Peptide carbocyclic derivatives as inhibitors of the viroporin function of RNA-containing viruses

Article abstract of DOI:10.1134/S1068162017050132

New carbocyclic derivatives of amino acids, peptides, and other compounds have been synthesized, and their antiviral activity toward the influenza A/H5N1 and hepatitis C viruses has been studied in vitro. It has been shown that the aminoacyl derivatives of aminoadamantane are capable of inhibiting the replication of the highly virulent strain of the avian influenza virus (H5N1), which is resistant to aminoadamantanes amantadine and rimantadine. The effect of the configuration of the carbocyclic moiety of the dipeptide H-Pro-Trp-OH on the antiviral properties toward the hepatitis C virus has been studied. The cyclohexyloxycarbonyl derivative of the H-Pro-Trp-OH dipeptide strongly inhibited the replication of HCV in vitro. Some compounds have been found to exhibit a high virucidal activity toward influenza A/H5N1 and HCV virions.

Full text of DOI:10.1134/S1068162017050132

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 5, pp. 517–525. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © V.A. Shibnev, P.G. Deryabin, T.M. Garaev, M.P. Finogenova, A.G. Botikov, D.V. Mishin, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43,
No. 5, pp. 491–500.
Peptide Carbocyclic Derivatives as Inhibitors of the Viroporin
Function of RNA-Containing Viruses
V. A. Shibnev, P. G. Deryabin, T. M. Garaev1, M. P. Finogenova, A. G. Botikov, and D. V. Mishin
Ivanovskii Institute of Virology, Gamaleya Federal Research Center for Epidemiology and Microbiology,
Ministry of Health of Russia, Moscow, 123098 Russia
Received September 2, 2016; in final form, April 10, 2017
Abstract⎯New carbocyclic derivatives of amino acids, peptides, and other compounds have been synthe-
sized, and their antiviral activity toward the influenza A/H5N1 and hepatitis C viruses has been studied in
vitro. It has been shown that the aminoacyl derivatives of aminoadamantane are capable of inhibiting the rep-
lication of the highly virulent strain of the avian influenza virus (H5N1), which is resistant to aminoadaman-
tanes amantadine and rimantadine. The effect of the configuration of the carbocyclic moiety of the dipeptide
H-Pro-Trp-OH on the antiviral properties toward the hepatitis C virus has been studied. The cyclohexyloxy-
carbonyl derivative of the H-Pro-Trp-OH dipeptide strongly inhibited the replication of HCV in vitro. Some
compounds have been found to exhibit a high virucidal activity toward influenza A/H5N1 and HCV virions.
Keywords: A/H5N1 influenza virus, adamantane derivatives, peptides, hepatitis C virus, antiviral activity
DOI: 10.1134/S1068162017050132
INTRODUCTION
gen ions into the virion, whereas the majority of viro-
porins exhibit moderate ion selectivity, i.e., do not dis-
play preference for a particular ion. The pentameric
viroporin, the protein Vpu from HIV-1, shows moder-
ate cation selectivity in NaCl and KCl electrolyte solu-
tions [2]. This holds true also for the HCV hexameric
viroporin p7, which transports monovalent cations
(Na+ and K+) [3].
The high ion selectivity of the influenza A virus M2
viroporin is due to a special structure of its TM
domain, which contains histidine residues at position
37 (His37) in the form of the tetrameric conjugation of
their imidazole rings. Protons from the intracellular
environment protonate three or all four imidazole
rings of histidines. This leads to a conformational
change due to electrostatic repulsion, which widens
the channel in the region of His37 and enables the pro-
tons to penetrate the virus envelope [4]. The indole
group of tryptophan 41 plays an important role in the
stabilization of the open state of the channel pore.
Thus, the molecular model of the functioning of the
M2 ion channel represents a “sluice” mechanism [5].
Among the first inhibitors of the influenza A virus
M2 proton channel to be investigated was amantadine
(Symmetrel, 1-adamantylamine hydrochloride). It
has been used in clinical practice against influenza A
virus since 1966 [6]. Later, in 1985 it was shown that
some mutations in the hydrophobic sequence of the
M2 protein can cause the resistance of the virus to
amantadine and its analogue Rim (Flumantadine,
1-(1-adamantyl)ethylamine hydrochloride). These
findings suggested that the M2 protein may be a target
Viral ion-selective channels proteins are involved
in many stages of the virus replication cycle. These
channels are localized in the virus envelope or the
membranes of the host cell and play an important role
in the penetration of viruses into the cell, as well as the
assembly and exit of newly formed virions. Viral ion
channels are not always required for virus replication,
although their involvement usually significantly con-
tributes to an increase in the number of virions. The
family of these proteins received the name viroporins;
it involves proteins containing at least one hydropho-
bic transmembrane (TM) domain in the form of the
α-helix [1]. As a rule, viroporins are small proteins
(50–120 amino acid residues long), which tend to
form homooligomers. The studies of the molecular
architecture of viral ion channels showed that most of
them are formed by tetramers although penta- and
hexameric structures are also characteristic of viropo-
rins. A representative of tetrameric ion-selective viro-
porins is the channel M2 of the influenza A virus. This
unique viroporin is specific to the transport of hydro-
1
Corresponding author: phone: +7 (499) 190-30-43; e-mail:
tmgaraev@gmail.com.
Abbreviations: Amt, 1-aminoadamantane; Ad, 1-adamantane-
carboxylic acid; Cho, (cyclohexyloxy)carboxylic acid; IBCF,
isobutylchlorformiate; Met(O ), methionine sulfone; Nor,
2
2-norbornene carboxylic acid; Nrb, 2-norbornan carboxylic
acid;NMM,N-methylmorpholine;Rim,rimantadine(1-(1-аda-
mantyl)ehylamine hydrochloride); TEA, 3-(2-thienyl)prope-
noic acid; Tha, 2-tetrahydrofuran carboxylic acid; Qln, quino-
line-2-carboxylic acid; HCV, hepatitis C virus.
517

518
SHIBNEV et al.
for the preparations of the adamantane series [7]; in
1992, this suggestion was confirmed [8].
RESULTS AND DISCUSSION
The resistance of the virus to amantadine and Rim
results from a mutation in the amino acid sequence of
proteins in the M2 proton channel of the influenza A
virus. It should be noted that Rim and amantadine
preparations exhibit cross-resistance. One of the ways
of restoring the antiviral properties of adamantane
preparations is the introduction of additional func-
tionally active groups into their structure. In the pres-
ent study, substituted amino acids, peptides, and TEA
were proposed as the sources of these groups. The res-
idues of amino acids and peptides can be attached to
Rim or amantadine molecules, which have one amino
group, by the method of mixed anhydrides [13, 14].
Recent investigations have shown that the ion
activity of the hexameric channel protein p7 of HCV
can also be inhibited by adamantane preparations in
vitro [9, 10]. This gives promise that the p7 channel
can be an adequate target for adamantane-based anti-
HCV drugs. The nonstructural HCV p7 protein con-
sists of 63 amino acid residues and has two TM
domains (TM1 and TM2) [11]. This viroporin is pre-
dominantly localized in intracellular membranes; in
experiments in vitro, it displays the function of ion
channels necessary for the virus assembly and optimal
exit from infected cells by changing the acid–alkaline
equilibrium in intracellular vesicles. The membrane
protein p7 is a new target for the antiviral therapy of
HVC [12].
As a result, we synthesized several derivatives of
adamantane and some other carbocycles, such as nor-
bornene, cyclohexane, and Qln (Scheme 1):
IBCF
NMM
R-OH +
NH
HCl* NH2
R
(II), (IV), (V), (VI)
4 M HCl
ethyl acetate
(III)
Cbz-Trp- (V);
S
R =
(II);
O
NH
S
H-Met(O2)-
(I)
(III);
O
(VI)
-Ala-Pro-
N
Boc-Met(O2)-OH
Boc-Met(O2)-
(IV);
(IVа)
O
Scheme 1. A general scheme of the synthesis of rimantadine derivatives. Compound (I) was synthesized in a similar way
but with the use of 1-aminoadamantane as an amino component instead of rimantadine.
N-[3-(2-thienyl)propenoyl]-(1-adamantyl)amide
(TEA-Amt (I));
N-[3-(2-thienyl)propenoyl]-1-adamantaylethylamide
(TEA-Rim (II)); methionyl(dioxide)-(1-adamantayl-
ethyl)amide hydrochloride (HCl*Met(O2)-Rim (III));
Antiviral activity of compounds toward the
A/duck/Novosibirsk/56/05 (H5N1) virus. The antivi-
ral activity of a compound as a rule involves both its
virucidal and antiviral properties, i.e., the capacity to
inhibit a particular stage of virus replication, from the
adsorption of a virus on the cell and its penetration
into the cell to the influence on the assembly and exit
of the virus from the infected cell. Therefore, the anti-
viral activity of a compound being examined is tested
by treating a cell monolayer with this compound prior
to the infection with the virus (the prophylactic effect
of the compound), immediately at the time of infec-
tion (the treatment-and-prophylactic effect), and
some time after the infection (the treatment effect).
The antiviral effect of the compound is estimated from
the fraction of viable infected cells. In the experiment,
N-Boc-methionyl(dioxide)-(1-adamantaylethyl)-
amide (Boc-Met(O2)-Rim (IV));
N-Z-L-tryptophyl-(1-adamantylethyl)amide (Z-
Trp-Rim (V));
quinaldine-2-oyl-L-alanyl-L-prolyl-(1-adamantyl-
ethyl)amide (Qln-Ala-Pro-Rim (VI)).
The antiviral activity of these compounds was
assessed in vitro using a highly pathogenic strain of the
avian influenza virus A/H5N1, which is resistant to
Rim, on models of the African green monkey kidney
(Vero-E6) and the porcine embryo kidney (SPEV) cell the viability (percent of surviving cells) of Vero-E6 (or
lines.
SPEV) cells infected with a highly virulent strain of the
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PEPTIDE CARBOCYCLIC DERIVATIVES AS INHIBITORS
519
Table 1. Antiviral activity of compounds toward the infection caused by the highly pathogenic influenza virus A/H5N1
Effective concentration (μg/mL) for compounds:
Time
of administration
of compound
Indicator
of the effect
of compound
rim
hydrochloride
(I)
(II)
(III)*
(IV)*
(V)*
(VI)
CD50
ID50
ID95
CD50
ID50
ID95
CD50
ID50
ID95
>6.25
0.2
>50.0
25.0
<3.12
3.12
100.0
0.2
100.0
0.2
>12.5
<1.56
1.56
>12.5
>12.5
>12.5
25.0
6 h prior to infection
1.56
0.4
3.12
>50.0
<6.25
6.25
0.4
0.4
>6.25
0.4
>50.0
<3.12
3.12
>25.0
0.4
>100.0
0.4
>25.0
1.56
At the instant of infec-
tion of cells
>25.0
>25.0
25.0
0.8
0.8
0.8
3.12
>12.5
6.25
12.5
>50.0
<6.25
1.56
50.0
100.0
<6.25
12.5
>100.0
0.8
>12.5
1.56
6 h after the infection
of cells
12.5
25.0
25.0
1.56
3.12
50.0
* Experiment was performed using the SPEV cell culture.
ID , the inhibitory dose 50: a minimal dilution of a preparation that provides the protection of 50% of monolayer cells;
50
CD , the cytotoxic dose 50: a dilution of a preparation that causes the death of 50% of monolayer cells;
50
ID , the inhibitory dose 50: a minimal concentration of a compound that provides the protection of 95% of cells.
95
influenza virus A/duck/Novosibirsk/56/05 (H5N1) incubate a mixture of a virus and the compound for a
was studied using different schemes of the administra- particular time and to test then the infective capacity
tion of the compounds synthesized.
of the virus without the compound by the titration in
cell cultures. A titer of a virus for cell cultures is a mini-
mal dilution of the virus that causes the death of 50% of
cells of the monolayer, expressed as the common loga-
rithm (logTCID50). A reliable decrease in the infectivity
of a virus in the scale of common logarithms by 1.0 and
more or its complete loss as compared with the virus
without a substance indicates the virucidal activity of the
compound being tested. The virucidal activity of the syn-
thetic compounds is demonstrated in Table 2.
It is easily seen from Table 2 that compounds (I)
and (II) possess a high virucidal activity. When the
virus-containing material came in contact with the
compounds, the virus completely lost its infectivity
within 20 min. Compound (V) also exhibited a high viru-
cidal effect: the decrease in the infectious titer was by four
and more logarithmic units (10000 times) relative to the
viral control (logTCID50/0.2 = 7.0 per 200.0 μL of the
virus-containing material). Compound (IV) had a mod-
erate virucidal activity: the decrease in the infectious titer
was approximately by four units (10000 times) relative to
the control (logTCID50/0.2 = 7.7). The virucidal effect
of compound (III) was much weaker. We failed to
reveal the virucidal effect of compound (VI). The
decrease in the infectious titer was less than one loga-
rithmic unit.
It is seen from the data in Table 1 that the Rim
derivative with thienylpropenoic acid (II) effectively
protected a monolayer of Vero-E6 cells with all
schemes of the administration of the compound:
ID50 < 0.8 μg/mL prior to, and at the time of infection
and ID50 = 6.25 μg/mL with the treatment scheme of
administration. The Rim derivative with TEA (I)
showed a similar efficacy with all schemes of the
administration; ID50 = 1.56 μg/mL with the prophy-
lactic scheme and less than 6.25 μg/mL with the treat-
ment-and-prophylactic and treatment schemes.
The antiviral activity of synthetic compounds (III),
(IV), and (V) was estimated from their capacity to pro-
tect a monolayer of SPEV cells from the cytopathic
action of the virus. The compounds were highly effec-
tive when being administered according to the treat-
ment-and-prophylactic and treatment schemes. The
ID50 value was less than 0.8 μg/mL for compounds
(III) and (V) and less than 3.12 μg/mL for (IV). If
these compounds were administered according to the
treatment scheme, the inhibitory activity decreased.
The N-quinaldinoyl derivative of Rim, dipeptide
amide (VI), effectively protected Vero-E6 cells with all
schemes of administration: the ID50 value was
1.56 μg/mL with the treatment-and-prophylactic
scheme and somewhat less with the prophylactic and
treatment schemes.
The antiviral activity of the derivatives of dipeptide
H-Pro-TRP-OH toward the infection caused by HCV
with different schemes of administration. We have
shown earlier that the adamantane carbocyclic peptide
derivatives of Rim are capable of inhibiting the repli-
cation of HCV in vitro [15]. These compounds repre-
sented Boc-lysine coupled to the adamantane carbo-
Virucidal activity of compounds toward the
A/duck/Novosibirsk/56/05 (H5N1) virus. The viru-
cidal activity of a compound is associated with the
direct inactivating effect on virions incorporated into
the viral population with the result that the infectivity
of a virus is partially or completely lost. To test the cycle through a spacer of glycine residues (from one to
virucidal properties of a compound, it is sufficient to three in a chain). The compound with three glycine
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SHIBNEV et al.
residues in the spacer Boc-Lys(Boc)-Gly3-Rim the ion channel; they are responsible for the selectivity
of the ion pump and unidirectivity of the ion flow [4].
The compound Boc-Lys(Boc)-Gly3-Rim has a bent
configuration, and this stereometry and the length of
the spacer composed of glycine residues probably con-
tribute to the formation of a stable complex of the
inhibitor molecule with the target protein.
In the present work, we made an attempt to study
how the carbocyclic backbone of the peptide deriva-
tive affects its capacity to inhibit the replication of
HCV in vitro. The following carbocyclic carboxylic
acids were used: 1-adamantanecarboxylic (Ad-OH),
2-norbornene carboxylic (Nor-OH), 2-norbornan
carboxylic (Nrb-OH), quinoline-2-carboxylic (Qln-
OH), 2-tetrahydrofuran carboxylic (Tha-OH), and
cyclohexyloxycarboxylic (Cho-OH) acids. With the
showed the highest activity.
The studies performed in silico showed that, in the
majority of models, Boc-Lys(Boc)-Gly3-Rim is
bound to several chains of the p7 ion channel. The inter-
action of this ligand with the p7 target protein was simu-
lated using the program Patchdock v.1.3. Molecular
Docking Algorithm (BioInfo3D), which makes it possi-
ble to perform the docking of molecules in silico. The
model of the p7 channel was obtained from the open
database of 3D protein structures (Protein Data Bank,
code 2M6X), and the structure of the ligand was gener-
ated by molecular simulation methods using the program
HyperChem v.8.0.0 (Hypercube, Inc.) (Figs. 1c, 1d).
This model was built by the PM3 semiempirical
method of quantum mechanical calculations.
The results of the solution presented in the figure use of these acids, the dipeptide H-Pro-Trp-OH was
show that there are four points of binding to the amino acylated by the methods of classical peptide synthesis
acid residues of the viral channel proteins. In all, the in solution (Scheme 2). The construction of the
protein has six chains (a hexameric model was used), dipeptide was chosen not accidentally: the unique
and four of these chains are involved in the docking, electron properties of the indole group of tryptophan
namely: His57 residues from different chains interact; in combination with the conformational features of
His17 interacts with the α-amino group of lysine, and the proline residue should have to create a multifunc-
Gly22 interacts with the methyl group of Rim. Histi- tional group with the hydrophobic properties of the
dine residues are very important for the operation of carbocyle.
Boc-Tro-OH + HCl*H-Trp-OMe
IBCF
2NMM
4 M HCl
Ethyl acetate
Boc-Pro-Trp-OMe
(XIII)
0.25 M NaOH
IBCF
R-Pro-Trp-OH
R-OH + HCl*H-Pro-Trp-OMe
R-Pro-Trp-OMe
2NMM
Acetone
(VII)−(XI)
(XIV)
0.25 M NaOH
Acetone
IBCF
CH
Cho-Pro-Trp-OH
Cho-Pro-Trp-OMe
Cho-Pro-OH + HCl*H-Trp-OMe
2NMM
(XII)
(VII)−(XI)
(VII) R =
(VIII) R =
(IX) R =
(X) R =
N
O
O
(XI) R =
O
O
O
O
(XII) Cho =
O
Scheme 2. A general scheme of the synthesis of carbocyclic derivatives of dipeptide H-Pro-Trp-OH. Compound (XII)
was synthesized based on Cho-Pro-OH (see the Experimental section).
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521
(а)
O
O
O
NH
NH
O
NH
NH
HN
O
O
NH
O
O
Boc-Lys(Boc)Gly-Gly-Gly-Rim
(c)
(d)
N
O
O
O
O
N
NH
HO
Cho-Pro-Trp-OH
Fig. 1. Molecular models of the inhibitors Boc-Lys(Boc)-Gly -Rim [15] (c) and Cho-Pro-Trp-OH (d) and their complexes with
3
the hexameric protein channel p7 of HCV ((a) and (b), respectively).
Thus, the following compounds were synthesized:
O
Tha-Pro-Trp-OH (XI)
Cho-Pro-Trp-OH (XII)
O
Pro-Trp-OH
Ad-Pro-Trp-OH (VII)
Pro-Trp-OH
O
Pro-Trp-OH
O
O
Nor-Pro-Trp-OH
(VIII)
Pro-Trp-OH
It is seen from Table 3 that all compounds were
ineffective or little effective with the treatment scheme
of the administration. In the prophylactic and treat-
ment-and-prophylactic schemes of the administration
of the virus and compounds, only one combination of
the carbocycle with the dipeptide Cho-Pro-Trp-OH
(XII) merits notice. The ID50 values for this com-
pound were 0.3, 0.6, and 12.0 μg/200 μL according to
the three schemes of administration. Among the other
compounds, the derivative Ad-Pro-Trp-OH (VII) is
O
Nrb-Pro-Trp-OH (IX)
Qln-Pro-Trp-OH (X)
Pro-Trp-OH
Pro-Trp-OH
O
N
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SHIBNEV et al.
Table 2. Virucidal activity of compounds (I)–(VI) toward the highly pathogenic influenza virus A/H5N1
Indicator
logTCID50/0.2 mL
(I)
(II)
(III)
(IV)
(V)
(VI)
Rim
0
7.7
7.7
0
6.6
6.6
7.0
1.5
8.5
4.8
3.7
8.5
2.9
4.1
7.0
8.0
0.5
8.5
8.5
0
8.5
log of decrease in infectivity
Without preparation (control)
logTCID , the designation of the infectious titer of a virus for cell cultures: a minimal dilution of a virus that causes the death of 50% of
50
monolayer cells, expressed as the common logarithm.
Table 3. Antiviral activity of carbocyclic derivatives of dipeptide H-Pro-Trp-OH toward the infection caused by HCV in
different schemes of administration
Indicator of the effect of compound (μg/200 μL)
Scheme of administration
Compound
of compounds
CD50
ID50
ID95
(VII)
(VIII)
(IX)
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
1.2
2.5
2.5
5.0
<10.0
2.5
10.0
10.0
10.0
0.6
6 h prior to infection of cells
(X)
(XI)
2.5
(XII)
(VII)
(VIII)
(IX)
<0.3
5.0
10.0
10.0
10.0
10.0
10.0
1.2
5.0
5.0
At the instant of infection of cells
(X)
<5.0
5.0
(XI)
(XII)
(VII)
(VIII)
(IX)
0.6
5.0
10.0
>10.0
>10.0
10.0
>10.0
10.0
>10.0
>10.0
<5.0
10.0
1.2
6 h after the infection of cells
Designations as in Table 1.
(X)
(XI)
(XII)
also worthy of notice with the ID50 values of 1.2, 5.0, Table 4 that the structurally different compounds
and 5.0 μg/200 μL according to the scheme of admin-
Boc-Lys(Boc)-Gly3-Rim and (XII) (Figs. 1c and 1d),
istration.
have similar values of antiviral activity in the schemes
of the administration of compounds. According to the
results of molecular docking in silico, the molecule of
compound (XII) (similar to the calculations for Boc-
Lys(Boc)-Gly3-Rim) is bound to several helices of
protein p7 in the external space of the conducting
channel (Figs. 1a and 1b). Presumably, the presence of
carbocyclic compounds in the interhelical space of the
channel protein mechanically disturbs the mobility of
chains for the opening or closing of the pore, which in
turn leads to the disturbance of ion transport. This has
yet to be elucidated.
Attempts to improve compound (XII) by substitut-
ing L-alanine, L-cysteine, or L-lysine for L-trypto-
phan, as well as the introduction of a spacer of glycine
residues (from one to three residues) between the
cyclohexyloxycarbonyl residue and the dipeptide
H-Pro-Trp-OH, led to the loss of the activity of the
compound.
Finally, the antiviral activity of compound (XII)
was compared with the results obtained earlier for the
adamantylethylamide tetrapeptide Boc-Lys(Boc)-
Gly3-OH [15] (Table 4). It follows from the data in
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PEPTIDE CARBOCYCLIC DERIVATIVES AS INHIBITORS
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Table 4. Comparison of antiviral activity of compounds of dipeptide Cho-Pro-Trp-OH (XII) and Boc-Lys(Boc)-Gly3-
Rim toward the infection caused by HCV in SPEV cells
Indicator of the activity of compound (μg/200 μL)
Compounds and the scheme of administration
of compounds into cell culture
CD50
ID50
ID95
Boc-Lys(Boc)-Gly3-Rim
>2.5
>2.5
>2.5
>2.5
>5.0
>5.0
0.5
1.0
1.0
0.5
0.5
0.5
0.312
0.312
0.156
<0.5
24 h prior to infection
(XII)
Boc-Lys(Boc)-Gly3-Rim
(XII)
At the instant of infection of cells
Boc-Lys(Boc)-Gly3-Rim
(XII)
<0.5
24 h after infection
<0.5
Designations as in Table 1.
EXPERIMENTAL
from hepatitis C patients (SCV, Ivanovskii Institute of
Virology, Gamaleya Research Center for Epidemiol-
ogy and Microbiology, Ministry of Public Health of
Russia). The virus-containing material represented a
sample of the HCV-containing liquid collected from
infected SPEV cultures. The infectious titer of the viral
strain for the given cell type was 7 logTCID50/mL.
The following reagents were used: racemic Rim
(Zhejiang Kangyu Pharmaceutical Co., China);
amantadine hydrochloride, TEA, Nor, Nrb, Qln, Ad,
Tha, NMM, and L-amino acids (Sigma-Aldrich,
United States); and IBCF (Fluka, Switzerland). All
solvents used for the condensation and removal of
protective groups were preliminarily dried and dis-
tilled by standard methods. The resulting compounds
were identified by TLC on Merck-Kieselgel 60 F (254)
plates in systems: methanol–chloroform 13 : 60 (A),
sec-butanol–3% ammonia 100 : 44 (B), n-butanol–
acetic acid–water–pyridine 30 : 3 : 12 : 10 (C), which
permitted us to ascertain the complete absence of ini-
tial reagents in samples being tested. The molecular
weight was determined on a Bruker UltraFlex II
MALDI-TOF mass spectrometer using the programs
flexControl 1.1 and flexAnalys 2.2 for the retrieval and
processing of mass spectra. IR spectra were recorded
on an InfraLUM FT-10 IR-Fourier spectrometer.
Melting temperatures were measured on an SMP20
Stuart Scientific digital apparatus. The specific optical
rotation of the resulting compounds was determined
under standard conditions on an A1-EPL automatic
polarimeter (a 1% solution of a compound in ethyl
ether; cuvette length 0.5 dm).
A highly virulent strain of the avian influenza A
(H5N1) virus A/duck/Novosibirsk/56/05 (H5N1)
was used, which was isolated during the epizootia
among domestic poultry in July 2005 in Novosibirsk
oblast [16]. Viruses were obtained from the State Col-
lection of Viruses (SCV) at the Ivanovskii Institute of
Virology (Gamaleya Research Center for Epidemiol-
ogy and Microbiology, the Ministry of Public Health
of Russia). The virus-containing material represented
a liquid collected from A(H5N1)-infected SPEV cell
cultures at the peak of the development of cytopathic
effects. The infectious titer of viral strains for cell cul-
tures varied from 5.5 to 6.0 logTCID50.
The culture of SPEV cells was grown as a two-day-
old monolayer in 48-well plastic plates in medium 199
(production of the Chumakov Research Institute of
Poliomyelitis and Viral Encephalites, Russian Acad-
emy of Medical Sciences) supplemented with
100 U/mL of penicillin, 100 U/mL of streptomycin,
and 10% bovine serum. The growth of culture cells was
controlled using a light optical microscope.
In experiments, the Vero-E6 clonal line sensitive to
the reproduction of the influenza A/H5N1 virus was
used, which is recommended by the WHO as a sub-
strate for the production of cultured inactivated vac-
cines. Vero-E6 cells were grown at 37°C in an atmo-
sphere of CO2 to a complete monolayer in 96-well
plastic plates using the Eagle’s MEM (PanEko, Mos-
cow) supplemented with glutamine and antibiotics
(100 U/mL of penicillin and 100 U/mL of streptomy-
cin) and 7% fetal calf serum (PanEko, Moscow) pre-
liminarily heated in a water bath at 56°C for 20 min.
The supporting medium after the adsorption of the
virus was Eagle’s MEM supplemented with glutamine
and antibiotics at the same concentrations and 1%
fetal calf serum (Sigma, United States). The peptide
bond formation was carried out in one stage at the
equimolar ratio by the reaction of mixed anhydrides
(Scheme 1).
Boc-Met(O2)-OH (IVa). Boc2O (1.4 g, 6.42 mmol)
was added in three portions under stirring within 1 h at
45°С to a solution of Met(O2) (1.0 g, 5.5 mmol) and
NaOH (0.22 g, 5.5 mmol) in H2O (5.0 mL) and But-OH
(4.0 mL). After the termination of the reaction, But-OH
was distilled, and the residue was diluted with H2O
The HCV strain D-1 was obtained from the work-
ing collection of cytopathogenic HCV strains isolated 1.5 times, washed with hexane (3 × 15.0 mL), and acidi-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 5 2017

524
SHIBNEV et al.
fiedwitha1МKHSO4 solution to pH 3–3.5. The product
was isolated by extraction with ethyl acetate (4 × 15.0 mL).
The extract was dried over anhydrous Na2SO4, and the
solvent was removed in vacuo. The remaining oil sponta-
neously crystallized. Yield: 1.37 g (88%); mp 112°С; Rf
20
Qln-Ala-Pro-Rim (VI) was obtained similarly to
(IV), starting from Qln-Ala-Pro-OH and Rim. Yield:
0.66 g. (90.4%); mp 145–147°С; Rf 0.81 (A), 0.92 (B),
20
0.88 (C);
+16°; IR: ν(NH) 3386, 3290 cm–1;
[α]D
ν(СH) CH3-groups 2974, 2900, 2850 cm–1; ν(С=О)
1675, 1628 cm–1; ν(С=С) 1620–700 cm–1; m/z: found
[M + H]+: 503.33; calculated M(C30H38N4O3) 502.64 Da.
0.55 (A), 0.81 (B), 0.78 (C);
+8°.
[α]D
Boc-Met(O2)-Rim (IV). NMM (0.40 mL, 3.5 mmol)
was added to compound (IVa) (1.0 g, 3.5 mmol) in a
mixture (25.0 mL) of CHCl3 and THF (1 : 1.5) after Trp-OMe (0.34 g, 0.69 mmol) in acetone (10.0 mL),
Ad-Pro-Trp-OH (VII). To a solution of Ad-Pro-
0.25 М NаOH (16.0 mL, 4.0 mmol) was added. The
mixture was allowed to stand for 45 min at 20°С. Ace-
tone was removed in vacuo; the aqueous fraction was
acidified with 10% citric acid to pH 4 after which the
product was extracted with ethyl acetate (20.0 mL)
and washed with water (2 × 4 mL). The product was
dried over anhydrous Na2SO4. Ethyl acetate was
removed in vacuo until an oily product formed. Yield:
20
which IBCF (0.47 mL, 3.5 mmol) was added under
stirring at –25°C. The mixture was stirred for 10 min,
and a solution of Rim (0.77 g, 3.5 mmol) in CHCl3
(10.0 mL), cooled to –20°C, and NMM (0.40 mL,
3.5 mmol) were added. The mixture was stirred for
30 min at –20°C, for 1 h at 0°C, and for 10 h at 20°C. The
solvent was removed in vacuo, and the residue was dis-
solved in ethyl acetate (35.0 mL) and H2O (10.0 mL)
and washed successively with 0.25 M H2SO4 (4.0 mL),
0.25 M KHCO3 (2 × 10.0 mL), and H2O (5.0 mL).
The organic layer was separated and dried over anhy-
drous Na2SO4. Ethyl acetate was removed in vacuo to
obtain an oily product. Yield: 1.31 g (83%); Rf 0.90 (A),
20
0.33 g (97%), R 0.59 (A), 0.47 (B), 0.59 (C);
–23°;
[α]D
f
IR: ν(NH) at 3298 cm–1; ν(С=О) 1708, 1663 and
1618 cm–1; m/z: found [M + H]+: 464.25; calculated
M(C27H33N3O4) 463.56 Da.
Compounds (VII)–(XI) were obtained by the
method of mixed anhydrides in a similar way as (IV),
starting from the corresponding carbocyclic acids and
HCl*H-Pro-Trp-OMe (XIV) followed by the saponi-
fication with the ether group, as it was shown for (VII)
(Scheme 2).
0.89 (B), 0.91 (C);
+7°; IR: ν(NH) at 3356 cm–1;
[α]D
ν(C=O) 1668 cm–1; m/z: found [M + Na]+: 465.22;
[M + K]+: 481.185; calculated M(C22H38N2O5S)
442.61.
HCl*H-Met(O2)-Rim (III). To a solution of com-
pound (IV) (1.0 g, 2.26 mmol) in ethyl acetate
(10.0 mL), 4 H HCl (4.4 mL) in ethyl acetate was
added at 5°C. The reaction mixture was allowed to
stand for 1 h at 20°C with stirring at regular intervals.
The product was precipitated and washed with ether.
The residue was dried in vacuo. Upon grinding in
ether, the remaining oil crystallized. Yield: 0.85 g
20
Nor-Pro-Trp-OH (VIII). Yield: 0.29 g (83%); Rf
20
0.42 (А), 0.47 (B), 0.51 (C);
–8°; IR: ν(NH) at
[α]D
3368 and 3324 cm–1; ν(С=О) 1725, 1657, and
1619 cm–1; m/z: found [M + H]+: 422.20; calculated
M(C24H27N3O4) 421.48 Da.
Nrb-Pro-Trp-OH (IX). Yield: 0.36 g (73%); Rf 0.45 (A),
20
0.53 (B), 0.53 (C);
–8°; IR: ν(NH) at 3305 cm–1;
(96%); mp 138–140°C; R 0.53 (A), 0.63 (B);
+6°;
[α]D
[α]D
Na]+: 443.25; calculated
M(C22H38N2O5S) 442.61.
f
ν(С=О) 1707, 1652, and 1620 cm–1; m/z: found [M +
H]+: 424.22; calculated M(C24H29N3O4) 423.50 Da.
m/z: found [M
+
Qln-Pro-Trp-OH (X). Yield: 0.25 g (47%); Rf 0.57
TEA-Amt (I) was obtained in a similar way as (IV),
starting from TEA and amantadine hydrochloride.
Yield: 1.68 g (90%); Rf 0.90 (А), 0.81 (B), 0.93 (C); IR:
20
(A), 0.68 (B), 0.57 (C);
–36°; IR: ν(NH) at
[α]D
3288 cm–1; ν(С=О) 1728, 1663, 1617 cm–1; m/z: found
[M + H]+: 457.19; calculated M(C26H24N4O4) 456.49 Da.
ν(NH) at 3304 cm–1; ν(С=О) 1652 cm–1; m/z: found
[M + H]+: 288.15; calculated M(C17H21NOS) 287.42 Da.
Tha-Pro-Trp-OH (XI). Yield: 0.28 g (93%); Rf 0.42
TEA-Rim (II) was obtained in a similar way as (IV),
starting from TEA and Rim. Yield: 1.98 g (97%); Rf
20
(A), 0.48 (B), 0.51 (C);
–30°; IR: ν(NH) at 3368
[α]D
and 3324 cm–1; ν(С=О) 1726, 1659, and 1618 cm–1;
0.90 (A), 0.83 (B), 0.92 (C); IR: ν(NH) at 3304 cm–1;
ν(С=О) 1652 cm–1; m/z: found [M + H]+: 316.19; cal-
culated M(C20H29NOS) 315.47 Da.
m/z: found [M
+
H]+: 394.17; calculated
M(C22H25N3O4) 399.43 Da.
Cho-Pro-Trp-OH (XII) was synthesized similarly
to (IV), starting from Cho-Pro-OH, which was
obtained as described in [17], and HCl*H-Trp-OMe
followed by the saponification as described for (VII).
Yield: 1.05 g (91%); Rf 0.80 (A), 0.58 (B), 0.60 (C);
Z-Trp-Rim (V) was obtained in a similar way as
(IV), starting from Z-Trp-OH and Rim. Yield: 1.31 g
20
(83%); R 0.85 (A), 0.93 (B), 0.69 (C);
+5°; IR:
[α]D
f
ν(NH) at 3388 cm–1; ν(C=O) 1668 cm–1; m/z: found
[M + Na]+: 522.34; calculated M(C31H37N3O3) 499.64 Da.
[α]2D0
–32°; IR: ν(NH) at 3312 cm–1; ν(С=О) 1739 and
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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PEPTIDE CARBOCYCLIC DERIVATIVES AS INHIBITORS
525
1675 cm–1; m/z: found [M + H]+: 428.21; calculated
M(C23H29N3O5) 427.49 Da.
A/duck/Novosibirsk/56/05 (H5N1) or HVC viruses
was estimated.
Thus, the results obtained can be used for other
viroporins, such as Vpu of HIV-1, pore-forming pro-
teins 2B of the poliomyelitis virus and 6K of the alpha-
virus, protein E of coronaroviruses, E5 of the papil-
loma virus and viroporin of bovine diarrhea virus
(BVDV), and other viral ion channels.
Boc-Pro-Trp-OMe (XIII) was synthesized simi-
larly to (IV), starting from Boc-Pro-OH and HCl*H-
Trp-OMe. Yield: 4.58 g (95%); Rf 0.89 (A), 0.88 (B),
20
0.84 (C);
–41°; IR: ν(NH) at 3397 and 3143 cm–1;
[α]D
ν(С=О) 1729, 1679 cm–1; m/z: found [M + H]+:
417.16; calculated M(C22H29N3O5) 415.48 Da.
REFERENCES
HCl*Н-Pro-Trp-OMe (XIV) was synthesized sim-
ilarly to (III). Yield: 0.21 g (96%); Rf 0.46 (А), 0.49
1. Gonzalez, M.E. and Carrasco, L., FEBS Lett., 2003,
20
vol. 552, pp. 28–34.
(B), 0.57 (C);
–36°; IR: ν(NH) at 3374 and
[α]D
3196 cm–1; ν(С=О) 1741, 1679 cm–1; m/z: found
[M + H]+: 316.16; calculated M(C17H21N3O3) 315.37 Da.
2. Ewart, G.D., Sutherland, T., Gage, P.W., and
Cox, G.B., J. Virol., 1996, vol. 70, pp. 7108–7115.
3. Premkumar, A., Wilson, L., Ewart, G.D., and
The antiviral activity of compounds. The antiviral
activity of the compounds synthesized was studied
using three schemes of the administration of com-
pounds into a cell culture: 6 h prior to the infection of
cells, at the time of infection, and 6 h after the infec-
tion of Vero-6 and SPEV cells. The multiplicity of the
infection of cells with the influenza A virus and HCV
was 0.1–0.01 TCID50/cell. The compounds at con-
centrations 100.0, 50.0, 25.0, 12.5, 6.25, 3.12, 1.56,
and 0.8 μg/mL were added to the wells of 96-well
plates (25 μL to each well) with a monolayer of Vero-6 or
SPEV cells and supporting medium (200 μL). The cell
cultures infected with a virus and treated with the
compounds were incubated at 37°C in a CO2 incuba-
tor. After 72 h of incubation, the cell monolayer was
stained with methylene blue, the percent of viable cells
was determined, and the cytotoxic effect (CD50, a
minimal concentration of a preparation that induces
the death of 50% of monolayer cells) was estimated
using a cytometer (Invitrogen). In the second part of
the experiment, by day 6 after the infection, when the
complete destruction of monolayer cells in control
wells occurred, the inhibitory dose (ID50), a concen-
tration of compounds capable of protecting 50% of
cells against the cytopathic action of the virus, and
ID95, a minimal concentration of compounds capable
of protecting 95% and more of monolayer cells was
determined.
Gage, P.W., FEBS Lett., 2004, vol. 557, pp. 99–103.
4. Gazzarrini, S., Kang, M., Abenavoli, A., Romani, G.,
Olivari, C., Gaslini, D., Ferrara, G., Etten, J.L.,
Kreim, M., Kast, S.M., Thiel, G., and Moroni, A.,
Biochem. J., 2009, vol. 420, pp. 295–303.
5. Miura, T. and Takeuchi, H., Biochemistry, 2001,
vol. 40, no. 20, pp. 6053–6060.
6. Oxford, J.S. and Galbraith, A., Pharmacol. Ther., 1980,
vol. 11, pp. 181–262.
7. Hay, A.J., Wolstenholme, A.J., Skehel, J.J., and
Smith, M.H., EMBO J., 1985, vol. 4, pp. 3021–3024.
8. Kendal, A.P. and Klenk, H.D., Arch. Virol., 1991,
vol. 119, pp. 265–273.
9. Griffin, S.D., Beales, L.P., Clarke, D.S., Worsfold, O.,
Evans, S.D., Jaeger, J., Harris, M.P., and Rowlands, D.J.,
FEBS Lett., 2003, vol. 535, pp. 34–38.
10. Griffin, S., Stgelais, C., Owsianka, A.M., Patel, A.H.,
Rowlands, D., and Harris, M., Hepatology, 2008,
vol. 48, pp. 1779–1790.
11. Lin, C., Lindenbach, B.D., and Pragai, B.M., J. Virol.,
1994, vol. 68, pp. 5063–5073.
12. Griffin, S.D., Proc. Natl. Acad. Sci. U. S. A., 2009,
vol. 106, pp. 12567–12568.
13. Shibnev, V.A., Garaev, T.M., Finogenova, M.P.,
Shevchenko, E.S., and Burtseva, E.I., Khim. Farm. Zh.,
2012, vol. 46, no. 1, pp. 3–4.
14. Shibnev, V.A., Garaev, T.M., Deryabin, P.G., Finog-
enova, M.P., Botikov, A.G., and Mishin, D.V., Byull.
Eksp. Biol. Med., 2016, vol. 161, no. 2, pp. 247–251.
15. Shibnev, V.A., Deryabin, P.G., Finogenova, M.P.,
Garaev, T.M., and Mishin, D.V., RF Patent no. RU
2524216 C1, 2014.
16. L’vov, D.K., Shchelkanov, M.Yu., Deryabin, P.G.,
Grebennikova, T.V., and Prilipov, A.G., RF Patent
no. RU 2309983 C1, 2005.
Determination of the virucidal activity of com-
pounds. Solutions of compounds at a concentration of
0.1 μg/mL in saline were used in experiments. A solu-
tion of the compound was mixed with the virus as fol-
lows. A virus-containing material (100 μL) at the ini-
tial concentration (8.0 logTCID50/0.2) was added to a
solution of a compound (200 μL). The exposure to the
virus-containing material was carried out at 24°C for
20 min after which the residual virus infectivity was
titrated in each variant of the experiment with SPEV
cells. From the difference of titers in the control and
experiment, the virucidal activity of a compound, i.e.,
its capacity to suppress the infectivity of the
17. Poroshin, K.T., Shibnev, V.A., and Grechishko, V.S.,
Izv. Akad. Nauk, Ser. Khim., 1956, no. 7, pp. 1294–
1295.
Translated by S. Sidorova
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 5 2017