New Monoterpenoid Coumarins from Clausena anisum-olens

Two new monoterpenoid coumarins: anisucumarin A (1) and B (2), a pair of epimers, were isolated from Clausena anisum-olens. Their structures were established based on extensive spectroscopic analyses.

Clausena anisum-olens is a shrub found growing in Hekou County of the Yunnan Province and the leaves and twigs of this plant are used for the treatment of dysentery and arthritis [7]. In a preliminary pharmacological study, the EtOH extract of the leaves and twigs of Clausena anisum-olens exhibited antifungal activities against three Candida species: C. albicans, C. tropicalis, and C. krusei. Previous studies on Clausena anisum-olens resulted in the isolation of a novel cyclopeptide [10]. In the present study, an epimer pair of new monoterpenoid coumarins anisucumarin A (1) and B (2) were isolated. Herein, we report the isolation and structural elucidation of these two new coumarins.

Results and Discussion
The powdered leaves and twigs of Clausena anisum-olens, collected from Hekou County, Yunnan province, were extracted with 90% ethanol. The concentrated extract suspended in water was successively extracted with petroleum ether, AcOEt and n-BuOH. The AcOEt extract was subjected to chromatography on silica gel, Sephadex LH-20 and RP C-18 to yield compounds 1 and 2 as a pair of inseparable epimers.  Structural elucidation of the new coumarins was mainly determined by spectroscopic 1D-and 2D-NMR experiments ( 1 H, 13 C, 1 H-1 H COSY, HMQC and HMBC; see Table 1), HR-ESI-MS, UV and IR. The molecular formula of compounds 1/2 was determined to be C 20 H 20 O 8 by HRESI-MS exhibiting the quasimolecular ion at m/z 389.1249 [M+H] + , which indicated eleven degrees of unsaturation. The UV spectra of 1/2 displayed typical absorption bands at λ max 211, 256, and 318 nm, respectively, accompanied with some minor bands. This feature was similar to that of a 7,8-dioxygenated coumarin with a C-10 terpenoid side chain containing a γ-lactone [5]. The IR bands at 3439 and 1730 cm -1 indicated the presence of hydroxyl groups and α,β-unsaturated-γ-lactone group in these molecules. The EI-MS spectra showed fragment ion at m/z 100, which was characteristic of 8-OMe coumarin and prominent fragment ion at m/z 370 corresponding to loss of H 2 O [11].
Through careful analysis of 1 H-NMR spectra the presence of a 7,8-dioxygenated coumarin backbone as a common structural unit in 1/2 was further deduced by a methoxy singlet signal at δ 3.95 and two sets of 1 H AB doublets at δ H 6.26 and 7.86 (each d, J = 9.6Hz) and δ H 7.31 and 7.08 (each d, J = 8.7 Hz), which were easily assignable to H-3 and H-4 and to H-5 and H-6 on the coumarin skeleton, respectively ( Table 1). Analysis of the 1 H-and 13 C-NMR spectra, including COSY, HMQC and HMBC, suggested the presence of a C 10 terpenoid side chain in 1/2. Two olefinic protons on a terminal methylene at δ 5.24, 5.42 (each d, J = 6.3Hz) were attributed to H-10′ according to signal complexity and chemical shift. The other olefinic proton at δ 7.55 (1H, d, J = 1.7Hz) and a lone 2H-broad singlet at δ 4.31 were observed in the 1 H spectrum, and the long-distance correlations between a 2H-broad singlet at The difference between 1 and 2 was due to the stereochemistry of hydroxyl group at C-2′. The NMR peaks of C-1′, C-2′, C-3′ and C-10′ appeared as pairs (Table 1), indicating the presence of 1 and its C-2′ stereoisomer 2. The 1 H-and 13 C-NMR spectra established that 1 and 2 consisted of two epimers in a 3:2 ratio. The compound pair resulted in a single spot by multiple solvent systems HPLC. Attempts in the separation of the epimers by HPLC, however, failed to split the products. A reason for this might be a small difference in the interactions between a pair of epimers and the column material for achieving their separation. An analysis of ROESY experiments showed significant NOE correlations between H-2′ and H-4b′ in the major epimers ( Figure 1). However, the same NOE correlation was not observed in the minor epimers. The evidences support the presence of a pair of epimers 1/2 instead of different conformations of one compound.
The configuration of these O-terpenoidal coumarins 1 and 2 remained to be determined. So far, the stereochemistry of this type of O-terpenoidal coumarins reported previously has not been resolved [5,[12][13][14]. Further structure elucidation on the stereochemistry pertaining to the C-2′ and C-5′ of 1/2 is in progress. In summary, the 1 H-NMR and 13 C-NMR spectra (Table 1), HMQC, HMBC data established the structures of 1 and 2 as a pair of epimers of monoterpenoid coumarins (Figure 1).
In a preliminary study, the EtOH extract of Clausena anisum-olens and the two isolated compounds were screened for antifungal activity against C. albicans, C. tropicalis and C. krusei, using the broth microdilution method described in [15]. To validate the MIC end points for antifungal testing of plant extracts, a classification of MIC values used is as follows: strong inhibitors -MIC up to 0.5 mg/mL; moderate inhibitors -MIC between 0.6 and 1.5 mg/mL and weak inhibitors -MIC above 1.6 mg/mL [16]. The EtOH extract of C. anisum-olens exhibited in vitro antifungal activities against C. albicans, C. tropicalis and C. krusei, with MIC values of 1.0, 0.25, 0.5 mg/mL. However, the new compound pair 1 and 2 didn't show antifungal activities in vitro in this bioassay. The fractionation of Clausena anisum-olens EtOH extract guided by the bioassays may lead to the isolation of the inhibitor compounds.

Conclusions
Two new monoterpenoid coumarins anisucumarin A (1) and B (2), whose separation was not successfully achieved were isolated as a pair of epimers from Clausena anisum-olens. Their structures were established based on extensive spectroscopic studies. The EtOH extract of Clausena anisum-olens and the monoterpenoid coumarins anisucumarin A (1) and B (2) were screened for antifungal activity against C. albicans, C. tropicalis and C. krusei. The EtOH extract of Clausena anisum-olens exhibited in vitro antifungal activities against above bioassays but the monoterpenoid coumarins anisucumarin A (1) and B (2) failed to show detectable inhibitory activity against the fungus.

General
Commercial silica-gel plates (Qing Dao Marine Chemical Group Co.) were used for TLC analyses. Melting points was measured on XRC-1 micro-melting point apparatus and uncorrected. UV/VIS Spectra was measured on Shimadzu UV-2401PC spectrophotometer; λ max in nm. IR spectra were obtained on Bio-Rad FTS-135 infrared spectrophotometer, ν max in cm -1 . 1 H-and 13 C-NMR as well as 2D-NMR spectra were recorded on Brucker DRX-500 spectrometer with TMS as internal standard, coupling constant J in Hz. MS spectra was performed on VG Autospec-3000 mass spectrometers.

Plant material
The

Assay for biological activity
The broth microdilution test M27-A2 [15] was used for the assessment of in vitro antifungal activity of the EtOH extract of Clausena anisum-olens and the compounds against Candida albicans ATCC 90028, Candida tropicalis ATCC 750, Candida krusei ATCC 6258. Amphotericin B was used as a reference drug. The procedure was performed in RPMI 1640 medium buffered to pH 7.0 with 3-morpholinopropane-1-sulfonic acid (0.165mol). Drug-free controls were included. The minimal inhibitory concentrations (MICs) were determined after 24 h and 48 h of static incubation at 35 °C.