Design and synthesis of novel bicalutamide and enzalutamide derivatives as antiproliferative agents for the treatment of prostate cancer

Article abstract of DOI:10.1016/j.ejmech.2016.04.052

Prostate cancer (PC) is one of the major causes of male death worldwide and the development of new and more potent anti-PC compounds is a constant requirement. Among the current treatments, (R)-bicalutamide and enzalutamide are non-steroidal androgen receptor antagonist drugs approved also in the case of castration-resistant forms. Both these drugs present a moderate antiproliferative activity and their use is limited due to the development of resistant mutants of their biological target. Insertion of fluorinated and perfluorinated groups in biologically active compounds is a current trend in medicinal chemistry, applied to improve their efficacy and stability profiles. As a means to obtain such effects, different modifications with perfluoro groups were rationally designed on the bicalutamide and enzalutamide structures, leading to the synthesis of a series of new antiproliferative compounds. Several new analogues displayed improved in vitro activity towards four different prostate cancer cell lines, while maintaining full AR antagonism and therefore representing promising leads for further development. Furthermore, a series of molecular modelling studies were performed on the AR antagonist conformation, providing useful insights on potential protein-ligand interactions.

Full text of DOI:10.1016/j.ejmech.2016.04.052

European Journal of Medicinal Chemistry 118 (2016) 230e243
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and synthesis of novel bicalutamide and enzalutamide
derivatives as antiproliferative agents for the treatment of prostate
cancer
,
Marcella Bassetto 1, Salvatore Ferla* 1, Fabrizio Pertusati 1, Sahar Kandil,
Andrew D. Westwell, Andrea Brancale, Christopher McGuigan
School of Pharmacy and Pharmaceutical Sciences, Redwood Building, King Edward VII Avenue, CF10 3NB, Cardiff, Wales, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 November 2015
Received in revised form
20 April 2016
Accepted 21 April 2016
Available online 22 April 2016
This work is dedicated to the memory of
Prof. Chris McGuigan, a great colleague and
scientist, invaluable source of inspiration
and love for research.
Prostate cancer (PC) is one of the major causes of male death worldwide and the development of new
and more potent anti-PC compounds is a constant requirement. Among the current treatments, (R)-
bicalutamide and enzalutamide are non-steroidal androgen receptor antagonist drugs approved also in
the case of castration-resistant forms. Both these drugs present a moderate antiproliferative activity and
their use is limited due to the development of resistant mutants of their biological target.
Insertion of fluorinated and perfluorinated groups in biologically active compounds is a current trend
in medicinal chemistry, applied to improve their efficacy and stability profiles. As a means to obtain such
effects, different modifications with perfluoro groups were rationally designed on the bicalutamide and
enzalutamide structures, leading to the synthesis of a series of new antiproliferative compounds. Several
new analogues displayed improved in vitro activity towards four different prostate cancer cell lines, while
maintaining full AR antagonism and therefore representing promising leads for further development.
Furthermore, a series of molecular modelling studies were performed on the AR antagonist confor-
mation, providing useful insights on potential protein-ligand interactions.
Keywords:
Bicalutamide
Enzalutamide
Prostate cancer
Perfluoroalkyl
© 2016 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
Androgen receptor
Antiproliferative activity
1. Introduction
bicalutamide (Casodex®) (3), nilutamide (Niladron®) (4) and
enzalutamide (previously called MDV3100) (Xtandi®) (5) are all
Prostate cancer (PC) is a leading cause of male death worldwide
and it is the most frequently diagnosed cancer among men aged
65e74 [1]. The prognosis varies greatly, being highly dependent on
a number of factors such as stage of diagnosis, race and age.
Currently, PC treatment includes androgen deprivation, surgery,
radiation, endocrine therapy and radical prostatectomy.
PC cell growth is strongly dependent on androgens, therefore
blocking their effect can be beneficial to the patient's health. Such
outcomes can be achieved by antagonism of the androgen receptor
(AR) using anti-androgen drugs, which have been extensively
explored either alone or in combination with castration [2]. Fluta-
mide (Eulexin®) (1) (in its active form as hydroxyflutamide (2)),
non-steroidal androgen receptor antagonists approved for the
treatment of PC (Fig. 1). In many cases, after extended treatment
over several years, these anti-androgens become ineffective and
the disease may progress to a more aggressive and lethal form,
known as castration resistant prostate cancer (CRPC). The major
cause of this progressive disease is the emergence of different
mutations on the AR, which cause the anti-androgen compounds to
function as agonists, making them tumour-stimulating agents [3].
Among the drugs used for the treatment of PC, bicalutamide and
enzalutamide selectively block the action of androgens while pre-
senting fewer side effects in comparison with other AR antagonists
[4e6]. The structure of these molecules is characterised by the
presence of a trifluoromethyl substituted anilide, which appears to
be critical for biological activity (Fig. 1). As a means to improve the
anti-proliferative activity of these compounds, and in order to
exploit the well established potential of the fluorine atom in
enhancing the pharmacological properties and drug-like
* Corresponding author.
E-mail address: Ferlas1@cardiff.ac.uk (S. Ferla).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.04.052
0223-5234/© 2016 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
231
Fig. 1. Structure of anti-androgen small molecules approved by FDA or in clinical development for the treatment of PC.
physicochemical characteristics of candidate compounds [7e9], a
wide array of diverse new structures has been rationally designed
and synthesised, through the introduction of fluoro-, tri-
fluoromethyl- and trifluoromethoxy groups in diverse positions of
both aromatic rings of the parent scaffolds. Our modifications
resulted in a marked improvement of in vitro anti-proliferative
activities on a range of human PC cell lines (VCap, LNCaP, DU-145
and 22RV1). In addition, we probed full versus partial AR antago-
nism for our new compounds.
groups (nitro, cyano, trifluoromethyl) in different positions, ani-
lines 7e11 were in some cases (9 and 10 in particular) of low
reactivity towards nucleophilic displacement. Synthetic efforts
were made to achieve good yields (Supplementary data). Phenyl-
acrylamides 12e16 were converted into the corresponding epox-
ides 17e21 in the presence of a large excess of hydrogen peroxide
and trifluoroacetic anhydride in dichloromethane [11]. Opening of
the epoxide rings of 17e21 with commercially available phenols
and thiophenols gave a series of ethers (27e31) and thioether de-
rivatives (22e26), respectively, in good yields after purification by
column chromatography [11]. Thioethers 22e25 were finally oxi-
dised to the corresponding sulfones 32e35 using mCPBA, main-
taining the temperature at 25 ꢀC [12]. Racemic bicalutamide (3) was
prepared as a positive control following this route.
Since R-bicalutamide is known to be the most active enantiomer
[13], chiral synthesis of selected R-bicalutamide analogues was
carried out as shown in Scheme 2. (R)-N-Methacryloylproline 37,
prepared using (R)-proline (36) and methacryloyl chloride (6), was
reacted with N-bromosuccinimide in DMF to afford bromolactone
38 as a single enantiomer [14]. Acid hydrolysis of 38 resulted in the
formation of bromohydrin acid 39, which was then converted into
the corresponding chiral anilide (40e41) [14]. Amide derivatives
40e41 were reacted with the sodium salt of different commercial
thiophenols in tetrahydrofuran to give, after purification by silica
gel chromatography, the desired (R)-thioethers (42e44) [15]. Re-
action of amide 40 with the sodium salt of 4-cyanophenol gave the
reference compound (S)-enobosarm (44e). Oxidation of thioethers
42e43 with mCPBA provided sulfones 45e46, with the desired R
absolute configuration [12]. Reference (R)-bicalutamide (45a) was
prepared following this synthetic route.
2. Results and discussion
Trifluoromethyl and trifluoromethoxy functions were system-
atically inserted on both aromatic rings of bicalutamide and enza-
lutamide, with the aim to explore the effect of perfluoro groups on
their biological activity. As a means to further expand structure-
activity relationship studies, the insertion of different linkers was
also envisaged, along with the replacement of the bicalutamide
methyl group with a trifluoromethyl function.
The main proposed modifications are summarised in Fig. 2.
2.1. Chemistry
Several reported methods for the synthesis of racemic bicalu-
tamide were explored to find a rapid methodology that would
allow the preparation of a wide range of new derivatives [10e12].
Modification and optimization of these procedures led to the
development of the synthetic pathway shown in Scheme 1. Phe-
nylacrylamides 12e16 were prepared by reacting the correspond-
ing aniline (7e11) with methacryloyl chloride (6) in
dimethylacetamide (DMA), modifying a reported methodology
[10]. In particular, due to the presence of electron withdrawing
For one of the most active compounds initially found, 23d,
replacement of the central methyl group with an extra tri-
fluoromethyl function was planned and carried out (Scheme 3).
3-Bromo-1,1,1-trifluoroacetone (48) was coupled with thio-
phenol 47 to afford 49, which was then converted into cyano de-
rivative 50 using potassium cyanide and 25% sulfuric acid [16].
Intermediate 51 was obtained after refluxing 50 in concentrated
HCl and glacial acetic acid. Coupling of 51 with commercially
available 4-nitro-3-(trifluoromethyl)aniline 8 yielded the desired
amide 52.
Several methods have been reported for the preparation of
enzalutamide, all showing as a synthetic challenge the formation of
the N-substituted thiohydantoin ring [17e19]. The method selected
in this study was a three-step synthesis involving the preparation
of different isothiocyanates (54e58), obtained in quantitative yield
after treating the corresponding aniline (7e10, 53) with thio-
phosgene (Scheme 4) [17]. Reaction conditions were optimised for
the different anilines used, with 9 and 10 requiring higher
Fig. 2. Proposed modifications on bicalutamide and enzalutamide scaffolds.

232
M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
Scheme 1. Synthetic pathway for the synthesis of racemic bicalutamide analogues adapted from reported procedures [10e12]. Reagents and conditions: (a) 6 (8 equiv.), DMA, RT,
3 h; (b) H2O2 (4 equiv.)/(CF3CO)2O (5 equiv.), DCM, RT, 24 h; (c) NaH (1.2 equiv.), 17e21 (1.2 equiv.), THF, RT, 24 h; (d) mCPBA (1.4 equiv.), DCM, RT, 4e6 h.
Scheme 2. Synthesis of (R)-bicalutamide derivatives [12,14,15]. Reagents and conditions: (a) 6 (1 equiv.), 2N NaOH, acetone, 0 ꢀC to 10e11 ꢀC, 3 h; (b) NBS, DMF, argon, 3 days, RT
followed by H2O, RT, 12e24 h; (c) 24% HBr, reflux, 1 h; (d) 39 (1 equiv.), SOCl2 (1.2 equiv.), DMA, ꢁ10 ꢀC, 2 h followed by 7/9 (1 equiv.), DMA, RT, o.n.; (e) 40/41 (1 equiv.), phenol/
thiophenol (1.2 equiv.), 60% NaH (1.2 equiv.), THF, RT, 24 h; (f) mCPBA (1.4 equiv.), DCM, RT, 4e6 h.
temperature, longer reaction time and the addition of extra
equivalents of thiophosgene due to their low reactivity. Strecker
reaction of substituted anilines (11, 53) with acetone and trime-
thylsilyl cyanide generated the desired cyanomines 59e60. Reac-
tion of 59e60 with isothiocyanates 54e58 in DMF followed by the
addition of HCl and MeOH gave the desired thiohydantoins 61e65.
The last reaction step was characterized by poor yields possibly due
to low reactivity and the formation of several side products (mainly
due to the degradation of the reagents into the original starting
anilines). Moreover, attempted preparation of enzalutamide de-
rivatives 71b and 71d, in which the nitro and cyano groups are
removed, was unsuccessful following this route.
An alternative method reported for the preparation of enzalu-
tamide was investigated to obtain 71b and 71d (Scheme 5) [19].

M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
233
Scheme 3. Synthetic strategy used in the synthesis of 52. Reagents and conditions: (a) NaH (1 equiv.), THF, 0 ꢀC to RT, 3 h; (b) KCN (1.2 equiv.), 25% H2SO4, 0 ꢀC to RT, 20 h; c) HCl,
AcOH, reflux, 24 h; (d) 8, SOCl2 (1.3 equiv.), DMA, RT, 72 h.
derivative 67, which was then converted into its methyl ester 68
after treatment with iodomethane. Reaction of 68 with different
isothiocynates (70b, d) yielded the desired thiohydantoins 71b and
71d. Following this procedure, standard enzalutamide (5) was also
prepared.
2.2. Antiproliferative assay
Antiproliferative activity of the newly synthesized compounds
was initially evaluated with an in vitro 2D monolayer assay using
four human prostate cancer cell lines (LNCaP, 22Rv1, VCaP, and
DU145). LNCaP, VCaP and 22Rv1 exhibit some androgen sensitivity,
whereas DU145 is hormone-insensitive. The assay allows deter-
mination of the compound's capacity to inhibit survival and/or cell
Scheme 4. Synthetic strategy toward enzalutamide derivatives. Adapted from re-
ported procedure [17]. Reagents and conditions: (a) CSCl2 (1.5 equiv.), NaHCO3, H2O,
DCM, RT, 24 h; (b) TMSCN (5.1 equiv.), acetone, 80 ꢀC, 12 h; (c) DMF, RT 48 h, followed
proliferation. The antiproliferative results are reported in Tables 1
and 2 (absolute IC50 in mM).
The overall activity of the four reference compounds (3, 5, 45a,
44e) is consistent with previous reported data for some of these
specific cell lines and it represents a confirmation of the reliability
of the test [20e22]. 70% of our new derivatives performed better
than bicalutamide, either as a racemic mixture or pure R-isomer (3,
45a), significantly improving its antiproliferative activity up to 50-
fold (overall antiproliferative activity in the four cell lines reported
as geometric mean).
by HCl, MeOH, reflux, 6 h.
Most inhibitors showed concentration-dependent activity
against the four prostate cancer cell lines, with mean IC50 values
ranging from 1.6
mM to >100 mM. Active inhibitors showed
sigmoidal concentrationeeffect curves with total cell kill at high
concentrations (Supplementary data). Although the overall anti-
proliferative activity seems to be the result of the whole structure of
each single derivative, a general SAR can be identified, considering
three main structural components: linker X, aromatic ring A and
aromatic ring B. Thioether or ether compounds (X ¼ S or O) are
associated with better antiproliferative activity than the corre-
sponding sulfone derivatives (X ¼ SO2), with a decrease of effect in
the sulfones up to eight fold (i.e. 25l and 28l vs 35l; 23d & 28d vs
33d; 42d vs 45d; 22d & 27d vs 32d; 23c & 28c vs 33c). These data
are consistent with previous work, in which thioether derivatives of
SARMs (selective androgen receptor modulators) were found to be
more active than the corresponding sulfones [20]. However,
different newly prepared sulfones showed improved anti-
proliferative activity in comparison with the standard bicaluta-
mide, with a decrease of IC50 values up to two-fold (i.e. 33c, 33b,
45b, etc.). Replacement of the sulfur atom with oxygen is in general
associated with retained activity (i.e. 23c vs 28c; 22c vs 27c), with
only few cases of decreased activity in comparison with the thio-
ether analogues (i.e. 25l vs 28l; 23d vs 28d), probably due to the
combined effect of the substituent on aromatic ring B and the
linker. Several new ether derivatives performed better than the
Scheme 5. Synthetic strategy toward enzalutamide derivatives 71b-d. Adapted from a
reported procedure [19]. Reagents and conditions: (a) 2-aminoisobutyric acid
(1.5 equiv.), K2CO3 (6.5 equiv.), CuCl (0.2 equiv.), 2-acetylcyclohexanone (0.2 equiv.),
DMF, H2O, 105 ꢀC, 14 h; (b) MeI (1.2 equiv.), K2CO3 (1.2 equiv.), H2O, DMF, 40 ꢀC, 1 h; c)
CSCl2 (1.5 equiv.), NaHCO3, H2O, DCM, RT, 24 h; d) DMSO, IPAc, 84 ꢀC, 14 h.
Commercially available 3-bromobenzotrifluoride (66) was coupled
with 2-aminoisobutyric-acid to obtain amino propionic acid

234
M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
Table 1
Antiproliferative activity for bicalutamide/enobosarm derivatives. All data are mean values from triplicate experiments.
Compound
Ar (B ring)
X
R (A ring)
Absolute IC50
LNCaP
(mM)
22Rv1
DU-145
VCaP
Geo.mean
3 (Bic.)
44e (S-Eno)
45a (R-Bic.)
22b
22c
22d
22h
22o
23b
23c
23d
24a
24b
24c
24d
25a
25b
25c
4-F-Ph
4-CN-Ph
4-F-Ph
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
4-OCF3-Ph
4-CF3-2-Pyridine
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
4-F-Ph
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
4-F-Ph
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
3,4-F-Ph
2,4-F-Ph
4-OCF3-Ph
3-OCF3-Ph
2-OCF3-Ph
4-CF3-2-Pyridine
5-CF3-2-Pyridine
3-CF3-Ph
2-CF3-Ph
3-OCF3-Ph
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
3,4-F-Ph
2,4-F-Ph
4-OCF3-Ph
3-OCF3-Ph
4-CF3-2-Pyridine
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
SO2
O
SO2
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-CF3
49.58
24.77
46.25
NA
5.56
5.06
46.62
53.91
16.43
5.07
4.6
30.08
NA
15.16
11.64
17.63
14.63
12.58
11.97
83.59
14.93
61.92
15.80
4.64
13.58
11.52
18.57
5.20
15.90
6.91
49.20
44.55
45.41
45.27
20.90
45.20
68.37
24.47
51.61
52.42
27.41
47.05
10.58
7.73
64.01
84.10
22.52
8.73
5.03
4.91
42.81
80.72
28.71
6.44
9.23
7.51
40.33
89.27
21.17
8.64
7.23
6.16
47.64
75.60
21.78
7.04
7.57
43.28
4.78
26.45
4.45
45.48
5.17
35.37
21.05
18.42
26.40
19.20
14.35
17.53
100
21.43
69.59
21.63
6.57
19.74
14.03
30.71
8.76
27.84
16.28
10.84
17.32
45.19
44.26
18.36
16.36
100
11.26
10.72
11.93
8.66
10.07
11.66
83.04
11.00
100
9.15
3.31
11.06
5.52
22.56
7.22
23.48
18.27
21.10
17.07
16.51
12.79
100
16.64
100
15.88
5.27
17.18
17.36
19.50
5.86
18.82
9.65
17.04
14.32
18.50
14.28
13.16
13.30
91.28
15.56
81.02
14.93
4.80
15.02
11.15
22.44
6.63
18.44
9.15
25d
25f
25g
25h
25i
25l
25o
25p
26c
26d
26i
27b
27c
27d
27f
27g
27h
27i
27o
28b
28c
28d
28e
28f
28g
28h
28i
S
S
S
4-CF3
4-CF3
13.88
6.45
8.31
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-CF3
5.89
9.04
8.32
11.55
35.54
34.00
17.20
9.79
100
16.19
8.58
20.16
30.92
18.54
20.16
7.53
9.68
16.60
25.64
38.69
17.59
9.91
13.39
29.73
38.39
16.57
11.17
100
18.98
37.30
13.56
9.83
100
17.24
9.91
22.60
18.69
14.50
10.06
17.66
9.95
100
25.93
10.02
35.20
32.68
21.61
22.39
10.43
13.14
9.88
10.91
10.28
27.16
26.88
18.92
20.92
19.55
9.60
9.61
8.38
12.39
100
23.02
21.59
21.93
61.370
47.18
50.62
22.37
56.34
43.85
17.70
77.20
100
18.56
19.34
18.64
11.49
4.28
16.76
9.68
25.69
26.69
18.21
17.56
12.83
10.40
8.15
4-CN-Ph
3,4-F-Ph
2,4-F-Ph
4-OCF3-Ph
3-OCF3-Ph
2-OCF3-Ph
4-CN,2-CF3-Ph
4-CN,3-F-Ph
4-CF3-2-Pyridine
4-CF3-Ph
9.31
6.60
5.55
28l
7.04
5.69
7.71
28m
28n
28o
29b
29c
29d
29e
29f
7.65
16.39
100
6.71
11.77
35.22
29.91
18.70
19.60
42.935
37.03
60.92
27.99
28.65
16.68
18.45
45.34
100
11.65
59.88
28.26
21.08
21.15
55.00
39.95
55.37
27.83
43.76
28.61
17.04
65.89
100
36.51
28.61
15.23
14.03
34.722
34.80
30.64
27.65
63.39
33.83
9.39
78.27
100
13.57
12.93
18.16
6.98
32.39
32.12
33.17
100
41.93
99.48
34.64
35.86
27.09
27.52
68.78
100
22.08
18.14
27.45
26.41
29.40
3-CF3-Ph
2-CF3-Ph
4-CN-Ph
3,4-F-Ph
29g
29h
29i
2,4-F-Ph
4-OCF3-Ph
3-OCF3-Ph
2-OCF3-Ph
4-CN,2-CF3-Ph
4-CN,3-F-Ph
4-CF3-2-Pyridine
4-CF3-Ph
3-CF3-Ph
3-CF3-Ph
2-CF3-Ph
3-OCF3-Ph
29l
29m
29n
29o
30b
30c
31c
31d
31i
18.93
15.17
25.68
6.42
18.01
16.20
22.100
10.80
8.05
4-CF3
4-CF3
15.43
2.16

M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
235
Table 1 (continued )
Compound
Ar (B ring)
X
R (A ring)
Absolute IC50
LNCaP
(mM)
22Rv1
DU-145
VCaP
Geo.mean
32b
32c
32d
32h
32o
33b
33c
33d
34a
34b
34c
34d
35a
35b
35c
35d
35f
35g
35h
35i
35l
35o
35p
42b (R)
42c (R)
42d (R)
42g (R)
42h (R)
43a (R)
45b (R)
45c (R)
45d (R)
45g (R)
45h (R)
46a (R)
52
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
4-OCF3-Ph
4-CF3-2-Pyridine
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
4-F-Ph
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
4-F-Ph
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
S
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 2-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 2-CF3
4-NO2, 3-CF3
21.54
20.954
46.55
17.02
100
32.84
39.112
55.03
27.28
100
24.68
31.62
37.51
100
44.56
45.94
100
60.73
40.91
32.01
46.09
48.09
46.58
100
20.05
14.94
42.74
31.64
100
29.11
39.66
58.55
32.81
100
31.53
30.83
27.55
65.22
43.29
44.92
100
58.21
46.89
38.66
38.58
37.26
57.96
100
20.27
51.74
100
25.345
26.40
50.32
26.35
100
18.66
16.14
18.56
77.00
27.87
34.35
98.82
46.67
25.39
18.14
28.33
33.58
30.24
100
17.96
28.79
100
46.46
9.75
13.14
5.30
17.15
28.81
NA
17.94
16.20
25.70
91.59
32.93
32.08
74.02
38.65
29.30
16.77
20.46
19.47
17.92
100
12.23
17.77
100
45.03
14.45
14.31
7.90
22.59
22.47
26.50
82.36
36.48
38.83
92.48
50.25
34.56
24.77
31.86
32.90
34.78
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
3,4-F-Ph
2,4-F-Ph
4-OCF3-Ph
3-OCF3-Ph
2-OCF3-Ph
4-CF3-2-Pyridine
5-CF3-2-Pyridine
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
2,4-F-Ph
4-OCF3-Ph
4-F-Ph
4-CF3-Ph
3-CF3-Ph
2-CF3-Ph
2,4-F-Ph
100
18.74
31.87
27.72
38.97
100
57.66
17.18
19.45
7.69
100
58.47
9.28
8.91
2.95
18.29
17.39
51.54
12.24
13.43
5.55
21.78
23.32
S
S
S
S
28.46
29.78
25.19
19.83
S
SO2
SO2
SO2
SO2
SO2
SO2
S
20.19
25.66
34.75
42.25
18.82
100
26.76
45.19
44.31
60.20
31.56
100
17.47
13.17
32.23
42.26
18.91
85.55
0.72
31.42
39.92
37.30
60.90
31.195
100
23.34
27.95
36.89
50.58
24.33
96.17
1.57
4-OCF3-Ph
4-F-Ph
2-CF3-Ph
1.28
2.40
2.78
Table 2
Antiproliferative activity for enzalutamide derivatives. All data are mean values from triplicate experiments.
Compound
R1
R2
IC50 (mM)
22Rv1
DU-145
LNCaP
VCaP
Geo.mean
61b
62b
62c
63b
63c
64b
64c
65b
65c
71b
71d
5 (Enzal.)
4-CN, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-CN, 2-CF3
4-CN, 2-CF3
4-NO2, 2-CF3
4-NO2, 2-CF3
3-CF3
3-CF3
4-CF3
2-CF3
e
4-CF3
4-CF3
3-CF3
4-CF3
3-CF3
4-CF3
3-CF3
4-CF3
3-CF3
3-CF3
3-CF3
e
63.58
100
100
100
100
100
100
100
100
7.88
100
100
100
100
32.27
9.37
2.12
15.16
8.36
18.3
3.73
4.07
5.95
5.61
30.06
73.13
100
100
100
100
13.26
72.40
75.66
100
100
53.04
36.58
35.28
62.40
53.78
57.40
27.96
6.80
45.56
40.05
57.34
77.79
28.10
100
59.31
16.39
5.01
100
60.68
92.47
100
16.59
28.28
11.47
31.76
standard (S)-enobosarm (44e), improving its activity up to 4-fold
(i.e. 28m, 28l, 27c, etc.). Modifications on aromatic ring A do not
appear to significantly affect antiproliferative activity.
Structural modifications on the B ring profoundly influence the
antiproliferative effect, improving or totally abolishing activity. In
particular, replacement of the original fluorine in the para position
with a more lipophilic and bulkier trifluoromethyl moiety is asso-
ciated with an improvement of anti-proliferative activity of an
average of two fold (i.e. 3 vs 32b; 45a vs 45b; 25a vs 25b; 34a vs
34b; 35a vs 35b). Introduction of the trifluoromethoxy group in the

236
M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
para position does not have a significant effect or slightly decreases
the activity if compared with eCF3 analogues. The 4-CN group
present in (S)-enobosarm (44e) slightly improves or leaves unal-
tered the antiproliferative activity, depending on the X linker and
the substituent in the A ring (44e, 28e, 29e). Repositioning of the
trifluoromethyl group from the para to the meta and the ortho
position greatly improves antiproliferative activity up to ten-fold.
The best results were obtained with a substituent in position
ortho, either a CF3 (23d) or an OCF3 (25l) group. The IC50 values for
each new derivative appear to be a combination of the substituent
in the meta or ortho position of aromatic ring B, the linker (thio-
ether analogues are the most active) and the substituent in the A
ring (nitro derivatives perform slightly better than cyano ana-
logues). Generally, all meta and ortho substitutions on the B ring
performed better than standard bicalutamide, leading to a signifi-
cant activity improvement. Replacement of the phenyl ring B with
either a 4-trifluoromethyl pyridine or a 5-trifluoromethyl pyridine
abolishes activity with only few exceptions (25o, 25p), which are
probably a consequence of the presence of a 4-NO2-2-CF3 aromatic
substituent in the A ring. Introduction of a second fluoro substit-
uent in the B ring, such as a 2,4-fluoro, 2,3-fluoro, 4-CN,3-F or 4-
CN,2-CF3, is in general associated with a better activity profile if
compared with standard bicalutamide and enobosarm. In partic-
ular, the introduction of an extra trifluoromethyl group in the ortho
improve the biological effects of the parent molecules.
2.3. Androgen receptor (AR) agonist/antagonist assay
Twenty-two derivatives were selected for the evaluation of their
AR antagonist/agonist effect using the GeneBLAzer® Betalactamase
reporter technology for Nuclear receptors (NRs), to assess whether
the antiproliferative activity found is related to any interference
with the AR function [23].
All tested compounds were found to be antagonist of the
androgen receptor in a single concentration antagonism experi-
ment (10
mM concentration, antagonistic effect > 80%), which
measures their ability to reduce the receptor activation induced by
the known androgen receptor agonist R1881 [24,25]. A 10-
concentration antagonism assay (Supplementary data) showed
that the new molecules possess an antagonistic IC50 in the same
range of reference (R)-bicalutamide and enzalutamide (Table 3).
The (R)-bicalutamide IC50 is comparable with previously reported
values in similar assays [26,27].
In the bicalutamide-derived series of analogues, the antago-
nistic effect is not influenced by the substituents in aromatic ring A
(28d vs 23d vs 25d vs 22d), with a small beneficial effect found for
both the O and S linkers (28d vs 23d vs 33d) in the racemic de-
rivatives, whereas the SO2 linker is the most effective found for the
(R) structures (42b vs 45b and 45h). Structural modifications on
aromatic ring B have a major influence for antagonistic activity;
substitutions in the meta position are associated with improved
IC50 values (i.e. 28d, 28l, 23d), substitutions in the ortho position are
well tolerated (i.e. 23c), while substitutions in the para position
result in higher IC50 values (i.e. 42b) if compared with (R)-bicalu-
tamide. Overall, the results obtained fall in a similar range, with no
significant changes in the anti-androgenic activity in comparison
with control bicalutamide. The presence of a cyano group in the
para position of ring B (28m, 28n), as in control (S)-enobosarm, is
associated with the best antagonistic activity, while the
position of enobosarm aromatic ring
B improved its anti-
proliferative activity up to 4-fold (28m and 29m vs 44e).
No substantial differences were found between racemic and
chiral (R)-bicalutamide derivatives (22d vs 42d; 22c vs 42c), con-
firming that the observed superior efficacy of the R-bicalutamide is
mainly due to metabolic properties [13].
Interestingly, the best-performing new compound was 52, in
which the central methyl group is replaced with a trifluoromethyl
function. The IC50 (geometric mean of the overall antiproliferative
activity in the four cell lines) is 3.5-fold better than its methyl
analogue (23d), and the improvement is evident if compared with
bicalutamide (50-fold).
Considering antiproliferative activity for each individual cell
line, most of our compounds showed interesting results in all four
cell types, with the best results found for those cells expressing the
androgen receptor, such as LNCaP (the most sensitive cell line to the
new derivatives). This evidence suggests that the newly syn-
thesised structures retain an antagonistic effect on the androgen
receptor. Significant antiproliferative activity was also found in the
DU-145 cell line (the least sensitive to our new molecules), which
does not express the androgen receptor and is insensitive to
androgen activity, suggesting that also a different antiproliferative
mechanism could be involved, along with the canonical anti-
androgen receptor action. This potential off-target effect seems to
be present also in the parent bicalutamide (3, 45a), which shows
similar IC50 values across the four cell lines, and might have been
enhanced by the newly introduced modifications.
Newly synthesized enzalutamide analogues showed reduced
activity in the antiproliferative assay in comparison with standard
enzalutamide, with the only exception being 64c. However, the
new modifications clearly influenced the activity on LNCaP cells,
improving enzalutamide IC50 up to five-fold (62b). The total
absence of activity in the DU-145 cell line could be an indication of
a pure anti-androgenic effect for these new compounds. Replace-
ment of the original enzalutamide substituents in aromatic ring A
with a para trifluoromethyl group is the most effective modifica-
tion, while modifications on the B ring do not appear to signifi-
cantly affect activity.
Table 3
AR antagonist/agonist assay results. * Compounds were considered full antagonist if
at a single concentration of 10
greater than 80%; ** Compound at 10
used; *** Compounds tested at 10 different concentrations; **** Due to poor solu-
bility, the highest concentration used was 1 M; N.E, no antagonistic effect.
m
M the reduction of receptor activation by R1881 was
mM concentration in absence of R1881 was
m
Compound
Antagonistic effect (%)* Agonistic effect (%)** IC50 M)***
(m
45a (R-Bic.)
44e (S-Enob.) 59
83
5
0.490
0.0364
0.361
0.425
0.625
0.280
0.332
0.183
0.625
0.407
0.198
0.0722
0.0492
0.736
0.811
1.07
26
N.E.
11
N.E.
4
5 (Enzal.)
22d
23c
23d
25d
28d
28h
28i
95
93
93
91
94
94
96
106
103
108
65****
99
85
104
92
2
7
e
e
28l
N.E.
N.E.
N.E.
e
28m
28n
29m
33d
33c
42b
45b
45h
52
62b
63b
64b
64c
1
e
9
e
2.170
0.775
0.777
0.245
0.0803
0.402
0.265
0.111
0.123
93
61****
110
91
96
102
87
N.E.
e
e
e
e
As a general consideration on the antiproliferative results, the
introduction/change of position of trifluoromethyl and tri-
fluoromethoxy groups emerges as an interesting strategy to
e
65c
100
e

M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
237
introduction of a second substituent is tolerated. Compound 52, the
best performing in the antiproliferative assay, showed an antago-
nistic IC50 comparable to the one of (R)-bicalutamide. Considering
the enzalutamide series, no significant change in the antagonistic
effect was observed for the new derivatives tested.
For 14 compounds, a further analysis was performed in order to
verify any potential agonistic effect on the androgen receptor. The
compounds were tested at a single concentration (10 mM) under the
same experimental conditions used for the antagonistic assay, in
the absence of R1881. All new derivatives showed either total
absence or an agonistic effect <10% (not significant), confirming
their full antagonistic behaviour. The partial agonist nature of (S)-
enobosarm (44e) was also confirmed in our assay: after an initial
antagonistic activity at low concentration (best antagonistic IC50), it
acts as an agonist, activating the androgen receptor (agonistic effect
>20%). Interestingly, the new ether analogues, which could be
considered as enobosarm derivatives, were found to be full AR
antagonists (i.e. 28m, 28n, 25d, 28l).
The AR antagonist/agonist assay confirmed a pure AR antago-
nistic nature for the newly prepared compounds, which showed a
concentration-dependent antagonistic activity. Moreover, an ac-
tivity switch from partial agonist to full antagonist can be observed
for our enobosarm analogues.
No correlation between AR assay and antiproliferative data can
be identified, implying that other modes of action or other pa-
rameters could play an important role in the antiproliferative ac-
tivity found for our new compounds.
Fig. 3. Superposition between the crystal structure of the AR in the closed form (PDB
ID: 1Z95) (pink) and the new open AR model (light blue). Mutated Leu741 (pink) re-
duces the steric hindrance in comparison with the non-mutated Trp741 (light blue).
Co-crystallised bicalutamide (carbon atoms in salmon) is shown as stick model. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
2.4. Molecular modelling studies
Due to the retained activity of the newly synthesised com-
pounds on the AR, and in an attempt to investigate the switch from
partial agonist to full antagonist found for the newly prepared
enobosarm derivatives, a series of molecular modelling analyses
was designed and carried out.
2.4.1. AR antagonist conformation homology model
The functional switch from the open (antagonist) to the closed
(agonist) conformation of the AR is caused by the movement of
helix 12 (AR-H12), one of the 12 helices that define the ligand-
binding domain (LBD) [28,29]. The crystal structure of the com-
plex bicalutamide-AR is only available in the closed conformation
due to the W741L mutation (PDB ID: 1Z95), which causes bicalu-
tamide to occupy the AR binding site in an agonist fashion, allowing
the closure movement of helix 12 and therefore the activation of
the AR [28,29]. For the purpose of this part of the study, a homology
model of the human WT-AR open conformation was built, using a
single template approach in the MOE2014 homology modelling
tool following a previously reported methodology [30]. The crystal
structure of the progesterone receptor in its open antagonistic form
(PDB ID: 2OVM) was used as template, considering the good
sequence identity (54%) with the human AR [31]. The final 3D
model was obtained as the Cartesian average of 10 generated in-
termediate models [32]. Validation of the new model was per-
formed using the Rampage Server, the Errat plot and calculating the
RMSD with the 2AM9 crystal structure [33e35]. Fig. 3 shows the
shift of helix 12 between the new model and the mutated AR-
bicalutamide crystal (PDB ID: 1Z95) [28].
Fig. 4. Docking of (R)-bicalutamide (carbon atoms in pink) in the AR homology model
(light blue). Bicalutamide occupies the binding site in proximity of Trp741. The occu-
pational molecular surface around the active site is shown in grey. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
through a second H-bond with its cyanide nitrogen [36]. A direct
comparison with the bicalutamide-resistant AR crystal (Fig. 5)
suggests that the sulfone ring orientation of the docking pose is the
most plausible considering the presence of Trp741 and the position
of helix 12. In fact, in the crystal, due to a W741L mutation, the
sulfone aromatic ring is pointing down towards the centre of the
protein, lying in the position that would be occupied by the Trp741
indole ring, allowing helix 12 closure [36]. The presence of Trp741
in the model does not allow this disposition (steric clash), orienting
aromatic ring B toward helix 12, thus blocking its closure. Docking
2.4.2. Docking studies
Docking of (R)-bicalutamide was performed in the LBD of the
new model to evaluate its predicted antagonistic binding mode. As
shown in Fig. 4, docked bicalutamide occupies the binding site in
proximity of Trp741, forming a H-bond with Asn705 through its
hydroxyl group, and most importantly interacting with Arg752

238
M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
made possible by the more flexible and less bulky oxygen linker,
which allows a binding conformation similar to the one found for
bicalutamide in the W741L AR mutant crystal structure (Fig. 9).
Docking results show that the replacement of the cyano substituent
in aromatic ring B with a larger group (i.e. 2-OCF3 in 28l or 2-CF3 in
28d), or the introduction of a second aromatic substituent (i.e. 2-
CF3 in 28m), might lead to structural clashes with helix-12, con-
firming that these new enobosarm derivatives could impede the
movement of helix 12 to the AR-closed conformation, hence the
absence of any residual AR agonistic activity (Fig. 10).
The results obtained for our new enobosarm analogues might
represent a further indication that the newly inserted structural
modifications could play a role in overcoming the problem of
bicalutamide-induced AR mutation W741L.
Fig. 5. Comparison between our AR model (light blue) and bicalutamide-resistant AR
crystal 1Z95 (pink). Crystallised (carbon atoms in salmon) and docked bicalutamide
(carbon atoms in pink) highlighted. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
2.4.3. Molecular dynamics simulations
To further confirm the reliability of the new homology model,
and consequently support the speculations on the binding mode
proposed for the newly synthesized compounds, 5 ns molecular
dynamics (MD) simulations for selected derivatives (45a, 23d, 25d,
28d, 33d, 62b), either free in solution or in complex with the AR,
were performed. These structures were selected in order to cover
the range of activities found in the AR antagonist assay. The MD
of the more rigid enzalutamide (Fig. 6) confirms the proposed
antagonistic binding mode (comparable to the crystal structure of
AR-LDB in complex with an enzalutamide-like compound, PDB ID:
3V49) [37].
output results were used to compute DGbinding energy calculations
of the ligand-protein complexes (Table 4).
Fig. 7 shows the docking of two newly designed compounds
(28m, 33d, 52) in the AR homology model. The three compounds
show a consistent binding mode, occupying the LBD in the same
manner as bicalutamide, maintaining the same key interactions,
consistently with the AR assay results.
Replacement of the fluorine substituent in the B ring with a
bigger group such as trifluoromethyl or trifluoromethoxy,
increasing the steric hindrance of this part of the molecule, could
allow the overcoming of bicalutamide resistance mechanism. This
speculation is supported by the docking of 33d in the mutant AR
crystal structure 1Z95: as shown in Fig. 8, the bulkier substituent in
the B ring does not fit in the AR closed conformation, even in the
presence of the adaptive mutation W741L, as indicated by the close
proximity of ring B to the protein surface. Biological confirmation of
this effect will be the focus of future studies.
The stability showed by most of the ligand-protein systems
during the simulations could be a consequence of the active site
composition, mainly formed by hydrophobic amino acids (Met745,
Met787, Leu707, Leu704, Leu712, Val746, Trp741, Phe764, etc.),
which provides an ideal environment for the lipophilic nature of
these fluorinated molecules. Moreover, the formation of two
hydrogen bonds in the bicalutamide/enobosarm derivatives, both
present during the entire MD duration, with Arg752 and Asn705,
further contribute to the system stability, allowing the molecules to
maintain a stable conformation in the active site. The lowest
calculated
DGbinding was obtained for 62b, whereas 33d, a deriva-
tive with one of the highest antagonistic IC50 values, showed the
highest energy result. Interestingly, a similar correlation between
the calculated
found for all the compounds studied. Enzalutamide derivative 62b
showed the best result in terms of Gbinding, confirming the best
antagonistic IC50 value obtained from the assay and highlighting
the high affinity of the rigid enzalutamide-like structure for the
DGbinding energy and the AR antagonistic IC50 was
Molecular docking of the new enobosarm derivatives (i.e. 28d,
28m, 28n, 28l, 25d) was performed on the crystal structure of
closed-conformation WT-AR in complex with (S)-enobosarm (PDB
ID: 3RLJ) [38], in order to explain the observed activity switch from
partial AR agonist to full antagonist. (S)-Enobosarm, after binding
the AR and acting as antagonist, activates the receptor by favouring
the closure movement of helix 12 even in the absence of the W741L
mutation. This movement, as shown in the crystal structure, is
D
androgen receptor. The
DGbinding/AR antagonistic IC50 correlation
might represent a further confirmation of the reliability of the new
AR homology model and of the proposed binding mode for the new
fluorinated compounds.
2.5. In vitro metabolic stability, cell permeability and cytotoxicity
studies
The significant improvement obtained with the newly synthe-
sized analogues in the antiproliferative assay, which is associated
with retained AR antagonistic activity in comparison with the
reference compounds, could be a consequence of an off-target ef-
fect, already present in the parent structures and enhanced by the
new modifications, or a consequence of altered cellular meta-
bolism, stability and/or cell permeability of the new molecules, due
to their high fluorine content.
In order to address these two points, additional in vitro studies
on a selection of our most active compounds (22c, 22d, 23c, 23d,
27b, 28m, 33d, 42b, 42c) were performed. Metabolic stability, cell
permeability and general cytotoxicity were evaluated. (R)-Bicalu-
tamide (45a) and (S)-enobosarm (44e) were used in control
experiments.
Fig. 6. Docking of enzalutamide (carbon atoms in salmon) in the AR homology model
(light blue). The occupational molecular surface around the active site is shown in grey.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
239
Fig. 7. Predicted binding mode of new derivatives 28m, 33d and 52 in the AR homology model.
Fig. 9. Superimposition between the crystal structure of WT-AR co-crystallised with
(S)-enobosarm (green) and the W741L-AR co-crystallized with bicalutamide (salmon).
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Fig. 8. Docking of 33d (carbon atoms in light pink) in the 1Z95 crystal structure. The
red circle highlights how the ring B of the new compound is in close proximity to the
protein surface, potentially causing steric clashes that might impede the helix 12
movement to the closed conformation of the W741L AR mutant. The occupational
molecular surface of the binding site is shown in grey. (For interpretation of the ref-
erences to colour in this figure legend, the reader is referred to the web version of this
article.)
type of biotransformation) in human liver microsomes (phase I
drug metabolism). The tested compounds were incubated for
45 min with pooled liver microsomes (Supplementary data), and
the intrinsic clearance (CLint) and half-life (t1/2) values were
calculated based on 5 time points (Table 5). Bicalutamide, as re-
ported in the literature, mainly undergoes oxidation in humans by
2.5.1. Metabolic stability in liver microsomes
The selected compounds together with the controls were tested
for their stability (oxidative metabolism being the predominant

240
M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
Fig. 10. A) (S)-Enobosarm co-crystallized in the WT-AR closed conformation (PDB ID: 3RLJ). B) Docking of 28d (carbon atoms in turquoise) and 28l (carbon atoms in white) in the
3RLJ crystal structure. Replacement of the cyano substituent with a bulkier group might cause structural clashes (red circles) with helix-12. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Table 4
new compounds. The data obtained are reported in Table 6 as
apparent permeability (Papp) and efflux ratio. Generally, com-
pounds with a Papp A / B between 2 and 20 ꢂ 10ꢁ6 cm sꢁ1 are
considered as medium permeability drugs, whereas an efflux ratio
(BA/AB) higher than 2 is an indication that the compound could be
a substrate for a drug transporter (i.e. P-gp, BCRP) and undergo
active efflux to the extracellular compartment. These transporters
are all over-expressed in cancer cells and are commonly respon-
sible for drug resistance [21,39].
Calculated ligand-interaction energies for the compounds analysed by Molecular
Dynamics and antagonistic IC50 values.
Compound
D
Gbinding (kJ/mol)
IC50 (mM)
62b
28d
23d
25d
45a (R-bic.)
33d
ꢁ13.425
ꢁ9.586
ꢁ8.909
ꢁ6.793
ꢁ2.521
0.781
0.0803
0.183
0.280
0.332
0.490
0.811
(R)-Bicalutamide (45a) shows
a Papp A / B value of
32.0 ꢂ 10ꢁ6 cm sꢁ1, which is comparable to the one reported in
literature (22.1 ꢂ 10ꢁ6 cm sꢁ1), as both of these values would fall
into the rank order category of good permeability [21]. Under the
cytochrome P450 enzymes [26].
As expected, intrinsic clearance (Clint) is high and, as conse-
quence, t1/2 is very low for 22c, 22d, 23c, 23d, 42b, 42c. These
compounds are thioethers and therefore they might undergo rapid
oxidation of their sulfide linker to the corresponding sulfoxide and
eventually to the sulfone. Ether derivatives 28m, 27b and (S)-
enobosarm and sulfones 33d and (R)-bicalutamide show lower Clint
and higher t1/2, with some of them showing complete metabolic
stability (no loss of the parent detected during the assay). Oxidative
metabolism seems to affect mainly the thioether linker with a small
effect on the aromatic systems.
Interestingly, introduction of a trifluoromethyl group in the ar-
omatic ring (33d) completely abolishes any oxidative metabolism
on the ring itself, doubling the t1/2 in comparison with (R)-bicalu-
tamide. The significant antiproliferative activity found for thioether
and ether derivatives does not seem to be caused by any oxidative
metabolite, as indicated by the high stability of ether analogues and
by the decrease in antiproliferative activity found for the respective
sulfone derivatives. Moreover, these results suggest that ether de-
rivatives have the best antiproliferative and stability (low intrinsic
clearance) profile, making them good potential drug candidates.
test conditions the Papp
B / A value was found to be
38.1 ꢂ 10ꢁ6 cm sꢁ1, indicating membrane permeability in both
directions for (R)-bicalutamide. The calculated efflux ratio (BA/AB)
of 1.19 indicates that (R)-bicalutamide is not an efficient substrate
for transporters, in discordance with published data, in which
bicalutamide shows an efflux ratio (BA/AB) of 5.4 [21]. This
discrepancy could be a consequence of using (R)-bicalutamide
(45a), since bicalutamide enantiomers have different metabolism,
permeability and absorption properties in vivo, which influence
their biological activity [13] and possibly the interaction with the P-
glycoprotein. Moreover, several experimental variables can influ-
ence the transport of compounds in both membrane directions.
Due to this variability, the Caco-2 cell data have been used only to
verify if any relevant difference in cell permeability is present
among the tested compounds. The reliability of our assay and the
functional presence of the protein transporters were evaluated
using two standard reference compounds (atenolol for low
permeability and propranolol for high permeability) and one
known P-glycoprotein substrate (talinolol) (Supplementary data).
The new molecules appear to be moderately permeable (2<Papp
A / B>20 ꢂ 10ꢁ6 cm sꢁ1), with a low tendency to efflux from the
cell. Differences between racemic and (R) derivatives are evident
comparing compounds 22c and 42c, in which the pure (R) enan-
tiomer has an increased efflux ratio, still lower than 2. Overall, the
2.5.2. Caco-2 cell permeability
An in vitro Caco-2 bi-directional permeability assay was per-
formed to gain information on membrane penetration, efflux from
cells and possible interaction with the P-glycoprotein (P-gp) for the

M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
241
Table 5
Metabolic stability in human liver microsomes; * CLint: theoretical unrestricted maximum clearance of unbound drug by an eliminating organ, in absence of blood or plasma
protein binding limitations; ** SE: standard error; ***Compounds metabolically stable with no loss of parent detected for the duration of the assay.
Compound
Ar (B ring)
X
R (A ring)
Metabolic stability
SE CLint**
CLint
6.48
(
m
L/min/mg protein)*
t1/2 (min)
214
45a (R-Bic.)
44e (S-Eno)
22c
4-F-Ph
4-CN-Ph
3-CF3-Ph
SO2
O
S
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
2.52
Metabolically Stable***
232
5.89
12.8
2.33
46.8
5.97
3.66
6.87
3.39
22d
23c
23d
2-CF3-Ph
3-CF3-Ph
2-CF3-Ph
S
S
S
4-CN, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
379
202
409
27b
28m
4-CF3-Ph
4-CN,2-CF3-Ph
O
O
4-CN, 3-CF3
4-NO2, 3-CF3
Metabolically Stable***
2.59
2.45
534
33d
42b
2-CF3-Ph
4-CF3-Ph
SO2
S
4-NO2, 3-CF3
4-CN, 3-CF3
Metabolically Stable***
248
11.8
5.66
5.60
4.59
42c
3-CF3-Ph
S
4-CN, 3-CF3
302
Table 6
Caco-2 cell permeability test; * (A / B): apical-basolateral flux; ** (B / A): basolateral-apical flux; this information was used for the apparent permeability (Papp) evaluation;
***Efflux Ratio: Mean Papp B / A/Mean Papp A / B; yCompounds tested under standard conditions using HBSS buffer at pH ¼ 7.4. All other compounds were tested under the
same conditions but the cell media contained BSA (1% w/v). All compounds were tested an initial concentration of 10 mM.
Compound
Ar (B ring)
X
R (A ring)
Permeability data
Mean Papp (A / B) (x 10ꢁ6 cm s1)*
Mean Papp (B / A) (x 10ꢁ6 cm sꢁ1)**
Efflux ratio BA/AB***
45a (R-Bic.)y
44e (S-Eno.)y
22c
22d
23c
4-F-Ph
SO2
O
S
S
S
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-CN, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
32.0
31.5
3.92
3.08
5.00
3.44
5.09
4.35
13.3
5.80
6.84
38.1
26.7
4.73
7.12
4.27
2.90
4.80
3.49
17.2
4.74
12.2
1.19
0.848
1.21
4-CN-Ph
3-CF3-Ph
2-CF3-Ph
3-CF3-Ph
2-CF3-Ph
4-CF3-Ph
4-CN,2-CF3-Ph
2-CF3-Ph
4-CF3-Ph
3-CF3-Ph
2.31
0.853
0.843
0.943
0.803
1.29
23d
27b
S
O
O
SO2
S
28m
33dy
42b
42c
0.817
1.78
S
results obtained suggest that these compounds do not show any
significant difference in cell permeability in comparison with the
standards, therefore no correlation can be identified between
antiproliferative data and cell permeability properties.
compounds showed a concentration-dependent decrease in for-
mazan production across the range studied (example plots in the
Supplementary data), indicating a decrease in cell viability associ-
ated with some growth inhibitory and toxic effects. Compared with
control (R)-bicalutamide (45a), the new compounds are in general
characterized by a higher cytotoxicity, with the lowest MEC and
AC50 values obtained for 23d, which is also one of the most active
compounds in the antiproliferative assay (antiproliferative data
Table 7).
These results might represent an important indication that the
newly prepared analogues, besides the canonical AR antagonist
activity, possess an extra effect, which positively contributes to
their antiproliferative action. Such effect seems to be particularly
evident for 23d, while other derivatives (e.g. 28m, 42b) do not
2.5.3. Cell viability assay (MTT)
A MTT cell viability assay was performed in a human hep-
atocarcinoma (HepG2) cell line, in order to evaluate the cytotoxicity
profile and to further explore the possible presence of an off-target
effect for the new compounds. The MTTassay measures the effect of
molecules on cell proliferation and other events that eventually
lead to cell death [40].
Data are reported in Table 7 as minimum effective concentration
(MEC) related to a vehicle control (DMSO) and AC50. All tested

242
M. Bassetto et al. / European Journal of Medicinal Chemistry 118 (2016) 230e243
Table 7
MTT assay; all the compounds have been tested at 8 different concentrations. * MEC: minimum effective concentration that significantly crosses vehicle control threshold; **
AC50: concentration at which 50% of maximum effect is observed; ***Geometric mean.
Compound
Ar (B ring)
X
R (A ring)
MTT test
Antiproliferative data
MEC (
m
M)*
AC50
(
mM)**
Abs. IC50 (mM)***
45a (R-Bic.)
44e (S-Eno.)
22c
22d
23c
23d
27b
28m
33d
4-F-Ph
SO2
O
S
S
S
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-CN, 3-CF3
4-NO2, 3-CF3
4-NO2, 3-CF3
4-CN, 3-CF3
4-CN, 3-CF3
19.2
21.8
18.4
14.7
13.2
1.71
11.3
10.6
22.7
49.6
26.6
54.3
32.8
36.6
25.8
26.1
2.73
23.9
20.2
32.6
61.2
34.6
47.05
27.41
7.23
6.16
7.04
5.17
9.15
6.71
4-CN-Ph
3-CF3-Ph
2-CF3-Ph
3-CF3-Ph
2-CF3-Ph
4-CF3-Ph
4-CN,2-CF3-Ph
2-CF3-Ph
4-CF3-Ph
3-CF3-Ph
S
O
O
SO2
S
26.50
12.24
13.43
42b
42c
S
show a significant increase in cytotoxicity, maintaining instead a
remarkable improvement in the antiproliferative assay in com-
parison with bicalutamide. The mechanism and effect (necrosis,
apoptosis, reduction of cellular proliferation or effect on mito-
chondria) causing this cytotoxicity, which seems to be a conse-
quence of the new structural modifications, will be the main focus
of future studies.
to the replacement of the central methyl group of bicalutamide
with a CF3 (52).
Future biological studies will aim to understand the mechanism
of action and the biological consequences of potential off target
effects, along with the evaluation of our novel AR antagonists in a
W741L mutant AR. Finally, the most promising compound for each
structural family (23d, 27b, 33d) has been selected for in vivo pre-
clinical studies in mouse models.
3. Conclusions
4. Experimental
The structures of bicalutamide and enzalutamide, two non-
steroidal androgen receptor antagonists in use to treat prostate
cancer, have been chemically modified leading to the synthesis of
new and more potent antiproliferative compounds. Different
chemical modifications have been planned in order to benefit from
the properties of fluorine in improving the activity and pharma-
cological profile of drugs. The novel chemical entities were found to
be full androgen receptor (AR) antagonists, with no residual agonist
effect found also for structural analogues of enobosarm (ether de-
rivatives), a well-known AR partial agonist. Introduction of extra
perfluoro groups (CF3 and OCF3) confers metabolic stability to
phase I drug metabolizing enzymes, maintaining the same good
membrane permeability of the parent compounds. A very notable
improvement in the in vitro anti-proliferative effect on four
different PC cell lines (VCap, LNCaP, DU-145 and 22RV1) has been
achieved, improving the activity of the parent structures up to 50
folds, thus making some of the new molecules potential drug
candidates for the treatment of prostate cancer. This marked ac-
tivity improvement did not parallel an enhanced AR antagonistic
effect. Moreover, increased cytotoxicity found for some of the new
analogues in the MTT assay could be an indication that the com-
pounds developed in this work, while certainly binding to AR LBD
and exhibiting full antagonist behaviour, might possess another
biological effect, which positively contributes to their anti-
proliferative action in the prostatic cancer cell lines. The reliable AR
homology model prepared and all the docking information pro-
vided can offer useful insights for the development of pure AR
antagonists. The new model appears well validated by all observed
data.
All chemistry, biology and molecular modelling experimental
procedures, along with compound characterisation, are fully
described in the Supplementary Data. All final compounds were
purified by column chromatography or recrystallization, fully
characterised by NMR (1H, 13C, 19F) and LRMS. HRMS is reported for
representative final compounds (one example for each synthetic
method). All final compounds were found to be >95% pure by HPLC.
Acknowledgments
The authors would like to thank Oncotest (Freiburg, Germany)
for provision of human prostate cancer cell line testing as an out-
sourced service. The AR binding assays were carried out by Invi-
trogen (location), and Cyprotex (U.K.) were responsible for
performing the metabolic stability, cell permeability and HepG2
cytotoxicity assays as an outsourced service. Mr Derek Angus is
warmly acknowledged for many valuable comments on the ADME
section of the manuscript. The Welsh Government is acknowledged
for funding (A4B-Academic Expertise for Business grant), in
collaboration with Catylix, Inc (Burbank, CA, USA) as industrial
advisers to the project).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.052.
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structural modifications will be further investigated, giving priority
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