Lactam Triterpenoids from the Bark of Toona sinensis

Three new limonoid-type triterpenoids, namely toonasins A–C (1–3) with a rare lactam E ring, along with six known compounds (4–9) were isolated from the barks of Toona sinensis. The structures of new compounds were elucidated by interpretation of spectroscopic data, and the relative configuration of compound 1 was further characterized by X-ray crystallographic analyses. The isolated compounds were evaluated for their cytotoxic activities against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480), and compounds 3 and 5 showed weak cytotoxicities.


Introduction
Toona sinensis is a shrub of Meliaceae distributed widely in Asian countries [1]. The leaves of T. sinensis, which contain a distinct flavor, are very popular in vegetarian cuisine and have long been used as a nutritious food in China and Malaysia [2]. In folk, almost every part of T. sinensis, including seeds, bark, root bark, petioles, and leaves, can be used to treat cold, rheumatic pain, stomach pain, and diarrhea without any irreversible side effects [3,4]. Modern pharmacological researches also demonstrated that this plant showed wide spectrum of biological activities, such as antioxidant [5], anti-diabetes [6], antiinflammatory [7], antimicrobial [8], antinociceptive [9] and anti-tumor [10], due to its plentiful chemical constituents (limonoids, flavonoids, phytols, coumarins and norcyteine derivatives) [11][12][13][14].
Compound 2 was obtained as a colorless needle with a molecular ion peak at m/z 592.2269 [M ? Na] ? (calcd 569.2261) in the HRESIMS, coincided with the molecular formula of C 30 H 35 NO 10 . The IR spectrum exhibited absorption bands for NH (3435 cm -1 ), carbonyl (1745 cm -1 ) and amide (1657 cm -1 ) groups. Detailed comparison of the 1D NMR data between 1 and 2 ( Table 1) showed that their main difference was an additional acetyl group [d C 170.1 (s), 21.2 (q)] in 2 instead of a methylene in 1. Furthermore, the HMBC correlations of H-6 (d H 5.27,   Fig. 4). Hence, the structure of 3 was established and named as toonasin C (3). Compounds 3, 4, 5, 7 and 9 were evaluated for their cytotoxicities against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480). The results ( Table 2) showed that toonasin C (3) and bourjotinolone B (5) have weak inhibition activities against above cell lines with IC 50 values of 12.00-25.00 lM.
To the best our knowledge, the spire leaves of T. sinensis were used for a long time as health food and traditional medicine to treat rheumatoid arthritis, cevicitis, ruethritis, gastric ulcers, enteritis, and cancer [1]. However,  phytochemical investigation mainly focused on flavonoids and polyphenols [28][29][30]. Pharmacological studies documented that the different parts of T. sinensis, including bark, seed, flower and root barks, also had various bioactivities [3,4]. Meanwhile, previous research on the stem bark and leaves of this plant resulted in the isolation of a series of limonoids and the part of them exhibited significant cytotoxic potential [10,31]. In the present paper, three novel limonoids with a unique lactam E ring were identified from the bark of T. sinensis, and toonasin C (3) showed comparable cytotoxic effect, with the positive control (Cisplatin), which indicated that limonoids from this plant should be paid more attention in order to explore and develop T. sinensis in more depth.

General
The optical rotations were taken on a JASCO P-1020 polarimeter. UV spectra were record using a Shimadzu UV2401PC spectrophotometres. 1 Fuji) and Sephadex LH-20 (20-150 lm, Pharmacia) were used for column chromatography. The crystal structure of 1 was solved by direct method SHELXS-97 (Sheldrich, G. M. University of Gottingen; Gottingen, Germany, 1997) and the full-maxtrix leastsquares deposited in the Cambridge Crystallographic Data Centre. Copies of these data can be obtained free of charge on application to CCDC via the Internet at www.ccdc.cam. ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax (?44) 1223-336-033; or e-mail: deposit @ccdc.cam.ac.uk).

Plant Material
The barks of T. sinensis were purchased from Beijing, China in May 2011, and identified by Prof. Jian Lou. A voucher specimen has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

Cytotoxicity Assays
There are five cancer cell lines including MCF-7, SMMC7721, HL-60, SW480 and A549, which were obtained from Shanghai cell bank in China. Cells were cultured in DMEM medium (Hyclone, USA), supplemented with 10 % fetal bovine serum (Hyclone, USA), in 5 % CO 2 at 37°C. Cytotoxicity was measured by standard MTT assay [27]. After the treatment of samples and positive control, cell viability was detected and a cell growth curve was graphed. The IC 50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves and expressed as mean ± standard deviation.