A New β-Lapachone Derivative from Distictella elongata ( Vahl ) Urb

O presente trabalho descreve a elucidação estrutural de uma nova β-lapachona tipo naftoquinona, isolada a partir das raízes de Distictella elongata. A nova substância, apresentando fórmula molecular C 16 H 16 O 6 , foi identificada como sendo 4,7-diidróxi-10-metóxi-2,2-dimetil3,4-diidro-2H-benzo[h]cromeno-5,6-diona, de acordo com os dados obtidos por meio de análises espectrométricas. A elucidação estrutural foi realizada utilizando-se as técnicas espectrométricas [HRESIMS, 1D RMN (H e C) e 2D RMN (g-DQF-COSY, g-HMQC e g-HMBC]. O extrato clorofórmico das raízes de D. elongata demonstrou significante inibição no crescimento de células do tipo SK-MEL (melanoma) e SK-OV-3 (carcinoma de ovário), com valores de IC 50 de 40 μg mL e 56 μg mL, respectivamente. Entretanto, a naftoquinona não foi responsável pela atividade citotóxica exibida pelo extrato.


Introduction
3][4][5][6] The Bignoniaceae family, represented by more than 100 genera and about 800 species, including Distictella elongata, is mainly tropical, with most accessions dispersed in tropical America. 7This species occurs in Cerrado areas, within the states of Goiás, São Paulo and Minas Gerais. 8The Cerrado is a highly endangered ecosystem, due to the intensive introduction of soybean farming and cattle ranching in this area over the last two or three decades.It represents the second largest biome in South America after the rain forests and originally covered one fourth of Brazil.No phytochemical or pharmacological investigation has yet been carried out on D. elongata.

Results and Discussion
Fractionation of chloroform root extract from D. elongata resulted in the isolation of a new naphthoquinone (1).Compound 1 was obtained as reddish oil.The molecular formula of 1 was determined as C 16 H 16 O 6 by HRESIMS, which exhibited ions at m/z 305.1031 [M+H] + and 327.0846 [M+Na] + (in the positive-ion mode).
The 1 H NMR (CDCl 3 ) spectrum of 1 exhibited a deshielded hydrogen at d 12.7 (s, O-H), two aromatic hydrogens overlapping at d 7.29, an O-methyl at d 3.97 (s, 3H), as well as a triplet at d 4.96 (t, J 6.0 Hz).In addition, the 1 H NMR spectrum showed two tertiary methyl hydrogens at d 1.45 and d 1.55, and a gem-methylene signals at d 2.05 and d 2.12 (dd, J 6.0 and 14.2 Hz, 2H).The 13 C NMR spectrum displayed 16 signals including six aromatic, two olefinic and two carbonyl signals.Total 6 double bonds, attributable to aforementioned carbon signals, when considered with 9 unsaturation degrees, the remaining 3 degrees required that compound 1 consists of tri-ring systems.[11] Two overlapping aromatic hydrogens (7.29, 2H) did not provide any information about substitution pattern of the aromatic ring.Thus, the 1 H NMR spectrum of compound 1 was obtained using acetone-d 6 as a solvent, in which aromatic hydrogens were observed as doublets at d 7.52 (d, 9.0 Hz, 1H), and d 7.30 (d, 9.0 Hz, 1H), indicative of the presence of ortho-coupling hydrogens.
The combined use of g-DQF-COSY, g-HMQC and g-HMBC techniques permitted the complete assignment of the pyranonaphthoquinone skeleton and the substitution pattern for the compound 1.Thus, the deshielded 1 H NMR signal at d 12.7 [C-7(OH)] showed correlations with a methine carbon at d 127.9 and two quaternary carbons at d 156.6 and d 114.1, attributed to C-8, C-7 and C-6a, respectively.Also, a methine carbon at d 127.9 (C-8) showed correlation with the aromatic hydrogen at d 7.30 (d, 9.0 Hz, in acetone-d 6 ), which is assigned to H-8.The aromatic hydrogen observed at d 7.52 (in acetone-d 6 , H-9) indicated a long-range correlation in the G-HMBC spectrum with a quaternary carbon at d 117.5, attributed to C-10a.The carbon signal at d 155.1 displayed a correlation with O-methyl hydrogen (d 3.97, s) in the g-HMBC spectrum, hence allowing it to be assigned unambiguously to C-10, and locating the methoxyl group to C-10.Moreover, the position of the hydroxyl group located on pyrane ring was deduced from g-HMBC correlations from quaternary carbons C-10b (155.9) and C-2 (80.4) to a methine proton at d 4.96 (t, J 6.0 Hz) attributed to H-4.
Based on these results, the structure of 1, a new natural product, was established as 4,7-dihydroxy-10-methoxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6dione.Having established the structure of 1, the relative stereochemistry of chiral center (C-4) remained to be solved.The absolute stereochemistry of 1 was established by NMR data and molecular modeling analysis after calculation of the minimal conformational energy for each of the two possible enantiomers using Accelrys Discovery Studio v2.0.0.7264.Only the stereoisomer, (S)-4-hydroxy  isomer of 1 showed agreement with the coupling constants observed in the 1 H NMR spectrum and the dihedral angles measured after the calculation of the minimal conformation energy for each of the two possible stereoisomers.The key coupling constant analyzed was that measured between H3a-H4 = H3b-H4 = 6.0 Hz.Therefore, the dihedral angles H3a-C3-C4-H4 and H3b-C3-C4-H4 must be identical.Whereas, the dihedral angles for the R-isomer should be non identical and should have at least two different coupling constants.When a comparison was made of the current analysis data with reported spectral data of similar systems, 12,13 we concluded that the absolute stereochemistry at C-4 position must be in S configuration.

Experimental
General NMR spectra were recorded on a Bruker  Avance DRX 500 FT spectrometer operating at 500 and 125 MHz for 1 H and 13 C NMR, respectively.The chemical shift values are reported as parts per million (ppm) units relative to tetramethylsilane (TMS); and the coupling constants are in Hz (in parentheses).For the 13 C NMR spectra, multiplicities were determined by a distortionless enhancement by polarization transfer (DEPT) experiment.HRESIMS (High Resolution Electrospray Ionization Mass Spectrometry) were obtained using a Bruker BioApex FT-MS in ESI mode.For TLC, precoated Si 250F layers (Baker) were used.Column chromatography was performed on silica gel 230-400 mesh (Merck).

Plant material
Roots of Distictella elongata (Bignoniaceae) were collected from preserved areas of a Brazilian Cerrado, at the Biological Reserve of Mogi-Guaçu Ecological and Experimental Station, São Paulo, Brazil.A voucher specimen was deposited in the herbarium of the University of Ribeirão Preto (HPM-482).The roots were dried at 50 ºC, powdered and kept until ready for extraction.

Extraction and isolation
Powdered roots (330 g) were extracted in CHCl 3 for 7 days.The CHCl 3 extract was concentrated to a small volume at reduced pressure to yield 1.3 g of oily residue.This residue was fractionated on a silica gel column eluting with hexane, and hexane/ethyl acetate gradient elution to ethyl acetate 100%.Using 50% hexane/ethyl acetate solvent a red crystal was obtained (naphthoquinone) and further purified by preparative TLC using ethyl acetate/hexane (4:1) as solvent system.The amount of 30 mg of the naphthoquinone was removed from the layer.The final purification performed on Sephadex column (LH-20, 30 mg) eluted with MeOH, to afford 9 mg naphthoquinone 1.

Biological activity
The in vitro anticancer activity was tested against a panel of four human cancer cell lines that included SK-MEL (malignant, melanoma), KB (epidermal carcinoma, oral), BT-549 (ductal carcinoma, breast), and SK-OV-3 (ovary carcinoma). 14All the cell lines were from ATCC (Manassas, VA).The cells were cultured in 75 cm 2 culture flasks in RPMI-1640 medium (Gibco TM , Invitrogen Corp.) supplemented with bovine calf serum (10%) and amikacin (60 mg L -1 ), at 37 o C, 95% humidity, 5% CO 2 using standard cell culture techniques.The assay was performed in 96-well microplates.Cells were seeded to the wells of the plate at a density of 25,000 cells/ well and grown for 24 hours at 37 o C. Samples were added to the cells and again incubated for 48 h.The number of viable cells was determined according to Neutral Red assay procedure. 15IC 50 (the concentration of the test sample that caused a growth inhibition of 50% after 48 h exposure of the cells) was calculated from the dose curves generated by plotting percent growth versus the test concentration on a logarithmic scale using Microsoft Excel®.

Table 1 .
In vitro anticancer activity of Distictella elongata root extract and compound 1