Cytotoxic Phenanthrenes from the Rhizomes of Tamus communis

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

From the fresh rhizomes of Tamus communis five phenanthrenes (1 - 5) were isolated under the guidance of cytotoxic assays in HeLa cells. The compounds were obtained from the highly active CHCl₃ fraction of the MeOH extract by using multistep chromatographic purifications, including VLC, preparative TLC, HPLC and gel filtration. The compounds were identified by means of EI-mass, UV and NMR spectroscopy as 7-hydroxy-2,3,4-trimethoxyphenanthrene (1), 2,7-dihydroxy-3,4-dimethoxyphenanthrene (nudol) (2), 2,7-dihydroxy-3,4,8-trimethoxyphenanthrene (3), 3,7-dihydroxy-2,4,8-trimethoxyphenanthrene (confusarin) (4), and 3,7-dihydroxy-2,4-dimethoxyphenanthrene (5). Compound 1 is a new natural product, and 2 - 4 were isolated for the first time from T. communis. In the cytotoxic assays, compounds 1 - 3 and 5 significantly inhibited the growth of HeLa cells (IC₅₀ = 0.97 - 20.18 μM). Compound 3, with an IC₅₀ value of 0.97 μM, is of special interest because of its high activity.

In recent years, the cis-stilbene combretastatins have attracted great interest because of their potential use in cancer chemotherapy. The most potent member, combretastatin A-4 in sodium phosphate prodrug (CA4P) form, is currently undergoing testing in Phase II clinical trials, and has been found to be effective against different solid tumours, including multidrug-resistant cancers [1]. The aim of the present work was to perform an antitumour evaluation of structurally related compounds: phenanthrenes, which are the conformationally restricted congeners of cis-stilbenes. Previous phytochemical examinations of Tamus communis L. (Dioscoreaceae) have demonstrated the presence of alkoxy-substituted phenanthrenes [2], [3], [4], [5], [6], but no data have been reported on their antitumour potency. The present paper deals with the bioguided isolation, structure elucidation and cytotoxic activity of phenanthrenes 1 - 5 from the fresh rhizomes of T. communis.[*]

Compound 1, obtained as yellowish-white crystals, possessed the molecular formula C₁₇H₁₆O₄, as confirmed by the HR-EI-MS. Its UV spectrum [λ_max (log ε) = 259 (3.94), 283 (3.24), 292 (3.13), 303 (2.92) nm] was characteristic of a phenanthrene derivative [7], [8]. The ¹H-NMR spectrum showed resonances for three methoxy groups [δ = 4.01 s (6H, s), 4.03 (3H, s)] and six aromatic protons. One set of aromatic protons comprised an ABX spin system [δ = 9.39 (1H, d, J = 9.2 Hz), 7.19 (1H, dd, J = 9.2, 2.6 Hz), 7.22 (1H, d, J = 2.6 Hz)]. The most deshielded aromatic proton signal (δ = 9.39) of this system was typical for H-5 of a phenanthrene [7], [8], and therefore a 7-substituted ring C was concluded. The signals of a pair of ortho-coupled aromatic protons [δ = 7.56, 7.52 (each 1H, each d, J = 8.8 Hz)] were attributed on the basis of literature values to H-9 and H-10 [5], [7], [8]. Moreover, an isolated aromatic proton at δ = 7.07 (1H, s) indicated a trisubstituted ring A. The substitution pattern of 1 was further studied in a NOESY experiment. Overhauser effects were detected between the signals of H-1 and H-10; H-10 and H-9; H-9 and H-8; and H-5 and H-6, demonstrating oxygen functionalities at C-2, C-3, C-4 and C-7. Besides the three methoxy groups, the presence of one hydroxy group was deduced from the molecular composition. The location of two methoxy groups at C-2 and C-4 was evident from the NOESY cross-peaks observed between the methoxy signals and the signals of H-1 and H-5. The third methoxy group was placed at C-3, and the hydroxy group at C-7, with regard to the absence of diagnostic NOEs between the methoxy group and H-6 and H-8. Furthermore, the chemical shifts of the aromatic protons were in good agreement with those published for phenanthrenes having the same functionalities on ring A or C [8], [9]. On the basis of these spectral data, the structure of 1 was elucidated as 7-hydroxy-2,3,4-trimethoxyphenanthrene. This compound is a new natural product.

By comparison of their physical and spectral data (UV, MS, NMR and NOESY) with those reported in the literature, compounds 2 - 4 were identified as nudol (2) [7], [9], [10], confusarin (3) [11], [12], and 3,7-dihydroxy-2,4,8-trimethoxyphenanthrene (4) [7], [13]. These compounds were previously described from different Orchidaceae species, but have been obtained for the first time from T. communis. Compound 5 proved to be identical with 3,7-dihydroxy-2,4-dimethoxyphenanthrene (= ”TaVIII”), isolated earlier from the rhizomes of this plant [2], [14].

The cytotoxic assays of the isolated phenanthrenes in HeLa cells, using the MTT test, revealed that compounds 1 - 3 and 5 possess marked cell growth inhibitory activity if compared with those of the positive controls cisplatin and doxorubicin (Table [1]). Primarily 2,7-dihydroxy-3,4,8-trimethoxyphenanthrene (3) is worthy of interest because of its high activity (IC₅₀ = 0.97 μM). Similarly, as in the case of the chemically closely related combretastatins [15], [16], the different cytotoxicities of compounds 1 - 5 demonstrated that the positions of the hydroxy and methoxy groups on the carbon skeleton are crucial for inhibition of cancer cell growth.

A large variety of combretastatin analogues have been investigated in order to elucidate the structure-activity relationship. It is generally accepted that a diaryl system linked by a carbon-carbon double bond is essential for the cytostatic effect together with the three methoxy groups on one of the rings [17]. However, the two most active members of the currently tested phenanthrenes, compounds 3 and 5, have only two methoxy groups on one of the rings. Moreover, the substance closest to combretastatin regarding the three methoxy groups, compound 1, has only a limited cytostatic effect. These findings do not fit into the previously published structure-activity relationship as concerns combretastatin analogues [17]. On the other hand, the olefinic part can be considered as an additional target for modification of the chemical structure. Many conformationally restricted analogues, including sulfonate and azetidinone derivatives of combretastatin, have clearly revealed that inhibition of the rotation favours antiproliferative action [18], [19]. Accordingly, it is conceivable that the structure-activity relationship observed for the substituted stilbenes is not directly applicable to the conformationally constrained phenanthrene skeleton.

Materials and Methods

The rhizomes of Tamus communis L. were collected in the Mecsek Hills (Hungary) in June 2003. A voucher specimen (No. 619) has been deposited at the Herbarium of the Department of Pharmacognosy, University of Szeged, Szeged, Hungary.

Chemicals used in the isolation protocol were purchased from Molar Chemicals Kft (Budapest, Hungary) except for petroleum ether and diethyl ether (Merck; Darmstadt, Germany). Resins for chromatography were from Merck (Darmstadt, Germany) (TLC Silica gel and LiChrospher) and from Pharmacia (Upsala, Sweden) (Sephadex LH 20).

The fresh rhizomes of the plant material (1.2 kg) were crushed and percolated with MeOH (10 L). The MeOH extract was concentrated to 400 mL and extracted with petroleum ether (5 × 400 mL) and CHCl₃ (5 × 400 mL). The CHCl₃ fraction (2.03 g) was chromatographed by vacuum liquid chromatography (VLC) on silica gel (GF₂₅₄ 15 μm), using a gradient system of cyclohexane-EtOAc-EtOH (9 : 1:0, 8 : 2:0, 7 : 3:0, 70 : 30 : 1, 70 : 30 : 2, 70 : 30 : 5, 50 : 50 : 10, 15 × 20 mL of each), yielding thirteen main fractions (I - XIII) after combinations. Fraction IV was fractionated by gel chromatography on Sephadex LH-20 with MeOH, and then further chomatographed by HPLC on a LiChrospher RP-18 (5 μm, 250 × 4 mm) column, using MeOH-H₂O (7 : 3), with detection at 250 nm, flow rate 2 mL/min. In this way, compounds 1 (t_R = 10.65 min, 6.0 mg) and 3 (t_R = 25.52 min, 7.4 mg) were isolated. Fraction V was separated by VLC on silica gel, using mixtures of n-hexane-CHCl₃-MeOH with increasing polarity. The subfractions containing the main component were subjected to TLC with benzene-diethyl ether-petroleum ether (2 : 1:1) as solvent system (R_f = 0.50), and finally purified by RP-HPLC under the above conditions, affording compound 2 (t_R = 25.97 min, 0.0015 g). Fractions VII and VIII were processed in the same manner, first by VLC on silica gel, using CHCl₃, CHCl₃-MeOH (99 : 1 and 98 : 2) and MeOH as eluents (30 mL of each), and then by TLC on silica gel, with the use of CHCl₃-Me₂CO (19 : 1) as mobile phase. These separations yielded compounds 4 (R_f = 0.53, 5.2 mg) and 5 (R_f = 0.37, 20.6 mg). Upon standing, β-sitosterine crystallized from fraction III.

7-Hydroxy-2,3,4-trimethoxyphenanthrene (1): Yellowish-white crystals; m. p. 185 - 187 °C; UV (MeOH): see text; HR-EI-MS: m/z 284.10505 [M]⁺ (calcd. for C₁₇H₁₆O₄ : 284.10486); ¹H-NMR (500 MHz, CDCl₃): see text.

2,7-Dihydroxy-3,4-dimethoxyphenanthrene ( = nudol) (2): Yellowish-white amorphous solid; UV (MeOH): λ_max (log ε) = 259 (3.89), 283 (3.28), 292 (3.19), 303 (2.96), 347 (2.48), 364 nm (2.48); ¹H-NMR (500 MHz, CDCl₃): δ = 7.17 (1H, s, H-1), 9.36 (1H, d, J = 9.2 Hz, H-5), 7.17 (1H, dd, J = 9.1, 2.8 Hz, H-6), 7.21 (1H, d, J = 2.8 Hz, H-8), 7.50 (1H, d, J = 8.7 Hz, H-9), 7.53 (1H, d, J = 8.7 Hz, H-10), 4.11 (3H, s, OCH₃), 3.98 (3H, s, OCH₃).

3,7-Dihydroxy-2,4,8-trimethoxyphenanthrene (4): Yellowish white crystals; UV (MeOH): λ_max (log ε) = 263 (3.96), 282 (3.30), 299 (3.41), 312 (3.08), 347 (2.54), 363 nm (2.58); ¹H-NMR (500 MHz, CDCl₃): δ = 7.07 (1H, s, H-1), 9.18 (1H, d, J = 9.3 Hz, H-5), 7.33 (1H, d, J = 9.3 Hz, H-6), 7.84 (1H, d, J = 8.9 Hz, H-9), 7.62 (1H, d, J = 8.9 Hz, H-10), 6.00, 6.14 (each 2H, each brs, 2 × OH), 3.95, 3.98, 4.02, (each 3H, each s, 3 × OCH₃); ¹³C-NMR (125 MHz, CDCl₃): δ = 104.9 (C-1), 146.8, 145.5 (C-2, C-4), 140.8, 139.3 (C-3, C-8), 119.2 (C-4a), 125.7 (C-4b), 117.9 (C-5), 116.1 (C-6), 144.0 (C-7), 124.2 (C-8a), 123.9 (C-9), 127.4 (C-10), 126.6 (C-10a), 56.1, 59.7, 61.8 (3 × OCH₃); assignments were made by comparison with previously assigned spectra [5], [10], [20].

Cytotoxic assay: Cytotoxic effects were measured in vitro in a HeLa (cervix adenocarcinoma) cell line (ECACC; Salisbury, UK), using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Human cancer cells (5000/well) were seeded into a 96-well microplate. During an overnight preincubation, the cells attached to the bottom of the well. On the second day of the procedure, the original medium was removed and 200 μL new medium containing the test substances were added. The tested extracts and compounds were dissolved in DMSO. After an incubation period of 72 hours, living cells were assayed by the addition of 20 μL 5 mg/mL MTT solution (Sigma-Aldrich; Budapest, Hungary). MTT was converted by intact mitochondrial reductase and precipitated as blue crystals during a 4-hour contact period. The medium was then removed and the precipitated crystals were dissolved in 100 μL DMSO during a 60-minute period of shaking. Finally, the reduced MTT was assayed at 545 nm by using a microplate reader. All in vitro experiments were carried out on two microplates with at least 5 parallel wells.

Acknowledgements

The authors thank Dr. Pál Szabó (Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary) for mass spectroscopic measurements. The authors are grateful to Prof. László Gy. Szabó (Department of Botany, Institute of Biology, University of Pécs, Pécs, Hungary) for the identification and collection of the plant material. Financial support of the Council for Health Research (ETT-382/2003) is gratefully acknowledged.

References

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Prof. Dr. Judit Hohmann

Department of Pharmacognosy

University of Szeged

Eötvös u. 6

6720 Szeged

Hungary

Phone: +36-62-546-453

Fax: +36-62-545-704

Email: hohmann@pharma.szote.u-szeged.hu

References

• 1 Cirla A, Mann J. Combretastatins: from natural products to drug discovery.  Nat Prod Rep. 2003;  20 558-64
  CrossrefPubMedSearch in Google Scholar
• 2 Reisch J, Báthory M, Szendrei K, Novák I, Minker E. Weitere Phenanthrene aus dem Rhizom von Tamus communis .  Phytochemistry. 1973;  12 228-9
  CrossrefPubMedSearch in Google Scholar
• 3 Reisch J, Báthory M, Novák I, Szendrei K. Természetes fenantrén vegyületek szerkezete és szintézise.  Herba Hung. 1970;  9 43-8
  PubMedSearch in Google Scholar
• 4 Reisch J, Báthory M, Novák I, Szendrei K, Minker E. Stickstofffreie Phenathren-Derivate als Pflanzeninhaltsstoffe.  Herba Hung. 1972;  11 61-71
  PubMedSearch in Google Scholar
• 5 Aquino R, Behar I, De Simone F, Pizza C, Senatore F. Phenanthrene derivatives from Tamus communis .  Biochem Syst Ecol. 1985;  13 251-2
  PubMedSearch in Google Scholar
• 6 Aquino R, Behar I, De Simone F, Pizza C. Natural dihydrophenanthrene derivatives from Tamus communis .  J Nat Prod. 1985;  48 811-3
  CrossrefPubMedSearch in Google Scholar
• 7 Tuchinda P, Udchachon J, Khumtaveeporn K, Taylor W C, Engelhardt L M, White A H. Phenanthrenes of Eulophia nuda .  Phytochemistry. 1988;  27 3267-71
  CrossrefPubMedSearch in Google Scholar
• 8 Leong Y W, Harrison L J, Powell A D. Phenanthrene and other aromatic constituents of Bulbophyllum vaginatum .  Phytochemistry. 1999;  50 1237-41
  CrossrefPubMedSearch in Google Scholar
• 9 Bhandari S R, Kapadi A H, Mujumder P L, Joardar M, Shoolery J N. Nudol, a phenanthrene of the orchids Eulophia nuda, Eria carinata and Eria stricta .  Phytochemistry. 1985;  24 801-4
  CrossrefPubMedSearch in Google Scholar
• 10 Leong Y W, Kang C C, Harrison L J, Powell A D. Phenanthrenes, dihydrophenanthrenes and bibenzyls from the orchid Bulbophyllum vaginatum .  Phytochemistry. 1997;  44 157-65
  CrossrefPubMedSearch in Google Scholar
• 11 Majumder P L, Kar A. Confusarin and confusaridin, two phenanthrene derivatives of the orchid Eria confusa .  Phytochemistry. 1987;  26 1127-9
  CrossrefPubMedSearch in Google Scholar
• 12 Majumder P L, Pal S, Majumder S. Dimeric phenanthrenes from the orchid Bulbophyllum reptans .  Phytochemistry. 1999;  50 891-7
  CrossrefPubMedSearch in Google Scholar
• 13 Estrada S, Rojas A, Mathison Y, Israel A, Mata R. Nitric oxide cGMP mediates the spasmolytic action of 3,4′-dihydroxy-5,5′-dimethoxybibenzyl from Scaphyglottis livida .  Planta Med. 1999;  65 109-14
  Thieme ConnectPubMedSearch in Google Scholar
• 14 Letcher R M, Wong K M. Structure and synthesis of the phenanthrenes TaIV and TaVIII from Tamus communis . J Chem Soc Perkin Trans 1 1979: 2449-50
  Search in Google Scholar
• 15 Lin C M, Singh S B, Chu P S, Dempcy R O, Schmidt J M, Pettit G R. et al . Interactions of tubulin with potent natural and synthetic analogs of the antimitotic agent combretastatin - a structure-activity study.  Mol Pharmacol. 1988;  34 200-8
  PubMedSearch in Google Scholar
• 16 Cushman M, Nagarathnam D, Gopal D, He H M, Lin C M, Hamel E. Synthesis and evaluation of analogs of (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene as potential cytotoxic and antimitotic agents.  J Med Chem. 1992;  35 2293-306
  CrossrefPubMedSearch in Google Scholar
• 17 Srivastava V, Negi A S, Kumar J K, Gupta M M, Khanuja S PS. Plant-based anticancer molecules: a chemical and biological profile of some important leads.  Bioorg Med Chem. 2005;  13 5892-908
  CrossrefPubMedSearch in Google Scholar
• 18 Gwaltney SL I I, Imade H M, Barr K J, Li Q, Gehrke L, Credo R B. et al . Novel sulfonate analogues of combretastatin A-4: potent antimitotic agents.  Bioorg Med Chem Lett. 2001;  11 871-4
  CrossrefPubMedSearch in Google Scholar
• 19 Sun L, Vasilevich N I, Fuselier J A, Hocart S J, Coy D H. Examination of the 1,4-disubstituted azetidinone ring system as a template for combretastatin A-4 conformationally restricted analogue design.  Bioorg Med Chem Lett. 2004;  14 2041-6
  CrossrefPubMedSearch in Google Scholar
• 20 Pettit G R, Singh S B, Niven M L, Schmidt J M. Cell-growth inhibitory dihydrophenanthrene and phenanthrene constituents of the African tree Combretum caffrum .  Can J Chem. 1988;  66 406-13
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Judit Hohmann

Department of Pharmacognosy

University of Szeged

Eötvös u. 6

6720 Szeged

Hungary

Phone: +36-62-546-453

Fax: +36-62-545-704

Email: hohmann@pharma.szote.u-szeged.hu

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