Discovery of diarylhydantoins as new selective androgen receptor modulators

Article abstract of DOI:10.1021/jm300249m

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

Full text of DOI:10.1021/jm300249m

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Article
Discovery of Diarylhydantoins as New Selective
Androgen Receptor Modulators (SARMs)
François Nique, Severine Hebbe, Christophe Peixoto, Denis Annoot, Jean-Michel
Lefrançois, Eric Duval, Laurence Michoux, Nicolas Triballeau, Jean-Michel Lemoullec,
Patrick Mollat, Maxime Thauvin, Thierry Prangé, Dominique Minet, Philippe Clément-
Lacroix, Catherine Robin-Jagerschmidt, Damien Fleury, Denis Guédin, and Pierre Deprez
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm300249m • Publication Date (Web): 16 Aug 2012
Downloaded from http://pubs.acs.org on September 2, 2012
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Discovery of Diarylhydantoins as New Selective
Androgen Receptor Modulators (SARMs)
François Nique,§ Séverine Hebbe,§ Christophe Peixoto,§ Denis Annoot,§ Jean-Michel Lefrançois,§ Eric
Duval,‡,§ Laurence Michoux,#,§ Nicolas Triballeau,§ Jean-Michel Lemoullec,**,§ Patrick Mollat, §
Maxime Thauvin,† Thierry Prangé,† Dominique Minet,§ Philippe Clément-Lacroix,§ Catherine Robin-
Jagerschmidt,§ Damien Fleury,§ Denis Guédin,**,§ Pierre Deprez.*,§
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§ GALAPAGOS, Parc Biocitech, 102 Avenue Gaston Roussel, 93230 Romainville, France
† UMR 8015 CNRS, Université Paris Descartes, 4 Avenue de l’Observatoire, 75006 Paris, France
‡ Present address: Directorate Organic Chemistry, European Patent Office, Patentlaan 2, 2288 EE
Rijswijk, The Netherlands
# Present address: Isochem, 4 avenue Philippe Lebon, 92230 Gennevilliers, France
** Present address: Institut de Recherche Servier, 125 chemin de ronde, 78290 Croissy-sur-Seine, France
* To whom correspondence should be addressed. Phone: +33 149424687. Fax: +33 149424658. E-mail:
pierre.deprez@glpg.com
Abstract
A novel selective androgen receptor modulator (SARM) scaffold has been discovered through structural
modifications of hydantoin antiandrogens. Several 4-(4-hydroxyphenyl)-N-arylhydantoins displayed
partial agonism with nanomolar in vitro potency in transactivation experiments using androgen receptor
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(AR) transfected cells. In a standard castrated male rat model, several compounds showed good anabolic
activity on levator ani muscle, dissociated from the androgenic activity on ventral prostate, after oral
dosing at 30 mg/kg. (+)-4-[3,4-dimethyl-2,5-dioxo-4-(4-hydroxyphenyl)imidazolidin-1-yl]-2-
trifluoromethylbenzonitrile ((+)-11b) displayed anabolic potency with a strong dissociation between
levator ani muscle and ventral prostate (A50 = 0.5 mg/kg vs 70 mg/kg). The binding mode of two
compounds including (+)-11b within the AR ligand binding domain has been studied by co-
crystallization experiments using a coactivator-like peptide. Both compounds bound to the same site and
the overall structure of the AR was very similar.
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Introduction
In males, testosterone (T) and 5α-dihydrotestosterone (DHT) are the endogenous hormones which
drive sexual development at puberty, maintenance of bone and muscle mass and strength, body
composition and sexual function in adulthood. The physiological effects of androgens are mediated by
the androgen receptor (AR), which belongs to the nuclear hormone receptor superfamily of intracellular
ligand-dependent transcription factors. Deficiencies in circulating androgen levels in male serum can be
caused either by hypogonadism or more often as a result of the aging process, which leads to
musculoskeletal functional decline and other effects.1 Cognitive disorders like Alzheimer’s disease have
also been linked to testosterone deficiency.2 It has been shown clinically that administration of
testosterone at physiological doses in elderly men improves both muscle strength and body
composition.3 However, unlike estrogen replacement therapy which has found extensive use in
menopausal women, androgen replacement therapy is not widely used due to potential side effects. The
main concern about testosterone replacement therapy is the possible impact on the prostate, i.e. increase
in benign prostatic hypertrophy and/or stimulation of a non-diagnosed prostate cancer.4 Moreover,
because of its steroidal structure, testosterone is not orally available due to rapid first pass metabolism in
the liver which produces DHT and estrogens.
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Journal of Medicinal Chemistry
Many efforts were initiated a decade ago to find orally available non-steroidal androgens.5 The
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improvement in safety that were achieved in non-steroidal selective estrogen receptor modulators
(SERMs) in post-menopausal women6 prompted the search for selective androgen receptor modulators
(SARMs) for male use.7 These compounds should offer a better distinction between desired anabolic
and unwanted androgenic properties. Turning non-steroidal androgen receptor antagonists into orally
active agonists has already been accomplished with several chemical scaffolds as exemplified by the
bicalutamide-derived propionamides 1 (andarine)8 and 2 (ostarine)9 or the design of the fused bicyclic
hydantoin 3 (BMS-564929)10 shown in Figure 1. Compounds from both series have been developed,
and some have reached the clinical stage.11
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HO
H
HO
O
HO
O
H
H
N
O
O
O
N
N
N
NHAc
CN
O2N
NC
O
CF3
CF3
NC
Cl
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OH
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O
NH
N
N
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N
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O
O2N
O
NC
CF3
CF3
(R)-(+)-11b
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Figure 1. Non-steroidal androgen receptors ligands
Our own early discovery program led us to investigate the N-arylhydantoin core of the antiandrogen
nilutamide 412 as a starting point. We have identified (R)-(+)-4-[3,4-dimethyl-2,5-dioxo-4-(4-
hydroxyphenyl)imidazolidin-1-yl]-2-trifluoromethylbenzonitrile (R)-(+)-11b as a new SARM from
structure-activity relationship (SAR) studies in a series of 1-aryl-4-hydroxyphenylhydantoins.
Compound (+)-11b displayed tissue selectivity in animal models upon oral administration with reduced
androgenic effect on prostate as compared to anabolic activity on muscle. Structural analyses from this
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new series have been used to obtain insight into the features driving tissue selectivity. Herein we report
the discovery of this new biarylhydantoin SARM scaffold.
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Chemistry
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The general synthesis of hydantoins (±)-11a-f is depicted in Scheme 1. These compounds were
prepared by condensation of amino ester 8 and commercially available isocyanate 10a or isocyanates
10b-f, obtained from the corresponding anilines 9b-f (96-100%). The amino ester 8 was obtained from
the hydroxyphenyl propionic acid 6 through successive esterification, acetylation and bromination α to
the aryl ring to provide the bromo ester 7 (quantitative overall yield). Treatment of 7 with an ethanolic
solution of methylamine gave rise to the amino ester 8 (78%) through substitution of the bromo atom
and in situ deprotection of the acetate group. The ring forming condensation of 8 on the isocyanates
10a-f provided the diarylhydantoins 11a-f as racemic mixtures (45-96%).
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Scheme 1a
OH
OAc
OH
O
O
O
HO
O
O
a, b, c
d
OH
Br
NHMe
6
7
8
N
O
O
N
N
Cl
f
Cl 10a
O
R
NH2
N
11a-f
a R = 3Cl,4Cl
b R = 3CF3,4CN
O
e
R
R
9b-f
10b-f
c R = 3OMe,4CN
d R = 2Me,3Cl,4CN
e R = 2Me,3CF3,4CN
f R = 2Me,4CN,5CF3
a Reagents and conditions: (a) SOCl2, MeOH, 0 °C; (b) Ac2O, K2CO3, THF; (c) NBS, AIBN, CCl4,
rfx, 100% (3 steps); (d) MeNH2, EtOH, 78%; (e) triphosgene, toluene/dioxane, 110 °C, (96-100%); (f)
THF, rfx, 45-96%.
The racemate (±)-11b was resolved by chiral HPLC to provide the single enantiomers (+)-11b and
(−)-11b. Assignment of the absolute stereochemistry (R and S respectively) was done by comparison of
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Journal of Medicinal Chemistry
these enantiomers with the authentic compounds prepared by enantioselective synthesis as described
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below.
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Analogous thiohydantoins (±)-13a-b were prepared in a similar manner by coupling the amino ester 8
with the appropriate thioisocyanates 12a-b. Thioisocyanate 12a is commercially available and 12b was
prepared by reacting aniline 9b and thiophosgene (Scheme 2).
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Scheme 2a
OH
N
S
Cl
O
Cl
8
N
N
12a
N
b
NH2
S
S
R
13a- b
NC
NC
a
a R = 3Cl,4Cl
CF3
CF3
9b
b R = 3CF3,4CN
12b
a Reagents and conditions: (a) CSCl2, H2O, rt, 86%; (b) NEt3, THF, rt, 18 h, 75-77%.
Substitution of the bromo atom of 7, by either allylamine or propargylamine, provided the amino
esters 14 and 16 respectively (Scheme 3). Coupling of isocyanate 10b and 14 or 16 produced the
hydantoins (±)-15b (73%) and (±)-17b (89%).
Scheme 3a
OH
OH
O
O
10b
7
N
R
N
O
a
b
NHR
O
NC
CF3
15b R = -CH2-CH=CH2
CH
14 R = -CH2-CH=CH2
CH
16 R = -CH2-C
17b R = -CH2-C
a Reagents and conditions: (a) allylamine or propargylamine, THF, rt, 30 min; (b) THF, rfx, 73-89%.
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The individual enantiomers (−)-11b, (+)-11b and (+)-11c have been prepared enantiopure according
to an analogous route, coupling the appropriate isocyanates with the suitable enantiomerically pure
amino ester. As the synthesis of the chiral N-methyl amino esters (S)-8 and (R)-8 was not obvious, we
used methyl 2-amino-2-(4-methoxyphenyl)propionates, (S)-(+)-20 and (R)-(−)-20, as the starting
materials (Scheme 4). The synthesis of the ethyl ester homologue of (S)-(+)-20 has been described in the
literature.13 We used a similar process to prepare (S)-(+)-20 by simply replacing ethanol with methanol
in the final esterification step. (R)-(−)-20 was obtained by the same sequence using (R)-(+)-α-
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methylbenzylamine as the resolving base instead of its (S)-(−)-enantiomer.
The amino ester (R)-(−)-20 was coupled to the isocyanates 10b and 10c to produce hydantoins (R)-
(+)-21b and (R)-(+)-21c in excellent yields (95-97%). These compounds were further subjected to an N-
methylation, O-demethylation sequence to provide the hydantoins (R)-(+)-11b (77%) and R-(+)-11c
(54%). The enantiomer (S)-(−)-11b was prepared the same way using the amino ester (S)-(+)-20.
Scheme 4.a Synthesis of Pure Enantiomers (R)-(+)-11b-c.
OMe
OMe
OMe
b
O
O
OMe
O
R
HO
HO
HN
NH
NH
NH
a
c
d
O
O
O
O
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19
(-)-19
OMe
OH
OMe
R
10b-c
O
R
O
O
R
O
NH
N
e
f, g
N
N
NH2
O
O
R
R
(-)-20
(+)-21b-c
(+)-11b-c
b R = 3CF3,4CN
c R = 3OMe,4CN
a Reagents and conditions: (a) (NH4)2CO3·H2O, KCN, EtOH, H2O, 55 °C, 3 h, 99%; (b) NaOH, Ac2O,
rfx, 64 h, 93%; (c) (R)-(+)-α-methylbenzylamine, EtOH, 40 °C, then HCl 2 N, 31%; (d) HCl 4 N, rfx, 4
h then HCl, MeOH, rfx, 15 h, then NaHCO3, 84% (2 steps); (e) THF, NEt3, rfx 2 h, 95-97%; (f) NaH,
MeI, DMF, rt, 20 min; (g) BF3.Me2S, rt, 54-77% (2 steps).
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Journal of Medicinal Chemistry
It was not possible to prepare N-alkyl thiohydantoins using this method due to the higher reactivity of
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the thiocarbonyl vs the intracyclic nitrogen towards alkylation. To prepare the N-methylated
thiohydantoin (R)-(+)-13a, the amino ester (R)-(−)-20 was converted to its o-nitrophenyl sulfonyl
derivative 22 (40%) to allow an easy N-alkylation (Scheme 5). Methylation of 22 followed by
sulfonamide cleavage proceeded in good yield (85%). The amino ester R-(−)-23 thus obtained was then
subjected to the coupling with thioisocyanate 12a (95%) and then O-demethylation to yield the
thiohydantoin R-(+)-13a (70%).
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Scheme 5.a Synthesis of Pure Enantiomer (R)-(+)-13a.
OMe
O
OMe
R
OMe
O
O
O
R
12a
R
NH
O
O
S O
O
a
b, c
d
NH
NH2
(-)-20
NO2
22
(-)-23
OMe
OH
R
R
O
O
N
N
N
e
N
S
S
Cl
Cl
Cl
Cl
(+)-13a
(+)-24a
a Reagents and conditions: (a) 2-NO2PhSO2Cl, pyridine, 1.5 h, rfx, 40%; (b) NaH, MeI, DMAC, 3 h,
rt; (c) K2CO3, thiophenol, DMAC, 2 h, rt, 85% (2 steps); (d) THF, NEt3, rt, 1 h, 95%; (e) BF3.Me2S, rt,
24 h, 70%.
As described below, the R absolute configurations of compounds (+)-11b and (+)-13a have been
further confirmed by solving the co-crystal structure of each compound bound to the ligand binding
domain of the androgen receptor (AR LBD).
Results and Discussion
In order to identify a selective androgen receptor modulator (SARM), we needed to choose
compounds that display a strong dissociation between the prostate (where ideally the compound should
not be an agonist) and muscle and/or bone (where ideally it should be a stronger agonist). Since the
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same androgen receptor is expressed in all three tissues, the origin of a SARM profile is not likely to be
traced back by a strong differential tissue potency. Conversely, partial activation of the AR might lead
to the desired tissue selectivity. The reason for this prospect is explainable within the framework of the
Monod-Wyman-Changeux model,14 where the receptor is in equilibrium between two states, the resting
state (R) and the active state (A). We believed this model provided a perfect framework for evaluation
of partial agonism. Any ligand of the receptor actually has two molecular affinities for the receptor: one
for the active state A (denoted KA) and one for the inactive state R (denoted KR). Applying the law of
mass action to each of the two states leads to the potency equation.
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In the absence of ligand, the equilibrium constant L is the ratio between the resting and active state of
the receptor, and this equilibrium is tissue dependent. In a given tissue, a ligand will shift the
equilibrium constant L by a factor depending on the ratio of its two affinities for the two states KR and
KA. We call this ratio KR/KA the molecular efficacy since it directly reflects the amplitude of the
discrimination the ligand exerts between the two states of the androgen receptor. To bring the activation
in a range where it will have an effect, the discrimination (i.e. molecular efficacy of the ligand) must
overwhelm the tissue dependent initial position L of the equilibrium: a smaller L will allow a compound
with a smaller efficacy to be active whereas a larger L will require a stronger molecular efficacy. In
other words, if two tissues differ by a factor of 10 in their L constants (ratio between the resting and
active state of the receptor), the tissue with the smallest L will allow a partial agonist to display activity
whereas the tissue with a 10 fold larger L will see it as neutral antagonist. Highly discriminated
compound (i.e. high molecular efficacy, also meaning full agonist) will show agonist activity anyway in
the two tissues. Therefore, the success of the approach using the Monod-Wyman-Changeux lies in the
hope that tissues may differ in their initial position of the equilibrium between the resting and the active
state, with prostate having an equilibrium shifted more towards the inactive state than bone and muscle,
thus allowing compounds with a moderate molecular efficacy to display their activity in one tissue
(bone and muscle) and not in the other (prostate). This hypothesis, although not formally established,
offers the great advantage to discriminate the parameters linked to the compound (i.e. the molecular
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Journal of Medicinal Chemistry
efficacy KR/KA) and the parameter linked to the tissue (i.e. equilibrium constant L). Of course, the
position of the equilibrium between the active and inactive state of the androgen receptor in the different
tissues is not the only explanation to the tissue selectivity. Densities of androgen receptor, spare
receptors, concentration of co-activator and co repressor are also varying from one tissue to the other
and all these factors play a role in the overall tissue selectivity. In our approach, we focused essentially
on the identification of partial agonists with limited activity even at saturation. For this purpose, we used
the Monod-Wyman-Changeux model heuristically, combining in vitro data (partial agonism) and in
vivo data (in vivo tissue selectivity), and actually found it convenient and reliable to progress along the
exploration of the chemical series.
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The scale we chose ascribes a molecular efficacy of 4000 to DHT (full agonist) and 1 to nilutamide
(neutral antagonist). Viewed as a log scale, the middle of this window (between 30 and 300) appeared to
us as defining the compounds having intermediate molecular efficacy, thus qualifying them for the
nomination of partial agonists, potentially capable to display tissue selectivity. Then, on selected
compounds, we confirmed in vivo that the in vitro partial agonism profile converts to tissue selectivity
with limited activity on the prostate and higher activity on muscle or bone in the Hershberger model.
Practically, in order to evaluate in vitro potency and molecular efficacy of our compounds, we set-up
a transcriptional assay using Hela cell line transfected with hAR receptor and with an Androgen
Responsive Element (ARE) coupled with luciferase. For each SARM compound prepared, we generated
dose-response curves, crossing a range of concentrations of DHT with a range of concentration of our
SARM compound. In this transcriptional assay, potency for DHT is 8 nM and molecular efficacy 4000.
We observed modifications of the DHT dose-response curve when different concentrations of SARM
are added. This led to the identification of SARM compounds with potency of 1-10 nM and efficacy of
30-150. When looking at the shape of the curves, the Hill coefficient of our SARM compounds in the in
vitro transactivation assay, it appears to be close to 1, thus indicating no apparent cooperativity. This
assay was also validated using reference compound that progressed in clinical development such as
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compound ostarine 2 known for its tissue-selective anabolic effect.7c In our assay, 2 had good potency
(1.5 nM) and a molecular efficacy of 60, which in our assay is indicating partial agonism.
4,4-Dimethyl hydantoins15 have long been known as antagonists to the androgen receptor, as
exemplified by nilutamide 4.12 Replacement of one methyl of this hydantoin core by a phenyl ring did
not change the antagonistic nature of the molecule in vitro (potency = 37 nM, molecular efficacy = 3),
so did the corresponding 4-methoxyphenyl analogue (N-methyl analogue of 21b, potency = 150 nM,
molecular efficacy = 1). However, the introduction of a 4-hydroxyphenyl group as in compounds 11a-f
turned their profile to partial agonism (potency = 1-10 nM, molecular efficacy = 100-300).
In vitro
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The in vitro functional activity of 1-aryl-4-(4-hydroxyphenyl)hydantoins has been assessed in cells
expressing human androgen receptor (hAR). The results of transcriptional activities of compounds 11a-
f, 13a-b, 15b and 17b are listed in Table 1 as potency and molecular efficacy values.
Table 1. In Vitro and in Vivo Activities of 1-Aryl-4-(4-hydroxyphenyl)hydantoins.
OH
O
N
R
N
Ar
X
Anabolic in
Androgenic
In vitro
potency molecular
(nM)
In vitro
vivo activity in vivo activity
Chiral
center
Cpda
Ar
X
R
levator ani
muscle
(%)
ventral
prostate
(%)
efficacy
TPb
-
-
-
-
1.5
10.9
1.6
600
150
114
48
99
12
84
-5
73
0
(±)-11a
(±)-11b
(−)-11b
(+)-11b
3Cl,4Cl Ph
3CF3,4CN Ph
3CF3,4CN Ph
3CF3,4CN Ph
O
O
O
O
O
O
Me
Me
Me
Me
Me
Me
RS
RS
S
14
5
200
0.9
R
132
141
200
75
96
132
25
51
30
(±)-11c 3OMe,4CN Ph
(+)-11c 3OMe,4CN Ph
RS
R
2.1
1.5
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(±)-11d 2Me,3Cl,4CN Ph
O
O
O
S
Me
Me
RS
RS
RS
RS
R
1.5
3
300
100
10
82
nd
nd
13
nd
12
15
95
12
nd
nd
0
1
2
3
4
5
6
7
8
(±)-11e 2Me,3CF3,4CN Ph
(±)-11f 2Me,4CN,5CF3 Ph
Me
200
2
(±)-13a
(+)-13a
(±)-13b
(±)-15b
3Cl,4Cl Ph
3Cl,4Cl Ph
Me
122
200
80
S
Me
0.8
4
nd
4
9
10
11
12
13
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15
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17
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60
3CF3,4CN Ph
3CF3,4CN Ph
3CF3,4CN Ph
S
Me
RS
RS
O
O
Allyl
2
89
-3
19
(±)-17b
Propargyl RS
2.4
110
a
All the test compounds except (+)-11c have been administered orally with a dose of 30 mg/kg; (+)-
b
11c has been administered orally with a dose of 10 mg/kg. Testosterone propionate (TP) has been
administered subcutaneously at 1 mg/kg.
Most of the compounds displayed a partial agonist profile. The N-substituents evaluated at position 3
of the hydantoin core did not influence the activity in the cell-based assay: N-methyl (±)-11b, N-allyl
(±)-15b and N-propargyl (±)-17b displayed similar potencies (potency = 1.6, 2 and 2.4 nM
respectively), in the range of that of DHT. However, as compared to DHT, the molecular efficacy was
much lower for these three compounds (around 100 vs 4000), in agreement with our goal of identifying
partial agonists. As a consequence, further optimization was carried out around similar compounds as
described in Table 1, with the simplest methyl substituent on the nitrogen in position 3. Substituents of
the N-phenyl in position 1 had a pronounced effect on potency, but for the most part maintained the
profile of a partial agonist. One of the most potent compounds of the series, (±)-11b, contained a 4-
cyano-3-trifluoromethylphenyl, which is present in many AR ligands. Replacement of the electron-
withdrawing trifluoromethyl group in (±)-11b by the electron-donating methoxy group led to (±)-11c,
with a similar in vitro profile (potency = 2.1 nM). On the other hand, compound (±)-11a bearing a 3,4-
dichlorophenyl was ten times less potent than (±)-11b (potency = 10.9 nM) due to a lack of a H-bond
acceptor group like the cyano. Several trisubstituted phenyl derivatives were explored with (±)-11d and
(±)-11e which retained similar potency as (±)-11b indicating that some steric hindrance in position 2 of
that phenyl ring was tolerated. On the other hand, the 4-cyano-2-methyl-5-trifluoromethylphenyl (±)-11f
was much less potent (potency = 200 nM) than the isomeric (±)-11e and behaves more like an
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antagonist with a molecular efficacy of 10. In (±)-11f the steric hindrance was much more pronounced
than in (±)-11e as two substituents are on opposite sides of the phenyl ring, precluding a correct fit in
the binding pocket of the AR. The configuration at the chiral center had a strong effect on the activity in
the cell-based assay. Compound (±)-11b has been resolved into its pure enantiomers which were tested
for AR transcriptional activity. Compound (+)-11b appeared to be >200-fold more potent (potency = 0.9
nM) than its antipode (−)-11b (potency = 200 nM) and 2-fold more potent than the racemate, as
expected. The compound (+)-11b was also confirmed as a partial agonist with a molecular efficacy of
132, allowing a theoretical differentiation between prostate, muscle and bone tissue (confirmed in the in
vivo experiments). After both R and S stereoselective syntheses had been performed (scheme 4), the R
configuration was attributed to the more potent (+)-11b and the S configuration to (−)-11b. This was
confirmed by structural analysis of (+)-11b co-crystallized with hAR LBD. The R enantiomer of 11c
has been similarly prepared giving rise to (+)-11c which showed an almost 2-fold higher activity as
compared to the racemic mixture (±)-11c (potency = 1.5 and 2.1 nM, respectively). In competitive
binding assays (+)-11b was specific for AR with an IC50 of 9.6 nM. A weak binding was recorded for
the progesterone receptor (IC50 = 8.8 µM) with no significant binding of the estrogen and glucocorticoid
receptors up to 10 µM.
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Replacement of the urea carbonyl of the hydantoin ring by a thiocarbonyl gave rise to the
thiohydantoins (±)-13a and (±)-13b. These two compounds are potent and partial agonists (molecular
efficacy = 122 and 80), but detailed effect of this replacement is compound dependent. A 5-fold increase
in transcriptional activity was recorded with thiohydantoin (±)-13a (potency = 2 nM) as compared to the
hydantoin (±)-11a, while a 2-fold decrease was noticed with thiohydantoin (±)-13b (potency = 4 nM)
versus the hydantoin (±)-11b. Nevertheless, the R enantiomer (+)-13a prepared stereospecifically was
shown to be twice as active as the racemate (±)-13a (potency = 0.8 nM) and partial agonist.
In vivo
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As several of these compounds displayed activity profiles of partial agonists in vitro with potencies
close to that of testosterone, we evaluated the ability of these compounds to be tissue selective by
stimulating muscle anabolism with limited effect on prostate growth in an orchidectomized (ORX) rat
model. In this model, young castrated rats are left untreated for one week and then treated orally for four
successive days with a partial agonist at 30 mg/kg. Untreated ORX rats and sham-operated animals were
used as controls. Another control group was treated subcutaneously with 1 mg/kg testosterone
propionate (TP). The weight of the levator ani muscle (LA) was considered a marker of anabolic
activity whereas the weight of the ventral prostate (VP) was regarded as a marker of androgenic
activity.16 Results of these experiments are shown in Table 1. At 30 mg/kg, partial agonists (±)-11b, (±)-
11c, (±)-11d and (±)-17b restored the weight of LA around that of sham control animals while the
prostate weights remained far below those of intact animals, in accordance with their in vitro activities.
This illustrates a significant distinction between the anabolic (muscle) and androgenic (prostate) effects
and correlation with the in vitro profiling of the compounds. The pure enantiomers (+)-11b and (+)-11c
behaved similarly and the antipode molecule (−)-11b was inactive, as expected from its lower in vitro
activity. Another less potent compound, the dichlorophenyl (±)-11a, did not show any activity either.
Despite good profiles, N-allyl derivative (±)-15b and the thiohydantoin (±)-13b were devoid of any in
vivo activity. The reasons were not investigated and could be due to lack of oral bioavailability and/or a
higher susceptibility to metabolism caused by the presence of the sulfur atom (for the thiohydantoins).
On the other hand, the N-propargyl (±)-17b and N-methyl (±)-11b with very similar in vitro profile
(potency and molecular efficacy) also behaved in the same way in vivo with a potent anabolic activity
on muscle and strong tissue differentiation versus prostate (95-84% on LA muscle compared to 19-14%
effect on VP). 11b was preferred to 17b for further studies due to a potential toxicity of the triple bond
in 17b (linked to possible oxidation of the triple bond and formation of further reactive metabolites).
In order to further explore the anabolic potency and tissue selectivity of (+)-11b, a wide dose-range
experiment was performed in comparison with TP. TP showed similar dose-response curves for both
LA and VP with A50 = 0.30 and 0.20 mg/kg respectively, A50 being the active dose that induced 50% of
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activity (sham = 1, ORX = 0) (Figure 2) and a maximal effect on both LA and VP of 130% (vs sham).
Conversely, (+)-11b demonstrated an A50 similar to TP on LA (A50 = 0.50 mg/kg) with a maximal effect
on LA around 80%. As expected, (+)-11b had a weak effect on VP (A50 = 70 mg/kg), showing a strong
dissociation between anabolic and androgenic effects (factor 140). Unlike TP, (+)-11b did not show an
increase in LA weight over that of sham animals even at very high doses (300 mg/kg). In contrast to TP
at this high dose, (+)-11b caused only a 50% increase of the VP weight, reflecting the partial agonist
profile of (+)-11b. These data clearly demonstrate the tissue selectivity of (+)-11b and warrant its
classification as a SARM.
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Figure 2. Comparison of the anabolic versus androgenic activities of (+)-11b and testosterone
propionate in ORX rats. A50, dose displaying 50% activity on levator ani or ventral prostate.
Effect of compound (+)-11b on bone was also evaluated in an ORX rat model. In this model, six
month-old male rats were castrated and treated with 30 mg/kg (+)-11b, once-daily, oral administration
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for three months in a preventive manner. As expected, the ORX surgery induced a significant bone loss
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2
3
4
as indicated by BMD (Bone Mineral Density, pQCT, Stratec) of −27% and a trabecular BV/TV (Bone
5
6
7
8
volume; µCT, Scanco) of −42% vs sham. The bone loss observed in ORX animals was fully prevented
by (+)-11b on both BMD and BV/TV parameters. This protection was correlated with bone
biomechanic properties of trabecular and cortical bones. Detailed bone data will be reported elsewhere.
This indicates that the partial agonist (+)-11b is also effective in preventing ORX-induced bone loss,
indicating that (+)-11b is active on bone in addition to muscle. Activity on prostate and LA after three
months of treatment were 30 and 124% respectively after oral administration of (+)-11b 30 mg/kg/day.
Total cholesterol was significantly decreased by 2-fold in ORX rats treated with (+)-11b (measure on
HDL is not relevant in rats). Triglycerides were significantly decreased by 30% in ORX rats treated
with (+)-11b.
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Structural biology, crystallography
In an attempt to compare binding modes of DHT and our hydantoin compounds, co-crystals of the
hAR LBD linked to a coactivator-like peptide17 were obtained with (+)-11b and (+)-13a.18 It has been
shown that the binding of different ligands (agonist, antagonist) critically affects the position of helix
H12, with respect to the interaction with different co-regulators.19 Both crystals generated in our work
showed the agonist conformation of the AR LBD, with helix H12 in the position observed upon binding
of DHT.18 We considered the possibility that the presence of a coactivator peptide in the crystals might
affect the dynamic of the helix H12. But actually, in the absence of a coactivator peptide, the AR LBD
adopted the same agonist conformation in the crystal structure with the partial agonist 1.20 This tends to
indicate that the coactivator peptide does not force the agonist conformation but participates to the
stabilization of the complex.The structures of the two hydantoin complexes are very similar with a root-
mean-square deviation (RMSD) of 0.39 Å calculated on all the atoms. The hydantoin molecules were
positioned in exactly the same way in the binding pocket, the R configuration being thus confirmed at
their asymmetric centers.
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Figure 3 displays a comparison of (+)-11b and (+)-13a to DHT in the hAR LBD binding pocket. The
cyano group of (+)-11b is in the same position as the 3-keto group of DHT allowing hydrogen bonding
with residue Arg752 of the receptor protein (2.89 Å), like DHT (Figure 3A). The dichlorophenyl
compound (+)-13a lacks the cyano group (Figure 3B), but remains as active as the cyano derivative (+)-
11b. In both compounds, the hydroxyphenyl group extends from the core of the molecule at the same
place as the 18-methyl of DHT. Conversely to what has been described for DHT,18, 21 no interaction
with Thr877 and Asn705 was possible for (+)-11b and (+)-13a, but a further hydrogen bond was evident
with His874 (2.77 Å for (+)-11b and 2.75 Å for (+)-13a respectively). This has already been observed
with the propionamide SARM 1 which was described to interact with His874, albeit through a water
molecule.20 In all three cases, the binding of the compound induced a widening of the pocket so that the
receptor could accommodate the protruding aryl groups.
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Figure 3. Superimposition of co crystals of (+)-11b (A) and (+)-13a (B) with DHT. Each complex was
superimposed on the co-crystal structure with DHT (PDB code: 2AMA) in Discovery Studio22 by
minimizing the RMSD of the Cα traces. (A) Superimposition of (+)-11b with DHT. The complex of
hAR LBD with (+)-11b is represented in green and DHT is pink. The cyano group of (+)-11b maintains
the same hydrogen bond with Arg752 that is observed in the complex with DHT. Conversely, at the
opposite side of the compound, there is no equivalent of the hydrogen bond of DHT with Asn705 and
Thr877. The hydroxyphenyl moiety of (+)-11b occupies a pocket almost perpendicular DHT’s hydroxyl
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group, which is involved in a direct hydrogen bond with His874. (B) Superposition of (+)-13a with
DHT. The complex of hAR LBD with (+)-13a is represented in orange and DHT is pink. Like in the
complex with (+)-11b, the hydroxyphenyl moiety occupies a pocket almost perpendicular to the DHT
plane and its hydroxyl group is involved in a direct hydrogen bond with His874.
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When comparing the peptide chain of hAR LBD co-crystallized with either (+)-11b, (+)-13a and
DHT, few differences were found. In the least-squares superposition of the coordinates, the average
RMSD is 0.73 Å for (+)-11b and 0.62 Å for (+)-13a for all the main-chain atoms. In both cases, we
were not able to observe electron density for the loop of five residues (845-849), as previously
described.18 As compared to DHT, we also observed that the helix H11 was slightly kinked from Thr877
to its end and the loop connecting helices H11 and H12 (residues 883-891) is disordered, with a poor
density fit. So no marked change was evident in the protein following binding with a full agonist (DHT)
or partial agonist (+)-11b, (+)-13a, making it difficult to explain on the protein level the observed partial
agonism profile resulting in the tissue selectivity of (+)-11b. Nonetheless it is possible that small
changes around the ligand may alter the cofactor interaction profile. Moreover, domains of the AR other
than the LBD were not included in this analysis and could play a major role in the selectivity of the
ligands.
Conclusion
In conclusion, a new series of hydroxyphenyl substituted arylhydantoins has been synthesized and
characterized in vitro as partial androgen agonists. Their partial agonism profile and high affinity for the
AR translates in vivo to tissue selective pharmacologic activity when orally administered in a castrated
rat model. Particularly, (+)-11b maintained levator ani muscle weight with potency similar to
testosterone while showing only weak stimulation of the prostate. The strong distinction between
anabolic and androgenic activity in vivo is typical of a SARM. Moreover, (+)-11b prevented
degradation of bone loss after ORX. The new scaffold described herein constitutes a significant advance
in the search of efficient and highly tissue-selective non-steroidal SARMs. Compound (+)-11b proved
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to be an excellent tool validating our approach towards in vivo tissue selectivity from our in vitro tools
around the hydantoin series. Further optimization around the hydantoin scaffold, keeping similar in vivo
selective profile as compound (+)-11b will be reported soon.
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8
Experimental Section
9
Unless otherwise stated, all procedures were performed under a nitrogen atmosphere using anhydrous
solvents purchased from commercial sources. Thin layer chromatography was performed using
precoated Merck silica gel 60 F254 plates, visualized under UV light or after staining by spraying a
solution of 20% phosphomolybdic acid in ethanol, then heating. Preparative chromatography was
conducted on Merck silica gel 60 (0.040-0.063 mm). All 1H NMR spectra were recorded using a Bruker
DPX (300 MHz) or Advance (400 MHz) spectrometer, in solution in the deuterated solvent mentioned;
chemical shifts were expressed in ppm as a value δ relative to the shift of the tetramethylsilane used as a
reference. LCMS were performed on a Waters HPLC with a 2996 photodiode array detector coupled to
a LCT TOF mass spectrometer with electrospray ionization. The HPLC method was the following,
column used: Waters Xterra RP18 (3.5 µm; 50 × 2.1 mm i.d.); solvents: (A) water + 0.01% of formic
acid and (B) CH3CN + 0.01% of formic acid, gradient method: from 95/5 A/B at time 0 to 100% of B
after 4 min, then 100% of B for 3 min. The purity of the described compounds was > 98% according to
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23
HPLC analysis. Optical rotations ([α]D were recorded on a Propol polarimeter. Specific rotations are
given as deg/dm and the concentrations c are reported as g/100 mL of the specific solvent and were
recorded at 23 °C.
Preparation of (±)-11b:
Methyl 2-(4-acetoxyphenyl)-2-bromopropionate (7).
Step 1: Methyl 2-(4-hydroxyphenyl)propionate: Thionyl chloride (25 mL, 0.35 mol) was added
dropwise for 15 min at 0 °C to a solution of 2-(4-hydroxyphenyl)propanoic acid 6 (25 g, 0.15 mol) in
methanol (500 mL); an exothermic reaction occurred. After 3.5 h at 0 °C, the mixture was concentrated
and quenched with water, extracted with ethyl acetate, washed with water and dried over MgSO4. After
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concentration, methyl 2-(4-hydroxyphenyl)propionate was obtained as a brown oil (34.7 g, > 100%) and
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3
1
1
used directly for the next step. H-NMR (CDCl3): H-NMR (400 MHz, CDCl3): δ 1.48 (d, J = 7.2 Hz,
4
5
6
3H), 3.67 (m, 4H), 6.78 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H).
7
8
9
Step 2: Methyl 2-(4-acetoxyphenyl)propionate: K2CO3 (31.05 g, 0.22 mol) was added at rt to a
solution of the product obtained in step 1 (34.7 g) in THF (450 mL). Acetic anhydride (21 mL, 0.22
mol) was added dropwise for 10 min at 0 °C, giving rise to an exothermic reaction. After one night at rt,
the mixture was filtered and rinsed with THF, and the filtrate was concentrated to provide methyl 2-(4-
acetoxyphenyl)propionate as a colourless oil (42.3 g, > 100%), used as such for the next step. 1H-NMR
(400 MHz, CDCl3): δ 1.50 (d, J = 7.2 Hz, 3H), 2.30 (s, 3H), 3.67 (s, 3H), 3.73 (q, J = 7.2 Hz, 1H), 7.05
(d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H). LCMS: m/z 223 [M+H]+, 240 [M+H2O]+, 264
[M+CH3CN]+.
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Step 3: Methyl 2-(4-acetoxyphenyl)-2-bromopropionate: N-bromosuccinimide (32.04 g, 0.18 mol)
and AIBN (2.95 g, 0.018 mol) were added to a solution of the compound obtained in step 2 (42.3 g) in
carbon tetrachloride (500 mL). After 7 h under reflux, another batch of N-bromosuccinimide (13.35 g,
75 mmol) and AIBN (1.23 g, 7.5 mmol) were added and the reflux was maintained overnight. The
mixture was then filtered, the solid was washed with dichloromethane and the filtrate was concentrated.
The residue was purified by chromatography (eluting with dichloromethane) to yield the title compound
1
as an oil (48 g, 100%). H-NMR (400 MHz, CDCl3): δ 2.30 (s, 6H), 3.79 (s, 3H), 7.09 (d, J = 8.8 Hz,
2H), 7.59 (d, J = 8.9 Hz, 2H). LCMS: m/z 179 [M+H−Br−MeCO]+.
Methyl 2-(4-hydroxyphenyl)-2-methylaminopropionate (8). A saturated solution of methylamine
in ethanol (40 mL) was added at rt to bromo ester 7 (8.12 g, 27 mmol) dissolved in THF (160 mL). The
mixture became pale yellow with formation of an insoluble substance. After 30 min at rt, the mixture
was filtered and concentrated. The residue (yellow oil, 9.1 g) was purified by chromatography (eluting
with 95/5 dichloromethane/methanol) to yield a pale yellow oil which was crystallized in isopropyl
ether. After filtration and drying, the title compound was obtained (2.46 g, 43%) as a white powder.
After evaporation, the filtrate provided a second crop of amorphous 8 (2 g, 35%) as pale yellow oil. 1H-
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NMR (400 MHz, CDCl3): δ 1.65 (s, 3H), 2.30 (s, 3H), 3.72 (s, 3H), 6.76 (d, J = 8.8 Hz, 2H), 7.29 (d, J
= 8.8 Hz, 2H). LCMS: m/z 179 [M−MeNH]+.
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8
4-cyano-3-trifluoromethylphenyl isocyanate (10b). Triphosgene (21.16 g, 71.3 mmol) was
dissolved in toluene (100 mL). A solution of aniline 9b (20 g, 0.107 mol) in dioxan (125 mL) was added
dropwise at rt over 1.5 h. The mixture was heated to 110 °C, a pronounced release of gas took place
then an insoluble brown substance appeared. After 2 h under reflux, stirring was maintained at rt
overnight. The insoluble matter was filtered off and washed with toluene. The filtrate was concentrated
under vacuum to provide the title compound (21.8 g, 96%). It was stored at 4 °C as a 2 M solution in
THF to be used quickly without purification for the next step.
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4-[3,4-Dimethyl-2,5-dioxo-4-(4-hydroxyphenyl)imidazolidin-1-yl]-2-trifluoromethylbenzonitrile
((±)-11b). A 2 M solution of isocyanate 10b in THF (940 µL, 1.88 mmol) was introduced in 4 portions
to amino ester 8 (196 mg, 0.94 mmol) dissolved in THF (4 mL), over 5 h at rt. The mixture was then
heated to reflux for 1 h. The insoluble matter was filtered and rinsed with THF, and the filtrate was
evaporated. The residue was purified by chromatography while eluting with 30-40% ethyl acetate in
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heptane to provide the title compound (350 mg, 96%) as a white solid; mp = 136 °C. H-NMR (400
MHz, CDCl3): δ 1.96 (s, 3H), 2.97 (s, 3H), 6.92 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 7.94 (d, J
= 8.4 Hz, 1H), 8.02 (dd, J = 1.8 and 8.5 Hz, 1H), 8.17 (d, J = 1.6 Hz, 1H). LCMS: m/z 388 [M−H]−, 777
[2M−H]−.
1-(3,4-dichlorophenyl)-3,4-dimethyl-4-(4-hydroxyphenyl)-2-thioxoimidazolidin-5-one ((±)-13a).
Using isothiocyanate 12a (Aldrich) and the amino ester 8 (0.48 g , 2.3 mmol), the procedure described
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above to prepare (±)-11b gave rise to the title compound (0.69 g, 77%); mp = 194 °C. H-NMR (400
MHz, CDCl3): δ 1.97 (s, 3H), 3.23 (s, 3H), 6.87 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.3 Hz, 2H), 7.25 (m,
1H), 7.50 (m, 1H), 7.58 (m, 1H). LCMS: m/z 379/381 [M−H]−.
Preparation of (+)-11b and (−)-11b:
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Resolution of (±)-11b by chiral HPLC. The racemic (±)-11b has been resolved at 25°C by chiral
HPLC over a column of Chiralpak AD 10 µm (250 × 4.6 mm i.d.). Elution was performed with a
mixture of heptane/ethanol/methanol 80/10/10 (flow rate 1 mL/min, detection UV 220 nm). Following
six consecutive injections and recycling the unresolved fraction, 1.39 g of racemate provided 595 mg (−
)-11b eluted first (retention time 10.8 min) and 501 mg (+)-11b eluted second (retention time 13.9 min).
Synthesis of (R)-(+)-11b:
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(R)-4-[2,5-dioxo-4-(4-methoxyphenyl)-4-methylimidazolidin-1-yl]-2-trifluoromethylbenzonitrile
((R)-(+)-21b). A 2 M solution of isocyanate 10b (9.5 mL, 19 mmol) in THF was added to a solution of
the amino ester (R)-(−)-20 (3.1 g, 14.8 mmol) in THF (60 mL) under argon atmosphere. After 20 min at
rt, triethylamine (2 mL, 14.8 mmol) was added and the mixture was heated under reflux for 2 h. After
cooling, the mixture was evaporated to dryness, and the residue was purified by chromatography,
eluting with heptane/ethyl acetate (2/1). The title compound was obtained as a white resin (5.5 g, 95%);
[α]D23 = + 26.4 (c 1%, MeOH). 1H-NMR (400 MHz, CDCl3): δ 1.97 (s, 3H), 3.84 (s, 3H), 6.22 (s, 1H),
6.97 (d, J = 8.9 Hz, 2H), 7.47 (d, J = 8.9 Hz, 2H), 7.92 (d, J = 8.4 Hz, 1H), 7.98 (dd, J = 2 and 8.4 Hz,
1H), 8.12 (d, J = 1.6 Hz, 1H). LCMS: m/z 388 [M−H]−, 390 [M+H]+.
(R)-4-[3,4-dimethyl-2,5-dioxo-4-(4-hydroxyphenyl)imidazolidin-1-yl]-2-
trifluoromethylbenzonitrile ((R)-(+)-11b).
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(R)-4-[3,4-dimethyl-2,5-dioxo-4-(4-methoxyphenyl)imidazolidin-1-yl]-2-
trifluoromethylbenzonitrile. To a solution of (R)-(+)-21b (5.4 g, 13.87 mmol) in DMF (100 mL) under
argon, a suspension of sodium hydride, 55% in oil (908 mg, 20.8 mmol), was added batchwise at 0 °C.
The mixture was stirred at 0 °C for 15 min, then methyl iodide (1.75 mL, 28 mmol) was added and the
mixture was stirred at rt for further 2 h. The mixture was poured on iced water and the product is
extracted with ethyl acetate. The organic phases were washed with a saturated aqueous NaHCO3
solution then with brine. After drying over MgSO4, filtration and concentration, the residue was purified
by chromatography, eluting with 2/1 heptane/ethyl acetate mixture. The title compound (4.5 g, 80%)
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was obtained in the form of a white solid; [α]D23 = + 28.2 (c 1%, MeOH). 1H-NMR (400 MHz, CDCl3):
δ 1.95 (s, 3H), 2.95 (s, 3H), 3.85 (s, 3H), 6.98 (d, J = 8.9 Hz, 2H), 7.28 (d, J = 8.9 Hz, 2H), 7.91 (d, J =
8.5 Hz, 1H), 8.02 (dd, J = 2 and 8.5 Hz, 1H), 8.18 (d, J = 1.8 Hz, 1H). LC/MS: m/z 404 [M+H]+.
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trifluoromethylbenzonitrile ((R)-(+)-11b). Boron trifluoride-dimethyl sulfide complex (7.83 mL, 744
mmol) was added dropwise to a solution of the product of step 1 (3 g, 7.44 mmol) in dichloromethane
(70 mL). After 24 h at rt, a saturated aqueous NaHCO3 solution was added until pH 8 then the product
was extracted with dichloromethane. The organic phase was washed with brine and dried over MgSO4.
After filtration and concentration to dryness, the residue was purified by chromatography, eluting with a
2/1 heptane/ethyl acetate mixture. The title compound was obtained in the form of a white solid (2.8 g,
96%); mp = 150.7 °C; [α]D23 = + 29.2 (c 1%, MeOH). 1H-NMR (400 MHz, CDCl3): δ 1.96 (s, 3H), 2.97
(s, 3H), 6.93 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.4 Hz, 1H), 8.02 (dd, J = 2 and
8.5 Hz, 1H), 8.18 (d, J = 1.5 Hz, 1H). LCMS: m/z 388 [M−H]−.
Synthesis of (S)-(−)-11b.
(S)-4-[2,5-dioxo-4-(4-methoxyphenyl)-4-methylimidazolidin-1-yl]-2-trifluoromethylbenzonitrile
((S)-(−)-21b). Starting from the aminoester (S)-(+)-20 (540 mg, 2.58 mmol) and the isocyanate 10b
according to the protocol described above to prepare (R)-(+)-21b, the title compound was obtained in
the form of a white solid (965 mg, 96%); [α]D23 = − 26.4 (c 1%, MeOH). 1H-NMR (400 MHz, CDCl3):
δ 1.95 (s, 3H), 3.85 (s, 3H), 6.98 (d, J = 8.9 Hz, 2H), 7.27 (d, J = 8.9 Hz, 2H), 7.91 (d, J = 8.5 Hz, 1H),
8.02 (dd, J = 2 and 8.5 Hz, 1H), 8.17 (s, 1H). LCMS: m/z 388 [M−H]−, 431 [M+H+CH3CN]+.
(S)-4-[3,4-dimethyl-2,5-dioxo-4-(4-hydroxyphenyl)imidazolidin-1-yl]-2-
trifluoromethylbenzonitrile ((S)-(−)-11b).
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trifluoromethylbenzonitrile. (S)-(−)-21b (915 mg, 2.35 mmol) was treated according to the protocol
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described above to prepare (R)-(+)-11b (MeI, NaH, DMF), to provide the title compound (1.15 g, >
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trifluoromethylbenzonitrile ((S)-(−)-11b). The compound obtained above (1.15 g) was treated following
the procedure used to prepare (R)-(+)-11b (BF3.Me2S, CH2Cl2). The title compound was obtained as a
white solid (867 mg, 95%); mp = 150.5 °C; [α]D23 = − 28 (c 1%, MeOH). 1H-NMR (400 MHz, CDCl3):
δ 1.96 (s, 3H), 2.97 (s, 3H), 6.92 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 8.6 Hz, 1H),
8.02 (dd, J = 1.1 and 8.6 Hz, 1H), 8.18 (bs, 1H).
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Synthesis of (R)-(+)-13a:
(R)-1-(3,4-dichlorophenyl)-3,4-dimethyl-4-(4-methoxyphenyl)-2-thioxoimidazolidin-5-one (24a).
Triethylamine (0.185 mL, 1.33 mmol) and isothiocyanate 12a (Aldrich, 0.230 g, 1.13 mmol) were
added to a solution of 23 (0.246 g, 1.10 mmol) in THF (10 mL). The mixture was stirred at rt for 1 h,
then evaporated to dryness. The residue was diluted with water and extracted with ethyl acetate, dried
over MgSO4, filtered and evaporated. The crude product was purified by chromatography while eluting
with 20% ethyl acetate in heptane. The title compound was obtained as a white solid (0.41 g, 95%). 1H-
NMR (400 MHz, CDCl3): δ 1.96 (s, 3H), 3.22 (s, 3H), 3.84 (s, 3H), 6.99 (d, J = 7.8 Hz, 2H), 7.20 to
7.27 (m, 3H), 7.49 (d, J = 2.4 Hz, 1H), 7.57 (d, J = 8.6 Hz, 1 H). LCMS: m/z 395/397 [M+H]+.
(R)-1-(3,4-dichlorophenyl)-3,4-dimethyl-4-(4-hydroxyphenyl)-2-thioxoimidazolidin-5-one ((R)-
(+)-13a).
Boron trifluoride-dimethylsulfide complex (1 mL, 9.5 mmol) was added to a solution of 24a (0.4 g,
1.01 mmol) in dichloromethane (30 mL). The mixture was stirred at rt for 24 h then poured into a
saturated aqueous NaHCO3 solution and extracted with ethyl acetate. The organic layer was dried over
MgSO4, filtered and evaporated. The residue was purified by crystallisation in a mixture of
dichloromethane/methanol/ether/pentane and then crystallised again in dichloromethane. The title
23
compound was obtained as white crystals (0.27 g, 70%); mp = 192 °C; [α]D = + 39.6 (c 1.05%,
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MeOH). 1H-NMR (400 MHz, CDCl3): δ 1.96 (s, 3H), 3.24 (s, 3H), 6.90 (d, J = 8.8 Hz, 2H), 7.17 (d, J =
8.8 Hz, 2H), 7.25 (dd, J = 2.4 and 8.6 Hz, 1H), 7.50 (d, J = 2.3 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H).
LCMS: m/z 379/381 [M−H] −, 381/383 [M+H]+.
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The in vitro molecular efficacy and potency have been assessed using a stable cell line derived from
Hela cells expressing hAR and a reporter gene placed under the transcriptional control of the probasin
AREs. This transactivation assay allows the identification of pure agonists (such as testosterone and
DHT), partial agonists as well as antagonists.
In the Monod-Wyman-Changeux model,14 the receptor is constantly in inter-conversion equilibrium
between two states (active A and inactive R), and any ligand of the receptor actually has two molecular
affinities for the receptor: one for the active state A (denoted KA) and one for the inactive state R
(denoted KR). The ratio of the affinities of the compound for the two states KR/KA (is called the
molecular efficacy of the compound. The position of the equilibrium in the absence of ligand is defined
by the ratio [R]/[A] and is equal to a constant classically denoted L0, L[X] being the value of this ratio in
the presence of the ligand at a concentration [X]. Applying the law of mass action to each of the states:
[RX] = [R] × [X]/KR and [AX] = [A] × [X]/KA leads to the key expression: L[X] = L0 × (1 + [X]/KR) / (1
+ [X]/KA). At saturating concentrations of the ligand a limit value is obtained: L∞ = L0 × KA/KR. This is
precisely the equation separating, in the expression of molecular efficacy, the contribution of the tissue
from that of the compound: the ratio of the affinities of the compound for the two states KR/KA (i.e., the
way the compound does or does not discriminate between the two states) is called the molecular
efficacy of the compound and determines its maximal effect on the equilibrium of active and inactive
receptor states, whereas L0 determines the contribution of the tissue. Efficacies reported in the paper are
ratio KR/KA related to the compound contribution. Applying the law of mass action to each of the two
states also leads to the potency equation: 1/Kd = L0/(1 + L0) × 1/KR + 1/(1 + L0) × 1/KA, expressing that
the apparent affinity is simply the harmonic mean of the two molecular affinities, weighed by the initial
position of the equilibrium.
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We therefore used a Schild-type curve-shift paradigm approach by crossing a range of concentrations
of our compound and a range of concentrations of DHT as reference agonist.23 We analysed at both the
rightward shift of the DHT curve induced by the antagonist activity of our compound, and the upward
shift of its lower plateau, induced by the agonist activity of our compound. To reach the two molecular
affinities KA and KR of our SARM compounds, we built the mathematical theoretical behaviour of our
system with parameterized equations where the KA and KR are the parameters identified by fitting the
bundle of curves from the curve-shift paradigm, as a whole. From these two parameters KA and KR
identified, potency and molecular efficacy were calculated as indicated.
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In vivo pharmacology
All procedures were performed in accordance with national and European legislations on animal
experimentation.
The dissociated activity of compounds was tested in an adapted model of castrated immature young
rats which is widely recognized for evaluating the anabolic and androgenic effects of androgens on
muscles and on genitalia.16b Castrated rats are left untreated for one week and then treated for four days
with test compound. Castration resulted in a rapid atrophy of the ventral prostate (VP) as well as of the
anus-lifting muscle levator ani (LA). These effects are completely compensated by an exogenous
administration of androgens. Stringency of therapeutic setting is greater than prophylactic setting and
allows detection of more potent myotrophic/anabolic compounds. After four days of treatment, the
activity of compound was expressed as a percentage and calculated using the following formula:
(W/BW treated – W/BW ORX)/(W/BW sham – W/BW ORX) × 100, where W is weight and BW is
body weight. The dissociated activity of test compound was expressed as the ratio between the dose
displaying 50% activity (A50) on VP and LA.
The bone activity of compounds was tested using rats castrated at six months of age and treated for 3
months, immediately after castration, either with test compound or vehicle (EtOH/corn oil, 10/90).
Sham-operated animals received vehicle treatment. Densitometric analysis was performed on tibias
using the Stratec peripheral quantitative computerized tomography (pQCT) XCT Research SA+
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(version 5.4B; Norland Medical Systems, White Plains, NY) at 70 µm resolution. Trabecular volumetric
bone mineral density (BMD) was measured by metaphyseal pQCT scans positioned 1 mm from the
distal growth plate and corresponding to 6% of the total length of the tibia. µCT scans of the
metaphyseal region of tibias were performed at an isotropic resolution of 9 µm, to obtain trabecular
bone structural parameter (BV/TV), using the Scanco Medical µCT scanner CT (µCT 20; Scanco
Medical AG, Bassersdorf, Switzerland). The mean tissue of the scanned area was between 1.0 and 3.0
mm from the growth plate in the trabecular bone of the tibial proximal metaphysis.
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After removal of fibula, a proximal tibia compression test was performed using axial compression of
the tibia plateau, the shaft being fixed in methylmethacrylate cement (Technovit 4071, Heraeus Kulzer
GmbH, Wehrheim, Germany). A three-point bending test of the femur was also performed. The femur
was placed in the material testing machine on two supports separated by a distance of 20 mm and load
was applied on the middle of the shaft. The mechanical resistance to failure was tested using a servo-
controlled electromechanical system (Instron 1114, Instron Corp., High Wycombe, UK) with the
actuator displaced at 2 mm/min. Both displacement and load were recorded. Ultimate strength (maximal
load, N) was calculated.
Structure determination of hAR LBD complexes with (+)-11b and (+)-13a
Expression, purification and crystallization have been reported previously.17 More details are
available in the supporting information. Briefly, structures were solved by the molecular replacement
method, using the model of hAR LBD complexed to DHT (pdb code: 2AMA) as a search model.18
Refinement was conducted with SHELXL24 and rebuilding was done with Coot.25 The final model for
the complex with (+)-11b has a Rfactor of 0.192 (Rfree = 0.230), and contains 2339 atoms (2006
protein atoms, 215 water molecules, 90 undecapeptide atoms and the 28 atoms of (+)-11b). The final
model for the complex with (+)-13a has a Rfactor of 0.173 (Rfree = 0.211) and contains 2388 atoms
(2024 protein atoms, 250 water molecules, 90 undecapeptide atoms and the 24 atoms of (+)-13a). The
coordinates have been deposited in the PDB with accession codes 3V49 and 3V4A.
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Acknowledgment
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Authors wish to thank Gaëtan Gitton and Karen Séchet for their excellent technical assistance during
the syntheses. They also thank Kara Bortone for her careful review of the manuscript. The chiral
chromatographic separations have been efficiently performed by Serge Droux and his team (Kyralia
SAS, Parc Biocitech, Romainville, France). Philippe Bénas is thanked for assistance during
crystallographic data collection. The biomechanical experiments on bone have been efficiently realized
by Patrick Ammann and his team (Fondation Recherches Médicales, Geneva, Switzerland).
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Associated content
Supporting Information Available: Preparation and characterization data for intermediates and final
compounds (±)-10c-f, (±)-11a,c-f, 12b, (±)-13b, 14, (±)-15b, 16, (±)-17b, (−)-19, (+)-20, (−)-20, (+)-
21c, (+)-11c, 22 and (−)-23 along with X-ray data processing and refinement statistics table are
available free of charge via the Internet at http://pubs.acs.org.
Accession codes
Compound (+)-11b, code 3V49 and compound (+)-13a, code 3V4A.
Abbreviations used
SARM, selective androgen receptor modulator; AR, androgen receptor; ORX, orchidectomized; T,
testosterone; TP, testosterone propionate; DHT, 5α-dihydrotestosterone; SERMs, non-steroidal selective
estrogen receptor modulators; SAR, structure-activity relationship; AR LBD, ligand binding domain of
the androgen receptor; ARE, androgen responsive element; LA, levator ani muscle; VP, ventral
prostate; BMD, Bone Mineral Density; HDL, high-density lipoprotein.
References
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and Therapeutic Implications. Endocr. Rev. 2005, 26, 833-876.
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(2) Rosario, E. R.; Pike, C. J. Androgen Regulation of β-Amyloid Protein and the Risk of Alzheimer’s
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Composition and Muscle Strength in Men over 65 of Age. J. Clin. Endocrinol. Metab. 1999, 84, 2647-
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E.; Sinha-Hikim, I.; Dzekov C.; Dzekov, J.; Magliano, L.; Storer, T. W. Older Men are as Responsive as
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Androgens. Biochem. Biophys. Res. Commun. 1998, 244, 1-4. (b) Hamann, L. G.; Mani, N. S.; Davis, R.
L.; Wang, X.N.; Marschke, K. B.; Jones, T. K. Discovery of a Potent, Orally Active, Nonsteroidal
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