HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 6, 2011, pp. 1405-1408
Received, 9th March, 2011, Accepted, 5th April, 2011, Published online, 7th April, 2011.
DOI: 10.3987/COM-11-12204

■ Purinium Derivatives with Antitumor Activities from Heterostemma alatum Wight

Wen-Wei Fu, Chang-Heng Tan,* Peng-Yu Zhuang, Shu-Min Yang, Hong-Feng Luo, Jun-Jie Tan, Ping Liu, and Da-Yuan Zhu

Department of Natural Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China

Abstract

Heteromine I (1), a new purinium derivative, together with six known compounds, were isolated from the aerial parts of Heterostemma alatum Wight. The structure of 1 has been established as 2-methylamino-6-methoxy-7,9-dimethyl-8-purinone by means of spectroscopic analysis. Two puriniums, heteromines J (2) and K (3) were isolated as natural products for the first time. Heteromine D (4) showed strong cytotoxicity against HL-60 cell line with IC50 of 4.04 nmol/mL.

Heterostemma alatum Wight (Asclepiadaceae) is a liana that is distributed over the south and the southwest of China, Nepal, and India. Its roots or whole plant were used as a Chinese folk medicine for expelling dampness and detoxifying.1 A few phytochemical studies on plants of the genus Heterostemma reported the isolation of steroids, fatty acids, flavonoids, flavonoid glycosides, adenines, uridines, puriniums and pyrimidines from H. brownii 2-4 and H. alatum.5 Further exploration of the title plant led to the isolation of a new purinium derivative, heteromine I (1), two first obtained natural puriniums, heteromines J (2) and K (3), and four know compounds. The structure of 1 is elucidated as 2-methylamino-6-methoxy-7,9-dimethyl-8-purinone on the basis of spectroscopic analysis. Those known compounds are identified to be 2-dimethylamino-6-methoxy-7,9-dimethyl-8-purinone (2),4 6-methoxy-9- methyl-2-dimethylaminopurinium (3),4,6,7 11-oxo-α-amyrin acetate,8 α-amyrin acetate,9 sitgmast-4-ene- 1,3-dione,10 and stigmast-4-en-3-one.11 An assay on cytotoxicity showed that heteromine D (4) had strong cytotoxicity against HL-60 cell line with IC50 of 4.04 nmol/mL in contrast with 6-mercaptopurine of 3.96 nmol/mL.

Heteromine I (1) was isolated as white amorphous powders. The molecular formula was determined to be C9H13N5O2 based on the pseudomolecular ion peak [M+Na]+ at m/z 246.0959 in the HR-ESI-MS. The IR spectrum exhibited absorptions at 3385 (N-H), 1703 (C=O), 1635 (C=C), 1608 (C=N), and 1294 (-NH-CH3) cm-1. The UV appeared the maximum absorptions at 205, 250 and 298 nm. The above evidences indicated a purinium derivative.3,4 The 1H-NMR spectrum of 1 showed signals for a methylamino group (δ 2.78, 3H, br s), two methyl groups attached on two quaternary amines (δ 3.18 and 3.31, each s), and a typical methoxy group (δ 3.92, s). The four methyls were easily assigned by the HMBC and ROESY cross-peaks (Figure 2), the structure of heteromine I (1) was assigned as 2-methyl-amino-6-methoxy-7,9-dimethyl-8- purinone.

Compounds 2 and 3, named heteromines J and K, were obtained from plant resource for the first time. As an analogue of 1, 2 was easily assigned its 1H and 13C signals in contrast with those of 1. The original assignments for C-2 (δ 153.6) and C-8 (δ 157.9) in the literature4 were reversed and revised. 2 and 3 had been reported as derivatives from heteromine A under treatment of NaBH4, following air oxidation.4 3 was also a key intermediate in the course of synthesis of heteromine A.6,7
All puriniums isolated from the title plant, including 1-3 and heteromines A, B, C, D (4), and F, were evaluated their cytotoxicities against tumor cell line HL-60 (human promyelocytic leukemia cell). The results showed that 4 had a comparable cytotoxic activity (IC50 = 4.04 nmol/mL) with that of 6-MP (6-mercaptopurine, IC50 = 3.96 nmol/mL).

EXPERIMENTAL
General Experimental Procedures: Optical rotations were measured using a Perkin-Elmer 341 polarimeter. IR spectra were recorded on a Nicolet Magna 750 FTIR (KBr) spectrometer. ESI-MS and HR-ESI-MS were obtained on an Esquire 3000plus and a Q-TOF-Ultima mass spectrometer, respectively. NMR spectra were recorded on a Bruker AM-400 NMR spectrometers, the chemical shift values are reported in ppm (δ) and coupling constants (J) are given in Hz. Silica gel (200-300, 400 mesh) and precoated plates of silica gel (HSGF-254) (Qingdao Haiyang Chemical Group Co., Qingdao) were used for column chromatography (CC) and TLC, respectively.
Plant Material: The aerial parts of H. alatum were collected in Xishuangbanna County, Yunnan Province, China, in July 2006. The plant were identified by Prof. J.-Y. Cui of Xishuangbanna Tropical Botanical Garden, CAS, China. A voucher specimen (No. 2006-64) was deposited in the Herbarium of Shanghai Institute of Materia Medica.
Extraction and Isolation: The dried aerial parts of H. alatum (5.0 kg) was extracted with 95% EtOH (25 L×3, each 3 d) at room temperature. The solvents were evaporated under reduced pressure to give 524 g residue. The concentrated extract was suspended in H2O (3 L) and partitioned successively with petroleum ether (PE), CHCl3, EtOAc to provide the PE- (80 g), CHCl3- (50 g), and EtOAc-soluble fraction (30 g). The EtOAc-soluble fraction (30 g) was subjected to CC of silica gel eluted with EtOAc-acetone (1:0 0:1) to afford Fr.E1−Fr.E8. Fr.E6 (2.1 g) was repeatedly chromatographed on silica gel columns with EtOAc-acetone (10:1‒5:1) to give 1 (11 mg), 2 (27 mg) and 3 (35 mg). By the similar procedures, 11-oxo-α-amyrin acetate (255 mg), α-amyrin acetate (50 mg), sitgmast-4-ene-1,3- dione (40 mg), and stigmast-4-en-3-one (9 mg) were obtained from the CHCl3-soluble fraction.
Assay of Cytotoxic Activity: HL-60 cells (ATCC, Rockville, MD) were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum at 37 °C in humidified air with 5% CO2. Cytotoxicity in vitro was measured by the MTT method. Briefly, cells (1 × 104 per well) were seeded into 96-well plates and exposed to various concentrations of test compounds. After treatment for 72 h, 10 μL MTT at 5 mg/ml was added to each well. The cells were then incubated at 37 °C for another 4 h and then 10% sodium dodecyl sulfate (SDS) in 0.01 M HCl was added to each well. The absorbance was detected at 570 nm with a microplate reader (KLx808, Bio-Tek, USA). Cytotoxicity was expressed as a percentage of that of the control culture, and IC50 was given as the concentration (nmol/mL) required for 50% inhibition of cell growth.
Heteromine I (1): White amorphous powders. UV (MeOH) λmax: 205, 250, 298 nm; IR (KBr) νmax: 3385, 2945, 1703, 1635, 1608, 1562, 1504, 1398, 1294, 1196, 1132, 771, 582 cm-1; 1H-NMR (DMSO-d6, 400 MHz) δ: 3.92 (6-Me, 3H, s), 3.31 (7-Me, 3H, s), 3.18 (9-Me, 3H, s), 2.78 (2-Me, 3H, br s); 13C-NMR (DMSO-d6, 100 MHz) δ: 158.1 (C-2), 152.9 (C-6), 152.7 (C-8), 150.8 (C-4), 98.6 (C-5), 53.1 (6-Me), 29.0 (7-Me), 28.1 (2-NMe), 26.0 (9-Me); ESI-MS m/z 234 ([M+H]+), 246 ([M+Na]+), 469 ([2M+Na]+); HR-ESI-MS m/z: 246.0959 (calcd. for C9H13N5O2Na: 246.0967).
Heteromine J (2): White amorphous powders. UV (MeOH) λmax: 205, 250, 298 nm; IR (KBr) νmax: 2929, 1618, 1574, 1462, 1390, 1329, 1298, 1261, 1068, 787, 638 cm-1; 1H-NMR (DMSO-d6, 400 MHz) δ: 3.94 (6-Me, 3H, s), 3.43 (7-Me, 3H, s), 3.31 (9-Me, 3H, s), 3.11 (2-Me2, 6H, s); 13C-NMR (DMSO-d6, 100 MHz) δ: 157.8 (C-2), 153.5 (C-8), 153.3 (C-6), 150.9 (C-4), 98.7 (C-5), 52.9 (6-Me), 29.2 (7-Me), 37.1 (2-NMe2), 26.2 (9-Me); ESI-MS m/z: 238 ([M+H]+), 260 ([M+Na]+), 497 ([2M+Na]+).

ACKNOWLEDGEMENTS
This study was financial supported by grants from the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (Nos. 2009ZX09301-001 and 2009ZX09311-003), the National Science Foundation of China (No. 81072545), and China Postdoctoral Science Foundation (No. 20090460644).

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