Lycopodine-Type Alkaloids from Lycopodium japonicum

Three new lycopodine-type alkaloids, 4α-hydroxyanhydrolycodoline (1), 4α,6α-dihydroxyanhydrolycodoline (2), and 6-epi-8β-acetoxylycoclavine (3), and an artifact, lycoposerramine G nitrate (4), along with seventeen related known compounds, were isolated from the club moss Lycopodiumjaponicum Thunb. ex Murray (Lycopodiaceae). Their structures were elucidated by extensive spectroscopic methods as well as X-ray analysis. Compounds 1–4 were evaluated for their acetylcholine esterase inhibitory activity. Electronic supplementary material The online version of this article (doi:10.1007/s13659-014-0027-1) contains supplementary material, which is available to authorized users.

Compound 1 was obtained as a colorless crystal. Its molecular formula, C 16 13 C NMR and DEPT spectra of 1 ( Table 2) exhibited 16 carbon resonances due to four quaternary carbons (one oxygenated at d C 75.2, one olefinic at d C 139.9, and one carbonyl at d C 210.5), three methines (one olefinic at d C 118.8), eight methylenes, and one methyl group at d C 22.1. The 1 H-1 H COSY and HSQC data revealed three partial structures: a CH 2 CH 2 CH 2 , b CH 2 CH 2 CH, and c CH 2 CHCH 2 CH(CH 3 )CH 2 (Fig. 2). Further detailed 2D NMR analysis indicated compound 1 was closely related to anhydrolycodoline [19]. The only difference was that 1 possessed an OH additional group, which was suggested to be connected to C-4 as inferred from the HMBCs of d   In the ROESY spectrum of 1, the correlation of H-14a with Me-16 was observed (Fig. 2). However, due to overlapped signals of H-1b with H-10a and H-9a with H-7, the ROESY spectrum could not provide more sufficient information to elucidate the stereochemistry of 1. The relative configuration of 1 was established by X-ray analysis (Fig. 3), which validated the a-orientition of OH-4, H-7, and Me-16. Therefore, the structure of compound 1 was established as 4a-hydroxyanhydrolycodoline.
Compound 2 was isolated a colorless crystal. The HRESIMS displayed an [M?H] ? peak at m/z 278.1748 (corresponding to a molecular formula C 16 H 23 NO 3 ), 16 mass unit higher than that of 1. Comparison of the 1D NMR data (Tables 1 and 2) with those of 1, compound 2 was readily identified as 6-hydroxy derivative of 1 as deduced from the HMBCs of d H 3.86 (1H, br. s, H-6) with d C 39.4 (t, C-8), 77.0 (s, C-4), and 207.3 (s, C-5). The relative configuration of 2 was also established by X-ray analysis, which validated the a-orientation of OH-4, OH-6, H-7, and Me-16 (Fig. 3). Thus, the structure of 2 was elucidated as 4a,6a-dihydroxyanhydrolycodoline.
The molecular formula of compound 3 was determined as C 20 H 31 NO 5 on the basis of its HRESIMS ([M?H] ? at m/z 366.2270), indicating 6°of unsaturation. IR absorption bands implied the presence of ketone (1738 cm -1 ) and OH (3472 cm -1 ) groups. The 1 H and 13 C NMR (Tables 1 and 2) spectra revealed the existence of two OAc groups, seven sp 3 methylenes, seven sp 3 methines (three oxygenated at d C 69.2, 76.4, and 79.6), one sp 3 quaternary carbon, and one methyl group. The above data indicated that 3 had a similar structure to that of lycoclavine [20], except for the existence of an additional OAc group which was located at C-8 according to the HMBCs of d H 2.11 (1H, m, H-12), 2.64 (1H, dd, J = 13.2, 6.5 Hz, H-14a), and 0.87 (3H, d, J = 6.2 Hz, Me-16) with d C 79.6 (d, C-8) as well as d H 4.55 (1H, dd, J = 11.0, 5.2 Hz, H-8) with d C 170.6. To establish the relative configuration, an X-ray experiment was evolved, which suggested the relative configuration of H-4, H-5, H-6, H-8, H-12, and H-15 to be a, a, b, a, b, and b, respectively (Fig. 4). Thus, the structure of compound 3 was elucidated and named as 6-epi-8b-acetoxylycoclavine.
Compound 4 had a molecular formula of C 16 H 25 NO 3 , the same as that of lycoposerramine G [18], a known compound also isolated this time. The NMR data and detailed 2D analysis indicated the two compounds had the same planar structure. However, according to the ROESY spectrum, the two compounds also possessed the same relative configuration which indicated 4 should be a salt form of lycoposerramine G. Therefore, a X-ray experiment was implemented that confirmed 4 was lycoposerramine G nitrate ( Fig. 4), which was produced during the isolation as verified by the TLC (Al 2 O 3 ).
The compounds 1-4 were tested for acetylcholine esterase (AChE) inhibitory activity, yet no positive results were observed.

General Experimental Procedures
Melting points were obtained on an WRX-4 micro melting point apparatus (Shanghai Yice Instrument Co., Ltd., Shanghai, China). Optical rotations were measured with

Extraction and Isolation
Air-dried, powdered sample (50 kg) of L. japonicum was dealt as the method reported before to obtain an base extract (67 g) [15]. This extract was subjected to a silica gel column chromatography (CC) with a gradient elution system of petroleum ether-actone (1:0-0:1) to give 7 fractions (I-VI). Fraction I (7 g) was chromatographed over several silica gel CC eluted with petroleum ether-EtOAc (9:1-1:1) to give three sub-fractions, I-a, I-b, and I-c. I-b was purified by (CHCl 3 -MeOH, 1:1) to yield 1

Acetylcholinesterase Inhibition
Acetylcholinesterase (AChE) inhibitory activity of the compounds 1-4 isolated was assayed by the spectrophotometric method developed by Ellman et al. [27] with slightly modification. S-Acetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5 0 -dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman's reagent), acetylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical. Compounds were dissolved in DMSO. The reaction mixture (totally 200 lL) containing phosphate buffer (pH 8.0), test compound (50 lM), and acetyl cholinesterase (0.02 U/mL), was incubated for 20 min (30°C). Then, the reaction was initiated by the addition of 40 lL of solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) for AChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 s for 1 h. Tacrine was used as positive control with final concentration of 0.333 lM. All the reactions were performed in triplicate.
The percentage inhibition was calculated as follows: % inhibition = (E -S)/E 9 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with test compounds).     (8)