In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2
Aim. To establish anti-influenza activities for 8 compounds of adamantyl (alkyl, cycloalkyl) de-rivatives of aminopropanol-2 in vitro and in vivo investigations. Methods. The antiviral action of compounds was determined in vitro by reduction of infectious titer of the influenza virus in the Madin-Da...
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| Cite this: | In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 / O.M. Voloshchuk, Y.V. Korotkiy, S.L. Rybalko, D.B. Starosila, V.P. Shirobokov // Вiopolymers and Cell. — 2017. — Т. 33, № 6. — С. 453-462. — Бібліогр.: 21 назв. — англ. |
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Voloshchuk, O.M. Korotkiy, Y.V. Rybalko, S.L. Starosila, D.B. Shirobokov, V.P. 2019-06-13T14:17:13Z 2019-06-13T14:17:13Z 2017 In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 / O.M. Voloshchuk, Y.V. Korotkiy, S.L. Rybalko, D.B. Starosila, V.P. Shirobokov // Вiopolymers and Cell. — 2017. — Т. 33, № 6. — С. 453-462. — Бібліогр.: 21 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000968 https://nasplib.isofts.kiev.ua/handle/123456789/153122 578.832.1:57.044:57.085.1/.2 Aim. To establish anti-influenza activities for 8 compounds of adamantyl (alkyl, cycloalkyl) de-rivatives of aminopropanol-2 in vitro and in vivo investigations. Methods. The antiviral action of compounds was determined in vitro by reduction of infectious titer of the influenza virus in the Madin-Darby Canine Kidney (MDCK) cell culture and in vivo on the model of influenza pneu-monia in mice. Results. Three of eight studied compounds inhibit the reproduction of the influ-enza virus strain A/FM/1/47(H1N1) by more than 2 lg ID50 in the concentration range from < 0.39 µg/ml (the compounds 30 and 33) to 1.56 µg/ml (the compound 5), their chemotherapeutic indices are 256 and 16, respectively. The experiments in vivo showed that in prophylactic scheme the efficiency index (EI) of the compounds 5 and 30 is 60 %; in therapeutic scheme EI of the compounds 5 and 33 is 100 % that is two times higher, than for rimantadine. Conclusions. High chemotherapeutic indices of the compounds 30, 33 and their ability to suppress the influenza virus reproduction in the MDCK cell culture by more than 2 lg ID50 indicate the anti-influenza activity of these compounds. It has been also confirmed by in vivo experiment. Мета. Визначити антигрипозну активність у 8 сполук адамантан (алкіл, циклоалкіл) похідних амінопропанолу-2 в дослідах in vitro та in vivo. Методи. Оцінку противірусної дії сполук проводили in vitro за зниженням інфекційного титру вірусу грипу на культурі клітин, та in vivo на моделі грипозної пневмонії у мишей. Результати. В досліді in vitro 3 сполуки серед 8 досліджених пригнічують репродукцію вірусу грипу штаму А/FM/1/47(H1N1) на 2 lg ID50 і більше в концентраціях від < 0,39 мкг/мл (сполуки №№ 30 і 33) до 1,56 мкг/мл (сполука № 5), їх хіміотерапевтичні індекси стосовно цього вірусу становлять 256 та 16, відповідно. В досліді in vivo при профілактичній схемі досліду сполуки №№ 5 і 30 мають індекс ефективності (ІЕ) 60 %, а при лікувальній схемі досліду ІЕ сполук №№ 5 та 33 більше ніж в два рази перевищує цей показник у ремантадину і становить 100 %. Висновки. Високі хіміотерапев-тичні індекси та здатність пригнічувати репродукцію вірусу грипу на 2 lg ID50 і більше дозволяє віднести сполуки №№ 30, 33 і 5 до таких, що проявляють протигрипозну активність. Це також підтверджено в досліді in vivo. Цель. Определить антигриппозную активность у 8 соединений адамантан (алкил, циклоалкил) производных аминопропанола-2 в экспериментах in vitro и in vivo. Методы. Противовирусное действие исследуемых соединений определяли in vitro по снижению инфекционного титра вируса гриппа в культуре клеток MDCK и in vivo на модели гриппозной пневмонии у мышей. Результаты. 3 соединения среди 8 исследованных подавляют репродукцию виру-са гриппа штамм А/FM/1/47(H1N1) на 2 lg ID50 и больше в концентрациях от < 0,39 мкг/мл (соединения №№ 30 и 33) до 1,56 мкг/мл (соединение № 5), их химиотерапевтические индексы (ХТИ) в отношении этого вируса состав-ляют 256 и 16, соответственно. В эксперименте in vivo при профилактической схеме опыта соединения №№ 5 и 30 имеют индекс эффективности (ИЭ) 60 %. ИЭ соединений №№ 5 и 33 при лечебной схеме опыта больше чем в два раза превышает этот показатель у Ремантадина и составляет 100 %. Выводы. Высокие ХТИ и способность подав-лять репродукцию вируса гриппа на 2 lg ID50 и более позволяют отнести соединения №№ 30, 33 и 5 к таким, что проявляют противогриппозную активность. Это также подтверждено в експриментах in vivo. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Viruses and Cell In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 Антигрипозна дія адамантан (алкіл, циклоалкіл) похідних амінопропанолу-2 в дослідах in vitro та in vivo Антигриппозное действие адамантан (алкил, циклоалкил) производных аминопропанола-2 в экспериментах in vitro и in vivo Article published earlier |
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In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 |
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In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 Voloshchuk, O.M. Korotkiy, Y.V. Rybalko, S.L. Starosila, D.B. Shirobokov, V.P. Viruses and Cell |
| title_short |
In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 |
| title_full |
In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 |
| title_fullStr |
In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 |
| title_full_unstemmed |
In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 |
| title_sort |
in vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 |
| author |
Voloshchuk, O.M. Korotkiy, Y.V. Rybalko, S.L. Starosila, D.B. Shirobokov, V.P. |
| author_facet |
Voloshchuk, O.M. Korotkiy, Y.V. Rybalko, S.L. Starosila, D.B. Shirobokov, V.P. |
| topic |
Viruses and Cell |
| topic_facet |
Viruses and Cell |
| publishDate |
2017 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Антигрипозна дія адамантан (алкіл, циклоалкіл) похідних амінопропанолу-2 в дослідах in vitro та in vivo Антигриппозное действие адамантан (алкил, циклоалкил) производных аминопропанола-2 в экспериментах in vitro и in vivo |
| description |
Aim. To establish anti-influenza activities for 8 compounds of adamantyl (alkyl, cycloalkyl) de-rivatives of aminopropanol-2 in vitro and in vivo investigations. Methods. The antiviral action of compounds was determined in vitro by reduction of infectious titer of the influenza virus in the Madin-Darby Canine Kidney (MDCK) cell culture and in vivo on the model of influenza pneu-monia in mice. Results. Three of eight studied compounds inhibit the reproduction of the influ-enza virus strain A/FM/1/47(H1N1) by more than 2 lg ID50 in the concentration range from < 0.39 µg/ml (the compounds 30 and 33) to 1.56 µg/ml (the compound 5), their chemotherapeutic indices are 256 and 16, respectively. The experiments in vivo showed that in prophylactic scheme the efficiency index (EI) of the compounds 5 and 30 is 60 %; in therapeutic scheme EI of the compounds 5 and 33 is 100 % that is two times higher, than for rimantadine. Conclusions. High chemotherapeutic indices of the compounds 30, 33 and their ability to suppress the influenza virus reproduction in the MDCK cell culture by more than 2 lg ID50 indicate the anti-influenza activity of these compounds. It has been also confirmed by in vivo experiment.
Мета. Визначити антигрипозну активність у 8 сполук адамантан (алкіл, циклоалкіл) похідних амінопропанолу-2 в дослідах in vitro та in vivo. Методи. Оцінку противірусної дії сполук проводили in vitro за зниженням інфекційного титру вірусу грипу на культурі клітин, та in vivo на моделі грипозної пневмонії у мишей. Результати. В досліді in vitro 3 сполуки серед 8 досліджених пригнічують репродукцію вірусу грипу штаму А/FM/1/47(H1N1) на 2 lg ID50 і більше в концентраціях від < 0,39 мкг/мл (сполуки №№ 30 і 33) до 1,56 мкг/мл (сполука № 5), їх хіміотерапевтичні індекси стосовно цього вірусу становлять 256 та 16, відповідно. В досліді in vivo при профілактичній схемі досліду сполуки №№ 5 і 30 мають індекс ефективності (ІЕ) 60 %, а при лікувальній схемі досліду ІЕ сполук №№ 5 та 33 більше ніж в два рази перевищує цей показник у ремантадину і становить 100 %. Висновки. Високі хіміотерапев-тичні індекси та здатність пригнічувати репродукцію вірусу грипу на 2 lg ID50 і більше дозволяє віднести сполуки №№ 30, 33 і 5 до таких, що проявляють протигрипозну активність. Це також підтверджено в досліді in vivo.
Цель. Определить антигриппозную активность у 8 соединений адамантан (алкил, циклоалкил) производных аминопропанола-2 в экспериментах in vitro и in vivo. Методы. Противовирусное действие исследуемых соединений определяли in vitro по снижению инфекционного титра вируса гриппа в культуре клеток MDCK и in vivo на модели гриппозной пневмонии у мышей. Результаты. 3 соединения среди 8 исследованных подавляют репродукцию виру-са гриппа штамм А/FM/1/47(H1N1) на 2 lg ID50 и больше в концентрациях от < 0,39 мкг/мл (соединения №№ 30 и 33) до 1,56 мкг/мл (соединение № 5), их химиотерапевтические индексы (ХТИ) в отношении этого вируса состав-ляют 256 и 16, соответственно. В эксперименте in vivo при профилактической схеме опыта соединения №№ 5 и 30 имеют индекс эффективности (ИЭ) 60 %. ИЭ соединений №№ 5 и 33 при лечебной схеме опыта больше чем в два раза превышает этот показатель у Ремантадина и составляет 100 %. Выводы. Высокие ХТИ и способность подав-лять репродукцию вируса гриппа на 2 lg ID50 и более позволяют отнести соединения №№ 30, 33 и 5 к таким, что проявляют противогриппозную активность. Это также подтверждено в експриментах in vivo.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/153122 |
| citation_txt |
In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2 / O.M. Voloshchuk, Y.V. Korotkiy, S.L. Rybalko, D.B. Starosila, V.P. Shirobokov // Вiopolymers and Cell. — 2017. — Т. 33, № 6. — С. 453-462. — Бібліогр.: 21 назв. — англ. |
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453
O. M. Voloshchuk, Y. V. Korotkiy, S. L. Rybalko
© 2017 O. M. Voloshchuk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf
of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any
medium, provided the original work is properly cited
UDC 578.832.1:57.044:57.085.1/.2
In vitro and in vivo investigations on anti-influenza effect of adamantyl
(alkyl, cycloalkyl) derivatives of aminopropanol-2
O. M. Voloshchuk1, Y. V. Korotkiy2, S. L. Rybalko3, D. B. Starosila3, V. P. Shirobokov1
1 Bogomolets National Medical University
13, Shevchenko Blvd., Kyiv, Ukraine, 01601
2 Institute of organic chemistry NAS of Ukraine
5, Murmanska Str., Kyiv, Ukraine, 02660
3 Gromashevsky L. V. Institute of Epidemiology and Infection Diseases, NAMS of Ukraine
5, Amosova Str., Kyiv, Ukraine, 03038
post-ua@yandex.ua
Aim. To establish anti-influenza activities for 8 compounds of adamantyl (alkyl, cycloalkyl)
derivatives of aminopropanol-2 in vitro and in vivo investigations. Methods. The antiviral ac-
tion of compounds was determined in vitro by reduction of infectious titer of the influenza virus
in the Madin-Darby Canine Kidney (MDCK) cell culture and in vivo on the model of influ-
enza pneumonia in mice. Results. Three of eight studied compounds inhibit the reproduction
of the influenza virus strain A/FM/1/47(H1N1) by more than 2 lg ID50 in the concentration
range from < 0.39 µg/ml (the compounds 30 and 33) to 1.56 µg/ml (the compound 5), their
chemotherapeutic indices are 256 and 16, respectively. The experiments in vivo showed that in
prophylactic scheme the efficiency index (EI) of the compounds 5 and 30 is 60 %; in therapeu-
tic scheme EI of the compounds 5 and 33 is 100 % that is two times higher, than for rimantadine.
Conclusions. High chemotherapeutic indices of the compounds 30, 33 and their ability to sup-
press the influenza virus reproduction in the MDCK cell culture by more than 2 lg ID50 indicate
the anti-influenza activity of these compounds. It has been also confirmed by in vivo experiment.
K e y w o r d s: influenza virus, adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2,
antiviral activity.
Introduction
For today in the world and in Ukraine in par-
ticular, is unstable epidemic situation for many
infectious diseases, including viral. According
to the WHO estimates by 2030, infection dis-
eases will be one of the major causes of mortal-
ity among people (Antimicrobial resistance:
global report on surveillance 2014, http://apps.
who.int/iris/bitstream/10665/112642/1/97892
41564748_eng.pdf?ua=1). Influenza is a lea der
among the respiratory tract infections by the
Viruses and Cell ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2017. Vol. 33. N 6. P 453–462
doi: http://dx.doi.org/10.7124/bc.000968
454
O. M. Voloshchuk, Y. V. Korotkiy, S. L. Rybalko et al.
number of cases and distribution. Airborne
droplets way of distribution of flu infection
leads to the rapid spread of the pathogen, and
high antigenic variability of the influenza virus
results in the appearance of new strains causing
epidemics and pandemics. According to the
WHO data, the flu and flu-like illnesses affect
annually 5 – 30 % of the world population. The
flu and acute respiratory viral infections annu-
ally affect about 15 million people in Ukraine
[1]. Despite the recent achievements in diag-
nostics and therapy, the problem of treatment
of influenza remains a relevant problem, as well
as the establishment of effective chemothera-
peutic agents for prevention and therapy of this
infection. Nowadays the process of creating
new medicines commonly starts from finding
new drug candidates – structural prototypes of
future chemotherapeutic agents with the desired
pharmacological effect [2]. The search for an-
timicrobial compounds is carried out among
new chemical classes as well as among new
derivatives of already known compounds af-
fecting certain targets. Since discovery of ada-
mantane its derivatives have proven to be the
most promising as viral inhibitors [3–6]. The
best of them is aminoadamantane (amantadine,
rimantadine) [3, 7], which entered the pharma-
ceutical market and is successfully used as an
anti-influenza drug.
The adamantane molecule is highly reactive
compared with other hydrocarbons, which al-
lows obtaining a wide range of poly-function-
al derivatives. The molecule can be modified
by the C–H bond activation for the direct nu-
cleophilic substitution or radical reactions
[8–12]. It can be also modified by the introduc-
tion of substituents or heterocyclic atoms for
designing new derivatives [13, 14].
It is known from literature that amino alco-
hols and the products of their substitution in
the amino and hydroxyl groups have polyvalent
pharmacological effects (stimulators or block-
ers of α and β-adrenaline, adrenaline mimetics,
antibiotics, etc.) [15, 16]. It is also known that
adamantane containing amino alcohols dem-
onstrates antiviral action [17]. The modification
of β-adrena blockers structures via the introduc-
tion of various adamantyl fragments as amino
groups results in the appearance of antivirus
effects [18]. Based on this knowledge it seemed
advisable to study an antiviral effect of new
adamantane derivatives containing amino al-
cohols and their related alkyl and cycloalkyl
structural analogs [19, 20].
Materials and Methods
The synthesized compounds
Investigated adamantanyl (alkyl, cycloalkyl)
derivatives of aminopropanol-2 were synthe-
sized in Institute of Organic Chemistry of the
NAS of Ukraine according to the procedures
developed in [15]. The structure and purity of
the synthesized compounds were proved via
elemental analysis, proton magnetic resonance
spectroscopy and chromatography mass spec-
trometry methods. The common formula of the
tested compounds is
ROCH2CH(OH)CH2R1
where: R – Ad(CH2)2, R1 – cyclohexylamin
(the compound 5); R – Ad(CH2)2, R1 – 1,1,3,3-
tetramethylbutylamine (the compound 6); R –
tretbutyl, R1 – 2,2,6,6-tetraethyl piperidin
amine (the compound 33); R – allyl, R1 –
1,1,3,3-tetramethylbutyl-amine (the compound
40); R – tret-amyl, R1 – 2,2,6,6-tetramethyl-
4-hydroxy piperidine amine (the compound
455
In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2
47); R – cyclopentyl, R1 – 2,2,6,6-tetramethyl-
4-hydroxy piperidine amine (the compound
48); R – bornyl, R1 – 2,2,6,6-tetramethyl-4-hy-
droxy piperidine amine (the compound 51).
The compounds 48 and 51 were investigated
in the alkali form.
Depending on the radicals structure all test-
ed compounds were divided into the adaman-
tane containing (compounds 5 and 6), bornyl
containing (the compound 51), the compounds
with cyclic substituents in alkoxy group (the
compounds 30 and 48) and the compounds
with alicyclic substituents in alkoxy group (the
compounds 33, 40, 47). All synthesized com-
pounds are colorless and odorless crystalline
substances with different solubility in water
and 96 % alcohol. The 1 mg/ml matrix solu-
tions of the studied compounds were prepared
using a medium for cell cultures without se-
rum. These solutions were sterilized via filtra-
tion through cellulose filter with 0.22 µm pore
diameter. The solution was stored in sterile
bottles at –20 °C before using.
Virus and cell culture
The mouse-adapted influenza virus A/
FM/1/47(H1N1) was cultivated in Madin-
Darby Canine Kidney (MDCK) cells (ATCC,
CCL-34). MDCK cells were cultured in
Dulbecco’s modified Eagle’s medium
(DMEM), supplemented with 5 % heat inac-
tivated fetal bovine serum (FBS) in 37 °C
incubator with 5 % CO2. The influenza A/
FM/1/47(H1N1) and MDCK cells were ob-
tained from the Bank of cell cultures of the
laboratory of experimental chemotherapy of
viral infections of the L. V. Gromashevsky
Institute of epidemiology and infectious dis-
eases of NAMS of Ukraine.
Virological tests in vitro
Anti-influenza activity of the tested compounds
was evaluated by chemotherapeutic index
(CTI) determined as the ratio of the maximum
tolerated dose (MTD) to the minimum active
concentration (MAC).
MTD was determined as the greatest con-
centration of the tested compound that did not
cause the cytopathic effect (CPE) in the mono-
layer of MDCK cells [16, 17]. For this pur-
pose, the growing MDCK cells were plated at
a density of 1 × 105 cells/ml and incubated in
96-well plates for 24 h (37 °C, 5 % CO2). Then
the cells were treated with the tested com-
pounds of different concentration (3.12, 6.25,
12.5, 25, 50, 100 μg/ml) for 5 days. The wells
containing no compounds were used as a neg-
ative control. In the experiments, we used at
least ten holes for each dilution of the sub-
stance. The CPE was determined by changed
cell morphology (degeneration or exfoliation
of cells from the hole surface).
MAC of the studied compounds was deter-
mined by the degree of reduction of infectious
titer of the virus according to the General es-
tablished procedure [16, 17]. The virus titers
were calculated as 50 % of tissue culture infec-
tious dose (TCID50) using the Kerber formula
(Manual for the virological investigation of
polio, http://apps. who.int/iris/bitstream/10665/
68762/1/WHO_IVB_04.10.pdf):
Lg TCID50= L-d(S-0.5),
where L – the initial dilution, d – the difference
between Lg of serial dilutions, S – the sum of
the proportions of tested objects, which gave
a positive result.
The hemagglutination inhibition test (HAI)
was carried out to study the influence of tested
compounds on the influenza virus hemagglu-
http://apps
456
O. M. Voloshchuk, Y. V. Korotkiy, S. L. Rybalko et al.
tination activity (HA). The tested substances
diluted in the range of 50 – 0.36 mkg/ml were
incubated with the virus (4 agglutination units)
for 30 min at room temperature; 0.75 % eryth-
rocytes suspension of Guinea pigs were added,
shaken and left at room temperature for
60 min. As a titer of HAI was taken a limiting
dilution, which gives a complete delay of the
hemagglutination reaction, that is ++++.
Virological studies in vivo
The anti-influenza activity of tested compounds
was studied via simulation of influenza pneu-
monia in mice using prophylactic and therapeu-
tic schemes of the compound introduction. In
these studies, we used white outbred mice
weighing 18–22 g, which were on the standard
diet for at least 7 days. All experiments were
performed with ten mice per group.
For the prophylactic scheme the mice were
intranasally (i.n.) inoculated with 0.2 ml of the
tested compounds at 10×ID50 (50 %-mouse in-
fectious dose; sub-lethal) 24 h before infection
with the influenza virus. For the therapeutic
scheme this procedure was carried out 24 h after
infection of mice with the influenza virus. At the
same time for both schemes, the control was a
group of animals infected with the influenza
virus without administration of the tested com-
pounds. The influenza virus strain A/
FM/1/47(H1N1) with infectious titer in the
MDCK cell culture 4.5 lg ID50, caused 100 %
mortality of control group mice within 5 days.
Observation of experimental animals was carried
out for 7 days. It was also studied the effect of
reference drugs in different regimens of admin-
istration to mice with experimental influenza
pneumonia. As the reference drugs we used the
known flu drugs Tamiflu® of pharmaceutical
company F. Hoffman-La Roche, Switzerland (75
mg per capsule) and Rimantadine of pharma-
ceutical company “Darnitsa”, Ukraine (50 mg
tablets), which were administered at a dose of
10 mg/kg of animal weight.
The efficiency of protective action of the
compounds was evaluated by the efficiency
index (EI) of inhibition of lethality. EI of the
tested substances was determined according to
the formula [21]:
EI =
PC – 1
×100 %,
PC
where PC — protection coefficient defined as
the ratio of the number of animals at the begin-
ning to that at the end of experiment.
Results and Discussion
The anti-influenza activity of new synthesized
adamantyl (alkyl, cycloalkyl) derivatives of
aminopropanol-2 was studied against the in-
fluenza virus strain A/FM/1/47(H1N1). The
antiviral activity of tested compounds 5, 6, 30,
33, 40, 47, 48, 51 was evaluated in vitro ex-
periments by their chemotherapeutic index
(CTI) determined as the ratio of the maximum
tolerated dose (MTD) to the minimum active
concentration (MAC). MTD of the tested com-
pounds was determined by their cytotoxic ef-
fect on a monolayer of the MDCK cells cul-
ture. For this purpose, after 24-h cell growth
the nutrient medium was replaced with the
medium containing the tested compound in the
concentration from 3.12 µg/ml to 100 µg/ml.
In the control, no replacement was carried out.
For the experiments at least ten wells with the
cell culture were taken for each dilution of the
compound. The level of CPE was determined
457
In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2
by the change in cell morphology (degenera-
tion or exfoliation of cells from the hole sur-
face). MTD of the tested compound was taken
as its highest concentration that did not cause
degeneration of cells in all ten wells. The re-
sults of MTD determination for the studied
compounds are presented in figure 1.
The MAC index of the tested substances
was determined in vitro in a monolayer of the
MDCK cells cultures by the level of reduction
of infectious titer of the virus, in accordance
with the General established procedure [16].
With this purpose the test-virus in a dose of
100 ID50 in 0.1 ml was introduced into the
MDCK cells culture and incubated for 1 h at
37 °C. Next, the virus was removed by wash-
ing with medium RPMI-1640, and the support
medium (RPMI-1640 + 2% fetal serum) with
the tested compounds in concentration from
0.39 mcg/ml to 50 µg/ml was added. A reduc-
tion of infectious titer in the cell cultures trea-
ted with different concentrations of the tested
compounds not less than 2 lg ID50 in the ex-
periment compared with control allowed us to
establish MAC of the tested compounds for
the influenza virus (see figure 2).
According to the data in fig. 2 the com-
pounds 47 and 48 have no influence on the
reproduction of influenza virus in the studied
concentrations (0.36 µg/ml to 50 µg/ml), they
inhibit the development of virus-specific CPE
in concentrations less than 2.0 lg ID50. The
compounds 5, 6, 30, 33, 40, 51 inhibited the
influenza virus reproduction more than 2.0
lg ID50 in concentrations from of 1.56 µg/ml
(compound 5) to 6.25 µg/ml (the compound
40), which gives grounds to consider them to
possess the antiviral activity. The compounds
30 and 33 were the most active against the
influenza virus, their MAC was the smallest
and amounted to < 0.39 µg/ml.
Fig. 2. The MAC of test
compounds for influenza
virus.
Fig. 1. The MTD of test compounds adamantyl (alkyl,
cycloalkyl) of aminopropanol-2 derivatives
458
O. M. Voloshchuk, Y. V. Korotkiy, S. L. Rybalko et al.
MTD of anti- influenza drugs Rimantadine
and Tamiflu was determined by CPE on the
monolayer of the MDCK cell culture, and the
value of their MAC was established by the
degree of reduction of infectious titer of the
influenza virus (Table 1). The data in the tab-
le 1 show that Rimantadine and Tamiflu were
non-toxic to the MDCK cell cultures in a dose
of 62.0 µg/ml and 250.0 µg/ml respectively,
that corresponds to the value of MTD of these
anti-influenza drugs.
The value of ratio of MTD to MAC of the
studied compounds, and that of anti-influenza
drugs Tamiflu and Rimantadine allowed us to
determine their CTI. These data are shown in
table 2.
Table 1. The value of CPE and MAC of anti-influenza drugs in relation to the influenza virus
Concentration of drugs µg/ml
anti-influenza drugs
Rimantadine Tamiflu
CPE Infectious titer of influenza
virus (lg ID50)
CPE Infectious titer of influenza
virus (lg ID50)
500.0 0 NT 0 NT
250.0 0 NT 100 < 1.0
125.0 0 NT 100 < 1.0
62.0 100 4.0 100 < 1.0
31.0 100 4.0 100 < 1.0
15.5 100 4.0 100 < 1.0
7.75 100 4.0 100 4.0
Control of virus 100 4.0 100 4.0
* NT – not tested
Table 2. MTD, МАC and CТІ of the tested
compounds, anti-influenza drugs Tamiflu and
Rimantadine
compounds MTD,
mcg/ml
МАC,
mcg/ml CТІ
5 25.0 1.56 16
6 12.5 3.125 4
30 > 50.0 0.36 256
33 > 50.0 0.36 256
40 > 50.0 6.25 8
47 12.5 0 –
48 < 100 0 –
51 12.5 3.125 4
Rimantadine 6200 0.36 172.2
Tamiflu 25 000 0.02 12 500
Table 3. The titer of HAI for compounds 5, 30, 33
Concentration
of compounds
μg/ml
The titer of HAI* tested compounds
5 30 33
50.0 – – –
25.0 – ++++(1/512) –
12.5 – ++++(1/1024) –
6.25 – ++++(1/1024) –
3.1 – ++++(1/1024) –
1.55 – ++++(1/1024) –
0.72 ++++(1/512) ++++(1/1024) –
0.36 ++++(1/1024) ++++(1/1024) –
Control of
virus ++++(1/1024) ++++(1/1024) ++++(1/512)
HAI * «-» – absence of hemagglutination ; «++++» –
presence of hemagglutination,titer of HAI
459
In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2
The values of MTD, МАC and CТІ of the
compounds 30 and 33 correspond to the sub-
stances highly active against the influenza virus.
CTI of these compounds was equal to 256 and
was higher than the same index of the anti-in-
fluenza drug Rimantadine, CTI of which was
172.2. The compound 5 is active against the
influenza virus, its CTI equal to 16. The value
of CTI of the compounds 6, 40 and 51 was also
more than 3 (4, 8 and 4 respectively) (Table 2).
It is known that cells are sensitive to the
influenza virus, bearing on their surface the
receptors to viral glycoprotein of hemaggluti-
nin (HA). The braking hemagglutination activ-
ity of the viruses in some cases is associated
with a loss of the virus infectivity; therefore,
it is appropriate to evaluate the influence of
compounds on the hemagglutinin properties
of viruses. We examined the influence of the
most active compounds 5, 30 and 33 on the
hemagglutinate activity of the influenza virus
using hemagglutination inhibition test (HAI)
(Table 3).
The titer of hemagglutination inhibition was
the approved limiting dilution, which gives a
complete delay of the hemagglutination reac-
tion. The results of determination of the effect
of various concentrations of the tested com-
pounds on hemaglutinin properties of the in-
fluenza virus are shown in table 3. The data in
the table 3 show that the compounds 30 and
33 with the same value of MAC (< 0.39 µg/
ml) make different effects on the hemaglutina-
tion properties of the influenza virus. The com-
pound 33 influences the virus hemagglutinin
in much smaller concentrations than the com-
pound 30. The HAI titers for the compound №
33 is 0,36 µg/ml and the HAI titer for com-
pound № 30 is only 50 µg/ml.
The anti-influenza activity of the tested
compounds 5, 30 and 33 was determined in
the in vivo experiments on the model of mice
Table 4. Anti-influenza action of the compounds 5, 30, 33 and the anti-influenza drugs in vivo experiment.
Substances Concentration of
substances mcg/ml
The number of mice Protection
coefficient
Efficiency index
(EI), %the total number of them died, (%)
prophylactic scheme
5 1.55 10 4 (40 %) 2.5 60
30 0.36 10 4 (40 %) 2.5 60
33 0.36 10 8 (80 %) 1.25 20
Rimantadine 0.5 10 5 (50 %) 2 50
Tamiflu 0.02 10 2 (20 %) 5 80
therapeutic scheme
5 1.55 10 0 > 10 100
30 0.36 10 4 (40 %) 2.5 60
33 0.36 10 0 > 10 100
Rimantadine 0.5 10 6 (60 %) 1,66 39.7
Tamiflu 0.02 10 2 (20 %) 5 80
Control of virus 0 10 10 (100 %) – –
460
O. M. Voloshchuk, Y. V. Korotkiy, S. L. Rybalko et al.
influenza pneumonia. The efficiency of the
protective action of the tested compounds and
anti-influenza drugs in the prophylactic and
therapeutic schemes of their administration is
shown in table 4.
Among the studied substances only the
compounds 5 and 30 protect mice from the
lethal influenza infection in vivo experiments
in the prophylactic scheme of administration
(Table 6). Their efficiency indices are equal
(60 %) and higher than the Remantadine EI by
10 %. Unlike the substances 5 and 30 the
compound 33 did not provide a significant
protective effect in the prophylactic scheme of
administration. EI of the compound 33 was
only 20 %, which is much less than that of
Tamiflu (80 %). The compounds 5 and 33
showed a much greater protective effect in the
therapeutic scheme of administration than in
the prophylactic scheme of in vivo experiment.
EI of these compounds was 100 % in the ther-
apeutic scheme of administration, compared
to the prophylactic scheme, where their EI
values were 60 % and 20 % respectively.
Comparison of the protective effects of newly
synthesized compounds 5, 30, 33 and anti-
influenza drugs Rimantadine and Tamiflu on
the model of influenza pneumonia of mice in
the prophylactic and therapeutic schemes of
their administration is shown in figure 3.
The EI values of tested compounds 5, 30,
and 33 were significantly different for different
schemes of the in vivo experiment (Fig. 3). The
tested compounds were more effective in ther-
apeutic scheme of their introduction, when
influenza drugs showed the same effect in both
variants of the experiment. Complete protec-
tion against experimental influenza infection
in mice was observed for compounds 5 and 33
in therapeutic scheme of their introduction.
Their EI values were 100 %, that is more than
twice higher than Rimantadine EI. The com-
pound 30 showed identical efficiency in dif-
ferent schemes of the in vivo experiment, its
Fig. 3. Anti-influenza
activity of the compounds
5, 30, 33 and anti-influen-
za drugs Rimantadine and
Tamiflu on the model of
influenza pneumonia of
mice in the prophylactic
(1) and therapeutic (2)
schemes of their adminis-
tration.
461
In vitro and in vivo investigations on anti-influenza effect of adamantyl (alkyl, cycloalkyl) derivatives of aminopropanol-2
EI in both cases was 60 %. Noteworthy, for
the compounds 30 and 33 an equally high CTI
(256) was identified in vitro experiment, but
these substances showed a different level of
protective effect in vivo experiment.
Conclusions
Among eight tested adamantyl (alkyl, cycloal-
kyl) derivatives of aminopropanol-2 in vitro
and in vivo experiments anti-influenza activity
was found for the compounds 5, 30 and 33.
CTI of the compounds 30 and 33 was equally
high in vitro experiment on the MDCK cell
culture, but these compounds showed different
degree of protective effect in vivo experiment
on the influenza pneumonia mice model, as
well as different influence on hemagglutinin
properties of the influenza virus. The com-
pounds 5 and 33 were more effective in the
therapeutic scheme of the in vivo experiment,
whereas the anti-influenza drugs showed equal
effect in both variants of the experiment (the
therapeutic and prophylactic schemes of their
administration). We suggest that these results
may be explained by different mechanisms of
action of the tested compounds on the flu virus.
The data obtained give ground to further
research of the compounds 5, 30 and 33 for
possible elaboration of new antimicrobial
drugs on their basis.
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Антигрипозна дія адамантил (алкіл,
циклоалкіл) похідних амінопропанолу-2
в дослідах in vitro та in vivo
О. М. Волощук, Ю. В. Короткий, С. Л. Рибалко,
Д. Б. Старосила, В. П. Широбоков
Мета. Визначити антигрипозну активність у 8 сполук
адамантил (алкіл, циклоалкіл) похідних амінопропа-
нолу-2 в дослідах in vitro та in vivo. Методи. Оцінку
противірусної дії сполук проводили in vitro за знижен-
ням інфекційного титру вірусу грипу на культурі клі-
тин, та in vivo на моделі грипозної пневмонії у мишей.
Результати. В досліді in vitro 3 сполуки серед 8 до-
сліджених пригнічують репродукцію вірусу грипу
штаму А/FM/1/47(H1N1) на 2 lg ID50 і більше в кон-
центраціях від < 0,39 мкг/мл (сполуки №№ 30 і 33) до
1,56 мкг/мл (сполука № 5), їх хіміотерапевтичні індек-
си стосовно цього вірусу становлять 256 та 16, відпо-
відно. В досліді in vivo при профілактичній схемі до-
сліду сполуки №№ 5 і 30 мають індекс ефективності
(ІЕ) 60 %, а при лікувальній схемі досліду ІЕ сполук
№№ 5 та 33 більше ніж в два рази перевищує цей по-
казник у ремантадину і становить 100 %. Висновки.
Високі хіміотерапевтичні індекси та здатність при-
гнічувати репродукцію вірусу грипу на 2 lg ID50 і
більше дозволяє віднести сполуки №№ 30, 33 і 5 до
таких, що проявляють протигрипозну активність. Це
також підтверджено в досліді in vivo.
К л юч ов і с л ов а: вірус грипу, адамантил (алкіл,
циклоалкіл) похідні амінопропанолу-2, антивірусна дія.
Антигриппозное действие адамантил (алкил,
циклоалкил) производных аминопропанола-2
в экспериментах in vitro и in vivo
Е. М. Волощук, Ю. В. Короткий, С. Л. Рыбалко,
Д. Б. Старосила, В. П. Широбоков
Цель. Определить антигриппозную активность у 8 со-
единений адамантил (алкил, циклоалкил) производных
аминопропанола-2 в экспериментах in vitro и in vivo.
Методы. Противовирусное действие исследуемых
соединений определяли in vitro по снижению инфек-
ционного титра вируса гриппа в культуре клеток
MDCK и in vivo на модели гриппозной пневмонии у
мышей. Результаты. 3 соединения среди 8 исследо-
ванных подавляют репродукцию вируса гриппа штамм
А/FM/1/47(H1N1) на 2 lg ID50 и больше в концентра-
циях от < 0,39 мкг/мл (соединения №№ 30 и 33) до
1,56 мкг/мл (соединение № 5), их химиотерапевтиче-
ские индексы (ХТИ) в отношении этого вируса состав-
ляют 256 и 16, соответственно. В эксперименте in vivo
при профилактической схеме опыта соединения №№
5 и 30 имеют индекс эффективности (ИЭ) 60 %. ИЭ
соединений №№ 5 и 33 при лечебной схеме опыта
больше чем в два раза превышает этот показатель у
Ремантадина и составляет 100 %. Выводы. Высокие
ХТИ и способность подавлять репродукцию вируса
гриппа на 2 lg ID50 и более позволяют отнести соеди-
нения №№ 30, 33 и 5 к таким, что проявляют проти-
вогриппозную активность. Это также подтверждено в
експриментах in vivo.
К л юч е в ы е с л ов а: вирус гриппа, адамантил (ал-
кил, циклоалкил) производные аминопропанола-2,
антивирусное действие.
Received 05.01.2017
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