Synthesis of Usnic Acid Derivatives and Evaluation of Their

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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-Felczykowska,†,¶ Rajeshwar Narlawar,‡,¶ Anna Pawlik,§,⊥ Beata Guzow-Krzeminś ka,∥ Damian Artymiuk,# Aleksandra Hac,́ § Kamil Ryś,§ Louis M. Rendina,‡ Tristan A. Reekie,‡ Anna Herman-Antosiewicz,*,§ and Michael Kassiou*,‡ †

Department of Physiology, Medical University of Gdańsk, 80-211 Gdańsk, Poland School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia § Faculty of Biology, Department of Medical Biology and Genetics, University of Gdańsk, 80-308 Gdańsk, Poland ⊥ Department of Biochemistry, Gdańsk University of Physical Education and Sport, 80-336 Gdańsk, Poland ∥ Faculty of Biology, Department of Plant Taxonomy and Nature Conservation, University of Gdańsk, 80-308 Gdańsk, Poland # Faculty of Biology, University of Gdańsk, 80-308 Gdańsk, Poland

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‡

S Supporting Information *

ABSTRACT: 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 timedependent 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.

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properties.9,10 Therefore, lichens can be a potential source of lead compounds useful in the development of pharmaceuticals.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-8,9bdimethyldibenzo[b,d]furan-1,3(2H,9bH)-dione, 1) is a dibenzofuran 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

ancer has become the second leading cause of human death worldwide. Despite continuous efforts to treat the 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 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 8, 2018

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DOI: 10.1021/acs.jnatprod.8b00980 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Scheme 1. Tautomeric Forms of Usnic Acid

Scheme 2. General Synthesis of Usnic Acid-Derived Isoxazoles 2a

Reagents and conditions: (a) NH2OH·HCl, pyridine, abs. ethanol, 80 °C, 1.5 h, 64−70%.

a

diaminooctane.26 Furthermore, synthetic and enamine derivatives 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

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 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 1,8B



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Scheme 3. General Synthesis of Usnic Acid-Derived Pyrazoles 3a

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%.

a

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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 development of structure−activity relationships for usnic acids. Therefore, it was hypothesized that replacement of the 1,3dicarbonyl 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 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.

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. NSubstituted 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-4methoxyphenyl (3e,f), N-4-fluorophenyl (3g,h), N-4-methylphenyl (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 C



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Table 1. Sensitivity (IC50) of Different Cancer Cell Lines to Usnic Acid and Its Derivatives IC50 [μM]a MCF-7 (−)-usnic acid (+)-usnic acid 2a 2b 3a 3b

HeLa

24 h

48 h

>10 >10 3.1 ± 0.3 3.2 ± 0.5 >10 >10

>10 >10 2.9 ± 0.3 2.5 ± 0.3 >10 >10

24 h >10 >10 1.0 ± 1.0 ± 2.7 ± 2.7 ±

0.3 0.3 0.5 0.5

PC-3 48 h >10 >10 1.0 ± 1.0 ± 1.1 ± 1.2 ±

0.1 0.1 0.2 0.2

HDFa

24 h

48 h

24 h

48 h

>10 >10 3.4 ± 0.6 3.2 ± 0.4 >10 >10

>10 >10 2.8 ± 0.3 3.1 ± 0.4 >10 >10

>10 >10 >10 9.2 ± 0.5 >10 >10

>10 >10 5.9 ± 0.3 3.0 ± 0.4 >10 >10

Results are expressed as the means ± SE of three independent experiments.

a

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-phenylsubstituted 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 D



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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 cell cycle phase [% of cells]a compound control (−)-usnic acid (+)-usnic acid 2a 2b

concentration [μM]

G0/G1

S

G2/M

2.9 8.7 2.9 8.7 2.9 8.7 2.9 8.7

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 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

Results are expressed as the means ± SE of three independent experiments.

a

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 *.

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

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 E



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determine an IC50 value using the same range of concentrations. 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 F



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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×).

survival by about 40%, lack of potentiation of the 2a or 2b antiproliferative activities may actually indicate its protective activity (Figure 5B). 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 hyperstimulation 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

endocytosis, mainly macropinocytosis, but also accumulates in caveolar vesicles.33 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 fluidphase 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 macropinocytosis 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 derivativeinduced vacuolization. On the other hand, dynasore, an inhibitor of dynamin necessary for clathrin- and caveolindependent endocytosis,36,37 protected cells against vacuolization 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 G



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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 syringeseptum 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 caveolemediated endocytosis) and not by inhibitors of macropinocytosis. 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.

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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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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). 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). 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

(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). (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); 13 C 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). 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-dihydro4H-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). (S)-8-Acetyl-5,7-dihydroxy-1,3,4a,6-tetramethyl-1,4a-dihydro-4H-benzofuro[3,2-f ]indazol-4-one (3b). This was synthesized by method A to give the desired product as a yellow solid: 340 I



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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. Statistical Analysis. All data are shown as means ± standard error (SE) of at least three independent experiments. Significance of differences between the control and treated cells in viability tests was analyzed with ANOVA and Dunett’s multiple comparison post hoc test using Statistica 12 (Statsoft). Differences were considered significant at p < 0.05 and are marked with an asterisk.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00980.

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Physical properties of nonactive compounds (3c−3n); original NMR spectra for the new compounds; NMR spectra and chiral HPLC spectra of starting usnic acid samples; effect of 3a and 3b on viability of the HeLa cancer cells; cell cycle distribution of the HeLa cells treated with usnic acid or its derivatives 2a,b and 3a,b for 24 h (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +48 58 523 6034. E-mail: anna.herman-antosiewicz@ biol.ug.edu.pl. *Tel: +61 2 9351 2745. E-mail: [email protected]. au. ORCID

Rajeshwar Narlawar: 0000-0002-2439-3502 Anna Herman-Antosiewicz: 0000-0003-0526-2168 Michael Kassiou: 0000-0002-6655-0529 Author Contributions ¶

A. Pyrczak-Felczykowska and R. Narlawar contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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 Biology, University of Gdańsk) for technical assistance.

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REFERENCES

(1) Stewart, B. W.; Wild, C. P. World Cancer Report 2014; WHO’s International Agency for Research on Cancer (IARC): Geneva, 2015. (2) McGuire, S. Adv. Nutr. 2016, 7, 418−419. J



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Synthesis of Usnic Acid Derivatives and Evaluation of Their

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