Introduction

Reinwardtiodendron, a genus belonging to the Meliaceae family, is composed of about seven species distributed mainly in Malaysia and Indonesia [1]. In our search for structurally and biologically interesting natural products from tropical plants [2,3,4,5,6,7,8,9,10,11,12,13], we screened Malaysian plants extracts for cytotoxic activity against human promyelocytic leukemia HL-60 cells, and the bark extract of R. cinereum was found to be cytotoxic. To date, chemical constituents of the plants of this genus have never been reported. Thus, we proceeded with bioactivity guided investigation of the extract, yielding two new onocerane triterpenoids, reinereins A and B (1 and 2, Fig. 1), together with known compounds lansic acid dimethyl ester (3) [14], methyl lansionate (4) [15, 16], and lansic acid (5) [14, 17]. The structure elucidation and cytotoxic activity of 1 and 2 are described herein.

Fig. 1
figure 1

Structures of reinereins A and B (1 and 2), lansic acid dimethyl ester (3), methyl lansionate (4), and lansic acid (5)

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Results and discussion

Reinerein A (1) was isolated as an optically active, [α] 21D +26.5 (c 1.0, CHCl3), white amorphous solid. The molecular formula of 1 was determined by HRESIMS [m/z 483.3471 ((M + H)+; ∆ − 0.3 mmu)] to be C31H46O4, and the presence of carbonyl groups in 1 was suggested by the IR absorption at 1740, 1704 and 1669 cm−1. The 1H and 13C NMR data of 1 (Table 1) were similar to 4, suggesting 1 was an onocerane triterpenoid related to 4. Further analysis of the NMR data suggested 1 was different from 4 only in the structure of ring C. The structure of 1, especially that of ring C was elucidated from 2D (HSQC, 1H-1H COSY and HMBC) NMR data (Fig. 2). The HMBC correlations of H2-12 to C-13 and C-14, H3-27 to C-13, C-14 and C-15, H3-28 to C-13, and H-17 to C-15 suggested the presence of a double bond between C-13 and C-14, and a carbonyl C-15, forming an α, β-unsaturated carbonyl moiety in ring C. The relative configuration of 1 was deduced from the value of 3JH-16a/H-17 (8.7 Hz) and NOESY correlations (Fig. 3), and was confirmed to be similar to 4. Thus, the structure of 1 was deduced to be a new compound, reinerein A (Fig. 1).

Table 1 1H & 13C NMR data of 1 and 2 in CDCl3
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Fig. 2
figure 2

Selected 2D NMR (1H-1H COSY and HMBC) correlations of 1 and 2

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Fig. 3
figure 3

Selected NOESY correlations of 1

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Reinerein B (2) was isolated as an optically active white amorphous solid, [α] 21D − 6.5 (c 1.0, CHCl3). The molecular formula of 2 was determined by HRESIMS (m/z 493.3277 ([M + Na]+; ∆ − 1.7 mmu) to be the same as 5 (C30H46O4). However, only 15 carbon signals were observed in the 13C NMR spectrum of 2, indicating the presence of an internal symmetry in 2. Analysis of the 1H and 13C NMR data of 2 (Table 1) showed that they were similar to those of C-12 to C-22 and C-27 to C30 of 5. Therefore, the structure of 2 was assumed to be comprised of two methylcyclohexene moieties instead of a methylenecyclohexane and a methylcyclohexene as in 5. Analysis of the 2D NMR (1H-1H COSY, HSQC, and HMBC, Fig. 2) data further supported this assumption, and the NOESY correlations (H-5/H-9, H-5/H-6b and H-6a/H3-25) suggested the relative configuration of 2 to be similar to 5. Thus, the structure of 2 was deduced to be a new compound, reinerein B (Fig. 1).

Reinwardtiodendron is morphologically and genetically closely related to Lansium [1, 18]. In general, previous phytochemical investigation on the members of Lansium resulted in the isolation of onocerane triterpenoids and tetranortriterpenoids [16, 19, 20]. Thus, our results shows that Lansium and Reinwardtiodendron are also closely related phytochemically.

Compounds 1–5 were tested for cytotoxic activity against HL-60 cells (Table 2). These onocerane triterpenoids, 3–5 in particular, were assumed to be responsible for the cytotoxic activity shown by the bark MeOH extract. In addition, the cytotoxic activity of 1–5 against A549, MCF-7, and HepG2 cells was also tested, and 1 and 5 showed cytotoxic activities against all three cancer cell lines (Table 2). Several onocerane triterpenoids have been reported to show anti-bacterial activity [19] and toxicity against brine shrimp (Artemia salina) [16]. However, to our knowledge, this is the first report on the cytotoxicity of 3–5 against human cancer cell lines.

Table 2 Cytotoxic activity of the isolated compounds
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Experimental section

General experimental procedures

Optical rotations were measured on a JASCO DIP-1000 polarimeter. UV spectra were recorded on a Shimadzu UVmini-1240 spectrophotometer and IR spectra on a JASCO FT/IR-4100 spectrophotometer. High-resolution ESI MS data were obtained on a LTQ Orbitrap XL (Thermo Scientific). 1H and 2D NMR spectra were measured on a 400 or a 600 MHz spectrometers at 300 K, while 13C NMR spectra were measured on a 100 or a 150 MHz spectrometers. The residual CDCl3 chemical shift is used as an internal standard that are δH 7.26 and δC 77.0. Standard pulse sequences were used for the 2D NMR experiments. Merck silica gel 60 (40–63 μm) and Nacalai Tesque Cosmosil 140C18-OPN were used for the column chromatography, and the separations were monitored by Merck silica gel 60 F254, or Merck silica gel RP C-18 F254 TLC plates.

Material

The barks of R. cinereum were collected in Pahang, Malaysia in September 1998. The botanical identification was made by Mr. Teo Leong Eng, Faculty of Science, University of Malaya. Voucher specimens (Herbarium No. 4809) are deposited in the Herbarium of the Chemistry Department, University of Malaya.

Extraction and isolation

The barks of R. cinereum (100 g) were extracted with methanol to obtain 10.4 g of extract. The methanol extract was successively partitioned with n-hexane, ethyl acetate, n-butanol and water, and the hexane-soluble materials (3.25 g), which showed cytotoxic activity, were further separated with a silica gel column (n-hexane/EtOAc, 1:0–1:1, CHCl3, MeOH) to obtain 20 fractions (Fraction A-T). Fractions showing cytotoxic activity were further separated as follows. Further separation of fraction D by a silica gel column (toluene/EtOAc, 1:0–20:1) yielded lansic acid dimethyl ester (3, 46.1 mg, 0.0461%) and methyl lansionate (4, 66.1 mg, 0.0661%). Fraction J was further separated by an ODS column (60–100% MeOH(aq)), followed by a silica gel column (benzene/EtOAc, 10:1) to obtain 1 (1.9 mg, 0.0019%). Further separation of fraction T by a silica gel column (CHCl3/MeOH, 30:1 with 0.1% HCO2H) and an ODS column (60–100% MeCN(aq)) yielded lansic acid (5, 51.2 mg, 0.0512%), and a mixture of 2 and 5 (4.7 mg). The mixture were then separated by HPLC (Nacalai tesque Cholester 4.6 × 250 mm, 70% MeCN(aq) with 0.1% HCO2H, flow rate 0.5 mL/min, UV detection at 210 nm) to obtain 2 (tR 80 min, 1.2 mg, 0.0012%).

Reinerein A (1)

White, amorphous solid; [α] 21D +26.5 (c 1.0, CHCl3); IR (neat) νmax 1740, 1704 and 1669 cm−1; UV (MeOH) λmax (ε) 200.5 (15,000) nm; 1H and 13C NMR data (Table 1); ESIMS m/z 483 (M + H)+; HRESIMS m/z 483.3471 (M + H)+; calcd. for C31H47O4, 483.3474).

Reinerein B (2)

White, amorphous solid; [α] 25D -6.5 (c 1.0, CHCl3); IR (neat) νmax 3405 and 1743 cm−1; UV (MeOH) λmax (ε) 201 (16000) nm; 1H and 13C NMR data (Table 1); ESIMS m/z 493 (M + Na)+; HRESIMS m/z 493.3277 (M + Na)+; calcd. for C30H46O4Na, 493.3294).

Cytotoxicity

HL-60, human promyelocyctic leukemia cells were maintained in RPMI-1640 medium; MCF-7, human breast adenocarcinoma; A549, human lung adenocarcinoma; and HepG2, human hepatocellular carcinoma cells were maintained in DMEM medium. Both growth media were supplemented with 10% fetal calf serum and 1% Penicillin–Streptomycin. The cells (5 × 103 cells/well) were cultured in Nunc disposable 96-well plates containing 90 µl of growth medium per well and were incubated at 37 °C in a humidified incubator of 5% CO2. Serially diluted samples (10 µl, final concentration; 50, 25, 12.5, and 6.25 µM) were added to the cultures at 24 h of incubation. After 48 h of incubation with the samples, MTT solution (15 µl, 5 mg/ml) was added to each of the wells. The cultures were incubated for another 3 h before the cells supernatant was removed. After the removal of the cells supernatant, DMSO (50 µl) was added to each well. The formed formazan crystal was dissolved by re-suspension by pipette. The optical density was measured using a microplate reader (Bio-Rad) at 550 nm with reference wavelength at 700 nm. In all experiments, three replicates were used.