The discovery of a potent orally efficacious indole androgen receptor antagonist through in vivo screening

Article abstract of DOI:10.1016/j.bmcl.2006.09.086

A series of novel 2-(1H-indol-2-yl)-propan-2-ols have been designed, synthesized, and screened for their ability to inhibit testosterone-induced prostate weight increases in immature rats. Through the use of this paradigm, we were able to identify compoun

Full text of DOI:10.1016/j.bmcl.2006.09.086

Bioorganic & Medicinal Chemistry Letters 17 (2007) 123–126
The discovery of a potent orally efficacious indole androgen
receptor antagonist through in vivo screening
*
James C. Lanter, James J. Fiordeliso, Weiqin Jiang, George F. Allan, Muh-Tsann Lai,
Olivia Linton, Do Won Hahn, Scott G. Lundeen and Zhihua Sui
Johnson & Johnson Pharmaceutical Research and Development L.L.C., 666 Stockton Drive, Exton, PA 19341, USA
Received 1 August 2006; revised 25 September 2006; accepted 27 September 2006
Available online 30 September 2006
Abstract—A series of novel 2-(1H-indol-2-yl)-propan-2-ols have been designed, synthesized, and screened for their ability to inhibit
testosterone-induced prostate weight increases in immature rats. Through the use of this paradigm, we were able to identify
Ò
compounds that exhibited in vivo potency equal to that of the marketed antiandrogen Casodex when orally administered.
Ó 2006 Elsevier Ltd. All rights reserved.
1
The androgen receptor (AR) belongs to a subclass of
the nuclear hormone receptor superfamily known as
the nuclear steroid hormone receptors. The principal cir-
culating endogenous AR ligand testosterone (T) and its
O2N
F3C
O
N
H
R
1: R = H (Flutamide)
: R = OH
more potent tissue metabolite 5a-dihydrotestosterone
DHT) are the compounds responsible for both the
2
2
(
development of male sex characteristics and the modula-
tion of androgen receptor mediated myotropic events.
The stimulatory effects of T and DHT have been identi-
fied as key facilitators of proliferative disease states such
as benign prostatic hyperplasia (BPH) and prostate car-
cinoma (PC). Side effects attributed to the non-specific
O2N
F3C
NC
F
O
O
O
S
OH O
N
F C
N
H
3
NH
O
3
3: Nilutamide
4: Bicalutamide
effects of steroidal antiandrogens prompted research
into non-steroidal scaffolds as a means of mitigating
the potential for crosstalk within the endocrine system.
The first compound to be commercialized from these ef-
forts was the anilide flutamide (1, Eulexin ), the active
metabolite of which, hydroxy anilide 2, was demonstrat-
Figure 1. Currently marketed non-steroidal antiandrogens.
Ò
molecules, the latter has become the gold standard in
antiandrogen therapy. Research in the field has contin-
4
ed to have strong androgen receptor binding affinity
(
ued with a number of interesting leads but to date no
third generation non-steroidal antiandrogens have made
it to market.
Fig. 1).
Subsequent work in the area leading to marketed prod-
ucts involved two major thrusts: bioisosteric replace-
ment of the hydroxy anilide culminating in the launch
5
As part of our interest in the area, we prepared a series
of indole carbinols (5) to investigate their potential as
Ò
6
of nilutamide (3, Nilandron ) and elaboration of one
of the methyl groups leading to the discovery of
bicalutamide (4, Casodex ). Of these second generation
bioisosteric replacements for the hydroxy amide of
bicalutamide (Fig. 2). During our SAR studies on these
structures we found that intermediate 6a was active not
only in our in vitro binding assay but also in vivo when
dosed orally. Noting its structural similarity to 2, we
began to explore the SAR of these lower molecular
weight compounds. As an alternative to the in vitro
screening we conducted on the other series, we used a
Ò
Keywords: Androgen receptor; Bioisostere; In vivo screening.
*
Corresponding author. Tel.: +1 610 458 4133; fax: +1 610 458
249; e-mail: jlanter@prdus.jnj.com
8
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.086

124
J. C. Lanter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 123–126
R2
R2
R2
I
F
b, c
a
O
S
NC
R1
R1
O
NH2
NH2
O
Z
R1
7
8
F3C
N
H
N
H
OH
OH
R2
R1
R4
4
5
R2
R1
I
d, e
R5
OH
Ms
O2N
F3C
O N
2
N
H
N
H
O
N
H
F C
3
N
H
Cl
9
6a-e,o
OH
OH
6a
2
Scheme 1. Synthesis of the indole carbinols. Reagents and conditions:
a) NIS, pTsOH (cat.), MeOH–THF, rt; (b) 5 equiv MsCl/pyridine,
heat; (c) NaOH/MeOH–H O; (d) HCCC(OH)R , 10% CuI, 5%
Pd(PPh Cl , Et N/THF rt; (e) NaOMe/MeOH.
(
Figure 2. Bioisosteric replacement of the amide moiety in 2.
2
4 5
R
3
)
2
2
3
modified Hersherberger assay as an in vivo screening
tool to evaluate our analogs. We found that in this par-
ticular program the in vivo screening paradigm provided
a substantial time savings because it did not require in vi-
tro binding assays, functional assays, and in vivo
ADME screening before moving to an oral efficacy
model. This model required only about 40 mg of mate-
rial and delivered our screening results in less than a
week. In our experience, this ability to screen com-
pounds for oral efficacy offset the substantially more
complex data analysis.
Table 1. In vivo screening results for compounds 6a–k
R2
R4
R5
OH
R1
N
H
1
R
2
R
4
R
5
R
Compound
% Pros
Wt. Redn
a
6a
CF3
NO2
CH Cl
2
CH3
38
32
na
63
66
58
na
78
82
80
76
30
55
75
6
b
6c
CF
CF
CF
CF
CF
CF
CF
CF
CF
CF
3
3
3
3
3
3
3
3
3
3
NO
NO
NO
NO
NO
NO
NO
CN
CN
CN
Cl
2
2
2
2
2
2
2
CH
H
2
H
CH
CH
CH
CH
CH
O
3
3
3
3
3
7
In our hands a five-day Hershberger assay provided the
6
6
6
6
6
6
d
e
f
CH
CH
CH
O
2
2
2
CH
3
best balance between turnaround time, compound
requirements, and assay resolution. We found that the
measurement of prostate rather than the seminal vesicle
weights provided more accurate data due to the highly
variable water retention of the latter tissue. When using
such a screening protocol, it is important to bear in
mind that the results are a combination of a test com-
pound’s ADME properties and intrinsic efficacy. As a
result, this method of screening compounds is most use-
ful when working in a series that is analogous to a com-
pound of known molecular mechanism or the molecular
mechanism of a series has been established. To mitigate
differences in the test compounds’ rate of dissolution, we
selected 30% hydroxy propyl b-cyclodextrin (HPBCD)
as our oral dosing vehicle as it dissolved all compounds
in the series.
SMe
OH
g
h
i
CF
CF
CF
CF
3
CH
CH
CH
CH
3
3
3
3
3
(
(
+)-6i
À)-6i
3
3
6j
6k
4
CF3
H
CF3
CH3
CN
—
CF
—
3
CH
—
3
—
a
Values are means of three animals, adjusted for the body weight of each
animal; standard deviation is given in parentheses (na, not active).
group (6e) and, surprisingly, a hydroxyl moiety (6f) all
increased the efficacy relative to the parent structure.
Further oxidation of 6f to the carboxylic acid (6g) abol-
ished activity while perfluorination of one methyl group
in 6b led to a compound (6h) with as good efficacy as 4
at the screening dose. The nitro group could be replaced
with a nitrile (6i) without loss of potency but replace-
ment with a chlorine (6j) was not as well tolerated. Like-
wise removal of the indole 6-trifluoromethyl group from
6i resulted in erosion of the potency (6k). After separa-
tion of the enantiomers of 6i by chiral HPLC (ChiralPak
AD column), we noted that both compounds reached
the same level of efficacy as 4 at the testing dose. This
is in contrast to the results for optically resolved 4 in
which one enantiomer was reported to be active while
the other is essentially inactive. Finally, it had been
We initiated our efforts by examining the SAR of the
indole 5- and 6-positions as well as substitution of the
pendant carbinol. To prepare these analogs, we iodinat-
8,9
ed the aniline ring using acid catalyzed reaction with
N-iodosuccinimide to afford an iodoaniline 8 (Scheme
1
). Conversion to the iodo mesylamide 9 was achieved
in a one-pot two-step fashion by reacting 8 with an ex-
cess of mesyl chloride in hot pyridine followed by mono
demesylation with aqueous hydroxide. Compound 9 was
converted to the target structures by Sonogashira cou-
1
0
pling-cyclization with the appropriate propargyl alco-
hol followed by N-deprotection.
1
6
Simplifying the initial lead by replacement of the chlo-
rine atom with hydrogen (6b) did not improve the effica-
cy and removal of the chloromethyl group entirely (6c)
abolished activity (Table 1). Replacement of the chlorine
atom with the isosteric methyl group (6d), a thiomethyl
reported in the literature that replacement of one of
the methyl groups in compound 2 with a trifluoromethyl
moiety led to partial agonism in vivo. Given the struc-
tural similarity between the indole carbinols and 2, we
screened compounds 6h–k using an agonist format

J. C. Lanter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 123–126
125
Hershberger assay by dosing the castrated rats in the ab-
Table 2. In vivo screening results for compounds 6l–w
sence of testosterone propionate. In contrast to the
reported results for fluorinated analogs of 2, we found
no agonist activity in our group of screened compounds.
R3
R2
F C
R4
N
OH
3
R6
We next turned our attention to the indole 1- and 3-po-
sitions. Installation of a 3-methyl group was achieved
starting from iodoanilines 8 by use of Larock’s cou-
2
3
R
4
6
R
Compound
R
R
% Pros
Wt. Redn
a
1
1
pling-annulation protocol (Scheme 2). N-Methylation
of the indoles was achieved by treatment with a slight
6
6
6
6
b
NO
NO
NO
NO
2
2
2
2
H
Cl
CH
3
3
3
3
H
H
H
H
H
H
H
H
H
H
H
H
CH
—
32
81
71
59
37
56
na
55
81
na
66
79
90
75
l
CH
CH
CH
1
2
excess of sodium hydride in DMF followed by the
addition of methyl iodide. Halogenation of the 3-posi-
tion could be accomplished by treating the unsubstituted
indoles 6 with the corresponding N-halosuccinimide in
m
n
Br
I
6o
6p
6q
NO2
CH3
CN
CHCH
CH3
NO
NO
CN
CN
Cl
2
CH
CH
CH
CH
CH
3
3
3
3
3
1
3
the presence of sodium methoxide in methanol. Treat-
4
2
2
1
6
6
r
s
CH
I
3
ment of 3-bromoindoles with CuCN in heated DMF
delivered the analogous 3-cyano compound. Palladium
1
5
6t
I
catalyzed coupling of 3-iodo structures with triethoxy
vinyl silane in the presence of TBAF produced 3-vinyl
indoles.
6
6
6
4
u
CN
CN
CN
—
Cl
I
CF
CF
CF
—
3
v
3
w
H
—
3
3
Installation of a chlorine atom at the 3-position of 6b in-
creased efficacy dramatically (6l) but increasing the atom
size to bromine (6m) and iodine (6n) caused moderate
erosion in potency (Table 2). For the corresponding ni-
trile 3-iodo (6s) and chloro (6u) compounds then trend
seemed to be reversed. Replacement of the 3-chloro moi-
ety in 6l with the isosteric methyl group (6o) led to a sub-
stantial loss in potency, while the corresponding change
in 6u produced an equipotent compound (6r). Installa-
tion of a nitrile (6p) at the indole C-3 position of 6b im-
proved potency somewhat while introduction of a vinyl
moiety (6q) abolished activity. Finally, introduction of
an iodine to the three position of 6i produced an equipo-
tent compound (6v) while methylation of the indole
nitrogen (6w) afforded a compound that was toxic,
though potent. As a result, indole nitrogen alkylation
was not further pursued.
a
Values are means of four animals, body weight adjusted (na, not
active).
With the screening results in hand we brought the SAR of
the 5-cyano-6-trifluoromethyl indole substitution pattern
into clearer focus by generating ID50 data for a select
group of compounds (Table 3). From this exercise we
were able to determine that the screening provided rea-
sonable guidance for selecting potent compounds. We
also determined the ID50 of each enantiomer of our lead
compound 6 (Fig. 3). From examining the curves, it is
apparent that both compounds had a potency similar in
Ò
to 4 (Casodex ) with (+)-6i the more potent of the two.
In summary, through an in vivo screening protocol we
identified the indole carbinol as a viable bioisosteric
replacement of the amide moiety in non-steroidal anilide
antiandrogens. Through systematic modification of this
scaffold, we have identified a compound with higher
potency in vivo when dosed orally in an immature rat
model. These data provide an impetus for further inves-
R2
I
R2
a
b
N
H
F3C
NH2
F C
3
OH
8
Table 3. Single dose and ID50 comparison
X
R2
R4
R2
R4
R3
R4
NC
F C
F3C
N
H
OH
F C
3
N
H
OH
N
H
OH
3
c
e
d
R2
R4
3
4
Compound
R
R
ID50 (mpd)
% Pros
Wt. Redn
a
R4
R2
F C
N
OH
3
6
x
Cl
Br
I
CH
CH3
3
>3.00
0.80
1.10
>3.00
6.80
nt
nt
F C
3
N
OH
6y
6t
nt
CN
R4
H
CH
CH
81
55
66
82
80
76
75
R2
F3C
3
6
s
CH
Cl
H
3
3
N
H
OH
6j
CF3
6
i
CF
CF
CF
—
3
3
3
(
(
+)-6i
À)-6i
H
0.13
0.43
0.29
Scheme 2. Elaboration of the indole 1- and 3-positions. Reactions and
conditions: (a) 5% Pd(OAc) , 10% PPh , MeCCC(OH)Me , KOAc,
LiCl/DMF, heat; (b) NXS, NaOMe/MeOH, rt; (c) NaH/DMF, then
MeI; (d) X = Br: CuCN/DMF, heat; (e) X = I: (CH @CH)Si(OEt)
Pd (dba) (cat.), TBAF, DMF, heat.
H
—
2
3
2
4
a
2
3
,
Values are means of four animals, body weight adjusted (nt, not
tested).
2
3

1
26
J. C. Lanter et al. / Bioorg. Med. Chem. Lett. 17 (2007) 123–126
15, 1565; (b) Chen, J.; Kim, J.; Dalton, J. T. Mol.
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1
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9
8
7
0
0
0
%
Pros.
Wt.
Inh 60
5
4
3
2
1
0
0
0
0
0
0
6. (a) Lima, L. M.; Barreiro, E. J. Curr. Med. Chem. 2005,
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J. Chem. Rev. 1996, 96, 3147.
-
-
-
10
20
30
7
. A typical procedure: A modified Hershberger assay
(
Hershberger, L. G.; Shipley, E. G.; Meyer, R. K.;
Myotrophic activity of 19-nortestosterone and other
steroids determined by modified levator ani muscle
method. Proc. Soc. Exp. Biol. Med. 1953 83, 175) was
performed to assess androgen antagonist activity in
orchidectomized rats. Sprague–Dawley rats (Charles Riv-
er Laboratories, Wilmington, MA) were castrated at three
weeks old. They were then dosed once daily for five days
with test compound (oral dosing by gavage needle; vehicle:
0.03
0.1
0.3
1
3
Dose Compound (mg/d)
(
(
+)-6i
-) -6i
4
(Casodex ®)
3
0% (w/v) hydroxypropyl-b-D-cyclodextrin [Cargil Inc.,
Cedar Rapids, IA]) in the presence of 0.1 mg/day (approx-
imately 1.3 mg/kg) testosterone propionate (subcutaneous
dosing; vehicle: sesame oil). At necropsy, ventral prostates
and seminal vesicles were removed and their wet weights
were measured. Tissue weights were divided by whole
body weight. Data were expressed as percentage of
inhibition relative to testosterone alone (0% inhibition)
and cyclodextrin vehicle alone (100% inhibition).
Figure 3. Dose–response curves of prostate weight reduction for
bicalutamide, (+)-6i, and (À)-6i in immature rats.
tigation of the indole carbinol nucleus for the purpose of
uncovering novel antiandrogens. This approach also
highlights the utility of the Hershberger assay as a
means of rapidly developing in vivo androgen receptor
SAR.
8
9
. Gladiali, S.; Pinna, L. Tetrahedron: Asymmetry 1991, 2,
6
23.
. Boger, D. L.; McKie, J. A. J. Org. Chem. 1995, 60,
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Sakamoto, Takao; Kondo, Yoshinori; Iwashita, Shigeki;
Nagano, Tatsuo; Yamanaka, Hiroshi. Pharm. Inst.,
Tohoku Univ., Sendai, Japan. Chem. Pharm. Bull. 1988,
Acknowledgment
The authors thank Professor Dale Boger for his sugges-
tions regarding the iodination of electron deficient
anilines.
3
6, 1305.
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