compound
TC50/μg mL–1
IC50/ μg mL–1
SI (TC50/IC50
4.39 0.45a
)
1a
1b
1c
1d
1e
1f
1g
1h
64.80 9.36a
478.33 27.15a,b
203.00 2.04a,b
0.201
14.58 0.63a
4.07 0.13a,b
1.29 0.09a,b
N.a.
118.53 10.38a,b
159.73 11.58a,b
—
1.06
N.a.
—
1.434
N.a.
—
<0.16
N.a.
—
—
<0.16
N.a.
Cidofovir
(control sample)
275.72 15.54a,b
10.03 0.63a,b
27.60 1.56a,b
Note. TC50 is the toxic concentration of the agent causing 50% death of uninfected monolayer cells; IC50 is the virus-
inhibitory concentration of the agent required to inhibit 50% of infected monolayer cells; SI is the selectivity index of
the agent defined as the TC50/IC50 ratio; N.a., not active.
a The mean values and standard errors are given, the number of repeated measurements of TC50, IC50, and SI is 4.
b The difference from 1a according to Student's t-test at р < 0.001.

636
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 3, March, 2019
Nikitina et al.
Table 2. Antiviral activity of imidazole 3-oxides 4a—c, 1-meth-
oxyimidazoles 5a—c, and 1H-imidazoles 6a—c against vaccinia
virus (Copenhagen strain) in Vero cell culture*
pure product 1d was obtained as yellowish powder with m.p.
1
262—264 °C in a yield of 1.06 g (57%). H NMR (DMSO-d6),
δ: 13.94 (br.s, 1 H, NH); 12.04 (br.s, 2 H, OH); 7.73—7.85
(m, 4 H, Ar); 7.67 (d, 2 H, Ar, J = 8.1 Hz); 7.42—7.57 (m, 4 H, Ar);
7.36 (m, 2 H, Ar); 7.12 (m, 1 H, Ar); 6.98—7.06 (m, 2 H, Ar).
13C NMR (DMSO-d6), δ: 156.8; 155.5; 137.3; 136.4; 133.3; 132.4;
132.1; 129.2; 129.0; 128.9; 128.5; 127.4; 123.5; 119.5; 119.3; 118.7;
118.5; 111.2. MS, found: m/z 372.1343 [M + H]+; C22H17N3O3;
calculated: [М + H]+ = 372.1348.
Compound
TC50
IC50
SI (TC50/IC50)
μg mL–1
4a
4b
4c
5a
5b
5c
6a
6b
6c
>100
>100
>100
56.061
>100
>100
>100
5.231
>100
N.a.
N.a.
N.a.
N.a.
42.926
N.a.
N.a.
N.a.
N.a.
—
—
N-Phenyl-2-(5-chloro-2-hydroxyphenyl)-1-hydroxy-4-phenyl-
1H-imidazole-5-carboxamide (1g). A mixture of aldehyde 2b
(0.16 g, 1.0 mmol), oxime 3d (0.27 g, 1.0 mmol), and ammonium
acetate (0.12 g, 1.6 mmol) in glacial acetic acid (3 mL) was stirred
at reflux for 3 h and then cooled to room temperature. The pre-
cipitate was filtered off, washed on a filter with diethyl ether, and
purified by refluxing in acetonitrile. Chromatographically pure
product 1g was obtained as white powder with m.p. 268—269 °C
in a yield of 0.24 g (59%). 1H NMR (DMSO-d6), δ: 13.95 (br.s,
2 H, 2 OH); 11.86 (br.s, 1 H, NH); 7.94 (s, 1 H, Ar); 7.81 (d, 2 H,
Ar, J = 7.0 Hz); 7.69 (d, 2 H, Ar, J = 8.0 Hz); 7.45—7.58 (m, 4 H,
Ar); 7.38 (t, 2 H, Ar, J = 7.8 Hz); 7.15 (t, 1 H, Ar, J = 7.4 Hz);
7.06 (d, 1 H, J = 8.8 Hz). 13C NMR (DMSO-d6), δ: 156.3; 155.9;
137.9; 135.7; 133.9; 133.6; 132.2; 132.1; 132.0; 129.4; 129.0; 128.0;
124.1; 122.7; 120.9; 120.8; 119.9; 113.2. MS, found: m/z 406.0953
[M + H]+; C22H16ClN3O3; calculated: [М + H]+ = 406.0958.
N-Phenyl-2-(5-bromo-2-hydroxyphenyl)-1-hydroxy-4-phenyl-
1H-imidazole-5-carboxamide (1h). A mixture of aldehyde 2d
(0.08 g, 0.4 mmol), oxime 3d (0.10 g, 0.4 mmol), and ammo-
nium acetate (0.08 g, 1.0 mmol) in glacial acetic acid (2 mL) was
stirred at reflux for 2 h and then cooled to room temperature.
The precipitate was filtered off, washed on a filter with diethyl
ether, and purified by refluxing in acetonitrile. Chromatographically
pure product 1h was obtained as white powder with m.p.
—
—
>2.32
—
—
—
—
* For notations, see Table 1.
It was demonstrated that compounds 4a—c, 5a—c, and
6a—c exhibited no significant activity, that proved the
N-hydroxy group to be an essential moiety. This factor
should be taken into account in the development of
chemotherapeutic agents against smallpox based on imid-
azoles containing a carbonyl group in position 5.
Experimental
Commercially available reagents were used as received. The
1H and 13C NMR spectra were recorded on a Bruker AvanceTM300
spectrometer (operating at 300 and 75.47 MHz, respectively) in
deuterated DMSO using residual signals of the solvent as the
internal standard. High-resolution mass spectra (HRMS) were
obtained on a BrukerMicrOTOF ESI-TOF mass spectrometer.
Starting diketone monooximes 3a—d were prepared accord-
ing to known procedures.14,15 1-Hydroxyimidazoles 1a—c,f,
imidazole 3-oxides 4a—c, 1-methoxyimidazoles 5a—c, and
1H-imidazole 6a were characterized previously.13,16
1-[2-(5-Chloro-2-hydroxyphenyl)-1-hydroxy-4-methyl-1H-
imidazol-5-yl]ethanone (1e). A mixture of aldehyde 2b (0.78 g,
5.0 mmol), oxime 3a (0.65 g, 5.0 mmol), and ammonium acetate
(0.45 g, 5.8 mmol) in glacial acetic acid (6 mL) was stirred at
room temperature and then left for a week. The precipitate was
filtered off and purified by refluxing in hexane. Chromatograph-
ically pure product 1e was obtained as beige powder with m.p.
176—178 °C in a yield of 0.61 g (46%). 1H NMR (DMSO-d6), δ:
13.60 (br.s, 2 H, 2 OH); 7.70 (s, 1 H, H(6´)); 7.43 (dd, 1 H,
H(4´), J = 8.9 Hz; J = 2.6 Hz); 6.95 (d, 1 H, H(3´), J = 8.8 Hz);
2.66 (s, 3 H, CH3); 2.48 (s, 3 H, CH3). 13C NMR (DMSO-d6),
δ: 181.3; 157.2; 135.7; 131.9; 127.1; 126.7; 122.1; 121.0; 119.5; 113.3;
30.1; 21.2. MS, found: m/z 267.0531 [M + H]+; C12H11ClN2O3;
calculated: [М + H]+ = 267.0536.
1
263—265 °C in a yield of 0.11 g (66%). H NMR (DMSO-d6),
δ: 13.99 (br.s, 2 H, OH); 11.88 (br.s, 1 H, NH); 8.06 (s, 1 H, Ar);
7.80 (d, 2 H, Ar, J = 6.7 Hz); 7.69 (d, 2 H, Ar, J = 8.1 Hz); 7.61
(d, 1 H, Ar, J = 8.9 Hz); 7.46—7.57 (m, 3 H, Ar); 7.39 (t, 2 H,
Ar, J = 7.8 Hz); 7.16 (d, 1 H, Ar, J = 7.5 Hz); 7.00 (d, 1 H, Ar,
J = 8.8 Hz). 13C NMR (DMSO-d6), δ: 156.7; 156.0; 137.9; 135.7;
135.2; 135.0; 133.9; 129.6; 129.4; 129.0; 128.0; 127.4; 124.1; 121.4;
120.9; 119.9; 113.8; 110.1. MS, found: m/z 450.0448 [M + H]+.
C22H16BrN3O3, calculated: [М + H]+ = 450.0453.
Ethyl 2-(2-hydroxyphenyl)-4-methyl-1H-imidazole-5-carb-
oxylate (6b). A mixture of 1-hydroxyimidazole 1b (1.05 g,
4.0 mmol) and triphenylphosphine (1.69 g, 6.4 mmol) in glacial
acetic acid (7 mL) was stirred at reflux for 4 h. Then approxi-
mately two-thirds of the solvent was distilled off under reduced
pressure. The residue was diluted with diethyl ether (15 mL). The
precipitate was filtered off and purified by refluxing twice in di-
ethyl ether. Chromatographically pure product 6b was obtained
as beige powder with m.p. 177—178 °C in a yield of 0.40 g (41%).
1H NMR (DMSO-d6), δ: 13.13 (br.s, 1 H, NH); 12.58 (br.s,
1 H, OH); 7.87 (br.s, 1 H, H(2´)); 7.29 (t, 1 H, Ar, J = 7.7 Hz);
6.88—7.01 (m, 2 H, Ar); 4.29 (q, 2 H, OCH2CH3, J = 7.1 Hz);
2.52 (s, 3 H, CH3); 1.32 (t, 3 H, OCH2CH3, J = 7.1 Hz). 13C NMR
(DMSO-d6), δ: 161.9; 156.6; 145.0; 131.5/131.4*; 130.5;
128.8/128.7*; 125.1; 118.9; 116.8; 112.5; 59.7; 14.2; 11.1. MS,
N-Phenyl-1-hydroxy-2-(2-hydroxyphenyl)-4-phenyl-1H-imid-
azole-5-carboxamide (1d). A mixture of aldehyde 2a (0.61 g,
5.0 mmol), oxime 3d (1.34 g, 5.0 mmol), and ammonium acetate
(0.62 g, 8.1 mmol) in glacial acetic acid (10 mL) was stirred at
reflux for 5 h and then cooled to room temperature. The pre-
cipitate was filtered off, washed on a filter with diethyl ether, and
purified by refluxing in acetonitrile. Chromatographically
* In the 13C NMR spectra of 1H-imidazoles 6b,c, the 13C NMR
signals of the carbon atoms in positions 4 and 5 of imidazole are
doubled due to prototropic tautomerism of the heterocycle.

1-Hydroxy-2-(2-hydroxyphenyl)imidazoles
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 3, March, 2019
637
found: m/z 247.1077 [M + H]+; C13H14N2O3; calculated:
[М + H]+ = 247.1083.
and within the framework of the State task No. 8/18 of the
State Research Center of Virology and Biotechnology
VECTOR.
2-(2-Hydroxyphenyl)-6,6-dimethyl-3,5,6,7-tetrahydro-4H-
benzimidazol-4-one (6c). A mixture of 1-hydroxyimidazole 1c
(0.54 g, 2.0 mmol) and triphenylphosphine (0.84 g, 3.2 mmol)
in glacial acetic acid (4 mL) was stirred at reflux for 4 h, cooled
to room temperature, and diluted with diethyl ether (10 mL).
The precipitate was filtered off, washed on a filter with diethyl
ether, and purified by refluxing in diethyl ether. Chromato-
graphically pure product 6c was obtained as pale-yellow powder
with m.p. 282—284 °C in a yield of 0.42 g (82%). 1H NMR
(DMSO-d6), δ: 13.56 and 13.26 (both br.s, 1 H, NH); 12.52 (br.s,
1 H, OH); 8.11 and 7.88 (both br.d, 1 H, H(2´)); 7.33 (t, 1 H,
Ar, J = 7.8 Hz); 6.90—7.04 (m, 2 H, Ar); 2.84 and 2.78 (both s,
2 H, CH2); 2.42 (s, 2 H, CH2); 1.10 (s, 6 H, 2 CH3). 13C NMR
(DMSO-d6), δ: 188.4; 172.0; 157.1; 131.5/131.4*; 131.2;
128.8/128.7*; 125.6; 119.1; 117.0; 112.3; 51.6; 35.5; 28.0; 21.0.
MS, found: m/z 257.1285 [M + H]+; C15H16N2O2; calculated:
[М + H]+ = 257.1290.
Evaluation of antiviral activity. Antiviral activity and toxicity
of the synthesized compounds were evaluated in Vero cell culture.
Vaccinia virus (Copenhagen strain), as a typical representative
of orthopoxviruses, was used as model. A Vero cell monolayer
was grown in wells of 96-well plates. Solutions of the compounds
to be tested were prepared in dimethyl sulfoxide at a concentra-
tion of 20 mg mL–1. These solutions were used to prepare a series
of dilutions in a culture medium. These dilutions were placed in
wells of the plates containing a cell monolayer. In one-half of the
wells, the virus was added, and another half was used to evaluate
the toxicity of the compounds. The dilutions were made in five-
or three-fold steps; the initial concentration in the wells of the
plates was 100 μg mL–1. Commercially available Cidofovir
(Vistide) purchased from Gilead Sciences Inc. (USA) was em-
ployed as the control. Cidofovir proved to be active against or-
thopoxviruses in in vitro and in vivo experiments.3
After incubation for four days, the vital dye neutral red that
is absorbed only by living cells was added to the wells. In the
wells, in which the cells are destroyed by the virus or damaged
by toxicity of the compound, the dye uptake does not occur. The
monolayer was washed with a physiological solution to remove
the unabsorbed dye. Then a lysis buffer was added to the wells to
dissolve the dye absorbed by the cells. The absorbance of the
solutions in the wells was measured with an E-Max microplate
reader (Molecular Devices, USA) at a wavelength of 490 nm.
The absorbance is proportional to the number of surviving cells
and characterizes the antiviral activity and toxicity of the com-
pound compared to the virus control and the cell control, re-
spectively. The data were processed with the Soft Max Pro 4.0
software, which calculated the 50% toxic concentration
(TC50, μg mL–1) and the 50% virus-inhibitory concentration
(IC50, μg mL–1). The selectivity index (SI) defined as SI =
= TC50/IC50 was determined based on the values of TC50 and
IC50. The value of SI smaller than 8 is regarded as unacceptable for
compounds that can be considered as promising antiviral agents.3
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Received October 24, 2018;
in revised form November 11, 2018;
accepted November 14, 2018
This study was financially supported by the Russian
Foundation for Basic Research (Project No. 18-33-00442)