Tetrahedron Letters 52 (2011) 4126–4128

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Tetrahedron Letters

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Lycotetrastine A, a novel hexacyclic alkaloid from Huperzia tetrasticha

Yusuke Hirasawa a, Adil Astulla a, Motoo Shiro b, Hiroshi Morita a,⇑a Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41 Shinagawa-ku, Tokyo 142-8501, Japanb X-ray Research Laboratory, Rigaku Corporation, Akishima, Tokyo 196-8666, Japan

a r t i c l e i n f o

Article history:Received 11 May 2011Revised 23 May 2011Accepted 27 May 2011Available online 12 June 2011

0040-4039/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.tetlet.2011.05.133

⇑ Corresponding author.E-mail address: moritah@hoshi.ac.jp (H. Morita).

a b s t r a c t

A novel C20N-type Lycopodium alkaloid with an unprecedented fused-hexacyclic ring system consisting ofa c-lactone, an aza-cycloheptene, an aza-cyclohexane, a cyclohexane, a cyclopentane, and tetrahydrofu-ran rings, lycotetrastine A (1) was isolated from the club moss of Huperzia tetrasticha. The structure andabsolute stereochemistry were elucidated on the basis of 2D NMR correlations and X-ray analysis.

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Lycopodium alkaloids1 with unique heterocyclic frameworks ofC11N, C16N, C16N2, C22N2, and C27N3 types have attracted greatinterest from biogenetic1,2 and biological3 points of view. A com-mon feature in all Lycopodium alkaloids is a polycyclic carbon skel-eton with different levels of oxidation. These unique skeletonshave also been challenging targets for total synthesis.4 Amongthem, huperzine A is a highly specific and potent inhibitor of ace-tylcholinesterase (AChE).3 The inherent inhibition of AChE hasprompted the pursuit of the total synthesis5 and SAR6 studies ofhuperzine A. Recently, we isolated new types of alkaloids such aslycochinine A7 from Lycopodium chinense, malycorin A8 from Lyco-podium phlegmaria, and lycoparin A9 from Lycopodium casuarino-ides. During our continuing search for biogenetically interestingintermediates and new alkaloids with a novel skeleton from Lyco-podium and Huperzia species, lycotetrastine A (1), a novel alkaloidwith an unprecedented fused-hexacyclic ring system consisting ofa c-lactone, an aza-cycloheptene, an aza-cyclohexane, a cyclohex-ane, a cyclopentane, and tetrahydrofuran rings was isolated fromthe club moss Huperzia tetrasticha (Kunze ex Alderw.) Holub. Inthis Letter we describe the isolation and structure elucidation of 1.

The club moss of H. tetrasticha (10 g) was extracted with MeOH

(100 mL � 3) at rt, and the extract (430 mg) was partitioned be-tween EtOAc and 3% aq tartaric acid. The water-soluble fraction

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was adjusted to pH 9 with saturated Na2CO3 and was extractedwith CHCl3. The CHCl3-soluble fraction (28.6 mg) was subjectedto amino silica gel column chromatography (elution, hexane/EtOAc, 1:1 and then CHCl3/MeOH, 1:0 ? 0:1). The fraction elutedwith CHCl3/MeOH (50:1) was purified by a silica gel column (CHCl3

saturated by NH4OH/MeOH, 1:0 ? 0:1) to afford lycotetrastine A(1, 1.5 mg, 0.015%) and huperzine A (2.1 mg, 0.021%).

Lycotetrastine A10 {1, ½a�20D �4 (c 1.0, MeOH)} showed a pseudo-

molecular ion peak at m/z 346 (M+H)+ in the ESI-MS. The molecularformula was established to be C20H27NO4 by the HR-ESI-TOF-MS[m/z 346.2022 (M+H)+, D +0.4 mmu] and its structure was estab-lished mainly on the basis of the NMR data. Its 13C NMR spectrum(Table 1) revealed twenty carbon signals due to one ester carbonyl,one quaternary, and one methine sp2 carbons, two sp3 quaternarycarbons, six sp3 methines, eight sp3 methylenes, and one methylgroup. Among them, two sp3 methylenes (dC 51.2; dH 2.97, 3.37and dC 48.1; dH 2.82, 3.40) were ascribed to those bearing a nitro-gen atom, two sp3 methines (dC 90.4; dH 4.50 and dC 68.3; dH 4.21)and one sp3 methylene (dC 72.3; dH 3.89, 4.05) to those bearing anoxygen atom, and one sp3 quaternary carbon (dC 96.8) to that bear-ing both an oxygen and nitrogen atoms.

Partial structures a (C-1–C-3), b (C-9–C-11), and c (C-5–C-8, C-14–C-16, and C-18–C-20) shown in Figure 1 were deduced from adetailed analysis of the 1H-1H COSY spectrum. The HMBC cross-peaks of H-3/C-5 and C-12, H-5/C-4, H-7/C-12, H-11b/C-12, andH-14a/C-12 indicated that the three units a, b, and c were con-nected through a C-4–C-12 bond. The HMBC correlations of H-1a/C-13 and H-9a/C-13, and the 13C chemical shifts of C-1, C-9,and C-13 implied that C-1, C-9, and C-13 were all connected to anitrogen atom. The presence of a tetrahydrofuran ring (C-13–C-16 and O) was indicated by the HMBC correlations of H-16a/C-13and H-14a/C-13. Furthermore, the HMBC correlation of H-18/C-17 and the low chemical shifts for 1H and 13C at C-6 in partial struc-ture c established the connection between C-6 and C-18 throughan ester bridge, constructing a c-lactone ring (C-5–C-6, C-17–C-18, and O). Thus, the gross structure of lycotetrastine A was eluci-dated to be 1, possessing a fused hexa-cyclic skeleton.

Table 11H and 13C NMR data of lycotetrastine A (1) in CD3OD

dH dc HMBC

1a 2.97 (1H, dd, 14.2, 5.3) 51.2 3, 91b 3.37 (1H, ddd, 14.2, 13.4, 4.3)2a 2.09 (1H, ddd, 18.2, 7.9, 4.3) 30.6 32b 2.78 (1H, m)3 5.72 (1H, br d, 7.9) 126.2 1a, 2a, 54 151.0 2a, 3, 5, 6, 7, 11b, 185 3.70 (1H, br s) 47.5 3, 7, 196 4.50 (1H, d, 6.5) 90.4 7, 8a, 187 2.35 (1H, dd, 12.7, 7.9) 50.3 8a8a 1.23 (1H, dd, 12.9, 12.7) 34.3 6, 7, 14, 168b 1.92 (1H, m)9a 2.82 (1H, dd, 14.0, 4.7) 48.1 1b9b 3.40 (1H, ddd, 14.1, 14.0, 4.1)10a 1.37 (1H, m) 23.2 3b10b 1.87 (1H, m)11a 1.70 (1H, br d, 13.4) 35.7 7, 9a11b 2.18 (1H, ddd, 13.7, 13.4, 4.1)12 58.1 3, 6, 11b, 14a13 96.8 1a, 9a, 11a, 14a, 16a14a 1.75 (1H, ddd, 10.2, 5.2, 1.7) 41.3 16a14b 2.29 (1H, d, 10.2)15 2.52 (1H, m) 37.6 8a, 14b, 16a16a 3.89 (1H, d, 7.3) 72.3 8a, 14b16b 4.05 (1H, dd, 7.3, 4.8)17 181.3 5, 1818 2.48 (1H, dd, 3.0, 1.9) 61.6 5, 2019 4.21 (1H, qd, 6.4, 3.0) 68.3 5, 18, 2020 1.34 (3H, d, 6.4) 22.1

Figure 1. Selected 2D NMR correlations for lycotetrastine A (1).

Figure 3. X-ray structure of lycotetrastine A (1).

Scheme 1. Plausible biogenetic pathway for lycotetrastine A (1).

Y. Hirasawa et al. / Tetrahedron Letters 52 (2011) 4126–4128 4127

The relative stereochemistry of 1 was elucidated by the NOESYcorrelations (Fig. 2). The NOESY correlation of H-1b/H2-14 indi-cated both C-1 and C-14 were oriented on the same side, and therelative configurations of C-12 and C-13 were assigned to be S⁄

and R⁄, respectively. The a-configuration of H-7 was elucidated

Figure 2. Selected NOESY correlations for lycotetrastine A (1).

by the NOESY correlation between H-7 and H-16a. The NOESY cor-relations of H-5/H-8a and H-6/H-8a, and H-11a/H-18 suggested b-configuration of H-5 and H-6, and a-configuration of H-18.

Finally, the absolute stereochemistry of 1 was assigned as5R,6R,7R,12S,13R,15R,18R,19S by use of Flack parameter [v = 0.04(15)]11–13 of X-ray analysis of lycotetrastine A (1) in Figure 3.

A plausible biogenetic pathway of lycotetrastine A (1) was pro-posed as shown in Scheme 1. Biogenetically, 1 may be derived fromlycodoline14 through a fawcettidane skeleton1 followed by cycliza-tion with acetoacetic acid at C-5 and C-6. Lycotetrastine A (1) is thefirst example of a new type of hybrid structure consisting of huper-zine Q15 and megastachine.16

Lycotetrastine A (1) inhibited acetylcholinesterase (from bovineerythrocyte) with IC50, 85 lM,17 in comparison with that (IC50,53 nM) of (�)-huperzine A.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Re-search from the Ministry of Education, Culture, Sports, Science,and Technology of Japan, The Open Research Center Project, andTakeda Science Foundation.

References and notes

1. For reviews of the Lycopodium alkaloids, see: (a) Hirasawa, Y.; Kobayashi, J.;Morita, H. Heterocycles 2009, 77, 679–729; (b) Kobayashi, J.; Morita, H. In TheAlkaloids; Cordell, G. A., Ed.; Academic Press: New York, 2004; 61, p 1; (c) Ayer,

4128 Y. Hirasawa et al. / Tetrahedron Letters 52 (2011) 4126–4128

W. A.; Trifonov, L. S. In The Alkaloids; Cordell, G. A., Brossi, A., Eds.; AcademicPress: New York, 1994; 45, p 233; (d) Ayer, W. A. Nat. Prod. Rep. 1991, 8, 455–463; (e) MacLean, D. B. In The Alkaloids; Brossi, A., Ed.; Academic Press: NewYork, 1985; 26, p 241; (f) MacLean, D. B. In The Alkaloids; Manske, R. H. F., Ed.;Academic Press: New York, 1973; 14, p 348; MacLean, D. B. In The Alkaloids;Manske, R. H. F., Ed.; Academic Press: New York, 1968; 10, p 305.

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) Snider, B. B.; Grabowski, J. F. J. Org. Chem. 2007, 72, 1039–1042; (b) Beshore,D. C.; Smith, A. B., III J. Am. Chem. Soc. 2007, 129, 4148–4149; (c) Canham, S. M.;France, D. J.; Overman, L. E. J. Am. Chem. Soc. 2010, 132, 7876–7877; (d)Nakamura, Y.; Burke, A. M.; Kotani, S.; Ziller, J. W.; Rychnovsky, S. D. Org. Lett.2010, 12, 72–75; (e) Fischer, D. F.; Sarpong, R. J. Am. Chem. Soc. 2010, 132, 5926–5927; (f) Yuan, C.; Chang, C.-T.; Axelrod, A.; Siegel, D. J. Am. Chem. Soc. 2010,132, 5924–5925. and references cited therein.

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10. Lycotetrastine A (1): colorless platelet; mp 182–183 �C (from MeOH–H2O); ½a�20D

�4 (c 1.0, MeOH); IR (neat) mmax 3390, 2930, 1760, 1030, and 750 cm�1; CD(MeOH) kext 201 (h 6400), 220 (1300), and 228 (1600) nm; 1H and 13C NMRdata (Table 1); ESI-MS m/z 346 (M+H)+; HR-ESI-TOF-MS m/z 346.2022 (M+H;calcd for C20H28NO4, 346.2018).

11. Crystal data for lycotetrastine A (1): mp 182–183 �C (from MeOH–H2O),C20H27NO4, Mr = 345.44, monoclinic, space group P21 (#4), a = 7.23584 (13)Å, b = 16.7433 (3) Å, c = 7.3010 (5) Å, V = 832.08 (6) Å3, Z = 2, Dcalc = 1.379 g/cm3, The final R value was 0.0302 (wR2 = 0.0777) for 2981 reflections [I >2.00r(I)].

12. Flack, H. D. Acta Cryst. 1983, A39, 876–881.13. CCDC 820390 contain the supplementary crystallographic data for this Letter.

These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/deposit, or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +441223 336 033; e-mail: deposit@ccdc.cam.ac.uk).

14. Ayer, W. A.; Iverach, G. G. Can. J. Chem. 1964, 42, 2514–2522.15. (a) Huperzine Q consists of a fawcettidane skeleton with an ether bridge at C-

13 and C-16.; (b) Tan, C. H.; Ma, X. Q.; Chen, G. F.; Zhu, D. Y. Helv. Chim. Acta2002, 85, 1058–1061.

16. (a) Megastachine consists of a fawcettidane skeleton with an additional C4 unitat C-5 and C-6.; (b) Braekman, J. C.; Hootele, C.; Miller, N.; Declercq, J. P.;Germain, G.; Van Meerssche, M. Can. J. Chem. 1979, 57, 1691–1693.

17. Ellman, G. L.; Courtney, K. D.; Anders, V.; Featherstone, R. M. Biochem.Pharmacol. 1961, 7, 88–90.