New cytotoxic dimeric and trimeric coumarins from Chimonanthus salicifolius

Phytochemistry Letters 16 (2016) 115–120

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New cytotoxic dimeric and trimeric coumarins from Chimonanthus salicifolius Kui-Wu Wanga,* , Dan Lia , Bin Wub , Xiao-Ji Caoc a b c

School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, PR China Ocean College, Zhejiang University, Hangzhou 310058, PR China Research Center of Analysis and Measurement, Zhejiang University of Technology, Hangzhou 310014, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 September 2015 Received in revised form 23 March 2016 Accepted 24 March 2016 Available online xxx

Two new dicoumarins named chimsalicifoliusins A (1) and B (2), a new tricoumarin, chimsalicifoliusin C (3), and nine known coumarin constituents have been isolated from Chimonanthus salicifolius. Their structures were identified by 1D, 2D NMR spectra and HR-ESI–MS. Compounds 1–3 showed modest cytotoxicity against Hela and HL-60 cell lines, with IC50 values ranging from 14.2 to 29.6 mM, while only chimsalicifoliusin C (3) had the cytotoxicity against PC-3 cell line. This study represents the first report of tricoumarin, C

C linked dicoumarins and coumarinolignoids from Calycanthaceae. ã 2016 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.

Keywords: Chimonanthus salicifolius Calycanthaceae Coumarin Phytochemical Cytotoxicity

1. Introduction Calycanthaceae included two genera Calycanthus and Chimonanthus (Ye et al., 2006; Fan et al., 2010; Dai et al., 2012). The genus Chimonanthus, comprises only six species, is widely cultivated in China. The chemical components of Chimonanthus praecox, Chimonanthus nitens and Chimonanthus salicifolius are much investigated. Essential oils, alkaloids and aromatic compounds are main classes of secondary metabolites (Kitajima et al., 2006; Ueyama et al., 1990; Liu and Liu, 2008; Lv et al., 2012; Takayama et al., 2004; Zhang et al., 2009, 2013; Wang et al., 2011a, 2011b; Ma et al., 2015). C. salicifolius Hu, a species endemic to China, has been used as Chinese traditional medicine to treat cough, vomiting, rheumatic arthritis, heatstroke, and measles (Ye et al., 2006; Ueyama et al., 1990; Zhang and Liu, 1998). But the chemical constituents of this plant were seldom reported before (Wang et al., 2011a, 2011b; Zhang et al., 2013). As part of serial research work on novel and bio-active compounds from the Chinese medicinal plants, we investigated the methanol extract of C. salicifolius, resulted in the isolation of twelve coumarin constituents including two new dicoumarins, named chimsalicifoliusins A (1), B (2) and a new tricoumarin, chimsalicifoliusin C (3). The structures of nine known compounds were identified as hymenain

* Corresponding author. E-mail address: [email protected] (K.-W. Wang).

(4) (Simoes et al., 2009), 3,30 -biisofraxidin (5) (Xu et al., 2008), cleomiscosin B (6) (Ray et al., 1985; Liu et al., 2007), cleomiscosin A (7) (Yun et al., 2001), cleomiscosin C (8) (Cottiglia et al., 2002), scoparone (9) (Xu et al., 2008), sopoletin (10) (Xu et al., 2008), isofraxidin (11) (Takemoto et al., 1975; Xu et al., 2008), fraxetin (12) (Xu et al., 2008) (Fig. 1). 2. Results and discussion Chimsalicifoliusin A (1) was obtained as yellowish needles. It gave ion peak at 413 [M+H]+ in the positive mode ESI–MS. The molecular formula C21H16O9 was tentatively determined by negative HR-ESI–MS (m/z 411.0726 [C21H16O9-H]
, calcd. 411.0722). The 1H NMR (Table 1) spectrum of 1 showed three methoxy groups at dH 3.80, 3.86, 3.88, and six aromatic proton signals. The doublets proton signals at dH 6.42 (1H, d, J = 9.6 Hz) and 8.04 (1H, d, J = 9.6 Hz) were characteristic of H-30 and H-40 of coumarin, respectively (Simoes et al., 2009; Xu et al., 2008; Yun et al., 2001). Based on 1H–1H-COSY and HMBC correlations (Fig. 2), the other four singlets at dH 6.98, 7.27, 7.48 and 7.60 were assigned to H-5, H-80 , H-50 , and H-4, respectively. There were 21 carbons in the 13C NMR spectrum including three methoxy groups, six aromatic CH and twelve quaternary carbon atoms. These information indicated that compound 1 is a diconmarin with three methoxy substituted groups. One coumarin moiety was determined as 7-hydroxy-6,8-dimethoxy-3-O-substituted, and the other coumarin unit was assigned to be 60 -methoxy-70 -O-

http://dx.doi.org/10.1016/j.phytol.2016.03.009 1874-3900/ ã 2016 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.

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5' H3CO

4'

6'

5'

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3'

4' 10'

6'

3'

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Fig. 1. The chemical structures of new compounds 1–12 from C. Salicifolius.

O

K.-W. Wang et al. / Phytochemistry Letters 16 (2016) 115–120 Table 1 NMR spectral data of compounds of 1–3 (DMSO-d6, 500 MHz for 1H, 125 MHz for 13 C). Position

2 3 4 5 6 7 8 9 10 OCH3-6 OCH3-8 OH-7 OH-6, 60 20 30 40 50 60 70 80 90 100 OCH3-8, 80 200 300 400 500 600 700 800 900 1000 OCH3-600 OH-700

Compound 1

Compound 2

Compound 3

dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

– – 7.60 (s) 6.98 (s) – – – – – 3.80 (s) 3.86 (s) 9.78 (s) – – 6.42 (d, 9.6) 8.04 (d, 9.6) 7.48 (s) – – 7.27 (s) – – 3.88 (s)

156.3 137.2 127.6 104.0 146.0 142.7 134.7 140.4 110.0 56.1 60.7 – – 160.1 114.6 143.9 110.8 146.4 147.9 106.2 148.4 1146 56.2

– 6.23 (d, 9.4) 8.01 (d, 9.4) 7.35 (s) – – – – – 3.92 (s) – – 9.83 (br, s) – 6.23 (d, 9.4) 8.01 (d, 9.4) 7.35 (s) – – – – – 3.92 (s)

160.6 111.4 144.9 108.7 149.5 107.7 144.9 147.8 110.2 56.1 – – – 160.6 111.4 144.9 108.7 149.5 107.7 144.9 147.8 110.2 56.1

– – 7.56 (s) 7.24 (s) – – – – – 3.76 3.91 – – – 6.48 (d, 9.6) 8.08 (d, 9.6) 7.56 (s) – – 7.40 – – 3.88 (s) – – 7.06 (s) 7.10 (s) – – 6.82 (s) – – 3.73 (s) 10.02 (s)

156.0 141.0 124.5 105.4 149.4 137.2 140.4 139.4 117.4 56.8 62.2 – – 160.5 115.7 144.4 111.6 147.3 147.0 108.2 148.8 116.1 56.8 156.8 140.1 119.4 109.3 146.1 149. 103.1 145.8 110.6 56.3 –

substituted based on analysis of the 1D, 2D NMR data of compound 1 and comparison of its 13C NMR data with those of scopoletin and isofraxidin, repectively (Xu et al., 2008). The two coumarin moieties were connected at C-3 and C-70 via an oxygen bridge, which was supported by the lowfield-shifted of C-3 (dC 137.2) (Simoes et al., 2009) and further confirmed by HMBC and ESI–MS/ MS experiments (Fig. 2 and 3) of 1. The HMBC correlations from OH (dH 9.78) to C-6 (dC 146.0) and C-8 (dH 134.7) defined that the OH group substitutes at C-7, so the two coumarin moieties couldn’t linked at C-7 and C-70 via an oxygen bridge. The positive ESI–MS (Fig. 3) showed fragment ion at m/z 221 [M+H-C10H8O4]+ from the protonated ion of m/z 413 [M+H]+ in the MS2 experiment. Therefore, compound 1 was elucidated as 7-hydroxy-6,8-dimethoxy-3-(60 -methoxy-70 - coumarinyloxy)coumarin, a new dicoumarin, and named chimsalicifoliusin A. Chimsalicifoliusin B (2) was isolated as yellowish amorphous powder, and suggested as a dicoumarin from the molecular formula of C20H14O8 by HR-ESI–MS (m/z 381.0610, calcd. for [C20H14O8-H]
, 381.0610). In the 1H NMR (Table 1) spectrum, only one singlet methoxy proton resonance at dH 3.92 (6H, OCH3-6, 60 ), two doublet aromatic proton resonances at dH 6.23 (2H, d, J = 9.4 Hz, H-3, 30 ), 8.01 (2H, d, J = 9.4 Hz, H-4, 40 ), and a singlet aromatic proton resonance at dH 7.35 (2H, s, H-5, 50 ) were observed. The 13C NMR (Table 1) spectrum of 2 showed ten carbon signals, which were assigned by DEPT experiments as a methoxy, three aromatic methines, and six quaternary carbons. These spectroscopic data suggested that 2 has a symmetric structure with 6(60 ), 7 (70 ), 8(80 )-trisubstituted coumarin moieties. Compared the 13C NMR data of 2 with those of isofraxidin (11) (Takemoto et al., 1975; Xu et al., 2008), the upfield-shifted of C-7(7') and lowfield-shifted of C-6(60 ), C-8(80 ) indicated that the two symmetric coumarin

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moieties was connected at 7-70 through a C

C bond. Thus, compound 2 was identified as 7,70 -di-(6- hydroxy-8-methoxycoumarin) and named chimsalicifoliusin B. This is the first report C

C linked dicoumarin from Calycanthaceae family to our best knowledge. Chimsalicifoliusin C (3) was also isolated as yellowish amorphous powder and determined to have the molecular formula of C31H22O13 by its negative mode HR-ESI–MS (m/z 601.0979, calcd. for [C31H22O13-H]
, 601.0982). In the 1H NMR (Table 1) spectrum, four singlet methoxy proton resonances at dH 3.73 (3H, OCH3-6”), 3.76 (3H, OCH3-6), 3.88 (3H, OCH3-60 ), 3.91 (3H, OCH3-8), two doublet aromatic proton resonances at dH 6.48 (1H, d, J = 9.6 Hz, H30 ), 8.08 (1H, d, J = 9.6 Hz, H-40 ), and seven singlet aromatic proton resonances at dH 6.82 (1H, H-800 ), 7.06 (1H, H-400 ), 7.10 (1H, H-500 ), 7.24 (1H, H-5), 7.40 (1H, H-80 ), 7.56 (2H, H-4, 50 ) were observed. The 13 C NMR (Table 1) spectrum of 3 showed the presence of thirty-one carbon signals, which were assigned by DEPT experiment as four methoxys, nine aromatic methines, and eighteen quaternary carbons (fifteen aromatic and three lactone carbon atoms). Apart from the carbon signals due to four methoxy groups (dC 56.3, 56.8, 56.8 and 62.2), there remain 24 aromatic carbons including three lactone carbons in the 13C NMR spectrum, suggesting that 3 possesses a tricoumarin skeleton. Only two doublet aromatic proton resonances at dH 6.48, 8.08 and the chemical shift of three lactone carbons at dC 156.0, 156.8 and 160.5 indicated that 3 has two 3-O-substituted coumarin moieties (Simoes et al., 2009; Liu et al., 2007). The positive ESI–MS (Fig. 3) showed prominent fragments at m/z 397 [M + H-C10H6O5]+, 411 [M + H-C10H8O4]+ from the protonated ion of m/z 603 [M + H]+ in the MS2 experiment. In MS3 experiments, the mother ions at m/z 397 and 411 can loss the same fragment of 204, produce the fragment ion of m/z 193 and 207. These MS spectral data indicated the link of the three coumarin moieties as shown in Fig. 1. This skeleton was confirmed by the HMBC spetrum of 3 (Fig. 2). Thus, compound 3 was identified as 6,8-dimethoxy-3-(60 -methoxy-70 -coumarinyloxy)-7(600 -methoxy-700 -hydroxy-300 -coumarinyloxy)coumarin, the first tricoumarin isolated from Calycanthaceae family, and named chimsalicifoliusin C. Coumarins have variety of biological activities such as antimicrobial, anti-cancer, anti-inflammatory, and anti-HIV (Ito et al., 2005; Song et al., 2010; Wang et al., 2011a, 2011b; Gao et al., 2014, 2015; Emami and Dadashpour, 2015; Lv et al., 2015; Sribuhom et al., 2015). Therefore, the new isolates (1–3) were evaluated for their inhibitory effects on Hela (human cervical squamous carcinoma), PC-3 (human prostate cancer), and HL-60 (human promyelocytic leukemia) human tumor cell lines using the MTT assay (Su et al., 2015). The results (Table 2) demonstrated that the three new compounds had modest cytotoxicity against Hela and HL-60 cell lines, with IC50 values ranging from 14.2 to 29.6 mM. However, only the new tricoumarin, chimsalicifoliusin C (3) had the cytotoxicity against PC-3 cell line (IC50 28.6 mM). Twelve coumarin constituents including three new natural compounds (1–3) have been isolated and identified from C. salicifolius. This study represents the first report of tricoumarin, C

C linked dicoumarins and coumarinolignoids from Calycanthaceae, suggesting that there is an unique biosynthetic route in C. salicifolius. Furthermore, this is the first report of the cytotoxic effects on Hela, HL-60 and PC-3 human tumor cells of the di- and tricoumarin components of C. salicifolius. 3. Experimental 3.1. General experimental procedures Melting points were measured on a BUCHI M565 instrument. Mass spectra were performed on a LCQ FLEET (Thermo scientific,

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H

H

H

H3CO H

H

H3CO

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3 Fig. 2. Key HMBC correlations of compounds 1–3.

San Jose, CA, USA) mass spectrometer and accurate masses were measured on Agilent 6210 TOF-MS spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an ESI source in the positive ion mode. The NMR spectra were recorded on a Bruker AVANCE III 500 MHz spectrometer, using TMS as internal standard. Column chromatography was performed with silica gel (100– 200 mesh, 200–300 mesh, 300–400 mesh, Qingdao Haiyang Co., Ltd.), polyamide (100–200 mesh, Sinopharm Group Co., Ltd.). Semi-preparative HPLC was performed on Waters 600 series, with a SunFire Prep C18 (250  19 mm 10 mm) column. TLC was performed on precoated plates (GF254, 0.2 mm, Qingdao Haiyang Co., Ltd.), and compounds were viewed under a UV lamp and sprayed with 10% H2SO4, followed by heating to 110  C. 3.2. Plant material The aerial parts of C. salicifolius were collected in Sep. 2011, from Jinfoshan, Chongqing Province, PR China and was authenticated by Prof. Hong-Xiang Sun (Zhejiang University). A voucher specimen (No. Zjgsu-20110901) was deposited in the School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, PR China.

3.3. Extraction and isolation The aerial parts of C. salicifolius (12 kg) were shade dried, powdered and extracted with methanol at room temperature for 3 times. The resulting methanol extract was evaporated to dryness under reduced pressure, affording gummy residue (880 g). Then this residue was partitioned in H2O and extracted at room temperature with petroleum ether (PE), EtOAc (EA) and BuOH, successively. The EA fraction (150 g) was subjected to column chromatography on a silica gel column (100–200 mesh, 150  12 cm) and eluted with a step wise gradient of CHCl3CH3OH (9:1, 7:1, 5:1, 3.5:1, 2:1, 1:1, 1:3.5, 1:5, 1:7, 1:9 by volume) to afford a total of 40 fractions. Column Fractions were analyzed by TLC (silica gel GF254), and fractions with similar TLC patterns were combined to give 7 major fractions (EA-Fr. 1–EA-Fr. 7). EA-Fr. 2 was rechromatographed on a silica gel column (100–200 mesh) with an isocratic elution using solvent system (hexane: ethylacetate, 3:1, by volume) to give pale yellow solids, which were identified as scoparone (9, 46 mg). EA-Fr. 3 was purified by repeated flash chromatography on silica gel (100–200 mesh) by eluting with PE/ EA (1:1) to yield the compounds scopoletin (10, 37 mg), isofraxidin (11, 60 mg), respectively. EA-Fr. 4 was chromatographed on a silica

K.-W. Wang et al. / Phytochemistry Letters 16 (2016) 115–120

119

H+ H3CO

m/z 221 O

H3CO

HO

O

O

O

O

OCH3

1 m/z 411

H+ H3CO

m/z 207 H3CO

H3CO

O

O

O

O

O

m/z 193

O

OCH3 HO

O

m/z 397

O

3 Fig. 3. Proposed fragmentation pathways and characteristic ions of compounds 1 and 3.

gel column (100–200 mesh, 70  5 cm) and, eluted with a step wise gradient of PE/EA (from 1:2 to 1:5, by volume) to give fraxetin (12, 34 mg) and 3,30 -biisofraxidin (5, 15 mg). EA-Fr. 5 was purified by polyamide column chromatography (100–200 mesh, CH3CH2OH/ H2O, 1:7–1:9) to give cleomiscosin B (6, 9 mg), cleomiscosin A (7, 14 mg). EA-Fr. 6 was achieved by the Semi-preparative HPLC (CH3OH/H2O 40:60) to give chimsalicifoliusin A (1, 21 mg), hymenain (4, 45 mg) and cleomiscosin C (8, 32 mg). EA-Fr. 7 was achieved by the Semi-preparative HPLC (CH3OH/H2O 50:50) to give chimsalicifoliusins B (2, 19 mg) and C (3, 16 mg).

Table 2 Cytotoxicity of compounds 1–3 against on Hela, PC-3 and HL-60 tumor cells (72 h). Compound

1 2 3 Cisplatina a

IC50 (mM) Hela

PC-3

HL-60

18.3 21.2 15.5 6.8

>50 >50 28.6 5.9

23.4 29.6 14.2 8.6

Cisplatin as a positive control.

3.3.1. Chimsalicifoliusin A (1) Yellowish needles. mp 150–151  C. HR-ESI–MS m/z 411.0726 [M
H]
, (calcd. for [C21H16O9-H]
, 411.0722); ESI–MS: m/z 413 [M +H]+, 435 [M+Na]+, 411 [M
H]
; ESI–MS2: m/z [M+H]+ 413 ! 320, 300, 211, 207. UV(MeOH) lmax (loge): 257 (3.55), 303 (3.75); 1H and 13 C NMR (Table 1). 3.3.2. Chimsalicifoliusin B (2) Yellowish powder. mp 356–357  C. HR-ESI–MS: m/z 381.0610 [M
H]
(calcd. for [C20H14O8-H]
, 381.0610); ESI–MS m/z 383 [M +H]+, 381 [M-H]
. UV(MeOH) lmax (loge): 248 (3.83), 335 (3.95); 1H and 13C NMR (Table 1). 3.3.3. Chimsalicifoliusin C (3) Yellowish powder. mp 288–289  C. HR-ESI–MS: m/z 601.0979 [M
H]
(calcd. for [C31H24O13-H]
, 601.0982); ESI–MS m/z 603 [M +H]+, 601 [M
H]
; ESI–MS2: m/z [M+H]+ 603 ! 397, 411; ESI–MS3: m/z [M+H]+ 397 ! 206, 193; 411 ! 383, 207. UV(MeOH) lmax (loge): 248 (4.02), 334 (3.91); 1H and 13C NMR (Table 1). Acknowledgments This work was supported by the NSFC of China (No. 31171701) and the Graduate Innovation Fund of Zhejiang Gongshang University.

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