Metabolically stable diphenylamine derivatives suppress androgen receptor and BET protein in prostate cancer

Article abstract of DOI:10.1016/j.bcp.2020.113946

Androgen receptor (AR) is a crucial driver of prostate cancer (PC). AR-relevant resistance remains a major challenge in castration-resistant prostate cancer (CRPC). Bromodomain and extra-terminal domain (BET) family are critical AR coregulators. Here, we

Full text of DOI:10.1016/j.bcp.2020.113946

BiochemicalPharmacology177(2020)113946
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Metabolically stable diphenylamine derivatives suppress androgen receptor
and BET protein in prostate cancer
Jiang Yua, Peiting Zhoua, Wu Dub, Ruixue Xua, Guoyi Yanc, Yufang Denga, Xinghai Lib,
⁎
Yuanwei Chena,b,
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu 610041,
China
b Hinova Pharmaceuticals Inc., 4th Floor, Building RongYao A, No. 5, Keyuan South Road, Chengdu 610041, China
c Department of Hepatobiliary Pancreatic Surgery, Henan Province People's Hospital, Zhengzhou 450003, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Androgen receptor
Bromodomain and extra-terminal domain
Diphenylamine derivatives
Castration-resistant prostate cancer
Antiandrogen resistance
Androgen receptor (AR) is a crucial driver of prostate cancer (PC). AR-relevant resistance remains a major
challenge in castration-resistant prostate cancer (CRPC). Bromodomain and extra-terminal domain (BET) family
are critical AR coregulators. Here, we developed several diphenylamine derivatives and identified compound 7d
that disrupted the functions of AR and BET family in prostate cancer and exhibited favorable metabolic stability
in vitro and high drug exposure in vivo. We showed 7d not only bound to AR, suppressed transactivation of wild-
type AR (wt-AR) and the mutant that mediates Enzalutamide resistance, but also reduced c-Myc protein ex-
pression through BET inhibition. In addition, 7d inhibited the proliferation of AR-positive PC cells with favorable
selectivity and suppressed AR-V7-expressing VCaP and 22Rv1 xenografts growth in vivo. Collectively, these
results indicate the potential of lead compound 7d as an orally available AR and BET inhibitor to treat CRPC and
overcome antiandrogen resistance.
1. Introduction
responsible for the secondary resistance, for example, AR-F876L mutant
confers resistance to the second-generation AR antagonist, En-
Androgen receptor, a member of nuclear receptor family, is a li-
gand-dependent transcription factor activated by dihydrotestosterone
(DHT). The dysregulation of AR signaling is crucial for the occurrence
and progression of PC [1–3]. Androgen deprivation therapy (ADT) by
medical or surgical castration is the standard first-line treatment for
men with advanced prostate cancer. However, most patients acquire
resistance to long-term treatment and result in CRPC, which is pre-
dominantly driven by deregulated AR signaling [4–7]. Since 2011,
several antiandrogens including Abiraterone Acetate, Enzalutamide
(Enza) and Apalutamide have been approved by FDA for CRPC [8–10]
and most recently Darolutamide (formerly known as ODM-201) has
also been approved [11]. Other AR antagonists like HC-1119 targeting
ligand binding domain of AR (AR-LBD) has entered phase III clinical
trials [12]. Despite the success achieved with these antiandrogens
[13–14], acquired resistance has been observed in patients, which is
partially mediated by constitutively active AR splicing variants. Among
them, truncated AR variant 7 (AR-V7) lacking ligand binding domain is
best studied [15–21]. Point mutations happened in AR-LBD are also
zalutamide [22–23].
Given the important role of AR mutation in disease progression, a
number of studies have been conducted to inhibit AR variants in
prostate cancer. Since N-terminal domain (NTD) is retained in most AR
variants, EPI-001 and its derivatives, Niphatenone B as well as KCI807
have been developed to bind AR-NTD and inhibit AR variants [24–26].
However, the phase 1/2 clinical trial (NCT02606123) testing the use of
EPI in patients with metastatic CRPC who had progressed after En-
zalutamide or Abiraterone treatments was terminated due to high pill
burden [27]. As with NTD, most AR mutants retain DNA binding do-
main (DBD). Consequently, VPC-14449 and VPC-14228 were identified
as AR-DBD inhibitors, which suppressed the transactivation of AR
variants in preclinical studies [28,29]. Meanwhile, AR variants dimer-
ization mediated by DNA-binding domain interactions is required to
genes transcription [30]. Although molecules disturbing AR dimeriza-
tion like resveratrol (RSV) and a fluorinated dialkylaminostilbene
(FIDAS) were able to block AR signaling and downregulate tumor
growth in vivo, their systemic bioavailability is very low [31]. Recently,
⁎ Corresponding author at: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for
Biotherapy, Chengdu 610041, China.
E-mail address: ywchen@scu.edu.cn (Y. Chen).
https://doi.org/10.1016/j.bcp.2020.113946
Received 20 December 2019; Accepted 31 March 2020
Availableonline02April2020
0006-2952/©2020ElsevierInc.Allrightsreserved.

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
virtual screening led to the discovery of VPC-17005 that inhibited AR
dimerization and transactivation through LBD-independent me-
chanism. However, VPC-17005 still requires further modifications to
improve the stability and it is currently sub-optimal for in vivo use [32].
Another strategy to impede AR variants activity is AR protein down-
regulation. Niclosamide is a helminthic drug that induces AR-V7 pro-
tein degradation and re-sensitizes antiandrogen-resistant prostate
cancer cells to Enzalutamide and Abiraterone treatments [33]. Other
drugs such as ASC-J9 [34], UT-155 and UT-69 [35] were developed to
reduce AR variants expression in preclinical studies. However, one of
the trials investigating Enzalutamide in combination with Niclosamide
(NCT02532114) was discontinued because of poor prostate specific
antigen (PSA) response [36]. Therefore, there is unmet and urgent
needs for new strategies to block aberrant AR signaling and overcome
antiandrogen resistance in CRPC.
BET family is an important class of chromatin readers. It consists of
bromodomain-containing protein 2 (BRD2), BRD3, BRD4 and testis-
specific BRDT. BET family recognize acetylated histones by tandem
domains (BD1 and BD2), recruit transcriptional regulatory complexes
and then regulate gene transcription [37]. BRD2/3/4 physically inter-
acts with AR-NTD, which can be disrupted by BET inhibitors (BETi),
resulting in abrogation of AR-mediated transcription and growth in-
hibition of CRPC xenografts [38–42]. As NTD is essential for the tran-
scriptional activity of both full-length AR and truncated AR-V7, BET
inhibition represents a promising opportunity to block the aberrant AR
signaling and overcome antiandrogen resistance. Besides, AR-positive
cells are preferentially sensitive to BETi and Enzalutamide-resistant
CRPC cell lines maintain sensitivity to BET inhibitors [43,44]. However,
Pawar et al reported that BETi-resistant CRPC cells display BRD4-in-
dependent transcription but increased sensitivity to AR blockade, which
highlights the limitation of BET inhibition as monotherapy for CRPC
[45]. Combination treatment of antiandrogen with BETi led to greater
growth inhibition than antiandrogen alone [44]. Currently, several BET
inhibitors, such as ZEN003694 and GS-5829 are being evaluated in
clinical trials for CRPC as a single agent or in combination with AR-
antagonists [46,47]. These findings indicate that AR antagonists with
inhibitory activity against BET family hold promise to block the aber-
rant AR signaling and overcome antiandrogen resistance in prostate
cancer.
AMX 400 spectrometer (1H = 400 MHz, 13C = 101 MHz). Chemical
shifts were reported in parts per million (ppm) and referenced to tet-
ramethylsilane (TMS) or residual deuterated solvent (DMSO‑d6 or
CDCl3). Purity of synthesized compounds was determined by high-
performance liquid chromatography (HPLC), which was performed on
Dionex ultimate 3000 HPLC instrument using Waters e2695 Series
chromatographs
and
Waters
xBridge
column
(5
μm,
4.6 mm × 150 mm). All compounds for biological evaluation were at
least 95% pure. HRMS was recorded on a Q-TOF Premier mass spec-
trometer (Micromass, Manchester, UK). The HRMS, NMR spectra and
purity data of compounds that were synthesized in our laboratory are
available online [61].
2.1.2. Synthetic route
As shown in Scheme 1, intermediate 2 was synthesized following
previously described procedures [48]. Then palladium-catalyzed C-N
coupling reaction and subsequent C–C coupling reaction yielded in-
termediates 4–5. Compounds 6d, 6f and 6e were synthesized through N-
alkylation reaction. The synthetic route of compounds 7b, 7d–7f, 7l,
7m, 7o-7q and 7s was outlined in Scheme 2. Starting from commercially
available material 6, intermediate 7 was obtained through nucleophilic
substitution reaction. And then 8 or 9 underwent palladium-catalyzed
C–C coupling reaction to achieve intermediates 10–12. The subsequent
palladium-catalyzed cross-coupling reaction between aromatic iodide
(7, 13–15) and aryl amine (10–12) followed by N-alkylation reaction
afforded the desired compounds. Analogues 7m and 7l with different
substituents at R3 position were also successfully synthesized via similar
methods (c, e, b) as illustrated in Scheme 2. Detailed procedures were
provided through Mendeley Data [61].
2.2. Biological experiments
2.2.1. Cell culture
Human PC cells LNCaP/AR were gifts from KangCheng Biotech (KC
BIO, Sichuan, China) in 2012. HEK293 cells were gifts from Yinlan Zhao
group from Sichuan University. Other cells were purchased from the
Cell Bank of Chinese Academy of Sciences (Shanghai, China). LNCaP/
AR, LNCaP (TCHu173), 22Rv1 (TCHu100) cells were cultured in RPMI-
1640 medium. SK-OV-3 (TCHu185), HT-29 (SCSP-5032), WPMY-1
(GNHu36), DU 145 (TCHu222) and VcaP (TCHu220) cells were cul-
tured in DMEM (high glucose) and PC-3 (SCSP-532) cells were in a
mixture (1:1) of DMEM and F12 nutrient medium. And all medium was
supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 1%
(v/v) penicillin/strep-tomycin (HyClone, SV30010).
Previously, we developed a series of AR antagonists (4a, 3e, 3d)
bearing diphenylamine scaffold (Fig. 1a) [48]. However, the most po-
tent compound 4a featuring 3, 5-dimethylisoxazole moiety exhibited
less satisfying pharmacokinetic (PK) profiles and poor anti-tumor ac-
tivity in vivo [48]. Thus, further structural optimization was conducted
to improve metabolic stability and anti-tumor activity of 4a. Besides,
we noticed the structural similarity between our diphenylamine deri-
vatives and some BET inhibitors of which 3, 5-dimethylisoxazole sub-
stituent is able to occupy the ε-N-lysine acetylation (KAc) binding
pocket of BRD4 as a KAc motif mimic [49,50]. Here, we show that
newly-synthesized diphenylamine derivatives bearing 3, 5-dimethyli-
soxazole moiety exhibit inhibitory activity against both BET family and
AR. In addition, we also report on the structural mechanism, improved
pharmacokinetic profiles and therapeutic effects in prostate cancer.
2.2.2. Cell proliferation assay
2 × 104 VCaP cells or 1–3 × 103 other cells in 100 μL medium were
seeded in 96-well plates per well. After 24 h, 100 μL medium containing
test compounds was added. Cells were incubated in 37% for 1 day
(HEK293) or 6 days. Cell counting kit-8 (CCK-8) reagent (Signalway
Antibody, Maryland, USA) was added and the absorbance value was
measured according to the manufacturers’ instructions. IC50 was cal-
culated by GraphPad Prism 5 software. Enzalutamide was a gift from
Hinova Pharmaceuticals Inc. (Chengdu, Sichuan, China). Cell viability
(% of maximum) = (X − Avgmin)/(Avgmax − Avgmin) * 100. The
Avgmax is the mean value of DMSO wells and the Avgmin is the mean
value of wells without cells.
2. Materials and methods
2.1. Chemistry
2.1.1. General methods
2.2.3. Reverse transcription-polymerase chain reaction (RT-PCR) analysis
The RT-PCR analysis for PSA was performed as previously described
[48]. Cells were maintained in 6-well plates in RPMI 1640 or DMEM
supplemented with 5% charcoal stripped FBS and 1% penicillin-strep-
tomycin. After 48 h, cells were treated with combination of compounds
and 0.2 nM R1881 for 24 h. Total RNA was isolated by TRIzol reagent
(Invitrogen, 15596026, USA). The cDNA was synthesized using reverse
conversion kit (Applied Biological Materials, G492, Canada). Real-time
Starting materials and reagents were purchased from Energy
Chemical (Shanghai, China) and Bidepharm (Shanghai, China).
Anhydrous solvents with molecular sieves were from Energy Chemical
(Shanghai, China). Other analytically pure solvents were bought from
Zhiyuanhx (Tianjing, China) and used as received. Flash column chro-
matography was carried out using Biotage Isolera One apparatus with
Agela flash column silica-CS. NMR spectra were recorded on a Bruker
2

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
(a)
(b)
wt-AR
wt-AR
100
50
0
100
50
0
7d IC50
7e IC50
7f IC50
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
***
-50
1
2
3
4
5
7l
6f
7f
7m
7s
6e
7e
4a
7o
Enza
3d 6d
7d 7b
7p
7q
Log [nM]
DMSO
Compounds
Negative
(c)
BRD4 (1,2)
BRD4
NS
NS
NS
NS
NS
100
50
0
*
100
50
0
**
***
***
7d IC50 = 1.87
7e IC50 = 4.69
***
***
***
***
***
***
***
7l
6f
7f
7s
7q
6e
7e
4a
7o
Enza
3d 6d
7d 7b
7p
7m
-10 -9 -8 -7 -6 -5 -4
Log [M]
DMSO
Compounds
Fig. 1. Diphenylamine derivatives suppress the function of BRD4 and AR. (a) The structures of compounds. Structural changes are highlighted in red. (b) AR
luciferase reporter assay. HEK293 cells were treated with 5 μM compounds or DMSO in the presence of 2 nM testosterone. No reporter gene plasmid was transfected
to cells in negative group. Data are expressed as the mean
sd (n = 3). Enza, Enzalutamide. (c) HTRF assay of BRD4 (BD1 and BD2). The final concentration of
compounds was 10 μM. Data are expressed as the mean
sd (n = 3). (d) Inhibitory activities towards BRD4 and wt-AR. Molecules that inhibited both BRD4 and wt-
AR with P < 0.05 were considered as dual-action inhibitors. (e) Western blot of c-Myc and grayscale analysis. 22Rv1 cells were treated with test compounds at a
concentration of 5 μM or DMSO for 24 h. Data are expressed as the mean
is referred to the web version of this article.)
sd (n = 3). (For interpretation of the references to colour in this figure legend, the reader
PCR was performed on CFX96™ Real-Time PCR Detection Systems (BIO-
RAD, USA) using EvaGreen qPCR MasterMix (Applied Biological Ma-
terials, MasterMix-S, Canada). The primers were purchased from Gen-
eCopoeia Inc (USA, PSA: HQP009633, GAPDH: HQP006940).
Protein concentration was determined using the BCA Protein Assay Kit
(Beyotime, P0012, Shanghai, China). The mixture of supernatant and
1/4 (v/v) 5 × SDS loading buffer (Beyotime, P0015, Shanghai, China)
was heated at 100 °C for 5 min. The cell extracts were subjected to SDS-
PAGE and then transferred to polyvinylidene fluoride (PVDF) mem-
branes (Millipore, ISEQ00010, USA). The membranes were incubated at
4 °C overnight with primary antibodies in 5% (w/v) milk/TBST and
then washed 3 times with TBST buffer before being incubated with 1/
4000 secondary antibodies. Images were acquired by chemilumines-
cence system (CLiNX, NO.810060). Antibodies included androgen re-
ceptor antibody (Cell Signaling Technology, 5153S, USA), c-Myc anti-
body (Cell Signaling Technology, 5650S, USA), PSA antibody (Cell
Signaling Technology, D11E1, USA), tublin antibody (Zen BioScience,
2.2.4. Western blot
Cells seeded in medium supplemented with 10% (v/v) FBS were
treated with different concentrations of compounds or DMSO for 24 h.
Then cells were washed with cold PBS and lysed in RIPA buffer
(Beyotime, P0013B, Shanghai, China) with protease inhibitors cock-
tails. For the analysis of tumor samples, homogenized tumors were
lysed in RIPA buffer with cocktails (Beyotime, P1005, Shanghai,
China). Lysates was centrifuged at 12,000 rpm for 15 min at 4 °C.
3

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
Fig. 1. (continued)
Scheme 1. General synthetic route of diphenylamine analogues 5a-f and 6a-g. Reagents and conditions: (a) H2SO4, NaNO2, H2O, KI, MeCN, 0 ℃-rt, overnight; (b)
Pd2(dba)3, DavePhos, Cs2CO3, MeCN, reflux, 5 h; (c) boronic acid/ester, Pd(PPh3)4, K2CO3, H2O, 1,4-Dioxane , 90 ℃, 6 h; (d) Haloalkane, NaH, DMF, 0 ℃-rt, 2 h.
EE1029, China) and goat anti-rabbit or anti-mouse secondary antibody
(Zen BioScience, China). Image J was used to calculate the grayscale.
The specificity of primary antibodies can be observed in the manu-
facturer’s instructions and our Fig. 5d.
methyltrienolone [3H]R1881 (PerkinElmer, USA, Cat: NET590250UC,
Lot: 2133648) and LNCaP cytosol as previously described [48].
2.2.6. Homogeneous time resolved fluorescence (HTRF) assay
Activity against bromodomain containing protein 4 and 9 (BRD4 (1,
2), BPS bioscience, USA, Cat. No. 31044; BRD9, Active Motif, USA, Cat.
No. B1048) was assessed through HTRF assay. Compounds were serially
diluted into 10 concentrations to determine IC50. Compounds or DMSO
2.2.5. Competitive AR binding assay
The competitive AR binding assay was performed at WuXi Apptec
Co., Ltd (Shanghai, China) using the synthetic androgen
4

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
Scheme 2. General synthetic routes of compounds 7a-s. Reagents and conditions: (a) Imidazole, NaH, DMSO, 120 ℃, 24 h; (b) boronic ester, Pd(PPh3)4, K2CO3, H2O,
1,4-Dioxane, 90 ℃, 6 h; (c) Pd(OAc)2, Xant-phos, Cs2CO3, 1,4-Dioxane, 110 ℃, overnight; (d) Pd2(dba)3, DavePhos, Cs2CO3, MeCN, 80 ℃, overnight; (e) NaH, DMF, 0
℃-rt, 2 h.
(high control) was transfered into 384-well assay plate followed by
addition of 2x Protein (only buffer was added as low control) and
Peptide Mix. Bromosporine (MedChemExpress, NJ, USA, Cat. No. HY-
15815) was used as positive control. The final concentration of DMSO
was 0.1%. Then 2x Detection Mix was added to the assay plate. After
2 h at room temperature, the HTRF signals (Ex at 340 nm, Em at
615 nm & 665 nm) was read on Envision. The data was fitted in Excel to
obtain inhibitory activity using equation: Inh % = (Avgmax − Signal)/
(Avgmax − Avgmin)*100. And then fit the data in GraphPad Prism 5 to
obtain IC50 values. % of maximum = 1-Inh %. This assay was com-
pleted by ChemPartner Co., Ltd (Shanghai, China).
2.2.8. AR reporter assay
Wt-AR or AR-F876L expression vector were transiently transfected
into HEK293 or PC-3 cells, respectivly. AR-F876L plasmid was estab-
lished at Sangon Biotech Co., Ltd. (Shanghai, China). The reporter gene
plasmid (QIAGEN, Germany, CCS-1019l and Promega, WI, USA,
E1360), transfection reagent (GeneCopoeia, USA, EF003 and Promega,
WI, USA, E2311), Luciferase Kit (Promega, WI, USA, E1910 and E2550)
were used according to the manufacturer’s instructions. The wt-AR
reporter gene assay was accomplished at WuXi Apptec Co., Ltd
(Shanghai, China). The general procedures were previously described
[48]. In order to rule out the false-positive results caused by direct
luciferase inhibition, PC-3 cells were transfected with positive control
plasmid with CMV promoter (QIAGEN, Germany, CCS-1019l). After
treatment of test compound at 5 μM for 24 h, the luminescence was
detected.
2.2.7. Amplified luminescent proximity homogeneous assay screen
(AlphaScreen)
Inhibitory activities on bromodomain containing proteins (BRD2,
BRD3, BRD4, CREBBP, EP300, SMARCA2, FALZ, TAF1(D2)) were as-
sessed by AlphaScreen (PerkinElmer). Compounds were resolved in
DMSO and serially diluted into 10 concentrations or 1 concentration in
duplicate. Transfer compounds to 384-well plates (PerkinElmer, USA)
and final concentration of DMSO was 0.1%. And then protein and
biotinylated peptide were respectively added into 1X buffer to obtain
protein solution and substrate solution. Protein solution (only buffer
was added as low control) was transferred to each well of assay plate,
which was incubated at room temperature for 15 min. Substrate solu-
tion was next added to start reaction and the resulting mixture was
incubated at room temperature for 60 min. Subsequently, acceptor and
donor solution was added. The mixture was incubated at room tem-
perature for another 60 min. The plate was read on EnSpire plate reader
(PerkinElmer, USA) with Alpha mode. Fit the data in Excel to obtain
2.2.9. AR nuclear translocation
As previously described [48], WPMY-1 cells were seeded in 48-well
plate in DMEM (high glucose) medium supplemented with 5% charcoal
stripped FBS. The AR-F876L-EGFP plasmid was established at Sangon
Biotech Co., Ltd. (Shanghai, China). Within 6 h, EndoFectin™-Max
transfection reagent (GeneCopoeia, USA, EF003) was used to transfect
200 ng AR-F876L-EGFP plasmid per well into cells. The second day,
compounds and 0.5 nM R1881 were added. After another 24 h, Hoechst
33342 (Sigma-Aldrich, MO, USA, B-2261) was used to label DNA fol-
lowing the manufacturers instructions. Pictures were obtained on Zeiss
Observer D1 fluorescence microscope (AX10 cam HRC).
2.2.10. Docking study
The predicted binding modes of compound 7d to AR (PDB ID:3V49)
[51] and BRD4(3SVG) [52] were analyzed by Autodock 4.0 [53]. The
crystal structure of AR and BRD4 were extracted from Protein Database
(www.rcsb.org). The binding site of small molecule was chosen as ac-
tive site. The grid dimensions were set to 20 × 20 × 20 Å3 and other
parameters were as default. A total of 20 conformations of compound
7d were generated for molecular docking.
inhibition values using Equation: Inh
% = (Avgmax − Signal)/
(Avgmax − Avgmin)*100. IC50 value was obtained through GraphPad
Prism 5. (+)-JQ1 (BPS bioscience, USA, Cat. No. 27402), Bromosporine
(MedChemExpress, NJ, USA, Cat. No. HY-15815) and PFI-3 (Cayman,
Michigan, USA, Cat. No. 15267) were used as positive control. This
assay was completed by Shanghai ChemPartner Co., Ltd. BRD2(1,2)
(BPS bioscience, USA, Cat. No. 31024); BRD3(1,2) (Cat. No. 31035);
CREBBP (Active Motif, USA, Cat. No. 31873); EP300, (Active Motif,
USA, Cat. No. 31801; FALZ (Active Motif, USA, Cat. No. 31447).
2.2.11. Pharmacokinetic studies
Male SD rats were orally administrated with compounds formulated
5

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
in poly ethylene glycol 200 (PEG200)/N,N-dimethylacetamide (DMA)
(9:1, v/v) at a single dose of 30 mg/kg (n = 3). The procedure was
described previously [48].
scanned on a computerized image system equiped with Pannoramic
DESK (3D HISTECH) and Pannoramic Scanner. The image was analyzed
on Caseviewer software. This assay was performed by Servicebio
technology Co. Ltd, Wuhan, China.
2.2.12. Liver microsome stability test
This assay was performed by Pharmaron Beijing Limited Co. Firstly,
10 μL of human/rat/mouse liver microsomes (Corning, USA, Cat.
452117 and 452501; XENOTECH, Cat. M1000) were incubated with
40 μL of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) or
ultra-pure H2O, respectively. The reaction was started with addition of
4 μL control compound (Verapamil) or test compound solution. The
final concentration of test compound and Verapamil was 2 μM. The
reaction was then evaluated at 0, 15, 30, 45, and 60 min and was
stopped by addition of cold acetonitrile. Samples were centrifuged for
40 min at 3220 rpm and the supernatant was mixed with ultra-pure
H2O and then used for LC-MS/MS analysis. Remaining percentage was
calculated and the results at 0 min were regarded as 100%.
2.2.16. Statistical analysis
The two-tailed t-test was used to determine the significance of re-
sults. GraphPad Prism 6 was used for statistical analysis. ∗ indicates
significance as p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. p < 0.05 was
considered as statistically significant. Quantification of Western blot
bands was performed on Image-J software. The image in IHC analysis
was analyzed on Caseviewer software. All calculations in hepatocyte
metabolical stability study were carried out in Microsoft Excel.
3. Results
3.1. Diphenylamine derivatives suppress the functions of AR and BRD4,
leading to proliferative inhibition of PC cells.
2.2.13. Metabolical stability in hepatocyte
Hepatocytes (BioIVT) was diluted to the working density (0.5 × 106
cells/mL). Cells in medium were added into a 96-well non-coated plate.
2 μL of 100 μM test compound (final concentration is 1 μM) or positive
control were added into wells to start the reaction, which was incubated
at 37℃. 25 μL of sample was collected at time points of 0.5, 15, 30, 60,
90 and 120 min in duplicate. The aliquots were then mixed with 12
volumes of acetonitrile containing internal standard to terminate the
reaction. Samples were vortexed for 5 min and centrifuged for 45 min at
3220 g. 100 µL of supernatant was diluted by 100 µL ultra-pure water.
The resulting mixture was used for LC/MS/MS analysis. All calculations
were carried out using Microsoft Excel. The peak areas were determined
from extracted ion chromatograms. The in vitro half-life of parent
compound was determined by regression analysis of the percent parent
disappearance vs. time curve using the formula T1/2 = 0.693/k. The in
vitro intrinsic clearance was calculated according to the formula
CLint = kV/N, wherein N was number of hepatocytes per well
(0.1 × 106 cells).
In order to develop molecules with improved PK property and anti-
tumor activity, we tried to modify the structure of previously reported
4a. The substituent on nitrogen bridge of 4a and the position of methyl
group on benzene ring were respectively changed. Recently, it has been
reported that the introduction of alkoxy or aromatic heterocycle group
to Galeterone improved antiproliferative activity and the resulting
compounds were metabolically stable [54]. Inspired by this result,
various substituents including alkyl, alkoxy, aryl and heteroaryl groups
were introduced to the similar position of diphenylamine derivatives.
The effects of these compounds (Fig. 1a) on BRD4 and AR were eval-
uated through Homogeneous time resolved fluorescence (HTRF) assay
and luciferase reporter gene assay, respectively. As exhibited in Fig. 1b-
1c, although compounds featuring CN group on benzene ring (4a, 3d,
6d, 6f, 6e) strongly suppressed wt-AR transactivation, they were less
potent against BRD4 than compounds 7d and 7e featuring imidazole
group on benzene ring. In comparison with 7 m, introduction of imi-
dazole group (7d) obviously enhanced the inhibitory activity against
AR and BRD4 (Fig. 1b-c). Compared to 7d featuring imidazole and 2-Me
groups on benzene ring, compounds 7l, 7p, 7s, 7q, 7o or 7f with 6-Me
were less potent towards BRD4 and AR. Replacing 3, 5-dimethylisox-
azole (6f, 7d) with 1-methylpyrazol (6e, 7b) led to decreased activity
toward BRD4 and AR. These results indicate that 3, 5-dimethylisoxazole
and imidazole groups are crucial for BRD4 and AR inhibition. In ad-
dition, introducing ethyl group on nitrogen bridge (7d) slightly im-
proved activities against BRD4 and AR, compared to analogue 7e.
Considering derivatives 7d, 7e and 7f featuring similar structures ex-
hibited comparable activity at the same concentration, we next mea-
sured the IC50 values. As exhibited in Fig. 1b-1c, 7d was identified as
the most potent molecule with IC50 value of 1.23 μM and 1.87 μM for
wt-AR and BRD4, respectively. These results indicate that the position
of methyl on benzene ring and ethyl on nitrogen bridge slightly affect
activities towards AR and BRD4 but imidazole and 3, 5-dimethylisox-
azole groups are crucial for BRD4 and AR inhibition.
Interestingly, BET inhibitors were reported to suppress AR trans-
activation in luciferase reporter assay [59,60], but we didn’t observe
decreased AR transcriptional activity caused by JQ1 or OTX-015 in our
system (not shown). Therefore, dual-action inhibitors were identified
through amplified luminescent proximity homogeneous assay screen
(AlphaScreen) on BRD4 and wt-AR luciferase reporter assay. As shown
in Fig. 1d, molecules that inhibited both BRD4 and wt-AR with
P < 0.05 were considered as dual-action inhibitors.
To rule out false-positive result in luciferase reporter assay caused
by direct luciferase inhibition or cell death, molecules were also tested
in reporter assay using positive control plasmid with cytomegalovirus
(CMV) promoter and HEK293 cytotoxicity assay. As presented in Figure
S1 (provided in Mendeley Data) [61], compounds except for 7e didn’t
inhibit the catalytic activity of firefly luciferase. Meanwhile, all these
2.2.14. In vivo xenograft studies
BALB/c nude male mice (5 weeks old from HFK Bioscience Co. ltd.,
Beijing, China) were subcutaneously injected with 1 × 106 22Rv1 cells
in 150 μL mixture (1:1) of RPMI-1640 medium and Matrigel (BD, NJ,
USA, 354234). When tumor volume reached about 150 mm3, the mice
were castrated or SHAM-operated under Nembutal anesthesia and then
randomized into four groups (n = 7–8). After 7 days, the mice were
orally treated with 7d (50 mg/kg, qd), enzalutamide (50 mg/kg, qd) or
vehicle (90% PEG200: 10% DMA) for 24 days. Procedures for VCaP
model were described previously [48]. All animal experiments have
been approved by Institutional Animal Care and Treatment Committee
of Sichuan University in China (IACUC number: 20100318).
2.2.15. Immunohistochemical (IHC) analysis
At the end of treatment, VCaP tumors were collected and fixed in
formalin. The paraffin-embedded tumors were sectioned at 4–8 μm
thickness and arranged on glass slides for IHC analysis. The slides were
baked at 60℃ for 6 h, followed by xylene deparaffinization and ethanol
rehydrating. 3% Hydrogen peroxide was added to block the en-
dogenous peroxidase activity. The sections were submerged in EDTA
buffer or citrate and microwaved for antigen retrieval. 3% BSA
(Servicebio, Wuhan, China, G5001) was used to block nonspecific
background. The samples were incubated at 4℃ overnight with specific
primary antibodies against c-Myc (R&D, USA, MAB3696) and PSA
(Signalway Antibody, Maryland, USA, D11E1) followed by incubation
with secondary antibody at room temperature for 50 min. The DAB
detection kit (Servicebio, Wuhan, Chian, G1211) was used to produce
brown stain. The sections were counterstained with hematoxylin
(Servicebio, Wuhan, Chian, G1004) and dehydrated. The slides were
6

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
7e
7d
100
50
0
LNCaP/AR
VCaP
LNCaP/AR
VCaP
100
50
0
*
*
*
22Rv1
22Rv1
*
*
*
*
*
*
**
PC-3
PC-3
**
**
**
*
**
**
**
**
**
**
**
**
**
**
**
0
0
370
370
1111
3333
1111
3333
10000
10000
Log (nM)
Log (nM)
7s
7f
100
50
0
100
LNCaP/AR
VCaP
*
LNCaP/AR
VCaP
*
*
*
*
22Rv1
22Rv1
*
*
*
**
**
**
**
PC-3
PC-3
*
*
50
0
**
**
**
**
**
**
**
**
**
0
0
370
370
1111
3333
10000
1111
3333
10000
Log (nM)
Log (nM)
Enzalutamide
6f
100
50
0
LNCaP/AR
VCaP
LNCaP/AR
VCaP
100
50
0
*
*
*
*
*
22Rv1
22Rv1
*
PC-3
PC-3
*
*
**
*
**
**
**
**
**
**
**
**
**
**
**
**
**
0
0
370
370
1111
3333
1111
3333
10000
10000
Log (nM)
Log (nM)
Fig. 2. Diphenylamine derivatives inhibit the proliferation of prostate cancer cells. Compounds were tested in LNCaP/AR, VCaP, 22Rv1 and PC-3 cells in at least two
independent experiments with duplicate measurements. Data are expressed as the mean sd (n = 2) and n means two independent experiments.
molecules had little effect on HEK293 viability (Figure S2) [61], sug-
gesting the decreased luminescence in wt-AR reporter gene assay was
not caused by cytotoxicity.
BRD2/3/4 physically interacts with AR-NTD and AR-positive cells are
preferentially sensitive to BET inhibitors [38]. Therefore, although 7d
can reduce c-Myc level, it’s understandable why it exhibited poor an-
tiproliferative activity in AR-negative PC-3 cells.
We next examined the c-Myc protein expression, a downstream ef-
fector of BRD4. As illustrated in Fig. 1e, western blot and the corre-
sponding quantified analysis suggest that introducing imidazole group
on benzene ring (7d, 7e) decreased the c-Myc expression compared to
compounds 3e and 4a. Replacing 3, 5-dimethylisoxazole (7d) with 1-
methylpyrazol (7b) resulted in increased c-Myc expression in 22Rv1
cells. These results also indicate that imidazole and 3, 5-dimethylisox-
azole groups are crucial for BRD4 inhibition, which were consistent
with the inhibitory activities on BRD4 in HTRF assay.
Subsequently, potent dual-action derivatives 7d, 7e, 7f, 7s and 6f
were selected and evaluated in antiproliferative assay toward prostate
cancer cells LNCaP/AR [55], VCaP and 22Rv1 cells [56]. To rule out
non-AR-mediated cytotoxicity, AR-negative PC-3 cells were also tested.
As shown in Fig. 2, these compounds suppressed AR-positive PC cells
proliferation in concentration-dependent manner but exhibited weak
efficacy against PC-3 cells. However, Enzalutamide that potently in-
hibited wt-AR transactivation but didn’t affect BRD4 (Fig. 1b-1c) ex-
hibited poor activity against androgen-independent 22Rv1 cells, sug-
gesting BRD4 inhibition contributes to antiproliferative activity toward
AR-V7-positive 22Rv1 cells. In addition, we analyzed the effect of
compounds on c-Myc in PC-3 cells. As shown in Figure S3, 7d weakly
reduced c-Myc protein compared to that in AR-positive VCaP and
22Rv1 cells (Fig. 4b). On the other hand, it has been reported that
3.2. Diphenylamine derivatives featuring imidazole group exhibit favorable
metabolic stability in vitro and drug exposure in vivo
To investigate whether the structural modification lead to improved
metabolic stability, compounds were next tested in liver microsomes
and human hepatocyte using Verapamil as reference. As presented in
Fig. 3a and 3c, more than 50% of compound 7d can be detected in liver
microsomes from human, rat, mouse and human hepatocyte after 1 h
and 2 h incubation, respectively. The pharmacokinetic parameters ex-
hibited in Fig. 3b and 3d suggest that 7d is metabolically stable with
long T1/2 and slow clearance, especially in human liver microsome.
Subsequently, male rats were orally administrated with compounds
formulated in PEG200/DMA (9:1, v/v) at a single dose of 30 mg/kg.
Blood were withdrawn at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h from
retrobulbar vein to obtain the concentration–time curves (Fig. 3e).
Fortunately, compounds 7d and 7e bearing imidazole group exhibited
high drug exposure in vivo with area under the curve (AUC0-t) values of
44433.9 ng*h/ml and 36546.7 ng*h/ml, which were 23-fold and 9-fold
higher than those of compounds 3e and 4a, respectively (Fig. 3f).
However, derivatives without imidazole substituent (3e, 4a, 6d, 6f)
showed poor pharmacokinetic profiles. These results suggest that
7

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
(a)
(b)
Clearance
T1/2
Liver Microsomes
Compd
7d
Species
(μL/min/mg
(min)
100
80
60
40
20
0
protein)
7d
Human
Rat
533.73
139.80
80.02
7.17
2.60
9.91
6d
Verapamil
Mouse
Human
Rat
17.32
193.26
438.42
125.64
94.39
229.78
186.96
6d
3.16
Mouse
Human
Rat
11.03
14.68
6.03
e
e
s
e
s
s
Rat
Mou
Rat Rat
Mou Mou
Human
Verapamil
Human
Human
Species
Mouse
7.41
(c)
(d)
Human Hepatocytes
Compounds Verapamil
7d
100
50
0
Verapamil
7d
Species
Human
25.20
Human
141.46
T1/2 (min)
Clearance Rate
(μL/min/106
cells)
55.07
0.80
15
30
60
90
0.5
120
Time (min)
(e)
(f)
Concentration-time curves
T1/2
(h)
Tmax
(h)
Cmax
AUC0-t
Compd
10000
8000
6000
4000
2000
0
(ng/ml) (ng*h/ml)
7e
7d
6f
6d
4a
3e
4.4
6.5
4.8
5.8
2.8
1.9
0.5
2320.0
841.0
89.9
4041.9
1917.0
158.9
4a
3e
6d
6f
0.5
0.3
0.3
0.8
2.2
368.7
8496.7
5320.0
916.5
44433.9
36546.7
7d
7e
0
4
8
12
16
20
24
Time(h)
Fig. 3. Diphenylamine derivatives featuring imidazole group exhibit superior metabolic stability. (a) Remaining percentage of compounds in liver microsomes after
60 min incubation. Verapamil was used as reference. Data are expressed as the mean sd (n = 3) (b) Half-life and clearance in different species of liver
microsomes. (c) Metabolic stability in human hepatocyte. Verapamil was used as reference (n = 3). (d) Half-life and clearance in human hepatocyte. (e)
Concentration-time curves in male rats. Compounds were formulated in a solvent of 10% DMA and 90% PEG200 for an oral dose of 30 mg/kg (n = 3). (f)
Pharmacokinetic parameters in male rats.
introducing imidazole group on benzene ring improves metabolic sta-
bility of compounds in vitro and oral drug exposure in vivo. Interest-
ingly, the half-life of 7d in male rats (T1/2 = 2.8 h) was almost equal to
that in rat liver microsome (T1/2 = 2.3 h). Since the metabolic stability
study in vivo measured both phase I and II metabolisms but only phase I
metabolism was accessed in liver microsome study, it’s not strange to
see such results. Despite the moderate half-life, high AUC value was
observed in male rats by 7d (Fig. 3e-3f). The reason could be that 7d
was weakly reduced by the first-pass effect in gastrointestinal tract and
liver during the process of absorption, which was consistent with the
high Cmax observed in male rats. Therefore, the most potent and stable
derivative 7d was selected for further evaluation.
3.3. Derivative 7d inhibits BET family with moderate selectivity
To investigate the effect of 7d on BET family, AlphaScreen was
performed. As exhibited in Fig. 4a, compound 7d inhibited BRD2/3/4
with IC50 values of 290 nM, 360 nM and 1870 nM, respectively. In
western blot analysis, 7d reduced BRD4-regulated c-Myc protein ex-
pression in 22Rv1, VCaP and LNCaP cells in a concentration-dependent
manner (Fig. 4b). As expected, Enzalutamide had no effect on c-Myc
level under the same conditions. Utilizing X-ray cocrystal structures of
8

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
(a)
IC50 on BRD3
IC50 on BRD4
IC50 on BRD2
120
80
40
0
120
80
40
0
120
80
40
0
7d
7d
7d
(IC50 = 1870 nM)
( IC50 = 360 nM)
(IC50 = 290 nM)
Enza
(+)-JQ1
(+)-JQ1
(+)-JQ1
-11 -10 -9 -8 -7 -6 -5
Log [Cpd] (M)
-11 -10 -9 -8 -7 -6 -5
Log [Cpd] (M)
-10 -9 -8 -7 -6 -5 -4
Log [Cpd] (M)
(b)
LNCaP
22Rv1
VCaP
7d
Enza Gal
Gal Enza
7d
Gal Enza
7d
c-Myc
Tublin
c-Myc
Tublin
c-Myc
Tublin
LNCaP
VCaP
22Rv1
NS
NS
1.0
0.5
0.0
*
NS
NS
NS
1.0
0.5
0.0
NS
1.0
0.5
0.0
*
**
***
****
****
****
****
****
l
1
5
1
5
10
10
1
5
Ga
Gal
10
Enza
Enza
Gal
DMSO
7d
Enza
7d
DMSO
7d
DMSO
Compounds
Compounds
Compounds
(c)
IC50 of 7d
(nM)
Protein
Classification
Reference
FALZ
BRD2
I
II
30000
Bromosporine
(+)-JQ1
290
BRD3
II
360
(+)-JQ1
BRD4
II
1870
6080
14700
30000
10000
8920
(+)-JQ1
CREBBP
EP300
III
III
IV
VII
VIII
I-CBP-112
I-CBP-112
Bromosporine
Bromosporine
PFI-3
BRD9
TAF1(D2)
SMARCA2
(d)
Fig. 4. Derivative 7d inhibits BET family with moderate selectivity. (a) Inhibitory activities on BRD2/3/4 (BD1 and BD2). The IC50 was calculated from the
AlphaScreen or HTRF assay. (b) Western blot analysis of c-Myc and grayscale analysis. 22Rv1, VCaP and LNCaP cells were treated with compounds or DMSO for 24 h.
Data are expressed as the mean
sd (n = 3). Enza, Enzalutamide. Gal, Galeterone. (c) The selectivity profile of 7d on bromodomain-containing proteins. IC50 was
determined by AlphaScreen or HTRF assay. Compounds were serially diluted to 10 concentrations. (d) The predicted binding mode and 2D diagram of 7d in complex
with BRD4 (PDB ID: 3SVG).
9

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
BRD4 [52], molecular docking simulation [53] was conducted to ex-
plore the possible binding mode of compound 7d in complex with
BRD4. As shown in Fig. 4d, the oxygen and nitrogen atoms of isoxazole
formed two hydrogen bonds with the amine group of Asn140 residue in
KAc-binding pocket of BRD4. In addition, the isoxazole ring of 7d es-
tablished a sigma-pi interaction with Ile146 residue. Subsequently,
molecular dynamic (MD) simulation was conducted to evaluate the
stability of 7d in complex with BRD4 using amber 12 package. As
shown in Figure S4 (provided in Mendeley Data) [61], the root means
square deviation (RMSD) was analysed during 50 ns. The small fluc-
tuations suggest BRD4 and 7d complex is stable. To investigate the
selectivity profile, 7d was next tested on other bromodomain-con-
taining proteins. As shown in Fig. 4c, 7d exhibited poor effects on FALZ,
EP300, BRD9 and TAF1(D2) with IC50 values over 30 μM or over 10 μM.
Although CREBBP and SMARCA2 inhibition was also observed by 7d
with IC50 values of 6.08 μM and 8.92 μM, 7d was over 20-fold, 16-fold
and 3-fold more potent towards BRD2/3/4, respectively. Taken to-
gether, these data demonstrate that 7d downregulates the intracellular
c-Myc expression and inhibits BET proteins with moderate selectivity.
MD simulation was conducted to evaluate the stability of 7d in complex
with AR. As shown in Figure S4 [61], RMSD was analysed during 50 ns.
The small fluctuations indicate that AR and 7d complex is stable. Taken
together, these data suggest that 7d competitively binds to AR and in-
hibits the activities of wt-AR and the mutant, leading to blockade of AR
signaling in prostate cancer.
3.5. Derivative 7d inhibits prostate cancer growth in vitro and in vivo
The in vitro antiproliferative activity and cell selectivity of 7d were
next evaluated through CCK-8 cytotoxicity assay. As shown in Fig. 6a,
compound 7d was more efficacious than Enzalutamide against AR-V7-
positive VCaP and 22Rv1 cells with IC50 values of 0.63 μM and 1.9 μM,
respectively. In AR-negative PC cells (PC-3, Du145), normal prostate
cells (WPMY-1), ovarian cancer cells (SK-OV-3) and colonic cancer cells
(HT-29), 7d exhibited poor antiproliferative activity with IC50 values
over 10 μM, suggesting 7d suppresses AR-positive PC cells with favor-
able selectivity.
To evaluate the in vivo anti-tumor activity of 7d, castrated male
BALB/c nude mice with VCaP or 22Rv1 xenografts were treated orally
once daily for 40 days and 24 days, respectively. As shown in Fig. 6b,
the growth of castration-resistant VCaP tumors was significantly in-
hibited by 7d with tumor growth inhibition (TGI) rates of 81.5%
(p = 0.0016) and 74.7% (p = 0.0074) for 50 mg/kg and 30 mg/kg,
which were more potent than Enzalutamide (43.0%, P = 0.1622) at the
same dosage. For Enza-resistant 22Rv1 model, combination of orch-
iectomy and 7d treatment (50 mg/kg, qd) inhibited tumor growth with
TGI of 53.9% (P = 0.029). As expected, Enzalutamide was less potent
(TGI = 24.8%) than 7d with average tumor volume equal to that of
castrated vehicle group (TGI = 21.6%), suggesting AR antagonism
didn’t contribute to 22Rv1 growth inhibition. As shown in Fig. 6c,
tumor weight was recorded at the end of treatment. In addition, both 7d
and Enzalutamide did not cause obvious weight loss during the treat-
ment (Figure S6) [61]. We next assessed the effects of 7d on AR and
BRD4 in tumors. Immunohistochemical analysis of VCaP tumors har-
vested from mice treated with 7d exhibited decreased c-Myc and PSA
levels compared to that from untreated animals (Fig. 6d). In western
blot analysis, decreased c-Myc protein was observed in 22Rv1 tumors
from 7d-treated mice (Fig. 6e). We have also made attempt to measure
the PSA protein through western blot but it failed possibly due to the
low expression of PSA in VCaP and 22Rv1 cells as shown in Fig. 5d.
3.4. Derivative 7d competitively binds to AR and disrupts the functions of
wt-AR and the mutant
The direct interaction between 7d and AR was next demonstrated by
a radioligand binding assay [48] in which 7d was measured in com-
petition with [3H] R1881. As displayed in Fig. 5a, 7d prevented [3H]
R1881 from binding to AR-LBD with higher efficacy than Enzalutamide
did. Recently, a missense mutation (F876L) happened in AR-LBD was
identified to confer resistance to Enzalutamide [22,23]. Therefore, lu-
ciferase reporter gene assay [48] was used to investigate the effect of 7d
on AR transactivation. As shown in Fig. 5b, compound 7d potently
suppressed androgen-induced transcriptional activities of wt-AR and
AR-F876L mutant. As expected, for AR-F876L, Enzalutamide treatment
caused increase in luminescence at 10 μM and 20 μM compared to those
at low concentration (0.1 μM and 1 μM), which was in accord with
Moilanen’s result [58]. Subsequently, the fluorescence-tagged AR was
used to evaluate 7d on AR nuclear localization [48]. As represented in
Fig. 5c, 7d prevented wt-AR and AR-F876L mutant from translocating
into nucleus. However, Enzalutamide was less efficacious towards AR-
F876L under the same conditions.
In order to evaluate the effect of 7d on AR signaling in prostate
cancer, we first measured PSA protein expression in AR-positive PC
cells (LNCaP, VCaP, 22Rv1) and AR-negative PC cells (PC-3, DU145) as
well as normal prostate cells (WPMY-1). Considering the low protein
level of intracellular PSA in VCaP and 22Rv1 cell (Fig. 5d), RT-PCR
analysis was performed to measure AR-regulated genes expression. As
illustrated in Fig. 5f, compound 7d inhibited R1881-induced PSA mRNA
expression in androgen-dependent LNCaP and VCaP cells. In AR-V7-
positive 22Rv1 cells, PSA expression was not sensitive to androgen
(Fig. 5e) but was significantly reduced by 7d (Fig. 5f), which might be
due to BET inhibition by 7d. Since BET family co-regulates AR-medi-
ated gene transcription through interacting with AR-NTD [38–41], it’s
reasonable why PSA expression of AR-V7-expressing 22Rv1 cells are
more sensitive to 7d than Enzalutamide. Besides, we also made attempt
to measure AR-V7 inhibition through luciferase reporter assay in AR-
V7-possitive 22Rv1 cells. Unexpected, extremely low luminescence
signals can be detected in the absence of androgen but the luminescence
can be induced by R1881 and decreased by Enzalutamide (Figure S4)
[61], so this experimental system can’t be used for AR-V7 evaluation.
Utilizing the X-ray cocrystal structures of AR (PDB ID: 3V49) [51],
molecular docking simulation [53] was performed to investigate the
possible binding mode. As shown in Fig. 5g, the oxygen atom of iso-
xazole formed a hydrogen bond interaction with His874 residue of AR.
3-Me substituent on isoxazole moiety and N-ethyl group of 7d occupied
the hydrophobic grooves formed by Ile898, Met895, Trp741 and
Ieu704, Met780, Phe876 residues of AR, respectively. Subsequently, the
4. Discussion
Resistance to traditional AR antagonists targeting LBD remains a
major challenge in the treatment of CRPC. NTD is essential for the
transcriptional activity of full-length AR as well as truncated AR var-
iants. Although BET family physically interacts with AR-NTD and co-
regulates AR-related gene expression, BETi-resistant CRPC cells display
BRD-independent transcription and increased sensitivity to AR
blockade [45]. Therefore, molecules showing simultaneous AR and BET
inhibition hold promise to disrupt the functions of full-length AR as
well as splicing AR variants lacking LBD and overcome antiandrogen
resistance.
In this study, thirteen diphenylamine derivatives were developed
and evaluated towards BRD4 and wt-AR. Structure-activity relationship
analysis indicates that imidazole and 3, 5-dimethylisoxazole groups are
crucial for BRD4 and AR inhibition. The position of methyl on benzene
ring and ethyl on nitrogen bridge slightly affect inhibitory activities
towards AR and BRD4. In addition, the imidazole group contributes to
improved metabolic stability in vitro and oral drug exposure, which
makes 7d potentially druggable. The understanding of structure me-
chanisms is of great help for further structural modification of 7d.
Mechanism studies demonstrated that compound 7d suppressed the
functions of AR and BET in prostate cancer cells. 7d competitively
bound to AR, suppressed AR nuclear localization and transcriptional
10

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
(a)
(b)
AR-F876L
wt-AR
Competitive AR binding
7d
Enza
7d
Enza
80
60
40
20
0
100
100
50
0
NS
Enza
7d
**
*
*
*
**
*
**
***
50
0
*
***
**
**
***
***
**
***
***
***
**
***
***
***
1
-20
10
20
1
3
0.1
39
10
30
625
156
0.1
2500
10000
DMSO
DMSO
Concentration (nM)
AR-F876L-EGFP
(c)
Nucleus
Merge
wt-AR-EGFP
Nucleus
Merge
DMSO
DMSO
+ R1881
Enza
+ R1881
7d
+ R1881
Fig. 5. Derivative 7d binds to AR and inhibits wt-AR and the mutant. (a) Competitive AR binding assay. 1 nM [3H] R1881 and LNCaP cytosol were used. Data are
expressed as the mean sd (n = 3). (b) Luciferase reporter assay of wt-AR and AR-F876L mutant. Data are expressed as the mean sem (n = 3). (c) Nuclear
localization of wt-AR and AR-F876L mutant. WPMY-1 cells were treated with DMSO or compounds (10 μM) in the presence or absence of 0.5 nM R1881 for 24 h.
Hoechst 33,342 was used to label the nuclei (blue). (d) Western blot analysis of AR and PSA in different cells. Cells were tested in three independent experiments. (e)
The mRNA expression of PSA in different prostate cancer cells. The concentration of R1881 is 0.2 nM. Data are expressed as the mean
stripped FBS. (f) RT-PCR analysis for 0.2 nM R1881-induced PSA mRNA expression. LNCaP, VCaP and 22Rv1 cells were treated with vehicle (DMSO) or compounds
for 24 h. Data are expressed as the mean sd (n = 3). (g) The predicted binding mode and 2D diagram of compound 7d in complex with AR (PDB ID: 3 V49). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
sd (n = 3). CSS, charcoal
activities of wt-AR and F876L mutant that confers resistance to
Enzalutamide, leading to decreased PSA mRNA expression in LNCaP,
VCaP and 22Rv1 cells. Notably, 7d simultaneously inhibited BET family
with moderate selectivity and decreased downstream c-Myc protein
expression in three different PC cells, which is totally distinct from
traditional AR antagonists. In antiproliferative assay, 7d selectively
suppressed AR-positive prostate cancer cells with higher potency than
Enzalutamide. 22Rv1 cells are androgen-independent that express AR-
V7 mutant and exhibit resistance to antiandrogens. Our findings suggest
the antiproliferative activity of 7d towards 22Rv1 could result from BET
inhibition. Finally, castration-resistant VCaP and 22Rv1 xenografts
were significantly suppressed by 7d with higher efficacy than
Enzalutamide. Therefore, 7d represents a promising lead compound to
address antiandrogen-resistant prostate cancer expressing truncated AR
variants. By far, we haven’t seen any other molecules that simulta-
neously suppress the functions of AR and BET family in prostate cancer,
so 7d might be a first-in-class inhibitor.
Collectively, we developed a new class of diphenylamine derivatives
featuring important imidazole and 3, 5-dimethylisoxazole substituents,
which can simultaneously inhibit ARs and BET family in prostate
cancer. Structural modification improves the PK property and anti-
tumor activity in vitro and in vivo, making 7d potentially druggable.
Compared to Enzalutamide, 7d decreases c-Myc expression, exhibits
higher AR binding affinity, better AR-F876L inhibiton and suppresses
PSA expression of AR-V7-expressing 22Rv1 cell more potently.
Importantly, it shows enhanced anti-tumor activity towards VCaP and
22Rv1 in vitro and in vivo. Therefore, derivative 7d represents a po-
tential alternative for the development of novel structures to treat CRPC
and overcome antiandrogen resistance. Our further interests would be
focused on improving the activity of 7d towards BET family through
structural optimization and further demonstrating the advantages of
dual inhibitor through comparing to combination of antiandrogens and
BET inhibitors. However, it’s definitely hard to maintain inhibitory
activity towards both AR and BET family as well as metabolic stability
11

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
(d)
(e)
P S A
1 2
1 0
8
ARs
(low exposure)
ARs
6
(high exposure)
4
PSA
(low exposure)
2
PSA
(high exposure)
0
P
P
P
P
v 1
a
Tublin
C
R V 1
2
V C a
N C a
V C a
2 2 R
2
L N
L
F B S
C S S + R 1881
(f)
22Rv1
VCaP
LNCaP
1.5
1.2
0.9
0.6
0.3
0.0
1.2
0.9
0.6
0.3
0.0
7d
7d
1.2
0.9
0.6
0.3
7d
NS
Enza
Enza
NS
Enza
NS
*
**
**
***
**
*
*
**
**
***
***
**
***
**
***
***
***
***
***
10
0.0
0
0
0.2
1
5
0
0.2
1
5
0.2
1
5
(g)
Fig. 5. (continued)
Declaration of Competing Interest
at the same time during structural modificaiton. Perhaps, a hetero-bi-
functional PROTAC (Proteolysis Targeting Chimera) derived from 7d
that recruits BRDs protein and AR to the E3 ubiquitin ligase would be a
promising stragegy to enhance and prolong activity.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
CRediT authorship contribution statement
Acknowledgments
Jiang Yu: Conceptualization, Methodology, Validation, Formal
analysis, Investigation, Writing - original draft, Writing - review &
editing, Project administration. Peiting Zhou: Validation, Formal
analysis, Investigation. Wu Du: Conceptualization. Ruixue Xu:
Investigation. Guoyi Yan: Investigation. Yufang Deng: Investigation.
Xinghai Li: Conceptualization, Methodology, Writing - review &
editing. Yuanwei Chen: Conceptualization, Supervision, Funding ac-
quisition.
We gratefully acknowledge the financial support from the National
Natural Science Foundation of China (81472418 and 81672951). We
thank Dr. Lei Fan, Zhilin Tu, Yongxu Huo in Hinova Pharmaceuticals
Inc. for their suggestions to this research. Authors also thank Drs.
Qinhuai Lai and Weirong Lai for their assistance for HPLC analysis of
compounds.
12

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
(a)
7d
22Rv1
VCaP
WPMY-1
DU 145
SK-OV-3
HT-29
7d
100
50
0
100
50
0
100
50
0
7d
Enza
Enza
PC-3
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Log (nM)
Log (nM)
Log (nM)
(b)
(c)
Growth inhibition of VCaP cells in vivo
Growth inhibition of 22Rv1 cells in vivo
2 .5
2 .0
1 .5
1 .0
0 .5
0 .0
Vehicle
1500
2.5
2.0
1.5
1.0
0.5
Sham
2500
NS
Enza 30 mg/kg
Vehicle
7d 30 mg/kg
2000
1500
1000
500
0
Enza 50 mg/kg
7d 50 mg/kg
NS
NS
7d 50 mg/kg
1000
NS
*
500
**
**
e
g
g
k
a
l
m
0
k
z
c
/
/
i
g
g
7d
h
0
10
20
30
40
0
4
8
12
16
20
24
E n
S h a
Enza
V e
Shame
Vehicle
Days after treatment
5 0
m
3 0 m
Days after treatment
7 d
7 d
(d)
(e)
Vehicle
7d (50 mg/kg)
G ra y sca le an a ly sis
PSA
N S
1 .0
0 .5
0 .0
7d
20
c-Myc
Tublin
20
****
e
l
m
7 d
c
c-Myc
i
h
S h a
V e
20
20
Fig. 6. Derivative 7d inhibits prostate cancer growth in vitro and in vivo. (a) survival curves of different cells. Data are expressed as the mean
sd (n = 2).
Compounds are tested in at least two independent experiments with duplicate measurements. (b) In vivo xenografts study. Castrated BALB/c male nude mice with
VCaP and 22Rv1 xenografts were treated orally once daily. The tumor volume is expressed as the mean sem (n = 7–8). Sham, sham-operated group. (c) Tumor
weight of VCaP and 22Rv1 xenografts at the end of treatment. Data are expressed as the mean sem. Statistical significance was determined by two-tailed Student’s
t test. NS, no significant difference. (d) Immunohistochemical staining of PSA and c-Myc in VCaP xenografts. (e) Western blot and grayscale analysis of c-Myc
expression in 22Rv1 tumors. Samples from four tumors were combined together to get the total protein for each group. Data are expressed as the mean sd (n = 3).
References
increased survival in metastatic prostate cancer, N. Engl. J. Med. 364 (2011)
1995–2005.
[9] H.I. Scher, K. Fizazi, F. Saad, M.E. Taplin, C.N. Sternberg, K. Miller, R. De Wit,
P. Mulders, K.N. Chi, N.D. Shore, A.J. Armstrong, T.W. Flaig, A. Fléchon,
P. Mainwaring, M. Fleming, J.D. Hainsworth, M. Hirmand, B. Selby, L. Seely, J.S. De
Bono, Increased survival with enzalutamide in prostate cancer after chemotherapy,
N. Engl. J. Med. 367 (2012) 1187–1197.
[10] M.R. Smith, F. Saad, S. Chowdhury, S. Oudard, B.A. Hadaschik, J.N. Graff,
D. Olmos, P.N. Mainwaring, J.Y. Lee, H. Uemura, A. Lopez-Gitlitz, G.C. Trudel,
B.M. Espina, Y.Y. Shu, Y.C. Park, W.R. Rackoff, M.K. Yu, E.J. Small, Apalutamide
treatment and metastasis-free survival in prostate cancer, N. Engl. J. Med. 378
(2018) 1408–1418.
[11] U.S. FDA approves darolutamide, a new treatment for men with non-metastatic
castration-resistant prostate cancer (nmCRPC). July 31, 2019, https://www.orion.
fi/en/Orion-group/media/press-releases/2019/u.s.-fda-approves-darolutamide-a-
new-treatment-for-men-with-non-metastatic-castration-resistant-prostate-cancer-
nmcrpc/ (accessed Sep 17, 2019).
[1] C.J. Brown, S.J. Goss, D.B. Lubahn, D.R. Joseph, E.M. Wilson, F.S. French,
H.F. Willard, Androgen receptor locus on the human X chromosome: regional lo-
calization to Xq11-12 and description of a DNA polymorphism, Am. J. Hum. Genet
44 (1989) 264–269.
[2] J.L. Mohler, C.W. Gregory, O.H. Ford III, D. Kim, C.M. Weaver, P. Petrusz,
E.M. Wilson, F.S. French, The androgen axis in recurrent prostate cancer, Clin.
Cancer Res. 10 (2004) 440–448.
[3] C.A. Heinlein, C. Chang, Androgen receptor in prostate cancer, Endocr. Rev. 25
(2004) 276–308.
[4] C.D. Chen, D.S. Welsbie, C. Tran, S.H. Baek, R. Chen, R. Vessella, M.G. Rosenfeld,
C.L. Sawyers, Molecular determinants of resistance to antiandrogen therapy, Nat.
Med. 10 (2004) 33–39.
[5] H.I. Scher, C.L. Sawyers, Biology of progressive, castration-resistant prostate cancer:
directed therapies targeting the androgen-receptor signaling axis, J. Clin. Oncol. 23
(2005) 8253–8261.
[12] HC-1119 Versus Enzalutamide in Metastatic Castration-Resistant Prostate Cancer
(mCRPC). https://www.clinicaltrials.gov/ct2/results?cond=Prostate+Cancer&
term=HC1119&cntry=&state=&city=&dist= (accessed Sep 17, 2019).
[13] N. D. James, J. S. De Bono, M. R. Spears, N. W. Clarke, M. D. Mason, D. P.
Dearnaley, A. W. Ritchie, C. L. Amos, C. Gilson, R. J. Jones, D. Matheson, R.
Millman, G. Attard, S. Chowdhury, W. R. Cross, S. Gillessen, C. C. Parker, J. M.
Russell, D. R. Berthold, C. Brawley, F. Adab, S. Aung, A. J. Birtle, J. Bowen, S.
Brock, P. Chakraborti, C. Ferguson, J. Gale, E. Gray, M. Hingorani, P. J. Hoskin, J. F.
Lester, Z. I. Malik, F. Mckinna, N. Mcphail, J. Money – Kyrle, J. O’sullivan, O.
Parikh, A. Protheroe, A. Robinson, N. N. Srihari, C. Thomas, J. Wagstaff, J. Wylie, A.
Zarkar, M. K. Parmar, M. R. Sydes. Abiraterone for prostate cancer not previously
treated with hormone therapy. N. Engl. J. Med. 2017, 377, 338–351.
[14] T.M. Beer, A.J. Armstrong, D.E. Rathkopf, Y. Loriot, C.N. Sternberg, C.S. Higano,
[6] S.P. Balk, Androgen receptor as a target in androgen-independent prostate cancer,
Urology. 60 (2002) 132–138.
[7] B.S. Taylor, N. Schultz, H. Hieronymus, A. Gopalan, Y. Xiao, B.S. Carver, V.K. Arora,
P. Kaushik, E. Cerami, B. Reva, Y. Antipin, N. Mitsiades, T. Landers, I. Dolgalev,
J.E. Major, M. Wilson, N.D. Socci, A.E. Lash, A. Heguy, J.A. Eastham, H.I. Scher,
V.E. Reuter, P.T. Scardino, C. Sander, C.L. Sawyers, W.L. Gerald, Integrative
genomic profiling of human prostate cancer, Cancer Cell 18 (2010) 11–22.
[8] J.S. de Bono, C.J. Logothetis, A. Molina, K. Fizazi, S. North, L. Chu, K.N. Chi,
R.J. Jones, O.B. Goodman, F. Saad, J.N. Staffurth, P. Mainwaring, S. Harland,
T.W. Flaig, T.E. Hutson, T. Cheng, H. Patterson, J.D. Hainsworth, C.J. Ryan,
C.N. Sternberg, S.L. Ellard, A. Fléchon, M. Saleh, M. Scholz, E. Efstathiou, A. Zivi,
D. Bianchini, Y. Loriot, N. Chieffo, T. Kheoh, C.M. Haqq, H.I. Scher, Abiraterone and
13

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
agents for the degradation of androgen receptor variants to treat castration-resistant
prostate cancer, Cancer Res. 77 (2017) 6282–6298.
P. Iversen, S. Bhattacharya, J. Carles, S. Chowdhury, I.D. Davis, J.S. de Bono,
C.P. Evans, K. Fizazi, A.M. Joshua, C.S. Kim, G. Kimura, P. Mainwaring,
H. Mansbach, K. Miller, S.B. Noonberg, F. Perabo, D. Phung, F. Saad, H.I. Scher,
M.E. Taplin, P.M. Venner, B. Tombal, Enzalutamide in metastatic prostate cancer
before chemotherapy, N. Engl. J. Med. 371 (2014) 424–433.
[36] M.T. Schweizer, K. Haugk, R. Gulati, H.H. Cheng, J.L. Maes, R.F. Dumpit,
P.S. Nelson, B. Montgomery, J.S. McCune, S.R. Plymate, E.Y. Yu, A phase I study of
niclosamide in combination with enzalutamide in men with castration-resistant
prostate cancer, PLoS ONE 13 (2018) e0198389.
[37] V. Sahai, A.J. Redig, K.A. Collier, F.D. Eckerdt, H.G. Munshi, Targeting BET bro-
modomain proteins in solid tumors, Oncotarget 7 (2016) 53997–54009.
[38] I.A. Asangani, V.L. Dommeti, X. Wang, R. Malik, M. Cieslik, R. Yang, J. Escara-
Wilke, K. Wilder-Romans, S. Dhanireddy, C. Engelke, M.K. Iyer, X. Jing, Y. Wu,
X. Cao, Z. Qin, S. Wang, F. Feng, A.M. Chinnaiyan, Therapeutic targeting of BET
bromodomain proteins in castration-resistant prostate cancer, Nature 510 (2014)
278–282.
[15] Z. Guo, X. Yang, F. Sun, R.C. Jiang, D.E. Linn, H. Chen, X. Kong, J. Melamed,
C.G. Tepper, H.J. Kung, A.M. Brodie, J. Edwards, Y. Qiu, A novel androgen receptor
splice variant is up-regulated during prostate cancer progression and promotes
androgen depletion-resistant growth, Cancer Res. 69 (2009) 2305–2313.
[16] R. Hu, T.A. Dunn, S.Z. Wei, S. Isharwal, R.W. Veltri, E. Hum-phreys, M. Han,
A.W. Partin, R.L. Vessella, W.B. Isaacs, G. Steven Bova, J. Luo, Ligand-independent
androgen receptor variants derived from splicing of cryptic exons signify hormone-
refractory prostate cancer, Cancer Res. 69 (2009) 16–22.
[17] Y. Li, S. Chan, L.J. Brand, T.H. Hwang, K.A. Silverstein, S.M. Dehm, Androgen re-
ceptor splice variants mediate enzalutamide resistance in castration-resistant
prostate cancer cell lines, Cancer Res. 73 (2013) 483–489.
[18] M. Nakazawa, C. Lu, Y. Chen, C.J. Paller, M.A. Carducci, M.A. Eisenberger, J. Luo,
E.S. Antonarakis, Serial blood-based analysis of AR-V7 in men with advanced
prostate cancer, Ann Oncol. 26 (2015) 1859–1865.
[19] E.S. Antonarakis, C. Lu, H. Wang, B. Luber, M. Nakazawa, J.C. Roeser, Y. Chen,
T.A. Mohammad, Y. Chen, H.L. Fedor, T.L. Lotan, Q. Zheng, A.M. De Marzo,
J.T. Isaacs, W.B. Isaacs, R. Nadal, C.J. Paller, S.R. Denmeade, M.A. Carducci,
M.A. Eisenberger, J. Luo, AR-V7 and resistance to enzalutamide and abiraterone in
prostate cancer, N. Engl. J. Med. 371 (2014) 1028–1038.
[20] L.B. Guedes, C.L. Morais, F. Almutairi, M.C. Haffner, Q. Zheng, J.T. Isaacs,
E.S. Antonarakis, C. Lu, H. Tsai, J. Luo, A.M. De Marzo, T.L. Lotan, Analytic vali-
dation of RNA in situ hybridization (RISH) for AR and AR-V7 expression in human
prostate cancer, Clin. Cancer Res. 22 (2016) 4651–4663.
[21] F. Qu, W. Xie, M. Nakabayashi, H. Zhang, S.H. Jeong, X. Wang, K. Komura,
C.J. Sweeney, O. Sartor, G.M. Lee, P.W. Kantoff, Association of AR-V7 and prostate-
specific antigen RNA levels in blood with efficacy of abiraterone acetate and en-
zalutamide treatment in men with prostate cancer, Clin Cancer Res. 23 (2017)
726–734.
[22] M. Korpal, J.M. Korn, X. Gao, D.P. Rakiec, D.A. Ruddy, S. Doshi, J. Yuan,
S.G. Kovats, S. Kim, V.G. Cooke, J.E. Monnahan, F. Stegmeier, T.M. Roberts,
W.R. Sellers, W. Zhou, P. Zhu, An F876L mutation in androgen receptor confers
genetic and phenotypic resistance to MDV3100 (enzalutamide), Cancer Discov. 3
(2013) 1030–1043.
[23] J.D. Joseph, N. Lu, J. Qian, J. Sensintaffar, G. Shao, D. Brigham, M. Moon,
E.C. Maneval, I. Chen, B. Darimont, J.H. Hager, A clinically relevant androgen re-
ceptor mutation confers resistance to second-generation antiandrogens en-
zalutamide and ARN-509, Cancer Discov. 3 (2013) 1020–1029.
[24] R. Andersen, et al., Regression of castrate-recurrent prostate cancer by a small-
molecule inhibitor of the amino-terminus domain of the androgen receptor, Cancer
Cell 17 (2010) 535–546.
[39] P. Filippakopoulos, J. Qi, S. Picaud, Y. Shen, W.B. Smith, O. Fedorov, E.M. Morse,
T. Keates, T.T. Hickman, I. Felletar, M. Philpott, S. Munro, M.R. McKeown, Y. Wang,
A.L. Christie, N. West, M.J. Cameron, B. Schwartz, T.D. Heightman, N. La Thangue,
C.A. French, O. Wiest, A.L. Kung, S. Knapp, J.E. Bradner, Selective inhibition of BET
bromodomains, Nature 468 (2010) 1067–1073.
[40] A. Wyce, Y. Degenhardt, Y. Bai, B. Le, S. Korenchuk, M.C. Crouthamel,
C.F. McHugh, R. Vessella, C.L. Creasy, P.J. Tummino, O. Barbash, Inhibition of BET
bromodomain proteins as a therapeutic approach in prostate cancer, Oncotarget 4
(2013) 2419–2429.
[41] S.C. Chan, L.A. Selth, Y. Li, M.D. Nyquist, L. Miao, J.E. Bradner, G.V. Raj,
W.D. Tilley, S.M. Dehm, Targeting chromatin binding regulation of constitutively
active AR variants to overcome prostate cancer resistance to endocrine-based
therapies, Nucleic Acids Res. 43 (2015) 5880–5897.
[42] J. Welti, A. Sharp, W. Yuan, D. Dolling, D.N. Rodrigues, I. Figueiredo, V. Gil,
A. Neeb, M. Clarke, G. Seed, M. Crespo, S. Sumanasuriya, J. Ning, E. Knight,
J.C. Francis, A. Hughes, W.S. Halsey, A. Paschalis, R.S. Mani, G.V. Raj, S.R. Plymate,
S. Carreira, G. Boysen, A.M. Chinnaiyan, A. Swain, J.S. de Bono, Targeting
Bromodomain and Extra-Terminal (BET) family proteins in castration-resistant
prostate cancer (CRPC), Clin. Cancer Res. 24 (2018) 3149–3162.
[43] A. Urbanucci, S.J. Barfeld, V. Kytölä, H.M. Itkonen, I.M. Coleman, D. Vodák,
L. Sjöblom, X. Sheng, T. Tolonen, S. Minner, C. Burdelski, K.K. Kivinummi,
A. Kohvakka, S. Kregel, M. Takhar, M. Alshalalfa, E. Davicioni, N. Erho, P. Lloyd,
R.J. Karnes, A.E. Ross, E.M. Schaeffer, G. Vander, J. Donald, S. Knapp, E. Corey,
F. Feng, P.S. Nelson, F. Saatcioglu, K.E. Knudsen, T.L. Tammela, G. Sauter,
T. Schlomm, M. Nykter, T. Visakorpi, I.G. Mills, Androgen receptor deregulation
drives bromodomain-mediated chromatin alterations in prostate cancer, Cell Rep.
19 (2017) 2045–2059.
[44] I.A. Asangani, K. Wilder-Romans, V.L. Dommeti, P.M. Krishnamurthy, I.J. Apel,
J. Escara-Wilke, S.R. Plymate, N.M. Navone, S. Wang, F. Feng, A.M. Chinnaiyan,
BET, bromodomain inhibitors enhance efficacy and disrupt resistance to ar an-
tagonists in the treatment of prostate cancer, Mol. Cancer Res. 14 (2016) 324–331.
[45] A. Pawar, P.N. Gollavilli, S.M. Wang, I.A. Asangani, Resistance to BET inhibitor
leads to alternative therapeutic vulnerabilities in castration-resistant prostate
cancer, Cell Rep. 22 (2018) 2236–2245.
[46] Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of GS-5829 as a
Single Agent and in Combination with Enzalutamide in Participants with Metastatic
Castrate-Resistant Prostate Cancer. Clinical Trials, Nov 17, 2015, https://
clinicaltrials.gov/ct2/show/NCT02607228 (accessed Oct 11, 2018).
[47] A Study of ZEN003694 in Patients with Metastatic Castration Resistant Prostate
Cancer. Clinical Trials, Mar 10, 2016, https://clinicaltrials.gov/ct2/show/
NCT02705469 (accessed Oct 11, 2018).
[48] J. Yu, L. Zhang, G. Yan, P. Zhou, C. Cao, F. Zhou, X. Li, Y. Chen, Discovery and
biological evaluation of novel androgen receptor antagonist for castration-resistant
prostate cancer, Eur. J. Med. Chem. 171 (2019) 265–281.
[25] L.G. Meimetis, D.E. Williams, N.R. Mawji, C.A. Banuelos, A.A. Lal, J.J. Park,
A.H. Tien, J.G. Fernandez, N.J. de Voogd, M.D. Sadar, R.J. Andersen, Niphatenones,
glycerol ethers from the sponge Niphates digitalis block androgen receptor tran-
scriptional activity in prostate cancer cells: structure elucidation, synthesis, and
biological activity, J. Med. Chem. 55 (2011) 503–514.
[26] R. Rosati, et al., Strategy for Tumor-selective disruption of androgen receptor
function in the spectrum of prostate cancer, Clin. Cancer Res. 24 (2018)
6509–6522.
[27] Safety and anti-tumor study of oral EPI-506 for patients with metastatic castration-
resistant prostate cancer. NLM Identifier: NCT02606123, https://www.
clinicaltrials.gov/ct2/results?term=NCT02606123&cond=prostate+cancer&
rank=1#rowId0 (accessed Sep 17, 2019).
[28] H. Li, et al., Discovery of small-molecule inhibitors selectively targeting the DNA-
binding domain of the human androgen receptor, J. Med. Chem. 57 (2014)
6458–6467.
[29] K. Dalal, et al., Selectively targeting the DNA-binding domain of the androgen re-
ceptor as a prospective therapy for prostate cancer, J. Biol. Chem. 289 (2014)
26417–26429.
[30] D. Xu, Y. Zhan, Y. Qi, B. Cao, S. Bai, W. Xu, S.S. Gambhir, P. Lee, O. Sartor,
E.K. Flemington, H. Zhang, C. Hu, Y. Dong, Androgen receptor splice variants di-
merize to transactivate target genes, Cancer Res. 75 (2015) 3663–3671.
[31] M. Wo Streicher, A. Luedeke, F. Azoitei, A. Zengerling, F. Herweg, M.G. Genze,
A.J. Schrader, M.V. Schrader, Cronauer, Stilbene induced inhibition of androgen
receptor dimerization: implications for AR and ARΔLBD-Signaling in Human
Prostate Cancer Cells, PLoS ONE 9 (2014) e98566.
[32] K. Dalala, F. Bana, H. Lia, H. Morina, M. Roshan-Moniria, K.J.T.A. Shepherda,
A. Sharmaa, J. Peacocka, M.L. Carlsonb, E. LeBlanca, C. Pereza, F. Duongb,
C.J. Onga, P.S. Renniea, A. Cherkasov, Selectively targeting the dimerization in-
terface of human androgen receptor with small-molecules to treat castration-re-
sistant prostate cancer, Cancer Lett. 437 (2018) 35–43.
[49] D.S. Hewings, M. Wang, M. Philpott, O. Fedorov, S. Uttarkar, P. Picaud,
S. Filippakopoulos, C. Vuppusetty, B. Marsden, S. Knapp, S.J. Conway,
T.D. Heightman, 3,5-dimethylisoxazoles act as acetyl-lysine-mimetic bromodomain
ligands, J. Med. Chem. 54 (2011) 6761–6770.
[50] Z. Liu, P. Wang, H. Chen, E.A. Wold, B. Tian, A.R. Brasier, J. Zhou, Drug discovery
targeting bromodomain-containing protein 4, J. Med. Chem. 60 (2017) 4533–4558.
[51] F. Nique, S. Hebbe, C. Peixoto, D. Annoot, J. Lefrançois, E. Duval, L. Michoux,
N. Triballeau, J. Lemoullec, P. Mollat, M. Thauvin, T. Prangé, D. Minet, P. Clément-
Lacroix, C. Robin-Jagerschmidt, D. Fleury, D. Guédin, P. Deprez, Discovery of
diarylhydantoins as new selective androgen receptor modulators, J. Med. Chem. 55
(2012) 8225–8235.
[52] D.S. Hewings, M. Wang, M. Philpott, O. Fedorov, S. Uttarkar, P. Filippakopoulos,
S. Picaud, C. Vuppusetty, B. Marsden, S. Knapp, S.J. Conway, T.D. Heightman, 3,5-
dimethylisoxazoles act as acetyl-lysine-mimetic bromodomain ligands, J. Med.
Chem. 54 (2011) 6761–6770.
[53] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Comput. Chem. 30 (2009) 2785–2791.
[33] C. Liu, W. Lou, Y. Zhu, N. Nadiminty, C.T. Schwartz, C.P. Evans, A.C. Gao,
Niclosamide inhibits androgen receptor variants expression and overcomes en-
zalutamide resistance in castration-resistant prostate cancer, Clin. Cancer Res. 20
(2014) 3198–3210.
[54] P. Purushottamachar, A.K. Kwegyir-Afful, M.S. Martin, V.P. Ramamurthy,
S. Ramalingam, V.C. Njar, Identification of novel steroidal androgen receptor de-
grading agents inspired by galeterone 3β-imidazole carbamate, ACS Med. Chem.
Lett. 7 (2016) 708–713.
[34] S. Yamashita, K. Lai, K. Chuang, D. Xu, H. Miyamoto, T. Tochigi, S. Pang, L. Li,
Y. Arai, H. Kung, S. Yeh, C. Chang, ASC-J9 suppresses castration-resistant prostate
cancer growth through degradation of full-length and splice variant androgen re-
ceptors, Neoplasia 14 (2012) 74–83.
[35] S. Ponnusamy, C.C. Coss, T. Thiyagarajan, K. Watts, D. Hwang, Y. He, L.A. Selth,
I.J. McEwan, C.B. Duke, J. Pagadala, G. Singh, R.W. Wake, C. Ledbetter,
W.D. Tilley, T. Moldoveanu, J.T. Dalton, D.D. Miller, R. Narayanan, Novel selective
[55] C. Tran, S. Ouk, N.J. Clegg, Y. Chen, P.A. Watson, V. Arora, J. Wongvipat,
P.M. Smith-Jones, D.W. Yoo, A. Kwon, T. Wasielewska, D. Welsbie, C. Chen,
C.S. Higano, T.M. Beer, D.T. Hung, H.I. Scher, M.E. Jung, C.L. Sawyers,
Development of a second-generation antiandrogen for treatment of advanced
prostate cancer, Science 324 (2009) 787–790.
[56] R. Hu, T.A. Dunn, S.Z. Wei, S. Isharwal, R.W. Veltri, E. Humphreys, M. Han,
A.W. Partin, R.L. Vessella, W.B. Isaacs, G.S. Bova, J. Luo, Ligand-independent
14

J. Yu, et al.
BiochemicalPharmacology177(2020)113946
androgen receptor variants derived from splicing of cryptic exons signify hormone-
prostate cancer (CRPC), J. Med. Chem. 61 (2018) 3037–3058.
refractory prostate cancer, Cancer Res. 69 (2009) 16–22.
[60] J. Shaw, M. Leveridge, C. Norling, J. Karén, D.M. Molina, D. O’Neill, J.E. Dowling,
P. Davey, S. Cowan, M. Dabrowski, M. Main, D. Gianni, Determining direct binders
of the Androgen receptor using a high throughput cellular thermal shift assay, Sci.
Rep. 8 (2018) 1–11.
[61] Y. Chen, J. Yu, P. Zhou, W. Du, R. Xu, G. Yan, Y. Deng, Supporting information for
metabolically stable diphenylamine derivatives suppress androgen receptor and
BET protein in prostate cancer, Mendeley Data 1 (2020), https://doi.org/10.17632/
pvrjnfr9zj.1.
[58] A.M. Moilanen, R. Riikonen, R. Oksala, L. Ravanti, E. Aho, G. Wohlfahrt,
P.S. Nykänen, O.P. Törmäkangas, J.J. Palvimo, P.J. Kallio, Discovery of ODM-201, a
new-generation androgen receptor inhibitor targeting resistance mechanisms to
androgen signaling-directed prostate cancer therapies, Sci. Rep. 5 (2015) 12007.
[59] M. Zhang, Y. Zhang, M. Song, X. Xue, J. Wang, C. Wang, C. Zhang, C. Li, Q. Xiang,
L. Zou, X. Wu, C. Wu, B. Dong, W. Xue, Y. Zhou, H. Chen, D. Wu, K. Ding, Y. Xu,
Structure-based discovery and optimization of Benzo[d]isoxazole derivatives as
potent and selective BET inhibitors for potential treatment of castration-resistant
15