Minor secoiridoid aglycones from the low-polarity part of the traditional Chinese herb: Swertia mileensis

Introduction

Secoiridoids, are biogenetically derived from the cleavage of the cylcopentane ring of iridiods (generally between C-7 and C-8), and represent a large and still expanding group of monoterpenoids. Secoiridoids are widely present in mainly in Gentianales, Dipsacales, Cornales and Oleaceae.^{1, 2} They have attracted significant interest from scientists for their diverse bioactivities, and conferred a diverse array of actions such as antiviral, cardiovascular, choleretic, hypoglycemic, antiinflammatory and purgative activities, and chemotaxonomic significance.³ Secoiridoids consistently have a hemiacetal hydroxy group at C-1 position, which is unstable and apt to be glycosylated, thus most of the secoiridoids from natural resources are secoiridoid glycosides.⁴ According to the carbon numbers involved in the aglycone part, secoiridoids can be classified into three main types: Ⅰ) the normal skeleton with ten carbons; Ⅱ) skeletons with more than ten carbons in the aglycone part; and Ⅲ) skeletons with less than ten carbons. Presently, quite a few cases of secoiridoids other than C₁₀ skeleton have been reported.^5-7 Subsequently, the discovery of more secoiridoid aglycones with dissimilar skeletons will help to formulate a greater understanding of these compounds.

Previous investigations of Swertia mileensis ('Qing-YeDan' in Mandarin), which is a well-known traditional Chinese herb, endemic to the Yunnan province and documented in every edition of the Chinese Pharmacopoeia from 1977–2010, has shown that secoiridoid glycosides, xanthones and triterpenoids were their active constituents.^8-10 As an ongoing search for anti-hepatitis B virus (anti-HBV) active compounds from natural resources, our recent investigation on the active part of S. mileensis resulted in a series of novel lactones, swerilactones A–O and swerilactosides A–C.^11-17 From a biosynthetic perspective, swerilactones A and B (C₁₈ skeleton) were derived from two C₉ secoiridoids, and swerilactones H– K (C₂₉ skeleton) were condensed by two C₁₀ and one C₉ secoiridoids. In addition, swerilactone G and swerilactosides A and B also contained the characteristic C₉ secoiridoid moieties in their structures. The above analysis suggests that S. mileensis may be rich in secoiridoid aglycones, especially for the unusual C₉ skeletons. Secoiridoids without glycosylation should be present in the low-polarity part of S. mileensis, however this part was ignored in the previous investigation, for its lowcontent and less-activity. Therefore, further investigation was focused on secoiridoid aglycones and was performed on the low-polarity part of S. mileensis, resulting in eleven new (1–11), as well as six pre-existing compounds (12–17). This paper will discuss their isolation, structural elucidation, and anti-HBV properties on HepG 2.2.15 cell line in vitro.

Results and Discussion

Compound 1 had the molecular formula of C₉H₁₂O₄, which was determined by negative HRESIMS ([M – H]^–, detected at m/z 183.0686, calcd for 183.0663), indicating four degrees of unsaturation. In the ¹³C NMR (DEPT) spectrum, three quaternary carbons, one methine and five methylenes, were observed. An α, β-unsaturated δ-lactone was proposed from the characteristic carbons at δ_C 163.0, 125.3, 149.5, 65.9 and 26.8, and HMBC correlations of H-7/C-5 and C-11, and H-6/C-4, which was in accordance with the UV (222 nm) and IR (1701 and 1631 cm⁻¹) absorptions. Therefore, four residual carbons (three O-bearing methylenes and one methine) required an additional ring to fulfill the unsaturation. The connectivity of O-C(8)-C(9)-C(1)-O was constructed by the ¹H-¹H COSY experiment (H-8/H-9/H-1), of which C-9 was proposed to be attached with C-5 by HMBC correlations from H-1 and H-8 to C-5 (Figure 1). In HMBC spectrum, correlations from δ_H 4.43 and 4.29 (H-3) to δ_C 149.5 (C-5), 163.0 (C-11) and 66.5 (C-1) suggested that C-3 was directly linked to C-4 and further cyclized with C-1 by an oxygen atom. The configuration at C-9 was determined as R-form based on its optical rotation ([α]_D¹⁸ – 12.4) and in comparison to the known compounds with the similar chiral center.^{18, 19} Thus, the structure of compound 1 was determined and named as swerimilegenin A.

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Compound 2 was assigned the molecular formula of C₉H₁₄O₃ by HREIMS which showed [M]⁺ at m/z 170.0944 (calcd. 170.0943), with three degrees of unsaturation. The existence of hydroxy and carbonyl groups was verified by IR absorptions at 3430 and 1711 cm^–1. Nine carbons involving three quaternary ones, five methylenes and one methyl were detected in the ¹³C NMR (DEPT) spectrum. The characteristic signals at δ_C 166.2 (s, C-11), 156.8 (s, C-5), 126.2 (s, C-4), 65.5 (t, C-7) and 28.6 (t, C-6) enabled the α, β-unsaturated ^δ-lactone moiety. A propyl group was proposed at C-5 based on ¹ H-¹H COSY (H-10/H-8/H-9) and HMBC experiments (H-9/C-4, C-6 and H-8/C-5). One remaining oxygenated methylene (C-3) was recognized with a hydroxmethyl group and assigned at C-4 according to the HMBC correlations of H-3/C-4 and C-5. Thus, the structure of compound 2 was constructed.

Compound 3 possessed the molecular formula of C₉H₁₂O₃ (four unsaturations) deduced from the pseudo-molecular ion peak of [M + Na]⁺ (m/z 191) in ESIMS and [M + H]⁺ (m/z 169.0866) in HRESIMS. The ¹H and ¹³C NMR spectra of compound 3 were similar to those of 2, except that the hydroxymethyl (C-3) and methylene (C-9) in 2 were changed to be methyl and carbonyl in 3. Two groups of adjacent protons at δ_H 1.15 (t, J = 7.2 Hz, H-10) and 2.64 (q, J = 7.2 Hz, H-8) in ¹H NMR spectrum, together with the carbons at δ_C 204.6 (s), 35.1 (t) and 7.4 (q) in ¹³C NMR (DEPT) spectrum suggested a propionyl group which was proposed at C-5 based on the HMBC correlations of H-8/C-5 and H-6/C-9. The remaining singlet methyl was assigned at C-4 by the long range correlations of H-3 with C-4, C-5 and C-11 in HMBC experiment.

Compound 4 was obtained as a hemiacetal analogue with the molecular formula of C₉H₁₂O₄ by a positive HRESIMS experiment ([M + Na]⁺ at m/z 207.0624 and [2M + Na]⁺ at m/z 391.1245). The existence of hydroxy (3385 cm^–1) and carbonyl (1686 cm^–1) groups was specified in IR spectrum. A characteristic δ-lactone fragment was recognized from its ¹H and ¹³C NMR data (Table 1). Besides the δ-lactone part, four residual carbons involving one di-oxygenated methine (C-3), one mono-oxygenated methine (C-8), one methylene (C-9), and one methyl (C-10), were proposed to occupy a cyclic pattern in agreement with the unsaturation degree. The proton signals at δ_H 1.17 (3H, d, J = 6.3 Hz, H-10), 4.28 (1H, m, H-8), 2.24 (1H, dd, J = 18.8, 3.6 Hz, H-9a) and 2.12 (1H, dd, J = 18.8, 10.8 Hz, H-9b) in ¹H NMR spectrum, as well as the ¹ H-¹H COSY correlations of H-10/H-8/H-9 manifested the connectivity of CH₃-CH(O-)-CH₂-, which was allocated at C-5 by HMBC cross-peaks from H-8 to C-5, and from H-9 to C-4, C-5 and C-6. The hemiacetal methine (C-3) was determined to be directly linked with C-4 and further cyclized with C-8 by an O-bridge based on the HMBC experiment (H-3/C-5, C-8 and C-11; HO-3/C-4; H-8/C-3).

In order to determine the stereochemistry of C-3 and C-8, a ROESY experiment was performed, however the key correlation of H-8 or H-10 with H-3 was not detected. This phenomenon is frequently encountered in secoiridoids whose structure contains a six-member ring fused with a δ-lactone fragment. The connection of C(10)-C(8)-O-C(3)-H bears a stable W configuration which makes a homolateral H-3 and H-10 departure from each other. Meanwhile, the hydroxyl group at C-3 was apt to form intramolecule hydrogen bond with O=C(11), resulted in OH-3 apart from H-8. Therefore, no correlation of H-3/H-10 or OH-3/H-8 was observed in the ROESY experiment, despite having been located at the same orientation. In this case, the dihedral angles of H-9a/H-8 and H-9b/H-8 are about 56.8° and 176.4° according to the Chem 3D model calculation (Figure 2), which are consistent with the coupling constants of 3.6 Hz (J_H-9a/H-8) and 10.8 Hz (J_H-9b/H-8). In addition, compound 7, the dehydrated dimer of 4, was also obtained and unambiguously determined by X-ray single crystal diffraction. Compounds 4 and 7 had the almost identical NMR spectral data and similar specific rotation value (–2.06° for 4, about half of –6.65° for 7) indicating the same stereochemistry.

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Compound 5 was proposed the molecular formula of C₁₀H₁₄O₄ based on positive HRESIMS, 14 mass units (CH₂) higher than swerimilegenin D (4). Its ¹H and ¹³C NMR spectroscopic data were similar to those of 4 except for an additional methoxy group (δ_H 3.48, δ_C 55.7). With the aid of HMBC analysis, the methoxy group was deduced at C-3 by the correlation of OMe with C-3. Similar to swerimilegenin D, no correlation between H-3 (or OMe) and H-8 (or H-10) was detected in the ROESY experiment due to the W configuration. The stereocenters of C-3 and C-8 in compound 5 were deduced to be the same as those of 4 by comparing their coupling constant in ¹H NMR spectrum and [α]_D values.

Compound 6 was designated the molecular formula of C₁₀H₁₂O₅ by positive HRESIMS ([M + Na]⁺, as detected at m/z 235.0586, calcd. for 235.0582). A methoxy group was speculated by the ion peak at m/z 181 (M⁺ – OMe) in the EIMS spectrum. Its 1H and ¹³C NMR data were similar to those of 5, except for the additional carbonyl group (196.6, s) instead of methylene [δ_C 36.8 (t, C-9)] in 5. The C(9)=O was evidenced based on the HMBC correlations of H-10/C-9, H-8/C-9 and C-5. Similar to compounds 4 and 5, neither H-8/H-3 nor Me-10/H-3 was detected in its ROESY spectrum due to the stable W configuration. The stereochemistry of compound 6 was proposed to be the same as compounds 4 and 5 by the coupling constant (J_H-10/H-8 = 6.5 Hz) and optical rotation value ([α]_D²⁴ = – 4.0).

Compound 7 was determined with the molecular formula of C₁₈H₂₂O₇ from positive HRESIMS (detected at m/z 373.1258). Only nine carbons and 11 protons were displayed in the ¹³C and ¹H NMR spectra, indicating a symmetrical skeleton. Its ¹H and ¹³C NMR spectral data were almost identical with those of 4, indicating the similar structures. The above analysis together with its molecular formula of C₁₈H₂₂O₇ (18 mass units less than two molecules of 4) suggested that compound 7 should be the dehydrated dimer of 4, which was finally confirmed by extensive 2D NMR experiment and X-ray single crystal diffraction (Figure 3).

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Compound 8 possessed the molecular formula of C₁₀H₁₂O₄ in accordance with the ion peaks of M⁺ at m/z 196 in EIMS and [M + Na]⁺ at m/z 219.0641 in HRESIMS. The presence of OH and C=O groups was evidenced by the absorption bands at 3362 and 1699 cm^–1 in IR spectrum. An obvious α, β-unsaturated δ-lactone fragment was revealed from its 1H and ¹³C NMR spectroscopic data (Table 2). In addition, one CH₃CH=C moiety was proposed to be linked with C-5 and C-1 from the HMBC correlations of H-8 with C-5, H-6 with C-9 and H-1 with C-5 and C-8. The HMBC correlations of H-3 with C-4, C-5 and C-1 revealed the linkage of C(4)-C(3)-OC(1). The Z-form of the double bond between C-8 and C-9 was determined by the correlation of H-8 with H-6 in the ROESY spectrum.

Compound 9 had the molecular formula of C₁₁H₁₄O₄ as a result of a positive HRESIMS experiment, with an additional CH₂ moiety compared to compound 8. Its IR, UV and NMR spectroscopic data were similar to 8, except for an additional methoxy group. In HMBC spectrum, the correlations of OCH₃/C-1 and H-1/OCH₃ indicated that the methoxy group was located at C-1 position. Finally, its structure was determined by an X-ray single crystal diffraction experiment.

Compound 10 was proposed with a molecular formula of C₁₀H₁₄O₄ by HREIMS at m/z 198.0897. The IR spectrum displayed the absorption bands at 3472, 1679, 1595 and 1404 cm ^–1 denoting the presence of OH, C=O and C=C groups. Its NMR spectroscopic data was close to that of dihydroepinaucledal (14), indicating a similar structure. From its ¹H-¹H COSY and HMBC experiments, compound 10 was established the same planar structure as 14. Therefore, their ¹H and ¹³C NMR spectral differences (Table 3) might be ascribed to the stereochemical variation. In the ROESY spectrum, the correlations of H-5 with H-9 and H-10, and H-1 with H-8 suggested the same orientation of H-5 with H-9 and H-10, which display an obvious difference from the ROESY experiment of 14, in which the correlations of H-5/H-1 and H-10, and H-9/H-8 were observed (see Electronic Supplementary Material).

Compound 11 possessed the molecular formula of C₁₀H₁₄O₄ identical with swerimilegenin J from HRESIMS. The same planar structures of 11 with 10 and 14 were constructed based on extensive ¹H-¹H COSY and HMBC analysis. In the ROESY spectrum, the correlations of H-5 with H-1 and H-8, and H-9 with H-10 manifested the α-orientation of H-5, H-8 and hydroxymethyl (C-1), and the β-orientation of H-9 and Me-10.

The known dihydroepinaucledal (14) was determined by comparing its NMR data with that of a previous report²⁰ and further confirmed by a 2D NMR experiment (see Electronic Supplementary Material). The other known compounds were determined as erythrocentaurin (12), ²¹ gentiolactone (13), ²² gentiogenal (15), ²³ secostrychnosin (16)²⁴ and angelone (17)⁵ by comparing their spectroscopic data to existing report data.

In order to evaluate their anti-HBV properties, namely inhibiting the secretions of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), as well as HBV DNA replication, the isolates were assayed on HepG 2.2.15 cell line in vitro (Table 4). Most of the tested compounds exhibited low activity and cytotoxicity at the highest tested concentration, except for erythrocentaurin and gentiogenal. Erythrocentaurin could inhibit the HBsAg secretion and HBV DNA replication with the IC₅₀ values of 1.39 ± 0.54 and 0.96 ± 0.45 mM, respectively. Gentiogenal showed activity inhibiting the secretion of HBsAg and HBeAg with the IC₅₀ values of 3.92 ± 0.82 and 2.99 ± 0.72 mM.

Previously, most of the secoiridoids isolated from Swertia plants were secoiridoid glycosides and always contained a C₁₀ aglycone part. Interestingly, this investigation resulted in eleven new secoiridoid aglycones including unusual C₉-skeleton (1–6), bis-C₉-skeleton (7) and C₁₀-skeleton (₈–₁₁) from the low-polarity part of S. mileensis. Structurally, compound 1 belonged to 10-nor-secoiridoid, and compounds 2–6 were attributed to 1-nor-secoiridoids, which further enriched the skeleton types of secoiridoids. It is concluded that diverse secoiridoid aglycones are contained in S. mileensis, although these compounds may be not corresponding to the anti-HBV activity in vitro.

Experimental Section

General Experimental Procedures. Melting points were measured on a SGWX-4B instrument (Shanghai Jingke, Shanghai, China). 1D and 2D NMR spectra were recorded on Bruker AV-400 NMR, DRX-500 or AVANCE Ⅲ-600 spectrometers (Bruker, Bremerhaven, Germany) corrected by deuterated solvent signals. MS data was collected on LCMSIT-TOF (Shimadzu, Kyoto, Japan) or VG Auto Spec-3000 (VG, Manchester, UK) spectrometers. IR (KBr) spectra was recorded on a Bruker Tensor 27 FT-IR (Bruker Optics GmbH, Ettlingen, Germany). UV data was collected on a Shimadzu UV-2401A spectrophotometer (Shimadzu, Kyoto, Japan). Optical rotations were collected on a Jasco model 1020 polarimeter (Horiba, Tokyo, Japan). Sephadex LH-20 (20–150 μm) was purchased from Pharmacia Fine Chemicals Co. Ltd. (Pharmacia, Uppsala, Sweden). Silica gel (200–300 mesh) for column chromatography was obtained from Qingdao Makall Chemical Company (Makall, Qingdao, China). CHP20P MCI gel (Mitsubishi Chemical Corporation, Tokyo, Japan) and Merck Lichrosorb RP-18 (Merck KGaA, Darmstadt, Germany) were applied for medium pressure liquid chromatographic (MPLC) preparation, which was performed on a Dr-Flash-S MPLC system (Lisui, Suzhou, China). Agilent Eclipse XDBC18 column (Agilent Technologies, Santa Clara, USA) was used for semi-preparation, which was conducted on a Waters 600-2695 HPLC instrument (Waters, Milford, MA, USA), equipped with a photodiode array detector (Waters 2996).

Plant Material. The whole plants of Swertia mileensis T. N. Ho et W. L. Shi were collected in Mile County, Yunnan Province, China, in November, 2008, and were identified by Prof. Li-Gong Lei. A voucher specimen (No. 2008-11-01) was deposited in the Laboratory of Antivirus and Natural Medicine Chemistry, Kunming Institute of Botany, CAS.

Extraction and Isolation. The air-dried and powered whole plant (5.0 kg) of S. mileensis was extracted with ethanol and condensed to provide a residue (1.3 kg). The residue was partitioned between water and petroleum ether (PE, 1 L × 2), ethyl acetate (EtOAc, 1 L × 3) and n-butanol (1 L × 3) successively. The EtOAc part (170 g) was chromatographed on silica gel column (2 kg, 110 mm id × 500 mm) eluted with CHCl₃-MeOH to provide fractions A−J. Fraction A (29 g) was separated by silica gel column chromatography (CC, 300 g, 50 mm id × 240 mm) with an eluent of CHCl₃-Me₂CO system (0:100 → 50:50, v/v) to yield Frs. A₁−A₅.¹¹ Frs. A₁ and A₂ were combined based on TLC detection which displayed many identical spots both under UV detector and treated with H₂SO₄ regent, and subjected to silica gel CC (200 g, 25 mm id × 600 mm) eluted with CHCl₃/MeOH gradient (100:0, 95:5, 90:10 and 80:20, with 2 L, respectively), to obtain three fractions A_1A–A_1C. Fraction A_1B (5 g) was separated by CHP20P MCI gel column chromatography (100.0 g, 25 mm id × 300 mm) on a MPLC system eluted with MeOH/H₂O (from 20:80 to 80:20, 10 L) to yield seven sub-fractions (Frs. A_1B-1 to A_1B-7). Fr. A_1B-1 (300 mg) was purified with a silica gel column (50 g, 25 mm id × 150 mm) eluting with PE/acetone (from 80:20 to 50:50, 800 mL) to afford compounds 3 (6 mg), 6 (8 mg), 12 (150 mg) and 15 (9 mg). Compounds 4 (5.0 mg), 5 (5.0 mg), 7 (6.0 mg), 8 (9.0 mg) and 9 (6.0 mg) were isolated from Fr. A_1B-2 (100 mg) by Sephadex LH-20 (50.0 g, 14 mm id × 1450 mm, MeOH, 300 mL) and repeated silica gel CC (20 g, 14 mm id × 250 mm, PE/acetone = 80:20, 500 mL). Fr. A_1B-3 (400 mg) was separated by RP-18 column (50 g, 25 mm id × 130 mm) eluted with MeOH/H₂O (40:60, 1000 mL) on MPLC system and further purified by repeated silica gel CC to give compounds 1 (3 mg), 2 (3 mg) and 13 (6 mg). Compounds 17 (7 mg) and 16 (3 mg) were obtained from Fr. A_1B-4 (60 mg) by repeated silica gel CC (20 g, 14 mm id × 250 mm) eluted with PE/acetone (70:30, 300 mL) and CHCl₃/acetone (85:15, 300 mL) systems. Fr. A_1B-5 (50 mg) was loaded on a HPLC apparatus using an Eclipse XDB-C18 column (94 mm id × 250 mm, 5 μm) and eluted with MeOH-H₂O (25:75, flow rate = 3 mL/min) providing compounds 10 (4 mg), 11 (6 mg) and 14 (8 mg). The purity of all the isolates was proposed to be higher than 95% based on TLC (only one spot under UV radiation and iodine atmosphere) and NMR (smooth baseline without impurity peaks) methods.

Swerimilegenin A (1): white amorphous powder; [α]_D¹⁸ – 12.4 (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 222 (3.54) nm; IR (KBr) ν_max 3418, 2926, 1701, 1631, 1413, 1385, 1048 cm^–1; for ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 183.0686 [M – H]^– (calcd. for C₉H₁₁O₄, 183.0663).

Swerimilegenin B (2): white amorphous powder; UV (MeOH) λ_max (log ε) 223 (3.82) nm; IR (KBr) ν_max 3430, 2963, 1711, 1469, 1404, 1314, 1170, 1142, 1074, 1008 cm^–1; ¹H and ¹³C NMR data, see Table 1; HREIMS m/z 170.0944 M⁺ (calcd. for C₉H₁₄O₃, 170.0943).

Swerimilegenin C (3): white amorphous powder; UV (MeOH) λ_max (log ε) 229 (3.83) nm; IR (KBr) ν_max 2939, 1720, 1403, 1309, 1173, 1130, 1072, 1130, 795 cm^–1; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 191 [M + Na]⁺; HRESIMS m/z 169.0866 [M + H]⁺ (calcd. for C₉H₁₃O₃, 169.0864).

Swerimilegenin D (4): white amorphous powder; [α]_D²⁵ – 2.0 (c 0.2, MeOH); UV (MeOH) λ_max (log ε) 209 (3.89) nm; IR (KBr) ν_max 3385, 2976, 1686, 1405, 1279, 1169, 1086, 1056, 1023, 986 cm^–1; ¹H and ¹³C NMR data, see Table 1; EIMS m/z 184 M⁺ (17), 183 (100), 167 (53), 140 (76), 125 (55), 94 (50); HRESIMS m/z 207.0624 [M + Na]⁺ (calcd. for C₉H₁₂O₄Na, 207.0633).

Swerimilegenin E (5): white amorphous powder; [α]_D²² – 6.1 (c 0.2, MeOH); UV (MeOH) λ_max (log ε) 207 (3.87) nm; IR (KBr) ν_max 2908, 1712, 1410, 1279, 1150, 983, 966 cm^–1; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 221.0763 [M + Na]⁺ (calcd. for C₁₀H₁₄O₄Na, 221.0784).

Swerimilegenin F (6): colorless gum; [α]_D²⁴ – 4.0 (c 0.2, MeOH); UV (MeOH) λ_max (log ε) 227 (3.87) nm; IR (KBr) ν_max 2937, 1727, 1700, 1415, 1214, 1083, 1060, 975, 851 cm^–1; ¹H and ¹³C NMR data, see Table 2; EIMS m/z 181 (35), 168 (100), 151 (25), 140 (20), 110 (17), 82 (55); ESIMS m/z 235 [M + Na]⁺; HRESIMS m/z 235.0586 [M + Na]⁺ (calcd. for C₁₀H₁₂O₅Na, 235.0582).

Swerimilegenin G (7): colorless cubic crystals; mp 228– 230 ℃; [α]_D²⁶ – 6.6 (c 0.3, MeOH/CHCl₃); UV (MeOH) λ_max (log ε) 210 (4.22) nm; IR (KBr) ν_max 2976, 2936, 2908, 1721, 1707, 1473, 1414, 1323, 1281, 1156, 1060, 962, 809, 661, 561 cm^–1; ¹H and ¹³C NMR data, see Table 2; ESIMS m/z 373 [M + Na]⁺; HRESIMS m/z 373.1258 [M + Na]⁺ (calcd. for C₁₈H₂₂O₇Na, 373.1263).

Swerimilegenin H (8): colorless gum; [α]_D²⁵ – 3.9 (c 0.4, MeOH); UV (MeOH) λ_max (log ε) 273 (3.78), 237 (3.82) nm; IR (KBr) ν_max 3362, 2954, 1699, 1637, 1419, 1286, 1064, 1030, 912, 773, 747 cm^–1; ¹H and ¹³C NMR data, see Table 2; EIMS m/z 196 M⁺ (10), 178 (90), 167 (20), 149 (24), 121 (30), 105 (26), 91 (100), 79 (25), 77 (45); ESIMS m/z 219 [M + Na]⁺; HRESIMS m/z 219.0641 [M + Na]⁺ (calcd. for C₁₀H₁₂O₄Na, 219.0633).

Swerimilegenin I (9): colorless needles; mp 144–145 ℃; [α]_D¹⁶ – 2.4 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 273 (4.21), 212 (3.66), 191 (3.90) nm; IR (KBr) ν_max 2915, 1700, 1637, 1426, 1304, 1246, 1160, 1124, 1054, 1041 cm^–1; ¹H and ¹³C NMR data, see Table 2; ESIMS m/z 233 [M + Na]⁺; HRESIMS m/z 211.0964 [M + H]⁺ (calcd. for C₁₁H₁₅O₄, 211.0970).

Swerimilegenin J (10): colorless gum; [α]_D¹³ – 90.6 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 250 (3.98) nm; IR (KBr) ν_max 3472, 2893, 1679, 1595, 1404, 1272, 1209, 1100, 1041, 962, 760 cm^–1; ¹H and ¹³C NMR data, see Table 3; EIMS m/z 199 [M + H]⁺ (40), 198 M⁺ (50), 168 (70), 127 (95), 123 (45), 97 (50), 57 (100); HREIMS m/z 198.0897 M⁺ (calcd. for C₁₀H₁₄O₄, 198.0892).

Swerimilegenin K (11): colorless gum; [α]_D¹⁹ + 2.2 (c 0.2, MeOH); UV (MeOH) λ_max (log ε) 249 (3.60) nm; IR (KBr) ν_max 3431, 2921, 1695, 1614, 1409, 1277, 1110, 1053 cm^–1; ¹H and ¹³C NMR data, see Table 3; HRESIMS m/z 233.0567 [M + Cl]^– (calcd. for C₁₀H₁₄O₄Cl, 233.0586).

Crystallographic Data of Compounds 7 and 9: Swerimilegenin G (7): C₁₈H₂₂O₇, MW = 350.14; monoclinic, space group p2₁; a = 9.5350(17) Å, b = 8.4098(15) Å, c = 21.881(4) Å, β = 101.266(2), V = 1720.8(5) ų, Z = 7, d = 1.315 g/cm³, crystal dimensions 0.28 × 0.19 × 0.08 mm³ were used for measurement on a Rigaku D/MAX-3B X-ray diffractometer with a graphite monochromater, Mo Kα radiation. The total number of reflections measured was 10830, of which 4045, were observed, I > 2σ(I). Final indices: R₁= 0.0523, wR₂ = 0.1341.

Swerimilegenin I (9): C₁₁H₁₄O₄, MW = 210.22; monoclinic, space group p2₁; a = 4.287(2) Å, b = 9.320(5) Å, c = 27.001(14) Å, β = 100.268(9), V = 1061.6(9) ų, Z = 2, d = 1.315 g/cm³, crystal dimensions 0.20 × 0.13 × 0.06 mm³ were used for measurement on a Rigaku D/MAX-3B X-ray diffractometer with a graphite monochromater, Mo Kα radiation. The total number of reflections measured was 5809, of which 1253, were observed, I > 2σ(I). Final indices: R₁= 0.1011, wR₂ = 0.4433.

Anti-HBV Assay. The anti-HBV procedure was performed in accordance with our previous report.¹⁵ Tenofovir (Lot No. 200904009, purity > 97.6%) was used as the positive control, which was purchased from Jiangxi Chenyang Pharmaceutical Co. Ltd..

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-013-0059-y and is accessible for authorized users.

Notes

Acknowledgments

This work was funded by the National Natural Science Foundation of China and Yunnan Province (U0832603), the National Science Foundation of China for Distinguished Young Scholars (81025023), the West Light Foundation of the Chinese Academy of Sciences, the International Foundation for Science (No. F/5202-1), and the Youth Innovation Promotion Association, CAS.