Phenolic Constituents Isolated from the Twigs of Cinnamomum cassia

Compounds 3, 5, 10, 11, 12, 20, 36, and 56 showed statistically significant ... (2−8) As part of a program to study the chemical diversity of tradit...
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Cite This: J. Nat. Prod. 2018, 81, 1333−1342

Phenolic Constituents Isolated from the Twigs of Cinnamomum cassia and Their Potential Neuroprotective Effects Xin Liu,† Jing Fu,† Xiao-Jun Yao,† Ji Yang,† Liang Liu,† Tang-Gui Xie,‡ Ping-Chuan Jiang,‡ Zhi-Hong Jiang,*,†,§ and Guo-Yuan Zhu*,†

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†

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, People’s Republic of China ‡ Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Chinese Medicine and Pharmaceutical Science, Nanning 530022, People’s Republic of China § International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, People’s Republic of China S Supporting Information *

ABSTRACT: Seven new α,β-diphenyl-γ-butyrolactones (1−7), three new lignans (8−10), five new neolignans (11−15), two new 1,3-biphenylpropanoids (16 and 17), and a new flavonol galactoside-lignan ester (18), together with 43 known compounds (19−61), were isolated from the twigs of Cinnamomum cassia. Their structures were elucidated by spectroscopic data analysis as well as chemical methods. The α,β-diphenyl-γ-butyrolactones are a class of unique natural compounds that have only been isolated from C. cassia. Compounds 11 and 12 are rare examples of neolignans possessing a 1,2-dioxetane moiety. Compound 13 is a new oxyneolignan possessing a unique C-9−O−C-9′ linkage between the benzopyran and cinnamyl alcohol moieties. Compound 15 is the first example of a natural neolignan possessing a 2-styryl-3-phenyltetrahydrofuran skeleton. The isolated compounds were evaluated for their neuroprotective activities against tunicamycin-induced cytotoxicity in SH-SY5Y cells. Compounds 3, 5, 10, 11, 12, 20, 36, and 56 showed statistically significant neuroprotective activity with EC50 values ranging between 21 and 75 μM. 43 known (19−61) compounds from the EtOAc-soluble fraction of the EtOH extract of C. cassia.

Cinnamomum cassia Presl. (Lauaceace) is a traditional medicinal plant in southern China and is widely cultivated in many countries of southern and eastern Asia including India, Malaysia, Thailand, Vietnam, and Laos. The dried tender stems of C. cassia are commonly used for the treatment of influenza, diarrhea, arthritis, and gastrointestinal neurosis in traditional Chinese medicine.1 Previous phytochemical studies on the bark and twigs of C. cassia have led to the isolation of several types of bioactive compounds such as flavonoids, proanthocyanidins, cinnamic acids, coumarins, protocatechuic acids, and lignans.2−8 As part of a program to study the chemical diversity of traditional Chinese medicines and their biological effects, an ethanol extract of the twigs of C. cassia was investigated. Herein, we describe the isolation, structure elucidation, and biological assessment of 18 new (1−18) and © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Compound 1 was isolated as a yellow, amorphous powder. Its molecular formula was established as C18H18O6 from the 13C NMR data and an HRESIMS ion at m/z 331.1179 [M + H]+ (calcd for [M + H]+, 331.1176), indicating 10 indices of hydrogen deficiency. The 1H NMR data of 1 (Table 1) revealed two sets of ABX coupled phenyl protons at δH 6.74 (1H, d, J = 1.8 Hz, H-2′), 6.72 (1H, d, J = 8.4 Hz, H-5′), 6.63 (1H, dd, J = 1.8, 8.4 Hz, H-6′), and 6.77 (1H, d, J = 2.4 Hz, H-2″), 6.85 Received: November 2, 2017 Published: June 8, 2018 1333

DOI: 10.1021/acs.jnatprod.7b00924 J. Nat. Prod. 2018, 81, 1333−1342

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Chart 1

Table 1. 1H NMR Spectroscopic Data of 1−7 in Methanol-d4 (δ in ppm, J in Hz) position 3 4 5a 5b 2′ 3′ 5′ 6′ 2″ 5″ 6″ 3′-OCH3 4′-OCH3 3″-OCH3 −OCH2O−

1 3.96 3.78 4.64 4.29 6.74

d (12.0) m brt (8.4) dd (8.4, 10.8) d (1.8)

6.72 6.63 6.77 6.85 6.72 3.78

d (8.4) dd (1.8, 8.4) d (2.4) d (8.4) dd (2.4, 8.4) s

2 3.97 3.70 4.62 4.27 7.10 6.86 6.86 7.10 6.69 6.70 6.60

d (13.2) m brt (8.4) dd (8.4, 10.8) d (8.4) d (8.4) d (8.4) d (8.4) d (2.4) d (8.4) dd (1.8, 8.4)

3.75 s 3.81 s

3 4.06 3.78 4.65 4.29 7.11 6.87 6.87 7.11 6.86 6.72 6.72

d (12.0) m brt (8.4) dd (8.4, 10.8) d (8.4) d (8.4) d (8.4) d (8.4) brs (overlapped) (overlapped)

4 3.92 3.72 4.63 4.27 6.73

d (12.0) m brt (9.0) brt (10.8) (overlapped)

6.72 6.63 6.70 6.72 6.61 3.78

(overlapped) dd (1.8, 8.4) (overlapped) (overlapped) dd (1.8, 8.4) s

3.76 s 3.79 s

5 4.04 3.81 4.64 4.29 7.11 6.85 6.85 7.11 6.87 6.73 6.72

d (13.2) m brt (8.4) dd (9.0, 12.8) d (8.4) d (8.4) d (8.4) d (8.4) brs (overlapped) (overlapped)

6 3.97 3.78 4.64 4.28 7.00 6.73 6.73 7.00 6.86 6.72 6.72

7

d (12.6) m brt (8.4) dd (8.4, 10.2) d (8.4) d (8.4) d (8.4) d (8.4) d (1.2) (overlapped) (overlapped)

3.99 3.83 4.63 4.28 6.75

d (12.0) m dd (7.8, 9.0) dd (9.0, 10.8) d (1.8)

6.72 6.63 6.87 6.73 6.73

d (7.8) dd (1.8, 7.8) brs (overlapped) (overlapped)

3.76 s 5.91 q (1.2)

(1H, d, J = 8.4 Hz, H-5″), 6.72 (1H, dd, J = 2.4, 8.4 Hz, H-6″). Four aliphatic proton signals were evident at δH 4.64 (1H, brt, J

5.91 q (1.2)

5.91 brs

= 8.4 Hz, H-5a), 4.29 (1H, dd, J = 8.4, 10.8 Hz, H-5b), 3.96 (1H, d, J = 12.0 Hz, H-3), and 3.78 (1H, m, H-4), and the 1334

DOI: 10.1021/acs.jnatprod.7b00924 J. Nat. Prod. 2018, 81, 1333−1342

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Table 2. 13C NMR Spectroscopic Data of 1−7 in Methanol-d4 (δ in ppm) position

1

2

3

4

5

6

7

2 3 4 5 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 3′-OCH3 4′-OCH3 3″-OCH3 −OCH2O−

179.7 54.1 51.4 73.3 128.5 113.3 149.1 147.1 116.3 122.4 131.4 115.4 148.6 147.9 112.9 120.0 56.3

179.7 53.8 51.7 73.5 129.2 130.9 115.1 160.6 115.1 130.9 129.8 115.6 146.6 145.9 116.6 119.9

179.7 53.8 52.0 73.4 129.3 130.9 115.1 160.6 115.1 130.9 129.8 112.1 149.3 147.2 116.5 121.2

179.8 54.2 51.4 73.4 128.6 113.3 149.0 147.1 116.3 122.4 130.1 115.6 146.6 145.9 116.6 119.9 56.4

179.4 53.8 52.0 73.3 129.0 130.8 115.1 160.6 115.1 130.8 132.2 108.6 149.6 148.6 109.3 122.4

179.6 53.8 52.0 73.2 127.8 130.9 116.5 158.0 116.5 130.9 132.3 108.6 149.6 148.6 109.3 122.3

179.5 54.1 51.7 73.2 128.3 113.3 149.1 147.2 116.3 122.5 132.4 108.6 149.1 148.6 109.3 122.3

55.7

55.7 56.4

102.5

102.5

56.4

55.7 102.5

Figure 1. Key 1H−1H COSY and HMBC correlations of compounds 1, 9, 11, 13−16, and 18.

protons of two methoxy groups resonated at δH 3.78 (3H, s) and 3.81 (3H, s). The 13C NMR data of 1 (Table 2) showed 18 carbon signals comprising two benzene rings, two methoxy groups, an ester carbonyl group, an oxygenated methylene, and two methines. The 1H−1H COSY correlations of H-3/H-4/H5 as well as HMBC correlations of H-3 and H-5 with C-2 revealed the presence of the γ-butyrolactone ring in 1 (Figure 1). Two aromatic units are located at C-3 and C-4 that were

indicated by the HMBC correlations from H-3 to C-1′, C-2′, and C-6′ and from H-4 to C-1″, C-2″, and C-6″. The HMBC correlations between the methoxy protons (δH 3.78 and 3.81) with C-3′ and C-3″ assigned the linkage positions of two methoxy groups. Furthermore, the coupling constant (J = 12.0 Hz) of H-3 and H-4 suggested that they are trans-oriented. The (3R,4R) absolute configuration was defined via the negative Cotton effects at 210 and 230 nm in the electronic circular 1335

DOI: 10.1021/acs.jnatprod.7b00924 J. Nat. Prod. 2018, 81, 1333−1342

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Table 3. 1H NMR Spectroscopic Data of 8−12 in Methanol-d4 (δ in ppm, J in Hz) position 2 6 7 8 9a 9b 11 2′ 6′ 7′ 8′ 9′a 9′b 3-OCH3 3,5-OCH3 3′-OCH3 3′,5′-OCH3 7′-OCH3

8 6.51 6.49 4.66 3.12 4.24 3.86

d (1.8) d (1.8) d (4.2) m (overlapped) (overlapped) (overlapped)

6.65 6.65 4.70 3.12 4.26 3.86 3.83

s s d (4.8) m (overlapped) (overlapped) (overlapped) s

3.84 s

9 6.58 6.58 4.78 2.37 4.34 4.13 1.93 6.61 6.61 4.30 2.85 4.15 4.09

10

s s d (6.0) m dd (6.0, 10.8) (overlapped) s s s d (6.6) m (overlapped) dd (7.8, 8.4)

6.59 6.59 4.88 2.67 4.56 4.33 2.02 6.61 6.61 4.12 2.80 3.57 3.53

11

s s (overlapped) m dd (4.8, 10.8) dd (8.4, 10.8) s s s d (10.8) m m m

3.83 s

3.83 s

3.84 s 3.20 s

3.85 s 3.14 s

12

6.72 6.72 4.86 4.00 3.72 3.50

s s (overlapped) ddd (2.4, 4.2, 7.8) dd (2.4, 12.6) dd (4.8, 12.6)

6.69 6.69 5.20 4.48 3.57 3.43

s s d (3.0) ddd (2.4, 3.6, 7.8) dd (8.4, 12.6) dd (3.6 12)

6.44 6.41 3.28 5.94 5.07 5.03

d (1.8) d (1.8) (overlapped) m dq (1.8, 17.4) dq (1.2, 10.2)

6.47 6.45 3.29 5.96 5.08 5.04

d (1.8) d (1.8) (overlapped) m dq (1.8, 17.4) dq (1.2, 10.2)

3.86 s 3.85 s

3.80 s 3.85 s

Table 4. 1H NMR Spectroscopic Data of 13−17 in Methanol-d4 (δ in ppm, J in Hz) position 2 3 4 5 6 7 8 9a 9b 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′a 9′b 3-OCH3 2′-OCH3 4′-OCH3 OCH2O

13 7.41 7.30 7.24 7.30 7.41 6.69 6.37 4.53 4.40 7.41 6.99 7.21 6.87

m t (8.4) m t (8.4) (overlapped) d (15.6) dt (6.0, 15.6) ddd (1.2, 5.4, 14.4) ddd (1.2, 6.6, 12.6) (overlapped) t (7.8) m d (7.8)

4.51 d (6.0) 3.83 brt (6.0) 5.16 d (4.8)

14

15

7.43 7.32 7.26 7.32 7.43 6.81 6.25 5.33

d (8.4) t (8.4) m t (8.4) d (8.4) d (15.6) dd (4.8, 15.6) dd (1.2, 4.8)

7.32 7.24 7.18 7.24 7.32 6.53 6.25 5.00

(overlapped) t (7.8) t (7.2) t (7.8) (overlapped) d (15.6) dd (7.2, 15.6) dd (7.2, 11.2)

7.47 7.37 7.25 7.37 7.47 4.43 3.66 4.23 3.65

d (8.4) t (8.4) m t (8.4) d (8.4) d (8.2) (overlapped) dd (2.4, 6.6) (overlapped)

7.41 7.31 7.23 7.31 7.41 3.44 4.10 5.30

d (7.8) t (7.8) t (7.2) t (7.8) d (7.8) dd (4.2, 11.2) d (4.2) s

16

17

7.15 d (1.8)

7.04 d (1.2)

6.79 6.91 4.89 4.21 2.91 2.88

6.80 6.95 4.90 4.19 2.91 2.75

d (7.8) dd (1.8, 7.8) (overlapped) m dd (4.2, 16.8) dd (1.8, 15.6)

d (8.4) dd (1. 2, 8.4) (overlapped) m dd (4.2, 12.8) dd (2.4, 12.8)

6.13 d (2.4)

6.13 brs

6.14 d (2.4)

6.13 brs

3.87 s 3.79 s 3.75 s

3.79 s 3.75 s 5.94 d (4.2)

4′. For compound 3, a methoxy group replaced the hydroxy group located at C-3″ in 2, which was confirmed by the HMBC correlations of the methoxy protons (δH 3.79) with C-3″ (δC 149.3). Compound 4 has similar NMR spectroscopic features to 1, except that the NMR resonances of the 3″-hydroxy-4″methoxyphenyl group in 1 were replaced by those of a 3″,4″dihydroxyphenyl group. The ECD spectra of 2, 3, and 4 (Figures S17, S26, and S35, Supporting Information) are similar to those of 1, indicating that 2, 3, and 4 have the same absolute configuration as 1. From the above data, the structures of 2, 3, and 4 were defined as shown and named cinncassins A2, A3, and A4, respectively. Cinncassins A5 (5), A6 (6), and A7 (7) are also α,β-diphenylγ-butyrolactones. The 1H and 13C NMR data of 5−7 (Tables 1 and 2) showed a methylenedioxy group at δH 5.91 and δC 102.5. The HMBC correlations between the methylenedioxy

dichroism (ECD) spectrum of 1, which are consistent with the reported ECD data of the similar known compound cinncassin A (36).6 Thus, cinncassin A1 (1) was identified as (3R,4R)-3,4di(3-methoxy-4-hydroxyphenyl) dihydrofuran-2-one. Compounds 2, 3, and 4 were obtained as yellow, amorphous powders and showed their protonated molecular ions at m/z 301.1170, 315.1217, and 317.1011 in the HRESIMS data, corresponding to the molecular formulas C17H16O5, C18H18O5, and C17H16O6, respectively. The 1H and 13C NMR data (Tables 1 and 2) are similar to those of 1 except for chemical shift differences due to the variation of substituents in the aromatic rings, indicating that compounds 2, 3, and 4 are also α,βdiphenyl-γ-butyrolactones. The 1H and 13C NMR spectra of 2 revealed the presence of a methoxy group located at C-4′ in 2, based on the HMBC correlations from the methoxy protons (δH 3.75) to C-4′ (δC 160.6) and from H-2′, 6′ (δH 7.10) to C1336

DOI: 10.1021/acs.jnatprod.7b00924 J. Nat. Prod. 2018, 81, 1333−1342

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Table 5. 13C NMR Spectroscopic Data of 8−17 in Methanol-d4 (δ in ppm) position

8

9

10

11

12

13

14

15

16

17

1 2 3 4 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 7′- OCH3 3-OCH3 3,5-OCH3 2′- OCH3 3′- OCH3 3′5′-OCH3 4′- OCH3 OCH2O

133.1 102.5 149.7 134.7 146.4 107.8 87.5 55.4 72.9

134.1 104.3 149.2 136.0 149.2 104.3 85.7 49.3 64.0 172.7 20.8 132.0 105.3 149.4 136.3 149.4 105.3 83.2 48.5 70.8 56.4

132.3 104.1 149.2 135.9 149.2 104.1 85.9 49.5 64.5 172.9 20.9 134.5 105.0 149.5 136.5 149.5 105.0 83.2 49.1 71.1 56.0

128.7 105.9 149.4 137.2 149.4 105.9 77.9 79.9 62.1

128.5 104.9 149.4 136.6 149.4 104.9 77.0 79.0 60.0

138.1 127.6 129.6 128.8 129.6 127.6 134.3 126.2 70.6

137.5 127.8 129.7 129.3 129.7 127.8 134.6 126.3 102.0

138.0 127.5 129.5 128.7 129.5 127.5 133.2 131.1 84.7

132.2 111.9 148.7 147.1 115.7 120.6 80.1 67.4 29.5

134.6 108.6 149.0 148.4 108.7 121.1 80.0 67.3 29.3

133.8 106.2 150.0 132.8 145.6 110.8 40.9 139.0 115.8

134.2 106.7 150.4 131.4 145.1 110.9 40.9 139.0 115.9

125.8 130.1 122.7 130.2 117.7 152.1 70.1 72.6 101.4

140.4 128.8 129.2 129.1 129.2 128.8 85.7 67.6 72.5

136.8 130.9 129.2 127.9 129.2 130.9 54.8 80.5 104.1

102.1 160.6 92.4 161.1 94.6 157.2

102.0 160.6 92.5 161.1 94.6 157.0

56.8

56.8

56.8

56.8

56.7

56.8

133.11 104.4 149.3 136.1 149.3 104.4 87.7 55.5 72.6 56.6

56.8

56.4

56.7

55.9

55.9

55.7

55.7 102.3

56.8

dimethoxyphenyl)methyl]tetrahydrofuran10 except for the presence of a methoxy group at δH 3.20 and 3.14 in 9 and 10, respectively. The methoxy group is linked at C-7′ on the basis of HMBC correlations between the methoxy protons and C-7′ (Figure 1). The resonances for H-7, H-8, and H-9 in 10 are deshielded, while those of H-7′, H-8′, and H-9′ are shielded by comparison to those of 9, which agrees well with those of the 7′R and 7′S isomers of 3-acetoxymethyl-2-(4-hydroxy-3,5dimethoxyphenyl)-4-[hydroxy-(4-hydroxy-3,5dimethoxyphenyl)methyl]tetrahydrofuran.10 The coupling constants of H-7′ in compounds 9 and 10 (J = 6.6 and 10.8 Hz, respectively) also supported that the relative configurations of C-7′ in these compounds are opposite. H-7 was assigned an αorientation on the basis of its coupling constant (J = 6.0 Hz), the same as that of 3-acetoxymethyl-2-(4-hydroxy-3,5-dimethoxyphenyl)-4-[hydroxy-(4-hydroxy-3,5-dimethoxyphenyl)methyl]tetrahydrofuran. The NOESY correlations of H-7 with H-9 and H-8 with H-8′ suggested that H-8 and H-8′ are βoriented. Similar NOESY correlations were also observed in 10, suggesting that compounds 9 and 10 have the same relative configurations at C-7, C-8, and C-8′. The absolute configurations of 9 and 10 were defined as (7R,8S,7′S,8′S)-9 and (7R,8S,7′R,8′S)-10 by comparing their experimental and calculated ECD spectra (Figures S82 and S92, Supporting Information). Cinnacassin I (11) was obtained as a yellow, amorphous solid with a molecular formula of C21H24O7 based on a protonated molecular ion at m/z 389.1589 (calcd for 389.1595) in the HRESIMS data. The 1H NMR data (Table 3) showed two sets of 1,3,4,5-tetrasubstituted aromatic rings at δH 6.72 (2H, s), 6.44 (1H, d, J = 1.8 Hz), and 6.41 (1H, d, J = 1.8 Hz),

protons and C-3″ and C-4″ indicated that the methylenedioxy group is attached to C-3″ and C-4″ in 5−7. Detailed NMR data analysis revealed that 4′-methoxyphenyl, 4′-hydroxyphenyl, and 4′-hydroxy-3′-methoxyphenyl moieties are located at C-3 in 5, 6, and 7, respectively. Compound 7 has the same 2D structure as cinnamomumolide,7 but with different Cotton effects in their ECD spectra. The negative Cotton effects at 205 and 235 nm in the ECD spectrum of 7 (Figure S62, Supporting Information) indicated that 7 has a (3R,4R) absolute configuration, i.e., enantiomeric with cinnamomumolide.7 Compound 8 was obtained as a yellow oil and was assigned a molecular formula of C21H24O8 on the basis of the protonated molecule at m/z 405.1673 (calcd 405.1644) in the HRESIMS data. Its NMR spectroscopic data (Tables 3 and 5) are similar to those of syringaresinol,6 except for the absence of a methoxy group in 8. Three methoxy groups were assigned to C-3, C-3′, and C-5′ on the basis of HMBC correlations of the proton at δH 3.83 with C-3 (δC 149.7) and the proton at δH 3.84 with C3′ and C-5′ (δC 149.3). Based on the oxymethine proton coupling constants [δH 4.66 (J = 4.2 Hz) and δH 4.70 (J = 4.8 Hz)], H-7/H-8 and H-7′/H-8′ were deduced to be in trans orientations. In addition, the ECD spectrum of 8 (Figure S72, Supporting Information) showed a positive Cotton effect at 203 nm and a negative Cotton effect at 195 nm, which were similar to those of (7S,8R,7′S,8′R)-sesamin.9 Therefore, the structure of cinnacassin F (8) was identified as shown. According to the HRESIMS and NMR data (Tables 3 and 5), cinnacassins G (9) and H (10) had similar molecular formulas (C25H32O10) and 2D structures. Their 1H and 13C NMR data are similar to those of 3-acetoxymethyl-2-(4hydroxy-3,5-dimethoxyphenyl)-4-[hydroxy-(4-hydroxy-3,51337

DOI: 10.1021/acs.jnatprod.7b00924 J. Nat. Prod. 2018, 81, 1333−1342

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three methoxy group protons at δH 3.86 (6H, s) and 3.85 (3H, s), three allylic protons at δH 3.28 (1H, H-7′), 5.94 (1H, m, H8′), 5.03 (1H, dq, J = 1.2, 10.2 Hz, H-9′a), and 5.07 (1H, dq, J = 1.8, 17.4 Hz, H-9′b), two hydroxymethyl protons [δH 3.72 (1H, dd, J = 2.4, 12.6 Hz, H-9a) and 3.50 (1H, dd, J = 4.8, 12.6 Hz, H-9b)], and two oxymethine protons at δH 4.86 (H-7) and 4.00 (1H, ddd, J = 2.4, 4.2, 7.8 Hz, H-8). Twenty-one carbon signals were observed in the 13C NMR spectrum of 11, comprising 12 aromatic carbons, three carbons of an allyl group, three oxygenated aliphatic carbons, and three methoxy carbons. By comparison of the molecular formula and the NMR data of 11 with those of mansoxetane,11 the presence of a 1,2peroxy-3-hydroxypropyl moiety was deduced, which was supported by the 1H−1H COSY correlations of H-7 with H-8 and H-8 with H-9. HMBC correlations (Figure 1) between H-7 and C-1, C-2, C-6 and between H-2, H-6, and C-7 indicated that the 1,2-peroxy-3-hydroxypropyl moiety is attached at C-1. The 1H−1H COSY correlations of H-7′/H-8′/H-9′ and HMBC correlations from H-7′ to C-1′, C-2′, and C-6′ and from H-2′ and H-6′ to C-7′ suggested that the allyl group is connected to C-1′. The HMBC correlations from the methoxy group protons to C-3, C-5, and C-3′ indicated that the methoxy groups were attached to C-3, C-5, and C-3′, respectively. Two C6C3 units are linked by a C-4−C-4′ bond based on the HMBC correlations from H-2 and H-6 to C-4 and from H-2′ and H-6′ to C-4′. Analysis of the HRESIMS and NMR data (Tables 3 and 5) showed that cinnacassin J (12) shared a 2D structure with 11. The major NMR differences between the two compounds are the chemical shifts of H-7, H-8, and H-9 due to the different configurations of C-7 and C-8. The experimental ECD spectra of 11 and 12 showed negative and positive Cotton effects at 205 and 230 nm, which were consistent with the calculated ECD spectra of (7R,8S)-11 and (7S,8R)-12, respectively (Figures S101 and S110, Supporting Information). Compound 13 was isolated as a yellow, amorphous solid with a molecular formula of C18H18O4 as established by the HRESIMS data ([M + Na]+, m/z 321.1108, calcd for 321.1097). Detailed NMR data analysis (Tables 4 and 5) revealed the presence of a trans-cinnamyl alcohol moiety in 13, which was suggested by the coupling constants of olefinic protons at δH 6.69 (1H, d, J = 15.6 Hz, H-7) and 6.37 (1H, dd, J = 6.0, 15.6 Hz, H-8), the monosubstituted aromatic ring protons at δH 7.41 (2H, d, H-2,6), 7.30 (2H, t, J = 8.4 Hz, H3,5), and 7.24 (1H, m, H-4), the 1H−1H COSY correlations of H-7/H-8/H-9 and H-2,6/H-3,5/H-4, and the key HMBC correlations from H-7 to C-1, C-2, and C-6 (Figure 1). Furthermore, a 3,4-chromandiol moiety was readily assigned by the 1H−1H COSY correlations of H-7′/H-8′/H-9′ and H-3′/ H-4′/H-5′/H-6′ and the HMBC correlations from H-7′ to C-2′ and C-6′ and from H-9′ to C-2′. Finally, the HMBC correlations of H-9′ with C-9 and H-9 with C-9′ indicated that the C6C3 units were linked via a C-9−O−C-9′ bond. The coupling constants of H-7′ and H-8′ (J = 6.0 Hz) indicated that 7′-OH and 8′-OH are trans oriented. The NOESY cross-peak between H-7′ and H-9′ suggested that H-7′ and H-9′ were cofacial. The (7′R,8′S,9′S) absolute configuration was defined by comparing the experimental and calculated ECD spectra (Figure S120, Supporting Information). Therefore, the structure of cinnacassin K (13) was assigned as shown. Compound 14, a white, amorphous solid, was found to have a molecular formula of C18H18O3, by the HRESIMS ion at m/z 305.1147 [M + Na]+ (calcd for 305.1148). 1H and 13C NMR data of 14 (Tables 4 and 5) showed signals for two

monosubstituted aromatic systems, a trans double bond, an oxygenated methylene, and three oxymethine groups. These NMR data are similar to those of (E)-4-(4-hydroxy-3,5dimethoxyphenyl)-2-styryl-1,3-dioxan-5-ol,12 except that a syringyl group in (E)-4-(4-hydroxy-3,5-dimethoxyphenyl)-2styryl-1,3-dioxan-5-ol was replaced by a phenyl group in 14. Additionally, HMBC correlations (Figure 1) of H-9 with C-7′ and C-9′ supported that C-9 was linked to C-7′ and C-9′ by oxygen bonds to form a 1,3-dioxane unit. Thus, the structure of cinnacassin L (14) was assigned as shown. The coupling constant of H-7′/H-8′ (J = 8.2 Hz) suggested their trans-diaxial relationship, and the NOESY correlation of H-9/H-7′ indicated that H-9 and H-7′ are cofacial. The (9S,7′S,8′R) absolute configuration was defined by a comparison of the experimental and calculated ECD spectra (Figure S130, Supporting Information). Compound 15 was obtained as a colorless oil, and its molecular formula was assigned as C18H18O3 based on the HRESIMS ion at m/z 327.1223 [M + COOH]− (calcd for 327.1238). The similar HRESIMS and NMR data (Tables 4 and 5) of 14 and 15 indicated that 15 is an isomer of 14. The 1 H−1H COSY correlations between H-9 and H-7′ and between H-7′ and H-8′ and HMBC correlations of H-9′ with C-9 and C-7′ (Figure 1) indicated the presence of a tetrahydrofuran unit in 15 instead of the 1,3-dioxane moiety in 14. The styryl and phenyl groups were assigned at C-9 and C-7′ based on HMBC correlations from H-9 to C-7 and C-8 and from H-7′ to C-2′ and C-6′, respectively. According to the coupling constants of H-9/H-7′ (J = 11.2 Hz), H-9/H-7′ were deduced to be trans orientated. Owing to the broad singlet of H-9′ in the 1H NMR spectrum, H-8′ and H-9′ were cis orientated. The NOESY correlations of H-9/H-8′ and H-9/H-9′ indicated that H-9, H8′, and H-9′ were cofacial. Comparing the experimental and calculated ECD spectra, the (9R,7′S,8′S,9′R) absolute configuration was defined (Figure S140, Supporting Information). Thus, the structure of cinnacassin M (15) was assigned as (9R,7′S,8′S,9′R)-(E)-7′-phenyl-9-styryltetradrofuran-8′,9′-diol. Compound 16 was obtained as a yellow oil. Its molecular formula was assigned as C18H20O6 on the basis of the HRESIMS ion at m/z 333.1341 [M + H]+. The 1H and 13C NMR data of 16 (Tables 4 and 5) are similar to those of katsumadin,13 a 1,3-biphenylpropanoid isolated from the seeds of Alpinia katsumadai, except for the variation of substituents in the aromatic moieties. Detailed 1D and 2D NMR data analyses revealed that the structure of 16 comprised a 4-hydroxy-3methoxyphenyl moiety, a 6′-hydroxy-2′,4′-dimethoxyphenyl group, and an oxypropanoid unit. These moieties were connected via the C-1−C-7 and C-9−C-1′ bonds based on the HMBC correlations of H-7/C-1, C-2, C-6 and H-9/C-1′, C-2′, C-6′ (Figure 1). Comparing the experimental and calculated ECD spectra, the (7S,8S) absolute configuration of cinnacassin N (16) was defined (Figure S150, Supporting Information). The 1H and 13C NMR data of 17 (Tables 4 and 5) are similar to those of 16, except that 17 possesses a 3,4methylenedioxyphenyl moiety instead of the 4-hydroxy-3methoxyphenyl moiety in 16. This was confirmed by the observation of the methylenedioxy proton resonances at δH 5.94 and HMBC correlations from the methylenedioxy protons to C-3 and C-4 in 17. The ECD curve of compound 17 was similar to that of 16 (Figure S160, Supporting Information), indicating 17 has the same configuration as 16. Hence, the structure of cinnacassin O (17) was identified as shown. 1338

DOI: 10.1021/acs.jnatprod.7b00924 J. Nat. Prod. 2018, 81, 1333−1342

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Figure 2. Neuroprotective effects of compounds 3, 5, 10, 11, 12, 20, 36, and 56 against tunicamycin (TM)-induced cell death in SH-SY5Y cells. The data (cell viability, measured by MTT assay) are expressed as means ± SEM. Three independent experiments were performed. aP < 0.05. bP < 0.01. P < 0.001. cSalubrinal was used as the positive control at 40 μM.

The α,β-diphenyl-γ-butyrolactones (1−7) are a class of uncommon natural compounds that have only been isolated from C. cassia.4,7,8 These compounds could be used as potential chemotaxonomic markers for C. cassia. Compounds 11 and 12 belong to the biphenylneolignans that possess a 1,2-dioxetane moiety. This type of neolignan is rarely found in nature, and only one analogue has been reported from Mansonia gagei.11 Compound 13 is the first example of an oxyneolignan possessing a unique C-9−O−C-9′ linkage between the benzopyran and cinnamyl alcohol moieties. Compound 15 is the first example of a natural neolignan possessing a 3-phenyl-2styryltetrahydrofuran skeleton. 1,3-Biphenylepoxypropanoids (16 and 17) are also rarely found in nature.13,16 Compound 18 is an unexpected flavonol galactosidic truxinate ester. According to the previous studies, this type of compound often existed as dimers.14,15 This is the first time that a truxinate flavonol galactosidic monoester has been identified from a natural source. By comparing the experimental and reported spectroscopic data, the known compounds were identified as (E)-cinnamyl(E)-cinnamate (19),17 trans-cinnamyl-3-phenylpropionate (20),18 1-(2-phenylcarbonyloxyacetyl)benzene (21),19 1,4diphenyl-1,4-butanedione (22),19 cis-3-phenyl-4-[(E)-styryl]-γbutyrolactone (23),20 trans-3-phenyl-4-[(E)-styryl]-γ-butyrolactone (24),20 (E)-3-phenyl-2-propenic acid-2-phenylethtyl ester (25),21 9,9′-dihydroxy-3,4-methylenedioxy-3′-methoxy[7-O4′,8,5′]neolignan (26),22 balanophonin (27),23 zhebeiresinol (28),24 evofolin B (29),25 5-methoxybalanophonin (30),26 (7R,8S)-ficusal (31),27 hierochin B (32),28 threo-(7R,8R)guaiacylglycerol-8-canillin ether (33),29 herpetal (34),30 salvinal (35), 31 cinncassin A (36), 4 cinnamomulactone (37), 8 (+)-(7S,8R,8’R)-5,5′-dimethoxylariciresinol (38),32 (7S,8R)3,3′,5-trimethoxy-4′,7-epoxy-8,5′-neolignan-4,9,9′-triol (39),33 dehydrodiconiferyl alcohol (40),34 (7′S,8S,8′R)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy-7′,9-epoxylignan-9′-ol-7-one (41),35 (7S,8R)-dihydrodehydroconiferyl alcohol (42),36 (7S,8R)-3,3′-diethoxy-4,7,9,9′-tetrahydroxy-8-O-4′-neolignan

Compound 18 has a molecular formula of C40H36O16 based on the sodium adduct ion at m/z 795.1906 [M + Na]+ in the HRESIMS data. The UV absorption maxima at 265 and 355 nm were characteristic of a flavonol skeleton. In the 1H NMR spectrum, proton signals at δH 6.23 (1H, d, J = 1.2 Hz), 6.40 (1H, brs), 8.07 (2H, d, J = 9.0 Hz), and 6.86 (2H, d, J = 9.0 Hz) were typical for a kaempferol moiety. The remaining four aromatic proton signals resonating as AA′BB′ spin systems [δH 6.45/6.51 (each 2H, d, J = 8.4 Hz); 6.56/6.63 (each 2H, d, J = 8.4 Hz)] were consistent with the protons of two phydroxyphenyl moieties. An anomeric proton at δH 5.25 (1H, d, J = 7.8 Hz), along with four oxygenated methines and an oxygenated methylene, suggested that 18 contained a sugar moiety. Four methine protons at δH 3.94 (1H, dd, J = 7.2, 9.6 Hz, H-7a), 3.77 (1H, dd, J = 6.0, 11.2 Hz, H-7b), 3.55 (1H, overlapped, H-8a), and 3.49 (1H, dd, J = 6.6, 11.2 Hz, H-8b) were also observed. The 1H−1H COSY data showed these four methines in the sequence of H-7a−H-7b−H-8b−H-8a−H-7a (Figure 1), indicating the presence of a cyclobutane moiety. These data were similar to those of potentilin A, a diflavonol ester of μ-truxinic acid.14 The most obvious difference in the NMR data between these compounds was that 18 has a methoxy group instead of a trifolin moiety of potentilin A. The HMBC correlations (Figure 1) between the methoxy protons (δH 3.58) and the ester carbonyl carbon (δC 175.2) suggested that the methoxy group was linked to C-9b. The NOESY correlations of H-2a(6a)/H-8a and H-2b(6b)/H-8b established a trans-geometry between H-7a and H-8a and between H-7b and H-8b. The NOESY correlations of H-7a/H-8b and H-7b/ H-8a supported a δ-truxinate arrangement of the cyclobutane ring in compound 18.15 Acid hydrolysis of 18 yielded a sugar moiety, which was identified as D-galactose by HPLC analysis. The anomeric proton of D-galactose was identified as having a β-orientation from the coupling constant of 7.8 Hz. Therefore, the structure of cinnamomoside A (18) was identified as δtruxinic acid 9a-(6′)-trifolin-9b-methyl diester. 1339

DOI: 10.1021/acs.jnatprod.7b00924 J. Nat. Prod. 2018, 81, 1333−1342

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(43),36 (7R,8R)-3,3′-diethoxy-4,7,9,9′-tetrahydroxy-8-O-4′-neolignan (44),36 simulanol (45),37 erythro-1,2-bis(4-hydroxy-3methoxyphenyl)-1,3-propanediol (46),38 threo-1,2-bis(4-hydroxy-3-methoxyophenyl)-1,3-propanediol (47),38 threo-1-(4hydroxy-3,5-dimethoxyphenyl)-2-(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (48),39 1-(4-hydroxy-3-methoxyphenyl)-2[3-(3-hydroxy-1-propenyl)-5-methoxyphenoxy]-1,3-propanediol (49),40 5,9-dimethoxyguaiacylglycerol (50),41 gualacylglycerol-β-O-4′-synapyl ether (51),42 erythro-guaiacylglycerol-β-O4′-dehydrodisinapyl ether (52),43 acemiko (53),44 erythrosyringylglycerol-β-O-4′-sinapyl ether (54),45 hedyotisol A (55),46 rosin (56),47 cinnacasside A (57),48 cinnacasside C (58),48 cinnacasside E (59),48 erythro-buddlenol B (60),49 and buddlenol A (61),50 respectively. It has been reported that lignans from different plant sources showed neuroprotective activities and may be useful in the treatment and prevention of neurodegenerative diseases.51−53 In the present study, the isolated compounds were tested for their neuroprotective effects against tunicamycin-induced cell death in SH-SY5Y cells, using salubrinal (40 μM) as a positive control (Figure S171, Supporting Information). As shown in Figure 2, compounds 3, 5, 10, 11, 12, 20, 36, and 56 showed neuroprotective effects in a dose-dependent manner with EC50 values ranging between 21 and 75 μM.

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mg), 44 (2.7 mg), 45 (1.4 mg), 46 (2.1 mg), 47 (3.3 mg), 48 (5.7 mg), 49 (2.2 mg), 50 (1.3 mg), and 51 (4.4 mg). Fr.6 (7.3 g) was separated in a similar manner to Fr.5 to give 52 (3.7 mg), 53 (4.2 mg), 54 (1.5 mg), 55 (3.2 mg), 56 (2.7 mg), 57 (4.2 mg), 58 (1.2 mg), 59 (0.9 mg), 60 (1.0 mg), and 61 (2.0 mg). Cinncassin A1 (1): yellow amorphous powder; [α] 25 D −182 (c 0.6, MeOH); UV (MeOH): λmax (log ε) 205 (4.27), 235 (3.98), 280 (3.78); ECD (MeOH): 207 (Δε − 3.13), 235 (Δε − 0.68), 285 (Δε − 0.30) nm; IR νmax 3388, 2942, 2841, 1765, 1604, 1519, 1450, 1376, 1273, 1026 cm−1; 1H and 13C NMR (methanol-d4), see Table 1 and Table 2; HRESIMS m/z 331.1179 [M + H]+ (calcd for 331.1176). Cinncassin A2 (2): yellow, amorphous powder; [α]25 D −172 (c 1.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.28), 230 (3.97), 280 (3.70); ECD (MeOH) 207 (Δε −1.98), 235 (Δε −1.45), 285 (Δε −0.18) nm; IR νmax 3386, 2961, 2840, 1759, 1609, 1517, 1453, 1380, 1249, 1176, 1021 cm−1; 1H and 13C NMR (methanol-d4), see Table 1 and Table 2; HRESIMS m/z 301.1070 [M + H]+ (calcd for 301.1071). Cinncassin A3 (3): yellow, amorphous powder; [α]25 D −134 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (4.32), 225 (4.11), 280 (4.08); ECD (MeOH) 212 (Δε −1.56), 232 (Δε −3.04), 285 (Δε −0.33) nm; IR νmax 3421, 2925, 2848, 1766, 1608, 1516, 1458, 1378, 1248, 1134, 1025 cm−1; 1H and 13C NMR (methanol-d4), see Table 1 and Table 2; HRESIMS m/z 315.1217 [M + H]+ (calcd for 315.1227). Cinncassin A4 (4): yellow, amorphous powder; [α]25 D −65 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (4.28), 230 (3.98), 280 (4.23); ECD (MeOH) 207 (Δε −2.75), 236 (Δε −0.66), 280 (Δε −0.46) nm; IR νmax 3438, 2842, 2038, 1762, 1609, 1524, 1460, 1391, 1122, 1013 cm−1; 1H and 13C NMR (methanol-d4), see Table 1 and Table 2; HRESIMS m/z 317.1047 [M + H]+ (calcd for 315.1020). Cinncassin A5 (5): yellow, amorphous powder; [α]25 D −195 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 205 (4.33), 225 (4.24), 285 (3.74); ECD (MeOH) 205 (Δε −3.36), 233 (Δε −1.85), 285 (Δε −0.34) nm; IR νmax 3421, 2911, 1772, 1611, 1510, 1447, 1361, 1249, 1151, 1030 cm−1; 1H and 13C NMR (methanol-d4), see Table 1 and Table 2; HRESIMS m/z 313.1079 [M + H]+ (calcd for 313.1071). Cinncassin A6 (6): yellow, amorphous powder; [α]25 D −203 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 205 (4.33), 225 (4.24), 285 (3.74); ECD (MeOH) 205 (Δε −3.29), 233 (Δε −1.14) nm; IR νmax 3390, 2921, 2382, 1760, 1611, 1511, 1445, 1363, 1249, 1155, 1016 cm−1; 1H and 13C NMR (methanol-d4), see Table 1 and Table 2; HRESIMS m/z 299.0909 [M + H]+ (calcd for 299.0914), 321.0729 [M + Na]+ (calcd for 321.0733). Cinncassin A7 (7): yellow, amorphous powder; [α]25 D −193 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 200 (4.02), 235 (4.16), 285 (4.09); ECD (MeOH) 207 (Δε −2.45), 240 (Δε −0.60), 285 (Δε −0.34) nm; IR νmax 3442, 2927, 1768, 1607, 1514, 1446, 1367, 1321, 1251, 1155, 1126, 1031 cm−1; 1H and 13C NMR (methanol-d4), see Table 1 and Table 2; HRESIMS m/z 329.1020 [M + H]+ (calcd for 329.1020). Cinnacassin F (8): yellow oil; [α]25 D +6 (c 1.2, MeOH); UV (MeOH) λmax (log ε) 205 (4.31), 235 (4.32), 275 (3.47); ECD (MeOH) 203 (Δε +1.15) nm; IR νmax 3365, 2944, 2846, 1730, 1612, 1518, 1461, 1431, 1328, 1215, 1112 1058, 1029 cm−1; 1H and 13C NMR (methanol-d4), see Table 3 and Table 5; HRESIMS m/z 405.152 [M + H]+ (calcd for 405.1544). Cinnacassin G (9): yellow oil; [α]25 D −1 (c 0.7, MeOH); UV (MeOH) λmax (log ε) 205 (4.40), 215 (4.38), 235 (4.16), 275 (3.34); ECD (MeOH) 213 (Δε +1.07) nm; IR νmax 3393, 2930, 2851, 1733, 1609, 1515, 1460, 1428, 1370, 1327, 1218, 1115 cm−1; 1H and 13C NMR (methanol-d4), see Table 3 and Table 5; HRESIMS m/z 515.1915 [M + Na]+ (calcd for 515.1888). Cinnacassin H (10): yellow oil; [α]25 D +5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (4.02), 235 (3.60), 275 (3.47); ECD (MeOH) 214 (Δε −1.87), 235 (Δε −1.39) nm; 1H and 13C NMR (methanol-d4), see Table 3 and Table 5; HRESIMS m/z 515.1918 [M + Na]+ (calcd for 515.1888). Cinnacassin I (11): yellow, amorphous solid; [α]25 D −15 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 205 (3.81), 245 (3.54), 275 (3.18); ECD (MeOH) 207 (Δε −2.17) nm; IR νmax 3396, 2947, 2838, 2522, 2371, 1652, 1512, 1458, 1218, 1118, 1026 cm−1; 1H and 13C

EXPERIMETNAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Research Autopol I automatic polarimeter. UV and ECD spectra were recorded on a JASCO high-performance J1500 CD spectrometer. NMR spectra were obtained at 600 MHz for 1 H NMR and 150 MHz for 13C NMR, respectively, on a Bruker Ascend 600 spectrometer. HRESIMS data were acquired on an Agilent Technologies 6230 Accurate Mass Q-ToF UHPLC/MS spectrometer. Plant Material. The dried tender twigs of C. cassia were purchased from Yulin, Guangxi Province, China, in January 2016. Plant identification was verified by one of the authors (G.-Y.Z.). A voucher specimen (CC-201601) was deposited at the State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology. Extraction and Isolation. The dried tender twigs of C. cassia (5.0 kg) were ground to a powder and extracted with 80% EtOH (4 × 10 L) under reflux. After filtration, the filtrate was concentrated in vacuo and partitioned with petroleum ether, EtOAc, and n-BuOH. After removal of the solvent, the EtOAc fraction (102.6 g) was subjected to silica gel column chromatography (CC) eluted with a gradient of increasing acetone (0−100%) in petroleum ether to afford six fractions (Fr.1−Fr.6) on the basis of TLC analysis. Fr.1 (1.5 g) was subjected to CC over RP C18 gel and eluted with MeOH−H2O (30:70 to 100:0) to give six subfractions (Fr.1−1−Fr.1− 6). Fr.1−3, Fr.1−4, and Fr.1−6 were initially subjected to an RP C8 column eluted with MeCN−H2O and further purified by semipreparative HPLC eluted with MeOH−H2O to obtain 3 (3.0 mg), 5 (1.7 mg), 6 (4.4 mg), 7 (5.2 mg), 13 (1.5 mg), 14 (1.2 mg), 15 (2.1 mg), 16 (1.7 mg), 17 (2.0 mg), 19 (1.5 mg), 20 (2.0 mg), 21 (2.5 mg), 22 (1.5 mg), 23 (1.0 mg), 24 (2.5 mg), and 25 (1.5 mg). Fr.4 (7.6 g) was subjected to CC over RP C18 and eluted with MeOH− H2O in a step gradient manner (20:80 to 100:0) to afford seven subfractions. These subfractions were further separated by semipreparative HPLC eluted with MeOH−H2O to give 37 (10.9 mg), 1 (2.7 mg), 2 (5.6 mg), 4 (3.3 mg), 8 (6.2 mg), 9 (3.4 mg), 10 (1.1 mg), 11 (1.3 mg), 12 (0.9 mg), 26 (2.4 mg), 27 (3.0 mg), 28 (1.5 mg), 29 (1.2 mg), 30 (2.2 mg), 31 (3.7 mg), 32 (2.3 mg), 33 (2.1 mg), 34 (1.2 mg), 35 (1.3 mg), 36 (2.8 mg), and 37 (10.9 mg). Fr.5 (13.6 g) was first subjected to RP C18 CC eluted with MeOH−H2O (20:80−100:0, v/v) and further purified by semipreparative HPLC repeatedly eluting with MeCN−H2O and MeOH−H2O to obtain 18 (5.5 mg), 38 (2.0 mg), 39 (1.7 mg), 40 (2.2 mg), 41 (1.5 mg), 42 (2.3 mg), 43 (3.6 1340

DOI: 10.1021/acs.jnatprod.7b00924 J. Nat. Prod. 2018, 81, 1333−1342

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NMR (methanol-d4), see Table 3 and Table 5; HRESIMS m/z 389.1589 [M + H]+ (calcd for 389.1595), 411.1408 [M + Na]+ (calcd for 411.1414). Cinnacassin J (12): yellow, amorphous solid; [α]25 D +9 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 210 (3.75), 245 (3.34), 275 (3.07); ECD (MeOH) 207 (Δε +1.90), 225 (Δε +3.36) nm; IR νmax 3397, 2947, 2838, 2371, 1652, 1512, 1459, 1218, 1118, 1026 cm−1; 1H and 13C NMR (methanol-d4), see Table 3 and Table 5; HRESIMS m/ z 389.1593 [M + H]+ (calcd for 389.1595), 411.1417 [M + Na]+ (calcd for 411.1414). Cinnacassin K (13). yellow, amorphous solid; [α]25 D +9 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 200 (4.13), 245 (4.32); ECD (MeOH) 217 (Δε −0.53), 243 (Δε +0.89) nm; IR νmax 3391, 2929, 1720, 1669, 1596, 1489, 1458, 1374, 1222, 1155, 1117, 1032 cm−1; 1H and 13C NMR (methanol-d4), see Table 4 and Table 5; HRESIMS m/ z 321.1108 [M + Na]+ (calcd for 321.1097). Cinnacassin L (14): white, amorphous solid; [α]25 D −7 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 205 (4.33), 215 (4.25), 245 (4.05); ECD (MeOH) 215 (Δε −2.95), 245 (Δε +0.88) nm; IR νmax 3426, 3060, 3020, 2926, 2956, 1712, 1664, 1603, 1495, 1455, 1395 1294, 1248, 1143, 1070, 1021 cm−1; 1H and 13C NMR (methanol-d4), see Table 4 and Table 5; HRESIMS m/z 305.1147 [M + Na]+ (calcd for 305.1148). Cinnacassin M (15): colorless oil; [α]25 D +14 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 205 (4.25), 255 (3.90); ECD (MeOH) 210 (Δε −1.75), 255 (Δε −0.28) nm; IR νmax 3445, 2925, 1640, 1400, 1245, 1105, 1020 cm−1; 1H and 13C NMR (methanol-d4), see Table 4 and Table 5; HRESIMS m/z 327.1223 [M + COOH]− (calcd for 327.1238). Cinnacassin N (16): yellow oil; [α]25 D −18 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 200 (4.22), 230 (3.95), 280 (3.70); ECD (MeOH) 208 (Δε −1.86), 230 (Δε −0.64) nm; IR νmax 3419, 2926, 2850, 1719, 1615, 1595, 1516, 1458, 1274, 1205, 1148, 1115, 1033 cm−1; 1H and 13C NMR (methanol-d4), see Table 4 and Table 5; HRESIMS m/z 333.1341 [M + H]+ (calcd for 333.1333). Cinnacassin O (17): yellow oil; [α]25 D −61 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 200 (4.33), 235 (3.83), 285 (3.35); ECD (MeOH) 208 (Δε −2.29), 230 (Δε −0.57) nm; IR νmax 3443, 2924, 2854, 1624, 1497, 1456, 1248, 1205, 1145, 1113, 1028 cm−1; 1H and 13 C NMR (methanol-d4), see Table 4 and Table 5; HRESIMS m/z 331.1175 [M + H]+ (calcd for 331.1176). Cinnamomoside A (18): yellow, amorphous powder; [α]25 D +4 (c 1.4, MeOH); UV (MeOH) λmax (log ε) 225 (3.78), 265 (3.48), 355 (3.20); IR νmax 3441, 1644, 1512, 1447, 1364, 1179, 1060, 1021 cm−1; HRESIMS m/z 773.2060 [M + H]+ (calcd for 773.2076), 795.1906 [M + Na]+ (calcd for 795.1896); 1H NMR (methanol-d4, 600 MHz) δH 6.23 (1H, d, J = 1.2 Hz, H-6), 6.40 (1H, brs, H-8), 8.07 (2H, d, J = 9.0 Hz, H-2′,6′), 6.86 (2H, d, J = 9.0 Hz, H-3′,5′), 5.25 (1H, d, J = 7.8 Hz, H-1″), 3.80 (1H, dd, J = 7.8, 9.6 Hz, H-2″), 3.55 (1H, m, H-3″), 3.79 (1H, overlapped, H-4″), 3.75 (1H, dd, J = 3.6, 8.4 Hz, H-5″), 4.23 (1H, dd, J = 8.4, 11.4 Hz, H-6″a), 4.19 (1H, dd, J = 3.6, 11.4 Hz, H6″b), 6.56 (2H, d, J = 8.4 Hz, H-2a, 6a), 6.45 (2H, d, J = 8.4 Hz, H-3a, 5a), 3.94 (1H, dd, J = 7.2, 9.6 Hz, H-7a), 3.55 (1H, overlapped, H-8a), 6.63 (2H, d, J = 8.4 Hz, H-2b, 6b), 6.51 (2H, d, J = 8.4 Hz, H-3b, 5b), 3.77 (1H, dd, J = 6.0, 11.2 Hz, H-7b), 3.49 (1H, dd, J = 6.6, 11.2 Hz, H-8b), 3.58 (3H, s, −OCH3); 13C NMR (methanol-d4, 150 MHz) δC 158.9 (C-2), 135.6 (C-3), 179.6 (C-4), 163.1 (C-5), 100.1 (C-6), 166.2 (C-7), 95.0 (C-8), 158.5 (C-9), 105.7 (C-10), 122.7 (C-1′), 132.4 (C-2′, 6′), 116.2 (C-3′, 5′), 161.6 (C-4′), 104.4 (C-1″), 73.0 (C2″), 75.0 (C-3″), 70.3 (C-4″), 74.6 (C-5″), 65.2 (C-6″), 131.0 (C-1a), 130.1 (C-2a, 6a), 115.5 (C-3a, 5a), 156.7 (C-4a), 45.5 (C-7a), 44.3 (C-8a), 174.5 (C-9a), 131.0 (C-1b), 130.0 (C-2b, 6b), 115.7 (C-3b, 5b), 156.5 (C-4b), 45.8 (C-7b), 44.6 (C-8b), 175.2 (C-9b), 52.5 (−OCH3). Determination of the Absolute Configuration of Galactose in Compound 18. The absolute configuration of the sugar moiety of compound 18 was identified according to the protocol described in a previous report with a slight modification.54 Compound 18 (0.5 mg) was hydrolyzed in the presence of 1 M HCl in a hot water bath at 100 °C for 1 h. The reaction mixture was air-dried, dissolved in pyridine

containing L-cysteine methyl ester hydrochloride (0.5 mg), and incubated at 60 °C for 1 h. The o-tolyl isothiocyanate (0.5 mg) was added to the mixture and heated at 60 °C for 1 h. The reaction mixture was directly analyzed by reversed-phase HPLC [column: Waters Symmetry C18, 4.6 × 250 mm; mobile phase: MeCN−H2O (22:78); flow rate: 1 mL/min; UV detection at 250 nm]. The retention time of the sugar derivative obtained from the acid hydrolysate of compound 18 was 20.85 min. Treated in the same way, standard D-galactose and L-galactose gave peaks at 20.90 and 22.45 min, respectively. Thus, the sugar was identified as D-galactose. Neuroprotective Activity Assay. SH-SY5Y cells were cultured in a 1:1 mixture of DMEM/High Glucose and F12 medium containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and were incubated at 37 °C with 5% CO2. Cells were plated at 4 × 103 per well in a 96-well plate and were incubated for 12 h. Cells were treated with the compounds at concentrations of 25, 50, and 100 μM for 6 h. After incubation, tunicamycin was added and incubated for 48 h. After a 48 h treatment, the supernatant was changed with fresh medium and thiazolyl blue tetrazolium bromide was added at a final concentration of 0.5 mg/mL. After incubation at 37 °C for 4 h, the absorbance was measured at 570 nm with a microplate reader. Data were evaluated for statistical significance with one-way ANOVA followed by least significant difference testing using a computerized statistical package.

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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.7b00924. HRESIMS, IR, UV, CD, 1H and 13C NMR, DEPT, HSQC, HMBC, COSY, and NOESY spectra of compounds 1−18 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(Z.-H. Jiang) E-mail: [email protected]. *(G.-Y. Zhu) E-mail: [email protected]. ORCID

Zhi-Hong Jiang: 0000-0002-7956-2481 Guo-Yuan Zhu: 0000-0002-4355-894X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by a grant from Macao Science and Technology Development Fund (033/2015/A1), Guangxi Science and Technology Bases and Talents Program (Guike AD16380013), and the Ministry of Science and Technology of China (Grant No. 2015DFM30010).

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