HETEROCYCLES

Note | Regular issue | Vol. 81, No. 6, 2010, pp. 1493-1502
Received, 11th March, 2010, Accepted, 8th April, 2010, Published online, 9th April, 2010.
DOI: 10.3987/COM-10-11942

■ Pyrrolizidine Alkaloids from the Roots of Paris verticillata

Ki Hyun Kim, Kyu Ha Lee, Sang Un Choi, Kyung Ran Kim, and Kang Ro Lee*

Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, 300 Chunchun-dong, Jangan-ku, Suwon 440-746, Korea

Abstract

Three new pyrrolizidine alkaloids, verticillatins A (1), B (2), and C (3), together with seven known pyrrolizidine alkaloids (4-10), were isolated from the roots of Paris verticillata (Liliaceae). Their structures were determined by spectroscopic data and chemical evidences. Verticillatin A (1), verticillatin B (2), verticillatin C (3), heliovinine N-oxide (4), and indicine N-oxide (5) showed cytotoxicity against the HCT15 cell line, with IC50 values of 67.6, 92.4, 35.7, 36.2, and 74.7 μM, respectively.

INTRODUCTION
Pyrrolizidine alkaloids (PAs) are deterrent and toxic to most vertebrates and insects and provide plants with defensive chemicals against herbivores.1,2 Their toxicity is based primarily on metabolism by means of cytochrome P-450 oxidases. This metabolism results in bioactivation via the formation of highly reactive pyrrole intermediates, causing cytotoxicity, mutagenicity, and genotoxicity.3,4 Individual PA patterns may be genetically controlled as a result of evolution under selective pressure.5 Because of this evolutionary advantage for the structural diversity of PAs, it is worthwhile to study PAs from Korean traditional plants used in folk medicine.
We have performed cytotoxic activity screening on a methanol extract from the roots of Paris veriticillata BIEB. (Liliaceae) that showed cytotoxicity against A549 and HCT15 cell lines. The perennial herb P. veriticillata is distributed in Korean valleys and its root have been used as a Korean traditional medicine against asthma, boils, and chronic bronchitis.6 Several compounds such as saponins, sterols, and flavonoids were reported from the aerial parts of P. verticillata,7 and we have reported a phenolic amide and phenolic constituents from the roots of P. verticillata.8 In continuing investigation of chemical constituents from the roots of P. verticillata, we further isolated three new pyrrolizidine alkaloids, verticillatins A (1), B (2), and C (3), together with seven known pyrrolizidine alkaloids (4-10). We then examined the cytotoxic activity of these isolated compounds (1-10) against four human tumor cells, A549, SK-OV-3, SK-MEL-2, and HCT15. We describe herein the isolation, structure assignment, and bioactivity of these compounds.

RESULTS AND DISCUSSION
Compound 1 was obtained as a colorless gum. The molecular formula of 1 was determined to be C15H25NO6 by the molecular ion peak [M + H]+ at m/z 316.1760 (calcd. for C15H26NO6: 316.1760) in HRFABMS. Its IR spectrum showed bands at 3388 cm-1 (OH) and 1720 cm-1 (C=O). The 1H NMR spectrum of 1 (Table 1) displayed two methyl signals for an isopropyl group at δH 0.94 (d, J = 7.0 Hz, H-7′) and 0.96 (d, J = 7.0 Hz, H-6′), two methine signals at δH 1.99 (m, H-5′) and 4.08 (q, J = 6.5 Hz, H-3′), and one methyl signal at δH 1.18 (d, J = 6.5 Hz, H-4′), which were correlated with carbon signals at δC 16.5 (C-7′), 16.1 (C-6′), 33.0 (C-5′), 69.3 (C-3′), and 16.0 (C-4′) in the HMQC experiment, respectively, indicating that 1 has a trachelanthate ester unit (ΔδC-6′/C-7′ = 0.4).9,10 Three methine protons were observed at δH 4.73 (br s, H-7, and H-8) and 5.94 (br s, H-2), which were correlated with carbon signals at δC 69.6 (C-7), 95.8 (C-8), and 122.4 (C-2) in the HMQC experiment. The downfield signal at δH 5.94 (H-2) in the 1H NMR spectrum and the signals at δC 77.6 (C-3), 68.7 (C-5), 34.4 (C-6), 69.6 (C-7), and 95.8 (C-8) in the 13C NMR spectrum suggest that the pyrrolizidine base is to be unsaturated pyrrolizidine N-oxide.10 These assignments were also confirmed by further analyses of HMQC, HMBC, and 1H-1H COSY experiments (Figure 2). Specifically, HMBC correlation from H-9 to C-1′ indicated the position of the trachelanthic acid at C-9.
Acidic hydrolysis of 1 gave trachelanthic acid and 1-hydroxymethyl-7-hydroxypyrrolizidine N-oxide. The absolute streochemistry of 1b, trachelanthic acid, was determined o be 2′R, 3′S by comparison of NMR data and negative optical rotation value; [α]25D-4.5 (c 0.05, MeOH) to those of its synthetic stereoisomer.9 The pyrrolizidine base 1a was identified as (-)-heliotridine N-oxide based on the NOESY experiment and a convenient Mosher ester procedure carried out in an NMR tube.11-14 Analysis of the chemical shift differences between MTPA ester derivatives of 1 (Figure 3) revealed R-configured C-7 and S- configured C-3′.

The NOESY spectrum of 1 showed correlations between H-8 and H-6β or H3-6′, H3-7′, and between H-7 and H-5α, which indicated S-configured C-8 (Figure 4). Thus, the base was identified as (-)-heliotridine N-oxide.15 Furthermore, the base 1a was reduced with zinc in dilute H2SO4 to give (-)-heliotridine.10 A negative optical rotation value of [α]25D -10.5 (c 0.01 in MeOH) and the 1H NMR data were in agreement with those of synthetic (-)-heliotridine.15
In the NOE difference spectra, a NOE enhancement between H-7, H-6α, and H-6β protons differed in magnitude, suggesting the conformation of pyrrolizidine base is an endo-buckled form in which H-7 and H-6β protons approximate a trans-configuration, resulting in different NOEs.16,17 The new pyrrolizidine alkaloid, named verticillatin A, is thus 9-(-)-trachelanthyl-(-)-heliotridine N-oxide.

Compound 2 was obtained as a colorless gum, whose molecular formula was determined as C15H25NO6 from the molecular ion peak [M + H]+ at m/z 316.1759 in HRFABMS. IR, MS, and NMR data of compound 2 were similar to those of compound 1. NMR spectral analyses, including HMQC and HMBC experiments, led to the chemical structure of 2, implying that compounds 1 and 2 were mutual stereoisomer. This was confirmed by a convenient Mosher ester procedure11-14 (Figure 5) and the NOESY correlations between H-7 and H-5β and between H-8 and H-6β or H3-6′, H3-7′ (Figure 4), which indicated S-configured C-7 and S-configured C-8. Acidic hydrolysis of 2 afforded side residue 2b, viridifloric acid. The absolute stereochemistry of viridifloric acid was determined to be 2′R and 3′R by comparison of NMR data and positive optical rotation value; [α]25D +3.5 (c 0.05, MeOH).9 Thus, compound 2 was determined to be 9-(+)-viridifloryl-(-)-retronecine N-oxide, named verticillatin B.

Compound 3 was isolated as a colorless gum, whose molecular formula was determined from the molecular ion peak [M + H]+ at m/z 300.1811, corresponding to a molecular formula of C15H26NO5 (calcd. for C15H26NO5: 300.1811). The 1H and 13C NMR spectra were also similar to those of verticillatin A (1), except for values assigned to C-7 in the 1H and 13C NMR spectra, in which the methine (δH 4.73/δC 69.6) at C-7 of verticillatin A (1) was replaced by methylene (δH 2.00 and 2.44/δC 27.3) at C-7 of 3. The 1H and 13C NMR assignments (Table 1) of 3 were confirmed by detailed analyses of 2D-NMR data, including HMQC, HMBC, and NOESY. Key HMBC showed correlations from H-8 to C-1, C-2, C-6, C-7, and C-9, and from H-9 to C-1′ (Figure 6). In NOESY spectrum of 3, the key correlation of H-2 with H3-4′ supported the proposed configuration at C-3′. Acidic hydrolysis of 3 afforded side residue, (+)-viridifloric acid, which was confirmed by co-TLC along with the produced 2b and direct comparison of its 1H NMR data and optical rotation value; [α]25D +5.2 (c 0.02, MeOH) with those of 2b.9 From the above evidence, compound 3 was identified as coromandalinine N-oxide, named verticillatin C.

Isolated known compounds were identified as heliovinine N-oxide (4),18 indicine N-oxide (5),10 (+)-viridifloryl heliotridine N-oxide (6),19 (-)-indicine N-oxide (7),19 (+)-viridifloryl-(+)-retronecine N-oxide (8),9 (7S)-indicine N-oxide (9),18 and isatinecine (10)9 by comparison with published data. This is the first report of pyrrolizidine alkaloids from this plant.
The cytotoxicities of the isolated compounds (1-10) against A549, SK-OV-3, SK-MEL-2, and HCT15 human tumor cell lines were evaluated using the SRB assay in vitro. Verticillatin A (1), verticillatin B (2), verticillatin C (3), heliovinine N-oxide (4), and indicine N-oxide (5) showed weak cytotoxicity against the HCT15 cell line, with IC50 values of 67.6, 92.4, 35.7, 36.2, and 74.7 μM, respectively, but were inactive against other tested cell lines (IC50 > 100 μM). Compounds 6-10 showed little cytotoxicity against any tested cell line (IC50 > 100 μM). The roots of P. verticillata have often been used as Korean traditional medicine, but we suggest here that its application should be more carefully monitored due to the isolation of various toxic pyrrolizidine alkaloids.3,4

EXPERIMENTAL DETAILS
General. Optical rotations were measured on a Jasco P-1020 polarimeter in MeOH. IR spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer. FAB and HRFAB mass spectra were obtained on a JEOL JMS700 mass spectrometer. NMR spectra, including 1H-1H COSY, HMQC, HMBC and NOESY experiments, were recorded on a Varian UNITY INOVA 500 NMR spectrometer operating at 500 MHz (1H) and 125 MHz (13C) with chemical shifts given in ppm (δ). Preparative HPLC was conducted using a Gilson 306 pump with Shodex refractive index detector and Apollo Silica 5μ column (250 × 10 mm). Silica gel 60 (Merck, 70-230 mesh and 230-400 mesh) was used for column chromatography. Merck precoated Silica gel F254 plates and RP-18 F254s plates were used for TLC. Spots were detected on TLC under UV light or by heating after spraying with 10% H2SO4 in EtOH (v/v).

Plant material. The roots of P. verticillata were collected at Mt. O-Dae, Gangwon Province in August, 2002, and the plants were identified by one of the authors (K.R.L.). A voucher specimen (SKKU 2002-08) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Extraction and isolation. The roots of P. verticillata (1.5 kg) were extracted using 80% MeOH at room temperature over a period of 3 days three times. The solvent was evaporated under reduced pressure to give a crude extract (180 g). The systematic solvent partitioning of the MeOH extract afforded n-hexane soluble (5.2 g), methylene chloride soluble (3.5 g), EtOAc soluble (4.4 g), and n-BuOH soluble (20 g) fractions. The n-BuOH soluble fraction (10 g) was subjected to column chromatography over HP-20 resin with 100% H2O and 100% MeOH to give two fractions (B1 - 100% H2O and B2 - 100% MeOH). The B2 fraction (3 g) was subjected to normal phase column chromatography over a silica gel (CHCl3-MeOH-H2O = 10:10:0.5) to give six fractions (B21 to B26). B24 fraction (1.2 g) was further separated over a silica gel column (CHCl3-MeOH-H2O = 10:4:0.5) to give two subfractions (B241 to B242). The B241 fraction (600 mg) was purified by semi-preparative HPLC, using a solvent system of CHCl3-EtOAc-MeOH-H2O-NH4OH (10:2:4:0.5:0.2) for 30 min at a flow rate of 2.0 mL/min (Apollo Silica 5μ column; 250 × 10 mm; 5 μm particle size; Shodex refractive index detector) to obtain 2 (20 mg, tR = 16.5 min), 3 (7 mg, tR = 14.9 min), 4 (6 mg, tR = 14.5 min), 6 (8 mg, tR = 17.3 min), 8 (8 mg, tR = 18.5 min), and 9 (10 mg, tR = 18.0 min). The B242 fraction (800 mg) was also purified by semi-preparative HPLC as above, using a solvent system of CHCl3-EtOAc-MeOH-H2O-NH4OH (10:2:4:0.5:0.2) to yield 1 (10 mg, tR = 16.2 min), 7 (4 mg, tR = 15.5 min) and 10 (5 mg, tR = 17.5 min). The B25 fraction (50 mg) was purified by semi-preparative HPLC as above, using a solvent system of CHCl3-MeOH-H2O (9:3.5:0.5) to afford 5 (5 mg, tR = 18.0 min).

Verticillatin A (1). colorless gum; [α]25D +2.5 (c 0.20, MeOH); CD (MeOH) λmax (Δε) 204 (+38.5), 213 (+25.1), 223 (+12.3) nm; IR (KBr) νmax 3388 (OH), 2948 (CH), 1720 (C=O), 1667 (C=C), 1453 (CH3), 1230 (C-O) cm-1; 1H (500 MHz) and 13C (125 MHz) NMR data, see Table 1; FABMS m/z 316 [M + H]+; HR-FABMS m/z 316.1760 [M + H]+ (calcd for C15H26NO6, 316.1760).

Verticillatin B (2). colorless gum; [α]25D +4.0 (c 0.60, MeOH); CD (MeOH) λmax (Δε) 202 (+31.2), 212 (+17.5), 223 (+5.1) nm; IR (KBr) νmax 3388 (OH), 2947 (CH), 1720 (C=O), 1667 (C=C), 1452 (CH3), 1230 (C-O) cm-1; 1H (500 MHz) and 13C (125 MHz) NMR data, see Table 1; FABMS m/z 316 [M + H]+; HR-FABMS m/z 316.1759 [M + H]+ (calcd for C15H26NO6, 316.1760).

Verticillatin C (3). colorless gum; [α]25D -2.8 (c 0.10, MeOH); CD (MeOH) λmax (Δε) 203 (+33.1), 213 (+22.0), 223 (+22.8) nm; IR (KBr) νmax 3387 (OH), 2948 (CH), 1721 (C=O), 1666 (C=C), 1450 (CH3), 1231 (C-O) cm-1; 1H (500 MHz) and 13C (125 MHz) NMR data, see Table 1; FABMS m/z 300 [M + H]+; HR-FABMS m/z 300.1811 [M + H]+ (calcd for C15H26NO5, 300.1811).

Preparation of the (R)- and (S)-MTPA Ester Derivatives of 1 and 2 by a Convenient Mosher Ester Procedure. Compound 1 (1.8 mg) in deuterated pyridine (0.75 mL) was transferred into a clean NMR tube. (S)-(+)-α-Methoxy-α-(trifluoromethyl)phenylacetyl chloride, (S)-MTPA-Cl (5 μL) was added immediately into the NMR tube under a stream of N2 gas and the NMR tube was shaken carefully to mix the sample and (S)-MTPA-Cl evenly. The reaction NMR tube stood at room temperature overnight. The completed reaction afforded the (R)-MTPA ester derivative (1r) of 1. The (S)-MTPA ester derivative of 1 (1s) and the (R)- and (S)-MTPA ester derivatives of 2 (2r and 2s) were obtained in the same manner as described for 1r. The 1H NMR spectra of 1r, 1s, 2r and 2s were directly measured with the reaction NMR tubes.
1s. 1H NMR (500 MHz, pyridine-d5): δ 5.934 (1H, br s, H-7), 5.895 (1H, br s, H-2), 5.181 (1H, d, J = 14.0 Hz, H-9a), 5.178 (1H, d, J = 14.0 Hz, H-9b), 5.109 (1H, br s, H-8), 5.097 (1H, br d, J = 16.5 Hz, H-3a), 4.926 (1H, br d, J = 16.5 Hz, H-3b), 4.747 (1H, m, H-5a), 4.548 (1H, q, J = 6.5 Hz, H-3′), 4.193 (1H, m, H-5b), 2.752 (1H, m, H-6a), 2.282 (1H, m, H-5′), 2.168 (1H, m, H-6b), 1.487 (3H, d, J = 6.5 Hz, H-4′), 1.167 (3H, d, J = 7.0 Hz, H-6′), 1.001 (3H, d, J = 7.0 Hz, H-7′).

1r. 1H NMR (500 MHz, pyridine-d5): δ 5.933 (1H, br s, H-7), 5.895 (1H, br s, H-2), 5.203 (1H, d, J = 14.0 Hz, H-9a), 5.199 (1H, d, J = 14.0 Hz, H-9b), 5.126 (1H, br s, H-8), 5.098 (1H, br d, J = 16.5 Hz, H-3a), 4.927 (1H, br d, J = 16.5 Hz, H-3b), 4.726 (1H, m, H-5a), 4.543 (1H, q, J = 6.5 Hz, H-3′), 4.179 (1H, m, H-5b), 2.725 (1H, m, H-6a), 2.298 (1H, m, H-5′), 2.160 (1H, m, H-6b), 1.484 (3H, d, J = 6.5 Hz, H-4′), 1.168 (3H, d, J = 7.0 Hz, H-6′), 1.003 (3H, d, J = 7.0 Hz, H-7′).

2s. 1H NMR (500 MHz, pyridine-d5): δ 5.910 (1H, br s, H-7), 5.885 (1H, br s, H-2), 5.252 (1H, d, J = 14.0 Hz, H-9a), 5.245 (1H, d, J = 14.0 Hz, H-9b), 5.180 (1H, br s, H-8), 5.122 (1H, br d, J = 16.5 Hz, H-3a), 4.914 (1H, br d, J = 16.5 Hz, H-3b), 4.519 (1H, m, H-5a), 4.396 (1H, q, J = 6.5 Hz, H-3′), 4.135 (1H, m, H-5b), 2.650 (1H, m, H-6a), 2.459 (1H, m, H-5′), 2.113 (1H, m, H-6b), 1.544 (3H, d, J = 6.5 Hz, H-4′), 1.222 (3H, d, J = 7.0 Hz, H-6′), 1.081 (3H, d, J = 7.0 Hz, H-7′).

2r. 1H NMR (500 MHz, pyridine-d5): δ 5.924 (1H, br s, H-7), 5.885 (1H, br s, H-2), 5.229 (1H, d, J = 14.0 Hz, H-9a), 5.211 (1H, d, J = 14.0 Hz, H-9b), 5.154 (1H, br s, H-8), 5.119 (1H, br d, J = 16.5 Hz, H-3a), 4.910 (1H, br d, J = 16.5 Hz, H-3b), 4.600 (1H, m, H-5a), 4.402 (1H, q, J = 6.5 Hz, H-3′), 4.195 (1H, m, H-5b), 2.700 (1H, m, H-6a), 2.458 (1H, m, H-5′), 2.161 (1H, m, H-6b), 1.552 (3H, d, J = 6.5 Hz, H-4′), 1.119 (3H, d, J = 7.0 Hz, H-6′), 1.080 (3H, d, J = 7.0 Hz, H-7′).

Acidic Hydrolysis of Compounds 1-3. Compounds (1: 5.0 mg; 2: 3.5 mg; and 3: 3.0 mg) were hydrolyzed with 2N HCl (2 mL) at 40 °C for 3 h. Then H2O (5 mL) was added and the mixture was extracted twice with EtOAc. The EtOAc layer was evaporated under reduced pressure to give an EtOAc extract. Each EtOAc extract was purified over a silica gel Waters Sep-Pak Vac 6cc [CHCl3-MeOH, 10:1] to give pyrrolizidine bases 1a (1.5 mg), 2a (0.8 mg), and 3a (0.8 mg). The aqueous layer of each reaction mixture was neutralized with dilute NH4OH (monitored with indicator paper) and, then filtered to afford the side residues 1b (0.8 mg) and 2b (0.6 mg), respectively from reaction mixtures of 2 and 3. The compounds, (-)-trachelanthic acid 1b and (+)-viridifloric acid 2b were identified by 1H NMR, MS data and optical rotation values. The aqueous layer of the reaction mixture of 3 contained the side residue, (+)-viridifloric acid, which were separated by HPLC analysis (CHCl3-MeOH-NH4OH = 9:1:0.1) and confirmed by co-TLC and direct comparison of its 1H NMR data and optical rotation value with those of 2b obtained above. The optical rotation values of the pyrrolizidine bases 1a, 2a, and 3a obtained from hydrolysis of 1-3 were [α]25D -20.1 (c 0.02, MeOH), [α]25D -9.5 (c 0.01, MeOH), and [α]25D -12.5 (c 0.01, MeOH), respectively. The optical rotation value of the side residue, (+)-viridifloric acid obtained from hydrolysis of 3 was [α]25D +5.2 (c 0.02, MeOH) and its 1H NMR and MS data were identical to those of 2b.

1b. colorless gum; [α]25D -4.5 (c 0.05, MeOH); 1H NMR (500 MHz, CD3OD): δ 4.12 (1H, q, J = 6.5 Hz, H-3), 2.05 (1H, m, H-5), 1.20 (3H, d, J = 6.5 Hz, H-4), 0.97 (3H, d, J = 7.0 Hz, H-6), 0.95 (3H, d, J = 7.0 Hz, H-7); FAB-MS m/z 163 [M + H]+.

2b. colorless gum; [α]25D +3.5 (c 0.05, MeOH); 1H NMR (500 MHz, CD3OD): δ 4.00 (1H, q, J = 6.5 Hz, H-3), 2.12 (1H, m, H-5), 1.27 (3H, d, J = 6.5 Hz, H-4), 0.92 (3H, d, J = 7.0 Hz, H-6), 0.90 (3H, d, J = 7.0 Hz, H-7); FAB-MS m/z 163 [M + H]+.

Reduction of 1a. The pyrrolizidine base 1a (1.5 mg) obtained from hydrolysis of 1 was reduced with zinc in dilute H2SO4, as described in previous paper.10 The aqueous layer of the reaction mixture was then neutralized with dilute NH4OH and extracted with CHCl3 three times. The CHCl3 extract was purified by HPLC analysis (CHCl3-MeOH-NH4OH, 5:1:0.1) to afford (-)-heliotridine (0.3 mg). The (-)-heliotridine was identified by its negative optical rotation value, [α]25D -10.5 (c 0.01, MeOH) and comparison of its 1H NMR data with that of synthetic (-)-heliotridine.15

Cytotoxicity Assay. A sulforhodamin B bioassay (SRB) was used to determine the cytotoxicity of each compound against four cultured human cancer cell lines. The assays were performed at the Korea Research Institute of Chemical Technology as described in previous paper.20 The cell lines used were A549 (non-small cell lung carcinoma), SK-OV-3 (ovarian malignant ascites), SK-MEL-2 (skin melanoma), and HCT15 (colon adenocarcinoma). Doxorubicin was used as a positive control. Doxorubicin had IC50s against A549, SK-OV-3, SK-MEL-2, and HCT15 of 0.007, 0.056, 0.017, and 0.024 μM, respectively.

ACKNOWLEDGEMENTS
The authors would like to thank Mr. Do Kyun Kim, Dr. Eun Jung Bang, and Dr. Jung Ju Seo at the Korea Basic Science Institute for the measurements of NMR and MS spectra.

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