Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators

Article abstract of DOI:10.1016/j.bmcl.2008.09.002

Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

Full text of DOI:10.1016/j.bmcl.2008.09.002

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5567–5570
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effect of B-ring substitution pattern on binding mode of propionamide
selective androgen receptor modulators
Casey E. Bohl a, Zengru Wu a, Jiyun Chen a, Michael L. Mohler b, Jun Yang a, Dong Jin Hwang b,
Suni Mustafa b, Duane D. Miller b, Charles E. Bell c, James T. Dalton a,
*
a Division of Pharmaceutics, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, 242 L.M. Parks Hall, Columbus, OH 43210, USA
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis, TN 38163, USA
c Department of Molecular and Cellular Biochemistry. College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2008
Revised 28 August 2008
Accepted 2 September 2008
Available online 5 September 2008
Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which pro-
vide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we
report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a ser-
ies of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We
found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding
not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conforma-
tion and aromatic interactions with Trp741. This information elucidates interactions important for high
AR binding affinity and provides new insight for structure-based drug design.
Keywords:
Androgen receptor
Crystallography
Prostate cancer
Cachexia
Ó 2008 Elsevier Ltd. All rights reserved.
SARM
Pharmacological agents that mimic the effects of endogenous
androgens provide treatment options for numerous diseases
involving AR regulation. Testosterone replacement therapy is given
to patients with androgen deficiencies often as a patch or gel due
to its poor oral bioavailability.1–3 Side effects from testosterone
treatment including hair loss, gynecomastia, and prostate hyper-
plasia stem from metabolism to dihydrotestosterone (DHT) by
sulted in agonist activity as witnessed by AR-mediated
transcription.12–14 In vivo experiments showed that the ether-
linked compounds offered more favorable responses due to meta-
bolic and pharmacokinetic profiles.13,15,16 Results also indicated
that ether-linked bicalutamide analogs elicit greater effects in ana-
bolic tissue (e.g., muscle and bone) than androgenic tissue (e.g.
prostate and seminal vesicles) as compared to DHT treated, cas-
trated rats.7,15,16 Furthermore, we found that hydrophilic substitu-
ents on the para-position of the B-ring including an acetamido, a
cyano, or a nitro group15 increase binding affinities and that further
modification of the B-ring with electron-withdrawing groups8,17
was also favorable (Table 1). Recently, we reported crystal struc-
tures of nonsteroidal ligands complexed to the AR, which provided
a basis for how the ether-linked bicalutamide agonist derivative,
S-1 could be accommodated in the AR.18 To advance our under-
standing as to why B-ring substituted S-1 derivatives resulted in
significantly higher AR binding affinities, we solved the X-ray crys-
tal structures of the AR LBD complexed to ligands containing a
para-substituted acetamido (S-4), nitro (S-21), and cyano group
(S-24), as well p-chloro-m-fluoro (C-31) and penta-fluoro (C-23)
B-ring substituted analogs. Herein we describe the AR interactions
that distinguish these ligands in terms of high binding affinities,
which progresses the understanding of the AR binding pocket
and provides new information for rational drug design.
5a-reductase or aromatization to estrogenic metabolites, limiting
its use in other AR regulated disorders.4–6 Nonsteroidal propiona-
mide antiandrogens such as bicalutamide are commonly used in
treatment of prostate cancer and provide advantages over steroidal
antagonists due to oral bioavailability and lack of cross-reactivity
with other steroid receptors.6 Nonsteroidal AR agonists would pro-
vide similar benefits over traditional testosterone therapy as well
as avoid conversion to DHT and estradiol. In vivo studies conducted
in our laboratory demonstrate that a series of nonsteroidal andro-
gens selectively increase muscle size relative to prostate and sem-
inal vesicles offering clinical potential for treating acute muscle
wasting and sarcopenia.1,7–10
Design, synthesis, and evaluation of a large number of aryl pro-
pionamides led to refinement of structure–activity relationships
for these SARMs.11–13 We discovered that modification of the sul-
fonyl group of bicalutamide to a thio- or ether-linkage group re-
The R-3-bromo-N-(4-cyano (or 4-nitro)-3-trifluoromethyl-
phenyl)-2-hydroxy-2-methylpropionamides were synthesized as
* Corresponding author. Tel.: +1 614 688 3797; fax: +1 614 292 7766.
E-mail address: dalton.1@osu.edu (J.T. Dalton).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.002

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C. E. Bohl et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5567–5570
Table 1
Chemical structures and binding affinities
Ki (nM)
OH
DHT
0.27
O
H
NC
F
O
O
S
A
B
R-bicalutamide
11 2
F3C
N
H
OH
O
O2N
F3C
F
O
O
O
S-1
6.1 0.2
4.0 0.7
2.5 0.2
0.54 0.03
1.7 0.2
1.4 0.3
N
H
O
O
O
OH
OH
OH
O2N
F3C
NHCOCH3
S-4
N
H
NC
NO2
S-21
S-24
C-31
C-23
F3C
N
H
NC
I
CN
O
N
O
H
OH
NC
Cl
F
O
F3C
N
O
H
OH
F
F
O2N
F3C
F
F
F
O
N
O
H
OH
Binding affinity determined by competitive inhibition of to rat prostate AR. Values previously reported by our laboratory include R-bicalutamide23, S-113, S-47, S-2124, and C-
23.22
previously reported by Kirkovsky et al.19 Target compounds S-1, S-
4, S-21, C-23, and C-31 were synthesized by modifications of the
methods previously published by Marhefka et al .13 and Nair
et al.,20 respectively (see Supplementary Data for details). Crystals
of the AR LBD SARM complexes were obtained using previously
published methods18,21 as described in the Supplementary Data.
Crystallography statistics are shown in Table 2.
on the para position of the A-ring are important for high binding
affinity to the AR.11,12 The nitro and cyano groups on hydroxyfluta-
mide and R-bicalutamide, respectively, were therefore preserved in
our optimization of nonsteroidal AR ligands. X-ray crystal struc-
tures demonstrate hydrogen bonds between the A-ring cyano
groups of S-21, S-24, and C-31 to Arg752 of helix 5, and to a water
molecule (Fig. 1) resembling the interactions with the cyano group
of R-bicalutamide binding to the W741L AR.21 The nitro group on
S-4 and C-23 are however within hydrogen bond range to
Previous reports of structure–activity relationships for nonste-
roidal AR ligands demonstrate that hydrogen bond acceptor groups

C. E. Bohl et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5567–5570
5569
Table 2
Crystallographic data and refinement statistics
Drug
S-4
S-21
S-24
C-31
C-23
PDB Code
Spacegroup
3b68
P212121
3b66
P212121
3b65
P212121
3b5r
P212121
3b67
P212121
Unit cell
a
b
c
54.73
66.57
68.64
54.74
66.60
69.10
54.64
66.71
69.04
54.89
66.80
69.16
54.86
66.20
69.74
Resolution range (Å)
Number of unique reflections
Average redundancy
% completeness
23.75–1.90 (1.97–1.90)a
20,381
23.98–1.65 (1.71–1.65)a
30,834
26.32–1.80 (1.86–1.80)a
23,944
22.01–1.80 (1.86–1.80)a
23,820
29.90–1.90 (1.97–1.90)a
20,501
7.03 (7.00)
100.0 (100.0)
0.159 (0.443)
6.6 (3.4)
0.234 (0.390)
0.274 (0.400)
0.007
5.98 (4.59)
99.2 (95.1)
0.089 (0.424)
10.9 (3.2)
0.213 (0.333)
0.242 (0.351)
0.006
13.85 (13.92)
99.5 (98.6)
0.144 (0.382)
12.1 (6.7)
0.242 (0.425)
0.292 (0.424)
0.006
9.12 (5.58)
98.3 (86.8)
0.129 (0.328)
10.4 (4.6)
0.233 (0.393)
0.272 (0.409)
0.006
6.93 (6.50)
99.3 (97.2)
0.141 (0.441)
9.2 (4.0)
0.226 (0.375)
0.273 (0.393)
0.006
b
Rmerge
I/
r
R factorc
Rfree
rmsd bonds (Å)
rmsd angles
mean B value (Å2)
1.1
23.0
1.1
20.0
1.1
21.9
1.1
20.2
1.1
23.2
a
Values for data in the last resolution shell shown in parenthesis.
P
P
b
c
Rmerge
=
|Ih À <I>h|/ Ih, where <I>h is average intensity over symmetry equivalents.
P
P
R-factor = |Fobs À Fcalc|/ Fobs. The free R-factor was calculated from 10% of the reflections that were omitted from the refinement.
Arg752 and the water molecule as well as Gln711 on helix 3 mim-
icking interactions seen with S-1.18 Hydrogen bonds involving the
amide nitrogen and chiral hydroxyl group interacting with Leu704
and Asn705 are conserved in this series of nonsteroidal ligands
resembling hydroxyflutamide and bicalutamide and provide the
basis for the stereo-selective binding of these compounds.
Presence of a hydrophobic substituent on the meta-position of
the A-ring, which has been shown important for maintaining high
binding affinity,11,12 was maintained with a trifluoromethyl group
on compounds S-4, S-21, C-31, and C-23 and an iodine atom on
S-24. The iodine at this position on S-24 mimics the trifluoro-
methyl substituted ligands as seen from similar binding affinities
of these derivatives11 and similar Van der Waals contacts in the
X-ray crystal structures.
Screening of B-ring substituted propionamide SARMs demon-
strated that halogens could be placed anywhere on the B-ring of
SARMs, while hydrophilic electron withdrawing substituents were
only found favorable on the para position.11,22 Crystal structures of
S-4, S-21, and S-24 reveal that the hydrophilic B-ring substituent
hydrogen bonds to a water molecule located in a kink between
helices 4 and 5, which also hydrogen bonds to His874 (Fig. 1a–c).
The improved binding affinities of these compounds over S-1 is
therefore likely attributed to the stronger hydrogen bonding prop-
erties of the acetamido, cyano, and nitro substitutions compared to
a fluorine. The nitro and cyano groups of compounds S-21 and S-
24, respectively, act as hydrogen bond acceptors, while the acet-
amide on S-4 can act as both a hydrogen bond donor and acceptor.
The lone pair of electrons on the acetamide nitrogen of S-4 thus
presumably accepts a hydrogen from the water molecule mimick-
ing the interactions seen with the nitro and cyano substituents,
which would explain the poor binding affinity of amine substitu-
tions on the para position of the B-ring.11,12 The significantly higher
binding affinity of S-24 and S-21 relative to S-4 appears to be a re-
sult of the extra bulk on the acetamido group. Steric clash of the S-
4 acetamide with Val903 causes a turn in the B-ring. In addition,
the acetyl portion of the acetamido group on S-4 is oriented such
that the oxygen atom displaces the Ile898 side chain relative to
the other structures (Fig. 2a) to allow its accommodation within
the binding pocket. This interaction pushes helix 12 away from
the binding pocket approximately 2 Å relative to the other AR
LBD-SARM complexes. Also, the methyl group of the acetamide is
unfavorably situated only 3.1 Å from the backbone oxygen of
Gln738. Further optimization of the para substituent therefore
may be possible to exploit a hydrogen bond to the backbone oxy-
gen of Gln738. The B-ring of S-21 and S-24 share nearly the same
orientation. However, the nitro group on the para position of S-21
Figure 1. Comparison of SARM binding conformations to the AR. (a) S-4 (carbons—
purple), (b) S-24 (carbons—yellow), (c) S-21 (carbons—salmon), (d) C-31 (carbons—
orange), (e) C-23 (carbons—green) fit into Fo À Fc similated annealing omit maps. AR
carbons—gray; nitrogens—blue; oxygens—red; sulfurs—orange; fluorines—
magenta; chlorine—green. Possible hydrogen bonds within 3.5 Å depicted as
dashed lines. Each of the compounds forms hydrogen bonds to Arg752, Leu704,
and Asn705. SARMs with a nitro substituent on the A-ring (i.e., S-4, S-21, and C-23)
also form a hydrogen bond to Gln711. SARMs with a hydrophilic B-ring substituent
(i.e. S-4, C-21, and C31) form a hydrogen bond to a water molecule that is stabilized
by His874 and backbone residues of helices 4 and 5. (f) Overlap of the bound
conformations of these SARMs portrays the different B-ring orientations.

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C. E. Bohl et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5567–5570
is observed almost parallel and aligned for p–p stacking. Moreover,
the ortho fluorine on the side facing Met745 was observed 3.1 Å
from the amide nitrogen suggesting that the B-ring may be pulled
toward the amide by an intramolecular hydrogen bond (Figs. 1e
and 2b).
The crystal structures presented herein demonstrate favorable
interactions in a subpocket of the AR unoccupied by steroidal
androgens. An additional region of hydrogen bonding not available
to endogenous AR ligands provides the potential for increasing
nonsteroidal ligand binding affinities beyond that of the most
potent endogenous androgen, DHT. Higher binding affinities
observed with SARMs containing multiple halogen substituted
B-rings may be attributed to p–p stacking and edge-to-face inter-
actions with Trp741, as well as increased Van der Waals contacts.
Further optimization of propionamide SARMs will however con-
ceivably require exploitation of increased hydrophobic interactions
and hydrogen bonding to Thr877 similar to steroidal ligands in
addition to interactions with Trp741, His874, and potentially even
Gln738.
Acknowledgment
Figure 2. AR-SARM complexes seen in multiple orientations. (a) Overlay of S-4
(purple), S-21 (salmon), and S-24 (yellow) demonstrating interactions between the
B-ring para substituent and residues His874, Ile898, Val903, and the backbone of
Gln738. Notice that each of these ligands hydrogen bonds to a conserved water
molecule in this region. The acetamido group on S-4 forms close contacts with the
side chains of Ile898 and Val903 inducing a slight displacement of helix 12. (b)
Overlay of S-1 (black, PDB code 2AXA18), C-31 (orange), and C-23 (green)
demonstrating differences in B-ring orientations with respect to Trp741. Substitu-
tion of a fluorine on the meta position of the B-ring in C-31 causes the B-ring to turn
relative to S-1. This orientation of the B-ring appears to result in an edge-to-face
aromatic ring interaction with Trp741 possibly due to the increased acidity of the
remaining hydrogen atom on the meta position. C-23 contains a completely fluorine
substituted B-ring, which orients nearly parallel to the Trp741 indole ring creating a
Supported by NIH Grants R01 DK59800 and R01 DK065227.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.002.
References and notes
1. Gao, W.; Reiser, P. J.; Coss, C. C.; Phelps, M. A.; Kearbey, J. D.; Miller, D. D.;
Dalton, J. T. Endocrinology 2005.
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p–p interaction and results in displacement of the Met745 side chain. Additionally,
the fluorine on the ortho position seems to be acting as a hydrogen bond acceptor
pulling the B-ring of C-23 towards the amide nitrogen of the compound.
4. Tan, R. S.; Culberson, J. W. Maturitas 2003, 45, 15.
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is observed less than 3.0 Å from the Val903 side chain in the X-ray
crystal structure. This steric interaction is alleviated in the S-24
bound AR in which the cyano group is situated over 3.5 Å from
the Val903 side-chain. However, structure–activity relationships
have shown very little difference in binding affinity between a cy-
ano and nitro at this position.15 Instead, the iodine on the meta po-
sition of the A ring appears to be the reason for the increased
affinity of S-24 over S-21.
C-31 and C-23 exhibit significantly higher AR binding affinity
than S-1 as a result of multiple halogen substitutions on the B-ring.
The electron withdrawing effect and/or increased Van der Waals
contacts with these analogs therefore cause increases in AR bind-
ing affinities. The B-ring was observed in a slightly different orien-
tation in each of the halogen substituted compounds when bound
to the AR (Figs. 1d, e, f and 2b). The C-31 B-ring is rotated compared
with compound S-1 to accommodate the meta fluorine in a cavity
between Thr877, Ile899, and Val903. Conversely, the B-ring of C-23
is not rotated relative to S-1, but instead is positioned towards
Met745 resulting in its displacement (Fig. 2b). A plausible explana-
tion is that the higher binding affinities of these multi-substituted
halogen derivatives results in favorable interactions with the
Trp741 side chain. Interestingly, the B-ring of C-31 turns relative
to S-1 to become nearly perpendicular to the indole ring of
Trp741. The additional electron-withdrawing effect of the meta
halogen would increase the acidity of the aromatic hydrogen for
12. Yin, D.; He, Y.; Perera, M. A.; Hong, S. S.; Marhefka, C.; Stourman, N.; Kirkovsky,
L.; Miller, D. D.; Dalton, J. T. Mol. Pharmacol. 2003, 63, 211.
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formation of
a edge-to-face aromatic ring interaction with
Trp741. Conversely, the B-ring of C-23 is completely substituted
abolishing the capability for this edge interaction. The C-23 B-ring