Di- and Triterpenoids from the Leaves of Casearia ... - ACS Publications

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Di- and Triterpenoids from the Leaves of Casearia balansae and Neurite Outgrowth Promoting Effects of PC12 Cells Jing Xu,† Jing Kang,† Xiaocong Sun,† Xiangrong Cao,† Kasimu Rena,§ Dongho Lee,⊥ Quanhui Ren,† Shen Li,† Yasushi Ohizumi,∥ and Yuanqiang Guo*,† †

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People’s Republic of China § College of Pharmacy, Xinjiang Medical University, Urumuqi 830011, People’s Republic of China ⊥ Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Korea ∥ Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan S Supporting Information *

ABSTRACT: A bioassay-guided phytochemical investigation of the leaves of Casearia balansae led to the isolation of six new cucurbitane-type triterpenoid derivatives (balanterpenes A−F, 1−6) and four new clerdoane-type diterpenoids (balanterpenes G−J, 7−10). The structures of 1−10 were established on the basis of extensive analysis of NMR spectroscopic data, X-ray crystallography, and experimental and calculated electronic circular dichroism spectra. Compound 1 features a ring-expanded triterpenoid skeleton with the C-19 methyl involved in the ring formation, compound 6 possesses a rare hexanortriterpenoid scaffold, and compounds 7−10 may be four new diterpenoid artifacts presumably formed during the extraction and purification processes. Compounds 3 and 7−10 showed promoting effects on neurite outgrowth of PC12 cells with EC50 values in the range 2.9−10.0 μM.

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growth may be expected to be potentially useful for the treatment of AD and other neurological disorders.28−30 The investigation to obtain bioactive compounds led to the isolation of six new triterpenoids (balanterpenes A−F, 1−6) and four new diterpenoids (balanterpenes G−J, 7−10). Herein, the isolation and structural elucidation of these compounds as well as their promoting effects on neurite outgrowth of PC12 cells are described.

lzheimer’s disease (AD), a neurodegenerative disorder, is the most common form of dementia worldwide. Patients with AD usually have the characteristic symptoms of progressive memory loss and cognitive decline.1 For this disease, some remedies, such as donepezil, rivastigmine, tacrine, and galantamine, have been approved for the treatment of AD. However, all of these drugs provide only symptomatic treatment, and the effectiveness has been questioned since they cannot prevent or delay neurodegeneration.2,3 Therefore, there is still an urgent need to develop new agents to treat AD effectively.4,5 Natural products play an important role in the research and development of new drugs, and many natural bioactive substances have been found from plants.6,7 Casearia balansae Gagnep., a small tree belonging to the Flacourtiaceae plant family, is a nontraditional medicinal plant mainly distributed in southern mainland China.8 Previous phytochemical investigations on this species and other species of the genus Casearia revealed that the major constituents of this genus include terpenoids, phenylethanoids, flavonoids, phenolics, steroids, and volatile oils,9−27 which showed diverse biological effects, such as antifungal, cytotoxic, antimalarial, antimicrobial, and DNAmodifying activities.9 In a search to discover neuroactive metabolites from natural products, an EtOAc-soluble part of the MeOH extract of the leaves of C. balansae showed moderate promoting effects on neurite growth of PC12 cells. Neurites have been proven to be important for maintenance of the central nervous system, and bioactive compounds to promote neurite © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The EtOAc-soluble part of the MeOH extract of the leaves of C. balansae afforded 10 new (1−10) terpenoids. Compound 1 was obtained as colorless crystals from MeOH. Its molecular formula, C31H48O4, was deduced from the 13C NMR data and the HRESIMS ion m/z 507.3450 [M + Na]+ (calcd for C31H48NaO4, 507.3450). The molecular formula indicated eight indices of hydrogen deficiency. From the 1H NMR spectrum, seven methyl groups [δH 0.80 (3H, s, H3-18), 0.89 (3H, d, J = 7.2 Hz, H3-21), 1.64 (3H, s, H3-27), 1.30 (3H, s, H3-28), 1.13 (3H, s, H3-29), 0.87 (3H, s, H3-30), and 1.05 (3H, d, J = 6.9 Hz, H3-31)], three oxymethine protons [δH 3.94 (1H, dd, J = 10.1, 4.2 Hz, H-3), 3.08 (1H, t, J = 3.1 Hz, H-11), and 3.60 (1H, brd, J = 10.5 Hz, H-22)], and two olefinic protons [δH 4.76 (2H, s, H-26)] were observed. The 13C NMR spectrum of 1 Received: September 15, 2015

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DOI: 10.1021/acs.jnatprod.5b00815 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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were performed. The HMBC spectrum showed long-range couplings of H-3 to C-1, C-2, C-4, C-5, C-28, and C-29, H-8 to C-6, C-7, C-9, C-11, C-13, C-14, C-19, and C-30, H2-19 to C-1, C-5, and C-8−C-11, H-11 to C-8, C-9, C-12, C-13, and C-19, H3-18 to C-12−C-14, and C-17, H3-30 to C-8 and C-13−C-15, and H3-28/29 to C-3−C-5. These HMBC correlations, together with the corresponding cross-peaks in the 1H−1H COSY spectrum (Figure 1), disclosed the presence of a 6/7/6/5 tetracyclic fused ring system.36−39 In addition to this fused ring system, a side chain consisting of C-20−C-27 and C-31 was deduced from the

showed 31 carbon resonances (Table 1), of which one carbonyl (δC 195.5) and four olefinic carbons (δC 166.7, 132.2, 149.2, and 110.8) were apparent. The remaining 26 signals were assigned as seven methyls [δC 18.2 (C-18), 12.3 (C-21), 18.1 (C-27), 24.0 (C-28), 19.6 (C-29), 16.0 (C-30), and 20.7 (C-31)], eight methylenes [δC 42.3 (C-2), 30.1 (C-6), 25.3 (C-7), 34.5 (C-12), 34.7 (C-15), 27.2 (C-16), 31.6 (C-19), and 34.8 (C-23)], seven methines [δC 73.7 (C-3), 47.1 (C-8), 63.3 (C-11), 49.2 (C-17), 42.3 (C-20), 71.0 (C-22), and 38.0 (C-24)], three quaternary carbons [δC 42.1 (C-4), 45.8 (C-13), and 47.0 (C-14)], and one oxygenated tertiary carbon [δC 61.1 (C-9)] with the aid of DEPT and HMQC experiments. The 31 carbons and the seven methyl groups implied compound 1 is a triterpenoid derivative.31−35 To elucidate its structure, HMBC and 1H−1H COSY experiments

Figure 1. 1H−1H COSY and key HMBC correlations of compounds 1, 2, 6, and 7.

Table 1. 13C NMR Data for Compounds 1−6 (δ in ppm) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

1 195.5 42.3 73.7 42.1 166.7 30.1 25.3 47.1 61.1 132.2 63.3 34.5 45.8 47.0 34.7 27.2 49.2 18.2 31.6 42.3 12.3 71.0 34.8 38.0 149.2 110.8 18.1 24.0 19.6 16.0 20.7

2 C CH2 CH C C CH2 CH2 CH C C CH CH2 C C CH2 CH2 CH CH3 CH2 CH CH3 CH CH2 CH C CH2 CH3 CH3 CH3 CH3 CH3

23.9 38.1 214.4 50.9 140.6 120.0 22.9 42.5 48.9 36.0 213.9 48.8 48.8 49.7 33.8 21.7 50.8 18.8 19.4 74.9 26.1 44.1 24.5 124.1 131.9 17.7 25.7 22.9 28.5 18.2

3 CH2 CH2 C C C CH CH2 CH C CH C CH2 C C CH2 CH2 CH CH3 CH3 C CH3 CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3

36.1 71.6 213.2 50.2 140.4 120.6 23.8 42.6 49.7 33.8 213.4 48.7 48.8 48.2 33.7 21.7 50.9 18.7 19.9 74.9 26.1 44.1 22.9 124.1 132.0 17.7 25.7 21.2 29.4 18.2

4 CH2 CH C C C CH CH2 CH C CH C CH2 C C CH2 CH2 CH CH3 CH3 C CH3 CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3

B

36.0 71.6 212.7 50.2 140.4 120.6 23.8 42.3 50.3 33.8 213.1 48.8 48.9 48.2 34.0 21.3 48.4 18.9 19.9 79.8 24.4 211.9 35.1 115.5 135.9 25.7 18.2 21.2 29.4 18.3

5 CH2 CH C C C CH CH2 CH C CH C CH2 C C CH2 CH2 CH CH3 CH3 C CH3 CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3

36.0 71.6 212.8 50.2 140.4 120.5 23.8 42.4 48.9 33.8 213.1 48.8 48.2 50.4 33.9 21.0 48.0 18.5 19.9 78.8 24.0 201.9 117.9 156.6 71.4 29.6 29.6 21.2 29.4 19.0

6 CH2 CH C C C CH CH2 CH C CH C CH2 C C CH2 CH2 CH CH3 CH3 C CH3 C CH CH C CH3 CH3 CH3 CH3 CH3

36.0 71.6 213.0 49.8 140.4 120.4 24.0 42.8 48.6 33.8 211.6 47.1 50.3 49.1 34.3 21.7 58.0 18.2 19.9 208.7 31.4

CH2 CH C C C CH CH2 CH C CH C CH2 C C CH2 CH2 CH CH3 CH3 C CH3

21.3 29.4 18.7

CH3 CH3 CH3



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Figure 2. Conformations and key NOESY correlations of compounds 1, 2, 7, and 9.

HMBC correlations of H3-21 to C-17, C-20, and C-22, H3-27 to C-24−C-26, H2-26 to C-24, C-25, and C-27, and H3-31 to C-23− C-25, as well as the corresponding 1H−1H COSY correlations shown in Figure 1.40,41 This side chain was shown to be at C-17 by the 1H−1H COSY correlation of H-17/H-20 and HMBC correlations (Figure 1). Further analysis of the HMQC, HMBC, and 1H−1H COSY data permitted the assignments of all the proton and carbon signals, and a ring-expanded triterpenoid skeleton with an additional methyl (Me-31) attached at C-24 for 1 seemed to be established. However, the molecular formula based on this 2D structure was incompatible with the HRESIMS data, indicating the presence of another ring according to the index of hydrogen deficiency. The chemical shifts of C-9 and C-11 and the HRESIMS data of compound 1 strongly pointed toward a 9,11-epoxy structural moiety. Thus, the 2D structure of compound 1 was defined. The relative configuration of compound 1 was deduced from the NOESY spectrum and Chem3D modeling (Figure 2). NOESY correlations of H-2β/H3-29, H-3/H3-28, H3-29/H-6β, H-6β/H-8, H-8/H3-18, H-8/H-19β, H-19α/H-11, H-7α/H3-30, H-12α/H3-30, H3-30/H-15α, H3-30/H-17, H3-18/H-15β, H3-18/H-16β, and H-15β/H-20, together with Chem3D modeling, implied a conformation for compound 1 as shown in Figure 2. According to this molecular arrangement, the B/C and C/D rings were both trans-fused with H-8 and Me-18 β-oriented and Me-30 in an α-position, ring A existed in a twistchair conformation with the C-3 hydroxy group in a β-equatorial position supported by the coupling constants (J3,2α/β = 4.2, 10.1 Hz) between H-3 and H2-2, ring C is also in a twist-chair conformation with the epoxy moiety occupying its α-face, and ring D had an envelope conformation with H-17 in an α-position. The Me-21 group was inferred to be in an α-position from the corresponding correlations. However, it is not feasible to deduce the orientations of the C-22 hydroxy and the C-31 methyl group from the NOESY spectrum. The X-ray crystallography data of crystals of compound 1 confirmed the ring-expanded triterpenoid skeleton and allowed the absolute configuration to be assigned. An ORTEP drawing of 1 with the atom numbering indicated is shown in Figure 3, and the absolute configuration was assigned as 3S, 8S, 9S, 11R, 13R, 14S, 17R, 20S, 22R, and 24S

Figure 3. ORTEP drawing of 1. Thermal ellipsoids are drawn at the 30% probability level.

according to the Flack parameter [0.17(8)]. The structure of 1 was therefore elucidated as a ring-expanded 24-homotriterpenoid with a new skeleton, and the compound was named balanterpene A. Compound 2, a colorless oil, possessed a molecular formula of C30H46O3 based on its 13C NMR and HRESIMS (m/z 477.3347 [M + Na]+, calcd for C30H46NaO3, 477.3345) data. The 1H NMR spectrum of 2 displayed eight methyl singlets including a pair of allylic methyls [δH 1.62 (H3-26) and 1.68 (H3-27)], a pair of geminal aliphatic methyls [δH 1.26 (H3-28) and 1.23 (H3-29)], and two olefinic protons [δH 5.76 (1H, brs, H-6) and 5.09 (1H, t, J = 6.8 Hz, H-24)] (Table 2). The 13C NMR data of 2 showed 30 skeletal resonances including two carbonyl, one oxygenated, and four olefinic carbons (Table 1). The remaining 23 aliphatic carbons were classified into eight methyls, eight methylenes, three methines, and four quaternary carbons based on DEPT and HMQC experiments (Table 1). The 30 skeletal carbon resonances and the above spectroscopic features, especially the eight methyl singlets, implied compound 2 had a cucurbitanetype triterpenoid skeleton according to the triterpenoids C



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Table 2. 1H NMR Data for Compounds 1−6 (δ in ppm, J in Hz) position

1

position

2

3

4

5

6

2α 2β 3 6α 6β 7α 7β 8 11 12α 12β 15α 15β 16α 16β 17 18 19

2.72 dd (16.2, 4.2) 2.53 dd (16.2, 10.1) 3.94 dd (10.1, 4.2) 2.70 dd (12.8, 8.4) 2.34 t (12.8) 1.74 m 1.45 m 1.47 ma 3.08 t (3.1) 2.04 d (3.1) 1.90 d (3.1) 1.39 m 1.28 m 1.74 m 1.45 m 2.06 dd (11.9, 2.9) 0.80 s 2.94 d (14.6) 2.36 d (14.6) 1.65 ma 0.89 d (7.2) 3.60 brd (10.5) 1.36 m 1.24 m 2.47 m 4.76 s 1.64 s 1.30 s 1.13 s 0.87 s 1.05 d (6.9)

1α 1β 2α 2β 6 7α 7β 8 10 12α 12β 15α 15β 16α 16β 17 18 19 21 22

2.40 m 1.96 m 2.49 m 2.35 m 5.76 brs 2.42 m 2.00 m 2.00 ma 2.57 ma 2.60 d (14.4) 3.00 d (14.4) 1.45 m 1.35 m 1.89 m 1.82 m 2.04 ma 0.94 s 1.07 s 1.29 s 1.49 m 1.39 m 1.85 m 1.50 m 5.09 t (6.8) 1.62 s 1.68 s 1.26 s 1.23 s 1.10 s

2.28 m 1.22 m 4.41 dd (12.8, 5.6)

2.28 m 1.24 m 4.43 dd (12.9, 6.0)

2.29 m 1.23 m 4.43 dd (12.9, 6.0)

2.26 m 1.25 m 4.43 dd (13.0, 6.2)

5.79 d (5.7) 2.41 m 2.01 m 2.03 ma 2.70 brd (13.2) 2.64 d (14.5) 3.02 d (14.5) 1.43 m 1.28 m 1.89 m 1.65 m 2.06 ma 0.95 s 1.06 s 1.30 s 1.48 m 1.39 m 2.00 m 1.90 m 5.10 t (7.0) 1.62 s 1.68 s 1.34 s 1.27 s 1.11 s

5.79 d (5.5) 2.41 m 1.98 m 2.04 d (8.4) 2.72 brd (14.4) 2.71 d (14.4) 3.14 d (14.4) 2.30 m 1.37 m 1.87 m 1.65 m 2.36 ma 0.99 s 1.08 s 1.44 s

5.78 d (5.7) 2.41 m 1.99 m 2.04 d (8.0) 2.71 brd (14.4) 2.70 d (14.4) 3.15 d (14.4) 1.47 m 1.34 m 1.68 m 1.42 m 2.37 ma 1.11 s 1.08 s 1.44 s

5.80 d (5.8) 2.43 m 2.02 m 2.01 ma 2.74 brd (13.0) 2.57 d (14.3) 3.22 d (14.3) 1.51 m 1.44 m 2.36 m 1.83 m 3.08 t (8.8) 1.16 s 1.08 s 2.13 s

3.24 d (6.8) 2.73 ma 5.28 dt (6.8, 1.3) 1.76 s 1.64 s 1.34 s 1.28 s 1.13 s

6.67 d (15.2)

20 21 22 23 24 26 27 28 29 30 31 a

23 24 26 27 28 29 30

7.15 d (15.2) 1.39 s 1.41 s 1.34 s 1.28 s 1.00 s

1.35 s 1.28 s 0.69 s

Signals were in overlapped regions of the spectra, and the multiplicities could not be discerned.

reported from the genus Casearia.9 From the 2D NMR spectra, the 6/6/6/5 tetracyclic fused ring system and the C-17 side chain comprising C-20−C-27 and, hence, a characteristic cucurbitanetype triterpenoid were inferred.42−45 Correspondingly, two carbonyl, one oxygenated, and four olefinic carbons signals at δC 214.4, 213.9, 74.9, 140.6, 120.0, 124.1, and 131.9 were attributed to C-3, C-11, C-20, C-5, C-6, C-24, and C-25, respectively. Several other proton and carbon signals were also assigned by the HMBC and 1H−1H COSY correlations shown in Figure 1. A NOESY spectrum allowed the relative configuration to be assigned via the correlations of H-2α/H3-28, H-2α/H-10, H-10/H3-28, H3-29/H-6, H-7β/H3-19, H3-19/H-8, H3-19/ H-1β, H3-19/H3-18, H3-18/H-8, H3-18/H-12β, H3-30/H-12α, H3-30/H-15α, H3-30/H-17, H3-18/H-15β, and H-12β/H3-21. On the basis of these correlations and Chem3D modeling, rings B and C were cis-fused with H-8 and Me-19 both in β-positions, and rings C and D were trans-fused with Me-18 and Me-30 in β- and α-positions, respectively. H-17 was assigned as α-oriented, and the relative configuration of C-20 was rel-20S. The absolute configuration was determined via experimental and calculated electronic circular dichroism (ECD) data, a powerful tool to determine the absolute configuration of natural products.46,47 Through systematic conformational search and geometry optimizations,48−50 the ECD spectra were calculated at the B3LYP/SVP level with the CPCM model in acetonitrile. The calculated ECD spectrum of 2 (Figure 4A) matched the experimental spectrum closely, which suggested an absolute

configuration of 8S, 9R, 10R, 13R, 14S, 17S, and 20S. The structure of 2 was therefore elucidated as shown, and the compound was named balanterpene B. The molecular formula of compound 3 was determined as C30H46O4 based on the 13C NMR and HRESIMS (m/z 493.3285 [M + Na]+, calcd for C30H46NaO4, 493.3294) data. Its 1H and 13 C NMR data (Tables 1 and 2) resembled those of compound 2, which implied that 3 is also a cucurbitane-type triterpenoid. From the 1H NMR spectrum, the characteristic methyl singlets and oxygenated and olefinic protons as present in compound 2 were observed. The 13C NMR spectrum of 3 showed 30 carbon resonances (Table 1). Upon comparison of 1H and 13C NMR data of 3 with those of 2, an additional oxymethine carbon at δC 71.6 (C-2) and the corresponding proton at δH 4.41 (1H, dd, J = 12.8, 5.6 Hz, H-2) suggested that 3 is an oxygenated derivative of 2. HMQC, HMBC, and 1H−1H COSY data revealed that the additional hydroxy group was located at C-2. The 2D structure was therefore elucidated. The NOESY spectrum disclosed that 3 had the same molecular conformation as 2. The C-2 hydroxy group occupied a β-equatorial position, which was also supported by the coupling constants (J2,1α/β = 5.6, 12.8 Hz) between H-2 and H2-1. TDDFT ECD calculations were performed to elucidate the absolute configuration of 3. By comparison of its experimental and calculated ECD spectra (Figure 4B), the absolute configuration of 3 was determined as 2S, 8S, 9R, 10R, 13R, 14S, 17S, and 20S. The structure of compound 3 (balanterpene C) was therefore elucidated. D



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Figure 4. Calculated and experimental ECD spectra of compounds 2−10 (A−I) in acetonitrile.

The 1H and 13C NMR data (Tables 1 and 2) of 4 resembled those of compound 3, which implied that 4 is also a cucurbitane-type

triterpenoid. The main difference in their 13C NMR data is that one more ketocarbonyl resonance occurred in the 13C NMR E



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spectrum of 4. HMQC, HMBC, and 1H−1H COSY experiments permitted the ketocarbonyl signal at δC 211.9 to be assigned to C-22. By further interpretation of the 2D NMR spectra, all of the proton and carbon signals were assigned and the 2D structure was established. A NOESY spectrum allowed the relative configuration of 4 to be elucidated, which was the same as that of 3. The absolute configuration of 4 was determined by comparison of its calculated and experimental ECD spectra (Figure 4C), which suggested an absolute configuration of 2S, 8S, 9R, 10R, 13R, 14S, 17S, and 20R. Thus, the structure of compound 4 (balanterpene D) was defined. The molecular formula of compound 5 (balanterpene E) was deduced as C30H44O6 from the 13C NMR and HRESIMS (m/z 523.3035 [M + Na]+, calcd for C30H44NaO6, 523.3036) data. The 1H NMR spectrum showed eight characteristic methyl singlets, an oxymethine, and three olefinic protons. The 13C NMR spectrum revealed total 30 carbons including four olefinic and three ketocarbonyl carbons. These spectroscopic features indicated a cucurbitane-type triterpenoid structure for 5 based on the comparison of its NMR data with those of compounds 2−4. The cucurbitane-type triterpenoid structure was further substantiated by the subsequent DEPT, HMQC, HMBC, and 1 H−1H COSY experiments. By interpretation of the 1D and 2D NMR data, all the protons and carbons were assigned and a cucurbitane-type triterpenoid structure for 5 was disclosed. It possessed an additional hydroxy group at C-25 and a Δ23,24 trans-double bond compared to the structure of compound 4. Using the same NOESY experiment as for compounds 2−4, the relative configuration was determined. The absolute configuration was assigned as 2S, 8S, 9R, 10R, 13R, 14S, 17S, 20R, and 23E based on its relative configuration and the comparison of experimental and calculated ECD spectra (Figure 4D). On the basis of the above analysis, the structure of compound 5 was established. Compound 6, an oil, had a molecular formula of C24H34O4 deduced from the 13C NMR and HRESIMS (409.2352 [M + Na]+, calcd for C24H34NaO4, 409.2355) data. Six methyl singlets, one olefinic proton, and one oxymethine proton were evident in the 1H NMR spectrum. The 13C NMR spectrum showed 24 carbon resonances including one oxygenated, two olefinic, and three ketocarbonyl carbons. Apart from these, the remaining 18 carbons comprised six methyl, five methylene, three methine, and four quaternary carbons based on DEPT and HMQC experiments (Table 1). These spectroscopic features implied a hexanortriterpenoid structure for 6 according to the comparison of its NMR data with those of compounds 2−5. The HMBC and 1 H−1H COSY spectra (Figure 1) disclosed the presence of a 6/6/6/5 tetracyclic fused ring system, which carried five methyls at C-13, C-9, C-4, C-4, and C-14, respectively. In turn, the two olefinic and two carbonyl carbons at δC 140.4 (C-5), 120.4 (C-6), 213.0 (C-3), and 211.6 (C-11) were also assigned. The residual two carbon signals at δC 208.7 and 31.4 represented an acetyl group, which was attached at C-17 by the corresponding HMBC correlations. On the basis of the above analysis, the 2D structure of 6 was established to be a cucurbitane-type hexanortriterpenoid. A NOESY spectrum revealed that the 6/6/6/5 tetracyclic fused ring system had the same conformation as those of compounds 2−5. The C-2 hydroxy group and H-17 were β- and α-oriented, respectively, by the corresponding correlations. The absolute configuration of 6 was established as 2S, 8S, 9R, 10R, 13R, 14S, and 17S by comparison of its experimental and calculated ECD spectra (Figure 4E). On the basis of the above evidence, the structure of compound 6 (balanterpene F) was

characterized as a rare hexanortriterpenoid derived from a cucurbitane-type triterpenoid. Compound 7 was obtained as a colorless oil. Its molecular formula was determined as C24H36O6 by HRESIMS (m/z 443.2404 [M + Na]+, calcd for C24H36NaO6, 443.2410). The 1 H NMR spectrum of 1 exhibited five olefinic protons, four oxymethine protons, two methyl singlets, one methyl doublet, and two methoxy groups (Table 3). The 13C NMR spectrum of 1 showed 24 carbon resonances (Table 3), of which four signals were indicative of two methoxy groups (δC 57.5 and 55.4) and an acetoxy moiety (δC 170.1 and 21.7). Apart from these carbon signals for the substituent groups, there were 20 additional resonances (Table 3), indicating a diterpenoid framework.51−54 These 20 typical skeletal carbons, especially the two acetal and the four olefinic carbons forming two terminal double bonds (Table 3), implied that compound 7 possessed a characteristic clerodane-type diterpenoid skeleton as shown in Figure 1.25 This was further confirmed by HMBC and 1H−1H COSY experiments. After defining the scaffold, the two methoxy groups located at C-6 and C-18 and the C-19 acetoxy group were also deduced from the HMBC spectrum via the correlations of H-6 with one methoxy carbon, H-18 with the other methoxy carbon, and H-19 with the carbonyl carbon of the acetoxy group. The C-2 substituent was assigned as a hydroxy group based upon the HRESIMS data. Further analysis of the HMQC, HMBC, and 1 H−1H COSY spectra (Figure 1) led to the assignments of all the proton and carbon signals and the establishment of the 2D structure for 7. NOESY correlations observed for H-1β/H-6, H-1β/H-19, H1β/H-8, H-6/H-8, H-1α/H3-20, H-10/H-19, H-7/H3-17, H-10/ H-11a(b), H-19/H-11b, H-7/H-11b, and H-3/H-18, together with Chem3D modeling, implied a conformation for compound 7 as depicted in Figure 2. On the basis of this conformation, two six-membered rings, A and B, were cis-fused with C-19 and H-1, both in α-positions, ring A had a twisted boat conformation with an α-orientation for the C-2 hydroxy group, ring B had a normal chair conformation with an α-equatorial orientation for Me-17 and an α-axial orientation for the C-9 side chain, and ring C adopted an envelope conformation with H-18 and H-19 in β- and α-positions, respectively. Using the same TDDFT ECD calculations as for compounds 2−6 and comparison of its experimental and calculated ECD spectra (Figure 4F), the absolute configuration of 7 was determined as 2R, 5S, 6S, 8R, 9R, 10S, 18S, and 19R. Thus, the structure of compound 7 (balanterpene G) was identified as (2R,5S,6S,8R,9R,10S,18S,19R)-19-acetoxy-18,19epoxy-6,18-dimethoxycleroda-3,13(16),14-trien-2-ol. Compound 8 (balanterpene H) had a molecular formula of C29H42O8 based on the HRESIMS data (m/z 536.3218 [M + NH4]+, calcd for C29H46NO8, 536.3223). From the 1H and 13 C NMR spectra, one methoxy and two acetoxy groups were apparent. In addition, a butanoyl moiety was deduced and defined from observation of carbon signals at δC 172.8, 36.4, 18.3, and 13.6 and the corresponding methyl and methylene proton signals (Table 3).20 Apart from these carbon signals, there were 20 additional carbons for the framework in the 13C NMR spectrum, suggesting a diterpenoid skeleton.55−57 Comparison of the chemical shifts of skeletal carbons with those of compound 7 suggested compounds 8 and 7 shared the same clerodane-type scaffold. This skeletal type was substantiated by the 2D NMR data. The locations of the substituents were determined by interepretation of the HMBC spectrum, which disclosed the butanoyloxy group located at C-18, the methoxy group at C-6, and the two acetoxy groups at C-2 and C-19, respectively. F



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Table 3. NMR Data for Compounds 7−10 (δ in ppm, J in Hz)a 7 position

13

1α 1β 2 3 4 5 6α 7α 7β 8 9 10 11a b 12 13 14 15

29.6

23.8 144.6 140.3 112.5

16

115.3

17 18 19 20 OAc-2

15.9 104.2 98.1 25.5

OCH3-6 OR-18

OR-19 a

63.7 124.6 145.4 52.9 82.5 31.1 36.9 37.4 35.3 27.8

1 2 2 1 2 3 4 1 2

57.5 55.4

170.1 21.7

8 1

C

H

1.94 m 1.75 m 4.38 brs 6.07 d (3.7)

3.28 dd (11.7, 4.0) 1.48 m 1.83 m 1.72 ma 2.32 dd (13.6,3.5) 1.24 m 1.49 m 2.08 m 6.42 dd (17.6, 10.8) 5.23 d (17.6) 5.02 d (10.8) 5.03 s 4.94 s 0.95 d (6.8) 5.39 s 6.40 s 0.97 s

3.28 s 3.41 s

1.85 s

13

26.5 71.0 123.4 145.5 53.0 83.0 31.3 37.0 38.2 41.1 27.4 23.7 145.0 140.3 112.5 115.5 15.9 95.4 97.9 25.7 170.8 21.7 57.5 172.8 36.4 18.3 13.6 169.8 21.3

9 1

C

13

H

2.17 m 1.70 m 5.60 t (7.6) 5.82 brs

3.51 dd (12.0, 3.2) 1.49 m 1.88 m 1.76 ma 2.35 d (14.0) 1.20 m 1.51 m 2.07 m 6.43 dd (17.6, 10.9) 5.21 d (17.6) 5.04 d (10.9) 5.04 s 4.92 s 0.93 d (6.2) 6.65 s 6.38 s 0.95 s 1.86 s 3.34 s

10

C

1

30.7

2.07 m 1.57 m 4.49 dd (8.8, 7.4) 5.97 brs

69.0 126.5 147.3 54.2 83.9 31.8 37.0 38.0 41.0 28.3 23.4 145.8 140.2 112.6 113.6 15.9 103.3 104.7 25.8

H

3.45 dd (8.8, 3.8) 1.44 m 1.84 m 1.76 ma 2.33 dd (12.4,4.2) 1.23 m 1.51 m 2.38,m 6.41 dd (17.6, 11.0) 5.20 d (17.6) 4.99 d (11.0) 5.00 s 4.97 s 0.94 d (6.0) 5.33 s 4.93 s 0.93 s

13

C

1

30.8

2.15 m 1.62 m 4.53 dd (8.8, 7.1) 6.00 brs

68.7 126.8 145.2 53.1 83.6 31.4 37.0 38.0 41.4 27.5 23.8 144.5 140.4 112.4 115.4 15.9 103.9 97.6 25.8

H

3.50 dd (12.1, 3.8) 1.49 m 1.86 m 1.79 ma 2.27 dd (13.8,2.5) 1.18 m 1.46 m 2.05 m 6.42 dd (17.6, 10.8) 5.18 d (17.6) 5.02 d (10.8) 5.03 s 4.93 s 0.94 d (7.3) 5.36 s 6.34 s 0.95 s

57.8 55.7

3.33 s 3.49 s

57.5 55.7

3.31 s 3.41 s

54.5

3.16 s

170.2 21.8

1.85 s

2.30 t (7.4) 1.63 m 0.93 t (7.4) 2.10 s

Signals were in overlapped regions of the spectra, and the multiplicities could not be discerned.

H-7/H3-17, and H-3/H-18, together with Chem3D modeling, suggested a cis-fusion of rings A and B with H-10, C-19, and H-2 all in α-positions and H-6 in a β-position. The C-18 and C-19 methoxy groups, relative to ring C, were determined as both α-oriented by the correpsonding NOESY correlations. The absolute configuration of 9 was established as 2S, 5S, 6S, 8R, 9R, 10S, 18S, and 19R by comparing the experimental ECD spectra with those calculated by the TDDFT method (Figure 4H). On the basis of the above evidence, compound 9 was characterized as (2S,5S,6S,8R,9R,10S,18S,19R)-18,19-epoxy-6,18,19-trimethoxycleroda-3,13(16),14-trien-2-ol. The 1H and 13C NMR spectra of compound 10 (balanterpene J) were similar to those of 9, except that one methoxy group in 9 was replaced by an acetoxy group in 10. This acetoxy group was located at C-19 via 2D NMR data. The residual two methoxy groups were located at C-6 and C-18, respectively, by the HMBC correlations of H-6 and H-18 to the corresponding methoxy carbons. The C-2 hydroxy group was verified by the HRESIMS data and supported by the chemical shifts of C-2 and H-2. The same relative configuration was inferred for balanterpene J (10) and balanterpene I (9) on the basis of comparison of their NOESY spectra. This deduced relative configuration and experimental and calculated ECD spectra (Figure 4I) permitted

A NOESY spectrum and Chem3D modeling revealed a similar molecular conformation to that of compound 7, and the only configurational difference is the change of H-2 from a β-orientation in 7 to an α-orientation in 8, which was supported by the coupling constants (J2,1α/β = 7.6 Hz) between H-2 and H2-1. The absolute configuration of 8 was determined as 2S, 5S, 6S, 8R, 9R, 10S, 18R, and 19R by comparison of its experimental and calculated ECD spectra (Figure 4G). Compound 8 was therefore elucidated as (2S,5S,6S,8R,9R,10S,18R,19R)-2,19diacetoxy-18-butyryloxy-18,19-epoxy-6-methoxycleroda-3,13(16),14-triene. The molecular formula of compound 9 was deduced as C23H36O5 through the presence of an ion at m/z 415.2461 [M + Na]+ (calcd for C23H36NaO5, 415.2460) from its HRESIMS. The 1H and 13C NMR spectra indicated that compound 9 had the same clerodane-type diterpenoid skeleton as compound 8.18−20 Using the same NMR experiments as for compounds 7 and 8, the skeleton of 9 was elucidated and the three methoxy groups appropriately located to suport a structure of 6,18,19-trimethoxy18,19-epoxycleroda-3,13(16),14-trien-2-ol for 9.20 NOESY correlations between H-2/H-10, H-2/H-1α, H-1α/H3-20, H-1β/H-6, H-1β/H-8, H-6/H-8, H-10/H-11a(b), H-19/H-7, H-19/H-11b, H-18/H-19, H-19/H-7, H-7/H-11b, G



Article

equipped with a Shodex RI-102 detector (Showa Denko Co., Ltd., Tokyo, Japan) and a YMC-pack ODS-AM (20 × 250 mm) column (YMC Co. Ltd., Kyoto, Japan). X-ray crystallographic analysis was carried out on a Rigaku Saturn 944 CCD diffractometer equipped with a multilayer monochromator and Cu Kα radiation (λ = 1.541 87 Å) (Rigaku Co. Ltd., Japan). The structure was solved by direct methods (SHELXL-97), expanded using Fourier techniques, and refined with full-matrix least-squares on F2 (SHELXL-97). Silica gel was used for column chromatography (200−300 mesh, Qingdao Haiyang Chemical Group Co., Ltd., Qingdao, People’s Republic of China). Chemical reagents for isolation were of analytical grade and purchased from Tianjin Yuanli Co., Ltd., Tianjin, People’s Republic of China. Biological reagents were from Sigma Chemical Co. The PC12 cell line was from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, People’s Republic of China). Plant Material. The leaves of C. balansae were collected from Xishuangbanna, Yunnan Province, People’s Republic of China, in August 2014. The botanical identification was made by Prof. ShunCheng Zhang (Xishuangbanna Botanical Garden, Chinese Academy of Sciences, People’s Republic of China), and a voucher specimen (No. 20140919) was deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University. Extraction and Isolation. The leaves of C. balansae (5.1 kg) were extracted with MeOH (3 × 40 L) under reflux. The organic solvent was evaporated to afford a crude extract (1150.0 g). The extract was suspended in H2O (1.5 L) and partitioned with EtOAc (3 × 1.5 L). The EtOAc-soluble portion (263 g) was subjected to silica gel column chromatography, using a gradient of acetone in petroleum ether (0−30%), to yield 10 fractions (F1−F10) based on TLC analysis. Fraction F9 was subjected to MPLC over ODS eluting with a step gradient of 62−90% MeOH in H2O to give eight subfractions (F9‑1−F9‑8). Compound 1 (tR = 30 min, 7.2 mg) was isolated from the above subfraction F9‑3 by preparative HPLC (86% MeOH in H2O), and the purification of subfraction F9‑2 with the same HPLC (86% MeOH in H2O) afforded compound 5 (tR = 22 min, 9.8 mg). Fraction F4, with the same procedure as for F9, gave subfractions F4‑1−F4‑10. Using the above HPLC system, compound 2 (tR = 28 min, 12.4 mg) was obtained from F4‑6 (93% MeOH in H2O), and compound 8 (tR = 36 min, 7.2 mg) was isolated from subfraction F4‑3 (86% MeOH in H2O). Fraction F7 was fractionated by the above MPLC to yield five subfractions, F7‑1−F7‑5. Compounds 3 (tR = 21 min, 8.7 mg) and 4 (tR = 20 min, 13.5 mg) were obtained from F7‑3 (86% MeOH in H2O), and compound 6 (tR = 23 min, 6.9 mg) was obtained from F7‑1 (82% MeOH in H2O) with the same HPLC system. Using the same MPLC system as for the above fractions, F8 provided subfractions F8‑1−F8‑15, and the subsequent purification of F8‑2 by the same HPLC system (82% MeOH in H2O) resulted in the isolation of compounds 7 (tR = 25 min, 15.2 mg), 9 (tR = 35 min, 12.4 mg), and 10 (tR = 23 min, 13.9 mg). Balanterpene A (1): colorless crystals (MeOH); mp 242−243 °C; [α]29 D −3 (c 0.1, CH2Cl2); IR (KBr) νmax 3447, 2973, 2941, 1742, 1749, 1652, 1456, 1393, 1339 cm−1; 1H NMR (400 MHz, CDCl3) and 13 C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 507 [M + Na]+; HRESIMS m/z 507.3450 [M + Na]+ (calcd for C31H48NaO4, 507.3450). X-ray crystal data of balanterpene A (1): C31H48O4, Mr = 484.69, orthorhombic, space group P2(1)2(1)2(1), a = 11.738(3) Å, b = 13.856(3) Å, c = 17.106(3) Å, α = 90°, β = 90°, γ = 90°, V = 2782.1(11) Å3, T = 173(2) K, Z = 4, μ(Cu Kα) = 0.580 mm−1, Dcalc = 1.157 g/cm3, F(000) = 1064, crystal dimensions 0.24 × 0.22 × 0.20 mm were used for measurements. The total number of reflections measured was 26 375, of which 5917 were unique (R(int) = 0.0464). Final R1 = 0.0424, wR2 = 0.1094 (I > 2σ(I)), Flack parameter = 0.17(8). Crystallographic data of this compound have been deposited in the Cambridge Crystallographic Data Centre (CCDC 1424453). Balanterpene B (2): colorless oil; [α]29 D +61 (c 0.2, CH2Cl2); ECD (CH3CN) 207 (Δε +8.8), 299 (Δε +2.6) nm; IR (KBr) νmax 3448, 2972, 2938, 1773, 1701, 1651, 1510, 1395, 1316 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 477 [M + Na]+; HRESIMS m/z 477.3347 [M + Na]+ (calcd for C30H46NaO3, 477.3345).

the absolute configuration of 2S, 5S, 6S, 8R, 9R, 10S, 18S, and 19S for 10 to be assigned. Thus, compound 10 was characterized as (2S,5S,6S,8R,9R,10S,18S,19S)-19-acetoxy-18,19-epoxy-6,18dimethoxycleroda-3,13(16),14-trien-2-ol. It should be noted that compounds 7−10 possess methoxy and/or acetoxy groups in their structures. Owing to contact with methanol and ethyl acetate during the extraction and purification processes, they may be four new diterpenoid artifacts presumably formed during these processes. Pharmacological studies of nerve growth factor (NGF) have demonstrated that bioactive substances to promote neurite outgrowth of nerve cells against neuron degeneration may be potentially useful for the treatment of Alzheimer’s disease.28,29 Compounds 1−10 were thus evaluated for their promoting effects on neurite outgrowth as described previously.58 NGF was used as the positive control.30,58 Compounds 1−3 and 5−10 showed promoting effects on NGF-mediated neurite outgrowth of PC12 cells. The EC50 values of these active terpenoids to promote neurite outgrowth dose-dependently are shown in Table 4. Table 4. EC50 Values of Compounds 1−3 and 5−10 Promoting NGF-Mediated Neurite Outgrowth of PC12 Cells compound

EC50 (μM)a

compound

EC50 (μM)

1 2 3 5 6

>50 >50 10.0 >50 >50

7 8 9 10

2.9 5.2 5.4 8.3

NGF was used as a positive control (EC50 value, 5.2 × 10−2 μg/mL). Data are presented based on three experiments. a

However, compound 4 had no effects on neurite outgrowth of PC12 cells in either the absence or presence of NGF (20 ng/mL). In conclusion, this phytochemical investigation on C. balansae has led to the isolation and structure elucidation of 10 new terpenoids including six new triterpenoids (1−6) and four new diterpenoids (7−10). Besides their relative configurations, the absolute configurations of the new terpenoids were determined on the basis of NMR data analysis, X-ray diffraction, and their experimental and calculated ECD spectra. Compound 1 is a ringexpanded 24-homotriterpenoid with a new skeleton, and compound 6 possessed a rare hexanortriterpenoid skeleton. Compounds 1−3 and 5−10 showed promoting effects on NGFmediated neurite outgrowth of PC12 cells, with compound 7 exerting the most significant promotion of NGF-mediated neurite outgrowth from PC12 cells. These bioactive diterpenoids may be useful for the development of antineurodegenerative agents to combat Alzheimer’s disease and other neurological disorders.28,29

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured in CH2Cl2 using an Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). ECD spectra were obtained on a Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, UK). IR spectra were recorded on a Bruker Tensor 27 FT-IR spectrometer with KBr disks. 1D and 2D NMR spectra were recorded on a Bruker AV 400 instrument (Bruker, Switzerland, 400 MHz for 1H and 100 MHz for 13C) with tetramethylsilane as an internal standard. ESIMS spectra were acquired on a Thermo Finnigan LCQ-Advantage mass spectrometer. HRESIMS spectra were recorded by an IonSpec 7.0 T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA, USA). HPLC separations were performed on a CXTH system, H



Article

Balanterpene C (3): colorless oil; [α]29 D +37 (c 0.2, CH2Cl2); ECD (CH3CN) 201 (Δε +6.6), 305 (Δε +1.7) nm; IR (KBr) νmax 3447, 2969, 2938, 1776, 1701, 1651, 1509, 1396, 1316 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 493 [M + Na]+; HRESIMS m/z 493.3285 [M + Na]+ (calcd for C30H46NaO4, 493.3294). Balanterpene D (4): colorless oil; [α]29 D +32 (c 0.2, CH2Cl2); ECD (CH3CN) 200 (Δε +8.9), 305 (Δε +2.1) nm; IR (KBr) νmax 3446, 2973, 2937, 1773, 1701, 1651, 1509, 1396, 1317 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 507 [M + Na]+; HRESIMS m/z 507.3085 [M + Na]+ (calcd for C30H44NaO5, 507.3086). Balanterpene E (5): colorless oil; [α]29 D +14 (c 0.2, CH2Cl2); ECD (CH3CN) 199 (Δε +6.3), 302 (Δε +1.0) nm; IR (KBr) νmax 3447, 2972, 2938, 1734, 1707, 1686, 1651, 1509, 1458, 1396, 1316 cm−1; 1 H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 523 [M + Na]+; HRESIMS m/z 523.3035 [M + Na]+ (calcd for C30H44NaO6, 523.3036). Balanterpene F (6): colorless oil; [α]29 D +36 (c 0.2, CH2Cl2); ECD (CH3CN) 212 (Δε +3.5), 299 (Δε +1.7) nm; IR (KBr) νmax 3446, 2971, 2941, 1775, 1706, 1647, 1517, 1456, 1387 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Tables 1 and 2; ESIMS m/z 409 [M + Na]+; HRESIMS m/z 409.2352 [M + Na]+ (calcd for C24H34NaO4, 409.2355). Balanterpene G (7): colorless oil; [α]28 D +20 (c 0.2, CH2Cl2); ECD (CH3CN) 201 (Δε +9.2), 222 (Δε −4.9) nm; IR (KBr) νmax 3447, 2882, 1749, 1647, 1489, 1374, 1230 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 3; ESIMS 443 [M + Na]+; HRESIMS m/z 443.2404 [M + Na]+ (calcd for C24H36NaO6, 443.2410). Balanterpene H (8): colorless oil; [α]28 D −40 (c 0.2, CH2Cl2); ECD (CH3CN) 195 (Δε −7.7), 221 (Δε −4.2) nm; IR (KBr) νmax 2923, 1741, 1639, 1458, 1372, 1229 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 3; ESIMS m/z 536 [M + NH4]+; HRESIMS m/z 536.3218 [M + NH4]+ (calcd for C29H46NO8, 536.3223). Balanterpene I (9): colorless oil; [α]28 D −41 (c 0.1, CH2Cl2); CD (CH3CN) 212 (Δε −2.6) nm; IR (KBr) νmax 3452, 2921, 1639, 1459, 1373, 1107 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 3; ESIMS 415 [M + Na]+; HRESIMS m/z 415.2461 [M + Na]+ (calcd for C23H36NaO5, 415.2460). Balanterpene J (10): colorless oil; [α]28 D −5 (c 0.08, CH2Cl2); CD (CH3CN) 215 (Δε −16.0) nm; IR (KBr) νmax 3446, 2928, 1748, 1646, 1456, 1374, 1106 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see Table 3; ESIMS m/z 465 [M + COOH]−; HRESIMS m/z 465.2494 [M + COOH]− (calcd for C25H37O8, 465.2488). Computations. Conformational searches were performed by the MOE software using the MMFF94S force field.42 The single-crystal X-ray diffraction data and the obtained conformers were used for geometry reoptimizations at the B3LYP/6-31+G(d,p) level in the Gaussian 09 package.43 The ECD spectra for the optimized conformers were calculated at the CAM-B3LYP/SVP level with a CPCM solvent model in acetonitrile, and the calculated ECD spectra of different conformers were simulated with a half-bandwidth of 0.3−0.4 eV. The ECD curves were extracted by SpecDis 1.6 software.44 The overall ECD curves of all the compounds were weighted by Boltzmann distribution after UV correction. Bioassay for Neurite Outgrowth. PC12 cells were cultured at 37 °C in DMEM supplemented with 5% (v/v) inactivated fetal bovine serum (FBS), 5% (v/v) inactivated horse serum (HS), and 100 U/mL penicillin/streptomycin under a water-saturated atmosphere of 95% air and 5% CO2. The cells were disassociated by incubation with 1 mM ethylene glycolbis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid in phosphate-buffered saline for 15 min and then seeded in 24-well culture plates (3 × 104 cells/well) coated with poly-L-lysine. After 24 h, the medium was changed to test medium containing various concentrations of NGF (100 ng/mL for positive control, 20 ng/mL for test samples and significant difference control), 1% FBS, 1% HS, and various

concentrations of test compounds. After a continuous incubation of 96 h, the neurite outgrowth was assessed under a phase contrast microscope. Neurite processes with a length equal to or greater than the diameter of the neuron cell body were scored as neurite-bearing cells. The ratio of the neurite-bearing cells to total cells (with at least 100 cells examined/viewing area; three viewing areas/well; six wells/sample) was determined and expressed as a percentage. Each sample was performed in three replicates. The EC50 values were determined on the basis of linear or nonlinear regression analysis of the concentration−response data curves.

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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.5b00815. NMR spectra of compounds 1−10 (PDF) X-ray data of compound 1 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax (Y. Guo): 86-22-23502595. E-mail: victgyq@nankai. edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (No. 21372125). REFERENCES

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