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Feb 25, 2016 - Zephyranthes candida and Their Anti-acetylcholinesterase and Anti- ... known analogues (17−24), were isolated from Zephyranthes candi...
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Galanthamine, Plicamine, and Secoplicamine Alkaloids from Zephyranthes candida and Their Anti-acetylcholinesterase and Antiinflammatory Activities Guanqun Zhan, Junfei Zhou, Rong Liu, Tingting Liu, Guoli Guo, Jianping Wang, Ming Xiang, Yongbo Xue, Zengwei Luo, Yonghui Zhang, and Guangmin Yao* Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China S Supporting Information *

ABSTRACT: Sixteen new alkaloids belonging to the galanthamine (1−6), plicamine (7−14), and secoplicamine (15 and 16) classes, together with eight known analogues (17−24), were isolated from Zephyranthes candida. The structures of 1−16 were determined by extensive spectroscopic analyses, and the absolute configurations of 1, 2, 7, 8, and 17 were confirmed by singlecrystal X-ray diffraction analysis. The orientation of 3-OCH3 in N-methyl-5,6-dihydroplicane (22) was revised. Alkaloids 3, 12− 14, and 18−21 exhibited anti-acetylcholinesterase activities with IC50 values ranging from 0.48 to 168.7 μM. Compounds 10−12, 14, and 16 showed in vitro anti-inflammatory activities with IC50 values ranging from 7.50 to 23.55 μM.

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the fact that alkaloid content and composition in plants may show seasonal changes, the whole plant of Z. candida was recollected in the fall of 2011 at the same region, leading to the isolation of 16 new alkaloids belonging to the galanthamine (1−6), plicamine (7−14), and secoplicamine (15 and 16) classes and eight known alkaloids, 11β-hydroxygalanthamine (17),7 galanthamine (18),8 sanguinine (19),9 lycoramine (20),10 O-demethylycoramine (21),11 N-methyl-5,6-dihydroplicane (22),5 obliquine (23),12 and (+)-plicane (24).13 This is the first report of the presence of obliquine (23) and (+)-plicane (24) in the Zephyranthes genus. Herein, the isolation, structure determination, and anti-acetylcholinesterase and anti-inflammatory activities of alkaloids 1−24 are described.

lzheimer’s disease (AD), an age-related neurodegenerative disorder, is the fourth leading cause of death in developed countries and is the most prevalent cause of dementia in the elderly.1 Recent studies revealed that neuroinflammation causes progression of AD pathology, and inhibition of neuroinflammatory pathways would reduce AD progression and neurodegeneration.2 To date, only five drugs including memantine and four cholinesterase inhibitors, tacrine, donepezil, rivastigmine, and galanthamine, have been approved to treat AD by the Food and Drug Administration (FDA) of the United States of America.3 Among them, galanthamine, a well-known Amaryllidaceae alkaloid, widely occurs in plants of the family Amaryllidaceae.4 Thus, it is essential to search for compounds possessing not only anti-acetylcholinesterase but also anti-inflammatory activities to treat AD. As a folk medicine in mainland China, the whole plant of Zephyranthes candida (Lindl.) Herb. has been used to treat infantile convulsions and epilepsy,5 suggesting the presence of alkaloids with potent effects on the central nervous system. In a previous study on the chemical constituents of the whole plant of Z. candida collected in the summer of 2010 at Shiyan, China, 15 alkaloids were isolated,5 and some of them showed antitumor activities in vitro5 and in vivo.6 In consideration of © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION 11β-Hydroxylycoramine (1) was isolated as colorless needles. Its molecular formula was assigned to be C17H23NO4 by the HRESIMS ion at m/z 306.1654 [M + H]+ (calcd for Received: August 3, 2015

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DOI: 10.1021/acs.jnatprod.5b00681 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Data for Compounds 1−6 in Methanol-d4 (100 MHz)

C17H24NO4, 306.1705) and the 13C NMR data. The NMR data of 1 (Tables 1 and 2) were similar to those of lycoramine (20),10 and the major difference was that the chemical shift of C-11 (δC 73.0) in 1 was shifted downfield by 40 ppm when compared with that (δC 33.0) in 20, suggesting that C-11 in 1 is oxygenated. Thus, 1 is an 11-OH derivative of 20. The COSY correlations of H-11 to H2-12 and HMBC correlations of H-11 to C-1, C-4a, C-10a, C-10b, and C-12 supported this deduction. A NOESY correlation between H-11 and H-1α assigned the αorientation of H-11. The absolute configuration of 1 was determined to be 1S,3S,10bS,11R by single-crystal X-ray diffraction analysis (Figure 1). Compound 2 possessed a molecular formula of C16H21NO4 as established by the [M + H]+ HRESIMS ion at m/z 292.1539 and the 13C NMR data. The NMR data of 2 (Tables 1 and 2) resemble those of 1, except for the absence of resonances for

position

1

2

3

4

5

6

1 2 3 4 4a 6 6a 7 8 9 10 10a 10b 11 12 NCH3 OCH3

91.1 35.5 66.9 28.8 22.7 59.7 130.0 123.3 113.3 145.8 149.0 131.9 53.7 73.0 63.0 44.1 56.8

90.7 35.3 66.9 28.8 22.5 59.7 128.7 123.3 116.8 142.3 148.1 131.4 53.9 72.9 62.9 44.1

89.0 34.0 63.0 131.9 125.4 60.4 128.7 123.3 117.0 142.6 147.6 130.5 54.7 73.0 62.8 44.2

91.1 35.3 66.4 28.6 21.8 74.1 121.2 125.0 114.1 147.6 149.5 131.0 52.2 72.0 75.4 54.3 56.7

89.9 34.8 62.8 134.6 123.4 74.6 121.1 124.9 114.2 147.8 149.1 129.8 53.1 72.3 75.1 54.5 56.8

91.9 74.8 69.4 133.0 125.3 59.7 129.5 123.5 113.7 146.0 148.3 130.1 57.4 72.9 62.1 43.7 56.8

the 9-O-methyl group. Thus, 2 is a 9-de-O-methyl derivative of 1, which was evidenced by the chemical shift of C-9 (δC 142.30) in 2 compared to δC 145.8 in 1. 2D NMR including COSY, HSQC, HMBC, and NOESY data determined the structure of 2 to be 9-de-O-methly-11β-hydroxylycoramine. Finally, single-crystal X-ray diffraction analysis established the absolute configuration of 2 as shown in Figure 1. The molecular formula of 9-de-O-methyl-11β-hydroxygalanthamine (3) was determined to be C16H19NO4 by the HRESIMS and the 13C NMR data. The NMR data of 3 (Tables 1 and 2) were similar to those of 11β-hydroxygalanthamine (17),7 and the major difference was the absence of the 9-Omethyl group in 3. Thus, 3 is the 9-de-O-methyl analogue of 17. On the basis of their HRESIMS and NMR data, the molecular formulas of compounds 4 and 5 were assigned as

Table 1. 1H NMR [δ, mult, (J in Hz)] Data for Compounds 1−6 in Methanol-d4 (400 MHz) position

1

2

1 2α

4.72, t (3.0) 2.26, m

4.72, t (2.8) 2.25, m

2β

2.26, m

2.25, m

3

4.06, m

4α

4aα 4aβ 6α 6β 7 8 11 12α

1.98, dddd (13.5, 7.2, 5.6, 2.7) 1.22, dddd (13.5, 13.3, 9.0, 3.0) 1.80, ddd (13.6, 13.3, 2.7) 1.71, ddd (13.6, 5.6, 3.0) 3.53, dd (14.5, 0.9) 3.86, d (14.5) 6. 63, d (8.1) 6.76, d (8.1) 3.96, dd (10.9, 3.4) 2.88, ddd (13.6, 3.4, 1.1)

4.09, dddd (9.0, 7.7, 5.5, 2.7) 2.00, dddd (13.4, 7.7, 5.2, 2.5) 1.19, dddd (13.5, 13.4, 9.0, 3.0) 1.78, ddd (13.6, 13.5, 2.5) 1.66, ddd (13.6, 5.2, 3.0) 3.50, br d (14.5) 3.84, d (14.5) 6.51, d (8.0) 6.60, d (8.0) 3.97, dd (10.9, 3.4) 2.87, ddd (13.6, 3.4, 1.0)

12β NCH3 OCH3

3.07, dd (13.6, 10.9) 2.38, s 3.83, s

4β

3.06, dd (13.6, 10.9) 2.39, s

3

4

4.77, dd (3.3, 2.9) 2.40, ddd (15.4, 2.9, 2.1) 2.48, ddd (15.4, 5.0, 3.3) 4.20, ddd (5.0, 4.6, 2.1) 6.09, dd (10.5, 4.6)

6.04, d (10.5) 3.57, br d (14.8) 3.92, d (14.8) 6.51, d (8.2) 6.59, d (8.2) 3.99, dd (11.1, 3.5) 2.91, ddd (13.7, 3.5, 1.1) 3.19, dd (13.7, 11.1) 2.43, s

B

5

6

4.78, dd (2.8, 2.7) 2.26, m

4.91, t (4.0) 2.38, t (4.0)

2.26, m

2.38, t (4.0)

4.10, m

4.24, dt (4.4,4.0)

2.02, dddd (13.5, 7.0, 5.5, 2.4) 1.22, dddd (13.5, 13.2, 9.0, 2.7) 1.87, ddd (13.4, 13.2, 2.4) 1.76, ddd (13.4, 5.5, 2.7) 4.22, dd (14.2, 1.8) 4.75, d (14.2) 6.80, d (8.2) 6.90, d (8.2) 4.02, dd (1.5, 2.9) 3.50, ddd (13.3, 2.9, 2.9)

6.21, dd (10.3, 4.4)

4.17, ddd (5.2, 3.1, 1.9) 5.98, dd (10.3, 3.1)

6.12, d (10.3)

6.10, br d (10.3)

4.25, dd (14.2, 1.8) 4.83, d (14.2) 6.79, d (8.3) 6.88, d (8.3) 4.07, dd (11.5, 3.0) 3.52, ddd (13.4, 3.0, 3.0) 3.90, dd (13.4, 11.5) 2.99, s 3.84, s

3.54, br d (14.6) 4.03, d (14.6) 6.63, d (8.1) 6.76, d (8.1) 3.96, dd (10.9, 3.6) 2.91, ddd (13.9, 3.6, 1.0) 3.16, dd (13.9, 10.9) 2.35, s 3.81, s

3.80, dd (13.3, 11.5) 2.96, s 3.86, s

4.77, d (4.9) 3.91, dd (5.2, 4.9)

DOI: 10.1021/acs.jnatprod.5b00681 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. ORTEP drawing of alkaloids 1, 2, and 17.

Figure 2. ORTEP drawing of alkaloids 7 and 8.

and the 13C NMR data. Its NMR (Tables S1 and S2, Supporting Information) data exhibited resonances for an isopentyl group.5 The NMR data of 7 were similar to those of obliquine (23),12 with the major difference being the presence of an N-isopentyl group in 7, instead of the N-phydroxyphenethyl group in 23. The HMBC correlations from H2-13 to C-6 and C-12 and from H2-6 and H-12 to C-13 in 7 established the location of the isopentyl group at N-5. Similar to that in 23, the NOESY correlation between H-1 and H-12 in 7 suggested the α-orientation of H-12. NOESY correlation between H-10 and H-4a established the β-orientation of H-4a. The α-orientation of H-3 was deduced from the small coupling constants of H-3 (J = 3.9, 3.4 Hz) with H2-4 and the large coupling constant of H-4a, J = 11.5 Hz, with one of the hydrogens of the C-4 methylene group. The absolute configuration of 7 was defined as 3S,4aS,10bS,12S based on single-crystal X-ray diffraction analysis (Figure 2). The molecular formulas of compounds 8−12 were determined as C 23H 30 N 2O 4, C 24 H32 N2 O4 , C22 H26N 2 O6 , C26H28N2O4, and C28H29N3O4, respectively, by their 13C NMR and HRESIMS data. Comparison of their NMR data (Tables S1−S4, Supporting Information) with those of 7 revealed that 8−12 and 7 had the same parent structures, and the main differences were the presence of N-s-pentyl, N-hexyl, N-hydroxycarbonylpropyl, N-phenethyl, and N-3-indolylethyl15 groups in 8−12, respectively, replacing the N-isopentyl group in 7. HMBC correlations from H2-13 to C-6 and C-12 and from H2-6 and H-12 to C-13 established the connectivity of these groups in 8−12 at N-5. The relative configurations of 8− 12 were determined to be the same as that of 7 based on their

C17H23NO5 and C17H21NO5, respectively, which had one more oxygen atom than those of 1 and 17, respectively. Comparison of their NMR data (Tables 1 and 2) revealed that H-6, H2-12, and NCH3 in 4 and 5 were deshielded compared to those in 1 and 17, respectively. In turn, C-6, C-12, and NCH3 in 4 and 5 were deshielded by 10 ppm more than those in 1 and 17, respectively. Therefore, 4 and 5 are the N-oxides14 of 1 and 17, respectively. The NOESY correlations of H-11 to H-1α and NCH3 established the α-orientations of the NCH3 group in 4 and 5. 2β,11β-Dihydroxygalanthamine (6) was isolated as a colorless oil. Its molecular formula, C17H21NO5, was determined by the HRESIMS ion at m/z 320.1446 [M + H]+ and the 13C NMR data. The NMR data of 6 (Tables 1 and 2) were similar to those of 17, and the main differences were that H-2 (δH 3.91) and C-2 (δC 74.8) in 6 were deshielded compared to those (δH 2.45, H-2α; 2.39, H-2β; δC 34.2, C-2) in 17. Thus, 6 is a 2-OH derivative of 17. The COSY correlations from H-2 to H-1 and H-3 of 6 supported this conclusion. The α-orientation of H-2 in 6 was deduced from the NOESY correlations from H2 to H-1α and H-3α. Accordingly, 2-OH in 6 is β-orientated. The absolute configuration of 11β-hydroxygalanthamine (17) was determined to be 1S,3R,10bS,11R by single-crystal X-ray diffraction analysis (Figure 1). By comparing their electronic circular dichroism (ECD) spectra (Figure S1, Supporting Information) with those of 1 and 17, the absolute configurations of 3−6 were determined. N-Isopentyl-5,6-dihydroplicane (7) was isolated as colorless cubic crystals. The molecular formula of 7 was established as C23H30N2O4 by the HRESIMS ion at m/z 399.2216 [M + H]+ C

DOI: 10.1021/acs.jnatprod.5b00681 J. Nat. Prod. XXXX, XXX, XXX−XXX

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NOESY data and 1H NMR coupling constants. Thus, the structures of compounds 8−12 were determined to be N-spentyl-5,6-dihydroplicane (8), N-hexyl-5,6-dihydroplicane (9), N-hydroxycarbonylpropyl-5,6-dihydroplicane (10), N-phenethyl-5,6-dihydroplicane (11), and N-3-indolylethyl-5,6-dihydroplicane (12), respectively. The absolute configuration of 8 was confirmed as 3S,4aS,10bS,12S,14S by single-crystal X-ray diffraction analysis (Figure 2). On the basis of their HRESIMS and NMR data, the molecular formulas of 13 and 14 were assigned as C23H30N2O5 and C26H28N2O6, respectively, which had one more oxygen atom than those of 7 and 23, respectively. By comparing their NMR data (Tables S3 and S4, Supporting Information), 13 and 14 were suggested to be the N-oxide14 derivatives of 7 and 23, respectively. Subsequent 2D NMR data analyses defined their structures as N-isopentyl-5,6-dihydroplicane N-oxide (13) and bliquine N-oxide (14), respectively. The NOESY correlations of H-12 and H2-13 assigned the orientation of the N-oxide group in 13 and 14. N-Methyl-11,12-seco-5,6-dihydroplicane (15) was assigned a molecular formula of C19H22N2O5 by the HRESIMS ion at m/z 359.1599 [M + H]+ and the 13C NMR data. The NMR data of 15 (Tables S3 and S4, Supporting Information) were similar to those of secoplicamine,16 a known Amaryllidaceae alkaloid, the difference being the absence of an N-p-hydroxyphenethyl group in secoplicamine and its replacement by an N-methyl group in 15, as evidenced by HMBC correlations from the N-CH3 (δH 3.05) to C-6 (δC 53.1) and C-12 (δC 169.1). Similar to 7, the NOESY correlation between H-10 (δH 6.69, s) and H-4a (δH 3.91, dd) and the large coupling constant (J = 13.1 Hz) of H-4a with H-4 suggested that H-4a was β-oriented in 15. The small coupling constants of H-3 (δH 4.08, ddd, J = 5.1, 4.0, 2.3 Hz) with H2-4 and lack of NOESY correlation between H-3 and H4a established the α-orientation of H-3. The molecular formula of N-isopentyl-11,12-seco-5,6-dihydroplicane (16) was determined as C23H30N2O5 by the HRESIMS ion at m/z 415.2220 [M + H]+ and the 13C NMR data. The NMR spectra of 16 (Tables S3 and S4, Supporting Information) resembled those of 15, except that resonances for the N-methyl group in 15 were replaced by resonances for an N-isopentyl group in 16. The location of the N-isopentyl group at N-5 was verified by the HMBC correlations from H-13 to C6 and C-12 and from H2-6 to C-13 in 16. The relative configuration of 16 was determined to be the same as that of 15 based on the 1H NMR coupling constants and NOESY data. Compounds 15 and 16 are the second examples of 11,12secoplicamine-type alkaloids. In the previous study on the structure of N-methyl-5,6dihydroplicane (22),5 the α-orientation of the 3-OCH3 group was defined via the coupling constant of J2,3 = 4.9 Hz,5 referring to a paper published by Hesse and co-workers.16 In fact, as reported by Campbell and co-workers,12 the orientation of H-3 in plicamine-type alkaloids could be deduced from the coupling constants of H-3 with H-4α and of H-4a with H-4α. The small coupling constants of H-3 with H2-4 (J = 4.0, 3.5 Hz) and the large coupling constants of H-4a (J = 10.8, 4.4 Hz) with H2-4 established the α-orientation of H-3 in 22. Single-crystal X-ray diffraction analyses of 7 and 8 (Figure 2) further supported this conclusion. Thus, the orientation of 3-OCH3 in 22 should be revised to be β. The absolute configurations of 9−16 and 22 were determined to be the same as that of 7 by their similar ECD spectra (Figure S2, Supporting Information).

Plicamine-type alkaloids are rare in nature; heretofore, only four compounds, plicamine,10 plicane,13 obliquine,12 and Nmethyl-5,6-dihydroplicane,5 are known. Interestingly, only one secoplicamine-type alkaloid, secoplicamine,16 was reported from nature. The isolation of eight new plicamine-type alkaloids (7−14) and two new 11,12-secoplicamine-type alkaloids (15 and 16) from Z. candida expands the chemistry diversity of the plicamine and secoplicamine alkaloids. Importantly, singlecrystal X-ray diffraction analysis and ECD data of 7 and 8 provided an important basis to determine the absolute configuration of plicamine and secoplicamine alkaloids. Since the whole plants of Z. candida have been used to treat infantile convulsions, epilepsy, and tetanus,5 and acetylcholinesterase (AChE) inhibitors and anti-inflammation drugs could be combined to treat AD, isolates 1−24 were evaluated for their anti-AChE and anti-inflammatory activities. The four known galanthamine-type alkaloids 18−21 exhibited significant AChE inhibitory activities (Table S5 and Figure S3, Supporting Information). However, among the six new galanthamine-type alkaloids (1−6), only 3 showed weak AChE inhibitory activity, with an IC50 value of 168.7 μM. The structure−activity relationship of galanthamine-type alkaloids 1−6 and 18−21 revealed that the 4,4a double bond and 9-OH are essential structural features for the AChE inhibitory activity; however, the presence of the 11-OH group dramatically decreases the AChE inhibitory activity. Furthermore, among the 13 plicamine- and secoplicamine-type alkaloids, only the new plicamine alkaloids 12−14 exhibited weak AChE inhibitory activities, with IC50 values of 110.6, 57.26, and 75.3 μM, respectively. The in vitro anti-inflammatory activities of alkaloids 1−24 were evaluated by monitoring the inhibition of lipopolysaccaride (LPS)-induced nitric oxide (NO) production in RAW 264.7 mouse macrophages. Plicamine-type alkaloids 10−12, 14, and 16 exhibited significant inhibitory activities on NO production in LPS-induced RAW 264.7 cells with IC50 values of 18.77, 10.21, 18.01, 7.50, and 23.55 μM, respectively (Table S6 and Figure S4, Supporting Information). However, the other alkaloids were inactive at concentrations of 200 μM. Furthermore, the general cytotoxicity of 10−12, 14, and 16 was evaluated, and none of them showed general cytotoxicity against the RAW 264.7 cells at a concentration of 200 μM (Table S6, Supporting Information). Therefore, the inhibitory activities of 10−12, 14, and 16 on the NO production in the LPS-induced RAW 264.7 cells do not involve general cytotoxicity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured with a Beijing Tech X-5 microscopic melting point apparatus and are uncorrected. Optical rotations were measured on a PerkinElmer 341 polarimeter. UV spectra were recorded using a Varian Cary 50 instrument. The ECD spectra were recorded on a JASCO J-810 spectrometer. FT-IR spectra were recorded on a Bruker Vertex 70 instrument. NMR spectra were recorded on a Bruker AM400 spectrometer, and the residual peaks of methanol-d4 (δH 3.31 and δC 49.15) were used as references. HRESIMS data were obtained on a Thermo Fisher LC-LTQ-Orbitrap XL spectrometer. Single-crystal Xray diffraction data were collected on a Bruker SMART APEX-II CCD diffractometer using graphite-monochromatized Cu Kα radiation (λ = 1.541 78 Å). HPLC was performed on an Agilent 2100 quaternary system with a UV detector using a reversed-phase (RP) C18 column (5 μm, 10 × 250 mm, Weltch Ultimate XB-C18, Weltch AQ-C18, and YMC-pack ODS-A). D

DOI: 10.1021/acs.jnatprod.5b00681 J. Nat. Prod. XXXX, XXX, XXX−XXX

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801 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 320.1446 [M + H]+ (calcd for C17H22NO5, 320.1498). N-Isopentyl-5,6-dihydroplicane (7): colorless cubes; mp 158 °C; [α]25 D +233 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 207 (4.60), 238 (3.88), 291 (3.69) nm; ECD (MeOH) 207 (Δε, +77.88), 227 (Δε, +14.87), 258 (Δε, −1.47), 290 (Δε, +2.16) nm; IR (KBr) νmax 3028, 2950, 2867, 2819, 1689, 1484, 1386, 1273, 1238, 1189, 1082, 1039 cm−1; 1H and 13C NMR data, Tables S1 and S2, Supporting Information; HRESIMS m/z 399.2216 [M + H]+ (calcd for C23H31N2O4, 399.2284). N-(S)-s-Pentyl-5,6-dihydroplicane (8): colorless cubes; mp 141 °C; [α]25 D +208 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 207 (4.54), 238 (3.86), 294 (3.69) nm; ECD (MeOH) 206 (Δε, +69.87), 227 (Δε, +14.32), 256 (Δε, −2.14), 291 (Δε, +2.25) nm; IR (KBr) νmax 2955, 2925, 1687, 1483, 1386, 1235, 1080, 1037 cm−1; 1H and 13C NMR data, Tables S1 and S2, Supporting Information; HRESIMS m/z 399.2217 [M + H]+ (calcd for C23H31N2O4, 399.2284). N-Hexyl-5,6-dihydroplicane (9): colorless oil; [α]25 D +219 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 207 (4.66), 238 (3.92), 294 (3.89) nm; ECD (MeOH) 207 (Δε, +79.56), 227 (Δε, +15.57), 260 (Δε, −1.28), 290 (Δε, +2.22) nm; IR (KBr) νmax 3028, 2926, 2856, 1689, 1483, 1432, 1387, 1239, 1082, 1037, 934, 872, 838 cm−1; 1H and 13C NMR data, Tables S1 and S2, Supporting Information; HRESIMS m/z 413.2369 [M + H]+ (calcd for C24H33N2O4, 413.2440). N-Hydroxycarbonylpropyl-5,6-dihydroplicane (10): colorless oil; [α]25 D +187 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.49), 238 (4.05), 294 (3.77) nm; ECD (MeOH) 207 (Δε, +83.60), 227 (Δε, +18.18), 258 (Δε, −0.41), 290 (Δε, +2.58) nm; IR (KBr) νmax 3386, 2929, 2823, 1688, 1484, 1393, 1087, 1037 cm−1; 1H and 13C NMR data, Tables S1 and S2, Supporting Information; HRESIMS m/z 415.1858 [M + H]+ (calcd for C22H27N2O6, 415.1869), 437.1690 [M + Na]+ (calcd for C22H26N2O6Na, 437.1689). N-Phenethyl-5,6-dihydroplicane (11): colorless oil; [α]25 D +211 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 208 (4.68), 238 (3.88), 291 (3.76) nm; ECD (MeOH) 207 (Δε, +82.27), 222 (Δε, +17.67), 257 (Δε, −1.60), 294 (Δε, +2.20) nm; IR (KBr) νmax 2929, 2892, 1689, 1501, 1483, 1454, 1387, 1238, 1085, 1037 cm−1; 1H and 13C NMR data, Tables S1 and S2, Supporting Information; HRESIMS m/z 433.2054 [M + H]+ (calcd for C26H29N2O4, 433.2127). N-3-Indolylethyl-5,6-dihydroplicane (12): colorless oil; [α]25 D +138 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.77), 223 (4.63), 291 (4.02) nm; ECD (MeOH) 207 (Δε, +51.54), 227 (Δε, +10.54), 258 (Δε, −0.71), 291 (Δε, +1.50) nm; IR (KBr) νmax 2918, 1674, 1501, 1481, 1455 1432, 1386, 1235, 1084, 1036, 932, 742 cm−1; 1H and 13C NMR data, Tables S3 and S4, Supporting Information; HRESIMS m/z 472.2180 [M + H]+ (calcd for C28H30N3O4, 472.2236). N-Isopentyl-5,6-dihydroplicane N-oxide (13): colorless oil; [α]25 D +275 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.55), 238 (3.98), 294 (3.78) nm; ECD (MeOH) 207 (Δε, +55.45), 227 (Δε, +20.44), 268 (Δε, +0.61), 294 (Δε, +2.67) nm; IR (KBr) νmax 3037, 2952, 2926, 2868, 1696, 1483, 1388, 1367, 1239, 1093, 1077, 1036, 933, 802 cm−1; 1H and 13C NMR data, Tables S3 and S4, Supporting Information; HRESIMS m/z 415.2160 [M + H]+ (calcd for C23H31N2O5, 415.2233). Bliquine N-oxide (14): colorless oil; [α]25 D +174 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.63), 228 (4.28), 288 (3.89) nm; ECD (MeOH) 208 (Δε, +42.56), 223 (Δε, +17.85), 268 (Δε, +0.55), 291 (Δε, +1.79) nm; IR (KBr) νmax 3332, 2924, 1672, 1621, 1513, 1482, 1448, 1391, 1367, 1239, 1081, 1036, 931, 817 cm−1; 1H and 13C NMR data, Tables S3 and S4, Supporting Information; HRESIMS m/z 465.1973 [M + H]+ (calcd for C26H29N2O6, 465.2026). N-Methyl-11,12-seco-5,6-dihydroplicane (15): colorless oil; [α]25 D +21 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.63), 230 (3.96), 288 (3.82) nm; ECD (MeOH) 206 (Δε, +19.26), 221 (Δε, +2.40), 233 (Δε, +5.58), 257 (Δε, +0.16), 289, (Δε, +2.48) nm; IR (KBr) νmax 2929, 2824, 1672, 1644, 1504, 1485, 1405, 1262, 1236, 1090, 1038, 933, 854 cm−1; 1H and 13C NMR data, Tables S3 and S4, Supporting Information; HRESIMS m/z 359.1599 [M + H]+ (calcd for C19H23N2O5, 359.1607).

Plant Material. The whole plants of Z. candida were collected in October 2011 at Shiyan, Hubei Province, China, and authentificated by Prof. C. G. Zhang at this institute. A voucher specimen (No. 20111023) has been deposited at Tongji School of Pharmacy, HUST. Extraction and Isolation. The dried whole plants of Z. candida (50 kg) were extracted with 95% EtOH at room temperature (25 L × 4, each for 1 week). Each filtrate was concentrated and combined to afford a residue (3.78 kg), which was suspended in H2O (6 L), adjusted to pH = 2 with 0.3 M HCl, and extracted with CHCl3 (10 × 3 L). The aqueous phase was adjusted to pH = 10 using an ammonia solution and extracted with CHCl3 (4 × 3 L) to afford 40 g of CHCl3 extract. Finally, the aqueous phase was partitioned with n-BuOH to get 215 g of n-BuOH extract. The CHCl3 extract (40 g) was subjected to silica gel column chromatography (CC) (CHCl3−MeOH, from 1:0 to 1:1) to afford five fractions (Fr. 1−5). Fr. 1 was fractionated on a silica gel column eluted with petroleum ether−acetone (from 15:1 to 2:1) and then separated by RP HPLC (MeOH−H2O−Et2NH, 69.8:30:0.2) to afford compounds 12 (3.9 mg, tR 44.2 min), 23 (2.3 mg, tR 24.4 min), and 14 (3.5 mg, tR 29.6 min). Fractionation and purification of the other fractions of the CHCl3 extract using repeated silica gel CC, Sephadex LH-20, RP C18, and RP HPLC yielded compounds 1 (5.8 mg), 4 (3 mg), 5 (4 mg), 7 (11.4 mg), 8 (5.6 mg), 9 (10.1 mg), 10 (5.7 mg), 11 (4.7 mg), 13 (3.0 mg), 15 (2.8 mg), 16 (11 mg), 17 (258 mg), 18 (57.8 mg), 20 (48.2 mg), 22 (2.8 mg), and 24 (9.2 mg). The n-BuOH extract (215 g) was subjected to silica gel CC (CHCl3− MeOH, from 30:1 to 1:1) to afford four fractions (Fr. I−IV). Using repeated silica gel CC, Sephadex LH-20, RP C18, and RP HPLC, compounds 2 (5.8 mg), 3 (4.3 mg), 6 (2.3 mg), 19 (28.2 mg), and 21 (38.7 mg) and lycorine (50 g) were isolated. The detailed isolation procedure is shown in the Supporting Information, Extraction and Isolation. 11β-Hydroxylycoramine (1): colorless needles (MeOH); mp 149 °C; [α]25 D −71 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 210 (4.47), 228 (3.78), 288 (3.31) nm; ECD (MeOH) 202 (Δε, +3.30), 210 (Δε, −18.23), 218 (Δε, −2.06), 224, (Δε, −2.22) nm; IR (KBr) νmax 3339, 2935, 1625, 1592, 1507, 1445, 1281, 1032 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 306.1654 [M + H]+ (calcd for C17H24NO4, 306.1705). 9-De-O-methyl-11β-hydroxylycoramine (2): colorless needles (MeOH); mp 255 °C; [α]25 D −75 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.55), 230 (3.98), 288 (3.58) nm; ECD (MeOH) 207 (Δε, −7.28), 228 (Δε, −2.49) nm; IR (KBr) νmax 3331, 2939, 1599, 1508, 1453, 1295, 1179, 1157, 1092, 1026 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 292.1539 [M + H]+ (calcd for C16H22NO4, 292.1549). 9-De-O-methyl-11β-hydroxygalanthamine (3): colorless oil; [α]25 D −44 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (4.27), 228 (3.78), 288 (3.23) nm; ECD (MeOH) 207 (Δε, −15.06), 212 (Δε, −20.08), 225 (Δε, −3.59), 254 (Δε, −0.15), 286 (Δε, +1.21) nm; IR (KBr) νmax 3356, 2919, 2850, 1598, 1508, 1294, 1085, 1032, 1199 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 290.1382 [M + H]+ (calcd for C16H20NO4, 290.1392). 11β-Hydroxylycoramine N-oxide (4): colorless oil; [α]25 D −91 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 212 (4.49), 237 (3.80), 288 (3.46) nm; ECD (MeOH) 206 (Δε, −10.35), 223 (Δε, −0.62), 240 (Δε, −0.90) nm; IR (KBr) νmax 3324, 2940, 1624, 1592, 1512, 1447, 1286, 1098, 1030 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 322.1646 [M + H]+ (calcd for C17H24NO5, 322.1655). 11β-Hydroxygalanthamine N-oxide (5): colorless oil; [α]25 D −95 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 213 (4.57), 237 (3.95), 292 (3.56) nm; ECD (MeOH) 204 (Δε, −20.06), 216 (Δε, −20.28), 228 (Δε, −1.88), 288 (Δε, +2.94) nm; IR (KBr) νmax 3310, 2932, 2843, 1625, 1594, 1512, 1446, 1285, 1103, 982, 849, 769 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 320.1490 [M + H]+ (calcd for C17H22NO5, 320.1498). 2β,11β-Dihydroxygalanthamine (6): colorless oil; [α]25 D −52 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (4.31), 228 (3.78), 288 (3.26) nm; ECD (MeOH) 210 (Δε, −14.68), 225 (Δε, −1.84), 240 (Δε, +0.10), 249 (Δε, −028), 286 (Δε, +0.90) nm; IR (KBr) νmax 3328, 2919, 2850, 1508, 1449, 1281, 1095, 1077, 1033, 1176, 1036, E

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N-Isopentyl-11,12-seco-5,6-dihydroplicane (16): colorless oil; [α]25 D +4 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 205 (4.66), 238 (3.71), 291 (3.81) 329 (3.25) nm; ECD (MeOH) 207 (Δε, +20.63), 218 (Δε, +2.69), 231 (Δε, +5.94), 256 (Δε, +0.40), 290, (Δε, +3.31) nm; IR (KBr) νmax 3481, 2955, 2931, 1672, 1644, 1482, 1424, 1403, 1234, 1078, 1037, 935, 761 cm−1; 1H and 13C NMR data, Tables S3 and S4, Supporting Information; HRESIMS m/z 415.2220 [M + H]+ (calcd for C23H31N2O5, 415.2233). Single-Crystal X-ray Diffraction Analysis. Diffraction intensity data collection, structure solution, refinement, and hydrogen atom treatment for compounds 1, 2, 7, 8, and 17 were performed as described in a previous paper.17 The ORTEP crystal structures for compounds 1, 2, 17, 7, and 8 are shown in Figures 1 and 2, respectively. Crystallographic data of 1: C17H22NO4·CH3OH, M = 337.41, orthorhombic, P2(1)2(1)2(1), a = 7.9947(2) Å, b = 8.6002(2) Å, c = 24.7362(5) Å, α = β = γ = 90°, V = 1700.76(7) Å3, T = 100(2) K, Z = 4, Dcalcd = 1.318 Mg/m3, crystal size 0.12 × 0.10 × 0.10 mm3, F(000) = 728, absorption coefficient 0.783 mm−1, reflections collected 8115, independent reflections 2648 [R(int) = 0.1519], final R indices [I > 2σ(I)] R1 = 0.0475, wR2 = 0.1316, R indices (all data) R1 = 0.0547, wR2 = 0.1332. Flack parameter: −0.3(3) for 1633 Friedel pairs. Hooft parameter: −0.3 (1) for 1142 Bijvoet pairs. Crystallographic data of 2: 2C16H21NO4·H2O, M = 600.69, orthorhombic, P2(1)2(1)2(1), a = 8.03720(10) Å, b = 15.6818(2) Å, c = 22.5648(3) Å, α = β = γ = 90°, V = 2844.02(6) Å3, T = 100(2) K, Z = 4, Dcalcd = 1.403 Mg/m3, crystal size 0.12 × 0.10 × 0.01 mm3, F(000) = 1288, absorption coefficient 0.842 mm−1, reflections collected 14 530, independent reflections 4917 [R(int) = 0.0413], final R indices [I > 2σ(I)] R1 = 0.0440, wR2 = 0.1382, R indices (all data) R1 = 0.0595, wR2 = 0.1976. Flack parameter: 0.0(2) for 2091 Friedel pairs. Hooft parameter: 0.06(4) for 2092 Bijvoet pairs. Crystallographic data of 7: C23H30N2O4, M = 398.49, monoclinic, P2(1), a = 9.6195(2) Å, b = 11.6392(2) Å, c = 9.64410(10) Å, α = γ = 90°, β = 94.2290(10), V = 1076.85(3) Å3, T = 298(2) K, Z = 2, Dcalcd = 1.229 Mg/m3, crystal size 0.15 × 0.12 × 0.10 mm3, F(000) = 428, absorption coefficient 0.678 mm−1, reflections collected 5579, independent reflections 2576 [R(int) = 0.0686], final R indices [I > 2σ(I)] R1 = 0.0418, wR2 = 0.1131, R indices (all data) R1 = 0.0451, wR2 = 0.1161. Flack parameter: 0.0(3) for 1006 Friedel pairs. Hooft parameter: −0.1(1) for 1005 Bijvoet pairs. Crystallographic data of 8: C23H30N2O4, M = 398.49, monoclinic, P2(1), a = 9.4965(2) Å, b = 11.4906(3) Å, c = 9.9133(3) Å, α = γ = 90°, β = 95.2470(10), V = 1077.21(5) Å3, T = 298(2) K, Z = 2, Dcalcd = 1.229 Mg/m3, crystal size 0.15 × 0.10 × 0.10 mm3, F(000) = 428, absorption coefficient 0.678 mm−1, reflections collected 18 180, independent reflections 3545 [R(int) = 0.0315], final R indices [I > 2σ(I)] R1 = 0.0424, wR2 = 0.1192, R indices (all data) R1 = 0.0441, wR2 = 0.1216. Flack parameter: −0.1(3) for 1577 Friedel pairs. Hooft parameter: −0.08(7) for 1576 Bijvoet pairs. Crystallographic data of 17: C17H21NO4·0.15H2O, M = 307.60, orthorhombic, P2(1)2(1)2(1), a = 9.7421(2) Å, b = 9.7391(2) Å, c = 16.0471(3) Å, α = β = γ = 90°, V = 1522.54(5) Å3, T = 298(2) K, Z = 4, Dcalcd = 1.342 Mg/m3, crystal size 0.30 × 0.20 × 0.20 mm3, F(000) = 657, absorption coefficient 0.790 mm−1, reflections collected 31 996, independent reflections 2741 [R(int) = 0.0269], final R indices [I > 2σ(I)] R1 = 0.0289, wR2 = 0.0798, R indices (all data) R1 = 0.0290, wR2 = 0.0799. Flack parameter: 0.06(17) for 1151 Friedel pairs. Hooft parameter: 0.03(2) for 1151 Bijvoet pairs. Crystallographic data for compounds 1, 2, 7, 8, and 17 have been deposited in the Cambridge Crystallographic Data Centre with deposition numbers CCDC 1415212−1415216, respectively. AChE Inhibitory Activity Testing. The AChE inhibitory activities of 1−24 were evaluated by Ellman’s method with slight modification.18 Galanthamine was used as a positive control. Anti-inflammatory Activity Evaluation. The inhibitory activities of compounds 1−24 on the NO production in LPS-induced RAW 264.7 mouse macrophages were evaluated as previously described.19 Dexamethasone was used as a positive control.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00681. NMR data for 7−16, bioassay data for 1−24, comparison of ECD spectra of 1−6 and 17, 7−16, and 22, data fitting curves for IC50 calculation, detailed isolation procedure, and HRESIMS, IR, UV, ECD, and NMR spectra for 1− 16 (PDF) X-ray crystallographic data for 1 (CIF) X-ray crystallographic data for 2 (CIF) X-ray crystallographic data for 7 (CIF) X-ray crystallographic data for 8 (CIF) X-ray crystallographic data for 17 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 86-15171484550. Fax: 86-2783692762. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the staff at the Analysis and Measurement Centre of HUST for IR and ECD data. This work was financially supported by the National Natural Science Foundation of China (31170323 and 81001368), Wuhan Youth Chenguang Program of Science and Technology (201271031389), and Major New Drugs Innovation Project of China (2013ZX09103001-020).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00681 J. Nat. Prod. XXXX, XXX, XXX−XXX