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<p>Steroids 74 (2009) 172–197</p><p>Contents lists available at ScienceDirect</p><p>Steroids</p><p>journa l homepage: www.e lsev ier .com/ locate /s tero ids</p><p>Review</p><p>Structural characteristics of anabolic androgenic steroids contributing to</p><p>binding to the androgen receptor and to their anabolic and androgenic activities</p><p>Applied modifications in the steroidal structure</p><p>A.G. Fragkakia,b, Y.S. Angelisa, M. Koupparisb, A. Tsantili-Kakoulidouc,</p><p>G. Kokotosd, C. Georgakopoulosa,∗</p><p>a Doping Control Laboratory of Athens, Olympic Athletic Center of Athens “Spyros Louis”, Kifisias 37, 15123 Maroussi, Greece</p><p>b Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis, Zografou, 15771 Athens, Greece</p><p>c Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimioupolis, Zografou, 15771 Athens, Greece</p><p>d Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis, Zografou, 15771 Athens, Greece</p><p>a r t i c l e i n f o</p><p>Article history:</p><p>Received 30 June 2008</p><p>Received in revised form 21 October 2008</p><p>Accepted 29 October 2008</p><p>Available online 5 November 2008</p><p>Keywords:</p><p>a b s t r a c t</p><p>Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone introduced for therapeutic</p><p>purposes providing enhanced anabolic potency with reduced androgenic effects. Androgens mediate their</p><p>action through their binding to the androgen receptor (AR) which is mainly expressed in androgen target</p><p>tissues, such as the prostate, skeletal muscle, liver and central nervous system. This paper reviews some</p><p>of the wide spectrum of testosterone and synthetic AAS structure modifications related to the intended</p><p>enhancement in anabolic activity. The structural features of steroids necessary for effective binding to</p><p>the AR and those which contribute to the stipulation of the androgenic and anabolic activities are also</p><p>Androgen receptor</p><p>Structure–activity</p><p>Androgens</p><p>A</p><p>D</p><p>presented.</p><p>© 2008 Elsevier Inc. All rights reserved.</p><p>C</p><p>0</p><p>d</p><p>nabolic steroids</p><p>esigner steroids</p><p>ontents</p><p>1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173</p><p>2. The androgen receptor as member of the nuclear receptor superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174</p><p>2.1. Structural organization of the androgen receptor gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175</p><p>2.2. Structure of the androgen receptor protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175</p><p>2.3. Molecular mechanisms of androgen receptor action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176</p><p>2.4. Ligands bound to the androgen receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177</p><p>3. Structural features of steroids contributing to their androgenic and anabolic activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178</p><p>4. Applied modifications in the steroidal structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179</p><p>4.1. C-1 substitution of steroids. 1-Ene steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179</p><p>4.2. C-2 substitution of steroids. 2-Ene steroids. 3-Deoxy steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190</p><p>4.3. C-4 substitution of steroids. 4-ene steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190</p><p>4.4. 5-Ene steroids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191</p><p>4.5. C-6 substitution of steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191</p><p>4.6. C-7 substitution of steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191</p><p>4.7. C-17 alkyl substitution of steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .</p><p>4.8. Steroids with conjugated double bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . .</p><p>4.9. Steroids with heteroatoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .</p><p>4.10. Steroids with heterocyclic rings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .</p><p>∗ Corresponding author. Tel.: +30 210 6834567 fax: +30 210 6834021.</p><p>E-mail address: oaka@ath.forthnet.gr (C. Georgakopoulos).</p><p>039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.</p><p>oi:10.1016/j.steroids.2008.10.016</p><p>. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192</p><p>. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193</p><p>. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193</p><p>. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193</p><p>http://www.sciencedirect.com/science/journal/0039128X</p><p>http://www.elsevier.com/locate/steroids</p><p>mailto:oaka@ath.forthnet.gr</p><p>dx.doi.org/10.1016/j.steroids.2008.10.016</p><p>A.G. Fragkaki et al. / Steroids 74 (2009) 172–197 173</p><p>5. Side effects of anabolic androgenic steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194</p><p>6. Selective androgen receptor modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194</p><p>7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194</p><p>Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .</p><p>myotropic activity as high as that of testos-</p><p>erone. It is quite similar to the corresponding 2-oxa analog (Table 2,</p><p>ow 29) [135].</p><p>Substitution of the methylene group at C-2 position of DHT and</p><p>7�-methyl-DHT structure by a nitrogen atom has been reported</p><p>Table 2, row 30) with no considerable results in activity [136].</p><p>•</p><p>s 74 (2009) 172–197 193</p><p>The oxygen atom has also replaced the C-7 methylene group.</p><p>he 7-oxa steroids referred in Table 2 (row 31) have been reported</p><p>or interesting antigonadotropic, androgenic and anabolic activities</p><p>137].</p><p>On the other hand, the replacement of the 11-methylene group</p><p>f testosterone by an oxygen atom diminishes the progestational,</p><p>ndrogenic, anabolic and estrogenic activities and this effect is par-</p><p>icularly strong in the case of estrogenic activity (Table 2, row 32)</p><p>138].</p><p>Even androgens having three heteroatoms in ring A have been</p><p>ynthesized (Table 2, row 33) [139]. When three of the six atoms in</p><p>ing A are replaced by heteroatoms, the electronic characteristics</p><p>f the ring are extremely different from DHT, providing convinc-</p><p>ng support for the concept that it is neither the electronic nor</p><p>ydrophobic bonding characteristics of the atoms in ring A but</p><p>heir steric properties which are dominant factors in engendering</p><p>iological activity in the androgen molecule.</p><p>.10. Steroids with heterocyclic rings</p><p>Many studies aimed to alter the nucleophilic environment of</p><p>he steroid nucleous by the attachment of a pyrazole, an isoxa-</p><p>ole or a furazan ring at the C-2, -3 position. Several of these novel</p><p>teroidal heterocycles possess interesting, and often unpredictable,</p><p>ndocrinological activity.</p><p>Steroids with a pyrazole ring: Stanozolol (Fig. 1, 10) is a typical</p><p>example of this class of compounds. It was synthesized in 1961</p><p>and was the first member of a series of anabolically active steroids</p><p>which had a heterocyclic ring fused to ring A of the steroidal</p><p>nucleus [79]. Clinically, it is used in cases of osteoporosis and a</p><p>deficiency in protein synthesis [140]. Slight modifications in the</p><p>molecule of stanozolol such as the introduction of double bonds</p><p>had a great effect on the general activity pattern; the derivative</p><p>with a C-4, -5 double bond did not promote nitrogen retention,</p><p>although it was myotrophic, weakly androgenic and estrogenic.</p><p>The derivative with two double bonds, at C-4, -5 and C-6, -7 posi-</p><p>tions, was typically estrogenic without anabolic or androgenic</p><p>activity (Table 2, rows 34 and 35) [79].</p><p>The series of androst-4-eno[3,2-c]pyrazoles and androsta-</p><p>4,6-dieno[3,2-c]pyrazoles had anabolic to androgenic ratios</p><p>comparable to their saturated analogs in the androstano[3,2-</p><p>c]pyrazole series; that is high ratios for the lower members of the</p><p>series and a rapid decrease in activities above 17�-ethyl [141,142].</p><p>Both anabolic and androgenic activities fell off abruptly in</p><p>the 17�-alkyl-17�-hydroxyandrostano[3,2-c]pyrazoles when the</p><p>17�-alkyl group was larger than ethyl, indicating rather severe</p><p>restrictions on fit to the cellular receptor site in the presence of the</p><p>[3,2-c]pyrazole ring. N-methylation and acylation of the pyrazole</p><p>ring did not affect the anabolic to androgenic ratios.</p><p>The more conspicuous effect of the [3,2-c]pyrazole moiety on</p><p>endocrinological activity was observed when substitutions were</p><p>made in the steroid nucleus in the androstano- and androst-</p><p>4-eno[3,2-c]pyrazoles such as the introduction of a 6�-methyl</p><p>substituent or of the 9�-fluoro-11�-hydroxy groups; 6�,17�-</p><p>dimethyl-17�-hydroxyandrost-4-eno[3,2-c]pyrazole (Table 2,</p><p>row 36) and 11�,17�-dihydroxy-9�-fluoro-17�-methylandrost-</p><p>4-eno[3,2-c]pyrazole (Table 2, row 37) were both less active,</p><p>androgenically and anabolically, than the parent 17�-hydroxy-</p><p>17�-methylandrost-4-eno[3,2-c]pyrazole. Analogous decrease</p><p>in the observed androgenic and anabolic activities were noted</p><p>with 4-methylation and with 19-nor-steroidal[3,2-c]pyrazoles</p><p>[141,142].</p><p>Steroids with an isoxazole ring: Danazol (Fig. 1, 45) is a synthetic</p><p>anabolic steroid which contains an isoxazole ring. It is the</p><p>1 teroid</p><p>•</p><p>a</p><p>o</p><p>S</p><p>c</p><p>e</p><p>a</p><p>a</p><p>d</p><p>s</p><p>M</p><p>b</p><p>5</p><p>p</p><p>f</p><p>i</p><p>k</p><p>[</p><p>r</p><p>•</p><p>•</p><p>•</p><p>•</p><p>•</p><p>•</p><p>•</p><p>•</p><p>6</p><p>i</p><p>o</p><p>a</p><p>d</p><p>n</p><p>a</p><p>a</p><p>t</p><p>s</p><p>w</p><p>p</p><p>n</p><p>p</p><p>d</p><p>i</p><p>b</p><p>[</p><p>p</p><p>i</p><p>p</p><p>a</p><p>o</p><p>n</p><p>c</p><p>p</p><p>[</p><p>7</p><p>a</p><p>94 A.G. Fragkaki et al. / S</p><p>isoxazole derivative of 17�-methyltestosterone with isolated</p><p>weak androgenic activity and no estrogenic or progestagenic</p><p>effects. In clinical use, danazol is applied for the treatment of</p><p>endometriosis. Danazol inhibits ovarian steroidogenesis result-</p><p>ing in decreased secretion of estradiol and may, also, increase</p><p>androgens production [143].</p><p>In regard to anabolic/androgenic properties, the androstano-</p><p>isoxazoles exhibited a pattern of activity similar to that reported</p><p>for their corresponding androstano[3,2-c]pyrazoles [144]. The</p><p>most interesting member of the isoxazole series in terms of</p><p>separation of anabolic and androgenic activities was 17�-</p><p>hydroxy-17�-methyl-androstano[2,3-d]isoxazole. Extension of</p><p>the isoxazole series to the higher homologs (e.g. 17�-hydroxy-</p><p>17�-ethylandrostano[2,3-d]isoxazole) resulted in a decline of</p><p>activity (Table 2, row 38). The 17�-hydroxy-17�-methylandrost-</p><p>4,6-dieno[2,3-d]isoxazole and the analogs with larger 17�-alkyl</p><p>chain resulted in a decline of activity. 17�-Ethynyl-17�-hydroxy-</p><p>androstano[2,3-d]isoxazole and 17�-ethynyl-17�-hydroxy-</p><p>androst-4-eno-[2,3-d]isoxazole (danazol) showed considerable</p><p>myotrophic to androgenic activity (Table 2, row 38).</p><p>The effect of substituents on the androstano-isoxazole</p><p>molecule other than at the 17-position was examined by prepar-</p><p>ing various derivatives containing 4,4-dimethyl, 6�-methyl</p><p>and 3′-alkyl substituents (Table 2, rows 39–41). All derivatives</p><p>showed minimal myotrophic activity [144].</p><p>In the 19-nor series, 19-nor-17�-hydroxy-17�-methyl-</p><p>androst-4-eno[2,3-d]isoxazole proved to be the most interesting.</p><p>In general, the 19-nor-unsaturated isoxazoles was somewhat</p><p>more myotrophic and androgenic than the 19-nor-saturated</p><p>isoxazoles (Table 2, row 42) [144].</p><p>Steroids with a furazan ring: Furazabol (Fig. 1, 46) is a represen-</p><p>tative synthetic anabolic steroid of this class of compounds. It</p><p>differs from stanozolol by having a furazan ring system in place</p><p>of the pyrazole ring. The synthesis was reported in 1965 [79].</p><p>Furazabol is used as a lipid reducer and is suitable for long-term</p><p>treatment of atheriosclerosis and hypercholesterolemia. The</p><p>modified furazabol molecule, with a C-4, -5 double bond, showed</p><p>more or less the same activity as the parent compound; this is</p><p>very strange since the 4,5-unsaturated derivative of stanozolol</p><p>has practically no activity (Table 2, row 43).</p><p>Having had an almost 20 years experience in doping control</p><p>nalysis, the successful undertaken of the Sixth World Champi-</p><p>nship of Athletics in Athens, in 1997 [145] and of the XXVIII</p><p>ummer Olympic Games in Athens, in 2004 [146,147], it is con-</p><p>luded that the “old fashioned synthetic steroids” are still abused,</p><p>.g. stanozolol, methandienone, methyltestosterone, methenolone</p><p>nd oxandrolone. Doping control analyses statistics from the WADA</p><p>ccredited laboratories confirm this conclusion [148]. The use of</p><p>oping agents is no longer restricted to competing athletes as young</p><p>portspeople in schools and non-competing amateurs use them.</p><p>isuse of AAS is also increasing among gym customers for whom</p><p>odily appearance is a priority [149].</p><p>. Side effects of anabolic androgenic steroids</p><p>AAS have been associated with several side effects necessitating</p><p>hysicians to use caution when prescribing these agents. Due to the</p><p>act that clinical trials for many AAS are not feasible, much of the</p><p>nformation on adverse reactions is anecdotal or is assumed from</p><p>nown problems associated with therapeutic use of these agents</p><p>150]. A summary of side effects associated with the use of AAS is</p><p>eferred below [151,152]:</p><p>Cardiovascular; the unfavorable changes in blood lipid profiles</p><p>caused by AAS include increased low-density lipoprotein (LDL)</p><p>i</p><p>h</p><p>a</p><p>s</p><p>f</p><p>s 74 (2009) 172–197</p><p>concentration, a decrease in high-density lipoprotein (HDL) con-</p><p>centration by 30–50% and a reduction</p><p>in the concentration of</p><p>apoprotein A1. These metabolic changes explain the many reports</p><p>of cardiovascular disease and hypertension in people who misuse</p><p>AAS [153,154]. Other cardiovascular problems are erythrocytosis,</p><p>myocardial hypertrophy, arrhythmias and thrombosis.</p><p>Hepatic; hepatotoxicity, jaundice, neoplasia, cholestasis, peliosis</p><p>hepatic. Elevated liver function tests (aspartate aminotransferase,</p><p>alanine aminotransferase, bilirubin, lactic dehydrogenase, alka-</p><p>line phosphatase) have been associated with the abuse of AAS,</p><p>particularly of the 17�-alkylated steroids [154].</p><p>Dermatologic; acne, gynecomastia, striae, alopecia.</p><p>Reproductive-endocrine; libido changes, subfertility, decreased</p><p>luteinizing hormone (LH) and follicle-stimulating hormone</p><p>(FSH).</p><p>Male specific; prostate hypertrophy, prostatic carcinoma, impo-</p><p>tence, impaired spermatogenesis, testicular atrophy, gynecomas-</p><p>tia, priapism [155].</p><p>Female specific; hirsutism/masculinization, voice deepening,</p><p>menstrual irregularities, clitoral enlargement, breast atrophy, ter-</p><p>atogenicity.</p><p>Children specific; premature epiphyseal closure, precocious</p><p>puberty.</p><p>Behavioral; mood swings, aggression, mania, depression, with-</p><p>drawal, dependence, anxiety [156].</p><p>. Selective androgen receptor modulators</p><p>The clinical application of the steroidal AR ligands has been lim-</p><p>ted by poor oral bioavailability, potential hepatotoxicity and lack</p><p>f tissue selectivity. Therefore, non-steroidal AR ligands, known</p><p>s selective androgen receptor modulators (SARMs), have been</p><p>eveloped to overcome these problems [157,158]. SARMs are a</p><p>ovel class of AR ligands with improved pharmacokinetic char-</p><p>cteristics, high specificity for AR, improved oral bioavailability</p><p>nd tissue-selective pharmacological activities, which are expected</p><p>o extend the clinical applications of androgens in osteoporo-</p><p>is, osteopaenia, sarcopaenia in elderly men and postmenomausal</p><p>omen, muscle wasting, male contraception and diseases of the</p><p>rostate [159]. Several structural classes of non-steroidal AR ago-</p><p>ists have been discovered and developed, most of which adopt the</p><p>harmacophores from the non-steroidal anti-androgens that were</p><p>eveloped in the 1970s and 1980s. The four major structural classes</p><p>nclude the quinolinone analogs, aryl propionamide analogs, the</p><p>icyclic hydantoin analogs and the tetrahydro-quinoline analogs</p><p>160]. All representatives from the four classes share similar</p><p>harmacological profiles, which include strong anabolic activity</p><p>n skeletal muscle and bone, and partial agonist activity in the</p><p>rostate. Since 2008, SARMs have been categorized as anabolic</p><p>gents and prohibited by the WADA [1]. Suitable detection meth-</p><p>ds for SARMs are based on mass spectrometric approaches which</p><p>ecessitated the elucidation of dissociation pathways in order to</p><p>haracterize and identify the target analytes in doping control sam-</p><p>les as well as potential metabolic products and synthetic analogs</p><p>161].</p><p>. Conclusions</p><p>Overall, the design and synthesis of steroids with increased</p><p>nabolic and decreased androgenic activity, mainly through mod-</p><p>fications in the structure of the natural androgen testosterone,</p><p>as been a challenge for research teams for many decades. Indeed,</p><p>s time passes, the AR is better explored and the biochemistry of</p><p>teroids is better understood. This paper summarized the main</p><p>unctional and structural aspects of the AR and those structural</p><p>teroid</p><p>f</p><p>A</p><p>t</p><p>i</p><p>m</p><p>c</p><p>e</p><p>m</p><p>A</p><p>p</p><p>A</p><p>A</p><p>A</p><p>A</p><p>A</p><p>A</p><p>A</p><p>D</p><p>E</p><p>F</p><p>G</p><p>G</p><p>H</p><p>I</p><p>L</p><p>L</p><p>L</p><p>M</p><p>N</p><p>P</p><p>R</p><p>S</p><p>T</p><p>W</p><p>R</p><p>A.G. Fragkaki et al. / S</p><p>eatures of a steroid which contribute to effective binding to the</p><p>R and stipulate its androgenic and anabolic activities. Some of</p><p>he numerous modifications in the structure of steroids found</p><p>n the literature were presented in the sections of the paper,</p><p>aking apparent the problem of designer steroids for doping</p><p>ontrol. WADA accredited laboratories should probably focus their</p><p>fforts on identification of substances where the lack of reference</p><p>aterials exists, by adopting more generic screening procedures.</p><p>cknowledgement</p><p>The authors wish to thank WADA for the financial support of the</p><p>roject.</p><p>ppendix A. 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J Mass Spectrom 2008;43:865–76.</p><p>http://www.wada-ama.org/en/dynamic.ch2%3FpageCategory.id=335</p><p>Structural characteristics of anabolic androgenic steroids contributing to binding to the androgen receptor and to their anabolic and androgenic activities</p><p>Introduction</p><p>The androgen receptor as member of the nuclear receptor superfamily</p><p>Structural organization of the androgen receptor gene</p><p>Structure of the androgen receptor protein</p><p>Molecular mechanisms of androgen receptor action</p><p>Ligands bound to the androgen receptor</p><p>Structural features of steroids contributing to their androgenic and anabolic activities</p><p>Applied modifications in the steroidal structure</p><p>C-1 substitution of steroids. 1-Ene steroids</p><p>C-2 substitution of steroids. 2-Ene steroids. 3-Deoxy steroids</p><p>C-4 substitution of steroids. 4-ene steroids</p><p>5-Ene steroids</p><p>C-6 substitution of steroids</p><p>C-7 substitution of steroids</p><p>C-17 alkyl substitution of steroids</p><p>Steroids with conjugated double bonds</p><p>Steroids with heteroatoms</p><p>Steroids with heterocyclic rings</p><p>Side effects of anabolic androgenic steroids</p><p>Selective androgen receptor modulators</p><p>Conclusions</p><p>Acknowledgement</p><p>Abbreviations</p><p>References</p><p>. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195</p><p>Appendix A. Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195</p><p>. . . . . .</p><p>1</p><p>o</p><p>s</p><p>d</p><p>a</p><p>i</p><p>h</p><p>s</p><p>i</p><p>H</p><p>f</p><p>b</p><p>s</p><p>h</p><p>i</p><p>o</p><p>a</p><p>e</p><p>a</p><p>a</p><p>s</p><p>m</p><p>a</p><p>c</p><p>r</p><p>A</p><p>s</p><p>t</p><p>p</p><p>o</p><p>i</p><p>r</p><p>c</p><p>d</p><p>2</p><p>t</p><p>(</p><p>u</p><p>o</p><p>u</p><p>[</p><p>t</p><p>T</p><p>a</p><p>T</p><p>a</p><p>a</p><p>o</p><p>t</p><p>w</p><p>t</p><p>a</p><p>s</p><p>a</p><p>t</p><p>o</p><p>t</p><p>l</p><p>l</p><p>o</p><p>t</p><p>o</p><p>a</p><p>f</p><p>r</p><p>fi</p><p>i</p><p>b</p><p>n</p><p>i</p><p>a</p><p>l</p><p>t</p><p>tor modulators (SARMs), that are currently developed instead</p><p>of steroidal androgens, are briefly discussed. The effect of the</p><p>route of AAS administration is beyond the scope of this review</p><p>paper.</p><p>Table 1</p><p>Steroids declared on labels of nutritional supplements found on the Internet market</p><p>[5].</p><p>Claimed substances</p><p>1-androstene-3�,17�-diol</p><p>1,4-androstadiene-3,17-dione</p><p>1,4,6-androstatriene-3,17-dione</p><p>2�,3�-epithio-17�-methyl-17�-hydroxy-5�-androstane</p><p>2�,17�-dimethyl-5�-androstane-3-one-17�-ol</p><p>3�-hydroxy-androstan ethyl ester</p><p>3�-hydroxy-androst-5-ene-17-one</p><p>[3,2-c]pyrazole-5�-androstane-17�-tetrahydropyranol</p><p>4-chloro-17�-methyl-androst-1,4-diene-3,17�-diol</p><p>4-chloro-17�-methyl-androst-4-ene-3,17�-diol</p><p>5�-androstane-[2,3-c]furazan-17�-tetrahydropyranol</p><p>5�-androstane-[2,3-c]furazan-17�-tetrahydropyranol ether</p><p>6-bromo-androstenedione</p><p>6-oxo-androstenedione</p><p>6,17-dioxo-4-androstene-3-ol</p><p>7-keto-DHEA</p><p>11-oxo-androsterone</p><p>17�-methyl-1,4-androstadien-3,17-diol</p><p>17�-methyl-4-hydroxy-nandrolone</p><p>17�-methyl-17�-hydroxy-androst-2,4,6-triene-3-one</p><p>17�-methyl-17�-hydroxy-estra-4,9(10)-dien-3-one</p><p>17-alkyl-5�-dehydro-androsta-1,6-diene-3-one</p><p>(25R)-4-spirosten-3�,6�-diol</p><p>References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .</p><p>. Introduction</p><p>Doping control analysis has become a multidisciplinary field</p><p>f analytical chemistry primarily utilizing chromatographic mass</p><p>pectrometric techniques. These strategies aim to the detection of</p><p>rug abuse in sports covering a wide range of xenobiotics (anabolic</p><p>gents, stimulants, narcotics, diuretics and other prohibited med-</p><p>cations) as well as synthetic analogs to endogenously produced</p><p>ormones such as testosterone. The use of anabolic androgenic</p><p>teroids (AAS) has been prohibited in sports since 1974 by the Med-</p><p>cal Commission of the International Olympic Committee (IOC).</p><p>owever, these medications which are in use in clinical practice</p><p>or medical purposes for many decades are still commonly abused</p><p>y athletes.</p><p>In the last years a new phenomenon closely connected to the</p><p>uperior efficiency of doping control to detect these substances</p><p>as been arisen. Chemically modified steroids that are not used</p><p>n clinical practice and either have been synthesized in the past</p><p>r have been specifically developed to circumvent doping control</p><p>re detected from doping authorities. Currently there is a great</p><p>ffort from the World Anti-Doping Agency (WADA) and WADA</p><p>ccredited laboratories to discover these preparations, known</p><p>s designer steroids, and include them in the list of prohibited</p><p>ubstances [1]. Many designer steroids have been derived from</p><p>odifications of testosterone structure (Fig. 1, 1), the main natural</p><p>ndrogenic and anabolic steroid formed in the interstitial (Leydig)</p><p>ells of the testes, which acts on both the reproductive and non-</p><p>eproductive target tissues [2]. Many designer steroids, aside from</p><p>AS prohormones, have been detected as ingredients in nutritional</p><p>upplements, although most of the times they are not declared on</p><p>he labels [3,4]. Research teams continue the analyses of such sup-</p><p>lements, found on the Internet, for which steroids are declared</p><p>n their labels (Table 1) [5]. Many of these steroids are neither</p><p>ncluded in the WADA list of prohibited substances nor are they</p><p>eferred in the Anabolic Steroid Control Act 2004 [6]. Notable suc-</p><p>ess have been made in the development of reliable methods to</p><p>etect some of these compounds, such as norbolethone (Fig. 1,</p><p>) [7], tetrahydrogestrinone (Fig. 1, 3) [8], desoxymethyltestos-</p><p>erone (Fig. 1, 4) [9], methyltrienolone (Fig. 1, 5) [10], methasterone</p><p>Fig. 1, 6) [11] and prostanozol (Fig. 1, 7) [12]. The detection of</p><p>nknown designer steroids includes methods which are based</p><p>n mass spectrometry [13–15]. As an alternative screening for</p><p>nknown synthetic steroids one might consider bioactivity testing</p><p>16].</p><p>Known modifications of testosterone molecule include alkyla-</p><p>ion at the 17�-position and/or modification of the ring structure.</p><p>he goal of these modifications is the production of derivatives that</p><p>re more anabolic and less androgenic than the parent molecule.</p><p>he esterification of the 17�-hydroxyl group by carboxylic acids</p><p>lso increases the steroid activity due to the prolongation of the</p><p>ction, as the steroid gets lipophilic properties and the capability</p><p>f retaining in fat tissue.</p><p>Androgens exert their effects by binding to the androgen recep-</p><p>or (AR) [17]. AR function is regulated by the binding of androgens,</p><p>hich initiates sequential conformational changes of the receptor</p><p>hat affect receptor–protein interactions and receptor–DNA inter-</p><p>(</p><p>A</p><p>A</p><p>E</p><p>N</p><p>. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195</p><p>ctions. In addition to natural androgens, AR binds a variety of</p><p>ynthetic molecules with different affinities. The known AR ligands</p><p>re classified as steroidal or non-steroidal based on their struc-</p><p>ure or as agonist or antagonist based on their ability to activate</p><p>r inhibit transcription of AR target genes.</p><p>Considerable attention has been directed to the elucidation of</p><p>he effect of structural changes on the action of AAS. However,</p><p>ittle information is available concerning the principles control-</p><p>ing the effect of substitution. Moreover, there is not a uniform</p><p>pinion about the mode by which the steroid molecules exert</p><p>heir action. As part of our research interests on the development</p><p>f generic screening methods that will include designer steroids</p><p>nd in an attempt to reorient ours and others research efforts</p><p>or detection of novel or possible designer steroids targets, we</p><p>eview in this paper the literature concerning the main modi-</p><p>cations in the structure of testosterone and of other steroids</p><p>ncluded in the WADA list of prohibited substances that are proba-</p><p>le to create new molecules/designer steroids. Structural elements</p><p>eeded for effective binding to the AR and aspects concern-</p><p>ng the biology, the structure and the mechanism of AR action</p><p>re also discussed. Structure–activity studies of testosterone ana-</p><p>ogues are summarized. The side effects of the AAS abuse and</p><p>he non-steroidal androgens, known as selective androgen recep-</p><p>25R)-5�-spirostan-6-one-3�-ol</p><p>ndrostane-3�-ol-17-one</p><p>ndrostane-3�-ol-17-one</p><p>pihydroxyandrostane-17-one ethyl ester</p><p>orandrostene-3�-ol-17-one</p><p>1 teroid</p><p>2</p><p>r</p><p>s</p><p>s</p><p>r</p><p>F</p><p>t</p><p>74 A.G. Fragkaki et al. / S</p><p>. The androgen receptor as member of the nuclear</p><p>eceptor superfamily</p><p>The androgen receptor is a member of the nuclear receptor</p><p>uperfamily, members of which function as ligand-inducible tran-</p><p>cription factors that mediate the expression of target genes, in</p><p>r</p><p>o</p><p>e</p><p>c</p><p>[</p><p>ig. 1. General steroidal structure (top) and molecular structures of steroids referred in th</p><p>he parentheses. Carbon atoms of the steroids are numbered according to the standard st</p><p>s 74 (2009) 172–197</p><p>esponse to ligands, specific to each receptor [18]. The nuclear</p><p>eceptors are subdivided into three general types [19,20], the first</p><p>f which include the classical steroid receptors such as the AR, the</p><p>strogen receptor (ER), the progesterone receptor (PR), the gluco-</p><p>orticoid receptor (GR) and the mineralocorticoid receptor (MR)</p><p>21,22]. These receptors bind to DNA response elements organized</p><p>e paper. Alternative names of the steroids and/or abbreviations are included inside</p><p>eroid nomenclature and the cycles designated by letters.</p><p>A.G. Fragkaki et al. / Steroids 74 (2009) 172–197 175</p><p>(Conti</p><p>a</p><p>r</p><p>i</p><p>M</p><p>a</p><p>2</p><p>m</p><p>(</p><p>d</p><p>e</p><p>p</p><p>p</p><p>t</p><p>s</p><p>s</p><p>b</p><p>n</p><p>t</p><p>2</p><p>Fig. 1.</p><p>s inverted repeats [19]. Extensive research in the field of steroid</p><p>eceptors for the last 3 decades provides considerable understand-</p><p>ng of the physiological and pathological roles of steroid receptors.</p><p>any functional and structural similarities are shared, yet display</p><p>pparent differences among steroid receptors.</p><p>.1. Structural organization of the androgen receptor gene</p><p>In 1981, the AR gene was found to be localized to the X chro-</p><p>osome by genetic analysis of androgen insensitivity syndromes</p><p>AIS) in humans and mice [17,23]. The AR cDNA was cloned in 1988,</p><p>espite the difficulty in obtaining sufficient purified protein to gen-</p><p>rate antibodies to screen cDNA expression libraries or to generate</p><p>artial amino acid sequences to design synthetic oligonucleotide</p><p>robes [24,25].</p><p>t</p><p>p</p><p>T</p><p>s</p><p>nued ).</p><p>AR is a single copy gene composed of 8 exons (Fig. 2) (see Sec-</p><p>ion 2.2) [26–28]. The existence of additional AR genes has been</p><p>uggested, possibly encoding a membrane bound AR [29–31]. This</p><p>uggestion is based on the observations in osteoblasts and in the</p><p>rain in which testosterone exerts an effect on calcium influx or</p><p>euronal activity in 2–30 s, too rapid to be the result of transcrip-</p><p>ional activity of the classical AR [29,30].</p><p>.2. Structure of the androgen receptor protein</p><p>The AR is a protein of approximately 918 amino acids due to</p><p>he variation in AR length from the length polymorphisms of a</p><p>oly-glutamine and poly-glycine tract in the NH2-terminal [32–36].</p><p>he AR has a molecular weight of 110 kDa [37]. Within 10 min of</p><p>ynthesis, AR becomes phosphorylated, resulting in an additional</p><p>176 A.G. Fragkaki et al. / Steroids 74 (2009) 172–197</p><p>with</p><p>b</p><p>a</p><p>r</p><p>a</p><p>l</p><p>(</p><p>•</p><p>•</p><p>•</p><p>•</p><p>o</p><p>t</p><p>a</p><p>f</p><p>i</p><p>i</p><p>l</p><p>b</p><p>s</p><p>t</p><p>i</p><p>t</p><p>r</p><p>t</p><p>A</p><p>i</p><p>d</p><p>A</p><p>b</p><p>i</p><p>r</p><p>u</p><p>m</p><p>p</p><p>M</p><p>h</p><p>d</p><p>2</p><p>s</p><p>c</p><p>l</p><p>p</p><p>l</p><p>r</p><p>c</p><p>p</p><p>t</p><p>r</p><p>c</p><p>Fig. 2. Structural organization of the AR gene and protein. Reprinted</p><p>and of 112 kDa [38]. A smaller translation product of 87 kDa is</p><p>lso detected in some tissues [39]. This smaller AR isoform (AR-A)</p><p>esults from translation initiating at the first internal methionine</p><p>nd is generally expressed at less than 20% of the level of the full</p><p>ength AR in tissues in which it is found [40].</p><p>The AR protein can be divided into four functional domains</p><p>Fig. 2) [17,41]:</p><p>The N-terminal domain (NTD), which serves a modulatory func-</p><p>tion, is encoded by exon 1 [27,42]. This domain shows the greatest</p><p>degree of variability (less than 25% identity) among nuclear</p><p>receptors both in length and in sequence [43]. The AR NTD</p><p>has been characterized to interact with several transcriptional</p><p>coregulators [44] and, also, contributes to the ligand-induced</p><p>three-dimensional structure of AR by interaction with the Ligand</p><p>Binding Domain (LBD) [45,46].</p><p>The DNA-binding domain (DBD). This domain of steroid recep-</p><p>tors consists of two zinc fingers, which are encoded by exons 2</p><p>and 3 [27,42]. This is the most highly conserved domain among</p><p>receptors, reflecting the common need to bind to DNA, while</p><p>the variation is responsible for the selection of different target</p><p>sequences [32,47]. AR binds to DNA as a dimer to the inverted</p><p>repeat androgen response element (ARE), as well as to more com-</p><p>plex response elements [48]. The AR DBD has between 56% and</p><p>79% overall amino acid identity to the DBD of PR, MR, GR and ER</p><p>[33,47].</p><p>The hinge region, which may function as an interaction site for</p><p>other proteins. Exon 4 encodes the hinge region which contains</p><p>part of the AR nuclear localization signal [27,42]. The AR hinge</p><p>region is phosphorylated in the absence of androgen [37]. The</p><p>dephosphorylation of the AR hinge region contributes to the reg-</p><p>ulation of AR transcriptional activity, although the mechanism of</p><p>this effect has not yet been described.</p><p>The Ligand Binding Domain (LBD). It forms a ligand binding</p><p>pocket which consists of 11–13 �-helices [49,50]. The first two</p><p>�-helices of the LBD are encoded by exon 4. The remainder of the</p><p>LBD is encoded by exons 5–8 [27,42]. The main function of the</p><p>LBD is to bind ligand. The LBD of different steroid receptors shows</p><p>sequence identity with a range from 22% to 55%, which reflects</p><p>receptor specificity for individual hormones [51]. Even though the</p><p>LBD of steroid receptors share a common relatively low sequence</p><p>identity, they all assume a similar three-dimensional structure</p><p>with certain highly conserved structural features. These confor-</p><p>mational similarities provide the structural basis for the cross</p><p>reactivity that is commonly observed in synthetic steroids.</p><p>Two transcriptional activation functions have been identified</p><p>n the basis of deletion and mutational analyses [49]. The NH2-</p><p>D</p><p>e</p><p>n</p><p>s</p><p>t</p><p>permission from [17]. Copyright 2008 American Chemical Society.</p><p>erminal activation function (AF-1) is composed of sequences that</p><p>llow ligand independent transcription when artificially separated</p><p>rom the LBD [52]. The second activation function (AF-2) is located</p><p>n the LBD. The mutation or deletion of the AF-2 domain dramat-</p><p>cally reduces transcriptional activation in response to activating</p><p>igands [45,53]. Recently, the crystal structure of the AR LBD has</p><p>een determined [50]. The AR and PR LBDs share 77% sequence</p><p>imilarity and as expected, the three-dimensional structure of the</p><p>wo LBDs is very similar.</p><p>A unique property of the AR in the regulation of gene expression</p><p>s its ability to provide intra-domain interaction and communica-</p><p>ion between the amino- and the carboxy-terminal domains of the</p><p>eceptor [45,46]. Interaction between NH2- and COOH-termini of</p><p>he AR was shown to be necessary for the testosterone-induced</p><p>R stabilization and an antiparallel arrangement of AR monomers</p><p>n the AR dimer [17,54]. The importance of this interaction was</p><p>emonstrated in a mutational analysis of the AF-2 domain of the</p><p>R, which showed that disruption of the functional interaction</p><p>etween NH2- and COOH-termini of the AR is linked to androgen</p><p>nsensitivity syndrome [55–57].</p><p>AR is extensively studied due to its involvement in the male</p><p>eproductive system, Kennedy’s disease and prostate cancer. Reg-</p><p>lation of AR activity can be achieved in several different ways:</p><p>odulation of AR gene expression, androgen binding to AR, AR</p><p>rotein stability, AR nuclear translocation and AR transactivation.</p><p>olecular and animal/clinical studies provide an understanding of</p><p>ow the androgen-AR signaling pathway play key roles in the above</p><p>iseases [58–60].</p><p>.3. Molecular mechanisms of androgen receptor action</p><p>The primary mechanism of action of AR is direct gene tran-</p><p>cription. The binding of a ligand to the AR induces specific</p><p>onformational changes in the LBD, which could further modu-</p><p>ate the surface topology of the protein and induce subsequent</p><p>rotein–protein interactions between the receptor and other cel-</p><p>ular proteins [61,62]. More specifically, the binding of a ligand</p><p>esults in a conformational change in the receptor which in turn</p><p>auses dissociation of heat shock proteins, dimerization and trans-</p><p>ort from the cytosol to the cell nucleus where the AR dimer binds</p><p>o AREs [61]. The ligand stimulation of AR transcriptional activity</p><p>equires AR interaction with a variety of cellular proteins, known as</p><p>oregulators that facilitate AR conformation, nuclear localization,</p><p>NA binding and interaction with the basal transcriptional machin-</p><p>ry. Coregulators are broadly defined as proteins that interact with</p><p>uclear receptors to enhance (coactivators) or reduce (corepres-</p><p>ors) transactivation of target genes but do not significantly alter</p><p>he basal transcriptional rate [63–65].</p><p>teroid</p><p>n</p><p>m</p><p>g</p><p>c</p><p>r</p><p>s</p><p>g</p><p>w</p><p>H</p><p>s</p><p>2</p><p>f</p><p>a</p><p>t</p><p>f</p><p>d</p><p>a</p><p>w</p><p>g</p><p>i</p><p>t</p><p>m</p><p>t</p><p>t</p><p>t</p><p>n</p><p>(</p><p>t</p><p>p</p><p>1</p><p>w</p><p>t</p><p>f</p><p>t</p><p>t</p><p>b</p><p>m</p><p>A</p><p>b</p><p>H</p><p>F</p><p>C</p><p>p</p><p>i</p><p>G</p><p>w</p><p>T</p><p>c</p><p>b</p><p>o</p><p>l</p><p>h</p><p>p</p><p>a</p><p>s</p><p>o</p><p>a</p><p>A</p><p>i</p><p>m</p><p>a</p><p>i</p><p>m</p><p>a</p><p>i</p><p>o</p><p>a</p><p>t</p><p>o</p><p>a</p><p>g</p><p>p</p><p>l</p><p>s</p><p>s</p><p>b</p><p>w</p><p>a</p><p>t</p><p>•</p><p>A.G. Fragkaki et al. / S</p><p>Besides the genomic pathway, a second mode of action or the</p><p>on-genomic pathway has been reported for the AR, in skeletal</p><p>uscle cells [66] and prostate cancer cells [59,67]. The non-</p><p>enomic pathway induces direct interactions between the AR and</p><p>ytosolic proteins from different signaling pathways and involves</p><p>apid changes</p><p>in cell function independent of changes in gene tran-</p><p>cription, such as changes in ion transport [68]. Separation of the</p><p>enomic and non-genomic functions of AR using specific ligands</p><p>as proposed as a new strategy to achieve tissue selectivity [68,69].</p><p>owever, structural features that are essential for achieving the</p><p>eparation have not been determined.</p><p>.4. Ligands bound to the androgen receptor</p><p>The various ligands bind AR with different affinities. The terms</p><p>or the quantitative estimation of the receptor–ligand interaction</p><p>re either the Ki values, ranging from low nanomolar concentra-</p><p>ions for the most potent androgens to micromolar concentrations</p><p>or the weaker ones, or the relative binding affinities (RBAs). It is</p><p>ifficult to predict the strength of the interaction between a lig-</p><p>nd and a receptor only on the basis of its structure, since steroids</p><p>ith very similar structures can possess different affinities for a</p><p>iven receptor while structurally different ligands could have sim-</p><p>lar affinities [70]. Research has been directed to the estimation of</p><p>he RBAs of several anabolic steroids when tested as competitors to</p><p>ethyltrienolone (Fig. 1, 5) for binding to the AR in rat, rabbit skele-</p><p>al muscle and rat prostate. Methyltrienolone binds AR so strongly</p><p>hat is often used in studies as a reference substance to estimate</p><p>he RBAs of other steroids. Steroids are characterized as strong (19-</p><p>ortestosterone, Fig. 1, 8 and methenolone, Fig. 1, 9) or weak ligands</p><p>stanozolol, Fig. 1, 10 and methandienone, Fig. 1, 11) for binding to</p><p>he AR according to the estimated values of the RBAs. Other com-</p><p>ounds had RBAs too low to be determined (oxymetholone, Fig. 1,</p><p>2 and ethylestrenol, Fig. 1, 13). It is believed that for those steroids</p><p>ith anabolic–androgenic activity in vivo that do not bind to AR,</p><p>here must be an indirect mechanism of action (e.g. via biotrans-</p><p>ormation to active compounds) [71,72].</p><p>The binding of 5�-dihydrotestosterone (5�-DHT, Fig. 1, 14),</p><p>estosterone and methyltrienolone has been studied using crys-</p><p>allography data and important information can be deduced. The</p><p>inding mechanism of these steroids (Fig. 3, for DHT) involves</p><p>ainly hydrophobic interactions which play an essential role in</p><p>R ligand binding as the steroid skeleton interacts with the ligand</p><p>inding pocket largely through hydrophobic interactions [73,74].</p><p>ydrogen bonding in some regions of the ligand binding pocket also</p><p>ig. 3. DHT-AR hydrogen bonding network. Reprinted with permission from [73].</p><p>opyright 2008 American Chemical Society.</p><p>•</p><p>s 74 (2009) 172–197 177</p><p>lays a critical role in steroidal ligand binding. The 3-keto group</p><p>n ring A forms hydrogen bonds with the side chains of residues</p><p>ln711 and Arg752 directly or indirectly (through a H2O molecule),</p><p>hile the ring C is in close contact with the side chain of Asn705.</p><p>he hydrogen atom of the 17�-OH group forms bonds with the side</p><p>hains of Asn705 and Thr877. The side chains of the LBD hydropho-</p><p>ic residues of these steroids can easily adopt variable positions in</p><p>rder to better fit the hydrophobic core of the steroid and stabi-</p><p>ize it. LBD is also composed of polar amino acids able to establish</p><p>ydrogen bonds at both extremities of the steroid nucleus of all</p><p>otent androgens. It is more likely that the hydrophobic residues</p><p>re of paramount importance, not only for the stabilization of the</p><p>teroid in its pocket but also for the high selectivity and specificity</p><p>bserved for all members of the nuclear superfamily.</p><p>The importance of these interactions have been revealed</p><p>lso from mutation studies [70]. For example, a mutation at</p><p>rg752Gln711 affects the AR functional activity and males bear-</p><p>ng this mutation suffer from the genetic disorder called AIS, the</p><p>ost common form of male pseudohermaphroditism and present</p><p>completely female phenotype. Moreover and in agreement with</p><p>ts role in steroid binding deduced from crystal structures, replace-</p><p>ent of residue Gln711 does not apparently affect AR binding</p><p>ctivity. Finally the importance of Asn705 and Thr877 residues</p><p>n the AR steroid binding is well documented since the change</p><p>f Asn705 for a serine residue results in a complete absence of</p><p>ndrogen binding. Replacement of Thr877 has been found many</p><p>imes in specimens obtained from men with metastatic carcinoma</p><p>f the prostate. Moreover, the threonine-to-alanine substitution</p><p>t position 877 is known to alter the selectivity toward andro-</p><p>ens and estrogen, and can be transcriptionally activated in the</p><p>resence of any other steroids. In the mutant form of the AR</p><p>igand binding domain (Thr877), the mutation introduces more</p><p>pace around the D-ring of 5�-DHT to accommodate a larger sub-</p><p>tituent at the 17-position, which allows ligands with greater steric</p><p>ulk at this position, like progesterone and cortisol, to bind AR as</p><p>ell.</p><p>Important conclusions have been also deduced from structure–</p><p>ctivity studies that complementary supports the above observa-</p><p>ions:</p><p>A 3-keto group in the A-ring (Fig. 4): The oxygen atom of the</p><p>keto group at position C-3 has a lone pair of electrons and</p><p>thus could act as a hydrogen bond acceptor able to establish a</p><p>strong interaction with polar or charged amino acids [70]. The</p><p>reduction of the keto group at position C-3 to an alcohol (either</p><p>� or � isomers) is not favorable for binding [73]. For example,</p><p>both 3�-androstanediol and 3�-androstanediol show weaker</p><p>binding than 5�-DHT. Similarly, the reduction of the keto group</p><p>at position C-3 in testosterone molecule to the 3�-hydroxy iso-</p><p>mer (4-androstenediol) reduces binding affinity about 39-fold.</p><p>However, elimination of the 3-keto group of DHT causes only a</p><p>5-fold reduction of binding.</p><p>A 17ˇ-hydroxyl in the D-ring (Fig. 4): The hydrogen atom of the</p><p>17�-hydroxyl group bears a positive charge that allows its inter-</p><p>action with a highly electronegative atom born by an adjacent</p><p>amino acid residue [70]. This interaction is observed between the</p><p>17�-hydroxyl group and the residues Asn705 and Thr877, which</p><p>maintains the steroid firmly inside the ligand binding pocket. Any</p><p>modification or elimination of the 17�-hydroxyl group reduces</p><p>the AR binding affinity. A reduction in binding affinity was also</p><p>observed by esterifying the 17�-hydroxyl in testosterone and in</p><p>DHT with benzoic acid [73]. The 17�-hydroxyl group does not</p><p>favor AR binding, either. Moving the hydroxyl group from the</p><p>17�- to the 17�-position resulted in almost 190-fold activity loss</p><p>of testosterone. As the number of carbon atoms of the 17�-side</p><p>178 A.G. Fragkaki et al. / Steroids 74 (2009) 172–197</p><p>of 5�</p><p>•</p><p>i</p><p>t</p><p>•</p><p>•</p><p>t</p><p>i</p><p>g</p><p>m</p><p>b</p><p>s</p><p>h</p><p>i</p><p>h</p><p>l</p><p>o</p><p>t</p><p>w</p><p>s</p><p>3</p><p>a</p><p>n</p><p>t</p><p>a</p><p>m</p><p>v</p><p>i</p><p>a</p><p>a</p><p>e</p><p>t</p><p>s</p><p>a</p><p>h</p><p>d</p><p>A</p><p>i</p><p>s</p><p>o</p><p>s</p><p>m</p><p>r</p><p>e</p><p>i</p><p>a</p><p>•</p><p>•</p><p>Fig. 4. Steroidal structure and significant sites for binding</p><p>chain increases, e.g. in ethynyl group, the binding affinity is</p><p>reduced, as happens in testosterone molecule for which a 7-fold</p><p>loss in binding affinity has been estimated when introducing a</p><p>17�-ethynyl group in the place of a 17�-methyl group.</p><p>A steroid hydrophobic backbone: The 5�-steroidal framework</p><p>favors binding, which is illustrated by the fact that 5�-DHT has</p><p>about a 173-fold higher binding affinity than the 5�-DHT [73].</p><p>Other structural features of the steroidal backbone for which</p><p>nformation is known about their contribution to binding to AR are</p><p>he following:</p><p>The 11-oxo substitution reduces affinity (6-fold binding affinity</p><p>loss from testosterone to 11-keto testosterone) [73].</p><p>A small steric substitution at the 7�-position enhances affin-</p><p>ity; 10-fold increase from methyltestosterone (Fig. 1, 15) to</p><p>mibolerone (Fig. 1, 16), but large substituents reduce affinity. The</p><p>combined removal of the 19-methyl group and 7�-methylation</p><p>of 17�-hydroxy-4-androstenes can enhance both the binding</p><p>affinity and the androgenicity of the steroid. Studies on steroids</p><p>without an oxygen function on ring D but with a methyl group</p><p>at position C-7, have enhanced binding affinity due to the pres-</p><p>ence of the C-7-methyl group which contribute to the steroids</p><p>bind firmly to the AR, and with major effect on the enhance-</p><p>ment of the</p><p>androgenicity and presumably of the anabolic activity</p><p>[75].</p><p>It is also of interest to note that the 4-ene double bond of testos-</p><p>erone is not critical for binding since 5�-reduction to DHT results</p><p>n a significant increase in binding affinity. Likewise, the 19-methyl</p><p>roup is also not critical for binding since the absence of the 19-</p><p>ethyl group results in 19-nortestosterone which also has a higher</p><p>inding affinity for AR. Interestingly, the compound resulting from</p><p>imultaneous 5�-reduction and removal of the 19-methyl group</p><p>as a lower binding affinity than testosterone. This special bind-</p><p>ng behavior of testosterone made it possible to find steroids with</p><p>igher myotropic activity than that of testosterone. A survey of the</p><p>ist of anabolic steroids has revealed examples where either the</p><p>mission of the 19-methyl group or certain changes introduced in</p><p>he structure of ring A of testosterone or 17�-methyltestosterone</p><p>as essential to obtain such steroids [76].</p><p>A large number of chemicals that bind AR have been recently</p><p>ummarized elsewhere [73,77].</p><p>•</p><p>-DHT (as a representative steroidal structure) to the AR.</p><p>. Structural features of steroids contributing to their</p><p>ndrogenic and anabolic activities</p><p>The actions in reproductive tissues, including prostate, semi-</p><p>al vesicle and testis, are known as the androgenic effects, while</p><p>he nitrogen-retaining effects in muscle and bone are known as the</p><p>nabolic effects [78,79]. The androgenic activity of steroids is deter-</p><p>ined by increased weight of the ventral prostate (v.p.) or seminal</p><p>esicles (s.p.). The anabolic or myotrophic activity of synthetic AAS</p><p>s evaluated in several ways such as the nitrogen balance (protein</p><p>nabolic activity) and the growth weight of the kidney (renotropic</p><p>ctivity). One method for determining myotrophic activity is by</p><p>stimating the growth weight in levator ani of rats. However,</p><p>he levator ani increase test has some limitations, as the growth</p><p>timulation is caused by not only anabolic activity, but also by</p><p>ndrogenic effects of the steroids. For an integral evaluation of</p><p>ormone anabolic properties the anabolic index is used, which is</p><p>efined by the ratio of myotrophic activity to androgenic activity.</p><p>n anabolic index greater than one indicates a steroid to be anabolic</p><p>n nature, while an index less than one represents an androgenic</p><p>teroid. There are significant differences in the relative potencies</p><p>f compounds even when comparisons were made with the same</p><p>tandard preparation [80,81]. Relative potencies have been esti-</p><p>ated by a variety of techniques. This fact resulted in a literature</p><p>eplete with inconsistent data and has made the evaluation of the</p><p>ffects of structural modifications on activity difficult for those</p><p>nterested in structure–activity relationships.</p><p>The structural elements that contribute to the stipulation of the</p><p>ndrogenic effect of steroids are:</p><p>A 17ˇ-OH in the D-ring (Fig. 5): In the absence of a 17-oxygen</p><p>function, as in 4,16-androstandien-3-one (Fig. 1, 17), the andro-</p><p>genic activity is completely lost [82]. By the oxidation of the</p><p>17�-hydroxyl group with the formation of 17-oxo-steroids (e.g.</p><p>5�-androstane-3�,17�-diol, Fig. 1, 18, to 5�-androstane-3�-</p><p>ol,17-one), the androgenic activity is considerably reduced or</p><p>disappears [78].</p><p>The presence of a C-4, -5 double bond: The bioreduction of the</p><p>C-4, -5 double bond of testosterone by 5�-reductase occurs selec-</p><p>tively from the alpha-face. The product of this reduction, 5�-DHT,</p><p>assumes a chairlike conformation and has a higher AR affinity</p><p>relative to testosterone [83]</p><p>A 3-keto group in the A-ring (Fig. 5): The keto group at posi-</p><p>tion C-3 is also necessary for androgen activity. For example,</p><p>A.G. Fragkaki et al. / Steroids 74 (2009) 172–197 179</p><p>dal str</p><p>k</p><p>p</p><p>e</p><p>p</p><p>f</p><p>m</p><p>fl</p><p>g</p><p>t</p><p>S</p><p>1</p><p>c</p><p>a</p><p>o</p><p>•</p><p>a</p><p>•</p><p>•</p><p>•</p><p>g</p><p>4</p><p>d</p><p>s</p><p>e</p><p>o</p><p>4</p><p>t</p><p>a</p><p>g</p><p>i</p><p>c</p><p>b</p><p>a</p><p>f</p><p>l</p><p>r</p><p>w</p><p>l</p><p>c</p><p>s</p><p>Fig. 5. Significant sites of testosterone (as a representative steroi</p><p>5�-androstane-3�,17�-diol (Fig. 1, 19) has 60% of the testos-</p><p>terone activity, although the 17�-OH group remains. A reduction</p><p>of testosterone androgenic properties is possible by introducing</p><p>an additional double bond in ring A which leads to ring flattening</p><p>[78,83].</p><p>Most of the highly active synthetic androgens are �4-3-</p><p>etosteroids. The bulkiness and flatness of the steroid molecule</p><p>lay a more important role in receptor binding than the detailed</p><p>lectronic structure of the C-4, -5 double bond of ring A. For exam-</p><p>le, potent androgens with conjugated double bonds extending</p><p>rom rings A and B to C (e.g. methyltrienolone and 2-oxa-17�-</p><p>ethyl-17�-hydroxy-estra-4,9,11-triene-3-one, Fig. 1, 20) are very</p><p>at molecules and bind to the androgen receptor firmly.</p><p>The 17�-methyl substitution had very little effect on the andro-</p><p>enic activity of testosterone and 5�-DHT, but enhanced slightly</p><p>he androgenic activity of 19-nortestosterone and its derivatives.</p><p>ome androgens, such as 17�-hydroxy-5�-androstane (Fig. 1, 21),</p><p>7�-methyl-17�-hydroxy-5�-androstane (Fig. 1, 22) and related</p><p>ompounds, do not have an oxygen at C-3 or C-17 positions, or both,</p><p>nd their androgenic activity is believed to be dependent on their</p><p>xygenation in animals.</p><p>The removal of the C-19 methyl group: This greatly reduces andro-</p><p>genic properties [75,84] and offers, partially, dissociation of the</p><p>androgenic and anabolic activities of a molecule. The difference</p><p>in the effects of testosterone and 19-nortestosterone is caused</p><p>by the fact that 5�-reduction potentiates binding to the AR and</p><p>increases the affinity of testosterone, while 5�-reduction reduces</p><p>binding and decreases the affinity of 19-nortestosterone [84,85].</p><p>The structural elements that contribute to the stipulation of the</p><p>nabolic effect of steroids are:</p><p>The modification of ring A: The modification of ring A by the junc-</p><p>tion with a pyrazol ring, as in stanozolol (see also Section 4.10),</p><p>or by the introduction of an oxygen atom at position C-2, as in</p><p>oxandrolone (Fig. 1, 23), changes the stereoelectronic environ-</p><p>ment of the molecule, leading to a considerable increase of the</p><p>anabolic activity. Moreover, the introduction of alkyl substituents</p><p>into ring A at position C-1 (e.g. methenolone) or at position C-2</p><p>d</p><p>o</p><p>a</p><p>a</p><p>a</p><p>ucture) for the expression of androgenic and anabolic activities.</p><p>(stenbolone, Fig. 1, 24) [86], in combination or not with a dou-</p><p>ble bond at position C-1, -2, leads to intensified steroid anabolic</p><p>activity.</p><p>The removal of the C-19 methyl group (see Section 3, fourth bullet).</p><p>The 17˛-alkylation (see Section 4.7).</p><p>The structural features influencing the expression of the andro-</p><p>enic and anabolic activities are summarized in Fig. 5.</p><p>. Applied modifications in the steroidal structure</p><p>In this section, important examples of synthetic molecules</p><p>erived from the wide spectrum of modifications in testosterone</p><p>tructure are presented (Table 2), together with representative</p><p>xamples of synthetic anabolic steroids included in the WADA list</p><p>f prohibited substances [1].</p><p>.1. C-1 substitution of steroids. 1-Ene steroids</p><p>Methylation at position C-1 is a common modification in syn-</p><p>hetic anabolic steroids [87]. An example of C-1 methyl substituted</p><p>nabolic steroid is mesterolone (Fig. 1, 25) [88], which has andro-</p><p>enic properties but is reported to have less inhibitory effect on</p><p>ntrinsic testicular function than testosterone [2]. Methenolone also</p><p>ombines the presence of a C-1 methyl substituent and a dou-</p><p>le bond at C-1 position. Methenolone was synthesized in 1960</p><p>nd was the first anabolic steroid not alkylated at 17�-position but</p><p>ound to be active by oral administration [79,88]. The 19-nor ana-</p><p>ogue of methenolone acetate was almost devoid of activity (Table 2,</p><p>ow 1) [79].</p><p>In 1940, it was demonstrated that the isomer of testosterone</p><p>ith a C-1, -2 double bond (1-testosterone, Fig. 1, 26) was much</p><p>ess androgenic than testosterone itself. 1-Testosterone can be</p><p>haracterized as a typical anabolic steroid which binds with high</p><p>electivity to the AR and has a high potency to stimulate AR-</p><p>ependent transactivation [89]. Since this compound possessed</p><p>all</p><p>f the structural features generally associated with high anabolic</p><p>ctivity, various C-2- and C-6 methyl substituted steroids in the 5�-</p><p>ndrost-1-ene series were prepared (Table 2, row 2). Methylation</p><p>t C-2 of these 1-ene steroids appeared to have little effect on the</p><p>180</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>Table 2</p><p>Modifications of anabolic steroids included in bibliographic references.</p><p>Row ID Parent compound Modification of the parent</p><p>compound</p><p>Effect in activity after</p><p>modification</p><p>Structure of parent molecule Structure of modified molecule Reference</p><p>1 Methenolone Removal of the 19-methyl</p><p>group</p><p>Devoid of activity [79]</p><p>2 1-Testosterone Addition of methyl group</p><p>at C-2 and C-6 positions</p><p>Methylation at C-2</p><p>appeared to have little</p><p>effect on the relative</p><p>anabolic and androgenic</p><p>potencies; a slight decrease</p><p>in activity was observed for</p><p>the C-6 methylated analogs</p><p>[90]</p><p>3 5�-Androstane steroids 1-Alkyl substitution Decreased androgenic and</p><p>possibly increased anabolic</p><p>activity</p><p>[91]</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>181</p><p>4</p><p>Testosterone and DHT</p><p>derivatives</p><p>2-Methyl and 2-</p><p>hydroxymethyl-androstane</p><p>derivatives in the</p><p>17-alkylated or 19-nor</p><p>analogues of testosterone</p><p>and DHT</p><p>*Enhanced anabolic</p><p>activity</p><p>[92]</p><p>** No reported data for</p><p>activity</p><p>5</p><p>5�-androstane-2-ene</p><p>steroids</p><p>2-Methyl-androst-2-enes High anabolic activity with</p><p>low androgenicity</p><p>[94,95]</p><p>6 2-Methylene androstanes High anabolic activity with</p><p>low androgenicity</p><p>7 2-Formyl-androst-2-enes</p><p>and related compounds</p><p>High anabolic activity with</p><p>low androgenicity</p><p>[96,97]</p><p>8 Fluoxymesterone Oxidation of 11-hydroxyl to</p><p>11-keto group</p><p>No difference in activity [79]</p><p>182</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>Table 2 (Continued )</p><p>Row ID Parent compound Modification of the parent</p><p>compound</p><p>Effect in activity after</p><p>modification</p><p>Structure of parent molecule Structure of modified molecule Reference</p><p>9 Testosterone</p><p>4-alkyl and their 19-nor</p><p>analogues</p><p>4-methyltestosterone but</p><p>not its 19-nor analogue</p><p>possessed satisfactory, but</p><p>still low, anabolic and</p><p>androgenic activity. Larger</p><p>groups at C-4 and</p><p>4,4-dialkylsteroids were</p><p>inactive</p><p>[101]</p><p>4,4-dialkyl derivatives</p><p>10 Norethandrolone 17�-Methyl analogue Good anabolic and</p><p>androgenic activities but</p><p>with high progestational</p><p>activity</p><p>[79]</p><p>11 Substitution of the</p><p>17�-ethyl group by propyl</p><p>and allyl groups</p><p>Decreased myotrophic</p><p>activity when tested in</p><p>castrated rats</p><p>12 Ethylestrenol Substitution of the</p><p>17�-ethyl group by ethynyl</p><p>and allyl groups</p><p>High progestational</p><p>activity and are in use in</p><p>clinical practice</p><p>[79]</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>183</p><p>13 Norbolethone Substitution of the</p><p>13�-ethyl group by propyl</p><p>group</p><p>No anabolic or androgenic</p><p>activity</p><p>[79]</p><p>14</p><p>19-nor-androst-5,6-ene</p><p>steroids</p><p>Addition of: 17�-methyl;</p><p>17�-ethyl; 17�-ethynyl</p><p>groups in 19-nor-androst-</p><p>5,6-ene-3�,17�-diols and</p><p>the corresponding ketones</p><p>1) 19-nor-androst-5,6-ene-</p><p>3�,17�-diols: highly</p><p>favorable anabolic to</p><p>androgenic ratios</p><p>[104]</p><p>2) 19-nor-androst-5,6-ene-</p><p>3-keto compounds:</p><p>anabolic and androgenic</p><p>ratios similar to those of</p><p>the androst-4,5-ene-3-keto</p><p>compounds</p><p>15 Testosterone Addition of 6�- and</p><p>6�-methyl group</p><p>Less androgenic and</p><p>anabolic activity than the</p><p>parent compound</p><p>[105]</p><p>184</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>Table 2 (Continued )</p><p>Row ID Parent compound Modification of the parent</p><p>compound</p><p>Effect in activity after</p><p>modification</p><p>Structure of parent molecule Structure of modified molecule Reference</p><p>16 Methyl-testosterone Addition of 6�- and</p><p>6�-methyl group</p><p>Less androgenic and</p><p>anabolic activity than the</p><p>parent compound</p><p>17 5�-DHT Addition of 6�-methyl</p><p>group</p><p>About 8-fold higher</p><p>anabolic activity than</p><p>testosterone</p><p>[106]</p><p>18 Testosterone Addition of a 6�-F Lack of aromatization of</p><p>6�-fluorotestosterone to</p><p>the corresponding</p><p>estrogen. Positive effect on</p><p>androgenic property is</p><p>possible.</p><p>[107]</p><p>19 Bolasterone Removal of the 19-methyl</p><p>group</p><p>18 times the androgenic</p><p>activity of</p><p>methyl-testosterone and 41</p><p>times the myotrophic</p><p>activity</p><p>[79]</p><p>20 5�-DHT Addition of 7�-methyl</p><p>group</p><p>Only weakly androgenic</p><p>compared to 5�-DHT</p><p>[112]</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>185</p><p>21 5�-DHT and</p><p>19-nor-5�-DHT</p><p>Addition of 7�-I and -F in</p><p>5�-DHT and</p><p>19-nor-5�-DHT and their</p><p>17�-methylated analogues</p><p>7�-F-17�-methyl-5�-DHT</p><p>had the highest AR binding</p><p>affinity and androgenic</p><p>potency</p><p>[113]</p><p>22 19-Nor-testosterone Addition of 7�-methyl</p><p>group</p><p>Enhanced anabolic activity [114], [115]]</p><p>23 Addition of methyl groups</p><p>at 7�-, 11�-positions</p><p>Enhanced androgen</p><p>receptor binding and</p><p>anabolic/androgenic ratio</p><p>over its two monomethyl</p><p>homologs</p><p>186</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>Table 2 (Continued )</p><p>Row ID Parent compound Modification of the parent</p><p>compound</p><p>Effect in activity after</p><p>modification</p><p>Structure of parent molecule Structure of modified molecule Reference</p><p>24 Methyl-testosterone Addition of 7�- and</p><p>7�-methyl group into</p><p>methyl-testosterone and its</p><p>11�-hydroxy analogue</p><p>No reported data on</p><p>androgenic and anabolic</p><p>activity</p><p>[117], [118]</p><p>25</p><p>Testosterone</p><p>Addition of 7�-methyl</p><p>group and C-14,15 double</p><p>bond</p><p>Both modifications in</p><p>testosterone have a</p><p>surprisingly synergistic</p><p>effect.</p><p>7�-methyl-14-dehydro-19-</p><p>nortestosterone is 1000</p><p>times as active as</p><p>testosterone in the chick</p><p>comb assay and 100 times</p><p>as active as testosterone in</p><p>either the rat ventral</p><p>prostate or levator ani</p><p>assay</p><p>[119]</p><p>26 Addition of: 7�-methyl,</p><p>7�-ethyl, 7�-n-propyl,</p><p>7�-n-butyl group</p><p>Decreased activities</p><p>(androgenic and anabolic)</p><p>as the alkyl group was</p><p>homologated</p><p>[120]</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>187</p><p>27 7-Chloro-4,6-dien-3-one</p><p>system or �7-7-chloro</p><p>system</p><p>No anabolic or androgenic</p><p>activity</p><p>[121]</p><p>28 5�-Androstane steroids Replacement of the 2-, 3-</p><p>and 4-methylene groups by</p><p>an oxygen atom in</p><p>5�-androstane series</p><p>Gradually decreased</p><p>androgenic activity</p><p>[134]</p><p>29 5�-Androstane steroids Replacement of the</p><p>2-methylene group by a</p><p>sulfur atom in</p><p>5�-androstane series</p><p>Similar to the 2-oxa analog</p><p>referred above</p><p>[135]</p><p>188</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>Table 2 (Continued )</p><p>Row ID Parent compound Modification of the parent</p><p>compound</p><p>Effect in activity after</p><p>modification</p><p>Structure of parent molecule Structure of modified molecule Reference</p><p>30 5�-DHT and</p><p>17�-methyl-DHT</p><p>Replacement of the</p><p>2-methylene group by a</p><p>nitrogen atom</p><p>No considerable results in</p><p>activity</p><p>[136]</p><p>31 Replacement of</p><p>7-methylene group by</p><p>oxygen</p><p>Antigonadotropic,</p><p>androgenic and anabolic</p><p>activities</p><p>[137]</p><p>32 Testosterone Replacement of</p><p>11-methylene group by</p><p>oxygen</p><p>Decreased progestational,</p><p>androgenic, anabolic and</p><p>estrogenic activities</p><p>[138]</p><p>33 5�-Androstane steroids Three heteroatoms in ring</p><p>A</p><p>50-100% the</p><p>androgenic–myotrophic</p><p>effect of testosterone</p><p>[139]</p><p>34</p><p>Stanozolol</p><p>Addition of double bond at</p><p>C-4, -5 position</p><p>No promotion of nitrogen</p><p>balance although it was</p><p>myotrophic, weakly</p><p>androgenic and estrogenic</p><p>[79]</p><p>35 Addition of double bonds</p><p>at C-4, -5 and C-5, -6</p><p>positions</p><p>Estrogenic properties</p><p>without anabolic or</p><p>androgenic activities</p><p>[79]</p><p>A</p><p>.G</p><p>.Fragkakiet</p><p>al./Steroids</p><p>74</p><p>(2009)</p><p>172–197</p><p>189</p><p>36 Androst-4-eno[3,2-</p><p>c]pyrazoles</p><p>6�-Methylation Less androgenic and</p><p>anabolic activities</p><p>[141], [142]</p><p>37 9�-Fluoro-11�-hydroxy</p><p>groups</p><p>Less androgenic and</p><p>anabolic activities</p><p>[141], [142]</p><p>38</p><p>Anadrostano[2,3-</p><p>d]isoxazoles</p><p>Various 17�-R</p><p>substituents</p><p>Less androgenic and</p><p>anabolic activities when R</p><p>is increased</p><p>[144]</p><p>The same for: Androst-4-</p><p>eno-[2,3-d]isoxazole series</p><p>and androst-4,6-dieno-</p><p>[2,3-d]isoxazole</p><p>series</p><p>39 4,4-Dimethyl analogues Decreased activity [144]</p><p>40 6�-Methyl analogue Decreased activity [144]</p><p>190 A.G. Fragkaki et al. / Steroid</p><p>Ta</p><p>bl</p><p>e</p><p>2</p><p>(C</p><p>on</p><p>ti</p><p>nu</p><p>ed</p><p>)</p><p>R</p><p>ow</p><p>ID</p><p>Pa</p><p>re</p><p>n</p><p>t</p><p>co</p><p>m</p><p>p</p><p>ou</p><p>n</p><p>d</p><p>M</p><p>od</p><p>ifi</p><p>ca</p><p>ti</p><p>on</p><p>of</p><p>th</p><p>e</p><p>p</p><p>ar</p><p>en</p><p>t</p><p>co</p><p>m</p><p>p</p><p>ou</p><p>n</p><p>d</p><p>Ef</p><p>fe</p><p>ct</p><p>in</p><p>ac</p><p>ti</p><p>vi</p><p>ty</p><p>af</p><p>te</p><p>r</p><p>m</p><p>od</p><p>ifi</p><p>ca</p><p>ti</p><p>on</p><p>St</p><p>ru</p><p>ct</p><p>u</p><p>re</p><p>of</p><p>p</p><p>ar</p><p>en</p><p>t</p><p>m</p><p>ol</p><p>ec</p><p>u</p><p>le</p><p>St</p><p>ru</p><p>ct</p><p>u</p><p>re</p><p>of</p><p>m</p><p>od</p><p>ifi</p><p>ed</p><p>m</p><p>ol</p><p>ec</p><p>u</p><p>le</p><p>R</p><p>ef</p><p>er</p><p>en</p><p>ce</p><p>41</p><p>3′ -</p><p>A</p><p>lk</p><p>yl</p><p>an</p><p>al</p><p>og</p><p>u</p><p>es</p><p>D</p><p>ec</p><p>re</p><p>as</p><p>ed</p><p>ac</p><p>ti</p><p>vi</p><p>ty</p><p>[1</p><p>4</p><p>4]</p><p>42</p><p>19</p><p>-N</p><p>or</p><p>an</p><p>al</p><p>og</p><p>u</p><p>e</p><p>D</p><p>ec</p><p>re</p><p>as</p><p>ed</p><p>ac</p><p>ti</p><p>vi</p><p>ty</p><p>.T</p><p>h</p><p>e</p><p>ac</p><p>ti</p><p>vi</p><p>ty</p><p>in</p><p>cr</p><p>ea</p><p>se</p><p>d</p><p>fo</p><p>r</p><p>th</p><p>e</p><p>u</p><p>n</p><p>sa</p><p>tu</p><p>ra</p><p>te</p><p>d</p><p>an</p><p>al</p><p>og</p><p>u</p><p>es</p><p>:</p><p>4-</p><p>en</p><p>o</p><p>an</p><p>d</p><p>4,</p><p>6-</p><p>d</p><p>ie</p><p>n</p><p>o</p><p>is</p><p>ox</p><p>az</p><p>ol</p><p>es</p><p>[1</p><p>4</p><p>4]</p><p>43</p><p>Fu</p><p>ra</p><p>za</p><p>bo</p><p>l</p><p>A</p><p>d</p><p>d</p><p>it</p><p>io</p><p>n</p><p>of</p><p>d</p><p>ou</p><p>bl</p><p>e</p><p>bo</p><p>n</p><p>d</p><p>at</p><p>C</p><p>-4</p><p>,-</p><p>5</p><p>p</p><p>os</p><p>it</p><p>io</p><p>n</p><p>Th</p><p>e</p><p>sa</p><p>m</p><p>e</p><p>ac</p><p>ti</p><p>vi</p><p>ti</p><p>es</p><p>as</p><p>th</p><p>e</p><p>p</p><p>ar</p><p>en</p><p>t</p><p>co</p><p>m</p><p>p</p><p>ou</p><p>n</p><p>d</p><p>[7</p><p>9]</p><p>r</p><p>i</p><p>a</p><p>a</p><p>v</p><p>p</p><p>o</p><p>4</p><p>a</p><p>a</p><p>a</p><p>n</p><p>s</p><p>d</p><p>4</p><p>h</p><p>O</p><p>c</p><p>2</p><p>l</p><p>a</p><p>-</p><p>g</p><p>o</p><p>A</p><p>m</p><p>a</p><p>t</p><p>t</p><p>s</p><p>2</p><p>C</p><p>w</p><p>[</p><p>s</p><p>s</p><p>p</p><p>w</p><p>t</p><p>i</p><p>r</p><p>a</p><p>a</p><p>s</p><p>T</p><p>m</p><p>a</p><p>s</p><p>e</p><p>a</p><p>t</p><p>i</p><p>4</p><p>c</p><p>t</p><p>s 74 (2009) 172–197</p><p>elative anabolic and androgenic potencies, while a slight decrease</p><p>n activity was observed for the C-6 methylated analogs [90].</p><p>The biological action of a series of androstane derivatives was</p><p>lso estimated and revealed that the C-1 alkyl substitution is</p><p>ccompanied by a decrease of activity in the prostate and seminal</p><p>esicles, i.e. a decrease in the androgenic activity, and possibly has a</p><p>ositive effect on anabolic activity. Some representative examples</p><p>f the compounds tested are presented in Table 2 (row 3) [91].</p><p>.2. C-2 substitution of steroids. 2-Ene steroids. 3-Deoxy steroids</p><p>Representative example of a C-2 methyl substituted synthetic</p><p>nabolic steroid is drostanolone (Fig. 1, 27), a substance with high</p><p>nabolic activity. It was synthesized in 1959 by Ringold et al. [92]</p><p>nd was formerly used in the treatment of advanced malignant</p><p>eoplasms of the breast in postmenopausal women [2]. In the</p><p>tudy for drostanolone synthesis [92], a number of 2-methyl, 2,2-</p><p>imethyl and 2-hydroxymethylene steroid derivatives (Table 2, row</p><p>) were also prepared. Among them, methasterone was found to</p><p>ave increased anabolic activity compared to testosterone or DHT.</p><p>ne possible explanation of the increased activity of the 2�-methyl</p><p>ompounds lies in their inability to undergo in vivo aromatization.</p><p>-Hydroxymethylene substitution, particularly of DHT derivatives</p><p>ed to oxymetholone [79,92], which was found to be a potent oral</p><p>nabolic agent with minimal androgenic activity.</p><p>Some 2�-halogeno (-F, -Cl and -Br) and 2�-alkyl (-methyl and</p><p>ethyl) analogs of 4-androstenedione (Fig. 1, 28) are known as</p><p>ood aromatase inhibitors (enzyme which inhibit the conversion</p><p>f androgens to estrogens) in human placental microsomes [93].</p><p>lthough the structure–activity relationships of steroids for aro-</p><p>atase inhibition activity is beyond the scope of this paper, these</p><p>ndrostenedione analogs are referred as an example of modifica-</p><p>ions in a steroid molecule which lead to neither the expression of</p><p>he androgenic nor of the anabolic activity.</p><p>Studies on the expansion of the unsaturation of the A-ring of the</p><p>teroidal structure at C-2 and C-3 positions, led to the synthesis of</p><p>-methyl-androst-2-enes (Table 2, row 5) and the corresponding</p><p>-2 exocyclic methylene analogs (Table 2, row 6). These derivatives</p><p>ere found to have high anabolic activity and low androgenicity</p><p>94,95]. On the other hand, the 2-methyl-androst-3-ene analogues</p><p>howed no considerable anabolic and androgenic activity [95].</p><p>The absence of the oxygen atom at position C-3 (3-deoxy</p><p>teroids) in certain androstane derivatives was found to be com-</p><p>atible with high anabolic to androgenic activities. Further studies</p><p>ith variable electron density patterns led to the investigation of</p><p>he system with a C-2, -3 double bond and a 2-formyl group and</p><p>ts analogs with a favorable anabolic to androgenic ratio (Table 2,</p><p>ow 7) [96,97]. Due to this observation that pronounced anabolic</p><p>ctivity is in association with 3-deoxy steroids, a series of 19-nor-</p><p>nd 19-substituted 5�-androst-2-ene derivatives were synthe-</p><p>ized in an attempt to prepare potentially useful anabolic steroids.</p><p>he study revealed that, 17�-methyl-5�-androst-2-ene-17�-ol or</p><p>adol (Fig. 1, 4) exhibited a favorable separation of anabolic and</p><p>ndrogenic activities and has been recently detected as designer</p><p>teroid, as referred in the introductory section of this paper. How-</p><p>ver, conversion to its 19-nor analog decreased both androgenic and</p><p>nabolic activities [98]. Recent studies for the characterization of</p><p>he pharmacological profile confirmed these early findings; madol</p><p>s a potent AR agonist which binds the AR with high selectivity [99].</p><p>.3. C-4 substitution of steroids. 4-ene steroids</p><p>Oral turinabol (Fig. 1, 29) is a synthetic anabolic steroid which</p><p>ombines the simultaneous presence of a C-4 Cl substitution and</p><p>wo double bonds at C-1 and C-4 positions. Oral turinabol was first</p><p>teroid</p><p>s</p><p>i</p><p>s</p><p>(</p><p>t</p><p>a</p><p>[</p><p>i</p><p>a</p><p>t</p><p>w</p><p>i</p><p>t</p><p>fl</p><p>g</p><p>t</p><p>n</p><p>s</p><p>f</p><p>9</p><p>a</p><p>1</p><p>T</p><p>4</p><p>o</p><p>n</p><p>d</p><p>i</p><p>g</p><p>a</p><p>E</p><p>a</p><p>n</p><p>a</p><p>i</p><p>o</p><p>m</p><p>c</p><p>n</p><p>s</p><p>(</p><p>i</p><p>a</p><p>g</p><p>a</p><p>m</p><p>d</p><p>f</p><p>t</p><p>A</p><p>1</p><p>g</p><p>e</p><p>h</p><p>p</p><p>4</p><p>o</p><p>t</p><p>c</p><p>b</p><p>a</p><p>3</p><p>a</p><p>r</p><p>s</p><p>p</p><p>4</p><p>b</p><p>o</p><p>h</p><p>p</p><p>d</p><p>t</p><p>a</p><p>s</p><p>c</p><p>a</p><p>3</p><p>1</p><p>d</p><p>p</p><p>v</p><p>i</p><p>t</p><p>s</p><p>f</p><p>m</p><p>t</p><p>o</p><p>r</p><p>4</p><p>s</p><p>s</p><p>s</p><p>a</p><p>a</p><p>r</p><p>a</p><p>t</p><p>b</p><p>a</p><p>t</p><p>T</p><p>w</p><p>m</p><p>m</p><p>3</p><p>p</p><p>M</p><p>A.G. Fragkaki et al. / S</p><p>ynthesized in 1960 and was the most misused steroid by athletes</p><p>n East Germany in 1990 [100].</p><p>Another type of C-4 substitution is hydroxylation as occurs in the</p><p>ynthetic anabolic steroids oxymesterone (Fig. 1, 30) and oxabolone</p><p>Fig. 1, 31). Oxymesterone was synthesized in 1961 and was proved</p><p>o be 3.3 times as active as 17�-methyltestosterone and only 0.48</p><p>s androgenic, hence giving a myotrophic–androgenic index of 6.9</p><p>79]. Oxabolone cypionate (Fig. 1, 31) was synthesized in 1962 dur-</p><p>ng a search for androstane and estrane derivatives with potential</p><p>nabolic activity.</p><p>A 4-ene steroid derived from testosterone with three modifica-</p><p>ions (9�-F, 11�-ol and 17�-methyl) is fluoxymesterone (Fig. 1, 32),</p><p>hich was synthesized in 1956. Biological tests showed that the</p><p>ntroduction of a fluorine atom at the C-9� position increased both</p><p>he anabolic and the androgenic activity. The attempt to modify the</p><p>uoxymesterone molecule led to the oxidation of the 11-hydroxyl</p><p>roup to 11-keto; no difference in activity was observed between</p><p>he two derivatives (Table 2, row 8) [79].</p><p>A number of 4-alkyl substituted androstane and 19-</p><p>orandrostane derivatives have been prepared in an earlier</p><p>tudy [101], in an attempt to possess high anabolic potency and</p><p>avorable ratios of anabolic to androgenic activity (Table 2, row</p><p>). The results showed that 4-methyltestosterone was 30% as</p><p>nabolic and 10% as androgenic compared to testosterone. Its</p><p>9-nor analogue possessed even lower results in both activities.</p><p>he analogous substances with larger alkyl groups at C-4 and the</p><p>,4-dialkylsteroids were inactive.</p><p>Many studies on synthetic anabolic steroids production focused</p><p>n the modification of the 17�-alkyl side chain of certain 4-en-19-</p><p>or steroids. Among them the 17�-methyl analogue of norethan-</p><p>rolone (Fig. 1, 33) possessed good anabolic and androgenic activ-</p><p>ties but high progestational activity. Substitution of the 17�-ethyl</p><p>roup by propyl and allyl groups resulted in decreased myotrophic</p><p>ctivity when tested in castrated rats (Table 2, rows 10 and 11) [79].</p><p>thylestrenol (Fig. 1, 13), a structurally related to norethandrolone</p><p>nabolic steroid, was first synthesized in 1959. Compared to</p><p>orethandrolone, ethylestrenol was found to have an anabolic to</p><p>ndrogenic ratio of 20. Modifications in ethylestrenol molecule</p><p>n order to increase its anabolic potency, led to the substitution</p><p>f the 17�-ethyl group by ethynyl and allyl groups; the resulted</p><p>olecules possessed high progestational activity and are in use in</p><p>linical practice (Table 2, row 12) [79]. Another type of 4-en-19-</p><p>or steroids replaced the C-13 and C-17 methyl group of a classical</p><p>teroidal structure by an ethyl group, leading to nor-bolethone</p><p>Fig. 1, 2), a synthetic anabolic steroid with decreased androgenic-</p><p>ty. Nor-bolethone showed a marked separation between anabolic</p><p>nd androgenic effects. However, substitution of the 13�-ethyl</p><p>roup of nor-bolethone by propyl group led to a molecule with no</p><p>nabolic or androgenic activity (Table 2, row 13) [79].</p><p>Many synthetic steroids with high myotrophic activity exhibit</p><p>yotrophic–androgenic dissociation, since, due to changes intro-</p><p>uced in the structure of ring A, they will probably not be substrates</p><p>or the 5�-reductases [85]. 5�-Reduction</p><p>does not always amplify</p><p>he androgenic potency in spite of high RBA of androgens to the</p><p>R. This is the case for norethisterone (Fig. 1, 34), a synthetic</p><p>9-nor-17�-ethynyl testosterone derivative, which also under-</p><p>oes enzyme-mediated 5�-reduction and exerts potent androgenic</p><p>ffects in target organs. 5�-Reduced norethisterone displays a</p><p>igher AR binding but shows a significantly lower androgenic</p><p>otency than unchanged norethisterone [102,103].</p><p>.4. 5-Ene steroids</p><p>The synthesis of 19-nortestosterone (Fig. 1, 8) by Birch, in 1950,</p><p>pened a new field of biologically interesting compounds and paved</p><p>b</p><p>a</p><p>r</p><p>i</p><p>L</p><p>s 74 (2009) 172–197 191</p><p>he way for the preparation of the 19-nor analogs of most of the</p><p>ommon steroid hormones containing the 3-keto and a C-4, -5 dou-</p><p>le bond system [104]. Later work led to various derivatives, such</p><p>s the 17�-methyl, -ethyl and -ethynyl-19-nor-androst-5,6-ene-</p><p>�,17�-diols (Table 2, row 14), which exhibited highly favorable</p><p>nabolic–androgenic ratios in the experimental animals. Their cor-</p><p>esponding 19-nor-3-keto compounds with a C-5, -6 double bond</p><p>howed the anabolic and androgenic properties of the 3-keto com-</p><p>ounds with a C-4, -5 double bond.</p><p>.5. C-6 substitution of steroids</p><p>Methyl substitution at position C-6 of the steroidal structure has</p><p>een described since 1958 [105]. The 6�- and 6�-methyl derivatives</p><p>f testosterone and methyl-testosterone (Table 2, rows 15 and 16)</p><p>as been found to be somewhat less active than their parent com-</p><p>ounds as androgenic and anabolic agents. However, these results</p><p>iffer from those reported by Ringold et al. [106] who claimed</p><p>hat 6�-methyltestosterone has about 4-fold higher the myotropic</p><p>ctivity of testosterone. This study also revealed that 6�-methyl</p><p>ubstitution of 5�-DHT increases its anabolic activity about 8-fold</p><p>ompared to testosterone (Table 2, row 17).</p><p>An other type of modification is the presence of a fluorine atom</p><p>t the 6�-position of testosterone (6�-fluorotestosterone, Fig. 1,</p><p>5), which interferes with the aromatization reaction (Table 2, row</p><p>8) [107]. The mechanism by which this happens has not been</p><p>etermined, but the inhibitory action on estrogen formation may</p><p>otentiate the androgenic properties of 6�-fluorotestosterone in</p><p>ivo due to a lowering of estrogen levels. Two possible contribut-</p><p>ng factors to the non-aromatizability of 6�-fluorotestosterone are</p><p>he increased size and increased electronegativity of the fluorine</p><p>ubstituent. A fluorine substituent will withdraw electron density</p><p>rom neighboring bonds due to its high electronegativity which</p><p>ight render aromatization unfavorable. As referred in Section 4.2,</p><p>he aromatase inhibitory activity of steroids in beyond the scope</p><p>f this paper; this type of modification in testosterone molecule is</p><p>eferred as a factor for affecting its androgenic potency.</p><p>.6. C-7 substitution of steroids</p><p>Methylation is a common substitution at C-7 position of the</p><p>teroidal structure. A representative example of a 7�-methylated</p><p>ynthetic anabolic steroid is bolasterone (Fig. 1, 36). Bolasterone</p><p>ynthesis was reported in 1959 and is the 7�,17�-dimethyl</p><p>nalogue of testosterone, causing increased protein synthesis,</p><p>mino acid consumption and catabolism. It is used for relief and</p><p>ecovery from common injuries, rehabilitation, weight control,</p><p>nti-insomnia, and regulation of sexuality, aggression, and cogni-</p><p>ion. The activity by oral administration of the compound found to</p><p>e over 13 times as active as 17�-methyltestosterone in the lev-</p><p>tor ani assay for myotrophic activity, while having only 3 times</p><p>he androgenic activity, as indicated by the seminal vesicles assay.</p><p>he 19-nor analogue of bolasterone is mibolerone (Fig. 1, 16) which</p><p>as found to have about 18 times the androgenic activity of 17�-</p><p>ethyltestosterone when assayed orally in the rat, and 41 times its</p><p>yotrophic activity (Table 2, row 19) [79].</p><p>However, 7�-methylation of 19-nortestosterone (MENT, Fig. 1,</p><p>7) reduces the androgenic effect and amplifies the anabolic</p><p>otency compared to testosterone. This probably happens since</p><p>ENT does not undergo 5�-reduction in a large extent, probably</p><p>ecause the 7�-methyl group hinders the action of 5�-reductase</p><p>nd, thus, a reduction in the androgenic potency is caused. The 5�-</p><p>educed MENT is slightly stimulatory to both prostate and muscle</p><p>n agreement with its low binding affinity to the AR [108–111,116].</p><p>ike MENT, other nor-androgens were tested for their resistance to</p><p>1 teroid</p><p>5</p><p>w</p><p>p</p><p>t</p><p>a</p><p>b</p><p>t</p><p>s</p><p>a</p><p>b</p><p>i</p><p>c</p><p>g</p><p>i</p><p>t</p><p>M</p><p>r</p><p>m</p><p>a</p><p>t</p><p>s</p><p>c</p><p>t</p><p>a</p><p>D</p><p>b</p><p>o</p><p>t</p><p>o</p><p>i</p><p>s</p><p>m</p><p>t</p><p>c</p><p>r</p><p>g</p><p>F</p><p>s</p><p>e</p><p>W</p><p>t</p><p>1</p><p>n</p><p>p</p><p>o</p><p>t</p><p>1</p><p>m</p><p>s</p><p>f</p><p>t</p><p>7</p><p>m</p><p>i</p><p>b</p><p>m</p><p>o</p><p>m</p><p>1</p><p>o</p><p>7</p><p>i</p><p>4</p><p>a</p><p>p</p><p>r</p><p>a</p><p>i</p><p>l</p><p>l</p><p>p</p><p>o</p><p>t</p><p>t</p><p>s</p><p>p</p><p>r</p><p>C</p><p>a</p><p>4</p><p>t</p><p>s</p><p>i</p><p>s</p><p>t</p><p>a</p><p>i</p><p>l</p><p>s</p><p>m</p><p>t</p><p>a</p><p>s</p><p>m</p><p>d</p><p>a</p><p>n</p><p>o</p><p>b</p><p>s</p><p>t</p><p>b</p><p>p</p><p>i</p><p>t</p><p>s</p><p>r</p><p>g</p><p>i</p><p>s</p><p>c</p><p>92 A.G. Fragkaki et al. / S</p><p>�-reduction due to 7�-methylation. Results indicated that they</p><p>ere, also, more anabolic relative to testosterone [109]. The high</p><p>otency of 7�-methylated androgens was shown to correlate with</p><p>heir higher binding affinity to androgen receptors. The substitution</p><p>t the 7�-position with a methyl group is important for increased</p><p>inding affinity and increased biological activity.</p><p>In comparison, the effects of 7�-methyl substitution on testos-</p><p>erone, 5�-DHT or 19-nor-5�-DHT (Fig. 1, 38) were surprisingly</p><p>mall [83]. The substitution at the 7�-position with a cyano or</p><p>cetylthio moiety resulted in lower binding affinity and decreased</p><p>iological activity compared to the 7�-methyl substitution. The</p><p>mportance of the �-configuration of the methyl group was further</p><p>onfirmed since a �-configuration led to a decrease in its andro-</p><p>enic potency. This suggested that the conformational changes for</p><p>ncreased receptor binding are specific for 7�-methylated deriva-</p><p>ives [116]. Studies on the conformation of the 7-methyl group of</p><p>ENT also showed that methyl substitution at the 7�-position</p><p>esulted in 8-fold decrease in binding affinity compared to 7�-</p><p>ethyl derivative.</p><p>The 7�-methyl analogue of 5�-DHT (Table 2, row 20) was tested</p><p>nd found to be weakly androgenic [112]. The 7�-methyl substitu-</p><p>ion may also be important in connection with antitumor activity,</p><p>ince the introduction of this group into methyltestosterone (e.g.</p><p>alusterone, Fig. 1, 39) enhanced the antitumor efficacy in the</p><p>reatment of advanced female breast cancer while decreasing the</p><p>ndrogenic activity.</p><p>More recently, several 7�-fluoro and 7�-iodo analogues of 5�-</p><p>HT and 19-nor-5�-DHT have been synthesized and tested for</p><p>inding to the AR and for their biological activity in vitro [113]. Some</p><p>f these analogues designed to have a 17�-methyl group in order</p><p>o protect from oxidation of the 17�-hydroxyl group. The syntheses</p><p>f the mentioned analogues was based on the assumption that C-7</p><p>s sufficiently distant from C-3 and C-17 in order the halogen sub-</p><p>titution to have minimal steric or stereoelectronic effects which</p><p>ight interfere with the important interactions between the recep-</p><p>or and the carbonyl and hydroxyl groups of the steroid. From all the</p><p>ompounds tested, 7�-fluoro-17�-methyl-5�-DHT had the highest</p><p>eceptor affinity as well as androgenic potency (Table 2, row 21).</p><p>Previous studies indicate the 7�-methylation of 19-nor andro-</p><p>ens with a 17-methyl group (17�-methyl-19-nortestosterone,</p><p>ig. 1, 40) causes a many fold increase in androgenicity due to a</p><p>ufficient protection of the 17�-methyl group that permits deliv-</p><p>ry of the highly active 19-nor steroid to the end organ [114].</p><p>hen the 7�-methyl group is added to the 19-nor compounds,</p><p>here is a many-fold increase in androgenicity which is least for</p><p>9-nor-androst-4-ene-3,17-dione and greatest for 17�-methyl-19-</p><p>ortestosterone (Table 2, rows 22-23). This shows the additional</p><p>rotective activity of the 17�-methyl group. The synergistic effect</p><p>f the three changes (19-nor, 7�-methyl and 17�-methyl) is indica-</p><p>ive of sufficient protection to permit delivery of the highly active</p><p>9-nor steroid to the end organ [114,115]. Substitution with a</p><p>ethyl group in 7�- and/or 17�-positions results in flattening of the</p><p>teroid molecule which leads to conformation that is less hindered</p><p>or receptor binding. However,</p><p>other reports showed no correla-</p><p>ion between the receptor binding affinity and bioactivity of some</p><p>�-methylated androgens. Thus it was suggested that structural</p><p>odifications of a compound can lead to changes in its biolog-</p><p>cal activity based on binding affinity, differences in absorption,</p><p>inding to plasma proteins and/or susceptibility to the action of</p><p>etabolizing enzymes [116].</p><p>The synthesis of some 7�- and 7�-methyl derivatives</p><p>f testosterone, methyl-testosterone and 11�-hydroxy-17-</p><p>ethyltestosterone (Table 2, row 24) has been reported since</p><p>958, but no data about the androgenic and/or the anabolic activity</p><p>f these compounds accompanied these studies [117,118].</p><p>t</p><p>t</p><p>f</p><p>s</p><p>b</p><p>s 74 (2009) 172–197</p><p>Two modifications in the molecule of 19-nortestosterone; the</p><p>�-methylation and a C-14,15 double bond, revealed that the mod-</p><p>fied molecule (7�-methyl-14-dehydro-19-nortestosterone, Fig. 1,</p><p>1) was 1000 times as active as testosterone in the chick comb assay</p><p>nd 100 times as active as testosterone in either the rat ventral</p><p>rostate or levator ani assay (Table 2, row 25) [119].</p><p>Many 7�-alkyltestosterone derivatives were tested (Table 2,</p><p>ow 26) [120] and were found to lose androgenic and anabolic</p><p>ctivity rapidly as the size of the group at the C-7 position</p><p>ncreased. 7�-Alkyltestosterone derivatives show a progressive</p><p>oss in activity, as competitive inhibitors of aromatase, as chain</p><p>ength increases. The change in binding affinity of these com-</p><p>ounds seems too gradual to be the result of an absolute lack</p><p>f bulk tolerance by the receptor. The similar affinities found for</p><p>he 7�-hydroxypropyl and the isosteric 7�-butyl derivatives of</p><p>estosterone indicate that the reduction in affinity cannot arise</p><p>olely as a consequence of a hydrophobic chain into a hydrophobic</p><p>ocket.</p><p>A steroid incorporating a 7-chroro-4,6-dien-3-one system rep-</p><p>esents a series of steroids with interesting biological activity, but</p><p>-7 chloro substitution revealed no antigonadotropic, anabolic or</p><p>ndrogenic activity (Table 2, row 27) [121].</p><p>.7. C-17 alkyl substitution of steroids</p><p>17�-Alkylation is a structural feature of steroids which con-</p><p>ributes to the prolongation of the anabolic effect. Methyl and ethyl</p><p>ubstitution at 17�-position makes a valuable contribution to the</p><p>ncrease of anabolic effects, as it is associated with increase of the</p><p>tability to enzymatic oxidation of the D-ring and the conversion</p><p>o low active 17-keto steroids [122–124]. The effectiveness of 17�-</p><p>lkylated androgens when given orally is due to a slower hepatic</p><p>nactivation that occurs with unmodified hormone. The intracel-</p><p>ular metabolism is limited and the transformation of this part of</p><p>teroid does not take place, leading to possible disturbances in liver</p><p>etabolism [123].</p><p>The 17�-methyl substituted analogue of testosterone, methyl-</p><p>estosterone (Fig. 1, 15), was among the first 17�-alkylated</p><p>ndrogens with increased androgenic and anabolic potencies,</p><p>ynthesized in 1935 [88]. Other 17�-methyl derivatives include</p><p>ethandienone (Fig. 1, 11), oxymetholone (Fig. 1, 12) and oxan-</p><p>rolone (Fig. 1, 23). In other 17�-alkylated androgens, such</p><p>s nor-bolethone (Fig. 1, 2), ethylestrenol (Fig. 1, 13) and</p><p>orethandrolone (Fig. 1, 33), an ethyl group is introduced instead</p><p>f a methyl group.</p><p>In an early study [125], a variety of other alkyl groups have</p><p>een also introduced at the 17�-position of the 19-nor androstane</p><p>eries, such as ethyl-, propyl-, butyl-, octyl- and vinyl-groups. Of</p><p>he compounds studied, 17-ethyl-19-nortestosterone appeared to</p><p>e the most applicable for clinical use because of its high anabolic</p><p>otency (comparable to testosterone) and its low androgenic activ-</p><p>ty.</p><p>�-Alkylation at position C-17 also prevents aromatization of</p><p>he A-ring to estrogens [126]. The lower affinity of 17�-methyl</p><p>teroids as compared to their unsubstituted counterparts could</p><p>esult from the conformational effect that weakens the hydro-</p><p>en bond with the 17-oxygen. However, the receptor site at C-17</p><p>s large enough to accommodate a 17�-methyl group since this</p><p>ubstituent does not hinder the interaction with the receptor. In</p><p>ontrast, epitestosterone (Fig. 1, 42) has virtually no affinity for</p><p>he receptor. Perhaps this is because the 17�-position corresponds</p><p>o a hydrophobic pocket on the receptor close enough to account</p><p>or this loss of affinity. On the other hand, ethynyl and ethyl sub-</p><p>tituents which are bulkier than a hydroxyl are still compatible with</p><p>inding.</p><p>teroid</p><p>4</p><p>f</p><p>n</p><p>a</p><p>1</p><p>d</p><p>s</p><p>r</p><p>H</p><p>t</p><p>c</p><p>�</p><p>(</p><p>t</p><p>s</p><p>[</p><p>i</p><p>t</p><p>t</p><p>k</p><p>a</p><p>d</p><p>n</p><p>a</p><p>c</p><p>s</p><p>p</p><p>4</p><p>m</p><p>s</p><p>i</p><p>s</p><p>(</p><p>e</p><p>a</p><p>r</p><p>e</p><p>n</p><p>a</p><p>H</p><p>t</p><p>a</p><p>s</p><p>p</p><p>s</p><p>w</p><p>(</p><p>i</p><p>o</p><p>t</p><p>r</p><p>1</p><p>(</p><p>T</p><p>f</p><p>[</p><p>o</p><p>a</p><p>t</p><p>[</p><p>s</p><p>r</p><p>o</p><p>i</p><p>h</p><p>t</p><p>b</p><p>4</p><p>t</p><p>z</p><p>s</p><p>e</p><p>•</p><p>A.G. Fragkaki et al. / S</p><p>.8. Steroids with conjugated double bonds</p><p>Many compounds with conjugated double bonds extending</p><p>rom rings A and B to C are clearly more active than 19-</p><p>ortestosterone or 5�-DHT. The androgenicities of these estratriens</p><p>re also increased further by a methyl substitution at the 7�- or</p><p>7�-positions [83].</p><p>The combination of a 17�-side chain and conjugated �4,9,11-</p><p>ouble bonds, in the presence of hydrophobic substituents leads to</p><p>teroids able to bind firmly not only to the androgen and progestin</p><p>eceptors, but also to the mineralo- and glucocorticoid receptors.</p><p>ydrophobic substituents are -ethyl or -propyl substituents at posi-</p><p>ion C-13 and -ethynyl substituents at position C-17, of various</p><p>ompounds in the androsta, estra, pregnane or gonane series. The</p><p>4,9,11 steroids are more flexible than the corresponding dienes</p><p>�4,9) and monoenes (�4). This lack of receptor specificity of</p><p>rienes is due to their conformational mobility [127].</p><p>Methyltrienolone (Fig. 1, 5) is a potent, toxic, non-aromatising</p><p>teroid with conjugated double bonds extending from ring A to C</p><p>79]. Methyltrienolone synthesis was first described in 1963. Stud-</p><p>es indicated that the anabolic effect of methyltrienolone was 100</p><p>imes that of 19-nor-methyltestosterone (Fig. 1, 43) and 300 times</p><p>hat of methyltestosterone (Fig. 1, 15). Although it was never mar-</p><p>eted for use in humans due to its highly toxic effects, it is both an</p><p>nabolic and androgenic agent, very active when given orally.</p><p>The “little-cousin” of methyltrienolone, is known to be methyl-</p><p>ienolone (Fig. 1, 44), which is highly orally bioavailable,</p><p>on-aromatising 19-nortestosterone derivative that boasts a very</p><p>nabolic and moderately androgenic profile [128]. Little data exists</p><p>oncerning the use of methyldienolone in humans. Given its close</p><p>tructural similarities to methyltrienolone, methyldienolone is</p><p>robably among the most hepatotoxic 17�-methyl AAS.</p><p>.9. Steroids with heteroatoms</p><p>The replacement of one or more carbon atoms in a steroid</p><p>olecule by a heteroatom affects the chemical properties of a</p><p>teroid and often results in useful alterations to its biological activ-</p><p>ty. Numerous publications have described the synthesis of novel</p><p>teroids with heteroatoms [129–131].</p><p>A representative example of a 2-oxasteroid is oxandrolone</p><p>Fig. 1, 23). Oxandrolone is structurally similar to 17�-methyl-DHT</p><p>xcept that it contains an oxygen atom instead of a methylene group</p><p>t the C-2 position. Such a structural modification is unique and</p><p>epresents the first anabolic steroid in which a substitution of a het-</p><p>roatom has been made for a carbon atom within the basic steroid</p><p>ucleus. Oxandrolone, indeed, possesses considerable myotropic</p><p>nd nitrogen-retaining activity with little androgenicity [132,133].</p><p>owever, 2-oxasteroids containing a 4,5-unsaturated 3-keto sys-</p><p>em (e.g. 2-oxa-17�-methyltestosterone) are about equivalent in</p><p>ctivity to normal steroids [133].</p><p>Comparison of 2-oxa, 3-oxa and 4-oxa-5�-androstan-17�-ol</p><p>teroids revealed that the introduction of an oxygen atom at C-2</p><p>osition gives rise to the most active compound of this series. Sub-</p><p>titution at C-3 position results in a much less active compound,</p><p>hereas, substitution at C-4 results in a compound which is inactive</p><p>Table 2, row 28) [134].</p><p>The corresponding steroid with a sulfur atom at C-2 position has</p><p>nteresting activities; it has about one-fifth the androgenic activity</p><p>f testosterone and</p>