Synthesis and in vitro study of novel borneol derivatives as potent inhibitors of the influenza A virus

Article abstract of DOI:10.1039/c6md00657d

Herein, we present the design and synthesis of a series of novel heterocyclic derivatives of (-)-borneol and (-)-isoborneol as potent inhibitors of the influenza A virus. All compounds were tested for their toxicity against MDCK cells and for virus-inhibiting activity against the influenza virus A/Puerto Rico/8/34 (H1N1). Compounds 7, 16 and 26 containing a morpholine fragment exhibited the highest efficiency as agents inhibiting the replication of the influenza virus A(H1N1) with selectivity indices of 82, 45 and 65, correspondingly. Derivatives 9 (SI = 23) and 18 (SI = 25) containing a 1-methylpiperazine motif showed moderate antiviral activity. Structure-activity analysis of this new series of borneol derivatives revealed that a 1,7,7-trimethylbicyclo[2.2.1]heptan scaffold is required for the antiviral activity.

Full text of DOI:10.1039/c6md00657d

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DOI: 10.1039/C6MD00657D
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ARTICLE
Synthesis and in vitro study of novel borneol derivatives as potent
†
inhibitors of the influenza A virus
a,b
a,b
a
c
c
A. S. Sokolova* , О. I. Yarovaya , M. D. Semenova , A. A. Shtro , I. R. Orshanskaya ,
Received 00th January 20xx,
Accepted 00th January 20xx
c
a,b
V. V. Zarubaev , N. F. Salakhutdinov
DOI: 10.1039/x0xx00000x
Herein, we present the design and synthesis of a series of novel heterocyclic derivatives of (−)-borneol and (-)-isoborneol as
potent inhibitors of influenza A virus. All compounds were tested for their toxicity against MDCK cells and for virus-inhibiting
activity against influenza virus A/Puerto Rico/8/34 (H1N1). Compounds 7, 16 and 26 containing a morpholine fragment
exhibited the highest efficiency as an agent inhibiting the replication of influenza virus A(H1N1) with selectivity indices of
www.rsc.org/
8
2, 45 and 65, correspondingly. Derivatives 9 (SI = 23) and 18 (SI = 25) containing a 1-methylpiperazine motif showed
moderate antiviral activity. Structure–activity analysis of this new series of borneol derivatives revealed that a 1,7,7-
trimethylbicyclo[2.2.1]heptan scaffold is required for the antiviral activity.
development of novel virus-inhibiting compounds with
alternative targets and mechanisms of activity.
Introduction
Borneol, a bicyclic monoterpenoid alcohol, exists as two
Influenza A viruses are important pathogens which are capable
of causing significant morbidity and mortality in humans. The
influenza virus is a negative-sense segmented single-strand
RNA-genome virus belonging to the Orthomyxoviridae family.
Two main approaches, vaccination and chemotherapy, are used
to control influenza in humans. Due to the short life cycle and
lack of error-correcting activity of polymerase, influenza viruses
are able to rapidly evolve. On one hand, this results in escape
from immune response and antigenic drift that, in turn, makes
necessary annual checking of efficacy of vaccine strain
composition against circulating viruses. On the other hand,
drug-resistant strains of influenza virus can quickly emerge and
spread. In particular, since the mid-1990s, mutation of
resistance to adamantane derivatives (amantadine and
rimantadine) has emerged, and currently the vast majority of
influenza isolates are rimantadine-resistant.1 In 2009, a novel
triple reassortant influenza A(H1N1)pdm09 virus of swine origin
that was also naturally resistant to adamantanes emerged and
enantiomers, D and L forms. Both the borneol forms occur in
the essential oils of numerous medicinal plants, such as valerian
(Valeriana officinalis), chamomile (Matricaria chamomilla) and
lavender (Lavandula officinalis). Generally, there are two
isomers of borneol: borneol and isoborneol, which vary with the
location of the hydroxyl group. These substances have a broad
spectrum of biological activity. Recently, it has been established
that isoborneol shows antiviral activity against herpes simplex
7
virus type 1, and both enantiomers of borneol were found to
have a highly efficacious positive modulating action at
mammalian
GABA
(γ-aminobutyric
acid)
inhibitory
8
neurotransmission receptors. In addition, borneol shows
inhibitory effects on several Gr (−) and Gr (+) pathogenic
9
microorganisms.
It can be assumed that cage compounds like borneol
derivatives containing 1,7,7-trimethylbicyclo[2.2.1]heptan
scaffold have high potential in the synthesis of antiviral agents.
Our previous works discovered the scaffold as a promising anti-
,2
3
quickly spread worldwide. Similarly, oseltamivir-resistant
H1N1 viruses appeared in 2007 and spread worldwide with
10,11,12
influenza agent.
The target compound was identified as an
inhibitor of H1N1 influenza virus with a selectivity index (SI)
value of 500, this camphor-derivative was called Camphecene
4
,5,6
almost 100% drug resistance in 2008/2009
due to mutations
7
in the NA gene. Drug resistance substantially restricts the use
of antivirals and makes necessary the search for and
13
(
Fig 1). In contrast to deitiforine that targets the M2 proton
channel, Camphecene was shown to directly inhibit the acid-
induced membrane-disrupting activity of the viral
14
hemagglutinin of influenza A viruses.
Moreover there are
another cage compounds with antiviral activity, for example
amino camphor derivatives were identified as a new class of M2
inhibitors with moderate activity. Also compounds based on
a.Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian
Academy of Sciences, Lavrentjev Avenue 9, 630090 Novosibirsk, Russia.
b.
15
Novosibirsk State University, Pirogova St. 2, 630090 Novosibirsk, Russia
Laboratory of Chemotherapy, Influenza Research Institute, Prof. Popova St.
c.
1
- and 2-adamantylamines exhibited high M2 inhibitor
1
5/17, 197376 St. Petersburg, Russia
Corresponding author E-mail: asokolova@nioch.nsc.ru
The authors declare no competing interests.
16,17
activity.
The ability to inhibit the influenza virus H3N2 was
†
18
found in amino and imine derivatives with a pinene scaffold;
compounds containing a pinanamine scaffold with secondary
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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ARTICLE
Journal Name
chloropropanoate
4–21. The compounds were obtained in good yields ranging
3
were transformed into target compounds
View Article Online
DOI: 10.1039/C6MD00657D
from 52% to 91%.
It is well recognised that the stereochemistry of molecules
Fig 1. Structural formula and anti-influenza A(H1N1)pdm09 activity of anti-
influenza drugs and camphor derivative (CC50 - the median cytotoxic dose; IC50
the 50% virus-inhibiting concentration; SI - the selectivity index).
-
amine and imidazole may increase inhibition of A/M2 channel
19
activity.
In view of the above, in the present work several series of
borneol derivatives containing cage structure based on the
1
,7,7-trimethylbicyclo[2.2.1]heptan scaffold were synthesized and
tested against influenza virus. According to literature data, the
general direction in the synthesis of biologically active borneol
derivatives include preparation of different borneol esters.
These esters were shown to be inhibitors of cyclooxygenase
20
enzymes and replication of coxsackievirus B3. Also the bornyl ester
derivatives showed cytotoxic activity against tumor cell lines.21,22
Therefore in this work the general approach to the synthesis of
the target compounds included synthesis of different borneol
ester containing various heterocycles. Nitrogen heterocycles
are attractive structural units in medicinal chemistry. There are
a lot of highly effective drugs with the active ingredient
containing nitrogen heterocyclic fragments, such as piperazine,
23
piperidine and morpholine. In our search for antiviral agents,
we screened several different nitrogen-containing heterocyclic
fragments for their ability to inhibit reproduction. Moreover,
we studied the influence of the length of the aliphatic linker
between ester group and heterocyclic scaffold.
Scheme 1. Reagents and conditions: (i) 2-chloroacetyl chloride, Et
5 °C; (ii) 3-chloropropanoyl chloride, Et N, CH Cl (dry), 25 °C; (iii) Et
dry), 25 °C.
3
N, CH
2
Cl
2
(dry),
Cl
2
(
3
2
2
3
N, CH
2
2
is often crucial in deriving a SAR. The next step in borneol based
SAR was using as the starting materials (-)-isoborneol 22 with
Results and discussion
Chemistry
different stereochemistry of hydroxyl group. Compounds
7 and
1
6
with morpholine fragment showed the highest antiviral
The borneol derivatives were prepared according to the
synthetic route as outlined in Scheme 1. In our work we use (-)
activity, so we synthesised analogies 23 and 24 based on (-)-
isoborneol (Scheme 2).
borneol
described in a patent. Reaction between the (−)-borneol and
chlorides, α or β chlorinated acids, led to esters and . By
nucleophilic substitution reaction which involved different
nitrogen-containing nucleophiles, (1S,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-yl 2-chloroacetate
1, obtained from natural material by the method
24
2
3
Fig 2. Compounds without the 1,7,7-trimethylbicyclo[2.2.1]heptan fragment.
2
and
3-
(1S,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl
To establish the key structural requirements for antiviral
25
26
activity we synthesized derivatives 27 and 28 without the
,7,7-trimethylbicyclo[2.2.1]heptan fragment (Fig. 2). As the
nitrogen-containing nucleophile we chose morpholine because
borneol derivatives and 16 with the morpholine fragment
1
7
showed considerable antiviral activity.
The structures of new synthesized compounds were
1
13
confirmed by NMR spectra ( H, C NMR), HR-MS and IR spectra.
Scheme 2. Reagents and conditions: (i) 2-chloroacetyl chloride, Et
3
N, CH
2
Cl
N, CH
2
(dry),
Cl
1
The H NMR spectrum of compounds 13
–21
, 26 revealed upfield
25 °C; (ii) 3-chloropropanoyl chloride, Et
3
N, CH Cl
2 2
(dry), 50 °C; (iii) Et
3
2
2
(
dry), 25 °C.
signals of the methylene protons N–CH
at δ 2.6–2.8 ppm in
2
2
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Pleas eM de od Cn oh te am d Cj u os mt mm argins
ARTICLE
contrast to the protons of starting compound
Also, in the H NMR spectra of compounds 4–12, 25 an upfield in their structures. To study the structure–activity relationship
3
at δ 3.74 ppm. compounds under investigation in having rigid cage fragments
View Article Online
DOI: 10.1039/C6MD00657D
1
singlet at δ 3.1 ppm was observed in comparison with the we examined changes of aliphatic linker and heterocyclic
singlet of compound
Study of antiviral activity.
2
that was revealed at δ 4.03 ppm.
fragment in terms of their effect on toxicity and virus-inhibiting
activity. In general, compounds with longer linkers (C2) were
slightly more toxic compared to their C1 analogues. The
The obtained compounds 4–21, 25, 26 were studied as
potential antiviral agents. (Table 1).
piperazine derivatives
9 and 18 exhibited high antiviral activity
with the IC50 values 4,4±0.5 and 1,9±0.4 respectively. Together
with a potent activity this compounds showed a high toxicity
which results in a relatively low therapeutic index. It was found
that the morpholine substituent resulted in a strong reduction
of toxicity compared to piperazine derivatives. This led to higher
SI’s, although the IC50 values of morpholine and piperazine
Table 1. Antiviral activity of camphor-based compounds against influenza virus
A/Puerto Rico/8/34 (H1N1) in MDCK cells
Compound
CC50, µM
IC50, μM
SI
LogPa
2.71
4.29
4.85
5.34
3.53
3.68
3.26
3.79
4.11
6.78
4.45
5.02
5.51
3.36
3.51
3.09
3.62
3.94
6.94
2.71
3.53
3.36
0.11
0.64
1
4
5
6
7
8
9
> 2142.9
155.8 ± 18.3
34.0 ± 4.2
15.1 ± 1.0
10.2 ± 1.4
7.1 ± 0.8
7.1 ± 0.5
4.4 ± 0.5
3.2 ± 0.4
683.1 ± 62.8
> 34.1
> 14
9
301.9 ± 21.4
137.6 ± 9.9
93.9 ± 7.3
580.1 ± 41.1
102.9 ± 8.2
100.0 ± 7.3
51.6 ± 4.1
> 852.0
derivatives did not differ (compounds
7 and 8, 16 and 17).
9
Acylation of one of the nitrogen atoms of the piperazine moiety
led to strongly decreased toxicity of the compound although its
antiviral properties did not increase. The derivatives containing
9
82
14
23
16
1
1
-methylpiperazine and 1-ethylpiperazine motifs showed
moderate antiviral activity.
It was also demonstrated that in case of short linker,
stereochemistry of the compound does not effect on
cytotoxicity of compounds but is of critical significance for anti-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
5
6
7
8
viral activity. Indeed, compounds
optical isomers of borneol, and their values of IC50 differ tenfold
7.1 and 73.7 correspondingly). This, however, was not
7 and 25 are based on two
69.3 ± 4.9
124.7 ± 10.1
69.6 ± 5.2
56.4 ± 3.8
552.5 ± 31.1
12.6 ± 1.0
48.1 ± 3.3
104.7±8.7
149.5 ± 11.7
41.8 ± 2.2
>974
> 2
9
(
14.3 ± 1.6
7.8 ± 0.8
15.3 ± 2.0
12.2 ± 1.6
6.8 ± 0.9
1.9 ± 0.4
8.4 ± 0.9
> 98.4
important for other two isomers, 16 and 26, with longer linker
that have similar values of both toxicity and activity and,
therefore, similar SI’s (45 and 65 respectively). This
phenomenon should be further studied, in particular using the
means of computer simulation of ligand-target interaction.
Also, the structure–activity analysis of this new series of
9
4
45
2
borneol
derivatives
revealed
that
the
1,7,7-
25
12
2
trimethylbicyclo[2.2.1]heptan scaffold is required for the
antiviral activity, as compounds 27 and 28 lacking this cage
fragment did not possess virus-inhibiting activity. Among all
compounds tested, the highest activity was found in substances
7.1 ± 0.9
>974
6
7, 16 and 26 combining a morpholine fragment and 1,7,7-
1
trimethylbicyclo[2.2.1]heptan
correspondingly).
(SI
82,
45,
and
65
>533,8
73,7±8,5
7,8±1,1
7
Lipophilicity is
a physicochemical property of principal
508,5±36,1
> 1886.8
65
1
27
importance in drug discovery and development. The quantitative
descriptor of lipophilicity, the partition coeficient P, is defined as the
ratio of the concentrations of a neutral compound in organic and
aqueous phases under equilibrium conditions. To correlate the
antiviral activity of the present series of compounds with their
> 1886.8
982.7 ± 75.3
67.0 ± 4.9
64.2 ± 4.7
208.6 ± 15.4
24.6±3,1
0,2±0,03
> 1734.1
> 2
5
Rimantadine
Amantadine
Deitiforine
Ribavirin
335.2 ± 26.8
284.1 ± 21.4
1266.2 ± 81.5
> 2000.0
4
liphophilicity,
log
p
values
were
calculated
using
6
ACDLabs/ChemSketch 12.01. All the new examined compounds have
the optimum lipophilicity range with a log p value 3.09 – 3.79.
> 81.0
781
Oseltamivir
160,3 ± 11,4
Conclusions
a
LogP, the octanol-water partitioning coefficient, was calculated using
ACDLabs/ChemSketch 12.01 (www.acdlabs.com).
All values are presented as mean±SD from at least three independent
experiments.
In summary, we presented a novel series of borneol derivatives
containing different heterocyclic fragments. All of the
compounds were investigated against influenza virus A/Puerto
Rico/8/34 (H1N1) in MDCK cells, and the structure–activity
relationship was studied. Among these novel derivatives,
Adamantane- and norbornane-based derivatives were used
as reference compounds due to their close similarity to the
compounds
7
,
16
and
26
with
1,7,7-
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ARTICLE
Journal Name
trimethylbicyclo[2.2.1]heptan and morpholine fragments were
found to possess the highest efficacy in virus inhibition.
Structure–activity analysis showed that an increase of linker
length leads to enhancement of the toxicity. The results
obtained suggest that a bulky lipophilic unit such as the 1,7,7-
trimethylbicyclo[2.2.1]heptan scaffold was necessary for
activity.
Acknowledgements
This work has been supported by Russian Scientific Foundation
grant 15-13-00017.
View Article Online
DOI: 10.1039/C6MD00657D
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