Clauemarazoles A–G, seven carbazole alkaloids from the stems of Clausena emarginata

    Clauemarazoles A-G, seven carbazole alkaloids from the stems of Clausena emarginata Hong-Min Xia, Guo-Qing Ou Yang, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma, Dan Zhang, Yan Li, Li Li, Dong-Ming Zhang PII: DOI: Reference:

S0367-326X(15)00070-2 doi: 10.1016/j.fitote.2015.03.016 FITOTE 3148

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

2 February 2015 11 March 2015 14 March 2015

Please cite this article as: Xia Hong-Min, Yang Guo-Qing Ou, Li Chuang-Jun, Yang Jing-Zhi, Ma Jie, Zhang Dan, Li Yan, Li Li, Zhang Dong-Ming, Clauemarazoles A-G, seven carbazole alkaloids from the stems of Clausena emarginata, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.03.016

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Clauemarazoles A−G, seven carbazole alkaloids from the stems

T

of Clausena emarginata

State Key Laboratory of Bioactive Substance and Function of Natural Medicines,

MA

a

NU

SC R

Dan Zhang a, Yan Li a, Li Li a, Dong-Ming Zhang a,*

IP

Hong-Min Xia a,b, Guo-Qing Ou Yang a, Chuang-Jun Li a, Jing-Zhi Yang a, Jie Ma a,

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union

Shandong Academy of Chinese Medicine, Jinan 250014, China

TE

b

D

Medical College, Beijing 100050, China

CE P

E-mail: [email protected] (Hong-Min Xia)

AC

E-mail: [email protected] (Dong-Ming Zhang)

* Corresponding author: Prof. Dong-ming Zhang Address: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No.1 Xiannongtan Street, Xicheng District, Beijing, 100050, P. R. China. E-mail: [email protected] Tel/Fax: +86-10-63165227

ACCEPTED MANUSCRIPT ABSTRACT Seven new carbazole alkaloids, clauemarazoles A−G, together with 19 known

T

analogues were isolated from the stems of Clausena emarginata. Their structures

IP

were established on the basis of spectroscopic analyses, and the absolute

SC R

configurations of compounds 1a, 1b, 5a, and 5b were confirmed by their ECD spectroscopy. Compounds 4, 13, 15, and 17 exhibited inhibitory abilities on LPS-induced NO production. Compounds 10−12, 20, 22, and 24 displayed

NU

hepatoprotective effects against DL-galactosamine-induced damage in WB-F344

MA

cells.

AC

CE P

TE

hepatoprotective effects

D

Keywords: Clausena emarginata, carbazole alkaloids, anti-inflammatory activity,

ACCEPTED MANUSCRIPT 1. Introduction Carbazole alkaloids are widely presented in the genus Clausena (Rutaceae), which

T

possess a large number of biological activities, such as antibacterial, neuroprotective,

IP

cytotoxicity, and antimalarial activities [1−3]. Clausena emarginata Huang, a bush

SC R

widely distributed in the southern part of China, was historically used as a folk medicine for the treatment of coughs, headache, gastrointestinal diseases (such as gastrointestinal inflammation), and rheumatic arthritis [4]. Previously, our research

NU

group have reported twelve new A,D-seco-limonoids [5] and two new amide alkaloids [6] with anti-inflammatory properties isolated from this plant. In addition, our

MA

previous study has revealed that the 95% EtOH extract of the stems of C. emarginata exhibited hepatoprotective effects in vitro. Based on the clinical use and to discover bioactive metabolites contributing to anti-inflammatory and hepatoprotective

D

activities, we performed systematical chemical and pharmacological investigations.

TE

Ultimately, clauemarazoles A−G, and 19 known analogues (6−24) were obtained (Fig. 1). Their structures were established by extensive spectroscopic analyses and ECD

CE P

spectroscopy. The inhibitory abilities on LPS-induced NO production in microglia BV2 cells and hepatoprotective effects against DL-galactosamine-induced WB-F344

AC

cells damage of compounds 1−24 were also evaluated.

2. Experimental 2.1. General experimental procedures Optical rotations were measured with a JASCO P2000 polarimeter. The UV spectra were recorded with a JASCO V-650 spectrophotometer, the ECD spectra were measured with a JASCO J-815 spectropolarimeter, and the IR spectra were obtained on a Nicolet 5700 FT-IR spectrometer using a transmission microscope method. The NMR spectra were collected on INOVA-500 and VNS-600 spectrometers. The HRESIMS spectra were taken on an Agilent 1100 series LC/MSD Trap SL mass spectrometer. The MPLC was performed with a UV detector C-635 (Buchi), two pumps C-605 (Buchi), a fraction collector C-660 (Buchi), and an ODS column (450

ACCEPTED MANUSCRIPT mm × 60 mm, 50 μm, 400 g; YMC). The preparative HPLC was performed with a Shimadzu LC-6AD instrument using a reversed-phase column (YMC-Pack ODS-A, 250 × 20 mm, 5 μm) and an SPD-20A detector. Silica gel (160−200 mesh, 200−300

IP

T

mesh, Qingdao Marine Chemical Inc., Qingdao, China) and ODS (50 μm, YMC, Japan) were used for column chromatography. TLC was conducted with GF254

SC R

plates. 2.2. Plant material

NU

The stems of C. emarginata were collected in Xishuangbanna, Yunnan Province, China, in August 2010 and identified by Professor Jing-yun Cui from the

MA

Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (ID-22254) was deposited at the herbarium of the Institute of Material

2.3. Extraction and isolation

D

Medica, Chinese Academy of Medical Sciences, China.

TE

The air-dried, powdered stems of C. emarginata (18 kg) were macerated with 95%

CE P

EtOH (100 L) and refluxed for 6 h (100 L × 3). A dark brown residue (570 g) was obtained after concentration under reduced pressure. The residue was subjected to kieselguhr (1.14 kg) eluting with petroleum ether (15 L), CHCl3 (15 L), EtOAc (15 L),

AC

acetone (20 L), and EtOH (18 L), successively. The CHCl3 fraction (80 g) was subjected to silica-gel column chromatography (200−300 mesh, 7 × 60 cm, 500 g) and eluted with petroleum ether−acetone (10: 0, 9: 1, 4: 1, 7: 3, 3: 2, and 1: 1, each 3 L) to afford six fractions (F1−F6). Fraction F2 (7 g) was separated by reversed-phase MPLC (25 mL/min, 6 h, 210 nm) using MeOH−H2O (30−90%) as the mobile phase, and followed by semipreparative HPLC (8 mL/min, 210 nm) to give compounds 1 (8 mg), 5 (2 mg), 12 (7.6 mg), 15 (3.6 mg), 16 (2.7 mg), 17 (16.9 mg), 20 (3.6 mg), 21 (4 mg), 23 (2.8 mg), and 24 (7 mg). Compound 1 was resolved using a chiral semipreparative column (n-hexane: 2-propanol, 4: 1, 2 mL/min) to afford compounds 1a (3.5 mg) and 1b (3.5 mg). Compound 5 was isolated by a chiral semipreparative column (n-hexane: 2-propanol, 3: 1, 2 mL/min) to obtain compounds 5a (0.8 mg) and 5b (0.8 mg). Fraction F3 (15 g) was chromatographed over silica gel (200−300 mesh, 7 × 55 cm,

ACCEPTED MANUSCRIPT 450 g) eluted with petroleum ether−acetone (9: 1, 17: 3, 4: 1, 3: 1, and 7: 3, each 2.5 L) to give five subfractions (F2S1−F2S5). Subfraction F2S1 (4.0 g) was further purified by reversed-phase MPLC (25 mL/min, 5 h, 210 nm, 30−90% MeOH−H2O) and

IP

T

semipreparative HPLC (8 mL/min, 210 nm) to afford compounds 2 (2.4 mg), 3 (1 mg), 4 (3.3 mg), 6 (2.5 mg), 7 (6.4 mg), 8 (4 mg), 9 (1.6 mg), 10 (1.1 mg), 11 (2.7 mg), 14

SC R

(2 mg), 18 (37 mg), and 19 (1 mg). Subfraction F2S2 (2.8 g) was purified by MPLC (25 mL/min, 4 h, 210 nm, 30−90% MeOH−H2O) and semipreparative HPLC (40% MeOH−H2O, 8 mL/min, 210 nm) to yield compound 22 (3 mg). Fraction F4 (22 g)

NU

was passed over silica gel (200−300 mesh, 7 × 110 cm, 900 g) eluting with petroleum ether−acetone (10: 0, 19: 1, 9: 1, 17: 3, 4: 1, 3: 1, 7: 3, 13: 7, 3: 2, 11: 9, and 1: 1, each

MA

4 L) to give eleven subfractions (F4S1−F4S11). According to HPLC analysis, subfractions F4S7−F4S11 (16 g) were combined and separated by MPLC (25 mL/min,

D

6 h, 210 nm) using MeOH−H2O (20−90%) as the mobile phase, followed by

TE

semipreparative HPLC (37% MeOH−H2O, 8 mL/min, 210 nm) to obtain compound 13 (2 mg).

CE P

Clauemarazoles A and B (1, racemic mixture): yellow amorphous powder; UV (MeOH) λmax (log ε): 206 (4.34), 242 (4.28), 276 (4.31), 292 (4.22), 356 (3.86) nm; IR (microscope) νmax 3305, 2854, 1627, 1587, 1329, 1225, 1024, 724 cm-1; 1H NMR

AC

(DMSO-d6, 500 MHz) and

13

C NMR (DMSO-d6, 125 MHz), see Tables 1 and 2;

HRESIMS m/z 312.1229 [M + H]+ (calcd for C18H18NO4, 312.1230). Clauemarazole A (1a): yellow amorphous powder; [α] 25D +8.6 (c 0.15, MeOH); Rh2(OCOCF3)4-induced CD (CHCl3) 354 (Δε −0.25) nm. Clauemarazole B (1b): yellow amorphous powder; [α] 25D −8.6 (c 0.15, MeOH); Rh2(OCOCF3)4-induced CD (CHCl3) 354 (Δε +0.48) nm. Clauemarazole C (2): white amorphous powder; UV (MeOH) λmax (log ε): 236 (4.37), 269 (4.44) nm; IR (microscope) νmax 3344, 3267, 2974, 1679, 1606, 1336, 1257, 1096, 744 cm-1; 1H NMR (DMSO-d6, 500 MHz) and 13C NMR (DMSO-d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 310.1435 [M + H]+ (calcd for C19H20NO3, 310.1438). Clauemarazole D (3): white amorphous powder; UV (MeOH) λmax (log ε): 229 (4.16), 269 (4.01), 290 (4.36), 333 (3.61), 345 (3.60) nm; IR (microscope) νmax 3274, 2926,

ACCEPTED MANUSCRIPT 1684, 1605, 1347, 1247, 1090, 742 cm-1; 1H NMR (DMSO-d6, 600 MHz) and

13

C

NMR (DMSO-d6, 150 MHz), see Tables 1 and 2; HRESIMS m/z 252.1026 [M + H]+ (calcd for C16H14NO2, 252.1019).

T

Clauemarazole E (4): white amorphous powder; UV (MeOH) λmax (log ε): 203 (4.43),

IP

240 (4.48), 286 (4.52), 297 (4.50), 342 (4.05) nm; IR (microscope) νmax 3428, 3330,

and

13

SC R

2963, 1602, 1583, 1343, 1238, 1169, 1064, 799 cm-1; 1H NMR (DMSO-d6, 500 MHz) C NMR (DMSO-d6, 125 MHz), see Tables 1 and 2; HRESIMS m/z 310.1437

[M + H]+ (calcd for C19H20NO3, 310.1438).

NU

Clauemarazoles F and G (5, racemic mixture): yellow amorphous powder; UV (MeOH) λmax (log ε): 207 (4.57), 242 (4.70), 276 (4.74), 292 (4.56), 355 (4.29) nm; IR

(DMSO-d6, 500 MHz) and

13

MA

(microscope) νmax 3237, 2918, 1731, 1621, 1468, 1377, 1224, 721 cm-1; 1H NMR C NMR (DMSO-d6, 125 MHz), see Tables 1 and 2;

D

HRESIMS m/z 330.1341 [M + H]+ (calcd for C18H20NO5, 330.1336). 25

TE

Clauemarazole F (5a): yellow amorphous powder; [α] D +7.8 (c 0.08, MeOH); Rh2(OCOCF3)4-induced CD (CHCl3) 352 (Δε −0.36) nm.

CE P

Clauemarazole G (5b): yellow amorphous powder; [α] 25D −7.8 (c 0.08, MeOH); Rh2(OCOCF3)4-induced CD (CHCl3) 350 (Δε +0.23) nm. 2.4. Anti-inflammatory activity assay

AC

The murine microglial BV2 cell line was purchased from the Cell Culture Centre at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cells were preincubated for 24 h in 96-well cell culture plates. Then they were treated with isolated carbazoles at various concentrations in triplicate for 1 h, followed by incubation with LPS (0.3 μg/mL) (Sigma-Aldrich) for 24 h. After incubation, the supernatants (100 μL) were added to a solution of Griess reagent (100 μL) (a 1:1 mixture of 0.1% naphthyl ethylenediamine in distilled H2O and 1% sulfanilamide in 5% H3PO4) at room temperature for 20 min. The amount of NO was quantified by the concentration of nitrite produced in the cell culture supernatants by a microplate reader at 540 nm. Curcumin was used as the positive control [7]. 2.5. Hepatoprotective activity assay The WB-F344 cell lines were maintained in DMEM medium with FBS (10%),

ACCEPTED MANUSCRIPT penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C in 5% CO2. Then, the cell suspension (100 μL) was placed in a 96-well microplate for 24 h, treated with various concentrations of test compounds and bicyclol (positive control) in three

IP

T

parallel wells for 1 h, and continuously cultured with DL-galactosamine (50 mM/L) for 24 h. Then, the cells were incubated in the culture medium containing MTT (0.5

SC R

mg/mL) for 4 h. After treatment, the resulting formazan was dissolved in DMSO and was measured on a microplate reader at 490 nm [8].

NU

3. Results and discussion

MA

Compound 1 was obtained as a yellow amorphous powder. Its molecular formula, C18H17NO4, was assigned from the HRESIMS quasi-molecular ion peak at m/z 312.1229 [M+H]+ (calcd for 312.1230), corresponding to 11 indices of hydrogen

D

deficiency. The UV spectrum showed absorbances at λmax 356, 292, 276, 242, and 206

TE

nm, and the IR spectrum indicated the presence of amino (3305 cm-1) and aromatic ring (1627 and 1587 cm-1) functionalities. The 1H NMR spectrum (Table 1) showed

CE P

resonances for three mutually coupling aromatic protons [δH 7.50 (1H, d, J = 8.0 Hz), 7.01 (1H, t, J = 8.0 Hz), and 6.82 (1H, d, J = 8.0 Hz)]; a lone aromatic proton [δH 8.31 (1H, s)] in the aromatic region; two olefinic protons [δH 4.82, 4.70 (each 1H, s)]; an

AC

aldehyde proton at δH 9.94; a broad NH singlet at δH 11.06. The

13

C NMR spectrum

(Table 2), classified by the HSQC spectrum, exhibited 12 aromatic carbons, an aldehyde group, two olefinic carbons, a methyl, a methylene, and a methine. The NMR data implied that compound 1 was a carbazole alkaloid. In the HMBC spectrum (Fig. 2), correlations from the aldehyde proton to C-3 (δC 114.8) and C-4 (δC 125.7) located the aldehyde group at C-3. Correlations from H2-1′ to C-1 (δC 107.9) and C-1a (δC 145.5), from H-2′ to C-1′ (δC 30.5) and C-5′ (δC 18.1), from H2-4′ to C-5′, and from CH3-5′ to C-3′ (δC 148.2) and C-4′ (δC 109.7) indicated that a modified isoprenyl unit was attached to C-1, and a hydroxy group was placed at C-2′ (δC 73.8). H2-1′ and the aldehyde proton showed correlations with C-2 (δC 157.4), suggested the presence of a hydroxy group at C-2. The attachment of a hydroxy group to C-8 (δC 143.1) was

ACCEPTED MANUSCRIPT demonstrated by the correlations from H-6 and H-7 to C-8. Thus, the planar structure of 1 was confirmed. Because of the low specific rotation and the absence of distinct Cotton effect in the

IP

T

Rh2(OCOCF3)4-induced CD spectrum, compound 1 was confirmed to be a racemic mixture. It was resolved by Chiral HPLC to afford the enantiomers (1a and 1b). The

SC R

absolute configuration of C-2′ of 1a and 1b were established by CD spectra using bulkiness rule for secondary alcohols [9, 10]. The Rh2(OCOCF3)4-induced CD spectra displayed a negative Cotton effect of 1a and a positive Cotton effect of 1b at 354 nm

NU

(Fig. 3) indicated the 2′R-configuration of 1a and 2′S-configuration of 1b. Therefore,

clauemarazole B, respectively.

MA

structures 1a and 1b were determined as depicted and named clauemarazole A and

Compound 2 was isolated as a white amorphous powder. It had a molecular

D

formula of C19H19NO3, as determined by the HRESIMS. The 1D NMR spectra of 2

TE

indicated a carbazole alkaloid skeleton having a ester carbonyl, an oxygenated quaternary, two methylenes, two methyls, and a methoxyl. In the HMBC experiment,

CE P

correlations from H-4 to the ester carbonyl carbon (δC 169.8) located the ester carbonyl at C-3 (δC 124.9). A series of correlations from H-1′ to C-1 (δC 141.4), C-2 (δC 128.3), C-3, and C-3′ (δC 80.6), from H-2′ to C-1′ (δC 21.9) and C-3′, from CH3-4′

AC

and CH3-5′ to C-2′ (δC 40.4), C-3′, and the C-3 ester carbonyl, taking the chemical shifts and the degree of unsaturation into consideration, confirmed that a modified isoprenyl unit from C-1′ to C-5′ was placed at C-2 and an oxygen atom at C-3′ was connected to the C-3 ester carbonyl to form a seven-membered lactone ring. The methoxyl was attached to C-1 due to the correlations from the methoxyl protons to C-1. Hence, the structure of 2 was established and named clauemarazole C. Compound 3 possessed the molecular formula C16H13NO2 according to the HRESIMS. Analyses of the NMR spectra indicated that 3 was also a 1-oxygenated 2,3-substituted carbazole alkaloid, similar to the known compound claulansine E [2]. The only difference between them was the absence of the 1′-OH in 3, which was confirmed by the HMBC correlations from H2-1′ to C-2 (δC 140.7), C-2′ (δC 36.4), and the C-3 keto carbonyl (δC 204.9), from H2-2′ to C-2 and the C-3 keto carbonyl, and

ACCEPTED MANUSCRIPT from H-4 to the C-3 keto carbonyl. Thus, structure 3 was confirmed and named clauemarazole D. The molecular formula of compound 4 was determined to be C19H19NO3

IP

T

established by the HRESIMS. The 1H NMR spectrum (Table 1) showed an ABX system [δH 7.91 (1H, d, J = 8.0 Hz), 6.90 (1H, d, J = 2.0 Hz), and 6.68 (1H, dd, J = 8.5,

SC R

2.0 Hz)], which indicated that one substituent on the ring A was attached to either C-6 or C-7. HMBC correlations from H-5 and H-8 to C-7 (δC 157.2) demonstrated the presence of a hydroxy group at C-7. Correlations from the aldehyde proton to C-2 (δC

NU

126.4), C-3 (δC 131.0), and C-4 (δC 120.3) placed the aldehyde group at C-3. Correlations from H2-1′ to C-2, C-2′ (δC 124.3), C-3, and C-3′ (δC 130.4), from CH3-4′

MA

and CH3-5′ to C-2′ and C-3′ suggested that an isoprenyl group was located at C-2. The OCH3 was attached to C-1 (δC 142.5) due to the correlations from the protons of the

D

OCH3 to C-1. Therefore, the structure of clauemarazole E was characterized as 4.

TE

Compound 5 had a molecular formula of C18H19NO5, as deduced from the HRESIMS. Comparison of the NMR spectra of 5 with those of 1 revealed a similar

CE P

carbazole skeleton and substitution patterns, with the only difference being the absence of the olefinic bond between C-3′ and C-4′ and the presence of a hydroxy group at C-3′. It was supported by the chemical shifts of C-3′ (δC 72.0) and C-4′ (δC

AC

26.5) as well as the HMBC correlations from CH3-4′ and CH3-5′ to C-2′ (δC 77.8) and C-3′. Therefore, the planar structure of 5 was established as the same as the known compound clauszoline-D [11]. However, the absolute configuration of clauszoline-D was not determined in the literature 9. Compound 5 was also a racemic mixture on the basis of the low specific rotation and the absence of distinct Cotton effect in the Rh2(OCOCF3)4-induced CD spectrum. Chiral HPLC was used to afford the enantiomers (5a and 5b). According to the bulkiness rule for secondary alcohols, a negative Cotton effect at 352 nm of 5a in the Rh2(OCOCF3)4-induced CD spectrum (Fig. 4) assigned the 2′R-configuration to 5a and 2′S-configuration to 5b. Thus, clauemarazole F and clauemarazole G were elucidated as 5a and 5b, respectively. The nineteen known carbazole alkaloids were identified to be mafaicheenamine C (6) [12], mafaicheenamine E (7) [13], claulansine G (8) [2], claulamine E (9) [14],

ACCEPTED MANUSCRIPT claulansine I (10) [2], clausine-F (11) [15], clauszoline-E (12) [11], clausine K (13) [16], lansine (14) [17], clausine-O (15) [18], glycozolidal (16) [19], murrayanine (17) [20], clausine E (18) [16], clausine-L (19) [21], methyl 6-methoxycarbazole [22],

3-formyl-6-methoxycarbazole

(21)

T

(20)

[22],

O-

IP

-3-carboxylate

demethylmurrayanine (22) [23], mukonal (23) [24], 3-formylarbazole (24) [22], based

SC R

on the spectroscopic profiles and comparison with literature values.

Compounds 1−24 were evaluated for their inhibitory activities on NO production in LPS-activated murine microglia BV2 cells as well as hepatoprotective activities

NU

against DL-galactosamine-induced toxicity in WB-F344 cells. As shown in Table 3, compounds 4, 13, 15, and 17 showed anti-inflammatory activities, and no influence

MA

on cell viability was observed by the MTT method. Compounds 10−12, 20, 22, and 24 exhibited hepatoprotective activities (Table 4), the other compounds showed weak

D

effects in the same assay.

TE

Conflict of Interest

The authors declare no conflict of interest.

CE P

Acknowledgements

We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,

AC

for the spectroscopic data measurements and bioactivity tests. This project was financially supported by the National Natural Science Foundation of China (No. 21272278), the Program for Changjiang Scholars and Innovative Research Team in the University (No. IRT1007), and the National Megaproject for Innovative Drugs (No. 2012ZX09301002-002). Appendix A. Supplementary data Supplementary data to this article can be found online. References [1] Maneerat W, Ritthiwigrom T, Cheenpracha S, Promgool T, Yossathera K, Deachathai S, et al. Bioactive carbazole alkaloids from Clausena wallichii roots. J Nat Prod 2012; 75: 741−746. [2] Liu H, Li CJ, Yang JZ, Ning Na, Si YK, Li L, et al. Carbazole alkaloids from the

ACCEPTED MANUSCRIPT stems of Clausena lansium. J Nat Prod 2012; 75: 677−682. [3] Songsiang U, Thongthoom T, Boonyarat C, Yenjai C. Claurailas A-D, cytotoxic carbazole alkaloids from the roots of Clausena harmandiana. J Nat Prod 2011; 74:

IP

T

208−212.

[4] Chen SK, Chen BY, Li H. Flora of Reipublicae Popularis Sinicae (Zhongguo

SC R

Zhiwu Zhi) (Science Press, Beijing) 1997; 43: 135.

[5] Xia HM, Li CJ, Yang JZ, Ma J, Chen XG, Zhang D, et al. A,D-seco-limonoids from the stems of Clausena emarginata. J Nat Prod 2014; 77: 784−791.

NU

[6] Xia HM, Li CJ, Yang JZ, Ma J, Li L, Chen XG, et al. Anti-inflammatory amide alkaloids from the stems of Clausena emarginata. J Asian Nat Prod Res 2014; 16:

MA

971−975.

[7] Zhang Y, Liu YB, Li Yong, Ma SG, Li L, Qu J, et al. Sesquiterpenes and alkaloids

D

from the roots of Alangium chinense. J Nat Prod 2013; 76: 1058–1063.

TE

[8] Shi J, Li CJ, Yang JZ, Ma J, Wang C, Tang J, et al. Hepatoprotective coumarins and secoiridoids from Hydrangea paniculata. Fitoterapia 2014; 96: 138–145.

CE P

[9] Frelek J, Szczepek WJ. [Rh< sub> 2(OCOCF< sub> 3 )< sub> 4 ] as an auxiliary chromophore in chiroptical studies on steroidal alcohols. Tetrahedron: Asymmetry 1999; 10: 1507−1520

AC

[10] Jadwiga F, Klimek A, Ruskowska P. Dinuclear transition metal complexes as auxiliary chromophores in chiroptical studies on bioactive compounds. Curr Org Chem 2003; 7: 1081−1104. [11] Ito C, Ohta H, Tan HTW, Furukawa H. Constituents of Clausena excavate. Isolation and structural elucidation of seven new carbazole alkaloids and a new courmarin. Chem Pharm Bull 1996; 44: 2231−2235. [12] Maneerat W, Laphookhieo S. Antitumoral alkaloids from Clausena lansium. Heterocycles 2010; 81: 1261–1269. [13] Maneerat W, Ritthiwigrom T, Cheenpracha S, Laphookhieo S. Carbazole alkaloids andcoumarins from Clausena lansium. Phytochemistry letters 2012; 5: 26–28. [14] Shen DY, Chan YY, Hwang TL, Juang SH, Huang SC, Kuo PC, et al.

ACCEPTED MANUSCRIPT Constituents of the roots of Clausena lansium and their potential antiinflammatory activity. J Nat Prod 2014; 77: 1215–1223. [15] Wu TS, Huang SC. Clausine-D and -F, two new 4-prenyl carbazole alkaloids

IP

T

from Clausena excavata. Chem Pharm Bull 1992; 40: 1069–1071. [16] Wu TS, Huang SC, Wu PL, Teng CM. Carbazole alkaloids from Clausena

SC R

excavate and their biological activity. Phytochemistry 1996; 43: 133–140. [17] Lange GL, Organ MG. Three new carbazole alkaloids isolated from Murraya siamensis. J Nat Prod 1990; 53: 946–952.

NU

[18] Wu TS, Huang SC, Wu PL, Kuoh CS. Alkaloidal and other constituents from the root bark of Clausena excavate. Phytochemistry 1999; 52: 523–527.

MA

[19] Dhan P, Kanwal R, Kapil RS, Popli SP. Chemical constituents of Clausena lansium: Part I. Structure of lansamide-I and lansine. Ind J Chem Section B 1980;

D

19B: 1075–1076.

TE

[20] Wen-Shyong L, McChesney JD, El-Feraly FS. Carbazole alkaloids from Clausena lansium. Phytochemistry 1991; 30: 523–527.

CE P

[21] Wu TS, Huang SC, Lai JS, Teng CM, Ko FN, Kuoh CS. Chemical and antiplatelet aggregative investigation of the leaves of Clausena excavata. Phytochemistry 1993; 32: 449–451.

AC

[22] Wen-Shyong L, McChesney JD, El-Feraly FS. Carbazole alkaloids from Clausena lansium. Phytochemistry 1991; 30: 343–346. [23] Ngadjui BT, Ayafor JF, Sondengam BL, Connolly JD. Quinolone and carbazole alkaloids from Clausena anisata. Phytochemistry 1989; 28: 1517–1519. [24] Bhattacharyya P, Chakraborty A. Mukonal, a probable biogenetic intermediate of pyranocarbazole alkaloids from Murraya koenigii. Phytochemistry 1984; 23: 471–472.

ACCEPTED MANUSCRIPT Appendix

Fig. 1

IP

T

Structures of compounds 1–24. Fig. 2

SC R

Key HMBC correlations of 1–4. Fig. 3

Rh2(OCOCF3)4-induced CD (in CHCl3) spectrum of compounds 1a and 1b.

NU

Fig. 4.

Rh2(OCOCF3)4-induced CD (in CHCl3) spectrum of compounds 5a and 5b.

1

MA

Table 1

H NMR spectroscopic data for compounds 1–5 in DMSO-d6.

D

Table 2 13

TE

C NMR spectroscopic data for compounds 1–5 in DMSO-d6.

Table 3

CE P

Inhibitory effects of compounds 4, 13, 15, and 17 against LPS-induced NO production in microglia BV2 cells. Table 4

AC

Hepatoprotective effects of compounds 10−12, 20, 22, and 24 against DLgalactosamine-induced toxicity in WB-F344 cells (10 μM).

ACCEPTED MANUSCRIPT

5a

AC

CE P

TE

D

MA

NU

SC R

IP

T

Table 1 1H NMR spectroscopic data for compounds 1–5 in DMSO-d6. pos a 1 2a 3b 4a ition 1 1a 2 3 4 8.31 s 8.25 s 8.22 s 8.26 s 4a 5 7.50 d (8.0) 8.18 d (8.0) 8.21d (7.8) 7.91 d (8.0) 5a 7.20 td (7.8, 6.68 dd (8.5, 6 7.01 t (8.0) 7.20 t (7.5) 0.6) 2.0) 7.43 td (7.8, 7 6.82 d (7.5) 7.43 t (8.0) 0.6) 8 7.54 d (8.0) 7.52 d (7.8) 6.90 d (2.0) 8a 1′ 3.15 dd 3.04 t (6.5) 3.27 m (2H) 3.87 d (6.5) (14.0, 4.5) (2H) (2H) 3.04 dd (14.0, 8.5) 4.31 dd (8.0, 2.12 t (6.5) 2′ 2.69 m (2H) 5.14 t (6.5) 4.0) (2H) 3′ 4′ 4.82 s, 4.70 s 1.16 s 1.63s 5′ 1.82 s 1.16 s 1.78 s 13.92 s 4.05 s 3.88 s OCH3 39.94 s 10.13 s CHO N 11.06 brs 11.71 brs 11.69 brs 11.49 brs H a1

8.30 s 7.50 d (8.0)

7.01 t (7.5) 6.81 d (8.0)

3.24 d (13.5) 2.80 dd (14.0, 10.5) 3.50 d (9.5) 1.19 s 1.19 s

9.96 s 10.89 brs

H NMR data (δH) were measured at 500 MHz. b 1H NMR data (δH) were measured at 600 MHz.

ACCEPTED MANUSCRIPT Table 2 13C NMR spectroscopic data for compounds 1–5 in DMSO-d6.

MA D

TE

59.9 204.9

195.8

a 13

AC

CE P

C NMR data (δH) were measured at 125 MHz.

MHz.

b 13

5a 109.3 145.7 157.3 115.0 125.2 117.3 110.6 124.9 120.9 111.0 143.1 129.8 26.5 77.8 72.0 26.5 24.9

191.8

195.7

T

4a 142.5 136.3 126.4 131.0 120.3 123.1 121.2 115.5 109.7 157.2 97.2 142.2 23.4 124.3 130.4 25.4 17.9 60.8

IP

3b 141.0 129.9 140.7 137.3 111.1 125.2 120.9 123.1 119.6 126.6 111.6 140.9 22.4 36.4

SC R

2a 141.4 135.3 128.3 124.9 119.1 122.9 120.6 122.8 119.5 126.3 111.6 140.3 21.9 40.4 80.6 30.6 30.6 61.5 169.8

NU

1a 107.9 145.5 157.4 114.8 125.7 117.3 110.6 124.8 121.0 111.0 143.1 129.8 30.5 73.8 148.2 109.7 18.1

position 1 1a 2 3 4 4a 5 5a 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 1-OCH3 3-CO3-CHO

C NMR data (δH) were measured at 150

ACCEPTED MANUSCRIPT Table 3 Inhibitory effects of compounds 4, 13, 15, and 17 against LPS-induced NO production in microglia BV2 cells. 13 4.61

15 6.37

17 9.94

curcumina 0.48

Positive control.

MA

NU

SC R

IP

a

4 10.91

T

Compounds IC50 (μM)

Table 4 Hepatoprotective effects of compounds 10−12, 20, 22, and 24 against

AC

TE

CE P

Croup Control Model Bicyclola 10 11 12 20 22 24

D

DL-galactosamine-induced toxicity in WB-F344 cells (10 μM). OD value 1.484±0.138 0.522±0.095 0.902±0.084 0.832±0.001 0.802±0.001 0.801±0.124 0.753±0.061 0.595±0.058 0.788±0.035

Survival rate (%) 100 33### 59*** 54* 52* 52* 49* 38* 51*

* P < 0.05, ** P < 0.01, *** P < 0.001 vs. model, ### P < 0.001 vs. control. a Positive control.

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 1. Structures of compounds 1–24.

ACCEPTED MANUSCRIPT

TE

D

MA

NU

SC R

IP

T

Fig. 2. Key HMBC correlations of 1–4.

AC

CE P

Fig. 3. Rh2(OCOCF3)4-induced CD (in CHCl3) spectrum of compounds 1a and 1b.

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 4. Rh2(OCOCF3)4-induced CD (in CHCl3) spectrum of compounds 5a and 5b.

ACCEPTED MANUSCRIPT Graphical abstract

Seven new carbazole alkaloids, clauemarazoles A−G, together with 19 known

IP

T

analogues were isolated from the stems of Clausena emarginata. Compounds 4, 13, 15, and 17 exhibited inhibitory abilities on LPS-induced NO production. Compounds

SC R

10−12, 20, 22, and 24 displayed hepatoprotective effects against DL-galactosamine-

AC

CE P

TE

D

MA

NU

induced damage in WB-F344 cells.