Diphenyl ethers as androgen receptor antagonists for the topical suppression of sebum production

Article abstract of DOI:10.1016/j.bmcl.2009.02.104

A series of diphenyl ethers was prepared and evaluated for androgen receptor antagonist activity in human androgen receptor binding and cellular functional assays. Analogs with potent in vitro activities were evaluated for topical in vivo efficacy in the

Full text of DOI:10.1016/j.bmcl.2009.02.104

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2176–2178
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diphenyl ethers as androgen receptor antagonists for the topical
suppression of sebum production
b
b
c
d
e
Lorna H. Mitchell a, , Lain-Yen Hu , Maria Nguyen , Stephen Fakhoury , Yvonne Smith , Donna Iula ,
Catherine Kostlan f, Matthew Carroll b, Danielle Dettling g, Daniel Du b, David Pocalyko h,
Kimberly Wade i, Bruce Lefker b
*
a Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Sandwich, CT13 9NJ, UK
b Pfizer Global Research and Development, Groton Laboratories, CT 06340, USA
c Pfizer Global Research and Development, Michigan Laboratories, MI 48105, USA
d Decision Informatics, Ltd, Ann Arbor, MI 48104, USA
e Cayman Chemical, Ann Arbor, MI 48108, USA
f IDSC, LLC, Chelsea MI 48118, USA
g Pfizer Global Research and Development, Research Technology Center, MA 02139, USA
h Pfizer Global Research and Development, La Jolla Laboratories, CA 92121, USA
i Pfizer Global Research and Development, St Louis Laboratories, MO 63141, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of diphenyl ethers was prepared and evaluated for androgen receptor antagonist activity in
human androgen receptor binding and cellular functional assays. Analogs with potent in vitro activities
were evaluated for topical in vivo efficacy in the Golden Syrian Hamster ear model. Several compounds
showed reduction in wax esters in this validated animal model.
Received 1 February 2009
Revised 25 February 2009
Accepted 26 February 2009
Available online 3 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Androgen receptor
Antagonist
Sebum
Topical
Diphenyl ether
AR
The androgen receptor (AR) is responsible for the activation of
genes involved in the pathogenesis of acne and alopecia.1 The onset
of acne vulgaris infections in the pilosebaceous unit is associated
with excess sebum production. The production of sebum is regu-
antagonist activity in a cellular assay (ARCELL)7 (Table 1). Unfortu-
nately, the chlorobenzonitriles exhibited photoinstability which is
not desirable in a topical agent. We therefore examined the 3-trif-
luoromethylbenzonitrile template based on precedence of known
AR binders.8 Gratifyingly the initial tolyl analog, 3a, retained AR
binding activity, a full antagonist profile, and was photostable
but, unfortunately, it lost appreciable cellular activity.
The compounds were synthesized by phenol displacement of
the desired para-fluorobenzonitrile template under basic condi-
tions (Scheme 1).9
lated by the androgens testosterone and 5a-dihydrotestosterone
(5a-DHT). Androgen receptor antagonists, such as cyproterone
acetate2,3 and RU-588414 have been shown to suppress sebum pro-
duction when applied topically.
Mining of data from our AR agonist program identified a number
of compounds that bound to the androgen receptor but did not pos-
sess agonist activity. A reevaluation showed that some of these were
full antagonists, for example diphenyl ethers such as 1, and we have
used them as starting points for our program to identify AR antago-
nists (Fig. 1) for the topical suppression of sebum production.5
The chlorine of hit 1 could be relocated from the 2-position to
the 3-position (2) with retention of AR binding (ARB)6 and full
NC
O
Cl
Me
1
* Corresponding author. Tel.: +44 1304 649599.
E-mail address: lorna.mitchell@pfizer.com (L.H. Mitchell).
Figure 1. Initial AR antagonist lead compound.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.104

L. H. Mitchell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2176–2178
2177
Table 1
Table 3
Initial SAR explorationa
In vivo efficacy in Golden Syrian hamster modela
Compound
R1
R2
ARB (nM)
ARCELL (nM)
cLogP
4
NC
4'
1
2
2-Cl
3-Cl
30-Me
20-Me
20-Me
30-Me
40-Me
20-Cl
64
30
92
770
4000
123
2720
6650
83
46
57
743
—
—
—
—
—
72
—
—
4.66
4.76
5.06
5.06
5.06
5.04
5.27
5.27
3.64
3.89
3.89
3
R1
3'
R2
O
2
2'
3a
3b
3c
4a
4b
4c
5a
5b
5c
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
Compound
R1
R2
WE reduction (%)
CE reduction (%)
1
2
6i
6r
6hb
2-Cl
3-Cl
3-CF3
3-CF3
3-CF3
20-Me
20-Me
20-OEt
20-OMe, 60-F
20-SMe
90
87
82
41
36
76
71
67
37
32
30-Cl
40-Cl
20-OMe
30-OMe
40-OMe
>10,000
>10,000
a
All compounds were tested at dose of 3% in a polyethylene glycol/transcutol/
ethanol 20/20/60 v/v/v% formulation unless otherwise stated.
a
Values (IC50) are given as an average of P2 experiments; —, not tested.
b
Polyethylene glycol/ethanol 30/70 v/v% formulation.
A variety of disubstituted analogs possessing either an ortho-
hydroxy, methoxy, or ethoxy substituent (6k–q) lost AR binding
and/or cellular activity when compared to the compounds with
only one ortho-substituent. Analogs 6k and 6n were not cellularly
active despite their lower cLogP values. The only disubstituted
analog which retained good cellular activity was 6r.
NC
NC
4'
3'
R2
a
3
R1
R1
O
F
2
2'
Scheme 1. Reagents and conditions: (a) ArOH, K2CO3, DMF, 60 °C.
Selected compounds with good in vitro profiles were tested
in vivo in Golden Syrian Hamsters for their ability to reduce wax
and cholesterol esters (Table 3). Wax and cholesterol esters consti-
tute 28% of total human sebum10 and it has been shown that there
is a direct correlation between reduction in wax esters and reduc-
tion in total sebum production in a clinical trial with oral cyproter-
one acetate.11 The Hamster Ear model is a widely used animal
model to test drug effects on sebaceous glands.12
Compounds 1, 2, and 6i exhibited the best in vivo profiles with
>80% reduction of wax esters when applied topically as a 3% for-
mulation. By comparison a 1% formulation of the positive control,
RU-58841, reduced wax esters by 95%. Disappointingly, the
methylthio analog 6h showed only marginal reduction of wax es-
ters in vivo despite possessing one of the best in vitro profiles:
we had considered that it may have been able to act as a soft-
drug13 by being metabolized prior to systemic exposure, though
this result suggests metabolism may have been too rapid.
In summary, we have prepared a series of diphenylethers as
androgen receptor antagonists and demonstrated that compounds
from this series exhibit reduction in sebum when applied topically
in a validated animal model.
Within the trifluoromethylbenzonitrile series, we explored sub-
stitution of the second phenyl ring. We found that when the R2
substituent was held constant and stepped around the ring AR
binding followed the trend ortho > meta > para (Table 1).
Having determined that ortho-placement of R2 was preferred
we looked at a variety of substitution in this position (Table 2).
Increasing the size of the ortho R2 substituent from methyl to ethyl
(6a) was tolerated while a decrease in AR binding was seen with
the propyl (6b), phenyl (6c), and isopropyl (6d) analogs.
Introducing a second ortho-methyl (6e) was tolerated. However,
further substitution, as in the mesityl analog 6f, was detrimental to
AR binding.
Replacement of the ortho-methyl with hydroxy (6g), methoxy
(5a), and methylthio (6j) was tolerated in terms of AR binding
and in all cases gave good cellular activity (ARCELL < 100 nM).
These analogs have lower cLogP than compounds 6a–f which
may account for the observed cellular activity. Cellular activity
was retained in the change from methoxy (5a) to ethoxy (6i). How-
ever, the ethylthio analog (6j) lost cellular activity compared to the
methylthio analog (6h).
References and notes
Table 2
SAR of trifluoromethylbenzonitrilesa
1. Redmond, G. P.; Bergfeld, W. F. Cleveland Clinic J. Med. 1990, 57, 428.
2. Bingham, K. D.; Low, M.; Wyatt, E. H. Lancet 1979, 314, 304.
3. Gruber, D. M.; Sator, M. O.; Joura, E. A.; Kokoschka, E. M.; Heinza, G.; Huber, J. C.
Arch. Dermatol. 1998, 134, 459.
Compound
R2
ARB (nM)
ARCELL (nM)
cLogP
6a
6b
6c
6d
6e
6f
6g
5a
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
20-Et
20
202
430
2280
10
198
34
83
356
303
—
5.58
6.11
5.88
5.58
5.55
6.05
3.64
4.13
4.72
5.15
5.24
3.86
4.62
4.62
3.09
5.15
4.14
4.14
4.21
4. Gao, W.; Bohl, C. E.; Dalton, J. T. Chem. Rev. 2005, 105, 3352.
20-Pr
5. (a) Hu, L. Y.; Du, D.; Hoffman, J.; Smith, Y.; Fedij, V.; Kostlan, C.; Johnson, T. R.;
Huang, Y.; Kesten, S.; Harter, W.; Yue, W. S.; Li, J. J.; Barvian, N.; Mitchell, L. H.;
Lei, H. J.; Lefker, B.; Carroll, M.; Dettling, D.; Krieger-Burke, T.; Samas, B.;
Yalamanchili, R.; Lapham, K.; Pocalyko, D.; Sliskovic, D.; Ciotti, S.; Stoller, B.;
Hena, M. A.; Ding, Q.; Maiti, S. N.; Stier, M.; Welgus, H. Bioorg. Med. Chem. Lett.
2007, 17, 5983; (b) Van Camp, J. A.; Hu, L.-Y.; Kostlan, C.; Lefker, B.; Li, J. J.;
Mitchell, L. H.; Wang, Z.; Yue, W.-S.; Carroll, M.; Dettling, D.; Du, D.; Pocalyko,
D.; Wade, K. Bioorg. Med. Chem. Lett. 2007, 17, 5529; (c) Hu, L.-Y.; Lei, H. J.; Du,
D.; Johnson, T. R.; Fedij, V.; Kostlan, C.; Yue, W.-S.; Lovdahl, M.; Li, J. J.; Carroll,
M.; Dettling, D.; Asbill, J.; Fan, C.; Wade, K.; Pocalyko, D.; Lapham, K.;
Yalamanchili, R.; Samas, B.; Vrieze, D.; Ciotti, S.; Krieger-Burke, T.; Sliskovic, D.;
Welgus, H. Bioorg. Med. Chem. Lett. 2007, 17, 5693; (d) Mitchell, L.; Wang, Z.;
Hu, L-Y.; Kostlan, C.; Carroll, M.; Dettling, D.; Du, D.; Pocalyko, D.; Wade, K.
Bioorg. Med. Chem. Lett. 2009, 19, 1310.
6. The androgen receptor binding assay run was a modification of that described
in Liao, S.; White, D.; Schilling, K.; Chang, C. J. Steroid Biochem. 1984, 1, 11 The
human AR cDNA cloned in baculovirus was expressed in Sf9 cells. Cell lysates
from transfected Sf9 cells were isolated and used as the source of human AR in
the radio-ligand binding assay. Different concentrations of test compounds
(10,000, 1000, 200, 40, 8, 1.6, and 0.16 nM) were incubated in the presence of
human AR extract, hydroxylapatite, and 1 nM 3H-DHT for one hour at 4 °C with
20-Ph
20-iPr
—
20, 60-di-Me
20, 40, 60-tri-Me
20-OH
681
812
97
72
22
20-OMe
20-SMe
74
20-OEt
120
229
157
69
103
216
257
354
127
158
47
20-SEt
545
>1000
299
482
>1000
—
—
811
42
20,60-di-OMe
20-OMe, 40-Me
20-OMe, 50-Me
20-OMe, 50-CH2OH
20-OEt, 4-Me
20-OH, 4-Me
20-OH, 5-Me
20-OMe, 60-F
a
Values (IC50) are given as an average of P2 experiments; —, not tested.

2178
L. H. Mitchell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2176–2178
gentle rocking. After incubation, plates were placed on a filter apparatus and
heated at 60 °C until consumption of starting material was seen and then
allowed to cool to room temperature. The reaction mixture was then poured
into ice water and the resulting precipitate collected by filtration. In examples,
where no precipitate was observed ethyl acetate was added and the organic
phase was washed with water followed by brine. The organic phase was dried
over MgSO4 and concentrated. The resulting residue was then purified by
column chromatography on silica using ethyl acetate/heptane mixtures as the
eluent.
the reaction mixture was removed under 15 psi vacuum pressure, then washed
three times with cold buffer. The plates were then dried at room temperature
overnight. The next day, scintillation fluid was added to each well and the
plates were counted using a Microbeta Trillux. To determine non-specific
binding, 1000-fold concentration of cold DHT was added to each well.
Triplicate wells were tested for each concentration. The experimental results
were expressed as the concentration of compound at which 50% of maximum
binding was inhibited (IC50). The AR antagonist RU-58841 was run as
a
10. Thiboutot, D. J. Invest. Dermatol. 2004, 123, 1.
standard revealing an inter-run variability within twofold and an average IC50
of 16 nM.
11. Miller, C. G.; Wojnarowska, F. T.; Dowd, P. M.; Ashton, R. E.; O’Brien, T. J.;
Griffiths, W. A.; Jacobs, H. S. Brit. J. Dermatol. 1986, 114, 705.
7. The androgen receptor cellular functional assay was conducted in a human
breast tumor cell line expressing androgen receptor (MDA-MB453-MMTV
clone 54-19). The cell line is a stably transfected cell line with MDA-MB453 cell
12. Male Syrian Hamsters aged 9–10 weeks were introduced into the laboratory
environment and acclimated for 2 weeks prior to use in each study. Each
experimental group consisted of five animals. Vehicle control and a reference
AR antagonist (RU-58841) were included in each experiment. Animals were
topically dosed twice daily (BID) for 2 weeks, 5 days a week (Monday–Friday).
background.
element (ARE) was first cloned in front of a firefly luciferase receptor gene.
Then the cascade was cloned into transfection vector pUV120-puro.
A MMTV minimal promoter containing androgen response
a
Each dose consisted of 25 lL of vehicle control or formulated test article, which
Electroporation was used for transfecting MDAMB-453 cells and a puromycin
resistant stable cell line was selected. See: Chang, C.; Wang, C.; DeLuca, H. F.;
Ross, T. F.; Shih, C.C.-Y. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5946. Compounds
were tested in this whole cell functional assay for their ability to antagonize the
effects of 1 nM DHT on the androgen receptor. The AR antagonist RU-58841
was run as a standard revealing an inter-run variability within twofold and an
average IC50 of 32 nM.
was evenly applied to ꢀ3 cm2 of the ventral surfaces of both the right and left
ears. Animals were sacrificed approximately 18–24 h after the final dose. The
ears were collected from each animal for sebum analysis. The ear samples were
prepared for sebum analysis as follows. One 8 mm distal biopsy punch was
taken just above the anatomical ‘V’ mark in the aural cartilage to normalize
sample area. The punch was then split into ventral and dorsal layers. The
ventral layer, where the topical dose was applied, was retained for sebum
analysis. Each ventral side sample was placed in a 1-dram glass vial, flushed
with nitrogen gas (N2), sealed, and stored at ꢁ80 °C until for sample lipid
extraction and HPLC lipid analysis. See: Plewig, G.; Luderschmidt, C. J. invest.
Dermatol. 1977, 68, 171.
8. (a) Koch, H. Drugs Today 1984, 20, 561; (b) Fradet, Y. Exp. Rev. Anticancer Ther.
2004, 4, 37.
9. Generalized synthetic procedure: To
a
stirring solution of the 4-
fluorobenzonitrile template (1 mmol) and phenolic derivative (1.5 equiv) in
DMF (10 mL) was added solid K2CO3 (2 equiv). The reaction mixture was
13. Bodor, N.; Buchwald, P. Mol. Biotechnol. 2004, 26, 123.