Anti-viral activity of (-)- and (+)-usnic acids and their derivatives against influenza virus A(H1N1)2009

Article abstract of DOI:10.1016/j.bmcl.2012.09.084

Influenza is a widespread respiratory infection. Every year it causes epidemics, quickly spreading from country to country, or even pandemics, involving a significant part of the human population of the earth. Being a highly variable infection, influenza

Full text of DOI:10.1016/j.bmcl.2012.09.084

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7060–7064
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-viral activity of (ꢀ)- and (+)-usnic acids and their derivatives against
influenza virus A(H1N1)2009
Dmitriy N. Sokolov a, Vladimir V. Zarubaev b, , Anna A. Shtro b, Marina P. Polovinka a, Olga A. Luzina a,
⇑
Nina I. Komarova a, Nariman F. Salakhutdinov a, Oleg I. Kiselev b
a Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia
b Department of Chemotherapy, Influenza Research Institute, 15/17 Prof. Popova St., 197376 St. Petersburg, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Influenza is a widespread respiratory infection. Every year it causes epidemics, quickly spreading from
country to country, or even pandemics, involving a significant part of the human population of the earth.
Being a highly variable infection, influenza easy accumulates the resistance mutations to many antivirals.
Usnic acid, a dibenzofuran originally isolated from lichens belongs to the secondary metabolites and
has a broad spectrum of biological activity. In humans, it can act as an anti-inflammatory, antimitotic,
antineoplasic, antibacterial, and antimycotic agent. In this work we studied for the first time the antiviral
activity of usnic acid and its derivatives against the pandemic influenza virus A(H1N1)pdm09. A total of
26 compounds representing (+) and (ꢀ) isomers of usnic acid and their derivates were tested for cytotox-
icity and anti-viral activity in MDCK cells by microtetrazolium test and virus yield assay, respectively.
Based on the results obtained, 50% cytotoxic dose (CTD50) and 50% effective dose (ED50) and selectivity
index (SI) were calculated for each compound. Eleven of them were found to have SI higher than 10
(highest value 37.3). Absolute configuration was shown to have critical significance for the anti-viral
activity. With minor exceptions, in the pair of enantiomers, (ꢀ)-usnic acid was more active comparing
to (+)-isomer, but its biological activity was reversed after the usnic acid was chemically modified. Based
on the obtained results, derivatives of usnic acid should be considered as prospective compounds for
further optimization as anti-influenza substances.
Received 6 August 2012
Revised 22 September 2012
Accepted 25 September 2012
Available online 4 October 2012
Keywords:
Usnic acid
Enantiomer
Influenza
Pandemic
Antiviral
Ó 2012 Elsevier Ltd. All rights reserved.
Influenza is a widespread respiratory infection. Every year it
causes epidemics, quickly spreading from country to country, or
even pandemics, involving a significant part of the human popula-
tion of the earth. It causes 20,000–40,000 deaths per year in the
United States.1,2 Despite the success in chemotherapy, vaccination
prevention measures and immunology of influenza, this infection
remains difficult to control due to high genetic variability of the
virus and various long-term complications after the acute stage,
which leads to ‘latent’ or secondary mortality caused by virus-in-
duced secondary processes, but not by the influenza virus itself.3
The influenza virions are enveloped, mostly spherical particles,
containing an outer lipid membrane with integrated surface
proteins of three types (hemagglutinin, neuraminidase and M2
protein). The genome of influenza virus is represented by eight
separate segments of single-strand negative RNA associated with
nucleoprotein and three subunits of the polymerase complex.
Unlike eukaryotic RNA-polymerase, viral polymerase complex does
not have error correction mechanism. For this reason influenza
virus demonstrates a very high rate of mutations leading to escape
of adaptive immune response and the fast selection of drug-resis-
tant strains. The life cycle of influenza virus consists of absorption
to the plasma membrane, penetration into cytoplasm, decapsida-
tion and release of nucleoprotein, and its transport into a nucleus
where transcription and replication of the viral genome occur.
Nascent virus-specific RNAs are transported into the cytoplasm
following by translation of viral mRNA and assembly of nascent
proteins with newly synthesized vRNAs. This results in formation
of progeny virions followed by their budding from the plasma
membrane.
Despite numerous steps in the viral life cycle that are potential
targets for drug intervention, only two of them are now available
for clinical administration. Currently, two main classes of chemical
compounds are used for the treatment of influenza. They differ in
their viral targets and mechanism of action.4 Amantadine and
rimantadine block viral M2 protein, which is necessary for the re-
lease of viral RNA into infected cells.5 Neuraminidase inhibitors
zanamivir, oseltamivir, peramivir and laninamivir6 interfere with
the activity of viral neuraminidase, which plays an essential role
in the release of progeny virus particles from the host cell. Despite
obvious advantages, derivatives of adamantane inhibit influenza
viruses of type A but not type B. Inhibitors of neuraminidase are
⇑
Corresponding author.
E-mail address: zarubaev@influenza.spb.ru (V.V. Zarubaev).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.084

D. N. Sokolov et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7060–7064
7061
expensive and drug-resistant strains have already been discovered,
particularly for the highly pathogenic viruses of H5N1 subtype and
seasonal H1N1 subtype.7
Since the absolute configuration of chiral compounds is gener-
ally critical to their biological activity,26 we synthesized pairs of
enantiomer derivatives from (+)- and (ꢀ)-usnic acids and tested
their antiviral activity. Only the structures of compounds derived
from R-(+)-usnic acid are shown on schemes. Enantiomers of these
compounds were obtained on the basis of S-(ꢀ)-usnic acid in the
same manner.
Compounds 2 were synthesized by the previously described
procedure27 in the reaction of usnic acid (1) with phenylhydrazine
hydrochloride (Scheme 1). Compounds 3 were synthesized by the
previously described procedure28 (Scheme 1).
Ribavirin is a ribosyl purine analog, the carboxamide group of
which can resemble adenosine or guanosine. As a prodrug, it is
phosphorylated by cellular kinases, and all its intermediates such
as ribavirin mono-(RMP), di-(RDP) and triphosphate (RTP) are
inhibitors of viral RNA-dependent RNA polymerases. RTP is incor-
porated into RNA and pairs with uracil and cytosine, inducing
lethal hypermutations. In addition, RMP has been shown to target
the cellular inosine monophosphate dehydrogenase (IMPDH) and
thereby depletes intracellular pools of GTP. When delivered intra-
venously, ribavirin eliminated virus from and produced improve-
ment in patients with severe influenza or parainfluenza virus
infection.8 However, the drug’s clinical utility may be limited by
the risk of hemolytic anemia, and its teratogenic properties pro-
scribing its use in pregnant women.9
Favipiravir (T-705), which is currently in Phase III trials in Japan,
also displays broad-spectrum antiviral activity, but only among
RNA viruses. Its in vivo efficacy was demonstrated for influenza
viruses (A, B, and C), flavi-, bunya-, arena- and picornaviruses.
T-705 is converted to the ribofuranosyl triphosphate form by host
cell enzymes, but the mechanism of T-705 is not fully understood.
It does not affect the synthesis of cellular DNA or RNA, and may
therefore target viral RNA-dependent RNA polymerases directly
or may be preferentially incorporated into viral RNA, thereby caus-
ing hypermutations.10
Compounds 4, 5a, 8, 9, 10, 11, and 12 were synthesized by the
procedures described by Tazetdinova et al .29 (Scheme 2).
Experimental details and data for synthesis procedure compounds
4, 8, 11, 12 are cited in the References and notes.30,31 Compounds
5b was synthesized by the method described previously29 in the
reaction of 5a with methyl iodide (Scheme 2). Compounds
6 and 7 were obtained by the procedure developed by Luzina
et al.32 in the reactions of usnic acid
(Scheme 2).
1 with amino acids
Compounds 13 were synthesized by the previously described
procedure33 in the reaction of usnic acid 1 with peracetic acid
(Scheme 3).
Studies34–36 of the biological activity of 1–13 with respect to the
influenza virus revealed high efficiency of many compounds as
virus reproduction inhibitors. As expected, the activity depended
strongly on the absolute configuration of the resulting compounds
(Table 1). As a result, these studies revealed that in the pair of sub-
strates, (-)-usnic acid (ꢀ)-1 was more active as an inhibitor of virus
reproduction, but its biological activity was reversed after the
chemical modification of usnic acid. (ꢀ)-Usnic acid (ꢀ)-1 exhibited
higher antiviral activity than its (+)-enantiomer (+)-1, but in the
pairs of enantiomer derivatives 3–5b, 7–10, 13, the (+)-enantio-
mers were much more potent inhibitors of the virus (according
to ED50). For compounds 2, 6, 11, and 12, the inhibiting activities
of the enantiomers were comparable.
Additionally, several studies describe the virus-inhibiting prop-
erties of synthetic compounds targeting viral RNA polymerase,11–16
fusion protein,17,18 and the pool of intracellular GTP19 or pyrimi-
dines in total.20 Nevertheless, none of these compounds have been
developed far enough to become a clinically available drug. There
is thus a need for the search and further development of new po-
tent antivirals against influenza, which have a broad spectrum of
activity and alternative mechanisms of action.
Natural compounds, including the secondary metabolites of
lichens, are a promising source of new antiviral compounds.21 Usnic
acid is a unique and accessible plant metabolite available in Russia.
Optically active usnic acid with high optical purity and rotation
angles of different signs can be extracted in sufficient quantities
from lichens of various species. Each enantiomer has a wide and
specific spectrum of bioactivity. The usnic acids were shown to
possess antibacterial, insecticidal, fungicidal and anti-viral proper-
ties. The commercially available (+)-usnic acid inhibits the cyto-
pathic effect of type 1 herpes virus.22 The Zn-usnic acid complex
drug was tested as a remedy against papilloma virus and was shown
to prevent virus replication within six months after therapeutic
Analyzing the structure–activity relationship, we noticed that
pyrazole derivatives 2 were less toxic and inactive against the
virus; their inhibiting activity increased dramatically when the
carbonyl group (C1@O) of 2 was reduced to the hydroxyl group
(compounds 3, Scheme 1). The cytotoxicity of the compounds,
however, increased substantially, leading to equal selectivity indi-
ces of ( )-2 and ( )-3. In the series of enamines 4, 5b and enamino
acid derivatives of usnic acid 6, 7 (Scheme 2), our studies revealed
both a pronounced general tendency of higher inhibiting activity of
(+)-enantiomers except derivative 6. Being more toxic than their
(ꢀ)-counterparts, (+)-isomers had much lower values of EC50’s
leading to higher SI’s.
To study the effect of the nature and position of the substituent
in the aromatic ring on the biological activity, we synthesized a
series of usnic acid derivatives8–12, which were obtained by reac-
tions with halogen-substituted anilines (Scheme 2). In this group
of compounds the effect of the absolute configuration on the bio-
logical activity was the weakest. None of the synthesized com-
pounds exhibited a selectivity index higher than that of ribavirin.
The nature of halogen (F, Cl, Br) and its position in the aromatic
ring (para-, meta-) did not substantially affect the anti-viral
activity.
course.23 (+)-Usnic acid at concentrations of 1.0
ited the replication of Epstein–Barr virus; (ꢀ)-usnic acid appeared to
be less active with effective concentration 5.0 g/ml (15
M).24 In
lg/ml (3 lM) inhib-
l
l
this work, we studied for the first time the antiviral activity of usnic
acid and its derivatives against the pandemic influenza virus
A(H1N1)pdm09.
Compounds (R)-(+)-usnic acid [(+)-1] (½a D
ꢁ
+478 (c 0.1, CHCl3))
and (S)-(ꢀ)-1 (½aꢁD ꢀ458 (c 0.1, CHCl3)) were isolated by the proce-
dure of Salakhutdinov et al.25 (see Supplementary data) from a
mixture of lichens of the genus Usnea [(+)-1] and Cladonia stellaris
[(ꢀ)-1], respectively.
OH
O
OH
O
OH HO
O
N
PhNHNH2
N
N
NaBH4
A
C
N
OH
HO
HO
HO
Ph
Ph
O
O
O
O
O
(+)-1
(+)-2
O
(+)-3
Scheme 1. Synthesis of pyrazole derivatives 2 and 3.

7062
D. N. Sokolov et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7060–7064
R
OH
O
NH
(+)-4 R = CH2CF2CF2H
(+)-5aR = CH2CH2NEt2
(+)-6 R = CH2COOH
RNH2
O
HO
O
(+)-7 R = CH2CH2COOH
O
(+)-4 - (+)-7
12
15 O
OH
10
7
11
2
O
8
9
1
R'
9b
9a
OH
HO
3
6
5a
4a
4
O
OH
O
13
O
(+)-1
14
NH
(+)-8 R' = p-F
R'ArNH2
(+)-9 R' = p-Cl
(+)-10 R' = p-Br
(+)-11 R' = m-F
(+)-12 R' = m-Br
HO
O
O
O
(+)-8 - (+)-12
I
OH
O
NH
N
O
OH
NH
N
MeI
O
HO
O
HO
O
O
O
O
(+)-5a
(+)-5b
Scheme 2. Synthesis of enamino derivatives 4–12.
Table 1
OH
O
OH
OH
O
Activity of compounds against influenza A(H1N1)pdm09 virusa
O
O
MeCOOOH
Compound
CTD50
(lM)
ED50
(lM)
SI
OH
OH
HO
O
(+)-1
305.2
209.3
768.9
522.2
78.9
51.7
14.5
62.2
57.8
6.0
5.9
O
O
O
(ꢀ)-1
14.4
12.4
9.0
O
O
(+)-1
(+)-13
(+)-2
(ꢀ)-2
Scheme 3. Synthesis of oxidized derivatives 13.
(+)-3
13.2
6.8
19.4
10.2
21.9
2.7
(ꢀ)-3
71.8
31.1
396.7
120.7
34.2
10.5
1.6
38.9
5.5
(+)-4
The most interesting results were obtained for the oxidized
derivatives of usnic acid 13 synthesized in the reactions of usnic
acid with organic peracids. The oxidized derivative of (+)-usnic
acid (+)-13 exhibited high inhibiting activity and low cytotoxicity,
and its selectivity index appeared the highest among all derivatives
of usnic acid tested so far (Table 1).
In the present study we investigated the anti-viral activity of
derivatives of usnic acid against recent pandemic influenza virus
A/California/07/09 (H1N1)pdm09. The activity of compounds was
compared with that of ribavirin that has been successfully used
as a reference compound in anti-influenza studies.37,38
(ꢀ)-4
(+)-5b
(ꢀ)-5b
(+)-6
12.5
11.2
10.7
6.9
400.0
23.6
90.4
25.1
13.1
20.2
30.4
12.7
4.0
10.4
9.8
5.1
27.7
24.6
56.9
67.3
6.0
(ꢀ)-6
197.0
162.2
400.0
208.2
350.1
172.4
152.7
193.3
154.2
61.3
18.4
23.5
1.0
8.8
3.9
6.9
11.7
9.6
5.1
4.8
(+)-7
(ꢀ)-7
(+)-8
(ꢀ)-8
(+)-9
(ꢀ)-9
(+)-10
(ꢀ)-10
(+)-11
(ꢀ)-11
(+)-12
(ꢀ)-12
(+)-13
(ꢀ)-13
Ribavirin
Rimantadine
The obtained data demonstrated the high activity of compounds
(ꢀ)-1, (+)-3, (+)-4, (+)-5b, (+)- and (ꢀ)-6, (+)-7, (+)- 8, (+)- and
(-)-11, (+)- and (-)-12, and (+)-13 against the virus in MDCK cells.
Having the highest values of SI’s, compounds (ꢀ)-1, (+)-5b, (ꢀ)-6,
and (+)-13 were the most active among the synthesized derivatives
(SI up to 37.3). Based on the values of SI, none of the compounds
appeared to be more active than the reference compound ribavirin.
Nevertheless, the vast majority of compounds were more active
than ribavirin based on their EC50’s, and their lower SI’s resulted
from their relatively high toxicity.
35.3
33.6
97.8
8.8
3.2
10.0
37.3
8.0
>81.0
5.0
190.2
220.7
>2000.0
284.4
a
The values of EC50 and CTD50 are mean of three different experiments, four
parallels in each.
Previously, usnic acid and its derivatives were shown to possess
the inhibiting activity against herpes virus type 122, papilloma
virus23 and Epstein–Barr virus.24 In a study of anti-HIV activity of
usnic acid it was shown to be inactive against human immunode-
ficiency virus (SI = 3).39 In another study the moderate activity of
usnic acid against mycobacteria was shown.40 As for our knowl-
edge, there is no information about anti-influenza virus activity
of usnic acid and its mechanisms. No chemical compounds of
similar structure were shown to possess direct influenza virus-
suppressing properties.
The virus we used in our experiments was A/California/07/09
(H1N1)pdm09 which is, similar to majority of currently circulating
human influenza
A viruses, resistant to adamantane-based

D. N. Sokolov et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7060–7064
7063
14. Parkes, K. E.; Ermert, P.; Fassler, J.; Ives, J.; Martin, J. A.; Merrett, J. H.; Obrecht,
D.; Williams, G.; Klumpp, K. J. Med. Chem. 2003, 46, 1153.
15. Cianci, C.; Chung, T. D. Y.; Meanwell, N.; Putz, H.; Hagen, M.; Colonno, R. J.;
Krystal, M. Antivir. Chem. Chemother. 1996, 7, 353.
16. Nakazawa, M.; Kadowaki, S.; Watanabe, I.; Kadowaki, Y.; Takei, M.; Fukuda, H.
Antiviral Res. 2008, 78, 194.
17. Plotch, S. J.; O’Hara, B.; Morin, J.; Palant, O.; LaRocque, J.; Bloom, J. D.; Lang, S.
A.; DiGrandi, M. J.; Bradley, M.; Nilakantan, R.; Gluzman, Y. J. Virol. 1999, 73,
140.
antivirals amantadine and rimantadine. As a reference compound,
rimantadine appeared to be inactive against this virus (SI = 5).
Having the activity regarding the influenza strain used, usnic acid
and its derivatives should target a component of the virus other
than M2 protein.
The results of Campanella et al.41 show that the synthesis of
RNA of mouse polyomavirus in 3T6 cells is severely inhibited in
the presence of usnic acid. Virus’ DNA replication was also severely
suppressed at noncytotoxic concentrations of the compound
18. Luo, G.; Torri, A.; Harte, W. E.; Danetz, S.; Cianci, C.; Tiley, L.; Day, S.; Mullaney,
D.; Yu, K. L.; Ouellet, C.; Dextraze, P.; Meanwell, N.; Colonno, R.; Krystal, M. J.
Virol. 1997, 71, 4062.
(5–10 lg/ml, or 15–30 lM). Taken together, the results of the
19. Colacino, J. M.; DeLong, D. C.; Nelson, J. R.; Spitzer, W. A.; Tang, J.; Victor, F.;
Wu, C. Y. Antimicrob. Agents Chemother. 1990, 34, 2156.
20. Hoffmann, H. H.; Kunz, A.; Simon, V. A.; Palese, P.; Shaw, M. L. Proc. Natl. Acad.
Sci. U.S.A. 2011, 108, 5777.
21. Ingolfsdottir, K. Phytochemistry 2002, 61, 729.
22. Perry, N. B.; Benn, M. H.; Brennan, N. J.; Burgess, E. J.; Ellis, G.; Galloway, D. J.;
Lorimer, S. D.; Tangney, R. S. Lichenologist 1999, 31, 627.
23. Scirpa, P.; Scambia, G.; Masciullo, V.; Battaglia, F.; Foti, E.; Lopez, R.; Villa, P.;
Malecore, M.; Mancuso, S. Minerva Ginecol. 1999, 51, 255.
24. Yamamoto, Y.; Miura, Y.; Kimoshita, Y.; Higuchi, M.; Yamada, Y.; Murakami, A.;
Ohigashi, H.; Koshimizu, K. Chem. Pharm. Bull. 1995, 43, 1388.
25. Salakhutdinov, N. F.; Polovinka, M. P.; Panchenko, M.; Yu. Byull. Izobret., 2008,
5, RF Patent No. 2317,076C1.
26. Farina, V.; Reeves, J. T.; Senanayake, C. H.; Song, J. J. Chem. Rev. 2006, 106,
2734.
27. Luzina, O. A.; Polovinka, M. P.; Salakhutdinov, N. F.; Tolstikov, G. A. Russ. J. Org.
Chem. 2009, 45, 1783.
study suggest that the target of the drug is viral RNA production.
This fact could serve as indirect evidence for similar mechanism
of anti-viral activity of usnic acid and its derivatives against influ-
enza virus. If these compounds are able to interact with and inhibit
the polymerase complex of the virus, they can represent a novel
class of low-molecular anti- influenza compounds with novel
mechanism of activity.
On the other hand, some results suggest an anti-inflammatory
activity of usnic acid. Particularly, in LPS-stimulated RAW264.7
macrophages, where it was shown to decrease the TNF-alpha level
(IC50 = 12.8
iNOS protein (IC50 = 4.7
l
M) as well as to attenuate LPS-induced synthesis of
M) and nuclear translocation of
l
28. Sokolov, D. N.; Luzina, O. A.; Polovinka, M. P.; Salakhutdinov, N. F. Chem. Nat.
Comp. 2011, 47, 203.
29. Tazetdinova, A. A.; Luzina, O. A.; Polovinka, M. P.; Salakhutdinov, N. F.;
Tolstikov, G. A. Chem. Nat. Comp. 2010, 45, 800.
NF-kappaB p65. Taken together, these facts suggest that an anti-
inflammatory effect of usnic acid could be explained by inhibiting
TNF-alpha and iNOS expression, possibly through suppression of
nuclear translocation of NF-kB p65 and I-kB
a
degradation.42
30. Experimental: Reactions were monitored by thin layer chromatography (TLC)
on silica gel plates (Sorbfil plates, Imid Ltd.) with products visualization by UV
light. Flash chromatography was performed on silica gel Merck (60–200 mesh)
using distilled chloroform, dichloromethane and ethanole. All compounds
were analyzed for purity by HPLC and characterized by 1H and 13C NMR using
AV-400 spectrometer (Bruker) at operating frequencies of 400.13 MHz (1H)
and 100.61 MHz (13C) for the CDCl3 solutions of the substances. Chemical shifts
are reported in ppm (d) relative to the residual solvent peak in the
corresponding spectra; chloroform 7.24 and d76.90, DMSO d2.6 and d43.5
and coupling constants (J) are reported in hertz (Hz) (where, s = singlet, br
s = broad singlet, d = doublet, dd = double doublet, t = triplet, q = quartet,
m = multiplet). The HPLC analyses were carried out on a Milichrom A-02,
using a ProntoSIL 120-5-C18 AQ column (BISCHOFF, 2.0 ꢂ 75 mm column,
NF-kB is known to play an important stimulatory role in the repro-
duction of the influenza virus in cells and its suppression strongly
decreases the replication activity and infectious titers of the
virus.43 Having NF-kB—suppressing activity, usnic acid may, there-
fore, indirectly reduce virus reproduction by inhibiting cellular
proviral pathways.
Comparing to the results of mentioned studies, the values of
EC50’s obtained in our experiments are for most of active com-
pounds are in the same range or lower. So, they might possess
any mechanism of activity, either direct anti-viral or indirect,
anti-inflammatory, or have another, not yet determined, mecha-
nism of action. Further experiments should be therefore performed
to determine the exact stage of viral life cycle and mode of action
of these substances as well as to optimize their structure to achieve
optimal activity.
grain size 5.0
lm). Mobile phase: Millipore purified water with 0.1%
trifluoroacetic acid at a flow rate of 150
l
L/min at 35 °C and UV detection at
280 nm. A typical run time was 25 min with a linear gradient of 0–100%
methanole. The mass spectra were recorded on a DFS Thermo Scientific high-
resolution mass spectrometer (the energy of ionizing electrons 70 eV). Melting
points were measured on a Kofler heating stage. The IR spectra were recorded
on a Vector 22 spectrometer. Optical rotation parameters were as follows:
polAAr 3005 spectrometer, CHCl3 solution. All of the target compounds
reported in this paper have a purity of P95% (HPLC).
31. General Method for synthesis of compounds 4, 8, 11 and 12: Compound
1(1 mmol) was treated with corresponding aniline (1.1 mmol), dissolved in
ethanol (12 mL), refluxed on a water bath for 3 h, cooled, and treated with
distilled water (10 mL) with formation of white precipitate. The precipitate
was filtered, washed with water and dried in air. The products were separated
by chromatography, and this afforded compounds 4, 8, 11 and 12.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
084.
(+)-(R,E)-6-Acetyl-7,9-dihydroxy-8,9b-dimethyl-2-(1-(20,20,30,30-tetrafluoropro-
pylamino)ethylidene)dibenzo[b,d]furan-1,3-dione (4), yield 92%; mp 155 °C,
½aꢁD +324 (c 0.34, CHCl3). IR spectrum (KBr, t
, cmꢀ1): 1061, 1103, 1288, 1373,
1460, 1560, 1630, 1699, 2989, 3016. 1H NMR (CDCl3) d (ppm): 1.74 (s, 3H,
H15), 2.13 (s, 3H, H10), 2.65 (s, 3H, H14), 2.68 (s, 3H, H12), 3.75 (s, 1H, NH),
4.11 (m, 2H, H10), 5.85 (s, 1H, H4), 5.97 (m,1H, H30), 11.57 (s, 1H, C9-OH), 13.34
(s, 1H, C7-OH). 13C NMR (CDCl3) d (ppm): 7.4 (C10), 17.9 (C12), 31.1 (C15), 31.7
(C14), 41.9 (m, C10), 57.5 (C9b), 101.3 (C4), 102.0 (C6), 103.1 (C9a), 108.1 (C8),
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1
1
109.6 (C2), 115.2 (C30, tt, JC-F = 240), 110.2 (C20, tt, JC-F = 250), 155.6 (C5a),
157.9 (C9), 163.4 (C7), 174.5 (C4a), 176.5 (C11), 190.9 (C3), 198.7 (C1), 200.6
(C13). HRMS: M+ 457 (C21H19O6N1F4); obsd: m/z 457.1141; calcd: 457.1143.
(-)-4, ½aꢁD26ꢀ320 (c 0.3; CHCl3);
(+)-(R,E)-6-Acetyl-2-(1-(4-fluorophenylamino)ethylidene)-7,9-dihydroxy-8,9b-
dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione (8), light yellow solid, 70%
8. Hayden, F. G.; Sable, C. A.; Connor, J. D.; Lane, J. Antivir. Ther. 1996, 1, 51.
9. Beigel, J.; Bray, M. Antiviral Res. 2008, 78, 91.
yield; mp 72–74 °C; ½a D31
ꢁ
+257 (c 0.2; CHCl3); IR (KBr) 1061, 1133, 1200,
10. Furuta, Y.; Takahashi, K.; Shiraki, K.; Sakamoto, K.; Smee, D. F.; Barnard, D. L.;
Gowen, B. B.; Julander, J. G.; Morrey, J. D. Antiviral Res. 2009, 82, 95.
11. Tomassini, J. E.; Davies, M. E.; Hastings, J. C.; Lingham, R.; Mojena, M.;
Raghoobar, S. L.; Singh, S. B.; Tkacz, J. S.; Goetz, M. A. Antimicrob. Agents
Chemother. 1996, 40, 1189.
12. Tomassini, J.; Selnick, H.; Davies, M. E.; Armstrong, M. E.; Baldwin, J.; Bourgeois,
M.; Hastings, J.; Hazuda, D.; Lewis, J.; McClements, W. Antimicrob. Agents
Chemother. 1994, 38, 2827.
1286, 1365, 1452, 1546, 1628, 1699, 2921, 3434; 1H NMR (CDCl3) d (ppm): 1.72
(s, 3H, H15), 2.06 (s, 3H, H10), 2.54 (s, 3H, H12), 2.65 (s, 3H, H14), 5.84 (s, H4),
7.15–7.17 (m, 4H, H2 and H60, H30027 and H50), 11.75 (s, C9-OH), 13.34 (s, C7-
OH), 15.00 (s, NH); 13C NMR (CDCl3) d (ppm): 7.4 (C10), 20.4 (C12), 31.2 (C14),
31.8 (C15), 57.5(C9b), 101.3, 102.7, 104.8 (C6, C9a, C2), 102.1 (C4), 108.1 (C8),
116.7 (d, 2JC-F = 23 Hz, C30 and C50), 127.5 (d, 3JC-F = 8 Hz, C20 and C60), 161.9 (d,
1JC-F = 248 Hz, C40), 132.1 (d, JC-F = 3 Hz, C10), 155.7 (C5a), 158.1 (C9), 163.5
4
(C7), 174.2 (C11), 174.8 (C4a), 191.0 (C3), 198.8 (C1), 200.6 (C13); HRMS: M+
437 (C24H20O6NF); obsd: 437.1263; calcd: 437.1269.
13. Hastings, J. C.; Selnick, H.; Wolanski, B.; Tomassini, J. E. Antimicrob. Agents
Chemother. 1996, 40, 1304.
(ꢀ)-8, ½a 3D1
ꢀ253 (c 0.2; CHCl3).
ꢁ

7064
D. N. Sokolov et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7060–7064
(+)-(R,E)-6-Acetyl-2-(1-(3-fluorophenylamino)ethylidene)-7,9-dihydroxy-8,9b-
dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione (11), light yellow solid, 75%
MEM. The dilutions were applied to the MDCK cells and incubated for 48 h at
37 °C. Cells were washed twice with PBS and the number of survived cells was
evaluated by a microtetrazolium test (MTT44). Briefly, 3-(4,5-dimethyltiazolyl-
2) 2,5-diphenyltetrazolium bromide (ICN Biochemicals Inc., Aurora, Ohio,
0,5 mg/ml, 0,1 ml per well) was added with subsequent incubation of plates at
37 °C in 5% CO2 for 60 min. Colored deposit was dissolved in 100 microliters of
DMSO. The plates were swirled gently and left in darkness at room
yield; mp 89–91 °C. ½a D31
ꢁ
+248 (c 0.2; CHCl3); IR 1063, 1132, 1188, 1282,
1366, 1461, 1545, 1629, 1699, 2927, 3434; 1H NMR (CDCl3) d (ppm): 1.73 (s,
3H, H15), 2.07 (s, 3H, H10), 2.58 (s, 3H, H10), 2.66 (s, 3H, H14), 5.86 (s, H4),
6.95 (d, J = 8.5 Hz, H20), 7.01 (d, J = 8 Hz, H60), 7.10 (dd, J = 8.5 Hz and J = 8 Hz,
H40), 7.44 (dd, J = 8 Hz and J = 8 Hz, H50), 11.69 (s, C9-OH), 13.34 (s, C7-OH),
15.14 (s, NH). 13C NMR (CDCl3) d (ppm): 7.3 (C10), 20.4 (C12), 31.1 (C14), 31.7
(C15), 57.5 (C9b), 101.2, 102.7 and 104.7 (C6, C9a and C2), 102.0 (C4), 108.1
temperature for 30 min. Optical density was measured on
a
spectrophotometer (Victor 1420, Perkin Elmer, Finland) at wavelength
535 nm. Based on these data, the CTD50 (compound concentration required
to reduce 50% cell viability) value was estimated for each compound.
2
2
(C8), 113.2 (d, JC-F = 23 Hz, C40), 115.2 (d, JC-F = 23 Hz, C20), 121.5
4
3
3
(d, JC-F = 3 Hz, C60), 130.9 (d, JC-F = 9 Hz, C50), 137.4 (d, JC-F = 9 Hz, C10),
1
162.8 (d, JC-F = 249 Hz, C30), 155.6 (C5a), 157.9 (C9), 163.4 (C7), 173.8 (C11),
36. Antiviral assay: For evaluation of anti-viral activity of usnic acid derivatives, the
assay was performed by quantifying virus yield by the end-point dilution
method. Briefly, serial twofold dilutions of compounds under investigation
were prepared in MEM with addition of arginine (2 mM), glutamine (2 mM)
174.7 (C4a), 190.9 (C3), 198.8 (C1), 200.4 (C13); HRMS: M+ 437 (C24H20O6NF);
obsd: 437.1267; calcd: 437.1269. (-)-11, ½a D31
ꢀ256 (c 0.2; CHCl3);
ꢁ
(+)-(R,E)-6-Acetyl-2-(1-(3-bromophenylamino)ethylidene)-7,9-dihydroxy-8,9b-
dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione (12), light yellow solid, 84%
and trypsin (2
for 1 h at 37 °C followed by infecting with influenza virus in a dose 1–106 50%
infecting dose (ID50 in the medium. Control wells did not contain the
compounds. Virus was cultivated at 36 °C for 48 h followed by
hemagglutination assay. 100 L of supernatant was transferred into round-
bottom wells and mixed with 100 L of 1% chicken erythrocytes followed by
lg/mL). The solutions were applied to MDCK cells and incubated
yield; mp 176–178 °C; ½a D30
ꢁ
+218 (c 0.2; CHCl3); IR 1062, 1136, 1188, 1286,
1365, 1459, 1541, 1624, 1695, 2921, 3440; 1H NMR (CDCl3) d (ppm): 1.74 (s,
3H, H15), 2.09 (s, 3H, H10), 2.58 (s, 3H, H12), 2.68 (s, 3H, H14), 5.87 (s, H4),
7.15 (d, J = 8 Hz, H60), 7.35 (dd, J = 8 Hz and J = 8 Hz, H50), 7.39 (s, H20), 7.53 (d,
J = 8 Hz, H40), 11.68 (s, C9-OH), 13.34 (s, C7-OH), 15.14 (s, NH). 13C NMR
(CDCl3) d (ppm): 7.4 (C10), 20.4 (C12), 31.1 (C14), 31.7 (C15), 57.5(C9b), 101.2,
102.7 and 104.7 (C6, C9a and C2), 102.0 (C4), 108.1 (C8), 123.0 (C30), 124.4 and
128.8 (C20 and C60), 130.8 and 131.2 (C50 and C40), 137.3 (C10), 155.6 (C5a),
157.9 (C9), 163.5 (C7), 173.7 (C11), 174.8 (C4a), 190.9 (C3), 198.8 (C1), 200.4
(C13); HRMS: M+ 497 (C24H20O6NBr); obsd: 497.0466; calcd: 497.0469. (ꢀ)-12,
)
l
l
1 h incubation at room temperature. Virus titer was considered as reciprocal to
the final dilution of the inoculum able to cause positive hemagglutination in
50% of wells and expressed in ID50’s. Anti-viral activity of the compounds was
evaluated by their ability to decrease the virus titer. Based on the results, 50%
effective dose (concentration of compound that decreases the virus production
twofold comparing to control) and the selectivity index (SI, relation of CTD50 to
ED50) were calculated.
½ ꢁ ꢀ228 (c 0.2; CHCl3).
a 3D0
32. Luzina, O. A.; Polovinka, M. P.; Salakhutdinov, N. F.; Tolstikov, G. A. Russ. Chem.
Bull. 2007, 56, 1249.
37. Vanderlinden, E.; Göktas, F.; Cesur, Z.; Froeyen, M.; Reed, M. L.; Russell, C. J.;
Cesur, N.; Naesens, L. J. Virol. 2010, 84, 4277.
38. Selvam, P.; Murugesh, N.; Chandramohan, M.; Sidwell, R. W.; Wandersee, M.
K.; Smee, D. F. Antivir. Chem. Chemother. 2006, 17, 269.
39. http://chemdb.niaid.nih.gov/CellularDetails.aspx?AIDSNO=028613&pn=1.
40. König, G. M.; Wright, A. D.; Franzblau, S. G. Planta Med. 2000, 66, 337.
41. Campanella, L.; Delfini, M.; Ercole, P.; Iacoangeli, A.; Risuleo, G. Biochimie 2002,
84, 329.
42. Jin, J. Q.; Li, C. Q.; He, L. C. Phytother. Res. 2008, 22, 1605.
43. Pinto, R.; Herold, S.; Cakarova, L.; Hoegner, K.; Lohmeyer, J.; Planz, O.; Pleschka,
S. Antiviral Res. 2011, 92, 45.
33. Sokolov, D. N.; Luzina, O. A.; Polovinka, M. P.; Korchagina, D. V.; Gatilov, Yu. V.;
Salakhutdinov, N. F. Chem. Nat. Comp. 2010, 46, 730.
34. Viruses and cells: For in vitro experiments MDCK cells (ATCC CCL 34) were used.
Experiments were performed with influenza virus A/California/07/09
(H1N1)pdm09 from the collection of viral strains from Center for Disease
Control and Prevention (CDC, Atlanta, GA). Virus was propagated in the
allantoic cavity of 10–12 days old chicken embryos for 48 h at 36 °C.
35. Toxicity assay: For compound’s cytotoxity evaluation, MDCK cells were seeded
onto 96-wells plates and cultivated in Eagle’s minimal essential medium
(MEM) with addition of 5% fetal calf serum. After the cell monolayer formation
cells were washed by serum-free MEM. The compounds were dissolved in
44. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
DMSO to 5 mg/ml, and serial twofold dilutions (1000–4 l/ml) were prepared in