Cytotoxic Amides from Piper sintenense

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Isolates
• Cell culture
• Cytotoxicity assay
• Lactate dehydrogenase (LDH) release assay
• Cell proliferation assay
• Statistics
• Results and Discussion
• Acknowledgements
• References

Abstract

A new alkaloid, pipersintenamide (1), together with fourteen known compounds, have been isolated from the whole plant of Piper sintenense. The structures of these compounds were elucidated by spectroscopic analysis. Pipersintenamide, sintenpyridone, sarmentine, and 1-(3,4-methylenedioxyphenyl)-1E-dodecene at 20 μg/ml exhibited effective cytotoxicities (cell survival < 15 %) against CCRF-CEM, HL-60, PC-3, and HA22T cell lines.

Introduction

In our continuing studies on the cytotoxic constituents of Formosan plants, over 700 species have been screened for in vitro cytotoxicity to date, and Piper sintenense Hatusima (Piperaceae) has been found to be one of the active species. P. sintenense is an endemic scandent plant growing in medium altitude forests throughout Taiwan [1]. Its leaves and stems have been used as folk medicine against snake bite and wounds [2]. A former study of this plant isolated two compounds, sesamin and piperarboricoline, without testing for any biological activity [3]. Subsequent investigation of the chloroform-soluble fraction of the whole plant of this species has led to the isolation of a new alkaloid, pipersintenamide (1), together with fourteen known compounds, including a piperidine: piperarboricoline (2) [3]; a 5,6-dihydro-2(1H)-pyridone: sintenpyridone (3) [3]; two pyrrolidides: sarmentine (4), nigrinodine (5); two amides: 3-(3′,4′-dimethoxyphenyl)-propionamide (6), (2E,4E)-N-isobutyldodecadienamide (7); three benzenoids: methyl 3,4-dimethoxyhydrocinnamate (8), tetracosyl ferulate (9) and 1-(3,4-methylenedioxyphenyl)-1E-dodecene (10); five steroids: β-sitosterol (11), mixture of β-sitostenone (12) and stigmasta-4,22-dien-3-one (13), a mixture of stigmast-4-ene-3,6-dione (14) and stigmasta-4,22-diene-3,6-dione (15). The structures of these isolates were elucidated by spectroscopic analysis. In this paper, we report on the isolation and structural elucidation of the new amide alkaloid (Fig. [1]), together with the fourteen known compounds and on their cytotoxic activities.

Materials and Methods

General experimental procedures

All melting points were determined on a Yanaco micro-melting point apparatus and were uncorrected. IR spectra were taken on a Hitachi 260-30 (KBr) spectrophotometer. UV spectra were obtained on a Shimadzu UV-160A spectrophotometer in EtOH. EI-mass spectra were recorded on a VG Biotech Quattro 5022 spectrometer. HR-mass spectra were recorded on a JEOL JMX-HX 110 spectrometer. ¹H-NMR and NOESY spectra were measured on a Varian Unity 400 spectrometer, and are given in ppm (δ). Silica gel (60 - 230, 230 - 400 mesh) (Merck) was used for CC and silica gel 60 F-254 (Merck) for TLC and preparative TLC. Optical rotations were measured using a Jasco DIP-370 polarimeter in CHCl₃ or MeOH.

Plant material

Piper sintenense was collected from Lai-I, Pingtung County, Taiwan, in August 2000. A voucher sample (Chen 5595) was deposited in the Herbarium of School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China.

Extraction and isolation

Dried whole plant (8.2 kg) was extracted with cold MeOH, and the extract concentrated under reduced pressure. The MeOH extract (870 g), when partitioned between H₂O-CHCl₃ (1 : 1), afforded a CHCl₃-soluble fraction (fr. A, 320 g). Part (30 g) of fr. A was rechromatographed on silica gel (980 g), eluting with n-hexane, gradually increasing the polarity with EtOAc to obtain 29 frs: fr. A1∼A6 (each 1000 ml, n-hexane), fr. A7 (1500 ml, n-hexane-EtOAc, 19 : 1), fr. A8∼A9 (each 1500 ml, n-hexane-EtOAc, 9 : 1), fr. A10∼A16 (each 1500 ml, n-hexane-EtOAc, 5 : 1), fr. A17∼A20 (each 1500 ml, n-hexane-EtOAc, 5 : 1), fr. A21∼A24 (each 1000 ml, n-hexane-EtOAc, 1 : 1), fr. A25 (2000 ml, EtOAc), fr. A26∼A29 (each 1500 ml, MeOH). Fr. A3 (73 mg) was further purified by preparative TLC on SiO₂ (n-hexane) to yield 10 (4.8 mg) (R_f = 0.42). Fr. A13 (479 mg) was rechromatographed on silica gel (16 g) using CH₂Cl₂-EtOAc (30 : 1) as eluent to give frs A13 - 1∼A13 - 4, and fr. A13 - 3 (113 mg) was further purified by preparative TLC on SiO₂ (n-hexane-EtOAc, 10 : 1) to obtain a mixture of 12 and 13 (10.8 mg) (R_f = 0.69). Frs A15 and A16 (201 mg) were further purified by preparative TLC (CH₂Cl₂) to yield 9 (11.4 mg) (R_f = 0.75) and 11 (16.8 mg) (R_f = 0.38), respectively. Fr. A17 (574 mg) was rechromatographed on silica gel (18 g) using CHCl₃-Me₂CO (60 : 1) as eluent to give frs A17 - 1∼A17 - 4, and fr. A17 - 3 (96 mg) was further purified by preparative TLC (CH₂Cl₂-EtOAc, 30 : 1) to obtain a mixture of 14 and 15 (7.6 mg) (R_f = 0.87). Fr. A20 (2.7 g) was rechromatographed on silica gel (83 g) using n-hexane-EtOAc (2 : 1) as eluent to give frs A20 - 1∼A20 - 7, and fr. A20 - 3 (364 mg) was further purified by preparative TLC (CH₂Cl₂-EtOAc, 20 : 1) to obtain 7 (13.9 mg) (R_f = 0.59) and 8 (7.5 mg) (R_f = 0.84). Fr. A22 (1.41 g) was rechromatographed on silica gel (44 g) using CH₂Cl₂-EtOAc (30 : 1) as eluent to give frs A22 - 1∼A22 - 5, and fr. A22 - 5 (172 mg) was further purified by preparative TLC (CH₂Cl₂-EtOAc, 10 : 1) to obtain 1 (5.2 mg) (R_f = 0.68). Fr. A24 (3.63 g) was rechromatographed on silica gel (108 g) using CHCl₃-Me₂CO (10 : 1) as eluent to give frs A24 - 1∼A24 - 7. Fr. A24 - 1 (276 mg) was further purified by preparative TLC (CHCl₃) to obtain 3 (15.2 mg) (R_f = 0.41). Fr. A24 - 3 (321 mg) was further purified by preparative TLC (CHCl₃-MeOH, 30 : 1) to obtain 2 (3.2 mg) (R_f = 0.65). Fr. A24 - 4 (197 mg) was further purified by preparative TLC (CHCl₃-Me₂CO, 20 : 1) to obtain 4 (6.4 mg) (R_f = 0.75). Fr. A26 (1.72 g) was rechromatographed on silica gel (51 g) using CHCl₃-Me₂CO (15 : 1) as eluent to give frs A26 - 1∼A26 - 5. Fr. A26 - 2 (351 mg) was further purified by preparative TLC (CHCl₃-Me₂CO, 15 : 1) to obtain 5 (5.2 mg) (R_f = 0.44). Fr. A26 - 3 (274 mg) was further purified by preparative TLC (CHCl₃-Me₂CO, 10 : 1) to obtain 6 (5.2 mg) (R_f = 0.37).

Isolates

Pipersintenamide (1): Colorless needles from MeOH, m. p. 116 - 118 °C. UV: λ_max (log ε) = 215 (4.27), 261 nm (4.06). IR: υ_max = 1657 (C = O), 1608, 1491, 1440 (aromatic ring C = C stretch), 1033, 926 (cm^-1) (OCH₂O). EI-MS: m/z (rel. int.) = 313 ([M]⁺, 28), 201 (7), 161 (97), 135 (66), 131 (100), 115 (11), 103 (85), 84 (39), 77 (43), 69 (19), 55 (25); HR-EI-MS: C₁₉H₂₃O₃N, found: 313.1672 [M]⁺, calcd: 313.1678. ¹H-NMR (CDCl₃, 400 MHz): see Table [1]. ¹³C-NMR (CDCl₃, 100 MHz): δ = 24.6 (C-4), 25.5 (C-3), 26.6 (C-5), 31.7 (C-11), 32.4 (C-10), 43.1 (C-2), 46.9 (C-6), 100.9 (OCH₂O), 105.4 (C-15), 108.1 (C-18), 120.3 (C-19), 121.1 (C-8), 127.6 (C-12), 130.2 (C-13), 132.0 (C-14), 144.4 (C-9), 146.7 (C-17), 147.9 (C-16), 165.5 (C-7).

Piperarboricoline (2): Colorless oil. UV: λ_max (log ε) = 205 (3.90), 266 nm (4.04). IR: υ_max = 1657 (cm^-1) (C = O). EI-MS: m/z (rel. int.) = 313 ([M]⁺, 25), 201 (6), 178 (12), 164 (10), 161 (10), 136 (31), 135 (100), 131 (12), 112 (15), 105 (15), 84 (35), 77 (49); HR-EI-MS: C₁₉H₂₃O₃N, found: 313.1673 [M]⁺, calcd: 313.1678. ¹H-NMR (CDCl₃, 400 MHz): see Table [1].

Sintenpyridone (3): Colorless needles from CHCl₃-MeOH, m. p. 125 - 127 °C. UV: λ_max (log ε) = 220 (4.68), 276 nm (4.03). IR: υ_max = 1697 (C = O), 1685 (C = O), 1588, 1518, 1468 (cm^-1) (aromatic ring C = C stretch). EI-MS: m/z (rel. int.) = 289 ([M]⁺, 73), 192 (42), 165 (13), 164 (100), 161 (11), 151 (44), 149 (22), 124 (12), 121 (11), 91 (12), 77 (11); HR-EI-MS: C₁₆H₁₉O₄N, found: 289.1307 [M]⁺, calcd: 289.1314. ¹H-NMR (CDCl₃, 400 MHz): see Table [1]. ¹H-NMR (CD₃OD, 400 MHz): δ = 2.39 (2H, tdd, J = 6.4, 4.4, 1.8 Hz, H-5), 2.88 (2H, t, J = 7.7 Hz, H-9), 3.18 (2H, t, J = 7.7 Hz, H-8), 3.79 (3H, s, OMe-12), 3.81 (3H, s, OMe-13), 3.92 (2H, t, J = 6.4 Hz, H-6), 5.94 (1H, dt, J = 9.6, 1.8 Hz, H-3), 6.77 (1H, dd, J = 8.0, 2.0 Hz, H-15), 6.85 (1H, d, J = 2.0 Hz, H-11), 6.85 (1H, d, J = 8.0 Hz, H-14), 7.01 (1H, dt, J = 9.6, 4.4 Hz, H-4). ¹³C-NMR (CDCl₃, 100 MHz): δ = 24.6 (C-5), 30.7 (C-9), 41.0 (C-6), 41.0 (C-8), 55.8 (OMe-13), 55.9 (OMe-12), 111.3 (C-11), 112.0 (C-14), 120.4 (C-15), 125.9 (C-3), 133.8 (C-10), 145.1 (C-4), 147.3 (C-12), 148.8 (C-13), 165.3 (C-2), 175.6 (C-7). ¹³C-NMR (CD₃OD, 125 MHz): δ = 25.6 (C-5), 31.9 (C-9), 42.3 (C-6), 42.1 (C-8), 56.6 (OMe-13), 56.4 (OMe-12), 113.6 (C-11), 113.2 (C-14), 121.7 (C-15), 126.0 (C-3), 135.5 (C-10), 147.9 (C-4), 150.4 (C-12), 148.9 (C-13), 167.3 (C-2), 177.2 (C-7).

β-Sitosterol (11): [α]_D ²⁴: -36.1 (c 0.21, CHCl₃).

Mixture of β-sitostenone (12) and stigmasta-4,22-diene-3-one (13): [α]_D ²⁴: + 82.4 (c 0.25, CHCl₃).

Mixture of stigmast-4-ene-3,6-dione (14) and stigmast-4,22-diene-3,6-dione (15): [α]_D ²⁴: -38.4 (c 0.17, CHCl₃).

Cell culture

Experiments were conducted using rat primary vascular smooth muscle cells (VSMCs) and four human cancer cell lines: CCRF-CEM (acute lymphoblastic leukemia), HL-60 (acute promyelocytic leukemia), PC-3 (prostate carcinoma), and HA22T (hepatoma). CCRF-CEM, HL-60, PC-3 and HA22T cells, and rat VSMCs were maintained in RPMI-1640 medium and Dulbecco’s modified Eagle’s medium (DMEM), respectively. The medium contained 10 % fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. Cells were incubated at 37 °C under 5 % CO₂ in cell culture flasks. The isolates were dissolved in dimethyl sulfoxide (DMSO) and further diluted in culture medium. The final DMSO concentration in the cell supernatant was less than 0.1 %, which did not interfere with the testing systems.

Cytotoxicity assay

The cytotoxicity assay was carried out as previously described [4]. Cell viability was determined by the enzymatic reduction of the yellow dye, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), to a blue formazan product in intact cells. Cells at a density of 1 - 5 × 10⁵/ml were treated with different concentrations of the isolates for 24 h. MTT dye was later added, and the cells were then incubated for 1 h at 37 °C. After removal of the supernatant, the cells were lysed and absorbance was detected at 550 nm. Moreover, taxol, an antineoplastic agent, was used as a positive control [5].

Lactate dehydrogenase (LDH) release assay

Plasma membrane integrity was determined by measuring lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis. After treatment with the isolates for 24 h, the supernatants were collected and measured using CytoTox® 96 non-radioactive cytotoxicity assay kit (Promega, Madison, USA). Taxol was used as a positive control [6].

Cell proliferation assay

The cell proliferation assay was slightly modified compared to that described by Guh et al. [7]. Rat vascular smooth muscle cells (10⁴/0.1 ml/well) were grown in 96-well plates overnight and then serum-deprived for 24 h. The medium was replaced with fresh medium containing 10 % fetal bovine serum in the absence or presence of the isolates for 48 h. The cells treated without stimulus (10 % FBS) were used as a control. After treatment, MTT dye (10 μl of 5 mg/ml) was added and the cells were further incubated for 2 h at 37 °C. The supernatants were removed and the cells were lysed with DMSO. The absorbance at 550 nm was measured in an automated microplate reader.

Statistics

The bioassay results are expressed as the means of at least two independent experiments.

Results and Discussion

Pipersintenamide (1) was obtained as colorless needles, m.p. 116 - 118 °C. Its molecular formula was established as C₁₉H₂₃O₃N by HR-EI-MS spectrometry. The IR spectrum showed a amidocarbonyl absorption at 1657 cm^-1 and a methylenedioxy group at 1033, 926 cm^-1. The UV absorption bands at 215 and 261 nm suggested the presence of a benzenoid moiety. The ¹H-NMR spectrum of 1 was similar to that of sarmentosine [8] except that piperidine protons of 1 replaced the pyrrolidine protons of sarmentosine. In addition, a methylenedioxy group at δ 5.94 (2H, s) and three mutually coupling aromatic protons at δ 6.88, 6.73, and 6.75 were similar to those of sarmentosine [8] and suggested the presence of a 3,4-methylenedioxybenzyl moiety. This was supported by the EI-mass spectrum, which gave an ion as the base peak having m/z 131 [8]. The presence of a piperidine moiety was easily revealed by the signals at δ 1.55 (4H), δ 1.63 (2H), and δ 3.47, 3.59 (each 2H), along with the fragment at m/z 84 in the EI-mass spectrum. The structure of 1 could be reasonably established as N-[2E,6E,7-(3,4-methylenedioxyphenyl)heptadienoyl]-piperidine (1) on the basis of the above data, which was further supported by ¹H-¹H COSY, NOESY experiments (Fig. [2]). The assignments of ¹³C-NMR of 1 were confirmed by DEPT, HETCOR, and HMBC experiments (Fig. [2]).

Piperarboricoline (2) was obtained as a colorless oil. The HR-MS revealed a molecular ion peak at m/z 313.1673 leading to the molecular formula C₁₉H₂₃O₃N (calcd 313.1678). The presence of a conjugated carbonyl group was indicated by IR spectrum at 1657 cm^-1, as well as ¹³C-NMR spectrum at δ 165.6. The UV absorption bands at 205 and 266 nm suggested the presence of a benzenoid moiety. Analysis of the ¹H-NMR spectrum of 2 exhibited the signals at δ 1.57 (4H), δ 1.64 (2H), and δ 3.49, 3.61 (each 2H), characteristic of the protons of piperidine moiety. This could be substantiated by the fragment ion at m/z 84 in the EI-mass spectrum. The presence of a 3,4-methylenedioxybenzyl moiety was easily revealed by three mutually coupling aromatic protons at δ 6.66, 6.72, and 6.61 and a methylenedioxy signal at δ 5.92, together with the base peak at m/z 135 in the EI-mass spectrum. The signals at δ 6.05, 6.18, 6.26, and 7.22 were attributed to the protons of trans-conjugated double bonds. In addition, the signals at δ 2.42 and δ 2.66 were due to the mutually coupling methylene protons. The above assignments were identified by a comparison of ¹H-NMR (Table [1]) with the reference compound [3] and further confirmed by ¹H-¹H COSY and NOESY experiments (Fig. [3]). From the above evidence, 2 was characterized as N-[2E,4E,7-(3,4-methylenedioxyphenyl)heptadienoyl]-piperidine. This compound had previously been isolated as new compound from the same plant [3], but this finding has never before been published in any journal.

Sintenpyridone (3) was obtained as colorless needles, m.p. 125 - 127 °C. The EI-MS afforded the molecular [M]⁺ ion at m/z 289, implying a molecular formula of C₁₆H₁₉O₄N, which was confirmed by the HR-EI-MS. The UV absorption bands at 220 and 276 nm suggested the presence of a benzenoid moiety. The IR spectrum showed two carbonyl absorptions at 1697 and 1685 cm^-1. The ¹H-NMR spectrum (CD₃OD) of 3 showed four mutually coupled protons at δ 5.94, 7.01, 2.39, and 3.92 were similar to those of N-(12,13,14-trimethoxydihydrocinnamoyl)-Δ³-pyridin-2-one [9] and suggested the presence of a 5,6-dihydro-2(1H)-pyridone moiety. In addition, two methoxy groups at δ 3.79, 3.81 (each 3H, s), three mutually coupled protons at δ 6.85, 6.85, and 6.77, and four methylene protons at δ 2.88, 3.18 (each 2H) suggested the presence of an N-12,13,14-trimethoxydihydrocinnamoyl moiety in 3. The above assignments were identified by a comparison of ¹H-NMR (Table [1]) with the reference compound [3]. Past literature has made wrong assignments of H-8 and H-9, which have since been corrected by HMBC experiments (Fig. [4]). According to the above data, the structure of 3 was elucidated as N-(3,4-dimethoxydihydrocinnamoyl)-5,6-dihydro-2(1H)-pyridone, named sintenpyridone; this was further confirmed by ¹H-¹H COSY and NOESY experiments (Fig. [4]). The assignments of ¹³C-NMR of 3 were confirmed by DEPT, HETCOR, and HMBC experiments (Fig. [4]). This compound was also previously isolated from the leaves of Piper arborescens [3], but this finding has never before been published in any journal.

The known compounds, including 4 amides: sarmentine (4) [10], nigrinodine (5) [11]; 3-(3′,4′-dimethoxyphenyl)-propionamide (6) [12], (2E,4E)-N-isobutyldodecadienamide (7) [13]; three benzenoids: methyl 3,4-dimethoxyhydrocinnamate (8) [14], tetracosyl ferulate (9) [15] and 1-(3,4-methylenedioxyphenyl)-1E-dodecene (10) [16]; five steroids: β-sitosterol (11) [17], a mixture of β-sitostenone (12) and stigmasta-4,22-dien-3-one (13), a mixture of stigmast-4-ene-3,6-dione (14) and stigmasta-4,22-diene-3,6-dione (15) [17], [18] were identified by comparison of physical and spectroscopic data (UV, IR, ¹H-NMR, [α]_D, and mass spectrometry data) with the literature values.

The cytotoxic effects of the isolates were tested in vitro against CCRF-CEM, HL-60, PC-3, and HA22T cell lines. The cytotoxicity data are shown in Table [2]. The clinically applied anticancer agent taxol was used as the reference compound. Pipersintenamide (1), sintenpyridone (3), sarmentine (4) and 1-(3,4-methylenedioxyphenyl)-1E-dodecene (10) at 20 μg/ml exhibited effective cytotoxicites (cell survival &λ< 15 %) against the CCRF-CEM cell line. Of these, 1-(3,4-methylenedioxyphenyl)-1E-dodecene (10) at 20 μg/ml also exhibited effective cytotoxicities (cell survival &λ< 9 %) against HL-60, PC-3, and HA22T cell lines. Sintenpyridone (3) at 20 μg/ml displayed effective cytotoxicities (cell survival ≤ 10 %) against HL-60 and HA22T cells. Pipersintenamide (1), a new isolated alkaloid, seems to be more cytotoxic in CCRF-CEM and HL-60 leukemia cells. However, (2E,4E-N-isobutyldodecadienamide) (7) and β-sitosterol (11) at 5 μg/ml apparently showed cytotoxic effects (cell survival < 35 %) in CCRF-CEM cells rather than other cells. Compounds 1, 3, 4, and 10 showed the obvious release of lactate dehydrogenase (LDH) at the concentration of inhibitory growth in CCRF-CEM, HL-60, PC-3, or HA22T cell lines (Table [3]). Among three benzenoids, 1-(3,4-methylenedioxyphenyl)-1E-dodecene (10) displayed the most effective cytotoxicity (IC₅₀ value = 1.95 and 2.13 μg/ml against CCRF-CEM and HL-60, respectively) (Table [4]). Compounds 1, 3, and 10 also showed the inhibitory effect of cell proliferation induced by serum in vascular smooth muscles (Table [5]). Moreover, compound 1, 3, and 10 exhibited apparent LDH releasing effects (75.5 %, 63.2 % and 69.5 %, respectively) in vascular smooth muscle cells. The above facts implicated that the cytotoxicity induced by these compounds was possibly due to disturbance of the cellular integrity and correlated to cellular LDH release. Finally, the presence of the 3,4-methylenedioxyphenylvinyl group seems to play an important role in the cytotoxicities of 1 and 10 in contrast to the lack of such activity in 2 and 5.

Acknowledgements

This work was supported by a grant (NSC 89-2320-B-127-009) from the National Science Council of the Republic of China.

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Prof. Ph. D. I. S. Chen

School of Pharmacy

Kaohsiung Medical University

Kaohsiung, Taiwan

Republic of China

Email: m635013@cc.kmu.edu.tw

Fax: +886-7-3210683

References

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Prof. Ph. D. I. S. Chen

School of Pharmacy

Kaohsiung Medical University

Kaohsiung, Taiwan

Republic of China

Email: m635013@cc.kmu.edu.tw

Fax: +886-7-3210683