Identification and Synthesis of Selected In Vitro Generated Metabolites of the Novel Selective Androgen Receptor Modulator (SARM) 2f

Among anabolic agents, selective androgen receptor modulators (SARMs) represent a new class of potential drugs that can exhibit anabolic effects on muscle and bone with reduced side effects due to a tissue-selective mode of action. Besides possible medical applications, SARMs are used as performance-enhancing agents in sports. Therefore, they are prohibited by the World Anti-Doping Agency (WADA) in and out of competition. Since their inclusion into the WADA Prohibited List in 2008, there has been an increase in not only the number of adverse analytical findings, but also the total number of SARMs, making continuous research into SARMs an ongoing topic in the field of doping controls. 4-((2R,3R)-2-Ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile (SARM 2f) is a novel SARM candidate and is therefore of particular interest for sports drug testing. This study describes the synthesis of SARM 2f using a multi-step approach, followed by full characterization using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). To provide the first insights into its biotransformation in humans, SARM 2f was metabolized using human liver microsomes and the microsomal S9 fraction. A total of seven metabolites, including phase I and phase II metabolites, were found, of which three metabolites were chemically synthesized in order to confirm their structure. Those can be employed in testing procedures for routine doping controls, further improving anti-doping efforts.


Introduction
In recent years, selective androgen receptor modulators (SARMs) have received an increasing interest in drug development as well as in doping controls. The specific binding of SARMs to the androgen receptor (AR) leads to a conformational change in the ligand binding domain, resulting in an activation of the receptor and, therefore, exhibits comparable effects on muscle tissue as endogenous androgenic anabolic steroids such as testosterone and dihydrotestosterone [1,2]. The administration of steroidal therapeutics can result in undesirable effects such as cardiovascular diseases or prostate cancer and therefore poses a health risk to patients [3][4][5]. In contrast to steroidal AR agonists, SARMs show a tissue-selective mechanism of action and thus exhibit reduced side effects. To date, numerous SARMs are under research for various diseases such as osteoporosis, cachexia and muscular dystrophy [6][7][8][9].
Due to their anabolic effects on muscle and bone, SARMs are further used as performanceenhancing substances in sports. Therefore, the World Anti-Doping Agency (WADA) has prohibited the use of SARMs in and out of competition explicitly since 2008 [10], and since their inclusion into the Prohibited List, the number of adverse analytical findings (AAFs) concerning SARMs has risen. In addition, the structural diversity and the total number of (WADA) has prohibited the use of SARMs in and out of competition explicitly since 2008 [10], and since their inclusion into the Prohibited List, the number of adverse analytical findings (AAFs) concerning SARMs has risen. In addition, the structural diversity and the total number of SARMs are also increasing, which calls for the continued implementation of new substances and respective characteristic metabolites into doping control testing procedures [2,11,12].
SARM 2f (4-((2R,3R)-2-ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl) benzonitrile) (Figure 1) represents another potential candidate as a drug of abuse in sports. First developed for clinical applications in 2017, SARM 2f acts as an agonist at the AR and shows tissue selectivity in Hershberger assays [13]. In vivo studies using rodents or cynomolgus monkey models confirmed the occurrence of anabolic effects by showing an increase in lean body mass, as well as suppression of blood lipid levels compared to testosterone [14][15][16]. However, no information on the metabolism of SARM 2f is known to date. To tackle the limited availability as a reference material, SARM 2f was synthesized using a multi-step approach. Furthermore, a suitable internal standard was synthesized using an analogous method. SARM 2f produced in this study was used for in vitro experiments to gain first insights into its metabolism. Metabolites found in this study were identified using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). The selected metabolites found in this study were synthesized and their structure confirmed via nuclear magnetic resonance spectroscopy (NMR) in order to provide critical information for the implementation of SARM 2f and its metabolites into routine doping control test methods.

Synthesis of SARM 2f
Due to its limited availability, SARM 2f was synthesized in-house, using the method shown in Scheme 1. The method partially described by Bisol et al. involves six reaction steps starting from commercially available 3-trans-hexenoic acid 1 [17]. After methylation to obtain 2a, meta-chloroperbenzoic acid (mCPBA) was used to produce the corresponding epoxide 3a [18]. Epoxide opening was realized using LiBr and Mg(ClO4)2 in acetonitrile (ACN) to produce the anti-configured bromohydrin 4a [18]. In order to invert the C4 stereocenter, NaN3 was used to create the corresponding syn conformer 5a. In the next step, γ-azido-β-hydroxy ester 5a underwent reductive cyclization to produce the desired γ-hydroxy lactam 6a as a white solid [17]. Finally, SARM 2f was synthesized using Buchwald-Hartwig amination with a catalyst system of Bis-(dibenzylidenacetone)-palladium(0) (Pd(dba)2) and xantphos [19]. These reaction steps resulted in the formation of unidentified by-products; therefore, it was necessary to investigate a suitable workup method to obtain a clean product. Final purification was obtained using a fractionated reverse-phase (RP) solid-phase extraction (SPE) with a gradient using ACN/Water (H2O). The same reaction methodology was applied to methyl (E)-pent-3-enoate to obtain 4-((2R,3R)-3-hydroxy-2-methyl-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile 7b (Figure 2), which can be used as an internal standard for the analysis of SARM 2f in doping control samples. To tackle the limited availability as a reference material, SARM 2f was synthesized using a multi-step approach. Furthermore, a suitable internal standard was synthesized using an analogous method. SARM 2f produced in this study was used for in vitro experiments to gain first insights into its metabolism. Metabolites found in this study were identified using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). The selected metabolites found in this study were synthesized and their structure confirmed via nuclear magnetic resonance spectroscopy (NMR) in order to provide critical information for the implementation of SARM 2f and its metabolites into routine doping control test methods.

Synthesis of SARM 2f
Due to its limited availability, SARM 2f was synthesized in-house, using the method shown in Scheme 1. The method partially described by Bisol et al. involves six reaction steps starting from commercially available 3-trans-hexenoic acid 1 [17]. After methylation to obtain 2a, meta-chloroperbenzoic acid (mCPBA) was used to produce the corresponding epoxide 3a [18]. Epoxide opening was realized using LiBr and Mg(ClO 4 ) 2 in acetonitrile (ACN) to produce the anti-configured bromohydrin 4a [18]. In order to invert the C4 stereocenter, NaN 3 was used to create the corresponding syn conformer 5a. In the next step, γ-azidoβ-hydroxy ester 5a underwent reductive cyclization to produce the desired γ-hydroxy lactam 6a as a white solid [17]. Finally, SARM 2f was synthesized using Buchwald-Hartwig amination with a catalyst system of Bis-(dibenzylidenacetone)-palladium(0) (Pd(dba) 2 ) and xantphos [19]. These reaction steps resulted in the formation of unidentified byproducts; therefore, it was necessary to investigate a suitable workup method to obtain a clean product. Final purification was obtained using a fractionated reverse-phase (RP) solid-phase extraction (SPE) with a gradient using ACN/Water (H 2 O). The same reaction methodology was applied to methyl (E)-pent-3-enoate to obtain 4-((2R,3R)-3-hydroxy-2methyl-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile 7b (Figure 2), which can be used as an internal standard for the analysis of SARM 2f in doping control samples.
In order to produce an authentic reference substance and to confirm the synthetic methodology described above, all reaction steps are characterized by means of 1 H, 13 C APT and HSQC-NMR spectroscopy. In the following, the mass spectrometric characterization of SARM 2f is described in detail. Full scan acquisition of SARM 2f (C 14 H 12 F 3 N 2 O 2 − m/z 297.0858 (0.7 ppm)) in negative ionization mode showed the appearance of formate adducts at m/z 343.0917 (1.7 ppm) as well as in-source dehydration at m/z 279.0758 (2.5 ppm). In positive ionization mode, neither adduct ions nor reaction products were found.  In order to produce an authentic reference substance and to confirm the synthetic methodology described above, all reaction steps are characterized by means of 1 H, 13 C APT and HSQC-NMR spectroscopy. In the following, the mass spectrometric characterization of SARM 2f is described in detail. Full scan acquisition of SARM 2f (C14H12F3N2O2 -m/z 297.0858 (0.7 ppm)) in negative ionization mode showed the appearance of formate adducts at m/z 343.0917 (1.7 ppm)) as well as in-source dehydration at m/z 279.0758 (2.5 ppm). In positive ionization mode, neither adduct ions nor reaction products were found. MS 2 data acquired in negative ionization mode revealed the appearance of dehydration to form the base peak of the product ion spectra at m/z 279.0751. This peak, assigned to the deprotonated 4-(2-ethyl-5-hydroxy-1H-pyrrol-1-yl)-2-(trifluoromethyl)benzonitrile, further dissociated to form a peak at m/z 250.0361 by releasing an ethane radical (29 u). This ion was proposed to be the deprotonated 4-(2-hydroxy-1H-pyrrol-1-yl)-2-(trifluoromethyl)benzonitrile radical. Further, pseudo MS 3 experiments of the ion at m/z 279.0751 showed the dissociation to form the signals at m/z 209.0332 and m/z 185.0332. While the product ion at m/z 209.0332 was tentatively identified as the deprotonated ((4-cyano-3-(trifluoromethyl)phenyl)amino)ethyn-1-ide, the ion at m/z 185.0332 was assigned to the deprotonated (4-cyano-3-(trifluoromethyl)phenyl)amide. The signal at m/z 185.0332 is of particular interest for the analysis of various SARMs, since it is commonly found in analogous structures [20]. The deprotonated intact compound at m/z 297.0866 further yielded a product ion at m/z 253.0960, which was tentatively assigned to the deprotonated 1-(4cyano-3-(trifluoromethyl)phenyl)-2-ethylazetidin-2-ide. An overview of the proposed dissociation pathway of SARM 2f is shown in Scheme 2a.
The proposed dissociation pathway in positive ionization mode is shown in Scheme 2b. The parent compound at m/z 299.0597 undergoes dehydration (18 u) to form a peak at m/z 281.0890, which was assigned to the protonated 4-(5-ethyl-2-oxo-2,3-dihydro-1H-pyrrol-1-yl)-2-(trifluoromethyl)benzonitrile. Pseudo MS 3 experiments of the ion at m/z 281.0890 gave rise to further dissociation products at m/z 239.0780 and m/z 213.0267. These  In order to produce an authentic reference substance and to confirm the synthetic methodology described above, all reaction steps are characterized by means of 1 H, 13 C APT and HSQC-NMR spectroscopy. In the following, the mass spectrometric characterization of SARM 2f is described in detail. Full scan acquisition of SARM 2f (C14H12F3N2O2 -m/z 297.0858 (0.7 ppm)) in negative ionization mode showed the appearance of formate adducts at m/z 343.0917 (1.7 ppm)) as well as in-source dehydration at m/z 279.0758 (2.5 ppm). In positive ionization mode, neither adduct ions nor reaction products were found. MS 2 data acquired in negative ionization mode revealed the appearance of dehydration to form the base peak of the product ion spectra at m/z 279.0751. This peak, assigned to the deprotonated 4-(2-ethyl-5-hydroxy-1H-pyrrol-1-yl)-2-(trifluoromethyl)benzonitrile, further dissociated to form a peak at m/z 250.0361 by releasing an ethane radical (29 u). This ion was proposed to be the deprotonated 4-(2-hydroxy-1H-pyrrol-1-yl)-2-(trifluoromethyl)benzonitrile radical. Further, pseudo MS 3 experiments of the ion at m/z 279.0751 showed the dissociation to form the signals at m/z 209.0332 and m/z 185.0332. While the product ion at m/z 209.0332 was tentatively identified as the deprotonated ((4-cyano-3-(trifluoromethyl)phenyl)amino)ethyn-1-ide, the ion at m/z 185.0332 was assigned to the deprotonated (4-cyano-3-(trifluoromethyl)phenyl)amide. The signal at m/z 185.0332 is of particular interest for the analysis of various SARMs, since it is commonly found in analogous structures [20]. The deprotonated intact compound at m/z 297.0866 further yielded a product ion at m/z 253.0960, which was tentatively assigned to the deprotonated 1-(4cyano-3-(trifluoromethyl)phenyl)-2-ethylazetidin-2-ide. An overview of the proposed dissociation pathway of SARM 2f is shown in Scheme 2a.
The proposed dissociation pathway in positive ionization mode is shown in Scheme 2b. The parent compound at m/z 299.0597 undergoes dehydration (18 u) to form a peak at m/z 281.0890, which was assigned to the protonated 4-(5-ethyl-2-oxo-2,3-dihydro-1H-pyrrol-1-yl)-2-(trifluoromethyl)benzonitrile. Pseudo MS 3 experiments of the ion at m/z 281.0890 gave rise to further dissociation products at m/z 239.0780 and m/z 213.0267. These MS 2 data acquired in negative ionization mode revealed the appearance of dehydration to form the base peak of the product ion spectra at m/z 279.0751. This peak, assigned to the deprotonated 4-(2-ethyl-5-hydroxy-1H-pyrrol-1-yl)-2-(trifluoromethyl)benzonitrile, further dissociated to form a peak at m/z 250.0361 by releasing an ethane radical (29 u). This ion was proposed to be the deprotonated 4-(2-hydroxy-1H-pyrrol-1-yl)-2-(trifluoromethyl) benzonitrile radical. Further, pseudo MS 3 experiments of the ion at m/z 279.0751 showed the dissociation to form the signals at m/z 209.0332 and m/z 185.0332. While the product ion at m/z 209.0332 was tentatively identified as the deprotonated ((4-cyano-3-(trifluoromethyl) phenyl)amino)ethyn-1-ide, the ion at m/z 185.0332 was assigned to the deprotonated (4-cyano-3-(trifluoromethyl) phenyl)amide. The signal at m/z 185.0332 is of particular interest for the analysis of various SARMs, since it is commonly found in analogous structures [20]. The deprotonated intact compound at m/z 297.0866 further yielded a product ion at m/z 253.0960, which was tentatively assigned to the deprotonated 1-(4-cyano-3-(trifluoromethyl)phenyl)-2-ethylazetidin-2-ide. An overview of the proposed dissociation pathway of SARM 2f is shown in Scheme 2a.

Scheme 2.
Proposed dissociation pathway of SARM 2f: (a) in negative ionization mode; (b) in positive ionization mode.

In Vitro Generated Metabolites
To investigate its metabolism, SARM 2f was incubated using human liver microsomes (HLM) and S9 fractions. Five metabolic pathways, including oxidation (M1), hydroxylation (M2 a-c), hydration (M3), sulfation (M4), glucuronidation (M5) and further glucuronidation of the metabolites M2 and M3 led to a total of nine metabolites being found in this study. Product ions obtained for each metabolite are shown in Table 1 (spectra and predicted structure can be found in the Supplementary Materials). The extracted ion chromatogram (EIC) for M2 showed the appearance of three distinct peaks with similar MS 2 spectra, indicating the formation of different stereo-or regiomers. The metabolites M2 and M3 were also detected in the sample incubated without the addition of enzyme mixture ('enzyme blank'). That observation was explained by the partial non-enzymatic Scheme 2. Proposed dissociation pathway of SARM 2f: (a) in negative ionization mode; (b) in positive ionization mode.

In Vitro Generated Metabolites
To investigate its metabolism, SARM 2f was incubated using human liver microsomes (HLM) and S9 fractions. Five metabolic pathways, including oxidation (M1), hydroxylation (M2 a-c), hydration (M3), sulfation (M4), glucuronidation (M5) and further glucuronidation of the metabolites M2 and M3 led to a total of nine metabolites being found in this study. Product ions obtained for each metabolite are shown in Table 1 (spectra and predicted structure can be found in the Supplementary Materials). The extracted ion chromatogram (EIC) for M2 showed the appearance of three distinct peaks with similar MS 2 spectra, indicating the formation of different stereo-or regiomers. The metabolites M2 and M3 were also detected in the sample incubated without the addition of enzyme mixture ('enzyme blank'). That observation was explained by the partial non-enzymatic conversion of SARM 2f under the existing conditions. Further, in the case of M2, the peak areas determined for the enzyme blank were decreased compared to the peak areas found in the enzymemixture-containing in vitro metabolism sample. Those findings indicate the existence of a non-enzymatic metabolic pathway contributing to the formation of M2, which is further enhanced by metabolic enzymes. Conversely, for M3, no differences in peak areas were found between these two scenarios.

Synthesis of Selected In Vitro Generated Metabolites
In order to confirm the structure of the metabolites described above, the metabolites M3, M4 and M5 were synthesized using SARM 2f as a starting material (Scheme 3). M3 was prepared using alkaline hydrolysis conditions, as it was predicted to see hydration at the amide group of the parent compound [21]. For purification, RP-SPE was used and a gradient of ACN and H 2 O (10% to 60% ACN) was applied to elute the product. The resulting substance showed coincident chromatographic and mass spectrometric properties with the metabolite produced in vitro (Figure 3a). Further, the predicted structure was confirmed by means of 1 H, 13 C APT and HSQC-NMR spectroscopy. Therefore, M3 was identified as (3R,4R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-3-hydroxyhexanoic acid. Purified M3 was then used for mass spectrometric characterization.

Synthesis of Selected In Vitro Generated Metabolites
In order to confirm the structure of the metabolites described above, the metabolites M3, M4 and M5 were synthesized using SARM 2f as a starting material (Scheme 3). M3 was prepared using alkaline hydrolysis conditions, as it was predicted to see hydration at the amide group of the parent compound [21]. For purification, RP-SPE was used and a gradient of ACN and H2O (10% to 60% ACN) was applied to elute the product. The resulting substance showed coincident chromatographic and mass spectrometric properties with the metabolite produced in vitro (Figure 3a). Further, the predicted structure was confirmed by means of 1 H, 13 C APT and HSQC-NMR spectroscopy. Therefore, M3 was identified as (3R,4R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-3-hydroxyhexanoic acid. Purified M3 was then used for mass spectrometric characterization.

Synthesis of Selected In Vitro Generated Metabolites
In order to confirm the structure of the metabolites described above, the metabolites M3, M4 and M5 were synthesized using SARM 2f as a starting material (Scheme 3). M3 was prepared using alkaline hydrolysis conditions, as it was predicted to see hydration at the amide group of the parent compound [21]. For purification, RP-SPE was used and a gradient of ACN and H2O (10% to 60% ACN) was applied to elute the product. The resulting substance showed coincident chromatographic and mass spectrometric properties with the metabolite produced in vitro (Figure 3a). Further, the predicted structure was confirmed by means of 1 H, 13 C APT and HSQC-NMR spectroscopy. Therefore, M3 was identified as (3R,4R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-3-hydroxyhexanoic acid. Purified M3 was then used for mass spectrometric characterization.    4 ppm)). It is worth mentioning that the resulting product ion corresponds to the mass of deprotonated SARM 2f. However, the product ion spectra differ, which can lead to misinterpretation of the metabolic pathways of SARM 2f. The proposed dissociation pathway of M3 is illustrated in Scheme 4. The base peak was found at m/z 255.0752, which was correlated with the deprotonated N-(4-cyano-3-(trifluoromethyl)phenyl)-Npropyl formamide. This ion was proposed to undergo a homolytic cleavage to release an ethyl radical (29 u) to form a peak at m/z 226.0360, which was suggested to be the deprotonated N-(4-cyano-3-(trifluoromethyl)phenyl)-N-methyl formamide. Further, pseudo MS 3 experiments indicated that this product ion dissociated into the ions at m/z 185.0334 and m/z 170.0223. These ions were assigned to the deprotonated 4-cyano-3-(trifluoromethyl)phenyl)amide and 4-cyano-3-(trifluoromethyl)benzen-1-ide, respectively. In addition, the parent compound underwent decarboxylation (44 u) and dehydration (18 u) to form the signals at m/z 271.1067 and m/z 297.0858, respectively. While the product ion at m/z 271.1067 was tentatively identified as the deprotonated 4-((2-hydroxypentan-3yl)amino)-2-(trifluoromethyl)benzonitrile, the peak at m/z 297.0858 was assigned to the deprotonated 4-((4-cyano-3-(trifluoromethyl)phenyl)amino)hex-3-enoic acid. The dehydrated parent ion at m/z 297.0858 further dissociated to form a product ion at m/z 253.0959, which was assigned to the deprotonated 4-(pent-2-en-3-ylamino)-2-(trifluoromethyl)benzonitrile.
ionization mode showed the appearance of in-source dehydration (C14H12O2N2F3m/z 297.0866 (3.4 ppm)). It is worth mentioning that the resulting product ion corresponds to the mass of deprotonated SARM 2f. However, the product ion spectra differ, which can lead to misinterpretation of the metabolic pathways of SARM 2f. The proposed dissociation pathway of M3 is illustrated in Scheme 4. The base peak was found at m/z 255.0752, which was correlated with the deprotonated N-(4-cyano-3-(trifluoromethyl)phenyl)-Npropyl formamide. This ion was proposed to undergo a homolytic cleavage to release an ethyl radical (29 u) to form a peak at m/z 226.0360, which was suggested to be the deprotonated N-(4-cyano-3-(trifluoromethyl)phenyl)-N-methyl formamide. Further, pseudo MS 3 experiments indicated that this product ion dissociated into the ions at m/z 185.0334 and m/z 170.0223. These ions were assigned to the deprotonated 4-cyano-3-(trifluoromethyl)phenyl)amide and 4-cyano-3-(trifluoromethyl)benzen-1-ide, respectively. In addition, the parent compound underwent decarboxylation (44 u) and dehydration (18 u) to form the signals at m/z 271.1067 and m/z 297.0858, respectively. While the product ion at m/z 271.1067 was tentatively identified as the deprotonated 4-((2-hydroxypentan-3yl)amino)-2-(trifluoromethyl)benzonitrile, the peak at m/z 297.0858 was assigned to the deprotonated 4-((4-cyano-3-(trifluoromethyl)phenyl)amino)hex-3-enoic acid. The dehydrated parent ion at m/z 297.0858 further dissociated to form a product ion at m/z 253.0959, which was assigned to the deprotonated 4-(pent-2-en-3-ylamino)-2-(trifluoromethyl)benzonitrile. The sulfated metabolite M4 was synthesized in accordance with the literature [22]. SO 3 ·py in DMF and 1,4-dioxane was used as sulfation reagent (Scheme 3b). The purification of the metabolite M4 was facilitated using RP-SPE with a gradient of 0.1% FA in ACN and 0.1% FA in H 2 O. The product obtained from this reaction was compared to the corresponding metabolite found in vitro. Both compounds showed agreement in chromatographic retention time and spectra (Figure 3b). In addition, synthetic M4 was submitted to NMR analysis in order to confirm its structure. Therefore, M4 was identified as (2R,3R)-1-(4-cyano-3-(trifluoromethyl)phenyl)-2-ethyl-5-oxopyrrolidin-3-yl sulfate.
The spectra obtained from synthesized M4 (C 14 H 13 O 5 N 2 F 3 S − m/z 377.0425 (0.0 ppm)) showed the occurrence of one product ion at m/z 96.9601, which was assigned to the hydrogen sulfate ion (Scheme 5). It should be mentioned that this ion is a non-specific product ion. But due to the fact that its appearance was confirmed with the reference material, it may be applicable to doping control analysis.
tion of the metabolite M4 was facilitated using RP-SPE with a gradient of 0.1% FA in ACN and 0.1% FA in H2O. The product obtained from this reaction was compared to the corresponding metabolite found in vitro. Both compounds showed agreement in chromatographic retention time and spectra (Figure 3b). In addition, synthetic M4 was submitted to NMR analysis in order to confirm its structure. Therefore, M4 was identified as (2R,3R)-1-(4-cyano-3-(trifluoromethyl)phenyl)-2-ethyl-5-oxopyrrolidin-3-yl sulfate.
The spectra obtained from synthesized M4 (C14H13O5N2F3S -m/z 377.0425 (0.0 ppm)) showed the occurrence of one product ion at m/z 96.9601, which was assigned to the hydrogen sulfate ion (Scheme 5). It should be mentioned that this ion is a non-specific product ion. But due to the fact that its appearance was confirmed with the reference material, it may be applicable to doping control analysis.

Scheme 5.
Proposed dissociation pathway of the deprotonated metabolite M4.
M5 was synthesized after glucuronidation according to the reaction conditions of Königs-Knorr, which uses AGME to produce the protected glucuronide, which is subsequently deprotected under alkaline conditions (Scheme 3c,d). The hydrolysis described in the literature leads to a cleavage of the glycosidic bond [23]. KCN in MeOH at 0 °C was therefore used for hydrolysis [24]. Even under these conditions, hydrolysis of the conjugate was not entirely excluded, yielding a product where both the desired glucuronide plus the deconjugated SARM 2f was obtained. Hence, identification of the target compound was conducted using only chromatographic and mass spectrometric methods that showed agreement with data obtained from the metabolite found in vitro (Figure 3).
The MS 2 spectra, obtained for M5 (Scheme 6) (C20H20O8N2F3 -= 473.1181 m/z (0.9 ppm)), showed cleavage of the glycosidic bond to form the base peak at m/z 193.0353, which was assigned to the deprotonated glucuronic acid. This ion undergoes sequential dehydration (18 u) to form the peaks at m/z 175.0249 and m/z 157.0143. These ions were presumably identified as the deprotonated 3,4,5-trihydroxy-3,4-dihydro-2H-pyran-2-carboxylate and 3,5-dihydroxy-2H-pyran-2-carboxylate, respectively. Further, the peak at m/z 175.0249 showed decarboxylation (44 u) followed by dehydration (18 u) to form the peaks at m/z 131.0350 and m/z 113.0244. While the peak at m/z 131.0350 was tentatively identified as the deprotonated 3,4,5-trihydroxy-3,4-dihydro-2H-pyran-2-ide, the peak at m/z 113.0244 likely represented 3,5-dihydroxy-2H-pyran-2-ide. M5 was synthesized after glucuronidation according to the reaction conditions of Königs-Knorr, which uses AGME to produce the protected glucuronide, which is subsequently deprotected under alkaline conditions (Scheme 3c,d). The hydrolysis described in the literature leads to a cleavage of the glycosidic bond [23]. KCN in MeOH at 0 • C was therefore used for hydrolysis [24]. Even under these conditions, hydrolysis of the conjugate was not entirely excluded, yielding a product where both the desired glucuronide plus the deconjugated SARM 2f was obtained. Hence, identification of the target compound was conducted using only chromatographic and mass spectrometric methods that showed agreement with data obtained from the metabolite found in vitro (Figure 3).
The MS 2 spectra, obtained for M5 (Scheme 6) (C 20 H 20 O 8 N 2 F 3 − = 473.1181 m/z (0.9 ppm)), showed cleavage of the glycosidic bond to form the base peak at m/z 193.0353, which was assigned to the deprotonated glucuronic acid. This ion undergoes sequential dehydration (18 u) to form the peaks at m/z 175.0249 and m/z 157.0143. These ions were presumably identified as the deprotonated 3,4,5-trihydroxy-3,4-dihydro-2H-pyran-2carboxylate and 3,5-dihydroxy-2H-pyran-2-carboxylate, respectively. Further, the peak at m/z 175.0249 showed decarboxylation (44 u) followed by dehydration (18 u) to form the peaks at m/z 131.0350 and m/z 113.0244. While the peak at m/z 131.0350 was tentatively identified as the deprotonated 3,4,5-trihydroxy-3,4-dihydro-2H-pyran-2-ide, the peak at m/z 113.0244 likely represented 3,5-dihydroxy-2H-pyran-2-ide. The results of this work provide the first insights into the metabolic pathways of SARM 2f, a novel selective androgen receptor modulator. A total of seven metabolites were found in this study, including phase-I and phase-II metabolites that are applicable to routine doping control analysis. Furthermore, selected metabolites were synthesized to confirm their structures and be used as reference materials. However, in vitro models are simple simulations of the human organism. Further research, such as the use of an organ-on-a-chip model or micro dosing studies, is needed to gain a deeper understanding The results of this work provide the first insights into the metabolic pathways of SARM 2f, a novel selective androgen receptor modulator. A total of seven metabolites were found in this study, including phase-I and phase-II metabolites that are applicable to routine doping control analysis. Furthermore, selected metabolites were synthesized to confirm their structures and be used as reference materials. However, in vitro models are simple simulations of the human organism. Further research, such as the use of an organ-on-a-chip model or micro dosing studies, is needed to gain a deeper understanding of the metabolism of SARM 2f.

In Vitro Metabolic Assay
In vitro assays were conducted according to a protocol described by Kuuranne et al. [25]. S9 fraction and HLM were used together to optimize the enzymatic conversion and to produce a wider range of different metabolites. As described in the literature, HLM showed a higher concentration of CYP enzymes compared to S9 fractions, but some metabolites may only be produced by S9 fractions [26,27]. In total, 1 mg/mL SARM 2f in DMSO was diluted in 50 mM phosphate buffer (pH 7.4) containing 5 mM MgCl 2 to produce a stock solution (100 µM). For phase-I-metabolism, 10 µL of stock solution, 10 µL of NADPH (50 mM), 5 µL of S9 fraction (20 mg/mL) and 5 µL of HLM (20 mg/mL) were added to 20 µL of phosphate buffer for a total volume of 50 µL. Samples were incubated at 37 • C for 24 h. For phase-II-metabolism, an additional 10 µL of NADPH, 5 µL of S9 fraction, 5 µL of HLM, as well as 10 µL of UDGPA (50 mM), 10 µL of SL (50 mM) and 10 µL of PAPS (20 µM) were added, and the mixture was incubated at 37 • C for 24 h. In addition, two negative control samples were prepared, one excluding HLM and S9 fraction and one excluding SARM 2f, to confirm whether any detected metabolites of interest were genuine metabolites of the incubated compound and to differentiate between possible enzymatic and nonenzymatic reaction pathways. Following incubation, each metabolism step was quenched by the addition of 150 µL of ice-cold ACN. The supernatant was obtained after centrifugation (17,000× g, 5 min) and transferred into a fresh tube. After drying using a vacuum centrifuge (45 • C, 45 min), the samples were reconstituted in 100 µL of ACN:H 2 O (90:10 v/v).

LC-HRMS
LC-MS data were generated using a Vanquish UHPLC system coupled with an Orbitrap Exploris 480 mass spectrometer, both from Thermo Fisher (Bremen, Germany). The HPLC system was equipped with an EC 4/2 Nucleodur C-18 Pyramid 3 µm pre-column from Macherey-Nagel (Düren, Germany) and a Poroshell 120 EC-C18 column (3.0 × 50 mm, 2.7 µm) from Agilent (Santa Clara, CA, USA). For gradient elution, 0.1% FA in H 2 O was used as Eluent A and 0.1% FA in ACN was used as Eluent B. The gradient started with 0% B, increasing to 100% within 10 min where it was held for 1 min. After returning to starting conditions within 0.01 min, the column was re-equilibrated for 4 min. A flow of 0.3 mL/min was applied. The injection volume was 10 µL.
MS data were collected using a heated ESI source in negative ionization mode with an ionization voltage of −2600 V. The ion transfer tube was heated to 320 • C, and the vaporizer temperature was 420 • C. Full scan acquisition mode in positive and negative ionization mode was used with a resolution of 60,000 and a mass range of m/z 50-800. MS 2 data for SARM 2f and its metabolites were acquired in negative ionization mode, using parallel reaction monitoring (PRM) with a resolution of 30,000 and an extraction window of 1 m/z. The normalized collision energies were optimized for each analyte, using 15% for m/z 377.0409, 20% for m/z 295.0695, 411.0468 and 473.1161, 25% for m/z 297.0845, 313.0790, 315.0951, 409.0317 and 491.1283 and 35% for m/z 489.1111. For SARM 2f, MS 2 data were also collected in positive ionization mode with a normalized collision energy of 45%. Pseudo MS 3 experiments were performed via in-source fragmentation with a source voltage of 35 V in positive and negative ionization mode. Nitrogen was used as the collision gas and was generated by a CMC nitrogen generator (Eschborn, Germany). The MS was regularly calibrated using the Pierce Flex Mix calibration solution from Thermo Fisher (Bremen, Germany).