HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 8, 2011, pp. 1903-1907
Received, 16th May, 2011, Accepted, 6th June, 2011, Published online, 9th June, 2011.
DOI: 10.3987/COM-11-12260

■ Metacolchicine, a New Colchicinoid from Sandersonia aurantiaca

Mariko Kitajima, Akiko Tanaka, Noriyuki Kogure, and Hiromitsu Takayama*

Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

Abstract

Metacolchicine is a new colchicinoid isolated from the tubers of Sandersonia aurantiaca. Its unique structure featuring an additional carbon unit at C-10 position was deduced from spectroscopic analyses.

Colchicine (1) is a well-known bioactive alkaloid that is used for the treatment of gout. It also acts as an antimitotic compound by binding to tubulin.1 The structure elucidation of the tubulin-colchicine complex by X-ray crystallographic study2 has contributed to progress in structure-activity relationship studies that are geared toward the development of new antitumor agents based on colchicine (1). In our continuing studies on novel and bioactive alkaloids, new colchicinoids, including 4-oxygenated ones named gloriosamines A and B, were isolated from Gloriosa rothschildiana.3 Recently, we also found semi-synthetic 4-halo colchicine derivatives that exhibited higher activity against cancer cell lines than colchicine (1).4 Sandersonia aurantiaca Hook (Liliaceae) is known to produce some colchicinoids.5 Then, to discover new alkaloids, we investigated the alkaloidal constituents in the tubers of S. aurantiaca and isolated one new colchicinoid, metacolchicine (2). In this paper, we report the structure elucidation of this new colchicinoid.

From the MeOH extract of the tubers of S. aurantiaca, one new colchicinoid 2 (5.8 mg) was isolated together with six known alkaloids, colchicine (1, 44.4 mg), 2-demethylcolchicine (27.5 mg),6 3-demethylcolchicine (126.8 mg),6 colchifoline (28.1 mg),7 demecolcine (7.0 mg),8 and 2-demethyldemecolcine (4.3 mg),9 by a combination of column chromatographies.

New alkaloid 2, named metacolchicine, was found to have the same molecular formula C22H25NO6 as colchicine (1) from HRFABMS [m/z 400.1752 (MH+)]. Its 1H NMR spectrum (Table 1) showed signals assignable to three methoxy groups on the aromatic ring at δH 3.94 (2-OCH3), 3.91 (3-OCH3), and 3.67 (1-OCH3) and a signal assignable to an acetyl group at δH 2.00, along with three proton signals on a tropolone ring at δH 7.30 (s, H-8), 7.50 (d, H-11), and 7.31 (d, H-12). These spectral data are similar to those of colchicine (1), the exceptions being the existence of signals of benzylic methylene protons of the aromatic alcohol at δH 4.72 (dd) and 4.60 (overlapped) instead of the signal for the 10-methoxy group, and chemical shift differences of H-8 and H-11 on the tropolone ring. In the 13C NMR spectrum, a hydroxymethyl carbon signal at δC 65.8, as well as the higher-field-shifted C-10 (δC 150.5) and the lower-field-shifted C-9 (δC 186.9) and C-11 (δC 134.4) carbon signals compared with those of 1 (C-9, δC 179.5; C-10, δC 164.0; C-11, δC 112.9), was observed. HMBC cross-peaks between methylene protons at δH 4.72 and 4.60 and both carbons at δC 186.9 (C-9) and 134.4 (C-11) and between H-11 at δH 7.50 and carbon at δC 65.8 indicated that a hydroxymethyl group was attached to C-10. This was supported by the differential NOE correlations from H-11 to the protons of H-12 and the hydroxymethyl group.

Acetylation of 2 with Ac2O and pyridine gave acetate 3 quantitatively. Its NMR spectra showed signals due to an acetoxy group at δH 2.19, δC 170.4, and δC 21.0 and the lower-field-shifted methylene signals at δH 5.20 and 5.12 (each d), confirming the existence of a hydroxymethyl group in 2. Therefore, the structure of metacolchicine was deduced to be that shown as formula 2. The absolute configuration at C-7 was deduced to be the same as that of colchicine (1) by comparison of their CD spectra.
This is the first example of natural colchicinoid having an additional carbon unit at C-10 position.

EXPERIMENTAL
General Experimental Procedures
1H and 13C NMR spectra were recorded on a JEOL JNM A-500 at 500 MHz (1H) and 125 MHz (13C) and a JEOL JNM ECP-400 at 400 MHz (1H), respectively. UV spectra were recorded on a JASCO V-560. EI-MS data were obtained on a JEOL GC mate. HR-FAB-MS data were acquired with a JEOL JMS-HX110 at the Chemical Analysis Center, Chiba University. Optical rotations were measured with a JASCO P-1020 polarimeter. CD spectra were measured with a JASCO J-720WI. Column chromatography was carried out over Silica gel 60 [Merck, 70-230 mesh (for open column chromatography)], Silica gel 60N [Kanto Chemical, 40-50 mm (for flash column chromatography)], and Chromatorex NH [Fuji Silysia Chemical, 100-200 mesh (for amino-silica gel open column chromatography)]. Medium pressure liquid column chromatography (MPLC) was carried out with C.I.G. prepacked column CPS-HS-221-05 (Kusano Kagakukikai, SiO2).

Plant Material
The tubers of Sandersonia aurantiaca were provided by Mr. Eichi Seki from the Crop Breeding Institute, Chiba Prefectural Agriculture and Forestry Research Center.

Extraction and Isolation
The tubers (4150 g, wet weight) of S. aurantiaca were extracted with MeOH (7.2 L, once at room temperature and twice under reflux) to give the extract (181.1 g). The MeOH extract was partitioned with 10% MeOH/CHCl3 and H2O to afford the 10% MeOH/CHCl3 extract (21.8 g). The extract was chromatographed on a silica gel column with MeOH/CHCl3 gradient to give four fractions: fr. A, 0-5% MeOH/CHCl3 (920 mg); fr. B, 5% MeOH/CHCl3 (946 mg); fr. C, 5-30% MeOH/CHCl3 (16.65 g); and fr. D, 30% MeOH/CHCl3 and 10% H2O/MeOH (2.98 g). Fr. C was separated by amino-silica gel column chromatography (0-30% MeOH/CHCl3). The fraction that was eluted with 8% MeOH/CHCl3 was purified by a combination of chromatographies [silica gel flash column (10-30% MeOH/AcOEt), MPLC (5% MeOH/AcOEt, and then 3-5% MeOH/CHCl3)] to afford metacolchicine (2, 5.8 mg). Colchicine (1, 44.4 mg) and colchifoline (28.1 mg) were obtained from fraction C. 2-Demethylcolchicine (27.5 mg), 3-demethylcolchicine (126.8 mg), and demecolcine (7.0 mg) were isolated from fractions C and D. 2-Demethyldemecolcine (4.3 mg) was obtained from fraction D.

Metacolchicine (2)
Amorphous, [α]D21 –160 (c 0.39, CHCl3). UV (EtOH) λmax nm: (log ε): 339 (3.95), 236 (4.29), 204 (4.35). 1H and 13C NMR data, see Table 1. EI-MS m/z (%): 399 (M+, 41), 371 (33), 340 (22), 312 (100). HR-FAB-MS (NBA/PEG) m/z: 400.1752 (MH+, calcd for C22H26NO6 400.1760). CD (c = 0.25 mmol/L, MeOH, 19 °C) Δε (λ nm): –0.9 (397), –6.6 (345), –3.9 (301), –6.6 (251), 0 (239), +19.3 (218), +7.0 (204).

Acetylation of Metacolchicine (2)
To a solution of metacolchicine (2, 1.2 mg, 0.003 mmol) in dry pyridine (0.1 mL) was added acetic anhydride (0.05 mL) and the mixture was stirred at room temperature for 3 h under Ar atmosphere. After removal of the volatiles, the residue was purified by SiO2 open column chromatography (20% MeOH/CHCl3) to give acetate 3 (1.8 mg, y. quant.).
Acetate 3: Amorphous, [α]D21 –95 (c 0.12, CHCl3). UV (EtOH) λmax nm: (log ε): 336 (3.77), 237 (sh, 4.20), 206 (4.40). 1H NMR (400 MHz, CDCl3) δ: 7.40 (1H, d, J = 10.3 Hz, H-11), 7.23 (2H, overlapped, H-8 and H-12), 6.52 (1H, s, H-4), 6.25 (1H, br-s, -NH), 5.20 and 5.12 (each 1H, d, J = 15.9 Hz, -CH2OAc), 4.60 (1H, ddd, J = 13.1, 6.6, 6.6 Hz, H-7), 3.94, 3.90 (each 3H, s, 2-OCH3, 3-OCH3), 3.67 (3H, s, 1-OCH3), 2.56 (1H, dd, J = 13.2, 6.6 Hz, H-5β), 2.44 (1H, ddd, J = 12.7, 12.7, 6.9 Hz, H-5α), 2.20 (1H, m, H-6β), 2.19 (3H, s, -CH2OCOCH3), 2.00 (3H, s, -NHCOCH3), 1.77 (1H, ddd, J = 11.6, 11.6, 6.1 Hz, H-6α). 13C NMR (125 MHz, CDCl3) δ: 185.0 (C-9), 170.4 (-CH2OCOCH3), 169.4 (-NHCOCH3), 153.8, 151.4, 150.6, 146.5, 142.8, 141.8 (C-1, C-2, C-3, C-7a, C-10, C-12a), 135.5, 134.4, 134.0, 133.1 (C-4a, C-8, C-11, C-12), 125.4 (C-12b), 107.3 (C-4), 63.8 (-CH2OCOCH3), 61.7, 61.4, 56.1 (1-OCH3, 2-OCH3, 3-OCH3), 52.0 (C-7), 36.5 (C-6), 29.7 (C-5), 23.1 (-NHCOCH3), 21.0 (-CH2OCOCH3). EI-MS m/z (%): 441 (M+, 48), 413 (33), 354 (39), 311 (83), 294 (100). HR-FAB-MS (NBA/PEG) m/z: 442.1842 (MH+, calcd for C24H28NO7 442.1866). CD (c = 0.25 mmol/L, MeOH, 20 °C) Δε (λ nm): –0.6 (399), –3.6 (340), –2.1 (296), –3.3 (253), 0 (240), +11.4 (217), +2.8 (206).

ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.

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