2
E. Suslov et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 1
Antiviral activity and cytotoxicity of compounds 7a–g, 8a–c, e against influenza virus
A/Puerto Rico/8/34 (H1N1) in MDCK cells.
Compound
CC50a, mM
IC50b, mM
SIc
7a
7b
7c
7d
7e
7f
>1000
>1000
113 15
>99
1
8
2
3
3
1
907 62
242 18
178 11
>904
66
8
260 14
>943
>943
7g
14
8a
8c
533 36
>1823
196 11
>1041
>331
143 16
<2
13
3
Fig. 1. Structures of adamantane derivatives with anti-influenza activity.
64
6
>1041
1
and then to bispidinone hydrochloride 11 (Scheme 1). The interac-
tion between bispidinone 11 and monoterpenoid aldehydes 12a–g
yielded compounds 7a-g.16 Monoterpenoids (À)-myrtenal 12a,
citral 12c (a 1: 1 mixture of cis- and trans-isomers), citronellal
12d, and 7-hydroxycitronellal 12e were commercially available.
Ketoaldehyde 12b was synthesized from monoterpenoid (À)-ver-
benone via multistep synthesis in accordance with the published
procedure.17 Monocyclic aldehydes 12f and 12g were prepared
8d
8e
239 21
979 55
360 21
8
2
30
7
8
134 12
42
Rimantadine
6
a
CC50 is the median cytotoxic concentration, i.e. the concentration causing 50%
cell death.
IC50 is the 50% inhibiting concentration, i.e. the concentration causing 50%
decrease of virus replication.
b
c
SI is the selectivity index, the CC50/IC50 ratio.
by ozonolysis of (À)-
a-pinene according to the previously estab-
lished reaction pathway18 and by isomerization of (+)-
epoxide,19 respectively.
a-pinene
pene moiety (compound 7e) led to no anti-antiviral effect. Com-
pounds 7f,g containing monocyclic substituents had no activity.
For comparison, the antiviral activity of amine 14 (a structural
analog of amino-adamantane 1 with known antiviral properties)
was tested. It was found to have moderate activity and an SI of 13.
With the addition of a bicyclic monoterpene moiety to com-
pound 14, resulting in 8a, a slight increase in the activity was seen,
but also with a concomitant increase in cytotoxicity and a decrease
in the SI. Compound 8c, which contains a citral moiety, was com-
pletely inactive. However, the citronellal derivative 8d, which dif-
fers from 8c by the lack of one of the double bonds, exhibited high
To produce compounds of the 8 series, compound 10 was used
to synthesize oxime 13, further reduction of which yielded amino-
diaza-adamantane 14 (Scheme 1). The reaction of compound 14
with several aldehydes of the monoterpenoid series, followed by
the reduction of intermediate imines, led to the target compounds
8a,c-e.15,20 The 8 derivatives of the 12b,f,g aldehydes were not syn-
thesized due to their poor availability.
The synthesized compounds were studied21 for their antiviral
activity against the pandemic influenza virus A/Puerto Rico/8/34
(H1N1) cultivated in cell culture using the procedure described
by Sokolov et al.22 Cytotoxicity of the compounds was evaluated23
in uninfected MDCK cells as described previously.24 The obtained
data were used to calculate the selectivity index (SI) for each
derivative; compounds with SI = 10 and higher were considered
as active and the results are presented in Table 1.
Compound 7a, which contains a bicyclic monoterpene sub-
stituent, had no antiviral effect. The introduction of a keto group
(7b) resulted in a moderate antiviral effect (IC50 = 113 lM), low
cytotoxicity, and an SI of 8. Interestingly, the same SI and a compa-
rable activity were observed for the reference drug rimantadine 2.
Compounds 7c,d containing citral and citronellal moieties also
antiviral activity (IC50 of 8 lM) and moderate cytotoxicity, leading
to the highest SI of 30. Previously, compound 5 (Fig. 1), a structural
analog of 8d but lacking nitrogen atoms in the cycle, was found to
have a SI of 22 with IC50 of 18 l
M.10 Therefore, transition from
compound 5 to its diaza-adamantane analog 8d significantly
increased both the antiviral activity and the selectivity index. It
is worth noting that the introduction of an additional double bond
into derivatives of compound 5 led to a tenfold decrease in their
activity, as seen here for 8c and 8d.10 Addition of a hydroxy group
to the monoterpene moiety (8e) caused a sharp decrease in the
antiviral activity.
Based on the structures, the M2 channel is the most plausible
target for these compounds. The thirteen molecules (2, 7a–g, 8a–
c,e, 14) were docked against an influenza virus M2 protein channel
((PDB ID: 3C9J, resolution 3.5 Å),25 which was obtained from the
Protein Data Bank (PDB).26,27 The Scigress version FJ 2.6 program28
was used to prepare the crystal structure for docking, i.e., hydrogen
atoms were added and the co-crystallised amantadine (1) was
removed. The mutant was prepared by changing the Ser31 amino
acid residues to Asn31. The configuration of Asn31’s side chain
was taken from the 5C02 crystal structure of the mutant.29 The
centre of the binding pocket was defined as the nitrogen atom in
the amantadine (x = À14.735, y = 14.685, z = À1.856) with a radius
of 10 Å. The GoldScore (GS),30 ChemScore (CS),31,32 ChemPLP,33 and
Astex statistical potential (ASP)34 scoring functions were imple-
mented to validate the predicted binding modes and relative ener-
gies of the ligands using the GOLD v5.4 software suite.
exhibited moderate activity (IC50 of 60–100 lM), but were more
toxic than 7b. The insertion of a hydroxy group into the monoter-
The M2 protein channel of influenza A virus is a pH dependent
channel. It mediates protein-protein dissociation, which takes
place during a viral uncoating process when the virus is entrapped
in the acidic portion of the lumen of endosomes.35 This channel is a
tetrameric protein bundle with a pore that the anti-influenza drug
amantadine targets.36,37 Due to the high genetic variability of the
Scheme 1. Synthesis of diazaadamantane derivatives.