176
Regular Article
Chem. Pharm. Bull. 61(2) 176–183 (2013)
Vol. 61, No. 2
Cytotoxic Evaluation of Phenolic Compounds from Lichens against Melanoma Cells Luiz Fabrício Gardini Brandão,a Glaucia Braz Alcantara,a Maria de Fátima Cepa Matos,b Danielle Bogo,b Deisy dos Santos Freitas,a Nathália Mitsuko Oyama,a and Neli Kika Honda*,a a
Department of Chemistry, Universidade Federal de Mato Grosso do Sul; Campo Grande, MS 79074–460, Brazil: and b Laboratory of Molecular Biology and Cell Culture, Universidade Federal de Mato Grosso do Sul; Campo Grande, MS 79070–900, Brazil. Received August 21, 2012; accepted November 14, 2012; advance publication released online December 3, 2012 Atranorin, lichexanthone, and the (+)-usnic, diffractaic, divaricatic, perlatolic, psoromic, protocetraric, and norstictic acids isolated from the lichens Parmotrema dilatatum (Vain.) H ale, Usnea subcavata Motyka, Usnea sp., Ramalina sp., Cladina confusa (Sant.) Folmm. & Ahti, Dirinaria aspera H äsänen, and Parmotrema lichexanthonicum Eliasaro & A dler were evaluated against UACC-62 and B16-F10 melanoma cells and 3T3 normal cells. Sulforhodamine B assay revealed significant cytotoxic activity in protocetraric, divaricatic, and perlatolic acids on UACC-62 cells (50% growth inhibitory concentration (GI50) 0.52, 2.7, and 3.3 µg/mL, respectively). Divaricatic and perlatolic acids proved the most active on B16-F10 cells (GI50 4.4, 18.0 µg/mL, respectively) and the most cytotoxic to 3T3 normal cells. Diffractaic, usnic, norstictic, and psoromic acids were cytotoxic to UACC-62 cells in the 24.7 to 36.6 µg/mL range, as were protocetraric and diffractaic acids to B16-F10 cells (GI50 24.0, 25.4 µg/mL, respectively). Protocetraric acid was highly selective (selectivity index (SI*) 93.3) against UACC-62 cells, followed by norstictic, perlatolic, psoromic, and divaricatic acids, while norstictic and divaricatic acids were more selective against B16-F10 cells. The high SI* value obtained for protocetraric acid on UACC-62 cells makes it a potential candidate for the study of melanomas in experimental models. Chemometric analysis was performed to evaluate the general behavior of the compounds against the cell lines tested. Key words lichen; melanoma; chemometric analysis; cytotoxic activity; phenolic compound
Cancer is the term used to designate all malignant tumors. The disorder can be hereditary and in such cases is caused by a defective gene inherited from a parent or activated by environmental factors, and has been described as the silent epidemic of the 21st century.1) A total of 1638910 new cases of cancer and 577190 deaths were projected in the United States in 2012. Among these new cases, 162480, or roughly 10%, were skin cancer (excluding basal and squamous) and expected to account for 24380 deaths. Melanoma represents 46.9% of skin cancers and 4.7% of all types of cancer. In Brazil, projections for 2012 indicated 134170 new cases of non-melanoma skin cancer and 6230 of melanoma, the most dangerous of skin cancer types, given its high risk of metastasis. Prognosis for this variety, for which chemotherapy is one of the available treatments, is linked to the socioeconomic status of each country.2,3) Many drugs for cancer treatment are obtained from natural sources. Examples include taxol (Paclitaxel), a diterpene isolated from Taxus breviofolia, and vincristine and vinblastine, alkaloids extracted from Catharanthus roseus.4–6) Searching for new antitumor drugs, however, is not limited to higher plants. Lichens, complex structures composed of two organisms (an alga and a fungus) living in symbiosis, can be promising sources of these drugs. The action of lichen-derived compounds on tumor cells has been a focus of evaluations for some decades. Cain evaluated the antileukemia activity of polyporic acid and derivatives,7–10) while Kupchan and Kopperman assessed the inhibition of Lewis lung carcinoma by S-(−)-usnic acid.11) Taking into account the moderate antitumor activity of S-(−)-usnic acid, The authors declare no conflict of interest.
Takai et al. employed it to synthesize 11 derivatives in an attempt to potentiate its activity against Lewis lung carcinoma and P388 leukemia cells.12) Unfortunately, none of these synthetic derivatives proved more potent than the parent compound. The relationship between the antineoplastic activity of usnic acid and p53 protein activation was investigated by Mayer et al.13) They concluded that usnic acid has antiproliferative activity against wild-type p53 (MCF7) and non-functional p53 (MDA-MB-231) breast cancer cells, as well as against the H1299 lung cancer cell line, which is null for p53. Usnic acid is therefore a non-genotoxic anticancer agent that works in a p53-independent manner. The cellular mechanisms of the anticancer effects of usnic acid were investigated by Einarsdóttir et al.,14) who concluded that usnic acid has a marked inhibitory effect on the growth and proliferation of T-47D breast cancer cells and Capan-2 pancreatic cancer cells, leading to loss of mitochondrial membrane potential. Usimine C, a natural derivative of usnic acid, exhibits type I procollagen induction activity in human dermal fibroblast cells.15) Among the compounds resulting from the insertion of amine compounds into a carbonyl group of usnic acid, the diaminooctane derivative displayed significant cytotoxicity against L1210 and 3LL murine cancer cells, K-562, DU145, MCF7, and U251 human cancer cells, and Chinese hamster ovary (CHO) and CHO-MG hamster cell lines.16) Pannarin, a depsidone, was shown to inhibit growth of DU-145 prostate carcinoma and M14 human melanoma cells.17,18) Protolichesterinic acid showed an inhibitory effect against 12 cell lines, with EC50 values of 2.4–18.1 µg/mL
© 2013 The Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail:
[email protected]
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Fig. 1. Structures of the Compounds Evaluated against UACC, B16-F10, and 3T3 Cells
(7.4–55.8 µm).19) The depsides resulting from decarboxylation of baeomycesic and squamatic acids showed antiproliferative effects on PC-3 prostate cancer cells (50% growth inhibitory concentration (GI50) 70.06, 79.37 µm, respectively).20) Ambewelamide A exhibited in vitro cytotoxicity against P388 murine leukemia cells (IC50 8.6 ng/mL) and in vivo antineoplastic activity against P388 cells (%T/C 140 at 160 µg/ kg).21) Bačkorová et al.22) reported the antiproliferative/cytotoxic effects of atranorin and usnic acid, both of which efficiently induced apoptosis and inhibited cell proliferation in all cell lines tested. The mechanisms of cytotoxicity of these compounds were investigated in the A2780 and HT-29 cancer cell lines, demonstrating the ability of atranorin and usnic acid to induce massive loss in mitochondrial membrane potential, along with caspase-3 activation in HT-29 cells and
phosphatidylserine externalization in both cell lines tested.23) Lichen-derived compounds, albeit poorly explored, are promising agents in the inhibition of cell proliferation. The search for new substances for the treatment of melanoma is the focus of our research group and in this article we report the results of the evaluation of nine lichen compounds tested against UACC-62 and B16-F10 melanoma cell lines.
Results and Discussion
The sulforhodamine B (SRB) assay was employed to evaluate the cytotoxic activity of the depsides atranorin and diffractaic, divaricatic, and perlatolic acids; the depsidones psoromic, protocetraric, and norstictic acids; R-(+)-usnic acid and xanthone lichexanthone (Fig. 1) on B16-F10 murine melanoma and UACC-62 human melanoma cells and NIH/3T3 fibroblasts. Doxorubicin, an anticancer drug, was the positive
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Fig. 2. Dendrogram Built from GI50 Data for the Compounds Tested against UACC-62 and B16-F10 Cells HCA, performed with Euclidean distances, employed an incremental linkage method to generate clusters. Branches represent compounds with stronger activity (upper portion, near doxorubicin) and intermediate and weaker activity.
Fig. 3. PCA (a) Score and (b) Loading Plots from LC50 Data for UACC-62, B16-F10, and 3T3 Cells 88.97 and 10.61% of total explained variance on PC1 and PC2, respectively.
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control. SRB assay results were expressed as GI50 and LC50, according to Holbeck,24) and also in terms of selectivity index (SI). SI values greater than 3 indicate that the neoplastic cell is more sensitive to a given compound than normal cells.25) Chemometric analysis allowed the general behavior of all compounds to be evaluated against all cell lines. Considering the concentration required to inhibit the growth (GI50) of B16-F10 and UACC-62 cells by 50%, hierarchical cluster analysis (HCA) (Fig. 2) showed that divaricatic acid is closest to the doxorubicin standard, followed by protocetraric and perlatolic acids. These three compounds can therefore be considered the most promising for inhibiting growth of both cell lines. Usnic, norstictic, and diffractaic acids defined an intermediate cluster in terms of activity against both B16-F10 and UACC-62 cells, while lichexanthone, psoromic acid, and atranorin were the least active substances tested, delineating a very distinct cluster relative to the doxorubicin standard. LC50 data were employed to construct Fig. 3, in which large loadings in principal component analysis (PCA) correspond to low cytotoxicity. The PC1 axis, comprising 88.97% of explained variance, compares the cytotoxic effects of compounds on melanoma and normal cells, while the PC2 axis, with 10.61% of explained variance, compares the cytotoxicity effects on two melanoma cell lines (B16-F10 and UACC-62) (Fig. 3). Compounds assigned to high values along the PC1 axis in Fig. 3a have no marked cytotoxicity against normal cells (3T3). At low values on the PC1 axis, loadings are subdivided (as seen along PC2) into cytotoxicity against UACC-62 and B16-F10 melanoma cells (Fig. 3b). Therefore, compounds assigned more positive scores on PC2 (doxorubicin and protocetraric acid) were more cytotoxic to B16-F10 cells than other compounds, whereas protocetraric acid was less cytotoxic to normal cells (3T3) than doxorubicin. Compounds assigned more negative scores along the PC2 axis (divaricatic and perlatolic acids) were the most cytotoxic against UACC-62 cells. Divaricatic and perlatolic acids killed fewer normal cells compared to doxorubicin, despite being more cytotoxic than protocetraric acid, as shown by their lower PC1 scores than protocetraric acid and higher PC1 scores than doxorubicin. Although chemometric analysis outlines the general behavior of the compounds against all cell lines tested, individual analyses provide valuable information on the specific action of each compound. UACC-62 cells proved most sensitive to protocetraric acid (GI50 0.52 µg/mL, 1.4 µm), followed by divaricatic acid (2.7 µg/mL, 7.0 µm) and perlatolic acid (3.3 µg/mL, 7.4 µm) (Table 1). Protocetraric acid was as active as doxorubicin. UACC-62 cells exhibited intermediate sensitivity to diffractaic, usnic, norstictic, and psoromic acids (GI50 24.7 µg/ mL, 66.0 µm; 31.5 µg/mL, 91.5 µm; 32.9 µg/mL, 88.4 µm; and 36.6 µg/mL, 102.2 µm, respectively). UACC-62 showed low sensitivity to atranorin. Lichexanthone proved inactive. B16-F10 melanoma cells were highly sensitive to divaricatic acid (GI50 4.4 µg/mL, 11.3 µm) and exhibited intermediate sensitivity to perlatolic, protocetraric, and diffractaic acids (GI50 18.0, 24.0, 25.4 µg/mL, respectively), followed by usnic and norstictic acids. Atranorin, lichexanthone, and psoromic acid were inactive on B16-F10 cells. Only divaricatic and norstictic acids showed SI* values higher than 3.0, being therefore more selective than doxorubicin (SI* 2.2) for B16-F10 melanoma
cells. Based on LC50 values calculated according to Holbeck,24) the majority of compounds exhibited values in the vicinity of 250.0 µg/mL, indicative of their low cytotoxicity. A noteworthy feature, despite the lower cytotoxic activity of all compounds relative to the positive control (doxorubicin), is that some of the compounds showed meaningful SI* values. Above 3.0, SI* values indicate that a given compound is at least three times more cytotoxic to cancer cells (in this case, melanoma) than to normal cells.25) Protocetraric acid had a high SI* of 93.3, while psoromic and norstictic acids had SI* values of 6.8 and >7.6 for UACC-62 cells, respectively. The depsidones protocetraric, psoromic, and norstictic acids are structurally similar in the A ring, and therefore differences in activity on the cells tested are possibly related to differences in the B ring. Among the depsides, divaricatic and perlatolic acids had high SI* values, indicating strong cytotoxicity to UACC-62 human melanoma cells. The structural difference between them lies at the alkyl chains. Divaricatic acid has two n-propyl groups linked at C-6 and C-6′, whereas in perlatolic acid the alkyl chains are n-pentyl groups (Fig. 1). UACC-62 cells exhibited higher sensitivity to perlatolic acid (SI* 7.9) than to divaricatic acid (SI* 5.4). Nevertheless, perlatolic acid was less active (SI* 1.4) than divaricatic acid (SI* 3.3) on B16-F10 cells. These differences in activity, indicated by SI* values, possibly reflect differences in target sites in UACC-62 and B16-F10 melanoma cells and 3T3 normal cells. In summary, the depsidones protocetraric, norstictic, and psoromic acids and the depsides divaricatic and perlatolic acids exhibited stronger activity against UACC-62 melanoma cells and proved more selective against melanoma cells than against 3T3 normal cells. Norstictic and divaricatic acids were the most selective of the compounds evaluated on B16-F10 melanoma cells. Considering that an ideal drug should have potent activity at lower concentrations, a high degree of selectivity, and low cytotoxicity,26) protocetraric acid can be viewed as a potential candidate for the study of melanoma in experimental models.
Experimental
General Procedures Si-gel (Merck, 230–400 mesh) was used in chromatography columns. Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker DPX-300 spectrometer using the solvent as an internal reference. Melting points were determined on a Uniscience Melting Point apparatus without corrections. Optical rotation measurements were recorded on a Perkin Eklmer, model 341, polarimeter at 589 nm. Thin-layer chromatography (TLC) was performed on plates precoated with silica gel 60 F254 (Merck) and spots were visualized by spraying the plates with 10% H2SO4-methanol solution, followed by heating. Lichens Parmotrema dilatatum (Vain.) Hale, Usnea subcavata Motika, and Dirinaria aspera Häsänen, were collected near Piraputanga village in Aquidauana county, Mato Grosso do Sul state, Brazil (20°27′21.2″S, 55°29′00.9″W; altitude approx. 200 m; corticicolous substrate in open forests). Usnea sp., Ramalina sp., Cladina confusa (Sant.) Folmm. & Ahti, and Parmotrema lichexanthonicum Eliasaro & Adler were obtained from home decor stores. The specimens were identified by Dr. Mariana Fleig of the Universidade Federal do Rio Grande do Sul and Dr. Marcelo P. Marcelli of the Botany
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Table 1. GI50, LC50, SI*, and SI** Values for Atranorin, Diffractaic Acid, Divaricatic Acid, Perlatolic Acid, Psoromic Acid, Protocetraric Acid, Norstictic Acid, Usnic Acid, Lichexanthone, and Doxorubicin Tested against UACC-62, B16-F10, and 3T3 Cells UACC-62
Cell lines
GI50
B16-F10 LC50
a)
GI50
LC50 µm
SIb)
>250.0 198.2
>667.8 529.4
>1.0 1.1
3.3
>250.0
>643.7
250.0
>697.8
250.0
>697.8
>1.0
>1.0
24.0
64.0
2.0
190.7
509.5
>1.3
646.0
>1.0
62.4
167.5
>4.0
>250.0
>671.5
>1.0
184.0 >250.0
534.4 >873.3
>1.4 >1.0
47.7 >250.0
137.7 >873.3
0.87 >1.0
>250.0 >250.0
>726.1 >873.3
>1.0 >1.0
17.8
30.7
1.2
3.2
11.7
µm
Atranorin 147.2 Diffractaic 24.7 acid Divaricatic 2.7 acid Perlatolic 3.3 acid Psoromic 36.6 acid Protocetraric 0.52 acid Norstictic 32.9 acid Usnic acid 31.5 Lichexan- >250.0 thone Doxorubicin 0.47
393.3 66.0
>1.7 1.3
>250.0 176.8
>667.8 472.2
>1.0 1.2
>250.0 25.4
>667.8 67.8
>1.0 1.2
7.0
5.4
19.5
50.2
9.2
4.4
11.3
7.4
7.9
27.6
62.1
5.9
18.0
102.2
6.8
>250.0
>697.8
>1.0
1.4
93.3
>250.0
>667.9
88.4
>7.6
240.5
91.5 >873.3
1.3 >1.0 1.2
SI
0.81
SI
µg/mL
0.25
µm
0.43
SI
2.2
µg/mL
1.83
3T3
Cell lines
LC50
GI50 µg/mL
µm
Atranorin >250.0 Diffractaic 31.2 acid Divaricatic 14.5 acid Perlatolic 26.0 acid Psoromic 248.6 acid Protocetraric 48.5 acid Norstictic >250.0 acid Usnic acid 41.4 Lichexan- >250.0 thone Doxorubicin 0.55
>667.8 83.3
>250.0 211.9
>667.8 566.0
37.3
178.4
459.3
58.5
162.2
364.9
693.8
>250.0
>697.8
129.6
>250.0
>667.9
>671.5
>250.0
>671.5
120.2 >873.3
>250.0 >250.0
>726.1 >873.3
21.5
37.0
Compounds
µm
a)
µg/mL
Compounds
µg/mL
b)
0.95
µg/mL
µm
a) SI* was the GI50 value for a compound on 3T3 cells divided by the GI50 value for the compound on a line of cancer cells.25) b) SI** was the LC50 value for a compound on 3T3 cells divide by the LC50 value for the compound on a line of cancer cells.26)
Institute of the Universidade de São Paulo. A voucher specimen of each species has been retained in our laboratory for future reference. Extraction and Isolation of Compounds Extraction of atranorin and protocetraric acid (from P. dilatatum), usnic and diffractaic acids (U. subcavata), lichexanthone (P. lichexanthonicum), and norstictic acid (Ramalina sp.) was conducted according to Honda et al.27) Perlatolic acid (C. confusa) and divaricatic acid (D. aspera) were isolated according to Gianini et al.28) Talli (20–40 g) were powdered and exhaustively extracted with hexane, followed by acetone, at room temperature. The extracts were concentrated in vacuo. The hexane extract from each species was fractionated by silica gel column and eluted with hexane and hexane–ethyl acetate mixtures in increasing polarity to yield atranorin (P. dilatatum), usnic acid (U. subcavata, Usnea sp., and Ramalina sp.), diffractaic acid (U. subcavata), lichexanthone (P. lichexanthonicum),
divaricatic acid (D. aspera), and perlatolic acid (C. confusa). Psoromic, protocetraric, and norstictic acids were isolated from the acetone extracts of Usnea sp., P. dilatatum, and Ramalina sp., respectively. Purification of these compounds was conducted by treating each concentrated extract with a small volume of acetone in an ice bath, followed by centrifugation at 3000 rpm for 5 min. The procedure was repeated until pure compounds were obtained. The structures were confirmed through analysis of 1H, 13C and distortionless enhancement by polarization transfer (DEPT) 135° spectra, and data were in agreement with references.29–35) The degree of purity of all compounds surpassed 95%, as determined by TLC and NMR. 3-Hydroxy- 4-(methoxycarbonyl)-2,5-dimethylphenyl3-formyl-2,4-dihydroxy-6-methylbenzoate (Atranorin): mp 196–198°C. 1H-NMR (CDCl3) δ: 2.09 (3H, s, CH3-9′), 2.52 (3H, s, CH3-8′), 2.67 (3H, s, CH3-8), 3.96 (3H, s, OCH37′), 6.38 (1H, s, H-5), 6.50 (1H, s, H-5′), 10.33 (1H, s, CHO-9),
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11.95 (1H, s, OH-2′), 12.49 (1H, s, OH-2), 12.53 (1H, s, OH-4). 13 C-NMR (CDCl3) δ: 9.3 (C-9′), 24.0 (C-8′), 25.6 (C-8), 52.3 (OCH3), 102.8 (C-1), 108.5 (C-3), 110.2 (C-3′), 112.8 (C-5), 116.0 (C-5′), 116.8 (C-1′), 139.9 (C-6′), 151.9 (C-4′), 152.4 (C-6), 162.9 (C-2′), 167.4 (C-4), 169.1 (C-2), 169.7 (C-7), 172.2 (C-7′), 193.8 (C-9).29) 2,6-Diacetyl-3,7,9-trihydroxy-8,9b-dimethyldibenzo[b,d] furan-1(9bH)-one (Usnic Acid): mp 202–203°C. [α]D +495.5° at 23°C (CHCl3). 1H-NMR (CDCl3) δ: 1.73 (3H, s, CH3-13), 2.07 (1H, s, CH3-16), 2.63 (3H, s, CH3-15), 2.64 (1H, s, CH3-18), 5.95 (1H, s, H-4), 11.00 (1H, s, OH-10), 13.28 (1H, s, OH-8), 18.82 (1H, s, OH-3). 13C-NMR (CDCl3) δ: 7.5 (C-16), 27.9 (C-15), 31.3 (C-18), 32.1 (C-13), 59.0 (C-12), 98.3 (C-4), 101.5 (C-7), 103.9 (C-11), 105.2 (C-2), 109.2 (C-9), 155.2 (C-6), 157.4 (C-10), 163.8 (C-8), 179.3 (C-5), 191.7 (C-3), 198.0 (C-1), 200.3 (C-17), 201.8 (C-14).29,30) 4-[(2,4-Dimethoxy-3,6-dimethylbenzoyl)oxy]-2-hydroxy-3,6dimethylbenzoic Acid (Diffractaic Acid): mp 194–195°C. 1 H-NMR (acetone-d6) δ: 2.05 (3H, s, CH3-9), 2.13 (1H, s, CH3-9′), 2.44 (3H, s, CH3-8), 2.61 (3H, s, CH3-8′), 3.83 (3H, s, OCH3-2), 3.89 (3H, s, OCH3-4), 6.67 (1H, s, H-5′), 6.75 (1H, s, H-5). 13C-NMR (acetone-d6) δ: 9.0 (C-9), 9.3 (C-9′), 19.9 (C-8), 23.9 (C-8′), 56.9 (OCH3-4), 62.3 (OCH3-2), 109.1 (C-5), 110.3 (C-3′), 117.1 (C-5′), 117.4 (C-1′), 117.8 (C-3), 120.9 (C-1), 136.0 (C-6), 141.0 (C-6′), 154.4 (C-4′), 157.8 (C-2), 160.9 (C-4), 164.0 (C-2′), 166.4 (C-7), 174.3 (C-7′).31) 1-Hydroxy-3,6-dimethoxy-8-methyl-9H-xanthen-9-one (Lichexanthone): mp 188–190°C. 1H-NMR (DMSO-d6) δ: 2.80 (3H, s, CH3), 3.84 (3H, s, OCH3-3), 3.86 (3H, s, OCH3-6), 6.26 (1H, d, J=2.3 Hz, H-2), 6.27 (1H, d, J=2.3 Hz, H-4), 6.60 (1H, d, J=2.3 Hz, H-7), 6.62 (1H, d, J=2.3 Hz, H-5), 13.35 (1H, s, OH). 13C-NMR (DMSO-d6) δ: 23.4 (CH3), 55.6 (OCH3-3), 55.7 (OCH3-6), 92.0 (C-4), 96.7 (C-2), 98.4 (C-5), 104.1 (C-9a), 112.9 (C-8a), 115.4 (C-7), 143.4 (C-8), 156.9 (C-4a), 159.4 (C-10a), 163.7 (C-1), 163.75 (C-6), 165.8 (C-3), 182.3 (C-9).32) 2-Hydroxy-4-[(2-hydroxy-4-methoxy-6-propylbenzoyl) oxy]-6-propylbenzoic Acid (Divaricatic Acid): mp 137–138°C. 1 H-NMR (acetone-d6) δ: 0.93–1.00 (6H, m, –CH3-3″, –CH3-3‴), 1.61–1.77 (4H, m, –CH2-2″, –CH2-2‴), 2.93–3.00 (4H, m, –CH2-1″, –CH2-1‴), 3.86 (3H, s, OCH3), 6.41 (1H, d, J=2.5 Hz, H-3), 6.46 (1H, d, J=2.5 Hz, H-5), 6.77 (1H, d, J=2.3 Hz, H-5′), 6.79 (1H, d, J=2.3 Hz, H-3′). 13C-NMR (acetone-d6) δ: 14.4 (C-3″*), 14.5 (C-3‴*), 25.7 (C-2″*), 25.9 (C-2‴*), 38.7 (C-1‴), 39.3 (C-1″), 55.9 (OCH3-4), 99.9 (C-3), 105.4 (C-1), 109.3 (C-3′), 111.2 (C-1′), 111.6 (C-5), 116.6 (C-5′), 148.6 (C-6*), 149.1 (C-6′*), 155.0 (C-4′), 165.2 (C-2′), 165.6 (C-4), 166.4 (C-2), 169.9 (C-7), 173.3 (C-7′).33) * Signals may be interchanged. 2-Hydroxy-4-[(2-hydroxy-4-methoxy-6-pentylbenzoyl) oxy]-6-pentylbenzoic Acid (Perlatolic Acid): mp 106–108°C. 1 H-NMR (CDCl3) δ: 0.83–0.91 (6H, m, H-5″, H-5‴), 1.23–1.62 (12H, m, H-2″, H-2‴, H-3‴, H-3‴, H-4″, H-4‴), 3.82 (3H, s, OCH3), 2.91–3.01 (4H, m, H-1″, H-1‴), 6.36 (2H, H-3, H-5), 6.61 (1H, d, J=2.2 Hz, H-5′), 6.73 (1H, d, J=2.2 Hz, H-3′). 13 C-NMR (CDCl3) δ: 14.0 (C-5″, C-5‴), 22.4 (C-4″*), 22.6 (C-4‴*), 29.7 (C-3″*), 31.3 (C-3‴*), 31.91 (C-2″*), 31.99 (C-2‴*), 36.5 (C-1‴), 37.2 (C-1″), 55.4 (OCH3), 99.0 (C-3), 103.6 (C-1), 108.9 (C-3′), 111.4 (C-5), 116.1 (C-5′), 148.4 (C-6), 149.4 (C-6′), 154.9 (C-4′), 164.9 (C-4), 165.2 (C-2′), 166.5 Signals may be inter(C-2), 169.4 (C-7), 174.9 (C-7′).33) * changed.
4-Formyl-3-hydroxy-8-methoxy-1,9-dimethyl-11-oxo-11Hdibenzo[b,e][1,4]dioxepine-6-carboxylic Acid (Psoromic Acid): mp 264–265°C (dec.). 1H-NMR (DMSO-d6) δ: 2.20 (3H, s, CH3-8′), 2.46 (3H, s, CH3-8), 3.84 (3H, s, OCH3), 6.84 (1H, s, H-5), 7.10 (1H, s, H-1′), 10.46 (1H, s, CHO), 12.15 (1H, s, OH).13C-NMR (DMSO-d6) δ: 9.6 (C-8′), 21.6 (C-8), 56.2 (OCH3), 107.9 (C-1′) 110.9 (C-3), 111.9 (C-1), 117.3 (C-5), 122.8 (C-3′), 123.3 (C-6′), 142.6 (C-5′), 143.4 (C-4′), 152.8 (C-6), 154.7 (C-2′), 161.0 (C-7), 164.2 (C-4), 164.8 (C-2), 166.1 (C-7′), 194.2 (C-9).34) 4-Formyl-3,8-dihydroxy-9-(hydroxymethyl)-1,6-dimethyl11-oxo-11H-dibenzo[b,e][1,4]dioxepine-7-carboxylic Acid (Protocetraric Acid): mp 244-250°C (dec.). 1H-NMR (DMSO-d6): δ: 2.38 (3H, s, CH3-8). 2.42 (3H, s, CH3-8′), 4.60 (2H, s, CH2-9′), 6.83 (1H, s, H-5), 10.57 (1H, s, CHO). 13C-NMR (DMSO-d6) δ: 14.4 (C-8′), 21.4 (C-8), 52.8 (C-9′), 111.8 (C-3), 112.4 (C-1), 116.7 (C-1′), 117.1 (C-5), 118.4 (C-3′), 129.3 (C-6′), 141.9 (C-5′), 144.6 (C-4′), 152.0 (C-6), 154.4 (C-2′), 161.2 (C-7), 163.8 (C-4), 163.9 (C-2), 170.2 (C-7′), 191.7 (C-9).34) 1 , 4 , 10 -T r i h y d r o x y - 5 , 8 - d i m e t h y l -3 ,7 - d i o x o -1 , 3 dihydro-7H-2,6,12-trioxabenzo[5,6]cyclohepta[1,2-e]indene-11carbaldehyde (Norstictic Acid): mp 285–287°C (dec.). 1HNMR (DMSO-d6) δ: 2.20 (3H, s, CH3-9′), 2.45 (3H, s, CH3-8), 6.78 (1H, d, J=7.5 Hz, H-8′), 6.87 (1H, s, H-5), 8.30 (1H, d, J=7.5 Hz, OH-8′), 10.26 (1H, s, OH-2′), 10.45 (1H, s, CHO), 12.06 (1H, s, OH-4). 13C-NMR (DMSO-d6) δ: 9.7 (C-9′), 21.5 (C-8), 95.0 (C-8′), 109.2 (C-1′), 110.7 (C-3), 111.9 (C-1), 117.4 (C-5), 121.0 (C-3′), 135.9 (C-6′), 137.4 (C-5′), 147.9 (C-4′), 152.0 (C-2′), 152.4 (C-6), 160.4 (C-7), 163.6 (C-7′), 164.0 (C-4), 166.2 (C-2), 192.7 (C-9).34,35) Melanoma Cell Assay Evaluation of cytotoxic activity was carried out in cultures of B16-F10 melanoma cells (ATCC-CRL-6322, murine melanoma, donated by Dr. Auro Nomizo of the School of Pharmaceutical Sciences of the Universidade de São Paulo at Ribeirão Preto) UACC-62 cells (human melanoma, donated by Dr. João Ernesto de Carvalho of CPQBA, the Chemical, Biological and Agricultural Pluridisciplinary Research Center of the Universidade Estadual de Campinas), and NIH 3T3 mouse embryonic fibroblast cells (ATCC-CRL 1658, purchased from the Rio de Janeiro Cell Bank). The cells were maintained in complete medium in a CO2 incubator until reaching exponential growth and were divided into aliquots, kept for 24 h at −86°C and then transferred to liquid nitrogen (−196°C) for storage. For the tests, they were rapidly thawed to 37°C and grown in RPMI-1640 medium (Sigma Chemical, St. Louis, MO, U.S.A.) supplemented with 10% v/v fetal bovine serum (FBS) and 50 µg/ mL of gentamicin (Schering–Plough) at 37°C in a 5% CO2 atmosphere. The cells were detached from the culture flasks by adding trypsin solution (0.25%+1 m m ethylenediaminetetraacetic acid (EDTA)) in phosphate buffered saline (PBS) buffer at pH 7.4. Cells were then transferred to conical tubes containing complete culture medium and centrifuged at 1000 rpm for 5 min. Subsequently, culture medium and trypsin were discarded and cells resuspended in complete medium.36) Cytotoxicity Assay The SRB assay was performed as described by Skehan et al.37) After counting, the cells were inoculated in 96-well microtiter plates (10000 cells/well) with a volume of 100 µL per well and incubated for 24 h. Subsequently, 100 µL of compound diluted in dimethyl sulfoxide (DMSO) was added to the appropriate microtiter wells,
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resulting in the required final concentrations of 0.25, 2.5, 25.0, and 250.0 µg/mL, with assays carried out in triplicate. The final content of DMSO was lower than 0.4%. Doxorubicin was used as the positive control at concentrations of 0.025, 0.25, 2.5, and 25.0 µg/mL. The control cells were grown in 100 µL of medium, and a 100 µL volume of medium containing DMSO was added after 24 h. In the blank, the same final concentrations of the compounds were employed without cells. The plates were incubated with the test compounds for 48 h, after which the cells were fixed with 20% TCA (100 µL/ well) at 4°C for 30 min. The supernatant was discarded and the plates washed five times with tap water. The cells were stained for 30 min with 0.1% SRB in 1% acetic acid (50 µL/ well) and subsequently washed four times with 1% acetic acid to remove the unbound dye. The plates were then air-dried and protein-bound dye was solubilized with 10 m m Tris buffer. The plates were then shaken for 10 min and absorbance was read at 540 nm. In order to measure cell density at time zero (the time at which the samples were added), the SRB assay also included an extra plate containing cells alone, which were fixed with TCA prior to addition of a compound. Absorbance measurements—i.e., time zero (T0), growth of control cells (C), and cell growth in the presence of a compound (T)—were thus calculated and cell proliferation (%) was calculated by the following equations:38) 100 ×[(T T0 ) / (C T0 )] , for T T0 100 ×[(T T0 ) / T0 ] , for T T0
From these measurements, a dose–response curve was plotted and nonlinear regression (sigmoidal type, Microcal Origin 6) was employed to calculate two levels of effect—namely, GI50 (the concentration of test drug required to inhibit the growth of a cell line by 50%, relative to untreated control cells) and LC50 (the concentration of test drug that reflects the concentration needed to kill 50% of the cells).24) Selectivity Index Calculation SI values were calculated as follows: SI* was the GI50 value for a compound on 3T3 cells divided by the GI50 value for the compound on a line of cancer cells.25) SI** was the LC50 value for a compound on 3T3 cells divided by the LC50 value for the compound on a line of cancer cells.26) SI was considered of interest when higher than 3.25) Chemometric Treatments PCA and HCA drew on biological activity data. Two chemometric matrices were evaluated: first, GI50 values were interpreted to outline the general behavior of each compound against UACC-62 and B16-F10 melanoma cell lines; finally, LC50 data were evaluated to specify cytotoxicity against normal and melanoma cells. PCA was performed using autoscaled preprocessing (mean-centering and variance scaling), which assigns the same loading to variables. For HCA, matrices were also autoscaled and Euclidean distances among compounds were calculated. The incremental linkage method (a sum-of-squares approach in calculating intercluster distances) was applied to generate clusters.39) Acknowledgments The authors wish to acknowledge Dr. Marcelo P. Marcelli and Dr. Mariana Fleig for the identification of lichens, Dr. Auro Nomizo for donating the B16-F10 cells, and Dr. João Ernesto de Carvalho for providing the UACC-62 cells. L.F.G.B thanks CAPES for the grant awarded. D.S.F. and N.M.O. appreciate the student research fellowships
from PET and PIBIC-CNPq, respectively. This investigation was funded by the Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT).
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