Synthesis of Usnic Acid Derivatives and Evaluation of Their Antiproliferative Activity against Cancer Cells

Article abstract of DOI:10.1021/acs.jnatprod.8b00980

Usnic acid is a secondary metabolite abundantly found in lichens, for which promising cytotoxic and antitumor potential has been shown. However, knowledge concerning activities of its derivatives is limited. Herein, a series of usnic acid derivatives were synthesized and their antiproliferative potency against cancer cells of different origin was assessed. Some of the synthesized compounds were more active than usnic acid. Compounds 2a and 2b inhibited survival of all tested cancer cell lines in a dose- and time-dependent manner. Their IC₅₀ values after 48 h of treatment were ca. 3 μM for MCF-7 and PC-3 cells and 1 μM for HeLa cells, while 3a and 3b revealed antiproliferative activity only against HeLa cells. All active usnic acid derivatives induced G0/G1 arrest and a drop in the fraction of HeLa cells in the S and G2/M phases. Compounds 2a and 2b decreased the clonogenic potential of the cancer cells evaluated and induced cell cycle arrest at the G0/G1 phase and apoptosis in MCF-7 cells. Moreover, they induced massive cytoplasmic vacuolization, which was associated with elevated dynein-dependent endocytosis, a process that has not been reported for usnic acid and indicates a novel mechanism of action of its synthetic derivatives. This work also shows that naturally occurring usnic acids are promising lead compounds for the synthesis of derivatives with more favorable properties against cancer cells.

Full text of DOI:10.1021/acs.jnatprod.8b00980

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Synthesis of Usnic Acid Derivatives and Evaluation of Their
Antiproliferative Activity against Cancer Cells
Agnieszka Pyrczak-Felczyk∥owska,†,¶ Rajeshwar Narlawar,‡,¶ Anna Pawlik,§,⊥
Tristan A. Reekie,‡ Anna Herman-Antosiewicz,*,§ and Michael Kassiou*,‡
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Beata Guzow-Krzeminska, Damian Artymiuk, Aleksandra Hac, Kamil Rys, Louis M. Rendina,
†
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Department of Physiology, Medical University of Gdansk, 80-211 Gdansk, Poland
‡School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
§
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Faculty of Biology, Department of Medical Biology and Genetics, University of Gdansk, 80-308 Gdansk, Poland
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Department of Biochemistry, Gdansk University of Physical Education and Sport, 80-336 Gdansk, Poland
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Faculty of Biology, Department of Plant Taxonomy and Nature Conservation, University of Gdansk, 80-308 Gdansk, Poland
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Faculty of Biology, University of Gdansk, 80-308 Gdansk, Poland
S
* Supporting Information
ABSTRACT: Usnic acid is a secondary metabolite abun-
dantly found in lichens, for which promising cytotoxic and
antitumor potential has been shown. However, knowledge
concerning activities of its derivatives is limited. Herein, a
series of usnic acid derivatives were synthesized and their
antiproliferative potency against cancer cells of different origin
was assessed. Some of the synthesized compounds were more
active than usnic acid. Compounds 2a and 2b inhibited
survival of all tested cancer cell lines in a dose- and time-
dependent manner. Their IC50 values after 48 h of treatment
were ca. 3 μM for MCF-7 and PC-3 cells and 1 μM for HeLa
cells, while 3a and 3b revealed antiproliferative activity only
against HeLa cells. All active usnic acid derivatives induced
G0/G1 arrest and a drop in the fraction of HeLa cells in the S and G2/M phases. Compounds 2a and 2b decreased the
clonogenic potential of the cancer cells evaluated and induced cell cycle arrest at the G0/G1 phase and apoptosis in MCF-7
cells. Moreover, they induced massive cytoplasmic vacuolization, which was associated with elevated dynein-dependent
endocytosis, a process that has not been reported for usnic acid and indicates a novel mechanism of action of its synthetic
derivatives. This work also shows that naturally occurring usnic acids are promising lead compounds for the synthesis of
derivatives with more favorable properties against cancer cells.
ancer has become the second leading cause of human
death worldwide. Despite continuous efforts to treat the
properties.9,10 Therefore, lichens can be a potential source of
lead compounds useful in the development of pharmaceut-
icals.11−14 One of the most widely studied lichen secondary
metabolites is usnic acid (1) (for reviews, see, e.g., refs 15−
17). Usnic acid (2,6-diacetyl-7,9-dihydroxy-8,9b-
dimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione, 1) is a diben-
zofuran derivative found in numerous lichen species. It occurs
in Nature as both its (−) and (+) isomers as well as a racemic
mixture9 and is one of the few lichen secondary metabolites
that are commercially available. It exhibits antibacterial,
antiviral, antiprotozoal, and cytotoxic activities (e.g., refs 15,
16, 18). Both the (−) and (+) isomers showed antiproliferative
activity against a wide range of cancer cell lines (e.g., refs 19−
22), although in a majority of cells tested effective
C
disease over the past few decades, human mortality rates for
numerous types of cancers have not been significantly reduced.
In 2012, the worldwide number of new cases of cancer had
increased to 14 million per year, and cancer deaths were
estimated to be 8.2 million annually; therefore improved
treatments are needed.1,2 Natural products are important
sources of anticancer drugs. Many clinically useful anticancer
drugs are of plant origin; for example, Taxol (paclitaxel) is a
diterpene isolated from Taxus brevifolia, while vincristine and
vinblastine are alkaloids extracted from Catharanthus roseus.3−7
Lichens are the symbiotic phenotype of nutritionally
specialized fungi (mycobiont) that derive fixed carbon from
green algae and/or cyanobacteria (photobionts).8 They are
known to produce numerous secondary metabolites; many of
them exhibit antibiotic, antitumor, antimutagenic, antifungal,
antiviral, enzyme inhibitory, and plant growth inhibitory
Received: December 8, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Scheme 1. Tautomeric Forms of Usnic Acid
a
Scheme 2. General Synthesis of Usnic Acid-Derived Isoxazoles 2
a
Reagents and conditions: (a) NH2OH·HCl, pyridine, abs. ethanol, 80 °C, 1.5 h, 64−70%.
concentrations were quite high: the IC50 values after 48 h of
treatment were in the range 18−92 μM, depending on the
cancer cell line.20,23
diaminooctane.26 Furthermore, synthetic and enamine deriv-
atives of both enantiomers of usnic acid showed cytotoxic
effects on blood tumor cell lines, especially the cyanoethyl
derivative, for which the activity against human T cell leukemia
(MT-4) cells was 2 times higher than that of (+)-usnic acid,
while derivatives with a quaternized nitrogen atom were
inactive against all cell lines tested.27,28 Naturally occurring in
Usnea longissima, the usnic acid derivatives usenamines and
isousone were isolated and showed inhibitory effects on the
growth of human hepatoma HepG2 cells with IC50 values of
3.3−6.0 μM.29 Zakharenko and co-workers reported that usnic
acid enamines revealed cytotoxicity against human MCF-7
cells with IC50 values in the range of 0.16−2.0 μM. Moreover,
these compounds enhanced the cytotoxicity of camptothecin
by an order of magnitude.30 Thus, some modifications to the
Despite reports on the biological activity of usnic acid,
current knowledge on its derivatives, both synthetic and
naturally occurring, is very limited. In previous studies, Takai
et al.24 synthesized a series of usnic acid derivatives to improve
their bioavailability by increasing water solubility and indicated
the importance of its lipophilicity and the β-triketone moiety of
usnic acid on cytotoxicity. Acylhydrazones of usnic acid
coordinated with Pd(II) and Cu(II) were found to be
cytotoxic in vitro toward HeLa cells with IC50 values ranging
from 1.8 to 86.0 μM.25 Synthetic usnic acid derivatives
obtained by conjugation of the acetyl group to a polyamine
chain were shown to be more active than usnic acid in cancer
cells, with IC50 values from 3 to 14 μM in the case of 1,8-
B
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a
Scheme 3. General Synthesis of Usnic Acid-Derived Pyrazoles 3
a
Reagents and conditions: (a) RNHNH2, abs. ethanol, reflux, 3 h, 50−66%; (b) RNHNH2·HCl, pyridine, abs. ethanol, 50 °C 10 min then reflux,
16 h, 33−60%.
structure of usnic acid have been shown to improve the
potency of its antiproliferative activity toward cancer cells.
The triketo functionality of ring C of usnic acid 1 results in
multiple tautomeric forms31 (Scheme 1). Tautomerism
between the enolic forms is usually very rapid and with a
very low interconversion barrier. Most of the biological effects
of usnic acid are believed to be related to ring C; therefore a
clear and defined structure of ring C is important to
understand activity.24 The possibility of multiple tautomeric
forms and E/Z-enol stereoisomeric forms hinder the develop-
ment of structure−activity relationships for usnic acids.
Therefore, it was hypothesized that replacement of the 1,3-
dicarbonyl functionality by bioisosteric heterocycles such as
isoxazole or pyrazole would lock the accessible conformation
into more rigid and defined arrangements, which would then
decrease pleiotropic effects derived from rotational freedom
and equilibria of stereoisomers. Such replacements of a
dicarbonyl functionality have been shown to improve activity
in the case of other molecules.32
RESULTS AND DISCUSSION
■
Synthesis of Usnic Acid Derivatives. The isoxazole
derivatives 2 of racemic usnic acid ( )- and (+)-usnic acid (1)
were prepared as depicted in Scheme 2, by reacting with
hydroxylamine hydrochloride and pyridine in ethanol. N-
Substituted pyrazole derivatives were prepared by heating to
reflux in ethanol the corresponding hydrazine and ( )- or
(+)-usnic acid (Scheme 3). Slight changes to the experimental
method depended on the availability of the reacting hydrazine
and whether it was used as the free base or hydrochloride, with
the latter requiring the inclusion of pyridine before the
addition of usnic acid. N-Methyl (3a,b), N-phenyl (3c,d), and
N-3,4-dichlorophenyl (3i,j) analogues of usnic acid used the
free base hydrazine and a reaction time of 3 h and required
flash column chromatography for purification. The N-4-
methoxyphenyl (3e,f), N-4-fluorophenyl (3g,h), N-4-methyl-
phenyl (3k,l) analogues of usnic acids were prepared from the
respective hydrazine hydrochlorides with pyridine and required
a reaction time of 16 h. All spectroscopic data matched with
the expected structure. Thus, mass spectrometry showed the
loss of two water molecules with the inclusion of the requisite
hydrazine mass. 1H NMR spectroscopy showed the loss of the
highly downfield shifted signal (∼18.8 ppm), and two signals
corresponding to carbonyl carbons were no longer observed in
the 13C NMR spectrum. The expected signals corresponding to
The major objectives of this study were to synthesize
isoxazole and pyrazole derivatives of usnic acid and evaluate
the antiproliferative activity of these derivatives toward cancer
cells of different origin, using breast (MCF-7), cervical (HeLa),
and prostate cancer (PC-3) cell lines, and then get some
insight into their mechanism of action.
C
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Table 1. Sensitivity (IC50) of Different Cancer Cell Lines to Usnic Acid and Its Derivatives
a
IC50 [μM]
MCF-7
HeLa
PC-3
HDFa
24 h
>10
48 h
>10
24 h
>10
48 h
>10
24 h
>10
48 h
>10
24 h
>10
48 h
(−)-usnic acid
>10
(+)-usnic acid
>10
>10
>10
>10
>10
>10
>10
>10
2a
2b
3a
3b
3.1 0.3
3.2 0.5
>10
2.9 0.3
2.5 0.3
>10
1.0 0.3
1.0 0.3
2.7 0.5
2.7 0.5
1.0 0.1
1.0 0.1
1.1 0.2
1.2 0.2
3.4 0.6
3.2 0.4
>10
2.8 0.3
3.1 0.4
>10
>10
9.2 0.5
>10
5.9 0.3
3.0 0.4
>10
>10
>10
>10
>10
>10
>10
a
Results are expressed as the means SE of three independent experiments.
Figure 1. Effect of compounds 2a and 2b on the viability of the MCF-7, HeLa, and PC-3 cancer cell lines. Cells were treated with different
concentrations of usnic acid derivatives for 24 h (A) or 48 h (B). Each value is the mean ( SE) of three experiments performed in duplicate.
Statistical significance was determined with ANOVA and Dunnett’s post hoc test and is marked with *.
the various R groups associated with the hydrazine were also
observed in both 1H and 13C NMR spectra. Regiochemistry of
the pyrazole and isoxazole were determined and confirmed
through two-dimensional NMR experiments including HSQC
and HMBC (Figures S1−S32, Supporting Information).
Cytostatic and Cytotoxic Activity of Usnic Acid
Derivatives toward Cancer Cells. It has been shown
previously that usnic acid enantiomers possess moderate
cytotoxic activity; thus the present research has been
performed to test whether usnic acid-derived isoxazoles
(2a,b) and pyrazoles (3a−l) affect the viability of different
cancer cell lines. MCF-7 breast cancer cells were used in a
preliminary screening test. The viability was estimated after 24
or 48 h of treatment using an MTT assay. All the N-phenyl-
substituted usnic acid pyrazoles (3c−l) did not show
significant activity, whereas the N-methyl analogues of
( )-usnic acid 3a and (+)-usnic acid 3b showed some activity
against MCF-7 cells. Replacing the diketo functionality of usnic
acids by isoxazole (2a,b) resulted in significant improvement
of the activity (Table 1). Consequently, 2a, 2b, 3a, and 3b
derivatives were selected for further experiments.
To elucidate whether the cytotoxic effect of selected usnic
acid derivatives is cell line specific or more general, in addition
to the MCF-7 cell line, two other cancer cell lines, HeLa
cervical cancer cells and PC-3 prostate cancer cells, were used.
As shown in Figure 1, 2a and 2b inhibited the viability of all
tested cancer cell lines in a dose- and time-dependent manner,
and HeLa was the most sensitive cell line. Interestingly, 3a and
3b demonstrated marked antiproliferative activity only against
HeLa cells, especially when treated for 48 h (Figure S38,
Supporting Information).
Activities of the selected usnic acid derivatives, i.e., 2a, 2b,
3a, and 3b, with those determined for usnic acid enantiomers
against cancer cells as well as HDFa normal human dermal
fibroblasts were compared. Calculated IC50 values are shown in
Table 1.
Compounds 2a and 2b revealed antiproliferative activity,
especially against cervical cancer, as their IC50 values for the
HeLa cell line were ca. 1 μM, while for the MCF-7 and PC-3
cell lines they were ca. 2.5−3 μM after 48 h of treatment. For
comparison, IC50 values of both (+)- and (−)-usnic acids for
both MCF-7 and HeLa cell lines were above 10 μM. Brisdelli
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Table 2. Cell Cycle Distribution of MCF-7 Cells Treated with Usnic Acid Enantiomers and Compounds 2a and 2b for 24 h
a
cell cycle phase [% of cells]
compound
control
(−)-usnic acid
concentration [μM]
G0/G1
S
G2/M
47.8 0.42
46.95 0.6
52.35 0.21
46 4.1
50.6 0.28
71.85 5.86
75.45 2.61
70.25 2.62
75.7 3.39
23.75 1.87
22.9 1.27
20.6 2.26
23 4.94
22.4 2.54
10.1 4.52
8.35 2.62
11.15 3.6
7.25 2.61
27 2.97
2.9
8.7
2.9
8.7
2.9
8.7
2.9
8.7
28.25 2.37
24.95 3.6
28.8 2.54
24.8 3.81
16.2 0.56
14.02 2.33
16.4 3.53
14.65 1.62
(+)-usnic acid
2a
2b
a
Results are expressed as the means SE of three independent experiments.
Figure 2. Compounds 2a (A) and 2b (B) decrease clonogenicity of different cancer cell lines. MCF-7, HeLa, and PC-3 cells were treated with
different concentrations of usnic acid derivatives for 24 h and allowed to grow for 2 weeks in medium free of usnic acid derivatives. Each value is a
mean ( SE) of three experiments performed in duplicate. Statistical significance was determined with ANOVA and Dunnett’s post hoc test and is
marked with *.
and co-workers also reported quite modest antiproliferative
activity of (+)-usnic acid against HeLa and MCF-7 cells (IC50
values after 48 h treatment of HeLa and MCF-7 were 23.7 and
75.7 μM, respectively).20 Thus, the newly synthesized
derivatives 2 and 3 are more active than the usnic acid
enantiomers, for which in some cases it was not possible to
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determine an IC50 value using the same range of concen-
trations.
The compounds 3a and 3b inhibited the viability of HeLa
cells only, and their activity was much higher than the activity
of usnic acid (IC50 values for both 3a,b and both (+)- and
(−)-usnic acids after 48 h of treatment were ca. 1 and above 10
μM, respectively). Generally toxic compounds are not
expected to show variations in IC50 between different cell
lines. Thus, the selective activity of 3a and 3b compounds
toward HeLa cells is interesting and warrants further
investigation. Importantly, cancer cells were found to be
more sensitive to the new usnic acid derivatives than normal
skin fibroblasts, except in the case of PC-3 cells treated with 2b
for 48 h.
As the doubling time of the cancer cell lines is about 24 h,
results of MTT tests after 24 h of treatment with usnic acid
derivatives indicated cytotoxic rather than cytostatic activity.
However, to elucidate whether usnic acid derivatives affect cell
proliferation, the cell cycle distribution of the MCF-7 cells
treated with 2a or 2b and HeLa cells treated with the panel of
usnic acid derivatives for 24 h has been investigated.
Compounds 2a and 2b induced in the MCF-7 cell line cell
cycle arrest at the G0/G1 phase at a low concentration, 2.9 μM
(about 71−72% in treated cells compared to 48% of control
cells in the G0/G1 phase). Higher concentrations (8.7 μM) of
these compounds increased the percentage of arrested cells to
76% and were accompanied by a drop in the percentage of
cells in S and G2/M phases (Table 2). Usnic acid enantiomers
at the same experimental conditions had no effect on MCF-7
cell cycle progression at 2.9 μM concentration and at higher
concentrations only moderately increased the fraction of G0/
G1 cells (51−52% vs 48% in control samples).
Interestingly, both usnic acid enantiomers induced a slight
accumulation of HeLa cells in the S and G2/M phases,
especially at higher concentrations. On the other hand, usnic
acid derivatives induced evident G0/G1 arrest (51−65%
compared to 44% in control samples) and a drop in the
fraction of HeLa cells in S or G2/M phases (Table S1,
Supporting Information). The percentage of G0/G1 cells was
dose-dependent only in the case of treatment with 3a.
Compounds 2a, 2b, and 3b at higher concentrations did not
potentiate the effects observed at lower concentrations, which
might indicate that more cells died, although they could not be
detected by the assay used in this study.
Further studies concentrated on 2a and 2b derivatives, as
they were active against a wider panel of cancer cells. A
clonogenic assay was performed to elucidate whether usnic
acid derivatives have a long-term effect and to what extent the
cells are able to restart proliferation after culturing without the
test compounds. Cells were treated with derivatives 2a and 2b
for 24 h and allowed to recover for 2 weeks. As shown in
Figure 2, both derivatives significantly inhibited the clonogenic
potential of MCF-7, HeLa, and PC-3 cancer cells. Less than
50% of cells treated with the highest test concentrations were
able to survive the treatment and proliferate to form colonies.
They probably represent the fraction of cells reversibly arrested
in the cell cycle.
The results of the clonogenic and MTT tests suggested that
compounds 2a and 2b induce death in the majority of cells. To
confirm this, we looked at apoptotic and necrotic fractions
using flow cytometric detection of cells labeled with annexin V,
which detects apoptotic cells, and 7-AAD dye, staining cells
with a disintegrated membrane. As can be seen in Figure 3A,
Figure 3. Compounds 2a and 2b induce apoptosis of MCF-7 cells.
Cells were treated with test usnic acid derivatives for 48 h (A) or 24 h
(B). (A) The amounts of apoptotic (A) and necrotic (N) cells were
determined by flow cytometry after staining with the Muse annexin V
and dead cell kit. Results are presented as means
SE of three
independent experiments. Statistical significance was determined with
ANOVA and Bonferroni’s post hoc test and is marked with *. (B)
Immunoblots for caspase-cleaved PARP, Bax, and Bcl-2. The blots
were stripped and reprobed with an anti-β-actin antibody to ensure
equal protein loading. Densitometric scanning data after correction
for loading control are above the immunoreactive bands.
compounds 2a and 2b induced apoptosis of MCF-7 cells: the
fraction of apoptotic cells was 2 times higher after treatment
with 2.9 μM and 3 times higher after treatment with 8.7 μM
compounds 2a and 2b than in control cells. These observations
were accompanied by characteristic cleavage of PARP and a
drop in the levels of antiapoptotic Bcl-2, which might indicate
a mitochondrial pathway of apoptosis (Figure 3B).
The morphology of MCF-7 cells treated with DMSO or 2a
or 2b derivatives was also analyzed. Surprisingly, massive
vacuolization of cells treated with these compounds was
evident (Figure 4A). This was accompanied by elevation of
LAMP1, a marker of lysosomes and late endosomes (Figure
4B). The possibility that vacuoles might be of autophagosomal
origin was excluded, since wortmannin, an inhibitor of the
early steps of autophagy, had no effect on vacuolization and did
not protect against a drop in survival of cells exposed to the
compounds tested (data not shown). Interestingly, cells treated
with compounds 2a and 2b accumulated Lucifer Yellow
(Figure 4C). The dye is used to label endocytic compartments
and has been shown to be transported into cells by fluid-phase
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Figure 4. Compounds 2a and 2b induce massive vacuolization of MCF-7 cells. Cells were treated with 8.7 μM of each usnic acid derivative for 24
or 48 h. (A) Morphology of cells was examined using transmission electron microscopy. Representative photographs of cells at 1650×
magnification. (B) Levels of LAPM1 were assessed by Western blot, which was stripped and reprobed with anti-β-actin antibody to ensure equal
protein loading. Densitometric scanning data after correction for loading control are shown above respective bands. (C) Vacuolization of cells
treated with derivative 2a or 2b is associated with increased endocytosis. Cells were treated with 2.9 or 8.7 μM 2a or 2b for 24 h, stained with 75
μM Lucifer Yellow, and analyzed under a fluorescent microscope (magnification 1000×).
endocytosis, mainly macropinocytosis, but also accumulates in
caveolar vesicles.33
survival by about 40%, lack of potentiation of the 2a or 2b
antiproliferative activities may actually indicate its protective
activity (Figure 5B).
Several forms of endocytosis have been described depending
on the type of cargo and molecular machinery involved in its
internalization and include clathrin-mediated, caveole/lipid
raft-mediated, clathrin- and caveole-independent, bulk fluid-
phase endocytosis (macropinocytosis), and phagocytosis.34 To
elucidate which mode of endocytosis is induced by derivatives
2a and 2b in MCF-7 cells, different inhibitors of macro-
pinocytosis were used. Cytochalasin D is an inhibitor of actin
dynamics and thus inhibits actin-dependent formation of
membrane ruffles, which are necessary to surround the
extracellular fluid. Amiloride is an inhibitor of Na+/H+
exchange, which is necessary for membrane closure and a
decrease of intravesicular pH.35 As shown in Figure 5A, neither
cytochalasin D nor amiloride blocked the usnic acid derivative-
induced vacuolization. On the other hand, dynasore, an
inhibitor of dynamin necessary for clathrin- and caveolin-
dependent endocytosis,36,37 protected cells against vacuoliza-
tion induced by derivative 2a or 2b. All inhibitors had no effect
on the usnic acid derivative-induced drop in cell survival.
However, considering that dynasore by itself decreased cell
This study shows that a series of usnic acid derivatives 2a,b
and 3a,b exhibited more potent antiproliferative activities in
comparison to enantiomers of usnic acid (1). Interestingly, the
configuration of these compounds seems to be less important
for their activity than the introduced structural modifications.
Derivatives 2a and 2b, which are active against cancer cells of
different origin, effectively decreased the clonogenic potential
of these cells. This was found to be due to the induction of
apoptosis and cell death connected with massive vacuolization,
which resulted from perturbations in endosomal trafficking or
function. It has been reported that overexpression of the Ras
oncogene or treatment with some small molecules, such as
indolyl chalcones, induces a caspase-independent cancer cell
death process called methuosis.38−40 This relies on hyper-
stimulation of macropinocytosis in which macropinosomes are
transported not to lysosomes but to the late endosomes, which
expand to form cytoplasmic vacuoles. The results of the
present work indicated that the new usnic acid derivatives
tested also stimulated endocytosis, but not via a bulk fluid
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Figure 5. Inhibition of dynamin but not actin dynamics or Na+/H+ exchanger protects against vacuolization induced by derivatives 2a and 2b.
MCF-7 cells were treated for 24 h with 8.7 μM of each usnic acid derivative with or without 0.5 μM cytochalasin D, 10 μM amiloride, or 50 μM
dynasore. (A) Morphology and vacuolization of cells were monitored using a light microscope (magnification 200×). (B) Cell viability was assessed
by an MTT assay. Each value is the mean ( SE) of three experiments performed in triplicate. Statistical significance was determined with ANOVA
and Bonferroni’s post hoc test (*p < 0.001 between cells treated with an inhibitor and respective control). There was no statistical significance
between viability of cells treated with 2a or 2b alone or together with inhibitors.
(+)-usnic acid (1), are commercially available (Ark Pharm, Inc. and
Sigma-Aldrich, respectively) (see Figures S33−S35 in the Supporting
Information for NMR spectra). Though ( ) is used for simplicity, the
ratio of (−)-usnic acid to (+)-usnic acid is 71:29 (Figures S36 and
S37, Supporting Information). All other chemicals used were
purchased from Sigma-Aldrich (St. Louis, MO, USA) and were of
highest commercially available purity. All solvents were distilled by
standard techniques prior to use. Where stated, reactions were
performed under an inert atmosphere of nitrogen using syringe-
septum cap techniques or a manifold. 1H NMR spectra were recorded
at 300 MHz using a Bruker NMR spectrometer. Chemical shifts (δ
units) are stated in parts per million (ppm), and tetramethylsilane
(TMS) was used as an internal reference at 0 ppm. All coupling
constants (J) are given in hertz, and the splitting patterns are
designated as follows: s (singlet), d (doublet), dd (doublet of
doublets), t (triplet), dt (doublet of triplets), q (quartet), and m
uptake process but rather a dynamin-dependent endocytosis,
as cytoplasm vacuolization was blocked by the inhibitors of
dynamin-dependent endocytosis (such as clathrin- or caveole-
mediated endocytosis) and not by inhibitors of macro-
pinocytosis. At present, it is difficult to establish an
unequivocal cause−effect relationship between vacuolization
and cell death, because pharmacological agents blocking
endocytic pathways, such as dynasore, compromise cell
viability. Mechanisms underlying this activity are currently
under investigation.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All glass apparatuses were
oven-dried prior to use. The starting materials, ( )-usnic acid and
H
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(multiplet). 13C NMR spectra were recorded at 75 MHz using a
Bruker 300 NMR spectrometer. Chemical shifts (δ units) are stated in
ppm and are assigned with standard 2D HSQC and HMBC
experiments. High-resolution electrospray ionization MS (ESIMS)
was carried out using a Bruker (USA) Daltronics BioApex II with a
7T superconducting magnet and an analytical ESI source. Thin-layer
chromatography was performed on Merck aluminum-backed plates,
precoated with silica gel (0.2 mm, 60F254), which were developed
using either UV fluorescence (254 nm) or iodine vapor. Flash
chromatography was performed on silica gel (Merck silica gel 60H,
particle size 5−40 μm).
Procedures for the Synthesis of Usnic Acid Isoxazoles. A
suspension of ( )-usnic acid or (+)-usnic acid (1 equiv) in dry
pyridine (5 mL) and absolute ethanol (5 mL) was treated with
hydroxylamine hydrochloride (1.1 equiv) and heated at 80 °C under a
nitrogen atmosphere for 1.5 h. Upon cooling, a deep yellow crystalline
product was formed, and the reaction mixture was diluted with cold
distilled water (20 mL), acidified with 1 N hydrochloric acid, and
extracted with ethyl acetate (3 × 50 mL). The combined organic layer
was washed with water, dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography (10% ethyl acetate in hexane) to afford
the desired product as a yellow crystalline solid.
8-Acetyl-5,7-dihydroxy-3,4a,6-trimethylbenzo[2,3]-
benzofuro[5,6-d]isoxazol-4(4aH)-one (2a): yellow solid, 630 mg
(64%); 1H NMR (CDCl3, 300 MHz) δ 13.28 (1H, s, OH-15), 10.52
(1H, s, OH-14), 6.40 (1H, s, H-10), 2.69 (3H, s, CH3-2), 2.53 (3H, s,
CH3-18), 2.10 (3H, s, CH3-13), 1.80 (3H, s, CH3-12); 13C NMR
(CDCl3, 75 MHz) δ 200.4 (C-1), 195.7 (C-4), 178.9 (C-10a), 178.1
(C-9a), 164.0 (C-7), 157.6 (C-3), 157.1 (C-5), 156.1 (C-8a), 109.3
(C-6), 107.9 (C-3a), 103.4 (C-4b), 101.8 (C-8), 89.5 (C-10), 62.4
(C-4a), 31.4 (C-2), 31.2 (C-12), 10.7 (C-18), 7.6 (C-13);
(+)-HRESIMS m/z 342.0971 [M + H]+ (calcd for C18H16NO6,
342.0978).
mg (66%); 1H NMR (CDCl3, 300 MHz) δ 13.27 (1H, s), 11.15 (1H,
s), 6.11 (1H, s), 3.80 (3H, s), 2.64 (3H, s), 2.46 (3H, s), 2.06 (3H, s),
1.69 (3H, s); 13C NMR (CDCl3, 75 MHz) δ 200.7, 195.8, 172.8,
163.8, 157.9, 156.6, 150.7, 149.2, 110.1, 108.4, 104.3, 101.8, 87.9,
60.4, 36.2, 31.5, 30.7, 13.3, 7.7; (+)-HRESIMS m/z 377.1107 [M +
Na]+ (calcd for C19H18NaN2O5, 377.1113).
Reagents for Biological Experiments. F12-K Nutrient Mixture
medium and fetal bovine serum were purchased from GIBCO (Grand
Island, NY, USA). RPMI 1640, DMEM, DMSO, penicillin/
streptomycin antibiotic mixture, and thiazolyl blue tetrazolium
bromide (MTT) were purchased from Sigma (St. Louis, MO,
USA). Antibodies against Bcl-2, Bax, were from Santa Cruz
Biotechnology (Santa Cruz, CA, USA); antibody against PARP was
from Cell Signaling Technology (Danvers, MA, USA).
Cell Culture Conditions. Monolayer cultures of MCF-7 and
HeLa cells were maintained in RPMI 1640 medium, PC-3 cells in
F12-K Nutrient Mixture medium, and HDFa cells in DMEM medium.
Basic media were supplemented with 10% (v/v) fetal bovine serum
and a penicillin−streptomycin mixture. Each cell line was maintained
at 37 °C in a humidified atmosphere with 5% CO2.
Cell Viability Assay. Cell viability was determined by an MTT
method. Cells were seeded at a density of 4 × 103 (all cell lines for 24
h of incubation and for the PC-3 cell line) and 2 × 103 (MCF-7 and
HeLa cell lines for 48 h of incubation) per well of a 96-well plate and
allowed to attach overnight. The medium was replaced with fresh
medium supplemented with desired concentrations of each
investigated compound for 24 or 48 h. Before the end of treatment,
25 μL of MTT solution (4 mg mL−1) was added to each well. After 3
h of incubation, medium was removed, and formazan crystals were
dissolved in 100 μL of DMSO. Absorbance was measured at 570 nm
(with reference wavelength 660 nm) in a Victor3 microplate reader.
Data were obtained from at least three independent experiments
performed in duplicate. IC50 values were calculated using the
GraphPad Prism software.
Cell Cycle and Cell Death Assays. The effect of the test
compounds on cell cycle distribution was determined by flow
cytometry after staining the cells with propidium iodide. A total of 2
×105 cells were seeded in six-well plates. After treatment, both the
medium and trypsinized cells were collected altogether, centrifuged
for 10 min at 300g, stained using the Muse cell cycle kit, and analyzed
by a Muse cell analyzer (Millipore).
(S)-8-Acetyl-5,7-dihydroxy-3,4a,6-trimethylbenzo[2,3]-
benzofuro[5,6-d]isoxazol-4(4aH)-one (2b): yellow solid, 350 mg
(70%); 1H NMR (CDCl3, 300 MHz) δ 13.29 (1H, s), 10.51 (1H, s),
6.38 (1H, s), 2.68 (3H, s), 2.52 (3H, s), 2.09 (3H, s), 1.78 (3H, s);
13C NMR (CDCl3, 75 MHz) δ 200.4, 195.7, 178.9, 178.1, 164.0,
157.6, 157.1, 156.1, 109.3, 107.9, 103.4, 101.8, 89.5, 62.4, 31.4, 31.2,
10.7, 7.6; (+)-HRESIMS m/z 364.0792 [M + Na]+ (calcd for
C18H15NaNO6, 364.0797).
Clonogenic Assay. Cells (1 × 106) were plated in 10 cm plates.
After 24 h, the medium was removed and fresh medium containing
derivative 2a or 2b at concentrations of 1, 2, 3, or 4 μg mL−1 was
added. Following 24 h of treatment, the cells from each plate were
trypsynized, counted, and plated (8 × 102) into two new plates in
medium free of usnic acid derivatives. After 2 weeks, the cells were
fixed with glutaraldehyde (6.0%, v/v) and stained with 0.5% crystal
violet solution. Colonies consisting of at least 50 cells were counted.
Immunoblotting. Cells were treated with 2a or 2b and lysed
using a solution containing 50 mM Tris (pH 7.5), 1% Triton X-100,
150 mM NaCl, 0.5 mM EDTA, and protease and phosphatase
inhibitor cocktails (Roche Diagnostics). The lysates were cleared by
centrifugation. Proteins were separated by SDS-PAGE and transferred
onto a PVDF membrane. The membrane was blocked with 5% nonfat
dry milk in phosphate-buffered saline (PBS) and incubated with the
desired primary antibody overnight at 4 °C. The membrane was then
treated with the appropriate secondary antibody for 1 h at room
temperature. Immunoreactive bands were detected with an enhanced
chemiluminescence reagent (Thermo Scientific). Blots were stripped
and reprobed with anti-actin antibodies to normalize for differences in
protein loading. Each protein was detected two or three times in
independently prepared lysates. Densitometry analysis was carried out
using Quantity One 1-D Analysis software (Bio-Rad).
Procedures for the Synthesis of Usnic Acid Pyrazoles.
Method A. A suspension of ( )-usnic acid or (+)-usnic acid (1 equiv)
in absolute ethanol (10 mL) was treated with hydrazine (1.1 equiv)
and heated at reflux temperature under nitrogen for 3 h. Upon
cooling, a yellow precipitate was formed, which was filtered and
washed with cold ethanol and n-hexane to afford a crude product.
This was purified by flash column chromatography (15% ethyl acetate
in hexane) to afford the desired product.
Method B. A mixture of phenylhydrazine hydrochloride (1.1 equiv)
and pyridine (1.1 equiv) in absolute ethanol (10 mL) was heated at
50 °C until it became homogeneous. To this was added ( )-usnic
acid or (+)-usnic acid (1 equiv), and the mixture was heated at reflux
temperature under nitrogen for 16 h and then allowed to cool.
8-Acetyl-5,7-dihydroxy-1,3,4a,6-tetramethyl-1,4a-dihydro-
4H-benzofuro[3,2-f]indazol-4-one (3a). This was synthesized by
method A to give the desired product as a yellow solid: 630 mg
(62%); 1H NMR (CDCl3, 300 MHz) δ 13.28 (1H, s, OH-15), 11.15
(1H, s, OH-14), 6.11 (1H, s, H-10), 3.83 (3H, s, CH3-17), 2.65 (3H,
s, CH3-2), 2.47 (3H, s, CH3-18), 2.07 (3H, s, CH3-13), 1.70 (3H, s,
CH3-12); 13C NMR (CDCl3, 75 MHz) δ 200.5 (C-1), 195.6 (C-4),
172.6 (C-8a), 163.6 (C-7), 157.8 (C-5), 156.4 (C-9a), 150.4 (C-3),
149.0 (C-10a), 109.9 (C-3a), 108.3 (C-6), 104.1 (C-4b), 101.6 (C-8),
87.8 (C-10), 60.2 (C-4a), 36.0 (C-17), 31.3 (C-12), 30.6 (C-12),
13.2 (C-18), 7.59 (C-13); (+)-HRESIMS m/z 377.1108 [M + Na]+
(calcd for C19H18NaN2O5, 377.1113).
Transmission Electron Microscopy (TEM) and Fluorescence
Microscopy. TEM of MCF-7 cells was performed essentially as
described previously.41 Briefly, cells (2 × 105) were plated in 12-well
plates and allowed to attach overnight. Next, cells were treated with
either DMSO (control) or 1 or 3 μg mL−1 2a or 2b for 24 or 48 h at
37 °C. For TEM, cells were fixed in ice-cold 2.5% electron microscopy
(S)-8-Acetyl-5,7-dihydroxy-1,3,4a,6-tetramethyl-1,4a-dihy-
dro-4H-benzofuro[3,2-f ]indazol-4-one (3b). This was synthe-
sized by method A to give the desired product as a yellow solid: 340
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grade glutaraldehyde (Polysciences) in 0.1 M PBS (pH 7.4). The
samples were rinsed with PBS, postfixed in 1% osmium tetroxide with
0.1% potassium ferricyanide, dehydrated through a graded series of
ethanol washes (30−100%), and embedded in Epon (Fluka).
Semithin (300 nm) sections were cut using an RMC Power Tome
XL ultramicrotome, stained with 0.5% toluidine blue, and examined
under a light microscope. Ultrathin sections (45 nm) were stained
with 2% uranyl acetate and Reynold’s lead citrate and examined on a
Philips CM100 transmission electron microscope. To investigate the
level of endocytosis, cells treated with 2a or 2b were stained with
Lucifer Yellow (75 μM) for 24 h, washed, and analyzed under a Nikon
Eclipse E800 fluorescence microscope.
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ASSOCIATED CONTENT
* Supporting Information
■
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The Supporting Information is available free of charge on the
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treated with usnic acid or its derivatives 2a,b and 3a,b
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +48 58 523 6034. E-mail: anna.herman-antosiewicz@
biol.ug.edu.pl.
■
(23) Nguyen, T. T.; Yoon, S.; Yang, Y.; Lee, H. B.; Oh, S.; Jeong, M.
H.; Kim, J. J.; Yee, S. T.; Crisan, F.; Moon, C.; Lee, K. Y.; Kim, K. K.;
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*Tel: +61 2 9351 2745. E-mail: michael.kassiou@sydney.edu.
au.
ORCID
Rajeshwar Narlawar: 0000-0002-2439-3502
Anna Herman-Antosiewicz: 0000-0003-0526-2168
Michael Kassiou: 0000-0002-6655-0529
(25) Natic, M.; Tesic, Z.; Andelkovic, K.; Brceski, I.; Radulovic, S.;
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P.; Boustie, J.; Corbel, J. C.; Tomasi, S. Bioorg. Med. Chem. 2008, 16,
6860−6866.
Author Contributions
¶A. Pyrczak-Felczykowska and R. Narlawar contributed
equally.
(27) Rakhmanova, M. E.; Luzina, O. A.; Pokrovskii, M. A.;
Pokrovskii, A. G.; Salakhutdinov, N. F. Russ. Chem. Bull. 2016, 65,
566−569.
Notes
The authors declare no competing financial interest.
(28) Luzina, O. A.; Salakhutdinov, N. F. Russ. J. Bioorg. Chem. 2016,
42, 249−268.
ACKNOWLEDGMENTS
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(29) Yu, X. L.; Guo, Q.; Su, G. Z.; Yang, A. L.; Hu, Z. D.; Qu, C. H.;
Wan, Z.; Li, R. Y.; Tu, P. F.; Chai, X. Y. J. Nat. Prod. 2016, 79, 1373−
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This study was financially supported by the Australian
Research Council (ARC) (DP110104237) and National
Science Centre, Poland (project no. 2017/26/M/NZ7/
00668). This research was also financially supported from
University of Gdansk statutory funds nos. 530-L140-D242-14
and 580-R300-0-5280-15. We are grateful to M. Richert and
M. Narajczyk (Laboratory of Electron Microscopy, Faculty of
(30) Zakharenko, A.; Luzina, O.; Koval, O.; Nilov, D.; Gushchina, I.;
Dyrkheeva, N.; Svedas, V.; Salakhutdinov, N.; Lavrik, O. J. Nat. Prod.
2016, 79, 2961−2967.
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2015, 3, 1014−1018.
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