Synthesis and Antiviral Properties of Ethyl(3-ethyladamant-1-yl)carbamate

Article abstract of DOI:10.1007/s11094-017-1548-9

The new adamantine derivative ethyl(3-ethyladamant-1-yl)carbamate was found to have high antiviral activity against herpes simplex virus and vaccinia viruses, as well as adenovirus (EC₅₀ = 0.62, 5.15, and 48.5 μg/ml respectively). Activity against the herpesvirus was demonstrated in experiments in various cell cultures using different virus strains, including a variant resistant to acycloguanosine (ACG). Simultaneous application of ethyl(3-ethyladamant-1-yl)carbamate and ACG increased the inhibition of herpesvirus reproduction in cell cultures and decreased the mortality of laboratory animals with experimental herpesvirus neuroinfection as compared with the use of each substance alone. 3-Ethyladamant-1-yl)carbamate was a low-toxicity compound, did not inhibit DNAsynthesis in uninfected cell cultures, and its acute intragastric LD₅₀ for white mice was 1831.8 mg/kg.

Full text of DOI:10.1007/s11094-017-1548-9

DOI 10.1007/s11094-017-1548-9
Pharmaceutical Chemistry Journal, Vol. 51, No. 1, April, 2017 (Russian Original Vol. 51, No. 1, January, 2017)
SYNTHESIS AND ANTIVIRAL PROPERTIES
OF ETHYL(3-ETHYLADAMANT-1-YL)CARBAMATE
Yu. N. Klimochkin,1 I. K. Moiseev,1 M. V. Leonova,1
S. N. Nikolaeva,2 and E. I. Boreko2
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 51, No. 1, pp. 15 – 19, January, 2017.
Original article submitted November 10, 2014.
The new adamantine derivative ethyl(3-ethyladamant-1-yl)carbamate was found to have high antiviral activ-
ity against herpes simplex virus and vaccinia viruses, as well as adenovirus (EC50 = 0.62, 5.15, and 48.5 mg/ml
respectively). Activity against the herpesvirus was demonstrated in experiments in various cell cultures using
different virus strains, including a variant resistant to acycloguanosine (ACG). Simultaneous application of
ethyl(3-ethyladamant-1-yl)carbamate and ACG increased the inhibition of herpesvirus reproduction in cell
cultures and decreased the mortality of laboratory animals with experimental herpesvirus neuroinfection as
compared with the use of each substance alone. 3-Ethyladamant-1-yl)carbamate was a low-toxicity com-
pound, did not inhibit DNA synthesis in uninfected cell cultures, and its acute intragastric LD50 for white mice
was 1831.8 mg/kg.
Keywords: adamantine, ethylcarbamate, virus infections, antiviral activity.
Herpesvirus infections are the second most common af-
ter influenza and other acute respiratory viral infections. Her-
pes simplex virus currently infects 60 – 90% of the popula-
tion [1]. Genital herpes is one of the most widespread infec-
tious pathologies in humans, with all the signs of an
epidemic [2]. Resistant variants of herpes simplex virus, de-
spite decades of use of acycloguanosine (ACG – acyclovir,
Virolex, Zovirax) and its analogs, account for a few per cent
of isolates, and are somewhat more common in patients with
immunodeficiencies (4 – 7%) [3, 4]. It should therefore be
noted that chronic recurrent herpes is the most frequently
seen form of endogenous infection, and that such patients
have repeated episodes of using ACG. According to our ad-
mittedly limited data, herpes simplex virus, usually type 2,
isolated from all the patients with urogenital pathology stud-
ied have some degree of resistance to ACG [5].
also from the existence of types of herpesvirus infection in
which ACG lacks adequate efficacy. The use of combina-
tions of antiviral drugs with different mechanisms of action
leads to additive or synergistic action; realization of this po-
tential in medical practice requires widening of the range of
available drugs.
We describe here the synthesis of a new adamantine de-
rivative, ethyl(3-ethyladamant-1-yl)carbamate, and present
the results of studies of its antiviral properties.
The main method used for preparing N-substituted esters
of carbamic acid consists of attaching alcohols to isocyanates
[6, 7]; the reaction can use isocyanates formed in situ from
carboxylic acid acylazides or amides in the Curtius or
Hofmann reactions. N-substituted esters of carbamic acid
were also synthesized by the reaction of alcohols with
isocyanic acid or chloroformates with amines [8 – 10].
Carbamic acid esters whose structures contain an amide
fragment are known to be able to undergo adamantylation in
nitric or sulfuric acid [11, 12]. Nitroxy and hydroxy deriva-
tives of adamantine in sulfuric acid easily generates the
adamantyl cation, which can attack the unshared electron
pair in nitrogen-containing nucleophiles.
The importance of finding and developing new sub-
stances effectively inhibiting herpesviruses results not only
from cases of resistance to ACG as the most widely drug, but
1
Samara State Technical University, 244 Molodogvardeiskaya Street,
443100 Samara, Russia; e-mail: orgchem@samgtu.ru
2
Republican Scientific Applied Center for Epidemiology and Microbiol-
We have previously shown that interaction of hydroxy or
nitroxy derivatives of adamantine with potassium cyanate
ogy, Ministry of Health of the Republic of Belarus, 23 Filimonov Street,
220114 Minsk, Belarus; e-mail: bei@mail.by
13
0091-150X/17/5101-0013 © 2017 Springer Science+Business Media New York

14
Yu. N. Klimochkin et al.
Scheme
C2H5
C2H5
C2H5
HNO3
Ac2O
NH2COOC2H5
H2SO4
ONO2
NHCOOC2H5
I
II
III
and alcohols in concentrated sulfuric acid leads to the pro-
duction of N-adamantylcarbamates 10]. In sulfuric acid, in
situ formed isocyanic acid quickly produces the carbamic
acid ester, which then undergoes alkylation by the 1-adaman-
tylation. The results showed that the reaction was partially
inhibited by addition of alkyl groups into the adamantane
carcass, and in this situation the method does not yield ada-
mantylated carbamates at sufficient yield. The starting
3-ethyladamant-1-yl nitrate (II) was synthesized from
1-ethyladamantane (I) as described in [13]. Ethyl(3-ethylada-
mantan-1-yl)carbamate (III) was obtained by the reaction of
compound II with ethylcarbamate in 94% sulfuric acid at
high yield (84%).
with TMS as the internal standard and CDCl3 as solvent. IR
spectra were recorded on a Shimadzu IRAffinity-1 spectro-
photometer (Japan) in a thin layer on a KBr plate. Mass spec-
tra were taken on a Thermo Finnigan DSQ (USA) chromato-
mass spectrometer with a mass-selective detector in the elec-
tron ionization regime (70 eV), a B-5MS 30 m ´ 0.32 mm
quartz column at a column temperature of 80 – 340°C (heat-
ing rate 20°C/min), an evaporator temperature of 300°C, and
helium as the carrier gas. UV spectra were recorded on a
Shimadzu UV mini 1240 (Japan) UV spectrometer. Elemen-
tal analysis was run on a EuroVector EA-3000 CHNS-O ele-
mental analyzer (Italy) with L-cysteine as the standard. Ele-
mental analysis data were consisted with calculated values.
3-Ethyladamant-1-yl nitrate (II) was prepared from
1-ethyladamantane (I) as described in [13] with a yield of
90% and nD20 = 1.4957.
1
The H NMR spectrum of compound III contained an
NH proton signal as a broad singlet with d = 4.51 ppm. Pro-
ton signals as triplets and quartets with d = 0.76 ppm and
1.13 ppm and a spin-spin coupling constant (SSCC) of
7.8 Hz corresponded to signals from the ethyl group attached
to the adamantine carcass. Ethyl group proton signals from
the ethyl carbamate fragment were present as a triplet with
d = 1.9 ppm for the methyl group and a quartet with
d = 4.0 ppm for the methylene group, with SSCC = 7.1 Hz.
Ethyl(3-ethyladamant-1-yl)carbamate (III). Nitrate II
(2.6 g, 0.01 mol) was dissolved in 20 ml of 94% sulfuric acid
with cooling, 8 g (0.09 mol) of ethylcarbamate was added,
and the mix was left overnight. The reaction was poured onto
ice and extracted with toluene; the toluene extract was
washed with saturated sodium bicarbonate solution and wa-
ter, and dried over anhydrous Na2SO4. The toluene was then
removed by evaporation in a vacuum rotary evaporator and
the residue was distilled in vacuo. The yield was 2.4 g
The 13C NMR spectrum contained
a
signal with
d = 154.76 ppm, corresponding to the carbon atom of the
carbonyl group.
(84%), the melting temperature was 164°C/15 mm; nD20
=
1.4998. The IR spectrum, nmax, cm-1, was: 3385 (NH), 3010,
1710 (C=O), 1520 (C-N). The mass spectrum, m/z (Irel, %),
was: 251 [M]+ (59), 222 (14), 194 (100), 176 (10), 166 (58),
148 (40), 138 (20), 93 (19), 79 (20). The atomic formula was
EXPERIMENTAL CHEMICAL SECTION
1H and 13C NMR spectra were recorded on a JEOL JNM
ECX-400 (Japan) spectrometer at a frequency of 400 MHz,
TABLE 1. Inhibition of the Multiplication of Herpes and Vaccinia Viruses in CEF Cell Cultures in the Presence of Ethyl(3-ethylada-
mant-1-yl)carbamate (II)
Zone diameter, screening test, mm
Plaque reduction assay
Virus
Concentration,
EC50 (I95),
suppression of plaque
formation
virus titer,
log pfu/ml
difference from con-
trol, log pfu/ml
toxicity
mg/ml
mg/ml
Herpes
9
22
50 – 6
< 3.9
4.9
> 1.6
0.6
–
0.62 (0.89 – 0.42)
3
0
5.5
Vaccinia
18
18
50 – 12
< 2.5
3.2
> 1.6
0.9
–
5.15 (6.63 – 3.99)
6
0
4.1
pfu – plaque-forming units; I95 – confidence interval

Ynthesis and Antiviral Properties of Ethyl(3-ethyladamant-1-yl)carbamate
15
1
C H NO . The H NMR spectrum (CDCl ), d, ppm, was:
program based on probit analysis using the Finney algorithm
[14].
15 25
2
3
0.76 (t, J 7.8 Hz, 3H, CH CH ), 1.13 (q, J 7.8 Hz, 2H,
2
3
Toxic properties were determined in terms of the ability
to induce cytodestructive changes to cells at 48 – 96 h of in-
cubation and impairment of the ability to take up neutral red.
The maximum tolerable concentration (MTC) was taken as
the highest concentration of substance not producing these
changes. In addition, 50% suppression of DNA synthesis in
uninfected Vero cell cultures was determined in terms of
3H-thymidine uptake [15].
CH CH ), 1.9 (t, J 7.1 Hz, 3H, CH CH ), 1.36 – 1.37 (m, 4H,
2
3
2
3
2CH ), 1.53 – 1.56 (m, 4H, 2CH ), 1.79 – 1.86 (m, 4H,
2Ad
2Ad
2CH ), 2.08 – 2.09 (m, 2H, 2CH ), 4.00 (q, J 7.1 Hz, 2H,
2Ad
Ad
CH CH ), 4.51 (broad s, 1H, NH). The 13C NMR spectrum,
2
3
d , ppm, was: 7.10, 14.69, 29.71, 34.55, 35.90, 36.02, 40.79,
C
41.60, 46.20, 51.54, 59.99, 154.76. The UV spectrum was
(C H OH), l , nm (lg e), was: 257 (5.0).
2
5
max
Study substances were prepared by dilution in mainte-
nance medium from stock solution (5 mg/ml) in double-dis-
tilled water; 10% ethanol was added for compound III. ACG
was used as the sodium salt of acyclovir (Virolex powder for
intravenous infusions, substance manufactured by The Well-
come Foundation Ltd.).
EXPERIMENTAL BIOLOGICAL SECTION
Cell culture studies. Studies were performed using mo-
nolayer cultures of primary chick embryo fibroblasts (CEF)
and human embryo kidney (HEK), transformed green mon-
key kidney cells (Vero), herpes simplex virus type 1 (strain
1C) and its variant resistant to the known antiherpetic agent
ACG obtained by serial passage of the starting virus in the
presence of the drug, vaccinia virus, and type 3 adenovirus.
Herpes and vaccinia viruses were used in a screening test in
CEF cells and plaque reduction assay in Vero cells under
agar; adenovirus was used for assessment of the effects of
study drug on the development of the cytoplasmic effect
(CPE) in HEK cells. Medium 199 was used as maintenance
and nutritive medium, based on a concentrate supplemented
with 1% bacteriological agar and 0.005% neutral red (all re-
agents from Sigma). Multiplicities of infection were
0.00002 cfu/cell in the screening test and the plaque reduc-
Laboratory animal studies. Mongrel white mice
weighing 6 – 7 g were infected with herpesvirus and its
ACG-resistant variant intracerebrally at a dose correspond-
ing to 1000 – 10,000 LD /0.1 ml or i.p. at a dose of 100
50
LD /0.1 ml. Both virus variants, the initial and resistant,
50
were preadapted by passaging by intracerebral infection of
white mice.
Drug III was given to animals intragastrically as a sus-
pension in isotonic sodium chloride solution containing 10%
Tween-80, starting 3 h after infection twice daily to comple-
tion of the experiment (or for seven days for i.p. infection).
Animals were observed until death in controls (5 – 6 days
from infection) or for 14 days after i.p. infection.
tion assay and 0.001 TCID /cell (tissue cytopathic infec-
50
The acute toxicity of compound III was tested on mice of
both genders weighing 18 – 20 g; compound III was given
intragastrically as a suspension in 10% Tween-80 in five in-
creasing doses starting at 1.6 g/kg with a step of 0.1 g/kg
(2 – 4 animals per dose, total 16 animals). Animals were ob-
served for 10 days after inoculation. Results were processed
tious dose).
The criteria for antiviral activity were the presence and
size of the plaque suppression zone (screening test) and the
decrease in the CPE and infectious titer of viruses in the
presence of different test substance concentrations. The con-
centration giving 50% suppression of virus replication in the
presence of study compound (EC ) was calculated using a
as described for calculation of EC .
50
50
TABLE 2. Inhibition of the Reproduction of Herpes Virus and its ACG-Resistant Variant by Ethyl(3-ethyladamant-1-yl)carbamate (III), ACG,
and the Combination of Compound III and ACG in Vero Cells
Herpesvirus
ACG-resistant herpesvirus
Concentration,
Group
virus titer,
difference from control,
virus titer,
difference from control,
lg TCID50/ml
mg/ml
lg TCID50/ml
lg TCID50/ml
lg TCID50/ml
III
30
15
4.0
4.9
5.7
4.8
5.6
0
2.1
1.2
0.4
1.3
0.5
6.1
3.1
–
3.8
4.5
5.6
6.2
6.2
4.0
5.1
6.2
2.4
1.7
0.6
0
7.5
ACG
0.4
0.2
0
ACG/III
0.4/15
0.2/7.5
0
2.2
1.1
–
3.0
6.1
Control virus

16
Yu. N. Klimochkin et al.
RESULTS AND DISCUSSION
In addition, studies in HEK showed that III suppressed
the development of the CPE of adenovirus by 75% at the
Cell culture toxicity studies showed that the MTC of
compound III was 50 mg/ml. A 50% level of suppression of
DNA synthesis was obtained in uninfected Vero cell cultures
MTC and ½MTC (EC = 48.5 (79.5 – 29.6) mg/ml).
50
Comparison of the antiviral actions of III and ACG
against herpesviruses was performed using Vero cells (Ta-
ble 2). ACG had no inhibitory activity against the resistant
variant of the virus. At the same time, compound III at con-
centrations of 30 and 15 mg/ml suppressed the multiplication
3
(suppression of H-thymidine uptake) at 92 mg/ml, which is
evidence that III has low toxicity (the value for ACG is
2 mg/ml [14], while the MTC is greater than 1600 mg/ml).
The antiviral activity of III was determined at the MTC
of this variant by 2.4 and 1.7 lg TCID respectively.
50
Use of low concentrations of III and ACG led to a
marked synergistic action in relation to herpesvirus. Thus,
the combination of 0.4 mg/ml acycloguanosine and 15 mg/ml
III produced 100% arrest ofvirus reproduction. At the same
time, individual use of substances at these concentrations de-
and lower concentrations (Table 1). These results show that
compound III has a marked ability to suppress herpes and
vaccinia virus multiplication in cell cultures.
A decrease in the infective titer of herpesvirus by 1.6 lg
cfu/ml or more in the concentration range 50 – 6 mg/ml was
creased the virus titer by no more than 1.3 log TCID /ml.
50
demonstrated. Calculated EC
values for III were
Lower concentrations of both substances, inactive when used
50
0.62 mg/ml for herpesvirus and 5.15 mg/ml for vaccinia virus.
alone, i.e., 0.2 mg/ml ACG and 7.5 mg/ml compound III, as
TABLE 3. Efficacies of Ethyl(3-ethyladamant-1-yl)carbamate (III), ACG, and their Combination in Experimental Herpes Meningoencep-
halitis in White Mice
Indicator of efficacy
herpesvirus
ACG-resistant herpesvirus
Dose,
Compound
mg/kg
Mortality,
Mortality,
number of animals
(died/total)
ED50 (I95),
mg/kg
number of animals
(died/total)
ED50 (I95),
mg/kg
± Sx, %
± Sx, %
Intracerebral infection
III
200
100
50
25
0
8/13
5/13
14/15
15/15
9/9
167.0
6/23
4/16
79.6
61.5 ± 13.4
26.1 ± 9.2
25.0 ± 10.8
56.2 ± 12.4
100.0
(287.5 – 97.0)
(109.2 – 58.0)
38.5 ± 13.5
93.3 ± 6.4
100.0
9/16
16/16
6/6
100.0
100.0
ACG
50
0
3/20
8/8
<< 50
17/17
20/21
100.0
> 50
15.0 ± 8.0
100.0
95.2 ± 4.7
I.p. infection
III
100
2/35
7/29
5/24
7/21
33/36
0/12
1/12
4/7
< 0.1
2/21
< 0.1
3.9 ± 3.3
7.9 ± 5.0
20.8 ± 8.3À
33.3 ± 10.3
91.7 ± 4.6
0
9.5 ± 6.4
10
5/25
20.0 ± 8.0
1
0.1
9/34
26.5 ± 7.6
9/25
36.0 ± 9.6
0
39/42
92.8 ± 4.0
ACG
10
0.13 (0.4 – 0.04)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
8.3 ± 2.6
57.1 ± 18.7
91.7 ± 8.0
0
0.1
0
11/12
0/3
ACG/III
1/0.1
0.1/0.1
0.01/0.1
~ 0.013/-
-
0/4
0
3/4
3
“-” – not determined

Ynthesis and Antiviral Properties of Ethyl(3-ethyladamant-1-yl)carbamate
17
tures and decreased mortality in laboratory animals with ex-
perimental herpesvirus neuroinfection as compared with use
of each substance alone.
well as 0.1 mg/ml ACG and 3.75 mg/ml compound III, sup-
pressed virus multiplication by 3.1 and 1.7 lg TCID /ml re-
50
spectively.
compound III was a low-toxicity compound. Its LD for
The level of suppression of the ACG-resistant
herpesvirus variant in the presence of these combinations of
ACG and compound III was no greater than the antiviral ef-
fect of III used alone.
50
single intragastric doses was 1831.8 (1754.1-1912.9) mg/kg.
Thus, the LD /ED ratio (k) was 11.0 – 23.0 in infections
50
50
induced by intracerebral administration of herpesvirus and
more than 18,318 in infection following i.p. administration
of virus.
The antiviral properties of compound III demonstrated in
cell cultures were confirmed in experimental herpesvirus in-
fection using different routes of infection. The results (Ta-
ble 3) provide evidence that compound III can reduce the
mortality of mice with infections due to viruses including the
ACG-resistant variant by amounts ranging from over 50% to
85%.
These results were obtained in the framework of carrying
out the project part of Russian Ministry of Education and
Science State Contract (No. 4.1440.2014/K) and a grant from
the Russian Foundation for Basic Research (No.
15-43-02536).
The mean effective dose of III was 79.6 mg/kg in infec-
tion with the ACG-resistant variant and 167 mg/kg on infec-
tion with the initiation herpes variant. Solvent (10%
tween-80 in isotonic saline, virus control group, dose 0 in ta-
ble 3) had no effect on the course of infection.
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