Cucurbitane triterpenoids from Hemsleya penxianensis

Introduction

The genus Hemsleya(Cucurbitaceae), consisting of more than twenty species, are mainly distributed in Yunnan and Sichuan Provinces.¹ The tubers of Hemsleya plants are known as famous herbal medicine in China, used in the treatment of bacillary dysentery, bronchitis, and tuberculosis etc.¹ The Hemsleya species distributed at Jinfu Mountain was initially recorded as H. jinfushanensis, and then as H. penxianensis var. jinfushanensis by Zhang W. J. and Shen L. T., but was ascribed to H. penxianensis by Li D. Z.^{2, 3} Previously, the phytochemical and bioactive studies of this plant have been reported.^{4, 5} In continuation of these study, nine new cucurbitane triterpenoids and nine known analogues were obtained. Herein, we describe the isolation and structural elucidation of the new ones.

Results and Discussion

Jinfushanencin A (1) was obtained as colorless powder with the empirical molecular formula of C₃₀H₄₆O₈, in agreement with the negative ion HRESIMS (m/z 533.3145 [M – H]^-, calcd for C₃₀H₄₅O₈, 533.3114) and ¹³C NMR spectroscopic data. The IR spectrum revealed absorptions at 3436, 1696, and 1648 cm^–1, suggestive of hydroxyl and conjugated carbonyl groups. Obvious signals observed in the ¹H NMR spectrum were eight methyl singlets at δ_H 1.24 (3H, s), 1.27 (3H, s), 1.36 (3H×2, s), 1.40 (3H, s), 1.48 (3H, s), 1.57 (3H, s), and 1.63 (3H, s), an olefinic proton singlet at δ_H 6.50 (1H, s), as well as two doublets at δ_H 3.54 (1H, d, J=9.1 Hz) and 2.92 (1H, d, J=7.0 Hz). The ¹³C NMR and DEPT spectra revealed 30 carbon signals due to eight methyls, five methylenes, seven methines, and ten quaternary carbons (including three carbonyl carbons and one olefinic carbon), which were assigned to a triterpene skeleton. Considering the fact that the tetracyclic triterpenoids isolated thus far from the genus Hemsleya are cucurbitane-type compounds, in combination with four characteristic quaternary carbons (δ_C 44.4 (C-4), 49.2 (C-9), 48.5 (C-13), and 50.7 (C-14)) at high field, compound 1 was tentatively proposed to be a cucurbitacin.⁶ Further, comparison of the NMR data of 1 with those of 2β, 3β, 16α, 20(R), 25-pentahydroxy-9-methyl-19-norlanost-5-en-7, 11, 22-trione indicated that these two compounds were structurally almost identical with exception of the configuration of the hydroxyl group at C-3.⁷ Detailed comparison of the NMR data of these two compounds disclosed that two proton signals at δ_H 4.22 (1H, m) and 3.54 (1H, d, J=9.1 Hz), ascribed to H-2 and H-3 by 2D NMR, in 1, instead of the signals at δ_H 4.61 (1H, br. d, J=10.9 Hz, H-2) and 3.98 (1H, br. s, H-3) in the known compound. All evidences mentioned above suggested that compound 1 had a 2β, 3α-diol moiety.

The ROESY correlation observed between δ_H 1.40 (3H, s, H-28) and δ_H 3.24 (1H, m, H-10) established an α configuration for C-28, together with the ROESY correlations between δ_H 4.22 (1H, m, H-2) and H-10, and between δ_H 1.48 (3H, s, H-29) and δ_H 3.54 (1H, d, J=9.1 Hz, H-3), confirmed the 2β and 3α substituents. The rings BD and side chain of 1 were identical to those of 2β, 3β, 16α, 20(R), 25-pentahydroxy-9-methyl-19-norlanost-5-en-7, 11, 22-trione by detailed analysis of the ¹³C, ¹H, HMBC, HSQC, and ₁H-₁H COSY spectra of 1.

The molecular formula of jinfushanencin B (2) was determined to be C₃₀H₄₈O₄ by negative ion HRESIMS (found [M - H]^-471.3487, calcd for 471.3474). From the ¹³C NMR data in combination with DEPT spectrum, a ketone carbon, two olefinic methines, two olefinic quaternary carbons, one oxygenated methine, two oxygenated methylenes, four methines, eight methylenes, four quaternary carbons, and six methyls were observed. The NMR data of 2 were very similar with those of carnosiflogenin A (10).⁸ The difference was that the methyl signal for C-27 in 10 was replaced by an oxygenated methylene (δ_C 58.5) in 2, in accordance with the olefinic quaternary carbon resonance at δ_C136.1 (C-25) in 10 being shifted downfield to δ_C 140.9 in 2. These changes suggested that the methyl at C-27 in 10 was oxygenated to be a hydroxymethyl in 2. The HMBC correlations from δ_H 4.73 (s, H-27) to δ_C 65.4 (t, C-26), 140.9 (s, C-25), and 127.5 (d, C-24), were also supporting evidences for the above deduction.

Jinfushanoside E (3) had a molecular formula of C₄₂H₆₆O₁₂ determined by HRESIMS, ¹³C NMR, and DEPT experiments. The molecular weight of 3 was 144 mass units more than that of delavanoside D (11).⁹The ¹³C NMR and DEPT data (Tables 1-5) of 3 were very similar with those of 11 exception of six additional resonances observed at δ_C 171.8 (s, C-1″), 46.4 (t, C-2″), 70.2 (s, C-3″), 46.8 (t, C-4″), 174.9 (s, C-5″), and 28.8 (q, C-6″). In the HMBC spectrum, correlations revealed from δ_H 3.14 (H-2″) to C-1″, 3″, 4″, 6″ and from δ_H 3.21 (2H, br. d, J=14.2 Hz, H-4″) to C-2″, 3″, 5″, 6″ (Figure 1) manifested the additional structural feature in 3 being a hydroxymethyl glutaryl (HMG) group.¹⁰ Additionally, HMBC correlation140 J. C. CHEN et al. Nat. Prod. Bioprospect. 2012, 2, 138–144 observed from δ_H 5.03 (d, J=11.1 Hz, H_a-6′) and δ_H 4.71 (m, H_b-6′) to δ_C 171.8 (s, C-1″) revealed that the HMG group was linked to C-6′ of the sugar unit. After alkaline hydrolysis of 3 with 5 M NaOH solvent, 11 was detected in reaction mixture by TLC. The rings A-D and side chain of 3 were identical to those of 11 by detailed analysis of the ¹³C, ¹H, HMBC, HSQC, and ¹H-¹H COSY spectra of 3.

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Jinfushanoside F (4) displayed a quasimolecular ion peak at m/z=777.4436 [M - H]^-(cald for C₄₂H₆₅O₁₃, 777.4425), which was consistent with the molecular formula C₄₂H₆₆O₁₃. Comparison of NMR data of 4 (Tables 1-5) with those of jinfushanoside B revealed great similarity.⁴ The difference was that the C-6′ position of the sugar unit in 4 was ester-linked to a HMG functionality, on the basis of the HMBC correlations from δ_H 5.04 (d, J=11.3 Hz, H_a-6′) and δ_H 4.72 (m, H_b-6′) to δ_C 171.7 (s, C-1″), from δ_H 3.11 (d, J=14.0 Hz, H_a-2″) and δ_H 3.08 (d, J=14.0 Hz, H_b-2″) to δ_C 171.7 (s, C-1″), 70.2 (s, C-3″), 47.0 (t, C-4″), and 28.4 (q, C-6″), and from δ_H 3.17 (br. d, J=13.8 Hz, H-4″) to δ_C 46.7 (t, C-2″), 70.2 (s, C-3″), 175.6 (s, C-5″), and 28.4 (q, C-6″). After alkaline hydrolysis of 4 with 5 M NaOH solvent, jinfushanoside B was detected in reaction mixture by TLC. The rings A-D and side chain of 4 were identical to those of jinfushanoside B by detailed analysis of the ¹³C, ¹H, HMBC, HSQC, and ¹H-¹H COSY spectra of 4.

The negative ion HRESIMS of jinfushanoside G (5) showed a molecular ion peak at 619.4183 [M - H]^-, in according with an empirical molecular formula of C₃₆H₆₀O₈. The molecular weight of 5 was 162 mass units more than that of 3β, 11α, 26-trihydroxycucurbita-5, 24-diene, ⁸ which implied an additional hexose unit in 5. Comparison of the ¹³C NMR spectrum of 5 with that of the known compound, an additional glucose signals at δ_C 107.3 (d), 75.5 (d), 78.7 (d), 71.8 (d), 78.1 (d), and 63.1 (t), as well as the signal of C-3 being shifted downfield by 11.4 ppm to δ_C 87.9 (d) in 5, were observed, which indicated an additional glucopyranosyl moiety being linked at C-3 in 5. This deduction was confirmed by the HMBC correlations from the anomeric proton signal at δ_H4.86 (d, J=7.8 Hz, H-1′) to δ_C 87.9 (d, C-3). The coupling value (J=7.8 Hz) of anomeric proton suggested the presence of a β-glucopyranosyl moiety, and the sugar was determined to be a D-glucose by comparison with authentic sample on GC analysis after acid hydrolysis of 5.

Jinfushanoside H (6) was determined to have the molecular formula C₄₂H₆₈O₁₂ by negative ion HRESIMS (found [M - H]^- 763.4648, calcd for 763.4632). Comparison of NMR data (Tables 1-5) of 6 with those of 5 displayed great similarity. The difference was that an additional HMG group was esterlinked to the C-6′ position of the sugar unit in 6, which was proved by the HMBC correlations from δ_H 5.03 (d, J=11.3 Hz, Ha-6') and δ_H 4.72 (m, H_b-6′) to δ_C 171.7 (s, C-1″) and from δ_H 3.14 (2H, m, H-2″) to δ_C 70.2 (s, C-3″), 46.8 (t, C-4″), and 28.3 (q, C-6″). After alkaline hydrolysis of 6 with 5 M NaOH solvent, 5 was detected in reaction mixture by TLC. The rings A-D and side chain of 6 were identical to those of 5 by detailed analysis of the ¹³C, ¹H, HMBC, HSQC, and ¹H-¹H COSY spectra of 6.

Jinfushanoside I (7) had the molecular formula C₅₄H₈₆O₂₂ based on HRESIMS ([M - H]^-1085.5546, calcd for 1085.5532) data. The molecular weight of 7 was 144 mass units more than that of carnosifloside Ⅲ (12), ⁸ which implied an additional HMG group in 7. Comparison of the ¹³C NMR spectrum of 7 with that of the known compound, an additional HMG signals at δ_C 171.6 (s), 47.3 (t), 70.6 (s), 47.4 (t), 174.8 (s), and 28.3 (q), and the signal of C-6′ being shifted downfield to δ_C 64.6 (t) in 7, were observed, which indicated that the HMG functionality was ester-linked at C-6′ in 7. This proposal was proved by the observed HMBC correlation from δ_H 4.98 (m, H_a-6′) and 4.69 (m, H_b-6′) to δ_C 171.6 (s, C-1″). After alkaline hydrolysis of 7 with 5 M NaOH solvent, carnosifloside Ⅲ (12) was detected in reaction mixture by TLC.

Jinfushanoside J (8) displayed a quasimolecular ion peak at m/z=957.5050 [M - H]^-(cald for C₄₈H₇₇O₁₉, 957.5059), in accordance with the molecular formula C₄₈H₇₈O₁₉. The molecular weight of 8 was 162 mass units more than that of jinfushanoside D, ⁴ which implied an additional hexose unit in 8. In the HMQC-TOCSY spectrum, the anomeric proton at δ_H 4.83 (1H, d, J=7.8 Hz, H-1′) was correlated with six carbons at δ_C 107.3 (d), 75.5 (d), 78.7 (d), 71.8 (d), 78.4 (d), and 63.1 (t). This glucose was attached to C-3 based on the HMBC correlation between the anomeric proton at δ_H 4.83 and the carbon signal at δ_C 87.2 (d, C-3). During the hydrolysis process of 8, jinfushanosides B-D were detected in reaction mixture by TLC. The configurations of the anomeric protons of the three glucoses were established to be β on the basis of the coupling constants of the anomeric protons, and the sugars were determined to be a D-glucose by comparison with authentic sample on GC analysis after acid hydrolysis of 8.

Jinfushanoside K (9) displayed a quasimolecular ion peak at m/z=927.5336 [M - H]^-(cald for C₄₈H₇₇O₁₇, 927.5317), consistent with the molecular formula C₄₈H₇₈O₁₇. The molecular weight of 9 was 14 mass units less than that of carnosifloside Ⅲ (12).⁸ The ¹³C NMR data (Tables 2 and 4) of 9 was very similar with that of 12 with the exception of a methylene (β_C 32.7) instead of the carbonyl functionality (β_C 213.7) at C-11 in 9, in accordance with the disappearance of the AB coupling system of H-12 in ¹H NMR spectrum. In HMBC spectrum, correlations observed from δ_H 0.85 (3H, s, H-19) to δ_C 44.0 (d, C-8), 38.7 (d, C-10), 35.1 (s, C-9), and 32.7 (t), also proved the presence of a methylene at C-11. The configurations of the anomeric protons of the three glucoses were established to be β on the basis of the coupling constants of the anomeric protons, and the sugars were determined to be a D-glucose by comparison with authentic sample on GC analysis after acid hydrolysis of 9. Exception of ring B, the other parts of 9 were identical to those of 12 by detailed analysis of the ¹³C, ¹H, HMBC, HSQC, and ¹H-¹H COSY spectra of 9. Compound 9 was also the first occurrence of cucurbitane triterpene without carbonyl or hydroxyl functionalities at C-11 from the genus Hemsleya.

Known compounds were identified as carnosiflogenin A (10), ⁸ delavanoside D (11), ⁹ carnosifloside Ⅲ (12), ⁸ 3β, 27-dihydroxycucurbita-5, 24-dien-11-on-3-O-β-D-glucopyranosyl-27-O-β-D-glucopyranoside (13), ⁸ delavanoside A (14), ⁹ 11α, 26-dihydroxycucurbita-5, 24-dien-26-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranoside (15), ⁸ carnosifloside Ⅵ (16), ⁸ hexanorcucurbitacin F (17), ¹² and scandenogenin A (18)¹⁰ by comparison with literature data. Among the known compounds, 13, 15, and 18 were firstly isolated as natural products, which had been obtained by acid hydrolysis of carnosifloside Ⅳ, ⁸ carnosifloside Ⅳ, ⁸ and scandenoside R1, ¹¹ respectively.

Compounds 2, 3, 5, 7, 8, 9, 13, and 15, were tested for in vitro inhibitory effects against HIV replication in C8166 cells. All the compounds were no or weak active (Table 6).

Experimental Section

General Experimental Procedures. Melting points were obtained on an XRC-1 apparatus and are uncorrected. Optical rotations were carried out on a Perkin-Elmer model 241 polarimeter. UV spectrum was measured in a UV 210A spectrometer. IR spectra were measured in a Bio-Rad FTS-135 spectrometer with KBr pellets. MS were recorded on a Finnigan MAT 90 instrument. 1D and 2D NMR spectra were measured on a Bruker DRX-500 instrument with TMS as internal standard. Column chromatography was performed either on silica gel (200-300 mesh; Qingdao Marine Chemical Inc., Qingdao, China), or Lichroprep RP-18 gel (40-63 μm; Merck, Darmstadt, Germany). Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 15% H₂SO₄ in H₂O.

Plant Material. The tubers of H.penxianensis were collected in Jinfu Mountain, Congqing City, China, in 2000. A voucher specimen (No. KIB 2000-9-4) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy Sciences. The plant material was identified by Prof. Wen-Jin Zhan, Penzhou Institute for Pharmaceutical Control, Sichuan.

Extraction and Isolation. Air-dried and powdered tubers (2.0 kg) of H. penxianensis were extracted with 95% ethanol under reflux (3 × 8 L), and filtered. After concentration of the combined filtrate under vacuum, 411 g residue was gotten. The residue (322 g) was absorbed on silica gel and chromatographed on silica gel column, eluting with a gradient system of CHCl₃, CHCl₃-Me₂CO (15:1, 9:1), and CHCl₃-EtOH (9:1, 7:3) to give six fractions (Frs. 1-6). Fraction 3 (2.83 g) was fractioned on silica gel, developing with a gradient system of CHCl₃-MeOH (20:1, 15:1, 12:1) to furnish three parts (Fr. 3.1-3.3). Fr. 3.3 (1.1 g) was rechromatographed on silica gel, eluting with CHCl₃-CH₃OH (15:1, 12:1), then purified on LH-20 using MeOH as eluent to give 1(12 mg), 2 (57 mg), 10 (17 mg), 17 (32 mg), and 18 (15 mg). Fr. 4 (12.8 g) was subjected to CC (20 g) to generate four fractions (Frs. 4.1-4.4). Fr. 4.3 (8.43 g) was repeatedly subjected to silica gel column eluting with CHCl₃-CH₃OH(10:1) and RP₁₈ silica gel developing with aqueous MeOH (60%→70%), to generate 3 (174 mg), 4 (19 mg), 5 (47 mg), 6 (23 mg), and 11 (86 mg). Compounds 13 (42 mg), 14 (31 mg), and 15 (19 mg) were isolated from Fr.4.4 (1.5 g) by subjected to column chromatography over silica gel (CHCl₃-MeOH, from 6:1 to 9:1) and RP-18 (aqueous MeOH, from 55% to 60%), then Sephadex LH-20 (MeOH). Fr. 5 (131 g) was rechromatographed on silica gel developing with a gradient system of CHCl₃-MeOH-H₂O (8:1:0.1, 8:2:02, 7:3:1, 7:3:0.5) to yield Fr. 5.1-Fr. 5.5. Fr.5.3 (9 g) was further rechromatographed over RP₁₈ silica gel developing with aqueous MeOH (40%→50%) to afford compounds 9 (39 mg), 12 (451 mg), and 16 (320 mg). Compounds 7 (150 mg) and 8 (164 mg) were isolated from Fr.5.4 (4 g) by subjected to column chromatography over RP-18 silica gel, eluting with a gradient aqueous MeOH system from 40% to 45%.

Jinfushanencin A (1): colorless powder; mp 217-219℃; [α]_D²⁰+22.0 (c 0.05, MeOH); IR (KBr)v_max: 3436, 2973, 1696, 1648, 1302 cm^–1; ¹H and ¹³C NMR data, see Tables 1 and 2; negative FABMS: m/z 533 [M - H]^–; negative HRESIMS: m/z 533.3145 [M - H]^–(calcd for C₃₀H₄₅O₈, 533.3114).

Jinfushanencin B (2): colorless powder; mp 149-151℃; [α]_D²⁰+15.8(c 0.12, MeOH); IR (KBr)v_max: 3372, 2952, 2929, 2874, 1693, 1464, 1382, 1028, 1006, 979 cm^–1; ¹H and ¹³C NMR data, see Tables 1 and 2; negative FABMS: m/z 471 [M - H]^–; negative HRESIMS: m/z 471.3487 [M - H]^–(calcd for C₃₀H₄₇O₄, 471.3474).

Jinfushanoside E (3): white powder; [α]_D²⁰+ 8.6(c 0.36, MeOH); IR (KBr)v_max: 3425, 2962, 2878, 1728, 1633, 1383, 1206, 1078, 978 cm^–1; ¹H and ¹³C NMR data, see Tables 1-5; negative FABMS: m/z 761 [M - H]^–; negative HRESIMS: m/z 761.4491 [M - H]^– (cald for C₄₂H₆₅O₁₂, 761.4476).

Jinfushanoside F (4): white powder; [α]_D²⁰+9.2(c 0.24, MeOH); IR (KBr)v_max: 3424, 3369, 2962, 2877, 1726, 1692, 1383, 1207, 1077, 1038 cm^–1; ¹H and ¹³C NMR data, see Tables 1-5; negative FABMS: m/z 777 [M - H]^–; negative HRESIMS: m/z777.4436 [M - H]^– (cald for C₄₂H₆₅O₁₃, 777.4425).

Jinfushanoside G (5): white powder; [α]_D²⁰+12.0(c 0.09, MeOH); IR (KBr) v_max: 3409, 3368, 2942, 2928, 1638, 1464, 1381, 1075, 1030 cm^–1; ¹H and ¹³C NMR data, see Tables 1-4; negative FABMS: m/z 619 [M - H]^–; negative HRESIMS: m/z 619.4183 [M - H]^– (cald for C₃₆H₅₉O₈, 619.4209).

Jinfushanoside H (6):white powder; [α]_D²⁰+8.1 (c 0.09, MeOH); IR (KBr) v_max: 3431, 2928, 2874, 1722, 1453, 1076, 981 cm^–1; ¹H and ¹³C NMR data, see Tables 1-5; negative FABMS: m/z 763 [M - H]^–; negative HRESIMS: m/z 763.4648 [M - H]^–(cald for C₄₂H₆₇O₁₂, 763.4632).

Jinfushanoside I (7): white powder; [α]_D²⁰+5.9 (c 0.09, MeOH); IR (KBr) v_max: 3431, 2945, 2875, 1729, 1579, 1458, 1076, 1021 cm^–1; ¹H and ¹³C NMR data, see Tables 1-5; negative FABMS: m/z 1085 [M - H]–; negative HRESIMS: m/z 1085.5546 [M - H]^– (cald for C₅₄H₈₅O₂₂, 1085.5532).

Jinfushanoside J (8): white powder; [α]_D²⁰+12.6 (c 0.11, MeOH); IR (KBr) v_max: 3452, 3283, 2926, 2877, 1690, 1467, 1075, 1020 cm^–1; ¹H and ¹³C NMR data, see Tables 1-4; negative FABMS: m/z 957 [M - H]^–; negative HRESIMS: m/z 957.5050 [M - H]^– (cald for C₄₈H₇₇O₁₉, 957.5059).

Jinfushanoside K (9): white powder; [α]_D²⁰-5.3 (c 0.17, MeOH); IR (KBr) v_max: 3409, 2935, 2872, 1640, 1460, 1379, 1075, 1038 cm^–1; ¹H and ¹³C NMR data, see Tables 1-4; negative FABMS: m/z 927 [M - H]^–; negative HRESIMS: m/z 927.5336 [M - H]^-(cald for C₄₈H₇₇O₁₇, 927.5317).

Acid Hydrolysis of 5, 8, and 9: Each of the compounds 5, 8, and 9 (2 mg) dissolved in 2 mL HCl-CH₃OH (1 mol L^–1 HCl-CH₃OH, 1:1, v/v) was placed in water bath at 90℃ for 6 hrs. After the hydrolysis was finished, the water-soluble part was evaporated to dryness. The dry sugar residue and authentic sample of D-(+)-glucose (Sigma Company, G5250) were respectively diluted in 1 ml pyridine without water and treated with 0.4 mL hexamethyl disilazane and 0.2 mL trimethylchlorosilane (TMCS, Fluka) at 0℃. After that, the upper layer of the reaction mixture was analyzed by GC. (GC condition: AC-5 capillary column (30 m × Ø0.25 mm); column temperature: 180-260℃; column head pressure: 12 Pa; carrier gas: N₂). GC of all samples showed same retention time, R_t (m): 11.521.

Partial Alkaline Hydrolysis of 3, 4, 6, and 7: A solution of the triterpene glycoside (each 0.5 mg dissolved in 1 ml MeOH) was added 0.5 % aq. NaOH (20 mL). After 2 h shaking at room temperature, the reaction mixture was acidified to PH 5 (5 M HCl) and was concentrated to dry. The resulting residue was dissolved in MeOH, and then was checked by using TLC. As a result, delavanoside D (11), jinfushanoside B, jinfushanoside G (5), and carnosifloside Ⅲ (12), were detected in the reaction mixture of 3, 4, 6, and 7, by TLC, respectively.

Anti-HIV-1 and Cytotoxicity Assay. The anti-HIV-1 activity was evaluated by the inhibition assay for the cytopathic effects of HIV-1 (EC₅₀), and cytotoxicity assay against C8166 cells (IC₅₀) was assessed using the MTT method as described in the literature.¹³

Notes

Acknowledgments

This work was financially supported by the Natural Science Foundation of Yunnan Province (2008CD158), the Western Doctoral Foundation of Chinese Academy of Sciences (J.C. Chen), the Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences (P2008ZZ23, P2010ZZ14), and the Cooperative Project of Guangdong Province and CAS (2009B091300135).