5-(4-Hydroxyphenethenyl)-4,7-dimethoxycoumarin, a New Constituent of Monotes engleri

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

A new coumarin, 5-(4-hydroxyphenethenyl)-4,7-dimethoxycoumarin (1) was isolated from the combined ethyl acetate extracts of the root bark, root wood and stem bark of Monotes engleri, and found to be cytotoxic against two cell lines in a human tumor panel. Its structure was determined on the basis of spectroscopic methods.

In a previous study performed as part of a collaborative search for novel antineoplastic agents from plants, the leaves of Monotes engleri Gilg (Dipterocarpaceae) were found to contain cytotoxic prenylated flavonoids [1]. Further investigation on the ethyl acetate extracts of the root bark, root wood, and stem bark of M.engleri was carried out in the present study, leading to the isolation of a new compound, 5-(4-hydroxyphenethenyl)-4,7-dimethoxycoumarin (1), which was absent from M.engleri leaves.

The molecular formula of 1 was deduced as C₁₉H₁₆O₅ from the molecular ion peak at m/z 324.1000 in its high-resolution EI mass spectrum. Compound 1 showed a strong blue color under UV (365nm) light, and the IR spectrum showed absorptions at 3370cm^–1 for a hydroxy group and 1714cm^–1 for a carbonyl functionality [2]. The ¹H- and ¹³C-NMR spectra of 1 displayed signals consistent with the presence of a coumarin skeleton at δ_H = 5.62/d_C 88.8 (C-3), 6.79/100.7 (C-8) and 7.07/111.6 (C-6), and at δ_C = 107.3 (C-10), 157.5 (C-9) and 162.2 (C-2) [3] [4] [5] [6]. In addition, signals for two methoxy groups, one para-substituted phenyl ring and two olefinic CH functionalities were observed. The relative positions of these functionalities were deduced by the selective- INEPT NMR experiment ([Fig. 1]) [7]. All NMR assignments for 1 were made unambiguously by appropriate 1D-NOE difference, ¹H-¹³C HETCOR and selective-INEPT NMR experiments. Thus, the new isolate 1 was assigned structurally as 5-(4- hydroxyphenethenyl)-4,7-dimethoxycoumarin.

Compound 1 exhibited partial selective cytotoxicity against fibrosarcoma (HT-1080: EC₅₀ 5.3 mg/ml) and hormone-dependent prostate cancer (LNCaP: EC₅₀ 1.2 mg/ml) cell lines, and was inactive against all other cell lines represented in the tumor panel [8].

Materials and Methods

The melting point was obtained with a Fisher-Johns melting point apparatus and is uncorrected. The UV spectrum was recorded on a Beckman model DU-7 spectrometer, and the IR spectrum was measured on a Midac Collegian FT-IR spectrometer. ¹H- and ¹³C-NMR spectra were obtained using a Varian XL-300 (300MHz and 75MHz, respectively) NMR spectrometer. 1D-NOE and ¹H-¹³C HETCOR NMR experiments were also performed on the Varian XL-300 instrument, and the selective-INEPT NMR experiments were run on a Nicolet NMC-360 NMR spectrometer. The LREIMS was obtained on a Finnigan MAT 90 instrument operating at 70eV. HRFABMS was obtained using a Kratos MS-50 spectrometer.

The root bark, root wood and stem bark of M.engleri were collected in a tropical rain forest at Chinhoi, Zimbabwe, in December 1991, and identified by one of the authors (T.E.C.). Voucher specimens (A861, A859, A860) of the root bark, root wood and stem bark have been deposited in the Field Museum of Natural History, Chicago, Illinois, U.S.A.

The EtOAc extracts of the root bark (180g), root wood (330g) and stem bark (160g) of M.engleri were combined due to their similar TLC profiles. The combined ethyl acetate extract (6g) was separated by silica gel column chromatography (silica gel, 300 g; 70–230mesh) using gradient mixtures of CHCl₃-MeOH (500 : 1 → 1 : 1) as eluents to afford fractions F003-F008.Fraction F006 (400mg) eluted with CHCl₃-MeOH (100 : 1) was further separated by preparative TLC (0.5 mm thick) using CHCl₃-MeOH (100 : 1) as solvent system, affording pure compound 1 (8 mg, 0.001 % w/w of the combined root bark, root wood and stem bark, Rf 0.30). Compound 1 was detected as a strong blue fluorescent spot in long-wave (365 nm) UV light, and did not require the use of a chromogenic visualization reagent. The compound did not exhibit any color in visible light after spraying with H₂SO₄.

5-(4-Hydroxyphenethenyl)-4,7-dimethoxycoumarin (1): Light yellow powder; m. p. 237–239 °C (dec). UV (CHCl₃): λ_max =242 (4.1), 285 (4.2), 315 (4.3), 325 (4.3) nm; IR (film): ν_max =3370, 2922, 2851, 2311, 1714, 1690, 1601, 1514, 1445, 1379, 1250, 1155, 1065, 970, 812cm^–1. EIMS: m/z (%) = 324 (100) [M]⁺, 281 (31), 231 (8); HREIMS: m/ z = 324.1000 [M] ⁺ calcd. 340.0998 for C₁₉H₁₆O₅. ¹H-NMR (acetone-d ₆, 300MHz): δ = 7.88 (1H, d, J = 8.6 Hz, H-α), 7.48 (2H,d, J = 8.6 Hz, H-2′ and H-6′), 7.07 (1H, d, J = 2.6 Hz, H-6), 6.95 (1H, d, J = 16.0 Hz, H-β), 6.88 (2H, d, J = 8.6 Hz, H-3′ and H-5′), 6.79 (1H, d, J = 2.6 Hz, H-8), 5.62 (1H, s, H-3), 4.07 (3H, s, OCH₃-4), 3.95 (3H, s, OCH₃-7). ¹³C- NMR (acetone-d ₆, 75 MHz): d = 169.8 (C-4), 163.1 (C-7), 162.2 (C-2), 158.5 (C-4′), 157.5 (C-9) 140.0 (C-5), 132.6 (C-β), 130.0 (C-1′), 129.1 (C-2′ and C-6′), 126.4 (C-a), 116.5 (C-3′ and C-5′), 111.6 (C-6), 107.3 (C-10), 100.7 (C-8), 88.8 (C-3), 57.1 (OCH₃-4), 56.2 (OCH₃-7). NOE correlations (acetone-d ₆, 300 MHz): H-3 → OCH₃-4, OCH₃-7 → H-8, H-α → H-2′ and H-6′.

Cytotoxicity testing. Data for compound 1 were obtained using a panel of human cancer cell lines and established protocols [8].

Acknowledgements

This study was supported by grant U19-CA-52956 from the National Cancer Institute, NIH, Bethesda, Maryland. We thank the Nuclear Magnetic Resonance Laboratory of the Research Resources Center, University of Illinois at Chicago, for provision of the highfield NMR spectrometer used in this study. Mr. R. B. Dvorak and Dr. K. Htin of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, are acknowledged for the MS data and for valuable input concerning the NMR studies, respectively.

References

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Prof. A. Douglas Kinghorn, Ph.D., D.Sc.

Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy

College of Pharmacy

University of Illinois at Chicago

833 South Wood Street (M/C 781)

Chicago, IL 60612

U.S.A.

Email: Kinghorn@uic.edu

Fax: +1-312-996-7107

Phone: +1-312-996-0914

References

• 1 Seo E -K, Silva G L, Chai H -B, Chagwedera T E, Farnsworth N R, Cordell G A, Pezzuto J M, Kinghorn A D. Phytochemistry 1997 45: 509-515
  PubMedSearch in Google Scholar
• 2 Silverstein R M, Bassler G C, Morrill T C. Spectroscopic Identification of Organic Compounds Fifth Edition. New York; John Wiley & Sons 1991: pp. 91-164
  PubMedSearch in Google Scholar
• 3 Vilegas W, Pozetti G L, Vilegas J HY. J. Nat. Prod 1993 56: 416-417
  PubMedSearch in Google Scholar
• 4 Abaul J, Philogéne É, Bourgeois P, Poupat C, Ahond A, Potier P. J Nat Prod 1994 57: 846-848
  PubMedSearch in Google Scholar
• 5 Debenedetti S L, Palacios P S, Nadinic E L, Coussio J D, de Kimpe N, Boeykens M, Feneau-Dupont J, Declercq J -P. J Nat Prod 1994 57: 1539-1542
  PubMedSearch in Google Scholar
• 6 Laakso J A, Narske E D, Gloer J B, Wicklow D T, Dowd P F. 1994 57: 128-133
  PubMedSearch in Google Scholar
• 7 Bax A. J Magn Res 1984 57: 314-318
  PubMedSearch in Google Scholar
• 8 Likhitwitayawuid K, Angerhofer C K, Cordell G A, Pezzuto J M, Ruangrungsi N. J Nat Prod 1993 56: 30-38
  PubMedSearch in Google Scholar

Prof. A. Douglas Kinghorn, Ph.D., D.Sc.

Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy

College of Pharmacy

University of Illinois at Chicago

833 South Wood Street (M/C 781)

Chicago, IL 60612

U.S.A.

Email: Kinghorn@uic.edu

Fax: +1-312-996-7107

Phone: +1-312-996-0914