Anti-inflammatory and cytotoxic carbazole alkaloids from Murraya kwangsiensis

Phytochemistry 170 (2020) 112186

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Anti-inflammatory and cytotoxic carbazole alkaloids from Murraya kwangsiensis

T

Yuemei Chena, Nankai Caoa, Haining Lva, Kewu Zenga, Jingquan Yuanb, Xiaoyu Guoa, Mingbo Zhaoa, Pengfei Tua, Yong Jianga,∗ a b

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China Guangxi Medicinal Plant Garden, Nanning 530023, China

ARTICLE INFO

ABSTRACT

Keywords: Murraya kwangsiensis (C.C. Huang) C.C. Huang Rutaceae Biscarbazole alkaloids Atropisomers Anti-inflammation Cytotoxicity

Chemical investigation of the traditional Chinese medicine, Murraya kwangsiensis, led to the isolation of 16 undescribed biscarbazole alkaloids, kwangsines A–M, two undescribed natural products, (+/−)-bispyrayafoline C, and 19 known monomeric analogues. ( ± )-Bispyrayafoline C and ( ± )-kwangsines A–C are four pairs of biscarbazole atropisomers, and they were separated by chiral HPLC to obtain the optically pure compounds. The structures of the undescribed compounds were elucidated on the basis of HRESIMS and NMR data analysis. Their absolute configurations were assigned via comparison of the specific rotation, ECD exciton coupling method, as well as comparison of experimental and calculated ECD data. A compound showed significant inhibition on NO production in lipopolysaccharide-stimulated BV-2 microglial cells, and four compounds exhibited moderate cytotoxicities against HepG2 cells, with IC50 values less than 20 μM.

1. Introduction The genus Murraya comprises approximately 14 species and two varieties, mainly distributed in the tropical and subtropical regions. There are nine species and one variety in China (Editorial Committee of Flora of China, 1997). Previous phytochemical investigations of Murraya species indicated the presence of carbazole alkaloids, coumarins, flavonoids, etc. (Liu et al., 2015; Lv et al., 2015a; Zhou et al., 2014). Carbazole alkaloids from Murraya plants were reported to possess novel structures and potent anticancer, antimicrobial, anti-inflammatory, anti-diabetic, analgesic, and antimicrobial activities (Knölker and Reddy, 2002; Nandy et al., 2014; Schmidt et al., 2012). Murraya kwangsiensis (C.C. Huang) C.C. Huang (Rutaceae) is a shrub growing up to 1–2 m high, distributed widely in Guangxi and Yunnan provinces of China (Editorial Committee of Flora of China, 1997). The leaves and stems of this plant have been used as a folk medicine for the treatment of cold, measles, bronchitis, keratitis, injuries, and fractures (Xie et al., 2000; Xie and Liao, 2004). Previously, five carbazole alkaloids were obtained from the leaves and stems of this plant (Xie et al., 2000). As part of an ongoing search for bioactive natural products from the Murraya plants (Liu et al., 2015; Lv et al., 2015a, 2015b, 2016; Zhou et al., 2014; Zeng et al., 2015), the 95% aqueous ethanol extract of the leaves and

stems of M. kwangsiensis was investigated to afford 16 undescribed carbazole alkaloid dimers (2a/2b−4a/4b, 5–14), two undescribed natural products (1a/1b) (Fig. 1), and 19 known monomeric carbazole alkaloids (15–33). The bispyranocarbazole alkaloid atropisomers (1a/1b−4a/4b) were separated by chiral HPLC and their absolute configurations were determined by comparison of the specific rotation, the electronic circular dichroism (ECD) exciton coupling method, and comparison of experimental and calculated ECD data. Herein, we described the isolation and structural characterization of compounds 1–33, as well as their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2 microglial cells and their cytotoxicities on HepG2 cells. 2. Results and discussion 2.1. Structural elucidation Compound 1 was isolated as a white amorphous powder, [α]25D 0 (c 0.1, MeOH). Its HRESIMS showed a pseudomolecular ion at m/z 555.2292 [M – H]− (calcd 555.2284), together with its 13C NMR data, indicating that the molecular formula of 1 was C36H32N2O4, with 22 indices of hydrogen deficiency. Analysis of the 1D and 2D NMR data (Tables 1 and 2) resulted in determination of the planar structure of 1 to

∗ Corresponding author. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Haidian District, Beijing 100191, China. E-mail address: [email protected] (Y. Jiang).

https://doi.org/10.1016/j.phytochem.2019.112186 Received 17 June 2019; Received in revised form 20 October 2019; Accepted 22 October 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Structures of compounds 1–14. Table 1 1 H NMR data of compounds 1–10 (δ in ppm, J in Hz, 500 MHz). Position 1 4 5 6 9 1′ 2′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 4″ 5″ 6″ 7″ 9″ 10″ 1‴ 2‴ 4‴ 5‴ 6‴ 7‴ 9‴ 10‴ CH3-3 CH3-3′ CHO-3′ OH-2 OH-2′ OH-7′ OCH3-1′ CH2-3′ a

1a 7.75, 7.74, 6.62, 9.77,

s d (8.3) d (8.3) br s

2a

3a

7.77, s 7.89, d (8.4) 6.73, d (8.4) 10.46, br s

7.78, 7.75, 6.61, 9.75,

4a s d (8.3) d (8.3) br s

7.75, 7.74, 6.62, 9.73,

5a s d (8.3) d (8.3) br s

7.75, s 7.74, d (8.3) 6.62, d (8.3)

8.40, s 7.74, d (8.4) 6.60, d (8.4)

7.70, s 7.65, d (8.3) 6.76, d (8.3)

7.75, s 7.74, d (8.3) 6.62, d (8.3)

9.77, 6.65, 5.53, 1.37, 1.37,

br s d (9.9) d (9.9) s s

9.73, 6.65, 5.61, 1.39, 1.39,

br s d (9.9) d (9.9) s s

8.48, 6.68, 5.53, 1.36, 1.37,

br s d (9.8) d (9.8) s s

9.73, 6.65, 5.51, 1.37, 1.37,

br s d (9.9) d (9.9) s s

6.65, 5.53, 1.37, 1.37,

d (9.9) d (9.9) s s

6.56, 5.52, 1.36, 1.36,

d (9.9) d (9.9) s s

3.40, 5.08, 1.38, 1.32,

d (7.1) t (7.1) s s

6.69, 5.51, 1.35, 1.67, 2.11, 5.08, 1.53, 1.61, 2.42, 2.42,

d (9.9) d (9.9) d (3.6) m m m s s s s

2.43, s 2.43, s 7.14, s 7.14, s

2.43, s 10.05, s 7.24, s 11.73, s

2.44, s 2.42, s

6a

7a

8a

6.69, 7.71, 7.77, 6.79,

s s d (8.2) d (8.2)

6.68, 7.71, 7.77, 6.78,

s s d (8.3) d (8.3)

6.70, 7.71, 7.76, 6.77,

s s d (8.3) m

6.73, 7.47, 7.90, 7.15, 7.37, 7.41, 8.24, 6.78, 5.47, 1.44, 1.44,

s s d (7.8) t (7.8) t (7.8) d (7.8) s d (9.9) d (9.9) s s

7.30, 7.17, 7.87, 7.94, 7.18, 7.38, 7.37, 7.99, 6.74, 5.45, 1.43, 1.43,

d (8.3) overlapped s d (7.8) overlapped overlapped overlapped br s d (9.9) d (9.9) s s

7.39, 7.22, 7.89, 7.96, 7.18, 7.39, 7.39, 8.04, 6.77, 5.43, 1.38, 1.70, 2.08, 5.05, 1.53, 1.62,

m d (7.8) s d (8.0) t (8.0) overlapped overlapped br s m d (10.0) s m m t (7.7) s s

2.39, s

2.38, s

2.38, s

7.07, s 7.99, s

3.87, s 5.62, s

5.66, s

5.68, s

9a

7.62, 7.65, 6.67, 7.63,

s d (8.3) d (8.3) br s

6.82, 7.60, 7.98, 7.19, 7.39, 7.44, 8.22, 6.34, 5.56, 1.43, 1.43,

s s d (7.5) t (7.5) t (7.5) d (7.5) br s d (9.8) d (9.8) s s

10a

7.62, 7.66, 6.68, 7.59, 7.31, 7.31, 7.99, 7.99, 7.20, 7.39, 7.39,

s d (8.3) d (8.3) s overlapped overlapped overlapped overlapped t (7.5) overlapped overlapped

6.38, 5.58, 1.43, 1.43,

d (9.7) d (9.7) s s

7.61, s 7.64, d (8.3) 6.66, d (8.3) 6.82, 7.60, 7.98, 7.19, 7.39, 7.44, 8.23, 6.37, 5.53, 1.38, 1.67, 2.10, 5.06, 1.54, 1.63,

s s d (7.8) t (7.8) t (7.8) d (7.8) br s d (9.8) d (9.8) s m m t s s

2.42, s

2.41, s

2.42, s

4.86, s

4.80, s

4.84, s

3.90, s 4.46, s

4.45, s

3.91, s 4.46, s

Assignments were based on HSQC and HMBC experiments. Compounds 1–4 were measured in acetone-d6 and 5–10 were measured in CDCl3.

be the same with that of a synthetic bispyranocarbazole, bispyrayafoline C (Brütting et al., 2018), but the stereochemistry of bispyrayafoline C has not yet been reported.

Analysis of the structural characteristics of 1 showed that there is a high steric hindrance at its central biaryl axis, indicating an axial chirality existed. However, its specific rotation approached zero and no 2

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Table 2 13 C NMR data of compounds 1–10 (δ in ppm, 125 MHz). Position

1a

2a

3a

4a

5a

6a

7a

8a

9a

10a

1 2 3 4 4a 4b 5 6 7 8 8a 9a 1′ 2′ 3′ 4′ 4a′ 4b′ 5′ 6′ 7′ 8′ 8a′ 9a′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ 10‴ CH3-3 CH3-3′ CHO-3′ OCH3-1′ CH2-3′

102.6, C 152.7, C 118.1, C 121.7, CH 117.5, C 119.3, C 119.6, CH 109.5, CH 151.2, C 106.0, C 137.5, C 140.7, C 102.6, C 152.7, C 118.1, C 121.7, CH 117.5, C 119.3, C 119.6, CH 109.5, CH 151.2, C 106.0, C 137.5, C 140.7, C 118.8, CH 129.7, CH 76.0, C 27.7, CH3 27.7, CH3

102.6, C 152.6, C 118.2, C 121.6, CH 119.1, C 118.7, C 120.6, CH 111.2, CH 152.6, C 106.8, C 138.6, C 140.3, C 103.8, C 160.0, C 116.8, C 127.1, CH 117.5, C 119.1, C 119.7, CH 109.5, CH 151.2, C 106.0, C 137.6, C 147.5, C 118.1, CH 130.7, CH 76.6, C 27.8, CH3 27.8, CH3

102.7, C 152.4, C 118.2, C 121.6, CH 117.6, C 119.3, C 119.7, CH 109.6, CH 151.2, C 106.1, C 137.6, C 140.3, C 102.9, C 152.3, C 118.0, C 121.6, CH 117.8, C 118.3, C 117.6, CH 109.2, CH 152.9, C 110.0, C 141.4, C 140.1, C 118.9, CH 129.6, CH 76.1, C 27.8, CH3 27.8, CH3

102.6, C 152.7, C 118.1, C 121.7, CH 117.5, C 119.3, C 119.6, CH 109.5, CH 151.2, C 106.0, C 137.6, C 140.7, C 102.6, C 152.7, C 118.1, C 121.7, CH 117.5, C 119.3, C 119.6, CH 109.4, CH 151.4, C 106.0, C 137.6, C 140.7, C 118.8, CH 129.7, CH 76.0, C 27.7, CH3 27.8, CH3

95.7, CH 152.3, C 116.6, C 120.9, CH 117.5, C 118.5, C 119.8, CH 109.7, CH 151.8, C 106.4, C 137.9, C 142.2, C 146.3, C 103.9, CH 129.9, C 110.0, CH 124.7, C 123.4, C 120.9, CH 119.6, CH 126.0, CH 111.1, CH 139.6, C 129.2, C 118.7, CH 129.0, CH 75.0, C 27.1, CH3 27.1, CH3

95.6, CH 152.5, C 116.6, C 120.9, CH 117.5, C 118.5, C 119.8, CH 109.7, CH 151.8, C 106.4, C 137.7, C 142.0, C 111.2, CH 123.7, CH 129.0, C 117.6, CH 124.0, C 123.1, C 120.7, CH 119.8, CH 126.2, CH 110.8, CH 140.0, C 139.0, C 118.7, CH 129.1, CH 75.0, C 27.1, CH3 27.1, CH3

108.8, C 150.3, C 116.8, C 119.2, CH 117.8, C 118.2, C 119.6, CH 109.5, CH 150.6, C 105.0, C 136.3, C 139.2, C 146.3, C 106.9, CH 130.9, C 112.2, CH 124.6, C 123.5, C 120.8, CH 119.6, CH 126.0, CH 111.1, CH 139.6, C 129.0, C 117.0, CH 129.7, CH 75.9, C 27.5, CH3 27.8, CH3

108.8, C 150.2, C 116.7, C 119.1, CH 117.5, C 118.5, C 119.6, CH 109.5, CH 150.6, C 105.0, C 136.3, C 139.1, C 111.2, CH 126.4, CH 130.1, C 119.9, CH 124.0, C 123.1, C 120.6, CH 119.1, CH 126.1, CH 110.8, CH 140.0, C 138.6, C 117.1, CH 129.7, CH 75.9, C 27.6, CH3 27.6, CH3

118.8, CH 129.7, CH 76.0, C 27.7, CH3 27.7, CH3

118.7, CH 129.7, CH 76.1, C 27.7, CH3 27.8, CH3

24.4, CH2 123.6, CH 132.0, C 17.4, CH3 25.5, CH3

108.8, C 150.2, C 116.7, C 119.1, CH 117.8, C 118.1, C 119.6, CH 109.4, CH 150.8, C 104.9, C 136.3, C 139.2, C 146.3, C 106.8, CH 130.9, C 112.2, CH 124.3, C 123.5, C 120.8, CH 119.6, CH 126.0, CH 111.1, CH 139.6, C 129.0, C 117.4, CH 128.8, CH 78.2, C 26.0, CH3 40.9, CH3 22.9, CH2 124.3, CH 131.8, C 17.8, CH3 25.8, CH3

17.4, CH3 17.4, CH3

17.4, CH3

17.4, CH3 17.4, CH3

119.3, CH 128.8, CH 78.4, C 26.1, CH3 41.4, CH2 23.4, CH2 125.1, CH 131.8, C 17.6, CH3 25.8, CH3 17.4, CH3 17.4, CH3

95.6, CH 152.4, C 116.4, C 120.9, CH 117.5, C 118.4, C 119.7, CH 109.7, CH 151.9, C 106.3, C 137.7, C 142.0, C 111.2, CH 123.8, CH 129.0, C 117.6, CH 124.1, C 123.1, C 120.7, CH 119.7, CH 126.2, CH 110.8, CH 140.0, C 139.0, C 119.0, CH 129.3, CH 77.1, C 25.2, CH3 40.2, CH3 22.8, CH2 124.3, CH 131.8, C 17.8, CH3 25.8, CH3

16.2, CH3

16.2, CH3

16.2, CH3

16.8, CH3

16.8, CH3

16.8, CH3

55.7, CH3 50.2, CH2

50.0, CH2

49.8, CH2

55.7, CH3 32.3, CH2

31.6, CH2

55.7, CH3 32.3, CH2

a

196.6, CH

Assignments were based on HSQC and HMBC experiments. Compounds 1–4 were measured in acetone-d6 and 5–10 were measured in CDCl3.

Fig. 2. The chiral HPLC separation (A) and experimental and calculated ECD data (B) of compounds 1a and 1b.

Cotton effects were observed in its ECD spectrum, suggesting 1 as an atropisomer mixture (Bringmann et al., 1995, 2001; Cao et al., 2018). Chiral HPLC resolution of 1 afforded a pair of enantiomers, 1a and 1b, in

a ratio of 1:1, and their opposite specific rotations (1a, −144; 1b, +144) and ECD curves supported their enantiomeric relationship (Fig. 2). The ECD spectrum of 1a exhibited sequential negative and positive Cotton 3

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Fig. 3. UV and ECD spectra and exciton coupling assignment of 1a.

effects at 252 and 218 nm, i.e., a negative couplet derived from the electronic transition of the two carbazole monomers as shown in Fig. 3. Therefore, the absolute configuration of (−)-bispyrayafoline C (1a) was determined as Ra (Harada and Nakanishi, 1972; Lo et al., 2000), and correspondingly, (+)-bispyrayafoline C (1b) was assigned as Sa configuration. Furthermore, the ECD spectra of (Ra)- and (Sa)-1 were calculated using the TDDFT method at the B3LYP/6-311 + G(d) level to confirm the result of ECD exciton coupling (Fig. 2B). During the chiral separation of 1a and 1b, we found that they could interchange in methanol solution at room temperature. After six days, pure compound 1a or 1b converted to a mixture of 1a and 1b in a ratio of 1:1 (Fig. 4), and the interconversion was accelerated at a higher temperature (40 °C, Fig. S2, Supporting Information). Thus, the pure atropisomer could only be obtained below the room temperature. ( ± )-Kwangsine A (2) was isolated as a brown amorphous powder, [α]25D −2 (c 0.1, MeOH). Its molecular formula was determined as C36H30N2O5 via its 13C NMR and HRESIMS data (m/z 569.2083 [M − H]−). The UV spectrum showed typical pyranocarbazole absorptions at λmax 222, 242, 296, and 320 nm (Chokkalingam et al., 2013). Analysis of the 1H and 13C NMR data (Tables 1 and 2) indicated that 2 is an unsymmetrical pyranocarbazole dimer, and its structure is similar to that of 1, except that the methyl at C-3′ in one unit was replaced by a formyl group [δH 10.05 and δC 196.6], which was supported by the HMBC correlations from the formyl proton to C-2′ (δC 160.0) and C-3′ (δC 116.8). Therefore, the structure of kwangsine A (2) was established as shown. Similar to 1, 2 was also a pair of atropisomers. The isolation of its individual conformers (2a and 2b) was accomplished by a chiral HPLC separation, and the pure enantiomers were obtained in a ratio of 1:1. Their configurations were determined as Ra for 2a and Sa for 2b by comparison of their ECD (Fig. S8, Supporting Information) and specific rotation data with those of 1a and 1b. ( ± )-Kwangsine B (3) was obtained as a brown amorphous powder, [α]25D −4 (c 0.1, MeOH). Its molecular formula was assigned as C36H34N2O4 from the 13C NMR and HRESIMS data (m/z 557.2429 [M − H]−, calcd for C36H33N2O4, 557.2440). Analysis of the NMR data of 3 indicated that it is an unsymmetrical dimeric carbazole, consisting of a pyrayafoline C unit and an isomurrayafoline B analogue unit (Ito et al., 1987; Rehman et al., 2014). Compared with isomurrayafoline B, the C-7′ methoxy group (δH 3.90, 3H, s) was replaced by a hydroxy group (δH

7.99, 1H, s) in 3, which was deduced to be located at C-7′ from the HMBC correlations of the hydroxy proton with C-6′ (δC 109.2), C-7′ (δC 152.9), and C-8′ (δC 110.0). The absence of H-1 and H-1′ signals indicated that these two units were linked through C-1−C-1′. The optical inactivity of 3 indicated that it is also a pair of atropisomer mixture in a ratio of 1:1. After chiral separation and ECD and specific rotation determination, the absolute configurations of 3a and 3b were determined as Ra and Sa, respectively (Fig. S14, Supporting Information), by comparison of their ECD and specific rotation data with those of 1a and 1b. ( ± )-Kwangsine C (4) was obtained as a brown amorphous powder, [α]25D −4 (c 0.1, MeOH). Its molecular formula, C41H40N2O4, was deduced from the HRESIMS (m/z 623.2902 [M − H]−, calcd for C41H39N2O4, 623.2910) and 13C NMR spectroscopic data. The NMR data of 4 indicated that it was also a dimeric carbazole consisting of a pyrayafoline C unit and a pyrayafoline D unit (Hesse et al., 2014; Ito et al., 1991), and these two units were linked through C-1−C-1′, the same linkage as that of 1−3. The lack of optical activity and Cotton effect indicated that 4 is also a pair of atropisomers. Chiral HPLC separation of 4 gave two enantiomers, 4a and 4b in a ratio of 1:1, which have opposite ECD curves and specific rotations (Fig. S20, Supporting Information). The absolute configurations of 4a and 4b were established as Ra and Sa, respectively, by comparison of their ECD and specific rotation data with those of 1a and 1b. The experimental ECD spectra of 4a/4b are almost similar to those of 1a/1b. In addition, the calculated ECD spectrum of 3‴R-(Ra)-4 is also similar to that of 3‴S(Ra)-4. These observations suggested that the configuration at C-3‴ does not contribute much to the ECD curves of compounds 4a and 4b, which leads to the inability to determine the configuration at C-3‴. Kwangsine D (5) was obtained as a brown amorphous powder. Its molecular formula was determined as C32H28N2O3 by the HRESIMS (m/ z 487.2024 [M − H]−, calcd for C32H27N2O3, 487.2022) and 13C NMR spectroscopic data. The NMR data (Tables 1 and 2) indicated 5 as a dimeric carbazole alkaloid consisting of a pyrayafoline C and a murrayafoline A units (Ding et al., 2014). Comparison of the 1H NMR data of 5 with those of murrayafoline A, the methyl group at δH 2.53 (3H, s) in murrayafoline A was replaced by a methylene group at δH 5.62 (2H, s) in 5. The two units were deduced to be linked via an N–C-3′-methyl linkage from the HMBC correlations of the methylene protons with C-8a (δC 137.9), C-9a (δC 142.2), C-2′ (δC 103.9), C-3′ (δC 129.9), and C-4′ 4

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Fig. 4. The chiral HPLC analysis of the interconversion of compounds 1a and 1b.

(δC 110.0) (Fig. 6). Therefore, the structure of kwangsine D (5) was elucidated as shown. Kwangsine E (6) was obtained as a brown amorphous powder. Its molecular formula was assigned as C31H26N2O2 on the basis of the 13C NMR and negative-ion HRESIMS data [m/z 457.1910 [M − H]− (calcd for C31H25N2O2, 457.1916)]. Its 1D and 2D NMR data were similar to those of 5, except for the absence of a methoxy singlet, and the replacement of an aromatic singlet in 5 by two aromatic signals in 6 [δH 7.30 (1H, d, J = 8.3 Hz, H-1′) and 7.17 (1H, overlapped, H-2′)]. Further 2D NMR analysis suggested that 6 was a demethoxy derivative of compound 5, thus, the structure of kwangsine E (6) was defined as shown. Kwangsine F (7) was obtained as a brown amorphous powder, [α]25D‒7 (c 0.1, MeOH). Its negative-ion HRESIMS data showed a deprotonated molecular ion at m/z 525.2560 [M − H]− (calcd for C36H33N2O2, 525.2542), which in conjunction with the 13C NMR data assigned 7 a molecular formula of C36H34N2O2. Interpretation of the NMR data revealed that the structure of 7 was similar to that of 6, except for the presence of an additional prenyl unit in 7 as evidenced by the resonances at δH 1.70 (2H, m, H-5″), 2.08 (2H, m, H-6″), 5.05 (1H, t, J = 7.7 Hz, H7″), 1.53 (3H, s, H-9″), and 1.62 (3H, s, H-10″). The HMBC correlations of H-5′′ (δH 1.70) with C-7′′ (δC 124.3), and H-4′′ (δH 1.38) with C-2′′ (δC 129.3), C-3′′ (δC 77.1), and C-5′′ (δC 40.2) suggested that the prenyl unit is linked at C-5′′. The absolute configuration of 7 was assigned as (3″S) from its similar ECD curve and optical rotation sign with those of 17 (Fig. 5; 7, [α]25D −7; 17, [α]25D −16), one of the biosynthetic precursor units of 7. Therefore, the structure of kwangsine F (7) was deduced as shown. Kwangsine G (8) was obtained as a brown amorphous powder and showed a deprotonated molecular ion [M – H]− at m/z 487.2021 (calcd 487.2022) in the HRESIMS, corresponding to a molecular formula of C32H28N2O3. Analysis of NMR data of 8 (Tables 1 and 2) showed a close structural resemblance to 5. The main difference between these two compounds was that the linkage pattern between the two carbazole units changed from the N–C-3′-methyl linkage in 5 to the C-1−C-3′-methyl linkage in 8, which was supported by the HMBC correlations of the methylene protons at δH 4.46 (2H, s) with C-1 (δC 108.8), C-2 (δC 150.3), C-9a (δC 139.2), C-2′ (δC 106.9), C-3′ (δC 130.9), and C-4′ (δC 112.2) (Fig. 6). From these data, the structure of kwangsine G (8) was characterized as shown.

Kwangsine H (9) was obtained as a brown amorphous powder. Its molecular formula was defined as C31H26N2O2 based on the 13C NMR and HRESIMS (m/z 457.1910 [M – H]−, calcd for C31H25N2O2, 457.1916) data. The NMR data of 9 resembled those of 8, except for the absence of a methoxy group and an aromatic singlet [δH 6.82] in 8 by two aromatic signals in 9 [δH 7.31 (2H, overlapped)]. Further 2D NMR analysis confirmed that 9 is a demethoxy derivative of 8 as shown. The molecular formula of 10 was determined as C37H36N2O3 based on the 13C NMR and negative-ion HRESIMS data (m/z 555.2646 [M − H]−, calcd for C37H35N2O3, 555.2648). Comparing the NMR data of 10 with those of 8 revealed their structural similarity, and the only difference is the presence of an additional prenyl group in 10. In combination with the relevant HMBC correlation analysis, the prenyl group was deduced to be attached at C-5′′. A (3″S) configuration for 10 was deduced from its similar ECD curves (Fig. 5B) and optical rotation sign with those of 7 (10, [α]25D −4; 7, [α]25D −7). Thus, the structure of kwangsine I (10) was established as depicted. Kwangsine J (11) was obtained as a brown amorphous powder. Its molecular formula was assigned as C36H32N2O4 via the 13C NMR spectroscopic data and the observed deprotonated molecular ion at m/z 555.2268 [M − H]− (calcd for C36H31N2O4, 555.2284) in the negativeion HRESIMS. The NMR spectroscopic data suggested 11 to be an unsymmetrical carbazole dimer consisting of two pyrayafoline C units (Hesse et al., 2014; Ito et al., 1991). These two units were linked via a C-1−C-3′-methyl pattern deduced from the HMBC correlations of the CH2-3′ protons [δH 4.40 (2H, s)] with C-1 (δC 111.3), C-2 (δC 151.6), C9a (δC 140.2), C-2′ (δC 152.8), C-3′ (δC 120.5), and C-4′ (δC 121.4). Therefore, the structure of kwangsine J (11) was elucidated as shown. Kwangsine K (12) gave a molecular formula of C32H28N2O3 determined from its 13C NMR and HRESIMS data (m/z 487.2023 [M − H]−, calcd for C32H27N2O3, 487.2022). Its 1H and 13C NMR data were similar to those of 5 owing to the same constituent units, pyrayafoline C and murrayafoline A moieties (Ding et al., 2014). However, the linkage pattern of the two units changed from N–C-3′-methyl linkage in 5 to C6−C-3′-methyl linkage in 12, deduced from the HMBC correlations of the CH2-3′ protons [δH 4.23 (2H, s)] with C-5 (δC 120.3), C-6 (δC 122.7), C-7 (δC 148.5), C-2′ (δC 108.0), C-3′ (δC 134.0), and C-4′ (δC 113.2), and 5

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Fig. 5. Calculated and experimental ECD spectra of compound 17 (A) and experimental ECD spectra of 7 and 10 (B).

of H-5 [δH 7.56 (1H, s)], H-2′ [δH 6.87 (1H, s)], and H-4′ [δH 7.63 (1H, s)] with the methylene carbon (Fig. 6). Thus, the structure of kwangsine K (12) was identified as depicted. Kwangsine L (13) was obtained as a brown amorphous powder with a molecular formula of C31H26N2O2, as deduced from the 13C NMR and HRESIMS data (m/z 457.1906 [M − H]−, calcd for C31H25N2O2, 457.1916). The NMR data (Tables 3 and 4) of 13 resembled those of 12, with the exception that one methoxy group was absent in 13, and the replacement of an aromatic singlet in 12 by two aromatic doublets at δH 7.38 (1H, d, J = 8.2 Hz, H-1′) and 7.39 (1H, d, J = 8.2 Hz, H-2′). These data revealed that 13 is a demethoxy derivative of 12. In combination with the HMBC correlations, the structure of kwangsine L (13) was characterized as shown. Kwangsine M (14) was obtained as a brown amorphous powder. Its molecular formula, C36H32N2O4, was deduced from the HRESIMS (m/z 555.2296 [M - H]−, calcd for C36H31N2O4, 555.2284) and 13C NMR spectroscopic data. Its NMR data were similar to those of 11, with the exception that the linkage pattern between the two carbazole units changed from the C-1−C-3′-methyl linkage in 11 to a C-6−C-3′-methyl linkage in 14, which was supported by the corresponding HMBC correlations. Therefore, the structure of kwangsine M (14) was defined as shown. By comparing the spectroscopic data with literature, the remaining 19 known compounds (Fig. S1, Supporting Information) were identified as pyrayaquinone B (15) (Furukawa et al., 1985), pyrayafoline C (16) (Hesse et al., 2014; Ito et al., 1991), pyrayafoline D (17) (Ito et al., 1991), 3-formylcarbazole (18) (Jiang et al., 2013), murrayanine (19) (Bhosale et al., 2012), O-demethylmurrayanine (20) (Bringmann et al., 1998), euchrestine-A (21) (Ito et al., 1991), euchrestine-C (22) (Ito et al., 1991), 2-hydroxy-3-methylcarbazole (23) (Dhara et al., 2015), 1hydroxy-3-methyl-9H-carbazole (24) (Lin and Zhang, 2000), 3-hydroxymethyl-9H-carbazole (25) (Ito et al., 1992), 3-(methoxymethyl)carbazole (26) (Yan et al., 2001), 1-methoxy-3-(methoxymethyl)carbazole (27) (Yan et al., 2001), claulansine Q (28) (Du et al., 2015), claulansine R (29) (Du et al., 2015), 3-carboxylicacid carbazole (30) (Humne et al., 2014), clausine E (31) (Tohyama et al., 2009), 3-methyl-9H-carbazole (32) (Alt and Plietker, 2016), and murrayafoline A (33) (Ding et al., 2014). The 3′R configuration of 17 was determined for the first time via

Table 3 1 H NMR data of compounds 11–14 (δ in ppm, J in Hz, 500 MHz). Position 1 4 5 6 9 1′ 2′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 4″ 5″ 1‴ 2‴ 4‴ 5‴ CH3-3 CH3-3′ OH-2 OH-2′ OCH3-1′ CH2-3′

11a 7.56, s 7.65, d (8.2) 6.57, d (8.2) 7.02, s 7.93, s 7.51, d (8.3) 6.51, d (8.3) 10.07, br s 6.97, d (9.8) 5.75, d (9.8) 1.43, s 1.43, s 6.82, d (9.8) 5.72, d (9.8) 1.40, s 1.40, s 2.37, s

4.40, s

12a

13a

14a

6.55, s 7.55, s 7.56, s

6.89, s 7.61, s 7.68, s

6.87, s 7.54, s 7.67, s

6.87, 7.63, 8.01, 7.19, 7.38, 7.41, 8.18, 6.45, 5.61, 1.37, 1.37,

9.90, br s 7.38, d (8.2) 7.39, d (8.2) 8.08, s 8.06, d (7.5) 7.12, t (7.5) 7.33, t (7.5) 7.46, d (7.5) 10.15, br s 6.86, d (9.8) 5.72, d (9.8) 1.35, s 1.35, s

9.90, br s 6.93, s

s s d (7.5) t (7.5) t (7.5) d (7.5) s d (9.6) d (9.6) s s

2.31, s

3.92, s 4.23, s

2.30, s

7.77, s 7.59, d (8.3) 6.54, d (8.3) 10.02, br s 6.87, d (9.8) 5.75, d (9.8) 1.45, s 1.45, s 6.87, d (9.8) 5.73, d (9.8) 1.41, s 1.41, s 2.28, s 8.05, br s 8.05, br s

4.20, s

4.16, s

a

Assignments were based on HSQC and HMBC experiments. Compounds 11, 13, and 14 were measured in acetone-d6, and 12 was measured in CDCl3.

comparison of the calculated and experimental ECD data (Fig. 5A). 2.2. Bioactivities All the isolated compounds (1–33) were evaluated for their inhibitory effects on NO production in the LPS-stimulated BV-2 microglial

Fig. 6. Key HMBC correlations of compounds 5, 8, and 12. 6

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Table 4 13 C NMR Data of Compounds 11–14 (δ in ppm, 125 MHz). Position

11a

12a

13a

14a

1 2 3 4 4a 4b 5 6 7 8 8a 9a 1′ 2′ 3′ 4′ 4a′ 4b′ 5′ 6′ 7′ 8′ 8a′ 9a′ 1″ 2″ 3‴ 4″ 5″ 1‴ 2‴ 3‴ 4‴ 5‴ CH3-3 CH3-3′ OCH3-1′ CH2-3′

111.3, C 151.6, C 118.4, C 119.1, CH 117.7, C 119.3, C 119.9, CH 109.5, CH 151.2, C 105.8, C 137.6, C 140.2, C 97.7, CH 152.8, C 120.5, C 121.4, CH 118.4, C 118.5, C 119.9, CH 109.4, CH 151.4, C 105.7, C 137.6, C 140.9, C 118.7, CH 129.9, CH 76.2, C 27.8, CH3 27.8, CH3 118.3, CH 130.1, CH 76.3, C 27.8, CH3 27.8, CH3 17.4, CH3

96.9, CH 152.0, C 116.3, C 121.1, CH 117.9, C 117.2, C 120.3, CH 122.7, C 148.5, C 104.9, C 135.0, C 139.2, C 145.4, C 108.0, CH 134.0, C 113.2, CH 123.9, C 124.2, C 120.6, CH 119.2, CH 125.5, CH 111.0, CH 139.5, C 128.3, C 117.4, CH 129.7, CH 75.9, C 27.5, CH3 27.5, CH3

97.3, CH 154.5, C 117.4, C 121.4, CH 117.6, C 118.2, C 120.7, CH 122.9, C 148.9, C 105.7, C 136.2, C 140.9, C 111.2, CH 128.0, CH 133.8, C 121.1, CH 123.9, C 124.1, C 120.7, CH 119.4, CH 126.1, CH 111.6, CH 141.3, C 139.4, C 118.8, CH 129.9, CH 76.3, C 27.7, CH3 27.7, CH3

16.2, CH3

16.7, CH3

97.3, CH 154.5, C 117.3, C 121.3, CH 117.5, C 118.3, C 121.0, CH 121.9, C 148.6, C 105.5, C 136.1, C 140.9, C 97.6, CH 154.4, C 121.9, C 121.8, CH 117.5, C 119.0, C 119.7, CH 109.3, CH 151.1, C 105.7, C 137.5, C 141.0, C 118.5, CH 129.8, CH 76.7, C 27.8,CH3 27.8, CH3 119.7, CH 130.0, CH 76.2, C 27.8, CH3 27.8, CH3 16.6, CH3

55.6, CH3 36.8, CH2

37.0, CH2

31.1, CH2

25.5, CH2

Table 5 Inhibitory effects of compounds from M. kwangsiensis on LPS-activated NO production in BV-2 microglial cells. Compounda

IC50 (μM)b

18 19 20 27 Dexamethasonec

78.2 12.2 79.2 65.1 14.5

± ± ± ± ±

2.6 0.2 2.1 1.7 0.31

a

Other compounds were inactive (< 50% inhibition at 80 μM). b Values are represented as means ± SD based on three independent experiments. c Positive control. Table 6 Cytotoxicities of compounds isolated from M. kwangsiensis in HepG2 cells. Compounda

IC50 (μM)b

Compounda

IC50 (μM)b

1 3 5 6 8 9 11

9.9 ± 0.7 44.3 ± 2.6 42.4 ± 1.2 15.8 ± 2.2 33.6 ± 2.3 25.4 ± 0.1 44.1 ± 1.4

12 13 14 22 24 33 Taxolc

28.2 ± 0.3 21.0 ± 2.6 15.5 ± 1.1 26.6 ± 1.2 22.9 ± 0.5 16.7 ± 2.8 0.032 ± 0.014

a

Other compounds are inactive (< 50% inhibition at 50 μM). Values are represented as means ± SD based on three independent experiments. c Positive control. b

UV–visible spectrophotometer (Shimadzu Co., Tokyo, Japan). IR spectra were recorded with a Thermo Nicolet Nexus 470 FT-IR spectrometer (MA, USA). NMR measurements were performed on a Varian INOVA-500 NMR spectrometer (Varian Co., USA), using CDCl3 or acetone-d6 as solvent, and the chemical shifts were referenced to the solvent residual peak. The HRMS data were acquired on a Waters Xevo G2 Q-TOF mass spectrometer fitted with an ESI source (Waters Co., Milford, MA, USA). CC was performed on silica gel (200–300 mesh, Qingdao Marine Chemical, Co., Ltd., China), Sephadex LH-20 (Pharmacia) and ODS (50 μm, YMC, Japan), respectively. Thin layer chromatography (TLC) was carried out on glass precoated silica gel GF254 plates (Qingdao Marine Chemical Co., Ltd., China). Semipreparative RP-HPLC was performed on an Agilent 1260 system with a Zorbax Eclipse XDB-C18 (9.4 mm × 250 mm, 5 μm) column. Chiral separations were performed on a Chiralpak AD-H column (4.6 × 250 mm, 5 μm, Daicel, China).

a Assignments were based on HSQC and HMBC experiments. Compounds 11, 13, and 14 were measured in acetone-d6, and 12 was measured in CDCl3.

cells and cytotoxic activities on HepG2 cells. As shown in Table 5, compounds 18–20 and 27 showed inhibitory activities against NO production with IC50 values of 78.2, 12.2, 79.2, and 65.1 μM, respectively. Compounds 1, 3, 5, 6, 8, 9, 11–14, 22, 24, and 33 exhibited cytotoxicities against HepG2 cells with IC50 values of 9.9−44.3 μM (Table 6). 3. Conclusions

4.2. Plant material

In summary, 16 previously undescribed biscarbazole (2a/2b−4a/ 4b, 5–14), two undescribed natural bispyranocarbazoles (1a/1b), and 19 known monomeric carbazole (15–33) alkaloids were isolated. Compounds 1–4 are four pairs of biscarbazole atropisomers, and their chiral resolution, configuration determination, and stability were systematically reported here. Compound 19 showed significant inhibition on NO production in lipopolysaccharide-stimulated BV-2 microglial cells, and compounds 1, 6, 14, and 33 exhibited moderate cytotoxicities against HepG2 cells, all with IC50 values less than 20 μM. All these above data supply a proof for the traditional use and reference for the future research and development of M. kwangsiensis.

The leaves and stems of Murraya kwangsiensis (C.C. Huang) C.C. Huang (Rutaceae) were collected in April 2013 from Guangxi Medicinal Plant Garden, Guangxi Province, China (GPS coordinates: 22°52′ N, 108°09′ E). The plant was identified by Prof. Peng-Fei Tu, and a voucher specimen (no. MK201304) has been deposited at the Herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine. 4.3. Extraction and isolation Air-dried and finely powdered leaves and stems of M. kwangsiensis (2.2 kg) were extracted three times with 95% aqueous EtOH (20 L × 2 h). The filtrate was evaporated under reduced pressure to yield a dark brown residue (150 g). The residue was suspended in water (1 L) and then partitioned with CHCl3 (1 L × 3), EtOAc (1 L × 3), and n-BuOH (1 L × 3), successively. After removing the solvents, the CHCl3 extract (80 g) was subjected to silica gel CC, eluting with petroleum etheracetone by gradually increasing the polarity of the elution solvents to

4. Experimental section 4.1. General experimental procedures Optical rotations were obtained on a Rudolph Autopol IV automatic polarimeter. CD spectra were measured on a J-815 spectropolarimeter (JASCO, Japan). UV spectra were recorded on a Shimadzu UV-2450 7

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1706, 1623, 1118, 1034, 899, 579 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 569.2083 [M − H]− (calcd for C36H29N2O5, 569.2076). (−)-Kwangsine A (2a): Brown amorphous powder; [α]25D −20 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 214 (+10.11), 240 (−5.66), 280 (−2.56) nm. (+)-Kwangsine A (2b): Brown amorphous powder; [α]25D +20 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 214 (−9.13), 242 (+6.68), 284 (+3.78) nm.

afford six fractions A−F. Fraction B (4.7 g) was chromatographed over Sephadex LH-20 eluting with CH2Cl2–MeOH (1:1) to give five subfractions (B1–B5). Compounds 15 (5.1 mg, tR 14.8 min), 26 (5.5 mg, tR 7.8 min), 27 (6.2 mg, tR 8.8 min), 28 (4.1 mg, tR 10.3 min), 29 (4.5 mg, tR 11.6 min), 32 (5.6 mg, tR 12.7 min), and 33 (6.5 mg, tR 14.2 min) were obtained from the subfraction B5 by semipreparative RP-HPLC using a mobile phase of MeCN–H2O (80:20, v/v, 3 mL/min). Purification of the fraction C (5.6 g) was performed on Sephadex LH-20 CC and eluted with CH2Cl2–MeOH (1:1) to give six subfractions (C1–C6). The carbazolecontaining fraction C5 was purified by MPLC (50–100% MeOH–H2O) and semipreparative RP-HPLC (detection at 238 nm, 3 mL/min), successively, to yield 1 (6.5 mg, tR 11.3 min) and 4 (2.6 mg, tR 9.5 min) using a mobile phase of MeCN–H2O (65:35, v/v) and MeCN–H2O (90:10, v/v), respectively. Fraction D (10.4 g) was chromatographed over Sephadex LH-20 eluting with CH2Cl2–MeOH (1:1) to afford five subfractions (D1−D5). D3 was further purified by semipreparative RP-HPLC using a mobile phase of MeCN–H2O (65:35, v/v, 3 mL/min) to afford 2 (1.1 mg, tR 30.6 min). Compounds 5 (4.2 mg, tR 33.7 min), 7 (1.2 mg, tR 35.4 min), and 10 (1.0 mg, tR 39.5 min) were purified from subfraction D4 by semipreparative RP-HPLC using a mobile phase of MeCN–H2O (65:35, v/ v, 3 mL/min). Subfraction D5 was separated by MCI (50–100% MeOH–H2O) to afford six fractions (D5a−D5f). Fraction D5a was further purified by semipreparative RP-HPLC using a mobile phase of MeCN–H2O (60:40, v/v, 3 mL/min) to afford 20 (2.5 mg, tR 4.8 min), 21 (2.1 mg, tR 6.0 min), 23 (1.1 mg, tR 6.5 min), 24 (1.3 mg, tR 7.3 min), and 25 (1.0 mg, tR 4.4 min). Fractions D5c was purified by semipreparative RP-HPLC to yield 16 (1.5 g, tR 11.6 min), 18 (8.2 mg, tR 6.2 min), 19 (6.5 mg, tR 5.8 min), and 22 (3.2 mg, tR 15.0 min), using a mobile phase of MeCN–H2O (60:40, v/v, 3 mL/min). Separation of the subfraction D5e by semipreparative RP-HPLC (isocratic 70% MeCN in H2O, 3 mL/min) yielded 3 (4.2 mg, tR 14.1 min) and 17 (10.8 mg, tR 20.5 min). Compounds 6 (4.7 mg, tR 29.7 min), 8 (10.3 mg, tR 27.0 min), and 9 (3.1 mg, tR 24.4 min) were obtained from subfraction D5f by semipreparative RP-HPLC using MeCN–H2O (80:20, v/v) as mobile phase. Fraction E was separated by Sephadex LH-20 eluting with CH2Cl2–MeOH (1:1) to give six subfractions (E1−E6). Subfraction E6 was further purified by semipreparative RP-HPLC using a mobile phase of MeCN–H2O (80:20, v/v, 3 mL/min) to afford compounds 11 (6.1 mg, tR 20.2 min), 12 (8.7 mg, tR 16.3 min), 13 (6.3 mg, tR 14.8 min), and 14 (6.6 mg, tR 12.9 min). Compounds 30 (2.0 mg, tR 5.2 min) and 31 (2.2 mg, tR 6.1 min) were isolated from fraction E5 by semipreparative RP-HPLC using MeCN–H2O (50:50, v/v, 3 mL/min). Chiral separations of 1, 2, 3, and 4 were performed on semipreparative NP-HPLC eluting with nhexane-iPrOH in a ratio of 85:15 (v/v), 75:25 (v/v), 75:25 (v/v), and 95:5 (v/v), respectively. The detection wavelength was 238 nm and the f1ow rate was 1 mL/min. Finally, compounds 1a (3.1 mg, tR 4.8 min), 1b (3.1 mg, tR 6.4 min), 2a (0.5 mg, tR 5.8 min), 2b (0.2 mg, tR 7.3 min), 3a (1.6 mg, tR 4.9 min), 3b (1.7 mg, tR 6.3 min), 4a (0.9 mg, tR 9.0 min), and 4b (1.2 mg, tR 11.9 min) were yielded, respectively.

4.3.3. ( ± )-Kwangsine B (3) Brown amorphous powder; UV (MeOH) λmax (log ε) 222 (4.37), 235 (4.39), 296 (4.16), 321 (3.86), 333 (3.76) nm; IR (KBr) νmax 3423, 2964, 2917, 2850, 1611, 1449, 1418, 1259, 1186, 1156, 1135, 1116, 1042, 803, 580, 437 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 557.2429 [M − H]− (calcd for C36H33N2O4, 557.2440). (−)-Kwangsine B (3a): Brown amorphous powder; [α]25D −100 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 216 (+8.87), 248 (−6.95), 296 (−1.37) nm. (+)-Kwangsine B (3b): Brown amorphous powder; [α]25D +100 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 214 (−8.23), 250 (+6.39), 294 (+1.07) nm. 4.3.4. ( ± )-Kwangsine C (4) Brown amorphous powder; UV (MeOH) λmax (log ε) 224 (4.86), 238.8 (4.83), 296.6 (4.80), 334 (4.26) nm; IR (KBr) νmax 3525, 3459, 3363, 2969, 2923, 2855, 1706, 1622, 1450, 1420, 1186, 1047, 580 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 623.2902 [M − H]− (calcd for C41H39N2O4, 623.2910). (−)-Kwangsine C (4a): Brown amorphous powder; [α]25D −80 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 218 (+18.61), 252 (−12.80), 296 (−4.78) nm. (+)-Kwangsine C (4b): Brown amorphous powder; [α]25D +80 (c 0.01, MeOH); ECD (MeOH) λmax (Δε) 218 (−19.45), 254 (+12.11), 298 (+4.63) nm. 4.3.5. Kwangsine D (5) Brown amorphous powder; UV (MeOH) λmax (log ε) 226 (4.76), 242 (4.81), 293 (4.56), 325 (4.13) nm; IR (KBr) νmax 3625, 3405, 2967, 2921, 2852, 1700, 1617, 1585, 1499, 1449, 1420, 1229, 1118, 1037, 581 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 487.2024 [M − H]− (calcd for C32H27N2O3, 487.2022). 4.3.6. Kwangsine E (6) Brown amorphous powder; UV (MeOH) λmax (log ε) 237 (4.77), 258 (4.53), 297 (4.65), 318 (3.92), 327 (3.96) nm; IR (KBr) νmax 3403, 2970, 2919, 2851, 1626, 1590, 1156, 1119, 579, 478 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 457.1910 [M - H]− (calcd for C31H25N2O2, 457.1916).

4.3.1. ( ± )-Bispyrayafoline C (1) Brown amorphous powder; UV (MeOH) λmax (log ε) 222 (3.85), 242 (3.86), 296 (3.73), 320 (3.73) nm; IR (KBr) νmax 3433, 2967, 2917, 2850, 1638, 1622, 1449, 1418, 1262, 1187, 1117, 1046, 898, 580 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 555.2292 [M − H]− (calcd for C36H31N2O4, 555.2284). (−)-Bispyrayafoline C (1a): White amorphous powder; [α]25D −144 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 218 (+15.79), 252 (−9.99), 296 (−3.74) nm. (+)-Bispyrayafoline C (1b): White amorphous powder; [α]25D +144 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 218 (−13.68), 252 (+8.30), 296 (+3.59) nm.

4.3.7. Kwangsine F (7) Brown amorphous powder; [α]25D −7 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 218 (−0.58), 262 (+0.31) nm; UV (MeOH) λmax (log ε) 237 (3.95), 297 (3.74), 323 (3.31) nm; IR (KBr) νmax 3417, 2919, 2850, 1624, 1155, 1039, 901, 579 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 525.2560 [M − H]− (calcd for C36H33N2O2, 525.2542). 4.3.8. Kwangsine G (8) Brown amorphous powder; UV (MeOH) λmax (log ε) 225 (4.65), 241 (4.66), 293 (4.53), 329 (3.91), 339 (3.91) nm; IR (KBr) νmax 3421, 2967, 2918, 2850, 1629, 1586, 1425, 1188, 1117, 1048, 581 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 487.2021 [M − H]− (calcd for C32H27N2O3, 487.2022).

4.3.2. ( ± )-Kwangsine A (2) Brown amorphous powder; UV (MeOH) λmax (log ε) 222 (3.85), 242 (3.86), 296 (3.73), 320 (3.73) nm; IR (KBr) νmax 3410, 2918, 2848, 8

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4.3.9. Kwangsine H (9) Brown amorphous powder; UV (MeOH) λmax (log ε) 236 (4.51), 259 (4.24), 293 (4.38), 330 (3.69), 341 (3.69) nm; IR (KBr) νmax 3452, 2850, 1637, 1446, 1238, 1186, 1164, 1115, 1046, 805 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 457.1910 [M − H]− (calcd for C31H25N2O2, 457.1916).

4.5. NO inhibitory assay The murine BV-2 microglial cells were purchased from Peking Union Medical College (PUMC) Cell Bank (Beijing, China). Cell maintenance, experimental procedures, and data determination for the inhibition of NO production and the viability assay were the same as previously described (Zeng et al., 2013). Dexamethasone was used as a positive control.

4.3.10. Kwangsine I (10) Brown amorphous powder; [α]25D −4 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 218 (−0.40), 251 (+0.42) nm; UV (MeOH) λmax (log ε) 241 (4.65), 293 (4.29), 320 (4.05) nm; IR (KBr) νmax 3434, 2992, 2851, 1623, 1451, 1175, 1036, 896, 579 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative-ion HRESIMS m/z 555.2646 [M − H]− (calcd for C37H35N2O3, 555.2648).

4.6. Cytotoxicity assay HepG2 cells (PUMC Cell Bank, Beijing, China) were used for the cytotoxicity assays. Cytotoxic activities were determined using the MTT method. Cell culture, experimental procedures, and data processing were performed according to the literature report (Ma et al., 2014), with taxol serving as a positive control.

4.3.11. Kwangsine J (11) Brown amorphous powder; UV (MeOH) λmax (log ε) 226 (4.44), 238 (4.47), 294 (4.48), 332 (3.84) nm; IR (KBr) νmax 3549, 3472, 3410, 2918, 1621, 1450, 1420, 1161, 1043, 582 cm−1; 1H and 13C NMR data, see Tables 3 and 4; negative-ion HRESIMS m/z 555.2268 [M − H]− (calcd for C36H31N2O4, 555.2284).

Declaration of competing interest The authors declare no competing financial interest. Acknowledgments This work was financially supported by National Natural Science Foundation of China (NSFC; Nos. 81973199 and 81773864), and The Drug Innovation Major Project of China (No. 2018ZX09711001-008003).

4.3.12. Kwangsine K (12) Brown amorphous powder; UV (MeOH) λmax (log ε) 223 (5.10), 242 (5.16), 294 (4.91), 343 (4.50) nm; IR (KBr) νmax 3432, 2920, 1629, 1149, 1125, 1035, 580 cm−1; 1H and 13C NMR data, see Tables 3 and 4; negative-ion HRESIMS m/z 487.2023 [M − H]− (calcd for C32H27N2O3, 487.2022).

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112186.

4.3.13. Kwangsine L (13) Brown amorphous powder; UV (MeOH) λmax (log ε) 237 (4.40), 297 (4.26), 332 (3.65), 343 (3.65) nm; IR (KBr) νmax 3391, 2914, 1628, 1606, 1147, 1125, 1038, 924, 872 cm−1; 1H and 13C NMR data, see Tables 3 and 4; negative-ion HRESIMS m/z 457.1906 [M − H]− (calcd for C31H25N2O2, 457.1916).

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