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

Abstract

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.

Full text

Citation: Möller, T.; Wen, H.-C.;
Naumann, N.; Krug, O.; Thevis, M.
Identification and Synthesis of
Selected In Vitro Generated
Metabolites of the Novel Selective
Androgen Receptor Modulator
(SARM) 2f. Molecules 2023,28, 5541.
https://doi.org/10.3390/
molecules28145541
Academic Editor: Simona Francese
Received: 29 June 2023
Revised: 14 July 2023
Accepted: 19 July 2023
Published: 20 July 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
molecules
Article
Identification and Synthesis of Selected In Vitro Generated
Metabolites of the Novel Selective Androgen Receptor
Modulator (SARM) 2f
Tristan Möller 1, Hui-Chung Wen 2, Nana Naumann 1, Oliver Krug 1,3 and Mario Thevis 1, 3, *
1Center for Preventive Doping Research, Institute of Biochemistry, German Sport University Cologne,
Am Sportpark Müngersdorf 6, 50933 Cologne, Germany
2Faculty of Chemistry, University of Cologne, Greinstraße 4-6, 50939 Cologne, Germany
3European Monitoring Center for Emerging Doping Agents (EuMoCEDA), 50933 Cologne, Germany
*Correspondence: thevis@dshs-koeln.de
Abstract:
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.
Keywords: selective androgen receptor modulators; LC-HRMS; metabolite synthesis
1. 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 bind-
ing 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
–
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 [6–9].
Due to their anabolic effects on muscle and bone, SARMs are further used as performance-
enhancing 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
Molecules 2023,28, 5541. https://doi.org/10.3390/molecules28145541 https://www.mdpi.com/journal/molecules

Molecules 2023,28, 5541 2 of 14
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) ben-
zonitrile) (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
–
16
]. However, no information on the metabolism of SARM 2f is known
to date.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 14
(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
ndings (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-(triuoromethyl)
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 cyno-
molgus monkey models conrmed the occurrence of anabolic eects by showing an in-
crease in lean body mass, as well as suppression of blood lipid levels compared to testos-
terone [14–16]. However, no information on the metabolism of SARM 2f is known to date.
Figure 1. Structure of SARM 2f.
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 exper-
iments to gain rst insights into its metabolism. Metabolites found in this study were iden-
tied using liquid chromatography coupled with high-resolution mass spectrometry (LC-
HRMS). The selected metabolites found in this study were synthesized and their structure
conrmed via nuclear magnetic resonance spectroscopy (NMR) in order to provide criti-
cal information for the implementation of SARM 2f and its metabolites into routine dop-
ing control test methods.
2. Results and Discussion
2.1. 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-congured bromohydrin 4a [18]. In order to invert the C4 ste-
reocenter, 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-hy-
droxy 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 unidenti-
ed by-products; therefore, it was necessary to investigate a suitable workup method to
obtain a clean product. Final purication was obtained using a fractionated reverse-phase
(RP) solid-phase extraction (SPE) with a gradient using ACN/Water (H2O). The same re-
action methodology was applied to methyl (E)-pent-3-enoate to obtain 4-((2R,3R)-3-hy-
droxy-2-methyl-5-oxopyrrolidin-1-yl)-2-(triuoromethyl)benzonitrile 7b (Figure 2),
which can be used as an internal standard for the analysis of SARM 2f in doping control
samples.
Figure 1. Structure of SARM 2f.
To tackle the limited availability as a reference material, SARM 2f was synthesized us-
ing 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 informa-
tion for the implementation of SARM 2f and its metabolites into routine doping control
test methods.
2. Results and Discussion
2.1. 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 ob-
tain
2a
,meta-chloroperbenzoic acid (mCPBA) was used to produce the corresponding epox-
ide
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
-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 (H
2
O). 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.
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/z343.0917 (1.7 ppm) as well as in-source dehydration at m/z279.0758 (2.5 ppm). In
positive ionization mode, neither adduct ions nor reaction products were found.

Molecules 2023,28, 5541 3 of 14
Molecules 2023, 28, x FOR PEER REVIEW 3 of 14
Scheme 1. Synthetic route to produce SARM 2f. Reaction conditions: (a) H2SO4, methanol (MeOH),
room temperature (RT), 12 h; (b) mCPBA, NaHCO3, dichloromethane (DCM), 0 °C to RT, 12 h; (c) LiBr,
Mg(ClO4)2, ACN, RT, 3 h; (d) NaN3, dimethyl sulfoxide (DMSO), 40 °C, 72 h; (e) Pd/C, H2, MeOH, RT,
4 h; (f) 4-iodo-2-(trifluoromethyl)benzonitrile, Pd(dba)2, xantphos, 1,4-dioxane, 120 °C, 12 h.
Figure 2. Internal standard 4-((2R,3R)-3-hydroxy-2-methyl-5-oxopyrrolidin-1-yl)-2-(triuorome-
thyl)benzonitrile 7b.
In order to produce an authentic reference substance and to conrm the synthetic
methodology described above, all reaction steps are characterized by means of 1H, 13C 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 ad-
ducts 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.
MS2 data acquired in negative ionization mode revealed the appearance of dehydra-
tion 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-(triuoromethyl)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-(triuoro-
methyl)benzonitrile radical. Further, pseudo MS3 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 identied as the deprotonated ((4-cyano-3-
(triuoromethyl)phenyl)amino)ethyn-1-ide, the ion at m/z 185.0332 was assigned to the
deprotonated (4-cyano-3-(triuoromethyl)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 anal-
ogous 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-(triuoromethyl)phenyl)-2-ethylazetidin-2-ide. An overview of the proposed dis-
sociation 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-pyr-
rol-1-yl)-2-(triuoromethyl)benzonitrile. Pseudo MS3 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
Scheme 1.
Synthetic route to produce SARM 2f. Reaction conditions: (
a
) H
2
SO
4
, methanol (MeOH),
room temperature (RT), 12 h; (
b
)mCPBA, NaHCO
3
, dichloromethane (DCM), 0
◦
C to RT, 12 h; (
c
) LiBr,
Mg(ClO
4
)
2
, ACN, RT, 3 h; (
d
) NaN
3
, dimethyl sulfoxide (DMSO), 40
◦
C, 72 h; (
e
) Pd/C, H
2
, MeOH,
RT, 4 h; (f) 4-iodo-2-(trifluoromethyl)benzonitrile, Pd(dba)2, xantphos, 1,4-dioxane, 120 ◦C, 12 h.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 14
Scheme 1. Synthetic route to produce SARM 2f. Reaction conditions: (a) H2SO4, methanol (MeOH),
room temperature (RT), 12 h; (b) mCPBA, NaHCO3, dichloromethane (DCM), 0 °C to RT, 12 h; (c) LiBr,
Mg(ClO4)2, ACN, RT, 3 h; (d) NaN3, dimethyl sulfoxide (DMSO), 40 °C, 72 h; (e) Pd/C, H2, MeOH, RT,
4 h; (f) 4-iodo-2-(trifluoromethyl)benzonitrile, Pd(dba)2, xantphos, 1,4-dioxane, 120 °C, 12 h.
Figure 2. Internal standard 4-((2R,3R)-3-hydroxy-2-methyl-5-oxopyrrolidin-1-yl)-2-(triuorome-
thyl)benzonitrile 7b.
In order to produce an authentic reference substance and to conrm the synthetic
methodology described above, all reaction steps are characterized by means of 1H, 13C 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 ad-
ducts 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.
MS2 data acquired in negative ionization mode revealed the appearance of dehydra-
tion 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-(triuoromethyl)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-(triuoro-
methyl)benzonitrile radical. Further, pseudo MS3 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 identied as the deprotonated ((4-cyano-3-
(triuoromethyl)phenyl)amino)ethyn-1-ide, the ion at m/z 185.0332 was assigned to the
deprotonated (4-cyano-3-(triuoromethyl)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 anal-
ogous 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-(triuoromethyl)phenyl)-2-ethylazetidin-2-ide. An overview of the proposed dis-
sociation 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-pyr-
rol-1-yl)-2-(triuoromethyl)benzonitrile. Pseudo MS3 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
Figure 2.
Internal standard 4-((2R,3R)-3-hydroxy-2-methyl-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)
benzonitrile 7b.
MS
2
data acquired in negative ionization mode revealed the appearance of dehydra-
tion to form the base peak of the product ion spectra at m/z279.0751. 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/z250.0361 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/z279.0751 showed
the dissociation to form the signals at m/z209.0332 and m/z185.0332. While the product ion
at m/z209.0332 was tentatively identified as the deprotonated ((4-cyano-3-(trifluoromethyl)
phenyl)amino)ethyn-1-ide, the ion at m/z185.0332 was assigned to the deprotonated
(4-cyano-3-(trifluoromethyl) phenyl)amide. The signal at m/z185.0332 is of particular
interest for the analysis of various SARMs, since it is commonly found in analogous struc-
tures [
20
]. The deprotonated intact compound at m/z297.0866 further yielded a product
ion at m/z253.0960, 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.
The proposed dissociation pathway in positive ionization mode is shown in Scheme 2b.
The parent compound at m/z299.0597 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/z281.0890
gave rise to further dissociation products at m/z239.0780 and m/z213.0267. These peaks
were proposed to represent the protonated 4-(2-ethyl-1H-azirin-1-yl)-2-(trifluoromethyl)
benzonitrile and N-(4-cyano-3-(trifluoromethyl)phenyl)formamide, respectively. The peak
at m/z213.0267 further dissociated to the ion at m/z185.0320, which was tentatively corre-
lated with an 2-amino-5-cyano-4-(trifluoromethyl)benzene-1-ylium ion. In addition, the in-
tact and protonated compound at m/z299.0597 was found to dissociate into a product ion at
m/z187.0475, which was identified as the protonated 4-amino-2-(trifluoromethyl)benzonitrile.

Molecules 2023,28, 5541 4 of 14
Molecules 2023, 28, x FOR PEER REVIEW 4 of 14
peaks were proposed to represent the protonated 4-(2-ethyl-1H-azirin-1-yl)-2-(triuoro-
methyl)benzonitrile and N-(4-cyano-3-(triuoromethyl)phenyl)formamide, respectively.
The peak at m/z 213.0267 further dissociated to the ion at m/z 185.0320, which was tenta-
tively correlated with an 2-amino-5-cyano-4-(triuoromethyl)benzene-1-ylium ion. In ad-
dition, the intact and protonated compound at m/z 299.0597 was found to dissociate into
a product ion at m/z 187.0475, which was identied as the protonated 4-amino-2-(triuo-
romethyl)benzonitrile.
Scheme 2. Proposed dissociation pathway of SARM 2f: (a) in negative ionization mode; (b) in posi-
tive ionization mode.
2.2. In Vitro Generated Metabolites
To investigate its metabolism, SARM 2f was incubated using human liver micro-
somes (HLM) and S9 fractions. Five metabolic pathways, including oxidation (M1), hy-
droxylation (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 (spec-
tra 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 sim-
ilar MS2 spectra, indicating the formation of dierent 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.
2.2. 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 glucuronida-
tion 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 enzyme-
mixture-containing
in vitro
metabolism sample. Those findings indicate the existence of
a n
on-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.

Molecules 2023,28, 5541 5 of 14
Table 1.
List of
in vitro
metabolites detected for SARM 2f, including the product ions obtained for
each metabolite. The data shown in this table are the experimental data obtained in this study, with
an error acceptance of 5 ppm. The base peaks are highlighted in bold.
Metabolic
Reaction
Precursor Ion
[M−H]−[m/z]
Elemental
Composition
Retention
Time [min]
Product Ions
[m/z]
Elemental
Composition
SARM 2f 297.0866 C14H12 O2N2F36.58 279.0751 C14H10ON2F3
253.0960 C13H12 N2F3
250.0361 C12H5ON2F3
209.0332 C10H4N2F3
185.0332 C8H4N2F3
M1
Oxidation
295.0702 C14H10 O2N2F35.51 265.0597 C13H8ON2F3
250.0359 C12H5ON2F3
186.0172 C8H3ONF3
185.0333 C8H4N2F3
M2 a
Hydroxylation
313.0794 C14H12 O3N2F35.94 185.0332 C8H4N2F3
127.0402 C6H7O3
M2 b 6.17 185.0332 C8H4N2F3
127.0402 C6H7O3
101.0608 C5H9O2
M2 c 6.74 185.0332 C8H4N2F3
127.0402 C6H7O3
101.0608 C5H9O2
M3
Hydration
315.0965 C14H14 O3N2F36.70 297.0858 C14H12 O2N2F3
271.1067 C13H14 ON2F3
255.0752 C12H10 ON2F3
253.0959 C13H12 N2F3
226.0360 C10H5ON2F3
185.0334 C8H4N2F3
170.0223 C8H3NF3
M4 Sulfation 377.0425 C14H12 O5N2F3S 6.17 96.9601 HSO4
M5
Glucuronidation
473.1181 C20H20 O8N2F35.70 193.0353 C6H9O7
175.0249 C6H7O6
157.0143 C6H5O5
131.0350 C5H7O4
113.0244 C5H5O3
M6
Hydroxylation +
Glucuronidation
489.1143 C20H20 O9N2F34.93 471.1033 C20H18 O8N2F3
277.0597 C14H8ON2F3
193.0353 C6H9O7
175.0249 C6H7O6
157.0143 C6H5O5
131.0350 C5H7O4
113.0244 C5H5O3
M7
Hydration +
Glucuronidation
491.1283 C20H22 O9N2F35.97 315.0965 C14H14O3N2F3
255.0753 C12H10 ON2F3
193.0353 C6H9O7
175.0249 C6H7O6
157.0143 C6H5O5
131.0350 C5H7O4
113.0244 C5H5O3
2.3. 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 gra-
dient of ACN and H
2
O (10% to 60% ACN) was applied to elute the product. The resulting

Molecules 2023,28, 5541 6 of 14
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.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 14
2.3. Synthesis of Selected In Vitro Generated Metabolites
In order to conrm 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 purication, RP-SPE was used and a
gradient of ACN and H2O (10% to 60% ACN) was applied to elute the product. The re-
sulting substance showed coincident chromatographic and mass spectrometric properties
with the metabolite produced in vitro (Figure 3a). Further, the predicted structure was
conrmed by means of 1H, 13C APT and HSQC-NMR spectroscopy. Therefore, M3 was
identied as (3R,4R)-4-((4-cyano-3-(triuoromethyl)phenyl)amino)-3-hydroxyhexanoic
acid. Puried M3 was then used for mass spectrometric characterization.
Scheme 3. Synthesis of the metabolites M3, M4 and M5. Reaction conditions: (a) LiOH, THF, MeOH,
H2O, RT 2 h [21]; (b) sulfur trioxide pyridine (SO3·py), N,N-dimethylformamide (DMF), 1,4 dioxane,
RT, 2 h [22]; (c) (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyl tri-
acetate (AGME), Ag2CO3, toluene, RT, 12 h [23]; (d) KCN, MeOH, 0 °C, 1.5 h [24].
Figure 3. Comparative analysis of in vitro generated metabolites with synthetic metabolites: (a)
chromatographic and MS2 results obtained for M3; (b) chromatographic and MS2 results obtained
for M4; (c) chromatographic and MS2results obtained for M5.
Scheme 3.
Synthesis of the metabolites
M3
,
M4
and
M5
. Reaction conditions: (
a
) LiOH, THF,
MeOH, H
2
O, RT 2 h [
21
]; (
b
) sulfur trioxide pyridine (SO
3·
py), N,N-dimethylformamide (DMF),
1,4 dioxane, RT, 2 h [
22
]; (
c
) (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)-tetrahydro-2H-pyran-
3,4,5-triyl triacetate (AGME), Ag2CO3, toluene, RT, 12 h [23]; (d) KCN, MeOH, 0 ◦C, 1.5 h [24].
Molecules 2023, 28, x FOR PEER REVIEW 6 of 14
2.3. Synthesis of Selected In Vitro Generated Metabolites
In order to conrm 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 purication, RP-SPE was used and a
gradient of ACN and H2O (10% to 60% ACN) was applied to elute the product. The re-
sulting substance showed coincident chromatographic and mass spectrometric properties
with the metabolite produced in vitro (Figure 3a). Further, the predicted structure was
conrmed by means of 1H, 13C APT and HSQC-NMR spectroscopy. Therefore, M3 was
identied as (3R,4R)-4-((4-cyano-3-(triuoromethyl)phenyl)amino)-3-hydroxyhexanoic
acid. Puried M3 was then used for mass spectrometric characterization.
Scheme 3. Synthesis of the metabolites M3, M4 and M5. Reaction conditions: (a) LiOH, THF, MeOH,
H2O, RT 2 h [21]; (b) sulfur trioxide pyridine (SO3·py), N,N-dimethylformamide (DMF), 1,4 dioxane,
RT, 2 h [22]; (c) (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)-tetrahydro-2H-pyran-3,4,5-triyl tri-
acetate (AGME), Ag2CO3, toluene, RT, 12 h [23]; (d) KCN, MeOH, 0 °C, 1.5 h [24].
Figure 3. Comparative analysis of in vitro generated metabolites with synthetic metabolites: (a)
chromatographic and MS2 results obtained for M3; (b) chromatographic and MS2 results obtained
for M4; (c) chromatographic and MS2results obtained for M5.
Figure 3.
Comparative analysis of
in vitro
generated metabolites with synthetic metabolites:
(
a
) chromatographic and MS
2
results obtained for
M3
; (
b
) chromatographic and MS
2
results ob-
tained for M4; (c) chromatographic and MS2results obtained for M5.
The full scan acquisition of
M3
(C
14
H
14
O
3
N
2
F
3−
m/z 315.0965 (1.0 ppm)) in negative
ionization mode showed the appearance of in-source dehydration (C
14
H
12
O
2
N
2
F
3−
m/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/z255.0752,
which was correlated with the deprotonated N-(4-cyano-3-(trifluoromethyl)phenyl)-N-
propyl formamide. This ion was proposed to undergo a homolytic cleavage to release

Molecules 2023,28, 5541 7 of 14
an ethyl radical (29 u) to form a peak at m/z226.0360, 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/z170.0223. 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/z271.1067 and m/z297.0858, respectively. While the product
ion at m/z271.1067 was tentatively identified as the deprotonated 4-((2-hydroxypentan-3-
yl)amino)-2-(trifluoromethyl)benzonitrile, the peak at m/z297.0858 was assigned to the de-
protonated 4-((4-cyano-3-(trifluoromethyl)phenyl)amino)hex-3-enoic acid. The dehydrated
parent ion at m/z297.0858 further dissociated to form a product ion at m/z253.0959, which
was assigned to the deprotonated 4-(pent-2-en-3-ylamino)-2-(trifluoromethyl)benzonitrile.
Molecules 2023, 28, x FOR PEER REVIEW 7 of 14
The full scan acquisition of M3 (C14H14O3N2F3− m/z 315.0965 (1.0 ppm)) in negative
ionization mode showed the appearance of in-source dehydration (C14H12O2N2F3- m/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 dier, which can
lead to misinterpretation of the metabolic pathways of SARM 2f. The proposed dissocia-
tion 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-(triuoromethyl)phenyl)-N-
propyl 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 depro-
tonated N-(4-cyano-3-(triuoromethyl)phenyl)-N-methyl formamide. Further, pseudo
MS3 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-(triuorome-
thyl)phenyl)amide and 4-cyano-3-(triuoromethyl)benzen-1-ide, respectively. In addi-
tion, 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 identied as the deprotonated 4-((2-hydroxypentan-3-
yl)amino)-2-(triuoromethyl)benzonitrile, the peak at m/z 297.0858 was assigned to the
deprotonated 4-((4-cyano-3-(triuoromethyl)phenyl)amino)hex-3-enoic acid. The dehy-
drated 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-(triuoromethyl)ben-
zonitrile.
Scheme 4. Proposed dissociation pathway of the deprotonated metabolite M3.
Scheme 4. Proposed dissociation pathway of the deprotonated metabolite M3.
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 purifi-
cation 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

Molecules 2023,28, 5541 8 of 14
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.
Molecules 2023, 28, x FOR PEER REVIEW 8 of 14
The sulfated metabolite M4 was synthesized in accordance with the literature [22].
SO3·py in DMF and 1,4-dioxane was used as sulfation reagent (Scheme 3b). The purica-
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 corre-
sponding metabolite found in vitro. Both compounds showed agreement in chromato-
graphic retention time and spectra (Figure 3b). In addition, synthetic M4 was submied
to NMR analysis in order to conrm its structure. Therefore, M4 was identied as (2R,3R)-
1-(4-cyano-3-(triuoromethyl)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 hy-
drogen sulfate ion (Scheme 5). It should be mentioned that this ion is a non-specic prod-
uct ion. But due to the fact that its appearance was conrmed 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 subse-
quently 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 conju-
gate was not entirely excluded, yielding a product where both the desired glucuronide
plus the deconjugated SARM 2f was obtained. Hence, identication of the target com-
pound was conducted using only chromatographic and mass spectrometric methods that
showed agreement with data obtained from the metabolite found in vitro (Figure 3).
The MS2 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 identied as the deprotonated 3,4,5-trihydroxy-3,4-dihydro-2H-pyran-2-car-
boxylate 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
identied 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.
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 subse-
quently 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/z193.0353,
which was assigned to the deprotonated glucuronic acid. This ion undergoes sequen-
tial dehydration (18 u) to form the peaks at m/z175.0249 and m/z157.0143. 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/z175.0249 showed decarboxylation (44 u) followed by dehydration (18 u) to form the
peaks at m/z131.0350 and m/z113.0244. While the peak at m/z131.0350 was tentatively
identified as the deprotonated 3,4,5-trihydroxy-3,4-dihydro-2H-pyran-2-ide, the peak at
m/z113.0244 likely represented 3,5-dihydroxy-2H-pyran-2-ide.
Molecules 2023, 28, x FOR PEER REVIEW 9 of 14
Scheme 6. Proposed dissociation pathway of the deprotonated metabolite M5.
The results of this work provide the rst 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
conrm 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 or-
gan-on-a-chip model or micro dosing studies, is needed to gain a deeper understanding
of the metabolism of SARM 2f.
3. Materials and Methods
3.1. Reagents and Chemicals
Trans-3-hexenoic acid, methyl-trans-3-pentenoate, H2SO4, mCPBA, magnesium per-
chlorate, MgSO4, Pd/C (10%), 4-iodo-2-(triuoromethyl)-benzonitrile, tris(dibenzyli-
deneacetone)dipalladium(0), xantphos, Cs2CO3, 1,4 dioxane, Celite®, LiOH · H2O, THF,
SO3·py, DMF, silver-(I)-carbonate, toluene, uridine diphosphate glucuronic acid
(UDGPA), D-saccharic acid-1,4-lactone (SL) and 3′-phosphoadenosine-5′-phosphosulfate
(PAPS) were purchased from Sigma Aldrich (St. Louis, Missouri, USA). AGME, formic
acid (FA), S9 fraction and HLM were obtained from Thermo Scientic (Bremen, Germany).
LiBr, DCM and DMSO were obtained from Carl Roth (Kalsruhe, Germany). ACN,
ethylacetate (EtOAc), n-pentane and NaN3 were purchased from VWR (Radnor, Pennsyl-
vania, USA). Sodium acetate and nicotinamide adenine dinucleotide phosphate (NADPH)
were obtained from Merck (Darmstadt, Germany). MeOH was purchased from J.T.Baker
(Phillipsburg, NJ, USA). Hydrogen gas (99.999%) was obtained from Praxair (Düsseldorf,
Germany). Ultrapure water was received from a Barnstead GenPure xCAD Plus from
Thermo Scientic (Bremen, Germany).
Column chromatography was performed using silica gel (63–200 µm) from Supelco
(Sigma Aldrich, St. Louis, MI, USA)). For reaction control and control of the column chro-
matography, thin layer chromatography (TLC) plates were used from Merck (Darmstadt,
Germany). Chromabond® C18 6cc SPE cartridges were purchased from Macherey-Nagel
(Düren, Germany), and Oasis® WAX 3cc cartridges were obtained from Waters (Milford,
MA, USA).
Scheme 6. Proposed dissociation pathway of the deprotonated metabolite M5.
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

Molecules 2023,28, 5541 9 of 14
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.
3. Materials and Methods
3.1. Reagents and Chemicals
Trans-3-hexenoic acid, methyl-trans-3-pentenoate, H
2
SO
4
,mCPBA, magnesium perchlo-
rate, MgSO
4
, Pd/C (10%), 4-iodo-2-(trifluoromethyl)-benzonitrile, tris(dibenzylideneacetone)
dipalladium(0), xantphos, Cs
2
CO
3
, 1,4 dioxane, Celite
®
, LiOH
·
H
2
O, THF, SO
3·
py, DMF,
silver-(I)-carbonate, toluene, uridine diphosphate glucuronic acid (UDGPA), D-saccharic
acid-1,4-lactone (SL) and 3
0
-phosphoadenosine-5
0
-phosphosulfate (PAPS) were purchased
from Sigma Aldrich (St. Louis, MI, USA). AGME, formic acid (FA), S9 fraction and HLM
were obtained from Thermo Scientific (Bremen, Germany). LiBr, DCM and DMSO were
obtained from Carl Roth (Kalsruhe, Germany). ACN, ethylacetate (EtOAc), n-pentane
and NaN
3
were purchased from VWR (Radnor, PA, USA). Sodium acetate and nicoti-
namide adenine dinucleotide phosphate (NADPH) were obtained from Merck (Darmstadt,
Germany). MeOH was purchased from J.T.Baker (Phillipsburg, NJ, USA). Hydrogen gas
(99.999%) was obtained from Praxair (Düsseldorf, Germany). Ultrapure water was received
from a Barnstead GenPure xCAD Plus from Thermo Scientific (Bremen, Germany).
Column chromatography was performed using silica gel (63–200
µ
m) from Supelco
(Sigma Aldrich, St. Louis, MI, USA)). For reaction control and control of the column chro-
matography, thin layer chromatography (TLC) plates were used from Merck (Darmstadt,
Germany). Chromabond
®
C18 6cc SPE cartridges were purchased from Macherey-Nagel
(Düren, Germany), and Oasis
®
WAX 3cc cartridges were obtained from Waters (Milford,
MA, USA).
3.2. NMR Spectroscopy
NMR-spectra were acquired using a Bruker Avance I 300 and Bruker Avance III 499.
1H NMR-spectra were acquired at a frequency of 300.1 MHz or 499.9 MHz, while 13C
NMR-spectra were acquired at a frequency of 125.7 MHz. Peak assignments were assisted
with two-dimensional spectra (H,H-COSY, H,C-HMBC, H,C-HMQC). The chemical shift
σ
and the coupling constant 3Jor 4Jare indicated in ppm and in Hz, respectively. The
multiplicity is classified as singlet (s), doublet (d), triplet (t), doublet doublet (dd), triplet
triplet (tt) and multiplet (m).
3.3. Synthesis
methyl (E)-hex-3-enoate (2a)
Conc. H
2
SO
4
(2.3 mL; 0.026 mL/mmol) was added to
a solution of (E)-hex-3-enoic acid (10.0 g; 87.6 mmol; 1 eq) in MeOH (227 mL) at RT, and
then the mixture was stirred overnight. The excess of MeOH was removed under reduced
pressure. The residue was dissolved in DCM, washed with NaHCO
3
solution and H
2
O and
dried over MgSO
4
. After removal of the solvent under reduced pressure, pure
2a
(10.98 g;
97%) was obtained as a yellow oil.
1
H-NMR (CDCl
3
; 500 MHz):
δ
0.99 (t; J= 7.5 Hz, 3H),
δ
2.05 (m, 2H),
δ
3.03 (dd, J= 6.8,
1.0; 2H), δ3.68 (s, 3H), δ5.52 (m, 1H), δ5.61 (m, 1H).
13
C-NMR (CDCl
3
; 125 MHz):
δ
13.4 ppm (CH
3
),
δ
25.5 ppm (CH
2
),
δ
37.9 ppm (CH
2
),
δ51.7 ppm (CH3), δ120.5 ppm (CH), δ136.4 ppm (CH), δ172.6 ppm (Cquat.).
methyl 2-((2R,3R)-3-ethyloxiran-2-yl)acetate (3a) 2a
(4.73 g; 36.90 mmol; 1 eq) was
dissolved in DCM (250 mL), followed by the addition of NaOAc (9.08 g; 109.38 mmol; 3 eq).
The suspension was cooled to 0
◦
C and mCPBA (10.94 g; 63.40 mmol; 1.32 eq) was added.
After 2 h, the ice bath was removed, and the mixture was stirred for an additional 3 h. The
reaction mixture was washed with NaHCO
3
solution (x3) and H
2
O, dried over MgSO
4
and

Molecules 2023,28, 5541 10 of 14
then the solvent was removed under reduced pressure. After column chromatography
(n-pentane:EtOAc 20:1), the product 3a (3.74 g; 70.2%) was obtained as a yellow oil.
1
H-NMR (CDCl3; 500 MHz):
δ
1.00 (t; J= 7.5 Hz, 3H),
δ
1.60 (m, 2H),
δ
2.57 (ddd, ABX:
J
AB
= 16.3 Hz, J
AX
= 5.9 Hz, 2H),
δ
2.74 (dt, J= 8.3, 2.1 Hz; 1H),
δ
3.05 (dt, J= 8.8, 2.1 Hz;
1H), δ3.73 (s, 3H).
13
C-NMR (CDCl
3
; 125 MHz):
δ
9.66 ppm (CH
3
),
δ
24.8 ppm (CH
2
),
δ
37.5 ppm (CH
2
),
δ51.8 ppm (CH3), δ53.6 ppm (CH), δ59.6 ppm (CH), δ170.9 ppm (Cquat.).
methyl (3R,4S)-4-bromo-3-hydroxyhexanoate (4a)
A stirred solution of
3a
(3.00 g;
20.8 mmol; 1 eq) in ACN (40 mL) was cooled to 0
◦
C using an ice bath. At this temperature,
LiBr (5.62 g, 62.4 mmol; 2 eq) and Mg
2
(ClO
4
) (9.29 g; 41.6 mmol; 3 eq) were added. The
mixture was allowed to heat up to RT and was stirred until complete conversion was
achieved (controlled by TLC). The reaction mixture was diluted in DCM and washed with
1 M HCl (x2). The aqueous phase was extracted with DCM (x2). The combined organic
phases were dried over MgSO
4
, and the solvent was removed under reduced pressure.
Pure
4a
(4.20 g; 89.7%) was obtained as a yellowish oil after column chromatography
(n-pentane:EtOAc 10:1).
1
H-NMR (CDCl
3
; 500 MHz):
δ
1.09 (t; J= 7.3 Hz, 3H),
δ
1.81 (ddq, ABX: J
AX
= 24.0 Hz,
J
AB
= 14.6 Hz, 7.3 Hz 1H)),
δ
2.00 (ddq, ABX: J
BX
= 22.2 Hz, J
AB
= 14.7 Hz, J= 7.4 Hz 1H),
δ
2.67 (dd, ABX: J
AB
= 16.5, J
AX
= 8.8 Hz; 1H),
δ
2.78 (dd, ABX: J
AB
= 16.5, J
BX
= 3.1 Hz;
1H),
δ
3.74 (s, 3H),
δ
4.03 (ddd, ABX: J
AX
= 9.4, J
BX
= 3.5 Hz; J= 5.8 1H),
δ
4.11 (ddd,
ABX: JAX = 8.8, JBX = 3.1 Hz; J= 5.7 1H).
13
C-NMR (CDCl
3
; 125 MHz):
δ
12.15 ppm (CH
3
),
δ
27.53 ppm (CH
2
),
δ
38.29 ppm
(CH2), δ52.04 ppm (CH3), δ62.54 ppm (CH), δ70.76 ppm (CH), δ172.88 ppm (Cquat.).
methyl (3R,4R)-4-azido-3-hydroxyhexanoate (5a) 4a
(4.00 g; 17.8 mmol; 1 eq) was dis-
solved in DMSO (80 mL), and NaN
3
(3.46 g; 53.3 mmol; 3 eq) was added. The reaction was
heated up to 40
◦
C and stirred for 72 h. The mixture was diluted in EtOAc and subsequently
washed with H
2
O (3x). The organic phase was dried over MgSO
4
, and the solvent was
removed under reduced pressure. After column chromatography (
n-pentane:EtOAc 5:1
),
the product 5a (2.32 g; 73.2%) was isolated as a pale yellow oil.
1
H-NMR (CDCl
3
; 500 MHz):
δ
1.06 (t; J= 7.4 Hz, 3H),
δ
1.73 (m, 2H),
δ
2.53 (dd, ABX:
J
AB
= 16.4, J
AX
= 3.5 Hz; 1H),
δ
2.65 (dd, ABX: J
AB
= 16.4, J
BX
= 9.1 Hz; 1H),
δ
3.00 (d,
J= 4.9 Hz, 1H), δ3.12 (m, 1H), δ3.73 (s, 3H), δ4.09 (m, 1H).
13
C-NMR (CDCl
3
; 125 MHz):
δ
10.75 ppm (CH
3
),
δ
23.43 ppm (CH
2
),
δ
38.37 ppm
(CH2), δ52.00 ppm (CH3), δ67.00 ppm (CH), δ69.63 ppm (CH), δ172.84 ppm (Cquat.).
(4R,5R)-5-ethyl-4-hydroxypyrrolidin-2-one (6a)
Pd/C (10% MW; 0.23 g; 10 mol-%)
was added to a solution of
5a
(2.30 mg; 12.4 mmol; 1 eq) in MeOH (25 mL). The reaction
was charged with H
2
and was stirred at room temperature. After 4 h, the reaction mixture
was filtered over Celite
®
, and the solvent was removed under reduced pressure. Pure
6a
(1.11 g, 69.4%) was obtained as a white solid after column chromatography (EtOAc).
1
H-NMR (CDCl
3
; 500 MHz):
δ
0.99 (t; J= 7.5 Hz, 3H),
δ
1.39 (m, 1H),
δ
1.56 (m, 1H),
δ
1.95 (dd, ABX: J
AB
= 16.5, J
AX
= 2.7 Hz; 1H),
δ
2.40 (dd, ABX: J
AB
= 16.52, J
BX
= 6.1 Hz;
1H), δ3.32 (m, 1H), δ4.20 (m, 1H), δ4.93 (d, J= 5.0 Hz, 1H), δ7.64 (s, 1H).
13
C-NMR (CDCl
3
; 125 MHz):
δ
9.89 ppm (CH
3
),
δ
21.41 ppm (CH
2
),
δ
40.41 ppm
(CH2), δ61.24 ppm (CH), δ67.52 ppm (CH), δ177.03 ppm (Cquat.).
4-((2R,3R)-2-ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile
(SARM 2f) 5a
(0.15 g; 1.16 mmol; 1 eq), 4-Iodo-2-(trifluoromethyl)-benzonitrile (0.38 g;
1.27 mmol
; 1.09 eq) and cesium carbonate (0.45 g; 1.40 mmol; 1.2 eq) were placed in
a S
chlenk tube under inert atmosphere and dissolved in 1,4 dioxane (5 mL). After the addi-
tion of tris(dibenzylideneacetone)dipalladium(0) (0.05 g; 0.06 mmol; 5 mol-%) and xantphos
(0.08 g; 0.12 mmol; 12 mol-%), the mixture was stirred at 100
◦
C for 10 h. The reaction was
cooled to room temperature and filtered over Celite
®
. The solvent was removed under
reduced pressure. For purification, a chromabond C18 SPE-cartridge (6cc) was used. The
SPE was conditioned with MeOH (3 mL) and H
2
O (3 mL). The crude product was dissolved

Molecules 2023,28, 5541 11 of 14
in H
2
O (3 mL) and was then eluted with an ACN/H
2
O gradient (10% to 60% ACN). SARM
2f (0.23 g; 64.5%) was isolated as a yellow oil.
1
H-NMR (CDCl
3
; 500 MHz):
δ
1.02 (t; J= 7.4 Hz, 3H),
δ
1.76 (m, 2H),
δ
2.46 (s, 1H),
δ
2.69 (dd, ABX: J
AB
= 17.4, J
AX
= 3.9 Hz; 1H),
δ
2.85 (dd, ABX: J
AB
= 17.4, J
BX
= 6.5 Hz; 1H),
δ4.19 (m, 1H), 4.68 (m, 1H), δ7.70 (dd, J= 8.4, 2.0 Hz, 1H), δ7.86 (m, 2H).
13
C-NMR (CDCl
3
; 125 MHz):
δ
9.91 ppm (CH
3
),
δ
19.81 ppm (CH
2
),
δ
41.26 ppm
(CH
2
),
δ
64.22 ppm (CH),
δ
65.24 ppm (CH),
δ
105.66 ppm (C
quat.
),
δ
115.38 ppm (C
quat.
),
δ
121.11 ppm (CH),
δ
123.21 ppm (C
quat.
),
δ
125.92 ppm (CH),
δ
133.65 ppm (C
quat.
),
δ135.39 ppm (CH), δ141.82 ppm (Cquat.), δ172.91 ppm (Cquat. ).
4-((2R,3R)-3-hydroxy-2-methyl-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile
(7b)
1
H-NMR (CDCl
3
; 500 MHz):
δ
1.22 (d; J= 6.5 Hz, 3H),
δ
2.24 (s, 1H),
δ
2.71 (dd, ABX:
J
AB
= 17.3, J
AX
= 5.3 Hz; 1H),
δ
2.87 (dd, ABX: J
AB
= 17.3, J
BX
= 6.8 Hz; 1H),
δ
4.44 (dq,
J= 12.6, 6.3, 1H), δ4.63 (m, 1H), δ7.82 (m, 2H), δ7.97 (d, J= 1.6 Hz, 1H).
13
C-NMR (CDCl
3
; 125 MHz):
δ
12.60 ppm (CH
3
),
δ
40.46 ppm (CH
2
),
δ
58.77 ppm
(CH),
δ
66.23 ppm (CH), 105.24 ppm (C
quat.
),
δ
115.44 ppm (C
quat.
),
δ
119.87 ppm (CH
.
),
δ121.38 ppm
(C
quat.
),
δ
124.76 ppm (CH
.
),
δ
133.86 ppm (C
quat.
),
δ
135.49 ppm (CH
.
),
δ141.78 ppm (Cquat.), δ172.25 ppm (Cquat. ).
(3R,4R)-4-((4-cyano-3-(trifluoromethyl)phenyl)amino)-3-hydroxyhexanoic acid (M3)
SARM 2f
(0.05 g; 0.17 mmol; 1 eq) was dissolved in THF (3.0 mL), MeOH (0.4 mL) and H
2
O
(1.5 mL), and LiOH
·
H
2
O (0.7 g; 1.68 mmol; 10 eq) was added at RT. After 3 h, the reaction
was stopped by the addition of 50% acidic acid in H
2
O (2 mL). The mixture was dissolved
in EtOAc, and the organic phase was washed with brine and water. After drying over
MgSO
4
, the solvent was removed under reduced pressure. The crude product was purified
using a chromabond C18 SPE-cartridge (6cc). After conditioning with MeOH (
3 mL
) and
H
2
O (
3 mL
), the crude product was dissolved in H
2
O (3 mL) and was then eluted with an
ACN/H
2
O gradient (10% to 60% ACN). Following this, the solvent was removed under
reduced pressure and M3 (0.05 g; 96.7%) was isolated as a colorless oil.
1
H-NMR (CDCl
3
; 500 MHz):
δ
0.99 (t; J= 7.4 Hz, 3H),
δ
1.70 (m, 2H),
δ
2.52 (dd, ABX:
J
AB
= 17.0, J
AX
= 3.2 Hz; 1H),
δ
2.70 (dd, ABX: J
AB
= 17.0, J
BX
= 9.6 Hz; 1H),
δ
3.35 (s, 1H),
δ
4.26 (dt, J= 9.4, 2.7, 1H),
δ
6.70 (dd, J= 8.6, 2.3, 1H),
δ
6.86 (d,2.2, 2H),
δ
7.53 (d, J= 8.6 Hz,
1H).
13
C-NMR (CDCl
3
; 125 MHz):
δ
10.72 ppm (CH
3
),
δ
25.07 ppm (CH
2
),
δ
38.30 ppm
(CH
2
),
δ
57.41 ppm (CH),
δ
67.95 ppm (CH
.
),
δ
95.43 ppm (C
quat.
),
δ
110.39 ppm (CH
.
),
δ
113.84 ppm (CH
.
),
δ
117.01 ppm (C
quat.
),
δ
134.37 ppm (C
quat.
),
δ
134.62 ppm (C
quat.
),
δ136.41 ppm (CH.), δ151.09 ppm (Cquat.), δ172.25 ppm (Cquat. ).
(2R,3R)-1-(4-cyano-3-(trifluoromethyl)phenyl)-2-ethyl-5-oxopyrrolidin-3-yl sulfate
(M4)
SO
3·
py (0.27 g; 1.68 mmol; 5 eq) was added to a solution of
SARM 2f
(0.10 g;
0.34 m
mol; 1 eq) in DMF (10 mL) and 1,4-Dioxane (10 mL). After 4 h, the reaction was
stopped by the addition of H
2
O (2 mL). For purification, RP-SPE was used. The SPE was
conditioned using MeOH (3 mL) and H
2
O (3 mL). The crude product was dissolved in
H
2
O (3 mL) and diluted with a gradient of 0.1% FA in ACN and 0.1% FA in H
2
O (20% to
30% of FA in ACN). After removal of the solvent under reduced pressure, the residue was
dissolved in 5 mM ammonium acetate solution (2 mL). After drying,
M4
(58.0 mg; 43.1%)
was isolated as a white solid.
1
H-NMR (CDCl
3
; 500 MHz):
δ
0.91 (t; J= 7.3 Hz, 3H),
δ
1.61 (m, 2H),
δ
2.81 (dd, ABX:
J
AB
= 17.4, J
AX
= 6.4 Hz; 1H),
δ
2.89 (dd, ABX: J
AB
= 17.2, J
BX
= 3.5 Hz; 1H),
δ
4.56 (m, 1H),
4.93 (m, 1H), δ7.85 (dd, J= 8.5, 1.8 Hz, 1H), δ8.12 (m, 2H).
13
C-NMR (CDCl
3
; 125 MHz):
δ
9.43 ppm (CH
3
),
δ
20.02 ppm (CH
2
),
δ
39.33 ppm
(CH
2
),
δ
62.90 ppm (CH),
δ
69.35 ppm (CH),
δ
103.94 ppm (C
quat.
),
δ
116.02 ppm (C
quat.
),
δ
121.76 ppm (CH),
δ
123.93 ppm (C
quat.
),
δ
126.50 ppm (CH),
δ
133.60 ppm (C
quat.
),
δ136.53 ppm (CH), δ142.63 ppm (Cquat.), δ173.39 ppm (Cquat. ).
(2S,3S,4S,5R)-6-(((2R,3R)-1-(4-cyano-3-(trifluoromethyl)phenyl)-2-ethyl-5-oxopyrrolidin
-3-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (M6) SARM 2f
(0.05 g;

Molecules 2023,28, 5541 12 of 14
0.17 mmol; 1 eq) was placed in a preheated Schlenk tube and was then diluted in toluene
(
1 mL
). AGME (0.08 g; 0.21 mmol; 1.2 eq) and Ag
2
CO
3
(0.05 g; 0.17 mmol; 1 eq) were
added. The reaction was covered with aluminum foil and was then stirred for 24 h at RT.
The suspension was filtered, and the precipitate was washed with EtOAc. The solvent was
then removed under reduced pressure, and the residue was dissolved in MeOH (6 mL).
KCN (5.4 mg; 0.08 mmol; 0.5 eq) was added at 0
◦
C, and the reaction was stirred for 2 h.
The reaction was extracted with EtOAc (3x), dried over MgSO
4
and then the solvent was
removed in vacuo. Due to the low yields of the reaction, no pure product was obtained.
3.4. 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
µ
M). For phase-I-metabolism, 10
µ
L of stock solution, 10
µ
L of NADPH
(
50 mM
), 5
µ
L of S9 fraction (20 mg/mL) and 5
µ
L of HLM (20 mg/mL) were added to
20
µ
L of phosphate buffer for a total volume of 50
µ
L. Samples were incubated at 37
◦
C
for 24 h. For phase-II-metabolism, an additional 10
µ
L of NADPH, 5
µ
L of S9 fraction,
5
µ
L of HLM, as well as 10
µ
L of UDGPA (50 mM), 10
µ
L of SL (50 mM) and 10
µ
L of
PAPS (20
µ
M) were added, and the mixture was incubated at 37
◦
C for 24 h. In addition,
t
wo n
egative 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
µ
L 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 vacu
um centrifuge (45
◦
C, 45 min), the samples were reconstituted in 100
µ
L of ACN:H
2
O
(90:10 v/v).
3.5. LC-HRMS
LC-MS data were generated using a Vanquish UHPLC system coupled with an Orbi-
trap 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
µ
m pre-column
from Macherey-Nagel (Düren, Germany) and a Poroshell 120 EC-C18 column (
3.0 ×50 mm
,
2.7
µ
m) 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/z50-800.
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

Molecules 2023,28, 5541 13 of 14
regularly calibrated using the Pierce Flex Mix calibration solution from Thermo Fisher
(Bremen, Germany).
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules28145541/s1, Figure S1: (Figures 1–18):
1
H NMR,
13
C
APT NMR of compounds; Figure S2: (Figures 19–26): mass spectra of the metabolites, found
in vitro
,
with predicted structures.
Author Contributions:
Conceptualization, M.T. and T.M.; synthesis, T.M.;
in vitro
analysis, N.N.,
O.K. and T.M.; LC-HRMS analysis, O.K. and T.M.; NMR measurements, H.-C.W. All authors have
read and agreed to the published version of the manuscript.
Funding:
This research was conducted with support of the Federal Ministry of the Interior and
Community of the Federal Republic of Germany.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
This project was conducted with the support of the Manfred-Donike-Institute
for Doping Analysis (Cologne, Germany).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not applicable.
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