J. Braz. Chem. Soc., Vol. 23, No. 2, 349-354, 2012. Printed in Brazil - ©2012 Sociedade Brasileira de Química Short Report 0103 - 5053 $6.00+0.00 S Lycopodium Alkaloids from Palhinhaea cernua Fu-Wei Zhao,a,b Qian-Yun Sun,c Fu-Mei Yang,c Ji-Feng Luo,a Guang-Wan Hu,a Fang Liu,d Yue-Hu Wang*,a and Chun-Lin Long*,a,e aKey Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China bGraduate University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China cKey Laboratory of Chemistry for Natural Products, Guizhou Province and Chinese Academy of Sciences, Guiyang 550002, People’s Republic of China dCollege of Landscape and Horticulture, Yunnan Agricultural University, Kunming 650201, People’s Republic of China eCollege of Life and Environmental Sciences, Minzu University of China, Beijing 100081, People’s Republic of China Dois novos alcaloides tipo-licopódio, acetil-licoposerramina M e palcernina A foram isolados de extratos de plantas inteiras de Palhinhaea cernua L. juntamente com dez compostos previamente identificados. As estruturas dos dois novos compostos foram elucidadas por métodos espectroscópicos e difratometria de Raios X de monocristal usando o parâmetro Flack. Two new Lycopodium alkaloids, acetyllycoposerramine M and palcernine A were isolated from whole plant extracts of Palhinhaea cernua L. together with ten previously identified compounds. The structures of the new compounds were elucidated by spectroscopic methods and single-crystal X-ray diffraction analyses using the Flack parameter. Keywords: Lycopodiaceae, Palhinhaea cernua, Lycopodium alkaloids Introduction N-oxide (3)8, lycoflexine (4),11 lycodine (5),12 α-obscurine (6),12 dehydroisofawcettiine (7),13,14 lycopodine (8),14 Palhinhaea cernua L. (syn. Lycopodium cernuum (L.); 5-acetyllycofoline (9),15 acetylfawcettiine (10),16,17 cernuine Lepidotis cernua (L.) P. Beauv.; Lycopodiella cernua (L.) Pic. (11)18 and lycocernuine (12).18 Ser.), found in both the Flora of North America and the Flora 1 H 16 of China, is a member of the Lycopodiaceae family. The O 16 H species is traditionally used to heal contusions, scalding and 1415 O 8 15 8 O 13 rheumatism by the Chinese people and is broadly distributed 11 7 12 6 OH 7 11 2 10 12 in Eastern and Southern China. The chemical constituents 10 14 H 6 9 13 5 9 5 of the genus Lycopodium (sensu lato) plants are mainly N 17 O N 4 O 4 1 3 Lycopodium alkaloids which are quinolizine or pyridine and 2 1 3 2 α-pyridone type alkaloids.3-10 Some of these alkaloids inhibit acetylcholinesterase (AChE) and provide a challenge for 12 total synthesis.9-10 In our previous research on this plant, Figure 1. The structures of compounds 1-2. 9 palhinine A, a novel C16N-type alkaloid was isolated. We now report the isolation and structural elucidation of two Results and Discussion new Lycopodium alkaloids (1-2) along with ten previously identified compounds (Figure 1), namely, lycoflexine Acetyllycoposerramine M (1) was obtained as a colorless columnar crystal (mp 197-198 °C). A molecular formula *e-mail: [email protected], [email protected]. (C18H27NO3) was assigned by HREIMS (electrospray 350 Lycopodium Alkaloids from Palhinhaea cernua J. Braz. Chem. Soc. ionization mass spectrometric) analysis (observed [M]+ Table 2. 13C NMR data of compounds 1-2 (d in ppm) at m/z 305.1995, calcd. [M]+: 305.1991), implying six degrees of unsaturation. The IR (infrared) spectrum showed No. 1a 2b absorptions for ester (1695 cm−1) and keto (1733 cm−1) 1 47.2 t 52.7 t 2 19.4 t 21.1 t carbonyl groups. One singlet (d 2.09, 3H) and one doublet H 3 20.0 t 29.3 t ( 0.88, 3H, J 6.3 Hz, H -16) for two methyl groups dH 3 4 45.4 d 54.0 s 1 were observed in H NMR (nuclear magnetic resonance) 5 214.4 s 203.8 s spectrum of 1 (Table 1). Meanwhile, signals of 18 carbons 6 44.4 t 149.1 s c 13 were recorded in the C NMR and DEPT (distortionless 7 35.1 d 145.0 s enhancement by polarization) spectra (Table 2), including 8 44.3 t 28.4 t the presence of one ester carbonyl (dC 170.3), one keto 9 42.1 t 57.7 t 3 3 10 31.0 t 26.1 t carbonyl (dC 214.4, C-5), one sp quaternary, five sp methine, eight sp3 methylene and two methyl groups. 11 72.3 d 34.0 t Specifically, the signals of an oxygenated methine group 12 46.8 d 61.7 s 13 58.9 s 210.0 s were recorded at dH 5.23 (ddd, J 2.9, 2.9, 2.9 Hz, H-11) and 14 43.8 t 48.9 t d 72.3 (C-11). The above mentioned analyses suggested C 15 25.5 d 27.4 d that 1 was a lyopodine alkaloid, and shared a similar 16 22.8 q 21.7 q planar structure with the known Lycopodium alkaloid, 17 57.8 t lycoposerramine M.19 Differentiations between the two OCCH3 170.3 s compounds were observed in that the additive signals ( dH OCCH3 22.0 q a b 2.09, 3H, s; dC 22.0 and 170.3) for an acetyl group appeared Recorded at 125 MHz in CDCl3; recorded at 125 MHz in C5D5N; cdetected by HMBC. Table 1. 1H NMR data of compounds 1-2 (d in ppm, J in Hz) in the 1H and 13C NMR spectra of 1, and the presence of No. 1a 2b a chemical shift of H-11 in 1 was downshifted compared 1a 3.42 (dd, 14.2, 3.4) 3.09 (m) 19 1b 2.64 (m) 2.93 (m) with that of the known compound (dH 4.22). Therefore, it 2a 1.89 (m) 2.51 (dd, 14.8, 4.0) could be speculated that 1 was the acetylated derivative of 2b 1.51 (m) 1.34 (d, 13.4) lycoposerramine M. This was confirmed by the presence 3a 2.13 (m) 2.29 (m) of the cross peak between H-11 and ester carbon in the 3b 1.59 (ddd, 13.6, 13.6, 4.4) 1.99 (dd, 14.8, 9.4) HMBC (heteronuclear multiple bond correlation) spectrum 4 3.24 (dd, 12.0, 2.9) of 1 (Figure 2). 6a 2.76 (dd, 16.6, 6.7) O 16 O 6b 2.23 (d, 16.6) H 7 2.33 (s) O O 15 8 11 8a 1.69 (br. d, 13.0) 2.90 (m; β) 12 7 8b 1.35 (ddd, 12.7, 12.7, 3.4) 2.66 (dd, 15.1, 8.0; α) 10 14 6 9 13H 5 9a 3.37 (m) 2.99 (m) 1 1 N 4 O N O H- H COSY 1 3 9b 2.61 (m) 2.82 (m) 2 HMBC 10a 2.10 (m) 2.15 (m) 10b 1.98 (m) 1.41 (m) Figure 2. 1H-1H COSY and HMBC correlations of 1. 11a 5.23 (ddd, 2.9, 2.9, 2.9) 2.28 (m; α) 11b 1.65 (dd, 14.7, 11.8; β) To elucidate the absolute configuration, the crystals 12 1.85 (m) of 1 were isolated from a mixed solution of petroleum 14a 2.66 (m) 2.49 (dd, 14.8, 4.0; α) ether/Me CO (9:1), and single-crystal X-ray crystallographic 14b 1.09 (dd, 13.1,13.1) 1.97 (dd, 14.8, 9.4; β) 2 15 1.54 (m) 2.19 (m) analysis was performed (Figure 3). The results of the 16 0.88 (3H, d, 6.3) 0.90 (3H, d, 6.7) analysis revealed the absolute configuration of1 to be 4R, 17a 3.10 (m) 7R, 11R, 12R, 15R in light of the Flack parameter of 0.0(2), 20-22 17b 2.99 (m) using anomalous dispersion with copper radiation. OCCH3 2.09 (3H, s) Finally, 1 was elucidated as acetyllycoposerramine M. 6-OH 12.19 (br. s) Palcernine A (2) was obtained as a colorless block a b Recorded at 500 MHz in CDCl3; recorded at 500 MHz in C5D5N. crystal (mp 98-101 °C). Its molecular formula (C17H24NO3) Vol. 23, No. 2, 2012 Zhao et al. 351 located at C-6, and two ketone groups were located at C-5 and C-13, respectively. After analysis of the molecular formula of 2 the presence of a hydroxy functionality in 2 was confirmed, possibly being located at C-6. The relative configuration of partial chiral center of 2 could be elucidated, integrating ROESY (rotating-frame Overhauser effect spectroscopy) experiment and analysis on coupling constant. In ROESY spectrum (Figure 4), the cross peaks of H-8α (dH 2.66, dd, J 15.1, 8.0 Hz)/H-11β (dH 1.65, dd, J 14.7, 11.8 Hz), H-8α/H-14α (dH 2.49, dd, J 14.8, 4.0 Hz) and H-11β/H-14α were observed. H-14α and H-15 should be in cis configuration due to the coupling constant of JH-14α/H-15 4.0 Hz (Table 1). Therefore, the 16- Figure 3. ORTEP drawing of the asymmetric unit of compound 1. CH3 in 2 was β-oriented, which is rare because the 16-CH3 is α-oriented in most of the Lycopodium alkaloids.3-5 Based was established on the basis of HRESIMS for the [M + H]+ on the single-crystal X-ray crystallographic analysis ion at m/z 290.1754 (calcd.