Fitoterapia 99 (2014) 72–77
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Huperserines A–E, Lycopodium alkaloids from Huperzia serrata Wei-Wei Jiang a,b, Fei Liu a,b, Xiu Gao a,b, Juan He a, Xiao Cheng a, Li-Yan Peng a, Xing-De Wu a,⁎, Qin-Shi Zhao a,⁎ a b
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China University of the Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 15 July 2014 Accepted in revised form 3 September 2014 Available online 16 September 2014 Keywords: Huperzia serrate Lycopodium alkaloids Huperserines A–E Anti-AChE activity
a b s t r a c t A phytochemical study on Huperzia serrata led to the isolation of four new 5-deoxyfawcettiminerelated Lycopodium alkaloids, huperserines A–D (1–4), and one new lycodine-type alkaloid, huperserine E (5). Their structures were elucidated based on spectroscopic data, including 1D and 2D NMR techniques. 5-Carbonyl or 5-hydroxyl group is a typical characteristic of lycopodine- and fawcettimine-type alkaloids. This is the first report of the 5-deoxyfawcettimine type Lycopodium alkaloids. In vitro acetylcholinesterase (AChE) inhibitory activity assay showed that huperserine E exhibited moderate anti-AChE activity with an IC50 value of 6.71 μM. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Huperzia serrata (Thunb.) Trev. is a traditional Chinese herbal medicine used for the treatment of contusion, strain, swelling, and schizophrenia [1]. Many Lycopodium alkaloids have been isolated from this plant [2,3]. Among them, huperzine A [4], a highly potent, specific, and reversible acetylcholinesterase inhibitor [5], has attracted great interest from synthetic and biological points of view [6–9]. Now, more than 300 Lycopodium alkaloids have been reported [10,11], which have been classified into four structural types, lycopodine, lycodine, fawcettimine, and miscellaneous following the work by the chemist, W. Ayer [12]. As our continuing efforts to search for structurally interesting and bioactive Lycopodium alkaloids [13–18], four 5deoxyfawcettimine-related Lycopodium alkaloids, huperserines A–D (1–4), and one new lycodine-type alkaloid, huperserine E (5), together with five known compounds, were isolated from H. serrata. The known compounds were readily identified as casuarinines A and B [19], huperzinine [20], Ndemethylhuperzinine [20], and huperzine B [4] by analysis of ⁎ Corresponding authors. Tel.: +86 871 65223058; fax: +86 871 65215783. E-mail addresses: [email protected] (X.-D. Wu), [email protected] (Q.-S. Zhao).
http://dx.doi.org/10.1016/j.fitote.2014.09.005 0367-326X/© 2014 Elsevier B.V. All rights reserved.
their NMR spectra and by comparison with the data reported in the literature. 5-Carbonyl or 5-hydroxyl group is a typical characteristic of lycopodine- and fawcettimine-type Lycopodium alkaloids. Up to now, only a few 5-deoxylycopodine-type Lycopodium alkaloids have been isolated [21–27]. This is the first report of the 5-deoxyfawcettimine-type Lycopodium alkaloids. Herein, we described the isolation, structural elucidation, and anti-AChE activity evaluation of the isolates. 2. Experimental 2.1. Experimental procedures Melting points were obtained on an X-4 micro-melting point apparatus. IR spectra were obtained on a Tensor 27 spectrometer with KBr pellets. UV spectra were recorded using a Shimadzu UV-2401A spectrophotometer. Optical rotations were measured on a JASCO-20C digital polarimeter. Circular dichroism spectra were measured on an Agilent Applied Photophysics. ESIMS and HRESIMS were recorded on an API QSTAR Pulsar i spectrometer. EIMS and HREIMS were measured using a Waters Auto Premier P776 spectrometer. 1D NMR and 2D NMR were performed on Bruker AM-400, DRX-500, or AVANCE III-600 spectrometers with TMS as an internal standard. X-ray
W.-W. Jiang et al. / Fitoterapia 99 (2014) 72–77
diffraction was performed on a Bruker APEX DUO diffractometer using graphite-monochromated MoKα radiation. Column chromatography (CC) was performed over silica gel (100–200 or 200–300 mesh; Qingdao Marine Chemical Co. Ltd., Qingdao, China), MCI gel (CHP 20P, 75–150 μm; Mitsubishi Chemical Corporation, Japan), and Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden). Thin-layer chromatography (TLC) was carried out on silica gel GF254 on glass plates (Qingdao Marine Chemical Inc.) using various solvent systems and spots were visualized by spraying improved Dragendorff's reagent to the silica gel plates. 2.2. Plant material The club moss H. serrata used in this study was collected from Jinping, Yunnan province, PR China, in June 2012. The plant was identified by Prof. Xiao Cheng and a voucher specimen (201206H01) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The air-dried whole plant of the club moss H. serrata (20 kg) was extracted with 3% HCl/H2O (24 h × 3), and the extract was partitioned with EtOAc. The water-soluble fraction was adjusted to pH 10 with NaOH and was extracted with CHCl3. The CHCl3soluble fraction (40 g) was subjected to MPLC over RP-C18 (MeOH/H2O, 5% → 100%) to give fractions I–VII. Fr. I (4 g) was chromatographed over repeated silica gel columns (CHCl3/ MeOH, 90:10 → 50:50 and then petroleum ether/acetone, 85:15 → 50:50) to yield 3 (2 mg). Fr. II (6 g) was subjected to a silica gel column (CHCl3/MeOH, 95:5 → 50:50) to afford four subfractions: Fr. II–I to Fr. II–IV. Fr. II–I (1.5 g) was subjected to a Sephadex LH-20 column (MeOH) to afford casuarinine B (13 mg). Fr. II–II (2 g) was subjected to a silica gel column (petroleum ether/acetone/diethylamine, 85:25:1 → 20:80:1) to afford N-demethylhuperzinine (28 mg) and huperzine B (24 mg). Fr. III (8 g) was subjected to a silica gel column (CHCl3/MeOH, 90:10 → 50:50) to afford three subfractions: Fr. III–I to Fr. III–III. Fr. III–II (2 g) was subjected to a silica gel column (petroleum ether/acetone/diethylamine, 80:20:1 → 20:80:1), and a Sephadex LH-20 column (MeOH) to afford 2 (12 mg). Fr. III–III (1 g) yielded crude crystals, recrystallized with MeOH to give 1 (25 mg). Fr. IV (5 g) was chromatographed over repeated silica gel columns (CHCl3/MeOH, 95:15 → 10:90 and then petroleum ether/acetone/diethylamine, 85:15:1 → 50:50:1) to yield casuarinine A (4 mg) and huperzinine (10 mg). Purification of Fr. V (8 g) over a silica gel column (CHCl3/MeOH, 90:10 → 10:90) to give Fr. V–I, Fr. V–II, and Fr. V–III. Fr. V–II was chromatographed over silica gel columns (petroleum ether/ EtOAc/diethylamine, 85:15:1 → 50:50:1) and a Sephadex LH20 column (MeOH) to obtain 4 (3 mg). Compound 5 (6 mg) was obtained from Fr. V–III after purification through a silica gel column (petroleum ether/EtOAc/diethylamine, 80:20:1 → 20:80:1). 2.3.1. Huperserine A (1) + Colorless crystal (MeOH); mp: 115 °C–116 °C; [α]21.3 D 233.7 (c 0.89, MeOH); CD (c 0.18, MeOH): 239 (+31.2); UV (MeOH) λmax (log ε): 194 (4.16) and 243 (4.55) nm. IR (KBr)
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νmax 3442, 2932, 1640 and 1445 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 262 [M + H]+; HRESIMS: m/z 262.1806 ([M + H]+, calcd. for C16H24NO2, 262.1807). Crystal data for: C16H23NO2, M = 261.35, monoclinic, a = 12.7750 (8) Å, b = 12.4253 (7) Å, c = 35.128 (2) Å, α = 90.00°, β = 90.066 (2)°, γ = 90.00°, V = 5575.9 (6) Å3, T = 100 (2) K, space group P21, Z = 16, μ (CuKα) = 0.641 mm−1, 30,264 reflections measured, 15,174 independent reflections (Rint = 0.0377). The final R1 values were 0.0488 (I N 2σ(I)). The final wR (F2) values were 0.1383 (I N 2σ(I)). The final R1 values were 0.0492 (all data). The final wR (F2) values were 0.1388 (all data). The goodness of fit on F2 was 1.036. Flack parameter = 0.00 (15). The Hooft parameter is 0.06 (7) for 5486 Bijvoet pairs. Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number CCDC 997339). Copies of the data can beobtained free of charge from the CCDC via www.ccdc.cam.ac.uk. 2.3.2. Huperserine B (2) + Colorless crystal (MeOH); mp: 132 °C–131 °C; [α]21.3 D 41.9 (c 0.88, MeOH); CD (c 0.19, MeOH): 243 (+27.8); UV (MeOH) λmax (log ε): 195 (3.43), 243 (3.83), and 380 (1.95) nm. IR (KBr) νmax 3426, 2932, 1639, and 1455 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 276 [M + H]+; HRESIMS: m/ z 276.1958 ([M + H]+, calcd for C17H26NO2, 276.1964). 2.3.3. Huperserine C (3) + 45.4 (c 0.82, MeOH); Colorless amorphous solid; [α]21.3 D CD (c 0.16, MeOH): 242 (+25.1); UV (MeOH) λmax (log ε): 197 (3.53), 242 (3.82), and 380 (2.28) nm. IR (KBr) νmax 3431, 2927, 1631, and 1454 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 246 [M + H]+; HREIMS: m/z 245.1781 ([M]+, calcd for C16H23NO, 245.1780). 2.3.4. Huperserine D (4) + 58.3 (c 0.81, MeOH); Colorless amorphous solid;[α]21.3 D CD (c 0.32, MeOH): 247 (+38.6); UV (MeOH) λmax (log ε): 196 (3.41), 237 (3.69), and 462 (1.65) nm. IR (KBr) νmax 3425, 2927, 1639, and 1458 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 262 [M + H]+; HREIMS: m/z 261.1732 ([M]+ calcd for C16H23NO2, 261.1729). 2.3.5. Huperserine E (5) Colorless amorphous powder;[α]20.9 D – 63.2 (c 0.75, MeOH); UV (MeOH) λmax (log ε): 202 (4.17), 231 (4.07), and 312 (3.92) nm. IR (KBr) νmax 3432, 2927, 1657, 1608, and 1440 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 315 [M + H]+; HREIMS: m/z 314.1634 ([M]+ calcd for C18H22N2O3, 314.1630). 2.4. Anti-AChE assay Acetylcholinesterase (AChE) inhibitory activity of the new compounds were assayed by the spectrophotometric method developed by Ellman et al. [28], with slight modifications. SAcetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5′dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman's reagent), acetylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical. Compounds were dissolved in DMSO. The reaction mixture (total 200 μL) containing phosphate buffer (pH 8.0), test compound (50 μM), and acetyl cholinesterase (0.02 U/mL), was incubated for 20 min (37 °C). Then, the
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Table 1 1 HNMR data of 1–5 (δ in ppm, J in Hz). No.
1a
2b
3c
4d
1a 1b 2a 2b 3 5 6a 6b 7 8a 8b 9a 9b 10a 10b 11a 11b 14a 14b 15 16 OMe 1′a 1′b
3.82 (br d, 19.9) 3.33 (td, 19.9, 3.8) 5.39 (dd, 12.1, 3.8)
3.67 (br d, 20.1) 3.32 (dd, 20.1, 4.8) 5.36 (dd, 12.1, 4.8)
3.78 (br d, 20.0) 3.26 (dd, 20.0, 4.8) 5.40 (dd, 12.1, 4.8)
6.30 (d, 12.1) 5.61 (br s) 2.53 (d,17.0) 2.18 (dd, 17.0, 3.0) 1.81 (dd, 9.6, 3.0) 2.71 (t, 9.6)
6.22 (dd, 12.0, 2.3) 5.54 (br s) 2.43 (d, 17.0) 2.14 (dd,17.0, 3.3) 1.73(m) 2.64 (t, 10.2)
3.67 (td, 13.8, 3.9) 2.61 (dd, 13.8, 4.5) 1.87 (m) 1.24 (m) 2.06 e 1.46 (br d, 12.0) 2.15 (m) 1.56 (m) 2.06 e 0.92 (d, 6.5, 3H)
3.09 (td, 13.8, 3.9) 2.50 (dd, 13.8, 5.1) 1.76 (m) 1.10 (m) 2.05 (ddd, 13.9, 12.0, 4.4) 1.32 (dt, 12.0, 2.8) 1.65 (dd, 12.8, 2.9) 1.21 (t, 12.8) 1.59 (m) 0.85 (d, 6.4) 2.97 (s, 3H)
6.28 (dd, 12.1, 1.8) 5.59 (br s) 2.47 (d, 16.7) 1.55 (dd,16.7, 3.3) 1.93 (m) 1.46 (m) 0.71 (d, 11.7) 3.59 (td, 13.7, 3.8) 2.53 (dd, 13.7, 4.8) 1.72 (m) 1.18 (m) 2.02 (ddd, 13.9, 12.0, 4.3) 1.36 (br d, 12.0) 1.42 (br d, 11.9) 1.34 (t, 11.9) 1.95 (m) 0.74 (d, 6.6)
3.50 (ddd, 15.1, 12.5, 4.4) 3.01 (dd,15.1,5.8) 2.63 (m) 2.08 (m) 5.70 (d, 5.8) 6.12 (d, 5.5) 6.80 (dd, 5.5, 2.9)
a b c d
Table 2 13 C NMR data of 1–5 (δ in ppm). No.
1a
2b
3c
4d
5a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 -OMe 1′ 2′
53.6 130.3 129.0 148.9 133.1 35.1 55.4 79.9 50.8 23.8 32.9 58.7 85.6 42.1 35.5 19.7
53.5 131.2 128.4 149.0 132.5 35.0 55.3 79.5 49.9 23.6 32.4 59.0 88.2 35.3 34.9 19.6 45.9
56.5 130.5 127.4 146.7 131.2 36.8 44.9 39.7 49.3 23.1 31.5 56.5 83.8 42.3 26.3 22.7
47.9 31.0 122.2 150.1 134.7 142.2 61.8 85.6 46.2 24.0 33.6 57.2 85.2 43.2 34.6 20.0
165.8 118.4 142.5 121.4 146.0 35.3 34.5 125.9
b c d
4.06 (td, 13.8, 4.6) 2.68 e 1.94 (m) 1.37 (m) 2.68 e 1.67 (dd, 12.0, 4.4) 2.18 (t, 12.3) 1.71 (dd, 12.3, 2.5) 2.78 (m) 1.21 (d, 6.6)
6.38 (d, 9.5) 7.72 (d, 9.5) 2.78 (dd, 17.0, 4.9) 2.58 (dd, 17.0, 1.7) 3.64 (m) 5.42 e
1.70 (d, 6.8) 5.42 e 2.29 (d, 16.5) 2.11 (d, 16.5) 1.55 (s) 3.70 (s) 3.26 (d, 17.5) 2.96 (d, 17.5)
Recorded at 400 MHz in CD3OD. Recorded at 600 MHz in acetone-d6. Recorded at 600 MHz in DMSO-d6. Recorded at 600 MHz in C5D5N; eOverlapping signals.
reaction was initiated by the addition of 40 μL of solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) for AChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 seconds for one hour. Tacrine was used as positive control with a final concentration of 0.333 μM. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E − S)/E × 100 (E is the activity of the
a
2.85 (dd, 8.6, 2.9) 3.14 (dd, 10.1, 8.6)
5a
Recorded at 100 MHz in CD3OD. Recorded at 150 MHz in acetone-d6. Recorded at 150 MHz in DMSO-d6. Recorded at 150 MHz in C5D5N.
12.7 114.2 138.5 60.6 50.7 135.8 22.9 52.6 45.9 174.4
enzyme without test compound and S is the activity of enzyme with test compound). 3. Results and discussion 3.1. Chemistry Huperserine A (1), a colorless crystal, had the molecular formula C16H23NO2 as established by the HRESIMS at m/z 262.1806 [M + H]+ (calc. 262.1807), corresponding to six degrees of unsaturation. IR absorption band at 3442 cm−1 implied the presence of the hydroxy group. Its 1H and 13C NMR spectra (Tables 1 and 2) displayed 16 carbon signals due to three quaternary carbons, six methines, six methylenes, and one methyl group, which included one disubstituted olefinic function [δH 6.30 (1H, d, J = 12.1 Hz), 5.39 (1H, dd, J = 12.1, 3.8 Hz), δC 129.0 and 130.3], one trisubstituted olefinic function [δH 5.61 (1H, br s), δC 133.1 and 148.9], one oxygenated carbon (δC 79.9), and one carbinolamine moiety (δC 85.6). The above information suggested that 1 should be a fawcettimine-related Lycopodium alkaloid. Analysis of the 2D NMR data of 1 established the gross structure (Fig. 1). The 1H–1H COSY and HSQC spectra of 1 (Fig. 2) disclosed the presence of three spin systems: a (C-1–C-3), b (C-5–C-8, C-14–C-16, and C-8–C-15), and c (C-9–C-11). On the basis of the HMBC cross-peaks of H2-1 with C-9 and C-13, and H2-9 with C-1 and C-13, the connections of C-1, C-9, and C-13 through a nitrogen atom were established. The HMBC correlations of H-3 and H-5 with C-4 and C-12, and H-3 with C-5, suggested that C-4 was connected to C-3, C-5, and C-12. Furthermore, the connectivity between C-7 and C-12 was inferred from the HMBC crosspeaks of H-7 and H-8 with C-12, which indicated the presence
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75
Fig. 1. Structures of the new compounds 1 5.
of a cyclopentene ring. At the same time, the HMBC cross-peaks of H2-10 and H2-11 with C-12 constructed the connectivity of C-11 and C-12. Finally, to fulfill the degrees of unsaturation, the linkage of C-13 with C-12 was assigned, which was further confirmed by the HMBC correlations. Thereby, the planar structure of compound 1 was elucidated as 8,13-dihydroxy-5deoxyfawcettimine-type alkaloid, and named as huperserine A. 5-Carbonyl or 5-hydroxyl group was a typical characteristic of lycopodine- and fawcettimine-type alkaloid. This is the first report of a 5-deoxyfawcettimine-type alkaloid. The relative configuration of 1 was established by ROESY spectrum (Fig. 2). The ROESY correlations of H-1a/H-14b and H-1b/H-9 indicated the decahydroquinoline ring system (C/Dring) was trans fused, which implied C-13 taking an opposite configuration compared with that in fawcettimine. Whereas the ROESY correlations of H-6b/H-8 and H-8/H3-16 suggested that H-8 and Me-16 were β-oriented. Up to now, only a few fawcettimine-type alkaloids possessing a trans-C/D ring junction with β-oriented Me-16 have been reported [29,30]. To elucidated the absolute configuration of 1, a single X-ray diffraction was made, which established the absolute configuration of 1 as 7S, 8S, 12S, 13R, 15R (Fig. 3).
Huperserine B (2) was obtained as a colorless crystal. Its molecular formula C17H25NO2 was established by the HRESIMS (m/z 276.1958 [M + H]+, calc. 276.1964). Comparing the 1D NMR data (Tables 1 and 2) of 2 with those of 1 suggested that they were similar, except for the existence of one additional methoxy group [δH 2.97 (3H), δC 45.9], which was attached to C-13 in 2. This deduction was inferred from the HMBC crosspeak between OMe and C-13. The relative configuration of 2 was determined to be the same as 1 on the basis of ROESY correlations of H-8/H-14b, H-8/H-6b, H-8/Me-16, and H-15/HOMe. Thus, the structure of 2 was elucidated as shown in Fig. 1. Huperserine C (3) possessed a molecular formula of C16H23NO, as deduced from the HREIMS analysis (m/z 245.1781 [M]+, calc. 245.1780). The 13C NMR and DEPT spectra exhibited 16 carbon signals due to a methyl, seven methylenes, five methines, and three quaternary carbons. The above data revealed that 3 shared the same skeleton as that of 1. The only difference was that the oxygenated methine of C-8 in 1 was replaced by a methylene in 2, which was established by the 1 H–1H COSY cross-peaks of H-7/H2-8/H-15, as well as the HMBC correlations from H2-8 to C-7, C-14, C-15, and Me-16. ROESY correlations of H-1a/H-14b and H-1b/H-9 indicated the
Fig. 2. The key 2D NMR correlations of compound 1. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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Fig. 3. The X-ray structure of compound 1. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
decahydroquinoline ring system take the same configuration as that of 1. Whereas the ROESY correlations of H3-16/H-8a, H316/H-14b, and H-8a/H-14b suggested that 3 had the β-oriented methyl group at C-15. Therefore, the structure of 3 was established. Huperserine D (4) was obtained as a colorless solid. HREIMS analysis gave a molecular ion peak at m/z 261.1732 [M]+ (calc. m/z 261.1729) and established the molecular formula as C16H23NO2. The 1D NMR spectra (Tables 1 and 2) showed the presence of one disubstituted olefinic function [δH 6.80 (dd, J = 5.5, 2.9 Hz), 6.12 (d, J = 5.5 Hz), δC; 142.2 and 134.7], one trisubstituted olefinic function [δH 5.70 (d, J = 5.8 Hz), δC 122.2 and 150.1], one oxygenated carbon (δC 85.6), and one
carbinolamine moiety (δC 85.2), which implied that 4 was an analogue of 1. The 1H–1H COSY analysis suggested that the two olefinic functions were located at C-3/C-4 and C-5/C-6, which was further confirmed by the HMBC correlations of H-3/C-4, H5/C-4, and H-6/C-5. ROESY correlations among H-1a/H-14a, H1b/H-9b, H-8/H-14a, and H-8/H3-16 indicated that 4 shares the same relative configuration as that of 1. Thus, the structure of 4 was established as shown in Fig. 1. The CD spectra of 1 4 were almost the same, as shown in Fig. 4, allowing us to determine the same absolute configuration of 2 4 as that of 1. Huperserine E (5), a colorless amorphous powder, has a molecular formula C18H22N2O3 as established by HREIMS at m/z 314.1634 [M]+ (calcd. 314.1630), suggesting nine degrees of unsaturation. IR absorptions implied the existence of amino (3432 cm−1) and carbonyl (1657 cm−1) groups. The UV absorption at 231 and 312 nm indicated the presence of a pyridone ring. Analysis of the 1D and 2D NMR spectra revealed the existence of 18 carbons due to two carbonyl carbons (δC 174.4 and 165.8), four sp2 quaternary carbons (δC 146.0, 138.5, 135.8, and 121.4), one sp3 quaternary (δC 60.6), four sp2 methines (δC 142.5, 125.9, 118.4, and 114.2), three sp3 methylenes (δC 50.7, 45.9, and 35.3), two methyl groups (δH 1.55, 1.70 and δC 12.7, 22.9), and one methoxy group [δH 3.70 (3H), δC 52.6]. Based on the above data, the structure of 5 was concluded to be a lycodine-type alkaloid, which resembled those of huperzine A [4]. The differences were that 5 had one CH3OCOCH2 group, which was connected to C-13 (δC 60.6) through a nitrogen atom as revealed by HMBC cross-peaks of H-1′ (δH 3.26 and 2.96) with C-13 and C-2′ (s, 174.4) and of OMe with C-2′. The ROESY correlation H-7/H-10 indicated that the geometry of C(10) = C(11) double bond is E. Thus, the structure of huperserine E was elucidated.
3.2. Biological activity The isolated huperserines A E were tested for AChE inhibitory activities using the Ellman method reported previously (huperzine A as positive control, IC50 = 0.03 μM). Finally, huperserine E showed a moderate AChE inhibitory activity (IC50 = 6.71 μM).
60
Δε
40 20
0 -20 -40 190
290
390
QP Fig. 4. CD spectra of compounds 1 4 in MeOH. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
W.-W. Jiang et al. / Fitoterapia 99 (2014) 72–77
Conflict of interest The authors declare that there is no conflict of interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. U09326024) and the National Basic Research Program of China (973 Program No. 2011CB915503). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.09.005. References [1] The Dictionary of Traditional Chinese Medicine. Shanghai: Shanghai Science and Technology Press; 1985 284. [2] Kobayashi J, Morita H. The Alkaloids. In: Cordell GA, editor. New York, NY: Academic; 2005. p. 1. [3] Ma XQ, Gang DR. The Lycopodium alkaloids. Nat Prod Rep 2004;21: 752–72. [4] Liu JS, Zhu YL, Yu CM, Zhou YZ, Han YY, Wu FW, et al. The structures of huperzine A and B, two new alkaloids exhibiting marked anticholinesterase activity. Can J Chem 1986;64:837–9. [5] Ashani Y, Peggins JO, Doctor BP. Mechanism of inhibition of cholinesterases by huperzine A. Biochem Biophys Res Commun 1992;184:719–26. [6] Wang R, Tang XC. Neuroprotective effects of huperzine A. A natural cholinesterase inhibitor for the treatment of Alzheimer's disease. Neurosignals 2005;14:71–82. [7] Jin G, Luo X, He X, Jiang H, Zhang H, Bai D. Synthesis and docking studies of alkylene-linked dimers of (-)-huperzine A. Arzneimittelforschung 2003; 53:753–7. [8] Yu D, Thakor Devang K, Han I, Ropper Alexander E, Haragopal H, Sidman Richard L, et al. Alleviation of chronic pain following rat spinal cord compression injury with multimodal actions of huperzine A. Proc Natl Acad Sci U S A 2013;110:E746–55. [9] Tun MKM, Wuestmann DJ, Herzon SB. A robust and scalable synthesis of the potent neuroprotective agent (−)-huperzine A. Chem Sci 2011;2: 2251–3. [10] Siengalewicz P, Mulzer J, Rinner U. Lycopodium alkaloids—synthetic highlights and recent developments. Alkaloids 2013;72:1–151. [11] Hirasawa Y, Kobayashi J, Morita H. The Lycopodium alkaloids. Heterocycles 2009;77:679–729.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Huperserines A–E, Lycopodium alkaloids from Huperzia serrata Wei-Wei Jiang a,b, Fei Liu a,b, Xiu Gao a,b, Juan He a, Xiao Cheng a, Li-Yan Peng a, Xing-De Wu a,⁎, Qin-Shi Zhao a,⁎ a b
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China University of the Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e
i n f o
Article history: Received 15 July 2014 Accepted in revised form 3 September 2014 Available online 16 September 2014 Keywords: Huperzia serrate Lycopodium alkaloids Huperserines A–E Anti-AChE activity
a b s t r a c t A phytochemical study on Huperzia serrata led to the isolation of four new 5-deoxyfawcettiminerelated Lycopodium alkaloids, huperserines A–D (1–4), and one new lycodine-type alkaloid, huperserine E (5). Their structures were elucidated based on spectroscopic data, including 1D and 2D NMR techniques. 5-Carbonyl or 5-hydroxyl group is a typical characteristic of lycopodine- and fawcettimine-type alkaloids. This is the first report of the 5-deoxyfawcettimine type Lycopodium alkaloids. In vitro acetylcholinesterase (AChE) inhibitory activity assay showed that huperserine E exhibited moderate anti-AChE activity with an IC50 value of 6.71 μM. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Huperzia serrata (Thunb.) Trev. is a traditional Chinese herbal medicine used for the treatment of contusion, strain, swelling, and schizophrenia [1]. Many Lycopodium alkaloids have been isolated from this plant [2,3]. Among them, huperzine A [4], a highly potent, specific, and reversible acetylcholinesterase inhibitor [5], has attracted great interest from synthetic and biological points of view [6–9]. Now, more than 300 Lycopodium alkaloids have been reported [10,11], which have been classified into four structural types, lycopodine, lycodine, fawcettimine, and miscellaneous following the work by the chemist, W. Ayer [12]. As our continuing efforts to search for structurally interesting and bioactive Lycopodium alkaloids [13–18], four 5deoxyfawcettimine-related Lycopodium alkaloids, huperserines A–D (1–4), and one new lycodine-type alkaloid, huperserine E (5), together with five known compounds, were isolated from H. serrata. The known compounds were readily identified as casuarinines A and B [19], huperzinine [20], Ndemethylhuperzinine [20], and huperzine B [4] by analysis of ⁎ Corresponding authors. Tel.: +86 871 65223058; fax: +86 871 65215783. E-mail addresses: [email protected] (X.-D. Wu), [email protected] (Q.-S. Zhao).
http://dx.doi.org/10.1016/j.fitote.2014.09.005 0367-326X/© 2014 Elsevier B.V. All rights reserved.
their NMR spectra and by comparison with the data reported in the literature. 5-Carbonyl or 5-hydroxyl group is a typical characteristic of lycopodine- and fawcettimine-type Lycopodium alkaloids. Up to now, only a few 5-deoxylycopodine-type Lycopodium alkaloids have been isolated [21–27]. This is the first report of the 5-deoxyfawcettimine-type Lycopodium alkaloids. Herein, we described the isolation, structural elucidation, and anti-AChE activity evaluation of the isolates. 2. Experimental 2.1. Experimental procedures Melting points were obtained on an X-4 micro-melting point apparatus. IR spectra were obtained on a Tensor 27 spectrometer with KBr pellets. UV spectra were recorded using a Shimadzu UV-2401A spectrophotometer. Optical rotations were measured on a JASCO-20C digital polarimeter. Circular dichroism spectra were measured on an Agilent Applied Photophysics. ESIMS and HRESIMS were recorded on an API QSTAR Pulsar i spectrometer. EIMS and HREIMS were measured using a Waters Auto Premier P776 spectrometer. 1D NMR and 2D NMR were performed on Bruker AM-400, DRX-500, or AVANCE III-600 spectrometers with TMS as an internal standard. X-ray
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diffraction was performed on a Bruker APEX DUO diffractometer using graphite-monochromated MoKα radiation. Column chromatography (CC) was performed over silica gel (100–200 or 200–300 mesh; Qingdao Marine Chemical Co. Ltd., Qingdao, China), MCI gel (CHP 20P, 75–150 μm; Mitsubishi Chemical Corporation, Japan), and Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden). Thin-layer chromatography (TLC) was carried out on silica gel GF254 on glass plates (Qingdao Marine Chemical Inc.) using various solvent systems and spots were visualized by spraying improved Dragendorff's reagent to the silica gel plates. 2.2. Plant material The club moss H. serrata used in this study was collected from Jinping, Yunnan province, PR China, in June 2012. The plant was identified by Prof. Xiao Cheng and a voucher specimen (201206H01) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The air-dried whole plant of the club moss H. serrata (20 kg) was extracted with 3% HCl/H2O (24 h × 3), and the extract was partitioned with EtOAc. The water-soluble fraction was adjusted to pH 10 with NaOH and was extracted with CHCl3. The CHCl3soluble fraction (40 g) was subjected to MPLC over RP-C18 (MeOH/H2O, 5% → 100%) to give fractions I–VII. Fr. I (4 g) was chromatographed over repeated silica gel columns (CHCl3/ MeOH, 90:10 → 50:50 and then petroleum ether/acetone, 85:15 → 50:50) to yield 3 (2 mg). Fr. II (6 g) was subjected to a silica gel column (CHCl3/MeOH, 95:5 → 50:50) to afford four subfractions: Fr. II–I to Fr. II–IV. Fr. II–I (1.5 g) was subjected to a Sephadex LH-20 column (MeOH) to afford casuarinine B (13 mg). Fr. II–II (2 g) was subjected to a silica gel column (petroleum ether/acetone/diethylamine, 85:25:1 → 20:80:1) to afford N-demethylhuperzinine (28 mg) and huperzine B (24 mg). Fr. III (8 g) was subjected to a silica gel column (CHCl3/MeOH, 90:10 → 50:50) to afford three subfractions: Fr. III–I to Fr. III–III. Fr. III–II (2 g) was subjected to a silica gel column (petroleum ether/acetone/diethylamine, 80:20:1 → 20:80:1), and a Sephadex LH-20 column (MeOH) to afford 2 (12 mg). Fr. III–III (1 g) yielded crude crystals, recrystallized with MeOH to give 1 (25 mg). Fr. IV (5 g) was chromatographed over repeated silica gel columns (CHCl3/MeOH, 95:15 → 10:90 and then petroleum ether/acetone/diethylamine, 85:15:1 → 50:50:1) to yield casuarinine A (4 mg) and huperzinine (10 mg). Purification of Fr. V (8 g) over a silica gel column (CHCl3/MeOH, 90:10 → 10:90) to give Fr. V–I, Fr. V–II, and Fr. V–III. Fr. V–II was chromatographed over silica gel columns (petroleum ether/ EtOAc/diethylamine, 85:15:1 → 50:50:1) and a Sephadex LH20 column (MeOH) to obtain 4 (3 mg). Compound 5 (6 mg) was obtained from Fr. V–III after purification through a silica gel column (petroleum ether/EtOAc/diethylamine, 80:20:1 → 20:80:1). 2.3.1. Huperserine A (1) + Colorless crystal (MeOH); mp: 115 °C–116 °C; [α]21.3 D 233.7 (c 0.89, MeOH); CD (c 0.18, MeOH): 239 (+31.2); UV (MeOH) λmax (log ε): 194 (4.16) and 243 (4.55) nm. IR (KBr)
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νmax 3442, 2932, 1640 and 1445 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 262 [M + H]+; HRESIMS: m/z 262.1806 ([M + H]+, calcd. for C16H24NO2, 262.1807). Crystal data for: C16H23NO2, M = 261.35, monoclinic, a = 12.7750 (8) Å, b = 12.4253 (7) Å, c = 35.128 (2) Å, α = 90.00°, β = 90.066 (2)°, γ = 90.00°, V = 5575.9 (6) Å3, T = 100 (2) K, space group P21, Z = 16, μ (CuKα) = 0.641 mm−1, 30,264 reflections measured, 15,174 independent reflections (Rint = 0.0377). The final R1 values were 0.0488 (I N 2σ(I)). The final wR (F2) values were 0.1383 (I N 2σ(I)). The final R1 values were 0.0492 (all data). The final wR (F2) values were 0.1388 (all data). The goodness of fit on F2 was 1.036. Flack parameter = 0.00 (15). The Hooft parameter is 0.06 (7) for 5486 Bijvoet pairs. Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number CCDC 997339). Copies of the data can beobtained free of charge from the CCDC via www.ccdc.cam.ac.uk. 2.3.2. Huperserine B (2) + Colorless crystal (MeOH); mp: 132 °C–131 °C; [α]21.3 D 41.9 (c 0.88, MeOH); CD (c 0.19, MeOH): 243 (+27.8); UV (MeOH) λmax (log ε): 195 (3.43), 243 (3.83), and 380 (1.95) nm. IR (KBr) νmax 3426, 2932, 1639, and 1455 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 276 [M + H]+; HRESIMS: m/ z 276.1958 ([M + H]+, calcd for C17H26NO2, 276.1964). 2.3.3. Huperserine C (3) + 45.4 (c 0.82, MeOH); Colorless amorphous solid; [α]21.3 D CD (c 0.16, MeOH): 242 (+25.1); UV (MeOH) λmax (log ε): 197 (3.53), 242 (3.82), and 380 (2.28) nm. IR (KBr) νmax 3431, 2927, 1631, and 1454 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 246 [M + H]+; HREIMS: m/z 245.1781 ([M]+, calcd for C16H23NO, 245.1780). 2.3.4. Huperserine D (4) + 58.3 (c 0.81, MeOH); Colorless amorphous solid;[α]21.3 D CD (c 0.32, MeOH): 247 (+38.6); UV (MeOH) λmax (log ε): 196 (3.41), 237 (3.69), and 462 (1.65) nm. IR (KBr) νmax 3425, 2927, 1639, and 1458 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 262 [M + H]+; HREIMS: m/z 261.1732 ([M]+ calcd for C16H23NO2, 261.1729). 2.3.5. Huperserine E (5) Colorless amorphous powder;[α]20.9 D – 63.2 (c 0.75, MeOH); UV (MeOH) λmax (log ε): 202 (4.17), 231 (4.07), and 312 (3.92) nm. IR (KBr) νmax 3432, 2927, 1657, 1608, and 1440 cm−1. 1H and 13C NMR (Tables 1 and 2). ESIMS: m/z 315 [M + H]+; HREIMS: m/z 314.1634 ([M]+ calcd for C18H22N2O3, 314.1630). 2.4. Anti-AChE assay Acetylcholinesterase (AChE) inhibitory activity of the new compounds were assayed by the spectrophotometric method developed by Ellman et al. [28], with slight modifications. SAcetylthiocholine iodide, S-butyrylthiocholine iodide, 5,5′dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman's reagent), acetylcholinesterase derived from human erythrocytes were purchased from Sigma Chemical. Compounds were dissolved in DMSO. The reaction mixture (total 200 μL) containing phosphate buffer (pH 8.0), test compound (50 μM), and acetyl cholinesterase (0.02 U/mL), was incubated for 20 min (37 °C). Then, the
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W.-W. Jiang et al. / Fitoterapia 99 (2014) 72–77
Table 1 1 HNMR data of 1–5 (δ in ppm, J in Hz). No.
1a
2b
3c
4d
1a 1b 2a 2b 3 5 6a 6b 7 8a 8b 9a 9b 10a 10b 11a 11b 14a 14b 15 16 OMe 1′a 1′b
3.82 (br d, 19.9) 3.33 (td, 19.9, 3.8) 5.39 (dd, 12.1, 3.8)
3.67 (br d, 20.1) 3.32 (dd, 20.1, 4.8) 5.36 (dd, 12.1, 4.8)
3.78 (br d, 20.0) 3.26 (dd, 20.0, 4.8) 5.40 (dd, 12.1, 4.8)
6.30 (d, 12.1) 5.61 (br s) 2.53 (d,17.0) 2.18 (dd, 17.0, 3.0) 1.81 (dd, 9.6, 3.0) 2.71 (t, 9.6)
6.22 (dd, 12.0, 2.3) 5.54 (br s) 2.43 (d, 17.0) 2.14 (dd,17.0, 3.3) 1.73(m) 2.64 (t, 10.2)
3.67 (td, 13.8, 3.9) 2.61 (dd, 13.8, 4.5) 1.87 (m) 1.24 (m) 2.06 e 1.46 (br d, 12.0) 2.15 (m) 1.56 (m) 2.06 e 0.92 (d, 6.5, 3H)
3.09 (td, 13.8, 3.9) 2.50 (dd, 13.8, 5.1) 1.76 (m) 1.10 (m) 2.05 (ddd, 13.9, 12.0, 4.4) 1.32 (dt, 12.0, 2.8) 1.65 (dd, 12.8, 2.9) 1.21 (t, 12.8) 1.59 (m) 0.85 (d, 6.4) 2.97 (s, 3H)
6.28 (dd, 12.1, 1.8) 5.59 (br s) 2.47 (d, 16.7) 1.55 (dd,16.7, 3.3) 1.93 (m) 1.46 (m) 0.71 (d, 11.7) 3.59 (td, 13.7, 3.8) 2.53 (dd, 13.7, 4.8) 1.72 (m) 1.18 (m) 2.02 (ddd, 13.9, 12.0, 4.3) 1.36 (br d, 12.0) 1.42 (br d, 11.9) 1.34 (t, 11.9) 1.95 (m) 0.74 (d, 6.6)
3.50 (ddd, 15.1, 12.5, 4.4) 3.01 (dd,15.1,5.8) 2.63 (m) 2.08 (m) 5.70 (d, 5.8) 6.12 (d, 5.5) 6.80 (dd, 5.5, 2.9)
a b c d
Table 2 13 C NMR data of 1–5 (δ in ppm). No.
1a
2b
3c
4d
5a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 -OMe 1′ 2′
53.6 130.3 129.0 148.9 133.1 35.1 55.4 79.9 50.8 23.8 32.9 58.7 85.6 42.1 35.5 19.7
53.5 131.2 128.4 149.0 132.5 35.0 55.3 79.5 49.9 23.6 32.4 59.0 88.2 35.3 34.9 19.6 45.9
56.5 130.5 127.4 146.7 131.2 36.8 44.9 39.7 49.3 23.1 31.5 56.5 83.8 42.3 26.3 22.7
47.9 31.0 122.2 150.1 134.7 142.2 61.8 85.6 46.2 24.0 33.6 57.2 85.2 43.2 34.6 20.0
165.8 118.4 142.5 121.4 146.0 35.3 34.5 125.9
b c d
4.06 (td, 13.8, 4.6) 2.68 e 1.94 (m) 1.37 (m) 2.68 e 1.67 (dd, 12.0, 4.4) 2.18 (t, 12.3) 1.71 (dd, 12.3, 2.5) 2.78 (m) 1.21 (d, 6.6)
6.38 (d, 9.5) 7.72 (d, 9.5) 2.78 (dd, 17.0, 4.9) 2.58 (dd, 17.0, 1.7) 3.64 (m) 5.42 e
1.70 (d, 6.8) 5.42 e 2.29 (d, 16.5) 2.11 (d, 16.5) 1.55 (s) 3.70 (s) 3.26 (d, 17.5) 2.96 (d, 17.5)
Recorded at 400 MHz in CD3OD. Recorded at 600 MHz in acetone-d6. Recorded at 600 MHz in DMSO-d6. Recorded at 600 MHz in C5D5N; eOverlapping signals.
reaction was initiated by the addition of 40 μL of solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM) for AChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 seconds for one hour. Tacrine was used as positive control with a final concentration of 0.333 μM. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E − S)/E × 100 (E is the activity of the
a
2.85 (dd, 8.6, 2.9) 3.14 (dd, 10.1, 8.6)
5a
Recorded at 100 MHz in CD3OD. Recorded at 150 MHz in acetone-d6. Recorded at 150 MHz in DMSO-d6. Recorded at 150 MHz in C5D5N.
12.7 114.2 138.5 60.6 50.7 135.8 22.9 52.6 45.9 174.4
enzyme without test compound and S is the activity of enzyme with test compound). 3. Results and discussion 3.1. Chemistry Huperserine A (1), a colorless crystal, had the molecular formula C16H23NO2 as established by the HRESIMS at m/z 262.1806 [M + H]+ (calc. 262.1807), corresponding to six degrees of unsaturation. IR absorption band at 3442 cm−1 implied the presence of the hydroxy group. Its 1H and 13C NMR spectra (Tables 1 and 2) displayed 16 carbon signals due to three quaternary carbons, six methines, six methylenes, and one methyl group, which included one disubstituted olefinic function [δH 6.30 (1H, d, J = 12.1 Hz), 5.39 (1H, dd, J = 12.1, 3.8 Hz), δC 129.0 and 130.3], one trisubstituted olefinic function [δH 5.61 (1H, br s), δC 133.1 and 148.9], one oxygenated carbon (δC 79.9), and one carbinolamine moiety (δC 85.6). The above information suggested that 1 should be a fawcettimine-related Lycopodium alkaloid. Analysis of the 2D NMR data of 1 established the gross structure (Fig. 1). The 1H–1H COSY and HSQC spectra of 1 (Fig. 2) disclosed the presence of three spin systems: a (C-1–C-3), b (C-5–C-8, C-14–C-16, and C-8–C-15), and c (C-9–C-11). On the basis of the HMBC cross-peaks of H2-1 with C-9 and C-13, and H2-9 with C-1 and C-13, the connections of C-1, C-9, and C-13 through a nitrogen atom were established. The HMBC correlations of H-3 and H-5 with C-4 and C-12, and H-3 with C-5, suggested that C-4 was connected to C-3, C-5, and C-12. Furthermore, the connectivity between C-7 and C-12 was inferred from the HMBC crosspeaks of H-7 and H-8 with C-12, which indicated the presence
W.-W. Jiang et al. / Fitoterapia 99 (2014) 72–77
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Fig. 1. Structures of the new compounds 1 5.
of a cyclopentene ring. At the same time, the HMBC cross-peaks of H2-10 and H2-11 with C-12 constructed the connectivity of C-11 and C-12. Finally, to fulfill the degrees of unsaturation, the linkage of C-13 with C-12 was assigned, which was further confirmed by the HMBC correlations. Thereby, the planar structure of compound 1 was elucidated as 8,13-dihydroxy-5deoxyfawcettimine-type alkaloid, and named as huperserine A. 5-Carbonyl or 5-hydroxyl group was a typical characteristic of lycopodine- and fawcettimine-type alkaloid. This is the first report of a 5-deoxyfawcettimine-type alkaloid. The relative configuration of 1 was established by ROESY spectrum (Fig. 2). The ROESY correlations of H-1a/H-14b and H-1b/H-9 indicated the decahydroquinoline ring system (C/Dring) was trans fused, which implied C-13 taking an opposite configuration compared with that in fawcettimine. Whereas the ROESY correlations of H-6b/H-8 and H-8/H3-16 suggested that H-8 and Me-16 were β-oriented. Up to now, only a few fawcettimine-type alkaloids possessing a trans-C/D ring junction with β-oriented Me-16 have been reported [29,30]. To elucidated the absolute configuration of 1, a single X-ray diffraction was made, which established the absolute configuration of 1 as 7S, 8S, 12S, 13R, 15R (Fig. 3).
Huperserine B (2) was obtained as a colorless crystal. Its molecular formula C17H25NO2 was established by the HRESIMS (m/z 276.1958 [M + H]+, calc. 276.1964). Comparing the 1D NMR data (Tables 1 and 2) of 2 with those of 1 suggested that they were similar, except for the existence of one additional methoxy group [δH 2.97 (3H), δC 45.9], which was attached to C-13 in 2. This deduction was inferred from the HMBC crosspeak between OMe and C-13. The relative configuration of 2 was determined to be the same as 1 on the basis of ROESY correlations of H-8/H-14b, H-8/H-6b, H-8/Me-16, and H-15/HOMe. Thus, the structure of 2 was elucidated as shown in Fig. 1. Huperserine C (3) possessed a molecular formula of C16H23NO, as deduced from the HREIMS analysis (m/z 245.1781 [M]+, calc. 245.1780). The 13C NMR and DEPT spectra exhibited 16 carbon signals due to a methyl, seven methylenes, five methines, and three quaternary carbons. The above data revealed that 3 shared the same skeleton as that of 1. The only difference was that the oxygenated methine of C-8 in 1 was replaced by a methylene in 2, which was established by the 1 H–1H COSY cross-peaks of H-7/H2-8/H-15, as well as the HMBC correlations from H2-8 to C-7, C-14, C-15, and Me-16. ROESY correlations of H-1a/H-14b and H-1b/H-9 indicated the
Fig. 2. The key 2D NMR correlations of compound 1. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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Fig. 3. The X-ray structure of compound 1. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
decahydroquinoline ring system take the same configuration as that of 1. Whereas the ROESY correlations of H3-16/H-8a, H316/H-14b, and H-8a/H-14b suggested that 3 had the β-oriented methyl group at C-15. Therefore, the structure of 3 was established. Huperserine D (4) was obtained as a colorless solid. HREIMS analysis gave a molecular ion peak at m/z 261.1732 [M]+ (calc. m/z 261.1729) and established the molecular formula as C16H23NO2. The 1D NMR spectra (Tables 1 and 2) showed the presence of one disubstituted olefinic function [δH 6.80 (dd, J = 5.5, 2.9 Hz), 6.12 (d, J = 5.5 Hz), δC; 142.2 and 134.7], one trisubstituted olefinic function [δH 5.70 (d, J = 5.8 Hz), δC 122.2 and 150.1], one oxygenated carbon (δC 85.6), and one
carbinolamine moiety (δC 85.2), which implied that 4 was an analogue of 1. The 1H–1H COSY analysis suggested that the two olefinic functions were located at C-3/C-4 and C-5/C-6, which was further confirmed by the HMBC correlations of H-3/C-4, H5/C-4, and H-6/C-5. ROESY correlations among H-1a/H-14a, H1b/H-9b, H-8/H-14a, and H-8/H3-16 indicated that 4 shares the same relative configuration as that of 1. Thus, the structure of 4 was established as shown in Fig. 1. The CD spectra of 1 4 were almost the same, as shown in Fig. 4, allowing us to determine the same absolute configuration of 2 4 as that of 1. Huperserine E (5), a colorless amorphous powder, has a molecular formula C18H22N2O3 as established by HREIMS at m/z 314.1634 [M]+ (calcd. 314.1630), suggesting nine degrees of unsaturation. IR absorptions implied the existence of amino (3432 cm−1) and carbonyl (1657 cm−1) groups. The UV absorption at 231 and 312 nm indicated the presence of a pyridone ring. Analysis of the 1D and 2D NMR spectra revealed the existence of 18 carbons due to two carbonyl carbons (δC 174.4 and 165.8), four sp2 quaternary carbons (δC 146.0, 138.5, 135.8, and 121.4), one sp3 quaternary (δC 60.6), four sp2 methines (δC 142.5, 125.9, 118.4, and 114.2), three sp3 methylenes (δC 50.7, 45.9, and 35.3), two methyl groups (δH 1.55, 1.70 and δC 12.7, 22.9), and one methoxy group [δH 3.70 (3H), δC 52.6]. Based on the above data, the structure of 5 was concluded to be a lycodine-type alkaloid, which resembled those of huperzine A [4]. The differences were that 5 had one CH3OCOCH2 group, which was connected to C-13 (δC 60.6) through a nitrogen atom as revealed by HMBC cross-peaks of H-1′ (δH 3.26 and 2.96) with C-13 and C-2′ (s, 174.4) and of OMe with C-2′. The ROESY correlation H-7/H-10 indicated that the geometry of C(10) = C(11) double bond is E. Thus, the structure of huperserine E was elucidated.
3.2. Biological activity The isolated huperserines A E were tested for AChE inhibitory activities using the Ellman method reported previously (huperzine A as positive control, IC50 = 0.03 μM). Finally, huperserine E showed a moderate AChE inhibitory activity (IC50 = 6.71 μM).
60
Δε
40 20
0 -20 -40 190
290
390
QP Fig. 4. CD spectra of compounds 1 4 in MeOH. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)
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