Cytotoxic Arylnaphthalide Lignan Glycosides from the Aerial Parts of Phyllanthus taxodiifolius

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The arylnaphthalide lignan glycosides, taxodiifoloside (1), cleistanthoside A (2), cleistanthin A (3) and cleistanthin A methyl ether (4), together with a triterpene, glochidone (5), have been isolated from the aerial parts of Phyllanthus taxodiifolius. The structures were established using spectral and chemical methods. Compounds 3 and 4, as well as the derivatives 2a and 3a exhibited potent cytotoxic activities with GI₅₀ values in the range of 10^-7 - 10^-9 M in five cultured mammalian cancer cell lines while the new compound 1 showed moderate activity (GI₅₀ in the order of 10^-6 M). Compounds 2 and 5 were inactive in all tested cell lines.

Phyllanthus taxodiifolius Beille (Euphorbiaceae), a shrub found in the central and Northeastern parts of Thailand [1], is used in Thai traditional medicine as a diuretic. In our ongoing search for anticancer agents from plants, an investigation of the cytotoxic EtOAc fraction obtained from the partitioning of the crude MeOH extract of the aerial parts of this plant has led to the isolation of a new arylnaphthalide lignan glycoside, taxodiifoloside (1), along with three known arylnaphthalide lignan glycosides, cleistanthoside A (2) [2], cleistanthin A (3) [3], [4], [5], cleistanthin A methyl ether (4) [3], [4] and a known triterpene, glochidone (5) [6], [7]. Compound 4, previously prepared from natural 3, was isolated for the first time as a natural product. NMR assignments of 1 are reported (Table [1]) while the revised data of other glycosides are included in the Supporting Information [8]. Both natural and modified compounds 1, 2, 2a, 3a, 4 and 5 were evaluated for cytotoxic effects against five cultured mammalian cancer cell lines for the first time.[]

Taxodiifoloside (1) (C₃₆H₄₀O₁₇) displayed typical ¹H-NMR signals of a substituted arylnaphthalene nucleus and a disaccharide portion (Table [1]). The doubling of some signals, due to restricted rotation around the aryl-naphthalene bond [9], is indicated by an asterik (*). The presence of signals corresponding to two aromatic protons, a 1,3,4-trisubstituted phenyl moiety, a lactone methylene, a methylenedioxy group and two aromatic methoxy groups suggested that 1 was a diphyllin analogue. The sugars in the disaccharide unit were identified as 3,4-di-O-methyl-β-D-xylopyranose and 6-acetoxy-β-D-glucopyranose by analyses of the J values, ¹³C-NMR (Table [1]) and 2D-NMR data (Table [1] in Supporting Information). The locations of the methoxy groups at C-3′′, C-4′′ and the acetoxy group at C-6′′′ were confirmed with HMBC correlations. The connectivities of the two sugars and to the diphyllin moiety were deduced from the NOESY and HMBC correlations. Therefore, the structure of 1 was established as 4-O-[6-acetoxy-β-D-glucopyranosyl(1→2)-3,4-di-O-methyl-β-D-xylopyranosyl]diphyllin. The stereochemistry within the sugars was confirmed by conversion to the tetraacetate 2a, which was found identical to the compound derived from acetylation of 2.

Cleistanthoside A (2), previously isolated from Cleistanthus patulus [2], was characterized as its tetraacetate 2a. Cleistanthin A (3) [10], [11] was identical to that reported from Cleistanthus collinus [3], [4], [5]. The preparation of acetate 3a [3], [4] further confirmed the structure 3 and provided material for biological evaluation. Methylation of 3 gave 4, which was identical to natural 4. Glochidone (5) was identified by comparison of the physical and spectral data to those reported for the substance isolated from Glochidion hohenackeri [6] and Glochidion heyneanum [7].

The MeOH extract, fractions and pure compounds were screened for cytotoxicity against five cultured mammalian cancer cell lines according to an established protocol [12]. Ellipticine was used as a positive control (Table 2 in Supporting Information). Compounds 2a, 3, 3a and 4 exhibited potent activities with GI₅₀ values in the range of 10^-7 - 10^-9 M. Compound 1 was less active with a GI₅₀ value in the order of 10^-6 M. Compounds 2 and 5 were inactive in all tested cell lines. It is noteworthy that 2a was very active (3.4 nM in P-388, KB and Col-2, 4.6 nM in MCF-7 and 46.0 nM in Lu-1) while its precursor 2 was inactive. The cytotoxic values of 3a are comparable to those of 3 and 4. Overall, the present work represents a significant contribution to the chemical and biological aspects of arylnaphthalide lignan glycosides.

Materials and Methods

Melting points (°C): uncorrected; NMR spectra: Bruker AV 500 spectrometer; HR-TOF-MS: Micromass model VQ-Tof2. CC: silica gel 60 (63 - 200 μm); PLC silica gel 60 PF₂₅₄ (5 - 40 µm, 0.5 mm). Plant materials: collected in January 2002 from Amnartchareon, Thailand. Voucher specimen (BKF no. 127 614): deposited at the Forest Herbarium in Bangkok.

Dried, powdered aerial parts (14.3 kg) were extracted with MeOH (6 × 33 L) to give a crude MeOH extract (835 g). After suspension in H₂O (2.5 L), it was sequentially partitioned with hexane (5 × 4 L), EtOAc (5 × 4 L) and n-BuOH (4 × 3 L), Removal of solvents yielded the hexane, EtOAc, n-BuOH and H₂O fractions in 230, 159, 140 and 283 g, respectively. The cytotoxic EtOAc fraction (158 g) was subjected to CC (SiO₂, 1.5 kg), eluting with CH₂Cl₂-hexane (0, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 %, 4 L each), followed by MeOH-CH₂Cl₂ (1, 2, 3, 5, 7, 10, 15, 20, 30, 50, 70 and 100 %, 6 L each). Frs. (500 mL each) were combined on the basis of TLC behaviour to give frs. A1 - A12. Fr. A4 (14.0 g, eluted with 30 - 50 % CH₂Cl₂-hexane), after recrystallization gave 5 {8.8 g, colourless needles, m. p. 166.0 - 166.6 °C (CH₂Cl₂ - MeOH), [α]_{58 9} ³⁰: + 72.38° (c 0.1 CHCl₃)}.

Purification of fr. A9 (22.2 g, eluted with 2 - 5 % MeOH-CH₂Cl₂) by CC (SiO₂, 230 g) afforded frs. B1 - B3. Fr. B1 (13.0 g, eluted with 0 - 30 % Me₂CO-hexane) was separated by CC (SiO₂, 325 g) to give frs. C1 - C5. Further separation of fr. C1 (2.6 g, eluted with 0 - 15 % Me₂CO-hexane) by CC (SiO₂, 60 g), yielded frs. D1 - D8. Fr. D7 (173.2 mg, eluted with 40 - 50 % EtOAc-hexane) afforded 4 {11.3 mg, white powder, m. p. 211 - 215 °C (EtOH), [α]_{58 9} ²⁷: -50° (c 1.0, CHCl₃)} after PLC (40 % EtOAc-hexane as eluent).

Fr. B2 (7.0 g, eluted with 30 - 35 % Me₂CO-hexane) yielded 3 {2.9 g, white powder, m. p. 140.0 - 141.3 °C (EtOH), [α]_{58 9} ²⁷: -69.6° (c 1.02, CHCl₃)} after recrystallization.

Fr. A10 (13.5 g, eluted with 5 - 7 % MeOH-CH₂Cl₂) was purified by CC (SiO₂, 170 g) to provide frs. E1 - E3. Separation of Fr. E2 (4.7 g, eluted with 1 - 15 % MeOH-CH₂Cl₂) by CC (SiO₂, 226 g) gave frs. F1 - F7. Fr. F4 (1.5 g, eluted with 6 % MeOH-CH₂Cl₂) was separated by CC (Sephadex LH20, 4.8 g, MeOH as eluent) to afford frs. G1 - G4. Further separation of fr. G2 (834.5 mg) by CC (SiO₂, 20 g) gave frs. H1 and H2. Fr. H2 (402.3 mg, eluted with 60 - 100 % EtOAc-hexane), after PLC (SiO₂, 2 % MeOH-EtOAc, 3 elutions) and recrystallization (EtOAc-hexane), afforded 1 (27.2 mg).

Fr. F5 (410 mg, eluted with 6 - 20 % MeOH-CH₂Cl₂) was further purified by CC (SiO₂, 26 g) to give frs. I1 - I4. Fr. I4 (60.3 mg, eluted with 1 - 20 % Me₂CO-MeOH), after recrystallization, afforded 2 {14.7 mg, white powder, m. p. 237 - 240 °C (EtOH), [α]_{58 9} ²⁶: -5.7^o (MeOH, c 1.0)}.

Fr. A11 (25.9 g, eluted with 7 - 10 % MeOH-CH₂Cl₂) after separation on CC (SiO₂, 500 g) yielded frs. J1 - J4. Fr. J2 (6.13 g, eluted with 4 - 8 % MeOH-CH₂Cl₂) was separated by CC (SiO₂, 200 g) to provide frs. K1 - K3. Separation of fr. K2 (1.95 g, eluted with 1 - 20 % MeOH-CH₂Cl₂) by CC (SiO₂, 62 g) gave frs. L1 - L3. Fr. L2 (336.5 mg, eluted with 6 - 9 % MeOH-CH₂Cl₂) was separated by PLC (SiO₂, 0.5 % MeOH-EtOAc, 3 elutions) to give 2 (150.1 mg).

Fr. J3 (14.34 g, eluted with 8 - 10 % MeOH-CH₂Cl₂) was separated by CC (SiO₂, 320 g) to yield frs. M1 - M3. Fr. M3 (9.47 g, eluted with 3 % MeOH-CH₂Cl₂) crystallized in EtOH to give 2 (5.62 g).

Taxodiifoloside (1): White powder, m. p. 142.3 - 143.1 °C (EtOAc-hexane); [α]_{58 9} ²⁷: -34° (CHCl₃, c 0.5); UV (EtOH): λ_max (log ε) = 223 (3.92), 258 (4.31), 294 (3.67), 314 (3.70), 349 (3.46) nm; CD (5.38 × 10^-5 M, EtOH): λ_max (Δε) = 315 (-2. 37), 263 (-9.08) nm; IR (KBr): ν_max = 3419, 1756, 1742, 1507, 930 cm^-1; EI-MS: m/z (rel. int.) = 744 [M]⁺ (0.5), 394 (100), 203 (29), 97 (24); HR-TOF-MS (ESI positive): m/z = 767.2163 (calcd. for C₃₆H₄₀O₁₇Na: 767.2163).

Acknowledgements

We thank the Thailand Research Fund for financial support (BRG/22/2544) to P.T. and M.P. and the award of a Senior Research Scholar to V.R. We also thank the Higher Education Development Project: Postgraduate Education and Research Program in Chemistry (PERCH) for support.

References

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Dr. Patoomratana Tuchinda

Department of Chemistry

Faculty of Science

Mahidol University

Rama 6 Road

Bangkok 10400

Thailand

Fax: +66-02-3547151

Email: scptc@mahidol.ac.th

References

• 1 Smitinand T. Thai Plant Names. Revised Edition by the Forest Herbarium, Bangkok Pra Cha Chon Co Ltd 2001: p 412
  Search in Google Scholar
• 2 Sastry K V, Rao E V. Isolation and structure of cleistanthoside A.  Planta Med. 1983;  47 227-9
  PubMedSearch in Google Scholar
• 3 Govindachari T R, Sathe S S, Viswanathan N, Pai B R, Srinivasan M. Chemical constituents of Cleistanthus collinus (Roxb.)  Tetrahedron. 1969;  25 2815-21
  CrossrefPubMedSearch in Google Scholar
• 4 Anjaneyulu A SR, Atchuta Ramaiah P, Ramachandra Row L, Venkateswarlu R. New lignans from the heartwood of Cleistanthus collinus .  Tetrahedron. 1981;  37 3641-52
  CrossrefPubMedSearch in Google Scholar
• 5 Ramesh C, Ravindranath N, Ram T S, Das B. Arylnaphthalide lignans from Cleistanthus collinus .  Chem Pharm Bull. 2003;  51 1299-300
  CrossrefPubMedSearch in Google Scholar
• 6 Ganguly A K, Govindachari T R, Mohamed P A, Rahimtulla A D, Viswanathan N. Chemical constituents of Glochidion hohemnackeri .  Tetrahedron. 1966;  22 1513-9
  CrossrefPubMedSearch in Google Scholar
• 7 Srivastava R, Kulshreshtha D K. Triterpenoids from Glochidion heyneanum .  Phytochemistry. 1988;  27 3575-8
  CrossrefPubMedSearch in Google Scholar
• 8 Copies of the original spectra are obtainable from the author of correspondence . . 
• 9 Charlton J L, Oleschuk C J, Chee G -L. Hindered rotation in arylnaphthalene lignans.  J Org Chem. 1996;  61 3452-7
  CrossrefPubMedSearch in Google Scholar
• 10 Pradheepkumar C P, Shanmugam G. Anticancer potential of cleistanthin A isolated from the tropical plant Cleistanthus collinus .  Oncol Res. 1999;  11 225-32
  PubMedSearch in Google Scholar
• 11 Pradheepkumar C P, Panneerselvam N, Shanmugam G. Cleistanthin A causes DNA strand breaks and induces apoptosis in cultured cells.  Mutat Res. 2000;  464 185-93
  PubMedSearch in Google Scholar
• 12 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren J T, Bokesch H, Kenney S, Boyd M R. New colorimetric cytotoxic assay for anticancer-drug screening.  J Natl Cancer Inst. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar

Dr. Patoomratana Tuchinda

Department of Chemistry

Faculty of Science

Mahidol University

Rama 6 Road

Bangkok 10400

Thailand

Fax: +66-02-3547151

Email: scptc@mahidol.ac.th

• Supplementary Material