(+)-Camphor and (?)-borneol derivatives as potential anti-orthopoxvirus agents

Article abstract of DOI:10.1002/ardp.202100038

Although the World Health Organisation had announced that smallpox was eradicated over 40 years ago, the disease and other related pathogenic poxviruses such as monkeypox remain potential bioterrorist weapons and could also re-emerge as natural infections

Full text of DOI:10.1002/ardp.202100038

Received: 28 January 2021
Accepted: 30 January 2021
|
DOI: 10.1002/ardp.202100038
F U L L P A P E R
(+)‐Camphor and (−)‐borneol derivatives as potential
anti‐orthopoxvirus agents
Anastasiya S. Sokolova1
| Kseniya S. Kovaleva1 | Olga I. Yarovaya1
|
Nikolay I. Bormotov2 | Larisa N. Shishkina2 | Olga A. Serova2
|
Alexander A. Sergeev2 | Alexander P. Agafonov2 | Rinat A. Maksuytov2
|
Nariman F. Salakhutdinov1
1N. N. Vorozhtsov Novosibirsk Institute of
Organic Chemistry, Siberian Branch Russian
Academy of Sciences, Novosibirsk, Russian
Federation
Abstract
Although the World Health Organisation had announced that smallpox was eradi-
cated over 40 years ago, the disease and other related pathogenic poxviruses such
as monkeypox remain potential bioterrorist weapons and could also re‐emerge as
natural infections. We have previously reported (+)‐camphor and (−)‐borneol deri-
vatives with an antiviral activity against the vaccinia virus. This virus is similar to the
variola virus (VARV), the causative agent of smallpox, but can be studied at BSL‐2
facilities. In the present study, we evaluated the antiviral activity of the most potent
compounds against VARV, cowpox virus, and ectromelia virus (ECTV). Among
the compounds tested, 4‐bromo‐Nʹ‐((1R,4R)‐1,7,7‐trimethylbicyclo[2.2.1]heptan‐2‐
ylidene)benzohydrazide 18 is the most effective compound against various ortho-
poxviruses, including VARV, with an EC50 value of 13.9 μM and a selectivity index of
206. Also, (+)‐camphor thiosemicarbazone 9 was found to be active against VARV
and ECTV.
2State Research Centre of Virology and
Biotechnology VECTOR, Rospotrebnadzor,
Novosibirsk, Russian Federation
Correspondence
Anastasiya S. Sokolova, N. N. Vorozhtsov
Novosibirsk Institute of Organic Chemistry,
Siberian Branch Russian Academy of Sciences,
9, Lavrent'ev Ave, Novosibirsk, Russian
Federation 630090.
Email: asokolova@nioch.nsc.ru
Funding information
Russian Foundation for Basic Research,
Grant/Award Number: 20‐33‐70067
K E Y W O R D S
cowpox virus, ectromelia virus, vaccinia virus, variola virus, (+)‐camphor, (−)‐borneol
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1
INTRODUCTION
repositories: the State Research Centre for Virology and Bio-
technology VECTOR, Koltsovo, Russian Federation, and the Centers
Forty years ago, smallpox was officially declared eradicated by the
World Health Organisation (WHO). Smallpox has struck humanity
throughout its history. In the 20th century alone, smallpox outbreaks
resulted in the death of about 400 million people. Therefore, the
eradication of smallpox was a landmark event. Moreover, it is still the
only case in human history when the world's community managed to
implement a program for the global eradication of a virus, thereby
freeing itself from a severe infection. Variola virus (VARV), the
causative agent of smallpox, belongs to the genus Orthopoxvirus, with
this genus also including the cowpox virus (CPV), monkeypox virus,
camelpox virus, ectromelia virus (ECTV), and vaccinia virus (VV),
among others. To date, VARV strains have only been kept in two
for Disease Control and Prevention (CDC), Atlanta, United States.
There are no known zoonotic hosts of VARV, whereas other ortho-
poxviruses (OPVs) may be transmitted to humans by animal hosts. In
2017 and 2018, human monkeypox outbreaks were reported in the
Democratic Republic of Congo, Central African Republic, Cameroon,
Republic of Congo, Liberia, and Nigeria.[1] From 1970 to 2018, the
total number of suspected monkeypox cases was 24,399.[2] The in-
creasing number of zoonotic OPV infections, the potential of using
OPVs as a bioterrorism weapon, and the weak vaccination coverage
of the population (by 1984, all countries had ceased vaccinating) are
all potential threats requiring effective anti‐poxvirus agents to be
developed.
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Arch Pharm. 2021;e2100038.
wileyonlinelibrary.com/journal/ardp
© 2021 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202100038

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SOKOLOVA ET AL.
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OPVs are large, linear, double‐stranded DNA viruses that re-
plicate using several virus‐encoded enzymes. The complex replica-
tion mechanism of OPVs provides a number of targets for drug
intervention. To date, several compounds have been identified to be
effective both in vitro and in vivo. Isatin β‐thiosemicarbazone 1
(Figure 1, methisazone, Marboran®) was the first compound identi-
fied to possess a marked antiviral activity in mice infected with the
smallpox virus.[3] To date, however, there has been limited clinical
experience with methisazone in humans.[4] Methisazone is thought to
inhibit the transcription process, but the mechanism is incompletely
understood. Currently, CMX001 and ST‐246 appear to have the
greatest potential to be approved for the treatment of OPV infec-
tions. The possible target of nucleoside analog CMX001 is the viral
DNA polymerase. It has been shown in vitro that the inhibitory
concentration of CMX001 against VARV is 0.1 μM, which varies from
0.5 to 0.9 μM against CPV, VV, mousepox, and rabbitpox.[5] The
molecular target of ST‐246 is the viral protein p37, required to
produce the extracellular virus envelope.[6] ST‐246 is active against
many OPVs, including VARV, monkeypox virus, camelpox virus, and
mousepox virus. A team of scientists from the Novosibirsk Institute
of Organic Chemistry and the State Research Center VECTOR has
developed a structural analog of the drug ST‐246: the agent NIOCH‐
14. This compound is effective against OPVs in both in vivo and in
vitro experiments.[7] The mechanism of action of this antiviral agent
is the same as that of ST‐246, as NIOCH‐14 is a pro‐drug and is
converted into its active metabolite ST‐246 in mammals. Despite
CMX001, ST‐246, and NIOCH‐14 being promising candidates, there
is a need to further develop antiviral agents against OPVs.
9–14,[12] and (+)‐camphor‐based N‐acylhydrazones 15–18[13] were
found as good inhibitors of the VV (Figure 2). Derivatives 1–18 had IC50
(the 50% inhibitory concentration) values in the range of 2.5–70 µM
against VV and CC50 (the 50% cytotoxic concentration) values in the
range of 120–1430 μM. On the basis of the antiviral data obtained,
derivatives 1–18 were identified as promising OPV inhibitors, which
may be candidates for further optimization. As the genus Orthopoxvirus
includes several virus species pathogenic for humans, an important
aspect in the development of OPV inhibitors is the extent of drug
action. Therefore, in the present work, we evaluated the antiviral ac-
tivity of monoterpenoid derivatives 1–18 against the different OPVs,
including the VARV known to be highly pathogenic for humans.
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2
RESULTS AND DISCUSSION
To study the effect of derivatives 1–18 on various OPVs replication,
we determined antiviral activity against zoonotic viruses CPV and
ECTV and the exclusively anthroponotic agent VARV. The results are
shown in Table 1. Among esters 1–4, only derivative 2 demonstrated
moderate antiviral activity against VARV and ECTV with an IC50
value of 38.0 and 47.7 µM, respectively, and low cytotoxicity with a
CC50 value of 413.0 µM (selectivity index [SI] equal to 11 and 9,
respectively). Esters 1 and 4 are devoid of inhibitory activity against
VARV, ECTV, and CPV. Ester 3, different from ester 2 by an addi-
tional methylene group, showed a similar IC50 value but was sig-
nificantly more toxic. Among amides 5–8, only derivative 8 bearing a
4‐methylpiperidine fragment demonstrated inhibitory activity
against VARV with an IC50 value of 14.5 µM (SI = 16). An accordance
of antiviral activity was found against VV and VARV in the series of
compounds 1–8. Compounds 2 and 8, showing the low IC50 value
against VV (3.5 and 2.5 µM respectively), also possessed moderate
antiviral activity against VARV. However, in the series, no compound
was found with satisfactory antiviral activity against all viruses tes-
ted (VV, VARV, CPV, and ECTV).
Monoterpenoid derivatives have been extensively studied as
therapeutic agents against the proliferation of cancer cells, for treating
neurodegenerative disorders, and against virus replication and bacterial
infections.[8] Our research group focused on the synthesis of camphor
and borneol derivatives reported to be inhibitors of the influenza
virus,[9] filoviruses,[10] and OPVs. In particular, we evaluated libraries of
compounds for the ability to interfere with the replication of VV in
vitro. This virus is similar to the VARV but can be studied at BSL‐2
facilities. Over 300 monoterpenoid derivatives were screened, among
which esters 1–4 based on (−)‐borneol, amides 5–8 with N‐containing
heterocycles,[11] the (+)‐camphor thiosemicarbazone derivatives
An extended study of the anti‐OPV activity of camphor thiose-
micarbazone 9 and its derivatives 10–14 indicated thiosemicarba-
zone 9 to possess inhibitory activity against VARV, with an IC50
value of 8.8 µM, along with an SI of 26, and against ECTV, with an
FIGURE 1 Chemical structures of inhibitors of orthopoxvirus replication

SOKOLOVA ET AL.
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FIGURE 2 Structure and antiviral activity against vaccinia virus of derivatives 1–18. IC50: 50% inhibitory concentration, ensuring 50% cell
survival in an infected monolayer
IC50 value of 12.4 µM (SI = 19). Transformation of the ═N–NH–C
(S)–NH2 group in the thiazole group led to an increased toxicity and
decreased antiviral potency. Thus, thiazole 11 displayed an IC50 va-
lue similar to that of compound 9, whereas the toxicity was almost
threefold higher than the CC50 value of 66.8 and 229.0 µM, re-
spectively. Thiazoles 10 and 12 demonstrated low antiviral potency
(IC50 = 14.7 and 10.3 μM) against VARV and higher cytotoxicity
(CC50 = 75.9 and 137.6 μM) than thiosemicarbazone 9. Thiazolidi-
nones 13 and 14 were found not to be active against VARV, CPV,
and ECTV.
propyl substituent, turned out to be inactive against the OPVs tes-
ted. Also, acylhydrazone 17 can be seen as being inactive against VV
(IC50 = 70 μM; Figure 2).
In general, there was good agreement with the IC50 values for a
compound between the VV and the VARV assays, as shown in
Figure 2 and Table 1. However, discrepancies were observed for
derivatives 4, 7, 13, and 14 that showed an antiviral activity against
VV with IC50 13, 12, 14.4, and 9.5 µM, respectively, and were not
active in the antiviral assay toward VARV, which is not surprising as
VARV and VV differ in their biological properties. It may also be
noted that the antiviral activity of the studied compounds against
CPV is generally lower than against VV, VARV, and ECTV. This result
may be due to different amino acid sequences in the domains of
conserved proteins, which could provide different sensitivities of
OPVs to the chemical compounds.[15]
Among the series of acylhydrazones 15–18, aroyl hydrazones 16
and 18 showed a good inhibitory activity against VARV, CPV, and
ECTV. Moreover, derivatives 16 and 18 demonstrated low cyto-
toxicity and high values of SI. The antiviral activity of compound 18
against CPV and ECTV with SI values of 460 and 854, respectively, is
worth noting. Compound 15, bearing an epoxyisoindole moiety,
showed moderate inhibition of OPV replication, with IC50 values in
the range of 13–29 μM and low cytotoxicity. Compound 17, with a
As the camphor thiosemicarbazone 9 displayed an inhibitory ac-
tivity against VARV and ECTV, and ═N–NH–C(S)–NH2 group allows for
chemical modifications, we implemented structural modifications on

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SOKOLOVA ET AL.
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TABLE 1 Cytotoxicity and anti‐orthopoxviruses activities of (+)‐camphor and (−)‐borneol derivatives 1–18 in Vero cells
Compound
CC50a, µM
IC50VARVb, µM (SIe)
166.0 77.9 (2)
38.0 6.8 (11)
38.5 6.9 (4)
NA
IC50CPVc, µM (SIe)
IC50ECTVd, µM (SIe)
168.8 79.2 (2)
47.7 8.5 (9)
NA
1
363.6 75.0
413.0 81.2
139.6 71.4
319.4 75.1
331.4 68.0
339.3 70.3
396.2 74.5
135.4 75.2
229.0 53.3
75.9 37.0
NA
2
145.6 104.9 (3)
3
NA
4
NA
NA
5
215.1 34.3 (2)
125.1 20.2 (3)
NA
177.6 76.1 (2)
130.8 47.2 (3)
NA
42.6 6.8 (8)
44.2 7.1 (8)
NA
6
7
8
14.5 7.9 (16)
8.8 6.0 (26)
14.7 10.5 (5)
3.1 1.0 (11)
10.3 1.8 (13)
NA
62.2 21.5 (2)
49.3 7.1 (5)
NA
33.2 18.1 (4)
12.4 8.9 (19)
NA
9
10
11
66.8 31.9
NA
15.1 4.7 (4)
42.8 7.6 (3)
NA
12
137.6 25.6
50.7 31.1
NA
13
NA
14
147.8 27.8
476.5 71.5
1638.5 466.0
853.8 206.4
2904.9 49.9
923.0 321.6
59.2 10.5 (2)
26.5 9.3 (18)
21.3 7.5 (76)
271.2 96.4 (3)
13.9 2.4 (206)
29.4 13.9 (31)
NA
NA
15
28.8 14.5 (17)
14.4 5.2 (117)
NA
13.1 4.6 (37)
10.4 3.7 (157)
NA
16
17
18
6.3 1.7 (460)
89.1 11.8 (10)
3.4 0.6 (854)
23.3 11.1 (40)
Cidofovir
Note: CC50, IC50VARV, IC50CPV, and IC50ECTV are presented as M SD, where M is the mean and SD is the standard deviation; n = 3 is the number of
measurements of CC50, IC50VARV, IC50CPV, and IC50ECTV
.
aCC50 is the cytotoxic concentration of a compound causing 50% cell death in an uninfected monolayer.
bIC50VARV is the concentration of a compound ensuring 50% cell survival in a monolayer infected with VARV.
cIC50CPV is the concentration of a compound ensuring 50% cell survival in a monolayer infected with CPV.
dIC50ECTV is the concentration of a compound ensuring 50% cell survival in a monolayer infected with ECTV.
eSI is the drug selectivity index (CC50/IC50). Compounds with SI <8 are considered practically inactive according to the recommendations.[14]
compound 9 to possibly improve toxicity and potency by adding an
isatin fragment and modifying the 1,7,7‐trimethylbicyclo[2.2.1]heptane
region, as outlined in Scheme 1. To study the effect of the 1,7,7‐
trimethylbicyclo[2.2.1]heptane scaffold on antiviral activity, cam-
phorquinone and norcamphor were coupled with thiosemicarbazide to
provide compounds 19 and 20. The conjugates of camphor hydrazine
and isatin 21 or 1‐methylisatin 22 were prepared, because the isatin
nucleus could be considered as a privileged scaffold for designing bio-
logically active agents.[16] Moreover, it is the constitutive fragment of
the anti‐OPV drug methisazone (Figure 1).
exhibited lower toxicity (СС50 values of 460.4 and 544.0 µM, re-
spectively) than thiosemicarbazone 9 (CC50 = 229.0 µM). The con-
jugation of camphor hydrazone with isatin scaffolds decreased the
inhibitory activity against VV and, according to the CC50 value,
compounds 21 and 22 were found to be quite cytotoxic (CC50 values
of 16.8 and 35 µM, respectively).
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3
CONCLUSION
The synthesized compounds were tested in the preliminary an-
tiviral study against VV. The results are summarized in Table 2. The
commercially available agents cidofovir and methisazone were used
as a positive control.
In conclusion, we have screened a series of (+)‐camphor and (−)‐
borneol derivatives against different OPV infections. The SAR in-
vestigation indicated the 1,7,7‐trimethylbicyclo[2.2.1]heptane core
structure, N‐acylhydrazone motif, and thiosemicarbazone group to
be favorable for the anti‐OPV activity. Among the compounds tested,
we have identified acylhydrazones 16 and 18 with a broad‐spectrum
activity (SI values of 76, 117, and 157; 206, 460, and 854, respec-
tively) against various OPVs, including VARV, CPV, and ECTV.
Biological evaluation of the thiosemicarbazones of camphorqui-
none 19 and norcamphor 20 against VV indicated a reduced activity
as compared with the camphor thiosemicarbazone 9 (IC50 = 18 µM;
Figure 2). At the same time, thiosemicarbazones 19 and 20

SOKOLOVA ET AL.
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SCHEME 1 Synthesis of the derivatives 19–21
Several other compounds show promise, including camphor thiose-
micarbazone 9 with an IC50VARV value of 8.8 µM and high SI of 26.
The transformation of the ═N–NH–C(S)–NH2 group in compound 9
to thiazole or thiazolidin‐4‐one cycle in compounds 10–14 led to a
loss of anti‐OPV activity. Also, conjugation of the isatin scaffold with
1,7,7‐trimethylbicyclo[2.2.1]heptane via a hydrazine linker resulted
in a marked increase in cytotoxicity and loss of anti‐OPV activity.
However, the results indicate that monoterpenoids with the 1,7,7‐
trimethylbicyclo[2.2.1]heptane core could be a potential scaffold for
developing compounds for anti‐orthopoxviral therapy.
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4
EXPERIMENTAL
Chemistry
TABLE 2 The antiviral activities of compounds 19–22 against
vaccinia virus
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4.1
Compound
CC50, µMa
IC50, µMb
171.7 53.0
212.8 64.9
NA
SIc
2
19
460.4 156.7
544.0 132.6
16.8 8.7
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4.1.1
General
20
2
The chemical compounds library contains 18 agents (1–18) that were
screened against OPVs including VARV and four agents (19–22) that
were tested against VV. The synthesis of compounds 1 and 2,[17] 3,
5–8,[11] 4,[18] 9–14,[12] and 15–18[13] was previously described. For the
synthesis of derivatives 19, 21, and 22, camphorquinone[19] and camphor
hydrazine[13] were prepared according to the procedure reported in the
literature from (+)‐camphor. Reagents and solvents were purchased from
commercial suppliers and used as received. 1H and 13C nuclear magnetic
21
22
34.9 16.3
923.0 321.6
192.5 8.9
NA
Cidofovir
Methisazone
37.9 18.9
8.1 4.7
24
23
Note: Data are presented as M SD, where M is the mean and SD is the
standard deviation; n = 3 is the number of CC50 and IC50 measurements.
aCC50 is the cytotoxic concentration causing 50% cell death in an
uninfected monolayer.
resonance (NMR) spectra were recorded with
a Bruker AV‐300
bIC50 is the concentration ensuring 50% cell survival in a monolayer
infected with VV.
cSI is the drug selectivity index (CC50/IC50). Compounds with SI <8 are
considered practically inactive in accordance with the
recommendations.[14]
(1H: 300.13 MHz, 13C: 75.47 MHz), AV‐400 (1H: 400.13 MHz, 13C:
100.78 MHz) in CDCl3; chemical shifts δ are expressed in ppm, relative to
residual [δ(CHCl3) 7.24, δ(CDCl3) 76.90]. Elemental analysis was carried
out using a Euro EA3000 C, H, N, S analyzer.

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SOKOLOVA ET AL.
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The original spectra and the InChI codes of the investigated
compounds, together with some biological activity data, are provided
as Supporting Information.
1.91–1.93 (1H, m), 1.99–2.06 (1H, m), 2.51–2.59 (1H, m), 3.23 (3H, s),
6.79 (1H, d, J = 7.7), 6.98–7.02 (1H, m), 7.32–7.36 (1H, m), 7.91 (1H, d,
J = 7.7). NMR 13C (100 MHz, CDCl3, δ, ppm): 178.5 s (C(N)), 164.2 s (C
(O)), 146.4 s, 145.6 s, 132.3 d (CH–Ar), 128.2 d (CH–Ar), 122.6 d
(CH–Ar), 116.7 s, 108.2 d (CH–Ar), 53.3 s, 48.1 s, 43.8 d, 35.6 t, 32.4 t,
26.9 t, 25.8 q, 19.4 q, 18.6 q, 11.1 q. HRMS (ESI) (m/z): [M+] calcd. for
|
4.1.2
Synthesis of 2‐((1S,4R)‐4,7,7‐trimethyl‐3‐
oxobicyclo[2.2.1]heptan‐2‐ylidene)‐
hydrazinecarbothioamide (19)
C19H23O1N3: 309.1836, found: 309.1838.
|
A solution of thiosemicarbazide (0.01 mol) in ethanol was added to a
solution of camphorquinone (0.01 mol) in ethanol and several drops of
H2SO4. The mixture was heated at reflux for 24 h and then washed with
brine and extracted with CHCl3, dried (Na2SO4), filtered, and con-
centrated. The crude product was purified by recrystallization from the
mixture hexane/CHCl3 5:1. Yield: 51%, a yellow solid; mp: 122.6°C;
NMR 1H (400 MHz, CDCl3, δ, ppm): 0.82 (3H, s), 0.98 (3H, s), 0.99 (3H,
s), 1.41–1.57 (2H, m), 1.73–1.85 (1H, m), 1.99–2.12 (1H, m), 2.97–3.04
(1H, m), 6.85 (1H, br s), 7.54 (1H, br s), 9.21 (1H, br s). NMR 13C
(100 MHz, CDCl3, δ, ppm): 204.7 s (C(O)), 180.2 s (C(S)), 152.1 s (C(N)),
58.3 s, 47.4 d, 45.3 s, 30.4 t, 23.8 t, 20.6 q, 17.6 q, 8.9 q. High‐resolution
electrospray ionization mass spectrometry (HRMS [ESI]) (m/z): [M+]
calcd. for C11H17O1N3S1: 239.1087, found: 239.1089.
4.1.5
Synthesis of 3‐(((1R,4R)‐1,7,7‐
trimethylbicyclo[2.2.1]heptan‐2‐ylidene)hydrazono)‐
indolin‐2‐one (22)
To a solution of camphor hydrazone (3 mmol) in 5 ml of ethanol, isatin
(3 mmol) was added, and the mixture was stirred at room temperature
for 8 h. The mixture was then washed with brine and extracted with
CHCl3, dried (Na2SO4), filtered, and concentrated. The crude product
was purified by silica gel column chromatography (eluent: CHCl3/MeOH)
to give a yellow solid. Yield: 42%; mp: 76.4°C; 1H NMR (400 MHz, CDCl3,
δ, ppm, J/Hz): 0.84 (3H, s), 0.97 (3H, s), 1.21 (3H, s), 1.22–1.26 (1H, m),
1.48–1.55 (1H, m), 1.78–1.91 (2H, m), 1.94–1.96 (1H, m), 2.03–2.11 (1H,
m), 2.57–2.65 (1H, m), 6.89 (1H, d, J = 7.8), 6.98–7.02 (1H, m), 7.28–7.33
(1H, m), 7.94 (1H, d, J = 7.8), 9.21 (1H, br s). NMR 13C (100 MHz, CDCl3,
δ, ppm): 179.1 s (C(N)), 166.1 s (C(O)), 146.9 s, 143.1 s, 132.5 d (CH–Ar),
128.5 d (CH–Ar), 122.7 d (CH–Ar), 117.19 s, 110.6 d (CH–Ar), 53.3 s,
47.8 s, 43.8 d, 35.7 t, 32.5 t, 26.9 t, 19.4 q, 18.6 q, 11.1 q. HRMS (ESI) (m/
z): [M+] calcd. for C18H21O1N3: 295.1679, found: 295.1680.
|
4.1.3
Synthesis of 2‐((1R,4S)‐bicyclo[2.2.1]heptan‐
2‐ylidene)hydrazinecarbothioamide (20)
A solution of thiosemicarbazide (0.006 mol) in ethanol was added to a
solution of norcamphor (0.006 mol) in ethanol and several drops of
H2SO4. The mixture was heated at reflux in 10 h and then washed with
brine and extracted with CHCl3, dried (Na2SO4), filtered, and con-
centrated. The crude product was purified by silica gel column chro-
matography (eluent: hexane/ethyl acetate). Yield: 45%, a white solid,
mp: 73.7°C; NMR 1H (400 MHz, CDCl3, δ, ppm, J/Hz): 1.19–1.31 (1H,
m), 1.36–1.52 (3H, m), 1.61–1.79 (2H, m), 1.87–1.95 (1H, m), 2.09–2.17
(1H, m), 2.55–2.60 (1H, m), 2.72–2.79 (1H, m), 6.57 (1H, br s), 7.11 (1H,
br s), 8.48 (1H, br s). NMR 13C (100 MHz, CDCl3, δ, ppm): 178.3 s (C(S)),
164.3 s (C(N)), 44.2 d, 38.5 t, 35.6 d, 35.4 t, 27.3 t, 26.7 t. HRMS (ESI)
(m/z): [M+] calcd. for C8H13N3S1: 183.0825, found: 183.0823.
|
4.2
Biological assays
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4.2.1
Cells and viruses
Vaccinia virus (VV, strain Copenhagen), cowpox virus (CPV, strain
Grishak), mousepox virus—ectromelia (ECTV, strain K‐1), and variola
virus (VARV, strain Ind‐3a), obtained from the state collection of pa-
thogens of viral infections and rickettsioses of SRC VB Vector, were
used in the work. Virus‐containing suspensions with concentrations
ranging from 5.6 to 6.7 log10 PFU/ml were prepared in Vero cell culture
medium using these strains. Virus‐containing material was packaged in
individual tubes and stored at a temperature of −70°C. Vero cell
monolayer was grown in Dulbecco's Modified Eagle's medium (DMEM;
OJSC BioloT) in the presence of 10% fetal bovine serum (FBS; HyClone)
supplemented with penicillin (100 IU/ml) and streptomycin (100 mg/ml).
The same medium supplemented with 2% FBS, penicillin (100 IU/ml),
and streptomycin (100 mg/ml) was used to support virus cultivation.
|
4.1.4
Synthesis of 1‐methyl‐3‐(((1R,4R)‐1,7,7‐
trimethylbicyclo[2.2.1]heptan‐2‐ylidene)hydrazono)‐
indolin‐2‐one (21)
To a solution of camphor hydrazone (3 mmol) in 5 ml of ethanol, 1‐
methylisatin (3 mmol) was added, and the mixture was stirred at room
temperature for 8 h. The mixture was then washed with brine and
extracted with CHCl3, dried (Na2SO4), filtered, and concentrated. The
crude product was purified by silica gel column chromatography (elu-
ent: CHCl3/MeOH) to give a yellow solid. Yield: 54%; mp: 82.3°C; 1H
NMR (400 MHz, CDCl3, δ, ppm, J/Hz): 0.81 (3H, s), 0.95 (3H, s), 1.19
(3H, s), 1.18–1.26 (1H, m), 1.47–1.54 (1H, m), 1.75–1.88 (2H, m),
|
4.2.2
Cytotoxicity assay and determination of
anti‐OPV activities
All experiments with live VARV were conducted at SRC VB Vector
in
a maximum containment facility (BSL‐4) using insulating

SOKOLOVA ET AL.
7 of 8
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pneumatic suits. Viruses were produced in Vero cell culture in
DMEM. The virus concentration in the culture liquid was de-
termined by plaque titration in Vero cell culture, calculated, and
expressed in decimal logarithms of plaque‐forming units in 1 ml
(log10 PFU/ml).[20] The concentration of the virus in the samples
used in the work ranged from 5.6 to 6.1 log10 PFU/ml. The series
of viruses with the indicated titer was stored and used in work
at −70°C.
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The antiviral efficacy of the compounds was evaluated as
follows. In wells of 96‐well plates containing a monolayer of Vero
cells in 100 μl of DMEM medium with 2% fetal serum, 50 μl of
serial dilutions of the test compounds were first introduced and
then 50 μl of a dilution of OPV at a dose of 1000 PFU/well was
added.
[5] K. Y. Hostetler, Antiviral Res. 2009, 82, 84. https://doi.org/10.1016/
j.antiviral.2009.01.005
[6] G. Yang, D. C. Pevear, M. H. Davies, M. S. Collett, T. Bailey,
S. Rippen, L. Barone, C. H. Burns, G. Rhodes, S. Tohan,
J. W. Huggins, R. O. Baker, R. L. M. Buller, E. Touchette, K. Waller,
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13149.2005
The toxicity of the compounds was determined by the Vero
cell death caused by the drug in the wells of the plate, into which
the virus was not introduced. Monolayers of cells were used as
controls in the wells of the plate, into which virus without com-
pounds (virus control) and monolayers of cells in wells into
which neither the virus nor the compound was introduced (cell
culture control) were introduced. After incubation for 4 days, the
monolayer of cells was stained with vital dye neutral red for 2 h.
After removing the dye and washing the wells from its unbound
fraction, a lysis buffer was added. The amount of dye adsorbed by
the living cells of the monolayer was evaluated by optical density
(OD), which is an indication of the number of cells undisturbed
under the influence of the virus in a monolayer. The OD was
measured on an EMax spectrophotometer (Molecular Devices) at
a wavelength of 490 nm. Results were processed using the Soft
Max Pro 4.0 program, which computed the 50% toxic con-
centration (CC50 in μM) and 50% inhibitory concentration (IC50
in μM). The SI was determined as SI = CC50/IC50 using the
corresponding concentrations.
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M. O. Skarnovich, N. I. Bormotov, M. A. Skarnovich,
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A. A. Sergeev, O. S. Taranov, B. A. Selivanov, A. Y. Tikhonov,
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1229. https://doi.org/10.1099/jgv.0.000422
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2017, 89, 1105. https://doi.org/10.1515/pac-2017-0109
[9] A. S. Sokolova, O. I. Yarovaya, A. V. Shernyukov, Y. V. Gatilov,
Y. V. Razumova, V. V. Zarubaev, T. S. Tretiak, O. I. Kiselev,
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[10] A. S. Sokolova, O. I. Yarovaya, A. V. Zybkina, E. D. Mordvinova,
N. S. Shcherbakova, A. V. Zaykovskaya, D. S. Baev, T. G. Tolstikova,
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[11] A. S. Sokolova, O. I. Yarovaya, N. I. Bormotov, L. N. Shishkina,
N. F. Salakhutdinov, Chem. Biodivers. 2018, 15, e180015. https://
doi.org/10.1002/cbdv.201800153
[12] A. S. Sokolova, O. I. Yarovaya, N. I. Bormotov, L. N. Shishkina,
N. F. Salakhutdinov, MedChemComm 2018, 9, 1746. https://doi.org/
10.1039/C8MD00347E
[13] K. S. Kovaleva, F. I. Zubkov, N. I. Bormotov, R. A. Novikov,
P. V. Dorovatovskii, V. N. Khrustalev, Y. V. Gatilov, V. V. Zarubaev,
O. I. Yarovaya, L. N. Shishkinad, N. F. Salakhutdinov,
MedChemComm 2018, 9, 2072. https://doi.org/10.1039/C8MD
00442K
ACKNOWLEDGMENTS
The authors would like to acknowledge the Multi‐Access Chemical
Service Centre SB RAS for spectral and analytical measurements.
This study has been supported by a Russian Foundation for Basic
Research (RFBR) Grant 20‐33‐70067. The experiments with live
VARV supported the state assignment of State Research Centre of
Virology and Biotechnology VECTOR.
[14] R. U. Khabriev, in Guidelines for Experimental (Preclinical) Study of
New Pharmacological Substances, Izdatelstvo Meditsina, Mos-
cow 2005.
[15] S. Duraffour, R. Snoeck, R. de Vos, J. van Den Oord, J. Crance,
D. Garin, D. Hruby, R. Jordan, D. E. Clercq, A. Graciela, Antivir. Ther.
2007, 12(8), 1205.
CONFLICTS OF INTERESTS
[16] C. Melis, R. Meleddu, A. Angeli, S. Distinto, G. Bianco, C. Capasso,
F. Cottiglia, R. Angius, C. T. Supuran, E. Maccionia, J. Enzyme Inhib.
Med. Chem. 2017, 32, 68. https://doi.org/10.1080/14756366.2016.
1235042
The authors declare that there are no conflicts of interests.
ORCID
[17] A. Sokolova, O. Yarovaya, M. Semenova, A. Shtro, Y. Orshanskay,
V. Zarubaev, N. Salakhutdinov, MedChemComm 2017, 8, 960.
https://doi.org/10.1039/C6MD00657D
Anastasiya S. Sokolova
https://orcid.org/0000-0001-5227-9996
[18] A. Sokolova, O. Yarovaya, A. Shtro, M. Borisova, E. Morozova,
T. Tolstikova, V. Zarubaev, N. Salakhutdinov, Chem. Heterocycl.
Compd. 2017, 53, 371. https://doi.org/10.1007/s10593-017-
2063-3
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How to cite this article: 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,
Arch. Pharm. 2021, e2100038.
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