Novel derivatives of usnic acid effectively inhibiting reproduction of influenza A virus

Article abstract of DOI:10.1016/j.bmc.2014.10.033

Influenza virus is serious human pathogen leading to high morbidity and mortality all over the world. Due to high rate of mutation, it is able to fast development of drug resistance that makes necessary to search novel antivirals with broad range and alternative targets. In the present study we describe synthesis and anti-viral activity of novel derivatives of usnic acid (2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione). It is shown that anti-viral activity of usnic acid can be increased by side moieties introduction. The modification with chalcones appeared to be the most effective. Our study revealed that (-)-usnic acid exhibited higher antiviral activity than its (+)-enantiomer, but in the pairs of enantiomer derivatives such as enamines, pyrazoles and chalcones, the (+)-enantiomers were more potent inhibitors of the virus. For other groups of compounds the inhibiting activities of the enantiomers were comparable. Further optimization of the structure could therefore result in development of novel anti-influenza compound with alternative target and mechanism of virus-inhibiting action.

Full text of DOI:10.1016/j.bmc.2014.10.033

Accepted Manuscript
Novel derivatives of usnic acid effectively inhibiting reproduction of influenza
A virus
Anna A. Shtro, Vladimir V. Zarubaev, Olga A. Luzina, Dmitry N. Sokolov, Oleg
I. Kiselev, Nariman F. Salakhutdinov
PII:
S0968-0896(14)00764-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.10.033
BMC 11879
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 August 2014
21 October 2014
24 October 2014
Please cite this article as: Shtro, A.A., Zarubaev, V.V., Luzina, O.A., Sokolov, D.N., Kiselev, O.I., Salakhutdinov,
N.F., Novel derivatives of usnic acid effectively inhibiting reproduction of influenza A virus, Bioorganic &
Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.10.033
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Novel derivatives of usnic acid effectively inhibiting reproduction of influenza
A virus
Anna A. Shtro1, Vladimir V. Zarubaev1*, Olga A. Luzina2, Dmitry N. Sokolov2, Oleg I. Kiselev1,
Nariman F. Salakhutdinov2
1Influenza Research Institute, 15/17 prof. Popova st., St. Petersburg, Russia
2Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of
Sciences, 630090 Novosibirsk, Russia
*
Corresponding author. Tel. +7 812 499 1527, Fax +7 812 499 1526,
Email:zarubaev@influenza.spb.ru

1. Introduction
Influenza is a global public health problem causing annual epidemics and sometimes
pandemics. Influenza is responsible for approximately 5–20 percent morbidity of the population,
and 3,300–48,600 (average 23,600) lethal outcomes per year1. Mild infection is characterized by
upper respiratory distress along with high fever, myalgia, headache and severe malaise,
nonproductive cough, sore throat and rhinitis. Severe illness and death are mainly associated
with the young, elderly, and persons with compromised immune system2. Occasionally influenza
virus creates substantial threat, for instance associated with ‘swine’ influenza H1N1, avian
influenza H5N1 and recent H7N9 viruses in humans that resulted in significant morbidity and
mortality. The 2009 pandemic influenza A (H1N1) virus, a new strain of virus identified in
Mexico in April 2009, spread rapidly across the globe3. According to WHO statistics, this
pandemic virus had killed more than 18000 people around the world since 2009, but in fact the
mortality was estimated at more than 284000, exceeding the number of laboratory-confirmed
cases about 15 times mainly because many people who die of flu-related causes were not tested
for the disease.4
There are two ways of protection against influenza – vaccination and chemotherapy.
Immunization against influenza is considered an essential public health intervention to control
both seasonal epidemics and pandemic influenza. Unfortunately, vaccination cannot make
absolute protection because of rapid antigenic change in circulating influenza viruses. New
vaccine composition is required every year. For example, the effectiveness of seasonal vaccine in
2009-2010 years was ranging between 59%–79%5,6 and the monovalent H1N1/09 vaccine
between 56–93%7,8,9,10
.
Chemotherapy can`t give absolute guarantee, too. At present there are two active working
groups of anti-influenza drugs – M2 protein inhibitors (amantadine and rimantadine) and
neuraminidase inhibitors (oseltamivir, zanamivir, peramivir and laninamivir). The third group
includes polymerase inhibitors – ribavirin, which possesses numerous side effects, and
favipiravir (T-705) which is currently at Phase III trials in Japan11,12,13
.
Amantadine and rimantadine block viral M2 protein, which is necessary for the release of
viral RNA into infected cells. These compounds were very effective, but now the group is “out
of work” because of a high level of resistance among the circulating strains of influenza viruses.
For example 100% (17/17) of influenza A (H3N2) and 100% (259/259) of 2009 influenza A
(H1N1) samples were resistant to amantadine in China14, 100% of strains isolated in Russia in
2009 were resistant too15. According to the global surveillance data, the frequency of
amantadine-resistant variants was 62.2%16.

The neuraminidase inhibitors are rather expensive and drug-resistant strains had already
been discovered, for example among A(H1N1)pdm09 isolates17.
Influenza virus frequently undergoes amino acid substitutions, which result in the
emergence of drug-resistant viruses. To control influenza viruses that are resistant to currently
available drugs, it is essential to develop new antiviral drugs with a novel molecular target. In
these conditions creation of novel compounds becomes evident.
Usnic acid (2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione) is
obtained from lichen Usnea sp. It has been shown to work as a growth regulator in higher
plants18. This substance has a broad spectrum of biological activity – antibacterial, anti-parasitic,
anti-mycotic, anti-inflammatory, anti-viral, anti-pyretic, and anesthetic effects19. Anti-tumor
activity of UA was first reported against lung carcinoma in mice and P388 leukemia20. Further
usnic acid has been shown to demonstrate anti-mitotic effects on human cancer cell lines21 and to
cause a loss of viable cells in leukemia, lung and breast cancer cells22,23. However, exposure to
UA does not activate p53 and has not been proposed to be involved in DNA damage24,25
.
Usnic acid has a good antimicrobial activity against Gram-positive bacteria26,27 such as
Staphylococcus epidermidis, S. aureus, Enterococcus faecalis, Mycobacterium tuberculosis and
some pathogenic fungi28. Usnic acid was also recently shown to be able to prevent biofilm
formation by Staphylococcus spp. when incorporated in polyurethanes29 or in bone cements30.
Therefore, this compound is under investigation for the prevention of medical device-related
infections as an antimicrobial agent to be adsorbed on the surface of medical devices as an
alternative to conventional antibiotics. Usnic acid also shows in vitro inhibitory and fungicidal
activity against environmental isolates of C. orthopsilosis and C. parapsilosis, but mechanisms
of this activity still remain unknown28.
Antiviral activity of usnic acid is investigated in less extent. Commercially obtained (+)-
usnic acid was shown to inhibit cytopathic effects of Herpes simplex type 1 and polio type 1
viruses in vitro31. In a clinical study performed in Italy with the participation of 100 female
patients (aged 18–45) infected with genital human papillomavirus, the effects of an intravaginal
formulation containing usnic acid and zinc sulphate were evaluated as adjuvant therapy to
radiosurgical treatment32. Its application showed favorable results for patients receiving the Zn–
usnic acid vaginal formulation, both with regard to re-epithelization of lesions and recurrence of
infection over a 6 month period, as compared to a control group receiving no adjuvant treatment.
In a cancer chemoprevention assay designed to detect potential inhibitors of tumour promotion,
(+)-usnic acid isolated from U. longissima showed potent inhibitory effects (ED50=1.0 mg/ml)
against Epstein–Barr virus activation induced by teleocidin B-4, a potent tumour promoter33.

Usnic acid is a potent inhibitor of the proliferation of mouse polyomavirus. Its anti-viral activity
is not exerted at the level of virion entry into the host cell. Moreover, the abolition of viral DNA
replication is an indirect consequence of the drastic inhibition of RNA transcription34.
In previous work35 we have studied the antiviral activity of usnic acid and its derivatives
against the pandemic influenza virus A (H1N1)pdm09 and showed inhibitory activity of some
compounds in some cases exceeding that of usnic acid itself. In the present work we continued to
study the antiviral activity of newly synthesized derivatives of (+) and (-) usnic acid.
2. Materials and methods
2.1 Chemicals
Compounds (R)-(+)-1 and (S)-(-)-1 were isolated by the procedure36 from mixture of
lichens of the genus Usnea ((+)-1) and Cladonia stellaris ((-)-1), respectively.
All chemicals and solvents were analytical grade and were purchased from Sigma–
Aldrich, Acros, Alfa Aesar, Panreac and Chimmed (Russia). All solvents were purified by
general methods before experiments. Peracetic acid CH3CO3H was prepared by conventional
procedure37. Merck silica gel (63—200m) was used for column chromatography. Thin-layer
chromatography was performed on Sorbfil plates (UV-254).
2.2. Physical-chemical methods
Analytical and spectral studies were carried out in the Chemical Collective Use Service
Center of the SB RAS. PMR spectra and 13C NMR spectra of compounds were recorded on an
AV-400 spectrometer (operating frequencies 400.13 MHz for 1H and 100.61 MHz for 13C)
(Bruker). The internal standards were the CDCl3 resonances (7.24 ppm and 76.90 ppm). The
structure was established using 1D and 2D spectra (with tuning on 1JH,C = 160 Hz for HSQC
and nJH,C = 7 Hz for HMBC). Mass spectra (energy of ionizing electrons 70 eV) were recorded
on mass spectrometer of high resolution Thermo Scientific DFS. Melting point was measured on
a Kofler heating stage. IR spectra were recorded on a Vector 22 spectrometer.
2.3. Synthetic usnic derivatives
Since the absolute configuration of chiral compounds is generally critical to a definite
biological activity38, we synthesized pairs of enantiomeric derivatives from (+)- and (-)-usnic
acids and tested their antiviral activity.
Compounds (+)-2, ( )-3, ( )-4, (+)-5, ( )-6, ( )-7, (+)-9, ( )-10, ( )-11, (+)-12, (+)-13,
( )-14, ( )-15, ( )-16, ( )-17, (+)-25, (+)-26, ( )-27, (+)-28, (+)-29, (+)-34, (+)-35, (-)-36, (+)-

37, (+)-38, +)-39, (+)-40, (+)-41, ( )-43, (+)-44 are known and were synthesized by the
procedures described (see below).
Spectral data of new compounds (+)-8, (+)-18, (+)-19, (+)-21, (+)-22, (+)-23, (+)-24, (+)-
30, (+)-31, (+)-32, (+)-33 and (+)-42 are provided below. The atom numeration in new
compounds is given for the assignment of signals in the NMR spectra and sometimes differs
from the atom numeration in the nomenclature names. All target compounds reported in this
paper had a purity of ≥95% (HPLC).
Compound 2 was synthesized by the procedure39 in the reaction of usnic acid (1) with
hydrogen on the catalyzer 10% Pd/C, with following condition: using flow reactor H-Cube,
0
atmospheric pressure, flow – 1 ml/min, 50 C, solution of 100 mg (+)-usnic acid 1 in 20 ml THF
(Scheme 1). Yield 90%.
Compounds 3-9 were obtained by the procedure40, 41 in the reactions of usnic acid 1 with
amino acids (Scheme 2). Yields 88%, 55%, 47%, 73%, 59%, 86% and 17% respectively.
(2S)-2-({1-[(2R,4E)-10-acetyl-11,13-dihydroxy-2,12-dimethyl-3,5-dioxo-8-
oxatricyclo[7.4.0.02,7]trideca-1(9),6,10,12-tetraen-4-ylidene]ethyl}amino)-3-(1H-indol-3-
yl)propanoic acid (+)-8.
0
20
Yield 86%. Mp: 165 – 167 C. [α]D +270 (c 0.14,
H
N
21
19
18
22
24
1
CHCl3). H NMR 400 MHz (DMSO-d6): 1.57 (s, 3H,
HOOC
23
OH
20
O
16 17
CH3-15), 1.92 (s, 3H, CH3-10), 2.34 (s, 3H, CH3-12),
2.59 (s, 3H, CH3-14), 3.32 (m, 2H, CH-17, CH-17` and
NH
25
(+)-8
HO
O
O
O

1H, NH), 5.02 (m, 1H, CH-16), 5.79 (s, 1H, CH-4), 6.91 (t, 1H, CH-23, J = 7.4 Hz), 7.01 (t, 1H,
CH-24, J = 7.4 Hz), 7.11 (d, 1H, CH-19, J = 2.0 Hz), 7.29(d, 1H, CH-25, J = 8.0 Hz), 7.43 (d,
1H, CH-22, J = 8.0 Hz), 10.96(s, 1H, OH-9), 12.08 (s, 1H, COOH), 13.36 (s, 1H, OH-7), 13.64
(bs, 1H, NH).
13C NMR 100 MHz (DMSO-d6) 7.5 (C-10), 18.7 (C-15), 28.4 (C-17), 31.1 (C-12), 31.7
(C-14), 56.4 (C-16), 57.0 (C-9b), 100.9 (C-2), 101.8 (C-6), 102.4 (C- 4), 105.1 (C-9a), 106.3 (C-
8), 107.6 (C-18), 111.5 (C-22), 118.1 (C-25), , 118.6 (C-23), 121.1 (C-24), 124.6 (C -19), 127.1
(C-20), 136.1 (C-21), 155.7 (C-7), 157.6 (C-5a), 162.5 (C-9), 171.3 (COOH), 172.9 (C-11),
174.4 (C-4a), 188.8 (C-3), 197.6 (C-1), 200.9 (C-13). HRMS, found: m/z 530.1574 [M]+.
C29H26N2O8. Calculated: M = 530.1684.
Compounds 10, 12 were synthesized by the procedure42 in the reaction of usnic acid (1)
with phenylhydrazine hydrochloride (Scheme 3). Yields 96% and 91% respectively.
Compound 11 was synthesized by the procedure43 (Scheme 3). Yield 96%.
Compound 13 was synthesized by the procedure44 in the reaction of 10 with
diazomethane (Scheme 3). Yield (50%).
Compounds 14-23, 25 were synthesized by the procedure45 (Scheme 4). Yields 61%,
68%, 60%, 79%, 70%, 58%, 38%, 55%, 46% for 14-23 respectively and 21% for 25.
(2E)-1-[(1S,16R)-5,16-dihydroxy-3-methoxy-1,4,14-trimethyl-12-phenyl-8-oxa-12,13-
diazatetracyclo[7.7.0.02,7.011,15]hexadeca-2(7),3,5,9,11(15),13-hexaen-6-yl]-3-(2,3-
dimethoxyphenyl)prop-2-en-1-one (+)-18.
20
The yield was 79%, viscous yellow amorphous
O
substance, [α]D20 +458 (c 0.30, CHCl3). 1H NMR: 1.55 (s, 3 H,
OH
HO
H-15); 2.19 (s, 3 H, H-10); 2.50 (s, 3 H, H-12); 3.89, 3.94,
N
N
16
(+)-18
O
O
4.00 (all s, 3 H each, OCH3); 5.44 (s, 1H, OH-1); 5.48 (s, 1H,
H-1); 5.95 (s, 1 H, H-4); 6.92 (d, 1H, H-25, J = 8.0 Hz);
7.15—7.60 (m, 7H, H-arom.); 7.86 (d, 1 H, H-14, J = 15.4
17
18
28
21
22
OMe
23
19
27
26
OMe
24
29
25

Hz); 7.92 (d, 1 H, H-21, J = 15.4 Hz); 14.06 (s, 1 H, OH-7). 13C NMR: 9.1 (C-10); 12.5 (C-12);
17.8 (C-15); 52.0 (C-9b); 55.1 and 55.6 (C-28 and C-29); 61.5 (C-20); 74.2 (C-1); 89.5 (C-4);
104.3 (C-6); 109.7 (C-25); 110.7 (C-27); 112.1 (C-2); 112.4 (C-9a); 114.7 (C-8); 122.5 (C-14);
122.7 (C-17, C-17´); 123.2 (C-26); 126.6 (C-19); 127.5 (C-22); 128.8 (C-18, C-18´); 135.9 (C-
16); 138.9 (C-3); 144.8 (C-21), 147.6 (C-23); 148.7 (C-11); 151.3 (C-24); 156.8 (C-5a); 159.2
(C-9); 164.2 (C-7); 166.7 (C-4a); 190.6 (C-13). HRMS, found: m/z 580.2209 [M]+. C34H32N2O7.
Calculated: M = 580.2204.
(2E)-1-[(1S,16R)-5,16-dihydroxy-3-methoxy-1,4,14-trimethyl-12-phenyl-8-oxa-12,13-
diazatetracyclo[7.7.0.02,7.011,15]hexadeca-2(7),3,5,9,11(15),13-hexaen-6-yl]-3-(2,3,4-
trimethoxyphenyl)prop-2-en-1-one (+)-19.
20
OMe
The yield was 70%, viscous yellow amorphous substance, [α]D
OH
+523 (c 0.14, CHCl3). 1H NMR: 1.51 (s, 3 H, H-15); 2.16 (s, 3 H, H-10);
HO
N
O
O
N
2.47 (s, 3 H, H-12); 3.85, 3.89, 3.94, 3.97 (all s, 3 H each, OCH3); 5.40
(s, 1H, OH-1); 5.43 (s, 1H, H-1); 5.98 (s, 1 H, H-4); 6.71 (d, 1H, H-25, J
= 8.7 Hz); 7.26—7.57 (m, 6H, H-arom.); 7.93 (d, 1 H, H-14, J = 15.6
Hz); 8.14 (d, 1 H, H-21, J = 15.6 Hz); 14.08 (s, 1 H, OH-7). 13C NMR:
Ph
OMe
(+)-19
OMe
OMe
9.3 (C-10); 12.8 (C-12); 18.1 (C-15); 52.3 (C-9b); 56.0 (C-20); 60.8, 61.4 and 61.8 (C-28, C-29
and C-30); 74.5 (C-1); 89.9 (C-4); 104.8 (C-6); 107.6 (C-26); 112.4 (C-2); 112.6 (C-9a); 115.0
(C-8); 121.8 (C-22); 123.0 (C-14); 123.1 (C-17, C-17´); 123.7 (C-19); 126.8 (C-27); 128.8 (C-
18, C-18´); 136.4 (C-16); 139.3 (C-3); 140.6 (C-21), 142.4 (C-24); 147.9 (C-23); 154.0 (C-11);
156.1 (C-25); 157.2 (C-5a); 159.4 (C-9); 164.5 (C-7); 167.2 (C-4a); 191.4 (C-13). HRMS, found:
m/z 610.2294 [M]+. C35H34N2O8. Calculated: M = 610.2310.
(2E)-1-[(1S,16R)-5,16-dihydroxy-3-methoxy-1,4,14-trimethyl-12-phenyl-8-oxa-12,13-
diazatetracyclo[7.7.0.02,7.011,15]hexadeca-2(7),3,5,9,11(15),13-hexaen-6-yl]-3-(2,5-
dimethoxyphenyl)prop-2-en-1-one (+)-21.
The yield was 38%, viscous yellow amorphous
OMe
OH
substance, [α]D20 +337 (c 0.08, CHCl3). 1H NMR: 1.51 (s, 3
HO
N
H, H-15); 2.16 (s, 3 H, H-10); 2.46 (s, 3 H, H-12); 3.75,
O
(+)-21
O
N
3.84, 3.96 (all s, 3 H each, OCH3); 4.05 (s, 1 H, OH-1);
5.44 (s, 1 H, H-1); 5.95 (s, 1 H, H-4); 6.84 (d, 1H, H-24, J
= 8.97 Hz); 6.92 (dd, 2 H, H-25, J = 8.97 Hz and 3.00 Hz);
OMe
MeO
7.15 (d, 1H, H-27, J = 3.00 Hz); 7.29—7.58 (m, 5H, H-arom.); 8.01 (d, 1 H, H-14, J = 15.6 Hz);
8.20 (d, 1 H, H-21, J = 15.6 Hz); 14.00 (s, 1 H, OH-7). 13C NMR: 9.4 (C-10); 12.8 (C-12); 18.0
(C-15); 52.2 (C-9b); 55.4 and 55.8 (C-28 and C-29); 61.8 (C-20); 74.4 (C-1); 89.9 (C-4); 104.6
(C-6); 112.2 (C-2); 112.6 (C-9a); 112.3 and 113.3 (C-24, C-25); 115.0 (C-8); 117.7 (C-27),
122.8 (C-17, C-17´); 124.2 (C-22); 125.5 (C-14); 126.8 (C-19); 129.0 (C-18, C-18´); 136.2 (C-

16); 139.1 (C-3); 140.2 (C-21); 147.8 (C-23), 153.1 (C-26); 153.3 (C-11); 157.2 (C-5a); 159.5
(C-9); 164.4 (C-7); 167.0 (C-4a); 191.5 (C-13). HRMS, found: m/z 580.2206 [M]+ C34H32N2O7.
Calculated: M = 580.2204.
3.1.5 (2E)-1-[(1S,16R)-5,16-dihydroxy-3-methoxy-1,4,14-trimethyl-12-phenyl-8-oxa-
12,13-diazatetracyclo[7.7.0.02,7.011,15]hexadeca-2(7),3,5,9,11(15),13-hexaen-6-yl]-3-(2,4-
dimethoxyphenyl)prop-2-en-1-one (+)-22.
20
The yield was 55%, viscous yellow amorphous substance, [α]D
OMe
OH
1
+209 (c 0.19, CHCl3). H NMR: 1.51 (s, 3 H, H-15); 2.16 (s, 3 H, H-10);
HO
N
O
2.46 (s, 3 H, H-12); 3.84, 3.87, 3.96 (all s, 3 H each, OCH3); 4.09 (s, 1 H,
O
N
Ph
OH-1); 5.44 (s, 1 H, H-1); 5.99 (s, 1 H, H-4); 6.44 (d, 1H, H-24, J = 2.3
Hz); 6.53 (dd, 2 H, H-26, J = 2.3 Hz and 8.5 Hz); 7.29—7.53 (m, 5H, H-
arom.); 7.57 (d, 1H, H-27, J = 8.5 Hz); 7.94 (d, 1 H, H-14, J = 15.6 Hz);
OMe
(+)-22
OMe
8.24 (d, 1 H, H-21, J = 15.6 Hz); 14.24 (s, 1 H, OH-7). 13C NMR: 9.4 (C-10); 12.7 (C-12); 18.1
(C-15); 52.2 (C-9b); 55.3 and 55.4 (C-28 and C-29); 61.7 (C-20); 74.4 (C-1); 89.8 (C-4); 98.1
(C-24), 104.7 (C-6); 105.5 (C-26), 112.3 (C-2); 112.5 (C-9a); 114.8 (C-8); 116.9 (C-22), 122.4
(C-14); 123.0 (C-17, C-17´); 126.8 (C-19); 129.1 (C-18, C-18´); 131.0 (C-27); 136.4 (C-16);
139.2 (C-3); 141.0 (C-21); 147.8 (C-23), 157.1 (C-5a); 159.2 (C-11); 160.5 (C-9); 163.3 (C-25),
164.4 (C-7); 167.3 (C-4a); 191.5 (C-13). HRMS, found: m/z 580.2210 [M]+ C34H32N2O7.
Calculated: M = 580.2204.
3.1.6 (2E)-1-[(1S,16R)-5,16-dihydroxy-3-methoxy-1,4,14-trimethyl-12-phenyl-8-oxa-
12,13-diazatetracyclo[7.7.0.02,7.011,15]hexadeca-2(7),3,5,9,11(15),13-hexaen-6-yl]-3-[4-
(methylsulfanyl)phenyl]prop-2-en-1-one (+)-23.
20
The yield was 46%, viscous yellow amorphous substance, [α]D
OMe
OH
1
+417 (c 0.14, CHCl3). H NMR: 1.52 (s, 3 H, H-15); 2.17 (s, 3 H, H-10);
HO
N
O
2.46 (s, 3 H, H-12); 2.49 (s, 3H, SCH3), 3.96 (s, 3 H, OCH3); 4.07 (s, 1 H,
OH-1); 5.44 (s, 1 H, H-1); 6.00 (s, 1 H, H-4); 7.22—7.55 (m, 9H, H-
arom.); 7.88 (bs, 2 H, H-14, H-21); 14.01 (s, 1 H, OH-7). 13C NMR: 9.4
(C-10); 12.8 (C-12); 14.8 (C-28), 18.1 (C-15); 52.2 (C-9b); 61.8 (C-20);
O
N
Ph
(+)-23
SMe
74.4 (C-1); 90.1 (C-4); 104.6 (C-6); 112.3 (C-2); 112.6 (C-9a); 115.0 (C-8); 122.8 (C-17, C-17´);
123.5 (C-14); 125.6 (C-24, C-24´); 126.8 (C-19); 129.0 (C-23, C-23´); 129.2 (C-18, C-18´);
131.0 (C-22); 136.3 (C-16); 139.1 (C-3); 142.8 (C-25); 144.8 (C-21), 147.8 (C-5a), 157.2 (C-
11); 159.6 (C-9); 164.4 (C-7); 167.0 (C-4a); 191.0 (C-13). HRMS, found: m/z 566.1865 [M]+
C33H30N2O5S. Calculated: M = 566.1870.
Compound 24 was synthesized by the following procedure. A 5.5 M solution of t-
BuOOH (0.3 mL) in hexane and seven crystalline grains of VO(acac)2 were added to a solution

of compound 18 (163 mg, 0.3 mmol) in dry toluene (10 mL). The reaction mixture was stirred at
55 °C for 2 h, cooled to 20 °C, and diluted with water. The product was extracted with
chloroform and the organic extract was concentrated. The residue was purified by column
chromatography (Scheme 4). Yield 73%.
(1R)-6-[(2E)-3-(2,3-dimethoxyphenyl)prop-2-enoyl]-5-hydroxy-3-methoxy-1,4,14-
trimethyl-12-phenyl-8-oxa-12,13-diazatetracyclo[7.7.0.02,7.011,15]hexadeca-
2(7),3,5,9,11(15),13-hexaen-16-one (+)-24.
The yield was 73%. Viscous yellow amorphous substance,
OMe
O
20
1
[α]D +631 (c 0.14, CHCl3). H NMR: 1.89 (s, 3 H, H-15); 2.20 (s,
3 H, H-10); 2.55 (s, 3 H, H-12); 3.87, 3.87, 4.10 (all s, 3 H each,
OCH3); 6.16 (s, 1 H, H-4); 6.95 (d, 1H, H-25, J = 8.0 Hz); 7.06 (t, 2
H, H-26, J = 8.0 Hz and 3.00 Hz); 7.24 (d, 1H, H-27, J = 8.0 Hz);
7.29—7.58 (m, 5H, H-arom.); 7.92 (d, 1 H, H-14, J = 15.7 Hz); 8.22
HO
O
N
O
N
Ph
OMe
(+)-24
OMe
(d, 1 H, H-21, J = 15.7 Hz); 13.85 (s, 1 H, OH-7). 13C NMR: 10.3 (C-10); 13.8 (C-12); 30.6 (C-
15); 56.6 (C-20); 61.9 (C-9b); 62.1 and 63.2 (C-28 and C-29); 89.6 (C-4); 105.1 (C-6); 111.8 (C-
2); 112.1 (C-9a); 115.0 (C-25); 116.0 (C-8); 120.5 (C-27), 124.4 (C-17, C-17´); 124.9 (C-14);
127.1 (C-26); 128.9 (C-19); 129.8 (C-22); 130.3 (C-18, C-18´); 139.0 (C-16); 140.7 (C-21);
146.3 (C-3), 149.8 (C-11); 151.5 (C-23); 153.9 (C-24); 156.9 (C-5a); 163.1 (C-9); 165.5 (C-7);
172.9 (C-4a); 192.1 (C-13); 193.6 (C-1). HRMS, found: m/z 578.2040 [M]+ C34H30N2O7.
Calculated: M = 578.2048.
Compound (+)-26 was synthesized by the procedure46 (Scheme 4). Yield 82%.

O
O
HO
OH HO
O
OH
O
N
N
N
Ph
a, b
c
N
HO
HO
OH
HO
Ph
O
O
O
O
O
(+)-1
(+)-11
(+)-11a
a, c, d
d
O
HO
O
HO
O
O
N
N
Ph
N
N
Ph
f
HO
HO
HO
N
N
O
O
O
O
O
N
Ph
(+)-14 - (+)-23
R1
HN
R2
(+)-26
(+)-25
e
R3
O
O
R4
(+)-14
R1 =R2 =R3 = R4 = H
O
(+)-15 R1 =R2 =R4= H, R3 = Cl
(+)-16 R1 =R3 =R4= H, R2 = OMe
(+)-17 R1 =R4 = H, R2 = R3 = OMe
(+)-18 R3= R4 = H, R1 = R2 = OMe
(+)-19 R1 =R2 =R3= OMe, R4 = H
(+)-20 R2 =R3 =R4= OMe, R1 = H
N
N
Ph
HO
O
O
O
(+)-21
R2 =R3= H, R1 = R4 = OMe
(+)-24
(+)-22 R1 =R3 = H, R2 = R4 = OMe
(+)-23 R1 =R2 =R4= H, R3 = SMel
O
Scheme 4
a: PhNHNH2, EtOH, reflux; b: NaBH4, THF, -20 oC; c: CH2N2, CHCl3, r.t.; d: ArCHO, MeOH/H2O, KOH,
70oC; e: t-BuOOH, VO(acac)2, PhMe, r.t.; f: NH2NH2*H2O, AcOH, EtOH, reflux.
Compounds 28-31 were synthesized by the procedure47 (Scheme 5). Yields 65%, 53%,
60% and 66% respectively.
O
O
O
OH
O
OH
O
OH
O
a
b
OH
HO
OH
HO
OH
O
O
O
O
O
O
(+)-1
(+)-27
Br (+)-1a
O
c
O
O
OH
(+)-28 R = 2-MeOPh-
(+)-29 R = 4-BrPh-
R = 4-Py-
(+)-31 R = 3-Py-
OH
O
(+)-30
O
R
O
Scheme 5
a: 2Br2, HBr, dioxane, r.t.; b: AcOK, acetone, reflux; c: aldehyde, MeOH/H2O, 70oC.

(4Z,10R)-12-acetyl-8,13-dihydroxy-7,10-dimethyl-4-(pyridin-4-ylmethylidene)-5,16-
dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione (+)-30.
20
The yield was 60%, mp decomposition >240°C, [α]D
O
O
1
OH
+1107 (c 0.08, CHCl3). H NMR (CDCl3, δ, ppm): 1.76 (3H, s,
H-15), 2.29 (3H, s, H-10), 2.65 (3H, s, H-12), 6.06 (1H, s, H-4),
6.65 (1H, s, H-16), 7.67 (2H, d, H-18,21, J = 5.0 Hz), 8.68 (2H,
d, H-19,20, J = 5.0 Hz), 11.55 (1H, s, 9-OH), 18.88 (1H, s, 3-
O
OH
O
O
N
(+)-30
OH). 13C NMR (CDCl3, δ, ppm): 7.52 (C-10), 27.84 (C-12), 31.86 (C-15), 58.49 (C-9b), 99.39
(C-4), 100.21 (C-6), 105.07 (C-2), 106.00 (C-8), 108.10 (C-9a), 108.64 (C-16), 124.32 (C-
18,21), 139.35 (C-17), 150.18 (C-19,20), 149.75 (C-14), 150.03 (C-5a), 160.06 (C-9), 165.63 (C-
7), 179.07 and 179.11 (C-13, C-4a), 191.53 (C-3), 197.69 (C-1), 201.82 (C-11). HRMS, found:
m/z 431.1009 [M]+ C24H17NO7. Calculated: M = 431.1000.
(4Z,10R)-12-acetyl-8,13-dihydroxy-7,10-dimethyl-4-(pyridin-3-ylmethylidene)-5,16-
dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione (+)-31.
20
Yield 66%, mp decomposition >200°C, [α]D +1084 (c
O
O
OH
1
0.18, CHCl3). H NMR (CDCl3, δ, ppm): 1.75 (3H, s, H-15), 2.26
O
OH
(3H, s, H-10), 2.64 (3H, s, H-12), 6.04 (1H, s, H-4), 6.72 (1H, s,
H-16), 7.38 (1H, dd, H-19, J = 3.4 and 7.8 Hz), 8.18 (1H, d, H-
18, J = 7.8 Hz), 8.56 (1H, d, H-20, J = 3.4 Hz), 8.99 (1H, s, H-21),
O
N
O
(+)-31
11.47 (1H, s, 9-OH), 18.85 (1H, s, 3-OH). 13C NMR (CDCl3, δ, ppm): 7.47 (C-10), 27.84 (C-12),
31.88 (C-15), 58.54 (C-9b), 99.24 (C-4), 100.46 (C-6), 105.05 (C-2), 105.87 (C-8), 107.80 (C-
16), 108.45 (C-9a), 123.67 (C-18), 128.41 (C-17), 137.38 (C-19), 148.57 (C-14), 149.70 (C-20),
149.88 (C-5a), 151.80 (C-21), 159.69 (C-9), 165.52 (C-7), 178.97 and 179.25 (C-13, C-4a),
191.54 (C-3), 197.71 (C-1), 201.78 (C-11). HRMS, found: m/z 431.1006 [M]+ C24H17NO7.
Calculated: M = 431.1000
Compounds 32, 33, 36, 37 were synthesized by the following procedure. 1 mmol (344
mg) of compound (+)-1 in 20 ml of DMF (previously distilled over BaO and passed through a
column of Al2O3 before the reaction). The solution was mixed with 500 mg of K2CO3 for two
hours at a room temperature before appearance of the dark green color of solution. 0.5 ml of
methyl iodide were added and stirred for two days at room temperature. The reaction mixture
was diluted with water; the precipitate was filtered through paper filter, washed with water,
dried, washed with chloroform and concentrated on a rotary evaporator. The resulting residue
was separated on silica gel and eluted with methylene chloride to obtain compound (+)-1a. 1
mmol of compound (+)-1a (358 mg) in 20 mL of dioxane was added to the previously prepared
bromine-dioxane complex (1 mmol of bromine (0.05 ml) dissolved in 10 mL dioxane) with few
drops of HBr, and stirred for 20 min at room temperature. After concentrating of the reaction

mixture on a rotary evaporator, the resulting residue was separated on silica gel (60 -200µ) and
eluted with methylene chloride to obtain compound (+)-1b. 1.1 mmol of potassium hydroxide, 6
ml of methanol and 1.1 mmol of the corresponding thiol were mixed and stirred at room
temperature for 10-15 minutes. Then the resulting solution was added to a solution of 1 mmol of
compound (+)-1b in 2 ml of methylene chloride and stirred at room temperatutre for 2-3 hours.
The course of reaction was controlled by TLC. The reaction mixture was washed 2 times with
distilled water, dried over MgSO4 and concentrated. The resulting residue was separated on silica
gel (60 -200µ) and eluted with methylene chloride to obtain compounds 32, 33, 36, 37 (Yield
53%, 56%, 63% and 58% respectively).
(2R)-4-acetyl-10-[2-(4,5-dihydro-1,3-thiazol-2-ylsulfanyl)acetyl]-5,13-dihydroxy-11-
methoxy-2,12-dimethyl-8-oxatricyclo[7.4.0.02,7]trideca-1(9),4,6,10,12-pentaen-3-one (+)-32.
Viscous yellow amorphous substance. Yield 53%. [α]29
O
D
O
OH
+129 (c 0.21; CHCl3). 1Н NMR (CDCl3, δ, ppm, J Hz): 1.77 (3Н, s,
O
OH
Н-15), 2.18 (3Н, s, Н-10), 2.65 (3Н, s, Н-12), 3.39 (2H, t, J = 8, H-
19) 3.77 (3H, s, OMe), 4.12 (2H, t, J = 8, H-18), 4.52 (2Н, dd, J =
16.7, J = 18.0, Н-14), 5.95 (1H, s, H-4), 10.90 (1H, s, ОН-9), 18.83
(1H, s, ОН-3). 13С NMR (CDCl3, δ, ppm): 8.36 (С-10), 27.52 (С-
O
O
S
(+)-32
S
N
12), 31.48 (С-15), 35.62 (C-19), 43.66 (С-14), 58.71 (С-9b), 61.99 (C-16), 63.45 (C-18), 97.70
(С-4), 104.72 (С-9a), 108.12 (С-6), 109.83 (С-2), 116.07 (С-8), 152.72 (С-5a), 155.10 (С-9),
159.26 (С-7), 179.42 (С-4a), 191.16 (С-13), 192.26 (С-3), 197.38 (С-1), 201.29 (С-11). HRMS:
Obtained: m/z 475.0758 [M]+ C22H21NO7S2. Calculated: М = 475.0754.
(2R)-4-acetyl-5,13-dihydroxy-11-methoxy-10-{2-[(5-methoxy-1H-1,3-benzodiazol-2-
yl)sulfanyl]acetyl}-2,12-dimethyl-8-oxatricyclo[7.4.0.02,7]trideca-1(9),4,6,10,12-pentaen-3-
one (+)-33.
Viscous yellow amorphous substance. Yield 56%.
O
O
OH
[α]29D +229 (c 0.27; CHCl3). 1Н NMR (CDCl3, δ, ppm, J Hz):
1.64 (3Н, s, Н-15), 2.11 (3Н, s, Н-10), 2.59 (3Н, s, Н-12),
3.70 (3H, s, OMe), 3.73 (3H, s, OMe), 4.55 (2Н, dd, J = 16.0,
J = 18.0, Н-14), 5.77 (1H, s, H-4), 6.70 (1H, m, H-arom.),
6.90 (1Н, m, H-arom.), 7.29 (1Н, m, H-arom.), 10.92 (1H, s,
ОН-9), 18.67 (1H, s, ОН-3). 13С NMR (CDCl3, δ, ppm): 8.63
O
OH
O
O
S
(+)-33
N
HN
O
(С-10), 27.74 (С-12), 31.67 (С-15), 43.57 (С-14), 55.50 (C-24) 58.66 (С-9b), 62.29 (C-16),
95.80 (C-19), 98.11 (С-4), 104.90 (С-9a), 108.48 (С-6), 109.66 (С-2), 111.48 (C-21), 115.50 (C-
22), 116.51 (С-8), 127.10 (C-23), 133.10 (C-18), 147.46 (C-17), 153.24 (С-5a), 155.89 (С-9),

156.04 (C-20), 159.73 (С-7), 179.07 (С-4a), 191.34 (С-13), 193.10 (С-3), 197.45 (С-1), 201.53
(С-11). HRMS: Obtained: m/z 536.1245 [M]+ C27H24N2O8S. Calculated: М = 536.1248.
Compounds 34, 35, 38-40 were synthesized by the following procedure (Scheme 6). 1.1
mmol of potassium hydroxide, 6 ml of methanol, 1.1 mmol of the corresponding thiol, were
placed into a flask which was stirred at room temperature for 10-15 minutes. Then, the resulting
solution was added a solution of 1 mmol 1d (obtained from 1 in procedure of47) in 2 ml of
methylene chloride and stirred at room temperature for 2-3 hours to complete the reaction
(control by TLC). The reaction mixture was washed 2 times with distilled water, dried over
MgSO4, concentrated and the resulting residue was chromatographed on silica gel – methylene
chloride to obtain compounds 34, 35, 38-40 (Yields 16%, 16%, 51%, 82 and 61% respectively).
Compound (+)-41 was synthesized by the procedure48 (Scheme 7). Yield 87%.

Compound (+)-42 was synthesized by the following procedure (Scheme 7). The 2-
bromobenzoyl chloride (3 mmol) were added to usnic acid (+)-1(1 mmol). The mixture was
dissolved in sulfuric ether (15 ml). Triethylamine (1.95 ml) and five crystals of
dimethylaminopyridine (DMAP) were added. The reaction mixture was stirred at room
temperature for 5 h. The solvent was distilled off on a rotary evaporator. The resulting mixture
was separated by silica gel column chromatography. Yield 69%.
(1R)-6,12-diacetyl-3-(2-bromobenzoyloxy)-11-hydroxy-1,4-dimethyl-13-oxo-8-
oxatricyclo[7.4.0.02,7]trideca-2(7),3,5,9,11-pentaen-5-yl 2-bromobenzoate (+)-42.
1
Yield 69 %, [α]D +446 (c 0.14; CHCl3). H
Br
NMR spectrum (CDCl3, δ, ppm, J Hz): 1.86 s (3Н, H-
15), 2.19 s (3Н, H-10), 2.39 s (3Н, H-12), 2.64 s (3Н,
O
O
O
O
Br
O
(+)-42
H-14), 5.90 s (1Н, H-4), 7.44 m (4H -Ar), 7.76 m
(2H-Ar), 8.12 m (2H-Ar), 18.19 s (1Н, ОН-3). 13C
NMR (CDCl3, δ, ppm): 10.6 (С-10), 25.3 (С-12),
O
OH
O
O
30.5 (С-14), 31.6 (С-15), 59.1 (С-9b), 98.4 (С-4), 105.5 (С-2), 115.9 (С-9а), 122.1 (С-6), 122.1
(С-8), 122.5 and 123.5 (2C-2`), 127.1 (2C-5`), 129.9 (2C-1`), 131.8 (2C-3`), 133.1 (2C-6`),
134.4 (2C-4`), 147.8 (C-5а), 148.4 (С-9), 153.2 (С-7), 162.8 and 163.1 (2С-16), 177.1 (С-4а),
190.4 (С-3), 192.1 (С-1), 194.5 (С-13), 197.7 (С-11). IR spectrum (ν, cm–1): 843, 1091, 1218,
1690, 2929, 3017. Found: m/z 707.9628 [M]+ C32H22O9Br2. Calcd: М = 707.9625.
Compounds 43, 44 were synthesized by the procedure49 (Scheme 8). Yields 41% and
43% respectively.

2.4. Inhibitory effect of usnic acid derivatives against influenza virus
The evaluation of anti-viral activity of usnic acid derivatives was performed by a
quantifying virus yield using the end-point dilution method. Briefly, serial twofold dilutions of
compounds were prepared in minimal essential medium (MEM) containing 2 mM arginine, 2
mM glutamine and 2 microgram/mL trypsin and applied to MDCK cells. After incubation for 1 h
at 37°C the cells were inoculated with influenza virus in a dose 1–106 of 50% infecting dose
(ID50). Cells that not treated by the compounds were used as control. After cultivation for 48 h at
36°C the hemagglutination assay was carried out as follows. 100 microliters of supernatant was
transferred into round-bottom wells and mixed with 100 microliters of 1% suspension of chicken
erythrocytes followed by 1 h incubation at room temperature. The virus titer was considered as a
reciprocal to the final dilution of the inoculum able to cause positive hemagglutination in 50% of
wells and expressed in 50% infecting doses. Anti-viral activity of the compounds was evaluated
by their ability to decrease the virus titer. Based on the results, 50% of the effective dose
(concentration of compound that decreases the virus production two- fold comparing to control)
was calculated.
2.5. Cell viability assay
For the compound’s cytotoxity evaluation, the compounds were dissolved in DMSO to 5
mg/ml, and serial twofold dilutions (1000–4 microgram/ml) were prepared in MEM. MDCK
cells were seeded in 96-wells plates and cultivated in Eagle’s minimal essential medium (MEM)
with addition of 5% fetal calf serum. After the monolayer formation the cells were washed by
serum-free MEM, the dilutions of compounds were applied to the MDCK cells and incubated for
48 h at 37°C. After incubation, the cells were washed twice with PBS and the number of viable
cells were evaluated by a microtetrazolium test (MTT38). 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 37ºС in 5% CO2 for 60 min. The colored
deposit was dissolved in 100 microliters of DMSO. The plates were swirled gently and left in the
dark at room temperature for 30 min. Optical density was measured by using a
spectrophotometer (Victor 1420, Perkin Elmer, Finland) at wavelength 535 nm. Based on this
data, the CTD50 (compound concentration required to reduce 50% cell viability) value was
estimated for each compound.
2.6. Selectivity
Selectivity index (SI) was calculated as relation of CTD50 to ED50. The compounds
having SI≥10 were considered active.
3. Results
3.1. Virus-inhibiting activity of usnic acid derivatives.
In present study anti-influenza activity of native (-)-usnic acid is four times higher than
the one of its (+)-counterpart. ED50 of (-)-UA was 13.1 µM. These data are in line with our
previous investigations. (-)-UA also had higher antiviral activity against herpes simplex virus
type I and Epstein-Barr virus33. The difference between (+)- and (-)-enantiomers in the last case
was approximately fivefold that is in a good agreement with our previous results35.
However, activity of newly synthesized derivatives of (+)-UA appeared equal
(compounds 3, 6, 7, 10, 16, 27) or higher than their (-)- counterpart (compounds 4, 11, 14, 15,
20, 43).
All the (+)-UA conjugates with amino acids are more active than (+)-UA itself with one
exception – gamma-aminobutyric acid conjugate (equal activity). ED50’s of compounds (+)-3 –
(+)-6, (+)-9 range from 4.2 to 11.2 µM. All the (-)-UA conjugates with amino acids are
comparable in activity with (-)-UA itself (ED50’s of compounds (-) 3- (-)-7 range from 10.7 to
14.5 µM) with the same exception – gamma amino butyric acid conjugate appeared less active.
Pyrazolic derivatives of both UA isomers ((+)-10, (-)-10, (+)-12) are less active than UA,
transformation of carbon group in C-ring being the most important in this phenomenon.
Transformation into the oxyran cycle reduces the activity by an order (ED50 of compound (+)-13
is 262 µM, whereas transformation into OH-group (compounds (+)-11 and (-)-11), in contrast,
increases the activity (ED50 2.5-12 µM).
The follow-up transformation of pyrazols into chalcones (+)-4 – (+)-23, (-)-14 – (-)-17
(chalcone fragment is made inside the ring A of usnic acid) again reduces activity, which doesn`t
depend on the chalcone fragment structure (ED50 25.9-82.7 µM). The only exception is
compound (+)-20 with thrimetoxybenzolic ring (ED50 8.2 µM). Transformation of OH-group in

C ring into the oxyran (compound (+)-25) or carbonyl (+)-24)) group reduces ED50 to 5.5 and 8.7
µM, respectively.
Compounds close to UA - (+)-27 и (-)-27 have close anti-viral activity which is less than
that of native UA. Furanone-based compounds (+)-28 – (+)-31 were much more active (ED50
4.8-7.7 µM).
Among compounds containing different pharmacophors linked to the A-ring of (+)-UA
((+)-34, 35, 38-40), three derivatives can effectively inhibit the virus replication at as low
concentration as 2 µM. These contain benzoxasolic, benzyl and para-chlorphenyl substituting
groups. Structurally close compounds based on UA-methyl ether (32, 33, 36, 37) are
significantly less active (ED50 14.7-160.7 µM).
Mono- and diethers of (+)-UA, compounds 41 and 42, regardless of ether fragment
structure, were more active then (+)-UA itself, ED50 was 7.2 and 7.8 µM, respectively.
Both dextrorotary oxyran derivatives (+)-43 (10.6 µM) and (+)-44 (13.4 µM) were more
active than primary compounds (+)-1 (51.7 µM) и (+)-10 (69.5 µM). The only (-)-oxyran
derivative (-)-43 (ED50 =28.7 µM), conversely, less active then (-)-UA (ED50 = 13.1 µM).
Generally, derivatives of (+) UA tend to have higher antiviral activity than (+)-UA itself,
and levorotating compounds were less active then native (-)-UA. The most active virus-
inhibiting compounds belong to auronic, sulfide and oxyran structure groups. None of the
compounds appeared to be more active than the reference compound ribavirin.
3.2. Cell toxicity
Toxicity of (+)-UA (CTD50 = 424 µM) was three times lower than that of (-)-UA (133.7
µM). After hydrogenation of double link in C-ring (compound 2) toxicity increases
approximately 30-fold.
All the amino acid derivatives of (+)-UA (compounds 3 – 9) were more toxic
(CTD50=13.1 -149.6 µM) then initial substance, among them substances with bulky substituents
such as L-leucine (+)-6, L-methionine (+)-9, L-tryptophan (+)-8 (CTD50=13.1-21.1 µM) possess
the highest toxicity.
Diastereomeric compounds based on (-)-UA had comparable toxicity, although being
compared with the (-)-UA, they are either less or equally toxic.
The addition of the pyrazole ring annelated to the ring C of UA markedly reduces the
toxicity (CTD50’s of compounds 10, 13 are 564-831.7 µM), which is in agreement with
published data20 about the role of ring C`s triketone system in the cytotoxicity of UA. The
presence of the trifluoromethyl group in an aromatic substituent of the pyrazole ring moiety
reduces the toxicity, (compound 12, CTD50=1095 µM). Recovery of endocyclic keto ring C

above pyrazoles (+)-3 and (-)-3 leads to increase of toxicity ten-fold and more, CTD50’s for
compounds (+)-11 and (-)-11 are 26.3 and 47.8 µM, respectively.
Chalcones (+)-14 and (+)-25 synthesized from compounds (+)-10 and (+)-11 by aldol-
crotonic condensation with unsubstituted benzaldehyde were quite toxic (CTD50 36.5-105.8 µM).
Introduction of substituent into the aromatic ring of chalcone fragment reduces toxicity
approximately ten times (CTD50`s of compounds (+)-16, (+)-17, (+)-18 were 431 - 909 µM),
among them methoxy-substituted compounds possessed the lowest toxicity (compound (+)-16,
CTD50=909 µM). Among mono-, di-and tri-metoxy substituted derivatives the lowest toxicity
was observed for compounds with metoxy-group in ortho-position. Oxidation of hydroxyl group
in the C ring to the carbonyl group (Compound (+)-24) again increases the toxicity. Interestingly,
in pairs of stereoisomers of compounds 3 and 11, the opposite effect was observed.
Aurone-like compounds (+)-28 - (+)-31 were quite toxic (CTD50’s 32.3-53.4 µM) and in
this case the presence of the metoxygroup in the aromatic ring did not affect the level of toxicity.
All compounds with various substituents linked to the acetyl fragment of ring A by the
sulfide bridge ((+)-34 – (+)-40) were more toxic than (+)-UA (CTD50’s 2-177 µM). Sulfide
derivatives synthesized on the base of the methyl ester of UA were considerably less toxic
(CTD50’s 105-937 µM).
Both (+)-UA ethers were more toxic than initial UA. Indeed, CTD50’s of compounds (+)-
41 and (+)-42 were 100 and 31.1 µM respectively.
Oxirane compounds 43 and 44 were more toxic than UA. At the same time the
introduction of the pyrazole fragment did not reduce the toxicity of the compound, although this
effect was observed for compounds 10 and 12.
3.3. Selectivity
For evaluation of therapeutic potential of novel compounds, the relation between activity
and toxicity is important issue in addition to their absolute values. Based on the values of
CTD50’s and ED50’s, selectivity index (SI) was calculated for each compound. In the present
study 56 compounds were tested, among them 21 (37.5%) were considered prospective (SI≥10).
In spite of broad range of inhibiting activity (2-262 µM) and toxicity (2-1016 µM) of the studied
compounds, their modifications resulting in increased inhibitory activity leaded also to increased
toxicity, so the variability among the values of selective index was not very high.
The highest rate of active compounds was found in the group of chalcones, this group
being maximally far from native usnic acid structure. Among them it was possible to indicate the
dependence of the activity on the methoxy groups in the aromatic ring of chalcone fragment.

Compounds with a methoxy substituent at the ortho-position of the aromatic ring appeared more
prospective (SI = 14-20).
Several members of other structural groups were also active: compound (+)-4 - derivative
(+)-UA with an amino acid β-alanine (SI = 20), compound (+)-12 - pyrazole derivative (+)-UA
with a trifluoromethyl substituent (SI = 16) and compound (+)-33 – 5-metoxybenzimidaziole
linked with the ring A of (+) UA and an oxyrane compound (+)-43, SI=22.
4. Discussion
In this study, we have demonstrated that antiviral activity of UA can be improved by its
conjugation with side chemical groups. Similar to previous results, the activity depended on the
absolute configuration of the resulting compounds. (-)-UA exhibited higher antiviral activity
than its (+)-enantiomer, but in the pairs of enantiomer derivatives such as enamines, pyrazoles
and chalcones the (+)-enantiomers were more potent inhibitors of the virus (according to ED50).
For other groups of compounds the inhibiting activities of the enantiomers were comparable.
Amino acid derivatives of usnic acid possessed the increased antiviral activity
(compounds 3, 4), if they did not contain large substituents (compound (+)-5).
Conjugation with pyrazole increased antiviral activity of (+)-UA (compounds (+)-10, (+)-
11, (+)-12) and reduced their cytotoxicity comparatively to initial UA. However, introduction of
the oxyran-group dramatically decreased its inhibiting activity (compound (+)-3).
Presence of methoxy-group in para-, ortho- position or both, in chalcone derivatives of
usnic acid extremely reduced the cytotoxicity of compounds without effect on anti-viral activity,
thus making selective index to increase (compounds (16, 17, 26). The same effect of methoxy-
group can be seen in sulphids - (+)-33.
Promising results were obtained for compound (+)-43, the oxidized derivative of (+)-UA.
It exhibited high inhibiting activity and low cytotoxicity, and its selectivity index appeared to be
high. Nevertheless the activity of its counterpart, derivative of (-)-UA (compound (-)-43) was
four-fold lower.
Despite various biological activities of UA have been studied in details, data about its
derivatives are limited. Most of the UA derivatives described in the literature belong to the two
group - enamines and imines. Imino- derivatives were not used in our study. Bruno et al.41
investigated the cicatrizing properties of enamine derivatives of UA. Cytotoxic effect of usnic
acid against tumor cell lines has been shown to enhance after the addition of polyamine chain18.

In fact, such compounds are also enamines, but no compound described in the article did
coincide with those studied in our study.
As far as we know, no antiviral properties of any derivatives of usnic acid had been
studied for today. At the same time the usnic acid itself showed activity against Epstein-Barr
virus33 and polio virus31,34, human papillomavirus32 and arenavirus Junin50. The presented
results, therefore, suggest that usnic acid can be chemically modified to achieve high values of
virus-inhibiting activity, at least against influenza virus. Noteworthy, the virus strain we used in
the study is resistant to currently available antivirals – adamantane derivatives, amantadine and
rimantadine. As several UA derivatives have demonstrated high SI’s, one can conclude that they
have alternative target(s) in viral life cycle and might be therefore considered as starting point for
further optimization and development of new class of anti-influenza drugs.
5. Conclusion
In general we demonstrated that the previously shown anti-viral activity of usnic acid
derivatives can be increased by side moieties introduction. The modification with chalcones
appeared to be the most effective.
As a result, these studies revealed that in the pair of enantiomers, in accordance with our
previous results35, (-)-usnic acid was more active as an inhibitor of virus reproduction. At the
same time, in the pairs of enantiomer derivatives such as enamines, pyrazoles and chalcones, the
(+)-enantiomers were more potent inhibitors of the virus (according to the values of ED50). For
other groups of compounds the inhibiting activities of the enantiomers were comparable.
Further optimization of the structure could therefore result in development of novel anti-
influenza compound with alternative target and mechanism of virus-inhibiting action.
Acknowledgment
The authors thank the Russian Foundation for Basic Research for the financial support
(grant # 13-03-00810).
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Table 1. Activity of usnic acid derivatives against influenza virus A/California/7/09
(H1N1)pdm09.
Compound CTD50,
µM
424,4
ED50,
µM
51,7
SI Compound CTD50,
µM
ED50,
µM
25,9
SI
8,2
10,2
1,8
517,2
530,0
34,6
20,0
10,0
4,0
(+)-1
(+)-22
(+)-23
(+)-24
(+)-25
(+)-26
(-)-14
(-)-15
(-)-16
(-)-17
(-)-27
(+)-27
(+)-28
(+)-29
133,7
13,1
8,1
53,0
8,7
(-)-1
14,5
(+)-2
67,3
11,2
10,7
7,2
6,0
18,3
5,5
3,3
(+)-3
149,6
14,0
20,3
10,0
3,4
1016,3
105,8
115,5
909,1
862,1
131,6
117,0
32,6
101,6 10,0
(-)-3
147,0
60,6
79,4
64,9
76,4
64,3
64,3
4,8
1,7
1,5
(+)-4
144,6
14,5
5,9
(-)-4
20,4
14,0
11,3
2,0
(+)-5
13,1
6,6
2,0
(+)-6
32,8
6,6
5,0
(-)-6
130,5
35,0
28,0
18,9
3,7
1,8
(+)-7
83,9
3,0
6,8
(-)-7
20,8
1,1
39,4
4,7
8,3
(+)-8

21,1
831,7
26,3
4,2
69,5
2,4
5,0
12,0
11,0
16,0
2,6
46,4
53,4
58,9
2,0
7,7
7,0
6,1
7,7
2,0
1,0
3,0
1,0
3,1
7,1
23,1
7,5
5,8
14,0
4,0
22,0
5,3
5,0
(+)-9
(+)-10
(+)-11
(+)-12
(+)-13
(-)-10
(-)-11
(+)-14
(+)-15
(+)-16
(+)-17
(+)-18
(+)-19
(+)-20
(+)-21
(+)-30
(+)-31
(+)-34
(+)-35
(+)-38
(+)-39
(+)-40
(+)-32
(+)-33
(-)-36
(+)-37
(+)-41
(+)-42
(+)-43
(-)-43
(+)-44
29,5
2,0
1095,0
681,2
564,9
47,8
68,4
262,0
56,3
12,0
32,7
55,1
82,7
34,5
50,7
32,8
8,2
6,4
2,1
10,0
4,0
2,1
2,1
177,1
105,3
559,7
177,5
937,5
100,0
31,1
234,0
151,6
67,0
56,3
14,7
24,3
23,7
160,7
7,2
36,5
1,1
252,7
909,1
431,0
862,1
278,7
98,4
4,6
11,0
12,5
17,0
8,5
7,8
12,0
20,0
10,6
28,7
13,4
517,2
25,9