Pergamon J. SteroidBiochem. Molec. Biol. Vol. 60, No. 1-2, pp. 131 136, 1997 Copyright ~) 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0960-0760(96)00164-1 0960-0760/97 $17.00 + 0.00 Non-steroidal L-245,976 Acts as a Classical Antiandrogen In Vitro Jeffrey H. Toney, 1. Yuli Chen, 2 Sue-Jane Rutledge, 3 Azriel Schmidt 3 and Alex Elbrecht 2 'Department of Biochemistry, Merck Research Laboratories, Rahway, NJ, U.S.A.; 2Department of Molecular Endocrinology, Merck Research Laboratories, Rahway, NJ, U.S.A. and SDepartment of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, PA, U.S.A. Non-steroidal antiandrogens have been employed in the management of prostate cancer, but the mechanism of action is unclear due to a lack of good tissue culture models. The growth of a hamster ductus deferens cell line (DDT1) is highly dependent upon the addition of 10 nM testosterone to syn- thetic serum-free media. We describe a non-steroidal compound N-(4-chlorophenyl)-(Z,Z)-2,3-bis(- cyclopropylmethylene) cyclopentanecarboxamide (L-245976) which antagonizes the action of testosterone on DDT1 cells at 10 pM but exhibits little or no effect on cell growth by itself. This com- pound also blocks the binding of 3H-dihydrotestosterone (DHT) to the human androgen receptor (AR) with an ICs0 of ~28 pM. In addition, L-245976 was found to antagonize DHT-dependent trans- activation of the AR via the probasin gene promoter at comparable doses with no agonist activity. Copyright © 1997 Elsevier Science Ltd. J. Steroid Biochem. Molec. Biol., Vol. 60, No. 1-2, pp. 131-136, 1997 INTRODUCTION Non-steroidal antiandrogens such as ICI-176344 (Casodex) or (hydro~r)flutamide (Scheme 1) have been employed in the management of prostate can- cer ([1] and references therein) with modest success. Although extensive clinical data is available on these compounds, the mechanism of action has been diffi- cult to elucidate due to a paucity of androgen-depen- dent cell lines. Stirrmlation of cell growth by androgens was reported 20 years ago using the human breast cancer cell line MCF-7 in serum-free media [2] and was inhibited by antiandrogens such as cyproterone acetate. Hydroxyflutamide has also been shown to inhibit androgen-stimulated growth of the breast cancer cell line EVSA-T [3]. The human pro- static carcinoma cell line LNCaP has been well stu- died [4-12], and is known to contain a point mutation of the androgen receptor (AR) within the ligand binding domain [8, 10]. Thus agents such as hydroxyflutamide can act aberrantly and can promote *Correspondence to Dr J. Toney, Department of Biochemistry, Building 80M-136, P.O. Box 2000, Merck Research Laboratories, Rahway, NJ 07065-0900, U.S.A. Fax: +1(908) 594 3664. Received 15 Apr. 1996; accepted 29 Jul. 1996. cell growth of LNCaP cells instead of acting as an antiandrogen [11, 12]. In addition, the maintenance of LNCaP in serum-free media is difficult [4]. Other human prostatic cell lines such as DU145 and PC3 have been employed to study the effect of antiandro- gens [13]. However, these cell lines have been reported to be both hormone-sensitive and hormone= insensitive depending upon growth conditions, and cannot be maintained in synthetic media. An andro- gen-dependent hamster ductus deferens cell line (DDT1) has been described that can be maintained in serum-free synthetic media in which proliferation is highly dependent upon the addition of testosterone [14]. The DDT1 cell line thus presents a good model for studying the biological action of putative andro- genic/antiandrogenic agents. We describe a 96-well colorimetric assay for cell proliferation using the DDT1 cells in the presence of androgens which led to the identification of the non- steroidal compound N-(4-chlorophenyl)-(Z,Z)-2,3- bis (cyclopropylmethylene) cyclopentanecarboxamide (L-245976; see Fig. 1). An assay for androgen-depen- dent transactivation of the AR using the rat probasin gene promoter [15] is also described to allow dis- crimination between agonists and antagonists. Taken together, these assays should prove useful for the 131