HETEROCYCLES

Paper | Special issue | Vol. 86, No. 1, 2012, pp. 497-503
Received, 21st June, 2012, Accepted, 13th July, 2012, Published online, 31st July, 2012.
DOI: 10.3987/COM-12-S(N)47

■ THREE NEW BISABOLANE-TYPE SESQUITERPENOIDS FROM CREMANTHODIUM rHODOCEPHALUM (ASTERACEAE)

Yoshinori Saito, Koji Takiguchi, Xun Gong, Chiaki Kuroda, and Motoo Tori*

Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihamabouji, Yamashiro-machi, Tokushima, 770-8514, Japan

Abstract

From Cremanthodium rhodocephalum (Asteraceae) collected in Yunnan Province of China, four bisabolane-type (three of them were new) and one oplopane-type sesquiterpenoid were isolated. Two new hydroperoxides were the first example with the bisabolane-type skeleton. This is the first report about the study on C. rhodocephalum.

INTRODUCTION
Plants belonging to the genus Cremanthodium grow on high mountains (usually more than 4000 m in elevation) of Tibet and adjacent areas.1 Because their physical size is too small to collect large amounts,2,3 chemical studies on these plants have been limited so far.4-7 Several papers have appeared since 1996,4-9 but those studies were limited to C. ellisii,4 C. discoideum,5 C. potaninii,6 and C. pleurocaule.7 We reported the chemical constituents of C. helianthus8 and C. stenactinium9 in the course of our investigation on Ligularia and related plants in the Hengduan Mountains area for more than 10 years.10,11 In 2007 we had a chance to collect one sample of C. rhodocephalum in Yunnan Province, China. From 146 mg of the EtOAc extract obtained from 6.8 g of dried roots, we isolated five compounds, three of which were new. Here we describe the details of our work.

RESULTS AND DISCUSSION
The EtOAc extract of C. rhodocephalum was separated by silica gel column chromatography and HPLC to afford three new compounds 1-3 and two known compounds 412 and 5.13 (Figure 1).

Compound 1 showed a quasi-molecular ion peak at m/z 259 (M+Na)+ in the FABMS spectrum and the molecular formula was determined to be C15H24O2 by HRMS. This compound indicated a positive reaction with starch-potassium iodide, suggesting the presence of a hydroperoxy group. This was supported by a signal of -OOH at δ 7.10 (1H, s) in 1H NMR, as well as IR absorption at 3350 cm-1. The 1H NMR spectrum showed the presence of two doublet methyls (δ 1.606 and 1.609, each J = 1.6 Hz), two sets of exomethylenes (δ 4.94, 4.87; 4.85, 4.81), and an oxymethine proton (δ 4.26, br t, J = 6.6 Hz). The exomethylene protons (δ 4.85, 4.81) correlated with a methylene at C-8 (HMBC), which connected with a methylene H2-9. The C-9 was correlated with a methine at δ 4.26 (H-10) (HMBC), which also coupled with H2-9 (COSY) (Figure 2). The H3-13 correlated with C-10, C-11, and C-12 (HMBC). Thus an eight-carbon side chain unit was revealed. The exomethylene H-14 also correlated with C-6 (HMBC), and H-6 was found in the proton connectivity H-2/H2-1/H-6/H2-5/H2-4 by the COSY spectrum. The H3-15 correlated with C-2, C-3, and C-4 (HMBC), and hence a six-carbon unit was connected to a cyclohexane ring connected with the aforementioned eight-carbon unit at C-6 and C-7. Therefore, this compound has a bisabolane-type skeleton with three double bonds and a hydroperoxy group (Figure 2). The structure of 1 was established to be 10-hydroperoxybisabola-2,7(14),11-triene. The relative configuration for compound 1 was not determined.

The molecular formula of compound 2 was determined to be C15H24O2 by HRFABMS. The 1H and 13C NMR spectra indicated the presence of three double bonds (one exomethylene, one disubstituted, and one trisubstituted), three singlet methyl groups, and a quaternary carbon bearing an oxygen function. This compound also indicated a positive reaction with starch-potassium iodide, and the presence of a hydroperoxy group was also suggested by a signal of -OOH at δ 6.73 (1H, s) in 1H NMR, as well as IR absorption (3400 cm-1). The bisabolane-type skeleton was indicated by COSY and HMBC spectra as shown in Figure 2. The position of the hydroperoxy group was determined by the HMBC correlation between H-12 and C-11 (δ 81.6). The geometry of the double bond at C-9 and 10 was determined to be E by the coupling constant (J = 15.6 Hz). Thus, compound 2 was established to be 11-hydroperoxybisabola-2,7(14),9E-triene.
The molecular formula of compound 2 was determined to be C15H24O2 by HRFABMS. The 1H and 13C NMR spectra indicated the presence of three double bonds (one exomethylene, one disubstituted, and one trisubstituted), three singlet methyl groups, and a quaternary carbon bearing an oxygen function. This compound also indicated a positive reaction with starch-potassium iodide, and the presence of a hydroperoxy group was also suggested by a signal of -OOH at δ 6.73 (1H, s) in 1H NMR, as well as IR absorption (3400 cm-1). The bisabolane-type skeleton was indicated by COSY and HMBC spectra as shown in Figure 2. The position of the hydroperoxy group was determined by the HMBC correlation between H-12 and C-11 (δ 81.6). The geometry of the double bond at C-9 and 10 was determined to be E by the coupling constant (J = 15.6 Hz). Thus, compound 2 was established to be 11-hydroperoxybisabola-2,7(14),9E-triene.

The relative configuration of the cyclohexane ring in compound 3 was deduced from the JHH values and the NOEs (Figure 3). When H-6 is assumed to be in α-orientation, H-1 must be β-quasi-axial due to the large J value (J = 13.0 Hz). The other two couplings for H-6 (J = 11.0, 7.8 Hz) are due to two protons at C-5. The quasi-diaxial nature of H-1 and H-5β was revealed by the NOESY spectrum. The H-5α proton had an NOE with H-4 and the J4,5β value was nearly 0 Hz, hence, the epoxide ring should be in β-orientation. The configuration at C-8 was not determined. The CD spectrum exhibited a positive Cotton effect at 297 nm and hence the absolute configuration of the cyclohexane ring was established as shown in the formula.14
We isolated three new and two known sesquiterpenoids from C. rhodocephalum. Four of them were bisabolane-type and one was an oplopane-type. Compounds 1 and 2 were the first examples of hydroperoxides with a bisabolane skeleton. Compound 4 was reported in 1981 by Bohlmann as a mixture with the corresponding senecionate.12 We have isolated 4 in pure form, and it is worth recording the spectral data in Experimental. Compound 5 was an oplopane-type sesquiterpenoid previously isolated from Senecio implexus by Bohlmann.13
Bisabolane sesquiterpenoids have been isolated from C. ellisii4 and C. discoideum,5 and oplopane sesquiterpenoids from C. ellisii.4 We previously isolated eremophilane sesquiterpenoids from C. helianthus8 and C. stenactinium.9 The genus Cremanthodium is taxonomically very close to Ligularia.3 Our research on Ligularia in the Hengduan Mountains area clarified that most of the Ligularia species produce sesquiterpenes such as eremophilanes, bisabolanes, and oplopanes.11 Thus, the present and the preceding results on the Cremanthodium species indicate that the two genera, Ligularia and Cremanthodium, are closely related not only morphologically but also chemically. We continue to search for more Cremanthodium plants in Hengduan Mountains area.

EXPERIMENTAL
General Specific rotations and CD spectra were measured on a JASCO P-1030 and a JASCO J-725 auto recording polarimeter; IR spectra, on a Shimadzu FT/IR-8400S spectrophotometer; 1H and 13C NMR spectra (400 MHz and 100 MHz, respectively), on a Varian 400-MR spectrometer. Mass spectra, including high-resolution ones, were recorded on a JEOL JMS-700 MStation. A Chemcopak Nucleosil 50-5 column (4.6 × 250 mm) and a hexane-ethyl acetate solvent system was used for HPLC (JASCO pump system). Silica gel BW127ZH (100–270 mesh, Fuji Silysia) was used for column chromatography. Silica gel 60 F254 plates (Merck) were used for TLC.
Plant Material The plant was collected at Daxueshan in Yunnan Province of China (28˚34'48''N, 99˚49'47''E) at 4300 m altitude and identified by Xun Gong, one of the authors (voucher specimen No. 2007118 was deposited in the herbarium at Kunming Institute of Botany).
Extraction and Isolation The washed and dried roots (6.8 g) were cut into pieces and were extracted with EtOAc at rt to give a residue (145.6 mg). The extract was separated by silica gel column chromatography (hexane-EtOAc, gradient) followed by HPLC (hexane-EtOAc) to afforded compounds 1 (1.5 mg), 2 (0.9 mg), 3 (0.6 mg), 4 (0.4 mg), and 5 (4.2 mg).
10-Hydroperoxybisabola-2,7(14),11-triene (1): [α]D21 -33.6 (c 0.15, EtOH); FT-IR (KBr) 3350, 1643 cm-1; 1H NMR (C6D6) δ 7.10 (1H, s, OOH), 5.42-5.38 (1H, m, H-2), 4.95-4.93 (1H, m, H-12a), 4.87 (1H, quint, J = 1.6 Hz, H-12b), 4.85 (1H, br s, H-14a), 4.81 (1H, br s, H-14b), 4.26 (1H, br t, J = 6.6 Hz, H-10), 2.14 (1H, ddd, J = 15.6, 9.8, 5.6 Hz, H-8a), 2.14-2.00 (2H, m, H-8b, 1a), 2.10-2.02 (1H, m, H-6), 1.98-1.90 (1H, m, H-1b), 1.92-1.79 (2H, m, H-4a, 4b), 1.81-1.75 (1H, m, H-9a), 1.77-1.70 (1H, m, H-5a), 1.609 (3H, d, J = 1.6 Hz, H-13 or 15), 1.606 (3H, d, J = 1.6 Hz, H-15 or 13), 1.62-1.56 (1H, m, H-9b), 1.44 (1H, dtd, J = 12.5, 11.2, 5.7 Hz, H-5b); 13C NMR (C6D6) δ 153.7 (C-7), 144.3 (C-11), 133.5 (C-3), 121.2 (C-2), 113.9 (C-12), 108.0 (C-14), 89.0 (C-10), 40.1 (C-6), 31.8 (C-1), 31.1 (C-8), 30.9 (C-4), 29.9 (C-9), 28.6 (C-5), 23.6 (C-15), 17.2 (C-13); MS (FAB) m/z 259 [M+Na]+ (base); HRMS (FAB) obs m/z 259.1675 [M+Na]+ (calcd for C15H24O2Na 259.1674).
11-Hydroperoxybisabola-2,7(14),9E-triene (2): [α]D19 –55.1 (c 0.10, EtOH); FT-IR (KBr) 3400, 1637 cm-1; 1H NMR (C6D6) δ 6.73 (1H, s, OOH), 5.65 (1H, dt, J = 15.6, 6.6 Hz, H-9), 5.55 (1H, dt, J = 15.6, 1.2 Hz, H-10), 5.43-5.40 (1H, m, H-2), 4.88 (1H, br s, H-14a), 4.86 (1H, q, J = 1.4 Hz, H-14b), 2.71 (1H, br dd, J = 15.6, 6.6 Hz, H-8a), 2.68 (1H, br dd, J = 15.6, 6.6 Hz, H-8b), 2.16-2.10 (1H, m, H-1a), 2.15-2.08 (1H, m, H-6), 1.99-1.92 (1H, m, H-1b), 1.92-1.85 (1H, m, H-4a), 1.87-1.80 (1H, m, H-4b), 1.79-1.73 (1H, m, H-5a), 1.62 (3H, br s, H-15), 1.50-1.40 (1H, m, H-5b), 1.24 (6H, s, H-12, 13); 13C NMR (C6D6) δ 152.8 (C-7), 136.1 (C-10), 133.5 (C-3), 129.0 (C-9), 121.1 (C-2), 109.1 (C-14), 81.6 (C-11), 40.1 (C-6), 38.4 (C-8), 31.7 (C-1), 30.8 (C-4), 28.6 (C-5), 24.6 (C-12), 24.6 (C-13), 23.6 (C-15); MS (FAB) m/z 259 [M+Na]+ (base); HRMS (FAB) obs m/z 259.1687 [M+Na]+ (calcd for C15H24O2Na 259.1674).
8-Acetoxy-3,4-epoxy-1-((E)-3-methylpent-2-enoyloxy)bisabola-7(14),10-dien-2-one (3): [α]D22 –152.3 (c 0.04, CHCl3); CD: +4000 (297 nm) (EtOH); FT-IR (KBr) 1733, 1716 cm-1; 1H NMR (C6D6) δ 6.16 (1H, d, J = 13.0 Hz, H-1), 5.87 (1H, sept, J = 1.2 Hz, H-2'), 5.24-5.20 (1H, m, H-10), 5.19 (1H, dd, J = 7.3, 5.1 Hz, H-8), 5.11 (1H, br s, H-14a), 4.95 (1H, br s, H-14b), 2.65 (1H, d, J = 3.9 Hz, H-4), 2.55 (1H, ddd, J = 13.0, 11.0, 7.8 Hz, H-6), 2.43-2.33 (2H, m, H-9a, 9b), 2.09 (3H, d, J = 1.2 Hz, H-6'), 1.97 (1H, ddd, J = 15.7, 7.8, 3.9 Hz, H-5a), 1.91 (1H, dd, J = 15.7, 11.0 Hz, H-5b), 1.73 (3H, s, H-2''), 1.71 (2H, qd, J = 7.4, 1.2 Hz, H-4'), 1.65 (3H, br s, H-12), 1.56 (3H, br s, H-13), 1.21 (3H, s, H-15), 0.70 (3H, t, J = 7.4 Hz, H-5'); 13C NMR (C6D6) δ 201.5 (C-2), 169.5 (C-1''), 165.3 (C-1'), 162.8 (C-3'), 148.5 (C-7), 134.2 (C-11), 120.0 (C-10), 114.1 (C-2'), 112.3 (C-14), 75.6 (C-8), 74.3 (C-1), 64.0 (C-4), 61.5 (C-3), 44.8 (C-6), 33.6 (C-4'), 32.6 (C-9), 31.8 (C-5), 25.8 (C-12), 20.7 (C-2''), 19.0 (C-6'), 18.0 (C-13), 15.0 (C-15), 11.6 (C-5'); MS (CI) m/z 405 [M+H]+, 345, 231 (base), 203, 97; HRMS (CI) obs m/z 405.2278 [M+H]+ (calcd for C23H33O6 405.2277).
8-Acetoxy-3,4-epoxy-1-angeloyloxybisabola-7(14),10-dien-2-one (4): [α]D23 –193.0 (c 0.02, CHCl3); 1H NMR (C6D6) δ 6.18 (1H, d, J = 13.0 Hz, H-1), 5.69 (1H, qq, J = 7.4, 1.5 Hz, H-2'), 5.21-5.17 (1H, m, H-10), 5.16 (1H, dd, J = 7.8, 4.9 Hz, H-8), 5.08 (1H, br s, H-14a), 4.89 (1H, br s, H-14b), 2.63 (1H, d, J = 4.2 Hz, H-4), 2.46 (1H, ddd, J = 13.0, 10.6, 7.6 Hz, H-6), 2.39-2.30 (2H, m, H-9a, 9b), 2.00 (3H, dq, J = 7.4, 1.5 Hz, H-4'), 1.99 (1H, ddd, J = 15.6, 7.6, 4.2 Hz, H-5a), 1.94 (3H, quint, J = 1.5 Hz, H-5'), 1.90 (1H, dd, J = 15.6, 10.6 Hz, H-5b), 1.68 (3H, s, H-2''), 1.63 (3H, br s, H-12), 1.54 (3H, br s, H-13), 1.19 (3H, s, H-15); 13C NMR (C6D6) δ 201.3 (C-2), 169.5 (C-1''), 166.5 (C-1'), 148.4 (C-7), 138.4 (C-3'), 134.4 (C-11), 127.8 (C-2'), 119.9 (C-10), 111.9 (C-14), 75.0 (C-8), 74.4 (C-1), 63.9 (C-4), 61.5 (C-3), 45.4 (C-6), 32.8 (C-9), 31.4 (C-5), 25.8 (C-12), 20.8 (C-5'), 20.7 (C-2''), 18.0 (C-13), 15.9 (C-4'), 14.9 (C-15).

ACKNOWLEDGEMENTS
We thank Dr. Yasuko Okamoto, Tokushima Bunri University, for measurements of MS spectra. We are grateful to Mrs. Guowen Hu of Kunming Institute of Botany for research coordination. This work was partly supported by a Grant-in-Aid for Scientific Research from JSPS (No. 21404009).

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