Discovery of deshydroxy bicalutamide derivatives as androgen receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2019.01.054

Deshydroxy propioanilides were synthesised by Michael addition reaction between substituted thiophenols onto four different phenylacrylamide derivatives to give twenty-three novel deshydroxy bicalutamide derivatives lacking the central hydroxyl group. The antiproliferative activities of these compounds were evaluated against human prostate cancer cell lines and thirteen compounds showed better inhibitory activities (IC₅₀ = 2.67–13.19 μM) compared to bicalutamide (IC₅₀ = 20.44 μM) in LNCaP. Remarkably, novel double branched bicalutamide analogues (27 and 28) were isolated as major by-products and found to have the best activity across three human prostate cancer cell lines (LNCaP, VCaP and PC3). The most active compound 28 shows sub-micromolar activity (IC₅₀ = 0.43 μM in LNCaP), which represents more than 40-fold improvement over the clinical anti-androgen bicalutamide (IC₅₀ = 20.44 μM) and a more than 3 fold improvement over enzalutamide (IC₅₀ = 1.36 μM). Moreover, strong reduction of PSA expression in LNCaP cells upon treatment with compounds 27, 28 and 33 was observed during qPCR analysis, confirming their AR antagonist activity. Molecular modelling studies revealed a novel binding mode of these structurally distinct double branched analogues within the ligand binding domain (LBD) of the androgen receptor.

Full text of DOI:10.1016/j.ejmech.2019.01.054

Accepted Manuscript
Discovery of deshydroxy bicalutamide derivatives as androgen receptor antagonists
Sahar Kandil, Kok Yung Lee, Laurie Davies, Sebastiano A. Rizzo, D. Alwyn Dart,
Andrew D. Westwell
PII:
S0223-5234(19)30067-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.054
EJMECH 11061
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 November 2018
Revised Date: 9 January 2019
Accepted Date: 22 January 2019
Please cite this article as: S. Kandil, K.Y. Lee, L. Davies, S.A. Rizzo, D.A. Dart, A.D. Westwell,
Discovery of deshydroxy bicalutamide derivatives as androgen receptor antagonists, European Journal
of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.01.054.
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ACCEPTED MANUSCRIPT
Discovery of deshydroxy bicalutamide derivatives as androgen receptor
antagonists
1
*
1
2
1
2
1
Sahar Kandil , Kok Yung Lee , Laurie Davies , Sebastiano A. Rizzo , D. Alwyn Dart and Andrew D. Westwell
1
2
School of Pharmacy & Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB, Wales, UK. Cardiff
China Medical Research Collaborative, Cardiff University School of Medicine, Cardiff, CF14 4XN, Wales, UK.
Keywords: Androgen receptor (AR), prostate cancer (PC), castration-resistant prostate cancer (CRPC), bicalutamide,
deshydroxy, double branched propioanilide.
Graphical abstract
Highlights





Synthesis of 23 novel deshydroxy bicalutamide derivatives.
Identification of novel and potent double branched AR antagonists.
Enhanced anticancer activity against PC compared to bicalutamide and enzalutamide.
Strong downregulation of PSA expression in qPCR analysis of LnCaP cell line.
Molecular modelling provides a rational explanation of the SAR observed.
Abstract
Deshydroxy propioanilides were synthesised by Michael addition reaction between substituted
thiophenols onto four different phenylacrylamide derivatives to give twenty-four novel deshydroxy
bicalutamide derivatives lacking the central hydroxyl group. The antiproliferative activities of these
compounds were evaluated against human prostate cancer cell lines and eleven compounds showed
better inhibitory activities (IC50 = 2.67-13.19 uM) compared to bicalutamide (IC50 = 20.44 uM) in
LNCaP. Remarkably, novel double branched bicalutamide analogues (27 and 28) were isolated as
major by-products and found to have the best activity across three human prostate cancer cell lines
(LNCaP, VCaP and PC3). The most active compound 28 shows sub-micromolar activity (IC50 = 0.43 uM
in LNCaP), which represents more than 40-fold improvement over the clinical anti-androgen
bicalutamide (IC50 = 20.44 uM) and a more than 3 fold improvement over enzalutamide (IC50 = 1.36
uM). Moreover, strong reduction of PSA expression in LNCaP cells upon treatment with compounds
*
Corresponding author
E-mail address: kandils1@cardiff.ac.uk (S. Kandil)

ACCEPTED MANUSCRIPT
27, 28 and 33 was observed during qPCR analysis, confirming their AR antagonist activity. Molecular
modelling studies revealed a novel binding mode of these structurally distinct double branched
analogues within the ligand binding domain (LBD) of the androgen receptor.
1
. Introduction
The androgen receptor (AR) is expressed in many cell types and plays critical anabolic and
reproductive roles in men and women. Androgen receptor (AR) signalling has been found to play
crucial functions in modulating tumourigenesis and metastasis in several types of cancers including
1
prostate, bladder, kidney, lung, breast and liver. The initiation and progression throughout the
different stages of prostate cancer (PC) is uniquely dependent on the androgen receptor (AR)
2,3
signalling pathway. Androgen receptor (AR), like other members of the nuclear receptor family, is
comprised of three main functional domains: a variable N-terminal domain, a highly conserved DNA-
4
binding domain (DBD) and a conserved ligand binding domain (LBD). Binding of endogenous
hormones; testosterone and dihydrotestosterone (DHT) to the LBD induces conformational changes
in the AR that results in its translocation into the nucleus, interaction with DNA, and modulation of
5
specific gene transcription (e.g. prostate specific antigen, PSA). Androgen receptor antagonists, so-
called anti-androgens, are designed to inhibit these processes and are clinically used for the
6,7
treatment of advanced prostate cancer (PC). Several non-steroidal anti-androgens (NSAA) have
been approved for the treatment of PC. The first generation NSAAs include flutamide,
hydroxyflutamide, nilutamide and bicalutamide (Figure 1). They decrease androgenic effects by
competitively inhibiting the binding of androgens (testosterone and DHT) to the AR and induce
conformational change of H12 via steric clashes. However, these antiandrogens eventually fail to
inhibit the AR with the development of castration resistant prostate cancer (CRPC). The development
of AR-LBD point mutants (e.g. AR-T877A and AR-W741L) upon long term treatment with NSAAs
ultimately result in switching these AR antagonists to AR agonists leading to the relapse of CRPC,
which is a more aggressive form of the disease associated with poor prognosis. Similar to the first-
generation androgen receptor (AR) antagonists, resistance to the new second generation anti-
androgens (enzalutamide, apalutamide) are developing in PC patients, despite the fact that these
8
drugs have better affinity for the AR. Anti-androgen resistance can also be triggered by the
upregulation of the androgen receptor expression, which promotes signalling from low levels of
8
residual hormone. More recently, darolutamide (ODM-201, Figure 1) is under evaluation in phase 3
9
clinical trials in patients with non-metastatic CRPC. The discovery of new AR antagonists is urgently
needed to improve anti-androgen efficacy and to avoid cross-resistance with the clinically used

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compounds.
First generation
O
O
O
HO
N
HN
NO2
HN
NO2
CF3
HN
NO2
O
CF3
Flutamide
CF3
Hydroxyflutamide
Nilutamide
O
F
S
HN
CN
CF3
O
OH O
Bicalutamide
Second generation
O
N
O
N
F
H
N
N
N
F
N
CN
H
CN
S
N
S
CF3
O
CF3
Enzalutamide
O
Apalutamide
Phase III
O
HO
N
H
N
HN
N
N
CN
Cl
Darolutamide
Figure 1. Chemical structures of the non-steroidal anti-androgens (NSAA); flutamide, hydroxyflutamide, nilutamide, and
bicalutamide (first generation), enzalutamide and apalutamide (second generation) and darolutamide (phase III clinical
trials).
2
. Results and discussion
Unfortunately, resistance to clinical anti-androgens has plagued the development of effective
therapeutics for advanced prostateꢀcancer (PC). Flutamide and bicalutamide (Figure 1) were first
generation non-steroidal AR antagonists. However, these antiandrogens lose their activity with time
upon the development of AR-LBD point mutants (AR-T877A and AR-W741L) triggered by flutamide
and bicalutamide, respectively. On the other hand, AR-F876L mutation causes enzalutamide and
apalutamide (ARN-509) to function as AR agonists and confers drug resistance across multiple AR
10
models both in vitro and in vivo. The X- ray structure of bicalutamide inside the LBD site of the AR -
W741L mutant, (PDB 1Z95), provides a significant insight into the protein−drug interactions and
10,
explains the structural basis for the conversion of bicalutamide from AR antagonist into AR agonist.
11
The binding mode of bicalutamide as an antagonist of the AR-wild type compared to its binding
mode as an agonist of the AR-W741L mutant is shown in Figure 2. It is observed that helix 12 (H12)
has changed its position from an open antagonist conformation (AR- wild type, blue) to a closed

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agonist conformation (AR- W741L mutant, green). In the case of the antagonist mode, ring B of
741
bicalutamide is pointing outwards away from Trp and towards helix 12, thus preventing AR from
adapting the closed AR agonist conformation. The loss of the steric bulk of the indolyl side chain of
741
741
tryptophan to the smaller size side chain of Leucine lead to the elimination of the steric clash
and hence the incorporation of bicalutamide molecule into the closed activated conformation of AR.
10-12
Figure 2. Proposed structural basis for the conversion of bicalutamide from AR antagonist into AR agonist by the gain-of-
function mutation (W741L); the X-ray structure of bicalutamide (green) inside the closed agonist conformation of AR LBD-
W741L, (green, PDB 1Z95) compared to its binding mode (blue) inside the open antagonist conformation of AR LBD-wt,
showing ring B orientation to hinder helix 12 from closing to an agonist conformation.
From previous studies, we have learnt that the size and structure of ring B determine the activity of
the anti-androgen and if possesses the correct volume and orientation, it will prevent the anti-
androgen molecule from being completely confined in the AR-LBD pocket cavity, leaving its bulky
substituents protruding against helix 12 (H12) and stopping the latter from adopting a position
1
0-12
essential for coactivator interaction (agonist closed conformation).
Previously, we have found that the introduction of 3,5-bis‐trifluoromethyl (3,5-bis-CF
3
) substituents
into ring B of bicalutamide, enobosarm and umbelliferone derivatives, has profoundly modified their
anti-proliferative activity, pharmacokinetic and tissue distribution profiles by providing the geometric
bulk needed to keep ring B towards Helix 12 while keeping the crucial interactions of the nitrile/nitro
1
3-15
group of ring A with Arg 752.
Noting that both flutamide and darolutamide (ODM-201) are lacking
the linker OH group, Figure 1, in this work we studied whether the central hydroxyl group of
bicalutamide is necessary for maintaining the anti-androgen activity and if the successive increase in
the bulk size of ring B substituents (from 4-F  4-CF
smaller size of the deshydroxy linker (lacking the central OH group). We used four different variations
of aromatic ring A (Figure 3); all containing the necessary 4-CN or 4-NO for the interaction with Arg
3
 3,5-bis-CF
3
) would compensate for the
2

ACCEPTED MANUSCRIPT
7
52 in addition to either 2-CF
3
or 3-CF substituent to understand better the structure activity
3
relationship.
2 3 3
Figure 3. Four different variations of ring A containing 4-CN or 4-NO group in addition to either 2-CF or 3-CF
2
.1 Chemistry
The novel series of deshydroxy propioanilide analogues (29-41) were synthesised in three steps. An
acylation reaction between the corresponding substituted trifluoromethyl anilines (1-4) with
methacryloyl chloride (5) in dimethylacetamide (DMA) to obtain phenylmethacrylamide derivatives
1
6,17
(
6-9)
was followed by Michael addition of the corresponding substituted thiophenols (10-13) to
obtain the thioether intermediates (14-26, 19-56% yield), which were subsequently oxidised to the
corresponding sulfone derivatives (29-41, 19-95% yield) using 3-chloroperbenzoic acid (mCPBA)
(
Scheme 1). It is worth noting that the thioethers (14-20 and 24-26) were prepared using sodium
hydride (NaH) in tetrahydrofuran (THF) at room temperature (method A), whereas the thioethers
21-23) corresponding to the N-(4-cyano-2-(trifluoromethyl)phenyl) methacrylamide (8) were
prepared using aqueous sodium hydroxide solution and tetrabutylammonium chloride in 1,4-dioxane
(
1
8
and reflux for 3 hours instead (method B), as the first method did not yield any products. The
intermediate sulphides (15, 16, 20-22) were used directly into the next oxidation step since further
purification was not feasible. Interestingly, alongside the thioether compounds (15 and 26) we
obtained two unusual disubstituted (double branched) acrylamide derivatives (27 and 28,
respectively) as major by-products in an approximate ratio of 2 (double branched 27 and 28) : 1
(single branched 15, 26), scheme 1.

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Scheme 1. Synthesis of racemic deshydroxy propioanilide derivatives. Reagents and conditions: (i) methacryloyl chloride 5
(
8 equiv), DMA, rt, 3 h; (ii) thiophenol derivatives (10-13, 1.2 equiv), method A: NaH (1.2 equiv), THF, rt, 24 h (6,7,9) or
method B: NaOH, 1,4-dioxane, tetrabutylammonium chloride reflux 3 hr (8); (iii) mCPBA (1.4 equiv), DCM, rt, 4-6 h.
The mechanism of the formation of the double branched products remains to be fully clarified. All
compounds were purified by column chromatography and/or recrystallised. The structures of all the
1
13
synthesised compounds were confirmed using analytical and spectroscopic data ( H NMR, C NMR,
19
F NMR and mass spectrometry), which were in full accordance with their depicted structures.
ID
R
1
R
2
R
4-F
4-CF
3,5-BisCF
3-OCF
4-F
4-CF
3
3,5-BisCF
4-F
4-CF
3
X
S
S
S
S
S
S
S
S
S
S
S
IC50 (uM)
8.69
1
1
1
1
1
1
2
2
2
2
2
4
5
6
7
8
9
0
1
2
3
4
CN
CN
CN
CN
NO
NO
NO
CN
CN
CN
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
2-CF
2-CF
2-CF
2-CF
3
3
3
3
3
3
3
3
3
3
3
3
-
3
-
114.97
27.16
4.41
-
3
2
2
2
3
3
-
-
3
3,5-BisCF
4-F
4.57
43.19
NO
2

ACCEPTED MANUSCRIPT
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
NO
NO
CN
2
2
2-CF
2-CF
3-CF
2-CF
3
3
3
3
4-CF
3,5-BisCF
4-CF
3,5-BisCF
4-F
3
S
S
-
54.33
43.68
1.68
3
3
3
NO
CN
CN
CN
CN
NO
NO
NO
CN
CN
CN
NO
NO
NO
-
2
-
0.43
3-CF3
SO2
114.74
57.98
6.16
13.19
2.67
3-CF
3-CF
3-CF
3-CF
3-CF
3-CF
2-CF
2-CF
2-CF
2-CF
2-CF
2-CF
-
3
3
3
3
3
3
3
3
3
3
3
3
4-CF
3,5-BisCF
3
SO
SO
SO
SO
SO
SO
SO
SO
SO
SO
SO
SO
-
2
2
2
2
2
2
2
2
2
2
2
2
3
3-OCF
4-F
3
2
2
2
4-CF
3
3.31
3,5-BisCF
4-F
3
3
3
12.23
137.7
28.79
9.56
4.05
26.34
3.08
4-CF
3
3,5-BisCF
4-F
2
2
2
4-CF
3
3,5-BisCF
Bical
Enzal
-
-
20.44
1.31
-
-
-
Table 1. Chemical structure and in vitro anti-proliferative activity (IC50 in M) of single (14-26, 29-41) and double (27, 28)
branched deshydroxy propioanilide analogues compared to bicalutamide and enzalutamide in the LNCaP cell line. IC50
values presented are the mean of three independent experiments.
2
.2 Cell growth inhibition activity
The twenty-four novel deshydroxy antiandrogen compounds 14, 17-19, 23-41, were tested for their
anti-cancer activity as racemic mixtures against human androgen-sensitive prostate cancer cell line
LNCaP at 9 concentrations in half-log increments up to 100 μM for 96 hours in triplicate. Bicalutamide
and enzalutamide were used as positive controls. Potency is expressed as absolute IC50 values,
calculated by non-linear regression analysis. The results summarised in Table 1 indicated that
thirteen compounds (14, 19, 23, 27, 28, 31-35, 38, 39 and 41) showed better anti-proliferative activity
than bicalutamide with IC50 values in the range of 0.43-13.19 μM, while the positive controls,
bicalutamide and enzalutamide exhibited IC50 values of 20.44 and 1.31 μM, respectively.
Interestingly, the double branched compounds 27 and 28 exhibited significantly potent activity
compared to bicalutamide and enzalutamide in LNCaP cell line (Table 2). The most active compounds
27, 28 and 33 (Figure 4) were selected to be further tested against VCaP and PC3 human prostate
19
cancer cell lines, which represent metastatic and more aggressive forms of prostate cancer (CRPC).
Compound ID
LNCaP IC50 (uM)
VCaP IC50 (uM)
0.13
PC3 IC50 (uM)
5.45
2
2
3
7
8
3
1.68
0.43
2.67
20.44
1.31
0.18
8.21
5.96
3.66
0.91
112.30
92.63
Bicalutamide
Enzalutamide
15.07
Table 2. In vitro anti-proliferative activity (IC50 in M) of compounds (33, 27 and 28) compared to bicalutamide and
enzalutamide in human prostate cancer cell lines; LNCaP, VCaP and PC3

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Figure 4. Dose-response relationship of LNCAP cells treated with 0-100 μM of compounds; (27, A), (29, B) or (33, C) (red),
bicalutamide (green) and enzalutamide (blue) for 96 h. Data is presented as the mean ± SD of three replicates at each
concentration.
2
.3 Downregulation of PSA Expression in LNCaP Cells
Prostate specific antigen (PSA) is a serine protease that is synthesised by both normal and malignant
epithelial cells of the human prostate. The expression of PSA is mainly induced by androgens and
2
0,21
regulated primarily by the androgen receptor (AR) at the transcriptional level
. The effect of
compounds 27, 28 and 33 on prostate specific antigen (PSA, an AR-regulated gene) expression was
analysed using qPCR after incubation of LNCaP cells with 5, 10, 50 and 100 μM of bicalutamide,
compounds 27, 28 and 33, for 24-hours. The analysis shows that the three compounds 27, 28 and 33
exhibited dose dependent inhibition of PSA expression with compound 28 showing the best activity
significantly exceeding that of bicalutamide (Figure 5). The significant reduction of the AR-regulated
PSA expression in the qPCR analysis, suggests that these compounds have strong AR-antagonist
activity.

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Figure 5. The relative change in PSA expression (PSA/HK) in LNCaP cells upon treatment with increasing doses of
bicalutamide (A), compounds; 27 (B), 28 (C) and 33 (D). HK: housekeeping genes (Bactin and GAPDH)
2.4 Docking studies reveal a structural basis for anti-androgen activity
Previous X-ray crystallographic studies of androgen receptor have elucidated the structures of the AR
ligand binding domain (LBD) bound to agonists, and of mutant AR bound to antagonists in an agonist
like conformation. However, there are no crystal structures of the AR-LBD in an antagonist
2
2,23
conformation reported so far.
The human progesterone receptor (hPR residues 683-931) has 56%
primary sequence homology to the hAR (residues 669-918) and 32% of these residues are
24
conservative mutations, giving an overall sequence homology of 87%. Building on this information,
25
the hPR crystal structure [PDB 2OVH] was used as a template to build an hAR homology model (HM)
26
using the sequence of the hAR in the agonist conformation [PDB 2AMA]. Applying the default
parameters of MOE (Molecular Operating Environment - Chemical Computing Group, Montreal,
Canada) homology modelling module and using the AMBER99 force field, a total of 10 homology
models were generated. The quality of each model was assessed within MOE, and the best model
was chosen for the docking studies.
Computational docking studies were performed to explore the binding modes of the deshydroxy
propioanilide analogues (14-41) within the AR-LBD binding site. The chemical structures of our
compounds (14-41) were constructed, rendered and minimized with the MMFF94x force field in
MOE. Docking simulations were performed using Glide SP in Maestro (Glide, version 9.5, Schrödinger,

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LLC, New York, NY. http://www.schrodinger.com). The putative docking modes of the most active
single branched deshydroxy propioanilide compounds 41, 34 and 33 (IC50 = 3.08, 3.31 and 2.67 M,
respectively) are shown in Figure 6 (A-C, respectively). All three compounds show the crucial H-bond
interaction between their nitro group (4-NO , ring A) and the guanidine group of the Arg-752 residue.
2
Other key interactions of compound 41 include an H-bond between the aromatic ring B (ArH) and
carbonyl group of Asn 705 and the carbonyl group and Met 742 (Figure 5A). Compound 34 shows an
additional H-bond between the nitro group (4-NO2, ring A) and the amino group of Gln 711, and
another H-bond between the amide NH group and the carbonyl group of Leu 704. Also, a H-bond
between the sulphone group and the side chain of Met 745 residue was observed (Figure 5B).
Compound 33 shows a hydrogen bond between the aromatic ring A and the side chain of Met 742
residue, and another H-bond between the sulphone group and the side chain of Met 745 residue.
Figure 6. The predicted binding modes of compounds (41, A), (34, B) and (33, C) inside the hAR-LBD showing H-bond
interactions with key amino acids; Arg752, Gln711, Met 742, Met 745, Leu704, Thr877 and Asn705.
On the other hand, the docking of the double branched deshydroxy compounds (27 and 28) revealed
an interesting binding mode where the ring A aryl nitrile (4-CN, 27) and aryl nitro (4-NO , 28) groups
2
form a hydrogen bond with the guanidine group of Arg752 in the AR ligand binding pocket, mimicking
the interaction between the 3-keto functionality of the hormone dihydrotestesterone (DHT) and the
androgen receptor. The two branches of ring B occupy simultaneously the two sub-pockets that are
27
known to be involved in the binding of bicalutamide and enzalutamide which may explain well the
high potency observed with this type of distinctive scaffold (Figure 7).

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Figure 7. The predicted binding modes of bicalutamide (green), enzalutamide (blue) compared to the double-branched
compounds (pink); 27 (I) and 28 (II) within the hAR-LBD (grey ribbon). Left panel (top) is showing the 2D representation of
compound 27 (I) and compound 28 (II).
Generally, our SAR analysis of this family of compounds indicates that the lack of the linker hydroxy
group of the propioanilide scaffold seems to be tolerated in terms of the anti-proliferative activity in
PC cell lines and in some cases showed enhanced activity compared to bicalutamide; compounds (14,
19, 23, 27, 28, 31-35, 38, 39 and 41). Compounds 27 and 28 were significantly more potent than both
bicalutamide and enzalutamide in our models. Docking studies suggest that this enhanced activity
can be attributed to the steric bulk added by the two branches of the aromatic ring B which occupy
both the proposed binding pockets of bicalutamide and enzalutamide simultaneously and would
741
provide the geometric bulk needed to keep ring B pointing outwards away from Trp and towards
helix 12, thus preventing AR from adapting the AR agonist (closed) conformation (Figure 7). These

ACCEPTED MANUSCRIPT
double branched compounds (27 and 28) are distinct from the structures of other nonsteroidal anti-
androgens, which is likely to result in differences in biology and resistance mechanisms.
3
. Conclusion
A series of twenty-four novel deshydroxy bicalutamide analogues (29-41) were synthesised using a
Michael addition reaction between four different acrylamide derivatives (6-9) and various fluorinated
thiophenols (10-13) as a key step. Eleven compounds showed anti-proliferative activity (IC50 = 0.43-
1
3.19 μM) better than bicalutamide (IC50 = 20.44 μM). Interestingly, compounds 27 and 28 exhibited
very potent activity with IC50 values of 1.68 and 0.43 μM, respectively, which is comparable or better
than enzalutamide (IC50 = 1.31 μM) in the LNCaP cell line. Further testing for the in vitro anti-
proliferative activity in VCaP and PC3 cell lines showed sub-micromolar activity. Furthermore,
compounds 27, 28 and 33 showed remarkable reduction of PSA expression in LNCaP cells confirming
their anti-androgenic activity. Molecular modelling studies indicated that the enhanced anti-
androgen activity of compounds 27 and 28 appears to be a result of the extra bulk conferred by the
two aromatic rings B, which ensure the occupation of the two sub-pockets of the AR-LBD involved in
the binding interaction of bicalutamde and enzalutamide simultaneously. This may forestall the drug
resistance seen with current clinical anti-androgens. Compounds 27 and 28 have distinctive chemical
structure and represents promising lead for further development of AR antagonists. Overall, this
study provides, for the first time, the double-branched configuration into the non-steroidal anti-
androgen library and lays a foundation for the development of alternative AR antagonist therapies
capable of combating prostate cancer.
Acknowledgment
The authors would like to thank the Welsh Government (A4B-Academic Expertise for Business), the
Life Sciences Research Network of Wales (LSRNW), and the Cardiff China Medical Research
Collaborative. We also thank the EPSRC National Mass Spectrometry centre (Swansea, U.K.) for
provision of accurate mass spectrometry.
Supplementary data
1
19
13
H, F and C NMR spectra, and mass spectrometry data, for representative compounds 25, 28, and
35
4
. Experimental section
4
.1 Chemistry
All chemicals were purchased from Sigma-Aldrich or Alfa Aesar and were used without further

ACCEPTED MANUSCRIPT
purification. Thin Layer Chromatography (TLC): pre-coated aluminium backed plates (60 F254, 0.2 mm
thickness, Merck) were visualized under both short and long wave UV light (254 and 366 nm). Flash
1
column chromatography was carried out using silica gel supplied by Fisher (60A, 35-70 m). H NMR
13
19
(
500 MHz), C NMR (125 MHz) and F NMR (470 MHz) spectra were recorded on a Bruker Avance
5
00 MHz spectrometer at 25 C. Chemical shifts (ä) are expressed in parts per million (ppm) and
coupling constants (J) are given in hertz (Hz). The following abbreviations are used in the assignment
of NMR signals: s (singlet), bs (broad singlet); d (doublet), t (triplet), q (quartet), qn (quintet), m
(
multiplet), dd (doublet of doublet), dt (doublet of triplet), td (triple doublet); dq (double quartet), m
multiplet), dm (double multiplet). Mass spectrometry was performed as a service through the EPSRC
(
National Mass Spectrometry centre (Swansea, UK).
.1.1 General method for the preparation of intermediates 6-9
4
Methacryloyl chloride 5 (8.4 mL, 85.96 mmol) was added over the course of 10 minutes to a stirring
solution of the appropriate trifluoromethyl-substituted aniline 1-4 (10.75 mmol) in N, N-
dimethylacetamide (10 mL) at room temperature for 24h. After the reaction was complete, the
mixture was diluted with ethyl acetate (100 mL), extracted with saturated NaHCO
3
solution (2 x 50
SO and
mL) then cold brine (2 x 50 mL). The combined organic layer was dried over anhydrous Na
2
4
the solvent was removed under reduced pressure. The crude oil residue was purified by flash column
chromatography eluting with chloroform-ethyl acetate 95:5 v/v to obtain the titled compounds.
16
N-(4-cyano-3-(trifluoromethyl)phenyl)methacrylamide (6) . Yield; 92%
1
H NMR (CDCl
3
) δ 8.10 (d, J = 2Hz, 1H, ArH), 8.06 (bs, 1H, NH), 8.01 (dd, J = 2, 8.5 Hz, 1H, ArH), 7.81 (d,
J = 8.5Hz, 1H, ArH), 5.89 (d, J = 1Hz, 1H, CH
2
), 5.62 (q, J = 1.5Hz, 1H, CH ), 2.10 (dd, J = 0.5, 1.5 Hz, 3H,
2
19
CH
3
). F-NMR: (CDCl ) δ -62.23.
3
1
6
N-(4-nitro-3-(trifluoromethyl)phenyl)methacrylamide (7) . Yield; 80%
1
H-NMR (CDCl
3
): δ 7.99-7.95 (m, 2H, ArH), 7.93 (d, J = 9 Hz, 1H, ArH), 7.8 (bs, 1H, NH), 5.81 (q, J = 1Hz
19
1
H, CH ), 5.55 (q, J = 1.5 Hz, 1H, CH
2
2
), 2.02 (dd, J = 1, 1.5 Hz, 3H, CH
3
). F-NMR (CDCl ): δ -60.09.
3
17
N-(4-cyano-2-(trifluoromethyl)phenyl)methacrylamide (8) . Yield; 88 %
1
H-NMR (CDCl
3
):  8.69 (d, J= 9 Hz, 1H, ArH), 8.07 (bs, 1H, NH), 7.84 (d, J= 1.5 Hz, 1H, ArH), 7.78 (dd,
J= 2, 9 Hz, 2 Hz, 1H, ArH), 5.83 (q, J = 1 Hz, 1H, CH
2
), 5.56 (q, J= 1.5 Hz, 1H, CH ), 2.02 (dd, J= 1, 1.5 Hz,
2
19
3
H, CH
3
). F-NMR (CDCl
3
):  -61.35.
1
7
N-(4-nitro-2-(trifluoromethyl)phenyl)methacrylamide (9) Yield; 94 %

ACCEPTED MANUSCRIPT
1
H NMR (CDCl
3
) δ 8.73 (d, J= 9 Hz, 1H, ArH), 8.46 (d, J= 3 Hz, 1H, ArH), 8.37 (dd, J= 9 Hz, 2.5 Hz, 1H,
ArH), 8.17 (bs, 1H, NH), 5.85 (q, J= 0.5 Hz, 1H, CH
2
), 5.58 (q, J= 1.5 Hz, 1H, CH ), 2.15-2.13 (dd, J= 1, 1.5
2
1
9
Hz, 1H, CH
3
). F-NMR: (CDCl )  -61.31.
3
4
4
.1.2 General method for the preparation of sulphide intermediates;
.1.2.1 Method A: (14-20 and 24-28)
To a mixture of 60% sodium hydride in mineral oil (94.43 mg, 2.36 mmol) in anhydrous
tetrahydrofuran (5 mL) at 0°C under anhydrous THF under nitrogen atmosphere, was added dropwise
the corresponding thiophenol 10-13 (2.05 mmol). This mixture was stirred at room temperature for
2
0 minutes. A solution of the appropriate intermediate 6-9 (1.57 mmol in 5 mL anhydrous
tetrahydrofuran) was added slowly to the thiophenol mixture and stirred at room temperature for
4h. The mixture was concentrated under vacuum then diluted with ethyl acetate (30 mL), washed
2
with brine (20 mL) and water (30 mL), dried over anhydrous sodium sulfate and concentrated under
vacuum. The crude residue was purified by column chromatography eluting with chloroform-ethyl
acetate gradually increasing from 95:5 to 90:10 v/v.
4
.1.2.2 Method B: (21-23)
The corresponding thiophenol 10-13 (2.05 mmol) was added dropwise to an aqueous sodium
hydroxide solution (79 mg in 1 ml water) for 15 min at room temperature. Then a solution of
compound 8 (2.05 mmol) in 1,4- dioxan (5 mL) was added at room temperature followed by a
catalytic amount of tetrabutylammonium bromide. The reaction mixture was refluxed for 3 h and
cooled to room temperature, followed by addition of 26% aqueous acetic acid. This mixture was
extracted with ethyl acetate (2 x 200 mL). The ethyl acetate layer was separated, washed with water,
and concentrated under reduced pressure. The crude residue was purified by column
chromatography eluting with chloroform-ethyl acetate gradually increasing from 95:5 to 90:10 v/v.
N-(4-cyano-3-(trifluoromethyl)phenyl)-3-((4-fluorophenyl)thio)-2-methylpropanamide (14). Yield;
50%
1
H-NMR: (CDCl
3
) δ 8.01 (s, 1H, ArH), 7.90 (d, J = 8.5Hz, 1H, ArH), 7.85 (s, 1H, NH), 7.79 (d, J = 9 Hz, 1H,
), 3.04
). F-NMR: (CDCl ) δ -
ArH), 7.38 (dd, J = 5.5, 8 Hz, 2H ArH), 7.00 (t, J = 8.5Hz, 2H, ArH), 3.28 (dd, J = 8, 14Hz, 1H, CH
2
19
(dd, J = 5, 13.5Hz, 1H, CH
2
), 2.64 (sext, J = 7Hz, 1H, CH), 1.37 (d, J = 7Hz, 3H, CH
3
3
13
1
6
2.23, -114.26, C-NMR: (CDCl
3
) δ 173.59 (C=O), 162.04 (d, JC-F = 246.4Hz, ArC), 142.10 (ArC), 135.79
2
3
4
(
ArCH), 133.99 (q, JC-F = 32.9Hz, ArC), 132.64 (d, JC-F = 8Hz, ArCH), 130.04 (d, JC-F = 3.6Hz, ArC),
1
3
2
1
22.10 (q, JC-F = 272.5Hz, CF
3
), 121.85 (ArCH), 117.31 (q, JC-F = 4.6Hz, ArCH), 116.32 (d, JC-F = 21.8Hz,

ACCEPTED MANUSCRIPT
ArCH), 115.63 (CN), 104.08 (ArC), 42.62 (CH), 38.57 (CH
M+H], 383.0836; found, 383.0836.
N-(4-cyano-3-(trifluoromethyl)phenyl)-2-methyl-3-((3-(trifluoromethoxy)phenyl)thio) propanamide
17). Yield; 47%
2
), 17.66 (CH
3
). HRMS calcd for C18
H
14
F
4
N OS
2
[
(
1
H-NMR: (CDCl
3
) δ 8.54 (s, 1H, NH), 8.08 (d, J = 1.5Hz, 1H, ArH), 7.94 (dd, J = 2, 8.5Hz, 1H, ArH), 7.74
(d, J = 8.5Hz, 1H, ArH), 7.27 (m, 2H, ArH), 7.16 (s, 1H, ArH), 6.99 (d, J = 7.5Hz, 1H, ArH), 3.35 (dd, J = 9,
1
3.5Hz, 1H, CH
2
), 3.09 (dd, J = 5.5, 13.5Hz, 1H, CH
2
), 2.83 – 2.74 (m, 1H, CH), 1.40 (d, J = 7Hz, 3H, CH
3
).
19
13
F-NMR: (CDCl
3
) δ -57.86, -62.34, C-NMR: (CDCl
3
) δ 173.93 (C=O), 149.50 (ArC), 142.58 (ArC),
2
1
38.06 (ArC), 135.73 (ArCH), 133.91 (q, JC-F = 32.4Hz, ArC), 130.21(ArCH), 127.10 (ArCH), 122.12 (q,
1
1
J
C-F = 272.5Hz, OCF
3
), 121.93 (ArCH), 121.11 (ArCH), 120.33 (q, JC-F = 253.5Hz, CF
3
), 118.53 (ArCH),
3
1
17.42 (q, JC-F = 4.8Hz, ArCH), 115.89 (CN), 103.41 (ArC), 42.49 (CH), 36.72 (CH
2
), 17.76 (CH ). HRMS
3
+
calcd for C19
H
14
F
6
N
2
O
2
S
,
449.0753; found, 449.0752 [M+H], 466.1015 [M+NH
4
].
3-((4-Fluorophenyl)thio)-2-methyl-N-(4-nitro-3-(trifluoromethyl)phenyl)propanamide (18). Yield; 45
%
1
H NMR (CDCl
3
) δ 8.36 (s, 1H, NH), 8.02 (d, J = 1.5 Hz, 1H, ArH), 7.98-7.95 (m, 2H, ArH), 7.38-7.33 (m,
2
2
H, ArH), 6.99-6.94 (m, 2H, ArH), 3.27 (dd, J = 8.5, 13.5 Hz, 1H, CH
2
), 3.03 (dd, J = 5, 13.5 Hz,1H, CH ),
2
13
19
.72-2.64 (m, 1H, CH), 1.36 (d, J = 6.5 Hz, 3H, CH
3
); F NMR (CDCl
3
) δ -60.14, -114.70; C NMR (CDCl )
3
1
3
ä 173.99 (C=O), 161.95 (d, JC-F = 245.8 Hz, ArC), 142.68 (ArC), 142.36 (ArC), 132.52 (d, JC-F = 7.8 Hz,
4
2
ArCH), 130.12 (d, JC-F = 2.9 Hz, ArC), 127.12 (ArCH), 125.15 (q, JC-F = 34.3 Hz, ArC), 122.23 (ArCH),
1
3
2
1
21.74 (q, JC-F = 271.9 Hz, CF
3
), 118.16 (q, JC-F = 6.1 Hz, ArCH), 116.23 (d, JC-F = 21.9 Hz, ArCH), 42.56
(
CH), 38.42 (CH
2
), 17.72 (CH
3
). MS [ESI, m/z]: HRMS calcd for C17 S [M+H], 403.0739; found,
H
14
F
4
N
2 3
O
4
03.0746.
-Methyl-N-(4-nitro-3-(trifluoromethyl)phenyl)-3-((4-(trifluoromethyl)phenyl)thio)propanamide
2
(
19). Yield; 52 %
1
H NMR (CDCl
3
) δ 8.00-7.92 (m, 3H, ArH), 7.74 (s, 1H, NH), 7.53 (d, J = 8 Hz, 2H, ArH), 7.41 (d, J = 8 Hz,
2
H, ArH), 3.27 (dd, J = 9, 13.5 Hz, 1H, CH
2
), 3.16 (dd, J = 5, 14 Hz,1H, CH ), 2.75-2.67 (m, 1H, CH), 1.44
2
1
3
19
(d, J = 7 Hz, 3H, CH
3
); F NMR (CDCl
3
) δ -60.13, -62.56; C NMR (CDCl
3
) ä 173.16 (C=O), 143.13 (ArC),
2
1
41.71 (ArC), 140.58 (ArC), 128.25 (q, JC-F = 32.6 Hz, ArC), 127.94 (ArCH), 127.10 (ArCH), 125.95 (q,
3
2
1
J
C-F = 3.8 Hz, ArCH), 125.30 (q, JC-F = 33.9 Hz, ArC), 122.13 (ArCH), 123.90 (q, JC-F = 270.3 Hz, CF
3
),
1
3
1
21.66 (q, JC-F = 272.4 Hz, CF
3
), 118.22 (q, JC-F = 6 Hz, ArCH), 42.73 (CH), 35.92 (CH
2
), 17.90 (CH
3
). MS
[
ESI, m/z]: HRMS calcd for C18
H
14
F
6
N
2
O S [M+H], 453.0702; found, 453.0699.
3

ACCEPTED MANUSCRIPT
3-((3,5-Bis(trifluoromethyl)phenyl)thio)-N-(4-cyano-2-(trifluoromethyl)phenyl)-2-methyl
propanamide (23). Yield; 41 %
1
H NMR (CDCl
3
) δ 8.54 (d, J = 9 Hz, 1H, ArH), 7.92 (d, J= 1.5 Hz, 1H, ArH), 7.84 (dd, J= 2, 9 Hz, 1H, ArH),
7
5
.75 (s, 1H, ArH), 7.70 (s, 1H, NH), 7.67 (s, 1H, ArH), 3.47 (dd, J= 8.5, 13.5 Hz, 1H, CH ), 3.19 (dd, J=
2
19
.5, 13.5 Hz, 1H, CH
2
), 2.77-2.69 (m, 1H, , CH), 1.45 (d, J = 7 Hz, 3H, CH
3
); F NMR (CDCl
3
) δ -61.15, -
1
3
2
6
3.10; C NMR (CDCl
3
) ä 172.43 (C=O), 139.27 (ArC), 138.79 (ArC), 136.69 (ArCH), 132.34 (q, JC-F
=
3
3
3
3.3 Hz, ArC), 130.25 (q, JC-F = 5.4 Hz, ArCH), 128.38 (q, JC-F = 3.3 Hz, ArCH), 123.64 (ArCH), 122.89 (q,
1
1
3
J
C-F = 271.9 Hz, CF
3
), 122.24 (ArC), 118.64 (q, JC-F = 304 Hz, CF
3
), 120.01 (sept, JC-F = 3.8 Hz, ArCH),
1
17.43 (CN), 108.08 (ArC), 43.06 (CH), 36.62 (CH
2
), 17.63 (CH
3
). C20H
13
F
9
N OS, MS (ES+) m/z: 523.0491
2
+
+
[
M+Na ], 518.0943 [M+NH
4
].
3-((4-Fluorophenyl)thio)-2-methyl-N-(4-nitro-2-(trifluoromethyl)phenyl)propanamide (24). Yield; 49
%
1
H NMR (CDCl
3
) δ 8.65 (d, J = 9 Hz, 1H, ArH), 8.52 (d, J = 2 Hz, 1H, ArH), 8.42 (dd, J= 2, 9 Hz, 1H, ArH),
7
1
.84 (s, 1H, NH), 7.40 (dd, J = 5.5, 8.5 Hz, 2H, ArH), 7.01 (t, J = 8.5 Hz, 2H, ArH), 3.28 (dd, J = 8.5, 14 Hz,
19
H, CH
2
), 3.28 (dd, J = 5, 13.5 Hz, 1H, CH
2
), 2.65 (sext, J = 7 Hz, 1H, CH), 1.38 (d, J = 7 Hz, 3H, CH ); F
3
1
3
1
NMR (CDCl
3
) δ -61.03, -114.95; C NMR (CDCl
3
) ä 173.15 (C=O), 162.14 (d, JC-F = 245.8 Hz, ArC),
3
4
1
1
42.89 (ArC), 140.76 (ArC), 133.03 (d, JC-F = 8 Hz, ArCH), 129.83 (d, JC-F = 3.4 Hz, ArC), 128.26 (ArCH),
1
3
2
23.36 (ArCH), 122.89 (q, JC-F = 272.1 Hz, CF
3
), 122.32 (q, JC-F = 6 Hz, ArCH), 119.14 (q, JC-F = 31 Hz,
2
ArC), 116.36 (d, JC-F = 22 Hz, ArCH), 43.05 (CH), 38.76 (CH
2
), 17.41 (CH ). MS [ESI, m/z]: HRMS calcd
3
for C17
H
14
F
4
N
2
O
3
S [M+H], 403.0734; found, 403.0733.
-Methyl-N-(4-nitro-2-(trifluoromethyl)phenyl)-3-((4-(trifluoromethyl)phenyl)thio)propanamide
25). Yield; 56 %
2
(
1
H NMR (CDCl
3
) δ 8.62 (d, J = 9 Hz, 1H, ArH), 8.52 (d, J = 2.5 Hz, 1H, ArH), 8.47 (dd, J= 2.5, 9 Hz, 1H,
ArH), 7.81 (s, 1H, NH), 7.54 (d, J = 8 Hz, 2H, ArH), 7.43 (d, J = 8 Hz, 2H, ArH), 3.42 (dd, J = 8.5, 13.5 Hz,
1
H, CH
2
), 3.16 (dd, J = 5.5, 14 Hz, 1H, CH
2
), 2.74 (sextet of triplet, J = 7, 1.5 Hz, 1H, CH), 1.44 (d, J = 7
19
13
Hz, 3H, CH
3
); F NMR (CDCl
3
) δ -61.05, -62.58; C NMR (CDCl
3
) ä 172.84 (C=O), 140.58 (ArC), 140.49
2
3
(
ArC), 128.36 (ArCH), 128.29 (q, JC-F = 32.5 Hz, ArC), 128.25 (ArCH), 125.91 (q, JC-F = 3.6 Hz, ArCH),
1
1
3
1
23.94 (q, JC-F = 270 Hz, CF
3
), 122.85 (q, JC-F = 271.8 Hz, CF
3
), 122.31 (q, JC-F = 5.6 Hz, ArCH), 119.25
2
(
q, JC-F = 31.5 Hz, ArC), 43.03 (CH), 36.18 (CH
2
), 17.51 (CH ). MS [ESI, m/z]: HRMS calcd for
3
C
18
H
14
F
6
N
2
O S [M+H], 453.0708; found, 453.0711.
3

ACCEPTED MANUSCRIPT
3-((3,5-Bis(trifluoromethyl)phenyl)thio)-2-methyl-N-(4-nitro-2-(trifluoromethyl)phenyl)
propanamide (26). Yield; 24%
1
H NMR (CDCl
3
) δ 8.64 (d, J = 9.5 Hz, 1H, ArH), 8.54 (d, J = 3 Hz, 1H, ArH), 8.43 (dd, J= 2.5, 9.5 Hz, 1H,
ArH), 7.79 (s, 1H, NH), 7.76 (s, 2H, ArH), 7.01 (s, 1H, ArH), 3.48 (dd, J = 8, 13.5 Hz, 1H, CH
2
), 3.21 (dd, J
19
=
5.5, 13.5 Hz, 1H, CH ), 2.76 (sextet of triplet, J = 7, 1.5 Hz, 1H, CH), 1.47 (d, J = 7 Hz, 3H, CH ); F
2
3
1
3
NMR (CDCl
3
) δ -61.10, -63.11; C NMR (CDCl
3
) ä 172.47 (C=O), 143.09 (ArC), 140.41 (ArC), 139.21
2
4
(
ArC), 132.38 (q, JC-F = 33.3 Hz, ArC), 128.40 (d, JC-F = 3.3 Hz, ArCH), 128.31 (ArCH), 123.30 (ArCH),
1
1
3
1
22.87 (q, JC-F = 271.1 Hz, CF
3
), 122.85 (q, JC-F = 271.9 Hz, CF
3
), 122.34 (q, JC-F = 5.9 Hz, ArCH), 119.30
2
3
(
q, JC-F = 31.1 Hz, ArC), 120.06 (sept, JC-F = 3.6 Hz, ArCH), 43.13 (CH), 36.62 (CH
2
), 17.63 (CH ). MS
3
[
ESI, m/z]: HRMS calcd for C19
H
13
F
9
N
2
O S [M+H], 521.0573; found, 521.0576.
3
N-(4-cyano-3-(trifluoromethyl)phenyl)-2-methyl-2,3-bis((4-(trifluoromethyl)phenyl)thio)
propanamide (27). Yield; 42%
1
H-NMR: (CDCl ) δ 9.06 (s, 1H, NH), 7.97 (d, J = 2Hz, 1H ArH), 7.85 (dd, J = 2, 8.5Hz, 1H, Ar-H), 7.79 (d,
3
J = 8.5Hz, 1H, ArH), 7.59 (d, J = 8.5Hz, 2H, ArH), 7.51 (d, J = 8Hz, 2H, ArH), 7.44 (dd, J = 8.5, 13.5Hz, 4H,
19
ArH), 3.60 (d, J = 13.5Hz, 1H, CH
2
), 3.52 (d, J = 13.5Hz, 1H, CH
2.23, -62.65, -63.00. C-NMR: (CDCl ) δ 170.74 (C=O), 141.22 (ArC), 140.60 (ArC), 135.86 (ArCH),
34.35 (ArCH), 134.23 (ArC), 134.11 (q, JC-F = 31Hz, ArC), 131.69 (q, JC-F = 33.1Hz, ArC), 131.26
2
), 1.73 (s, 3H, CH
3
). F-NMR: (CDCl
3
) δ -
1
3
6
1
3
2
2
2
3
(
ArCH), 129.00 (ArCH), 128.64 (q, JC-F = 32.6Hz, ArC), 126.35 (q, JC-F = 3.6Hz, ArCH), 125.89 – 125.74
1
1
1
(
m, ArCH), 123.77 (q, JC-F = 270Hz, CF
3
), 123.51 (q, JC-F = 270.8Hz, CF
3
), 122.00 (q, JC-F = 272.3Hz,
3
CF
3
), 117.14 (q, JC-F = 5Hz, ArCH), 115.30 (CN), 105.04 (ArC), 59.51 (-C-CH
3
), 42.06 (CH
2
), 23.87 (CH ).
3
+
HRMS calcd for C26
H
17
F
9
2
N OS2, 609.0711; found, 609.0708 [M+H], 626.0965 [M+NH
4
].
2
,3-Bis((3,5-bis(trifluoromethyl)phenyl)thio)-2-methyl-N-(4-nitro-2-(trifluoromethyl)phenyl)
propanamide (28). Yield; 48 %
1
H NMR (CDCl
3
) δ 9.19 (s, 1H, NH), 8.59 (d, J = 3 Hz, 1H, ArH), 8.54 (d, J = 9.5 Hz, 1H, ArH), 8.45 (dd, J=
2
1
.5, 9 Hz, 1H, ArH), 7.92 (s, 1H, ArH), 7.89 (s, 2H, ArH), 7.78 (s, 2H, ArH), 7.67 (s, 1H, ArH), 3.63 (d, J =
19
3.5 Hz, 1H, CH
2
), 3.49 (d, J = 13.5 Hz, 1H, CH
2
), 1.80 (s, 3H, CH
3
); F NMR (CDCl ) δ -61.05, -63.14,
3
1
3
6
3.19; C NMR (CDCl
3
) ä 169.75 (C=O), 143.34 (ArC), 139.89 (ArC), 138.78 (ArC), 132.30 (ArC), 132.87
2
2
3
(
q, JC-F = 33.9 Hz, ArC), 132.42 (q, JC-F = 33.3 Hz, ArC), 134.69, 134.66 (ArCH), 129.58 (d, JC-F = 3.6 Hz,
1
3
ArCH), 128.53 (ArCH), 122.44 (q, JC-F = 271.6 Hz, CF
3
), 123.87 (septet, JC-F = 3.6 Hz, ArCH), 122.53 (q,
3
1
1
J
C-F = 5.5 Hz, ArCH), 122.88 (q, JC-F = 272.3 Hz, CF
3
), 122.75 (q, JC-F = 274.4 Hz, CF
3
), 122.31 (ArCH),

ACCEPTED MANUSCRIPT
3
2
1
20.72 (septet, JC-F = 3.9 Hz, ArCH), 119.29 (q, JC-F = 31.5 Hz, ArC), 57.94 (CH
3
C), 42.43 (CH ), 14.15
2
(
CH
3
). MS [ESI, m/z]: HRMS for C27
H
15
F
N
15 2
3
O S
2
[M+H], 765.0371.
4
.1.3 General method for the preparation of sulfones derivatives 29-41
To a stirring solution of the corresponding sulfide 14-26 (0.7 mmol) in 5 mL anhydrous
dichloromethane, was added 3-chloroperbenzoic acid (MCPBA) (1.4 mmol). The solution was stirred
at room temperature for 24 h. The reaction mixture was neutralized with 1M sodium hydroxide. 50
mL distilled water was added to the reaction mixture and was extracted with 2 x 50mL of
dichloromethane. The combined organic layers were washed, dried over anhydrous sodium sulfate,
and concentrated in vacuo. The crude residue was purified by column chromatography, preparative
TLC or crystallization from methanol.
N-(4-cyano-3-(trifluoromethyl)phenyl)-3-((4-fluorophenyl)sulfonyl)-2-methylpropanamide (29).
Yield; 69%
1
H-NMR: ((CD
3
) SO) δ 10.82 (s, 1H, NH), 8.12 (d, J = 2Hz, 1H, ArH), 8.09 (d, J = 8.5Hz, 1H, ArH), 7.94
2
(dd, J = 5, 9Hz, 2H, ArH), 7.82 (dd, J = 2, 8Hz, 1H, ArH), 7.38 (t, J = 9Hz, 2H, ArH), 3.81 (dd, J = 9.5,
1
4.5Hz, 1H, CH
2
), 3.50 (dd, J = 4, 14.5Hz, 1H, CH
2
), 3.06 – 2.98 (m, 1H, CH), 1.23 (d, J = 7.5Hz, 3H, CH ).
3
1
3
19
1
F-NMR: ((CD
3
)
2
SO) δ -61.34, -104.87, C NMR ((CD
3
)
2
SO) ä 173.24 (C=O), 165.56 (d, JC-F = 250 Hz,
2
3
ArC), 143.76 (ArC), 136.92 (ArCH), 135.71 (ArC), 132.13 (q, JC-F = 31.5 Hz, ArC), 131.61 (d, JC-F = 9.9
1
2
Hz, ArCH), 122.86 (q, JC-F = 273.3 Hz, CF
3
), 122.49 (ArCH), 116.93 (d, JC-F = 22.6 Hz, ArCH), 116.84 (m,
ArCH), 116.21 (CN), 102.26 (ArC), 57.72 (CH
2
), 36.81 (CH), 18.76 (CH
3
). HRMS calcd for C18
H
14
F
4
N
2
O S
3
+
[
M+H], 415.0734; found, 415.0735, [M+NH ], 432.0999.
4
N-(4-cyano-3-(trifluoromethyl)phenyl)-2-methyl-3-((4-(trifluoromethyl)phenyl)sulfonyl)
propanamide (30). Yield; 21%
1
H-NMR: (CDCl
3
) δ 8.88 (s, 1H, NH), 8.12 (d, J = 8.5Hz, 2H ArH), 7.96 (d, J = 2Hz, 1H, ArH), 7.88 – 7.83
(m, 3H, ArH), 7.72 (d, J = 8.5Hz, 1H, ArH), 3.99 (dd, J = 9.5, 14Hz, 1H, CH
2
), 3.43 – 3.35 (m, 1H, CH),
1
9
13
3
.22 (dd, J = 3, 14Hz, 1H, CH
2
), 1.43 (d, J = 7.5Hz, 3H, CH
3
). F-NMR: (CDCl
3
) δ -62.31, -63.42. C-
2
NMR: (CDCl
3
) δ 172.27 (C=O), 142.37 (ArC), 142.25 (ArC), 136.04 (q, JC-F = 33Hz, ArC), 135.75 (ArCH),
2
3
1
1
33.72 (q, JC-F = 32.5Hz, ArC), 128.65 (ArCH), 126.67 (q, JC-F = 3.6Hz, ArCH), 122.87 (q, JC-F = 271.6Hz,
1
3
CF
3
), 122.07 (q, JC-F = 272.5Hz, CF
), 36.69 (CH), 18.88 (CH
65.0708; found, 465.0703.
-((3,5-Bis(trifluoromethyl)phenyl)sulfonyl)-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-methyl
3
), 121.81 (ArCH), 117.18 (q, JC-F = 4.9Hz, ArCH), 115.52 (CN),
1
4
3
04.40 (ArC), 58.88 (-CH
2
3
). HRMS calculated for C19 S [M+H],
H
14
F
6 2 3
N O
propanamide (31). Yield; 19%

ACCEPTED MANUSCRIPT
1
H-NMR: (CDCl
3
) δ 8.38 (s, 1H, NH), 8.30 (s, 2H, ArH), 8.06 (s, 1H, ArH), 7.88 (d, J = 2Hz, 1H, ArH), 7.74
(
dd, J = 2, 8.5Hz, 1H, ArH), 7.68 (d, J = 8.5Hz, 1H, ArH), 3.91 (dd, J = 9.5, 14.5Hz, 1H, CH ), 3.29 – 3.21
2
1
9
(m, 1H, CH), 3.17 (dd, J = 3.5, 14.5Hz, 1H, CH
2
), 1.37 (d, J = 7Hz, 3H, CH
3
). F-NMR: (CDCl
3
) δ -62.38, -
1
3
2
6
3
1
3.04. C-NMR: (CDCl
3
) δ 171.78 (C=O), 141.89 (ArC), 141.81 (ArC), 135.82 (ArCH), 134.03 (q, JC-F
=
2
3
2.6Hz, ArC), 133.43 (q, JC-F = 34.6Hz, ArC), 128.43 (q, JC-F = 3Hz, ArCH), 128.01 – 127.77 (m, ArCH),
1
1
3
22.14 (q, JC-F = 271.8Hz, CF
3
), 122.00 (q, JC-F = 272.4Hz, CF
3
), 121.79 (ArCH), 117.23 (q, JC-F = 4.6Hz,
ArCH), 115.52 (CN), 104.40 (ArC), 58.88 (CH
2
), 36.69 (CH), 18.88 (CH ). HRMS calculated for
3
C
20
H
13
F
9
N
2 3
O S [M+H], 533.0576; found, 533.0572.
N-(4-cyano-3-(trifluoromethyl)phenyl)-2-methyl-3-((3-(trifluoromethoxy)phenyl)sulfonyl)
propanamide (32). Yield; 95%
1
H-NMR: (CDCl
3
) δ 8.67 (s, 1H, NH), 7.88 (d, J = 2Hz, 1H, ArH), 7.82 (d, J = 7.5Hz, 1H, ArH), 7.77 (dd, J =
2
=
1
, 8.5Hz, 1H, ArH), 7.72 (s, 1H, ArH), 7.63 (d, J = 8.5Hz, 1H, ArH), 7.57 (t, J = 8Hz, 1H, ArH), 7.45 (d, J
8.5Hz, 1H, ArH), 3.86 (dd, J = 9.5, 14Hz, 1H, CH
2
), 3.34 – 3.25 (m, 1H, CH), 3.12 (dd, J = 3.5, 14.5Hz,
1
9
13
H, CH
2
), 1.34 (d, J = 7Hz, 3H, CH
3
). F-NMR: (CDCl
3
) δ -58.04, -62.34. C-NMR: (CDCl
3
) δ 171.69
2
(
C=O), 149.10 (ArC), 141.72 (ArC), 140.37 (ArC), 135.21 (ArCH), 133.28 (q, JC-F = 32.8Hz, ArC), 130.95
1
(
ArCH), 126.19 (ArCH), 125.65 (ArCH), 121.55 (q, JC-F = 272.3Hz, OCF
3
), 121.30 (ArCH), 119.92 (ArCH),
1
3
1
3
3
19.68 (q, JC-F = 258.3Hz, CF
3
), 116.76 (q, JC-F = 4.8Hz, ArCH), 115.05 (CN), 103.58 (ArC), 58.43 (CH
S [M+H], 481.0657; found, 481.0654.
-((4-Fluorophenyl)sulfonyl)-2-methyl-N-(4-nitro-3-(trifluoromethyl)phenyl)propanamide (33).
2
),
5.82 (-CH), 18.46 (CH
3
). HRMS calculated for C19
H
14
F
6
N
O
2 4
Yield; 41 %
1
H NMR (CD
3
OD) δ 8.14 (d, J = 2 Hz, 1H, ArH), 8.14 (d, J = 2 Hz, 1H, ArH), 8.01-7.97 (m, 2H, ArH), 7.88
), 3.34 (dd, J = 3,
); F NMR (CD OD) δ -61.67, -
(dd, J = 2, 9 Hz, 1H, ArH), 7.32-7.26 (m, 2H, ArH), 3.91 (dd, J = 9.5, 14.5 Hz, 1H, CH
2
19
1
4.5 Hz, 1H, CH
2
), 3.17-3.08 (m, 1H, CH), 1.34 (d, J = 7.5 Hz, 3H, CH
3
3
1
3
1
1
1
05.84; C NMR (CD
3
OD) ä 175.65 (C=O), 168.31 (d, JC-F = 253.5 Hz, ArC), 145.40 (ArC), 145.01 (ArC),
4
3
2
37.66 (d, JC-F = 3.3 Hz, ArC), 133.52 (d, JC-F = 9.9 Hz, ArCH), 129.05 (ArCH), 126.38 (q, JC-F = 33.5 Hz,
1
3
2
ArC), 124.53 (ArCH), 124.46 (q, JC-F = 270.9 Hz, CF
3.3 Hz, ArCH), 60.25 (CH ), 39.09 (CH), 20.14 (CH
M+H], 435.0638; found, 435.0643.
3
), 119.95 (q, JC-F = 5.8 Hz, ArCH), 118.53 (d, JC-F
=
2
2
3
). MS [ESI, m/z]: HRMS calcd for C17 O S
H
14
F
4
N
2
5
[
2-Methyl-N-(4-nitro-3-(trifluoromethyl)phenyl)-3-((4-(trifluoromethyl)phenyl)sulfonyl)
propanamide (34). Yield; 78 %
1
H NMR (CDCl
3
) δ 8.77 (s, 1H, NH), 8.15-8.11 (m, 3H, ArH), 7.96 (dd, J = 9, 2.5 Hz, 1H, ArH), 7.94-7.91
(m, 1H, ArH), 7.90-7.87 (m, 2H, ArH), 3.97 (dd, J = 9.5, 14 Hz, 1H, CH
2
), 3.45-3.35 (m, 1H, CH), 3.22

ACCEPTED MANUSCRIPT
1
3
19
(dd, J = 3, 14 Hz,1H, CH
2
), 1.45 (d, J = 7 Hz, 3H, CH
3
); F NMR (CDCl
3
) δ -60.18, -63.35; C NMR (CDCl )
3
2
ä 172.02 (C=O), 143.06 (ArC), 142.22 (ArC), 141.96 (ArC), 136.20 (q, JC-F = 33.3 Hz, ArC), 128.58
3
2
1
(
ArCH), 127.02 (ArCH), 126.79 (q, JC-F = 3.6 Hz, ArCH), 125.15 (q, JC-F = 33.8 Hz, ArC), 122.87 (q, JC-F =
1
3
2
71.4 Hz, CF
3
), 122.12 (ArCH), 121.65 (q, JC-F = 271.9 Hz, CF
3
), 118.22 (q, JC-F = 5.6 Hz, ArCH), 58.98
(
CH
found, 502.0857.
-((3,5-Bis(trifluoromethyl)phenyl)sulfonyl)-2-methyl-N-(4-nitro-3-(trifluoromethyl) phenyl)
2
), 36.22 (CH), 18.94 (CH
3
). MS [ESI, m/z]: HRMS calcd for C18
H
14
F
6
N
2
O
5
S [M+NH ], 502.0866;
4
3
propanamide (35). Yield; 69%
1
H NMR (CD
3
OD) δ 8.48 (s, 2H, ArH), 8.25 (s, 1H, ArH), 8.04 (d, J = 1.5 Hz, ArH), 8.01 (d, J = 9 Hz, 1H,
ArH), 7.78 (dd, J = 1.5, 9 Hz, 1H, ArH), 4.13 (dd, J = 10.5, 15 Hz, 1H, CH
2
), 3.51 (dd, J = 3, 15 Hz, 1H,
1
3
19
CH
2
), 3.23-3.15 (m, 1H, CH), 1.36 (d, J = 7 Hz, 3H, CH
3
); F NMR (CD
3
OD) δ -61.79, -64.50; C NMR
2
(
CD
3
OD) ä 172.90 (C=O), 142.79 (ArC), 142.71 (ArC), 142.01 (ArC), 132.65 (q, JC-F = 38.8 Hz, ArC),
3
3
1
28.44 (q, JC-F = 3Hz, ArCH), 127.44 (q, JC-F = 3 Hz ArCH), 126.67 (ArCH), 125.58 (m, ArC), 124.31 (m,
1
1
ArC), 123.33 (q, JC-F = 270.3 Hz, CF
7.70 (CH ), 36.74 (CH), 18.23 (CH
found, 570.0733.
3
), 122.01 (ArCH), 121.9 (q, JC-F = 270.9 Hz, CF
3
), 117.68 (m, ArCH),
5
2
3
). MS [ESI, m/z]: HRMS calcd for C19
H
13
F
N
9 2
5
O S [M+NH ], 570.0740;
4
N-(4-cyano-2-(trifluoromethyl)phenyl)-3-((4-fluorophenyl)sulfonyl)-2-methylpropanamide (36).
Yield; 68 %
1
H NMR (CDCl
3
) δ 8.41 (d, J = 8.5 Hz, 1H, ArH), 8.00-7.95 (m, 3H, ArH), 7.86 (dd, J= 1.5, 8.5 Hz, 1H,
), 3.21-3.14 (m, 2H, CH, CH ), 1.45
) δ -61.04, -102.36; C NMR (CDCl ) ä 171.66 (C=O), 166.08 (d,
C-F = 256.3 Hz, ArC), 138.82 (ArC), 136.59 (ArCH), 135.19 (ArC), 135.16 (ArC), 130.89 (d, JC-F = 9.4 Hz,
ArH), 7.83 (s, 1H, NH), 7.28-7.24 (m, 2H, ArH), 3.85-3.79 (m, 1H, CH
2
2
13
19
(d, J = 7 Hz, 3H, CH
3
); F NMR (CDCl
3
3
1
3
J
3
1
ArCH), 130.28 (q, JC-F = 5.5 Hz, ArCH), 124.51 (ArCH), 122.84 (q, JC-F = 271.6 Hz, CF
3
), 117.25 (CN),
2
1
16.88 (d, JC-F = 22.6 Hz, ArCH), 108.42 (ArC), 58.99 (CH
2
), 37.06 (CH), 18.50 (CH
3
). MS [ESI, m/z]:
HRMS calcd for C18
H
14
F
4
2 3
N O S [M+H], 415.0734; found, 415.0733.
N-(4-cyano-2-(trifluoromethyl)phenyl)-2-methyl-3-((4-(trifluoromethyl)phenyl)sulfonyl)
propanamide (37). Yield; 71 %
1
H NMR (CD
3
OD) δ 8.20 (d, J = 8 Hz, 2H, ArH), 8.16 (d, J = 2 Hz, 1H, ArH), 8.02-7.99 (m, 3H, ArH), 7.76
), 3.43 (dd, J = 3.5, 14.5 Hz, 1H, CH ), 3.32-3.25
OD) ä 173.92
(d, J= 8.5 Hz, 1H, ArH), 3.92 (dd, J = 9.5, 14 Hz, 1H, CH
2
2
13
19
(m, 1H, CH), 1.34 (d, J = 7 Hz, 3H, CH
3
); F NMR (CD
3
OD) δ -62.52, -64.66; C NMR (CD
3
3
2
(
C=O), 143.13 (ArC), 139.04 (q, JC-F = 1.5 Hz ArC), 136.03 (ArCH), 134.96 (q, JC-F = 32.9 Hz, ArC),

ACCEPTED MANUSCRIPT
3
3
1
30.44 (q, JC-F = 5.4 Hz, ArCH), 129.84 (ArCH), 128.85 (ArCH), 126.31 (q, JC-F = 3.8 Hz, ArCH), 125.35
2
1
1
(
q, JC-F = 30.8 Hz, ArC), 123.39 (q, JC-F = 270.9 Hz, CF
10.13 (ArC), 57.47 (CH ), 35.43 (CH), 17.56 (CH
M+H], 465.0702; found, 465.0699.
3
), 122.61 (q, JC-F = 271.6 Hz, CF
3
), 116.82 (CN),
1
2
3
). MS [ESI, m/z]: HRMS calcd for C19
H
14
F
6
N
2
O S
3
[
3-((3,5-Bis(trifluoromethyl)phenyl)sulfonyl)-N-(4-cyano-2-(trifluoromethyl)phenyl)-2-methyl
propanamide (38). Yield; 59 %
1
H NMR (CDCl
3
) δ 8.40 (s, 1H, ArH), 8.38 (d, J= 9 Hz, 1H, ArH), 8.18 (s, 1H, ArH), 7.96 (s, 1H, ArH), 7.86
(
(
(
d, J= 9 Hz, 1H, ArH), 7.81 (s, 1H, NH), 3.47 (dd, J= 10, 15 Hz, 1H, CH
2
), 3.31-3.23 (m, 2H, CH , CH), 1.50
2
1
3
13
19
d, J = 7 Hz, 3H, CH
3
); F NMR (CDCl
3
) δ -61.04, -62.95; C NMR (CDCl
3
) C NMR (CDCl
3
-d ) ä 171.21
1
2
3
C=O), 142.14 (ArC), 138.55 (ArC), 136.65 (ArCH), 133.40 (q, JC-F = 35 Hz, ArC), 132.32 (q, JC-F = 5.5 Hz,
3
3
1
ArCH), 128.44 (q, JC-F = 3.4 Hz, ArCH), 127.81 (sept, JC-F = 3.8 Hz, ArCH), 124.27 (ArCH), 122.84 (q, JC-F
1
=
271.8 Hz, CF
3
), 122.66 (ArC), 117.56 (q, JC-F = 281.5 Hz, CF
3
), , 117.16 (CN), 108.64 (ArC), 58.77
+
+
(
CH ), 37.12 (CH), 18.56 (CH
2
3
). MS (ES+) m/z: C20
H
13
F
9
N
2
O
3
S, 550.084 [M+NH
4
], 555.0395 [M+Na ].
3-((4-Fluorophenyl)sulfonyl)-2-methyl-N-(4-nitro-2-(trifluoromethyl)phenyl)propanamide (39).
Yield; 72 %
1
H NMR (CDCl
3
) δ 8.53 (d, J = 2 Hz, 1H, ArH), 8.44-8.39 (m, 2H, ArH), 8.04 (s, 1H, NH), 7.97 (dd, J= 5, 9
2
Hz, 2H, ArH), 7.25 (t, J = 8.5 Hz, 2H, ArH), 3.84 (dd, J = 8.5, 14 Hz, 1H, CH ), 3.30-3.22 (m, 1H, CH), 3.17
1
3
19
(
dd, J = 4, 14 Hz, 1H, CH
CDCl
2
), 1.44 (d, J = 7.5 Hz, 3H, CH
3
); F NMR (CDCl
3
) δ -60.99, -102.41; C NMR
1
4
(
3
) ä 170.91 (C=O), 165.02 (d, JC-F = 255 Hz, ArC), 143.40 (ArC), 140.49 (ArC), 135.13 (d, JC-F = 3
3
1
Hz, ArC), 130.89 (d, JC-F = 9.9 Hz, ArCH), 128.05 (ArCH), 124.67 (ArCH), 122.73 (q, JC-F = 271.9 Hz,
3
2
2
CF ), 122.33 (q, JC-F = 5.8 Hz, ArCH), 120.50 (q, JC-F = 31.1 Hz, ArC), 116.86 (d, JC-F = 22.6 Hz, ArCH),
3
5
4
2
8.21 (CH
2
), 36.88 (CH), 18.53 (CH
3
). HRMS calcd for C17
H
14
F
4
N
2
O S [M+H], 435.0632; found,
5
35.0631.
-Methyl-N-(4-nitro-2-(trifluoromethyl)phenyl)-3-((4-(trifluoromethyl)phenyl)sulfonyl)
propanamide (40). Yield; 74 %
1
H NMR (CDCl ) δ 8.56 (d, J = 2Hz, 1H, ArH), 8.48-8.40 (m, 2H, ArH), 8.10 (d, J= 8.5 Hz, 2H, ArH), 7.91 -
3
7
=
.84 (m, 2H, ArH, 1H, NH), 3.89 (dd, J = 9.5, 15 Hz, 1H, CH
2
), 3.26-3.16 (m, 1H, CH , 1H, CH), 1.48 (d, J
2
19
13
7 Hz, 3H, CH
3
); F NMR (CDCl
3
) δ -60.93, 60.97, -63.28; C NMR (CDCl
3
) ä 171.51 (C=O), 143.41
2
(
ArC), 142.58 (ArC), 140.32 (ArC), 135.88 (q, JC-F = 32.9 Hz, ArC), 128.70 (ArCH), 128.19 (ArCH), 126.64
3
1
1
(
q, JC-F = 3.6 Hz, ArCH), 124.15 (ArCH), 122.95 (q, JC-F = 271.8 Hz, CF
3
), 122.79 (q, JC-F = 271.9 Hz, CF
3
),

ACCEPTED MANUSCRIPT
3
2
1
22.36 (q, JC-F = 5.5 Hz, ArCH), 120.12 (q, JC-F = 30.9 Hz, ArC), 58.67 (CH), 37.08 (CH
2
), 18.57 (CH ). MS
3
[
ESI, m/z]: HRMS calcd for C18
H
14
F
6
N
2
O
5
S [M+H], 485.0600; found, 485.0595.
3-((3,5-Bis(trifluoromethyl)phenyl)sulfonyl)-2-methyl-N-(4-nitro-2-(trifluoromethyl)phenyl)
propanamide (41). Yield; 58 %.
1
H NMR ((CD
3
)
2
SO) δ 10.10 (s, 1H, NH), 8.65 (s, 1H, ArH), 8.55 (s, 2H, ArH), 8.52 (d, J = 8.5 Hz, 1H,
ArH), 8.46 (d, J = 2 Hz, 1H, ArH), 7.72 (d, J = 8.5 Hz, 1H, ArH), 4.01 (dd, J = 9, 14.5 Hz, 1H, CH
2
), 3.84
19
2
(dd, J = 3, 14 Hz, 1H, CH ), 3.34 (m, 1H, CH), 1.26 (d, J = 7 Hz, 3H, CH
3
); F NMR ((CD
3
)
2
SO) δ -59.84, -
1
3
2
6
3
1.32; C NMR ((CD
3
)
2
SO) ä 173.28 (C=O), 144.86 (ArC), 142.40 (ArC), 141.27 (ArC), 131.92 (q, JC-F
=
4
4 Hz, ArC), 130.21 (ArCH), 129.29 (d, JC-F = 3.4 Hz, ArCH), 128.65 (m, ArCH), 128.33 (ArCH), 124.06
2
1
1
3
(
q, JC-F = 31.4 Hz, ArC), 122.79 (q, JC-F = 275.3 Hz, CF
5.5 Hz, ArCH), 56.86 (CH ), 35.22 (CH), 18.70 (CH
M+NH ], 570.0740; found, 570.0736.
3
), 123.01 (q, JC-F = 271.8 Hz, CF
3
), 122.68 (q, JC-F
=
2
3
). MS [ESI, m/z]: HRMS calcd for C19
H
13
F
9
N
2
O S
5
[
4
4
.2 In vitro cell based assay
4
.2.1 Cell culture
All cells were cultured in T75 flasks in a humidified atmosphere of 5% CO at 37°C. LNCaP cells were
2
cultured in RPMI-1640 medium, PC3 and VCaP cells in DMEM, all supplemented with 10% FCS and
antibiotics (penicillin 100U/ml and streptomycin 100μg).
4
.2.2 Anti-androgen treatment in vitroꢀ
Bicalutamide and enzalutamide were used as positive controls (used as the clinical standard) in
LNCaP, PC3 and VCaP cells, and DMSO was used as a vehicle control. Nine 1⁄2 serial dilutions of 10
mM bicalutamide stock were made in 50 μL of DMSO to give nine increasing concentrations from 0.1
μM to 100 μM. All novel deshydroxy compounds were tested in LNCaP cells, and compounds 27, 28
and 33 were subsequently tested in PC3 and VCaP cells because of their low IC50 values.
PC3 and LNCaP cells were plated in a 96- well plate (4000cells/well) and then incubated overnight to
adhere. Each drug concentration (0-100uM) was then added in sequence to ten tubes of fresh media
to achieve a 1:100 dilution. The old medium was then gently removed and replaced with 200 μL of
fresh media containing the nine increasing drug concentrations and the DMSO control, in triplicate.
Following 96 hours of incubation the samples were tested with the MTT or MTS assay. The IC50 was
then calculated from the mean of these triplicate values and dose-response curves were plotted.
VCaP cells were treated as above, with the following exceptions. The novel compounds were diluted
1
:50, only 100 μL of the old medium was extracted and it was replaced with 100 μL of the novel

ACCEPTED MANUSCRIPT
compound in media. This was done to avoid removing the cells from the 96-well plates in view of
their poor adherence.
4
.2.3 MTT and MTS assays
Colorimetric MTT (LNCaP and PC3) and MTS (VCaP) assays were used to determine cell viability
following in vitro anti-androgen treatment. 20μL (1/10th well volume) of MTT or MTS were added to
the wells as appropriate. MTS absorbance was recorded at 490 nm by the Elx800TM microplate
reader after 1 and 2-hours of incubation. For the MTT assay, following the appearance of purple
formazan crystals under the light microscope, the medium was then carefully removed and the
formazan crystals dissolved in 200 μL of acidified isopropanol and MTT absorbance was recorded at
5
40nm on the Elx800TM microplate reader. Percentage cell viability was normalised to the DMSO
control.
.2.4 Statistics
4
Cell number-MTT correlations and IC50 values were calculated from the mean of three replicates
using Microsoft Excel and GraphPad Prism v6.01 (GraphPad Software, Inc., San Diego, CA). Cell
number-MTT correlations were analysed by linear regression, whereas IC50 values were produced
through non-linear regression. qPCR results were analysed using the ΔΔCT method in Excel. Mean IC50
values were then analysed for statistical significance (p=<0.05) compared to bicalutamide and
enzalutamide using GraphPad Prism v6.01 with consideration to the OECD guidelines.
One-way analysis of variance (ANOVA) followed by Tukey’s Honest Significant Difference test
analysed the dose-response data for compounds 27, 28 and 33 in LNCaP cells, which satisfied the
assumptions of normality and homogeneity of variance according to the D'Agostino- Pearson
omnibus, Brown-Forsythe and Bartlett’s tests. The Kruskal-Wallis test followed by the Dunn’s test
analysed the dose-response data for compound 28 in LNCaP cells and compounds 27, 28 and 33 in
PC3 cells.
4
.3 PSA Expression and androgen inhibition qPCR experiments
LNCaP cells were trypsinised and re-suspended in 13ml of RPMI. 0.5ml of this cell suspension was
then added to individual wells in 6-well plates with 3 ml of fresh RPMI, and the cells incubated for 24-
hours to adhere. Two individual wells in each plate received no treatment and DMSO at a 1:100
dilution as negative controls. The 6-well plates used to analyse PSA expression by qPCR then were
treated with 5, 10, 50 and 100 μM of bicalutamide, compounds 27, 28 and 33, followed by 24-hours
incubation.

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4.3.1 RNA Isolation
RNA isolation was carried out in the six-well plates using the Sigma procedure. The media was gently
removed, 1ml of TRI-reagent added per sample and the viscous lysate containing DNA, RNA and
proteins transferred to 1.5ml Eppendorf tubes. 200μl of 1-bromo-3-chloropropane was then added,
the Eppendorf tubes vortexed, left to stand for 5 minutes and stored at -80°C. The samples were then
warmed to room temperature, vortexed and centrifuged at 12,000 x g for 15 minutes at 4°C to
solubilise the RNA in the upper, colourless aqueous phase. The samples were then kept on ice, the
aqueous phase transferred to fresh Eppendorf tubes and 0.5ml of isopropanol added. The tubes were
then vortexed, left to stand for 5 minutes and centrifuged at 12,000 x g for 10 minutes at 4°C in order
to recover the RNA pellet. After removing the supernatant, the pellet was washed with 1ml of 75%
ethanol, vortexed and centrifuged at 7500 x g for 5 minutes at 4°C. After this, ethanol was carefully
removed, the samples briefly air-dried for 5-10 minutes and the RNA pellet dissolved with 20μl of
RNAse-free PCR water. The RNA concentration was then measured in ng/μl by recording the
absorption of each 1μl sample at 260nm on the Implen NanoPhotometer against nuclease-free water
as a control. The samples were then stored at -80°C for 24 hours.
4.3.2 Complementary DNA synthesis (cDNA)
The RNA samples were uniformly diluted to 50ng/μl on ice. 10 μl of RNA and 10 μl of RT mix were
then centrally added to a 96-well non-skirted PCR plate on ice, thermosealed and centrifuged up to
1
000rpm in the Grant bio LMC-3000. The components of the RT mix are shown below: The samples
were then placed in the Simpli-Amp thermal cycler under the following conditions: 25°C for 5
minutes, 42°C for 60 minutes, 70°C for 15 minutes, and 4°C. The samples were then stored at -20°C
overnight.
4.3.3 Quantitative polymerase chain reaction (qPCR)
qPCR was performed in triplicate to analyse the LNCaP cells for relative PSA gene expression. The PSA
primer was run alongside the AR, GAPDH, RPL19 and B-actin as housekeeping genes in order to
normalise the data and validate the results. These forward and reverse primer pairs were briefly
thawed and vortexed, diluted 1:40 with RNAse-free PCR water in four 1.5ml Eppendorf tubes, and
returned to ice. Following this, the cDNA was diluted in 20μl of RNAse-free PCR water. 40ul of this
diluted cDNA was then transferred into 0.5ml Eppendorf tubes and kept on ice. 2ul of this diluted
cDNA was added to individual wells of an optical qPCR 96-well plate, followed by 8μl of the PCR mix.
The qPCR plate was then thermosealed, centrifuged up to 1000rpm in the Grant bio LMC-3000, and
loaded into the StepOnePlus real-time PCR system. The PCR reaction volume was 10μl, and 50 cycles

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were performed within the standard 2-hour cycle.
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1
H NMR spectrum of compound 25

19
F NMR spectrum of compound 25