Terpenoids from the Root Bark of

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Terpenoids from the Root Bark of Pterolobium macropterum Jittra Suthiwong,† Siripit Pitchuanchom,‡ Wareeporn Wattanawongdon,§ Chariya Hahnvajanawong,§ and Chavi Yenjai*,† †

Natural Products Research Unit, Center of Excellence for Innovation in Chemistry, Department of Chemistry, Faculty of Science, and §Department of Microbiology, Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand ‡ Laboratory of Natural Products, Faculty of Science and Center of Excellence for Innovation in Chemistry, Lampang Rajabhat University, Lampang 52100, Thailand S Supporting Information *

ABSTRACT: Five new compounds, including pteroloterins A−C (1, 3, and 4), 1β-acetoxytaepeenin C (2), and 8aαhydroxycadinenal (5), and 11 known compounds were isolated from the root bark of Pterolobium macropterum. All compounds were evaluated for cytotoxicity against the cholangiocarcinoma cell lines. Compound 9 showed weak cytotoxicity against the KKU-M139 cell line with an IC50 value of 23.24 ± 0.18 μM and showed no activity against normal cells.

C

observed that crude extracts (hexanes, EtOAc, and MeOH) of the root bark of P. macropterum exhibited cytotoxicity with IC50 values ranging between 9 and 45 μg/mL. Thus, investigating the root bark of P. macropterum resulted in the isolation of four new cassane diterpenoids (1−4) and a new cadinane derivative (5). All of the pure compounds were evaluated for cytotoxicity against the CCA, KKU-M136, KKU-M159, and KKU-M216 cell lines.

holangiocarcinoma (CCA) is one of the major health problems that affects northeast Thailand. Liver fluke infection and hepatolithiasis have been identified as the etiological factors that influence the regional frequency of CCA. The liver fluke, Opisthorchis viverrini, is endemic to northeast Thailand and is closely related to the high incidence of bile duct cancer.1 The infection of O. viverrini occurs primarily in the liver, extrahepatic bile ducts, and gall bladder. The only potentially curative treatment for CCA is a surgery that is appropriate in less than 50% of cases.2 In patients with irresectable and metastatic CCA, chemotherapy has been used to control the disease and improve the patients’ survival rates. However, a relatively poor response rate of CCA to chemotherapy has been demonstrated in clinical studies.3 Therefore, a search for new and effective therapeutic agents is still needed. This has led to screening chemical constituents from Thai medicinal plants for cytotoxicity against cholangiocarcinoma cell lines.4−6 Pterolobium macropterum, which belongs to the family Leguminosae, is a medicinal plant that is found throughout China, Bhutan, India, Indonesia, and Southeast Asia. In Thailand, this plant has been used to treat toothache and fever and to promote wound healing.7 In northeast Thailand, the plant is known as “nam kra jai”. In a study on the isolation of cytotoxic agents against CCA from natural sources, it was © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Crude hexanes and EtOAc extracts from the root bark of P. macropterum were separated by column chromatography and preparative TLC, leading to five new compounds, pteroloterins A−C (1, 3, and 4), 1β-acetoxytaepeenin C (2), and 8aαhydroxycadinenal (5). In addition, 11 known compounds, including taepeenin A (6),8 taepeenin C (7),8 epi-benthaminin 2 (8),9 nortaepeenin A (9),8 neocaesalpin I methyl ester (10),10 neocaesalpin H methyl ester (11),10 neocaesalpin H (12),10 4norcadin-5-en-4-one (13),11 7-hydroxy-4-norcadin-5-en-4-one (14),11 cyperotundone (15),12 and n-hexacosyl ferulate (16),13 were isolated (Figure 1). The known compounds were Received: June 11, 2014

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Figure 1. Structures of compounds 1−16.

proton at δ 7.32 (H-11) and C-8, C-10, C-12, and C-13 were observed in the HMBC experiment, along with correlations between H-16 (δ 7.52) and C-12 and C-13 and between H-15 (δ 6.72) and C-12. In the HMBC spectrum, two methyl ester protons at δ 3.77 and 3.75 correlated with carbons at δ 172.4 and 173.6, respectively, confirming the presence of two ester groups. The 1H−1H COSY spectrum displayed correlations in the aliphatic region, representing H-1/H-2/H-3 and H-5/H-6/ H-7. The methine proton at δ 2.42 (H-5) correlated with C-4, C-6, C-10, C-18, and C-19. The 1H and 13C NMR data were similar to those of taepeenin A (6),8 except the methyl group at C-4 in 6 was replaced by a methyl ester group in 1. The relative configuration of 1 was determined based on the coupling constant between H-5 and H-6β (J = 11.6 Hz), which indicated a diaxial orientation of these two protons. The NOESY spectrum showed a cross-peak between Me-20 and CO2Me-19, which indicated that these groups were cofacial. Thus, the structure of compound 1, named pteroloterin A, was established as shown. Compound 2 was obtained as colorless needles (MeOH). A molecular formula of C23H28O6 was determined from the 13C NMR data and its quasi-molecular ion peak at m/z 423.1784 [M + Na]+ in the HRESIMS spectra, corresponding to 10 indices of hydrogen deficiency. In the IR spectrum, the absorption bands at 3447 and 1731 cm−1 indicated the

identified by spectroscopic and spectrometric data, including those obtained from 1D and 2D NMR, IR, and MS, and by comparison with literature values. Compound 1 was obtained as colorless needles (MeOH). A molecular formula of C22H26O5 was determined from the 13C NMR data and its quasi-molecular ion peak at m/z 393.1664 [M + Na]+ in the HRESIMS spectra, corresponding to 10 indices of hydrogen deficiency. The carbonyl group showed an absorption band at 1726 cm−1 in the IR spectrum. The 13C NMR and DEPT spectra showed 22 carbon signals, including two methyl, two methyl ester, five methylene, four methine (one aliphatic and three aromatic), seven quaternary (two aliphatic and five aromatic), and two carbonyl carbons. The 1H NMR data showed two singlets at δ 7.52 and 6.72, which were assigned as H-16 and H-15, respectively (Table 1). These protons were connected to carbons at δ 144.5 and 105.1 in the HMQC spectrum, which suggested a furan moiety. The singlet at δ 7.32 was assigned as H-11 and correlated to a carbon at δ 105.6 in the HMQC experiment. The 13C NMR data displayed five other aromatic carbons at δ 128.1 (C-8), 145.3 (C-9), 153.7 (C-12), 125.8 (C-13), and 128.4 (C-14) (Table 1). In the HMBC spectrum, the methyl protons at δ 2.36 (Me-17) correlated with C-8 and C-13, and correlations between the methyl protons at δ 1.13 (Me-20) and C-1, C-5, C-9, and C-10 were observed (Figure 2). Correlations between the aromatic B

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Table 1. 1H and 13C NMR Data (400 MHz, CDCl3) for Compounds 1−3 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OCH3-18 OCH3-19 OCOCH3 OCOCH3

δC, type 39.8 20.0 34.2 57.8 47.0 23.8 29.3 128.1 145.3 38.5 105.6 153.7 125.8 128.4 105.1 144.5 16.1 173.6 172.4 23.8 52.8 52.0

CH2 CH2 CH2 C CH CH2 CH2 C C C CH C C C CH CH CH3 C C CH3 CH3 CH3

2 δH (J in Hz)

1.50, 2.32, m 1.75, 2.07, m 1.47, 2.49, m 2.42, br d (11.6) 1.94, 2.11, m 2.75, 2.90, m

7.32, s

6.72, br s 7.52, br s 2.36, s

1.13, s 3.75, s 3.77, s

δC, type 77.1 24.7 40.2 53.1 48.1 68.5 39.2 123.9 145.1 37.7 105.5 154.0 126.8 128.9 105.1 144.7 16.3 176.8 13.4 27.6 52.6

CH CH2 CH2 C CH CH CH2 C C C CH C C C CH CH CH3 C CH3 CH3 CH3

21.2 CH3 170.2 C

3 δH (J in Hz)

5.19, dd (10.8, 5.6) 1.74, 1.99, m 2.28, 2.31, m 2.28, br s 4.21, br d (5.6) 2.91, d (17.2); 3.08, dd (17.2, 5.6)

7.35, s

6.73, br d (2.0) 7.54, br d (2.0) 2.36, s 1.68, s 1.68, s 3.69, s

δC, type 38.8 19.1 34.6 57.5 50.0 25.4 30.6 36.8 51.8 37.2 22.9 152.2 119.0 142.5 106.5 141.6 104.2 173.6 172.5 13.5 52.8 52.0

CH2 CH2 CH2 C CH CH2 CH2 CH CH C CH2 C C C CH CH CH2 C C CH3 CH3 CH3

δH (J in Hz) 1.50, 1.75, m 1.39, 2.40, m 2.02, 1.10, 2.31, 2.21, 1.51,

br d (12.0) 1.79, m 2.41, m br t (11.8) m

2.74, dd (16.8, 5.6); 2.47, dd (16.8, 10.8)

6.44, br d (1.6) 7.23, br s 4.87/5.07, br s

0.79, s 3.70, s 3.75, s

2.03, s

(br d, J = 5.6 Hz) was assigned as H-6 and attached to the carbon at δ 68.5 in the HMQC spectrum. This proton displayed correlations to H-5 and H-7 in the 1H−1H COSY spectrum. In the HMBC spectrum, correlations between H-7 and C-5, C-8, and C-9 were observed. The coupling constant between H-1 and H-2β was 10.8 Hz (diaxial orientation), and it was 5.6 Hz between H-1 and H-2 α (axial−equatorial orientation), indicating that the acetoxy substituent at C-1 was in the β-equatorial orientation. The orientations of H-5 and H-6 were deduced from the small coupling constants of H-5 (δ 2.28, br s), indicating that H-5 and H-6 were oriented in α-axial and α-equatorial positions, respectively. Thus, the structure of compound 2, named 1β-acetoxytaepeenin C, was established as shown. Compound 3 was obtained as an amorphous powder. A molecular formula of C22H28O5 was determined from the 13C NMR data and its quasi-molecular ion peak at m/z 395.1888 [M + Na]+ in the HRESIMS spectra, corresponding to nine indices of hydrogen deficiency. The 13C NMR and DEPT spectra showed 22 carbon signals, including a methyl, two methyl ester, seven methylene (six aliphatic and one olefinic), five methine (three aliphatic and two olefinic), five quaternary (two aliphatic, one olefinic, and two aromatic), and two carbonyl carbons (Table 1). The 1H and 13C NMR data showed the presence of a furan moiety, as observed for compounds 1 and 2. In the HMQC spectrum, two protons at δH 4.87 and 5.07 correlated with the same carbon at δC 104.2, indicating the presence of an exocyclic double bond. These protons displayed correlations with C-8 and C-13 in the HMBC spectrum (Figure 2). The methylene protons (H-11) gave rise to two doublets of doublets at δ 2.74 (dd, J = 16.8, 5.6 Hz) and 2.47 (dd, J = 16.8, 10.8 Hz). These protons displayed correlations with C-8, C-9, and C-13. Signals at δH/C 0.79/13.5 were assigned to the Me-20 group and correlated with C-1, C-5,

Figure 2. HMBC correlations of compounds 1−5.

presence of a hydroxy group and a carbonyl group, respectively. The 13C NMR and DEPT spectra showed 23 carbon signals, including four methyl, one methyl ester, three methylene, six methine (three aliphatic and three aromatic), seven quaternary (two aliphatic and five aromatic), and two carbonyl carbons. The 1H NMR data showed similar signals of aromatic protons to those of compound 1, which indicated a benzofuran moiety. The doublet of doublets at δ 5.19 (J = 10.8, 5.6 Hz) assigned to H-1 correlated with the carbon at δ 77.1 in the HMQC experiment (Table 1). The methyl group Me-20 (δH/C 1.68/ 27.6) showed correlations with C-1, C-5, C-9, and C-10 in the HMBC experiment (Figure 2). The methyl group at δH/C 2.03/ 21.2 correlated with the carbonyl at δ 170.2, indicating the presence of an acetyl group. The methyl protons, Me-19, and CO2Me-18 correlated with the ester carbonyl at δ 176.8 in the HMBC spectrum. In addition, the correlation of H-19 with C-3 and C-4 was observed. In the 1H NMR data, the signal at δ 4.21 C

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C-9, and C-10. Two singlets at δ 3.75 and 3.70 were assigned to two groups of methyl ester protons, and these protons correlated with carbons at δ 172.5 and 173.6, respectively. In the HMBC spectrum, correlations between H-5 (δC 50.0) and C-4, C-6, C-7, C-10, C-18, C-19, and C-20 were observed. The relative configuration of this compound is the same as 1, which showed trans ring fusion and an α-axial orientation of H-5 (δ 2.02, br d, J = 12.0 Hz). The β-axial orientation of H-8 was determined from the large coupling constant (J = 11.8 Hz), and the NOESY spectrum showed a cross-peak between H-8 and Me-20, which indicated that these groups were cofacial. The αorientation of H-9 was also determined from a cross-peak with H-5 in the NOESY experiment. Thus, the structure of 3, named pteroloterin B, was determined as shown. Compound 4 was obtained as an amorphous powder. It was assigned a molecular formula of C19H28O3, based on its 13C NMR data and quasi-molecular ion peak at m/z 327.1930 [M + Na]+, corresponding to six indices of hydrogen deficiency. The IR spectrum displayed the absorption band of the conjugated carbonyl group at 1667 cm−1. The 13C NMR and DEPT spectra exhibited 19 carbon signals, including three methyl, one methyl ester, six methylene, four methine (three aliphatic and one olefinic), three quaternary (one olefinic and two aliphatic), and two carbonyl carbons. The 1H NMR displayed a singlet at δ 5.83, which was assigned as H-13 and correlated with the carbon at δ 126.8 in the HMQC spectrum (Table 2). This proton showed correlations to C-8 and C-15 in the HMBC experiment (Figure 2). The methylene protons at δ 2.42 (dd, J = 15.6, 3.2 Hz, H-11a) and 2.07 (t, J = 15.6 Hz, H-11b)

correlated with C-8 and C-12 in the HMBC experiment. The correlations of the methyl protons at δ 1.89 (Me-15) to C-8 and C-14 were observed in the HMBC spectrum. Two methyl groups resonating at δ 1.19 and 0.93 were assigned as Me-17 and Me-18, respectively, and showed correlations to carbons at δ 16.7 and 14.8, respectively. Correlations between Me-17 and C-3, C-5, and C-16 were observed in the HMBC spectrum. The Me-18 protons showed correlations with C-1, C-5, C-9, and C10. The singlet at δ 3.65 correlated with the carbonyl carbon at δ 179.1 in the HMBC experiment, indicating the presence of a methyl ester group. The large coupling constant (br d, J = 12.4 Hz) of H-5 indicated the α-axial orientation of this proton. The NOESY experiment showed a correlation of the Me-18 with H8, which confirmed the cofacial orientation of these protons. Thus, the structure of 4, named pteroloterin C, was determined as shown. Compound 5 was obtained as a colorless oil with [α]23D −121 (c 0.001, CHCl3). A molecular formula of C15H24O2 was established based on the 13C NMR and HRESIMS data, corresponding to four indices of hydrogen deficiency. The absorption bands of the hydroxy group at 3418 cm−1 and the carbonyl group at 1691 cm−1 were observed in the IR spectrum. This compound contained 15 carbons, including three methyl, four methylene, five methine (four aliphatic and one olefinic), a carbonyl, an oxygenated, and an olefinic carbon. The 1H NMR data showed a singlet at δ 9.48, which was assigned as a formyl proton. It also showed an olefinic proton at δ 6.78 that correlated with a carbon at δ 148.2 (C-1) (Table 2). The 13C NMR signal at δ 144.0 was assigned as the C-2 olefinic carbon. The 1H−1H COSY spectrum showed correlations between H3/H-4/H-4a and H-5/H-6/H-7/H-8. Correlations of H-13 with C-2 and C-3 were observed in the HMBC spectrum (Figure 2). In this spectrum, the correlations of H-1 with C-3, C-13, C-4a, and C-8a confirmed the unsaturated cyclic system. The 1H NMR data displayed three methyl doublets at δ 1.06 (J = 6.8 Hz), 0.97 (J = 7.2 Hz), and 0.93 (J = 6.4 Hz), which were assigned as Me-11, Me-10, and Me-12, respectively. The HMBC spectrum exhibited correlations of H-10 and H-11 with C-8 and C-9. Correlations between H-9 and C-7, C-8, and C-8a were also observed. The COSY spectrum showed correlations between the methine proton (H-9) and Me-10 and Me-11, which confirmed the presence of an isopropyl group at C-8. The 13C NMR data showed a signal at δ 73.0, which was assigned as an oxygenated carbon (C-8a). The HMBC spectrum displayed the correlations of H-12 with C-5, C-6, and C-4a. The coupling constants and data from the NOESY experiment were used to elucidate the relative configuration of 5. The large coupling constant (J = 12.8 Hz) of H-8 is characteristic of a diaxial relationship with H-7 and indicates the equatorial orientation of the C-8 isopropyl group. The axial orientation of H-4a was confirmed by the correlation between H-4a and H-8 in the NOESY experiment. Thus, the structure of 5, named 8aα-hydroxycadinenal, was established as shown. The cytotoxicity against human CCA, KKU-M156, KKUM213, and KKU-M139 cell lines as well as normal cells (Vero cells) was tested using the modified Skehan method.14 Compound 9 showed weak cytotoxicity against the KKUM139 and KKU-M213 cell lines, with IC50 values of 23.24 ± 0.18 and 34.83 ± 0.18 μM, respectively (Table 3). In addition, this compound was inactive against normal cells. Compounds 1, 4, 5, and 10−13 exhibited cytotoxicity against the KKUM213 cell line, with IC50 values ranging from 43 to 88 μM. Compounds 6 and 11−13 exhibited cytotoxicity against KKU-

Table 2. 1H and 13C NMR Data (400 MHz, CDCl3) for Compounds 4 and 5 4 position

δC, type

5 δH (J in Hz)

1.03, 1.73, m 1.59, m 1.56, m

δC, type 148.2 CH 144.0 C 17.8 CH2

1 2 3

37.5 CH2 18.0 CH2 36.9 CH2

4

47.5 C

4a 5 6

49.1 CH 24.4 CH2

1.78, br d (12.4) 1.27, 1.56, m

50.0 CH 31.1 CH 34.9 CH2

7 8

30.1 CH2 40.0 CH

1.17, 2.19, m 2.28, m

25.1 CH2 53.1 CH

8a 9 10

53.3 CH 36.4 C

1.63 m

73.0 C 25.8 CH 20.5 CH3

11

37.5 CH2

2.07, t (15.6); 2.42, dd (15.6, 3.2)

25.0 CH3

12

200.4 C

13 14 15 16 17 18 OCH3-16

126.8 165.2 22.0 179.1 16.7 14.8 51.9

CH C CH3 C CH3 CH3 CH3

18.6 CH2

20.3 CH3 5.83, s

194.9 CH

δH (J in Hz) 6.78, s 1.97, 2.31, m 1.89, 2.04, m 1.29, m 0.89, m 1.04, 1.68, m 1.64, m 1.39, br d (12.8) 2.29, m 0.97, d (7.2) 1.06, d (6.8) 0.93, d (6.4) 9.48, s

1.89, s 1.19, s 0.93, s 3.65, s D

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Table 3. Cytotoxicity of Isolated Compounds (μM)a

a

compound

KKU-M156

KKU-M213

KKU-M139

Vero cells

crude hexanes crude EtOAc crude MeOH 1 2 4 5 6 7 9 10 11 12 13 14 15 ellipticine

45.15 ± 1.76b 8.94 ± 0.05b 38.70 ± 0.76b 83.6 ± 2.3 115.36 ± 1.79 inactivec inactivec 80.08 ± 1.44 112.72 ± 0.44 147.69 ± 0.73 inactivec 61.25 ± 0.47 82.94 ± 0.40 88.75 ± 7.66 138.58 ± 2.02 inactivec 37.92 ± 6.74

16.10 ± 0.23b 27.69 ± 0.70b 9.16 ± 0.48b 44.16 ± 0.67 98.23 ± 1.49 66.78 ± 0.09 88.77 ± 0.97 144.23 ± 9.93 105.28 ± 0.93 34.83 ± 0.18 47.58 ± 0.58 43.95 ± 0.47 68.85 ± 5.37 87.34 ± 10.81 inactivec 138.28 ± 0.96 6.58 ± 1.74

27.58 ± 0.6b 15.09 ± 0.51b 10.29 ± 0.002b 80.20 ± 0.94 155.04 ± 1.87 196.80 ± 1.47 inactivec 70.61 ± 1.10 121.51 ± 1.17 23.24 ± 0.18 inactivec 76.28 ± 11.42 77.26 ± 10.39 70.81 ± 8.68 148.16 ± 4.86 inactivec 18.27 ± 1.14

69.18 ± 3.72b 75.56 ± 0.26b 133.67 ± 0.88b 65.68 ± 6.8 inactivec 50.48 ± 1.28 inactivec 57.56 ± 3.00 inactivec inactivec 94.41 ± 4.62 59.18 ± 2.87 97.09 ± 4.85 153.89 ± 2.96 153.52 ± 5.13 inactivec 15.51 ± 2.11

Data shown are from triplicate experiments. bμg/mL. cInactive at >200 μM.

M156, with IC50 values ranging from 61 to 88 μM. Compounds 1, 6, and 11−13 displayed cytotoxicity against KKU-M139, with IC50 values ranging from 70 to 80 μM. Comparison between 13 and 14 suggested that the C-7 hydroxy group may be detrimental to cytotoxicity. In conclusion, four new cassanes (1−4) and a new cadinane (5) were isolated from the root bark of P. macropterum. Cytotoxicity against three CCA cell lines and normal cells were evaluated. Cytotoxicity results showed that 9 had the highest cytotoxicity against the KKU-M139 cell line, with an IC50 value of 23.24 ± 0.18 μM.

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EtOAc, and MeOH. On the basis of their TLC characteristics, the fractions that contained the same major compounds were combined to give five fractions, HF1 to HF5. Fraction HF2 was purified by silica gel FCC, and 10% EtOAc−hexanes was used as an eluent to give two subfractions, HF2.1 and HF2.2. Further purification of these two subfractions by preparative thin layer chromatography (PLC) and developing with 1% acetone−hexanes afforded 8 (7.4 mg, 0.0004%) and 6 (15.4 mg, 0.0008%), respectively. Fraction HF3 was purified by silica gel FCC and eluted with an isocratic system of 50% CH2Cl2− hexanes to give three subfractions, HF3.1−HF3.3. Subfractions HF3.1 and HF3.2 were further purified by PLC using 50% CH2Cl2−hexanes and 10% EtOAc−hexanes as eluting solvents to give 3 (3.5 mg, 0.0002%) and 13 (10.7 mg, 0.00005%), respectively. The purification of subfraction HF3.3 by silica gel FCC (50% CH2Cl2−hexanes) gave three subfractions, HF3.3.1−HF3.3.3. Further purification of HF3.3.1 with PLC (100% CH2Cl2) afforded 15 (13.6 mg, 0.0007%). Purifications of HF3.3.2 and HF3.3.3 were carried out on PLC using 10% EtOAc− hexanes as the eluting solvent to give 9 (33.7 mg, 0.0017%) and 10 (13.8 mg, 0.0007%), respectively. Fraction HF4 was purified by silica gel column chromatography and eluted with a gradient of 20% EtOAc−hexanes to give three subfractions, HF4.1−HF4.3. Subfraction HF4.1 was purified by Sephadex LH-20 column chromatography and eluted with 100% MeOH to afford 1 (13.2 mg, 0.0007%). Subfraction HF4.2 was purified using PLC, and 30% acetone−hexanes was used as developing solvent to give 5 (5.3 mg, 0.0003%). Subfraction HF4.3 was purified by Sephadex LH-20 column chromatography (100% MeOH) followed by PLC (100% CH2Cl2) to yield 4 (9.5 mg, 0.0005%). The crude EtOAc extract was subjected to silica gel FCC and eluted with a gradient system of hexanes, EtOAc, and MeOH to afford five subfractions, EF1−EF5. Subfraction EF2 was subjected to silica gel FCC and eluted with a gradient of 20% EtOAc−hexanes to give three subfractions, EF2.1−EF2.3. Subfraction EF2.2 was purified by PLC (10% acetone−hexanes) to yield 13 (10.7 mg, 0.0005%). Further purification of EF2.3 by silica gel FCC and elution with a gradient of 50% CH2Cl2−hexanes gave 11 (9.8 mg, 0.0005%) and 16 (9.6 mg, 0.0005%). Fraction EF3 was purified by silica gel FCC (20% EtOAc− hexanes as eluent) to give subfractions EF3.1 and EF3.2. Further purification of EF3.1 with PLC (100% CH2Cl2) yielded 7 (18.3 mg, 0.0009%). Purification of EF3.2 by reversed-phase column chromatography and elution with 20% H2O−MeOH afforded 2 (7.3 mg, 0.0004%). Fraction EF4 was purified by silica gel FCC and eluted with 30% EtOAc−hexanes to give two subfractions, EF4.1 and EF4.2. Subfraction EF4.2 was subjected to gel filtration over Sephadex LH-20 (MeOH) to afford two subfractions, EF4.2.1 and EF4.2.2. After purification of subfraction EF4.2.1 with PLC (20% EtOAc−hexanes), 14 (11.4 mg, 0.0006%) was obtained. Subfraction EF4.2.2 was

EXPERIMENTAL SECTION

General Experimental Procedures. A SANYO Gallenkamp (UK) melting point apparatus was used to determine melting points. A JASCO DIP-1000 digital polarimeter was used to identify the optical rotation. An Agilent 8453 UV−visible spectrophotometer (Germany) was used to record the UV spectra. IR spectra were recorded as thin films using a PerkinElmer Spectrum One FT-IR spectrophotometer (UK). The NMR spectra were recorded on a Varian Mercury plus spectrometer (UK) operating at 400 MHz (1H) and at 100 MHz (13C). The solvent residual peak was used for chemical shift referencing (δH 3.31, δC 49.0 for methanol-d4 and δH 7.26, δC 77.2 for CDCl3). Mass spectra were obtained on a Micromass Q-TOF 2 hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer with a Z-spray ES source (Micromass, UK). Column chromatography (CC) was carried out using silica gel 60 (100−200 mesh, Merck). TLC was performed on silica gel 60 F254 (Merck) precoated aluminum sheets. The compounds were visualized under UV light and by spraying with acidic anisaldehyde solution followed by heating. Gel filtration was carried out over Sephadex LH-20 (Pharmacia) suspended in MeOH. Distilled solvents were used throughout the separation process. Plant Material. The roots of Pterolobium macropterum were collected from Khon Kaen Province, Thailand, in August 2012. The plant was identified by Prof. Dr. Pranom Chantaranothai, Faculty of Science, Khon Kaen University, where a voucher specimen (KKU032012) is deposited. Extraction and Isolation. Air-dried roots (2.0 kg) of P. macropterum were ground and successively extracted at room temperature with hexanes (3 × 12 L), EtOAc (3 × 5 L), and MeOH (3 × 5 L). After the evaporation of solvents, the crude hexanes (29 g), EtOAc (48 g), and MeOH (125 g) extracts were obtained. The crude hexanes extract was separated by silica gel flash column chromatography (FCC) and eluted with a gradient system of hexanes, E

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recrystallized with EtOAc−hexanes (1:1) and gave 12 (14.2 mg, 0.0007%) as a colorless solid. Cytotoxicity Assay. Cells ((1−2) × 104 cells/well) were seeded in 96-well plates at 37 °C. After 24 h incubation, cells were treated with 0.1% DMSO (as solvent-control cells) and the compounds by adding 10 μL/well of each concentration in triplicate to obtain a final concentration of 0.025−20 μg/well at 37 °C. After incubation for 1 h (starting cells) and 72 h, cell growth was determined using the sulforhodamine B (SRB) assay.14 The percentage of cell viability was calculated as [(OD treated cells on day 3 − OD starting cells)/(OD control on day 3 − OD starting cells)] × 100. The 50% growth inhibitory concentrations (IC50) of the compounds on the CCA cell lines were calculated from the dose−response curves. The IC50 values were calculated through computation using the CalcuSyn software. Pteroloterin A (1): colorless needles (MeOH); mp 148−150 °C; [α]23D +51 (c 0.001, CHCl3); UV (CHCl3) λmax (log ε) 255 (4.10) nm; IR (neat) νmax 2951, 1726, 1435, 1247, 1141, 1016, 765 cm−1; 1H NMR (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Table 1; HRESIMS m/z 393.1664 [M + Na]+ (calcd for C22H26O5Na, 393.1678). 1β-Acetoxytaepeenin C (2): colorless needles (MeOH); mp 179− 183 °C; [α]22D −60 (c 0.001, CHCl3); UV (MeOH) λmax (log ε) 208 (4.33), 252 (3.92) nm; IR (neat) νmax 3447, 2924, 2854, 1731, 1668, 1245, 1140, 1032, 766 cm−1; 1H NMR (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Table 1; HRESIMS m/z 423.1784 [M + Na]+ (calcd for C23H28O6Na, 423.1784). Pteroloterin B (3): colorless, amorphous powder; [α]22D +24 (c 0.001, CHCl3); UV (CHCl3) λmax (log ε) 253 (3.71) nm; IR (neat) νmax 2951, 1724, 1436, 1249, 1225, 1141, 754 cm−1; 1H NMR (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Table 1; HRESIMS m/z 395.1888 [M + Na]+ (calcd for C22H28O5Na, 395.1834). Pteroloterin C (4): colorless, amorphous powder; [α]23D −32 (c 0.001, CHCl3); UV (CHCl3) λmax (log ε) 244 (3.89) nm; IR (neat) νmax 2932, 1723, 1667, 1437, 1380, 1247, 1177, 1142 cm−1; 1H NMR (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Table 2; HRESIMS m/z 327.1930 [M + Na]+ (calcd for C19H28O3Na, 327.1936). 8aα-Hydroxycadinenal (5): colorless oil; [α]23D −121 (c 0.001, CHCl3); UV (CHCl3) λmax (log ε) 243 (3.66) nm; IR (neat) νmax 3418, 2925, 1691, 1455, 1373, 1242, 1003 cm−1; 1H NMR (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Table 2; HRESIMS m/z 237.1834 [M + H]+ (calcd for C15H24O2Na, 237.1855).

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S Supporting Information *

1

H and 13C NMR spectra for 1−5 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +66-4320-2222-41, ext 12243. Fax: +66-4320-2373. Email: [email protected] (C. Yenjai). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Royal Golden Jubilee Scholarship (PHD/0020/ 2556) and the National Research University Project of Thailand through the Advanced Functional Materials Cluster of Khon Kaen University for financial support. The Center of Excellence for Innovation in Chemistry (PERCH-CIC), Office of the Higher Education Commission, Ministry of Education, is gratefully acknowledged. F

dx.doi.org/10.1021/np500476h | J. Nat. Prod. XXXX, XXX, XXX−XXX