Synthesis, structure-activity relationships, and characterization of novel nonsteroidal and selective androgen receptor modulators

Article abstract of DOI:10.1021/jm901149c

Herein we describe the discovery of ACP-105 (1), a novel and potent nonsteroidal selective androgen receptor modulator (SARM) with partial agonist activity relative to the natural androgen testosterone. Compound 1 was developed from a series of compounds found in a HTS screen using the receptor selection and amplification technology (R-SAT). In vivo, 1 improved anabolic parameters in a 2-week chronic study in castrated male rats. In addition to compound 1, a number of potent antiandrogens were discovered from the same series of compounds whereof one compound, 13, had antagonist activity at the AR T877A mutant involved in prostate cancer.

Full text of DOI:10.1021/jm901149c

7186 J. Med. Chem. 2009, 52, 7186–7191
DOI: 10.1021/jm901149c
Synthesis, Structure-Activity Relationships, and Characterization of Novel Nonsteroidal and Selective
Androgen Receptor Modulators
Nathalie Schlienger,† Birgitte W. Lund,† Jan Pawlas,† Fabrizio Badalassi,† Fabio Bertozzi,† Rasmus Lewinsky,† Alma Fejzic,†
Mikkel B. Thygesen,† Ali Tabatabaei,‡ Stefania Risso Bradley,‡ Luis R. Gardell,‡ Fabrice Piu,‡ and Roger Olsson*,†
†ACADIA Pharmaceuticals AB, Medeon Science Park, S-205 12 Malmo€, Sweden, and ‡ACADIA Pharmaceuticals, Inc., 3911 Sorrento Valley
Boulevard, San Diego, California 92121
Received August 1, 2009
Herein we describe the discovery of ACP-105 (1), a novel and potent nonsteroidal selective androgen
receptor modulator (SARM) with partial agonist activity relative to the natural androgen testosterone.
Compound 1 was developed from a series of compounds found in a HTS screen using the receptor
selection and amplification technology (R-SAT). In vivo, 1 improved anabolic parameters in a 2-week
chronic study in castrated male rats. In addition to compound 1, a number of potent antiandrogens were
discovered from the same series of compounds whereof one compound, 13, had antagonist activity at the
AR T877A mutant involved in prostate cancer.
Chart 1
Introduction
The design of nonsteroidal androgen receptor (ARa) li-
gands with specific pharmacological profiles will expand the
clinical applications of androgens in diseases of the prostate,
hormone-replacement therapy, osteoporosis, and male con-
traception.1 Physiologically, AR is predominantly activated
by testosterone (T) and its metabolite dihydrotestosterone
(DHT) (Chart 1). In addition, various synthetic androgen
steroidal ligands have been developed for the treatment of, for
example, male hypogonadism, muscle wasting, anemia, be-
nign prostate hyperplasia, and prostate cancer.2 However, the
use of steroidal androgens has been limited because of the risk
of serious side effects, e.g., cardiovascular events, hepatotoxi-
city, and the potential of prostatic hyperplasia or cancer.2,3 In
particular, the lack of discrimination between anabolic and
androgenic effects of the current forms of testosterone is of
major concern and has driven the search for selective AR
modulators (SARMs).
A number of nonsteroidal selective AR agonists that are
under preclinical or clinical development has been reported,
e.g., BMS-564929, S-4 (andarine), and LGD-2941.1,4 Several
of these compounds have revealed potent and efficacious
anabolic activity and acted as partial agonists in androgenic
tissues in animal models.5
Recently we reported on the pharmacological characteriza-
tion of compound 2 (AC-262536) as a novel, potent, and
selective AR ligand that shows partial agonistic activity
relative to T.6 The partial agonistic activity was suggested to
*To whom correspondence should be addressed. Phone:
þ46406013466. Fax: þ46406013405. E-mail: roger@acadia-pharm.
com.
a Abbreviations: AR, androgen receptor; DHT, dihydrotestosterone;
F, flutamide; HF, hydroxyflutamide; LBD, ligand binding domain; o/n,
overnight; PTSA, p-toluenesulfonic acid; rt, room temperature; R-SAT,
receptor selection and amplification technology; SAR, structure-activ-
ity relationship; SARM, selective androgen receptor modulator; SLR;
structure-liability relationship; T, testosterone.
have beneficial implications, (e.g., differences in tissue selec-
tive actions could be enhanced because of distinct receptor
reserve contents in androgen-responsive tissues).Further-
more, in tissues with high natural androgen content, such as
the prostate, a partial AR agonist could act as a functional
r
pubs.acs.org/jmc
Published on Web 10/26/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7187
Scheme 1a
antagonist of androgen action by effectively competing with
both T and DHT. Tropanol 2 was developed in a hit-to-lead
optimization effort starting from the hit compound 3 identi-
fied in an in-house screen using the receptor selection and
amplification technology (R-SAT).7 R-SAT is a functional
cell-based assay that allows one to monitor receptor-depen-
dent proliferative responses of various receptor classes, in-
cluding G-protein-coupled receptors and nuclear receptors.8
In vivo experiments in castrated rats showed that 2 signifi-
cantly improved anabolic parameters in these animals while
only having minimal effects on the androgenic tissues.
Herein we report on the structure-activity relationship
(SAR) studies first leading to 2 and further to the discovery
of ACP-105 (1), an orally available, selective, and potent
SARM. Furthermore, we also describe how small changes
in the structures led to intrinsic receptor activities ranging
from full agonistic to antagonistic activities including an
antiandrogen with antagonist activity at the AR T877A
mutant involved in prostate cancer.
Chemistry
The syntheses of naphthalene 4 and a series of tropane
analogues are outlined in Scheme 1. Compounds 2 and 4 were
synthesized from 4-fluoro-1-naphthonitrile and nortropine or
pyrrolidine, respectively, in a microwave assisted nucleophilic
aromatic substitution. The methoxytropanol analogue 7 was
obtained by alkylation of 2 with methyl iodide and sodium
hydride. The exo-tropanol analogue 8 was obtained from the
endo-derivative 2 using Mitsunobu conditions. Swern oxida-
tion of 2 afforded the ketone 5 which served as a useful
synthetic intermediate to prepare a number of different
derivatives. Ketone 5 was converted to the corresponding
p-toluenesulfonylhydrazone derivative and subsequently
reduced using sodium cyanoborohydride and p-toluenesulfo-
nic acid to yield tropane 6. Compounds 9a-g were synthe-
sized using the protocol developed by Knochel and co-
workers wherein Grignard reagents were added to ketones
a (a) Nortropine or pyrrolidine, pyridine, microwave, 220 °C, 5 min,
92% and 62% yields, respectively; (b) (i) (COCl)2, DMSO, dichloro-
methane, -60 °C; (ii) NEt3, rt, o/n, 86% yield; (c) pTsNHNH2, EtOH,
reflux, 1 h, 92% yield; (d) NaBH3CN, PTSA, DMF/sulfolane, 1:1,
cyclohexane, 110 °C, 7 h, 40% yield; (e) MeI, NaH, 60 °C DMF, o/n,
8% yield; (f) DIAD, PPh3, p-nitrobenzoic acid, THF, rt, o/n, then 40 °C, 3
h, 50% yield; (g) 2 M LiOH, THF, o/n, 89% yield; (h) RMgX,
CeCl3 2LiCl, THF, -10 °C to rt, o/n, 23-63% yields; (j) (CH3)3SOI,
3
DMSO, NaH, rt, o/n, 61% yield; (k) 0.2 M H2SO4, THF, rt, 3 h, 31% yield.
Scheme 2a
using CeCl3 2LiCl as an additive.9 This protocol gave both
3
high chemoselectivity and regioselectivity, with a preferential
exo-attack on the ketone, dr> 9:1 (9a-g).9b Tropanone 5was
subjected to a Corey-Chaykovsky reaction to obtain epoxide
10 with complete diastereoselectivity. Aqueous H2SO4
was used to ring-open epoxide 10, affording diol 11. Com-
pound 12 was obtained by a nucleophilic aromatic substitu-
tion between nortropine and 2,3-dimethyl-4-nitrophenol tri-
flate (Scheme 2). Syntheses of compounds 13 and 14 were
carried out by a nucleophilic aromatic substitution of nortro-
pine with 2-chloro-4-fluorobenzonitrile and 2-chloro-4-
fluoro-3-methylbenzonitrile, respectively, in pyridine. An ef-
ficient convergent synthesis was developed to prepare com-
pound 1 from 2-chloro-4-fluoro-3-methylbenzonitrile and
endo-3-exo-methyl-8-azabicyclo[3.2.1]octan-3-ol hydrochlor-
ide (15). Tropanol 15 was synthesized in three steps from
N-Boc-tropinone via a complete diastereoselective epoxida-
tion followed by a reduction and a subsequent trituration
as HCl salt.
a (a) (CF3SO2)2O, NEt3, CH2Cl2, 0 °C to rt, 98% yield; (b) nortropine,
pyridine, 110 °C, 16 h, 7% yield; (c) (CH3)3SOI, DMSO, NaH, rt, 20 h, used
without further purification; (d) superhydride, THF, 0 °C, rt, 77% yield; (e)
4 M HCl in dioxane, Et2O, rt,2h,77%yield;(f) Br2,Fe(0), 30°C, 1 h; (g) Pd-
(PPh3)4, Zn(CN)2, DMF, 120 °C, 2 h, 40% yield, two steps; (h) 15 or nortro-
pine, pyridine, 110 °C, 18 h, 63% yield (13), 18% yield (14), 40% yield (1).
Results and Discussion
In vitro functional activity determined with R-SAT assays
and radioligand binding data at the AR receptor and the
intrinsic clearance values in human and rat liver microsomes
of compounds 1-14 are summarized in Table 1. The first step
in the hit-to-lead optimization was to turn our attention to the
potential toxic functionality of 3. The aromatic nitro group
was exchanged with a cyano group to give compound 4. Other
functionalgroupexchanges (e.g., chlorine, amides, acetamide,

7188 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Schlienger et al.
Table 1. Ligand Activities at AR Wild Type and T877A Mutant and Intrinsic Microsomal Clearancea
AR
AR, mutation T877A
% eff
Clint (μL/(mL mg))
3
compd
pEC50
% eff
pKi
pEC50
pKi
ClInt,human
ClInt,rat
T
DHT
8.7 ( 0.2
8.4 ( 0.2
102 ( 11
107 ( 19
NA
8.9 ( 0.3
4.7 ( 0.2
7.0 ( 0.4
9.4 ( 0.3
100 ( 5
36 ( 5
97 ( 8
37 ( 5
F
HF
6.2 ( 0.6
7.3 ( 0.2
6.4 ( 0.3
NA
NA
1 (ACP-105)
2
9.0 ( 0.3
7.9 ( 0.3
8.2 ( 0.5
7.8 ( 0.6
7.0 ( 0.3
8.4 ( 0.3
7.7 ( 0.3
7.0 ( 0.4
8.7 ( 0.1
8.4 ( 0.4
8.7 ( 0.1
81 ( 6
73 ( 11
96 ( 22
67 ( 14
66 ( 10
95 ( 11
60 ( 24
47 ( 3
88 ( 14
44 ( 2
48 ( 3
NA
5.5 ( 0.4
28
70
72
148
3
4
528
49
1177
189
5
6
114
591
7
8
7
22
57
95
9a
9b
9c
9d
9e
9f
9g
10
11
12
13
14
7.0 ( 0.5
8.1 ( 0.2
66 ( 7
6.4 ( 0.2
19
97
8.7 ( 0.1
8.3 ( 0.8
7.8 ( 0.6
56 ( 1
42 ( 6
42 ( 6
Na
7.4 ( 0.3
6.9 ( 0.1
7.9 ( 0.3
7.4 ( 0.1
39 ( 8
58 ( 10
6.8 ( 0.2
NA
Na
76 ( 8
NA
28
69
35
7.9 ( 0.2
8.7 ( 0.1
7.0 ( 0.1
NA
7.0 ( 0.2
81 ( 3
214
a R-SAT was performed as described in ref 6. Agonist efficacies were compared to that of DHT (100%). Values represent the mean ( SEM of three or more
independent experiments (n > 3). NA = not active. T = testosterone. DHT = dihydrotestosterone. F = flutamide. HF = hydroxyflutamide.
and carboxylic acid) were also screened; however, the replace-
ment with any of these groups gave compounds devoid of
activity in R-SAT (results not shown). Pyrrolidine 4 showed a
somewhat lower activity than 3 and turned out to have an
unacceptably high metabolic in vitro clearance. In general,
cyclic amines are known to be readily subject to metabolic
transformations, one of those paths being oxidation at the
R-positions. We therefore looked at the possibility of blocking
this path by introducing another ring system, which would
inhibit the metabolism at the two positions neighboring the
nitrogen. Replacing the pyrrolidine with a tropane (6) gave, in
addition to an increased stability in both human and rat liver
microsomes, a significant increase in potency compared to 4.
Although a clear improvement was achieved, the in vitro
clearance for 6 was still suboptimal. The synthetic intermedi-
ate 5 provided the knowledge that the AR receptor pharma-
cophore shows a certain tolerance toward the presence
of polar groups, i.e., carbonyl. Whereas 5 was approximately
50 times less potent than 6 (the pEC50 decreases from 8.4 for 6
to 7.0 for 5), a significant increase in metabolic stability was
observed. Therefore, insertion of a hydroxyl group at the
3-position of the tropane ring could have several benefits, first
blocking the 3-position toward metabolism and second by
potentially improving binding interactions by capturing
H-bonds with asparagine 705 (N705) and threonine 877
(T877) at the ligand binding domain (LBD) of the AR, as
has been suggested for the endogenous ligands.10 Tropanol 2
was therefore synthesized and indeed showed an improved
liver microsomal stability. The AR agonistic activity of 2 was
slightly lower than for 6, suggesting that hydrogen bond
interactions with the LBD are not crucial for the activity for
this class of compounds.
tropane, were investigated, but all were devoid of activity
(results not shown). Epoxide 10 and diol 11 were prepared to
test whetheradditional polargroups may beacceptable. These
compounds showed significant antagonistic activityinstead of
the expected agonistic activity. Whereas methylation of the
3-hydroxyl gave a compound (7) that was approximately
equipotent with the parent tropanol 2, the exo-tropanol 8
showed a significant decrease in both potency and efficacy
compared to the endo-tropanol 2. Nevertheless, 8 displayed a
further improvement in microsomal stability compared to 2.
The reduction of clearance seen with exo-alcohol 8 could be
due to steric hindrance by the tropane bridge limiting the
accessibility of hydrogen on the methine carbon. Thus, by
removal of the exo-hydrogen, a potential oxidation path may
be avoided. Furthermore, although the accessibility to the
endo-alcohol group is limited by the carbon bridge of the
tropane, phase II metabolism, i.e., conjugation, may still
represent a potential problem. Hence, introducing a sterically
crowded environment around the alcohol may decrease both
potential phase I and phase II metabolic pathways. Therefore,
a series of tropanol derivatives (9a-g) bearing varying
alkyl substituents in 3-exo-position was synthesized. The
exo-methyl tropanol 9a displayed about 10-fold increase in
potency (pEC=8.7) compared to 2 (pEC=7.9). In addition,
9a showed increased stability in both rat and human liver
microsomes compared to 2.
The increased microsomal stability for 9a and 2 corrobo-
rated with the results from stability studies in cryopreserved
rat hepatocytes, where half-lives of 96 and 3 min, respectively,
were observed. When the chain length was extended with one
carbon from methyl to ethyl (9b), a decrease in both potency
andefficacy was observed comparedto9a. Further elongation
of the alkyl chain from ethyl (9b) to propyl (9g) denoted a
trend of decreased activity with longer alkyl chain. Thus, in
this series a steric component can be observed going from
To improve the metabolic stability further and to explore
changes in the tropane, other functional groups, e.g., amine,
hydroxylamine, and amide derivatives in the 3-position of the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7189
mutant T877A. Antiandrogens are used clinically for the
treatment of cancer, e.g., flutamide (Euxlin). However, these
compounds often give rise to mutations in the AR, resulting in
drug resistance.12 The AR T877A mutation has been found in
cases of advanced prostate cancer and provide a selective
growth and survival advantage in a cell-based model.13 The
T877A mutant, resulting from replacement of threonine 877
with alanine, possesses a larger LBD that compensates for a
bulkier antagonist compared to an agonist compound. Thus,
the antagonist allows the adoption of an AR-LBD agonist
conformation in the T877A mutant. Hydroxyflutamide (HF)
is an antagonist at the wild type AR and an agonist at the
T877A AR mutant. In line with this, ethyne 9d, speculated to
be an antagonist due to steric reasons, was the most potent
agonist at the AR T877A mutant with a pEC50 of 8.1 ( 0.2,
whereas the benzonitrile 13 retained its antagonist activity at
the mutant. The result for 13 may be expected, as we hypothe-
sized that its antagonist activity does not result from bulkiness
but rather from a slight conformation change. Both 10 and 11
showed partial agonist activity at the T877A mutant. These
results could provide an insight in designing antiandrogens
based on agonists that retain antagonist activity at the AR
T877A mutant.
AR agonists 1 and 2 together with testosterone were also
tested in an AR transcriptional activity luciferase repor-
ter gene assay (for testosterone, pEC50 = 8.5 ( 0.6, efficacy
104 ( 24%; for 2, pEC50=8.8 ( 0.1, efficacy 72 ( 9%; for 1,
pEC50 = 9.6 ( 0.1, efficacy 86 ( 11%), and the results ob-
tained corroborated those seen using R-SAT.
As part of establishing a structure-liability relationship
(SLR) investigating off-target activities, 1 was further evalu-
ated in a number of in vitro receptor assays and no agonism
activity at the other 47 nuclear receptors and no significant
binding affinity for 31 selected G-protein-coupled receptors
were observed.
Figure 1. Receptor activity for DHT and the partial agonists 1 and
9e. Each compound was tested in concentration-response experi-
ments in R-SAT assay: (9 ) DHT, (2) 1, and (b) 9e.
methyl (9a, pEC50=8.7) to ethyl (9b, pEC50=8.4) to propyl
(9g, pEC50=7.8). The vinyl analogue 9c showed potency com-
parable with the methyl analogue 9a and somewhat higher
than the ethyl derivative 9b. The introduction of a cyclopropyl
group (9e) led to a more active compound than 9f bearing an
isopropyl group. The acetylene analogue 9d on the other hand
showed no AR agonistic activity; instead 9d displayed antago-
nistic activity. The reversal of activity for ethyne 9d, going
from agonistic to antagonistic activity, could be due the
rigidity of the ethyne part. What might occur is that the ethyne
part interacts unfavorably, for agonistic activity, with T877 in
the AR. With the exception of compound 9a and the antago-
nist 9d, all 3-alkyl substituted compounds prepared in this
series showed partial agonist activity in the cell-proliferation
assay R-SAT.
Next, we focused on the aromatic moiety, with the purpose
of replacing the naphthalene scaffold with a substituted
phenyl.11 A number of substituted phenyl derivatives were
synthesized, anda representativeset of analogues(12-14, 1) is
outlined in Scheme 2. 2,3-Dimethyl substituted nitrobenzene
12 was prepared as the first representative of the phenyl series
and showed potent ARagonistic activity, whichindicated that
the established SAR in the naphthalene series may be applic-
able to the phenyl series as well. Indeed, exchanging the nitro
group in 12 with cyano functionality and the 2-methyl with
chlorine gave compound 14, which displayed an in vitro
activity (pEC50=8.7, 81% eff) comparable to 9a and signifi-
cantly higher than the activity observed for 12 or the corre-
sponding naphthalene analogue 2. Furthermore, introduction
of the 3-exo-methyl group on the tropanol (1) led to a highly
potent and metabolically stable compound (Figure 1). Inter-
estingly, the 2-chlorobenzonitrile 13, not having a substituent
in the 3-position of the phenyl, was a potent antagonist
and did not display any agonistic activity. This may be
explained by the absence of a steric interaction between a
substituent at the neighboring position with the tropane ring,
leading to small conformation changes necessary for agonist
activity. This result also confirms that small modifications of
the compound, in particular introduction of more polar
groups on the tropane moiety, switch the intrinsic receptor
activity from full agonist via partial agonism to antagonist
activity.
In addition to the metabolic clearance determined in liver
microsomes, the half-lives of 9a and 1 in human hepatocytes
were measured and found to be 8.4 and 5.0 h, respectively.
Pharmacokinetic studies of both compounds were performed
showing high oral bioavailability in both rats and dogs
(Foral,rat = 41% and 38% in rats and Foral,dog = 52% and
56% in dogs, 9a and 1, respectively). Compound 1 was further
assessed for efficacy and selectivity in the stimulation of
muscle vs prostate tissue growth in castrated male rats. This
pharmacological model has been widely used for the assess-
ment of anabolic and androgenic activities. 1 was delivered via
subcutaneous osmotic pumps for 14 days at doses of 0.1-3
(mg/kg)/day to ORX rats. Testosterone propionate was ad-
ministered at a dose of 0.75 (mg/kg)/day. After 2 weeks of
dosing, levator ani muscle and ventral prostate tissues were
weighed and compared to those of a sham-operated vehicle-
treated control group. The principal finding from this study
was that 1 produced a robust anabolic effect on the levator ani
muscle (67% reversal of ORX-induced atrophy at 1 (mg/kg)/
day) while having lesser androgenic effects on prostate (21%
reversal; T, 48%).14 Furthermore, to test if 1 was a partial AR
agonist in vivo, the ability of 1 to reverse the androgenic
actions of testosterone was evaluated. ORX rats were dosed
daily with either testosterone propionate, 1 mg/kg sc, alone or
together with 1, 10 mg/kg po, for 14 days. Twenty-four hours
after the lastdosing, animalsweresacrificed and the weightsof
their prostate measured. Testosterone propionate produced
androgenic effects in the prostate that were attenuated by
1 (Figure 2).
The antiandrogens 9d, 10, 11, and 13 found in this SAR
study were further examined for their activities at the AR

7190 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Schlienger et al.
19.0 mmol), and K2CO3 (6.67 g, 48.2 mmol) were dissolved in
dimethyl sulfoxide (40 mL), and the mixture was stirred under
argon at 80 °C for 18 h. The reaction mixture was poured into
water (200 mL) and stirred for 30 min. The off-white solid was
filtered off and recrystallized twice from toluene, giving a white
powder (1.53 g). The mother liquor was evaporated and the
residue recrystallized to yield a second batch of compound
(210 mg), giving an overall yield of 40%. The hydrochloride
salt was prepared by dissolving the product in diethyl ether and
adding HCl (1 equiv, 4 M solution in 1,4-dioxane). The mixture
was allowed to stir for 15 min and the precipitated salt
was filtered off, washed with diethyl ether, and dried. The title
compound was obtained as a colorless solid. Mp = 160 °C.
1H NMR (CDCl3, 400 MHz) δ 7.39 (d, J = 8.6 Hz, 1H), 6.84 (d,
J = 8.6 Hz, 1H), 3.82 (m, 2H), 2.36 (s, 3H), 2.32-2.22 (m, 2H),
2.17-1.98 (m, 2H), 1.92-1.77 (m, 4H), 1.26 (s, 3H). 13C NMR
(CDCl3, 100 MHz) δ 155.8, 138.4, 132.0, 129.7, 117.9, 115.5,
105.1, 69.6, 59.3, 45.9, 34.7, 27.4, 17.9. LCMS m/z 291 [M þ H]þ.
Anal. (C16H19ClN2O) C, H, N.
Figure 2. Compound 1 attenuated the androgenic effects of testo-
sterone propionate (TP) in an animal model of orchidectomy (ORX).
General Procedure (GP) for Compounds 9a-g. In a flame-
dried Schlenk flask under argon atmosphere was placed a
CeCl3 2LiCl solution in anhydrous THF (∼0.6 M, 1.0 mL,
3
Conclusion
0.6 mmol). Ketone 5 (0.50 mmol) was added neat, and the
resulting mixture was stirred for 1 h at rt. The reaction mixture
was cooled to 0 °C, Grignard reagent (0.6 mmol) was added
dropwise, and the reaction mixture was allowed to stir at the
same temperature. Upon full conversion of starting material
(checked by TLC and/or analytical HPLC/MS), saturated
ammonium chloride (1.0 mL) and ethyl acetate (2.0 mL) were
added. The aqueous layer was extracted with more ethyl acetate
(2 ꢀ 10 mL), and the combined extracts were dried over sodium
sulfate, filtered, and concentrated to dryness. The residues were
purified by column chromatography on silica gel to give pure
products.
In conclusion, we have presented herein the discovery of a
new class of highly potent, nonsteroidalSARMs. Inparticular
compound 1 showed promising overall pharmacological,
pharmacokinetic, and in vivo properties, making it suitable
for further clinical development.
Experimental Section
All procedures were carried out under a nitrogen atmosphere
unless otherwise indicated using anhydrous solvents purchased
from commercial sources without further purification. The mi-
crowave-assisted reactions were carried out using a SmithCreator
or Initiator 60EXP single mode cavity, producing continuous
irradiation at 2450 MHz. Reaction temperatures and pressures
were determined using the built-in, online IR and pressure
sensors. Thin layer chromatography (TLC) was performed on
precoated Merck silica gel 60 F254, which was visualized by UV
light or by staining with a solution of KMnO4 (1%) and Na2CO3
(5%). Flash chromatography was performed using SiO2 60
(0.040-0.063 mm). All 1H and 13C NMR spectra were recorded
using a Varian XL 400 MHz spectrometer. NMR spectra were
recorded in CD3OD, CDCl3, or DMSO-d6; chemical shifts are
given in ppm relative to CH3OH (1H, 3.31 ppm; 13C, 49.00 ( 0.01
ppm), CHCl3 (1H, 7.26 ppm; 13C, 77.16 ( 0.06 ppm), or DMSO
(1H, 2.50 ppm; 13C, 39.52 ( 0.06 ppm), respectively. Liquid
4-(3-endo-Hydroxy-3-exo-methyl-8-azabicyclo[3.2.1]oct-8-yl)-
naphthalene-1-carbonitrile (9a). The title compound was pre-
pared according to GP from 4-(3-oxo-8-azabicyclo[3.2.1]oct-8-
yl)naphthalene-1-carbonitrile (5) (138 mg, 0.50 mmol) and
methylmagnesium bromide (3.0 M in diethyl ether, 0.2 mL,
0.6 mmol). The crude product was purified by preparative TLC
(DCM/EtOAc, 3:1, two runs) to yield the title compound (42
1
mg, 29%) as a colorless solid. H NMR (CDCl3, 400 MHz) δ
8.16 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.1 Hz, 1H), 7.66-7.59 (m,
1H), 7.56-7.49 (m, 1H), 6.89 (d, J = 8.1 Hz, 1H), 4.17-4.08 (m,
2H), 2.35-2.23 (m, 4H), 2.02-1.88 (m, 4H), 1.37 (s, 3H). 13C
NMR (CDCl3, 100 MHz) δ 153.0, 134.6, 133.8, 128.5, 128.0,
126.2, 126.1, 125.6, 119.3, 111.2, 102.2, 69.9, 60.6, 46.2, 34.7,
26.9. LCMS m/z 293 [M þ H]þ. Anal. (C19H20N2O) C, H, N.
4-(3-endo-Hydroxy-3-exo-cyclopropyl-8-azabicyclo[3.2.1]oct-
8-yl)naphthalene-1-carbonitrile (9e). The title compound was
prepared according to GP from 4-(3-oxo-8-azabicyclo[3.2.1]-
oct-8-yl)naphthalene-1-carbonitrile (5) (138 mg, 0.50 mmol)
and cyclopropylmagnesium bromide (0.5 M in THF, 1.2 mL,
0.60 mmol). Purification by reverse phase preparative HPLC
yielded the title compound (39 mg, 25%) as a pale-yellow solid.
1H NMR (CDCl3, 400 MHz) δ 8.19-8.14 (m, 2H), 7.73 (d, J =
8.1 Hz, 1H), 7.63 (ddd, J = 1.2, 6.9, 8.4 Hz, 1H), 7.57-7.50 (m,
1H), 6.89 (d, J = 8.1 Hz, 1H), 4.17-4.12 (m, 2H), 2.31-2.22 (m,
4H), 1.98-1.91 (m, 2H), 1.80 (dd, J = 1.8, 15.7, 2H), 1.02-0.95
(m, 2H), 0.46-0.33 (m, 4H). 13C NMR (100 MHz, CDCl3) δ
152.9, 134.4, 133.6, 128.3, 127.7, 125.9, 125.8, 125.4, 119.1,
110.8, 101.8, 70.0, 60.1, 44.1, 26.9, 24.7, 0.15. LCMS m/z 319
[MþH]þ. Anal. (C21H22N2O) C, H, N.
chromatography/mass spectrometry was performed on
a
Waters/Micromass ZQ2000 LC/MS instrument consisting of a
ZQ single quadropole mass spectrometer equipped with an
electrospray ionization interface, and a Waters Alliance HT with
a 2795 separation module and 996 photodiode array detector
(PDA). HPLC method and parameters were as follows. Mobile
phase: (A) 10 mM NH4OAc H2O; (B) 10 mM NH4OAc,
CH3CN-H2O (95:5). Column: Waters Xterra MS C18 3.5 μm,
30 mm ꢀ 4.6 mm i.d. with a guard column cartridge system.
Program: 5 min gradient starting at 30% B (initial hold for
0.5 min), to 100% B, hold for 1.5 min, over 0.5 min to 30% B,
hold for 2.5 min. The flow rate was 1 mL/min. PDA range:
190-450 nm. HRMS analyses were recorded in FAB(þ) mode
using direct inlet, at the University of Lund, Sweden. Elemental
analysis was determined on a “2400 CHN elemental analyzer” by
€
Perkin-Elmer in the microanalytical laboratory of the Fakultat
R-SAT Assays. Receptor selection and amplification technol-
ogy (R-SAT) is a functional cell-based assay that allows one to
monitor receptor-dependent proliferative responses of various
receptor classes including nuclear receptors. This process is
achieved by partial cellular transformation via loss of contact
inhibition and growth factor dependency. Monitoring is
achieved by transfecting the cells with a β-galactosidase reporter
€
€
fur Chemie, Universitat Wien, Austria. The purity of compounds
1-14 are >98% based on HPLC analysis and elemental analysis.
2-Chloro-4-(3-endo-hydroxy-3-exo-methyl-8-azabicyclo[3.2.1]-
oct-8-yl)-3-methylbenzonitrile Hydrochloride (1). 2-Chloro-4-
fluoro-3-methylbenzonitrile (2.48 g, 14.6 mmol), endo-3-exo-
methyl-8-azabicyclo[3.2.1]octan-3-ol hydrochloride (15) (3.37 g,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7191
gene vector whose expression is under a constitutively active
promoter. Briefly, mouse NIH-3T3 fibroblasts were plated
overnight in 96-well plates in DMEM 10% calf serum
(Hyclone) and grown to 60-70% confluency prior to transfec-
tion. Transient transfections were performed using Polyfect
(Qiagen) according to the manufacturer’s instructions. Typi-
cally, a transfection mix would consist of expression vectors
encoding the androgen receptor (200 ng), β-galactosidase (500
ng), and the coactivators SRC1, DRIP205, and GRIP1 (10 ng
each). Such a transfection mix would be sufficient to transfect 30
96-wells. Sixteen hours after transfection, cells were incubated
with different doses of ligand in DMEM containing 30% ultra-
culture (Hyclone) and 0.4% calf serum (Hyclone) to generate a
dose-response curve. After 5 days, plates were developed by
adding onto the washed cells a solution containing the β-galacto-
sidase substrate o-nitrophenyl β-galactose (ONPG) (in phos-
phate-buffered saline with 5% Nonidet P-40 detergent). Plates
were read using a microplate reader at 420 nm. Data from R-SAT
assays were fit to the equation r=A þ B(x/(x þ c)), where A=
minimum response, B = maximum response minus minimum
response, c = EC50, r = response, and x = concentration of
ligand. Curves were generated using the curve fitting softwares
Excel Fit and GraphPad Prism (San Diego, CA).
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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 character-
ization of a novel selective androgen receptor modulator: potent
hyperanabolic stimulation of skeletal muscle with hypostimulation of
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of a potent, orally available, and isoform-selective retinoic acid β2
receptor agonist. J. Med. Chem. 2005, 48, 7517–7519. (b) Piu, F.;
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Acknowledgment. We thank Agnete Dyssegaard, Sine
Mandrup Bertozzi, and Susanna Henriksson for expert ana-
lytical services, Thomas Son for R-SAT data, Nathalie Gau-
thier and Steve Ohrmund for technical assistance with the
luciferase experiments, and Richard Barido and Linda
Pounds for assistance with the in vivo studies.
Supporting Information Available: Synthetic procedures for
all compounds and biological methods. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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