Unexpected binding orientation of bulky-B-ring anti-androgens and implications for future drug targets

Article abstract of DOI:10.1021/jm2000097

Several new androgen receptor antagonists were synthesized and found to have varying activities across typically antiandrogen resistant mutants (Thr877 → Ala and Trp741 → Leu) and markedly improved potency over previously reported panantagonists. X-ray crystallography of a new anti-androgen in an androgen receptor mutant (Thr877 → Ala) shows that the receptor can accommodate the added bulk presented by phenyl to naphthyl substitution, casting doubt on previous reports of predicted binding orientation and the causes of antagonism in bulky-B-ring antagonists.

Full text of DOI:10.1021/jm2000097

BRIEF ARTICLE
pubs.acs.org/jmc
Unexpected Binding Orientation of Bulky-B-Ring Anti-Androgens and
Implications for Future Drug Targets†
Charles B. Duke, III,‡ Amanda Jones,§ Casey E. Bohl,§ James T. Dalton,§ and Duane D. Miller*,‡
‡Department of Pharmaceutical Sciences, College of Pharmacy, Health Science Center, The University of Tennessee,
847 Monroe Avenue, Johnson Building, Room 227C, Memphis, Tennessee 38163, United States
§GTx, Inc., 3 North Dunlap Street, Memphis, Tennessee 38163, United States
S Supporting Information
b
ABSTRACT: Several new androgen receptor antagonists were synthesized and found to have varying activities across typically anti-
androgen resistant mutants (Thr877 f Ala and Trp741 f Leu) and markedly improved potency over previously reported pan-
antagonists. X-ray crystallography of a new anti-androgen in an androgen receptor mutant (Thr877 f Ala) shows that the receptor
can accommodate the added bulk presented by phenyl to naphthyl substitution, casting doubt on previous reports of predicted
binding orientation and the causes of antagonism in bulky-B-ring antagonists.
’ INTRODUCTION
Prostate cancer, the second most frequent cause of cancer
death in men, is treated with nonsteroidal anti-androgens such as
bicalutamide (1, Figure 1) and hydroxyflutamide that antagonize
the androgen receptor (AR) pathway, preventing androgen-
dependent cell growth. In AR wild type (AR(wt)), 1 and
hydroxyflutamide act as antagonists. Long-term treatment with
anti-androgens results in anti-androgen resistance in prostate
cancer cells or androgen withdrawal syndrome in patients. This
phenomenon is thought to arise from, among other mechanisms,
mutations to several key residues in the ligand-binding domain.
Commonly studied mutations in AR include Thr877 f Ala
(AR(T877A)), Trp741 f Leu (AR(W741L)), and Trp741 f
Cys (AR(W741C)), in which varying agonism and antagonism
are observed with use of those drugs. Specifically, 1 is an agonist
in AR(W741L) and AR(W741C)1 while hydroxyflutamide is an
agonist in AR(T877A).2
Figure 1. Structures of 1 and 2, McGinley and Koh’s analogues 3À5,5
In contrast, AR(wt) agonists such as S-22 (2)3 are probably
and new analogues 6À8.
agonists in all of these mutants, since they will geometrically fit
no worse with the bulk-reducing mutations than in AR(wt). The
difference in AR(wt) activity between agonists such as 2 and
antagonists such as 1 seems to arise from the linker oxygen’s
smaller size relative to the sulfonyl group of 1. This bulk in
antagonist drugs, though small, is thought to push the AR helix
12 (H12) away from the binding pocket, disturbing the agonist
conformation of the receptor, the current theoretical key to
antagonism.4
In a recent communication to Journal of the American Chemical
Society on the use of molecular design to circumvent anti-
androgen resistance in prostate cancer, McGinley and Koh
predicted the orientation of bulky nonpolar substituents in
analogues of the nonsteroidal anti-androgen 1 in molecular
modeling and the apparent contact with H12.5 This was the
case in AR(wt) and all mutants for three reported compounds 3,
4, and 5 (Figure 1), all of which had Ki in the 2À5 μM range and
IC50 in the 3À20 μM range. The study utilized our X-ray
structure of 1 bound in agonist mode to AR(W741L) with
H12 deleted to predict binding of their antagonists in the
antagonist mode.6
Here we report findings of X-ray crystallographic structures
and biological evaluation of several new compounds (6, 7, and 8,
Figure 1) with markedly increased binding affinity but in some
cases similar pan-antagonism as those reported by McGinley and
Koh.5 The X-ray structure of 7 in AR(T877A) has an orientation
of the bulky naphthyl substituent that disagrees strikingly with
the previous researchers’ predictions.
Noting the value of the findings with 3, we sought to improve
upon these structures by varying the linker group of 3 from SO2
to O (6) and improving binding by adding a CN group (7). We
then attempted an improved activity SO2-linked compound (8).
Ether and amine linkages have been used in our labs in the past to
Received: February 21, 2011
Published: April 20, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 1. Synthesis of 4-Cyano-1-naphthothionitrile (12)a
a Conditions: (a) dabco, N,N-dimethylthiocarbamoyl chloride, DMF,
70 °C; (b) 210 °C, argon atmosphere, 2 h; (c) THF, KH, MeOH, rt, 3 h.
Scheme 2. Synthesis of 6À8a
a Conditions: (a) (i) K2CO3, acetone, reflux 3 h; (ii) 1-naphthol,
2-propanol, K2CO3 reflux 18 h, 6 or 4-hydroxynaphtho-1-nitrile (7);
(b) (i) sodium 4-cyanonaphthylthiolate, THF, 25 °C; (ii) MCPBA,
dichloromethane.
Table 1. AR(wt) Relative Binding Affinitiesa
compd
RBA (%)
DHT
100 ( 11.6
0.69 ( 0.07
5.5 ( 1.8
1
2
6
7
8
0.97 ( 0.03
1.84 ( 0.7
0.37 ( 0.02
a Binding affinities were determined in a radiolabeled competitive assay
and expressed relative to DHT.11
produce several potent AR modulators including 2 (agonist,
1 nM).3,7
’ CHEMISTRY
A previously reported method was used for preparation of
4-hydroxy-1-naphthonitrile (9).8 A reported synthesis of 4-cya-
nothiophenol was adapted to obtain 4-mercapto-1-naphthonitrile
(12) from 9 via thermal isomerization of a N,N-dimethylthiocar-
bamoyl fragment.9 (Scheme 1)
Compounds 6À8 were all synthesized from previously known
1310 using varying coupling methods (Scheme 2). Epoxide
formation from 13 using K2CO3 followed by treatment with
1-naphthol or 4-hydroxy-1-naphthonitrile in the presence of
K2CO3 afforded 6 and 7, respectively. Treatment of 13 with
sodium 4-cyano-1-thionaphthylate in THF at room temperature
followed by oxidation with mCPBA afforded 8.
Figure 2. In vitro activity of anti-androgens. The functional activity of each
compound was determined by the ability of each ligand to induce or suppress
AR(wt), AR(W741L), or AR(T877A)-mediated transcriptional activation
in a cotransfection system.11 The activity of each compound (final concen-
trations of 1À1000 nM) was determined by incubating cells in the absence
(agonist assays) or presence (antagonist assays) of DHT and expressed as
the percentage of that induced by DHT. (A) Agonist and antagonist activity
with or without the presence of 1 nM DHT of each compound in AR(wt).
(B) Agonist and antagonist activity with or without the presence of 100 nM
DHT in AR(W741L). (C) Agonist and antagonist activity with or without
the presence of 1 nM DHT of each compound in AR(T877A).
’ IN VITRO RESULTS
well as relative binding affinities (RBA) compared to dihydro-
testosterone (DHT).11 1 bound the AR(wt) with an RBA of 0.69
Compounds 6À8 were evaluated in vitro for agonist and
antagonist effects on AR(wt), AR(W741L), and AR(T877A) as
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(Table 1). Addition of the naphthyl ring and 4-CN (8) decreased
the binding affinity. However, replacing the sulfonyl group with
an ether linkage and adding the naphthyl ring (6) increased
affinity. Binding affinity was further enhanced by addition of a
4-CN group (7).
Where 2 is a full agonist, 6À8 were antagonists in the AR(wt)
with varying activity (Figure 2A). 7 is also a partial agonist in
AR(wt), while 8 is not. 8 displays weak antagonist activity at the
highest concentration tested (1 μM), by inhibiting DHT-in-
duced transactivation by 15% (Figure 2B). In the AR(T877A)
mutant, 8 inhibited the activity of DHT by 61% (Figure 2C). 1
inhibited the activity of DHT by 84% in the AR(T877A) (not
shown). In the AR(W741L) mutant, 1 (not shown) and 7
(Figure 2B) are full agonists.
Of the studied compounds, 7 displayed the highest affinity but
did not exhibit pan-antagonism. As such, a crystal structure was
obtained for the 7/AR(T877A) complex. The scaffold of 7
overlays closely with that of 2 in AR(wt), reported here for the
first time (Figure 3). To our surprise, 7/AR(T877A) did not
show the orientation of the distal naphthyl ring predicted by
McGinley and Koh.5 Rather, the distal ring of the naphthyl group
experiences an edge-to-face πÀπ interaction with the A-ring and
is situated between W741 and M742 opposite H12. In addition,
M745 shifts relative to the structure of 2/AR(WT) to accom-
modate the bulk (Figure 4). Interestingly, there is a cascade of
effects that are set off by the bulk accommodation; the distal
naphthyl ring causes M742 to reorient (not shown), which
pushes M745 toward the A-ring of 7, causing a steric interaction.
This steric interaction explains the lowered binding affinity of 7
compared to 2.
Considering the similarities of the structures, the orientation
of the naphthyl ring is expected to be highly conserved across 3
and 6À8. The 7/AR(T877A) complex is in transcriptionally
active form, which does not explain how the naphthyl moiety
causes antagonism in AR(WT), since the ring does not appear to
appreciably interact with the T877/T877A space. This does,
however, suggest that the binding of 7 in AR(WT) in antagonist
mode would not be different with respect to the naphthyl group’s
orientation and that the naphthyl group orientation should be
conserved across the ligands considered here. Therefore, the
linker must be responsible for the observed differences in
agonism and antagonism. Further supporting this is the fact that
7 is a partial agonist in AR(wt) while 8 shows no agonist activity.
The differences observed across the range of linkers are
highlighted by the differences in the O-linked complexes 7 and
2 from 1 and its SO2 linker. The molecules are separated with
respect to binding not because of their B-ring size or shape but
because of their linker size and orientation. In the structure of
1/AR(W741L) the sulfonyl group is directed away from the rest
of the ligand and forced to impinge on the space occupied by
H12, causing antagonism.6 In contrast are the intramolecular
H-bond to the amide experienced by the 2 and 7 ether linkers.
Results of 3, 4, and 5 were predicted by the deletion of H12 in
silico, which provided ample space for the distal naphthyl ring of
3, but in the presence of H12, the ring’s true preferred position is
opposite the modeled results. This approach of H12 deletion is
predicated on the assumption that AR in its inactive form is
somewhat similar to ER, but it is well established in other
receptors of the same family that the movement of H12 away
from the active conformation can be much more subtle as in the
case of PR.4 This unfortunately suggests that deletion of H12 in
silico is not as effective a predictive tool as it once appeared.
In sum, it is apparent that antagonism can be controlled by
bulk extending from not only the B-ring but also an appropriate
linker. Work is currently underway in our hands to explore the
role of bulkier, branched linker groups.
Figure 3. Overlaid X-ray structures of 2 (green carbons) and 7 (orange
carbons) cocrystallized with AR(T877A) with (A) and without (B)
electron density maps. The orientation of 7 is closely overlaid with 2, and
the distal naphthyl ring extends directly away from H12 toward M745.
Figure 4. Stereoview of the X-ray structure of 7 (orange) cocrystallized with AR(T877A). There is an intermolecular πÀπ interaction between the
naphthyl ring of 7 and the W741 indolyl ring, which is 3.1 Å from the naphthyl ring.
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BRIEF ARTICLE
’ ASSOCIATED CONTENT
(10) Kirkovsky, L.; Mukherjee, A.; Yin, D.; Dalton, J. T.; Miller,
D. D. Chiral nonsteroidal affinity ligands for the androgen receptor. 1.
Bicalutamide analogues bearing electrophilic groups in the B aromatic
ring. J. Med. Chem. 2000, 43 (4), 581–590.
(11) Jones, A.; Hwang, D. J.; Duke, C. B., III; He, Y.; Siddam, A.;
Miller, D. D.; Dalton, J. T. Nonsteroidal selective androgen receptor
modulators enhance female sexual motivation. J. Pharmacol. Exp. Ther.
2010, 334 (2), 439–448.
S
Supporting Information. Synthesis procedures and char-
b
acterization data, including NMR, MS, and CHN results; X-ray
crystallographic data for complexes 2/AR(WT) and 7/AR-
(T877A). This material is available free of charge via the Internet
at http://pubs.acs.org.
Accession Codes
†PDB codes: WT-S-22, 3RLJ; T877A-7, 3RLL.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (901) 448-6026. Fax: (901) 448-3446. E-mail: dmiller@
uthsc.edu.
’ ACKNOWLEDGMENT
The authors thank GTx, Inc. for supporting this project.
’ ABBREVIATIONS USED
AR,androgenreceptor; AR(T877A), Thr877fAla; AR(W741C),
Trp741 f Cys; AR(W741L), Trp741 f Leu; AR(wt), wild-type
androgen receptor; Dabco, 1,4-diazabicyclo[2.2.2]octane; DHT,
dihydrotestosterone; DMF, dimethylformamide; H12, androgen
receptor helix 12; MCPBA, m-chloroperoxybenzoic acid;
RBA, relative binding affinity; rt, room temperature; THF,
tetrahydrofuran
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