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Notes Lyconadin A, a Novel Alkaloid from Lycopodium complanatum
Table 1.
1H
and
13C
NMR Data of Lyconadin A (1) in CD3OD at 300 K
δH
Jun’ichi Kobayashi,* Yusuke Hirasawa, Naotoshi Yoshida, and Hiroshi Morita Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
[email protected] Received April 16, 2001
Lycopodium alkaloids1 such as serratinine and lycodoline with a common formula of C16N and lycodine with that of C16N2 possess unique tetracyclic frameworks with five to seven stereogenic centers and have attracted great interest from biogenetic1,2 and biological3 points of view. These unique skeletons have also been challenging targets for total synthesis.4 Recently, we have isolated serratezomines A, B, and C with a seco-serratinine-type, a serratinine-type, and a lycodoline-type skeleton, respectively, from the club moss Lycopodium serratum var. serratum5 and complanadine A with a lycodine dimeric skeleton from L. complanatum.6 In our search for biogenetically interesting intermediates of Lycopodium alkaloids, lyconadin A (1), a novel alkaloid with an unprecedented skeleton consisting of three six-membered, one five-membered, and one R-pyridone rings, and a new alkaloid, 11-hydroxy lycodine (2), were isolated from the club moss Lycopodium complanatum. In this paper, we describe the isolation and structure elucidation of 1 and 2.
The club moss L. complanatum collected in Hokkaido was extracted with MeOH, and the MeOH extract was (1) For reviews of the Lycopodium alkaloids, see: (a) Ayer, W. A.; Trifonov, L. S. In The Alkaloids; Cordell, G. A., Brossi, A., Ed.; Academic Press: New York, 1994; Vol. 45, p 233. (b) Ayer, W. A. Nat. Prod. Rep. 1991, 8, 455. (c) MacLean, D. B. In The Alkaloids; Brossi, A., Ed.; Academic Press: New York, 1985; Vol. 26, p241. (d) MacLean, D. B. In The Alkaloids; Manske, R. H. F., Ed.; Academic Press: New York, 1973; Vol. 14, p348. (e) MacLean, D. B. In The Alkaloids; Manske, R. H. F., Ed.; Academic Press: New York, 1968; Vol. 10, p305. (2) (a) Hemscheidt, T.; Spenser, I. D. J. Am. Chem. Soc. 1996, 118, 1799-1800. (b) Hemscheidt, T.; Spenser, I. D. J. Am. Chem. Soc. 1993, 115, 3020-3021. (3) Liu, J. S.; Zhu, Y. L.; Yu, C. M.; Zhou, Y. Z.; Han, Y. Y.; Wu, F. W.; Qi, B. F. Can J. Chem. 1986, 64, 837-839. (4) (a) Sha, C.-K.; Lee, F.-K.; Chang, C.-J. J. Am. Chem. Soc. 1999, 121, 9875-9876. (b) Williams, J. P.; St. Laurent, D. R.; Friedrich, D.; Pinard, E.; Roden, B. A.; Paquette, L. A. J. Am. Chem. Soc. 1994, 116, 4689-4696. (c) Hirst, G. C.; Johnson, T. O.; Overman, L. E. J. Am. Chem. Soc. 1993, 115, 2992-2993 and references therein. (5) Morita, H.; Arisaka, M.; Yoshida, N.; Kobayashi, J. J. Org. Chem. 2000, 65, 6241-6245.
1 2 3 4 5 6 7 8a 8b 9a 9b 10 11a 11b 12 13 14a 14b 15 16
6.35 (1H, d, 8.9) 7.42 (1H, d, 8.9) 4.19 (1H, brs) 2.25 (1H, brd, 4.4) 1.05 (1H, t, 13.0) 1.94 (1H, m) 2.88 (1H, d, 13.7) 3.55 (1H, dd, 3.1, 13.7) 2.81 (1H, m) 1.74 (1H, brd, 13.9) 2.14 (1H, ddd, 3.9, 5.6, 13.9) 2.04 (1H, m) 3.54 (1H, d, 2.7) 1.18 (1H, t, 12.1) 2.07 (1H, m) 1.86 (1H, m) 0.95 (3H, d, 6.4)
HMBC (1H)
δC 165.32 116.64 141.60 126.23 148.76 64.56 50.41 40.24
3 2, 6, 9b 3, 6 8a, 9b, 12, 13 6, 8a, 11b 6, 16
61.39
6, 11a
33.63 34.02
11a 9a
48.06 73.11 40.24
6, 8, 11b 7, 9a, 11a 16
26.11 21.91
7, 8a, 13, 14a, 16 14a
partitioned between EtOAc and 3% tartaric acid. Watersoluble materials, adjusted at pH 10 with saturated Na2CO3, were partitioned with CHCl3. CHCl3-soluble materials were subjected to an amino silica gel column (Hex/ EtOAc, 1:0 f 0:1 and then CHCl3/MeOH, 1:0 f 0:1), in which fractions eluted with CHCl3/MeOH (10:1) were purified by silica gel columns (CHCl3/MeOH/EtOAc, 10: 1:0.5) to afford lyconadin A (1, 0.0003% yield) and 11hydroxylycodine (2, 0.0003% yield) together with complanadine A6 and lycodine.7 The molecular formula, C16H20N2O, of lyconadin A (1) was established by HRFABMS [m/z 257.1659, (M + H)+, ∆ +0.5 mmu]. The IR absorption implied the presence of conjugated carbonyl (1660 cm-1) functionality. 1H and 13C NMR data (Table 1) disclosed the existence of one amide carbonyl, one disubstituted olefin, one tetrasubstituted olefin, four sp3 methylenes, six sp3 methines, and one methyl. Among them, the signals due to two methines (δC 64.56; δH 4.19 and δC 73.11; δH 3.54) and one methylene (δC 61.39; δH 2.88 and 3.55) were ascribed to those bearing a nitrogen, while those due to one amide carbonyl (δC 165.32), one disubstituted olefin (δC 116.64 and 141.60), and one tetrasubstituted olefin (δC 126.23 and 148.76) indicated that the presence of a 4,5-disubstituted pyridone ring. Since four out of eight unsaturations were accounted for, 1 was inferred to possess five rings. The gross structure of 1 was elucidated by analyses of 2D NMR data including 1H-1H COSY, HOHAHA, HMQC, and HMBC spectra in CD3OD (Figure 1). The connectivity of C-7 to C-8, C-9-C-16, C-7 to C-12, and C-8 to C-15 was revealed by the 1H-1H COSY and HOHAHA spectra. The HMBC cross-peak of Ha-9 to C-13 indicated the connection between C-9 (δC 61.39) and C-13 (δC 73.11) through a nitrogen. On the other hand, the (6) Kobayashi, J.; Hirasawa, Y.; Yoshida, N.; Morita, H. Tetrahedron Lett. 2000, 41, 9069-9073. (7) Ayer, W. A.; Iverach, G. G. Can. J. Chem. 1960, 38, 1823-1826.
10.1021/jo0103874 CCC: $20.00 © 2001 American Chemical Society Published on Web 07/26/2001
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Notes
Figure 1. Selected 2D NMR correlations of lyconadin A (1). Figure 3. Selected 2D NMR correlations of 11-hydroxylycodine (2). Table 2.
Figure 2. Selected NOESY correlations (dotted arrows) and relative configurations for lyconadin A (1).
presence of a 4,5-disubstituted pyridone ring was inferred by the mutually coupled olefinic protons (δH 6.35 and 7.42, J ) 8.9 Hz) whose attached carbon resonances were observed at δC 116.64 and 141.60, respectively. HMBC correlations from H-3 to C-10 (δC 33.63) and Hb-9 to C-4 (δC 126.23) established the connection between C-4 and C-10. HMBC correlations of Hb-9 and H-13 to C-6 (δC 64.56) indicated that C-6 was attached to C-9 and C-13 through N-9, while HMBC correlations of H-6 to C-4 and C-5 suggested the C-5 and C-6 linkage. The final connectivity between C-6 and C-7 was supported by HMBC correlations of Ha-8 and H-12 to C-6. Thus, the gross structure of lyconadin A was assigned as 1. The relative stereostructure of 1 as shown in a computer-generated 3D drawing (Figure 2) was deduced from cross-peaks observed in the phase-sensitive NOESY spectrum and 3J proton coupling constants. NOESY correlations of H-12/Ha-8 and H-12/Ha-14 and large 1H1H couplings of H -8/H-15 (13.0 Hz) and H -14/H-15 (12.1 a a Hz), and small ones of H-12/H-13 (2.7 Hz) and H-7/H-12 (ca. 2 Hz), indicated that Ha-8, H-12, and Ha-14 had a 1,3-diaxial relationship. It was found that the cyclohexane ring (C-7-C-8 and C-12-C-15) took a chair form, which was consistent with a W-type long-range coupling between H-7 and H-13, both equatorial. On the other hand, NOESY cross-peaks of Ha-9/H-13, Ha-9/Ha-11, and Ha-11/H-13 indicated that the piperidine ring took a chair form as well. Both six-membered rings were cis fused to form the decahydroquinoline ring system by the NOESY correlation of H-12/H-13 and each small coupling constant (2.7 Hz). The relative stereochemistry at C-10 was elucidated by the NOESY correlation of H-3/H-10, and a small coupling of H-10 (10 µg/mL, respectively) in vitro. Experimental Section General Procedures. 1H and 13C NMR spectra were recorded in CD3OD and CDCl3 on 600 MHz and 500 MHz spectrometers (Bruker AMX600 and ARX500, respectively) at
Figure 4. Selected NOESY correlations (dotted arrows) and relative configurations for 11-hydroxylycodine (2).
300 K equipped with an X32 computer and an Eurotherm temperature control unit. 1D NMR spectra were measured at 300 K and were multiplied by a Gaussian filter and zero filled to 32K data points before Fourier transformation. 2D NMR spectra were measured at 300 K. NOESY and HOHAHA spectra in the phase-sensitive mode were recorded using the TPPI method. HOHAHA spectra were recorded by spin-lock field preceded and followed by 2.5 ms trim pulses. NOESY spectra were measured with mixing times of 800 ms. Typically, 256 FIDs of 2K data points and 32 scans each were employed. Chemical shifts were presented using residual CD3OD (δH 3.31 and δC 49.00) and CDCl3 (δH 7.26 and δC 77.03) as internal standards. Standard pulse sequences were employed for 2D NMR experiments. HMBC spectra were recorded using a 50 ms delay time for long-range C-H coupling with Z-axis PFG. FABMS was measured by using glycerol as a matrix. Material. The club moss L. complanatum was collected in Hokkaido in 1999. The botanical identification was made by Mr. N. Yoshida, Graduate School of Pharmaceutical Sciences, Hokkaido University. A voucher specimen has been deposited in the herbarium of Hokkaido University. Isolation. The club moss L. complanatum (300 g) was crushed and extracted with MeOH (1 L × 3). The MeOH extract (15 g) was treated with 3% tartaric acid (pH 2) and then partitioned with EtOAc. The aqueous layer was treated with saturated Na2CO3(aq) to pH 10 and extracted with CHCl3 to give a crude alkaloidal fraction (303 mg). This fraction was subjected to an amino silica gel column chromatography (Hexane/EtOAc, 1:0 f 1:1, and then CHCl3/MeOH, 1:0 f 0:1), in which fractions eluted with CHCl3/MeOH solvent system were purified by silica gel columns (CHCl3/MeOH/EtOAc, 10:1:0.5) to afford lyconadin A (1, 0.0003% yield) and 11-hydroxylycodine (2, 0.0003%), respectively, as colorless solid, together with complanadine A (0.003%) and lycodine (0.0005%). Lyconadin A (1): colorless solid; [R]D +14° (c 0.35, MeOH); IR (neat) νmax 2920, 1660, 1615, and 1455 cm-1; UV (MeOH) λmax 235 ( 6300), 323 nm (4800); 1H and 13C NMR data (Table 1); FABMS m/z 257 (M + H)+; HRFABMS m/z 257.1659 (M + H; calcd for C16H21N2O, 257.1654). (10) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092-4096. (11) Hemscheidt, T.; Spenser, I. D. J. Am. Chem. Soc. 1996, 118, 1799-1800. (12) Nyembo, L.; Goffin, A.; Hootele, C.; Braekman, J.-C. Can. J. Chem. 1978, 56, 851-856. (13) There are reports on two types of uncommon Lycopodium alkaloids, alopecurine14 (C16N-type) and fastigiatine15 (C16N2-type), with an extra carbon-carbon bond between C-4 and C-10. (14) Ayer, W. A.; Altenkirk, B.; Valverde-Lopez, S.; Douglas, B.; Raffauf, R. F.; Weisbach, J. A. Can. J. Chem. 1968, 46, 15-20. Ayer, W. A.; Altenkirk, B.; Masaki, N.; Valverde-Lopez, S. Can. J. Chem. 1969, 47, 2449-2455. (15) Gerard, R. V.; MacLean, D. B. Phytochemistry 1986, 25, 11431150. Gerard, R. V.; MacLean, D. B.; Fagianni, R.; Lock, C. J. Can. J. Chem. 1986, 64, 943-949.
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11-Hydroxylycodine (2): colorless solid; [R]D -47° (c 0.5, MeOH); IR (neat) νmax 3255, 2915, 1570, and 1430 cm-1; UV (MeOH) λmax 269 ( 3500); 1H and 13C NMR data (Table 2); FABMS m/z 259 (M + H)+; HRFABMS m/z 259.1811 (M + H; calcd for C16H23N2O, 259.1810). (R)- and (S)-MTPA Ester of 11-Hydroxylycodine (2). To a solution of 2 (0.1 mg) in CH2Cl2 (50 µL) were added (+)MTPACl (0.8 µL), triethylamine (0.5 µL), and N,N-dimethylaminopyridine (20 µg). The mixture was allowed to stand at room temperature for 6 h. N,N-Dimethylamino-1,3-propandiamine (1.0 µL) was added, and after evaporation of solvent, the residue was applied to a silica gel column (CHCl3-MeOH, 10:1) to give the (S)-MTPA ester of 3 (0.1 mg, 54%). The (R)-MTPA ester of 2 was prepared according to the same procedure as described above. (S)-MTPA ester of 2: 1H NMR (CDCl3) δ 8.45 (dd, 1.3, 4.7, H-1), 7.15 (m, H-2), 7.95 (dd, 1.1, 7.8, H-3), 3.13 (dd, 7.4, 19.2, H-6a), 2.63 (d, 19.2, H-6b), 3.22 (brt, 13.3 H-9a), 3.30 (brd, 13.3, H-9b), 2.33 (m, H-10a), 1.91 (m, H-10b), 4.89 (dt, 11.3, 5.1, H-11), 2.28 (dd, 3.0, 11.2, H-12), 1.82 (t, 12.0, H-14a), 1.52 (brd, 12.0,
Notes H-14b), 0.77 (d, 6.1, H-16); FABMS m/z 475 (M + H)+; HRFABMS m/z 475.2173 (M + H; calcd for C26H30N2O3F3, 475.2209). (R)-MTPA ester of 2: 1H NMR (CDCl3) δ 8.43 (brd, 4.7, H-1), 7.15 (dd, 4.7, 7.8, H-2), 7.95 (brd, 7.8, H-3), 3.20 (m, H-6a), 2.79 (d, 19.1, H-6b), 3.20 (m, H-9a), 3.26 (m, H-9b), 2.20 (m, H-10a), 1.85 (m, H-10b), 4.89 (dt, 11.3, 4.6, H-11), 2.30 (dd, 3.0, 11.2, H-12), 1.83 (m, H-14a), 1.55 (m, H-14b), 0.82 (d, 6.6, H-16); FABMS m/z 475 (M + H)+; HRFABMS m/z 475.2186 (M + H; calcd for C26H30N2O3F3, 475.2209).
Acknowledgment. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Supporting Information Available: 1D and 2D NMR spectra for compounds 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. JO0103874
