A New Cytotoxic Naphthoquinone and Other Chemical Constituents of Sinningia reitzii

Phytochemical study of Sinningia reitzii (Gesneriaceae) led to the isolation of three new naphtoquinones, 6,7-dimethoxydunnione, 7-hydroxy-6-methoxydunnione, and 5-hydroxy6,7-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-methoxy2-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-hydroxy6,7-dimethoxy-α-dunnione. Compounds 6,7-dimethoxydunnione, 8-hydroxydunnione, 5-hydroxy6,7-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), and was inactive against 3T3 fibroblasts (IC50 > 100 μmol L). Compounds 8-hydroxydunnione, 5-hydroxy-6,7-dimethoxyα-dunnione and 8-hydroxydeydrodunnione were inactive against all tested cell lines.


General experimental procedures
Optical rotations were measured in CHCl 3 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 ( 1 H, 13 C) 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 1 H at 200, 400 or 600 MHz and 13 C at 50, 100 or 150 MHz.CDCl 3 or CD 3 OD were used as solvents, and the chemical shifts are given in ppm (d) 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. 11,12Theoretical optical activities were calculated after geometry optimization. 13High performance liquid chromatography (HPLC) separations were performed in a Waters apparatus equipped with photodiode array (PDA) detector, and a semi-preparative Nucleosil 100-5 C 18 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 UV 254/366 light and spraying with 5% (v/v) H 2 SO 4 in ethanol solution, followed by heating on a hot plate.

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. 14,15he 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 CO 2 , 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 (IC 50 ) was calculated in µmol L -1 by nonlinear regression using the GraphPad program. 16

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 C 17 H 18 O 5 , which is consistent with nine indices of hydrogen deficiency.In the 1 1).Therefore, compound 1 was identified as 6,7-dimethoxydunnione (Figure 1).
Compound 2 was isolated as purple solid and has the molecular formula C 16 H 16 O 5 , corresponding to nine indices of hydrogen deficiency, which was deduced from an ion at m/z 289.10808 [M + H] + in HRESIMS.The   6 Analysis of HSQC and HMBC data (Table 2) led to the structure 3 (Figure 1).The aromatic proton (d H 7.36) was located in C-8 due to cross-peaks of this signal in the HMBC spectrum with C-1 (d C 180.5), C-6 (d C 142.3), and C-10 (d C 106.9).On the other hand, the position of hydroxy group in C-5 was determined from cross-peaks of the signal at d H 7.69 with C-5 (d C 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. 17As 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. 6 Considering that naphthoquinones frequently display cytotoxic activity, 18 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 (IC 50 < 10 µmol L -1 ), and a smaller activity against SKMEL 103 (IC 50 26.2µmol L -1 ).Compound 1 was more active against HeLa cells (IC 50 4.47 µmol L -1 ) than the standard drug doxorubicin (IC 50 14.2 µmol L -1 ); it was also less toxic towards the non-cancer cell line 3T3 (IC 50 157 µmol L -1 ) in comparison to doxorubicin (IC 50 27.9µmol L -1 ).Compounds 5-7 were considered inactive against all tested cells since they showed IC 50 > 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%, GI 50 0.54-5.2µmol L -1 ), including HeLa cells (GI 50 1.9 µmol L -1 ), at roughly the same compound concentrations. 19This 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 structureactivity relationship was found. 19Anti-inflammatory and antinociceptive activities were previously reported for compound 7. 5 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.

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, 20 S. canescens, 21 S. leucotricha, 21 and S. hatschbachii, 22 and can be considered as typical of Sinningia.Many biological activities are associated with naphthoquinones, including antibacterial, cytotoxic, antitumoral, insecticidal, and anti-inflammatory. 18Accordingly, a new naphthoquinone, reported in this work, displays selective cytotoxic activity against three human tumor cell lines.

13 C
NMR spectrum showed 17 signals, including three that were typical of C-1 (d C 180.5), C-2 (d C 175.3), and C-4 (d C 168.1) in the dunnione framework.
6 NMR data, signals of two para-coupled aromatic protons, (d H 7.07 and 7.55), two singlets of methoxy groups (d H 3.97 and 4.01), and the typical signals of a 2,3-dihydro-2,3,3-trimethylfuran group (quartet at d H 4.63, two singlets at d H 1.25 and 1.44, and a doublet at d H 1.47) were observed (Table1).The 13 C NMR data showed 17 peaks, including two of carbonyl groups at d C 175.8 and 180.5.These data indicate a 1,2-naphthoquinone type dunnione.6Analysis of HSQC and HMBC spectra confirmed the structure 1 H NMR data of 2 (Table 1) were very similar to those of compound 1, showing signals for two aromatic protons in para relationship (d H 7.07 and 7.59), one singlet of a methoxy group (d H 4.03), and the signals for a 2,3-dihydro-2,3,3-trimethylfuran group.In the HMBC spectrum the methoxy group (d H 4.03) and H-8 (d H 7.59) showed cross-peaks with a carbon at d C 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 C 17 H 18 O 6 , with nine indices of hydrogen deficiency, as deduced from an ion at m/z 319.1184 [M + H] + in the HRESIMS.The 1 H NMR data (Table 2) showed signals for one hydroxy group (d H 7.68), one aromatic proton (d H 7.36), two methoxy groups (d H 3.96 and 3.97), and the signals for a 2,3-dihydro-2,3,3-trimethylfuran group.The 1

Table 3 .
Cytotoxic activity of compounds 1
a Doxorubicin was used as positive control.