Chemical components of Dysoxylum densiflorum

Introduction

The plants of genus Dysoxylum, with about 200 species, is distributed naturally in India and south-east Asia. About 10 species of this genus have been found in Yunnan province.1 According to the literatures, this genus have provided sorts of compounds, such as limonoids, ^{1, 2} steroids, ³ sesquiterpenoids, ⁴ diterpenes, ⁵ triterpenes, ⁶ triterpene glycosides, ^6d and alkaloids.⁷ Many plants of this genus have been used as traditional medicine by the indigenous.⁸ D. densiflorum, is mainly distributed in southern China, Malaysia, and Philippines. Phytochemical research on D. densiflorum led to the isolation of terpenoids, steroids and flavonoids.9 In the course of our ongoing investigation of genus Dysoxylum provided a series of bioactive chemical constituents by our lab, ^{1, 2c, 6b, 10} including nineteen compounds from the EtOAc extracts of D. densiflorum. In the present research, three new diterpenoids including two new halimanes, 5(10), 13E-halimadiene-3α, 15-diol (1) and 5(10), 14-halimadiene-3α, 13ξ-diol (2), one new labdane, 12-(3-methyl-furan)-labd-8(17)-en-19-oic acid (3), were isolated and characterized by extensive spectroscopic analysis. Compounds 1 and 2 possessed 5(10)-halimane skeleton were rare in nature since the 20-methyl rearranged labdane skeleton does not conform to the biogenetic 'isoprene rule'.¹¹ The known compounds were determined as piscidinol A, ¹² 3-oxotirucalla-7, 24-dien-23-ol, ¹³ 3β-acetoxy-betulin, ¹⁴ isocupressic acid, ¹⁵ 12-oxo-15-hydroxylabda-8(17), 13E-dien-19-oic acid, ¹⁶ 14(R), 15-dihydroxy-8(17), 12(E)-labdadien-19-oic acid, ¹⁷ 15-nor-14-oxolabda-8(17), 12E-dien-19-oic acid, ¹⁸ cryptotrienolic acid, ¹⁹ 7-hydroxy-cupressic acid, ²⁰ (+)-labda-8(17), 13(Z)-dien-15, 16-diol, ²¹ 4β-hydroxy-15-(3-methyl-2-butenyl)-aromadendr-△¹⁰⁽¹²⁾-en, ²² 4(15)-eudesmene-1β, 7α-diol, ²³ β-sitosterol, 7α-hydroxysitosterol, ²⁴ 3, 4, 5-trihydroxycinnamate, ²⁵ and 5, 6-dihydroxy-6-methyl-3-en-2-one.²⁶ Herein, we report the isolation and structural elucidation of the isolated compounds.

Results and Discussion

Compound 1, as an optically active white amorphous powder {[α] _D¹⁹ + 68.3 (c 0.172, Me₂CO)}, possessed the molecular formula C₂₀H₃₄O₂ by HREIMS at m/z 306.2566 [M]⁺, indicating four degrees of unsaturation. The IR spectrum revealed absorption bands for hydroxyl (3430 cm^-1) and olefinic bond (1631 cm^-1). The ¹H, ¹³C NMR and DEPT spectra of 1 exhibited 20 carbon resonances, assigned to one tetrasubstituted double bond [δ_C 132.4 (s), 138.0 (s)]; one trisubstituted double bond [δ_C 125.5 (d), 138.4 (s)] with a corresponding proton at δ_H 5.33 (t, J = 6.2 Hz, H-14); five methyls with corresponding four tertiary methyl protons at δ_H 1.62 (Me-16), 1.04 (Me-18), 0.94 (Me-19), and 0.82 (Me-20), and a secondary methyl protons at δ_H 0.85 (d, J = 6.9 Hz, Me-17); seven methylenes (one oxygenated); two methines (one oxygenated); and two quaternary carbons. Besides a tetrasubstituted double bond and a trisubstituted double bond, the degrees of unsaturation required two rings for the structure. Similarities of the chemical shifts and coupling constants of 1 with known compound 3ξ-hydroxy-5(10), 13E-halimadien-15-al11b revealed that 1 possesses a halimane-type diterpenoid skeleton (Tables 1 and 2). The difference was the presence of an oxygenated methylene in 1 with the chemical shift of δ_C 59.1 (t), instead of an aldehyde group in 3ξ-hydroxy-5(10), 13E-halimadien-15-al [δ_C 189.8 (d)] at C-15. This was further confirmed by the HMBC correlation from δ_H 5.33 (t, J = 6.2 Hz, H-14) and 3.47 (15-OH) to δ_C 59.1 (t, C-15), and from δ_H 4.04 (t, J = 5.7 Hz, H-15) to δ_C 138.4 (s, C-13).

According to literatures, ^{11c, 11h, 11j} the Me-20 of 1 was positioned at axial bond for reducing steric hindrance of C-9 side chain, and assigned as β-orientation temporarily. In the ROESY spectrum of 1, ROESY correlations of δ_H 0.82 (Me-20)/2.15 (H-1a) assigned the β-position of H-1a. Furthermore, cross-peaks of 1.95 (H-1b)/3.59 (3-OH), 3.59 (3-OH)/1.04 (Me-18), 1.04 (Me-18)/2.00 (H-6b), 2.00 (H-6b)/1.63 (H-8) suggested they were all located on the same face and assigned as α-position. Accordingly, the Me-17 and Me-19 were elucidated to be β-oriented. In addition, ROESY correlation of δ_H 4.04 (H-15)/1.62 (Me-16) indicated an E-configuration of △^13/14. Thus, compound 1 was elucidated to be 5(10), 13E-halimadiene-3α, 15-diol as shown in Figure 1.

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Compound 2 had an identical molecular formula C₂₀H₃₄O₂ as 1 according to its HREIMS at m/z 306.2550 [M]⁺. The ¹H and ¹³C NMR spectra of 2 (Tables 1 and 2) were close similarities to those of 1, except that the allylic alcohol moiety (R₂C=CHCH₂OH, C-13-C-16) in 1 was changed to be an oxygenated quaternary carbon (δ_C 72.8) at C-13 and a terminal double bond [δ_C 147.2 (d), 111.1 (t)] at △^14/15 in 2. The assumption was further supported by the HMBC correlations of δ_H 5.18 (dd, J = 17.3, 1.8 Hz, H-15a) and 4.94 (dd, J = 10.7, 1.8 Hz, H-15b) with δ_C 72.8 (s, C-13), of δ_H 1.13 (m, H-12b) and 1.20 (s, Me-16) with δ_C 147.2 (d, C-14). Other parts of 2 were identical to those of 1, as supported by HSQC, HMBC, and ROESY spectra. Thus, 2 was deduced as 5(10), 14-halimadiene-3α, 13ξ-diol.

Compound 3, as a white amorphous powder, exhibited the molecular formula C₂₀H₂₈O₃ by HREIMS at m/z 316.2039 [M]⁺, indicating seven degrees of unsaturation. The ¹H, ¹³C NMR and DEPT spectra of 3 (Tables 1 and 2) exhibited 20 carbon signals, ascribed to one carbonyl group [δ_C 178.7 (s)]; one exocylic double bond [δ_C 149.0 (s), 107.3 (t)], with corresponding protons at δ_H 4.75, 4.60 (s, H-17); four olefinic carbons [δ_C 113.6 (d), 114.0 (s), 140.2 (d), 151.4 (s)], with corresponding protons at δ_H 6.12 (d, J = 1.6 Hz, H-14) and 7.22 (d, J = 1.6 Hz, H-15); three tertiary methyls with corresponding methyl protons at δ_H 1.94 (Me-16), 1.22 (Me-18), and 0.72 (Me-20); six methylenes, two methines, and two quaternary carbons. As four degrees of unsaturation were accounted by one carbonyl group and three C-C double bonds, the remaining three degrees of unsaturation were attributed to three rings for 3. Comparison of the ¹H and ¹³C NMR data of 3 with those of 12-oxo-15-hydroxylabda-8(17), 13E-dien-19-oic acid16 suggested that 3 possesses a labdane skeleton. A furan moiety was suggested on the basis of four olefinic carbons at δ_C 113.6 (d), 114.0 (s), 140.2 (d), 151.4 (s), and the corresponding protons at δ_H 6.12 (d, J = 1.6 Hz, H-14) and 7.22 (d, J = 1.6 Hz, H-15). The HMBC correlations of δ_H 2.31 (overlap, H-9), 1.94 (s, Me-16), 6.12 (d, J = 1.6Hz, H-14) and 7.22 (d, J = 1.6Hz, H-15) with δ_C 151.4 (s, C-12), of δ_H 2.75 (dd, J = 15.4, 2.9 Hz, H-11a), 2.67 (d, J = 10.4 Hz, H-11b) with δ_C 114.0 (s, C-13), and of δ_H 1.94 (s, Me-16) with δ_C 113.6 (d, C-14) further permitted the assignment of a 3-methyl-furan moiety at C-12.

In the ROESY spectrum of 3, ROESY correlations of δ_H 1.46 (H-5)/1.25 (H-1b), 2.31 (H-9) suggested that they all located on the same side. Me-20 (δ_H 0.72) did not show ROESY correlation to any of above protons, but showed correlations with the proton signals at δ_H 2.75, 2.67 (2H, H-11) and 1.89 (H-1a), which placed them at another side. The A/B ring was deduced to be trans-fused, which was consistent with labdane-type diterpenoids reported.^{15, 16, 18, 27} According to the literatures, H-5 was assigned at α-position, which further assigned the H-9 to be α-oriented and Me-20 to be β-oriented. Moreover, ROESY cross-peak of δ_H 1.46 (H-5)/1.22 (Me-18) suggested the α-orientation of Me-18. Hence, compound 3 was established as 12-(3-methyl-furan)-labd-8(17)-en-19-oic acid (Figure 2).

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Experimental Section

General Experimental Procedures

Optical rotations were obtained with a Jasco P-1020 Automatic Digital Polariscope. UV spectrua was measured with a Shimadzu UV2401PC in MeOH solution. IR spectra (KBr) were obtained on a Bruker tensor-27 infrared spectrophotometer. ¹H, ¹³C, and 2D NMR spectra were recorded on a Bruker AM-400, a DRX-500 NMR and an Avance Ⅲ 600 spectrometer with TMS as internal standard. MS data were obtained on a Waters Autospec Premier P776 for HREI. Column chromatography (CC) was performed on Silica gel (200-300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China) and RP-18 gel (20-45 μm, Fuji Silysia Chemical Co., Ltd., Tokyo, Japan). Fractions were monitored by TLC (GF 254, Qingdao Marine Chemical Co., Ltd., Qingdao, China), and spots were visualized by 10% H₂SO₄-ethanol reagent.

Plant Material

The barks of Dysoxylum densiflorum was collected from Xishuangbanna Autonomous Prefecture, Yunnan Province, China, and identified by Jingyun Cui of Xishuangbanna Botanic Garden. A voucher specimen (Cui200811-18) has been deposited at Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and Isolation

The air-dried barks (5.0 kg) of Dysoxylum densiflorum was extracted with MeOH three times under normal temperature. After removal of the solvent, the extract suspended in H₂O and extracted with ethyl acetate four times. The EtOAc fraction (129.0 g) was subjected to CC on Si gel, eluted with gradient mixtures of CHCl₃-Me₂CO (1:0-0:1). According to differences in composition monitored by TLC, five fractions were obtained. Fraction Ⅲ (15.6 g) was separated to MPLC with RP-18 CC (MeOH-H₂O, 3:7-8:2), then followed by Si gel CC (petroleum ether-EtOAc, 5:1-1:1) to afford 3 (5.0 mg), 3, 4, 5-trihydroxycinnamate (12.0 mg), and two subfractions Ⅲa and Ⅲb. Subfraction Ⅲa was chromatographed on a Si gel CC (CHCl₃-Me₂CO, 12:1-8:1) to yield 7-hydroxy-cupressic acid (9.0 mg) and 5, 6-dihydroxy-6-methyl-3-en-2-one (6.0 mg). Subfraction Ⅲb was separated with a Si gel CC (petroleum ether-Me₂CO, 8:1-5:1) to get piscidinol A (47.0 mg), 4(15)-eudesmene-1β, 7α-diol (13.0 mg), and a mixture. The mixture was further chromatographed on a Si gel CC (CHCl₃-Me₂CO, 10:1-6:1) to yield isocupressic acid (20.0 mg). Fraction IV (5.3 g) was subjected to MPLC with RP-18 CC (MeOH-H₂O, 3:7-7:3) to afford a mixture. The mixture was lately separated by Si gel CC (petroleum etherMe₂CO, 4:1-2:1) to yield 15-nor-14-oxolabda-8(17), 12E-dien-19-oic acid (4.0 mg) and 3β-acetoxy-betulin (26.0 mg). Fraction V (24.0 g) was isolated with MPLC RP-18 CC (MeOH-H₂O, 2:8-7:3) to obtained different subfractions Va-c. Subfraction Va was chromatographed on a Si gel CC (CHCl₃-Me₂CO, 8:1-5:1) to yield 2 (33.0 mg) and 3-oxotirucalla-7, 24-dien-23-ol (22.0 mg). Then, subfraction Vb was purified by a Si gel CC (petroleum ether-EtOAc, 3:1-2:1) to the isolation of 1 (18.0 mg), (+)-labda-8(17), 13(Z)-dien-15, 16-diol (7.0 mg), and cryptotrienolic acid (2.0 mg). With MeOH-H₂O (4:6-6:4) as elution solvent, subfraction Vc was separated with RP-18 CC into two mixtures, one of which was further purified by a Si gel CC (petroleum ether-EtOAc, 1:1) to give the separation of 12-oxo-15-hydroxylabda-8(17), 13E-dien-19-oic acid (14.0 mg), 7α-hydroxysitosterol (24.0 mg), and β-sitosterol (425.0 mg). The other one was subjected through a Si gel CC (petroleum ether-Me₂CO, 3:1-2:1) to afford 4β-hydroxy-15-(3-methyl-2-butenyl)-aromadendr-△¹⁰⁽¹²⁾-en (14.0 mg) and 14(R), 15-dihydroxy-8(17), 12(E)-labdadien-19-oic-acid (26.0 mg).

5(10), 13E-halimadiene-3α, 15-diol (1)

a white amorphous powder; [α]_D¹⁹ + 68.3 (c 0.172, Me₂CO); IR (KBr) υ_max 3430, 2965, 2931, 1631, 1467, 1381, 1050, 1006 cm¹; ¹H (400 MHz) and ¹³C NMR (150 MHz) data (Me₂CO), see Tables 1 and 2, respectively; HREIMS m/z 306.2566 (calcd for C₂₀H₃₄O₂ [M]⁺, 306.2559).

5(10), 14-halimadiene-3α, 13ξ-diol (2)

a white amorphous powder; [α]_D²⁰ + 61.0 (c 0.120, Me₂CO); IR (KBr) υ_max 3431, 2965, 2925, 1634, 1457, 1380, 1049 cm^-1; ¹H (400 MHz) and 13 C NMR (150 MHz) data (Me₂CO), see Tables 1 and 2, respectively; HREIMS m/z 306.2550 (calcd for C₂₀H₃₄O₂ [M]⁺, 306.2559).

12-(3-methyl-furan)-labd-8(17)-en-19-oic acid (3)

a white amorphous powder; [α]_D¹⁹ -4.7 (c 0.114, Me₂CO); UV (MeOH) λ_max (log ε) 218 (3.03), 202 (3.16) nm; IR (KBr) υ_max 3440, 2958, 2934, 1693, 1632, 1449, 1266, 1181, 1151 cm^-1; ¹H (500 MHz) and ¹³C NMR (150 MHz) data (Me₂CO), see Tables 1 and 2, respectively; HREIMS m/z 316.2039 (calcd for C₂₀H₂₈O₃ [M]⁺, 316.2038).

Notes

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-013-0025-8 and is accessible for authorized users.

Acknowledgments

The authors are grateful to the Natural Science Foundation of China (81225024, 31170334, 21072198), the National Basic Research Program of China (973 Program 2009CB522300), for partly financial support, and to the analytical group of the Laboratory of Phytochemistry, Kunming Institute of Botany, for the spectral measurement.