Introduction

The wide-ranging activities of limonoids are of interest in the field of plant natural products. Limonoids with insecticidal, insect antifeedant, antibacterial, antifungal, antimalarial, anticancer, and antiviral activities [1–5] are known to be especially bountiful in plants from the Meliaceae family [1].

In our continued effort to gain a deeper understanding of secondary metabolites from plants of the Meliaceae family [6–8], the studies of Chisocheton ceramicus bark extract have yielded three new limonoids, ceramicines J–L (1–3). In this paper, we describe the isolation, structure elucidation, and cytotoxic activity of 1–3 isolated from C. ceramicus.

Results and discussion

The methanol extract from the bark of C. ceramicus was sequentially partitioned between hexane, ethyl acetate, n-butanol, and water. Subsequent purification by a silica gel column and HPLC led to the isolation of three new limonoids, ceramicines J (1, 1.0 mg, 0.0002% yield), K (2, 0.6 mg, 0.00012% yield), and L (3, 0.8 mg, 0.00016% yield).

Ceramicine J {1, [α] 25D −88 (c 0.5, CHCl3)} was isolated as a colorless amorphous solid and had molecular formula, C26H32O5 as determined by high-resolution–electrospray ionization–time-of-flight mass spectroscopy (HR-ESI-TOF-MS) [m/z 425.2328 (M + H)+, Δ −0.3 mmu]. IR absorptions suggested the presence of two carbonyls (1724 and 1675 cm−1). 1H and 13C NMR data (Tables 1, 2) revealed 26 carbon resonances due to two carbonyls, one sp 2 quaternary carbons, four sp 3 quaternary carbons, five sp 2 methines, six sp 3 methines, four sp 3 methylenes, and four methyls. Among them, two sp 2 methines (δ C 140.2 and 142.8), two sp 3 methines (δ C 73.6 and 69.4), and one sp 3 methylene (δ C 79.8) are connected to an oxygen atom (Fig. 1).

Table 1 1H (J, Hz) data of ceramicines J–L (1–3) in CDCl3 at 300 K
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Table 2 13C NMR data of ceramicines J–L (1–3) in CDCl3 at 300 K
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Fig. 1
figure 1

Structures of ceramicines J–L (1–3)

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1H and 13C NMR data (Tables 1, 2) of 1 were analogous to those of previously reported ceramicine B [7], but instead an olefin between C-14 and C-15 was replaced by a carbonyl. On the basis of data from COSY analysis of 1 in CDCl3 (Fig. 2), five partial structures a (from C-2 to C-3), b (from C-5 to C-7), c (from C-9 to C-12), d (from C-16 to C-17), and e (from C-22 to C-23) were deduced. The HMBC correlations for H3-29 of C-3 to C-5; H-2 of C-4 and C-10; H3-19 of C-1, C-5, C-9; H3-30 of C-7, C-9, and C-14; and H3-18 of C-12 to C-14 bridged the partial structure of a, b, and c through C-1, C-8, and C-10, thus forming a phenanthrene ring system. The relation between partial structure c and d could be assigned by HMBC correlations for H3-18 of C-12 to C-14, and C-17; H2-16 and H-14 of C-15, such that a cyclopentanone was deduced to be attached to the phenanthrene ring system through C-13 and C-14. The connectivity of partial structure e (β-furyl ring) to d was shown by HMBC correlations for H-20 of C-17, H-21 of C-20, and C-22. Further analysis of HMBC correlations of H3-29 of C-3 and C-5, and H2-28 of C-6, indicated the presence of a tetrahydrofuran ring at C-4–C6 and C-28. Therefore, ceramicine J (1) was established as a new limonoid with a cyclopentanone[α]phenanthrene ring system with a β-furyl ring at C-17, and a tetrahydrofuran ring.

Fig. 2
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Selected 2D NMR correlations for ceramicine J (1)

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The relative stereochemistry of 1 was elucidated by ROESY correlations as shown in the computer-generated 3D rendering (Fig. 2). ROESY correlations of H3-29/H-6, and H3-19 suggested the stereochemistry of the C-29 methyl group as shown in Fig. 2. The 3 J proton coupling constants (3 J H-5/H-6 = 12.5 Hz and 3 J H-6/H-7 ≈ 0 Hz) as well as ROESY correlations of H-7/H-6 and H-14, and H3-18/H-14 indicated that H-6 and H-7 adopted β-configurations, and H-5, H-9, and C-18 adopted α-configurations as shown in Fig. 2.

Ceramicine K {2, [α] 26D +6 (c 0.4, CHCl3)} was isolated as a colorless amorphous solid and had molecular formula of C26H32O6 as determined by HR-ESI-TOF-MS [m/z 463.2136 (M + Na)+, Δ +3.9 mmu], which contained one oxygen more than ceramicine J (1). IR absorptions showed the presence of two carbonyls (1747 and 1691 cm−1). 1H and 13C NMR data (Tables 1, 2) revealed 26 carbon resonance due to two carbonyls, two sp 2 quaternary carbons, three sp 3 quaternary carbons, six sp 2 methines, six sp 3 methines, three sp 3 methylenes, and four methyls. Of these, two sp 2 methines (δ C 139.7 and 142.7) and three sp 3 methines (δ C 65.2, 65.6, and 74.8) are attached to an oxygen atom. Further observation analysis of the 13C NMR data showed the presence of an olefin on C-14 and C-15 (δ C 160.7 and 120.0, respectively) in place of the carbonyl on C-15 (δ C 219.1) as seen in ceramicine J (1). In addition, HMBC correlations for H-4 and H3-2′ of C-1′ included the presence of an acetate at C-4 (δ C 65.2). On the basis of these NMR data including 2D NMR (COSY, HSQC, and HMBC) as well as HR-ESI-TOF-MS, the planar structure of 2 was elucidated as shown in Fig. 3.

Fig. 3
figure 3

Selected 2D NMR correlations for ceramicine K (2)

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The relative stereochemistry of 2 was elucidated by ROESY correlations as shown in the computer-generated 3D drawing (Fig. 3). The relative stereochemistry was similar to that of 1. ROESY correlations of H-6/H3-19, and H3-30; H-7/H-15, and H3-30 suggested the presence of the hydroxyls attached to C-6 and C-7 adopted the α-configuration. In contrast, the 3 J proton coupling constants (3 J H-4/H-5 = 4.6 Hz), along with ROESY correlation of H-4/H-5 implied, the acetate attached to C-4 adopted the β-configuration.

Ceramicine L {3, [α] 27D +14 (c 0.3, CHCl3)} was isolated as a colorless amorphous solid with molecular formula of C25H32O5 as established by HR-ESI-TOF-MS [m/z 435.2147 (M + Na)+, Δ +2.6 mmu], which was smaller than that of ceramicine K (2) by a CO unit. 1H and 13C NMR data (Tables 1, 2) of 3 were analogous to those of 2, but instead of an acetate attached at C-4 in 2, the presence of an sp 3 quaternary carbon for C-4 with a methyl and a hydroxy group was suggested by the HMBC correlations H3-29 to C-3, C-4 (δ C 71.5), and C-5. The ROESY correlations between H-6/H3-29 and H3-30, H-7/H3-30, and H3-29/H3-19 suggested that all three hydroxyls adopted the α-configuration (Fig. 4).

Fig. 4
figure 4

Selected 2D NMR correlations for ceramicine L (3)

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Ceramicines J–L (1–3) from C. ceramicus were subjected to cytotoxic activity studies using the MTT assay [9]. Results showed that 1, 2, and 3 exhibited dose-dependent but weak cytotoxicity against the HL-60 cell line (36, 33, and 25% inhibition at 50 μM, respectively). Cisplatin was used as a positive control (IC50 at 0.87 μM for HL-60).

Experimental

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 Fourier transform/infrared (FT/IR)-4100 spectrophotometer. CD spectra were recorded on a JASCO J-820 polarimeter. High-resolution ESI-MS were obtained on an LTQ Orbitrap XL (Thermo Scientific, USA). 1H and 2D NMR spectra were measured on 700-MHz spectrometers at 300 K, whereas 13C NMR spectra were measured with a 175-MHz spectrometer. Each ceramicine’s NMR sample was prepared by dissolving with CDCl3 in 2.5-mm microcells (Kanto Chemicals Co., Inc., Japan) and the residual CHCl3 chemical shifts used as an internal standard are δ H 7.26 and δ C 77.0. Standard pulse sequences were used for the 2D NMR experiments. 1H–1H COSY and NOESY spectra were measured with spectral widths of both dimensions of 4800 Hz, and 8 scans with two dummy scans were accumulated into 1 K data points for each of 256 t 1 increments. ROESY spectra in the phase-sensitive mode were measured with a mixing time of 800 and 30 ms. For the HSQC spectra in the phase-sensitive mode and HMBC spectra, a total of 256 increments of 1 K data points were collected. The (HMBC) spectra with Z-axis pulsed field gradient (PFG), a 50-ms delay time was used for long-range C–H coupling. Zero-filling to 1 K for F 1 and multiplication with squared cosine-bell windows shifted in both dimensions were performed prior to 2D Fourier transformation.

Material

The barks of C. ceramicus were collected from Pahang, Malaysia, in 1996. The botanical identification was made by Mr. Teo Leong Eng, Faculty of Science, University of Malaya. Voucher specimens (herbarium no. KL4648) are deposited in the Herbarium of the Chemistry Department, University of Malaya.

Extraction and isolation

The dried ground barks of C. ceramicus (500 g) were extracted successively with methanol and 49 g of extract was obtained. The total extract was successively partitioned with hexane, ethyl acetate, n-butanol, and water. The hexane-soluble materials were preliminary partitioned with a silica gel column (hexane/EtOAc, 1:0 → 0:1). The fraction eluted from the silica gel column with hexane/EtOAc (1:9) was further purified a silica gel column (toluene/EtOAc, 6:1 → 4:1). The fraction eluted with toluene/EtOAc (4:1) was finally purified using HPLC with Cosmosil πNAP, 10 × 250 mm, under isocratic elution [MeOH/H2O (78%/22%)], flow rate 2.0 ml min−1, UV 210 nm, to yield ceramicine J (1, 1.0 mg, 0.0002% yield) as a colorless amorphous solid. The fraction eluted with 100% EtOAc from the preliminary silica gel column was purified with another silica gel column with toluene/EtOAc (6:1) under isocratic condition. Eluent nos. 12 and 13 were finally purified using HPLC with Cosmosil πNAP, 10 × 250 mm, under isocratic elution [MeOH/H2O (78%/22%)], flow rate 2.0 ml min−1, UV 210 nm, to yield ceramicine K (2, 0.6 mg, 0.00012% yield) and ceramicine L (3, 0.8 mg, 0.00016% yield) both also as colorless amorphous solids.

Ceramicine J (1): colorless amorphous solid. [α] 25D −88 (c 0.5, CHCl3). IR (CCl4) cm−1: 2928, 1724, 1675, 1574. UV λ max (MeOH) nm (ε): 204 (13600). 1H and 13 C NMR (Tables 1, 2). ESI-MS m/z: 425 (M + H)+. HR-ESI-MS m/z: 425.2325 (M + H: calcd for C26H33O5, 425.2328).

Ceramicine K (2): colorless amorphous solid. [α] 26D +6 (c 0.4, CHCl3). IR (CCl4) cm−1: 2927, 1747, 1691, 1653. UV λ max (MeOH) nm (ε): 203 (8760). 1H and 13 C NMR (Tables 1, 2). ESI-MS m/z: 463 (M + Na)+. HR-ESI-MS m/z: 463.2136 (M + Na: calcd for C26H32O6, Na, 463.2097).

Ceramicine L (3): colorless amorphous solid. [α] 27D +14 (c 0.3, CHCl3). IR (CCl4) cm−1: 2926, 2854, 1689, 1653. UV λ max (MeOH) nm (ε): 203 (13240). 1H and 13 C NMR (Tables 1, 2). ESI-MS m/z: 435 (M + Na)+. HR-ESI-MS m/z: 435.2173 (M + Na: calcd for C25H32O5Na, 435.2147).

Cytotoxicity

Human promyelocytic leukemia cells (HL-60) were maintained in RPMI-1640 medium. Growth medium were supplemented with 10% fetal calf serum and 1% penicillin–streptomycin. The cells (5 × 103 cells/well) were cultured in Nunc disposable 96-well plate containing 90 μl of growth medium per well and were incubated at 37°C in a humidified incubator of 5% CO2. At 24 h of incubation, 10-μl aliquots of serially diluted samples (50, 25, 12.5, 6.25, and 3.125 μM) were added to the cultures. After 48 h of incubation with the samples, 15 μl of MTT (5 mg ml−1) was added to each of the wells. The cultures were incubated for another 3 h before the cells’ supernatant was removed. Thereafter 50 μl of dimethyl sulfoxide (DMSO) 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, USA) at 550 nm with reference wavelength at 700 nm. In all experiments, three replicates were used. Cisplatin was used as positive control (IC50 0.87 μM for HL-60).