Cytotoxic Compounds from Brucea mollis

Ten compounds, including soulameanone (1), isobruceine B (2), 9-methoxy-canthin-6-one (3), bruceolline F (4), niloticine (5), octatriacontan-1-ol (6), bombiprenone (7), α-tocopherol (8), inosine (9), and apigenin 7-O-β-D-glucopyranoside (10), were isolated from the leaves, stems, and roots of Brucea mollis Wall. ex Kurz. Their structures were determined using one-and two-dimensional NMR spectroscopy and mass spectrometry. All compounds were evaluated for their cytotoxic activity against KB (human carcinoma of the mouth), LU-1 (human lung adenocarcinoma), LNCaP (human prostate adeno-carcinoma), and HL-60 (human promyelocytic leukemia) cancer cell lines. Compound 2 showed significant cytotoxic activity against KB, LU-1, LNCaP, and HL-60 cancer cells with IC50 values of 0.39, 0.40, 0.34, and 0.23 μg/mL, respectively. In addition, compounds 3 and 5 showed significant cytotoxic activity against KB, LU-1, LNCaP, and HL-60 cancer cells with IC50 values around 1–4 μg/mL. Compounds 9-methoxycanthin-6-one (3) and niloticine (5) have been discovered for the first time from the Brucea genus.


Introduction
Brucea is a genus belonging to the family Simaroubaceae. It contains about six species mainly distributed in the tropical Eastern Hemisphere and found in Vietnam, China, northern Australia, and other Asian countries [1].
There are six Brucea species that have been chemically and biologically characterized: Brucea javanica, Brucea mollis var. tonkinensis, Brucea mollis, Brucea sumatrana, Brucea antidysenterica, and Brucea amarissima. Many compounds have been isolated from this genus, including quassinoids, alkaloids, triterpenoids, and flavonoids. Among these, quassinoids, a chemical class isolated only from the Simaroubaceous species, are the dominant constituents [2]. According to Ho, 2000, the Brucea genus in Vietnam consists of three species: B. javanica, B. mollis, and B. tonkinensis [3]. Up until now, there were some reports on the chemistry and bioactivity of Brucea mollis in Japan [4] and China [5]. However, the plant Brucea mollis collected in Vietnam has not been studied so far.
The results of the present study demonstrated that the methanol extract and n-hexane fraction from B. mollis leaves showed strong cytotoxic activity against three cancer cell lines: LU-1 (human lung adenocarcinoma), Hep G2 (liver hepatocellular carcinoma), and MCF-7 (human breast adenocarcinoma) [6].
The 13 C and DEPT NMR spectra of 1 revealed 20 carbon signals, including four methyls, one methylene, seven methines, and eight non-hydrogenated carbons. The 13 C-NMR, HSQC, and HMBC spectra showed that 1 was a quassinoid with a picrasane skeleton. As compared with data reported by Polonsky et al., 1980, 1 was determined to be soulameanone, a quassinoid isolated from Soulamea muelleri [7].
Compound 2 was obtained as a yellow powder. The 13 C-NMR spectra of 2 were similar to those of soulameanone, with additional signals due to the methyleneoxy bridge between the C-8 and C-13 carbons and two substituted groups at the C-13 and C-15 carbons.
Compound 2 was a C-20 pentacyclic quassinoid with two side chains at the C-13 and C-15 carbons, as indicated in the HMBC spectra. The mass spectrometric (ESI-MS) data for 2 showed a molecular ion peak at m/z 480.7 [M+H] + . Based on these data and a comparison with data reported by Fukamiya, 1988 [8], cpd. 2 was determined to be isobruceine B.
Compound 3 was obtained as a yellow powder from the CH 2 Cl 2 fraction of the stems and roots of B. mollis. The molecular formula of 3 was determined to be C 15 -17). In addition, the HMBC correlation of H 3 -17 methoxyl protons to aromatic carbon C-9 suggested that the position of the methoxyl group was C-9. Based on the HMBC, COSY spectra of 3, and comparisons with previously reported data [9], compound 3 was determined to be 9-methoxycanthin-6-one. Compound 5 was identified as niloticine by comparing its 1D, 2D-NMR spectral data with those in the literature [11].
Some other compounds were also isolated from the leaves, stem, and roots of B. mollis, including 1-octatriacontan-1-ol (6), bombiprenone (7), α-tocopherol (8), one nucleoside, inosine (9), and one flavonoid, apigenin 7-O-β-D-glucopyranoside (10). Structures of all of the isolated compounds were determined on the basis of 1 H, 13 C NMR, and ESI-MS spectra, and were compared with references [12][13][14]  The inhibitory activity of the isolated compounds was assessed using a cytotoxic assay against four cancer cell lines: KB (human carcinoma of the mouth), LU-1 (human lung adenocarcinoma), LNCaP (human prostate adenocarcinoma), and HL-60 (human promyelocytic leukemia). Those cell lines were selected based on the reason that KB cells were usually the classical cells used for all cytotoxic assays. Also, the others were employed in order to discover new activities of the tested compounds. The results showed that isobruceine B (2), 9-methoxycanthin-6-one (3), and niloticine (5) possessed strong inhibitory activities toward the tested cancer cell lines, with low IC 50 values ranging from 0.23-0.4 μg/mL, 0.91-3.73 μg/mL, and 1.00-2.22 μg/mL, respectively (see Table 1, Fig. 2). In particular, the cancerous cellular growth inhibitory activity of isobruceine B (2) was stronger than that of ellipticine, the positive control (IC 50 values ranging from 0.66-0.89 μg/mL). Isobruceine B was isolated from the wood of Brucea antidysenterica [15] with no biological activity reported, and from the stems of the Simaroubaceae Picrolemma sprucei plant, which was used in the Amazon regions of Peru, Brazil, and French Guiana as antimalarials [16]. Isobruceine B exhibited in vitro cytotoxic activities against SF-295, HCT-8, and HL-60 human tumor cells lines (IC 50 = 5-27 μg/L), and against the malarial multidrug-resistant Plasmodium falciparum K1 strain (IC 50 = 1.0-4.0 μg/L) [17].
Compound 2 had structural patterns of an anticancer quassinoid, including a four-ring skeleton with a lactone D-ring, an α,β-unsaturated ketone in the A-ring, two free hydroxyl groups, an oxygen-methylene bridge in the C-ring, and an ester group at either the C-13 or C-15 position [18].

Extraction and isolation
Dried powder from the leaves (2.6 kg), stems, and roots (8.6 kg) of B. mollis were extracted at room temperature with MeOH (three times × 72 h). The solvent was removed under reduced pressure to obtain a residue (191 g) of leaves and a residue (300 g) of the stem and roots. These residues were suspended in water and partitioned with n-hexane, CH 2 Cl 2 , and EtOAc, successively.