A New Cytotoxic Naphthoquinone and Other Chemical Constituents of Sinningia reitzii

Adson S. Silva Magali S. Amorim Mariana M. Fonseca Marcos J. Salvador Eduardo L. de Sá Maria Élida A. Stefanello About the authors

Abstract

Phytochemical study of Sinningia reitzii (Gesneriaceae) led to the isolation of three new naphtoquinones, 6,7-dimethoxydunnione, 7-hydroxy-6-methoxydunnione, and 5-hydroxy-6,7-dimethoxydunnione from the tubers, together with four known compounds, 7-hydroxydunnione, 8-hydroxydunnione, 5-hydroxy-6,7-dimethoxy-α-dunnione, and 8-hydroxydeydrodunnione. Aerial parts furnished five known compounds, 5-hydroxy-6,7-dimethoxydunniol, 6,8-dihydroxy-7-methoxy-2-O-methyldunniol, loureirin B, jacaranone, and methyl 4-hydroxyphenylacetate. Compounds 8-hydroxydunnione, 5-hydroxy-6,7-dimethoxy-α-dunnione, 8-hydroxydeydrodunnione, 5-hydroxy-6,7-dimethoxydunniol and 6,8-dihydroxy-7-methoxy-2-O-methyldunniol had been previously reported in the tubers of S. reitzii. Density functional theory was used to assign the absolute configuration of compounds 6,7-dimethoxydunnione, 7-hydroxy-6-methoxydunnione 5-hydroxy-6,7-dimethoxydunnione, 7-hydroxydunnione, 8-hydroxydunnione, 5-hydroxy-6,7-dimethoxy-α-dunnione. Compounds 6,7-dimethoxydunnione, 8-hydroxydunnione, 5-hydroxy-6,7-dimethoxy-α-dunnione and 8-hydroxydeydrodunnione were evaluated for cytotoxicity against PC3 (prostate), SKMEL 103 (melanoma), and HeLa (cervix) human cancer, and 3T3 fibroblast cell lines, using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Compound 6,7-dimethoxydunnione displays cytotoxic activity against all the tumor cell lines tested (half maximal inhibitory concentration, IC50, 4.47-26.2 µmol L-1), and was inactive against 3T3 fibroblasts (IC50 > 100 µmol L-1). Compounds 8-hydroxydunnione, 5-hydroxy-6,7-dimethoxy-α-dunnione and 8-hydroxydeydrodunnione were inactive against all tested cell lines.

Keywords:
Gesneriaceae; Sinningia reitzii; naphthoquinones; cytotoxic activity


Introduction

The family Gesneriaceae (150 gen / 3000 spp) comprises herbs, lianas and shrubs, distributed in the tropics around the world. Several species are used as ornamental plants due their attractive flowers. Aeschynanthus spp. (“basket plant”, “lipstick plant”), Columnea spp., Nematanthus spp. (both known as “goldfish plant”), Saintpaulia ionantha (“African violet”), and Sinningia speciosa (“gloxinia”) are widely cultivated species. The chemical profile of Gesneriaceae includes flavonoids, terpenoids, phenolic glycosides, phenyl propanoids, quinones and essential oils. The production of caffeoyl phenylethanoid glycosides together with the lack of iridoids is considered characteristic of the family.11 Zaitlin, D.; AoB Plants 2012, pls039, DOI: 10.1093/aobpla/pls039; Verdan, M. H.; Stefanello, M. E. A.; Chem. Biodiversity. 2012, 9, 2701; Lorenzi, H.; Souza, H. M.; Plantas Ornamentais do Brasil: Arbustivas, Herbáceas e Trepadeiras, 3a ed.; Instituto Plantarum: Nova Odessa, Brasil, 2003; Jensen, S. R.; Phytochemistry 1996, 43, 777.
https://doi.org/10.1093/aobpla/pls039...

Gesneriaceae is represented in Brazil by 28 genera and 221 species. Sinningia is a neotropical genus comprising ca. 70 species distributed from Southern Mexico to Northern Argentina. This is the major genus of Gesneriaceae in Brazil, followed by Nematanthus (32 spp) and Besleria (21 spp).22 Ferreira, G. E.; Ferreira, P. M. A.; Chautems, A.; Waechter, J. L.; Flora 2016, 222, 86.,33 Araujo, A. O.; Chautems, A.; Ferreira, A. In Flora do Brasil 2020 em Construção, Jardim Botânico do Rio de Janeiro, available at http://floradobrasil.jbrj.gov.br/reflora/floradobrasil/FB119, accessed in December 2018.
http://floradobrasil.jbrj.gov.br/reflora...

S. reitzii (Hoehne) L. E. Skog is a perennial subshrub, with numerous small tubers, endemic to Brazil, where it is found in the States of São Paulo, Paraná and Santa Catarina.33 Araujo, A. O.; Chautems, A.; Ferreira, A. In Flora do Brasil 2020 em Construção, Jardim Botânico do Rio de Janeiro, available at http://floradobrasil.jbrj.gov.br/reflora/floradobrasil/FB119, accessed in December 2018.
http://floradobrasil.jbrj.gov.br/reflora...
Several Sinningia species, including S. reitzii, are known as “cachimbo” in Santa Catarina.44 Reitz, R.; Sellowia 1959, 11, 9. No uses for S. reitzii were found in traditional medicine. Eight naphthoquinones were previously reported from the tubers of this plant, including one with anti-inflammatory and antinociceptive activities.55 Soares, A. S.; Barbosa, F. L.; Rüdiger, A. L.; Hughes, D. L.; Salvador, M. J.; Zampronio, A. R.; Stefanello, M. E. A.; J. Nat. Prod. 2017, 80, 1837. The isolation of additional compounds from tubers, the first study of aerial parts, and the cytotoxic evaluation of four naphthoquinones are reported herein.

Tubers of S. reitzii yielded three new naphthoquinones (1-3) and four known compounds (4-7), while aerial parts furnished five known compounds (8-12). All isolated compounds were analyzed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, and the data were compared to the literature. Thus, the known compounds were identified as 7-hydroxydunnione (4),66 Inoue, K.; Ueda, S.; Nayeshiro, H.; Inouye, H.; Phytochemistry 1983, 22, 737. 8-hydroxydunnione (5),66 Inoue, K.; Ueda, S.; Nayeshiro, H.; Inouye, H.; Phytochemistry 1983, 22, 737. 5-hydroxy-6,7-dimethoxy-α-dunnione (6),55 Soares, A. S.; Barbosa, F. L.; Rüdiger, A. L.; Hughes, D. L.; Salvador, M. J.; Zampronio, A. R.; Stefanello, M. E. A.; J. Nat. Prod. 2017, 80, 1837. 8-hydroxydehydrodunnione (7),55 Soares, A. S.; Barbosa, F. L.; Rüdiger, A. L.; Hughes, D. L.; Salvador, M. J.; Zampronio, A. R.; Stefanello, M. E. A.; J. Nat. Prod. 2017, 80, 1837. 5-hydroxy-6,7-dimethoxydunniol (8),55 Soares, A. S.; Barbosa, F. L.; Rüdiger, A. L.; Hughes, D. L.; Salvador, M. J.; Zampronio, A. R.; Stefanello, M. E. A.; J. Nat. Prod. 2017, 80, 1837. 6,8-dihydroxy-7-methoxy-2-O-methyldunniol (9),77 Zhong, Y.-J.; Wen, Q.-F.; Li, C.-Y.; Su, X.-H.; Yuan, Z.-P.; Li, Y.-F.; Helv. Chim. Acta 2013, 96, 1750. loureirin B (10),88 Meksuriyen, D.; Cordell, G. A.; J. Nat. Prod. 1988, 51, 1129. jacaranone (11),99 Massaoka, M. H.; Matsuo, A. L.; Figueiredo, C. R.; Farias, C. F.; Girola, N.; Arruda, D. C.; Scutti, J. A. B.; Romoff, P.; Favero, O. A.; Ferreira, M. J. P.; Lago, J. H. G.; Travassos, L. R.; PloS One 2012, e38698. and methyl 4-hydroxyphenylacetate (12)1010 Fleming, P.; O'Shea, D. F.; J. Am. Chem. Soc. 2011, 133, 1698. (Figure 1). Compounds 5-9 had been previously reported in the tubers.55 Soares, A. S.; Barbosa, F. L.; Rüdiger, A. L.; Hughes, D. L.; Salvador, M. J.; Zampronio, A. R.; Stefanello, M. E. A.; J. Nat. Prod. 2017, 80, 1837. The cytotoxic activity of compounds 1 and 5-7 against human tumor cell lines was evaluated.

Figure 1
Chemical structures of the compounds 1-12.

Experimental

General experimental procedures

Optical rotations were measured in CHCl3 on a Rudolph Research polarimeter. The UV spectrum was obtained in MeOH on a Shimadzu UV-2401PC spectrophotometer. Optical density (λ = 570 nm) was measured using a Synergy 2 spectrophotometer (Bio-Tek). One-dimensional (1H 13C) and two-dimensional (gradient selected heteronuclear multiple bond coherence (gHMBC), gradient selected heteronuclear single quantum coherence (gHSQC)) NMR spectra were recorded on Bruker spectrometers (AC 200, Avance 400 and/or Avance 600), observing 1H at 200, 400 or 600 MHz and 13C at 50, 100 or 150 MHz. CDCl3 or CD3OD were used as solvents, and the chemical shifts are given in ppm (ẟ) using tetramethylsilane (TMS) as internal reference, with coupling constants (J) in Hz. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were obtained on a Micromass ESI Q-TOF mass spectrometer. Geometry optimization and density functional theory calculations on the electronic structure of the compounds employed B3LYP functional having Los Alamos ECP as basis set as implemented in Gaussian09 suite program.1111 Hay, P. J.; Wadt, W. R.; J. Chem. Phys. 1985, 82, 270.,1212 Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery Jr., J. A.; Peralta, J. E. Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, J.; Tomasi, S. S.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J.; Gaussian 09, Revision C01; Gaussian Inc., Wallingford, CT, 2016. Theoretical optical activities were calculated after geometry optimization.1313 Pedersen, T. B.; Hansen, A. E.; Chem. Phys. Lett. 1995, 246, 1. High performance liquid chromatography (HPLC) separations were performed in a Waters apparatus equipped with photodiode array (PDA) detector, and a semi-preparative Nucleosil 100-5 C18 column (250 × 10 mm). The mobile phase was acetonitrile:water 80:20 (isocratic elution), applied for 15 min. The flow rate was 2.8 mL min-1 at room temperature. Silica gel (Merck, 230-400 mesh) was used for column chromatographic separations (CC), while precoated silica gel plates (Macherey-Nagel) were used for thin layer chromatography (TLC) and preparative TLC (PTLC). Compounds were visualized by exposure under UV254/366 light and spraying with 5% (v/v) H2SO4 in ethanol solution, followed by heating on a hot plate.

Plant material

Specimens of Sinningia reitzii (Hoehne) L. E. Skog were collected from a natural population in Jundiaí do Sul, Paraná State, Brazil (23o26’45” S, 50o15’33” W) in March 2010. The plant was identified by Clarice B. Poliquesi, who deposited a voucher specimen in the herbarium of Museu Botânico Municipal (MBM 253150). The plants were cultivated in Curitiba, Paraná State, Brazil (25o26’34.85” S, 49o14’22.58” W). Botanical material for phytochemical study was harvested from the cultivated plants in August 2012 (tubers, sample A) and April 2015 (aerial parts and tubers, sample B).

Extraction and isolation

Dried and powdered tubers (sample A, 30.9 g; sample B 46.6 g) were extracted with hexanes (HEX, mixture of isomers), CH2Cl2, EtOAc and EtOH, successively (50 mL of each solvent for 10 g of material, three times each solvent) at room temperature. The solvent was removed at reduced pressure using a rotatory evaporator, yielding the extracts in HEX (A = 0.30 g; B = 0.34 g), CH2Cl2 (A = 0.27 g; B = 0.28 g); EtOAc (A = 0.25 g; B = 0.26 g), and EtOH (A = 1.03 g; B = 1.22 g). Extracts from sample A were kept at -4 °C until fractionation. After TLC analyses, HEX and CH2Cl2 extracts from both samples were pooled, and an aliquot (0.14 g) was reserved. The remaining extract (1.05 g) was submitted to CC (HEX:EtOAc 95:5, 9:1, 8:2 and 0:1) to give 20 fractions (F1-20) after TLC analyses. Compounds 7 (10.2 mg), 6 (10.9 mg), and 5 (13.6 mg) were obtained from F4 (127.7 mg, eluted with HEX:EtOAc 9:1) after PTLC (CH2Cl2:MeOH 99:1). Fraction F16 (44.0 mg, eluted with HEX:EtOAc 8:2) yielded 1 (6.6 mg) after PTLC (HEX:EtOAc 1:1). Fraction F19 (163.1 mg) was submitted to CC (CH2Cl2:EtOAc 95:5, 9:1, 8:2, 7:3 and 0:1) to give eight subfractions (F19.1-8) after TLC analyses. Subfraction F19.2 (12.3 mg, eluted with CH2Cl2:EtOAc 95:5) yielded 1 (1.7 mg) and 3 (6.1 mg) after PTLC (CH2Cl2:EtOAc 95:5). Subfraction F19.3 (eluted with CH2Cl2:EtOAc 95:5) yielded 3 (4.3 mg). Subfraction F19.6 (eluted with CH2Cl2:EtOAc 9:1) yielded 2 (5.2 mg). Subfraction F19.7 (15.9 mg, eluted with CH2Cl2:EtOAc 8:2), yielded 4 (3.7 mg) after preparative TLC (HEX:EtOAc 1:1).

Dried and powdered aerial parts (29.6 g) were extracted with HEX, CH2Cl2, EtOAc and EtOH as was described for the tubers, yielding the respective extracts after solvent removal. The HEX extract (0.162 g) was submitted to CC (HEX:CH2Cl2 8:2, 1:1 and 0:1) to give eight fractions (FH1-8), after TLC analyses. Fraction FH4 (20.3 mg, eluted with HEX:CH2Cl2 1:1) yielded 8 (2.6 mg), after PTLC (CH2Cl2). Fraction FH6 (10.3 mg, eluted with CH2Cl2) yielded 9 (0.8 mg), after PTLC (CH2Cl2:acetone 95:5). The CH2Cl2 extract (0.209 g) was submitted to CC (CH2Cl2:EtOAc 1:0, 99:1, 8:2, 0:1) to give five fractions (FD1-5). Fraction FD2 (27.9 g, eluted with CH2Cl2) yielded 9 (3.2 mg) after repeated PTLC (CH2Cl2). Fraction FD4 (24.9 mg, eluted with CH2Cl2:EtOAC 8:2) yielded 10 (2.1 mg) after PTLC (CH2Cl2:MeOH 99:1). The EtOAC extract (0.240 g) was submitted to CC (CH2Cl2:MeOH 99:1, 95:5, 9:1, 0:1) to give eight fractions (FA1-8). Fraction FA4 (47.4 mg, eluted with CH2Cl2:MeOH 99:1) yielded a mixture (8.5 mg), which was separated by HPLC to give 11 (tR7.8 min, 2.7 mg) and 12 (tR 8.1 min, 3.2 mg).

Cytotoxic activity assay

The cytotoxic activity of compounds 1 and 5-7 was evaluated against the human tumor cell lines PC-3 (prostate), SKMEL 103 (melanoma), HeLa (epitheloid cervix carcinoma), and the non-cancer cell line 3T3 (fibroblast), all from the American Type Culture Collection (ATCC). The assays were done as previously described.1414 Mosmann, T.; J. Immunol. Methods 1983, 16, 55.,1515 Pascoal, A. C. R. F.; Lourenço, C. C.; Sodek, L.; Tamashiro, J. Y.; Franchi Jr., G. C.; Nowill, A. E.; Stefanello, M. E. A.; Salvador, M. J.; J. Essent. Oil Res. 2011, 23, 34. The cells were distributed in 96-well plates (100 µL cells per well), and exposed to various concentrations of the tested compounds (0.25, 2.5, 25.0 and 250 µg mL-1) in dimethyl sulfoxide (DMSO) (0.1%) at 37 °C, with 5% of CO2, for 48 h. The final concentration of DMSO did not affect cell viability. Doxorubicin (0.025, 0.25, 2.5 and 25 µg mL-1) was used as the positive control. The proliferation of tumor cells was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product. At the end of 48 h incubation, the medium was replaced by fresh medium containing 0.5 mg mL-1 of MTT. Three hours later, the formazan product was dissolved in DMSO, and the optical density was measured by spectrophotometry at 570 nm. The experiments were carried at least in triplicate and the concentration needed to achieve 50% inhibition of cell viability (IC50) was calculated in µmol L-1 by nonlinear regression using the GraphPad program.1616 GraphPad Prism, version 5.00; Intuitive Software for Science, San Diego, 2007.

6,7-Dimethoxydunnione (1)

Dark purple solid; experimental [α]D20 -228 (c 0.27, CHCl3), calculated [α]D -268; UV-Vis (MeOH) λmax / nm (log ε) 214 (3.99), 280 (3.95), 332 (3.32); IR (KBr) νmax / cm-1 2922 and 2840 (C-H), 1640 (C=O), 1600, 1560, and 1520 (C=C, aromatic), 1270, 1161 and 1022 (C-O); for 1H and 13C NMR data, see Table 1; HRESIMS m/z, calcd. for C17H19O5 [M + H]+: 303.12299, found: 303.12349.

Table 1
NMR data (CDCl3) for the compounds 1 (600 MHz) and 2 (400 MHz)

7-Hydroxy-6-methoxydunnione (2)

Purple solid; experimental [α]D20 -79 (c 1.0, CHCl3), calculated [α]D -224; UV-Vis (MeOH) λmax / nm (log ε) 211 (4.07), 277 (4.01), 283 (4.02), 316 (3.62); IR (KBr) νmax / cm-1 3539 (OH, free), 3431 (OH, associated), 2916 and 2848 (C-H), 1629 (C=O), 1607, 1576, and 1529 (C-C, aromatic), 1274 (C-O); for 1H and 13C NMR data, see Table 1; HRESIMS m/z, calcd. for C16H17O5 [M + H]+: 289.10808, found: 289.10760.

5-Hydroxy-6,7-dimethoxydunnione (3)

Dark red solid; experimental [α]D20 -146 (c 0.21, CHCl3), calculated [α]D -440; UV-Vis (MeOH) λmax / nm (log ε) 212 (4.30), 267 (4.03), 300 (3.91); IR (KBr) νmax / cm-1 3421 (OH), 2958, 2927 and 2848 (C-H), 1643 (C=O), 1604 and 1585 (C=C, aromatic), 1260 (C-O); for 1H and 13C NMR data, see Table 2; HRESIMS m/z, calcd. for C17H19O6 [M + H]+: 319.11847, found: 319.11816.

Table 2
NMR data (400 MHz, CDCl3) for 3

Results and Discussion

Compound 1 was isolated as dark purple solid. HRESIMS in positive mode displays an ion at m/z 303.12299 [M + H]+, indicating the molecular formula C17H18O5, which is consistent with nine indices of hydrogen deficiency. In the 1H NMR data, signals of two para-coupled aromatic protons, (ẟH 7.07 and 7.55), two singlets of methoxy groups (ẟH 3.97 and 4.01), and the typical signals of a 2,3-dihydro-2,3,3-trimethylfuran group (quartet at ẟH 4.63, two singlets at ẟH 1.25 and 1.44, and a doublet at ẟH 1.47) were observed (Table 1). The 13C NMR data showed 17 peaks, including two of carbonyl groups at ẟC 175.8 and 180.5. These data indicate a 1,2-naphthoquinone type dunnione.66 Inoue, K.; Ueda, S.; Nayeshiro, H.; Inouye, H.; Phytochemistry 1983, 22, 737. Analysis of HSQC and HMBC spectra confirmed the structure 1 through the following cross-peaks: H-5 (ẟH 7.07) with carbons at ẟC 125.0 (C-9), 151.5 (C-7), and 168.2 (C-4); H-8 (ẟH 7.55) with carbons at ẟC 122.5 (C-10), 153.9 (C-6), and 180.5 (C-1); and H-14 (ẟH 1.25) with carbons at ẟC 25.8 (C-15), 44.1 (C-11), 92.9 (C-12), and 121.4 (C-3) (Table 1). Therefore, compound 1 was identified as 6,7-dimethoxydunnione (Figure 1).

Compound 2 was isolated as purple solid and has the molecular formula C16H16O5, corresponding to nine indices of hydrogen deficiency, which was deduced from an ion at m/z 289.10808 [M + H]+ in HRESIMS. The 1H NMR data of 2 (Table 1) were very similar to those of compound 1, showing signals for two aromatic protons in para relationship (ẟH 7.07 and 7.59), one singlet of a methoxy group (ẟH 4.03), and the signals for a 2,3-dihydro-2,3,3-trimethylfuran group. In the HMBC spectrum the methoxy group (ẟH 4.03) and H-8 (ẟH 7.59) showed cross-peaks with a carbon at ẟC 151.1, which must be C-6. These and remaining correlations in the HSQC and HMBC spectra (Table 1) led to identification of 2 as 7-hydroxy-6-methoxydunnione (Figure 1).

Compound 3, a dark red solid, had the molecular formula C17H18O6, with nine indices of hydrogen deficiency, as deduced from an ion at m/z 319.1184 [M + H]+ in the HRESIMS. The 1H NMR data (Table 2) showed signals for one hydroxy group (ẟH 7.68), one aromatic proton (ẟH 7.36), two methoxy groups (ẟH 3.96 and 3.97), and the signals for a 2,3-dihydro-2,3,3-trimethylfuran group. The 13C NMR spectrum showed 17 signals, including three that were typical of C-1 (ẟC 180.5), C-2 (ẟC 175.3), and C-4 (ẟC 168.1) in the dunnione framework.66 Inoue, K.; Ueda, S.; Nayeshiro, H.; Inouye, H.; Phytochemistry 1983, 22, 737. Analysis of HSQC and HMBC data (Table 2) led to the structure 3 (Figure 1). The aromatic proton (ẟH 7.36) was located in C-8 due to cross-peaks of this signal in the HMBC spectrum with C-1 (ẟC 180.5), C-6 (ẟC 142.3), and C-10 (ẟC 106.9). On the other hand, the position of hydroxy group in C-5 was determined from cross-peaks of the signal at ẟH 7.69 with C-5 (ẟC 148.9), C-6 and C-10. Therefore, compound 3 was identified as 5-hydroxy-6,7-dimethoxydunnione.

In order to determine the absolute configuration of compounds 1-6, the optical rotations were calculated for the isomers using density functional theory (DFT). The results indicate that S isomers are levorotatory, in agreement with previous X-ray monocrystal diffraction studies with a dunnione derivative.1717 Cooke, R. G.; Ghisalberti, E. L.; Johnson, B. L.; Raston, C. L.; Skelton, B. W.; White, A. H.; Aust. J. Chem. 2006, 59, 925. As the experimental optical rotations of compounds 1-6 were negative, their absolute configuration was assigned as 12S (Figure 1). Chiral natural products are generally biosynthesized as one pure enantiomer. However, naphthoquinones have been isolated with various optical purity degree. Compound 4 was previously isolated optically pure (optical rotation +350), while 5 was obtained only as racemic mixture.66 Inoue, K.; Ueda, S.; Nayeshiro, H.; Inouye, H.; Phytochemistry 1983, 22, 737. In the present work, compound 4 showed [α]D -23, indicating a mixture of enantiomers, with predominance of the levorotatory isomer (53:47). The optical purity of the remaining compounds was not experimentally determined. The high optical rotation of 1 ([α]D -228), near from value calculated by DFT ([α]D -268) suggest that it is enantiomerically pure. On the other hand, the optical rotations of 2 ([α]D -79), 3 ([α]D -146), 5 ([α]D -78), and 6 ([α]D -13) were relatively low in comparison with calculated values ([α]D > -200), suggesting that these compounds were isolated as scalemic mixtures.

Considering that naphthoquinones frequently display cytotoxic activity,1818 Babula, P.; Adams, V.; Havel, L.; Kizek, R.; Curr. Pharm. Anal.. 2009, 5, 47; López, J.; Cruz, F.; Alcaraz, Y.; Delgado, F.; Vásquez, M. A.; Med. Chem. Res. 2015, 24, 3599. compounds 1 and 5-7, which were obtained in sufficient quantity and purity, were assayed in vitro against three human tumor cell lines (PC3, SKMel 103 and HeLa) and the 3T3 (fibroblast) cell line. Compound 1 exhibited strong cytotoxicity against HeLa and PC3 cell lines (IC50 < 10 µmol L-1), and a smaller activity against SKMEL 103 (IC50 26.2 µmol L-1). Compound 1 was more active against HeLa cells (IC50 4.47 µmol L-1) than the standard drug doxorubicin (IC50 14.2 µmol L-1); it was also less toxic towards the non-cancer cell line 3T3 (IC50 157 µmol L-1) in comparison to doxorubicin (IC50 27.9 µmol L-1). Compounds 5-7 were considered inactive against all tested cells since they showed IC50 > 50 µmol L-1 (Table 3). Strong cytotoxic activity had previously been reported for a racemic mixture of 5 against four tumor cell lines (average growth inhibition of 50%, GI50 0.54-5.2 µmol L-1), including HeLa cells (GI50 1.9 µmol L-1), at roughly the same compound concentrations.1919 Duchowicz, P. R.; Bennardi, D. O.; Bacelo, D. E.; Bonifazi, E. L.; Rios-Luci, C.; Padrón, J. M.; Burton, G.; Misico, R. I.; Eur. J. Med. Chem.. 2014, 77, 176; Sheridan, H.; Nestor, C.; O'Driscoll, L.; Hooke, I.; J. Nat. Prod. 2011, 74, 82. This striking difference in cytotoxicity between the two studies may be a consequence of the different methods employed for estimating cell viability. The cytotoxicity of other α-dunnione derivatives (similar, but not identical, to compound 6) has also been evaluated previously, but no clear pattern of structure-activity relationship was found.1919 Duchowicz, P. R.; Bennardi, D. O.; Bacelo, D. E.; Bonifazi, E. L.; Rios-Luci, C.; Padrón, J. M.; Burton, G.; Misico, R. I.; Eur. J. Med. Chem.. 2014, 77, 176; Sheridan, H.; Nestor, C.; O'Driscoll, L.; Hooke, I.; J. Nat. Prod. 2011, 74, 82. Anti-inflammatory and antinociceptive activities were previously reported for compound 7.55 Soares, A. S.; Barbosa, F. L.; Rüdiger, A. L.; Hughes, D. L.; Salvador, M. J.; Zampronio, A. R.; Stefanello, M. E. A.; J. Nat. Prod. 2017, 80, 1837. The small number of assayed compounds does not allow generalizations about structure-activity relationship; however, a comparison of the structures of the active compound (1) to the inactive compounds (5-7) suggests that the absence of substituents in C-5 and C-8 may be important for activity.

Table 3
Cytotoxic activity of compounds 1 and 5-7

Conclusions

Less polar extracts of S. reitzii are characterized by the presence of several prenylated naphthoquinones, mainly dunnione-type. This compound class has been previously reported in other Sinningia species, such as S. allagophylla,2020 Scharf, D. R.; Verdan, M. H.; Ribeiro, M. A.; Simionatto, E. L.; Sá, E. L.; Salvador, M. J.; Barison, A.; Stefanello, M. E. A.; J. Nat. Prod. 2016, 79, 792.S. canescens,2121 Verdan, M. H.; Scharf, D. R.; Barison, A.; Salvador, M. J.; Stefanello, M. E. A.; Phytochem. Lett. 2017, 22, 205.S. leucotricha,2121 Verdan, M. H.; Scharf, D. R.; Barison, A.; Salvador, M. J.; Stefanello, M. E. A.; Phytochem. Lett. 2017, 22, 205. and S. hatschbachii,2222 Amorim, M. S.; Serain, A. F.; Salvador, M. J.; Stefanello, M. E. A.; Nat. Prod. Commun. 2017, 12, 1763. and can be considered as typical of Sinningia. Many biological activities are associated with naphthoquinones, including antibacterial, cytotoxic, antitumoral, insecticidal, and anti-inflammatory.1818 Babula, P.; Adams, V.; Havel, L.; Kizek, R.; Curr. Pharm. Anal.. 2009, 5, 47; López, J.; Cruz, F.; Alcaraz, Y.; Delgado, F.; Vásquez, M. A.; Med. Chem. Res. 2015, 24, 3599. Accordingly, a new naphthoquinone, reported in this work, displays selective cytotoxic activity against three human tumor cell lines.

Supplementary Information

Supplementary information (spectra of compounds 1-12, and data of known compounds 4-12) is available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

We are grateful to CAPES (Finance Code 001), FAEPEX-UNICAMP, FAPESP (processes: 15/03726-8 and 17/03598-5), and CNPq for scholarships and financial support, and to Clarisse B. Poliquesi, from Museu Botânico Municipal de Curitiba, for the collection and identification of the plant material.

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Publication Dates

  • Publication in this collection
    21 Oct 2019
  • Date of issue
    Oct 2019

History

  • Received
    9 Dec 2018
  • Accepted
    4 Apr 2019
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