Anti-influenza activity of diazaadamantanes combined with monoterpene moieties

Article abstract of DOI:10.1016/j.bmcl.2017.08.062

The antiviral activity of several diaza-adamantanes containing monoterpenoid moieties against a rimantadine-resistant strain of the influenza A/Puerto Rico/8/34 (H1N1) virus was studied. Hetero-adamantanes containing monoterpene moieties at the aminal position of the heterocycle were found to exhibit lower activity compared to compounds with a diaza-adamantane fragment and a monoterpene moiety linked via an amino group at the 6-position of the hetero-adamantane ring. The highest selectivity index (a ratio of the 50% cytotoxic concentration to the 50% inhibitory concentration) out of 30 was observed for compound 8d, which contains a citronellal monoterpenoid moiety. Diaza-adamantane 8d was superior to its adamantane-containing analog 5 both in its anti-influenza activity and selectivity. Furthermore, 8d has more balanced physicochemical properties than 5, making the former a more promising drug candidate. Modelling these compounds against an influenza virus M2 ion channel predicted plausible binding modes to both the wild-type and the mutant (S31N).

Full text of DOI:10.1016/j.bmcl.2017.08.062

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-influenza activity of diazaadamantanes combined with
monoterpene moieties
b
a
a
Evgeniy Suslov a, Vladimir V. Zarubaev b, , Alexander V. Slita , Konstantin Ponomarev , Dina Korchagina ,
⇑
Daniel M. Ayine-Tora c, Jóhannes Reynisson c, Konstantin Volcho a,d, Nariman Salakhutdinov a,d
a Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia
b Pasteur Institute of Epidemiology and Microbiology, 14 Mira Str., 197101 St. Petersburg, Russia
c School of Chemical Sciences, University of Auckland, New Zealand
d Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
The antiviral activity of several diaza-adamantanes containing monoterpenoid moieties against a riman-
tadine-resistant strain of the influenza A/Puerto Rico/8/34 (H1N1) virus was studied. Hetero-adaman-
tanes containing monoterpene moieties at the aminal position of the heterocycle were found to
exhibit lower activity compared to compounds with a diaza-adamantane fragment and a monoterpene
moiety linked via an amino group at the 6-position of the hetero-adamantane ring. The highest selectivity
index (a ratio of the 50% cytotoxic concentration to the 50% inhibitory concentration) out of 30 was
observed for compound 8d, which contains a citronellal monoterpenoid moiety. Diaza-adamantane 8d
was superior to its adamantane-containing analog 5 both in its anti-influenza activity and selectivity.
Furthermore, 8d has more balanced physicochemical properties than 5, making the former a more
promising drug candidate. Modelling these compounds against an influenza virus M2 ion channel pre-
dicted plausible binding modes to both the wild-type and the mutant (S31N).
Received 14 June 2017
Revised 27 August 2017
Accepted 29 August 2017
Available online xxxx
Keywords:
Diaza-adamantane
Terpene
Influenza
Antivirals
Citronellal
Myrtenal
Ó 2017 Published by Elsevier Ltd.
Influenza is one of the most common infectious diseases
known; the infection or its complications cause up to 500,000
deaths every year.1 The high variability of the influenza virus
(aerosol dissemination mechanism, natural reservoirs of its
circulation (birds, pigs, etc.), and possibility of interspecific trans-
mission) makes vaccination strategies a challenge, so their effec-
tiveness largely depends on the prediction of strains that will
circulate in a given epidemiological season. Its high variability
enabled the influenza virus to develop resistance to the few known
drugs that are currently used (neuraminidase inhibitors, M2 chan-
nel blockers).2 Thus, developing novel anti-influenza drugs is of a
paramount importance.
Some of the first effective low-molecular weight antiviral agents
were adamantane derivatives, amantadine 1 and rimantadine 2
(Fig. 1).3 These compounds exert their antiviral effect by blocking
the virus-specific proton channel M2, impairing the virus’s ability
to enter the cell.4 However, due to the developed resistance of influ-
enza A virus to adamantyl-containing drugs,5 all of the viral strains
(H3N2 and H1N1) isolated in 2009 were not susceptible to these
drugs.2 The antiviral activity of adamantane derivatives is
known6–8 to be recovered by introducing substituents on the nitro-
gen atom. For example, compound 3 with heteroaromatic sub-
stituents at the nitrogen atom of 1-amino-adamantane (Fig. 1)
exhibited high activity.9 Furthermore, addition of a hydroxyl on
the adamantine scaffold (4) resulted in
a more drug-like
derivative.9
Previously, we demonstrated10 that the introduction of
a
monoterpenoid moiety to a 1- or 2-amino-adamantane fragment
resulted in high activity against the remantadine-resistant influ-
enza virus A(H1N1)pdm09, for compounds containing both acyclic
(5) and bicyclic (6) monoterpenoid moieties (Fig. 1). It should be
noted that these compounds are quite lipophilic, barring them from
in vivo experiments.
The insertion of nitrogen atoms into the adamantane core has a
significant effect on the physical and chemical properties of the
molecule, most notably reducing its lipophilicity.11,12 We decided
to prepare analogs of compounds 5 and 6 that contain two nitrogen
atoms in the cycle and study their antiviral activity for the first
time.
Compounds 7 and 8 were synthesized according to the previ-
ously described procedures.13–15 For this purpose, hexam-
ethylenetetramine 9 was first converted to the intermediate 10
⇑
Corresponding author.
E-mail address: zarubaev@gmail.com (V.V. Zarubaev).
http://dx.doi.org/10.1016/j.bmcl.2017.08.062
0960-894X/Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Suslov E., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.08.062

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.
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E. Suslov et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 2
Criteria of lead-like (LLS), drug-like (DLS) and known drug space (KDS) in terms of molecular descriptors and calculated molecular descriptors of compounds 5 and 8d.
Molecular descriptors
Criteria of spaces
LLS
Compounds
DLS
KDS
5
8d
Molecular weight (g mol-1
Lipophilicity (Log P)
Hydrogen bond donors (HD)
Hydrogen bond acceptors (HA)
Polar surface area (Å2) (PSA)
Rotatable bonds (RB)
)
300
3
3
3
60
3
500
5
5
10
140
10
800
6.5
7
15
180
17
289.5
5.3
1.0
1.5
10.3
7.0
319.5
2.9
1.0
5.5
19.8
7.0
influenza virus, most of the current isolates are resistant to the
adamantane derivatives. The resistance is mainly conferred by
amino acid substitutions in M2 proteins L26F, V27A, S31N and
G34E38,39, S31N being the most important and widely distributed.
In our study, the amantadine-resistant virus A/Puerto Rico/8/34
(H1N1) was used with a transmembrane domain sequence 22-
SSDPLTIAANIIGILHLTLWILDRL-46 that bears asparagine in position
31.
The modeling shows that the polar diazaadamantane moiety of
8d occupies the inner portion of the channel where the tetrameric
proteins converge, while the bulky substituent occupies the outer
wider divergent opening of the channel as shown in Fig. 2A. The
binding mode also shows that the side chain carbonyl group of
asparagine (Asn31) forms a non-classical hydrogen bond to C–H
in the diazaadamantane ring, and the backbone carbonyl oxygen
of alanine (Ala30) forms a hydrogen bond with the amine hydro-
gen on the ligand, as shown in Fig. 2B. This is a plausible binding
mode which is consistent with a possible orientation of the aman-
tadine drug molecule, which indicates that bulky groups can be
substituted on the amino group of the drug.25 All the substituted
ligands show similar binding modes, though unsubstituted ligands
2 and 14 are oriented in the opposite direction for the wild-type
structure. It is worth noting that ligands can flip in this channel,
as shown in NMR derived structures.40 The modelling to the
mutated structure gave similar results for the wild-type for GS
and CS. The binding scores are similar for all the ligands, including
reference compounds 2 and 14 with the exception of ASP giving
lower scores for the wild-type structure (see Table S1 in the SI).
Interestingly, using ChemPLP, 8d has the highest score for both
wild-type and the mutant. ChemPLP is reported to be the best or
one of the best performing scoring functions available.41,42 Fur-
thermore, 8d is in second place using CS for both structures and
had the best predicted score for the mutant and was in third place
for the wild-type using GS, indicating its tight binding to M2.
As can be seen from the model, 8d can theoretically bind with
both wild-type (S31) and mutated, rimantadine/amantadine resis-
tant (N31) M2. The spectrum of 8d’s activity will be determined in
separate study using a panel of influenza viruses differing in their
M2 structure. In addition to M2 inhibition, interaction of 8d with
other viral and/or cellular targets that are unrelated to M2 cannot
be ruled out. Further experiments are therefore needed to decipher
the specific mode of action of diazaadamantanes against influenza
virus, as well as to evaluate their ability to induce viral drug
resistance.
Next, the drug-like properties of 5 and its diazaadamantane
analogue 8d were compared. The definitions of lead-like (LLS),
drug-like (DLS), and known drug space (KDS) are given in Table 2.
The calculated mainstream molecular descriptors of molecular
weight (MW), water/octanol partition coefficient (log P), hydrogen
bond donors (HD), hydrogen bond acceptors (HA), polar surface
area (PSA), and rotatable bonds (RB) for 5 and 8d are also shown
in the Table 243
Both compounds are within the drug-like chemical space,
though, notably, 5 is placed in KDS by its predicted log P value of
5.3. Log P is often considered to be the most important molecular
descriptor because it is linked to toxicity issues and failure in clin-
ical trials.44–46 The PSA for the derivatives is quite low, both are in
the LLS with 5, with only half of 8d’s polar surface area. In all, it can
be argued that 8d conforms well to the DLS whereas 5 is less bal-
anced, with properties in all three defined areas of chemical space.
The QikProp 3.247 software package was used to calculate the
molecular descriptors of the compounds. The reliability of the pre-
diction power of QikProp is established for the molecular descrip-
tors used in this study.48
B
A
In conclusion, we studied the anti-influenza virus activity of
compounds combining hetero-adamantane and monoterpenoid
moieties. Although most of the tested compounds showed moder-
ate activity and a low selectivity index, we found that compound
8d had a high activity against the rimantadine-resistant strain of
the influenza A/Puerto Rico/8/34 (H1N1) virus and moderate cyto-
toxicity, which both led to a very favorable SI of 30. Compound 8d
had much higher activity than both amino-diazaadamantane 14
and its structural analog 5, without heteroatoms in the adaman-
tane scaffold. Finally, 8d has excellent drug-like properties,
whereas compound 5 is much less balanced. Finally, compound
8d demonstrated high affinity to an influenza virus M2 protein
channel, based on the molecular modelling results.
C
D
Fig. 2. The docked configuration of 8d in the binding site of Mutated M2 protein
channel as predicted by ChemScore (A). The ligand occupies the binding pocket. The
protein surface is rendered. Red depicts a negative partial charge on the surface,
blue depicts positive partial charge and grey shows neutral/lipophilic areas. (B)
Hydrogen bond is shown as a green dotted line between 8d and the amino acid
Ala30 whilst non-classical hydrogen bond is shown as a blue dotted line to Asn31.
(C) Close up of 8d in the binding pocket. (D) Hydrogen bonds are shown as green
lines between ligand 8d and the amino acids Ala30 and Ser31 in the wild-type
structure.
Please cite this article in press as: Suslov E., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.08.062

4
E. Suslov et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Na2SO4. Then drying agent was filtered off, and the solvent was distilled off on
Acknowledgments
a rotary evaporator.
21. The compounds in appropriate concentrations were dissolved in 0.1 ml DMSO
and final solutions were prepared by adding MEM with 1 mg/ml trypsin.
This work was supported by Russian Scientific Foundation
(grant 15-13-00017). Spectral and analytical investigations were
carried out at Collective Chemical Service Center of Siberian Branch
of the Russian Academy of Sciences.
Compounds were incubated with MDCK cells for
1 h at 36 °C. Each
concentration of the compounds was tested in triplicate. The cell culture was
then infected with influenza virus A/Puerto Rico/8/34 (H1N1) (moi 0.01) for 24
h at 36 °C in the presence of 5% CO2. A virus titer in the supernatant was
determined by hemagglutination test after cultivating of the virus progeny in
MDCK cells for 48 h at 36 °C in the presence of 5% CO2. The 50% inhibiting
concentrations (IC50) and the selectivity index (SI, the ratio of CC50 to IC50)
were calculated from the data obtained.
A. Supplementary data
22. Sokolov DN, Zarubaev VV, Shtro AA, et al. Anti-viral activity of (À)- and (+)-
usnic acids and their derivatives against influenza virus A(H1N1)2009. Bioorg
Med Chem Lett. 2012;22:7060–7064.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08.062.
23. The microtetrazolium test (MTT) was used to study the cytotoxicity of the
compounds. Briefly, a series of three-fold dilutions of each compound in MEM
were prepared. MDCK cells were incubated for 48 h at 36°C in 5% CO2 in the
presence of the dissolved substances. The cells were washed twice with
phosphate-buffered saline (PBS), and a solution of 3-(4,5-dimethylthiazolyl-2)
2,5-diphenyltetrazolium bromide (ICN Biochemicals Inc., Aurora, Ohio) (0.5 mg/
ml) in PBS was added to the wells. After 1h incubation, the wells were washed
and the formazan residue was dissolved in DMSO (0.1 ml per well). The optical
density of cells was then measured on a Victor 2 1440 multifunctional reader
(Perkin Elmer, Finland) at wavelength of 535 nm and plotted against the
concentration of compounds. Each concentration was tested in three parallels.
The 50% cytotoxic concentration (CC50) of each compound was calculated from
the data obtained.
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