Synthesis and biological activity of ferrocenyl derivatives of the non-steroidal antiandrogens flutamide and bicalutamide

Article abstract of DOI:10.1016/j.jorganchem.2010.10.051

A series of ferrocenyl derivatives of the two non steroidal antiandrogens flutamide and bicalutamide have been prepared. Ferrocenyl bicalutamide complexes were initially synthesized in their racemic forms, and subsequently prepared as pure (R) and (S) enantiomers, and their structure was determined by X-ray crystallography. Most of the complexes retain a modest affinity for the androgen receptor and show an antiproliferative effect on both hormone-dependent (LNCaP) and -independent (PC-3) prostate cancer cells. Ferrocenyl derivatives of bicalutamide are the most cytotoxic (IC₅₀ values on PC-3 around 15 μM); however, they are less potent than the ferrocenyl derivatives of ethynyltestosterone or nilutamide (IC₅₀ around 5 μM).

Full text of DOI:10.1016/j.jorganchem.2010.10.051

Journal of Organometallic Chemistry 696 (2011) 1049e1056
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis and biological activity of ferrocenyl derivatives of the non-steroidal
antiandrogens flutamide and bicalutamide
,
a
a,
Olivier Payen a, Siden Top a , Anne Vessières , Emilie Brulé c, Agnès Lauzier a, Marie-Aude Plamont a,
*
Michael J. McGlinchey b, Helge Müller-Bunz b, Gérard Jaouen a
a Chimie ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire Charles Friedel, UMR CNRS 7223, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France
b UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
c Université Pierre et Marie Curie, 4 Place Jussieu, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ferrocenyl derivatives of the two non steroidal antiandrogens flutamide and bicalutamide
have been prepared. Ferrocenyl bicalutamide complexes were initially synthesized in their racemic
forms, and subsequently prepared as pure (R) and (S) enantiomers, and their structure was determined
by X-ray crystallography. Most of the complexes retain a modest affinity for the androgen receptor and
show an antiproliferative effect on both hormone-dependent (LNCaP) and -independent (PC-3) prostate
cancer cells. Ferrocenyl derivatives of bicalutamide are the most cytotoxic (IC50 values on PC-3 around
Received 29 July 2010
Received in revised form
13 October 2010
Accepted 21 October 2010
Keywords:
15
m
M); however, they are less potent than the ferrocenyl derivatives of ethynyltestosterone or niluta-
M).
Anticancer agents
Antiandrogen
Ferrocene
mide (IC50 around 5
m
Ó 2010 Elsevier B.V. All rights reserved.
Prostate cancer
Flutamide
Bicalutamide
1. Introduction
years most patients relapse with advanced metastatic disease. The
cancer usually becomes hormone refractory, possibly because of
mutations of the androgen receptor [9], and the prognosis for these
advanced metastatic prostate cancers is poor. Therefore, there is
a crucial need to develop novel therapeutic agents effective on both
hormone dependent and -independent prostate cancer cells.
Moreover the antiandrogens described above are also antago-
nists of the AR in other peripheral tissues such as in muscle and
bone in addition to prostate. In recent years, selective androgen
receptor modulators (SARMs) have emerged whose aim is to target
the AR in a tissue selective way, thus leading to selective biological
effects [10,11]. For example, a SARM can be an antagonist or weak
agonist in the prostate and fully agonistic in the pituitary and
muscle. This concept of a SARM is thought to have therapeutic
promise in the use of androgens [12]. The first generation of SARMs
was discovered by Dalton and Miller [13,14] by replacing the
sulfone of bicalutamide by an ether function, leading to SARM S-1
(Chart 1). Since then, many SARMs have been reported in the
literature such as arylpropionamide analogues [15] (derivatives of
bicalutamide) and bicyclic hydantoins [16], and some of them are in
now in the clinical phase [17].
Prostate cancer is a major societal problem which affects one
man in eight and is the cause of 200,000 deaths per year worldwide
[1]. The growth and maintenance of the prostatic cells are stimu-
lated by androgens, testosterone and
5a-dihydrotestosterone
(DHT), the male sex hormones. The majority of these cancers are
hormone dependent, and their development involves an interac-
tion between the androgen receptor (AR) and the androgen [2]. The
most important treatment of these hormone dependent cancers
involves androgen withdrawal using surgical or chemical castration
with steroidal or non-steroidal antiandrogens. Cyproterone acetate
was one of the first steroidal antiandrogen clinically used but its
side-effects, especially the interaction with the progestin and
glucocorticoid receptor, made this drug less popular than the non-
steroidal antiandrogens such as nilutamide [3,4], flutamide [5e7]
and bicalutamide [8] (Chart 1). However, within a period of 1e3
* Corresponding author. Tel: þ33 1 44 27 66 99.
E-mail address: siden-top@chimie-paristech.fr (S. Top).
0022-328X/$ e see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2010.10.051

1050
O. Payen et al. / Journal of Organometallic Chemistry 696 (2011) 1049e1056
OH
OH
CF3
CF3
O2N
O2N
CH3
CH3
O
O
CH3
CH3
N
H
N
O
NH
O
O
dihydrotestosterone
testosterone
CF3
Flutamide
Nilutamide
CN
CF3
CF3
F3C
NC
O
NC
F
O2N
F
O
O
5
O
S
O
N
NH
N
H
N
H
O
Fe
OH
O
H3C
OH
H3C
Bicalutamide
SARM S-1
5-Fc-hydantoin
Chart 1.
We have previously shown that the incorporation of a ferrocenyl
unit into non steroidal antiestrogens such as hydroxytamoxifen can
lead to complexes showing a strong antiproliferative effect on both
hormone dependent and -independent breast cancer cells [18e20].
Following the same strategy, we recently prepared ferrocenyl
derivatives of nilutamide [21], (5-Fc-hydantoin) as well as of
testosterone and dihydrotestosterone [22]. These new ferrocenyl
derivatives showed strong cytotoxicity on hormone-independent
synthesis of 1 bearing a nitro substituent on the aromatic ring has
recently been described [25] and the cyano analogue 2 was
prepared following the same procedure, i.e. by treating the acid
chloride formed from ferrocene carboxylic acid and oxalyl chloride
with 4-cyano-3-trifluoromethyl-aniline in the presence of trie-
thylamine to give N-(4-cyano-3-trifluoro-phenyl)-ferrocenylamide
2 in 48% yield (Scheme 1). Treatment of aminoferrocene with 2-
methylpropanoyl chloride furnished N-(2-methylpropanoyl)-ami-
noferrocene, 3 in 43% yield.
PC-3 cell lines (IC50 around 5 mM). These very promising results
prompted us to extend the study to ferrocenyl derivatives of flu-
tamide and bicalutamide. Recently the synthesis of rhenium and
technetium derivatives of flutamide was reported by Benny et al. as
a novel class of single photon emission computer tomography
(SPECT) imaging agents [23,24]. Our group also reported the
synthesis of cyclopentadienyl-M-tricarbonyl derivatives (M ¼ Re or
Tc) of flutamide using the novel ferrocenyl derivative of flutamide 1
as the starting material (Chart 2) [25].
We here describe the syntheses of ferrocenyl derivatives of
flutamide 2 and 3, as well as ferrocenyl derivatives of SARM S-1: the
racemate 4 and 5, as well as the two pure enantiomers (R)-5 and
(S)-5 (Chart 2). We also report some of their biological properties
including the determination of their affinity for the androgen
receptor, and an evaluation of their cytotoxic activities on hormone
dependent (LNCaP) and hormone independent (PC-3) prostate
cancer cells.
The design of the ferrocenyl derivatives of SARM S-1 involved
the exchange of the fluorophenyl group by a ferrocenyl methyl unit
while varying the para-substituent on the remaining aromatic with
NO2 and CN, thus leading to 4 and 5, respectively. They were first
obtained in racemic form before the independent syntheses of the
two enantiomers (S)-5 and (R)-5 were carried out for comparison of
their activity on prostate cancer cells.
The new compounds 4 and 5 were prepared via the reaction of
sodium ferrocenylmethoxide with epoxides 8 and 9, respectively,
which were themselves obtained by following the literature
procedure reported by Chen and coworkers [26] (Scheme 2). The
reaction of 4-nitro-3-trifluoromethyl-fluorobenzene with sodium
methacrylamide, generated in situ from the addition of NaH to
methacrylamide, secured 6 in 88% yield. The epoxidation of 6,
performed in the presence of hydrogen peroxide and trifluoroacetic
anhydride, furnished 8 in 61% yield. Analogously, 7 was obtained
CF3
CF3
R
R
O
O
O
N
H
N
H
O
CH3
CH3
N
H
OH
H3C
Fe
Fe
Fe
1: R= NO2
2: R= CN
3
4: R= NO2
5, (S)-5, (R)-5: R= CN
Chart 2.
2. Results and discussion
from 4-cyano-3-trifluoromethyl-fluorobenzene. The reaction of 7
with trifluoroacetic anhydride and hydrogen peroxide gave 9 in an
improved yield of 87%. Finally, addition of 8 to sodium ferroce-
nylmethoxide, obtained from the reaction of NaH with ferroce-
nylmethanol in THF at 0 ꢀC, led to the formation of 4 in 31% yield.
Similarly, 5 was obtained in 28% yield by reaction of sodium fer-
rocenylmethoxide with 9. The ring-opening of the epoxide was
confirmed by the appearance in the 1H NMR spectrum of
2.1. Synthesis of the ferrocenyl derivatives of flutamide and
bicalutamide 1e5
A ferrocenyl moiety could be incorporated in the flutamide
skeleton either by replacing its isopropyl group leading to the nitro
and cyano compounds,1 and 2, or the aromatic ring to obtain 3. The

O. Payen et al. / Journal of Organometallic Chemistry 696 (2011) 1049e1056
1051
Scheme 1. Synthesis of ferrocenyl derivatives of flutamide. (a) (i) (COCl)2, CH2Cl2, room temperature, 3 h, (ii) 4-nitro-3-fluoromethyl-aniline or 4-cyano-3-fluoromethyl-aniline,
NEt3, CH2Cl2, room temperature, 2 h, 40e48%; (b) NEt3, CH2Cl2, room temperature, 2 h, 43%.
CF3
CF3
CF3
R
R
R
R
c
b
a
O
O
O
O
N
H
F3C
N
H
O
N
H
F
OH
CH3
H3C
CH3
Fe
R= NO2
R= CN
6: R= NO2
7: R= CN
8: R= NO2
9: R= CN
4: R= NO2
5: R= CN
Scheme 2. Synthesis of the racemic derivatives of ferrocenyl SARM: (a) methacrylamide, NaH, DMF, room temperature, 3 h, 49e88%; (b) (CF3CO)2O, H2O2, CH2Cl2, reflux, 3 h,
61e87%; (c) (i) NaH, ferrocenylmethanol, THF, 0 ꢀC, 5 min, (ii) 8 or 9, reflux, 3 h, 31e28%.
diastereotopic protons adjacent to the ferrocenyl unit. Thus, in 5,
the CH2Fc protons appeared as 11.3 Hz doublets at 4.41 and
4.31 ppm.
for the preparation of (R)-2-hydroxy-3-bromo-propanoic acid (R)-
10, but starting this time from (S)-Proline (Scheme 3). The bromi-
nated derivative (S)-11 was then formed from (S)-10 by a slightly
different procedure in that THF was replaced by DMA, and no
triethylamine was added, as reported for the synthesis of (R)-
bicalutamide [29]. The final step, the reaction of (S)-11 with sodium
ferrocenylmethoxide to form (R)-5 proceeded in 37% yield. The
formation of the two enantiomers (S)-5 and (R)-5 was confirmed by
the measurement of their optical rotations which gave specific
rotations of ꢁ10ꢀ and þ11ꢀ, respectively.
Following the successful preparation of 5 as a racemate, the
synthesis of its two enantiomers was explored. (S)-5 was prepared
from (R)-proline, which was transformed into (R)-2-hydroxy-3-
bromo-propanoic acid (R)-10 in a three-step process following the
method used to prepare (R)-bicalutamide, as described by Kirkov-
sky et al. [27] (Scheme 3). Treatment of (R)-10 with thionyl chloride
in THF gave the corresponding acyl chloride; subsequent reaction
with 4-cyano-3-trifluoromethyl-aniline and triethylamine led to
(R)-11, as reported by Miller and coworkers for the formation of
SARM S-1 [28]. Finally, the reaction of (R)-11 with sodium ferro-
cenylmethoxide completed the synthesis of the ferrocenyl deriva-
tive (S)-5 in 31% yield. Analogously, the route to the pure
enantiomer (R)-5, followed the same synthetic method as was used
It is noteworthy that during the formation of the brominated
compounds (R)-10 and (S)-10, the corresponding chlorinated
molecules are also formed to the extent of 7e14%, as reported by
Kirkovsky [27]. This mixture of halogenated compounds subsists
for (R)-11 and (S)-11 but does not interfere in the formation of (S)-5
and (R)-5.
O
CF3
NC
CF3
NC
b
HO
H3C OH
Br
Br
O
a
c
O
N
H
O
O
N
H
Br
Br
H3C OH
H3C OH
Fe
Fe
(R)-10
(R)-11
(S)-5
O
CF3
CF3
NC
NC
b
HO
O
O
H3C OH
N
H
N
H
H3C OH
H3C OH
(S)-10
(S)-11
(R)-5
Scheme 3. Synthesis of the two enantiomers of ferrocenyl SARM: (a) (i) SOCl2, THF, ꢁ5 ꢀC, 2 h, and (ii) 4-amino-2-trifluoromethylbenzonitrile, NEt3, THF, reflux, 12 h, 55%; (b) (i)
K2CO3, acetone, reflux, 4 h, (ii) ferrocenylmethanol, NaH, THF, room temperature, 12 h, 31e37%; (c) (i) SOCl2, DMA, ꢁ5 ꢀC, 30 min, (ii) 4-amino-2-trifluoromethylbenzonitrile, DMA,
room temperature, 3 h, 32%.

1052
O. Payen et al. / Journal of Organometallic Chemistry 696 (2011) 1049e1056
2.3. Biochemical studies
The biological properties of the ferrocenyl derivatives 1e5
were tested and compared with those obtained with the male
steroids dihydrotestosterone (DHT), and the non-steroidal anti-
androgens bicalutamide and hydroxy-flutamide, the active
metabolite of flutamide. The relative binding affinity (RBA) values
of the compounds for the androgen receptor (AR) were assayed in
a standard competitive radio ligand assay, using full-length
human recombinant androgen receptor (AR) and [3H]-DHT as
a tracer. The RBA values obtained are reported in Table 2, and are
shown to be very low (<0.3%) even for bicalutamide and hydroxy
flutamide, the antiandrogens used for the treatment of prostate
cancer. This is the case for most of the non-steroidal anti-
androgens and could be explained by the fact that their chemical
structures have only a poor analogy with those of the natural male
steroids, testosterone and DHT. In both series, RBA values are
higher for the nitro than the cyano complexes and those of the
ferrocenyl complexes are lower than that of their organic coun-
terparts. Decreases are limited in the bicalutamide series but
significant in the flutamide one where complex 2 has completely
lost its recognition for the AR. This is also the case for complex 3
where a nonsubstituted ferrocenyl replaces a flat disubstituted
aromatic ring. This confirms the observation that CF3 and NO2
groups (or CN) play an important role in the recognition of the
ligand by its specific receptor. Indeed, it has been shown that the
cyano group of bicalutamide forms H bonds with Gln711 and
Arg752 of androgen receptor ligand binding domain while the CF3
occupies a hydrophobic pocket [2,14].
Fig. 1. X-ray crystal structure of 4; thermal ellipsoids are drawn at the 50% probability
level. Selected bond lengths (Å) and angles (deg): Fe-Cent(1) 1.645(2); Fe-Cent(2) 1.635
(2); C9eC10 1.423(3); C1eC2 1.419(3); C10eC11 1.487(3); C11eO1 1.435(2); O1eC12
1.420(2); C12eC13 1.522(3); C13eC15 1.542(3); C15eN1 1.357(3); N1eC16 1.408(2);
C10eC11eO1 106.92(15); C11eO1eC12 112.79(14); O1eC12eC13 107.64(15);
C12eC13eC15 107.48(15); C13eC15eN1 115.71(16); C15eN1eC16 124.16(16).
2.2. X-ray crystal structure of 4
The effect of 10 mM of the newly synthesized molecules was
then studied on the growth of both hormone-dependent (LNCaP)
and hormone-independent (PC-3) prostate cancer cells, and the
results are also shown in Table 2. On LNCaP cells, DHT and
hydroxy-flutamide have a proliferative effect, bicalutamide has
almost no effect, while all the ferrocenyl complexes show an
antiproliferative effect. The proliferative effect found for hydroxy
flutamide described as an antiandrogen is quite surprising, and it
was also found with nilutamide on this cell line [30e32]. This
could be attributable to the fact that the AR present in LNCaP cells
contains a point mutation in the ligand-binding domain [33].
Unfortunately, however, it is the only available human cell line
that shows both hormone dependency and continuous growth in
vitro.
Owing to the low RBA values found for the complexes, the
observed antiproliferative effect might be associated with a cyto-
toxic rather to an anti-hormonal effect. On the hormone-inde-
pendent prostate cancer cells PC-3, five ferrocenyl complexes show
an antiproliferative effect higher than 15%, while the other
compounds, including the organic molecules hydroxy-flutamide,
bicalutamide and SARM S-1, and two of three ferrocenyl derivatives
of flutamide (2 and 3) have almost no effect. IC50 values were
determined for the five most cytotoxic complexes and are reported
The structure of racemic 4 was confirmed by X-ray crystallog-
raphy, and Fig. 1 shows only the (S) enantiomer. Crystallographic
data are collected in Table 1. The structural parameters for the
ferrocenyl group are within the normal ranges, and the iron atom is
sandwiched almost perfectly centrally between the two cyclo-
pentadienyl rings. The side-chain bearing the nitro-tri-
fluoromethyl-phenyl adopts a distal position with respect to the
C5H5 ring.
Table 1
Crystal data and structure refinement for compound 4.
Empirical formula
Formula weight
Temperature
C22H21N2O5F3Fe
506.26
100(2) K
Wavelength
Crystal system
Space group
0.71073 Å
Triclinic
Pꢁ1 (#2)
Unit cell dimensions
a ¼ 9.5084(9) Å,
a
¼ 67.137(2)ꢀ.
b ¼ 11.3147(11) Å,
c ¼ 11.3550(11) Å,
1072.53(18) Å3
2
b
g
¼ 76.914(2)ꢀ.
¼ 74.081(2)ꢀ.
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
Reflections collected
Independent reflections
Completeness to theta ¼ 26.00ꢀ
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
Largest diff. peak and hole
1.568 Mg/m3
0.767 mmꢁ1
520
in Table 2. They range from 30.5
mide derivative 1 to an average of 13
m
M for the nitro ferrocenyl fluta-
M for the three cyano fer-
0.40 ꢂ 0.30 ꢂ 0.28 mm3
m
1.96e26.00ꢀ
rocenyl bicalutamide derivatives 5 (racemic, (S) and (R) forms). The
ferrocenyl complex 4 is significantly more cytotoxic than its cor-
responding organic molecule SARM S-1, but it seems that the
stereochemistry of the asymmetric carbon plays no role in the
cytotoxic effect of the complexes as 5, (S)-5 and (R)-5 exhibit
similar IC50 values. This result is different from what is observed in
vivo for the antiandrogenic effect of the two bicalutamide enan-
tiomers. In that case, the enantiomer with the (R) absolute
configuration has been described as being 60-fold more potent
than the (S) enantiomer [29], even if prescribed as the racemic
mixture [34].
ꢁ11 ꢃ h ꢃ11, ꢁ13 ꢃ k ꢃ13, ꢁ13 ꢃ l ꢃ 13
8771
4116 [R(int) ¼ 0.0168]
97.7%
Semi-empirical from equivalents
0.8139 and 0.6331
Full-matrix least-squares on F2
4116/0/307
1.068
R1 ¼ 0.0352, wR2 ¼ 0.0902
R1 ¼ 0.0384, wR2 ¼ 0.0931
0.827 and ꢁ0.321 eÅꢁ3

O. Payen et al. / Journal of Organometallic Chemistry 696 (2011) 1049e1056
1053
Table 2
Relative Binding Affinity (RBA) for the androgen receptor (AR), effect on the growth of LNCaP (hormone-dependent prostate cancer cells) and of PC-3 (hormone-independent
prostate cancer cells) and log Po/w of the compounds.
Compound
RBA (%) on AR
Cell viability on LNCaP (%)a
Cell viability on PC-3a (%)
IC50 on PC-3 (
m
M)b
log Po/w
DHT
100
148
99
e
3.2
OH-flutamide
1
2
3
Bicalutamide
SARM S-1
4
5
0.20 ꢄ 0.01
0.007 ꢄ 0.004
0.00
181 ꢄ 47
78 ꢄ 7
83 ꢄ 12
89 ꢄ 2
93 ꢄ 4
nd
73 ꢄ 11
73 ꢄ 18
55 ꢄ 1.5
nd
87 ꢄ 3
71 ꢄ 2
86.2 ꢄ 0.6
92.4 ꢄ 0.6
85 ꢄ 6
94 ꢄ 3
65 ꢄ 3
76 ꢄ 2
83 ꢄ 1
81 ꢄ 4
e
3.75
4.42
4.10
2.64
3.40
e
30 ꢄ 1
e
0.00
e
0.29 ꢄ 0.02
e
nd
e
0.084 ꢄ 0.02
0.040 ꢄ 0.006
nd
17 ꢄ 2
13.5 ꢄ 0.3
12.8 ꢄ 0.5
14 ꢄ 2
5.01
4.63
4.82
4.69
(S)-5
(R)-5
nd
a
Incubation in the presence of 10 mM of the compounds [except for DHT (10 nM), OH-flutamide (1 mM) on LNCaP], after 5 days of culture. Non-treated cells are used as the
control (100%). An effect higher than 100% indicates a proliferative effect. Mean of two separate experiments (6 measurements for each one) ꢄ range.
b
IC50 values are determined when cell viability in the presence of 10 mM of the complex is lower than 85% (with the control set at 100%).
Finally, the lipophilicity values found for the new molecules
are higher than the log Po/w values of bicalutamide or hydroxy
flutamide (except for 3) (Table 2). This was to be expected as they
all possess ferrocenyl substituents which are known to be
lipophilic.
4.2.1. N-(4-cyano-3-trifluoromethyl-phenyl)-ferrocenyl-
carboxamide (2)
Ferrocenecarboxylic acid (0.460 g, 2.00 mmol) was partially
dissolved in 20 mL of dichloromethane. Oxalyl chloride (0.35 mL,
4.00 mmol) was added dropwise followed by one drop of DMF. After
stirring for 3 h at room temperature, dichloromethane and excess of
oxalyl chloride were removed under reduced pressure. Dichloro-
methane (20 mL) was added to dissolve the acid chloride formed. In
another Schlenk tube, 4-cyano-3-trifluoromethyl-aniline (0.372,
2.00 mmol) was dissolved in 20 mL of dichloromethane and trie-
thylamine (0.28 mL, 2.00 mL) was added. The solution was cooled to
0 ꢀC, then the solution of acid chloride was added dropwise. The
mixture was stirred at room temperature for 2 h before being
poured in water. The reaction mixture was extracted with
dichloromethane. The organic layer was washed with water, dried
over MgSO4, filtered, and solvents evaporated. The crude product
obtained was purified by chromatography on silica gel column
(eluent:ethyl acetate/petroleum ether 1:3). Compound 2 was iso-
lated as an orange solid which was crystallized in ethyl acetate:-
pentane (0.383 g, 48% yield): mp: 205 ꢀC; 1H NMR (300 MHz, [d6]
3. Conclusions
The results found here confirm that the ferrocenyl derivatives of
flutamide and of bicalutamide are only weakly recognized by the
androgen receptor, but this is not a complete surprise considering
that there is not a close analogy between their structures and those
of the natural androgens, testosterone and dihydrotestosterone.
The ferrocenyl complexes of bicalutamide are noticeably more
cytotoxic towards the hormone-independent cell line PC-3 than are
those of ferrocenyl flutamide. With an IC50 value of 17 mM, the
ferrocenyl complex 4 is considerably more cytotoxic than the cor-
responding organic compound SARM S-1. In contrast, however, the
absolute stereochemistry (R or S) of these complexes does not
appear to play any significant role. Finally, the IC50 values found for
the ferrocenyl bicalutamide derivatives 4 and 5, the most active
compounds of the series, are significantly higher than the values
acetone):
d
¼ 9.45 (s,1H, NH), 8.43 (d, J ¼ 2.0 Hz,1H, Ar H2,), 8.27 (dd,
J ¼ 8.4 , J ¼ 2.0 Hz, 1H, Ar H6,), 7.99 (d, J ¼ 8.4 Hz, 1H, Ar H5), 5.01 (t,
J ¼ 2.0 Hz, 2H, C5H4), 4.51 (t, J ¼ 2.0 Hz, 2H, C5H4), 4.25 (s, 5H, C5H5);
(around 5 mM) found previously for the ferrocenyl complexes of
ethynyl testosterone or nilutamide.
4. Experimental section
4.1. General comments
13C NMR (75 MHz, [d6]acetone):
d
¼ 69.7 (C5H4), 70.7 (C5H5), 72.2
(C5H4), 76.1 (C5H4, Cip), 104.0 (Ar C4), 116.5 (CN), 118.0 (Ar C2), 123.0
(Ar C6), 124.0 (q, CF3, J ¼ 262.5 Hz), 134.0 (Ar C3), 137.0 (Ar C5), 144.9
(Ar C1), 171.2 (CO); IR (CH2Cl2): n
x 2230 (CN), 1687 (CO) cmꢁ1; MS
(EI) m/z: 398 [Mþ.]; Anal. Calcd for C19H13F3FeN2O: C 57.31, H 3.29, N
7.04, found: C 57.16, H 3.43, N 7.22.
All reactions were carried under an atmosphere of argon.
Diethyl ether, ethyl acetate and toluene were distilled from sodium/
benzophenone. All other chemical reagents and solvents were used
as purchased without further purification. Column flash chroma-
4.2.2. N-(ferrocenyl)-isobutyramide (3)
Aminoferrocene (0.310 g, 1.54 mmol) was dissolved in 20 mL of
dichloromethane. Triethylamine (0.22 mL, 1.54 mmol) was added
into the solution. The mixture was cooled to 0 ꢀC, then isobutyryl
chloride (0.16 mL,1.54 mmol) was added dropwise. After stirring for
2 h at room temperature, the mixture was poured in water and
extracted with dichloromethane. The organic layer was thenwashed
with water, dried over MgSO4, filtered, and evaporated. The crude
product obtained was purified by chromatography on silica gel
column (eluent:dichloromethane/petroleum ether 3:1). Compound
3 was isolated as an orange solid which was crystallized in dichlor-
omethane:pentane (0.179 g, 43% yield): mp: 172e174 ꢀC; 1H NMR
tography was performed on silica gel Merck 60 (40e63 mm).
Melting points were measured with a Kofler device. Infrared
spectra were recorded on an IR-FT BOMEM Michelson-100 spec-
trometer. 1H and 13C NMR spectra were recorded on a 300 MHz
Bruker spectrometer. Elemental analyses were performed by the
Service de Microanalyse I.C.S.N., Gif sur Yvette, France. Analytical
HPLC was performed on a C18 Kromasil column 10
D ¼ 4.6 mm, eluent: acetonitrile:water 80:20, flow rate ¼ 1 mL /
min,
¼ 254 nm.
mm, L ¼ 25 cm,
l
(300 MHz, CDCl3):
C5H5), 4.03 (s, 2H, C5H4), 2.39 (septuplet, J ¼ 6.9 Hz, 1H, CH), 1.23 (s,
3H, CH3),1.20 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3):
¼ 19.8 (CH3),
36.4 (CH), 61.4 (C5H4), 64.8 (C5H4), 69.6 (C5H5), 95.9 (C5H4, Cip); IR
(CH2Cl2):
x 1733 cmꢁ1 (CO); MS (EI) m/z: 271 [Mþ.]; Anal. Calcd for
C14H17FeNO: C 62.02, H 6.32, N 5.17, found: C 61.96, H 6.31, N 5.29.
d
¼ 6.43 (s,1H, NH), 4.65 (s, 2H, C5H4), 4.16 (s, 5H,
4.2. Synthesis
d
The synthesis of SARM S-1 was carried out following the liter-
ature procedure [14] as well as (R)-10 and (S)-10 [27,29], 1 [25], 6
and 7 [8,26] and hydroxy-flutamide [35].
n

1054
O. Payen et al. / Journal of Organometallic Chemistry 696 (2011) 1049e1056
4.2.3. N-(4-nitro-3-trifluoromethyl-phenyl)-3-ferrocenylmethoxy-
2-hydroxy-2-methyl- propanamide (4)
dissolved in distilled THF (2 mL) was added. The mixture was stirred at
reflux for 2 h before it was left stirring at room temperature for 12 h.
The mixture was then quenched by addition of water (30 mL) and was
then extracted with ethyl acetate (4 ꢂ 30 mL). After drying over
magnesium sulfate, the organic phase was concentrated under
vacuum to give a brown oil. It was then purified by column chroma-
tography on silica gel (eluent:petroleum ether/ethyl acetate 3:2) to
A solution of ferrocenylmethanol (0.610 g, 2.82 mmol) in THF
(10 mL) was added to a suspension of NaH in THF (0.226 g,
5.64 mmol) at 0 ꢀC. The mixture was stirred for 5 min. A solution of
8 (0.500 g, 2.17 mmol) in THF (15 mL) was added slowly. The
mixture was stirred at reflux for 3 h, and then cooled to room
temperature. THF was then distilled off followed by addition of
ethyl acetate. The organic layer was washed with water, dried over
MgSO4, filtered and evaporated. The crude product was purified by
chromatography on silica gel column (eluent:ethyl acetate/petro-
leum ether 1:3, then 1:2). Compound 4 was obtained as a red oil
(0.273 g, 31 % yield). The oil was crystallized from dichloro-
methane/hexane to give an orange solid: mp: 146e148 ꢀC; 1H NMR
give the pure product as a light brown solid (0.128 g, 31% yield): mp:
20
degradation above 50 ꢀC; [
a]
D
¼ ꢁ10 (c ¼ 1.9 in acetone); 1H NMR
(300 MHz, CDCl3):
d
¼ 9.11 (sl, 1H, NH); 7.98 (d, J ¼ 1.3 Hz, 1H, Ar H2),
7.83 (d, J ¼ 8.7, J ¼ 1.7, Hz, 1H, Ar H6), 7.69 (d, J ¼ 8.5 Hz,1H, Ar H5), 4.40
(d, J ¼ 11.1 Hz,1H, CH2Fc), 4.31 (1H, d, J ¼ 11.1 Hz, CH2Fc), 4.20e4.14 (m,
4H, C5H4), 4.11 (s, 5H, C5H5), 4.01 (d, J ¼ 9.0 Hz, 1H, CH2O), 3.60 (s, 1H,
OH), 3.43 (d, J ¼ 9.0 Hz, 1H, CH2O), 1.38 (s, 3H, CH3); 13C NMR (75 MHz,
(300 MHz, CDCl3):
d
¼ 9.10 (s, 1H, NH), 7.95e7.99 (m, 3H, Ar H2, H5,
CDCl3):
d
¼ 23.0 (CH3), 68.6 (C5H5), 69.0 (C5H4), 69.6 (C5H4), 70.1 (CH2),
H6), 4.24e4.34 (m, 6H, C5H4 þ CH2Fc), 4.21 (s, 5H, C5H5), 3.84 (d,
74.1 (CH2), 75.4 (C(OH)), 82.0 (C5H4, Cip), 104.1 (Ar C4), 115.5 (CN), 117.2
J ¼ 9.0 Hz, 1H, CH2O), 3.46 (s, 1H, OH), 3.41 (d, J ¼ 9.0 Hz, 1H, CH2O),
(Ar C2),120.3 (CF3),121.7 (Ar C6),133.8 (q, Ar C3,),135.7 (Ar C5),141.6 (Ar
1.39 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3):
d
¼ 23.2 (CH3), 69.4
C1), 173.9 (CO); FT-IR: n x 3497 (OH), 3324 (NH), 2231 (CN), 1700 (CO)
(C5H5), 69.8 (C5H4), 70.2 (CH2), 70.3 (C5H4), 73.9 (CH2), 75.4 (C(OH)),
82.9 (C5H4, Cip), 118.3 (Ar C2), 121.8 (q, CF3, J ¼ 266.5 Hz), 122.1 (Ar
C6), 125.2 (q, Ar C3), 127.2 (Ar C5), 141.8 (Ar C1) 143.2 (Ar C4), 174.0
cmꢁ1; MS (ESI) m/z: 486 [Mþ.]; Anal. Calcd for C23H213F3FeN2O3: C
56.81, H 4.35, N 5.76, found: C 56.80, H 4.54, N 5.66.
(CO); IR (KBr):
n
x 3523 (OH), 3306 (NH), 1696 (CO) cmꢁ1; MS (EI)
4.2.6. (2R)-N-(4-cyano-3-trifluoromethylphenyl)-3-
ferrocenylmethoxy-2-hydroxy-2-methylpropionamide ((R)-5)
m/z: 506 [Mþ.]. Anal. Calcd for C22H21F3FeN2O5: C 52.19, H 4.18, N
5.53, found: C 52.23, H 4.24, N 5.49.
Under argon, (2S)-3-bromo-N-(4-cyano-3-trifluoromethylphenyl)-
2-hydroxy-2-methylpropionamide (S)-11 (0.296 g, 0.84 mmol) and
potassium carbonate (0.261 g, 1.89 mmol) were dissolved in acetone
(3 mL) and the reaction mixture was refluxed for 4 h. After dilution in
40 mL of water, the mixture was extracted with ethyl acetate
(3 ꢂ 30 mL), the organic phase was dried over sodium sulfate and
solvents evaporated under vacuum. A yellow-brownish oil corre-
sponding to the epoxide was obtained and used without further
purification. In the meantime, a solution of ferrocenylmethanol(0.28 g,
1.30 mmol) in distilled THF (4 mL) was added to a suspension of
sodium hydride (0.063 g, 2.63 mmol) in THF (2 mL) under argon. The
reaction mixture was stirred at 60 ꢀC for 1 h before the epoxide dis-
solved in distilled THF (2 mL) was added. The mixture was stirred at
reflux for 2 h before it was left stirring at room temperature for 12 h.
The mixture was then quenched by addition of water (30 mL) and was
then extracted with ethyl acetate (4 ꢂ 30 mL). After drying over
magnesium sulfate, the organic phase was concentrated under
vacuum to give a brown oil. It was then purified by column chroma-
tography on silica gel (eluent:petroleum ether/ethyl acetate 3:2) to
4.2.4. N-(4-cyano-3-trifluoromethyl-phenyl)-3-ferrocenylmethoxy-
2-hydroxy-2-methyl-propanamide (5)
A solution of ferrocenylmethanol (0.520 g, 2.4 mmol) in THF
(10 mL) was added toa suspension of NaH (0.192 g; 4.8 mmol) inTHF
at 0 ꢀC. The mixture was stirred for 5 min. A solution of 9 (0.500 g;
1.85 mmol) in THF (12 mL) was added slowly. The mixture was
stirred at reflux for 2.5 h, then cooled to room temperature. THF was
then distilled off before ethyl acetate was added. The organic layer
was washed with water, dried over MgSO4, filtered and evaporated.
The crude solid obtained was purified by chromatography on silica
gel column (eluent:ethyl acetate/petroleum ether 1:2). Compound 5
was obtained as a yellow solid (0.250 g, 28.0% yield). The solid was
recrystallized from dichloromethane/hexane: mp: 102e104 ꢀC; 1H
NMR (300 MHz, CDCl3):
d
¼ 9.09 (s,1H, NH), 8.01 (d, J ¼ 1.6 Hz,1H, Ar
H2), 7.88 (dd, J ¼ 8.5, J ¼ 1.6 Hz, 1H, Ar H6), 7.77 (d, J ¼ 8.5 Hz, 1H, Ar
H5), 4.41 (d, J ¼ 11.3 Hz, 1H, CH2Fc), 4.31 (d, J ¼ 11.3 Hz, 1H, CH2Fc),
4.21 (s, 2H, C5H4), 4.19 (s, 2H, C5H4), 4.14 (s, 5H, C5H5), 3.87 (d,
J ¼ 9.0 Hz, 1H, CH2O), 3.49 (s, 1H, OH), 3.44 (d, J ¼ 9.0 Hz, 1H, CH2O),
give the pure product as a light brown solid (0.15 g, 37% yield): mp:
20
1.40 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3):
d
¼ 23.3 (CH3), 68.8
degradation above 50 ꢀC; [
a]
D
¼ þ11ꢀ (c ¼ 1.9 in acetone); 1H NMR
(C5H5), 69.2 (C5H4), 69.8 (C5H4), 70.3 (CH2), 74.0 (CH2), 75.6 (C(OH)),
82.3 (C5H4, Cip), 104.6 (Ar C4), 115.8 (CN), 117.4 (Ar C2), 122.0 (Ar C6),
134.0 (q, Ar C3), 136.0 (Ar C5), 141.8 (Ar C1), 174.1 (CO); IR (KBr):
(300 MHz, CDCl3):
d
¼ 9.06 (broad singlet, 1H, NH), 7.99 (s, 1H, Ar H2),
7.85 (d, J ¼ 8.1 Hz, 1H, Ar H6), 7.73 (d, J ¼ 8.0 Hz, 1H, Ar H5), 4.20e4.09
(m, 6H, C5H4, CH2Fc), 4.06 (s, 5H, C5H5), 3.84 (d, J ¼ 9.0 Hz, 1H, CH2O),
3.45 (1H, s, OH), 3.41 (d, J ¼ 9.0 Hz, 1H, CH2O), 1.37 (s, 3H, CH3);
n
x 3410 (OH), 3311 (NH), 2232 (CN), 1691 (CO) cmꢁ1; MS (EI) m/z:
486 [Mþ.]; Anal. Calcd for C23H21F3FeN2O3: C 56.81, H 4.35, N 5.76,
found: C 56.41, H 4.72, N 5.31.
13C NMR (75 MHz, CDCl3):
d
¼ 23.0 (CH3), 69.5 (C5H5), 69.9 (C5H4), 70.0
(C5H4), 70.4 (C9), 73.9 (C8), 75.3 (C(OH)), 82.9 (C5H4, Cip), 104.3 (Ar C4),
115.5 (CN), 117.2 (Ar C2), 120.3 (Ar CF3), 121.7 (Ar C6), 133.8 (q, Ar C3,),
4.2.5. (2S)-N-(4-cyano-3-trifluoromethylphenyl)-3-
135.8 (Ar C5), 141.6 (Ar C1), 173.8 (CO); FT-IR: n x 3473 (OH), 3319
ferrocenylmethoxy-2-hydroxy-2-methylpropionamide ((S)-5)
Under argon, (2R)-3-bromo-N-(4-cyano-3-trifluoromethylphenyl)-
2-hydroxy-2-ethylpropionamide (R)-11 (0.296 g, 0.843 mmol) and
potassium carbonate (0.261 g, 1.89 mmol) were dissolved in acetone
(3 mL) and the reaction mixture was refluxed for 4 h. After dilution in
40 mL of water, the mixture was extracted with ethyl acetate
(3 ꢂ 30 mL), the organic phase was dried over sodium sulfate and
solvents evaporated under vacuum. A yellow-brownish oil corre-
sponding to the epoxide was obtained and used without further
purification. In the meantime, a solution of ferrocenylmethanol (0.28 g,
1.30 mmol) in distilled THF (4 mL) was added to a suspension of
sodium hydride (0.063 g, 2.63 mmol) in THF (2 mL) under argon. The
reaction mixture was stirred at 60 ꢀC for 1 h before the epoxide
(NH), 2231 (CN), 1700 (CO) cmꢁ1; MS (ESI) m/z: 486 [Mþ.]; Anal. Calcd
for C23H213F3FeN2O3: C 56.81, H 4.35, N 5.76, found: C 56.88, H
4.72, N 5.37.
4.2.7. N-(4-nitro-3-trifluoromethyl-phenyl)-methyl-oxiranyl-
carboxamide (8)
To a stirred solution of 6 (2.00 g, 7.30 mmol) in dichloro-
methane (20 mL) was added an aqueous solution of 30% hydrogen
peroxide H2O2 (1.4 mL; 43.8 mmol). Trifluoroacetic anhydride
(5.2 mL; 36.5 mmol) was added slowly and the reaction mixture
was stirred to reflux for 3 h. The mixture was poured in
dichloromethane. The organic layer was then washed with water,
saturated aqueous sodium bisulfite NaHSO3, saturated sodium

O. Payen et al. / Journal of Organometallic Chemistry 696 (2011) 1049e1056
1055
bicarbonate, brine, dried over MgSO4, filtered, and evaporated. The
crude product obtained was purified by chromatography on silica
gel column (eluent:dichloromethane/petroleum ether 3:1).
Compound 8 was obtained as a pale yellow solid (1.30 g, 61%
yield): mp: 120e122 ꢀC (literature [8]: 120e121 ꢀC); 1H NMR
0.983 mmol) was added rapidly to the solution containing the acid
chloride formed in situ. The reaction mixture was left stirring for 3 h
at room temperature. DMA was then removed under vacuum and
the dark residue obtained was diluted with saturated aqueous
NaHCO3 (5 mL) and the solution was extracted with ether
(3 ꢂ10 mL). The organic phase was dried over magnesium sulfate
and concentrated under vacuum. The dark oil obtained was purified
by column chromatography on silica gel (eluent:petroleum ether/
ethyl acetate, 3:2) to give a brown solid (0.112 g, 32% yield): mp:
106 ꢀC (literature [29]: 106e107 ꢀC); The NMR spectrum shows the
presence of 86% brominated compound and 14% of chlorinated one:
(300 MHz, CDCl3):
H6), 3.01 (s, 2H, CH2O), 1.68 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3):
¼ 16.9 (CH3), 54.4 (CH2), 56.9 (C(CH3)), 118.5 (Ar C2), 121.9 (q,
J ¼ 273 Hz, CF3),122.3 (Ar C6), 125.6 (q, J ¼ 34.2 Hz, Ar C3),127.3 (Ar
C5), 141.5 (Ar C1),143.5 (Ar C4),169.5 (CO); IR (KBr): x 3339 (NH),
1708 (CO) cmꢁ1
d
¼ 8.48 (s,1H, NH), 8.01e7.96 (m, 3H, Ar H2, H5,
d
n
.
1H NMR (300 MHz, CDCl3):
d
¼ 9.08 (broad singlet, 1H, NH), 8.11 (s,
4.2.8. N-(4-cyano-3-trifluoromethyl-phenyl)-methyl-oxiranyl-
carboxamide [26] (9)
1H, Ar H2), 7.96 (d, J ¼ 8.7 Hz, 1H, Ar H6), 7.80 (d, J ¼ 8.1 Hz, 1H, Ar
H5), 4.10 (d, J ¼ 10.8 Hz, 1H, CH2Cl), 4.05 (d, J ¼ 10.8 Hz, 1H, CH2Br),
3.69 (d, J ¼ 10.8 Hz, 1H, CH2Cl), 3.59 (d, J ¼ 10.8 Hz, 1H, CH2Br), 3.09
(s, 1H, OH(Cl)), 3.03 (1H, s, OH(Br)), 1.64 (s, 3H, CH3(Br)), 1.59 (s, 3H,
CH3(Cl)).
To a stirred solution of 7 (0.500 g,1.97 mmol) in dichloromethane
(10 mL) was added an aqueous solution of 30% hydrogen peroxide
H2O2 (0.40 mL; 6.00 mmol). Trifluoroacetic anhydride (1.4 mL;
9.85 mmol) was added slowly and the reaction mixture was stirred
at room temperature for 2 h 30 s. The mixture was poured in
dichloromethane. The organic layer was then washed with water,
saturated aqueous sodium bisulfite NaHSO3, saturated sodium
bicarbonate, brine, dried over MgSO4, filtered, and concentrated
under vacuum. Compound 9 was isolated as a white solid (0.465 g;
87% yield): mp: 147e148 ꢀC (literature [26]: 149e150 ꢀC); 1H NMR
4.3. X-ray crystal structure determination for 4
Suitable crystals of 4 were obtained by recrystallization from
dichloromethane:hexane. Crystal data were collected using
a Bruker SMART APEX CCD area detector diffractometer, and are
listed in Table 1. A full sphere of reciprocal space was scanned by
phi-omega scans. Pseudoempirical absorption correction based
on redundant reflections was performed by the program SADABS
[36]. The structures were solved by direct methods using
SHELXS-973 [37] and refined by full-matrix least-squares on F2
for all data using SHELXL-97 [38]. Hydrogen atoms attached to
oxygen or nitrogen were located in the difference fourier map
and allowed to refine freely with isotropic thermal displacement
parameters. All other hydrogen atoms were added at calculated
positions and refined using a riding model. Their isotropic
thermal displacement parameters were fixed to 1.2 times (1.5
times for methyl groups) the equivalent one of the parent atom.
Anisotropic thermal displacement parameters were used for all
non-hydrogen atoms.
(300 MHz, CDCl3):
d
¼ 8.44 (s, 1H, NH), 8.02 (d, J ¼ 2.1 Hz, 1H, Ar H2),
7.90 (dd, J ¼ 8.7, J ¼ 2.1 Hz, 1H, Ar H6), 7.78 (d, J ¼ 8.7 Hz, 1H, Ar H5),
2.99 (s, 2H, CH2O), 1.67 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3):
d
¼ 16.8 (CH3), 54.2 (CH2), 56.8 (C(CH3)), 104.8 (Ar C4), 115.5 (CN),
117.4 (Ar C2), 122.0 (Ar C6), 122.1 (q, J ¼ 273.9 Hz, CF3), 134.0 (q,
J ¼ 33.0 Hz, Ar C3), 136.0 (Ar C5), 141.4 (Ar C1), 169.4 (CO); IR (KBr):
n
x 3334 (NH), 2230 (CN), 1706 (CO) cmꢁ1
.
4.2.9. (2R)-N-(4-cyano-3-trifluoromethylphenyl)-3-bromo-2-
hydroxy-2-methylpropionamide [28] ((R)-11)
Under argon, a solution of (2R)-3-bromo-hydroxy-2-methyl-
propanoic acid (0.502 g, 2.74 mmol) in distilled THF (5 mL) was
cooled to ꢁ5 ꢀC before thionyl chloride (0.21 mL, 2.88 mmol) was
added dropwise. The reaction mixture was left stirring at ꢁ5 ꢀC for
2 h. A solution of 4-amino-2-trifluoromethylbenzonitrile (0.470 g,
2.28 mmol) in distilled THF (5 mL) was then added at ꢁ5 ꢀC fol-
lowed by distilled triethylamine (0.87 mL, 6.24 mmol). The reac-
tion was warmed to room temperature and was then refluxed for
12 h. The mixture was then diluted in ethyl acetate (50 mL),
washed with diluted HCl 5% (3 ꢂ 30 mL), saturated aq. NaHCO3
(3 ꢂ 30 mL) and water (3 ꢂ 30 mL). The organic phase was dried
over magnesium sulfate and the solvents evaporated. The dark oil
obtained was purified by column chromatography on silica gel
(eluent:petroleum ether/ethyl acetate, 3:2) to give a brown solid
(0.441 g, 55% yield): mp ¼ 135 ꢀC (literature [27]: 132e134 ꢀC).
The NMR spectrum shows the presence of 93% brominated
compound and 7% of chlorinated one: 1H NMR (300 MHz, CDCl3):
4.4. Biochemical experiments
4.4.1. Materials
DHT, and protamine sulfate were obtained from SigmaeAldrich
(France). Stock solutions (1 ꢂ10ꢁ3 M) of the compounds to be
tested were prepared in DMSO and were kept at ꢁ20 ꢀC. Under
these conditions, they are stable for at least two weeks. Serial
dilutions in DMSO were prepared just prior to use. Dulbecco’s
modified eagle medium (DMEM) for PC-3 and RPMI 1640 for LNCaP
were purchased from Invitrogen. Fetal bovine serum, glutamine
and kanamycine were obtained from Invitrogen. Prostate cancer
cells LNCaP and PC-3 cells were from American Type Culture
Collection (ATCC e LGC Promochem). [1,2-3H]-DHT was purchased
from NEN Life Science Product.
d
¼ 9.02 (sl, 1H, NH). 8.07 (d, J ¼ 1.8 Hz, 1H, Ar H2), 7.97 (dd, J ¼ 8.7,
J ¼ 2.1 Hz, 1H, Ar H6), 7.77 (d, J ¼ 8.4 Hz, 1H, Ar H5), 4.12 (d,
J ¼ 10.8 Hz, 1H, CH2Cl), 4.02 (d, J ¼ 10.8 Hz, 1H, CH2Br), 3.67 (d,
J ¼ 10.8 Hz, 1H, CH2Cl), 3.57 (d, J ¼ 10.8 Hz, 1H, CH2Br), 3.07 (s, 1H,
OH(Cl)), 3.02 (s, 1H, OH(Br)), 1.62 (s, 3H, CH3(Br)), 1.57 (s, 3H,
CH3(Cl)).
4.4.2. Determination of the RBA of the compounds for the androgen
receptor (AR)
RBA values were measured on PanVera AR (750 pmol)
purchased from Invitrogen. This AR is a recombinant rat protein
expressed in Escherichia coli. The amino sequence of the ligand-
binding domain of this AR is identical to that of the human AR LBD.
4.2.10. (2S)-N-(4-cyano-3-trifluoromethylphenyl)- 3-bromo-2-
hydroxy-2-methylpropionamide [29] ((S)-11)
ARs were aliquoted in 15 mL fractions and kept in liquid nitrogen
Under argon, a solution of (2S)-3-bromo-hydroxy-2-methyl-
propanoic acid (0.2 g, 1.09 mmol) in anhydrous DMA (3 mL) was
cooled to 5 ꢀC before thionyl chloride was added dropwise. The
reaction mixture was stirred for 30 min at this temperature and
until used. For each experiment, 10 mL of buffer containing 10%
glycerol, 50 mM Tris pH 7.5, 0.8 M NaCl, 2 mM DTT and 0.1% BSA
was added to one aliquot. Fractions of 200 mL of the AR solution
were incubated in polypropylene tubes for 3 h and 30 s at 4 ꢀC with
a
solution of 4-amino-2-trifluoromethylbenzonitrile (0.183 g,
[1,2-3H]-DHT (2 ꢂ 10ꢁ9 M, specific activity 1.6 TBq/mmol) in the

1056
O. Payen et al. / Journal of Organometallic Chemistry 696 (2011) 1049e1056
presence of nine concentrations of the compounds to be tested
(between 1 ꢂ10ꢁ5 M and 6 ꢂ 10ꢁ7 M) or of non-radioactive DHT
(between 8 ꢂ 10ꢁ8 M and 7.5 ꢂ10ꢁ10 M). At the end of the incu-
bation period, the fractions of [3H]-DHT bound to the androgen
Appendix A. Supplementary data
Crystallographic data for the structures reported in this paper
have been deposited with the Cambridge Crystallographic Data
Centre as supplementary publication nos. CCDC-785377. Copies of
this information may be obtained free of charge from The Director,
CCDC, 12 Union Road, Cambridge CB2 1EZ (Fax: þ44 1223 336033;
e-mail: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
receptor (Y values) were precipitated by addition of a 200 mL of
a cold solution of protamine sulfate (1 mg/mL in water). After
a 10 min period of incubation at 4 ꢀC, the precipitates were recov-
ered by filtration on 25 mm diameter glass microfibre filters GF/C
filters using a Millipore 12 well filtration ramp. The filters were
rinsed twice with cold phosphate buffer and then transferred into
20 mL plastic vials. After addition of 5 mL of scintillation liquid (BCS
Amersham) the radioactivity of each fraction was counted in
a Packard tricarb 2100TR liquid scintillation analyzer. The concen-
tration of unlabeled steroid required to displace 50% of the bound
[3H]-DHT was calculated for DHT and for each complex by plotting
the logit values of Y (logit Y ¼ ln(Y/100 ꢁ Y)) versus the mass of the
competing complex. The RBA was calculated as follows: RBA of
a compound ¼ concentration of DHT required to displace 50% of
[3H]-DHT ꢂ 100/concentration of the compound required to
displace 50% of [3H]-DHT. The RBA value of DHT is by definition
equal to 100%.
References
[1] F. Labrie, L. Cusan, J. Gomez, V. Luu-The, B. Candas, A. Belanger, C. Labrie,
J. Steroid Biochem. Mol. Biol. 92 (2004) 327.
[2] W. Gao, C.E. Bohl, J.T. Dalton, Chem. Rev. 105 (2005) 3352.
[3] A.U. Decensi, F. Boccardo, D. Guarneri, N. Positano, M.C. Paoletti, M. Costantini,
G. Martorana, L. Giuliani, J. Urol. 146 (1991) 377.
[4] J.P. Raynaud, C. Bonne, M. Moguilewsky, F.A. Lefebvre, A. Bélanger, F. Labrie,
Prostate 5 (1984) 299.
[5] R.A. Airhart, T.F. Barnett, J.W. Sullivan, R.L. Levine, J.U. Schlegel, South J. Med.
Chem. 71 (1978) 798.
[6] R. Neri, K. Florance, P. Koziol, S. Van Cleave, Endocrinology 91 (1972) 427.
[7] R. Neri, E. Peets, A. Watnick, Biochem. Soc. Trans. 7 (1979) 565.
[8] H. Tucker, J.W. Crook, G.J. Chesterson, J. Med. Chem. 31 (1988) 954.
[9] T. Hara, J. Miyazaki, H. Araki, N. Kanzaki, M. Kusaka, M. Miyamoto, Cancer Res.
63 (2003) 149.
[10] W. Gao, J.T. Dalton, Drug Discovery Today 12 (2007) 241.
[11] M.L. Mohler, C.E. Bohl, A. Jones, C.C. Coss, R. Naarayanan, Y. He, D.J. Hwang,
J.T. Dalton, D.D. Miller, J. Med. Chem. 52 (2009) 3598.
[12] A. Negro-Vilar, J. Clin. Endocrinol. Metabol. 84 (1999) 3459.
[13] W.Q. Gao, J.D. Kearbey, V.A. Nair, K.W. Chung, A.F. Parlow, D.D. Miller,
J.T. Dalton, Endocrinology 145 (2004) 5420.
[14] C.A. Marhefka, W. Gao, K. Chung, J. Kim, Y. He, D. Yin, C. Bohl, J.T. Dalton,
D.D. Miller, J. Med. Chem. 47 (2004) 993.
[15] D.J. Hwang, J. Yang, H.P. Xu, I.M. Rakov, M.L. Mohler, J.T. Dalton, D.D. Miller,
Bio. Med. Chem. 14 (2006) 6525.
[16] J.J. Li, J.C. Sutton, A. Nirschl, Y. Zou, H.X. Wang, C.Q. Sun, Z.L. Pi, R. Johnson,
S.R. Krystek, R. Seethala, R. Golla, P.G. Sleph, B.C. Beehler, G.J. Grover, A. Fura,
V.P. Vyas, C.Y. Li, J.Z. Gougoutas, M.A. Galella, R. Zahler, J. Ostrowski,
L.G. Hamann, J. Med. Chem. 50 (2007) 3015.
[17] R. Narayanan, M.L. Mohler, C.E. Bohl, D.D. Miller, J.T. Dalton, Nucl. Recept.
Signal 6 (2008) 1.
[18] G. Jaouen, S. Top, A. Vessières, G. Leclercq, J. Quivy, L. Jin, A. Croisy, C.R. Acad.
Sci. Paris Série II C (2000) 89.
[19] S. Top, A. Vessières, G. Leclercq, J. Quivy, J. Tang, J. Vaissermann, M. Huché,
G. Jaouen, Chem. Eur. J. 9 (2003) 5223.
4.4.3. Measurement of octanol/water partition coefficient (log Po/w
of the compounds
)
The log Po/w values of the compounds were determined by
reverse phase HPLC on a C-8 column (Kromasil C8 from AIT)
according to the method previously used [21]. Measurement of the
chromatographic capacity factors (k0) for each molecule was done
at various concentrations in the range 95e80% methanol (con-
taining 0.25% octanol) and an aqueous phase consisting of 0.15% n-
decylamine in 0.02 M MOPS (3-morpholinopropanesulfonic acid)
buffer pH 7.4 (prepared in 1-octanol-saturated water). These
capacity factors (k0) are extrapolated to 100% of the aqueous
component given the value of kw0. The log Po/w is then obtained by
0
the formula log Po/w ¼ 0.13418 þ 0.98452 log kw
.
4.4.4. Culture conditions
[20] A. Vessières, S. Top, P. Pigeon, E.A. Hillard, L. Boubeker, D. Spera, G. Jaouen,
J. Med. Chem. 48 (2005) 3937.
Cells were maintained in monolayer culture in DMEM or RPMI
1640 with phenol red/Glutamax I, supplemented with 9% of
decomplemented fetal bovine serum and 0.9% kanamycine, at
37 ꢀC in a 5% CO2 air humidified incubator. For proliferation assays,
PC-3 cells were seeded at a density of 15,000e25,000 cells per mL
and 25,000e35,000 cells per mL for LNCaP in 24-well sterile
plates in 1 mL of medium, supplemented with 9% of fetal bovine
serum desteroided on dextran charcoal, 0.9% Glutamax I and 0.9%
kanamycine, and were incubated 24 h. The following day (D0),
1 mL of the same medium containing the compounds to be tested
diluted in DMSO, was added to the plates (three wells for each
product). After three days (D3), the incubation medium was
removed and 2 mL of fresh medium containing the compounds
was added. At different days (D3, D4, D5), the protein content of
each well was quantified by methylene blue staining as follows.
Cell monolayers were fixed and stained for 1 h in methanol with
methylene blue (2.5 mg/mL), and then washed thoroughly with
water. Two milliliters of HCl (0.1 M) was then added, and the plate
was incubated for 1 h at 37 ꢀC. Then the absorbance of each well
was measured at 655 nm with a Biorad spectrophotometer
(microplate reader). The results are expressed as the percentage of
proteins versus the control. Experiments were performed at least
in duplicate.
[21] O. Payen, S. Top, A. Vessières, E. Brulé, M.-A. Plamont, M.J. McGlinchey,
H. Müller-Bunz, G. Jaouen, J. Med. Chem. 51 (2008) 1791.
[22] S. Top, C. Thibaudeau, A. Vessières, E. Brulé, F. Le Bideau, J.-M. Joerger,
M.-A. Plamont, S. Samreth, A. Edgar, J. Marrot, P. Herson, G. Jaouen,
Organometallics 28 (2009) 1414.
[23] H. He, J.E. Morely, E. Silva-Lopez, B. Bottenus, M. Montajano, G.A. Fugate,
B. Twamley, P.D. Benny, Bioconjug. Chem. 20 (2009) 78.
[24] A.L.Moore, D.K.Bucar, L.R. MacGillivray,P.D.Benny,DaltonTrans. 39(2010)1926.
[25] T. Dallagi, S. Top, S. Masi, G. Jaouen, M. Saidi, Metallomics 2 (2010) 289.
[26] B.C. Chen, R. Zhao, S. Gove, B. Wang, J.E. Sundeen, M.E. Salvati, J.C. Barrish,
J. Org. Chem. 68 (2003) 10181.
[27] L. Kirkovsky, A. Mukherjee, D. Yin, J.T. Dalton, D.D. Miller, J. Med. Chem. 43
(2000) 581.
[28] R. Patil, W. Li, C.R. Ross, E. Kraka, D. Cremer, M.L. Mohler, J.T. Dalton,
D.D. Miller, Tetrahedron Lett. 47 (2006) 3941.
[29] H. Tucker, G.J. Chesterson, J. Med. Chem. 31 (1988) 885.
[30] A.L.G. Schuurmans, J. Bolt, M.M. Voorhorst, R.A. Blankenstein, E. Mulder, Int.
J. Cancer 42 (1988) 917.
[31] A.L.G. Schuurmans, J. Bolt, J. Veldscholte, E. Mulder, J. Steroid Biochem. Mol.
Biol. 37 (1990) 849.
[32] G. Wilding, M. Chen, E.P. Gelmann, Prostate 14 (1989) 103.
[33] J. Veldscholte, C. Risstalpers, G.G.J.M. Kuiper, G. Jenster, C. Berrevoets,
E. Claassen, H.C.J. Vanrooij, J. Trapman, A.O. Brinkmann, E. Mulder, Biochem.
Biophys. Res. Commun. 173 (1990) 534.
[34] K.D. James, N.N. Ekwuribe, Tetrahedron 58 (2002) 5905.
[35] J.J. Morris, L.R. Hughes, A.T. Glen, P.J. Taylor, J. Med. Chem. 34 (1991) 447.
[36] G.M. Sheldrick, SADABS. Bruker AXR Inc., Madison, WI 53711, 2000.
[37] G.M. Sheldrick, SHELXS-97. University of Göttingen, 1997.
[38] G.M. Sheldrick, SHELXL-97-2. University of Göttingen, 1997.