Identification of Novel Steroidal Androgen Receptor Degrading Agents Inspired by Galeterone 3β-Imidazole Carbamate

Article abstract of DOI:10.1021/acsmedchemlett.6b00137

Degradation of all forms of androgen receptors (ARs) is emerging as an advantageous therapeutic paradigm for the effective treatment of prostate cancer. In continuation of our program to identify and develop improved efficacious novel small-molecule agents designed to disrupt AR signaling through enhanced AR degradation, we have designed, synthesized, and evaluated novel C-3 modified analogues of our phase 3 clinical agent, galeterone (5). Concerns of potential in vivo stability of our recently discovered more efficacious galeterone 3β-imidazole carbamate (6) led to the design and synthesis of new steroidal compounds. Two of the 11 compounds, 3β-pyridyl ether (8) and 3β-imidazole (17) with antiproliferative GI₅₀ values of 3.24 and 2.54 μM against CWR22Rv1 prostate cancer cell, are 2.75- and 3.5-fold superior to 5. In addition, compounds 8 and 17 possess improved (～4-fold) AR-V7 degrading activities. Importantly, these two compounds are expected to be metabolically stable, making them suitable for further development as new therapeutics against all forms of prostate cancer.

Full text of DOI:10.1021/acsmedchemlett.6b00137

Letter
pubs.acs.org/acsmedchemlett
Identification of Novel Steroidal Androgen Receptor Degrading
Agents Inspired by Galeterone 3β-Imidazole Carbamate
Puranik Purushottamachar,†,‡ Andrew K. Kwegyir-Afful,†,‡ Marlena S. Martin,†,‡,§
Vidya P. Ramamurthy,†,‡ Senthilmurugan Ramalingam,†,‡ and Vincent C. O. Njar*,†,‡,∥
†Department of Pharmacology, ‡Center for Biomolecular Therapeutics, and ∥Marlene Stewart Greenbaum Cancer Center, University
of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, Maryland 21201-1559, United States
S
* Supporting Information
ABSTRACT: Degradation of all forms of androgen receptors (ARs) is emerging as an advantageous therapeutic paradigm for
the effective treatment of prostate cancer. In continuation of our program to identify and develop improved efficacious novel
small-molecule agents designed to disrupt AR signaling through enhanced AR degradation, we have designed, synthesized, and
evaluated novel C-3 modified analogues of our phase 3 clinical agent, galeterone (5). Concerns of potential in vivo stability of our
recently discovered more efficacious galeterone 3β-imidazole carbamate (6) led to the design and synthesis of new steroidal
compounds. Two of the 11 compounds, 3β-pyridyl ether (8) and 3β-imidazole (17) with antiproliferative GI50 values of 3.24 and
2.54 μM against CWR22Rv1 prostate cancer cell, are 2.75- and 3.5-fold superior to 5. In addition, compounds 8 and 17 possess
improved (∼4-fold) AR-V7 degrading activities. Importantly, these two compounds are expected to be metabolically stable,
making them suitable for further development as new therapeutics against all forms of prostate cancer.
KEYWORDS: Androgen receptor (AR), splice variant AR (AR-V7), androgen receptor degrading agents (ARDAs), prostate cancer
lobally, prostate cancer (PC) is the second most
Gcommonly diagnosed human cancer in men, accounting
for ∼260,000 deaths yearly.1 Almost 80% of cases are diagnosed
as localized disease, and radiation or surgery can be curative.
However, despite current treatment options, there is still a
relapse rate of 30−60%.1
Androgens and androgen receptor (AR) play crucial roles in
the development and progression of PC.2,3 As a consequence,
for locally advanced or metastatic disease, hormonal treatment
with androgen deprivation therapy, which blocks the
production (CYP17 inhibitors: abiraterone; 1)4 and/or activity
of androgen (antiandrogens, bicalutamide; 2)5 (Figure 1), is a
standard approach for the majority of patients, but, for most
cases, the duration of response is limited to 12−24 months, and
the disease will become castration-resistant prostate cancer
(CRPC) with no treatment options.6 Approximately 85% of
CRPC patients die within 5 years and docetaxel (3) is currently
the only treatment shown to provide even minimal survival
benefit.7
Figure 1. Chemical structures of compounds 1−5 (clinical anti-PC
agents) and selective ARDAs (6−7).
AR isoforms (AR-Vs) have been identified in CRPC.11−14
Unlike full-length AR (fAR), AR-Vs lack the ligand-binding
domain (LBD) and are androgen refractory.12,14 The AR-Vs are
expressed at higher levels in various tumors and are 3- to 5-fold
more potent than f-AR in transactivating activity.15 It has
In castration-resistant environment, aberrant AR reactivation
is implicated through numerous mechanisms, which leads to
overexpression of mutated AR, AR amplification, and local
androgen synthesis.8−10 Recently, multiple alternative spliced
Received: April 1, 2016
Accepted: May 23, 2016
© XXXX American Chemical Society
A
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recently been clearly demonstrated that patients expressing
constitutively active AR-Vs do not benefit from antiandrogens
and therapies that inhibit androgen biosynthesis.16,17 In fact,
not all CRPC patients respond to novel antiandrogen
(enzalutamide; 4) or CYP17 inhibitors (1), and even those
who do subsequently relapse within a few months.18−20 Based
on these findings, we envision that effective treatment of CRPC
patients will require new drugs that can modulate all forms of
AR such as AR degrading/down-regulating agents
(ARDAs).21,22 The substantial anti-PC efficacy of phase 3
development agent galeterone (Figure 1; 5) in comparison to
abiraterone (1)23 or casodex (2)24 may be attributed to
galeterone’s AR degrading (ARD) activity.
Scheme 1. Synthesis of Androstene Derivatives (8−13, 15−
a
17)
For the development of new ARDAs, we recently showed,
using rational structure−activity relationship (SAR) studies that
modifications of the C-3 hydroxyl, unlike modifications at C-16
and C-17 of galeterone (5), yield novel analogues (e.g.,
imidazole carbamate, 6 and pyridine carboxylate, 7) with
enhanced PC antiproliferative and AR modulating (AR
antagonism and AR/AR-Vs degrading) activities.22 Mechanistic
examination suggests that compounds 5 and 6 induce
proteasomal degradation of AR/AR-V7, which involves
complex formation with E3 ligases Mdm2 and CHIP (C-
terminus of Hsp70-interacting protein) concomitant with
enhanced ubiquitination.25
In recent antitumor efficacy studies, we showed that 6 was
moderately more efficacious than galeterone (70 versus 60%
growth inhibition, respectively, compared to control) in
inhibiting the growth of CRPC CWR22Rv1 tumor xeno-
grafts.25 Although we are yet to conduct in vivo pharmacoki-
netics study of these lead ARDAs, the presence of metabolically
labile functions such as carbamate and ester groups in
compound 6 and 7, respectively, may be a matter of concern.
Therefore, the present study aimed to develop novel ARDAs
with metabolically stable chemical moieties tethered at C-3 of
androstene and estrogen steroidal cores, but with retention of
the C-17 benzimidazole moiety.
a
Reagents and conditions: (i) NaH, DMF, R-X, rt or heat, 1−12 h; (ii)
1,1′-thiocarbonyldiimidazole, CH3CN, DCM, reflux, 5 h; (iii) 1,1′-
carbonyldiimidazole, CH3CN, rt, 2 h; (iv) dry heat at 195 °C, 1 h; (v)
mesyl chloride, pyridine, ice (5 h), then rt (5 h); (vi) toluene,
imidazole, reflux, overnight.
possible elimination product (Rf value similar to 13, not
isolated) (Scheme 1). These three positional isomers were
separated by preparative HPLC (see Supporting Information
for method and chromatogram).
As an androstene core alternate, estrogen was selected, where
three derivatives, including C-3 hydroxy (22), C-3 methyl-
carboxylate (27), and C-3 carboxy (28) were designed and
synthesized (Scheme 2). Synthesis of these compounds was
achieved by slightly modifying our routine method for synthesis
for galeterone and related compounds.22
Starting from previously identified 4-pyridylester (7) with
potent ARD activity,22 we decided to keep the pyridyl ring fixed
and modify the ester-link to obtain metabolically stable
compounds. This modification also serves as probe for the
role/significance of the ester moiety toward ARD activity.
Considering the relative metabolic stability of ether function
over ester, compounds 8 and 9 were designed. Similarly, to
probe influence of the pyridyl ring toward ARD activity, the
simple methyl (10) and benzyl (11) ethers were designed
(Scheme 1).
These four C-3 ethers (8−11, Scheme 1) were prepared by
following Williamson’s etherification method of treating alcohol
(5) with respective aryl/alkyl halide in the presence of sodium
hydride as base in DMF with yields of 12, 43, 77, and 87%,
respectively. Considering the potency of 6, we also prepared
the thiocarbamate derivative 12, 54.5% yield (Scheme 1), by
refluxing 5 and 1,1′-thiocarbonyldiimidazole in acetonitrile and
dichloromethane. To probe the influence of C3 substituent the
Δ3,5-diene (13), without any C3 substituent was obtained (13%
yield) by neat pyrolysis of 622 at 195 °C (just above its mp).26
In our previous investigation, an attempt to synthesize 3β-
imidazole derivative of galeterone yielded C-3 imidazole
carbamate (6).22 An attempt to synthesize 3β-imidazole
derivative by refluxing 3β-mesyl compound 14,22 with
imidazole under anhydrous condition in toluene resulted in
compounds 15 (35%), 16 (3%), and 17 (11%) along with a
The synthesis of estrone-3-hydroxy-17-1H-benzimidazole
compound (22, Scheme 2) started with 3-acetylation of estrone
with acetic anhydride in pyridine to give 18 in 98% yield.
Compound 18 was subjected to Vilsmeier−Haack reaction by
treating with phosphorus tribromide in DMF and CHCl3 to
give 16-formy-17-bromo derivative 19 in 26% yield. An attempt
to append a benzimidazole (BzIm) to C-17 position of 19
following our routine K2CO3 in DMF method22,27 resulted into
mixture of four compounds (partial 3-deacetylation with or
without BzIm substitution, acetylated BzIm product and
substrate). Therefore, we first hydrolyzed 3-acetyl group of
19 by treating with 10% ethanolic-KOH to obtain 20 (28%).
Thereafter, the BzIm group was appended using K2CO3 in
DMF to give 21 in 84% yield and finally 16-deformylation by
refluxing with 10% Pd/C in benzonitrile to obtain desired
compound 22 in very low isolated 6% yield.
The synthesis of 17-benzimibazole estrone-3-carboxlate (28,
Scheme 2) was initiated by activation of estrone via treatment
with triflic anhydride in the presence of organic base (TEA)28
to give the corresponding triflate 23 in 37.4% yield. Treatment
of 23 with Pd-catalyzed carbonylation using Pd(OAc)2 as
catalyst, 1,c-bis(diphenylphospheno)propane as the phosphine
ligand, in the presence of gaseous carbon monoxide and
methanol in DMF29 gave the corresponding methyl carboxylate
24 in 32% yield. The remaining four steps for the synthesis of
desired 28 followed the above-described method for 22,22
B
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a
Scheme 2. Synthesis of Estrogen-3-hydroxy (22) and 3-Carboxyl Derivative (28)
a
Reagents and conditions: (i) pyridine, acetic anhydride, rt, 12 h, (ii) PBr3, DMF, CHCl3, reflux, 5 h; (iii) ethanol, 10% ethanolic-KOH, rt, 12 h; (iv)
BzIm, K2CO3, DMF, 80 °C, 1−5 h; (v) benzonitrile, 10% Pd/C, 185 °C, 12 h; (vi) triflic anhydride, TEA, DCM, 0 °C, 1 h; (vii) palladium(II)
acetate, dppp, TEA, MeOH, DMF, carbon monoxide, 0 °C, 9 h; (viii) MeOH, 10% methanolic-KOH, reflux, 2 h.
Figure 2. (A) Effects of compounds at 10 μM on dihydrotestosterone (DHT)-stimulated transcription of AR. LNCaP cells were transfected with the
AAR2 reporter construct + the Renilla luciferase reporting vector pRL-null and treated with novel compounds for 24 h in the presence of 10 nM
dihydrotestosterone (DHT). Control, baseline activity without androgen stimulation. Androgen-stimulated luciferase activity (luminescence) was
measured in a Victor 1420 plate reader. The results are presented as the fold induction (i.e., the relative luciferase activity of the treated cells divided
by that of the control) normalized to that of the Renilla luciferase activity. *, P < 0.01; #, P < 0.05 compared with DHT treated cells. (B) Inhibitory
effects of ARDAs/MDV3100 on DHT induced AR transcriptional activity in LNCaP cells. EC50 = the concentration of inhibitor (ARDAs/
MDV3100) required to inhibit the DHT-induced AR transcriptional activity by 50%. EC50 values of the compounds were determined from dose−
response curves. Points, mean of replicates from three independent experiments; bars, SE. Solid line, best-fit sigmoidal dose response (variable
slope).
involving three intermediate steps: formation of 16-formyl-17-
bromo derivative 25 (52%) by Vilsmeier−Haack reaction, then
C-17 BzIm condensation to give 26 (95%), followed by Pd
catalyzed 16-deformylation to give 27 (57%) and finally basic
hydrolysis of methyl ester group to obtain the desired
compound 28 in 93% yield.
DHT treatment for 24 h. The ability of the novel compounds
(10 μM, each) to modulate DHT-induced AR transactivation
was assessed, using enzalutamide (4) and galeterone (5) as
positive controls. Gratifyingly, nine of the 12 new compounds
tested were more potent than galeterone and compounds 12
and 17 were equipotent to enzalutamide. The order of potency
for the new analogues was 12, 17 > 8, 15 > 10 > 13 > 16 > 27
> 22 > 9, 11 > 28. Based on these results, we selected the top
four compounds and determined the effective concentrations
that caused 50% inhibition of DHT-induced AR transactivation
(EC50 values) from dose−response curves (Figure 2B).
As expected, these compounds exhibited impressive EC50
values ranging from <100 to 1650 nM. The EC50 of compound
17 (190 nM) was comparable to that of enzalutamide (180
nM), while that of compound 12 (<100 nM; determined via
extrapolation) was superior to enzalutamide.
Structural integrity of all the compounds in the study were
1
characterized and confirmed by H and 13C NMR and HRMS
spectroscopy. Purity of the all the compounds used for
biological studies are analyzed by UPLC method and are
>93% pure (see Supporting Information). Synthesis and purity
check for the compounds 6−7 and 14 was reported
previously.22
To determine whether our new compounds modulate f-AR
transcriptional activation, we performed a luciferase experiment
utilizing LNCaP cells dual-transfected with the probasin
luciferase reporter construct AAR2-luc and the Renilla luciferase
reporting vector pRL-null as we previously described22,27 and
reported in the Supporting Information. As shown in Figure
2A, luciferase expression was significantly increased after 10 nM
To determine whether our compounds induce AR
degradation, LNCaP cells were treated with each of the
compounds (5, 6, 8, 10, 12, 13, 15−17, 27, and 28) of interest
(15 μM each) for 24 h, followed by Western blotting analysis.
C
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Figure 3. (A−D) Differential effect of compounds on suppressing AR expression in LNCaP and CWR22Rv1 prostate cancer cells: Western blot
analysis of f-AR/AR-V7 expression in LNCaP or CWR22Rv1 cells treated with various compounds. Cells were exposed to individual compounds (15
μM) for 24 h, and the protein lysates were subjected to Western blot analysis. (E) EC50 values (for fAR and AR-V7) for compounds were
determined from dose−response curves following compound treatments (0 → 7.5 μM) of CWR22Rv1 cells for 72 h followed by Western blot
analysis of lysates.
As depicted in Figure 3A, B, most of the new compounds
significantly caused AR degradation in LNCaP cells, com-
parable to or better than galeterone (5). We note that two
compounds 12 and 16 were ineffective. It is also unclear why
compound 28 caused significant AR degradation when it
showed no effect on inhibition of AR transactivation as
depicted in Figure 2A.
Based on our previous reports, which show that AR-Vs drive
the progression of CRPC, we next determined the effect of our
compounds on the down-regulation of AR-V7 (also called AR-
3). As depicted in Figure 3C, we observed that compound 5
and some of our new compounds 6, 8, and 28 tested in this
assay (CWR22Rv1, prostate cancer cell line) caused significant
down-regulation AR-V7. Figure 3D shows f-AR and AR-V7
depletion caused by compounds 8 and 17. Whereas their EC50
values for f-AR are similar, compounds 6, 8, and 17 exhibited
>4-fold increased efficacy in AR-V7 degradation activity as
compared to galeterone (5) (Figure 3E).
Table 1. IC50 of Select Compounds for Inhibition of CYP17
(17α-Hydroxylase Activity) and GI50 Values of Novel ARDA
Compounds
a
b
IC50 (μM)
CYP17
>15
GI50 values (μM)
compd
LNCaP
CWR22Rv1
8
2.45
4.26
8.71
5.12
9.57
3.24
0.81
7.50
1.2
9
10
11
12
13
15
16
17
22
27
28
39
7.52
6.61
8.91
1.64
3.89
6.91
12.30
9.88
2.54
0.50
8.74
0.480
25.9
>15
for comparison
In response to some concerns of an astute reviewer, we note
the following: (i) most of the AR biology studies reported here
were conducted in LNCaP prostate cancer cells (expressing
T877A mutant AR-FL), the dominant model system in the field
that is used widely to study the general biology of AR, to screen
for novel AR antagonists;30,31 (ii) we did not conduct AR
ligand binding studies, based on our previous findings that this
class of compounds does not have to bind to the AR ligand
binding domain to cause modulation/degradation of AR. It is
important to note that this class of compounds also causes
degradation of AR splice variants that are devoid of the
LBD;22,25 (iii) for the compounds tested (see Figure 2A),
agonistic activities were not observed; (iv) we have previously
reported on the selectivity of galeterone (5) and compound 6,
where we clearly showed that these AR degrading agents do not
affect other nuclear receptors,25 which is likely to be the case
with these related new analogues.
1
4
5
0.048
0.140
4.85
3.93
8.91
a
IC50 value is the concentration of inhibitor that inhibits the CYP17
enzyme activity by 50% each in duplicate. IC50 values were each
determined from dose−response curve. The GI50 were determined
from dose−response curves (by nonlinear regression analysis using
GraphPad Prism) compiled from at least three independent
experiments using LNCaP cells, SEM < 10%, and represents the
compound concentration required to inhibit cell growth by 50%. Blank
cells = not tested.
b
expected, (except for 17, IC50 = 0.48 μM) the compounds were
weak inhibitors of CYP17 (IC50 = 2.59 to >15 μM), buttressing
established SAR data for potent steroidal CYP17 inhibitors.22
Finally, we assessed the antiproliferative activities of our
novel compounds in two human prostate cancer cell lines
(LNCaP and CWR22Rv1). The inhibitory concentrations that
caused 50% growth inhibition of cell proliferation (GI50 values)
Since these compounds are analogues of galeterone (5), we
evaluated their ability to inhibit CYP17 enzyme (Table 1). As
D
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were determined from dose−response curves using MTT
assays as we have previously described,22 presented in Table 1.
Although there is a modest correlation between the
antiproliferative activities GI50 values and inhibition of AR-
transactivations, AR degrading activities, compounds 8, 9, 11,
17, and 22, are 2.4- (for LNCaP) to 18-fold (for CWR22Rv1)
more potent than lead compound 5, representing a significant
improvement. Their full potential may become evident
following comparative in vivo antitumor efficacy assessments.
It is important to note here that we have previously reported
that galeterone and its analogues also inhibit the growth of PC-
3 and DU-145 prostate cancer cells,32 which may also be the
case for these new analogues.
some related compounds. A patent application to protect these
novel compounds has been filed.
ABBREVIATIONS
■
AR, androgen receptor; ARD, AR down-regulation; ARDAs,
AR down-regulating agents; CRPC, castration resistant prostate
cancer; gal, galeterone; GI50, compound concentration required
to inhibit cell growth by 50%; IC50, compound concentration
required to inhibit cell growth by 50%; MTT, 3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide;
PC, prostate cancer; Pd, palladium; TEA, triethylamine; TLC,
thin layer chromatography
In conclusion, a small library of galeterone analogues was
designed and synthesized with modifications of substituents at
C3 and C6 and the architecture of ring A. The substituent at
C3 could be varied somewhat (see compounds 8 and 17), but a
basic heterocycle seems important for bioactivity. Because the
yields for these promising compounds (8 and 17) are low, we
have embarked on new studies to develop facile and robust
synthesis for these compounds. This will enable biological
studies to assess their efficacies in clinically relevant models of
prostate cancer, in vitro and in vivo. Importantly, the most
effective compounds are predicted to be metabolically more
stable (by virtues of their C-3 moieties) than compound 6,
making them suitable for further development as new
therapeutics against prostate cancer.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00137.
Synthetic experimental details, analytical and further
biological data of compounds and biological assay
protocols. UPLC chromatograms and high resolution
mass spectral data for final compound (PDF)
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 410-706-6364. Fax: 410-706-0032. E-mail: vnjar@
som.umaryland.edu.
Present Address
§(M.S.M.) Bernard J. Dunn School of Pharmacy, Shenandoah
University, 45085 University Drive, Suite 202, Ashburn,
Virginia 20147, United States.
Funding
This work was supported in part by a grant from NIH and NCI
(RO1CA129379) and start-up funds from University of
Maryland School of Medicine and the Center for Biomolecular
Therapeutics (CBT), and Marlene Stewart Greenbaum Cancer
Center (Philanthropic Funds), Baltimore, USA to V.C.O.N.
A.K.K.-A. was supported in part by University of Maryland
School of Medicine Toxicology Program.
Notes
The authors declare the following competing financial
interest(s): V.C.O.N. is the lead inventor of galeterone and
new analogues, patents, and technologies thereof owned by the
University of Maryland, Baltimore, and licensed to Tokai
Pharmaceuticals, Inc. A.K.K.-A. and P.P. are co-inventors of
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Kong, X.; Melamed, J.; Tepper, C. G.; Kung, H. J.; Brodie, A. M.;
Edwards, J.; Qiu, Y. A novel androgen receptor splice variant is up-
regulated during prostate cancer progression and promotes androgen
depletion-resistant growth. Cancer Res. 2009, 69, 2305.
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DOI: 10.1021/acsmedchemlett.6b00137
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