Culcitiolides E–J, Six New Eremophilane-Type Sesquiterpene Derivatives from Senecio culcitioides

Abstract

Culcitiolides E–J (1–6), six new eremophilane-type sesquiterpenes, were isolated from the stem of Senecio culcitioides SCH. BIP. (Asteraceae). The structures were determined by detailed NMR spectral analysis. The inhibitory activities of these compounds against nuclear factor κB (NF-κB)-dependent gene expression were assessed. Culcitiolides E, H, and I potently inhibited NF-κB-induced gene expression at 20 µM.

Senecio culcitioides SCH. BIP. is distributed throughout the Peruvian Andes in South America at an altitude of 4500–5000 m. S. culcitioides belongs to the family Asteraceae and has been used as a traditional folk medicine to treat cough, bronchitis and asthma.^1–3) Nuclear factor κB (NF-κB) is a transcription factor and functions as a key regulatory element in inflammatory and immune responses and in cancer. During our search for new classes of inhibitors of NF-κB derived from natural products, we previously isolated culcitiolides A–D, eremophilane-type sesquiterpenoids from the stem of the previously uninvestigated plant S. culcitioides, and we showed that culcitiolides C and D are potent NF-κB inhibitors.⁴⁾ Our continuous effort to identify NF-κB inhibitors in S. culcitioides led to the isolation of six new eremophilane-type sesquiterpenoids exhibiting potent NF-κB inhibitory activity. Herein, we describe the isolation and structural elucidation of culcitiolides E–J and the evaluation of their inhibitory activities against NF-κB-dependent gene expression.

Results and Discussion

Culcitiolide E (1), obtained as a brownish oil, was determined to have a molecular formula of C₂₀H₂₆O₆ by high resolution (HR)-FAB-MS (m/z 385.1625 [M+Na]⁺; Calcd: 385.1627), and the IR spectrum exhibited absorption bands corresponding to carbonyl (1713 cm⁻¹) and α,β-unsaturated γ-lactone (1761 cm⁻¹) groups.^4–6) The ¹H-NMR spectrum of 1 contained signals corresponding to a tertiary methyl group [δ_H 0.81 (3H, s)], a secondary methyl group [δ_H 1.01 (3H, d, J=6.8 Hz)], three vinyl methyl groups [δ_H 1.84 (3H, d, J=1.2 Hz), 1.87 (3H, s), 2.26 (3H, d, J=1.2 Hz)], an oxymethine group [δ_H 6.29 (1H, br s)] and a trisubstituted olefinic proton [δ_H 5.95 (1H, br s)]. The ¹³C-NMR spectrum showed twenty carbon signals, including signals for four olefinic carbons (δ_C 160.4, 156.4, 124.4, 115.5), an acetal carbon (δ_C 104.0), an oxycarbon (δ_C 77.0), a ketone (δ_C 208.6) and two ester carbonyl carbons (δ_C 171.8, 165.6). The presence of a senecioyl group was deduced based on the typical ¹H- and ¹³C-NMR signals observed in the spectra for culcitiolide A [δ_H 1.84 (3H, d, J=1.2 Hz), 2.26 (3H, d, J=1.2 Hz), 5.95 (1H, s); δ_C 20.4, 27.3, 115.5, 160.4, 165.6].^4,5,7) The NMR signals of 1 had chemical shifts and couplings similar to those of culcitiolides A–D,⁴⁾ suggesting that culcitiolide E (1) is an eremophilane-type sesquiterpenoid. The planar structure of 1 was elucidated based on the heteronuclear multiple bond connectivity (HMBC) spectrum. Long-range ¹³C–¹H correlations (H-13/C-7, C-11 and C-12) indicated the presence of an α-substituted α,β-unsaturated γ-lactone moiety. The positions of the acetal and α-substituted α,β-unsaturated γ-lactone moiety were determined by the HMBC correlations of H-9/C-7, C-8 and H-6/C-7. Additionally, HMBC correlations (H-6/C-1′, H-9/C-1 and H-10/C-1) indicated that the senecioyl and ketone groups were positioned at C-6 and C-1, respectively. Thus, the planar structure of 1 was elucidated and is shown in Fig. 2. The assignments of the NMR signals are shown in Tables 1 and 2. The relative configuration of 1 was determined by the nuclear Overhauser effect spectroscopy (NOESY) spectrum (Fig. 3). The trans-decalin ring was established by the NOESY correlations from H-14 to H-3_β, H-9_β and H-10 to H-2_α, H-4, H-6, H-9_α. NOESY cross peak (H-4/H-6) suggested that the senecioyl group was directed to be β-orientation. Additionally, vinyl methyl proton (H-13) showed homoallylic coupling (J=2.0 Hz) with oxymethine proton (H-6), indicating that the mutual dihedral angle between H-6 and H-13 is approximately 90°. These evidences confirmed that hydroxy group at C-8 was α-orientation.^8,9) Based on these results, the relative configuration of 1 was determined as shown in Fig. 1. Furthermore, the absolute stereochemistry of 1 was elucidated by applying the empirical rule for the α,β-unsaturated γ-lactone of eremophilane-type sesquiterpenoids in circular dichroism (CD) spectrum. The negative Cotton effects, observed at 245 nm, substantiated the absolute configuration of C-8 to be R.^8,10)

Fig. 1. Structures of Culcitiolides A–J from Senecio culcitioides SCH. BIP.

Fig. 2. ¹H–¹H COSY (Bold), HMBC (Solid Arrows) Correlations of Culcitiolides E–I (1–5)

Fig. 3. Key NOESY Correlations of Culcitiolides E–G (1–3)

Table 1. ¹H-NMR Data of Culcitiolides E–J (1–6) (400 MHz) in C₅D₅N (δ in ppm, Multiplicities, J in Hz)

Position	1	2	3	4	5	6a)
1						2.14 (1H, m)
						1.54 (1H, m)
2	2.34 (1H, m)	2.23 (1H, m)	2.23 (1H, m)	3.45 (1H, m)	3.21 (1H, m)	1.96 (1H, m)
	2.46 (1H, m)	2.59 (1H, m)	2.59 (1H, m)	2.32 (1H, m)	2.28 (1H, m)	1.47 (1H, m)
3	1.55 (1H, m)	1.54 (1H, m)	1.54 (1H, m)	1.68 (1H, m)	1.80 (1H, m)	3.71 (1H, td, 10.2, 5.0)
	1.66 (1H, m)	1.72 (1H, m)	1.72 (1H, m)	1.61 (1H, m)	1.62 (1H, m)
4	2.15 (1H, m)	1.85 (1H, m)	1.85 (1H, m)	3.18 (1H, m)	3.18 (1H, m)
5						2.10 (1H, m)
6	6.29 (1H, br d, 2.0)	5.85 (1H, s)	5.85 (1H, s)	7.07 (1H, d, 2.0)	7.07 (1H, d, 2.0)	1.51 (1H, m)
7						2.61 (1H, m)
8						2.37 (1H, m)
						2.40 (1H, m)
9	2.12 (1H, m)	2.35 (1H, td, 13.2, 4.4)	2.35 (1H, td, 13.2, 4.4)	6.69 (1H, s)	6.67 (1H, s)
	2.82 (1H, dd, 14.0, 3.6)	2.48 (1H, dd, 13.2, 4.4)	2.48 (1H, dd, 13.2, 4.4)
10	3.24 (1H, dd, 13.0, 3.6)	3.11 (1H, dt, 13.2, 4.4)	3.11 (1H, dt, 13.2, 4.4)			2.26 (1H, br s)
11
12						4.06 (2H, s)
13	1.87 (3H, d, 2.0)	2.17 (3H, s)	2.17 (3H, s)	1.79 (3H, d, 2.0)	1.86 (3H, 2.0)	5.11 (1H, s)
						4.95 (1H, s)
14	0.81 (3H, s)	0.97 (3H, s)	0.95 (3H, s)	0.93 (3H, s)	0.93 (3H, s)	1.09 (3H, s)
15	1.01 (3H, d, 6.8)	0.81 (3H, d, 7.2)	0.81 (3H, d, 7.2)	1.00 (3H, d, 6.8)	0.94 (3H, d, 6.8)	0.97 (3H, d, 6.8)
–OMe		3.04 (3H, s)	2.97 (3H, s)
1′						4.32 (1H, d, 7.6)
2′	5.95 (1H, br s)	5.81 (1H, s)		5.97 (1H, s)		3.11 (1H, dd, 9.2, 7.6)
3′			7.01 (1H, qd, 6.8, 1.2)		7.15 (1H, q, 7.2)	3.26b) (1H, m)
4′	1.84 (3H, d, 1.2)	1.81 (3H, s)	1.67 (3H, d, 6.8, 1.2)	2.29 (3H, s)	1.75 (3H, d, 7.2)	3.31b) (1H, m)
5′	2.26 (3H, d, 1.2)	2.22 (3H, s)	1.85 (3H, s)	1.85 (3H, s)	1.95 (3H, s)	3.34b) (1H, m)
6′						3.67 (1H, dd, 11.6, 5.2)
						3.86 (1H, dd, 12.0, 2.0)

a) Measured in CD₃OD. b) Signals may be interchanged within same column.

Table 2. ¹³C-NMR Data of Culcitiolides E–J (1–6) (100 MHz in C₅D₅N)

Position	1	2	3	4	5	6a)
1	208.6	210.5	210.4	209.1	209.1	19.6
2	41.0	36.7	36.7	36.5	36.5	28.7
3	32.5	30.7	30.8	31.7	31.6	78.5
4	41.7	28.3	28.3	33.5	33.5	40.7
5	48.8	45.6	45.4	51.5	51.4	43.0
6	77.0	70.6	69.7	72.4	71.4	43.6
7	156.4	149.8	149.8	145.8	145.6	35.6
8	104.0	105.5	105.4	150.6	150.6	47.4
9	34.9	37.3	37.5	106.3	106.3	213.6
10	53.3	53.0	52.9	78.6	78.6	56.2
11	124.4	131.7	131.8	125.0	124.9	153.1
12	171.8	170.5	170.4	170.4	170.4	65.1
13	8.2	9.2	9.2	8.7	8.7	109.6
14	7.9	15.3	15.3	9.6	9.6	23.2
15	17.7	15.5	15.3	17.3	17.3	10.6
–OMe		50.4	50.4
1′	165.6	165.8	166.9	166.5	167.4	101.3
2′	115.5	115.3	128.3	115.5	128.4	75.1
3′	160.4	159.8	139.2	160.9	140.3	77.8b)
4′	27.3	27.2	12.0	27.4	14.6	71.7
5′	20.4	20.3	14.4	20.5	12.1	78.1b)
6′						62.9

a) Measured in CD₃OD. b) Signals may be interchanged within same column.

Culcitiolides F and G (2, 3) were purified as an inseparable mixture of two structural isomers. The molecular formulas of these isomers were established by HR-FAB-MS as C₂₁H₂₈O₆ (m/z 377.1942 [M+H]⁺; Calcd: 377.1964). The IR spectrum exhibited absorption bands corresponding to carbonyl (1710 cm⁻¹) and α,β-unsaturated γ-lactone (1767 cm⁻¹) groups. The NMR signals of compounds 2 and 3 were similar to those of culcitiolide E (1) except for the methoxy signal. The ¹H-NMR spectrum contained characteristic vinyl proton signals [δ_H 7.01 (1H, qd, J=6.8, 1.2 Hz) for 2/δ_H 5.81 (1H, s) for 3] that were identical to the signals for senecioyl and tigloyl groups^4,5,7,11) with the ratio of 1 to 1. These signals suggest that compound 2 is an eremophilane-type sesquiterpenoid with a senecioyl group and that 3 is a structural isomer of 2 at the tigloyl group (C-2′ and C-3′). The positions of the methoxy and acyl groups were determined based on HMBC correlations (H-16/C-8 and H-6/C-1′) (Fig. 2). The HMBC cross peaks for H-2/C-1, H-10/C-1, H-9/C-1 and H-13/C-7, H-13/C-11, H-13/C-12 confirmed the positions of the ketone and α-methyl α,β-unsaturated γ-lactone. Thus, the planar structures of 2 and 3 were determined (Fig. 2). The assignments of the NMR signals are listed in Tables 1 and 2. The relative stereochemistries of 2 and 3 were deduced from the NOESY spectrum (Fig. 3). The NOE enhancements for H-14/H-3_β, H-14/H-6, H-14/H-10, H-15/H-3_β and H-4/H-6 indicated that these compounds contain a cis-decalin ring system with a steroidal-like conformation^7,10) and that each acyl group was in the β-orientation. Furthermore, the direction of methoxy group (C-16) was determined to be β-orientation according to the above mentioned rules reported by Naya et al.^8–10) Based on these results, the relative stereochemistries of culcitiolides F and G were determined as shown in Fig. 2.

Culcitiolides H and I (4, 5) were also obtained as two isomeric compounds that could not be separated. They have the same molecular formula of C₂₀H₂₄O₆ according to the HR-FAB-MS data (m/z 361.1665 [M+H]⁺; Calcd: 361.1651). The IR spectrum exhibited absorption bands for hydroxy (3420 cm⁻¹), ketone carbonyl (1716 cm⁻¹) and α,β-unsaturated γ-lactone groups (1775 cm⁻¹). ¹H-NMR spectrum contained two characteristic signals for vinyl protons [δ_H 5.97 (1H, s) for 4 and δ_H 7.15 (1H, q, J=7.2 Hz) for 5], indicating that compounds H and I were a mixture of eremophilane-type sesquiterpenoids with a ratio of 1 : 1, with a senecioyl moiety and a tigloyl moiety in each. Further, the NMR spectra of these compounds indicated the presence of a ketone (δ_C 209.1), an oxygen-bearing quaternary carbon (δ_C 78.6) and a trisubstituted double bond group [δ_H 6.67 (1H, s); δ_C 150.6, 106.3] conjugated with an α,β-unsaturated γ-lactone [δ_C 124.9 (125.0), 145.6 (145.8), 150.6, 170.4], which was supported by the UV absorption at 276 nm.¹²⁾ These spectral data were very close to those for culcitiolide D except for the absence of a 3-methyl-2-pentenoyl group in culcitiolide D.⁴⁾ Detailed analysis of the HMBC spectra (H-6/C-1′, H-2/C-10, H-14/C-10) revealed that the senecioyl and tigloyl moieties were substituted at C-6 and that a hydroxy group was located at C-10 (Fig. 2). The 2D-NMR spectra allowed the assignments of all proton and carbon signals, as shown in Tables 1 and 2. Therefore, the planar structures of 4 and 5 were proposed (Fig. 1). The relative configurations of 4 and 5, except for the configuration of the hydroxyl group at C-10, were deduced (Fig. 2) based on the chemical shifts in the NOESY spectrum and the co-occurrence of other culcitiolides in this plant.^4,6,13)

Culcitiolide J (6), obtained as a yellow powder, was a major constituent (approximately 5%) of this plant, and its molecular formula was determined to be C₂₁H₃₄O₈ by HR-FAB-MS. The IR spectrum showed absorption bands attributed to a hydroxy group (3404 cm⁻¹) and a ketone (1697 cm⁻¹) group. The ¹H- and ¹³C-NMR spectra contained signals corresponding to a secondary methyl group [δ_H 0.97 (3H, d, J=6.8 Hz); δ_C 10.6], a tertiary methyl group [δ_H 1.09 (3H, s); δ_C 23.2], an oxymethylene group [δ_H 4.06 (2H, s); δ_C 65.1] and a ketone (δ_C 213.6) group. The presence of a sugar moiety was deduced based on the characteristic signals at δ_H 3.11 (1H, dd, J=9.2, 7.6 Hz), 3.26 (1H, m), 3.31 (1H, m), 3.34 (1H, m), 3.67 (1H, dd, J=11.6, 5.2 Hz), 3.86 (1H, dd, J=12.0, 2.0 Hz) and 4.32 (1H, d, J=7.6 Hz) in the ¹H-NMR spectrum and at δ_C 62.9, 71.7, 75.1, 77.8, 78.1 and 101.3 in the ¹³C-NMR spectrum. The acetylation of 6 yielded a pentaacetate (6a), indicating the presence of five hydroxy groups. Compound 6 was hydrolyzed with β-glucosidase to afford a sugar and an aglycon (6b), and the acetylation of the latter gave a diacetate (6c). The sugar was determined to be D-glucose by comparison with an authentic sample, and its anomeric configuration was deduced to be the β-form on the basis of the coupling constant (J=7.6 Hz, H-1′) and the hydrolysis of 6 with β-glucosidase. The ¹³C-NMR spectrum displayed 15 signals for the aglycon and 6 signals for the glycoside. Together with the molecular formula and NMR data, these data indicate that compound 6 is a bicyclic sesquiterpene glucoside. The structure of 6 was deduced mainly from the HMBC spectrum. The H-1′/C-3, H-8/C-9 and H-10/C-9 correlations indicated that a ketone and a glucose were present at the C-9 and C-3 positions, respectively. The position of the 3-hydroxy 1-isopropenyl group was determined from the HMBC correlations of H-13 with C-7, C-11 and C-12. Thus, 6 was determined to be an eremophilane-type sesquiterpene glucoside, as shown in Fig. 4a. The relative stereochemistry was elucidated based on the NOE experiment. NOE enhancements were observed between H-14/H3, H-14/H-10, H-14/H-15 and H-4/H-7, suggesting that H-3, H-10, H-14 and H-15 were β-oriented and H-4 and H-7 were α-oriented. Based on these data, the relative stereostructure was determined (Fig. 4b). The absolute configuration of 6 was determined using a modified Mosher’s method for the aglycon (6b). The (R)- and (S)-methoxytrifluoromethylphenyl acetate (MTPA) esters of 6b were prepared using conventional procedures, and the distribution patterns of the Δδ_S–R values for 6b demonstrated that 6 has a 3R-configuration¹⁴⁾ (Fig. 5).

Fig. 4. (a) Key ¹H–¹H COSY (Bold), HMBC (Solid Arrows) Correlations of Culcitiolide J (6); (b) Key NOE Correlations of Culcitiolide J (6)

Fig. 5. Differences of Chemical Shifts between (S)-MTPA Ester and (R)-MTPA Ester of Aglycon in Culcitiolide J (6b) Were Shown in ppm (500 MHz) in C₆D₆

Nuclear factor κB (NF-κB) is a transcription factor that plays a critical role in inflammatory and immune responses and in cancer. NF-κB regulates the expression of inflammation-related genes, including cytokines. We evaluated the inhibitory effects of culcitiolides E–J (1–6) on NF-κB-regulated gene expression using HeLa_NF-κB-3 cells that express the secreted alkaline phosphatase (SEAP) reporter enzyme under the control of an NF-κB responsive promoter. Culcitiolides E–J (1–6) were evaluated at 20 µM using this HeLa_NF-κB-3 reporter gene system, and each value was obtained from at least three individual assays. Culcitiolides E, H, and I potently inhibited NF-κB-responsive gene expression [for E (1); 20 µM: 100%, 5.0 µM: 37%, 2.5 µM: 5% inhibition and for a mixture of H and I (4, 5); 20 µM: 100%, 5.0 µM: 22%, 2.5 µM: 6% inhibition], but no inhibitory activity was observed for culcitiolide J. These active compounds (culcitiolides E, H, and I) have an α-substituted α,β-unsaturated γ-lactone as the common moiety. Therefore, the presence of the α,β-unsaturated γ-lactone moiety is presumed to play a crucial role in the observed inhibitory activities, as in the case of parthenolide, a known potent inhibitor of NF-κB.¹⁵⁾ These results are consistent with the activities of culcitiolides C and D reported previously.⁴⁾ Culcitiolides F and G, bearing a cis-decalin structure, weakly inhibited NF-κB activity [less than 25% inhibition at 20 µM], suggesting that the conformation of the decalin ring is an important factor affecting the inhibitory activity.

Experimental

General Procedures

General: The melting points were determined using a YANACO MP-500D melting point apparatus and are uncorrected. The optical rotations were measured on a JASCO DIP-140 polarimeter. The IR and UV spectra were recorded on HORIBA FT-720 and HITACHI U-2810 spectrometers, respectively. The HR-FAB-MS spectrum was taken on a JEOL JMS 700 MS spectrometer. The NMR spectra were recorded on a JEOL ECS400 NMR spectrometer using tetramethylsilane (TMS) as an internal standard. Silica gel, 230–400 mesh (Merck silica gel 60), and octadecyl silica (ODS), 100–200 mesh (Fuji Silisia ODS), were used for column chromatography and medium-pressure liquid chromatography, respectively. Preparative TLC (Merck 25 silica gel 60 F₂₅₄) was also used for purification. HPLC was performed on a JASCO 880-PU instrument (column: YMC PA-03, 4.6×250 mm; YMC Co., Ltd., Japan). β-Glucosidase was purchased from Wako Pure Chemical Industries, Ltd. (R)-(−)- and (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride were purchased from Wako Pure Chemical Industries, Ltd.

Plant Material

Stems of S. culcitioides SCH. BIP. were collected in June 2005 from the Peruvian Andes and were identified by Dr. Goro Hashimoto, Centro de Pesquica de Historia Natural, São Paulo, Brazil. A voucher specimen (No. OUS-1105) was deposited in the Herbarium of Okayama University of Science.

Extraction and Isolation

The shade-dried plant material (940 g) was extracted with methanol at room temperature for 3 weeks and then filtered. The filtrate was concentrated under vacuum to give 200 g of crude residue, which was subjected to silica gel column chromatography and were eluted with a gradient mixture of chloroform–MeOH to afford six fractions (F1 to F6). F2 (22.4 g) was chromatographically separated using silica gel and eluted with a mixture of n-hexane–EtOAc with increasing polarity to yield seven fractions (F21 to F27). F23 (2.57 g) was fractionated by silica gel column chromatography with a mixture of chloroform–EtOAc (30 : 1) to give five fractions (F231 to F235). F232 (0.94 g) was further subjected to column chromatography on ODS using with MeOH–H₂O (3 : 1) to give ten fractions (F2320 to F2329). F2324 (108.9 mg) was separated by column chromatography on silica gel and eluted with n-hexane–EtOAc (3 : 1) to give four fractions (F23241 to F23244). F23243 (55.8 mg) was purified by preparative TLC using chloroform–EtOAc (15 : 1) to afford a mixture (13.4 mg) of culcitiolides F (2) and G (3). F234 (0.26 g) was further separated by column chromatography on silica gel eluting with n-hexane–acetone (3 : 1) to give four fractions (F2341 to F2344). Further purification of F2342 (6.4 mg) was performed by column chromatography on silica gel using n-hexane–acetone (2 : 1) to yield a mixture (5.3 mg) of culcitiolides H (4) and I (5). F24 (6.37 g) was fractionated by silica gel column chromatography and eluted with benzene–MeOH (8 : 1) to give five fractions (F241 to F245). F243 (1.2 g) was further separated by column chromatography on ODS using MeOH–H₂O (2 : 1) to give five fractions (F2431 to F2435). F2433 (285.0 mg) was separated by silica gel column chromatography, eluting with n-hexane–EtOAc (2 : 1), to give eight fractions (F24331 to F24338). F243342 (51.2 mg) was purified by column chromatography on silica gel column using benzene–EtOAc (3 : 1) to yield culcitiolide E (1) (10.8 mg). F5 (9.8 g) was separated by column chromatography on ODS silica gel using H₂O with increasing volumes of MeOH (1 : 1, 3 : 2, 3 : 1, 5 : 1) as the eluent to give five fractions (F51 to F55). F52 (5.01 g) was purified by silica gel column chromatography using chloroform–MeOH–H₂O (20 : 1 : 0.1, 10 : 1 : 0.1, 4 : 1 : 0.1, 1 : 1 : 0.1, 1 : 2 : 0.1) to give culcitiolide J (6) (3.56 g).

Assay for NF-κB Inhibitory Activity

The NF-κB inhibitory activities of culcitiolides E–J were evaluated using HeLa_NF-κB-3 cells as described previously.¹⁶⁾ In brief, 0.5×10⁴ cells were seeded into a 96-well tissue culture plate. After 2 h, an aliquot of a test sample solution was added to each well. After 1 h, 10 µL of 800 ng/mL tumor necrosis factor-α (TNF-α) was also added to each well. After 24 h, the medium from each well was removed and assayed for secreted alkaline phosphatase (SEAP) activity according to the manufacturer’s protocol. The NF-κB inhibitory activity was defined as the percent inhibition (%) of the TNF-α-induced expression of the SEAP reporter gene, which was under the control of NF-κB transcriptional activity. The inhibitory data are presented as the average value (n=3) of three independent experiments. The SD of each population was lower than 5% of the average value. Parthenolide (1 µM), a known NF-κB inhibitor,¹⁵⁾ was used as a positive control.

Culcitiolide E (1): Brownish oil. [α]_D²⁰: −46° (c=0.43, CHCl₃). IR (neat) ν_max 1761, 1713 cm⁻¹. UV λ_max (MeOH) nm (log ε): 218 (4.22). ¹H-NMR: Table 1. ¹³C-NMR: Table 2. CD (MeOH): [θ]₂₄₅=−10607° cm²/dmol, [θ]₂₈₆=+1587° cm²/dmol. HR-FAB-MS: m/z [M+Na]⁺ Calcd for C₂₀H₂₄O₆Na: 385.1627; Found 385.1625.

Culcitiolides F (2) and G (3): Brownish oil. [α]_D²⁰: +2° (c=0.87, CHCl₃). IR (neat) ν_max 1767, 1710, 1648 cm⁻¹. UV λ_max (MeOH) nm (log ε): 218 (4.28). ¹H-NMR: Table 1. ¹³C-NMR: Table 2. HR-FAB-MS: m/z [M+H]⁺ Calcd for C₂₁H₂₇O₆: 377.1964; Found 377.1942.

Culcitiolides H (4) and I (5): Yellow oil. [α]_D²⁰: −85° (c=0.27, CHCl₃). IR (neat) ν_max 3420, 1775, 1716, 1644 cm⁻¹. UV λ_max (MeOH) nm (log ε): 220 (4.16), 276 (4.11). ¹H-NMR: Table 1. ¹³C-NMR: Table 2. HR-FAB-MS: m/z [M+H]⁺ Calcd for C₂₀H₂₅O₆: 361.1651; Found 361.1665.

Culcitiolide J (6): Light yellow powder. mp: 99–101°C [α]_D²⁰: −29° (c=0.41, MeOH). IR (KBr) ν_max 3404, 2945, 1697 cm⁻¹. ¹H-NMR: Table 1. ¹³C-NMR: Table 2. CD (MeOH): [θ]₂₉₄=−2872° cm²/dmol. HR-FAB-MS: m/z [M+Na]⁺ Calcd for C₂₁H₂₆O₆Na: 437.2152; Found 437.2154.

Acetylation of 6

A solution of 6 (29.0 mg) in dry pyridine was added to acetic anhydride (in large excess). The solution was stirred at room temperature overnight and worked up, followed by column chromatography to give the acetate (6a) (18.5 mg, yield 43%) as a white powder.

Acetate (6a) of 6: White powder. mp: 169–172°C. [α]_D²⁰: +36° (c=0.38, CHCl₃). IR (KBr) ν_max 2947, 1755 cm⁻¹. ¹H-NMR (400 MHz in CDCl₃) δ: 0.89 (3H, d, J=6.3 Hz), 1.05 (3H, s), 1.38 (1H, m), 1.44 (1H, m), 1.49 (1H, m), 1.53 (1H, m), 1.83 (1H, m), 2.00 (3H, s), 2.03 (3H, s), 2.04 (3H, s), 2.07 (3H, s), 2.10 (3H, s), 2.14 (overlapped), 2.20 (overlapped), 2.23 (1H, br s), 2.51 (1H, br d, J=14.2 Hz), 2.61 (1H, br t, J=12.7 Hz), 3.64 (1H, m), 3.48 (1H, m), 4.14 (1H, dd, J=11.7, 2.4 Hz), 4.23 (1H, dd, J=12.2, 4.9 Hz), 4.50 (1H, d, J=7.8 Hz), 4.56 (2H, s), 4.92 (1H, m), 5.02 (1H, br s), 5.07 (1H, m), 5.15 (1H, br s), 5.19 (1H, m). ¹³C-NMR (100 MHz in CDCl₃) δ: 10.0, 18.6, 20.6, 20.7, 20.7, 20.7, 20.9, 22.7, 22.7, 27.7, 34.4, 39.4, 41.7, 42.1, 46.2, 55.0, 62.1, 65.9, 68.8, 71.4, 71.4, 72.9, 79.1, 98.6, 112.9, 146.1, 169.4, 169.5, 170.3, 170.5, 170.6, 210.0. HR-FAB-MS: m/z [M+H]⁺ Calcd for C₃₁H₄₅O₁₃: 625.2861; Found 625.2833.

Enzymatic Hydrolysis of 6

A solution of 6 (75.6 mg) in 1.0 mL of acetate buffer (pH 5.0) was reacted with β-glucosidase (15.5 mg) at 38°C for 6 h. The resulting solution was extracted with water and ethyl acetate. The organic layer was evaporated under reduced pressure to give the aglycon (6b) as a yellow oil (39.5 mg, 86% yield). The aqueous layer (10 µL) was subjected to HPLC analysis using the following conditions: column temperature, 35°C; mobile phase, CH₃CN–H₂O (75 : 25, v/v); flow rate 1.0 mL/min; and column pressure, 60 kg/cm².

Aglycon (6b) of Culcitiolide J (6): Yellow oil. [α]_D²⁰: +13° (c=0.50, CHCl₃). IR (KBr) ν_max 3358, 2941, 1699 cm⁻¹. ¹H-NMR (400 MHz in CDCl₃) δ: 0.96 (3H, d, J=6.4 Hz), 1.06 (3H, s), 1.27 (1H, m), 1.43 (1H, t, J=13.6 Hz), 1.53 (1H, m), 1.56 (1H, m), 1.86 (1H, m), 2.12 (1H, m), 2.14 (1H, m), 2.25 (1H, t, J=13.6 Hz), 2.49 (1H, br dt, J=13.6, 2.4 Hz), 2.62 (1H, br t, J=13.0 Hz), 3.48 (1H, td, J=10.0, 4.4 Hz), 4.12 (2H, s), 4.92 (1H, s), 5.12 (1H, s). ¹³C-NMR (100 MHz in CDCl₃) δ: 9.9, 18.8, 22.6, 31.8, 34.0, 41.6, 41.8, 42.3, 46.5, 55.3, 64.8, 71.8, 109.5, 151.1, 211.5. CD (MeOH): [θ]₂₉₅ (−8678). HR-FAB-MS: m/z [M+Na]⁺ Calcd for C₁₅H₂₄O₃Na: 275.1623; Found 275.1617.

Acetylation of 6b

A solution of 6 (29.0 mg) in dry pyridine was added to acetic anhydride. The solution was stirred at room temperature, overnight and work-up followed by column chromatography to give an acetate (6c) (18.5 mg, yield 43%) as a white powder.

Acetate (6c) of Culcitiolide J Aglycon (6b): Light yellow oil. [α]_D²⁰: −16° (c=0.43, CHCl₃). IR (KBr) ν_max 2962, 1736 cm⁻¹. ¹H-NMR (400 MHz in CDCl₃) δ: 0.83 (3H, d, J=6.3 Hz), 1.12 (3H, s), 1.45 (1H, m), 1.52 (1H, m), 1.53 (1H, m), 1.56 (1H, m), 1.87 (1H, br d, J=10.7 Hz), 2.03 (3H, s), 2.10 (3H, s), 2.15 (overlapped), 2.17 (overlapped), 2.27 (1H, br t, J=13.5 Hz), 2.53 (1H, m), 2.57 (1H, m), 4.58 (2H, dd, J=17.9, 13.0 Hz), 4.78 (1H, m), 5.04 (1H, br s), 5.17 (1H, br s). ¹³C-NMR (100 MHz in CDCl₃) δ: 9.9, 18.5, 20.9, 21.2, 22.6, 27.8, 34.6, 38.4, 41.7, 42.0, 46.2, 54.9, 65.9, 74.6, 112.9, 145.9, 170.5, 170.7, 210.0. HR-FAB-MS: m/z [M+Na]⁺ Calcd for C₁₉H₂₈O₅Na: 359.1835; Found 359.1858.

Preparation of (R)- and (S)-MTPA Esters from 6b

A solution of 6b (5.9 mg) in CH₂Cl₂ (1.0 mL) was reacted with (R)-(−)-methoxytrifluoromethylphenyl chloride (MTPACl) (12 µL) in presence of dimethylaminopyridine (DMAP) (8.8 mg), and then the mixture was stirred at room temperature for 40 min. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel eluting with n-hexane : EtOAc=2 : 1, to give 3-(S)-MTPA ester (4.1 mg, 26% yield) of 6b.

(S)-MTPA Ester of 6b: Colorless oil. [α]_D²⁰: −51° (c=0.28, MeOH). IR (KBr) ν_max 2921, 2849, 1746, 1705, 1451 cm⁻¹. ¹H-NMR (400 MHz in C₆D₆) δ: 0.34 (3H, d, J=6.4 Hz), 0.56 (3H, s), 0.72 (1H, t, J=14.1 Hz), 1.50 (1H, br d, J=14.1 Hz), 1.60 (1H, t, J=12.8 Hz), 1.67 (1H, br q, J=12.5 Hz), 1.81 (1H, ddd, J=25.9, 12.1, 4.7 Hz), 1.96 (1H, m), 2.13 (2H, br d, J=13.1 Hz), 3.35 (3H, s), 3.45 (3H, s), 4.23 (1H, d, J=13.1 Hz), 4.44 (1H, d, J=13.2 Hz), 4.54 (1H, s), 4.88 (1H, s), 4.97 (1H, td, J=11.3, 4.9 Hz), 7.03–7.10 (6H, m, aromatic protons), 7.56 (2H, d, J=6.7 Hz), 7.73 (2H, d, J=7.9 Hz). HR-FAB-MS: m/z [M+Na]⁺ Calcd for C₃₅H₃₈F₆O₇Na: 707.2419; Found 707.2396.

Through a similar procedure, 3-(R)-MTPA ester (8.8 mg, 61% yield) of culcitiolide J aglycon was prepared from 6b (5.3 mg) by using of (S)-(+)-MTPACl (12 µL), DMAP (7.3 mg) for an hour.

(R)-MTPA Ester of 6b: Colorless oil. [α]_D²⁰: +15° (c=0.33, MeOH). IR (KBr) ν_max 2920, 2848, 1738, 1705, 1451 cm⁻¹. ¹H-NMR (400 MHz in C₆D₆) δ: 0.57 (3H, s), 0.62 (3H, d, J=6.7 Hz), 0.79 (1H, t, J=13.6 Hz), 1.17 (1H, m), 1.32 (1H, m), 1.36 (1H, m), 1.53 (1H, m), 1.59 (1H, m), 1.69 (1H, m), 1.84 (1H, br t, J=13.0 Hz), 2.03 (1H, m), 2.13 (1H, br d, J=13.7 Hz), 3.37 (3H, s), 3.39 (3H, s), 4.28 (1H, d, J=13.1 Hz), 4.47 (1H, d, J=12.8 Hz), 4.59 (1H, s), 4.91 (1H, s), 4.94 (1H, td, J=11.2, 4.6 Hz), 6.99–7.04 (2H, m, aromatic protons), 7.07–7.11 (4H, m, aromatic protons), 7.61 (2H, d, J=8.0 Hz), 7.70 (2H, d, J=8.0 Hz). HR-FAB-MS: m/z [M+Na]⁺ Calcd for C₃₅H₃₈F₆O₇Na: 707.2419; Found 707.2427.

References

• 1)  Hammond G. B., Fernandez I. D., Villegas L. F., Vaisberg A. J., J. Ethnopharmacol., 61, 17–30 (1998).
• 2)  Yang Y., Zhao L., Wang Y. F., Chang M. L., Huo C. H., Gu Y. C., Shi Q. W., Kiyota H., Chem. Biodivers., 8, 13–72 (2011).
• 3)  Hashimoto G., Nishimoto S., “Illustrated Cyclopedia of Brazilian Medicinal Plants,” Aboc-sha & Co., Ltd., Kanagawa, 2002, pp. 324–325.
• 4)  Nozaki H., Hayashi K., Kawai M., Mitsui T., Kido M., Tani H., Takaoka D., Uno H., Ohira S., Kuboki A., Matsuura N., Nat. Prod. Commun., 7, 427–430 (2012).
• 5)  Wang W. S., Gao K., Jia Z. J., J. Nat. Prod., 65, 714–717 (2002).
• 6)  Wu Q. X., Yang A. M., Shi Y. P., Tetrahedron, 61, 10529–10535 (2005).
• 7)  Bodensieck A., Kunert O., Haslinger E., Bauer R., Helv. Chim. Acta, 90, 183–195 (2007).
• 8)  Naya K., Kanazawa R., Sawada M., Bull. Chem. Soc. Jpn., 48, 3220–3225 (1975).
• 9)  Naya K., Nogi N., Makiyama Y., Takashima H., Imagawa T., Bull. Chem. Soc. Jpn., 50, 3002–3006 (1977).
• 10)  Naya K., Shimizu M., Nishino H., Takeda M., Oka S., Hirota K., Bull. Chem. Soc. Jpn., 64, 1071–1080 (1991).
• 11)  Mustakerova E., Todorova M., Tsankova E., Z. Naturforsch., C, 57, 568–570 (2002).
• 12)  Kinoshita T., Haga Y., Narimatsu S., Shimada M., Goda Y., Chem. Pharm. Bull., 53, 717–720 (2005).
• 13)  Zhao Y., Wang P., Hao X. J., Parsons S., Rankin D. W. H., Shi H., Yu H., Sevenet T., Gueritte F., Chin. Chem. Lett., 13, 754–757 (2002).
• 14)  Kusumi T., Ooi T., Ohkubo Y., Yamaguchi T., Bull. Chem. Soc. Jpn., 79, 965–980 (2006).
• 15)  Bork P. M., Schmitz M. L., Kuhnt M., Escher C., Heinrich M., FEBS Lett., 402, 85–90 (1997).
• 16)  Matsuura N., Murakami R., Katayama T., Hibino S., Choshi T., Yamada M., J. Health Sci., 55, 311–313 (2009).