Small Molecule Antagonists of the Nuclear Androgen Receptor for the Treatment of Castration-Resistant Prostate Cancer

Article abstract of DOI:10.1021/acsmedchemlett.6b00186

After a high-throughput screening campaign identified thioether 1 as an antagonist of the nuclear androgen receptor, a zone model was developed for structure-activity relationship (SAR) purposes and analogues were synthesized and evaluated in a cell-based luciferase assay. A novel thioether isostere, cyclopropane (1S,2R)-27, showed the desired increased potency and structural properties (stereospecific SAR response, absence of a readily oxidized sulfur atom, low molecular weight, reduced number of flexible bonds and polar surface area, and drug-likeness score) in the prostate-specific antigen luciferase assay in C4-2-PSA-rl cells to qualify as a new lead structure for prostate cancer drug development.

Full text of DOI:10.1021/acsmedchemlett.6b00186

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Letter
pubs.acs.org/acsmedchemlett
Small Molecule Antagonists of the Nuclear Androgen Receptor for
the Treatment of Castration-Resistant Prostate Cancer
James K. Johnson,† Erin M. Skoda,† Jianhua Zhou,§ Erica Parrinello,§ Dan Wang,§ Katherine O’Malley,§
Benjamin R. Eyer,† Mustafa Kazancioglu,† Kurtis Eisermann,§ Paul A. Johnston,# Joel B. Nelson,§
Zhou Wang,*,§ and Peter Wipf*,†,#
†Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
§Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15232, United States
#Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, United States
S
* Supporting Information
ABSTRACT: After a high-throughput screening campaign
identified thioether 1 as an antagonist of the nuclear androgen
receptor, a zone model was developed for structure−activity
relationship (SAR) purposes and analogues were synthesized
and evaluated in a cell-based luciferase assay. A novel thioether
isostere, cyclopropane (1S,2R)-27, showed the desired
increased potency and structural properties (stereospecific
SAR response, absence of a readily oxidized sulfur atom, low
molecular weight, reduced number of flexible bonds and polar
surface area, and drug-likeness score) in the prostate-specific
antigen luciferase assay in C4-2-PSA-rl cells to qualify as a new lead structure for prostate cancer drug development.
KEYWORDS: Androgen receptor, CRPC, advanced prostate cancer, luciferase assay, isoxazoles, thioether isostere
he steroidal hormones testosterone and dihydrotestoster-
Tone are the major endogenous androgens that cause
nuclear translocation and subsequent activation of androgen
receptor (AR).1 In prostate cancer, AR shows a higher nuclear
concentration in the presence of androgens,2,3 and androgen-
deprivation therapy (ADT) is one of the primary treatments.4
Figure 1. Structures of clinically used AR antagonists enzalutamide
Unfortunately, even with ADT, almost all patients eventually
progress to the stage of castration-resistant prostate cancer
(CRPC, formerly known as hormone-refractory prostate
cancer), a fatal condition that makes prostate cancer the
second most deadly cancer type in men in the U.S.5 Despite a
high survival rate with early detection and treatment with
surgery or radiation, prostate cancer is responsible for the death
of 30,000 patients each year in the U.S.5,6
CRPC is postulated to arise through either adaption or
selection of cancer cells in a low androgen environment7 as a
result of the initial ADT.4,8 In the laboratory setting, studies of
overexpression9 and knockdown10 of AR have shown that this
receptor plays a key role in the progression of CRPC.11,12
Enzalutamide (MDV3100) and bicalutamide are AR antago-
nists that are currently used as treatments for CRPC and can
extend the lifespan of patients for 3−5 months (Figure 1).
Enzalutamide, in particular, attenuates nuclear translocation of
AR but does not seem to reduce nuclear levels of AR in
prostate cancer cells.13 Since these therapeutics are only
partially effective, there is a definite need for new regimens
that extend life expectation beyond several months.14
Unfortunately, there are no known therapies that decisively
and bicalutamide.
inhibit nuclear localized AR in CRPC cells.15−22 Herein, we
investigate a novel series of small molecules identified by their
ability to reduce the nuclear level of AR and, subsequently, AR
activity.
Prior to the onset of our medicinal chemistry efforts, a high-
throughput screening (HTS) campaign for antagonists of AR
nuclear localization identified compounds 1 and 2 that also
reduced levels of prostate-specific antigen (PSA), a key marker
for CRPC, in a PSA luciferase reporter assay performed in
CRPC cell lines (Figure 2).23 Both HTS hits demonstrated low
micromolar potency with little to no cytotoxicity or activity in
AR negative cell lines. Close structural analogues of 3-phenyl-
6,7-dihydro-5-pyrrolo[1,2-a]imidazole (2) were previously
found to have antifungal effects, which raised off-target
concerns.24 In contrast, 2-((isoxazol-4-ylmethyl)thio)-1-(4-
phenylpiperazin-1-yl)ethanone (1) had not yet been bio-
Received: May 2, 2016
Accepted: May 27, 2016
© XXXX American Chemical Society
A
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Table 1. Structures of Amine Building Blocks 4 and
Analogues 5, 7−11, and 16 (Schemes 1 and 2)
Figure 2. HTS hits that reduced PSA levels in a luciferase assay in
CRPC cell lines.23
logically annotated, and this structural novelty led us to
prioritize this scaffold over 2. In an effort to determine a
structure−activity relationship and identify more potent
antagonists of CRPC, we designed and synthesized analogues
of 1 in a series of structural modifications of subunits 1−5
(Figure 3).
Figure 3. Zones of planned structural modifications of 1.
Our goal was to probe five key moieties in compound 1: the
benzene substitution pattern (zone 1), modifications at the
piperazine (zone 2), carbonyl replacements (zone 3), a sulfur-
atom exchange in the 3-atom linker and the use of less flexible
linkers (zone 4), and variations of the 3,5-dimethylisoxazole
(3,5-DMI) ring (zone 5) (Figure 3).
In the synthesis of zone 1−3 analogues, we used the amide
bond as the lynchpin disconnection. Compounds 5a−h were
synthesized directly from commercially available carboxylic acid
3a and N-arylated piperazines 4a−h under amide coupling
conditions with T3P (Scheme 1 and Table 1).25 We also
examined the diamine linker in zone 2 in more detail through
the synthesis of analogues 5i−5m. For these target molecules,
the requisite diamines 4i−m were prepared by a Buchwald−
Hartwig cross-coupling of mono-Boc-protected diamines with
bromoarenes.26,27 Reduction of amide 5b with lithium
aluminum hydride led to diamine 6. For an initial set of zone
Scheme 1. Synthesis of DMI-Containing Analogues 5, 6, 12,
a
and 13
4 analogues, thioether 5b was also oxidized to sulfoxide 12 and
sulfone 13 in good yields with sodium periodate and m-
chloroperbenzoate, respectively (Scheme 1).
Additional zone 4 and zone 5 analogues with a phenyl group
in place of the isoxazole ring were obtained from carboxylic
acids 3b−3g (Scheme 2 and Table 1). Coupling to piperazine
4b provided amides 7−11 and 16 in high yields. Alkynyl amide
10 was further hydrogenated to cis-alkene 14 using a Lindlar
a
Reagents and conditions: (a) T3P, Et3N, CH2Cl2, rt, overnight, 52−
98%; (b) LiAlH4, dry THF, 0 °C, 1 h, 42%; (c) NaIO4, MeOH, H2O,
rt, 15 h, 68%; (d) m-CPBA, CH2Cl2, rt, 15 h, 44%.
B
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ACS Medicinal Chemistry Letters
Letter
a
a
Scheme 2. Synthesis of Piperazines 7−11 and 14−16
Scheme 4. Synthesis of Bridged Analogues 26a and 26b
a
Reagents and conditions: (a) Boc2O, DMAP, CH2Cl2, rt, overnight,
a
78%; (b) NaHMDS, PhNTf2, THF, −78 °C to rt, 4 h, 78%; (c)
Pd(PPh3)4, LiCl, Na2CO3, (2-Me)PhB(OH)2, DME, H2O, 60 °C, 3 h,
78%; (d) H2, Pd/C, EtOH, rt, 14 h, 90%; (e) TFA, CH2Cl2, rt, 16 h,
quant.; (f) 2-chloroacetyl chloride, Et3N, THF, rt, 22 h, 79%; (g) 25,
NaH, THF, rt, 1 d, 30%.
Reagents and conditions: (a) T3P, Et3N, CH2Cl2, rt, overnight, 62−
96%; (b) Lindlar’s catalyst, quinoline, H2, EtOAc, quant.; (c) CrCl2,
CH2ICl, THF, reflux, overnight, 57%.
catalyst. The cis-cyclopropane 15 was prepared by a Simmons−
Smith cyclopropanation of cis-alkene 14,28 whereas the trans-
cyclopropane 16 was obtained by coupling of commercially
available trans-2-phenylcyclopropanecarboxylic acid 3g with
piperazine 4b.
Further modifications in zones 3−4 were accomplished by
acylation of piperazine 4b with either 2-chloroacetyl chloride or
chloromethanesulfonyl chloride to form the corresponding
amide 17a or sulfonamide 17b in good yields (Scheme 3). SN2
phenyltriflimide provided vinyl triflate 22 in good yield. A
Suzuki coupling was used to install the o-tolyl group, and the
styrene double bond was reduced with Pd/C to afford 23 as a
mixture of diastereomers. Without separation, this mixture was
deprotected and acylated with α-chloroacetyl chloride. Finally,
the chloride was displaced using thiol 25 and sodium hydride to
afford the thioether. Diastereomers 26a and 26b were separated
by chromatography on SiO2 to afford both analogues in modest
yields.
Scheme 3. Alkylation of 17a and 17b To Give Analogues
The biological activity of analogs 5−16, 18, 20, and 26 was
determined and compared to HTS hit 1 (EC50 7.3 μM) and
enzalutamide (EC50 1.1 μM) using the Dual-Glo luciferase
system (Promega, WI, USA) in the presence of 1 nM synthetic
androgen R1881 in C4-2-PSA-rl cells, which were generated by
stable cotransfection of C4-2 cells with a PSA promoter driven
luciferase reporter vector (pPSA6.1) and a Renilla luciferase
reporter vector as a control. Relative luciferase activity was
calculated as the quotient of androgen-induced PSA-firefly/
Renilla luciferase activity. Since PSA promoter activity
correlates to AR transcriptional activity, inhibition of AR will
result in decreased PSA-luciferase activity. EC50 values were
calculated using graphpad prism, and data represent the mean
and SD of 2−6 independent experiments (Table 2). To verify
that these compounds did not have undesirable electrophilic
properties, their stability was tested in the presence of thiols.
Neither thiophenol in CDCl3 nor 2-mercaptoethanol in PBS
18a−18c and Conversion of Isocyanate 19 To Give
a
Thioethers 20a/b
1
resulted in any trapping products by H NMR and LCMS
analysis.
Simple modifications of the substituents on the benzene ring
in zone 1 revealed that methyl groups in the 3- and 4-positions
(5c, 5d) led to loss of activity, while the 2-methyl analogue 5b
(EC50 14.5 μM) retained about half of the activity of the 2,3-
dimethylated 1 (Table 2). Removal of the 2-methyl group in 5a
deleted activity. In agreement with this trend in zone 1, the
bulky 1-naphthyl substituent (5g) recovered activity (EC50 11.1
μM). Analogues with electron-withdrawing substituents at the
benzene 2-position (2-NC, 5e, and 2-F, 5f) also maintained or
slightly increased activity (EC50 12−13 μM); however, the
electron-donating 2-methoxy substituted 5h was not tolerated
and resulted in a complete loss of activity, possibly due to an
increase in the pKa of the aniline and/or an unfavorable
increase in the π-electron density of the aromatic ring.30 To
potentially reduce the expected rapid metabolism of benzylic
methyl groups by cytochrome P450 enzymes,31 we selected the
minimally required substitution in zone 1, e.g., the 2-methyl
a
Reagents and conditions: (a) 2-chloroacetyl chloride, Et3N, CH2Cl2,
rt, overnight, 99%; (b) chloromethanesulfonyl chloride, Et3N, CH2Cl2,
rt, overnight, 85%; (c) NaH, THF, rt, 1−2 d, 4−99%; (d) DPPA,
Et3N, toluene, reflux, overnight, 17−65%.
reaction of 17a and 17b led to ether 18a, amine 18b, and
thioether 18c. Starting with carboxylic acid 3a, urea 20a and
carbamate 20b were obtained in moderate yields via a Curtius
rearrangement and addition of the intermediate isocyanate 19
to amine 4b and alcohol 4n, respectively (Scheme 3).29
A bridged bicyclic ring was introduced to add a strong
conformational constraint in zone 2 (Scheme 4). Boc-
protection of nortropinone hydrochloride 21 followed by
enolization with NaHMDS and trapping of the enolate with N-
C
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ACS Medicinal Chemistry Letters
Letter
an EC50 of 2.9 μM (Table 2). More significantly, chiral
resolution of the bis-halogenated cyclopropane 27 (EC50 2.7
μM, entry 34) provided a more potent enantiomer (1S,2R)-27
(EC50 1.7 μM, entry 35) and the ca. 10-fold less potent
(1R,2S)-27 (EC50 15.2 μM, entry 36), and supporting specific
contact of this scaffold at a still to be defined AR binding site
(Figure 4).
Table 2. In Vitro Activity of Analogues in the PSA Luciferase
Assay in C4-2-PSA-rl Cells
entry compd
EC50 (μM)
entry
compd
EC50 (μM)
c
a
1
1
7.3 2.5
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
10
11
12
13
14
15
16
20.3 11.6
a
a
2
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
6
>25
>25
b
b
3
14.5 3.2
>25
a
b
4
>25
16.1 3.3
a
a
5
>25
12.7 0.8
b
b
6
12.0 1.6
2.9 1.0
b
b
7
12.6 7.7
>25
b
b
8
11.1 5.3
18a
>25
a
b
9
>25
18b
>25
b
c
10
11
12
13
14
15
16
17
18
18.4 9.2
18c
7.2 2.7
a
a
11.1 3.3
20a
>25
a
c
3.1 1.1
20b
>25
a
b
Figure 4. More potent enantiomer of cyclopropane 27.
14.7 4.4
26a
7.7 1.6
b
a
16.6 4.8
26b
7.9 2.8
b
e
10.8 5.7
enzalutamide
27
1.1 0.5
In summary, 35 analogues were synthesized, and the
resulting SAR evaluated 5 zones of modification in the starting
hit, compound 1. We discovered several attributes that proved
essential for activity. Zone 1 modifications showed that the
ortho-substituent on the phenyl ring was important for activity.
In zone 2, the sterically encumbered 2,6-dimethylpiperazine
proved superior to flexible, unsubstituted, and bridged
analogues. In zone 3, a carbonyl group was not required, and
a sulfonamide and even the reduced amine were well tolerated.
In zone 4, thioether oxidation reduced activity, and only the cis-
cyclopropane significantly improved the EC50. Limited
substitutions were performed in zone 5, but in general,
analogues with a phenyl group were equipotent with their
3,5-dimethylisoxazole congeners (see, for example, 7 vs 5b).
The cis-cyclopropane (1S,2R)-27 was found to be substantially
equipotent to the commercial AR antagonist, enzalutamide.
Compound (1S,2R)-27 is of particular interest in comparison
to 1 due to the isosteric replacement of the thioether linker
with the metabolically more stable cyclopropane, a reduction of
the topological polar surface area (TPSA) from 49.6 to 23.6 Å2,
a reduction of the number of rotatable bonds from 3 to 2, and
an improvement in the drug-likeness score from 6.3 to 8.0.33
Further modifications of lead structure 27 based on these SAR
results as well as in vivo tumor xenograft data will be reported in
due course.
b
d
7
13.7 0.8
2.7 1.1
b
a
8
14.4 3.7
(1S,2R)-27
(1R,2S)-27
1.7 + 0.2
a
a
9
>25
36
15.2 3.3
a
b
c
d
e
Assay repeats. n = 2. n = 3. n = 4. n = 5. n = 6. For assay
description and complete structural information, please see the
Supporting Information and Table S1.
group, for further structure−activity relationship (SAR)
investigations.
The piperazine core (zone 2) was queried through
substitutions with flexible as well as constrained acyclic and
cyclic diamines. The flexible N,N′-dimethylethylenediamine
linker in 5i (EC50 18.4 μM) and the 7-membered diazepane 5j
(EC50 11.1 μM) both dropped off in activity. The dimethylated
piperazines 5l and 5m (EC50 15−17 μM) were also less active
than the initial hit. In contrast, the conformationally more
highly constraint 2,6-dimethylpiperazine 5k was more active
with an EC50 of 3.1 μM. Installment of an ethylene bridge and a
carbon-linked (2-Me)Ph group decreased activity again since
both diastereomers of the bicyclo[3.2.1] ring systems 26a and
26b showed an EC50 of 8 μM.
Reduction of amide 5b to amine 6 resulted in a 1.3-fold
increase in activity to an EC50 of 10.8 μM. Sulfonamide 18c
(EC50 7.2 μM) was as active as the initial hit 1, but urea 20a
and carbamate 20b were inactive.
The replacement of the thioether linkage in zone 2 with an
ether group abolished activity in 18a. Substituting the thioether
with the N-methylated amine in 18b also abolished activity. In
contrast, in an analogous system with a phenyl group in place of
the isoxazole, both thioether 7 as well as the all-carbon chain
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00186.
■
S
containing 8 showed decreased yet consistent activity (EC50
14 μM).
≈
Methods for all assays, cell cultures, treatment con-
ditions, and compound synthesis (PDF)
In order to verify that the biological effect in the thioether
series was not a result of S-oxidation in the cellular assay,
common products of thioether oxidation, i.e., sulfoxide 12 and
sulfone 13, were tested. While sulfone 13 retained some activity
(EC50 16.1 μM), sulfoxide 12 was inactive. Shortening the
three-atom chain to afford the two-atom thioether-linked 9 also
abolished activity. The rigidified alkyne 10 and the correspond-
ing (E)-alkene 11 and its cyclopropane isostere 16 were also
found to be essentially inactive. In contrast, we were pleasantly
surprised to find that the (Z)-alkene 14 showed an EC50 of 12.7
μM and that the corresponding cis-fused cyclopropane
isostere32 15 was even more potent than analogue 1, showing
AUTHOR INFORMATION
Corresponding Authors
*E-mail: wangz2@upmc.edu (Z.W.).
*E-mail: pwipf@pitt.edu (P.W.).
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
D
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ACS Medicinal Chemistry Letters
Letter
pyrrolidine derivatives as novel androgen receptor antagonists. Bioorg.
Med. Chem. 2013, 21, 70−83.
ACKNOWLEDGMENTS
■
This research was supported by the Department of Urology,
University of Pittsburgh and the Johnson & Johnson Corporate
Office of Science and Technology (COSAT) − University of
Pittsburgh Translational Innovation Partnership Program. The
authors thank Ms. Taber Lewis (University of Pittsburgh) for
LCMS analyses, Dr. Marianne Sadar (University of British
Columbia) for the PSA6.1--luc plasmid, and Dr. Leland W. K.
Chung (Cedars-Sinai Medical Center) for the C4-2 cell line.
(17) Balog, A.; Rampulla, R.; Martin, G. S.; Krystek, S. R.; Attar, R.;
Dell-John, J.; Dimarco, J. D.; Fairfax, D.; Gougoutas, J.; Holst, C. L.;
Nation, A.; Rizzo, C.; Rossiter, L. M.; Schweizer, L.; Shan, W.; Spergel,
S.; Spires, T.; Cornelius, G.; Gottardis, M.; Trainor, G.; Vite, G. D.;
Salvati, M. E. Discovery of BMS-641988, a novel androgen receptor
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(18) Bradbury, R. H.; Acton, D. G.; Broadbent, N. L.; Brooks, A. N.;
Carr, G. R.; Hatter, G.; Hayter, B. R.; Hill, K. J.; Howe, N. J.; Jones, R.
D. O.; Jude, D.; Lamont, S. G.; Loddick, S. A.; Mcfarland, H. L.;
Parveen, Z.; Rabow, A. A.; Sharma-Singh, G.; Stratton, N. C.;
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ABBREVIATIONS
■
AR, androgen receptor; 3,5-DMI, 3,5-dimethylisoxazole;
CRPC, castration-resistant prostate cancer; HTS, high-
throughput screen; MW, molecular weight; PSA, prostate-
specific antigen; SAR, structure−activity relationship; SD,
standard deviation
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