合蕊五味子中两个新的三萜内酯及其在生物合成途径中的意义(英文)

1Chinese Journal of Natural Medicines 2010, 8(1): 0001−0005doi: 10.3724/SP.J.1009.2010.00001ChineseJournal ofNaturalMedicines ·Original papers·Propinic Lactones A and B, Two New Triterpenoids from Schisandra propinqua var. propinqua and the Significance inBiosythesis PathwayLEI Chun, PU Jian-Xin, XIAO Wei-Lie, YANG Li-Bin, LIU Jing-Ping,CHEN Ji-Jun, SUN Han-Dong *State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, ChinaAvailable online 20 Jan 2010[ABSTRACT]AIM: To study the chemical constituents of the aerial parts of Schisandra propinqua var. propinqua. METHOD: Silica gel, C18 reversed phase silica gel and HPLC were used. The structures were elucidated by extensive spectroscopic methods. RESULT: Two new triterpenoids, propinic lactone A (1) with 3, 4-seco-cycloartane skeleton, and propinic lactone B (2) possessing 3, 4:9, 10-seco-cycloartane skeleton, together with a known typical cycloartane triterpenoid, schizandronic acid (3), have been isolated and identified. CONCLUSION: These two new triterpenoids serve as two important bridge intermediates from cycloartane triterpenoids to Schisandra nortriterpenoids biogenetically.[KEY WORDS] Schisandraceae;Schisandra; Schisandra propinqua var. propinqua; Triterpenoids[CLC Number]R284.1 [Document code] A [Article ID] 1672-3651(2010)01-0001-051 IntroductionSchisandra nortriterpenoids are a group of structurallyintriguing triterpenoids with promising bioactivities producedby the medicinally important genus schisandra in Schi-sandraceae family. They mainly consist of schiartane[1],18(13→14)-abeo-schiartane[2], 18-nor-schiartane[3], pre-schisanartane[4], schisanartane[5], and wuweiziartane skele-tons[6]. Compounds with these skeletons were theoreticallyconsidered to be biogenetically related and all derived fromcycloartane backbone precursors[2, 4, 7]. However, there wasno obvious evidence to prove this biological pathway due tothe lack of convincible intermediates discovered to co-existwith cycloartane and those nortriterpenoids[8]. From our re-cent investigations, a new 3, 4-seco-cycloartane triterpenoid,propinic lactone A (1), and a new 3, 4:9, 10-seco-cycloartane[Received on] 02-May-2009[Research Funding]This project was supported by the NationalNatural Science Foundation (No. 30830115 and 20802082), theMajor State Basic Research Development Program of China (No.2009CB522300 and 2009CB940900).[*Corresponding author] SUN Han-Dong: Prof., Tel: 86-871-5223251, Fax: 86-871-5216343, E-mail: hdsun@triterpenoid, propinic lactone B (2), were isolated togetherwith a known cycloartane triterpenoid, schizandronic acid(3)[9] and many Schisandra nortriterpenoids from the stems ofSchisandra propinqua var. propinqua[9-12]. The discovery ofthese two new triterpenoids could fill the biosynthesis gapsfrom cycloartane to schiartane (3, 4:9, 10-seco-28-norcycloartane) skeleton through 3,4-seco-and 3, 4:9, 10-seco-cycloartane triterpenoid as key intermediates in the ge-nus Schisandra.Herein, we report the identification of thenew compounds, and discuss the putative biosynthetic path-ways from cycloartane to schiartane skeleton.2 Materials and Methods2.1 GeneralPetroleum ether (PE, 60-90°C), EtOAc, CHCl3, acetone,methanol, and i-prOH were analytical grade and produced bySinopharm Chemical Reagent Co. Ltd., China. Silica gel(200-300 mesh and 10-40 μm; Qingdao Marine Chemical,Inc., China) and sephadex (General Electric Company, USA)for column separation. Fractions were monitored by thinlayer chromatography (TLC), and spots were visualized byspraying with 8% H2SO4 in EtOH, followed by heating. Op-tical rotations: Horiba SEP A-300 spectropolarimeter. IRspectra: BioRad FTS-135 spectrophotometer, KBr discs; in2 Chin J NatMed Jan. 2010 V ol. 8 No.12010年1月 第8卷 第1期cm -1. 1D- and 2D-NMR Spectra: DRX-500 instruments; J in Hz. HR-ESI-MS: VG AutoSpec-3000 spectrometers; in m/z . 2.2 Plant MaterialStems of S. propinqua var. propinqua were collected in Tengchong Country, Yunnan Province, China, in July 2006, and were identified by Prof. LI Xi-Wen, Kunming Institue of Botany, Chinese Academy of Sciences. A voucher specimen (No.20050823) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kun-ming Institute of Botany, Chinese Academy of Sciences, China.2.3 Extraction and IsolationThe air-dried stems of S. propinqua var. propinqua (8 kg) were extracted with 70% aqueous acetone (4 × 15 L, 3d each) at room temperature. The solvent was removed in vacuo to afford a crude extract (560 g), which was dissolved in H 2O, and then extracted successively with petroleum ether and EtOAc. The EtOAc-soluble part (250 g) was purified by CC (on SiO 2 with CHCl 3/acetone 1 → 0) to obtain six main frac-tions (Fr. A–F). Fr.B was placed in acetone to crystallize compound 3 (5 g). Fr.C (CHCl 3/acetone 9: 1-8: 2, 29 g) was purified by repeated CC, first on Sephadex LH-20 eluted with MeOH, then on silica gel eluted by PE/i-prOH in gradient system and followed by recrystallization in acetone to afford 1 (30 mg). Fr.D (CHCl 3/acetone 8: 2-2: 1, 45 g) was purified first by CC on silica gel with CHCl 3/acetone 4:1 to obtain small fractions of D1, D2 and D3. D2 was then subjected to RP-18 in 30%-60% aqueous MeOH gradient system after purified on Sephadex LH-20 eluted with MeOH to afford five mixtures (D2-1-D2-5). D2-3 (40% aqueous MeOH) was pu-rified on silica gel with PE/i-PrOH 5: 1 and followed by semi-prep. HPLC (40% aqueous MeOH) to yield 2 (3 mg).Propinic lactone A (1). Colorless needles. mp 172–173°C. [α]22.9 D+ 86.73 (c 2.13, CH 3OH/CHCl 3 2:1); CD (MeOH) λmax nm (Δε): 260.0 (+6.00), 247.5 (+5.20). IR (KBr) νmax : 3 550, 2 970, 2 953, 1 736, 1 715, 1 446, 1 380, 1 359, 1 194, 1 130 cm –1. 1H and 13C NMR data, see Table 1, OCH 3: 3.60 (s ), 51.3 (q ); HR-ESI-MS (neg.): 499.3413 ([M-H]–, C 31H 47O 5–, calc. 499.342 3).Propinic lactone B (2). Colorless solid. [α]19.8 D +28.90 (c 0.15, MeOH); CD (MeOH) λmax nm (Δε): 220.0 (– 10.2). IR (KBr) νmax : 3468, 2927, 1757, 1684, 1456, 1385, 1260, 1205, 1103, 1063, 1034, 914, 801 cm –1; 1H and 13C NMR data, see Table 1. HR-ESI-MS (neg.): 531.296 6 ([M-H]–, C 30H 43O 8–, calcd. 531.295 7).3 Results and Discussion3.1 Structure elucidationCompounds 1–3 (Fig.1) were isolated from 70% aqueous acetone extract of the stems of S. propinqua var. propinqua . Compound 3 was identified to be schizandronic acid, a typi-cal cycloartane triterpenoid in genus schisandra , by compar-ing its 13C NMR and DEPT spectra with the literature [9]. Compounds 1 and 2 are new triterpenoids related to cycloar-tane as determined by comprehensive spectroscopic meansincluding 1D and 2D NMR.Propinic lactone A (1) was obtained as colorless needles. Its formula was established as C 31H 48O 5 from HR-ESI-MS (m/z 499.341 3 ([M-H]–, calcd. 499.342 3)) and 13C NMR spectroscopic data (Table 1), requiring eight degrees of un-saturation. The 1H NMR spectrum of 1 (Table 1) exhibited signals of one typical AB coupled system at δH 0.58 (ABd, J = 5.5 Hz, H-19α), 0.79 (ABd, J = 5.5 Hz, H-19β), and seven methyl signals including one oxygenated singlet at δH 3.60 (s, H 3-OCH 3), five aliphatic singlets at δH 1.94, 1.42, 1.40, 0.95, 0.92 and a doublet at δH 0.97 (d, J = 8.5 Hz, H 3-21). The 13C NMR (Table 1) and DEPT spectra showed the presence of two ester carbons (δC 174.8,s; 166.3, s), one trisubstituent double bond (δC 140.2, d) and 128.1(s), and seven methyl groups including an oxygenated one at δC 51.3. All these signals, plus eight degrees of unsaturation, suggested that compound 1 was a methylated triterpenoid with five rings including a three-member ring, as that of 3 possessing cyc-loartane skeleton.However, the 13C NMR data of 1 differed greatly from those of compound 3. Three spin systems, H-24/H 2-23/ H-22/H-20/H-17,21/H 2-16/H 2-15, H 2-12/H 2-11, and H-8/H 2- 7/H 2-6/H-5 in 1H,1H-COSY spectrum of 1 (Fig. 2 ), together with five groups of HMBC correlations (Fig. 2 ) from H 3-27 to C-24, C-25 and C-26, from H-22 to C-17, C-20, C-21, C-23, C-24 and C-26, from H 3-18 to C-12, C-13 and C-17, from H 3-28 to C-8, C-13, C-14 and C-15, and from H 2-19 to C-5, C-8, C-9, C-10 and C-11 building up the five rings of 1 as shown. This partial structure was almost the same as that of kadsudilactone [13] with A ring cracked and a six-member α-methyl-α, β-unsaturated-δ-lactone ring in the side chain which was remarkably distinct from that of 3. In the HMBC experiment (Fig. 2), correlations from H 2-19 to C-1, from both H 2-1, H 2-2 and H 3-OCH 3 to C-3, and from H-5 to C-4, C-29 and C-30 indicated that the bond of C-3/C-4 in 1 broke down followed by a methyl esterification at C-3. Thus, the planar structure of compound 1 was established as a 3, 4-seco cycloartane derivative.The relative stereochemistry of compound 1 was deter-mined to be the same as that of 3 and kadsudilactone on the biosynthetic consideration, which was proved by ROESY experiment. C-22 of 1 was assigned to be R configuration as the CD spectrum showed a positive Cotton effect near 260 nm (Δε + 6.0), similar to that of kadsulactone.Propinic lactone B (2) was analyzed to be C 30H 44O 8 from HR-ESI-MS (m/z 531.296 6 [M-H] –, calcd. 531.295 7) and it required nine degrees of unsaturation. The 1H NMR spectrum (Table 1) of 2 displayed five tertiary methyls (δH 1.80, 1.62, and 1.24, 1.08, 0.99), a secondary methyl (δH 1.36, d, J = 6.0 Hz) and a characteristic ABX spin system ( δ 4.27 (d, J = 4.5 Hz, H(1)), 2.74 (d, J = 18.0 Hz, H α(2)), and 2.99 (dd, J = 4.5, 18.0 Hz, H β(2)). The 13C NMR spectrum (Table 1) showed signals for 30 carbons including six methyl groups, seven3 Table 1 1H and 13C NMR data of 1 and 2 (recorded with a Bruker DRX-500 MHz in C5D5N; J in Hz.) Position 1 2 δH (mult, J in Hz) δC (mult) δH (mult, J in Hz) δC (mult)1 1.60–1.64 (m, H a) 31.1(t) 4.27 (d, J = 4.5, Hβ) 81.9 (d)3.20–3.28 (m, H b)2 3.07 – 3.11 (m, H a) 32.6(t) 2.74 (d, J =18.0, Hα) 36.6 (t)2.43 – 2.47 (m, H b) 2.99 (dd, J = 4.5, 18.0, Hβ)3 174.8 (s) 175.3 (s)4 75.1 (s) 84.9 (s)5 2.10 (dd, J = 7.5, 17.5, Hα) 45.9 (d) 2.55 (dd, J = 4.0, 13.5, Hα ) 60.0 (d)6 1.72 – 1.79 (overlapped, Hα) 25.5 (t) 1.26 – 1.32 (m, Hα ) 28.8 (t)0.72 – 0.76 (m, Hβ) 1.59 – 1.65 (overlapped, Hβ)7 1.00 – 1.04 (m, Hα) 26.2 (t) 2.80 – 2.85 (overlapped, Hα) 24.7 (t)1.23 – 1.41 (overlapped, Hβ)2.02 – 2.12 (overlapped, Hβ)8 1.30 – 1.36 (overlapped, Hβ) 48.8 (d) 1.82 – 1.87 (overlapped, Hβ) 54.0 (d)9 22.6 (s) 73.8 (s)10 27.3 (s) 100.3 (s)11 2.17 – 2.26 (overlapped, Hα) 26.7 (t) 1.79 – 1.86 (overlapped, Hα) 39.5 (t)1.20 – 1.28 (overlapped, Hβ) 1.69 – 1.72 (overlapped, Hβ)12 1.51 – 1.59 (overlapped, 2H) 33.2 (t) 2.28 – 2.34 (overlapped, Hα) 30.8 (t)1.55 – 1.61 (overlapped, Hβ)13 45.6 (s) 47.3 (s)14 48.8 (s) 51.0 (s)15 1.27 – 1.35 (overlapped, 2H) 36.3 (t) 4.30 – 4.36 (m, Hβ ) 77.1 (d)16 1.68 – 1.75 (overlapped, Hα ) 27.1 (t) 2.28 – 2.34 (overlapped, Hα ) 40.4 (t)1.29 – 1.34 (overlapped, Hβ )2.14 – 2.24 (overlapped, Hβ )17 1.51 – 1.58 (overlapped, Hα ) 48.4 (d) 2.17 – 2.26 (overlapped, Hα ) 46.0 (d)18 0.95 (s) 18.5 (q) 0.99 (s) 15.7 (q)19 0.58 (ABd, J = 5.5, Hα) 31.5 (t) 1.98 – 2.07 (overlapped, 2H ) 48.1 (t)0.79 (ABd, J = 5.5, Hβ )20 1.95 – 2.01 (m) 39.5 (d) 2.14 – 2.24 (overlapped) 42.3 (d)21 0.97(d,J = 8.5) 13.1 (q) 1.36 (d, J = 6.0) 15.6 (q)22 4.45(ddd,J = 4.0, 4.5, 16.5) 80.5 (d) 4.10 (br. s) 73.9 (d)23 2.20 – 2.27 (overlapped, H a) 23.6 (t) 5.27 (br. s) 82.6 (d)2.00 – 2.08 (m, H b)24 6.53(d,J = 8.0) 140.2 (d) 7.19 (br. s) 148.7 (d)25 128.1 (s) 130.2 (s)26 166.3 (s) 174.9 (s)27 1.94 (s)17.3 (q) 1.80 (s) 10.7 (q)28 0.92 (s) 19.8 (q) 1.62 (s) 13.1 (q)29 1.42 (s) 32.0 (q) 1.08 (s) 23.0 (q)30 1.40 (s) 26.7 (q) 1.24 (s) 29.5 (q)OCH3 3.60 (s) 51.3 (q)Fig. 1 Structures of 1–34 Chin J NatMed Jan. 2010 V ol. 8 No.12010年1月 第8卷 第1期Fig. 2 1H-1H COSY and selected HMBC correlations of 1 and 2methylene carbons, eight methine carbons, two ester groups, a tri-attributed double bond, three oxygenated tertiary car-bons and two aliphatic tertiary carbons. Comparing these signals with those of micrandilactone B [1,10] with the schiar-tane carbon skeleton, it was found that signals of rings A-C, the side chain, as well as that of ring F in 2 were almost the same in the case of micrandilactone B, which was further confirmed by the key 1H,1H-COSY and HMBC data as shown in Fig. 2.Detailed comparisons of 1D-NMR (Table 1) indicated the main difference restricted in rings D and E. As an oxy- genated tertiary carbon in micrandilactone B replaced by an aliphatic one in 2 and one more methyl group appearing in 2, there could be a methyl attributed at C-14 or C-15. Two key HMBC correlations (Fig. 2) from H 3-18 to C-12, C-13 and C-17 and from H 3-28 to C-8, C-13, C-14 and C-15 proved that the methyl was attributed at C-14. Thus, compound 2 was actually a 3, 4: 9, 10-seco -cycloartane triterpenoid.The configuration of CH 3-18 was biogenetically β direc- tion while H-17 was α direction. Thus CH 3-28 was α oriented, which was established by the key ROESY correlation of H 3-28/H-7α/H-16α /H-17α (Fig. 3). The H-15 was assignedto be β orientation by the cross-peak of H 3-18/H-8/H-11β/H- 15β in the ROESY experiment (Fig. 3). And the other con-figuration was the same as that of micrandilactone B, which was deduced by the similar proton coupling constants and ROESY correlation data, as well as CD experiment.3.2 Putative biogenetic pathway from cycloartane to schiartane skeletonAs the oxidation degree deepening, cycloartane back- bone was presumed to be oxygenated into schiartane skeletonFig. 3 SelectedROESY correlations of 2Scheme 1 Proposed biosynthetic pathway from 3 to micrandilactone B5 through ring A opened at bond of C-3/C-4, three-member ringcleavage occurred at C-9/C-10, and then oxidative de-carboxylation occurred of CH3-28. Compounds 3, 1, 2, andmicrandilactone B were representive compound possessingcycloartane, 3,4-seco-cycloartane, 3, 4:9, 10-seco-cycloartaneand schiartane (3, 4:9, 10-seco-28-norcycloartane) skeleton,respectively. Their co-existence in S. propinqua var. propin-qua brought obvious evidence to fill in the gap between theputative biosynthetic sequences from cycloartane to schiar-tane skeleton (Fig. 3). First, compound 3 may be hydroxyl-ized at C-22 followed by dehydration, producing thesix-member α-methyl-α, β-unsaturated-δ-lactone ring in theside chain to produce kadsulactone[13]. Then, ring A of kad-sulactone disconnected at the bonds of C-3/C-4 followed bymethyl esterified at COOH-3 and hydroxylized at C-4 toproduce compound 1. After a few hydroxylation modifica-tions happened on 1 (intermediate A), the three-member ringwas broken at C-9/C-10 to build the seven-member ring C(intermediate B). Ester-exchange reaction formed twofive-member lactone rings A and F, and a Michael additionreaction (intermediate C) was finished to form the furan ringB; compound 2 was thus obtained. Micrandilactone B wasfinally produced through an oxidative decarboxylation ofCH3-28 (intermediate D). It’s the first time to discover com-pounds with 3, 4-seco-cycloartane and 3, 4: 9,10-seco-cycloartane skeletons co-existing with cycloartaneand schiartane backbones in the same source.References[1] Li RT, Han QB, Zheng YT, et al.Structure and anti-HIV ac-tivity of micrandilactones B and C, new nortriterpenoidspossessing a unique skeleton from Schisandra micrantha [J].Chem Commun, 2005, 23: 2936-2938.[2] Huang SX, Yang LB, Xiao WL, et al. WuweizidilactonesA-F: novel highly oxygenated nortriterpenoids with unusualskeletons isolated from Schisandra chinensis [J]. Chem EurJ,2007, 13(17): 4816-4822.[3] Li RT, Li SH, Zhao QS, et al. Lancifodilactone A, a novelbisnortriterpenoid from Schisandra lancifolia [J]. Tetrahe-dron Lett, 2003, 44(17): 3531-3534.[4] Huang SX, Li RT, Liu JP, Lu Y, et al. Isolation and charac-terization of biogenetically related highly oxygenated nor-triterpenoids from Schisandra chinensis [J]. Org Lett,2007,9(11): 2079-2082.[5] Li RT, Zhao QS, Li SH, et al. Micrandilactone A: a noveltriterpene from Schisandra micrantha [J]. Org Lett, 2003,5(7): 1023-1026.[6] Huang SX, Yang J, Huang H, et al. Structural characteriza-tion of schintrilactone, a new class of nortriterpenoids fromSchisandra chinensis [J]. Org Lett, 2007, 9(21): 4175-4178.[7] Li RT, Xiao WL, Shen YH, et al. Structure characterizationand possible bioggenesis of three new families of nortriter-penoids: Schisanartane, schiartane, and 18-norschiartane[J].Chem Eur J, 2005, 11(10): 2989-2996.[8] Huang SX, Han QB, Lei C, et al. Isolation and characteriza-tion of miscellaneous terpenoids of Schisandra chinensis [J].Tetrahedron, 2008, 64(19): 4260-4267.[9] Takahashi K, Tasako M. Studies on constituents of medicinalplants.XIV. constituents of Schisandra nigra Max. [J]. ChemPharm Bull, 1975, 23(3): 538-542.[10] Lei C, Huang SX, Chen JJ, et al. Propindilactones E–J,schiartane nortriterpenoids from Schisandra propinqua var.propinqua [J]. J Nat Prod, 2008, 71(7): 1228-1232.[11] Lei C, Pu JX, Huang SX, et al. A class of 18(13→14)-abeo-schiartane skeleton nortriterpenoids from Schisandrapropinqua var. propinqua [J]. Tetrahedron,2009, 65(1):164-170.[12] Lei C, Huang SX, Chen JJ, et al. Four new schisanartane-type nortriterpenoids from Schisandra propinqua var.propinqua [J]. Helv Chim Acta, 2007,90(7): 1399-1405.[13] Tan R, Xue H, Li LN. Kadsulactone and kadsudilactone, twonew triterpenoid lactones from Kadsura species [J]. PlantaMedica, 1991, 57(1): 87-88.合蕊五味子中两个新的三萜内酯及其在生物合成途径中的意义雷春, 普建新, 肖伟烈, 杨黎彬, 刘靖平, 陈纪军, 孙汉董*中国科学院昆明植物研究所植物化学与西部植物资源持续利用国家重点实验室, 昆明 650204【摘要】 目的：研究合蕊五味子(Schisandra propinqua var. propinqua) 地上部分化学成分。