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Two new alkaloids from Portulaca oleracea and their cytotoxic activities ab

ab

c

ab

ab

Jin-Long Tian , Xiao Liang , Pin-Yi Gao , Dan-Qi Li , Qian Sun , ab

Ling-Zhi Li

& Shao-Jiang Song

ab

a

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, China b

Key Laboratory of Structure-Based Drug Design and Discovery (Ministry of Education), Shenyang Pharmaceutical University, Shenyang, 110016, China c

College of Chemical Engineering, Shenyang University of Chemical Technology, Shenyang, 110142, China Published online: 10 Dec 2013.

To cite this article: Jin-Long Tian, Xiao Liang, Pin-Yi Gao, Dan-Qi Li, Qian Sun, Ling-Zhi Li & ShaoJiang Song (2014) Two new alkaloids from Portulaca oleracea and their cytotoxic activities, Journal of Asian Natural Products Research, 16:3, 259-264, DOI: 10.1080/10286020.2013.866948 To link to this article: http://dx.doi.org/10.1080/10286020.2013.866948

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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 3, 259–264, http://dx.doi.org/10.1080/10286020.2013.866948

Two new alkaloids from Portulaca oleracea and their cytotoxic activities Jin-Long Tianab, Xiao Liangab, Pin-Yi Gaoc, Dan-Qi Liab, Qian Sunab, Ling-Zhi Liab* and Shao-Jiang Songab*

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a

School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China; bKey Laboratory of Structure-Based Drug Design and Discovery (Ministry of Education), Shenyang Pharmaceutical University, Shenyang 110016, China; cCollege of Chemical Engineering, Shenyang University of Chemical Technology, Shenyang 110142, China (Received 14 September 2013; final version received 14 November 2013) Two new alkaloids named (3R)-3,5-bis(3-methoxy-4-hydroxyphenyl)-2,3-dihydro-2(1H)pyridinone (1) and 1,5-dimethyl-6-phenyl-1,2-dihydro-1,2,4-triazin-3(2H)-one (2), together with two known compounds (70 R)-N-feruloyl normetanephrine (3) and N-trans-feruloyl tyramine (4) were isolated from the air-dried aerial parts of Portulaca oleracea L. Their structures and configurations were elucidated by spectroscopic methods including 1D NMR, 2D NMR, and HR-MS techniques. In addition, compounds 1–4 were tested for in vitro cytotoxic activities against human lung (K562 and A549) and breast (MCF-7 and MDA-MB-435) cancer cell lines. Keywords: alkaloid; Portulaca oleracea; chemical constituent; cytotoxicity

1. Introduction Portulaca oleracea L. (machixian in Chinese and purslane in English), belonging to Portulacaceae, is widely used as a potherb in the Mediterranean, Central European, and Asian countries. It is listed in the World Health Organization as one of the most used medicinal plants, and it has been given the term ‘Global Panacea’ [1]. As a traditional Chinese folk medicine, it has been used as a diuretic, antiseptic, antiscorbutic, febrifuge, antispasmodic, and vermifuge agent with a long history [2]. Previously, pharmacological researches have indicated that P. oleracea produces a wide range of pharmacological effects such as anti-inflammatory, [3] antibacterial, [4] skeletal muscle relaxant, [5] wound-healing, [6] and in vitro antitumor [7] activities. Many chemical constituents including terpenoids, [8,9]

alkaloids, [10] cerebrosides, [11] coumarins, and flavonoids [12] have recently been isolated from this plant. Meanwhile, alkaloids have been reported to be biologically active chemical constituents of this plant [13]. So for all these reasons, it seemed intriguing to investigate the important secondary metabolites, alkaloids, from the P. oleracea L. in more detail. In this paper, we report the structural characterization of two new alkaloids, (3R)-3,5-bis(3-methoxy4-hydroxyphenyl)-2,3-dihydro-2(1H)-pyridinone (1), 1,5-dimethyl-6-phenyl-1,2dihydro-1,2,4-triazin-3(2H)-one, along with two known alkaloids (70 R)-N-feruloyl normetanephrine (3) and N-trans-feruloyl tyramine (4) (Figure 1). The cytotoxicities of compounds 1–4 were evaluated against human lung (K562 and A549) and breast (MCF-7 and MDA-MB-435) cancer cell lines by MTT methods.

*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis

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Figure 1. Structures of compounds 1 –4.

2. Results and discussion Compound 1 was obtained as a yellow amorphous powder (7 mg). Its molecular formula was determined to be C19H19O5N by HR-ESI-MS at m/z 342.1318 [M þ H]þ, indicating 11 degrees of unsaturation. The 1 H NMR spectrum of compound 1 (Table 1) displayed two 1,3,4-trisubstituted aromatic rings with protons at d 6.84 (d, 1H, J ¼ 1.7 Hz), d 6.86 (dd, 1H, J ¼ 8.2 and 1.7 Hz), d 6.68 (d, 1H, J ¼ 8.2 Hz) and at d 6.81 (d, 1H, J ¼ 1.9 Hz), d 6.55 (dd, 1H, J ¼ 8.1 and 1.9 Hz), d 6.66 (d, 1H, J ¼ 8.1 Hz), respectively. One olefinic proton at d 7.22 (d, 1H, J ¼ 1.9 Hz), two methyl groups at d 3.48 (s, 3H) and 3.65 (s, 3H), one methine group at d 4.51 (dd, 2H, J ¼ 7.9 and 1.8 Hz) and one methylene group at d 3.02 (d, 1H, J ¼ 9.8 Hz), 3.77 (t, 1H, J ¼ 8.2 Hz), and one amino proton at d 7.95 (s, 1H) were present. The 13C NMR spectrum of compound 1 (Table 1) showed 19 signals, which were classified as one carbonyl carbon (d 171.6), two sets of aromatic carbons (d 131.5, 113.5, 147.9, 147.7, 115.7, and 124.9) and (d 134.7, 111.7, 148.2, 145.7, 116.1, and 119.2), one methylene carbon (d 42.3) and one methine

carbon (d 49.5). HMBC (Figure 2) correlations from H-1 to C-2, C-3, C-4, and C-6; from H-2 (d 3.02) to C-3, C-6, and C-100 ; from H-2 (d 3.77) to C-3 and C-100 ; from H3 to C-2, C-4, C-6, C-100 , C-200 and C-600 ; and from H-4 to C-3, C-5, C-6, C-10 , C-20 , and C-60 established the structure of a 5,6dihydropyridin-2(1H)-one with 3,5-substitution. Consequently, key HMBC correlations and HMQC spectrum revealed that two hydroxyl groups at d 9.37 (s, 1H) and d 8.89 (s, 1H) were attached to the C-40 and C-400 , respectively. Similarly, two methyl groups at d 3.48 (s, 3H) and d 3.65 (s, 3H) were attached to the C-30 and C-300 . Based on the above analysis, the positions of the substituents were identified. Meanwhile, the HMBC correlations of H-2 with C-100 and H-3 with C-100 /200 /600 clearly suggested the linkage between C-100 and C-3. In addition, correlations of H-4 with C-10 /20 /60 , H-20 with C-4/5, and H-60 with C-4 indicated the linkages between C-10 and C-5 or C-4. However, the 1H NMR spectrum shows that the proton at d 7.22 (1H) is a doublet peak (d) and the proton at d 4.51 (1H) is a double–doublet peak (dd). So the linkage between C-10 and C-5 is

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Table 1.

1

H NMR (600 MHz) and 13C NMR (150 MHz) spectral data of compound 1 in DMSO-d6.

Position

1

1 2 3 4 5 6 10 20 30 40 50 60 100 200 300 400 500 600 40 -OH 400 -OH 30 -OCH3 300 -OCH3

7.95, 1H, 3.02, 1H, 4.51, 1H, 7.22, 1H,

H NMR (dH, mult, J in Hz) s d, 9.8; 3.77, 1H, t, 7.9 dd, 7.9, 1.9 d, 1.9

6.84, 1H, d, 1.7 6.68, 1H, d, 8.2 6.86, 1H, dd, 8.2, 1.7 6.81, 1H, d, 1.9 6.66, 1H, 6.55, 1H, 9.37, 1H, 8.89, 1H, 3.48, 3H, 3.65, 3H,

d, 8.1 dd, 8.1, 1.9 s s s s

more reasonable. Its absolute configuration was determined by [a ]20 D value 251.0 (c 0.45, CHCl3), the value was in good agreement compared with the values of similar compounds reported in the literature, [14] so for this reason the absolute configuration at C-3 was R. Thus, the structure of compound 1 was elucidated as (3R)-3,5-bis(3-methoxy-4-hydroxyphenyl)2,3-dihydro-2(1H)-pyridinone. Compound 2 was obtained as a deep yellow amorphous powder (29 mg). The Table 2. Position 3 5 6 1-CH3 5-CH3 10 20 , 60 30 , 50 40

1

261

13

C NMR (d C)

HMBC C-2, C-3, C-4, C-6 C-3, C-6, C-100 , C-3, C-100 C-2, C-4, C-6, C-100 , C-200 , C-600 C-3, C-5, C-6, C-10 , C-20 , C-60

49.5 42.3 131.3 126.5 171.6 131.5 113.5 147.9 147.7 115.7 124.9 134.7 111.7 148.2 145.7 116.1 119.2

C-4, C-5, C-30 , C-40 , C-60 C-5, C-30 , C-40 C-4, C-20 , C-30 , C-40 C-3, C-100 , C-300 , C-400 , C-600 C-100 , C-300 , C-400 C-3, C-200 , C-400 C-30 C-40 , C-50 C-300 , C-400 , C-500 C-40 C-300

55.7 56.0

molecular formula was established as C11H13N3O by HR-ESI-MS at m/z 226.0948 [M þ Na]þ, indicating seven degrees of unsaturation. The analysis of 1 H NMR and 13C NMR data (Table 2) of compound 2 revealed one mono-substituted benzene ring [dH: 7.39 (2H), 7.42 (2H), 7.20 (1H); dC: 134.7, 126.7, 123.9 £ 2, 128.9 £ 2] and two methyl groups [dH 2.20 (s), 2.89 (s)]. The locations of these groups were confirmed by the HMBC (Figure 2) correlations of 5-CH3 with C-5 and C-6; 1-

H NMR (600 MHz) and 13C NMR (150 MHz) spectral data of compound 2 in DMSO-d6. 1

H NMR (dH, mult, J in Hz)

2.89, 3H, s 2.20, 3H, s 7.39, 2H, m, overlapped 7.42, 2H, m, overlapped 7.20, 1H, m, overlapped

13

C NMR (dC) 161.9 117.7 139.4 36.1 8.6 134.7 123.9 128.9 126.7

HMBC

C-6 C-1, C-6 C-40 , C-30 /50 , C-6 C-10 , C-20 /60 , C-40 C-30 /50 , C-20 /60

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Figure 2. Key HMBC ( ! ) correlations of compounds 1 and 2.

CH3 with C-6; H-20 , 60 with C-40 , C-30 /50 , and C-6; H-30 , 50 with C-40 , C-20 /60 , and C10 ; and H-40 with C-30 /50 and C-20 /60 . The IR spectrum showed vibration peaks due to an amide group (1649 cm21). On the basis of evidence above, the structure of 2 was determined to be 1,5-dimethyl-6-phenyl1,2-dihydro-1,2,4-triazin-3(2H)-one. Compound 3 was obtained as a yellow amorphous powder (9 mg). 1H NMR and 13 C NMR data are consistent with the literature values [15]. However, the absolute configuration of this compound had not been determined. In order to determine the absolute configuration at C70 , its optical rotation was determined. The value of ½a20 D – 46.7 (c 0.03, MeOH) indicated the absolute configuration of C70 as R [14]. Based on the above evidence, the structure of compound 3 was deduced as (70 R)-N-feruloyl normetanephrine. Compound 4 was obtained as a faint yellow amorphous powder (10 mg). 1H NMR and 13C NMR data are in good agreement with the published data [16]. 3. Experimental 3.1 General experimental procedures Optical rotations were measured on a JASCO P-1020 polarimeter (Jasco Co., Tokyo, Japan). IR spectra were obtained on a Shimadzu ftir-8400s spectrophotometer (Shimadzu Corporation, Tokyo, Japan). UV spectra were detected on a Shimadzu UV2401 PC spectrophotometer (Shimadzu

Corporation). NMR spectra were recorded on Bruker ARX-600 instruments (Bruker Co., Billerica, MA, USA). HR-ESI-TOFMS experiments were carried out on a MicroTOF spectrometer (Bruker Co., Karlsruhe, Germany). HPLC was carried out on a HITACHI preparative HPLC system (Hitachi Ltd, Tokyo, Japan) equipped with Refractive Index Detector (L-2490) and prep-ODS (10 £ 250 mm). Sephadex LH-20 (20–100 mm, Pharmacia Fine Chemical Co. Ltd, Piscataway, NJ, USA), silica gel (200–300 mesh, Qingdao Marine Chemistry Ltd, Qingdao, China), and Cosmosil ODS (40–80 mm, Nacalai Tosoh Inc., Uetikon, Switzerland) were used for column chromatography (CC). TLC was carried out on silica gel GF254 (Qingdao Marine Chemistry Ltd). 3.2 Plant material The air-dried aerial parts of P. oleracea L. were collected from Hebei Province, China, in June 2011. Samples were authenticated by Prof. Jin-cai Lu from the Department of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. The voucher specimen (No. 20110701) is lodged in the herbarium of Shenyang Pharmaceutical University, Liaoning, China. 3.3

Extraction and isolation

The air-dried aerial parts of P. oleracea (5.0 kg) were extracted with 75% (v/v) EtOH (15 L) three times, each time for 2 h. The ethanol extract was concentrated under reduced pressure to yield a brownish-dark crude extract (500 g), which was subjected to the macroporous resin (ethanol:water 0:100, 30:70, 70:30, 95:5, v/v) to yield four main fractions: Frs A – D. According to chemical monitoring by TLC, Fr. D (50 g) was further chromatographed on silica gel column with a CH2Cl2:MeOH gradient system (100:0, 95:5, 90:10, 80:20, 50:50, 0:100, v/v) to

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Table 3. Cytotoxicities of compounds 1 – 4 against tumor cell lines (IC50 value, mmol L21). Cell lines Compounds

K562

MDA-MB-435

MCF-7

A549

1 2 3 4 Adriamycin

222.77 66.94 90.09 41.52 3.74

. 400 – . 400 120.75 4.19

.400 .400 .400 328.78 3.33

28.80 21.76 24.54 37.20 1.22

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Notes: –, no cytotoxic activities; .400, IC50 .400 mmol L21.

provide eight fractions [Frs D1 (4.3 g); D2 (2.1 g); D3 (3.5 g); D4 (10 g); D5 (2.7 g); D6 (4.4 g); D7 (5.5 g); D8 (3.7 g)], among which Fr. D4 (10 g) was purified by CC (Sephadex LH-20, MeOH) to yield four fractions: Frs D4-1 (1.6 g), D4-2 (2.2 g), D4-3 (1.8 g), and D4-4 (2.9 g), and Fr. D42 (2.2 g) was further chromatographed on reversed-phase silica gel CC (RP-18; MeOH:H2O 60:40, 100:0, v/v) to provide two fractions: Frs D4-2-1 (430 mg) and D4-2-2 (710 mg), then the Fr. D4-2-2 (710 mg) was further chromatographed on CC (SiO2; CH2Cl2:MeOH 100:0, 95:5, 90:10, 80:20, 50:50, 0:100, v/v) to provide five fractions: Frs D4-2-2-1 (87 mg), D4-22-2 (64 mg), D-4-2-2-3 (113 mg), D-4-2-24 (66 mg), and D4-2-2-5 (150 mg), and then D4-2-2-5 (150 mg) was subjected to semipreparative HPLC eluted with aqueous methanol (75%, v/v; 1.5 ml/min) to yield compounds 1 (7 mg; tR 17.7 min), 3 (9 mg; tR 27.7 min), and 4 (10 mg; tR 33.2 min). Fr. D6 (15 g) was subjected to an C18 silica gel column chromatography (MeOH:H2O 60:40, 80:20, 100:0, v/v) to provide three fractions: Frs D6-1 (3.3 g), D6-2 (3.1 g), and D6-3 (4.2 g), and Fr. D63 (4.2 g) was further purified by silica gel (CH2Cl2:MeOH 100:0, 95:5, 90:10, 80:20, 50:50, 0:100, v/v) to yield six fractions: Frs D6-3-1 (470 mg), D6-3-2 (230 mg), D6-3-3 (450 mg), D-6-3-4 (590 mg), D6-35 (770 mg); and D6-3-6 (870 mg), then Fr. D6-3-6 (870 mg) was further chromatographed on Sephadex LH-20 (MeOH:H2O

80:20, v/v) to provide three fractions: Frs D6-3-6-1 (177 mg), D6-3-6-2 (124 mg), and D6-3-6-3 (289 mg), and Fr. D6-3-6-1 (177 mg) was chromatographed by reversed-phase silica gel (MeOH:H2O 40:60, 60:40, 80:20, 100:0, v/v) to yield compound 2 (29 mg; 60:40, v/v). 3.3.1 (3R)-3,5-bis(3-methoxy-4hydroxyphenyl)-2,3-dihydro-2(1H)pyridinone (1) Yellow amorphous powder; ½a25 D – 51.0 (c 0.45, CHCl3). UV (MeOH) lmax: 231 and 323 nm. IR (KBr) vmax (cm21): 3439, 2920, 1643, 1552, 1384, and 1120. For 1H NMR (600 MHz) and 13C NMR (150 MHz) spectral data, see Table 1. HR-ESI-MS: m/z 342.1318 [M þ H] þ (calcd for C19H20O5N, 342.1336). 3.3.2 1,5-Dimethyl-6-phenyl-1,2dihydro-1,2,4-triazin-3(2H)-one (2) Deep yellow amorphous powder; ½a25 D – 28.1 (c 0.10, MeOH). UV (MeOH) lmax: 386 nm. IR (KBr) vmax (cm21): 3452, 2919, 1649, 1380, and 1129. For 1H NMR (600 MHz) and 13C NMR (150 MHz) spectral data, see Table 2. HR-ESI-MS: m/z 226.0948 [M þ Na]þ (calcd for C11H13N3ONa, 226.0951). 3.4 Cytotoxicity assay Cytotoxicities of the target compounds were evaluated by the MTT assay. Cells

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were seeded at a density of 5 £ 104 cells ml21 in 96-well microplate (100 ml per well). After 24 h of incubation for K562, A549, MCF-7, and MDA-MB-435 cells, media containing tested compounds were added in triplicate. After 48 h of incubation, the media were replaced by phosphate-buffered saline medium containing 0.5 mg ml21 MTT and incubated for another 4 h. Then, the medium was removed and 100 ml of DMSO was added in each well to dissolve formazan. The absorbances at 490 nm were measured for the cells using a Thermo microplate reader. The untreated controls were calculated as a cell viability value of 100%. The IC50 values were obtained by nonlinear regression using SPSS 19.0. IC50 measurements for each compound were carried out three times. The results (Table 3) indicated that compounds 1– 4 have moderate cytotoxic activities against A549 with the IC50 of 28.80, 21.76, 24.54, and 37.20 mmol L21, and weak cytotoxic activities against K562 with the IC50 of 222.77, 66.94, 90.09, and 41.52 mmol L21, respectively. However, compounds 1– 4 have almost no cytotoxic activities against MCF-7 and MDA-MB-435 cells. Acknowledgments The authors thank Drs Ying Peng, Wen Li, and Yi Sha of Shenyang Pharmaceutical University for recording HR-ESI-MS and NMR spectra.

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Two new alkaloids from Portulaca oleracea and their cytotoxic activities.

Two new alkaloids named (3R)-3,5-bis(3-methoxy-4-hydroxyphenyl)-2,3-dihydro-2(1H)-pyridinone (1) and 1,5-dimethyl-6-phenyl-1,2-dihydro-1,2,4-triazin-3...
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