Two Polycyclic Geranylhydroquinone-Derived Metabolites from Roots of Arnebia hispidissima (Lehm.) DC.

A phytochemical investigation of the least polar organic extracts of Arnebia hispidissima (Lehm.) DC. roots has led to the isolation of two unique polycyclic geranylhydroquinone-derived metabolites, arnebacene (1) and arnebidin (2), along with some known phenolic metabolites viz., arnebin-7 (3) and vanillic acid (4). The chemical identification of the new isolated compounds, including their relative stereochemistry, was achieved via spectroscopic analyses, including 2D NMR, and spectral comparison with related compounds. A biosynthetic pathway is proposed for the new compounds on the basis of their structure-relationship with previously isolated metabolites.


Introduction
Arnebia hispidissima (Lehm.) DC. (Arabian primrose), which belongs to the family Boraginaceae, is widely distributed in the northern Africa area, through Egypt, to northern India [1]. To date, the roots of eight Arnebia species were studied phytochemically, and several naphthoquinone metabolites possessing various biological activities, have been isolated [2][3][4][5][6][7]. The root of A. hispidissima has been used as a food colorant whereas the flowering shoot has been employed in diseases of the tongue and OPEN ACCESS throat as well as fevers and cardiac disorders [4,8]. Moreover, the organic extracts of A. hispidissima exhibited antibacterial [3,9] and antitumor activity [10] which were attributable to the presence of naphthoquinones, triterpenoids, and pyrrolizidine alkaloids. Our phytochemical investigation on the root of Arnebia hispidissima growing in Sinai Peninsula, has led to the isolation of new furanohexahydroanthracene-based metabolite arnebacene (1) and the arnebin-7-related dimer arnebidin (2), along with two known phenolic compounds 3 and 4. The structures of the new metabolites, including their stereochemistry, were elucidated on the basis of extensive spectroscopic analyses (including 1D and 2D NMR) and by comparison of their spectral data with those of related compounds. Compound 2 is reported herein as a novel heptacyclic arnebin-7 dimer with an unprecedented tricyclo[3.3.0.0 1,3 ]octane core.

Results and Discussion
The dried root of A. hispidissima was powdered, sequentially extracted with light petroleum and then MeOH. The MeOH extract was further partitioned with H 2 O/n-hexane, H 2 O/CH 2 Cl 2 , and then with H 2 O/EtOAc to yield MHF, MCF, and MEF fractions, respectively. The light petroleum extract and MeOH-derived fractions were separately subjected to chromatographic fractionation and purification, utilizing a series of normal-phased chromatographic systems, to afford compounds 2 and 3 from the light petroleum extract, 1 from MCF, and 4 from MEF ( Figure 1).

Figure 1. Phenolic constituents (1-4) isolated from A. hispidissima roots and shikometabolin D (5).
Compounds 3 and 4 were identified by comparison of their physical and spectroscopic (MS and NMR) data with those of the previously isolated compounds as arnebin-7 [11] (syn. deoxyalkannin [12] or deoxyshikonin [7]) and vanillic acid [13], respectively. Compound 4 is reported herein from genus Arnebia for the first time, although it was isolated once from the genus Onosma [14] of the same family (Boragenaceae O+H] + indicated the presence in the structure of 1 of at least two hydroxyl groups, a methyl and a methoxy group. The 13 C-NMR spectrum displayed 17 carbon signals (Table 1) which were assigned, by the assistance of DEPT spectra, into two methyls, two methylenes, seven methines (including two sp 3 oxymethines), and six quaternary carbons (including one sp 3 and two sp 2 oxygenated carbons). The eight sp 2 carbons resonating at δ C 111.4-151.3 indicated the presence of four double bonds. Thus, the remaining four degrees of unsaturation indicated a tetracyclic skeleton for compound 1. The HMQC spectrum showed δ H /δ C correlations at 2.67 (2H, s)/33.2; 3.92 and 4.08 (each 1H, d, J = 10.0 Hz)/77.9; 0.85 (3H, s)/19.5; and 3.79 (3H, s)/56.1 attributable to the presence of an isolated methylene (10-CH 2 ), an isolated oxymethylene (12-CH 2 ), a ring-junction tertiary methyl (10a-CH 3 ) and an aromatic methoxy group (4-OCH 3 ), respectively. The 1 H-1 H COSY experiment established three partial proton spin systems structure ( Figure 2 6 and H-7) exhibited 3 J correlations to C-9a (qC), C-4a (qC), C-10a (qC) and C-8a (CH), respectively. Since the isolated methylene protons H 2 -10 and the oxymethine proton H-9 were found correlated to C-4a and C-10a and to C-9a and C-8a, respectively, therefore ring B should be fused with rings A and C through C-4a/C-9a and C-10a/C-8a, respectively (Figures 1 and 2). The HMBC correlations observed from upfield shifted protons at δ H 0.85 (3H, s) to C-5, C-8a, C-10, and C-10a positioned the single methyl group in the molecule at the ring juncture carbon C-10a. Furthermore, the HMBC correlations exhibited from H 2 -12 to C-8 (δ C 78.8, qC) and C-9 (δ C 73.9, CH) and from H-8a (δ H 2.59, 1H, d, J = 6.0 Hz), to C-12 indicated a trisubstituted furan ring to be located at C-8, C-8a, and C-9. Acetylation of compound 1 afforded diacetate and triacetate derivatives 1a and 1b. The latter compound 1b displayed ester carbonyl absorptions at 1770 and 1734 cm −1 and no hydroxyl absorptions in the IR spectrum, whereas it exhibited successive ions peaks at m/z 370 [M-AcOH] + , 310 [M-2AcOH] + , and 250 [M-3AcOH] + in the EI-MS due to elimination of three acetoxyl groups. Therefore, the three hydroxyl groups in 1 were determined. The C-1, C-5, and C-8 positions of these hydroxyl groups were suggested by detailed analysis of HMBC correlations of compound 1 ( Figure 2). Moreover, the NMR data of 1b showed an upfield shift at C-1 (Δδ C -5.9) and downfield shifts at 5-CH (Δδ H + 1.06 and Δδ C + 0.8) and C-8 (Δδ C + 8.3), relative to those of compound 1, which further supported the locations of hydroxyl groups to be at C-1, C-5, and C-8, respectively. It is noteworthy to mention that the 2H singlet of the methylene protons at C- 10 Hz) derivatives as a result of acetylation of 5-OH in compound 1. On the basis of the above findings, the gross structure of compound 1 was thus deduced as illustrated in Figure 2.  The relative stereochemistry of compound 1 was interpreted from the NOE interactions ( Figure 2) observed in the NOESY spectra of 1, 1a and 1b. Supposing the β-orientation of the ring-juncture methyl at C-10a, the significant NOESY correlations observed between Me-11 and H-5 revealed the α-orientation of the 5-OH group in 1. Although the methine protons H-8a in 1, 1a, and 1b displayed a strong NOE interaction with H-9, both protons lacked NOE cross-peaks with Me-11. The α-orientation for H-8a and H-9 in 1 was thus suggested. One of the methylene protons at C-10 in 1a (δ H 2.73) or 1b (δ H 2.73) displayed NOE interactions with Me-11 and H-5 and designated as Hβ-10 whereas the other one (δ H 2.49 in 1a or δ H 2.54 in 1b) was found to be NOE correlated with H-8a. The α-orientation for H-8a and consequently for H-9 was thus confirmed. Moreover, one oxymethylene proton at C-12 in 1a (δ H 3.92) or 1b (δ H 4.05) exhibited significant NOE response with Me-11 of 1a or 1b, respectively, disclosing the β-configuration of the furan ring and thus 8-OH should be α-oriented. From the above findings, the structure and relative configuration of 1 was unambiguously established as (5S*,8R*,8aS*,9S*,10aR*)-1,5,8-trihydroxy-4-methoxy-10a-methyl-8,9-oxymethylene-5,10a,8,8a,9,10-hexahydroanthracene and named arnebacene.
The new metabolite 1 is proposed to be biosynthetically derived from a monoterpenoid benzoquinone (rhizonone) [18], obtained by cyclization of an intermediate precursor (geranylhydroquinone), through hydroxylation of C-5, oxidation of C-8, and monomethylation of the p-quinonoid moiety (pathway a). The production of monomeric metabolites from geranylhydroquinone e.g., shikonin and arnebin-7 (3) (pathway b), followed by condensation of two molecules of 3 from their side chain was suggested to yield the intermediate 3a then the hexacyclic dimeric metabolite shikometabolin D (5) with a bicylic pentalene core. Protonation of metabolite 5 at C-2 affords the carbonium ion 5a. The subsequent deprotonation at C-3 of 5a and the following electrophilic aliphatic substitution at C-11could generate the heptacyclic derivative (2) with a tricyclo[3.3.0.0 1,3 ]octane core. The presence of 2,11-double bond is thought to catalyze the intramolecular rearrangement (cyclopropanation) in metabolite 5 to yield compound 2. Although they are few in number, cyclopropyl-containing natural products, in which the double bonds of their precursors involved in the cyclopropanation by a similar mechanism, have been reported. These can be exemplified by the biosynthesis of crythanthemic acid from dimethylallyl diphosphate [19] and sabinene or thujone from terpinen-4-yl cation [20] and substantiated the proposed biosynthetic pathway for arnebidin (2) ( Figure 5).

General
IR and UV spectra were recorded on Hitachi I-2001 infrared and Hitachi U-3210 spectrophotometers, respectively. Mass spectral data were obtained by EI and FAB with a VG Quattro GC/MS spectrometer. HRMS spectra were obtained by ESI on a Bruker APEX II mass spectrometer. NMR spectra were recorded on Brukers Avance DPX-300 and 400 and Varian INOVA-500 NMR spectrometers at 300, 400, and 500 MHz for 1 H, and at 75, 100, and 125 MHz for 13 C, respectively, in CDCl 3 or CD 3 OD. Silica gel 60 (Merck, 230-400 mesh) were used for column chromatography. Precoated Si gel plates (Merck, Kieselgel 60 F 254 , 0.25 mm) were used for analytical thin layer chromatography (TLC). Preparative high-performance liquid chromatography (HPLC) was performed on a Hitachi L-7100 apparatus using a Merck Hibar Si-60 column (250 × 21 mm, 7 μm) and Hitachi L-7400 UV detector (detection wavelength = 280 nm).

Plant Materials
The roots of A. hispidissima were collected from Wady Khashaba, South of Sinai Peninsula, Egypt, during spring, and identified by Ibrahim A. Mashaly, Department of Botany, Faculty of Science, MU. A voucher sample (S-98-1) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, MU. All freshly prepared organic extracts and fractions of the air-dried roots were kept in freezer at −20 °C until use.

Extraction and Isolation of Compounds
The air-dried roots of A. hispidissima (1 Kg) were powdered and exhaustively extracted with light petroleum (boiling point 60-80 °C). The light petroleum extract was concentrated under vacuum to afford a reddish brown viscous residue (13.8 g). The marc was exhaustively extracted with MeOH and the solvent-free extract (106.7 g) was then successively partitioned with H 2 O/n-hexane, H 2 O/CH 2 Cl 2 and then with H 2 O/EtOAc. The three organic partitions were separately evaporated under vacuum to give the MeOH-derived n-hexane, CH 2 Cl 2 , and EtOAc fractions (MHF: 4.9 g, MCF: 3.6 g, and MEF: 6.5 g, respectively). The light petrol extract was chromatographed on a column of Si gel and eluted with benzene in n-hexane then CH 2 Cl 2 in benzene (0%-100%, gradient) to afford six fractions (P1-P6). The orange red fractions P2 eluted with benzene-n-hexane (1:1 to 7:3, gradient) and P5 eluted with benzene-n-hexane (1:0) were purified separately on preparative Si gel TLC using benzene-n-hexane (2:3, double run) or CH 2 Cl 2 -benzene-acetone (50:50:1) to obtain 3 (6 mg) from P2 and 2 (7 mg) from P5. MCF was chromatographed on Si gel column using MeOH in CH 2 Cl 2 (0% to 2%) to give a UV-absorbing fraction. This fraction, eluted with 2% MeOH in CH 2 Cl 2 , was then purified on Si gel column using 1.5% MeOH in CH 2 Cl 2 followed by preparative Si gel HPLC using 1.5% MeOH in CH 2 Cl 2 (flow rate 2.5 mL/min) to yield 1 (3.2 mg). MEF was fractionated on Si gel column using EtOAc-MeOH-H 2 O (95:5:0.5 to 60:40:4, gradient) to obtain a UV-absorbing fraction which was further purified by preparative Si gel TLC using 12% MeOH in CH 2 Cl 2 to afford 4 (2.5 mg). Acetylation of compound 1: A solution of 1 (2.2 mg, 0.007 mM) in pyridine (0.3 mL) was mixed with Ac 2 O (0.2 mL), and the mixture was stirred at RT for 24 h. The mixture was diluted to 5 mL with distilled water, neutralized with sodium carbonate powder, extracted with ether (2 × 3 mL). The ether extract was then evaporated to dryness, dissolved in acetone-n-hexane (1:3) and subjected to normal phase HPLC using the same solvent system at a flow rate of 1 mL/min. Two UV-absorbing products: a diacetyl derivative 1a (1.2 mg, 0.0031 mmol, 42.7%) and a triacetyl derivative 1b (0.6 mg, 0.0014 mmol, 19.3%) were separately collected.