Small-molecule androgen receptor downregulators as an approach to treatment of advanced prostate cancer

Article abstract of DOI:10.1016/j.bmcl.2011.06.122

Chemical starting points were investigated for downregulation of the androgen receptor as an approach to treatment of advanced prostate cancer. Although prototypic steroidal downregulators such as 6a designed for intramuscular administration showed insuff

Full text of DOI:10.1016/j.bmcl.2011.06.122

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5442–5445
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Small-molecule androgen receptor downregulators as an approach to treatment
of advanced prostate cancer
,
⇑
Robert H. Bradbury , Neil J. Hales , Alfred A. Rabow , Graeme E. Walker , David G. Acton,
David M. Andrews, Peter Ballard, Nigel A. N. Brooks, Nicola Colclough, Alan Girdwood,
Urs J. Hancox, Owen Jones, David Jude, Sarah A. Loddick, Andrew A. Mortlock
Oncology iMed, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical starting points were investigated for downregulation of the androgen receptor as an approach
to treatment of advanced prostate cancer. Although prototypic steroidal downregulators such as 6a
designed for intramuscular administration showed insufficient cellular potency, a medicinal chemistry
program derived from a novel androgen receptor ligand 8a led to 6-[4-(4-cyanobenzyl)piperazin-1-yl]-
3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (10b), for which high plasma levels following oral
administration in a preclinical model compensate for moderate cellular potency.
Received 31 May 2011
Revised 27 June 2011
Accepted 28 June 2011
Available online 2 July 2011
Keywords:
Prostate cancer
Ó 2011 Elsevier Ltd. All rights reserved.
Androgen receptor downregulator
Androgen antagonist
Prostate cancer is the second leading cause of death from cancer
among men in developed countries, and was projected to account
for 28% of newly-diagnosed cases and 11% of deaths due to cancer
in the USA in 2010.1 The androgen receptor (AR), a ligand binding
transcription factor in the nuclear hormone receptor super family,
is a key molecular target in the etiology and progression of prostate
cancer.2–4 Binding of the endogenous AR ligand dihydrotestoster-
one (DHT, 1) (Fig. 1) stabilizes and protects the AR from rapid pro-
teolytic degradation. The early stages of prostate cancer tumor
growth are androgen dependent and respond well to androgen
ablation,2–4 either via surgical castration or by chemical castration
with a luteinizing hormone releasing hormone agonist in combina-
tion with an AR antagonist, such as hydroxyflutamide (2a) or bica-
lutamide (2b).
Although introduction of androgen deprivation therapy repre-
sented a major advance in prostate cancer treatment, recurrence
within 1–2 years typically marks transition to the so-called cas-
trate-resistant state, in which the tumor continues to grow in the
presence of low circulating endogenous ligand and is no longer
responsive to classical AR antagonists.2–4 Castrate-resistant pros-
tate cancer is a largely unmet medical need with a 5 year survival
rate of less than 15%, and docetaxel is currently the only treatment
shown to provide even minimal survival benefit.5
a majority of castrate-resistant prostate tumors.3,4 It is also well
established that the functional AR in castrate-resistant tumors is
frequently mutated or amplified, and that over-expression can
convert hormone responsive cell lines to hormone refractory.2–4
Recent second-generation AR antagonists have been designed that
retain antagonism in over-expressing cell lines, and among these
agents MDV3100 has now progressed to late-stage clinical trials
in patients with advanced prostate cancer.6–8
By analogy with fulvestrant (3),9 an estrogen receptor (ER)
downregulator approved by the FDA in 2002 for treatment of
advanced breast cancer and initially characterized as a pure ER
antagonist, a ligand which downregulates the AR represents one
OH
OH
H
N
R
H
H
O
H
H
X
O
2a X = NO2, R = H
2b X = CN, R =
1
CF3
H
O
S
O
OH
F
3
O
S
F
F
Recent evidence from both preclinical and clinical studies is
consistent with the importance of reactivation of AR signaling in
H
H
F
F
HO
F
⇑
Corresponding author. Tel.: +44 (0) 1625 515708.
E-mail address: rob.bradbury@astrazeneca.com (R.H. Bradbury).
These authors contributed equally.
Figure 1. Chemical structures of DHT (1, endogenous AR ligand), hydroxyflutamide
(2a) and bicalutamide (2b) (AR antagonists) and fulvestrant (3, ER receptor
antagonist and downregulator).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.122

R. H. Bradbury et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5442–5445
5443
of a number of potential approaches3,4 to treatment of hormone-
OAc
OAc
refractory prostate cancer via a sustained reduction in tumor AR
content. In contrast with direct intervention at the AR, several of
these approaches involve indirect mechanisms such as HDAC or
HSP90 inhibition.3,4 At the outset of our work, we were aware of
publications and patent applications describing steroidal10,11 and
non-steroidal12 derivatives as AR destabilizers or downregulators,
but to our knowledge no definitive data have been published that
characterize the mode of action of these compounds. More re-
cently, androgen receptor inactivation has been shown to contrib-
ute to the antitumor efficacy of steroidal derivative VN/124-1, a
potent inhibitor of steroidal biosynthesis.13
R
R
H
H
4a R = H
4b R = Me
H
H
H
H
O
O
(a), (b)
5a R = H
O
S
F
F
5b R = Me
F
F
F
(c)
OH
R
H
6a R = H
Fulvestrant is a highly potent downregulator of the ER, formally
derived from attachment of a long side chain containing a terminal
pentafluoropentyl sulfoxide at the 7a-position of the endogenous
O
S
F
F
6b R = Me
H
H
F
O
F
F
ER ligand estradiol. The low oral bioavailability and high presys-
temic metabolism of fulvestrant preclude conventional routes of
administration, and a long-acting intramuscular depot formulation
was developed that provides the sustained exposure required for
clinical efficacy.14
Scheme 1. Synthesis of compounds 6a–b. Reagents and conditions: (a)
Br(CH2)9S(CH2)3CF2CF3,17 Mg, CuCl, THF, À30 °C; (b) NaIO4, MeOH/H2O, 20 °C; (c)
NaOH, MeOH/H2O, 20 °C.
In anticipation that AR downregulators of comparable potency
to fulvestrant could be identified, we initially sought a steroidal
derivative that would be suitable for intramuscular administration.
In this letter we describe how the modest downregulatory potency
of prototype steroidal compounds led us to seek a chemical start-
ing point commensurate with high oral exposure in order to com-
pensate for compromised potency.
X
Cl
(a)
N
N
F
N
F
N
N
N
N
N
N
F
8a X = CH2
8b X = NCO2Bu-t
8c X = NH
F
7
F
F
(b)
Illustrative compounds prepared during the course of this work
are listed in Table 1, and synthetic routes are outlined in Schemes 1
and 2.15 Prototype steroidal compounds 6a–b derived from the po-
tent AR ligands nortestosterone16 and testosterone were obtained
as outlined in Scheme 1, via a sequence involving as the key step
copper-catalyzed conjugate 1,6-addition to precursor dienones
4a–b of the Grignard reagent derived from the side-chain building
block used in the process synthesis of fulvestrant.17 Oxidation and
deprotection of the intermediate thioethers then gave 6a–b.
Novel AR binder 8a and the corresponding piperazine derivative
8c were obtained (Scheme 2) from 6-chloro-3-(trifluoro-
methyl)[1,2,4]triazolo[4,3-b]pyridazine 7.18 As part of the synthe-
sis of wider compound libraries, AR downregulators 9a–d and
10a–b were also obtained from 7 by direct displacement with
the appropriate secondary amine.
Binding of compounds listed in Table 1 to the isolated ligand-
binding domain of recombinant rat AR was determined using a
fluorescence polarization assay.15,19 Also included as standards in
Table 1 are functionally active AR ligands DHT and bicalutamide.
Central to evaluation of compounds listed in Table 1 as AR down-
regulators was development of a novel and innovative microtitre
plate-based mode of action assay.15 By use of a fluorescent goat
(d)
(c)
N
X
Y
N
N
R
N
N
F
N
N
N
N
N
N
F
F
F
F
F
9a X-Y = CHCH2
9b X-Y = CH (R-enantiomer)
9c X-Y = CH (S-enantiomer)
9d X-Y = NCH2
10a R = H
10b R = CN
Scheme 2. Synthesis of compounds 8a, c, 9a–d, 10a–b. Reagents and conditions:
(a) Piperidine or t-butyl piperazine-1-carboxylate, DIPEA, EtOH, 70 °C; (b) TFA,
DCM, 20 °C; (c) 4-Phenylpiperidine, R or S 3-phenylpyrrolidine or 1-phenylpiper-
azine, DIPEA, EtOH, 70 °C; (d) N-Benzyl piperazine or N-(4-cyanobenzyl)piperazine,
DIPEA, DMF, 70 °C.
anti-mouse IgG secondary antibody to detect the immunoreactivity
of a mouse anti-human AR monoclonal antibody (clone 441) that is
specific for nuclear AR,20 levels of AR in human LNCaP prostate can-
cer cells in response to compound were quantified by immuno-
fluorescence detected by a TTP Acumen Explorer HTS Reader.
In contrast with binding of fulvestrant to the ER,9 prototypic
steroidal derivatives 6a–b were significantly weaker AR binders
than the endogenous ligand DHT, and these compounds were only
moderately potent AR downregulators in LNCaP cells. As men-
tioned later for compound 10b, more detailed studies with 6a were
consistent with a mechanism of action involving downregulation
of the AR. We anticipated that variation of the length and fluorina-
Table 1
AR binding15,19 and cellular downregulation15 data for 1, 2b, 6a–b, 8a, 8c, 9a–d,
10a–b
a
a
Entry
AR binding pIC50
AR downregulation pIC50
1 (DHT)
2b (Bicalutamide)
6a
6b
8a
8.39
6.55
5.1
<3.5
<3.5
6.02
5.66
<3.5
<4.3
5.95
6.22
<4.5
5.36
5.72
5.82
4.8
6.20
<4.1
4.9
4.48
4.88
<4.2
<4.2
<4.1
tion of the pendant 7a-sulfoxide substituent might lead to a signif-
8c
9a
icant improvement in downregulatory potency, but an initial set of
approximately 20 compounds derived from testosterone and nor-
testosterone gave flat cellular SAR (data not shown).
Before progression to anti-tumor models, efficacy in vivo was
assessed using the Hershberger assay,21 a longstanding model used
in the discovery of the AR antagonist bicalutamide,22 in which
effects on accessory sex organ weight in immature castrated
9b
9c
9d
10a
10b
a
n P 3, SEM values are available in the Supplementary data.

5444
R. H. Bradbury et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5442–5445
rats stimulated with testosterone propionate serve as a marker for
intervention via the AR. On intramuscular administration for
4 days at the highest dose compatible with formulation
To explore the effect on AR downregulation, libraries compris-
ing several hundred compounds in total were synthesized, either
from 6-chloro-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine
7 and available cyclic secondary amines, or via reductive amination
of piperazine 8c (data not shown). Appending a phenyl substituent
in the piperidine ring (9a) reduced AR binding but gave moderately
potent downregulation. A significant difference in downregulation
potency was seen for the corresponding pair of enantiomeric phe-
nyl pyrrolidine dervatives (9b–c), implying the directionality of the
aryl group is crucial for the interaction that leads to downregula-
tion of the AR.
(82
model in which bicalutamide (4.6
control, an observation consistent with analysis of plasma samples
that showed a mean steady state concentration of 6a (0.4 M) be-
low the cellular downregulatory IC50
By way of comparison, the ER downregulator fulvestrant is typ-
ically active in preclinical rodent models at doses of 0.4–4 mol kg/
day,23 and monthly 412
mol intramuscular depot administration
lmol/kg/day), compound 6a gave no response in a Hershberger
lmol/kg) was used as positive
l
.
l
l
in breast cancer patients gives sustained exposure that exceeds the
minimum efficacious concentration for 28 days.14 We concluded
that the cellular potency of compound 6a was approximately 2–3
orders of magnitude lower than required for intramuscular admin-
istration to patients, and that flat SAR implied the feasibility of
attaining the required potency was low.
Although bicalutamide is only a moderately potent AR binder
and antagonist, high plasma levels following oral administration
in preclinical models and patients compensate for modest
potency.24 We therefore initiated a new medicinal chemistry pro-
gram to seek downregulators of comparable potency to prototype
6a, but with ADME properties commensurate with high exposure
after oral dosing.
In order to identify novel, drug-like AR binding cores, a directed
high-throughput screening set was generated. A computational
approach was used that focused on covering a drug-like productive
subspace with low complexity compounds identified via a two-
dimensional pharmacophore model chosen to correspond to
known AR ligands such as DHT and bicalutamide, in which a termi-
nal hydrogen bond acceptor is located in or adjacent to a ring.
A hit rate of 1.7% was obtained25 when the resulting set of
Although the unsubstituted piperazine (8c) showed no detect-
able AR binding, appending a phenyl substituent (9d) again con-
verted the motif to a downregulator. From a further synthesis
iteration, benzyl piperazine derivatives (10a–b) were obtained that
are comparable in downregulatory potency to prototype steroid
6a. Although binding of 10a–b could not be detected in the fluores-
cence polarization assay, data determined from an assay using the
full length AR binding domain27 were consistent with weak but
reproducible binding (66% inhibition at 10 lM concentration for
10b). Data from human PC3 cell reporter assays28 showed that
whereas the novel AR binder 8a is a functional AR agonist (pIC50
5.0), downregulators such as 10b are devoid of agonist activity. A
proposed binding mode for 10b is similar to that of 8a (Fig. 2),
but with the cyanobenzyl moiety orientated towards the Helix
12 region of the AR ligand binding domain, an interaction that is
believed to play a key role in modulating receptor function.26,29
Compound 10b showed low clearance and high oral bioavail-
ability in a low dose rat pharmacokinetic study (Table 2). Exposure
scaled well to higher doses and the compound binds only moder-
ately to plasma proteins. As illustrated in Figure 3, compound
10b dosed orally at 258 lmol/kg twice daily in the Hershberger
100,000 compounds was evaluated at a concentration of 10
lM
model for 7 days caused a significant inhibition of testosterone-in-
in a directed high-throughput screen that measured affinity for
the rat AR-ligand binding domain by fluorescence polarization.15,19
Determination of IC50 values identified 6-(1-piperidinyl)-3-(tri-
fluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazine 8a as a novel and
moderately potent AR binder (pIC50 6.2) that appeared an attrac-
tive chemical starting point (MWt 271, measured LogD 3.0). Excel-
lent selectivity was seen versus a core panel of nuclear hormone
duced growth of rat seminal vesicles, the magnitude of effect being
comparable to that seen with bicalutamide dosed at 4.6 lmol/kg.
Analysis of plasma samples 18 h subsequent to administration
of the final dose of 10b in the Hershberger model showed a con-
centration of compound 10b (17 lM) that significantly exceeds
the steady state exposure seen after intramuscular administration
of prototypic steroidal downregulator 6a, and corresponds to a ter-
minal free plasma concentration of 10b that is comparable to the
IC50 for AR downregulation. Pharmacological studies confirming
the mechanism of action of compound 10b will be detailed in a fu-
ture publication, along with data from rodent tumor models of hu-
man prostate cancer. We believe that although compounds like
10b bind only weakly to the AR, the binding interaction promotes
modulation of AR levels.
receptor binding assays (pIC50 <4 vs ERa, PR and GR).
A plausible binding mode for novel AR binder 8a is obtained by
overlaying the hydrogen bond acceptor of the fused 1,2,4-triazole
moiety and the trifluoromethyl substituent with the corresponding
nitro and trifluoromethyl groups in the published structure of
hydroxyflutamide 2a bound to the AR (Fig. 2).26
In summary, we have investigated chemical starting points for
downregulation of the AR, an approach to treatment of advanced
prostate cancer analogous to the clinically precedented use of the
ER downregulator fulvestrant to combat advanced breast cancer.
Prototypic steroidal downregulators such as 6a designed for intra-
muscular administration showed insufficient cellular potency.
However, a medicinal chemistry program derived from a novel AR
ligand led to 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoro-
methyl)[1,2,4]triazolo[4,3-b]pyridazine (10b), in which high plas-
ma levels following oral administration in the Hershberger in vivo
model compensate for moderate cellular potency. Further work
leading to a clinical candidate30 will be described in due course.
Table 2
Rat pharmacokinetic parameters for 10ba
Species
Rata
% Free
9.2
Vdss l/kg
2.4
Cl ml/min/kg
9.0
Bioavailability %
100
Figure 2. Stereo-diagram of potential binding mode for compound 8a (thick orange
lines) superimposed on reference structure of hydroxyflutamide 2a bound to the AR
ligand binding domain (thin black lines, pdb code 2ax626). Potential hydrogen
bonds are shown in dotted magenta lines.
a
Male Alderley Park Han Wistar rats dosed at 4 lmol/kg i.v. and 10 lmol/kg p.o.

R. H. Bradbury et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5442–5445
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lmol/kg po bid
l
Acknowledgements
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