New monoterpene phenyl ethers from Illicium micranthum

Graphical abstract Seven new monoterpene phenyl ethers, namely micranthumnins A-G (1–7), were isolated from the stem bark of Illicium micranthum (Illiciaceae). Their structures were elucidated by comprehensive spectroscopic analyses including MS, IR, 1D and 2D NMR. All compounds were evaluated for their anti-AChE activities. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-013-0007-x and is accessible for authorized users.


Introduction
Illicium species, which belong to the only genus of the family Illiciaceae, are mainly distributed in East Asia and the southeast of North America. 1 Twenty eight species (over 60% of the family) in Illiciaceae are mainly distributed in southern and eastern China, and twelve species of Illicium L. are found in Yunnan province. 2,3 Phytochemical investigation showed that this genus produced monoterpenoids, 4 sesquiterpene lactones, 5-8 diterpenoids, 9 triterpenoids, 10 lignans and neolignans, [11][12][13] which showed various bioactivities, such as insecticidal activity, 14 cancer chemopreventive activity, 15 and neurotrophic activity. [16][17][18] Illicium micranthum was a poisonous shrub used as a traditional pesticide. 3 Previous studies on this plant have resulted in the isolation of eight secoprezizaane sesquiterpene lactones, 5 seven phenylpropanoids 19 and several other compounds. 20 As an on-going search for neurotrophic active compounds from natural resources, our investigation on I. micranthum led to the isolation of seven new monoterpene phenyl ethers (1)(2)(3)(4)(5)(6)(7). This paper deals with the isolation, structure characterization, and anti-AChE activity of these compounds.

Results and Discussion
A 90% aqueous MeOH extract the stem bark of I. micranthum was partitioned between CHCl 3 and H 2 O. The CHCl 3 solubles were dried and subjected to silica gel, Sephadex LH-20 and RP-18 gel column chromatography (CC) and semipreparative HPLC to afford seven new compounds.
The relative configurations of 1 and 2 were further confirmed by the derivative reaction. In the reactions, the vicinal diol parts in 1 and 2 react with DMP to yield ketal products 1a and 2a, respectively, which makes the carboncarbon bond between C-7 and C-8 rotate unfreely. The relative configurations of 1 and 2 were determined depending on the two ketal products 1a and 2a whose relative configurations were clarified by ROESY experiments (Figure 3). ROESY correlations of H-7/H-1′′ and H-8/H-1′′ were detected while the correlation of H-7/H-9 was disappeared in 1a, which determined the erythro configuration of 1a. The relative configuration of 2a was determined to be threo by ROESY correlations of H-7/H-1′′, H-8/H-3′′, and H-7/H-9. Consequently, the relative configurations of 1 and 2 were determined to be erythro and threo, respectively ( Figure 1).
The mass spectrum of compound 3 (HREIMS m/z 362.2098, clacd for 362.2093, [M] + ) was 14 amu higher than that of 1, indicating that there was probably one more methyl group in 3. The similarities of the spectroscopic data (Tables 2 and 3) between 3 and 1 suggested that 3 was identical to 1 except for the methoxyl group at C-7, causing a significant downfield chemical shift from δ C 77.4 to δ C 89.2 due to C-7. HMBC correlation ( Figure 2) from OCH 3 -7 at δ H 3.23 to C-7 at δ C 89.2 further confirmed the above assignment. Without an isomer as a comparison, as well as the significant difference of C-7 chemical shift between 3 and 1, the relative configuration of 3 was not clarified. Finally, the structure of 3 was established as shown in Figure 1, and named as micranthumnin C.
The mass spectrum showed that compound 7 was 34 mass amu lower than that of 1, indicating the probable loss of two hydroxyl groups in 7. The NMR data indicated that 7 was similar to 1. Extensive analyses of its 1D and 2D NMR data suggested that the 1,2-propanediol group in 1 was replaced by a propenyl group. The signals corresponding to a propenyl group at δ H 6.32 (1H, dq, J = 15.6, 1.2 Hz, H-7), 6.13 (1H, dq, J = 15.6, 6.6 Hz, H-8), and 1.85 (3H, dd, J = 6.6, 1.2 Hz, H-9) were evident in the 1 H NMR data (Table 2) of 7. The structure was further confirmed by HMBC correlations (Figure 2) of H-7/C-1, C-2, C-6 and H-8/C-1, C-9. Moreover, the analysis of the ROESY spectrum as well as the coupling constant (J = 15.6 Hz) between H-7 and H-8, indicating an E-geometry double bond. Therefore, the structure of 7 was determined and named as micranthumnin G.
The acetyl cholinesterase (AChE) inhibitory activities of all compounds were assayed using the Ellman method. 24 Compound 5 showed weak inhibitory activity (27.4%) at a concentration of 50 μM, using tacrine (0.33 μM) as the positive control (50.56% inhibition). The remaining compounds were inactive at 50 μM.

Experimental Section
General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter. IR and UV spectra were recorded on a Bruker Tensor 27 FT-IR spectrometer and a Shimadzu UV2401PC spectrometer, respectively. 1D and 2D NMR spectra were recorded on Bruker Avance Ⅲ-600, DRX-500 or AM-400 MHz spectrometers with TMS as internal standard at room temperature. HRESIMS were recorded on a  Extraction and Isolation. The air-dried stem bark of I. micranthum (14 kg) was powdered and extracted with MeOH (3 × 25 L) at room temperature, and concentrated in vacuo to give a crude extract. The extract was successively fractionated with CHCl 3 and EtOAc. A portion of the CHCl 3 extract (310 g) was separated by silica gel column chromatography, using CHCl 3 /MeOH (20:1 to 2:1) as a gradient solvent system to afford fractions I-VI.
Fraction II was isolated by silica gel eluting with a gradient of petroleum ether/EtOAc (40:1, 20:1, 10:1, 5:1, to 2:1) to afford four subfractions (A1-A4). Fraction A2 was chromatographed over Sephadex LH-20 with MeOH to yield 7 (31 mg). Fraction III was subjected to RP-18 column chromatography   Anti-AChE Assay. AChE inhibitory activities of the compounds isolated were assayed by the spectrophotometric method developed by Ellman et al. 11 Acetylthiocholine iodide (Sigma) was used as substrate in the assay. Compounds were dissolved in DMSO. The mixture contained 110 μL phosphate buffer (pH 8.0), 10 μL of test compound solution (50 μM), and 40 μL AChE solution (0.04 U/100 μL), and the mixture was incubated for 20 min (30 °C). The reaction was initiated by the addition of 20 μL of DTNB (6.25 mM) and 20 μL of acetylthiocholine iodide (6.25 mM). The hydrolysis of acetylthiocholine was monitored at 405 nm after 30 min. Tacrine was used as positive control. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E -S)/E × 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound).

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/ 10.1007/s13659-013-0007-x and is accessible for authorized users.