1. Introduction

Caesalpinia sappan L. (Fabaceae) is widely distributed in tropical and subtropical regions, including Southeast Asia (Burma, Vietnam and Indonesia) and Southwest China [1]. The dried heartwood of the plant, named Sappan Lignum, has been used as anti-malarial, anti-inflammatory and analgesic agent in traditional Chinese medicine [2, 3]. The seeds of this plant are rich in cassane-type diterpenoids, characterized with tricyclic framework with fused furan or butenolide ring [4-6]. These diterpenoids were reported with wide bioactivities, including cytotoxicity, anti-inflammation and anti-malaria [7]. Our previous study led to the isolation of a series of cassane-type diterpenoids from the seeds of C. sappan [8-10]. As a part of our ongoing investigation on the discovery of new anti-cancer agents, five new diterpenoids (Fig. 1) were isolated from the seeds of C. sappan. Herein, the isolation and structure elucidation of the new compounds, as well as their cytotoxic effect on human breast cancer MCF-7 and human colon cancer HCT116 cell lines, were described.

2. Results and discussion

Compound 1, a white amorphous powder, had a molecular formula of C₂₁H₂₆O₆ as determined by the protonated ion peak in the HR-ESIMS (m/z 397.1634 for C₂₁H₂₆O₆Na, calcd. m/z 397.1627, see Supporting information Fig. S7), indicating nine degrees of unsaturation. The ¹H and ¹³C NMR spectra of 1 displayed resonances for a 1, 2-disubstituted furan ring [δ_H 7.50 (d, 1H, J = 1.6 Hz) and 6.36 (d, 1H, J = 1.6 Hz); δ_C 147.3, 146.8, 143.8 and 110.5], an oxygenated methylene [δ_H 4.35 (dd, 1H, J = 11.7, 2.3 Hz) and 3.73 (d, 1H, J = 11.9 Hz); δ_C 61.3], an oxygenated methine [δ_H 5.72 (s, 1H); δ_C 95.3], a methoxyl [δ_H 3.68 (s, 3H); δ_C 51.6], a secondary methyl [δ_H 1.14 (d, 3H, J = 7.2 Hz); δ_C 13.9], and two carbonyl carbons (δ_C 187.2 and 175.6) (Table 1). These data revealed that 1 possessed a cassane-type furanoditerpene framework [11], and its structure was closely related to that of phanginin A [12]. The major difference was a methylene [δ_H 2.60 (dd, J = 15.9, 11.7 Hz), 2.72 (dd, J = 15.9, 5.7 Hz); δ_C 22.5] in phanginin A replaced by a carbonyl carbon (δ_C 187.2) in 1. The HMBC correlations of this carbonyl carbon with H-9 (δ_H 2.26) and H-8 (δ_H 3.08) assigned it as C-11 (Fig. 2). The relative configuration of 1 was inferred by ROESY spectrum (Fig. 2). The correlations of H-5/H-9, H-9/H-7a and H-9/ H₃-17 indicated these protons and H₃-17 were axial and α-oriented. The hydroxy group at C-20 was determined to be β-oriented based on the NOE correlations between H-20 (δ_H 5.72) and H-1β (δ_H 3.01) (Fig. 2). Accordingly, the structure of compound 1 was established, and it was named 11-oxophanginin A.

Compound 2 was obtained as a white amorphous powder. Its molecular formula was assigned as C₂₃H₃₂O₇ according to the HRESIMS ion peak at m/z 421.2247 [M+H]⁺ (calcd. m/z 421.2226, see Supporting information Fig. S14). The olefinic proton signal at δ_H 5.80 (s, 1H) in the ¹H NMR spectrum, and the downfield carbon signals at δ_C 171.2, 170.0, 115.6, 107.7 in the ¹³C NMR spectrum (Table 1), indicated the presence of an α, β-unsaturated butenolide ring [13]. Besides, the ¹H NMR spectrum exhibited signals for one methyl [δ_H 1.10 (d, 3H, J = 7.4 Hz)], three methoxyls [δ_H 3.17 (s, 3H), 3.32 (s, 3H) and 3.69 (s, 3H)], an oxygenated methylene [δ_H 3.30 (d, 1H, J = 11.2 Hz) and 3.90 (dd, 1H, J = 11.2, 2.7 Hz)], and one oxygenated methine [δ_H 4.74 (br s, 1H)]; and the ¹³C NMR spectrum exhibited 19 resonances attributed to three quaternary carbons (δ_C 174.7, 43.9 and 38.0), five methines (δ_C 102.4, 49.5, 42.3, 40.8 and 37.3), seven methylenes (δ_C 62.0, 37.4, 36.2, 35.6, 29.9, 23.8 and 21.0) and four methyls (δ_C 55.5, 51.6, 50.9 and 11.4) (Table 1). The ¹H NMR and ¹³C NMR data were quite similar to those of caesalsappanin F [13]. After careful comparison, the ethoxy group in caesalsappanin F was replaced by a methoxy group in 2. In the HMBC spectrum, the correlations from OCH₃-19 (δ_H 3.32, s) and H-5 (δ_H 1.68, m) to C-19 (δ_C 102.4), H-9 (δ_H 1.54, m) to C-20 (δ_C 62.0), and H-20 (δ_H 3.90, dd, J = 11.2, 2.7 Hz) to C-10 (δ_C 38.0), assigned the oxygenated methine and methylene as C-19 and C-20, respectively, and a methoxy group at C-19. The relative configuration of 2 was assigned by ROSEY spectrum. The NOE correlations of H-1α /H-5, H-5/H-9 and H-9/H₃-17 indicated these protons and H₃-17 were axial and α-oriented. H-19 was determined as α-oriented based on the correlation of H-19/H-3β in the ROESY spectrum. Therefore, the structure of 2 was established, named caesalsappanin O.

The molecular formula of compound 3 was determined by its HR-ESIMS (m/z 407.2081 [M+H]⁺, calcd. for C₂₂H₃₁O₇, 407.2070, see Supporting information Fig. S21). The ¹H NMR and ¹³C NMR data (Table 1) of 3 closely resembled those of caesalsappanin G [13]. The main difference was an additional methoxy group in 3. The HMBC correlation from the methoxy (δ_H 3.19) to C-12 (δ_C 108.0) suggested it was attached at C-12. H-19 was determined as α-oriented according to the NOE correlations of H-19/H-1β and H-19/H-2β. The NOE correlations between OCH₃-12 and H₃-17 indicated the methoxy group was α-faced. Thus, the structure of 3 was characterized, named caesalsappanin P.

Compound 4 was obtained as a white amorphous powder. It molecular formula was deduced as C₂₃H₃₄O₇ from the ion peak in the HR-ESIMS (m/z 445.2199 [M+Na]⁺, calcd. 445.2202, see Supporting information Fig. S28). The ¹H NMR spectrum of 4 (Table 1) showed the presences of four methyls [δ_H 1.01 (d, 3H, J = 7.3 Hz), 3.18 (s, 3H), 3.50 (s, 3H) and 3.66 (s, 3H)], an oxygenated methylene [δ_H 3.67 (d, 1H, J = 11.8 Hz) and 4.33 (dd, 1H, J = 11.8, 2.6 Hz)], two oxygenated methines [δ_H 4.83 (d, 1H, J = 2.1 Hz) and 5.35 (d, 1H, J = 1.4 Hz)], and an olefinic proton [δ_H 5.58 (d, 1H, J = 1.3 Hz)]. The ¹³C NMR spectrum (Table 1) displayed 23 carbon signals attributed to one methyl (δ_C 12.2), three methoxyls (δ_C 50.3, 51.6 and 56.1), seven methylenes (δ_C 20.9, 23.9, 29.2, 35.7, 37.3, 38.7 and 61.5), seven methines (δ_C 35.9, 41.2, 41.7, 45.0, 97.2, 105.7 and 120.4), and five quaternary carbons (δ_C 38.3, 45.5, 110.2, 148.4 and 175.7). The NMR data of 4 showed high similarity with those of 3, except that an oxygenated methine (δ_H 5.35, d, J = 1.4 Hz/δ_C 105.7) and a methoxyl (δ_H 3.50, s/δ_C 56.1) were observed in 4 rather than the carbonyl group (δ_C 170.3) in 3. In the HMBC spectrum of 4 (Fig. 3), the correlations of H-16 (δ_H 5.35) with C-12 (δ_C 110.2), C-13 (δ_C 148.4) and C-14 (δ_C 35.9), and H-15 (δ_H 5.58) with C-8 (δ_C 41.2), C-12 (δ_C 110.2) and C-13 (δ_C 148.4), demonstrated their proximity as caesalminaxin H [14]. The correlations between H-20 and H-1β, as well as OCH₃-12 and H₃-17, in the ROESY spectrum indicated that H-20 and OCH₃-12 were α-oriented (Fig. 3). OCH₃-16 was determined as α-oriented according to the reference [14]. Thus, the structure of 4 was determined, named caesalsappanin Q.

Compound 5 and phanginin B [11] were isolated as a pair of isomers, which were inseparable. To separate them, the mixture was methylated by methyl iodide. The reaction mixture was further purified by preparative HPLC to yield the methyl derivatives of compound 5 and phanginin B, 5a and phanginin D. The IR absorptions at 1722, 1239 and 1144 cm^-1 suggested the presence of a carboxylic ester group in 5a. The 1H and ¹³C NMR spectra of 5a (Table 2) were quite similar to those of phanginin D [11]. The HMBC correlations of the oxymethine proton at δ_H 5.02 (s, 1H) with C-3 (δ_C 29.1), C-4 (δ_C 49.5), C-5 (δ_C 48.0) and C-20 (δ_C 67.4) assigned it as H-19. The relative configuration of 5a was confirmed through ROESY experiment. The NOE correlations between H-19 and H-6β suggested that H-19 was β-oriented. Thus, the structure of 5a was determined. Compound 5 was proposed as a demethylated derivative of 5a, and it was named as phanginin U.

Five known diterpenoids were identified as tomocin B (6) [15], tomocin G (7) [15], phanginin J (8) [11], 20-nor-10α-hydroperoxyphanginin K (9) [16], and 19α-hydroxy-20-O-methylphanginin A (10) [16] by comparing their spectroscopic data with those reported in the literature.

Compounds 1-4 and 5a were tested for their cytotoxic activity against MCF-7 and HCT116 cancer cell lines. All of them showed moderate cytotoxic activity against two cell lines (Table 3). Especially, compound 4 showed inhibitory effect on MCF-7 (28.8 ± 0.5%) and HCT116 (46.1 ± 5.4%) cells at the concentration of 20mmol/L.

3. Conclusion

Five new cassane-type diterpenoids, 11-oxo-phanginin A (1), caesalsappanins O-Q (2-4) and phanginin U (5), together with five known analogues (6-10), were isolated from the seeds of C. sappan. Compounds 1-3 and 5 possess a common cassane-type skeleton with a furano or an α, β-unsaturated butenolide ring, and compound 1 contains a rare carbonyl group at C-11. Compound 4 represents the first example of cassane-type diterpenoid with a 19, 20-epoxide linkage and a methoxy group at C-16 from C. sappan.

4. Experimental 4.1. General experimental procedures

Optical rotation data were obtained using an Autopol VI polarimeter. UV data were recorded with a Varian CARY 50 spectrophotometer. IR spectra were recorded on a PerkinElmer spectrum-100 FTIR spectrometer using KBr disks. NMR spectra were recorded on a Bruker Avance-600 (Bruker, Switzerland, 600 MHz for ¹H NMR, 150 MHz for ¹³C NMR) NMR spectrometers with the chemical shift values presented as δ values having TMS as an internal standard. The coupling constants (J) were given in Hz. HR-ESIMS spectra were recorded on an LTQ-Orbitrap XL spectrometer. All solvents were analytical grade (TianJing Chemical Plant, Tianjing, China). Silica gel used for column chromatography and precoated silica gel GF254 plates used for TLC were produced by Qingdao Haiyang Chemical Co., Ltd. TLC spots were viewed at 254 nm and visualized by spraying with 1% vanillin in H₂SO₄. MCI gel (CHP20P, 75-150mm, Mitsubishi Chemical Industries Ltd.) and ODS C18 gel (50 mm, YMC, Kyoto, Japan) was used for column chromatography (CC). Preparative HPLC was performed on a Shimadzu LC-20AP instrument with a SPD-M20A PDA detector. Chromatographic separation was carried out on a C18 column (19 × 250 mm, 5mm, Waters, SunFire^TM), using a gradient solvent system comprised of H₂O (A) and CH₃CN (B) at a flow rate of 10 mL/min.

4.2. Plant material

The seeds of Caesalpinia sappan Linn. were collected in Baise City, Guangxi Province, People's Republic of China, and authenticated by Professor Jingui Shen from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. A specimen was deposited at the herbarium of Institute of Chinese Medical Sciences, University of Macau (LL-20140601).

4.3. Extraction and isolation

The air-dried seeds of C. sappan (15.1 kg) were ground into powder and extracted with petroleum ether for three times to remove lipids. Then the residues were extracted three times with 80% ethanol under reflux and evaporated to afford a crude extract (500.4 g). The extract was suspended in 2.0 L H₂O and extracted with chloroform (1.5 L × 3) to yield a chloroform-soluble extract (370.0 g). The chloroform extract was subjected to CC over silica gel and eluted with petroleum ether-acetone (19:1, 15:1, 10:1, 7:1, 5:1, 1:1, v/v), to yield ten major fractions (C1 to C10). Fraction C1 was were subjected to an MCI gel column eluted with CH₃OH-H₂O (60:40, 70:30, 80:20, 90:10, 100:0, v/v) to yield subfractions C1A-C1J. Subfraction C1C was purified by preparative HPLC eluted with gradient H₂O/CH₃CN (2:3 to 0:1, v/v) to obtain compound 9 (1.3 mg). Fraction C6 (19.6 g) was subjected to CC over silica gel and eluted with petroleum ether-acetone (60:1, 40:1, 30:1, 20:1, 10:1, 0:1, v/v) to yield subfractions C6A-C6F. Fraction C6C was subjected to a silica gel column eluted with gradient petroleum etheracetone (30:1 to 10:1, v/v), and further purified using preparative HPLC to obtain 2 (3.2 mg), 3 (1.0 mg), 4 (1.2 mg) and 10 (2.6 mg). C6D was subjected to an ODS column eluted with CH₃OH/H₂O (40:60, 60:80, 80:20, 90:10, 100:0, v/v), followed by preparative HPLC to yield 1 (11.2 mg), 6 (0.8 mg) and 7 (8.9 mg). C6E was further chromatographed over silica gel eluted with gradient petroleum ether-acetone (30:1, 20:1, 10:1, v/v) to obtain 5 (120 mg) and 8 (1.1 mg).

11-Oxophanginin A (1): White amorphous powder; [α]_D²⁰ + 18.7 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 271 nm (3.93); IR ν_max (KBr) 3431 (strong, broad), 2928, 1727, 1667, 1430, 1258, 1101, 1056 cm^-1; ¹H NMR and ¹³C NMR data see Table 1; HR-ESIMS m/z 397.1634 [M+Na]⁺ (calcd. for C₂₁H₂₆O₆Na, 397.1627).

Caesalsappanin O (2): White amorphous powder; [α]_D²⁰ -6.8 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 209 nm (3.76); IR ν_max (KBr) 3430 (strong, broad), 2924, 1768, 1734, 1631, 1383, 1118, 1053 cm^-1; ¹H NMR and ¹³C NMR data see Table 1; HR-ESIMS m/z 421.2247 [M +H]⁺ (calcd. for C₂₃H₃₃O₇, 421.2226).

Caesalsappanin P (3): White amorphous powder; [α]_D²⁰ -124 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 213 nm (3.53); IR ν_max (KBr) 3448 (strong, broad), 2992, 1757, 1643, 1262, 1099, 1043 cm^-1; ¹H NMR and ¹³C NMR data see Table 1; HRSEIMS m/z 407.2081 [M+H]⁺ (calcd. for C₂₂H₃₁O₇, 407.2070).

Caesalsappanin Q (4): White amorphous powder; [α]_D²⁰ + 17.4 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 203 nm (4.07); IR ν_max (KBr) 3431 (strong, broad), 2926, 1729, 1632, 1383, 1101, 1058 cm^-1; ¹H NMR and ¹³C NMR data see Table 1; HR-ESIMS m/z 445.2199 [M +Na]⁺ (calcd. for C₂₃H₃₄O₇Na, 445.2202).

Methylphanginin U (5a): White amorphous powder; [α]_D²⁰ + 35.0 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 217 nm (3.77); IR ν_max (KBr) 3443 (strong, broad), 2926, 1731, 1632, 1383, 1254, 1101, 1090 cm^-1; ¹H NMR and ¹³C NMR data see Table 2; HRESIMS m/z 375.2162 [M+H]⁺ (calcd. for C₂₂H₃₁O₅, 375.2171).

4.4. Methylation of mixture of phanginins B and U (5)

The mixture of phanginins B and U (5) (58 mg, 0.16 mmol) was dissolved in dehydrated acetone (3 mL), and the solution was treated with powdered potassium carbonate (0.6 g, 4.3 mmol) and methyl iodide (0.6 mL, 9.6 mmol). After stirred at room temperature for 17 h under an N₂ atmosphere, the reaction mixture was evaporated under reduced pressure to remove the solvent. The residue was suspended in the ice-cooled H₂O (10 mL) and extracted with EtOAc (3 ×10 mL). The combined organic phase was washed with satd aq NaCl (3 ×15 mL), dried over MgSO₄, filtered, and evaporated in vacuo to give an oily residue, which by preparative HPLC (CH₃OH/H₂O = 85:15) gave 5a (27 mg, 45%).

4.5. Cytotoxicity bioassays

The isolates were evaluated their cytotoxic effects against MCF-7 and HCT116 cancer cell lines using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method, as described previously [8]. The human breast cancer MCF-7 cells and human colon cancer HCT116 cells were acquired from American Type Culture Collection (Manassas, VA, USA). MCF-7 and HCT116 cell lines were cultured in DMEM and RPMI1640 medium, respectively, supplemented with 10% (v/v) FBS and 1% (v/v) PenicillinStreptomycin in a standard humidified incubator with 5% CO2. Cells were dispensed in a 96 well sterile microplate (1 ×10⁴ cells/ well), and incubated with series different concentrations of tested compound or Doxorubicin^® (positive control) for 48 h at 37 ℃. After incubation, MTT reagent (1 mg/mL) was added to each well and then the 96-well plate was incubated for an additional 4 h. The purple formazan dye crystals were solubilized by the addition of 100mL of DMSO. The absorption at 570 nm was measured using a microplate reader (Perkin Elmer, 1420 Multilabel Counter Victor 3, Wellesley, MA, USA).

Competing financial interests

The authors declare no competing financial interests.

Acknowledgments

Financial support by Science and Technology Development Fund, Macao S.A.R (No. FDCT 120/2013/A3) and the Research Fund of University of Macau (Nos. MYRG2014-00020-ICMS-QRCM and MYRG2015-00153-ICMS-QRCM) are gratefully acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.04.023.