Synthesis and Structure Activity Relationship of Organometallic

Feb 6, 2009 - The ferrocenyl complexes described here are the first examples of organometallic steroidal androgens possessing a strong antiproliferati...
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Organometallics 2009, 28, 1414–1424

Synthesis and Structure Activity Relationship of Organometallic Steroidal Androgen Derivatives Siden Top,*,† Ce´line Thibaudeau,† Anne Vessie`res,† Emilie Brule´,† Franck Le Bideau,† Jean-Michel Joerger,† Marie-Aude Plamont,† Soth Samreth,‡ Alan Edgar,‡ Je´roˆme Marrot,§ Patrick Herson,⊥ and Ge´rard Jaouen*,† Ecole Nationale Supérieure de Chimie de Paris, Laboratoire Charles Friedel, UMR 7223, 11 Rue Pierre et Marie Curie, 75231 Paris Cedex 05, France, Laboratoire Fournier, 50 Rue de Dijon, 21121 Daix, France, Institut LaVoisier de Versailles, UMR 8180, UniVersite´ de Versailles-Saint-Quentin-en-YVelines, 45 AVenue des Etats-Unis, 78035 Versailles Cedex, France, and Laboratoire de Chimie Inorganique et Mate´riaux Mole´culaires, UMR 7071, UniVersite´ Pierre et Marie Curie, 4 Place Jussieu, 75252 Paris Cedex 05, France ReceiVed July 23, 2008

We present here the synthesis and the structure activity relationship of a series of organometallic complexes of the steroidal androgens testosterone and dihydrotestosterone (DHT) substituted at the C-17 position of the steroid skeleton with an ethynyl substituent grafted with various sandwich or semisandwich organometallic units [ferrocenyl, (η5-C5H4)-Re(CO)3, (η5-C5H4)-Mn(CO)3, (η6-C6H5)-Cr(CO)3] and of 3β-androstanediol substituted at C-16 and C-17 respectively by a ferrocenyl vinyl and a ferrocenyl ethynyl unit. In contrast to the estradiol series, there are currently very few examples of organometallic steroidal androgens in the literature. The ethynyltestosterone derivatives were obtained via a Stille coupling reaction between the appropriate iodo-organometallics and 17β-ethynyltestosterone stannyl derivatives. The ethynylDHT derivatives were synthesized by addition of the corresponding acetylide to the C-17 carbonyl of the steroid. The crystal structures of two ferrocenyl and one rhenium complexes were determined by X-ray diffraction and had confirmed that the organometallic moiety points toward the R face of the steroid skeleton. All the complexes retain a modest affinity for the androgen receptor. The ferrocenyl derivatives of ethynyl testosterone, 8 and 12, show a strong antiproliferative effect on the hormoneindependent prostate cancer cells PC-3 with IC50 values of respectively 4.7 and 8.3 µM. These values are very similar, for 12, or slightly better, for 8, than those found recently for the most active ferrocenyl derivative of the nonsteroidal antiandrogen nilutamide (IC50 value of 5.4 µM). The ferrocenyl complexes described here are the first examples of organometallic steroidal androgens possessing a strong antiproliferative activity on prostate cancer cells. Introduction Prostate cancer is a major cause of mortality in men.1 The number of deaths caused by this cancer is similar to breast cancer deaths for women. However prostate cancer tends to appear at a more advanced age, whereas breast cancer targets a younger population. The majority of human prostatic tumors are initially androgen-dependent. The proliferation of cancer cells in this case is controlled by androgens, which bind to the androgen receptor (AR).1 It was found that hormone-dependent prostate cancer cells are programmed to die when they are deprived of androgens.2 Huggins et al. demonstrated in 1941 that the suppression by castration of circulating testosterone and consequently of dihydrotestosterone (DHT), its active metabolite in cells, led to a therapeutic response. Radical prostatectomy as well as chemotherapy and radiotherapy are now used clinically in the treatment of early stage prostate cancer3 and * Corresponding author. E-mail: [email protected]. Tel: 01 33 1 43 26 95 55. Fax: 33 1 43 26 00 61. † Ecole Nationale Supe´rieure de Chimie de Paris. ‡ Laboratoire Fournier. § Universite´ de Versailles-Saint-Quentin-en-Yvelines. ⊥ Universite´ Pierre et Marie Curie. (1) Jemal, A.; Siegel, R.; Ward, E.; Murray, T.; Xu, J.; Thun, M. J. CA Cancer J. Clin. 2007, 57, 43. (2) Kyprianou, N.; Isaacs, J. T. Endocrinology 1988, 122, 552.

can be combined with hormonal therapy.4 The latter therapy consists in using antiandrogens, which block the androgeninduced hormonal effect by binding to the AR instead of testosterone. There are two types of commercially available antiandrogens,5 namely, steroidal and nonsteroidal antiandrogens.6 A few steroidal ligands have been used as antiandrogens, for example megestrol acetate7 and, most commonly, cyproterone acetate8 (Chart 1). However, due to several unwanted sideeffects (loss of libido, gynecomastia) cyproterone acetate has not been approved for use in the United States.9 For this reason, nonsteroidal antiandrogens are preferred, and three of them are presently in clinical use for treatment of prostate cancer: flutamide,10 nilutamide,4,11 and bicalutamide12 (Chart 1). However, after a period of 1-3 years, most cancers become resistant (3) Denmeade, S. R.; Isaacs, J. T. Prostate 2004, 58, 211. (4) Raynaud, J. P.; Bonne, C.; Moguilewsky, M.; Lefebvre, F. A.; Belanger, A.; Labrie, F. Prostate 1984, 5, 299. (5) Wirth, M. P.; Hakenberg, O. W.; Froehner, M. Eur. Urol. 2007, 51, 306. (6) Gao, W.; Bohl, C. E.; Dalton, J. T. Chem. ReV. 2005, 105, 3352. (7) Osborn, J. L.; Smith, D. C.; Trump, D. L. Am. J. Clin. Oncol. 1997, 20, 308. (8) (a) Goldenberg, S. L.; Bruchovsky, N. Urol. Clin. North Am. 1991, 18, 111. (b) Appu, S.; Lawrentschuck, N.; Grills, R. J.; Neerhut, G. J. Urol. 2005, 174, 140. (9) Anderson, J. BJU Int. 2003, 91, 455.

10.1021/om800698y CCC: $40.75  2009 American Chemical Society Publication on Web 02/06/2009

Organometallic Steroidal Androgen DeriVatiVes Chart 1. Natural Androgens (T and DHT) and Steroidal and Nonsteroidal Antiandrogens

to hormonal therapy, possibly due to mutations of the androgen receptor, and the prognosis at this stage is poor.13 As a consequence of the inefficiency of some of the current treatments for prostate cancer, an important research interest resides in the development of new antiandrogens, which could be effective on both hormone-dependent and hormoneindependent prostate cancers. We have recently developed ferrocenyl derivatives of tamoxifen, the leading drug in the treatment of hormone-dependent breast cancer.14 Interestingly, some of these complexes show antiproliferative activity on both hormone-dependent and hormone-independent breast cancer cells. One of the most active compounds, Fc-OHTam (Chart 2), is characterized by an antiestrogenic effect as well as a strong cytotoxic effect.15,16 Chart 2. Ferrocenyl Anti-estrogen and Anti-androgen

The similarity in the hormonal activity of estrogens and androgens, both of which act via an interaction with specific (10) (a) Airhart, R. A.; Barnett, T. F.; Sullivan, J. W.; Levine, R. L.; Schlegel, J. U. South Med. J. 1978, 71, 798. (b) Neri, R.; Peets, E.; Watnick, A. Biochem. Soc. Trans. 1979, 7, 565. (11) (a) Decensi, A. U.; Boccardo, F.; Guarneri, D.; Positano, N.; Paoletti, M. C.; Costantini, M.; Martorana, G.; Giuliani, L. J. Urol. 1991, 146, 377. (b) Kuhn, J. M.; Billebaud, T.; Navratil, H.; Moulonget, A.; Fiet, J.; Grise, P.; Louis, F.; Costa, P.; Husson, J. P.; Dahan, R.; Bertagna, C.; Eldelstein, R. New Engl. J. Med. 1989, 321, 413. (12) (a) Tucker, H.; Crook, J. W.; Chesterson, G. J. J. Med. Chem. 1988, 31, 954. (b) Blackledge, G. R. Eur. Urol. 1996, 29 Suppl 2, 96. (13) Hara, T.; Miyazaki, J.; Araki, H.; Yamaoka, M.; Kanzaki, N.; Kusaka, M.; Miyamoto, M. Cancer Res. 2003, 63, 149. (14) (a) Jaouen, G.; Top, S.; Vessie`res, A.; Leclercq, G.; Quivy, J.; Jin, L.; Croisy, A. C. R. Acad. Sci. Paris 2000, Se´rie IIc, 89. (b) Top, S.; Vessie`res, A.; Cabestaing, C.; Laios, I.; Leclercq, G.; Provot, C.; Jaouen, G. J. Organomet. Chem. 2001, 637, 500. (c) Top, S.; Vessie`res, A.; Leclercq, G.; Quivy, J.; Tang, J.; Vaissermann, J.; Huche´, M.; Jaouen, G. Chem.Eur. J. 2003, 9, 5223. (d) Vessie`res, A.; Top, S.; Pigeon, P.; Hillard, E. A.; Boubeker, L.; Spera, D.; Jaouen, G. J. Med. Chem. 2005, 48, 3937. (15) (a) Jaouen, G.; Top, S.; Vessie`res, A.; Leclercq, G.; McGlinchey, M. J. Curr. Med. Chem. 2004, 11, 2505. (b) Jaouen, G.; Top, S.; Vessie`res, A. Organometallics targeted to specific biological sites: The development of new therapies. In Bioorganometallics; Jaouen, G., Ed.; Wiley-VCH: Weinheim, 2006; p 65. (c) Vessie`res, A.; Top, S.; Beck, W.; Hillard, E. A.; Jaouen, G. Dalton Trans. 2006, 4, 529. (16) Nguyen, A.; Vessieres, A.; Hillard, E. A.; Top, S.; Pigeon, P.; Jaouen, G. Chimia 2007, 61, 761.

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receptors (ER or AR), led us to explore the potential offered by ferrocenyl complexes in the area of prostate cancer. Thus, we recently synthesized a series of ferrocenyl derivatives of the nonsteroidal antiandrogen nilutamide.17 We found that the sterically hindered ferrocenyl hydantoin, 5-Fc-hydantoin (Chart 2), showed a significant cytotoxicity on hormone-independent prostate cancer cells PC-3 with an IC50 value of 5.4 µM. Following the same idea, we felt that it would be interesting to prepare organometallic derivatives of the two main steroidal androgens, testosterone and DHT. A glance at the literature shows that many organometallic derivatives of estradiol have been published.18-26 For example, 17R-derivatives of estradiol bearing Co2(CO)6,24,25 CpRe(CO)3,18,23 CpMn(CO)3,22 PhCr(CO)3,19 or ferrocenyl units21,26 have been synthesized with the aim of obtaining compounds that retain affinity for the estrogen receptor and show interesting and, ideally, new biological properties. In contrast, very few examples of organometallic steroidal androgens are to be found. To the best of our knowledge, only three publications on this topic have been reported in the literature.27-29 They report the synthesis of organometallic complexes derived from 17R-ethynyltestosterone (ethisterone) and bearing ferrocenyl,27 Co2(CO)6,28 or gold substituents.29 This encouraged us to take advantage of our expertise acquired in the estrogen series to prepare some of their androgen analogues. Here we report the synthesis of organometallic complexes of testosterone and DHT substituted at the C-17 position with various sandwich or semisandwich organometallic units [ferrocenyl, CpRe(CO)3, CpMn(CO)3, PhCr(CO)3]. Because androstane-3β,17β-diol (3β-androstanediol), a metabolite of DHT, has recently been found to induce inhibition of DU-145 prostate cancer cell migrations,30 and to bind to ERβ, (17) Payen, O.; Top, S.; Vessie`res, A.; Brule´, E.; Plamont, M.-A.; McGlinchey, M. J.; Mu¨ller-Bunz, H.; Jaouen, G. J. Med. Chem. 2008, 51, 1791. (18) Top, S.; El Hafa, H.; Vessie`res, A.; Huche´, M.; Vaissermann, J.; Jaouen, G. Chem.-Eur. J. 2002, 8, 5241. (19) El Amouri, H.; Vessie`res, A.; Vichard, D.; Top, S.; Gruselle, M.; Jaouen, G. J. Med. Chem. 1992, 35, 3130. (20) (a) Morel, P.; Top, S.; Vessie`res, A.; Ste´phan, E.; Laı¨os, I.; Leclercq, G.; Jaouen, G. C. R. Acad. Sci. Paris, Chim./Chem. 2001, 4, 201. (b) Vessie`res, A.; Top, S.; Ismail, A. A.; Butler, I. S.; Lou¨er, M.; Jaouen, G. Biochemistry 1988, 27, 6659. (c) Le Bideau, F.; Kaloun, E. B.; Haquette, P.; Kernbach, U.; Marrot, J.; Ste´phan, E.; Top, S.; Vessie`res, A.; Jaouen, G. Chem. Commun. 2000, 211. (d) Ramesh, C.; Bryant, B.; Nayak, T.; Revankar, C. M.; Anderson, T.; Carlson, K. E.; Katzenellenbogen, J. A.; Sklar, L. A.; Norenberg, J. P.; Prossnitz, E. R.; Arterburn, J. B. J. Am. Chem. Soc. 2006, 128, 14476. (e) Arterburn, J. B.; Corona, C.; Rao, K. V.; Carlson, K. E.; Katzenellenbogen, J. A. J. Org. Chem. 2003, 68, 7063. (21) Osella, D.; Nervi, C.; Gaelotti, F.; Cavigiolio, G.; Vessie`res, A.; Jaouen, G. HelV. Chim. Acta 2001, 84, 3289. (22) Top, S.; El Hafa, H.; Vessie`res, A.; Quivy, J.; Vaissermann, J.; Hughes, D. W.; McGlinchey, M. J.; Mornon, J. P.; Thoreau, E.; Jaouen, G. J. Am. Chem. Soc. 1995, 117, 8372. (23) Top, S.; Vessie`res, A.; Jaouen, G. J. Chem. Soc., Chem. Commun. 1994, 453. (24) Vessie`res, A.; Jaouen, G.; Gruselle, M.; Rossignol, J. L.; Savignac, M.; Top, S.; Greenfield, S. J. Steroid Biochem. 1988, 30, 301. (25) Vessie`res, A.; Top, S.; Vaillant, C.; Osella, D.; Mornon, J. P.; Jaouen, G. Angew. Chem., Int. Ed. Engl. 1992, 31, 753. (26) Vessie`res, A.; Spera, D.; Top, S.; Misterkiewicz, B.; Heldt, J. M.; Hillard, E. A.; Huche´, M.; Plamont, M. A.; Napolitano, E.; Fiaschi, R.; Jaouen, G. ChemMedChem 2006, 1, 1275. (27) Jones, C. M.; Mocellin, E.; Tiekink, E. R. T. Z. Kristallogr.: New Cryst. Struct. 2000, 215, 89. (28) Osella, D.; Gaelotti, F.; Cavigiolio, G.; Nervi, C.; Hardcastle, K. I.; Vessie`res, A.; Jaouen, G. HelV. Chim. Acta 2002, 85, 2918. (29) Stockland, R. A.; Kohler, M. C.; Guzei, I. A.; Kastner, M. E.; Bawiec, J. A.; Labaree, D. C.; Hochberg, R. B. Organometallics 2006, 25, 2475. (30) Weihua, Z.; Makela, S.; Andersson, L. C.; Salmi, S.; Saji, S.; Webster, J. I.; Jensen, E. V.; Nilsson, S.; Warner, M.; Gustafsson, J. A. Proc. Natl. Acad. Sci. USA 2001, 98, 6330.

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the second form of the estrogen receptor,31 we also decided to prepare its C-17 and C-16 ferrocenyl derivatives. The affinity of these newly synthesized complexes for the androgen receptor was measured, and the effect of the ferrocenyl complexes on the growth of hormone-independent (PC-3) and hormonedependent prostate cancer cells (LNCaP) was studied.

Results and Discussion Synthesis. Substitution at the 17r Position of Testosterone. Synthesis of 6-8, the 17R organometallic derivatives of testosterone, involved a Stille coupling reaction between the appropriate iodo-organometallic 3-5 and the 17β-ethynyltestosterone stannyl derivative 2, which was obtained by heating ethynyltestosterone 1 with n-Bu3SnOMe at 150 °C for 5 h (Scheme 1). Scheme 1. Synthesis of 17r-Ethynyltestosterone Organometallic Derivatives

The iodo-organometallics 3,32 4,32 and 533 were synthesized according to literature procedures. The Stille coupling reaction using a catalytic amount of (MeCN)2PdCl2 was normally completed after approximately 3 h stirring at room temperature. The isomer possessing the hydroxy group in the β-position was the sole isomer isolated after purification. Its epimer was not even observed in the crude reaction mixture. Compounds 6 [Testo-17RE-CpRe(CO)3], 7 [Testo-17RE-CpMn(CO)3], and 8 [Testo-17RE-Fc] were obtained with yields of 90%, 75%, and 36%, respectively. It is noteworthy that in the series of iodoorganometallics (C5H4I)Re(CO)3 and (C5H4I)Mn(CO)3 were more reactive than the iodoferrocene counterpart. A similar result has been obtained when a Stille coupling reaction was used with 17R-(tributylstannylethynyl)estradiol.34 It has also been explained by electrochemical study why iodocymantrene and iodocyrhetrene are suitable in Stille reaction conditions, while iodoferrocene gives a better yield under Sonogashira reaction conditions.35 The difference in reactivity can be related to the rate constant of the oxidative addition of the species. Substitution at the 17r-Position of Dihydrotestosterone. As DHT is a major metabolite of testosterone and has an affinity toward the androgen receptor 5 times higher than testosterone, it was important to prepare its organometallic complexes; therefore we decided to anchor a ferrocenyl substituent at the 17R-position as for testosterone. Androstanedione, which has a carbonyl function on C-17, was the starting material of choice, but because the carbonyl on C-3 is the most reactive function, (31) Guerini, V.; Sau, D.; Scaccianoce, E.; Rusmini, P.; Ciana, P.; Maggi, A.; Martini, P. G.; Katzenellenbogen, B. S.; Martini, L.; Motta, M.; Poletti, A. Cancer Res. 2005, 65, 5445. (32) Lo Sterzo, C.; Stille, J. K. Organometallics 1990, 9, 687. (33) Guillaneux, D.; Kagan, H. B. J. Org. Chem. 1995, 60, 2502. (34) Ferber, B.; Top, S.; Vessieres, A.; Welter, R.; Jaouen, G. Organometallics 2006, 25, 5730.

Top et al.

it had to be protected to give the acetal 9, according to a literature procedure.36 Ethynylferrocene 10 was lithiated to give the corresponding acetylide, which was allowed to react with 9 at -78 °C to produce exclusively the 17R-ferrocenyl complex isomer 11 in 39% yield (Scheme 2). The deprotection of the C-3 ketone was carried out quantitatively in an acetone/dichloromethane mixture using a catalytic amount of PTSA to give the ferrocenyl DHT derivative 12 [DHT-17RE-Fc], in an excellent yield of 90%. Following the same procedure involving the phenylacetylene 13, the corresponding organic derivative 14 bearing a phenyl group on the alkyne was prepared in 80% yield to produce the DHT derivative 15 [DHT-17RE-Ph], in 89% yield. Scheme 2.

Synthesis of Ferrocenyl and Organic 17rEthynyl-DHT Derivatives

The addition of a phenyl group to DHT gives the possibility of anchoring a transition metal unit on the arene. Chromium is the most commonly used metal for this kind of complexation. Although complexation is normally performed by heating the arene with chromium hexacarbonyl, this was unsuccessful in this case. However, when 14 was heated with Cr(CO)3(NH3)337 in dioxane for 8 h, the desired chromium compound 16 was obtained in 32% yield after purification. Its deprotection using PTSA produced the chromium DHT derivative 17 in a moderate 34% yield (Scheme 3). Substitution at the 17r-Position of Androstane-3β,17βdiol (3β-androstanediol). It was first decided to anchor a ferrocene on the C-17 of 3β-androstanediol, as we already had on hand the DHT ferrocenyl derivative 12, which could be reduced in one step to give the androstanediol derivative. Cuilleron et al.38 reported the reduction of a similar DHT derivative substituted in the 17R-position by a (5-azidonitrobenzoyl) amido group under mild conditions using sodium borohydride at 0 °C in methanol. The sole product obtained was the 3β-hydroxy epimer. The same procedure was applied to our ferrocenyl derivative 12, and similarly, 17R-[ferroceneethynyl]androstane-3β,17β-diol 18 (3β-androstanediol-17E-Fc) was (35) Amatore, C.; Godin, B.; Jutand, A.; Ferber, B.; Top, S.; Jaouen, G. Organometallics 2007, 26, 3887. (36) Liu, A.; Carlson, K. E.; Katzenellenbogen, J. A. J. Med. Chem. 1992, 35, 2113. (37) Vebrel, J.; Mercier, R.; Belleney, J. J. Organomet. Chem. 1982, 235, 197. (38) Mappus, E.; Chambon, C.; Fenet, B.; Rolland de Ravel, M.; Grenot, C.; Cuilleron, C. Y. Steroids 2000, 65, 459.

Organometallic Steroidal Androgen DeriVatiVes

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Scheme 3. Synthesis of Cyclopentadienyl Chromium Tricarbonyl DHT Derivative

Scheme 4. Synthesis of C-17 Ferrocenyl Androstane-3β,17βdiol Derivative

NOE correlation between the ethylenic proton and H17 strongly supports this result (Figure 1).

Figure 1. NOE experiment supporting the E configuration of 21.

Scheme 5. Synthesis of C-16 Ferrocenyl Androstane-3β,17βdiol Derivative

formed in 60% yield as the single product characterized by NMR (Scheme 4). The formation of the 3β-epimer was confirmed by X-ray analysis. Substitution at the C-16 Position of Androstane-3β,17βdiol. Some derivatives of 3β-androstanediol substituted at the 16-position by an arylidene have been reported in the literature, and their androgenic activities were evaluated.39 This prompted us to synthesize a ferrocenyl analogue starting from the readily available compound 9 using a Knoevenagel condensation (Scheme 5). The enolate of 9 was formed by deprotonation with sodium hydride and was then reacted with ferrocenecarboxaldehyde for 3 h in refluxing THF to give 19 in 75% yield. The acetal was then quantitatively deprotected by stirring 19 in dilute acetic acid at 55 °C for 30 min to yield androstane-3,17-dione 20. The final step consisted in the reduction of the two ketones to the corresponding alcohols according to the procedure described above using NaBH4. The characterization of the molecule 21 [3β-androstanediol-16-CdC-Fc], obtained as a single diastereomer in 60% yield, was carried out by analogy with compound 18, which presented two hydroxyl groups on the β face. In order to determine the configuration of the double bond in 21, NOESY correlations were used. It was predicted that the alkene was in E configuration, as reported for similar compounds bearing an aryl instead of the ferrocene. Significant

X-ray Structure of 6, 12, and 18. Crystal data and structure refinement parameters for 6, 12, and 18 are summarized in Table 1. Crystallization of 6 [Testo-17RE-CpRe(CO)3)] from diethyl ether produced colorless crystals that were suitable for X-ray structural determination. Compound 6 crystallizes under two forms in the monoclinic space group P2(1). Figure 2 shows the molecular structure representation of one of the two forms of 6. This structure with the piano stool CpRe(CO)3 unit on the R face of the steroid confirms the stereoselectivity of the addition reaction on C-17. It is worth noting that the bulky CpRe(CO)3 unit is positioned beneath the steroidal C and D rings. A similar structure was previously observed for an estradiol-17R-ethynylCpRe(CO)3 complex.22 Compound 12 (DHT-17RE-Fc) crystallized from CDCl3 in the NMR tube as red crystals in the orthorhombic space group P212121 (Figure 3). The X-ray structure shows that, as in 6, the organometallic unit, here ferrocenyl, is positioned on the R face of the testosterone, but this time points away from the steroidal skeleton, not under the C and D ring. Compound 18 (3β-androstanediol-17E-Fc) was crystallized from dichloromethane/pentane as red crystals in the tetragonal space group I4 (Figure 4). The X-ray structure of 18 is similar to that of 6 with the organometallic unit, here ferrocenyl, positioned on the R face of the steroid and beneath the C and D rings. The X-ray also confirms that the hydroxy group on C-3 points toward the β face. This different positioning of the ferrocenyl unit in 12 and 18 might be explained by differences of hydrogen bonding between molecules. In fact, the packing diagram of 12 shows that the molecules form a chain by making a hydrogen bond between the 17β-OH of one molecule and the oxygen of the C-3 carbonyl group of the adjacent molecule (O · · · O ) 2.793(3) Å) (Figure 5). In the case of 18, the 3β-OH units of four steroids aggregate to form a square with O1 · · · O1′ ) 2.739(4) Å, while the four 17β-OH groups bind to one molecule of water with two different distances, O100 · · · O2′ ) 2.849(5) Å and O100 · · · O2′′ ) 2.799(5) Å (Figure 6). Biochemical Studies. The biological properties of the organometallic steroidal androgen derivatives have been tested and compared with the results obtained with testosterone and DHT, the natural androgens. The relative binding affinity (RBA) values for the compounds were assayed in a standard competitive radioligand assay, using full-length recombinant AR and [3H]-

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Table 1. Crystal Data and Structure Refinement Parameters for 6, 12, and 18 empirical formula fw cryst syst space group a (Å) b (Å) c (Å) R (deg) β (deg) γ (deg) V (Å3) Z T (K) λ (Å) Dcalc (g cm-3) µ (mm-1) Flack parameter R1 (wR2) (all data) R1 (wR2) (with cutoff) largest diff peak and hole (e · Å-3)

6

12

18 0.5H2O

C29H31O5Re 645.74 monoclinic P2(1) 9.4184(2) 9.1764(2) 30.1753(4) 90 92.537(1) 90 2605.40(9) 4 296(2) 0.71073 1.646 4.700 -0.02(1) 0.0730 (0.1162)a,b 0.0505 (0.1070)a,b 0.908 and -0.993

C31H38FeO2 498.49 orthorhombic P212121 7.6130(6) 16.644(2) 20.723(3) 90 90 90 2625.8(5) 4 250 0.71073 1.261 0.599 0.11(2) 0.0610 (0.0588)c,d 0.0312 (0.0350)c,d 0.20 and -0.28

C31H40FeO2.5 508.50 tetragonal I4 25.967(3) 25.967(4) 8.183(1) 90 90 90 5518(1) 8 250 0.71073 1.22 0.573 0.04(3) 0.0951 (0.0670)a,c 0.0536 (0.0569)a,c 1.16 and -0.62

a Cutoff ) I > 2σ(I). b wR2 ) [∑[w(Fo2 - Fc2)2]/∑[w(Fo2)2)]]1/2, w ) 1/[σ2(Fo2) + (aP)2 + bP) where P ) [max(Fo2 or O) + 2(Fc2)]/3. c wR2 ) w′[1 - ((||Fo | - |Fc||)/6σ(Fo))2]2 with w′ ) 1/∑rArTr(X) with coefficients 0.921, 0.0220, and 0.543 for a Chebyshev series for which X ) Fc/Fc(max). d Cutoff ) I > 3σ(I).

Figure 2. ORTEP diagram of the molecular structure of 6. Selected bond lengths (Å) and angles (deg): C17A-C20A 1.457(15); C20A-C21A 1.181(13); C21A-C22A 1.457(16); C22A-C23A 1.410(17); C22A-ReA 2.328(12); ReA-C27A, 1.896(14); C27A-O27A 1.150(13); C17A-C20A-C21A 177.6(12); C20A-C21A-C22A 179.2(13).

Figure 3. ORTEP diagram of the molecular structure of 12. Displacement ellipsoids are plotted for 50% probability. Selected bond lengths (Å) and angles (deg): C3-O1 1.226(3); C17-O2 1.437 (3); C17-C20 1.494(3); C20-C21, 1.199(3); C21-C22 1.443(3); C22-C23 1.442(4); C23-Fe 2.042(22); Fe-C27 2.056(3); C2-C3-O1 123.2(3); C17-C20-C21 178.8(3); C13-C17-C20, 110.90(18); C16-C17-C20 110.1(2); C20-C21-C22 174.2(3).

DHT as a tracer. The RBA values obtained for the compounds are reported in Table 2. The RBA values found for all the complexes are low (less than 1% except for 17). This result is quite unexpected for the 17R-ethynyl derivatives 6-8, 12, 15, and 17, which possess both the 3-keto and 17β-OH functions, which are considered to be essential for the recognition of a ligand by the androgen receptor. They are also lower than the values found for norethindrone (RBA ) 2.59) and norgestrel (RBA ) 16.59), two ethisterone derivatives (Chart 3).40 Quite surprisingly, a simple phenyl substituent causes a dramatic decrease in the RBA value (RBA ) 0.12 for 15), greater than that caused by the phenyl chromium tricarbonyl substituent (RBA ) 1.31 for 17). This means that there is not enough space in the binding pocket of the receptor to accommodate practically any substituent in the 17R-position, whether a bulky substituent like an organometallic piano stool or a sandwich unit or a

simple phenyl group. Recent publication of the crystal structure of the androgen receptor with one molecule of DHT bound at the active site41 has given access to docking experiments. Osella and his co-workers have done calculations where 17R-[Co2(CO)6]-ethisterone, a compound with a low RBA value for the AR (0.5%), replaced DHT in the binding site of the androgen receptor.28 Their results confirmed that the architecture of the binding site is incompatible with the insertion of almost any substituent at the 17R-position. This observation differs totally from what has been previously observed for steroidal estrogen derivatives.19,21-24 In this series, bulky organometallic 17Rderivatives of estradiol, and more precisely the corresponding derivatives of 6-8, 12, and 17, possess high RBA values (between 15% and 28%). On the other hand, this result is not very surprising for the androstane and androstene derivatives, 18 and 21, that lack the 3-keto group. Finally,

Organometallic Steroidal Androgen DeriVatiVes

Figure 4. ORTEP diagram of the molecular structure of 18. Displacement ellipsoids are plotted for 50% probability. Selected bond lengths (Å) and angles (deg): C3-O1 1.456(5); C17-O2 1.466(5); C17-C20 1.488(6); C20-C21, 1.215(6); C21-C22 1.443(6); C22-C23 1.426(7); C23-Fe 2.062(5); Fe-C27 2.053(5); C2-C3-O1 109.7(4); C17-C20-C21 171.6(5); C13-C17-C20, 110.9 (3); C16-C17-C20 110.0(4); C20-C21-C22 175.6(5). The molecule crystallizes with 0.5 equiv of water (see Figure 6).

Organometallics, Vol. 28, No. 5, 2009 1419

Figure 6. Packing of 18 showing hydrogen bonding between four 3β-OH (O1 · · · O1′ ) 2.739(4) Å) and between four 17β-OH and one molecule of water (O2′ · · · O100 ) 2.849(5) Å and O2′′ · · · O100 ) 2.799(5) Å). Table 2. Relative Binding Affinity Values (RBA) for the Androgen Receptor (AR) and Lipophilicity (log Po/w) of the steroidal complexes no.

compound

RBA (%)on ARa

log Po/w

6 7 8 12 15 17 18 21

DHT Testo-17RE-CpRe(CO)3 Testo-17RE-CpMn(CO)3 Testo-17RE-Fc DHT-17RE-Fc DHT-17RE-Ph DHT-17RE-Ph-Cr(CO)3 3β-androstanediol-17E-Fc 3β-androstanediol-16CdC-Fc

100 0.073 ( 0.007 0.068 ( 0.002 0.57 ( 0.05 0.32 ( 0.05 0.120 ( 0.005 1.31 ( 0.04 0.032 ( 0.004 0.020 ( 0.002

3.2 5.15 5.24 5.28 5.4 n.d. n.d. 5.4 n.d.

a

Mean of two experiments ( range; n.d.: not determined.

Chart 3. Ethisterone Derivatives

Figure 5. Hydrogen bonding in 12 between the 17β-OH and the oxygen of the 3-CO group of an adjacent molecule (O · · · O ) 2.793(3) Å).

as 3β-androstanediol has been described as the endogeneous ligand for the beta form of the estrogen receptor (ERβ) in the prostate, we also measured the RBA value on ERβ for the two corresponding complexes 18 and 21. Within our experimental conditions 18 and 21 show no affinity for ERβ. The values found for the lipophilicity of the new molecules (log Po/w, Table 2) are higher than the value found for DHT. This is expected, as all the added organometallic units are known to be lipophilic.22 The in Vitro antiproliferative effect of the 17R- and 16ferrocenyl androgen derivatives (8, 12, 18, 21) was then studied

on PC-3 (hormone-independent prostate cancer cells) and compared to the results obtained with testosterone and DHT (Figure 7). As expected, testosterone and DHT have no effect on this cell line lacking androgen receptor. Regarding the ferrocenyl complexes, their antiproliferative effect is quite modest at 1 µM but becomes high at 10 µM, especially for the 17R-complexes 8, 12, and 18. Thus, the IC50 values obtained are low (respectively 4.7, 8.3, and 5.5 µM) (Table 3). These values are significantly lower than that found for the corresponding 17R-ferrocenyl-estradiol derivative (IC50 ) 13.4 µM).26 This result was actually obtained on a different cell line (MDA-MB-231; hormone-independent breast cancer cells), but we have previously shown that other ferrocenyl complexes

1420 Organometallics, Vol. 28, No. 5, 2009

Top et al.

Figure 7. Effect of 10 nM of testosterone (Testo) and dihydrotestosterone (DHT) and of 1 or 10 µM of the ferrocenyl steroidal derivatives 8, 12, 18, and 21 on the growth of hormone-independent prostate cancer cells PC-3 after 5 days of culture. Nontreated PC-3 cells are used as the control. Mean of two separate experiments (six measurements for each one) ( range. Table 3. IC50 Values of the Ferrocenyl Complexes on PC-3 (hormone-independent prostate cancer cells) 8 12 18 21 a

compound

IC50 (µM)a

Testo-17RE-Fc DHT-17RE-Fc 3β-androstanediol-17E-Fc 3β-androstanediol-16CdC-Fc

4.7 ( 0.3 8.3 ( 0.2 5.5 ( 0.2 12.2 ( 0.2

Mean of two experiments ( range.

behaved similarly on these two cell lines.42 On the other hand, these IC50 values are very close, for 18, and even slightly better, for 8, than those found for the most active ferrocenyl derivative of the nonsteroidal antiandrogen nilutamide, 5-Fc-hydantoin (IC50 ) 5.4 µM).17 Consequently, this ferrocenyl-testosterone complex 8 is the most cytotoxic organometallic complex found so far in the steroidal/nonsteroidal androgen series. However, these IC50 values are significantly (about 10 times) higher than the values found for ferrocifens and conjugates but are in the same range as those of ferrocenyl diphenols linked by an sp3 carbon.16,42 One hypothesis is that the cytotoxicity of these two series of complexes might arise from Fenton-type chemistry associated with oxidation of ferrocene complexes to ferriceniumtype radical ions.16,43 These complexes have also been tested on the hormone-dependent prostate cancer cell line LNCaP (Figure 8). (39) Safwat, H. M.; El Gamal, M. H.; Hussein, M. M.; Abdullah, M. M. Bull. Fac. Pharm. 2002, 40, 47. (40) Fang, H.; Tong, W.; Branham, W. S.; Moland, C. L.; Dial, S. L.; Hong, H.; Xie, Q.; Perkins, R.; Owens, W.; Sheehan, D. M. Chem. Res. Toxicol. 2003, 16, 1338. (41) Sack, J. S.; Kish, K. F.; Wang, C.; Attar, R. M.; Kiefer, S. E.; An, Y.; Wu, G. Y.; Scheffler, J. E.; Salvati, M. E.; Krystek, S. R., Jr.; Weinmann, R.; Einspahr, H. M. Proc. Natl. Acad. Sci. USA 2001, 98, 4904. (42) Hillard, E. A.; Vessie`res, A.; Le Bideau, F.; Plazuk, D.; Spera, D.; Huche´, M.; Jaouen, G. ChemMedChem 2006, 1, 551. (43) (a) Tamura, H.; Miwa, M. Chem. Lett. 1997, 1177. (b) Osella, D.; Ferrali, M.; Zanello, P.; Laschi, F.; Fontani, M.; Nervi, C.; Cavigiolio, G. Inorg. Chim. Acta 2000, 306, 42. (c) Tabbi, G.; Cassino, C.; Cavigiolio, G.; Colangelo, D.; Ghiglia, A.; Viano, I.; Osella, D. J. Med. Chem. 2002, 45, 5786. (d) Ko¨pf-Maier, P.; Ko¨pf, H.; Neuse, E. W. Angew. Chem., Int. Ed. Engl. 1984, 23, 456.

Figure 8. Effect of 10 nM of testosterone (Testo) and dihydrotestosterone (DHT) and of 1 or 10 µM of the ferrocenyl steroidal derivatives 8, 12, and 18 on the growth of hormone-dependent prostate cancer cells LNCaP after 5 days of culture. Nontreated LNCaP cells are used as the control. Mean of two separate experiments (six measurements for each one) ( range.

On this cell line testosterone and DHT have an expected proliferative effect that occurs via their interaction with their specific androgen receptor. Complexes 8 and 12, which have low RBA values for this receptor, are slightly proliferative on these cells at a concentration of 1 µM and become antiproliferative at higher concentration (10 µM). However, these effects are less pronounced than those observed on PC-3 cells. This could be explained by the fact that the effects observed on this androgen receptor positive cell line is the net result of a positive proliferative effect mediated by an interaction with AR, which is expressed at low concentrations (1nM-1 µM), and of a negative antiproliferative effect due to the inherent cytotoxicity of the ferrocenyl unit that starts at 1 µM.

Conclusion A variety of organometallic steroids based on the androgen skeleton were prepared by modification of position 17 or 16. The organometallic moieties to be used were chosen on the basis of specific objectives as follows. The CpRe(CO)3 moiety will be useful as a nonradioactive model for future studies on radiopharmaceutical compounds based on 188Re and 99mTc.44 It should be noted, however, that all the modifications carried out in this study led to a serious decrease in affinity toward the androgen receptor. Fixation of the CpMn(CO)3 moiety was motivated by our discovery that the photochemical decomplexation of the Mn(CO)3 in a protic media allowed the liberation of a cyclopentadiene to serve as a stable intermediate in the synthesis of other organometallics.45 Modification by a ferrocenyl group was explored due to its previously demonstrated utility in medicinal chemistry. This metallocene can act either via its bioisosterism relative to the aryl groups46 or via its redox properties.47 It is shown here that such modifications give compounds with a significant antiproliferative effect on hormoneindependent PC-3 prostate cancer cells. This behavior is most likely linked to the Fenton-type properties of ferrocene. Although affinity for the androgen receptor is low, it should

Organometallic Steroidal Androgen DeriVatiVes

also be noted that the medications used to combat prostate cancer also show weak affinity for the androgen receptor. Still, the concept of employing the unusual properties of ferrocene labeling to provide access to cytotoxic compounds is demonstrated here for the first time on steroids with an androstanic skeleton.

Experimental Section General Procedures. All reactions were performed under a dry argon atmosphere using standard Schlenk techniques. Solvents were purified by conventional distillation techniques under argon. IR spectra were recorded on a Bomem Michelson 100 spectrophotometer. 1H NMR and 13C NMR spectra were recorded on a Bruker AM-200, 300, or 400 MHz Bruker spectrometer. 13C chemical shifts of 8 were used as a reference for the carbon attributions for 2, 6, 7, 11, 12, and 18. Mass spectra were obtained by the “Service de Spectrome´trie de Masse” of the ENSCP, Paris. Melting points were measured with a Kofler device. Elemental analyses were performed by the Regional Microanalysis Department of Universite´ Pierre et Marie Curie or by the Service de Microanalyse ICSN, Gif sur Yvette, France. High-resolution mass spectroscopy was carried out by the “Groupe de Spectroscopie de Masse” of the laboratory “Structure et Fonction de Mole´cules Bioactives” at the University of Pierre et Marie Curie, Paris. The compounds (η5-C5H4I)Re(CO)3 3,17 (η5-C5H4I)Mn(CO)3 4,17 iodoferrocene 5,18 and 3,3-(ethylendioxy)androstan-17-one 920 were prepared following literature procedures.

17r-(Tributylstannylethynyl)-4-androsten-17β-ol-3-one, 2. A mixture of 17β-ethynyl-4-androsten-17β-ol-3-one (1.875 g, 6.00 mmol) and Bu3SnOMe (4.009 g, 12 mmol) was heated for 5 h at 130 °C. After cooling to room temperature, pentane (100 mL) was added to precipitate the unreacted 17β-ethynyl-4-androsten-17βol-3-one. The solid was separated from the solution by filtration. The solution was then evaporated, and the crude product obtained was purified by chromatography on silica gel plates using ether/ pentane (1:1) as eluent to give 2, 2.040 g, 56% yield. Mp: 70 °C. 1 H NMR (CDCl3, 400 MHz): δ 5.72 (s, 1H, H(4)), 2.41-0.89 (m, 19H), 1.53 (m, 6H, 3 CH2, Bu), 1.31 (m, 6H, 3 CH2, Bu), 1.19 (s, 3H, CH3, H(19)), 0.96 (m, 6H, 3 CH2, Bu), 0.89 (s, 3H, CH3, H(18)), 0.87 (t, 9H, 3 CH3, Bu) ppm. 13C NMR (CDCl3, 100 MHz): δ 199.6 (C(3)), 171.3 (C(5)), 123.9 (C(4)), 113.9 and 88.3 (CtC), 80.2 (C(17)), 53.8 (C(9)), 50.0 (C(14)), 46.6 (C(13)), 39.2 (C(16)), 38,7 (C(10)), 36.3 (C(8), 35.8, 34.0, 32.9, 32.6, 31.7 (C(1), C(2), C(6), C(7) and C(12), 29.0, 26.9, and 11.2 (CH2CH2CH2 of SnBu3); 23.1 and 20.9 (C(15) and C(11)), 17.5 (C(19)), 13.6 (CH3 of SnBu3), 12.8 (C(18)) ppm. Anal. Calcd for C33H54O2Sn: C, 65.89; H, 9.05. Found: C, 66.00; H, 8.94. 17r-[(Cyclopentadienyltricarbonylrhenium)ethynyl]-4-androsten-17β-ol-3-one, 6. (η5-C5H4I)Re(CO)3 (0.348 g, 0.75 mmol) and (MeCN)2PdCl2 (0.009 g, 0.02 mmol) were dissolved in DMF (10 mL). 2 (0.460 g, 0.75 mmol) was then added, and the solution was stirred at room temperature for 3 h. Dichloromethane (40 mL) and an aqueous solution of 50% KF (15 mL) were added, and the stirring was maintained for 30 min. After hydrolysis with water (120 mL), extraction with dichloromethane (2 × 60 mL), and solvent removal, the residue was chromatographed on silica gel

Organometallics, Vol. 28, No. 5, 2009 1421 plates using ether/pentane (1:1) as eluent to give 6 as a yellowish solid, 0.435 g, 90% yield. Recrystallization in diethyl ether produced white crystals, mp 174 °C. IR (CH2Cl2) νCO: 2026, 1931, 1672 cm-1. 1 H NMR (CDCl3, 300 MHz): δ 5.72 (s, 1H, H(4)), 5.58 (t, 2H, J ) 2.4 Hz, C5H4), 5.29 (t, 2H, J ) 2.4 Hz, C5H4), 0.91-2.42 (m, 19H), 1.22 (s, 3H, CH3, (H19)), 0.93 (s, 3H, CH3, H(18)) ppm. 13C NMR (75 MHz, CDCl3): δ 199.5 (C(3)), 193.2 (Re(CO)), 171.0 (C(5)), 123.9 (C(4)), 87.6, 87.7 (C5H4), 92.8 (Ct), 84.0, 84.1 (C5H4), 85.4 (Ct), 79.9 (C(17)), 53.2 (C(9)), 50.1 (C(14)), 47.4 (C(13)), 38.6 (C(10)), 36,2 (C(8)), 38.8, 35.6, 33.9, 32.7, 32.6, 31.3 (C(1), C(2), C(6), C(7), C(12), C(16)), 23.1, 20.7, (C(11), C(15)); 17.4 (C(19)), 12.8 (C(18)) ppm. MS (EI, 70 eV) (m/z): 646 [M]+; 618 [M - CO]+; 562 [M - 3CO]+; 402. Anal. Calcd for C29H31O5Re: C, 53.94; H, 4.84. Found: C, 53.94; H, 4.81. 17r-[(Cyclopentadienyltricarbonylmanganese)ethynyl]-4-androsten-17β-ol-3-one, 7. (η5-C5H4I)Mn(CO)3 (0.253 g, 0.75 mmol) and (MeCN)2PdCl2 (0.009 g, 0.02 mmol) were dissolved in DMF (10 mL). 2 (0.460 g, 0.75 mmol) was then added, and the solution was stirred at room temperature for 3 h and 30 min. Dichloromethane (40 mL) and an aqueous solution of 50% KF (15 mL) were added, and the stirring was maintained for 30 min. After hydrolysis with water (120 mL), extraction with dichloromethane (2 × 60 mL), and solvent removal, the residue was chromatographed on silica gel plates using diethyl ether/pentane (1:1) as eluent to give 7 as a yellow solid: 0.291 g; 75% yield. Recrystallization in diethyl ether produced yellow crystals, mp 180 °C. IR (CH2Cl2) νCO: 2024, 1940, 1674 cm-1. 1H NMR (CDCl3, 300 MHz): δ 5.75 (s, 1H, H(4)), 4.68 (m, 2H, C5H4), 4.97 (m, 2H, C5H4), 2.40-0.91 (m, 19H), 1.19 (s, 3H, CH3, H(19)), 0.91 (s, 3H, CH3, H(18)) ppm. 13C NMR (75 MHz, CDCl3): δ 199.5 (C(3)), 171.1 (C(5)), 123.9 (C(4)), 92.4 (Ct), 86.2, 86.4 (C5H4), 82.0, 82.1 (Ct + C5H4), 80.0 (C(17)), 78.3 (C5H4 Cip), 53.3 (C(9)), 50.1 (C(14)), 47.2 (C(13)), 38.6 (C(10)), 38.8, 35.6, 33.9, 32.7, 32.6, 31.4 (C(1), C(2), C(6), C(7), C(12), C(16)), 23.1, 20.7 (C(11), C(15)), 17.4 (C(19)), 12.8 (C(18)) ppm. MS (EI, 70 eV) (m/z): 514 [M]+; 430 [M - 3CO]+. Anal. Calcd for C29H31O5Mn: C, 67.70; H, 6.07. Found: C, 67.65; H, 6.13. 17r-[Ferrocenylethynyl]-4-androsten-17β-ol-3-one, 8. Iodoferrocene (0.232 g, 0.75 mmol) and (MeCN)2PdCl2 (0.038 g, 0.15 mmol) were dissolved in DMF (10 mL). 2 (0.460 g, 0.75 mmol) was added, and the solution was stirred at room temperature for 1 h and 15 min. Diethyl ether (40 mL) and an aqueous solution of 50% KF (20 mL) were added, and the stirring was maintained for 30 min. After hydrolysis with water (120 mL), extraction with diethyl ether (2 × 40 mL), and solvent removal, the residue was chromatographed on silica gel plates using ether/pentane (9:1) as eluent to give 8 as an orange oil: 0.136 g, 36% yield. Recrystallization in diethyl ether produced orange crystals, mp 191 °C. IR (CH2Cl2) νCO: 1666 cm-1. The attribution of proton and carbon chemical shifts was obtained by using HMBC and HMQC NMR 2D techniques. Chemical shifts of H (1, 2, 6-9, 11-12, 14-16) appear as overlapping multiplets and cannot be attibuted with precise value. 1H NMR (CDCl3, 400 MHz): δ 5.73 (s, 1H, H(4)), 4.42 (t, 2H, C5H4, HR), 4.20 (s, 5H, C5H5), 4.20 (t, 2H, C5H4, Hβ), 2.48-2.30 (m, 2H, H(2)), 2.45-2.25 (m, 2H, H(6)), 2.40-2.25 and 2.10-2.00 (m, m, 1H, 1H, H(16)), 2.10-2.00 and 1.80-1.68 (m, m, 1H, 1H, H(1)), 1.89 and 1.10-1.05 (m, m, 1H, 1H, H(7)), (44) Masi, S.; Top, S.; Boubeker, L.; Jaouen, G.; Mundwiler, S.; Spingler, B.; Alberto, R. Eur. J. Inorg. Chem. 2004, 10, 2013. (45) (a) Top, S.; Kaloun, E. B.; Jaouen, G. J. Am. Chem. Soc. 2000, 122, 736. (b) Top, S.; Kaloun, E. B.; Vessie`res, A.; Laı¨os, I.; Leclercq, G.; Jaouen, G. J. Organomet. Chem. 2002, 643-644, 350. (c) Top, S.; Kaloun, E. B.; Toppi, S.; Herrbach, A.; McGlinchey, M. J.; Jaouen, G. Organometallics 2001, 20, 4554. (46) Metzler-Nolte, N.; Salmain, M. The Bioorganometallic Chemistry of Ferrocene. In Ferrocenes: Ligands, Materials and Biomolecules; Stepnicka, P., Ed.; John Wiley & Sons: Chichester, 2008; p 499. (47) Hillard, E. A.; Vessie`res, A.; Thouin, L.; Jaouen, G.; Amatore, C. Angew. Chem., Int. Ed. 2006, 45, 285.

1422 Organometallics, Vol. 28, No. 5, 2009 1.80-1.65 (m, 2H, H(12)), 1.80-1.70 and 1.40-1.32 (m, m, 1H, 1H, H(15)), 1.75-1.64 and 1.50-1.40 (m, m, 1H, 1H, H(11)), 1.62-1.55 (m, 1H, H(8)), 1.60-1.50 (m, 1H, H(14)), 1.00-0.89 (m, 1H, H(9)), 1.21 (s, 3H, CH3, H(19)), 0.93 (s, 3H, CH3, H(18)) ppm. 13C NMR (CDCl3, 100 MHz): δ 199.6 (C(3)), 171.2 (C(5)), 124.1 (C(4)), 88.7 (C(20)), 84.6 (C(21)), 80.2 (C(17)), 71.7 and 71.6 (C5H4, 2 CR), 70.0 (Cp), 68.9 (C5H4, 2 Cβ), 65.1 (C5H4, Cip), 53.9 (C(9)), 50.2 (C(14)), 47.1 (C(13)), 39.1 (C(16)), 38.7 (C(10)), 36.4 (C(8)), 35.9 (C(1)), 34.1 (C(2)), 32.8 (C(6) and C(12)), 31.7 (C(7)), 23.2 (C(15)), 20.9 (C(11)), 17.5 (C(19)), 13.0 (C(18)) ppm. MS (EI, 70 eV) (m/z): 496 [M]+; 478 [M - H2O]+; 431[M - Cp]+. Anal. Calcd for C31H36O2Fe: C, 74.99; H, 7.31. Found: C, 74.68; H, 7.42. 3,3-(Ethylendioxy)-17r-[ferrocenylethynyl]androstan-17βol, 11. In a Schlenk tube, ethynylferrocene (0.947 g, 4.51 mmol) was dissolved in THF (10 mL). The solution was cooled to -78 °C and n-BuLi (2.0 M in hexane) (2.25 mL, 4.50 mmol) was added dropwise. After stirring for 1 h, a solution of 3,3-(ethylendioxy)androstan-17-one (9) (1.000 g, 3.01 mmol) in THF (6 mL) was added. The reaction mixture was stirred for 26 h at room temperature before being poured into a saturated sodium chloride solution (25 mL). The organic phase was extracted with dichloromethane (3 × 25 mL). After solvent removal, the residue was chromatographed on a silica gel column using dichoromethane/ethyl acetate (97:3) as eluent. Compound 11 was isolated as an orange solid: 0.616 g, 39% yield, mp 85 °C. IR (KBr) νOH: 3443, νCtC: 2222, cm-1. 1H NMR (CDCl3, 300 MHz): δ 4.41-4.39 (m, 2H, C5H4), 4.20-4.17 (m, 7H, Cp + C5H4), 3.93 (s, 4H, OCH2CH2O), 2.27-1.23 (m, 22H), 0.87 (s, 3H, CH3, H(19)), 0.84 (s, 3H, CH3, H(18)). 13C NMR (CDCl3, 50 MHz): δ 109.2 (C(3)), 88.9 (C5H4, Cip), 84.0, 82.8 (CtC), 80.3 (C(17)), 71.4 (C5H4), 69.8 (Cp), 68.6 (C5H4), 64.0 (OCH2CH2O), 53.9 (C(9)), 50.5 (C(14)), 47.2 (C(13)), 43.6 (C(5)), 38.96 (C(16)), 37.8 (C(4)), 36.1 (C(1)), 35.8 (C(10)), 35.46 (C(8)), 32.9 (C(12)), 31.5 (C(7)), 31.0 (C(2)), 28.3 (C(6)), 23.1 (C(15)), 20.8 (C(11)), 12.9 (C(18)), 11.3 (C(19)). HRMS (ESI): exact mass calcd for C33H42FeO3+ 542.24784, found 542.24665. 17r-[Ferrocenylethynyl]androstan-17β-ol-3-one, 12. 11 (0.500 g, 0.922 mmol) was dissolved in acetone (10 mL) before APTS monohydrate (0.010 g, 0.053 mmol) and water (50 µL) were added. The reaction mixture was stirred for 6 h at room temperature. After solvent removal, the residue was chromatographed on a silica gel column using petroleum ether/ethyl acetate (4:1) as eluent. Compound 12 was isolated as an orange solid: 0.413 g, 90% yield, mp 215 °C. IR (CHCl3) νOH: 3573 cm-1, νCH (ferrocene): 3097 cm-1, νCO: 1714 cm-1. 1H NMR (C6D6, 200 MHz): δ 4.34 (t, 2H, J ) 1.9 Hz, C5H4); 4.13 (s, 5H, Cp), 3.91 (t, 2H, J ) 1.9 Hz, C5H4), 2.34-1.34 (m, 22H), 0.90 (s, 3H, CH3, H(19)), 0.60 (s, 3H, CH3, H(18)) ppm. 13C NMR (CDCl3, 50 MHz): δ 212.0 (C(3)), 88.9 (C5H4, Cip), 84.1 (CtC), 80.2 (C(17)), 71.5 (C5H4), 69.9 (Cp), 68.7 (C5H4), 53.7 (C(9)), 50.4 (C(14)), 47.2 (C(13)), 46.6 (C(5)), 44.5 (C(4)), 38.9 (C(16)), 38.5 (C(1)), 38.0 (C(2)), 36.0 (C(8)), 35.6 (C(10)), 32.8 (C(12)), 31.3 (C(7)), 28.7 (C(6)), 23.1 (C(15)), 21.1 (C(11)), 12.9 (C(18)), 11.4 (C(19)) ppm. MS (EI, 70 eV) (m/z): 498 [M]+; 210 [ethynylferrocene]+, 121 [FeCp]+. HRMS (ESI): exact mass calcd for C31H38FeO2+ 498.22162, found 498.22052. 3,3-(Ethylendioxy)-17r-[phenylethynyl]androstan-17β-ol, 14. In a Schlenk tube, phenylacetylene (0.50 mL, 4.51 mmol) was dissolved in THF (10 mL). The solution was cooled to -78 °C and n-BuLi (2.5 M in hexane) (1.80 mL, 4.51 mmol) was added dropwise. After stirring for 1 h, a solution of 3,3-(ethylendioxy)androstan-17one (9) (1.00 g, 3.01 mmol) in THF (6 mL) was added. The reaction mixture was stirred for 28 h at room temperature before it was poured into a saturated sodium chloride solution (25 mL). The organic phase was extracted with dichloromethane (3 × 25 mL). After solvent removal, the residue was purified by chromatography on silica gel using dichloromethane/ethyl acetate (99:1) as eluent. Compound 14 was isolated as a white solid: 1.046 g, 80% yield. IR (CHCl3) νOH: 3599

Top et al. cm-1. 1H NMR (CDCl3, 200 MHz): δ 7.48-7.41 (m, 2H, Ph), 7.37-7.31 (m, 3H, Ph), 3.95 (bs, 4H, OCH2CH2O), 2.45-1.20 (m, 22H), 0.91 (s, 3H, CH3, H(19)), 0.86 (s, 3H, CH3, H(18)) ppm. 13C NMR (CDCl3, 50 MHz): δ 131.5 and 128.2 (Ph), 122.9 (Ph, Cip), 109.3, 92.8, 85.7, 80.2, 64.0, 53.8, 50.6, 47.3, 43.6, 38.9, 37.8, 36.1, 35.9, 35.4, 32.9, 31.4, 31.0, 28.3, 23.1, 20.8, 12.9, 11.3 ppm. MS (EI, 70 eV) (m/z): 434 [M]+. HRMS (ESI): exact mass calcd for C29H38O3Na+ 457.27132, found 457.27060. 17r-[Phenylethynyl]androstan-17β-ol-3-one, 15. 14 (0.500 g, 1.15 mmol) was dissolved in acetone (10 mL) before APTS monohydrate (0.010 g, 0.053 mmol) and water (50 µL) were added. The reaction mixture was stirred for 7 h at room temperature. After solvent removal, the residue was chromatographed on a silica gel column using petroleum ether/ethyl acetate (5:1) as eluent. Compound 15 was isolated as a white solid: 0.349 g, 89% yield, mp 112 °C. IR (CHCl3) νOH: 3603 cm-1, νCtC: 2244 cm-1, νCO: 1706 cm-1. 1H NMR (CDCl3, 200 MHz): δ 7.50-7.44 (m, 2H, Ph), 7.36-7.32 (m, 3H, Ph), 2.60-0.70 (m, 22 H), 0.94 (s, 3H, CH3, H(19)), 0.91 (s, 3H, CH3, H(18)) ppm. 13 C NMR (CDCl3, 50 MHz): δ 211.8, 131.5, 128.2, 122.9, 92.6, 80.2, 53.4, 50.4, 47.3, 46.5, 44.6, 38.9, 38.4, 38.0, 36.0, 35.6, 32.8, 31.1, 28.7, 23.1, 21.1, 12.9, 11.4 ppm. MS (EI, 70 eV) (m/z): 390 [M]+, 375 [M - CH3]+. Anal. Calcd for C27H34O2: C, 83.03; H, 8.77. Found: C, 82.81; H, 9.12. 3,3-(Ethylendioxy)-17r-[(phenyltricarbonylchromium)ethynyl]androstan-17β-ol, 16. In a Schlenk tube, 14 (0.434 g, 1 mmol) and Cr(CO)3(NH3)3 (0.243 g, 1.3 mmol) were dissolved in anhydrous dioxane (12 mL). The solution was heated at reflux for 8 h. After solvent removal, the residue was chromatographed on a silica gel column using petroleum ether/ethyl acetate (3:2) as eluent. Compound 16 was isolated as a yellow solid: 0.180 g, 32% yield. IR (CHCl3) νOH: 3594 cm-1, νCO: 1977 cm-1, 1907 cm-1. 1H NMR (C6D6, 400 MHz): δ 4.78 (d, 2H, J ) 6.2 Hz, Ph), 4.28 (t, 2H, J ) 6.2 Hz, Ph), 4.15 (t, 1 H, J ) 6.2 Hz, Ph), 3.60-3.50 (m, 4H, OCH2CH2O), 2.40-0.80 (m, 23H), 0.91 (s, 3H, CH3, H(19)), 0.85 (s, 3H, CH3, H(18)) ppm. 13C NMR (C6D6, 100 MHz): δ 232.6 (CO), 109.3, 94.6, 94.5, 91.7, 90.6, 90.1, 82.0, 80.1, 64.2, 64.1, 54.1, 51.2, 47.9, 43.8, 39.6, 38.7, 36.4, 35.8, 33.5, 31.8, 28.8, 23.5, 21.4, 13.1, 11.5 ppm. HRMS (ESI): exact mass calcd for C32H39CrO6+ 571.21463, found 571.21390. 17r-[(Phenyltricarbonylchromium)ethynyl]androstan-17β-ol3-one, 17. 16 (0.170 g, 0.297 mmol) was dissolved in acetone/ dichloromethane (5 mL:5 mL), and PTSA monohydrate (0.003 g, 0.015 mmol) was added. The reaction mixture was stirred for 7.5 h at room temperature before it was poured into water (10 mL). The organic phase was extracted with dichloromethane (3 × 10 mL) and dried over MgSO4. After solvent removal, the residue was chromatographed on a silica gel column using petroleum ether/ ethyl acetate (3:2) as eluent. Compound 17 was isolated as a yellow solid: 0.054 g, 34% yield, mp 138 °C. IR (C6D6) νOH: 3597, νCO: 1976, 1908 cm-1. 1H NMR (C6D6, 200 MHz): δ 4.82 (d, 2H, J ) 6.0 Hz, Ph), 4.31 (t, 2H, J ) 6.0 Hz, Ph), 4.18 (t, 1H, J ) 6.0 Hz, Ph), 2.50-0.50 (m, 23 H), 0.86 (s, 3H, CH3, H(19)), 0.61 (s, 3H, CH3, H(18)) ppm. MS (EI, 70 eV) (m/z): 526 [M]+; 442 [M (CO)3]+, 390 [M - Cr(CO)3]+. 13C NMR (C6D6, 100 MHz): δ 232.7, 208.7, 94.6, 94.5, 91.8, 90.5, 90.3, 82.0, 80.0, 65.8, 53.5, 50.9, 47.8, 46.3, 44.6, 39.6, 38.4, 38.0, 36.2, 35.6, 33.4, 31.3, 30.4, 28.8, 23.5, 21.4, 15.5, 13.1, 11.1 ppm. HRMS (ESI): exact mass calcd for C30H34CrO5Na+ 549.17036, found 549.16950. 17r-[Ferrocenylethynyl]androstan-3β,17β-ol, 18. 12 (0.100 g, 0.2 mmol) was suspended in methanol (22 mL), and the reaction mixture was shielded from light. NaBH4 (0.028 g 0.74 mmol) was then added in three portions at 0 °C, and it was left stirring 1 h at 0 °C before the solvents were evaporated. The crude yellow oil was purified by chromatography on silica gel using petroleum ether/ ethyl acetate (4:1), and compound 18 was isolated as a yellow solid: 0.061 g; 60% yield, mp 112 °C. IR (KBr) νOH: 3299, νCtC: 2222 cm-1. 1H NMR (CDCl3, 300 MHz): δ 4.34 (m, 2H, C5H4); 4.13 (s,

Organometallic Steroidal Androgen DeriVatiVes 5H, Cp), 4.11 (t, 2H, J ) 1.9 Hz, C5H4), 3.50 (quint, 1H, J ) 1.9 Hz, H(3)), 2.27-1.16 (m, 24H), 0.80 (s, 3H, CH3, H(19)), 0.76 (s, 3H, CH3, H(18)) ppm. 13C NMR (CDCl3, 75 MHz): δ 89.2 (C5H4, Cip), 84.0 (CtC), 80.3 (C(17)), 71.5, 71.4 (C5H4), 71.2 (C(3)), 69.8 (Cp), 68.6 (C5H4), 54.4 (C(9)), 50.6 (C(14)), 47.3 (C(5)), 47.2 (C(13)), 44.9 (C(4)), 39.0 (C(16)), 38.1 (C(1)), 37.1 (C(2)), 36.2 (C(8)), 35.6 (C(10)), 33.0 (C(12)), 31.5 (C(7)), 28.6 (C(6)), 23.2 (C(15)), 21.0 (C(11)), 13.0 (C(18)), 12.3 (C(19)) ppm. MS (CI) (m/z): 501 [M + H]+; 210 [ethynylferrocene]+. HRMS (CI): exact mass calcd for C31H40FeO2 500.2456, found 500.2402. 3,3-(Ethylendioxy)-16-[ferrocenylmethylene]androstan-17one, 19. 3,3-(ethylendioxy)androstan-17-one (9) (0.22 g, 0.66 mmol) was dissolved in THF (2 mL) and was added to a suspension of 60% dispersed NaH (0.033 g, 0.02 g) in THF (2 mL). The cloudy white reaction mixture was stirred for 45 min at room temperature before a solution of ferrocenecarboxaldehyde (0.26 g, 1.22 mmol) in THF (2 mL) was added. The reaction mixture was then stirred at reflux for 3 h before water was added to quench the reaction. The organic phase was extracted with ethyl acetate, washed with water, and dried over sodium sulfate, and the solvents were evaporated. The red oil was purified by column chromatography using dichloromethane as eluent to give pure 19 as a red solid: 0.26 g, 75% yield, mp 87 °C. IR (KBr) νCO: 1712, νCdC: 1622 cm-1. The attribution of proton and carbon chemical shifts was obtained by using HMBC and HMQC NMR 2D techniques. 1H NMR (CDCl3, 300 MHz): δ 7.26 (s, 1H, CHd); 4.51 (d, 2H, J ) 5.7 Hz, C5H4); 4.40 (d, 2H, J ) 9.6 Hz, C5H4), 4.11 (s, 5H, Cp); 3.92 (s, 4H, CH2O), 2.68 (dd, 1H, J ) 4.7 Hz, J ) 11.6 Hz, H(15)), 2.16-2.08 (m, 1H, H(14)), 1.90-1.05 (m, 18H), 0.91 (s, 3H, CH3, H(19)), 0.86 (s, 3H, CH3, H(18)) ppm. 13C NMR (CDCl3, 75 MHz): δ 209.3 (C(17)), 133.9 (CHd), 132.7 (C(16)), 109.2 (C(3)), 79.0 (C5H4, Cip), 71.6, 70.8, 69.6 (C5H4), 69.5 (Cp), 64.1 (CH2O), 54.3 (C(9)), 49.3 (C(14)), 47.6 (C(13)), 43.7 (C(5)), 38.0 (C(4)), 35.9 (C(1)), 35.7 (C(10)), 34.7 (C(8)), 31.7 (C(2)), 31.1 (C(7)), 31.0 (C(6)), 28.7 (C(15)), 28.3 (C(12)); 20.6 (C(11)), 14.7 (C(18)), 11.4 (C(19)) ppm. MS (CI) (m/z): 529 [M + H]+. Anal. Calcd for C32H40FeO3: C, 72.72; H, 7.63. Found: C, 72.21; H, 7.79. HRMS (ESI): exact mass calcd for C32H40FeO3+ 528.23214, found 528.23113. 16-[Ferrocenylmethylene]androstan-3,17-one, 20. A solution of 19 (0.10 g, 0.189 mmol) in a mixture of acetic acid/water (2.5 mL/1 mL) was heated at 55 °C for 30 min. After cooling to room temperature, dichloromethane was added and the phases separated. The organic phase was dried over sodium sulfate, and the solvents evaporated to give a red solid of pure compound 20: 0.91 g, 100%, mp 217 °C. IR (KBr) ν2CO: 1706, νCdC: 1619 cm-1. The attribution of proton and carbon chemical shifts was obtained by using HMBC and HMQC NMR 2D techniques. 1H NMR (CDCl3, 300 MHz): δ 7.28 (s, 1H, CH)), 4.51 (m, 2H, C5H4), 4.41 (t, 2H, J ) 4.4 Hz, C5H4), 4.11 (s, 5H, Cp), 2.69 (dd, 1H, J ) 5.4 Hz, J ) 15.4 Hz, H(15)), 2.35-1.18 (m, 19H), 1.07 (s, 3H, CH3, H(19)), 0.94 (s, 3H, CH3, H(18)) ppm. 13C NMR (CDCl3, 75 MHz): δ 211.5 (C(3)), 208.9 (C(17)), 134.2 (CH)), 132.4 (C(16)), 78.8 (C5H4, Cip), 71.6, 70.8, 69.7 (C5H4), 69.5 (Cp), 54.0 (C(9)), 49.2 (C(14)), 47.5 (C(13)), 46.6 (C(5)), 44.6 (C(4)), 38.3 (C(2)), 38.06 (C(1)), 35.9 (C(10)), 34.6 (C(8)), 31.6 (C(7)), 30.7 (C(6)), 28.7 (C(15), C(12)), 20.6 (C(11)), 14.7 (C(18)), 11.4 (C(19)). MS (CI) (m/z) ppm: 485 [M + H]+. HRMS (ESI): exact mass calcd for C30H36FeO2+ 484.20592, found 484.20500. 16-[Ferrocenylmethylene]androstan-3β,17β-ol, 21. 20 (0.07 g, 0.144 mmol) was suspended in methanol (20 mL), and the reaction mixture was shielded from light. NaBH4 (0.028 g, 0.72 mmol) was then added in three portions at room temperature, and it was left stirring 2 h at room temperature before the solvents were evaporated. Water was added, and the organic phase was extracted with dichloromethane. After drying on sodium sulfate, the solvents were evaporated to yield a yellow solid, which was purified by column chromatography on silica

Organometallics, Vol. 28, No. 5, 2009 1423 gel using dichloromethane/ethyl acetate (9:1), and compound 21 was isolated as a yellow solid: 0.041 g; 60% yield, mp 89 °C. IR (KBr) νOH: 3422, νCdC: 16242 cm-1. The attribution of proton and carbon chemical shifts was obtained by using HMBC and HMQC NMR 2D techniques. 1H NMR (CDCl3, 300 MHz): δ 6.17 (s, 1H, CHd), 4.35 (sl, 2H, C5H4), 4.19 (sl, 2H, C5H4), 4.08 (s, 5H, Cp), 3.92 (s, 1H, H(17)), 3.61 (quint, 1H, J ) 5.1 Hz, H(3)), 2.45 (dd, J ) 7.1 Hz, J ) 16.4 Hz, H(15)), 2.35-1.18 (m, 19H), 0.84 (s, 3H, CH3, H(19)), 0.65 (s, 3H, CH3, H(18)) ppm. 13C NMR (CDCl3, 75 MHz): 142.5 (C(16)), 119.6 (CHd), 109.2 (C(3)), 84.7 (C(17)), 83.0 (C5H4, Cip), 71.2 (C(3)), 68.4, 68.3 (C5H4), 68.9 (Cp), 54.6 (C(9)), 48.3 (C(14)), 44.9 (C(5)), 42.32 (C(13)), 38.1 (C(4)), 37.0 (C(1)), 36.41 (C(2)), 35.7 (C(10)), 35.0(C(8)), 31.8 (C(12)), 31.5 (C(7)), 30.5 (C(15)), 28.5 (C(6)), 20.9 (C(11)), 12.4 (C(18)), 11.1 (C(19)) ppm. MS (CI) (m/z): 488 [M + H]+. HRMS (CI): exact mass calcd for C30H40FeO2 488.2378, found 488.2372. X-ray Crystal Structure Determination for 6. Data were collected at 296 K with a Siemens SMART diffractometer equipped with a CCD two-dimensional detector with Mo KR radiation. Data processing was carried out with the program SAINT. Data were corrected for Lorentz and polarization effects, and a semiempirical absorption correction based on symmetry equivalent reflections was applied by using the SADABS program.48 All non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atoms were located with geometrical restraints in riding mode. Orientation matrix and lattice parameters were obtained by leastsquares refinement of the diffraction data of 7325 reflections. The index ranges of data collection were -11 e h e 7, -10 e k e 10, -35 e l e 32. Intensity data were collected in the θ range 0.68-25°, and all reflections (8787) were used in the refinement with 636 variables and 1 restraint. X-ray Crystal Structure Determination for 12 and 18. Data were collected at 250 K with a Kappa-CCD Enraf-Nonius diffractometer with Mo KR radiation. The structure was solved by direct methods and refined with full-matrix least-squares technique on F using the CRYSTALS1 programs.49 All non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atoms were located with geometrical restraints in riding mode. 12: Orientation matrix and lattice parameters were obtained by leastsquares refinement of the diffraction data of 258 reflections within the range 3° < θ < 21°. The index ranges of data collection were -10 e h e 10, -12 e k e 23, -29 e l e 20. Intensity data were collected in the θ range 2-30°, 4515 have (Fo)2 g 3σ(Fo)2, and these were used for the refinement with 309 variables and zero restraints. 18: Orientation matrix and lattice parameters were obtained by leastsquares refinement of the diffraction data of 166 reflections within the range 3° < θ < 21°. The index ranges of data collection were -29 e h e 32, -36 e k e 19, -11 e l e 7. Intensity data were collected in the θ range 2-30°, 3466 have (Fo)2 g 2σ(Fo)2, and these were used for the refinement with 313 variables and one restraint. Biochemical Experiments. Materials. DHT, testosterone, and protamine sulfate were obtained from Sigma-Aldrich (France). Stock solutions (1 × 10-3 M) of the compounds to be tested were prepared in DMSO and were kept at -20 °C. Under these conditions, they are stable for at least two weeks. Serial dilutions in DMSO were prepared just prior to use. Dulbecco’s modified Eagle medium (DMEM) for PC3 and RPMI 1640 for LNCaP were purchased from Invitrogen. Fetal bovine serum, glutamine, and kanamycin were obtained from Invitrogen. Prostate cancer cells LNCaP and PC3 cells were from American Type Culture Collection (ATCC, LGC Promochem). [1,2-3H]-DHT was purchased from NEN Life Science Product. Determination of the RBA of the Compounds for the Androgen Receptor (AR). RBA values were measured on a PanVera AR (750 pmol) purchased from Invitrogen. This AR is a (48) (a) Sheldrick, G. M. SADABS; University of Go¨ttingen Go¨ttingen: Germany, 1997. (b) Blessing, R. H. Acta Crystallogr. 1998, 51, 33.

1424 Organometallics, Vol. 28, No. 5, 2009 recombinant rat protein expressed in Escherichia coli. The amino sequence of the ligand binding domain of this AR is identical to that of the human AR LBD. ARs were aliquoted in 15 µL fractions and kept in liquid nitrogen until used. For each experiment, 10 mL of buffer containing 10% glycerol, 50 mM Tris pH 7.5, 0.8 M NaCl, 2 mM DTT, and 0.1% BSA was added to one aliquot. Fractions of 200 µL of the AR solution were incubated in polypropylene tubes for 3 h and 30 min at 4 °C with [1,2-3H]-DHT (2 × 10-9 M, specific activity 1.6 TBq/mmol) in the presence of nine concentrations of the compounds to be tested (between 1 × 10-5 and 6 × 10-7 M) or of nonradioactive DHT (between 8 × 10-8 and 7.5 × 10-10 M). At the end of the incubation period, the fractions of [3H]-DHT bound to the androgen receptor (Y values) were precipitated by addition of 200 µL of a cold solution of protamine sulfate (1 mg/ mL in water). After a 10 min period of incubation at 4 °C, the precipitates were recovered by filtration on 25 mm diameter glass microfiber GF/C filters using a Millipore 12 well filtration ramp. The filters were rinsed twice with cold phosphate buffer and then transferred into 20 mL plastic vials. After addition of 5 mL of scintillation liquid (BCS Amersham) the radioactivity of each fraction was counted in a Packard tricarb 2100TR liquid scintillation analyzer. The concentration of unlabeled steroid required to displace 50% of the bound [3H]-DHT was calculated for DHT and for each complex by plotting the logit values of Y (logit Y ) ln(Y/100 - Y) versus the mass of the competing complex. The RBA was calculated as follows: RBA of a compound ) concentration of DHT required to displace 50% of [3H]-DHT × 100/concentration of the compound required to displace 50% of [3H]-DHT. The RBA value of DHT is by definition equal to 100%. Measurement of Octanol/Water Partition Coefficient (log Po/w) of the Compounds. The log Po/w values of the compounds were determined by reversed-phase HPLC on a C-8 column (Kromasil C8 from AIT) according to the method previously used.17 Measurement of the chromatographic capacity factors (k′) for each molecule was done at various concentrations in the range 95-80% methanol (containing 0.25% octanol) and an aqueous phase consisting of 0.15% n-decylamine in 0.02 M MOPS (3-morpholinopropanesulfonic acid) buffer pH 7.4 (prepared in 1-octanolsaturated water). These capacity factors (k′) are extrapolated to 100% of the aqueous component given the value of kw′. The log Po/w is then obtained by the formula log Po/w ) 0.13418 + 0.98452 log kw′.

Top et al. Culture Conditions. Cells were maintained in monolayer culture in DMEM or RPMI 1640 with phenol red/Glutamax I, supplemented with 9% decomplemented fetal bovine serum and 0.9% kanamycine, at 37 °C in a 5% CO2 air humidified incubator. For proliferation assays, PC3 cells were seeded at a density of 15 000 to 25 000 cells per mL and 25 000 to 35 000 cells per mL for LNCaP in 24-well sterile plates in 1 mL of medium, supplemented with 9% fetal bovine serum desteroided on dextran charcoal, 0.9% Glutamax I, and 0.9% kanamycine, and were incubated 24 h. The following day (D0), 1 mL of the same medium containing the compounds to be tested diluted in DMSO was added to the plates (three wells for each product). After 3 days (D3), the incubation medium was removed and 2 mL of fresh medium containing the compounds was added. At different days (D3, D4, D5), the protein content of each well was quantified by methylene blue staining as follows. Cell monolayers were fixed and stained for 1 h in methanol with methylene blue (2.5 mg/mL) and then washed thoroughly with water. Two milliliters of HCl (0.1 M) was then added, and the plate was incubated for 1 h at 37 °C. Then the absorbance of each well was measured at 655 nm with a Biorad spectrophotometer (microplate reader). The results are expressed as the percentage of proteins versus the control. Experiments were performed at least in duplicate. Crystallographic data for the structural analyses have been deposited at the Cambridge Crystallographic Data Centre, CCDC Nos. 706482 (6), 707351 (12), and 707352 (18).

Acknowledgment. We thank the Centre National de la Recherche Scientifique and the French Ministry of Research and Technology for financial support, E. A. Hillard for helpful discussion, M.-N. Rager for NMR analysis, E. Salomon and A. Cordaville for technical assistance, and B. McGlinchey for correction of the manuscript. Supporting Information Available: Crystallographic data for the structural analyses are available free of charge via the Internet at http://pubs.acs.org. OM800698Y (49) Betteridge, P. W.; Carruthers, J. R.; Cooper, R. I.; Prout, K.; Watkin, D. J. J. Appl. Crystallogr. 2003, 36, 1487.