two unusual nitro-substituted hasubanan-type alkaloids from stephania longa

Chinese Journal of Chemistry, 2006, 24, 781—784 Full Paper

* E-mail: [email protected], Tel.: 0086-21-50806718, Fax: 0086-21-50806718 Received December 15, 2005; revised February 16, 2006; accepted February 27, 2006. Project supported by the National Natural Science Foundation of China (No. 30025044), Shanghai Municipal Scientific Foundation (No.

04XD14019), and the Foundation from the Ministry of Science and Technology (No. 2002CB512807) of China.

© 2006 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Two Unusual Nitro-substituted Hasubanan-type Alkaloids from Stephania longa

ZHANG, Hua(张华) WANG, Fang-Dao(王方道) YUE, Jian-Min*(岳建民)

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Zhangjiang Hi-Tech Park, Shanghai 201203, China

Two unusual nitro-substituted hasubanan-type alkaloids, stephalonines J (1) and K (2), together with ten known alkaloids, protostephanine, dehydrostephanine, (－)-stephanine, (－)-isolaureline, R-roemeroline, (＋)-pronucife-rine, (＋)-stepharine, (＋)-N-acetylstepharine, (＋)-lirioferine, and (＋)-norlirioferine, were isolated from the whole plant of Stephania longa. Their structures were characterized mainly by spectroscopic methods including IR, MS, and NMR experiments, and the structures of 1 and 2 were further confirmed through chemical correlations with the known alkaloids stephalonines A (1a) and B (2a), respectively.

Keywords Stephania longa, hasubanan-type alkaloid, stephalonine J, stephalonine K

Introduction

Among the thirty-nine species and a variety of the genus Stephania (Menispermaceae) distributed mainly in the south of China,1 many plants are used in tradi-tional Chinese medicine (TCM) or as folklore herbs.2,3 The whole plant of Stephania longa Lour., a TCM, has been applied to the treatment of fever, inflammation, and dysentery.3 Chemical investigations conducted pre-viously on this plant have led to the isolation of about thirty alkaloids and a few nonalkaloids.4,5 As a con-tinuation of our research work on this TCM, two un-common nitro-substituted hasubanan-type alkaloids, stephalonines J (1) and K (2), together with ten known other types of alkaloids, protostephanine,6 dehydrostephanine,7 (－)-stephanine,8 (－)-isolaureline,9 R-roemeroline,10 (＋)-pronuciferine,11 (＋)-stepharine,11 (＋)- N-acetylstepharine,11 (＋ )-lirioferine,12 and (＋ )-nor-lirioferine,13 were isolated from the whole plant of S. longa. The structures of these alkaloids were elucidated mainly by spectroscopic methods, and those of the new alkaloids 1 and 2 were further confirmed through chemical correlations with two structurally relevant known alkaloids stephalonines A (1a) and B (2a), re-spectively. We report herein the isolation and structural elucidation of these alkaloids.

Results and discussion

Stephalonine J (1) was isolated as yellow powder. Its IR spectrum revealed the presence of hydroxyl, car-bonyl, and conjugated nitro groups corresponding to the absorption bands at 3425, 1724, 1520 (NO2) and 1338

(NO2)14 cm－1, respectively. A molecular formula of

C25H34N2O9 was established for 1 by HREIMS at m/z 506.2241 (calcd 506.2264), which was 45 mass units more than that of stephalonine A (1a),5 indicating the substitution of a hydrogen by a nitro group. Comparison of its 1H and 13C NMR (Table 1) spectra with those of 1a showed that the structure of 1 was closely related to 1a only with the replacement of a proton by a nitro group at the aromatic ring. After the assignment of all the proton and carbon signals on the basis of 1H-1H COSY, HMQC, and HMBC (Figure 1) spectra, the H-10 at δH 5.89 (d, J＝6.6 Hz, 1H) of 1 was obviously downfield shifted from the H-10 at δH 4.87 (d, J＝6.2 Hz, 1H) of 1a, while the C-10 (δC 69.9) of 1 was upfield shifted from that (δC 77.0) of 1a. The occurrence of such phenomena was apparently caused by the effect of the nitro group, and hence the nitro group must be allot-ted to C-1 position vicinal to C-10. The HMBC (Figure 1) correlation between H-10 and C-1 (δC 139.2), and the downfield shifted C-1 carbon signal (compared with that of 1a at δC 115.9) due to the presence of the nitro group, were also good proof. Its structure was finally confirmed to be (6β,7β,8β,10β)-8,10-epoxy-4,6-dihydroxy-3,7,8-trimethoxy-17-methyl-1-nitrohasubanan 6-((2S)-methylbutyrate) by the conversion of 1a into 1 according to a method for phenol nitration.15

Stephalonine K (2) had a molecular formula of C29H32N2O9 as determined by HREIMS, indicating 45 mass units more than that of stephalonine B (2a).5 Nitro absorption bands at 1518 and 1338 cm−1 were also ob-served in the IR spectrum.14 Its NMR data (Table 1) were closely related to those of 2a with minor variation

782 Chin. J. Chem., 2006, Vol. 24, No. 6 ZHANG, WANG & YUE


Table 1 NMR data (δ) of 1 and 2 in CDCl3

1 2 No.

δC δH δC δH

1 139.2 139.4

2 103.9 7.26 (s, 1H) 103.6 6.95 (s, 1H)

3 145.6 145.5

4 147.9 147.6

5 32.2 2.99 (dd, J＝15.3, 2.9 Hz, 1H)

2.20 (dd, J＝15.3, 2.9 Hz, 1H) 31.3

3.21 (dd, J＝15.4, 3.8 Hz, 1H)

2.13 (dd, J＝15.4, 2.4 Hz, 1H)

6 66.9 5.33—5.37 (m, 1H) 68.1 5.34—5.38 (m, 1H)

7 81.6 3.70 (d, J＝4.4 Hz, 1H) 81.5 3.78 (d, J＝4.1 Hz, 1H)

8 103.4 103.6

9 28.0 2.74 (dd, J＝11.2, 6.6 Hz, 1H)

1.53 (d, J＝11.2 Hz, 1H) 27.6

2.78 (dd, J＝11.3, 6.7 Hz, 1H)

1.60 (d, J＝11.3 Hz, 1H)

10 69.9 5.89 (d, J＝6.6 Hz, 1H) 70.1 6.04 (d, J＝6.7 Hz, 1H)

11 131.0 130.2a

12 130.3 130.0

13 49.2 49.3

14 75.9 76.0

15 34.8 2.50—2.57 (m, 1H)

1.83 (ddd, J＝14.6, 12.6, 6.9 Hz, 1H) 34.5

2.44—2.51 (m, 1H)

1.79—1.87 (m, 1H)

16 53.9 3.38—3.44 (m, 1H)

2.48—2.54 (m, 1H) 53.9

3.34—3.51 (m, 1H)

2.49—2.56 (m, 1H)

17 38.3 2.57 (s, 1H) 38.2 2.58 (s, 1H)

1′ 176.6 134.3

2′ 40.5 1.62—1.68 (m, 1H) 128.1 7.38—7.43 (m, 2′—6′-H, 5H)

3′ 26.3 1.24—1.31 (m, 1H)

1.09—1.17 (m, 1H) 128.9

4′ 11.1 0.70 (t, J＝7.5 Hz, 3H) 130.3a

5′ 15.4 0.67 (d, J＝7.0 Hz, 3H) 128.9

6′ 128.1

7′ 143.1 7.22 (d, J＝16.1 Hz, 1H)

8′ 118.1 5.59 (d, J＝16.1 Hz, 1H)

9′ 166.7

3-OMe 56.5 3.93 (s, 3H) 55.7 3.25 (s, 3H)

7-OMe 57.5 3.35 (s, 3H) 57.4 3.41 (s, 3H)

8-OMe 51.7 3.52 (s, 3H) 51.8 3.57 (s, 3H) a Signals may be interchanged in the same vertical column.

at the aromatic part of the mother skeleton, also very similar to those of stephalonine J (1) with the difference only in the side ester chain moiety. The above analysis revealed that 2 was a nitro derivative of 2a, which was supported also by the following observations: the H-10 at δH 6.04 (d, J＝6.7 Hz, 1H) of 2 was downfield shifted from that of 2a at δH 4.96 (d, J＝6.2 Hz, 1H), while the C-10 at δC 70.1 of 2 was upfield shifted from that of 2a

at δC 77.3. The HMBC (Figure 1) correlation between H-10 and C-1 (δC 139.4), the latter was notably down-field shifted compared with that (δC 115.9) of 2a due to the existence of the nitro group, was also indicative. Sephalonine K (2) was therefore identified to be (6β,7β, 8β,10β)-8,10-epoxy-4,6-dihydroxy-3,7,8-trimethxy-17- methyl-1-nitrohasubanan 6-cinnamate. The structure of 2 was also verified through chemical correlation with 2a

Stephania longa Chin. J. Chem., 2006 Vol. 24 No. 6 783


via nitration.15

Figure 1 1H-1H COSY (bold) correlations of 1 and selected HMBC (single arrow, H→C) correlations of 1 and 2.

To the best of our knowledge, this is the first report of natural nitro-substituted hasubanan-type alkaloids. Though the mechanism of how the nitro group is intro-duced into the molecules of such hasubanan-type alka-loids still remains unclear, this report will certainly at-tract the interest of biosynthetic chemists.

Experimental

General procedures

UV spectra were measured on a Hitachi U-2010 spectrophotometer. IR spectra were recorded on a Perkin Elmer 577 spectrometer. Optical rotations were determined on a Perkin Elmer 341 polarimeter. NMR spectra were measured on a Bruker AM-400 spec-trometer. ESIMS data were determined on a Bruker Es-quire 3000 mass spectrometer while EIMS (70 eV) data were on a Finnigan MAT 95 mass spectrometer. All solvents used were of analytical grade (Shanghai Chemical Reagent Company Ltd.). Precoated silica gel GF254 plates (Yantai Huiyou Silica Gel Exploitation Company Ltd.) were used for TLC, and silica gel H (Qingdao Haiyang Chemical Company Ltd.) and neutral alumina (200—300 mesh, Shanghai Chemical Reagent Company Ltd.) were used for column chromatography. Sephadex LH-20 (Amersham Biosciences), amino silica gel (20—45 µm, Fuji Silysia Chemical Ltd.), and MCI gel CHP20P (75—150 µm, Mitsubishi Chemical Indus-try Ltd.) were also used for column chromatography.

Plant material

The whole plant of S. longa was collected from Guangxi province of China in the summer of 2002, and identified by Shi Su-Hua of the Institute of Botany, School of Life Sciences, Zhongshan University. A

voucher specimen has been deposited in Shanghai In-stitute of Materia Medica with accession number: SL-2002-1Y.

Extraction and isolation

The plant material was extracted and fractionated through the procedures reported previously5 to yield 37.0 g of crude alkaloids. The crude alkaloids were par-titioned on a neutral alumina column (Et2O/MeOH, 100∶1 to 1∶1, V∶V) to afford six fractions F1—F6. F1 was chromatographed on a silica gel column (CHCl3/ MeOH, 100∶1 to 40∶1, V∶V) to form protostephanine (1050 mg) and a major mixture of two alkaloids, which was further purified on an amino silica gel col-umn (cyclohexane/EtOAc, 10∶1, V∶V) to obtain de-hydrostephanine (5 mg) and (－)-stephanine (35 mg). F2 was first treated on a silica gel column (isopropyl ether/MeOH, 80∶1 to 40∶1, V∶V) to obtain several sub-fractions, and three of these sub-fractions were pu-rified by preparative TLC (CHCl3/MeOH, 50∶1, V∶V) to afford (－)-isolaureline (29 mg), (＋)-pronuciferine (11 mg), and (＋)-N-acetylstepharine (15 mg), respec-tively. F3 was first dealt with a silica gel column (pe-troleum ether/EtOAc/Et2NH, 10∶1∶0.3 to 2∶1∶0.3, V∶V) to collect several major parts, one of which was then run on a Sephadex LH-20 column (in MeOH) to produce a mixture of two alkaloids, which was further separated on a silica gel column (CHCl3/MeOH, 30∶1 to 20∶1, V∶V) to obtain R-roemeroline (26 mg) and (＋)-stepharine (40 mg). F4 was fractionated on a silica gel column (petroleum ether/EtOAc/Et2NH, 5∶1∶0.3 to 1∶1∶0.3, V∶V) to collect the major sub-fractions, and one of them was further applied on a Sephadex LH-20 column (in MeOH) to afford a mixture, which was further separated on an amino silica gel column (CHCl3/MeOH, 30∶1, V∶V) to obtain stephalonines J (1, 10 mg) and K (2, 11 mg). F5 was fractionated on an MCI column (MeOH/H2O, 3∶7 to 6∶4, V∶V) to yield several sub-fractions, one of which was purified on an amino silica gel column (CHCl3/MeOH, 20∶1, V∶V) to furnish (＋)-lirioferine (7 mg) and (＋)-norlirioferine (8 mg) in turn.

Nitration of stephalonines A (1a) and B (2a)

To a stirred mixture of water (2.0 mL), diethyl ether (2.0 mL), and excess potassium nitrate (5.0 mg), 1.0 mL of concentrated hydrochloric acid was added, followed by addition of 10.2 mg of stephalonine A (1a). The re-action mixture was kept over night, and basified with saturated NaHCO3 solution. The product was extracted with chloroform (3×3.0 mL), and then purified by preparative TLC (CHCl3/MeOH, 20∶1, V∶V) to af-ford 3.9 mg of stephalonine J (1, yield 35%). The alka-loid stephalonine B (2a, 14.0 mg) was transformed into stephalonine K (2, 4.7 mg) in a yield of 31% by the same procedure.

Stephalonine J (1)

Yellow powder with 21D[ ]α ＋ 181.2 (c 0.165,

784 Chin. J. Chem., 2006, Vol. 24, No. 6 ZHANG, WANG & YUE


CHCl3); UV (MeOH) λmax (log ε): 285 (3.76) nm; 1H and 13C NMR: see Table 1; IR (KBr) ν: 3425, 2939, 1724, 1619, 1583, 1520, 1483, 1338, 1284, 1096, 1065, 1028, 966, 922 cm－1; ESIMS m/z: 507.2 [M＋H]＋

(positive mode) and 505.2 [M－H]－ (negative mode); EIMS (70 eV) m/z (%): 506 (M＋, 5), 490 (25), 489 (100), 405 (9), 276 (90), 275 (61), 274 (76), 258 (45), 228 (50); HREIMS m/z: 506.2241 (M ＋ , calcd for C25H34N2O9, 506.2264).

Stephalonine K (2)

Yellow powder with 21D[ ]α ＋354.6 (c 0.13, CHCl3);

UV (MeOH) λmax (logε): 277 (4.22) nm; 1H and 13C NMR: see Table 1; IR (KBr) ν: 3400, 2941, 1705, 1635, 1518, 1481, 1441, 1338, 1286, 1175, 1090, 1063, 1028, 768 cm－1; ESIMS m/z: 553.2 [M＋H]＋(positive mode) and 551.3 [M－H]－ (negative mode); EIMS (70 eV) m/z (%): 552 (M＋, 3), 536 (34), 535 (100), 276 (27), 275 (23), 274 (43), 258 (22), 228 (28); HREIMS m/z: 552.2119 (M＋, calcd for C29H32N2O9, 552.2108).

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(E0512155 ZHAO, X. J.)

two unusual nitro-substituted hasubanan-type alkaloids from stephania longa

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