Adamantane derivatives as potential inhibitors of p37 major envelope protein and poxvirus reproduction. Design, synthesis and antiviral activity

Article abstract of DOI:10.1016/j.ejmech.2021.113485

Currently, smallpox, caused by the variola virus belonging to the poxvirus family, has been completely eradicated according to the WHO. However, other representatives of poxviruses, such as vaccinia virus, cowpox virus, ectromelia virus, monkeypox virus, mousepox virus and others, remain in the natural environment and can infect both animals and humans. The pathogens of animal diseases, belonging to the category with a high epidemic risk, have already caused several outbreaks among humans, and can, in an unfavorable combination of circumstances, cause not only an epidemic, but also a pandemic. Despite the fact that there are protocols for the treatment of poxvirus infections, the targeted design of new drugs will increase their availability and expand the arsenal of antiviral chemotherapeutic agents. One of the potential targets of poxviruses is the p37 protein, which is a tecovirimat target. This protein is relatively small, has no homologs among proteins of humans and other mammals and is necessary for the replication of viral particles, which makes it attractive target for virtual screening. Using the I-TASSER modelling and molecular dynamics refinement the p37 orthopox virus protein model was obtained and its was confirmed by ramachandran plot analysis and superimposition of the model with the template protein with similar function. A virtual library of adamantane containing compounds was generated and a number of potential inhibitors were chosen from virtual library using molecular docking. Several compounds bearing adamantane moiety were synthesized and their biological activity was tested in vitro on vaccinia, cowpox and mousepox viruses. The new compounds inhibiting vaccinia virus replication with IC₅₀ concentrations between 0.133 and 0.515 μM were found as a result of the research. The applied approach can be useful in the search of new inhibitors of orthopox reproduction. The proposed approach may be suitable for the design of new poxvirus inhibitors containing cage structural moiety.

Full text of DOI:10.1016/j.ejmech.2021.113485

European Journal of Medicinal Chemistry 221 (2021) 113485
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Adamantane derivatives as potential inhibitors of p37 major envelope
protein and poxvirus reproduction. Design, synthesis and antiviral
activity
,
Vadim A. Shiryaev a *, Michael Yu Skomorohov a, Marina V. Leonova a,
Nikolai I. Bormotov b, Olga A. Serova b, Larisa N. Shishkina b, Alexander P. Agafonov b,
Rinat A. Maksyutov b, Yuri N. Klimochkin a
a Department of Organic Chemistry, Samara State Technical University, 244 Molodogvardeyskaya St., Samara, Samara Region, 443100, Russia
b State Research Centre of Virology and Biotechnology VECTOR, Koltsovo, Novosibirsk Region, 630559, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Currently, smallpox, caused by the variola virus belonging to the poxvirus family, has been completely
eradicated according to the WHO. However, other representatives of poxviruses, such as vaccinia virus,
cowpox virus, ectromelia virus, monkeypox virus, mousepox virus and others, remain in the natural
environment and can infect both animals and humans. The pathogens of animal diseases, belonging to
the category with a high epidemic risk, have already caused several outbreaks among humans, and can,
in an unfavorable combination of circumstances, cause not only an epidemic, but also a pandemic.
Despite the fact that there are protocols for the treatment of poxvirus infections, the targeted design of
new drugs will increase their availability and expand the arsenal of antiviral chemotherapeutic agents.
One of the potential targets of poxviruses is the p37 protein, which is a tecovirimat target. This protein is
relatively small, has no homologs among proteins of humans and other mammals and is necessary for
the replication of viral particles, which makes it attractive target for virtual screening. Using the I-TASSER
modelling and molecular dynamics refinement the p37 orthopox virus protein model was obtained and
its was confirmed by ramachandran plot analysis and superimposition of the model with the template
protein with similar function. A virtual library of adamantane containing compounds was generated and
a number of potential inhibitors were chosen from virtual library using molecular docking. Several
compounds bearing adamantane moiety were synthesized and their biological activity was tested in vitro
on vaccinia, cowpox and mousepox viruses. The new compounds inhibiting vaccinia virus replication
Received 12 March 2021
Received in revised form
13 April 2021
Accepted 15 April 2021
Available online 29 April 2021
Keywords:
Adamantane
Orthopox viruses
Molecular modelling
Drug design
Antiviral activity
with IC50 concentrations between 0.133 and 0.515 mM were found as a result of the research. The applied
approach can be useful in the search of new inhibitors of orthopox reproduction. The proposed approach
may be suitable for the design of new poxvirus inhibitors containing cage structural moiety.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
Although smallpox is now globally eradicated, the infections
caused by zoonotic pox viruses may still be a threat. Monkeypox,
Poxviruses occupy a special place among DNA genomic viruses.
The variola virus, which belongs to the genus of orthopoxviruses of
the poxviridae family, is considered to be one of the most
dangerous viruses because of its pathogenic and epidemic prop-
erties (mortality exceeds 30%) and poses a serious threat as a bio-
logical agent for bioterrorism [1,2].
cowpox and parapox viruses are already causing outbreaks in
humans [3,4]. New zoonotic poxviruses are discovered lately [5].
The increase in the number of people with reduced immunity and
the fashion for the maintenance of exotic animals increases the
frequency of people contacts with undiscovered animal poxviruses
and, therefore, increases the probability of emergence of new
* Corresponding author.
E-mail address: shiryaev.va@samgtu.ru (V.A. Shiryaev).
https://doi.org/10.1016/j.ejmech.2021.113485
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
mutant poxvirus strain more pathogenic to humans, that put ani-
mal poxviruses in the range of high epidemic risk pathogens.
Moreover, the progress of modern biotechnology opens the way
to the creation of genetically modified microorganisms with
increased pathogenicity and drug resistance [6]. Since the structure
of the poxvirus genome is now well known, there is a danger of
creating an artificial poxvirus pathogen [7,8].
antiviral drug design.
Since the compounds [14e23] containing the adamantane core
frequently exhibit noticeable activity against poxviruses and the
adamantane fragment is highly similar in geometry and lip-
ophilicity to the cage fragment tecovirimat, we propose a number
of compounds based on the replacement of the hydrocarbon moi-
ety of tecovirimat with the adamantane residue.
Poxviruses are the largest and the most complex of all known
human and animal viruses. Their genome is represented by double-
stranded DNA, which contains information on the amino acid
sequence of several dozen polypeptides. The reproductive cycle of
the smallpox virus is very complex. The processes of poxvirus
maturation and exit from the cell, as well as the processes of
penetration into the cell, are multistage and include intermediate
stages [9,10].
Variability and mutation of viruses as well as the possibility of
the emergence of new mutagenic forms on the one hand, and the
limited arsenal of antiviral agents on the other, necessitate the
search for new effective antiorthopoxvirus agents.
2. Results and discussion
2.1. Design approach
Similarly to design of viral reproduction influenza viruses [35]
and hepatitis virus [36] we used the standard approach [37], that
includes the molecular modelling of spatial structure of target
protein by the means of molecular dynamics, the determination of
potential ligands using molecular docking, verification of potential
inhibitor by evaluation of biological activity.
To date only primary structure of p37 protein is determined
experimentally, as far as secondary and tertiary structures are not
investigated. The search for the structure of this protein gave no
experimental models and only several primary structures of several
strains in the UniProt database. The tertiary models of these pro-
teins are available through SWISS-MODEL Repository basing on
PDB structures 4urj and 4ggj or ModBase database basing on PDB
structure 1v0w. The SWISS-MODEL is only fragmentary as far as
each of the templates are at least twice smaller than p37 protein. On
the other side, the ModBase model is based on a homologous
Streptomyces phospholipase D [28] and its sequence almost fully
covers the sequence of p37, however this model is rather outdated.
We have chosen the sequence of protein P20638 (F13_VACCC)
from the UniProt database corresponding to the available experi-
mental vaccinia model. Since the available models of p37 are
unconfident, we used blind modelling of p37 protein structure by I-
TASSER web service without any predefined templates [38e40]. In
these conditions I-TASSER searches the best alignment of proteins
through the whole RCSB PDB database. The resulting 3D model is
constructed by clustering a large ensemble of similar proteins
structural conformations. The confidence and folding of the model
are evaluated by C-score and TM-score. The resulting model is
characterized by high C-score (0.63) what confirms the high quality
of the model. TM-score shows similarity in folding between two or
more proteins while low values of TM-score usually appear for
randomly chosen structures. The high value of TM-score
(0.80 0.09) for predicted model shows good folding, similar to
selected templates. The resulting model has very close TM scores
and rather small RMSD comparing to the existing experimental
models of phospholipase D from Streptomyces sp. (1v0w, TM 0.98,
RMSD 1.97) what correlates with the lipase activity of p37. The
modelling of molecular dynamics (MD) was used to refine the
predicted model in order to remove the steric clashes of the resi-
dues and to check the stability of the protein during thermal mo-
tion. The initial model for MD simulation was constructed using
CHARMM-GUI web service [41e43]. At the first stage, the protein
was palmytoilated on Cys185 and Cys186 residues [44], introduced
into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
bilayer and solvated with water containing Kþ and Clꢀ ions with a
concentration of 0.15 M. As far as no complexes of similar proteins
with metals are known, but on the contrary metal-free complexes
of substrates exist (for example 1v0w), no other ions were intro-
duced into the system. Next, the structure was optimized at 310 K
with restraints on the movement of protein backbone and side
chains using NAMD software [45]. Then the resulting structure
underwent a stage of equilibration at a time interval of 80 ns at
310 K. The analysis of RMSD during equilibration showed, that the
Anti-smallpox activity has been found among a wide range of
cage compounds [11e13]. Screening of a huge number of cage
compounds revealed a number of promising drug candidates with
pronounced antiviral activity. Inhibitors of orthopoxvirus repro-
duction have been found in the series of functionalized compounds
containing adamantane moiety [14e23].
Currently one of the most effective drugs against orthopoxvi-
ruses is 4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-
dioxo-4,6-ethenocycloprop[f]isoindole-2-(1H)-ylbenzamide (Teco-
virimat, ST-246) [24e26].
This drug, found by SIGA Technologies (USA), is effective against
a number of orthopoxviruses, including vaccinia virus, monkeypox
viruses, cowpox, camelpox and ectromelia virus and now is
approved by FDA (“Tpoxx”) for treatment of adult and pediatric
patients infected with variola virus [27].
The mechanism of action of tecovirimat is related to the for-
mation of enveloped forms of orthopoxviruses. It prevents the
wrapping of virion and so dramatically decreases the rate of
spreading of viruses in the tissue. The protein, that is believed to be
affected by tecovirimat, is a relatively small globular peripheral
membrane protein weighing 37 kDa (p37) that plays a central role
in the envelopment viral particles and is coded by a highly
conserved F13L gene [28] and has no mammalian homologs. The
viruses bearing artificially mutated variant of this gene were up to
10000 times less sensitive to tecovirimat comparing to wild type
strain [29]. Purified p37 shows lipase activity [30,31], however the
actual role of lipase activity in p37 function is unclear and may
involve p37-induced membrane alterations [31,32]. Several in-
vestigations evidence that p37 interacts with host cellular mem-
brane proteins Rab9 and TIP47. The tecovirimat may disrupt such
interactions preventing the formation of intracellular mature virus
[33]. The interaction of tecovirimat with p37 protein in is under no
hesitance [34], however the exact mechanism of interruption of
reproduction cycle of orthopox viruses by ST-246 is still not
investigated.
Taken together, these results validate p37 as potential target for
2

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
protein reached equilibrium in 40 ns and the further calculation
was carried out to improve reliability of the model.
VdW volume of other available cage fragments is either smaller, for
example bicyclo[2.2.2]octane (101.5 Å3), or noticeably bigger, for
example bornane (138,8 Å3). At the same time adamantane and
tecovirimate cage have very similar VdW areas (131.7 Å2 and
124.0 Å2 respectively). Thus, the presence of adamantane moiety in
the structure of the ligand increases the probability of suitable
binding of ligand to p37. Therefore, we decided to construct a set of
virtual compounds, containing structural fragments using ACD Labs
Chem Sketch software pack [51].
The geometry of virtual structures was optimized using the
molecular mechanics method in the MMFF94 force field using the
Avogadro 1.1.1 molecular editor [52]. The docking of a set of
structures to the p37 protein cavity revealed 19 compounds that are
promising for further research and optimization by varying their
structure. The structures of ligands, which showed the best binding
energy for protein p37 and their average values (kcal/mol), calcu-
lated using Autodock Vina, are shown in Table 1.
The analysis of docking results was performed with Discovery
Studio software package [53] and showed interactions of ligands
with Phe25, Tyr150, Asn312, Lys314 and Phe307 for both tecovir-
imat and proposed ligands. Moreover all ligands are positioned in
the cavity of protein similarly: the lipophilic hydrocarbon cage of
the molecule directed towards Phe307, trifluoromethyl group
directed to Lys314 and Asn312 and carbonyl group directed to
Asp361.
However, it should be noted, that all chosen structures have
close calculated affinities that are rather moderate. The most likely
cause of this phenomenon is rather big volume of cavity that binds
the ligand. The spatial structure of the protein allows the ligands
with close steric properties to enter the binding site and interact
with amino acid sidechains that directed into the cavity. On the
other side, the binding site is not very deep, therefore ligands are
not encapsulated by protein and remain rather agile, what can
explain moderate biding energies. According to the results of mo-
lecular docking we decided to build a model of pharmacophore of
the present protein. The best binding modes of the most active
compounds were submitted to PharmGist [54,55] web service
which produced the spatial dislocation of several features. The
average throughout the whole set of ligands dislocation of molec-
ular features are shown in Fig. 6: the bulky hydrocarbon cage
(white) should be linked to aromatic ring (blue) by a linker, con-
taining H-bond donors and acceptors (magenta).
The last 40 ns (40e80 ns interval, 4000 snapshots with 10 ps
increment) of molecular dynamics trajectory was analysed with
Chimera [46] and all snapshots were clustered basing on the mass-
weighted RSMD of the heavy atoms (see Fig. 1). The representative
frameshots of 10 most populated clusters were chosen for virtual
screening in order to increase the reliability of molecular docking.
The reliability of every model vas validated by analyzing its ram-
achandran plot and determination of compatibility of 3D model
with its sequence by Verify 3D and PROCHECK service [47] (Fig. 2).
The distributions of
f (psi) and j (phi) of the amino acid resi-
dues obtained by ramachandran plots showed that 84.9% were in
most favoured regions, 10.7% in additional allowed region and 3.6%
in generously allowed region. Only 3 residues (0.9%) were in dis-
allowed region, which are Cys185, Ser207 and Ile195 which can be
caused by proximity of lipid layer to these residues as Cys186 and
Cys186 are palmytoilated and act as anchor residues. The quality of
the model was also qualified by superimposition the XRD structure
of phospholipase 1v0w with modelled protein. As seen on Fig. 3
almost all helices, sheets and binding sites are overlapping
among these structures.
The binding site of most phospholipases D, which are similar to
p37, is known to be situated within the HKD domains that is highly
conserved among all phospholipases [48]. As a result, these do-
mains catalyzes the hydrolysis of phospholipids. The prediction of
binding site with COFACTOR module of I-TASSER gave the most
probable binding site similar to the phospholipase 1v0r with the C-
score 0.29. The residues that form the site are Phe52, Leu118,
Cys120, Ser135, Asn312, Lys314, Asn329 and Asp331. To define
other possible pockets we conducted a blind docking of the known
smallpox replication inhibitor tecovirimat within the whole protein
using the AutoDock Vina software [49].
Five potential binding sites were found with the binding en-
ergies of tecovirimat ranged from ꢀ6.1 to ꢀ9.3 kcal/mol. However,
the best cavity was the same as predicted by COACH with a calcu-
lated binding energy to protein ꢀ9.3 kcal/mol, which is 1.5 kcal/mol
lower than the next rated cavity.
The validation of docking modes of tecovirimat was performed
by simulation of molecular dynamics of ST-246 and p37 complex
for 80 ns. The procedure of building the starting model and simu-
lation parameters was the same as in modelling the lone protein.
The analysis of RMSD shows that the complex retains for the whole
time of simulation and reaches equilibrium after 60 ns of simula-
tion (Fig. 4). The simulation of molecular dynamics for complexes of
p37 with ST-246 in other cavities but all of them were unstable and
dissociate at time interval 10e20 ns (see Fig. 5).
2.2. Virtual screening of potential p37 inhibitors
In order to find new potential inhibitors of poxvirus replication,
we constructed a number of compounds containing adamantane
pharmacophore residue and a fragment of 4-substituted benzoic
acid that have structural similarity with the known active com-
pounds d tecovirimat. The selection of p-bromo and p-trifluoro
substituents is governed by the empiric dependencies of antiviral
activity from electronegativity of substituent [50]. The results of the
research showed that electron donating groups at 4- position as
well as electron withdrawing groups at 2- position increases the
EC50 of compound, whereas electron withdrawing groups at 3- and
4- positions decreases EC50. The most active compounds contained
4-Br and 4-CF3 substituted benzene ring. Adamantane cage was
chosen because of its spatial similarity to tecovirimat cage frag-
ment. While the Van de Waals volume of adamantane (124,5 Å3) is
slightly bigger than tecovirimat cage fragment (109,79 Å3), the
Fig. 1. RMSD of p37-POPC bilayer complex during production stage of molecular
dynamics.
3

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
Fig. 2. Ramachandran plot and results of Verify_3D analysis of p37 model.
29 by reaction with hydrazine hydrate and the subsequent acyla-
tion of the compounds obtained with p-trifluoromethylbenzoyl
chloride. The corresponding benzohydrazides 27, 28, 32 were ob-
tained by reduction of hydrazones 25, 26, 31 with sodium cyano-
borohydride in methanol (Scheme 3).
The corresponding benzhydrazides 35, 36 were obtained from
1-adamantylhydrazines 33, 34 and p-trifluoromethylbenzoyl
chloride (Scheme 4).
The data of the extensive testing of compounds showed that the
activity spectrum of most compounds extends to a wide range of
orthopoxviruses, pathogenic for humans. At the same time, various
representatives of orthopoxviruses differ in sensitivity to the same
drug. One can trace the tendency that the cowpox virus has the
least sensitivity to the antiviral compounds studied than the
vaccinia virus (Table 1).
Basing on the results obtained, it can be assumed that the tri-
fluoromethylbenzamides 5, 8 and 12 are the most promising for
further in vitro and in vivo studies. The effective concentration of N-
(1-adamantyl)-4-trifluoromethylbenzamide 5 against vaccinia vi-
rus is 0.133 mM, while the cytotoxic activity is more than 309 mM
therefore the value of the selectivity index (CC50/IC50 ratio) of 5 is
more than 2325. Also, the compound 5 effectively suppresses the
Fig. 3. Superimposition of modelled p37 (light blue) and phospholipase 1v0w. The
binding site of both proteins is shown by yellow sphere.
reproduction of mousepox virus at a concentration of 0.492
(selectivity index is more than 628) and cowpox virus at a con-
centration of 0.928
M (SI ¼ 333).
Compound 8 effectively inhibit reproduction of vaccinia virus at
concentration of 0.27 M and selectivity index more than 1098. In
addition, benzamide 8 showed effectiveness against cowpox virus
at a concentration of 1.24 M (SI > 239). However, benzamide 8 did
not show a virus inhibitory effect on the mousepox virus.
Trifluoromethylbenzamide 12 showed effectiveness against
vaccinia, mousepox and cowpox viruses. The effective concentra-
mM
2.3. Organic synthesis
m
Twenty compounds with the best binding were synthesized for
determination their antiviral activity. The N-adamantylated aryl-
carboxamides 5-9, 11, 12 were obtained by acylation of the amines
of the adamantane series 1-4, 10 with benzoyl chlorides, containing
electron-withdrawing substituents in the para-position (Scheme
1). The corresponding benzhydrazides 16-18 were synthesized by
the reaction of adamantanecarboxylic acids hydrazides 13e15 with
p-trifluoromethylbenzoyl chloride (Scheme 2). Reaction of 1-
adamantylsemicarbazide 19 with p-trifluoromethylbenzoyl chlo-
ride gave N-(adamantan-1-yl)-2-(4-(trifluoromethyl)benzoyl)
hydrazin-1-carboxamide 20.
m
m
tion of 12 against vaccinia virus is 0.216
activity is more than 309 M and the selectivity index is 1428.
Compound 12 inhibits reproduction of mousepox virus at a con-
centration of 0.569 M (SI > 543) and cowpox virus at a concen-
tration of 0.960 M (SI > 321).
mM, while the cytotoxic
m
m
m
Hydrazones of the adamantane series 25, 26, 31 were synthe-
sized from the corresponding ketones d 2-adamantanone 21, 5-
chloro-2-adamantanone 22 and 1-(adamantan-1-yl)ethan-1-one
According to the data acquired it should be noted that the
presence of alkyl groups at adamantyl cage or at side chain leads to
recession of antiviral activity and elevation of cytotoxicity. The
4

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
Fig. 4. RMSD of p37 e ST-246 e POPC complex during production stage of molecular dynamics (left) and the view of the complex (right). The surface of the protein is colored
according to residues hydrophilicity from brown (hydrophobic) to blue (hydrophilic), ST-246 is represented by spacefil model.
replacement of trifluoromethyl group by bromine also leads to
decrease in inhibitory activity.
used for further development of new inhibitors of orthopoxvirus
reproduction by means of molecular docking. series of
A
It can be noticed, that despite all chosen compounds have close
computed binding energies, their evaluated biological activities
vary a lot. Whereas the best compound 5 is only 50 times less active
than the reference compound e tecovirimat, the worst is totally
inactive. The most probable reason of this phenomenon the
complexity of interaction of a compound with the whole cell
comparing to isolated protein. The other explanation of the
observed phenomena may be the interaction between p37 and
other membrane proteins or covalent interaction between five
membered imide ring and active site of p37 that cannot be easily
modelled by means of molecular dynamics. However, the proposed
procedure can still be useful. As far as the first stage of inhibition of
a target protein includes formation of simple complex, primarily by
van der Waals forces and hydrogen bonds, and consequent for-
mation of covalent bond may occur only on the next stage, the
application of molecular docking can distinguish the potential
binding molecules from those that can not achieve the binding site
of p37.
adamantane-containing compounds have been synthesized and
their antiviral activities against vaccinia, cowpox and mousepox
viruses were defined in vitro. Although the results of molecular
docking do not show good correlation with experimental data,
probably due to complex interactions between p37 and other viral
and host proteins or drug-like molecules with lipophilic cell
membranes in native conditions, it still allows to reveal compounds
with high potential activities in vitro. In the structure of poxvirus
replication inhibitors the three main structural fragments can be
distinguished - a lipophilic cage residue and a fragment of a ben-
zene linked together by a hydrophilic fragment.
The discovery of several compounds, which IC50 lies in sub-
micromolecular range, indicates that adamantane scaffold has
considerable potential for creating new effective antiorthopoxvirus
drug candidates and there is an opportunity of finding such com-
pounds among tecovirimat analogues with different hydrocarbon
cage type.
4. Experimental
3. Conclusion
4.1. Protein modelling and virtual docking
The initial protein sequence was obtained from the UniProt
In summary, using methods of virtual modelling, we have ac-
quired a model of p37 orthopoxvirus membrane protein that can be
Fig. 5. The residues of p37 interacting with 5 (left) and tecovirimat (right) acquired by molecular docking. Visualized with Discovery Studio.
5

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
Table 1
Cytotoxicity, antiviral activity against orthopoxviruses (vaccinia, cowpox and mousepox viruses) and calculated binding affinities of compounds.
Compounds
CC50 (
m
M)
IC50 (
m
M)/SI (CC50/IC50)
Binding affinity (Vina, kcal/mol)
Vaccinia
Cowpox
Mousepox
>309.3
>280
0.133/>2325
0.928/>333
0.492/>628
ꢀ8.1
5
6
4.1/>68
11.06/>25
32.9/>9
11.1/>25
6.17/>48
ꢀ8.4
ꢀ8.1
>296.4
4.85/>61
7
8
9
>296.4
238
0.27/>1098
8.3/28
1.24/>239
n/a
n/a
ꢀ8.3
ꢀ7.5
ꢀ7.2
1.75/136
4.9/>61
>299.2
3.06/>98
11.3/>26.5
11
>309
0.216/>1428
0.96/>321
0.569/>543
ꢀ7.9
12
16
>273
1.07/>255
30.9/>8.2
4.77/>57
1.0/>273
ꢀ8.8
ꢀ9.1
>253.7
n/t
n/t
17
47
12.53/3.75
n/a
n/a
ꢀ9.0
18
20
94.3
36.26/2.6
n/t
n/t
n/t
n/t
ꢀ9.1
ꢀ8.6
>297
198/>1.5
25
27
>296
2.8/>103
4.06/>72
2.5/>118
ꢀ8.4
ꢀ8.5
~214.6
3.52/61
10.36/20.7
7.8/27.5
28
31
>274.4
14
10.55/>26
0.515/27
n/a
11.9/>23
n/t
10.9/>25
n/t
ꢀ8.7
ꢀ8.8
ꢀ8.4
32
35
2.8
n/t
n/t
6

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
Table 1 (continued)
Compounds
CC50 (
m
M)
IC50 (
m
M)/SI (CC50/IC50)
Binding affinity (Vina, kcal/mol)
Vaccinia
1.66/1.5
Cowpox
n/t
Mousepox
2.5
n/t
ꢀ8.7
ꢀ8.4
36
37
>296.4
8.98/>33
15.6/>19
4.1/>72
20.5
n/a
n/t
n/t
ꢀ8.4
ꢀ8.9
38
Tecovirimat
>266
0.00266/>100000
0.00532/>50000
0.00806/>33000
Scheme 2. Synthesis of compounds 16e18, 20.
Fig. 6. The pharmacophore model of p37 inhibitors.
Scheme 3. Synthesis of compounds 25e28, 31, 32.
Scheme 1. Synthesis of compounds 5e9, 11, 12.
Scheme 4. Synthesis of compounds 35, 36.
database P20638 (F13_VACCC). The I-TASSER web service was used
to model the initial tertiary structure [38e40]. The obtained model
proceeded the stage of refinement by molecular dynamics simu-
lation at neutral pH. The initial model was achieved by CHARMM-
GUI web service input generator [41e43]. The protein was palmi-
toylated on Cys185 and Cys186 residues, integrated into POPC
bilayer and embedded into rectangular periodic boundary box with
walls at least 20 Å away from the sides of the protein. The box was
infilled with water molecules (TIP3P model) and some of them
were replaced by chlorine and potassium ions to ensure electric
7

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
neutrality and KCl concentration of 0.15 M. The resulting system
contained about 100,000 atoms.
hydrazinecarboxamide 19 (12 mmol) and triethylamine (15 mmol)
in chloroform (6 mL) at 10 ꢂC and the mixture was stirred for 2e3 h
at r.t. The resulting solution was evaporated, and the residue was
filtered off and washed with cold chloroform, dried and finally
recrystallized from ethanol to give (59e72%) the product.
N-(Adamantan-1-yl)-4-(trifluoromethyl)benzamide (5). Colorless
crystals; yield 72%; mp 159e160 ꢂC (lit. 160e161 ꢂC [62]). MS, m/z
(%): 323 (Mþ, 44), 304 (8), 280 (3), 266 (76), 230 (5), 173 (100), 145
(64).
The molecular dynamics simulations were performed by NAMD
2.11 package using NVIDIA CUDA acceleration on two NVIDIA
GeForce GTX 1080 GPUs [45]. The CHARMM36 force field was used
for protein [56,57]. The initial minimization included 10000 steps
of steepest descent following equilibrating step of 25 ps using
Langevin thermostat at 310 K and Langevin barostat at 1.013 kPa.
The integration step value was 2 fs in combination with rigid
Bonds/SHAKE algorithm. The production step included 40 ns of
molecular dynamics at 1.013 kPa and 310 K using Langevin barostat
and thermostat.
The analysis and visualization of the results was performed with
UCSF Chimera package [46]. The last 40 ns of production step were
clustered basing on cross-pair RMSD values of protein backbone
atoms. The resulting pattern contained 40 clusters, the represent-
ing frames from 10 the most populated of which were chosen for
next stage of virtual screening.
The library of potential ligand structures was prepared using the
ACD ChemSketch 2015 software [51] and their geometries were
optimized with molecular mechanics in the MMFF94 force field
using Avogadro 1.2.0 [52]. The PDB files were converted to the
PDBQT format using the AutoDock Raccoon 1.0 software [58].
The p37 protein models were prepared for molecular docking
using the AutoDockTools 1.5.6 software [59]. First the chosen
models were cleared from water and ions and the grid box was set
to cover the entire protein. The docking was performed with
exhaustiveness set to 1000 and number of modes to 50. The
resulting binding modes divided into several groups. The area
occupied by the group with the lowest binding energies was
considered as binding site. Next the grid box size of approximately
18 ꢁ 28 ꢁ 16 Å was specified in order to cover the entire binding
site. Molecular docking was performed using the AutoDock Vina
1.1.2 software [49] with exhaustiveness 1000 and number of modes
50. The analysis of molecular docking results was performed with
Discovery Studio Visualiser (free version) [53]. The best calculated
affinities were averaged through 10 frames and the best com-
pounds were chosen using energy cut-off of ꢀ7.2 kcal/mol.
N-(3-Ethyl
а
damantan-1-yl)-4-(trifluoromethyl)benzamide
(6).
Colorless crystals; yield 70%; mp 125e127 ꢂC. IR (nmax, cmꢀ1): 3356
(N-H), 2910, 1639 (C¼O), 1541 (N-H), 1307, 1157, 1126, 1068. 1H NMR
(DMSO‑d6),
d
, ppm: 0.74 (t, 3J ¼ 7.6 Hz, 3H), 1.10 (q, 3J ¼ 7.6 Hz, 2H),
1.34e2.06 (m, 14Н), 7.75 (d, 3J ¼ 8.2 Hz, 2H), 7.85 (br. s, 1Н), 7.92 (d,
3J ¼ 8.2 Hz, 2H). 13C NMR (DMSO‑d6),
d, ppm: 7.51, 29.60, 34.45,
36.11, 36.19, 40.88, 40.97, 45.57, 53.16, 124.53 (q, 1JCF ¼ 270.8 Hz),
3
2
125.52 (q, JCF ¼ 3.8 Hz), 128.79, 131.19 (q, JCF ¼ 31.5 Hz), 140.24,
165.46. MS, m/z (%): 351 (Mþ, 26), 322 (8), 294 (68), 266 (12), 173
(100), 145 (52). EA calcd. for C20H24F3NO: C 68.36, H 6.88, N 3.99;
found: C 68.32, H 6.93, N 4.07.
N-(Adamantan-1-yl)-N-methyl-4-(trifluoromethyl)benzamide (7).
Light yellow crystals; yield 61%; mp 156e158 ꢂC. IR (nmax
/
cmꢀ1) ¼ 2910, 1693 (C¼O), 1622, 1514, 1319, 1120, 1101, 1062. 1H
NMR (DMSO‑d6), d, ppm: 1.58e2.26 (m, 15Н), 2.71 (s, 3Н), 7.84 (d,
3J ¼ 8.2 Hz, 2H), 8.10 (d, 3J ¼ 8.2 Hz, 2H). 13C NMR (DMSO‑d6),
d,
ppm: 29.83, 34.33, 36.51, 38.87, 57.78, 124.48 (q, 1JCF ¼ 271.4 Hz),
3
2
126.15 (q, JCF ¼ 3.8 Hz), 130.65, 132.98 (q, JCF ¼ 32.4 Hz), 135.17,
166.75. MS, m/z (%): 337 (Mþ, 97) 318 (7), 280 (100), 186 (10), 173
(70), 145 (44), 135 (70), 107 (6), 93 (12), 79 (12). EA calcd. for
C
19H22F3NO: C 67.64, H 6.57, N 4.15; found: C 67.59, H 6.65, N 4.11.
N-((Adamantan-1-yl)methyl)-4-(trifluoromethyl)benzamide (8).
Colorless crystals; yield 65%; mp 151e152 ꢂC. IR (nmax, cmꢀ1) ¼ 3356
(N-H), 2899, 1647 (C¼O), 1543 (N-H), 1332, 1165, 1124, 1068. 1H
NMR (DMSO‑d6), d, ppm: 1.33e1.69 (m, 12Н), 1.85e1.98 (m, 3Н),
2.97 (d, 3J ¼ 6.2 Hz, 2H), 7.79 (d, 3J ¼ 8.0 Hz, 2H), 8.00 (d, 3J ¼ 8.0 Hz,
2H), 8.46 (t,3J ¼ 6.2 Hz, 1H). 13C NMR (CDCl3),
d, ppm: 28.25, 34.14,
270.8 Hz), 125.74 (q,
1
36.95, 40.41, 51.61, 123.76 (q, JCF
¼
3JCF ¼ 3.8 Hz), 127.43, 133.14 (q, 2JCF ¼ 32.6 Hz), 138.40, 166.59. MS,
m/z (%): 337 (Mþ, 22), 318 (2), 173 (12), 145 (10), 135 (100), 107 (6),
93 (10), 79 (10). EA calcd. for C19H22F3NO: C 67.64, H 6.57, N 4.15;
found: C 67.61, H 6.63, N 4.12.
4.2. Chemistry
4.2.1. General information
N-((Adamantan-1-yl)methyl)-4-bromobenzamide (9). Colorless
crystals; yield 64%; mp 190e192 ꢂC. IR (nmax, cmꢀ1) ¼ 3269 (N-H),
2902, 1631 (C¼O), 1589, 1548, 1309, 1068, 1010. 1H NMR (DMSO‑d6),
Starting materials and solvents were purchased from commer-
cial suppliers and were used without further purification. The
infrared spectra were recorded in potassium bromide disks on
Shimadzu IRAfinity-1 FT IR spectrometer. The 1H and 13C NMR
spectra (400 and 100 MHz, respectively) were recorded on JEOL-
ECX 400 spectrometer using the residual signal of deuterated sol-
vent as internal standard. Chemical shifts are given in ppm; mul-
tiplicities are described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m) and broad (br); coupling constants are given in
Hertz (Hz). Mass spectra were recorded on a GC-mass Thermo-
Finnigan DSQ spectrometer (70 eV), a quartz column DB-5MS
30 m ꢁ 0.32 mm, column temperature 80e340 ꢂC (20 ꢂC/min).
Elemental analyses were carried out on CHNS-EuroVector EA-
d
, ppm: 1.45e1.82 (m, 12Н), 1.85e1.98 (m, 3Н), 2.97 (d, 3J ¼ 6.2 Hz,
2H), 7.59 (d, 3J ¼ 8.0 Hz, 2H), 7.75 (d, 3J ¼ 8.0 Hz, 2H), 7.95 (br. s, 1H).
13C NMR (DMSO‑d6),
d, ppm: 28.49, 34.78, 37.22, 40.78, 51.38,
124.95,129.89,131.62,134.96,166.49. MS, m/z (%): 347 (Mþ, 20), 212
(1), 183 (8), 157 (6), 135 (100), 107 (6), 93 (11), 79 (11). EA calcd. for
C
18H22BrNO: C 62.08, H 6.37, N 4.02; found: C 61.93, H 6.42, N 4.09.
N-(Adamantan-2-yl)-4-bromobenzamide (11). Colorless crystals;
yield 72%; mp 181e182 ꢂC. IR (nmax, cmꢀ1) ¼ 3348 (N-H), 2904, 1633
(C¼O), 1537 (N-H), 1321, 1105, 1068, 1012. 1H NMR (DMSO‑d6),
d,
ppm: 1.40e2.15 (m, 14Н), 3.92e4.05 (m, 1H), 7.61 (d, 3J ¼ 8.5 Hz,
2H), 7.75 (d, 3J ¼ 8.5 Hz, 2H), 7.96 (d, 3J ¼ 6.4 Hz, 1H). 13C NMR
3000 EA analyzer with
L-cystine as a standard. Melting points were
(DMSO‑d6), d, ppm: 27.33, 31.61, 31.68, 37.42, 37.69, 54.77, 125.06,
determined on the SRS OptiMelt MPA100 apparatus.
Compounds 33, 34, 37, 38 were synthesized according to known
methods [60,61].
130.35, 131.53, 134.81, 166.16. MS, m/z (%): 333 (Mþ, 100), 254 (5),
200 (7), 183 (55), 155 (24), 134 (44), 119 (14), 91 (20), 79 (16). EA
calcd. for C17H20BrNO: C 61.09, H 6.03, N 4.19; found: C 61.13, H 6.10,
N 4.14.
4.2.2. General procedure for the synthesis of compounds 5e9, 11, 12,
16e18, 20
A solution of 4-X-benzoyl chloride (X ¼ Br, CF3) (12 mmol) in
chloroform (5 mL) was added dropwise to a mixture of amino de-
rivative of adamantane 1-4 or carboxylic acid hydrazide 13-15 or
N-(Adamantan-2-yl)-4-(trifluoromethyl)benzamide (12). Color-
less crystals; yield 68%; mp 183e185 ꢂC. IR (nmax, cmꢀ1) ¼ 3334 (N-
H), 2904, 1635 (C¼O), 1533 (N-H), 1321, 1163, 1105, 1064, 1016. 1H
NMR (DMSO‑d6), d, ppm: 1.39e2.12 (m, 14Н), 4.00 (m, 1H), 7.78 (d,
3J ¼ 8.2 Hz, 2H), 7.96 (d, 3J ¼ 8.2 Hz, 2H), 8.17 (d, 6.6 Hz, 1H). 13
C
8

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
NMR (DMSO‑d6),
d
, ppm: 27.32, 31.61, 31.68, 37.41, 37.69, 54.89,
cmꢀ1) ¼ 3150 (N-H), 2908, 1657 (C¼N), 1649 (C¼O), 1631, 1539 (N-
124.54 (q, 1JCF ¼ 270.8 Hz), 125.53 (q, JCF ¼ 3.8 Hz), 129.09, 131.31
(q, 2JCF ¼ 31.4 Hz), 139.68, 166.15. MS, m/z (%): 323 (Mþ, 55), 304 (7),
280 (2), 266 (2), 207 (7), 173 (100), 145 (71). EA calcd. for
H), 1321, 1168, 1105, 1064, 1018. 1H NMR (DMSO‑d6),
d, ppm:
3
1.64e2.05 (m, 12Н), 2.52e2.66 (m, 1H), 3.15e3.23 (m, 1H), 7.80 (d,
3J ¼ 8.1 Hz, 2H), 7.96 (d, 3J ¼ 8.1 Hz, 2H), 10.78 (s, 1H). 13C NMR
C
18H20F3NO: C 66.86, H 6.23, N 4.33; found: C 66.81, H 6.30, N 4.28.
(DMSO‑d6), d, ppm: 27.68, 32.3, 36.31, 37.85, 39.19, 39.61, 124.49 (q,
N1-(4-(Trifluoromethyl)benzoyl)adamantane-1-carbohydrazide
1JCF ¼ 272.2 Hz), 125.68 (q, JCF ¼ 3.6 Hz), 129.14, 131.51 (q,
2JCF ¼ 32.6 Hz), 138.66, 163.08, 173.19. MS, m/z (%): 336 (65) [M]þ,
307 (10), 265 (12),190 (62),173 (100),145 (46),163 (36),137 (10), 91
(22), 79 (21). EA calcd. for C18H19F3N2O: C 64.28, H 5.69, N 8.33;
found: C 64.23, H 5.77, N 8.27.
3
(16). Colorless crystals; yield 63%; mp 227e229 ꢂC; IR (nmax, cmꢀ1):
3336 (N-H), 3184 (N-H), 2908, 1631(C¼O), 1548, 1514, 1319, 1165,
1128, 1060. 1H NMR (DMSO‑d6),
d, ppm: 1.58e1.89 (m, 12Н),
1.93e2.09 (m, 3H), 7.84 (d, 3J ¼ 8.2 Hz, 2H), 8.03 (d, 3J ¼ 8.2 Hz, 2H),
9.49 (s, 1H), 10.39 (s, 1H). 13C NMR (DMSO‑d6),
d
, ppm: 28.07, 36.60,
N1-(1-(Adamantan-1-yl)ethylidene)-4-(trifluoromethyl)benzohy-
1
3
39.05, 40.09, 124.41 (q, JCF ¼ 270.7 Hz), 126.09 (q, JCF ¼ 3.8 Hz),
128.81, 132.05 (q, 2JCF ¼ 32.4 Hz), 137.05, 164.94, 176.81. MS, m/z (%):
366 (Mþ, 2), 347 (2), 173 (9), 163 (12), 135 (100). EA calcd. for
drazide (31). Colorless crystals; yield 69%; m.p. 213e215 ꢂC. IR (nmax
,
cmꢀ1) ¼ 3280 (NH), 2904, 1659 (C¼N), 1651(C¼O), 1541 (N-H),
1325, 1128, 1103, 1066. 1H NMR (DMSO- d6),
d, ppm: 1.45e2.10 (m,
C
19H21F3N2O2: C 62.29, H 5.78, N 7.65; found: C 62.23, H 5.84, N
15Н), 1.86 (s, 3H), 7.81 (d, 3J ¼ 7.8 Hz, 2H), 7.97 (d, 3J ¼ 7.8 Hz, 2H),
7.61.
10.49 (s, 1H). 13C NMR (DMSO‑d6),
d
, ppm: 27.73, 30.81, 31.45, 37.12,
3,5-Dimethyl-N1-(4-(trifluoromethyl)benzoyl)adamantane-1-
37.93, 124.46 (q, JCF ¼ 271.7 Hz), 125.84 (q,3JCF ¼ 3.8 Hz), 128.60,
1
2
carbohydrazide (17). Colorless crystals; yield 66%; mp 183e184 ꢂC.
IR (nmax, cmꢀ1): 3244 (N-H), 2897, 1683 (C¼O), 1651 (C¼O), 1546
(NH), 1504 (NH), 1325, 1168, 1130, 1064, 1018. 1H NMR (DMSO- d6),
131.62 (q, JCF ¼ 32,4 Hz), 137.55, 160.46, 164.77. MS, m/z (%): 364
(Mþ, 1) 345 (2), 229 (100), 173 (13), 145 (10), 135 (3), 91 (5), 79 (5).
EA calcd. for C20H23F3N2O: C 65.92, H 6.36, N 7.69; found: C 65.87, H
6.41, N 7.65.
d
, ppm: 0.80 (s, 6H), 1.09e2.10 (m, 13Н), 7.84 (d, 3J ¼ 8.3 Hz, 2Н),
8.02 (d, 3J ¼ 8.3 Hz, 2Н), 9.57 (br. s, 1H), 10.36 (br. s, 1H). 13C NMR
(DMSO‑d6),
d
, ppm: 29.29, 30.96, 31.19, 37.67, 41.99, 42.86, 45.27,
4.2.4. General procedure for the synthesis of compounds 27, 28, 32
To the solution of hydrazine (25, 26, 31) (12 mmol) in 20 mL of
methanol 3 mL of glacial acetic acid were added followed by
addition of sodium cyanoborohydride (10 mmol). The reaction
mixture was then stirred at rt for 24 h. Aqueous NaOH solution
(15 mL, 10%) was then added and most of the CH3OH was removed
on a rotary evaporator. The reaction mixture was diluted with water
(30 mL) and the product was extracted with CH2Cl2 (3 ꢁ 10 ml). The
organic extract was dried with sodium sulfate and evaporated to
yield hydrazide (27, 28, 32).
50.76, 124.40 (q, 1JCF ¼ 271.7 Hz), 126.04 (q, JCF ¼ 3.8 Hz), 128.81,
3
2
132.02 (q, JCF ¼ 32.4 Hz), 137.09, 164.87, 176.53. MS, m/z (%): 394
(Mþ,1) 376 (2),191 (4),173 (7),163 (100),145 (6),107 (22), 91 (4), 79
(3). EA calcd. for C21H25F3N2O2: C 63.95, H 6.39, N 7.10; found: C
63.86, H 6.42, N 7.03.
3-Ethyl-N1-(4-(trifluoromethyl)benzoyl)adamantane-1-
carbohydrazide (18). Colorless crystals; yield 59%; mp 198e200 ꢂC.
IR (nmax, cmꢀ1) ¼ 3350 (N-H), 3190 (N-H), 2900, 1691 (C¼O), 1647
(C¼O), 1583, 1516, 1317, 1165, 1107, 1064, 1016. 1H NMR (DMSO‑d6),
d
, ppm: 0.75 (t, 3J ¼ 7.5 Hz, 3Н), 1.10 (q, 3J ¼ 7.5 Hz, 2H), 1.31e2.04
N1-(Adamantan-2-yl)-4-(trifluoromethyl)benzohydrazide
Colorless crystals; yield 83%; mp 218e220 ꢂC. IR
(27).
nmax
(m,14Н), 7.63 (d, 3J ¼ 8.0 Hz, 2H), 8.04 (d, 3J ¼ 8.0 Hz, 2H), 9.94 (br. s,
(
,
2Н). 13C NMR (DMSO‑d6),
d
, ppm: 7.38, 28.64, 32.81, 36.33, 36.37,
cmꢀ1) ¼ 3296 (NH), 2900, 1633 (C¼O), 1533, 1325, 1161, 1128, 1066.
1
39.15, 40.79, 41.07, 43.89, 124.92 (q, JCF ¼ 270.7 Hz), 125.00 (q,
1H NMR (DMSO‑d6),
d, ppm: 1.32e2.26 (m, 14Н), 2.99e3.04 (m, 1H),
2
JCF ¼ 3.8 Hz), 128.21, 129.47 (q, JCF ¼ 32.4 Hz), 141.89, 163.58,
5.03 (br. s, 1Н), 7.77 (d, 3J ¼ 8.0 Hz, 2H), 7.99 (d, 3J ¼ 8.0 Hz, 2H), 9.99
172.49. MS, m/z (%): 394 (Mþ,1), 376 (3), 347 (4),191 (3),173 (8),163
(100), 145 (6), 107 (8), 93 (8), 79 (7). EA calcd. for C21H25F3N2O2: C
63.95, H 6.39, N 7.10; found: C 63.91, H 6.44, N 7.06.
(br. s, 1H). 13C NMR (DMSO‑d6),
d
, ppm: 27.84, 31.04, 31.56, 37.29,
1
3
38.02, 63.74, 124.48 (q, JCF ¼ 270.8 Hz), 125.70 (q, JCF ¼ 3.8 Hz),
2
128.56, 131.73 (q, JCF ¼ 31.5 Hz), 137.77, 164.81. MS, m/z (%): 338
N1-(Adamantan-1-yl)-2-(4-(trifluoromethyl)benzoyl)hydrazine-
1-carboxamide (20). Colorless crystals; yield 61%; mp 173e175 ꢂC. IR
(Mþ, 4), 319 (4), 190 (35), 173 (28), 149 (100), 135 (64), 107 (11), 93
(21), 79 (20). EA calcd. for C18H21F3N2O: C 63.89, H 6.26, N 8.28;
found: C 63.83, H 6.32, N 8.24.
(
nmax, cmꢀ1) ¼ 3307 (N-H), 2906, 1687, 1641 (C¼O), 1552, 1323,
1165, 1128, 1064, 1018. 1H NMR (DMSO‑d6),
d, ppm: 1.46e1.92 (m,
N1-(5-Chloroadamantan-2-yl)-4-(trifluoromethyl)benzohydrazide
(28). Colorless crystals; yield 67%; mp 141e142 ꢂC; E:Z ¼ 2:1 (GC-
MS). IR (nmax, cmꢀ1) ¼ 3298 (NH), 2918, 1633 (C¼O), 1537 (NH),
12H), 1.93e2.05 (m, 3H), 5.99 (s, 1H), 7.69 (s, 1H)), 7.83 (d,
3J ¼ 8.2 Hz, 2H), 8.01 (d, 3J ¼ 8.2 Hz, 2H), 10.29 (s, 1H). 13C NMR
(DMSO‑d6),
d
,
ppm: 29.43, 36.54, 42.26, 50.48, 124.41 (q,
1321, 1163, 1130, 1066; 1H NMR (DMSO‑d6),
d, ppm: 1.29e3.10 (m,
1JCF ¼ 271,7 Hz), 125.94 (q, JCF ¼ 3.8 Hz), 128.85, 131.95 (q,
2JCF ¼ 32.4 Hz), 137.09, 157.05, 165.48. MS, m/z (%): 381 (Mþ, 1), 230
(2), 204 (96), 173 (86), 145 (28), 135 (100), 93 (24), 79 (23). EA calcd.
for C19H22F3N3O2: C 59.83, H 5.81, N 11.02; found: C 59.78, H 5.89, N
10.97.
28Н, E þ Z), 5.11 (d d, 3J ¼ 3.0 Hz, 3J ¼ 5.7 Hz) and 5.14 (d d,
3J ¼ 3.7 Hz, 3J ¼ 6.0 Hz) (2H, E þ Z), 7.78 (d, 3J ¼ 8.2 Hz, 4H, E þ Z),
7.99 (d, 3J ¼ 8.2 Hz, 4H, E þ Z), 10.08 (d, 3J ¼ 5.7 Hz) and 10.11 (d,
3
3J ¼ 6.0 Hz) (2H, E þ Z). 13C NMR (DMSO‑d6),
d, ppm: 29.37, 31.29,
33.94, 34.39, 34.65, 41.67, 46.81, 48.03, 48.11, 61.39, 60.69, 69.72,
70.43, 124.48 (q, 1JCF ¼ 276.1 Hz), 125.90 (q, JCF ¼ 3.8 Hz), 128.65,
3
2
4.2.3. General procedure for the synthesis of compounds 25, 26, 31
To the solution of hydrazone (23, 24, 30) (10 mmol) and trie-
thylamine (12 mmol) in 10 ml of methylene chloride a solution of p-
trifluoromethylbenzoic acid chloride (10 mmol) in 10 ml of meth-
ylene chloride was added dropwise under stirring at 10 ꢂC. The
reaction mixture was stirred for 3 h at rt. The solvent was evapo-
rated, the precipitate was filtered off, dried and recrystallized from
ethanol to give hydrazones (25, 26, 31) in a yield of 68e69%. The
hydrazone (26) was subjected to the reduction step without
purification.
131.68 (q, JCF ¼ 31.5 Hz), 137.52, 165.06, 165.31. MS, m/z (%): 372
(Mþ, 1), 353 (1), 190 (100), 173 (34), 145 (17), 133 (17), 91 (20), 79
(12). EA calcd. for C18H20ClF3N2O: C 57.99, H 5.41, N 7.51; found: C
57.91, H 5.48, N 7.47.
(rac)-N1-(1-(Adamantan-1-yl)ethyl)-4-(trifluoromethyl)benzohy-
drazide (32). Colorless crystals; yield 80%; mp 217e219 ꢂC. IR (nmax
,
cmꢀ1) ¼ 3189 (N-H), 2910, 1678 (C¼O), 1570, 1541, 1326, 1166, 1126,
1066, 1014. 1H NMR (DMSO‑d6),
d
, ppm: 1.11(d, 3J ¼ 6.9 Hz, 3H),
1.47e1.81 (m, 12H), 1.89e2.05 (m, 3H), 2.86 (q,3J ¼ 6.9 Hz, 1H), 6.7
(br. s, 1H), 7.87 (d, 3J ¼ 8.3 Hz, 2H), 8.09 (d, 3J ¼ 8.3 Hz, 2H), 11.69 (br.
N1-(Adamantan-2-ylidene)-4-(trifluoromethyl)benzohydrazide
(25). Colorless crystals; yield 68%; m.p. 225e227 ꢂC. IR (nmax
s, 1H). 13C NMR (DMSO‑d6),
d, ppm: 11.36, 28.17, 35.41, 36.85, 38.08,
3
,
67.28, 124.33 (q, 1JCF ¼ 270.8 Hz), 126.08 (q, JCF ¼ 3.8 Hz), 129.24,
9

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
132.53 (q, 2JCF ¼ 31.4 Hz), 135.68, 164.63. MS, m/z (%): 366 (Mþ, 2),
231 (100), 190 (8), 173 (20), 145 (10), 135 (7), 91 (6), 79 (7). EA calcd.
for C20H25F3N2O: C 65.56, H 6.88, N 7.65; found: C 65.45, H 6.96, N
7.59.
according to the method described by the authors [64], was used as
a reference compound.
Evaluation of antiviral efficacy of the compounds was carried
out according to an adapted and modified method [65]. To assess
the effectiveness of each sample 8 three-fold dilutions were used.
4.2.5. General procedure for the synthesis of compounds 35, 36
To a solution of 0.70 g adamant-1-ylhydrazine dihydrochloride
(33) or 3-ethyladamant-1-ylhydrazine dihydrochloride (34)
(3 mmol) and 0.42 g of sodium hydroxide(10 mmol) in 10 mL of
water a solution of 0.6 g (3 mmol) p-trifluoromethylbenzoyl chlo-
ride in 20 mL of ether was added under stirring at rt during 5 min
and the stirring continued for 1 h. The ether layer was separated,
washed with 10 ml of water, dried with sodium sulfate and evap-
orated. The residue was then recrystallized from the mixture of
hexane and toluene (1:1) to give the product.
The initial concentration of each compound was 1, 10 or 100 mg/ml,
depending on the preliminary screening of antiviral activity and
toxicity of the drug. For evaluating the antiviral activity firstly to the
wells of 96-well plates with a monolayer of cells containing 100
of DMEM medium with 2% fetal serum of a 50- l dilution of the
samples were added, and then 50 l of a 1000 pfu/well virus dose
ml
m
m
was added. Evaluation of antiviral activity was performed after 4
days. After incubation of the cell monolayers infected with ortho-
poxvirus and treated with tested compounds for 4 days, a vital red
dye “neutral red” was added to the culture medium for 1.5 h. Next,
the monolayer was washed twice with saline solution, the lysis
buffer was added and after 30 min the optical density (OD), which
is an indicator of the number of cells in the monolayer that is not
destroyed under the influence of virus, was measured on an Emax
plate reader (Molecular Devices, USA). The OD values were used to
N1-(Adamantan-1-yl)-4-(trifluoromethyl)benzohydrazide
Colorless crystals; yield 72%; mp 150e152 ꢂC. IR
(35).
nmax
(
,
cmꢀ1) ¼ 3199 (N-H), 3099 (N-H), 2912, 1658 (C¼O), 1585 (N-H),
1311, 1165, 1124, 1060. 1H NMR (DMSO‑d6),
d, ppm: 1.45e1.68 (m,
12Н), 1.95e2.05 (m, 3Н), 4.91 (s, 1H), 7.81 (d, 3J ¼ 8.2 Hz, 2H), 7.99
(d, 3J ¼ 8.2 Hz, 2H), 9.99 (s, 1H). 13C NMR (DMSO‑d6),
d
, ppm: 29.27,
calculate a 50% cytotoxic concentration (CC50
mM) and a 50% virus
1
36.64, 41.23, 55.25, 124.47(q, JCF
¼
270.7 Hz), 125.85 (q,
inhibiting concentration (IC50 M) using the SoftMax Pro-4.0
m
3JCF ¼ 3.8 Hz), 128.69, 131.58 (q, 2JCF ¼ 31.8 Hz), 137.43, 165.09. MS,
m/z (%): 338 (Mþ, 11), 173 (10), 145 (7), 135 (100), 107 (7), 93 (14), 79
(13). EA calcd. for C18H21F3N2O: C 63.89, H 6.26, N 8.28; found: C
63.81, H 6.32, N 8.22.
computer program. Based on these indicators, the therapeutic in-
dex (TI) (or selectivity index - SI) was calculated: SI ¼ CC50/IC50. The
results of experimental evaluation of antiorthopox activity are
represented in Table 1.
N1-(3-Ethyladamantan-1-yl)-4-(trifluoromethyl)benzohydrazide
(36). Colorless crystals; yield 74%; mp 133e135 ꢂC. IR (nmax, cmꢀ1):
3253 (N-H), 2905, 1666 (C¼O), 1581, 1537 (N-H), 1317, 1165, 1124,
Declaration of competing interest
1062, 1014. 1H NMR (DMSO‑d6),
d
, ppm: 0.72 (t, 3J ¼ 7.3 Hz, 3Н), 1.08
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.
(q, 3J ¼ 7.3 Hz, 2H),1.21e1.61 (m,12Н),1.99e2.09 (m, 2H), 5.05 (br. s,
1Н), 7.80 (d, 3J ¼ 8.3 Hz, 2H), 7.99 (d, 3J ¼ 8.3 Hz, 2H), 9.99 (br. s, 1Н).
13C NMR (DMSO‑d6),
d, ppm: 7.56, 29.52, 34.45, 36.08, 36.28, 40.87,
1
41.06, 45.53, 56.22, 124.46 (q, JCF
¼
270.7 Hz), 125.85 (q,
Acknowledgement
3JCF ¼ 3.8 Hz), 128.69, 131.61 (q, JCF ¼ 31.8 Hz), 137.41, 165.09. MS,
m/z (%): 366 (Mþ, 9), 163 (100), 145 (7), 135 (3), 107 (13), 93 (14), 79
(10). EA calcd. for C20H25F3N2O: C 65.56, H 6.88, N 7.65; found: C
65.49, H 6.95, N 7.63.
2
The authors gratefully acknowledge the support of this work by
the Russian Science Foundation (project 20-73-00254) for syn-
thesis of compounds and Russian Ministry of Education and Science
for a basic part of a research project 0778-2020-0005 for investi-
gation of properties of synthesized compounds with the use of
scientific equipment of the Center for Collective Use “Investigation
of the Physicochemical properties of Substances and Materials”.
4.3. Poxvirus inhibitory activity evaluation
Antiviral activity of compounds 5e9,11,12,16e18, 20, 25, 27, 28,
31, 32, 35e38 against orthopoxviruses was determined in vitro in
Vero cell culture. Vero cell monolayer was grown in DMEM (BioloT,
Russia) in the presence of 5% fetal bovine serum (HyClone, USA)
with the addition of gentamicin sulfate (40 units/ml) and ampho-
tericin B (2.5 units/ml) The same medium was used as a support for
the cultivation of cells with the virus, but with the addition of 2%
serum.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113485.
References
Vaccinia virus (strain Copenhagen), cowpox virus (strain
Grishak), mousepox virus - ectromelia (strain K-1), obtained from
the State collection of pathogens of viral infections and rickettsioses
of SRC VB Vector (Koltsovo, Novosibirsk region, Russia) were used
in the work. Viruses were produced in Vero cell culture in DMEM
medium (BioloT, Russia). The concentration of viruses in the culture
fluid was determined by the method of plaques by titration of
samples in a culture of Vero cells, calculated and expressed in
decimal logarithms of plaque-forming units per ml (lg PFU/ml) [63].
The virus concentration in the samples used in the work was
6.85 0.50 lg PFU/ml. The accumulated and used in work series of
viruses with the indicated titer were stored at ꢀ70ꢂС.
ꢀ
[1] N. Sklenovska, M. Van Ranst, Emergence of monkeypox as the most important
orthopoxvirus infection in humans, Front Public Health 6 (2018) 241, https://
doi.org/10.3389/fpubh.2018.00241.
[2] D. DiEuliis, K. Berger, G. Gronvall, Biosecurity implications for the synthesis of
horsepox, an orthopoxvirus, Health Security (5) (2017) 629e637, https://
doi.org/10.1089/hs.2017.0081, 016.
[3] S.N. Shchelkunov, An increasing danger of zoonotic orthopoxvirus infections,
PLoS Pathog.
e1003756.
9
(12) (2013), https://doi.org/10.1371/journal.ppat.1003756
ꢁ
[4] G. Bohelay, T.-A. Duong, Infections humaines a poxvirus, Ann. Dermatol.
Venereol.
146
(5)
(2019)
387e398,
https://doi.org/10.1016/
j.annder.2019.03.001.
[5] S. Sarker, C. Hannon, A. Athukorala, H. Bielefeldt-Ohmann, Emergence of a
novel pathogenic poxvirus infection in the endangered green sea turtle
(Chelonia mydas) highlights a key threatening process, Viruses 13 (2) (2021)
219, https://doi.org/10.3390/v13020219.
[6] R.C. Hendrickson, C. Wang, E.L. Hatcher, E.J. Lefkowitz, Orthopoxvirus genome
evolution: the role of gene loss, Viruses 2 (9) (2010) 1933e1967, https://
doi.org/10.3390/v2091933.
Compound 4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-
1,3-dioxo-4,6-ethenocycloprop(f)isoindol-2(1H)-yl)-benzamide
(tecovirimat), synthesized in N.N. Vorozhtsov Novosibirsk Institute
of Organic Chemistry, Siberian Branch of the Russian Academy
10

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
[7] G. Impelluso, F. Lentzos, The threat of synthetic smallpox: European per-
spectives, Health Security 16 (5) (2017) 582e586, https://doi.org/10.1089/
hs.2017.0045.
major membrane component of extracellular vaccinia virions, Virology 308
(2) (2003) 233e242, https://doi.org/10.1016/s0042-6822(03)00063-1.
€
[29] Duraffour S, Lorenzo MM, Zoller G, Topalis D, Grosenbach D, Hruby DE, Andrei
[8] R.S. Noyce, S. Lederman, D.H. Evans, Construction of an infectious horsepox
virus vaccine from chemically synthesized DNA fragments, PLoS 13 (1) (2018),
https://doi.org/10.1371/journal.pone.0188453 e0188453.
G, Blasco R, Meyer H, Snoeck R. ST-246 is a key antiviral to inhibit the viral
F13L phospholipase, one of the essential proteins for orthopoxvirus wrapping.
J Antimicrob Ther 70(5):1367-1380. doi: 10.1093/jac/dku545.
[9] B. Moss, Poxvirus DNA replication, Cold Spring Harb Perspect Biol 5 (9) (2013),
https://doi.org/10.1101/cshperspect.a010199 a010199.
[10] S.N. Shchelkunov, Orthopoxvirus genes that mediate disease virulence and
host tropism, Advanc Virol ID (2012), 524743, https://doi.org/10.1155/2012/
524743.
[30] S.H. Baek, J.Y. Kwak, S.H. Lee, T. Lee, S.H. Ryu, D.J. Uhlinger, J.D. Lambeth,
Lipase activities of p37, the major envelope protein of vaccinia virus, J. Biol.
Chem. 272 (1997) 32042e32049, https://doi.org/10.1074/jbc.272.51.32042.
[31] G. Hiller, H. Eibl, K. Weber, Characterization of intracellular and extracellular
vaccinia
virus
variants:
N1-isonicotinoyl-N2-3-methyl-4-
[11] G. Andrei, R. Snoeck, Cidofovir activity against poxvirus infections, Viruses 2
(12) (2010) 2803e2830, https://doi.org/10.3390/v2122803.
chlorobenzoylhydrazine interferes with cytoplasmic virus dissemination and
release, J. Virol. 39 (3) (1981) 903e913. https://jvi.asm.org/content/39/3/903.
long.
[12] A.S. Sokolova, K.S. Kovaleva, O.I. Yarovaya, N.I. Bormotov, L.N. Shishkina,
O.A. Serova, A.A. Sergeev, A.P. Agafonov, R.A. Maksuytov, N.F. Salakhutdinov,
þ)-Camphor and (ꢀ)-borneol derivatives as potential anti-orthopoxvirus
agents, Arch Pharm (2021), https://doi.org/10.1002/ardp.202100038
e2100038.
[13] M.N. Prichard, E.R. Kern, Orthopoxvirus targets for the development of new
antiviral agents, Antivir. Res. 94 (2) (2012) 111e125, https://doi.org/10.1016/
j.antiviral.2012.02.012.
[14] Y.N. Klimochkin, V.A. Shiryaev, M.V. Leonova, Antiviral properties of cage
compounds. New prospects, Russ. Chem. Bull. 64 (7) (2015) 1473e1496,
https://doi.org/10.1007/s11172-015-1035-y.
[15] I.K. Moiseev, S.A. Kon’kov, K.A. Ovchinikov, N.M. Kilyaeva, K.M. Bormasheva,
O.N. Nechaeva, M.V. Leonova, Y.N. Klimochkin, S.M. Balachin, N.I. Bormotov,
O.A. Serova, E.F. Belanov, Synthesis and antiviral activity of new adamantane
derivatives, Pharm. Chem. J. 45 (10) (2012) 588e592, https://doi.org/10.1007/
s11094-012-0686-3.
[32] T.C. Sung, R.L. Roper, Y. Zhang, S.A. Rudge, R. Ternel, S.M. Hammond,
A.J. Morris, B. Moss, J. Engebrecht, M.A. Frohman, Mutagenesis of phospholi-
pase D defines a superfamily including a trans-Golgi viral protein required for
poxvirus pathogenicity, EMBO J. 16 (1997) 4519e4530, https://doi.org/
10.1093/emboj/16.15.4519.
[33] Y. Chen, K.M. Honeychurch, G. Yang, C.M. Byrd, C. Harver, D.E. Hruby,
R. Jordan, Vaccinia virus p37 interacts with host proteins associated with LE-
derived transport vesicle biogenesis, Virol. J. 6 (2009) 44, https://doi.org/
10.1186/1743-422X-6-44.
[34] FDA Advisory Committee Briefing Document, Tecovirimat for the Treatment
of Smallpox Disease, Antimicrobial Division Advisory Committee Meeting,
May 1, 2018. https://www.fda.gov/media/112808/download.
[35] Y.N. Klimochkin, V.A. Shiryaev, P.V. Petrov, E.V. Radchenko, V.A. Palyulin,
N.S. Zefirov, Design of broad-spectrum inhibitors of influenza A virus M2
proton channels: a molecular modeling approach, Curr. Comput. Aided Drug
[16] Y.N. Klimochkin, M.V. Leonova, I.R. Korzhev, I.K. Moiseev, G.V. Vladyko,
L.V. Korobchenko, E.I. Boreko, S.N. Nikolaeva, Antiviral activity of adamantane
series hydroxy derivatives, Pharm. Chem. J. 26 (7e8) (1992) 616e618, https://
doi.org/10.1007/BF00777145.
[17] N. Kolocouris, A. Kolocouris, G.B. Foscolos, G. Fytas, J. Neyts, E. Padalko,
J. Balzarini, R. Snoeck, G. Andrei, E. De Clercq, Synthesis and antiviral activity
evaluation of some new aminoadamantane derivatives, J. Med. Chem. 39 (17)
(1996) 3307e3318, https://doi.org/10.1021/jm950891z.
[18] Klimochkin YuN, I.K. Moiseev, G.V. Vladyko, L.V. Korobchenko, E.I. Boreko,
Synthesis and investigation of viral-inhibitory activity of nitrogen-containing
derivatives of adamantane, Pharm. Chem. J. 25 (7) (1991) 485e488, https://
doi.org/10.1007/BF00772005.
Des.
12
(2)
(2016)
154e164,
https://doi.org/10.2174/
1573409912666160505113408.
[36] V.A. Shiryaev, E.V. Radchenko, V.A. Palyulin, N.S. Zefirov, N.I. Bormotov,
O.A. Serova, L.N. Shishkina, M.R. Baimuratov, K.M. Bormasheva, Y.A. Gruzd,
E.A. Ivleva, M.V. Leonova, A.V. Lukashenko, D.V. Osipov, V.A. Osyanin,
A.N. Reznikov, V.A. Shadrikova, A.E. Sibiryakova, Y.N. Klimochkin, Molecular
design, synthesis and biological evaluation of cage compound-based in-
hibitors of hepatitis C virus p7 ion channels, Eur. J. Med. Chem. 158 (2018)
214e235, https://doi.org/10.1016/j.ejmech.2018.08.009.
[37] W. Yu, A.D. MacKerell, Computer-aided drug design methods, in: P. Sass (Ed.),
Antibiotics. Methods in Molecular Biology vol. 1520, Humana Press, New York,
NY, 2017, https://doi.org/10.1007/978-1-4939-6634-9_5.
[19] Klimochkin YuN, I.K. Moiseev, O.V. Abramov, G.V. Vladyko, L.V. Korobchenko,
E.I. Boreko, Synthesis and antiviral activity of sulfur-containing derivatives of
adamantane, Pharm. Chem. J. 25 (7) (1991) 489e492, https://doi.org/10.1007/
BF00772006.
[20] Y.N. Klimochkin, I.K. Moiseev, E.I. Boreko, G.V. Vladyko, L.V. Korobchenko,
Synthesis and antiviral activity of nitrogen-containing adamantane de-
rivatives, Pharm. Chem. J. 23 (4) (1989) 304e307, https://doi.org/10.1007/
BF00758419.
[38] J. Yang, R. Yan, A. Roy, D. Xu, J. Poisson, Y. Zhang, The I-TASSER Suite: protein
structure and function prediction, Nat. Methods 12 (2015) 7e8, https://
doi.org/10.1038/nmeth.3213.
[39] A. Roy, A. Kucukural, Y. Zhang, I-TASSER: a unified platform for automated
protein structure and function prediction, Nat. Protoc. 5 (2010) 725e738,
https://doi.org/10.1038/nprot.2010.5.
[40] Y. Zhang, I-TASSER server for protein 3D structure prediction, BMC Bioinf. 9
(2008) 40, https://doi.org/10.1186/1471-2105-9-40.
[21] A. Kolocouris, K. Dimas, C. Pannecouque, M. Witvrouw, G.B. Foscolos,
G. Stamatiou, G. Fytas, G. Zoidis, N. Kolocouris, G. Andrei, R. Snoeck, E. De
Clercq, New 2-(1-adamantylcarbonyl)pyridine and 1-acetyladamantane thi-
osemicarbazonesethiocarbonohydrazones: cell growth inhibitory, antiviral
and antimicrobial activity evaluation, Bioorg. Med. Chem. Lett 12 (5) (2002)
723e727, https://doi.org/10.1016/S0960-894X(01)00838-1.
[41] J. Lee, X. Cheng, J.M. Swails, M.S. Yeom, P.K. Eastman, J.A. Lemkul, S. Wei,
J. Buckner, J.C. Jeong, Y. Qi, S. Jo, V.S. Pande, D.A. Case, C.L. Brooks,
A.D. MacKerell, J.B. Klauda, W. Im, CHARMM-GUI input generator for NAMD,
GROMACS, AMBER, OpenMM, and CHARMM/OpenMM simulations using the
CHARMM36 additive force field, J. Chem. Theor. Comput. 12 (1) (2016)
405e413, https://doi.org/10.1021/acs.jctc.5b00935.
[22] H. Banie, A. Sinha, R.J. Thomas, J.C. Sircar, M.L. Richards, 2-
Phenylimidazopyridines, a new series of golgi compounds with potent anti-
viral activity, J. Med. Chem. 50 (24) (2007) 5984e5993, https://doi.org/
10.1021/jm0704907.
[42] S. Jo, T. Kim, V.G. Iyer, W. Im, CHARMM-GUI: a Web-based graphical user
interface for CHARMM, J. Comput. Chem. 29 (11) (2008) 1859e1865, https://
doi.org/10.1002/jcc.20945.
ꢀ
[43] E.L. Wu, X. Cheng, S. Jo, H. Rui, K.C. Song, E.M. Davila-Contreras, Y. Qi, J. Lee,
[23] E.V. Suslov, E.S. Mozhaytsev, D.V. Korchagina, N.I. Bormotov, O.I. Yarovaya,
K.P. Volcho, O.A. Serova, A.P. Agafonov, R.A. Maksyutov, L.N. Shishkina,
V. Monje-Galvan, R.M. Venable, J.B. Klauda, W. Im, CHARMM-GUI Membrane
Builder toward realistic biological membrane simulations, J. Comput. Chem.
35 (27) (2014) 1997e2004, https://doi.org/10.1002/jcc.23702.
N.F.
Salakhutdinov,
New
chemical
agents
based
on
adamantaneemonoterpene conjugates against orthopoxvirus infections, RSC
Med Chem 11 (2020) 1185e1195, https://doi.org/10.1039/d0md00108b.
[24] T.R. Bailey, S.R. Rippin, E. Opsitnick, C.J. Burns, D.C. Pevear, M.S. Collett,
G. Rhodes, S. Tohan, J.W. Huggins, R.O. Baker, E.R. Kern, K.A. Keith, D. Dai,
G. Yang, D. Hruby, R. Jordan, N-(3,3a,4,4a,5,5a,6,6a-Octahydro-1,3-dioxo-4,6-
ethenocycloprop[f]isoindol-2-(1H)-yl)carbox-amides: identification of novel
orthopoxvirus egress inhibitors, J. Med. Chem. 50 (7) (2007) 1442e1444,
https://doi.org/10.1021/jm061484y.
[25] G. Yang, D.C. Pevear, M.H. Davies, M.S. Collett, T. Bailey, S. Rippen, L. Barone,
C. Burns, G. Rhodes, S. Tohan, J.W. Huggins, R.O. Baker, R.L. Buller,
E. Touchette, K. Waller, J. Schriewer, J. Neyts, E. DeClercq, K. Jones, D. Hruby,
R. Jordan, An orally bioavailable antipoxvirus compound (ST-246) inhibits
extracellular virus formation and protects mice from lethal orthopoxvirus
challenge, J. Virol. 79 (20) (2005) 13139e13149, https://doi.org/10.1128/
JVI.79.20.13139-13149.2005.
[44] P. Bryk, M.G. Brewer, B.M. Ward, Vaccinia virus phospholipase protein F13
promotes rapid entry of extracellular virions into cells, J. Virol. 92 (2018),
https://doi.org/10.1128/JVI.02154-17 e02154-17.
[45] J.C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C. Chipot,
R.D. Skeel, L. Kale, K. Schulten, Scalable molecular dynamics with NAMD,
J. Comput. Chem. 26 (2005) 1781e1802, https://doi.org/10.1002/jcc.20289.
[46] E.F. Pettersen, T.D. Goddar, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,
T.E. Ferrin, UCSF Chimera–a visualization system for exploratory research and
analysis, J. Comput. Chem. 25 (13) (2004) 1605e1612, https://doi.org/
10.1002/jcc.20084.
[47] Bowie Ju, R. Luthy, D. Eisenberg, A method to identify protein sequences that
fold into a known three-dimensional structure, Science 253 (5016) (1991)
164e170, https://doi.org/10.1126/science.1853201.
[48] McDermott M, Wakelam MJO, Morris AJ. Phosphlipase D. Biochem. Cell. Biol.
82:225-253. doi: 10.1139/o03-079.
[26] D.W. Grosenbach, R. Jordan, D.E. Hruby, Development of the small-molecule
[49] Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of
antiviral ST-246® as
a
smallpox therapeutic, Future Virol.
6
(5) (2011)
docking with a new scoring function, efficient optimization and multi-
653e671, https://doi.org/10.2217/fvl.11.27.
threading. J. Comput. Chem. 31(2):455e461. doi: 10.1002/jcc.21334.
[27] M. Merchlinsky, A. Albright, V. Olson, H. Schiltz, T. Merkeley, C. Hughes,
B. Petersen, M. Challberg, The development and approval of tecoviromat
(TPOXX®), the first antiviral against smallpox, Antivir. Res. 168 (2019)
168e174, https://doi.org/10.1016/j.antiviral.2019.06.005.
[50] R. Jordan, J.M. Leeds, S. Tyavanagimatt, D.E. Hruby, Development of ST-246 for
treatment of poxvirus infections, Viruses
doi.org/10.3390/v2112409.
2 (2010) 2409e2435, https://
[51] Acd ChemSketch 2015, Advanced Chemistry Development, Inc., Toronto,
Canada, 2015. www.acdlabs.com.
[28] M. Husain, A. Weisberg, B. Moss, Topology of epitope-tagged F13L protein, a
11

V.A. Shiryaev, M.Y. Skomorohov, M.V. Leonova et al.
European Journal of Medicinal Chemistry 221 (2021) 113485
[52] M.D. Hanwell, D.E. Curtis, D.C. Lonie, T. Vandermeersch, E. Zurek,
G.R. Hutchison, Avogadro: an advanced semantic chemical editor, visualiza-
A.J. Olson, Autodock4 and AutoDockTools4: automated docking with selective
receptor flexiblity, J. Comput. Chem. 30 (16) (2009) 2785e2791, https://
doi.org/10.1002/jcc.21256.
tion, and analysis platform, J. Cheminf.
4 (1) (2012) 17, https://doi.org/
10.1186/1758-2946-4-17.
[60] T.L. Thomas, M. Fedorchuk, B.V. Shetty, F.E. Anderson, Synthesis and activity of
some 3-substituted 1,2,3,4-pseudooxatriazol-5-ones and their precursors and
related compounds, J. Med. Chem. 13 (2) (1970) 196e203, https://doi.org/
10.1021/jm00296a007.
[61] A. Spasov, P.M. Vasil’ev, D.A. Babkov, Prokhorova TYu, E.A. Sturova,
Klimochkin YuN, M.V. Leonova, M.R. Baimuratov, New dipeptidyl peptidase 4
inhibitors among adamantane derivatives, Russ. J. Bioorg. Chem. 43 (4) (2017)
449e455, https://doi.org/10.1134/S1068162017040124.
ꢁ
[53] Dassault Systemes Biovia, Discovery Studio Visualiser Free, v19.1.0.18287,
ꢁ
Dassault Systemes, San Diego, 2019.
[54] D. Schneidman-Duhovny, O. Dror, Y. Inbar, R. Nussinov, H.J. Wolfson, Deter-
ministic pharmacophore detection via multiple flexible alignment of drug-like
molecules, J. Comput. Biol. 15 (7) (2007) 737e754, https://doi.org/10.1089/
cmb.2007.0130.
[55] D. Schneidman-Duhovny, O. Dror, Y. Inbar, R. Nussinov, H.J. Wolfson, Phar-
maGist:
a
webserver for ligand-based pharmacophore detection, Nucleic
[62] P. Mampuys, E. Ruijter, R.V.A. Orru, B.U.W. Maes, Synthesis of secondary
amides from thiocarbamates, Org. Lett. 20 (14) (2018) 4235e4239, https://
doi.org/10.1021/acs.orglett.8b01654.
[63] Brian W.J. Mahy, Hillar O. Kangro (Eds.), Virology Methods Manual, Academic
Press, 1996, p. 374.
Acids Res. 36 (2008) W223eW228, https://doi.org/10.1093/nar/gkn187.
[56] J. Huang, A.D. MacKerell, CHARMM36 all-atom additive protein force field:
validation based on comparison to NMR data, J. Comput. Chem. 34 (25) (2013)
2135e2145, https://doi.org/10.1002/jcc.23354.
[57] J.B. Klauda, R.M. Venable, J.A. Freites, J.W. O'Connor, D.J. Tobias, C. Mondragon-
Ramirez, I. Vorobyov, A.D. MacKerell, R.W. Pastor, Update of the CHARMM all-
atom additive force field for lipids: validation on six lipid types, J. Phys. Chem.
B 114 (23) (2010) 7830e7843, https://doi.org/10.1021/jp101759q.
[58] S. Forli, R. Huey, M.E. Pique, M.F. Sanner, D.S. Goodsell, A.J. Olson, Computa-
tional protein-ligand docking and virtual drug screening with the AutoDock
suite, Nat. Protoc. 11 (5) (2016) 905e919, https://doi.org/10.1038/
nprot.2016.051.
[64] Mazurkov OYu, A.S. Kabanov, L.N. Shishkina, A.A. Sergeev, M.O. Skarnovich,
N.I. Bormotov, M.A. Skarnovich, A.S. Ovchinnikova, K.A. Titova, D.O. Galahova,
L.E. Bulychev, ArA. Sergeev, O.S. Taranov, B.A. Selivanov, Tikhonov AYa,
E.L. Zavjalov, A.P. Agafonov, A.N. Sergeev, The new effective chemically syn-
thesized anti smallpox compound NIOCH-14, J. Gen. Virol. 97 (5) (2016)
1229e1239, https://doi.org/10.1099/jgv.0.000422.
[65] R.O. Baker, M. Bray, J.W. Huggins, Potential antiviral therapeutics for smallpox,
monkeypox and other orthopoxvirus infections, Antivir. Res. 57 (1e2) (2003)
13e23, https://doi.org/10.1016/S0166-3542(02)00196-1.
[59] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
12