Antiandrogens are commonly used to treat androgen-dependent disorders. The currently used drugs unfortunately possess very weak affinity for the human AR (hAR), thus indicating the need to develop new high-affinity steroidal antiandrogens. Our compounds are specially designed to impede repositioning of the mobile carboxyl-terminal helix 12, which blocks the ligand-dependent transactivation function (AF-2) located in the AR ligand-binding domain (ARLBD). Using crystal structures of the hARLBD, we first found that H12 could be directly reached from the ligand-binding pocket (LBP) by a chain positioned on the C18 atom of an androgen steroid nucleus. A set of 5α-dihydrotestosterone-derived molecules bearing various C18 chains were thus synthesized and tested for their capacity to bind hAR and act as antagonists. Although most of those having very high affinity for hAR were agonists, several very potent antagonists were obtained, confirming the structural importance of the C18 chain. To understand the role of the C18 chain in their agonistic/antagonistic properties, the structure of the hARLBD complexed with one of these agonists, EM5744, was determined at a 1.65-Å resolution. We have identified new interactions involving Gln738, Met742, and His874 that explain both the high affinity of this compound and the inability of its bulky chain to prevent the repositioning of H12. This structural information will be helpful to refine the structure of the chains placed on the C18 atom to obtain efficient H12-directed steroidal antiandrogens.

Skin disorders such as acne, seborrhea, hirsutism, and androgenic alopecia are secondary to excess local androgenic activity. Because the most potent androgen, dihydrotestosterone, is formed from testosterone by the action of 5α-reductase, the inhibition of 5α-reductase is a logical approach to interfere with androgenic action in the skin. In this study, we have investigated the inhibitory effect of a series of 17β-(N-alkyl/arylformamido)- and 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-one derivatives as 5α-reductase inhibitors following their topical application on the flank organs and ears of Golden Syrian hamsters. The parameters measured were mainly the size of the underlying sebaceous glands and 5α-reductase activity in the flank organs and ears. We found that 17β-(N-amylformamido)-4-methyl-4-aza-5α-androstan-3-one (EM-401), 17β-(N-hexylformamido)-4-methyl-4-aza-5α-androstan-3-one (EM-402), and 17β-(N-heptylformamido)-4-methyl-4-aza-5α-andro-stan-3-one (EM-540) are potent inhibitors of 5α-reductase activity. EM-402 decreases the size of treated flank organs by 22%, 31%, and 32% (p < 0.01 for all) after topical application at the doses of 30, 100, and 300 μg, respectively, twice daily for 4 wk. EM-402 also reduced the size of underlying sebaceous glands by 38%, 42%, and 59% of intact control values at the same doses. Comparable results were observed on the size of the sebaceous glands of the ears. In addition, we have observed a concentration-dependent 47%–80% (p < 0.01) and 46%–80% (p < 0.01) inhibition of 5α-reductase activity in the right flank organs and ears, respectively, using topical EM-402. EM-402 had no significant effect on the same parameters in the left contralateral flank organs or ears. In addition, EM-402 had no effect on prostatic and seminal vesicle weights whereas EM-401 and EM-540 showed some systemic effects. These data illustrate that EM-402 applied topically, at the concentrations used, exerts a potent local anti-androgenic effect without any systemic action in the hamster. Skin disorders such as acne, seborrhea, hirsutism, and androgenic alopecia are secondary to excess local androgenic activity. Because the most potent androgen, dihydrotestosterone, is formed from testosterone by the action of 5α-reductase, the inhibition of 5α-reductase is a logical approach to interfere with androgenic action in the skin. In this study, we have investigated the inhibitory effect of a series of 17β-(N-alkyl/arylformamido)- and 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-one derivatives as 5α-reductase inhibitors following their topical application on the flank organs and ears of Golden Syrian hamsters. The parameters measured were mainly the size of the underlying sebaceous glands and 5α-reductase activity in the flank organs and ears. We found that 17β-(N-amylformamido)-4-methyl-4-aza-5α-androstan-3-one (EM-401), 17β-(N-hexylformamido)-4-methyl-4-aza-5α-androstan-3-one (EM-402), and 17β-(N-heptylformamido)-4-methyl-4-aza-5α-andro-stan-3-one (EM-540) are potent inhibitors of 5α-reductase activity. EM-402 decreases the size of treated flank organs by 22%, 31%, and 32% (p < 0.01 for all) after topical application at the doses of 30, 100, and 300 μg, respectively, twice daily for 4 wk. EM-402 also reduced the size of underlying sebaceous glands by 38%, 42%, and 59% of intact control values at the same doses. Comparable results were observed on the size of the sebaceous glands of the ears. In addition, we have observed a concentration-dependent 47%–80% (p < 0.01) and 46%–80% (p < 0.01) inhibition of 5α-reductase activity in the right flank organs and ears, respectively, using topical EM-402. EM-402 had no significant effect on the same parameters in the left contralateral flank organs or ears. In addition, EM-402 had no effect on prostatic and seminal vesicle weights whereas EM-401 and EM-540 showed some systemic effects. These data illustrate that EM-402 applied topically, at the concentrations used, exerts a potent local anti-androgenic effect without any systemic action in the hamster. The relationship between androgenic hormones and skin physiology has been an attractive area of research for endocrinologists and dermatologists for several decades (Pochi and Straus, 1964Pochi P.E. Straus J.S. Sebum production, casual sebum levels, titrable acidity of sebum, and urinary fractional, 17-ketosteroid excretion in males with acne.J Invest Dermatol. 1964; 43: 383-388Abstract Full Text PDF PubMed Scopus (81) Google Scholar;Cunliffe and Shuster, 1969aCunliffe W.J. Shuster S. Pathogenesis of acne.Lancet. 1969 a; i: 685-687Abstract Google Scholar). It is now well recognized that acne, hirsutism, and male pattern baldness are all androgen-related disorders (Darley et al., 1982Darley C.R. Kirby J.D. Besser G.M. Munro D.D. Edwards C.R. Rees L.H. Circulating testosterone, sex hormone binding globulin and prolactin in women with the late onset or persistent acne vulgaris.Br J Dermatol. 1982; 106: 517-522Crossref PubMed Scopus (59) Google Scholar;Barth, 1988Barth J.H. Alopecia and hirsuties. Current concepts in pathogenesis and management.Drugs. 1988; 35: 83-91Crossref PubMed Scopus (6) Google Scholar). Moreover, patients suffering from acne usually present seborrhea or an excess of serum production (Cunliffe and Shuster, 1969bCunliffe W.J. Shuster S. The rate of sebum secretion in man.Br J Dermatol. 1969 b; 81: 697-704Crossref PubMed Scopus (160) Google Scholar). In addition to excess production of androgens by the ovaries and adrenals (through DHEA), a hyperandrogenic state can also result from an increase in the 5α-reduction of testosterone in the skin (Barth, 1988Barth J.H. Alopecia and hirsuties. Current concepts in pathogenesis and management.Drugs. 1988; 35: 83-91Crossref PubMed Scopus (6) Google Scholar). In fact, testosterone, the main androgen of testicular and ovarian origins, is converted into dihydrotestosterone (DHT) in the target cell by 5α-reductase (Labrie et al., 1991Labrie C. Trudel C. Li S. Martel C. Couet J. Labrie F. Combination of an antiandrogen and a 5α-reductase inhibitor: A further step towards total androgen blockade?.Endocrinology. 1991; 129: 566-568PubMed Google Scholar[BC1]). Blockade of androgenic action can be achieved through several mechanisms, e.g., by inhibiting the conversion of testosterone to DHT with 5α-reductase inhibitors, whereas the action of both testosterone and DHT may be efficiently prevented from binding to the androgen receptor by competition with a receptor-binding compound possessing no androgenic activity, i.e., a pure anti-androgen (Voigt and Hsia, 1973Voigt W. Hsia S.L. The antiandrogen action of 4-androsten-3-one 17a-carboxyl acid and its methyl ester in hamster flank organ.Endocrinology. 1973; 92: 1216-1222Crossref PubMed Scopus (78) Google Scholar;Neumann and Steinback, 1991Neumann F. Steinback H. Androgens and antiandrogens.in: Eichler O. Farh A. Herken H. Weleh A.D. Berlin Heidelberg. Springer-Verlag, New York1991: 236Google Scholar;Plewig and Luderschmidt, 1991Plewig G. Luderschmidt C. Effect of antiandrogen on sebaceous glands and acne vulgaris. Animal experiments and studies in acne patients.in: Hammerstein J. Lachnia-Fixon U. Androgenization in Women. Excerpta Medica, Amsterdam, Oxford1991: 96-102Google Scholar). In fact, a series of steroidal and nonsteroidal compounds have been tested in animals or humans (Lutsky et al., 1975Lutsky B.N. Budak M. Koziol P. Monahan M. Neri R.O. The effects of a nonsteroid antiandrogen, Flutamide, on sebaceous gland.J Invest Dermatol. 1975; 64: 412-417Abstract Full Text PDF PubMed Scopus (32) Google Scholar;La Vecchia et al., 1984La Vecchia C. Franceschi S. Decarli A. Gallus G. Tognoni G. Risk factors for endometrial cancer at different ages.J Natl Cancer Inst. 1984; 73: 667-671PubMed Google Scholar;Luderschmidt et al., 1984Luderschmidt C. Eiermann W. Jawny J. Bidlingmaier F. Ring J. 17α-propylmesterolone (SH-434): an antiandrogenic sebosuppressive substance not influencing circulating testosterone concentrations. Experimental studies in Syrian hamsters.Nauynyn Schmiedebergs Arch Pharmacol. 1984; 328: 214-218Crossref Scopus (13) Google Scholar;Weissmann et al., 1985Weissmann A. Bowden J. Frank B.L. Horwitz S.N. Frost P. Antiandrogenic effects of topically applied spironolactone on the hamster flank organ.Arch Dermatol. 1985; 121: 57-62Crossref PubMed Scopus (37) Google Scholar;Bouton et al., 1986Bouton M.M. Lecaque D. Secchi J. Tournemine C. Effect of a new topically active antiandrogen (RU38882) on the rat sebaceous gland: comparison with cyproterone acetate.J Invest Dermatol. 1986; 86: 163-167Crossref PubMed Scopus (17) Google Scholar;Brooks et al., 1991Brooks J.R. Primka R.L. Berman C. Krupa D.A. Reynolds G.F. Rasmusson G.H. Topical anti-androgenicity of a new 4-azasteroid in the hamster.Steroids. 1991; 56: 428-433Crossref PubMed Scopus (14) Google Scholar;Lookingbill et al., 1992Lookingbill D.P. Abrams B.B. Ellis C.N. et al.Inocoterone and acne.Arch Dermatol. 1992; 128: 1197-1200Crossref PubMed Google Scholar;Cusan et al., 1993Cusan L. Dupont A. Cossette M. Labrie F. Flutamide in the treatment of female androgenic alopecia.Can J Dermatol. 1993; 5: 421-427Google Scholar,Cusan et al., 1994Cusan L. Dupont A. Gomez J.L. Tremblay R.R. Labrie F. Comparison of flutamide and spironolactone in the treatment of hirsutism: a randomized controlled trial.Fertil Steril. 1994; 61: 281-287Abstract Full Text PDF PubMed Google Scholar;Matias and Gaillard, 1995Matias J.R. Gaillard M. Local inhibition of sebaceous gland growth by topically applied (RU 58841).Ann N Y Acad Sci. 1995; 761: 56-65Crossref Scopus (7) Google Scholar;Labrie et al., 1996Labrie F. Cusan L. Dupont A. et al.In: Androgen receptor.in: Adashi E.Y. Rock J.A. Rosenwaks Z. Reproductive Endocrinology, Surgery and Technology. Lippincott-Raven Publishers, Philadelphia1996: 560-583Google Scholar). Systemically administered anti-androgens, such as 17α-methyl-B-nortestosterone and flutamide as well as the partial androgen antagonists spironolactone and cyproterone acetate, have been found to improve acne, hirsutism, and androgenic alopecia in women (Zarate et al., 1966Zarate A. Mahesh V.B. Greenblatt R.B. Effect of an antiandrogen 17α-methyl-B-nortestosterone, on acne and hirsutism.J Clin Endocrinol Metab. 1966; 26: 1394-1398Crossref PubMed Scopus (22) Google Scholar;Lookingbill et al., 1992Lookingbill D.P. Abrams B.B. Ellis C.N. et al.Inocoterone and acne.Arch Dermatol. 1992; 128: 1197-1200Crossref PubMed Google Scholar;Cusan et al., 1993Cusan L. Dupont A. Cossette M. Labrie F. Flutamide in the treatment of female androgenic alopecia.Can J Dermatol. 1993; 5: 421-427Google Scholar,Cusan et al., 1994Cusan L. Dupont A. Gomez J.L. Tremblay R.R. Labrie F. Comparison of flutamide and spironolactone in the treatment of hirsutism: a randomized controlled trial.Fertil Steril. 1994; 61: 281-287Abstract Full Text PDF PubMed Google Scholar); however, these applications are limited because of the systemic action of these anti-androgens. The pure anti-androgenic compound flutamide has been found to be more potent than the mixed androgenic/anti-androgenic compound spironolactone (Cusan et al., 1993Cusan L. Dupont A. Cossette M. Labrie F. Flutamide in the treatment of female androgenic alopecia.Can J Dermatol. 1993; 5: 421-427Google Scholar). It would be of great therapeutic value to make available drugs that have pure local anti-androgenic activity without causing systemic side-effects. Two types of human 5α-reductase, chronologically identified as type I (Andersson and Russel, 1990Andersson S. Russel D.W. Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases.Proc Natl Acad Sci USA. 1990; 87: 3640-3644Crossref PubMed Scopus (482) Google Scholar;Harris et al., 1992Harris G. Azzolina B. Baginsky W. et al.Identification and selective inhibition of an isoenzyme of steroid 5α-reductase in human scalp.Proc Natl Acad Sci USA. 1992; 89: 10787-10791Crossref PubMed Scopus (126) Google Scholar) and type II (Andersson et al., 1991Andersson S. Bergman D.M. Jenkins E.P. Russel D.W. Deletion of steroid 5α-reductase 2 gene in male pseudophermaphroditism.Nature. 1991; 354: 159-161Crossref PubMed Scopus (600) Google Scholar;Labrie et al., 1992Labrie F. Sugimoto Y. Luu-the V. et al.Structure of human type II 5α-reductase.Endocrinology. 1992; 131: 1571-1573Crossref PubMed Scopus (171) Google Scholar) 5α-reductase have been isolated from human prostatic cDNA libraries, and the structure of the two isoenzymes has been elucidated. Type I 5α-reductase is predominately expressed in the skin (Andersson and Russel, 1990Andersson S. Russel D.W. Structural and biochemical properties of cloned and expressed human and rat steroid 5α-reductases.Proc Natl Acad Sci USA. 1990; 87: 3640-3644Crossref PubMed Scopus (482) Google Scholar;Harris et al., 1992Harris G. Azzolina B. Baginsky W. et al.Identification and selective inhibition of an isoenzyme of steroid 5α-reductase in human scalp.Proc Natl Acad Sci USA. 1992; 89: 10787-10791Crossref PubMed Scopus (126) Google Scholar;Luu-the et al., 1994Luu-the V. Sugimoto Y. Puy L. Labrie Y. Lopez I. Singh M. Labrie F. Characterization, expression and immunohistochemical localization of 5α-reductase in human skin.J Invest Dermatol. 1994; 102: 221-226Abstract Full Text PDF PubMed Google Scholar), whereas the type II enzyme is responsible for male pseudohermaphroditism when defect. We have previously reported that a series of 17β-(N-alkyl/arylformamido)- and 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-one derivatives strongly inhibit human type I 5α-reductase activity and show low potency on human type II 5α-reductase in transfected cells in vitro (Li et al., 1995Li X. Singh S.M. Labrie F. Synthesis and in vitro activity of 17β-(N-alkyl/arolformamido) – and 17β-[(N-alkyl/aryl) alkyl/arylamido]-4-methyl-4-aza-3-oxo-5α-androstan-3-ones as inhibitors of human 5α-reductases and antagonists of the androgen receptor.J Med Chem. 1995; 38: 1158-1173Crossref Scopus (23) Google Scholar). Because the androgen sensitivity of the Syrian hamster flank organs and ears have made this animal a useful model for the study of such compounds (Hamilton and Montagna, 1950Hamilton J.B. Montagna W. The sebaceous glands of the hamster. Morphological effects of androgens on integumentary structure.Am J Anat. 1950; 86: 191-233Crossref PubMed Scopus (70) Google Scholar;Plewig and Luderschmidt, 1982Plewig G. Luderschmidt C. Hamster ear model for sebaceous glands.J Invest Dermatol. 1982; 68: 171-176Crossref Scopus (72) Google Scholar;Vermorken et al., 1982Vermorken A.J.M. Goos C.M.A.A. Wirtz P. Evaluation of the hamster flank organ test for the screening of antiandrogens.Br J Dermatol. 1982; 106: 99-101Crossref PubMed Scopus (30) Google Scholar;Matias and Gaillard, 1995Matias J.R. Gaillard M. Local inhibition of sebaceous gland growth by topically applied (RU 58841).Ann N Y Acad Sci. 1995; 761: 56-65Crossref Scopus (7) Google Scholar), we have evaluated the anti-androgenic activity of these compounds on Syrian hamster flank organs and ears after a 4 wk topical administration. Male Syrian hamsters ≈8–10 wk old and weighing 110–120 g were purchased from Charles-River Laboratories (St. Constant, Québec, Canada) and housed 4–5 per cage in a light (14 h light per d, lights on at 07:15) and temperature (21 ± 3°C) controlled environment. The animals were housed in plastic boxes on sawdust and received hamster chow (Agway Country Food, Syracuse, NY) and tap water ad libitum. At the beginning of the experiment, the ear was marked for identification purposes and the area of the flank organ was shaved with electric clippers. The appropriate doses of the indicated compounds were dissolved in ethanol-propylene glycol (50:50, vol/vol) and administered in 10 μl on the right side of the hamster flank organs as well as the ears. Meanwhile, the left side of the flank organs and ears of the animals of the control and treated groups received the vehicle only. The three experiments were performed as follows: Experiment 1Intact hamsters (four per group) were treated with EM-423, 347, 401, 402, 422, 435, 336, 337, 436, 540, 541 at a dose of 50 μg, twice daily, on the right flank organ. Experiment 2Intact hamsters (four per group) were treated with EM-424, 486, 497, 494, 493, 498, 503, 580, 568, 606, 567, 682 at a dose of 100 μg, twice daily, on the right flank organ. Experiment 3Intact hamsters (12 per group) were treated with EM-401, 402, and 540 at doses of 30, 100, and 300 μg, twice daily, on the right flank organ and ear. The 5α-reductase inhibitors were administered twice daily for 28 consecutive days. The length and width of the darkly pigmented oval spot outlining each flank organ were carefully measured with Vernier calipers (Fisher Scientific, Pittsburgh, PA). Flank organ area was then calculated according to the following formula: area of ellipse = π(L/2 × W/2) = 3.14×(L × W)/4. The animals were killed by decapitation on the morning following the last dose. The flank organs and ears were then removed quickly and fixed for histologic examination or frozen in dry ice, and stored at –80°C for enzymatic assays. The ventral prostate as well as the seminal vesicles were removed, dissected, freed from fat and connective tissue, and rapidly weighed. The flank organs and ears were fixed in 4% paraformaldehyde for 48 h and then rinsed in 15% sucrose phosphate buffer for 24 h at 4°C. The flank organs were then cut in the middle in the rostral to caudal direction and the ears were cut between the first and second cartilage ridges. The tissues were then embedded in tissue-tek (Miles, Diagnostic Division, Elkhart, IN) and cut with a cryostat in 8 μm thick sections before staining with hematoxylin and eosin. The surface areas of the sebaceous glands of flank organs were estimated with a computer-assisted Image-Pro Plus program (Media Cybernetics, Silerspring, MD), and the surface areas of the sebaceous glands of the inner surface of the ears were estimated directly under optical microscopy using a grid. The surface area was counted as square units occupied by sebaceous glands in each section area. Six consecutive sections of each sample were analyzed for both flank organ and ear. The total number of arbitrary units was counted and the mean of six slides from each sample was calculated. Only the glands of the inner surface of the ear were measured. The results were calculated in units and expressed as a percentage of the control group that was taken as 100%. The enzymatic assay was performed as described previously (Martel et al., 1994Martel C. Melner M.H. Gagné D. Simard J. Labrie F. Widespread tissue distribution of steroid sulfatase, 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD), 17β-HSD 5α-reductase and aromatase activities in the rhesus monkey.Mol Cell Endocrinol. 1994; 104: 103-111Crossref PubMed Scopus (184) Google Scholar). In brief, 100 μl aliquots of the 1000 ×g supernatant of hamster flank organs and ears were incubated for 3 h at 37°C in a total volume of 0.5 ml phosphate buffer (12.5 mM KH2PO4, 1 mM ethylenediamine tetraacetic acid, pH 7.5) containing 0.5 μM [4–14C]-testosterone (S.A., 51.4 mCi per mmol) and the cofactor NADPH. Labeled radioactivity was purchased from New England Nuclear/Dupont (Markham, Canada) and purified by thin-layer chromatography before use. The enzymatic reaction was stopped by chilling the incubation mixture in an ice-water slurry and adding 3 ml of diethyl ether. The components were then mixed and frozen in a dry ice–ethanol bath. The organic phase was kept while the remaining frozen aqueous fraction was re-extracted once with ether. The organic phases were then pooled and evaporated to dryness under a nitrogen stream. All components were then separated on thin-layer chromatography (60 F254 silica gel, Merck, Darmstadt, Germany) using toluene:acetone (4:1, vol/vol) as solvent before autoradiography of the plates for 48 h. The metabolites revealed by autoradiography were identified by comparison with standard labeled steroids. The thin-layer chromatography areas corresponding to testosterone, DHT, and the DHT metabolites androstane-3α,17β-diol and androstane-3β,17β-diol were scraped and transferred into vials containing 0.5 ml ethanol and 10 ml scintillation liquid. The radioactivity was then measured in a liquid scintillation counter. Results are expressed as means ± SEM in pM product formed per mg protein per min. Statistical significance was measured according to the multiple-range test of Duncan-Kramer (Kramer, 1956Kramer C.Y. Extension of multiple range tests to group means with unique numbers of replications.Biometrics. 1956; 12: 307-310Crossref Google Scholar). Data are expressed as means ± SEM. Novel 17β-(N-alkyl/aryl formamido)-4-methyl-4-aza-5α-androstan-3-ones show topical anti-androgenic activityA series of novel 17β-(N-alkyl/arylformamido)-4-methyl-4-aza-5α-androstan-3-ones (Figure 1) were tested by application on hamster flank organs. The compounds were administered topically on the right flank organs of Golden Syrian hamsters at a dose of 50 μg, twice daily, for 4 wk, whereas the control animals and the left side of the animals of the treated groups received the vehicle only. As can be seen in Table 1, the surface area of the right side flank organs (treated side) of hamsters treated with EM-347, EM-402, EM-540, EM-337, EM-436, and EM-435 decreased by 27% (p < 0.01), 26% (p < 0.01), 24% (p < 0.01), 17% (p < 0.05), 21% (p < 0.05), and 25% (p < 0.01), respectively, whereas the surface area of the left side flank organs (the side treated with vehicle only) was unaffected. On the other hand, compounds EM-401 and EM-541 demonstrated systemic activity because they reduced the surface area of both side flank organs by 24% (p < 0.01), 26% (p < 0.01), 18% (p < 0.01), and 25% (p < 0.01), respectively. Treatment with EM-423, EM-422, and EM-336 did not affect flank organ size. It can be seen in the same table that the weights of the ventral prostates and seminal vesicles were not significantly reduced by treatment with any of the compounds used.Table IInhibitory effects of 17β-(N-alkylformanido)-4-methyl-4-aza-5α-androstan-3-one derivatives on the size of flank organs as well as ventral prostate and seminal vesicle weight. Compounds were applied on the right flank organaData are expressed as means ± SEM (n = 4). *p < 0.05, **p < 0.01 versus intact controls.EM(50 mg, BID)Ventral prostate weight (mg)Seminal vesicle weight (mg)Left sideRight sideControl33.5 ± 2.133.4 ± 1.353.9 ± 4.1228.4 ± 6.442334.9 ± 3.134.8 ± 1.355.3 ± 4.0242.7 ± 6.934732.0 ± 1.324.6 ± 1.0**55.0 ± 3.2208.0 ± 12.140125.3 ± 1.8**24.6 ± 2.0**54.1 ± 1.6249.5 ± 17.040232.1 ± 0.924.6 ± 1.2**50.0 ± 0.9226.5 ± 25.554032.9 ± 0.825.1 ± 0.7**50.7 ± 3.0248.9 ± 6.454127.5 ± 1.4*25.0 ± 0.5**55.5 ± 1.0256.0 ± 3.642230.4 ± 2.228.2 ± 1.858.2 ± 2.8227.3 ± 22.343529.3 ± 0.925.2 ± 1.3**59.0 ± 6.0223.9 ± 20.733632.3 ± 0.633.2 ± 1.149.3 ± 4.5212.1 ± 10.233732.4 ± 1.227.7 ± 0.8*55.4 ± 2.1221.7 ± 23.843636.1 ± 1.826.3 ± 1.7*58.4 ± 3.1214.3 ± 23.0a Data are expressed as means ± SEM (n = 4). *p < 0.05, **p < 0.01 versus intact controls. Open table in a new tab A series of 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-ones (Figure 1) were also tested on hamster flank organs. As illustrated in Table 2, the topical administration of EM-486, EM-568, EM-606, and EM-567, at a dose of 100 μg, twice daily, for 4 wk, caused respective 21% (p < 0.05), 30% (p < 0.01), 29% (p < 0.01), and 28% (p < 0.05) reductions in the left flank organ size and 26%, 25%, 29%, and 29% (p < 0.01 for all) reductions in the right side flank organ surface area compared with the intact control group. Ventral prostate weights were decreased by 18% (p < 0.05) and 27% (p < 0.01) following treatment with EM-568 and EM-606, respectively, whereas EM-486 did not affect this parameter. Moreover, treatment with EM-568 and EM-606, at the same dose, caused 23% and 33% (p < 0.01 for both) reductions in seminal vesicle weight, although EM-468 and EM-567 had no significant effect on this parameter. On the other hand, although EM-682 did not significantly affect flank organ surface area, ventral prostate and seminal vesicle weights were reduced by 23% and 22% (p < 0.01), respectively, with topical treatment at the twice daily dose of 100 μg. As can be seen in Table 2, treatment with EM-424, EM-497, EM-494, EM-498, EM-503, EM-493, and EM-580 did not affect the surface area of either flank organs or prostate and seminal vesicle weight.Table IIInhibitory effects of 17β-[N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-one series on the size of flank organ, ventral prostatic, and seminal vesicle weights. Compounds were applied on the right flank organaData are expressed as means ± SEM (n = 4). *p < 0.05, **p < 0.01 versus intact controls.EM(100 μg, BID)Ventral prostate weight (mg)Seminal vesicle weight (mg)Left sideRight sideControl33.2 ± 2.033.6 ± 1.655.4 ± 3.1236.3 ± 13.242431.9 ± 1.332.8 ± 1.656.5 ± 4.4265.4 ± 15.848626.3 ± 0.6*25.0 ± 1.3**59.3 ± 7.4267.1 ± 16.649730.4 ± 1.230.4 ± 0.859.1 ± 2.8249.3 ± 5.349431.1 ± 1.130.7 ± 0.953.8 ± 3.3240.1 ± 5.349331.7 ± 2.127.3 ± 1.457.8 ± 2.0238.3 ± 6.349830.4 ± 1.632.3 ± 2.153.1 ± 3.5248.8 ± 8.350331.9 ± 1.230.0 ± 1.055.1 ± 1.6277.1 ± 11.258030.2 ± 1.132.1 ± 1.253.7 ± 3.2224.2 ± 4.856823.2 ± 0.7**25.3 ± 1.5**45.7 ± 3.5*182.7 ± 7.5**60623.5 ± 2.3**23.8 ± 2.2**40.3 ± 3.4**157.7±11.6**56724.0 ± 1.6**24.0 ± 1.4**51.3 ± 1.4211.8 ± 6.968228.1 ± 1.831.1 ± 1.442.9 ± 2.0**183.3 ± 8.2**a Data are expressed as means ± SEM (n = 4). *p < 0.05, **p < 0.01 versus intact controls. Open table in a new tab In order to obtain more precise information on the activity of the compounds potentially suitable for topical use and devoid of systemic activity, we measured the effects of EM-401, EM-402, and EM-540 on the size of the flank organs and the size of the sebaceous glands of flank organs and ears, 5α-reductase activity of flank organs and ears, as well as the weight of the prostate and seminal vesicle. The compounds were applied topically on the right flank organ and ear at doses of 30, 100, or 300 μg, twice daily. As illustrated in Figure 2, the surface areas of the flank organs of intact control animals measured 32.7 ± 1.0 and 33.4 ± 1.22 mm2 for the left and right sides, respectively. In hamsters treated with EM-401 at doses of 30, 100, and 300 μg, twice daily, for 4 wk, left and right flank organs measured 28.1 ± 1.1 and 29.2 ± 1.7 mm2, 26.0 ± 1.4 and 25.0 ± 1.5 mm2, and 25.3 ± 2.0 and 21.9 ± 0.7 mm2. On the other hand, treatment with increasing doses of EM-402 decreased the size of the right flank organ to the values of 26.0 ± 1.2, 22.8 ± 1.3, and 22.6 ± 1.9 mm2, whereas the size of left flank organs remained within the range of intact controls. Similar results were observed in the animals treated with EM-540 where right flank organ size decreased to 26.7 ± 1.2, 25.3 ± 1.6, and 20.7 ± 1.0 mm2 following doses of 30, 100, and 300 μg, respectively, whereas the left flank organ did not change significantly. Figure 3 illustrates the changes in the size of the sebaceous glands underlying the hamster flank organs that may be considered as a more clinically relevant parameter than the change in size of the flank organ itself. Treatment with 30, 100, and 300 μg EM-401 reduced the size of the left flank sebaceous glands to 89.3% (not significant), 66.7% (p < 0.01), and 63.4% (p < 0.01) of control and that of the right flank sebaceous glands to 94.4% (not significant), 42.0% (p < 0.01), and 24.0% (p < 0.01) of control, respectively. In contrast, treatment with EM-402 and EM-540 reduced the size of the flank organ sebaceous glands on the treated side only, whereas contralateral glands were unaffected. In fact, EM-402 at the dose of 30 μg decreased right sebaceous gland size to 58% (p < 0.01) of control, whereas the doses of 100 and 300 μg twice daily, led to values of 61.6% and 40.8% (p < 0.01) of control, respectively. On the other hand, the inhibitory effect of EM-540 decreased right sebaceous gland size to 53% (p < 0.01) for the smallest dose used (30 μg), whereas the larger doses of 100 and 300 μg resulted in further decreases to 56% and 45% (p < 0.01) of control, respectively. The inhibitory effects of EM-401, EM-402, and EM-540 were also examined on the size of the sebaceous glands of ears, because the inner surface of the hamster ear contains large sebaceous glands that are highly sensitive to androgens. As can be seen in Figure 4, the effects of the three compounds were comparable with those obtained on the size of the sebaceous glands of the flank organs. In fact, topical treatment with EM-401 at the dose of 30 μg decreased the size of sebaceous glands of the right ear to 57.6 ± 6.9% (p < 0.01) of control, whereas the left side was unaffected. Following twice daily treatment with 100 and 300 μg of EM-401, 91.4 ± 4.3% and 73.1 ± 6.2% (p < 0.01) of control values were measured on the left side and 36.5 ± 3.1% and 31.9 ± 3.1% (p < 0.01) of control values were measured on the right side, respectively. On the other hand, EM-402 decreased right ear sebaceous gland size to 48.0 ± 3.6%, 38.9 ± 4.8%, and 48.8 ± 5.1% (p < 0.01) of control intact values and EM-540 decreased gland size to 73.8 ± 5.9%, 54.1 ± 4.7%, and 43.0 ± 2.2% at the doses of 30, 100 and 300 μg, respectively. Neither of the two compounds affected gland size in untreated ears. As we reported previously, 17β-(N-Alkyl/arylformamido)- and 17β-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-5α-androstan-3-ones are potent inhibitors of transfected human type I 5α-

The properties and regulation of the mammalian polyamine transport system are still poorly understood, In estrogen-responsive ZR-75-1 human breast cancer cells, which display low polyamine biosynthetic activity, putrescine and spermidine were internalized with high affinity (K-m = 3.7 and 0.5 mu M, respectively) via a single class of saturable transporter shared by both substrate types, or via distinct but closely similar carriers, The V-max, but not the K-m of polyamine transport was rapidly and synergistically up-regulated by estrogens and insulin, The steady decay in transport activity observed in hormone deprived cells was accelerated by retinoic acid, The enhancement of uptake activity resulting from polyamine depletion was amplified 3-fold by estrogens and insulin despite profound growth inhibition, indicating that the cooperative hormonal induction of polyamine transport is dissociated from cell growth status, Polyamine uptake was under feedback inhibition by at least three distinct mechanisms in these cells, namely (i) the induction of a short-lived protein not actively synthesized without ongoing uptake or upon polyamine deletion; (ii) a more latent, protein synthesis-independent ''trans-inhibition'' mechanism; and (iii) a post-carrier, cycloheximide-sensitive mechanism limiting substrate accumulation. The complexity of these multiple levels of feedback transport inhibition is in keeping with the cytotoxicity of excessive polyamine content.

Since evidence of 5 alpha-reductase activity in rabbit liver homogenate was discovered in 1954, the presence of this enzyme has been demonstrated in many other organs and tissues of mammalian species. 5 alpha-Reductase selectively transforms a 4-ene-3-oxosteroid (e.g., testosterone) irreversibly to the corresponding 5 alpha-3-oxosteroid (e.g., 5 alpha-dihydrotestosterone) in the presence of NADPH as an essential coenzyme at an optimal pH. However, excessive production of 5 alpha-dihydrotestosterone is the major cause of many androgen-related disorders, such as prostate cancer, benign prostatic hyperplasia, acne, female hirsutism, and male pattern baldness; therefore, inhibition of androgenic action by 5 alpha-reductase inhibitors is a logical treatment. During the past two decades, research has focused on understanding the biological functions and effects of 5 alpha-reductase and its 5 alpha-reduced metabolites: purification of the enzyme, substrates, and metabolites; characterization of their physical, chemical, and biochemical properties; analysis of the amino acid sequence of the enzyme; synthesis of various classes of molecules as potential inhibitors; and examination of the biological activity of the inhibitors in vitro and/or in vivo. This review summarizes the biochemical studies on this enzyme, suggests the mechanisms of action of the enzyme or inhibitors, and discusses the chemistry necessary for the preparation, structure-activity relationships, and in vitro and/or in vivo data obtained from the evaluation of nonsteroidal and steroidal compounds that have been tested as inhibitors of 5 alpha-reductase. In particular, IC50 and K-i values for relevant compounds will be compared according to molecular class. This review could function as a comprehensive working reference of what research has been accomplished so far and what problems remain to be solved in the future for those engaged in this interesting field.

The hamster flank organ is a widely used model of the control of sebaceous gland activity by androgens and anti-androgens. Finasteride, a 5 alpha-reductase inhibitor, was administered locally on the surface of the right flank organ and right ear twice daily for 4 weeks. The treatment caused similar 12% to 30% reductions in the size of the sebaceous glands in both flank organs. Moreover, relative mRNA levels of the androgen-regulated FAR-17a gene measured by in situ hybridization as well as [3H]-thymidine incorporation and 5 alpha-reductase activity were similarly decreased in the two flank organs after topical application. The pure anti-androgen flutamide, at the same doses, exerted a more potent effect on all the same parameters, and the effect was also comparable on both the treated and untreated sides of flank organs. Finasteride and flutamide significantly decreased ventral and dorsal prostatic weights after topical application. The present data show that the topical administration of finasteride, in analogy with flutamide, causes local inhibition of sebaceous gland growth in both the costovertebral organs and ears. However, as demonstrated by the similar inhibitory effect in the contralateral untreated side and the reduced weight of the dorsal and ventral lobes of the prostate and seminal vesicles, finasteride and flutamide both exert significant systemic effects.

Synthesis of 17β-hydroxy-17α-(ω-hydroxy/haloalkyn-1′-yl)-4-methyl-4-aza-(1-ene)-5α-steroids (7–22) was achieved by the addition of THP protected hydroxy alkynyllithium to 4-methyl-4-aza-(1-ene)-5α-androstan-3,17-diones (1 and 2), followed by deprotection and halogenation of 17α-(ω-hydroxy) compounds (7–10). Chloro- compounds 13 and 14, and iodo- compound 21 are potent antiandrogens. Introduction of a 1,2-double bond increased the potency by 2-fold compared to the parent compounds.

The stereochemistry of N-n-butyl-N-methyl-11-(16'alpha-chloro-3',17'beta-dihydroxyestra-1',3',5'(10')-trien-7'alpha-yl) undecanamide (4) and N-n-butyl-N-methyl-11-(16'alpha-chloro-3',17'alpha-dihydroxyestra-1',3',5'(10')-trien-7'alpha-yl) undecanamide (5) at the 17'-position was unambiguously established by one dimensional nuclear Overhauser enhancement (NOE difference spectroscopy). Irradiation of H-18' led to the increase in the signal of H-11'beta, H-12'beta, H-8'beta, H-15'beta, and H-16'beta for compound 4 and a very small increase in the signal of H-17' indicating the beta-orientation of the 17'-OH. In contrast, for compound 5, the increase in the signal of H-17' indicated the alpha-orientation of the 17'-hydroxy group. Complete assignment of the H-1 and C-13 resonances is facilitated by the following one- and two-dimensional NMR experiments. H-1 homonuclear correlated spectroscopy (COSY), H-1-C-13 heteronuclear shift correlation (HSC), H-1-C-13 heteronuclear shift correlation via long range couplings (COLOC), and distortionless enhancement by polarisation transfer (DEPT). Comparison of the H-1 and C-13 NMR chemical shifts indicates that the stereochemistry at the 17' position is more easy to determine by analysing the chemical shifts of C-17', C-12', and C-18'.

The stereochemistry of N-n-butyl-N-methyl-11-(16′α-chloro-3′,17′β-dihydroxyestra-1′,3′,5′(10′)-trien-7′α-yl) undecanamide (4) and N-n-butyl-N-methyl-11-(16′α-chloro-3′,17′α-dihydroxyestra-1′,3′,5′(10′)-trien-7′α-yl) undecanamide (5) at the 17′-position was unambiguously established by one dimensional nuclear Overhauser enhancement (NOE difference spectroscopy). Irradiation of H-18′ led to the increase in the signal of H-11′β, H-12′β, H-15′β, and H-16′β for compound 4 and a very small increase in the signal of H-17′ indicating the β-orientation of the 17′-OH. In contrast, for compound 5, the increase in the signal of H-17′ indicated the α-orientation of the 17′-hydroxy group. Complete assignment of the 1H and 13C resonances is facilitated by the following one- and two-dimensional NMR experiments: 1H homonuclear correlated spectroscopy (COSY), 1H-13C heteronuclear shift correlation (HSC), 1H-13C heteronuclear shift correlation via long range couplings (COLOC), and disoritionless enhancement by polarisation transfer (DEPT). Comparison of the 1H and 13C NMR chemical shifts indicated that the stereochemistry at the 17′ position is more easy to determine by analysing the chemical shifts of C-17′, C-12′, and C-18′. (Steroids 59:493–497, 1994)

Synthesis of sulfonium ion mimics 15 and 16 of the carbocationic intermediates 3 and 7, respectively, presumed to be involved in the squalene synthetase catalyzed rearrangement of farnesyl pyrophosphate (1), is reported. Synthesis of 15 involved combination of homogeranyl sulfide with with ethyl alpha-bromoacetate through use of the thallium salt or via the combination of the copper enolate of ethyl acetate and homogeranyl thiosulfonate. Alkylation of the resulting thioester with farnesyl bromide followed by reduction of the ester moiety provided the required alcohol. The sulfur was methylated with iodomethane in a solution of CH3CN and THF to yield 15. Dialkylation of acetylene with farnesyl bromide and homogeranyl thiosulfonate followed by reduction of the triple bond gave vinyl sulfides, which were methylated with iodomethane in the presence of silver perchlorate to give 16.

Solutions comprised of equimolar concentrations of methyllithium, trimethylstannyllithium and cuprous cyanide as well as reaction of Me2Cu(CN)Li2 with one equivalent of hexamethylditin give Me3Sn(Me)Cu(CN)Li2 which selectively delivers the R3Sn moviety to a variety of substrates.