Highly Conjugated Norditerpenoid and Pyrroloquinoline Alkaloids with

Mar 4, 2014 - The Key Laboratory of Plant Resources and Chemistry of Arid Zone and State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants ...
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Highly Conjugated Norditerpenoid and Pyrroloquinoline Alkaloids with Potent PTP1B Inhibitory Activity from Nigella glandulifera Qi-Bin Chen,†,‡ Xue-Lei Xin,† Yi Yang,† Shoei-Sheng Lee,§ and H. A. Aisa*,† †

The Key Laboratory of Plant Resources and Chemistry of Arid Zone and State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China § School of Pharmacy, College of Medicine, National Taiwan University, Taipei 10051, Taiwan, Republic of China S Supporting Information *

ABSTRACT: Three norditerpenoid alkaloids, nigelladines A−C (1−3), and one pyrroloquinoline alkaloid, nigellaquinomine (4), all possessing new skeletons with highly conjugated systems, were isolated from Nigella glandulifera. The 8aS-configuration for 1 and 2 was determined by comparison of the experimental and calculated electronic circular dichroism spectra. These alkaloids exhibited potent protein tyrosine phosphatase 1B (PTP1B) inhibitory activity but are devoid of cytotoxicity against the A431 cell line at 100 μM.

C

hemical constituents of the Nigella plants (Ranunculaceae) have been investigated for decades due to their broad spectrum of bioactivities, such as antidiabetic, antioxidant, antiulcer, and antimicrobial and their effects on the cardiovascular system and blood.1 Interestingly, from this class of edible plants some structurally specific alkaloids have been isolated from seeds, including indazole alkaloids2a−e and dolabellane-type diterpene alkaloids.3 Dolabellane diterpene alkaloids have been reported to possess potent promoting activity on lipid metabolism in high-glucose-pretreated HepG2 cells, equivalent to the PPAP-α agonist clofibrate.3a−c Thus this study was aimed at investigating potential antidiabetes alkaloids from the seeds of Nigella glandulifera Freyn. Following a bioassay-guided approach, three norditerpenoid alkaloids and one pyrroloquinoline alkaloid, all of them possessing new skeletons, were isolated by application of high-speed countercurrent chromatography (HSCCC). This report describes the isolation, structure elucidation, plausible biogenetic pathways, and biological activities of these compounds.

δMe‑16 1.17) and for an allylic residue (δMe‑12 2.25 ↔ δH‑3 5.97). The NOESY spectrum showed correlations of Me-10/-11 (δ 1.54, 1.55) ↔ H-3 (δ 5.97) ↔ Me-12 (δ 2.25) ↔ H-5 (δ 7.64) ↔ Me-15/-16 (δ 1.14, 1.17) and of H2-9 (δ 3.01) ↔ Me-13 (δ 1.36) ↔ H2-8 (δ 2.84), thereby designating the spatial location of these protons (Figure 1). The HMBC spectrum of 1 showed the following key correlations (Figure 1): Me-15 (-16)/C-6 (δ 154.7), thus designating the isopropyl group as linked to C-6; H-14/C-5 (δ 130.3) and C-7 (δ 197.6) and H2-8/H-5 to C-7, C-6, C-8a (δ 49.6), and C-4b (δ 165.8), thereby establishing the structure of the C-ring; Me-13 (δ 1.36)/C-8, C-8a, C-4b, and C-9 (δ 44.2) and H2-9 (δ 3.01)/C-8, C-8a, C-4b, C-4a (δ 126.0), and C-9a (δ 184.3), establishing the structure of the Bring; Me-12/C-4a, C-4 (δ 126.7), and C-3 (δ 136.4) and Me-10 (-11)/C-2 (δ 61.5) and C-3, establishing the fragment a (bold black carbon sequence in Figure 1) to be linked to ring B via the C-4a−C-4 bond. The MS data indicated 1 to contain a nitrogen atom, which was inserted between C-9a and C-2 to form the heterocyclic A-ring, based on the chemical shifts of C9a (δ 184.3) and C-2 (δ 61.5) and the absence of HMBC correlation between Me-10 (-11) and C-9a, and an NOE

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RESULTS AND DISCUSSION Nigelladine A (1) had a molecular formula of C19H25NO, based on 13C NMR and HRESI(+)MS data, implying eight indices of hydrogen deficiency. The 1H and 13C NMR spectroscopic data of 1 (Table 1) revealed the presence of six methyls (C-10, -11, -12, -13, -15, -16), two methylenes (C-8, -9), one alphatic methine (C-14), two olefinic methines (C-3, -5), and eight quaternary carbons including two aliphatic (C-2, -8a), one carbonyl (C-7), and five olefinic or aromatic carbons (C-4, -4a, -4b, -6, -9a). The resonances for the proton-attached carbons (Table 1) were designated by analysis of the HSQC spectrum. The 1H−1H COSY spectrum of 1 showed the proton correlations of an isopropyl residue (δMe‑15 1.14 ↔ δH‑14 3.05 ↔ © 2014 American Chemical Society and American Society of Pharmacognosy

Received: October 27, 2013 Published: March 4, 2014 807

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Table 1. 1H and 13C NMR Spectroscopic Data (δ in ppm and J in Hz) for 1−4a 1 no.

δC type

2 3 3a 4 4a 4b 5 5a 6 7 8

61.5 C 136.4 CH

8a 9 9a 10 11 12 13 14 15 16

126.7 126.0 165.8 130.3

C C C CH

154.7 C 197.6 C 51.6 CH2 49.6 C 44.2c CH2 184.3 C 29.5 CH3 29.2 CH3 20.6 CH3 27.8 CH3 29.1 CH 21.6 CH3 22.1 CH3

2 δH mult. (J)

5.97 q (1.2)

δC type 60.9 C 135.8 CH

3 δH mult. (J)

δC type 60.3 C 142.8 CH

6.20 brs

128.2 C 128.1 C 7.64 brs

2.84 s

3.01 s 1.55 1.54 2.25 1.36 3.05 1.14 1.17

s s d (1.2) s dhb (0.8, 6.9) d (7.2) d (6.6)

131.2 163.5 150.8 207.1 51.2

CH C C C CH2

41.6 43.7 173.8 29.4 28.1 18.1 27.4 27.0 20.8 21.4

C CH2 C CH3 CH3 CH3 CH3 CH CH3 CH3

4 δH mult. (J) 6.23 brs

128.6 C 131.0 C 7.60 s

α 2.53 d (18.6) β 2.49 d (18.6) 3.24 s 1.60 1.58 2.13 1.28 3.07 1.25 1.29

s s d (1.2) s hb (7.0) d (7.2) d (7.2)

118.6 169.5 136.4 209.9 43.9

C C C C CH2

49.1 C 44.6c CH2 162.2 C 30.7 CH3 29.8 CH3 18.1 CH3 7.8 CH3 33.0 CH 18.2 CH3 18.0 CH3

δC type 77.5 C 222.0 C 52.0 C 42.0c CH2 162.1 C

δH mult. (J)

2.67 brs

6.87 s

β 2.52 d (18.6) α 2.07 d (18.6) 3.10 s 1.29 1.32 1.98 1.86 1.80 0.58 1.02

s s d (1.2) s hb (6.8) d (6.6) d (6.6)

60.0 C 144.1 CH 130.0 C 131.3 131.2 178.8 15.9 26.7 25.0 22.1 29.9 30.6 23.6

C C C CH3 CH3 CH3 CH3 CH3 CH3 CH3

6.16 s

2.34 1.42 1.34 1.22 1.32 1.26 2.20

s s s s s s d (6.0)

Data were recorded in methanol-d4 at 600 MHz (1H) and 150 MHz (13C). b“h” stands for heptet. cAppear as a triplet in the BBD 13C NMR spectrum. a

carbon types with 1. The 1H NMR spectrum showed similar coupling patterns to that of 1 except for an AB system for a methylene pair in 2 (δ 2.49 and 2.53) instead of a singlet (δ 2.84) in 1. The 1H−1H COSY spectrum of 2 showed correlations for an isopropyl residue (δMe‑15 1.29 ↔ δH‑14 3.07 ↔ δMe‑16 1.25), an allylic residue (δMe‑12 2.13 ↔ δH‑3 6.20), and one pair of methylene protons (δH‑8 2.53/2.49). The NOESY spectrum showed correlations of Me-10/-11 (δ 1.60 and 1.58) ↔ H-3 (δ 6.20) ↔ Me-12 (δ 2.13) ↔ H-5 (δ 7.60) ↔ Me-15/-16 (δ 1.25 and 1.29) and of H2-9 (δ 3.24) ↔ Me-13 (δ 1.28) ↔ H-8β (δ 2.49), indicating the spatial location of these protons to be similar to that in 1. In the 13C NMR spectra, two larger chemical shift differences were observed, i.e., one for the C-7 carbonyl (δ 207.1 vs 197.6), thereby suggesting the cyclohexenone moiety (ring C) in 1 to be replaced by a cyclopentenone moiety in 2, and the other for C-9a, δ 173.8 vs 184.3, suggesting the 6/5-membered A/B rings in 1 to be replaced by the 6/6-membered rings in 2. The structure of 2 was finalized on the basis of the HMBC analysis, showing the following key correlations (Figure 3): Me-15 (-16)/C-6 (δ 150.8), designating the isopropyl group to be linked to C-6; H14/C-5a (δ 163.5) and C-7 (δ 207.1) and H2-8 to C-7, C-6, C8a (δ 41.6), and C-5a (δ 163.5), establishing the structure of ring C as a cyclopentenone moiety; Me-13 (δ 1.28)/C-8 (δ 51.2), C-8a, C-5a, and C-9 (δ 43.7) and H-5 (δ 7.60) together with H2-9 (δ 3.24) to C-8a, C-5a, C-4a (δ 128.1), and C-9a (δ 173.8), establishing the cyclohexenyl-type structure of ring B; Me-12/C-4a, C-4 (δ 128.2), and C-3 (δ 135.8) and Me-10 (-11)/C-2 (δ 60.9) and C-3, establishing the fragment a (bold black carbon sequence in Figure 3) to be linked to ring B via the C-4a−C-4 bond. As in compound 1, the nitrogen atom was inserted between C-9a and C-2 to form the heterocyclic A-ring concluded from MS data, chemical shifts of C-9a (δ 173.8) and C-2 (δ 60.9), and the absence of HMBC correlation between

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

correlation between Me-10 (-11) and H2-9. These analyses thus furnished the structure of 1 as shown, leaving the configuration at C-8a to be determined. In the aprotic DMSO-d6, the C-9 methylene resonance appeared as a singlet in both broad-band decoupled (BBD) and DEPT-135 13C NMR spectra of nigelladine A (1). In methanold4 (150 MHz), however, it appeared as a triplet in both spectra (Figure 2). This indicated that one of the C-9 methylene protons was deuterium-exchanged in the protic solvent. The HRESI(+)MS spectrum of 1 dissolved in methanol-d4 showed two quasi-molecular ions peak at m/z 285.2077 ([M + H]+, calcd for C19H25DNO, 285.2077) and 286.2130 ([M + H]+, calcd for C19H24D2NO, 286.2140), indicating mono- and dideuteration at C-9 of 1. Nigelladine B (2) had the same molecular formula as 1 based on 13C NMR and HRESI(+)MS data. Its 1H and 13C NMR spectroscopic data (Table 1) indicated that it had analogous 808

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Figure 2. DEPT-135 spectra of nigelladine A (1) in methanol-d4 (I) and DMSO-d6 (II).

Figure 3. Key 1H−1H COSY (blue bold) and HMBC (→) correlations of 2−4.

Figure 4. Experimental (exptl) and calculated (calcd) ECD spectra of 1 and 2.

pbe0/def-TZVP level, and the result was subsequently optimized by the Gaussian method of TmoleX 4.3 software. The overall pattern of the calculated ECD spectrum for 8aS-1 was in good agreement with the experimental ECD spectrum of 1, showing a positive Cotton effect at 233 nm and a negative Cotton effect at 372 nm (Figure 4). Therefore, the absolute configuration of the 8a stereogenic center was defined as S. Similarly, since the overall pattern of the calculated ECD spectrum for 8aS-2 was in good agreement with the experimental ECD spectrum of 2 (Figure 4), the absolute configuration of C-8a of 2 was also determined as S.

Me-10 (-11) and C-9a and NOE associations between Me-10 (-11) and H2-9. Accordingly, compound 2 possessed a 6/6/5tricyclic heterocyclic skeleton. The absolute configuration of 1 and 2 was established by comparison of their experimental electronic circular dichroism (ECD) spectra with those obtained by quantum chemical ECD calculation. 4 The geometrical optimization and energy calculation of conformers for ECD calculation were performed by TmoleX 4.3 software,5 using the TDDFT method at the pbe0/def-TZVP level. The stable conformer obtained was subjected to ECD calculation by the TDDFT method at the 809

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Scheme 1. Plausible Biosynthetic Pathways to 1−4

1.22), designating the spatial location of these protons. The HMBC spectrum showed the following correlations (Figure 3): Me-14 (-15)/C-6 (δ 60.0) and C-7 (δ 144.1); H-7/C-6, C-14 (δ 29.9), C-16 (δ 23.6), and C-8a (δ 131.3); and Me-16/C-8 (δ 130.0), C-8a, and C-7, designating the structure of fragment a (Figure 3) and its linkage to ring B via the C-8−C-8a bond; Me-10/C-9 (δ 131.2), C-8a, and C-9a (δ 178.8) and H2-4/C-3a (δ 52.0), C-4a (δ 162.1), C-8a, and C-9a, indicative of the structure of the six-membered B-ring; Me-11 (-12)/C-12 (-11), C-2 (δ 77.5), and C-3 (δ 222.0), designating the structure of fragment b (bold black carbon sequence in Figure 3); and Me13/C-3, C-3a, C-4 (δ 42.0), and C-9a, establishing both fragment b and Me-13 being linked to C-3a. The HRESI(+)MS data indicated that compound 4 contained two nitrogen atoms, one inserted between C-9a and C-2 to form the heterocyclic five-membered C-ring and the other one inserted between C-4a and C-6 to form the heterocyclic six-membered A-ring, elucidated on the basis of the chemical shifts of C-9a (δ 178.8), C-2 (δ 77.5), C-4a (δ 162.1), and C-6 (δ 60.0) and the absence of HMBC correlations of Me-11 (-12)/C-9a and Me14 (-15)/C-4a. These analyses thus furnished the structure of 4 as shown. Both nigelladine C (3) and nigellaquinomine (4) are optically inactive.

Nigelladine C (3) also had the same molecular formula, C19H25NO, as 1 and 2 based on 13C NMR and HRESI(+)MS data. The 1H and 13C NMR spectra of 3 were similar to those of 2 (Table 1). The NOESY spectrum of 3 was also similar to that of 2 except that the NOE relationships of Me-10/-11 (δ 1.29 and 1.32) ↔ H-3 (δ 6.23) ↔ Me-12 (δ 1.98) ↔ H-5 (δ 6.87) ↔ Me-13 (δ 1.86) and of H2-9 (δ 3.10) ↔ Me-15/-16 (δ 0.58 and 1.02) ↔ H-8β (δ 2.52) were observed, suggesting that C-6 and C-8a in 3 carried methyl and isopropyl groups, respectively. The suggested structure 3 was supported by the key HMBC correlations of Me-13/C-6 (δ 136.4), C-7 (δ 209.9), and C-5a (δ 169.5); Me-15 (−16)/C-8a (δ 49.1); and H-14/C-8 (δ 43.9), C-8a, C-9 (δ 44.6), and C-5a (Figure 3). Nigellaquinomine (4) had a molecular formula of C18H24N2O based on 13C NMR and HRESI(+)MS data, implying eight indices of hydrogen deficiency. Analysis of the 1 H and 13C NMR spectroscopic data of 4 (Table 1) indicated seven methyls, one methylene, one olefinic methine (C-7), and nine quaternary carbons comprising three aliphatic, one carbonyl, and five olefinic or aromatic carbons. The COSY spectrum of 4 showed correlations for an allylic residue (δMe‑16 2.20 ↔ δH‑7 6.16). The NOESY spectrum showed correlations of Me-14/-15 (δ 1.32 and 1.26) ↔ H-7 (δ 6.16) ↔ Me-16 (δ 2.20) ↔ Me-10 (δ 2.34) and of H2-4 (δ 2.67) ↔ Me-13 (δ 810

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organic stationary phase and the lower aqueous mobile phase, respectively. The fractions were further analyzed by HPLC (Phenomenex Gemini 5 μm C18 110 Å, 250 × 4.6 mm; MeOH (A)−0.05% Et3N in H2O (B), 40:60 to 60:40 in 10 min, 60:40 to 75:25 in 20 min, 75:25 for 7 min, 75:25 to 100:0 in 3 min; flow rate, 1 mL/min; column temperature, 30 °C; injection volume, 20 μL; detection at 365 nm). The crude alkaloid mixture (2.5 g) was fractionated by HSCCC into 10 fractions (A−J) based on detection at 254 nm. The aqueous layer of the solvent system, n-hexane−EtOAc−MeOH−H2O (4:5:4:5, v/v), was used as mobile phase with a flow rate of 1.5 mL/min, rotation speed of 850 rpm, and detection at 254 nm. Fraction I (250 to 380 min, 40 mg), being active against PTP1B, was further separated on silica gel CC (200−300 mesh), eluted by CHCl3−MeOH (40:1 to 15:1), to yield 4 (2.1 mg, 0.000042%). Nigelladine A (1): yellow, amorphous powder; [α]20D −53 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 357 (4.37), 276 (4.02) nm; ECD (MeOH) 237 (Δε +19.97), 352 (Δε −18.19); 1H and 13C NMR, see Table 1; HRESI(+)MS m/z 284.2009 [M + H]+ (calcd for C19H26NO 284.2014). Nigelladine B (2): yellow, amorphous powder; [α]20D +99 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 353 (4.04), 277 (3.81) nm; ECD (MeOH) 231 (Δε −30.51), 308 (Δε +25.13), 346 (Δε +2.67), 377 (Δε +11.02); 1H and 13C NMR, see Table 1; NOESY data Me-10/-11 ↔ H-3 ↔ Me-12 ↔ H-5 ↔ Me-15/-16, H2-9 ↔ Me-13 ↔ H-8β; HRESI(+)MS m/z 284.2017 [M + H]+ (calcd for C19H26NO 284.2014). Nigelladine C (3): yellow, amorphous powder; [α]20D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 346 (4.40), 276 (3.95) nm; 1H and 13C NMR, see Table 1; NOESY data Me-10/-11 ↔ H-3 ↔ Me-12 ↔ H-5 ↔ Me-13, H2-9 ↔ Me-15/-16 ↔ H-8β; HRESI(+)MS m/z 284.2020 [M + H]+ (calcd for C19H26NO 284.2014). Nigellaquinomine (4): yellow, flaky crystals (MeOH); [α]20D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 306 (4.01) nm; 1H and 13C NMR, see Table 1; NOESY data Me-14/-15 ↔ H-7 ↔ Me-16 ↔ H10, H2-4 ↔ Me-13; HRESI(+)MS m/z 285.1957 [M + H]+ (calcd for C18H25N2O, 285.1967). D−H Exchange Experiments of 1. Nigelladine A (1) was dissolved in methanol-d4 in an NMR tube (0.5 mm), and the solution was kept at room temperature for four days and subjected to DEPT135 and HRESI(+)MS measurements. The residue obtained after removal of methanol-d4 upon condensation was added to MeOH (2 mL), and the solution was stirred overnight at 40 °C, then evaporated. The residue was dissolved in DMSO-d6 and subjected to DEPT-135 measurement. PTP1B Inhibitory Assay. The protein tyrosine phosphatase 1B inhibitory activity of compounds 1−4 was evaluated as described.7 pNitrophenyl phosphate with a final concentration of 3.5 mM in the assay mixture (200 μL) was used as substrate. The mixture of tested compound, PTP1B, and substrate was incubated at 30 °C for 30 min and immediately subjected to a 96-well microplate spectrophotometer under 405 nm. The PTP1B inhibitor 3-(3,5-dibromo-4-hydroxybenzoyl)-2-ethylbenzofuran-6-sulfonic acid-[4-(thiazol-2-ylsulfamyl)phenyl]amide was used as positive control. Cytotoxicity Assay. See ref 8.

Nigelladines A−C (1−3) and nigellaquinomine (4) are new alkaloids with unprecedented skeletons, each containing a unique and highly cross-ring conjugated system. The plausible biosynthetic pathways of 1−4 are proposed as shown in Scheme 1. Compounds 1−3 could be produced biosynthetically via the intermediate of iphionane-type sesquiterpenoid 5,6 and compound 4 could originate from two units of isopentenyl pyrophosphate (IPP) as shown in Scheme 1. Nigelladines A−C (1−3) and nigellaquinomine (4) were evaluated for their inhibitory activity against protein tyrosine phosphatase 1B (PTP1B)7 using the PTP1B inhibitor 3-(3,5dibromo-4-hydroxybenzoyl)-2-ethylbenzofuran-6-sulfonic acid[4-(thiazol-2-ylsulfamyl)phenyl]amide as positive control, with an IC50 value of 6.81 ± 0.53 μM. The result indicated that compounds 1−4 possess good anti-PTP1B activity, with IC50 values of 9.97 ± 0.17, 9.71 ± 1.04, 16.85 ± 0.22, and 6.44 ± 0.15 μM, respectively. In addition the cell proliferation inhibitory assay of these compounds by the SRB method8 showed no apparent cytotoxicity on the A431 cell line at 100 μM. This study implies that these alkaloids could serve as lead compounds for the development of antidiabetic drugs.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a Rudolph Research Autopol VI polarimeter. UV spectra were obtained on a Shimadzu UV-2550 spectrophotometer. ECD spectra were measured in MeOH on a JASCO J-810 spectropolarimeter. 1D and 2D NMR data were obtained on a Varian Inova 600 spectrometer in methanol-d4 or DMSO-d6 with TMS as an internal standard. HRESIMSTOF data were recorded on a QSTAR Elite mass spectrometer. High-speed counter-current chromatography was carried out using a model TBE-300A (Shang-hai Tauto Biotech, Shanghai, China; 110 m multilayer coil and 1.8 mm i.d.) with a total capacity of 290 mL. CC was performed on silica gel (200−300 mesh, Haiyang Chemical Co. Ltd., Qingdao, P. R. China). The compounds were evaluated using a Dionex P680 HPLC apparatus. OD values of the bioassay were measured by a SpectraMax MD5 (Molecular Devices, USA). Solvents for HSCCC separation and HPLC analysis were HPLC grade. Plant Material. Seeds of N. glandulifera were purchased from the China Xinjiang Uyghur Pharmaceutical Co., Ltd. in October 2011. The specimen was authenticated by Prof. De Min of Xinjiang Uyghur Autonomous Region Food and Drug Inspection Office. A voucher specimen (XTIPC-NG-051011) was deposited at the Key Laboratory of Plant Resources and Chemistry of Arid Zone and State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, P. R. China. Extraction and Isolation. The powdered seeds (5 kg) were defatted with petroleum ether (10 L × 2, each 1 h) at 65 °C. The residue was treated with aqueous NH3 (25−28%)−acetone (1: 2, 3 L) overnight and dried at room temperature. The processed residue was extracted with CHCl3 (10 L × 2, each 1 h) at 60 °C and afforded extract (362.2 g) after solvent evaporation under reduced pressure. This extract was partitioned between CHCl3 (200 mL) and 0.5 N H2SO4 (200 mL × 10). The acidic aqueous layer was basified with aqueous NH3 (25−28%) in an ice−H2O bath to pH 10, and the suspension formed was filtered to give the alkaloid mixture (2.0 g). The filtrate was extracted with CHCl3 to obtain crude alkaloids (2.5 g) after solvent evaporation under reduced pressure. The precipitated alkaloids (2.0 g) were separated by pH-zonerefining counter-current chromatography (CCC), flow rate 2 mL/min, rotation speed 850 rpm, detection 254 nm, and 6 mL per fraction, to yield 1 (8.1 mg, 0.000 16% w/w dried seeds), 2 (10.5 mg, 0.000 21%), and 3 (2.2 mg, 0.000 044%). Methyl tert-butyl ether−nBuOH−H2O (2:1:4, v/v) was used as solvent system for pH-zone-refining CCC. Triethylamine (10 mM) and HCl (10 mM) were added to the upper

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ASSOCIATED CONTENT

S Supporting Information *

UV spectra, 1D and 2D NMR, and HRESI(+)MS of compounds 1−4; ECD spectra of compounds 1 and 2. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-0991-3835679. Fax: 86-0991-3835679. E-mail: haji@ ms.xjb.ac.cn. 811

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Prof. J.-M. Yue of Shanghai Institute of Materia Medica, Chinese Academy of Sciences, for ECD measurements. This research was supported by the National Science Fund for Distinguished Young Scholars (No. 30925045), the National Natural Science Foundation of China (No. 81173658), and the Projects of International Science & Techology Cooperation of the Xinjiang Uyghur Autonomous Region (No. 20136014).

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