Activity-Guided Isolation of Steroidal Alkaloid Antiestrogen-Binding Site Inhibitors from Pachysandra procumbens Leng Chee Chang, Krishna P. L. Bhat, Emily Pisha, Edward J. Kennelly, ² Harry H. S. Fong, John M. Pezzuto, and A. Douglas Kinghorn* Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612 Received April 24, 1998 Four novel steroidal alkaloids, (+)-(20S)-20-(dimethylamino)-3-(3′R-isopropyl)-lactam-5R-pregn-2-en-4- one (1), (+)-(20S)-20-(dimethylamino)-16R-hydroxy-3-(3′R-isopropyl)-lactam-5R-pregn-2-en-4-one (2), (+)- (20S)-3-(benzoylamino)-20-(dimethylamino)-5R-pregn-2-en-4-yl acetate (3), and (+)-(20S)-2R-hydroxy- 20-(dimethylamino)-3-phthalimido-5R-pregnan-4-yl acetate (4), as well as five known compounds, (-)- pachyaximine A (5), (+)-spiropachysine (6), (+)-axillaridine A (7), (+)-epipachysamine D (8), and (+)- pachysamine B (9), were isolated from Pachysandra procumbens, using a bioassay-guided fractionation based on inhibition of 3 H-tamoxifen binding at the antiestrogen binding site (AEBS). Compounds 1-7 and 9 demonstrated significant activity as AEBS-inhibitory agents, and compounds 3, 5 and 9 were found to potentiate significantly the antiestrogenic effect mediated by tamoxifen in cultured Ishikawa cells. The structure elucidation of compounds 1-4 was carried out by spectral data interpretation. Tamoxifen is currently the most widely used antiestro- genic agent for the therapy of hormone receptor-positive advanced breast cancer and as an adjuvant treatment in postmenopausal breast-cancer patients. 1 In addition, in a recent study conducted with more than 13 000 women at risk for developing breast cancer, it was concluded that tamoxifen can reduce the incidence of this disease by approximately 45%. 2 The mechanism of action has not been thoroughly delineated, but major effects include both competition with endogenous estradiol by binding to the estrogen receptor 3 and blockage of breast-cancer cells in the G 1 -phase of the cell cycle. 4,5 Apart from binding to the estrogen receptors, triphenylethylene antiestrogens have been shown to interact with high affinity and specificity to the microsomal antiestrogen binding site (AEBS). 6,7 AEBS receptors are found in a wide range of human tissues 8 and may play an important role in the mechanism of acquiring resistance to antiestrogen therapy. 9 Cancer chemoprevention can be defined as the preven- tion, delay, or reversal of cancer by ingestion of dietary or pharmaceutical agents capable of modulating the process of carcinogenesis. 10,11 As part of our current work in this area, a battery of mechanism-based in vitro assays is employed to facilitate the discovery of potential cancer chemoprevention agents. 11 As exemplified by tamoxifen, one method of preventing tumor progression associated with breast cancer is to exploit the hormone-dependence of this disease. 11 Accordingly, we have employed the Ishikawa cell line (an endometrial adenocarcinoma cell line) to search for new antiestrogens. 12 An ancillary approach involves the use of compounds that bind specif- ically to the AEBS, 9 because, in principle, combining such agents with tamoxifen could elevate the concentration of this drug available for binding to the estrogen receptor. 9 In this regard, we have examined the potential of test agents to interact with the AEBS. Pachysandra is a small genus of four species of shrubs and perennial herbs. Three species are indigenous to eastern Asia, occurring in mainland China, Japan, and Taiwan (P. terminalis Sieb. et Zucc., P. axillaris Franch., and P. stylosa Dunn.). 13 P. procumbens Michx. (Buxaceae), a native American ornamental, is a clump-forming adapt- able ground-cover plant, valued for its bluish or grayish green foliage. 14 There have been no previous phytochemi- cal or biological studies on this species. Several steroidal (3,20S)-diamino-5R-pregnane alkaloids, however, have been isolated previously from two related Asian species, P. terminalis Sieb. et Zucc. 15-19 and P. axillaris Franch. 20-23 In the current investigation, the entire plant of P. procumbens was chosen for activity-guided fractionation. The petroleum ether- and ethyl acetate-soluble extracts were found to exhibit significant activity in an AEBS assay, as measured by inhibition of 3 H-tamoxifen binding. 24 Four novel steroidal alkaloids, namely, (+)-(20S)-20-(dimethy- lamino)-3-(3′R-isopropyl)-lactam-5R-pregn-2-en-4-one (1), (+)-(20S)-20-(dimethylamino)-16R-hydroxy-3-(3′R-isopro- pyl)-lactam-5R-pregn-2-en-4-one (2), (+)-(20S)-3-(benzoy- lamino)-20-(dimethylamino)-5R-pregn-2-en-4-yl acetate (3), and (+)-(20S)-2R-hydroxy-20-(dimethylamino)-3-phthal- imido-5R-pregnan-4-yl acetate (4), have been isolated and characterized structurally. Five known compounds, (-)- pachyaximine A (5), (+)-spiropachysine (6), (+)-axillaridine A(7), (+)-epipachysamine D (8), and (+)-pachysamine B (9), were also isolated. The structure elucidation of 1-4 and the biological evaluation of 1-9 are reported in this communication. Results and Discussion A molecular formula of C 29 H 46 N 2 O 2 was determined from the HREIMS data (m/z 454.3568) for compound 1. Com- parison of its UV, IR, and 1 H NMR data with pachyster- mine A 17 and several more recently isolated analogues indicated that 1 was a steroidal alkaloid. 20,21,25,26 The functional groups present in the molecule of 1 could be assigned as an amide carbonyl (IR, ν max 1635 cm -1 ; δ C 169.5), a conjugated ketone (δ C 196.7), a four-membered nonfused -lactam ring (IR, ν max 1732 cm -1 ), and a gem- dimethyl group (ν max 1336-1231 cm -1 ; δ C 20.1, 19.9; δ H 1.06, 0.96). The 1 H and 13 C NMR data for the -lactam moiety [δ C 169.5, δ C 57.8; δ H 3.00, δ C 46.5; δ H 3.40 and 4.00] * Address reprint requests to A. D. Kinghorn. Tel.: (312) 996-0914. Fax: (312) 996-7107. E-mail Kinghorn@uic.edu. ² Present address: U.S. Food and Drug Administration, Center for Food and Nutrition, Office of Special Nutritionals, HFS-465, 200 C Street NW, Washington, DC 20204. 1257 J. Nat. Prod. 1998, 61, 1257-1262 10.1021/np980162x CCC: $15.00 © 1998 American Chemical Society and American Society of Pharmacognosy Published on Web 09/10/1998