A New Long-Chain Alkene and Antituberculosis ... - Wiley Online Library

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)

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A New Long-Chain Alkene and Antituberculosis Constituents from the Leaves of Pourthiaea lucida by Jih-Jung Chen* a ), Wen-Jiou Lin a ), Po-Chuen Shieh a ), Ih-Sheng Chen b ), Chien-Fang Peng c ), and Ping-Jyun Sung d ) a ) Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung 907, Taiwan (phone: þ 886-8-7624002 (ext. 2827); fax: þ 886-8-7624002 (ext. 5064); e-mail: [email protected]) b ) Faculty of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan c ) Faculty of Biomedical Laboratory Sciences, College of Health Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan d ) National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan

A new long-chain alkene, dotriacont-1-ene (1), was isolated from the leaves of Pourthiaea lucida, together with twelve known compounds. The structure of this new compound was determined by NMR and mass-spectrometric analyses. Among the isolated compounds, a-tocospiro A (2), a-tocopheryl quinone (4), and (E)-phytol (5) exhibited antituberculosis activities (MICs 30 mg/ml) against Mycobacterium tuberculosis H37Rv in vitro.

Introduction. – Pourthiaea lucida Decaisne (Rosaceae) is a small endemic deciduous tree which grows in thickets at low altitude throughout Taiwan [1]. Biphenyls, dibenzofurans, triterpenoids, steroids, flavonoids, and fatty acid derivatives have been identified as constituents of plants of the genus Pourthiaea [2 – 4]. Some of these compounds exhibit cytotoxic [2] and anti-inflammatory [4] activities. In our studies on the antituberculosis constituents of Formosan plants, many species have been screened for their in vitro antituberculosis activities, and P. lucida has been found to be an active species. The current phytochemical investigation of the leaves of this plant has led to the isolation of a new long-chain alkene, dotriacont-1-ene (1), along with twelve known compounds, including two a-tocopheroids, a-tocospiro A (2), and a-tocospiro B (3), a benzoquinone, a-tocopheryl quinone (4), a diterpenoid, (E)-phytol (5), a triterpenoid, friedelan-3-one (6), a fatty acid ester, methyl linoleate (7), three steroids, b-sitostenone (8), b-sitosterol (9), and 3b-hydroxystigmast-5-en-7-one (10), and a mixture of hexacosan-1-ol (11), octacosan-1-ol (12), and triacontan-1-ol (13). The structure elucidation of 1 and the antituberculosis properties of the isolated compounds are described herein. Results and Discussion. – 1. Structure Elucidation. Extensive chromatographic purification of the AcOEt-soluble fraction of the MeOH extract of the leaves of P. lucida afforded compounds 1 – 13. The new compound 1 was isolated as colorless needles. EI-MS Analysis of 1 showed the [M] þ ion peak at m/z 448, in agreement with the molecular formula C32H64 (one degree of unsaturation), as confirmed by HR-EI 2010 Verlag Helvetica Chimica Acta AG, Zrich

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MS and 13C-NMR. The presence of a long-chain terminal alkene group in the molecule was revealed by a band at 1651 cm 1 in the IR spectrum, which was confirmed by signals in the 1H-NMR spectrum.

The 1H-NMR spectrum of 1 showed the resonances for a terminal Me group (d 0.88 (t, J ¼ 6.4, MeCH2 ), a terminal alkene function (d 5.81 (m, CH¼CH2 ), 4.94 (d, J ¼ 12.4, 1 H, CH¼CH2 ), and 5.01 (d, J ¼ 17.2, 1 H, CH¼CH2 )), the CH2 group linked to the alkene function (2.04 (m, CH2CH¼CH2 )), and those for the remaining CH2 groups of the aliphatic chain (d 1.20 – 1.35 (br. s, 56 H, CH2(4)CH2(31)). The locations of the terminal alkene function and terminal Me group were determined by the HMBC experiment (Fig.), in which cross-peaks were observed between HC(1) (d(H) 4.94, 5.01) and both C(2) (d(C) 139.5) and C(3) (d(C) 34.1);


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Figure. NOESY (a) and HMBC (b) correlations of 1

and between HC(2) (d(H) 5.81) and both C(1) (d(C) 114.3) and C(4) (d(C) 29.2); between Me(32) (d(H) 0.88) and both C(30) (d(C) 32.2) and C(31) (d(C) 22.9). On the basis of the above data, the structure of compound 1 was identified as dotriacont-1-ene. This was further confirmed by 1H,1H-COSY and NOESY (Fig.) experiments. The assignment of the 13C-NMR resonances was confirmed by DEPT, HSQC, and HMBC (Fig.) techniques. The known isolates, including two a-tocopheroids, a-tocospiro A (2) [5] and atocospiro B (3) [6], a benzoquinone, a-tocopheryl quinone (4) [7], a diterpenoid, (E)phytol (5) [8], a triterpenoid, friedelan-3-one (6) [9], a fatty acid ester, methyl linoleate (7) [10], three steroids, b-sitostenone (8) [11], b-sitosterol (9) [12], and 3bhydroxystigmast-5-en-7-one (10) [13], and a mixture of hexacosan-1-ol (11), octacosan-1-ol (12), and triacontan-1-ol (13) [14], were readily identified by comparison of their physicochemical, NMR, and mass-spectrometric data with those of authentic samples or literature values. 2. Biological Studies. The biological activities of the isolated compounds from the leaves of P. lucida were tested in vitro against Mycobacterium tuberculosis H37Rv. The antituberculosis-activity data are collected in the Table. The clinically used antituberculosis agent, ethambutol, was used as positive control. From the results of our antituberculosis tests, the following conclusions can be drawn: a) a-Tocospiro A (2), a-

Table. Antituberculosis Activities of 1 – 13 against Mycobacterium tuberculosis H37Rv Compound

Name

MIC [mg/ml]

1 2 3 4 5 6 7 8 9 10 11 – 13 Ethambutol a )

Dotriacont-1-ene a-Tocospiro A a-Tocospiro B a-Tocopheryl quinone ( E )-Phytol Friedelan-3-one Methyl linoleate b-Sitostenone b-Sitosterol 3b-Hydroxystigmast-5-en-7-one Mixture of hexacosan-1-ol, octacosan-1-ol, and triacontan-1-ol

> 100 30 50 25 12.5 > 100 60 > 100 > 100 55 > 100 6.25

a

) Positive control.

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tocopheryl quinone (4), and (E)-phytol (5) exhibited antituberculosis activities (MICs 30 mg/ml) against M. tuberculosis H37Rv in vitro. b) a-Tocospiro A (2) with an (S)configuration at C(9) exhibited stronger antituberculosis activity than its diastereoisomer, a-tocospiro B (3) with (R)-configuration at C(9), against M. tuberculosis H37Rv. c) Among the steroid analogues 8 – 10, 3b-hydroxystigmast-5-en-7-one (10) with a C(7)¼O function exhibited stronger antituberculosis activity than its analogues 8 and 9 against M. tuberculosis H37Rv. d) (E)-Phytol (5) is the most effective among the isolates, with an MIC value of 12.5 mg/ml against M. tuberculosis H37Rv. e) Compounds 1, 6, 8, 9, and 11 – 13 showed basically no antituberculosis activities, and the isolates 3, 7, and 10 were only weakly active. This work was supported by a grant from the National Science Council of the Republic of China (NSC 95-2320-B-127-001-MY3). Experimental Part General. TLC: Silica-gel 60 F254 precoated plates (Merck). Column chromatography (CC): silica gel 60 (70 – 230 or 230 – 400 mesh, Merck). M.p.: Yanaco micro-melting-point apparatus; uncorrected. UV Spectra: Jasco UV-240 spectrophotometer; lmax (log e) in nm. IR Spectra: Perkin-Elmer-2000 FT-IR spectrophotometer; in cm 1. 1H-, 13C-, and 2D-NMR spectra: Varian Unity-Plus-400 and Varian Inova500 spectrometers; d in ppm rel. to Me4Si; J in Hz. EI-MS: VG-Biotech Quatro-5022 mass spectrometer; in m/z (rel. %). HR-EI-MS: Jeol JMX-HX 110 mass spectrometer; in m/z. Plant Material. The leaves of Pourthiaea lucida Decaisne were collected from Mutan, Pingtung County, Taiwan, in April 2004, and identified by Dr. I.-S. C. A voucher specimen (Chen 6116) was deposited with the Herbarium of the Faculty of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan. Extraction and Isolation. The dried leaves (10 kg) were extracted with cold MeOH (40 l) for one week. The extract was concentrated under reduced pressure, and the residue (1200 g) was extracted with H2O/AcOEt 1 : 1. The AcOEt-soluble fraction (Fr. A; 590 g) was separated. The H2O-soluble fraction was further extracted with BuOH, and the BuOH-soluble part (Fr. B; 265 g) and the H2O-soluble one (Fr. C; 310 g) were separated. A part (110 g) of Fr. A was purified by CC (3.3 kg of SiO2 , 70 – 230 mesh; hexane/AcOEt gradient) to afford ten fractions: Fr. A1 (eluted with 3 l of hexane), Fr. A2 (3.5 l; hexane/ AcOEt 50 : 1), Fr. A3 (5 l; hexane/AcOEt 30 : 1), Fr. A4 (5 l; hexane/AcOEt 20 : 1), Fr. A5 (6 l; hexane/ AcOEt 10 : 1), Fr. A6 (5 l; hexane/AcOEt 5 : 1), Fr. A7 (5.5 l; hexane/AcOEt 3 : 1), Fr. A8 (5 l; hexane/ AcOEt 1 : 1), Fr. A9 (4.5 l, AcOEt), Fr. A10 (4 l, MeOH). Fr. A1 (1.9 g) was further purified by CC (150 g of SiO2 , 230 – 400 mesh; hexane/CHCl3 20 : 1; 1.5-l fractions) to afford six subfractions: Frs. A1.1 – A1.6. Fr. A1.2 (119 mg) was washed with MeOH to yield 1 (15.6 mg) after recrystallization (acetone/MeOH). Fr. A4 (6.3 g) was subjected to CC (210 g of SiO2 , 230 – 400 mesh; hexane/acetone 30 : 1; 2-l fractions) to afford seven subfractions: Frs. A4.1 – A4.7. Fr. A4.1 (281 mg) was purified further by prep. TLC (SiO2 ; hexane/AcOEt 80 : 1) to yield 6 (12.4 mg; Rf 0.55). Fr. A5 (7.4 g) was subjected to CC (210 g of SiO2 , 230 – 400 mesh; hexane/AcOEt 5 : 1; 2-l fractions) to afford ten subfractions: Frs. A5.1 – A5.10. Fr. A5.3 (213 mg) was purified further by prep. TLC (SiO2 ; CHCl3/AcOEt 40 : 1) to yield 2 (10.2 mg; Rf 0.73) and 3 (9.8 mg; Rf 0.70 ). Fr. A5.4 (251 mg) was purified further by prep. TLC (SiO2 ; CHCl3/acetone 40 : 1) to provide 7 (8.6 mg; Rf 0.54). Fr. A5.6 (263 mg) was purified further by prep. TLC (SiO2 ; CHCl3/acetone 30 : 1) to afford a mixture of 11, 12, and 13 (12.5 mg; Rf 0.66). Fr. A6 (9.4 g) was further purified by CC (215 g of SiO2 , 230 – 400 mesh; CHCl3/acetone 20 : 1; 2-l fractions) to afford eleven subfractions: Frs. A6.1 – A6.11. Fr. A6.1 (186 mg) was purified further by prep. TLC (SiO2 ; hexane/acetone 3 : 1) to give 8 (16.1 mg; Rf 0.67). Fr. A6.2 (159 mg) was purified further by prep. TLC (SiO2 ; hexane/acetone 2 : 1) to provide 5 (13.2 mg; Rf 0.71). Fr. A6.4 (293 mg) was washed with MeOH and filtered to yield 9 (23.7 mg). Fr. A6.5 (278 mg) was purified further by prep. TLC (SiO2 ; hexane/acetone 2 : 1) to give 4 (8.1 mg; Rf 0.50). Fr. A6.6 (249 mg) was purified by prep. TLC (SiO2 ; hexane/acetone 2 : 1) to afford 10 (9.3 mg; Rf 0.51).


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Dotriacont-1-ene (1). Colorless needles. M.p. 59 – 618 (Me2CO/MeOH). IR (KBr): 1651 (C¼C). H-NMR (CDCl3 , 400 MHz): 0.88 (t, J ¼ 6.4, MeCH2 ); 1.25 (br. s, Me(CH2 )28 ); 2.04 (m, CH2CH¼CH2 ); 4.94 (d, J ¼ 12.4, 1 H, CH¼CH2 ); 5.01 (d, J ¼ 17.2, 1 H, CH¼CH2 ); 5.81 (m, CH¼CH2 ). 13C-NMR (CDCl3 , 100 MHz): 14.3 (C(32)); 22.9 (C(31)); 29.2 (C(4)); 29.4 (C(5)); 29.6 (C(29)); 29.8 (C(6)); 29.9 (C(7)C(28)); 32.2 (C(30)); 34.1 (C(3)); 114.3 (C(1)); 139.5 (C(2)). EI-MS: 448 (5, M þ ), 419 (6), 405 (8), 391 (12), 377 (17), 363 (28), 349 (30), 335 (34), 321 (39), 307 (46), 293 (53), 279 (65), 265 (77), 251 (86), 237 (93), 223 (100). HR-EI-MS: 448.5010 (C32H þ64 ; calc. 448.5008). Biological Assay. Antituberculosis activities were evaluated, and minimal inhibitory concentration (MIC) values were determined using the Mycobacterium tuberculosis H37Rv strain. Middlebrook-7 H10 agar was used to determine the MIC values, as recommended by the proportion method [15]. Briefly, each tested compound was added to agar supplemented with OADC (oleic acid-albumin-dextrosecatalase) at 50 – 568 by serial dilution to yield a final concentrations from 100 to 0.8 mg/ml. Then, 10 ml of each concentration of tested compound containing medium were dispensed into plastic quadrant Petri dishes. The inoculum of the tested isolate of M. tuberculosis was prepared by diluting the initial inoculum in Middlebrook-7 H9 broth until the turbidity was reduced to that of 1 equiv. of McFarland No. 1 standard. Final suspensions were prepared by adding Middlebrook-7 H9 broth and by preparing 100-fold dilutions of the standardized suspensions. After solidification of the Middlebrook-7 H10 agar, portions (33 ml) of the dilutions were placed on each quadrant of the agar plates, and the plates were incubated at 358 for 14 d under an atmosphere containing 10% CO2 .

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