Structural elucidation of major selective androgen receptor modulator (SARM) metabolites for doping control

Article abstract of DOI:10.1039/c7ob03030d

Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.

Full text of DOI:10.1039/c7ob03030d

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Structural elucidation of major selective androgen receptor
modulator (SARM) metabolites for doping control
Neeraj Garg,a Annelie Hansson,b,c Heather K. Knych,d,e Scott D. Stanley,d,e Mario Thevis,f
Ulf Bondessonb,c, Mikael Hedelandb,c and Daniel Globischa,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Selective androgen receptor modulators (SARMs) are a class of control and setting rules, whereas in horseracing the
androgen receptor drugs, which have a high potential to be International Federation of Horseracing Authorities (IFHA)
performance enhancers in human and animal sports. provides best practice guidelines and recommendations for
Arylpropionamides are one of the major SARM classes and get horseracing.1,
2
In horseraces prohibited substances are
rapidly metabolized signficantly complicating simple detection of categorized in two different compound classes: i) completely
misconduct in blood or urine sample analysis. Specific drug- banned substances, which are not supposed to be present in
derived metabolites are required as references due to a short half- the horse body at any time and ii) therapeutic substances,
life of the parent compound but are generally lacking. The which are allowed for animal caretaking but must be cleared
difficulty in metabolism studies is the determination of the correct from the horse body before a competition race.2, 3 One of the
regio and stereoselectivity during metabolic conversion processes. banned compound classes are the selective androgen receptor
In this study, we have elucidated and verified the chemical modulators (SARMs), which have selective anabolic effects on
structure of two major equine arylpropionamide-based SARM muscle and bone and were originally synthesized for
metabolites using a combination of chemical synthesis and liquid treatment of prostate cancer.4, 5 These properties make them
chromatography-mass spectrometry (LC-MS) analysis. These highly attractive for doping in sports events. Among a growing
synthesized SARM-derived metabolites can readily be utilized as variety of pharmacophores, four major compound classes of
reference standards for routine mass spectrometry-based doping SARMs are reported: arylpropionamides, bicyclic hydantoins,
control analysis of at least three commonly used performance- quinolones, and tetrahydroquinolines.5-7 The WADA prohibited
enhancing drugs to unambigously identify misconduct.
these compounds in 2008 from application in sport
competitions and since the first case in 2010 several athletes
8
have been tested positive.6, Recently, this compound class
has also been identified during animal sports events.9
Introduction
R2
Successful and efficient doping control in sports is a challenge
due to the rapidly changing development of new illegal drugs,
which affect the outcome and diminish the ethical integrity of
human and animal sports events. In human sports the World
Anti-Doping Agency (WADA) is the agency regulating doping
O2N
X
Y
R1
R3
R4
O
O
A
B
F3C
N
N
Z
H
H
OH
R5
1
Flutamide
X = NO2, CN, Halogen
Y = CF3, Halogen, CH3
Z = O, S, SO, SO2, NH
R1-R5 = Halogen, CN, NHCOMe
Figure 1: General structure of SARM analogues (1) with variable residues and
Flutamide. Highlighted in red are residues included in this study.
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Scheme 1: Overview of metabolic conversions of SARMs S1, S4 and S22 to the major metabolites identified in horse urine samples 3 or 6 h after drug
administration. Metabolic intermediate 3 is derived from ring A and the detectable part of SARM metabolites, while metabolites derived from 2 were
not detected. Compound 3 undergoes selective oxidation through P450 enzymes followed by sulphation to yield major detectable metabolites 5
and 7. The para-hydroxylation to the CF3-substituent of SARMs S1, S4 and S22-derived metabolites was prior unknown.
The focus of this study lies on the three arylpropionamide- i) Sample contaminations can be excluded as SARM-derived
based SARM compounds S1, S4 (Andarine) and S22 (Ostarine / metabolites must have been exposed to the metabolic system
Enobosarm). This SARM class was designed and developed of the horse; and ii) the detection time of urinary metabolites
from androgen receptor antagonists like flutamide (Figure 1).5 is significantly extended compared to that of the parent
More than 100 identified cases of misuse in human sport compound.15 Crucial for the confirmation of the presence of
events have been reported by the WADA including 60 cases of drug-derived or any other metabolite in any biological fluid are
S22 abuse in 2015 and 2016. S4 was identified for the first the availability of reference compounds for comparison of
time in an equine blood sample in 2016 during a routine chromatographic retention times and mass spectrometric
analysis.9 The number of SARM doping cases is expected to be properties.16 Mass spectrometric analyses include high-
significantly higher due to the lack of reference metabolites for resolution accurate mass and MS/MS fragmentation pattern,
routine urine analysis. Two different aromatic moieties A and B which are selective properties of each metabolite and
build up the core structure of this SARM class connected via important for the identification of the molecular structure.
modified propionamides, which have been synthesized at a However, reference compounds of these SARM-derived phase
high structural heterogeneity.7 Metabolism studies in II metabolites are not commercially available and no chemical
microsomes, fungal incubates as well as in urine samples from synthesis has been reported.
humans and horses described rapid and extensive metabolism We sought to overcome this limitation for arylpropionamide-
of these SARM compounds through a series of phase I and based SARM drugs and have synthesized the two major SARM-
phase II metabolic conversions.10-14 Four species-specific major derived phase II metabolites
5
and
(Scheme 1). Prior to our study, only the chemical
enzymatic hydrolysis of the amide bond, oxidation, sulphation, formula, MS/MS fragmentation pattern and the presence of a
glucuronidation and combinations of
these sulphate in the identified metabolite could be determined.12, 13
biotransformations. The hydrolysis of the amide bond yields The challenge was to identify and verify the correct
two separate metabolites and Scheme 1). Interestingly, hydroxylation site as several regioisomers are possible and
scaffold is conserved in most arylpropionamide drugs with cannot be determined by UHPLC-MS/MS analysis itself
nitro- or cyano-substitution in the ortho-position to the CF3- (Scheme 2a). In our present study, we have validated the
moiety embodying the two major modifications including structure of the modified para-position to CF3 using
drugs available on the black market.7 On the other hand, combination of chemical synthesis, UHPLC-MS and MS/MS
moiety has been synthesized with a high degree of different fragmentation experiments. The UHPLC-MS(/MS) data of the
substituents (Figure 1). synthesized molecules were compared to those of the
Mass spectrometry is the method of choice for doping control metabolites present in urine samples collected from the
investigations due to the high sensitivity and high-throughput corresponding SARM-treated horses or hours after
7 as well as intermediates 4
metabolic conversions have been described including and
6
2
3 (
A
a
B
3
6
analysis.1, 2, 6, 8 The variety of biotransformations complicates administration. The aim was to use the synthesized molecules
detection of misuse in doping control analysis resulting in a as reference standard metabolites for doping control
short half-life of these drugs. The structure of the investigations in equine sports events, which are required for
administered SARMs compound is only detectable in horse the unambiguous identification of SARMs S1, S4 and S22 drug
plasma up to 6 h post-administration and cannot be detected
in horse urine samples at all.12,
13
However, this rapid
metabolism can also be advantageous especially for highly
heterogeneous compound classes like arylpropionamide-based
SARMs. Especially, in the case of the three investigated SARM
drugs S1
conserved moiety
a cyano-substituent for S22 and a nitro-substituent for S1 and
,
S4 and S22 and their structural analogues with a
A
will yield the same metabolites class with
13
S4 12,
.
The analysis of metabolites instead of the parent
compound is advantageous due to two main reasons:
2 | J. Name., 2012, 00, 1-3
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Scheme 2: a) Possible regioisomers of hydroxylated SARM-metabolites. b) (i) NIS,
MeOH/THF (ii) CuI, Cs2CO3, 1,10-phenanthrolin-mono hydrate, MeOH (iii) BBr3, DCM
(iv) SO3-NMe3, NaHCO3, NaOH, H2O.
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1
steps by two H NMR signals, which were found to be either
5
two singlets or signals with a J long-range coupling constant
abuse in horses.
below 1 Hz for both aromatic protons. In case of hydroxylation
in the meta-position the signals would usually have a higher 4J-
Results and discussion
In previous studies, several metabolites derived from
arylpropionamide-based SARMs S1, S4 and S22 drugs have
been identified in horse urine and plasma samples.12, 13 In all
three cases, the chemical formula of the major metabolite was
identified as a monooxidized and sulphated metabolite of ring
A
upon amide hydrolysis of the parent compound through
ultra-high-performance liquid chromatography / quadrupole
time-of-flight mass spectrometry (UHPLC/QTOFMS) analysis.12,
13
This observation is consistent with reports that phase II
modification of phenolic xenobiotics in horses predominantly
undergo sulphation compared to glucuronidation in humans.3,
16
The most prevalent mass spectrometric peak can either be
the metabolite with the highest concentration, the highest
ionization properties or both.12,
13, 17
In the case of doping
analysis the highest signal selective for a prohibited substance
is an indication of misuse. Our study focuses on these ring A-
derived metabolites as newly developed analogues are mostly
modified in moiety
The difficulty for the design and synthesis of the main
metabolites of SARMs S1 S4 and S22 was the identification of
B, while aromatic ring A is conserved.
,
the oxidation site in these major metabolites. It is not possible
to assign the correct structure solely based on the chemical
formula and the mass spectrometric fragmentation pattern.
The high-resolution accurate mass allowed for the
determination of the chemical formula and four regioisomers
are plausible for SARM-derived compounds (Scheme 2a
,
depicted as hydroxylated phase I metabolites lacking the
sulphate group for simplification purposes). The three phenolic
compounds
as well as hydroxylamine 10 are possible. We identified a
specific product ion of [M+H 80 Da] in the MS/MS
4, 8 and 9 with different aromatic oxidation sites
-
fragmentation pattern for each metabolite class, which
demonstrates a sulphated phenolic compound (Figure 3b).18
The sulphated hydroxylamine of 10 can thus be excluded as
the fragmentation would yield a signal at m/z = 96 in negative
ionization mode analysis.18 To elucidate the correct structure
of this SARM-derived metabolite, we designed the synthesis
for the most likely monooxidized regioisomer in the para-
position of the CF3-moiety (Scheme 2b). Our design is based on
metabolic studies of the related drug flutamide in humans,
which represents a simplified scaffold of SARMs S1 and S4 19,20
Upon hydrolysis of the acetamide in flutamide, oxidation of
the para-position to the CF3-substituent ( ) was reported.
We initially focussed on the synthesis of cyano-metabolite
.
6
5,
one conceivable metabolite derived from the most prevalent
SARM drug.6, 7 The straightforward chemical synthesis of the
Figure 2: Comparison of the urinary natural and the synthesized purified cyano SARM-
derived metabolite 5: a) retention time comparison of the extracted ion
chromatograms on a biphenyl column. Sulfatase treatment hydrolysed metabolite 5
(bottom chromatogram); b) mass spectrometric fragmentation pattern. Urine samples
were collected 3h after drug administration; c) Comparison of synthesized metabolite 4
cyano-metabolite
4-amino-2-(trifluoromethyl)-benzonitrile 11 in the para-
position of CF3 using NIS in 90% yield intermediate 12 15, 21, 22
Cu(I)-catalyzed conversion of 12 into the corresponding
5 began with the regioselective iodination of
.
methylether 13 was accomplished prior ether cleavage using with the urinary metabolite after sulfatase treatment. (top right corner of each
chromatogram: signal intensities)
BBr3 to yield metabolite
4 in 34%. Hydroxylation in the para-
position of the CF3-moiety was confirmed for all synthetic
coupling constant of about 2 Hz. The synthesis of our target
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Figure 3: Comparison of the urinary natural and the synthesized purified nitro-SARM-derived metabolite 7 (SARM S1): a) UHPLC-MS co-injection using a biphenyl column (top right
corner of each chromatogram: signal intensities) and b) extracted mass spectrometric fragmentation pattern of the urinary and synthesized metabolite. Specific fragmentation
signals are highlighted with red arrows. Urine samples were collected 6h after drug administration.
molecule was finalized through selective O-sulphation using an enhanced mass signal of the metabolite. To provide
the sulfur trioxide trimethylamine complex. The high additional reference molecules for pretreated samples, we
selectivity of the reaction was accomplished due to the low have also analyzed the regiochemistry of the hydrolyzed
reactivity of the aniline amine, which was not sulphated metabolite
applying these conditions. The final compound was obtained As expected, also the synthesized metabolite
in 72% yield after HPLC-purification in highest purity (>99%) the hydrolyzed metabolite in equine urine samples had the
demonstrated by 19F NMR (Figure S1).
same retention time during UHPLC-MS experiments
This synthesized reference molecule Figure 2c).
the metabolite identified in urine samples collected from The subsequent analysis of the corresponding sulphated nitro-
SARM S22-treated horses 3h after administration. The correct SARM-derived metabolite paralleled our findings from
structure of this cyano-SARM-derived metabolite was cyano-metabolite and also confirmed hydroxylation of the
4, which is an intermediate in our synthetic route.
5
4
and the peak of
5
was then compared to
(
7
5
confirmed by UHPLC-MS analysis using a biphenyl-column. The para-position to CF3 for SARM S1 metabolism in samples
same retention time as the natural metabolite and the collected 6 h after administration. We adapted the same
matching accurate mass as deduced from the deprotonated synthetic route as for the cyano-SARM metabolites
ion at m/z = 280.9858 corroborated the correct structure of obtain arylpropionamide-based nitro-SARM metabolites
metabolite Figure 2a). Co-spiking experiments at a ratio of in comparable yields (Figure S3). This synthesized reference
1:1 resulted in one perfectly overlapping peak without molecule was also obtained in highest purity (> 99%) after
additional band broadening in the extracted ion HPLC purification as demonstrated by 19F NMR spectroscopy
chromatogram (Data not shown). As a second stationary Figure S4). UHPLC-MS analysis on two different stationary
4
and
5
to
6
and
5
(
7
7
(
phase, we tested the retention time of the natural metabolite phases (C18 and biphenyl) resulted in overlapping
and the synthesized molecule on a C18-column. Using this chromatographic peaks as shown by the extracted ion
column, we also obtained perfectly overlapping peaks to add a chromatogram (EIC) of the synthesized and the natural urinary
second level of confirmation to validate the correct drug metabolite (Figure 3a and Figure S5). Spiking of the
regioisomer for the sulphated cyano-metabolite
The presence of a sulfate-ester in urine metabolite
confirmed upon sulfatase treatment with the disappearing with the accurate mass deduced from the deprotonated ion at
mass signal Figure 2a). Furthermore, the fragmentation m/z = 300.9740. The fragmentation pattern for the synthesized
pattern of the synthesized metabolite perfectly aligns with the and the urinary metabolite perfectly match as shown in Figure
5
(
Figure S2). synthesized metabolite into the urine sample at a 1:1 ratio
5
was also resulted in a single peak in LC-MS co-injection experiments
(
identified metabolite in horse urine samples (Figure 2b).
Due to the lack of reference material, treatment of urine
samples with enzymatic mixtures (sulphatases and
glucuronidases) to hydrolyse phase II metabolites is
standardized in most laboratories prior mass spectrometric
3b.
Conclusions
Herein, we describe the elucidation and verification of the
chemical structure of major metabolites derived from SARM
23
doping control analysis.3, This pre-treatment is required to
gain higher quantities of metabolites at low concentrations for
analogues S1
,
S4 and S22 in equine urine samples collected
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after intravenous administration.12, 13 The analysis of specific
drug-derived metabolites significantly extends the detection
time for misconduct compared to analysis of the parent
compound. By applying a new and straightforward synthesis
11
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12
13
for the four SARM-derived phase II metabolites
as phase I metabolites and , we validated the correct
structures through UHPLC-MS co-injection experiments. The
sulphated metabolites and have not been synthesized
5 and 7 as well
4
6
5
7
14
15
16
before and can be analysed in urine samples without
enzymatic pre-treatment of the sample simplifying doping
analysis of these drugs. These compounds were used to
validate the correct metabolite structure in authentic urine
samples collected from SARM-treated horses. The high purity of
these synthesized compounds allows for the direct use
unambiguous identification of the consumption of illegal
SARM-drugs during sports events. These SARM-derived
metabolites cannot be endogenously produced by the equine
system due to the non-natural CF3-moiety. Highly
heterogeneous structures of arylpropionamide-based SARM
drugs have been developed and our synthesized metabolites
have a high potential to be applicable as reference compounds
for a series of new and unknown synthetic analogues.
17
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Conflict of interest
The authors declare no conflict of interest.
22
23
Acknowledgements
This study was supported by a starting grant by the Science for
Life Laboratories (to DG).
1
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