Design, synthesis, and biological evaluation of nonsteroidal cycloalkane[ d ]isoxazole-containing androgen receptor modulators

Article abstract of DOI:10.1021/jm300233k

We report here the design, preparation, and systematic evaluation of a novel cycloalkane[d]isoxazole pharmacophoric fragment-containing androgen receptor (AR) modulators. Cycloalkane[d]isoxazoles form new core structures that interact with the hydrophobic region of the AR ligand-binding domain. To systematize and rationalize the structure-activity relationship of the new fragment, we used molecular modeling to design a molecular library containing over 40 cycloalkane[d]isoxazole derivatives. The most potent compound, 4-(3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazol-3-yl)-2-(trifluoromethyl) benzonitrile (6a), exhibits antiandrogenic activity significantly greater than that of the most widely used antiandrogenic prostate cancer drugs bicalutamide (1) and hydroxyflutamide (2) in reporter gene assays measuring the transcriptional activity of AR (decreasing approximately 90% of the total AR activity) and in competitive AR ligand-binding assays (showing over four times higher potency to inhibit radioligand binding in comparison to bicalutamide). Notably, 6a maintains its antiandrogenic activity with AR mutants W741L and T877A commonly observed and activated by bicalutamide and hydroxyflutamide, respectively, in prostate cancer patients.

Full text of DOI:10.1021/jm300233k

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Nonsteroidal
Cycloalkane[d]isoxazole-Containing Androgen Receptor Modulators
Pekka K. Poutiainen,† Tuomas Oravilahti,‡ Mikael Perakyla,‡ Jorma J. Palvimo,‡ Janne A. Ihalainen,§
̈
̈
Reino Laatikainen,† and Juha T. Pulkkinen*,†
‡
†School of Pharmacy and Institute of Biomedicine, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
§Nanoscience Center, Department of Biological and Environmental Sciences, University of Jyvaskyla, P.O. Box 35, FI-40014
̈
̈
University of Jyvaskyla, Finland
̈
̈
S
* Supporting Information
ABSTRACT: We report here the design, preparation, and
systematic evaluation of a novel cycloalkane[d]isoxazole
pharmacophoric fragment-containing androgen receptor
(AR) modulators. Cycloalkane[d]isoxazoles form new core
structures that interact with the hydrophobic region of the AR
ligand-binding domain. To systematize and rationalize the
structure−activity relationship of the new fragment, we used
molecular modeling to design a molecular library containing
over 40 cycloalkane[d]isoxazole derivatives. The most potent
compound, 4-(3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazol-3-yl)-
2-(trifluoromethyl)benzonitrile (6a), exhibits antiandrogenic activity significantly greater than that of the most widely used
antiandrogenic prostate cancer drugs bicalutamide (1) and hydroxyflutamide (2) in reporter gene assays measuring the
transcriptional activity of AR (decreasing approximately 90% of the total AR activity) and in competitive AR ligand-binding
assays (showing over four times higher potency to inhibit radioligand binding in comparison to bicalutamide). Notably, 6a
maintains its antiandrogenic activity with AR mutants W741L and T877A commonly observed and activated by bicalutamide and
hydroxyflutamide, respectively, in prostate cancer patients.
binding sites and/or in interactions with coregulators whose
expression levels vary between tissues.4 Few nonsteroidal
antiandrogens have been used clinically for treatment of
prostate cancer,4,10,31,32 but during the treatment, they
eventually lose their ability to inhibit the AR, and the cancer
turns into a “refractory form”, i.e. “castration resistant” prostate
cancer (CRPC). This is suggested to result from, for example,
mutations in AR, altered expression of AR or its coregulator
proteins, aberrant AR posttranslational modifications, and gene
fusions resulting in abnormal androgen regulation of oncogenic
transcription factors and intracrine androgen production.33−37
Therefore, there is a continuous need for novel AR antagonists
which can tolerate these mutations.10,31,38−42
In the present work, a new class of nonsteroidal
antiandrogenic compounds have been designed, synthesized,
and biologically evaluated by utilizing computer-assisted
molecular modeling, flexible molecular libraries,43,44 and cell-
based transcription45 and binding assays with AR.46 The
developed lead compounds bind to and inhibit the activity of
the AR as potently as or better than the most widely used
nonsteroidal antiandrogenic drug bicalutamide (1)47 and
hydroxyflutamide (2, Figure 1).48 The novel cycloalkane[d]-
isoxazole pharmacophoric fragment-containing compounds
INTRODUCTION
■
Androgens are especially important for the maintenance and
development of the male sexual characteristics.1 The regulatory
effects of the androgens (testosterone and its metabolite 5α-
dihydrotestosterone, DHT) are mediated through the androgen
receptor (AR), which is a ligand-regulated transcription factor
and a member of the nuclear receptor superfamily.2 The AR is
prominently expressed in male reproductive organs but
moderately also in female genitalia and ovary as well as tissues
such as skin, sebaceous and sweat glands, hair follicles, cardiac
and skeletal muscle, and brain.3 AR has a supreme role in
development and progression of prostate cancer,4,5 which is the
second most common cause of cancer-related deaths among
men in developed countries.6 Competitive antiandrogens are
needed for maximal androgen blockade in treatment of both
early stage and progressed prostate cancer.7,8
Nonsteroidal selective androgen receptor modulators
(SARMs) have been developed to overcome side effects related
to the clinical use of steroidal compounds.4,9−29 The basic idea
of the SARMs is that they modulate the transcriptional activity
of AR in a tissue-selective fashion.9−11 Ligand binding induces
conformational changes in the ligand binding domain (LBD) of
the AR, which modulates its surface topology and the protein−
protein interactions between the receptor and other cellular
proteins.26,30 This all offers tools to ligand-selective gene
regulation, due to potential changes in recognition of AR DNA-
Received: February 22, 2012
Published: July 2, 2012
© 2012 American Chemical Society
6316
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
Figure 1. The skeletal structure of novel cycloalkane[d]isoxazole-containing androgen receptor modulators (5−8) and the structures of nonsteroidal
androgen antagonists 1 and 2. Chiral centers are marked with *. Due to cis-geometry of the cycloalkenes, only R,R- and S,S-enantiomers are formed
in the 1,3-dipolar cycloaddition. Ring C points out of the plane formed by rings A and B, giving the molecule a twisted shape (see Figure 2 for a
better view of the 3D-structure).
maintained their antagonistic activity with AR mutants
commonly observed in prostate cancer patients.
cycloalkane ring that fuses carbons 4 and 5 of isoxazole (ring B)
to form cycloalkane[d]isoxazoles (Figure 1).
Syntheses. All the present isoxazoles were synthesized
starting from appropriate aromatic aldoximes 3 and alkenes 4,
as shown in Scheme 1, using methods described previously in
detail by us.43 Slight modifications were made in an effort to
reduce nitrile oxide dimerization and, by that means, to
improve yields. The dimerization reaction was mainly a
problem with the aldoximes having the aromatic ring
substituted with an electron-withdrawing group (nitro or
cyano). The method was modified in such a way that the
aldoxime dissolved in dichloromethane and triethylamine was
added simultaneously dropwise to a dichloromethane/aqueous
sodium hypochlorite biphasic mixture containing a 10-fold
excess of the cycloalkene dipolarophile. We also tried
cycloaddition with N-chlorosuccinimide and chloramine-T
instead of the hypochlorite, but yields were lower. The in
situ-generated nitrile oxide underwent 1,3-dipolar cycloaddition
and led to isoxazoles 5−8 (d−t). With nitrile-substituted
aldoximes, the cycloaddition gave less than 3% yields, but with
fluorine substituted precursors, the desired nitrile products 5−8
(a−c) were achieved with satisfactory yields. In the latter case,
the aromatic fluorine of the cycloaddition product was
substituted with a cyano group by performing a cyano-
defluorination reaction with KCN.49,50 After preparation, all
the compounds were run through a semipreparative HPLC
system.43 For each successful separation, the enantiomer with a
shorter retention time is marked in the tables as ′ and the other
enantiomer as ″.
RESULTS AND DISCUSSION
■
Molecular Design. The structures of the earlier described
estrogen receptor (ER) agonists44 were used as a template for
the design of the new AR-binding compounds. The aromatic
ring (ring A, Figure 1) and the 4,5-dihydroisoxazole ring (ring
B) forming the main body of the ER active compounds were
left intact, while we focused on modifications at the substituents
of the phenyl ring and the size of the novel cycloalkane moiety
(ring C) fused with the heterocycle. The new compounds were
designed to fill the AR ligand-binding pocket efficiently (Figure
2), to be rigid enough to avoid entropic loss upon binding, and
to increase receptor selectivity. These goals were sought with a
Biological Evaluation. Our structure−activity relationship
(SAR) studies were carried out with luciferase reporter gene
activity and [3H]R1881 competitive receptor-binding assays.
The biological activities of the compounds were compared with
those of 1 and 2. The measured activity differences were
considered statistically significant when p < 0.05. The reporter
assays measured the transcriptional activity of AR. We used a
firefly luciferase (FLuc) gene construct driven by an AR-
regulated rat probasin promoter fragment which was
cotransfected with an AR expression construct into COS-1
cells. When the cells were exposed to an AR agonist
testosterone, the AR was activated, resulting in increased
transcription of the reporter gene and thereby augmented
synthesis and activity of luciferase. Addition of antiandrogens
Figure 2. R,R (orange) and S,S (dark gray) isomers of compound 6a
bound in the AR ligand-binding pocket. The receptor structure shown
in the figure is from the energy minimization with R,R-isomer. The
cyano group makes hydrogen bonds with Arg752 and Gln711. The
cyclohexane ring of the ligand fills the other corner of the pocket
contacting hydrophobic residues Leu701, Leu704, Trp741, and Met
742. Compound 6a lacks an aliphatic hydroxyl group, which in the
case of DHT connects Asn705 and Thr877 with hydrogen bonds, thus
stabilizing the helix 12.
6317
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
a
Scheme 1
a
Synthesis of cycloalkane[d]isoxazole-containing androgen receptor modulators: R1 = H, Cl, CN, F, OMe, NO2; R2 = H, Cl, CF3, F, OMe; R3 = H,
Cl, CF3, OMe; or R1 = H, R2 and R3 form a naphthalen-1-yl with the Ph. Reagents and conditions: (a) NaOCl, TEA, CH2Cl2, 0 °C to rt, 24 h. (b)
KCN, DMSO, 80−150 °C, overnight.
together with the agonist inhibited the AR activity leading to a
decreased luciferase activity, which was directly proportional to
the inhibitory activity of the antiandrogen.45 The results
obtained from these assays with wild type hAR are listed in
Tables 1−4.
Results from the whole cell binding studies showed that our
most efficient compound 6a inhibits the radioligand binding
with greater affinity than 1 and 2. The fitted curve of 6a
indicates that it has over inhibition potency 4-fold higher than
that of 1 and 8-fold higher than that of 2. Furthermore, 7a
which is a cycloheptane-containing analogue of 6a also binds to
AR significantly stronger than that of 1 and 2 and shows similar
antiandrogenic activity decreasing the total luc-reporter activity
by ca. 87%. Overall, compounds bearing 4-NO2 or 4-CN group
and 3-CF3 or 2-CF3 group attached to the aromatic moiety and
cyclohexane- or cycloheptane[d]isoxazole pharmacophoric
fragment have the best affinities for the hAR. The electron-
withdrawing group in ring A (Figure 1) seems to be crucial for
AR binding as well as for their antagonistic behavior. Similar
aromatic substituents are found also in 1 and 2.4
We also evaluated several other aromatic substituents to
characterize the main interactions between the receptor and the
ligand (Table 2). Compounds bearing a halogen, hydroxyl, or
methoxyl group as a substituent on ring A showed only weak
affinity for AR. We were not able to fit inhibition curves for all
the compounds presented here (marked with N.D. in Tables
1−4), but a few of these compounds showed inhibition in the
competitive binding assay at 10000 molar excess compared to
radioligand (inh% in Tables 1−4). Also the naphthalen-1-yl
group (6t′) showed weak affinity. Compounds having an
aromatic fluorine together with a trifluoromethyl group (5f, 5g′,
5g″, 6f, 6g′, 6g″, 7f, 7g′, 7g″, 8f′, 8f″, 8g′, and 8g″) showed
significant affinity to AR. Interestingly, compounds which have
para-nitro (6i) or para-cyano (6h′ and 6h″) group without
trifluoromethyl show very weak ability to inhibit the binding of
[3H]R1881. On the basis of these results, the polar
trifluoromethyl substituent seems to be essential for AR
binding affinity and antagonist activity. We wanted also to
determine the influence of the position of cyano group in the
ring A with compound 6a (para-cyano) and enantiomers 6c′
and 6c″ (meta-cyano). Although both isomers of 6c are very
active, racemic mixture 6a has significantly higher potency to
inhibit radioligand binding. For most ligands, the results of
FLuc reporter and whole cell binding assays seem to correlate
very well, i.e., the most active compounds also appear to bind to
Well known antiandrogens 1 and 2 (Table 1) reduced the
total luciferase activity by 86−87% when 10 μM antiandrogen
and 0.1 μM testosterone concentrations were used. The in vitro
activity screening revealed several compounds (6a, 6c″, 6d, 6e′,
7a, 7d, 8b′, 8e′, and 8e″) expressing antagonistic behavior
equal or stronger (Tables 1−4) than that of 1 and 2.
Compound 6a (R1 = CN, R2 = CF3, R3 = H, Figure 1)
reduced the total luciferase activity approximately 90% and 6e′,
8e′, and 8e″ (R1 = NO2, R2 = H, R3 = CF3) showed over 95%
reduction of the activity. Moreover, 6a and 6d (R1 = NO2, R2 =
CF3, R3 = H) were tested using AR LBD mutants W741L and
T877A commonly observed in prostate cancer patients,51−53
and both compounds were found to maintain their antagonist
behavior (Figure 3).
We did not detect any agonistic activity using a 10 μM
concentration of the ligands with AR construct. In addition, we
evaluated the possible partial agonist function of 6a and 6d
using various concentrations (from 1 nM to 10 μM) of the
ligands but did not see any activation of AR (Figure S1 in
Supporting Information). We also wanted to rule out the
possibility that the observed AR antagonistic behavior of our
compounds was due to misinterpreted inhibition of the
enzymatic activity of FLuc, which has been shown to be a
potential error source causing “false positives” in high-
throughput screens.54,55 However, none of the compounds
showed any inhibitory effect at 10 μM concentration when
incubated with purified-recombinant FLuc enzyme in vitro.
To further characterize the compounds’ biological activity log
IC50 values were determined. Relative binding inhibition (RBI)
was measured in COS-1 cells transfected with an AR expression
vector and exposed to synthetic AR agonist [3H]R1881 in the
absence (with vehicle, ethanol) and presence of AR antagonist.
Dose−response curve fitting (e.g., in Figure 4) was performed
with GrafPad Prism56 using least-squares fit with logarithmic
scale and variable slope.
6318
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
Table 1. In Vitro Biological Evaluation of Bicalutamide,
Hydroxyflutamide, Testosterone, and
Table 2. In Vitro Biological Evaluation of
Cyclohexane[d]isoxazole Derivatives
Cyclopentane[d]isoxazole Derivatives
a
AR antagonism activity was determined by FLuc gene construct
driven by an AR-regulated rat probasin promoter fragment which was
cotransfected with an AR expression construct (pSG5-hAR) into
b
COS-1 cells. Relative binding inhibition (RBI) was measured using a
whole COS-1 cell assay in which the cells were transfected with pSG5-
hAR, exposed to synthetic AR agonist [3H]R1881 in the absence (with
vehicle, ethanol) and the presence of a wide range of compound
c
concentrations. The relative transcriptional activity of AR in the
presence of 100 nM testosterone (set as 100), sample concentrations
10 μM. Results are presented as mean together with standard deviation
d
(SDant). Relative binding inhibition (log IC50) was measured using
competitive whole cell binding assay in the presence of 1.34 nM the
[3H]R1881 with a wide range of compound concentrations to obtain
their binding inhibition curves and to determine each chemical’s log
e
IC50 value (log M) with standard error (log SE). Inhibition
percentage (inh %) at the highest measured concentration (10000-
fold molar excess compared to [3H]R1881) are presented together
with standard deviation (SDinh). Nonspecific binding was measured,
and complete inhibition of the specific binding of the radioligand was
f
set as 100%. Symbol ′ = enantiomer with a shorter retention time in
the chiral separation and ″ = the enantiomer with a longer retention
time (retention times reported in Experimental Procedures). Entry
number without a symbol means that we were unable to separate the
enantiomers and that a racemic mixture was tested.
a−f
g
See Table 1. N.D. indicates a negligible functional potency that
could not be detected.
Table 3. In Vitro Biological Evaluation of
Cycloheptane[d]isoxazole Derivatives
the receptor with the highest potency. In many cases, the
enantiomers of the same compound differ significantly in the
potency of AR activation (e.g., 5a′ compared to 5a″).
To evaluate cycloalkane[d]isoxazole interactions, we system-
atically studied how the size of the C ring affects the binding
affinity of the compounds possessing both meta-trifluoromethyl
and para-cyano groups as aromatic substituents. Ligand
docking suggested that the C ring contacts hydrophobic side
chains of Leu701, Leu704, Trp41, and Met742 (Figure 2). In
this series of compounds the cyclohexane (6a) and cyclo-
heptane (7a) derivatives bind significantly stronger than the
other members of the cycloalkane homology series. Cyclo-
pentane-containing ligands 5a′ and 5a″ bind significantly
weaker than 6a and are the weakest binders of all the
homologues. On the other hand, the cycloheptane derivative 7a
expresses affinity to AR stronger than that of cyclooctane-
containing 8a. Reduction of the aliphatic ring size probably
a−f
See Table 1.
6319
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
Table 4. In Vitro Biological Evaluation of
Cyclooctane[d]isoxazole Derivatives
Figure 4. Competition of [3H]R1881 binding from AR in COS-1 cells.
Inhibition of specific [3H]R1881 binding by each compound is
expressed relative to the value in the absence of competition set to
100. Data are presented as mean SEM. Curve fitting and log IC50
calculations were performed using GraphPad Prism software.
a−f
g
See Table 1. See Table 2.
is essential for the maximal binding and activity of the novel AR
antagonists.
ECD Spectra and Absolute Configurations. The HPLC-
separated enantiomers of compounds 5a, 5d, 6e, and 7e were
selected as examples for the characterization of the absolute
configurations by using electronic circular dichroism (ECD)
spectroscopy and time-dependent density functional theory
calculations. Compound 5a possesses an aromatic substitution
similar to the most potent ligand 6a (4-CN, 3-CF3), and 5d is
their 4-NO2 analogue (Table 1). On the other hand, compound
6e was chosen for the experiment because the difference of the
transcriptional antagonistic effect between the enantiomers 6e′
and 6e″ is particularly significant (Table 2). The measured CD
spectra of the ′ and ″ forms and the theoretical spectra of the
S,S-enantiomers of the four compounds are shown in Figures
S2−S5 (Supporting Information). In each sample, the two
enantiomers show clear mirror images in the CD spectra.
Comparison of the spectra suggests that 5a′ and 5d′ are S,S-
enantiomers, whereas 6e′ and 7e′ have R,R-configurations. In
the case of 5a, 5d, and 6e, the prediction of the absolute
configuration can be considered reliable. In general, the
calculated spectra are red-shifted by 10−30 nm compared to
experimental spectra. For 7e, also the relative intensities of the
measured and calculated bands show notable deviation, making
the prediction less reliable but still indicative. Compound 6e′,
which according to our ECD prediction possesses the R,R-
configuration, is a remarkably stronger transcriptional antago-
nist compared with the S,S- enantiomer 6e″ (Table 2). Thus,
this also suggests that binding of (R,R)-6a (which differs from
6e only by the para substitution) to the LBP of AR shown in
Figure 2 represents the mode that leads to AR antagonism
higher than that of the alternative configuration.
Figure 3. Comparison of 6a, 6d, bicalutamide (1), and hydroxy-
flutamide (2) on the transcriptional activity of WT, W741L, or T877A
AR as assessed by luciferase reporter gene assay. COS-1 cells were
transfected with pSG5-hAR, pSG5-hARW741L, or pSG5-hART877A
with pProbasin(−285/+32)-Luc reporter, and the cells were treated
with vehicle (ethanol), 100 nM testosterone, or 10 μM 1, 2, 6a, or 6d
for 18 h before harvesting the cells for reporter analyses. Results are
shown as relative luciferase (rel luc) activity, with reporter activity in
the presence of testosterone set as 100%. Columns present the mean
SD.
leads to a loss of favorable contacts with the receptor, whereas a
larger ring size may be too bulky to optimally fill the binding
pocket (Figure 2). In addition, it is possible that changing the
number of the carbons in the aliphatic C ring alters its
orientation relative to the main body formed by the A and B
rings, thus also modifying the overall shape of the molecule.
Hydrogen bonds formed by the para-nitro or -cyano group of
the A ring are important for binding affinity. These groups are
supposed to bind to the hydrogen-bonding network formed by
residues Arg752 and Gln711 (Figure 2). Comparison of para-
cyano- and meta-trifluoromethyl-containing ligands to para-
nitro- and meta-trifluoromethyl-containing ligands shows that
the binding affinity significantly increases in most cases when
substituting the nitro group with a cyano group (5−8a in
comparison to 5−8d). Altogether, having para-cyano together
with a meta-trifluoromethyl group at the phenyl ring together
with a novel cyclohexane[d]isoxazole pharmacophoric fragment
CONCLUSIONS
■
In this report, we have described the cycloalkane[d]isoxazole
moiety as a novel pharmacophoric fragment interacting with
the hydrophobic region of the AR ligand-binding pocket. Our
SAR studies show that the key features for maximal biological
activity are an aromatic ring bearing an electron-withdrawing
group (para-cyano together with meta-trifluoromethyl) togeth-
6320
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
8.5), 5.31 (dd, 1 H, J = 8.9, 5.6), 4.13 (dd, 1 H, J = 8.9, 8.4), 2.19 (dd,
1 H, J = 12.2, 6.8), 1.70−1.45 (m, 5 H); 13C NMR δ 156.2, 148.0,
135.4 (3 s), 132.9 (d), 130.6 (q), 126.6 (d), 122.6 (q), 122.5 (dq),
88.5 (d), 54.9 (dq), 35.7, 30.6, 23.1 (3 t). HRMS (ESI+) calcd for
C16H18F3N2O3 [M + H]+ 301.0800, found 301.0812.
3-(4-Fluoro-3-(trifluoromethyl)phenyl)-4,5,6,6a-tetrahydro-3aH-
cyclopenta[d]isoxazole (5f). Yield 70%, a yellow wax; 1H NMR δ 7.94
(dd, 1 H, J = 7.0, 1.4), 7.88 (dd, 1 H, J = 9.0, 6.8, 1.4, 1 H), 7.24 (dd, 1
H, J = 9.4, 9.0), 4.28 (dd, 1 H, J = 8.5, 5.0), 4.03 (m, 1 H), 2.19 (m, 1
H), 1.95−1.87 (m, 2 H), 1.85−1.73 (m, 2 H), 1.53 (m, 1 H); 13C
NMR δ 160.7 (dd), 157.0 (s), 132.6 (dd), 126.6 (d), 126.0 (qd), 122.6
(q), 119.1 (qd), 117.8 (dd), 88.9, 52.1 (2 d), 36.1, 31.7, 23.8 (3 t).
HRMS (ESI+) calcd for C13H12F4NO [M + H]+ 274.0855, found
274.0858.
er with the novel cyclohexane[d]isoxazole moiety. The most
potent structure, 6a, fills the AR ligand-binding pocket by
making favorable hydrophobic contacts with the receptor. The
best ligand 6a shows four times higher potency to inhibit
radiolabeled ligand binding compared with that of 1. Our
reporter assays measuring transcriptional activity of AR indicate
that the compound exhibits strong antiandrogenic activity with
a potency significantly greater than that of the most widely used
antiandrogenic prostate cancer drugs 1 and 2. It is also notable
that the lead compound maintained its antiandrogenic activity
with AR mutants W741L and T877A that are activated by 1
and 2, respectively, and commonly observed in prostate cancer
patients. To summarize, our novel compounds form a library of
highly potential AR antagonists. The cyclohexane[d]isoxazole
pharmacophoric fragment interacts with the AR LBD and can
thus be considered as a unique lead for further development of
AR modulators with a high potency. The next logical step will
be the characterization of the in vivo behavior of the
compounds.
3-(4-Fluoro-2-(trifluoromethyl)phenyl)-4,5,6,6a-tetrahydro-3aH-
1
cyclopenta[d]isoxazole (5g). Yield 75%, a yellow wax; H NMR δ
7.43−7.38 (m, 2 H), 7.24 (ddd, 1 H, J = 8.2, 7.9, 2.6), 5.20 (m, 1 H),
4.02 (m, 1 H), 2.11 (m, 1 H), 1.72−1.65 (m, 2 H), 1.64−1.57 (m, 2
H), 1.47 (m, 1 H); 13C NMR δ 163.0 (dd), 157.2 (s), 133.9 (dd),
131.3 (qd), 125.3 (d), 123.3 (qd), 119.4 (dd), 115.0 (ddq), 87.9, 55.7
(2 d), 36.2, 30.7, 23.4 (3 t). Chiral separation in n-hexane/i-PrOH/
AcOH 98/2/0.5, retention times 8.8 min. (5g′) and 10.2 min. (5g″).
HRMS (ESI+) calcd for C13H12F4NO2 [M + H]+ 274.0855, found
274.0858 (5g′) and 274.0852 (5g″).
MATERIALS AND METHODS
■
Reagents and Equipment. Aldoximes 3 were synthesized from
the corresponding benzaldehydes and hydroxylamine hydrochloride.18
All the other starting materials, reagents, and solvents were commercial
products. DMSO was distilled prior to use. Preparative thin layer
chromatography was performed using 2.0 mm thick silica gel plates
with fluorescent indicator UV254 (Macherey-Nagel). HPLC purifica-
tions and chiral separations were performed on a Shimadzu
chromatography system using a Regis Technologies (R,R)-Whelk-O
2 (25 cm × 10 mm i.d.) column in n-hexane/i-PrOH/AcOH at flow
rate 5 mL/min, and the compounds were detected by UV absorption
at 254 nm. The purity of >95% of the compounds was confirmed by
GC/EI-MS data recorded on a Hewlett-Packard (Palo Alto, CA) gas
chromatography mass spectrometer. High-resolution electrospray
mass spectra were obtained on an Applied Biosystems/MDS Sciex
QSTAR XL spectrometer. 1H and 13C NMR spectra were recorded on
a Bruker Avance 500 spectrometer operating at 500.13 and 125.77
MHz, respectively. All J coupling constants are given in hertz. The CD
spectra were acquired with a Jasco J-700 spectropolarimeter and
processed using the J-700 program for Windows. The experiments
were carried out at rt using quartz cells (Hellma GmbH, Germany)
with Suprasil windows and an optical path length of 0.5 cm. The
HPLC-separated pure enantiomers were dissolved in acetonitrile to
100 μM concentration, and the spectra were recorded in the
wavelength range of 190−450 nm.
3-(4-Nitro-3-(trifluoromethyl)phenyl)-3a,4,5,6,7,7a-
1
hexahydrobenzo[d]isoxazole (6d). Yield 1%, a yellow wax; H NMR
δ 8.15 (d, 1 H, J = 1.7), 8.02 (dd, 1 H, J = 8.5, 1.7), 7.93 (d, 1 H, J =
8.5), 4.61 (m, 1 H), 3.31 (m, 1 H), 2.33 (m, 1 H), 2.00 (m, 1 H), 1.80
(m, 1 H), 1.78−1.63 (m, 2 H), 1.54 (m, 1 H), 1.32−1.21 (m, 2 H);
13C NMR (in acetone-d6) δ 163.1, 149.3, 135.9 (3 s), 133.1, 127.5 (2
d), 127.0 (dq), 124.8, 124.5 (2 q), 82.9, 44.4 (2 d), 27.3, 25.8, 23.0,
21.3 (4 t). HRMS (ESI+) calcd for C14H14F3N2O3 [M + H]+
315.0957, found 315.0941.
3-(4-Nitro-2-(trifluoromethyl)phenyl)-3a,4,5,6,7,7a-
1
hexahydrobenzo[d]isoxazole (6e). Yield 4%, yellow wiscous oil H
NMR: δ 8.62 (d, 1 H, J = 2.1), 8.45 (dd, 1 H, J = 8.3, 2.1), 7.76 (d, 1
H, J = 8.3), 4.69 (m, 1 H), 3.40 (m, 1 H), 2.15 (m, 1 H), 1.82 (m, 1
H), 1.72−1.62 (m, 2 H), 1.58 (m, 1 H), 1.34−1.22 (m, 2 H); 13C
NMR δ 161.8, 149.5, 135.6 (3 s), 133.1 (d), 130.7 (q), 126.9 (d),
123.9 (q), 122.6 (dq), 81.3 (d), 48.1 (dq), 25.6, 25.4, 22.1, 20.3 (4 t).
Chiral separation in n-hexane/i-PrOH/AcOH 90/10/0.5, retention
times 13.2 min (6e′) and 14.5 min (6e″). HRMS (ESI+) calcd for
C14H14F3N2O3 [M + H]+ 315.0957, found 315.0943 (6e′) and
315.0959 (6e″).
3-(4-Fluoro-3-(trifluoromethyl)phenyl)-3a,4,5,6,7,7a-
hexahydrobenzo[d]isoxazole (6f). Yield 55%, a yellow wax; 1H NMR
δ 7.94−7.88 (m, 2 H), 7.25 (dd, 1 H, J = 9.3, 9.2), 4.54 (m, 1 H), 3.72
(m, 1 H), 2.26 (m, 1 H), 1.97 (m, 1 H), 1.78 (m, 1 H), 1.77−1.59 (m,
2 H), 1.54 (m, 1 H), 1.38−1.15 (m, 2 H); 13C NMR δ 162.3 (s),
160.8, 138.7 (2 d), 132.6, 126.6 (2 dd), 126.1 (qd), 122.6 (q), 116.3
(dd), 81.4, 45.0 (2 d), 26.7, 25.3, 22.5, 20.4 (4 t). HRMS (ESI+) calcd
for C14H14F4NO [M + H]+ 288.1012, found 288.1017.
Synthesis of Cycloalkane[d]isoxazoles (compounds 5d−g,
6d−g, 6i−t, 7d−g, and 8d−g). Procedure A (Scheme 1). To a
vigorously stirred solution of alkene 4 (10 mmol) in 3 mL of DCM
was added 5% NaOCl solution (5 mL) at 0 °C. Then aldoxime 3 (1
mmol) in 2 mL of DCM and TEA (1.08 mmol) were added dropwise,
the temperature was allowed to rise to rt, and the mixture was stirred
for 24 h. The water layer was separated and washed twice with 5 mL of
DCM. Then the organic layers were combined and washed with 2 M
HCl and saturated NaHCO3 and H2O and evaporated to give the
crude product, which was purified by TLC using DCM as an eluent.
3-(4-Nitro-3-(trifluoromethyl)phenyl)-4,5,6,6a-tetrahydro-3aH-
3-(4-Fluoro-2-(trifluoromethyl)phenyl)-3a,4,5,6,7,7a-
hexahydrobenzo[d]isoxazole (6g). Yield 51%, a yellow wax; 1H NMR
δ 7.52 (dd, 1 H, J = 8.4, 5.5), 7.47 (dd, 1 H, J = 8.7, 2.6), 7.30 (ddd, 1
H, J = 8.5, 8.4, 2.6), 4.63 (m, 1 H), 3.31 (m, 1 H), 2.10 (m, 1 H), 1.81
(m, 1 H), 1.68 (m, 1 H) 1.59−1.52 (m, 3 H), 1.38−1.20 (m, 2 H); 13C
NMR δ 163.1 (dd), 162.7 (s), 133.8 (dd), 131.4 (qd), 125.3 (d), 123.4
(qd), 119.3 (dd), 115.0 (ddq), 80.8, 48.4 (2 d), 25.8, 25.4, 22.3, 20.5
(4 t). Chiral separation in n-hexane/i-PrOH/AcOH 98/2/0.5,
retention times 7.2 min (6g′) and 9.6 min (6g″). HRMS (ESI+)
calcd for C14H14F4NO [M + H]+ 288.1012, found 288.1007 (6g′) and
288.1002 (6g″).
1
cyclopenta[d]isoxazole (5d). Yield 16%, a yellow wax; H NMR δ
8.13 (d, 1 H, J = 1.6), 7.95 (dd, 1 H, J = 8.5, 1.6), 7.90 (d, 1 H, J =
8.5), 5.32 (m, 1 H), 4.01 (m, 1 H), 2.19 (m, 1 H), 1.97−1.83 (m, 2
H), 1.82−1.75 (m, 2 H), 1.50 (m, 1 H); 13C NMR δ 156.5, 148.2,
134.8 (3 s), 131.0 (d), 126.5 (q), 126.1 (d), 124.8 (dq), 122.1 (q),
90.0, 51.4 (2 d), 36.1, 31.8, 23.9 (3 t). Chiral separation in n-hexane/i-
PrOH/AcOH 98/2/0.5, retention times 19.4 min (5d′) and 22.1 min
(5d″). HRMS (ESI+) calcd for C13H12F3N2O3 [M + H]+ 301.0800,
found 301.0794 (5d′) and 301.0812 (5d″).
3-(4-Nitrophenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole (6i).
1
Yield 6%, a yellow viscous oil; H NMR δ 8.25 (m, 2 H), 7.88 (m, 2
H), 4.58 (m, 1 H), 3.30 (m, 1 H), 2.30 (m, 1 H), 2.00 (m, 1H), 1.81
(m, 1 H), 1.75−1.64 (m, 2H), 1.55 (m, 1 H), 1.33−1.21 (m, 2H). 13C
NMR δ 162.7, 148.8, 136.0 (3 s), 127.9, 124.5, 81.9, 44.3 (4 d), 26.7,
25.3, 22.4, 20.4 (4 t). HRMS (ESI+) calcd for C13H15N2O3 [M + H]+
247.1183, found 247.1077.
3-(4-Nitro-2-(trifluoromethyl)phenyl)-4,5,6,6a-tetrahydro-3aH-
cyclopenta[d]isoxazole (5e). Yield 13%, yellow viscous oil 1H NMR δ
8.61 (d, 1 H, J = 2.2), 8.41 (dd, 1 H, J = 8.5, 2.2), 7.70 (d, 1 H, J =
6321
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
3-(4-Fluorophenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
3-(3-Methoxyphenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
(6q). Yield 77%, a yellow wax; 1H NMR δ 7.29 (m, 1 H), 7.27 (d, 1 H,
J = 7.9), 7.20 (d, 1 H, J = 7.7), 6.92 (d, 1 H, J = 8.2), 4.47 (m, 1 H),
3.81 (s, 3 H), 3.21 (m, 1 H), 2.24 (m, 1 H), 1.96 (m, 1 H), 1.73 (m, 1
H), 1.65 (m, 1 H), 1.59 (m, 1 H), 1.51 (m, 1 H), 1.30−1.18 (m, 2 H);
13C NMR δ 164.3, 160.2, 131.1 (3 s), 130.1, 120.0, 116.7, 111.9, 80.9
(5 d), 55.8 (q), 45.0 (d), 26.9, 25.5, 22.8, 20.6 (4 t). Chiral separation
in n-hexane/i-PrOH/AcOH 98/2/0.5, retention times 14.5 min (6q′)
and 16.7 min (6q″). HRMS (ESI+) calcd for C14H18NO2 [M + H]+
232.1338, found 232.1334 (6q′) and 232.1332 (6q″).
1
(6j). Yield 11%, a yellow wax; H NMR δ 7.70 (m, 2 H), 7.09 (m, 2
H), 4.50 (m, 1 H), 3.23 (m, 1 H), 2.26 (m, 1 H), 1.96 (m, 1 H), 1.77
(m, 1 H), 1.71−1.60 (m, 2H), 1.54 (m, 1 H), 1.30−1.20 (m, 2H); 13C
NMR δ 164.1 (d), 163.1 (s), 129.2 (dd), 126.1 (d), 116.3 (dd), 80.9,
45.0 (2 d), 26.8, 25.4, 22.7, 20.6 (4 t). Chiral separation in n-hexane/i-
PrOH/AcOH 90/10/0.5, retention times 6.8 min (6j′) and 7.6 min
(6j″). HRMS (ESI+) calcd for C13H15FNO [M + H]+ 220.1138, found
220.1130 (6j′) and 220.1140 (6j″).
3-(3-Fluorophenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
1
(6k). Yield 11%, a yellow wax; H NMR δ 7.47 (d, 1 H, J = 7.9), 7.43
3-(2-Methoxyphenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
(6r). Yield 21%, a yellow wax; 1H NMR δ 7.61 (dd, 1 H, J = 7.6, 1.6),
7.34 (ddd, 1 H, J = 8.4, 7.5, 1.8), 6.94 (ddd, 1 H, J = 7.6, 7.5, 1.0), 6.90
(dd, 1 H, J = 8.4), 4.52 (m, 1 H), 3.81 (s, 3 H), 3.58 (m, 1 H), 2.02
(m, 1 H), 1.77 (m, 1 H), 1.72 (m, 1 H), 1.53−1.41 (m, 3 H), 1.29−
1.18 (m, 2 H); 13C NMR δ 163.5, 157.9 (2 s), 131.6, 130.5, 121.2 (3
d), 119.1 (s), 111.7, 80.3 (2 d), 55.9 (q), 47.1 (d), 26.2, 25.5, 22.6,
20.8 (4 t). HRMS (ESI+) calcd for C14H18NO2 [M + H]+ 232.1338,
found 232.1331.
(ddd, 1 H, J = 9.0, 2.5, 1.5), 7.37 (ddd, 1 H, J = 8.1, 8.1, 5.9), 7.09
(ddd, 1 H, J = 8.3, 8.1, 2.5), 4.52 (m, 1 H), 3.23 (m, 1 H), 2.27 (m, 1
H), 1.98 (m, 1 H), 1.77 (m, 1 H), 1.72−1.61 (m, 2H), 1.55 (m, 1 H),
1.30−1.20 (m, 2H); 13C NMR δ 163.5, 163.3, 132.0 (3 d), 130.6,
123.0, 117.2, 114.1 (4 dd), 81.1, 44.8 (2 d), 26.8, 25.4, 22.6, 20.5 (4 t).
HRMS (ESI+) calcd for C13H15FNO [M + H]+ 220.1138, found
220.1141.
3-(2-Fluorophenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
1
(6l). Yield 25%, a yellow viscous oil; H NMR δ 7.85 (ddd, 1 H, J =
3-Phenyl-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole (6s). Yield
1
7.6, 7.5, 1.7), 7.38 (m, 1 H), 7.17 (ddd, 1 H, J = 7.6, 7.5, 0.9), 7.11 (dd,
1 H, J = 11.3, 8.4), 4.54 (m, 1 H), 3.44 (m, 1 H), 2.19 (m, 1 H), 1.90
(m, 1 H), 1.78 (m, 1 H), 1.64−1.55 (m, 2H), 1.54 (m, 1 H), 1.30−
1.20 (m, 2H); 13C NMR δ 160.9, 160.7 (2 d), 132.0, 129.9, 124.9 (3
dd), 118.0 (d), 116.8 (dd), 80.9, 46.3 (2 d), 25.9, 25.6, 22.5, 20.6 (4 t).
Chiral separation in n-hexane/i-PrOH/AcOH 90/10/0.5, retention
times 6.1 min (6l′) and 6.9 min (6l″). HRMS (ESI+) calcd for
C13H15FNO [M + H]+ 220.1138, found 220.1140 (6l′) and 220.1138
(6l″).
46%, a yellow viscous oil; H NMR δ 7.71 (m, 2 H), 7.39 (m, 3
H), 4.49 (m, 1 H), 3.26 (m, 1 H), 2.26 (m, 1 H), 1.98 (m, 1 H), 1.76
(m, 1 H), 1.70−1.60 (m, 2 H), 1.55 (m, 1 H), 1.31−1.18 (m, 2 H);
13C NMR δ 163.9 (s), 129.0, 128.8 (2 d), 128.7 (s), 126.9, 80.4, 44.4
(3 d), 26.5, 25.1, 22.4, 20.2 (4 t). Chiral separation in n-hexane/i-
PrOH/AcOH 90/10/0.5, retention times 6.9 min (6s′) and 7.9 min
(6s″). HRMS (ESI+) calcd for C13H16NO [M + H]+ 202.1232, found
202.1228 (6s′) and 202.1239 (6s″).
3-Naphthalen-1-yl-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
(6t). Yield 14%, a colorless wax; 1H NMR δ 8.78 (d, 1 H, J = 8.5), 7.86
(m, 2 H), 7.57 (m, 2 H), 7.52 (dd, 1 H, J = 7.0, 6.9), 7.48 (dd, 1 H, J =
7.9, 7.5), 4.65 (m, 1 H), 3.53 (m, 1 H), 2.20 (m, 1 H), 1.84 (m, 1 H),
1.61 (m, 1 H), 1.60−1.52 (m, 2 H), 1.40 (m, 1 H), 1.30−1.20 (m, 2
H); 13C NMR δ 164.5, 134.5, 131.7 (3 s), 130.8 (d), 130.2 (s), 128.9,
128.1, 127.7, 127.1, 126.7, 125.3, 79.8, 48.1 (8 d), 26.1, 26.0, 22.7, 20.7
(4 t); Chiral separation in n-hexane/i-PrOH/AcOH 90/10/0.5,
retention times 9.9 min (6t′) and 17.2 min (6t″). HRMS (ESI+)
calcd for C17H18NO [M + H]+ 252.1388, found 252.1389 (6t′) and
252.1398 (6t″).
3-(4-Chlorophenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
(6m). Yield 25%, a colorless wax; 1H NMR δ 7.64 (m, 2 H), 7.37 (m, 2
H), 4.50 (m, 1 H), 3.22 (m, 1 H), 2.27 (m, 1 H), 1.95 (m, 1 H), 1.77
(m, 1 H), 1.72−1.60 (m, 2H), 1.54 (m, 1 H), 1.28−1.20 (m, 2H); 13C
NMR δ 163.4, 136.3 (2 s), 129.5, 128.5 (2 d), 128.3 (s), 81.0, 44.7 (2
d), 26.8, 25.4, 22.7, 20.6 (4 t). Chiral separation in n-hexane/i-PrOH/
AcOH 98/2/0.5, retention times 9.0 min (6m′) and 10.8 min (6m″).
HRMS (ESI+) calcd for C13H15ClNO [M + H]+ 236.0842, found
236.0838 (6m′) and 236.0849 (6m″).
3-(3-Chlorophenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
1
(6n). Yield 11%, a colorless wax; H NMR δ 7.68 (dd, 1 H, J = 1.7,
3-(4-Nitro-3-(trifluoromethyl)phenyl)-4,5,6,7,8,8a-hexahydro-
3aH-cyclohepta[d]-isoxazole (7d). Yield 58%, a yellow wax; 1H NMR
δ 8.14 (d, 1 H, J = 1.5), 7.95 (dd, 1 H, J = 8.5, 1.5), 7.94 (d, 1 H, J =
8.5), 4.99 (m, 1 H), 3.77 (m, 1 H), 2.09 (m, 1 H), 2.01 (m, 1 H), 1.81
(m, 1 H) 1.79−1.60 (m, 2 H), 1.60−1.52 (m, 3 H), 1.51−1.42 (m, 2
H); 13C NMR δ 157.9, 148.2, 134.8 (3 s), 131.2 (d), 126.6 (q), 126.2
(d), 124.5 (dq), 122.1 (q), 86.9, 50.9 (2 d), 31.3, 30.4, 28.3, 27.4, 24.0
(5 t). HRMS (ESI+) calcd for C15H16F3N2O3 [M + H]+ 329.1113,
found 329.1117.
1.6), 7.59 (ddd, 1 H, J = 7.5, 1.7, 1.6), 7.37 (ddd, 1 H, J = 7.5, 1.7, 1.7),
7.31 (ddd, 1 H, J = 8.4, 7.5), 4.51 (m, 1 H), 3.23 (m, 1 H), 2.27 (m, 1
H), 1.97 (m, 1 H), 1.77 (m, 1 H), 1.71−1.60 (m, 2H), 1.54 (m, 1 H),
1.27−1.22 (m, 2H); 13C NMR δ 163.3, 135.2 (2 s), 130.4, 130.3 (2 d),
128.3 (s), 127.3, 125.4, 81.1, 44.7 (4 d), 26.7, 25.4, 22.6, 20.5 (4 t).
Chiral separation in n-hexane/i-PrOH/AcOH 98/2/0.5, retention
times 8.7 min (6n′) and 9.6 min (6n″). HRMS (ESI+) calcd for
C13H15ClNO [M + H]+ 236.0842, found 236.0842 (6n′) and
236.0833 (6n″).
3-(4-Nitro-2-(trifluoromethyl)phenyl)-4,5,6,7,8,8a-hexahydro-
1
3-(2-Chlorophenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
3aH-cyclohepta[d]-isoxazole (7e). Yield 24%, yellow viscous oil H
1
(6o). Yield 24%, a colorless wax; H NMR δ 7.53 (dd, 1 H, J = 7.6,
NMR: δ 8.62 (d, 1 H, J = 2.1), 8.44 (dd, 1 H, J = 8.3, 2.1), 7.72 (d, 1
H, J = 8.3), 4.99 (m, 1 H), 3.80 (m, 1 H), 2.81 (m, 1 H), 2.08 (m, 1
H), 1.95 (m, 1 H), 1.87 (m, 1 H), 1.34−1.22 (m, 2 H); 13C NMR δ
159.9, 149.0, 136.5 (3 s), 133.0 (d), 130.7 (q), 126.5 (d), 122.5 (q),
122.4 (dq), 85.9, 54.2 (2d), 31.1, 30.3, 28.1, 27.2, 24.1 (5 t). Chiral
separation in n-hexane/i-PrOH/AcOH 98/2/0.5, retention times 12.5
min (7e′) and 13.6 min (7e″). HRMS (ESI+) calcd for C15H16F3N2O3
[M + H]+ 329.1113, found 329.1109 (7e′) and 329.1125 (7e″).
3-(4-Fluoro-3-(trifluoromethyl)phenyl)-4,5,6,7,8,8a-hexahydro-
3aH-cyclohepta[d]-isoxazole (7f). Yield 70%, a yellow wax; 1H NMR
δ 7.90 (dd, 1 H, J = 6.8, 1.8), 7.83 (dd, 1 H, J = 9.0, 6.9, 1.8), 7.24 (dd,
1 H, J = 9.0, 8.0), 4.91 (m, 1 H), 3.72 (m, 1 H), 2.05 (m, 1 H), 1.98
(m, 1 H), 1.83−1.47 (m, 6 H), 1.45−1.36 (m, 2 H); 13C NMR δ 160.7
(dd), 157.0 (s), 132.7 (dd), 126.6 (d), 126.2 (qd), 122.7 (q), 119.3
(qd), 117.9 (dd), 85.9, 51.5 (2 d), 31.4, 30.5, 28.4, 27.4, 24.1 (5 t).
HRMS (ESI+) calcd for C15H15F4NO [M + H]+ 302.1168, found
302.1180.
1.8), 7.43 (dd, 1 H, J = 8.1, 1.2), 7.35 (ddd, 1 H, J = 8.1, 7.5, 1.8), 7.30
(dd, 1 H, J = 7.6, 7.5, 1.2), 4.67 (m, 1 H), 3.70 (m, 1 H), 2.04 (m, 1
H), 1.83 (m, 1 H), 1.71 (m, 1 H), 1.57 (m, 1 H), 1.59−1.43 (m, 2H),
1.35 (m, 1 H), 1.26 (m, 1 H); 13C NMR δ 163.6, 133.5 (2 s), 131.5,
131.2, 130.6 (3 d), 129.5 (s), 127.3 (d), 80.6, 47.2 (2 d), 26.2, 25.2,
22.4, 20.7 (4 t). Chiral separation in n-hexane/i-PrOH/AcOH 98/2/
0.5, retention times 7.9 min (6o′) and 17.2 min (6o″). HRMS (ESI+)
calcd for C13H15ClNO [M + H]+ 236.0842, found 236.0845 (6o′) and
236.0838 (6o″).
3-(4-Methoxyphenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole
1
(6p). Yield 22%, a yellow wax; H NMR δ 7.61 (m, 2 H), 6.89 (m, 2
H), 4.43 (m, 1 H), 3.80 (s, 3 H), 3.19 (m, 1 H), 2.23 (m, 1 H), 1.93
(m, 1 H), 1.72 (m, 1 H), 1.67 (m, 1 H), 1.58 (m, 1 H), 1.51 (m, 1 H),
1.25−1.18 (m, 2 H); 13C NMR δ 164.0, 161.4 (2 s), 128.8 (d), 122.4
(s), 114.6, 80.5 (2 d), 55.7 (q), 45.1 (d), 26.9, 25.5, 22.9, 20.7 (4 t).
Chiral separation in n-hexane/i-PrOH/AcOH 98/2/0.5, retention
times 19.4 min (6p′) and 25.1 min (6p″). HRMS (ESI+) calcd for
C14H18NO2 [M + H]+ 232.1338, found 232.1338 (6p′) and 232.1338
(6p″).
3-(4-Fluoro-2-(trifluoromethyl)phenyl)-4,5,6,7,8,8a-hexahydro-
3aH-cyclohepta[d]-isoxazole (7g). Yield 73%, a yellow wax; 1H NMR
δ 7.49−7.43 (m, 2 H), 7.29 (ddd, 1 H, J = 8.9, 8.3, 2.6), 4.92 (m, 1 H),
6322
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
3.71 (m, 1 H), 2.07 (m, 1 H), 1.94 (m, 1 H), 1.85 (m, 1 H) 1.73−1.62
(m, 2 H), 1.59−1.45 (m, 3 H), 1.38 (m, 1 H), 1.27 (m, 1 H); 13C
NMR δ 163.0 (dd), 158.9 (s), 134.0 (dd), 131.5 (qd), 125.7 (d), 123.7
(qd), 119.4 (dd), 114.7 (ddq), 85.6, 55.0 (2 d), 31.6, 30.7, 28.6, 27.5,
24.6 (5 t). Chiral separation in n-hexane/i-PrOH/AcOH 98/2/0.5,
retention times 7.7 min (7g′) and 9.9 min (7g″). HRMS (ESI+) calcd
for C15H15F4NO [M + H]+ 302.1168, found 302.1173 (7g′) and
302.1172 (7g″).
2 H), 1.48 (m, 1 H); 13C NMR δ 157.8 (s), 135.6 (d), 134.0 (s), 132.8
(d), 131.0, 130.6, 123.1 (3 q), 117.3 (d), 114.3 (s), 88.7, 55.3 (2 d),
36.1, 30.9, 23.5 (3 t). HRMS (ESI+) calcd for C14H12F3N2O [M + H]+
281.0902, found 281.0910.
4-(3a,4,5,6,7,7a-Hexahydrobenzo[d]isoxazol-3-yl)-2-
(trifluoromethyl)benzonitrile (6a). Yield 93%, a yellow wax; 1H NMR
δ 8.12 (d, 1 H, J = 0.8), 7.97 (dd, 1 H, J = 8.1, 0.8), 7.88 (d, 1 H, J =
8.1), 4.59 (m, 1 H), 3.29 (m, 1 H), 2.31 (m, 1 H), 1.98 (m, 1 H), 1.81
(m, 1 H), 1.75−1.62 (m, 2 H), 1.55 (m, 1 H), 1.35−1.18 (m, 2 H);
13C NMR δ 162.0 (s), 135.6, 134.8 (2 d), 133.8 (q), 130.2 (d), 125.1,
3-(4-Nitro-3-(trifluoromethyl)phenyl)-3a,4,5,6,7,8,9,9a-
1
octahydrocycloocta[d]isoxazole (8d). Yield 17%, a yellow wax; H
NMR δ 8.10 (s, 1 H), 7.91−7.89 (m, 2 H), 4.58 (m, 1 H), 3.42 (m, 1
H), 2.12 (m, 1 H), 2.01 (m, 1 H), 1.82−1.70 (m, 4 H), 1.66 (m, 1 H),
1.55−1.44 (m, 2 H), 1.40−1.25 (m, 3 H); 13C NMR δ 160.1, 148.3,
134.7 (3 s), 131.1 (d), 126.6 (q), 126.2 (d), 124.9 (dq), 122.1 (q),
87.6, 49.7 (2 d), 30.3, 25.8, 25.6, 25.6, 25.2, 24.9 (6 t). HRMS (ESI+)
calcd for C16H18F3N2O3 [M + H]+ 343.1270, found 343.1283.
3-(4-Nitro-2-(trifluoromethyl)phenyl)-3a,4,5,6,7,8,9,9a-
octahydrocycloocta[d]isoxazole (8e). Yield 16%, yellow viscous oil
1H NMR δ 8.60 (d, 1 H, J = 2.2), 8.42 (dd, 1 H, J = 8.4, 2.2), 7.70 (d,
122.5 (2 q), 115.5 (d), 110.9 (s), 82.3, 43.9 (2 d), 26.6, 25.2, 22.3, 20.3
(4 t). C15H14F3N2O [M + H]+ 295.1058, found 295.1051.
4-(3a,4,5,6,7,7a-Hexahydrobenzo[d]isoxazol-3-yl)-3-
(trifluoromethyl)benzonitrile (6b). Yield 67%, a yellow wax; 1H NMR
δ 8.05 (s, 1 H), 7.89 (d, 1 H, J = 8.0), 7.69 (d, 1 H, J = 8.0), 4.67 (m, 1
H), 3.37 (m, 1 H), 2.13 (m, 1 H), 1.83 (m, 1 H), 1.69 (m, 1 H), 1.59−
1.45 (m, 3 H), 1.32−1.24 (m, 2 H); 13C NMR δ 162.1 (s), 135.6 (d),
134.0 (s), 132.6 (d), 130.9, 130.7, 123.1 (3 q), 117.4 (d), 114.5 (s),
81.3, 48.1 (2 d), 25.7, 25.5, 22.2, 20.4 (4 t). Chiral separation in n-
hexane/i-PrOH/AcOH 98/2/0.5, retention times 16.5 min (6b′) and
18.8 min (6b″). HRMS (ESI+) calcd for C14H14F4NO [M + H]+
295.1058, found 295.1067 (6b′) and 295.1070 (6b″).
1 H, J = 8.4), 4.70 (dd, 1 H, J = 10.4, 10.3), 3.51 (t, 1 H, J = 10.4),
2.08−1.94 (m, 2 H), 1.75−1.43 (m, 6 H), 1.43−1.20 (m, 3 H), 1.13
(m, 1 H); 13C NMR δ 159.7, 148.5, 135.5 (3 s), 133.4 (d), 130.7 (q),
126.5 (d), 122.6 (q), 122.4 (dq), 86.8, 53.3 (2d), 29.6, 26.2, 25.9, 25.3,
25.2, 24.1 (6 t). Chiral separation in n-hexane/i-PrOH/AcOH 98/2/
0.5, retention times 18.8 min. (8e′) and 21.6 min. (8e″). HRMS (ESI
+) calcd for C16H18F3N2O3 [M + H]+ 343.1270, found 343.1284 (8e′)
and 343.1286 (8e″).
3-(3a,4,5,6,7,7a-Hexahydrobenzo[d]isoxazol-3-yl)-2-
1
(trifluoromethyl)benzonitrile (6c). Yield 17%, yellow viscous oil H
NMR δ 7.92 (d, 1 H, J = 7.5), 7.73 (dd, 1 H, J = 8.3, 7.5), 7.71 (d, 1 H,
J = 8.3), 4.66 (m, 1 H), 3.25 (m, 1 H), 2.16 (m, 1 H), 2.01 (m, 1 H),
1.71 (m, 1 H), 1.66 (m, 1 H), 1.58 (m, 1 H), 1.51 (m, 1 H), 1.25−1.18
(m, 2 H); 13C NMR δ 162.7 (s), 136.0 (q), 135.9, 135.5, 132.2 (3 d),
128.8, 122.4 (2 q), 115.4 (s), 112.0 (q), 80.7, 48.4 (2 d), 30.4, 25.3,
22.0, 20.1 (4 t). Chiral separation in n-hexane/i-PrOH/AcOH 90/10/
0.5, retention times 20.8 min. (6c′) and 24.7 min. (6c″). HRMS (ESI
+) calcd for C15H14F3N2O [M + H]+ 295.1058, found 295.1048 (6c′)
and 295.1044 (6c″).
3-(4-Fluoro-3-(trifluoromethyl)phenyl)-3a,4,5,6,7,8,9,9a-
1
octahydrocycloocta[d]isoxazole (8f). Yield 63%, a yellow wax; H
NMR δ 7.92 (dd, 1 H, J = 7.0, 1.5), 7.84 (ddd, 1 H, J = 9.0, 6.8, 1.5),
7.26 (dd, 1 H, J = 9.2, 9.0), 4.54 (m, 1 H), 3.42 (m, 1 H), 2.06 (m, 1
H), 1.88−1.73 (m, 4 H), 1.68 (m, 1 H), 1.60−1.50 (m, 2 H), 1.42−
1.25 (m, 3 H); 13C NMR δ 160.6 (dd), 160.8 (s), 132.7 (dd), 126.6
(d), 126.2 (qd), 122.6 (q), 119.3 (qd), 117.9 (dd), 86.6, 50.3 (2 d),
30.4, 25.8, 25.7, 25.6, 25.2, 24.8 (6 t). Chiral separation in n-hexane/i-
PrOH/AcOH 98/2/0.5, retention times 7.6 min (8f′) and 8.5 min
(8f″). HRMS (ESI+) calcd for C16H18F4NO [M + H]+ 316.1325,
found 316.1315 (8f′) and 316.1312 (8f″).
4-(3a,4,5,6,7,7a-Hexahydrobenzo[d]isoxazol-3-yl)benzonitrile
(6h). Yield 30%, a colorless viscous oil; 1H NMR δ 7.81 (m, 2 H), 7.69
(m, 2 H), 4.56 (m, 1 H), 3.26 (m, 1 H), 2.30 (m, 1 H), 1.97 (m, 1H),
1.79 (m, 1 H), 1.75−1.64 (m, 2H), 1.55 (m, 1 H), 1.32−1.21 (m, 2H).
13C NMR δ 163.6 (s), 135.0, 133.5, 128.3 (s), 127.5, 81.9, 44.3 (3 d),
27.1, 25.5, 22.7, 21.0 (4 t). Chiral separation in n-hexane/i-PrOH/
AcOH 90/10/0.5, retention times 13.3 min (6 h′) and 14.3 min (6
h″). HRMS (ESI+) calcd for C14H15N2O [M + H]+ 227.1184, found
227.1184 (6 h′) and 227.1180 (6 h″).
3-(4-Fluoro-2-(trifluoromethyl)phenyl)-3a,4,5,6,7,8,9,9a-
1
octahydrocycloocta[d]-isoxazole (8g). Yield 30%, a yellow wax; H
NMR δ 7.44 (dd, 1 H, J = 8.5, 5.5), 7.42 (dd, 1 H, J = 9.1, 2.7), 7.26
(ddd, 1 H, J = 9.1, 8.5, 2.7), 4.63 (m, 1 H), 3.41 (m, 1 H), 2.00−1.91
(m, 2 H), 1.70−1.41 (m, 6 H), 1.40−1.10 (m, 4 H); 13C NMR δ 163.1
(dd), 160.5 (s), 134.4 (dd), 131.5 (qd), 125.4 (d), 123.3 (qd), 119.3
(dd), 115.0 (ddq), 86.1, 53.7 (2 d), 30.1, 26.7, 26.4, 25.7, 25.6, 24.2 (6
t). Chiral separation in n-hexane/i-PrOH/AcOH 98/2/0.5, retention
times 7.3 min (8g′) and 9.6 min (8g″). HRMS (ESI+) calcd for
C16H18F4NO [M + H]+ 316.1325, found 316.1321 (8g′) and 316.1325
(8g″).
4-(4,5,6,7,8,8a-Hexahydro-3aH-cyclohepta[d]isoxazol-3-yl)-2-
(trifluoromethyl)benzonitrile (7a). Yield 81%, a yellow wax; 1H NMR
δ 8.07 (d, 1 H, J = 0.8), 7.87 (dd, 1 H, J = 8.1, 0.8), 7.88 (d, 1 H, J =
8.1), 4.94 (m, 1 H), 3.71 (m, 1 H), 2.05 (m, 1 H), 1.98 (m, 1 H),
1.83−1.47 (m, 6 H), 1.45−1.36 (m, 2 H); 13C NMR δ 158.2 (s),
135.6, 134.9 (2 d), 133.8 (q), 130.4 (d), 125.3, 122.5 (2 q), 115.6 (d),
110.7 (s), 86.9, 50.8 (2 d), 31.3, 30.4, 28.4, 27.4, 24.0 (5 t). HRMS
(ESI+) calcd for C16H16F3N2O [M + H]+ 309.1215, found 309.1224.
4-(4,5,6,7,8,8a-Hexahydro-3aH-cyclohepta[d]isoxazol-3-yl)-3-
(trifluoromethyl)benzonitrile (7b). Yield 78%, a yellow wax; 1H NMR
δ 8.04 (s, 1 H), 7.88 (d, 1 H, J = 8.0), 7.64 (d, 1 H, J = 8.0), 4.97 (m, 1
H), 3.77 (m, 1 H), 2.07 (m, 1 H), 1.94 (m, 1 H), 1.85 (m, 1 H), 1.74−
1.25 (m, 5 H); 13C NMR δ 158.3 (s), 135.5 (d), 134.0 (s), 132.9 (d),
131.3, 130.9, 123.0 (3 q), 117.3 (d), 114.3 (s), 86.2, 54.6 (2 d), 31.5,
30.7, 28.5, 27.5, 24.5 (5 t). Chiral separation in n-hexane/i-PrOH/
AcOH 98/2/0.5, retention times 16.7 min (7b′) and 19.0 min (7b″).
HRMS (ESI+) calcd for C16H16F3N2O [M + H]+ 309.1215, found
309.1230 (7b′) and 309.1220 (7b″).
Synthesis of Cycloalkane[d]isoxazolylbenzonitriles (com-
pounds 5a, 5b, 6a−c, 6h, 7a, 7b, 8a, and 8b). Procedure B
(Scheme 1). A stirred solution of an appropriate fluoro compound (1
mmol) and KCN (4 mmol) in dry DMSO (5 mL) was heated at 80−
150 °C on a sand bath overnight. After the reaction, the mixture was
diluted with water and extracted with diethyl ether. The organic phase
was dried (MgSO4) and evaporated to give crude product, which was
purified by TLC using DCM as an eluent.
4-(4,5,6,6a-Tetrahydro-3aH-cyclopenta[d]isoxazol-3-yl)-2-
(trifluoromethyl)benzonitrile (5a). Yield 82%, a yellow wax; 1H NMR
δ 8.14 (s, 1 H), 7.95 (d, 1 H, J = 8.5), 7.87 (d, 1 H, J = 8.5), 5.35 (dd,
1 H, J = 9.0, 4.5), 4.03 (td, 1 H, J = 9.0, 2.0), 2.23 (m, 1 H), 2.00−1.78
(m, 4 H), 1.52 (m, 1 H); 13C NMR δ 156.9 (s), 135.5, 134.9 (2 d),
133.7 (q), 130.2 (d), 125.1, 122.6 (2 q), 115.6 (d), 110.6 (s), 90.0,
51.3 (2 d), 36.0, 31.8, 23.9 (3 t). Chiral separation in n-hexane/i-
PrOH/AcOH 98/2/0.5, retention times 27.1 min (5a′) and 29.8 min
(5a″). HRMS (ESI+) calcd for C14H12F3N2O [M + H]+ 281.0902,
found 281.0905 (5a′) and 281.0904 (5a″).
4-(3a,4,5,6,7,8,9,9a-Octahydrocycloocta[d]isoxazol-3-yl)-2-
(trifluoromethyl)benzonitrile (8a). Yield 98%, a yellow wax; 1H NMR
δ 8.07 (s, 1 H), 7.86−7.82 (m, 2 H), 4.56 (m, 1 H), 3.40 (m, 1 H),
2.15−2.08 (m, 1 H), 2.01 (m, 1 H), 1.81−1.70 (m, 4 H), 1.65 (m, 1
H), 1.55−1.43 (m, 2 H), 1.35−1.21 (m, 3 H); 13C NMR δ 160.4 (s),
135.6, 134.8 (2 d), 133.8 (q), 130.3 (d), 125.3, 122.5 (2 q), 115.5 (d),
110.7 (s), 87.6, 49.6 (2 d), 30.3, 25.8, 25.6, 25.6, 25.2, 24.9 (6 t).
HRMS (ESI+) calcd for C17H18F3N2O [M + H]+ 323.1371, found
323.1387.
4-(4,5,6,6a-Tetrahydro-3aH-cyclopenta[d]isoxazol-3-yl)-3-
(trifluoromethyl)benzonitrile (5b). Yield 37%, a yellow wax; 1H NMR
δ 8.00 (s, 1 H), 7.85 (d, 1 H, J = 8.0), 7.60 (d, 1 H, J = 8.0), 5.27 (m, 1
H), 4.08 (m, 1 H), 2.15 (m, 1 H), 1.77−1.69 (m, 2 H), 1.69−1.57 (m,
6323
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
4-(3a,4,5,6,7,8,9,9a-Octahydrocycloocta[d]isoxazol-3-yl)-3-
(trifluoromethyl)benzonitrile (8b). Yield 18%, a yellow wax; 1H NMR
δ 8.01 (s, 1 H), 7.86 (d, 1 H, J = 8.0), 7.62 (d, 1 H, J = 8.0), 4.68 (m, 1
H), 3.48 (m, 1 H), 2.05−1.93 (m, 2 H), 1.75−1.44 (m, 6 H), 1.43−
1.07 (m, 4 H); 13C NMR δ 159.9 (s), 135.5 (d), 134.2 (s), 133.2 (d),
130.9, 130.7, 123.0 (3 q), 117.3 (d), 114.4 (s), 86.7, 53.8 (2 d), 30.0,
26.6, 26.3, 25.7, 25.6, 24.4 (6 t). Chiral separation in n-hexane/i-
PrOH/AcOH 98/2/0.5, retention times 15.4 min (8b′) and 17.5 min
(8b″). HRMS (ESI+) calcd for C17H18F3N2O [M + H]+ 323.1371,
found 323.1378 (8b′) and 323.1370 (8b″).
LKB WALLAC 1214 racbeta counter. Nonspecific binding (NSB) was
subtracted from these data, using corresponding radioinert hormone at
200-fold compared to [3H]R1881. The results (% inhibition) were
calculated as % inhibition = 100 − [100 × (average compound/
average [3H]R1881)]. The dose−response log IC50 values were
analyzed with GraphPad Prism.56
Statistical Analysis. Student's t test was used for comparisons with
the aid of a software package GraphPad Prism.56 p < 0.05 is considered
as significant.
Molecular Modeling. The crystal structure of AR in complex with
DHT (PDB code 1T7T)57 was used for AR LBD coordinates. Ligand
docking was performed using the crystal structure of the AR−1
complex (PDB code 1Z95).58 Enantiomers (R,R)-6a and (S,S)-6a
were docked to the LBP by superimposing the A rings of the
compounds and 1. After that, the moiety formed by rings B and C was
rotated to fit to the binding pocket. This approach was chosen because
the A ring of 6a is identical to the aromatic ring of 1 and was therefore
assumed to bind similarly. In addition, binding of the aromatic ring of
1 is shared by other nonsteroidal AR agonists and antagonists having
identical or similar aromatic rings.59 The docked structures were
solvated in a box of water molecules and energy-minimized using the
AMBER 9.0 program60 and the Amber ff99SB force field.61 Accelrys
discovery studio software62 was used to model the new compounds in
the AR LBP.
Calculation of ECD Spectra. ECD spectra of the S,S-enantiomers
of 5a, 5d, 6e, and 7e were calculated using the TDDFT63,64 method at
the B3LYP/aug-cc-pVDZ level. The polarizable continuum model
using the integral equation formalism (acetonitrile)65 was included in
the TDDFT calculations. For the TDDFT calculations, geometries of
the molecules were optimized at the B3LYP/6-311G** level. SpecDis
1.53 program66 was used to sum up and visualize the ECD spectra.
The spectra were simulated by using the calculated velocity rotational
strengths, Gaussian bandshapes, and bandwidth σ = 0.3 eV. Quantum
mechanical calculations were perfromed using the Gaussian09
program revision C.01.67
Firefly Luciferase Enzyme Inhibition Assay. The HPLC-
purified samples were dissolved in ethanol at 0.01 mol/L
concentrations. They were further diluted to a concentration range
from 10−3 to 10−7 mol/L in 1x Reporter Lysis Buffer (Promega). The
purified luciferase protein was purchased from Sigma-Aldrich (FLuc
from the firefly Photinus pyralis). The luciferase was diluted to 0.1 μg/
mL concentration in 1x Reporter Lysis Buffer. To 30 μL of the
luciferase solution was added 1.2 μL of different compound
concentrations as three replicates. After incubation at rt for 20 min,
10 μL of the incubations was transferred to 96-well plates (Greiner
Microlon lumitrac 200) and 30 μL of luciferase substrate solution was
added (Promega Luciferase Assay Substrate). Also three blank (1x
Reporter Lysis Buffer-containing) and control (vehicle, ethanol-
exposed luciferase) samples were measured. Lumiscence produced
by the luciferase was measured with Thermo Luminoscan Ascent
scanner.
Cell Culture, Transfection, and Luciferase Reporter Assays.
Testosterone was purchased from Steraloids Inc., bicalutamide from
Molekula Ltd., and hydroxyflutamide from Chemos GmbH. One day
before transfection, COS-1 cells (from ATCC) were seeded onto 24-
̀
well plates in 1 mL of DMEM (Dulbeccos modified Eagle medium,
Gibco) containing 10% dextran-charcoal-treated fetal bovine serum
(FBS) and 0.25% (v/v) penicillin−streptomycin (Euroclone) at a
density of 70 × 103 cells/well. After medium change to DMEM
containing 2.5% FBS, the cells were transfected for 24 h with pSG5-
hAR (10 ng/well), pProbasin(−285/+32)-Luc (100 ng/well), and
internal control pCMVβ (10 ng/well) by using TransIT-LT1
Transfection Reagent (Mirus Bio LCC). After the transfection, the
cells were exposed to vehicle (ethanol) alone, testosterone (100 nM)
as a reference for a pure androgen agonist (100% activation), or test
compounds at 10 μM concentration. In the antagonism reporter
assays, the cells were exposed simultaneously to testosterone and test
compounds. After 18 h, cells were processed, luciferase and β-
galactosidase activities and protein concentrations were measured, and
relative luciferase activities were calculated as described previously.45
When studying mutated androgen receptors, pSG5-hARW741L or
pSG5-hART877A was used instead of pSG5-hAR.
ASSOCIATED CONTENT
* Supporting Information
■
S
A figure showing the concentration dependence of the agonistic
and antagonistic effect of 1, 6a, and 6d, figures showing the
comparison of the experimental and calculated ECD spectra of
5a, 5d, 6e, and 7e, and tables showing the atomic coordinates
of the optimized conformations of (3aS,6aS)-5a, (3aS,6aS)-5d,
(3aS,7aS)-6e, and (3aS,8aS)-7e. This material is available free
of charge via the Internet at http://pubs.acs.org.
Whole Cell Binding Assay. The ability of the test compounds to
AUTHOR INFORMATION
Corresponding Author
*Tel: +358 40 355 3247; e-mail, juha.t.pulkkinen@uef.fi.
Notes
■
bind to AR was measured by relative binding inhibition (RBI) assay
3
which measures their ability to displace H-labeled synthetic agonist
R1881 from AR expressed in COS-1 cells.46 One day before
transfection, COS-1 cells were seeded into 12-well plates in 2 mL of
DMEM (Dulbecco's modified Eagle medium, Gibco) containing 10%
dextran-charcoal-treated fetal bovine serum and 0.25% (v/v)
penicillin−streptomycin (Euroclone) at a density of 140 × 103 cells/
well. After medium change to DMEM containing 2.5% FBS, the cells
were transfected for 24 h with pSG5-hAR (100 ng/well) by using
TransIT LT1 transfection reagent. After 36 h, the medium was
aspirated, and the cells were washed with phosphate-buffered saline
(PBS) and then treated with test compounds at several concentrations:
1, 10, 100, 1000, and up to 10000-fold molarities compared to labeled
R1881 (1.34 nM) in 0.5 mL of DMEM. After 2 h incubation at 37 °C,
the medium was aspirated, and cells were transferred to Eppendorf
tubes with phosphate-buffered saline (PBS), centrifuged at 4 °C at
4000g for 5 min, and then washed twice with an excess of PBS. The
cell pellets were dissolved in 50 μL of 0.5 M NaOH and incubated for
15 min at 56 °C, after which the samples (three replicates for each
sample compound and concentration) were transferred to liquid
scintillation tubes with 3 mL of OptiPhase HiSafe 3 solution (Perkin-
Elmer), and radioactivity of AR-bound [3H]R1881 was measured with
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mrs. Merja Rasanen for expert technical assistance.
̈
̈
The Finnish IT Center for Science (CSC) is acknowledged for
providing computational resources. Financial support from
Graduate School of Organic Chemistry and Chemical Biology
(P.K.P.), National Graduate School in Informational and
Structural Biology (T.O.), The Cancer Society of Finland
(J.J.P.), and The Foundation for Finnish Innovations (J.T.P.,
project nr. 21092) is gratefully acknowledged.
ABBREVIATIONS USED
■
3D, three-dimensional; AR, androgen receptor; COS-1, African
green monkey kidney cells; CRPC, castration-resistant prostate
cancer; DFT, density functional theory; DHT, 5α-dihydrotes-
6324
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
androgen receptor modulator. J. Steroid Biochem. Mol. Biol. 2008, 109,
129−137.
(18) Ohta, K.; Goto, T.; Fijii, S.; Suzuki, T.; Ohta, S.; Endo, Y.
Design and synthesis of carborane-containing androgen receptor (AR)
antagonist bearing a pyridine ring. Bioorg. Med. Chem. 2008, 16, 8022−
8028.
tosterone; DMEM, Dulbecco's modified Eagle medium; ECD,
electronic circular dichroism; ER, estrogen receptor; FBS, fetal
bovine serum; FLuc, firefly luciferase; hAR, human androgen
receptor; N.D, not detected; NSB, nonspecific binding; p,
probability value of statistical significance; R1881, 17β-hydroxy-
17-methylestra-4,9,11-trien-3-one; RBI, relative binding inhib-
ition; SD, standard deviation; SE, standard error; SEM,
standard error of mean; TDDFT, time-dependent density
functional theory; TEA, triethylamine
(19) Nirschl, A. A.; Zou, Y.; Krystek, S. R., Jr; Sutton, J. C.; Simpkins,
L. M.; Lupisella, J. A.; Kuhns, J. E.; Seethala, R.; Golla, R.; Sleph, P. G.;
Beehler, B. C.; Grover, G. J.; Egan, D.; Fura, A.; Vyas, V. P.; Li, Y. X.;
Sack, J. S.; Kish, K. F.; An, Y.; Bryson, J. A.; Gougoutas, J. Z.; DiMarco,
J.; Zahler, R.; Ostrowski, J.; Hamann, L. G. N-Aryloxazolidin-2-imine
muscle selective androgen receptor modulators enhance potency
through pharmacophore reorientation. J. Med. Chem. 2009, 52, 2794−
2798.
(20) Schlienger, N.; Lund, B. W.; Pawlas, J.; Badalassi, F.; Bertozzi,
F.; Lewinsky, R.; Fejzic, A.; Thygesen, M. B.; Tabatabaei, A.; Bradley,
S. R.; Gardell, L. R.; Piu, F.; Olsson, R. Synthesis, structure−activity
relationships, and characterization of novel nonsteroidal and selective
androgen receptor modulators. J. Med. Chem. 2009, 52, 7186−7191.
(21) Miller, C. P.; Bhaket, P.; Muthukaman, N.; Lyttle, C. R.;
Shomali, M.; Gallacher, K.; Slocum, C.; Hattersley, G. Synthesis of
potent, substituted carbazoles as selective androgen receptor
modulators (SARMs). Bioorg. Med. Chem. Lett. 2010, 20, 7516−7520.
(22) Zilbermint, M. F.; Dobs, A. S. Nonsteroidal selective androgen
receptor modulator Ostarine in cancer cachexia. Fut. Oncol. 2009, 5,
1211−1220.
(23) Ostrowski, J.; Kuhns, J. E.; Lupisella, J. A.; Manfredi, M. C.;
Beehler, B. C.; Krystek, S. R., Jr; Bi, Y.; Sun, C.; Seethala, R.; Golla, R.;
Sleph, P. G.; Fura, A.; An, Y.; Kish, K. F.; Sack, J. S.; Mookhtiar, K. A.;
Grover, G. J.; Hamann, L. G. Pharmacological and x-ray structural
characterization of a novel selective androgen receptor modulator:
potent hyperanabolic stimulation of skeletal muscle with hypostimu-
lation of prostate in rats. Endocrinology 2007, 148, 4−12.
(24) Narayanan, R.; Coss, C. C.; Yepuru, M.; Kearbey, J. D.; Miller,
D. D.; Dalton, J. T. Steroidal androgens and nonsteroidal, tissue-
selective androgen receptor modulator, S-22, regulate androgen
receptor function through distinct genomic and nongenomic signaling
pathways. Mol. Endocrinol. 2008, 22, 2448−2465.
(25) Hanada, K.; Furuya, K.; Yamamoto, N.; Nejishima, H.; Ichikawa,
K.; Nakamura, T.; Miyakawa, M.; Amano, S.; Sumita, Y.; Oguro, N.
Bone anabolic effects of S-40503, a novel nonsteroidal selective
androgen receptor modulator (SARM), in rat models of osteoporosis.
Biol. Pharm. Bull. 2003, 26, 1563−1569.
REFERENCES
■
(1) Mooradian, A. D.; Morley, J. E.; Korenman, S. G. Biological
actions of androgens. Endocr. Rev. 1987, 8, 1−28.
(2) Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schutz,
G.; Umesono, K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon, P.;
Evans, R. M. The nuclear receptor superfamily: the second decade. Cell
1995, 83, 835−839.
(3) Sar, M.; Lubahn, D. B.; French, F. S.; Wilson, E. M.
Immunohistochemical localization of the androgen receptor in rat
and human tissues. Endocrinology 1990, 127, 3180−3186.
(4) Gao, W.; Bohl, C. E.; Dalton, J. T. Chemistry and structural
biology of androgen receptor. Chem. Rev. 2005, 105, 3352−3370.
(5) Heinlein, C. A.; Chang, C. Androgen receptor in prostate cancer.
Endocr. Rev. 2004, 25, 276−308.
(6) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman,
D. Global cancer statistics. Ca-Cancer J. Clin. 2011, 61, 69−90.
(7) Klotz, L. Maximal androgen blockade for advanced prostate
cancer. Best Pract. Res. Clin. Endocrinol. Metab. 2008, 22, 331−340.
(8) Labrie, F.; Dupont, A.; Belanger, A.; Cusan, L.; Lacourciere, Y.;
Monfette, G.; Laberge, J. G.; Emond, J. P.; Fazekas, A. T.; Raynaud, J.
P.; Husson, J. M. New hormonal therapy in prostatic carcinoma:
combined treatment with an LHRH agonist and an antiandrogen. Clin.
Invest. Med. 1982, 5, 267−275.
(9) Mohler, M. L.; Bohl, C. E.; Jones, A.; Coss, C. C.; Narayanan, R.;
He, Y.; Hwang, D. J.; Dalton, J. T.; Miller, D. D. Nonsteroidal selective
androgen receptor modulators (SARMs): dissociating the anabolic and
androgenic activities of the androgen receptor for therapeutic benefit.
J. Med. Chem. 2009, 52, 3597−3617.
(10) Haendler, B.; Cleve, A. Recent developments in antiandrogens
and selective androgen receptor modulators. Mol. Cell. Endocrinol.
2012, 352, 79−91.
(11) Narayanan, R.; Mohler, M. L.; Bohl, C. E.; Miller, D. D.; Dalton,
J. T. Selective androgen receptor modulators in preclinical and clinical
development. Nucl. Recept. Signaling 2008, 6, e010.
(26) Sun, C.; Robl, J. A.; Wang, T. C.; Huang, Y.; Kuhns, J. E.;
Lupisella, J. A.; Beehler, B. C.; Golla, R.; Sleph, P. G.; Seethala, R.;
Fura, A.; Krystek, S. R., Jr; An, Y.; Malley, M. F.; Sack, J. S.; Salvati, M.
E.; Grover, G. J.; Ostrowski, J.; Hamann, L. G. Discovery of potent,
orally active, and muscle-selective androgen receptor modulators based
on an N-arylhydroxybicyclohydantoin scaffold. J. Med. Chem. 2006, 49,
7596−7599.
(27) Allan, G. F.; Tannenbaum, P.; Sbriscia, T.; Linton, O.; Lai, M.
T.; Haynes-Johnson, D.; Bhattacharjee, S.; Zhang, X.; Sui, Z.;
Lundeen, S. G. A selective androgen receptor modulator with minimal
prostate hypertrophic activity enhances lean body mass in male rats
and stimulates sexual behavior in female rats. Endocrine 2007, 32, 41−
51.
(12) Brown, T. R. Nonsteroidal selective androgen receptors
modulators (SARMs): designer androgens with flexible structures
provide clinical promise. Endocrinology 2004, 145, 5417−5419.
(13) Zhang, X.; Li, X.; Allan, G. F.; Sbriscia, T.; Linton, O.; Lundeen,
S. G.; Sui, Z. Design, synthesis, and in vivo SAR of a novel series of
pyrazolines as potent selective androgen receptor modulators. J. Med.
Chem. 2007, 50, 3857−3869.
(14) Zhao, S.; Shen, Y.; van Oeveren, A.; Marschke, K. B.; Zhi, L.
Discovery of a novel series of nonsteroidal androgen receptor
modulators: 5- or 6-oxachrysen-2-ones. Bioorg. Med. Chem. Lett.
2008, 18, 3431−3435.
(28) Vajda, E. G.; Lopez, F. J.; Rix, P.; Hill, R.; Chen, Y.; Lee, K. J.;
O’Brien, Z.; Chang, W. Y.; Meglasson, M. D.; Lee, Y. H.
Pharmacokinetics and pharmacodynamics of LGD-3303 [9-chloro-2-
ethyl-1-methyl-3-(2,2,2-trifluoroethyl)-3H-pyrrolo-[3,2-f]quinolin-
7(6H)-one], an orally available nonsteroidal-selective androgen
receptor modulator. J. Pharmacol. Exp. Ther. 2009, 328, 663−670.
(29) Dayger, C.; Villasana, L.; Pfankuch, T.; Davis, M.; Raber, J.
Effects of the SARM ACP-105 on rotorod performance and cued fear
conditioning in sham-irradiated and irradiated female mice. Brain Res.
2011, 1381, 134−140.
(15) Zhou, C.; Wu, G.; Feng, Y.; Li, Q.; Su, H.; Mais, D. E.; Zhu, Y.;
Li, N.; Deng, Y.; Yang, D.; Wang, M. W. Discovery and biological
characterization of a novel series of androgen receptor modulators. Br.
J. Pharmacol. 2008, 154, 440−450.
(16) Long, Y. O.; Higuchi, R. I.; Caferro, T. R.; Lau, T. L.; Wu, M.;
Cummings, M. L.; Martinborough, E. A.; Marschke, K. B.; Chang, W.
Y.; Lopez, F. J.; Karanewsky, D. S.; Zhi, L. Selective androgen receptor
modulators based on a series of 7H-[1,4]oxazino[3,2-g]quinolin-7-
ones with improved in vivo activity. Bioorg. Med. Chem. Lett. 2008, 18,
2967−2971.
(17) Piu, F.; Gardell, L. R.; Son, T.; Schlienger, N.; Lund, B. W.;
Schiffer, H. H.; Vanover, K. E.; Davis, R. E.; Olsson, R.; Bradley, S. R.
Pharmacological characterization of AC-262536, a novel selective
(30) Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T.
Structural basis for antagonism and resistance of bicalutamide in
prostate cancer. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6201−6206.
6325
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
(31) Chen, Y.; Clegg, N. J.; Scher, H. I. Anti-androgens and
androgen-depleting therapies in prostate cancer: new agents for an
established target. Lancet Oncol. 2009, 10, 981−991.
(32) Taplin, M. E. Drug insight: role of the androgen receptor in the
development and progression of prostate cancer. Nat. Clin. Pract.
Oncol. 2007, 4, 236−244.
(33) Buchanan, G.; Greenberg, N. M.; Scher, H. I.; Harris, J. M.;
Marshall, V. R.; Tilley, W. D. Collocation of androgen receptor gene
mutations in prostate cancer. Clin. Cancer Res. 2001, 7, 1273−1281.
(34) Wilding, G.; Chen, M.; Gelmann, E. P. Aberrant response in
vitro of hormone-responsive prostate cancer cells to antiandrogens.
Prostate 1989, 14, 103−115.
(35) Chen, C. D.; Welsbie, D. S.; Tran, C.; Baek, S. H.; Chen, R.;
Vessella, R.; Rosenfeld, M. G.; Sawyers, C. L. Molecular determinants
of resistance to antiandrogen therapy. Nat. Med. 2004, 10, 33−39.
(36) Knudsen, K. E.; Penning, T. M. Partners in crime: deregulation
of AR activity and androgen synthesis in prostate cancer. Trends
Endocrinol. Metab. 2010, 21, 315−24.
(37) Attard, G; Cooper, C. S.; de Bono, J. S. Steroid hormone
receptors in prostate cancer: a hard habit to break? Cancer Cell 2009,
16, 458−462.
(38) Tran, C.; Ouk, S.; Clegg, N. J.; Chen, Y.; Watson, P. A.; Arora,
V.; Wongvipat, J.; Smith-Jones, P. M.; Yoo, D.; Kwon, A.;
Wasielewska, T.; Welsbie, D.; Chen, C. D.; Higano, C. S.; Beer, T.
M.; Hung, D. T.; Scher, H. I.; Jung, M. E.; Sawyers, C. L. Development
of a second-generation antiandrogen for treatment of advanced
prostate cancer. Science 2009, 324, 787−790.
(48) Neri, R.; Florance, K.; Koziol, P.; Van Cleave, S. A biological
profile of a nonsteroidal antiandrogen, SCH 13521 (4′-nitro-3′-
trifluoromethylisobutyranilide). Endocrinology 1972, 91, 427−437.
(49) Hankovsky, O. H.; Hideg, K.; Lovas, M. J. Synthesis and
reaction of ortho-fluoronitroaryl nitroxides. Novel versatile synthons
and reagents for spin-labelling studies. Can. J. Chem. 1989, 67, 1392.
(50) Li, H. Y.; DeLucca, I.; Boswell, G. A.; Billheimer, J. T.;
Drummond, S.; Gillies, P. J.; Robinson, C. Design, synthesis and
structure-activity relationship studies of novel 4,4-bis(trifluoromethyl)-
imidazolines as acyl-CoA: cholesterol acyltransferase (ACAT)
inhibitors and antihypercholesterolemic agents. Bioorg. Med. Chem.
1997, 5, 1345−1361.
(51) Bergerat, J. P.; Ceraline, J. Pleiotropic functional properties of
androgen receptor mutants in prostate cancer. Hum. Mutat. 2009, 30,
145−157.
(52) Brooke, G. N.; Bevan, C. L. The role of androgen receptor
mutations in prostate cancer progression. Curr. Genomics 2009, 10,
18−25.
(53) Koochekpour, S. Androgen receptor signaling and mutations in
prostate cancer. Asian J. Androl. 2010, 12, 639−657.
(54) Leitao, J. M.; Esteves da Silva, J. C. Firefly luciferase inhibition. J.
Photochem. Photobiol. B 2010, 101, 1−8.
(55) Bakhtiarova, A.; Taslimi, P.; Elliman, S. J.; Kosinski, P. A.;
Hubbard, B.; Kavana, M.; Kemp, D. M. Resveratrol inhibits firefly
luciferase. Biochem. Biophys. Res. Commun. 2006, 351, 481−484.
(56) GraphPad Prism version 5.03 for Windows, GraphPad Software,
San Diego, CA.
(39) Scher, H. I.; Beer, T. M.; Higano, C. S.; Anand, A.; Taplin, M.
E.; Efstathiou, E.; Rathkopf, D.; Shelkey, J.; Yu, E. Y.; Alumkal, J.;
Hung, D.; Hirmand, M.; Seely, L.; Morris, M. J.; Danila, D. C.; Humm,
J.; Larson, S.; Fleisher, M.; Sawyers, C. L. Prostate Cancer
Foundation/Department of Defense Prostate Cancer Clinical Trials
Consortium Antitumour Activity of MDV3100 in Castration-Resistant
Prostate Cancer: A Phase 1−2 Study. Lancet 2010, 375, 1437−1446.
(40) Yamamoto, S.; Matsunaga, N.; Hitaka, T.; Yamada, M.; Hara, T.;
Miyazaki, J.; Santou, T.; Kusaka, M.; Yamaoka, M.; Kanzaki, N.;
Furuya, S.; Tasaka, A.; Hamamura, K.; Ito, M. Design, synthesis, and
biological evaluation of 4-phenylpyrrole derivatives as novel androgen
receptor antagonists. Bioorg. Med. Chem. 2012, 20, 422−434.
(41) Guo, C.; Kephart, S.; Ornelas, M.; Gonzalez, J.; Linton, A.;
Pairish, M.; Nagata, A.; Greasley, S.; Elleraas, J.; Hosea, N.;
Engebretsen, J.; Fanjul, A. N. Discovery of 3-aryloxy-lactam analogs
as potent androgen receptor full antagonists for treating castration
resistant prostate cancer. Bioorg. Med. Chem. Lett. 2012, 22, 1230−
1236.
(42) Guo, C.; Pairish, M.; Linton, A.; Kephart, S.; Ornelas, M.;
Nagata, A.; Burke, B.; Dong, L.; Engebretsen, J.; Fanjul, A. N. Design
of Oxobenzimidazoles and Oxindoles as Novel Androgen Receptor
Antagonists. Bioorg. Med. Chem. Lett. 2012, 22, 2572−2578.
(43) Pulkkinen, J. T.; Honkakoski, P.; Perakyla, M.; Berczi, I.;
Laatikainen, R. Synthesis and evaluation of estrogen agonism of diaryl
4,5-dihydroisoxazoles, 3-hydroxyketones, 3-methoxyketones, and 1,3-
diketones: a compound set forming a 4D molecular library. J. Med.
Chem. 2008, 51, 3562−3571.
(44) Poutiainen, P. K.; Venalainen, T. A.; Perakyla, M.; Matilainen, J.
M.; Vaisanen, S.; Honkakoski, P.; Laatikainen, R.; Pulkkinen, J. T.
Synthesis and biological evaluation of phenolic 4,5-dihydroisoxazoles
and 3-hydroxy ketones as estrogen receptor alpha and beta agonists.
Bioorg. Med. Chem. 2010, 18, 3437−3447.
(45) Makkonen, H.; Jaaskelainen, T.; Rytinki, M. M.; Palvimo, J. J.
Analysis of androgen receptor activity by reporter gene assays. Methods
Mol. Biol. 2011, 776, 71−80.
(46) Thompson, J.; Saatcioglu, F.; Janne, O. A.; Palvimo, J. J.
Disrupted amino- and carboxyl-terminal interactions of the androgen
receptor are linked to androgen insensitivity. Mol. Endocrinol. 2001, 15,
923−935.
(47) Furr, B. J.; Valcaccia, B.; Curry, B.; Woodburn, J. R.; Chesterson,
G.; Tucker, H. ICI 176,334: a novel non-steroidal, peripherally
selective antiandrogen. J. Endocrinol. 1987, 113, R7−9.
(57) Hur, E.; Pfaff, S. J.; Payne, E. S.; Gron, H.; Buehrer, B. M.;
Fletterick, R. J. Recognition and accommodation at the androgen
receptor coactivator binding interface. PLoS Biol. 2004, 2, E274.
(58) Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T.
Structural basis for antagonism and resistance of bicalutamide in
prostate cancer. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6201−6206.
(59) Bohl, C. E.; Miller, D. D.; Chen, J.; Bell, C. E.; Dalton, J. T.
Structural basis for accommodation of nonsteroidal ligands in the
androgen receptor. J. Biol. Chem. 2005, 280, 37747−37754.
(60) Case, D. A.; Darden, T. A.; Cheatham T. E. I.; Simmerling, C.
L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Pearlman, D. A.;
Crowley, M.; Walker, R. C.; Zhang, W.; Wang, B.; Hayik, S.; Roitberg,
A.; Seabra, G.; Wong, K. F.; Paesani, F.; Wu, X.; Brozell, S.; Tsui, V.;
Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V.; Cui, G.:
Beroza. P.; Mathews, H. D.; Schafmeister, C.; Ross, W. S.; Kollman, P.
A. AMBER 9, 2006.
(61) Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.;
Simmerling, C. Comparison of multiple Amber force fields and
development of improved protein backbone parameters. Proteins 2006,
65, 712−725.
(62) Accelrys Discovery Studio software version 1.7, Anonymous
Accelrys Software Inc., 2005−06.
(63) Autschbach, J; Ziegler, T; van Gisbergen, S. J. A.; Baerends, E. J.
Chiroptical properties from time-dependent density functional theory.
I. Circular dichroism spectra of organic molecules. J. Chem. Phys. 2002,
116, 6930−40.
(64) Scalmani, G.; Frisch, M. J.; Mennucci, B.; Tomasi, J.; Cammi, R.;
Barone, V. Geometries and properties of excited states in the gas phase
and in solution: Theory and application of a time-dependent density
functional theory polarizable continuum model. J. Chem. Phys. 2006,
124, 1−15.
(65) Tomasi, J.; Mennucci, B.; Cammi, R. Quantum mechanical
continuum solvation models. Chem. Rev. 2005, 105, 2999−3093.
(66) Bruhn, T.; Schaumloffel, A.; Hemberger, Y.; Bringmann, G.
̈
SpecDis version 1.53, University of Wuerzburg, Germany, 2012.
(67) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Peralta, J. E.; Ogliaro,
F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V.
6326
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327

Journal of Medicinal Chemistry
Article
N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant,
J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.;
Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo,
J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi,
R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.;
Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;
̈
Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.;
Cioslowski, J.; Fox, D. J. Gaussian 09 Revision C.01, Gaussian, Inc.,
Wallingford, CT, 2011.
6327
dx.doi.org/10.1021/jm300233k | J. Med. Chem. 2012, 55, 6316−6327