Chinese Journal of

Natural Chinese Journal of Natural Medicines 2014, 12(9): 06890692 Medicines

Heteroclitins R-S: new dibenzocylooctadiene lignans from Kadsura heteroclita

CHEN Min1*, LUO You-Ping1, 2, ZOU Yan-Lin1, LANG Ling-Hu1, CHEN Dao-Feng3

1 Key Laboratory on Luminescence and Real-Time Analysis (Ministry of Education), College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, ; 2 College of Chemistry and Chemical Engineering, Normal University, Hai Kou 571158, China; 3 Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China

Available online September 2014

[ABSTRACT] AIM: To study the dibenzocylooctadiene lignans from the stems of Kadsura heteroclita. METHOD: Chromatographic separations of silica gel and semi-preparative HPLC were used. All of the structures were elucidated on the basis of spectroscopic analysis ,including 2D-NMR and HR-MS techniques. RESULTS: Four dibenzocylooctadiene lignans were isolated from K. heteroclita. Their structures were identified as heteroclitin R (1), heteroclitin S (2), gonisin O (3), and schisanlignone A (4). CONCLUSION: Heteroclitin R (1) and heteroclitin S (2) are new natural lignans.

[KEY WORDS] Dibenzocylooctadiene Lignan; Kadsura heteroclita; ; Heteroclitins R-S [CLC Number] R284.1 [Document code] A [Article ID] 2095-6975(2014)09-0689-04

ern area of China. Repeated column chromatography of the Introduction Et2O extract of the stems of K. heteroclita led to the isolation The stems of Kadsura heteroclita (Roxb.) Craib (Schi- and identification of a new dibenzocylooctadiene lignan named sandraceae) are commonly used in Chinese traditional medi- heteroclitin R (1), a new natural product named heteroclitin S [9] cine to promote vital energy and blood circulation, to expel (2), and two known compounds, gonisin O (3) and schi- [10] wind-evil, and to remove wetness-evil [1]. The are sanlignone A (4) . Compounds 3 and 4 were isolated from K. known to be a rich source of lignans and tripenternoids, heteroclita for the first time. Their structures and sterochemis- which have been found to possess various beneficial pharma- tries were elucidated by spectroscopic methods. cological effects [2-7]. In previous studies from this laboratory, some dibenzocyclooctadiene lignans were isolated from K. heteroclita [1, 8]. Efforts have continued to search for bioactive natural products from K. heteroclita indigenous to the south-

[Received on] 24-Aug.-2013 [Research funding] This investigation was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2011AA100607), the National Natural Science Foundation of China (No. 81102894), and the Key Technologies Fig. 1 Structures of compounds 1 and 2 Research and Development Program of China (No. 2011BAI 13B06). Results and Discussion [*Corresponding author] CHEN Min: Prof., Tel/Fax: 86-23-6825 1225, E-mail: [email protected] Compound 1, obtained as a yellow amorphous powder, These authors have no conflict of interest to declare. was assigned the molecular formula C27H28O9 by HR-EI-MS Published by Elsevier B.V. All rights reserved (m/z 519.163 3 [M + Na]+, Calcd. 519.163 1). The 1H NMR

– 689 – CHEN Min, et al. / Chin J Nat Med, 2014, 12(9): 689692 and 13C NMR data indicated that 1 is a dibenzocyclooctadiene showed the presence of a tigloxyl group. HMBC correlations lignan. The characteristic proton signals at δH 4.69 and 4.16 of H-11 with δC 78.4 (C-9), and δH 5.70 (1H, d, J = 5.1 Hz,

(2H, d, J = 8.8 Hz), and a quaternary carbon signal at δC 78.3, H-9) with the C-1′ (δC 167.1), C-7 (δC 43.7) and C-8 (δC 44.3) indicated that 1 possessed a spiroenone ring, similar to het- and C-17 (δC 10.9) in the HMBC spectrum, indicated an ester eroclitin H [8]. group substituted at C-9. The 1H NMR spectrum of 1 (Table 1) showed the pres- The CD spectrum of 1 with negative Cotton effect at 239 ence of two H-atoms at δH 7.38 (1H, s) and 6.53 (1H, s), nm and a positive Cotton effect at 223 nm indicated 1 had a which were assignable to H-4 and H-11, respectively. The S-biphenyl configuration [13]. The ketone group (C=O) at C-6 1H NMR spectrum also showed two doublet methyl groups indicated a twist-boat (TB) conformation was the only possi- [14-15] at δH 1.03 (3H, d, J = 6.6 Hz) and 0.94 (3H, d, J = 7.3 Hz), ble conformation for the cyclooctadiene ring . NOESY indicating the presence of two secondary methyl groups on correlations of H-4/CH3O-3, H-4/CH3-5', H-7/H-8, H-7/CH3- the cyclooctadiene ring. Two methoxy groups (δH 4.02, 17, H-8/CH3-18, CH3-18/CH3-17, H-9/H-8, H-9/H-11, and

3.78, each 3H, s), one methylenedioxy group (δH 6.07, H-9/CH3-5' strengthened the substituent positions and the 6.02, each 1H, d, J = 1.5 Hz), and two methine protons at stereochemical assignments (Fig. 2). Thus, 1 was elucidated 1 δH 3.14 and 2.01 (each 1H, m) also be observed in the H as shown in Fig. 1, and as a new compound, was named het- NMR spectrum. eroclitin R.

The cross peaks of H-4 (δH 7.38) with δC 135.7 and Compound 2, obtained as a colorless powder, has the

153.2, and of H-11 (δH 6.53) with δC 130.6 and 150.8 in the molecular formula C22H24O7, based on HR-ESI-MS data (m/z + 1 13 HMBC spectrum suggested that these four carbons were 423.141 7 [M + Na] , Calcd. 423.142 0). The H- and C- C-3, C-2, C-12, and C-13, respectively (Fig. 2). Two NMR spectra (Table 1) indicated that 2 is a dibenzocyclo-

H-atoms of a O-bearing CH2 group resonating at δH 4.69 octene lignan. 1 and 4.16 corre- lated with quaternary C-atoms at 143.5 The H NMR spectrum of 2 showed the presence of two aromatic protons at δ 7.49 and 6.38 (each 1H, s), assignable (C-14) and 141.2 (C-5), and the carbonyl group at δC 193.8 H in HMBC suggesting that 1 contained an α, β, γ, δ-dienone to H-4 and H-11, respectively. The resonances at δH 1.00 and structure, and a carbonyl group at C-1, similar to kad- 0.82 (each 3H, d, J = 6.6 Hz) could be assigned to the [11] cis-oriented CH -17 and CH -18 [15]. The 1H NMR spectrum sutherin C . The HMBC correlations of δH 6.07 and 6.02 3 3 also showed three methoxyl groups at δ 3.94, 3.92, and 3.81 with δC 130.6 and 150.8, and δH 4.02 (3H, s) with δC 153.2, (each 3H, s), a methylenedioxy group at δ 6.06, 6.03 (each and δH 3.78 (3H, s) with δC 135.7, suggested the substitu- tion of the two methylenedioxy groups at C-12 and C-13, 1H, d, J = 1.5 Hz), two methylene protons at δ 2.63 and 2.24 and that the two methoxyl groups were located in C-3 and (each 1H, m), and two methine protons at δ 2.63 and 1.79 C-2, respectively. (each 1H, m). The correlations of H-4 with δC 141.1 and 148.9, and of

H-11 with δC 152.2, 133.6, and 147.5 in the HMBC spectrum (Fig. 3), suggested that these five carbons were located as C-2, C-3, C-12, C-13, and C-14., respectively. The correlations of

the methylenedioxy protons (δ 6.06, 6.03) with δC 141.1 (C-2) and 148.9 (C-3) in the HMBC, indicated the methylenedioxy group was connected between C-2 and C-3. The cross peaks

of the methoxy groups at δH 3.81, 3.92, and 3.94 (each 3H, s)

with the carbons at δC 140.9, 152.2, and 133.6, respectively, revealed these three methoxy groups were located at C-1, Fig. 2 Key HMBC and ROESY correlations of 1 C-12, and C-13. The absence of a typical carbon signal for the methylenedioxy group at δ 100-102 in the 13C NMR spec- The signal at δH 7.38 (H-4) was shifted downfield by ca. C 1 ppm as compared with “common” aromatic H-atoms, and trum (Table 1) and the presence of one proton signals at δH showed HMBC correlation (Fig. 2) with the carbonyl group at 5.89 (1H, br s) without any correlation in HMQC spectrum, [16] δC 198.3, indicating that the carbonyl group was located at suggested the presence of hydroxyls on the aromatic rings . [12] C-6 and was conjugated with the aromatic ring . The Cross peaks of δH 5.89 (-OH) with δC 147.5 (C-14), 133.6 methyl protons at δH 1.03 correlated with δC 198.3 in the (C-13), and 115.4 (C-15) indicated that the hydroxy was lo- 13 HMBC suggesting that the methyl group was assignable to cated at C-14. The C NMR spectrum at δC 200.3 revealed C-17. the presence of a conjugated ketone moiety. HMBC cross

The proton signals at δH 5.78 (1H, q, J = 7.3 Hz), 1.70 peaks of H-4 and the methyl group at δH 1.00 with δC 200.3, (3H, dd, J = 7.3, 1.8 Hz), and 1.62 (3H, d, J = 1.8 Hz), and indicated that the C=O group was located at C-6, similar to the carbon signals at δC 167.1, 127.2, 137.1, 15.6, and 20.3 compound 1.

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University, Shanghai, China. Extraction and Isolation The stems (9 kg) of K. heteroclita were air-dried, ground and extracted five times with 95% ethanol (5 × 40 L) at room temperature and filtered. The alcoholic extract was evapo- rated in vacuo to yield a semisolid (1 200 g). The semisolid was suspended in water (2000 mL) and extracted seven times

with diethyl ether (7 × 3 L). This ether solution was concen- Fig. 3 Key HMBC and ROESY correlations of 2 trated to yield a residue (75 g). The residue was chroma- tographed on silica gel (1 500 g, 200−300 mesh), employing The CD spectrum of 2 with a negative Cotton effect at petroleum ether (60−90 °C) containing increasing amounts of 228, 280 nm and a positive Cotton effect at 247 nm indicated ethyl acetate as eluent (100% petroleum ether, 98 : 2, 95 : 5, [17] that 2 possessed an R-biphenyl configuration . NOESY 9 : 1, 8 : 2, 7 : 3, 6 : 4, 5 : 5, 100% ethyl acetate) to afford correlations of H-11 with CH3O-12, H-7β with H-8β, H-8β nine fraction. Fraction 6 [petroleum ether−EtOAc (7 : 3), 12.5 with CH3-17, CH3-18 with CH3-17, CH3-18 with H-9α, H-8β g] was subjected to repeated silica gel column chromato- with H-11, H-9β with H-11, and CH3O-13 with OH-14 graphy with petroleum ether-acetone (8 : 1 to 3 : 1). Fr. 6-3 strengthened the substituent positions and the stereochemical (72 mg) was purified with semi-preparative HPLC (65% assignments (Fig. 3). Thus, the structure of 2 was determined -1 MeOH−H2O, flow rate 2 mL·min ) to yield 1 (7 mg) and 2 as shown in Fig. 1. The structure of 2 was the same as the (8.5 mg). Fraction 5 [petroleum ether−EtOAc (8 : 2), 10.3 g] synthetic compound 2a [18], and is a new natural product, was also subjected to repeated silica gel column chroma- named as heterolitin S (Fig. 1). tography with petroleum ether−acetone (8 : 1). Semi- prepa- Compounds 3 and 4 were identified as gomisin O and rative HPLC (70% MeOH−H O, flow rate 2 mL·min-1) of schisanlignone A by comparison their UV, IR, EI-MS, 2 fraction 5-4 (112 mg) gave 3 (12 mg) and 4 (5 mg). 1H NMR, and 13C NMR data with those reported [9-10], and were isolated from K. heteroclita for the first time. Structure identification

Experimental Heteroclitin R (1) obtained as a yellow amorphous pow- der; [α] 25 +7.9° (c 0.29 MeOH); UV (MeOH) λ (log ε) General D max −1 The IR spectra were recorded as KBr pellets on the Ava- 200 nm (4.78); IR (KBr) max: 2 953, 1 715, 1 651, 1 117 cm ; 1 13 tar 360E.S.P spectrophotometer (Thermo Nicolet Co.). The H NMR and C NMR, see Table 1; HR-ESI-MS: C27H28O9 + UV spectra were measured on a Shimadzu UV-260 spectro- (m/z 519.163 3 [M + Na] , Calcd. 519.163 1); CD (c 0.29, 15 photometer in absolute MeOH. Optical rotations were meas- MeOH), (θ) (nm): +22477 (223), –16746 (239). ured with a JASCO P-1020 spectropolarimeter. The CD spec- Table 1 1H NMR and 13C NMR spectra for compounds 1 and tra were measured with a JASCO J-715 spectropolarimeter. 2 (400 MHz and 100 MHz, CDCl3) Mass spectra were determined on a HP5989A mass spec- 1 2 trometer for EI-MS and a Kratos Concept 1H series mass No. δC δH δC δH spectrometer for HR-EI-MS. 1H NMR and 13C NMR spectra 1 193.8 140.9 were measured on the Bruker DRX400 spectrometers with 2 153.2 141.1 TMS as internal standard and CDCl3 as solvent. Column chromatography (CC) was carried out on a silica gel (200− 3 135.7 148.9 300 or 300−400 mesh) (Qingdao Marine Chemical Factory, 4 127.3 7.38 (s) 104.1 7.49 (s) Qingdao, China). Semi-prep HPLC was carried out on Amer- 5 141.2 134.4 sham UV-900 with an ODS column (Rp-18, 250 × 10 mm, 6 198.3 200.3 YMC, 10 μm, detector: UV) with a flow rate of 2.0 mL·min-1. Analytical TLC was performed on silica gel plates (Yantai 7 43.7 3.14 (m) 44.6 2.63 (m) Institute of Chemical Technology) with petroleum ether― 8 44.3 2.01 (m) 41.2 1.79 (m) EtOAc (3 : 1). Spots on the plates were observed under UV 9 78.4 5.70 (d, 5.1) 40.1 2.63, 2.24 (m) light and visualized by spraying with 10% H SO , followed 2 4 10 128.7 136.1 by heating. 11 101.2 6.53 (s) 102.8 6.38 (s) material The stems of Kadsura heteroclita were collected in 12 130.6 152.2 Fengqing County, Province, China in July of 1997, 13 150.8 133.6 identified by CHEN Dao-feng. A voucher specimen (DFC- 14 143.5 147.5 JXT9707) is deposited in the Herbarium of Materia Medica, 15 120.5 115.4 Department of Pharmacognosy, School of Pharmacy, Fudan

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(Continue) [5] Xu LJ, Peng ZG, Chen HS, et al. Bioactive triterpenoids 1 2 from Kadsura heteroclita [J]. Chem Biodivers, 2010, 7 (9): No. 2289-2295. δ δ δ δ C H C H [6] Yang XW, Miyashiro H, Hattori M, et al. Isolation of novel 16 64.1 124.8 lignans, heteroclitin-F and heteroclitin-G, from the stems of 17 10.9 1.03 (d, 6.6) 15.4 1.00 (d, 6.6) Kadsura heteroclita, and antilipid peroxidative actions of heteroclitins A-G and related compounds in the in vitro 18 15.1 0.94 (d, 7.3) 15.3 0.82 (d, 6.6) rat-liver homogenate system [J]. Chem Pharm Bull, 1992, 40 6.06, 6.03 (d, 19 102.2 6.07, 6.02 (d, 1.5) 101.8 (6): 1510-1516. 1.5) [7] Yang XW, Hattori M, Namba T, et al. Anti-lipid peroxidative 20 78.3 4.69,4.16 (d, 8.8) - effect of an extract of the stems of Kadsura heteroclita and its major constituent, kadsurin, in mice [J]. Chem Pharm Bull, 2-OCH3 58.3 4.02 (s) 60 3.81 (s) 1992, 40 (2): 406-409. 3-OCH 59.1 3.78 (s) - 3 [8] Chen M, Jia ZW, Chen DF. Heteroclitin H, a new lignan from 12-OCH3 - 55.7 3.92 (s) Kadsura heteroclita [J]. J Asian Nat Prod Res, 2006, 8 (7): 643-648. 13-OCH3 61.1 3.94 (s) [9] Ikeya Y, Taguchi H, Yosioka I, et al. Constituents of Schizandra 14-OH - - 5.89 (br s) chinensis Baill. 5. Structures of 4 new lignans, gomisin-N, OTig:1' 167.1 - gomisin-O, epigomisin-O and gomisin-E, and transformation of 2' 127.2 - gomisin-N to deangeloylgomisin-B [J]. Chem Pharm Bull, 1979, 27 (11): 2695-2709. 3' 137.1 5.78 (q, 7.3) - [10] Liu JS, Zhou HX. Isolation and structures of schisanlignone-A, 4' 15.6 1.70 (dd, 7.3/1.8) - schisanlignone-B and butyryl binankadsurin-A [J]. Acta Chim 5' 20.3 1.62 (d,1.8) - Sin, 1991, 49 (4): 412-416. [11] Lu Y, Chen DF. Kadsutherins A-C: three new dibenzocyclooc- tane lignans from the stems of Kadsura species [J]. Helv Chim Heteroclitin S (2) obtained as a colorless amorphous 25 Acta, 2006, 89 (5): 895-901. powder; [α] D +9.6° (c 0.24, MeOH); UV (MeOH) λmax (log ε) [12] Luo G, Liu JS, Huang MF. Studies on the constituent of Schi-

195 nm (5.86); IR (KBr) max: 3 462, 2 950, 1 584, 1 484, 1 sandra viridis Ac Sm in the Northern Guangdong Province [J]. 072 cm-1; 1H NMR and 13C NMR, see Table 1; HR-ESI-MS: Acta Chim Sin, 1992, 50 (6): 620-624. + C22H24O7 (m/z 423.141 7 [M + H] , Calcd. 423.142 0); CD (c [13] Liu JS, Li L. Schisantherins L-O and acetylschisantherin-L 0.24, MeOH), (θ)15 (nm): +122 289 (247), –171 895 (228), from Kadsura coccinea [J]. Phytochemistry, 1993, 32 (5): –47 538 (280). 1293-1296. [14] Gottlieb HE, Mervic M, Ghera E, et al. Conformational References study of dibenzocyclo-octadiene systems related to the schizandrin- type lignans [J]. J Chem Soc Perk T, 1982, 1 [1] Chen DF, Xu G.J, Yang XW, et al. Dibenzocyclo-octadiene (10): 2353-2358. lignans from Kadsura heteroclita [J]. Phytochemistry, 1992, 31 [15] Ma WH, Ma XL, Lu Y, et al. Lignans and triterpenoids from the (2): 629-632. stems of Kadsura induta [J]. Helv Chim Acta, 2009, 92 (4): [2] Kangouri K, Miyoshi, T, Ikeda A, et al. Cholesterol- 709-715. biosynthesis inhibitory activities of novel triterpenoid acids [16] Chen M, Liao ZX, Chen DF. Four new dibenzocyclooctene from Kadsura heteroclita and Kadsura longipedunculata [J]. lignans from Kadsura renchangiana [J]. Helv Chim Acta, 2004, Agric Biol Chem Tokyo, 1990, 54 (4): 993-997. 87 (6): 1368-1376. [3] Pu JX, Yang LM, Xiao WL, et al. Compounds from Kadsura [17] Yang JH, Zhang HY, Wen J, et al. Dibenzocyclooctadiene heteroclita and related anti-HIV activity [J]. Phytochemistry, lignans with antineurodegenerative potential from Kadsura 2008, 69 (5): 1266-1272. ananosma [J]. J Nat Prod, 2011, 74 (5): 1028-1035. [4] Wang W, Liu JZ, Han J, et al. New triterpenoids from Kadsura [18] Luo G, Liu JS. Studies on the constituent of viridis heteroclita and their cytotoxic activity [J]. Planta Med, 2006, Ac Sm in the Northern Guangdong Province [J]. Acta Chim Sin, 72 (5): 450-457. 1992, 50 (5): 515-520.

Cite this article as: CHEN Min, LUO You-Ping, ZOU Yan-Lin, LANG Ling-Hu, CHEN Dao-Feng. Heteroclitins R-S: new dibenzocylooctadiene lignans from Kadsura heteroclita [J]. Chinese Journal of Natural Medicines, 2014, 12(9): 689-692

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