Two Unusual Methylidenecyclopropane Glucosides from Metaxya rostrata C.Presl

Two new natural compounds, (1R,2E)-2-(6-hydroxyhexylidene)cyclopropyl-β-D-glucopyranoside (1) and (6E)-6-[(2R)-2-(β-D-glucopyranosyloxy)cyclopropylidene]hexanoic acid (2), glucosides of a very rare methylidenecyclopropane alcohol, as well as two known glycosides of phenolic acids, namely 4-O-β-D-glucopyranosylcaffeic acid (3) and (E)-4-O-β-D-glucopyranosylcoumaric acid (4), and methyl α-fructofuranoside (5) were isolated for the first time from the rhizomes of the tree fern Metaxya rostrata C.Presl. The structures were elucidated on the basis of detailed spectroscopic data analysis, and the structure of 1 was additionally confirmed by X-ray crystal-structure analysis.

Introduction. -The tree fern Metaxya rostrata C.Presl (Metaxyaceae) is distributed widely in lowland rain forests of Central and South America, and is used in traditional medicine in Costa Rica against intestinal diseases such as ulcers or tumors. For the treatment of these diseases, a suspension of the dried rhizome of Metaxya rostrata in water is applied orally [1]. As tropical plants are a rich and promising source for new cytotoxic compounds [2], and no data were available on the chemical composition of this plant, the rhizomes of Metaxya rostrata were studied phytochemically. In our previous investigation, an aqueous extract of the rhizomes was fractionated. Within this earlier study, two cytotoxic proanthocyanidins (cinnamtannin B-1 and aesculitannin B), common sterols, and sugars had been isolated [1]. Further separation of other fractions from the aqueous extract which did not show significant cytotoxicity should give a more detailed insight into the chemical composition of this plant.
Results and Discussion. -Rhizomes of Metaxya rostrata were extracted with hot water according to the ethnomedicinal procedure and fractionated. After vacuum liquid chromatography and repeated column chromatography on silica gel or Sephadex LH-20, two new compounds 1 and 2, two phenolic acid glycosides 3 and 4, and a methyl glycoside 5 have been isolated (Fig. 1).
The characterization of 1, a white crystalline substance, was achieved by means of mass spectrometry and extensive 1D-and 2D-NMR experiments. From the ESI mass spectra, a molecular weight of 318 Da was deduced. Using flow injection analysis (FIA), the mass spectrum in the negative-ion mode displayed the [M À H] À ion peak at m/z 317, whereas in positive-ion mode not only the [M þ H] þ ion peak at m/z 319, but also the [M þ NH 4 ] þ and [M þ Na] þ peaks at m/z 336 and 341, respectively, were detected. Product ion peaks were recorded with MS/MS in both, the positive-and negative-ion mode at various collision energies. The [M þ H] þ ion showed mainly fragmentation of the glycosidic bond to yield the Y þ 0 ion (m/z 157) and the Z þ 0 ion (m/z 139). The [M À H] À ion produced the complementary C À 1 and B À 1 fragment ions (m/z 179 and 161, resp.), as well as the 0,2 A À and the 0,3 A À ions (m/z 119 and 89, resp.), resulting from cross-ring cleavage of the hexose. The HR-ESI-MS revealed a molecular weight of 318.3618 in accordance with the molecular formula C 15 H 26 O 7 .
In the NMR experiments, the sugar part was easily characterized as a bglucopyranose. The aglycon showed mainly signals for CH 2 groups in the aliphatic region, one C¼C bond CH signal, and an additional CH signal at 4.18 ppm in the 1 H-NMR. The 13 C-NMR additionally showed the resonance of a quaternary olefinic Catom at 123.9 ppm. The structure of the aglycon could be identified as a 2-(6hydroxyhexylidene)cyclopropanol moiety (for the structure, see Fig. 1), linked glycosidically with glucose via the alcohol attached to the cyclopropane ring. This result is based on the characteristic 1 H-and 13 C-NMR chemical shifts ( Table 1) of the cyclopropane signals together with rather large 1 H, 13 C one-bond coupling constants, namely 188.2 Hz for HÀC(9'), and 163.9 and 161.0 Hz for the CH 2 (8'), respectively [3]. The observed C¼C stretching frequency of 1775 cm À1 in the IR spectrum of 1 is in accordance with published data of similar methylidenecyclopropane structures [4]. Numerous HMBCs established all the necessary connectivities, e.g., the interglycosidic linkage by a cross-peak from HÀC(1) to C(9'), or correlations within the aglycon from HÀC(6') to the C(9'), C(8'), C(5'), and C(4'), and from HÀC(1') to C(2') and C(3'), respectively (Fig. 2). NOESY Cross-peaks from CH 2 (5') to CH 2 (8') within the cyclopropanol revealed the geometry of the C¼C bond. Attached to the C¼C bond is a C 5 aliphatic chain terminated by an alcohol function. From all data above, the structure of compound 1 was established as (2E)-2-(6-hydroxyhexylidene)cyclopropylb-d-glucopyranoside.   respecively, were observed again. The MS/MS measurements in the positive-ion mode revealed corresponding results.
The NMR data of compound 2 were very similar to those of compound 1. Again the sugar part was a b-glucopyranose with the glycosidic bond to the unusual methylidenecyclopropane alcohol. Differences were detected in the side chain: the signals for the terminal CH 2 ÀOH group were missing, one CH 2 signal was shifted to lower field, and a CO group signal at 182.9 ppm appeared in the 13 C-NMR spectrum showing a HMBC to the afore mentioned CH 2 group. Accordingly, the terminal alcohol in the alkane side chain of 1 should have been oxidized in 2 to give the corresponding carboxylic acid, (6E)-6-[2-(b-glucopyranosyloxy)cyclopropylidene]hexanoic acid.
Both compounds have, to the best of our knowledge, never been described in the literature, neither isolated from natural sources nor synthesized. The common characteristic structural feature of these new glycosidic compounds is the extremly rare methylidenecyclopropane alcohol as the aglycon moiety.
b-Glucopyranose was also determined as the sugar part in compounds 3 and 4, but their aglycons were different. Both genins are phenolic acids for which differing NMR signals in the aromatic region were observed: the 1 (Fig. 1). Their NMR spectroscopic data corresponded to those published [7]. Compounds 3 and 4 are not very widespread in plant kingdom. They have been isolated from several ferns such as Davillia mariesii Moore, Camptosorus sibiricus Rupr., and Matteuccia struthiopteris (L.) Tod. [7 -9]. Additionally, 4-O-b-d-glucopyranosylcaffeic acid (3) has been found in the Colchicaceae family [10] and (E)-4-O-b-d-glucopyranosylcoumaric acid (4) in Fabaceae [11] [12], and both only in few other plant species so far.
Compound 5 was identified as a methyl glycoside. Two CH 2 groups and one quarternary C-atom indicated a ketose. The detailed NMR analysis and comparison of the 13 C-NMR data with those in the literature [13] revealed compound 5 as methyl afructofuranoside. The new compounds 1 and 2 were tested for in vitro cytotoxic activity against the colon carcinoma cell line SW480. Cell viability was assessed by a neutral red uptake assay as described in [14]. The compounds did not exhibit significant activity (IC 50 > 250 mm).

Experimental Part
General. Anal.-grade solvents for extraction and fractionation were obtained from VWR (A-Vienna). TLC: on silica gel plates (Merck, Germany), mobile phase AcOEt/HCOOH/MeOH/H 2 O 70 : 8 : 8 : 11, and detection with anisaldehyde/sulfuric acid reagent [15]. Optical rotation: Perkin Elmer Polarimeter 341. UV/VIS Spectra: Beckman DU 640 spectrophotometer. IR Spectra: Perkin Elmer FT-IR 2000 instrument in attenuated total reflection mode using a Golden Gate ATR unit. All NMR spectra were recorded on a Bruker Avance DRX 600 NMR spectrometer using a 5 mm switchable quadruple probe (QNP, 1 H, 13 C, 19  Crystallographic Structure Determination. X-Ray diffraction measurement was performed on a Bruker X8 APEX II CCD diffractometer. Single crystal of 1 was mounted on glass fiber, coated with Parathone N oil and positioned at 35 mm from the detector. 890 frames were measured, each for 50 s over 18 scan width. The data were processed using SAINT software [16]. Crystal data, data collection parameters, and structure refinement details are compiled in Table 2. The structure was solved by direct methods and refined by full-matrix least-squares techniques. The non-H-atoms were refined with anisotropic displacement parameters, while the H-atoms were placed at geometrically calculated positions and refined as riding atoms in the subsequent least-squares model refinements. The isotropic thermal parameters were estimated to be 1.2 times the values of the equivalent isotropic thermal parameters of the atoms to which H-atoms were bonded. The following software programs and computer were used: structure solution, SHELXS-97 [17]; refinement, SHELXL-97 [18]; molecular diagrams, ORTEP-3 [19]; computer, Intel CoreDuo. Extraction and Isolation. Dried roots (800 g) of Metaxya rostrata C.Presl were pulverized and portions of 100 g each were extracted by sonification at 508 with 1 l H 2 O, each. The extraction was repeated five times with the same amount of solvent. After lyophilization, the residue (80 g) was extracted sequentially with AcOEt, BuOH, and MeOH. The fractions of different polarity (AcOEt fraction (0.75 g), BuOH fraction (26.35 g), and MeOH fraction (18.7 g)) were subjected to vacuum liquid chromatography (VLC) on silica (15 -40 mm, Merck) with AcOEt/MeOH/H 2 O mixtures of increasing polarity as mobile phases. All obtained fractions after these three fractionation steps were pooled according to their composition to 15 combined fractions [1]. Fr. 12 (2.96 g) was chosen for further separation in this study and subjected to column chromatography (CC) on Sephadex LH-20 (3 Â 65 cm) with 80% MeOH. Of 13 subfractions, Frs. 12/3 (0.50 g), 12/4 (0.69 g), and 12/5 (0.55 g) were further fractionated.
Financial support of the FWF (project-No. P20354) is gratefully acknowledged. We thank Alexander Roller (University of Vienna, Institute of Inorganic Chemistry) for single-crystal X-ray diffraction measurements.