Carbazole-pyranocoumarin conjugate and two carbazole alkaloids from the stems of Clausena excavata

Abstract A carbazole-pyranocoumarin conjugate, carbazomarin B (1), and two carbazole alkaloids, 6-methoxymukonidine (2) and 2-hydroxy-3-methoxycarbazole (3), together with 27 known compounds (4–30), were isolated from the stems of Clausena excavata. Their structures have been elucidated by spectroscopic analyses. Compound 2 showed moderate cytotoxicity to HuCCA-1, MOLT-3 and HepG2 cancer cell lines with IC50 values of 15.09–28.50 μg/mL, but none to A549 cell line. Heptaphylline (6) and nordentatin (23) were found to show moderate cytotoxic activity against HepG2 cell line with IC50 values of 12.33 and 11.33, respectively, while clausine K (27) exhibited strong cytotoxicity with IC50 value of 1.05 μg/mL, better than a standard drug (etoposide, IC50 13.40 μg/mL).


Introduction
Clausena excavata Burm. f. (Rutaceae) is a tall shrub or small-to medium-sized tree which is traditionally used as herbal medicine in South East Asia for the treatment of snakebite, abdominal pain, cold, malaria and as a detoxification agent (Wu & Furukawa 1982;Wu et al. 1993). The main chemical and bioactive constituents of this plant have been reported as carbazole alkaloids (Taufiq-Yap et al. 2007;Sripisut & Laphookhieo 2010;Sripisut et al. 2012;Peng, Zeng, et al. 2013), coumarins (Kumar et al. 2012;Peng, Zheng, et al. 2013;Santoni et al. 2014), along with a small group of flavonoids, limonoids (Sunthitikawinsakul et al. 2003) and triterpenoids (He et al. 2002(He et al. , 2004Peng et al. 2014;Trung et al. 2014). However, there was only one previous report on the isolation of a carbazole-pyranocoumarin conjugate from this plant . Some isolated compounds have been reported to possess antifungal (Kumar et al. 2012), antimycobacterial, antimalarial (Sripisut & Laphookhieo 2010), anti-HIV-1 (Kongkathip & Kongkathip 2009), anti-HBV and cytotoxic activities (Su et al. 2009) as well as inhibition of rabbit platelet aggregation and topoisomerase II (Xin et al. 2008). In continuing our investigation of bioactive compounds from Rutaceae plants (Chakthong et al. 2012;Sriyatep et al. 2014), we report herein the isolation and structural elucidation of a carbazole-pyranocoumarin conjugate (1) and carbazole alkaloids (2, 3) (Figure 1), together with 27 known compounds (4-30) from the stems of this plant. Compound 2 and most of isolated known compounds were evaluated for their potential cytotoxic and antibacterial activities.
Compound 2 was isolated as a light yellow solid and its molecular formula of C 15 H 13 NO 4 was obtained by HREIMS ( Figure 1). The IR spectrum showed absorption bands indicating a hydroxyl (3380 cm −1 ) and an aryl ester carboxyl (1660 cm −1 ). The uV, IR and 1 H NMR spectroscopic data (Table S2) of 2 were similar to those of mukonidine (8) (Knölker & Wolpert 2003) which was previously isolated from Murraya koenigii (Chakraborty et al. 1978) and also isolated from this study. The differences between them were shown as the presence of an additional methoxyl signal at δ H 3.89 and the downfield ABX system signals at δ H 7.69 (d, J = 2.4 Hz), 6.99 (dd, J = 8.7, 2.4 Hz) and 7.36 (d, J = 8.7 Hz) for H-5, H-7 and H-8, respectively in 2 which replaced a set of four mutually coupled proton signals of ring A in 8. The HMBC correlation between δ H 3.89 (6-OCH 3 ) and δ C 154.6 (C-6) as well as the correlation from δ C 154.6 (C-6) to H-8 (δ H 7.36) suggested the position of the methoxyl group at C-6 ( Figure S1). A carbazole core structure with an oxygenated substituent at C-6 or C-7 has been reported. Inspection of the chemical shift values of H-5 and H-8 of previously isolated carbazole core structures with either 6-or 7-oxygenated substituent revealed that these values of the chemical shift could be used to identify the position of the oxygenated substituent in ring A of C 3 -carbonylcarbazole structure. The signals at around δ H 8.0 and δ H 7.0 were characteristic peaks for H-5 and H-8, respectively, in an 7-oxygenated-3-carbonylcarbazole, whereas the chemical shift values of around δ H 7.7 (H-5) and around 7.4 (H-8) were those of 6-oxygenated carbonylcarbazole (Ruangrungsi et al. 1990;Wu, Huang, Wu, Teng 1996;Wu et al. 1999;Schmidt & Knölker 2009). Thus, the structure of 2 was assigned as 6-methoxymukonidine.
Compound 3 was isolated as yellow viscous liquid. The HREIMS gave the [M] + peak at m/z 213.0785, corresponding to the molecular formula C 13 H 11 NO 2 (Figure 1). The IR spectrum showed absorption bands at 3485 (N-H, O-H stretching) and 1629 (aromatic) cm −1 . The 1 H NMR spectral data displayed a characteristic broad singlet signal of 9-NH of a carbazole alkaloid at δ H 10.01 (Table S2). An unsubstituted ring C was proposed due to the four mutually coupled protons at δ H 7.93 (d, J = 8.0 Hz, H-5), 7.40 (d, J = 8.0 Hz, H-8), 7.24 (td, J = 8.0, 1.2 Hz, H-7) and 7.07 (td, J = 8.0, 1.2 Hz, H-6), leaving two aromatic proton singlet signals at δ H 7.51 and 7.09 for H-4 and H-1, respectively. A methoxyl group at δ H 3.93 was assigned on C-3 due to the HMBC correlation to the carbon at δ C 147.8 (C-3) as well as a NOESY correlation to H-4 (δ H 7.51). This suggested that a remaining phenolic hydroxyl group at δ H 7.14 was located at C-2 which was confirmed by the HMBC correlation of δ H 7.51 (H-4) to δ C 134.4 (C-2) and of δ H 7.14 (2-OH) to δ C 147.8 (C-3) ( Figure S1). It was remarkably that compound 3 was an unsubstituted-ring C carbazole which was rarely found in a natural source. Clausine-V (2,7-dimethoxycarbazole) was the first isolated decarboxylated carbazole alkaloid from this plant (Wu et al. 1999). Thus, compound 3 was assigned as 2-hydroxy-3-methoxycarbazole.

General procedure
Melting point was recorded in °C on a digital Electrothermal 9100 Melting Point Apparatus. ultraviolet spectra were measured in methanol solution on a uV-160A spectrophotometer (SHIMAdZu). The IR spectra were recorded in neat on a Perkin-Elmer FTS FT-IR spectrophotometer. The 1 H and 13 C NMR spectra were recorded on a FT-NMR Bruker ultra Shield TM 300 and 500 MHz and a unity Inova Varian 500 MHz spectrometer using tetramethylsilane (TMS) as the internal standard. EI and HREI mass spectra were measured on ThermoFinnigan MAT 95 XL spectrometer. Quick column chromatography (QCC) was carried out on silica gel 60 GF 254 (Merck). Column chromatography (CC) was performed using silica gel 100 (70-230 Mesh ASTM, Merck) or on Sephadex LH-20. Thin-layer chromatography (TLC) and preparative TLC were performed on silica gel 60 F 254 (Merck). Solvents for extraction and chromatography were distilled at their boiling ranges prior to use.

Plant material
The dried stems of C. excavata were collected from Songkhla province in the Southern part of Thailand in September 2009. Identification was made by Assoc. Prof. dr. Kitichate Sridith, department of Biology, Faculty of Science, Prince of Songkla university. The specimen (N. Bindulem 1) with Herbarium number (0013589) has been deposited in the Herbarium of department of Biology, Faculty of Science, Prince of Songkla university, Thailand.

Antibacterial assay
The isolated compounds from the stems of C. excavata were tested against both Grampositive and Gram-negative bacteria: B. subtilis, S. aureus TISTR517, E. faecalis TISTR459, methicillin-resistant S. aureus (MRSA) ATCC43300, vancomycin-resistant E. faecalis (VRE) ATCC51299, S. typhi, S. sonnei and P. aeruginosa. The micro-organisms were obtained from the culture collections, department of Industrial Biotechnology and department of Pharmacognosy and Botany, PSu, except for the TISTR and ATCC strains, which were obtained from Microbial Research Center (MIRCEN), Bangkok, Thailand. The antimicrobial assay employed was the same as described in Boonsri et al. (2006). Vancomycin, which was used as a standard, showed antibacterial activity against MRSA and VRE at 2.34 μg/mL.

Cytotoxicity assay
The cytotoxic assay was performed as previously described by Tengchaisri and co-workers (Tengchaisri et al. 1998). Briefly, cell lines suspended in RPMI 1640 containing 10% FBS were seeded at 1 × 104 cells (100 μl) per well in 96-well plate, and incubated in humidified air atmosphere (containing 5% CO 2 ) at 37 °C. After 24 h, additional medium (100 μl) containing the test compound and vehicle was added to a final concentration of 50 μg/mL, 0.2% dMSO, and further incubated for 3 days. After that, the cells were fixed with EtOH-H 2 O (95:5, v/v), stained with crystal violet solution, and lysed with a solution of 0.1 N HCl in MeOH, after which absorbance was measured at 550 nm. The number of surviving cells was determined from the absorbance. Etoposide and doxorubicin were used as the reference drugs.