Structure elucidation of four steroids from the soft coral sinularia nanolobata - Ninh Thị Ngọc

Vietnam Journal of Science and Technology 55 (3) (2017) 258-262 DOI: 10.15625/2525-2518/55/3/8800 STRUCTURE ELUCIDATION OF FOUR STEROIDS FROM THE SOFT CORAL SINULARIA NANOLOBATA Ninh Thi Ngoc1, 2, Nguyen Van Thanh1, Nguyen Xuan Cuong1, Nguyen Hoai Nam1, *, Do Cong Thung3, Chau Van Minh1 1Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 2Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, Ha Noi 3Institute of Marine Environment and Resources, VAST, 246 Da Nang, Ngo Quyen, Hai Phong *Email: namnguyenhoai@imbc.vast.vn Received: 20 October 2016, Accepted for publication: 10 January 2017 ABSTRACT Four steroids namely 3β-hydroxyergosta-5,24(28)-diene-7-one (1), dissesterol (2), 16α- hydroxysarcosterol (3), and sarcophytosterol (4) were isolated from the soft coral Sinularia nanolobata using various chromatographic methods. Their structures were elucidated by detailed analysis of the 1D and 2D NMR data and comparison with the reported values. Keywords: Sinularia nanolobata, soft coral, steroids. 1. INTRODUCTION Soft corals are marine invertebrates of the order Alcyonacea, subclass Octocorallia, class Anthozoa, and phylum Cnidaria. Among these marine invertebrates, the genera Cespitularia, Clavularia, Gersemia, Lobophytum, Nephthea, Sarcophyton, and Sinularia are the most prolific [1]. The Sinularia soft corals are a rich source of hydroxylated steroids having cytotoxic effects [2 – 8]. Within the frame of our recent investigations on chemical constituents and biological activities of Vietnamese Sinularia soft corals [9, 10], we have recently reported six steroids from the soft coral Sinularia nanolobata and evaluation of their cytotoxic activity [11]. The current paper deals with detailed structure elucidation of four steroids from this species. 2. EXPERIMENTAL 2.1. General methods The NMR spectra were recorded on a Bruker AVANCE III HD 500 spectrometer with TMS as an internal standard. Medium pressure liquid chromatography (MPLC) was carried out on a Biotage - Isolera One system. TLC was performed on Kieselgel 60 F254 (1.05715; Merck) or Structure elucidation of four steroids from the soft coral Sinularia nanolobata 259 RP-18 F254s plates. Spots were visualized by spraying with 10% aqueous H2SO4 solution, followed by heating for 3 - 5 minutes. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70 - 230 mesh and 230 - 400 mesh, Merck) and YMC*GEL resins (ODS-A, 12 nm S-150 µm, YMC Co., Ltd.). 2.2. Biological material The samples of the soft coral S. nanolobata Verseveldt, 1977 were collected in Lang Co, Hue, Vietnam, in April 2015 and identified by Prof. Do Cong Thung (Institute of Marine Environment and Resources, VAST). Voucher specimens (No SN201504) were deposited at the Institute of Marine Biochemistry, VAST, Vietnam. 2.3. Extraction and isolation Dried bodies of the soft coral S. nanolobata (1.5 kg) were extracted three times with methanol under ultrasonic condition. The resulting solutions were filtered, combined, and concentrated under reduced pressure to obtain the methanol residue (SNM, 140 g), which was suspended in water and extracted in turn with n-hexane and dichloromethane resulting in extracts of n-hexane (SNH, 47 g), dichloromethane (SND, 5 g), and an aqueous layer. Extract SNH (47 g) was crudely separated on silica gel MPLC using the mobile phase of n- hexane−acetone (gradient 50:1→1:1, v/v) to obtain six fractions, SNH1−SNH6. Fraction SNH4 (7.3 g) was further separated on silica gel MPLC using the mobile phase of n-hexane−ethyl acetate (gradient 5:1→1:1, v/v) to give seven subfractions, SNH4A-SNH4G. Subfraction SNH4B (2.1 g) was further separated into nine smaller fractions, SNH4B1-SNH4B9, by YMC CC eluting with methanol−water (2:1, v/v). Compounds 3 (5.0 mg) and 4 (3.5 mg) were obtained from subfraction SNH4B7 (300 mg) after purification by YMC CC eluted with acetone−water (3:1, v/v), followed by silica gel CC with n-hexane−acetone (3.5:1, v/v). Fraction SNH5 (5.0 g) was further separated on RP-18 MPLC with methanol−water (2:1, v/v) to give six subfractions, SNH5A-SNH5G. Finally, compounds 1 (6.0 mg) and 2 (2.6 mg) were purified from subfraction SNH5F (130 mg) by silica gel CC eluting with n-hexane−ethyl acetate (2:1, v/v). Figure 1. Chemical structures of compounds 1−4. Ninh Thi Ngoc, N. V. Thanh, N. X. Cuong, Nguyen Hoai Nam, Do Cong Thung, Chau Van Minh 260 3β-Hydroxyergosta-5,24(28)-diene-7-one (1): White powder; 1H-NMR (500 MHz, CDCl3) and 13C-NMR (125 MHz, CDCl3) see Table 1. Dissesterol (2): White powder; 1H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) see Table 1. Table 1. The NMR spectroscopic data of compounds 1 and 2. C aδC 1 eδC 2 δCb,c δH b,d mult. (J = Hz) δC c,f δHd,f mult. (J = Hz) 1 36.34 36.42 1.18 m /1.92 m 36.5 36.47 1.20 m/1.60 m 2 31.19 31.23 1.92 m/1.60 m 28.5 28.52 1.43 m/1.78 m 3 70.54 70.55 3.65 m 67.3 65.94 3.96 m 4 41.81 41.89 2.38 m/2.49 m 128.1 128.40 5.37 br s 5 165.07 165.34 - 147.1 145.17 - 6 126.13 126.14 5.66 s 73.6 72.10 4.02 br s 7 202.30 202.45 - 38.9 39.33 1.15 m/1.98 m 8 45.41 45.48 2.22 m 30.1 29.72 1.82 m 9 49.95 49.98 1.32 m 54.1 53.96 0.68 m 10 38.28 38.36 - 36.7 36.04 - 11 21.22 21.28 1.54 m/1.55 m 20.8 20.29 1.35 m/1.45 m 12 38.69 38.76 1.12 m/2.01 m 39.7 39.93 1.01 m/1.79 m 13 43.14 43.21 - 42.8 42.28 - 14 49.90 50.02 1.50 m 55.8 55.35 0.96 m 15 26.31 26.38 2.39 m/1.26 m 24.2 23.66 1.08 m/1.58 m 16 28.54 28.60 1.28 m/1.89 m 28.0 27.42 1.35 m/1.96 m 17 54.63 54.70 1.06 m 57.8 57.33 1.25 m 18 12.00 12.05 0.66 s 11.8 11.56 0.69 s 19 17.33 17.38 1.17 s 21.1 20.50 1.17 s 20 35.66 35.72 1.15 m 35.1 33.95 1.08 m 21 18.87 18.94 0.93 d (7.0) 21.1 20.53 0.98 d (7.0) 22 34.68 34.75 1.53 m 31.8 31.35 0.22 m 23 30.97 31.05 1.84 m/2.08 m 25.6 25.00 - 24 156.79 156.83 - 50.6 49.62 0.32 m 25 33.76 33.82 2.20 m 32.0 31.08 1.59 m 26 21.87 22.08 0.99 d (7.0) 21.2 20.45 0.85 d (7.0) 27 22.01 21.93 1.00 d (7.0) 21.8 21.60 0.94 d (7.0) 28 106.03 106.10 4.69 br s/4.62 br s 15.2 14.31 0.92 d (7.0) 29 14.0 13.82 0.91 s 30 20.9 20.12 −0.10 dd (4.5, 5.5) 0.45 dd (4.5, 9.0) aδC of 3β-hydroxyergosta-5,24(28)-diene-7-one [12], brecorded in CDCl3, c125 MHz, d500 MHz, eδC of dissesterol [13], frecorded in DMSO-d6. 3. RESULTS AND DISCUSSION Compound 1 was obtained as a white powder. The NMR features indicated an ergosterol derivative, one main constituent of Sinularia soft corals [2]. The 1H- and 13C-NMR spectra of 1 showed typical signals of one oxymethine [δC 70.55 (C-3)/δH 3.65 (1H, m, H-3)], one trisubstituted endocyclic double bond [δC 165.34 (C, C-5) and 126.14 (CH, C-6)/δH 5.66 (1H, s, Structure elucidation of four steroids from the soft coral Sinularia nanolobata 261 H-6)], one ketone [δC 202.45 (C, C-7)], one 1,1-disubstituted double bond [δC 156.83 (C, C-24) and 106.10 (CH2, C-28)/δH 4.62 and 4.69 (H-28), each 1H, br s], two tert-methyls [δC 12.05 (C- 18) and 17.38 (C-19)/δH 0.66 (H-18) and 1.17 (H-19), each 3H, s], and three sec-methyls [δC 18.94 (C-21), 22.08 (C-26), and 21.93 (C-27)/δH 0.93 (H-21), 0.99 (H-26), and 1.00 (H-27), each 3H, d, J = 7.0 Hz]. The HMBC correlations (Fig. 2) of H-6 with C-4, C-8, and C-10; H-8 with C-7, and H-19 with C-1, C-5, C-9, and C-10, clearly confirmed positions of the double bond C- 5/C-6 and ketone C-7. The detailed analysis of other HMBC correlations and an agreement of the 13C-NMR chemical shifts of 1 with the reported data led to assignment of 1 as 3β- hydroxyergosta-5,24(28)-diene-7-one [12]. Figure 2. Key HMBC correlations of compounds 1 and 2. Compound 2 was also obtained as a white powder. The 13C-NMR and HSQC spectra confirmed 30 carbon signals including seven methyls, eight methylenes, eleven methines, and four quaternary carbons. The signals at δC 11.56 (C-18), 20.50 (C-19), 20.53 (C-21), 20.45 (C- 26), 21.60 (C-27), 14.31 (C-28), and 13.82 (C-29) indicated the presence of seven methyl groups. Moreover, two oxymethine groups [δC 65.94 (C-3) and 72.10 (C-6)] and a trisubstituted double bond [δC 128.40 (CH, C-4) and 145.17 (C, C-5)] were also identified. In the 1H-NMR spectrum, the presence of four high-field protons at δH 0.22 (1H, m, H-22), 0.32 (1H, m, H-24), −0.10 (1H, dd, J = 4.5, 5.5 Hz, Hβ-30), and 0.45 (1H, dd, J = 4.5, 9.0 Hz, Hα-30) is characteristic of a gorgosterol-type side chain possessing a cyclopropane ring [14]. The HMBC cross peaks of H-19 (δH 1.17) with C-1 (δC 36.47)/C-5 (δC 145.17)/C-9 (δC 53.96)/C-10 (δC 36.04) and H-6 (δH 4.02) with C-4 (δC 128.40)/C-8 (δC 29.72)/C-10 (δC 36.04) confirmed positions of the double bond at C-4/C-5 and the hydroxy group at C-6. The detailed analyses of other HMBC correlations (Fig. 2) and the comparison of the 13C-NMR chemical shifts of 2 with those reported (Table 1) clearly identified compound 2 as dissesterol [13]. The other compounds were elucidated as 16α-hydroxysarcosterol (3) [15] and sarcophytosterol (4) [16] by detailed analysis of their 1D and 2D NMR data and in comparison with reported data. Acknowledgements. 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