New Insights Into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites
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molecules Article New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites Steffen Loke 1, Lingyu Liu 1 , Maxi Wenzel 1, Heike Scheffler 1, Michele Iannone 2, Xavier de la Torre 2 , Nils Schlörer 3 , Francesco Botrè 2,4, Annekathrin Martina Keiler 5,6 , Matthias Bureik 7 and Maria Kristina Parr 1,* 1 Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany; [email protected] (S.L.); [email protected] (L.L.); [email protected] (M.W.); heike.scheffl[email protected] (H.S.) 2 Laboratorio Antidoping FMSI, Largo Giulio Onesti 1, 00197 Rome, Italy; [email protected] (M.I.); [email protected] (X.d.l.T.); [email protected] (F.B.) 3 Institute for Organic Chemistry, Universität zu Köln, Grenstraße 4, 50939 Cologne, Germany; [email protected] 4 REDs–Research and Expertise in Antidoping Sciences, ISSUL–Institute del Sciences du Sport de l’Université de Lausanne, 1015 Lausanne, Switzerland 5 Institute of Doping Analysis & Sports Biochemistry Dresden, Dresdner Str. 12, 01731 Kreischa, Germany; [email protected] 6 Environmental Monitoring & Endocrinology, Faculty of Biology, Technische Universität Dresden, Zellescher Weg 20b, 01217 Dresden, Germany 7 School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, Nankai District, Citation: Loke, S.; Liu, L.; Wenzel, Tianjin 300072, China; [email protected] M.; Scheffler, H.; Iannone, M.; de la * Correspondence: [email protected]; Tel.: +49-30-838-57686 Torre, X.; Schlörer, N.; Botrè, F.; Keiler, A.M.; Bureik, M.; et al. New Insights Abstract: Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a into the Metabolism of 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Methyltestosterone and Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl- Metandienone: Detection of Novel 17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog de- A-Ring Reduced Metabolites. hydrochloromethyltestosterone (“oral turinabol”), in this study we investigated the formation of Molecules 2021, 26, 1354. https:// similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a doi.org/10.3390/molecules26051354 fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference com- pounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst- Academic Editors: Christoph Müller 13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. and Franz Bracher In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, Received: 17 February 2021 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides Accepted: 27 February 2021 the classical metabolites included in the screening procedures. Besides their applicability for anti- Published: 3 March 2021 doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations Publisher’s Note: MDPI stays neutral to the D-ring. with regard to jurisdictional claims in published maps and institutional affil- Keywords: 17α-methyl steroids; long-term metabolites; gas chromatography-mass spectrometry; iations. 17-hydroxymethyl-17-methyl-18-nor; D-ring alteration; doping control; metabolism Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. Metandienone (17β-hydroxy-17α-methyl-androsta-1,4-dien-3-one, MD, 12; list of steroids This article is an open access article available in supplement S1) and methyltestosterone (17β-hydroxy-17α-methylandrost-4-en- distributed under the terms and 3-one, MT, 18) are anabolic-androgenic steroids. They were introduced to the market in conditions of the Creative Commons 1960 (MD) [1] and 1939 (MT) [2] as orally active anabolic androgenic steroids. Although Attribution (CC BY) license (https:// there is no approved drug available anymore, they are still widely marketed and misused creativecommons.org/licenses/by/ as performance-enhancing drugs in sports, even though they are prohibited in and outside 4.0/). Molecules 2021, 26, 1354. https://doi.org/10.3390/molecules26051354 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 1354 2 of 18 Molecules 2021, 26, 1354 2 of 18 they are prohibited in and outside of competition by the World Anti-Doping Agency [3]. ofMany competition so-called by “adverse the World analytical Anti-Doping findings” Agency (AAFs) [3]. Many in doping so-called control “adverse have analytical been re- findings”ported, and (AAFs) their in numbers doping control in the havelast 17 been years reported, are displayed and their in numbers Figure 1 in Over the lastthe 17last years few areyears, displayed metandienone in Figure and1 Over methyltestosterone the last few years, represent metandienone 10% and and 1% methyltestosterone of all AAFs in the representclass of anabolic 10% and agents. 1% of In all 2018, AAFs only in thefive classother of substances anabolic agents.out of all In prohibited 2018, only compound five other substancesclasses were out identified of all prohibited more frequently compound than classes metandienone were identified (n = 131), more with frequently clenbuterol than metandienonegiving the highest (n = 131),number with (n clenbuterol = 320). giving the highest number (n = 320). Figure 1. Adverse analytical findings of methyltestosterone and metandienone between 2003 and Figure 1. Adverse analytical findings of methyltestosterone and metandienone between 2003 and 2018, according to [4]. 2018, according to [4]. Due to the 17α-methyl group, the steroids become orally active, because it prevents the first-passDue to the metabolism 17α-methyl by group, hindering the thesteroids oxidation become of the orally 17β active,-hydroxy because group it sterically, prevents whilethe first-pass the introduction metabolism of a by double hindering bond inthe position oxidation 1 was of the intended 17β-hydroxy to avoid group aromatization sterically, andwhile reduced the introduction the activity of of a double A-ring-reducing bond in po enzymessition 1 was [5–7 intended]. to avoid aromatization and Manyreduced metabolites the activity related of A-ring-reducing to the intake of enzymes metandienone [5–7]. are reported in the literature, and aMany number metabolites of these have related been to the known intake for of decades. metandienone These areare generatedreported in by the both literature, phase Iand and a phase number II drugof these metabolizing have been enzymes.known for Phase decades. I reactions These are include generated the introduction by both phase of aI doubleand phase bond II drug at position metabolizing 6, the reduction enzymes. ofPhas doublee I reactions bonds ininclude the A-ring, the introduction the reduction of a ofdouble the 3-oxo bond group,at position hydroxylations 6, the reduction in positions of double 6, bonds 11, 12, in 16,the orA-ring, 18, epimerization the reduction inof positionthe 3-oxo 17, group, and rearrangementhydroxylations ofin thepositions D-ring 6, [ 811,–15 12,]. With16, or respect 18, epimerization to phase II in reactions, position both17, and glucuronidation rearrangement and of the sulfonation D-ring [8–15]. have beenWith reportedrespect to [ 16phase,17]. II In reactions, recent years, both new glu- investigationscuronidation and on long-term sulfonation metabolites have been of reported MD (12) identified[16,17]. In further recent metabolitesyears, new investiga- with 17β- hydroxymethyl-17tions on long-termα -methyl-13-enemetabolites of MD structure (12) identified [18–21]. The further known metabolites metabolites with are 17 shownβ-hy- indroxymethyl-17 Figure2. Anti-dopingα-methyl-13-ene laboratories structure mostly [18–21]. target The the known parent metabolites compound are ( 12 shown), 6-OH- in metandienoneFigure 2. Anti-doping (13), epi-metandienone laboratories (mo14),stly epi-metendiol target the (parent15), nor-epi-metendiol compound (12 (),16 ),6-OH- and 20metandienoneβOH-nor-metandienone (13), epi-metandienone (17)[18,22,23 ].(14), epi-metendiol (15), nor-epi-metendiol (16), and The20βOH-nor-metandienone intake of methyltestosterone (17) [18,22,23]. leads to several metabolites, which derive from hydroxylationsThe intake in of positions methyltestosterone 2, 4, 6, 11, or leads 20, reduction to several of metabolites, the 4,5-double which bond, derive reduction from hy- of thedroxylations 3-oxo group, in positions oxidation 2, yielding 4, 6, 11, aor 1,2- 20, or reduction a 6,7-double of the bond, 4,5-double epimerization bond, reduction in position of 17,the and 3-oxo rearrangement group, oxidation of the yielding D-ring [ 24a 1,2-–28 ].or Subsequent a 6,7-double phase bond, II reactionsepimerization are also in position leading to17, both and glucuronidesrearrangement and of the sulfates D-ring [26 [24–28].,29]. The Subsequent structures phase of metabolites II reactions of are MT also (18 )lead- are showning to both in Figure glucuronides3. Laboratories and sulfates mainly [26,29 screen]. forThe the structures parent compound of metabolites itself of ( 18 MT) and (18 two) are reducedshown in derivatives Figure