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(±)-Sativamides A and B, Two Pairs of Racemic Nor-lignanamide Enantiomers from the Fruits of Cannabis sativa (Hemp Seed) Guo-Yuan Zhu, Ji Yang, Xiao-Jun Yao, Xing Yang, Jing Fu, Xin Liu, Li-Ping Bai, Liang Liu, and Zhi-Hong Jiang J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02765 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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The Journal of Organic Chemistry

(±)-Sativamides A and B, Two Pairs of Racemic Nor-lignanamide Enantiomers from the Fruits of Cannabis sativa (Hemp Seed)

Guo-Yuan Zhu,*,† Ji Yang,† Xiao-Jun Yao,† Xing Yang,‡ Jing Fu,† Xin Liu,† Li-Ping Bai,† Liang Liu,† and Zhi-Hong Jiang*,† †

State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied

Research in Medicine and Health, Macau University of Science and Technology, Macau, China ‡

Department of Chemistry, Lanzhou University, Lanzhou 730000, China

ACS Paragon Plus Environment

The Journal of Organic Chemistry 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 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: (±)-Sativamides A (1) and B (2), two pairs of nor-lignanamide enantiomers featuring a unique benzo-angular triquinane skeleton, were isolated from the fruits of Cannabis sativa (hemp seed). Their structures were elucidated by detailed spectroscopic analysis and ECD calculations. The resolution of (+)- and (−)-sativamides A and B were achieved by chiral HPLC. Pretreatment of neuroblastoma cells with 1 and 2 significantly reduced the endoplasmic reticulum (ER) stress-induced cytotoxicity.


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Cannabis sativa L. (Cannabaceae), an annual herbaceous plant, is native of western and central Asia and now cultivated commercially all over the world. It has been used by humans as food, fiber, medicine, and psychoactive drug for thousands of years.1 It is currently recognized as a highly variable species in the Cannabis genus. Two major varieties of C. sativa are marijuana (drug type) and hemp (nondrug type). Marijuana has psychoactivity because of the existence of high content of ∆9-tetrahydrocannabinol (THC, ranging from 1 to 20%) and is prohibited worldwide. Whereas, hemp with low THC content (< 0.3%) and no psychoactive property is an important industrial source of fiber and food with a global market for its products valued at $100−2000 million annually.2 In China, the fruits of C. sativa (hemp seed) has been used as food and traditional Chinese medicine for at least 3000 years.3 It has been reported that hemp seed possesses a wide range of biological activities including antiplatelet aggregation,4 alleviating functional constipation,5 lowering cholesterol,6 cardioprotective effects,7 and improving learning and memory function.8 Cannabinoids, a class of C21 meroterpenoids with psychoactivity, are the best-known and the most specific group of compounds found in C. sativa.9 However, the THC content in hemp seed is usually less than 1 ppm.10 Instead, lignanamides are the major secondary metabolites isolated from hemp seed. Up to now, 21 lignanamides have been reported from hemp seed.11 These lignanamides exhibited various bioactivities such as anti-oxidant, inducing autophagic cell death, inhibiting acetylcholinesterase, and anti-inflammation.11,12 Herein, we report the isolation and structure elucidation of two pairs of racemic lignanamide enantiomers, (±)-sativamides A (1) and B (2), which possess an unique 6/5/5/5 tetracyclic rearranged nor-lignan carbon skeleton (Figure 1) and exhibited potential neuroprotective activity on several cell models.



HO

OH HO

4'

OH

4''

1'

1'' 8''

8'

O O H 17 NH

7'

HN 2

HO

9

H

8

10 4

HN

O O H

14

NH H

15

R

11

OH

13

C

6 5

D

B

A

7''

16 7

1 3

HO

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OH

12

R

O R (+)-1: OH (+)-2: OCH3

O

R (−)-1: OH (−)-2: OCH3

Figure 1. Structures of compounds 1 and 2. (±)-Sativamide A (1) was obtained as a light-yellow powder. Its molecular formula was established as C33H30N2O8 on the basis of HR-TOF-MS analysis (m/z 583.2075 [M + H]+, calcd 583.2075), indicating that 1 has 20 degrees of unsaturation. The IR spectrum of 1 displayed strong absorptions at 1705, 1650 and 1613 cm−1, suggesting the presence of carbonyl groups and double bonds in the molecule. The 1H and

13

C NMR data of 1 (Table 1) showed resonances

assignable to two p-tyramine moieties (C-1′–C-8′ and C-1′′–C-8′′) that are typical groups in most of lignanamides.11 Besides, two conjugated olefinic protons at δH 6.16 (d, J = 5.9 Hz, H-12) and 7.66 (d, J = 5.9 Hz, H-13), an olefinic proton with long range J-coupling at δH 6.29 (d, J = 1.9 Hz, H-15), two aromatic singlets at δH 6.64 (H-2) and 6.93 (H-5), and two coupled methines at δH 3.42 (d, J = 4.4 Hz, H-7) and 3.74 (dd, J = 4.4, 1.9 Hz, H-8) were observed in the 1H NMR spectrum of 1. Except for those from two sets of p-tyramine moieties, the 13C NMR spectrum of 1 showed additional 17 carbon signals, including six benzene carbons, four olefinic carbons, a ketone, two carbonyls, two methines, as well as two quaternary carbons. Taking account of HRTOF-MS and NMR data, compound 1 should be a nor-lignanamide derivative with a


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tetrasubstituted benzene unit, an α,β-unsaturated ketone group, a carbonyl group and an α,βunsaturated carbonyl group. The above functional groups and two p-tyramine moieties occupied 17 degrees of unsaturation, indicating that the skeleton of 1 possesses an additional tricyclic ring system. The HMBC correlations (Figure 2) between H-7 and C-2/C-6, between H-2 and C-6/C-7, and between H-5 and C-1/C-10 suggested that a methine group at δC 57.1 (C-7) and the oxygenated quaternary carbon at δC 87.7 (C-10) are connected to the benzene ring at C-1 and C-6, respectively.

1

H-1H COSY correlation (Figure 2) between H-7 and H-8, as well as HMBC

correlations be-tween H-7 and C-1/C-9, between H-8 and C-1 allowed to the assignment of C7−C-8−C-9 chain. The α,β-unsaturated carbonyl group (C-15−C-17) was assigned at C-8 position on the basis of HMBC correlations between H-7 and C-16, and between H-8 and C15/C-17. Furthermore, HMBC correlations between H-12 and C-10 fixed the α,β-unsaturated ketone group (C-11−C-13) at C-10. Accordingly, a structural fragment (1F) of 1 was configured as shown in Figure 2. The last quaternary carbon at δC 75.5 (C-14) was assigned reasonably at the center of B, C and D rings (Figure 1), which form an angularly fused triquinane sharing a common vertex (C-14) and two fusion bonds (C-10−C-14 and C-7−C-14). The downfield chemical shift of C-14 (δC 75.5) is similar with that of sesquiterpenes possessing a triquinane skeleton.13 These connections were also confirmed by HMBC correlations between H-8/H-12 and C-14, between H-13 and C-10, between H-13 and C-15, as well as between H-15 and C-7. Finally, HMBC correlations between H-8′ and C-9, between H-8′′and C-17 placed two ptyramines at C-9 and C-17, respectively. Thus, the plain structure of 1 was established as shown.



HO

OH

1'

8'

O O

7'

HN

9

2

7 1

8

NH

15 13

10

HO

14

HO 11 O

7

1

15 6 5

10 5

16

2

HO

13

HO

17 9

7''

17 16

8

HO

O O

2''

8''

HO

11

12

O

12

1F

1 HMBC 1H-1H

H

C

COSY

Figure 2. Key HMBC and 1H-1H COSY correlations of compound 1. The relative configuration of 1 was determined by a NOESY experiment (Figure S1) and the coupling constant of proton signals. The NOE correlation between H-7 and H-13 indicated that H-7 and C-ring are above the A, B-rings and assigned as β-orientation. The coupling constant (J = 4.4 Hz) of H-7 and H-8, as well as the NOE correlation between H-7 and H-8 suggested that they are located on the same side. The

specific rotation of 1 is near zero, and no Cotton effect was observed in ECD spectrum of 1, indicating that 1 is a racemic mixture. Subsequent chiral resolution of 1 was performed by chiral HPLC separation (Figure 3) to afford the anticipated enantiomers (+)- and (–)-1, which showed same NMR data, opposite specific rotations ((+)-1: [α]20D +24.3; (–)-1: –25.4) and mirror imagelike ECD curves (Figure 4). To establish the absolute configuration of (+)- and (–)-1, ECD curves for the two possible enantiomers (7R,8S,10S,14S; 7S,8R,10R,14R) were calculated using the TD-DFT theory method.14 As shown in Figure 4, The theoretical ECD spectra of 7R,8S,10S,14S and 7S,8R,10R,14R were found to match well with the experimental ECD spectra of (+)- and (–)-1, respectively. Thus, the absolute configuration of chiral carbons of (+)- and (–)-1 were determined as 7R,8S,10S,14S and 7S,8R,10R,14R, respectively.


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Figure 3. HPLC chromatograms of 1 and 2 on a chiral OZ-H column (250 × 4.6 mm, 5 µm).

Figure 4. Experimental and theoretical ECD spectra of (+)-1 and (−)-1.



Table 1. 1H (600 MHz) and 13C (150 MHz) NMR data of 1 and 2 in CD3OD. 1 2 No. δC δH (J in Hz) δC δH (J in Hz) 1 136.4 137.4 2 111.7 6.64, br s 112.1 6.66, br s 3 149.6 150.1 4 147.3 147.3 5 112.0 6.93, s 112.5 6.84, s 6 132.4 128.4 7 57.1 3.42, d, 4.4 57.0 3.42, d, 5.0 8 61.2 3.74, dd, 4.4, 1.9 60.7 3.75, dd, 5.0, 2.1 9 175.6 175.3 10 87.7 93.3 11 208.3 206.8 12 131.8 6.16, d, 5.9 132.0 6.17, d, 5.9 13 166.3 7.66, d, 5.9 166.7 7.67, d, 5.9 14 75.4 74.7 15 137.6 6.29, d, 1.9 137.0 6.35, d, 2.1 16 142.2 143.0 17 166.6 166.3 1′ 131.2 131.2 2′, 6′ 130.9 7.10, d, 8.5 130.9 7.10, d, 8.5 3′, 5′ 116.3 6.71, d, 8.5 116.3 6.72, d, 8.5 4′ 156.9 156.8 7′ 35.8 2.79, m 35.7 2.80, m 3.54, dt, 13.2, 7.6; 3.53, dt, 13.4, 7.7; 8′ 42.6 42.5 3.38, m 3.40, m 1′′ 131.2 131.2 2′′, 6′′ 130.8 7.01, d, 8.5 130.8 7.01, d, 8.5 3′′, 5′′ 116.2 6.69, d, 8.5 116.2 6.68, d, 8.5 4′′ 156.9 156.8 7′′ 35.7 2.69, t, 7.4 35.5 2.71, m 3.38, m 3.46, dt, 13.5, 7.2; 8′′ 42.5 42.4 3.32, m 3.31, m OCH3 53.8 3.08, s Data were assigned by the DEPT, HSQC, HMBC, 1H-1H COSY, and NOESY spectra.


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(±)-Sativamide B (2) was isolated as a light yellow powder with molecular formula C34H32N2O8 (HR-TOF-MS: m/z 597.2240 [M + H]+, calcd 597.2231). The 1H and

13

C NMR spectroscopic

data of 2 (Table 1) are similar to those of 1, except for a methoxy group in 2 instead of a hydroxy group in 1 at C-10. This was confirmed by the additional 14 amu in the HRESIMS, by the downfield shifted carbon signal of C-10 (δC 93.3), and by the HMBC correlation between protons of methoxy group (δH 3.08, s) and C-10. Therefore, the structure of 2 was assigned as 10-methoxyl-sativamide A. Chiral HPLC analysis of 2 (Figure 3) indicated the presence of a pair of racemate and provided two enantiomers [(+)-2 and (−)-2]. NOESY correlations (Figure S1) between proton signals of methoxy group and H-15, between H-7 and H-13 suggested that the stereochemistry of 2 is same with that of 1, which was confirmed by CD data of (+)-2 and (−)-2 (Figure S5). Compounds 1 and 2 represent the first example of a new class of natural products with a benzo-angular triquinane core. A biogenetic pathway for 1 derived from N-transcaffeoyltyramine was proposed as shown in Figure S2. It has been revealed that endoplasmic reticulum (ER) stress plays an important role in neurodegenerative diseases.15 The pharmacological targeting of ER stress signaling pathway is emerging as a therapeutic strategy for several neurodegenerative diseases such as Parkinson’s disease and Alzheimer's disease.15 As a continuing interest in natural ER stress modulators,16 we tested the effects of 1 and 2 on ER stress-induced neurotoxicity on two neuroblastoma cell models (PC12 and SH-SY5Y cells). Pretreatment of PC12 cells with 50 µM of compounds 1 and 2 markedly rescued the tunicamycin- (Tm, a chemical inducer of ER stress) induced cell death and cell growth inhibition (Figure 5A). The results of MTT assay showed that compounds 1 and 2 mitigated the Tm-induced PC12 cell toxicity in a dose-dependent manner (Figure 5B). Cytoprotective activity of 50 µM 1 or 2 was similar with that of 25 µM salubrinal (Sal), an



inhibitor of ER stress (Figure 5B). Compounds 1 and 2 also suppressed ER stress-mediated cytotoxicity induced by thapsigargin (Tg) in a dose-dependent manner (Figure 5C). Data from these experiments indicated that 1 and 2 have no evident adverse effects on cell viability at final concentrations of 50 µM (Figure 5 and S24). Cytoprotection effects of 1 and 2 were also observed in SH-SY5Y cells undergoing ER stress (Figure S42). Compound (+)-1 and (–)-1 showed similar bioactivity with that of 1 (Figure S43). Taken together, these results indicated that compounds 1 and 2 have the ability to mitigate the ER stress-induced cytotoxicity, and could be developed as a new class of ER stress inhibitor and neuro-protective agent.

Figure 5. Compounds 1 and 2 protect PC12 cells from ER stress-induced cytotoxicity. (A) Original microscopic observations (magnification: 20×) showed that pre-treatment of cells with 1 and 2 reduced the Tm-induced cell death and cell growth. (B and C) MTT assays showed that pre-treatment of cells with salubrinal (Sal, a positive control), 1 and 2 significantly reduced Tm


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(B) and Tg (C)-induced cytotoxicity in PC12 cells. Values are means ± S.D. from three independent experiments; *P < 0.05, ** P < 0.01, compared with Tm (B) or Tg (C) treatment.

In summary, this study describes the discovery of two novel nor-lignanamides (1 and 2) with a unique benzo-angular triquinane ring skeleton from hemp seed. Compounds 1 and 2 are two pairs of racemic enantiomers, which were successfully separated by chiral HPLC. A great attention of synthetic chemistry community has been drawn to the angular triquinanes because they possess highly congested structures and are the biosynthetic precursors of pentalenolactone, an antibiotic drug.17 The discovery of compounds 1 and 2 provides a new insight into the structural diversity of natural products, and a new challenge for synthetic chemists. Furthermore, compounds 1 and 2 showed potential neuroprotective activity in cells undergoing ER stress, indicating that 1 and 2 might be potential leading compounds for the drug discovery of neurodegenerative diseases. EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations were obtained on a Rudolph Research Analytical Autopol I automatic polarimeter (Na 589 nm). UV and CD spectra were recorded on a JASCO J-1500 Circular Dichroism Spectrometer. IR spectra were determined on an Agilent Cary 600 series FTIR spectrometer (KBr). NMR spectra were recorded on a Bruker Ascend 600 NMR spectrometer (600 MHz for 1H and 150 MHz for

13

C) using standard Bruker pulse programs. Samples were dissolved in

CD3OD and the NMR spectra were recorded using the signals of CD3OD (1H, δ 3.31; 13C, δ 49.0) as an internal reference. HR-TOF-MS spectra were measured on an Agilent 6230 Accurate-Mass TOF-LC/MS system. UHPLC analyses were carried out on an Agilent 1290 Infinity LC system using an Extend-C18 column (1.8 µm, 50 × 2.1 mm, i.d., Agilent). Semi-preparative HPLC was performed on the Waters 1525



HPLC system using Grace Alltima C18 (5 µm, 250 × 10 mm, i.d.) and Waters XBridge C18 (5 µm, 250 × 10 mm, i.d.) columns, with gradient solvent system composed of H2O and CH3CN or MeOH, and with a flow rate of 3.0 mL/min. MPLC was conducted on the Sepacore Flash Chromatography System (Buchi, Switzerland) using a Siliabond® C18 ODS column (40-63 µm, 460 × 36 mm, i.d., Silicycle, Canada). Column chromatography (CC) was carried out with silica gel (40-63 µm, Grace, USA) as packing material. All solvents were spectroscopic grade or HLPC grade and purchased from Labscan Asia (Bangkok, Thailand) or distilled prior to use. CHIRALCEL® OZ-H chiral column (250 × 10 mm, 5 µm) was used for chiral separation on HPLC chromatographs.

Plant material. The fruits of Cannabis sativa were collected from Linzhou, Henan Province, China, in October 2015. The species was identified by the author, Dr. Zhu G.Y. A voucher specimen (CS-2011510) was deposited at the State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology.

Extraction and isolation. The air-dried and powdered fruits of C. sativa (9.0 kg) were extracted with 80% EtOH (40 L × 3) under reflux. The combined extracts were concentrated under reduced pressure to afford a brown residue, which was suspended in H2O (8 L) and partitioned with petroleum ether, EtOAc and n-BuOH successively. The EtOAc extract (98 g) was subjected to silica gel CC eluting with petroleum ether – acetone – MeOH (10:1:0 → 0:5:5, v/v) to obtain 36 fractions (Fr.1−Fr.36). Fr.28 (6 g) was isolated by MPLC with a reversed-phase RP-18 column eluting with MeOH−H2O (20:80 → 80:20, v/v), and further repeatedly purified by preparative HPLC eluting with MeCN−H2O (30:70, 25:75, v/v) to yield 2 (7 mg). Fr.29 (15 g) was re-subjected to silica gel column chromatography eluting with CHCl3−MeOH (20:1→4:1, v/v) to afford 20 subfractions (Fr.29.1−Fr.29.20). Fr.29.13 was isolated by MPLC with a reversed-phase RP-18 column eluting with MeOH−H2O (20:80 → 100:0, v/v), and further repeatedly purified by preparative HPLC eluting with MeCN−H2O (26:74, v/v) to yield 1 (22


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mg). Compound 1 (8 mg) was further separated by chiral HPLC eluting with n-hexane−EtOH (67:33) to give (–)-1 (Rt: 11.98 min; 3.5 mg) and (+)-1 (Rt: 14.52 min; 3.5 mg). Compound 2 (3 mg) was separated by chiral HPLC eluting with n-hexane−EtOH (60:40) to obtain (–)-2 (Rt: 10.57 min; 1.4 mg) and (+)-2 (Rt: 14.59 min; 1.4 mg) Characterization Data. (±)-Sativamide A (1): Light yellow powder; [α]25D −1.9 (c = 1.0, EtOH); UV (MeOH) λmax (log ε) 283 (3.70), 223 (4.48) nm; ECD (MeOH) λmax nm (∆ε) 206 (+3.9), 224 (+0.6), 250 (–2.4), 305 (+0.5); IR (KBr) νmax 3401, 1705, 1650, 1613, 1515, 1458, 1311, 1242 m−1; 1H and 13C NMR data, see Table 1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C33H31N2O8 583.2075; Found 583.2075. [M + Na]+ Calcd for C33H30N2O8Na 605.1897; Found 605.1894. (+)-1: Light yellow powder; [α]25D +23.5 (c = 0.5, EtOH); ECD (EtOH) λmax nm (∆ε) 220 (+29.9), 250 (–39.0), 306 (+6.1); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C33H31N2O8 583.2075; Found 583.2081. (–)-1: Light yellow powder; [α]25D –25.1 (c = 0.5, EtOH); ECD (EtOH) λmax nm (∆ε) 216 (–30.3), 253 (+24.0), 310 (–3.8); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C33H31N2O8 583.2075; Found 583.2078.

(±)-Sativamide B (2): Light yellow powder; [α]25D −2.8 (c = 1, EtOH); UV (MeOH) λmax (log ε) 283 (3.77), 224 (4.56) nm; ECD (MeOH) λmax nm (∆ε) 206 (+4.9), 224 (+0.7), 255 (–2.9), 312 (+0.9); IR (KBr) νmax 3418, 1709, 1650, 1613, 1515, 1456, 1317, 1245 cm−1; 1H and

13

C NMR

data, see Table 1; HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C34H33N2O8 597.2231; Found 597.2240. [M + Na]+ Calcd for C34H32N2O8Na 619.2051; Found 619.2059. (+)-2: Light yellow powder; [α]25D +22.0 (c = 0.5, EtOH); ECD (EtOH) λmax nm (∆ε) 207 (+16.7), 227 (+5.0), 258 (– 13.5), 310 (+1.7); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C34H33N2O8 597.2231; Found 597.2235. (–)-2: Light yellow powder; [α]25D –24.1 (c = 0.5, EtOH); ECD (EtOH) λmax nm (∆ε) 213 (–18.9), 250 (+12.3), 310 (–1.9); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C34H33N2O8 597.2231; Found 597.2228.



Quantum Chemical ECD Calculation. In theoretical calculations, the conformational search was applied using Spartan software with Molecular Mechanics method and MMFF force field. Forty conformers of two configurations were obtained for further optimizations using density of functional theory (DFT) at the B3LYP/6-31G (d, p)//M06-2X/def2-TZVP level, as implemented in the Gaussian 09 program package. Thus, five and six conformers which contributions are greater than one percent can be obtained for 7S,8R,10R,14R-1 and 7R,8S,10S,14S-1 (see supplementary Figures S44 and S45), respectively. All of them were true minima via vibrational frequency calculations, because all frequencies are positive. Then, the ECD spectra of all the obtained configurations were calculated with the solvent model with ethanol using the TDDFT method at the PBE0/def-TZVP level, and the Boltzmann-averaged ECD spectra of (+)- and (–)-1 were obtained as a comparison with the experimental data using SpecDis program through the above optimized configurations with the important contributions, the geometry of the molecules was optimized with Gaussian 09 package1 at B3LYP/6-31G (d) computational level.18 Cell cultures. PC-12 and SH-SY5Y cells were purchased from American Type Culture Collection (ATCC). PC-12 cells were cultured in DMEM medium (Invitrogen) supplemented with 100 U/ml penicillin, 100 µg/mL streptomycin, and 10% horse serum and 5% fetal bovine serum (Gibco, Carlsbad, USA). SH-SY5Y cells were cultured in 1:1 mixture of DMEM and F12 medium containing 10% fetal bovine serum (Gibco, Carlsbad, USA). Both of them were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. MTT assay. PC12 or SH-SY5Y cells were seeded into 96-well culture plates (5.0 × 103 cells/well) and cultured under standard conditions for 12 h. Cells were then treated with compounds 1 and 2 (12.5, 25, 50 µM) or vehicle for another 12 h. After test compounds or vehicle treatment, the whole medium was replaced with fresh medium containing Tm (0.1 or 1.0


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µM) or Tg (10 or 100 nM) and cells were incubated for additional 48 h. After incubation, supernatant was changed by fresh medium and thiazolyl blue tetrazolium bromide (MTT) were given at the concentration of 0.5 mg/mL. After incubation at 37℃ for 4 h, the absorbance was measured at 570nm with a micro-plate reader.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: The CD, UV, IR, MS, and NMR spectra of 1 and 2. A proposed biogenetic pathway for 1. Chiral HPLC chromatograms. Cell protective effects of 1 and 2 in SH-SY5Y cells. DFT computational optimized conformations (≥1%) of (–)-1 and (+)-1 and their cartesian coordinates and energies. (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] ORCID Guo-Yuan Zhu: 0000-0002-4355-894X Zhi-Hong Jiang: 0000-0002-7956-2481 Author Contributions



The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by Macao Science and Technology Development Fund, MSAR (033/2015/A1). REFERENCES (1) Brand, E. J.; Zhao, Z. Front Pharmacol. 2017, 8, 108. (2) (a) Ahmed, S. A.; Ross, S. A.; Slade, D.; Radwan, M. M.; Zulfiqar, F.; Matsumoto, R. R.; Xu, Y. T.; Viard, E.; Speth, R. C.; Karamyan, V. T.; ElSohly, M. A. J. Nat. Prod. 2008, 71, 536– 542. (b) Montserrat-de la Paz, S.; Marín-Aguilar, F.; García-Giménez, M. D.; Fernández-Arche, M. A. J. Agric. Food Chem. 2014, 62, 1105–1110. (3) Jiang, H. E.; Li, X.; Zhao, Y. X.; Ferguson, D. K.; Hueber, F.; Bera, S., Wang, Y. F.; Zhao, L. C.; Liu, C. J.; Li, C. S. J. Ethnopharmacol. 2006, 108, 414–422. (4) Prociuk, M. A.; Edel, A. L.; Richard, M. N.; Gavel, N. T.; Ander, B. P.; Dupasquier, C. M.; Pierce, G. N. Can. J. Physiol. Pharmacol. 2008, 86, 153–159. (5) Cheng, C. W.; Bian, Z. X.; Zhu, L. X.; Wu, J. C.; Sung, J. J. Am. J. Gastroenterol. 2011, 106, 120–129. (6) Schwab, U. S.; Callaway, J. C.; Erkkila, A. T.; Gynther, J.; Uusitupa, M. I. J.; Jarvinen, T. Eur. J. Nutr. 2006, 45, 470–477.


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