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Developing a dual-efficiency agent with antiresorptive and anabolic applications is a promising strategy for treating osteoporosis. This study reports the discovery of dual antiosteoporosis agents via a chemotype-assembly approach. Chemotype analysis identified 12 antiresorptive and 12 anabolic chemotypes and 7 dual-function chemotype-assembly rules. Based on these assembly rules, a dual-functional compound S24 was discovered. S24 exhibits osteoclastogenesis inhibition with an IC 50 value of 10.28 μM and osteoblast differentiation stimulation at 10 μM. S24 derivatives were designed and synthesized based on the activity relationship of the chemotypes. This yielded a more active compound, S24-14 , with an osteoclastogenesis inhibition IC 50 value of 0.40 μM and osteoblast differentiation stimulation at 1.0 μM; compound S24-14 also suppressed bone loss in vivo . These results prove that S24-14 can be a potential lead for antiosteoporosis drug development.

Selective androgen receptor (AR) modulators (SARMs) are potent anabolic agents with a high potential of misuse in horseracing and equestrian sports. In this study, we applied label-free proteomics to discover plasma protein biomarkers in geldings (castrated horses) after administration with a popular SARM named RAD140. Tryptic peptides were prepared from plasma samples and analyzed by nano-flow ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (nano-UHPLC-HRMS/MS) using data-independent acquisition (DIA) method. Orthogonal projection on latent structure-discriminant analysis (OPLS-DA) has led to the development of a predictive model that could discriminate RAD140-administered samples from control samples and could also correctly classify 18 out of 19 in-training horses as control samples. The model comprises 75 proteins with variable importance in projection (VIP) score above 1. Gene Ontology (GO) enrichment analysis and literature review have identified upregulation of AR-regulated clusterin, and proteins associated with inflammation (haptoglobin, cluster of differentiation 14 [CD14], and inter-alpha-trypsin inhibitor heavy chain 4 [ITIH4]) and erythropoiesis (glycosylphosphatidylinositol-specific phospholipase D1 [GPLD1]) after RAD140 administration. Their changes were confirmed by selected reaction monitoring (SRM) experiments. Similar effects have been reported by the use of androgens and other SARMs. This is the first reported study that describes the use of a proteomic biomarker approach to detect horses that have been administered with RAD140 by applying label-free proteomic profiling of plasma samples. These results support the concept of a biomarker-driven approach to enhance the doping control of RAD140 and potentially other SARMs in the future., (© 2020 John Wiley & Sons, Ltd.)

A photochemical approach to 18-nor-17β-hydroxymethyl-17α-methylandrost-13-ene unit of the long-term metabolites of 17-methylated androgenic anabolic steroids (AAS) is reported. It is based on a visible light-promoted radical decarboxylative alkynylation of steroidal redox-active ester. The developed method was used in synthesis of the long-term metabolite of AAS oxymesterone., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Rationale: Isoflavones are a group of flavonoids that may be of interest in sport doping because they can be used by athletes in the recovery periods after the administration of anabolic steroids, with the aim of increasing the natural production of luteinizing hormone (LH) and, consequently, the biosynthesis of endogenous androgens., Methods: The in vivo metabolism of methoxyisoflavone (5-methyl-7-methoxyisoflavone) and ipriflavone (7-isopropoxyisoflavone), respectively present in a dietary supplement and in a pharmaceutical preparation, was investigated. The study was carried out by the analysis of urinary samples collected from male Caucasian subjects before, during and after the oral administration of methoxyisoflavone or ipriflavone. After enzymatic hydrolysis and liquid-liquid extraction, all urinary samples were analyzed by gas chromatography/quadrupole time-of-flight (qTOF MS system/qTOF) electron ionization mass spectrometry (EI-MS)., Results: Eight metabolites of methoxyisoflavone and six metabolites of ipriflavone were isolated. The corresponding accurate mass spectra are specific for isoflavone structures and revealed also a retro-Diels-Alder fragmentation., Conclusions: When excreted in large amounts, the urinary metabolites of methoxyisoflavone and ipriflavone can be traced to potential confounding factors in doping analysis. As methoxyisoflavone and ipriflavone have been shown to inhibit the enzyme aromatase, thus interfering with the normal metabolic pathways of testosterone, the detection of their intake, by screening for the presence of their main metabolites in urine, might be helpful in routine doping control analysis., (© 2019 John Wiley & Sons, Ltd.)

Benzofuran moiety is an important pharmacophore showing positive effects on bone health. In the present study, sixteen benzofuran-pyran hybrids were synthesized and were evaluated for their osteogenic effects on primary osteoblast cells isolated from calvaria. Compounds 22 and 24 were found potent in stimulating osteoblast differentiation as assessed by the alkaline phosphatase activity. These compounds were also found to be nontoxic to osteoblast cells as compared to the control cells in MTT assay. Further, Alizarin Red-S staining for visualization of calcium nodules demonstrated compounds 22 and 34 as active in enhancing mineralization in osteoblast cells. Additionally, transcriptional analysis of these compounds on osteoblast cells revealed that compound 22 up-regulated the expression of osteogenic genes RUNX2, BMP-2, COL-1, thus substantiating that compound 22 having two geminal methyl groups in its R 3 position is a potent osteogenic agent. Additionally, compound 22 enhanced the ability of bone marrow stromal cells to differentiate towards osteoblast lineage and therefore can be further studied in vivo in bone loss model., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In an effort to find new and safer treatments for osteoporosis and frailty, we describe a novel series of selective androgen receptor modulators (SARMs). Using a structure-based approach, we identified compound 7, a potent AR (ARE EC 50 = 0.34 nM) and selective (N/C interaction EC 50 = 1206 nM) modulator. In vivo data, an AR LBD X-ray structure of 7, and further insights from modeling studies of ligand receptor interactions are also presented.

A series of new 6H-benzofuro[3, 2-c]chromenes (BFC, pterocarpans) with structure-activity relationships were investigated for their potential use in osteoporosis treatment. One of the BFCs 3-piperidylethoxypterocarpan 20 promotes osteoblast differentiation and mineralization at a dose as low as 1 pM via activation of ER/P38MAPK/BMP-2 pathway. When evaluated for in-vivo osteogenic activity in female Sprague-Dawley rats, BFC 20 increased bone mineral density and new bone formation, compared with control at 1.0 and 10.0 mg/kg/body weight by oral gavage for 30 days. The compound was devoid of any uterotrophic effect and led to the new bone formation in adult ovariectomized osteopenic rats. BFC 20 compound also inhibited bone resorption by reducing Ovx induced increase in urinary CTx, thus exhibiting both bone anabolic and anti-catabolic action. Finally, BFC 20 treatment to Ovx rats led to improved trabecular microarchitectural restoration and exhibited therapeutic potential as a dual acting anti-osteoporotic agent for the management of osteoporosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An imbalance between bone resorption by osteoclasts and bone formation by osteoblasts can cause bone loss and bone-related disease. In a previous search for natural products that increase osteogenic activity, we found that 5,6-dehydrokawain (1) from Alpinia zerumbet promotes osteoblastogenesis. In this study, we synthesized and evaluated series of 5,6-dehydrokawain analogs. Our structure-activity relationships revealed that alkylation of para or meta position of aromatic ring of 1 promote osteogenic activity. Among the potential analogs we synthesized, (E)-6-(4-Ethylstyryl)-4-methoxy-2H-pyran-2-one (14) and (E)-6-(4-Butylstyryl)-4-methoxy-2H-pyran-2-one (21) both significantly up-regulated Runx2 and Osterix mRNA expression at 10µM. These osteogenic activities could be mediated by bone morphogenetic protein (BMP) and activation of p38 MAPK signaling pathways. Compounds 14 and 21 also inhibited RANKL-induced osteoclast differentiation of RAW264 cells. These results indicated that novel 5,6-dehydrokawain analogs not only increase osteogenic activity but also inhibit osteoclast differentiation, and could be potential lead compounds for the development of anti-osteoporosis agents., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Palladium-catalyzed C-H acetoxylation has been proposed as a key transformation in the first chemical synthesis of steroids bearing a unique 17β-hydroxymethyl-17α-methyl-18-nor-13-ene D-fragment. This C-H functionalization step was crucial for inverting the configuration at the quaternary stereocenter of a readily available synthetic intermediate. The developed approach was applied to prepare the metandienone metabolite needed as a reference substance in anti-doping analysis to control the abuse of this androgenic anabolic steroid., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A transdermal SARM has a potential to have therapeutic benefit through anabolic activity in muscle while sparing undesired effects of benign prostate hyperplasia (BPH) and liver-mediated decrease in HDL-C. 2-Chloro-4-[(2-hydroxy-2-methyl-cyclopentyl)amino]-3-methyl-benzonitrile 6 showed the desired muscle and prostate effects in a preclinical ORX rat model. Compound 6 had minimal effect on HDL-C levels in cynomolgus monkeys and showed human cadaver skin permeability, thus making it an effective tool for proof-of-concept studies in a clinical setting.

Novel 16-hydroxymethyl-19-nortestosterone diastereomers were prepared by Birch reduction from the corresponding 3-methoxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol isomers with known configurations. The synthesized compounds are 16α- and 16β-hydroxymethyl-substituted 19-nortestosterone and their 17α-epimers. To prepare 17α-19-nortestosterone, the Mitsunobu inversion reaction of 19-nortestosterone with different alkyl and aryl carboxylic acids was chosen. Deacylation of the new compounds by the Zemplén method yielded the required 17α-19-nortestosterone. The antiproliferative activities of the structurally related compounds were determined in vitro through microculture tetrazolium assays on a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines. The 17α epimer of 19-nortestosterone demonstrated considerable activity, selectively for HeLa cells, with a calculated IC50 of 0.65 μM. The reference compound, cisplatin, displayed an order of magnitude higher IC50 (12.4 μM). The cancer selectivity of 17α-19-nortestosterone was tested by MTT assay performed with noncancerous human fibroblast cell line MRC-5. The results indicated that 17α-19-nortestosterone selectively disturbs the viability of HeLa cells without greatly affecting other cancer cell types and intact fibroblasts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To develop effective drugs for hypogonadism, sarcopenia, and cachexia, we designed, synthesized, and evaluated selective androgen receptor modulators (SARMs) that exhibit not only anabolic effects on organs such as muscles and the central nervous system (CNS) but also neutral or antagonistic effects on the prostate. Based on the information obtained from a docking model with androgen receptor (AR), we modified a hit compound A identified through high-throughput screening. Among the prepared compounds, 1-(4-cyano-1-naphthyl)-2,3-disubstituted pyrrolidine derivatives 17h, 17m, and 17j had highly potent AR agonistic activities in vitro and good tissue selectivity in vivo. These derivatives increased the weight of the levator ani muscle without influencing the prostate and seminal vesicle. In addition, these compounds induced sexual behavior in castrated rats, indicating that the compounds could also act as agonists on the CNS., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

For the first time, structural information regarding the role of simvastatin in bone anabolism is described, and a bone-specific statin is introduced. Polyaspartate-conjugated simvastatin was synthesized by solid-phase synthesis with the assistance of microwave irradiation. It displays significant bone targeting and bone formation with less toxicity than simvastatin.

In continuation of our previous work, fused steroidal derivatives with pyrane, pyridine, pyrimidine moieties were synthesized and evaluated as androgenic-anabolic agents. Some of the newly synthesized compounds are exhibited pronounced androgenic-anabolic activities.

Tryptophan hydroxylase 1 (Tph-1), the principal enzyme for peripheral serotonin biosynthesis, provides a novel target to design anabolic agents for osteoporosis. Here, we present a design, synthesis of a novel series of ursolic acid derivatives under the guidance of docking technique, and bioevaluation of the derivatives using RBL2H3 cells and ovariectomized (OVX) rats. Of the compounds, 9a showed a potent inhibitory activity on serotonin biosynthesis. Further investigations revealed that 9a, as an efficient Tph-1 binder identified by SPR (estimated KD: 6.82 μM), suppressed the protein and mRNA expressions of Tph-1 and lowered serotonin contents in serum and gut without influence on brain serotonin. Moreover, oral administration of 9a elevated serum level of N-terminal propeptide of procollagen type 1 (P1NP), a bone formation marker, and improved bone microarchitecture without estrogenic side effects in ovariectomized rats. Collectively, 9a may serve as a new candidate for bone anabolic drug discovery.

We present a novel series of selective androgen receptor modulators (SARMs) which shows excellent biological activity and physical properties. 1-(2-Hydroxy-2-methyl-3-phenoxypropanoyl)-indoline-4-carbonitriles showed potent binding to the androgen receptor (AR) and activated AR-mediated transcription in vitro. Representative compounds demonstrated diminished activity in promoting the intramolecular interaction between the AR carboxyl (C) and amino (N) termini. This N/C-termini interaction is a biomarker assay for the undesired androgenic responses in vivo. In orchidectomized rats, daily administration of a lead compound from this series showed anabolic activity by increasing levator ani muscle weight. Importantly, minimal androgenic effects (increased tissue weights) were observed in the prostate and seminal vesicles, along with minimal repression of circulating luteinizing hormone (LH) levels and no change in the lipid and triglyceride levels. This lead compound completed a two week rat toxicology study, and was well tolerated at doses up to 100 mg/kg/day, the highest dose tested, for 14 consecutive days.

The discovery and implementation of the long-term metabolite of metandienone, namely 17β-hydroxymethyl-17α-methyl-18-norandrost-1,4,13-trien-3-one, to doping control resulted in hundreds of positive metandienone findings worldwide and impressively demonstrated that prolonged detection periods significantly increase the effectiveness of sports drug testing. For oxandrolone and other 17-methyl steroids, analogs of this metabolite have already been described, but comprehensive characterization and pharmacokinetic data are still missing. In this report, the synthesis of the two epimeric oxandrolone metabolites-17β-hydroxymethyl-17α-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one and 17α-hydroxymethyl-17β-methyl-18-nor-2-oxa-5α-androsta-13-en-3-one-using a fungus (Cunninghamella elegans) based protocol is presented. The reference material was fully characterized by liquid chromatography nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. To ensure a specific and sensitive detection in athlete's urine, different analytical approaches were followed, such as liquid chromatography-tandem mass spectrometry (QqQ and Q-Orbitrap) and gas chromatography-tandem mass spectrometry, in order to detect and identify the new target analytes. The applied methods have demonstrated good specificity and no significant matrix interferences. Linearity (R(2) > 0.99) was tested, and precise results were obtained for the detection of the analytes (coefficient of variation <20%). Limits of detection (S/N) for confirmatory and screening analysis were estimated at 1 and 2 ng/mL of urine, respectively. The assay was applied to oxandrolone post-administration samples to obtain data on the excretion of the different oxandrolone metabolites. The studied specimens demonstrated significantly longer detection periods (up to 18 days) for the new oxandrolone metabolites compared to commonly targeted metabolites such as epioxandrolone or 18-nor-oxandrolone, presenting a promising approach to improve the fight against doping.

The concept of molecular hybridization led us to discover a novel series of coumarin-dihydropyridine hybrids that have potent osteoblastic bone formation in vitro and that prevent ovariectomy-induced bone loss in vivo. In this context, among all the compounds screened for alkaline phosphatase activity, four compounds 10, 14, 18, and 22 showed significant activity at picomolar concentrations. A series of other in vitro data strongly suggested compound 18 as the most promising bone anabolic agent, which was further evaluated for in vivo studies. From these studies compound 18 proved to be useful, which at low oral dose of 1 (mg/kg)/day body weight increased bone mass density and volume, expression of osteogenic genes (RUNX2, BMP-2, and ColI), bone formation rate (BFR), and mineral apposition rate (MAR), improved the trabecular microarchitecture, and decreased bone turn over markers in an ovariectomized rodent model for postmenopausal osteoporosis.

A novel selective androgen receptor modulator scaffold has been discovered through structural modifications of hydantoin antiandrogens. Several 4-(4-hydroxyphenyl)-N-arylhydantoins displayed partial agonism with nanomolar in vitro potency in transactivation experiments using androgen receptor (AR) transfected cells. In a standard castrated male rat model, several compounds showed good anabolic activity on levator ani muscle, dissociated from the androgenic activity on ventral prostate, after oral dosing at 30 mg/kg. (+)-4-[3,4-Dimethyl-2,5-dioxo-4-(4-hydroxyphenyl)imidazolidin-1-yl]-2-(trifluoromethyl)benzonitrile ((+)-11b) displayed anabolic potency with a strong dissociation between levator ani muscle and ventral prostate (A(50) = 0.5 mg/kg vs 70 mg/kg). The binding modes of two compounds, including (+)-11b, within the AR ligand-binding domain have been studied by cocrystallization experiments using a coactivator-like peptide. Both compounds bound to the same site, and the overall structures of the AR were very similar.

Structural modification performed on a 4-methyl-4-(4-hydroxyphenyl)hydantoin series is described which resulted in the development of a new series of 4-(hydroxymethyl)diarylhydantoin analogues as potent, partial agonists of the human androgen receptor. This led to the identification of (S)-(-)-4-(4-(hydroxymethyl)-3-methyl-2,5-dioxo-4-phenylimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile ((S)-(-)-18a, GLPG0492) evaluated in vivo in a classical model of orchidectomized rat. In this model, (-)-18a exhibited anabolic activity on muscle, strongly dissociated from the androgenic activity on prostate after oral dosing. (-)-18a has very good pharmacokinetic properties, including bioavailability in rat (F > 50%), and is currently under evaluation in phase I clinical trials.

Brassinosteroids are plant-derived polyhydroxylated derivatives of 5α-cholestane, structurally similar to cholesterol-derived animal steroid hormones and insect ecdysteroids. In this study, we synthesized a set of brassinosteroid analogues of a natural brassinosteroid (22S,23S)-homobrassinolide (HB, 1), including (22S,23S)-homocastasterone (2), (22S,23S)-3α-fluoro-homobrasinolide (3), (22S,23S)-3α-fluoro-homocastasterone (4), (22S,23S)-7-aza-homobrassinolide (5), and (22S,23S)-6-aza-homobrassinolide (6) and studied their anabolic efficacy in the L6 rat skeletal muscle cells in comparison to other synthetic and naturally occurring brassinosteroids (22R,23R)-homobrassinolide (7), (22S,23S)-epibrassinolide (8), and (22R,23R)-epibrassinolide (9). Presence of the 6-keto group in the B ring and stereochemistry of 22α,23α-vicinal hydroxyl groups in the side chain were critical for the anabolic activity, possibly due to higher cytotoxicity of the 22β,23β-hydroxylated brassinosteroids. All anabolic brassinosteroids tested in this study selectively activated PI3K/Akt signaling pathway as evident by increased Akt phosphorylation in vitro. Plant brassinosteroids and their synthetic derivatives may offer a novel therapeutic strategy for promoting growth, repair, and maintenance of skeletal muscles.

Testosterone is the principal male sex hormone. As with all natural steroids, it is biosynthesized from cholesterol. Phase I metabolism employs some very specific enzymes and pathways. Phase II metabolism and excretion follow more general patterns. The effects of testosterone are twofold: anabolic and androgenic. Because of its anabolic effects, testosterone is frequently abused in sports. Because of its endogenous nature, testosterone doping is difficult to detect. The standard procedure is based on the evaluation of the urinary steroid profile. Conspicuous samples then are submitted to compound-specific (13)C/(12)C analysis. Synthetic and endogenous steroids differ in this measure. Numerous xenobiotic compounds have been derived from testosterone. The modifications typically aim at a reduction of the androgenic properties while maintaining the anabolic potential. Most of these compounds have been withdrawn from the legal market. However, they are found to be illicitly added to otherwise inefficient nutritional supplements. These products represent a major problem to doping control. Recently, clinical trials with selective androgen receptor modulators have been started., (Copyright 2010 Elsevier Inc. All rights reserved.)

In this work, substituted benzothiophene and benzofuran compounds were found to be a new class of potential anabolic agents by enhancing BMP-2 expression. A series of benzothiophene and benzofuran derivatives have been synthesized, and their activities of up-regulating BMP-2 and bone loss prevention efficacies in SAMP6 mice and OVX rats have been studied. Benzothiophenes 1, 3, 14, 4a, 7a, 8a, and benzofuran analogue 2 showed higher BMP-2 up-regulation rates in vitro. Compound 8a was found to significantly affect the bone formation rate of tested SAMP6 mice. Compound 1 showed an improved bone quality in SAMP6 mice and also showed activity in OVX rats. We have demonstrated that substituted benzothiophene and benzofuran derivatives, especially compounds 1 and 8a, enhance BMP-2 expression in vitro and in vivo and stimulate bone formation and trabecular connectivity restoration in vivo. The compounds represent potential leads in the development of a new class of anabolic agents.

Anabolic steroids have been studied for over 50 years and during that time numerous compounds with a variety of functional groups have been produced and many have been published. Of these only a small number have been introduced to the pharmaceutical market. WADA has continued the work begun by the IOC banning the use of these agents within sport as performance enhancing substances. Athletes, however, continue to use these anabolic steroids but tighter testing and the introduction of unannounced sample collection has made this form of cheating harder.In order to try to evade detection, athletes who continue to dope are having to resort to the use of a far more dangerous form of drug - the designer steroid. These steroids are manufactured to closely resemble existing known compounds, but with sufficient chemical diversity to ensure that their detection by the WADA accredited laboratories is more difficult. A worrying feature of the use of these compounds is that no data is available to evaluate either the efficacy or the safety of these substances. Many such drugs are now being made in clandestine ways (as demonstrated by the recent BALCO case) and then passed on to athletes who become the guinea pigs determining the potential of the substances as doping agents.Methods for the detection of these new compounds are being developed using emerging techniques such as gas chromatography or liquid chromatography attached to a variety of mass spectrometry instruments. This technology as well as vigilance by laboratories and enforcement agencies can all help in early detection of designer steroids being used for doping.

We identified a novel synthetic steroid, S42, as a promising candidate of selective androgen receptor (AR) modulator. Results of the whole-cell binding assay using COS-7 cells exogenously expressing various steroid receptors indicated that S42 specifically binds to AR and progesterone receptor. When orchiectomized Sprague Dawley rats were administered with S42 for 3 wk, the muscle weight of the levator ani was increased as markedly as that induced by 5alpha-dihydrotestosterone (DHT), but the weight of the prostate was not elevated at any doses in contrast to DHT. The plasma concentrations of gonadotropin and adiponectin, those down-regulated by DHT, were unaffected by S42. In addition, although the plasma triglyceride level was unaffected by DHT, it was significantly reduced by S42. This effect of S42 was associated with suppression of the SRBP-1c-mediated lipogenic and insulin-desensitizing pathway in the liver and visceral fat. Taken together, S42 works as an AR agonist in muscle and as an AR antagonist in the prostate, pituitary gland, and liver, accompanying beneficial potentials on lipid metabolism.

The canine phase I and phase II metabolism of the synthetic anabolic-androgenic steroid stanozolol was investigated following intramuscular injection into a male greyhound. The major phase I biotransformation was hydroxylation to give 6alpha-hydroxystanozolol which was excreted as a glucuronide conjugate and was identified by comparison with synthetically derived reference materials. An analytical procedure was developed for the detection of this stanozolol metabolite in canine urine using solid phase extraction, enzyme hydrolysis of glucuronide conjugates and analysis by positive ion electrospray ionisation ion trap LC-MS.

3Beta,11beta-dihydroxy-9alpha-fluor-5alpha-androstane-17-one (2), 3beta-acetoxy-9alpha-fluor-11beta-hydroxy-5alpha-androstane-17-one (3), 3beta-acetoxy-9alpha-fluor-11beta,17beta-dihydroxy-5alpha-androstane (4), 3beta,17beta-diacetoxy-9alpha-fluor-11beta-hydroxy-5alpha-androstane (5), 3beta-acetoxy-9alpha-fluor-11beta-hydroxy-5alpha-androstane 17beta-propionate (6), 3beta-acetoxy-9alpha-fluor-11beta-hydroxy-5alpha-androstane 17beta-enanthate (7), 3beta-acetoxy-9alpha-fluor-11beta-hydroxy-5alpha-androstane 17beta-isobutyrate (8) were synthesized in the present study. Compounds 2 and 8 exhibited higher anabolic activity than the rest of the synthesized compounds. The structure of all these newly synthesized compounds was confirmed by analytic spectral data (mass, (1)H NMR and (13)C NMR).

Selective androgen receptor modulators represent an emerging class of therapeutics to counteract various diseases such as osteoporosis and muscle wasting. Numerous drug candidates have been developed and investigated including a group that comprises a tricyclic tetrahydroquinoline nucleus such as 2-methyl-2-(8-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]chinolin-4-yl)propan-1-ol. Due to their novelty and medicinal purpose, these compounds also possess great potential for misuse in sports, and studies on the mass spectrometric behavior of three synthesized model substances and drug candidates were conducted to provide information on typical dissociation pathways following electrospray ionization and collision-induced dissociation. Product ion mass spectra derived from protonated molecules were studied using high resolution/high accuracy orbitrap mass spectrometry, and characteristic fragmentation routes and product ions were elucidated. Major and general findings include the elimination of a hydroxyl radical from [M+H](+), the elimination of the 2-substituted side chain, and the gas-phase rearrangement of the investigated tricyclic tetrahydroquinolines to 6-nitroquinoline yielding a common product ion at m/z 175. Knowledge of these dissociation pathways supports the identification of related substances as well as metabolic products, which is of utmost importance to drug testing laboratories. The compounds were implemented into existing screening procedures, and detection limits (0.2-0.6 ng/mL), recoveries (92-97%), and intraday and interday precision (<22%) were evaluated., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Modification on a lead series of [1,4]oxazino[3,2-g]quinolin-7-ones at the 2-position led to selective androgen receptor modulators with improved in vivo activity. The most potent analog (-)-33a exhibited full maintenance of levator ani muscle at 3mg/kg and reduced activity on ventral prostate weight in a 2-week orally-dosed and orchidectomized rat maintenance assay.

In this study, we synthesized some new substituted steroidal derivatives using 3beta-hydroxyandrosten-17-one (dehydroepiandrosterone) as starting material. The synthesized steroidal derivatives 1-11 were evaluated for their androgenic-anabolic activities compared to testosterone as positive control. Details of the synthesis, spectroscopic data and toxicity (LD50) of synthesized compounds are reported.

Selective androgen receptor modulators (SARM) are a prominent group of compounds for being misused in sports owing to their advantageous anabolic properties and reduced side effects. To target the preventive doping control analysis in relevant compounds, the challenge is to predict the metabolic fate of a new compound. For aryl-propionamide-derived SARM, an in vitro assay employing microsomal and S9 human liver enzymes was developed to simulate phase-I and phase-II metabolic reactions. In vitro metabolic profiles and the structure-metabolic relationship were compared between four structurally modified substrates. Accurate mass measurements were used to characterize the synthesized metabolites, and also collision-induced dissociation was examined to suggest the methodological approach to monitor the prohibited use of aryl-propionamide-derived drug candidates. Subsequent phase-I and phase-II metabolic reactions were successfully combined in one in vitro assay. The main routes of phase-I modifications involved the hydrolysis of ether linkage, monohydroxylation, and hydrolytic cleavage of the amide bond. Nitro-reduction and deacetylation were reactions observed for substrates possessing the corresponding functionality. SARM metabolites were analyzed in negative ion electrospray ionization and detected as deprotonated species [M-H](-). The main metabolic modifications were observed to occur in the B-ring side, and collision-induced dissociation resulted in the product ions originating from the A-ring side of the compound. These structure-specific ions may be monitored as target ions in the routine doping control.

The elucidation of the metabolism of new therapeutics is a major task for pharmaceutical companies and of great interest for drug testing laboratories. The latter in particular need to determine the presence or absence of drugs or their metabolic products in urine to test for a misuse of these compounds. Commonly, in vitro or animal models are used to mimic the human metabolism and produce potential targets in amounts allowing for method development. An alternative route based on electrochemical reactions of drugs was reported to allow for the generation of selected metabolites. The utility of this approach for doping control purposes was demonstrated with a novel class of anabolic agents termed selective androgen receptor modulators (SARMs). An arylpropionamide- derived drug candidate was subjected to electrochemical "metabolism" and a major phase-I- metabolite, resulting from the elimination of a substituted phenol residue as identified in in vitro experiments, was generated and characterised using liquid chromatography/nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. The metabolite was included in routine doping control procedures based on liquid chromatography/tandem mass spectrometry and has served as a reference compound for 5000 doping control specimens.

The androgen receptor is a ligand inducible transcription factor that is involved in a broad range of physiological functions. Here we describe the discovery of a new class of orally available selective androgen receptor modulators. The lead compound, 6-[(2R,5R)-2-methyl-5-((R)-2,2,2-trifluoro-1-hydroxyethyl)pyrrolidin-1-yl]-4-trifluoromethylquinolin-2(1H)-one (6a), showed excellent anabolic activity in muscle with reduced effect on the prostate in a rat model of hypogonadism. The compound also improved bone strength in a rat model of post-menopausal osteoporosis.

A novel series of pyrazolines 2 have been designed, synthesized, and evaluated by in vivo screening as tissue-selective androgen receptor modulators (SARMs). Structure-activity relationships (SAR) were investigated at the R1 to R6 positions as well as the core pyrazoline ring and the anilide linker. Overall, strong electron-withdrawing groups at the R1 and R2 positions and a small group at the R5 and R6 position are optimal for AR agonist activity. The (S)-isomer of 7c exhibits more potent AR agonist activity than the corresponding (R)-isomer. (S)-7c exhibited an overall partial androgenic effect but full anabolic effect via oral administration in castrated rats. It demonstrated a noticeable antiandrogenic effect on prostate in intact rats with endogenous testosterone. Thus, (S)-7c is a tissue-selective nonsteroidal androgen receptor modulator with agonist activity on muscle and mixed agonist and antagonist activity on prostate.

A novel series of imidazolin-2-ones were designed and synthesized as highly potent, orally active and muscle selective androgen receptor modulators (SARMs), with most of the compounds exhibiting low nM in vitro potency in androgen receptor (AR) binding and functional assays. Once daily oral treatment with the lead compound 11a (AR Ki = 0.9 nM, EC50 = 1.8 nM) for 14 days induced muscle growth with an ED50 of 0.09 mg/kg, providing approximately 50-fold selectivity over prostate growth in an orchidectomized rat model. Pharmacokinetic studies in rats demonstrated that the lead compound 11a had oral bioavailability of 65% and a plasma half-life of 5.5 h. On the basis of their preclinical profiles, the SARMs in this series are expected to provide beneficial anabolic effects on muscle with minimal androgenic effects on prostate tissue.

Recent interest in orally available androgens has fueled the search for new androgens for use in hormone replacement therapy and as anabolic agents. In pursuit of this, we have discovered a series of novel androgen receptor modulators derived from 7H-[1,4]oxazino[3,2-g]quinolin-7-ones. These compounds were synthesized and evaluated in competitive binding assays and an androgen receptor transcriptional activation assay. A number of compounds from the series demonstrated single-digit nanomolar agonist activity in vitro. In addition, lead compound (R)-16e was orally active in established rodent models that measure androgenic and anabolic properties of these agents. In this assay, (R)-16e demonstrated full efficacy in muscle and only partially stimulated the prostate at 100 mg/kg. These data suggest that these compounds may be utilized as selective androgen receptor modulators or SARMs. This series represents a novel class of compounds for use in androgen replacement therapy.

A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed.

Selective androgen receptor modulators (SARMs) represent a class of emerging drugs with high potential for misuse in sports, and therefore members of this group are banned as anabolic agents by the World Anti-Doping Agency. Preventive approaches to restrict their use include early implementation of target analytes into doping control screening assays and evaluation of the mass spectrometric behavior of these drugs to allow their unequivocal identification as well as the characterization of structurally related compounds and metabolic products. Four model SARMs with the 6-alkylamino-2-quinolinone structure, including the advanced drug candidate LGD-2226, were synthesized. Fragmentation pathways after positive electrospray ionization and collision-induced dissociation were studied using an LTQ Orbitrap mass analyzer, and diagnostic product ions and common dissociation pathways were employed to establish a screening procedure targeting intact quinolinone-based SARMs as well as putative metabolic products such as dealkylated analogues. Therefore, features of a triple quadrupole mass analyzer such as multiple reaction monitoring and precursor ion scanning were utilized. Sample preparation based on commonly employed liquid-liquid extraction and subsequent liquid chromatographic/tandem mass spectrometric measurement allowed for detection limits of 0.01-0.2 ng/mL, and intra- and interday precisions between 3.2 and 8.5% and between 6.3 and 16.6%, respectively. Recoveries varied from 81 to 98%, and tests for ion suppression or enhancement effects were negative for all analytes., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Anabolic agents have been among the most frequently detected drugs in amateur and professional sport. A novel class of therapeutics presumably complementing anabolic steroids in the near future includes so-called selective androgen receptor modulators (SARMs) that have been under clinical investigations for several years. Although not yet commercially available, their potential for misuse in sports is high. Four aryl-propionamide-derived SARMs were synthesized in order to establish a fast and robust screening procedure using liquid chromatography/electrospray ionization tandem mass spectrometry. Synthesized compounds were characterized by high-resolution/high-accuracy mass analysis employing a linear ion trap-Orbitrap hybrid mass spectrometer while routine analyses were conducted on a triple-quadrupole mass spectrometer. Characteristic product ions obtained by collision-induced dissociation were found at m/z 289 and 261 as well as m/z 269 and 241 representing the bisubstituted aniline residues of selected model compounds. Assay validation was performed regarding lower limit of detection (1 ng/mL), recovery (85-105%), intraday precision (7.6-11.6%) and interday precision (9.9-14.4%), and precursor ion scan experiments on diagnostic product ions enabled the detection of a structurally related compound at 50 ng/mL., (Copyright 2006 John Wiley & Sons, Ltd.)

The use of steroids and other pharmaceuticals to gain a competitive edge in athletics has been present in the sports world for a long time. Over the past several years, scientific advances in the detection of sports doping agents and improved collaboration between sports organizations have enhanced the monitoring of fair athletic play. Many have suspected the illegal development of designer steroids by rogue scientists to avoid detection by the standard sports doping drug screen. In 2003, the Olympic Analytical Laboratory at the University of California, Los Angeles discovered the first designer steroid, tetrahydrogestrinone (THG), by using liquid chromatography with tandem mass spectrometry. Over the past year, the THG story continues to shock the sports world with its potential to discredit or terminate several high-profile athletic careers. While confirming the existence of designer steroids is credit to the sports antidoping movement, antidoping agencies will need to continue to invest in research and depend on honest athletic participants to maintain fairness and safety in sports.

A series of nonsteroidal ligands were synthesized as second-generation agonists for the androgen receptor (AR). These ligands were designed to eliminate metabolic sites identified in one of our first-generation AR agonists, which was inactive in vivo due to its rapid metabolism to inactive constituents. The binding affinity of these compounds was evaluated using AR isolated from rat ventral prostate. These second-generation compounds bound the AR in a high affinity and stereoselective manner, with K(i) values ranging from about 4 to 130 nM. The ability of these ligands to stimulate AR-mediated transcriptional activation was examined in cells transfected with the human AR and a hormone-dependent luciferase reporter gene. Although some compounds were unable to stimulate AR-mediated transcription, several demonstrated activity similar to that of dihydrotestosterone (DHT, an endogenous steroidal ligand for the AR). We also evaluated the in vivo pharmacologic activity of selected compounds in castrated male rats. Three compounds were identified as selective androgen receptor modulators (SARMs), exhibiting significant anabolic activity while having only moderate to minimal androgenic activity in vivo.

The syntheses of the 7beta-hydroxy metabolite of ORG OD14 (Livial), (3alpha,7beta, 17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-t riol (35), and its 7-epimer, (3alpha,7alpha, 17alpha)-7-methyl-19-norpregn-5(10)-en-20-yne-3,7,17-t riol (11), are described.

Hydroxylation at position 6 beta testosterone I (17 beta-hydroxyandrost-4-en-3-one) and the anabolic steroids 17 alpha-methyltestosterone II (17 beta-hydroxy-17 alpha-methylandrost-4-en-3-one), metandienone III (17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one), 4-chloro-1,2-dehydro-17 alpha-methyltestosterone IV (4-chloro-17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one), and fluoxymesterone V (9-fluoro-11 beta, 17 beta-dihydroxy-17 alpha-methylandrost-4-en-3-one) was achieved via light-induced autooxidation of the corresponding trimethysilyl 3,5-dienol ethers dissolved in isopropanol or ethanol. The reaction further yielded the 6 alpha-hydroxy isomer in low amounts. The 6 beta-hydroxy isomer of I-V and the 6 alpha-hydroxy isomers of I, III, and IV were isolated and characterized by 1H and 13C NMR, high-performance liquid chromatography, gas chromatography, and mass spectrometry. Human excretion studies with single administered doses of boldenone (17 beta-hydroxyandrosta-1,4-dien-3-one), 4-chloro-1,2-dehydro-17 alpha-methyltestosterone, fluoxymesterone, metandienone, 17 alpha-methyltestosterone, and [16,16,17-2H3] testosterone showed that 6 beta-hydroxylation is the major metabolic pathway in the metabolism of 4-chloro-1,2-dehydro-17 alpha-methyltestosterone, fluoxymesterone, and metandienone, whereas for boldenone, 17 alpha-methyltestosterone, and testosterone, 6 beta-hydroxylation is negligible.

The 17-epimers of the anabolic steroids bolasterone (I), 4-chlorodehydromethyltestosterone (II), fluoxymesterone (III), furazabol (IV), metandienone (V), mestanolone (VI), methyltestosterone (VII), methandriol (VIII), oxandrolone (IX), oxymesterone (X), oxymetholone (XI), stanozolol (XII), and the human metabolites 7 alpha,17 alpha-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (XIII) (metabolite of I), 6 beta-hydroxymetandienone (XIV) (metabolite of V), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (XV) (metabolite of V), 3'-hydroxystanozolol (XVI) (metabolite of XII), as well as the reference substances 17 beta-hydroxy-17 alpha-methyl-5 beta-androstan-3-one (XVII), 17 beta-hydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (XVIII) (also a metabolite of V), the four isomers 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (XIX) (also a metabolite of VI, VII, and XI), 17 alpha-methyl-5 alpha-androstane-3 beta,17 beta-diol (XX), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (XXI) (also a metabolite of V, VII, and VIII), 17 alpha-methyl-5 beta-androstane-3 beta,17 beta-diol (XXII), and 17 beta-hydroxy-7 alpha,17 alpha-dimethyl-5 beta-androstan-3-one (XXIII) were synthesized via a 17 beta-sulfate that spontaneously hydrolyzed in water to several dehydration products, and to the 17 alpha-hydroxy-17 beta-methyl epimer. The 17 beta-sulfate was prepared by reaction of the 17 beta-hydroxy-17 alpha-methyl steroid with sulfur trioxide pyridine complex. The 17 beta-methyl epimers are eluted in gas chromatography as trimethylsilyl derivatives from a capillary SE-54 or OV-1 column 70-170 methylen units before the corresponding 17 alpha-methyl epimer. The electron impact mass spectra of the underivatized and trimethylsilylated epimers are in most cases identical and only for I, II, and V was a differentiation between the 17-epimers possible. 1H nuclear magnetic resonance (NMR) spectra show for the 17 beta-methyl epimer a chemical shift for the C-18 protons (singlet) of about 0.175 ppm (in deuterochloroform) to a lower field. 13C NMR spectra display differences for the 17-epimeric steroids in shielding effects for carbons 12-18 and 20. Excretion studies with I-XII with identification and quantification of 17-epimeric metabolites indicate that the extent of 17-epimerization depends on the A-ring structure and shows a great variation for the different 17 alpha-methyl anabolic steroids.

The synthesis of 11-oxatestosterone from 11-oxa-5 alpha-androstane-3,17-dione, which is available from hecogenin, is described. The product shows, in comparison with the natural hormone, diminished androgenic and anabolic activities.

A new group of steroidal alkylating agents has been synthesized for potential anticancer activity. The compounds contain a sulfonic ester, a mono- or a bifunctional nitrogen mustard function attached to an ethyl chain and forming ether linkages at the 3- or 17 beta-position of the steroids selected. The products were tested for their in vitro anabolic and catabolic properties by measuring their effects on the bovine pancreatic ribonuclease.