Tick-borne flavivirus reproduction inhibitors based on isoxazole core linked with adamantane

Article abstract of DOI:10.1016/j.bioorg.2019.03.028

Infections caused by flaviviruses pose a huge threat for public health all over the world. The search for therapeutically relevant compounds targeting tick-borne flaviviruses requires the exploration of novel chemotypes. In the present work a large series

Full text of DOI:10.1016/j.bioorg.2019.03.028

Accepted Manuscript
Tick-borne flavivirus reproduction inhibitors based on isoxazole core linked
with adamantane
Dmitry A. Vasilenko, Evgenia V. Dueva, Liubov I. Kozlovskaya, Nikolay A.
Zefirov, Yuri K. Grishin, Gennady M. Butov, Vladimir A. Palyulin, Tamara S.
Kuznetsova, Galina G. Karganova, Olga N. Zefirova, Dmitry I. Osolodkin,
Elena B. Averina
PII:
S0045-2068(18)31444-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.028
YBIOO 2854
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 December 2018
22 February 2019
13 March 2019
Please cite this article as: D.A. Vasilenko, E.V. Dueva, L.I. Kozlovskaya, N.A. Zefirov, Y.K. Grishin, G.M. Butov,
V.A. Palyulin, T.S. Kuznetsova, G.G. Karganova, O.N. Zefirova, D.I. Osolodkin, E.B. Averina, Tick-borne
flavivirus reproduction inhibitors based on isoxazole core linked with adamantane, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.03.028
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Tick-borne flavivirus reproduction inhibitors based on isoxazole core
linked with adamantane
Dmitry A. Vasilenkoa, Evgenia V. Duevaa,b,†, Liubov I. Kozlovskayab,c, Nikolay A. Zefirova,d,
Yuri K. Grishina, Gennady M. Butove, Vladimir A. Palyulina,d, Tamara S. Kuznetsovaa,
Galina G. Karganovab,c, Olga N. Zefirovaa,d, Dmitry I. Osolodkina,b,c,*, and Elena B.
Averinaa,d,*
a Department of Chemistry Department, Lomonosov Moscow State University, Leninskie Gory
1 bd. 3, Moscow, 119991, Russia
b FSBSI “Chumakov FSC R&D IBP RAS”, Moscow, 108819, Russia
c Sechenov First Moscow State Medical University, Moscow, 119991, Russia
d Institute of Physiologically Active Compounds, Severny Proezd 1, Chernogolovka, Moscow
Region, 142432, Russia
e Volgograd State Technical University, VSTU Lenina avenue, 28, Volgograd, 400005, Russia
† Present address: Vancouver Prostate Centre, Department of Urologic Sciences, The
University of British Columbia, Vancouver V6H 3Z6, BC, Canada
* Corresponding authors. E-mail: elaver@med.chem.msu.ru (E.B. Averina),
dmitry_o@qsar.chem.msu.ru (D.I. Osolodkin)
Graphical Abstract
Abbreviations: β-OG, n-octyl-β-d-glucoside; TBEV, tick-borne encephalitis virus; OHFV,
Omsk hemorrhagic fever virus; POWV, Powassan virus; PEK, porcine embryo kidney; FBS,
fetal bovine serum.
Abstract
Infections caused by flaviviruses pose a huge threat for public health all over the
world. The search for therapeutically relevant compounds targeting tick-borne flaviviruses
requires the exploration of novel chemotypes. In the present work a large series of novel
polyfunctionalized isoxazole derivatives bearing substituents with various steric and
1

electronic effects was obtained by our unique versatile synthetic procedure and their antiviral
activity against tick-borne encephalitis, Omsk hemorrhagic fever, and Powassan viruses was
studied in vitro. The majority of studied isoxazoles showed activity in low micromolar range.
No appreciable cytotoxicity was observed for tested compounds. The lead compounds, 5-
aminoisoxazole derivatives containing adamantyl moiety, exhibited strong antiviral activity
and excellent therapeutic index.
Keywords: aminoisoxazoles, adamantane derivatives, tick-borne encephalitis virus, Omsk
hemorrhagic fever, Powassan virus, flavivirus
1. Introduction
Flaviviral (genus Flavivirus, family Flaviviridae) infections, such as dengue fever,
West Nile disease, Zika fever, yellow fever, Japanese encephalitis, tick-borne encephalitis,
etc., pose a huge threat for public health all over the world. Vaccines are available only
against yellow fever, Japanese encepahlitis, and tick-borne encephalitis, but they can be used
only as preventive measures and do not provide 100% protection. The situation is complicated
by the absence of any anti-flaviviral drugs, and only symptomatic treatment of flaviviral
diseases is practiced. Though mosquito-borne flaviviral diseases are more incident and spread
all over the world, tick-borne flaviviruses are especially important for public health in the
Northern hemisphere. Tick-borne encephalitis virus (TBEV) causes up to 10,000 cases every
year in the Northern Eurasia. Powassan virus (POWV) and Omsk hemorrhagic fever virus
(OHFV) are of local importance. With the global climate changes in the last decades, ticks
moved further north, and the incidence and area of tick-borne flaviviral diseases increased
dramatically [1-3], raising an urgent need for new antiflavivirals.
A widely used strategy of design of antivirals against enveloped viruses, including
flaviviruses, is the inhibition of virus entry into the host cells through prevention of the fusion
between cellular and virion membranes (reviewed in [3]). One of the possible molecular
targets for flavivirus fusion inhibitors is the envelope protein E, involved in this process.
Earlier we constructed a homology model for tick-borne encephalitis virus E protein based on
template structures with a detergent n-octyl-β-D-glucoside (β-OG) molecule bound in a
hydrophobic pocket [4]. Small molecules may occupy the pocket and prevent conformational
rearrangement of the E protein, required for membrane fusion and viral genome release into
the cytoplasm.
2

As far as protein composition and structure of flaviviruses, especially tick-borne ones,
are rather conserved [5], TBEV fusion inhibitors can be expected to inhibit the reproduction
of other group members as broad-spectrum antivirals.
Representatives of several chemical classes were assessed against flavivirus
reproduction at the stage of entry. Various heterocyclic compounds, rigid amphipathic fusion
inhibitors (RAFIs), organoselenium compounds showed activity in micromolar range and
lower (reviewed in [1]).
Figure 1. Examples of TBEV fusion inhibitors studied earlier (1–3) and in the present work
(4).
Previously, our search for structurally different inhibitors of TBEV reproduction based
on the results of virtual or exploratory screening [4, 6-12] revealed the pronounced antiviral
activity of derivatives of pyrido[2,1-b][1,3,5]thiadiazines (e.g., 1, Figure 1), while a search
among 1,4-dihydropyridines (e.g., 2) or 4-aminotetrahydroquinazoline N-oxides (e.g., 3) led
to less potent and rather toxic compounds.
As the enhancement of structural diversity provides opportunities for the development
of antiflavivirals with greater therapeutic potential, in the present work we aimed to
synthesize a novel series of compounds based on a scaffold never assessed against
flaviviruses and to study their activity against tick-borne flaviviruses (TBEV, POWV,
OHFV). We studied the isoxazolyl-based compounds of general formula 4 (Figure 1) due to
their structural compactness, synthetic availability, and possibility of diverse
functionalization.
2. Results and Discussion
2.1 Chemistry
The synthesis of a series of novel 5-aminoisoxazoles 4a-o with various structures was
performed using a three-step synthetic procedure based on our previously described method
[13-17]. The route to target compounds 4a-o includes the preparation of unsaturated esters 5a-
o, their subsequent heterocyclization under the treatment with activated tetranitromethane,
followed by the reduction of 5-nitroisoxazoles 6a-o (Table 1). The majority of starting
alkenes (5c-n) was synthesized by the acylation of corresponding alcohol in the presence of
3

base. The ester 5o was obtained by Wittig reaction from phosphonium ylide of 1-
adamantylmethyl iodoacetate and 3-phenylpropanal. All synthesized compounds were
1
characterized and identified using H and 13C NMR spectroscopy, and also HRMS or
elemental analysis.
Table 1. Three-step procedure for the synthesis of 5-aminoisoxazoles 4a-o.
R1
R2
Yield, %a
Compounds Compounds
7
4-6
5
6
4
b
t-Bu
H
H
-
-
75c
36c
45 (A)c
72 (A)c
a
a
b
PhCH2
b
b
H
H
84c
90c
57c
58c
62 (A)
67 (A)
c
c
d
d
H
52
44
55 (A)
e
e
H
H
61c
54c
33c
63c
68 (A)
34 (A)
f
f
g
g
4

R1
R2
Yield, %a
6
Compounds Compounds
7
4-6
5
4
H
H
43
32
65 (B)
h
h
50
44
49 (В)
i
i
H
H
H
H
83c
68c
75c (A)
j
j
78c
72c
82c
84c
55c
78 (A)
52 (A)
56 (A)
k
l
k
l
36c
54
m
m
Me
78
74 (B)
64 (B)
j
j
n
o
CH2CH2Ph
84d
37
a
b
c
Isolated yields after chromatographic purification. Commercially available. Compounds were
d
synthesized by known procedures [15]. Alkene 5o was synthesized by Wittig reaction (see
Supplementary data).
2.2 Assessment of cytotoxicity and antiviral activity
Based on the data obtained in our previous studies and aiming to find flavivirus fusion
inhibitors that may interact with the β-OG pocket of the E protein, we challenged isoxazole
derivatives 4a-o with different lipophilic moieties in positions 3 and 4 of heterocycle against
TBEV, POWV, and OHFV reproduction in in vitro plaque formation inhibition test, and 50%
effective concentrations were calculated (EC50). The viruses were studied in permissive
porcine embryo kidney (PEK) cell line and conditions. Cytotoxicity was estimated in the
5

same cell line for active compounds as the concentration causing death of 50% of the cell
monolayer (CC50) acutely (24 h exposition) and chronically (7 days exposition).
Compounds 4a-d bearing lipophilic tert-butyl, benzyl, 1-adamantyl or 2-adamantyl
moieties in the ester groups showed similar moderate anti-flavivirus activity (Table 2). The
EC50 values were the lowest for both 1-adamantyl and 2-adamantyl containing isoxazole
derivatives 4c,d and did not depend on the position of the adamantyl substitution. However,
the 1-adamantyl derivative 4c was less toxic than 4d.
Table 2. Cytotoxicity and antiviral activity of the compounds with variations of the lipophilic moiety
Cmpd.
R
anti-TBEV
EC50, M
17 ± 3
anti-OHFV
EC50, M
> 50
anti-POWV
EC50, M
13 ± 3
CC50, M
24 h
7 days
>500
324
t-Bu
PhCH2
>500
>500
>500
4a
4b
4c
19 ± 6
11 ± 4
> 50
26 ± 1
31 ± 7
12 ± 5
382
350
160
11 ± 2
12 ± 1
30 ± 1
ND
13 ± 6
ND
4d
N-Butyl-2-
methyl-
5,6,7,8-
> 50
> 50
Reference
[12]
tetrahydroq
uinazoline-
4-amine 1-
oxide
ND, not determined
To probe the additional modifications of the 1-adamantyl moiety, we introduced there
the substituents with different steric and electronic effects (compounds 4e-h, 6h) or replaced
it with a more flexible bicyclo[3.3.1]nonane moiety (compound 4i, analogous to adamantyl
containing heterocycle 4e, Table 3). The introduction of the substituents into adamantane
core, regardless of their type, bulkiness, number, and positions, did not lead to improvement
of antiviral properties as compared to the parent compound 4c. Both carbomethoxy
derivatives 4i and 4e with different conformational flexibility of the polycyclic moiety were
inactive against studied tick-borne flaviviruses. Addition on acetamide to the adamantyl also
rendered the compounds inactive (4h, 6h).
Table 3. Cytotoxicity and antiviral activity of the compounds with variations in the adamantane core.
Cmpd.
Y
anti-TBEV
EC50, M
anti-OHFV
EC50, M
anti-POWV
EC50, M
R
CC50, M
24 h 7 days
6

Cmpd.
Y
anti-TBEV
EC50, M
anti-OHFV
EC50, M
anti-POWV
EC50, M
R
CC50, M
24 h
7 days
4e
NH2
ND
ND
>50
>50
>50
4f
NH2
NH2
>500
354
>500
354
29 ± 3
13 ± 2
>50
18 ± 7
8 ± 2
4g
6h
22 ± 5
NO2
ND
ND
>50
ND
ND
4h
4i
NH2
NH2
ND
ND
ND
ND
>50
>50
>50
>50
>50
>50
ND, not determined
More successful against TBEV reproduction was the modification of the linker
between the isoxazole and adamantane fragments (Table 4). The elongation of the linker by
one methylene group (4j) led to a substantial increase in the antiviral activity, about one order
of magnitude. Further elongation and/or modification of the linker, including its
conformational restriction, did not improve the activity against TBEV (4k-m). The activity of
the studied subseries against OHFV and POWV reproduction was less prominent.
Table 4. Cytotoxicity and antiviral activity of the compounds with linker variations.
Cmpd.
X
Y
anti-TBEV
EC50, M
1.7 ± 0.1
anti-OHFV
EC50, M
24 ± 3
anti-POWV
EC50, M
24 ± 8
CC50, M
24 h
7 days
-CH2-
-CH2-
NH2
NO2
NH2
NH2
NH2
88
NDa
>500
ND
88
4j
6j
NDa
416
ND
273
17 ± 3
14 ± 2
>50
ND
13 ± 5
>50
ND
-CH2-CH2-
-O-CH2-CH2-
12 ± 2
38 ± 12
29 ± 5
4k
4l
386
20 ± 3
22 ± 20
4m
7

a СС50 was not determined due to the ability of 6j to cause the cytoskeleton disruption.
ND, not determined
The synthetic precursor of heterocycle 4j, corresponding 5-nitroisoxazole 6j, showed a
significant antiviral activity decrease, confirming the importance of the presence of the amino
group in the heterocycle. Finally, the introduction of a lipophilic β-phenylethyl substituent in
the position 4 of the isoxazole ring (4o) not only maintained reasonable antiviral properties
against TBEV, but markedly reduced the toxicity compared to heterocycle 4j. Moreover, the
activity of 4o against OHFV reproduction was the highest from the whole series. Thus, the
therapeutic index of 4o was the best in the studied series of isoxazole derivatives (Table 5).
Table 5. Cytotoxicity and antiviral activity of the compounds with variation of the isoxazole
core.
Cmpd.
R
anto-TBEV
EC50, M
22.4 ± 0.6
anti-OHFV,
EC50, M
25 ± 5
anti-POWV
EC50, M
14 ± 3
CC50, M
24 h
7 days
Me
118
62
4n
4o
CH2CH2Ph
>500
416
3.7± 0.2
12 ± 2
15 ± 2
2.3. Mechanism of action studies
A ‘time-of-addition’ experiment was performed to elucidate the stage of flavivirus
replication cycle inhibited by the compounds. During this experiment the compounds were
added to the infected cells at the different stages of viral replication cycle to elucidate the
inhibited stage that decreases viral reproduction the most. The experiment was performed
using TBEV only. We compared the effect of compound addition on the several stages of
viral replication cycle: before infection on cells or virions (viral adsorption and entry,
including fusion), on viral protein synthesis, and viral genome replication. To achieve that, we
estimated and compared with the control the viral yields after infection of the cells with
TBEV after pretreatment of the cells with solutions of the compounds with concentration of
5×EC50, virus preincubation with the compounds, or when a compound solution was added at
the start of viral protein synthesis at 2 hours post infection (h.p.i.) or at the start of viral
genome replication at 5 h.p.i. Two potent compounds with different EC50 values, 4b (EC50 19
8

µM) and 4o (EC50 3.7 µM), were studied, DMSO-treated cells were used as a control, and the
results were summarized in Figure 2.
Figure 2. ‘Time of addition’: Comparison of the viral yields on the control DMSO-
treated cells after infection with TBEV (MOI 0.1 PFU/cell) with viral yields on the cells,
treated with compounds 4b or 4o (5×EC50, 95 and 18.5 µM) on the various stages of
infection. (* - inhibition is statistically significant in comparison with a DMSO-treated
control)
Addition of a compound solution 2 or 5 h.p.i. did not inhibit viral reproduction. Virus
exposition to the compound before the interaction with the cells significantly decreased virus
growth in comparison with DMSO-treated control. The effect was the most pronounced upon
the preincubation of the virus with a compound, implying the direct interaction of the
compound with the virion. However, the preincubation of the cells with the compound
decreases viral yields as well. Thus, we can expect a compound to interact with the cellular
targets involved into viral attachment and entry. Nevertheless, compounds inhibit viral
replication cycle on the first stages, similarly to the results of the previous studies. Most
likely, the compounds do interact with the E protein molecule, as it is the only one fully
exposed on the flavivirus virion surface.
9

2.4. Docking studies
Docking studies were performed to investigate possible binding modes of all the
studied compounds in β-OG pocket of TBEV E protein. The homology model, structure
preparation and docking protocol were the same as in the previous study [12]. The β-OG
pocket has the shape of a funnel open to the space surrounding the virus particle (Figure 3).
Majority of the compounds, bearing isoxazole warhead unsubstituted in the position 4, were
docked in an orientation with the warhead buried deep in the pocket and forming hydrogen
bonds with Gln214 amide group and Val273 backbone carbonyl (Figure 3A,B). Adamantyl
substituent is largely exposed to the environment and may form hydrophobic interactions;
however, the entrance to the pocket is open enough to allow different substitutions without
substantial changes in the binding mode. That explains the limited influence of the adamantyl
modification on the activity.
Figure 3. Predicted binding modes of compounds 4j (A, B) and 4o (C, D) in β-OG pocket of
TBEV E protein. Panels A, C: side view (environment on the right, virus membrane on the
left). Panels B, D: view from outside. Hydrogen bonds predicted by VIDA ‘FRED view’ are
shown as green lines and participating amino acid residues are inscribed.
On the other hand, compound 4o bears a bulky substituent in the position 4 of the
isoxazole ring. Thus, realization of the common binding mode becomes impossible due to a
10

very limited volume of the funnel, and the compound binds mostly on the periphery of the
pocket (Figure 3C,D). In this binding mode, a hydrogen bond is predicted between the
isoxazole oxygen and Tyr132 phenolic hydroxyl. Such a binding mode appears to be more
susceptible to the amino acid variability, which is more common for the pocket periphery than
for its deep region. Additional substituent extending into the pocket could improve the
binding between the compound and the E protein.
The results of the docking modeling are consistent with the possibility of binding the
suggested compounds in the β-OG pocket.
2.5. Predicted ADMET profiles and physico-chemical properties of compounds
In order to assess the suitability of the synthesized compounds for the in vivo studies,
we have performed a preliminary computational estimation of certain ADMET and physico-
chemical properties, relevant in the drug development context. In particular, the human
intestinal absorption (HIA) [18], blood-brain barrier permeability (LogBB) [19], and hERG-
mediated cardiac toxicity risk (channel affinity pKi and inhibitory activity pIC50) [20] were
estimated using the predictive QSAR models based on the accurate and representative training
sets, fragmental descriptors, and artificial neural networks. The lipophilicity (logPow) for the
neutral compound and the aqueous solubility (pS) were estimated by the ALogPS 3.0 neural
network model based on the E-State descriptors implemented in the OCHEM platform [21].
The predicted properties of the compounds (Supplementary Table S1) seem to be reasonable
for early lead candidates, including excellent intestinal absorption, medium or low blood-
brain barrier permeability, acceptable hERG liability risks, as well as moderate lipohilicity
and good solubility. Moreover, the ADMET properties will be inevitably optimized at later
stages of the development.
3. Conclusion
A series of novel isoxazole derivatives was synthesized and tested for activity against
TBEV and POWV reproduction in vitro. These heterocycles represent a novel type of small-
molecule inhibitors of flaviviruses. The investigation led to the discovery of two potent and
synthetically accessible 5-aminoisoxazole derivatives containing adamantyl moiety, one of
them possessing an attractive therapeutic index. Realization of the antiviral activity on the
virus attachment and entry stages was confirmed by the ‘time-of-addition’ experiments.
Studied heterocycles may be considered as a promising and useful scaffold for the further
optimization of antiviral activity.
11

4. Experimental Section
4.1. Chemistry
4.1.1. General information
1
NMR spectra were recorded on spectrometer Agilent 400-MR (400.0 MHz for H;
100.6 MHz for 13C, 161.9 MHz for 31P) at room temperature; chemical shifts (δ) were
1
measured with reference to the solvent CDCl3 for Н (δ = 7.26 ppm) and 13C (δ = 77.1 ppm),
31
and 85% H3PO4 for P as external standard. Chemical shifts (δ) are given in ppm; J values
are given in Hz. When necessary, assignments of signals in NMR spectra were made using 2D
techniques. Accurate mass measurements (HRMS) were performed on a Bruker micrOTOF II
instrument using electrospray ionization (ESI). The measurements were done in a positive ion
mode (interface capillary voltage 4500 V) or in a negative ion mode (3200 V). Analytical thin
layer chromatography was carried out with Silufol silica gel plates (supported on aluminum);
the detection was done by UV lamp (254 and 365 nm) and chemical staining (5% aqueous
solution of KMnO4). Column chromatography was performed on silica gel (230–400 mesh,
Merck). Compounds 4a [16], 4b [16], 4j [15], 5c [16], 5d [16], 5f [16], 5g [16], 5j [16], 5k
[16], 5l [16], 5m [16], 6a [16], 6b [16], 6c [16], 6d [16], 6f [16], 6g [16], 6j [22], 6k [16], 6m
[16], 7c [23], 7d [24], 7e [25,26], 7f [25,26], 7g [27], 7h [28-30], 7i [31], 7j [32], 7k [33],
7l [34], and 7m [35] were synthesized by known procedures.
All other starting materials were commercially available.
All reagents except commercial products of satisfactory quality were purified by
literature procedures prior to use.
4.1.2. General procedure for the preparation of compounds 4 a-o
Method A: To a stirred solution of 5-nitroisoxazole (0.3 mmol) in 96% EtOH (1 mL),
SnCl2 (285 mg, 1.5 mmol) was added in one portion. The reaction mixture was stirred for 2 h
at r.t., then the solvent was evaporated in vacuo and the residue was dissolved in CH2Cl3 (20
mL) and washed with sat. aq NaHCO3 (resulting in a solution at pH 8). The aqueous phase
was extracted with CH2Cl2 (3 × 10 mL) and the combined organic layers were dried over
MgSO4. The solvent was evaporated in vacuo and the residue was purified by preparative
column chromatography on silica gel.
Method B: To a stirred solution of 5-nitroisoxazole (0.3 mmol) in a mixture (1:1)
THF-H2O (2 mL) Na2S2O4 (392 mg, 1.8 mmol) was added in one portion at room
o
temperature. The resulting mixture was stirred for 1 h at 90 C. Then the mixture was cooled
to room temperature, water (5 mL) was added and extracted with EtOAc (3×10 mL). The
12

combined organic layer was dried over anhydrous MgSO4. The solvent was evaporated in
vacuo; the residue was purified by preparative column chromatography on silica gel.
4.1.2.1. 1-Adamantyl 5-aminoisoxazole-3-carboxylate (4c). Colourless solid (49 mg, 62%, A),
1
mp: 188-190°С, Rf (petroleum ether/EtOAc 2:1): 0.20; H NMR (400.0 MHz, CDCl3) ( ,
ppm): 1.62–1.72 (m, 6H, 2CH2(Ad)), 2.16–2.20 (m, 3H, 3CH(Ad)), 2.20–2.24 (m, 6H,
3CH2(Ad)), 4.63 (br.s, 2H, NH2), 5.44 (s, 1H, CH); 13C NMR (100.6 MHz, CDCl3) ( , ppm):
30.9 (3CH(Ad)), 36.1 (3CH2(Ad)), 41.2 (3CH2(Ad)), 80.7 (CH), 83.2 (C(Ad)), 158.8 (C),
+
159.1 (C=O), 169.4 (CNH2); HRMS-ESI m/z [M+H]+ calcd for C14H19N2O3 : 263.1390,
found 263.1390; Anal. calcd for С14Н18N2O3: C 63.11, H 6.92, N 10.68, found: C 63.98, H
7.15, N 10.63.
4.1.2.2. 2-Adamantyl 5-aminoisoxazole-3-carboxylate (4d). Colourless solid (53 mg, 67%, A),
1
mp: 171-174°С, Rf (petroleum ether/EtOAc 3:1): 0.30; H NMR (400.0 MHz, CDCl3) ( ,
ppm): 1.52–1.60 (m, 2H, 2CH2(Ad)), 1.69–1.74 (m, 2H, 2CH2(Ad)), 1.74–1.81 (m, 2H,
2CH2(Ad)), 1.81–1.90 (m, 4H, 2CH+CH2(Ad)), 2.03–2.14 (m, 4H, 2CH2 (Ad)+2CH(Ad)),
4.86 (br.s, 2H, NH2), 5.11–5.15 (m, 1H, CHO), 5.50 (s, 1H, CH); 13C NMR (100.6 MHz,
CDCl3) ( , ppm): 26.9 (CH(Ad)), 27.1 (CH(Ad)), 31.7 (2CH2(Ad)), 31.8 (2CH(Ad)), 36.2
(2CH2(Ad)), 37.2 (CH2(Ad)), 78.9 (CHO), 80.7 (CH), 157.9 (C), 159.8 (C=O), 169.9 (CNH2);
HRMS-ESI m/z [M+Na]+ calcd for C14H18N2O3Na+: 283.1210, found: 285.1210. Anal. calcd
for С14Н18N2O3: C 63.11, H 6.92, N 10.68, found: C 63.42, H 7.23, N 10.36.
4.1.2.3. 3-(Methoxycarbonyl)-1-adamantyl 5-aminoisoxazole-3-carboxylate (4e). Colourless
1
solid (53 mg, 55%, A), mp: 166-167°С, Rf (petroleum ether/EtOAc 2:1): 0.20; H NMR
(400.0 MHz, CDCl3) ( , ppm): 1.57–1.68 (m, 2H, CH2(Ad)), 1.81–1.88 (m, 4H, 2CH2(Ad)),
2.14–2.20 (m, 2H, 2CH2(Ad)), 2.20–2.26 (m, 2H, 2CH2(Ad)), 2.27–2.32 (m, 2H, 2CH(Ad)),
2.34 (br.s, 2H, CH2(Ad)), 3.64 (s, 3H, CH3), 4.70 (br.s, 2H, NH2), 5.43 (s, 1H, CH); 13C NMR
(100.6 MHz, CDCl3) ( , ppm): 30.3 (2CH(Ad)), 35.0 (CH2(Ad)), 37.7 (2CH2(Ad)), 40.2
(2CH2(Ad)), 42.2 (CH2(Ad)), 44.1 (C(Ad)), 51.8 (C-O), 80.5 (CH), 82.5 (CH3), 158.5 (C),
+
159.0 (C=O), 169.6 (CNH2).176.4 (C=O); HRMS-ESI m/z [M+H]+ calcd for C16H21N2O6 :
321.1445, found: 321.1444; Anal. calcd for С16Н20N2O5: C 59.99, H 6.29, N 8.74, found: C
59.85, H 6.38, N 8.42.
4.1.2.4. 3-(i-Propoxycarbonyl)-1-adamantyl 5-aminoisoxazole-3-carboxylate (4f). Colourless
1
solid (71 mg, 68%, A), mp: 177-178°С, Rf (petroleum ether/EtOAc 2:1): 0.42; H NMR
(400.0 MHz, CDCl3) ( , ppm): 1.18 (d, J = 6.3 Hz, 6H, 2CH3), 1.58–1.70 (m, 2H, CH2(Ad)),
13

1.77–1.87 (m, 4H, 2CH2(Ad)), 2.13–2.20 (m, 2H, 2CH2(Ad)), 2.23–2.28 (m, 2H, 2CH2(Ad)),
2.28–2.33 (m, 4H, CH2(Ad)+2CH(Ad)), 4.64 (br.s, 2H, NH2), 4.95 (sept, J = 6.3 Hz, 1H,
OCH), 5.45 (s, 1H, CH); 13C NMR (100.6 MHz, CDCl3) ( , ppm): 21.7 (2CH3), 30.4
(2CH(Ad)), 35.0 (CH2(Ad)), 37.6 (2CH2(Ad)), 40.2 (2CH2(Ad)), 42.2 (CH2(Ad)), 44.0
(C(Ad)), 67.5 (CH-O), 80.6 (CH), 82.7 (C-O), 158.5 (C), 159.0 (C=O), 169.5 (CNH2).175.4
+
(C=O); HRMS-ESI m/z [M+H]+ calcd for C18H25N2O5 : 349.1758; found 349.1755; Anal.
calcd for С18Н22N2O5: C 62.05, H 6.94, N 8.04, found: C 61.93, H 6.71, N 7.78.
4.1.2.5. 3,5-Dimethyl-1-adamantyl 5-aminoisoxazole-3-carboxylate (4g). Colourless solid (30
1
mg, 34%, A), mp: 146-148°С, Rf (petroleum ether/EtOAc 4:1): 0.24; H NMR (400.0 MHz,
CDCl3) ( , ppm): 0.86 (s, 6H, 2CH3), 1.09–1.23 (m, 2H, CH2 (Ad)), 1.24–1.31 (m, 2H,
2CH2(Ad)), 1.35–1.41 (m, 2H, 2CH2(Ad)), 1.79–1.91 (m, 4H, 2CH2(Ad)), 2.05 (br.s, 2H,
CH2(Ad)), 2.20–2.25 (m, 1H, CH(Ad)), 4.77 (s, 2H, NH2), 5.41 (s, 1H, CH); 13C NMR (100.6
MHz, CDCl3) ( , ppm): 29.8 (2CH3), 31.2 (CH(Ad)), 34.1 (2C(Ad)), 39.6 (CH2(Ad)), 42.4
(2CH2(Ad)), 47.1 (2CH2(Ad)), 50.4 (CH2(Ad)), 80.5 (CH), 84.5 (C (Ad)), 158.7 (C), 159.2
(C=O), 169.6 (C-NH2). Anal. calcd for С18Н22N2O5: C 66.18, H 7.64, N 9.65, found: C 66.17,
H 7.31, N 9.53.
4.1.2.6. 3-(Acetylamino)-5,7-dimethyl-1-adamantyl 5-aminoisoxazole-3-carboxylate (4h).
1
Colourless solid (68 mg, 65%, B), mp: 119-122°С, Rf (CHCl3/MeOH 20:1): 0.35; H NMR
(400.0 MHz, CDCl3) ( , ppm): 0.93 (s, 6H, 2CH3), 1.12–1.20 (m, 2H, CH2(Ad)), 1.59–1.65
(m, 2H, 2CH2(Ad)), 1.67–1.73 (m, 2H, 2CH2(Ad)), 1.81–1.92 (m, 4H, 2CH2(Ad), 1.86 (s, 3H,
СH3), 2.35 (br.s, 2H, CH2(Ad)), 4.79 (br.s, 2H, NH2), 5.34 (br.s 1H, NH), 5.40 (s, 1H, CH);
13C NMR (100.6 MHz, CDCl3) ( , ppm): 24.5 (CH3), 29.0 (2CH3), 34.2 (2C(Ad)), 43.5
(CH2(Ad)), 46.1 (2CH2(Ad)), 46.4 (2CH2(Ad)), 49.3 (CH2(Ad)), 54.9 (C(Ad)), 80.4 (CH),
83.6 (C(Ad)), 158.4 (C), 159.1 (C=O), 169.6 (C), 169.7 (C); HRMS-ESI m/z [M+H]+ calcd
+
for C18H26N3O4 : 348.1918, found: 348.1918.
4.1.2.7. (3r,7r)-7-(Methoxycarbonyl)bicyclo[3.3.1]non-3-yl 5-aminoisoxazole-3-carboxylate
(4i). Colourless solid (45 mg, 49 %, B), mp: 177-178°С, Rf (petroleum ether/EtOAc 4:1):
1
0.10; H NMR (400.0 MHz, CDCl3) ( , ppm): 1.32–1.40 (m, 1H, CH2), 1.57–1.72 (m, 5H,
5CH2), 1.82–1.91 (m, 2H, 2CH2), 2.19–2.26 (m, 2H, 2CH), 2.25 (ddd, J = 14.6, 8.6, 6.9 Hz,
2H, 2CH2), 3.25 (J = 12.8, 5.1 Hz , 1H, CH-C=O), 3.70 (s, 3H, CH3), 4.64 (br. s, 2H, NH2),
5.29 (quint, J = 6.8 Гц, 1H, OCH), 5.57 (s, 1H, CH); 13C NMR (100.6 MHz, CDCl3) ( ,
ppm): 25.8 (2CH), 29.8 (CH2), 32.8 (2CH2), 34.1 (2CH), 34.4 (CH2), 51.5 (CH-O), 69.3
(CH3), 80.5 (CH), 157.7 (C), 159.8(C=O), 169.6 (C-NH2), 177.7 (C=O); HRMS-ESI m/z
14

[M+K]+ calcd for C15H20N2O5K+: 347.1004, found: 347.1003.
4.1.2.8. 2-(1-Adamantyl)ethyl 5-aminoisoxazole-3-carboxylate (4k). Colourless solid (68 mg,
1
78%, A), mp: 152-154°С, Rf (petroleum ether/EtOAc 3:1): 0.34; H NMR (400.0 MHz,
CDCl3) ( , ppm): 1.50–1.56 (m, 8H, 3CH2(Ad)+CH2), 1.57–1.64 (m, 3H, 3CH2(Ad)), 1.64–
1.72 (m, 3H, 3CH2(Ad)), 1.90–1.96 (m, 3H, 3CH(Ad)), 4.36 (t, J = 7.4 Hz, 2H, OCH2), 4.69
(br.s, 2H, NH2), 5.50 (s, 1H, CH); 13C NMR (100.6 MHz, CDCl3) ( , ppm): 28.5 (3CH(Ad)),
31.8 (C(Ad)), 36.9 (3CH2(Ad)), 42.1 (CH2), 42.4 (3CH2(Ad)), 62.4 (CH2O), 80.6 (CH), 157.5
(C), 160.5 (C=O), 169.6 (CNH2); HRMS-ESI m/z [M+Na]+ calcd for C16H22N2O3Na+:
313.1523, found: 313.1532; Anal. calcd for С16Н22N2O3: C 66.18, H 7.64, N 9.65, found: C
66.58, H 7.94, N 9.67.
4.1.2.9. 2-(1-Adamantyloxy)ethyl 5-aminoisoxazole-3-carboxylate (4l). Yellow solid (48 mg,
1
52 %, A), mp: 178-179°С, Rf (petroleum ether/EtOAc 2:1): 0.10; H NMR (400.0 MHz,
CDCl3) ( , ppm): 1.53–1.65 (m, 6H, 3CH2(Ad)), 1.70–1.75 (m, 6H, 3CH2(Ad)), 2.09–2.16
(m, 3H, 3CH(Ad)), 3.72 (t, J = 5.3 Hz, 2H, CH2), 4.40 (t, J = 5.3 Hz, 2H, CH2), 4.62 (br.s,
2H, NH2), 5.52 (s, 1H, CH); 13C NMR (100.6 MHz, CDCl3) ( , ppm): 30.5 (3CH(Ad)), 36.4
(3CH2(Ad)), 41.4 (3CH2(Ad)), 57.8 (CH2O), 65.5 (CH2O), 72.7 (C(Ad)), 80.7 (CH), 157.3
(C), 160.3 (C=O), 169.5 (C-NH2); HRMS-ESI m/z [M+Na]+ calcd for C16H22N2O4Na+:
329.1472, found: 329.1471; Anal. calcd for С16Н22N2O4: C 62.73, H 7.24, N 9.14, found: C
62.91, H 7.09, N 9.25.
4.1.2.10. 4-(1-Adamantyl)benzyl 5-aminoisoxazole-3-carboxylate (4m). Colourless solid (59
1
mg, 56%, A), mp: 168-170°С, Rf (petroleum ether/EtOAc 4:1): 0.11; H NMR (400.0 MHz,
CDCl3) ( , ppm): 1.73–1.86 (m, 6H, CH2(Ad)), 1.89–1.97 (m, 6H, CH2(Ad)), 2.08–2.16 (m,
3H, 3CH(Ad)) 4.66 (br.s, 2H, NH2), 5.36 (s, 2H, CH2O), 5.55 (s, 1H, CH), 7.34–7.45 (m, 4H,
4CH(Ar)); 13C NMR (100.6 MHz, CDCl3) ( , ppm): 28.9 (3CH(Ad)), 36.1 (C(Ad)), 36.7
(3CH2(Ad)), 40.1 (3CH2(Ad)), 67.4 (CH2O), 80.7 (CH), 125.2 (2CH(Ar)), 128.6 (2CH(Ar)),
132.0 (C(Ar)), 151.9 (C(Ar)), 157.3 (C), 160.3 (C=O), 169.7 (C-NH2); HRMS-ESI m/z
[M+Na]+ calcd for C21H24N2O3Na+: 375.1679, found: 375.1672.
4.1.2.11. 1-Adamantylmethyl 5-amino-4-methylisoxazole-3-carboxylate (4n). Colourless solid
1
(64 mg, 74%, B), mp: 189-190°С, Rf (petroleum ether/EtOAc 4:1): 0.16; H NMR (400.0
MHz, CDCl3) ( , ppm): 1.56–1.60 (m, 6H, 2CH2(Ad)), 1.60–1.67 (m, 3H, 3CH2(Ad)), 1.67–
1.73 (m, 3H, 3CH2(Ad)), 1.93–1.98 (m, 3H, 3CH(Ad)), 1.99 (s, 3H, CH3), 3.92 (s, 2H, CH2),
3.73 (br. .s, 2H, NH2); 13C NMR (100.6 MHz, CDCl3) ( , ppm): 6.8 (CH3), 27.9 (3CH(Ad)),
15

33.4 (C(Ad)), 36.8 (3CH2(Ad)), 39.2 (3CH2(Ad)), 74.9 (CH2), 89.8 (C), 155.7 (C), 161.3
(C=O), 166.9 (CNH2); HRMS-ESI m/z [M+K]+ calcd for C16H22N2O3K+: 329.1262, found:
329.1263; Anal. calcd for С16Н22N2O3: C 66.18, H 7.64, N 9.65, found: C 66.19, H 7.68, N
9.59.
4.1.2.12. 1-Adamantylmethyl 5-amino-4-(2-phenylethyl)isoxazole-3-carboxylate (4o).
Colourless solid (73 mg, 64%, B), mp: 155-157°С, Rf (petroleum ether/EtOAc 4:1): 0.20; 1H
NMR (400.0 MHz, CDCl3) ( , ppm): 1.59–1.63 (m, 6H, 2CH2(Ad)), 1.63–1.68 (m, 3H,
3CH2(Ad)), 1.68–1.75 (m, 3H, 3CH2(Ad)), 1.96–2.02 (m, 3H, 3CH(Ad)), 2.71–2.78 (m, 2H,
CH2), 2.78–2.84 (m, 2H, CH2), 3.67 (br. s, 2H, NH2), 3.95 (s, 2H, CH2), 7.09–7.14 (m, 2H,
2CH(Ar)), 7.16–7.23 (m, 1H, CH(Ar)), 7.24–7.29 (m, 2H, 2CH(Ar)); 13C NMR (100.6 MHz,
CDCl3) ( , ppm): 24.3 (CH2), 27.9 (3CH(Ad)), 33.4 (C(Ad)), 36.1 (CH2), 36.8 (3CH2(Ad)),
39.2 (3CH2(Ad)), 75.0 (CH2), 93.9 (C), 126.3 (CH(Ar)), 128.6 (2CH(Ar)), 128.8 (2CH(Ar)),
141.5 (C(Ar)), 155.4 (C), 161.2 (C=O), 167.7 (CNH2); HRMS-ESI m/z [M+K]+ calcd for
C23H28N2O3K+: 419.1732, found: 419.1727; Anal. calcd for С23Н28N2O3: C 72.60, H 7.42, N
7.36, found: C 73.77, H 7.76, N 7.58.
4.2. Protocols of biological tests
4.2.1. Cells and viruses
Porcine embryo kidney (PEK) cell line was maintained at 37 °C in medium 199
(FSBSI “Chumakov FSC R&D IBP RAS”, Russia) supplemented with 5% fetal bovine serum
(FBS, Invitrogen, South America).
TBEV strain Absettarov (GenBank access no. KU885457) from laboratory collection
was isolated in the Leningrad region, USSR, in 1951 from the blood of a patient with acute
TBE. POWV strain Powassan 24 (GenBank access no. KU160627) from laboratory collection
was isolated from Ixodes persulcatus ticks in Primorsky kray, USSR, in 1976. OHFV strain
Nikitina (GenBank access no. GU290187) from laboratory collection was isolated in the
Omsk region, USSR, in 1948 from the blood of a patient with Omsk hemorrhagic fever.
Viruses were stored as aliquots of infected cultural fluid at −70°C.
4.2.2. Cytotoxicity test in PEK cells
The test was performed as described previously [4] with some changes. Two-fold
dilutions of studied compounds were prepared in medium 199 on Earle solution (FSBSI
“Chumakov FSC R&D IBP RAS”, Russia) in 96-well plates to obtain final concentrations
starting from 500 μM. Equal 100 μL aliquots of compound dilutions were added in three
16

replicates to each well of 96-well plates with PEK cell monolayers. Negative control cells
were treated with the same sequential concentrations of DMSO, as it was in compound
dilutions, or cultural medium with no additions. After incubation at 37 °C in CO2-incubator
on days 1 or 7 cultural supernatant was gently removed and the cells were washed with
phosphate buffered saline (PBS, Sigma) twice. Solution of 0.0002% neutral red in PBS was
added to the washed cells, and cells were incubated for 30 minutes at 37 °C in CO2-incubator.
Afterwards cells were gently washed with PBS twice and 100 μL of 96% ethanol were added.
Absorption was counted in MultiScan FC (Thermo) at 450 nm. CC50 was calculated according
to Reed-and-Muench method [36].
4.2.3. 50% plaque reduction test
The test was performed as described previously [4] with some changes. Four 4-fold
dilutions of studied compounds were prepared in medium 199 on Earle solution (FSBSI
“Chumakov FSC R&D IBP RAS”, Russia) to obtain final concentrations in the wells starting
from 50 µM. Equal volume of TBEV or POWV suspension, containing approximately 20-30
PFU, were added to each compound dilution. Control virus was added to the same sequential
dilutions of DMSO, as it was in compounds dilutions. Virus+compound and virus+DMSO
mixtures were incubated at 37 °C in CO2-incubator for 1 hour and then added to 24-well
plates with PEK cell monolayers in 2 replicates, and incubated at 37 °C in CO2-incubator for
1 hour with gentle shaking. Then, each well was overlaid with 0.85-1.26% methylcellulose
(Sigma) containing 2% FBS (Invitrogen, South America). After incubation at 37 °C in CO2-
incubator for 6 days cells were fixed with 96% ethanol, and foci were stained with 0.4%
gentian violet and counted. EC50 values for TBEV were calculated according to Reed-and-
Muench method [36] and for OHFV and POWV were estimated from the linear fitting of the
inhibition curves with OriginLab Corporation OriginPro 8.
N-Butyl-2-methyl-5,6,7,8-tetrahydroquinazoline-4-amine
1-oxide,
described
previously [12], was used as the reference compound.
4.2.4. ‘Time of addition’ experiments.
PEK cells were infected with TBEV strain Absettarov (MOI 0.1 PFU/cell) and treated
with 5×EC50 compound or DMSO (negative control) solution addition according to one of the
schemes:
1) Cells were incubated with 5×EC50 compound / DMSO solution for 2 h, washed and
then infected with virus;
2) Virus was incubated with 5×EC50 compound / DMSO solution for 2 h prior to
infection of the cells;
17

3) 5 µM compound / DMSO solution was added to the cells 2 h.p.i.;
4) 5 µM compound / DMSO solution was added to the cells 5 h.p.i.
In all the schemes virus was left with the cells for 2 h for sorption and entry, and then
cell monolayers were washed from unsorbed virus. Each experimental scheme was performed
in two replicates. Infected cells culture supernates were harvested 21 h.p.i., and viral yields
were determined by plaque formation assay under 1.26% methylcellylose (Sigma),
supplemented with 2% FBS (Invitrogen, South America). On the 6th day post infection
plaques were stained with 0.6% gentian violet and counted; virus yields were expressed as
lgPFU. Inhibition of viral reproduction was estimated as the difference between DMSO-
treated control and compound-treated cells for each scheme.
4.3. Molecular Docking
Molecular docking was performed according to the protocol described in ref. [12].
Conformational generation for small molecules was performed in OMEGA 2.4.6 [37,38],
partial atomic charges were calculated in QUACPAC 1.5.0 [39], docking was performed with
FRED 2.2.5 [40,41]. Figure 3 was rendered in VIDA 4.3.0 [42].
4.4. Prediction of ADMET profiles and physico-chemical properties
The human intestinal absorption (HIA) [18], blood-brain barrier permeability (LogBB)
[19], and hERG-mediated cardiac toxicity risk (channel affinity pKi and inhibitory activity
pIC50) [20] were estimated using the integrated online service for ADMET properties
prediction [43]. It implements the predictive QSAR models based on the accurate and
representative training sets, fragmental descriptors, and artificial neural networks. The
lipophilicity (logPow) for the neutral compound and the aqueous solubility (pS) were
estimated by the ALogPS 3.0 neural network model based on the E-State descriptors
implemented in the OCHEM platform [21].
Acknowledgements
The antiviral activity studies were supported by the Russian Science Foundation (grant
no. 16-15-10307). Compound synthesis was supported by IPhAC RAS program 45.5 and the
Russian Foundation for Basic Research (projects 18-03-00651 and 18-03-00524). Free
academic license for QUACPAC, OMEGA, FRED, and VIDA was kindly provided by
OpenEye Scientific Software Inc. The authors appreciate technical assistance by Alexey A.
Orlov and calculation of ADMET parameters by Dr. Eugene V. Radchenko.
18

Appendix A. Supplementary data
Supplementary data contain the additional experimental data and copies of 1H and 13C
NMR spectra of synthesized compounds.
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Highlights
 Novel 5-aminoisoxazoles were synthesized via three-step synthetic procedure.
 Two adamantyl substituted 5-aminoisoxazoles show promising anti-TBEV activity.
 The most active compounds exhibit high therapeutic index.
 Omsk hemorrhagic fever and Powassan viruses' reproduction is inhibited by
compounds.
 Time-of-addition assay shows that compounds block the early steps of viral life cycle.
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