An Uncommon Naphthaquinoid and a New Lignan Derivative from the Roots of Cordia leucocephala Moric

Two uncommon compounds, a meroterpene naphthoquinoid (cordiaquinone P) and a new lignan derivative, were isolated from the hexane extract of roots of Cordia leucocephala Moric and their structures, including the relative stereochemistry, were determined by nuclear magnetic resonance (NMR) (H and C NMR, HSQC, HMBC and NOESY) and high resolution electrospray ionization mass spectrometry (HRESIMS) techniques.


Introduction
Cordia comprises one of the major and most important genus of the Boraginaceae family.For this genus are recognized ca.300 species, many of which are used in traditional medicine. 1It is known as a prolific source of terpenoids, phenolic compounds and saponins, [2][3][4][5][6] however, it is famous for its ability to biosynthesize meroterpenoid benzoquinone and naphthoquinones, including their reduced forms. 7,8][10][11] Interestingly, the occurrence of such secondary metabolites seems to be restrict to the roots.Previous work with the roots of C. polycephala resulted in the isolation of the new cordiaquinones P and Q, besides the known cordiaquinone J, whose cytotoxic properties were evaluated. 12Our efforts to find anticancer compounds led us to continue the chemical investigation of the EtOH extract from roots of C. polycephala.In the present work, it is described the isolation and characterization of two further new compounds, a naphthoquinoid and a lignan derivative (Figure 1).

Results and Discussion
The new compounds 1 and 2 were isolated from the root EtOH extract of C. leucocephala and their structures were unequivocally elucidated by spectrometric methods, including 1D and 2D nuclear magnetic resonance (NMR) experiments and high resolution electrospray ionization mass spectrometry (HRESIMS).
The combination of 13 C NMR, DEPT and HSQC spectra were consistent with the presence of one methylene, sixteen methines and ten nonhydrogenated carbons (Table 1).A detailed NMR data analysis allowed to identify two aromatic rings, three carboxyl carbons were easily identified, as well as an a, b, γ, d-conjugated carboxylic acid system, Table 1 and Figure 2. Comparison of the NMR data of 2 with those reported for the bicyclic [2.2.2] octane lignin (rufescenolide), an adduct of two caffeic acid molecules, previously isolated of C. rufescens, 13 revealed a close similarity among them, differing only by the substitution of the hydroxyl proton at C-4" of the bicyclic [2.2.2] octane lignan for another caffeic acid moiety, as well as by the presence of a carboxylic acid function instead the methyl ester.The COSY and HMBC contour map supported the structure of the new lignan derivative after the esterification with 2-hydroxy-dihydrocaffeic acid (Figure 2).The relative stereochemistry suggested for 2 was established by the NOESY analysis (Figure 3), which showed correlations between Ha-7' (d 3.14) and Ha-4'' (d 3.99, d, J 3.2 Hz) allowing to set the aromatic ring at C-7' and the 2-hydroxy-dihydrocaffeic acid moiety at C-4'', both b-positioned.This proposition also was supported by the NOE correlations observed for Hb-8' (d 2.67) with H-2' (d 6.42, d, J 1.6 Hz) and H-6' (d 6.33, dd, J 8.1, 1.6 Hz).
A speculative biogenetic route for the formation of 2, involving three caffeic acid units and common enzymatic reactions, such as reduction, oxidation, hydrogenation and cyclization, is suggested (Figure 4).

Conclusions
From C. leucocephala were isolated two new compounds, a meroterpene naphthoquinoid which was designated as cordiaquinone P, and a lignan trimmer-like of caffeic acid, the acid form of rufescenolide esterified with a 2-hydroxy-dihydrocaffeic acid moiety at C-4''.Based on the current results, rufescenolide, previously isolated from C. rufescens, could be speculated as being an artifact, since the solvent used during the extraction process was methanol. 13The cytotoxicity of both compounds 1 and 2 have being assayed against a panel of tumor cells, unfortunately, neither one showed any cytotoxic activity.

General methods
The optical rotations were measured on a Perkin-Elmer 341 digital polarimeter.The HRESIMS were acquired using a LCMS-IT-TOF (SHIMADZU) spectrometer.IR spectra (KBr pellets) were recorded using a Perkin-Elmer FT-IR 1000 spectrometer.All NMR experiments were recorded on a Bruker DRX-500 spectrometer operating at 500 and 125 MHz for 1 H and 13 C, respectively.The high performance liquid chromatography (HPLC) analysis was carried out using a Class LC-10 (SHIMADZU) system equipped with a SPD-M10Avp diode array UV-Vis detector, two LC10ATvp pumps and LC-Si column (250 mm × 10 mm i.d.× 5 µm) from Supelco.Chromatography columns were carried out on silica gel 60 (Merck) and Sephadex LH-20 (Pharmacia), while thin layer chromatography (TLC) was performed on  precoated silica gel polyester sheets (kieselgel 60 F254, 0.20 mm, Merck).The fractions and pure compounds were monitored by spraying with vanillin/perchloric acid/EtOH solution followed by heating at 105 °C.

Plant material
C. leucocephala, collected in May 2006, was harvested at Mossoró County, Rio Grande do Norte State, Brazil, and identified by the botanist Odaci Fernandes de Oliveira.A voucher specimen (MOSS 8827) has been deposited at the Herbarium Dárdano de Andrade Lima of the Universidade Federal Rural do Semi-Árido (UFERSA), Rio Grande do Norte, Brazil.

Extraction and isolation
Dried and powdered roots of C. leucocephala (650.0 g) were extracted, at room temperature, with hexane followed by a 70% aqueous ethanol solution to yield 9.3 and 57.0 g, respectively, of crude extracts.The crude hydroalcoholic extract was dissolved in H 2 O (700 mL) and partitioned with CHCl 3 , EtOAc and n-BuOH (3 × 200 mL, each), to yield, after the solvent evaporation, 28.5, 2.71 and 7.26 g, respectively.The CHCl 3 fraction (28.5 g) was subjected to chromatography column (CC) over silica gel, by elution with binary mixtures of CHCl 3 /MeOH of increasing degree of polarities to yield 57 fractions (20 mL each), which after TLC analysis were pooled together to six fractions (F1A to F6A).The F3A (170.2 mg) was subjected to a Sephadex LH-20 column, using MeOH as eluent, to give 19 subfractions of 8 mL, that after TLC analysis were grouped to 5 subfractions (F1B to F5B).The F3B (80.0 mg), was purified by semipreparative HPLC analysis using H 2 O (0.1% TFA)/ACN (42:58; v/v) at a flow rate of 1.9 mL min −1 to yield compound 1 (67.0 mg, t R = 16.2 min).The EtOAc fraction (2.71 g) was fractionated by silica gel CC, using as eluent a ternary mixture of AcOEt/MeOH/H 2 O (70:22:08 v/v), to give 29 fractions (8 mL).TLC analysis showed a major spot for fractions 11-14 (687.3 mg) that were pooled together.The final fraction was subjected to a Sephadex LH-20 column, followed by semipreparative HPLC using H 2 O (0.1% formic acid)/ACN (75:25; v/v) at flow rate of 2.9 mL min -1 to yield compound 2 (70.2 mg, t R = 22.1 min).