Benzyl isoquinoline and tetrahydroprotoberberine alkaloids from Stephania rotunda Lour

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Journal of Chemistry, Vol. 44 (3), P. 372 - 376, 2006 Benzyl isoquinoline and tetrahydroprotoberberine alkaloids from Stephania rotunda Lour. Received 16 February 2005 Trinh Thi Thuy1, Tran Van Sung1, K. Franke2 and L. Wessjohann2 1 Institute of Chemistry, Vietnamese Academy of Science and Technology 2 Department of Bioorganic Chemistry, Leibniz-Institute of Plant Biochemistry, Germany summary Chemical investigation of Stephania rotunda Lour. growing in Vietnam led to the isolation and structural elucidation of benzylisoquinolines, coclaurine (1), dehassiline (2), as well as tetrahydroprotoberberines, stepholidine (3) and corynoxidine (4), along with 24 other alkaloids. These structures were determined on the basis of MS, NMR spectroscopic data and comparison with reported data. Keywords: Stephania rotunda, Menispermaceae, benzylisoquinoline, coclaurine, dehassiline, tetrahydroprotoberberine, stepholidine, corynoxidine. I - Introduction In a previous paper [1], we have reported the isolation and structural determination of a new isoquinolone alkaloid, two novel tetrahydroprotoberberine alkaloids, seven quaternary protoberberines, four aporphines, three oxoaporphines, morphinane and proaporphine. In a continuing investigation of the alkaloid constituents from the tubers of this plant, we describe in this paper the isolation and structural determination of benzylisoquinolines, coclaurine (1), dehassiline (2), as well as tetrahydroprotoberberines, stepholidine (3) and corynoxidine (4). These structures were elucidated by MS, NMR techniques and comparison with reported data. II - Experimental 1. General Optical rotation [ ]D: Digital Polarimeter Jasco DIP 1000. EIMS: ADM 402, 70 eV, 372 Finigan TSQ 700. HR-ESI-MS: BRUKER BIOAPEX 70 eV. NMR: VARIAN MERCURY 400 MHz, UNITY 500 spectrometer at 499.83 MHz (1H) and 100, 125 MHz (13C, 13C APT). Chemical shifts were referenced to internal TMS ( = 0, 1H) and CDCl3 ( = 77.0, 13C) or CD3OD ( = 49.0, 13C). CC: Silica gel 60, 0.06 - 0.20 mm (Merck) for the first column, silica gel 60, 40 - 63 µm (Merck) for the following columns. TLC: Silica gel 60 F-254 (Merck). 2. Plant material Tubers of S. rotunda Lour. were collected in Hoa Binh province, North of Vietnam, in 1999. The species was identified by Dr. Vu Xuan Phuong, Institute of Ecology and Natural Resources, Vietnamese Academy of Science and Technology, Hanoi. A voucher specimen Nr. 5111 (19/08/2001) is deposited in the Herbarium at the same institute. 3. Extraction and Isolation The dried and powdered tubers of S. rotunda (2.4 kg) were extracted with 85% aq. EtOH at room temperature. EtOH was evaporated in vacuo at 45oC and the aq. solution (200 g) was partitioned with n-hexane followed by EtOAc and n-BuOH. The organic solvents were evaporated in vacuo to afford 9.5, 70 and 30 g extracts, respectively. The n-BuOH extract was separated on silica gel using CHCl3-MeOH (65 : 35) and then CHCl3-MeOH-H2O (65 : 35 : 5) to afford 58 fractions (F-1 F-58). The crude compound 1 was isolated from fraction 8 and further purified by chromatography on silica gel to yield 1 (77 mg, 0.0032%); compound 2 was isolated from fraction 12 (52 mg, 0.0022%); compound 3 from fraction 5 (140 mg, 0.0058%); compound 4 from fraction 14 (128 mg, 0.0053%). a) Coclaurine (1, 7-hydroxy-6-methoxy-1,2,3,4tetrahydroisoquinoline) The compound was purified by CC [silica gel, CHCl3/MeOH 95:5); [ ]22D -9o (MeOH, c 0.5); powder from MeOH; HR-ESI-MS (m/z): 286.1432 [M+H]+ (C17H20NO3, calc. 286.1438); EI-MS 70 eV, m/z (rel. int.): 285 [M]+ (1), 282 (3), 206 (4), 192 (8), 179 (14), 178 (100), 163 (26), 148 (2), 134 (5). 1H and 13C NMR data, see table 1. b) Dehassiline [2, (S)-1-(2'-OH-5'-methoxy-7hydroxy-6-methoxy-1,2,3,4-tetrahydroisoquinoline N-methyl] The compound was purified by CC [silica gel, CHCl3/MeOH/H2O 65 : 35 : 5); yellow powder from MeOH; [ ]22D + 62° (CHCl3, c 1.0); EI-MS 70 eV, m/z (rel. int.): 329 [M]+ (1), 193 (20), 192 (100), 177 (35), 149 (4), 148 (5); 1 H and 13C NMR data, see table 1. c) Stepholidine (3, 3,9-dihydroxy-2,10dimethoxy-tetrahydroprotoberberine, 5,8,13,13a-tetrahydro-3,9-dimethoxy-6Hdibenzo[a,g]quinolizine-2,10-diol) Yellow needles from MeOH/acetone; [ ]22D -295o (MeOH, c 0.5). EI-MS 70 eV, m/z (rel. int.): 327 [M]+ (80), 192 (5), 178 (100), 166 (15), 150 (60), 149 (55), 135 (28). 1H- and 13C NMR data, see table 2. d) Corynoxidine (4, 2,3,9,10-tetramethoxytetrahydroprotoberberine N-oxide) Yellow powder from MeOH/EtOAc; [ ]22D -81 (MeOH, c 0.5) (lit. for (S)-enantiomer [ ]D22 -57o). ESI-MS (positive ion): 372.2 [M+H]+ (C21H25NO5). ESI-MS (negative ion): 370.2 [M-H]-. 1H and 13C NMR data, see table 2. o III - Results and Discussion The residue of an ethanol extract of the tubers of S. rotunda was partitioned successively with n-hexane, ethyl acetate, and n-butanol. The n-butanol extract, after evaporation of the solvents, were subjected to crystallization from EtOAc/EtOH to remove most of tetrahydropalmatine. The mother liquid was further purified by column chromatography and recrystallization to give 1 - 4, together with other alkaloids [1, 2]. Compounds 1 - 4 showed a strong fluorescence under UV light (254 and 366 nm) and positive reactions with Dragendorff's and Mayer reagent. 5 MeO HO 6 7 4a 8 HO 13 3 1 NH H 15 14 4 9 MeO N HO H HO 10 11 OMe 12 1 2 The HR-ESI-MS of compound 1 gave the [M+H]+ peak at m/z 286.14324 leading to the molecular formula C17H19NO3 (M = 285). The EI-MS spectrum of compound 1 exhibited characteristic fragments of isoquinoline skeleton possessing one hydroxy and one methoxy (m/z 178) as well as benzylhydroxy fragment (m/z107). The 13C-NMR spectrum exhibited 12 aromatic and 8 aliphatic carbon signals, indicating the presence of two aromatic rings, as present in the benzylisoquinoline system. From the multiplicities of the signals at H7.04 and 6.74, which were parts of an AA'XX'-system 373 and the intensity from 13C-NMR, a 1,4-paradisubstituted benzene-ring was deduced. One substituent was identified as a hydroxy group by the high chemical shift of the connected carbon ( C 157.0), whereas the other substituent was a CH2 group with a signal at C 42.1. The 1HNMR spectrum confirmed the presence of CH2CH2 and CH-CH2 moieties and methoxy group ( 3.77). The aromatic region displayed two singlets at 6.61 and 6.67, which suggested two protons must be located at C-5 and C-8, thus two substituents are at C-6 and C-7. Analysis of MS and NMR spectra combined with the optical rotation ([ ]D22 +9o, MeOH) confirmed the structure (+)-coclaurine (sanjoinine K) for 1. Coclaurine is a hypotensive agent and was isolated earlier from Xylopia papuana and Alseodaphne archboldiana [3]. Table 1: 13C- and 1H-NMR data of compounds 1 and 2 [MeOH-d4, 100/400 MHz, (ppm), J (Hz)] 1 2 C 1 3 4 4a 5 6 7 8 8a 57.86 41.37 29.52 126.60 113.96 145.53 147.60 112.69 130.16 3.98 dd (9.3; 4.2) 3.14 - 3.05 m 2.76 - 2.69 m 9 42.10 2.69 ma; 3.06 dd (11.0; 4.4) 10 11 12 13 14 15 6-OMe 11-OMe N-CH3 130.94 131.22 116.36 157.00 116.36 131.22 56.25 C H C 6.67 s 6.61 s 7.04 d (8.6) 6.74 d (8.4) 6.74 d (8.4) 7.04 d (8.6) 3.78 s 65.79 47.50 26.07 133.63 121.70 144.82 147.59 112.46 125.19 40.96 130.49 147.08 117.40 115.50 147.25 112.46 56.21 56.34 H 3.65 dd (7.3; 4.9) 2.82 m; 3.11 m 2.65 m; 2.72 m 6.60 s 6.11 s 2.65 dd (13.7; 5.0) 3.04 dd (11.7; 4.9) 6.77 d (8.2) 6.49 dd (8.2; 2.2) 6.60 d (2.2) 3.77 s 3.79 s 2.42 s a Overlapping. The EI-MS spectral analysis of 2 indicated a close structural relationship to coclaurine, but with one methoxy group more than 1 (m/z 329 [M]+). The 1H- and 13C NMR spectra indicated the presence two methoxy, three methylene and an N-methyl groups. The aromatic region displayed two singlets, one doublet with a large coupling constant (J = 8.2 Hz), another doublet with a small coupling constant (J = 2.2 Hz) and a doublet of doublets (J = 8.2, 2.2 Hz), which suggested one aromatic ring possess two ortho protons and a third proton is in meta position to 374 one of the two protons. Combination of NMR, MS spectral data and optical rotation ([ ]D22 +62o, CHCl3) confirmed the structure of compound 2 as (+)-dehassiline (reported for the +147o, CHCl3). (S)-enantiomer [ ]D27 Dehassiline was isolated for the first time from the bark of Dehaasia kurzii (Lauraceae) [4]. The molecular formula of compound 3 (C19H21NO4) was deduced from combined analysis of ESI-MS with a peak at m/z 328 [M+H]+, 1H and 13C APT NMR spectra. The EI 4 MeO 4a 5 6 3 2 N HO 1 H 13 14 8 8a 12a OMe 9 10 12 OH 11 3 3 R MeO + N MeO O - H OMe OMe R 5: H R = OH 4: R =4 H; MS spectrum of compound 3 exhibited characteristic fragments due to a Retro Diels Alder cleavage of a tetrahydroprotoberberine Table 2: skeleton possessing one hydroxy and one methoxy groups in the A ring (m/z 178) and the same groups in the D ring (m/z 150) [1, 2]. The 1 H and 13C-NMR spectra indicated the presence of two aromatic rings, two methoxy and four methylene groups. There was no evidence for a carbonyl function. The aliphatic region of the 1 H-NMR spectrum displayed a pair of "AB" doublets at 4.19 and 3.57 ppm. The large coupling between these signals (JH-H = 15.2 Hz) suggested geminal protons (C-8) of the protoberberine alkaloid. The aromatic region of the 1H-NMR spectrum displayed a pair of doublets ("AB" pattern) suggesting the presence of two ortho protons in one of the aromatic ring and two singlets consisted with two para related protons in the second aromatic ring (C-1 and C4), which suggested that two protons can be located at the position C-1 and C-4 and thus two substituent groups are at C-2 and C-3. 13 C- and 1H-NMR data of compounds 3 and 4 [CDCl3, 100/400 MHz, (ppm), J (Hz)] 3 C 1 2 3 4 4a 5 6 8 8a 9 10 11 12 12a 13 14 14a 2-OMe 3-OMe 9-OMe 10-OMe a C 4 H C 110.79 6.81 s 108.39 146.59 147.90 145.23 147.48 114.43 6.59 s 111.46 126.06 122.51 29.52 3.20 m 24.07 52.06 3.92 m 64.09 54.23 4.19 d (15.2); 3.57 d (5.5) 66.88 127.46 124.84 146.59 150.09 144.06 145.02 111.52 6.78 d (8.2) 110.94 125.32 6.81 d (8.2) 123.24; 124.21 128.00 H 6.69 s 6.69 s 3.66 m; 3.49 m 3.84 m; 4.50 br d (15.5) 4.50 br d (15.5); 4.71 d (15.5) 6.88 d (8.4) 7.01 d (8.4) 2.73 br d (15.5) 3.31 dd (16.3; 4.0) 3.58 br d (11.0) 36.52 2.66 m; 3.08 m 29.25 59.70 130.53 3.54 m 67.45 124.84 56.27 3.87 s 55.60; 55.48 3.87 s 3.89 s 59.70 3.81 s 59.90; 55.91 3.86 s; 3.88 s Overlapping. 375 Placement of two other substituents groups at C-9 and C-10 in D ring has been confirmed by a pair of doublets with AB-system and ortho coupling (J = 8.2 Hz) between H-11 and H-12 in the 1H NMR spectrum. Combination of MS, NMR and comparison with reported data [1, 2] confirmed the structure of stepholidine (3,9dihydroxy-2,10-dimethoxy-tetrahydroprotoberberine) for 3. Stepholidine was isolated from Stephania glabra (Menispermaceae) [5]. The ESI-MS of compound 4 gave the [M+H]+ peak at m/z 372 (C21H25NO5, M = 371), with one oxygen atom more than tetrahydropalmatine. The EI-MS spectral analysis indicated a close structural relationship to tetrahydropalmatine, but displayed a major fragment ion at m/z 355 [M-O]+, attributed to a facile loss of an oxygen from the molecular ion, which is characteristic for protoberberine Noxides. The 1H- and 13C-NMR spectra of 4 were suggestive for a tetrahydroprotoberberine skeleton with the same substitution pattern as 3 and tetrahydropalmatine. However, comparison of the 13C-NMR data showed significant difference in the chemical shift to tetrahydropalmatine [2], especially at 5, 6, 8, 13 and 14 positions (table 2): 28.9, 51.3, 53.8, 36.1, 59.1 in (-)-tetrahydropalmatine [2] and 24.07, 64.09, 66.88, 29.25, 67.45 in 4, respectively. Based on spectral data, compared with the data of L-tetrahydropalmatine N-oxide [6] as well as [ ]D22 (-81o, CHCl3), compound 4 376 has been determined as L-tetrahydropalmatine N-oxide (corynoxidine). Corynoxidine and epicorynoxidine were isolated for the first time from Corydalis koidzumiana (Fumariaceae) [6]. A new similar compound, 4-hydroxycorynoxidine (thaicanine N-oxide, 5), was also isolated from this plant [1]. Acknowledgements: We are indebted to BMBF, Germany, for financial support in form of a project. We thank Dr. A. Porzel, Mrs. M. Suesse for the NMR spectra and Dr. J. Schmidt (Institute of Plant Biochemistry, Weinberg 3, D06120 Halle/Saale, Germany) for the mass spectra. References 1. T. T. Thuy, A. Porzel, F. Katrin, L. Wessjohann, T. V. Sung. Pharmazie 60 (9), 701 - 704 (2005). 2. J. T. Blanchfield, D. P. A. Sands, C. H. L. Kennard, K. A. Byriel, W. Kitching. Phytochemistry, 63, 711 - 720 (2003). 3. S. R. Johns, et al. Aust. J. Chem., 20, 1729 (1967). 4. A. Watanabe, et al. Heterocycles, 48, 1623 1630 (1998). 5. S. Wu, et al. Acta Cryst. C, 43, 2126 (1987). 6. C. Tani, N. Nagakura, S. Hattori, N. Masaki. Chemistry Letters, 1081 - 1084 (1975).
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