Division of Pharmaceutics and Pharmaceutical Chemistry, College of
Pharmacy, The Ohio State University, Columbus, Ohio (D.Y., M.A.P.,
J.T.D.); and Department of Pharmaceutical Sciences, College of
Pharmacy, University of Tennessee, Memphis, Tennessee (Y.H., S.S.H.,
C.M., N.S., L.K., D.D.M.)
The purposes of the present studies were to examine the androgen
receptor (AR) binding ability and in vitro functional activity of
multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a
radioligand competitive binding assay with the use of cytosolic AR
prepared from rat prostates. The AR agonist and antagonist activities
of high-affinity ligands were determined by the ability of the ligand
to regulate AR-mediated transcriptional activation in cultured CV-1
cells, using a cotransfection assay. Nonsteroidal compounds with
diverse structural features demonstrated a wide range of binding
affinity for the AR. Ten compounds, mainly from the
bicalutamide-related series, showed a binding affinity superior to the
structural pharmacophore from which they were derived. Several SARs
regarding nonsteroidal AR binding were revealed from the binding data,
including stereoisomeric conformation, steric effect, and electronic
effect. The functional activity of high-affinity ligands ranged from
antagonist to full agonist for the AR. Several structural features were
found to be determinative of agonist and antagonist activities. The
nonsteroidal AR agonists identified from the present studies provided a
pool of candidates for further development of selective androgen
receptor modulators (SARMs) for androgen therapy. Also, these studies
uncovered or confirmed numerous important SARs governing AR binding and
functional properties by nonsteroidal molecules, which would be
valuable in the future structural optimization of SARMs.
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Introduction |
The
role of androgens in the development and maintenance of male sexual
phenotype is mediated by the androgen receptor (AR), a member of the
steroid/thyroid hormone receptor superfamily (Tsai and O'Malley, 1994
;
Zhou et al., 1994). Upon androgen binding, AR undergoes a
conformational change, binds to specific DNA sequences called androgen
response elements, and modulates the transcription of target genes
(Zhou et al., 1994). For decades, AR has been a target of drug
development, aiming at the therapy of diseases caused by altered
androgen levels/responsiveness or the improvement of physical
performance and regulation of male fertility.
Chemicals that modulate the transcriptional activity of AR can be
divided into two structural (steroidal and nonsteroidal) and two
functional (androgenic and antiandrogenic) classes. Steroidal androgens, mainly testosterone and its derivatives, have been used
clinically as replacement therapy for androgen-deficiency (Wu, 1992;
Bagatell and Bremner, 1996). Antiandrogens are used to counteract the
undesirable actions of excessive androgens (e.g., to treat acne,
hirsutism, male-pattern baldness, and androgen-dependent prostatic
hyperplasia and carcinoma) (Neumann, 1982; McLeod, 1993). Nonsteroidal
antiandrogens, such as flutamide (Eulexin), nilutamide (Anandron), and
bicalutamide (Casodex), are often referred to as "pure
antiandrogens" because they bind exclusively to the AR and,
therefore, are devoid of antigonadotropic, antiestrogenic, and
progestational effects. These agents are advantageous over steroidal
antiandrogens (e.g., megestrol acetate, cyproterone acetate) in terms
of specificity, selectivity and pharmacokinetic properties (Neri et
al., 1979; Cockshott et al., 1990; Teutsch et al., 1994).
In recent years, there has been growing interest in the development of
nonsteroidal modulators for steroid hormone receptors as therapeutic
agents. In addition to the above-discussed nonsteroidal antiandrogens,
selective estrogen receptor modulators (SERMs) and nonsteroidal
modulators for progesterone receptor have been successfully developed
(Hamann et al., 1998; Mitlak and Cohen, 1999; Weryha et al., 1999; Zhi
et al., 1998, 2000). These nonsteroidal ligands are known for their
better receptor selectivity than steroidal ligands, and are more
flexible in structural modification for optimal physicochemical,
pharmacokinetic, and pharmacologic properties. More importantly, with
these nonsteroidal ligands, it is possible to achieve tissue-selective
actions and thus to generate agents with diverse activity profiles
meeting specific therapeutic needs. SERMs, for example, are well known
to demonstrate tissue-selectivity (Mitlak and Cohen, 1999; Weryha et
al., 1999). SERMs, such as tamoxifen and raloxifene, are nonsteroidal
estrogen receptor (ER) ligands that manifest distinctive agonist and
antagonist actions in various target tissues. Both tamoxifen and
raloxifene are ER antagonists in breast but agonists in bone. However,
unlike tamoxifen, raloxifene has no ER agonist activity in the uterus.
Whereas nonsteroidal antiandrogens have been used clinically for many
years, nonsteroidal androgens were only recently conceptualized. In the
search for AR affinity ligands, our laboratories discovered a group of
nonsteroidal androgens that are electrophilic derivatives of
bicalutamide and hydroxyflutamide (Dalton et al., 1998). Also, several
analogs of quinoline-based AR antagonists were reported by other
research groups to have AR agonist activity (Edwards et al., 1998,
1999; Hamann et al., 1999; Higuchi et al., 1999; Zhi et al., 1999).
These studies marked the emergence of a novel category of
pharmacological agents with potential applications in androgen therapy.
The discovery of nonsteroidal androgens not only provides an
opportunity to identify agents with superior therapeutic index and
pharmacokinetic profiles to steroidal androgens but also implicates the
possibility to obtain tissue-selective AR modulators (SARMs), the
counterpart of SERMs.
Although nonsteroidal androgens bearing different pharmacophores have
been reported, the general structural elements of nonsteroidal ligands
that lead to optimal agonist activity remain poorly defined. Systematic
studies to explore the structure-activity relationships (SARs) of
nonsteroidal ligands for AR binding and agonist activity are of crucial
importance for the optimization of chemical structures for maximal
functional activity and for the ultimate development of SARMs. Toward a
better understanding of the SARs, our laboratories designed and
synthesized several series of nonsteroidal compounds that incorporated
a variety of structural features known or unknown to influence the
ligand-AR interaction and evaluated their biological properties. We
present herein the results of our AR binding and in vitro functional
activity studies with these synthetic molecules and the SARs revealed
by these compounds.
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Materials and Methods |
Organic Synthesis
Compounds were prepared in our laboratories and the purities
were greater than 99% (He et al., 2002). The structures of synthesized compounds were confirmed using elemental analyses and spectroscopic data (1H and 13C NMR, mass
spectroscopy, and infrared spectroscopy).
Chemicals and Animals
[17
-methyl-3H]Mibolerone
([3H]MIB, 84 Ci/mmol) and unlabeled MIB were
purchased from PerkinElmer Life Sciences (Boston, MA). Triamicinolone acetonide, phenylmethylsulfonyl fluoride (PMSF), Tris
base, sodium molybdate, dihydrotestosterone (DHT),
o-nitrophenyl-
-D-galactopyranoside, and ATP were purchased from Sigma Chemical Co. (St. Louis, MO). Hydroxyapatite (HAP) was purchased from Bio-Rad Laboratories (Hercules, CA). EcoLite (+) scintillation cocktail was purchased from ICN Research
Products Division (Costa Mesa, CA). Ethyl alcohol (USP grade) was
purchased from AAPER Alcohol and Chemical Company (Shelbyville, KY).
Minimal essential medium (MEM), Dulbecco's modified Eagle's medium
(DMEM), penicillin-streptomycin, trypsin-EDTA, and LipofectAMINE reagent were purchased from Invitrogen (Carlsbad, CA). Fetal
bovine serum (FBS) was obtained from Atlanta Biologicals, Inc.
(Norcross, GA). Adult male Sprague-Dawley rats, weighing approximately
250 g, were purchased from Harlan Biosciences (Indianapolis, IN).
Buffers
Homogenization buffer contained 10 mM Tris, 1.5 mM disodium
EDTA, 0.25 M sucrose, 10 mM sodium molybdate, and 1 mM PMSF and was
adjusted to pH 7.4. PMSF was prepared as a stock solution of 200 mM in
ethanol and added to other components immediately before use. HAP wash
buffer contained 50 mM Tris and 1 mM
KH2PO4 and was adjusted to
pH 7.4.
-Galactosidase assay buffer was 200 mM sodium phosphate buffer, pH
7.3, containing 2 mM MgCl2, 100 mM
-mercaptoethanol, and 1.33 mg/ml
o-nitrophenyl--D-galactopyranoside.
Luciferase assay buffer consisted of 25 mM glycylglycine, 15 mM
MgCl2, 5 mM ATP, and 0.5 mg/ml bovine serum
albumin, and was adjusted to pH 7.8 with 1 M sodium hydroxide.
Preparation of Rat Prostate Cytosolic AR
Cytosolic AR was prepared from ventral prostates of castrated
male Sprague-Dawley rats as described previously (Mukherjee et al.,
1999). Briefly, rats were surgically castrated via a scrotal incision
before the removal of prostates. One day after castration, rats were
anesthetized with a mixture of ketamine/xylazine (87:13; v/v) at 1 ml/kg body weight. Ventral prostates were excised, weighed, and
immersed immediately in ice-cold homogenization buffer. The prostates
were minced with scissors and homogenized (Model Pro 200 homogenizer;
Pro Scientific, Monroe, CT) in homogenization buffer (1:2, w/v). The
homogenate was then centrifuged at 114,000g, 0°C for
1 h in an ultracentrifuge (Model L8-M; Beckman Instruments Inc.,
Palo Alto, CA). The supernatant (cytosol), containing AR proteins, was
collected and stored at 
80°C until use.
AR Competitive Binding Assay
The AR binding affinity of synthesized nonsteroidal compounds
was determined using a radioligand competitive binding assay. An
aliquot of AR cytosol preparation (50 µl) was incubated with a
saturating concentration (1 nM) of [3H]MIB and
1 µM of triamcinolone acetonide at 4°C for 18 h in the absence
or presence of increasing concentrations of the compound of interest
(10 different concentrations ranging from 10
1
nM to 104 nM). MIB is a synthetic high-affinity
ligand for the AR. Triamcinolone acetonide (1 µM) was included in the
incubate to block the interaction of [3H]MIB
with glucocorticoid and progesterone receptors. Nonspecific binding of
[3H]MIB was determined separately by adding an
excess of unlabeled MIB (1000 nM) to the incubate. After incubation,
the protein-bound radioactivity was separated from free radioactivity
by HAP precipitation. HAP was prepared as a slurry in 50 mM Tris, pH
7.2 (1:15, w/v) after being washed twice with HAP wash buffer (1:15,
w/v). An aliquot (500 µl) of HAP slurry was added to the incubate and
gently agitated for 15 min at 4°C. The mixture was centrifuged at
2000g, 4°C for 1 min, and the HAP pellet was obtained and
washed three times with 1 ml of 50 mM Tris, pH 7.2. The bound
radioactivity was then extracted from HAP by incubating the HAP pellet
with 1 ml of ethanol at room temperature for 1 h. After
centrifugation at 2500g for 1 min, 0.8 ml of the ethanolic supernatant
was added to 5 ml of scintillation cocktail. The radioactivity was
counted in a Beckman LS6800 liquid scintillation counter (Beckman
Coulter, Fullerton, CA).
Cell Culture
Monkey kidney fibroblast-like CV-1 cells were obtained from
American Type Culture Collection (Manassas, VA). The cells were grown
at 37°C in a humidified atmosphere with 5% carbon dioxide, and
maintained in minimal essential medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Cotransfection and Enzyme Assays
The in vitro functional activities of nonsteroidal ligands, as
assessed by the ability of each ligand to induce or repress AR-mediated
transcriptional activation of the luciferase reporter gene, were
examined in transiently transfected CV-1 cells. One day before
transfection, CV-1 cells were seeded into DMEM supplemented with 10%
fetal bovine serum at a density of 2 × 105
cells/well in 12-well tissue culture plates. For the following steps,
DMEM without phenol red was used. Transient transfection of plated
cells was carried out in serum-free medium using LipofectAMINE according to the manufacturer's instructions. Cells in each well were
transfected with 50 ng of a human AR expression construct (pCMVhAR;
generously provided by Dr. Donald J. Tindall, Mayo Clinic and Mayo
Foundation, Rochester, MN), 1 µg of an androgen-dependent luciferase
reporter construct (pMMTV-Luc; generously provided by Dr. Ronald Evans
at The Salk Institute, San Diego, CA), and 1 µg of a
-galactosidase expression construct (pSV--galactosidase; Promega
Corporation, Madison, WI) for constitutive expression of
-galactosidase using 6 µl of LipofectAMINE. After 10 h of transfection, cells were washed once with DMEM, and then recovered in
fresh DMEM supplemented with 0.2% FBS for 10 to 12 h before the
start of treatments.
To determine the AR agonist activity, the transfected cells were
treated with increasing concentrations of the ligand of interest. To
determine the AR antagonist activity, the cells were treated simultaneously with increasing concentrations of the ligand of interest
and 1 nM DHT. Controls, where cells were treated with 1 nM DHT alone or
vehicle alone, were included in each experiment. To measure any
AR-independent effect, a parallel experiment was also performed in
which cells cotransfected with pMMTV-Luc and pSV--galactosidase only
were treated with 500 nM of the ligand of interest. All drugs were
initially dissolved in 100% ethanol, and then serially diluted to the
desired concentration using DMEM containing 0.2% FBS. The volume of
ethanol in the final solutions was 
0.05%. The final concentrations
of the compound of interest were 1, 10, 100, and 500 nM. Drug
treatments continued for 48 h, during which all drug-containing
solutions were replaced at a 24-h interval to minimize the effect of
possible chemical degradation.
After treatment, the cells were washed twice with phosphate-buffered
saline and lysed with 200 µl/well of 1× Reporter Lysis Buffer
(Promega Corporation) at room temperature for 30 min. Cell lysates were
placed in 1.5 ml of polypropylene centrifuge tubes and centrifuged at
12,000g and 4°C for 2 min. For -galactosidase assays,
an aliquot (50 µl) of supernatant from each cell extract was
transferred to a 96-well plate and incubated with 50 µl of -galactosidase assay buffer at 37°C for 3 h. An aliquot (150 µl) of 1 M Na2CO3 was
then added to each incubate to stop the enzyme reaction, and absorbance
at 414 nm was measured using a 96-well plate reader (Titertek Multiskan
MCC/340; Labsystems Inc., Franklin, MA). For luciferase assays, an
aliquot (100 µl) of supernatant from each cell extract was added with
an equal volume of luciferase assay buffer. Luminescence was then
measured in an automated luminometer (AutoLumat LB953; PerkinElmer
Wallac Inc., Gaithersburg, MD) immediately after injecting 100 µl of
1 mM beetle luciferin to each sample.
Data Analyses
AR Binding.
The specific binding of
[3H]MIB at each concentration of the compound
of interest (B) was obtained after subtracting the nonspecific binding
of [3H]MIB, and expressed as the percentage of
the specific binding in the absence of the compound of interest
(B0). The concentration of compound that reduced
the specific binding of [3H]MIB
(B0) by 50% (IC50) was
determined by computer-fitting the data to the following equation using
WinNonlin (Pharsight Corporation, Mountain View, CA): B = B0 × [1 C/(IC50 + C)], where
C was the concentration of the compound of interest.
The equilibrium binding constant (Ki)
of the compound of interest was calculated by
Ki = Kd × IC50/(Kd + L), where Kd was the
equilibrium dissociation constant of [3H]MIB
(0.19 ± 0.01 nM; determined in preliminary experiments as described previously) (Mukherjee et al., 1996), and L was
the concentration of [3H]MIB used in the
experiment (1 nM). Statistical analyses were performed using single
factor analysis of variance, with p values less than 0.05 being considered as statistically significant differences.
Transcriptional Activation.
Transcriptional activation in
each well was calculated as the ratio of luciferase activity to
-galactosidase activity to normalize the variance in cell number and
transfection efficiency. Transcriptional activation induced by each
ligand of interest, in the absence or presence of 1 nM DHT, was
expressed as a percentage of that induced by 1 nM DHT. The efficacy of
agonist activity for a ligand of interest was the maximal percentage of
transcriptional activation induced by that ligand in the absence of
DHT. All experiments were performed in triplicate or greater, and data
were expressed as the mean ± S.D. in representative experiments.
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Results |
Androgen Receptor Binding of Nonsteroidal Compounds.
We
designed and synthesized several series of nonsteroidal compounds,
based on the SARs for nonsteroidal AR binding obtained from the
literature and previous studies in our laboratories (Glen et al., 1986;
Tucker et al., 1988; Morris et al., 1991; Mukherjee et al., 1996, 1999;
Dalton et al., 1998; Kirkovsky et al., 2000). Bicalutamide,
hydroxyflutamide, or nilutamide pharmacophores (Fig. 1) were investigated. The AR binding
affinities of these synthetic molecules, reported as
Ki values, were determined by a
radioligand competitive binding assay using rat prostates as an AR
source. A representative binding displacement curve (with
R-5) is shown in Fig. 2. The
lower the Ki value, the greater the
receptor binding affinity.
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Fig. 2.
A representative AR binding displacement curve with
R-5. Rat prostate cytosolic AR was incubated with 1 nM
of [3H]MIB, 1 µM of triamcinolone acetonide at 4°C
for 18 h, in the presence of increasing concentrations of testing
ligand or in the absence of testing ligand, as described under
Materials and Methods. Nonspecific binding was
determined separately by including 1000 nM of unlabeled MIB in the
incubate. Each data point ( ) represents the mean ± S.D. in a
representative experiment of triplicate measurements. The smooth line
is the fitted curve by the inhibitory effect sigmoid
Emax model.
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The binding results of a series of bicalutamide derivatives with
modifications in the aromatic B-ring or the X-linkage are shown in
Table 1. Previous studies from our
laboratories found that bicalutamide displayed enantioselective AR
binding; the binding affinity of the R-isomer was 30-fold
higher than that of the S-isomer (Mukherjee et al., 1996).
Thus, all compounds in this series were prepared as pure optical
isomers (R or S). Consistent with our previous
finding, all R-isomers showed at least 4-fold higher AR
binding affinity (lower Ki values)
than their corresponding S-isomers. Because the
S-isomers are essentially of no use for our purposes, the
following discussion of SARs for binding is focused entirely on
R-isomers. In this series, R-5, R-12,
and R-13 exhibited higher AR binding affinity than the lead
compound (R)-bicalutamide (R-1; p < 0.05). AR binding affinity in this series of compounds was
influenced by the X-linkage group and B-ring substituents. When the
para-substitution in the aromatic B-ring was an amino group,
the sulfone (X = SO2, R-4) showed
greater affinity than the sulfide (X = S, R-2), whereas
the sulfoxide (X = SO, R-3) had no binding affinity.
However, when the nitrogen molecule in the para-amino group
was further substituted, sulfides demonstrated higher binding than
sulfones (compare R-5 with R-7, and
R-12 with R-13). In compounds with the same
X-linkage, the B-ring substituent clearly played an important role in
AR binding. The overall effect seemed to be determined by a delicate
balance of factors, including nature, size, and position of the
substituent. Introduction of a chloroacetamido group at the
para- position (R-12 and R-13)
resulted in the highest binding affinity in each of the sulfide and
sulfone sets of compounds (compare R-12 with R-5,
R-8, R-10, and R-9; R-13
with R-1, R-4, R-6, and
R-16), indicating a beneficial role of an electrophilic
group at this position in AR binding. However, the beneficial effect of
an electrophilic group might also be offset by the negative effect of
its bulky size on receptor binding. For instance, R-10,
which bears a bromoacetamido group at the para-position,
showed lower binding affinity than R-5 (with a
para-acetamido substitution) and R-8 (with a
para-propionamido substitution), probably because of the
bulkiness of the bromoacetamido moiety. However, it is also important
to note that the bromoacetamido group is less electrophilic than the
chloroacetamido group. Excluding those compounds that bear strong
electrophilic substituents and R-4 (with an amino group),
there was a general observation that an increase in the size of the
substituent above a yet undefined limit decreased the binding affinity.
This effect can be illustrated by comparison of the binding affinities
of R-5, R-6, R-8, and R-9
in the sulfides and R-7 and R-16 in the sulfones.
In addition, as shown by comparisons of R-10 with
R-11 and R-13 with R-14, para-substitution of the B-ring was superior to
meta-substitution in terms of AR binding. Tucker et al.
(1988) proposed that the tertiary hydroxy group was involved in direct
interaction with the AR. This hypothesis was corroborated by our
observation that introduction of an acetyl group to that hydroxy moiety
in R-17 abolished the receptor binding of R-9.
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TABLE 1
AR binding affinity of bicalutamide B-ring derivatives
Ki values were determined from the competitive
binding assay, as described under Materials and Methods.
Values represent the mean ± S.D. of at least three
experiments.
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We previously reported that a nitro group, instead of a cyano group, in
the para-position of the anilide ring (corresponding to the
aromatic A-ring in bicalutamide derivatives) promoted AR binding and
agonist activity of some hydroxyflutamide analogs (Dalton et al.,
1998). We tested whether the same SAR could be extended to our
bicalutamide derivatives. Thus, the second series of bicalutamide
derivatives, with a para-nitro group in the A-ring and
various substituents in the para-position of the B-ring or a
modified X-linkage group, was evaluated for their AR binding affinity
(Table 2). Confirming the previous
results, these compounds showed enantioselective binding. All
R-isomers had receptor affinity at least 9-fold higher than
their corresponding S-isomers. As far as the
R-isomers were concerned, the introduction of a nitro group
in the A-ring generally improved, or at least maintained, the binding
affinity compared with analogs bearing the cyano moiety at this
position (compare R-18 with R-2, R-19
with R-5, R-20 with R-7, and
R-24 with R-13). A single exception to this
trend, where the nitro substitution slightly decreased the receptor
binding (compare R-23 with R-12), was noted.
Among the present series, a total of five R-isomers
(R-19, R-21, R-22, R-23,
and R-24) were superior to R-bicalutamide in
terms of AR binding (p < 0.05). Sulfides in most cases
showed at least a 2-fold higher binding affinity than corresponding
sulfones (compare R-19 with R-20, R-21
with R-22, and R-23 with R-24).
However, this relationship was reversed when the B-ring substituent was
a NHSO2CH3 group, where the
binding affinity of R-26 (sulfone) was 3-fold higher than
that of R-25 (sulfide). These results further indicated that the B-ring substituents largely determined the effect of the X-linkage on AR binding. A trifluoroacetamido, chloroacetamido, or acetamido substituent at the para-position of B-ring led to superior
AR binding, regardless of the X-linkage.
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TABLE 2
AR binding affinity of bicalutamide A- and B-ring derivatives
Ki values were determined from the competitive
binding assay, as described under Materials and Methods.
Values represent the mean ± S.D. of at least three
experiments.
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Morris et al. (1991) showed that hydroxyflutamide analogs require
strong hydrogen bond donor ability of the tertiary hydroxy group for
efficient AR binding. To examine the effect of an increased hydrogen
bond donor ability of the tertiary hydroxy group on the binding
affinity of our bicalutamide analogs, we synthesized and evaluated a
group of compounds bearing a trifluoromethyl group connected to the
chiral carbon (Table 3). These compounds
were prepared as racemic mixtures of R- and
S-isomers. Among this series, compounds 29, 30, and 31 demonstrated very high binding affinities for the AR, each similar to
the binding affinity of the corresponding R-isomer in the
previous series, in which a methyl group was connected to the chiral
carbon (i.e., R-19, R-23, and R-24,
respectively). Considering that the S-isomers generally have
poor receptor binding, the binding affinities of pure
R-isomers in these racemic compounds would thus be expected
to be even higher. Although a definitive conclusion can be made only by
directly comparing binding affinities of parallel pure isomers, it
seemed that the replacement of methyl group with trifluoromethyl group
improved AR binding affinity. Moreover, the introduction of an
electron-withdrawing nitro group into the B-ring (compounds 27 and 28)
was apparently not beneficial for AR binding in this series.
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TABLE 3
AR binding affinity of racemic bicalutamide derivatives
Ki values were determined from the competitive
binding assay, as described under Materials and Methods.
Values represent the mean ± S.D. of at least three
experiments.
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Our laboratories reported previously that hydroxyflutamide derivatives
32 and 33 (Table 4) were high- affinity
AR ligands, and that ligand 33 was a potent AR agonist (Dalton et al.,
1998). We thus further evaluated the effects of various combinations of
electron-withdrawing substitutions in the aniline ring on the binding
affinity (compounds 34 to 39). As shown in Table 4, different substitution patterns led to compounds with a wide range of binding affinities. However, none of the newly synthesized compounds showed higher binding than ligands 32 and 33. Nitro substitution in the 2-position of the aniline ring resulted in decreased AR binding (compare 34 with 35). The 3,5-dinitro substitution (compound 37) was
superior to the 2,4-dinitro substitution (compound 35) in terms of AR
binding. The binding affinity was improved with the presence of
electron-withdrawing groups in the para- and
meta-positions of the aniline ring (compare 33 with 34 and
32-33 with 38-39). We also determined the binding affinities of a group
of compounds (compounds 40-44) that differed from hydroxyflutamide in
the aromatic ring substituents. None of these compounds bound to the
AR, suggesting that bisubstitution in this ring was essential for high
AR binding affinity.
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TABLE 4
AR binding affinity of hydroxyflutamide derivatives
Ki values were determined from the competitive
binding assay, as described under Materials and Methods.
Ki values of compounds 32 to 39 represent the
mean ± S.D. of three experiments; Ki of
compounds 40 to 44 represent the average of two
experiments.
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Recently, a series of nonsteroidal compounds based on tricyclic
quinolinones (Fig. 3A) were shown to
produce AR agonist activity (Edwards et al., 1998; Hamann et al., 1999;
Higuchi et al., 1999; Zhi et al., 1999). Because the quinolinone
skeleton is structurally different from the previously discussed
pharmacophores, we were interested to determine whether hybrids of
these two sets of lead structures bound to the AR and elicited agonist
activity. Meanwhile, Ekena et al. (1998) showed that a proton-accepting
group in the ligand is important for AR binding. We hypothesized,
therefore, that replacement of the NH in the aromatic ring system
(carbostyril ring, Fig. 3B) of quinolinones with an oxygen molecule, a
proton acceptor in hydrogen bonding, might improve the receptor
binding. The change from NH to oxygen would convert the carbostyril
ring to a coumarin ring (Fig. 3C). This rationale led to our design and
synthesis of compounds bearing the coumarin ring and segments from
bicalutamide, hydroxyflutamide, or nilutamide analogs. To our surprise,
the introduction of a coumarin ring into the bicalutamide, hydroxyflutamide, or nilutamide derivatives was detrimental to AR
binding affinity. As shown in Table 5, in
the series of bicalutamide analogs whose aromatic A-ring was replaced
with a coumarin ring, the highest binding affinity was observed for
compound 47 (Ki = 80 ± 16 nM).
The replacement of aromatic A-ring with coumarin resulted in decreased
binding affinities (compare compounds 47, 51, and 52 with
R-1, R-2, and R-5, respectively), and
the relative magnitudes of reduction were related to the
para-substituent in the B-ring. Particularly, the largest
drop in binding was observed when there was a para-acetamido
group in the B-ring (compare 52 with R-5). Data in Table
6 showed that compounds combining a coumarin ring with segments from hydroxyflutamide or nilutamide derivatives also exhibited poor AR binding. Most of the compounds in
the latter series (series B) had no affinity for the AR.
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Fig. 3.
Chemical structures of the tricyclic quinolinone
skeleton (A), carbostyril ring (B), and coumarin ring (C).
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TABLE 5
AR binding affinity of bicalutamide derivatives bearing coumarin ring
Ki values were determined from the competitive
binding assay, as described under Materials and Methods.
Values represent the mean ± S.D. of at least three
experiments.
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In Vitro Functional Activity of Nonsteroidal AR Ligands.
Previous experiments in our laboratories determined that the potency of
DHT (i.e., the lowest DHT concentration that produced the maximal
transcriptional activation of the AR) was 1 nM (Dalton et al., 1998).
Therefore, in the present study, the transcriptional activation induced
by this concentration of DHT was set as 100%, and used as the
reference for quantifying the agonist and antagonist activity of other
testing ligands.
According to their pharmacological activity, receptor ligands can be
classified into full agonists, partial agonists, and antagonists. For
convenience of comparison, we adopted the following arbitrary standards
to define our AR ligands: 1) full AR agonists refer to those ligands
that induced a level of transcriptional activation not significantly
lower than that of 1 nM DHT in the agonist assay; 2) partial AR
agonists refer to those ligands that induced at least 10% of
transcriptional activation of 1 nM DHT but significantly lower than
that of 1 nM DHT in the agonist assay; 3) AR antagonists refer to those
compounds that induced less than 10% of transcriptional activation of
1 nM DHT in the agonist assay but significantly suppressed the
DHT-induced transcriptional activation in the antagonist assay.
Figure 4 shows the AR agonist (A) and
antagonist (B) activities of a series of (R)-bicalutamide
derivatives with modifications in the B-ring or the X-linkage.
R-2, with a para-amino group in the B-ring,
demonstrated the lowest AR binding affinity
(Ki = 91 ± 16 nM) in this series
of compounds in previous experiments. This compound was not able to
stimulate AR-mediated transcriptional activation with concentrations up
to 500 nM in the agonist assay. However, R-2 significantly
inhibited DHT-induced transcriptional activation at higher
concentrations. Therefore, R-2 was classified as an AR
antagonist. Bearing an N-alkylamido substituent in the para-amine of B-ring, all other ligands in this series
induced the expression of luciferase in a concentration-dependent
fashion in the agonist assay. The AR must mediate the activity of these ligands, because 500 nM of each ligand had no effect on luciferase expression when there was no AR expression plasmid transfected in the
cells. These results clearly demonstrated that R-5,
R-7, R-8, and R-13 function as AR
agonists in the mammalian cell context. R-5
(acetothiolutamide), the ligand with the highest AR binding affinity
(Ki = 4.9 ± 0.2 nM) in this
series, exhibited the most potent and efficacious agonist activity.
Nevertheless, R-7, which demonstrated higher AR binding
affinity than R-8, was inferior to R-8 in agonist
activity, indicating that higher AR binding affinity was not
necessarily a predictor of greater AR agonist activity. Interestingly,
a change of the linkage group from sulfur in R-5 to sulfone
in R-7 switched a full agonist to a partial agonist. In
accordance with previously identified SARs for AR binding, an increase
in the size of R1 resulted in decreased agonist activity (compare
R-5 with R-8). However, this SAR was not true when a strong electrophilic function was introduced at the
para-position of B-ring (compare R-7 with
R-13). It is also important to note that higher
concentrations of R-5 and R-13 resulted in a
lesser degree of AR-mediated transcriptional activation when
coincubated with 1 nM DHT during our studies for antagonist activity
(Fig. 4B). This phenomenon was previously reported for androgen
agonists (i.e., testosterone, R1881, and mibolerone) during in vitro
transcriptional activation assays (Kemppainen et al., 1999) and may be
related to squelching of intracellular factors, such as coactivators
and some transcriptional factors, in the cellular system under
excessive ligand stimulation. Similar results were observed with
R-19 and R-23 (Fig.
5B).
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Fig. 4.
AR-mediated transcriptional activation of
R-2, R-5, R-7,
R-8, and R-13. A, AR agonist activity. B,
AR antagonist activity. CV-1 cells plated at 2 × 105
cells/well in 12-well tissue culture plates were transfected with a
human AR expression construct, an androgen-responsive luciferase
reporter construct and a constitutively expressed -galactosidase
construct using LipofectAMINE, as described under Materials and
Methods. Transfected cells were incubated with 1 nM DHT alone,
vehicle, or increasing concentrations of ligand alone or together with
1 nM DHT for 48 h. Luciferase activity in each well was
standardized according to -galactosidase activity, and then
expressed as the percentage of that produced by 1 nM DHT. Values
represent mean ± S.D. (n = 3).
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Fig. 5.
AR-mediated transcriptional activation of
R-18, R-19, R-20,
R-23, and R-24. A, AR agonist activity.
B, AR antagonist activity. CV-1 cells plated at 2 × 105 cells/well in 12-well tissue culture plates were
transfected with a human AR expression construct, an
androgen-responsive luciferase reporter construct, and a constitutively
expressed -galactosidase construct using LipofectAMINE, as described
under Materials and Methods. Transfected cells were
incubated with 1 nM DHT alone, vehicle, or increasing concentrations of
ligand alone or together with 1 nM DHT for 48 h. Luciferase
activity in each well was standardized according to -galactosidase
activity, and then expressed as the percentage of that produced by 1 nM
DHT. Values represent mean ± S.D. (n = 3).
|
|
Dalton et al. (1998) previously showed that hydroxyflutamide analogs
with a para-nitro group in the anilide ring possess greater AR agonist activity than those with a para-cyano
substituent. In AR binding studies, we also observed that the change
from a para-cyano to a para-nitro in the A-ring
of bicalutamide derivatives in most cases improved AR binding affinity.
Here, we further examined the effect of this structural change on
functional activities of bicalutamide derivatives. Shown in Fig. 5 are
the agonist (A) and antagonist (B) activities of a series of
bicalutamide derivatives with a para-nitro group in the
A-ring. Similar to the observation with ligands shown in Fig. 4,
R-18, with a para-amino group in the B-ring,
functioned as an AR antagonist, whereas N-alkylamido substituents at this position conferred agonist activity to other ligands in this series. Among the four agonists, the change of a
sulfide to a sulfone in the X-linkage converted full AR agonists to
partial agonists, as shown by a comparison of R-19 with
R-20 and R-23 with R-24. Unlike those
ligands with a para-cyano in the A-ring, the presence of an
electrophilic group in the para-position of the B-ring
slightly decreased the agonist activity in this series of ligands
(compare R-19 with R-23, and R-20 with
R-24). An interseries comparison of ligands in Fig. 5 with
ligands in Fig. 4 indicated that the replacement of the
para-cyano with a para-nitro in the A-ring
increased AR agonist activity whenever the binding was improved
(compare R-19 with R-5, and R-20 with R-7). However, the agonist activity decreased when there was
no notable enhancement in AR binding (compare R-24 with
R-13).
Surprisingly, although the change from sulfide to sulfone led to
decreased agonist activity in the previous series of ligands, it
switched a full agonist (R-21) to an antagonist
(R-22) when the para-substituent in the B-ring
was a trifluoroacetamido group (Fig. 6).
This change in functional activity was accompanied by a more than
2-fold decrease in binding affinity. R-22 differs from
R-20, a partial agonist, only in the
para-substituent in the B-ring. Despite a 1.5-fold higher
binding affinity than R-20, R-22 functioned as an
antagonist, again demonstrating that higher AR binding affinity does
not necessarily predict greater agonist activity. The functional
activities of another pair of sulfide and sulfone ligands,
R-25 and R-26, are shown in Fig.
7. R-25 showed a minimal level
of agonist activity (15% at 500 nM), but antagonist activity at higher
concentrations. R-26 showed no agonist activity at
concentrations up to 500 nM and a weak antagonist activity. The change
of the carbonyl link of the B-ring substituent in R-21 and
R-22 to a sulfonyl link in R-25 and
R-26 not only impaired the AR binding but also reduced the
activities (compare agonist activity of R-21 with
R-25 and antagonist activity of R-22 with
R-26).
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Fig. 6.
AR-mediated transcriptional activation of
R-21 and R-22. A, AR agonist activity. B,
AR antagonist activity. CV-1 cells plated at 2 × 105
cells/well in 12-well tissue culture plates were transfected with a
human AR expression construct, an androgen-responsive luciferase
reporter construct and a constitutively expressed -galactosidase
construct using LipofectAMINE, as described under Materials and
Methods. Transfected cells were incubated with 1 nM DHT alone,
vehicle, or increasing concentrations of ligand alone or together with
1 nM DHT for 48 h. Luciferase activity in each well was
standardized according to -galactosidase activity and then expressed
as the percentage of that produced by 1 nM DHT. Values represent
mean ± S.D. (n = 3).
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Fig. 7.
AR-mediated transcriptional activation of
R-25 and R-26. A, AR agonist activity. B,
AR antagonist activity. CV-1 cells plated at 2 × 105
cells/well in 12-well tissue culture plates were transfected with a
human AR expression construct, an androgen-responsive luciferase
reporter construct and a constitutively expressed -galactosidase
construct using LipofectAMINE, as described under Materials and
Methods. Transfected cells were incubated with 1 nM DHT alone,
vehicle, or increasing concentrations of ligand alone or together with
1 nM DHT for 48 h. Luciferase activity in each well was
standardized according to -galactosidase activity, and then
expressed as the percentage of that produced by 1 nM DHT. Values
represent mean ± S.D. (n = 3).
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Ligands 29, 30, and 31 are racemic bicalutamide derivatives that bear a
trifluoromethyl connected to the chiral carbon, and they exhibited very
high binding affinity for the AR. As shown in Fig.
8, all three ligands acted as full AR
agonists in the agonist assay and none showed any antagonist activity
in the antagonist assay. Ligand 29, which demonstrated higher AR
binding affinity than the other two ligands, showed the greatest
agonist activity within this series. At 10 nM, ligand 29 was equally
efficient as 1 nM DHT in inducing the AR-mediated transcriptional
activation.
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Fig. 8.
AR-mediated transcriptional activation of
nonsteroidal ligands 29, 30, and 31. A, AR agonist activity. B, AR
antagonist activity. CV-1 cells plated at 2 × 105
cells/well in 12-well tissue culture plates were transfected with a
human AR expression construct, an androgen-responsive luciferase
reporter construct and a constitutively expressed -galactosidase
construct using LipofectAMINE, as described under Materials and
Methods. Transfected cells were incubated with 1 nM DHT alone,
vehicle, or increasing concentrations of ligand alone or together with
1 nM DHT for 48 h. Luciferase activity in each well was
standardized according to -galactosidase activity, and then
expressed as the percentage of that produced by 1 nM DHT. Values
represent mean ± S.D. (n = 3).
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Although the data are not shown, studies in our laboratories also
examined the specificity of these nonsteroidal agonists over other
steroidal hormone receptors. Results showed that these agonists had no
effect on the other steroid receptor-mediated transcriptional
activation and are therefore highly specific for the AR.
| |
Discussion |
By modifying the structures of known nonsteroidal AR ligands, we
obtained several series of compounds that covered a wide range of
binding affinity for the AR. These novel compounds were mainly based
upon three structurally related antiandrogen pharmacophores: bicalutamide, hydroxyflutamide, and nilutamide. Earlier studies with
these chemical scaffolds revealed several structural elements that are
important for nonsteroidal AR binding and antiandrogen activity (Glen
et al., 1986; Tucker et al., 1988; Morris et al., 1991). For compounds
based on the bicalutamide and hydroxyflutamide pharmacophores, Glen et
al. (1986) and Tucker et al. (1988) showed that an electron-deficient
aromatic ring separated from the tertiary carbinol by an amide link was
required for AR binding. Substitutions at the 3- and 4- positions of
the anilide ring with electron-withdrawing groups, particularly with
cyano or nitro as the 4-substituent and trifluoromethyl or chloro as
the 3-substituent, improved AR binding affinity. Physicochemical
studies also showed that a coplanar geometry between the amide bond and
the tertiary hydroxyl group, along with electron-withdrawing groups in
the anilide ring, enhanced the hydrogen bond donor ability of the
tertiary hydroxyl group; hydrogen bonding is regarded as another
crucial factor for receptor binding (Tucker et al., 1988; Morris et
al., 1991). Results from present binding studies with novel
nonsteroidal compounds were consistent with findings from these SAR
studies. Besides, the present binding data revealed new AR binding SARs
for the linkage group and the aromatic B-ring substitutions of the
bicalutamide pharmacophore. A number of our novel compounds, mainly
from the bicalutamide-related series, exhibited an AR binding affinity higher than their lead molecules. For the bicalutamide-related series,
the AR binding was generally enhanced by: 1) R-isomer; 2) an
electrophilic para-substituent in the aromatic B-ring; 3) a
nitro group in the para-position of A-ring; and 4) a
trifluoromethyl group linked to the chiral carbon. The AR binding of
hydroxyflutamide-related series was improved in the presence of
electrowithdrawing substituents at the para-and
meta-positions of the aniline ring. The introduction of a
coumarin ring decreased the AR binding of bicalutamide,
hydroxyflutamide, and nilutamide analogs.
The recently reported crystal structure of metribolone (R1881)-bound
human AR ligand binding domain (LBD) showed that a total of 18 amino
acid residues, scattering sparsely over two regions (amino acids 700 to
788, and 875 to 896) in the domain, interact directly with the ligand
(Matias et al., 2000). These amino acids may form a ligand-binding
pocket to accommodate the ligand. Most of these residues are
hydrophobic in nature and interact mainly with the hydrophobic moieties
in the ligand molecule, whereas a few residues are hydrophilic and may
form hydrogen bonds with polar atoms in the ligand (Matias et al.,
2000). Moreover, in vitro mutagenesis studies demonstrated that the
last 12 carboxyl-terminal amino acid residues in the receptor are also
required for ligand binding, because truncation of these residues
abolishes the binding of ligands to the human AR (Jenster et al., 1991;
Kuil et al., 1995). With the present nonsteroidal ligands, we found
that the AR binding is influenced by structural properties such as
stereoisomeric conformation, steric effect, and electronic effect in
the molecule. These findings indicated indirectly that accessibility to
the binding pocket, and interaction with amino acid residues in the pocket through hydrophobic interaction and hydrogen binding are also
critical for nonsteroidal AR binding. It would be very interesting to
determine whether these nonsteroidal ligands bind to the same binding
pocket and amino acids in the LBD as those steroid ligands.
The AR agonist and antagonist activities of identified high-affinity AR
ligands were determined in vitro using the cotransfection assay. These
nonsteroidal ligands demonstrated a range of functional activity
profiles, including antagonists, partial agonists, and full agonists.
No general correlation was observed between the receptor binding
affinity and functional activities with these ligands. In general,
agonist activity was most often observed in bicalutamide derivatives
with a sulfide linkage and an N-alkylamido substituent in
the para-position of B-ring. The change from sulfide to
sulfone in the linkage position resulted in attenuated agonist activity
or even switched a full agonist to an antagonist.
The present functional activity data demonstrated that minor structural
differences in these nonsteroidal molecules could greatly alter the
nature of receptor-ligand interaction and lead to completely different
pharmacological responses (i.e., agonist or antagonist activities). The
divergent event triggering the differentiated activities of these
structurally related molecules is most likely the ligand-induced
conformational change in the receptor, an essential step in steroid
hormone receptor signaling pathways. Compelling evidence shows that AR
agonists and antagonists induce distinct conformational changes in the
receptor, so that the receptor interacts differently with other
transcriptional factors and/or coactivator/corepressors in the
subsequent signaling pathway (Kallio et al., 1994; Kuil and Mulder,
1994, 1995; Kuil et al., 1995). It is possible, as was suggested for
other steroid receptors (Brzozowski et al., 1997), that the AR may
respond to agonists and antagonists by positioning helix H12, one of
the 12 helices contained in the secondary structure of its LBD, to either seal (in the case of agonists) or open (in the case of antagonists) the ligand binding pocket. The full spectrum of functional activities displayed by our series of nonsteroidal ligands suggests the
potential value of these compounds as tools in studying ligand-AR interactions at the molecular level.
Very importantly, the identification of numerous potent and efficacious
full AR agonists by in vitro assay represents another major progress in
our efforts toward defining the SAR necessary for AR agonist activity
of bicalutamide-based nonsteroidal ligands. These SARs provided
valuable guides for the eventual structural optimization for AR agonist
activity and development of a new generation of tissue-selective
nonsteroidal androgens, SARMs. With obvious advantages over steroid
androgens, SARMs have great therapeutic implications in that they could
not only be used as superior alternatives to steroidal androgens in
androgen replacement therapy for male hypogonadism but also expand the
scope of androgen therapy to treat wasting syndromes in critically ill
patients, to prevent aging-related disorders, and to regulate male fertility.
In summary, the present studies examined the AR binding and functional
activities of a group of novel nonsteroidal compounds with in vitro
systems. From these studies, high-affinity AR ligands with diverse
activity profiles were discovered, and SARs for AR binding and
transcriptional activation were identified. Because in vitro
cotransfection studies cannot account for the myriad of factors that
govern in vivo activity, later studies in our laboratories have focused
on characterization of the in vivo functional activity of selected full
agonists. These studies and others detailing the preclinical
pharmacology of SARMs are the topics of forthcoming reports from our laboratories.
These studies were supported by grants from the National
Institute of Child Health and Human Development (R15-HD35329), National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK59800), National Cancer Institute (R29-CA68096), the St. Francis of Assisi Foundation, and the Harriet S. Van Vleet Professorship in Pharmacy.
AR, androgen receptor;
SAR, structure-activity
relationship;
SARM, selective androgen receptor modulator;
SERM, selective estrogen receptor modulator;
ER, estrogen receptor;
MIB, mibolerone;
PMSF, phenylmethylsulfonyl fluoride;
DHT, dihydrotestosterone;
FBS, fetal bovine serum;
HAP, hydroxyapatite;
LBD, ligand binding domain.
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Abstract
Introduction
uses and abuses.
N Engl J Med
334:
707-714
