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Journal of Chemistry, Vol. 43 (4), P. 513 - 516, 2005 TWO NEW ALOCERAMIDES FROM ALOCASIA MACRORRHIZA Received 26th, July 2004 NGUYEN QUYET TIEN1, PHAM HOANG NGOC1, PHAM HONG MINH1, PHAN VAN KIEM2 AND YOUNG HO KIM3 1 Institute of Chemistry, Vietnamese Academy of Science and Technology 2 Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology 3 College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea summary Two new aloceramides alomacrorrhiza A and alomacrorrhiza B were isolated from the ethanolic extract of the plant Alocasia macrorrhiza (L.) Schott. Their chemical structures were elucidated as (2S,3S,4R)-2N-[(2'R)-2'-hydroxy-hexacosanoyl]-tetradecane-1,3,4-triol (1) and (2S,3S,4R)-2N-[(2'R)-2'-hydroxy-hexacosanoyl]-hexadecane-1,3,4-triol (2) based on extensive 1D, 2D NMR, EI-MS, FAB-MS, HR-FAB-MS spectroscopic data and chemical degradation studies. Keywords: Araceae, Alocasia macrorrhiza, ceramide, alomacrorrhiza A, alomacrorrhiza B. I - INTRODUCTION Alocasia macrorrhiza (L.) Schott (Araceae) is widely distributed in Vietnam, and used as a folk medicine to treat inflammation, eczema and abscess [2, 5]. Alocasin, an anti-fungal protein and trypsin inhibitor has been isolated from the giant taro A. macrorrhiza [1, 3, 7]. We report herein the isolation and structural elucidation of two new ceramides, (2S,3S,4R)-2N-[(2'R)-2'hydroxy-hexacosanoyl]-tetradecane-1,3,4-triol (1) and (2S,3S,4R)-2N-[(2'R)-2'-hydroxy-hexaco-sanoyl]-hexadecane-1,3,4-triol, (2) from the ethanolic extract of A. macrorrhiza. II - MATERIALS AND METHODS General experimental procedures Melting points were determined using an Electrothermal IA-9200. IR spectrum was obtained on a Hitachi 270-30 type spectrometer with KBr discs. Optical rotations were determined on a JASCO DIP-1000 KUY polarimeter. EI-MS was obtained using a Hewlett Packard 5989 B MS spectrometer. FABMS and HR-FAB-MS were obtained using a JEOL JMS-DX 300 spectrometer. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) were recorded on a Bruker AM500 FT-NMR spectrometer and TMS was used as an internal standard. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70 - 230 mesh and 230 - 400 mesh, Merck). Plant material The roots of A. macrorrhiza were collected in Hoabinh province, Vietnam in December 1999 and identified by Prof. Nguyen Tien Ban, Institute of Ecology, Biological Resources, Vietnamese Academy of Science and Technology (VAST). Voucher specimens are deposited at the Institute Chemistry, VAST. Extraction and isolation The dried and powdered roots of A. macrorrhiza (2.0 kg) were extracted three times 513 with hot EtOH repeatedly to give ethanolic extract (210.0 g), which was suspended in water and extracted using hexane, chloroform, ethyl acetate and n-butanol, respectively. The ethyl acetate extract (11.5 g) was subjected to chromatography on a silica gel column, using chloroform-methanol (9 : 1) as eluent to yield six fractions (Fr. A-F). Fraction C (1.2 g) was followed by CC on a YMC RP-8 column using a MeOH-H2O (10 : 1) as eluent to yield 1 (34.5 mg) and 2 (57 mg). (2S,3S,4R)-2N-[(2'R)-2'-hydroxyhexacosanoyl]-tetradecane-1,3,4-triol (1) White amorphous powders; mp 112 - 114oC; [ ]25D +32.5o (c 1.00, MeOH); positive FAB-MS m/z: 678.6 [M+Na]+; HR-FAB-MS m/z: 678.6013 [M+Na]+ (Calcd for C40H81NO5Na: 678.6012); 1H- and 13C-NMR (see table 1). (2S,3S,4R)-2N-[(2'R)-2'-hydroxyhexacosanoyl]-hexadecane-1,3,4-triol (2) White amorphous powders; mp 107 - 109oC; [ ]25D +42.5o (c 1.00, MeOH); positive FAB-MS m/z: 706.63 [M+Na]+; HR-FAB-MS m/z: 706.6321 [M+Na]+ (Calcd for C42H85NO5Na: 706.6325); 1H- and 13C-NMR (see table 1). NMR (500 MHz, CDCl3) : 4.18 (H, br s, H-2), 3.79 (3H, s, CH3-O), 0.89 (3H, t, 8.7); 13C-NMR (125 MHz, CDCl3) : 175.8 (C-1'), 70.4 (C-2'), 52.4 (CH3-O), 34.4 (C-3'), 21.4 - 30.9 (C-4' to C-13') and 14.1 (C-14'). Acetylation of 1 Compound 1 (4 mg) was added to dry pyridine (0.25 ml) and Ac2O (0.5 ml) and left overnight. After usual workup, the reaction mixture was chromatographed over silica gel [hexane-EtOAc (5 : 1)] to yield derivative 1a (1.4 mg) as white crystals; mp 105 - 108oC; [ ]25D +26.5o (c 0.1, MeOH); 1H-NMR (500 MHz, CDCl3) : 6.57 (d, J = 9.1 Hz, NH), 4.33 4.95 (m, 5H, carbinol protons), 2.18 (3H, s, OAc), 2.08 (3H, s, OAc), 2.05 (3H, s, OAc), 2.02 (3H, s, OAc) and 0.88 (6H, t, J = 8.7 Hz). Acetylation of 2 Compound 2 (4 mg) was acetylated as 1 to yield derivative 2a as white crystals; mp 108 - 111 oC; [ ]D +31.0o (c 0.1, MeOH); 1H-NMR (500 MHz, CDCl3) : 6.58 (d, J = 9.1 Hz, NH), 4.33-4.96 (m, 5H, carbinol protons), 2.19 (3H, s, OAc), 2.08 (3H, s, OAc), 2.06 (3H, s, OAc), 2.03 (3H, s, OAc) and 0.88 (6H, t, J = 8.7 Hz). 25 Acid hydrolysis 1 and 2 Each ceramide 1 (20 mg) and 2 (20 mg) were refluxed with 0.9 N HCl in 82% aqueous MeOH (15 ml) for 18 h. The resulting solutions were extracted with hexane, and combined organic phases were dried over Na2SO4. Evaporation of the hexane yielded the same fatty acid methyl ester 3 as a white amorphous powder. The H2O layers were neutralized with conc-NH4OH and extract with ether. The ether layers were dried over Na2SO4, filtered and then concentrated to yield the long chain bases. 2-hydroxy-hexacosanoic acid methyl ester (3) A white amorphous powder; mp 60 - 62oC; [ ] -1.5o (c 0.5, CHCl3); EI-MS (70 eV) m/z (%): 426 [M]+ (9.3), 412 (22.7), 398 (61.0), 367 (8.0), 159 (15.4), 145 (23.9), 126 (13), 111 (13), 97 (65.7), 83 (45.2), 57 (100) and 55 (82.3); 1H25 D 514 III - RESULTS AND DISCUSSION Repeated column chromatography on silica gel and YMC RP-8 of the EtOAc extract of the dried and powdered roots of A. macrorrhiza yielded two new ceramides 1 and 2. Compound 1 and 2 formed as white amorphous powders. The IR spectrum of 1 and 2 exhibited hydroxyl absorptions at 3434 cm-1 and amide functions at 1645 cm-1. The HR-FAB-MS of 1 provided the molecular formula C40H81NO5 (observed m/z: 678.6013 [M+Na]+; Calcd for C40H81NO5Na: 678.6012). The 1H- and 13C-NMR spectra of 1 were typical of a ceramide processing a long chain base and 2-hydroxy fatty acid (table 1). Assignments of all protons and carbons of 1 were made by 1H-1H COSY, HMQC and HMBC spectra. The 1H-NMR of 1 showed a doublet at 7.50 (d, J = 9.1 Hz) due to an NH proton, a broad singlet at 1.21 (methylene protons) and carbinol protons appearing between 3.40 and 3.86 suggesting it to be a ceramide. The 13CNMR spectrum of 1 showed carbonyl carbon signals at 173.2 (s), carbinol carbons at 60.4 (t), 71.0 (d), 71.0 (d) and 74.5 (d), methine carbon at 51.3 (d), methylene carbons at 34.0-21.5 and two methyl carbons at 13.2 (q). In the 1H-1H COSY spectrum (table 1), the NH doublet at 7.50 showed a cross peak at 4.00 attributed to the H-2 proton. The latter proton showed coupling with two doublet doublet at 3.72, 3.61 and a doublet doublet at 3.42, assigned to protons H-1 and H-3, respectively. The H-3 proton also showed coupling with the multiplet at 3.40 assigned to H-4. O 1' 3' 2' HN 26' 20 OR OR RO 2 1 4 14 n 3 Hb Ha OR 1 R = H, n = 7; 1a R = Ac, n = 7 2 R = H, n = 9; 2a R = Ac, n = 9 O H3C 1' O 2' 3' (CH2)21 26' OH 3 Figure 1. Structures of Compounds 1, 1a, 2, 2a and 3 The other carbinol proton appearing at 3.86 showed only one cross peak to 2.00 m. The above 1H-1H correlation studies suggested the placement of three hydroxyl groups in long chain base and one hydroxyl group in N-acetyl moiety. Moreover, the H-C long-range correlations between NH proton and carbon C-1' ( 173.2)/C-2' ( 71.0)/C-1 (60.4)/C-2 (51.3)/C3 (74.5), and between proton H-2 ( 4.00) and carbon C-3 ( 74.5)/C-4 (71.0) were observed in the HMBC. This confirmed the location of carbonyl group at C-1', three methine carbinol groups at C-2', C-3 and C-4, and methylene carbinol at C-1. The stereochemistry of ceramide 1 was determined as 2S, 3S, 4R, 2'R by comparison of the 1H, 13C-NMR data of 1 with that of the other (2S,3S,4R,2'R)cerebrosides [6] and (2S,3S,4R,2'R)-phytosphingosine moieties [4]. Compound 1 on acetylation with Py-Ac2O (see experimental part) gave peracetyl derivative 1a that showed four acetyl groups at 2.18, 2.08, 2.05 and 2.02 in the 1H-NMR. Furthermore, 1 was methanolyzed with methanolic hydrochloric acid, 2hydroxy-hexacosanoic acid methyl ester (3) was obtained together with long-chain base (see experimental part). Based on the above data, 1 was elucidated as (2S,3S,4R)-2N-[(2'R)-2'hydroxy-hexacosanoyl]-tetradecane-1,3,4-triol, which we named alomacrorrhiza A. The molecular formula of 2 was deduced to be C42H85NO5 from the HR-FAB-MS (observed Calcd for m/z: 706.6321 [M+Na]+; C40H81NO5Na: 706.6325). The 1H- and 13CNMR spectra of 2 were very similar to those of 1 (table 1). Compound 1 was methanolyzed with methanolic hydrochloric acid, 2-hydroxyhexacosanoic acid methyl ester (3) was obtained together with the long-chain base. This confirmed that compounds 1 and 2 had the same N-acyl moiety. In addition, compound 2 on acetylation with Py-Ac2O gave peracetyl derivative 2a that showed four acetyl groups at 2.19, 2.08, 2.06 and 2.03 in the 1H-NMR. The stereochemistry of ceramide 2 was determined as 2S, 3S, 4R, 2'R by comparison of the 1H-, 13 C-NMR data of 2 with that of 1 and the other (2S,3S,4R,2'R)-cerebrosides [6] and (2S,3S,4R,2'R)-phytosphingosine moieties [4]. These data led to the structure of 2 as (2S,3S,4R)-2N-[(2'R)-2'-hydroxyhexacosanoyl]-hexadecane-1,3,4-triol, which we named alomacrorrhiza B. Acknowledgements: We are grateful to the KBSI for measuring the MS spectra and NMR Lab., Institute of Chemistry, VAST for NMR experiments. Thanks are due to Prof. Nguyen 515 Table 1: 1H- and 13C-NMR spectral data for 1 and 2 1 C a,b C Long chain base NH 1a 60.4 t 1b 2 3 51.3 d 74.5 d 4 71.0 d 5a 31.8 t 5b 6 - 13 21.5 -30.8 t 14 13.2 q N-acyl moiety 1' 173.2 s 71.0 d 2' 3'a 3'b 4'-25' 26' 27' 28' 34.0 t 21.5 - 30.8 t 13.2 q a,c H (J, Hz) 2 1 H-1H COSY HMBC (H to C) 7.50 d (9.1) H-2 3.61 dd (6.1, 12.5) 3.72 dd (6.0, 12.8) 4.00 m H-1b, 2 C-1, 2, 3 1', 2' C-2, 3 H-1a, 2 C-2, 3 3.42 dd (3.2, 4.5) 3.40 m 1.55 m 1.28 m 1.26 br s 0.89 t (8.7) 3.86 dd (8.0, 3.7) 2.00 m, 2.20 m 1.26 br s 0.89 t (8.7) H-1a, 1b, 3, C-1’, 3, 4 NH H-2, H-4 C-1, 2, 4 H-3, 5a, 5b H-4, 6 H-4, 6 C-2, 3 a,b C H-3'a, 3'b C-1', 3' H-2', 4' H-2', 4' C-1', 2' C-1', 2' H-25' (J, Hz) - 7.50 d (9.1) 60.6 t 3.61 dd (6.1, 12.5) 3.72 dd (6.0, 12.8) 4.00 m 51.4 d 74.7 d 71.2 d 31.9 t 21.5-30.8 t 13.2 q H-13 a,c H 173.3 s 71.0 d 34.0 t 21.5-30.8 t 21.5-30.8 t 21.5-30.8 t 13.2 q 3.42 dd (3.2, 4.5) 3.40 m 1.55 m 1.28 m 1.26 br s 0.89 t (8.7) 3.86 dd (8.0, 3.7) 2.00 m 2.20 m 1.26 br s 1.26 br s 1.26 br s 0.89 t (8.7) a In DMSO, b125 MHz, c500 MHz, Chemical shift ( ) in ppm. 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