Anti-influenza activity of monoterpene-containing substituted coumarins

Article abstract of DOI:10.1016/j.bmcl.2017.04.091

Compounds simultaneously carrying the monoterpene and coumarin moieties have been tested for cytotoxicity and inhibition of activity against influenza virus A/California/07/09 (H1N1)pdm09. The structure of substituents in the coumarin framework, as well as the structure and the absolute configuration of the monoterpenoid moiety, are shown to significantly influence the anti-influenza activity and cytotoxicity of the compounds under study. The compounds with a bicyclic pinane framework exhibit the highest selectivity indices (the ratios between the cytotoxicity and the active dose). The derivative of (?)-myrtenol 15c, which is characterized by promising activity, low cytotoxicity, and synthetic accessibility, has the greatest potential among this group of compounds. It exhibited the highest activity when added to the infected cell culture at early stages of viral reproduction.

Full text of DOI:10.1016/j.bmcl.2017.04.091

Accepted Manuscript
Anti-influenza activity of monoterpene-containing substituted coumarins
Tatyana M. Khomenko, Vladimir V. Zarubaev, Iana R. Orshanskaya, Renata A.
Kadyrova, Victoria A. Sannikova, Dina V. Korchagina, Konstantin P. Volcho,
Nariman F. Salakhutdinov
PII:
S0960-894X(17)30476-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.091
BMCL 24944
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 March 2017
27 April 2017
28 April 2017
Please cite this article as: Khomenko, T.M., Zarubaev, V.V., Orshanskaya, I.R., Kadyrova, R.A., Sannikova, V.A.,
Korchagina, D.V., Volcho, K.P., Salakhutdinov, N.F., Anti-influenza activity of monoterpene-containing
substituted coumarins, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.04.091
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anti-influenza activity of monoterpene-containing substituted coumarins
Tatyana M. Khomenko,a,c Vladimir V. Zarubaev,b Iana R. Orshanskaya,b Renata A. Kadyrova,b
Victoria A. Sannikova,a,c Dina V. Korchagina,a Konstantin P. Volcho,a,c Nariman F.
Salakhutdinova,c
aNovosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of
Sciences, 630090, Novosibirsk, Russia
bDepartment of Chemotherapy, Influenza Research Institute, 15/17 Prof. Popova St., 197376 St.
Petersburg, Russia
cNovosibirsk State University, Pirogova 2, Novosibirsk, 630090, Russia
Abstract: Compounds simultaneously carrying the monoterpene and coumarin
moieties have been tested for cytotoxicity and inhibition of activity against
influenza virus A/California/07/09 (H1N1)pdm09. The structure of substituents in
the coumarin framework, as well as the structure and the absolute configuration of
the monoterpenoid moiety, are shown to significantly influence the anti-influenza
activity and cytotoxicity of the compounds under study. The compounds with a
bicyclic pinane framework exhibit the highest selectivity indices (the ratios between
the cytotoxicity and the active dose). The derivative of (-)-myrtenol 15c, which is
characterized by promising activity, low cytotoxicity, and synthetic accessibility,
has the greatest potential among this group of compounds. It exhibited the highest
activity when added to the infected cell culture at early stages of viral reproduction.
Keywords: terpene, coumarin, anti-influenza activity, antivirals, cytotoxicity, influenza A virus,
aurapten, myrtenal
Influenza A virus is the major cause of seasonal or pandemic influenza worldwide. These
annual epidemics are estimated to result in about 3 to 5 million cases of severe illness, and about
250,000 to 500,000 deaths.1 New influenza viruses are constantly evolving by mutation or by
reassortment, giving rise to new strains that can infect people who are immune only to the pre-
existing influenza strains.2 Although vaccination against the virus is quite effective, low-
molecular anti-influenza drugs are the first line of protection against the virus during an
epidemic outbreak, since an effective vaccine for the circulating strains usually takes at least 6
months to be developed.3 The ability of the influenza virus to develop resistance to the available
1

drugs is a serious problem4,5 that necessitates designing new structural types of drugs with novel
targets, improved antiviral effects, higher safety, and increased tolerability.
An important area in searching for novel antiviral agents is using natural compounds,
including marine natural products,6 monoterpenoid derivatives,7-13 phenylethanoid glycosides,14
etc., as starting compounds. Natural coumarins and their derivatives are attracting significant
attention as lead structures to search for orally bioavailable antiviral agents.15 Thus, it has
recently been suggested using the pharmacophore-based virtual screening of the library of
natural compounds taken from the Princeton database that some coumarin derivatives, for
example 1 (Fig. 1), may act as novel neuraminidase inhibitors.16 Coumarin derivative 2 was
identified as a promising anti-influenza agent by cell-based high-throughput screening of 20,000
compounds.17 The detailed studies focused on structure–activity relationship revealed that BPR2-
D2 (Fig. 1) exhibits an excellent antiviral efficacy against the oseltamivir-resistant virus.18 virus.
BPR2-D2 may target viral ribonucleoproteins that are responsible for viral RNA synthesis. A
promising group of sesquiterpene coumarins with anti-influenza activity was isolated from
Ferula assafoetida.19 The structure of one of these compounds (3) is presented in Fig. 1. This
compound contains a set of functional groups with fixed stereoconfiguration; synthesizing this
compound is very challenging. We supposed that replacement of the sesquiterpene moiety with a
monoterpene might give rise to new synthetically accessible compounds with anti-influenza
activity. Hence, this study was aimed at searching for novel agents that would possess activity
against H1N1 influenza virus among synthetic coumarin derivatives containing a monoterpenoid
moiety.
Fig. 1. Structures of anti-influenza active coumarin derivatives and umbelliferone.
2

The commercially available umbelliferone 4 (Fig. 1) and its analogues 5-7 synthesized
via interaction between resorcin 8 and esters of the corresponding β-keto acids 9-11 (Scheme 1)
as described previously20,21 were used as a coumarin component.
Scheme 1. Synthesis of coumarins 5-7 and their yields.
Aurapten 13a and its analogues 13b-e (Scheme 2) were obtained by interaction of
umbelliferone 4 with monoterpenoid bromides 12a-d and, for the sake of comparison, with aryl
bromide 12e using DBU in DMF.22 Bromides 12a, 12b, 12c, 12d and 12e were synthesized by
interaction between the corresponding alcohols and PBr3 with the yields of 91%, 55%, 60%,
24% and 65%, respectively.23 Compound 12d obtained by interaction between nopol and PBr3
was insufficiently pure, thus making it necessary to use column chromatography for purification
and abruptly decreasing its yield.
In a similar manner, compounds 14a-c,e, 15a-e, and 16a-e were prepared as described
previously21 using K2CO3, ethanol, and coumarins 5-7 as phenol components.24 The products
were purified by recrystallization or column chromatography (the yields of 29-56%). the reaction
of nopyl bromide 12d with methylcoumarin 14 was not successful due to the formation of a
complex reaction mixture with high level of resinification.
Scheme 2. Synthesis of substituted coumarins.
3

The water/octanol partition coefficient (Log P)) is often considered to be an important
molecular descriptor as it is linked to toxicity issues and oral bioavailability. The Calculated Log
P (cLog P) data are presented in Table 1. Although cLog P of all the compounds carrying the
monoterpene and coumarin moieties exceeds the Lipinski’s cLog P = 5 limit,25 most of them are
within the known drug chemical space according to this criterion (cLog P ≤ 6.5).26
The antiviral activity of the synthesized compounds was studied27 against the pandemic
influenza virus A/California/07/09 (H1N1)pdm09 cultivated in cell culture by the technique
described earlier.28 Cytotoxicity of the compounds was evaluated29 in uninfected MDCK cells as
described previously.30The selectivity index was calculated for each derivative based on the data
obtained. The compounds with SI = 10 and higher were regarded as active. The test results are
summarized in Table 1. Rimantadine was taken as a reference drug due to its polycyclic structure
being close to the pinane scaffold used in the study.
Table 1.
Antiviral activity and cytotoxicity of compounds 13a-e, 14a-c,e, 15a-e, and 16a-e
against influenza virus A/California/07/09 (H1N1)pdm09 in MDCK cells
Compound
R
cLogPa
5.69
CC50b, µM IC50b, µM
SId
1
7 1
7 0
7 2
>10
13a
13b
5.04
5.04
5.52
3.35
1
3
310 22
10 1
>101
0.5 0
>1170
13c
13d
20
1
>1170
13e
14a
14b
6.28
5.63
28 2
>10
3
130 9
10 2
13
5.63
3.94
30 2
4 1
9
1
14c
>1115
1014 113
14e
15a
15b
6.75
6.09
>888
>888
33 5
1
4
133 8
4

6.09
6.57
4.40
982 47
>857
36 5
21 3
>932
28
41
1
15c
15d
>932
15e
16a
16b
7.31
6.66
100 6
>85
1
3
415 29
149 18
6.66
7.14
4.97
857 71
18 1
64 5
3 1
13
6
16c
16d
>982
>982
1
16e
363 20
41
7
9
Rimantadine
a cLog P Calculated using the ACD/LogP ChemSketch 12 software
b CC50 is the median cytotoxic concentration; i.e., the concentration causing 50% cell death.
c
IC50 is the 50% inhibiting concentration; i.e., the concentration causing a 50% decrease in
virus replication.
d SI is the selectivity index, which is the CC50/IC50 ratio.
First, we studied the antiviral activity of aurapten 13a that is the most abundant natural
prenyloxycoumarin. Aurapten 13a exhibits versatile biological activities, but to the best of our
knowledge, no data on its antiviral activity have been reported.31 We found that aurapten 13a,
identically to its analogue 13b containing a (+)-myrtenol fragment, exhibits high cytotoxicity
against MDCK cells that is comparable to its antiviral activity and, therefore, possesses zero
selectivity (SI = 1). Compound 13c synthesized from (-)-myrtenol turned out to be both less
toxic and less active than its enantiomer 13b. The methylene moiety inserted between the
monoterpene and coumarin moieties when proceeding to compound 13d drastically increased its
activity: IC50 of this compound lay in the nanomolar concentration range, which made it possible
to achieve a good selectivity index (SI = 20) despite the significant cytotoxicity. The use of an
aromatic moiety (13e) resulted in complete disappearance of antiviral activity. Hence, compound
13d is the most promising umbelliferone derivative.
When proceeding to type 14 compounds, insertion of methyl group into the coumarin
framework decreased toxicity and increased the selectivity index in all compounds excluding
14c, while the level of activity remained the same. Compound 14b showed the greatest
5

selectivity index (SI = 13). Unfortunately, we did not succeed in obtaining an analogue of
compound 13d with an acceptable purity for compounds of this structural type.
Compound 15a, which has the cyclopentane ring annulated with the coumarin framework
and containing a geraniol moiety, was inactive. Meanwhile, compounds 15b-d containing the
bicyclic monoterpene moiety exhibited a significant antiviral activity. Due to their low
cytotoxicity, compounds 15c and 15d synthesized using (-)-myrtenol and nopol, respectively,
exhibited a selectivity index as high as 41.
After proceeding to type 16 compounds that contained the cyclohexane ring annulated
with the coumarin framework, we found that the selectivity index was higher than 10 only for
compound 16c. Although compound 16d exhibited good antiviral activity, its selectivity index
was low because of high cytotoxicity.
The compounds containing an aromatic moiety (13e, 14e, 15e, and 16e) exhibited no
anti-influenza activity regardless of the structure of the coumarin component in the molecule.
Hence, compounds 15c and 15d, which exhibit both high activity and low cytotoxicity
with the highest selectivity indices, are the most promising ones among the compounds under
study. Taking into account the availability of the starting compound ((-)-myrtenal) and simplicity
of its synthesis procedure, 15c shows greater potential among these two compounds. Compound
15c was earlier found21 to be a low-toxicity inhibitor of Tdp1, one of the enzymes of DNA repair
system.32 These inhibitors can increase the potency of some anti-tumor agents whose activity is
based on inhibition of topoisomerase 1 (Top1), even though they exhibit no intrinsic
cytotoxicity.33 It should be mentioned that inhibition of Tdp1 does not cause any problems
associated with toxicity in the absence of Top1 inhibitors and, therefore, is expected to cause no
adverse events when inhibitors are used in anti-influenza therapy. Indeed, it is known that Tdp1-
/- mice were fertile and had a normal life expectancy.34
As can be seen from the data presented, 5 of 19 (26%) tested compounds have
demonstrated good selectivity (SI>10). All 3-metoxybenzene derivatives of coumarin (13e, 14e,
15e, 16e) possessed low toxicity but no virus-inhibiting activity. Elongation of the linker
between the coumarine and pinane moieties in groups of 13’s and 16’s dramatically increased
cytotoxicity (13c and 13d, 16c and 16d).
Pinane stereoisomers conferred different toxicity levels to the compounds. In most cases,
(+)-isomers were more toxic than (-)-isomers, although to different degrees.
Compounds 15c and 15d possessed the highest selectivity among all the compounds
tested. This selectivity was provided by low toxicity rather than by very high anti-influenza
activity. Compound 13d had the best IC50 value but its high cytotoxicity resulted in moderate SI
(20).
6

In order to assess what stage of the viral life cycle is affected by compound 15c, we
carried out a series of experiments that differed in time when the compound was added.35 It
turned out (Fig. 2) that 15c exhibits the highest activity when added to the infected cell culture at
early stages of viral reproduction (1-2 hours after infection). The potency of the agent decreased
with time. Viral infectivity statistically did not differ from the control values starting with 6
hours after infection.
Fig. 2. Time-of-addition activity of 15c against influenza virus A/PR/8/34 (H1N1).
o
A/Puerto Rico/8/34 (H1N1) was absorbed to MDCK cells (m.o.i. 10) for 1 h at 4 C. After
removal of the non-absorbed virions, the plates were incubated for 8 h at 36oC in 5% CO2. The
starting point of incubation was denoted as 0. Cells were treated with 15c (500 µmol) for the
following time: (-2) – (-1) (before being infected); (-1) – 0 (simultaneously to absorption); 0 – 2;
2 – 4; 4 – 6; 6 – 8 and (-2) – 8 h. After 8 h of growth, the virus titer was determined in the culture
medium according to TCID50 in the MTT assay.
Hence, the specific target of 15c is critical for viral cycle at its early stages. The most
probable targets are viral hemagglutinin or proton channel M2. It should be mentioned that the
virus used for testing is resistant to adamantane derivatives due to S31N substitution in M2
protein. This results in low activity of rimantadine against this virus (SI=9, Table 1). Several
attempts have been made to develop the “universal” M2 inhibitor; i.e., a compound that would
inhibit both wild-type (with serine at position 31, M2wt) and resistant (bearing substitution
S31N, V27A, L26I or G34E) M2 channels.36 Thus far, no compound has been identified with
equal activity against M2wt and S31N viruses37, although a spiroadamantane amine derivative
has recently been shown to inhibit M2wt and V27A resistant mutant in in vitro and in vivo
models.38
Most of the compounds for inhibiting M2 ion channel are polycyclic amines.37 The
Coumarine derivatives studied here are of different chemical structures and should therefore be
considered as having different viral target(s). Computer simulation of the interaction between the
two most active compounds, 15c and 15d, suggests that they may bind to viral hemagglutinin
between the head and the stalk in the region of fusion peptide. Their bulky polycyclic moiety is
inserted into a pocket formed by amino acids 556-IEMNI-561 (15c), probably via hydrophobic
interaction with I556 and I561, or into a wide pocket formed by L48-K49-S81 and S118 (15d).
The tricyclic coumarine-derived moiety is stabilized by either serines 291 and 292 (15c) or T264
7

and F563 (15d) on one side and either E305 and K281 (15c) or F264 (15d) on another side.
Using this mechanism, compounds might suppress the conformational changes in an HA
molecule, thus interfering with membrane fusion.
On the other hand, one cannot rule out that these compounds are directed against cellular
target(s) important for viral life cycle at early stages, such as the components of cytoskeleton,
transport proteins, lysosomal components, etc. Further studies are therefore needed to identify
the exact mechanism of activity of coumarin derivatives, as well as their range of activity against
influenza viruses.
Hence, we have studied the anti-influenza activity of a series of compounds containing
both the coumarin and monoterpenoid moieties. The compounds having the bicyclic pinane
framework were shown to exhibit the highest selectivity index (the ratio between cytotoxicity
and the active dose). Both the structure of substituents in the coumarin framework and the
absolute configuration of the monoterpene moiety have a significant effect on the selectivity
index. The (-)-myrtenol derivative 15c, which is simultaneously characterized by promising
activity, low cytotoxicity, and synthetic accessibility, shows the greatest potential among this
group of compounds. It is found to exhibit the highest activity when added to the infected cell
culture at the early stages of viral reproduction.
8

Fig. 3. Model of binding of coumarine derivatives 15c (A, B) and 15d (C, D) to trimeric
hemagglutinin of influenza virus A/Puerto Rico/8/34 (H1N1). A, C – general view. Ligands are
shown in magenta and indicated with arrows. B, D – a close view with specific amino acids.
Acknowledgments
This work was supported by the Russian Scientific Foundation (grant no. 15-13-00017).
The Authors would like to acknowledge the Multi-Access Chemical Service Center (Siberian
Branch of the Russian Academy of Sciences) for spectral and analytical measurements.
9

References and notes
1. World Health
http://www.who.int/mediacentre/factsheets/fs211/en/; 2014 Accessed 24.04.2017.
Organization.
Influenza
(seasonal)
fact
sheet
N°211.
2. Spanakis N, Pitiriga V, Gennimata V, Tsakris A. A review of neuraminidase inhibitor
susceptibility in influenza strains. Expert Rev. Anti Infect. Ther. 2014;12:1325-1336.
3. Shen Z, Lou K, Wang W. New small-molecule drug design strategies for fighting
resistant influenza A. Acta Pharmaceut. Sinica B 2015;5:419-430.
4. Baranovich T, Bahl J, Marathe BM, Culhane M, Stigger-Rosser E, Darnell D, Kaplan
BS, Lowe JF, Webby RJ, Govorkova EA. Influenza A viruses of swine circulating in the
United States during 2009–2014 are susceptible to neuraminidase inhibitors but show
lineage-dependent resistance to adamantanes. Antiviral Res. 2015;117:10-19.
5. Palmer R. Drugs: Lines of defence. Nature Outlook, Influenza 2011;480:S9-S10.
6. Gogineni V, Schinazi RF, Hamann MT. Role of marine natural products in the genesis of
antiviral agents. Chem. Rev. 2015;115:9655-9706.
7. Dong J, Chen S, Li R, Cui W, Jiang H, Ling Y, Yang Z, Hu W. Imidazole-based
pinanamine derivatives: Discovery of dual inhibitors of the wild-type and drug-resistant
mutant of the influenza A virus. Eur. J. Med. Chem. 2016;108:605-615.
8. Teplov GV, Suslov EV, Zarubaev VV, Shtro AA, Karpinskaya LA, Rogachev AD,
Korchagina DV, Volcho KP, Salakhutdinov NF, Kiselev OI. Synthesis of new
compounds combining adamantanamine and monoterpene fragments and their antiviral
activity against influenza virus A(H1N1)pdm09. Lett. Drug Des. Discov. 2013;10:477-
485.
9. Zhao X, Jie Y, Rosenberg MR, Wan J, Zeng S, Cui W, Xiao Y, Li Z, Tu Z, Casarotto
MG, Hu W. Design and synthesis of pinanamine derivatives as anti-influenza A M2 ion
channel inhibitors. Antiviral Res. 2012;96:91-99.
10. Ardashov OV, Zarubaev VV, Shtro AA, Korchagina DV, Volcho KP, Salakhutdinov NF,
Kiselev OI. Antiviral activity of 3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol and
its derivatives against influenza A(H1N1)2009 virus. Lett. Drug Des. Discov.
2011;8:375-380.
11. Zarubaev VV, Garshinina AV, Tretiak TS, Fedorova VA, Shtro AA, Sokolova AS,
Yarovaya OI, Salakhutdinov NF. Broad range of inhibiting action of novel camphor-
based compound with anti-hemagglutinin activity against influenza viruses in vitro and in
vivo. Antiviral. Res. 2015;120:126-133.
10

12. Nazimova EV, Shtro AA, Anikin VB, Patrusheva OS, Il’ina IV, Korchagina DV,
Zarubaev VV, Volcho KP, Salakhutdinov NF. Influenza antiviral activity of Br-
containing
[2R,4R(S),4aR,7R,8aR]-4,7-dimethyl-2-(thiophen-2-yl)octahydro-2H-
chromen-4-ols prepared from (–)-isopulegol. Chem. Nat. Comp. 2017;53:260-264.
13. Sokolova AS, Yarovaya OI, Shernyukov AV, Gatilov YV, Razumova YV, Zarubaev VV,
Tretiak TS, Pokrovsky AG, Kiselev OI, Salakhutdinov NF. Discovery of a new class of
antiviral compounds: Camphor imine derivatives. Eur. J. Med. Chem. 2015;105:263-273.
14. Chen B-L, Wang Y-J, Guo H, Zeng G-Y. Design, synthesis, and biological evaluation of
crenatoside analogues as novel influenza neuraminidase inhibitors. Eur. J. Med. Chem.
2016;109:199-205.
15. Hassan MZ, Osman H, Ali MA, Ahsan MJ. Therapeutic potential of coumarins as
antiviral agents. Eur. J. Med. Chem. 2016;123:236-255.
16. Batool S, Mushtaq G, Kamal W, Kamal MA. Pharmacophore-Based Virtual Screening
for Identification of Novel Neuraminidase Inhibitors and Verification of Inhibitory
Activity by Molecular Docking. Med. Chem. 2016;12:63-73.
17. Yeh JY, Coumar MS, Horng JT, Shiao HY, Kuo FM, Lee HL, Chen IC, Chang CW,
Tang WF, Tseng SN, Chen CJ, Shih SR, Hsu JT, Liao CC, Chao YS, Hsieh HP. Anti-
influenza drug discovery: structure−activity relationship and mechanistic insight into
novel angelicin derivatives. J. Med. Chem. 2010;53:1519-1533.
18. Shih SR, Horng JT, Poon LL, Chen TC, Yeh JY, Hsieh HP, Tseng SN, Chiang C, Li WL,
Chao YS, Hsu JT. BPR2-D2 targeting viral ribonucleoprotein complex-associated
function inhibits oseltamivir-resistant influenza viruses. J. Antimicrob. Chemother.
2010;65:63-71.
19. Lee C-L, Chiang L-C, Cheng L-H, Liaw C-C, El-Razek MHA, Chang F-R, Wu Y-C.
Influenza A (H1N1) antiviral and cytotoxic agents from Ferula assa-foetida. J. Nat.
Prod. 2009;72:1568-1572.
20. General procedure for the synthesis of coumarins 5-7. Conc. H2SO4 (5 mL, 94 mmol)
was added dropwise to cooled (0-5 °C) solution of resorcin 8 (45 mmol) and appropriate
β-keto esters (9-11) (45 mmol) in dry ethanol (15 mL) with vigorously stirring. The
mixture was stirred until to be congealed, left overnight at r.t., and poured into ice water
(150 mL). The resulting solid was filtered off and crystallized from ethanol-water (75%).
The yields of 5-7 were 95%, 64% and 70% respectively.
21. Khomenko T, Zakharenko A, Odarchenko T, Arabshahi HJ, Sannikova V, Zakharova O,
Korchagina D, Reynisson J, Volcho K, Salakhutdinov N, Lavrik O. New Inhibitors of
11

tyrosyl-DNA phosphodiesterase I (Tdp 1) combining 7-hydroxycoumarin and
monoterpenoid moieties. Bioorg. Med. Chem. 2016;24:5573-5581.
22. Synthesis of compounds 13a-e. DBU (1.0 mmol) and corresponding bromide 12a-e (0.75
mmol) were added to compound 4 (0.5 mmol) in dry DMF (5 mL) at r.t. under stirring.
The reaction mixture was stirred at r.t. for 15 minutes, and then heated at 60 °C for 5
hours. H2O (15 ml) was added and the product was extracted with ethyl acetate. The
extracts were washed with brine, dried with Na2SO4 and evaporated.
23. Synthesis of compounds 12a-e. Geranyl bromide 12a was synthesized from geraniol via
the reaction with PBr3. PBr3 (8.9 mmol) was added to cooled (0-5°C) solution of geraniol
(26.7 mmol) in dry ether (30 ml) and the reaction mixture was stirred for 2 h at r.t.
Saturated aqueous NaHCO3 was added and the product was extracted with ether. The
extracts were washed with brine, dried with Na2SO4 and evaporated. Other used
bromides 12b-e were synthesized as described above. Compounds 12a, 12b, 12c and 12e
(the yields 91%, 55%, 60% and 65%, respectively) were sufficiently pure and used for
the next step without purification. The compound 12d was purified by column
chromatography on SiO2, eluent - hexane (yield 24%).
24. Synthesis of compounds 13a-e, 14a-c,e, 15a-e and 16a-e. K2CO3 (1.0 mmol) and
corresponding bromides 12a-e (0.75 mmol) were added to corresponding compound 5-7
(0.5 mmol) in dry ethanol (5 mL) at r.t. under stirring. The reaction mixture was stirred at
r.t. for 15 minutes, and then heated at 60 °C for 5 hours. The hot solution was filtered; the
filtrate was kept at -18 °C for 48 hours. The products were isolated in the individual form
by recrystallization from ethanol by column chromatography on silica gel, eluent -
hexane, solution containing from 25 to 100% chloroform in hexane, ethanol.
25. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational
approaches to estimate solubility and permeability in drug discovery and development
settings. Adv. Drug Deliv. Rev. 2001;46:3-26.
26. Bade R, Chan H-F, Reynisson J. Characteristics of known drug space. Natural products,
their derivatives and synthetic drugs. Eur. J. Med. Chem. 2010;45:5646-5652.
27. The compounds in appropriate concentrations (300 - 3 µg/ml) were dissolved in 0.1 ml
DMSO and final solutions were prepared by adding MEM with 1 µg/ml trypsin.
Compounds were incubated with MDCK cells for 1 h at 36⁰C. Each concentration of the
compounds was tested in triplicate. The cell culture was then infected with influenza
virus A/California/07/09 (H1N1)pdm09 (moi 0.01) for 24 h at 36⁰C in the presence of
5% CO2. A virus titer in the supernatant was determined by hemagglutination test after
cultivating of the virus in MDCK cells for 48 h at 36⁰C in the presence of 5% CO2. For
12

calculations, virus titer was expressed as per cent of the titer in control wells without
compounds. The 50% inhibiting concentrations (IC50) and the selectivity index (SI, the
ratio of CC50 to IC50) were calculated from the data obtained.
28. Sokolova AS, Yarovaya ОI, Baev DS, Shernyukov АV, Shtro AA, Zarubaev VV,
Salakhutdinov NF. Aliphatic and alicyclic camphor imines as effective inhibitors of
influenza virus H1N1. Eur. J. Med. Chem. 2017;127:661-670.
29. The microtetrazolium test (MTT) was used to study the cytotoxicity of the compounds.
Briefly, series of three-fold dilutions of each compound in MEM were prepared. MDCK
cells were incubated for 48 h at 36oC 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 µg/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.
30. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Methods 1983;65:55-63.
31. Genovese S, Epifano F. Auraptene: a natural biologically active compound with multiple
targets. Curr. Drug Targets 2011;12:381-386.
32. Laev SS, Salakhutdinov NF, Lavrik OI. Tyrosyl-DNA phosphodiesterase inhibitors:
Progress and potential. Bioorg. Med. Chem. 2016;24:5017-5027.
33. Zakharenko AL, Ponomarev KU, Suslov EV, Korchagina DV, Volcho KP, Vasil’eva IA,
Salakhutdinov NF, Lavrik OI. Inhibitory properties of nitrogen-containing adamantane
derivatives with monoterpenoid fragments against Tyrosyl-DNA Phosphodiesterase 1.
Russ. J. Bioorg. Chem. 2015;41:657-662.
34. Katya S, El-Khamisy SF, Russell HR, Li Y, Ju L, Caldecott KW, McKinnon PJ. TDP1
facilitates chromosomal single-strand break repair in neurons and is neuroprotective in
vivo. EMBO J. 2007;26:4720-4731.
35. To determine the stage of the viral life cycle that is affected with the compound, cells
were seeded into 24-wells plates and incubated with influenza virus A/Puerto Rico/8/34
(H1N1) (m.o.i. 10) for 1 hour at 4°C. After washing of non-absorbed virions for 5 min
with MEM, fresh portion of compound-containing medium was added and plates were
13

incubated for 8 hours at 36°C at 5% CO2. The starting point of this incubation was
referred as 0 hours. 15c (final concentration 500 µM) was dissolved in MEM and cells
were treated with 15c for the time periods as following: (-2) – (-1) (before infecting); (-1)
– 0 (simultaneously to absorption); 0 – 2; 2 – 4; 4 – 6; 6 – 8 hours post infection (hpi).
The treatment (-2) – 8 hpi was considered as a positive control. In each case after
incubation the medium was removed and cells were washed for 5 min with MEM. After
8 hours of growth, the infectious titer of the virus was determined in culture medium and
cells as described above.
36. Dong G, Peng C, Luo J, Wang C, Han L, Wu B, Ji G, He H. Adamantane-resistant
influenza a viruses in the world (1902-2013): frequency and distribution of M2 gene
mutations. PLoS On. 2015;10:e0119115.
37. Wang J, Li F, Ma C. Recent progress in designing inhibitors that target the drug-resistant
M2 proton channels from the influenza A viruses. Biopolymers 2015;104(4):291-309.
38. Hu Y, Musharrafieh R, Ma C, Zhang J, Smee DF, DeGrado WF, Wang J. An M2-V27A
channel blocker demonstrates potent in vitro and in vivo antiviral activities against
amantadine-sensitive and -resistant influenza A viruses. Antiviral Res. 2017;140:45-54.
39. For computer modeling, the crystal structure of hemagglutinin trimer was taken from
Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). The molecules of 15c and
15d were structurally optimized by Hyperchem software (Hypercube Inc., Gainesville,
FL, USA) followed by computer docking with Hex 8.0.0 (accessible at
http://hex.loria.fr/dist/index.php).
14

O
O
IC50=21 µM
SI=41
КВ
(-2) - (10) (-2) - (-1) (-1) - 0
0-1
1-2
2-4
4-6
6-8
Time of compound presence