Lycocasuarines A–C, Lycopodium alkaloids from Lycopodiastrum casuarinoides

Tetrahedron Letters 58 (2017) 4827–4831

Contents lists available at ScienceDirect

Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet

Lycocasuarines A–C, Lycopodium alkaloids from Lycopodiastrum casuarinoides Lu-Lu Wang a, Zhong-Bo Zhou b, Xin-Liu Zhu a, Fang-Yu Yuan a, Tomofumi Miyamoto c, Ke Pan a,⇑ a State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 639 Longmian Road, Nanjing 211198, People’s Republic of China b Engineering Laboratory of Chemical Resources Utilization in South Xinjiang of Xinjiang Production and Construction Corps, Tarim University, Alaer 843300, People’s Republic of China c Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582, Japan

a r t i c l e

i n f o

Article history: Received 13 October 2017 Revised 7 November 2017 Accepted 10 November 2017 Available online 11 November 2017 Keywords: Lycopodiastrum casuarinoides Lycopodium alkaloids Lycodine-type alkaloids Acetylcholinesterase

a b s t r a c t Three lycodine-type Lycopodium alkaloids (1–3) were isolated from Lycopodiastrum casuarinoides. Their structures were elucidated by spectroscopic analysis, single-crystal X-ray crystallography, and computational methods. Compound 1 possesses a rearranged five-membered ring D resulting from C-8/C-15 cleavage and a new C-7/C-15 linkage. Compound 2 is the first Lycopodium alkaloid found to bear an additional carbon (C-17) directly bonded to C-8, which is particularly unusual from a biogenetic point of view. Compounds 1–3 were evaluated for acetylcholinesterase inhibitory activities. Ó 2017 Elsevier Ltd. All rights reserved.

As a subclass of Lycopodium alkaloids, the lycodine-type alkaloids, such as the well-known huperzine A,1 usually possess potent acetylcholinesterase (AChE) inhibitory activities, attracting great interest from synthetic, biogenetic, and biological perspectives.2–4 Lycopodiastrum casuarinoides (Spring) Holub (Lycopodiaceae) is a medicinal plant that is widely distributed in South China and is used to treat contusion, strain, and swelling. Previous phytochemical studies on L. casuarinoides indicated that this plant was a rich source of lycodine-type alkaloids and thus considered to be a promising natural remedy for Alzheimer’s disease.5–9 As part of our efforts to explore structurally interesting and bioactive Lycopodium alkaloids, the whole plant of L. casuarinoides was investigated, leading to the isolation of three unusual lycodine-type alkaloids, namely, lycocasuarines A–C (1–3) (Fig. 1). Compound 1 possesses a rearranged five-membered ring D resulting from C-8/C-15 scission and a new C-7/C-15 linkage. Compound 2 is the first Lycopodium alkaloid found to bear an additional carbon (C-17) directly bonded to C-8. Additionally, compound 3 is a rare lycodine-type alkaloid with a rearranged five-membered ring C. We herein report the structure elucidation, possible biosynthetic pathway and biological evaluation of these compounds. ⇑ Corresponding author. E-mail address: [email protected] (K. Pan). https://doi.org/10.1016/j.tetlet.2017.11.019 0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.

Lycocasuarine A (1)10 was obtained as a colorless crystal. Its molecular formula, C17H24N2O3, was determined by HRESIMS at m/z 305.1868 ([M+H]+, calcd 305.1860), corresponding to seven degrees of unsaturation. The IR spectrum of 1 displayed an absorption for an a,b-unsaturated carbonyl (1665 cm 1) group. UV absorptions at kmax 234 and 316 nm indicated the presence of an a-pyridone ring. In the 1H NMR spectrum (Table 1), signals for a tertiary methyl at dH 1.49 (3H, s), two N-methyls at dH 2.43 (6H, s), and an exomethylene group at dH 5.11 (1H, dd, J = 9.9, 2.0 Hz) and 5.22 (1H, dd, J = 17.1, 2.0 Hz), were readily observed. The 13C NMR (Table 1) and HSQC spectra exhibited 17 carbon resonances and confirmed the presence of an a-pyridone ring (dC 117.1, 123.0, 142.3, 145.4 and 165.3) and an exocyclic double band (dC 119.1 and 138.1). The aforementioned evidence suggested that 1 was a ring C-cleaved lycodine-type alkaloid. The interpretation of 2D NMR data (Fig. 2) allowed the elucidation of the planar structure of 1. In addition to the C-2–C-3 fragment, another short partial structure (C-10–C-12) was deduced from the 1H–1H COSY spectrum, indicating that C-7 was a quaternary carbon. This deduction was also supported by the splitting patterns and coupling constants of H2-6 at dH 2.39 and 3.18 (each 1H, d, J = 18.1 Hz). The HMBC correlations from the oxygenated methylene at dH 3.47 and 3.67 (each 1H, d, J = 11.1 Hz) to C-7 (dC 52.7), C-12 (dC 50.4) and C-6 (dC 31.6) placed this oxygenated

4828

L.-L. Wang et al. / Tetrahedron Letters 58 (2017) 4827–4831

Fig. 1. Structures of Lycocasuarines A–C (1–3).

methylene at C-8, suggesting that 1 was an uncommon 8,15-seco product. Since the a-pyridone (ring A), the cyclohexene (ring B), and the C-10/C-11 double bond already accounted for six unsaturations, the remaining unsaturation must be due to an additional ring. The key HMBC correlations from Me-16 to C-7 and from H6, H-12 and H-8 to C-15 revealed that 1 possessed a rearranged five-membered ring D with a C-7/C-15 bond. The relative configuration of 1 was determined by a ROESY experiment (Fig. 2). Specifically, the ROESY correlations of H-12/ Me-16 and H-12/H-14a revealed that Me-16 was a-oriented. Finally, 1 was recrystallized from a mixture of MeOH/EtOAc (vol/ vol, 1/5). X-ray diffraction analysis using Cu Ka radiation not only confirmed the structures deduced above but also established the absolute configuration of 1, as shown (Fig. 3) [Flack parameter = 0.02(8)].11,12 Lycocasuarine B (2)13 was obtained as a white powder. Its molecular formula, C18H24N2O3, was deduced from HRESIMS. The 1 H NMR spectrum of 2 exhibited a pair of intercoupled aryl proton signals at dH 7.70 and 6.46 (each 1H, d, J = 9.4 Hz) for an a-pyridone ring; a set of olefinic proton signals at dH 6.07 (1H, dt, J = 17.0, 10.2 Hz), 5.26 (1H, d, J = 17.0 Hz), and 5.07 (1H, d, J = 10.2 Hz), attributed to a monosubstituted vinyl group (CH2@CHAR); and two Nmethyl resonances at dH 2.42 (6H, s). These data implied that 2, similar to 1, was a ring C-cleaved lycodine-type product with two N-methyl groups. The 13C NMR data (Table 1) of 2 displayed

Fig. 2. Selected 2D NMR correlations for 1.

resonances for a total of 18 carbons, which was one more carbon than 1 and other reported analogues,6,7 suggesting that 2 had a modified carbon skeleton. In addition, a methine resonance at dC 103.4 indicated the existence of a hemiacetal functionality in 2. Analysis of the 1H–1H COSY spectrum of 2 revealed the presence of three partial structures (Fig. 4). Although there was no obvious 1 H–1H COSY correlation of H-7/H-8, the HMBC correlations from H-8 to C-6, C-7, and C-12 established the C-7/C-8 linkage. The oxygenated methylene (H-17) showed 1H–1H COSY correlation with H-8, indicating its (C-17) direct attachment to C-8. This deduction was further corroborated by the HMBC correlations from H-17 to C-7 and C-8. The hemiacetal carbon (dC 103.4) was located at C16, as revealed by the HMBC correlations from H-16 (dH 5.05) to C-8, C-14 and C-15 and from H-15 to C-16. Finally, the HMBC correlations from H-16 to C-17 and from H-17 to C-16 indicated a linkage between C-16 and C-17 via an oxygen atom. In the NOESY spectrum, the correlation of H-12/H-14a suggested the b-orientation of H-12. No useful NOESY correlations were observed for H-15. However, the J14a–15 value (12.4 Hz) required H-15 to be trans-diaxial with H-14a. This observation, together with the NOESY correlation of H-8/H-6b, indicated that

Table 1 H and 13C NMR data for compounds 1–3 in CD3OD.

1

No.

1a dC

1 2 3 4 5 6a 6b 7 8a 8b 9 10a 10b 11 12 13 14a 14b 15 16 17a 17b N-MeA N-MeB a b

165.3 117.1 142.3 123.0 145.4 31.6 52.7 66.5

119.1 138.1 50.4 68.7 54.1 77.8 29.0

40.5

2b dH multi (J in Hz)

164.6 118.1 142.6 118.0 143.9 31.4

6.40, d (9.4) 7.69, d (9.4)

2.39, d (18.1) 3.18, d (18.1)

37.9 42.3

3.67, d (11.1) 3.47, d (11.1) 5.22, 5.11, 5.73, 2.92,

dd (17.1, 2.0) dd (9.9, 2.0) dt (17.1, 9.9) d (9.9)

2.33, d (13.6) 1.63, d (13.6)

116.1 140.8 43.5 60.4 38.5 44.4 103.4 69.8

1.49, s

2.43, s 2.43, s

Recorded at 500 MHz for 1H NMR and 125 MHz for Recorded at 400 MHz for 1H NMR and 100 MHz for

dC

39.0 13

C NMR. C NMR.

13

3b dH multi (J in Hz) 6.46, d (9.4) 7.70, d (9.4)

3.12, 2.33, 2.03, 2.58,

dd (18.1, 5.8) d (18.1) br s m

5.26, 5.07, 6.07, 3.02,

d (17.0) d (10.2) dt (17.0, 10.2) d (7.9)

1.81, 1.43, 1.63, 5.05, 3.88, 4.14, 2.42, 2.42,

t (12.4) dd (12.4, 5.8) m s t (9.3) t (8.8) s s

dC 165.8 118.7 142.5 120.3 143.7 29.5

dH multi (J in Hz) 6.39, d (9.4) 7.72, d (9.4)

32.3 127.2

2.93, 2.43, 2.83, 5.57,

48.3 66.0

2.75, d (7.5) 3.59, 2H, d (6.6)

42.0 49.2 62.8 45.6

2.05, m 1.73, dd (10.1, 3.9)

135.0 23.2

dd (17.9, 5.0) d (17.9) m d (5.4)

2.39, d (16.5) 2.13, d (16.5) 1.62, s

L.-L. Wang et al. / Tetrahedron Letters 58 (2017) 4827–4831

4829

Fig. 5. Experimental and calculated CD spectra of 2.

Fig. 3. X-ray crystal structure of 1.

Fig. 6. X-ray crystal structure of 3.

Fig. 4. Selected 2D NMR correlations for 2.

the hemiacetal-functional tetrahydrofuran ring is cis-fused to ring D. In addition, H-16 at dH 5.05 (1H, s) showed no recognizable vicinal coupling with H-15, implying that H-15 and H-16 were trans to each other with a dihedral angle of approximately 90°. The structure of 2, including its relative configuration, was thus established and further corroborated by computational methods (Supporting information S31–S54). Finally, the calculated CD spectrum of (7S,8R,12R,13R,15S,16S)-2 at the B3LYP/6-311G(d,p) level with the PCM in MeOH matched well with the experimental CD spectrum (Fig. 5), allowing the assignment of the absolute configuration of 2 as 7S,8R,12R,13R,15S,16S. Lycocasuarine C (3)14 was obtained as a colorless crystal. Its molecular formula, C16H20N2O2, was suggested by HRESIMS. A comparison between the 13C NMR data of 3 (Table 1) and those of casuarinine I revealed that 3 closely resembled casuarinine I.6 The marked differences were the absence of N-methyl and the existence of a hydroxymethyl instead of a secondary methyl in 3. Extensive analyses of 2D NMR data (Fig. S25, Supporting Information) allowed us to establish the planar structure of 3. The relative configuration of 3 was determined by ROESY correlations. In particular, the ROESY correlation of H-11/H-6a was indicative of the a-orientation of H-11. Finally, the absolute configuration of 3

was established by an X-ray diffraction experiment (Fig. 6) using Cu Ka radiation [Flack parameter = 0.10(6)].15 The possible biosynthetic pathways for 1–3 were proposed (Scheme 1). Huperzinine, the major alkaloid of this plant,8 undergoes C-8/C-15 oxidative cleavage to form the intermediate E, which can spontaneously convert to its enol form F. This enol intermediate cyclizes via an aldol reaction with the C-15 carbonyl group to afford a C-8 aldehyde intermediate G, which is finally reduced to compound 1. For compound 2, the biogenetic origin of C-17 is a major concern. Hypothetically, 2 can be tracked back to a methylated product of huperzinine (intermediate B, path A). However, considering that C-17 is directly linked to C-8, another hypothetical biosynthetic pathway (path B) for 2 should be stressed. Contrary to the existing understanding that the coupling of L-Lysinederived pelletierine and 4-(2-piperidyl)acetoacetate (4PAA) is accompanied by a requisite decarboxylation to afford phlegmarine, which is a common precursor for Lycopodium alkaloids,3,16 compound 2 could be derived from intermediate A, suggesting that the decarboxylase involved might be inactivated under certain conditions. Following this hypothesis, the isolation of 2 might also provide new evidence that the basic carbon skeleton of Lycopodium alkaloids originates from the condensation of pelletierine and 4PAA rather than two pelletierines, as proposed by Professor Ian Spenser based on feeding experiments.17–21 After casuarinine I, compound 3 is the second lycodine-type alkaloid with a rearranged five-membered ring C. It has been hypothesized that casuarinine I is generated from intermediate D, which is biosynthesized from huperzinine by a series of reactions, particularly intramolecular 1,3-dipolar cycloaddition.6 In

4830

L.-L. Wang et al. / Tetrahedron Letters 58 (2017) 4827–4831

Scheme 1. Hypothetical Biosynthetic Pathways for 1–3.

light of this hypothesis, a plausible biosynthetic pathway for 3, a key intermediate for casuarinine I, is outlined. All isolates were tested for AChE inhibitory activities by the Ellman method (huperzine A as positive control, IC50 = 0.05 lM).22,23 Compound 3 showed moderate activity (IC50 = 7.4 lM), while 1 and 2 showed no inhibitory activity (IC50 >100 lM). In summary, we have identified three new lycodine-type alkaloids (1–3) from L. casuarinoides. Each of these alkaloids has structurally interesting elements, representing new additions to the structure diversity of the Lycopodium alkaloids. Notably, lycocasuarine B (2) is the first Lycopodium alkaloid found to bear an additional carbon (C-17) bonded to C-8. To understand the exact biosynthetic mechanism of 2 and its implication on Lycopodium alkaloid biosynthesis, further genetic and biochemical investigations are awaited.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11.

Acknowledgement This research work was financially supported by the National Natural Science Foundation of China (No. 81403083). A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.tetlet.2017.11.019. These data include MOL files and InChiKeys of the most important compounds described in this article.

12. 13.

Liu JS, Zhu YL, Yu CM, et al. Can J Chem. 1986;64:837–839. Ayer WA. Nat Prod Rep. 1991;8:455–463. Ma XQ, Gang DR. Nat Prod Rep. 2004;21:752–772. Azuma M, Yoshikawa T, Kogure N, Kitajima M, Takayama H. J Am Chem Soc. 2014;136:11618–11621. Liu F, Wu XD, He J, et al. Tetrahedron Lett. 2013;54:4555–4557. Tang Y, Fu Y, Xiong J, et al. J Nat Prod. 2013;76:1475–1484. Hirasawa Y, Kato E, Kobayashi JI, et al. Bioorg Med Chem. 2008;16:6167–6171. Liu JS, Huang MF. Phytochemistry. 1994;37:1759–1761. Shen YC, Chen CH. J Nat Prod. 1994;57:824–826. Lycocasuarine A (1): colorless crystal; [a]25 43.1 (c 0.13, MeOH); UV (MeOH) D kmax (log e) 234 (3.90), 316 (3.83) nm; CD (c 1.0  10 3 M, MeOH) kmax (De) 236 (7.22), 316 ( 3.02) nm; IR mmax: 3272, 2923, 1665, 1618, 1466, 1034, and 846 cm 1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z 305.1868 [M+H]+ (calcd for C17H25N2O3, 305.1860). Crystal data for 1: C17H26N2O4, M = 322.40; monoclinic system, space group P21, a = 8.1854(2) Å, b = 14.7099(4) Å, c = 14.4459(3) Å, V = 1707.01(7) Å3, Z = 4, d = 1.254 g/cm3, F(000) = 696.0. A crystal of dimensions 0.25  0.12  0.09 mm3 was used for measurement with monochromatorgraphite, m (Cu Ka) = 0.810 mm 1. A total of 16855 reflections were collected in the range 6.234  2h 2234 tedn, of which 6043 unique reflections (Rint = 0.0349, Rsigma = 0.0381) were collected for the analysis. Using Olex2, the structure was solved with the ShelXT structure solution program using Intrinsic Phasing and refined with the ShelXL refinement package using Least Squares minimisation. The final R1 was 0.0357 (I > 2r(I)) and wR2 was 0.0954 (all data). Crystallographic data for the structure of 1 have been deposited at the Cambridge Crystallographic Data Centre under the reference number CCDC 1566943. Flack HD, Bernardinelli G. Acta Crystallogr, Sect A. 1999;55:908–915. Lycocasuarine B (2): white powder; [a]25 7.8 (c 0.11, MeOH); UV (MeOH) kmax D (log e) 231 (3.97), 312 (3.82) nm; ECD (c 1.0  10 3 M, MeOH) kmax (De) 233 (5.86), 313 ( 2.93) nm; IR mmax: 3388, 2929, 1670, 1618, 1460, 1054, and 910 cm 1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z 317.1870 [M+H]+ (calcd for C18H25N2O3, 317.1860).

L.-L. Wang et al. / Tetrahedron Letters 58 (2017) 4827–4831 14. Lycocasuarine C (3): colorless crystal; [a]25 112.5 (c 0.31, MeOH); UV (MeOH) D kmax (log e) 231 (3.99), 312 (3.87) nm; CD (c 1.0  10 3 M, MeOH) kmax (De) 231 (12.00), 308 ( 3.20) nm; IR mmax: 3267, 2934, 1648, 1622, 1457, 1120, and 852 cm 1; 1H and 13C NMR data, see Table 1; (+)-HRESIMS m/z 273.1607 [M+H]+ (calcd for C16H21N2O2, 273.1598). 15. Crystal data for 3: C16H22N2O3, M = 290.35; tetragonal system, space group P43212, a = 13.1884(4) Å, b = 13.1884(4) Å, c = 17.5109(7) Å, V = 3045.7(2) Å3, Z = 8, d = 1.266 g/cm3, F(000) = 1248.0. A crystal of dimensions 0.25  0.12  0.11 mm3 was used for measurement with monochromatorgraphite, m (Cu Ka) = 0.712 mm 1. A total of 16288 reflections were collected in the range 8.392  2h  124.534, of which 2332 unique reflections (Rint = 0.0377, Rsigma = 0.0203) were collected for the analysis. Using Olex2, the structure was solved with the ShelXT structure solution program using Intrinsic Phasing and refined with the ShelXL refinement package using Least Squares minimisation. The final R1 was 0.0286 (I > 2r(I)) and wR2 was 0.0757 (all data). Crystallographic

16. 17. 18. 19. 20. 21. 22. 23.

4831

data for the structure of 3 have been deposited at the Cambridge Crystallographic Data Centre under the reference number CCDC 1566944. Nyembo L, Goffin A, Hootelé C, Braekman JC. Can J Chem. 1978;56:851–856. Castillo M, Gupta RN, MacLean DB, Spenser ID. Can J Chem. 1970;48:1893–1903. Castillo M, Gupta RN, Ho YK, MacLean DB, Spenser ID. J Am Chem Soc. 1970;92:1074–1075. Braekman JC, Gupta RN, MacLean DB, Spenser ID. Can J Chem. 1972;50:2591–2602. Hemscheidt T, Spenser ID. J Am Chem Soc. 1993;115:3020–3021. Hemscheidt T, Spenser ID. J Am Chem Soc. 1996;118:1799–1800. Ellman GL, Courtney KD, Andres V, Featherstone RM. Biochem Pharmacol. 1961;7:88–95. Tsai SF, Lee SS. J Nat Prod. 2010;73:1632–1635.