Neuroprotective Dihydroagarofuran Sesquiterpene Derivatives from

Jan 22, 2018 - ... and other 1H NMR data (Table 1); 13C NMR (methanol-d4, 150 MHz) δC trans-Cin-1 (167.6, 146.3, 135.4, 131.5, 129.8 × 2, 129.0 × 2...
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Neuroprotective Dihydroagarofuran Sesquiterpene Derivatives from the Leaves of Tripterygium wilfordii Fang-You Chen, Chuang-Jun Li, Jie Ma, Jian Zhou, Li Li, Zhao Zhang, Nai-Hong Chen, and Dong-Ming Zhang* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Thirteen dihydroagarofuran derivatives, including 12 new sesquiterpenoid esters and one known sesquiterpenoid alkaloid, were obtained from the leaves of Tripterygium wilfordii. Spectroscopic techniques and the ECD method were used for the structure elucidation of the compounds. The structures of compounds 1 and 8 were confirmed by single-crystal X-ray crystallographic analyses. Compounds 8, 9, 11, 12, and 13 increased cell viability of the okadaic acid treated PC12 cells from 60.4 ± 23.0% to 72.4 ± 14.1, 71.5 ± 11.5, 75.7 ± 15.6, 81.2 ± 13.1, and 86.2 ± 25.5% at 10 μM, respectively. from 60.4 ± 23.0% to 72.4 ± 14.1, 71.5 ± 11.5, 75.7 ± 15.6, 81.2 ± 13.1, and 86.2 ± 25.5% at 10 μM, respectively.

Tripterygium wilfordii Hook. f. (Celastraceae) has been used for the treatment of skin disorders, rheumatoid arthritis, and autoimmune diseases for centuries.1−6 Based on the marked bioactivities of T. wilfordii, numerous studies have been carried out on this plant. Consequently, hundreds of effective substances have been acquired from this species, including diterpenoids, sesquiterpenoids, triterpenoids, and lignans.7−22 However, the most widespread and characteristic metabolites are a large class of highly oxygenated tricyclic sesquiterpenoids known as dihydroagarofurans, which can structurally be classified into sesquiterpenoid polyesters and macrolide sesquiterpenoid pyridine alkaloids. Dihydroagarofuran sesquiterpenoids possess up to nine ester groups. The number, position, and configuration of these substituents create a large structural diversity of sesquiterpenoids, which exhibit a broad range of biological activities including immunosuppressive, cytotoxic, insecticidal, anti-HIV, MDR-reversing, and antitumor activities.18−23 Continuation of the phytochemical and biological studies on the leaves of T. wilfordii afforded 13 dihydroagarofuran derivatives including 12 new sesquiterpenoid esters and a known sesquiterpenoid alkaloid (Figure 1). Spectroscopic data and the electronic circular dichroism (ECD) method were used for the structure elucidation of the compounds. The structures of compounds 1 and 8 were confirmed by single-crystal X-ray crystallographic analysis. Compounds 8, 9, 11, 12, and 13 increased cell viability of the okadaic acid-treated PC12 cells © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The molecular formula of compound 1 was established as C33H38O12 via HRESIMS (m/z 649.2256 [M + Na]+, calcd 649.2255), indicating 15 indices of hydrogen deficiency. The IR spectrum showed adsorption bands at 3497, 1732, and 1235 cm−1 indicating the presence of hydroxy and ester functionalities. The 1H NMR spectroscopic data of 1 showed the presence of two acetyl groups [δH 2.13, 2.23 (each 3H, s)], a trans-cinnamoyl group [δH 7.38 (d, J = 16.2 Hz, 1H), 7.36 (overlapped, 1H), 7.33 (overlapped, 2H), 7.26 (d, J = 6.6 Hz, 2H), 5.99 (d, J = 16.2 Hz, 1H)], a furanoyl group [δH 8.25 (s, 1H), 7.25 (overlapped, 1H), 6.67 (d, J = 1.8 Hz, 1H)], an oxymethylene group [δH 5.38 and 4.51 (each 1H, d, J = 13.2 Hz)], three methyl groups [δH 1.58, 1.56, 1.45 (s, each 3H)], and four oxymethine protons (δH 6.99, 5.45, 5.43, 4.29). The 13 C NMR data of 1 confirmed the presence of these moieties. The 1H−1H COSY spectrum (Figure 2) of 1 revealed the correlations of H-1/H-2/H-3 and H-6/H-7/H-8. Compound 1 was deduced to be a dihydroagarofuran sesquiterpenoid, substituted with a trans-cinnamoyl, a furanoyl, and two acetyl groups. Received: July 21, 2017

A

DOI: 10.1021/acs.jnatprod.7b00615 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Compounds isolated from the leaves of T. wilfordii.

Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations of compound 1.

(1S,4S,5R,6R,7R,8S,9S,10S)-5,11-diacetoxy-1-trans-cinnamoyl8-furanoyl-4,7-dihydroxydihydroagarofuran. Compound 2 was obtained as a white, amorphous powder and was found to have the same molecular formula as 1 based on its HRESIMS data (m/z 649.2283 [M + Na]+, calcd 649.2255). Its NMR spectra resembled those of compound 1, and the main differences were the locations of the acyl groups. The assignment of the ester groups was done via the HMBC correlations, in which the dihydroagarofuran signals of H-1 (δH 5.48), H-7 (5.56), H-8 (5.62), and H-11 (5.10) were correlated with the carbonyl carbons of t-Cin (δC 167.3), H-7/Ac (171.5), H-8/Fu (162.9), and H-11/Ac (171.9), respectively. The NOESY correlations of H-5/H-6, H-11, H-12 and H-8/H-1, H7, H-14 established the relative configuration of 2. The UV absorption spectrum of 2 showed a strong absorption at λmax 280 nm attributable to the ester carbonyls. Its ECD spectrum (Figure S18, Supporting Information) revealed a positive Cotton effect at 289 nm (Δε = 1.27) and a negative Cotton effect at 250 nm (Δε = −1.35). The ECD spectrum resembled that of 1 and allowed the absolute configuration of 2 to be

The assignment of the ester groups was done via the HMBC spectrum (Figure 2), in which the dihydroagarofuran signals of H-1 (δH 5.43), H-5 (6.99), H-8 (5.45), and H-11 (5.38) were correlated with the carbonyl carbons of t-Cin (δC 167.5), Ac (171.6), Fu (163.7), and Ac (172.5), respectively. The NOESY correlations of H-5/H-6, H-11, H-12 and H-8/H-1, H-7, H-14 established the relative configuration of 1. The UV spectrum of 1 showed a strong absorption at λmax 281 nm attributable to the ester carbonyls. Its ECD spectrum (Figure S9, Supporting Information) showed a Davydov split with a positive first Cotton effect at 286 nm (Δε = 0.78) and a negative second Cotton effect at 258 nm (Δε = −0.39), based on the coupling of the trans-cinnamoyloxy group at C-1 and the furanoyloxy functionality at C-8. In addition, the ECD spectrum of 1, calculated at the B3LYP/6-31G(d) level, agreed with the experimental ECD spectrum (Figure 3). A single-crystal X-ray crystallographic analysis of compound 1 (Figure 4) confirmed the absolute configuration as assigned by the ECD data. The structure of compound 1, triptersinine V, was thus defined as B

DOI: 10.1021/acs.jnatprod.7b00615 J. Nat. Prod. XXXX, XXX, XXX−XXX

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276 nm attributable to the ester carbonyls. Its ECD spectrum (Figure S27, Supporting Information) showed a Davydov split with a negative first Cotton effect at 276 nm (Δε = −6.48) and a positive second Cotton effect at 225 nm (Δε = 1.25), based on coupling of the cis-cinnamoyloxy group at C-1 and the furanoyloxy function at C-8. In addition, the ECD spectrum of 3, calculated at the B3LYP/6-31G(d) level, agreed with the experimental ECD spectrum (Figure 3). Thus, the structure of triptersinine X (3) was defined as (1S,4S,5R,6S,7R,8S,9S,10S)7,11-diacetoxy-1-cis-cinnamoyl-8-furanoyl-4,5-dihydroxydihydroagarofuran. Compound 4 was found to have a molecular formula of C31H36O11 based on its HRESIMS data (m/z 607.2151 [M + Na]+, calcd 607.2150). Analysis of the 1H NMR data of 4 showed the presence of a furanoyl, an acetyl, and a transcinnamoyl group. The assignment of the ester groups was done via the HMBC correlations, in which the dihydroagarofuran signals of H-1 (δH 5.42), H-5 (6.81), and H-8 (5.56) were correlated with the carbonyl carbons of t-Cin (δC 167.6), Ac (171.5), and Fu (163.4), respectively. The relative configuration of 4 was established via the NOESY correlations of H5/H-6, H-11, H-12 and H-8/H-1, H-7, H-14. The UV absorption spectrum of 4 showed a strong absorption at λmax 278 nm attributable to the ester carbonyls. Its ECD spectrum (Figure S36, Supporting Information) showed a positive Cotton effect at 296 nm and a negative Cotton effect at 254 nm. This spectrum resembled that of 1 and allowed the absolute configuration of 4 to be determined. Thus, the structure of triptersinine Y (4) was defined as (1S,4S,5R,6R,7R,8S,9S,10S)-5-acetoxy-1-trans-cinnamoyl-8-furanoyl-4,7,11-trihydroxydihydroagarofuran. Compounds 5 (triptersinine Z1) and 6 (triptersinine Z2) were isolated as amorphous powders and were both found to have molecular formulas of C31H34O11 based on their HRESIMS data. The 1H NMR data of 5 indicated the presence of an acetyl, a trans-cinnamoyl, and a furanoyl group. The 13C NMR spectroscopic data showed the presence of a ketocarbonyl (δC 201.5). The 1H and 13C NMR data of 6 resembled those of compound 5, except that the transcinnamoyl group at C-1 in 5 was replaced by a cis-cinnamoyl group in 6. Their relative configurations were determined by the correlations of H-5/H-6, H-11, H-12 and H-8/H-1, H-14. The UV absorption spectra of 5 and 6 showed strong absorptions at λmax 281 and 279 nm, respectively. In their ECD spectra, a positive first Cotton effect at 285 nm (Δε = 11.43) and a negative second Cotton effect at 227 nm (Δε = −3.17) were observed for 5 (Figure S45, Supporting Information), and a negative first Cotton effect at 251 nm (Δε = −17.86) and a positive second Cotton effect at 224 nm (Δε = −2.95) were observed for 6 (Figure S54, Supporting Information). These spectra resembled those of 2 and 3, respectively. In addition, the ECD spectrum of 6, calculated at the B3LYP/6-31G(d) level, agreed with the experimental ECD spectrum (Figure 3). Therefore, the structures of 5 and 6 were determined to be (1S,4S,5R,6R,8S,9S,10S)-11-acetoxy-1-transcinnamoyl-8-furanoyl-4,5-dihydroxy-7-oxodihydroagarofuran and (1S,4S,5R,6R,8S,9S,10S)-11-acetoxy-1-cis-cinnamoyl-8-furanoyl-4,5-dihydroxy-7-oxodihydroagarofuran, respectively. Compound 7 was found to have the molecular formula C28H36O10 based on its HRESIMS data (m/z 555.2206 [M + Na]+, calcd 555.2201). The NMR data of 7 showed that it was also a dihydroagarofuran sesquiterpenoid possessing a transcinnamoyl and two acetyl substituents. The acetyl groups were

Figure 3. Calculated and experimental ECD spectra of 1, 3, and 6.

determined. Consequently, the structure of compound 2 was defined as (1S,4S,5R,6S,7R,8S,9S,10S)-7,11-diacetoxy-1-transcinnamoyl-8-furanoyl-4,5-dihydroxydihydroagarofuran. Based on the HRESIMS and NMR data, the molecular formula of 3 was also established as C33H38O12. The NMR data of 3 indicated the presence of a furanoyl, a cis-cinnamoyl, and two acetyl groups. The NMR data of 3 were similar to those of 2 except that the trans-cinnamoyl group at C-1 in 2 was replaced by a cis-cinnamoyl group in 3. The relative configuration of 3 was established via the NOESY correlations of H-5/H-6, H-11, H-12 and H-8/H-1, H-7, H-14. The UV absorption spectrum of 3 showed a strong absorption at λmax C

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Figure 4. ORTEP drawings of compounds 1 and 8.

−0.27) and a positive Cotton effect at 224 nm (Δε = 3.10). This spectrum resembled that of 8 and allowed the absolute configuration of 9 to be determined. The structure of triptersinine Z5 (9) was thus defined as (1S,4R,5R,6R,7R,8S,9S,10S)-1,7,11-triacetoxy-5-furanoyl-8nicotinoyldihydroagarofuran. Triptersinine Z6 (10) was found to have a molecular formula of C35H37NO13 as established from its HRESIMS data (m/z 680.2352, [M + H]+, calcd 680.2338). The NMR data showed the presence of two furanoyl, a nicotinoyl, and two acetyl groups. The NMR data of 10 resembled those of 9 except for the acetyl group at C-1 in 9 being replaced by a furanoyl group in 10. The NOESY correlations of H-5/H-6, H-11, H-12 and H-8/H-1, H-7, H-14 established the relative configuration of 10. The UV absorption spectrum of 10 showed a strong absorption at λmax 268 nm attributable to the ester carbonyls. Its ECD spectrum (Figure S89, Supporting Information) showed sequential negative and positive Cotton effects at 268 nm (Δε = −1.02) and a positive Cotton effect at 229 nm (Δε = 1.99). This spectrum resembled that of 8 and allowed the absolute configuration of 10 to be assigned. The structure of triptersinine Z6 (10) was thus defined as (1S,4R,5R,6R,7R,8S,9S,10S)-7,11-diacetoxy-1,5-difuranoyl-8nicotinoyldihydroagarofuran. The molecular formula of 11 was found to be C36H41NO11 via its HRESIMS data (m/z 664.2765 [M + H]+, calcd 664.2752). The NMR data of 11 indicated the presence of three acetyl, a nicotinoyl, and a cis-cinnamoyl group. Its NMR spectra resembled those of 8 except that the furanoyl group at C-1 in 8 was replaced by a cis-cinnamoyl group in 11. The NOESY correlations of H-5/H-6, H-11, H-12 and H-8/H-1, H7, H-14 established the relative configuration of 11. The UV absorption spectrum of 11 showed a strong absorption at λmax 269 nm attributable to the ester carbonyls. Its ECD spectrum (Figure S98, Supporting Information) showed a negative Cotton effect at 281 nm (Δε = −13.02) and a positive Cotton effect at 250 nm (Δε = −4.59). The structure of triptersinine Z7 (11) was thus defined as (1S,4R,5R,6R,7R,8S,9S,10S)5,7,11-triacetoxy-1-cis-cinnamoyl-8-nicotinoyldihydroagarofuran. The molecular formula of 12 was found to be C36H41NO11 via its HRESIMS data (m/z 664.2742 [M + H]+, calcd 664.2752). Its 1H NMR spectrum indicated the presence of three acetyl, a nicotinoyl, and a trans-cinnamoyl group. The NMR data of 12 resembled those of 11, except that the ciscinnamoyl group at C-1 in 11 was replaced by a transcinnamoyl group in 12. The assignment of the ester groups was done via the HMBC correlations, in which the dihydroagarofuran signals of H-1 (δH 5.58), H-5 (6.72), H-7 (5.62), H-8

assigned to be at C-5 and C-11, while the trans-cinnamoyl group was located at C-1 based on the HMBC experiment. The NOESY correlations of H-5/H-6, H-11, H-12 and H-8/H-1, H7, H-14 established the relative configuration of 7. The UV absorption spectrum of 7 showed a strong absorption at λmax 276 nm attributable to the ester carbonyls. Consequently, the structure of triptersinine Z3 (7) was defined as 5α,11diacetoxy-1β-trans-cinnamoyl-4α,7β,8β-trihydroxydihydroagarofuran. The molecular formula of 8 was established to be C32H37NO12 via its HRESIMS data (m/z 628.2402 [M + H]+, calcd 628.2389). The methyl signals at δH 1.12 (d, J = 7.2 Hz, H-12) as well as the correlation between H-12 and H-4 in the 1H−1H COSY spectrum of 8 indicated that a C-4 hydroxy substituent was absent. The 1H NMR data indicated the presence of a nicotinoyl [δH 8.88 (s, 1H), 8.65 (d, J = 4.8 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 7.42 (dd, J = 7.8, 4.8 Hz, 1H)], three acetyl [δH 2.03, 2.14, 2.31 (s, each 3H)], and a furanoyl group [δH 7.67 (s, 1H), 7.15 (s, 1H), 6.32 (s, 1H)]. The assignment of the ester groups was done via the HMBC correlations of H-1 (δH 5.66), H-5 (6.73), H-7 (5.61), H-8 (5.74), and H-11 (5.21) with the carbonyl carbons of Fu (δC 163.3), Ac (171.4), Ac (171.6), Nic (164.6), and Ac (172.0), respectively. The relative configuration of 8 was established via the NOESY correlations of H-5/H-6, H-11, H-12 and H-8/H1, H-7, H-14. The UV absorption spectrum of 8 showed a strong absorption at λmax 265 nm attributable to the ester carbonyls. Its ECD spectrum (Figure S71, Supporting Information) showed a negative first Cotton effect at 268 nm (Δε = −0.80) and a positive second Cotton effect at 232 nm (Δε = 0.67). A single-crystal X-ray crystallographic analysis of compound 8 (Figure 4) confirmed the absolute configuration as assigned by the ECD data. The structure of triptersinine Z4 (8) was thus defined as (1S,4R,5R,6R,7R,8S,9S,10S)-5,7,11triacetoxy-1-furanoyl-8-nicotinoyldihydroagarofuran. The molecular formula of 9 was found to be C32H37NO12 via its HRESIMS data (m/z 628.2406 [M + H]+, calcd 628.2389). Its NMR spectra resembled those of compound 8, except for the locations of the acyl groups. The assignment of the ester groups was done via the HMBC correlations, in which the dihydroagarofuran signals of H-1 (δH 5.43), H-5 (6.89), H-7 (5.65), H-8 (5.72), and H-11 (5.16) were correlated with the carbonyl carbons of Ac (δC 171.4), Fu (163.3), Ac (171.6), Nic (164.8), and Ac (171.9), respectively. The NOESY correlations of H-5/H-6, H-11, H-12 and H-8/H-1, H-7, H-14 established the relative configuration of 9. The UV absorption spectrum of 9 showed a strong absorption at λmax 263 nm attributable to the ester carbonyls. Its ECD spectrum (Figure S80, Supporting Information) showed a negative Cotton effect at 253 nm (Δε = D

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(5.73), and H-11 (5.27) were correlated with the carbonyl carbons of t-Cin (δC 167.1), Ac (171.4), Ac (171.7), Nic (164.7), and Ac (172.0), respectively. The NOESY correlations of H-5/H-6, H-11, H-12 and H-8/H-1, H-7, H-14 established the relative configuration of 12. The UV absorption spectrum of 12 showed a strong absorption at λmax 278 nm attributable to the ester carbonyls. In its ECD spectra (Figure S107, Supporting Information), the observation of a negative Cotton effect at 279 nm (Δε = −0.59), and a positive Cotton effect at 261 nm (Δε = 1.57) allowed the determination of the absolute configuration of 12 based on similarities to the ECD spectrum of 8. Thus, the structure of triptersinine Z8 (12) was defined as (1S,4R,5R,6R,7R,8S,9S,10S)-5,7,11-triacetoxy-1-trans-cinnamoyl-8- nicotinoyldihydroagarofuran. Although compounds 2, 3, 5, and 6 exhibited identical relative configurations, they displayed opposite Cotton effects due to the different dipole moments of their trans-Cin and cisCin groups. Single-crystal X-ray crystallographic analyses of compounds 1 and 8 (Figure 3) confirmed the absolute configurations as assigned by the ECD data. Thus, we interpreted the absolute configurations of the other compounds via UV absorption maxima as well as by the ECD method. The one known sesquiterpene pyridine alkaloid was identified as euojaponine C (13),24 from a comparison of its NMR data with the reported data. Tripterygium wilfordii polyglycoside, a preparation derived from the roots of T. wilfordii, exhibited a positive effect on spatial learning and memory promotion.25 Moreover, previous findings revealed that the triptolide and its analogue tripchlorolide exhibited neuroprotective effects against various neurodegenerative diseases.26−28 Dihydroagarofuran-type sesquiterpenoids, the characteristic metabolites of Celastraceae plants, exhibit diverse biological activities, including memory-promoting and neuroprotective effects.29 Previous findings revealed that 21 of the 62 tested dihydroagarofuran-type sesquiterpenoids isolated from the genus Celastrus were found to possess neuroprotective effects against Aβ25−35-induced toxicity on SH-SY5Y cells.30 The structure−activity relationship revealed that compounds containing a C-2 ester group are more potent than those without a substituent at that position. Most of the compounds described in the literature lacked a hydroxy group at C-4. Compounds 1−13 were tested for their neuroprotective activities on PC12 cells treated with okadaic acid (OKA). Neuron growth factor (NGF) was used as a positive control. At 10 μM, 8, 9, 11, 12, and 13 increased the cell survival rate of the okadaic acid-treated group, but the other compounds were inactive (Figure 5). Several dihydroagarofuran sesquiterpene derivatives showed moderate activity. Comparison of the activities revealed that compounds without a hydroxy group at C-4 are more potent than those containing such a group. Although the leaves of T. wilfordii showed significant antiinflammatory effects,6 few chemical investigations have been reported. Dihydroagarofuran sesquiterpenoid derivatives are common characteristic metabolites of T. wilfordii. Compared with the majority of reported dihydroagarofuran sesquiterpenoids, the compounds reported herein have different ester groups; for example, benzoyl groups, the most common ester groups in previously reported compounds, are not present in the structures of the new compounds, and cis-cinnamoyl groups, which are rare in reported compounds, are present in the structures of some of the new compounds. T. wilfordii can

Figure 5. Neuroprotective effects of isolated compounds against okadaic acid-induced injury in PC12 cells (10 μM, means ± SD, n = 6). ###p < 0.001 vs control, ***p < 0.001 vs model, **p < 0.01 vs model, *p < 0.05 vs model.

be more sustainably used when the leaves are used efficiently since they can be collected every year.21 In summary, 13 dihydroagarofuran derivatives, including 12 new sesquiterpenoid esters and a known sesquiterpenoid alkaloid, were obtained from the leaves of T. wilfordii. Spectroscopic data and the ECD method were used for the structural elucidation of the compounds. The structures of compounds 1 and 8 were confirmed by single-crystal X-ray crystallographic analyses. Several of the compounds showed moderate neuroprotective activity toward okadaic acid-treated PC12 cells. Comparison of the activities showed that compounds without a hydroxy group at C-4 are more potent than those containing such a group. Certain dihydroagarofuran sesquiterpenoids have the potential to be developed into new chemical skeletons for drug leads for neurodegenerative diseases.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P2000 automatic digital polarimeter. UV spectra were acquired on a JASCO V-650 spectrophotometer, and IR spectra were measured on a Nicolet 5700 spectrometer. ECD spectra were recorded on a JASCO J-815 spectrometer. NMR experiments were conducted on VNS-600 and Bruker AV-600 spectrometers in methanol-d4. HRESIMS data were collected on an Agilent 1100 series LC/MSD ion trap mass spectrometer. MPLC analyses were conducted on a Büchi system consisting of two pumps (C-605), a UV detector (C-635), and a fraction collector (C-660). Preparative HPLC separations were performed on a Shimadzu LC-6AD instrument with a UV detector (SPD-20A) and an ODS column (5 μm, 250 × 20 mm, YMC). Silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), diatomite (60−100 mesh, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), SF-PRP 512A (100−200 mesh, Beijing Sunflower and Technology Development Co., Ltd., Beijing, China), and ODS (50 μm, YMC, Japan) were used for column chromatography. Precoated silica gel GF254 plates and UV light were used for TLC. PC12 cells (American Type Culture Collection, USA), DMSO, okadaic acid, MTT (St. Louis, MO, USA), fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and equine serum (Gibco BRL, USA) were used for bioassay experiments. Other chemicals were acquired from commercial sources. Single-crystal X-ray diffraction data were collected on an Agilent Xcalibur Eos Gemini diffractometer with Cu Kα radiation. Plant Material. Leaves of T. wilfordii were collected in September 2009 in Taining County, Fujian Province, China. A voucher specimen was identified by Prof. Lin Ma from the Institute of Materia Medica (IMM), Chinese Academy of Medical Sciences (CAMS), and deposited at the herbarium of the IMM, CAMS (No. 20090034). Extraction and Isolation. Leaves of T. wilfordii (50 kg) were extracted with EtOH−H2O (4:1, v/v, 400 L × 2 h × 3). The crude E

DOI: 10.1021/acs.jnatprod.7b00615 J. Nat. Prod. XXXX, XXX, XXX−XXX

a

5.45 5.38 4.51 1.45 1.56 1.58

d (5.4) d (13.2) d (13.2) s s s

5.44 s 2.32 d (4.2) 5.56 dd (5.4, 4.2) 5.62 d (5.4) 5.10 d (13.2) 4.76 d (13.2) 1.68 s 1.60 s 1.58 s

5.42 s 2.29 d (4.2) 5.55 dd (5.4, 4.2) 5.59 d (5.4) 4.82 d (13.8) 4.42 d (13.8) 1.61 s 1.60 s 1.56 s

6.81 s 2.33 d (4.2) 4.30 dd (5.4, 4.2) 5.56 d (5.4) 4.37 d (12.6) 4.23 d (12.6) 1.48 s 1.58 s 1.56 s

5.42 dd (11.4, 4.8) 1.68 2H m 2.00 m 1.71 m

δH (J in Hz)

4

For the signals of other ester groups see the Experimental Section.

12 14 15

8 11

4 5 6 7

3

6.99 s 2.29 d (4.2) 4.29 t (4.2)

1

2

δH (J in Hz) 5.38 dd (9.0, 7.2) 1.77 2H m 2.10 m 1.61 m

δH (J in Hz)

5.48 dd (12.0, 4.8) 1.91 m 1.83 m 2.15 m 1.64 m

δH (J in Hz)

5.43 dd (12.0, 4.8) 1.83 m 1.77 m 2.05 m 1.67 m

position

3

2

1

5.89 5.41 4.31 1.71 1.55 1.66

s d (13.2) d (13.2) s s s

5.36 s 2.99 s

5.61 dd (12.6, 4.2) 1.81 2H m 2.13 m 1.73 m

δH (J in Hz)

5

5.86 5.09 4.15 1.65 1.54 1.64

s d (12.6) d (12.6) s s s

5.33 s 2.97 s

5.52 dd (12.6, 4.2) 1.76 2H m 2.08 m 1.70 m

δH (J in Hz)

6

Table 1. 1H NMR Spectroscopic Data of Compounds 1−12 (in Methanol-d4 600 MHz)a

4.15 5.02 4.47 1.38 1.45 1.54

d (5.4) d (13.2) d (13.2) s s s

6.75 s 2.25 d (4.2) 4.06 d (5.4)

5.54 dd (12.0, 4.8) 1.87 m 1.70 m 2.02 m 1.68 m

δH(J in Hz)

7

5.74 5.21 4.62 1.12 1.59 1.49

d d d d s s

(5.4) (13.2) (13.2) (7.2)

5.66 dd (12.0, 4.8) 1.98 m 1.73 m 2.28 m 1.57 m 2.32 m 6.73 s 2.48 d (4.2) 5.61 t (5.4)

δH (J in Hz)

8

5.72 5.16 4.53 1.08 1.58 1.52

d d d d s s

(5.4) (13.2) (13.2) (7.8)

5.43 dd (12.0, 4.8) 1.83 m 1.69 m 2.23 m 1.53 m 2.39 m 6.89 s 2.58 d (4.2) 5.65 t (5.4)

δH (J in Hz)

9 5.69 dd (12.0, 4.8) 1.98 m 1.73 m 2.30 m 1.59 m 2.42 m 6.91 s 2.59 d (4.2) 5.66 dd (6.0, 4.2) 5.78 d (6.0) 5.26 d (13.2) 4.63 d (13.2) 1.12 d (7.8) 1.62 s 1.54 s

δH (J in Hz)

10 5.45 dd (12.0, 4.8) 1.77 m 1.68 m 2.18 m 1.54 m 2.25 m 6.67 s 2.45 d (4.2) 5.58 dd (6.0, 4.2) 5.69 d (6.0) 4.84 d (13.2) 4.28 d (13.2) 1.05 d (7.2) 1.57 s 1.47 s

δH (J in Hz)

11

5.58 dd (12.0, 4.8) 1.94 m 1.73 m 2.26 m 1.57 m 2.29 m 6.72 s 2.49 d (4.2) 5.62 dd (5.4, 4.2) 5.73 d (5.4) 5.27 d (13.2) 4.62 d (13.2) 1.13 d (7.8) 1.59 s 1.50 s

δH (J in Hz)

12

Journal of Natural Products Article

F

DOI: 10.1021/acs.jnatprod.7b00615 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 2. 13C NMR Spectroscopic Data of Compounds 1−12 (In Methanol-d4 150 MHz)a

a

1

2

3

4

5

6

7

8

9

10

11

position

δC

δC

δC

δC

δC

δC

δC

δC

δC

δC

δC

12 δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

79.5 26.1 38.4 72.0 76.5 57.3 69.2 76.7 54.4 93.5 60.7 23.4 83.8 24.3 29.8

79.8 26.1 36.1 73.9 75.9 55.5 71.5 74.0 53.0 92.4 61.4 24.5 83.7 24.9 29.9

79.6 25.8 36.0 73.8 75.8 55.6 71.5 73.8 52.8 92.3 61.2 24.5 83.6 24.9 29.9

78.5 26.2 38.8 71.8 76.3 57.5 68.8 76.9 56.0 93.3 59.0 24.2 83.9 24.2 29.9

77.4 26.1 36.2 73.6 77.3 68.5 201.5 81.1 53.5 94.0 61.2 26.0 85.5 25.3 29.8

77.1 26.0 36.1 73.5 77.2 68.5 201.6 81.0 53.3 94.0 61.2 25.9 85.4 25.3 29.8

78.7 26.5 38.7 71.9 76.7 57.0 71.4 74.1 55.0 93.3 61.1 23.4 83.3 24.3 29.9

80.3 24.3 27.4 34.7 75.8 54.1 71.2 74.9 52.5 92.4 60.7 15.4 82.5 24.6 30.8

80.8 24.0 27.4 34.9 76.0 54.2 71.2 74.7 52.3 92.4 60.8 15.6 82.4 24.6 30.9

80.3 24.4 27.5 35.0 76.0 54.2 71.2 74.9 52.6 92.6 60.7 15.5 82.5 24.6 30.9

80.5 23.9 27.3 34.7 75.7 54.0 71.2 74.7 52.2 92.2 60.6 15.4 82.4 24.7 30.8

80.6 24.3 27.4 34.8 75.8 54.1 71.2 74.9 52.5 92.3 60.8 15.5 82.5 24.6 30.8

For the signals of other ester groups see the Experimental Section.

extract was suspended in H2O and extracted with EtOAc (3 × 30 L). The EtOAc fraction (2500 g) was subjected to diatomite chromatography eluted with CHCl3, EtOAc, acetone, and MeOH to give fractions A1 (783 g), A2 (1052 g), A3 (583 g), and A4 (115 g). Fraction A1 (783 g) was subjected to a silica gel column chromatography with petroleum ether−acetone (1:0−1:1) to afford 10 fractions (B1−B10). B7 (87.2 g) was fractionated on a PRP-512A resin column eluted with 50%, 75%, and 95% EtOH in H2O to afford three fractions (C1−C3). Subfraction C2 (32.9 g) was subjected to silica gel (200−300 mesh) column chromatography eluted with CHCl3−MeOH (100:1−10:1) to afford four fractions (F1−F4). F2 (11.5 g) was fractionated using MPLC with MeOH−H2O (60−100%, 50 mL min−1, 6 h) to give 24 fractions (Z1−Z24). Fraction Z6 was successively separated using preparative HPLC (PHPLC) (55% CH3CN) to yield 1 (25.4 mg), 4 (1.1 mg), and 7 (4.1 mg). Compounds 2 (3.6 mg), 3 (2.5 mg), 8 (30.8 mg), and 9 (21.9 mg) were obtained from fraction Z7 via PHPLC (55% CH3CN). Fraction Z11 was subjected to PHPLC (detection at 210 nm, 65% CH3CN, 8 mL min−1) and afforded 8 (30.8 mg), 9 (21.9 mg), 10 (13.9 mg), and 13 (23.2 mg). Compounds 11 (3.3 mg) and 12 (3.6 mg) were obtained from fraction Z12 via PHPLC (65% CH3CN). Triptersinine V (1): colorless crystals (MeOH); [α]25 D −9.4 (c 0.1 CHCl3); UV (MeOH) λmax (log ε) 208 (3.04), 281 (2.97) nm; ECD (MeOH) λmax (Δε) 218 (+2.32), 258 (−0.39), 286 (+0.78) nm; IR (microscope) νmax 3497, 2920, 1732, 1370, 1235 cm−1; 1H NMR (methanol-d4, 600 MHz) δH trans-Cin-1 [7.38 (d, J = 16.2 Hz, 1H), 7.36 (overlapped, 1H), 7.33 (overlapped, 2H), 7.26 (d, J = 6.6 Hz, 2H), 5.99 (d, J = 16.2 Hz, 1H)], Fu-8 [8.25 (s, 1H), 7.25 (overlapped, 1H), 6.67 (d, J = 1.8 Hz, 1H)], 2.13 (s, 3H, Ac-5), 2.23 (s, 3H, Ac-11), and other 1H NMR data (Table 1); 13C NMR (methanol-d4, 150 MHz) δC trans-Cin-1 (167.5, 146.3, 135.3, 131.5, 129.8 × 2, 129.0 × 2, 118.3), Fu-8 (163.7, 150.3, 145.2, 120.6, 110.8), Ac-5 (171.6, 21.6), Ac-11 (172.5, 21.6), and other 13C NMR data (Table 2); HRESIMS m/z 649.2256 (calcd for C33H38NaO12, 649.2255). Triptersinine W (2): white, amorphous solid; [α]25 D −20.4 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 203 (3.09), 280 (2.94) nm; ECD (MeOH) λmax (Δε) 216 (+3.97), 250 (−1.35), 289 (+1.27) nm; IR (microscope) νmax 3402, 2948, 1739, 1369, 1307, 1240 cm−1; 1H NMR (methanol-d4, 600 MHz) δH trans-Cin-1 [7.36 (m, 1H), 7.34 (d, J = 15.6 Hz, 1H), 7.32 (m, 2H), 7.24 (m, 2H), 6.01 (d, J = 15.6 Hz, 1H)], Fu-8 [8.10 (s, 1H), 7.26 (t, J = 1.2 Hz, 1H), 6.59 (d, J = 1.2 Hz, 1H)], 2.03 (s, 3H, Ac-7), 2.25 (s, 3H, Ac-11), and other 1H NMR data (Table 1); 13C NMR (methanol-d4, 150 MHz) δC trans-Cin-1 (167.3, 146.4, 135.3, 131.5, 129.8 × 2, 129.0 × 2, 118.2), Fu-8 (162.9, 149.8, 145.6, 120.2, 110.4), Ac-7 (171.5, 20.9), Ac-11 (171.9, 21.8), and other

13

C NMR data (Table 2); HRESIMS m/z 649.2283 (calcd for C33H38NaO12, 649.2255). Triptersinine X (3): white, amorphous solid; [α]25 D −55.2 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 204 (3.06), 276 (2.73) nm; ECD (MeOH) λmax (Δε) 210 (−3.16), 225 (+1.25), 276 (−6.48) nm; IR (microscope) νmax 3398, 2934, 1733, 1370, 1304, 1238 cm−1; 1H NMR (methanol-d4, 600 MHz) δH cis-Cin-1 [7.40 (m, 2H), 7.32 (m, 1H), 7.31 (m, 2H), 6.66 (d, J = 12.6 Hz, 1H), 5.43 (d, J = 12.6 Hz, 1H)], Fu-8 [8.12 (s, 1H), 7.56 (t, J = 1.8 Hz, 1H), 6.68 (s, 1H)], 2.01 (s, 3H, Ac-7), 2.19 (s, 3H, Ac-11), and other 1H NMR data (Table 1); 13C NMR (methanol-d4, 150 MHz) δC cis-Cin-1 (166.5, 145.0, 136.1, 130.8 × 2, 130.1, 129.0 × 2, 119.9), Fu-8 (163.0, 150.1, 145.5, 120.1, 110.7), Ac-7 (171.5, 20.9), Ac-11 (171.5, 21.7), and other 13C NMR data (Table 2); HRESIMS m/z 649.2247 (calcd for C33H38NaO12, 649.2255). Triptersinine Y (4): white, amorphous solid; [α]25 D −17.4 (c 0.1 CHCl3); UV (MeOH) λmax (log ε) 208 (2.58), 278 (2.35) nm; ECD (MeOH) λmax (Δε) 216 (+0.35), 254 (−0.17), 296 (+0.05) nm; IR (microscope) νmax 3398, 2917, 1731, 1370, 1310, 1237 cm−1; 1H NMR (methanol-d4, 600 MHz) δH trans-Cin-1 [7.38 (d, J = 16.2 Hz, 1H), 7.36 (overlapped, 1H), 7.33 (overlapped, 2H), 7.26 (d, J = 7.2 Hz, 2H), 6.04 (d, J = 16.2 Hz, 1H)], 2.11 (s, 3H, Ac-5), Fu-8 [8.10 (s, 1H), 7.31 (overlapped, 1H), 6.62 (d, J = 1.2 Hz, 1H)], and other 1H NMR data (Table 1); 13C NMR (methanol-d4, 150 MHz) δC transCin-1 (167.6, 146.3, 135.4, 131.5, 129.8 × 2, 129.0 × 2, 118.5), Fu-8 (163.4, 149.6, 145.5, 120.6, 110.5), Ac-5 (171.5, 21.6), and other 13C NMR data (Table 2); HRESIMS m/z 607.2151 (calcd for C31H36NaO11, 607.2150). Triptersinine Z1 (5): white, amorphous solid; [α]25 D −55.8 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 204 (3.15), 281 (3.11) nm; ECD (MeOH) λmax (Δε) 227 (−3.17), 285 (+11.43) nm; IR (microscope) νmax 3387, 2950, 1730, 1392, 1313, 1229 cm−1; 1H NMR (methanold4, 600 MHz) δH trans-Cin-1 [7.42 (d, J = 16.2 Hz, 1H), 7.35 (m, 1H), 7.33 (m, 2H), 7.27 (m, 2H), 6.10 (d, J = 16.2 Hz, 1H)], Fu-8 [8.01 (brs, 1H), 7.24 (t, J = 1.2 Hz, 1H), 6.58 (d, J = 1.2 Hz, 1H)], 1.95 (s, 3H, Ac-11), and other 1H NMR data (Table 1); 13C NMR (methanold4, 150 MHz) δC trans-Cin-1 (167.2, 146.9, 135.3, 131.6, 129.8 × 2, 129.1 × 2, 118.1), Fu-8 (163.1, 149.9, 145.5, 119.8, 110.5), Ac-11 (171.7, 20.8), and other 13C NMR data (Table 2); HRESIMS m/z 605.2010 (calcd for C31H34NaO11, 605.1993). Triptersinine Z2 (6): white, amorphous solid; [α]25 D −71.1 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 203 (3.11), 279 (2.78) nm; ECD (MeOH) λmax (Δε) 211 (− 5.06), 224 (− 2.95), 251 (− 17.86) nm; IR (microscope) νmax 3386, 2950, 1731, 1392, 1312, 1228 cm−1; 1H NMR (methanol-d4, 600 MHz) δH cis-Cin-1 [7.44 (m, 2H), 7.33 (m, 1H), 7.32 (m, 2H), 6.71 (d, J = 12.6 Hz, 1H), 5.53 (d, J = 12.6 Hz, G

DOI: 10.1021/acs.jnatprod.7b00615 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Triptersinine Z8 (12): white, amorphous solid; [α]25 D −8.3 (c 0.1 CHCl3); UV (MeOH) λmax (log ε) 218 (2.95), 278 (2.89) nm; ECD (MeOH) λmax (Δε) 224 (+2.86), 242 (−1.18), 261 (+1.57), 279 (−0.59) nm; IR (microscope) νmax 2935, 1739, 1370, 1284, 1246 cm−1; 1H NMR (methanol-d4, 600 MHz) δH trans-Cin-1 [7.33 (m, 1H), 7.25 (overlapped, 2H), 7.22 (d, J = 16.2 Hz, 1H), 7.07 (d, J = 7.8 Hz, 2H), 5.94 (d, J = 16.2 Hz, 1H)], 2.15 (s, 3H, Ac-5), 2.03 (s, 3H, Ac-7), Nic-8 [9.04 (d, J = 1.8 Hz, 1H), 8.37 (d, J = 4.2 Hz, 1H), 8.24 (dt, J = 8.4, 1.8 Hz, 1H), 7.23 (overlapped, 1H)], 2.32 (s, 3H, Ac-11), and other 1H NMR data (Table 1); 13C NMR (methanol-d4, 150 MHz) δC trans-Cin-1 (167.1, 146.4, 134.9, 131.6, 129.8 × 2, 129.0 × 2, 118.2), Nic-8 (164.7, 153.9, 151.1, 139.0, 127.2, 125.3), Ac-5 (171.4, 21.2), Ac-7 (171.7, 21.0), Ac-11 (172.0, 21.8), and other 13C NMR data (Table 2); HRESIMS m/z 664.2742 (calcd for C36H42NO11, 664.2752). X-ray Crystallographic Analysis of Compound 1. Colorless crystals of 1 (CCDC: 1562383) were obtained from MeOH. Crystal data: C33H38O12, M = 626.63, monoclinic, a = 9.6865(2) Å, b = 11.9018(3) Å, c = 13.7460(4) Å, β = 104.952(3)°, U = 1531.09(8) Å3, T = 103.2, space group P21 (no. 4), Z = 2, μ(Cu Kα) = 0.867, 11 802 reflections measured and 5749 unique (Rint = 0.0283). These parameters were used in all calculations. The final wR(F2) was 0.0916. Flack parameter = 0.03(10). The data can be accessed via www.ccdc.cam.ac.uk. X-ray Crystallographic Analysis of Compound 8. Colorless crystals of 8 (CCDC: 1562382) were obtained from MeOH. Crystal data: C32H37NO12, M = 627.62, monoclinic, a = 10.5270(15) Å, b = 9.3108(5) Å, c = 15.753(2) Å, β = 102.012(13)°, U = 1510.2(3) Å3, T = 106.4, space group P21 (no. 4), Z = 2, μ(Cu Kα) = 0.890, 10 483 reflections measured and 5610 unique (Rint = 0.0239). These parameters were used in all calculations. The final wR(F2) was 0.1097. Flack parameter = −0.02(13). The data can be accessed via www.ccdc.cam.ac.uk. Neuroprotection Bioassays. Neuroprotection bioassays were carried out according to the described procedure,31 and compounds 1−13 were tested for neuroprotective activity against OKA-treated pheochromocytoma (PC12) cells. NGF was used as a positive control. Analysis of variance (ANOVA) followed by Newman−Keuls post hoc test were performed to assess the differences between the relevant control and each experimental group. p-Values of