Synthesis of 7α-(Fluoromethyl) dihydrotestosterone and 7α

Jun 22, 2007 - Although prostate cancer growth is regulated by androgens through the androgen receptor (AR), in vitro assays of AR levels in prostate ...
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Synthesis of 7r-(Fluoromethyl)dihydrotestosterone and 7r-(Fluoromethyl)nortestosterone, Structurally Paired Androgens Designed To Probe the Role of Sex Hormone Binding Globulin in Imaging Androgen Receptors in Prostate Tumors by Positron Emission Tomography Ephraim E. Parent, Kathryn E. Carlson, and John A. Katzenellenbogen* Department of Chemistry, UniVersity of Illinois, Urbana, Illinois 61801 [email protected] ReceiVed February 20, 2007

Although prostate cancer growth is regulated by androgens through the androgen receptor (AR), in vitro assays of AR levels in prostate tumors have limited prognostic value. This might be improved by direct measurement of tumor AR in vivo using positron emission tomography (PET) imaging with fluorine18-labeled androgens. Most AR PET imaging agents have been designed to limit steroid binding to serum proteins, but there is evidence that binding to sex hormone binding globulin (SHBG) might enhance tumor uptake. To probe the role of SHBG in prostate tumor uptake of PET imaging agents, we have synthesized two fluoro steroids, 7R-(fluoromethyl)dihydrotestosterone (7R-FM-DHT) and 7R-(fluoromethyl)nortestosterone (7R-FM-norT), by a route amenable to their labeling with [18F]fluoride ion. Both compounds have high affinity for AR, but 7R-FM-norT has much lower affinity for SHBG. Thus, these two fluoro steroids are well matched in terms of their site of fluorine labeling, similarity of structure, and equivalent AR binding affinitysbut contrasting SHBG bindingsand therefore can be used as agents for evaluating the role of SHBG binding in the target tissue uptake of AR PET imaging agents in humans.

Introduction The majority of prostate tumors express high levels of androgen receptor (AR), which is thought to be important for sustaining hormone-regulated tumor growth. The presence and concentration of this receptor, as currently measured by immunohistochemical or biochemical assays of tumor biopsy samples, is, however, often poorly correlated with the prognosis of prostate cancer or tumor responsiveness to endocrine therapies.1 To improve the predictive value of AR, efforts are being made to determine the AR content of tumors directly by noninvasive, in vivo imaging using radiolabeled AR ligands and positron * To whom correspondence should be addressed. Phone: (217) 333 6310.

(1) Blankenstein, M. A.; Bolt-de Vries, J.; van Aubel, O. G.; van Steenbrugge, G. J. Scand. J. Urol. Nephrol. Suppl. 1988, 107, 39-45.

emission tomography (PET). Biodistribution studies with a few radiolabeled androgens in animals and a limited number of PET imaging studies in baboons and human prostate cancer patients suggest that this approach has promise.2-4 The design of radiolabeled steroids for PET imaging of AR in prostate tumors has traditionally been based on the bioavail(2) Bonasera, T. A.; O’Neil, J. P.; Xu, M.; Dobkin, J. A.; Cutler, P. D.; Lich, L. L.; Choe, Y. S.; Katzenellenbogen, J. A.; Welch, M. J. J. Nucl. Med. 1996, 37, 1009-1015. (3) Larson, S. M.; Morris, M.; Gunther, I.; Beattie, B.; Humm, J. L.; Akhurst, T. A.; Finn, R. D.; Erdi, Y.; Pentlow, K.; Dyke, J.; Squire, O.; Bornmann, W.; McCarthy, T.; Welch, M.; Scher, H. J. Nucl. Med. 2004, 45, 366-373. (4) Zanzonico, P. B.; Finn, R.; Pentlow, K. S.; Erdi, Y.; Beattie, B.; Akhurst, T.; Squire, O.; Morris, M.; Scher, H.; McCarthy, T.; Welch, M.; Larson, S. M.; Humm, J. L. J. Nucl. Med. 2004, 45, 1966-1971. 10.1021/jo070328b CCC: $37.00 © 2007 American Chemical Society

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Published on Web 06/22/2007

Synthesis of Androgens 7R-FM-DHT and 7R-FM-norT

ability hypothesis, which assumes that only the unbound fraction of steroid in blood is available for uptake by AR-positive target cells.5 In humans, androgens are extensively bound in the blood by sex hormone binding globulin (SHBG), corticosteroid binding globulin, and albumin. In normal men and women, between 40% and 65% of the circulating testosterone is bound to SHBG6,7 and the remainder is bound to albumin, leaving only about 1-2% of the circulating testosterone free or unbound. It was believed that, for in vivo imaging, AR radioligands should possess high binding affinity for AR and low affinity for other steroid receptors, including SHBG. This would ensure that free steroid levels were adequate for good cellular uptake by ARpositive tumor cells. On the other hand, the binding of testosterone to SHBG is known to decrease the metabolic conversion rate of testosterone to androstenedione8 and thus slow the rate at which testosterone is cleared from the circulation. Prolongation of blood levels might elevate the target tissue uptake of SHBG-bound steroids such as testosterone by a passive process. Also, there are reports that SHBG facilitates target cell uptake of androgens more directly, through its own receptor, SHBG-R, also termed megalin.9 According to these reports, SHBG-R, a plasma membrane protein present in androgen target cells, mediates endocytosis of SHBG, carrying along bound steroids.10-15 This mechanism has been questioned directly and is still considered controversial.9,16,17 Nevertheless, should either of these SHBGmediated processes be operative, radiopharmaceuticals with a high affinity for SHBG might, in fact, be more effectively delivered to the AR target tissues and thus more effective for PET imaging than those with low SHBG binding. A number of AR ligands labeled with 18F,6,18,19 11C,20,21 76Br,22,23 and iodine radioisotopes24,25 have been prepared and (5) Mendel, C. M. Endocr. ReV. 1989, 10, 232-274. (6) Liu, A.; Carlson, K. E.; Katzenellenbogen, J. A. J. Med. Chem. 1992, 35, 2113-2129. (7) de Ronde, W.; van der Schouw, Y. T.; Muller, M.; Grobbee, D. E.; Gooren, L. J. G.; Pols, H. A. P.; de Jong, F. H. J. Clin. Endocrinol. Metab. 2005, 90, 157-162. (8) Vermeulen, A.; Ando, S. J. Clin. Endocrinol. Metab. 1979, 48, 320326. (9) Adams, J. S. Cell 2005, 122, 647-649. (10) Farnsworth, W. E. Prostate (N. Y.) 1996, 28, 17-23. (11) Hryb, D. J.; Khan, M. S.; Romas, N. A.; Rosner, W. J. Biol. Chem. 1990, 265, 6048-6054. (12) Krupenko, S. A.; Krupenko, N. I.; Danzo, B. J. J. Steroid Biochem. Mol. Biol. 1994, 51, 115-124. (13) Hammes, A.; Andreassen, T. K.; Spoelgen, R.; Raila, J.; Hubner, N.; Schulz, H.; Metzger, J.; Schweigert, F. J.; Luppa, P. B.; Nykjaer, A.; Willnow, T. E. Cell 2005, 122, 751-762. (14) Fortunati, N. J. Endocrinol. InVest. 1999, 22, 223-234. (15) Porto, C. S.; Lazari, M. F.; Abreu, L. C.; Bardin, C. W.; Gunsalus, G. L. J. Steroid Biochem. Mol. Biol. 1995, 53, 561-565. (16) Rosner, W. Cell 2006, 124, 455-456; author reply 456-457. (17) Willnow, T. E.; Nykjaer, A. Cell 2006, 124, 456-457. (18) Choe, Y. S.; Lidstroem, P. J.; Chi, D. Y.; Bonasera, T. A.; Welch, M. J.; Katzenellenbogen, J. A. J. Med. Chem. 1995, 38, 816-825. (19) Liu, A.; Katzenellenbogen, J. A.; VanBrocklin, H. F.; Mathias, C. J.; Welch, M. J. J. Nucl. Med. 1991, 32, 81-88. (20) Reiffers, S.; Vaalburg, W.; Wiegman, T.; Wynberg, H.; Woldring, M. G. Int. J. Appl. Radiat. Isot. 1980, 31, 535-539. (21) Berger, G.; Maziere, M.; Prenant, C.; Sastre, J.; Comar, D. Int. J. Appl. Radiat. Isot. 1981, 32, 811-815. (22) Ghanadian, R.; Waters, S. L.; Chisholm, G. D. Eur. J. Nucl. Med. 1977, 2, 155-157. (23) Eakins, M. N.; Waters, S. L. Int. J. Appl. Radiat. Isot. 1979, 30, 701-703. (24) Hoyte, R. M.; MacLusky, N. J.; Hochberg, R. B. J. Steroid Biochem. 1990, 36, 125-132. (25) Hoyte, R. M.; Borderon, K.; Bryson, K.; Allen, R.; Hochberg, R. B.; Brown, T. J. J. Med. Chem. 1994, 37, 1224-1230.

FIGURE 1. Two previously reported radiolabeled androgens for PET imaging and their binding affinities to AR and SHBG (relative to those of the standards R1881 and estradiol, respectively).

investigated as AR imaging agents, but in most cases, they have been designed to have low SHBG binding so as to have high bioavailability.6,18,26,27 A few of these androgens have been studied in vivo, both in rats, which have no SHBG,28,29 and in baboons, which do.2,30,31 In baboons (with SHBG), 16β-fluoro5R-dihydrotestosterone (FDHT, 1), which has high affinity for SHBG, showed somewhat greater specific prostate uptake than did 16β-fluoromibolerone (FMib, 2), which has a low affinity for SHBG, although both have similar affinities for AR (structures and affinities shown in Figure 1).32 By contrast, in the rat (no SHBG), FMib showed greater prostate uptake than FDHT.6,33 Also, FDHT, the only 18F-labeled androgen that has been studied in humans and the one with good SHBG binding, provided clear images of prostate tumors.3 Thus, contrary to the bioavailability hypothesis, it appears that greater SHBG binding might lead to increased uptake of the bound androgens into AR-rich target tissues in humans. The two compounds discussed above, FDHT and FMib, were not well matched structurally, because in addition to being a 19-nor steroid, FMib has two methyl groups, at C-7 and C-17R, which affect androgen metabolism, whereas FDHT has a classical C-19 steroidal skeleton. Furthermore, the two fluoro androgens differed markedly in their extent of metabolic defluorination in rodent tissue uptake studies, with FDHT showing considerable 18F activity in bone.19,33 Unfortunately, too few AR radioligands have progressed past preliminary biodistribution studies in rats to clarify the role that SHBG might be playing in target tissue delivery of steroids in primate systems. For this reason, as reported here, we have designed and synthesized two new fluorine-substituted AR ligands that could be developed into PET imaging agents; one of these has both high AR and SHBG binding affinity, and the other has high AR affinity but low affinity for SHBG. These two ligands are more structurally matched than FDHT and FMib, and thus, they should help to elucidate the role of SHBG in the (26) Carlson, K. E.; Katzenellenbogen, J. A. J. Steroid Biochem. 1990, 36, 549-561. (27) Brandes, S. J.; Katzenellenbogen, J. A. Mol. Pharmacol. 1987, 32, 391-403. (28) Hobbs, C. J.; Jones, R. T.; Plymate, S. R. J. Steroid Biochem. Mol. Biol. 1992, 42, 629-635. (29) Reventos, J.; Sullivan, P. M.; Joseph, D. R.; Gordon, J. W. Mol. Cell. Endocrinol. 1993, 96, 69-73. (30) Muntzing, J.; Myhrberg, H.; Saroff, J.; Sandberg, A. A.; Murphy, G. P. InVest. Urol. 1976, 14, 162-167. (31) Kaack, B.; Lewis, R. W.; Resnick, M. I.; Roberts, J. A. Arch. Androl. 1983, 11, 123-129. (32) Downer, J. B.; Jones, L. A.; Engelbach, J. A.; Lich, L. L.; Mao, W.; Carlson, K. E.; Katzenellenbogen, J. A.; Welch, M. J. Nucl. Med. Biol. 2001, 28, 613-626. (33) Liu, A.; Dence, C. S.; Welch, M. J.; Katzenellenbogen, J. A. J. Nucl. Med. 1992, 33, 724-734.

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FIGURE 2. T, DHT, MNT, and Mib and their binding affinities to AR and SHBG (relative to those of the standards R1881 and estradiol, respectively).

delivery of radioligands to AR target tissue cells, whether this might be due to increased metabolic stability afforded by binding to SHBG or whether cellular uptake mediated by SHBG-R plays a more direct role.12,34,35 Results and Discussion Design of Ligands. Testosterone (T) is rapidly transformed in vivo into two principal active hormones:36 to 5R-dihydrotestosterone (DHT) by 5R-reductase in male sex organs and skin and to estradiol by aromatase primarily in brain, liver, and adipose tissue. In prostate, DHT is inactivated by the 3-hydroxysteroid dehydrogenases37 and is then glucuronidated and excreted.38 In other tissues, notably the liver, T and DHT are also metabolized extensively at other sites.39 T binds well to SHBG, and DHT binds particularly well (Figure 2).6 Modifications to T and DHT markedly affect their metabolism, affinity, and potency. Nortestosterone analogues, such as 7R-methyl-19-nortestosterone (MNT, 3) and mibolerone (Mib, 4), with both 7R- and 17R-methyl groups (Figure 2), are poor substrates for 5R-reductase and aromatase,40 and they are relatively resistant to metabolic degradation to less active products. The 7R-group present in MNT and Mib enhances binding affinity for the AR, and the 17R-methyl in Mib blocks 17β-dehydrogenase activity, all of which increase nuclear retention in target cells. Thus, overall they are of higher affinity and potency than testosterone,41 but their binding to SHBG is weaker, at least compared to that of the C19 steroids T and DHT.19 Therefore, for a carefully controlled study to probe the role of SHBG in the uptake of androgens, we wanted structurally matched androgens with low probability of metabolism, matched AR binding affinity, and divergent SHBG binding affinity. We hypothesized that 7R-(fluoromethyl)dihydrotestosterone (7RFM-DHT, 5) and 7R-(fluoromethyl)nortestosterone (7R-FMnorT, 6) would be a good pair of compounds (Figure 3). These AR ligands have closely related structures, with high affinity for the AR and resistance to metabolic enzymes, and identical sites for fluorine radiolabeling (on the 7R-methyl group). This (34) Noe, G.; Cheng, Y. C.; Dabike, M.; Croxatto, H. B. Biol. Reprod. 1992, 47, 970-976. (35) ding, V. D. H.; Moller, D. E.; Feeney, W. P.; Didolkar, V.; Nakhla, A. M.; Rhodes, L.; Rosner, W.; Smith, R. G. Endocrinology 1998, 139, 213-218. (36) Cummings, D. E.; Kumar, N.; Bardin, C. W.; Sundaram, K.; Bremner, W. J. J. Clin. Endocrinol. Metab. 1998, 83, 4212-4219. (37) Span, P. N.; Swee, C. G. J.; Benraad, T. J.; Smals, A. G. H. J. Steroid Biochem. Mol. Biol. 1996, 58, 319-324. (38) Rizner, T. L.; Lin, H. K.; Peehl, D. M.; Steckelbroeck, S.; Bauman, D. R.; Penning, T. M. Endocrinology 2003, 144, 2922-2932. (39) Vajiala, G. E.; Dumitru, I. F. J. Med. Biochem. 1998, 2, 3-20. (40) LaMorte, A.; Kumar, N.; Bardin, C. W.; Sundaram, K. J. Steroid Biochem. Mol. Biol. 1994, 48, 297-304. (41) Kumar, N.; Sundaram, K.; Bardin, C. W. J. Steroid Biochem. Mol. Biol. 1995, 52, 105-112.

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FIGURE 3. The two steroidal 7R-fluoromethyl androgens 5 and 6.

is a site different from C-16β, from which there was considerable metabolic release of fluoride ion, in our previous studies.33 Significantly and most importantly, the DHT and norT steroids have a large difference in SHBG affinity because of the differences in their steroid skeletal structures: 7R-FM-DHT being a 5R-dihydrotestosterone (good SHBG binder) vs 7RFM-norT being a 19-nortestosterone (poor SHBG binder). A key consideration in the synthesis of PET imaging agents labeled with short-lived isotopes such as 18F is the introduction of the radioisotope at, or near, the end of the synthetic scheme. Additionally, incorporation of the radiolabel at high specific activity levels is limited to simple nucleophilic displacement using [18F]fluoride ion, which constrains the range of reagents that can be used for fluorination. The 7R-methyl group appears to be a suitable site for introducing 18F into both of these steroids and one that we hoped might also prove to be less prone to release of fluoride ion by metabolism. Synthesis of 7r-FM-DHT (5). Overall, the route to 7R-FMDHT involves introduction of a cyano functionality stereoselectively at the 7R-position by a 1,6-conjugate hydrocyanation of 6-dehydrotestosterone, progressive reduction to the desired 7R-hydroxymethyl group, and finally introduction of the fluorine group (Scheme 1).42 Initially, we examined protection of the enone functionality at C-3 with a dioxolane;43 however, the known isomerization of the enone double bond to the 5,6positions created a “doubly allylic” 7β-hydrogen that was unstable to the reduction conditions that followed and underwent epimerization at C-7. Therefore, we protected the enone as a dithiolane. Because later in the sequence we were unable to selectively activate the 7R-hydroxymethyl in the presence of the free 17β-hydroxyl, we protected the 17β-hydroxyl moiety as an MOM ether, the 17-acetate not being stable to the hydride reduction conditions. Testosterone acetate was converted to our 7R-cyano intermediate 7 starting point in three steps in a 60% yield by a known sequence.42 This cyano intermediate was protected as the dithiolane 8; the 17β-acetate protecting group in 8 was cleaved with base, and the resulting alcohol 944 was reprotected as the MOM ether. Treatment of the MOM ether 10 with DIBAL-H gave the aldehyde 11, which was reduced again with NaBH445 to generate the 7R-hydroxymethyl steroid 12, both reduction steps proceeding in good yields. When we subjected hydroxymethyl 12 to the dithiolane deprotection conditions, we obtained a C-5,7 cyclic ether instead of the desired enone, and attempts to reverse this formal (42) Rasmusson, G. H.; Chen, A.; Arth, G. E. J. Org. Chem. 1973, 38, 3670-3672. (43) De Munari, S.; Cerri, A.; Gobbini, M.; Almirante, N.; Banfi, L.; Carzana, G.; Ferrari, P.; Marazzi, G.; Micheletti, R.; Schiavone, A.; Sputore, S.; Torri, M.; Zappavigna, M. P.; Melloni, P. J. Med. Chem. 2003, 46, 3644-3654. (44) Reinhard, B.; Faillard, H. Liebigs Ann. Chem. 1994, 193-203. (45) Hirsch, J. A.; Vu Chi, T. J. Org. Chem. 1986, 51, 2218-2227.

Synthesis of Androgens 7R-FM-DHT and 7R-FM-norT SCHEME 1.

Synthesis of 7r-(Fluoromethyl)dihydrotestosterone

intramolecular Michael addition by treatment with base were unsuccessful. To prevent this cyclization, we protected the 7Rhydroxymethyl functionality of 12 as an acetate, which could be treated with Tl(NO3)3 to deprotect the dithiolane and produce the desired enone 14 in an 88% yield. Dissolving metal reduction of enone 14 to the corresponding saturated ketone 15 proceeded with simultaneous hydrolysis of the 7R-acetoxymethyl group. The 7R-hydroxymethyl group was converted to the corresponding methanesulfonate 16, the precursor for introducing the fluorine substituent.46 As noted above, for radiofluorination at high specific activity, one is limited to using 18F ion, but simple fluoride ion displacement proved more difficult than anticipated. Although the 7R-methyl group is a primary center, it is branched at the β-carbon, and the methanesulfonate can adopt geometry suitable for an E2 elimination. In this steroidal system, fluoride ion acted almost exclusively as a base, and initial fluoride incorporation attempts using tetrabutylammonium fluoride (TBAF) and Kryptofix 222/KF, common reagents for nucelophilic radiofluorination, led to complete elimination of the mesylate to form a C-7 exocyclic methylene elimination product, 26, identified by its mass spectrum and appearance of two additional alkene carbons and vinylic hydrogens in the NMR spectra (see the Supporting Information for further characterization). We were able, however, to obtain a 7% yield of the desired 7R-fluoromethyl product 17 by using CsF in an ionic liquid as solvent, as recently described,47 with the remainder forming the exocyclic methylene elimination product. In the final step, the 7R-fluoromethyl (46) Corey, E. J.; Burk, R. M. Tetrahedron Lett. 1987, 28, 6413-6416. (47) Kim Dong, W.; Choe Yearn, S.; Chi Dae, Y. Nucl. Med. Biol. 2003, 30, 345-350.

compound 17 was deprotected with acid to produce the final product 5 in a 95% yield. The synthesis of 7R-FM-DHT took place in 11 steps starting from the known 7R-cyano starting material 7, with an overall yield of 0.90%. The yield based on testosterone acetate as a precursor would be 0.54% in 14 steps. Synthesis of 7r-FM-norT (6). The synthesis of 7R-FM-norT, shown in Scheme 2, followed a route similar to that outlined above for the DHT analogue, with a few key differences. Nortestosterone was first converted to the known cyano intermediate 18,48 our starting point, in three steps and 64% yield by a known sequence.42 We returned to the dioxolane protecting group with the expectation that double bond isomerization would occur largely to the 5,10-positions, rather than the 5,6-positions, which had caused configurational instability of the C-7 center in the testosterone system (Scheme 1, above). Treatment of 7R-cyano starting material 18 with ethylene glycol gave the desired dioxolane 5,10-alkene 19, along with a lesser amount of the 5,6-isomer, from which it could be separated by column chromatography. Conversion to the 17βMOM ether 20 and sequential reduction as before gave the corresponding 7R-methyl alcohol 22, which was activated as the mesylate, giving the labeling precursor 23. As in the DHT series, fluorination of the mesylate precursor 23 was accomplished with CsF in an ionic liquid as solvent, and the crude 7R-fluoromethyl product was deprotected with HCl to give the final product 7R-FM-norT in 11% yield for the last two steps, with the remainder forming the C-7 exocyclic methylene elimination product 27, whose structure was supported by mass spectroscopy and 1H NMR (see the Supporting Information for (48) Ali, H.; Rousseau, J.; Ahmed, N.; Guertin, V.; Hochberg, R. B.; van Lier, J. E. Steroids 2003, 68, 1163-1171.

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Synthesis of 7r-(Fluoromethyl)nortestosterone

TABLE 1. Binding Affinities of Various Steroidal Ligands for the AR and SHBG Proteins

a RBAs were determined by a competitive radiometric binding assay. Values given are the average of 2-3 determinations ( the standard deviation and are expressed on a percentage scale relative to the affinity of the 3H-labeled tracer: R1881 for AR, Kd ) 0.6 nM; E2 for SHBG, Kd ) 1.6 nM. Details are given in the Supporting Information.

further characterization). The overall yield of 7R-FM-norT from the 7R-cyano starting material 18 was 3.6% over six linear steps. The yield based on 19-norT would be 2.3% in nine steps. Assays for Binding to the Androgen Receptor and Sex Hormone Binding Globulin. The binding of these compounds to the AR and to SHBG was determined using a competitive radiometric binding assay (Table 1). The AR assay used the high-affinity synthetic androgen [3H]R1881 ([3H]24) as a tracer, unlabeled R1881 as a standard, and purified recombinant rat AR ligand binding domain protein. The source of the AR had little effect on the relative binding affinity (RBA) values, since previous studies with the standard ligands, done in our labora5550 J. Org. Chem., Vol. 72, No. 15, 2007

tory, using rat prostate cytosol or recombinant full-length human AR gave essentially the same RBA values. Binding to SHBG was determined by a similar assay with [3H]estradiol as a tracer, estradiol as a standard, and charcoal-stripped third-trimester, human pregnancy serum as a source of SHBG.50 Affinities are expressed as RBA, with the standards set to 100% by definition. Details concerning these binding affinity measurements have been described elsewhere.49,50 The RBA values for the various (49) Katzenellenbogen, J. A.; Johnson, H. J., Jr.; Myers, H. N. Biochemistry 1973, 12, 4085-4092. (50) McElvany, K. D.; Carlson, K. E.; Katzenellenbogen, J. A.; Welch, M. J. J. Steroid Biochem. 1983, 18, 635-641.

Synthesis of Androgens 7R-FM-DHT and 7R-FM-norT

7R-halomethyl products, as well as several intermediates, are given in Table 1. Of greatest significance, both 7R-fluoromethyl compounds 5 and 6 have quite high AR binding affinities, being comparable to those of their unsubstituted parent steroids DHT and norT, respectively. By contrast, their affinities for SHBG differ by more than 22-fold, with the expected preference for the DHT skeleton of 7R-FM-DHT over the 19-nortestosterone skeleton of 7R-FM-norT; a somewhat greater differential in SHBG affinity is seen with the parent androgens DHT and 19-norT, and their overall affinities for SHBG are higher. The equivalent AR binding, but differential SHBG binding, of the structurally matched androgens 7R-FM-DHT and 7R-FM-norT makes them well suited to our intended use as probes of the function of SHBG binding in the target tissue uptake of AR radioligands in humans. The radiochemical syntheses of 7R-[18F]FM-DHT ([18F]5) and 7R-[18F]FM-norT ([18F]6) have been accomplished successfully and will be presented elsewhere, together with the results of their biodistribution in experimental animals. Conclusion 7R-(Fluoromethyl)dihydrotestosterone (5) and 7R-(fluoromethyl)nortestosterone (6) have been synthesized and tested in vitro for binding to the androgen receptor and the blood steroid binding protein SHBG. Both of these compounds have high affinity for the androgen receptor; 5 also has high affinity for the sex hormone binding globulin, whereas 6 does not. The two fluoro steroids 5 and 6 are well paired in terms of their identical site of fluorine labeling, their similarity of structure, and their equivalent AR binding affinitysbut their contrasting SHBG bindingsand therefore are appropriate agents to evaluate the role of SHBG binding in modulating target tissue uptake efficiency and selectivity of AR PET imaging agents in humans. The syntheses of these compounds are amenable to radiolabeling with 18F ion as the final step, so that they can be evaluated in vivo. In addition, the 7R-fluoromethyl site of 18F labeling, as hoped, proved to be likely less prone to fluoride ion release by metabolism in the animal test system than was the 16β site of labeling.19,33 Results of these experiments will be presented elsewhere. Experimental Section 17β-(Acetyloxy)-7r-cyano Cyclic 3-(1,2-Ethanediyl dithioacetal)androst-4-en-3-one (8). 7R-cyano 7 (12.403 g, 34.92 mmol) was placed in a flame-dried round-bottom flask, dissolved in dry CH2Cl2 (300 mL), and cooled to 0 °C. Ethane-1,2-dithiol (9.1 mL, 108 mmol) was added through a syringe followed by drop by drop addition of BF3‚OEt2 (8.2 mL, 65 mmol). The solution was warmed to room temperature and stirred for 12 h, during which time the solution turned a bright red. The volatile solvents were removed through heat and a stream of N2. The crude mixture was purified through column chromatography using 40% EtOAc/60% hexanes and produced pure product (13.383 g, 31.04 mmol) as a white solid in 89% yield: mp 229-232 °C; Rf ) 0.65 in 60% EtOAc/40% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.79 (s, 3H), 1.00 (s, 3H), 1.80 - 1.20 (m, 11H), 2.02 (s, 3H), 2.10 (td, J ) 12.97, 3.00 Hz, 1H), 2.25 - 2.14 (m, 2H), 2.26 (dd, J ) 2.36, 14.15 Hz, 1H), 2.43 (ddd, J ) 1.82, 4.93, 14.15 Hz, 1H), 2.85 (dt, J ) 6.54, 2.25 Hz, 1H), 2.88 (m, 1H), 3.19 (m, 1H), 3.35 (m, 3H), 4.61 (t, J ) 7.93 (51) Liu, A.; Carlson, K. E.; Katzenellenbogen, J. A. J. Med. Chem. 1992, 35, 2113-2129.

Hz, 1H), 5.69 (t, J ) 1.39 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 11. 9, 18.4, 20.5, 21.1, 22.9, 27.3, 32.9, 34.1, 35. 9, 36.3, 36.7, 36.9, 37.5, 39.7, 40.2, 42.6, 47.2, 49.1, 64.7, 82.0, 119.1, 129.5, 135.8, 170.9; HRMS (EI+) m/z calcd 431.1953, found 431.1959. 17β-Hydroxy-7r-cyano Cyclic 3-(1,2-Ethanediyl dithioacetal)androst-4-en-3-one (9). The acetate precursor 8 (13.383 g, 31.04 mmol) was dissolved in a 40:60 CH2Cl2/MeOH solution (800 mL) and heated to 60 °C. NaOH (8.225 g, 155 mmol) was added in one portion, and the heterogeneous mixture was stirred while being exposed to the atmosphere for 48 h. Product isolation (CHCl3, water, Na2SO4) followed by flash chromatography (40% EtOAc/60% hexanes) afforded product (8.647 g, 22.23 mmol) as a white solid in 72% yield: mp 252-253 °C; Rf ) 0.38 in 60% EtOAc/40% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.73 (s, 3H), 0.99 (s, 3H), 1.80-1.05 (m, 12H), 2.10 (m, 3H), 2.23 (dd, J ) 14.04, 2.25 Hz, 1H), 2.42 (ddd, J ) 14.15, 4.93, 1.72, Hz, 1H), 2.85 (dt, J ) 6.54, 2.25 Hz, 1H), 3.18 (m, 1H), 3.34 (m, 3H), 3.66 (t, J ) 8.47 Hz, 1H), 5.70 (t, J ) 1.29 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 11.0, 18.5, 20.6, 22.7, 30.2, 33.0, 34.2, 35.7, 36.4, 37.0, 37.6, 39.7, 40.2, 41.4, 43.15, 47.5, 49.3, 81.2, 119.4, 129.5, 139.0, 184.9; HRMS (EI+) m/z calcd 389.1847, found 389.1849. 17β-(Methoxymethyl)-7r-cyano Cyclic 3-(1,2-Ethanediyl dithioacetal)androst-4-en-3-one (10). 17β-Hydroxy 9 (8.565 g, 29.62 mmol) was added to a round-bottom flask and dissolved in THF (200 mL). DPEA (51.6 mL, 296 mmol) was added through a syringe and chilled to 0 °C. Chloromethyl methyl ether (22.5 mL, 296 mmol) was then added, and the reaction was stirred for 24 h. Product isolation (CH2Cl2, water, Na2SO4) followed by flash chromatography (40% EtOAc/60% hexanes) gave pure product (8.377 g, 19.35 mmol) as a white solid with a yield of 65%: mp 173 °C; Rf ) 0.29 in 60% EtOAc/40% hexanes; 1H NMR (400 MHz, CDCl3) δ 0.77 (s, 3H), 1.00 (s, 3H), 1.70-1.12 (m, 10H), 1.78 (dt, J ) 13.67, 3.05 Hz, 1H), 1.84 (dt, J ) 12.70, 3.17 Hz, 1H), 2.19-2.01 (m, 3H), 2.24 (dd, J ) 14.16, 2.32 Hz, 1H), 2.42 (ddd, J ) 14.04, 4.88, 1.71 Hz, 1H), 2.84 (dt, J ) 6.59, 2.32 Hz, 1H), 3.22-3.15 (m, 1H), 3.32 (s, 3H), 3.37-3.30 (m, 3H), 3.54 (t, J ) 8.55 Hz, 1H), 4.58 (d, J ) 6.47 Hz, 1H), 4.60 (d, J ) 6.59 Hz, 1H), 5.68 (t, J ) 1.22 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 11.6, 18.4, 20.6, 22.7, 27.8, 32.9, 34.2, 36.2, 36.3, 36.8, 37.0, 37.5, 39.7, 40.2, 42.7, 47.3, 49.2, 55.1, 64.7, 86.1, 96.1, 119.2, 129.3, 139.00; HRMS (EI+) m/z calcd 433.2109, found 433.2125. Cyclic 3-Oxo-3-(1,2-ethanediyl dithioacetal)-17β-(Methoxymethyl)androst-4-en-7r-al (11). 7R-cyano 10 (8.245 g, 19.03 mmol) was placed in a round-bottom flask, dissolved in toluene (500 mL), and cooled to -78 °C. DiBAL-H (1 M, 76 mL) was added slowly to the solution and the resulting mixture stirred for 2 h. The reaction was then quenched by addition of a saturated ammonium sulfate solution (100 mL). Product isolation (CH2Cl2, water, Na2SO4) followed by flash chromatography (30% EtOAc/70% hexanes) produced product (6.053 g, 13.88 mmol) as a white solid in 73% yield: mp 131-132 °C; Rf ) 0.48 in 30% EtOAc/70% hexanes; 1H NMR (500 MHz, CDCl ) δ 0.75 (s, 3H), 1.06 (s, 3H), 2.153 1.15 (m, 15H), 2.29 (dd, J ) 14.26, 2.14 Hz, 1H), 2.34 (m, 1H), 2.46 (ddd, J ) 14.15, 5.25, 1.82 Hz, 1H), 3.19-3.14 (m, 1H), 3.353.29 (m, 3H), 3.30 (s, 3H), 3.47 (t, J ) 8.36 Hz, 1H), 4.56 (d, J ) 6.54 Hz, 1H), 4.59 (d, J ) 6.54 Hz, 1H), 5.54 (s, 1H), 9.77 (d, J ) 3.75 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 11.0, 18.5, 21.0, 22.9, 27.7, 32.9, 36.5, 36.6, 37.2, 37.6, 37.9, 39.5, 40.0, 42.85, 46.4, 48.7, 49.0, 55.1, 65.0, 86.2, 96.0, 127.6, 141.0, 205.9; HRMS (EI+) m/z calcd 436.2106, found 436.2093. 17β-(Methoxymethyl)-7r-(hydroxymethyl) Cyclic 3-(1,2Ethanediyl dithioacetal)androst-4-en-3-one (12). Aldehyde 11 (6.012 g, 13.80 mmol) was added to a round-bottom flask and dissolved in EtOH (400 mL). NaBH4 (1.050 g, 27.60 mmol) was added in one portion, and the resulting mixture was stirred at room temperature for 2 h. The reaction was quenched by addition of a saturated sodium bicarbonate solution (50 mL) and diluted with brine (500 mL). Product isolation (CH2Cl2, water, Na2SO4) followed by flash chromatography (30% EtOAc/70% hexanes) produced pure J. Org. Chem, Vol. 72, No. 15, 2007 5551

Parent et al. product (6.030 g, 13.80 mmol) as a white solid in 100% yield: mp 98 °C; Rf ) 0.16 in 30% EtOAc/70% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.77 (s, 3H), 1.04 (s, 3H), 1.80-1.25 (m, 14H), 2.01 (m, 1H), 2.13 (dd, J ) 9.65, 3.43 Hz, 2H), 2.26 (dd, J ) 3.43, 0.75 Hz, 2H), 3.23-3.18 (m, 1H), 3.32 (s, 3H), 3.37-3.33 (m, 3H), 3.49 (t, J ) 8.47 Hz, 1H), 3.54 (d, J ) 10.18 Hz, 1H), 3.58 (dd, J ) 10.18, 3.54 Hz, 1H), 4.59 (d, J ) 6.54 Hz, 1H), 4.61 (d, J ) 6.65 Hz, 1H), 5.54 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 11.3, 18.9, 21.1, 22.9, 27.8, 34.1, 36.7, 36.8, 37.5, 37.6, 37.8, 38.8, 39.7, 40.0, 42.8, 46.2, 48.6, 55.1, 60.1, 65.5, 86.4, 96.0, 126.9, 143.1; HRMS (EI+) m/z calcd 438.2262, found 438.2267. 17β-(Methoxymethyl)-7r-[(acetyloxy)methyl] Cyclic 3-(1,2Ethanediyl dithioacetal)androst-4-en-3-one (13). 7-Hydroxymethyl 12 (6.030 g, 13.76 mmol) was added to a round-bottom flask and dissolved in CH2Cl2 (400 mL). Acetic anhydride (13.0 mL, 138 mmol) and pyridine (4.7 mL, 69 mmol) were added through a syringe followed by DMAP (839 mg, 6.88 mmol) in one portion. The reaction was stirred at room temperature for 8 h. Product isolation (CH2Cl2, water, Na2SO4) followed by flash chromatography (40% EtOAc/60% hexanes) gave product (6.530 g, 13.60 mmol) as a white solid in 99% yield: mp 67 °C; Rf ) 0.43 in 40% EtOAc/60% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.71 (s, 3H), 0.98 (s, 3H), 1.75-0.90 (m, 12H), 1.97 (s, 3H), 2.071.83 (m, 3H), 2.08 (dd, J ) 16.08, 2.36 Hz, 2H), 2.20 (dd, J ) 13.72, 4.72 Hz, 1H), 3.13 (m, 1H), 3.26 (s, 3H), 3.31-3.22 (m, 3H), 3.42 (t, J ) 8.36 Hz, 1H), 3.76 (t, J ) 10.50 Hz, 1H), 4.06 (dd, J ) 10.61, 3.64 Hz, 1H), 4.52 (d, J ) 6.54 Hz, 1H), 4.54 (d, J ) 6.54 Hz, 1H), 5.42 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 11.2, 18.7, 20.8, 20.9 20.9, 22.7, 27.6, 34.1, 35.5, 36.5, 37.2, 37.8, 39.3, 39.9, 42.6, 46.0, 47.9, 48.9, 54.9, 61.9, 65.2, 86.2, 95.8, 127.0, 142.1, 170.9; HRMS (EI+) m/z calcd 480.2368, found 480.2352. 17β-(Methoxymethyl)-7r-[(acetyloxy)methyl]androst-4-en-3one (14). 7R-(Acetyloxy)methyl 13 (5.904 g, 12.29 mmol) was placed in a round-bottom flask and dissolved in a 1:1 THF/MeOH solution (400 mL). To this solution was added H2O (100 mL), and the resulting solution was stirred at room temperature until homogeneous. Tl(NO3)3‚3H2O (13.113 g, 29.51 mmol) was dissolved in MeOH (50 mL), and the resulting Tl(NO3)3 solution was added slowly to the starting material solution with vigorous stirring for 10 min. The reaction was quenched by being poured into a 1 N NaOH (100 mL)/H2O-ice mixture (500 mL). Product isolation (CHCl3, brine, Na2SO4) followed by flash chromatography (40% EtOAc/60% hexanes) gave pure product (4.372 g, 10.81 mmol) as a white solid in 88% yield: mp 136-137 °C; Rf ) 0.37 in 40% EtOAc/60% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.75 (s, 3H), 1.14-1.00 (m, 3H), 1.15 (s, 3H), 1.27 (qd, J ) 12.22, 6.00 Hz, 1H), 1.37 (qd, J ) 13.08, 4.07 Hz, 1H), 1.85-1.45 (m, 6H), 1.96 (s, 3H), 2.05-1.98 (m, 3H), 2.27 (dt, J ) 16.94, 4.07 Hz, 1H), 2.33 (dd, J ) 14.58, 5.04 Hz, 1H), 2.38 (d, J ) 2.57 Hz, 1H), 2.42 (dd, J ) 14.26, 4.93 Hz, 1H), 3.27 (s, 3H), 3.45 (t, J ) 8.47 Hz, 1H), 3.67 (t, J ) 10.72 Hz, 1H), 4.11 (dd, J ) 10.93, 4.07 Hz, 1H), 4.53 (d, J ) 6.54 Hz, 1H), 4.55 (d, J ) 6.54 Hz, 1H), 5.66 (s, 1H); 13C NMR (125 MHz, CDCl3) δ 11.2, 17.7, 20.7, 20.8, 22.7, 27.6, 33.8, 35.1, 35.4, 35.7, 36.4, 37.2, 38.5, 42.6, 46.0, 47.8, 55.0, 62.1, 86.1, 96.0, 126.1, 167.9, 170.9, 198.6; HRMS (EI+) m/z calcd 404.2563, found 404.2573. 17β-(Methoxymethyl)-7r-(hydroxymethyl)-5r-androstan-3one (15). A three-neck round-bottom flask was cooled to -78 °C and filled with liquid ammonia (100 mL). Li(s) (700 mg, 100 mmol) was dissolved in the refluxing ammonia, 7R-(acetyloxy)methyl 14 (4.174 g, 10.32 mmol) was placed in the resulting blue solution, and the mixture was stirred at -78 °C for 75 min. The reaction was quenched with ammonium chloride (200 mg), and the solution was warmed to room temperature. Product isolation (EtOAc, water, Na2SO4) followed by flash chromatography (40% EtOAc/60% hexanes) yielded pure product (550 mg, 1.5 mmol) as a white solid in 15% yield. The bulk of the remaining matter was unreacted starting material (2.128 g, 5.26 mmol) and 7R-acetate-protected material (1.067 g, 2.63 mmol). One additional recycling of the 5552 J. Org. Chem., Vol. 72, No. 15, 2007

unreacted starting material and acetate-protected hydroxyl afforded product (2.584 g, 6.397 mmol) in 62% yield: mp 76 °C; Rf ) 0.10 in 40% EtOAc/hexanes; 1H NMR (500 MHz, CDCl3) δ 0.72 (s, 3H), 0.99 (s, 3H), 1.80-0.90 (m, 15H), 1.97 (m, 3H), 2.21 (m, 3H), 2.32 (td, J ) 15.44, 6.54, Hz, 1H), 3.28 (s, 3H), 3.45 (t, J ) 8.47 Hz, 1H), 3.58 (t, J ) 10.50 Hz, 1H), 3.62 (dd, J ) 10.61, 4.93, Hz, 1H), 4.54 (d, J ) 6.54 Hz, 1H), 4.57 (d, J ) 6.54 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 10.8, 11.2, 20.9, 23.0, 27.7, 29.2, 36.1, 36.6, 37.4, 38.0, 38.5, 40.0, 42.7, 44.3, 45.7, 47.2, 55.0, 59.3, 86.3, 95.8, 170.1, 212.2; HRMS (EI+) m/z calcd 364.2614, found 364.2622. 7β-(Methoxymethyl)-7r-[(mesyloxy)methyl]-5r-androstan-3one (16). 7R-Hydroxymethyl 15 (700 mg, 1.92 mmol) was placed in a round-bottom flask, dissolved in CH2Cl2 (150 mL), and cooled to 0 °C. Pyridine (1.6 mL, 19 mmol) and methanesulfonyl chloride (1.5 mL, 19 mmol) were added via syringe, and the reaction was allowed to warm to room temperature over 24 h. The volatile organics were removed under reduced pressure, and the crude mixture was purified with column chromatography using 50% EtOAc/50% hexanes to yield pure product (697 mg, 1.58 mmol) as a white solid in 82% yield: mp 169-170 °C; Rf ) 0.27 in 50% EtOAc/50% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.76 (s, 3H), 0.93 (td, J ) 11.68, 4.39 Hz, 1H), 1.02 (s, 3H), 1.32 (td, J ) 12.65, 6.22 Hz, 1H), 1.44 (dt, J ) 13.51, 4.50 Hz, 1H), 1.69-1.50 (m, 8H), 1.71 (dt, J ) 13.08, 3.43 Hz, 1H), 1.76 (dd, J ) 11.79, 4.93 Hz, 1H), 1.82 (td, J ) 12.54, 3.64 Hz, 1H), 2.04 (m, 4H), 2.23 (t, J ) 15.11 Hz, 1H), 2.30 (dd, J ) 5.57, 2.36 Hz, 1H), 2.35 (td, J ) 15.76, 6.54 Hz, 1H), 2.97 (s, 3H), 3.31 (s, 3H), 3.48 (t, J ) 8.58 Hz, 1H), 4.20 (t, J ) 10.61 Hz, 1H), 4.29 (dd, J ) 4.39, 9.97 Hz, 1H), 4.57 (d, J ) 6.54 Hz, 1H), 4.60 (d, J ) 6.65 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 10.8, 11.2, 20.9, 22.9, 27.6, 29.2, 34.9, 36.1, 36.4, 36.5, 37.4, 37.9, 38.2, 39.9, 42.9, 44.1, 45.9, 47.3, 55.1, 67.6, 86.1, 96.0, 211.0; HRMS (EI+) m/z calcd 442.2389, found 442.2385. 17β-(Methoxymethyl)-7r-(fluoromethyl)-5r-androstan-3one (17). 7R-(Mesyloxy)methyl 16 (206 mg, 0.466 mmol) was placed in a conical vial, and wet CH3CN (2 mL) was added followed by [bmim][BF4] (2 mL) and H2O (20 µL). CsF (351 mg, 2.33 mmol) was added, and the mixture was heated with stirring to 120 °C for 3 h. Product isolation (EtOAc, water, Na2SO4) followed by flash chromatography (20% EtOAc/78% hexanes/2% MeOH) produced product (12 mg, 0.033 mmol) as a white solid in 7% yield: mp 116-117 °C; Rf ) 0.26 in 20% EtOAc/78% hexanes/2% MeOH; 1H NMR (500 MHz, CDCl ) δ 0.78 (s, 3H), 1.04 (s, 3H), 1.803 0.95 (m, 12H), 1.80 (ddt, J ) 29.16, 12.76, 3.32 Hz, 2H), 2.101.97 (m, 4H), 2.24 (t, J ) 14.69 Hz, 1H), 2.30 (ddt, J ) 15.65, 5.25, 2.47 Hz, 1H), 2.37 (td, J ) 13.18, 6.22 Hz, 1H), 3.33 (s, 3H), 3.51 (t, J ) 8.47 Hz, 1H), 4.51 (dd, 2JHF ) 47.27 Hz, J ) 2.14 Hz, 1H), 4.52 (d, 2JHF ) 47.59 Hz, 1H), 4.60 (d, J ) 6.65 Hz, 1H), 4.62 (d, J ) 6.54 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 10.8, 11.3, 21.0, 23.2, 27.8, 30.2 (d, 3JCF ) 4.6 Hz), 35.8 (d, 2JCF ) 16.6 Hz), 36.3 (d, 3JCF ) 6.4 Hz), 36.4, 38.1, 38.4, 40.7, 42.9, 44.4, 46.1, 47.6, 55.1, 83.3 (d, 1JCF ) 166.6 Hz), 86.2, 96.0, 137.1, 211.5; 19F NMR (470 MHz, CDCl3) δ -11.83 (td, 2JHF ) 45.78, 3J + HF ) 18.31, Hz) (external reference C6F6 δ -163.0); HRMS (EI ) m/z calcd 366.2570, found 366.2577. 17β-Hydroxy-7r-(fluoromethyl)-5r-androstan-3-one (5). 7Rfluoromethyl 17 (12 mg, 0.033 mmol) was placed in a round-bottom flask and dissolved in MeOH (4 mL). Concentrated HCl (60 µL) was added to the solution, and the reaction was heated at 60 °C for 60 min. Product isolation (CHCl3, water, Na2SO4) followed by flash chromatography (40% EtOAc/58% hexanes/2% MeOH) gave product (10 mg, 0.031 mmol) as a white solid in 95% yield: mp 225-226 °C; Rf ) 0.62 in 75% EtOAc/25% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.75 (s, 3H), 1.04 (s, 3H), 1.80-0.97 (m, 16H), 2.01 (dt, J ) 6.54, 2.36 Hz, 1H), 2.04 (dd, J ) 6.22, 2.36 Hz, 1H), 2.07 (td, J ) 3.86, 1.93 Hz, 1H), 2.24 (t, J ) 14.26 Hz, 1H), 2.82 (dt, J ) 5.36, 2.25 Hz, 1H), 2.36 (ddd, J ) 13.61, 6.54, 0.64 Hz, 1H), 3.63 (t, J ) 8.47 Hz, 1H), 4.51 (dd, 2JHF ) 47.70

Synthesis of Androgens 7R-FM-DHT and 7R-FM-norT Hz, J ) 7.93 Hz, 1H), 4.52 (dd, 2JHF ) 47.27, J ) 5.47 Hz, 1H); 13C NMR (125 MHz, CDCl ) δ 10.7, 10.8, 21.0, 23.2, 30.2, 30.2 3 (d, 3JCF ) 4.6 Hz), 35.8 (d, 2JCF ) 16.6 Hz), 36.0, 36.1, 36.5 (d, 3J CF ) 5.5 Hz), 38.1, 38.5, 40.7, 43.2, 44.4, 46.2, 47.6, 81.5, 83.4 (d, 1JCF ) 166.6 Hz), 211.5; 19F (470 MHz, CDCl3) δ -11.58 (td, 2J 3 HF ) 45.78 Hz, JHF ) 21.36 Hz) (external reference C6F6 δ -163.0); HRMS (EI+) m/z calcd 322.2308, found 322.2304. 17β-Hydroxy-7r-cyano Cyclic 3-(1,2-Ethanediyl acetal)estr5,10-en-3-one (19). 17β-Hydroxy-7R-cyanoestr-4-en-3-one (1.906 g, 6.371 mmol) was placed in a round-bottom flask and dissolved in dry benzene (100 mL). Ethylene glycol (3.6 mL, 64 mmol) and p-toluenesulfonic acid (1.212, 6.371 mmol) were added, and the mixture was refluxed for 4 h with a Dean-Stark condenser. Product isolation (CH2Cl2, water, Na2SO4) followed by flash chromatography (50% EtOAc/50% hexanes) yielded pure product (1.105 g, 3.22 mmol) as a white solid in 51% yield: mp 157 °C; Rf ) 0.16 in 50% EtOAc/50% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.69 (s, 3H), 1.13 (td, J ) 12.65, 3.22 Hz, 1H), 1.25 (dd, J ) 11.47, 5.47 Hz, 1H), 1.33 (td, J ) 12.33, 6.97 Hz, 1H), 1.43 (m, 1H), 1.51 (td, J ) 11.15, 3.22 Hz, 1H), 2.30-1.57 (m, 15H), 2.81 (ddd, J ) 6.32, 3.22, 1.29 Hz, 1H), 3.68 (t, J ) 8.47 Hz, 1H), 3.973.87 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 11.1, 22.2, 24.4, 26.0, 27.7, 30.1, 30.8, 32.8, 36.3, 39.2, 40.0, 41.7, 43.6, 46.9, 64.1, 64.4, 80.9, 107.7, 120.6, 121. 8, 130.2; HRMS (EI+) m/z calcd 343.2147, found 343.2145. 17β-(Methoxymethyl)-7r-cyano Cyclic 3-(1,2-Ethanediyl acetal)estr-5,10-en-3-one (20). 7R-Cyano 19 (1.102 g, 3.21 mmol) was placed in a round-bottom flask and dissolved in THF (100 mL). DPEA (5.6 mL, 32 mmol) was added, and the solution was chilled to 0 °C. Chloromethyl methyl ether (2.4 mL, 32 mmol) was added, and the reaction was allowed to warm to room temperature and stirred for 24 h. Product isolation (CH2Cl2, water, Na2SO4) followed by flash chromatography (50% EtOAc/50% hexanes) produced pure product (971 mg, 2.51 mmol) as a clear viscous oil with a yield of 78%: Rf ) 0.52 in 50% EtOAc/50% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.75 (s, 3H), 2.31-1.21 (m, 19H), 2.82 (ddd, J ) 6.32, 3.11,1.39 Hz, 1H), 3.32 (s, 3H), 3.59 (t, J ) 8.25 Hz, 1H), 3.94 (m, 4H), 4.59 (d, J ) 6.54 Hz, 1H), 4.61 (t, J ) 6.54 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 11.8, 22.3, 24.5, 26.1, 27.7, 27.9, 30.9, 33.0, 36.9, 39.1, 40.1, 41.8, 43. 5, 46.9, 55.1, 64.2, 64.5, 86.1, 93.1, 107.8, 120.7, 121.9, 130.3; HRMS (EI+) m/z calcd 387.2406, found 387.2409. Cyclic 3-Oxo-3-(1,2-ethanediyl acetal)-17β-(Methoxymethyl)estr-5,10-en-7r-al (21). 7R-Cyano 20 (946 mg, 2.44 mmol) was placed in a round-bottom flask, dissolved in toluene (100 mL), and cooled to -78 °C. DiBAL-H (1 M, 14.7 mL) was added to the solution and the resulting mixture stirred for 90 min. The reaction was quenched with saturated ammonium sulfate (50 mL). Product isolation (CH2Cl2, water, Na2SO4) followed by flash chromatography (50% EtOAc/50% hexanes) gave pure product (810 mg, 2.08 mmol) as a clear viscous oil in 85% yield: Rf ) 0.58 in 50% EtOAc/50% hexanes; 1H NMR (400 MHz, CDCl3) δ 0.67 (s, 3H), 1.28 (qd, J ) 11.84, 5.74 Hz, 1H), 2.20-1.41 (m, 18H), 2.38 (m, 1H), 3.23 (s, 3H), 3.48 (t, J ) 8.55 Hz, 1H), 3.86 (m, 4H), 4.49 (d, J ) 6.47 Hz, 1H), 4.52 (d, J ) 6.59 Hz, 1H), 9.62 (d, J ) 1.83 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 11.1, 22.5, 25.1, 26.0, 27.7, 30.4, 30. 9, 37.0, 40.1, 40.3, 42.1, 43.4, 45.8, 46.3, 54.8, 64.0, 64.2, 86.0, 95.7, 107.7, 123.6, 129.8, 204.7; HRMS (EI+) m/z calcd 390.2406, found 390.2400. 17β-(Methoxymethyl)-7r-(hydroxymethyl) Cyclic 3-(1,2Ethanediyl acetal)estr-5,10-en-3-one (22). 17β-Methoxymethyl 21 (801 mg, 2.05 mmol) was added to a round-bottom flask and dissolved in EtOH (150 mL). NaBH4 (155 mg, 4.11 mmol) was added in one portion, and the resulting mixture was stirred at room temperature for 1 h. The reaction was quenched with saturated sodium bicarbonate (25 mL). Product isolation (CH2Cl2, brine, Na2SO4) followed by flash chromatography (50% EtOAc/50% hexanes) afforded pure product (801 mg, 2.04 mmol) as a white solid in 99% yield: mp 64 °C; Rf ) 0.26 in 50% EtOAc/50%

hexanes; 1H NMR (500 MHz, CDCl3) δ 0.74 (s, 3H), 2.30-1.10 (m, 21H), 3.32 (s, 3H), 3.42 (t, J ) 9.86 Hz, 1H), 3.54 (t, J ) 8.47 Hz, 1H), 3.62 (dd, J ) 10.18, 3.75 Hz, 1H), 3.94 (m, 4H), 4.58 (d, J ) 6.65 Hz, 1H), 4.60 (d, J ) 6.54 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 11.6, 22.4, 25.5, 26.1, 27.9, 31.3, 32.8, 35.9, 37.5, 39.9, 40.9, 41.5, 43.5, 46. 6, 55.04, 61.1, 64.2, 64.4, 86.5, 95. 9, 108.2, 124.0, 129.0; HRMS (EI+) m/z calcd 392.2563, found 392.2558. 17β-(Methoxymethyl)-7r-[(mesyloxy)methyl] Cyclic 3-(1,2Ethanediyl acetal)estr-5,10-en-3-one (23). 7R-Hydroxymethyl 22 (782 mg, 1.99 mmol) was placed in a round-bottom flask, dissolved in CH2Cl2 (150 mL), and cooled to 0 °C. Pyridine (1.6 mL, 19 mmol) and methanesulfonyl chloride (1.5 mL, 19 mmol) were added via syringe, and the reaction was allowed to warm to room temperature and stirred for 24 h. The volatile organics were removed under reduced pressure, and the crude mixture was purified with column chromatography using 50% EtOAc/50% hexanes to yield pure product (902 mg, 1.92 mmol) as a white solid in 96% yield: mp 64 °C; Rf ) 0.31 in 50% EtOAc/50% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.75 (s, 3H), 1.12 (td, J ) 12.97, 3.43 Hz, 1H), 2.30-1.19 (m, 19H), 2.95 (s, 3H), 3.32 (s, 3H), 3.54 (t, J ) 8.36 Hz, 1H), 3.94 (m, 4H), 4.00 (t, J ) 9.65 Hz, 1H), 4.22 (dd, J ) 9.43, 4.07 Hz, 1H), 4.58 (d, J ) 6.54 Hz, 1H), 4.61 (d, J ) 6.54 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 11.6, 22.3, 25.4, 26.1, 27.9, 31.2, 32.7, 33.0, 37.2, 37.4, 39.7, 40.7, 41.5, 43.6, 46.6, 55.1, 64.2, 64.4, 69.1, 86.3, 95.9, 108.0, 123.4, 129.2; HRMS (EI+) m/z calcd 470.2338, found 470.2328. 17β-Hydroxy-7r-(fluoromethyl)estr-4-ene-3-one (6). 7R-(Mesyloxy)methyl 23 (401 mg, 0.853 mmol) was placed in a pear flask and dissolved in CH3CN (4 mL). [bmim][BF4] (2 mL) and H2O (40 µL) were added, and the mixture was stirred vigorously. CsF (648 mg, 4.26 mmol) was added, and the mixture was heated to 105 °C for 10 h. Product isolation (Et2O, brine, Na2SO4) followed by flash chromatography (20% EtOAc/78% hexanes) gave the fluorinated product (40 mg, 0.10 mmol) in 12% yield, which was then deprotected without further characterization: Rf ) 0.23 in 20% EtOAc/80% hexanes. The fluorinated intermediate (40 mg, 0.10 mmol) was placed in a round-bottom flask and dissolved in MeOH (10 mL). Concentrated HCl (100 µL) was added, and the reaction was heated at 60 °C for 90 min. The reaction was quenched with saturated sodium bicarbonate (2 mL). Product isolation (CHCl3, water, Na2SO4) followed by flash chromatography (50% EtOAc/50% hexanes) produced pure product 5 (29 mg, 0.095 mmol) as a white solid in 92% yield: mp 86 °C; Rf ) 0.12 in 50% EtOAc/50% hexanes; 1H NMR (500 MHz, CDCl3) δ 0.80 (s, 3H), 2.50-1.02 (m, 19H), 2.62 (dd, J ) 14.69, 2.36 Hz, 1H), 3.66 (t, J ) 8.68 Hz, 1H), 4.29 (ddd, 3J 3 HF ) 47.22 Hz, J ) 9.22, 8.36 Hz, 1H), 4.44 (ddd, JHF ) 46.84 Hz, J ) 9.22, 5.15 Hz, 1H), 5.88 (t, J ) 1.39 Hz, 1H); 13C NMR (125 MHz, CDCl3) δ 10.7, 22.7, 26.8, 26.9, 30.2 (d, 3JCF ) 4.60 Hz), 36.1, 36.6, 37.7 (d, 2JCF ) 12.89 Hz), 37.7, 41.6 (d, 3JCF ) 5.52 Hz), 42.8, 44.0, 46.3, 49.0, 81.4, 82.9 (d, 1JCF ) 167.55 Hz), 126.8, 163.3, 199.4; 19F (470 MHz, CDCl3) δ -13.55 (td, 2JHF ) 45.78 Hz, 2JHF )18.31 Hz); HRMS (EI+) m/z calcd 306.1995, found 306.1999. 17β-Hydroxy-7r-(hydroxymethyl)-5r-androstan-3-one (25). 17β-(Methoxymethyl)-7R-(hydroxymethyl)-5R-androstan-3-one (208 mg, 0.571 mmol) was placed in a round-bottom flask and dissolved in THF (50 mL). H2SO4 (3 M, 2 mL) was added, and the solution was stirred at 60 °C for 3 h. The reaction was quenched with saturated sodium bicarbonate (20 mL). Product isolation (CH2Cl2, water, Na2SO4) followed by flash chromatography (5% MeOH/ 95% CH2Cl2) yielded pure product (136 mg, 0.425 mmol) as a white solid in 74% yield: mp 258-259 °C; Rf ) 0.17 in 5% MeOH/ 95% CH2Cl2; 1H NMR (500 MHz, DMSO-d6) δ 0.62 (s, 3H), 0.87 (td, J ) 12.86, 3.86 Hz, 1H), 0.98 (s, 3H), 1.85-0.92 (m, 16H), J. Org. Chem, Vol. 72, No. 15, 2007 5553

Parent et al. 1.89 (ddd, J ) 13.08, 6.86, 2.04 Hz, 1H), 2.07 (ddt, J ) 15.22, 4.50, 2.14 Hz, 1H), 2.27 (t, J ) 14.36 Hz, 1H), 2.38 (td, J ) 14.58, 6.43 Hz, 1H), 3.41 (t, J ) 8.36 Hz, 1H), 3.42 (m, 2H), 4.23 (t, J ) 5.47 Hz, 1H), 4.42 (d, J ) 4.82 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 10.5, 10.8, 20.7, 22.8, 29.1, 29.7, 35.8, 36.20 36.6, 37.2, 37.8, 38.1, 39.4, 42.7, 44.2, 45.5, 46.6, 57.5, 79.9, 210.7; HRMS (EI+) m/z calcd 320.2351, found 320. 2347.

5554 J. Org. Chem., Vol. 72, No. 15, 2007

Acknowledgment. This work was supported in part by grants from the Department of Energy (FG02 86ER60401) and the National Institutes of Health (PHS 5R37 CA25836). Supporting Information Available: 1H NMR spectra for compounds 5-23 and 25. This material is available free of charge via the Internet at http://pubs.acs.org. JO070328B