Hupercumines A and B, Lycopodium Alkaloids from Huperzia

Publication Date (Web): February 12, 2018. Copyright © 2018 American Chemical Society. *E-mail: [email protected]. Cite this:Org. Lett. 20, 5, 1384...
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Cite This: Org. Lett. 2018, 20, 1384−1387

Hupercumines A and B, Lycopodium Alkaloids from Huperzia cunninghamioides, Inhibiting Acetylcholinesterase Yusuke Hirasawa,† Chiho Mitsui,† Nahoko Uchiyama,‡ Takashi Hakamatsuka,‡ and Hiroshi Morita*,† †

Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41 Shinagawa-ku, Tokyo 142-8501, Japan National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan

‡

S Supporting Information *

ABSTRACT: A novel class of C38N4 Lycopodium alkaloid, hupercumine A (1), consisting of two octahydroquinolines, a decahydroquinoline, and a piperidine, and a new C27N3-type alkaloid, hupercumine B (2), were isolated from Huperzia cunninghamioides (Hayata) Holub. The structures and absolute configurations of 1 and 2 were elucidated on the basis of spectroscopic data, chemical means, and biogenetic point of view. Hupercumines A (1) and B (2) showed moderate inhibitory activity against acetylcholinesterase.

3.20, δC 56.0; δH 2.57, δC 56.0; δH 2.60, δC 48.7; δH 3.11, δC 49.7; δH 3.40, and δC 56.7; δH 2.91) and two sp3 methylenes (δC 46.4; δH 2.73 and 3.08 and δC 47.8; δH 2.69 and 3.12) were attributed to those attached to a nitrogen atom. The gross structure of 1 was elucidated by analysis of 2D NMR data including the 1H−1H COSY, TOCSY, HSQC, HMBC, and HSQC-TOCSY spectra in CDCl3 (Figure 1). The 1H−1H COSY, TOCSY, and HSQC-TOCSY spectra in CDCl3 revealed the presence of five partial structures, a (C-2−C-4), b (C-6−C-9 and C-12), c (C-11, C-13−C17, and C-11′), d (C-2′−C-4′ and C-2′′−C-12′′), and e (C-6′−C-9′ and C-12′), as shown in Figure 1. Connectivity of C-2 and C-6 through a nitrogen atom was revealed by the HMBC correlation of H-2b (δH 3.08)/C-6 (δC 56.8). HMBC cross-peaks of H-11b (δH 2.18)/C-5 (δC 132.3) and C-10 (δC 127.3), H-4b (δH 2.84)/C-5, C-6, and C-10, and H3-12 (δH 0.93)/C-10 (four bonds) indicated the connection among partial structures a, b, c, and tetrasubstituted olefinic carbons assigned to C-5 and C-10, constructing the octahydroquinoline ring (C-2−C-10 and N-1). Another octahydroquinoline moiety (C-2′−C-10′ and N-3) was elucidated in the same way as mentioned above. Also, a NOESY cross-peak of H-6′ (δH 3.40)/H-11′′b (δH 2.13) supported the connectivity of C-2′ and C-6′ through a nitrogen atom. Furthermore, the HMBC correlations of H-13 (δH 2.57)/ C-17 (δC 56.0), H-17 (δH 2.60)/C-13 (δC 56.0), and H-2′′b (δH 3.12)/C-6′′ (δC 56.7) revealed the connectivity between C-13 with C-17 and C-2′′ with C-6′′ through a nitrogen atom, respectively. Thus, the gross structure of hupercumine A was assigned as 1. The relative stereochemistry of 1 was elucidated by NOESY correlations and by comparison of chemical shifts with

Lycopodium alkaloids,1 with unique heterocyclic frameworks of C11N, C16N, C16N2, and C27N3 types, have attracted great interest from biogenetic2 and biological3 points of view and are challenging targets for total synthesis.4 Recently, we isolated new types of alkaloids, such as huperminone A,5 hupermine A,6 and lycobeline A,7 from various Huperzia spp. In our search for biogenetically interesting Lycopodium alkaloids, hupercumine A (1), a novel C38N4-type alkaloid consisting of two octahydroquinoline rings, a decahydroquinoline ring, and a piperidine ring, and hupermine B (2), a new C27N3-type alkaloid, were isolated from the club moss Huperzia cunninghamioides (Lycopodiaceae). In this paper, we describe the isolation and structure elucidation of 1 and 2. The club moss H. cunninghamioides (57 g) collected in Taiwan was extracted with MeOH, and the extract (6 g) was partitioned between EtOAc and 2% tartaric acid aq. Water-soluble materials, which were adjusted at pH 10 with satd Na2CO3 aq, were extracted with CHCl3. CHCl3-soluble materials (156 mg) were subjected to an amino silica gel column (hexane/EtOAc, 1:0 to 1:1, and then CHCl3/MeOH, 1:0 to 0:1) in which a fraction eluted with CHCl3/MeOH (160:1) was separated by a silica gel column (CHCl3/MeOH, 1:0 to 2:1, and then CHCl3/MeOH/ H2O/AcOH, 5:5:1:0 to 5:5:1:0.1), an ODS column (MeOH/ H2O/TFA, 0:1:0.01 to 1:0:0.01), and then an amino silica gel column (CHCl3/MeOH, 10:1) to afford hupercumines A (1, 5.9 mg) and B (2, 1.5 mg) together with huperzine A.8 Hupercumine A (1)9 was shown to have the molecular formula C38H64N4 by HRESIMS [m/z 577.5231, (M + H)+, Δ +2.2 mmu]. The IR spectrum was indicative of amine (3290 cm−1) functionality. 1H and 13C NMR data (Table 1) and the HSQC spectrum of 1 revealed 38 carbon signals due to 11 sp3 methines, 20 sp3 methylenes, three methyls, and four sp2 quaternary carbons. Among them, six sp3 methines (δC 56.8; δH © 2018 American Chemical Society

Received: January 16, 2018 Published: February 12, 2018 1384

DOI: 10.1021/acs.orglett.8b00152 Org. Lett. 2018, 20, 1384−1387

Letter

Organic Letters Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of Hupercumine A (1) in CDCl3 (δ in ppm, J in Hz) δH

no. 2a 2b 3a 3b 4a 4b 5 6 7a 7b 8 9a 9b 10 11a 11b 12 13 14a 14b 15a 15b 16a 16b 17

2.73 3.08 1.39 1.74 1.74 2.84

(1H, (1H, (1H, (1H, (1H, (1H,

ddd, 12.4, 12.4, 1.9) brd, 12.4) m) m) m) brd, 13.2)

3.20 0.95 1.92 1.60 1.70 1.92

(1H, (1H, (1H, (1H, (1H, (1H,

m) q, 12.4) m) m) m) m)

2.00 2.18 0.93 2.57 1.10 1.54 1.27 1.76 1.05 1.61 2.60

(1H, (1H, (3H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,

m) dd, 13.3, 8.4) d, 6.5) m) m) m) m) m) m) m) m)

δC

no.

δC

no.

46.4

2′

3.11 (1H, m)

48.7

28.6

3′a 3′b 4′a 4′b 5′ 6′ 7′a 7′b 8′ 9′a 9′b 10′ 11′a 11′b 12′

1.55 1.72 2.03 2.56

(1H, (1H, (1H, (1H,

m) m) m) m)

31.7

3.40 0.95 1.83 1.60 1.69 1.94

(1H, (1H, (1H, (1H, (1H, (1H,

m) q, 12.4) m) m) m) m)

2′′a 2′′b 3′′a 3′′b 4′′a 4′′b 5′′ 6′′ 7′′a 7′′b 8′′ 9′′a 9′′b 10′′ 11′′a 11′′b 12′′

27.6 132.3 56.8 39.9 27.9 39.9 127.3 40.4 22.0 56.0 32.8

δH

23.2

2.03 (1H, m) 2.14 (1H, m) 0.93 (3H, d, 6.5)

132.7 49.7 40.0 28.1 39.9 127.0 40.3 21.9

δH 2.69 3.12 1.33 1.51 1.41 2.03 1.21 2.91 1.16 1.62 1.73 0.54 1.84 1.93 0.82 2.13 0.83

(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (3H,

δC

ddd, 11.8, 11.8, 2.4) m) brd, 13.0) m) m) m) m) m) ddd, 13.4, 13.4, 3.5) m) m) ddd, 12.2, 12.2, 12.2) m) m) m) m) d, 6.5)

47.8 21.0 26.7 40.6 56.7 41.5 26.5 41.6 29.1 34.8 22.7

24.9 32.0 56.0

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

Figure 3. Selected NOESY correlations for hupercumine A (1).

were observed. These correlations indicated the α-orientation of H-6′, H-8′, and C-11′′ (Figure 3). In the decahydroquinoline moiety (C-2′′−C-10′′ and N-4), the correlations of H-5′′ (δH 1.21)/H-6′′ (δH 2.91) and H-9′′a (δH 0.54), H-7′′a (δH 1.16)/H-9′′a, and H-6′′/H-2′′a (δH 2.69) and H-4′′a (δH 1.41) suggested that both the cyclohexane ring and piperidine ring took a chair form and the junction of decahydroquinoline ring was cis. In addition, the NOESY crosspeaks of H-8′′ (δH 1.73)/H-10′′ (δH 1.93) indicated that these protons were β-oriented. In the piperidine moiety (C-13−C-17 and N-2), the chemical shifts of CH-13 (δC 56.0; δH 2.57), CH-17 (δC 56.0; δH 2.60), and CH2-15 (δC 24.9; δH 1.27 and 1.76) indicated that H-13 and H-17 were syn.10 In addition, the NOESY correlations of H-2′ (δH 3.11)/H-9′′b (δH 1.84), H-10″, and H-11′′a (δH 0.82) and H-6′/H-10′′ and H-11′′b revealed the relative configuration

Figure 2. NOESY correlation for hupercumine A (1).

tetrahydrodeoxylycoperine A.10 In the octahydroquinoline ring (C-2−C-10 and N-1), the NOESY correlation of H-6/H-8 suggested that H-6 and H-8 were oriented to the same side (Figure 2). In another octahydroquinoline ring (C-2′−C-10′ and N-3), the NOESY cross-peaks of H-6′/H-8′ (δH 1.60) and H-11′′b 1385

DOI: 10.1021/acs.orglett.8b00152 Org. Lett. 2018, 20, 1384−1387

Letter

Organic Letters Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Data of Hupercumine B (2) in CD3OD (δ in ppm, J in Hz) δH

no. 2a 2b 3a 3b 4a 4b 5 6 7a 7b 8 9a 9b 10 11a 11b 12 13 14a 14b 15a 15b 16a 16b 17 2′a 2′b 3′a 3′b 4′a 4′b 5′ 6′ 7′a 7′b 8′ 9′a 9′b 10′ 11′a 11′b 12′

2.72 3.04 1.46 1.76 1.80 2.88

(1H, (1H, (1H, (1H, (1H, (1H,

ddd, 12.0, 12.0, 2.4) brd, 12.0) qt, 13.0, 3.8) m) m) m)

3.22 1.01 1.94 1.62 1.72 1.99

(1H, (1H, (1H, (1H, (1H, (1H,

dd, 9.0, 9.0) m) m) m) m) brd, 16.2)

2.09 2.16 0.98 2.65 1.10 1.61 1.32 1.80 1.04 1.68 2.65 3.04 2.72 1.46 1.77 1.80 2.88

(1H, (1H, (3H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,

dd, 13.2, 6.6) m) d, 6.5) m) m) m) qt, 13.4, 4.1) m) m) m) m) brd, 12.0) ddd, 12.0, 12.0, 2.4) qt, 13.0, 3.8) m) m) m)

3.22 1.01 1.94 1.62 1.72 1.99

(1H, (1H, (1H, (1H, (1H, (1H,

dd, 9.0, 9.0) m) m) m) m) brd, 16.2)

2.17 (1H, m) 2.17 (1H, m) 0.98 (3H, d, 6.5)

between the octahydroquinoline ring (C-2′−C-10′ and N-3) and the decahydroquinoline ring (C-2′′−C-10′′ and N-4) was as shown in Figure 3. On the other hand, the molecular formula of hupercumine B (2)11 was determined to be C27H45N3 on the basis of HRESIMS, 1H NMR, 13C NMR, and HSQC data (Table 2). The 1H and 13C NMR data of 2 were analogous to those of tetrahydrodeoxylycoperine A,10 although two N-ethyl groups observed for tetrahydrodeoxylycoperine A were absent for 2. This conclusion was confirmed by 2D NMR spectra (1H−1H COSY, HMBC, and NOESY) of 2. Furthermore, the absolute configuration of hupercumine B was assigned to be 6R,8R,13S,17R,6′R,8′R from chemical conversion from 2 to lycoperine A by acetylation4e,10 (see the Supporting Information, S2). Hupercumine A (1) has a structure in which the C-2′ (or C-2) of hupercumine B (2) (6R,8R,13S,17R,6′R,8′R) is bonded with a decahydroquinoline unit. Considering that hupercumines A (1) and B (2) were isolated from the same plant, the absolute configuration of 1 was considered as 6R,8R,13S*,17R*,2′S,6′R,8′R,5′′R,6′′S,8′′R,10′′R. This presumption was supported by the similar CD data of 1 [203 (Δε −11.6) nm] and 2 [201 (Δε −7.1) nm]. A plausible biogenetic pathway for hupercumines A (1) and B (2) is proposed as shown in Scheme 1. Hupercumine A might be generated from three C11N units (A)10 and a Δ1-piperidine unit through hupercumine B (2). Hupercumines A and B inhibited acetylcholinesterase (from bovine erythrocyte) with IC50, 41.9 μM and 92.3 μM, respectively (huperzine A: IC50, 98.1 nM).12 In summary, we have described here the isolation and structure elucidation of the first C38N4-type Lycopodium alkaloid, hupercumine A (1), and biogenetically related alkaloid, hupercumine B (2). Hupercumine A consists of two octahydroquinoline rings, a decahydroquinoline ring, and a piperidine ring, which are connected by a methylene unit with each other. In addition, 1 and 2 showed moderate inhibitory effects against acetylcholinesterase.

δC 47.0 28.5 28.7 133.2 57.8 40.5 29.2 41.0 129.2 40.6 22.3 57.6 33.0 25.7 32.0 57.3 47.1 28.6 28.6 132.7 57.9 40.4

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29.3 40.9

ASSOCIATED CONTENT

S Supporting Information *

129.0 40.6

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00152.

22.3

Scheme 1. Plausible Biogenetic Pathway for Hupercumines A (1) and B (2)

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DOI: 10.1021/acs.orglett.8b00152 Org. Lett. 2018, 20, 1384−1387

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Organic Letters

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Detailed experimental procedures and 1D and 2D NMR spectra (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hiroshi Morita: 0000-0002-7950-7722 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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REFERENCES

(1) For reviews of the Lycopodium alkaloids, see: (a) Kitajima, M.; Takayama, H. Top. Curr. Chem. 2011, 309, 1−31. (b) Hirasawa, Y.; Kobayashi, J.; Morita, H. Heterocycles 2009, 77, 679−729. (c) Kobayashi, J.; Morita, H. In The Alkaloids; Cordell, G. A., Ed.: Academic Press: New York, 2005; Vol. 61, p 1. (d) Ayer, W. A. Nat. Prod. Rep. 1991, 8, 455−463 and references cited therein. (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) Ma, X.; Tan, C.; Zhu, D.; Gang, D. R.; Xiao, P. J. Ethnopharmacol. 2007, 113, 15−34. (4) (a) Azuma, M.; Yoshikawa, T.; Kogure, N.; Kitajima, M.; Takayama, H. J. Am. Chem. Soc. 2014, 136, 11618−11621. (b) Snider, B. B.; Grabowski, J. F. J. Org. Chem. 2007, 72, 1039−1042. (c) Beshore, D. C.; Smith, A. B., III J. Am. Chem. Soc. 2007, 129, 4148−4149. (d) Canham, S. M.; France, D. J.; Overman, L. E. J. Am. Chem. Soc. 2010, 132, 7876−7877. (e) Nakamura, Y.; Burke, A. M.; Kotani, S.; Ziller, J. W.; Rychnovsky, S. D. Org. Lett. 2010, 12, 72−75. (f) Fischer, D. F.; Sarpong, R. J. Am. Chem. Soc. 2010, 132, 5926−5927. (g) Yuan, C.; Chang, C.-T.; Axelrod, A.; Siegel, D. J. Am. Chem. Soc. 2010, 132, 5924−5925 and references cited therein. (5) Hirasawa, Y.; Kato, Y.; Wong, C. P.; Uchiyama, N.; Goda, Y.; Hadi, A. H. A.; Morita, H. Tetrahedron Lett. 2013, 54, 1593−1595. (6) Hirasawa, Y.; Kato, Y.; Wong, C. P.; Uchiyama, N.; Goda, Y.; Hadi, A. H. A.; Ali, H. M.; Morita, H. Tetrahedron Lett. 2014, 55, 1902−1904. (7) Hirasawa, Y.; Matsuya, R.; Shaari, K.; Lajis, N. H.; Uchiyama, N.; Goda, Y.; Morita, H. Tetrahedron Lett. 2012, 53, 3971−3973. (8) 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. (9) Hupercumine A (1): colorless amorphous solid; [α]D28 −15 (c 1.0, MeOH); IR (Zn−Se) νmax 3290, 2921, 2861, 1682, and 1445 cm−1; CD (MeOH) λmax 192 (Δε + 10.3), 196 (−14.9), 198 (+1.0), 200 (−9.7), 201 (−8.2), and 203 (−11.6) nm; 1H and 13C NMR (Table 1); HRESIMS m/z 577.5231 (M + H; calcd for C38H65N4, 577.5209). (10) Hirasawa, Y.; Kobayashi, J.; Morita, H. Org. Lett. 2006, 8, 123− 126. (11) Hupercumine B (2): colorless amorphous solid; [α]D29 −57 (c 0.75, MeOH); IR (Zn−Se) νmax 3290, 2924, 2861, 1654, 1447, and 1112 cm−1; CD (MeOH) λmax 193 (Δε − 1.5), 195 (−13.5), 197 (+4.1), 199 (−8.2), 200 (−6.7), and 201 (−7.1) nm; 1H and 13C NMR (Table 2); HRESIMS m/z 412.3700 (M + H; calcd for C27H46N3, 412.3692) (12) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88−95.

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