Efficient synthesis of an androgen receptor modulator

An efficient synthesis of the androgen receptor modulator (R)-4a having an 8H-[1,4]oxazino[2,3-f]quinolin-8-one skeleton is described. Synthesis of this ring system, not readily accessible by classical Knorr cyclization methodology, was accomplished by an ortho-metallation strategy. Thus, lithiation of a pivaloyl-protected 7-amino-3,4-dihydro-1,4-benzoxazine using n-butyllithium allowed the introduction of a trifluoroacetyl group regioselectively at the 8-position. Subsequent Wittig reaction and acid catalyzed cyclization afforded the desired 8H-[1,4]oxazino[2,3-f]quinolin-8-one (R)-4a in very good overall yield from the corresponding benzoxazine.     

The application of high-resolution mass spectrometry-based data-mining tools in tandem to metabolite profiling of a triple drug combination in humans

Patients are usually exposed to multiple drugs, and metabolite profiling of each drug in complex biological matrices is a big challenge. This study presented a new application of an improved high resolution mass spectrometry (HRMS)-based data-mining tools in tandem to fast and comprehensive metabolite identification of combination drugs in human. The model drug combination was metronidazole-pantoprazole-clarithromycin (MET-PAN-CLAR), which is widely used in clinic to treat ulcers caused by Helicobacter pylori. First, mass defect filter (MDF), as a targeted data processing tool, was able to recover all relevant metabolites of MET-PAN-CLAR in human plasma and urine from the full-scan MS dataset when appropriate MDF templates for each drug were defined. Second, the accurate mass-based background subtraction (BS), as an untargeted data-mining tool, worked effectively except for several trace metabolites, which were buried in the remaining background signals. Third, an integrated strategy, i.e., untargeted BS followed by improved MDF, was effective for metabolite identification of MET-PAN-CLAR. Most metabolites except for trace ones were found in the first step of BS-processed datasets, and the results led to the setup of appropriate metabolite MDF template for the subsequent MDF data processing. Trace metabolites were further recovered by MDF, which used both common MDF templates and the novel metabolite-based MDF templates. As a result, a total of 44 metabolites or related components were found for MET-PAN-CLAR in human plasma and urine using the integrated strategy. New metabolic pathways such as N-glucuronidation of PAN and dehydrogenation of CLAR were found. This study demonstrated that the combination of accurate mass-based multiple data-mining techniques in tandem, i.e., untargeted background subtraction followed by targeted mass defect filtering, can be a valuable tool for rapid metabolite profiling of combination drugs in vivo.     

Synthesis, characterisation, and mass spectrometric detection of a pegylated EPO-mimetic peptide for sports drug testing purposes

Erythropoietin (EPO) and other erythropoiesis‐stimulating agents possess a high misuse potential in elite sport due to their ability to increase the oxygen transport capacity, which plays a vital role in enhancing endurance performance. Currently, a new generation of EPO‐mimetic peptides is under development from which peginesatide (also referred to as Hematide?), a pegylated homodimeric peptide of approximately 45 kDa with no structural relationship to erythropoietin, is the most advanced drug candidate undergoing phase‐III clinical trials. Since preventive doping research aims at the development of detection methods before a drug receives clinical approval, a selective and sensitive assay has to be established knowing that conventional doping control assays for EPO will not succeed in detecting peginesatide. Thus, a pegylated EPO‐mimetic peptide simulating the structure and properties of the lead drug candidate peginesatide was synthesised as a model compound for this new class of emerging drugs and characterised by means of gel electrophoresis, matrix‐assisted laser desorption/ionisation (MALDI) mass spectrometry, as well as liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI‐MS/MS) after proteolytic digestion. Based on these results, a mass spectrometric detection method of the product in plasma was developed targeting a pentapeptide fragment after protein precipitation and subtilisin digestion. Its fitness for purpose was evaluated by the determination of selected method characteristics focusing particularly on specificity, recovery (ca. 60%), and limit of detection (1 ng/mL). Copyright © 2011 John Wiley & Sons, Ltd.     

Design and synthesis of an array of selective androgen receptor modulators

We describe the design, using shape comparison and fast docking computer algorithms, and rapid parallel synthesis of a 1300 member array based on GSK7721, a 4-aminobenzonitrile androgen receptor (AR) antagonist identified by focused screening of the GSK compound collection. The array yielded 352 submicromolar and 17 subnanomolar AR agonists as measured by a cell-based reporter gene functional assay. The rapid synthesis of a large number of active compounds provided valuable information in the optimization of AR modulators, which may be useful in treating androgen deficiency in aging males.     

Development and validation of a mass spectrometric detection method of peginesatide in dried blood spots for sports drug testing

As recently reported, dried blood spot (DBS) analysis is an advantageous technique for doping control purposes due to the minimal invasive sample collection, the simple and economic manner, as well as the low susceptibility to manipulation. Its general applicability to the sports drug testing arena has been shown for analytes of various substance classes, all of which comprise exclusively low molecular mass compounds. The aim of the present study was to investigate whether the technique of DBS analysis is applicable also to (pegylated) peptides with relevance for doping controls. As target analyte, peginesatide (Omontys, Hematide), a recently approved pegylated erythropoietin-mimetic peptide of approximately 45 kDa, tested for the treatment of anaemia in patients with renal failure, was chosen, which has been prohibited in elite sports due to its assumed endurance enhancing effects. Therefore, a detection method for peginesatide employing DBS was developed based on extraction, proteolytic digestion and cation-exchange purification followed by liquid chromatography-tandem mass spectrometry analysis. Eventually, the assay was validated for qualitative purposes and proved to be specific, sensitive (limit of detection, 10 ng/mL) and precise (relative standard deviations below 18%), demonstrating the general suitability of DBS analysis in sports drug testing also for (pegylated) peptides.     

Recent developments in the use of isotope ratio mass spectrometry in sports drug testing

According to the annual report of the World Anti-Doping Agency, steroids are the most frequently detected class of doping agents. Detecting the misuse of endogenously occurring steroids, i.e. steroids such as testosterone that are produced naturally by humans, is one of the most challenging issues in doping control analysis. The established thresholds for urinary concentrations or concentration ratios such as the testosterone/epitestosterone quotient are sometimes inconclusive owing to the large biological variation in these parameters.     

Mass spectrometric characterization of urinary metabolites of the selective androgen receptor modulator S-22 to identify potential targets for routine doping controls

Drugs that promote anabolic processes with limited undesirable effects are of considerable therapeutic interest; some notable examples include those for the treatment of cancer cachexia and muscle-wasting diseases. Anabolic properties are not only therapeutically beneficial to critically ill and debilitated patients, but are also desirable to athletes seeking artificial enhancements in endurance, strength and accelerated recovery. The use of anabolic agents in the clinical setting is being reconsidered with the emergence of a new class of drugs referred to as SARMs (selective androgen receptor modulators). SARMs have the potential to complement or even replace anabolic androgenic steroidal use with the benefit of a reduction of the undesirable side effects associated with steroid administration alone. Arylpropionamide-based SARMs such as andarine (S-4) and S-22 have shown promising therapeutic properties and have attracted the interest of elite and amateur athletes despite the absence of clinical approval, and evidence for trafficking and misuse in sport has been obtained by doping control authorities. In this communication, the elucidation of urinary metabolites of the SARM drug candidate S-22 is compared with earlier in vitro metabolism studies. Following oral administration of illicit S-22, urine samples were collected after 62 and 135 h and analyzed for the active drug and its major metabolic products. Liquid chromatography interfaced with high-resolution/high-accuracy (tandem) mass spectrometry was used to identify and/or confirm the predicted target analytes for sports drug testing purposes. S-22 was detected in both specimens accompanied by its glucuronic acid conjugate. This was the B-ring hydroxylated derivative of S-22 plus the corresponding glucuronide (with the phase-II metabolites being the more abundant analytes). In addition, the samples collected 62 h post-administration also contained the phase-I metabolite hydroxylated at the methyl residue (C-20) and the B-ring depleted degradation product ('dephenylated' S-22) together with the corresponding carboxy analog that was previously reported for canine metabolism. The obtained data supports future efforts to effectively screen for and confirm the misuse of the non-approved S-22 drug candidate in doping controls.     

Use of a five-channel multiplexed electrospray quadrupole time-of-flight hybrid mass spectrometer for metabolite identification

Metabolism data provided with reduced cycle time has become of increasing importance for the early evaluation of DMPK properties of drugs in discovery. In this regard, quadrupole time-of-flight hybrid mass spectrometers (Q-TOF) can provide very reliable metabolite identification via accurate mass measurement of ions and the consequent access to the elemental composition of the metabolite. However, due to their cost, they are often used for drug metabolism studies on later stage drug candidates or to address challenging metabolism questions. A new prototype, consisting of a five-channel multiplexed electrospray ionization (ESI) source on a Q-TOF with one channel used for lock-mass compound infusion, was evaluated for metabolite identification. The goal was to increase the sample throughput of a single ESI-MS system by a factor of 4, while maintaining efficient metabolite separation in high-performance liquid chromatography (HPLC) as well as adequate sensitivity and mass accuracy, and ultimately improve the speed and quality of metabolism studies supporting drug discovery. The analytical performance of the system was assessed by evaluating the sensitivity and mass accuracy (using real in vitro and in vivo samples), inter-channel differences in retention times, MS/UV response, and cross-talk among channels. The sensitivity using the multiplexed ESI source was on average 2-fold lower than with single ESI, correlating well with previous literature data. The mass accuracy was comparable to that obtained using single ESI in both MS and MS/MS modes, making the metabolite identification process using the multiplexed ESI source as reliable as with single ESI. Compound-dependent differences in ionization efficiencies were observed among channels, and were minimized by analyzing related samples on the same channel. Finally, the level of cross-talk among channels was acceptable (around 0.3%) and comparable to levels previously published for quantitative applications using multiplexed ESI. The paper also focuses on the advantages and disadvantages of this new approach compared to other approaches in the literature in the field of metabolite identification.     

Mass spectrometric characterization of urinary metabolites of the selective androgen receptor modulator andarine (S‐4) for routine doping control purposes

Selective androgen receptor modulators (SARMs) are potent anabolic agents with tissue‐selective properties. Due to their potential misuse in elite sport, the World Anti‐Doping Agency (WADA) has prohibited SARMs since 2008, and although no representative drug candidate has yet received full clinical approval, recent findings of SARMs illegally sold via the internet have further supported the need to efficiently test for these compounds in doping controls. In the present communication, the mass spectrometric characterization of urinary metabolites of the SARM Andarine (also referred to as S‐4) compared with earlier in vitro and animal studies is reported. Liquid chromatography interfaced to high‐resolution/high‐accuracy (tandem) mass spectrometry was used to identify phase I and II metabolites, confirming the predicted target analytes for sports drug testing purposes including the glucuronic acid conjugates of the active drug, its monohydroxylated and/or deacetylated product, the hydrolysis product resulting from the removal of the compound's B‐ring, as well as the sulfate of the monohydroxylated and the deacetylated phase I metabolite. The obtained data will support future efforts to effectively screen for and confirm the misuse of the non‐approved drug candidate Andarine. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of endogenous steroids in urine by means of multi-immunoaffinity chromatography and isotope ratio mass spectrometry for sports drug testing

Analytical and Bioanalytical Chemistry - Detecting the administration of naturally occurring but synthetically derived steroids (e.g., testosterone) in routine doping controls is particularly...     

Use of an electrochemically labeled nucleotide terminator for known point mutation analysis

A novel single nucleotide polymorphism (SNP) assay utilizing an electrochemically tagged chain terminator is described. The system employs the single-base extension (SBE) technique coupled to capillary gel electrophoresis with end-column electrochemical detection. A redox-labeled chain terminator, ferrocene-acycloATP, is used in the SBE reaction. When the mutation site corresponds to the labeled chain terminator, the extension product is rendered electroactive. The reaction mixture is subsequently separated by capillary gel electrophoresis and the extension product detected at the separation anode with sinusoidal voltammetry. This work demonstrates the first known SNP assay utilizing redox-active chain terminators coupled to electrochemical detection. The methodology presented could lead to a fast, simple, and cost-effective SNP scoring system.     

A highly selective rearrangement of a housane-derived cation radical: an electrochemically mediated transformation

To utilize housane-derived cation radicals as intermediates for the synthesis of the bicyclo (n.3.0) framework of natural products, a highly regioselective [1,2] shift of carbon to either a radical or an electron-deficient site is required. Herein we describe how this has been accomplished, provide a set of guidelines to assess housane oxidizability prior to its synthesis, and describe a synthesis of housane 18 that capitalizes upon the facility of [1,5] hydrogen shifts in substituted cyclopentadienes. The catalytic electrochemically mediated oxidation of 18 leads to a cation radical that engages in a rearrangement leading to the (4.3.0) adduct 23. The appearance of a catalytic current in the cyclic voltammogram of a solution containing the tris(aryl)amine and housane 18 is an excellent indicator that the amminium cation radical 14*+ is able to oxidize the housane and return the mediator to the original redox couple. DFT calculations show electron density on both the aryl and strained sigma framework in the HOMO of housane 18. From the spin density and electrostatic potential map for the cation radical, a picture where the spin resides on the side that is distal to the substituent emerges, while the hole is proximal to it. Both experiment and theory show that the rearrangement is best characterized as a [1,2] carbon shift toward an electron-deficient site and that migration toward the substituent-bearing carbon is much preferred over the alternative pathway.     

Enzyme-catalyzed asymmetric deacylation for the preparation of lasofoxifene (CP-336156), a selective estrogen receptor modulator

[structure] selective estrogen receptor modulator (SERM), Lasofoxifene (CP-336156), was prepared by an enzyme-catalyzed asymmetric deacylation with high optical purity and excellent yield even though the hydrolytic site is remote from the chiral centers.     

Doping control analysis of tricyclic tetrahydroquinoline-derived selective androgen receptor modulators using liquid chromatography/electrospray ionization tandem mass spectrometry

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.     

Recommended criteria for the mass spectrometric identification of target peptides and proteins (<8 kDa) in sports drug testing

Mass spectrometry has become an invaluable tool for the identification of prohibited peptide hormones and proteins in doping control analysis. Regulatory authorities have established criteria for identifying banned drugs in doping control specimens, but these criteria do not address the specific issues for high molecular weight protein drugs such as molecular weight determination of multiply charged molecules, analysis of chemically or enzymatically derived degradation products, identification of amino acid sequence tags, etc. Technical considerations such as sample preparation methods (e.g. immunoaffinity purification), resulting analytes (e.g. intact compounds vs. chemically or enzymatically derived peptides), ionization modes, analyzer resolution, and the information provided by respective techniques are discussed in light of sports drug testing requirements using typical application examples.