New class of hantaan virus inhibitors based on conjugation of the isoindole fragment to (+)-camphor or (?)-fenchone hydrazonesv

Article abstract of DOI:10.1016/j.bmcl.2021.127926

This work presents the design and synthesis of camphor, fenchone, and norcamphor N-acylhydrazone derivatives as a new class of inhibitors of the Hantaan virus, which causes haemorrhagic fever with renal syndrome (HFRS). A cytopathic model was developed fo

Full text of DOI:10.1016/j.bmcl.2021.127926

Bioorg. Med. Chem. Lett. 40 (2021) 127926
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New class of hantaan virus inhibitors based on conjugation of the isoindole
fragment to (+)-camphor or (ꢀ )-fenchone hydrazonesv
a,
*
a
b
b
Olga I. Yarovaya , Kseniya S. Kovaleva , Anna A. Zaykovskaya , Liudmila N. Yashina ,
b
b
c
d
Nadezda S. Scherbakova , Dmitry N. Scherbakov , Sophia S. Borisevich , Fedor I. Zubkov ,
d
e
e
b
Alexandra S. Antonova , Roman Yu. Peshkov , Ilia V. Eltsov , Oleg V. Pyankov ,
Rinat A. Maksyutov , Nariman F. Salakhutdinov
b
a
a
N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, Lavrent’ev av., 9, Novosibirsk 630090, Russia
State Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, Koltsovo, Novosibirsk Region 630559, Russia
Ufa Institute of Chemistry, Ufa Federal Research Center, RAS, Octyabrya pr., 71, Ufa 450054, Russia
b
c
d
e
Organic Chemistry Department, Faculty of Science, Peoples’ Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya St., Moscow 117198, Russia
Novosibirsk State University, Pirogova St. 1, Novosibirsk 630090, Russia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hantavirus
HFRS
This work presents the design and synthesis of camphor, fenchone, and norcamphor N-acylhydrazone derivatives
as a new class of inhibitors of the Hantaan virus, which causes haemorrhagic fever with renal syndrome (HFRS).
A cytopathic model was developed for testing chemotherapeutics against the Hantaan virus, strain 76–118. In
addition, a study of the antiviral activity was carried out using a pseudoviral system. It was found that the hit
compound possesses significant activity (IC50 = 7.6 ± 2 µM) along with low toxicity (CC50 > 1000 µM). Using
molecular docking procedures, the binding with Hantavirus nucleoprotein was evaluated and the correlation
between the structure of the synthesised compounds and the antiviral activity was established.
Antiviral
Terpene
Isoindole
Haemorrhagic fever with renal syndrome (HFRS) (synonyms: Hae-
Laguna Negra Hantaviruses. The first clinical form of the disease (HFRS)
is registered in Russia, and the circulation of 7 types, including 4
pathogenic Hantavirus types, is established. In the European region, the
HFRS pathogen in the vast majority of cases is the Hantaan- and
Puumala-type. HFRS is widespread mainly in China, the Republic of
Korea and the Russian Federation, as well as in Sweden, Finland, and
morrhagic nephritis, Churilov’s disease, epidemic nephritis, Far Eastern
haemorrhagic fever, Korean haemorrhagic fever, Manchurian haemor-
rhagic fever, Scandinavian epidemic nephropathy, Tula fever) is an
acute viral, natural-focal disease, characterised by systemic lesions of
small vessels, haemorrhagic diathesis, haemodynamic disorders, and a
1
4
peculiar lesion of kidneys with acute renal failure. The HFRS pathogen
western and central Europe. China has experienced approximately 90%
of the cases worldwide over the last few decades.5 From 2006 to 2015,
,6
belongs to the Orthohantaviruses, previously known as hantaviruses
family Hantaviridae, order Bunyavirales).2 Over the past decades,
(
>110,000 cases of HFRS were reported in China,7 and from 2000 to
8
Hantavirus diseases have been included in the range of topical and high-
priority problems around the world, as one of the so-called emerging
unpredictable) infections, threatening complex epidemic situations.3
2018 , >130,000 cases were registered in the Russian Federation. The
hantavirus prototype strain, HTNV, was first isolated from the striped
9
(
field mouse, Apodemus agrarius, in 1976. The discovery of the etiolog-
This is due to the variability of the Hantavirus genome, and, therefore, is
fraught with the emergence of new types and genetic variants in new
regions of the world, with high virulence for humans. To date, >30
serologically and genetically different Hantaviruses are known. Two
clinical forms of the Hantavirus infection in humans are described: HFRS
caused by Hantaan, Seul, Puumala, Dobrava/Belgrade, Seoul, Amur; and
Hantavirus pulmonary syndrome, first described in the USA in 1993, is
caused by Sin-Nombre, Black Creek, New York, Bayou, Andes, and
ical agent of HFRS in South Korea prompted research all over the world,
resulting in the discovery of other HFRS-associated novel viruses in the
Old World. Hantaviruses have since been discovered to circulate not
1
0,11
only in Asia and Europe, but also in both the Americas and Africa.
In addition to the serious epidemiological situation around the
world, viruses causing HFRS can pose a threat as biological weapons.
Depending on the severity of the disease they cause and their ability to
spread, biological agents are classified by the Center for Disease Control
*
Corresponding author.
https://doi.org/10.1016/j.bmcl.2021.127926
Received 6 December 2020; Received in revised form 5 February 2021; Accepted 25 February 2021
Available online 9 March 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

O.I. Yarovaya et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127926
and Prevention (CDC) into three categories: A–C. Category A agents
include haemorrhagic fever viruses (some filoviruses, flavoruses,
bunyaviruses and arenaviruses) and pox viruses, including Variola
major (smallpox is natural). Viruses causing HFRS are considered to be
potential weapons in bioterrorism attacks.12Fig 1.
In the search for new virus inhibitors, we recently identified a series
of various (+)-camphor N-acylhydrazone derivatives that are active
against vaccinia and influenza viruses.31 Among these, 6-methyl-1-oxo-
2-phenyl-N’-[1,7,7-trimethylbicyclo[2.2.1]hept-2-ylidene]-
1,2,3,6,7,7a-hexahydro-3a,6-epoxyisoindole-7-carbohydrazide, shown
in Fig. 2, exhibited significant activity against both the smallpox virus
and the influenza virus in the micromolar concentration range. As the
surface protein structure of smallpox and influenza viruses differs
significantly, we assume that this compound does not exhibit viral ac-
tivity in the early stages of viral replication. Given the high importance
of finding new agents with specific activity against Hantaan viruses, we
have synthesised analogues of the specified N-acylhydrazone in this
work. It was also of importance to find out the influence of both the
structure of a natural fragment and the structure of an artificial epox-
yisoindole scaffold. Thus, this work presents the synthesis and detailed
study of the antiviral activity of 3a,6-epoxyisoindole derivatives, in
which the four most important positions of the molecule vary; specif-
The main route of infection is airborne dust, where the virus, con-
tained in the biological excretions of rodents, in the form of an aerosol
enters through the upper respiratory tract to the lungs, where the con-
ditions for its reproduction are most favourable, and then is transferred
with blood to other organs and tissues.13 Infection is also possible
through damaged skin, when exposed to excreta of infected rodents or to
saliva when bitten by another human. Despite the severity of the disease
and its widespread occurrence, there is no specific antiviral therapy at
present. Treatment may be carried out with a wide range of drugs,
including ribavirin.14 Recent clinical studies (placebo-controlled,
double-blind) have shown no efficacy of ribavirin in the treatment of
1
5
HFRS. It has been shown that Triazavirin, which has been approved in
–
Russia as an anti-influenza agent, is active against the Hantavirus in
ically, the C4 C5 double bond, the terpene fragment, substituents at the
–
1
6
vitro, but has not demonstrated high activity in animal models. Arbidol
is a broad-spectrum antiviral compound that has been shown to have an
inhibitory effect on the influenza virus and hantavirus.17 Arbidol in-
hibits TLR4 expression and iNOS production induced by HTNV infection
in HUVECs, which represents one of the virus-sensing pattern-recogni-
tion receptors (PRRs) and its resulting inflammatory cytokines/chemo-
N-2 nitrogen atom and at the node C-6 carbon atom, as shown in Fig. 2.
The target compounds for this study were prepared according to the
general synthetic route depicted in Schemes 1 and 2, based on optically
active D-(+)-camphor or L-(ꢀ )-fenchone. In the first stage of the work,
camphor, fenchone and norcamphor hydrazones 1, 2 and 3 were ob-
tained by the reaction of the corresponding ketones with hydrazine
hydrate in the presence of acetic acid (AcOH), according to a reported
procedure.32 In the second step, the condensation of hydrazones 1–3
with heterocyclic carboxylic acids was performed. Note that the syn-
thesis of the carboxylic acids and compounds 4–7 and 10 was either
1
8
kines. The activity against Hantavirus of an analogue of ribavirin,
agent ETAR containing a terminal alkyne bond in its triazole cycle, was
shown. The dose-dependent effect was studied, and it was shown that
this agent exhibits pronounced activity against Hantavirus in vitro and in
1
9
31,33,34,35,36
vivo. Compounds belonging to the purine nucleoside class have been
synthesised and tested as inhibitors of Hantavirus infections, with
compound FPI, containing fluorine in the aromatic ring, showing the
greatest antiviral activity. In spite of the fact that the active concentra-
tion (EC50) for compound FPI was 94 µM and SI was not >3, the authors
described earlier or carried out using similar methods.
As the result of this section of experiments, a wide range of diverse
isoindole derivatives was obtained, including a) compounds 5–7, which
differ in substituents at the N-2 of the isoindole fragment; b) compounds
8–16, containing an additional methyl group at the C-6 position; c)
compounds 10–12, which differ from each other by the presence or
2
0
of this work considered this compound as promising. Using high-
throughput flow cytometry, it has been shown that Antimycin is active
against Hantavirus and binds directly to the viral particles.21 Several 2-
phenyl-benzotriazoles showed fairly potent inhibition of the Hantaan
virus in a chemiluminescence focus reduction assay (C-FRA), showing
–
absence of the C4 C5 double bond, or the presence or absence of an
–
additional 4,5-epoxy bridge; d) compounds 13 and 14, bearing sub-
stituents in the para-position of the aromatic moiety; e) compounds 15
and 16, which differ by the length of an aliphatic linker between the
benzene ring and the isoindole nucleus; and f) compound 17, possessing
an aromatic isoindole fragment.
2
2
EC50 between 4 and 5 µM, a ten-fold increase compared to ribavirin.
The strategy of searching for new antiviral agents based on terpenic
compounds is extremely promising.2
3,24
The use of available natural
According NMR data, compounds 4 and 17 were obtained as a single
isomer, while the other compounds 5–16 were a mixture of di-
astereomers. As the reaction occurs between the optically-pure hydra-
zone of camphor 1 and acid, which is a racemic mixture of isomers, a
mixture of diastereomers is formed in a 1:1 ratio, as shown in Fig. 3.
Attempts to separate these mixtures by preparative column chroma-
tography failed, due to close retention factors. Therefore, in these cases,
the diastereomer mixtures were used for subsequent biological tests.
substances as starting compounds allowed our team to discover new
classes of agents with a wide range of antiviral activity. Thus, we
discovered a new class of agents with high activity against the influenza
virus, specifically imino derivatives based on camphor;2
5,26
it was
shown that borneol derivatives are effective inhibitors of the entry of
Marburg and Ebola viruses;2
7,28
and it was found that compounds con-
taining a framework bicyclic fragment exhibit pronounced activity with
orthopoxviruses.2
9,30
The purpose of the presented work is synthesis and
identification of new agents, based on natural compounds of the terpene
series with specific activity to viruses causing HFRS.
Fig. 1. Chemical structure of inhibitors of Hantavirus replication.
Fig. 2. Design strategy for new heterocycle-containing N-acylhydrazones.
2

O.I. Yarovaya et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127926
Fig. 3. Chemical structure of diastereomers 5–16.
fragment of all four isomers was established based on 2D NOESY
experiments.
The natural bicyclic ketone (ꢀ )-fenchone differs from camphor by
the location of the gem-dimethyl groups. In order to study the effect of
the natural ketone type on biological activity, we synthesised N-acyl
hydrazones 18–21 on the basis of fenchone hydrazone 2 (Scheme 2).
Among these substances, compounds 18–20 have different substituents
in the aromatic fragment, and compound 21 has an ethyl substituent in
the 6th position of the epoxyisoindole fragment. Substances 18–21 are
also a mixture of diastereomers. In addition, compound 22 was syn-
thesised from a racemic mixture of norcamphor hydrazone 3, and,
hence, has a mixture of 4 stereoisomers.
Scheme 1. Reagents and conditions: (a) NH
2
NH
2
⋅H
2 3 2
O (4 eq.), CH CO H (1
eq.), EtOH, reflux 4 h; (b) Et
3
N (1.2 eq.), carboxylic acid chloride (1 eq.), CHCl
3
;
(
c) carboxylic acid (1 eq.), EtO
2
CCl/ Et N, CHCl
3
3
.
To study the biological activity of synthesised substances against the
Hantaan virus, we developed a cytopathic model. Hantaviruses do not
adapt to reproduction in laboratory animals as well as they do in cell
cultures, which likely explains over thirty s of unsuccessful attempts to
isolate the HFRS pathogen, whose viral nature has already been proven.
The laboratory model of hantavirus infection is only known for certain
types of hantaviruses.37 The possibility of using cell cultures to cultivate
hantaviruses was actively studied after the successful adaptation of the
Hantaan virus, isolated in laboratory mice, to human lung carcinoma
cell culture, A549.38 Later, various properties of hantaviruses, including
the pathogenesis of hantavirus infections and the development of inac-
tivated vaccines, were used as primary and transferable cell lines.
Despite numerous studies, the selection of a cell culture suitable for
the successful cultivation of HFRS viruses remains a current problem in
virology. Difficulties in the cultivation of hantaviruses are due to the fact
that under normal cultivation conditions they do not have a visible
cytopathic effect on cell culture, and they are accumulated in low titres
even with long incubation periods. Difficulties in selecting animal
models and cell cultures to visualise the results of exposure to hantavi-
ruses make it highly difficult to find antiviral drugs. The methods
described earlier are difficult and time-consuming.
Scheme 2. Reagents and conditions: (a) NH
2
NH
2
⋅H
2
O (4 eq.), CH
3
CO
2
H (1
Initially, 3 strains of HFRS virus deposited in the State Collection of
viral and rickettsiosis agents of the State Research Center of Virology
and Biotechnology VECTOR – Hantaan 76-118, Amur AP 94-415 and FE
HTN P-98-87 – were used. Viruses were cultivated in Vero cell culture
for between 14 and 36 d. The level of virus reproduction was assessed
using real-time quantitative PCR analysis, with a set of primers to the S
segment of the nucleocapse. The results obtained were confirmed using
ELISA using components of the VektoKhanta-IgG (Vector-Best CJSC) and
GLPS Diagnosticum of the culture, polyvalent for the indirect method of
immunofluorescence (Chumakov Immunofluorescence Department of
the Russian Academy of Medical Sciences).
eq.), EtOH, reflux 4 h; (b) carboxylic acid (1 eq.), EtO
2
CCl/ Et N, CHCl
3
3
.
The structures of compounds 8, 9, and 11–17 were elucidated using
the combination of 1D and 2D methods of NMR spectroscopy (COSY,
TOCSY, NOESY, HSQC, HMBC, and HSQC-COSY). It should be noted
here that the 1H NMR spectra of the derived N-acylhydrazones were
6
complex. For example, in DMSO‑d at room temperature, solutions of
5
–16 represented mixtures of four isomers, two of which (a and b, see
Fig. 3) were diastereomers and the other two of which (c and d) were
rotamers on the amide fragment. The (E)-configuration of the hydrazone
3

O.I. Yarovaya et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127926
We have assessed the possibility of using an MTT test to indicate the
replication of hantaviruses. The Hantaan 76-118 virus strain was used
for this, for which the results were more visible, i.e., the difference be-
tween the optical density values in wells with infected and non-infected
cells after colouration was the largest for this strain. We prepared suc-
cessive ten-fold dilutions of the virus, starting with 0.1 of the original
concentration. The 96-well culture plates were infected with the pre-
Table 1
Antiviral activity of compounds 4–22 against virus Hantaan 76–118 in MDCK
cells and recombinant vesicular stomatitis virus (rVSV-ΔG) particles, pseudo-
typed with Gn-Gc, in HEK293T cells.
b
c
c
Compound
CC50
IC50 (µM)
Hantaan
76–118
SI
CC50
IC50
SI
a
a
b
(
µM)
(µM)
HEK293T
(µM)
rVSV-
ΔG-Gn-
Gc
rVSV-
ΔG-Gn-
Gc
Vero
◦
pared virus dilutions, incubated at 37 C for 10 and 14 d, and then dyed
with MTT. The results were recorded on an automatic tablet photometer
at a wavelength of 540 nm and data was processed using the SOFTmax
PRO 4.0 program. The data obtained indicates that a replication of the
Hantaan 76-188 virus can be recorded as early as 10 d post-infection. In
this work, we also studied the biological activity of the synthesised
compounds, relating to the Hantaan virus.
d
4
5
6
7
8
9
>150
NA
–
–
–
–
3
9
1095 ±
780
790
>800
750
468
NT
–
–
–
–
5
4
6
250 ±
17
NA
1072
1029
860
250 ±
NA
1
8
250 ±
NA
In order to understand at what stage of the virus’s life cycle
chemotherapeutic agents are active, a time of addition test is often used.
However, in the study of hantavirus, this method is extremely difficult to
apply due to the length of the experiment. Thus, we have additionally
studied the action of the synthesised compounds on a pseudovirus sys-
tem with Hantaan virus Gn glycoprotein on its surface.
1
2
250 ±
15
92 ± 5
26 ± 3
14 ± 3
20 ± 3
37 ± 5
NA
>2500
e
246 ±
NT
1
5
1
0
142 ±
10
6
448
1452
1039
175
NT
96
4
3
5
–
1
3
The results of biological testing are presented in Table 1. We selected
two broad-spectrum antiviral drugs for reference – Ribavirin and Tri-
azavirin. Our research revealed Triazavirin’s activity in relation to the
Hantaan virus and showed that Ribavirin does not exhibit any activity at
all, being quite toxic under experimental conditions.
11
130 ±
375
195
120
NT
1
0
1
1
2
3
195 ±
5
1
2
78 ±
10
–
The data presented in Table 1 on the activity of synthesised com-
pounds against the Hantaan virus showed that compounds 8–12, 14–16,
14
120 ±
8
19 ± 3
33 ± 6
42 ± 7
6
1
1
5
6
245 ±
7
335
216
147
69
2
3
1
8 and 20–22 exhibited specific antiviral activity, while substance 18,
1
2
which we synthesised on the basis of fenchone hydrazine 2, proved to be
much more effective than the reference drug. It should be noted that a
similar compound 10, which has the camphor core, possesses compa-
rable activity, but its toxicity is significantly higher. The analysis of the
results led us to conclude that the presence of the isoindole fragment
attached to the nodal carbon atom is necessary for the target activity to
occur. For example, substances 5–7, which have no substituents in the
195 ±
17
4
1
1
7
8
>300
1064
NA
–
140
NT
NT
–
3
7.6 ± 2
1180
431
±
26
1
9
80 ±
10
NA
–
9
3
9
78
65
74
NT
NT
–
2
–
–
20
292 ±
30 ± 4
40 ± 6
28 ± 7
152
NT
NT
1
6
6
th position of the isoindole fragment, are completely inactive against
2
2
1
2
120 ±
7
Hantaan viruses.
Among compounds 10, 11 and 12, which differ from each other in
the presence/absence of the double bond in the C4-C5 position of the
isoindole fragment or the presence of the additional C4-C5 epoxy bridge,
compound 10 was the most active, with comparable toxicity. Compar-
ison of the biological properties of compounds based on camphor
hydrazone 10, 13, 14, and compounds based on fenchone hydrazone 18,
269 ±
17
Ribavirin
20 ± 3
368 ±
NA
–
26
NT
NT
–
4
Triazavirin
14 ± 3
1513
408
1
8
a
CC50 ꢀ 50% cytotoxic concentration; the concentration resulting in death of
0% of cells; M ± I95, n = 3.
5
1
9, 20, leads to the conclusion that the introduction of substituents in
b
IC50 ꢀ 50% inhibitory concentration; the concentration leading to 50% in-
the para-position of the N-phenyl moiety reduces the activity and in-
creases the toxicity of substances. An increase in the length of the linker
between the aromatic fragment and the N-2 nitrogen atom in camphor-
based compounds 10, 15 and 16 leads to a complete loss of activity.
Compound 17, which has an aromatic isoindole fragment, does not
exhibit any activity. Compound 22, which we synthesised on the basis of
norcamphor hydrazone 3, being active against the Hantaan virus, is a
diastereomeric mixture which is very poorly soluble in water, DMSO,
hibition of virus replication; M ± I95, n = 3.
c
d
e
.
SI – selectivity index, ratio CC50/IC50
NA – not active.
.
NT – not tested.
inactive against VSV-ΔG-Gn-Gc pseudoviruses.
It should be noted that the toxicity of the compounds was tested on
the HEK293T cell line, with a residence time of 72 h, whereas residence
time is up to 10 d with the live virus, which does not allow direct
comparison of these results.
3
CHCl , EtOH and other organic solvents, which makes it difficult to work
with. Therefore, compound 18 exhibits the highest activity and lowest
toxicity from the entire chemical library.
The results presented are consistent with the fact that we have pre-
viously shown marked activity against the smallpox virus and influenza
virus for compound 10. It appears that this class of compounds may not
be an inhibitor of virus entry, but an inhibitor of intracellular replica-
tion. Hantavirus nucleoprotein (N) may then be considered as a poten-
tial biological target. This protein N is involved in the processes of virus
transcription and replication, and, therefore, represents an attractive
target for therapeutic effects. The nucleoprotein structure describes a
highly conservative RNA binding site.41. It has been shown that N pro-
To study the mechanism of antiviral activity of the compounds, we
tested them using a pseudovirus system. Previously, the receipt and use
of such a system was described for the Puumala virus.3
9,40
In the first
stage, HEK293T cell culture was transfected with a plasmid containing
the gene Gn-Gc of the Hantaan virus strain 76–118. After 48 h, VSV-ΔG-
G was added to the cells. After another 48 h, a supernatant containing
pseudoviruses was collected. In the second stage, different concentra-
tions of substances were added to a suspension of pseudoviruses
(
500,000 RLU), incubated for 1 h, and then a HEK293T cell suspension
′
tein binds to the host mRNA cap and protects the 5 end of this molecule
was added. After 48 h, the luminescence level was measured. The results
presented in Table 1 show that the substances we describe are virtually
from degradation, which is later trapped and used as a viral RdRp primer
4

O.I. Yarovaya et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127926
during transcription initiation.42 Protein N also seems to be responsible
for switching the host’s broadcast machine for the preferred broadcast of
viral transcripts. Moreover, nucleoprotein N has been shown to delay the
induction of cell apoptosis and facilitate the transport and localisation of
viral ribonucleoproteins (RNP).43 Presumably, binding small molecules
in a given location may inhibit the binding of viral RNA and/or oligo-
merisation of the protein, and therefore block replication of the virus.
Assessment of the affinity of the studied compounds to the described site
binding was carried out using molecular modelling methods.
The crystalline structure of nucleoprotein (PDB code 5FSG)41 was
downloaded from the Protein Data Bank database.44 Model protein
structures were prepared using the Schrodinger Protein Preparation
Wizard tool: hydrogen atoms were added and minimised; missing amino
acid side chains were added; bond multiplicities were restored; solvent
molecules were removed; and the entire structure was restrained and
optimised in the OPLS3e force field45 at physiological pH values (7.0 ±
Fig. 4. A) Secondary protein structure (PDB code 5FSG); B) functional amino
acids located at the binding site of potential inhibitors.
0
.2). The geometric parameters of potential ligands were also optimised,
taking into account all permissible conformations. All biological ex-
periments were conducted for a mixture of diastereomers, although
calculations were made for each isomer individually.
The binding site of nucleoprotein binding with viral RNA was
considered as a potential binding site for the studied compounds. The
active site contains a number of functional amino acid residues: Ser180,
Asn183, Ser186, Ser187 and Thr 194 line the deduced RNA binding
grove, and could form hydrogen bridges; Lys189, Arg197, and Arg199
can form salt bridges;41 and Arg339, Arg367 and Arg368 neutralise the
charge of the RNA phosphate group (Fig. 4).
Molecular docking procedures were performed using Schrodinger
Suite (Release 2018-4) software. Compounds 8–12, 14–16, 18 and
2
0–22 were docked using the forced ligand positioning protocol (IFD),
with the following conditions: flexible protein and ligand; grid matrix
size of 15 Å; and amino acids (within a radius of 5 Å from the ligand)
restrained and optimised, taking into account the influence of the
ligand. Docking solutions were ranked by evaluating the following
calculation parameters: docking score (based on GlideScore minus
penalties); ligand efficiency (LE, which takes into account atomic dis-
tribution of scoring function); and parameter of model energy value
Fig. 5. Molecular docking results: A) the intended binding location of potential
nucleoprotein inhibitors; B) regression analysis of the results of biological ex-
periments and theoretical calculations; C) the location of two lead-compound
(
Emodel), including GlideScore value, energy unrelated interactions and
1
8 diastereomers in the inhibitor binding site (hydrogen bridges are shown
the parameters of the energy spent on the formation of the laying of the
compound in the binding site. According to the results of molecular
docking, all the compounds may bind in the place of binding viral RNA
with N and form a number of intermolecular interactions with the amino
acid residues described above (Fig. 5A).
by the yellow dotted line).
(
+)-camphor, (ꢀ )-fenchon and norcamphor, with all compounds fully
characterised using a set of physical and chemical methods. A cytopathic
model suitable for testing chemical compounds against the Hantaan
virus, strain 76-118, has been developed, and a study of the activity of
compounds 4–22 against this virus and with the pseudovirus system
used has been carried out. It has been shown that the agents we describe
have a pronounced activity against the Hantaan virus, and are not active
in the pseudovirus system. A synthesised compound 18, based on
hydrazone-(ꢀ )-fenchon with a fragment of epoxyisoidol, is the agent
with the highest activity and has low toxicity. The relationship between
the structure of the synthesised agents and their antiviral activity has
been studied. Based on a combination of biological experiments and
molecular modelling data, it can be assumed that the compounds under
study may be associated with the site of binding RNA of nucleoprotein N
Hantan virus. Such competitive binding of small molecules may lead to
blockages in the replication process of viral infection.
The docking score values range from ꢀ 7.2 to ꢀ 4.1 kcal/mol
(
Fig. 5B). There is a trend, namely that the compounds characterised by
low IC50 values, measured by us using the cytopathic model for the
Hantaan virus, show better affinity to the binding site than connections
with low antiviral activity. The statistical dependence of pIC50 and the
docking score is observed. To find a statistical relationship between the
values that characterise the antiviral activity of the compounds and the
affinity to the selected binding site, regression analysis was performed
using the method of least squares. The dependence of pIC50 (decimal
logarithm of half-maximal inhibition concentration) values and the
weighted average value of the docking score was plotted. The contri-
bution of each diastereomer was taken into account. The dependence
between these parameters can be considered as evidence of the correct
choice of a biological target.
It was shown that different diastereomers fold slightly differently in
the binding site. For example, one diastereomer of the lead-compound
Declaration of Competing Interest
1
8a is located in the site binding with the formation of hydrogen
bridges with amino acids Arg144, Gln147 and Thr148, and another
18b) forms hydrogen bridges with Thr194 and Gly196 (Fig. 5C). At the
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
(
same time, the energy parameters of diastereomers binding do not differ
significantly (detailed data are presented in Supplementary Material).
As a result of our research, we have synthesised a small library of
compounds based on hydrazones of frame ketones, namely
5

O.I. Yarovaya et al.
Bioorganic & Medicinal Chemistry Letters 40 (2021) 127926
Acknowledgements
22 Sanna G, Piras S, Madeddu S, et al. 5,6-Dichloro-2-phenyl-benzotriazoles: New
potent inhibitors of orthohantavirus. Viruses. 2020;12:122. https://doi.org/10.3390/
v12010122.
The reported study was funded by RFBR according to the research
project No. 18-03-00271. The synthesis of the epoxyisoindole acids was
provided by the Ministry of Education and Science of the Russian
Federation (award no. 075-03-2020-223 (FSSF-2020-0017)). The au-
thors would like to express their gratitude to the Collective Use Chemical
Service Center of the Siberian Branch of Russian Academy of Sciences for
the obtained spectra and analytical data.
2
2
2
2
2
2
2
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compounds: Camphor imine derivatives. Eur J Med Chem. 2015;105:263–273.
https://doi.org/10.1016/j.ejmech.2015.10.010.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127926.
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