Cytotoxic Cordiaquinones from the Roots of Cordia polycephala

A investigação química das raízes de Cordia polycephala resultou no isolamento de duas novas naftoquinonas terpenoídicas, 6-[10-(12,12-dimetil-13α-(22-metil-21-butenoilóxi)16-metenilciclohexil)etil]-naftaleno-1,4-diona e (6-[10-(12,12-dimetil-13α-(tigloilóxi)-16metenilciclohexil)etil]-naftaleno-1,4-diona, designadas de cordiaquinonas N (1) e O (2), respectivamente. As cordiaquinonas já conhecidas, B (3), L (4) e E (5), foram também isoladas. Suas estruturas foram elucidadas após detalhada análise dos dados de RMN H e C (1D e 2D) e EMAR (espectrometria de massas de alta resolução). Todas as cordiaquinonas (1-5) foram avaliadas contra quatro linhagens de células cancerígenas: HCT-8 (colo), HL-60 (leucemia), MDA-MB-435 (melanoma) e SF295 (glioblastoma), mostrando IC50 na faixa de 1,2 a 11,1 mmol L. As novas cordiaquinonas 1 e 2 foram as mais ativas contra todas as linhagens de câncer, enquanto 3 foi mais seletiva para células leucêmicas (IC50 2,2 μmol L ).


Introduction
][7][8][9][10] These quinoid compounds, known as cordiaquinones, could be considered as chemotaxinomical markers for the Cordia genus.Nevertheless, the occurrence in their producing plants is limited to the roots and rarely to the trunk wood.As part of a search for bioactive compounds from Cordia, the chemical composition of several Cordia species native to the Brazilian flora, including its pharmacological and biological effects have been performed. 11,12Previous investigations have demonstrated that cordiaquinones display anticancer activity, inducing reactive oxygen species generation and apoptosis in cancer cells. 13,14The present work describes the phytochemical analysis of the hexane extract from roots of C. polycephala (Lam.)I. M. Johnston (syn.: Cordia ulmifolia Juss, Cordia wickstroemii Steudel, Varronia polycephala Lam.Varronia paniculata Wakström), a flowering and aromatic shrub native to Brazil, Colombia, French Guiana and Suriname.In addition, the cytotoxic effect of all isolated compounds was also evaluated against four cancer cell lines.and NMR spectral analysis.The IR spectrum exhibited absorption bands for carbon-oxygen (1712 and 1666 cm -1 ) and carbon-carbon (1600 cm -1 ) double bonds.The 13 C NMR spectrum displayed 26 carbon signals, identified through DEPT and HSQC experiments as eight methines (six sp 2 and two sp 3 ), one methylidene, four methylenes, four methyls and nine non-hydrogenated carbons (eight sp 2 and one sp 3 ).The 1 H and 13 C NMR features of 1 (Table 1) suggested that this compound was a 1,4-naphthoquinone, especially due to the presence of the signals at d 185.6 and 185.1. 8,12Comparison of the 1 H and 13 C NMR data of 1 with those reported for cordiaquinone L, 8 showed high similarity, except for the presence of the additional signals at d 166.4,156.9, d 116.5/5.65 (br s), d 27.6/1.89(s) and d 20.4/2.17 (s) in 1, which were assigned to a 3-methyl-2-butenoyl moiety.The position of this group at C-13 was established by the HMBC correlation exhibited between H-13 (d 4.70) and C-20 (d 166.4) (Figure 1), while its equatorial position was deduced from the multiplicity and coupling constant values displayed by H-13 (dd, J 8.5 and 4.5 Hz).The relative stereochemistry of 1 was established by analysis of the NOESY spectrum, which showed NOE correlations among the axial protons H-13, H-11 and Me-18 (Figure 1).Thus, the structure of 1 was established as the 6-[10-(12,12-dimethyl-13α-(22-methyl-21-butenoyloxy)-16-methenylcyclohexyl)ethyl]-naphtalene-1,4-dione).Terpenoid naphthoquinones have been previously isolated from Cordia species and designated as cordiaquinones, an allusion to the genus.Since cordiaquinones A-M were already reported, 8 compound 1 was designated as cordiaquinone N.

Results and Discussion
Compound 2 was also obtained as a yellow resin.The molecular formula (C 26 H 30 O 4 ) determined by HRESIMS (observed m/z 407.2206 [M+H] + ; calc.for C 26 H 31 O 4 , 407.2222), was identical to that of 1.Its IR spectrum exhibited absorption bands for carbon-oxygen (1703 and 1667cm -1 ) and carbon-carbon (1600 cm -1 ) double bonds.Comparative analyses of the 1 H and 13 C NMR data of 2 with those of 1 (Table 1) showed very close structural similarity,    The MTT analysis showed that all compounds (1-5) exhibited cytotoxic activity against tested cancer cell lines (Table 2).Compounds 1 and 2 were the most active among all, with IC 50 values ranging from 1.2 to 3.4 μmol L -1 , whereas for the known cordiaquinones B, E and L, IC 50 ranged from 2.2 to over 15 μmol L -1 .It's worthwhile to mention that cordiaquinone E (3) was selective towards leukemia cells (IC 50 2.2 μmol L -1 ), since it displayed a weak activity to cells from different histological origins (IC 50 > 15 μmol L -1 in MDA-MB-435 and SF-295 cells, and IC 50 of 11.1 μmol L -1 in HCT-8 cells).None of the tested compounds (1-5) showed hemolytic activity in mice erythrocytes (EC 50 > 500 μmol L -1 ), suggesting that the cytotoxicity of cordiaquinones is not related to non-specific membrane damage (Table 2).Among cordiaquinones, one could speculate about the higher activity of compounds 1 and 2 referring to the presence of the extra α-β-conjugated carbonyl of the end tigloyloxy side chains.
Several molecules possessing a quinone moiety show antiproliferative effects on tumor cell growth. 15The cytotoxic activity of quinones is related to inhibition of electron transporters, 16 uncouple of oxidative phosphorylation, 17 ROS generation, 18 protein adducts formation, 19 especially with enzyme SH groups and DNA damage. 15In a previous work,

General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 digital polarimeter.IR (4000 to 650 cm -1 ) spectra were obtained on a Perkin-Elmer 100 FT-IR spectrum.The high resolution electrospray ionization mass spectra (HRESIMS) were acquired using a LCMS-IT-TOF (SHIMADZU) spectrometer. 1H (500 MHz) and 13 C NMR (125 MHz) spectra were performed on a Bruker DRX-500 spectrometer.HPLC analysis was carried out using a UFLC (SHIMADZU) system equipped with a SPD-M20A diode array UV-Vis detector and a Phenomenex LC-Silica column, 5 mm (4.6 × 250 mm).The mobile phase was consisted of hexane/CHCl 3 (65:35 v/v) with a 4.72 mL min -1 flow rate, and the chromatograms were acquired at 247 nm.Column chromatography was carried out on silica gel 60 (70-230 mesh, Vetec or 230-400 mesh, Merck), TLC was performed on precoated silica gel aluminium sheets (kieselgel 60 F 254 , 0.20 mm, Merck).Fractions and pure compounds were monitored by TLC, and the spots were visualized by the color reaction by spraying with a solution of vanillin/perchloric acid/EtOH followed by heating (ca. 100 °C).

Plant material
Roots of Cordia polycephala were collected in August 2009 along the road margins at Pico-Alto, Guaramiranga-CE, Brazil, and identified by Maria Iracema B. Loiola, botanist of the Federal University of Ceará, Brazil.The voucher specimen (IAC # 44.582) has been deposited at the Herbário Prisco Bezerra, at the Departamento de Biologia of the Universidade Federal do Ceará, Brazil.

Inhibition of cancer cells proliferation
The cytotoxicity of all cordiaquinones were tested against the HL-60 (promyelocytic leukemia), HCT-8 (colon), MDA-MB-435 (melanoma) and SF-295 (brain) cell lines.For all experiments, cells were plated in 96-well plates (10 5 cells per well for adherent cells or 0.3 × 10 5 cells per well for suspended cells in 100 μL of medium).After 24 h, all cordiaquinones (0.06-77.0 μmol L -1 ) dissolved in 1% DMSO were added to each well using a high throughput screening system (Biomek 3000-Beckman Coulter, Inc. Fullerton, CA, USA), and the cultures were incubated for 72 h.Doxorubicin (Zodiac) was used as a positive control.Control groups received the same amount of DMSO.Tumor cell growth was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) to a purple formazan product. 20t the end of the incubation, the plates were centrifuged and the medium was replaced with fresh medium (150 μL) containing MTT (0.5 mg mL -1 ).Three hours later, the plates were centrifuged, the MTT formazan product was dissolved in 150 μL DMSO, and the absorbance was measured using a multiplate reader (Spectra Count, Packard, Ontario, Canada).The drug effect was quantified as the percentage of the control absorbance of the reduced dye at 595 nm.

Hemolytic activity
Membrane disruption was performed in 96-well plates.Briefly, each well received 100 μL of a 0.85% NaCl solution containing 10 mmol L -1 CaCl 2 and 100 μL of a 2% suspension of mouse erythrocytes in the same medium.The cordiaquinones were tested at concentrations ranging from 12 to 500 μmol L -1 .Triton X-100 (Isofar, Brazil) at 0.1% (in 0.85% NaCl) was used as a positive control.After incubation for 60 min at room temperature, the plate was centrifuged, the supernatant was removed and the liberated hemoglobin was measured at 540 nm (DTX-880, Beckman Coulter®). 21

Conclusions
From roots of C. polycephala were isolated five terpenoid naphtoquinones, two of which are new.The isolation of additional new terpenoid quinones from Cordia species reinforces the importance of this type of compounds as possible chemomarkers for the genus.All naphthoquinones showed antiproliferative effects, particularly the new compounds (1 and 2).The investigation of plants from Cordia species as a source of anticancer agents could improve the development of new drugs.