Potent, nonsteroidal selective androgen receptor modulators (SARMs) based on 8H-[1,4]oxazino[2,3-f]quinolin-8-ones

Article abstract of DOI:10.1016/j.bmcl.2007.07.034

A series of androgen receptor modulators based on 8H-[1,4]oxazino[2,3-f]quinolin-8-ones was synthesized and evaluated in an androgen receptor transcriptional activation assay. The most potent analogues from the series exhibited single-digit nanomolar potency in vitro. Compound 18h demonstrated full efficacy in the maintenance of muscle weight, at 10 mg/kg, with reduced activity in prostate weight in an in vivo model of androgen action.

Full text of DOI:10.1016/j.bmcl.2007.07.034

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5442–5446
Potent, nonsteroidal selective androgen receptor
modulators (SARMs) based on 8H-[1,4]oxazino[2,3-f]
quinolin-8-ones
Robert I. Higuchi,* Anthony W. Thompson, Jyun-Hung Chen, Thomas R. Caferro,
Marquis L. Cummings, Charlotte P. Deckhut, Mark E. Adams, Christopher M. Tegley,
´
James P. Edwards, Francisco J. Lopez, E. Adam Kallel, Donald S. Karanewsky,
William T. Schrader, Keith B. Marschke and Lin Zhi
Discovery Research, Ligand Pharmaceuticals, 10275 Science Center Drive, San Diego, CA 92121, USA
Received 5 June 2007; revised 9 July 2007; accepted 10 July 2007
Available online 25 July 2007
Abstract—A series of androgen receptor modulators based on 8H-[1,4]oxazino[2,3-f]quinolin-8-ones was synthesized and evaluated
in an androgen receptor transcriptional activation assay. The most potent analogues from the series exhibited single-digit nanomolar
potency in vitro. Compound 18h demonstrated full efficacy in the maintenance of muscle weight, at 10 mg/kg, with reduced activity
in prostate weight in an in vivo model of androgen action.
Ó 2007 Elsevier Ltd. All rights reserved.
The androgen receptor (AR) is a member of the intracel-
lular receptor superfamily of ligand-dependent transcrip-
tion factors.1 The endogenous ligands for AR are the
steroids testosterone (T) and dihydrotestosterone
(DHT). When bound to AR, these ligands play impor-
tant roles in sexual development and function,2 and mus-
culo-skeletal growth.3 Steroidal androgen therapy is
effective for the treatment of androgen insufficiency.
However, the broader use of these androgens for addi-
tional treatments, such as osteoporosis or frailty, is lim-
ited by undesirable AR-mediated effects, such as
prostatic hypertrophy and hirsutism. A selective andro-
gen receptor modulator (SARM), with full anabolic
activity but reduced impact on the undesirable effects,
could have a large role on endocrine therapies to treat
muscle wasting and osteoporosis.4 Early studies on mod-
ified androgens explored alkylation at C-17, as in fluox-
ymesterone (1, Fig. 1).5 However, compounds from this
general class are associated with potential liver toxicity.2
Recent publications in the area of nonsteroidal andro-
gens are indicative of the high level of interest in discov-
ering novel safe, effective anabolic agents (2, 3).6–8
Our continued interest in SARMs is based on scaffolds
derived from quinolin-2-ones (4 and 5).9,10 During the
course of our studies on 7H-[1,4]oxazino[3,2-g]quino-
lin-7-ones (5a), we isolated as a minor by-product of
the Knorr quinolone reaction11 regioisomer (6a, <5%
yield, Scheme 1) which fortuitously possessed AR
OH
HO
HO
H
H
F3C
N
F
H
O
O
E
O
2
1
R = CN, NO2
R
CF3
O
CF3
N
N
CF3
OH
N
H
O
O
N
H
NC
4
3
R3
R2
R1
CF3
R1
CF3
O
R2
R3
N
N
O
N
O
Keywords: Androgen; SARM; Oxazino[2,3-f]quinolin-8-one; Testos-
terone; Anabolic; Androgenic; AR; Benzoxazine.
O
N
H
H
6
5
*
Corresponding author. Tel.: +1 858 550 7627; fax: +1 858 550
7249; e-mail: rhiguchi@ligand.com
Figure 1. Synthetic androgens.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.034

R. I. Higuchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5442–5446
5443
R2
NHBoc
CF3
CF3
CF3
O
CF3
N
O
N
O
N
CF3
N
O
a, b
+
Br
a
R2
CF3
H2N
O
N
H
N
H
O
HO
6a
< 5%
11 +
5a
42%
7
O
NHBoc
12
13
b
Scheme 1. Reagents and conditions: (a) 4,4,4-trifluoroacetoacetate,
PhH, reflux; (b) concd H2SO4, 100 °C.
R2
NH2
R2
NH
CF3
O
CF3
N
O
c
Br
agonist activity in a transcriptional activation assay
(78% agonist efficacy, 5 nM). Investigations into the
scaffold led to a series of novel androgens derived from
8H-[1,4]oxazino[2,3-f]quinolin-8-ones (6). The lead com-
pound from this series, 18h, demonstrated full efficacy in
the maintenance of levator ani weight (LA) muscle, an
anabolic endpoint in a castrated mature rat model.
O
N
O
14
15
R2
R1
R2
R1
CF3
O
CF3
O
e
d
N
N
15
O
N
H
i-Pr-O
N
f
18
Our previous investigations of quinolinone scaffolds 4
and 5 revealed that the substitution pattern proximal
to the tertiary amine group strongly affected AR activ-
ity. Consequently, we sought to probe the R1 and R2 re-
gion of 6. The yields obtained by isolating the minor
Knorr product were too low to be a practical method
of analogue preparation, so we developed a new synthe-
sis that would allow for modifications on the oxazine
portion of 6. A Knorr-type reaction of 5-chloro-1,3-phe-
nylene diamine proceeded by treatment with ethyl 4,4,4-
trifluoroacetoacetate in EtOH to afford the quinolin-2-
one (47%), followed by hydrogenation (100%) to
provide 8 (Scheme 2). Treatment of 8 with sodium ni-
trite in concd H2SO4 effects the Sandmeyer reaction with
dehydration to afford 9, and is an effective method to
prepare the 5-substituted 1H-quinolin-2-one, which is
not available by the direct Knorr cyclization of 3-amino-
phenol derivatives. Bromination with NBS afforded 10
in 77% yield.12 To prepare 11, it was necessary to benzy-
late the phenol, alkylate the quinolinone with isopropyl
iodide and CsF, then remove the benzyl group with
methanesulfonic acid to afford 11 in 55% yield over 3
steps. With 11 in hand, the N-Boc-protected aminoalco-
hol 12 was subject to Mitsunobu conditions13 to afford
13, followed by hydrolysis of the Boc group with TFA
to afford 14 (Scheme 3). Formation of the benzoxazine
subunit was achieved utilizing the Buchwald–Hartwig
aromatic amination conditions to afford intermediate
17
R2
NH
CF3
O
O
N
H
16
Scheme 3. Reagents and conditions: (a) diisopropyl azodicarboxylate,
Ph3P, N-methylmorpholine; (b) TFA, CH2Cl2; (c) Pd2(dba)3 (2–
5 mol%), ( )-BINAP (4–10 mol%), t-BuONa, toluene, reflux; (d)
R1CHO or R1CH(OH)OEt, NaBH3CN, HOAc or TFA; (e) HOAc/
concd HCl (3:1), 60–100 °C.
15.14 In addition to providing the requisite scaffold, this
synthesis represents a novel method to prepare 3,4-dihy-
dro-2H-1,4-benzoxazines. Reductive amination, fol-
lowed by acid hydrolysis, afforded 18.15 Compound 16
is obtained by acid hydrolysis of 15.
The compounds were evaluated in a transcriptional acti-
vation assay with hAR in a mammalian cell (CV-1) as
previously described.16 Both R1 and R2 positions are
important for AR agonist activity as compounds with
no R1 substituent showed no agonist activity regardless
of the R2 substituent (Table 1). Antagonist activity was
observed (16a–e), demonstrating that this position can
be used to switch the AR activity to antagonists. This
finding parallels series 5, in which substitution at the
R1 position enhances AR agonist activity. Replacement
of the 2,2,2-trifluoroethyl substituent of 6a with small al-
kyl groups (18a–d) resulted in a significant drop in
potency, even in the case of 18b, where R1 = Et. Re-
moval of the R2 substituent (18e) likewise resulted in
significant reduction of AR agonist activity. Substitu-
tion of R2 with an ethyl group resulted in a series of
analogues with AR activity that roughly parallels that
of the corresponding methyl substituted compounds.
Compound 18f demonstrates no AR agonist activity,
and the same significant difference in potency between
the ethyl (18g) and 2,2,2-trifluoroethyl substituent
(18h) exists. Compound 18h demonstrates activity com-
parable to that of DHT. Weaker agonist activity is seen
among the propyl, isobutyl, and 3-hydroxy-2-methyl-
CF3 OH
NH2
NH2
OH
F3C
c
a, b
O
O
N
H2N
Cl
O
N
9
H
H
8
CF3 OH
CF3 OH
Br
e, f, g
d
Br
N
O
N
H
11
10
Scheme 2. Reagents and conditions: (a) 4,4,4-trifluoroacetoacetate,
EtOH; (b) 10% Pd-C, H2, (1 atm), KOAc, EtOH; (c) NaNO2, concd
H2SO4, 0 °C, then 140 °C; (d) NBS, diisopropylamine, EtOAc, À10 °C;
(e) BnBr, CsF, DMF; (f) isopropyl iodide, CsF, DMF; (g) methane-
sulfonic acid, HOAc (1:1).

5444
R. I. Higuchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5442–5446
Table 1. Activity in the AR transcriptional activation assaya
Compoundc
R1
R2
hAR Agonist
EC50 (nM)
hAR Antagonist
Eff (%)
100
IC50 (nM)
Eff (%)
DHT
5a
6a
5.7 0.1
1.1 0.2
5.4 3.4
—
—
—
—
—
82
78
—
—
—
—
—
—
5
4
CH2CF3
H
Me
H
—
1500 100
—
62 16
64
16a
16b
16c
16d
16e
18a
18b
18c
18d
18e
18f
18g
18h
18i
H
Me
Et
—
—
72
84 14
1480 750
H
58
78
76
32
26
6
9
4
4
8
H
Ph
Pr
—
—
H
Me
113 58
nd
nd
Me
Me
Me
Me
H
—
Et
Pr
420 120b
406 56
228 84
190 66
—
31 12
45 22
97 12
nd
na
21 13
na
Allyl
CH2CF3
Me
Et
38
—
2
nd
nd
25 12
32 11
—
Et
Et
410 70
1.0 0.4
51 14
394 42
227 26
300 140
276 22
272 74
20 16
27 10
92
—
—
CH2CF3
Allyl
Pr
Et
Et
5
—
—
83 14
61 12
46 14
57 11
—
—
18j
Et
Et
—
38 15
18k
18l
i-Bu
—
—
Me2C(OH)CH2-
Me
Et
Et
26
31
9
9
18m
18n
18o
18p
18q
18rd
18s
18t
i-Pr
i-Pr
i-Pr
i-Pr
Bn
i-Bu
Ph
Ph
31
76
74
78
6
6
5
3
nd
—
21 10
—
—
CH2CF3
Allyl
—
—
35
6
CH2CF3
CH2CF3
CH2CF3
Cyclopropylmethyl
(S)-18h
( )-18h
43 14
98 32
63 14
—
nd
—
36
—
—
3
34
85
88
—
—
9
3
2
—
—
65 16
45 10
—
18
3.5 1.3
3
53 23
89
8
—
a AR transcriptional activation experimental results with at least three experiments in triplicate with SEM.
b Mean value from two experiments.
c For chiral compounds, the (R)-enantiomer was prepared except where indicated. For all tables, a hyphen is indicative of efficacy <20% or a potency
>10,000 nM, and nd means the IC50 could not be calculated.
d Isolated as a minor by-product of the Knorr reaction as in Scheme 1.
propyl analogues (18j–l). In the cases where R2 = isopro-
pyl, a slight reduction in potency is seen with 18o
compared to 18h, while allyl analogue 18p maintains
the moderate AR potency seen in 18i. Because they
tended to have the best AR activity, the subsequent
compounds focused more on analogues where
R1 = 2,2,2-trifluoroethyl. Compound 18q has compara-
ble activity to 18o, while the isobutyl analogue 18r
shows more partial agonist activity. Phenyl analogues
18s and 18t switch activity to full antagonists. Hence,
the R2 position can be utilized to provide the full spec-
trum of AR activity from full agonist, partial agonist,
and antagonist. To determine the effect of the other
enantiomer, lead compound ( )-18h was prepared as a
racemate and the distomer (S)-18h separated by chiral
HPLC. Compound (S)-18h was less active than the
(R)-enantiomer 18h.
To determine the steroid hormone transactivation selec-
tivity, selected compounds were evaluated in transacti-
vation assays for progesterone receptor (PR),
glucocorticoid receptor (GR), and mineralocorticoid
receptor (MR) activity.16 In no instance was agonist
activity detected for PR, GR, or MR. No MR antago-
nist activity was detected, while weak PR and GR antag-
onist activity is observed (Table 2).
Table 2. Steroid hormone selectivitya
Compound
AR agonist
EC50 (nM)
PR antagonist
IC50 (nM)
GR antagonist
IC50 (nM)
Eff (%)
Eff (%)
Eff (%)
6a
16c
18h
5.4 3.4
84 14b
1.0 0.4
78
58
92
4
6
5
nd
nd
40
42
3200
—
53
—
520 190
94
1
1510 80
98
1
a Steroid hormone selectivity experimental results with at least three separate experiments in triplicate with SEM. If no SEM is noted, value is from a
single determination.
b Antagonist activity with IC50 reported.

R. I. Higuchi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5442–5446
5445
Figure 2. Compound 18h with key contacts in the androgen binding
site based on 4-AR structure.
Modeling results based on the 4-AR structure17,9 indi-
cate that the fluorine atoms on the 2,2,2-trifluoroethyl
substituent of 18h interact with amino acid THR-877
which rationalizes the enhanced AR activity of the
2,2,2-trifluoroethyl substituent compared to the nonflu-
orinated side chains (Fig. 2).18 The A-ring also forms
bifurcated hydrogen bonds with ARG-752 and GLN-
711. The ethyl group on the C-ring fills a lipophilic
pocket delineated by MET-780, PHE-876, LEU-880,
and LEU-701(omitted for clarity), which also provides
increased binding interactions.
Figure 3. Compounds 18h and T (as testosterone propionate)9 in a
2-week castrated mature rat model. The dashed lines (—) represent
intact levels.
the possibility that 18h could potentially see selectivity
enhancement if conducted in the restoration mode.
We have described a series of orally-active, nonsteroidal
androgen receptor modulators based on an 8H-[1,4]-
oxazino[2,3-f]quinolin-8-one scaffold. These compounds
exhibit good potency in vitro, and compound 18h has a
favorable profile compared to T in an adult castrated
rat in vivo model that measures androgen action, and
hence can be regarded as a SARM.
Compound (R)-18h was tested in order to demonstrate an
in vivo proof of concept for the series. A 2-week castrated
mature rat assay was utilized to assess the AR-mediated
effect on organ weights.9,10 The male sexual accessory or-
gans, such as the ventral prostate (VP), are stimulated to
grow and are maintained in size and function by the pres-
ence of endogenous androgens. This model is used to
determine the androgen-dependent growth of the VP in
mature castrated rats. Over-stimulation of the VP is unde-
sirable because of its association with increased risk of
prostatic disorders. In addition to the VP, the LA muscle
demonstrates androgen-dependent growth.19 The LA is a
useful endpoint to evaluate the anabolic effects of the
compounds in muscle. A compound that has full activity
on LA but a reduced impact on VP represents a good pro-
file for SARM activity. Figure 3 demonstrates that com-
pound 18h has good anabolic activity in this model,
maintaining the LA weight at 10 mg/kg. At 100 mg/kg,
the full weight of the VP is not maintained, suggesting that
there is no over-stimulation of the VP. Compared to T,9
compound 18h has a muscle-selective profile based on
the separation of the LA weight dose–response compared
to the VP, and possesses at least 10-fold selectivity over
that T in this maintenance model. Castrated mature rat
assays can alternatively be conducted in a growth restora-
tion model by delaying the administration of androgens
after castration. Synthetic androgens, such as 1, demon-
strate significantly higher selectivity in a restoration mod-
el compared to this maintenance model.20 This suggests
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extracted with EtOAc, and then washed with saturated
NaHCO3 to afford 14 (CH2Cl2/MeOH). Compound 14 (1
equiv) in toluene (0.1–0.2 M) was added to ( )-BINAP (4–
10 mol %), Pd2(dba)3 (2–5 mol%), and t-BuONa (1.4
equiv). The solution was heated at 90–100 °C for 6–24 h,
poured into cold saturated NH4Cl, and extracted with
EtOAc. Compound 15 (R2 = Et) was prepared in 63%
yield (hexanes:EtOAc). 1H NMR (400 MHz, CDCl3) d
7.36 (d, 1H, J = 8.8), 7.18 (s, 1H), 7.03 (d, 1H, J = 8.8),
5.47 (septet, 1H, J = 6.2), 4.36 (dd, 1H, J = 10.6, 2.9), 3.87
(dd, 1H, J = 10.4, 7.5), 3.83 (br s, 1H), 3.48–3.40 (m, 1H),
1.63–1.53 (m, 2H), 1.38 (d, 6H, J = 6.2), 1.06 (t, 3H,
J = 7.4). Compound 18h was prepared in 67% yield from
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(R2 = Et),
and
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J = 8.9), 4.38 (d, 1H, J = 10.9), 3.98 (dd, 1H, J = 10.8, 2.4)
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15. Representative procedures. All reaction mixtures were
washed with brine, dried over MgSO4, and purified by
flash chromatography. Compound 13: Diisopropyl azodi-
carboxylate (1.6 equiv) was added to 11 (1 equiv), 12 (1.6
equiv), PPh3 (1.6 equiv), and N-methylmorpholine (10
equiv) in THF (0.1–0.2 M) at 0 °C, and after 5 min, the
reaction mixture was stirred at rt for 2–16 h. The solution
was poured into water, neutralized with 1.0 M HCl,
extracted with EtOAc, and then washed with of 0.1 M
HCl. Compound 13 (R2 = Et) was prepared in 74% yield
18. Calculations were performed using the Tripos forcefield
and Gasteiger-Huckel, and 18h was docked using the
¨
X-ray cocrystal structure of AR-lbd and 4 as a template.
Structures were refined using a simulated annealing
protocol as described in SYBYL 7.0, Tripos Inc., 1699
South Hanley Rd., St. Louis, MO 63144, USA.
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1
(hexanes:EtOAc). H NMR (400 MHz, CDCl3) d 7.80 (d,
1H, J = 8.9), 7.55 (d, 1H, J = 8.9), 7.29 (s, 1H), 5.52
(septet, 1H, J = 6.3), 4.80 (br s, 1H), 4.06–3.90 (m, 3H),
1.91–1.81 (m, 1H), 1.71–1.59 (m, 1H), 1.46 (s, 9H), 1.41 (d,
6H, J = 6.2), 1.01 (t, 3H, J = 7.4). Compound 15. Com-
pound 13 in 1:1 CH2Cl2/TFA (0.1 M) was stirred at rt for
´
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