Cytotoxic Alkaloids from the Flowers of Senna spectabilis

• Abstract
• Material and Methods
• References

Abstract

Three new alkaloids, 3(R)-benzoyloxy-2(R)-methyl-6(R)-(11′-oxododecyl)-piperidine (3), 5-hydroxy-2-methyl-6-(11′-oxododecyl)-pyridine (4) and 5-hydroxy-2-methyl-6-(11′-oxododecyl)-pyridine N-oxide (5), together with a known alkaloid, (-)-cassine (1), were isolated from the flowers of Senna spectabilis. A derivative, N,O-diacetylcassine (2), was semisynthesized. Their structures and stereochemistry were established on the basis of spectroscopic analysis. Cytotoxic activity and brine shrimp lethality of these compounds were evaluated. Compounds 2, 3 and 5 exhibited cytoxicity against KB cell lines with IC₅₀ values of 5.2, 3.7 and 2.0 μg/mL, respectively.

Senna spectabilis D.C. (Leguminosae-Caesalpinioideae) is an ornamental plant of Thailand. Its leaves and seeds contain 3-piperidinol alkaloids [1], [2]. Up to date, several piperidine alkaloids have been shown to possess cytotoxic activity [3], [4]. In our investigation, (-)-cassine (1), a new piperidine alkaloid (3), and two new pyridine alkaloids (4 and 5) were isolated from the flowers of this plant. (-)-Cassine (1), [α]_D ²⁵: -1.07°, was identified by comparing its physical and spectroscopic properties with those previously reported [5]. By acetylation of 1, its N,O-diacetyl derivative (2), [α]_D ²⁵: -19.43°, was semisynthesized.

Compound 3 has the molecular formula C₂₅H₃₉NO₃. Its NMR data (Tables [1] and [2]) exhibited five aromatic protons, six aromatic carbons and one carbonyl carbon more than compound 1 and IR spectrum indicated an ester carbonyl (1720 and 1280 cm^-1). Thus a benzoyl substitution was concluded. An NOE correlation between the aromatic protons (δ = 8.06 ppm) and 2-CH₃ (δ = 3.43 ppm) indicated that this functional group was at the C-3 position. An NOE from H-2 to H-3 and H-1′, but not to H-6, confirmed the contrary orientation of H-6. Hydrolysis of 3 yielded benzoic acid and a product whose spectral data and optical rotation was identical to those of (2R,3R,6R)-6-isocassine, [α]_D ²⁵: + 3.3°, in [1]. Therefore, the structure and absolute stereochemistry of 3 was established as 3(R)-benzoyl-2(R)-methyl-6(R)-(11′-oxododecyl)-piperidine.

The molecular weight of compound 4 was 6 a. m. u. less than compound 1. Its ¹H-NMR spectra characterized an aromatic pyridine system. The methyl protons of 2-CH₃ (δ = 2.41 ppm) displayed a three-bond correlation to a methine carbon (H-3, δ = 121.5 ppm). Its proton (H-3, δ = 6.81 ppm) exhibited ortho-coupling (J = 8.2 Hz) with H-4 (δ = 7.00 ppm). A hydroxy group was located at C-5 and this carbon was significantly deshielded (δ = 148.8 ppm). The tree-bond correlation from C-6 to H-3 and H-1′ and the above evidence indicated the structure of 4 as 5-hydroxy-2-methyl-6-(11′-oxododecyl)-pyridine.

Compound 5 exhibited NMR and MS data similar to those of compound 4. Its molecular ion at m/z = 307 indicated one more oxygen atom, and indicated it to be the N-oxide of 4. According to the mesomeric effect of an N-oxide, ¹³C-NMR signals of carbons at the α-position next to N-1 (C-2 and C-6) are shifted upfield, whereas those of β-carbons (C-3 and C-5) are shifted downfield. Accordingly, the structure of 5 was determined to be 5-hydroxy-2-methyl-6-(11′-oxododecyl)-pyridine N-oxide. Compounds 3 - 5 are new naturally occurring alkaloids.

All compounds were tested for brine shrimp lethality and cytotoxicity. Compound 1 showed a significant, strong toxicity against brine shrimp (Table [3]). This compound probably exhibits insecticidal activity like other piperidine alkaloids [6]. Substitution at positions 1 and 3 of compound 1 as in compounds 2 and 3, enhanced the cytotoxic activity. The pyridine alkaloid 4 was not cytotoxic. However, its N-oxide, compound 5, demonstrated high activity.[*]

Material and Methods

Melting points were determined on a Büchi melting point apparatus and are uncorrected. UV and IR spectra were obtained on a Hitachi U-2000 spectrophotometer and a Nicolet IR spectrophotometer 440, respectively. NMR spectra were recorded at 500 MHz for ¹H and 125 MHz for ¹³C on a JEOL JMN-A 500 spectrometer. EI-mass spectra were measured with a Bruker mass spectrometer and Hewlett Packard 5989 B mass spectrometer. Column chromatography was carried on Kieselgel 60 (grain size 0.063 - 0.2 mm).

The flowers of Senna spectabilis DC. were collected in 1999 in Saraburi Province, Thailand, and identified by comparison with a herbarium specimen in the Botany Section, Department of Agriculture, Ministry of Agriculture and Cooperative, Thailand. The voucher specimen (LS-050) was deposited at the Department of Pharmacognosy, Chulalongkorn University, Thailand. The 95 % ethanolic extract (120 g) was extracted from dried powered flowers (1 kg), and treated with 5 % H₂SO₄. The acid solution was extracted with CH₂Cl₂ (3 × 1 L) to yield a non-alkaloidal portion (500 g). Then, the residual acid solution was basified with NH₄OH, and extracted with CH₂Cl₂ (3 × 500 mL) to obtain an alkaloidal extract (12 g).

The alkaloidal extract was subjected to silica gel column chromatography (10 × 30 cm) and eluted with a gradient of CH₂Cl₂ with increasing amounts of MeOH to obtain 6 fractions (F1 - F6, each Fr 1000 mL). F2 (550 mg) was chromatographed on a silica gel column (4.5 × 15 cm), CH₂Cl₂/MeOH, 95 : 5, each Fr 25 mL; Fr 15 yielded 3 (50 mg). F3 (970 mg) was submitted to a silica gel column (2 × 20cm), CH₂Cl₂/MeOH, 50 : 1, each Fr 15 mL; Fr 10 yielded 4 (30 mg) and Fr 15 was further subjected to preparative TLC (silica gel, n-hexane/EtOAc/MeOH, 20 : 20 : 1) to obtain 5 (20 mg). Compound 1 (300 mg) was purified from F4 (2.5 g) on a silica gel column (4.5 × 25 cm), CH₂Cl₂/MeOH/NH₄OH, 20 : 1:0.5, each Fr 50 mL; Frs 16 - 17. Compound 2 (60 mg) was prepared from 50 mg of 1 by acetylation as described in [7]. The R_f values of 1 - 5 on TLC (SiO₂ GF₂₅₄, CHCl₃/MeOH/NH₃, 20 : 1:0.5; Dragendorff reagent) were 0.66, 0.76, 0.90, 0.80 and 0.73, respectively.

3(R)-Benzoyloxy-2(R)-methyl-6(R)-(11′-oxododecyl)-piperidine (3): C₂₅H₃₉NO₃; pale yellow oil; [α]_D ²⁵: + 2.64° (c 0.46, EtOH); UV (MeOH): λ_max (log ε) = 204.5 (5.2), 229.5 (5.4), 273.5 (4.5) nm; IR (KBr): ν_max = 2927, 2854, 1717, 1465, 1451, 1368, 1315, 1273, 1176, 1160, 1114, 1109, 1070, 1026, 714 cm^-1; EI-MS: m/z (rel. int.) = 402 ([M⁺ + 1], 37), 401 ([M⁺], 3), 358 (1), 344 (5), 308 (11), 280 (100), 279 (30), 218 (58), 96 (56).

5-Hydroxy-2-methyl-6-(11′-oxododecyl)-pyridine (4): C₁₈H₂₉NO₂; pale oil; UV (MeOH): λ_max (log ε) = 224.5 (3.9), 288.5 (3.8) nm; IR (KBr): ν_max = 2926, 2854, 1718, 1710, 1579, 1491, 1466, 1460, 1439, 1420, 1364, 1278, 1163, 1135, 1123 cm^-1; EI-MS: m/z (rel. int.) = 291 ([M⁺], 3), 248 (4), 234 (13), 220 (1), 206 (2), 192 (3), 178 (4), 164 (1), 150 (10), 136 (17), 123 (100).

5-Hydroxy-2-methyl-6-(11′-oxododecyl)-pyridine N-oxide (5): C₁₈H₂₉NO₃; white crystals; m. p. 71 - 72 °C; UV (MeOH): λ_max (log ε) = 222 (4), 260.5 (3.6), 303(3.2) nm; IR (KBr): ν_max = 2927, 2854, 1714, 1575, 1453, 1431, 1405, 1302, 1202, 1164, 1106, 1061, 423 cm^-1; EI-MS: m/z (rel. int.) = 307 ([M⁺], 3), 291 (6), 290 (14), 248 (5), 234 (14), 220 (3), 206 (2), 192 (11), 178 (5), 164 (6), 150 (12), 136 (21), 123 (100).

The brine shrimp lethality bioassay was performed according to Meyer et al. [8]. Cytotoxic activity was assessed out by the method of Skehan et al. [9].

References

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• 2 Christofidis I, Walter A, Jadot J. Spectalinine and iso-6-carnavaline, two unprecedented piperidine alkaloids from the seeds of Cassia spectabilis .  Tetrahedron. 1977;  33 3005-6
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• 3 Freyer A J, Patil A D, Killlmer L, Troupe N, Mentzer M, Carte B, Faucette L, Johnson R K. Three new pseudodistomin, piperidine alkaloids from ascidian Pseudodistoma megalarva .  Journal of Natural Products. 1997;  60 986-90
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• 5 Rice W Y, Coke J L. Structure and configuration of alkaloids. II. Cassine.  Journal of Organic Chemistry. 1966;  31 1010-2
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• 6 Premaratne bandara K AN, Kumar V, Jacobson U, Molleyers L P. Insecticidal piperidine alkaloid from Microcos paniculata stem bark.  Phytochemistry. 2000;  54 29-32
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• 7 Pavia D L, Lampman G M, Kriz G S. Introduction to organic laboratory techniques. Philadelphia; W.B. Saunders company 1976: pp 25-30
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• 8 Meyer B N, Ferrigni N R, Putnam J E, Jacobsen L B, McLaughlin J L, et al. Brine shrimp: convenient general bioassay for active plant constituents.  Planta Medica. 1982;  45 31-4
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• 9 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer drug screening.  Journal of the National Cancer Institute. 1990;  82 1107-12
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Associate Professor Lawan Sriphong

Department of Pharmaceutical Chemistry

Faculty of Pharmacy

Silpakorn University

Nakorn-Pathom 73000

Thailand

Phone: +66-34-255800

Fax: +66-34-255801

Email: lawan@email.pharm.su.ac.th

References

• 1 Chritofidis I, Watter A, Jadot J. Spectaline and iso-6-cassine, two new piperidin-3-ol alkaloids from the leaves of Cassia spectabilis .  Tetrahedron. 1977;  33 377-9
  PubMedSearch in Google Scholar
• 2 Christofidis I, Walter A, Jadot J. Spectalinine and iso-6-carnavaline, two unprecedented piperidine alkaloids from the seeds of Cassia spectabilis .  Tetrahedron. 1977;  33 3005-6
  CrossrefPubMedSearch in Google Scholar
• 3 Freyer A J, Patil A D, Killlmer L, Troupe N, Mentzer M, Carte B, Faucette L, Johnson R K. Three new pseudodistomin, piperidine alkaloids from ascidian Pseudodistoma megalarva .  Journal of Natural Products. 1997;  60 986-90
  CrossrefPubMedSearch in Google Scholar
• 4 Charan R D, Garson M J, Brereton I M, Willis A C, Hooper J NA. Haliclonacyclamines A and B, cytotoxic alkaloids from the tropical marine sponge Haliclona sp.  Tetrahedron. 1996;  52 9111-20
  CrossrefPubMedSearch in Google Scholar
• 5 Rice W Y, Coke J L. Structure and configuration of alkaloids. II. Cassine.  Journal of Organic Chemistry. 1966;  31 1010-2
  PubMedSearch in Google Scholar
• 6 Premaratne bandara K AN, Kumar V, Jacobson U, Molleyers L P. Insecticidal piperidine alkaloid from Microcos paniculata stem bark.  Phytochemistry. 2000;  54 29-32
  CrossrefPubMedSearch in Google Scholar
• 7 Pavia D L, Lampman G M, Kriz G S. Introduction to organic laboratory techniques. Philadelphia; W.B. Saunders company 1976: pp 25-30
  Search in Google Scholar
• 8 Meyer B N, Ferrigni N R, Putnam J E, Jacobsen L B, McLaughlin J L, et al. Brine shrimp: convenient general bioassay for active plant constituents.  Planta Medica. 1982;  45 31-4
  PubMedSearch in Google Scholar
• 9 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer drug screening.  Journal of the National Cancer Institute. 1990;  82 1107-12
  PubMedSearch in Google Scholar

Associate Professor Lawan Sriphong

Department of Pharmaceutical Chemistry

Faculty of Pharmacy

Silpakorn University

Nakorn-Pathom 73000

Thailand

Phone: +66-34-255800

Fax: +66-34-255801

Email: lawan@email.pharm.su.ac.th