Four new diterpenoids from Isodon eriocalyx var. laxiflora

Introduction

The Natural Products Library, is one of the primary sources for uncovering novel drug candidates, and is highly regarded in drug discovery.^1-3 The construction of an ent-kaurane diterpenoids library was initiated by our group in 1976, and more than 1000 pure ent-kauranoids, which includes more than 700 novel discoveries, have been identified from Isodon genus. ^{4, 5}

As one of the important plants of Isodon genus, I. eriocalyx var. laxiflora has been investigated phytochemically as it is a rich source of diterpenoids, such as 7, 20-epoxy-ent-kauranoids (laxiflorins H and I), ⁶ 3, 20-epoxy-ent-kauranoids (laxiflorins J–M), ^{7, 8} 6, 7-seco-ent-kauranoids (laxiflorins A–C), ⁹ 6, 7:8, 15-seco-ent-kauranoids (laxiflorins F and G), ¹⁰ ent-abietanoids (laxiflorin N), ¹¹ 15, 16-seco-16, 17-dinor-ent-kauranoids (neolaxiflorins D–F)¹², two unprecedented epimeric bishomoditerpene lactones with a unique C₂₂ framework (laxiflorolides A and B), ¹³ and two unprecedented ent-kaurane diterpenoids (neolaxiflorins A and B).¹⁴ Our further investigation of this plant led to the isolation of four novel ones, laxiflorins S–V (1–4), and one known compound, laxiflorin O (5)¹⁵ (Figure 1). These new compounds could be classified into 4 different types as below: tetrahomo-7, 20-epoxy-ent-kauranoid (1), 7, 20-epoxy-ent-kauranoid (2), 15, 16-seco-16, 17-dinor-entkauranoid (3), and 3, 4-seco-ent-abietane diterpenoid (4), respectively. In addition, compound 1 was the first example of ent-kauranoids bearing a unique C₂₄ carbon framework; compound 3 was the second 15, 16-seco-16, 17-dinor-entkauranoid obtained from the Isodon genus; compound 4 was the first example of 3, 4-seco-ent-abietane diterpenoid obtained from the Isodon genus plants. In this paper, we report the isolation and structure elucidation of compounds 1–5.

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Results and Discussion

Laxiflorin S (1) was obtained as an amorphous powder. The molecular formula C₂₄H₃₀O₇, has ten degrees of unsaturation, was established based on HREIMS ([M]⁺, 430.2003; calcd for C₂₄H₃₀O₇, 430.1992) and NMR spectroscopy (Tables 1 and 2). The IR spectrum of 1 indicated the presence of hydroxy groups (3480 cm^–1) and two types of carbonyl group for saturated ketone (1741 cm^–1), and conjugated ketone (1648 cm^–1), respectively.

The ¹H NMR spectrum (Tables 1 and 2) showed resonances attributed to an α, β-unsaturated ketone moiety δ_H 5.70 (¹H, ABd, J = 10.1 Hz, 6.41, ¹H, ABd, J = 10.1 Hz), two tertiary methyls at δ_H 1.08 (3H, s) and δ_H 0.80 (3H, s), and quaternary methyl at δ_H 1.75 (3H, s). In addition, the spectrum showed resonances due to a characteristic AB methylene group at (δ_H 4.21 and 3.82, each ¹H, d, J = 9.8 Hz), together with an oxygenated methine at δ_H 3.97 (dd, J = 11.3, 7.4 Hz). The analysis of the ¹³C NMR and DEPT spectra (Table 2) revealed the presence of 24 carbons, which were assigned as three methyls (δ_C 29.9, δ_C 24.1, and δ_C 29.7), six methylenes (one oxygenated at δ_C 65.6), seven methines (one oxygenated at δ_C 73.5 and two olefinic groups at δ_C 127.4 and δ_C 160.7), and eight quaternary carbons (one hemiacetal at δ_C 96.3 and four carbonyls at δ_C 197.4, δ_C 225.2, δ_C 207.5, and δ_C 206.1), which suggested that 1 is a highly oxygenated diterpenoid with a C₂₄ skeleton similar to the ent-kaurane skeletons previously reported.^{4, 9} Comparison of the NMR spectral data of 1 with those of eriocalyxin B (6)¹⁶ revealed that 1 was consistent with a 7, 20-epoxy-ent-kaurane similar to 6 by reducing the carboncarbon double bond between C-16 and C-17 of 6 and then adding a butyl-1, 3-dione group at C-17.

The HMBC spectrum of 1 showed obvious correlations from the geminal methyls Me-18 and Me-19 to C-3, C-4, and C-5, and from Me-4' to C-2' and C-3'. In addition, the AB methylene H₂-20 showed HMBC correlations with C-1, C-5, C-7, C-9, and C-10 respectively. Other HMBC correlations were noted between the AB methine H-2 and C-1, C-3, C-4, and C-10, and between H-3 and C-1, C-4, C-5, C-18, and C-19, between H-6 (δ_H 3.97) and C-4, C-5, C-7, C-8, and C-10, and between H-17 and C-13, C-15, C-16, C-1', and C-2'. These observed HMBC correlations coupled with three spin systems (i.e., (CHCH, H-2/H-3), (CHCH, H-5/H-6), and (CHCH₂CH₂CH(CHCH₂)CH₂, H-9/H₂-11/H₂-12/H-13(/H-16/H₂-17))/H₂-14)) established by ¹H-¹H COSY correlations and HSQC spectra suggested the gross structure of compound 1 (Figure 2).

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In the ROESY spectrum of compound 1, the NOE correlations for Me-19/H-6, H-6/H₂-20, H-20a/H-14a, H-13/H-16, and H-16/H₂-14 suggested that H-6, H-13, C-14, H-16, Me-19, and C-20 adopted the same α-orientation. The cross-peaks observed between H-5/H-9, H-5/Me-18, and H-9/H₂-17 in the ROESY spectrum demonstrated that H-5, H-9, C-17, and Me-18 all possessed the same β-orientation. (Figure 2) Therefore, compound 2 was 6β, 7β-dihydroxy-17-(butyl 1', 3'-dione)-7α, 20-epoxy-ent-kaur-2-en-1, 15-dione, and named laxiflorin S.

The HREIMS (m/z 448.2087, [M]⁺; calcd for C₂₄H₃₂O₈, 448.2097) of compound 2 suggested a molecular formula of C₂₄H₃₂O₈, with nine degrees of unsaturation. The ¹H and ¹³C NMR data of 2 (Tables 1 and 2) were consistent with a 7, 20-epoxy-ent-kaurane, similar to 7β-hydroxy-6β, 15β-diacetoxy-7, 20-epoxy-ent-kaur-16-en-1-one (7)¹⁷. The most notable difference is that the C-13 methine group in 7 was substituted by a hydroxy group in 2. This difference was supported by HMBC correlations n gcfgnffrom H-15 (δ_H 6.40, s) and H₂-17 (δ_H 5.81, d, J = 2.2 Hz and δ_H 5.44, d, J = 2.2 Hz) to C-13 (δ_C 75.4, s). The ROESY cross peaks correlations indicated that compounds 2 and 7 had the same relative configuration. Consequently, compound 2 was 7β, 13α-dihydroxy-6β, 15β-diacetoxy-7, 20-epoxy-ent-kaur-16-en-1-one, and it was named laxiflorin T.

The absolute configuration of neolaxiflorin D (8), a 15, 16-seco-16, 17-dinor-ent-kaurane diterpenoid isolated from the title plant¹², was confirmed by single-crystal X-ray diffraction using anomalous scattering of CuK_α radiation (CCDC 861510).^{18, 19} Using HREIMS (m/z 334.1416; calcd for C₁₈H₂₂O₆, 334.1416), the molecular formula of laxiflorin U (3) was determined to be C₁₈H₂₂O₆, indicating eight degrees of unsaturation. Its ¹H and ¹³C NMR data (Tables 1 and 2) indicated that it was structurally very similar to neolaxiflorin D (8).¹² The most notable difference was that the C-13 carbonyl group in compound 8 (δ_C 208.7, s)¹² changed into hydroxy group in compound 3 (δ_C 63.8, d). This structural assignment was supported by the observed HMBC correlations between H-13 (δ_H 5.30, m) with C-8, C-11, C-12, and C-14, coupled with three spin systems (i.e., (CHCH, H-2/H-3), (CHCH, H-5/H-6), and (CHCH₂CH₂CHCH₂, H-9/H₂-11/H₂-12/H-13/H₂-14)) established by ¹H-¹H COSY correlations and HSQC spectra. Thus compound 3 was 7, 20-epoxy-7β, 13β-dihydroxy-1-oxo-15, 16-seco-16, 17-dinor-ent-kaur-2-en-6β, 15-olide, and it was named laxiflorin U.

Laxiflorin O (5) was an ent-abietane diterpenoid isolated from I. eriocalyx var. laxiflora, and its structure was confirmed by NMR and single-crystal X-ray diffraction in 2002.¹⁵ The molecular formula of compound 4 was determined to be C₂₀H₃₂O₃ based on HREIMS (m/z 320.2343, [M]⁺; calcd. for C₂₀H₃₂O₃, 320.2351). A comparison of its corresponding ¹H and ¹³C NMR data (Tables 1 and 2) with those of laxiflorin O (5)¹⁵ indicates that compound 4 was the 3, 4-cleavage derivative of 5. The structural difference between these two compounds are that the oxygenated methine group at C-3 (δ_C 73.9, d) in 5 changed into a hydroxymethyl in 4 (δ_C 62.9, t), and saturated quaternary carbon at C-4 (δ_C 38.3, s) and C-19 (δ_C 23.5, q) in 5 changed into a double bond at C-4 (δ_C 153.5, s) and C-19 (δ_C 110.6, q) in 4. The HMBC correlations (Figure 3) of H₂-18 (δ_H 4.51, d, J = 11.6 Hz and δ_H 4.40, d, J = 11.6 Hz) with C-4, C-5, and C-19 and of H₂-19 (δ_H 5.70, br. s and δ_H 5.14, br. s) with C-4, C-5, and C-18, coupled with ¹H-¹H COSY correlations (Figure 3) (for the observed proton spin systems, H-9/H₂-11/H₂-12/H-13/H₂-14, H₂-1/H₂-2/H₂-3, and H-5/H₂-6/H-7) confirmed this result. Based on detailed analysis of ROESY data, the relative configuration of the stereogenic centers in compound 4 were determined to be the same as those in laxiflorin O (5). The correlations from Me-20 to H-11α; from H-11α to H-13α; from H-5 to H-9, and from H-9 to H-11β was observed in the ROESY spectrum, and thus H-13 and Me-20 were assumed to be α-oriented, and of H-5 and H-9 were assumed to be β-oriented. Therefore, compound 4 was 3, 16, 18-trihydroxy-3, 4-seco-ent-abieta-4(19), 7(8), 15-triene, and it was named as laxiflorin V.

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In summary, four new diterpenoids belonging to four different types as: tetrahomo-7, 20-epoxy-ent-kauranoid, 7, 20-epoxy-ent-kauranoid, 15, 16-seco-16, 17-dinor-ent-kauranoid, and 3, 4-seco-ent-abietane diterpenoid, have been isolated from I. eriocalyx var. laxiflora collected in the south-west of China. Their structures were determined by analyses of 1D and 2D NMR spectroscopic data.

Additionally, compound 1 was the first example of ent-kauranoids bearing a unique C₂₄ carbon framework and compound 4 was the first example of 3, 4-seco-ent-abietane diterpenoid obtained from the Isodon genus plants.

Experimental Section

General Experimental Procedures. Optical rotations were measured with a JASCO DIP-370 digital polarimeter. UV data were obtained on a Shimadzu UV-2401A spectrophotometer. A BioRad FtS-135 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D and 2D NMR spectra were recorded on DRX-500 and DRX-600 spectrometers. Unless otherwise noted, the chemical shifts (δ) are expressed in ppm with respect to the solvent signals. HREIMS was performed on a VG Autospec-3000 spectrometer at 70 eV. Column chromatography was performed with silica gel (100–200 mesh; Qingdao Marine Chemical, Inc., Qingdao, China). PPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column. The fractions were monitored by TLC, and the spots were visualized by heating silica gel plates sprayed with 8% H₂SO₄ in EtOH. All of the solvents including petroleum ether (60–90 ℃) were distilled prior to use.

Plant Material. The leaves of Isodon eriocalyx var. laxiflora were collected from Yunnan Province, China in September 2009. Voucher specimens (KIB20080028) were deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences and were identified by Prof. Xi-Wen Li.

Extraction and Isolation. The air-dried leaves of Isodon eriocalyx var. laxiflora (10 Kg) were extracted with 70% aqueous Me₂CO (3 × 40 L, 2 days each) at room temperature. The solvent was evaporated in vacuo to afford a crude extract, which was suspended in H₂O and then successively extracted with EtOAc and n-BuOH. The EtOAcsoluble fraction (600 g) was decolorized on an MCI gel with 90:10 MeOH:H₂O to obtain a yellow gum (427.5 g). The gum was purified by CC on silica gel column with a CHCl₃:Me₂CO gradient system consisting of 1:0, 9:1, 8:2, 7:3, 6:4, and 1:1 to yield six main fractions (A–F). Fraction C (CHCl₃:Me₂CO, 8:2; 30 g) was subjected to repeated chromatography over silica gel (CHCl₃:MeOH, from 90:1, 60:1, to 30:1) to yield fractions C1–C3. Fraction C2 (CHCl₃:MeOH, 60:1, 15 g) was separated by RP-18 CC (MeOH:H₂O, 15:85 to 1:0) to afford C2/1–C2/4. Compound 1 (10 mg) was isolated from fraction C2/3 (500 mg) using PPLC (MeCN:H₂O, 35:65, 15 mL/min) to achieve this separation. Fraction C3 (CHCl₃: MeOH, 5 g) was eluted with RP-18 CC (MeOH:H₂O, 10:90 to 1:0) yielding subfractions C3/1–C3/3. Subfraction C3/1 (1.06 g) was fractionated by repeated CC, first on silica gel column with a gradient elution with CHCl₃: isopropyl alcohol (60:1 to 20:1) to yield fractions C3/1/1– C3/1/3. Subsequently, fraction C3/1/1 (250 mg) was purified using RP-18 CC (MeOH:H₂O, 45:60) to give 4 (6 mg).

Fraction D (CHCl₃:Me₂CO 7:3, 50 g) was eluted with CHCl₃:MeOH (30:1, 20:1, and 10:1) yielding sub-fractions D1–D3. Sub-fraction D1 (CHCl₃:MeOH, 30:1, 20 g, ) was fractionated by repeated CC, first on RP-18 with a gradient elution with MeOH:H₂O (2:8 to 1:0) to yield fractions D1/1– D1/8. Subsequently, fraction D1/3 (2.27 g) was purified using a silica gel column (CHCl₃: MeOH, 50:1 to 10:1) to give subfractions D1/3/1–D1/3/8. Sub-fraction D1/3/3 was purified by PPLC (MeOH:H₂O, 40:60, 20 mL/min) to yield 5 (19 mg). Sub-fraction D2 (10 g, CHCl₃:MeOH, 20:1) was fractionated by repeated CC, first on RP-18 with gradient elution with MeOH:H₂O (2:8 to 1:0) to yield fractions D2/1-D2/5. Subsequently, fraction D2/2 (0.87 g) was purified using a silica gel column (CHCl₃: isopropyl alcohol, 30:1 to 10:1) to afford subfractions D2/2/1 (180 mg), D2/2/2 (105 mg), D2/2/3 (150 mg), and D2/2/4 (120 mg). Sub-fraction D2/2/3 was purified by RP-18 CC (MeOH:H₂O, 40:60) to yield 2 (2 mg). Sub-fraction D3 (10:1 CHCl₃:MeOH, 6 g) was purified by CC on RP-18 (15:85-1:0, MeOH-H₂O) to yield fractions D3/1–D3/6. Subsequently, fraction D3/3 (1.26 g) was purified by CC on silica gel column (30:1-10:1 CHCl₃:isopropyl alcohol) to yield subfractions D3/3/1 (750 mg), D3/3/2 (85 mg), and D3/3/3 (120 mg). compound 3 (6 mg) was precipitated from sub-fraction D3/3/1 by subsequent silica gel CC (20:1 CHCl₃:MeOH) and RP-18 (40:60 MeOH:H₂O).

Laxiflorin S (1): white, amorphous powder. [α]_D^21.0 – 53.1 (c 0.10, MeOH). UV (MeOH) λ_max (log ε): 199.2 (3.0), 237.2 (3.3) nm; IR (KBr) ν_max 3480, 2977, 2961, 2851, 1741, 1647, 1455, 1434, 1371, 1259, 1221, 1033 cm^–1; For ¹H and ¹³C spectroscopic data, see Tables 1 and 2; EIMS: m/z 430 [M]⁺; HREIMS [M]⁺ m/z 430.2003 (calcd for C₂₄H₃₀O₇, 430.1992).

Laxiflorin T (2): white, amorphous powder. [α]_D^24.5+ 1.2 (c 0.10, MeOH). UV (MeOH) λ_max (log ε): 202.6 (2.7) nm; IR (KBr) ν_max 3521, 2958, 2933, 1740, 1726, 1696, 1428, 1379, 1285, 1116, 1040 cm^–1; For ¹H and ¹³C spectroscopic data, see Tables 1 and 2; Positive ESIMS: m/z 471 [M + Na]⁺; HREIMS [M]⁺ m/z 448.2087 (calcd for C₂₄H₃₂O₈, 448.2097).

Laxiflorin U (3): white power. [α] ^22.0 _D + 21.6 (c 0.10, MeOH). UV (MeOH) λ_max (log ε): 227 (3.03) nm. IR (KBr) ν_max 3402, 2961, 2924, 1780, 1723, 1700, 1671, 1630, 1379, 1228, 1072 cm^–1; For ¹H and ¹³C spectroscopic data, see Tables 1 and 2; EIMS: m/z 334 [M]⁺; HREIMS [M]⁺ m/z 334.1416 (calcd for C₁₈H₂₂O₆, 334.1416).

Laxiflorin V (4): white, amorphous powder. [α]_D^18.3– 20.3 (c 0.10, MeOH). UV (MeOH) λ_max (log ε): 203.0 (3.1) nm; IR (KBr) ν_max 3440, 2926, 2867, 1708, 1690, 1641, 1459, 1449, 1411, 1378, 1307, 1237, 1052 cm^–1; For ¹H and ¹³C spectroscopic data, see Tables 1 and 2; Positive ESIMS: m/z 343 [M + Na]⁺; HREIMS [M]⁺ m/z 320.2343 (calcd for C₂₀H₃₂O₃, 320.2351).

Notes

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-013-0057-0 and is accessible for authorized users.

Acknowledgements

The authors are grateful to Prof. Xi-Wen Li of the Kunming Institute of Botany, Chinese Academy of Sciences, for the identification of the plant. This project was supported financially by the National Natural Science Foundation of China (No. 81172939), the West Light Foundation of the Chinese Academy of Sciences (J.X. Pu), the Major State Basic Research Development Program of China (No. 2009CB522300), the reservation talent project of Yunnan Province (2011CI043 to J.X. Pu), the Science and Technology Program of Yunnan Province (Nos. 2008IF010), and the Major Direction Projection Foundation of CAS Intellectual Innovation Project (No. KSCX2-EW-J-24 to J.X. Pu).