Cytotoxic clerodane diterpenes fromPolyalthia barnesii

Pergamon

0031-9422(94)00466-8

Phylochermstry, Vol. 37, No. 6, pp. 1659 1662, 1994 Copyright © 1994 Elsevier Soenee Ltd Printed in Great Britain. All rights reserved 0031 9422/94 $7.00 + 0.00

CYTOTOXIC CLERODANE DITERPENES F R O M P O L Y A L T H I A B A R N E S I I X. MA, I.-S. LEE,. H.-B. CHAI, K. ZAW, N. R. FARNSWORTH,D. D. SOEJARTO,G. A. CORDELL, J. M. PEZZUTO and A. n. KINGHORN* Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, U.S.A.

(Received 20 April 1994) Key Word lndex--Polyalthia barnesii; Annonaceae; stem bark; diterpenes; clerodanes; cytotoxicity assessment.

Abstract--Three cytotoxic clerodane diterpenes were purified from an ethyl acetate-soluble extract of the stem bark of Polyalthia barnesii, namely, 16~-hydroxycteroda-3,13(14)Z-dien-15,16-olide, a known compound, and two novel compounds, 3fl,16a-dihydroxycleroda-4(18),13(14)Z-dien-15,16-olide and 4fl,16ct-dihydroxyclerod-13(14)Z-en-15,16olide. These compounds were found to exhibit broad cytotoxicity against a panel of human cancer cell lines.

O

INTRODUCTION

The tropical genus Polyalthia has been found to be the source of a wide range of secondary metabolites, including aporphine and other alkaloids [1-7], clerodane and labdane diterpenes [8-13], flavonoids [ 14], indolosesquiterpenes [15, 16], a lactone [17], a proanthocyanidin [18], sterols [19], triterpenes [19, 20], and zinc-containing compounds [21]. Several of these compounds have been shown to exhibit interesting biological activities, including a labdane diterpene with leishmanicidal effects [ 12], an aporphine alkaloid [6] and clerodane diterpenes [11] with cytotoxic effects for cancer cell lines, and a lanostane-type triterpenoid with anti-HIV activity [20]. In the present communication, we report the occurrence of three cytotoxic clerodane diterpenes (1-3) from the stem bark of Polyalthia barnesii, which was collected in the Philippines as part of a collaborative search for naturally occurring antineoplastic agents. Two of these compounds, 2 and 3, are of novel structure. To date, there appears to have been no previous report on the phytochemical constituents or biological activity of P. barnesii.

O

1,

,1+..OH

...... OH

1 1 j 12 2

H ~1i , , , , 9+44'+444,,~,,~o417

3

~

7

H

4,,,4,+,44`44

,4

HO

~8

!

2

~

O

""OH

RESULTSAND DISCUSSION The ethyl acetate-soluble fraction of a methanolic extract of the stem bark of P. barnesii was chromatographed over silica gel, with cytotoxicity monitored by a human lung cancer cell line (Lul), leading to the isolation of three bioactive diterpenes 1-3. By direct comparison of the tH and 13C NMR spectral data of these compounds with reported data for clerodane-type diterpenes from other species in the genus Polyalthia [8-11, 13], it was

*Author to whom correspondence should be addressed.

HO

apparent that 1-3 belonged to this same compound class. Compound 1, C20H3oO3, the most abundant of the compounds obtained, is of known structure and its identity was established by IR, ~H NMR, t3C NMR and MS data comparison with published literature. This

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X. MA et al.

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compound, 16~-hydroxycleroda-3,13(14)Z-dien-15,16olide, was previously reported from the leaves of Polyalthia longifolia Thw. and its structure was confirmed by X-ray crystallography [8]. Compound 2, obtained as a minor constituent, exhibited a molecular formula of C2oH3oO4, as determined by HREIMS. Analysis of the IH and 13C N M R spectra of 2 indicated resonances for three methyl signals, with two being tertiary (6H0.78 s and 1.05 s; ,~c 17.9 and 21.17) and one secondary (6H0.82 d; 6c 15.8). It was apparent that the C-18 methyl group in 1 (diH1.59 s; 6c 18.2) was replaced by a 4,18-exo-methylene group [~H4.74 d ( J = 0 . 4 Hz) and 4.91 d (d = 1.2 Hz); 6c161.7 and 99.7] in 2. This observation was supported by previously reported data for model cterodane diterpenes with this same functionality [22, 23]. Its MS and IR data suggested that 2 had one more hydroxyl group present in the molecule than 1. The location of this secondary OH was established as occurring at C-3, using the selective I N E P T NMR technique [24]. Thus, irradiation of the proton signals at ~H4.91 (H-18a) and 4.74 (H-18b) enhanced the carbon signals at ~c69.7 (C-3), 161.7 (C-4) and 39.2 (C-5), and at 6c69.7 (C-3) and 39.2 (C-5), respectively, revealing carbons two or three bonds away from the selected protons. Furthermore, from the analysis of the ~H NMR spectrum of 2, the proton at position C-3 (6n4.32) was determined to be in the axial (~) orientation, on the basis of the multiplicity (dd, J = 5.8, 12.0 Hz) of the resonance peak arising from spin-spin interaction with its neighbouring protons H-2~ and H-2/L Accordingly, 2 was assigned the novel structure, 3fl,16zc-dihydroxycleroda-4(18),13(14)Zdien-15,16-olide. The elemental composition of a further minor compound, 3, was established by HREIMS as C2oH320 4. A comparison of the tH NMR and MS data measured for 3 to those of 1 provided evidence that the 3,4-double bond was saturated (6H1.38 m, 1.69 m) with addition of a hydroxyl group (prominent M+-18 ElMS fragment ion peak at m/z 318) at position C-4 [25]. This conclusion was supported by the ~3C NMR data of 3, in which the resonance signals at ~c120.3 (C-3) and 144.2 (C-4) of 1 were replaced by signals at C5c36.5(C-3) and 76.2 (C-4). In a selective I N E P T NMR irradiation experiment, application of soft proton pulses at 6H 1.08 (Me-18) and 0.99 (Me-

19) enhanced the carbon signals at 6c36.5 (C-3), 76.2 (C4), 42.7 (C-5), and at t~c76.2 (C-4), 42.7 (C-5), 32.7 (C-6) and 41.3 (C-10), respectively. The stereochemistry of the hydroxyl group at position C-4 was deduced as axial (fl) from the results of a 2D-NOESY NMR experiment, in which a cross-peak between Me-18 and Me-19 could be clearly observed. Thus, 3 was assigned the novel structure, 4fl,16ct-dihydroxyclerod-13(14)Z-en- 15,16-olide. Compounds 1-3 were evaluated against a panel of human and murine cancer cell lines [26] and the data obtained are summarized in Table 1. All of these compounds were broadly cytotoxic, exhibiting EDso values of < 4 # g m l - 1 in several cell lines. Among the human cancer cell lines, 1-3 were most potently cytotoxic for the human prostate (LNCaP) and human glioma (U373) cell lines represented. For most of the other cell lines, 3 was markedly less active than 1 and 2.

EXPERIMENTAL

Mps. uncorr.; UV: MeOH; IR: film; tH and 13C NMR spectra were recorded on 300 or 360 MHz instruments with TMS as int. standard; EIMS (70 eV) data were recorded with a direct probe. Low and high resolution data were obtained on a Finnigan MAT-90 instrument. Plant material. The stem bark of P. barnesii Merrill (Annonaceae) was collected in a tropical rain forest on the lower slopes of Mt Natib near Morong, Bataan, Philippines, in July 1992. Voucher specimens (Soejarto et al. 7775) have been deposited at the Philippine National Herbarium, Manila, Philippines, and at the John G. Searle Herbarium, Field Museum of Natural History, Chicago, Illinois, U.S.A. Extraction and isolation. The dried, powdered stem bark of P. barnesii (4.8 kg) was extracted with MeOH (2 x 18 1) at room temp. After filtration and evapn of the solvent, the dried MeOH extract was defatted with nhexane (4 x 250 ml), and then partitioned between EtOAc and H20, with the EtOAc extract evapd to dryness to yield 45.3 g of a brown powder. The EtOAc extract exhibited significant cytotoxic activity with a human lung cancer cell line (Lul; EDso 5.1/~g m l - 1). This residue was

Table 1. Evaluation of the cytotoxic potential of isolates obtained from P. barnesii with cancer cell lines Cell line*'l" Compound A431 1

2 3

8.7 4.1 12.4

BC1

Co12

HT

KB

KBV +

KBV

LNCaP

Lul

Mel2

P - 3 8 8 U 3 7 3 ZR75-1

4.7 2.9 8.7

7.4 3.2 5.1

4.5 5.3 16.0

4.0 9.0 13.8

3.4 6.8 > 20

4.1 18.5 > 20

2.6 3.2 3.9

4.7 2.0 6.0

1.2 6.8 12.0

0.5 2.5 2.2

1.7 2.7 1.1

4.5 3.9 9.1

*Key: A431 = human epidermoid carcinoma; BC1 = human breast cancer; Col2 = human colon cancer; HT = human fibrosarcoma; KB = human oral epidermoid carcinoma; KBV + = drug-resistant KB in the presence of vinblastine (1 #g ml-~); KBV = drug-resistant KB; LNCaP = human prostate cancer; Lul = human lung cancer; Mel2 = human melanoma; P-388 = murine lymphocytic leukemia: U373 = human glioma; ZR75-1 = hormone-dependent human breast cancer. tResults are expressed as EDs0 values (ggml-1), and were obtained using standard procedures [26].

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subjected to CC over Si gel, and eluted with a CHCI 3MeOH gradient to give 9 bulked frs. Cytotoxic fr. 3, eluted with n-hexane-CHCl 3 (4: 1), gave a major active compound, 1 (1.68 g, 0.035% w/w). Cytotoxic fr. 8 was further purified over Si gel by sequentially eluting with CHCI3-MeOH (97:3); CHC13-Me2CO (5:1); n-hexaneE t O A c - M e O H - H 2 0 (7: 3: 1.5:0.6, upper layer) to afford two minor active compounds. These were 2 (18 mg, 0.0004% w/w) from fr. 11, and 3 (14 mg, 0.0003% w/w) from fr. 13. 16ot-Hydroxycleroda- 3,13(14) Z-dien- 15,16-olide (1). Gum, [ ~ ] ~ ° - 4 1 . 3 ° (CHCI3; c5.44) (lit. [9]; [~t]25"5 - 48.7°). This compound exhibited UV, IR, MS, tH and 1aC spectral data comparable to those reported in the lit.

Herbarium, Manila, Philippines, for assistance in the collection of plant samples. We wish to thank Mr R. B. Dvorak of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago for running the mass spectra. We thank also Drs M. E. Wall and D. M. Brown, Research Triangle Institute, Research Triangle Park, North Carolina, U.S.A., and Drs M. J. O'Neill and R. M. Tait, Glaxo Research and Development Limited, Greenford, Middlesex, for the performance of certain enzyme inhibition and receptor binding assays on the isolates described herein.

[8].

1. Guinaudeau, H., Ramahatra, A., Leboeuf, M. and Cav6, A. (1978) Plantes M~d. Phytoth~r. 12, 166. 2. Jossang, A., Leboeuf, M. and Cav6, A. (1982) Tetrahedron Letters 23, 5147. 3. Abu Zarga, M. H. and Shamma, M. (1982) J. Nat. Prod. 45, 471. 4. Jossang, A., Leboeuf, M., Cav6, A., S6venet, T. and Padmawinata, K. (1984) J. Nat. Prod. 47, 504. 5. Wu, Y.-C. (1989) Heterocycles 29, 463. 6. Wu, Y.-C., Duh, C.-Y., Wang, S.-K., Chen, K.-S. and Yang, T.-H. (1990) J. Nat. Prod. 53, 1327. 7. Lavault, M., Guinaudeau, H., Bruneton, J., S6venet, T. and Hadi, H. A. (1990) Phytochemistry 29, 3845. 8. Phadnis, A. P., Patwardhan, S. A., Dhaneshwar, N. N., Tavale, S. S. and Guru Row, T. N. (1988) Phytochemistry 27, 2899. 9. Kijjoa, A., Pinto, M. M. M. and Herz, W. (1989) Planta Med. 55, 205. 10. Kijjoa, A., Pinto, M. M. M., Pinho, P. M. M., Tantisewie, B. and Herz, W. (1990) Phytochemistry 29, 653. 11. Zhao, G., Jung, J. H., Smith, D. L., Wood, K. V. and McLaughlin, J. L. (1991) Planta Med. 57, 380. 12. Richomme, P., Godet, M. C., Foussard, F., Toupet, L., S6venet, T. and Bruneton, J. (1991) Planta Med. 57, 552. 13. Chakrabarty, M. and Nath, A. C. 0992)J. Nat. Prod. 55, 256. 14. Seetharaman, T. R. (1986) Fitoterapia 57, 198. 15. Leboeuf, M., Hamonni6re, M., Cav6, A., Gottlieb, H. E., Kunesch, N. and Wenkert, E. (1976) Tetrahedron Letters 17, 3559. 16. Kunesch, N., Cav6, A., Leboeuf, M., Hocquemiller, R., Dubois, G., Guittet, E. and Lallemand, J.-Y. (1985) Tetrahedron Letters 26, 4937. 17. Loder, J. W. and Nearn, R. H. (1977) Heterocycles 7, 113. 18. Agrawal, S. and Misra, K. (1979) Curr. Sci. 48, 141. 19. Goyal, M. M. and Gupta, A. (1985) Ind. Drugs 22, 6~8. 20. Li, H.-Y., Sun, N.-J., Kashiwada, Y., Sun, L., Snider, J. V., Cosentino, L. M. and Lee, K.-H. (1993) J. Nat. Prod. 56, 1130. 21. Han, G.-Y., Xu, B.-X., Wang, X.-P., Liu, M.-Z., Xu, X.-Y., Meng, L.-N., Chen, Z.-L. and Zhu, D.-Y. (1981) Hua Hsueh Hsueh Pao 39, 433.

3 ~, 16ot-Dihydroxyeleroda-4 (18), 13 (14) Z-dien- 15,16olide (2). Powder; [ct]~° - 3 . 2 8 ° (CHC13; c0.21); UV 2~,~°n nm (loge): 206 (3.83); IR v,,,~ (film) cm-l: 3345 (OH), 2923, 2871, 1750 (C=O), 1647 (C=C), 1472, 1387, 1343, 1186, 1132, 1051,953,899,760; ~H N M R (300 MHz, CDC13 ): 60.78 (3H, s, Me-20), 0.82 (3H, d, J = 6.3 Hz, Me17), 1.05 (3H, s, Me-19), 2.20 (IH, dddd, J=2.9, 2.9, 5.8, 11.6 Hz, H-2a), 4.32 (1H, dd, J=5.8, 12.0 Hz, H-3~), 4.74 (1H, d, J = 0 . 4 Hz, H-18b), 4.91 (1H, d, J = 1.2 Hz, H- 18a), 5.81 (1H, s, H-14), 5.98 (1H, s, H-16); ~3CNMR (75.6 MHz, CDC13): 615.8 (C-17), 17.9 (C-20), 20.4 (C-l), 21.2 (C-19), 21.2 (C-11), 27.1 (C-V), 34.7 (C-12), 36.6 (C-8), 37.0 (C-2), 37.3 (C-6), 39.2 (C-5), 40.2 (C-9), 48.2 (C-10), 69.7 (C-3), 99.3 (C-16), 99.7 (C-18), 116.9 (C-14), 161.7 (C4), 170.6 (C-13), 171.9 (C-15); EIMS, m/z (rel. int.) 334 [M] ÷ (2), 318 (98), 299 (100), 281 (15), 247 (17), 207 (31), 189 (56), 161 (14), 147 (14), 135 (13), 121 (17), 107 (18); HREIMS, m/z 334.2143 (calcd for C2oH3oO4; 334.2144). 4 fl, 16a-Dihydroxyclerod- 13(14)Z-en- 15,16-olide (3). Powder; [~t]~° -4.08 ° (CHCI3; e0.25); UV "max~u'onnm (loge) 208 (3.66); IR Vm,x(film) c m - ' : 3351 (OH), 2945, 2878, 1755 (C=O), 1641 (C=C), 1462, 1377, 1342, 1189, 1123, 953, 916, 717; 1HNMR (CDC13) 60.79 (3H, s, Me20), 0.82 (3H, d, J = 6.1 Hz, Me-37), 0.99 (3H, s, Me-19), 1.08 (3H, s, Me-18), 1.38 (1H, m, H-3a), 1.69 (IH, n, H-3fl), 2.24 (2H, m, H-2), 5.80 (1H, s, H-14), 5.96 (1H, s, H-16); 13CNMR (75.6 MHz, CDCI3): 616.4 (C-17), 18.1 (C-19), 18.6 (C-20), 22.2 (C-I), 22.3 (C-2), 23.1 (C-12), 24.3 (C-18), 28.3 (C-11), 32.7 (C-6), 36.3 (C-7), 36.5 (C-3), 37.5 (C-8), 39.8 (C-9), 41.3 (C-10), 42.7 (C-5), 76.2 (C-4), 101.7 (C-16), 117.4 (C-14), 173.3 (C-13), 173.8 (C-15); ElMS m/z (rel. int.) 336 [M] + (2), 318 (80), 301 (18), 205 (11), 191 (100), 163 (11), 149 (24), 137 (27), 123 (45), 109 (45); HREIMS, m/z 336.2300 (calcd for C2oH3204; 336.2301). Cytotoxicity evaluation of 1-3. Compounds 1-3 were evaluated against a panel of human cancer cell lines and murine P-388 cell, according to established protocols [26]. Among the cell lines represented, a human lung cancer cell line (Lul) was used to guide the fractionations of 1-3 from the crude MeOH extracts of P. barnesii. Acknowledgements--This investigation was supported by grant U01-CA-52956 from the National Cancer Institute, NIH, Bethesda, Maryland. We are grateful to Mr O. Fernando and Mr E. Sagcal, Philippine National

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