11β-alkyl-Δ9-19-nortestosterone derivatives: High-affinity ligands and potent partial agonists of the androgen receptor

Article abstract of DOI:10.1021/jm0342515

We report the synthesis of novel steroidal androgen receptor ligands comprising 11β-alkyl-Δ⁹-derivatives of 19-nortestosterone. These compounds are structurally related to the antiprogestin, antiglucocorticoid, and antiandrogen drug mifepristone (RU486). Nortestosterone analogues bearing 11β-octyl and 11β-decyl side-chains bind tightly to recombinant AR protein (IC₅₀ = 6.6 nM and IC₅₀ = 0.8 nM), block AR dimerization, exhibit activity against LNCaP prostate cancer cells, and comprise partial AR agonists with low antiglucocorticoid activity.

Full text of DOI:10.1021/jm0342515

J . Med. Chem. 2004, 47, 4985-4988
4985
11â-Alk yl-∆9-19-Nor testoster on e
Der iva tives: High -Affin ity Liga n d s a n d
P oten t P a r tia l Agon ists of th e An d r ogen
Recep tor
Smita S. Muddana, Aimee M. Price,
Megan M. MacBride, and Blake R. Peterson*
Department of Chemistry, The Pennsylvania State
University, University Park, Pennsylvania 16802
Received December 11, 2003
Abstr a ct: We report the synthesis of novel steroidal androgen
receptor ligands comprising 11â-alkyl-∆9-derivatives of 19-
nortestosterone. These compounds are structurally related to
the antiprogestin, antiglucocorticoid, and antiandrogen drug
mifepristone (RU486). Nortestosterone analogues bearing 11â-
octyl and 11â-decyl side-chains bind tightly to recombinant
AR protein (IC50 ) 6.6 nM and IC50 ) 0.8 nM), block AR
dimerization, exhibit activity against LNCaP prostate cancer
cells, and comprise partial AR agonists with low antigluco-
corticoid activity.
The androgen receptor (AR) is a member of the
nuclear receptor superfamily of ligand-regulated tran-
scription factors.1 This steroid hormone receptor com-
prises a major drug target involved in prostate cancer,
acne, hirsutism, male pattern baldness, and androgen
insensitivity syndrome (AIS).2-5 Binding of the steroid
hormones testosterone (1) and the more potent 5R-
dihydrotestosterone (2) to the AR initiates a complex
series of events that result in translocation of the AR
into the nucleus, binding to specific DNA sites, recruit-
ment of components of the transcriptional machinery,
and activation of the expression of specific genes.1 In
many prostate cancers, these androgen-dependent pro-
cesses are required for cellular proliferation.6 Hence,
androgen antagonists such as cyproterone acetate (CPA,
3) that block AR-mediated gene expression are often
used as first-line therapeutics against prostate cancer.
However, many clinically employed antiandrogens are
limited by low relative binding affinities, low selectivity
across the nuclear hormone receptor superfamily, or
agonist activity toward AR mutants such as T877A that
can emerge in advanced prostate cancers.6,7 As a
consequence of these limitations, novel small molecule
antiandrogens are desired as improved prostate cancer
therapeutics.8
ifen,16 and ICI 164,384 (5).17 Despite the fact that
mifepristone is effective against prostate cancer cells in
vivo, the use of this drug as a chronically administered
anticancer agent may be limited by its potent antiglu-
cocorticoid activity.18
In an effort to overcome limitations of mifepristone
as an antiandrogen, we synthesized and evaluated the
structurally related analogues 6-11. Compounds 8-11
bear long aliphatic side chains reminiscent of the
antiestrogens ICI 182,78019 and ICI 164,384 (5), but
project these substituents from the 11â position analo-
gous to mifepristone. Examination of recent high-
resolution X-ray crystal structures of the AR bound to
ligands4,20,21 suggested that these 11â alkyl substituents
might similarly disrupt the conformation of the adjacent
helix 12 when bound to the AR.
Compounds 6-11 were synthesized as shown in
Scheme 1. â-Estradiol (12) was protected as the methyl
Sch em e 1a
Mifepristone (RU486, 4) is under investigation as a
potential anticancer agent effective against prostate
cancers.9-12 This drug is a highly potent antiprogestin
(IC50 ) 25 pM)13 but also exhibits potent antiglucocor-
ticoid (IC50 ) 2.2 nM)13 and antiandrogen (IC50 ) 10
nM)13 activities. The dimethylaniline substituent at the
11â position of mifepristone is thought to interfere with
gene expression by dislodging the activation helix 12 of
the progesterone receptor (PR), glucocorticoid receptor
(GR), and AR.14 This active antagonism mechanism is
also employed by the antiestrogens raloxifene,15 tamox-
a
(a) CH3I, K2CO3, CH3CN. (b) Na, NH3, i-PrOH, THF. (c) Oxalic
acid, H2O, acetone. (d) Polyvinylpyridinium bromide perbromide,
polyvinyl pyridine, pyridine. (e) Ethylene glycol, pyridinium
chloride. (f) H2O2, hexafluoroacetone, pyridine, CH2Cl2. (g) Alkyl-
magnesium bromide, CuCl, THF. (h) Amberlyst-15 H+, ethanol.
* Corresponding author. E-mail: brpeters@chem.psu.edu; Tel: (814)
865-2969; Fax: (814) 863-5319.
10.1021/jm0342515 CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/14/2004

4986 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Letters
F igu r e 1. Competition fluorescence polarization binding
assays with purified AR protein.
Ta ble 1. Compilation of Data for Synthetic and Control
Compoundsa-e
inhibition
AR
of AR
GR
LNCaP
ligand
binding dimerization antagonism cytotoxicity
testosterone (1)
CPA (3)
mifepristone (4)
6 (11â-H)
7 (11â-Et)
8 (11â-pentyl)
9 (11â-octyl)
10 (11â-decyl)
11 (11â-dodecyl)
3.9
22
-
ND
0.9
0.008
133
1294
125
979
1812
870
ND
61
23
F igu r e 2. Mammalian two-hybrid assays of ligand-mediated
dimerization of the AR. Panels A and B: Dose-response
curves that assess agonist potency of control compounds (Panel
A) and novel AR ligands (Panel B). Panels C and D: Competi-
tion assays in the presence of testosterone (1, 1 nM) to identify
inhibitors of AR dimerization.
4.7
7.8
2.2
0.2
2.0
0.1
6.6
0.8
0.2
-
-
0.009
0.9
85
>100
55
17
17
17
nanomolar affinity to 8. Similarly, the longest side
chains of compounds 10 and 11 provided subnanomolar
affinity.
-
19
a
AR binding: IC50 values (nM) quantified by in vitro fluores-
b
cence polarization assays. Inhibition of AR dimerization and GR
To compare the activities of synthetic compounds
6-11 with the androgen testosterone (1), the antian-
drogen cyproterone acetate (3), and the antiandrogen
mifepristone (4) in living cells, a previously reported30
mammalian two-hybrid assay in chinese hamster ovary
cells (CHO-K1) was employed. These cells were tran-
siently transfected with two expression vectors encoding
N-terminal (AR residues 1-660 fused to the VP16
activation domain) and C-terminal (AR residues 624-
919 fused to the Gal4 DNA-binding domain) fragments
of the human AR that undergo ligand-mediated dimer-
ization.5,31 These plasmids were cotransfected with a
luciferase reporter plasmid and a plasmid constitutively
expressing â-galactosidase to control for variations in
transfection efficiency.
Testosterone (1), the unsubstituted compound 6, and
the ethyl-appended compound 7 were agonists in this
whole-cell assay, promoting AR dimerization with EC50
values of 0.3 nM (7), 0.9 nM (6), and 1.1 nM (1) (Figure
2). In contrast, the pentyl side chain of 8 conferred only
weak agonist activity, and the longer analogues 9-11
did not appreciably enable AR dimerization. The two
known AR antagonists cyproterone acetate (3) and
mifepristone (4) did not activate reporter gene expres-
sion in this assay (Figure 2).
Compounds were evaluated in a competition assay
format to assess inhibition of AR dimerization promoted
by testosterone (1 nM) in CHO-K1 cells (Figure 2). In
this assay, the control antagonists cyproterone acetate
(3) and mifepristone (4) fully blocked reporter gene
expression with IC50 values of 4.7 nM and 7.8 nM (Table
1). The pentyl-appended compound 8 was a highly
potent dimerization inhibitor (IC50 ) 0.009 nM) whereas
the octyl-appended compound 9 was similar but less
potent (IC50 ) 0.9 nM). Despite their high affinities for
the AR, the decyl (10) and dodecyl (11)-substituted
compounds exhibited weaker (IC50(10) ) 85 nM) or
insignificant (11) inhibition of AR dimerization (Figure
2 and Table 1).
antagonism: IC50 values (nM) quantified by nonlinear regression
analysis of luciferase assay data obtained from transiently trans-
fected CHO-K1 cells. c Cytotoxicity: IC50 values (µM) determined
by quantifying viable LNCaP prostate cancer cells with a sulfor-
hodamine-binding assay36 after treatment with compounds for 72
d
h. ND: Not determined. e Typical 95% confidence intervals were
within 2-fold.
ether (13) and subjected to Birch reduction22 to afford
the unconjugated diene. Deprotection of the ether under
acidic conditions was followed by bromination and
dehydrobromination to yield dienone 6.23-25 This di-
enone was protected as cyclic acetal 14,23,26-28 followed
by epoxidation of the tetrasubstituted alkene to afford
15. Addition of appropriate Grignard-cuprate reagents
to epoxide 15 followed by deprotection of the acetal and
dehydration of the 5R-alcohol provided compounds
7-11.27-29 Two-dimensional COSY and NOESY NMR
experiments confirmed that the side-chains projected
exclusively from the 11â position of these steroids.
To compare the affinity of 19-nortestosterone deriva-
tives with other known ligands, competition fluorescence
polarization assays were employed. These experiments
used E. coli-expressed AR protein (PanVera Corp) bound
to a fluorescent tracer and treated with compounds 1,
3, 4, and 6-11. As shown in Figure 1, equilibrium-
binding isotherms were observed with all of these
compounds. As listed in Table 1, these data enabled
quantification of IC50 values by nonlinear regression
analysis. Mifepristone (4) proved to be the highest
affinity of the known ligands (IC50(4) ) 2.2 nM).
Moreover, compounds 6-11 bound specifically to the
AR with affinities higher than or comparable to mife-
pristone (e.g. IC50(8) ) 0.1 nM; IC50(9) ) 6.6 nM;
IC50(10) ) 0.8 nM), and all of the nortestosterone
analogues exhibited higher affinity for the AR than the
clinically employed antiandrogen cyproterone acetate
(CPA, IC50(3) ) 22 nM). Remarkably, increasing the
side-chain length from two (7) to five (8) carbon atoms
was found to increase affinity by 20-fold, conferring sub-

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 4987
F igu r e 4. Effects of compounds on AR-mediated gene expres-
sion from an MMTV reporter vector in transiently transfected
CV-1 cells. Panels A and B: Dose-response curves of control
compounds 2-4 (Panel A) compared with synthetic compounds
7-11 (Panel B). Panels C and D: Competition assays in the
presence of DHT (2, 0.1 nM).
F igu r e 3. Effects of compounds on GR-mediated gene expres-
sion in transiently transfected CHO-K1 cells. Panels A and
B: Dose-response curves of control compounds 3, 4, and
dexamethasone (16, Panel A) compared with synthetic com-
pounds 6-11 (Panel B). Panels C and D: Competition assays
in the presence of dexamethasone (16, 100 nM) to quantify
GR antagonism.
Compounds 3, 4, 6-11 were further investigated for
their ability to halt the growth of lymph node carcinoma
of the prostate (LNCaP) cells. These cells express a
mutant AR (T877A) that recognizes the clinically im-
portant antiandrogens hydroxyflutamide and cyproter-
one acetate (3) as agonists.12,34 In contrast, the antian-
drogen mifepristone (4) is an antagonist of this mutant
AR.12,35 Cell death curves were constructed from cell
density measurements in the presence of compounds,
and calculated IC50 values are shown in Table 1.
Compounds 8-11 exhibited IC50 values similar to
mifepristone (4) with 4-fold greater potency than cypro-
terone acetate (3).
These results indicate that 11â-alkyl-∆9-19-nortestos-
terone derivatives bind tightly to the AR LBD and
exhibit minimal antiglucocorticoid activity. Some of
these compounds potently inhibit AR dimerization,
exhibit partial AR agonist activity, and provide interest-
ing candidates for studies in animal models of human
prostate cancer.
Although mifepristone (4) is a potent AR antagonist,
this drug may be limited as a chronically administered
antiandrogen by its potent antagonism of the glucocor-
ticoid receptor.18 High-resolution X-ray crystal struc-
tures have demonstrated that this antagonism results
from the active displacement of the GR helix 12 by the
bulky 11â-dimethylaniline side chain.14 The clinically
employed antiandrogen cyproterone acetate (3) is a
somewhat less potent antiglucocorticoid that is thought
to antagonize the GR through a passive antagonism
mechanism.18 To investigate whether the more flexible
11â-alkyl side chains of compounds 7-11 might reduce
this undesirable cross reactivity with the GR, these
compounds were investigated in CHO-K1 cells tran-
siently transfected with a full-length GR expression
vector32 and a glucocorticoid responsive luciferase re-
porter vector.33 As shown in Figure 3, control experi-
ments demonstrated that both cyproterone acetate and
mifepristone (4) were potent GR antagonists (IC50(3) )
0.9 nM; IC50(4) ) 0.008 nM). In contrast, compounds
6-11 were only weak GR antagonists (Figure 3 and
Table 1).
Ack n ow led gm en t. We thank Dr. E. Wilson, Dr. M.
Carey, Dr. G. Tomaselli, Dr. R. Evans, and Dr. G.
Perdew for gene constructs, Dr. C. Smith for cytotoxicity
assays, and PanVera-Invitrogen Discovery Screening for
the FP AR assay kit. We thank the NIH (R01-CA83831)
and American Cancer Society (RSG-02-025-01) for fi-
nancial support.
To examine the ability of compounds 7-11 to affect
gene expression driven by full-length AR from an
androgen response element (ARE), these compounds
were compared with cyproterone acetate (3) and mife-
pristone (4) in CV-1 cells transfected with an AR
expression vector and an MMTV-luciferase reporter
vector. These experiments (Figure 4) revealed that the
octyl- and decyl-substituted compounds 9 and 10 com-
prise potent partial agonists (EC50(9) ) 2.2 nM;
IC50(9) ) 1.3 nM; EC50(10) ) 8.4 nM; IC50(10) ) 0.6 nM)
comparable to cyproterone acetate (3, EC50(3) ) 3.6 nM;
IC50(3) ) 2.1 nM). In contrast, the pentyl-substituted
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures and characterization data for new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org
Refer en ces
(1) Mangelsdorf, D. J .; Thummel, C.; Beato, M.; Herrlich, P.; Schutz,
G.; et al. The nuclear receptor superfamily: the second decade.
Cell 1995, 83, 835-839.
(2) Gottlieb, B.; Lehvaslaiho, H.; Beitel, L. K.; Lumbroso, R.; Pinsky,
L.; et al. The Androgen Receptor Gene Mutations Database.
Nucl. Acids Res. 1998, 26, 234-238.
(3) Yong, E. L.; Tut, T. G.; Ghadessy, F. J .; Prins, G.; Ratnam, S. S.
Partial Androgen Insensitivity and Correlations with the Pre-
dicted Three-Dimensional Structure of the Androgen Receptor
Ligand-Binding Domain. Mol. Cell. Biol. 1998, 137, 41-50.
compound
8
exhibited greater agonist activity
(EC50(8) ) 0.3 nM; IC50(8) ) 72 µM). Control experi-
ments confirmed that these compounds exhibit rela-
tively low toxicity to CHO-K1 and CV-1 cells at the
highest concentration studied (data shown in the Sup-
porting Information).

4988 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Letters
(4) Matias, P. M.; Donner, P.; Coelho, R.; Thomaz, M.; Peixoto, C.;
et al. Structural Evidence for Ligand Specificity in the Binding
Domain of the Human Androgen Receptor. J . Biol. Chem. 2000,
275, 26164-26171.
(5) Langley, E.; Kemppainen, J . A.; Wilson, E. M. Intermolecular
NH2-/carboxyl-terminal interactions in androgen receptor dimer-
ization revealed by mutations that cause androgen insensitivity.
J . Biol. Chem. 1998, 273, 92-101.
(6) Snyder, P. J . Androgens. Goodman and Gilman’s The Pharma-
cological Basis of Therapeutics, 10th ed.; McGraw-Hill: New
York, 2001; pp 1635-1648.
(7) Gaddipati, J . P.; McLeod, D. G.; Heidenberg, H. B.; Sesterhenn,
I. A.; Finger, M. J .; et al. Frequent detection of codon 877
mutation in the androgen receptor gene in advanced prostate
cancers. Cancer Res. 1994, 54, 2861-2864.
(8) Kuil, C. W.; Mulder, E. Mechanism of Antiandrogen Action:
Conformational Changes of the Receptor. Mol. Cell. Endocrinol.
1994, 102, R1-R5.
(9) Lin, M. F.; Kawachi, M. H.; Stallcup, M. R.; Grunberg, S. M.;
Lin, F. F. Growth inhibition of androgen-insensitive human
prostate carcinoma cells by a 19-norsteroid derivative agent,
mifepristone. Prostate 1995, 26, 194-204.
(10) El Etreby, M. F.; Liang, Y.; J ohnson, M. H.; Lewis, R. W.
Antitumor activity of mifepristone in the human LNCaP,
LNCaP-C4, and LNCaP-C4-2 prostate cancer models in nude
mice. Prostate 2000, 42, 99-106.
(11) Liang, Y.; Eid, M. A.; El Etreby, F.; Lewis, R. W.; Kumar, M. V.
Mifepristone-induced secretion of transforming growth factor
beta1-induced apoptosis in prostate cancer cells. Int. J . Oncol.
2002, 21, 1259-1267.
(12) Song, L. N.; Coghlan, M.; Gelmann, E. P. Antiandrogen effects
of mifepristone on coactivator and corepressor interactions with
the androgen receptor. Mol. Endocrinol. 2004, 18, 70-85.
(13) Fuhrmann, U.; Hess-Stumpp, H.; Cleve, A.; Neef, G.; Schwede,
W.; et al. Synthesis and Biological Activity of a Novel, Highly
Potent Progesterone Receptor Antagonist. J . Med. Chem. 2000,
43, 5010-5016.
the natural agonist dihydrotestosterone. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 4904-4909.
(21) Matias, P. M.; Carrondo, M. A.; Coelho, R.; Thomaz, M.; Zhao,
X. Y.; et al. Structural basis for the glucocorticoid response in a
mutant human androgen receptor (AR(ccr)) derived from an
androgen-independent prostate cancer. J . Med. Chem. 2002, 45,
1439-1446.
(22) Holland, H. L.; Taylor, G. J . Enzymic Aromatization of Deute-
rium Labelled Testosterone and Androst-4-ene-3,17-dione. Can.
J . Chem. 1981, 59, 2809-2819.
(23) Ottow, E.; Beier, S.; Elger, W.; Henderson, D. A.; Neef, G.; et
al. Synthesis of Ent-17-(prop-1-ynyl-17â-hydroxy-11â-(4-N,N-
dimethylamino)-phenyl)-4,9-estradien-3-one, the Antipode of
RU-38 486. Steroids 1984, 44, 519-530.
(24) Ponsold, K. Synthese von 3-Keto-4,9-diensteroiden mit poly-
meren Reagenzien. Z. Chem. 1986, 26, 371.
(25) Perelman, M.; Farkas, E.; Fornefeld, E. J .; Kraay, R. J .; Rapala,
R. T. A New Class of Active Steroids: The 19-Nor-∆4,9-3-
Ketosteroids. J . Am. Chem. Soc. 1960, 82, 2402-2403.
(26) Faraj, H.; Claire, M.; Rondot, A.; Aumelas, A.; Auzou, G.
Synthesis of New Steroidal 11â-Substituted Spirolactones. J .
Chem. Soc., Perkin Trans. 1 1990, 3045-3048.
(27) Napolitano, E.; Fiaschi, R.; Hanson, R. N. Epoxidation of Estra-5
(10), 9 (11)-diene Derivatives; A Convenient Synthesis of 11â-
Vinylestrone Acetate. Gazz. Chim. Ital. 1990, 120, 323-326.
(28) Teutsch, G.; Belanger, A.; Philibert, D.; Tournemine, C. Syn-
thesis of 11â-Vinyl-19-norsteroids as Potent Progestins. Steroids
1982, 39, 607-615.
(29) Belanger, A.; Philibert, D.; Teutsch, G. Regio and Stereospecific
Synthesis of 11â-Substituted 19-Norsteroids: Influence of 11â-
Substitution on Progesterone Receptor Affinity. Steroids 1981,
37, 361-382.
(30) He, B.; Kemppainen, J . A.; Wilson, E. M. FXXLF and WXXLF
sequences mediate the NH2-terminal interaction with the ligand
binding domain of the androgen receptor. J . Biol. Chem. 2000,
275, 22986-22994.
(14) Kauppi, B.; J akob, C.; Farnegardh, M.; Yang, J .; Ahola, H.; et
al. The three-dimensional structures of antagonistic and ago-
nistic forms of the glucocorticoid receptor ligand-binding do-
main: RU-486 induces a transconformation that leads to active
antagonism. J . Biol. Chem. 2003, 278, 22748-22754.
(15) Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R. E.; Bonn,
T.; et al. Molecular basis of agonism and antagonism in the
oestrogen receptor. Nature 1997, 389, 753-758.
(16) Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner, P.
J .; et al. The structural basis of estrogen receptor/coactivator
recognition and the antagonism of this interaction by tamoxifen.
Cell 1998, 95, 927-937.
(17) Pike, A. C.; Brzozowski, A. M.; Walton, J .; Hubbard, R. E.;
Thorsell, A.; et al. Structural Insights into the Mode of Action
of a Pure Antiestrogen. Structure 2001, 9, 145-153.
(18) Honer, C.; Nam, K.; Fink, C.; Marshall, P.; Ksander, G.; et al.
Glucocorticoid receptor antagonism by cyproterone acetate and
RU486. Mol. Pharmacol. 2003, 63, 1012-1020.
(19) Wakeling, A. E.; Bowler, J . ICI 182,780, a new antiestrogen with
clinical potential. J . Steroid Biochem. Mol. Biol. 1992, 43, 173-
177.
(31) Langley, E.; Zhou, Z.; Wilson, E. M. Evidence for an Antiparallel
Orientation of the Ligand-activated Human Androgen Receptor
Dimer. J . Biol. Chem. 1995, 270, 29983-29990.
(32) Danielsen, M.; Northrop, J . P.; Ringold, G. M. The mouse
glucocorticoid receptor: mapping of functional domains by
cloning, sequencing and expression of wild-type and mutant
receptor proteins. EMBO J . 1986, 5, 2513-2522.
(33) Nordeen, S. K. Luciferase reporter gene vectors for analysis of
promoters and enhancers. Biotechniques 1988, 6, 454-458.
(34) Veldscholte, J .; Ris-Stalpers, C.; Kuiper, G. G.; J enster, G.;
Berrevoets, C.; et al. A mutation in the ligand binding domain
of the androgen receptor of human LNCaP cells affects steroid
binding characteristics and response to anti-androgens. Biochem.
Biophys. Res. Commun. 1990, 173, 534-540.
(35) Kuil, C. W.; Mulder, E. Deoxyribonucleic acid-binding ability of
androgen receptors in whole cells: implications for the actions
of androgens and antiandrogens. Endocrinology 1996, 137,
1870-1877.
(36) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J .;
et al. New colorimetric cytotoxicity assay for anticancer-drug
screening. J . Natl. Cancer Inst. 1990, 82, 1107-1112.
(20) Sack, J . S.; Kish, K. F.; Wang, C.; Attar, R. M.; Kiefer, S. E.; et
al. Crystallographic structures of the ligand-binding domains
of the androgen receptor and its T877A mutant complexed with
J M0342515