TETRAHEDRON LETTERS Pergamon Tetrahedron Letters 42 (2001) 3685–3688 Novel stilbenes isolated from the root bark of Ekebergia benguelensis Daniel Chávez,a,† Hee-Byung Chai,a Tangai E. Chagwedera,b Qi Gao,c Norman R. Farnsworth,a Geoffrey A. Cordell,a John M. Pezzutoa and A. Douglas Kinghorna,* a Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy , College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA b Department of Pharmacy, University of Zimbabwe, Harare, Zimbabwe c Bristol-Myers Squibb, Pharmaceutical Research Institute, Wallingford, CT 06492, USA Received 1 March 2001; revised 23 March 2001; accepted 26 March 2001 Abstract—Four novel stilbenes (1–4) were isolated from the root bark of Ekebergia benguelensis. The structures were established by spectroscopic methods including 2D NMR analysis. The structure of 1, a new stilbene–coumarin hybrid representing a novel skeleton, was confirmed by single X-ray analysis. Compounds 1–4 were evaluated against a panel of human cancer cell lines. © 2001 Elsevier Science Ltd. All rights reserved. As part of an ongoing collaborative search for novel antineoplastic agents derived from plants, the root bark of Ekebergia benguelensis C. DC. (Meliaceae), collected in Zimbabwe, was investigated.1 In a previous study on the root bark of E. benguelensis collected in Tanzania, Jonker et al. isolated a coumarin, two oxygenated squalene derivatives, and two triterpenes.2 We report herein the bioassay-guided chromatographic separation of a CHCl3-soluble extract of E. benguelensis, which has led to the isolation of four novel stilbenes (1–4). Of the new compounds, 1 proved to be the only isolate with threshold cytotoxic activity (Lu1, ED50=5.1 mg/mL), while its glucoside (2) was isolated as a non-cytotoxic constituent. These compounds represent a novel carbon skeleton having a new stilbene–coumarin nucleus. The structure of 1 was confirmed by X-ray crystallographic analysis. Compounds 3 and 4 are resveratrol derivatives with an isobutyryl substituent. A methanolic extract (26 g) of the air-dried root bark of E. benguelensis 3 (1.2 kg) was partitioned with petroleum ether and CHCl3 to afford petroleum ether (3.1 g) and CHCl3 (5.5 g) residues. Bioassay-guided chromatographic separation of the CHCl3 extract, using the Lu1 human lung cancer cell line (ED50, 16.0 mg/mL) to monitor cytotoxicity, led to the isolation of four novel stilbenes [(14 (40 mg, 0.0033% w/w), 24 (20 mg, 0.0017% w/w), 34 (174 mg, 0.0145% w/w), and 44 (4.5 mg 0.0004% w/w)]. Additionally, two known compounds were isolated and identified by comparison of their published spectroscopic data as 5-(4-hydroxyphenethenyl)-4,7-dimethoxycoumarin5 (28 mg, 0.0023% w/w) and betulinic acid6 (12 mg, 0.0010% w/w). In this paper, we report the isolation and structural characterization of 1–4. Compound 1 was isolated as yellow needles. Its molecular formula was deduced as C20H18O5 by HREIMS. Its IR spectrum showed absorptions for hydroxyl (3300 cm−1), carbonyl (1698 cm−1), and aromatic (1597 cm−1) functionalities. The 1H NMR spectrum (Table 1) showed signals of a trans double bond at lH 8.01/7.11 (J=16.1 Hz), an A2B2 system at lH 7.69/7.27 (J=8.5 Hz) typical for a para-substituted phenyl ring, and an AB system with meta-correlated protons at lH 7.16/ * Corresponding author. Tel.: (312) 996-0914; fax: (312) 996-7107; e-mail: kinghorn@uic.edu † Present address: Centro de Graduados e Investigación, Instituto Tecnológico de Tijuana, Apdo. Postal 1166, Tijuana, BC 22000, Mexico. 0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 0 - 4 0 3 9 ( 0 1 ) 0 0 5 6 0 - 3 D. Chá6ez et al. / Tetrahedron Letters 42 (2001) 3685–3688 3686 Table 1. NMR data and HMBC correlations for 1 and 2a Position 1 1 H 2 3 4 5 6 7 8 9 10 1% 2% 1¦ 2¦, 6¦ 3¦, 5¦ 4¦ C-3 Me C-4 OMe C-7 OMe C-4¦ OH 1§ 2§ 3§ 4§ 5§ 6a§ 6b§ 7.16 d (2.3) 6.90 d (2.3) 8.01 d (16.1) 7.11 d (16.1) 7.69 d (8.5) 7.27 d (8.5) 2.17 s 3.62 s 3.79 s 11.94 s HMBCb 2 13 C 163.7 111.5 166.1 138.4 111.6 163.7 100.3 155.7 108.8 125.2 132.4 129.1 128.9 116.9 159.5 10.4 60.3 55.8 – 1 13 H 7.14 d (2.5) 6.94 d (2.5) 8.00 d (16.1) 7.05 d (16.1) 7.65 d (8.7) 6.47 d (8.7) 2.18 s 3.59 s 3.78 s 5.74 d (7.1) 4.38–4.44 m 4.38–4.44 m 4.38–4.44 m 4.20 m 4.61 dd (12.0, 1.9) 4.45 dd (12.0, 5.1) C 163.7 111.6 166.0 138.0 117.7 161.9 100.5 155.7 108.9 126.7 131.7 131.8 128.4 117.4 158.7 10.4 60.3 55.8 102.1 74.9 78.6 71.3 79.1 62.4 7, 8, 10, 1% 6, 7, 9, 10 5, 6, 10, 1¦ 5, 1¦, 2¦, 6¦ 2%, 3¦, 4¦, 5¦ 1¦, 2¦, 4¦, 6¦ 2, 3, 4 4 7 4¦ a1 b H NMR, 300 MHz; 13C NMR, 75 MHz, pyridine-d5; data in ppm (J in Hz). HMBC correlations for compounds 1 and 2. 6.90 (J=2.3 Hz). These chemical shifts were observed in the HMQC spectrum to correlate with their 13C NMR signals at lC 125.2/132.4, 128.9/116.9, and 111.6/ 100.3, respectively, supporting a stilbene type of nucleus.7–9 This compound showed NMR data very similar to values published for 5-(4-hydroxyphenethenyl)-4,7-dimethoxycoumarin, a compound previously isolated from Monotes engleri 5 and also isolated in the present study. The only differences observed were signals for a methine carbon at lH/C 5.81/88.9 (C-3) instead of a methyl group at lH/C 2.17/10.4, as in compound 1.10 Careful analysis of the HMBC spectrum allowed a stilbene–coumarin skeleton for 1 to be proposed. The cross-peaks observed for the meta-correlated aromatic protons (l 7.16/6.90) and the methoxy signal at lH 3.79 with the carbon resonance at lC 163.7 enabled the methoxy group to be placed at C-7 and the hydroxy group at C-4§. Thus, 1 was assigned as the new compound 5-[(1E)-2-(4-hydroxyphenyl)ethenyl]4,7-dimethoxy-3-methyl-2H-1-benzopyran-2-one. In order to support the proposed structure of 1, an X-ray analysis was performed on a single crystal obtained from pyridine/CH2Cl2. The crystal was found to be a 1:1 pyridine solvate of compound 1,11 and confirmed the novel carbon skeleton suggested (Fig. 1). Compound 2 was obtained as a white amorphous powder. Its molecular formula was determined as C26H28O10 by HRFABMS (positive mode). The NMR data indicated clearly that this compound is a glycoside of 1. Signals for the sugar moiety at lH 5.74 (H-1§), 4.61 (H-6a§), 4.45 (H-6a§), 4.38–4.44 (3H, H-2§, H-3§, H-4§), and 4.20 (H-5§) and its correlated signals in the HMQC spectrum at lC 102.1 (C-1§), 79.1 (C-5§), 78.6 (C-3§), 74.9 (C-2§), 71.3 (C-4§) and 62.4 (C-6§) indicated the presence of a glucose moiety.12 Treatment of 2 with b-D-glucosidase led to the aglycone, which showed identical spectroscopic properties to compound 1. The sugar unit was determined as glucose by co-TLC with an authentic sample. Analysis of the HMBC spectrum showed correlations for the anomeric proton (lH 5.74) as well as H-2¦/6¦ (lH 7.65) and H-3¦/5¦ (lH 6.47) with lC 158.7, indicating that glucose is attached to C-4¦. Thus, compound 2 was characterized as 5-[(1E)-2(4b - D - glucopyranosyloxyphenyl)ethenyl] - 4,7 - dimethoxy-3-methyl-2H-1-benzopyran-2-one. Figure 1. X-Ray crystallographic stereoviews of 1. D. Chá6ez et al. / Tetrahedron Letters 42 (2001) 3685–3688 Compound 3 was a major component of the CHCl3 extract. Its molecular formula was established as C19H20O4 by HREIMS. The 1H NMR data (Table 2) indicated a stilbenoid nucleus7–9 having a trans double bond at lH 7.13 (H-1¦)/6.77 (H-2¦), an A2B2 system for the para-substituted phenyl ring at lH 7.35/6.88 (J=8.5 Hz), and an AB system with meta-correlated protons at lH 6.55/6.39 (J=2.5 Hz). Additional coupled protons at lH 3.58 (H-2) and 1.15 (6H), confirming an AB6 system (J=6.7 Hz), were in agreement with an isopropyl moiety. Finally, a signal for a methoxy group at lH 3.83 and a hydroxy signal at lH 12.29 that disappeared with D2O were observed. The presence of a chelated proton at lH 12.29 and HMBC correlations for the protons in the isopropyl unit with a carbonyl at lC 212.8 led to the conclusion that an isobutyryl moiety was attached to the aromatic ring ortho to a hydroxyl group. Additional HMBC correlations for the methoxy group and the meta-correlated aromatic protons with the C-4% signal at lC 164.3 enabled the placements of the methoxy group at C-4% and the remaining hydroxyl group at C-4§. Thus, 3 was assigned as 1-{2-hydroxy-6[(1E)-2-(4-hydroxyphenyl)ethenyl]-4-methoxyphenyl}-2methyl-1-propanone. 3687 Compound 4 was isolated as a yellow amorphous powder. The NMR data were very similar to those of 3, with the only difference observed being the lack of the methoxy group. The molecular formula determined as C18H18O4 by HREIMS showed a difference of 14 amu, suggesting that 4 is the demethoxy derivative of 3. Permethylation of 4 with diazomethane gave a compound with an identical 1H NMR spectrum and Rf on TLC to the permethylated derivative of 3, confirming the structure of 4. Therefore, 4 was determined as 1{2,4 - dihydroxy - 6 - [(1E) - 2 - (4 - hydroxyphenyl)ethenyl]phenyl}-2-methyl-1-propanone. Isolates 1–4 were evaluated against a panel of human cancer cell lines.13 Compound 1 showed marginal cytotoxicity against a Lu1 cell line (human lung cancer, ED50=5.1 mg/mL), whereas glucoside 2 was inactive (ED50 >20 mg/mL). The most susceptible cell lines with 3 and 4 were KB (human oral epidermoid carcinoma, ED50=9.9 mg/mL) and LNCaP (hormone-dependent human prostate cancer, ED50=7.5 mg/mL), respectively. We have previously reported the selective cytotoxicity of 5-(4-hydroxyphenethenyl)-4,7-dimethoxycoumarin and betulinic acid against the LNCaP (hor- Table 2. NMR data for 3 and 4 and HMBC correlations for 3a 3b Position 1 13 H 1 2 3, C-2 CH3 1% 2% 3% 4% 5% 6% 1¦ 2¦ 1§ 2§, 6§ 3§, 5§ 4§ C-4% OCH3 C-2% OH C 212.8 39.3 19.7 113.8 164.1 99.9 164.3 108.4 142.4 125.8 132.3 129.2 128.2 115.9 156.3 55.5 3.58 sept. (6.7) 1.15 d (6.7) 6.39 d (2.5) 6.55 d (2.5) 7.13 d (15.9) 6.77 d (15.9) 7.35 d (8.5) 6.88 d (8.5) 3.83 s 12.29 s a1 H NMR, 300 MHz; In CDCl3. c In acetone-d6. 4c HMBC 1 3, 4 2 3.50 sept. (6.8) 1.11 d (6.8) 1%, 2%, 4%, 5% 6.33 d (2.2) 1%, 3%, 4%, 1¦ 6.65 d (2.2) 1%, 5%, 6%, 1§ 6%, 2§, 6§ 7.13 d (16.0) 6.89 d (16.0) 2¦, 3§, 4§, 5§ 2§, 4§, 6§ 7.41 d (8.6) 6.86 d (8.6) 13 H C 211.8 40.8 19.3 117.1 161.9 102.5 158.6 107.3 141.9 125.5 132.2 129.6 129.0 116.5 158.6 4% 1%, 2%, 3% 13 C NMR, 75 MHz; data in ppm (J in Hz). b OH OH O O O a) oxidation O OH OH O OH b) lactonization d) O-methylation O c) aromatization OH O [O] OH HO O O 3 Figure 2. Postulated biogenesis of 1. O O O 1 D. Chá6ez et al. / Tetrahedron Letters 42 (2001) 3685–3688 3688 mone-dependent human prostate cancer) and Mel-2 (human melanoma) cell lines, respectively.5,14 Based on the structures of the compounds isolated, it is proposed that the carbon skeleton of compound 1 is biosynthesized from 3, as shown in Fig. 2. This hypothesis relies on the fact that compound 3 with a stilbene nucleus was isolated in large amounts and may be the precursor of 1. If this is the case, then compound 1 should be considered a stilbene derivative, which undergoes subsequent modifications to the stilbene–coumarin type nucleus. 5. Acknowledgements 6. 7. We are grateful to Mr. R. B. Dvorak of the Department of Medicinal Chemistry and Pharmacognosy for recording the mass spectral data. D.C. thanks CONACyT, Mexico, for a postdoctoral fellowship. This investigation was supported by grant U19-CA-52956 funded by the National Cancer Institute, NIH, Bethesda, Maryland. 8. 9. 10. References 11. 1. Duvigneaud, P. In Flora Zambeziaca; Exell, A. W.; Wild, H., Eds.; Crown Agents: London, 1960; pp. 318–319. 2. Jonker, S. A.; Nkunya, M. H. H.; Mwamtobe, L.; Geenevase, J.; Koomen, G.-J. Nat. Prod. Lett. 1997, 10, 245–248. 3. This is a recollection of an initial sample deposited in the Field Museum of Natural History, Chicago, IL, under the reference number A1849. 4. Compound 1: Yellow needle crystals. Mp 192–194°C; UV (MeOH) umax (log m) 207 (4.41), 220 (4.38), 293 (4.31), 334 (4.46) nm; IR (film) wmax 3373, 2940, 2869, 1684, 1599, 1540, 1508, 1337, 1233, 1156 cm−1; 1H and 13C NMR, see Table 1; EIMS (70 eV) m/z [M]+ 338 (100), 310 (7), 295 (8), 231 (26), 218 (8), 190 (10), 107 (10); HREIMS m/z [M]+ 338.1152 (calcd for C20H18O5 338.1154). Compound 2: White amorphous powder. Mp 123–126°C; [h]20 D +9.0 (c 1.0, MeOH); UV (MeOH) umax (log m) 210 (3.22), 228 sh (3.04), 290 (2.85), 327 (2.96) nm; IR (film) wmax 3334, 2918, 2850, 1650, 1540, 1467, 1076 cm−1; 1H and 13C NMR, see Table 1; FABMS (glycerol) m/z [M+H]+ 501 (4), 460 (3), 338 (13), 277 (20), 105 (100); HRFABMS (glycerol) m/z [M+H]+ 501.1762 (calcd for C26H29O10 501.1761). Compound 3: Reddish amorphous powder. Mp 173–175°C; UV (MeOH) umax (log m) 207 (4.28), 230 sh (4.10), 280 (4.18), 309 sh (4.26), 321 (4.28) 12. 13. 14. . 15. nm; IR (film) wmax 3338, 2958, 2927, 1605, 1512, 1479, 1381, 1361, 1208, 1150 cm−1; 1H and 13C NMR, see Table 2; EIMS (70 eV) m/z [M]+ 312 (41), 297 (7), 269 (100), 241 (9); HREIMS m/z [M]+ 312.1371 (calcd for C19H20O4 312.1362). Compound 4: Pale yellow amorphous powder. Mp 80–72°C; UV (MeOH) umax (log m) 209 (4.34), 230 sh (4.14), 279 (4.20), 318 (4.25) nm; IR (film) wmax 3416, 2960, 2925, 1598, 1510, 1480, 1350, 1200, 1169 cm−1; 1H and 13C NMR, see Table 2; EIMS (70 eV) m/z [M]+ 298 (41), 255 (100), 227 (9); HREIMS m/z 298.1205 (calcd for C18H18O4 298.1200). Seo, E.-K.; Chai, H.-B.; Chagwedera, T. E.; Farnsworth, N. R.; Cordell, G. A.; Pezzuto, J. M.; Kinghorn, A. D. Planta Med. 2000, 66, 182–184. Sholichin, M.; Yamasaki, K.; Kasai, R.; Tanaka, O. Chem. Bull. Pharm. 1980, 28, 1006–1008. Seo, E.-K.; Chai, H.-B.; Constant, H. L.; Santisuk, T.; Reutrakul, V.; Beecher, C. W. W.; Farnsworth, N. R.; Cordell, G. A.; Pezzuto, J. M.; Kinghorn, A. D. J. Org. Chem. 1999, 64, 6976–6983. Lins, A. P.; Ribeiro, M. N. De S.; Gottlieb, O. R.; Gottlieb, H. E. J. Nat. Prod. 1982, 45, 755–761. Gonzalez-Laredo, R. F.; Chaidez-Gonzalez, J.; Ahmed, A. A.; Karchesy, J. J. Phytochemistry 1997, 46, 175–176. Kalinin, A. V.; Snieckus, V. Tetrahedron Lett. 1998, 39, 4999–5002. X-Ray crystal structure analysis of compound 1: Crystal data: C20H18O5·C5H5N, monoclinic, space group: P21/c, a=12.0355(4), b=10.8991(7), c=16.6204(8) A, , i= 103.043(4)°, V=2124.0(2) A, 3, Z=4, dx=1.304 g cm−3, Cu Ka radiation, absorption coefficient v=0.71 mm−1. A colorless plate crystal of dimensions 0.15×0.20×0.40 mm was used for X-ray measurements at 295 K on an Enraf– Nonius CAD4 diffractometer with a graphite monochromator. The total number of reflections measured was 4595, of which 3497 were consider to be observed (I4|). The absorption correction was 0.76–1.00 (Tmin−Tmax). The structure was solved by direct methods and refined by full-matrix least-squares.15 Final agreement factors were R(F 2)=0.081; wR(F 2)=0.180, where w=1/ [| 2(Fo2)+(0.123P)], S=1.057. Agrawal, P. K. Phytochemistry 1992, 31, 3307–3330. Likhitwitayawuid, K.; Angerhofer, C. K.; Cordell, G. A.; Pezzuto, J. M.; Ruangrungsi, N. J. Nat. Prod. 1993, 56, 30–38. Pisha, E.; Chai, H.; Chagwedera, T. E.; Farnsworth, N. R.; Cordell, G. A.; Beecher, C. W. W.; Fong, H. H. S.; Kinghorn, A. D.; Brown, D. M.; Wani, M. C.; Wall, M. E.; Hieken, T. J.; Das Gupta, T. K.; Pezzuto, J. M. Nature (Med.) 1995, 1, 1046–1051. Fair, C. K. MolEN. An Interactive Intelligent System for Crystal Structure Analysis; Enraf–Nonius: Delft, The Netherlands, 1990.